CROSS-REFERENCE This application claims the benefit of U.S. Provisional Application No. 62/445,570, filed Jan. 12, 2017; U.S. Provisional Application No. 62/445,557, filed Jan. 12, 2017; U.S. Provisional Application No. 62/447,889, filed Jan. 18, 2017; U.S. Provisional Application No. 62/467,032, filed Mar. 3, 2017; U.S. Provisional Application No. 62/566,199, filed Sep. 29, 2017; and U.S. Provisional Application No. 62/577,147, filed Oct. 25, 2017, which applications are incorporated herein by reference.
STATEMENT AS TO FEDERALLY SPONSORED RESEARCH This invention was made with the support of the United States government under SBIR grant 1520545 awarded by the National Science Foundation. The government has certain rights in the disclosed subject matter.
STATEMENT REGARDING SEQUENCE LISTING The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jan. 3, 2018, is named 47736-707_601_SL.txt and is ≈599 kb in size.
BACKGROUND OF THE INVENTION Plants are linked to the microbiome via a shared metabolome. A multidimensional relationship between a particular crop trait and the underlying metabolome is characterized by a landscape with numerous local maxima. Optimizing from an inferior local maximum to another representing a better trait by altering the influence of the microbiome on the metabolome may be desirable for a variety of reasons, such as for crop optimization. Economically-, environmentally-, and socially-sustainable approaches to agriculture and food production are required to meet the needs of a growing global population. By 2050 the United Nations' Food and Agriculture Organization projects that total food production must increase by 70% to meet the needs of the growing population, a challenge that is exacerbated by numerous factors, including diminishing freshwater resources, increasing competition for arable land, rising energy prices, increasing input costs, and the likely need for crops to adapt to the pressures of a drier, hotter, and more extreme global climate.
One area of interest is in the improvement of nitrogen fixation. Nitrogen gas (N2) is a major component of the atmosphere of Earth. In addition, elemental nitrogen (N) is an important component of many chemical compounds which make up living organisms. However, many organisms cannot use N2 directly to synthesize the chemicals used in physiological processes, such as growth and reproduction. In order to utilize the N2, the N2 must be combined with hydrogen. The combining of hydrogen with N2 is referred to as nitrogen fixation. Nitrogen fixation, whether accomplished chemically or biologically, requires an investment of large amounts of energy. In biological systems, an enzyme known as nitrogenase catalyzes the reaction which results in nitrogen fixation. An important goal of nitrogen fixation research is the extension of this phenotype to non-leguminous plants, particularly to important agronomic grasses such as wheat, rice, and maize. Despite enormous progress in understanding the development of the nitrogen-fixing symbiosis between rhizobia and legumes, the path to use that knowledge to induce nitrogen-fixing nodules on non-leguminous crops is still not clear. Meanwhile, the challenge of providing sufficient supplemental sources of nitrogen, such as in fertilizer, will continue to increase with the growing need for increased food production.
SUMMARY OF THE INVENTION An aspect of the invention provides a bacterial composition that comprises at least one genetically engineered bacterial strain that fixes atmospheric nitrogen in an agricultural system that has been fertilized with more than 20 lbs of Nitrogen per acre.
Another aspect of the invention provides a bacterial composition that comprises at least one bacterial strain that has been bred to fix atmospheric nitrogen in an agricultural system that has been fertilized with more than 20 lbs of Nitrogen per acre.
An additional aspect of the invention provides a bacterial composition that comprises at least one genetically engineered bacterial strain that fixes atmospheric nitrogen, the at least one genetically engineered bacterial strain comprising exogenously added DNA wherein said exogenously added DNA shares at least 80% identity to a corresponding native bacterial strain.
A further aspect of the invention provides a seed composition comprising a seed of a plant that is inoculated with a bacterial composition.
Another aspect of the invention provides a method of growing a crop using a plurality of seeds having a seed composition that is inoculated with a bacterial composition.
An additional aspect of the invention provides a method of applying a bacterial composition to a field.
A further aspect of the invention provides a fertilizer composition comprising a bacterial composition.
Another aspect of the invention provides a method of maintaining soil nitrogen levels. The method comprises planting, in soil of a field, a crop inoculated by a genetically engineered bacterium that fixes atmospheric nitrogen. The method also comprises harvesting said crop, wherein no more than 90% of a nitrogen dose required for producing said crop is administered to said soil of said field between planting and harvesting.
An additional aspect of the invention provides a method of delivering a probiotic supplement to a crop plant. The method comprises coating a crop seed with a seed coating, seed treatment, or seed dressing. Said seed coating, seed dressing, or seed treatment comprises living representatives of said probiotic. Additionally, the method comprises applying, in soil of a field, said crop seeds.
In a further aspect of the invention, the genetically engineered bacterial strain is a genetically engineered Gram-positive bacterial strain. In some cases, the genetically engineered Gram-positive bacterial strain has an altered expression level of a regulator of a Nif cluster. In some cases, the genetically engineered Gram-positive bacterial strain expresses a decreased amount of a negative regulator of a Nif cluster. In some cases, the genetically engineered bacterial strain expresses a decreased amount of GlnR. In some cases, the genome of the genetically engineered bacterial strain encodes a polypeptide with at least 75% identity to a sequence from the Zehr lab NifH database. In some cases, the genome of the genetically engineered bacterial strain encodes a polypeptide with at least 85% identity to a sequence from the Zehr lab NifH database. In some cases, the genome of the genetically engineered bacterial strain encodes a polypeptide with at least 75% identity to a sequence from the Buckley lab NifH database. In some cases, the genome of the genetically engineered bacterial strain encodes a polypeptide with at least 85% identity to a sequence from the Buckley lab NifH database.
Another aspect of the invention provides a method of increasing nitrogen fixation in a non-leguminous plant. The method comprises applying to the plant a plurality of non-intergeneric bacteria, said plurality comprising non-intergeneric bacteria that (i) have an average colonization ability per unit of plant root tissue of at least about 1.0×104 bacterial cells per gram of fresh weight of plant root tissue and (ii) produce fixed N of at least about 1×10−17 mmol N per bacterial cell per hour. Additionally, the plurality of non-intergeneric bacteria, in planta, produce 1% or more of the fixed nitrogen in the plant. Further, each member of the plurality of non-intergeneric bacteria comprises at least one genetic variation introduced into at least one gene, or non-coding polynucleotide, of the nitrogen fixation or assimilation genetic regulatory network, such that the non-intergeneric bacteria are capable of fixing atmospheric nitrogen in the presence of exogenous nitrogen.
An additional aspect of the invention provides a method of increasing nitrogen fixation in a non-leguminous plant. The method comprises applying to the plant a plurality of non-intergeneric bacteria, said plurality comprising non-intergeneric bacteria that (i) have an average colonization ability per unit of plant root tissue of at least about 1.0×104 bacterial cells per gram of fresh weight of plant root tissue and/or (ii) produce fixed N of at least about 1×10−17 mmol N per bacterial cell per hour. Additionally, the plurality of non-intergeneric bacteria, in planta, produce 1% or more of the fixed nitrogen in the plant. Further, each member of the plurality of non-intergeneric bacteria comprises at least one genetic variation introduced into at least one gene, or non-coding polynucleotide, of the nitrogen fixation or assimilation genetic regulatory network, such that the non-intergeneric bacteria are capable of fixing atmospheric nitrogen in the presence of exogenous nitrogen.
A further aspect of the invention provides a method of breeding microbial strains to improve specific traits of agronomic relevance. The method comprises providing a plurality of microbial strains that have the ability to colonize a desired crop. The method also comprises improving regulatory networks influencing the trait through intragenomic rearrangement. Further, the method comprises assessing microbial strains within the plurality of microbial strains to determine a measure of the trait. Additionally, the method comprises selecting one or more microbial strains of the plurality of microbial strains that generate an improvement in the trait in the presence of the desired crop.
Another aspect of the invention provides a method of breeding microbial strains to improve specific traits of agronomic relevance. The method comprises providing a plurality of microbial strains that have the ability to colonize a desired crop. The method also comprises introducing genetic diversity into the plurality of microbial strains. Additionally, the method comprises assessing microbial strains within the plurality of microbial strains to determine a measure of the trait. Further, the method comprises selecting one or more microbial strains of the plurality of microbial strains that generate an improvement in the trait in die presence of the desired crop.
Another aspect of the invention provides a method of increasing the amount of atmospheric derived nitrogen in a non-leguminous plant. The method comprises exposing said non-leguminous plant to engineered non-intergeneric microbes, said engineered non-intergeneric microbes comprising at least one genetic variation introduced into a nitrogen fixation genetic regulatory network or at least one genetic variation introduced into a nitrogen assimilation genetic regulatory network.
A further aspect of the invention provides a method of increasing an amount of atmospheric derived nitrogen in a corn plant. The method comprises exposing said corn plant to engineered non-intergeneric microbes comprising engineered genetic variations within at least two genes selected from the group consisting of nifL, glnB, glnE, and amtB.
Another aspect of the invention provides a method of increasing an amount of atmospheric derived nitrogen in a corn plant. The method comprises exposing said corn plant to engineered non-intergeneric microbes comprising at least one genetic variation introduced into a nitrogen fixation genetic regulatory network and at least one genetic variation introduced into a nitrogen assimilation genetic regulatory network, wherein said engineered non-intergeneric microbes, in planta, produce at least 5% of fixed nitrogen in said corn plant as measured by dilution of 15N in crops grown in fields treated with fertilizer containing 1.2% 15N.
An additional aspect of the invention provides a method of increasing nitrogen fixation in a non-leguminous plant. The method comprises applying to the plant a plurality of non-intergeneric bacteria, said plurality comprising non-intergeneric bacteria that (i) have an average colonization ability per unit of plant root tissue of at least about 1.0×104 bacterial cells per gram of fresh weight of plant root tissue and (ii) produce fixed N of at least about 1×10−17 mmol N per bacterial cell per hour. Additionally, the product of (i) the average colonization ability per unit of plant root tissue and (ii) produced fixed N per bacterial cell per hour, is at least about 2.5×10−8 mmol N per grain of fresh weight of plant root tissue per hour. Further, the plurality of non-intergeneric bacteria, in planta, produce 1% or more of the fixed nitrogen in the plant. Additionally, each member of the plurality of non-intergeneric bacteria comprises at least one genetic variation introduced into at least one gene, or non-coding polynucleotide, of the nitrogen fixation or assimilation genetic regulatory network, such that the non-intergeneric bacteria are capable of fixing atmospheric nitrogen in the presence of exogenous nitrogen.
Another aspect of the invention provides a method of increasing nitrogen fixation in a non-leguminous plant. The method comprises applying to the plant a plurality of bacteria, said plurality comprising bacteria that (i) have an average colonization ability per unit of plant root tissue of at least about 1.0×104 bacterial cells per gram of fresh weight of plant root tissue and/or (ii) produce fixed N of at least about 1×10−17 mmol N per bacterial cell per hour. Additionally, the plurality of bacteria, in planta, produce 1% or more of the fixed nitrogen in the plant.
An additional aspect of the invention provides a non-intergeneric bacterial population capable of increasing nitrogen fixation in a non-leguminous plant, comprising a plurality of non-intergeneric bacteria that (i) have an average colonization ability per unit of plant root tissue of at least about 1.0×104 bacterial cells per gram of fresh weight of plant root tissue and/or (ii) produce fixed N of at least about 1×10−17 mmol N per bacterial cell per hour. Additionally, the plurality of non-intergeneric bacteria, in planta, produce 1% or more of the fixed nitrogen in a plant grown in the presence of the plurality of non-intergeneric bacteria. Further, each member of the plurality of non-intergeneric bacteria comprises at least one genetic variation introduced into at least one gene, or non-coding polynucleotide, of the nitrogen fixation or assimilation genetic regulatory network, such that the non-intergeneric bacteria are capable of fixing atmospheric nitrogen in die presence of exogenous nitrogen.
A further aspect of the invention provides a bacterial population capable of increasing nitrogen fixation in a non-leguminous plant, the bacterial population comprising a plurality of bacteria that (i) have an average colonization ability per unit of plant root tissue of at least about 1.0×104 bacterial cells per gram of fresh weight of plant root tissue; and/or (ii) produce fixed N of at least about 1×10−17 mmol N per bacterial cell per hour. Additionally, the plurality of bacteria, in planta, produce 1% or more of the fixed nitrogen in the plant.
In another aspect of the invention, a bacterium is provided that (i) has an average colonization ability per unit of plant root tissue of at least about 1.0×104 bacterial cells per gram of fresh weight of plant root tissue and/or (ii) produces fixed N of at least about 1×10−17 mmol N per bacterial cell per hour.
In a further aspect of the invention, a non-intergeneric bacterium is provided that comprises at least one genetic variation introduced into at least one gene, or non-coding polynucleotide, of the nitrogen fixation or assimilation genetic regulatory network, such that the non-intergeneric bacterium is capable of fixing atmospheric nitrogen in the presence of exogenous nitrogen, and wherein said bacterium (i) has an average colonization ability per unit of plant root tissue of at least about 1.0×104 bacterial cells per gram of fresh weight of plant root tissue and/or (ii) produces fixed N of at least about 1×10−17 mmol N per bacterial cell per hour.
In an additional aspect of the invention provides a method for increasing nitrogen fixation in a plant, comprising administering to the plant an effective amount of a composition that comprises a purified population of bacteria that comprises bacteria with a 16S nucleic acid sequence that is at least about 97% identical to a nucleic acid sequence selected from SEQ ID NOs: 85, 96, 111, 121, 122, 123, 124, 136, 149, 157, 167, 261, 262, 269, 277-283; a purified population of bacteria that comprises bacteria with a nucleic acid sequence that is at least about 90% identical to a nucleic acid sequence selected from SEQ ID NOs: 86-95, 97-110, 112-120, 125-135, 137-148, 150-156, 158-166, 168-176, 263-268, 270-274, 275, 276, 284-295; and/or a purified population of bacteria that comprises bacteria with a nucleic acid sequence that is at least about 90% identical to a nucleic acid sequence selected from SEQ ID NOs: 177-260, 296-303; and wherein the plant administered the effective amount of the composition exhibits an increase in nitrogen fixation, as compared to a plant not having been administered the composition.
A further aspect of the invention provides an isolated bacteria comprising a 16S nucleic acid sequence that is at least about 97% identical to a nucleic acid sequence selected from SEQ ID NOs: 85, 96, 111, 121, 122, 123, 124, 136, 149, 157, 167, 261, 262, 269, 277-283; a nucleic acid sequence that is at least about 90% identical to a nucleic acid sequence selected from SEQ ID NOs: 86-95, 97-110, 112-120, 125-135, 137-148, 150-156, 158-166, 168-176, 263-268, 270-274, 275, 276, 284-295; and/or a nucleic acid sequence that is at least about 90% identical to a nucleic acid sequence selected from SEQ ID NOs: 177-260, 296-303.
Another aspect of the invention provides a method of detecting a non-native junction sequence, comprising: amplifying a nucleotide sequence that shares at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 372-405.
An additional aspect of the invention provides a method of detecting a non-native junction sequence, comprising: amplifying a nucleotide sequence that shares at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least a 10 contiguous base pair fragment contained in SEQ ID NOs: 372-405, said contiguous base pair fragment being comprised of nucleotides at the intersection of: an upstream sequence comprising SEQ ID NOs: 304-337 and downstream sequence comprising SEQ ID NOs: 338-371.
A further aspect of the invention provides a non-native junction sequence comprising a nucleotide sequence that shares at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 884/0, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 372-405.
An additional aspect of the invention provides a non-native junction sequence comprising a nucleotide sequence that shares at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least a 10 contiguous base pair fragment contained in SEQ ID NOs: 372-405, said contiguous base pair fragment being comprised of nucleotides at the intersection of: an upstream sequence comprising SEQ ID NOs: 304-337 and downstream sequence comprising SEQ ED NOs: 338-371.
A further aspect of the invention provides a bacterial composition comprising at least one remodeled bacterial strain that fixes atmospheric nitrogen, the at least one remodeled bacterial strain comprising exogenously added DNA wherein said exogenously added DNA shares at least 80% identity to a corresponding native bacterial strain.
An additional aspect of the invention provides a method of maintaining soil nitrogen levels. The method comprises planting, in soil of a field, a crop inoculated by a remodeled bacterium that fixes atmospheric nitrogen. The method also comprises harvesting said crop, wherein no more than 90% of a nitrogen dose required for producing said crop is administered to said soil of said field between planting and harvesting.
Another aspect of the invention provides a method of delivering a probiotic supplement to a crop plant. The method comprises coating a crop seed with a seed coating, seed treatment, or seed dressing, wherein said seed coating, seed dressing, or seed treatment comprise living representatives of said probiotic. The method also comprises applying said crop seeds in soil of a field.
An additional aspect of the invention provides a method of increasing the amount of atmospheric derived nitrogen in a non-leguminous plant. The method comprises exposing said non-leguminous plant to remodeled non-intergeneric microbes, said remodeled non-intergeneric microbes comprising at least one genetic variation introduced into a nitrogen fixation genetic regulatory network or at least one genetic variation introduced into a nitrogen assimilation genetic regulatory network.
A further aspect of the invention provides a method of increasing an amount of atmospheric derived nitrogen in a corn plant. The method comprises exposing said corn plant to remodeled non-intergeneric microbes comprising remodeled genetic variations within at least two genes selected from the group consisting of nifL, glnB, glnE, and amtB.
Another aspect of the invention provides a method of increasing an amount of atmospheric derived nitrogen in a corn plant. The method comprises exposing said corn plant to remodeled non-intergeneric microbes comprising at least one genetic variation introduced into a nitrogen fixation genetic regulatory network and at least one genetic variation introduced into a nitrogen assimilation genetic regulatory network, wherein said remodeled non-intergeneric microbes, in planta, produce at least 5% of fixed nitrogen in said corn plant as measured by dilution of 15N in crops grown in fields treated with fertilizer containing 1.2% 15N.
Additional aspects of the invention provide genus of microbes that are evolved and optimized for in planta nitrogen fixation in non-leguminous crops. In particular, methods of increasing nitrogen fixation in a non-leguminous plant are disclosed. The methods can comprise exposing the plant to a plurality of bacteria. Each member of the plurality comprises one or more genetic variations introduced into one or more genes of non-coding polynucleotides of the bacteria's nitrogen fixation or assimilation genetic regulatory network, such that the bacteria are capable of fixing atmospheric nitrogen in the presence of exogenous nitrogen. The bacteria are not intergeneric microorganisms. Additionally, the bacteria, in planta, produce 1% or more of the fixed nitrogen in the plant.
Further aspects of the invention provide beneficial isolated microbes and microbial compositions. In particular, isolated and biologically pure microorganisms that have applications, inter alia, in increasing nitrogen fixation in a crop are provided. The disclosed microorganism can be utilized in their isolated and biologically pure states, as well as being formulated into compositions. Furthermore, the disclosure provides microbial compositions containing at least two members of the disclosed microorganisms, as well as methods of utilizing said microbial compositions.
INCORPORATION BY REFERENCE All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:
FIGS. 1A-B depict enrichment and isolation of nitrogen fixing bacteria. (A) Nfb agar plate was used to isolate single colonies of nitrogen fixing bacteria. (B) Semi-solid Nfb agar casted in Balch tube. The arrow points to pellicle of enriched nitrogen fixing bacteria.
FIG. 2 depicts a representative nifH PCR screen. Positive bands were observed at ˜350 bp for two colonies in this screen. Lower bands represent primer-dimers.
FIG. 3 depicts an example of a PCR screen of colonies from CRISPR-Cas-selected mutagenesis. CI006 colonies were screened with primers specific for the nifL locus. The wild type PCR product is expected at ˜2.2 kb, whereas the mutant is expected at ˜1.1 kb. Seven of ten colonies screened unambiguously show the desired deletion.
FIGS. 4A-D depict in vitro phenotypes of various strains. The Acetylene Reduction Assay (ARA) activities of mutants of strain CI010 (FIG. 4A) and mutants of strain CI006 (FIG. 4B) grown in nitrogen fixation media supplemented with 0 to 10 mM glutamine. ARA activities of additional strains are shown in FIG. 4C, and the ammonium excretion profile across time of two strains is shown in FIG. 4D.
FIG. 5 depicts in culture expression profile of 9 different genes in strains CI006 involved in diazaotrophic nitrogen fixation. Numbers represent counts of each transcript. Various conditions (0, 1, 10 mM Glutamine and 0%, 10%, 20% atmospheric air in N2) are indicated.
FIG. 6 depicts CI006 colonization of corn roots. Corn seedlings were inoculated with CI006 harboring an RFP expression plasmid. After two weeks of growth and plasmid maintenance through watering with the appropriate antibiotic, roots were harvested and imaged through fluorescence microscopy. Colonization of the root intercellular space is observed.
FIG. 7 depicts nitrogen derived from microbe level in WT (CI050) and optimized (CM002) strain.
FIG. 8 shows an experimental setup for a Micro-Tom fruiting mass assay.
FIG. 9 shows a screen of 10 strains for increase in Micro-Tom plant fruit mass. Results for six replicates are presented. For column 3, p=0.07. For column 7, p=0.05.
FIGS. 10A-C depict additional results for ARA activities of candidate microbes and counterpart candidate mutants grown in nitrogen fixation media supplemented with 0 to 10 mM glutamine.
FIG. 11 depicts a double mutant that exhibits higher ammonia excretion than the single mutant from which it was derived.
FIG. 12 depicts NDFA obtained from 15N Gas Uptake experiment (extrapolated back using days exposed) to measure NDFA in Corn plants in fertilized condition.
FIG. 13 depicts NDFA value obtained from 15N Gas Uptake experiment (extrapolated back using days exposed) to measure NDFA in Setaria plants in fertilized condition.
FIG. 14A depicts rate of incorporation of 15N gas. Plants inoculated with evolved strain showed increase in 151 gas incorporation compared to uninoculated plants.
FIG. 14B depicts 4 weeks after planting, up to 7% of the nitrogen in plants inoculated with an evolved strain is derived from microbially fixed nitrogen.
FIG. 14C depicts leaf area (and other biomass measurement, data not shown) is increased in plants inoculated with an evolved strain when compared to uninoculated or wild type inoculated plants.
FIG. 15A depicts evolved strains that show significantly higher nifH production in the root tissue, as measured by in planta transcriptomic study.
FIG. 15B depicts that rate of fixed nitrogen found in plant tissue is correlated with the rate in which that particular plant is colonized by HoME optimized strain.
FIG. 16A depicts a soil texture map of various field soils tested for colonization. Soils in which a few microbes were originally source from are indicated as stars.
FIG. 16B depicts the colonization rate of Strain 1 and Strain 5 that are tested across four different soil types (circles). Both strains showed relatively robust colonization profile across diverse soil types.
FIG. 16C depicts colonization of Strain 1 as tested in a field trial over the span of a growing season. Strain 1 persists in the corn tissue up to week 12 after planting and starts to show decline in colonization after that time.
FIG. 17A depicts a schematic of microbe breeding, in accordance with embodiments.
FIG. 17B depicts an expanded view of the measurement of microbiome composition as shown in FIG. 17A.
FIG. 17C depicts sampling of roots utilized in Example 7.
FIG. 18 depicts the lineage of modified strains that were derived from strain CI006.
FIG. 19 depicts the lineage of modified strains that were derived from strain CI019.
FIG. 20 depicts a heatmap of the pounds of nitrogen delivered per acre-season by microbes of the present disclosure recorded as a function of microbes per g-fresh weight by mmol of nitrogen/microbe-hr. Below the thin line that transects the larger image are the microbes that deliver less than one pound of nitrogen per acre-season, and above the line are the microbes that deliver greater than one pound of nitrogen per acre-season. The table below the heatmap gives the precise value of mmol N produced per microbe per hour (mmol N/Microbe hr) along with the precise CFU per gram of fresh weight (CFU/g fw) for each microbe shown in the heatmap. The microbes utilized in the heatmap were assayed for N production in corn. For the WT strains CI006 and CI019, corn root colonization data was taken from a single field site. For the remaining strains, colonization was assumed to be the same as the % VT field level. N-fixation activity was determined using an in vitro ARA assay at 5 mM glutamine.
FIG. 21 depicts the plant yield of plants having been exposed to strain CI006. The area of the circles corresponds to the relative yield, while the shading corresponds to the particular MRTN treatment. The x-axis is the p value and the y-axis is the win rate.
FIG. 22 depicts the plant yield of plants having been exposed to strain CM029. The area of the circles corresponds to the relative yield, while the shading corresponds to the particular MRTN treatment. The x-axis is the p value and the y-axis is the win rate.
FIG. 23 depicts the plant yield of plants having been exposed to strain CM038. The area of the circles corresponds to the relative yield, while the shading corresponds to the particular MRTN treatment. The x-axis is the p value and the y-axis is the win rate.
FIG. 24 depicts the plant yield of plants having been exposed to strain CI019. The area of the circles corresponds to the relative yield, while the shading corresponds to the particular MRTN treatment. The x-axis is the p value and the y-axis is the win rate.
FIG. 25 depicts die plant yield of plants having been exposed to strain CM081. The area of the circles corresponds to the relative yield, while the shading corresponds to the particular MRTN treatment. The x-axis is the p value and the y-axis is the win rate.
FIG. 26 depicts the plant yield of plants having been exposed to strains CM029 and CM081. The area of the circles corresponds to the relative yield, while the shading corresponds to the particular MRTN treatment. The x-axis is the p value and the y-axis is the win rate.
FIG. 27 depicts the plant yield of plants as the aggregated bushel gain/loss. The area of the circles corresponds to the relative yield, while the shading corresponds to the particular MRTN treatment. The x-axis is the p value and the y-axis is the win rate.
FIGS. 28A-28E illustrate derivative microbes that fix and excrete nitrogen in vitro under conditions similar to high nitrate agricultural soils. FIG. 28A illustrates the regulatory network controlling nitrogen fixation and assimilation in PBC6.1 is shown, including the key nodes NifL, NifA, GS, GlnE depicted as the two-domain ATase-AR enzyme, and AmtB. FIG. 28B illustrates the genome of Kosakonia sacchari isolate PBC6.1 is shown. The three tracks circumscribing the genome convey transcription data from PBC6.1, PBC6.38, and the differential expression between the strains respectively. FIG. 28C illustrates the nitrogen fixation gene cluster and transcription data is expanded for finer detail. FIG. 28D illustrates nitrogenase activity under varying concentrations of exogenous nitrogen is measured with the acetylene reduction assay. The wild type strain exhibits repression of nitrogenase activity as glutamine concentrations increase, while derivative strains show varying degrees of robustness. Error bars represent standard error of the mean of at least three biological replicates. FIG. 28E illustrates temporal excretion of ammonia by derivative strains is observed at mM concentrations. Wild type strains are not observed to excrete fixed nitrogen, and negligible ammonia accumulates in the media. Error bars represent standard error of the mean.
FIGS. 29A-29C illustrate greenhouse experiments demonstrate microbial nitrogen fixation in corn. FIG. 29A illustrates microbe colonization six weeks after inoculation of corn plants by PBC6.1 derivative strains. Error bars show standard error of the mean of at least eight biological replicates. FIG. 29B illustrates in planta transcription of nifH measured by extraction of total RNA from roots and subsequent Nanostring analysis. Only derivative strains show WIN transcription in the root environment. Error bars show standard error of the mean of at least 3 biological replicates. FIG. 29C illustrates microbial nitrogen fixation measured by the dilution of isotopic tracer in plant tissues. Derivative microbes exhibit substantial transfer of fixed nitrogen to the plant. Error bars show standard error of the mean of at least ten biological replicates.
FIG. 30 illustrates PBC6.1 colonization to nearly 21% abundance of the root-associated microbiota in corn roots. Abundance data is based on 16S amplicon sequencing of the rhizosphere and endosphere of corn plants inoculated with PBC6.1 and grown in greenhouse conditions.
FIG. 31 illustrates transcriptional rates of nifA in derivative strains of PBC6.1 correlated with acetylene reduction rates. An ARA assay was performed as described in the Methods, after which cultures were sampled and subjected to qPCR analysis to determine nifA transcript levels. Error bars show standard error of the mean of at least three biological replicates in each measure.
FIG. 32 illustrates results from a summer 2017 field testing experiment. The yield results obtained demonstrate that the microbes of the disclosure can serve as a potential fertilizer replacement. For instance, the utilization of a microbe of the disclosure (i.e. 6-403) resulted in a higher yield than the wild type strain (WT) and a higher yield than the untreated control (UTC). The “−25 lbs N” treatment utilizes 25 lbs less N per acre than standard agricultural practices of the region. The “100% N” UTC treatment is meant to depict standard agricultural practices of the region, in which 100% of the standard utilization of N is deployed by the farmer. The microbe “6-403” was deposited as NCMA 201708004 and can be found in Table A. This is a mutant Kosakonia sacchari (also called CM037) and is a progeny mutant strain from CI006 WT.
FIG. 33 illustrates results from a summer 2017 field testing experiment. The yield results obtained demonstrate that the microbes of the disclosure perform consistently across locations. Furthermore, the yield results demonstrate that the microbes of the disclosure perform well in both a nitrogen stressed environment, as well as an environment that has sufficient supplies of nitrogen. The microbe “6-881” (also known as CM094, PBC6.94), and which is a progeny mutant Kosakonia sacchari strain from CI006 WT, was deposited as NCMA 201708002 and can be found in Table A. The microbe “137-1034,” which is a progeny mutant Klebsiella variicola strain from CI137 WT, was deposited as NCMA 201712001 and can be found in Table A. The microbe “137-1036,” which is a progeny mutant Klebsiella variicola strain from CI137 WT, was deposited as NCMA 201712002 and can be found in Table A. The microbe “6-404” (also known as CM38, PBC6.38), and which is a progeny mutant Kosakonia sacchari strain from CI006 WT, was deposited as NCMA 201708003 and can be found in Table A. The “Nutrient Stress” condition corresponds to the 0% nitrogen regime. The “Sufficient Fertilizer” condition corresponds to the 100% nitrogen regime.
FIG. 34 depicts the lineage of modified strains that were derived from strain CI006 (also termed “6”, Kosakonia sacchari WT).
FIG. 35 depicts the lineage of modified strains that were derived from strain CI019 (also termed “19”, Rahnella aquatilis WT).
FIG. 36 depicts the lineage of modified strains that were derived from strain CI137 (also termed (“137”, Klebsiella variicola WT).
FIG. 37 depicts the lineage of modified strains that were derived from strain 1021 (Kosakonia pseudosacchari WT).
FIG. 38 depicts the lineage of modified strains that were derived from strain 910 (Kluyvera intermedia WT).
FIG. 39 depicts the lineage of modified strains that were derived from strain 63 (Rahnella aquatilis WT).
FIG. 40 depicts a heatmap of the pounds of nitrogen delivered per acre-season by microbes of the present disclosure recorded as a function of microbes per g-fresh weight by mmol of nitrogen/microbe-hr. Below the thin line that transects the larger image are the microbes that deliver less than one pound of nitrogen per acre-season, and above the line are the microbes that deliver greater than one pound of nitrogen per acre-season. The Table C in Example 12 gives the precise value of mmol N produced per microbe per hour (mmol N/Microbe hr) along with the precise CFU per gram of fresh weight (CFU/g fw) for each microbe shown in the heatmap. The data in FIG. 40 is derived from microbial strains assayed for N production in corn in field conditions. Each point represents 1b N/acre produced by a microbe using corn root colonization data from a single field site. N-fixation activity was determined using in vitro ARA assay at 5 mM N in the form of glutamine or ammonium phosphate.
FIG. 41 depicts a heatmap of the pounds of nitrogen delivered per acre-season by microbes of the present disclosure recorded as a function of microbes per g-fresh weight by mmol of nitrogen/microbe-hr. Below the thin line that transects the larger image are the microbes that deliver less than one pound of nitrogen per acre-season, and above the line are the microbes that deliver greater than one pound of nitrogen per acre-season. The Table Din Example 12 gives the precise value of mmol N produced per microbe per hour (mmol N/Microbe hr) along with the precise CFU per gram of fresh weight (CFU/g fw) for each microbe shown in the heatmap. The data in FIG. 41 is derived from microbial strains assayed for N production in corn in laboratory and greenhouse conditions. Each point represents 1b N/acre produced by a single strain. White points represent strains in which corn root colonization data was gathered in greenhouse conditions. Black points represent mutant strains for which corn root colonization levels are derived from average field corn root colonization levels of the wild-type parent strain. Hatched points represent the wild type parent strains at their average field corn root colonization levels. In all cases, N-fixation activity was determined by in vitro ARA assay at 5 mM N in the form of glutamine or ammonium phosphate.
DETAILED DESCRIPTION OF THE INVENTION While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.
Increased fertilizer utilization brings with it environmental concerns and is also likely not possible for many economically stressed regions of the globe. Furthermore, many industry players in the microbial arena are focused on creating intergeneric microbes. However, there is a heavy regulatory burden placed on engineered microbes that are characterized/classified as intergeneric. These intergeneric microbes face not only a higher regulatory burden, which makes widespread adoption and implementation difficult, but they also face a great deal of public perception scrutiny.
Currently, there are no engineered microbes on the market that are non-intergeneric and that are capable of increasing nitrogen fixation in non-leguminous crops. This dearth of such a microbe is a missing element in helping to usher in a truly environmentally friendly and more sustainable 21st century agricultural system.
The present disclosure solves the aforementioned problems and provides a non-intergeneric microbe that has been engineered to readily fix nitrogen in crops. These microbes are not characterized/classified as intergeneric microbes and thus will not face the steep regulatory burdens of such. Further, the taught non-intergeneric microbes will serve to help 21st century farmers become less dependent upon utilizing ever increasing amounts of exogenous nitrogen fertilizer.
Definitions The terms “polynucleotide”, “nucleotide”, “nucleotide sequence”, “nucleic acid” and “oligonucleotide” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three dimensional structure, and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.
“Hybridization” refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues. The hydrogen bonding may occur by Watson Crick base pairing, Hoogstein binding, or in any other sequence specific manner according to base complementarity. The complex may comprise two strands forming a duplex structure, three or more strands forming a multi stranded complex, a single self-hybridizing strand, or any combination of these. A hybridization reaction may constitute a step in a more extensive process, such as the initiation of PCR, or the enzymatic cleavage of a polynucleotide by an endonuclease. A second sequence that is complementary to a first sequence is referred to as the “complement” of the first sequence. The term “hybridizable” as applied to a polynucleotide refers to the ability of the polynucleotide to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues in a hybridization reaction.
“Complementarity” refers to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types. A percent complementarity indicates the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary, respectively). “Perfectly complementary” means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. “Substantially complementary” as used herein refers to a degree of complementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions. Sequence identity, such as for the purpose of assessing percent complementarity, may be measured by any suitable alignment algorithm, including but not limited to the Needleman-Wunsch algorithm (see e.g. the EMBOSS Needle aligner available at www.ebi.ac.uk/Tools/psa/emboss_needle/nucleotide.html, optionally with default settings), the BLAST algorithm (see e.g. the BLAST alignment tool available at blast.ncbi.nlm.nih.gov/Blast.cgi, optionally with default settings), or the Smith-Waterman algorithm (see e.g. the EMBOSS Water aligner available at www.ebi.ac.uk/Tools/psa/emboss_water/nucleotide.html, optionally with default settings). Optimal alignment may be assessed using any suitable parameters of a chosen algorithm, including default parameters.
In general, “stringent conditions” for hybridization refer to conditions under which a nucleic acid having complementarity to a target sequence predominantly hybridizes with a target sequence, and substantially does not hybridize to non-target sequences. Stringent conditions are generally sequence-dependent and vary depending on a number of factors. In general, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes to its target sequence. Non-limiting examples of stringent conditions are described in detail in Tijssen (1993), Laboratory Techniques In Biochemistry And Molecular Biology-Hybridization With Nucleic Acid Probes Part I, Second Chapter “Overview of principles of hybridization and the strategy of nucleic acid probe assay”, Elsevier, N.Y.
As used herein, “expression” refers to the process by which a polynucleotide is transcribed from a DNA template (such as into and mRNA or other RNA transcript) and/or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides may be collectively referred to as “gene product.” If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.
The terms “polypeptide”, “peptide” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component. As used herein the term “amino acid” includes natural and/or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics.
As used herein, the term “about” is used synonymously with the term “approximately.” Illustratively, the use of the term “about” with regard to an amount indicates that values slightly outside the cited values, e.g., plus or minus 0.1% to 10%.
The term “biologically pure culture” or “substantially pure culture” refers to a culture of a bacterial species described herein containing no other bacterial species in quantities sufficient to interfere with the replication of the culture or be detected by normal bacteriological techniques.
“Plant productivity” refers generally to any aspect of growth or development of a plant that is a reason for which the plant is grown. For food crops, such as grains or vegetables, “plant productivity” can refer to the yield of grain or fruit harvested from a particular crop. As used herein, improved plant productivity refers broadly to improvements in yield of grain, fruit, flowers, or other plant parts harvested for various purposes, improvements in growth of plant parts, including stems, leaves and roots, promotion of plant growth, maintenance of high chlorophyll content in leaves, increasing fruit or seed numbers, increasing fruit or seed unit weight, reducing NO2 emission due to reduced nitrogen fertilizer usage and similar improvements of the growth and development of plants.
Microbes in and around food crops can influence the traits of those crops. Plant traits that may be influenced by microbes include: yield (e.g., grain production, biomass generation, fruit development, flower set); nutrition (e.g., nitrogen, phosphorus, potassium, iron, micronutrient acquisition); abiotic stress management (e.g., drought tolerance, salt tolerance, heat tolerance); and biotic stress management (e.g., pest, weeds, insects, fungi, and bacteria). Strategies for altering crop traits include: increasing key metabolite concentrations; changing temporal dynamics of microbe influence on key metabolites; linking microbial metabolite production/degradation to new environmental cues; reducing negative metabolites; and improving the balance of metabolites or underlying proteins.
As used herein, a “control sequence” refers to an operator, promoter, silencer, or terminator.
As used herein, “in planta” refers to in the plant, and wherein the plant further comprises plant parts, tissue, leaves, roots, stems, seed, ovules, pollen, flowers, fruit, etc.
In some embodiments, native or endogenous control sequences of genes of the present disclosure are replaced with one or more intrageneric control sequences.
As used herein, “introduced” refers to the introduction by means of modern biotechnology, and not a naturally occurring introduction.
In some embodiments, the bacteria of the present disclosure have been modified such that they are not naturally occurring bacteria.
In some embodiments, the bacteria of the present disclosure are present in the plant in an amount of at least 103 cfu, 104 cfu, 105 cfu, 106 cfu, 107 cfu, 108 cfu, 109 cfu, 1010 cfu, 1011 cfu, or 1012 cfu per gram of fresh or dry weight of the plant. In some embodiments, the bacteria of the present disclosure are present in the plant in an amount of at least about 103 cfu, about 104 cfu, about 105 cfu, about 106 cfu, about 107 cfu, about 108 cfu, about 109 cfu, about 1010 cfu, about 1011 du, or about 1012 cfu per gram of fresh or dry weight of the plant. In some embodiments, the bacteria of the present disclosure are present in the plant in an amount of at least 103 to 109, 103 to 107, 103 to 105, 105 to 109, 105 to 107, 106 to 1010, 106 to 107 cfu per gram of fresh or dry weight of the plant.
Fertilizers and exogenous nitrogen of the present disclosure may comprise the following nitrogen-containing molecules: ammonium, nitrate, nitrite, ammonia, glutamine, etc. Nitrogen sources of the present disclosure may include anhydrous ammonia, ammonia sulfate, urea, diammonium phosphate, urea-form, monoammonium phosphate, ammonium nitrate, nitrogen solutions, calcium nitrate, potassium nitrate, sodium nitrate, etc.
As used herein, “exogenous nitrogen” refers to non-atmospheric nitrogen readily available in the soil, field, or growth medium that is present under non-nitrogen limiting conditions, including ammonia, ammonium, nitrate, nitrite, urea, uric acid, ammonium acids, etc.
As used herein, “non-nitrogen limiting conditions” refers to non-atmospheric nitrogen available in the soil, field, media at concentrations greater than about 4 mM nitrogen, as disclosed by Kant et al. (2010. J. Exp. Biol. 62(4):1499-1509), which is incorporated herein by reference.
As used herein, an “intergeneric microorganism” is a microorganism that is formed by the deliberate combination of genetic material originally isolated from organisms of different taxonomic genera. An “intergeneric mutant” can be used interchangeably with “intergeneric microorganism”. An exemplary “intergeneric microorganism” includes a microorganism containing a mobile genetic element which was first identified in a microorganism in a genus different from the recipient microorganism. Further explanation can be found, inter alia, in 40 C.F.R. § 725.3.
In aspects, microbes taught herein are “non-intergeneric,” which means that the microbes are not intergeneric.
As used herein, an “intrageneric microorganism” is a microorganism that is formed by the deliberate combination of genetic material originally isolated from organisms of the same taxonomic genera. An “intrageneric mutant” can be used interchangeably with “intrageneric microorganism”.
As used herein, “introduced genetic material” means genetic material that is added to, and remains as a component of, die genome of the recipient.
In some embodiments, the nitrogen fixation and assimilation genetic regulatory network comprises polynucleotides encoding genes and non-coding sequences that direct, modulate, and/or regulate microbial nitrogen fixation and/or assimilation and can comprise polynucleotide sequences of the nif cluster (e.g., nifA, niFB, nifC, . . . , nifZ), polynucleotides encoding nitrogen regulatory protein C, polynucleotides encoding nitrogen regulatory protein B, polynucleotide sequences of the gin cluster (e.g. glnA and glnD), draT, and ammonia transporters/permeases. In some cases, the Nif cluster may comprise NifB, NifH, NifD, NifK, NifE, NifN, NifX, hesa, and NifV. In some cases, the Nif cluster may comprise a subset of NifB, NifH, NifD, NifK, NifE, NifN, NifX, hesa, and NifV.
In some embodiments, fertilizer of the present disclosure comprises at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 854/0, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% nitrogen by weight.
In some embodiments, fertilizer of the present disclosure comprises at least about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% nitrogen by weight.
In some embodiments, fertilizer of the present disclosure comprises about 5% to 50%, about 5% to 75%, about 10% to 50%, about 10% to 75%, about 15% to 50%, about 15% to 75%, about 20% to 50%, about 20% to 75%, about 25% to 50%, about 25% to 75%, about 30% to 50%, about 30% to 75%, about 35% to 50%, about 35% to 75%, about 40% to 50%, about 40% to 75%, about 45% to 50%, about 45% to 75%, or about 50% to 75% nitrogen by weight.
In some embodiments, the increase of nitrogen fixation and/or the production of 1% or more of the nitrogen in the plant are measured relative to control plants, which have not been exposed to the bacteria of the present disclosure. All increases or decreases in bacteria are measured relative to control bacteria. All increases or decreases in plants are measured relative to control plants.
As used herein, a “constitutive promoter” is a promoter, which is active under most conditions and/or during most development stages. There are several advantages to using constitutive promoters in expression vectors used in biotechnology, such as: high level of production of proteins used to select transgenic cells or organisms; high level of expression of reporter proteins or storable markers, allowing easy detection and quantification; high level of production of a transcription factor that is part of a regulatory transcription system; production of compounds that requires ubiquitous activity in the organism; and production of compounds that are required during all stages of development. Non-limiting exemplary constitutive promoters include, CaMV 35S promoter, opine promoters, ubiquitin promoter, alcohol dehydrogenase promoter, etc.
As used herein, a “non-constitutive promoter” is a promoter which is active under certain conditions, in certain types of cells, and/or during certain development stages. For example, tissue specific, tissue preferred, cell type specific, cell type preferred, inducible promoters, and promoters under development control are non-constitutive promoters. Examples of promoters under developmental control include promoters that preferentially initiate transcription in certain tissues.
As used herein, “inducible” or “repressible” promoter is a promoter which is under chemical or environmental factors control. Examples of environmental conditions that may affect transcription by inducible promoters include anaerobic conditions, certain chemicals, the presence of light, acidic or basic conditions, etc.
As used herein, a “tissue specific” promoter is a promoter that initiates transcription only in certain tissues. Unlike constitutive expression of genes, tissue-specific expression is the result of several interacting levels of gene regulation. As such, in the art sometimes it is preferable to use promoters from homologous or closely related species to achieve efficient and reliable expression of transgenes in particular tissues. This is one of the main reasons for the large amount of tissue-specific promoters isolated from particular tissues found in both scientific and patent literature.
As used herein, the term “operably linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is regulated by the other. For example, a promoter is operably linked with a coding sequence when it is capable of regulating the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in a sense or antisense orientation. In another example, the complementary RNA regions of the disclosure can be operably linked, either directly or indirectly, 5′ to the target mRNA, or 3′ to the target mRNA, or within the target mRNA, or a first complementary region is 5′ and its complement is 3′ to the target mRNA.
In aspects, “applying to the plant a plurality of non-intergeneric bacteria,” includes any means by which the plant (including plant parts such as a seed, root, stem, tissue, etc.) is made to come into contact (i.e. exposed) with said bacteria at any stage of the plant's life cycle. Consequently, “applying to the plant a plurality of non-intergeneric bacteria,” includes any of the following means of exposing the plant (including plant parts such as a seed, root, stem, tissue, etc.) to said bacteria: spraying onto plant, dripping onto plant, applying as a seed coat, applying to a field that will then be planted with seed, applying to a field already planted with seed, applying to a field with adult plants, etc.
As used herein “MRTN” is an acronym for maximum return to nitrogen and is utilized as an experimental treatment in the Examples. MRTN was developed by Iowa State University and information can be found at: http://cnrc.agron.iastate.edu/ The MRTN is the nitrogen rate where the economic net return to nitrogen application is maximized. The approach to calculating the MRTN is a regional approach for developing corn nitrogen rate guidelines in individual states. The nitrogen rate trial data was evaluated for Illinois, Iowa, Michigan, Minnesota, Ohio, and Wisconsin where an adequate number of research trials were available for corn plantings following soybean and corn plantings following corn. The trials were conducted with spring, sidedress, or split preplant/sidedress applied nitrogen, and sites were not irrigated except for those that were indicated for irrigated sands in Wisconsin. MRTN was developed by Iowa State University due to apparent differences in methods for determining suggested nitrogen rates required for corn production, misperceptions pertaining to nitrogen rate guidelines, and concerns about application rates. By calculating the MRTN, practitioners can determine the following: (1) the nitrogen rate where the economic net return to nitrogen application is maximized, (2) the economic optimum nitrogen rate, which is the point where the last increment of nitrogen returns a yield increase large enough to pay for the additional nitrogen, (3) the value of corn grain increase attributed to nitrogen application, and the maximum yield, which is the yield where application of more nitrogen does not result in a corn yield increase. Thus the MRTN calculations provide practitioners with the means to maximize corn crops in different regions while maximizing financial gains from nitrogen applications.
The term mmol is an abbreviation for millimole, which is a thousandth (10−3) of a mole, abbreviated herein as mol.
As used herein the terms “microorganism” or “microbe” should be taken broadly. These terms, used interchangeably, include but are not limited to, the two prokaryotic domains, Bacteria and Archaea. The term may also encompass eukaryotic fungi and protists.
The term “microbial consortia” or “microbial consortium” refers to a subset of a microbial community of individual microbial species, or strains of a species, which can be described as carrying out a common function, or can be described as participating in, or leading to, or correlating with, a recognizable parameter, such as a phenotypic trait of interest.
The term “microbial community” means a group of microbes comprising two or more species or strains. Unlike microbial consortia, a microbial community does not have to be carrying out a common function, or does not have to be participating in, or leading to, or correlating with, a recognizable parameter, such as a phenotypic trait of interest.
As used herein, “isolate,” “isolated,” “isolated microbe,” and like terms, are intended to mean that the one or more microorganisms has been separated from at least one of the materials with which it is associated in a particular environment (for example soil, water, plant tissue, etc.). Thus, an “isolated microbe” does not exist in its naturally occurring environment; rather, it is through the various techniques described herein that the microbe has been removed from its natural setting and placed into a non-naturally occurring state of existence. Thus, the isolated strain or isolated microbe may exist as, for example, a biologically pure culture, or as spores (or other forms of the strain). In aspects, the isolated microbe may be in association with an acceptable carrier, which may be an agriculturally acceptable carrier.
In certain aspects of the disclosure, the isolated microbes exist as “isolated and biologically pure cultures.” It will be appreciated by one of skill in the art, that an isolated and biologically pure culture of a particular microbe, denotes that said culture is substantially free of other living organisms and contains only the individual microbe in question. The culture can contain varying concentrations of said microbe. The present disclosure notes that isolated and biologically pure microbes often “necessarily differ from less pure or impure materials.” See, e.g. In re Bergstrom, 427 F.2d 1394, (CCPA 1970)(discussing purified prostaglandins), see also, In re Bergy, 596 F.2d 952 (CCPA 1979)(discussing purified microbes), see also, Parke-Davis & Co. v. H.K. Mulford & Co., 189 F. 95 (S.D.N.Y. 1911) (Learned Hand discussing purified adrenaline), aff'd in part, rev'd in part, 196 F. 496 (2d Cir. 1912), each of which are incorporated herein by reference. Furthermore, in some aspects, the disclosure provides for certain quantitative measures of the concentration, or purity limitations, that must be found within an isolated and biologically pure microbial culture. The presence of these purity values, in certain embodiments, is a further attribute that distinguishes the presently disclosed microbes from those microbes existing in a natural state. See, e.g., Merck & Co. v. Olin Mathieson Chemical Corp., 253 F.2d 156 (4th Cir. 1958) (discussing purity limitations for vitamin B12 produced by microbes), incorporated herein by reference.
As used herein, “individual isolates” should be taken to mean a composition, or culture, comprising a predominance of a single genera, species, or strain, of microorganism, following separation from one or more other microorganisms.
Microbes of the present disclosure may include spores and/or vegetative cells. In some embodiments, microbes of the present disclosure include microbes in a viable but non-culturable (VBNC) state. As used herein, “spore” or “spores” refer to structures produced by bacteria and fungi that are adapted for survival and dispersal. Spores are generally characterized as dormant structures; however, spores are capable of differentiation through the process of germination. Germination is the differentiation of spores into vegetative cells that are capable of metabolic activity, growth, and reproduction. The germination of a single spore results in a single fungal or bacterial vegetative cell. Fungal spores are units of asexual reproduction, and in some cases are necessary structures in fungal life cycles. Bacterial spores are structures for surviving conditions that may ordinarily be nonconducive to the survival or growth of vegetative cells.
As used herein, “microbial composition” refers to a composition comprising one or more microbes of the present disclosure. In some embodiments, a microbial composition is administered to plants (including various plant parts) and/or in agricultural fields.
As used herein, “carrier,” “acceptable carrier,” or “agriculturally acceptable carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the microbe can be administered, which does not detrimentally effect the microbe.
Regulation of Nitrogen Fixation In some cases, nitrogen fixation pathway may act as a target for genetic engineering and optimization. One trait that may be targeted for regulation by the methods described herein is nitrogen fixation. Nitrogen fertilizer is the largest operational expense on a farm and the biggest driver of higher yields in row crops like corn and wheat. Described herein are microbial products that can deliver renewable forms of nitrogen in non-leguminous crops. While some endophytes have the genetics necessary for fixing nitrogen in pure culture, the fundamental technical challenge is that wild-type endophytes of cereals and grasses stop fixing nitrogen in fertilized fields. The application of chemical fertilizers and residual nitrogen levels in field soils signal the microbe to shut down the biochemical pathway for nitrogen fixation.
Changes to the transcriptional and post-translational levels of components of the nitrogen fixation regulatory network may be beneficial to the development of a microbe capable of fixing and transferring nitrogen to corn in the presence of fertilizer. To that end, described herein is Host-Microbe Evolution (HoME) technology to precisely evolve regulatory networks and elicit novel phenotypes. Also described herein are unique, proprietary libraries of nitrogen-fixing endophytes isolated from corn, paired with extensive omics data surrounding the interaction of microbes and host plant under different environmental conditions like nitrogen stress and excess. In some embodiments, this technology enables precision evolution of the genetic regulatory network of endophytes to produce microbes that actively fix nitrogen even in the presence of fertilizer in the field. Also described herein are evaluations of the technical potential of evolving microbes that colonize corn root tissues and produce nitrogen for fertilized plants and evaluations of the compatibility of endophytes with standard formulation practices and diverse soils to determine feasibility of integrating the microbes into modern nitrogen management strategies.
In order to utilize elemental nitrogen (N) for chemical synthesis, life forms combine nitrogen gas (N2) available in the atmosphere with hydrogen in a process known as nitrogen fixation. Because of the energy-intensive nature of biological nitrogen fixation, diazotrophs (bacteria and archaea that fix atmospheric nitrogen gas) have evolved sophisticated and fight regulation of the nif gene cluster in response to environmental oxygen and available nitrogen. Nif genes encode enzymes involved in nitrogen fixation (such as the nitrogenase complex) and proteins that regulate nitrogen fixation. Shamseldin (2013. Global J. Biotechnol. Biochem. 8(4):84-94) discloses detailed descriptions of nif genes and their products, and is incorporated herein by reference. Described herein are methods of producing a plant with an improved trait comprising isolating bacteria from a first plant, introducing a genetic variation into a gene of the isolated bacteria to increase nitrogen fixation, exposing a second plant to the variant bacteria, isolating bacteria from the second plant having an improved trait relative to the first plant, and repeating the steps with bacteria isolated from the second plant.
In Proteobacteria, regulation of nitrogen fixation centers around the σ54-dependent enhancer-binding protein NifA, the positive transcriptional regulator of the nif cluster. Intracellular levels of active NifA are controlled by two key factors: transcription of the nifLA operon, and inhibition of NifA activity by protein-protein interaction with NifL. Both of these processes are responsive to intraceullar glutamine levels via the PII protein signaling cascade. This cascade is mediated by GlnD, which directly senses glutamine and catalyzes the uridylylation or deuridylylation of two PII regulatory proteins—GlnB and GlnK—in response the absence or presence, respectively, of bound glutamine. Under conditions of nitrogen excess, unmodified GlnB signals the deactivation of the nifLA promoter. However, under conditions of nitrogen limitation, GlnB is post-translationally modified, which inhibits its activity and Leads to transcription of the nifLA operon. In this way, nifLA transcription is tightly controlled in response to environmental nitrogen via the PII protein signaling cascade. On the post-translational level of NifA regulation, GlnK inhibits the NifL/NifA interaction in a matter dependent on the overall level of free GlnK within the cell.
NifA is transcribed from the nifLA operon, whose promoter is activated by phosphorylated NtrC, another σ54-dependent regulator. The phosphorylation state of NtrC is mediated by the histidine kinase NtrB, which interacts with deuridylylated GlnB but not undylylated GlnB. Under conditions of nitrogen excess, a high intracellular level of glutamine leads to deuridylylation of GlnB, which then interacts with NtrB to deactivate its phosphorylation activity and activate its phosphatase activity, resulting in dephosphorylation of NtrC and the deactivation of the nifLA promoter. However, under conditions of nitrogen limitation, a low level of intracellular glutamine results in uridylylation of GlnB, which inhibits its interaction with NtrB and allows the phosphorylation of NtrC and transcription of the nifLA operon. In this way, nifLA expression is tightly controlled in response to environmental nitrogen via the PII protein signaling cascade. nifA, ntrB, ntrC, and glnB, are all genes that can be mutated in the methods described herein. These processes may also be responsive to intracellular or extracellular levels of ammonia, urea or nitrates.
The activity of NifA is also regulated post-translationally in response to environmental nitrogen, most typically through Nil-mediated inhibition of NifA activity. In general, the interaction of NifL and NifA is influenced by the PII protein signaling cascade via GlnK, although the nature of the interactions between GlnK and NifL/NifA varies significantly between diazotrophs. In Klebsiella pneumoniae, both forms of GlnK inhibit the NifL/NifA interaction, and the interaction between GlnK and NifL/NifA is determined by the overall level of free GlnK within the cell. Under nitrogen-excess conditions, deuridylylated GlnK interacts with the ammonium transporter AmtB, which serves to both block ammonium uptake by AmtB and sequester GlnK to the membrane, allowing inhibition of NifA by NifL. On the other hand, in Azotobacter vinelandii, interaction with deuridylylated GlnK is required for the NifL/NifA interaction and NifA inhibition, while uridylylation of GlnK inhibits its interaction with Nil. In diazotrophs lacking the nifL gene, there is evidence that NifA activity is inhibited directly by interaction with the deuridylylated forms of both GlnK and GlnB under nitrogen-excess conditions. In some bacteria the Nif cluster may be regulated by glnR, and further in some cases this may comprise negative regulation. Regardless of the mechanism, post-translational inhibition of NifA is an important regulator of the nif cluster in most known diazotrophs. Additionally, nifL, amtB, glnK, and glnR are genes that can be mutated in the methods described herein.
In addition to regulating the transcription of the nif gene cluster, many diazotrophs have evolved a mechanism for the direct post-translational modification and inhibition of the nitrogenase enzyme itself, known as nitrogenase shutoff. This is mediated by ADP-ribosylation of the Fe protein (NifH) under nitrogen-excess conditions, which disrupts its interaction with the MoFe protein complex (NifDK) and abolishes nitrogenase activity. DraT catalyzes the ADP-ribosylation of the Fe protein and shutoff of nitrogenase, while DraG catalyzes the removal of ADP-ribose and reactivation of nitrogenase. As with nifLA transcription and NifA inhibition, nitrogenase shutoff is also regulated via the PII protein signaling cascade. Under nitrogen-excess conditions, deuridylylated GlnB interacts with and activates DraT, while deuridylylated GlnK interacts with both DraG and AmtB to form a complex, sequestering DraG to the membrane. Under nitrogen-limiting conditions, the uridylylated forms of GlnB and GlnK do not interact with DraT and DraG, respectively, leading to the inactivation of DraT and the diffusion of DraG to the Fe protein, where it removes the ADP-ribose and activates nitrogenase. The methods described herein also contemplate introducing genetic variation into the nifH, nifD, nifK, and draT genes.
Although some endophytes have the ability to fix nitrogen in vitro, often the genetics are silenced in the field by high levels of exogenous chemical fertilizers. One can decouple the sensing of exogenous nitrogen from expression of the nitrogenase enzyme to facilitate field-based nitrogen fixation. Improving the integral of nitrogenase activity across time further serves to augment the production of nitrogen for utilization by the crop. Specific targets for genetic variation to facilitate field-based nitrogen fixation using the methods described herein include one or more genes selected from the group consisting of nifA, ntrB, ntrC, glnA, glnB, glnK, draT, amtB, glnD, glnE, nifJ, nifH, nifD, nifK, nifY, nifE, nifN, nifU, nifS, nifV, nifW, nifZ, nifM, nifF, nifB, and nifQ.
An additional target for genetic variation to facilitate field-based nitrogen fixation using the methods described herein is the NifA protein. The NifA protein is typically the activator for expression of nitrogen fixation genes. Increasing the production of NifA (either constitutively or during high ammonia condition) circumvents the native ammonia-sensing pathway. In addition, reducing the production of NifL proteins, a known inhibitor of NifA, also leads to an increased level of freely active NifA. In addition, increasing the transcription level of the nifAL operon (either constitutively or during high ammonia condition) also leads to an overall higher level of NifA proteins. Elevated level of nifAL expression is achieved by altering the promoter itself or by reducing the expression of NtrB (part of ntrB and ntrC signaling cascade that originally would result in the shutoff of nifAL operon during high nitrogen condition). High level of NifA achieved by these or any other methods described herein increases the nitrogen fixation activity of the endophytes.
Another target for genetic variation to facilitate field-based nitrogen fixation using the methods described herein is the glnD/GlnB/GlnK PII signaling cascade. The intracellular glutamine level is sensed through the GlnD/GlnB/GlnK PII signaling cascade. Active site mutations in GlnD that abolish the uridylyl-removing activity of GlnD disrupt the nitrogen-sensing cascade. In addition, reduction of the GlnB concentration short circuits the glutamine-sensing cascade. These mutations “trick” the cells into perceiving a nitrogen-limited state, thereby increasing the nitrogen fixation level activity. These processes may also be responsive to intracellular or extracellular levels of ammonia, urea or nitrates.
The amtB protein is also a target for genetic variation to facilitate field-based nitrogen fixation using the methods described herein. Ammonia uptake from the environment can be reduced by decreasing the expression level of amtB protein. Without intracellular ammonia, the endophyte is not able to sense the high level of ammonia, preventing the down-regulation of nitrogen fixation genes. Any ammonia that manages to get into the intracellular compartment is converted into glutamine. Intracellular glutamine level is the major currency of nitrogen sensing. Decreasing the intracellular glutamine level prevents the cells from sensing high ammonium levels in the environment. This effect can be achieved by increasing the expression level of glutaminase, an enzyme that converts glutamine into glutamate. In addition, intracellular glutamine can also be reduced by decreasing glutamine synthase (an enzyme that converts ammonia into glutamine). In diazotrophs, fixed ammonia is quickly assimilated into glutamine and glutamate to be used for cellular processes. Disruptions to ammonia assimilation may enable diversion of fixed nitrogen to be exported from the cell as ammonia. The fixed ammonia is predominantly assimilated into glutamine by glutamine synthetase (GS), encoded by glnA, and subsequently into glutamine by glutamine oxoglutarate aminotransferase (GOGAT). In some examples, glnS encodes a glutamine synthetase. GS is regulated post-translationally by GS adenylyl transferase (GlnE), a bi-functional enzyme encoded by glnE that catalyzes both the adenylylation and deadenylylation of GS through activity of its adenylyl-transferase (AT) and adenylyl-removing (AR) domains, respectively. Under nitrogen limiting conditions, glnA is expressed, and GlnE's AR domain de-adynylylates GS, allowing it to be active. Under conditions of nitrogen excess, glnA expression is turned off, and GlnE's AT domain is activated allosterically by glutamine, causing the adenylylation and deactivation of GS.
Furthermore, the draT gene may also be a target for genetic variation to facilitate field-based nitrogen fixation using the methods described herein. Once nitrogen fixing enzymes are produced by the cell, nitrogenase shut-off represents another level in which cell downregulates fixation activity in high nitrogen condition. This shut-off could be removed by decreasing the expression level of DraT.
Methods for imparting new microbial phenotypes can be performed at the transcriptional, translational, and post-translational levels. The transcriptional level includes changes at the promoter (such as changing sigma factor affinity or binding sites for transcription factors, including deletion of all or a portion of the promoter) or changing transcription terminators and attenuators. The translational level includes changes at the ribosome binding sites and changing mRNA degradation signals. The post-translational level includes mutating an enzyme's active site and changing protein-protein interactions. These changes can be achieved in a multitude of ways. Reduction of expression level (or complete abolishment) can be achieved by swapping the native ribosome binding site (RBS) or promoter with another with lower strength/efficiency. ATG start sites can be swapped to a GTG, TTG, or CTG start codon, which results in reduction in translational activity of the coding region. Complete abolishment of expression can be done by knocking out (deleting) the coding region of a gene. Frameshifting the open reading frame (ORF) likely will result in a premature stop codon along the ORF, thereby creating a non-functional truncated product. Insertion of in-frame stop codons will also similarly create a non-functional truncated product. Addition of a degradation tag at the N or C terminal can also be done to reduce the effective concentration of a particular gene.
Conversely, expression level of the genes described herein can be achieved by using a stronger promoter. To ensure high promoter activity during high nitrogen level condition (or any other condition), a transcription profile of the whole genome in a high nitrogen level condition could be obtained and active promoters with a desired transcription level can be chosen from that dataset to replace the weak promoter. Weak start codons can be swapped out with an ATG start codon for better translation initiation efficiency. Weak ribosomal binding sites (RBS) can also be swapped out with a different RBS with higher translation initiation efficiency. In addition, site specific mutagenesis can also be performed to alter the activity of an enzyme.
Increasing the level of nitrogen fixation that occurs in a plain can lead to a reduction in the amount of chemical fertilizer needed for crop production and reduce greenhouse gas emissions (e.g., nitrous oxide).
Generation of Bacterial Populations Isolation of Bacteria Microbes useful in methods and compositions disclosed herein can be obtained by extracting microbes from surfaces or tissues of native plants. Microbes can be obtained by grinding seeds to isolate microbes. Microbes can be obtained by planting seeds in diverse soil samples and recovering microbes from tissues. Additionally, microbes can be obtained by inoculating plants with exogenous microbes and determining which microbes appear in plant tissues. Non-limiting examples of plant tissues may include a seed, seedling, leaf cutting, plant, bulb, or tuber.
A method of obtaining microbes may be through the isolation of bacteria from soils. Bacteria may be collected from various soil types. In some example, the soil can be characterized by traits such as high or low fertility, levels of moisture, levels of minerals, and various cropping practices. For example, the soil may be involved in a crop rotation where different crops are planted in the same soil in successive planting seasons. The sequential growth of different crops on the same soil may prevent disproportionate depletion of certain minerals. The bacteria can be isolated from the plants growing in the selected soils. The seedling plants can be harvested at 2-6 weeks of growth. For example, at least 400 isolates can be collected in a round of harvest. Soil and plant types reveal the plant phenotype as well as the conditions, which allow for the downstream enrichment of certain phenotypes.
Microbes can be isolated from plant tissues to assess microbial traits. The parameters for processing tissue samples may be varied to isolate different types of associative microbes, such as rhizopheric bacteria, epiphytes, or endophytes. The isolates can be cultured in nitrogen-free media to enrich for bacteria that perform nitrogen fixation. Alternatively, microbes can be obtained from global strain banks.
In planta analytics are performed to assess microbial traits. In some embodiments, the plant tissue can be processed for screening by high throughput processing for DNA and RNA. Additionally, non-invasive measurements can be used to assess plant characteristics, such as colonization. Measurements on wild microbes can be obtained on a plant-by-plant basis. Measurements on wild microbes can also be obtained in the field using medium throughput methods. Measurements can be done successively over time. Model plant system can be used including, but not limited to, Setaria.
Microbes in a plant system can be screened via transcriptional profiling of a microbe in a plant system. Examples of screening through transcriptional profiling are using methods of quantitative polymerase chain reaction (qPCR), molecular barcodes for transcript detection, Next Generation Sequencing, and microbe tagging with fluorescent markers. Impact factors can be measured to assess colonization in the greenhouse including, but not limited to, microbiome, abiotic factors, soil conditions, oxygen, moisture, temperature, inoculum conditions, and root localization. Nitrogen fixation can be assessed in bacteria by measuring 15N gas/fertilizer (dilution) with IRMS or NanoSIMS as described herein NanoSIMS is high-resolution secondary ion mass spectrometry. The NanoSIMS technique is a way to investigate chemical activity from biological samples. The catalysis of reduction of oxidation reactions that drive the metabolism of microorganisms can be investigated at the cellular, subcellular, molecular and elemental level. NanoSIMS can provide high spatial resolution of greater than 0.1 μm. NanoSIMS can detect the use of isotope tracers such as 13C, 15N, and 18O. Therefore, NanoSIMS can be used to the chemical activity nitrogen in the cell.
Automated greenhouses can be used for planta analytics. Plant metrics in response to microbial exposure include, but are not limited to, biomass, chloroplast analysis, CCD camera, volumetric tomography measurements.
One way of enriching a microbe population is according to genotype. For example, a polymerase chain reaction (PCR) assay with a targeted primer or specific primer. Primers designed for the nifH gene can be used to identity diazotrophs because diazotrophs express the nifH gene in the process of nitrogen fixation. A microbial population can also be enriched via single-cell culture-independent approaches and chemotaxis-guided isolation approaches. Alternatively, targeted isolation of microbes can be performed by culturing the microbes on selection media. Premeditated approaches to enriching microbial populations for desired traits can be guided by bioinformatics data and are described herein.
Enriching for Microbes with Nitrogen Fixation Capabilities Using Bioinformatics
Bioinformatic tools can be used to identify and isolate plant growth promoting rhizobacteria (PGPRs), which are selected based on their ability to perform nitrogen fixation. Microbes with high nitrogen fixing ability can promote favorable traits in plants. Bioinformatic modes of analysis for the identification of PGPRs include, but are not limited to, genomics, metagenomics, targeted isolation, gene sequencing, transcriptome sequencing, and modeling.
Genomics analysis can be used to identify PGPRs and confirm the presence of mutations with methods of Next Generation Sequencing as described herein and microbe version control.
Metagenomics can be used to identify and isolate PGPR using a prediction algorithm for colonization. Metadata can also be used to identify the presence of an engineered strain in environmental and greenhouse samples.
Transcriptomic sequencing can be used to predict genotypes leading to PGPR phenotypes. Additionally, transcriptomic data is used to identify promoters for altering gene expression. Transcriptomic data can be analyzed in conjunction with the Whole Genome Sequence (WGS) to generate models of metabolism and gene regulatory networks.
Domestication of Microbes Microbes isolated from nature can undergo a domestication process wherein the microbes are converted to a form that is genetically trackable and identifiable. One way to domesticate a microbe is to engineer it with antibiotic resistance. The process of engineering antibiotic resistance can begin by determining the antibiotic sensitivity in the wild type microbial strain. If the bacteria are sensitive to the antibiotic, then the antibiotic can be a good candidate for antibiotic resistance engineering. Subsequently, an antibiotic resistant gene or a counterselectable suicide vector can be incorporated into the genome of a microbe using recombineering methods. A counterselectable suicide vector may consist of a deletion of the gene of interest, a selectable marker, and the counterselectable marker sacB. Counterselection can be used to exchange native microbial DNA sequences with antibiotic resistant genes. A medium throughput method can be used to evaluate multiple microbes simultaneously allowing for parallel domestication. Alternative methods of domestication include the use of homing nucleases to prevent the suicide vector sequences from looping out or from obtaining intervening vector sequences.
DNA vectors can be introduced into bacteria via several methods including electroporation and chemical transformations. A standard library of vectors can be used for transformations. An example of a method of gene editing is CRISPR preceded by Cas9 testing to ensure activity of Cas9 in the microbes.
Non-Transgenic Engineering of Microbes A microbial population with favorable traits can be obtained via directed evolution. Direct evolution is an approach wherein the process of natural selection is mimicked to evolve proteins or nucleic acids towards a user-defined goal. An example of direct evolution is when random mutations are introduced into a microbial population, the microbes with the most favorable traits are selected, and the growth of the selected microbes is continued. The most favorable traits in growth promoting rhizobacteria (PGPRs) may be in nitrogen fixation. The method of directed evolution may be iterative and adaptive based on the selection process after each iteration.
Plant growth promoting rhizobacteria (PGPRs) with high capability of nitrogen fixation can be generated. The evolution of PGPRs can be carried out via the introduction of genetic variation. Genetic variation can be introduced via polymerase chain reaction mutagenesis, oligonucleotide-directed mutagenesis, saturation mutagenesis, fragment shuffling mutagenesis, homologous recombination, CRISPR/Cas9 systems, chemical mutagenesis, and combinations thereof. These approaches can introduce random mutations into the microbial population. For example, mutants can be generated using synthetic DNA or RNA via oligonucleotide-directed mutagenesis. Mutants can be generated using tools contained on plasmids, which are later cured. Genes of interest can be identified using libraries from other species with improved traits including, but not limited to, improved PGPR properties, improved colonization of cereals, increased oxygen sensitivity, increased nitrogen fixation, and increased ammonia excretion. Intrageneric genes can be designed based on these libraries using software such as Geneious or Platypus design software. Mutations can be designed with the aid of machine learning, Mutations can be designed with the aid of a metabolic model. Automated design of the mutation can be done using a la Platypus and will guide RNAs for Cas-directed mutagenesis.
The intra-generic genes can be transferred into the host microbe. Additionally, reporter systems can also be transferred to the microbe. The reporter systems characterize promoters, determine the transformation success, screen mutants, and act as negative screening tools.
The microbes carrying the mutation can be cultured via serial passaging. A microbial colony contains a single variant of the microbe. Microbial colonies are screened with the aid of an automated colony picker and liquid handler. Mutants with gene duplication and increased copy number express a higher genotype of the desired trait.
Selection of Plant Growth Promoting Microbes Based on Nitrogen Fixation The microbial colonies can be screened using various assays to assess nitrogen fixation. One way to measure nitrogen fixation is via a single fermentative assay, which measures nitrogen excretion. An alternative method is the acetylene reduction assay (ARA) with in-line sampling over time. ARA can be performed in high throughput plates of microtube arrays. ARA can be performed with live plants and plant tissues. The media formulation and media oxygen concentration can be varied in ARA assays. Another method of screening microbial variants is by using biosensors. The use of NanoSIMS and Raman microspectroscopy can be used to investigate the activity of the microbes. In some cases, bacteria can also be cultured and expanded using methods of fermentation in bioreactors. The bioreactors are designed to improve robustness of bacteria growth and to decrease the sensitivity of bacteria to oxygen. Medium to high TP plate-based microfermentors are used to evaluate oxygen sensitivity, nutritional needs, nitrogen fixation, and nitrogen excretion. The bacteria can also be co-cultured with competitive or beneficial microbes to elucidate cryptic pathways. Flow cytometry can be used to screen for bacteria that produce high levels of nitrogen using chemical, colorimetric, or fluorescent indicators. The bacteria may be cultured in the presence or absence of a nitrogen source. For example, the bacteria may be cultured with glutamine, ammonia, urea or nitrates.
Microbe Breeding Microbe breeding is a method to systematically identify and improve the role of species within the crop microbiome. The method comprises three steps: 1) selection of candidate species by mapping plant-microbe interactions and predicting regulatory networks linked to a particular phenotype, 2) pragmatic and predictable improvement of microbial phenotypes through intra-species crossing of regulatory networks and gene clusters, and 3) screening and selection of new microbial genotypes that produce desired crop phenotypes. To systematically assess the improvement of strains, a model is created that links colonization dynamics of the microbial community to genetic activity by key species. The model is used to predict genetic targets breeding and improve the frequency of selecting improvements in microbiome-encoded traits of agronomic relevance. See, FIG. 17A for a graphical representation of an embodiment of the process. In particular, FIG. 17A depicts a schematic of microbe breeding, in accordance with embodiments. As illustrated in FIG. 17A, rational improvement of the crop microbiome may be used to increase soil biodiversity, tune impact of keystone species, and/or alter timing and expression of important metabolic pathways. To this end, the inventors have developed a microbe breeding pipeline to identify and improve the role of strains within the crop microbiome. The method comprises three steps: 1) selection of candidate species by mapping plant-microbe interactions and predicting regulatory networks linked to a particular phenotype, 2) pragmatic and predictable improvement of microbial phenotypes through intragenomic crossing of gene regulatory networks and gene clusters, and 3) screening and selection of new microbial genotypes that produce desired crop phenotypes. To systematically assess the improvement of strains, the inventors employ a model that links colonization dynamics of the microbial community to genetic activity by key species. This process represents a methodology for breeding and selecting improvements in microbiome-encoded traits of agronomic relevance.
Production of bacteria to improve plant traits (e.g., nitrogen fixation) can be achieved through serial passage. The production of this bacteria can be done by selecting plants, which have a particular improved trait that is influenced by the microbial flora, in addition to identifying bacteria and/or compositions that are capable of imparting one or more improved traits to one or more plants. One method of producing a bacteria to improve a plant trait includes the steps of: (a) isolating bacteria from tissue or soil of a first plant; (b) introducing a genetic variation into one or more of the bacteria to produce one or more variant bacteria; (c) exposing a plurality of plants to the variant bacteria; (d) isolating bacteria from tissue or soil of one of the plurality of plants, wherein the plant from which the bacteria is isolated has an improved trait relative to other plants in the plurality of plants; and (e) repeating steps (b) to (d) with bacteria isolated from the plant with an improved trait (step (d)). Steps (b) to (d) can be repeated any number of times (e.g., once, twice, three times, four times, five times, ten times, or more) until the improved trait in a plant reaches a desired level. Further, the plurality of plants can be more than two plants, such as 10 to 20 plants, or 20 or more, 50 or more, 100 or more, 300 or more, 500 or more, or 1000 or more plants.
In addition to obtaining a plant with an improved trait, a bacterial population comprising bacteria comprising one or more genetic variations introduced into one or more genes (e.g., genes regulating nitrogen fixation) is obtained. By repeating the steps described above, a population of bacteria can be obtained that include the most appropriate members of the population that correlate with a plant trait of interest. The bacteria in this population can be identified and their beneficial properties determined, such as by genetic and/or phenotypic analysis. Genetic analysis may occur of isolated bacteria in step (a). Phenotypic and/or genotypic information may be obtained using techniques including: high through-put screening of chemical components of plant origin, sequencing techniques including high throughput sequencing of genetic material, differential display techniques (including DDRT-PCR, and DD-PCR), nucleic acid microarray techniques, RNA-sequencing (Whole Transcriptome Shotgun Sequencing), and qRT-PCR (quantitative real time PCR). Information gained can be used to obtain community profiling information on the identity and activity of bacteria present, such as phylogenetic analysis or microarray-based screening of nucleic acids coding for components of rRNA operons or other taxonomically informative loci. Examples of taxonomically informative loci include 16S rRNA gene, 23S rRNA gene, 5S rRNA gene, 5.85 rRNA gene, 12S rRNA gene, 18S rRNA gene, 28S rRNA gene, gyrB gene, rpoB gene, fusA gene, recA gene, coxl gene, nifD gene. Example processes of taxonomic profiling to determine taxa present in a population are described in US20140155283. Bacterial identification may comprise characterizing activity of one or more genes or one or more signaling pathways, such as genes associated with the nitrogen fixation pathway. Synergistic interactions (where two components, by virtue of their combination, increase a desired effect by more than an additive amount) between different bacterial species may also be present in the bacterial populations.
Genetic Variation—Locations and Sources of Genomic Alteration The genetic variation may be a gene selected from the group consisting of: nifA, nifL, ntrB, ntrC, glnA, glnB, glnK, draT, amtB, glnD, glnE, nifJ, nifH, nifD, nifK, nifY, nifE, nifN, nifU, nifS, nifV, nifW, nifZ, nifM, nifF, nifB, and nifQ. The genetic variation may be a variation in a gene encoding a protein with functionality selected from the group consisting of: glutamine synthetase, glutaminase, glutamine synthetase adenylyltransferase, transcriptional activator, anti-transcriptional activator, pyruvate flavodoxin oxidoreductase, flavodoxin, or NAD+-dinitrogen-reductase aDP-D-ribosyltransferase. The genetic variation may be a mutation that results in one or more of: increased expression or activity of NifA or glutaminase; decreased expression or activity of NifL, NtrB, glutamine synthetase, GlnB, GlnK, DraT, AmtB; decreased adenylyl-removing activity of GlnE; or decreased uridylyl-removing activity of GlnD. Introducing a genetic variation may comprise insertion and/or deletion of one or more nucleotides at a target site, such as 1, 2, 3, 4, 5, 10, 25, 50, 100, 250, 500, or more nucleotides. The genetic variation introduced into one or more bacteria of the methods disclosed herein may be a knock-out mutation (e.g. deletion of a promoter, insertion or deletion to produce a premature stop codon, deletion of an entire gene), or it may be elimination or abolishment of activity of a protein domain (e.g. point mutation affecting an active site, or deletion of a portion of a gene encoding the relevant portion of the protein product), or it may alter or abolish a regulatory sequence of a target gene. One or more regulatory sequences may also be inserted, including heterologous regulatory sequences and regulatory sequences found within a genome of a bacterial species or genus corresponding to the bacteria into which the genetic variation is introduced. Moreover, regulatory sequences may be selected based on the expression level of a gene in a bacterial culture or within a plant tissue. The genetic variation may be a pre-determined genetic variation that is specifically introduced to a target site. The genetic variation may be a random mutation within the target site. The genetic variation may be an insertion or deletion of one or more nucleotides. In some cases, a plurality of different genetic variations (e.g. 2, 3, 4, 5, 10, or more) are introduced into one or more of the isolated bacteria before exposing the bacteria to plants for assessing trait improvement. The plurality of genetic variations can be any of the above types, the same or different types, and in any combination. In some cases, a plurality of different genetic variations are introduced serially, introducing a first genetic variation after a first isolation step, a second genetic variation after a second isolation step, and so forth so as to accumulate a plurality of genetic variations in bacteria imparting progressively improved traits on the associated plants.
Genetic Variation—Methods of Introducing Genomic Alteration In general, the term “genetic variation” refers to any change introduced into a polynucleotide sequence relative to a reference polynucleotide, such as a reference genome or portion thereof, or reference gene or portion thereof. A genetic variation may be referred to as a “mutation,” and a sequence or organism comprising a genetic variation may be referred to as a “genetic variant” or “mutant”. Genetic variations can have any number of effects, such as the increase or decrease of some biological activity, including gene expression, metabolism, and cell signaling. Genetic variations can be specifically introduced to a target site, or introduced randomly. A variety of molecular tools and methods are available for introducing genetic variation. For example, genetic variation can be introduced via polymerase chain reaction mutagenesis, oligonucleotide-directed mutagenesis, saturation mutagenesis, fragment shuffling mutagenesis, homologous recombination, recombineering, lambda red mediated recombination, CRISPR/Cas9 systems, chemical mutagenesis, and combinations thereof. Chemical methods of introducing genetic variation include exposure of DNA to a chemical mutagen, e.g., ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), N-nitrosourea (EN U), N-methyl-N-nitro-N′-nitrosoguanidine, 4-nitroquinoline N-oxide, diethylsulfate, benzopyrene, cyclophosphamide, bleomycin, trimethylmelamine, acrylamide monomer, nitrogen mustard, vincristine, diepoxyalkanes (for example, diepoxybutane), ICR-170, formaldehyde, procarbazine hydrochloride, ethylene oxide, dimethylnitrosamine, 7,12 dimethylbenz(a)anthracene, chlorambucil, hexamethylphosphoramide, bisulfan, and the like. Radiation mutation-inducing agents include ultraviolet radiation, γ-irradiation, X-rays, and fast neutron bombardment. Genetic variation can also be introduced into a nucleic acid using, e.g., trimethylpsoralen with ultraviolet light. Random or targeted insertion of a mobile DNA element, e.g., a transposable element, is another suitable method for generating genetic variation. Genetic variations can be introduced into a nucleic acid during amplification in a cell-free in vitro system, e.g., using a polymerase chain reaction (PCR) technique such as error-prone PCR. Genetic variations can be introduced into a nucleic acid in vitro using DNA shuffling techniques (e.g., exon shuffling, domain swapping, and the like). Genetic variations can also be introduced into a nucleic acid as a result of a deficiency in a DNA repair enzyme in a cell, e.g., the presence in a cell of a mutant gene encoding a mutant DNA repair enzyme is expected to generate a high frequency of mutations (i.e., about 1 mutation/100 genes-1 mutation/10,000 genes) in the genome of the cell. Examples of genes encoding DNA repair enzymes include but are not limited to Mut H, Mut S, Mut L, and Mut U, and the homologs thereof in other species (e.g., MSH 1 6, PMS 1 2, MLH 1, GTBP, ERCC-1, and the like). Example descriptions of various methods for introducing genetic variations are provided in e.g., Stemple (2004) Nature 5:1-7; Chiang et al. (1993) PCR Methods Appl 2(3): 210-217; Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751; and U.S. Pat. Nos. 6,033,861, and 6,773,900.
Genetic variations introduced into microbes may be classified as transgenic, cisgenic, intragenomic, intrageneric, intergeneric, synthetic, evolved, rearranged, or SNPs.
Genetic variation may be introduced into numerous metabolic pathways within microbes to elicit improvements in the traits described above. Representative pathways include sulfur uptake pathways, glycogen biosynthesis, the glutamine regulation pathway, the molybdenum uptake pathway, the nitrogen fixation pathway, ammonia assimilation, ammonia excretion or secretion, nNitrogen uptake, glutamine biosynthesis, annamox, phosphate solubilization, organic acid transport, organic acid production, agglutinins production, reactive oxygen radical scavenging genes, Indole Acetic Acid biosynthesis, trehalose biosynthesis, plant cell wall degrading enzymes or pathways, root attachment genes, exopolysaccharide secretion, glutamate synthase pathway, iron uptake pathways, siderophore pathway, chitinase pathway, ACC deaminase, glutathione biosynthesis, phosphorous signaling genes, quorum quenching pathway, cytochrome pathways, hemoglobin pathway, bacterial hemoglobin-like pathway, small RNA rsmZ, rhizobitoxine biosynthesis, lapA adhesion protein, AHL quorum sensing pathway, phenazine biosynthesis, cyclic lipopeptide biosynthesis, and antibiotic production.
CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats)/CRISPR-associated (Cas) systems can be used to introduce desired mutations. CRISPR/Cas9 provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. The Cas9 protein (or functional equivalent and/or variant thereof, i.e., Cas9-like protein) naturally contains DNA endonuclease activity that depends on the association of the protein with two naturally occurring or synthetic RNA molecules called crRNA and tracrRNA (also called guide RNAs). In some cases, the two molecules are covalently link to form a single molecule (also called a single guide RNA (“sgRNA”). Thus, the Cas9 or Cas9-like protein associates with a DNA-targeting RNA (which term encompasses both the two-molecule guide RNA configuration and the single-molecule guide RNA configuration), which activates the Cas9 or Cas9-like protein and guides the protein to a target nucleic acid sequence. If the Cas9 or Cas9-like protein retains its natural enzymatic function, it will cleave target DNA to create a double-stranded break, which can lead to genome alteration (i.e., editing: deletion, insertion (when a donor polynucleotide is present), replacement, etc.), thereby altering gene expression. Some variants of Cas9 (which variants are encompassed by the term Cas9-like) have been altered such that they have a decreased DNA cleaving activity (in some cases, they cleave a single strand instead of both strands of the target DNA, while in other cases, they have severely reduced to no DNA cleavage activity). Further exemplary descriptions of CRISPR systems for introducing genetic variation can be found in, e.g. U.S. Pat. No. 8,795,965.
As a cyclic amplification technique, polymerase chain reaction (PCR) mutagenesis uses mutagenic primers to introduce desired mutations. PCR is performed by cycles of denaturation, annealing, and extension. After amplification by PCR, selection of mutated DNA and removal of parental plasmid DNA can be accomplished by: 1) replacement of dCTP by hydroxymethylated-dCTP during PCR, followed by digestion with restriction enzymes to remove non-hydroxymethylated parent DNA only; 2) simultaneous mutagenesis of both an antibiotic resistance gene and the studied gene changing the plasmid to a different antibiotic resistance, the new antibiotic resistance facilitating the selection of the desired mutation thereafter; 3) after introducing a desired mutation, digestion of the parent methylated template DNA by restriction enzyme Dpnl which cleaves only methylated DNA, by which the mutagenized unmethylated chains are recovered; or 4) circularization of the mutated PCR products in an additional ligation reaction to increase the transformation efficiency of mutated DNA. Further description of exemplary methods can be found in e.g. U.S. Pat. No. 7,132,265, U.S. Pat. No. 6,713,285, U.S. Pat. No. 6,673,610, U.S. Pat. No. 6,391,548, U.S. Pat. No. 5,789,166, U.S. Pat. No. 5,780,270, U.S. Pat. No. 5,354,670, U.S. Pat. No. 5,071,743, and US20100267147.
Oligonucleotide-directed mutagenesis, also called site-directed mutagenesis, typically utilizes a synthetic DNA primer. This synthetic primer contains the desired mutation and is complementary to the template DNA around the mutation site so that it can hybridize with the DNA in the gene of interest. The mutation may be a single base change (a point mutation), multiple base changes, deletion, or insertion, or a combination of these. The single-strand primer is then extended using a DNA polymerase, which copies the rest of the gene. The gene thus copied contains the mutated site, and may then be introduced into a host cell as a vector and cloned. Finally, mutants can be selected by DNA sequencing to check that they contain the desired mutation.
Genetic variations can be introduced using error-prone PCR. In this technique the gene of interest is amplified using a DNA polymerase under conditions that are deficient in the fidelity of replication of sequence. The result is that the amplification products contain at least one error in the sequence. When a gene is amplified and the resulting product(s) of the reaction contain one or more alterations in sequence when compared to the template molecule, the resulting products are mutagenized as compared to the template. Another means of introducing random mutations is exposing cells to a chemical mutagen, such as nitrosoguanidine or ethyl methanesulfonate (Nestmann, Mutat Res 1975 June; 28(3):323-30), and the vector containing the gene is then isolated from the host.
Saturation mutagenesis is another form of random mutagenesis, in which one tries to generate all or nearly all possible mutations at a specific site, or narrow region of a gene. In a general sense, saturation mutagenesis is comprised of mutagenizing a complete set of mutagenic cassettes (wherein each cassette is, for example, 1-500 bases in length) in defined polynucleotide sequence to be mutagenized (wherein the sequence to be mutagenized is, for example, from 15 to 100, 000 bases in length). Therefore, a group of mutations (e.g. ranging from 1 to 100 mutations) is introduced into each cassette to be mutagenized. A grouping of mutations to be introduced into one cassette can be different or the same from a second grouping of mutations to be introduced into a second cassette during the application of one round of saturation mutagenesis. Such groupings are exemplified by deletions, additions, groupings of particular codons, and groupings of particular nucleotide cassettes.
Fragment shuffling mutagenesis, also called DNA shuffling, is a way to rapidly propagate beneficial mutations. In an example of a shuffling process, DNAse is used to fragment a set of parent genes into pieces of e.g. about 50-100 bp in length. This is then followed by a polymerase chain reaction (PCR) without primers—DNA fragments with sufficient overlapping homologous sequence will anneal to each other and are then be extended by DNA polymerase. Several rounds of this PCR extension are allowed to occur, after some of the DNA molecules reach the size of the parental genes. These genes can then be amplified with another PCR, this time with the addition of primers that are designed to complement the ends of the strands. The primers may have additional sequences added to their 5′ ends, such as sequences for restriction enzyme recognition sites needed for ligation into a cloning vector. Further examples of shuffling techniques are provided in US20050266541.
Homologous recombination mutagenesis involves recombination between an exogenous DNA fragment and the targeted polynucleotide sequence. After a double-stranded break occurs, sections of DNA around the 5′ ends of the break are cut away in a process called resection. In the strand invasion step that follows, an overhanging 3′ end of the broken DNA molecule then “invades” a similar or identical DNA molecule that is not broken. The method can be used to delete a gene, remove exons, add a gene, and introduce point mutations. Homologous recombination mutagenesis can be permanent or conditional. Typically, a recombination template is also provided. A recombination template may be a component of another vector, contained in a separate vector, or provided as a separate polynucleotide. In some embodiments, a recombination template is designed to serve as a template in homologous recombination, such as within or near a target sequence nicked or cleaved by a site-specific nuclease. A template polynucleotide may be of any suitable length, such as about or more than about 10, 15, 20, 25, 50, 75, 100, 150, 200, 500, 1000, or more nucleotides in length. In some embodiments, the template polynucleotide is complementary to a portion of a polynucleotide comprising the target sequence. When optimally aligned, a template polynucleotide might overlap with one or more nucleotides of a target sequences (e.g. about or more than about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more nucleotides). In some embodiments, when a template sequence and a polynucleotide comprising a target sequence are optimally aligned, the nearest nucleotide of the template polynucleotide is within about 1, 5, 10, 15, 20, 25, 50, 75, 100, 200, 300, 400, 500, 1000, 5000, 10000, or more nucleotides from the target sequence. Non-limiting examples of site-directed nucleases useful in methods of homologous recombination include zinc finger nucleases, CRISPR nucleases, TALE nucleases, and meganuclease. For a further description of the use of such nucleases, see e.g. U.S. Pat. No. 8,795,965 and US20140301990.
Mutagens that create primarily point mutations and short deletions, insertions, transversions, and/or transitions, including chemical mutagens or radiation, may be used to create genetic variations. Mutagens include, but are not limited to, ethyl methanesulfonate, methylmethane sulfonate, N-ethyl-N-nitrosurea, triethylmelamine, N-methyl-N-nitrosourea, procarbazine, chlorambucil, cyclophosphamide, diethyl sulfate, acrylamide monomer, melphalan, nitrogen mustard, vincristine, dimethylnitrosamine, N-methyl-N′-nitro-Nitrosoguanidine, nitrosoguanidine, 2-aminopurine, 7,12 dimethyl-benz(a)anthracene, ethylene oxide, hexamethylphosphoramide, bisulfan, diepoxyalkanes (diepoxyoctane, diepoxybutane, and the like), 2-methoxy-6-chloro-9[3-(ethyl-2-chloro-ethyl)aminopropylamino]acridine dihydrochloride and formaldehyde.
Introducing genetic variation may be an incomplete process, such that some bacteria in a treated population of bacteria carry a desired mutation while others do not. In some cases, it is desirable to apply a selection pressure so as to enrich for bacteria carrying a desired genetic variation. Traditionally, selection for successful genetic variants involved selection for or against some functionality imparted or abolished by the genetic variation, such as in the case of inserting antibiotic resistance gene or abolishing a metabolic activity capable of converting a non-lethal compound into a lethal metabolite. It is also possible to apply a selection pressure based on a polynucleotide sequence itself, such that only a desired genetic variation need be introduced (e.g. without also requiting a selectable marker). In this case, the selection pressure can comprise cleaving genomes lacking the genetic variation introduced to a target site, such that selection is effectively directed against the reference sequence into which the genetic variation is sought to be introduced. Typically, cleavage occurs within 100 nucleotides of the target site (e.g. within 75, 50, 25, 10, or fewer nucleotides from the target site, including cleavage at or within the target site). Cleaving may be directed by a site-specific nuclease selected from the group consisting of a Zinc Finger nuclease, a CRISPR nuclease, a TALE nuclease (TALEN), or a meganuclease. Such a process is similar to processes for enhancing homologous recombination at a target site, except that no template for homologous recombination is provided. As a result, bacteria lacking the desired genetic variation are more likely to undergo cleavage that, left unrepaired, results in cell death. Bacteria surviving selection may then be isolated for use in exposing to plants for assessing conferral of an improved trait.
A CRISPR nuclease may be used as the site-specific nuclease to direct cleavage to a target site. An improved selection of mutated microbes can be obtained by using Cas9 to kill non-mutated cells. Plants are then inoculated with the mutated microbes to re-confirm symbiosis and create evolutionary pressure to select for efficient symbionts. Microbes can then be re-isolated from plant tissues. CRISPR nuclease systems employed for selection against non-variants can employ similar elements to those described above with respect to introducing genetic variation, except that no template for homologous recombination is provided. Cleavage directed to the target site thus enhances death of affected cells.
Other options for specifically inducing cleavage at a target site are available, such as zinc finger nucleases, TALE nuclease (TALEN) systems, and meganuclease. Zinc-finger nucleases (ZFNs) are artificial DNA endonucleases generated by fusing a zinc finger DNA binding domain to a DNA cleavage domain. ZFNs can be engineered to target desired DNA sequences and this enables zinc-finger nucleases to cleave unique target sequences. When introduced into a cell, ZFNs can be used to edit target DNA in the cell (e.g., the cell's genome) by inducing double stranded breaks. Transcription activator-like effector nucleases (TALENs) are artificial DNA endonucleases generated by fusing a TAL (Transcription activator-like) effector DNA binding domain to a DNA cleavage domain. TALENS can be quickly engineered to bind practically any desired DNA sequence and when introduced into a cell, TALENs can be used to edit target DNA in the cell (e.g., the cell's genome) by inducing double strand breaks. Meganucleases (homing endonuclease) are endodeoxyribonucleases characterized by a large recognition site (double-stranded DNA sequences of 12 to 40 base pairs. Meganucleases can be used to replace, eliminate or modify sequences in a highly targeted way. By modifying their recognition sequence through protein engineering, the targeted sequence can be changed. Meganucleases can be used to modify all genome types, whether bacterial, plant or animal and are commonly grouped into four families: the LAGLIDADG family (SEQ ID NO: 1), the GIY-YIG family, the His-Cyst box family and the HNH family. Exemplary homing endonucleases include I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII and I-TevIII.
Genetic Variation—Methods of Identification The microbes of the present disclosure may be identified by one or more genetic modifications or alterations, which have been introduced into said microbe. One method by which said genetic modification or alteration can be identified is via reference to a SEQ ID NO that contains a portion of the microbe's genomic sequence that is sufficient to identify the genetic modification or alteration.
Further, in the case of microbes that have not had a genetic modification or alteration (e.g. a wild type, WT) introduced into their genomes, the disclosure can utilize 16S nucleic acid sequences to identify said microbes. A 16S nucleic acid sequence is an example of a “molecular marker” or “genetic marker,” which refers to an indicator that is used in methods for visualizing differences in characteristics of nucleic acid sequences. Examples of other such indicators are restriction fragment length polymorphism (RFLP) markers, amplified fragment length polymorphism (AFLP) markers, single nucleotide polymorphisms (SNPs), insertion mutations, microsatellite markers (SSRs), sequence-characterized amplified regions (SCARs), cleaved amplified polymorphic sequence (CAPS) markers or isozyme markers or combinations of the markers described herein which defines a specific genetic and chromosomal location. Markers further include polynucleotide sequences encoding 16S or 18S rRNA, and internal transcribed spacer (ITS) sequences, which are sequences found between small-subunit and large-subunit rRNA genes that have proven to be especially useful in elucidating relationships or distinctions when compared against one another. Furthermore, the disclosure utilizes unique sequences found in genes of interest (e.g. nif H,D,K,L,A, glnE, amtB, etc.) to identify microbes disclosed herein.
The primary structure of major rRNA subunit 16S comprise a particular combination of conserved, variable, and hypervariable regions that evolve at different rates and enable the resolution of both very ancient lineages such as domains, and more modern lineages such as genera. The secondary structure of the 16S subunit include approximately 50 helices which result in base pairing of about 67% of the residues. These highly conserved secondary structural features are of great functional importance and can be used to ensure positional homology in multiple sequence alignments and phylogenetic analysis. Over the previous few decades, the 16S rRNA gene has become the most sequenced taxonomic marker and is the cornerstone for the current systematic classification of bacteria and archaea (Yarza et al. 2014. Nature Rev. Micro. 12:635-45).
Thus, in certain aspects, the disclosure provides for a sequence, which shares at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any sequence in Tables E, F, G, or H.
Thus, in certain aspects, the disclosure provides for a microbe that comprises a sequence, which shares at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 62-303. These sequences and their associated descriptions can be found in Tables F, G, and H.
In some aspects, the disclosure provides for a microbe that comprises a 16S nucleic acid sequence, which shares at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 85, 96, 111, 121, 122, 123, 124, 136, 149, 157, 167, 261, 262, 269, 277-283. These sequences and their associated descriptions can be found in Tables G and H.
In some aspects, the disclosure provides for a microbe that comprises a nucleic acid sequence, which shares at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 86-95, 97-110, 112-120, 125-135, 137-148, 150-156, 158-166, 168-176, 263-268, 270-274, 275, 276, 284-295. These sequences and their associated descriptions can be found in Tables G and H.
In some aspects, the disclosure provides for a microbe that comprises a nucleic acid sequence, which shares at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 177-260, 296-303. These sequences and their associated descriptions can be found in Tables G and H.
In some aspects, the disclosure provides for a microbe that comprises, or primer that comprises, or probe that comprises, or non-native junction sequence that comprises, a nucleic acid sequence, which shares at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 304-424. These sequences and their associated descriptions can be found in Table E.
In some aspects, the disclosure provides for a microbe that comprises a non-native junction sequence that shares at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 372-405. These sequences and their associated descriptions can be found in Table E.
In some aspects, the disclosure provides for a microbe that comprises an amino acid sequence, which shares at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 77, 78, 81, 82, or 83. These sequences and their associated descriptions can be found in Tables F and H.
Genetic Variation—Methods of Detection: Primers, Probes, and Assays The present disclosure teaches primers, probes, and assays that are useful for detecting the microbes taught herein. In some aspects, the disclosure provides for methods of detecting the WT parental strains. In other aspects, the disclosure provides for methods of detecting the non-intergeneric engineered microbes derived from the WT strains. In aspects, the present disclosure provides methods of identifying non-intergeneric genetic alterations in a microbe.
In aspects, the genomic engineering methods of the present disclosure lead to the creation of non-natural nucleotide “junction” sequences in the derived non-intergeneric microbes. These non-naturally occurring nucleotide junctions can be used as a type of diagnostic that is indicative of the presence of a particular genetic alteration in a microbe taught herein.
The present techniques are able to detect these non-naturally occurring nucleotide junctions via the utilization of specialized quantitative PCR methods, including uniquely designed primers and probes. In some aspects, the probes of the disclosure bind to the non-naturally occurring nucleotide junction sequences. In some aspects, traditional PCR is utilized. In other aspects, real-time PCR is utilized. In some aspects, quantitative PCR (qPCR) is utilized.
Thus, the disclosure can cover the utilization of two common methods for the detection of PCR products in real-time: (1) non-specific fluorescent dyes that intercalate with any double-stranded DNA, and (2) sequence-specific DNA probes consisting of oligonucleotides that are labelled with a fluorescent reporter which permits detection only after hybridization of the probe with its complementary sequence. In some aspects, only the non-naturally occurring nucleotide junction will be amplified via the taught primers, and consequently can be detected via either a non-specific dye, or via the utilization of a specific hybridization probe. In other aspects, the primers of the disclosure are chosen such that the primers flank either side of a junction sequence, such that if an amplification reaction occurs, then said junction sequence is present.
Aspects of the disclosure involve non-naturally occurring nucleotide junction sequence molecules per se, along with other nucleotide molecules that are capable of binding to said non-naturally occurring nucleotide junction sequences under mild to stringent hybridization conditions. In some aspects, the nucleotide molecules that are capable of binding to said non-naturally occurring nucleotide junction sequences under mild to stringent hybridization conditions are termed “nucleotide probes.”
In aspects, genomic DNA can be extracted from samples and used to quantify the presence of microbes of the disclosure by using qPCR. The primers utilized in the qPCR reaction can be primers designed by Primer Blast (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) to amplify unique regions of the wild-type genome or unique regions of the engineered non-intergeneric mutant strains. The qPCR reaction can be carried out using the SYBR GreenER qPCR SuperMix Universal (Thermo Fisher P/N 11762100) kit, using only forward and reverse amplification primers; alternatively, the Kapa Probe Force kit (Kapa Biosystems P/N KK4301) can be used with amplification primers and a TaqMan probe containing a RAM dye label at the 5′ end, an internal ZEN quencher, and a minor groove binder and fluorescent quencher at the 3′ end (Integrated DNA Technologies).
Certain primer, probe, and non-native junction sequences are listed in Table E. qPCR reaction efficiency can be measured using a standard curve generated from a known quantity of gDNA from the target genome. Data can be normalized to genome copies per g fresh weight using the tissue weight and extraction volume.
Quantitative polymerase chain reaction (qPCR) is a method of quantifying, in real time, the amplification of one or more nucleic acid sequences. The real time quantification of the PCR assay permits determination of the quantity of nucleic acids being generated by the PCR amplification steps by comparing the amplifying nucleic acids of interest and an appropriate control nucleic acid sequence, which may act as a calibration standard.
TaqMan probes are often utilized in qPCR assays that require an increased specificity for quantifying target nucleic acid sequences. TaqMan probes comprise a oligonucleotide probe with a fluorophore attached to the 5′ end and a quencher attached to the 3′ end of the probe. When the TaqMan probes remain as is with the 5′ and 3′ ends of the probe in close contact with each other, the quencher prevents fluorescent signal transmission from the fluorophore. TaqMan probes are designed to anneal within a nucleic acid region amplified by a specific set of primers. As the Taq polymerase extends the primer and synthesizes the nascent strand, the 5′ to 3′ exonuclease activity of the Taq polymerase degrades the probe that annealed to the template. This probe degradation releases the fluorophore, thus breaking the close proximity to the quencher and allowing fluorescence of the fluorophore. Fluorescence detected in the qPCR assay is directly proportional to the fluorophore released and the amount of DNA template present in the reaction.
The features of qPCR allow the practitioner to eliminate the labor-intensive post-amplification step of gel electrophoresis preparation, which is generally required for observation of the amplified products of traditional PCR assays. The benefits of qPCR over conventional PCR are considerable, and include increased speed, ease of use, reproducibility, and quantitative ability
Improvement of Traits Methods of the present disclosure may be employed to introduce or improve one or more of a variety of desirable traits. Examples of traits that may introduced or improved include: root biomass, root length, height, shoot length, leaf number, water use efficiency, overall biomass, yield, fruit size, grain size, photosynthesis rate, tolerance to drought, heat tolerance, salt tolerance, resistance to nematode stress, resistance to a fungal pathogen, resistance to a bacterial pathogen, resistance to a viral pathogen, level of a metabolite, and proteome expression. The desirable traits, including height, overall biomass, root and/or shoot biomass, seed germination, seedling survival, photosynthetic efficiency, transpiration rate, seed/fruit number or mass, plant grain or fruit yield, leaf chlorophyll content, photosynthetic rate, root length, or any combination thereof, can be used to measure growth, and compared with the growth rate of reference agricultural plants (e.g., plants without the improved traits) grown under identical conditions.
A preferred trait to be introduced or improved is nitrogen fixation, as described herein. In some cases, a plant resulting from the methods described herein exhibits a difference in the trait that is at least about 5% greater, for example at least about 5%, at least about 8%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 75%, at least about 80%, at least about 80%, at least about 90%, or at least 100%, at least about 200%, at least about 300%, at least about 400% or greater than a reference agricultural plant grown under the same conditions in the soil. In additional examples, a plant resulting from the methods described herein exhibits a difference in the trait that is at least about 5% greater, for example at least about 5%, at least about 8%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 75%, at least about 80%, at least about 80%, at least about 90%, or at least 100%, at least about 200%, at least about 300%, at least about 400% or greater than a reference agricultural plant grown under similar conditions in the soil.
The trait to be improved may be assessed under conditions including the application of one or more biotic or abiotic stressors. Examples of stressors include abiotic stresses (such as heat stress, salt stress, drought stress, cold stress, and low nutrient stress) and biotic stresses (such as nematode stress, insect herbivory stress, fungal pathogen stress, bacterial pathogen stress, and viral pathogen stress).
The trait improved by methods and compositions of the present disclosure may be nitrogen fixation, including in a plant not previously capable of nitrogen fixation. In some cases, bacteria isolated according to a method described herein produce 1% or more (e.g. 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, or more) of a plant's nitrogen, which may represent an increase in nitrogen fixation capability of at least 2-fold (e.g. 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 50-fold, 100-fold, 1000-fold, or more) as compared to bacteria isolated from the first plant before introducing any genetic variation. In some cases, the bacteria produce 5% or more of a plant's nitrogen. The desired level of nitrogen fixation may be achieved after repeating the steps of introducing genetic variation, exposure to a plurality of plants, and isolating bacteria from plants with an improved trait one or more times (e.g. 1, 2, 3, 4, 5, 10, 15, 25, or more times). In some cases, enhanced levels of nitrogen fixation are achieved in the presence of fertilizer supplemented with glutamine, ammonia, or other chemical source of nitrogen. Methods for assessing degree of nitrogen fixation are known, examples of which are described herein.
Microbe breeding is a method to systematically identify and improve the role of species within the crop microbiome. The method comprises three steps: 1) selection of candidate species by mapping plant-microbe interactions and predicting regulatory networks linked to a particular phenotype, 2) pragmatic and predictable improvement of microbial phenotypes through intra-species crossing of regulatory networks and gene clusters, and 3) screening and selection of new microbial genotypes that produce desired crop phenotypes. To systematically assess the improvement of strains, a model is created that links colonization dynamics of the microbial community to genetic activity by key species. The model is used to predict genetic targets breeding and improve the frequency of selecting improvements in microbiome-encoded traits of agronomic relevance.
Measuring Nitrogen Delivered in an Agriculturally Relevant Field Context In the field, the amount of nitrogen delivered can be determined by the function of colonization multiplied by the activity.
The above equation requires (1) the average colonization per unit of plant tissue, and (2) the activity as either the amount of nitrogen fixed or the amount of ammonia excreted by each microbial cell. To convert to pounds of nitrogen per acre, corn growth physiology is tracked over time, e.g., size of the plant and associated root system throughout the maturity stages.
The pounds of nitrogen delivered to a crop per acre-season can be calculated by the following equation:
Nitrogen delivered=∫Plant Tissue(t)×Colonization(t)×Activity(t)dt
The Plant Tissue(t) is the fresh weight of corn plant tissue over the growing time (t). Values for reasonably making the calculation are described in detail in the publication entitled Roots, Growth and Nutrient Uptake (Mengel. Dept. of Agronomy Pub.# AGRY-95-08 (Rev. May-95. p. 1-8.).
The Colonization (t) is the amount of the microbes of interest found within the plant tissue, per gram fresh weight of plant tissue, at any particular time, t, during the growing season. In the instance of only a single timepoint available, the single timepoint is normalized as the peak colonization rate over the season, and the colonization rate of the remaining timepoints are adjusted accordingly.
Activity(t) is the rate of which N is fixed by the microbes of interest per unit time, at any particular time, t, during the growing season. In the embodiments disclosed herein, this activity rate is approximated by in vitro acetylene reduction assay (ARA) in ARA media in the presence of 5 mM glutamine or Ammonium excretion assay in ARA media in the presence of 5 mM ammonium ions.
The Nitrogen delivered amount is then calculated by numerically integrating the above function. In cases where the values of the variables described above are discretely measured at set timepoints, the values in between those timepoints are approximated by performing linear interpolation.
Nitrogen Fixation Described herein are methods of increasing nitrogen fixation in a plant, comprising exposing the plant to bacteria comprising one or more genetic variations introduced into one or more genes regulating nitrogen fixation, wherein the bacteria produce 1% or more of nitrogen in the plant (e.g. 2%, 5%, 10%, or more), which may represent a nitrogen-fixation capability of at least 2-fold as compared to the plant in the absence of the bacteria. The bacteria may produce the nitrogen in the presence of fertilizer supplemented with glutamine, urea, nitrates or ammonia. Genetic variations can be any genetic variation described herein, including examples provided above, in any number and any combination. The genetic variation may be introduced into a gene selected from the group consisting of nifA, nifL, ntrB, ntrC, glutamine synthetase, glnA, glnB, glnK, draT, amtB, glutaminase, glnD, glnE, nifH, nifD, nifK, nifY, nifE, nifN, nifU, nifS, nifW, nifZ, nifM, nifF, nifB, and nifQ. The genetic variation may be a mutation that results in one or more of: increased expression or activity of nifA or glutaminase; decreased expression or activity of nifL, ntrB, glutamine synthetase, glnB, glnK, draT, amtB; decreased adenylyl-removing activity of GlnE; or decreased uridylyl-removing activity of GlnD. The genetic variation introduced into one or more bacteria of the methods disclosed herein may be a knock-out mutation or it may abolish a regulatory sequence of a target gene, or it may comprise insertion of a heterologous regulatory sequence, for example, insertion of a regulatory sequence found within the genome of the same bacterial species or genus. The regulatory sequence can be chosen based on the expression level of a gene in a bacterial culture or within plant tissue. The genetic variation may be produced by chemical mutagenesis. The plants grown in step (c) may be exposed to biotic or abiotic stressors.
The amount of nitrogen fixation that occurs in the plants described herein may be measured in several ways, for example by an acetylene-reduction (AR) assay. An acetylene-reduction assay can be performed in vitro or in vivo. Evidence that a particular bacterium is providing fixed nitrogen to a plant can include: I) total plant N significantly increases upon inoculation, preferably with a concomitant increase in N concentration in the plant; 2) nitrogen deficiency symptoms are relieved under N-limiting conditions upon inoculation (which should include an increase in dry matter); 3) N2 fixation is documented through the use of an 15N approach (which can be isotope dilution experiments, 15N2 reduction assays, or 15N natural abundance assays); 4) fixed N is incorporated into a plant protein or metabolite; and 5) all of these effects are not be seen in non-inoculated plants or in plants inoculated with a mutant of the inoculum strain.
The wild-type nitrogen fixation regulatory cascade can be represented as a digital logic circuit where the inputs O2 and NH4+ pass through a NOR gate, the output of which enters an AND gate in addition to ATP. In some embodiments, the methods disclosed herein disrupt the influence of NH4+ on this circuit, at multiple points in the regulatory cascade, so that microbes can produce nitrogen even in fertilized fields. However, the methods disclosed herein also envision altering the impact of ATP or O2 on the circuitry, or replacing the circuitry with other regulatory cascades in the cell, or altering genetic circuits other than nitrogen fixation. Gene clusters can be re-engineered to generate functional products under the control of a heterologous regulatory system. By eliminating native regulatory elements outside of, and within, coding sequences of gene clusters, and replacing them with alternative regulatory systems, the functional products of complex genetic operons and other gene clusters can be controlled and/or moved to heterologous cells, including cells of different species other than the species from which the native genes were derived. Once re-engineered, the synthetic gene clusters can be controlled by genetic circuits or other inducible regulatory systems, thereby controlling the products' expression as desired. The expression cassettes can be designed to act as logic gates, pulse generators, oscillators, switches, or memory devices. The controlling expression cassette can be linked to a promoter such that the expression cassette functions as an environmental sensor, such as an oxygen, temperature, touch, osmotic stress, membrane stress, or redox sensor.
As an example, the nifL, nifA, nifT, and nifX genes can be eliminated from the nif gene cluster. Synthetic genes can be designed by codon randomizing the DNA encoding each amino acid sequence. Codon selection is performed, specifying that codon usage be as divergent as possible from the codon usage in the native gene. Proposed sequences are scanned for any undesired features, such as restriction enzyme recognition sites, transposon recognition sites, repetitive sequences, sigma 54 and sigma 70 promoters, cryptic ribosome binding sites, and rho independent terminators. Synthetic ribosome binding sites are chosen to match the strength of each corresponding native ribosome binding site, such as by constructing a fluorescent reporter plasmid in which the 150 bp surrounding a gene's start codon (from −60 to +90) is fused to a fluorescent gene. This chimera can be expressed under control of the Ptac promoter, and fluorescence measured via flow cytometry. To generate synthetic ribosome binding sites, a library of reporter plasmids using 150 bp (−60 to +90) of a synthetic expression cassette is generated. Briefly, a synthetic expression cassette can consist of a random DNA spacer, a degenerate sequence encoding an RBS library, and the coding sequence for each synthetic gene. Multiple clones are screened to identify the synthetic ribosome binding site that best matched the native ribosome binding site. Synthetic operons that consist of the same genes as the native operons are thus constructed and tested for functional complementation. A further exemplary description of synthetic operons is provided in US20140329326.
Bacterial Species Microbes useful in the methods and compositions disclosed herein may be obtained from any source. In some cases, microbes may be bacteria, archaea, protozoa or fungi. The microbes of this disclosure may be nitrogen fixing microbes, for example a nitrogen fixing bacteria, nitrogen fixing archaea, nitrogen fixing fungi, nitrogen fixing yeast, or nitrogen fixing protozoa. Microbes useful in the methods and compositions disclosed herein may be spore forming microbes, for example spore forming bacteria. In some cases, bacteria useful in the methods and compositions disclosed herein may be Gram positive bacteria or Gram negative bacteria. In some cases, the bacteria may be an endospore forming bacteria of the Firmicute phylum. In some cases, the bacteria may be a diazatroph. In some cases, the bacteria may not be a diazotroph.
The methods and compositions of this disclosure may be used with an archaea, such as, for example, Methanothermobacter thermoautotrophicus.
In some cases, bacteria which may be useful include, but are not limited to, Agrobacterium radiobacter, Bacillus acidocaldarius, Bacillus acidoterrestris, Bacillus agri, Bacillus aizawai, Bacillus albolactis, Bacillus alcalophilus, Bacillus alvei, Bacillus aminoglucosidicus, Bacillus aminovorans; Bacillus amyloyticus (also known as Paenibacillus amylolyticus) Bacillus amyloliquefaciens, Bacillus aneurinolyticus, Bacillus atrophaeus, Bacillus azotoformans, Bacillus badius, Bacillus cereus (synonyms: Bacillus endorhythmos, Bacillus medusa), Bacillus chitinosporus, Bacillus circulans, Bacillus coagulans, Bacillus endoparasiticus Bacillus fastidiosus, Bacillus firmus, Bacillus kurstaki, Bacillus lacticola, Bacillus lactimorbus, Bacillus lactis, Bacillus laterosporus (also known as Brevibacillus laterosporus), Bacillus lautus, Bacillus lentimorbus, Bacillus lentus, Bacillus licheniformis, Bacillus maroccanus, Bacillus megaterium, Bacillus metiens, Bacillus mycoides, Bacillus natio, Bacillus nematocida, Bacillus nigrificans, Bacillus nigrum, Bacillus pantothenticus, Bacillus popillae, Bacillus psychrosaccharolyticus, Bacillus pumilus, Bacillus sianensis, Bacillus smithii, Bacillus sphaericus, Bacillus subtilis, Bacillus thuringiensis, Bacillus uniflagellatus, Bradyrhizobium japonicum, Brevibacillus brevis Brevibacillus laterosporus (formerly Bacillus laterosporus), Chromobacterium subtsugae, Delftia acidovorans, Lactobacillus acidophilus, Lysobacter antibioticus, Lysobacter enzymogenes, Paenibacillus alvei, Paenibacillus polymyxa, Paenibacillus popilliae (formerly Bacillus popilliae), Pantoea agglomerans, Pasteuria penetrans (formerly Bacillus penetrates), Pasteuria usgae, Pectobacterium carotovorum (formerly Erwinia carotovora), Pseudomonas aeruginosa, Pseudomonas aureofaciens, Pseudomonas cepacia (formerly known as Burkholderia cepacia), Pseudomonas chlororaphis, Pseudomonas fluorescens, Pseudomonas proradix, Pseudomonas putida, Pseudomonas syringae, Serratia entomophila, Serratia marcescens, Streptomyces colombiensis, Streptomyces galbus, Streptomyces goshikiensis, Streptomyces griseoviridis, Streptomyces lavendulae, Streptomyces prasinus, Streptomyces saraceticus, Streptomyces venezuelae, Xanthomonas campestris, Xenorhabdus luminescens, Xenorhabdus nematophila, Rhodococcus globerulus AQ719 (NRRL Accession No. B-21663), Bacillus sp. AQ175 (ATCC Accession No. 55608), Bacillus sp. AQ 177 (ATCC Accession No. 55609), Bacillus sp. AQ178 (ATCC Accession NO. 53522), and Streptomyces sp. strain NRRL Accession No. 13-30145. In some cases the bacterium may be Azotobacter chroococcum, Methanosarcina barker, Klesiella pneumoniae, Azotobacter vinelandii, Rhodobacter spharoides, Rhodobacter capsulatus, Rhodobcter palustris, Rhodosporillum rubrum, Rhizobium leguminosarum or Rhizobium etli.
In some cases the bacterium may be a species of Clostridium, for example Clostridium pasteurianum, Clostridium beijerinckii, Clostridium perfringens, Clostridium tetani, Clostridium acetobutylicum.
In some cases, bacteria used with the methods and compositions of the present disclosure may be cyanobacteria. Examples of cyanobacterial genuses include Anabaena (for example Anagaena sp. PCC7120), Nostoc (for example Nostoc punctiforme), or Synechocystis (for example Synechocystis sp. PCC6803).
In some cases, bacteria used with the methods and compositions of the present disclosure may belong to the phylum Chlorobi, for example Chlorobium tepidum.
In some cases, microbes used with the methods and compositions of the present disclosure may comprise a gene homologous to a known NifH gene. Sequences of known NifH genes may be found in, for example, the Zehr lab NifH database, (https://wwwzehr.pmc.aese.edu/nifH_Database_Public/, Apr. 4, 2014), or the Buckley lab NifH database (http://www.css.cornell.edu/faculty/buckley/nifh.htm, and Gaby, John Christian, and Daniel H. Buckley. “A comprehensive aligned nifH gene database: a multipurpose tool for studies of nitrogen-fixing bacteria.” Database 2014 (2014): bau001.). In some cases, microbes used with the methods and compositions of the present disclosure may comprise a sequence which encodes a polypeptide with at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 96%, 98%, 99% or more than 99% sequence identity to a sequence from the Zehr lab NifH database, (https://wwwzehr.pmc.ucsc.edu/nifH_Database_Public/, Apr. 4, 2014). In some cases, microbes used with the methods and compositions of the present disclosure may comprise a sequence which encodes a polypeptide with at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 96%, 98%, 99% or more than 99% sequence identity to a sequence from the Buckley lab NifH database, (Gaby, John Christian, and Daniel H. Buckley. “A comprehensive aligned nifH gene database: a multipurpose tool for studies of nitrogen-fixing bacteria.” Database 2014 (2014): bau001.).
Microbes useful in the methods and compositions disclosed herein can be obtained by extracting microbes from surfaces or tissues of native plants; grinding seeds to isolate microbes; planting seeds in diverse soil samples and recovering microbes from tissues; or inoculating plants with exogenous microbes and determining which microbes appear in plant tissues. Non-limiting examples of plant tissues include a seed, seedling, leaf, cutting, plant, bulb or tuber. In some cases, bacteria are isolated from a seed. The parameters for processing samples may be varied to isolate different types of associative microbes, such as rhizospheric, epiphytes, or endophytes. Bacteria may also be sourced from a repository, such as environmental strain collections, instead of initially isolating from a first plant. The microbes can be genotyped and phenotyped, via sequencing the genomes of isolated microbes; profiling the composition of communities in planta; characterizing the transcriptomic functionality of communities or isolated microbes; or screening microbial features using selective or phenotypic media (e.g., nitrogen fixation or phosphate solubilization phenotypes). Selected candidate strains or populations can be obtained via sequence data; phenotype data; plant data (e.g., genome, phenotype, and/or yield data); soil data (e.g., pH, N/P/K content, and/or bulk soil biotic communities); or any combination of these.
The bacteria and methods of producing bacteria described herein may apply to bacteria able to self-propagate efficiently on the leaf surface, root surface, or inside plant tissues without inducing a damaging plant defense reaction, or bacteria that are resistant to plant defense responses. The bacteria described herein may be isolated by culturing a plant tissue extract or leaf surface wash in a medium with no added nitrogen. However, the bacteria may be unculturable, that is, not known to be culturable or difficult to culture using standard methods known in the art. The bacteria described herein may be an endophyte or an epiphyte or a bacterium inhabiting the plant rhizosphere (rhizospheric bacteria). The bacteria obtained after repeating the steps of introducing genetic variation, exposure to a plurality of plants, and isolating bacteria from plants with an improved trait one or more times (e.g. 1, 2, 3, 4, 5, 10, 15, 25, or more times) may be endophytic, epiphytic, or rhizospheric. Endophytes are organisms that enter the interior of plants without causing disease symptoms or eliciting the formation of symbiotic structures, and are of agronomic interest because they can enhance plant growth and improve the nutrition of plants (e.g., through nitrogen fixation). The bacteria can be a seed-borne endophyte. Seed-borne endophytes include bacteria associated with or derived from the seed of a grass or plant, such as a seed-borne bacterial endophyte found in mature, dry, undamaged (e.g., no cracks, visible fungal infection, or prematurely germinated) seeds. The seed-borne bacterial endophyte can be associated with or derived from the surface of the seed; alternatively, or in addition, it can be associated with or derived from the interior seed compartment (e.g., of a surface-sterilized seed). In some cases, a seed-borne bacterial endophyte is capable of replicating within the plant tissue, for example, the interior of the seed. Also, in some cases, the seed-borne bacterial endophyte is capable of surviving desiccation.
The bacterial isolated according to methods of the disclosure, or used in methods or compositions of the disclosure, can comprise a plurality of different bacterial taxa in combination. By way of example, the bacteria may include Proteobacteria (such as Pseudomonas, Enterobacter, Stenotrophomonas, Burkholderia, Rhizobium, Herbaspirillum, Pantoea, Serratia, Rahnella, Azospirillum, Azorhizobium, Azotobacter, Duganella, Delftia, Bradyrhizobiun, Sinorhizobium and Halomonas), Firmicutes (such as Bacillus, Paenibacillus, Lactobacillus, Mycoplasma, and Acetabacterium), and Actinobacteria (such as Streptomyces, Rhodacoccus, Microbacterium, and Curtobacterium). The bacteria used in methods and compositions of this disclosure may include nitrogen fixing bacterial consortia of two or more species. In some cases, one or more bacterial species of the bacterial consortia may be capable of fixing nitrogen. In some cases, one or more species of the bacterial consortia may facilitate or enhance the ability of other bacteria to fix nitrogen. The bacteria which fix nitrogen and the bacteria which enhance the ability of other bacteria to fix nitrogen may be the same or different. In some examples, a bacterial strain may be able to fix nitrogen when in combination with a different bacterial strain, or in a certain bacterial consortia, but may be unable to fix nitrogen in a monoculture. Examples of bacterial genuses which may be found in a nitrogen fixing bacterial consortia include, but are not limited to, Herbaspirillum, Azospirillum, Enterobacter, and Bacillus.
Bacteria that can be produced by the methods disclosed herein include Azotobacter sp., Bradyrhizobium sp., Klebsiella sp., and Sinorhizobium sp. In some cases, the bacteria may be selected from the group consisting of: Azotobacter vinelandii, Bradyrhizobium japonicum, Klebsiella pneumoniae, and Sinorhizobium meliloti. In some cases, the bacteria may be of the genus Enterobacter or Rahnella. In some cases, the bacteria may be of the genus Frankia, or Clostridium. Examples of bacteria of the genus Clostridium include, but are not limited to, Clostridium acetobutilicum, Clostridium pasteurianum, Clostridium beijerinckii, Clostridium perfringens, and Clostridium tetani. In some cases, the bacteria may be of the genus Paenibacillus, for example Paenibacillus azotofixans, Paenibacillus borealis, Paenibacillus durus, Paenibacillus macerans, Paenibacillus polymyxa, Paenibacillus alvei, Paenibacillus amylolyticus, Paenibacillus campinasensis, Paenibacillus chibensis, Paenibacillus glucanolyticus, Paenibacillus illinoisensis, Paenibacillus larvae subsp. Larvae, Paenibacillus larvae subsp. Pulvifaciens, Paenibacillus lautus, Paenibacillus macerans, Paenibacillus macquariensis, Paenibacillus macquariensis, Paenibacillus pabuli, Paenibacillus peoriae, or Paenibacillus polymyxa.
In some examples, bacteria isolated according to methods of the disclosure can be a member of one or more of the following taxa: Achronobacter, Acidithiobacillus, Acidovorax, Acidovoraz, Acinetobacter, Actinoplanes, Adlercreutzia, Aerococcus, Aeromonas, Afipia, Agromyces, Ancylobacter, Arthrobacter, Atopostipes, Azospirillum, Bacillus, Bdellovibrio, Beijerinckia, Bosea, Bradyrhizobium, Brevibacillus, Brevundimonas, Burkholderia, Candidatus Haloredivivus, Caulobacter, Cellulomonas, Cellvibrio, Chryseobacterium, Citrobacter, Clostridium, Coraliomargarita, Corynebacterium, Cupriavidus, Curtobacterium, Curvibacter, Deinococcus, Delftia, Desemzia, Devosia, Dokdonella, Dyella, Enhydrobacter, Enterobacter, Enterococcus, Erwinia, Escherichia, Escherichia/Shigella, Exiguobacterium, Ferroglobus, Filimonas, Finegoldia, Flavisolibacter, Flavobacterium, Frigoribacterium, Gluconacetobacter, Hafnia, Halobaculum, Halomonas, Halosimplex, Herbaspirillum, Hymenobacter, Klebsiella, Kocuria, Kosakonia, Lactobacillus, Leclercia, Lentzea, Luteibacter, Luteimonas, Massilia, Mesorhizobium, Methylobacterium, Microbacterium, Micrococcus, Microvirga, Mycobacterium, Neisseria, Nocardia, Oceanibaculum, Ochrobactrum, Okibacterium, Oligotropha, Oryzihumus, Oxalophagus, Paenibacillus, Panteoa, Pantoea, Pelomonas, Perlucidibaca, Plantibacter, Polynucleobacter, Propionibacterium, Propioniciclava, Pseudoclavibacter, Pseudomonas, Pseudonocardia, Pseudoxanthomonas, Psychrobacter, Rahnella, Ralstonia, Rheinheimera, Rhizobium, Rhodococcus, Rhodopseudomonas, Roseateles, Ruminococcus, Sebaldella, Sediminibacillus, Sediminibacterium, Serratia, Shigella, Shigella, Sinorhizobium, Sinosporangium, Sphingobacterium, Sphingomonas, Sphingopyxis, Sphingosinicella, Staphylococcus, 25 Stenotrophomonas, Strenotrophomonas, Streptococcus, Streptomyces, Stygiolobus, Sulfurisphaera, Tatumella, Tepidimonas, Thermomonas, Thiobacillus, Variovorax, WPS-2 genera incertae sedis, Xanthomonas, and Zimmermanella.
In some cases, a bacterial species selected from at least one of the following genera are utilized: Enterobacter, Klebsiella, Kosakonia, and Rahnella. In some cases, a combination of bacterial species from the following genera are utilized: Enterobacter, Klebsiella, Kosakonia, and Rahnella. In some cases, the species utilized can be one or more of: Enterobacter sacchari, Klebsiella variicola, Kosakonia sacchari, and Rahnella aquatilis.
In some cases, a Gram positive microbe may have a Molybdenum-Iron nitrogenase system comprising: nifH, nifD, nifK, nifB, nifE, nifN, nifX, hesA, nifV, nifU, nifW, nifU, nifS, nifl1, and nifl2. In some cases, a Gram positive microbe may have a vanadium nitrogenase system comprising: vnfDG, vnfK, vnfE, vnfN, vupC, vupB, vupA, vnfV, vnfR1, vnfH, vnfR2, vnfA, (transcriptional regulator). In some cases, a Gram positive microbe may have an iron-only nitrogenase system comprising: anfK, anfG, anfD, anfH, anfA (transcriptional regulator). In some cases a Gram positive microbe may have a nitrogenase system comprising glnB, and glnK (nitrogen signaling proteins). Some examples of enzymes involved in nitrogen metabolism in Gram positive microbes include glnA (glutamine synthetase), gdh (glutamate dehydrogenase), bdh (3-hydroxybutyrate dehydrogenase), glutaminase, gltAB/gltB/gltS (glutamate synthase), asnA/asnB (aspartate-ammonia ligase/asparagine synthetase), and ansA/ansZ (asparaginase). Some examples of proteins involved in nitrogen transport in Gram positive microbes include amtB (ammonium transporter), glnK (regulator of ammonium transport), glnPHQ/glnQHMP (ATP-dependent glutamine/glutamate transporters), glnT/alsT/yrbD/yflA (glutamine-like proton symport transporters), and gltP/gltT/yhcl/nqt (glutamate-like proton symport transporters).
Examples of Gram positive microbes which may be of particular interest include Paenibacillus polymixa, Paenibacillus riograndensis, Paenibacillus sp., Frankia sp., Heliobacterium sp., Heliobacterium chlorum, Heliobacillus sp., Heliophilum sp., Heliorestis sp., Clostridium acetobutylicum, Clostridium sp., Mycobacterium flaum, Mycobacterium sp., Arthrobacter sp., Agromyces sp., Corynebacterium autitrophicum, Corynebacterium sp., Micromonspora sp., Propionibacteria sp., Streptomyces sp., and Microbacterium sp.
Some examples of genetic alterations which may be make in Gram positive microbes include: deleting glnR to remove negative regulation of BNF in the presence of environmental nitrogen, inserting different promoters directly upstream of the nif cluster to eliminate regulation by GlnR in response to environmental nitrogen, mutating glnA to reduce the rate of ammonium assimilation by the GS-GOGAT pathway, deleting amtB to reduce uptake of ammonium from the media, mutating glnA so it is constitutively in the feedback-inhibited (FBI-GS) state, to reduce ammonium assimilation by the GS-GOGAT pathway.
In some cases, glnR is the main regulator of N metabolism and fixation in Paenibacillus species. In some cases, the genome of a Paenibacillus species may not contain a gene to produce glnR. In some cases, the genome of a Paenibacillus species may not contain a gene to produce glnE or glnD. In some cases, the genome of a Paenibacillus species may contain a gene to produce glnB or glnK. For example Paenibacillus sp. WLY78 doesn't contain a gene for glnB, or its homologs found in the archaeon Methanococcus maripaludis, nifI1 and nifI2. In some cases, the genomes of Paenibacillus species may be variable. For example, Paenibacillus polymixa E681 lacks glnK and gdh, has several nitrogen compound transporters, but only amtB appears to be controlled by GlnR. In another example, Paenibacillus sp. JDR2 has glnK, gdh and most other central nitrogen metabolism genes, has many fewer nitrogen compound transporters, but does have glnPHQ controlled by GlnR. Paenibacillus riograndensis SBR5 contains a standard glnRA operon, an fdx gene, a main nil operon, a secondary nif operon, and an anf operon (encoding iron-only nitrogenase). Putative glnR/tnrA sites were found upstream of each of these operons. GlnR may regulate all of the above operons, except the anf operon. GlnR may bind to each of these regulatory sequences as a dimer.
Paenibacillus N-fixing strains may fall into two subgroups: Subgroup I, which contains only a minimal nif gene cluster and subgroup II, which contains a minimal cluster, plus an uncharacterized gene between nifX and hesA, and often other clusters duplicating some of the nif genes, such as nifH, nifHDK, nifBEN, or clusters encoding vanadaium nitrogenase (vnf) or iron-only nitrogenase (anf) genes.
In some cases, the genome of a Paenibacillus species may not contain a gene to produce g/n/3 or &K. In some cases, the genome of a Paenibacillus species may contain a minimal nif cluster with 9 genes transcribed from a sigma-70 promoter. In some cases a Paenibacillus nif cluster may be negatively regulated by nitrogen or oxygen. In some cases, the genome of a Paenibacillus species may not contain a gene to produce sigma-54. For example, Paenibacillus sp. WLY78 does not contain a gene for sigma-54. In some cases, a nif cluster may be regulated by glnR, and/or TnrA. In some cases, activity of a nif cluster may be altered by altering activity of glnR, and/or TnrA.
In Bacilli, glutamine synthetase (GS) is feedback-inhibited by high concentrations of intracellular glutamine, causing a shift in confirmation (referred to as FBI-GS). Nif clusters contain distinct binding sites for the regulators GlnR and TnrA in several Bacilli species. GlnR binds and represses gene expression in the presence of excess intracellular glutamine and AMP. A role of GlnR may be to prevent the influx and intracellular production of glutamine and ammonium under conditions of high nitrogen availability. TnrA may bind and/or activate (or repress) gene expression in the presence of limiting intracellular glutamine, and/or in the presence of FBI-GS. In some cases the activity of a Bacilli nif cluster may be altered by altering the activity of GlnR.
Feedback-inhibited glutamine synthetase (FBI-GS) may bind GlnR and stabilize binding of GlnR to recognition sequences. Several bacterial species have a GlnR/TnrA binding site upstream of the nif cluster. Altering the binding of FBI-GS and GlnR may alter the activity of the nif pathway.
Sources of Microbes The bacteria (or any microbe according to the disclosure) may be obtained from any general terrestrial environment, including its soils, plants, fungi, animals (including invertebrates) and other biota, including the sediments, water and biota of lakes and rivers; from the marine environment, its biota and sediments (for example, sea water, marine muds, marine plants, marine invertebrates (for example, sponges), marine vertebrates (for example, fish)); the terrestrial and marine geosphere (regolith and rock, for example, crushed subterranean rocks, sand and clays); the cryosphere and its meltwater; the atmosphere (for example, filtered aerial dusts, cloud and rain droplets); urban, industrial and other man-made environments (for example, accumulated organic and mineral matter on concrete, roadside gutters, roof surfaces, and road surfaces).
The plants from which the bacteria (or any microbe according to the disclosure) are obtained may be a plant having one or more desirable traits, for example a plant which naturally grows in a particular environment or under certain conditions of interest. By way of example, a certain plant may naturally grow in sandy soil or sand of high salinity, or under extreme temperatures, or with little water, or it may be resistant to certain pests or disease present in the environment, and it may be desirable for a commercial crop to be grown in such conditions, particularly if they are, for example, the only conditions available in a particular geographic location. By way of further example, the bacteria may be collected from commercial crops gown in such environments, or more specifically from individual crop plants best displaying a trait of interest amongst a crop grown in any specific environment: for example the fastest-growing plants amongst a crop grown in saline-limiting soils, or the least damaged plants in crops exposed to severe insect damage or disease epidemic, or plants having desired quantities of certain metabolites and other compounds, including fiber content, oil content, and the like, or plants displaying desirable colors, taste or smell. The bacteria may be collected from a plant of interest or any material occurring in the environment of interest, including fungi and other animal and plant biota, soil, water, sediments, and other elements of the environment as referred to previously.
The bacteria (or any microbe according to the disclosure) may be isolated from plant tissue. This isolation can occur from any appropriate tissue in the plant, including for example root, stem and leaves, and plant reproductive tissues. By way of example, conventional methods for isolation from plants typically include the sterile excision of the plant material of interest (e.g. root or stem lengths, leaves), surface sterilization with an appropriate solution (e.g. 2% sodium hypochlorite), after which the plant material is placed on nutrient medium for microbial growth. Alternatively, the surface-sterilized plant material can be crushed in a sterile liquid (usually water) and the liquid suspension, including small pieces of the crushed plant material spread over the surface of a suitable solid agar medium, or media, which may or may not be selective (e.g. contain only phytic acid as a source of phosphorus). This approach is especially useful for bacteria which form isolated colonies and can be picked off individually to separate plates of nutrient medium, and further purified to a single species by well-known methods. Alternatively, the plant root or foliage samples may not be surface sterilized but only washed gently thus including surface-dwelling epiphytic microorganisms in the isolation process, or the epiphytic microbes can be isolated separately, by imprinting and lifting off pieces of plant roots, stem or leaves onto the surface of an agar medium and then isolating individual colonies as above. This approach is especially useful for bacteria, for example. Alternatively, the roots may be processed without washing off small quantities of soil attached to the roots, thus including microbes that colonize the plant rhizosphere. Otherwise, soil adhering to the roots can be removed, diluted and spread out onto agar of suitable selective and non-selective media to isolate individual colonies of rhizospheric bacteria.
Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedures The microbial deposits of the present disclosure were made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure (Budapest Treaty).
Applicants state that pursuant to 37 C.F.R. § 1.808(a)(2) “all restrictions imposed by the depositor on the availability to the public of the deposited material will be irrevocably removed upon the granting of the patent.” This statement is subject to paragraph (b) of this section (i.e. 37 C.F.R. § 1.808(b)).
Biologically pure cultures of Rahnella aquatilis and Enterobacter sacchari were deposited on Jul. 14, 2015 with the American Type Culture Collection (ATCC; an International Depositary Authority), 10801 University Blvd., Manassas, Va. 20110, USA, and assigned ATTC Patent Deposit Designation numbers PTA-122293 and PTA-122294, respectively. The applicable deposit information is found below in Table A.
The Enterobacter sacchari has now been reclassified as Kosakonia sacchari, the name for the organism may be used interchangeably throughout the manuscript.
Many microbes of the present disclosure are derived from two wild-type strains, as depicted in FIG. 18 and FIG. 19. Strain CI006 is a bacterial species previously classified in the genus Enterobacter (see aforementioned reclassification into Kosakonia), and FIG. 19 identities the lineage of the mutants that have been derived from CI006. Strain CI019 is a bacterial species classified in the genus Rahnella, and FIG. 19 identifies the lineage of the mutants that have been derived from CI019. With regard to FIG. 18 and FIG. 19, it is noted that strains comprising CM in the name are mutants of the strains depicted immediately to the left of said CM strain. The deposit information for the CI006 Kosakonia wild type (WT) and CI019 Rahnella WT are found in the below Table A.
Some microorganisms described in this application were deposited on Jan. 6, 2017 or Aug. 11, 2017 with the Bigelow National Center for Marine Algae and Microbiota (NCMA), located at 60 Bigelow Drive, East Boothbay, Me. 04544, USA. As aforementioned, all deposits were made under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. The Bigelow National Center for Marine Algae and Microbiota accession numbers and dates of deposit for the aforementioned Budapest Treaty deposits are provided in Table A.
Biologically pure cultures of Kosakonia sacchari (WT), Rahnella aquatilis (WT), and a variant Kosakonia sacchari strain were deposited on Jan. 6, 2017 with the Bigelow National Center for Marine Algae and Microbiota (NCMA), located at 60 Bigelow Drive, East Boothbay, Me. 04544, USA, and assigned NCMA Patent Deposit Designation numbers 201701001, 201701003, and 201701002, respectively. The applicable deposit information is found below in Table A.
Biologically pure cultures of variant Kosakonia sacchari strains were deposited on Aug. 11, 2017 with the Bigelow National Center for Marine Algae and Microbiota (NCMA), located at 60 Bigelow Drive, East Boothbay, Me. 04544, USA, and assigned NCMA Patent Deposit Designation numbers 201708004, 201708003, and 201708002, respectively. The applicable deposit information is found below in Table A.
A biologically pure culture of Klebsiella variicola (WV was deposited on Aug. 11, 2017 with the Bigelow National Center for Marine Algae and Microbiota (NCMA), located at 60 Bigelow Drive, East Boothbay, Me. 04544, USA, and assigned NCMA Patent Deposit Designation number 201708001. Biologically pure cultures of two Klebsiella variicola variants were deposited on Dec. 20, 2017 with the Bigelow National Center for Marine Algae and Microbiota (NCMA), located at 60 Bigelow Drive, East Boothbay, Me. 04544, USA, and assigned NCMA Patent Deposit Designation numbers 201712001 and 201712002, respectively. The applicable deposit information is found below in Table A.
TABLE A
Microorganisms Deposited under the Budapest Treaty
Pivot Strain
Designation
(some strains
Depos- have multiple Accession Date of
itory designations) Taxonomy Number Deposit
ATCC Rahnella aquatilis PTA-122293 Jul. 14,
2015
ATCC Enterobacter PTA-122294 Jul. 14,
sacchari 2015
(taxonomically re-
classified after de-
posit as Kosakonia
sacchari)
NCMA CI006, Kosakonia sacchari 201701001 Jan. 6,
PBC6.1, 6 (WT) 2017
NCMA CI019, 19 Rahnella aquatilis 201701003 Jan. 6,
(WT) 2017
NCMA CM029, Kosakonia sacchari 201701002 Jan. 6,
6-412 2017
NCMA 6-403 Kosakonia sacchari 201708004 Aug. 11,
CM037 2017
NCMA 6-404, Kosakonia sacchari 201708003 Aug. 11,
CM38, 2017
PBC6.38
NCMA CM094, Kosakonia sacchari 201708002 Aug. 11,
6-881, 2017
PBC6.94
NCMA CI137, 137, Klebsiella variicola 201708001 Aug. 11,
PB137 (WT) 2017
NCMA 137-1034 Klebsiella variicola 201712001 Dec. 20,
2017
NCMA 137-1036 Klebsiella variicola 201712002 Dec. 20,
2017
Isolated and Biologically Pure Microorganisms The present disclosure, in certain embodiments, provides isolated and biologically pure microorganisms that have applications, infer alia, in agriculture. The disclosed microorganisms can be utilized in their isolated and biologically pure states, as well as being formulated into compositions (see below section for exemplary composition descriptions). Furthermore, the disclosure provides microbial compositions containing at least two members of the disclosed isolated and biologically pure microorganisms, as well as methods of utilizing said microbial compositions. Furthermore, the disclosure provides for methods of modulating nitrogen fixation in plants via the utilization of the disclosed isolated and biologically pure microbes.
In some aspects, the isolated and biologically pure microorganisms of the disclosure are those from Table A. In other aspects, the isolated and biologically pure microorganisms of the disclosure are derived from a microorganism of Table A. For example, a strain, child, mutant, or derivative, of a microorganism from Table A are provided herein. The disclosure contemplates all possible combinations of microbes listed in Table A, said combinations sometimes forming a microbial consortia. The microbes from Table A, either individually or in any combination, can be combined with any plant, active (synthetic, organic, etc.), adjuvant, carrier, supplement, or biological, mentioned in the disclosure.
Compositions Compositions comprising bacteria or bacterial populations produced according to methods described herein and/or having characteristics as described herein can be in the form of a liquid, a foam, or a dry product. Compositions comprising bacteria or bacterial populations produced according to methods described herein and/or having characteristics as described herein may also be used to improve plant traits. In some examples, a composition comprising bacterial populations may be in the form of a dry powder, a slurry of powder and water, or a flowable seed treatment. The compositions comprising bacterial populations may be coated on a surface of a seed, and may be in liquid form.
The composition can be fabricated in bioreactors such as continuous stirred tank reactors, batch reactors, and on the farm. In some examples, compositions can be stored in a container, such as a jug or in mini bulk. In some examples, compositions may be stored within an object selected from the group consisting of a bottle, jar, ampule, package, vessel, bag, box, bin, envelope, carton, container, silo, shipping container, truck bed, and/or case.
Compositions may also be used to improve plant traits. In some examples, one or more compositions may be coated onto a seed. In some examples, one or more compositions may be coated onto a seedling. In some examples, one or more compositions may be coated onto a surface of a seed. In some examples, one or more compositions may be coated as a layer above a surface of a seed. In some examples, a composition that is coated onto a seed may be in liquid form, in dry product form, in foam form, in a form of a slurry of powder and water, or in a flowable seed treatment. In some examples, one or more compositions may be applied to a seed and/or seedling by spraying, immersing, coating, encapsulating, and/or dusting the seed and/or seedling with the one or more compositions. In some examples, multiple bacteria or bacterial populations can be coated onto a seed and/or a seedling of the plant. In some examples, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more than ten bacteria of a bacterial combination can be selected from one of the following genera: Acidovorax, Agrobacterium, Bacillus, Burkholderia, Chryseobacterium, Curtobacterium, Enterobacter, Escherichia, Methylobacterium, Paenibacillus, Pantoea, Pseudomonas, Ralstonia, Saccharibacillus, Sphingomonas, and Stenotrophomonas.
In some examples, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more than ten bacteria and bacterial populations of an endophytic combination are selected from one of the following families: Bacillaceae, Burkholderiaceae, Comamonadaceae, Enterobacteriaceae, Flavobacteriaceae, Methylobacteriaceae, Microbacteriaceae, Paenibacillileae, Pseudomonnaceae, Rhizobiaceae, Sphingomonadaceae, Xanthomonadaceae, Cladosporiaceae, Gnomoniaceae, Incertae sedis, Lasiosphaeriaceae, Netriaceae, and Pleosporaceae.
In some examples, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least night, at least ten, or more than ten bacteria and bacterial populations of an endophytic combination are selected from one of the following families: Bacillaceae, Burkholderiaceae, Comamonadaceae, Enterobacteriaceae, Flavobacteriaceae, Methylobacteriaceae, Microbacteriaceae, Paenibacillileae, Pseudomonnaceae, Rhizobiaceae, Sphingomonadaceae, Xanthomonadaceae, Cladosporiaceae, Gnomoniaceae, Incertae sedis, Lasiosphaeriaceae, Netriaceae, Pleosporaceae.
Examples of compositions may include seed coatings for commercially important agricultural crops, for example, sorghum, canola, tomato, strawberry, barley, rice, maize, and wheat. Examples of compositions can also include seed coatings for corn, soybean, canola, sorghum, potato, rice, vegetables, cereals, and oilseeds. Seeds as provided herein can be genetically modified organisms (GMO), non-GMO, organic, or conventional. In some examples, compositions may be sprayed on the plant aerial parts, or applied to the roots by inserting into furrows in which the plant seeds are planted, watering to the soil, or dipping the roots in a suspension of the composition. In some examples, compositions may be dehydrated in a suitable manner that maintains cell viability and the ability to artificially inoculate and colonize host plants. The bacterial species may be present in compositions at a concentration of between 108 to 1010 CFU/ml. In some examples, compositions may be supplemented with trace metal ions, such as molybdenum ions, iron ions, manganese ions, or combinations of these ions. The concentration of ions in examples of compositions as described herein may between about 0.1 mM and about 50 mM. Some examples of compositions may also be formulated with a carrier, such as beta-glucan, carboxylmethyl cellulose (CMC), bacterial extracellular polymeric substance (EPS), sugar, animal milk, or other suitable carriers. In some examples, peat or planting materials can be used as a carrier, or biopolymers in which a composition is entrapped in the biopolymer can be used as a carrier. The compositions comprising the bacterial populations described herein can improve plant traits, such as promoting plant growth, maintaining high chlorophyll content in leaves, increasing fruit or seed numbers, and increasing fruit or seed unit weight.
The compositions comprising the bacterial populations described herein may be coated onto the surface of a seed. As such, compositions comprising a seed coated with one or more bacteria described herein are also contemplated. The seed coating can be formed by mixing the bacterial population with a porous, chemically inert granular carrier. Alternatively, the compositions may be inserted directly into the furrows into which the seed is planted or sprayed onto the plant leaves or applied by dipping the roots into a suspension of the composition. An effective amount of the composition can be used to populate the sub-soil region adjacent to the roots of the plant with viable bacterial growth, or populate the leaves of the plant with viable bacterial growth. In general, an effective amount is an amount sufficient to result in plants with improved traits (e.g. a desired level of nitrogen fixation).
Bacterial compositions described herein can be formulated using an agriculturally acceptable carrier. The formulation useful for these embodiments may include at least one member selected from the group consisting of a tackifier, a microbial stabilizer, a fungicide, an antibacterial agent, a preservative, a stabilizer, a surfactant, an anti-complex agent, an herbicide, a nematicide, an insecticide, a plant growth regulator, a fertilizer, a rodenticide, a dessicant, a bactericide, a nutrient, or any combination thereof. In some examples, compositions may be shelf-stable. For example, any of the compositions described herein can include an agriculturally acceptable carrier (e.g., one or more of a fertilizer such as a non-naturally occurring fertilizer, an adhesion agent such as a non-naturally occurring adhesion agent, and a pesticide such as a non-naturally occurring pesticide). A non-naturally occurring adhesion agent can be, for example, a polymer, copolymer, or synthetic wax. For example, any of the coated seeds, seedlings, or plants described herein can contain such an agriculturally acceptable carrier in the seed coating. In any of the compositions or methods described herein, an agriculturally acceptable carrier can be or can include a non-naturally occurring compound (e.g., a non-naturally occurring fertilizer, a non-naturally occurring adhesion agent such as a polymer, copolymer, or synthetic wax, or a non-naturally occurring pesticide). Non-limiting examples of agriculturally acceptable carriers are described below. Additional examples of agriculturally acceptable carriers are known in the art.
In some cases, bacteria are mixed with an agriculturally acceptable carrier. The carrier can be a solid carrier or liquid carrier, and in various forms including microspheres, powders, emulsions and the like. The carrier may be any one or more of a number of carriers that confer a variety of properties, such as increased stability, wettability, or dispersability. Wetting agents such as natural or synthetic surfactants, which can be nonionic or ionic surfactants, or a combination thereof can be included in the composition. Water-in-oil emulsions can also be used to formulate a composition that includes the isolated bacteria (see, for example, U.S. Pat. No. 7,485,451). Suitable formulations that may be prepared include wettable powders, granules, gels, agar strips or pellets, thickeners, and the like, microencapsulated particles, and the like, liquids such as aqueous flowables, aqueous suspensions, water-in-oil emulsions, etc. The formulation may include grain or legume products, for example, ground grain or beans, broth or flour derived from grain or beans, starch, sugar, or oil.
In some embodiments, the agricultural carrier may be soil or a plant growth medium. Other agricultural carriers that may be used include water, fertilizers, plant-based oils, humectants, or combinations thereof. Alternatively, the agricultural carrier may be a solid, such as diatomaceous earth, loam, silica, alginate, clay, bentonite, vermiculite, seed cases, other plant and animal products, or combinations, including granules, pellets, or suspensions. Mixtures of any of the aforementioned ingredients are also contemplated as carriers, such as but not limited to, pesta (flour and kaolin clay), agar or flour-based pellets in loam, sand, or clay, etc. Formulations may include food sources for the bacteria, such as barley, rice, or other biological materials such as seed, plant parts, sugar cane bagasse, hulls or stalks from grain processing, ground plant material or wood from building site refuse, sawdust or small fibers from recycling of paper, fabric, or wood.
For example, a fertilizer can be used to help promote the growth or provide nutrients to a seed, seedling, or plant. Non-limiting examples of fertilizers include nitrogen, phosphorous, potassium, calcium, sulfur, magnesium, boron, chloride, manganese, iron, zinc, copper, molybdenum, and selenium (or a salt thereof). Additional examples of fertilizers include one or more amino acids, salts, carbohydrates, vitamins, glucose, NaCl, yeast extract, NH4H2PO4, (NH4)2SO4, glycerol, valine, L-leucine, lactic acid, propionic acid, succinic acid, malic acid, citric acid, KH tartrate, xylose, lyxose, and lecithin. In one embodiment, the formulation can include a tackifier or adherent (referred to as an adhesive agent) to help bind other active agents to a substance (e.g., a surface of a seed). Such agents are useful for combining bacteria with carriers that can contain other compounds (e.g., control agents that are not biologic), to yield a coating composition. Such compositions help create coatings around the plant or seed to maintain contact between the microbe and other agents with the plant or plant part. In one embodiment, adhesives are selected from the group consisting of: alginate, gums, starches, lecithins, formononetin, polyvinyl alcohol, alkali formononetinate, hesperetin, polyvinyl acetate, cephalins, Gum Arabic, Xanthan Gum, Mineral Oil, Polyethylene Glycol (PEG), Polyvinyl pyrrolidone (PVP), Arabino-galactan, Methyl Cellulose, PEG 400, Chitosan, Polyacrylamide, Polyacrylate, Polyacrylonitrile, Glycerol, Triethylene glycol, Vinyl Acetate, Gellan Gum, Polystyrene, Polyvinyl, Carboxymethyl cellulose, Gum Ghatti, and polyoxyethylene-polyoxybutylene block copolymers.
In some embodiments, the adhesives can be, e.g. a wax such as carnauba wax, beeswax, Chinese wax, shellac wax, spermaceti wax, candelilla wax, castor wax, ouricury wax, and rice bran wax, a polysaccharide (e.g., starch, dextrins, maltodextrins, alginate, and chitosans), a fat, oil, a protein (e.g., gelatin and zeins), gum arables, and shellacs. Adhesive agents can be non-naturally occurring compounds, e.g., polymers, copolymers, and waxes. For example, non-limiting examples of polymers that can be used as an adhesive agent include: polyvinyl acetates, polyvinyl acetate copolymers, ethylene vinyl acetate (EVA) copolymers, polyvinyl alcohols, polyvinyl alcohol copolymers, celluloses (e.g., ethylcelluloses, methylcelluloses, hydroxymethylcelluloses, hydroxypropylcelluloses, and carboxymethylcelluloses), polyvinylpyrolidones, vinyl chloride, vinylidene chloride copolymers, calcium lignosulfonates, acrylic copolymers, polyvinylacrylates, polyethylene oxide, acylamide polymers and copolymers, polyhydroxyethyl acrylate, methyl acrylamide monomers, and polychloroprene.
In some examples, one or more of the adhesion agents, anti-fungal agents, growth regulation agents, and pesticides (e.g., insecticide) are non-naturally occurring compounds (e.g., in any combination). Additional examples of agriculturally acceptable carriers include dispersants (e.g., polyvinylpyrrolidone/vinyl acetate PVPIVA S-630), surfactants, binders, and filler agents.
The formulation can also contain a surfactant. Non-limiting examples of surfactants include nitrogen-surfactant blends such as Prefer 28 (Cenex), Surf-N(US), Inhance (Brandt), P-28 (Wilfann) and Patrol (Helena); esterified seed oils include Sun-It II (AmCy), MSO (UAP), Scoil (Agsco), Hasten (Wilfarm) and Mes-100 (Drexel); and organo-silicone surfactants include Silwet L77 (UAP), Silikin (Terra), Dyne-Amit (Helena), Kinetic (Helena), Sylgard 309 (Wilbur-Ellis) and Century (Precision). In one embodiment, the surfactant is present at a concentration of between 0.01% v/v to 10% v/v. In another embodiment, the surfactant is present at a concentration of between 0.1% v/v to 1% v/v.
In certain cases, the formulation includes a microbial stabilizer. Such an agent can include a desiccant, which can include any compound or mixture of compounds that can be classified as a desiccant regardless of whether the compound or compounds are used in such concentrations that they in fact have a desiccating effect on a liquid inoculant. Such desiccants are ideally compatible with the bacterial population used, and should promote the ability of the microbial population to survive application on the seeds and to survive desiccation. Examples of suitable desiccants include one or more of trehalose, sucrose, glycerol, and Methylene glycol. Other suitable desiccants include, but are not limited to, non reducing sugars and sugar alcohols (e.g., mannitol or sorbitol). The amount of desiccant introduced into the formulation can range from about 5% to about 50% by weight/volume, for example, between about 10% to about 40%, between about 15% to about 35%, or between about 20% to about 30%. In some cases, it is advantageous for the formulation to contain agents such as a fungicide, an antibacterial agent, an herbicide, a nematicide, an insecticide, a plant growth regulator, a rodenticide, bactericide, or a nutrient. In some examples, agents may include protectants that provide protection against seed surface-borne pathogens. In some examples, protectants may provide some level of control of soil-borne pathogens. In some examples, protectants may be effective predominantly on a seed surface.
In some examples, a fungicide may include a compound or agent, whether chemical or biological, that can inhibit the growth of a fungus or kill a fungus. In some examples, a fungicide may include compounds that may be fungistatic or fungicidal. In some examples, fungicide can be a protectant, or agents that are effective predominantly on the seed surface, providing protection against seed surface-borne pathogens and providing some level of control of soil-borne pathogens. Non-limiting examples of protectant fungicides include captan, maneb, thiram, or fludioxonil.
In some examples, fungicide can be a systemic fungicide, which can be absorbed into the emerging seedling and inhibit or kill the fungus inside host plant tissues. Systemic fungicides used for seed treatment include, but are not limited to the following: azoxystrobin, carboxin, mefenoxam, metalaxyl, thiabendazole, trifloxystrobin, and various triazole fungicides, including difenoconazole, ipconazole, tebuconazole, and triticonazole. Mefenoxam and metalaxyl are primarily used to target the water mold fungi Pythium and Phytophthora. Some fungicides are preferred over others, depending on the plant species, either because of subtle differences in sensitivity of the pathogenic fungal species, or because of the differences in the fungicide distribution or sensitivity of the plants. In some examples, fungicide can be a biological control agent, such as a bacterium or fungus. Such organisms may be parasitic to the pathogenic fungi, or secrete toxins or other substances which can kill or otherwise prevent the growth of fungi. Any type of fungicide, particularly ones that are commonly used on plants, can be used as a control agent in a seed composition.
In some examples, the seed coating composition comprises a control agent which has antibacterial properties. In one embodiment, the control agent with antibacterial properties is selected from the compounds described herein elsewhere. In another embodiment, the compound is Streptomycin, oxytetracycline, oxolinic acid, or gentamicin. Other examples of antibacterial compounds which can be used as part of a seed coating composition include those based on dichlorophene and benzylalcohol hemi formal (Proxel® from ICI or Acticide® RS from Thor Chemie and Kathon® MK 25 from Rohm & Haas) and isothiazolinone derivatives such as alkylisothiazolinones and benzisothiazolinones (Acticide® NIBS from Thor Chemie).
In some examples, growth regulator is selected from the group consisting of: Abscisic acid, amidochlor, ancymidol, 6-benzylaminopurine, brassinolide, butralin, chlormequat (chlormequat chloride), choline chloride, cyclanilide, daminozide, dikegulac, dimethipin, 2,6-dimethylpuridine, ethephon, flumetralin, flurprimidol, fluthiacet, forchlorfenuron, gibberellic acid, inabenfide, indole-3-acetic acid, maleic hydrazide, mefluidide, mepiquat (mepiquat chloride), naphthaleneacetic acid, N-6-benzyladenine, paclobutrazol, prohexadione phosphorotrithioate, 2,3,5-tri-iodobenzoic acid, trinexapac-ethyl and uniconazole. Additional non-limiting examples of growth regulators include brassinosteroids, cytokinines (e.g., kinetin and zeatin), auxins (e.g., indolylacetic acid and indolylacetyl aspartate), flavonoids and isoflavanoids (e.g., formononetin and diosmetin), phytoaixins (e.g., glyceolline), and phytoalexin-inducing oligosaccharides (e.g., pectin, chitin, chitosan, polygalacuronic acid, and oligogalacturonic acid), and gibellerins. Such agents are ideally compatible with the agricultural seed or seedling onto which the formulation is applied (e.g., it should not be deleterious to the growth or health of the plant). Furthermore, the agent is ideally one which does not cause safety concerns for human, animal or industrial use (e.g., no safety issues, or the compound is sufficiently labile that the commodity plant product derived from the plant contains negligible amounts of the compound).
Some examples of nematode-antagonistic biocontrol agents include ARF18; 30 Arthrobotrys spp.; Chaetomium spp.; Cylindrocarpon spp.; Exophilia spp.; Fusarium spp.; Gliocladium spp.; Hirsutella spp.; Lecanicillium spp.; Monacrosporium spp.; Myrothecium spp.; Neocosmospora spp.; Paecilomyces spp.; Pochonia spp.; Stagonospora spp.; vesicular-arbuscular mycorrhizal fungi, Burkholderia spp.; Pasteuria spp., Brevibacillus spp.; Pseudomonas spp.; and Rhizobacteria. Particularly preferred nematode-antagonistic biocontrol agents include ARF18, Arthrobotrys oligospora, Arthrobotrys dactyloides. Chaetomium globosum, Cylindrocarpon heteronema, Exophilia jeanselmei, Exophilia pisciphila, Fusarium aspergilus, Fusarium solani, Gliocladium catenulatum, Gliocladium roseum, Gliocladium vixens, Hirsutella rhossiliensis, Hirsutella minnesotensis, Lecanicillium lecanii, Monacrosporium drechsleri, Monacrosporium gephyropagum, Myrotehcium verrucaria, Neocosmospora vasinfecta, Paecilomyces lilacinus, Pochonia chlamydosporia, Stagonospora heteroderae, Stagonospora phaseoli, vesicular-arbuscular mycorrhizal fungi, Burkholderia cepacia, Pasteuria penetrans, Pasteuria thornei, Pasteuria nishizawae, Pasteuria ramosa, Pastrueia usage, Brevibacillus laterosporus strain G4, Pseudomonas fluorescens and Rhizobacteria.
Some examples of nutrients can be selected from the group consisting of a nitrogen fertilizer including, but not limited to Urea, Ammonium nitrate, Ammonium sulfate, Non-pressure nitrogen solutions, Aqua ammonia, Anhydrous ammonia, Ammonium thiosulfate, Sulfur-coated urea, Urea-formaldehydes, IBDU, Polymer-coated urea, Calcium nitrate, Ureaform, and Methylene urea, phosphorous fertilizers such as Diammonium phosphate, Monoammonium phosphate, Ammonium polyphosphate, Concentrated superphosphate and Triple superphosphate, and potassium fertilizers such as Potassium chloride, Potassium sulfate, Potassium-magnesium sulfate, Potassium nitrate. Such compositions can exist as free salts or ions within the seed coat composition. Alternatively, nutrients/fertilizers can be complexed or chelated to provide sustained release over time.
Some examples of rodenticides may include selected from the group of substances consisting of 2-isovalerylindan-1,3-dione, 4-(quinoxalin-2-ylamino) benzenesulfonamide, alpha-chlorohydrin, aluminum phosphide, antu, arsenous oxide, barium carbonate, bisthiosemi, brodifacoum, bromadiolone, bromethalin, calcium cyanide, chloralose, chlorophacinone, cholecalciferol, coumachlor, coumafuryl, coumatetralyl, crimidine, difenacoum, difethialone, diphacinone, ergocalciferol, flocoumafen, fluoroacetamide, flupropadine, flupropadine hydrochloride, hydrogen cyanide, iodomethane, lindane, magnesium phosphide, methyl bromide, norbormide, phosacetim, phosphine, phosphorus, pindone, potassium arsenite, pyrinuron, scilliroside, sodium arsenite, sodium cyanide, sodium fluoroacetate, strychnine, thallium sulfate, warfarin and zinc phosphide.
In the liquid form, for example, solutions or suspensions, bacterial populations can be mixed or suspended in water or in aqueous solutions. Suitable liquid diluents or carriers include water, aqueous solutions, petroleum distillates, or other liquid carriers.
Solid compositions can be prepared by dispersing the bacterial populations in and on an appropriately divided solid carrier, such as peat, wheat, bran, vermiculite, clay, talc, bentonite, diatomaceous earth, fuller's earth, pasteurized soil, and the like. When such formulations are used as wettable powders, biologically compatible dispersing agents such as non-ionic, anionic, amphoteric, or cationic dispersing and emulsifying agents can be used.
The solid carriers used upon formulation include, for example, mineral carriers such as kaolin clay, pyrophyllite, bentonite, montmorillonite, diatomaceous earth, acid white soil, vermiculite, and pearlite, and inorganic salts such as ammonium sulfate, ammonium phosphate, ammonium nitrate, urea, ammonium chloride, and calcium carbonate. Also, organic fine powders such as wheat flour, wheat bran, and rice bran may be used. The liquid carriers include vegetable oils such as soybean oil and cottonseed oil, glycerol, ethylene glycol, polyethylene glycol, propylene glycol, polypropylene glycol, etc.
Application of Bacterial Populations on Crops The composition of the bacteria or bacterial population described herein can be applied in furrow, in talc, or as seed treatment. The composition can be applied to a seed package in bulk, mini bulk, in a bag, or in talc.
The planter can plant the treated seed and grows the crop according to conventional ways, twin row, or ways that do not require tilling. The seeds can be distributed using a control hopper or an individual hopper. Seeds can also be distributed using pressurized air or manually. Seed placement can be performed using variable rate technologies. Additionally, application of the bacteria or bacterial population described herein may be applied using variable rate technologies. In some examples, the bacteria can be applied to seeds of corn, soybean, canola, sorghum, potato, rice, vegetables, cereals, pseudocereals, and oilseeds. Examples of cereals may include barley, Fonio, oats, palmer's grass, rye, pearl millet, sorghum, spelt, teff, triticale, and wheat. Examples of pseudocereals may include breadnut, buckwheat, cattail, chia, flax, grain amaranth, hanza, quinoa, and sesame. In some examples, seeds can be genetically modified organisms (GMO), non-GMO, organic or conventional.
Additives such as micro-fertilizer, PGR, herbicide, insecticide, and fungicide can be used additionally to treat the crops. Examples of additives include crop protectants such as insecticides, nematicides, fungicide, enhancement agents such as colorants, polymers, pelleting, priming, and disinfectants, and other agents such as inoculant, PGR, softener, and micronutrients. PGRs can be natural or synthetic plant hormones that affect root growth, flowering, or stem elongation. PGRs can include auxins, gibberellins, cytokinins, ethylene, and abscisic acid (ABA).
The composition can be applied in furrow in combination with liquid fertilizer. In some examples, the liquid fertilizer may be held in tanks. NPK fertilizers contain macronutrients of sodium, phosphorous, and potassium.
The composition may improve plant traits, such as promoting plant growth, maintaining high chlorophyll content in leaves, increasing fruit or seed numbers, and increasing fruit or seed unit weight. Methods of the present disclosure may be employed to introduce or improve one or more of a variety of desirable traits. Examples of traits that may introduced or improved include: root biomass, root length, height, shoot length, leaf number, water use efficiency, overall biomass, yield, fruit size, grain size, photosynthesis rate, tolerance to drought, heat tolerance, salt tolerance, tolerance to low nitrogen stress, nitrogen use efficiency, resistance to nematode stress, resistance to a fungal pathogen, resistance to a bacterial pathogen, resistance to a viral pathogen, level of a metabolite, modulation in level of a metabolite, proteome expression. The desirable traits, including height, overall biomass, root and/or shoot biomass, seed germination, seedling survival, photosynthetic efficiency, transpiration rate, seed/fruit number or mass, plant grain or fruit yield, leaf chlorophyll content, photosynthetic rate, root length, or any combination thereof, can be used to measure growth, and compared with the growth rate of reference agricultural plants (e.g., plants without the introduced and/or improved traits) grown under identical conditions. In some examples, the desirable traits, including height, overall biomass, root and/or shoot biomass, seed germination, seedling survival, photosynthetic efficiency, transpiration rate, seed/fruit number or mass, plant grain or fruit yield, leaf chlorophyll content, photosynthetic rate, root length, or any combination thereof, can be used to measure growth, and compared with the growth rate of reference agricultural plants (e.g., plants without the introduced and/or improved traits) grown under similar conditions.
An agronomic trait to a host plant may include, but is not limited to, the following: altered oil content, altered protein content, altered seed carbohydrate composition, altered seed oil composition, and altered seed protein composition, chemical tolerance, cold tolerance, delayed senescence, disease resistance, drought tolerance, ear weight, growth improvement, health enhancement, heat tolerance, herbicide tolerance, herbivore resistance improved nitrogen fixation, improved nitrogen utilization, improved root architecture, improved water use efficiency, increased biomass, increased root length, increased seed weight, increased shoot length, increased yield, increased yield under water-limited conditions, kernel mass, kernel moisture content, metal tolerance, number of ears, number of kernels per ear, number of pods, nutrition enhancement, pathogen resistance, pest resistance, photosynthetic capability improvement, salinity tolerance, stay-green, vigor improvement, increased dry weight of mature seeds, increased fresh weight of mature seeds, increased number of mature seeds per plant, increased chlorophyll content, increased number of pods per plant, increased length of pods per plant, reduced number of wilted leaves per plant, reduced number of severely wilted leaves per plant, and increased number of non-wilted leaves per plant, a detectable modulation in the level of a metabolite, a detectable modulation in the level of a transcript, and a detectable modulation in the proteome, compared to an isoline plant grown from a seed without said seed treatment formulation.
In some cases, plants are inoculated with bacteria or bacterial populations that are isolated from the same species of plant as the plant element of the inoculated plant. For example, an bacteria or bacterial population that is normally found in one variety of Zea mays (corn) is associated with a plant element of a plant of another variety of Zea mays that in its natural state lacks said bacteria and bacterial populations. In one embodiment, the bacteria and bacterial populations is derived from a plant of a related species of plant as the plant element of the inoculated plant. For example, an bacteria and bacterial populations that is normally found in Zea diploperennis Iltis et al., (diploperennial teosinte) is applied to a Zea mays (corn), or vice versa. In some cases, plants are inoculated with bacteria and bacterial populations that are heterologous to the plant element of the inoculated plant. In one embodiment, the bacteria and bacterial populations is derived from a plant of another species. For example, an bacteria and bacterial populations that is normally found in dicots is applied to a monocot plant (e.g., inoculating corn with a soybean-derived bacteria and bacterial populations), or vice versa. In other cases, the bacteria and bacterial populations to be inoculated onto a plant is derived from a related species of the plant that is being inoculated. In one embodiment, the bacteria and bacterial populations is derived from a related taxon, for example, from a related species. The plant of another species can be an agricultural plant. In another embodiment, the bacteria and bacterial populations is part of a designed composition inoculated into any host plant element.
In some examples, the bacteria or bacterial population is exogenous wherein the bacteria and bacterial population is isolated from a different plant than the inoculated plant. For example, in one embodiment, the bacteria or bacterial population can be isolated from a different plant of the same species as the inoculated plant. In some cases, the bacteria or bacterial population can be isolated from a species related to the inoculated plant.
In some examples, the bacteria and bacterial populations described herein are capable of moving from one tissue type to another. For example, the present invention's detection and isolation of bacteria and bacterial populations within the mature tissues of plants after coating on the exterior of a seed demonstrates their ability to move from seed exterior into the vegetative tissues of a maturing plant. Therefore, in one embodiment, the population of bacteria and bacterial populations is capable of moving from the seed exterior into the vegetative tissues of a plant. In one embodiment, the bacteria and bacterial populations that is coated onto the seed of a plant is capable, upon germination of the seed into a vegetative state, of localizing to a different tissue of the plant. For example, bacteria and bacterial populations can be capable of localizing to any one of the tissues in the plant, including: the root, adventitious root, seminal 5 root, root hair, shoot, leaf, flower, bud, tassel, meristem, pollen, pistil, ovaries, stamen, fruit, stolon, rhizome, nodule, tuber, trichome, guard cells, hydathode, petal, sepal, glume, rachis, vascular cambium, phloem, and xylem. In one embodiment, the bacteria and bacterial populations is capable of localizing to the root and/or the root hair of the plant. In another embodiment, the bacteria and bacterial populations is capable of localizing to the photosynthetic tissues, for example, leaves and shoots of the plant. In other cases, the bacteria and bacterial populations is localized to the vascular tissues of the plant, for example, in the xylem and phloem. In still another embodiment, the bacteria and bacterial populations is capable of localizing to the reproductive tissues (flower, pollen, pistil, ovaries, stamen, fruit) of the plant. In another embodiment, the bacteria and bacterial populations is capable of localizing to the root, shoots, leaves and reproductive tissues of the plant. In still another embodiment, the bacteria and bacterial populations colonizes a fruit or seed tissue of the plant. In still another embodiment, the bacteria and bacterial populations is able to colonize the plant such that it is present in the surface of the plant (i.e., its presence is detectably present on the plant exterior, or the episphere of the plant). In still other embodiments, the bacteria and bacterial populations is capable of localizing to substantially all, or all, tissues of the plant. In certain embodiments, the bacteria and bacterial populations is not localized to the root of a plant. In other cases, the bacteria and bacterial populations is not localized to the photosynthetic tissues of the plant.
The effectiveness of the compositions can also be assessed by measuring the relative maturity of the crop or the crop heating unit (CHU). For example, the bacterial population can be applied to corn, and corn growth can be assessed according to the relative maturity of the corn kernel or the time at which the corn kernel is at maximum weight. The crop heating unit (CHU) can also be used to predict the maturation of the corn crop. The CHU determines the amount of heat accumulation by measuring the daily maximum temperatures on crop growth.
In examples, bacterial may localize to any one of the tissues in the plant, including: the root, adventitious root, seminal root, root hair, shoot, leaf, flower, bud tassel, meristem, pollen, pistil, ovaries, stamen, fruit, stolon, rhizome, nodule, tuber, trichome, guard cells, hydathode, petal, sepal, glume, rachis, vascular cambium, phloem, and xylem. In another embodiment, the bacteria or bacterial population is capable of localizing to the photosynthetic tissues, for example, leaves and shoots of the plant. In other cases, the bacteria and bacterial populations is localized to the vascular tissues of the plant, for example, in the xylem and phloem. In another embodiment, the bacteria or bacterial population is capable of localizing to reproductive tissues (flower, pollen, pistil, ovaries, stamen, or fruit) of the plant. In another embodiment, the bacteria and bacterial populations is capable of localizing to the root, shoots, leaves and reproductive tissues of the plant. In another embodiment, the bacteria or bacterial population colonizes a fruit or seed tissue of the plant. In still another embodiment, the bacteria or bacterial population is able to colonize the plant such that it is present in the surface of the plant. In another embodiment, the bacteria or bacterial population is capable of localizing to substantially all, or all, tissues of the plant. In certain embodiments, the bacteria or bacterial population is not localized to the root of a plant. In other cases, the bacteria and bacterial populations is not localized to the photosynthetic tissues of the plant.
The effectiveness of the bacterial compositions applied to crops can be assessed by measuring various features of crop growth including, but not limited to, planting rate, seeding vigor, root strength, drought tolerance, plant height, dry down, and test weight.
Plant Species The methods and bacteria described herein are suitable for any of a variety of plants, such as plants in the genera Hordeum, Oryza, Zea, and Triticeae. Other non-limiting examples of suitable plants include mosses, lichens, and algae. In some cases, the plants have economic, social and/or environmental value, such as food crops, fiber crops, oil crops, plants in the forestry or pulp and paper industries, feedstock for biofuel production and/or ornamental plants. In some examples, plants may be used to produce economically valuable products such as a grain, a flour, a starch, a syrup, a meal, an oil, a film, a packaging, a nutraceutical product, a pulp, an animal feed, a fish fodder, a bulk material for industrial chemicals, a cereal product, a processed human-food product, a sugar, an alcohol, and/or a protein. Non-limiting examples of crop plants include maize, rice, wheat, barley, sorghum, millet, oats, rye triticale, buckwheat, sweet corn, sugar cane, onions, tomatoes, strawberries, and asparagus.
In some examples, plants that may be obtained or improved using the methods and composition disclosed herein may include plants that are important or interesting for agriculture, horticulture, biomass for the production of biofuel molecules and other chemicals, and/or forestry. Some examples of these plants may include pineapple, banana, coconut, lily, grasspeas and grass; and dicotyledonous plants, such as, for example, peas, alfalfa, tomatillo, melon, chickpea, chicory, clover, kale, lentil, soybean, tobacco, potato, sweet potato, radish, cabbage, rape, apple trees, grape, cotton, sunflower, Chale cress, canola, citrus (including orange, mandarin, kumquat, lemon, lime, grapefruit, tangerine, tangelo, citron, and pomelo), pepper, bean, lettuce, Panicum virgatum (switch), Sorghum bicolor (sorghum, sudan), Miscanthus giganteus (miscanthus), Saccharum sp. (energycane), Populus balsamifera (poplar), Zea mays (corn), Glycine max (soybean), Brassica napus (canola), Triticum aestivum (wheat), Gossypium hirsutum (cotton), Oryza sativa (rice), Helianthus annuus (sunflower), Medicago sativa (alfalfa), Beta vulgaris (sugarbeet), Pennisetum glaucum (pearl millet), Panicum spp. Sorghum spp., Miscanthus spp., Saccharum spp., Erianthus spp., Populus spp., Secale cereale (rye), Salix spp. (willow), Eucalyptus spp. (eucalyptus), Triticosecale spp. (triticum-25 wheat X rye), Bamboo, Carthamus tinctorius (safflower), Jatropha curcas (Jatropha), Ricinus communis (castor), Elaeis guineensis (oil palm), Phoenix dactylifera (date palm), Archontophoenix cunninghamiana (king palm), Syagrus romanzoffiana (queen palm), Linum usitatissimum (flax), Brassica juncea, Manihot esculenta (cassaya), Lycopersicon esculentum (tomato), Lactuca saliva (lettuce), Musa paradisiaca (banana), Solanum tuberosum (potato), Brassica oleracea (broccoli, cauliflower, brussel sprouts), Camellia sinensis (tea), Fragaria ananassa (strawberry), Theobroma cacao (cocoa), Coffea arabica (coffee), Vitis vinifera (grape), Ananas comosus (pineapple), Capsicum annum (hot & sweet pepper), Allium cepa (onion), Cucumis melo (melon), Cucumis sativus (cucumber), Cucurbita maxima (squash), Cucurbita moschata (squash), Spinacea oleracea (spinach), Citrullus lanatus (watermelon), Abelmoschus esculentus (okra), Solanum melongena (eggplant), Papaver somniferum (opium poppy), Papaver orientale, Taxus baccata, Taxus brevifolia, Artemisia annua, Cannabis saliva, Camptotheca acuminate, Catharanthus roseus, Vinca rosea, Cinchona officinalis, Coichicum autumnale, Veratrum californica, Digitalis lanata, Digitalis purpurea, Dioscorea 5 spp., Andrographis paniculata, Atropa belladonna, Datura stomonium, Berberis spp., Cephalotaxus spp., Ephedra sinica, Ephedra spp., Erythroxylum coca, Galanthus wornorii, Scopolia spp., Lycopodium serratum (Huperzia serrata), Lycopodium spp., Rauwolfia serpentina, Rauwolfia spp., Sanguinaria canadensis, Hyoscyamus spp., Calendula officinalis, Chrysanthemum parthenium, Coleus forskohlii, Tanacetum parthenium, Parthenium argentatum (guayule), Hevea spp. (rubber), Mentha spicata (mint), Mentha piperita (mint), Bixa orellana, Alstroemeria spp., Rosa spp. (rose), Dianthus caryophyllus (carnation), Petunia spp. (petunia), Poinsettia pulcherrima (poinsettia), Nicotiana tabacum (tobacco), Lupinus albus (lupin), Uniola paniculata (oats), Hordeum vulgare (barley), and Lolium spp. (rye).
In some examples, a monocotyledonous plant may be used. Monocotyledonous plants belong to the orders of the Alismatales, Arales, Arecales, Bromeliales, Commelinales, Cyclanthales, Cyperales, Eriocaulales, Hydrocharitales, Juncales, Lilliales, Najadales, Orchidales, Pandanales, Poales, Restionales, Triuridales, Typhales, and Zingiberales. Plants belonging to the class of the Gymnospermae are Cycadales, Ginkgoales, Gnetales, and Pinales. In some examples, the monocotyledonous plant can be selected from the group consisting of a maize, rice, wheat, barley, and sugarcane.
In some examples, a dicotyledonous plant may be used, including those belonging to the orders of the Aristochiales, Asterales, Batales, Campanulales, Capparales, Caryophyllales, Casuarinales, Celastrales, Cornales, Diapensales, Dilleniales, Dipsacales, Ebenales, Ericales, Eucomiales, Euphorbiales, Fabales, Fagales, Gentianales, Geraniales, Haloragales, Hamamelidales, Middles, Juglandales, Lamiales, Laurales, Lecythidales, Leitneriales, Magniolales, Malvales, Myricales, Myrtales, Nymphaeales, Papeverales, Piperales, Plantaginales, Plumbaginales, Podostemales, Polemoniales, Polygalales, Polygonales, Primulales, Proteales, Rafflesiales, Ranunculales, Rhamnales, Rosales, Rubiales, Salicales, Santales, Sapindales, Sarraceniaceae, Scrophulariales, Theales, Trochodendrales, Umbellales, Urticales, and Violates. In some examples, the dicotyledonous plant can be selected from the group consisting of cotton, soybean, pepper, and tomato.
In some cases, the plant to be improved is not readily amenable to experimental conditions. For example, a crop plant may take too long to grow enough to practically assess an improved trait serially over multiple iterations. Accordingly, a first plant from which bacteria are initially isolated, and/or the plurality of plants to which genetically manipulated bacteria are applied may be a model plant, such as a plant more amenable to evaluation under desired conditions. Non-limiting examples of model plants include Setaria, Brachypodium, and Arabidopsis. Ability of bacteria isolated according to a method of the disclosure using a model plant may then be applied to a plant of another type (e.g. a crop plant) to confirm conferral of the improved trait.
Traits that may be improved by the methods disclosed herein include any observable characteristic of the plant, including, for example, growth rate, height, weight, color, taste, smell, changes in the production of one or more compounds by the plant (including for example, metabolites, proteins, drugs, carbohydrates, oils, and any other compounds). Selecting plants based on genotypic information is also envisaged (for example, including the pattern of plant gene expression in response to the bacteria, or identifying the presence of genetic markers, such as those associated with increased nitrogen fixation). Plants may also be selected based on the absence, suppression or inhibition of a certain feature or trait (such as an undesirable feature or trait) as opposed to the presence of a certain feature or trait (such as a desirable feature or trait).
Concentrations and Rates of Application of Agricultural Compositions As aforementioned, the agricultural compositions of the present disclosure, which comprise a taught microbe, can be applied to plants in a multitude of ways. In two particular aspects, the disclosure contemplates an in-furrow treatment or a seed treatment
For seed treatment embodiments, the microbes of the disclosure can be present on the seed in a variety of concentrations. For example, the microbes can be found in a seed treatment at a cfu concentration, per seed of: 1×101, 1×102, 1×103, 1×104, 1×105, 1×106, 1×1017, 1×108, 1×109, 1×1010, or more. In particular aspects, the seed treatment compositions comprise about 1×104 to about 1×108 cfu per seed. In other particular aspects, the seed treatment compositions comprise about 1×105 to about 1×107 cfu per seed. In other aspects, the seed treatment compositions comprise about 1×106 cfu per seed.
In the United States, about 10% of corn acreage is planted at a seed density of above about 36,000 seeds per acre; ⅓ of the corn acreage is planted at a seed density of between about 33,000 to 36,000 seeds per acre; ⅓ of the corn acreage is planted at a seed density of between about 30,000 to 33,000 seeds per acre, and the remainder of the acreage is variable. See, “Corn Seeding Rate Considerations,” written by Steve Butzen, available at: https://www.pioneer.com/home/site/us/agronomy/librarykorn-seeding-rate-considerations/
Table B below utilizes various cfu concentrations per seed in a contemplated seed treatment embodiment (rows across) and various seed acreage planting densities (1st column: 15K-41K) to calculate the total amount of cfu per acre, which would be utilized in various agricultural scenarios (i.e. seed treatment concentration per seed×seed density planted per acre). Thus, if one were to utilize a seed treatment with 1×106 cfu per seed and plant 30,000 seeds per acre, then the total cfu content per acre would be 3×1010 (i.e. 30K*1×106).
TABLE B
Total CFU Per Acre Calculation for Seed Treatment Embodiments
Corn Population
(i.e, seeds per
acre) 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07 1.00E+08 1.00E+09
15,000 1.50E+06 1.50E+07 1.50E+08 1.50E+09 1.50E+10 1.50E+11 1.50E+12 1.50E+13
16,000 1.60E+06 1.60E+07 1.60E+08 1.60E+09 1.60E+10 1.60E+11 1.60E+12 1.60E+13
17,000 1.70E+06 1.70E+07 1.70E+08 1.70E+09 1.70E+10 1.70E+11 1.70E+12 1.70E+13
18,000 1.80E+06 1.80E+07 1.80E+08 1.80E+09 1.80E+10 1.80E+11 1.80E+12 1.80E+13
19,000 1.90E+06 1.90E+07 1.90E+08 1.90E+09 1.90E+10 1.90E+11 1.90E+12 1.90E+13
20,000 2.00E+06 2.00E+07 2.00E+08 2.00E+09 2.00E+10 2.00E+11 2.00E+12 2.00E+13
21,000 2.10E+06 2.10E+07 2.10E+08 2.10E+09 2.10E+10 2.10E+11 2.10E+12 2.10E+13
22,000 2.20E+06 2.20E+07 2.20E+08 2.20E+09 2.20E+10 2.20E+11 2.20E+12 2.20E+13
23,000 2.30E+06 2.30E+07 2.30E+08 2.30E+09 2.30E+10 2.30E+11 2.30E+12 2.30E+13
24,000 2.40E+06 2.40E+07 2.40E+08 2.40E+09 2.40E+10 2.40E+11 2.40E+12 2.40E+13
25,000 2.50E+06 2.50E+07 2.50E+08 2.50E+09 2.50E+10 2.50E+11 2.50E+12 2.50E+13
26,000 2.60E+06 2.60E+07 2.60E+08 2.60E+09 2.60E+10 2.60E+11 2.60E+12 2.60E+13
27,000 2.70E+06 2.70E+07 2.70E+08 2.70E+09 2.70E+10 2.70E+11 2.70E+12 2.70E+13
28,000 2.80E+06 2.80E+07 2.80E+08 2.80E+09 2.80E+10 2.80E+11 2.80E+12 2.80E+13
29,000 2.90E+06 2.90E+07 2.90E+08 2.90E+09 1.90E+10 2.90E+11 2.90E+12 2.90E+13
30,000 3.00E+06 3.00E+07 3.00E+08 3.00E+09 3.00E+10 3.00E+11 3.00E+12 3.00E+13
31,000 3.10E+06 3.10E+07 3.10E+08 3.10E+09 3.10E+10 3.10E+11 3.10E+12 3.10E+13
32,000 3.20E+06 3.20E+07 3.20E+08 3.20E+09 3.20E+10 3.20E+11 3.20E+12 3.20E+13
33,000 3.30E+06 3.30E+07 3.30E+08 3.30E+09 3.30E+10 3.30E+11 3.30E+12 3.30E+13
34,000 3.40E+06 3.40E+07 3.40E+08 3.40E+09 3.40E+10 3.40E+11 3.40E+12 3.40E+13
35,000 3.50E+06 3.50E+07 3.50E+08 3.50E+09 3.50E+10 3.50E+11 3.50E+12 3.50E+13
36,000 3.60E+06 3.60E+07 3.60E+08 3.60E+09 3.60E+10 3.60E+11 3.60E+12 3.60E+13
37,000 3.70E+06 3.70E+07 3.70E+08 3.70E+09 3.70E+10 3.70E+11 3.70E+12 3.70E+13
38,000 3.80E+06 3.80E+07 3.80E+08 3.80E409 3.80E+10 3.80E+11 3.80E+12 3.80E+13
39,000 3.90E+06 3.90E+07 3.90E+08 3.90E+09 3.90E+10 3.90E+11 3.90E+12 3.90E+13
40,000 4.00E+06 4.00E+07 4.00E+08 4.00E+09 4.00E+10 4.00E+11 4.00E+12 4.00E+13
41,000 4.10E+06 4.10E+07 4.10E+08 4.10E+09 4.10E+10 4.10E+11 4.10E+12 4.10E+13
For in-furrow embodiments, the microbes of the disclosure can be applied at a cfu concentration per acre of: 1×106, 3.20×1010, 1.60×1011, 3.20×1011, 8.0×1011, 1.6×1012, 3.20×1012, or more. Therefore, in aspects, the liquid in-furrow compositions can be applied at a concentration of between about 1×106 to about 3×1012 cfu per acre.
In some aspects, the in-furrow compositions are contained in a liquid formulation. In the liquid in-furrow embodiments, the microbes can be present at a cfu concentration per milliliter of: 1×101, 1×102, 1×103, 1×104, 1×105, 1×106, 1×107, 1×108, 1×109, 1×1010, 1×1011, 1×1012, 1×1013, or more. In certain aspects, the liquid in-furrow compositions comprise microbes at a concentration of about 1×106 to about 1×1011 cfu per milliliter. In other aspects, the liquid in-furrow compositions comprise microbes at a concentration of about 1×107 to about 1×1010 cfu per milliliter. In other aspects, the liquid in-furrow compositions comprise microbes at a concentration of about 1×108 to about 1×109 cfu per milliliter. In other aspects, the liquid in-furrow compositions comprise microbes at a concentration of up to about 1×1013 cfu per milliliter.
Examples The examples provided herein describe methods of bacterial isolation, bacterial and plant analysis, and plant trait improvement. The examples are for illustrative purposes only and are not to be construed as limiting in any way.
Example 1: Isolation of Microbes from Plant Tissue Topsoil was obtained from various agricultural areas in central California. Twenty soils with diverse texture characteristics were collected, including heavy clay, peaty clay loam, silty clay, and sandy loam. Seeds of various field corn, sweet corn, heritage corn and tomato were planted into each soil, as shown in Table 1.
TABLE 1
Crop Type and Varieties planted into soil with diverse characteristics
Crop Type
Field
Corn Sweet Corn Heritage Corn Tomato
Varieties Mo17 Ferry-Morse Victory Seeds Ferry-Morse Roma
‘Golden Cross ‘Moseby Prolific’ VF
Bantam T-51’
B73 Ferry-Morse ‘Silver Victory Seeds ‘Reid's Stover Roma
Queen Hybrid’ Yellow Dent’
DKC 66- Ferry-Morse ‘Sugar Victory Seeds Totally Tomatoes
40 Dots’ ‘Hickory King’ ‘Micro Tom Hybrid’
DKC 67- Heinz 1015
07
DKC 70- Heinz 2401
01
Heinz 3402
Heinz 5508
Heinz 5608
Heinz 8504
Plants were uprooted after 2-4 weeks of growth and excess soil on root surfaces was removed with deionized water. Following soil removal, plants were surface sterilized with bleach and rinsed vigorously in sterile water. A cleaned, 1 cm section of root was excised from the plant and placed in a phosphate buffered saline solution containing 3 mm steel beads. A slurry was generated by vigorous shaking of the solution with a Qiagen TissueLyser II.
The root and saline slurry was diluted and inoculated onto various types of growth media to isolate rhizospheric, endophytic, epiphytic, and other plant-associated microbes. R2A and Nfb agar media were used to obtain single colonies, and semisolid Nfb media slants were used to obtain populations of nitrogen fixing bacteria. After 2-4 weeks incubation in semi-solid Nfb media slants, microbial populations were collected and streaked to obtain single colonies on R2A agar, as shown in FIG. 1A-B. Single colonies were resuspended in a mixture of R2A and glycerol, subjected to PCR analysis, and frozen at −80° C. for later analysis. Approximately 1,000 single colonies were obtained and designated “isolated microbes.”
Isolates were then subjected to a colony PCR screen to detect the presence of the nifH gene in order to identify diazotrophs. The previously-described primer set Ueda 19F/388R, which has been shown to detect over 90% of diazotrophs in screens, was used to probe the presence of the nif cluster in each isolate (Ueda et al. 1995; J. Bacteriol. 177: 1414-1417). Single colonies of purified isolates were picked, resuspended in PBS, and used as a template for colony PCR, as shown in FIG. 2. Colonies of isolates that gave positive PCR bands were re-streaked, and the colony PCR and re-streaking process was repeated twice to prevent false positive identification of diazotrophs. Purified isolates were then designated “candidate microbes.”
Example 2: Characterization of Isolated Microbes Sequencing, Analysis and Phylogenetic Characterization Sequencing of 16S rDNA with the 515f-806r printer set was used to generate preliminary phylogenetic identities for isolated and candidate microbes (see e.g. Vernon et al.; BMC Microbiol. 2002 Dec. 23; 2:39.). The microbes comprise diverse genera including: Enterobacter, Burkholderia, Klebsiella, Bradyrhizobium, Rahnella, Xanthomonas, Raoultella, Pantoea, Pseudomonas, Brevundimonas, Agrobacterium, and Paenibacillus, as shown in Table 2.
TABLE 2
Diversity of microbes isolated from tomato plants
as determined by deep 16S rDNA sequencing.
Genus Isolates
Achromobacter 7
Agrobacterium 117
Agromyces 1
Alicyclobacillus 1
Asticcacaulis 6
Bacillus 131
Bradyrhizobium 2
Brevibacillus 2
Burkholderia 2
Coulobacter 17
Chryseobacterium 42
Comamonas 1
Dyadobacter 2
Flavobacterium 46
Halomonas 3
Leptothrix 3
Lysobacter 2
Neisseria 13
Paenibacillus 1
Paenisporosarcina 3
Pantoea 14
Pedobacter 16
Pimelobacter 2
Pseudomonas 212
Rhizobium 4
Rhodoferax 1
Sphingobacterium 13
Sphingobium 23
Sphingomonas 3
Sphingopyxis 1
Stenotrophomonas 59
Streptococcus 3
Variovorax 37
Xylanimicrobium 1
unidentified 75
Subsequently, the genomes of 39 candidate microbes were sequenced using Illumina Miseq platform. Genomic DNA from pure cultures was extracted using the QIAmp DNA mini kit (QIAGEN), and total DNA libraries for sequencing were prepared through a third party vendor (SeqMatic, Hayward). Genome assembly was then carried out via the AS pipeline (Tritt et al. 2012; PLoS One 7(9):e42304). Genes were identified and annotated, and those related to regulation and expression of nitrogen fixation were noted as targets for mutagenesis.
Transcriptomic Profiling of Candidate Microbes Transcriptomic profiling of strain 0010 was performed to identify promoters that are active in the presence of environmental nitrogen. Strain 0010 was cultured in a defined, nitrogen-free media supplemented with 10 mM glutamine. Total RNA was extracted from these cultures (QIAGEN RNeasy kit) and subjected to RNAseq sequencing via Illumina HiSeq (SeqMatic, Fremont Calif.). Sequencing reads were mapped to CI010 genome data using Geneious, and highly expressed genes under control of proximal transcriptional promoters were identified.
Tables 3A-C lists genes and their relative expression level as measured through RNASeq sequencing of total RNA. Sequences of the proximal promoters were recorded for use in mutagenesis of nif pathways, nitrogen utilization related pathways, or other genes with a desired expression level.
TABLE 3A
Name Minimum Maximum Length Direction
murein lipoprotein CDS 2,929,898 2,930,134 237 forward
membrane protein CDS 5,217,517 5,217,843 327 forward
zinc/cadmium-binding 3,479,979 3,480,626 648 forward
protein CDS
acyl carrier protein CDS 4,563,344 4,563,580 237 reverse
ompX CDS 4,251,002 4,251,514 513 forward
DNA-binding protein HU- 375,156 375,428 273 forward
beta CDS
sspA CDS 629,998 630,636 639 reverse
tatE CDS 3,199,435 3,199,638 204 reverse
LexA repressor CDS 1,850,457 1,851,065 609 forward
hisS CDS <3999979 4,001,223 >1245 forward
TABLE 3B
RNASeq_ RNASeq_ RNASeq_ RNASeq_
Differential nifL- nifL- WT- WT-
Expression Differential Raw Raw Raw Raw
Absolute Expression Read Transcript Read Transcript
Name Confidence Ratio Count Count Count Count
murein 1000 −1.8 12950.5 10078.9 5151.5 4106.8
lipoprotein
CDS
membrane 1000 −1.3 9522.5 5371.3 5400 3120
protein CDS
zinc/cadmium- 3.3 1.1 6461 1839.1 5318 1550.6
binding
protein CDS
acyl carrier 25.6 1.6 1230.5 957.6 1473.5 1174.7
protein CDS
ompX CDS 1.7 1.1 2042 734.2 1687.5 621.5
DNA-binding 6.9 −1.3 1305 881.7 725 501.8
protein HU-
beta CDS
sspA CDS 0.2 1 654 188.8 504.5 149.2
tatE CDS 1.4 1.3 131 118.4 125 115.8
LexA 0.1 −1.1 248 75.1 164 50.9
repressor
CDS
hisS CDS 0 −1.1 467 69.2 325 49.3
TABLE 3C
Prm (In Forward SEQ SEQ SEQ
direction, -250 ID Expressed ID Neighbor ID
Name to +10 region) NO: Sequence NO: Sequence NO:
murein GCCTCTCGGGGC 3 ATGAATCGTACT 13 ATGAAAAAGACC 23
lipoprotein GCTTTTTTTTATT AAACTGGTACTG AAAATTGTTTGC
CDS CCGGCACTAGCC GGCGCGGTAATC ACCATCGGTCCG
GCTATTAATAAA CTGGGTTCTACTC AAAACCGAATCC
AATGCAAATCGG TGCTGGCTGGTT GAAGAGATGTTG
AATTTACTATTTA GCTCCAGCAATG ACCAAAATGCTG
ACGCGAGATTAT CTAAAATCGATC GACGCGGGCATG
CTAAGATGAATC AGCTGTCTTCTGA AACGTTATGCGT
CGATGGAAGCGC CGTTCAGACTCT CTGAACTTCTCTC
GCTGTTTTCACTC GAACGCTAAAGT ACGGTGACTATG
GCCTTTTTAAAGT TGACCAGCTGAG CGGAACACGGTC
TACGTGATGATTT CAACGACGTGAA AGCGCATCCAGA
CGATGCTTCTTTG CGCAATGCGTTC ATCTGCGCAATG
AGCGAACGATCA CGACGTTCAGGC TGATGAGTAAAA
AAAATAAGCGTA TGCTAAAGATGA CCGGTAAGAAAG
TTCAGGTAAAAA CGCAGCTCGCGC CGGCAATCCTGC
AATATTCTCATCA TAACCAGCGTCT TGGACACCAAAG
CAAAAAAGTTTG GGACAACGCAGC GTCCGGAAATCC
TGTAATACTTGTA TACTAAATACCG GTACCATTAAGC
ACGCT--- TAAGTAA TGGAAGGCGGCA
ACATGGAGATTA ACGACGTCTCCC
ACTC TGAAAGCGGGCC
AGACCTTCACCTT
CACCACCGATAA
ATCCGTTGTCGGT
AATAACGAAATC
GTTGCGGTGACC
TATGAAGGCTTC
ACCAGCGACCTG
AGCGTTGGCAAC
ACGGTACTGGTT
GACGATGGTCTG
ATCGGTATGGAA
GTGACCGCTATC
GAAGGCAACAAA
GTTGTTTGTAAA
GTGCTGAACAAC
GGCGACCTCGGC
GAGAACAAAGGC
GTTAACCTGCCG
GGCGTATCTATC
GCGCTGCCGGCG
CTGGCTGAAAAA
GACAAACAGGAT
CTGATCTTCGGTT
GCGAACAGGGCG
TTGACTTTGTTGC
GGCATCCTTTATC
CGTAAGCGTTCT
GACGTTGTTGAA
ATCCGTGAGCAC
CTGAAAGCCCAC
GGCGGCGAGAAG
ATCCAGATCATC
TCCAAAATCGAA
AACCAGGAAGGC
CTGAACAACTTC
GACGAAATCCTC
GAAGCCTCTGAC
GGCATCATGGTA
GCCCGTGGCGAC
CTGGGCGTTGAA
ATCCCGGTTGAA
GAAGTTATCTTC
GCGCAGAAGATG
ATGATCGAGAAA
TGTATCCGCGCG
CGTAAAGTCGTT
ATCACCGCGACC
CAGATGCTGGAT
TCCATGATCAAA
AACCCGCGTCCG
ACCCGTGCGGAA
GCAGGCGACGTG
GCCAACGCCATC
CTCGACGGCACC
GACGCAGTTATG
CTGTCCGGCGAA
TCCGCGAAAGGT
AAATACCCGCTG
GAAGCGGTCACC
ATCATGGCGACC
ATCTGCGAACGT
ACCGACCGCGTC
ATGACCAGCCGT
CTTGAGTACAAC
AACGACAACCGT
AAGCTGCGCATC
ACCGAAGCGGTG
TGCCGCGGTGCG
GTAGAAACGGCT
GAAAAACTGGAA
GCGCCGCTGATC
GTTGTGGCAACC
CAGGGCGGTAAA
TCCGCGCGCGCC
GTACGTAAATAC
TTCCCGGATGCC
ACTATCCTGGCG
CTGACCACCAAC
GAAACCACCGCG
CGTCAGCTGGTG
CTGAGCAAAGGC
GTTGTGGCACAG
CTGGTTGAAGAT
ATCTCCTCTACCG
ATGCGTTCTACAT
CCAGGGTAAAGA
ACTGGCGCTGCA
GAGCGGTCTGGC
GCGTAAAGGCGA
CGTGGTTGTTATG
GTTTCCGGCGCG
TTAGTCCCGAGC
GGAACCACCAAT
ACCGCTTCCGTG
CACGTGCTGTAA
membrane GGTTCACATAAA 4 ATGGCCAACCGA 14 ATGTATTTAAGA 24
protein CATAATTATCGC GCAAACCGCAAC CCCGATGAGGTG
CDS CACGGCGATAGC AACGTAGAAGAG GCGCGTGTTCTTG
CGTACGCTTTTTG AGCGCTGAAGAT AAAAAGCCGGCT
CGTCACAACATC ATCCATAACGAT TCACCATGGATG
CATGGTGAAGCC GTCAGCCAATTA TTGTGACGCAAA
GGCTTTTTCAAG GCGGATACGCTG AAGCGTACGGCT
AACACGCGCCAC GAAGAGGTGCTG ATCGCCGTGGCG
CTCATCGGGTCTT AAATCGTGGGGC ATAATTATGTTTA
AATACATACTC AGCGACGCCAAA TGTGAACCGTGA
ATTCCTCATTATC GACGAAGCGGAG AGCTCGTATGGG
TTTTACCGCACGT GCCGCGCGCAAA GCGTACCGCGTT
TAACCTTACCTTA AAAGCGCAGGCG AATTATTCATCCG
TTCATTAAAGGC CTGCTGAAAGAG GCTTTAAAAGAG
AACGCTTTCGGA ACCCGCGCCCGG CGCAGCACAACG
ATATTCCATAAA CTTAACGGCAAC CTTGCGGAGCCC
GGGCTATTTACA AACCGCGTCCAG GCGTCGGATATC
GCATAATTCAAA CAGGCGGCGTGC AAAACCTGCGAT
ATCTTGTCCTACA GACGCCATGGGC CATTATGAGCAG
CTTATAGACTCA TGCGCTGACAGC TTCCCGCTCTATT
ATGGAATTAAGG TACGTGCGCGAC TAGCGCTGGGATG
GA AAACCGTGGCAA CTCAACACTCATT
AGCGTCGGCGCC ATGGTATTCCAC
GCAGCAGCCGTT ACGGGTTCAGTT
GGGGTATTTATT CGCGAATGGCGC
GGCGTATTACTG TTGAGCGTTTTCT
AATTTACGTCGA GAGTGGCCTGTT
TAA TGGCGAAACGCA
GTATAGCTGA
zinc/ GCGCGGAAAATC 5 ATGACCAAAAAG 15 ATGGATAGCGAC 25
cadmium- GACGCATAGCGC ATTTCCGCCCTAG ATTAATCAGGTC
binding ATTCTCAGAAGC CGTTTGGCATTG ATTGATTCTTTTG
protein CGGCCTGGTCTC GCATGGTAATGG TTAAAGGCCCGG
CDS GGTGGAAAAGCG CGAGCAGCCAGG CGGTCGTGGGAA
AATCTTTCCCACG CTTTTGCCCACGG AGATTCGCTTTTC
ACCGCCGGGCCT TCACCATAGTCA CACCGAGACCAG
TTAACAAAAGAA TGGCCCGGCGCT GCCGGCTTCTGA
TCAATGACCTGA GACCGAAGCGGA GAATGCGCTATG
TTAATGTCGCTAT ACAAAAGGCGAG CGTCGATTTTCCG
CCATTCTCTCTCC TGAAGGCATTTTT CGCCTCGAAATC
GCGTAATGCGAT GCTGACCAGGAC ATGCTTGCGGGT
CTTTTTTCATCAT CTTAAAGGACAGG CAGCTTCACGAT
ACCTAACAAACT GCGCTGAGCGAC CCGGCGATTAAA
GGCAGAGGGAAA TGGGAGGGGATC GCCGATCGCGCC
AGCCGCGCGGTT TGGCAGTCGGTT CAGCTCATGCCG
TTTCTGCGAAGT AACCCCTATCTG CACGATGTGCTG
GTATTGTAAGAT CTGAACGGGGAT TATATTCCCGCTG
TTGTTTGATATGT TTAGATCCGGTTC GCGGATGGAATG
TATATCGTAACA TGGAGCAGAAGG ACCCGCAATGGC
TATTATTGCAAA CCAAAAAGGCCG TGGCGCCCTCCA
CAT GTAAAAGCGTGG CTCTGCTCACTAT
CGGAATATCGGG CTTATTTGGTAAA
AATATTATAAGA CAGCAGCTGGAA
AGGGCTACGCTA TTCGTCCTGCGCC
CCGATGTCGACC ACTGGGACGGCA
AGATTGGTATCG GCGCGCTTAACG
AGGATAACGTCA TGCTGGATAAAC
TGGAGTTTCACG AGCAGGTTCCGC
TCGGGAAAACCG GCCGCGGTCCCC
TCAACGCCTGTA GGGTCGGCTCTTT
AGTACAGCTATT TCTGCTGCAGGC
CCGGTTACAAAA GCTGAATGAAAT
TTCTGACCTACGC GCAGATGCAGCC
ATCCGGTAAAAA GCGGGAGCAGCA
AGGCGTGCGCTA CACGGCCCGCTT
CCTGTTCGAATG TATTGTCACCAG
CCAGCAGGCGGA CCTGCTCAGCCA
TTCAAAAGCGCC CTGTGCCGATCT
GAAGTTTGTTCA GCTGGGCAGCCA
GTTTAGCGATCA GGTACAAACCTC
CACCATCGCGCC ATCGCGCAGCCA
ACGCAAGTCCCA GGCGCTTTTTGA
GCATTTCCACATC AGCGATTCGTAA
TTTATGGGCAAT GCATATTGACGC
GAGTCCCAGGAA CCACTTTGCCGA
GCGCTGCTGAAA CCCGTTAACCCG
GAGATGGATAAC GGAGTCGGTGGC
TGGCCAACCTAC GCAGGCGTTTTA
TATCCTTATGCGC CCTCTCGCCAAA
TGCATAAAGAGC CTATCTATCCCAC
AGATTGTCGACG CTGTTCCAGAAA
AAATGCTGCACC TGCGGGCCAATG
ACTAA GGCTTTAACGAG
TATCTGAATCAC
ATCCGCCTGGAG
CAGGCCAGAATG
CTGTTAAAAGGC
CACGATATGAAA
GTGAAAGATATC
GCCCACGCCTGC
GGTTTCGCCGAC
AGCAACTACTTC
TGCCGCCTGTTTC
GCAAAAACACCG
AACGCTCGCCGT
CGGAGTATCGCC
GTCAATATCACA
GCCAGCTGACGG
AAAAAACAGCCC
CGGCAAAAAACT
AG
acyl CTGACGAAGCGA 6 ATGAGCACTATC 16 ATGAGTTTTGAA 26
carrier GTFACATCACCG GAAGAACGCGTT GGAAAAATCGCG
protein GTGAAACTCTGC AAGAAAATTATC CTGGTTACCGGT
CDS ACGTCAACGGCG GGCGAACAGCTG GCAAGTCGCGGG
GAATGTATATGG GGCGTTAAGCAG ATTGGCCGCGCA
TCTGACCGAGAT GAAGAAGTTACC ATCGCTGAAACG
TTGCGCAAAACG AACAATGCTTCC CTCGTTGCCCGTG
CTCAGGAACCGC TTCGTTGAAGAC GCGCGAAAGTTA
GCAGTCTGTGCG CTGGGCGCTGAT TCGGGACTGCGA
GTTCACTGTAAT TCTCTTGACACCG CCAGCGAAAGCG
GTTTTGTACAAA TTGAGCTGGTAA GCGCGCAGGCGA
ATGATTTGCGTTA TGGCTCTGGAAG TCAGCGATTATTT
TGAGGGCAAACA AAGAGTTTGATA AGGTGCTAACGG
GCCGCAAAATAG CTGAGATTCCGG TAAAGGTCTGCT
CGTAAAATCGTG ACGAAGAAGCTG GCTGAATGTGAC
GTAAGACCTGCC AGAAAATCACTA CGATCCTGCATCT
GGGATTTAGTTG CTGTTCAGGCTG ATTGAATCTGTTC
CAAATTTTTCAAC CCATTGATTACAT TGGGAAATATTC
ATTTTATACACTA CAACGGCCACCA GCGCAGAATTTG
CGAAAACCATCG GGCGTAA GTGAAGTTGATA
CGAAAGCGAGTT TCCTGGTGAACA
TTGA ATGCCGGGATCA
CTCGTGATAACC
TGTTAATGCGCA
TGAAAGATGATG
AGTGGAACGATA
TTATCGAAACCA
ACCTGTCATCTGT
TTTCCGTCTGTCA
AAAGCGGTAATG
CGCGCTATGATG
AAAAAGCGTCAT
GGACGTATTATC
ACTATCGGTTCTG
TGGTTGGTACCA
TGGGAAATGCGG
GTCAGGCCAACT
ACGCTGCGGCGA
AAGCGGGTCTGA
TTGGCTTCAGTA
AATCACTGGCTC
GCGAAGTTGCGT
CCCGCGGTATTA
CTGTAAACGTTG
TTGCTCCGGGCTT
TATTGAAACGGA
CATGACGCGTGC
GCTGACCGATGA
GCAGCGTGCGGG
TACGCTGGCGGC
AGTTCCTGCGGG
GCGCCTCGGCTC
TCCAAATGAAAT
CGCCAGTGCGGT
GGCATTTTTAGCC
TCTGACGAAGCG
AGTTACATCACC
GGTGAAACTCTG
CACGTCAACGGC
GGAATGTATATG
GTCTGA
ompX ACGCCTGGGGCG 7 ATGAATAAAATT 17 ATGCCCGGCTCG 27
CDS CCGACCAGCGGG GCACGTTTTTCAG TCTCGTAAGGTA
AAGAGTGATTTG CACTGGCCGTTG CCGGCATGGTTG
GCCAACGAGGCG TTCTGGCTGCATC CCGATACTGGTT
CCGCTCTGAATG CGTAGGTACCAC ATTTTAATCGCCA
GAAATCATGGCG TGCTTTCGCTGCG TGATTTCCAT
ATTAAAATAACC ACTTCTACCGTTA
AGTATCGGCAAC CCGGTGGCTACG
CATGCCGGTACC CGCAGAGCGACA
TTACGAGACGAG TGCAGGGTGAAG
CCGGGCATCCTTT CGAACAAAGCTG
CTCCTGTCAATTT GCGGTTTCAACC
TGTCAAATGCGG TGAAGTACCGCT
TAAAGGTTCCAG ACGAGCAAGACA
TGTAATTGAATT ACAACCCGCTGG
ACCCCGCGCCGG GTGTTATCGGTTC
TTGAGCTAATGTT TTTCACCTACACC
GAAAAAAAGGGT GAAAAAGATCGT
CTTAAAAGCAGT TCTGAATCTGGC
ACAATAGGGCGG GTTTACAAAAAA
GTCTGAAGATAA GGCCAGTACTAC
TTTCA GGCATCACCGCA
GGTCCGGCTTAC
CGTCTGAACGAC
TGGGCTAGCATC
TACGGCGTAGTG
GGTGTTGGTTAC
GGTAAATTCCAG
GACAACAGCTAC
CCGAACAAATCT
GATATGAGCGAC
TACGGTTTCTCTT
ACGGCGCTGGTC
TGCAGTTCAACC
CGATCGAAAACG
TTGCCCTGGACTT
CTCCTACGAGCA
GTCTCGCATTCGT
AACGTTGACGTT
GGCACCTGGATT
GCTGGCGTAGGT
TACCGCTTCTAA
DNA- TCTGATTCCTGAT 8 GTGAATAAATCT 18 ATGAATCCTGAG 28
binding GAAAATAAACGC CAACTGATTGAC CGTTCTGAACGC
protein GACCTTGAAGAA AAAATTGCTGCC ATTGAAATCCCC
HU-beta ATTCCGGATAAC GGTGCGGACATT GTATTGCCGTTGC
CDS GTTATCGCCGATT TCTAAAGCCGCA GCGATGTGGTGG
TAGATATCCATC GCTGGACGTGCG TTTATCCGCACAT
CGGTGAAACGAA TTAGATGCTTTAA GGTCATACCCCT
TCGAGGAAGTTC TCGCTTCTGTTAC GTTTGTAGGGCG
TGGCACTTGCGC TGAATCTCTGCA GGAAAAATCTAT
TACAGAACGAAC GGCTGGAGATGA CCGTTGTCTCGA
CGTTTGGAATGG CGTTGCGCTGGT AGCAGCCATGGA
AAGTCGTCACGG AGGGTTTGGTAC CCATGATAAAAA
CAAAATAGTGAT TTTTGCTGTTAAA AATCATGCTGGT
TTCGCGCAAATA GAGCGCGCTGCC TGCGCAGAAAGA
GCGCTAAGAAAA CGTACTGGTCGC AGCCTCGACGGA
ATAGGGCTGGTA AATCCGCAAACA TGAGCCGGGTGT
AGTAAATTCGTA GGCAAAGAAATC AAACGATCTTTTC
CTTGCCAGCCTTT ACCATTGCTGCT ACCGTCGGGACC
TTTTGTGTAGCTA GCTAAAGTTCCG GTGGCGTCTATTT
ACTTAGATCGCT GGTTTCCGCGCA TGCAAATGCTGA
GGCAGGGGGGTC GGTAAAGCGCTG AGCTACCGGACG
AATT AAAGACGCGGTA GTACTGTTAAAG
AACTGA TGCTGGTCGAAG
GTTTGCAGCGCG
CGCGCATCTCTG
CGCTGTCTGATA
ATGGCGAACATT
TTTCGGCGAAGG
CGGAATACCTTG
AATCGCCGGCGA
TTGACGAACGCG
AGCAGGAAGTGC
TGGTTCGTACCG
CTATCAGCCAGT
TTGAAGGCTACA
TCAAGCTGAACA
AAAAAATCCCTC
CGGAAGTGCTGA
CGTCGCTGAATA
GCATCGACGATC
CGGCGCGTCTGG
CGGATACCATCG
CTGCGCATATGC
CGCTGAAGCTGG
CGGACAAACAGT
CCGTGCTGGAGA
TGTCCGACGTTA
ACGAGCGTCTGG
AATATCTGATGG
CGATGATGGAGT
CGGAAATCGATC
TGCTGCAGGTGG
AGAAGCGTATTC
GCAACCGCGTGA
AAAAGCAGATGG
AGAAATCTCAGC
GCGAGTACTATC
TGAATGAGCAAA
TGAAAGCCATTC
AAAAAGAGCTCG
GCGAGATGGACG
ACCTCCCCGGACG
AGAACGAAGCGC
TGAAGCGTAAGA
TCGACGCGGCGA
AAATGCCGAAAG
AGGCAAAAGAGA
AAACCGAAGCGG
AACTGCAAAAAC
TGAAAATGATGT
CCCCGATGTCGG
CGGAAGCGACCG
TCGTTCGCGGCT
ACATCGACTGGA
TGGTGCAGGTAC
CGTGGAACGCTC
GCAGCAAGGTTA
AAAAAGACCTGC
GTCAGGCTCAGG
AGATCCTCGATA
CCGATCACTACG
GCCTTGAGCGCG
TGAAGGATCGCA
TTCTTGAGTACCT
CGCGGTGCAGAG
CCGTGTTAACAA
GCTCAAAGGGCC
GATCCTGTGCCT
GGTTGGCTCCTCC
GGGGGTAGGTAA
AACCTCTCTCGG
CCAATCCATCGC
CAAAGCAACTGG
ACGCAAATATGT
GCGTATGGCGCT
GGGCGGCGTGCG
TGATGAAGCGGA
AATCCGCGGTCA
CCGCCGTACCTA
TATTGGCTCAAT
GCCGGGCAAACT
GATCCAGAAAAT
GGCTAAAGTGGG
CGTTAAAAACCC
GCTGTTCTTGCTG
GATGAGATCGAC
AAGATGTCTTCT
GACATGCGCGGC
GATCCGGCCTCG
GCGCTGCTGGAG
GTGTTGGATCCG
GAACAGAACGTG
GCCTTTAACGAC
CACTATCTGGAA
GTGGATTACGAT
CTCAGCGACGTG
ATGTTCGTTGCG
ACCTCTAACTCC
ATGAACATCCCG
GCGCCGCTGCTG
GATCGTATGGAA
GTGATCCGCCTCT
CCGGCTATACCG
AAGATGAGAAGC
TAAACATCGCCA
AACGCCATCTGC
TGTCAAAACAGA
TTGAGCGTAACG
CGCTCAAGAAAG
GCGAGCTGACGG
TGGATGACAGCG
CGATTATCGGCA
TCATTCGCTACTA
CACCCGTGAAGC
AGGCGTGCGTGG
TCTGGAGCGTGA
AATCTCGAAACT
GTGCCGCAAAGC
GGTGAAACAGCT
GCTGCTGGATAA
GTCGCTGAAACA
CATCGAGATTAA
CGGCGACAACCT
GCACGATTTCCTT
GGCGTGCAGCGC
TACGACTATGGT
CGTGCGGATAGC
GAAAACCGCGTA
GGTCAGGTGACC
GGACTGGCGTGG
ACGGAAGTGGGC
GGCGATCTGCTG
ACCATTGAAACC
GCCTGCGTTCCG
GGTAAAGGCAAA
CTGACCTACACC
GGTTCACTGGGT
GAAGTCATGCAG
GAATCCATCCAG
GCGGCGCTGACG
GTGGTTCGTTCAC
GTGCGGATAAGC
TGGGTATTAACT
CAGACTTTTACG
AAAAACGTGATA
TTCACGTTCACGT
GCCGGAAGGCGC
GACGCCGAAGGA
TGGTCCAAGCGC
CGGTATCGCGAT
GTGCACCGCGCT
GGTTTCCTGTCTG
ACGGGTAATCCG
GTACGCGCCGAC
GTGGCGATGACC
GGTGAGATTACC
CTCCGTGGCCAG
GTATTGCCGATT
GGTGGTCTGAAG
GAAAAACTGTTG
GCCGCGCATCGC
GGCGGCATTAAG
ACTGTTCTGATTC
CTGATGAAAATA
AACGCGACCTTG
AAGAAATTCCGG
ATAACGTTATCG
CCGATTTAGATA
TCCATCCGGTGA
AACGAATCGAGG
AAGTTCTGGCAC
TTGCGCTACAGA
ACGAACCGTTTG
GAATGGAAGTCG
TCACGGCAAAAT
AG
sspA CDS GTAAGAAAGTCG 9 ATGGCTGTCGCT 19 ATGGCTGAAAAT 29
GCCTGCGTAAAG GCCAACAAACGT CAATACTACGGC
CACGTCGTCGTC TCGGTAATGACG ACCGGTCGCCGC
CTCAGTTCTCCAA CTGTTTTCTGGTC AAAAGTTCCGCA
ACGTTAATTGTTT CTACTGACATCT GCTCGCGTTTTCA
TCTGCTCACGCA ATAGCCATCAGG TCAAACCGGGCA
GAACAATTTGCG TCCGCATCGTGCT ACGGTAAAATCG
AAAAAACCCGCT GGCCGAAAAAGG TTATCAACCAGC
TCGGCGGGTTTTT TGTTAGTTTTGAG GTTCTCTGGAAC
TTATGGATAAAT ATAGAGCACGTG AGTACTTCGGTC
TTGCCATTTTCCC GAGAAGGACAAC GTGAAACTGCCC
TCTACAAACGCC CCGCCTCAGGAT GCATGGTAGTTC
CCATTGTTACCAC CTGATTGACCTC GTCAGCCGCTGG
TTTTTCAGCATTT AACCCGAATCAA AACTGGTCGACA
CCAGAATCCCCT AGCGTACCGACG TGGTTGAGAAAT
CACCACAACGTC CTTGTGGATCGT TAGATCTGTACA
TTCAAAATCTGG GAGCTCACTCTG TCACCGTTAAAG
TAAACTATCATC TGGGAATCTCGC GTGGTGGTATCT
CAATTTTCTGCCC ATCATTATGGAA CTGGTCAGGCTG
AAATGCAGGTGA TATCTGGATGAG GTGCGATCCGTC
TTGTTCATTTTT CGTTTCCCGCATC ACGGTATCACCC
CGCCGCTCATGC GCGCTCTGATGG
CGGTTTACCCGG AGTACGACGAGT
TGGCGCGTGGGG CCCTGCGTGGCG
AAAGCCGTCTGT AACTGCGTAAAG
ATATGCAGCGTA CTGGTTTCGTTAC
TCGAAAAGGACT TCGTGATGCTCGT
GGTATTCGTTGAT CAGGTTGAACGT
GAATACCATTCA AAGAAAGTCGGC
GACCGGTACCGC CTGCGTAAAGCA
TGCGCAGGCTGA CGTCGTCGTCCTC
TACTGCGCGTAA AGTTCTCCAAAC
GCAGCTGCGTGA GTTAA
AGAACTACAGGC
GATTGCGCCAGT
TTTCACCCAGAA
GCCCTACTTCCTG
AGCGATGAGTTC
AGCCTGGTGGAC
TGCTACCTGGCA
CCACTGCTGTGG
CGTCTGCCGGTTC
TCGGCGTAGAGC
TGGTCGGCGCTG
GCGCGAAAGAGC
TTAAAGGCTATA
TGACTCGCGTATT
TGAGCGCGACTC
TTTCCTCGCTTCT
TTAACTGAAGCC
GAACGTGAAATG
CGTCTCGGTCGG
GGCTAA
tatE CDS GTCAAAGCCGTA 10 ATGGGTGAGATT 20 ATGTTTGTTGCTG 30
TTATCGACCCCTT AGTATTACCAAA CCGGACAATTTG
AGGGACAACGCT CTGCTGGTAGTC CCGTAACGCCGG
TGCCGGGGCGGG GCAGCGCTGATT ACTGGACGGGAA
AGAGCGGCCGCA ATCCTGGTGTTTG ACGCGCAGACCT
GTTGATTTTTGCC GTACCAAAAAGT GCGTCAGCATGA
GAACTTTCAGCT TACGCACGCTGG TGCGCCAGGCCG
GATTATATTCAG GTGGAGACCTGG CGGAGCGGGGGG
CAGGTACGCGAG GCTCGGCTATCA CGTCGCTTCTGGT
CGCCTGCCGGTG AAGGCTTTAAAA TCTGCCTGAGGC
TTGCGCAATCGC AAGCCATGAGCG GTTGCTGGCGCG
CGCTTTGCGCCA ATGACGATGACA AGACGATAACGA
CCGCAATTATTAT GTGCGAAGAAGA TGCGGATTTATC
GACGTTTTTTTAA CCAGTGCTGAAG GGTTAAATCCGC
ACAAGGCTTGAT AAGCGCCGGCAC CCAGCAGCTGGA
TCACCTTGTTACA AGAAGCTCTCTC TGGCGGCTTCTTA
GATTGCTATTGTG ATAAAGAGTAA CAGCTCTTGCTG
TCCGCGCGTCAA GCGGAGAGCGAA
ATAGCCGTTAAT AACAGCGCTTTG
TGTATGCGTGTAT ACGACGGTGCTG
GATGGCGTATTC ACCCTGCATATC
G CCTTCCGGCGAA
GGTCGAGCGACG
AATACGCTGGTG
GCCCTGCGTCAG
GGGAAGATTGTG
GCGCAATATCAG
AAACTGCATCTC
TATGATGCGTTC
AATATCCAGGAA
TCCAGGCTGGTC
GATGCCGGGCGG
CAAATTCCGCCG
CTGATCGAAGTC
GACGGGATGCGC
GTCGGGCTGATG
ACCTGCTACGAT
TTACGTTTCCCTG
AGCTGGCGCTGT
CGTTAGCGCTCA
GCGGCGCGCAGC
TCATAGTGTTGCC
TGCCGCGTGGGT
AAAAGGGCCGCT
GAAGGAACATCA
CTGGGCGACGCT
GCTGGCGGCGCG
GGCGCTGGATAC
AACCTGCTATATT
GTCGCCGCAGGA
GAGTGCGGGACG
CGTAATATCGGT
CAAAGCCGTATT
ATCGACCCCTTA
GGGACAACGCTT
GCCGGGGCGGGA
GAGCGGCCGCAG
TTGATTTTTGCCG
AACTTTCAGCTG
ATTATATTCAGC
AGGTACGCGAGC
GCCTGCCGGTGT
TGCGCAATCGCC
GCTTTGCGCCAC
CGCAATTATTAT
GA
LexA GAGGCGGTGGTT 11 ATGAAAGCGTTA 21 ATGGCCAATAAT 31
repressor GACCGTATCGGT ACGACCAGGCAG ACCACTGGGTTA
CDS CCCGAGCATCAT CAAGAGGTGTTT ACCCGAATTATT
GAGCTTTCGGGG GATCTCATTCGG AAAGCGGCCGGG
CGAGCGAAAGAT GATCATATCAGC TATTCCTGGAAA
ATGGGATCGGCG CAGACGGGCATG GGATTCCGTGCG
GCGGTACTGCTG CCGCCGACGCGT GCGTGGGTCAAT
GCGATTATCATC GCGGAGATTGCT GAGGCCGCATTT
GCGCTGATCGCG CAGCGCTTGGGG CGTCAGGAAGGC
TGGGGAACGCTG TTTCGCTCCCCAA ATCGCGGCCGTT
CTGTGGGCGAAC ACGCGGCGGAAG ATTGCCGTGGCG
TACCGCTAAGTC AGCATCTGAAAG ATCGCCTGCTGG
TTGTCGTAGCTGC CGCTGGCGCGTA TTGGACGTCGAT
TCGCAAAACGGA AAGGCGCAATCG GCCATCACGCGG
AAGAAACTCCTG AGATCGTTTCCG GTGCTGCTCATTA
ATTTTTGTGTGAA GCGCCTCCCGCG GCTCGGTCCTGTT
ATGTGGTTCCAA GTATTCGTCTGCT AGTGATGATAGT
AATCACCGTTAG GACGGAAGAAGA TGAAATTATCAA
CTGTATATACTCA AACCGGTCTGCC TAGCGCGATTGA
CAGCATAACTGT GCTTATTGGCCG GGCGGTGGTTGA
ATATACACCCAG CGTCGCGGCAGG CCGTATCGCTCC
GGGGC TGAGCCGCTGCT CGAGCATCATGA
AGCGCAGCAGCA GCTTTCGGGGCG
CATTGAAGGCCA AGCGAAAGATAT
CTACCAGGTGGA GGGATCGGCGGC
CCCGGCCATGTTT GGTACTGCTGGC
AAGCCGAACGCC GATTATCATCGC
GATTTTCTGCTGC GCTGATCGCGTG
GTGTTAGCGGTA GGGAACGCTGCT
TGTCGATGAAGG GTGGGCGAACTA
ATATCGGTATTCT CCGCTAA
CGATGGCGACCT
GCTGGCTGTCCA
TAAAACGCAGGA
TGTGCGCAATGG
TCAGGTGGTTGT
GGCGCGTATCGA
CGAAGAAGTGAC
CGTGAAGCGTCT
GAAAAAACAGGG
TAACGTCGTGGA
ATTGCTGCCGGA
AAACAGCGAATT
CTCGCCGATCGT
GGTCGACCTTCG
CGAACAAAGCTT
TACTATTGAAGG
CCTGGCCGTCGG
CGTTATCCGCAA
CGGCAACTGGCA
ATAA
hisS CDS TAAGAAAAGCGG 12 ...ATGAACGATTA 22 ATGCATAACCAG 32
CCTGTACGAAGA TCTGCCGGGCGA GCTCCGATTCAA
CGGCGTACGTAA AACCGCTCTCTG CGTAGAAAATCA
AGACACTGCTGGA GCAGCGCATTGA AAACGAATTTAC
TAACGACGATAT AGGCTCACTGAA GTTGGGAATGTG
GATCGATCAGCT GCAGGTGCTTGG CCGATTGGCGAT
GGAAGCGCGTAT TAGCTACGGTTA GGCGCCCCCATC
TCGCGCTAAAGC CAGCGAAATCCG GCCGTACAGTCG
ATCGATGCTGGA TTTGCCGATTGTA ATGACAAACACG
TGAGGCGCGTCG GAGCAGACCCCG CGCACCACCGAT
TATCGATATCCA TTATTCAAACGC GTGGCGGCGACG
GCAGGTTGAAGC GCTATCGGCGAA GTAAATCAAATT
GAAATAACGTGT GTGACCGACGTG AAAGCCCTCGAG
TGCTGAAGCGATA GTTGAAAAAGAG CGCGTTGGCGCG
CGCTTCCCGTGTA ATGTACACCTTTG GATATCGTGCGC
TGATTGAACCTG AGGACCGTAACG GTTTCGGTGCCG
CGGGCGCGAGGC GCGATAGCCTGA ACGATGGATGCG
GCCGGGGTTCAT CTCTACGTCCGG GCGGAAGCGTTC
TTTTGTATATATA AAGGCACGGCTG AAACTTATCAAA
AAGAGAATAAAC GCTGCGTACGCG CAGCAGGTTAAC
GTGGCAAAGAAC CCGGTATCGAAC GTCCCGCTGGTT
ATTCAA ATGGTCTCCTGTA GCCGATATCCAC
CAATCAAGAACA TTCGATTACCGC
GCGCCTGTGGTA ATTGCGCTGAAG
CATTGGGCCGAT GTAGCGGAATAC
GTTCCGCCACGA GGCGTTGATTGC
ACGTCCGCAAAA CTGCGTATTAAC
AGGCCGCTACCG CCGGGCAATATC
TCAGTTCCACCA GGCAACGAAGAG
GATTGGCGCCGA CGTATCCGCATG
AGCGTTTGGCCT GTGGTGGACTGC
GCAGGGGCCGGA GCTCGCGATAAA
TATCGATGCCGA AATATTCCTATCC
GCTGATTATGCT GTATCGGGGTAA
GACCGCCCGCTG ACGCCGGTTCTCT
GTGGCGCGAGCT GGAAAAAGATCT
GGGCATCTCCGG CCAGGAAAAATA
CCACGTTGCGCT CGGCGAACCGAC
GGAGCTGAACTC TCCGCAGGCGCT
TATCGGTTCGCTG GCTGGAATCGGC
GAGGCTCGCGCT AATGCGCCATGT
AACTATCGCGAC TGATCATCTCGAT
GCGCTGGTGGCC CGTCTCAACTTCG
TATCTTGAGCAG ATCAGTTTAAAG
TTTAAAGATAAG TCAGCGTAAAAG
CTGGACGAAGAC CCTCCGATGTGTT
TGCAAACGCCGC CCTCGCGGTTGA
ATGTACACCAAC ATCCTATCGCCTG
CCGCTGCGCGTG TTGGCGAAACAG
CTGGATTCTAAA ATCGATCAGCCT
AACCCGGACGTC CTGCACCTCGGG
CAGGCGCTGCTG ATCACCGAAGCG
AACGACGCCCCG GGCGGCGCGCGC
ACGCTGGGCGAC AGCGGCGCGGTG
TATCTTGATGAA AAGTCCGCGATC
GAGTCCAAAACG GGCCTCGGCCTG
CATTTTGCCGGG CTGCTGTCTGAA
CTGTGCGCGCTG GGGATTGGCGAT
CTGGATGATGCC ACGCTGCGCGTC
GGTATTCGCTAT TCTCTGGCGGCG
ACCGTGAATCAG GATCCCGTTGAA
CGTCTGGTACGC GAGATCAAAGTG
GGTCTCGACTAC GGCTTCGATATTC
TACAACCGCACC TCAAGTCGCTGC
GTGTTTGAGTGG GTATTCGCTCTCG
GTCACCACCAGC CGGGATCAACTT
CTCGGTTCCCAG TATTGCCTGCCCG
GGCACCGTCTGC ACCTGTTCACGTC
GCCGGAGGCCGT AGGAGTTTGACG
TACGATGGTCTG TTATCGGTACCGT
GTTGAGCAGCTT TAACGCGCTGGA
GGCGGTCGCGCT GCAGCGCCTGGA
ACCCCTGGCGTC AGATATCATTAC
GGCTTTGCGATG GCCGATGGATAT
GGGCTGGAACGT TTCGATCATTGGC
CTTGTTTTACTGG TGCGTGGTAAAC
TTCAGGCAGTGA GGTCCCGGCGAG
ATCCGGAATTTA GCGCTGGTTTCC
AAGCCGATCCTG ACCCTCGGCGTA
TTGTCGATATATA ACCGGCGGCAAT
CCTGGTAGCCTC AAGAAAAGCGGC
CGGAACTGACAC CTGTACGAAGAC
CCAGTCCGCAGC GGCGTACGTAAA
AATGCGTCTGGC GACAGGCTGGAT
TGAACAGGTACG AACGACGATATG
CGATGCGTTACC ATCGATCAGCTG
CGGCGTTAAGCT GAAGCGCGTATT
GATGACCAACCA CGCGCTAAAGCA
TGGCGGCGGCAA TCGATGCTGGAT
CTTTAAGAAGCA GAGGCGCGTCGT
GTTTGCGCGCGC ATCGATATCCAG
TGATAAATGGGG CAGGTTGAAGCG
CGCTCGCGTTGC AAATAA
GCTGGTGCTGGG
CGAATCAGAAAT
CGCCGACGGAAA
CGTGGTAGTGAA
AGATTTACGCTC
AGGTGAGCAAAC
TACCGTAACGCA
GGATAGCGTTGC
TGCGCATTTGCG
CACACTTCTGGG
TTAA
Table of Strains
First Current Universal Mutagenic DNA Gene 1 Gene 2
Sort Reference Name Name Lineage Description Genotype mutation mutation
1 Application CI006 CI006 Isolated None WT
text strain from
Enterobacter
genera
2 Application CI008 CI008 Isolated None WT
text strain from
Burkholderia
genera
3 Application CI010 CI010 Isolated None WT
text strain from
Klebsiella
genera
4 Application CI019 CI019 Isolated None WT
text strain from
Rahnella
genera
5 Application CI028 CI028 Isolated None WT
text strain from
Enterobacter
genera
6 Application CI050 CI050 Isolated None WT
text strain from
Klebsiella
genera
7 Application CM002 CM002 Mutant of Disruption of nifL ΔnifL::KanR SEQ ID
text CI050 gene with a NO: 33
kanamycin resistance
expression cassette
(KanR) encoding the
aminoglycoside O-
phosphotransferase
gene aph1 inserted.
8 Application CM011 CM011 Mutant of Disruption of nifL ΔnifL::SpecR SEQ ID
text CI019 gene with a NO: 34
spectinomycin
resistance expression
cassette (SpecR)
encoding the
streptomycin 3″-O-
adenylyltransferase
gene aadA inserted.
9 Application CM013 CM013 Mutant of Disruption of nifL ΔnifL::KanR SEQ ID
text CI006 gene with a NO: 35
kanamycin resistance
expression cassette
(KanR) encoding the
aminoglycoside O-
phosphotransferase
gene aph1 inserted.
10 FIG. 4A CM004 CM004 Mutant of Disruption of amtB ΔamtB::KanR SEQ ID
CI010 gene with a NO: 36
kanamycin resistance
expression cassette
(KanR) encoding the
aminoglycoside O-
phosphotransferase
gene aph1 inserted.
11 FIG. 4A CM005 CM005 Mutant of Disruption of nifL ΔnifL::KanR SEQ ID
CI010 gene with a NO: 37
kanamycin resistance
expression cassette
(KanR) encoding the
aminoglycoside O-
phosphotransferase
gene aph1 inserted.
12 FIG. 4B CM015 CM015 Mutant of Disruption of nifL ΔnifL::Prm5 SEQ ID
CI006 gene with a fragment NO: 38
of the region
upstream of the
ompX gene inserted
(Prm5).
13 FIG. 4B CM021 CM021 Mutant of Disruption of nifL ΔnifL::Prm2 SEQ ID
CI006 gene with a fragment NO: 39
of the region
upstream of an
unanotated gene and
the first 73 bp of that
gene inserted (Prm2).
14 FIG. 4B CM023 CM023 Mutant of Disruption of nifL ΔnifL::Prm4 SEQ ID
CI006 gene with a fragment NO: 40
of the region
upstream of the acpP
gene and the first
121 bp of the acpP
gene inserted (Prm4).
15 FIG. 10A CM014 CM014 Mutant of Disruption of nifL ΔnifL::Prm1 SEQ ID
CI006 gene with a fragment NO: 41
of the region
upstream of the lpp
gene and the first
29 bp of the lpp gene
inserted (Prm1).
16 FIG. 10A CM016 CM016 Mutant of Disruption of nifL ΔnifL::Prm9 SEQ ID
CI006 gene with a fragment NO: 42
of the region
upstream of the lexA
3 gene and the first
21 bp of the lexA 3
gene inserted (Prm9).
17 FIG. 10A CM022 CM022 Mutant of Disruption of nifL ΔnifL::Prm3 SEQ ID
CI006 gene with a fragment NO: 43
of the region
upstream of the mntP
1 gene and the first
53 bp of the mntP 1
gene inserted (Prm3).
18 FIG. 10A CM024 CM024 Mutant of Disruption of nifL ΔnifL::Prm7 SEQ ID
CI006 gene with a fragment NO: 44
of the region
upstream of the sspA
gene inserted (Prm7).
19 FIG. 10A CM025 CM025 Mutant of Disruption of nifL ΔnifL::Prm10 SEQ ID
CI006 gene with a fragment NO: 45
of the region
upstream of the hisS
gene and the first
52 bp of the hisS gene
inserted (Prm10).
20 FIG. 10B CM006 CM006 Mutant of Disruption of glnB ΔglnB::KanR SEQ ID
CI010 gene with a NO: 46
kanamycin resistance
expression cassette
(KanR) encoding the
aminoglycoside O-
phosphotransferase
gene aph1 inserted.
21 FIG. 10C CI028 CM017 Mutant of Disruption of nifL ΔnifL::KanR SEQ ID
nifL:KanR CI028 gene with a NO: 47
kanamycin resistance
expression cassette
(KanR) encoding the
aminoglycoside O-
phosphotransferase
gene aph1 inserted.
22 FIG. 10C CI019 CM011 Mutant of Disruption of nifL ΔnifL::SpecR SEQ ID
nifL:SpecR CI019 gene with a NO: 48
spectinomycin
resistance expression
cassette (SpecR)
encoding the
streptomycin 3″-O-
adenylyltransferase
gene aadA inserted.
23 FIG. 10C CI016 CM013 Mutant of Disruption of nifL ΔnifL::KanR SEQ ID
nifL:KanR CI006 gene with a NO: 49
kanamycin resistance
expression cassette
(KanR) encoding the
aminoglycoside O-
phosphotransferase
gene aph1 inserted.
24 FIG. 10C CI010 CM005 Mutant of Disruption of nifL ΔnifL::KanR SEQ ID
nifL:KanR CI010 gene with a NO: 50
kanamycin resistance
expression cassette
(KanR) encoding the
aminoglycoside O-
phosphotransferase
gene aph1 inserted.
25 FIG. 4C Strain 2 CI006 Isolated None WT
strain from
Enterobaeter
genera
26 FIG. 4C Strain 4 CI010 Isolated None WT
strain from
Klebsiella
genera
27 FIG. 4C Strain 1 CI019 Isolated None WT
strain from
Rahnella
genera
28 FIG. 4C Strain 3 CI028 Isolated None WT
strain from
Enterobacter
genera
29 FIG. 4B Strain 2 CI006 Isolated None WT
strain from
Enterobacter
genera
30 FIG. 4B High CM014 Mutant of Disruption of nifL ΔnifL::Prm1 SEQ ID
CI006 gene with a fragment NO: 51
of the region
upstream of the lpp
gene and the first
29 bp of the lpp gene
inserted (Prm1).
31 FIG. 4B Med CM015 Mutant of Disruption of nifL ΔnifL::Prm5 SEQ ID
CI006 gene with a fragment NO: 52
of the region
upstream of the
ompX gene inserted
(Prm5).
32 FIG. 4B Low CM023 Mutant of Disruption of nifL ΔnifL::Prm4 SEQ ID
CI006 gene with a fragment NO: 53
of the region
upstream of the acpP
gene and the first
121 bp of the acpP
gene inserted (Prm4).
33 FIG. 4D Strain 2 CI006 Isolated None WT
strain from
Enterobacter
genera
34 FIG. 4D Evolved CM029 Mutant of Disruption of nifL ΔnifL::Prm5 SEQ ID SEQ ID
CI006 gene with a fragment ΔglnE- NO: 54 NO: 61
of the region AR_KO1
upstream of the
ompX gene inserted
(Prm5) and deletion
of the 1287 bp after
the start codon of the
glnE gene containing
the adenylyl-
removing domain of
glutamate-ammonia-
ligase
adenylyltransferase
(ΔglnE-AR_KO1).
35 FIG. 14C Wild CI006 Isolated None WT
strain from
Enterobacter
genera
36 FIG. 14C Evolved CM014 Mutant of Disruption of nifL ΔnifL::Prm1 SEQ ID
CI006 gene with a fragment NO: 55
of the region
upstream of the lpp
gene and the first
29 bp of the lpp gene
inserted (Prm1).
37 FIG. 14B Wild CI019 Isolated None WT
strain from
Rahnella
genera
38 FIG. 14B Evolved CM011 Mutant of Disruption of nifL ΔnifL::SpecR SEQ ID
CI019 gene with a NO: 56
spectinomycin
resistance expression
cassette (SpecR)
encoding the
streptomycin 3″-O-
adenylyltransferase
gene aadA inserted.
39 FIG. 14A Evolved CM011 Mutant of Disruption of nifL ΔnifL::SpecR SEQ ID
CI019 gene with a NO: 57
spectinomycin
resistance expression
cassette (SpecR)
encoding the
streptomycin 3″-O-
adenylyltransferase
gene aadA inserted.
40 FIG. 15A Wild CI006 Isolated None WT
strain from
Enterobacter
genera
41 FIG. 15A Evolved CM013 Mutant of Disruption of nifL ΔnifL::KanR SEQ ID
CI006 gene with a NO: 58
kanamycin resistance
expression cassette
(KanR) encoding the
aminoglycoside O-
phosphotransferase
gene aph1 inserted.
42 FIG. 15B No CM011 Mutant of Disruption of nifL ΔnifL::SpecR SEQ ID
name CI019 gene with a NO: 59
spectinomycin
resistance expression
cassette (SpecR)
encoding the
streptomycin 3″-O-
adenylyltransferase
gene aadA inserted.
43 FIG. 16B Strain 5 CI008 Isolated None WT
strain from
Burkholderia
genera
44 FIG. 16B Strain 1 CM011 Mutant of Disruption of nifL ΔnifL::SpecR SEQ ID
CI019 gene with a NO: 60
spectinomycin
resistance expression
cassette (SpecR)
encoding the
streptomycin 3″-O-
adenylyltransferase
gene aadA inserted.
Table of Strains sequences
SEQ
ID
NO: Sequence
33 ATGAGCCATATTCAACGGGAAACGTCTTGCTCCAGGCCGCGATTAAATT
CCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGT
CGGGCAATCAGGTGCGACAATCTATCGATTGTATGGGAAGCCCGATGCG
CCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTCTCCAATGATGTTA
CAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCC
GACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCA
CTGCGATCCCCGGGAAAACAGCATTCCAGGTATTAGAAGAATATCCTGA
TTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGC
ATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTC
TCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGA
TTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAA
ATGCATAAGCTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGA
TTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTA
TTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCAT
CCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTT
TTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCAT
TTGATGCTCGATGAGTTTTTCTAATAAGCCTGCCTGGTTCTGCGTTTCCC
GCTCTTTAATACCCTGACCGGAGGTGAGCAATGA
34 ATGAGCATCACGGCGTTATCAGCATCATTTCCTGAGGGGAATATCGCCA
GCCGCTTGTCGCTGCAACATCCTTCACTGTTTTATACCGTGGTTGAACAA
TCTTCGGTGGCGAGCGTGTTGAGTCATCCTGACTAGCTGAGATGAGGGC
TCGCCCCCTCGTCCCGACACTTCCAGATCGCCATAGCGCACAGCGCCTC
GAGCGGTGGTAACGGCGCAGTGGCGGTTTTCATGGCTTGTTATGACTGT
TTTTTTGGGGTACAGTCTATGCCTCGGGCATCCAAGCAGCAAGCGCGTT
ACGCCGTGGGTCGATGTTTGATGTTATGGAGCAGCAACGATGTTACGCA
GCAGGGCAGTCGCCCTAAAACAAAGTTAAACATCATGAGGGAAGCGGT
GATCGCCGAAGTATCGACTCAACTATCAGAGGTAGTTGGCGTCATCGAG
CGCCATCTCGAACCGACGTTGCTGGCCGTACATTTGTACGGCTCCGCAG
TGGATGGCGGCCTGAAGCCACACAGTGATATTGATTTGCTGGTTACGGT
GACCGTAAGGCTTGATGAAACAACGCGGCGAGCTTTGATCAACGACCTT
TTGGAAACTTCGGCTTCCCCTGGAGAGAGCGAGATTCTCCGCGCTGTAG
AAGTCACCATTGTTGTGCACGACGACATCATTCCGTGGCGTTATCCAGCT
AAGCGCGAACTGCAATTTGGAGAATGGCAGCGCAATGACATTCTTGCAG
GTATCTTCGAGCCAGCCACGATCGACATTGATCTGGCTATCTTGCTGACA
AAAGCAAGAGAACATAGCGTTGCCTTGGTAGGTCCAGCGGCGGAGGAA
CTCTTTGATCCGGTTCCTGAACAGGATCTATTTGAGGCGCTAAATGAAA
CCTTAACGCTATGGAACTCGCCGCCCGACTGCTGCTGGCGATGAGCGAAA
TGTAGTGCTTACGTTGTCCCGCATTTGGTACAGCGCAGTAACCGGCAAA
ATCGCGCCGAAGGATGTCGCTGCCGACTGGGCAATGGAGCGCCTGCCGG
CCCAGTATCAGCCCGTCATACTTGAAGCTAGACAGGCTTATCTTGGACA
AGAAGAAGATCGCTTGGCCTCGCGCGCAGATCAGTTGGAAGAATTTGTC
CACTACGTGAAAGGCGAGATCACCAAGGTAGTCGGCAAATAATGTCTA
ACAATTCGTTCAAGCCGACGCCGCTTCGCGGCGCGGCTTAACTCAAGCG
TTAGATGCACTAAGCACATAATTGCTCACAGCCAAACTATCAGGTCAAG
TCTGCTTTTATTATTTTTAAGCGTGCATAATAAGCCCTACACAAATGGTA
CCCGACCGGTGGTGAATTTAATCTCGCTGACGTGTAGACATTCCCTTATC
CAGACGCTGATCGCCCATCATCGCGGTTCTTTAGATCTCTCGGTCCGCCC
TGATGGCGGCACCTTGCTGACGTTACGCCTGCCGGTACAGCAGGTTATC
ACCGGAGGCTTAAAATGA
35 CTGATCCTTCAACTCAGCAAAAGTTCGATTTATTCAACAAAGCCACGTT
GTGTCTCAAAATCTCTGATGTTACATTGCACAAGATAAAAATATATCAT
CATGAACAATAAAACTGTCTGCTTACATAAACAGTAATACAAGGGGTGT
TATGAGCCATATTCAACGGGAAACGTCTTGCTCCAGGCCGCGATTAAAT
TCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATG
TCGGGCAATCAGGTGCGACAATCTATCGATTGTATGGGAAGCCCGATGC
GCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTT
ACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTC
CGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACC
ACTGCGATCCCCGGGAAAACAGCATTCCAGGTATTAGAAGAATATCCTG
ATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTT
GCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTC
GTCTCCTCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAG
TGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAA
GAAATGCATAAGCTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGG
TGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTT
GTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGC
CATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGC
TTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTT
CATTTGATGCTCGATGAGTTTTTCTAATAAGCCTTGACCCTACGATTCCC
GCTATTTCATTCACTGACCGGAGGTTCAAAATGA
36 ATGAAGATAGCAACAATGAAAACAGGTCTGGGAGCGTTGGCTCTTCTTC
CCTGATCCTTCAACTCAGCAAAAGTTCGATTTATTCAACAAAGCCACGTT
GTGTCTCAAAATCTCTGATGTTACATTGCACAAGATAAAAATATATCAT
CATGAACAATAAAACTGTCTGCTTACATAAACAGTAATACAAGGGGTGT
TATGAGCCATATTCAACGGGAAACGTCTTGCTCCCGTCCGCGCTTAAAC
TCCAACATGGACGCTGATTTATATGGGTATAAATGGGCTCGCGATAATG
TCGGGCAATCAGGTGCGACAATCTATCGCTTGTATGGGAAGCCCGATGC
GCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTT
ACAGATGAGATGGTCCGTCTCAACTGGCTGACGGAGTTTATGCCTCTCC
CGACCATCAAGCATTTTATCCGTACTCCTGATGATGCGTGGTTACTCACC
ACCGCGATTCCTGGGAAAACAGCCTTCCAGGTATTAGAAGAATATCCTG
ATTCAGGTGAAAATATTTTTGATGCGCTGGCCGTGTTCCTGCGCCGGTTA
CATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGTGTATTTCGT
CTTGCTCAGGCGCAATCACGCATGAATAACGGTTTGGTTGATGCGAGTG
ATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGA
AATGCACAAGCTCTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTG
ATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGT
ATTGATGTTGGACGGGTCGGAATCGCAGACCGTTACCAGGACCTTGCCA
TTCTTTGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTT
TTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCA
TTTGATGCTCGATGAGTTTTTCTAATAAGCCTGTGAAGGGCTGGACGTA
AACAGCCACGGCGAAAACGCCTACAACGCCTGA
37 ATGACCCTGAATATGATGCTCGATAACGCCGTACCCGAGGCGATTGCCG
GCTGATCCTTCAACTCAGCAAAAGTTCGATTTATTCAACAAAGCCACGT
TGTGTCTCAAAATCTCTGATGTTACATTGCACAAGATAAAAATATATCAT
CATGAACAATAAAACTGTCTGCTTACATAAACAGTAATACAAGGGGTGT
TATGAGCCATATTCAACGGGAAACGTCTTGCTCCCGTCCGCGCTTAAAC
TCCAACATGGACGCTGATTTATATGGGTATAAATGGGCTCGCGATAATG
TCGGGCAATCAGGTGCGACAATCTATCGCTTGTATGGGAAGCCCGATGC
GCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTT
ACAGATGAGATGGTCCGTCTCAACTGGCTGACGGAGTTTATGCCTCTCC
CGACCATCAAGCATTTTATCCGTACTCCTGATGATGCGTGGTTACTCACC
ACCGCGATTCCTGGGAAAACAGCCTTCCAGGTATTAGAAGAATATCCTG
ATTCAGGTGAAAATATTGTTGATGCGCTGGCCGTGTTCCTGCGCCGGTTA
CATTCGATTCCTCTTTTGTAATTGTCCTTTTAACAGCGATCGTGTATTTCGT
CTTGCTCAGGCGCAATCACGCATGAATAACGGTTTGGTTGATGCGAGTG
ATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGA
AATGCACAAGCTCTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTG
ATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGT
ATTGATGTTGGACGGGTCGGAATCGCAGACCGTTACCAGGACCTTGCCA
TTCTTTGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTT
TTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCA
TTTGATGCTCGATGAGTTTTTCTAATAAGCCTTGGTTCTGCGTTTCCCGCT
CTTTAATACCCTGACCGGAGGTGAGCAATGA
38 ATGACCCTGAATATGATGATGGATGCCGGCGGACATCATCGCGACAAAC
AATATTAATACCGGCAACCACACCGGCAATTTACGAGACTGCGCAGGCA
TCCTTTCTCCCGTCAATTTCTGTCAAATAAAGTAAAAGAGGCAGTCTACT
TGAATTACCCCCGGCTGGTTGAGCGTTTGTTGAAAAAAAGTAACTGAAA
AATCCGTAGAATAGCGCCACTCTGATGGTTAATTAACCTATTCAATTAA
GAATTATCTGGATGAATGTGCCATTAAATGCGCAGCATAATGGTGCGTT
GTGCGGGAAAACTGCTTTTTTTTGAAAGGGTTGGTCAGTAGCGGAAACA
ACTCACTTCACACCCCGAAGGGGGAAGTTGCCTGACCCTACGATTCCCG
CTATTTCATTCACTGACCGGAGGTTCAAAATGA
39 ATGACCCTGAATATGATGATGGATGCCGGCTCACCACGGCGATAACCAT
AGGTTTTCGGCGTGGCCACATCCATGGTGAATCCCACTTTTTCCAGCACG
CGCGCCACTTCATCGGGTCTTAAATACATAGATTTTCCTCGTCATCTTTC
CAAAGCCTCGCCACCTTACATGACTGAGCATGGACCGTGACTCAGAAAA
TTCCACAAACGAACCTGAAAGGCGTGATTGCCGTCTGGCCTTAAAAATT
ATGGTCTAAACTAAAATTTACATCGAAAACGAGGGAGGATCCTATGTTT
AACAAACCGAATCGCCGTGACGTAGATGAAGGTGTTGAGGATATTAACC
ACGATGTTAACCAGCTCGAACTCACTTCACACCCCGAAGGGGGAAGTTG
CCTGACCCTACGATTCCCGCTATTTCATTCACTGACCGGAGGTTCAAAAT
GA
40 ATGACCCTGAATATGATGATGGATGCCGGCTGACGAGGCAGGTTACATC
ACTGGTGAAACCCTGCACGTCAATGGCGGAATGTATATGGTTTAACCAC
GATGAAAATTATTTGCGTTATTAGGGCGAAAGGCCTCAAAATAGCGTAA
AATCGTGGTAAGAACTGCCGGGATTTAGTTGCAAATTTTTCAACATTTTA
TACACTACGAAAACCATCGCGAAAGCGAGTTTTGATAGGAAATTTAAGA
GTATGAGCACTATCGAAGAACGCGTTAAGAAAATTATCGGCGAACAGCT
GGGCGTTAAGCAGGAAGAAGTTACCAACAATGCTTCCTTCGTTGAAGAC
CTGGGCGCTGATTCTCTTGACACCGAACTCACTTCACACCCCGAAGGGG
GAAGTTGCCTGACCCTACGATTCCCGCTATTTCATTCACTGACCGGAGGT
TCAAAATGA
41 ATGACCCTGAATATGATGATGGATGCCGGCCGTCCTGTAATAATAACCG
GACAATTCGGACTGATTAAAAAAGCGCCCTTGTGGCGCTTTTTTTATATT
CCCGCCTCCATTTAAAATAAAAAATCCAATCGGATTTCACTATTTAAACT
GGCCATTATCTAAGATGAATCCGATGGAAGCTCGCTGTTTTAACACGCG
TTTTTTAACCTTTTATTGAAAGTCGGTGCTTCTTTGAGCGAACGATCAAA
TTTAAGTGGATTCCCATCAAAAAAATATTCTCAACCTAAAAAAGTTTGT
GTAATACTTGTAACGCTACATGGAGATTAACTCAATCTAGAGGGTATTA
ATAATGAATCGTACTAAACTGGTACTGGGCGCAACTCACTTCACACCCC
GAAGGGGGAAGTTGCCTGACCCTACGATTCCCGCTATTTCATTCACTGA
CCGGAGGTTCAAAATGA
42 ATGACCCTGAATATGATGATGGATGCCGGCATATTGACACCATGACCYCG
CGTAATGCTGATTGGTTCTGTGACGCTGGTAATGATTGTCGAAATTCTGA
ACAGTGCCATCGAAGCCGTAGTAGACCGTATTGGTGCAGAATTCCATGA
ACTTTCCGGGCGGGCGAAGGATATGGGGTCCTGCGGCGGTGCTGATGTCC
ATCCTGCTGGCGATGTTTACCTGGATCGCATTACTCTGGTCACATTTTCG
ATAACGCTTCCAGAATTCGATAACGCCCTGGTTTTTTGCTTAAATTTGGT
TCCAAAATCGCCTTTAGCTGTATATACTCACAGCATAACTGTATATACAC
CCAGGGGGCGGGATGAAAGCATTAACGGCCAGGAACTCACTTCACACC
CCGAAGGGGGAAGTTGCCTGACCCTACGATTCCCGCTATTTCATTCACT
GACCGGAGGTTCAAAATGA
43 ATGACCCTGAATATGATGATGGATGCCGGCATCATATTGCGCTCCCTGG
TTATCATTTGTTACTAAATGAAATGTTATAATATAACAATTATAAATACC
ACATCGCTTTCAATTCACCAGCCAAATGAGAGGAGCGCCGTCTGACATA
GCCAGCGCTATAAAACATAGCATTATCTATATGTTTATGATTAATAACTG
ATTTTTGCGTTTTGGATTTGGCTGTGGCATCCTTGCCGCTCTTTTCGCAGC
GTCTGCGTTTTTGCCCTCCGGTCAGGGCATTTAAGGGTCAGCAATGAGTT
TTTACGCAATTACGATTCTTGCCTTCGGCATGTCGATGGATGCTTTAACT
CACTTCACACCCCGAAGGGGGAAGTTGCCTGACCCTACGATTCCCGCTA
TTTCATTCACTGACCGGAGGTTCAAAATGA
44 ATGACCCTGAATATGATGATGGATGCCGGCCGCGTCAGGTTGAACGTAA
AAAAGTCGGTCTGCGCAAAGCACGTCGTCGTCCGCAGTTCTCCAAACGT
TAATTGGTTTCTGCTTCGGCAGAACGATTGGCGAAAAAACCCGGTGCGA
ACCGGGTTTTTTTATGGATAAAGATCGTGTTATCCACAGCAATCCATTGA
TTATCTCTTCTTTTTCAGCATTTCCAGAATCCCCTCACCACAAAGCCCGC
AAAATCTGGTAAACTATCATCCAATTTTCTGCCCAAATGGCTGGGATTGT
TCATTTTTTGTTTGCCTTACAACGAGAGTGACAGTACGCGCGGGTAGTTA
ACTCAACATCTGACCGGTCGATAACTCACTTCACACCCCGAAGGGGGAA
GTTGCCTGACCCTACGATTCCCGCTATTTCATTCACTGACCGGAGGTTCA
AAATGA
45 ATGACCCTGAATATGATGATGGATGCCGGCCCTGTATGAAGATGGCGTG
CGCAAAGATCGCCTGGATAACAGCGATATGATTAGCCAGCTTGAAGCCC
GCATTCGCGCGAAAGCGTCAATGCTGGACGAAGCGCGTCGTATTCGATGT
GCAACAGGTAGAAAAATAAGGTTGCTGGGAAGCGGCAGGCTTCCCGTG
TATGATGAACCCGCCCGGCGCGACCCGTTGTTCGTCGCGGCCCCGAGGG
TTCATTTTTTGTATTAATAAAGAGAATAAACGTGGCAAAAAATATTCAA
GCCATTCGCGGCATGAACGATTATCTGCCTGGCGAACTCACTTCACACC
CCGAAGGGGGAAGTTGCCTGACCCTACGATTCCCGCTATTTCATTCACT
GACCGGAGGTTCAAAATGA
46 ATGAAAAAGATTGATGCGATTATTAAACCTTTCAAACTGGATGACGTGC
GCTGATCCTTCAACTCAGCAAAAGTTCGATTTATTCAACAAAGCCACGT
TGTGTCTCAAAATCTCTGATGTTACATTGCACAAGATAAAAATATATCAT
CATGAACAATAAAACTGTCTGCTTACATAAACAGTAATACAAGGGGTGT
TATGAGCCATATTCAACGGGAAACGTCTTGCTCCCGTCCGCGCTTAAAC
TCCAACATGGACGCTGATTTATATGGGTATAAATGGGCTCGCGATAATG
TCGGGCAATCAGGTGCGACAATCTATCGCTTGTATGGGAAGCCCGATGC
GCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTT
ACAGATGAGATGGTCCGTCTCAACTGGCTGACGGAGTTTATGCCTCTCC
CGACCATCAAGCATTTTATCCGTACTCCTGATGATGCGTGGTTACTCACC
ACCGCGATTCCTGGGAAAACAGCCTTCCAGGTATTAGAAGAATATCCTG
ATTCAGGTGAAAATATTGTTGATGCGCTCTGCCGTGTTCCTGCGCCGGTTA
CATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGTGTATTTCGT
CTTGCTCAGGCGCAATCACGCATGAATAACGGTTTGGTTGATGCGAGTG
ATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGA
AATGCACAAGCTCTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTG
ATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGT
ATTGATGTTGGACGGGTCGGAATCGCAGACCGTTACCAGGACCTTGCCA
TTCTTTGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTT
TTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCA
TTTGATGCTCGATGAGTTTTTCTAATAAGCCTCGCGCGTGATTCGTATCC
GCACCGGCGAAGAAGACGACGCGGCGATTTAA
47 ATGACCATGAACCTGATGACGGATGTCGTCTCAGCCACCGGGATCGCCG
GGTTGCTTTCACGACAACACCCGACGCTGTTTTTTACACTAATTGAACAG
GCCCCCGTGGCGATCACGCTGACGGATACCGCTGCCCGCATTGTCTATG
CCAACCCGGGCGTGTTGAGTCATCCTGACTAGCTGAGATGAGGGCTCGC
CTGATCCTTCAACTCAGCAAAAGTTCGATTTATTCAACAAAGCCACGTT
GTGTCTCAAAATCTCTGATGTTACATTGCACAAGATAAAAATATATCAT
CATGAACAATAAAACTGTCTGCTTACATAAACAGTAATACAAGGGGTGT
TATGAGCCATATTCAACGGGAAACGTCTTGCTCCAGGCCGCGATTAAAT
TCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATG
TTCGGGCAATCAGGTGCGACAATCTATCGATTGTATGGGAAGCCCGATGC
GCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTT
ACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTC
CGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACC
ACTGCGATCCCCGGGAAAACAGCATTCCAGGTATTAGAAGAATATCCTG
ATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTT
GCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTC
GTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAG
TGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAA
GAAATGCATAAGCTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGG
TGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTT
GTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGC
CATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGC
TTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTT
CATTTGATGCTCGATGAGTTTTTCTAATAAGCCTGACCGGTGGTGAATTT
AATCTCGCTGACGTGTAGACATTCATCGATCTGCATCCACGGTCCGGCG
GCGGTACCTGCCTGACGCTACGTTTACCGCTCTTTTATGAACTGACCGGA
GGCCCAAGATGA
48 ATGAGCATCACGCCGTTATCAGCATCATTTCCTGAGGGGAATATCGCCA
GCCGCTTGTCGCTGCAACATCCTTCACTGTTTTATACCGTGGTTGAACAA
TCTTCGGTGGCGAGCGTGTTGAGTCATCCTGACTAGCTGAGATGAGGGC
TCGCCCCCTCGTCCCGACACTTCCAGATCGCCATAGCGCACAGCGCCTC
GAGCGGTGGTAACGGCGCAGTGGCGGTTTTCATGGCTTGTTATGACTGT
TTTTTTGGGGTACAGTCTATGCCTCGGGCATCCAAGCAGCAAGCGCGTT
ACGCCGTGGGTCGATGTTTGATGTTATGGAGCAGCAACGATGTTACGCA
GCAGGGCAGTCGCCCTAAAACAAAGTTAAACATCATGAGGGAAGCGGT
GATCGCCGAAGTATCGACTCAACTATCAGAGGTAGTTGGCGTCATCGAG
CGCCATCTCGAACCGACGTTGCTGGCCGTACATTTGTACGGCTCCGCAG
TGGATGGCGGCCTGAAGCCACACAGTGATATTGATTTGCTGGTTACGGT
GACCGTAAGGCTTGATGAAACAACGCGGCGAGCTTTGATCAACGACCTT
TTGGAAACTTCGGCTTCCCCTGGAGAGAGCGAGATTCTCCGCGCTGTAG
AAGTCACCATTGTTGTGCACGACGACATCATTCCGTGGCGTTATCCAGCT
AAGCGCGAACTGCAATTTGGAGAATGGCAGCGCAATGACATTCTTGCAG
GTATCTTCGAGCCAGCCACGATCGACATTGATCTGGCTATCTTGCTGACA
AAAGCAAGAGAACATAGCGTTGCCTTGGTAGGTCCAGCGGCGGAGGAA
CTCTTTGATCCGGTTCCTGAACAGGATCTATTTGAGGCGCTAAATGAAA
CCTTAACGCTATGGAACTCGCCGCCCGACTGGGCTGGCGATGAGCGAAA
TGTAGTGCTTACGTTGTCCCGCATTTGGTACAGCGCAGTAACCGTGCAAA
ATCGCGCCGAAGGATGTCGCTGCCGACTGGGCAATGGAGCGCCTGCCGG
CCCAGTATCAGCCCGTCATACTTGAAGCTAGACAGGCTTATCTTGGACA
AGAAGAAGATCGCTTGGCCTCGCGCGCAGATCAGTTGGAAGAATTTGTC
CACTACGTGAAAGGCGAGATCACCAAGGTAGTCGGCAAATAATGTCTA
ACAATTCGTTCAAGCCGACGCCGCTTCGCGGCGCGGCTTAACTCAAGCG
TTAGATGCACTAAGCACATAATTGCTCACAGCCAAACTATCAGGTCAAG
TCTGCTTTTATTATTTTTAAGCGTGCATAATAAGCCCTACACAAATGGTA
CCCGACCGGTGGTGAATTTAATCTCGCTGACGTGTAGACATTCCCTTATC
CAGACGCTGATCGCCCATCATCGCGGTTCTTTAGATCTCTCGGTCCGCCC
TGATGGCGGCACCTTGCTGACGTTACGCCTGCCGGTACAGCAGGTTATC
ACCGGAGGCTTAAAATGA
49 CTGATCCTTCAACTCAGCAAAAGTTCGATTTATTCAACAAAGCCACGTT
GTGTCTCAAAATCTCTGATGTTACATTGCACAAGATAAAAATATATCAT
CATGAACAATAAAACTGTCTGCTTACATAAACAGTAATACAAGGGGTGT
TATGAGCCATATTCAACGGGAAACGTCTTGCTCCAGGCCGCGATTAAAT
TCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATG
TCGGGCAATCAGGTGCGACAATCTATCGATTGTATGGGAAGCCCGATGC
GCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTT
ACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTC
CGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACC
ACTGCGATCCCCGGGAAAACAGCATTCCAGGTATTAGAAGAATATCCTG
ATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTT
GCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTC
GTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAG
TGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAA
GAAATGCATAAGCTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGG
TGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTT
GTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGC
CATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGC
TTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTT
CATTTGATGCTCGATGAGTTTTTCTAATAAGCCTTGACCCTACGATTCCC
GCTATTTCATTCACTGACCGGAGGTTCAAAATGA
50 ATGACCCTGAATATGATGCTCGATAACGCCGTACCCGAGGCGATTGCCG
GCTGATCCTTCAACTCAGCAAAAGTTCGATTTATTCAACAAAGCCACGT
TGTGTCTCAAAATCTCTGATGTTACATTGCACAAGATAAAAATATATCAT
CATGAACAATAAAACTGTCTGCTTACATAAACAGTAATACAAGGGGTGT
TATGAGCCATATTCAACGGGAAACGTCTTGCTCCCGTCCGCGCTTAAAC
TCCAACATGGACGCTGATTTATATGGGTATAAATGGGCTCGCGATAATG
TCGGGCAATCAGGTGCGACAATCTATCGCTTGTATGGGAAGCCCGATGC
GCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTT
ACAGATGAGATGGTCCGTCTCAACTGGCTGACGGAGTTTATGCCTCTCC
CGACCATCAAGCATTTTATCCGTACTCCTGATGATGCGTGGTTACTCACC
ACCGCGATTCCTGGGAAAACAGCCTTCCAGGTATTAGAAGAATATCCTG
ATTCAGGTGAAAATATTGTTGATGCGCTGGCCGTGTTCCTGCGCCGGTTA
CATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGTGTATTTCGT
CTTGCTCAGGCGCAATCACGCATGAATAACGGTTTGGTTGATGCGAGTG
ATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGA
AATGCACAAGCTCTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTG
ATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGT
ATTGATGTTGGACGGGTCGGAATCGCAGACCGTTACCAGGACCTTGCCA
TTCTTTGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTT
TTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCA
TTTGATGCTCGATGAGTTTTTCTAATAAGCCTTGGTTCTGCGTTTCCCGCT
CTTTAATACCCTGACCGGAGGTGAGCAATGA
51 ATGACCCTGAATATGATGATGGATGCCGGCCGTCCTGTAATAATAACCG
GACAATTCGGACTGATTAAAAAAGCGCCCTTGTGGCGCTTTTTTTATATT
CCCGCCTCCATTTAAAATAAAAAATCCAATCGGATTTCACTATTTAAACT
GGCCATTATCTAAGATGAATCCGATGGAAGCTCGCTGTTTTAACACGCG
TTTTTTAACCTTTTATTGAAAGTCGGTGCTTCTTTGAGCGAACGATCAAA
TTTAAGTGGATTCCCATCAAAAAAATATTCTCAACCTAAAAAAGTTTGT
GTAATACTTGTAACGCTACATGGAGATTAACTCAATCTAGAGGGTATTA
ATAATGAATCGTACTAAACTGGTACTGGGCGCAACTCACTTCACACCCC
GAAGGGGGAAGTTGCCTGACCCTACGATTCCCGCTATTTCATTCACTGA
CCGGAGGTTCAAAATGA
52 ATGACCCTGAATATGATGATGGATGCCGGCGGACATCATCGCGACAAAC
AATATTAATACCGGCAACCACACCGGCAATTTACGAGACTGCGCAGGCA
TCCTTTCTCCCGTCAATTTCTGTCAAATAAAGTAAAAGAGGCAGTCTACT
TGAATTACCCCCGGCTGGTTGAGCGTTTGTTGAAAAAAAGTAACTGAAA
AATCCGTAGAATAGCGCCACTCTGATGGTTAATTAACCTATTCAATTAA
GAATTATCTGGATGAATGTGCCATTAAATGCGCAGCATAATGGTGCGTT
GTGCGGGAAAACTGCTTTTTTTTGAAAGGGTTGGTCAGTAGCGGAAACA
ACTCACTTCACACCCCGAAGGGGGAAGTTGCCTGACCCTACGATTCCCG
CTATTTCATTCACTGACCGGAGGTTCAAAATGA
53 ATGACCCTGAATATGATGATGGATGCCGGCTGACGAGGCAGGTTACATC
ACTGGTGAAACCCTGCACGTCAATGGCGGAATGTATATGGTTTAACCAC
GATGAAAATTATTTGCGTTATTAGGGCGAAAGGCCTCAAAATAGCGTAA
AATCGTGGTAAGAACTGCCGGGATTTAGTTGCAAATTTTTCAACATTTTA
TACACTACGAAAACCATCGCGAAAGCGAGTTTTGATAGGAAATTTAAGA
GTATGAGCACTATCGAAGAACGCGTTAAGAAAATTATCGGCGAACAGCT
GGGCGTTAAGCAGGAAGAAGTTACCAACAATGCTTCCTTCGTTGAAGAC
CTGGGCGCTGATTCTCTTGACACCGAACTCACTTCACACCCCGAAGGGG
GAAGTTGCCTGACCCTACGATTCCCGCTATTTCATTCACTGACCGGAGGT
TCAAAATGA
54 ATGACCCTGAATATGATGATGGATGCCGGCGGACATCATCGCGACAAAC
AATATTAATACCGGCAACCACACCGGCAATTTACGAGACTGCGCAGGCA
TCCTTTCTCCCGTCAATTTCTGTCAAATAAAGTAAAAGAGGCAGTCTACT
TGAATTACCCCCGGCTGGTTGAGCGTTTGTTGAAAAAAAGTAACTGAAA
AATCCGTAGAATAGCGCCACTCTGATGGTTAATTAACCTATTCAATTAA
GAATTATCTGGATGAATGTGCCATTAAATGCGCAGCATAATGGTGCGTT
GTGCGCTGAAAACTGCTTTTTTTTGAAAGGGTTGGTCAGTAGCGGAAACA
ACTCACTTCACACCCCGAAGGGGGAAGTTGCCTGACCCTACGATTCCCG
CTATTTCATTCACTGACCGGAGGTTCAAAATGA
55 ATGACCCTGAATATGATGATGGATGCCGGCCGTCCTGTAATAATAACCG
GACAATTCGGACTGATTAAAAAAGCGCCCTTGTGGCGCTTTTTTTATATT
CCCGCCTCCATTTAAAATAAAAAATCCAATCGGATTTCACTATTTAAACT
GGCCATTATCTAAGATGAATCCGATGGAAGCTCGCTGTTTTAACACGCG
TTTTTTAACCTTTTATTGAAAGTCGGTGCTTCTTTGAGCGAACGATCAAA
TTTAAGTGGATTCCCATCAAAAAAATATTCTCAACCTAAAAAAGTTTGT
GTAATACTTGTAACGCTACATGGAGATTAACTCAATCTAGAGGGTATTA
ATAATGAATCGTACTAAACTGGTACTGGGCGCAACTCACTTCACACCCC
GAAGGGGGAAGTTGCCTGACCCTACGATTCCCGCTATTTCATTCACTGA
CCGGAGGTTCAAAATGA
56 ATGAGCATCACGGCGTTATCAGCATCATTTCCTGAGGGGAATATCGCCA
GCCGCTTGTCGCTGCAACATCCTTCACTGTTTTATACCGTGGTTGAACAA
TCTTCGGTGGCGAGCGTGTTGAGTCATCCTGACTAGCTGAGATGAGGGC
TCGCCCCCTCGTCCCGACACTTCCAGATCGCCATAGCGCACAGCGCCTC
GAGCGGTGGTAACGGCGCAGTGGCGGTTTTCATGGCTTGTTATGACTGT
TTTTTTGGCTGTACAGTCTATGCCTCGGGCATCCAAGCAGCAAGCGCGTT
ACGCCGTGGGTCGATGTTTGATGTTATGGAGCAGCAACGATGTTACGCA
GCAGGGCAGTCGCCCTAAAACAAAGTTAAACATCATGAGGGAAGCGGT
GATCGCCGAAGTATCGACTCAACTATCAGAGGTAGTTGGCGTCATCGAG
CGCCATCTCGAACCGACGTTGCTGGCCGTACATTTGTACGGCTCCGCAG
TGGATGGCGCCCTGAAGCCACACAGTGATATTGATTTGCTGGTTACGGT
GACCGTAAGGCTTGATGAAACAACGCGGCGAGCTTTGATCAACGACCTT
TTGGAAACTTCGGCTTCCCCTGGAGAGAGCGAGATTCTCCGCGCTGTAG
AAGTCACCATTGTTGTGCACGACGACATCATTCCGTGGCGTTATCCAGCT
AAGCGCGAACTGCAATTTGGAGAATGGCAGCGCAATGACATTCTTGCAG
GTATCTTCGAGCCAGCCACGATCGACATTGATCTGGCTATCTTGCTGACA
AAAGCAAGAGAACATAGCGTTGCCTTGGTAGGTCCAGCGGCGGAGGAA
CTCTTTGATCCGGTTCCTGAACAGGATCTATTTGAGGCGCTAAATGAAA
CCTTAACGCTATGGAACTTCGCCGCCCGACTGGGCTGGCGATGAGCGAAA
TGTAGTGCTTACGTTGTCCCGCATTTGGTACAGCGCAGTAACCGGCAAA
ATCGCGCCGAAGGATGTCGCTGCCGACTGGGCAATGGAGCGCCTGCCGG
CCCAGTATCAGCCCGTCATACTTGAAGCTAGACAGGCTTATCTTGGACA
AGAAGAAGATCGCTTGGCCTCGCGCGCAGATCAGTTGGAAGAATTTGTC
CACTACGTGAAAGGCGAGATCACCAAGGTAGTCGGCAAATAATGTCTA
ACAATTCGTTCAAGCCGACGCCGCTTCGCGGCGCGGCTTAACTCAAGCG
TTAGATGCACTAAGCACATAATTGCTCACAGCCAAACTATCAGGTCAAG
TCTGCTTTTATTATTTTTAAGCGTGCATAATAAGCCCTACACAAATGGTA
CCCGACCGGTGGTGAATTTAATCTCGCTGACGTGTAGACATTCCCTTATC
CAGACGCTGATCGCCCATCATCGCGGTTCTTTAGATCTCTCGGTCCGCCC
TGATGGCGGCACCTTGCTGACGTTACGCCTGCCGGTACAGCAGGTTATC
ACCGGAGGCTTAAAATGA
57 ATGAGCATCACGGCGTTATCAGCATCATTTCCTGAGGGGAATATCGCCA
GCCGCTTGTCGCTGCAACATCCTTCACTGTTTTATACCGTGGTTGAACAA
TCTTCGGTGGCGAGCGTGTTGAGTCATCCTGACTAGCTGAGATGAGGGC
TCGCCCCCTCGTCCCGACACTTCCAGATCGCCATAGCGCACAGCGCCTC
GAGCGGTGGTAACGGCGCAGTGGCGGTTTTCATGGCTTGTTATGACTGT
TTTTTTGGGGTACAGTCTATGCCTCGGGCATCCAAGCAGCAAGCGCGTT
ACGCCGTGGGTCGATGTTTGATGTTATGGAGCAGCAACGATGTTACGCA
GCAGGGCAGTCGCCCTAAAACAAAGTTAAACATCATGAGGGAAGCGGT
GATCGCCGAAGTATCGACTCAACTATCAGAGGTAGTTGGCGTCATCGAG
CGCCATCTCGAACCGACGTTGCTGGCCGTACATTTGTACGGCTCCGCAG
TGGATGGCGGCCTGAAGCCACACAGTGATATTGATTTGCTGGTTACGGT
GACCGTAAGGCTTGATGAAACAACGCGGCGAGCTTTGATCAACGACCTT
TEGGAAACTTCGGCTTCCCCTGGAGAGAGCGAGATTCTCCGCGCTGTAG
AAGTCACCATTGTTGTGCACGACGACATCATTCCGTGGCGTTATCCAGCT
AAGCGCGAACTGCAATTTGGAGAATGGCAGCGCAATGACATTCTTGCAG
GTATCTTCGAGCCAGCCACGATCGACATTGATCTGGCTATCTTGCTGACA
AAAGCAAGAGAACATAGCGTTGCCTTGGTAGGTCCAGCGGCGGAGGAA
CTCTTTGATCCGGTTCCTGAACAGGATCTATTTGAGGCGCTAAATGAAA
CCTTAACGCTATGGAACTCGCCGCCCGACTGGGCTGGCGATGAGCGAAA
TGTAGTGCTTACGTTGTCCCGCATTTGGTACAGCGCAGTAACCGGCAAA
ATCGCGCCGAAGGATGTCGCTGCCGACTGGGCAATGGAGCGCCTGCCGG
CCCAGTATCAGCCCGTCATACTTGAAGCTAGACAGGCTTATCTTGGACA
AGAAGAAGATCGCTTGGCCTCGCGCGCAGATCAGTTGGAAGAATTTGTC
CACTACGTGAAAGGCGAGATCACCAAGGTAGTCGGCAAATAATGTCTA
ACAATTCGTTCAAGCCGACGCCGCTTCGCCTGCGCGGCTTAACTCAACTCG
TTAGATGCACTAAGCACATAATTGCTCACAGCCAAACTATCAGGTCAAG
TCTGCTTTTATTATTTTTAAGCGTGCATAATAAGCCCTACACAAATGGTA
CCCGACCGGTGGTGAATTTAATCTCGCTGACGTGTAGACATTCCCTTATC
CAGACGCTGATCGCCCATCATCGCGGTTCTTTAGATCTCTCGGTCCGCCC
TGATGGCGGCACCTTGCTGACGTTACGCCTGCCGGTACAGCAGGTTATC
ACCGGAGGCTTAAAATGA
58 CTGATCCTTCAACTCAGCAAAAGTTCGATTTATTCAACAAAGCCACGTT
GTGTCTCAAAATCTCTGATGTTACATTGCACAAGATAAAAATATATCAT
CATGAACAATAAAACTGTCTGCTTACATAAACAGTAATACAAGGGGTGT
TATGAGCCATATTCAACGGGAAACGTCTTGCTCCAGGCCGCGATTAAAT
TCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATG
TCGGGCAATCAGGTGCGACAATCTATCGATTGTATGGGAAGCCCGATGC
GCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTT
ACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTC
CGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATCTGTTACTCACC
ACTGCGATCCCCGGGAAAACAGCATTCCAGGTATTAGAAGAATATCCTG
ATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTT
GCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTC
GTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAG
TGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAA
GAAATGCATAAGCTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGG
TGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTT
GTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGC
CATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGC
TTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTT
CATTTGATGCTCGATGAGTTTTTCTAATAAGCCTTGACCCTACGATTCCC
GCTATTTCATTCACTGACCGGAGGTTCAAAATGA
59 ATGAGCATCACGGCGTTATCAGCATCATTTCCTGAGGGGAATATCGCCA
GCCGCTTGTCGCTGCAACATCCTTCACTGTTTTATACCGTGGTTGAACAA
TCTTCGGTGGCGAGCGTGTTGAGTCATCCTGACTAGCTGAGATGAGGGC
TCGCCCCCTCGTCCCGACACTTCCAGATCGCCATAGCGCACAGCGCCTC
GAGCGGTGGTAACGGCGCAGTGGCGGTTTTCATGGCTTGTTATGACTGT
TTTTTTGGGGTACAGTCTATGCCTCGGGCATCCAAGCAGCAAGCGCGTT
ACGCCGTGGGTCGATGTTTGATGTTATGGAGCAGCAACGATGTTACGCA
GCAGGGCAGTCGCCCTAAAACAAAGTTAAACATCATGAGGGAAGCGGT
GATCGCCGAAGTATCGACTCAACTATCAGAGGTAGTTGGCGTCATCGAG
CGCCATCTCGAACCGACGTTGCTGGCCGTACATTTGTACGGCTCCGCAG
TGGATGGCGGCCTGAAGCCACACAGTGATATTGATTTGCTGGTTACGGT
GACCGTAAGGCTTGATGAAACAACGCGGCGAGCTTTGATCAACGACCTT
TTGGAAACTTCGGCTTCCCCTGGAGAGAGCGAGATTCTCCGCGCTGTAG
AAGTCACCATTGTTGTGCACGACGACATCATTCCGTGGCGTTATCCAGCT
AAGCGCGAACTGCAATTTGGAGAATGGCAGCGCAATGACATTCTTGCAG
GTATCTTCGAGCCAGCCACGATCGACATTGATCTGGCTATCTTGCTGACA
AAAGCAAGAGAACATAGCGTTGCCTTGGTAGGTCCAGCGGCGGAGGAA
CTCTTTGATCCGGTTCCTGAACAGGATCTATTTGAGGCGCTAAATGAAA
CCTTAACGCTATGGAACTCGCCGCCCGACTGGGCTGGCGATGAGCGAAA
TGTAGTGCTTACGTTGTCCCGCATTTGGTACAGCGCAGTAACCGGCAAA
ATCGCGCCGAAGGATGTCGCTGCCGACTGGGCAATGGAGCGCCTGCCGG
CCCAGTATCAGCCCGTCATACTTGAAGCTAGACAGGCTTATCTTGGACA
AGAAGAAGATCGCTTGGCCTCGCGCGCAGATCAGTTGGAAGAATTTGTC
CACTACGTGAAAGGCGAGATCACCAAGGTAGTCGGCAAATAATGTCTA
ACAATTCGTTCAAGCCGACGCCGCTTCGCGGCGCGGCTTAACTCAAGCG
TTAGATGCACTAAGCACATAATTGCTCACAGCCAAACTATCAGGTCAAG
TCTGCTTTTATTATTTTTAAGCGTGCATAATAAGCCCTACACAAATGGTA
CCCGACCGGTGGTGAATTTAATCTCGCTGACGTGTAGACATTCCCTTATC
CAGACGCTGATCGCCCATCATCGCGGTTCTTTAGATCTCTCGGTCCGCCC
TGATGGCGGCACCTTGCTGACGTTACGCCTGCCGGTACAGCAGGTTATC
ACCCTGAGGCTTAAAATGA
60 ATGAGCATCACGGCGTTATCAGCATCATTTCCTGAGGGGAATATCGCCA
GCCGCTTGTCGCTGCAACATCCTTCACTGTTTTATACCGTGGTTGAACAA
TCTTCGGTGGCGAGCGTGTTGAGTCATCCTGACTAGCTGAGATGAGGGC
TCGCCCCCTCGTCCCGACACTTCCAGATCGCCATAGCGCACAGCGCCTC
GAGCGGTGGTAACGGCGCAGTGGCGGTTTTCATGGCTTGTTATGACTGT
TTTTTTGGGGTACAGTCTATGCCTCGGGCATCCAAGCACCAAGCGCGTT
ACGCCGTGGGTCGATGTTTGATGTTATGGAGCAGCAACGATGTTACGCA
GCAGGGCAGTCGCCCTAAAACAAAGTTAAACATCATGAGGGAAGCGGT
GATCGCCGAAGTATCGACTCAACTATCAGAGGTAGTTGGCGTCATCGAG
CGCCATCTCGAACCGACGTTGCTGGCCGTACATTTGTACGGCTCCGCAG
TGGATGGCGGCCTGAAGCCACACAGTGATATTGATTTGCTGGTTACGGT
GACCGTAAGGCTTGTATGAAACAACGCGGCGAGCTTTGATCAACGACCTT
TTGGAAACTTCGGCTTCCCCTGGAGAGAGCGAGATTCTCCGCGCTGTAG
AAGTCACCATTGTTGTGCACGACGACATCATTCCGTGGCGTTATCCAGCT
AAGCGCGAACTGCAATTTGGAGAATGGCAGCGCAATGACATTCTTGCAG
GTATCTTCGAGCCAGCCACGATCGACATTGATCTGGCTATCTTGCTGACA
AAAGCAAGAGAACATAGCGTTGCCTTGGTAGGTCCAGCGGCGGAGGAA
CTCTTTGATCCGGTTCCTGAACAGGATCTATTTGAGGCGCTAAATGAAA
CCTTAACGCTATGGAACTCGCCGCCCGACTGGGCTGGCGATGAGCGAAA
TGTAGTGCTTACGTTGTCCCGCATTTGGTACAGCGCAGTAACCGGCAAA
ATCGCGCCGAAGGATGTCGCTGCCGACTGGGCAATGGAGCGCCTGCCGG
CCCAGTATCAGCCCGTCATACTTGAAGCTAGACAGGCTTATCTTGGACA
AGAAGAAGATCGCTTGGCCTCGCGCGCAGATCAGTTGGAAGAATTTGTC
CACTACGTGAAAGGCGAGATCACCAAGGTAGTCGGCAAATAATGTCTA
ACAATTCGTTCAAGCCGACGCCGCTTCGCGGCGCGGCTTAACTCAAGCG
TTAGATGCACTAAGCACATAATTGCTCACAGCCAAACTATCAGGTCAAG
TCTGCTTTTATTATTTTTAAGCGTGCATAATAAGCCCTACACAAATGGTA
CCCGACCGGTGGTGAATTTAATCTCGCTGACGTGTAGACATTCCCTTATC
CAGACGCTGATCGCCCATCATCGCGGTTCTTTAGATCTCTCGGTCCGCCC
TGATGGCGGCACCTTGCTGACGTTACGCCTGCCGGTACAGCAGGTTATC
ACCGGAGGCTTAAAATGA
61 ATGTTTAACGATCTGATTGGCGATGATGAAACGGATTCGCCGGAAGATG
CGCTTTCTGAGAGCTGGCGCGAATTGTGGCAGGATGCGTTGCAGGAGGA
GGATTCCACGCCCGTGCTGGCGCATCTCTCAGAGGACGATCGCCGCCGC
GTGGTGGCGCTGATTGCCGATTTTCGCAAAGAGTTGGATAAACGCACCA
TTGGCCCGCGAGGGCGGCAGGTACTCGATCACTTAATGCCGCATCTGCT
CAGCGATGTATGCTCGCGCGACGATGCGCCAGTACCGCTGTCACGCCTG
ACGCCGCTGCTCACCGGAATTATTACCCGCACCACTTACCTTGAGCTGCT
AAGTGAATTTCCCGGCGCACTGAAACACCTCATTTCCCTGTGTGCCGCGT
CGCCGATGGTTGCCAGTCAGCTGGCGCGCTACCCGATCCTGCTTGATGA
ATTGCTCGACCCGAATACGCTCTATCAACCGACGGCGATGAATGCCTAT
CGCGATGAGCTGCGCCAATACCTGCTGCGCGTGCCGGAAGATGATGAAG
AGCAACAGCTTGAGGCGCTGCGGCAGTTTAAGCAGGCGCAGTTGCTGCG
CGTGGCGGCGGCGGATATTGCCGGTACGTTGCCAGTAATGAAAGTGAGC
GATCACTTAACCTGGCTGGCGGAAGCGATTATTGATGCGGTGGTGCAGC
AAGCCTGGGGGCAGATGGTGGCGCGTTATGGCCAGCCAACGCATCTGCA
CGATCGCGAAGGGCGCGCTTTTTGCGGTGGTCGGTTATGGCAAGCTGGGC
GGCTGGGAGCTGGGTTACAGCTCCGATCTGGATCTGGTATTCCTGCACG
ACTGCCCGATGGATGTGATGACCGATGGCGAGCGTGAAATCGATGGTCG
CCAGTTCTATTTGCGTCTCGCGCAGCGCGTGATGCACCTGTTTAGCACGC
GCACGTCGTCCGGCATCCTTTATGAAGTTGATGCGCGTCTGCGTCCATCT
GGCGCTGCGGGGATGCTGGTCACTACTACGGAATCGTTCGCCGATTACC
AGCAAAACGAAGCCTGGACGTGGGAACATCAGGCGCTGGCCCGTGCGC
GCGTGGTGTACGGCGATCCGCAACTGACCGCCGAATTTGACGCCATTCG
CCGCGATATTCTGATGACGCCTCGCGACGGCGCAACGCTGCAAACCGAC
GTGCGAGAAATGCGCGAGAAAATGCGTGCCCATCTTGGCAACAAGCAT
AAAGACCGCTTCGATCTGAAAGCCGATGAAGGCGGTATCACCGACATCG
AGTTTATCGCCCAATATCTGGTGCTGCGCTTTGCCCATGACAACTCCGAA
ACTGACGCGCTGGTCGGATAATGTGCGCATTCTCGAAGGGCTGGCGCAA
AACCTGCATCATGGAGGAGCAGGAAGCGCAGGCATTGACGCTGGCGTAC
ACCACATTGCGTGATGAGCTGCACCACCTGGCGCTGCAAGAGTTGCCGG
GACATGTGGCGCTCTCCTGTTTTGTCGCCGAGCGTGCGCTTATTAAAACC
AGCTGGGACAAGTGGCTGGTGGAACCGTGCGCCCCGGCGTAA
Assessment of Genetic Tractability Candidate microbes were characterized based on transformability and genetic tractability. First, optimal carbon source utilization was determined by growth on a small panel of relevant media as well as a growth curve in both nitrogen-free and rich media. Second, the natural antibiotic resistance of each strain was determined through spot-plating and growth in liquid culture containing a panel of antibiotics used as selective markers for mutagenesis. Third, each strain was tested for its transformability through electroporation of a collection of plasmids. The plasmid collection comprises the combinatorial expansion of seven origins of replication, i.e., p15a, pSC101, CloDF, colA, RK2, pBBR1, and pRO1600 and four antibiotic resistance markers, i.e., CmR, KmR, SpecR, and TetR. This systematic evaluation of origin and resistance marker compatibility was used to identify vectors for plasmid-based mutagenesis in candidate microbes.
Example 3: Mutagenesis of Candidate Microbes Lambda-Red Mediated Knockouts Several mutants of candidate microbes were generated using the plasmid pKD46 or a derivative containing a kanamycin resistance marker (Datsenko et al. 2000; PNAS 97(12): 6640-6645). Knockout cassettes were designed with 250 bp homology flanking the target gene and generated via overlap extension PCR. Candidate microbes were transformed with pKD46, cultured in the presence of arabinose to induce Lambda-Red machinery expression, prepped for electroporation, and transformed with the knockout cassettes to produce candidate mutant strains. Four candidate microbes and one laboratory strain, Klebsiella oxytoca M5A1, were used to generate thirteen candidate mutants of the nitrogen fixation regulatory genes nifL, glnB, and amtB, as shown in Table 4.
TABLE 4
List of single knockout mutants created through
Lambda-red mutagenesis
Strain nifL glnB amtB
M5A1 X X X
CI006 X X X
CI010 X X X
CI019 X X
CI028 X X
Oligo-Directed Mutagenesis with Cas9 Selection
Oligo-directed mutagenesis was used to target genomic changes to the rpoB gene in E. coli DH10B, and mutants were selected with a CRISPR-Cas system. A mutagenic oligo (ss1283: “G*T*T*G*ATCAGACCGATGTTCGGACCTTCcaagGTTTCGATCGGACATACGCGAC CGTAGTGGGTCGGGTGTACGTCTCGAACTTCAAAGCC” (SEQ ID NO: 2), where * denotes phosphorothioate bond) was designed to confer rifampicin resistance through a 4-bp mutation to the rpoB gene. Cells containing a plasmid encoding Cas9 were induced for Cas9 expression, prepped for electroporation, and then electroporated with both the mutagenic oligo and a plasmid encoding constitutive expression of a guide RNA (gRNA) that targets Cas9 cleavage of the WT rpoB sequence. Electroporated cells were recovered in nonselective media overnight to allow sufficient segregation of the resulting mutant chromosomes. After plating on selection for the gRNA-encoding plasmid, two out of ten colonies screened were shown to contain the desired mutation, while the rest were shown to be escape mutants generated through protospacer mutation in the gRNA plasmid or Cas9 plasmid loss.
Lambda-Red Mutagenesis with Cas9 Selection
Mutants of candidate microbes CI006 and CI010 were generated via lambda-red mutagenesis with selection by CRISPR-Cas. Knockout cassettes contained an endogenous promoter identified through transcriptional profiling (as described in Example 2 and depicted in Tables 3A-C) and ˜250 bp homology regions flanking the deletion target. CI006 and CI010 were transformed with plasmids encoding the Lambda-red recombination system (exo, beta, gam genes) under control of an arabinose inducible promoter and Cas9 under control of an IPTG inducible promoter. The Red recombination and Cas9 systems were induced in resulting transformants, and strains were prepared for electroporation. Knockout cassettes and a plasmid-encoded selection gRNA were subsequently transformed into the competent cells. After plating on antibiotics selective for both the Cas9 plasmid and the gRNA plasmid, 7 of the 10 colonies screened showed the intended knockout mutation, as shown in FIG. 3.
Example 4: In Vitro Phenotyping of Candidate Molecules The impact of exogenous nitrogen on nitrogenase biosynthesis and activity in various mutants was assessed. The Acetylene Reduction Assay (ARA) (Temme et. al. 2012; 10908): 7085-7090) was used to measure nitrogenase activity in pure culture conditions. Strains were grown in air-tight test tubes, and reduction of acetylene to ethylene was quantified with an Agilent 6890 gas chromatograph. ARA activities of candidate microbes and counterpart candidate mutants grown in nitrogen fixation media supplemented with 0 to 10 mM glutamine are shown in FIGS. 4A-B and FIGS. 10A-C.
Under anaerobic culture conditions, a range of glutamine and ammonia concentrations was tested to quantify impact on nitrogen fixation activity. In wild-type cells, activity quickly diminished as glutamine concentrations increased. However, in a series of initial knock-out mutations, a class of mutation was validated enabling expression of nitrogen fixation genes under concentrations of glutamine that would otherwise shut off activity in wild type. This profile was generated in four different species of diazotrophs, as seen in FIG. 4C. In addition, by rewiring the regulatory network using genetic parts that have been identified, the nitrogen fixation activity level was tuned predictably. This is seen in FIG. 4B, which illustrates strains CM023, CM021, CM015, and CI006. Strain CM023 is an evolved strain low; strain CM021 is an evolved strain high; strain CM015 is an evolved strain mid; strain CI006 is a wild-type (strain 2). Ammonia excreted into culture supernatants was tested using a enzymatic-based assay (MEGAZYME). The assay measures the amount of NADPH consumed in the absorbance of 340 nm. The assay was conducted on bacterial cultures grown in nitrogen-free, anaerobic environment with a starting density of 1E9 CFU/ml. Across a panel of six evolved strains, one strain excreted up to 100 μM of ammonia over a course of a 48 hour period, as seen in FIG. 4D. Further, a double mutant exhibited higher ammonia excretion than the single mutant from which it was derived, as seen in FIG. 11. This demonstrates a microbial capacity to produce ammonia in excess of its physiological needs.
Transcription Profiling of Pure Cultures Transcriptional activity of CI006 was measured using the Nanostring Elements platform. Cells were grown in nitrogen-free media and 10E8 cells were collected after 4 hours incubation. Total RNA was extracted using the Qiagen RNeasy kit. Purified RNA was submitted to Core Diagnostics in Palo Alto, Calif., for probe hybridization and Digital Analyzer analysis, as shown in FIG. 5.
Example 5: In Planta Phenotyping of Candidate Microbes Colonization of Plants by Candidate Microbes Colonization of desired host plants by a candidate microbe was quantified through short-term plant growth experiments. Corn plants were inoculated with strains expressing RFP either from a plasmid or from a Tn5-integrated RFP expression cassette. Plants were grown in both sterilized sand and nonsterile peat medium, and inoculation was performed by pipetting 1 mL of cell culture directly over the emerging plant coleoptile three days post-germination. Plasmids were maintained by watering plants with a solution containing the appropriate antibiotic. After three weeks, plant roots were collected, rinsed three times in sterile water to remove visible soil, and split into two samples. One root sample was analyzed via fluorescence microscopy to identify localization patterns of candidate microbes. Microscopy was performed on 10 mm lengths of the finest intact plant roots, as shown in FIG. 6.
A second quantitative method for assessing colonization was developed. A quantitative PCR assay was performed on whole DNA preparations from the roots of plants inoculated with the endophytes. Seeds of corn (Dekalb DKC-66-40) were germinated in previously autoclaved sand in a 2.5 inch by 2.5 inch by 10 inch pot. One day after planting, 1 ml of endophyte overnight culture (SOB media) was drenched right at the spot of where the seed was located. 1 mL of this overnight culture is roughly equivalent to about 10̂9 cfu, varying within 3-fold of each other, depending on which strain is being used. Each seedling was fertilized 3× weekly with 50 mL modified Hoagland's solution supplemented with either 2.5 mM or 0.25 mM ammonium nitrate. At four weeks after planting, root samples were collected for DNA extraction. Soil debris were washed away using pressurized water spray. These tissue samples were then homogenized using QIAGEN Tissuelyzer and the DNA was then extracted using QIAmp DNA Mini Kit (QIAGEN) according to the recommended protocol. qPCR assay was performed using Stratagene Mx3005P RT-PCR on these DNA extracts using primers that were designed (using NCBI's Primer BLAST) to be specific to a loci in each of the endophyte's genome. The presence of the genome copies of the endophytes was quantified. To further confirm the identity of the endophytes, the PCR amplification products were sequenced and are confirmed to have the correct sequence. The summary of the colonization profile of strain CI006 and CI008 from candidate microbes are presented in Table 5. Colonization rate as high as 10̂7×cfu/g fw of root was demonstrated in strain CI008.
TABLE 5
Colonization of corn as measured by qPCR
Strain Colonization Rate (CFU/g fw)
CI006 1.45 × 10{circumflex over ( )}5
CI008 1.24 × 10{circumflex over ( )}7
In Planta RNA Profiling Biosynthesis of nif pathway components in planta was estimated by measuring the transcription of nif genes. Total RNA was obtained from root plant tissue of CI006 inoculated plants (planting methods as described previously). RNA extraction was performed using RNEasy Mini Kit according to the recommended protocol (QIAGEN). Total RNA from these plant tissues was then assayed using Nanostring Elements kits (NanoString Technologies, Inc.) using probes that were specific to the nif genes in the genome of strain CI006. The data of nif gene expression in planta is summarized in Table 6. Expression of nifH genes was detected in plants inoculated by CM013 strains whereas nifH expression was not detectable in CI006 inoculated plants. Strain CM013 is a derivative of strain CI006 in which the nifL gene has been knocked out.
Highly expressed genes of CM011, ranked by transcripts per kilobase million (TPM), were measured in planta under fertilized condition. The promoters controlling expression of some of these highly expressed genes were used as templates for homologous recombination into targeted nitrogen fixation and assimilation loci. RNA samples from greenhouse grown CM011 inoculated plant were extracted, rRNA removed using Ribo-Zero kit, sequenced using Illumina's Truseq platform and mapped back to the genome of CM011. Highly expressed genes from CM011 are listed in Table 7.
TABLE 6
Expression of nifH in planta
Strains Relative Transcript Expression
CI006 9.4
CM013 103.25
TABLE 7
TPM
Raw (Transcripts
Gene Read Per Kilobase
Gene Name Location Direction Count Million)
rpsH CDS 18196-18588 reverse 4841.5 27206.4
rplQ CDS 11650-12039 reverse 4333 24536.2
rpsJ CDS 25013-25324 reverse 3423 24229
rplV CDS 21946-22278 reverse 3367.5 22333
rpsN CDS 18622-18927 reverse 2792 20150.1
rplN CDS 19820-20191 reverse 3317 19691.8
rplF CDS 17649-18182 reverse 4504.5 18628.9
rpsD CDS 13095-13715 reverse 5091.5 18106.6
rpmF CDS 8326-8493 forward 1363.5 17923.8
rplW CDS 23429-23731 reverse 2252 16413.8
rpsM CDS 14153-14509 reverse 2269 14036.2
rplR CDS 17286-17639 reverse 2243.5 13996.1
rplC CDS 24350-24979 reverse 3985 13969.2
rplK CDS 25526-25954 reverse 2648.5 13634.1
rplP CDS 20807-21217 reverse 2423 13019.5
rplX CDS 19495-19809 reverse 1824 12787.8
rpsQ CDS 20362-20616 reverse 1460.5 12648.7
bhsA 3 CDS 79720-79977 reverse 1464 12531.5
rpmC CDS 20616-20807 reverse 998.5 11485
rpoA CDS 12080-13069 reverse 4855 10830.2
rplD CDS 23728-24333 reverse 2916.5 10628.5
bhsA 1 CDS 78883-79140 reverse 1068 9141.9
rpsS CDS 22293-22571 reverse 1138.5 9011.8
rpmA CDS 2210-2467 forward 1028.5 8803.7
rpmD CDS 16585-16764 reverse 694.5 8520.8
rplB CDS 22586-23410 reverse 3132 8384
rpsC CDS 21230-21928 reverse 2574.5 8133.9
rplE CDS 18941-19480 reverse 1972.5 8066.9
rplO CDS 16147-16581 reverse 1551 7874.2
preprotein translocase 14808-16139 reverse 4657 7721.2
subunit SecY CDS
rpsE CDS 16771-17271 reverse 1671.5 7368
rpsK CDS 13746-14135 reverse 1223.5 6928.2
tufA CDS 27318-28229 reverse 2850 6901.3
rpmI CDS 38574-38771 forward 615 6859.5
rplU CDS 1880-2191 forward 935.5 6621.7
rplT CDS 38814-39170 forward 1045 6464.4
bhsA 2 CDS 79293-79550 reverse 754 6454.1
rpmB CDS 8391-8627 reverse 682 6355.1
rplJ CDS 23983-24480 reverse 1408 6243.9
fusA 2 CDS 481-2595 reverse 5832 6089.6
rpsA CDS 25062-26771 reverse 4613 5957.6
rpmJ CDS 14658-14774 reverse 314 5926.9
rpsR CDS 52990-53217 forward 603 5840.7
rpsG CDS 2692-3162 reverse 1243 5828.2
rpsI CDS 11354-11746 reverse 980.5 5509.8
cspC 1 CDS 8091-8300 reverse 509 5352.8
rpsF CDS 52270-52662 forward 916 5147.4
rpsT CDS 55208-55471 reverse 602 5035.9
infC CDS 38128-38478 forward 755 4750.3
cspG CDS 30148-30360 forward 446 4624.2
15N Assay The primary method for demonstrating fixation uses the nitrogen isotope 15N, which is found in the atmosphere at a set rate relative to 14N. By supplementing either fertilizer or atmosphere with enriched levels of 15N, one can observe fixation either directly, in heightened amounts of 15N fixed from an atmosphere supplemented with 15N2 gas (Yoshida 1980), or inversely, through dilution of enriched fertilizer by atmospheric N2 gas in plant tissues (Iniguez 2004). The dilution method allows for the observation of cumulative fixed nitrogen over the course of plant growth, while the 15N2 gas method is restricted to measuring the fixation that occurs over the short interval that a plant can be grown in a contained atmosphere (rate measurement). Therefore, the gas method is superior in specificity (as any elevated 15N2 levels in the plant above the atmospheric rate can be attributed unambiguously to fixation) but cannot show cumulative activity.
Both types of assay has been performed to measure fixation activity of improved strains relative to wild-type and uninoculated corn plants, and elevated fixation rates were observed in planta for several of the improved strains (FIG. 12, FIG. 14A, and FIG. 14B). These assays are instrumental in demonstrating that the activity of the strains observed in vitro translates to in vivo results. Furthermore, these assays allow measurement of the impact of fertilizer on strain activity, suggesting suitable functionality in an agricultural setting. Similar results were observed when setaria plants were inoculated with wild-type and improved strains (FIG. 13). In planta fixation activity shown in FIGS. 14A-14C is further backed up by transcriptomic data. Evolved strains exhibit increased nifH transcript level relative to wild-type counterparts. Furthermore, the microbe derived nitrogen level in planta is also correlated with the colonization level on a plant by plant basis. These results (FIG. 12, FIG. 13, FIGS. 14A-14C, FIG. 15A, and FIG. 15B) support the hypothesis that the microbe, through the improved regulation of the nif gene cluster, is the likely reason for the increase in atmospheric derived nitrogen seen in the plant tissue. In addition to measuring fixation directly, the impact of inoculating plants with the improved strains in a nitrogen-stressed plant biomass assay was measured. While plant biomass may be related to many possible microbe interactions with the plant, one would expect that the addition of fixed nitrogen would impact the plant phenotype when nitrogen is limited. Inoculated plants were grown in the complete absence of nitrogen, and significant increases in leaf area, shoot fresh and dry weight, and root fresh and dry weight in inoculated plants relative to untreated controls was observed (FIG. 14C). Although these differences cannot be attributed to nitrogen fixation exclusively, they support the conclusion that the improved strains are actively providing nitrogen to the plant. Corn and setaria plants were grown and inoculated as described above. Fertilizer comprising 1.2% 15N was regularly supplied to plants via watering. Nitrogen fixation by microbes was quantified by measuring the 15N level in the plant tissue. Fourth leaf tissue was collected and dried at 4 weeks after planting. Dried leaf samples were homogenized using beads (QIAGEN Tissuelyzer) and aliquoted out into tin capsules for IRMS (MBL Stable Isotope Laboratory at The Ecosystems Center, Woods Hole, Mass.). Nitrogen derived from the atmosphere (NDFA) was calculated, and nitrogen production by CI050 and CM002 are shown in FIG. 7.
Phytohormone Production Assay The dwarf tomato (Solanum lycopersicum) cultivar ‘Micro-Tom’ has previously been used to study the influence of indole-3-acetic acid on fruit ripening through an in vitro assay (Cohen 1996; J Am Soc Hortic Sci 121: 520-524). To evaluate phytohormone production and secretion by candidate microbes, a plate-based screening assay using immature Micro-Tom fruit was developed. Twelve-well tissue culture test plates were prepared by filling wells with agar medium, allowing it to solidify, and spotting 10 uL of overnight microbial cultures onto the agar surface, as shown in FIG. 8. Wells with agar containing increasing amounts of gibberellic acid (GA) but no bacterial culture were used as a positive control and standards. Flowers one day post-anthesis abscised from growing Micro-Tom plants were inserted, stem-first, into the agar at the point of the bacterial spot culture. These flowers were monitored for 2-3 weeks, after which the fruits were harvested and weighed. An increase in plant fruit mass across several replicates indicates production of plant hormone by the inoculant microbe, as shown in FIG. 9.
Example 6: Cyclical Host-Microbe Evolution Corn plants were inoculated with CM013 and grown 4 weeks to approximately the V5 growth stage. Those demonstrating improved nitrogen accumulation from microbial sources via 15N analysis were uprooted, and roots were washed using pressurized water to remove bulk soil. A 0.25 g section of root was cut and rinsed in PBS solution to remove fine soil particles and non-adherent microbes. Tissue samples were homogenized using 3 mm steel beads in QIAGEN TissueLyser II. The homogenate was diluted and plated on SOB agar media. Single colonies were resuspended in liquid media and subjected to PCR analysis of 16s rDNA and mutations unique to the inoculating strain. The process of microbe isolation, mutagenesis, inoculation, and re-isolation can be repeated iteratively to improve microbial traits, plant traits, and the colonization capability of the microbe.
Example 7: Compatibility Across Geography The ability of the improved microbes to colonize an inoculated plant is critical to the success of the plant under field conditions. While the described isolation methods are designed to select from soil microbes that may have a close relationship with crop plants such as corn, many strains may not colonize effectively across a range of plant genotypes, environments, soil types, or inoculation conditions. Since colonization is a complex process requiring a range of interactions between a microbial strain and host plant, screening for colonization competence has become a central method for selecting priority strains for further development. Early efforts to assess colonization used fluorescent tagging of strains, which was effective but time-consuming and not scalable on a per-strain basis. As colonization activity is not amenable to straightforward improvement, it is imperative that potential product candidates are selected from strains that are natural colonizers.
An assay was designed to test for robust colonization of the wild-type strains in any given host plant using qPCR and primers designed to be strain-specific in a community sample. This assay is intended to rapidly measure the colonization rate of the microbes from corn tissue samples. Initial tests using strains assessed as probable colonizers using fluorescence microscopy and plate-based techniques indicated that a qPCR approach would be both quantitative and scalable.
A typical assay is performed as follows: Plants, mostly varieties of maize and wheat, are grown in a peat potting mix in the greenhouse in replicates of six per strain. At four or five days after planting, a 1 mL drench of early stationary phase cultures of bacteria diluted to an OD590 of 0.6-1.0 (approximately 5E+08 CFU/mL) is pipetted over the emerging coleoptile. The plants are watered with tap water only and allowed to grow for four weeks before sampling, at which time, the plants are uprooted and the roots washed thoroughly to remove most peat residues. Samples of clean root are excised and homogenized to create a slurry of plant cell debris and associated bacterial cells. We developed a high-throughput DNA extraction protocol that effectively produced a mixture of plant and bacterial DNA to use as template for qPCR. Based on bacterial cell spike-in experiments, this DNA extraction process provides a quantitative bacterial DNA sample relative to the fresh weight of the roots. Each strain is assessed using strain-specific primers designed using Primer BLAST (Ye 2012) and compared to background amplification from uninoculated plants. Since some primers exhibit off-target amplification in uninoculated plants, colonization is determined either by presence of amplification or elevated amplification of the correct product compared to the background level.
This assay was used to measure the compatibility of the microbial product across different soil geography. Field soil qualities and field conditions can have a huge influence on the effect of a microbial product. Soil pH, water retention capacity, and competitive microbes are only a few examples of factors in soil that can affect inoculum survival and colonization ability. A colonization assay was performed using three diverse soil types sampled from agricultural fields in California as the plant growth medium (FIG. 16A). An intermediate inoculation density was used to approximate realistic agricultural conditions. Within 3 weeks, Strain 5 colonized all plants at 1E+06 to 1E+07 CFU/g FW. After 7 weeks of plant growth, an evolved version of Strain 1 exhibited high colonization rates (1E+06 CFU/g FW) in all soil types. (FIG. 16B).
Additionally, to assess colonization in the complexity of field conditions, a 1-acre field trial in San Luis Obispo in June of 2015 was initiated to assess the impacts and colonization of seven of the wild-type strains in two varieties of field corn. Agronomic design and execution of the trial was performed by a contract field research organization, Pacific Ag Research. For inoculation, the same peat culture seed coating technique tested in the inoculation methods experiment was employed. During the course of the growing season, plant samples were collected to assess for colonization in the root and stem interior. Samples were collected from three replicate plots of each treatment at four and eight weeks after planting, and from all six reps of each treatment shortly before harvest at 16 weeks. Additional samples were collected from all six replicate plots of treatments inoculated with Strain 1 and Strain 2, as well as untreated controls, at 12 weeks. Numbers of cells per gram fresh weight of washed roots were assessed as with other colonization assays with qPCR and strain-specific primers. Two strains, Strain 1 and Strain 2, showed consistent and widespread root colonization that peaked at 12 weeks and then declined precipitously (FIG. 16C). While Strain 2 appeared to be present in numbers an order of magnitude lower than Strain 1, it was found in more consistent numbers from plant to plant. No strains appeared to effectively colonize the stem interior. In support of the qPCR colonization data, both strains were successfully re-isolated from the root samples using plating and 16S sequencing to identify isolates of matching sequence.
Example 8: Microbe Breeding Examples of microbe breeding can be summarized in the schematic of FIG. 17A. FIG. 17A depicts microbe breeding wherein the composition of the microbiome can be first measured and a species of interest is identified. The metabolism of the microbiome can be mapped and linked to genetics. Afterwards, a targeted genetic variation can be introduced using methods including, but not limited to, conjugation and recombination, chemical mutagenesis, adaptive evolution, and gene editing. Derivative microbes are used to inoculate crops. In some examples, the crops with the best phenotypes are selected.
As provided in FIG. 17A, the composition of the microbiome can be first measured and a species of interest is identified. FIG. 17B depicts an expanded view of the measurement of the microbiome step. The metabolism of the microbiome can be mapped and linked to genetics. The metabolism of nitrogen can involve the entrance of ammonia (NW) from the rhizosphere into the cytosol of the bacteria via the AmtB transporter. Ammonia and L-glutamate (L-Glu) are catalyzed by glutamine synthetase and ATP into glutamine. Glutamine can lead to the formation of biomass (plant growth), and it can also inhibit expression of the nif operon. Afterwards, a targeted genetic variation can be introduced using methods including, but not limited to, conjugation and recombination, chemical mutagenesis, adaptive evolution, and gene editing. Derivative microbes are used to inoculate crops. The crops with the best phenotypes are selected.
Example 9: Field Trials with Microbes of the Disclosure—Summer 2016 In order to evaluate the efficacy of strains of the present disclosure on corn growth and productivity under varying nitrogen regimes, field trials were conducted.
Trials were conducted with (1) seven subplot treatments of six strains plus the control—four main plots comprised 0, 15, 85, and 100% of maximum return to nitrogen (MRTN) with local verification. The control (UTC only) was conducted with 10 100% MRTN plus, 5, 10, or 15 pounds. Treatments had four replications.
Plots of corn (minimum) were 4 rows of 30 feet in length, with 124 plots per location. All observations were taken from the center two rows of the plots, and all destructive sampling was taken from the outside rows. Seed samples were refrigerated until 1.5 to 2 hours prior to use.
Local Agricultural Practice:
The seed was a commercial corn without conventional fungicide and insecticide treatment. All seed treatments were applied by a single seed treatment specialist to assure uniformity. Planting date, seeding rate, weed/insect management, etc. were left to local agricultural practices. With the exception of fungicide applications, standard management practices were followed.
Soil Characterization:
Soil texture and soil fertility were evaluated. Soil samples were pre-planted for each replicate to insure residual nitrate levels lower than 50 lbs/Ac. Soil cores were taken from 0 cm to 30 cm. The soil was further characterized for pH, CEC, total K and P.
Assessments:
The initial plant population was assessed 14 days after planting (DAP)/acre, and were further assessed for: (1) vigor (1 to 10 scale, w/10=excellent) 14 DAP & V10; (2) recordation of disease ratings any time symptoms are evident in the plots; (3) record any differences in lodging if lodging occurs in the plots; (4) yield (Bu/acre), adjusted to standard moisture pet; (5) test weight; and (6) grain moisture percentage.
Sampling Requirements:
The soil was sampled at three timepoints (prior to trial initiation, V10-VT, 1 week post-harvest). All six locations and all plots were sampled at 10 grams per sample (124 plots×3 timepoints×6 locations).
Colonization Sampling:
Colonization samples were collected at two timepoints (V10 and VT) for five locations and six timepoints (V4, V8, V10, VT, R5, and Post-Harvest). Samples were collected as follows: (1) from 0% and 100% MRTN, 60 plots per location; (2) 4 plants per plot randomly selected from the outside rows; (3) 5 grams of root, 8 inches of stalk, and top three leaves—bagged and IDed each separately—12/bags per plot; (4) five locations (60 plots×2 timepoints×12 bags/plot); and one location (60 plots×6 timepoints×12 bags/plot. See, FIG. 17C illustrating colonization sampling.
Normalized difference vegetation index (NDVI) determination was made using a Greenseeker instrument at two timepoints (V4-V6 and VT). Assessed each plot at all six locations (124 plots×2 timepoints×6 locations).
Root analysis was performed with Win Rhizo from one location that best illustrated treatment differentiation. Ten plants per plot were randomly sampled (5 adjacent from each outside row; V3-V4 stage plants were preferred) and gently washed to remove as much dirt as reasonable. Ten roots were placed in a plastic bag and labelled. Analyzed with WinRhizo Root Analysis.
Stalk Characteristics were measured at all six locations between R2 and R5. The stalk diameter of ten plants per plot at the 6″ height were recorded, as was the length of the first internode above the 6″ mark. Ten plants were monitored; five consecutive plants from the center of the two inside rows. Six locations were evaluated (124 plots×2 measures×6 locations).
The tissue nitrates were analyzed front all plots and all locations. An 8″ segment of stalk beginning 6″ above the soil when the corn is between one and three weeks after black layer formation; leaf sheaths were removed. All locations and plots were evaluated (6 locations×124 plots).
The following weather data was recorded for all locations from planting to harvest: daily maximum and minimum temperatures, soil temperature at seeding, daily rainfall plus irrigation (if applied), and any unusual weather events such as excessive rain, wind, cold, or heat.
Yield data across all six locations is presented in Table 8. Nitrogen rate had a significant impact on yield, but strains across nitrogen rates did not. However, at the lowest nitrogen rate, strains CI006, CM029, and CI019 numerically out-yielded the UTC by 4 to 6 bu/acre. Yield was also numerically increased 2 to 4 bu/acre by strains CM029, CI019, and CM081 at 15% MRTN.
TABLE 8
Yield data across all six locations
Stalk
Vigor_ Vigor_ Diameter Internode NDVI_ NDVI_
MRTN % YLD (bu) E L (mm) Length (in) Veg Rep
0 143.9 7.0 5.7 18.87 7.18 64.0 70.6
15 165.9 7.2 6.3 19.27 7.28 65.8 72.5
85 196.6 7.1 7.1 20.00 7.31 67.1 74.3
100 197.3 7.2 7.2 20.23 7.37 66.3 72.4
Stalk
Vigor_ Vigor_ Diameter Internode NDVI_ NDVI_
Strain YLD (bu) E L (mm) Length (in) Veg Rep
CI006 (1) 176.6 7.2 6.6 19.56 18.78 66.1 72.3
CM029 (2) 176.5 7.1 6.5 19.54 18.61 65.4 71.9
CM038 (3) 175.5 7.2 6.5 19.58 18.69 65.7 72.8
CI019 (4) 176.0 7.1 6.6 19.51 18.69 65.5 72.9
CM081 (5) 176.2 7.1 6.6 19.57 18.69 65.8 73.1
CM029/ 174.3 7.1 6.6 19.83 18.79 66.2 72.5
CM081(6)
UTC (7) 176.4 7.1 6.6 19.54 18.71 65.9 71.7
Stalk
MRTN/ Vigor_ Vigor_ Diameter Internode NDVI_ NDVI_
Strain YLD (bu) E L (mm) Length (in) Veg Rep
0 1 145.6 7.0 5.6 19.07 7.12 63.5 70.3
0 2 147.0 7.0 5.5 18.74 7.16 64.4 70.4
0 3 143.9 7.0 5.5 18.83 7.37 64.6 70.5
0 4 146.0 6.9 5.7 18.86 7.15 63.4 70.7
0 5 141.7 7.0 5.8 18.82 7.05 63.6 70.9
0 6 142.2 7.2 5.8 19.12 7.09 64.7 69.9
0 7 141.2 7.0 5.8 18.64 7.32 64.0 71.4
15 1 164.2 7.3 6.1 19.09 7.21 66.1 71.5
15 2 167.3 7.2 6.3 19.32 7.29 65.5 72.7
15 3 165.6 7.3 6.3 19.36 7.23 64.8 72.5
15 4 167.9 7.3 6.4 19.31 7.51 66.1 72.3
15 5 169.3 7.2 6.2 19.05 7.32 66.0 72.8
15 6 161.9 7.1 6.3 19.45 7.20 66.2 72.2
15 7 165.1 7.3 6.4 19.30 7.18 66.0 73.3
85 1 199.4 7.3 7.2 19.70 7.32 67.2 74.0
85 2 195.1 7.1 7.2 19.99 7.09 66.5 74.4
85 3 195.0 7.0 7.0 20.05 7.26 67.3 74.6
85 4 195.6 7.2 7.1 20.04 7.29 66.4 74.4
85 5 196.4 7.2 7.0 19.87 7.39 67.3 74.5
85 6 195.1 7.0 6.9 20.35 7.34 67.4 74.4
85 7 199.5 6.9 7.2 19.97 7.48 67.4 74.1
100 1 197.1 7.2 7.3 20.38 7.68 67.5 73.4
100 2 196.5 7.0 7.1 20.11 7.21 65.3 70.2
100 3 197.6 7.5 7.3 20.08 7.42 66.3 73.4
100 4 194.6 7.1 7.1 19.83 7.40 66.1 74.1
100 5 197.4 7.2 7.3 20.53 7.36 66.2 74.3
100 6 198.1 7.2 7.4 20.40 7.16 66.6 73.6
100 7 199.9 7.2 7.2 20.26 7.39 66.2 68.1
Another analysis approach is presented in Table 9. The table comprises the four locations where the response to nitrogen was the greatest which suggests that available residual nitrogen was lowest. This approach does not alter the assessment that the nitrogen rate significantly impacted yield, which strains did not when averaged across all nitrogen rates. However, the numerical yield advantage at the lowest N rate is more pronounced for all strains, particularly CI006, CM029, and CM029/CM081 where yields were increased from 8 to 10 bu/acre. At 15% MRTN, strain CM081 outyielded UTC by 5 bu.
TABLE 9
Yield data across four locations
4 Location Average - SGS, AgIdea, Bennett, RFR
Stalk Internode
YLD Diameter Length
(bu) Vigor_E Vigor_L (mm) (in)
Table 16
MRTN %
0 137.8 7.3 5.84 18.10 5.36
15 162.1 7.5 6.63 18.75 5.40
85 199.2 7.4 7.93 19.58 5.62
100 203.5 7.5 8.14 19.83 5.65
Strain
CI006 (1) 175.4 7.5 7.08 19.03 5.59
CM029 (2) 176.1 7.4 7.08 19.09 5.39
CM038 (3) 175.3 7.5 7.05 19.01 5.59
CI019 (4) 174.8 7.5 7.16 19.02 5.45
CM081 (5) 176.7 7.4 7.16 19.00 5.53
CM029/ 175.1 7.4 7.17 19.33 5.46
CM081 (6)
UTC (7) 176.0 7.3 7.27 18.98 5.55
MRTN/Strain
0 1 140.0 7.3 5.69 18.32 5.28
0 2 140.7 7.4 5.69 18.19 5.23
0 3 135.5 7.3 5.63 17.95 5.50
0 4 138.8 7.3 5.81 17.99 5.36
0 5 136.3 7.3 6.06 18.05 5.34
0 6 141.4 7.5 6.00 18.43 5.30
0 7 131.9 7.3 6.00 17.75 5.48
15 1 158.0 7.6 6.44 18.53 5.34
15 2 164.1 7.5 6.56 19.13 5.42
15 3 164.3 7.6 6.63 18.68 5.51
15 4 163.5 7.6 6.81 18.84 5.34
15 5 166.8 7.5 6.63 18.60 5.39
15 6 156.6 7.4 6.56 18.86 5.41
15 7 161.3 7.5 6.81 18.62 5.42
85 1 199.4 7.6 8.00 19.15 5.63
85 2 199.0 7.4 8.09 19.49 5.46
85 3 198.2 7.4 7.75 19.88 5.69
85 4 196.8 7.4 8.00 19.65 5.60
85 5 199.5 7.4 7.75 19.26 5.70
85 6 198.7 7.3 7.81 19.99 5.61
85 7 202.8 7.2 8.13 19.66 5.65
100 1 204.3 7.4 8.19 20.11 6.10
100 2 200.6 7.3 8.00 19.53 5.46
100 3 203.3 7.7 8.19 19.55 5.67
100 4 200.2 7.6 8.00 19.59 5.49
100 5 203.9 7.4 8.19 20.08 5.68
100 6 203.8 7.5 8.31 20.05 5.52
100 7 208.1 7.4 8.13 19.90 5.63
The results from the field trial are also illustrated in FIGS. 21-27. The results indicate that the microbes of the disclosure are able to increase plant yield, which points to the ability of the taught microbes to increase nitrogen fixation in an important agricultural crop, i.e. corn.
The field based results further validate the disclosed methods of non-intergenerially modifying the genome of selected microbial strains, in order to bring about agriculturally relevant results in a field setting when applying said engineered strains to a crop.
FIG. 18 depicts the lineage of modified strains that were derived from strain CI006 (WT Kosakonia sacchari). The field data demonstrates that an engineered derivative of the CI006 WT strain, i.e. CM029, is able to bring about numerically relevant results in a field setting. For example, Table 8 illustrates that at 0% MRTN CM029 yielded 147.0 bu/acre compared to untreated control at 141.2 bu/acre (an increase of 5.8 bu/acre). Table 8 also illustrates that at 15% MRTN CM029 yielded 167.3 bu/acre compared to untreated control at 165.1 bu/acre (an increase of 2.2 bu/acre). Table 9 is supportive of these conclusions and illustrates that at 0% MRTN CM029 yielded 140.7 bu/acre compared to untreated control at 131.9 bu/acre (an increase of 8.8 bu/acre). Table 9 also illustrates that at 15% MRTN CM029 yielded 164.1 bu/acre compared to untreated control at 161.3 bu/acre (an increase of 2.8 bu/acre).
FIG. 19 depicts the lineage of modified strains that were derived from strain CI019 (WT Rahnella aquatilis). The field data demonstrates that an engineered derivative of the CI019 WT strain, i.e. CM081, is able to bring about numerically relevant results in a field setting. For example, Table 8 illustrates that at 15% MRTN CM081 yielded 169.3 bu/acre compared to untreated control at 165.1 bu/acre (an increase of 4.2 bu/acre). Table 9 is supportive of these conclusions and illustrates that at 0% MRTN CM081 yielded 136.3 bu/acre compared to untreated control at 131.9 bu/acre (an increase of 4.4 bu/acre). Table 9 also illustrates that at 15% MRTN CM081 yielded 166.8 bu/acre compared to untreated control at 161.3 bu/acre (an increase of 5.5 bu/acre).
Further, one can see in Table 9 that the combination of CM029/CM081 at 0% MRTN yielded 141.4 bu/acre compared to untreated control at 131.9 bu/acre (an increase of 9.5 bu/acre).
Example 10: Field Trials with Microbes of the Disclosure A diversity of nitrogen fixing bacteria can be found in nature, including in agricultural soils. However, the potential of a microbe to provide sufficient nitrogen to crops to allow decreased fertilizer use may be limited by repression of nitrogenase genes in fertilized soils as well as low abundance in close association with crop roots. Identification, isolation and breeding of microbes that closely associate with key commercial crops might disrupt and improve the regulatory networks linking nitrogen sensing and nitrogen fixation and unlock significant nitrogen contributions by crop-associated microbes. To this end, nitrogen fixing microbes that associate with and colonize the root system of corn were identified.
Root samples from corn plants grown in agronomically relevant soils were collected, and microbial populations extracted from the rhizosphere and endosphere. Genomic DNA from these samples was extracted, followed by 16S amplicon sequencing to profile the community composition. A Kosakonia sacchari microbe (strain PBC6.1) was isolated and classified through 16S rRNA and whole genome sequencing. This is a particularly interesting nitrogen fixer capable of colonizing to nearly 21% abundance of the root-associated microbiota (FIG. 30). To assess strain sensitivity to exogenous nitrogen, nitrogen fixation rates in pure culture were measured with the classical acetylene reduction assay (ARA) and varying levels of glutamine supplementation. The species exhibited a high level of nitrogen fixing activity in nitrogen-free media, yet exogenous fixed nitrogen repressed nif gene expression and nitrogenase activity (Strain PBC6.1, FIGS. 28C and 28D). Additionally, when released ammonia was measured in the supernatant of PBC6.1 grown in nitrogen-fixing conditions, very little release of fixed nitrogen could be detected (FIG. 28E).
We hypothesized that PBC6.1 could be a significant contributor of fixed nitrogen in fertilized fields if regulatory networks controlling nitrogen metabolism were rewired to allow optimal nitrogenase expression and ammonia release in the presence of fixed nitrogen. Sufficient genetic diversity should exist within the PBC6.1 genome to enable broad phenotypic remodeling without the insertion of transgenes or synthetic regulatory elements. The isolated strain has a genome of at least 5.4 Mbp and a canonical nitrogen fixation gene cluster. Related nitrogen metabolism pathways in PBC6.1 are similar to those of the model organism for nitrogen fixation, Klebsiella oxytoca m5al.
Several gene regulatory network nodes were identified which may augment nitrogen fixation and subsequent transfer to a host plant, particularly in high exogenous concentrations of fixed nitrogen (FIG. 28A). The niFL operon directly regulates the rest of the nif cluster through transcriptional activation by NifA and nitrogen- and oxygen-dependent repression of NifA by NifL. Disruption of tuff, can abolish inhibition of NifA and improve nif expression in the presence of both oxygen and exogenous fixed nitrogen. Furthermore, expressing nifA under the control of a nitrogen-independent promoter may decouple nitrogenase biosynthesis from regulation by the NtrB/NtrC nitrogen sensing complex. The assimilation of fixed nitrogen by the microbe to glutamine by glutamine synthetase (GS) is reversibly regulated by the two-domain adenylyltransferase (ATase) enzyme GlnE through the adenylylation and deadenylylation of GS to attenuate and restore activity, respectively. Truncation of the GlnE protein to delete its adenylyl-removing (AR) domain may lead to constitutively adenylylated glutamine synthetase, limiting ammonia assimilation by the microbe and increasing intra- and extracellular ammonia. Finally, reducing expression of AmtB, the transporter responsible for uptake of ammonia, could lead to greater extracellular ammonia. To generate rationally designed microbial phenotypes without the use of transgenes, two approaches were employed: creating markerless deletions of genomic sequences encoding protein domains or whole genes, and rewiring regulatory networks by intragenomic promoter rearrangement. Through an iterative mutagenesis process, several non-transgenic derivative strains of PBC6.1 were generated (Table 10).
TABLE 10
List of isolated and derivative K. sacchari strains used in this work.
Prm, promoter sequence derived from the PBC6.1 genome; ΔglnEAR1
and ΔglnEAR2, different truncated versions of glnE gene
removing the adenylyl-removing domain sequence.
Strain ID Genotype
PBC6.1 WT
PBC6.14 ΔnifL::Prm1
PBC6.15 ΔnifL::Prm5
PBC6.22 ΔnifL::Prm3
PBC6.37 ΔnifL::Prm1 ΔglnEAR2
PBC6.38 ΔnifL::Prm1 ΔglnEAR1
PBC6.93 ΔnifL::Prm1 ΔglnEAR2 ΔamtB
PBC6.94 ΔnifL::Prm1 ΔglnEAR1 ΔamtB
Several in vitro assays were performed to characterize specific phenotypes of the derivative strains. The ARA was used to assess strain sensitivity to exogenous nitrogen, in which PBC6.1 exhibited repression of nitrogenase activity at high glutamine concentrations (FIG. 28D). In contrast, most derivative strains showed a derepressed phenotype with varying levels of acetylene reduction observed at high glutamine concentrations. Transcriptional rates of nifA in samples analyzed by qPCR correlated well with acetylene reduction rates (FIG. 31), supporting the hypothesis that nifL disruption and insertion of a nitrogen-independent promoter to drive nifA can lead to nil cluster derepression. Strains with altered GlnE or AmtB activity showed markedly increased ammonium excretion rates compared to wild type or derivative strains without these mutations (FIG. 28E), illustrating the effect of these genotypes on ammonia assimilation and reuptake.
Two experiments were performed to study the interaction of PBC6.1 derivatives with corn plants and quantify incorporation of fixed nitrogen into plant tissues. First, rates of microbial nitrogen fixation were quantified in a greenhouse study using isotopic tracers. Briefly, plants are grown with 15N labeled fertilizer, and diluted concentrations of 15N in plant tissues indicate contributions of fixed nitrogen from microbes. Corn seedlings were inoculated with selected microbial strains, and plants were grown to the V6 growth stage. Plants were subsequently deconstructed to enable measurement of microbial colonization and gene expression as well as measurement of 15N/14N ratios in plant tissues by isotope ratio mass spectrometry (IRMS). Analysis of the aerial tissue showed a small, nonsignificant contribution by PBC6.38 to plant nitrogen levels, and a significant contribution by PBC6.94 (p=0.011). Approximately 20% of the nitrogen found in above-ground corn leaves was produced by PBC6.94, with the remainder coming from the seed, potting mix, or “background” fixation by other soilborne microbes (FIG. 29C). This illustrates that our microbial breeding pipeline can generate strains capable of making significant nitrogen contributions to plants in the presence of nitrogen fertilizer. Microbial transcription within plant tissues was measured, and expression of the nif gene cluster was observed in derivative strains but not the wild type strain (FIG. 29B), showing the importance of nif derepression for contribution of BNF to crops in fertilized conditions. Root colonization measured by qPCR demonstrated that colonization density is different for each of the strains tested (FIG. 29A). A 50 fold difference in colonization was observed between PBC6.38 and PBC6.94. This difference could be an indication that PBC6.94 has reduced fitness in the rhizosphere relative to PBC6.38 as a result of high levels of fixation and excretion.
Methods Media Minimal medium contains (per liter) 25 g Na2HPO4, 0.1 g CaCL2-2H2O, 3 g KH2PO4, 0.25 g MgSO4.7H2O, 1 g NaCl, 2.9 mg FeCl3, 0.25 mg Na2MoO4.2 H2O, and 20 g sucrose. Growth medium is defined as minimal medium supplemented with 50 ml of 200 mM glutamine per liter.
Isolation of Diazotrophs Corn seedlings were grown from seed (DKC 66-40, DeKalb, Ill.) for two weeks in a greenhouse environment controlled from 22° C. (night) to 26° C. (day) and exposed to 16 hour light cycles in soil collected from San Joaquin County, Calif. Roots were harvested and washed with sterile deionized water to remove bulk soil. Root tissues were homogenized with 2 mm stainless steel beads in a tissue lyser (TissueLyser II, Qiagen P/N 85300) for three minutes at setting 30, and the samples were centrifuged for 1 minute at 13,000 rpm to separate tissue from root-associated bacteria. Supernatants were split into two fractions, and one was used to characterize the microbiome through 16S rRNA amplicon sequencing and the remaining fraction was diluted and plated on Nitrogen-free Broth (NfB) media supplemented with 1.5% agar. Plates were incubated at 30° C. for 5-7 days. Colonies that emerged were tested for the presence of the nifH gene by colony PCR with primers Ueda19f and Ueda406r. Genomic DNA from strains with a positive nifH colony PCR was isolated (QIAamp DNA Mini Kit, Cat No. 51306, QIAGEN, Germany) and sequenced (Illumina MiSeq v3, SeqMatic, Fremont, Calif.). Following sequence assembly and annotation, the isolates containing nitrogen fixation gene clusters were utilized in downstream research.
Microbiome Profiling of Isolation Seedlings Genomic DNA was isolated from root-associated bacteria using the ZR-96 Genomic DNA I Kit (Zymo Research P/N D3011), and 16S rRNA amplicons were generated using nextera-barcoded primers targeting 799f and 1114r. The amplicon libraries were purified and sequenced with the Illumina MiSeq v3 platform (SeqMatic, Fremont, Calif.). Reads were taxonomically classified using Kraken using the minikraken database (FIG. 30).
Acetylene Reduction Assay (ARA) A modified version of the Acetylene Reduction Assay was used to measure nitrogenase activity in pure culture conditions. Strains were propagated from single colony in SOB (RPI, P/N S25040-1000) at 30° C. with shaking at 200 RPM for 24 hours and then subcultured 1:25 into growth medium and grown aerobically for 24 hours (30° C., 200 RPM). 1 ml of the minimal media culture was then added to 4 ml of minimal media supplemented with 0 to 10 mM glutamine in air-tight Hungate tubes and grown anaerobically for 4 hours (30° C., 200 RPM). 10% headspace was removed then replaced by an equal volume of acetylene by injection, and incubation continued for 1 hr. Subsequently, 2 ml of headspace was removed via gas tight syringe for quantification of ethylene production using an Agilent 6850 gas chromatograph equipped with a flame ionization detector (FED).
Ammonium Excretion Assay Excretion of fixed nitrogen in the form of ammonia was measured using batch fermentation in anaerobic bioreactors. Strains were propagated from single colony in 1 ml/well of SOB in a 96 well DeepWell plate. The plate was incubated at 30° C. with shaking at 200 RPM for 24 hours and then diluted 1:25 into a fresh plate containing 1 ml/well of growth medium. Cells were incubated for 24 hours (30° C., 200 RPM) and then diluted 1:10 into a fresh plate containing minimal medium. The plate was transferred to an anaerobic chamber with a gas mixture of >98.5% nitrogen, 1.2-1.5% hydrogen and <30 ppM oxygen and incubated at 1350 RPM, room temperature for 66-70 hrs. Initial culture biomass was compared to ending biomass by measuring optical density at 590 nm. Cells were then separated by centrifugation, and supernatant from the reactor broth was assayed for free ammonia using the Megazyme Ammonia Assay kit (P/N K-AMIAR) normalized to biomass at each timepoint.
Extraction of Root-Associated Microbiome Roots were shaken gently to remove loose particles, and root systems were separated and soaked in a RNA stabilization solution (Thermo Fisher P/N AM7021) for 30 minutes. The roots were then briefly rinsed with sterile deionized water. Samples were homogenized using bead beating with ½-inch stainless steel ball bearings in a tissue lyser (TissueLyser Qiagen P/N 85300) in 2 ml of lysis buffer (Qiagen P/N 79216). Genomic DNA extraction was performed with ZR-96 Quick-gDNA kit (Zymo Research P/N D3010), and RNA extraction using the RNeasy kit (Qiagen P/N 74104).
Root Colonization Assay Four days after planting, 1 ml of a bacterial overnight culture (approximately 109 cfu) was applied to the soil above the planted seed. Seedlings were fertilized three times weekly with 25 ml modified Hoagland's solution supplemented with 0.5 mM ammonium nitrate. Four weeks after planting, root samples were collected and the total genomic DNA (gDNA) was extracted. Root colonization was quantified using qPCR with primers designed to amplify unique regions of either the wild type or derivative strain genome. QPCR reaction efficiency was measured using a standard curve generated from a known quantity of gDNA from the target genome. Data was normalized to genome copies per g fresh weight using the tissue weight and extraction volume. For each experiment, the colonization numbers were compared to untreated control seedlings.
In Planta Transcriptomics Transcriptional profiling of root-associated microbes was measured in seedlings grown and processed as described in the Root Colonization Assay. Purified RNA was sequenced using the Illumina NextSeq platform (SeqMatic, Fremont, Calif.). Reads were mapped to the genome of the inoculated strain using bowtie2 using ‘--very-sensitive-local’ parameters and a minimum alignment score of 30. Coverage across the genome was calculated using samtools. Differential coverage was normalized to housekeeping gene expression and visualized across the genome using Circos and across the nif gene cluster using DNAplotlib. Additionally, the in planta transcriptional profile was quantified via targeted Nanostring analysis. Purified RNA was processed on an nCounter Sprint (Core Diagnostics, Hayward, Calif.).
15N Dilution Greenhouse Study 15N fertilizer dilution experiment was performed to assess optimized strain activity in planta. A planting medium containing minimal background N was prepared using a mixture of vermiculite and washed sand (5 rinses in DI 1120). The sand mixture was autoclaved for 1 hour at 122° C. and approximately 600 g measured out into 40 cubic inch (656 mL) pots, which were saturated with sterile DI H2O and allowed to drain 24 hours before planting. Corn seeds (DKC 66-40) were surface sterilized in 0.625% sodium hypochlorite for 10 minutes, then rinsed five times in sterile distilled water and planted 1 cm deep. The plants were maintained under fluorescent lamps for four weeks with 16-hour day length at room temperatures averaging 22° C. (night) to 26° C. (day).
Five days after planting, seedlings were inoculated with a 1 ml suspension of cells drenched directly over the emerging coleoptile. Inoculum was prepared from 5 ml overnight cultures in SOB, which were spun down and resuspended twice in 5 ml PBS to remove residual SOB before final dilution to OD of 1.0 (approximately 109 CFU/ml). Control plants were treated with sterile PBS, and each treatment was applied to ten replicate plants.
Plants were fertilized with 25 ml fertilizer solution containing 2% 15N-enriched 2 mM KNO3 on 5, 9, 14, and 19 days after planting, and the same solution without KNO3 on 7, 12, 16, and 18 days after planting. The fertilizer solution contained (per liter) 3 mmol CaCl2), 0.5 mmol KH2PO4, 2 mmol MgSO4, 17.9 μmol FeSO4, 2.86 mg H3BO3, 1.81 mg MnCl2.4H2O, 0.22 mg ZnSO4.7H2O, 51 μg CuSO4.5H2O, 0.12 mg Na2MoO4.2H2O, and 0.14 nmol NiCl2. All pots were watered with sterile DI H2O as needed to maintain consistent soil moisture without runoff.
At four weeks, plants were harvested and separated at the lowest node into samples for root gDNA and RNA extraction and aerial tissue for IRMS. Aerial tissues were wiped as needed to remove sand, placed whole into paper bags and dried for at least 72 hours at 60° C. Once completely dry, total aerial tissue was homogenized by bead beating and 5-7 mg samples were analyzed by isotope ratio mass spectrometry (IRMS) for δ15N by the MBL Stable Isotope Laboratory (The Ecosystems Center, Woods Hole, Mass.). Percent NDFA was calculated using the following formula: % NDFA=(δ15N of UTC average—δ15N of sample)/(δ15N of UTC average)×100.
Example 11: Field Trials with Microbes of the Disclosure—Summer 2017 In order to evaluate the efficacy of strains of the present disclosure on corn growth and productivity under varying nitrogen regimes, field trials were conducted. The below field data demonstrates that the non-intergeneric microbes of the disclosure are able to fix atmospheric nitrogen and deliver said nitrogen to a plant—resulting in increased yields—in both a nitrogen limiting environment, as well as a non-nitrogen limiting environment.
Trials were conducted at seven locations across the United states with six geographically diverse Midwestern locations. Five nitrogen regimes were used for fertilizer treatments: 100% of standard agricultural practice of the site/region, 100% minus 25 pounds, 100% minus 50 pounds, 100% minus 75 pounds, and 0%; all per acre. The pounds of nitrogen per acre for the 100% regime depended upon the standard agricultural practices of the site/region. The aforementioned nitrogen regimes ranged from about 153 pounds per acre to about 180 pounds per acre, with an average of about 164 pounds of nitrogen per acre.
Within each fertilizer regime there were 14 treatments. Each regime had six replications, and a split plot design was utilized. The 14 treatments included: 12 different microbes, 1 UTC with the same fertilizer rate as the main plot, and 1 UTC with 100% nitrogen. In the 100% nitrogen regime the 2nd UTC is 100 plus 25 pounds.
Plots of corn, at a minimum, were 4 rows of 30 feet in length (30 inches between rows) with 420 plots per location. All observations, unless otherwise noted, were taken from the center two rows of the plants, and all destructive sampling was taken from the outside rows. Seed samples were refrigerated until 1.5 to 2 hours prior to use.
Local Agricultural Practice:
The seed was a commercial corn applied with a commercial seed treatment with no biological co-application. The seeding rate, planting date, weed/insect management, harvest times, and other standard management practices were left to the norms of local agricultural practices for the regions, with the exception of fungicide application (if required).
Microbe Application:
The microbes were applied to the seed in a seed treatment over seeds that had already received a normal chemical treatment. The seed were coated with fermentation broth comprising the microbes.
Soil Characterization:
Soil texture and soil fertility were evaluated. Standard soil sampling procedures were utilized, which included soil cores of depths from 0-30 cm and 30-60 cm. The standard soil sampling included a determination of nitrate nitrogen, ammonium nitrogen, total nitrogen, organic matter, and CEC. Standard soil sampling further included a determination of pH, total potassium, and total phosphorous. To determine the nitrogen fertilizer levels, preplant soil samples from each location were taken to ensure that die 0-12″ and potentially the 12″ to 24″ soil regions for nitrate nitrogen.
Prior to planting and fertilization, 2 ml soil samples were collected from 0 to 6-12″ from the UTC. One sample per replicate per nitrogen region was collected using the middle of the row. (5 fertilizer regimes×6 replicates=thirty soil samples).
Post-planting (V4-V6), 2 ml soil samples were collected from 0 to 6-12″ from the UTC. One sample per replicate per nitrogen region was collected using the middle of the row. (5 fertilizer regimes×6 replicates=thirty soil samples).
Post-harvest (V4-V6), 2 ml soil samples were collected from 0 to 6-12″ from the UTC. One sample per replicate per nitrogen region was collected using the middle of the row. Additional post-harvest soil sample collected at 0-12″ from the UTC and potentially 12-24″ from the UTC (5 fertilizer regimes×6 replicates=thirty soil samples).
A V6-V10 soil sample from each fertilizer regime (excluding the treatment of 100% and 100%+25 lbs [in the 100% block] for all fertilizer regimes at 0-12″ and 12-24″. (5 fertilizer regimes×2 depths=10 samples per location).
Post-harvest soil sample from each fertilizer regime (excluding the treatment of 100% and 100%+25 lbs [in the 100% block] for all fertilizer regimes at 0-12″ and 12-24″. (5 fertilizer regimes×2 depths=10 samples per location).
Assessments:
The initial plant population was assessed at ˜50% UTC and the final plant population was assessed prior to harvest. Assessment included (1) potentially temperature (temperature probe); (2) vigor (1-10 scale with 10=excellent) at V4 and V8-V10; (3) plant height at V8-V10 and V14; (4) yield (bushels/acre) adjusted to standard moisture percentage; (5) test weight; (6) grain moisture percentage; (7) stalk nitrate tests at black layer (420 plots×7 locations); (8) colonization with 1 plant per plot in zip lock bag at 0% and 100% fertilizer at V4-V6 (1 plant×14 treatments×6 replicates×2 fertilizer regimes=168 plants); (9) transcriptomics with 1 plant per plot in zip lock bag at 0% and 100% fertilizer at V4-V6 (1 plant×14 treatments×6 replicates×2 fertilizer regimes=168 plants); (10) Normalized difference vegetative index (NDVI) or normalized difference red edge (NDRE) determination using a Greenseeker instrument at two time points (V4-V6 and VT) to assess each plot at all 7 locations (420 plots×2 time points×7 locations=5,880 data points); (11) stalk characteristics measured at all 7 locations between R2 and R5 by recording the stalk diameter of 10 plants/plot at 6″ height, record length of first internode above the 6″ mark, 10 plants monitored (5 consecutive plants from center of two inside rows) (420 plots×10 plants×7 locations=29,400 data points).
Monitoring Schedule:
Practitioners visited all trials at V3-V4 stage to assess early-season response to treatments and during reproductive growth stage to monitor maturity. Local cooperator visited research trial on an on-going basis.
Weather Information:
Weather data spanning from planting to harvest was collected and consisted of daily minimum and maximum temperatures, soil temperature at seeding, daily rainfall plus irrigation (if applied), and unusual weather events such as excessive wind, rain, cold, heat.
Data Reporting:
Including the data indicated above, the field trials generated data points including soil textures; row spacing; plot sizes; irrigation; tillage; previous crop; seeding rate; plant population; seasonal fertilizer inputs including source, rate, timing, and placement; harvest area dimensions, method of harvest, such as by hand or machine and measurement tools used (scales, yield monitor, etc.)
Results:
Select results from the aforementioned field trial are reported in FIG. 32 and FIG. 33.
In FIG. 32, it can be seen that a microbe of the disclosure (i.e. 6-403) resulted in a higher yield than the wild type strain (WT) and a higher yield than the untreated control (UTC). The “−25 lbs N” treatment utilizes 25 lbs less N per acre than standard agricultural practices of the region. The “100% N” UTC treatment is meant to depict standard agricultural practices of the region, in which 100% of the standard utilization of N is deployed by the farmer. The microbe “6-403” was deposited as NCMA 201708004 and can be found in Table A. This is a mutant Kosakonia sacchari (also called CM037) and is a progeny mutant strain from CI006 WT.
In FIG. 33, the yield results obtained demonstrate that the microbes of the disclosure perform consistently across locations. Furthermore, the yield results demonstrate that the microbes of the disclosure perform well in both a nitrogen stressed environment (i.e. a nitrogen limiting environment), as well as an environment that has sufficient supplies of nitrogen (i.e. a non-nitrogen-limiting condition). The microbe “6-881” (also known as CM094, PBC6.94), and which is a progeny mutant Kosakonia sacchari strain from CI006 WT, was deposited as NCMA 201708002 and can be found in Table A. The microbe “137-1034,” which is a progeny mutant Klebsiella variicola strain from CI137 WI, was deposited as NCMA 201712001 and can be found in Table A. The microbe “137-1036,” which is a progeny mutant Klebsiella variicola strain from CI137 WT, was deposited as NCMA 201712002 and can be found in Table A. The microbe “6-404” (also known as CM38, PBC6.38), and which is a progeny mutant Kosakonia sacchari strain from CI006 WT, was deposited as NCMA 201708003 and can be found in Table A.
Example 12: Genus of Non-Intergeneric Microbes Beneficial for Agricultural Systems The microbes of the present disclosure were evaluated and compared against one another for the production of nitrogen produced in an acre across a season. See FIG. 20, FIG. 40, and FIG. 41
It is hypothesized by the inventors that in order for a population of engineered non-intergeneric microbes to be beneficial in a modern row crop agricultural system, then the population of microbes needs to produce at least one pound or more of nitrogen per acre per season.
To that end, the inventors have surprisingly discovered a functional genus of microbes that are able to contribute, inter alia, to: increasing yields in non-leguminous crops; and/or lessening a farmer's dependence upon exogenous nitrogen application; and/or the ability to produce at least one pound of nitrogen per acre per season, even in non-nitrogen-limiting environments, said genus being defined by the product of colonization ability×mmol of N produced per microbe per hour (i.e. the line partitioning FIGS. 20, 40, and 41).
With respect to FIGS. 20, 40, and 41, certain data utilizing microbes of the disclosure was aggregated, in order to depict a heatmap of the pounds of nitrogen delivered per acre-season by microbes of the disclosure, which are recorded as a function of microbes per g-fresh weight by mmol of nitrogen/microbe-hr. Below the thin line that transects the larger images are the microbes that deliver less than one pound of nitrogen per acre-season, and above the line are the microbes that deliver greater than one pound of nitrogen per acre-season.
Field Data & Wild Type Colonization Heatmap:
The microbes utilized in the FIG. 20 heatmap were assayed for N production in corn. For the WT strains CI006 and CI019, corn root colonization data was taken from a single field site. For the remaining strains, colonization was assumed to be the same as the WT field level. N-fixation activity was determined using an in vitro ARA assay at 5 mM glutamine. The table below the heatmap in FIG. 20 gives the precise value of mmol N produced per microbe per hour (mmol N/Microbe hr) along with the precise CFU per gram of fresh weight (CFU/g fw) for each microbe shown in the heatmap.
Field Data Heatmap:
The data utilized in the FIG. 40 heatmap is derived from microbial strains assayed for N production in corn in field conditions. Each point represents lb N/acre produced by a microbe using corn root colonization data from a single field site. N-fixation activity was determined using in vitro ARA assay at 5 mM N in the form of glutamine or ammonium phosphate. The below Table C gives the precise value of mmol N produced per microbe per hour (mmol N/Microbe hr) along with the precise CFU per gram of fresh weight (CFU/g fw) for each microbe shown in the heatmap of FIG. 40.
Greenhouse & Laboratory Data Heatmap:
The data utilized in the FIG. 41 heatmap is derived from microbial strains assayed for N production in corn in laboratory and greenhouse conditions. Each point represents lb N/acre produced by a single strain. White points represent strains in which corn root colonization data was gathered in greenhouse conditions. Black points represent mutant strains for which corn root colonization levels are derived from average field corn root colonization levels of the wild-type parent strain. Hatched points represent the wild type parent strains at their average field corn root colonization levels. In all cases, N-fixation activity was determined by in vitro ARA assay at 5 mM N in the form of glutamine or ammonium phosphate. The below Table D gives the precise value of mmol N produced per microbe per hour (mmol N/Microbe hr) along with the precise CFU per gram of fresh weight (CFU/g fw) for each microbe shown in the heatmap of FIG. 41.
TABLE C
FIG. 40 - Field Data Heatmap
Peak
Activity Coloniza-
(mmol tion N Pro-
Strain N/Mi- (CFU/ duced/acre Taxonomic
Name crobe hr) g fw) season Designation
CI006 3.88E−16 1.50E+07 0.24 Kosakonia sacchari
6-404 1.61E−13 3.50E+05 2.28 Kosakonia sacchari
6-848 1.80E−13 2.70E+05 1.97 Kosakonia sacchari
6-881 1.58E−13 5.00E+05 3.20 Kosakonia sacchari
6-412 4.80E−14 1.30E+06 2.53 Kosakonia sacchari
6-403 1.90E−13 1.30E+06 10.00 Kosakonia sacchari
CI019 5.33E−17 2.40E+06 0.01 Rahnella aquatilis
19-806 6.65E−14 2.90E+06 7.80 Rahnella aquatilis
19-750 8.90E−14 2.60E+05 0.94 Rahnella aquatilis
19-804 1.72E−14 4.10E+05 0.29 Rahnella aquatilis
CI137 3.24E−15 6.50E+06 0.85 Klebsiella variicola
137-1034 1.16E−14 6.30E+06 2.96 Klebsiella variicola
137-1036 3.47E−13 1.30E+07 182.56 Klebsiella variicola
137-1314 1.70E−13 1.99E+04 0.14 Klebsiella variicola
137-1329 1.65E−13 7.25E+04 0.48 Klebsiella variicola
63 3.60E−17 3.11E+05 0.00 Rahnella aquatilis
63-1146 1.90E−14 5.10E+05 0.39 Rahnella aquatilis
1021 1.77E−14 2.69E+07 19.25 Kosakonia
pseudosacchari
728 5.56E−14 1445240.09 3.25 Klebsiella variicola
TABLE D
FIG. 41 Greenhouse & Laboratory Data Heatmap
N
Activity Peak Produced/
Strain (mmol N/ Colonization acre
Name Microbe hr) (CFU/g fw) season Taxonomic Designation
CI006 3.88E−16 1.50E+07 0.24 Kosakonia sacchari
6-400 2.72E−13 1.79E+05 1.97 Kosakonia sacchari
6-397 1.14E−14 1.79E+05 0.08 Kosakonia sacchari
CI137 3.24E−15 6.50E+06 0.85 Klebsiella variicola
137-1586 1.10E−13 1.82E+06 8.10 Klebsiella variicola
137-1382 4.81E−12 1.82E+06 354.60 Klebsiella variicola
1021 1.77E−14 2.69E+07 19.25 Kosakonia pseudosacchari
1021-1615 1.20E−13 2.69E+07 130.75 Kosakonia pseudosacchari
1021-1619 3.93E−14 2.69E+07 42.86 Kosakonia pseudosacchari
1021-1612 1.20E−13 2.69E+07 130.75 Kosakonia pseudosacchari
1021-1623 4.73E−17 2.69E+07 0.05 Kosakonia pseudosacchari
1293 5.44E−17 8.70E+08 1.92 Azospirillum lipoferum
1116 1.05E−14 1.37E+07 5.79 Enterobacter sp.
1113 8.05E−15 4.13E+07 13.45 Enterobacter sp.
910 1.19E−13 1.34E+06 6.46 Kluyvera intermedia
910-1246 2.16E−13 1.34E+06 11.69 Kluyvera intermedia
850 7.2301E−16 1.17E+06 0.03 Achromobacter spiritinus
852 5.96E−16 1.07E+06 0.03 Achromobacter marplatensis
853 6.42E−16 2.55E+06 0.07 Microbacterium murale
Conclusions:
The data in FIGS. 20, 40, 41, and Tables C and D, illustrates more than a dozen representative members of the described genus (i.e. microbes to the right of the line in the figures). Further, these numerous representative members come from a diverse array of taxonomic genera, which can be found in the above Tables C and D. Further still, the inventors have discovered numerous genetic attributes that depict a structure/function relationship that is found in many of the microbes. These genetic relationships can be found in the numerous tables of the disclosure setting forth the genetic modifications introduced by the inventors, which include introducing at least one genetic variation into at least one gene, or non-coding polynucleotide, of the nitrogen fixation or assimilation genetic regulatory network.
Consequently, the newly discovered genus is supported by: (I) a robust dataset, (2) over a dozen representative members, (3) members from diverse taxonomic genera, and (4) classes of genetic modifications that define a structure/function relationship, in the underlying genetic architecture of the genus members.
Example 13: Methods and Assays for Detection of Non-Intergeneric Engineered Microbes The present disclosure teaches primers, probes, and assays that are useful for detecting the microbes utilized in the various aforementioned Examples. The assays are able to detect the non-natural nucleotide “junction” sequences in the derived/mutant non-intergeneric microbes. These non-naturally occurring nucleotide junctions can be used as a type of diagnostic that is indicative of the presence of a particular genetic alteration in a microbe.
The present techniques are able to detect these non-naturally occurring nucleotide junctions via the utilization of specialized quantitative PCR methods, including uniquely designed primers and probes. The probes can bind to the non-naturally occurring nucleotide junction sequences. That is, sequence-specific DNA probes consisting of oligonucleotides that are labelled with a fluorescent reporter, which permits detection only after hybridization of the probe with its complementary sequence can be used. The quantitative methods can ensure that only the non-naturally occurring nucleotide junction will be amplified via the taught primers, and consequently can be detected via either a non-specific dye, or via the utilization of a specific hybridization probe. Another aspect of the method is to choose primers such that the primers flank either side of a junction sequence, such that if an amplification reaction occurs, then said junction sequence is present.
Consequently, genomic DNA can be extracted from samples and used to quantify the presence of microbes of the disclosure by using qPCR. The primers utilized in the qPCR reaction can be primers designed by Primer Blast (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) to amplify unique regions of the wild-type genome or unique regions of the engineered non-intergeneric mutant strains. The qPCR reaction can be carried out using the SYBR GreenER qPCR SuperMix Universal (Thermo Fisher P/N 11762100) kit, using only forward and reverse amplification primers; alternatively, the Kapa Probe Force kit (Kapa Biosystems P/N KK4301) can be used with amplification primers and a TaqMan probe containing a FAM dye label at the 5′ end, an internal ZEN quencher, and a minor groove binder and fluorescent quencher at the 3′ end (Integrated DNA Technologies).
Certain primer, probe, and non-native junction sequences—which can be used in the qPCR methods—are listed in the below Table E. Specifically, the non-native junction sequences can be found in SEQ ID NOs: 372-405.
TABLE E
Microbial Detection
up/down SEQ 100 bp SEQ 100 bp SEQ Junction SEQ F R
base Junction stream ID upstream ID downstream ID ″/″ indicating Junction primer primer Probe
CI Name junction NO of junction NO junction NO junction des. SEQ SEQ SEQ
1021 ds1131 up 304 TGGTGTCCGGGC 338 TTCTTGGTTCTCT 372 5′- disrupted N/A N/A N/A
GAACGTCGCCAG GGAGCGCTTTAT TGGTGTCCGGGC nifL gene/
GTGGCACAAATT CGGCATCCTGAC GAACGTCGCCAG PinfC
GTCAGAACTACG TGAAGAATTTGC GTGGCACAAATT
ACACGACTAACC AGGCTTCTTCCCA GTCAGAACTACG
GACCGCAGGAGT ACCTGGCTTGCA ACACGACTAACC
GTGCGATGACCC CCCGTGCAGGTA GACCGCAGGAGT
TGAATATGATGA GTTGTGATGAAC GTGCGATGACCC
TGGA AT TGAATATGATGA
TGGA/
TTCTTGGTTCTCT
GGAGCGCTTTAT
CGGCATCCTGAC
TGAAGAATTTGC
AGGCTTCTTCCCA
ACCTGGCTTGCA
CCCGTGCAGGTA
GTTGTGATGAAC
AT-3′
1021 ds1131 down 305 CGGAAAACGAGT 339 GCGATAGAACTC 373 5′- PinfC/ N/A N/A N/A
TCAAACGGCGCG ACTTCACGCCCC CGGAAAACGAGT disrupted
TCCCAATCGTATT GAAGGGGGAAGC TCAAACGGCGCG nifL gene
AATGGCGAGATT TGCCTGACCCTAC TCCCAATCGTATT
CGCGCCACGGAA GATTCCCGCTATT AATGGCGAGATT
GTTCGCTTAACAG TCATTCACTGACC CGCGCCACGGAA
GTCTGGAAGGCG GGAGGTTCAAAA GTTCGTTAACA
AGCACCTTGGTA TGACCCAGCGAA GGTCTGGAAGGC
TT C GAGCAGCTTGGT
ATT/
GCGATAGAACTC
ACTTCACGCCCC
GAAGGGGGAAGC
TGCCTGACCCTA
CGATTCCCGCTAT
TTCATTCACTGAC
CGGAGGTTCAAA
ATGACCCAGCGA
AC-3′
1021 ds1133 N/A 306 CGCCAGAGAGTT 340 TCCCTGTGCGCCG 374 5′- 5′ UTR N/A N/A N/A
GAAATCGAACAT CGTCGCCGATGG CGCCAGAGAGTF and ATG/
TTCCGTAATACCG TGGCCAGCCAAC GAAATCGAACAT truncated
CCATTACCCAGG TGGCGCGCTACC TTCCGTAATACC glnE gene
AGCCGTTCTGGTT CGATCCTGCTCG GCCATTACCCAG
GCACAGCGGAAA ATGAACTGCTCG GAGCCGTTCTGG
ACGTTAACGAAA ACCCGAACACGC TTGCACAGCGGA
GGATATTTCGCAT TCTATCAACCGA AAACGTTAACGA
G CGG AAGGATATTTCG
CATG/
TCCCTGTGCGCC
GCGTCGCCGATG
GTGGCCAGCCAA
CTGGCGCGCTAC
CCGATCCTGCTC
GATGAACTGCTC
GACCCGAACACG
CTCTATCAACCG
ACGG-3′
1021 ds1145 up 307 CGGGCGAACGTC 341 CGTTCTGTAATAA 375 5′- disrupted N/A N/A N/A
GCCAGGTGGCAC TAACCGGACAAT CGGGCGAACGTC nifL gene/
AAATTGTCAGAA TCGGACTGATTA GCCAGGTGGCAC Prm1
CTACGACACGAC AAAAAGCGCCCT AAATTGTCAGAA
TAACCGACCGCA CGCGGCGCTTTTT CTACGACACGAC
GGAGTGTGCGAT TTATATTCTCGAC TAACCGACCGCA
GACCCTGAATAT TCCATTTAAAATA GGAGTGTGCGAT
GATGATGGATGC AAAAATCCAATC GACCCTGAATAT
CAGC GATGATGGATGC
CAGC/
CGTTCTGTAATA
ATAACCGGACAA
TFCGGACTGATT
AAAAAAGCGCCC
TCGCGGCGCTTTT
TTTATATTCTCGA
CTCCATTTAAAAT
AAAAAATCCAAT
C-3′
1021 ds1145 down 308 TCAACCTAAAAA 342 AACTCACTTCAC 376 5′- Prm1/ N/A N/A N/A
AGTTTGTGTAATA GCCCCGAAGGGG TCAACCTAAAAA disrupted
CTTGTAACGCTAC GAAGCTGCCTGA AGTTTGTGTAAT nifL gene
ATGGAGATTAAC CCCTACGATTCCC ACTTGTAACGCT
TCAATCTAGAGG GCTATTTCATTCA ACATGGAGATTA
GTATTAATAATG CTGACCGGAGGT ACTCAATCTAGA
AATCGTACTAAA TCAAAATGACCC GGGTATTAATAA
CTGGTACTGGGC AGCGAACCGAGT TGAATCGTACTA
GC CG AACTGGTACTGG
GCGC/
AACTCACTTCAC
GCCCCGAAGGGG
GAAGCTGCCTGA
CCCTACGATTCCC
GCTATTTCATTCA
CTGACCGGAGGT
TCAAAATGACCC
AGCGAACCGAGT
CG-3′
1021 ds1148 up 309 CGGGCGAACGTC 343 CGCGTCAGGTTG 377 5′- disrupted N/A N/A N/A
GCCAGGTGGCAC AACGTAAAAAAG CGGGCGAACGTC nifL gene/
AAATTGTCAGAA TCGGTCTGCGCA GCCAGGTGGCAC Prm7
CTACGACACGAC AAGCACGTCGTC AAATTGTCAGAA
TAACCGACCGCA GTCCGCAGTTCTC CTACGACACGAC
GGAGTGTGCGAT CAAACGTTAATT TAACCGACCGCA
GACCCTGAATAT GGTTTCTGCTFCG GGAGTGTGCGAT
GATGATGGATGC GCAGAACGATTG GACCCTGAATAT
CAGC GC GATGATGGATGC
CAGC/
CGCGTCAGGTTG
AACGTAAAAAAG
TCGGTCTGCGCA
AAGCACGTCGTC
GTCCGCAGTTCTC
CAAACGTTAATT
GGTTTCTGCTTCG
GCAGAACGATTG
GC-3′
1021 ds1148 down 310 AATTTTCTGCCCA 344 AACTCACTTCAC 378 5′- Prm4/ N/A N/A N/A
AATGGCTGGGAT GCCCCGAAGGGG AATGGCTGCCCA disrupted
TGTTCATTTTTTG GAAGCTGCCFGA AATGGCTGGGAT nifL gene
TTTGCCTTACAAC CCCTACGATTCCC TGTTCATTTTTTG
GAGAGTGACAGT GCTATTTCATTCA TTTGCCTTACAAC
ACGCGCGGGTAG CTGACCGGAGGT GAGAGTGACAGT
TTAACTCAACATC TCAAAATGACCC ACGCGCGGGTAG
TGACCGGTCGAT AGCGAACCGAGT TTAACTCAACAT
CG CTGACCGGTCGA
T/
AACTCACTTCAC
GCCCCGAAGGGG
GAAGCTGCCTGA
CCCTACGATTCCC
GCTATTTCATTCA
CTGACCGGAGGT
TCAAAATGACCC
AGCGAACCGAGT
CG-3′
CI006 ds126 N/A 311 GTAACCAATAAA 345 CCGATCCCCATC 379 5′- 5′ UTR up N/A N/A N/A
GGCCACCACGCC ACTGTGTGTCTTG GTAACCAATAAA to ATG-
AGACCACACGAT TATTACAGTGCC GGCCACCACGCC 4 bp of
AGTGATGGCAAC GCTTCGTCGGCTT AGACCACACGAT amtB gene/
ACTTTCCAGCTGC CGCCGGTACGAA AGTGATGGCAAC disrupted
ACCAGCACCTGA TACGAATGACGC ACTTTCCAGCTGC amtB gene
TGGCCCATGGTC GTTGCAGCTCAG ACCAGCACCTGA
ACACCTTCAGCG CAACGAAAATTT TGGCCCATGGTC
AAA TG ACACCTTCAGCG
AAA/
CCGATCCCCATC
ACTGTGTGTCTTG
TATTACAGTGCC
GCTTCGTCGGCTT
CGCCGGTACGAA
TACGAATGACGC
CAACGAAAATTT
TG-3′
CI019 ds172 down 312 TGGTATTGTCAGT 346 CCGTCTCTGAAG 380 5′- Pnn1.2/ SEQ SEQ N/A
CTGAATGAAGCT CTCTCGGTGAAC TGGTATTGTCAGT disrupted ID ID
CTTGAAAAAGCT ATTGTTGCGAGG CTGAATGAAGCT nifL gene NO: NO:
GAGGAAGCGGGC CAGGATGCGAGC CTTGAAAAAGCT 406 407
GTCGATTTAGTAG TGGTTGTGTMG GAGGAAGCGGGC CAAG TGCC
AAATCAGTCCGA ACATTACCGATA GTCGATTTAGTA AAGT TCGC
ATGCCGAGCCGC ATGTGCCGCGTG GAAATCAGTCCG TCGC AACA
CAGTTTGTCGAAT AACGGGTGCGTT AATGCCGAGCCG CTCA ATGT
C ATG CCAGTTTGTCGA CAGG TCAC
ATC/
CCGTCTCTGAAG
CTCTCGGTGAAC
ATTGTTGCGAGG
CAGGATGCGAGC
TGGTTGTGTTTTG
ACATTACCGATA
ATGTGCCGCGTG
AACGGGTGCGTT
ATG-3′
CI019 ds172 up 313 ACCGATCCGCAG 347 TGAACATCACTG 381 5′- disrupted N/A N/A N/A
GCGCGCATTTGTT ATGCACAAGCTA ACCGATCCGCAG nifL gene/
ATGCCAATCCGG CCTATGTCGAAG GCGCGCATTTGTT Prm1.2
CATTCTGCCGCCA AATTAACTAAAA ATGCCAATCCGG
GACGGGTTTTGC AACTGCAAGATG CATTCTGCCGCC
ACTTGAGACACTT CAGGCATTCGCG AGACGGGTTTTG
TTGGGCGAGAAC TTAAAGCCGACT CACTTGAGACAC
CACCGTCTGCTGG TGAGAAATGAGA TTTTGGGCGAGA
AGAT ACCACCGTCTGC
TGG/
TGAACATCACTG
ATGCACAAGCTA
CCTATGTCGAAG
AATTAACTAAAA
AACTGCAAGATG
CAGGCATTCGCG
TTAAAGCCGACT
TGAGAAATGAGA
AGAT-3′
CI019 ds175 down 314 CGGGAACCGGTG 348 CCGTCTCTGAAG 382 5′- Prm3.1/ SEQ SEQ SEQ
TTATAATGCCGCG CTCTCGGTGAAC CGGGAACCGGTG disrupted ID ID ID
CCCTCATATTGTG ATTGTTGCGAGG TTATAATGCCGC nifL gene NO: NO: NO:
GGGATTTCTTAAT CAGGATGCGAGC GCCCTCATATTGT 408 409 410
GACCTATCCTGG TGGTTGTGTTTTG GGGGATTTCTTA CGCC GGCA /56-
GTCCTAAAGTTGT ACATTACCGATA ATGACCTATCCT CTCA TAAC FAM/
AGTTGACATTAG ATGTGCCGCGTG GGGTCCTAAAGT TATT GCAC TA
CGGAGCACTAAC AACGGGTGCGTT TGTAGTTGACATT GTGG CCGT ACC
ATG AGCGGAGCACTA GGAT TCA CGT
AC/ C/
CCGTCTCTGAAG ZEN/T
CTCTCGGTGAAC CTG
ATTGTTGCGAGG AAG
CAGGATGCGAGC CTC
TGGTTGTGTTTTG TCG
ACATTACCGATA GT/
ATGTGCCGCGTG 3IABkFQ/
AACGGGTGCGTT
ATG-3′
CI019 ds175 up 315 ACCGATCCGCAG 349 TACAGTAGCGCC 383 5c disrupted N/A N/A N/A
GCGCGCATTTGTT TCTCAAAAATAG ACCGATCCGCAG nifL gene/
ATGCCAATCCGG ATAAACGGCTCA GCGCGCATTTGTT Prm3.1
CATTCTGCCGCCA TGTACGTGGGCC ATGCCAATCCGG
GACGGGTTTTGC GTTTATTTTTTT CATTCTGCCGCC
ACTTGAGACACTT ACCCATAATCGG AGACGGGTTTTG
TTGGGCGAGAAC GAACCGGTGTTA CACTTGAGACAC
CACCGTCTGCTGG TAATGCCGCGCC TTTTGGGCGAGA
CTC ACCACCGTCTGC
TGG/
TACAGTAGCGCC
TCTCAAAAATAG
ATAAACGGCTCA
TGTACGTGGGCC
GTTTATTTTTTCT
ACCCATAATCGG
GAACCGGTGTTA
TAATGCCGCGCC
CTC-3′
CI006 ds20 down 316 TCAACCTAAAAA 350 AACTCACTTCAC 384 5′- Prm1/ SEQ SEQ SEQ
AGTTTGTGTAATA ACCCCGAAGGGG TCAACCTAAAAA disrupted ID ID ID
CTTGTAACGCTAC GAAGTTGCCTGA AGTTTGTGTAAT nifL gene NO: NO: NO:
ATGGAGATTAAC CCCTACGATTCCC ACTTGTAACGCT 411 412 413
TCAATCTAGAGG GCTATTTCATTCA ACATGGAGATTA TAAA CAAA 56-
GTATTAATAATG CTGACCGGAGGT ACTCAATCTAGA CTGG TCGA FAM
AATCGTACTAAA TCAAAATGACCC GGGTATTAAXAA TACT AGCG AAG
CTGGTACTGGGC AGCGAACCGAGT TGAATCGTACTA GGGC CCAG TTGC
GC CG AACTGGTACTGG GCAA ACGG CT/
GCGC/ CT TAT ZEN/G
AACTCACTTCAC ACC
ACCCCGAAGGGG CTAC
GAAGTTGCCTGA GATT
CCCTACGATTCCC CCC/
GCTATTTCATTCA 3IABkFQ/
CTGACCGGAGGT
TCAAAATGACCC
AGCGAACCGAGT
CG-3′
CI006 ds20 up 317 GGGCGACAAACG 351 CGTCCTGTAATA 385 5′- disrupted N/A N/A N/A
GCCTGGTGGCAC ATAACCGGACAA GGGCGACAAACG nifL gene/
AAATTGTCAGAA TTCGGACTGATTA GCCTGGTGGCAC Prm1
CTACGACACGAC AAAAAGCGCCCT AAATTGTCAGAA
TAACTGACCGCA TGTGGCGCTTTTT CTACGACACGAC
GGAGTGTGCGAT TTATATTCCCGCC TAACTGACCGCA
GACCCTGAATAT TCCATTTAAAATA GGAGTGTGCGAT
GATGATGGATGC AAAAATCCAATC GACCCTGAATAT
CGGC GATGATGGATGC
CGGC/
CGTCCTGTAATA
ATAACCGGACAA
TTCGGACTGATT
AAAAAAGCGCCC
TTGTGGCGCTTTT
TTTATATTCCCGC
CTCCATTTAAAAT
AAAAAATCCAAT
C-3′
CI006 ds24 up 318 GGGCGACAAACG 352 GGACATCATCGC 386 5′- disrupted SEQ SEQ SEQ
GCCTGGTGGCAC GACAAACAATAT GGGCGACAAACG nifL gene/ ID ID ID
AAATTGTCAGAA TAATACCGGCAA GCCTGGTGGCAC Prm5 NO: NO: NO:
CTACGACACGAC CCACACCGGCAA AAATTGTCAGAA 414 415 416
TAACTGACCGCA TTTACGAGACTG CTACGACACGAC GGTG GCGC /56-
GGAGTGTGCGAT CGCAGGCATCCT TAACTGACCGCA CACT AGTC FAM/
GACCCTGAATAT TTCTCCCGTCAAT GGAGTGTGCGAT CTTT TCGT CA
GATGATGGATGC TTCTGTCAAATAA GACCCTGAATAT GCAT AAAT GGA
CGGC AG GATGATGGATGC GGTT TGCC GTG
CGGC/ T/
GGACATCATCGC ZEN/G
GACAAACAATAT CGA
TAATACCGGCAA TGA
CCACACCGGCAA CCC
TTTACGAGACTG TGA
CGCAGGCATCCT AT/
TTCTCCCGTCAAT 3IABkFQ
TTCTGTCAAATA
AAG-3′
CI006 ds24 down 319 TAAGAATTATCTG 353 AACTCACTTCAC 387 5′- Prm5/ N/A N/A N/A
GATGAATGTGCC ACCCCGAAGGGG TAAGAATTATCT disrupted
ATTAAATGCGCA GAAGTTGCCTGA GGATGAATGTGC nifL gene
GCATAATGGTGC CCCTACGATTCCC CATTAAATGCGC
GTTGTGCGGGAA GCTATTTCATTCA AGCATAATGGTG
AACTGCTTTTTTT CTGACCGGAGGT CGTTGTGCGGGA
TGAAAGGGTTGG TCAAAATGACCC AAACTGCTTTTTT
TCAGTAGCGGAA AGCGAACCGAGT TTGAAAGGGTTG
AC CG GTCAGTAGCGGA
AAC/
AACTCACTTCAC
ACCCCGAAGGGG
GAAGTTGCCTGA
CCCTACGATTCCC
GCTATTTCATTCA
CTGACCGGAGGT
TCAAAATGACCC
AGCGAACCGAGT
CG-3′
CI006 ds30 N/A 320 CGCCAGAGAGTC 354 TTTAACGATCTGA 388 5′- 5′ UTR N/A N/A N/A
GAAATCGAACAT TTGGCGATGATG CGCCAGAGAGTC and ATG/
TTCCGTAATACCG AAACGGATTCGC GAAATCGAACAT truncated
CGATTACCCAGG CGGAAGATGCGC TTCCGTAATACC glnE gene
AGCCGTTCTGGTT TTTCTGAGAGCTG GCGATTACCCAG
GCACAGCGGAAA GCGCGAATTGTG GAGCCGTTCTGG
ACGTTAACGAAA GCAGGATGCGTT TTGCACAGCGGA
GGATATTTCGCAT GCAGGAGGAGGA AAACGTTAACGA
G TT AAGGATATTTCG
CATG/
TTTAACGATCTG
ATTGGCGATGAT
GAAACGGATTCG
CCGGAAGATGCG
CTTTCTGAGAGCT
GGCGCGAATTGT
GGCAGGATGCGT
TGCAGGAGGAGG
ATT-3′
CI006 ds31 N/A 321 CGCCAGAGAGTC 355 GCACTGAAACAC 389 5′- 5′ UTR N/A N/A N/A
GAAATCGAACAT CTCATTTCCCTGT CGCCAGAGAGTC and ATG/
TTCCGTAATACCG GTGCCGCGTCGC GAAATCGAACAT truncated
CGATTACCCAGG CGATGGTTGCCA TTCCGTAATACC glnE gene
AGCCGTTCTGGTT GTCAGCTGGCGC CFCGATTACCCAG
GCACAGCGGAAA GCTACCCGATCCT GAGCCGTTCTGG
ACGTTAACGAAA GCTTGATGAATT TTGCACAGCGGA
GGATATTTCGCAT GCTCGACCCGAA AAACGTTAACGA
G TA AAGGATATTTCG
CATG/
GCACTGAAACAC
CTCATTTCCCTGT
GTGCCGCGTCGC
CGATGGTTGCCA
GTCAGCTGGCGC
GCTACCCGATCC
TGCTTGATGAATT
GCTCGACCCGAA
TA-3′
CI019 ds34 N/A 322 GATGATGGATGC 356 GCGCTCAAACAG 390 5′- 5′ UTR N/A N/A N/A
TTTCTGGTTAAAC TTAATCCGTCTGT GATGATGGATGC and ATG/
GGGCAACCTCGT GTGCCGCCTCGC TTTCTGGTTAAAC truncated
TAACTGACTGACT CGATGGTCGCGA GGGCAACCTCGT glnE gene
AGCCTGGGCAAA CACAACTTGCAC TAACTGACTGAC
CTGCCCGGGCTTT GTCATCCTTTATT TAGCCTGGGCAA
TTTTTGCAAGGAA GCTCGATGAACT ACTGCCCGGGCT
TCTGATTTCATG GCTCGACCCGCG TTTTTTTGCAAGG
CA AATCTGATTTCAT
G/
GCGCTCAAACAG
TTAATCCGTCTGT
GTGCCGCCTCGC
CGATGGTCGCGA
CACAACTTGCAC
GTCATCCTTTATT
GCTCGATGAACT
GCTCGACCCGCG
CA-3′
CI019 ds70 up 323 ACCGATCCGCAG 357 AGTCTGAACTCA 391 5′- disrupted N/A N/A N/A
GCGCGCATTTGTT TCCTGCGGCAGT ACCGATCCGCAG nifL gene/
ATGCCAATCCGG CGGTGAGACGTA GCGCGCATTTGTT Prm4
CATTCTGCCGCCA TTTTTGACCAAAG ATGCCAATCCGG
GACGGGTTTTGC AGTGATCTACAT CATTCTGCCGCC
ACTTGAGACACTT CACGGAATTTTGT AGACGGGTTTTG
TTGGGCGAGAAC GGTTGTTGCTGCT CACTTGAGACAC
CACCGTCTGCTGG TAAAAGGGCAAA TTTTGGGCGAGA
T ACCACCGTCTGC
TGG/
AGTCTGAACTCA
TCCTGCGGCAGT
CGGTGAGACGTA
TTTTTGACCAAA
GAGTGATCTACA
TCACGGAATTTT
GTGGTTGTTGCTG
CTTAAAAGGGCA
AAT-3′
CI019 ds70 down 324 CATCGGACACCA 358 CCGTCTCTGAAG 392 5′- Prm4/ N/A N/A N/A
CCAGCTTACAAA CTCTCGGTGAAC CATCGGACACCA disrupted
TTGCCTGATTGCG ATTGTTGCGAGG CCAGCTTACAAA nifL gene
GCCCCGATGGCC CAGGATGCGAGC TTGCCTGATTGCG
GGTATCACTGAC TGGTTGTGTTTTG GCCCCGATGGCC
CGACCATTTCGTG ACATTACCGATA GGTATCACTGAC
CCTTATGTCATGC ATGTGCCGCGTG CGACCATTTCGT
GATGGGGGCTGG AACGGGTGCGTT GCCTTATGTCATG
G ATG CGATGGGGGCTG
GG/
CCGTCTCTGAAG
CTCTCGGTGAAC
ATTGTTGCGAGG
CAGGATGCGAGC
TGGTTGTGTTTTG
ACATTACCGATA
ATGTGCCGCGTG
AACGGGTGCGTT
ATG-3′
137 ds799 down 325 TCTTCAACAACTG 359 GCCATTGAGCTG 393 5′- PinfC/ SEQ SEQ SEQ
GAGGAATAAGGT GCTTCCCGACCG TCTTCAACAACT disrupted ID ID ID
ATTAAAGGCGGA CAGGGCGGCACC GGAGGAATAAGG nifL gene NO: NO: NO:
AAACGAGTTCAA TGCCTGACCCTGC TATTAAAGGCGG 417 418 419
ACGGCACGTCCG GTTTCCCGCTGTT AAAACGAGTTCA CTCG AGGG /56-
AATCGTATCAAT TAACACCCTGAC AACGGCACGTCC GCAG TGTT FAM/
GGCGAGATTCGC CGGAGGTGAAGC GAATCGTATCAA CATG AAAC AA
GCCCTGGAAGTT ATGATCCCTGAA TGGCGAGATTTCG GACG AGCG CGG
CGC TC CGCCCTGGAAGT TAA GGAA CAC
TCGC/ A G/
GCCATTGAGCTG ZEN/T
GCTTCCCGACCG CCG
CAGGGCGGCACC AAT
TGCCTGACCCTG CGT
CGTTTCCCGCTGT ATC
TTAACACCCTGA AA/
CCGGAGGTGAAG 3IABkFQ/
CATGATCCCTGA
ATC-3′
137 ds799 up 326 TCCGGGTTCGGCT 360 AGCGTCAGGTAC 394 5′- disrupted N/A N/A N/A
TACCCCGCCGCGT CGGTCATGATTC TCCGGGTTCGGC nifL gene/
TTTGCGCACGGTG ACCGTGCGATTCT TTACCCCGCCGC PinfC
TCGGACAATTTGT CGGTTCCCTGGA GTTTTGCGCACG
CATAACTGCGAC GCGCTTCATTGGC GTGTCGGACAAT
ACAGGAGTTTGC ATCCTGACCGAA TTGTCATAACTGC
GATGACCCTGAA GAGTTCGCTGGC GACACAGGAGTT
TATGATGCTCGA TTCTTCCCAACCT TGCGATGACCCT
G GAATATGATGCT
CGA/
AGCGTCAGGTAC
CGGTCATGATTC
ACCGTGCGATTC
TCGGTTCCCTGG
AGCGCTTCATTG
GCATCCTGACCG
AAGAGTTCGCTG
GCTTCTTCCCAAC
CTG-3′
137 ds809 N/A 327 ATCGCAGCGTCTT 361 GCGCTGAAGCAC 395 5′- 5′ UTR SEQ SEQ SEQ
TGAATATTTCCGT CTGATCACGCTCT ATCGCAGCGTCT and ATG/ ID ID ID
CGCCAGGCGCTG GCGCGGCGTCGC TTGAATATTTCCG truncated NO: NO: NO:
GCTGCCGAGCCG CGATGGTCGCCA TCGCCAGGCGCT glnE gene 420 421 422
TTCTGGCTGCATA GCCAGCTGGCGC GGCTGCCGAGCC GAGC GCCG /56-
GTGGAAAACGAT GCCACCCGCTGC GTTCTGGCTGCAT CGTT TCGG FAM
AATTTCAGGCCA TGCTGGATGAGC AGTGGAAAACGA CTGG CTGA TTAT
GGGAGCCCTTAT TGCTGGATCCCA TAATTTCAGGCC CTGC TAGA GGC
G ACA AGGGAGCCCTTA ATAG GG GC/
TG/ ZEN/T
GCGCTGAAGCAC GAA
CTGATCACGCTCT GCA
GCGCGGCGTCGC CCTG
CGATGGTCGCCA ATC
GCCAGCTGGCGC A/
GCCACCCGCTGC 3IABkFQ/
TGCTGGATGAGC
TGCTGGATCCCA
ACA-3′
137 ds843 up 328 TCCGGGTTCGGCT 362 GCCCGCTGACCG 396 5′- disrupted N/A N/A N/A
TACCCCGCCGCGT ACCAGAACTTCC TCCGGGTTCGGC nifL gene/
TTTGCGCACGGTG ACCTTGGACTCG TTACCCCGCCGC Prm1.2
TCGGACAATTTGT GCTATACCCTTGG GTTTTGCGCACG
CATAACTGCGAC CGTGACGGCGCG GTGTCGGACAAT
ACAGGAGTTTGC CGATAACTGGGA TTGTCATAACTGC
GATGACCCTGAA CTACATCCCCATT GACACAGGAGTT
TATGATGCTCGA CCGGTGATCTTAC TGCGATGACCCT
C GAATATGATGCT
CGA/
GCCCGCTGACCG
ACCAGAACTTCC
ACCTTGGACTCG
GCTATACCCTTG
GCGTGACGGCGC
GCGATAACTGGG
ACTACATCCCCA
TTCCGGTGATCTT
ACC-3′
137 ds843 down 329 TCACTTTTTAGCA 363 GCCATTGAGCTG 397 5′- Prm1.2/ N/A N/A N/A
AAGTTGCACTGG GCTTCCCGACCG TCACTTTTCAGCA disrupted
ACAAAAGGTACC CAGGGCGGCACC AAGTTGCACTGG nifL gene
ACAATTGGTGTA TGCCTGACCCTGC ACAAAAGGTACC
CTGATACTCGAC GTTTCCCGCTGTT ACAATTGGTGTA
ACAGCATTAGTG TAACACCCTGAC CTGATACTCGAC
TCGATTTTTCATA CGGAGGTGAAGC ACAGCATTAGTG
TAAAGGTAATTTT ATGATCCCTGAA TCGATTTTTCATA
G TC TAAAGGTAATTT
TG/
GCCATTGAGCTG
GCTTCCCGACCG
CAGGGCGGCACC
TGCCTGACCCTG
CGTTTCCCGCTGT
TTAACACCCTGA
CCGGAGGTGAAG
CATGATCCCTGA
ATC-3′
137 ds853 up 330 TCCGGGTTCGGCT 364 GCTAAAGTTCTC 398 5′- disrupted N/A N/A N/A
TACCCCGCCGCGT GGCTAATCGCTG TCCGGGTTCGGC nifL gene/
TTTGCGCACGGTG ATAACATTTGAC TTACCCCGCCGC Prm6.2
TCGGACAATTTGT GCAATGCGCAAT GTTTTGCGCACG
CATAACTGCGAC AAAAGGGCATCA GTGTCGGACAAT
ACAGGAGTTTGC TTTGATGCCCTTT TTGTCATAACTGC
GATGACCCTGAA TTGCACGCTTTCA GACACAGGAGTT
TATGATGCTCGA TACCAGAACCTG TGCGATGACCCT
GC GAATATGATGCT
CGA/
GCTAAAGTTCTC
GGCTAATCGCTG
ATAACATTDGAC
GCAATGCGCAAT
AAAAGGGCATCA
TTTGATGCCCTTT
TTGCACGCTTTCA
TACCAGAACCTG
GC-3′
137 ds853 down 331 GTTCTCCTTTGCA 365 GCCATTGAGCTG 399 5′- Prm6.2/ N/A N/A N/A
ATAGCAGGGAAG GCTTCCCGACCG GTTCTCCTTTGCA disrupted
AGGCGCCAGAAC CAGGGCGGCACC ATAGCAGGGAAG nifL gme
CGCCAGCGTTGA TGCCTGACCCTGC AGGCGCCAGAAC
AGCAGTTTGAAC GTTTCCCGCTGTT CGCCAGCGTTGA
GCGTTCAGTGTAT TAACACCCTGAC AGCAGTTTGAAC
AATCCGAAACTT CGGAGGTGAAGC GCGTTCAGTGTA
AATTTCGGTTTGG ATGATCCCTGAA TAATCCGAAACT
A TC TAATTTCGGTTTG
GA/
GCCATTGAGCTG
GCTTCCCGACCG
CAGGGCGGCACC
TGCCTGACCCTG
CGTTTCCCGCTGT
TTAACACCCTGA
CCGGAGGTGAAG
CATGATCCCTGA
ATC-3′
137 ds857 up 332 TCCGGGTTCGGCT 366 CGCCGTCCTCGC 400 5′- disrupted N/A N/A N/A
TACCCCGCCGCGT AGTACCATTGCA TCCGGGTTCGGC nifL gene/
TTTGCGCACGGTG ACCGACTTTACA TTACCCCGCCGC Prm8.2
TCGGACAATTTGT GCAAGAAGTGAT GTTTTGCGCACG
CATAACTGCGAC TCTGGCACGCAT GTGTCGGACAAT
ACAGGAGTTTGC GGAACAAATTCT TTGTCATAACTGC
GATGACCCTGAA TGCCAGTCGGGC GACACAGGAGTT
TATGATGCTCGA TTTATCCGATGAC TGCGATGACCCT
GAA GAATATGATGCT
CGA/
CGCCGTCCTCGC
ACTACCATTGCA
ACCGACTTTACA
GCAAGAAGTGAT
TCTGGCACGCAT
GGAACAAATTCT
TGCCAGTCGGGC
TTTATCCGATGAC
GAA-3′
137 ds857 down 333 GATATGCCTGAA 367 GCCATTGAGCTG 401 5′- Prm8.2/ N/A N/A N/A
GTATTCAATTACT GCTTCCCGACCG GATATGCCTGAA disrupted
TAGGCATTTACTT CAGGGCGGCACC GTATTCAATTACT nifL gene
AACGCAGGCAGG TGCCTGACCCTGC TAGGCATTTACTT
CAATTTTGATGCT GTTTCCCGCTGTT AACGCAGGCAGG
GCCTATGAAGCG TAACACCCTGAC CAATTTTGATGCT
TTTGATTCTGTAC CGGAGGTGAAGC GCCTATGAAGCG
TTGAGCTTGATC ATGATCCCTGAA TTTGATTCTGTAC
TC TTGAGCTTGATC/
GCCATTGAGCTG
GCTTCCCGACCG
CAGGGCGGCACC
TGCCTGACCCTG
CGTTTCCCGCTGT
TTAACACCCTGA
CCGGAGGTGAAG
CATGATCCCTGA
ATC-3′
63 ds908 down 334 TGGTATTGTCAGT 368 TCTTTAGATCTCT 402 5′- PinfC/ SEQ SEQ N/A
CTGAATGAAGCT CGGTCCGCCCTG TGGTATTGTCAGT disrupted ID ID
CTTGAAAAAGCT ATGGCGGCACCT CTGAATGAAGCT nifL gene NO: NO:
GAGGAAGCGGGC TGCTGACGTTAC CTTGAAAAAGCT 423 424
GTCGATTTAGTAG GCCTGCCGGTAC GAGGAAGCGGGC GGAA GGGC
AAATCAGTCCGA AGCAGGTTATCA GTCGATTTAGTA AACG GGAC
ATGCCGAGCCGC CCGGAGGCTTAA GAAATCAGTCCG AGTT CGAG
CAGTTTGTCGAAT AATGACCCAGTT AATGCCGAGCCG CAAC AGAT
C ACC CCAGTTTGTCGA CGGC CTAA
ATC/
TCTTTAGATCTCT
CGGTCCGCCCTG
ATGGCGGCACCT
TGCTGACGTTAC
GCCTGCCGGTAC
AGCAGGTTATCA
CCGGAGGCTTAA
AATGACCCAGTT
ACC-3′
63 ds908 up 335 TGCAAATTGCAC 369 TGAATATCACTG 403 5′- disrupted N/A N/A N/A
GGTTATTCCGGGT ACTCACAAGCTA TGCAAATTGCAC nifL gene/
GAGTATATGTGT CCTATGTCGAAG GGTFATTCCGGG PinfC
GATTTGGGTTCCG AATTAACTAAAA TGAGTATATGTG
GCATTGCGCAAT AACTGCAAGATG TGATTTGGGTTCC
AAAGGGGAGAAA CAGGCATTCGCG GGCATTGCGCAA
GACATGAGCATC TTAAAGCCGACT TAAAGGGGAGAA
ACGGCGTTATCA TGAGAAATGAGA AGACATGAGCAT
GC AGAT CACGGCGTTATC
AGC/
TGAATATCACTG
ACTCACAAGCTA
CCTATGTCGAAG
AATTAACTAAAA
AACTGCAAGATG
CAGGCATTCGCG
TTAAAGCCGACT
TGAGAAATGAGA
AGAT-3′
910 ds960 up 336 TCAGGGCTGCGG 370 CTGGGGTCACTG 404 5′- disrupted N/A N/A N/A
ATGTCGGGCGTTT GAGCGCTTTATC TCAGGGCTGCGG nifL gene/
CACAACACAAAA GGCATCCTGACC ATGTCGGGCGTT PinfC
TGTTGTAAATGCG GAAGAATTTGCC TCACAACACAAA
ACACAGCCGGGC GGTTTCTTCCCGA ATGTTGTAAATG
CTGAAACCAGGA CCTGGCTGGCCC CGACACAGCCGG
GCGTGTGATGAC CTGTTCAGGTTGT GCCTGAAACCAG
CTTTAATATGATG GGTGATGAATAT GAGCGTGTGATG
C CA ACCTTTAATATG
ATGC/
CTGGGGTCACTG
GAGCGCTTTATC
GGCATCCTGACC
GAAGAATTTGCC
GGTTTCTTCCCGA
CCTGGCTGGCCC
CTGTTCAGGTTGT
GGTGATGAATAT
CA-3′
910 ds960 down 337 CGGAAAACGAGT 371 GCAATAGAACTA 405 5′- PinfC/ N/A N/A N/A
TCAAACGGCACG ACTACCCGCCCT CGGAAAACGAGT disrupted
TCCGAATCGTATC GAAGGCGGTACC TCAAACGGCACG nifL gene
AATGGCGAGATT TGCCTGACCCTGC TCCGAATCGTAT
CGCGCCCAGGAA GATTCCCGTTATT CAATGGCGAGAT
GTTCGCTTAACTG TCATTCACTGACC TCGCGCCCAGGA
GTCTGGAAGGTG GGAGGCCCACGA AGTTCGCTTAACT
AGCAGCTGGGTA TGACCCAGCGAC GGTCTGGAAGGT
TT C GAGCAGCTGGGT
ATT/
GCAATAGAACTA
ACTACCCGCCCT
GAAGGCGGTACC
TGCCTGACCCTG
CGATTCCCGTTAT
TTCATTCACTGAC
CGGAGGCCCACG
CC-3′
TABLE F
Engineered Non-intergeneric Microbes
Strain Name Genotype SEQ ID NO
CI006 16S rDNA-contig 5 62
CI006 16S rDNA-contig 8 63
CI019 16S rDNA 64
CI006 nifH 65
CI006 nifD 66
CI006 nifK 67
CI006 nifL 63
CI006 nifA 69
CI019 nifH 70
CI019 nifD 71
CI019 nifK 72
CI019 nifL 73
CI019 nifA 74
CI006 Prm5 with 500 bp 75
flanking regions
CI006 nifLA operon-upstream 76
intergenic region plus
nifL and nifA CDSs
CI006 nifL (Amino Acid) 77
CI006 nifA (Amino Acid) 78
CI006 glnE 79
CI006 glnE_KO1 80
CI006 glnE (Amino Acid) 81
CI006 glnE_KO1 (Amino Acid) 82
CI006 GME ATase domain 83
(Amino Acid)
CM029 Prm5 inserted into nifL 84
region
TABLE G
Engineered Non-intergeneric Microbes
Associated
Novel
Strain SEQ ID Junction If
Strain ID NO Genotype Description Applicable
CI63; 63 SEQ ID 16S N/A N/A
CI063 NO 85
CI63; 63 SEQ ID nifH N/A N/A
CI063 NO 86
CI63; 63 SEQ ID nifD1 1 of 2 unique genes annotated as nifD N/A
CI063 NO 87 in 63 genome
CI63; 63 SEQ ID nifD2 2 of 2 unique genes annotated as nifD N/A
CI063 NO 88 in 63 genome
CI63; 63 SEQ ID nifK1 1 of 2 unique genes annotated as nifK N/A
CI063 NO 89 in 63 genome
CI63; 63 SEQ ID nifK2 2 of 2 unique genes annotated as nifK N/A
CI063 NO 90 in 63 genome
CI63; 63 SEQ ID nifL N/A N/A
CI063 NO 91
CI63; 63 SEQ ID nifA N/A N/A
CI063 NO 92
CI63; 63 SEQ ID glnE N/A N/A
CI063 NO 93
CI63; 63 SEQ ID amtB N/A N/A
CI063 NO 94
CI63; 63 SEQ ID PinfC 500 bp immediately upstrea of the ATG N/A
CI063 NO 95 start codon of the infC gene
CI137 137 SEQ ID 16S N/A N/A
NO 96
CI137 137 SEQ ID nifH1 1 of 2 unique genes annotated as nifH N/A
NO 97 in 137 genome
CI137 137 SEQ ID nifH2 2 of 2 unique genes annotated as nifH N/A
NO 98 in 137 genome
CI137 137 SEQ ID nifDl 1 of 2 unique genes annotated as nifD N/A
NO 99 in 137 genome
CI137 137 SEQ ID nifD2 2 of 2 unique genes annotated as nifD N/A
NO 100 in 137 genorne
CI137 137 SEQ ID nifK1 1 of 2 unique genes annotated as nifK N/A
NO 101 in 137 genome
CI137 137 SEQ ID nifK2 2 of 2 unique genes annotated as nifK N/A
NO 102 in 137 genome
CI137 137 SEQ ID nifL N/A N/A
NO 103
CI137 137 SEQ ID nifA N/A N/A
NO 104
CI137 137 SEQ ID glnE N/A N/A
NO 105
CI137 137 SEQ ID PinfC 500 bp immediately upstream of the N/A
NO 106 TTG start codon of infC
CI137 137 SEQ ID amtB N/A N/A
NO 107
CI137 137 SEQ ID Prm8.2 internal promoter located in nfpI gene; N/A
NO 108 299 bp starting at 81 bp after the A of
the ATG of the nlpI gene
CI137 137 SEQ ID Prm6.2 300 bp upstream of the secE gene N/A
NO 109 starting at 57 bp upstream of the A of
the ATG of secE
CI137 137 SEQ ID Prm1.2 400 bp immediately upstream of the N/A
No 110 ATG of cspE gene
none 728 SEQ ID 16S N/A N/A
No 111
none 728 SEQ ID nifH N/A N/A
NO 112
none 728 SEQ ID nifD1 1 of 2 unique genes annotated as nifD N/A
NO 113 in 728 genome
none 728 SEQ ID nifD2 2 of 2 unique genes annotated as nifD N/A
NO 114 in 728 genome
none 728 SEQ ID nifK1 1 of 2 unique genes annotated as nifK N/A
NO 115 in 728 genome
none 728 SEQ ID nifK2 2 of 2 unique genes annotated as nifK N/A
NO 116 in 728 genome
none 728 SEQ ID nifL N/A N/A
NO 117
none 728 SEQ ID nifA N/A N/A
NO 118
none 728 SEQ ID glnE N/A N/A
NO 119
none 728 SEQ ID amtB N/A N/A
NO 120
none 850 SEQ ID 16S N/A N/A
NO 121
none 852 SEQ ID 16S N/A N/A
NO 122
none 853 SEQ ID 16S N/A N/A
NO 123
none 910 SEQ ID 16S N/A N/A
NO 124
none 910 SEQ ID nifH N/A N/A
NO 125
none 910 SEQ ID Dinitrogenase iron- N/A N/A
NO 126 molybdenum
cofactor CDS
none 910 SEQ ID nifD1 N/A N/A
NO 127
none 910 SEQ ID nifD2 N/A N/A
NO 128
none 910 SEQ ID nifK1 N/A N/A
NO 129
none 910 SEQ ID nifK2 N/A N/A
NO 130
none 910 SEQ ID nifL N/A N/A
NO 131
none 910 SEQ ID nifA N/A N/A
NO 132
none 910 SEQ ID glnE N/A N/A
NO 133
MC 910 SEQ ID amtB N/A N/A
NO 134
none 910 SEQ ID PinfC 498 bp immediately upstream of the N/A
NO 135 ATG of the infC gene
none 1021 SEQ ID 16S N/A N/A
NO 136
none 1021 SEQ ID nifH N/A N/A
NO 137
none 1021 SEQ ID nifD1 1 of 2 unique genes annotated as nifD N/A
NO 138 in 910 genome
none 1021 SEQ ID nifD2 2 of 2 unique genes annotated as nifD N/A
NO 139 in 910 genome
none 1021 SEQ ID nifK1 1 of 2 unique genes annotated as nifK N/A
NO 140 in 910 genome
none 1021 SEQ ID nifK2 2 of 2 unique genes annotated as nifK N/A
NO 141 in 910 genome
none 1021 SEQ ID nifL N/A N/A
NO 142
none 1021 SEQ ID nifA N/A N/A
NO 143
none 1021 SEQ ID glnE N/A N/A
NO 144
none 1021 SEQ ID amtB N/A N/A
NO 145
none 1021 SEQ ID PinfC 500 bp immediately upstream of the N/A
NO 146 ATG start codon of the infC gene
none 1021 SEQ ID Prm1 348 bp includes the 319 bp immediately N/A
NO 147 upstream of the ATG start codon of the
lpp gene and the first 29 bp of the lpp
gene
none 1021 SEQ ID Prm7 339 bp upstream of the sspA gene, N/A
NO 148 ending at 46 bp upstream of the ATG of
the sspA gene
none 1113 SEQ ID 16S N/A N/A
NO 149
none 1113 SEQ ID nifH N/A N/A
NO 150
none 1113 SEQ ID nifD1 1 of 2 unique genes annotated as nifD N/A
NO 151 in 1113 genome
none 1113 SEQ ID nifD2 2 of 2 unique genes annotated as nifD N/A
NO 152 in 1113 genome
none 1113 SEQ ID nifK N/A N/A
NO 153
none 1113 SEQ ID nifL N/A N/A
NO 154
none 1113 SEQ ID nifA partial gene due to a gap in the sequence assembly, N/A
NO 155 we can only identify a partial gene
from the 1113 genome
none 1113 SEQ ID glnE N/A N/A
NO 156
none 1116 SEQ ID 16S N/A
NO 157
none 1116 SEQ ID nifH N/A
NO 158
none 1116 SEQ ID nifD1 1 of 2 unique genes annotated as nifD N/A
NO 159 in 1116 genome
none 1116 SEQ ID nifD2 2 of 2 unique genes annotated as nifD N/A
NO 160 in 1116 genome
none 1116 SEQ ID nifK1 1 of 2 unique genes annotated as nifK N/A
NO 161 in 1116 genome
none 1116 SEQ ID nifK2 2 of 2 unique genes annotated as nifK N/A
NO 162 in 1116 genome
none 1116 SEQ ID nifL N/A N/A
NO 163
none 1116 SEQ ID nifA N/A N/A
NO 164
none 1116 SEQ ID glnE N/A N/A
NO 165
none 1116 SEQ ID amtB N/A N/A
NO 166
none 1293 SEQ ID 16S N/A N/A
NO 167
none 1293 SEQ ID nifH N/A N/A
NO 168
none 1293 SEQ ID nifD1 1 of 2 unique genes annotated as nifD N/A
NO 169 in 1293 genome
none 1293 SEQ ID nifD2 2 of 2 unique genes annotated as nifD N/A
NO 170 in 1293 genome
none 1293 SEQ ID nifK 1 of 2 unique genes annotated as nifK N/A
NO 171 in 1293 genome
none 1293 SEQ ID nifK1 2 of 2 unique genes annotated as nifK N/A
NO 172 in 1293 genome
none 1293 SEQ ID nifA N/A N/A
NO 173
none 1293 SEQ ID glnE N/A N/A
NO 174
none 1293 SEQ ID amtB1 1 of 2 unique genes annotated as amtB N/A
NO 175 in 1293 genome
none 1293 SEQ ID amtB2 2 of 2 unique genes annotated as amtB N/A
NO 176 in 1293 genome
none 1021-1612 SEQ ID ΔnifL::PinfC starting at 24 bp after the A of the ATG ds1131
NO 177 start codon, 1375 bp of nifL, have been
deleted and replaced with the 1021
PinfC promoter sequence
none 1021-1612 SEQ ID ΔnifL::PinfC with starting at 24 bp after the A of the ATG ds1131
NO 178 500 bp flank start codon, 1375 bp of nifL have been
deleted and replaced with the 1021
PinfC promoter sequence; 500 bp
flanking the nifL gene upstream and
downstream are included
none 1021-1612 SEQ ID glnEΔAR-2 glnE gene with 1673 bp immediately ds1133
NO 179 downstream of the ATG start codon
deleted, resulting in a truncated glnE
protein lacking the adenylyl-removing
(AR) domain
none 1021-1612 SEQ ID glnEΔAR-2 with glnE gene with 1673 bp immediately ds1133
NO 180 500 bp flank downstream of the ATG start codon
deleted, resulting in a truncated glnE
protein lacking the adenylyl-removing
(AR) domain; 500 bp flanking the glnE
gene upstream and downstream are
included
none 1021-1615 SEQ ID ΔnifL::Prm1 starting at 24 bp after the A of the ATG ds1145
NO 181 start codon, 1375 bp of nifL have been
deleted and replaced with the 1021
Prm1 promoter sequence
none 1021-1615 SEQ ID ΔnifL:Prm1 with starting at 24 bp after the A of the ATG ds1145
NO 182 500 bp flank start codon, 1375 bp of nifL have been
deleted and replaced with the 1021 rm1
promoter sequence; 500 bp flanking the
nifL gene upstream and downstream
are included
none 1021-1615 SEQ ID glnEΔAR-2 glnE gene with 1673 bp immediately ds1133
NO 183 downstream of the ATG start codon
deleted, resulting in a truncated glnE
protein lacking the adenylyl-removing
(AR) domain
none 1021-1615 SEQ ID glnEΔAR-2 with glnE gene with 1673 bp immediately ds1133
NO 184 500 bp flank downstream of the ATG start codon
deleted, resulting in a truncated glnE
protein lacking the adenylyl-removing
(AR) domain; 500 bp flanking the glnE
gene upstream and downstream are
included
none 1021-1619 SEQ ID ΔnifL::Prm1 starting at 24 bp after the A of the ATG ds1145
NO 185 start codon, 1375 bp of nifL have been
deleted and replaced with the 1021
Prm1 promoter sequence
none 1021-1619 SEQ ID ΔnifL:Prm1 with starting at 24 bp after the A of the ATG ds1145
NO 186 500 bp flank start codon, 1375 bp of nifL have been
deleted and replaced with the 1021 rm1
promoter sequence; 500 bp flanking the
nifL gene upstream and downstream
are included
none 1021-1623 SEQ ID glnEΔAR-2 glnE gene with 1673 bp immediately ds1133
NO 187 downstream of the ATG start codon
deleted, resulting in a truncated glnE,
protein lacking the adenylyl-removing
(AR) domain
none 1021-1623 SEQ ID glnEΔAR-2 with glnE gene with 1673 bp immediately ds1133
NO 188 500 bp flank downstream of the ATG start codon
deleted, resulting in a truncated glnE
protein lacking the adenylyl-removing
(AR) domain; 500 bp flanking the glnE
gene upstream and downstream are
included
none 1021-1623 SEQ ID ΔnifL::Prm7 starting at 24 bp after the A of the ATG ds1148
NO 189 start codon, 1375 bp of nifL have been
deleted and replaced with the 1021
Prm7 promoter sequence
none 1021-1623 SEQ ID ΔnifL::Prm7 with starting at 24 bp after the A of the ATG ds1148
NO 190 500 bp flank start codon, 1375 bp of nifL have been
deleted and replaced with the 1021 rm7
promoter sequence; 500 bp flanking the
nifL gene upstream and downstream
are included
none 137-1034 SEQ ID glnEΔAR-2 glnE gene with 1290 bp immediately ds809
NO 191 downstream of the ATG start codon
deleted, resulting in a truncated glnE
protein lacking the adenylyl-removing
(AR) domain
none 137-1034 SEQ ID glnEΔAR-2 with glnE gene with 1290 bp immediately ds809
NO 192 500 bp flank downstream of the ATG start codon
deleted, resulting in a truncated glnE
protein lacking the adenylyl-removing
(AR) domain; 500 bp flanking the glnE
gene upstream and downstream are
included
none 137-1036 SEQ ID ΔnifL::PinfC starting at 24 bp after the A of the ATG ds799
NO 193 start codon, 1372 bp of nifL have been
deleted and replaced with the 137
PinfC promoter sequence
none 137-1036 SEQ ID ΔnifL::PinfC with starting at 24 bp after the A of the ATG ds799
NO 194 500 bp flank start codon, 1372 bp of nifL have been
deleted and replaced with the 137
PinfC promoter sequence; 500 bp
flanking the nifL gene upstream and
downstream are included
none 137-1314 SEQ ID glnEΔAR-2 36 bp glnE gene with 1290 bp immediately none
NO 195 deletion downstream of the ATG start codon
deleted AND 36 bp deleted beginning at
1472 bp downstream of the start codon,
resulting in a truncated glnE protein
lacking the adenylyl-removing (AR)
domain
none 137-1314 SEQ ID glnEΔAR-2 36 bp glnE gene with 1290 bp immediately none
NO 196 deletion downstream of the ATG start codon
deleted AND 36 bp deleted beginning at
1472 bp downstream of the start codon,
resulting in a truncated glnE protein
lacking the adenylyl-removing (AR)
domain; 500 bp flanking the nifL gene
upstream and downstream are included
none 137-1314 SEQ ID ΔnifL::Prm8.2 starting at 24 bp after the A of the ATG ds857
NO 197 start codon, 1372 bp of nifL have been
deleted and replaced with the 137
Prm8.2 promotor sequence
none 137-1314 SEQ ID ΔnifL::Prm8.2 with starting at 24 bp after the A of the ATG ds857
NO 198 500 bp flank start codon, 1372 bp of nifL have been
deleted and replaced with the 137
Prm8.2 promoter sequence: 500 bp
flanking the nifL gene upstream and
downstream are included
none 137-1329 SEQ ID glnEΔAR-2 36 bp glnE gene with 1290 bp immediately none
NO 199 deletion downstream of the ATG start codon
deleted AND 36 bp deleted beginning at
1472 bp downstream of the start codon
resulting in a truncated gluE protein
lacking the adenylyl-removing (AR)
domain
none 137-1329 SEQ ID glnEΔAR-2 36 bp glnE gene with 1290 bp immediately none
NO 200 deletion downstream of the ATG start codon
deleted AND 36 bp deleted beginning at
1472 bp downstream of the start codon,
resulting in a truncated glnE protein
lacking the adenylyl-removing (AR)
domain; 500 bp flanking the nifL gene
upstream and downstream are included
none 137-1329 SEQ ID ΔnifL::Prm6.2 starting at 24 bp after the A of the ATG ds853
NO 201 start codon, 1372 bp of nifL have been
deleted and replaced with the 137
Prm6.2 promoter sequence
none 137-1329 SEQ ID ΔnifL::Prm6.2 with starting at 24 bp after the A of the ATG ds853
NO 202 500 bp flank start codon, 1372 bp of nifL have been
deleted and replaced with the 137
Prm6.2 promoter sequence; 500 bp
flanking the nifL gene upstream and
downstream are included
none 137-1382 SEQ ID ΔnifL::Prm1.2 starting at 24 bp after the A of the ATG
NO 203 start codon, 1372 bp of nifL have been ds843
deleted and replaced with the 137
Prm1.2 promoter sequence
none 137-1382 SEQ ID ΔnifL::Prm1.2 with starting at 24 bp after the A of the ATG ds843
NO 204 500 bp flank start codon, 1372 bp of nifL have been
deleted and replaced with the 137
Prm1.2 promoter sequence; 500 bp
flanking the nifL gene upstream and
downstream are included
none 137-1382 SEQ ID glnEΔAR-2 36 bp glnE gene with 1290 bp immediately none
NO 205 deletion downstream of the ATG start codon
deleted AND 36 bp deleted beginning at
1472 bp downstream of the start codon,
resulting in a truncated glnE protein
lacking the adenylyl-removing (AR)
domain
none 137-1382 SEQ ID glnEΔAR-2 36 bp glnE gene with 1290 bp immediately none
NO 206 deletion downstream of the ATG start codon
deleted AND 36 bp deleted beginning at
1472 bp downstream of the start codon,
resulting in a truncated glnE protein
lacking the adenylyl-removing (AR)
domain; 500 bp flanking the nifL gene
upstream and downstream are included
none 137-1586 SEQ ID ΔnifL::PinfC starting at 24 bp after the A of the ATG ds799
NO 207 start codon, 1372 bp of nifL have been
deleted and replaced with the 137
PinfC promoter sequence
none 137-1586 SEQ ID ΔnifL::PinfC with starting at 24 bp after the A of the ATG ds799
NO 208 500 bp flank start codon, 1372 bp of nifL have been
deleted and replaced with the 137
PinfC promoter sequence; 500 bp
flanking the nifL gene upstream and
downstream are included
none 137-1586 SEQ ID glnEΔAR-2 glnE gene with 1290 bp immediately
NO 209 downstream of the ATG start codon
deleted, resulting in a truncated glnE ds809
protein lacking the adenylyl-removing
(AR) domain
none 137-1586 SEQ ID glnEΔAR-2 with glnE gene with 1290 bp immediately ds809
NO 210 500 bp flank downstream of the ATG start codon
deleted, resulting in a truncated glnE
protein lacking the adenylyl-removing
(AR) domain; 500 bp flanking the glnE
gene upstream and downstream are
included
none 19-594 SEQ ID glnEΔAR-2 glnE gene with 1650 bp immediately ds34
NO 211 downstream of the ATG start codon
deleted, resulting in a truncated glnE
protein lacking the adenylyl-removing
(AR) domain
none 19-594 SEQ ID glnEΔAR-2 with glnE gene with 1650 bp immediately ds34
NO 212 500 bp flank downstream of the ATG start codon
deleted, resulting in a truncated glnE
protein lacking the adenylyl-removing
(AR) domain; 500 bp flanking the glnE
gene upstream and downstream are
included
none 19-594 SEQ ID ΔnifL::Prm6.1 starting at 221 bp after the A of the ds180
NO 213 ATG start codon, 845 bp of nifL have
been deleted and replaced with the
CI019 Prm6.1 promoter sequence
none 19-594 SEQ ID ΔnifL::Prm6.1 with starting at 221 bp after the A of the ds180
NO 214 500 bp flank ATG start codon, 845 bp of nifL have
been deleted and replaced with the
CI019 Prm6.1 promoter sequence;
500 bp flanking the nifL gene upstream
and downstream are included
none 19-714 SEQ ID ΔnifL::Prm6.1 starting at 221 bp after the A of the ds180
NO 215 ATG start codon, 845 bp of nifL have
been deleted and replaced with the
CI019 Prm6.1 promoter sequence
none 19-714 SEQ ID ΔnifL::Prm6.1 with starting at 221 bp after the A of the ds180
NO 216 500 bp flank ATG start codon, 845 bp of nifL have
been deleted and replaced with the
CI019 Prm6.1promoter sequence;
500 bp flanking the nifL gene upstream
and downstream are included
none 19-715 SEQ ID ΔnifL::Prm7.1 starting at 221 bp after the A of the ds181
NO 217 ATG start codon, 845 bp of nifL have
been deleted and replaced with the
CI019 Prm7.1 promoter sequence
none 19-715 SEQ ID ΔnifL::Prm7.1 with starting at 221 bp after the A of the ds181
NO 218 500 bp flank ATG start codon, 845 bp of nifL have
been deleted and replaced with the
CI019 Prm76.1promoter sequence;
500 bp flanking the nifL gene upstream
and downstream are included
19-713 19-750 SEQ ID ΔnifL::Prm1.2 starting at 221 bp after the A of the ds172
NO 219 ATG start codon, 845 bp of nifL have
been deleted and replaced with the
CI019 Prm1.2 promoter sequence
19-713 19-750 SEQ ID ΔnifL::Prm1.2 with starting at 22l bp after the A of the ds172
NO 220 500 bp flank ATG start codon, 845 bp of nifL have
been deleted and replaced with the
CI019 Prm1.2 promoter sequence;
500 bp flanking the nifL gene upstream
and downstream are included
17-724 19-804 SEQ ID ΔnifL::Prm1.2 starting at 221 bp after the A of the ds172
NO 221 ATG start codon, 845 bp of nifL have
been deleted and replaced with the
CI019 Prm1.2 promoter sequence
17-724 19-804 SEQ ID ΔnifL::Prm1.2 with starting at 221 bp after the A of the ds172
NO 222 500 bp flank ATG start codon, 845 bp of nifL have
been deleted and replaced with the
CI019 Prm1.2 promoter sequence;
500 bp flanking the nifL gene upstream
and downstream are included
17-724 19-804 SEQ ID glnEΔAR-2 glnE gene with 1650 bp immediately ds34
NO 223 downstream of the ATG start codon
deleted, resulting in a truncated glnE
protein lacking the adenylyl-removing
(AR) domain
17-724 19-804 SEQ ID glnEΔAR-2 with glnE gene with 1650 bp immediately ds34
NO 224 500 bp flank downstream of the ATG start codon
deleted, resulting in a truncated glnE
protein lacking the adenylyl-removing
(AR) domain; 500 bp flanking the glnE
gene upstream and downstream are
included
19-590 19-806 SEQ ID ΔnifL::Prm3.1 starting at 22l bp after the A of the ds175
NO 225 ATG start codon, 845 bp of nifL have
been deleted and replaced with the
CI019 Prm3.1 promoter sequence
19-590 19-806 SEQ ID ΔnifL::Prm3.1 with starting at 221 bp after the A of the ds175
NO 226 500 bp flank ATG start codon, 845 bp of nifL have
been deleted and replaced with the
CI019 Prm3.1 promoter sequence;
500 bp flanking the nifL gene upstream
and downstream are included
19-590 19-806 SEQ ID glnEΔAR-2 glnE gene with 1650 bp immediately ds34
NO 227 downstream of the ATG start codon
deleted, resulting in a truncated glnE
protein lacking the adenylyl-removing
(AR) domain
19-590 19-806 SEQ ID glnEΔAR-2 with glnE gene with 1650 bp immediately ds34
NO 228 500 bp flank downstream of the ATG start codon
deleted, resulting in a truncated glnE
protein lacking the adenylyl-removing
(AR) domain; 500 bp flanking the glnE
gene upstream and downstream are
included
none 63-1146 SEQ ID ΔnifL::PinfC starting at 24 bp after the A of the ATG ds908
NO 229 start codon, 1375 bp of nifL have been
deleted and replaced with the 63 PinfC
promoter sequence
none 63-1146 SEQ ID ΔnifL::PinfC with starting at 24 bp after the A of the ATG ds908
NO 230 500 bp flank start codon, 1375 bp of nifL have been
deleted and replaced with the 63 PinfC
500 bp flank sequence; 500 bp flanking the
nifL gene upstream and downstream
are included
CM015; 6-397 SEQ ID ΔnifL::Prm5 starting at 31 bp after the A of the ATG ds24
PBC6.15 NO 231 start codon, 1375 bp of nifL have been
deleted and replaced with the CI006
Prm5 promoter sequence
CM015; 6-397 SEQ ID ΔnifL::Prm5 with starting at 31 bp after the A of the ATG ds24
PBC6.15 NO 232 500 bp flank start codon, 1375 bp of nifL have been
deleted and replaced with the CI006
Prm5 promoter sequence; 500 bp
flanking the nifL gene upstream and
downstream are included
CM014 6-400 SEQ ID ΔnifL:Prm1 starting at 31 bp after the A of the ATG ds20
NO 233 start codon, 1375 bp of nifL have been
deleted and replaced with the CI006
Prm1 promoter sequence
CM014 6-400 SEQ ID ΔnifL::Prm1 with starting at 31 bp after the A of the ATG ds20
NO 234 500 bp flank start codon, 1375 bp of nifL have been
deleted and replaced with the CI006
Prm1 promoter sequence; 500 bp
flanking the nifL gene upstream and
downstream are included
CM037; 6-403 SEQ ID ΔnifL::Prm1 starting at 31 bp after the A of the ATG ds20
PBC6.37 NO 235 start codon, 1375 bp of nifL have been
deleted and replaced with the CI006
Prm1 promoter sequence
CM037; 6-403 SEQ ID ΔnifL::Prm1 with starting at 3l bp after the A of the ATG ds20
PBC6.38 NO 236 500 bp flank start codon, 1375 bp of nifL have been
deleted and replaced with the CI006
Prm1 promoter sequence: 500 bp
flanking the nifL gene upstream and
downstream are included
CM037; 6-403 SEQ ID glnEΔAR-2 glnE gene with 1644 bp immediately ds31
PBC6.3-9 NO 237 downstream of the ATG start codon
deleted, resulting in a truncated glnE
protein lacking the adenylyl-removing
(AR) domain
CM037; 6-403 SEQ ID glnEΔAR-2 with glnE gene with 1644 bp immediately ds31
PBC6.40 NO 238 500 bp flank downstream of the ATG start codon
deleted, resulting in a truncated glnE
protein lacking the adenylyl-removing
(AR) domain; 500 bp flanking the glnE
gene upstream and downstream are
included
CM038; 6-404 SEQ ID glnEΔAR-1 glnE gene with 1287 bp immediately ds30
PBC6.38 NO 239 downstream of the ATG start codon
deleted, resulting in a truncated glnE
protein lacking the adenylyl-removing
(AR) domain
CM038; 6-404 SEQ ID ΔnifL::Prm1 starting at 31 bp after the A of the ATG ds20
PBC6.38 NO 240 start codon, 1375 bp of nifL have been
deleted and replaced with the CI006
Prm1 promoter sequence
CM038; 6-404 SEQ ID ΔnifL:Prm1 with starting at 31 bp after the A of the ATG ds20
PBC6.38 NO 241 500 bp flank start codon, 1375 bp of nifL have been
deleted and replaced with the CI006
Prm1 promoter sequence; 500 bp
flanking the nifL gene upstream and
downstream are included
CM038; 6-404 SEQ ID glnEΔAR4 with glnE gene with 1287 bp immediately ds30
PBC6.38 NO 242 500 bp flank downstream of the ATG start codon
deleted, resulting in a truncated glnE
protein lacking the adenylyl-removing
(AR) domain; 500 bp flanking the glnE
gene upstream and downstream are
included
CM029; 6-412 SEQ ID glnEΔAR-1 glnE gene with 1287 bp immediately ds30
PBC6.29 No 243 downstream of the ATG start codon
deleted, resulting in a truncated glnE
protein lacking the adenylyl-removing
(AR) domain
CM029; 6-412 SEQ ID glnEΔAR-1 with glnE gene with 1287 bp immediately ds30
PBC6.29 NO 244 500 bp flank downstream of the ATG start codon
deleted, resulting in a truncated glnE
protein lacking the adenylyl-removing
(AR) domain; 500 bp flanking the glnE
gene upstream and downstream are
included
CM029; 6-412 SEQ ID ΔnifL::Prm5 starting at 31 bp after the A of the ATG ds24
PBC6.29 NO 245 start codon, 1375 bp of nifL have been
deleted and replaced with the CI006
Prm5 promoter sequence
CM029; 6-412 SEQ ID ΔnifL::Prm5 with starting at 31 bp after the A of the ATG ds24
PBC6.29 NO 246 500 bp flank start codon, 1375 bp of nifL have been
deleted and replaced with the CI006
Prm5 promoter sequence; 500 bp
flanking the nifL gene upstream and
downstream are included
CM093; 6-848 SEQ ID ΔnifL::Prm1 starting at 31 bp after the A of the ATG ds20
PBC6.93 NO 247 start codon, 1375 bp of nifL have been
deleted and replaced with the CI006
Prm1 promoter sequence
CM093; 6-848 SEQ ID ΔnifL::Prm1 with starting at 31 bp after the A of the ATG ds20
PBC6.93 NO 248 500 bp flank start codon, 1375 bp of nifL have been
deleted and replaced with the CI006
Prm1 promoter sequence; 500 bp
flanking the nifL gene upstream and
downstream are included
CM093; 6-848 SEQ ID glnEΔAR-2 glnE gene with 1644 bp immediately ds31
PBC6.93 NO 249 downstream of the ATG start codon
deleted, resulting in a truncated glnE
protein lacking the adenylyl-removing
(AR) domain
CM093; 6-848 SEQ ID glnEΔAR-2 with glnE gene with 1644 bp immediately ds31
PBC6.93 NO 250 500 bp flank downstream of the ATG start codon
deleted, resulting in a truncated glnE
protein lacking the adenylyl-removing
(AR) domain; 500 bp flanking the glnE
gene upstream and downstream are
included
CM093; 6-848 SEQ ID ΔamtB First 1088 bp of amtB gene and 4 bp ds126
PBC6.93 NO 251 upstream of start codon deleted; 199 bp
of gene remaining lacks a start endow
no amtB protein is translated
CM093; 6-848 SEQ ID ΔamtB with 500 bp First 1088 bp of amtB gene and 4 bp ds126
PBC6.93 NO 252 flank upstream of start codon deleted; 199 bp
of gene remaining lacks a start codon;
no amtB protein is translated
CM094; 6-881 SEQ ID glnEΔAR-1 glnE gene with 1287 bp immediately ds30
PBC6.94 NO 253 downstream of the ATG start codon
deleted, resulting in a truncated glnE
protein lacking the adenylyl-removing
(AR) domain
CM094; 6-881 SEQ ID glnEΔAR-1 with glnE gene with 1287 bp immediately ds30
PBC6.94 NO 254 500 bp flank downstream of the ATG start codon
deleted, resulting in a truncated glnE
protein lacking the adenylyl-removing
(AR) domain; 500 bp flanking the gthE
gene upstream and downstream are
included
CM094; 6-881 SEQ ID ΔnifL::Prm1 starting at 31 bp after the A of the ATG ds20
PBC6.94 NO 255 start codon, 1375 bp of nifL have been
deleted and replaced with the CI006
Prm1 promoter sequence
CM094; 6-881 SEQ ID ΔnifL::Prm1 with starting at 31 bp after the A of the ATG ds20
PBC6.94 NO 256 500 bp flank start codon, 1375 bp of nifL have been
deleted and replaced with the CI006
Prm1 promoter sequence; 500 bp
flanking the nifL gene upstream and
downstream are included
CM094; 6-881 SEQ ID ΔamtB First 1088 bp of amtB gene and 4 bp ds126
PBC6.94 NO 257 upstream of start codon deleted; 199 bp
of gene remaining lacks a start codon;
no amtB protein is translated
CM094; 6-881 SEQ ID ΔamtB with 500 bp First 1088 bp of amtB gene and 4 bp ds126
PBC6.94 NO 258 flank upstream of start codon deleted; 199 bp
of gene remaining lacks a start codon;
no amtB protein is translated
none 910-1246 SEQ ID ΔnifL::PinfC starting at 20 bp after the A of the ATG ds960
NO 259 start codon, 1379 bp of nifL have been
deleted and replaced with the 910
PinfC promoter sequence
none 910-1246 SEQ ID ΔnifL::PinfC with starting at 20 bp after the A of the ATG ds960
NO 260 500 bp flank start codon, 1379 bp of nifL have been
deleted and replaced with the 910
PinfC promoter sequence; 500 bp
flanking the nifL gene upstream and
downstream are included
PBC6.1, CI006 SEQ ID 16S-1 1 of 3 unique 16S rDNA genes in the N/A
6, CI6 NO 261 CI006 genome
PBC6.1, CI006 SEQ ID 16S-2 2 of 3 unique 16S rDNA genes in the N/A
6, CI6 NO 262 CI006 genome
PBC6.1, CI006 SEQ ID nifH N/A N/A
6, CI6 NO 263
PBC6.1, CI006 SEQ ID nifD2 2 of 2 unique genes annotated as nifD N/A
6, CI6 NO 264 in CI006 genome
PBC6.1, CI006 SEQ ID nifK2 2 of 2 unique genes annotated as nifK N/A
6, CI6 NO 265 in CI006 genome
PBC6.1, CI006 SEQ ID nifL N/A N/A
6, CI6 NO 266
PBC6.1, CI006 SEQ ID nifA N/A N/A
6, CI6 NO 267
PBC6.1, CI006 SEQ ID glnE N/A N/A
6, CI6 NO 268
PBC6.1, CI006 SEQ ID 16S-3 3 of 3 unique 16S rDNA genes in the N/A
6, CI6 NO 269 CI006 genome
PBC6.1, CI006 SEQ ID nifD1 1 of 2 unique genes annotated as nifD N/A
6, CI6 NO 270 in CI006 genome
PBC6.1, CI006 SEQ ID nifK1 1 of 2 unique genes annotated as nifK N/A
6, CI6 NO 271 in CI006 genome
PBC6.1, CI006 SEQ ID amtB N/A N/A
6, CI6 NO 272
PBC6.1, CI006 SEQ ID Prm1 348 bp includes the 319 bp immediately N/A
6, CI6 NO 273 upstream of the ATG start codon of the
lpp gene and the first 29 bp of the lpp
gene
PBC6.1, CI006 SEQ ID Prm5 313 bp starting at 432 bp upstream of the N/A
6, CI6 NO 274 ATG start codon of the ompX gene and
ending 119 bp upstream of the ATG
start codon of the ompX gene
19, CI19 CI019 SEQ ID nifL N/A N/A
NO 275
19, CI19 CI019 SEQ ID nifA N/A N/A
NO 276
19, CI19 CI019 SEQ ID 16S-1 1 of 7 unique 16S rDNA genes in the N/A
NO 277 CI019 genome
19, CI19 CI019 SEQ ID 16S-2 2 of 7 unique 16S rDNA genes in the N/A
NO 278 CI019 genome
19, CI19 CI019 SEQ ID 16S-3 3 of 7 unique 16S rDNA genes in the N/A
NO 279 CI019 genome
19, CI19 CI019 SEQ ID 16S-4 4 of 7 unique 16S rDNA genes in the N/A
NO 280 CI019 genome
19, CI19 CI019 SEQ ID 16S-5 5 of 7 unique 16S rDNA genes in the N/A
NO 281 CI019 genome
19, CI19 CI019 SEQ ID 16S-6 6 of 7 unique 16S rDNA genes in the N/A
NO 282 CI019 genome
19, CI19 CI019 SEQ ID 16S-7 7 of 7 unique 16S rDNA genes in the N/A
NO 283 CI019 genome
19, CI19 CI019 SEQ ID nifH1 1 of 2 unique genes annotated as nifH N/A
NO 284 in CI019 genome
19, CI19 CI019 SEQ ID nifH2 2 of 2 unique genes annotated as nifH N/A
NO 285 in CI019 genome
19, CI19 CI019 SEQ ID nifD1 1 of 2 unique genes annotated as nifD N/A
NO 286 in CI019 genome
19, CI19 CI019 SEQ ID nifD2 2 of 2 unique genes annotated as nifD N/A
NO 287 in CI019 genome
19, CI19 CI019 SEQ ID nifK1 1 of 2 unique genes annotated as nifK N/A
NO 288 in CI019
19, CI19 CI019 SEQ ID nifK2 2 of 2 unique genes annotated as nifK N/A
NO 289 in CI019 genome
19, CI19 CI019 SEQ ID glnE N/A N/A
NO 290
19, CI19 CI019 SEQ ID prm4 449 bp immediately upstream of the N/A
NO 291 ATG of the dscC 2 gene
19, CI19 CI019 SEQ ID Prm1.2 500 bp immediately upstream of the N/A
NO 292 TTG start codon of the infC gene
19, CI19 CI019 SEQ ID Prm3.1 170 bp immediately upstream of the N/A
NO 293 ATG start codon of the rplN gene
19, CI20 CI020 SEQ ID Prm6.1 142 bp immediately upstream of the N/A
NO 294 ATG of a highly-expressed
hypothetical protein (annotated as
PROKKA_00662 in CI019 assembly
82)
19, CI21 CI021 SEQ ID Prm7.1 293 bp immediately upstream of the N/A
NO 295 ATG of the lpp gene
19-375, CM67 SEQ ID glnEΔAR-2 glnE gene with 1650 bp immediately ds34
19-417, NO 296 downstream of the ATG start codon
CM067 deleted, resulting in a truncated glnE
protein lacking the adenylyl-removing
(AR) domain
19-375, CM67 SEQ ID glnEΔAR-2 with glnE gene with 1650 bp immediately ds34
19-417, NO 297 500 bp flank downstream of the ATG start codon
CM067 deleted, resulting in a truncated g1nE
protein lacking the adenylyl-removing
(AR) domain; 500 bp flanking the glnE
gene upstream and downstream are
included
19-375, CM67 SEQ ID ΔnifL::null-v1 starting at 221 bp after the A of the none
19-417, NO 298 ATG start codon, 845 bp of nifL have
CM067 been deleted and replaced with the
31 bp sequence
“GGAGTCTGAACTCATCCTGCGA
TGGGGGCTG”
19-375, CM467 SEQ ID ΔnifL::null-v1 with starting at 221 bp after the A of the none
19-417, NO 299 500 bp flank ATG start codon, 845 bp of nifL have
CM067 been deleted and replaced with the
31 bp sequence
“GGAGTCTGAACTCATCCTGCGA
TGGGGGCTG”; 500 bp flanking the
nifL gene upstream and downstream
are included
19-377, CM69 SEQ ID ΔnifL::null-v2 starting at 221 bp after the A of the none
CM069 NO 300 ATG start codon, 845 bp of nifL have
been deleted and replaced with the 5 bp
sequence “TTAAA”
19-377 CM69 SEQ ID ΔnifL::null-v2 with starting at 221 bp after the A of the none
CM069 NO 301 500 bp flank ATG start codon, 845 bp of nifL have
been deleted and replaced with the 5 bp
sequence “TTAAA”; 500 bp flanking
the nifL gene upstream and
downstream are included
19-389, CM81 SEQ ID ΔnifL::Prm4 starting at 221 bp after the A of the ds70
19-418, NO 302 ATG start codon, 845 bp of nifL have
CM081 been deleted and replaced with the
CI19 Prm4 sequence
19-389, CM81 SEQ ID ΔnifL::Prm4 with starting at 221 bp after the A of the ds70
19-418, NO 303 500 bp flank ATG start codon, 845 bp of nifL have
CM081 been deleted and replaced with the
CI19 Prm4 sequence; 500 bp flanking
the nifL gene upstream and
downstream are included
TABLE H
SEQ ID
NO: Sequence
61 atgttttaacgatctgattggcgatgatgaaacggattcgccggaagatgcgctttctgagagctggcgcgaattgtggcaggatgcgttgcaggagga
ggattccacgcccgtgctggcgcatctctcagaggacgatcgccgccgcgtggtggcgctgattgccgattttcgcaaagagttggataaacgcacc
attggcccgcgagggcggcaggtactcgatcacttaatgccgcatctgctcagcgatgtatgctcgcgcgacgatgcgccagtaccgctgtcacgcc
tgacgccgctgctcaccggaattattacccgcaccacttaccttgagagctaagtgaatttcccggcgcactgaaacacctcatttccctgtgtgccgc
gtcgccgatggttgccagtcagctggcgcgctacccgatcctgcttgatgaattgctcgacccgaatacgctctatcaaccgacggcgatgaatgccta
tcgcgatgagctgcgccaatacctgctgcgcgtgccggaagatgatgaagagcaacagcttgaggcgctgcggcagtttaagcaggcgcagttgct
gcgcgtggcggcggcggatattgccggtacgttgccagtaatgaaagtgagcgatcacttaacctggctggcggaagcgattattgatgcggtggtg
cagcaagcctgggggcagatggtggcgcgttatggccagccaacgcatctgcacgatcgcgaagggcgcggttttgcggtggtcggttatggcaag
ctgggcggctgggagctgggttacagctccgatctggatctggtattcctgcacgactgcccgatggatgtgatgaccgatggcgagcgtgaaatcg
atggtcgccagttctatttgcgtctcgcgcagcgcgtgatgcacctgtttagcacgcgcacgtcgtccggcatcctttatgaagttgatgcgcgtctgcgt
ccatctggcgctgcggggatgctggtcactactacggaatcgttcgccgattaccagcaaaacgaagcctggacgtgggaacatcaggcgctggcc
cgtgcgcgcgtggtgtacggcgatccgcaactgaccgccgaatttgacgccattcgccgcgatattctgatgacgcctcgcgacggcgcaacgctgc
aaaccgacgtgcgagaaatgcgcgagaaaatgcgtgcccatcttggcaacaagcataaagaccgcttcgatctgaaagccgatgaaggcggtatca
ccgacatcgagtttatcgcccaatatctggtgctgugctttgcccatgacaagccgaaactgacgcgctggtcggataatgcgcattctcgaagggc
tggcgcaaaacggcatcatggaggagcaggaagcgcaggcattgacgctggcgtacaccacattgcgtgatgagctgcaccacctggcgctgcaa
gagttgccgggacatgtggcgctctcctgttttgtcgccgagcgtgcgcttattaaaaccagctgggacaagtggctggtggaaccgtgcgccccggc
gtaa
62 ttgaagagtttgatcatggctcagattgaacgctggcggcaggcctaacacatgcaagtcgaacggtagcacagagagcttgctctcgggtgacgagt
ggcggacgggtgagtaatgtctgggaaactgcctgatggagggggataactactggaaacggtagctaataccgcataacgtcgcaagaccaaaga
gggggaccttcgggcctcttgccatcagaggcccagatgggattagctagtaggtggggtaacggctcacctaggcgacgatccctagctggtag
agaggatgaccagccacactggaactgagacacggtccagactcctacgggaggcagcagtggggaatattgcacaatgggcgcaagcctgatgc
agccatgccgcggtgtgaagaaggccttcgggttgtaaagcactttcagcggggaggaagggagtaaggttaataaccttattcattgacgttacccg
cagaagaagcaccggctaactccgtgccagcagccgcggtaatacggagggtgcaagcgttaatcggaattactgggcgtaaagcgcacgcaggc
ggtctgtcaagtcggatgtgaaatccccgggctcaacctgggaactgcatccgaaactggcaggcttgagtctcgtagagggaggtagaattccagg
tgtagcggtgaaatgcgtagagatctggaggaataccggtggcgaaggcggcctcctggacgaagactgacgctcaggtgcgaaagcgtgggga
gcaaacaggattagataccctggtagtccacgccgtaaacgatgtctatttggaggttgtgcccttgaggcgtggcttccggagctaacgcgttaaata
gaccgcctggggagtacggccgcaaggttaaaactcaaatgaattgacgggggcccgcacaagcggcgagcatgtggtttaattcgatgcaacgc
gaagaaccttacctggtcttgacatccacagaactttccagagatggattggtgccttcgggaactgtgagacaggtgctgcatggctgtcgtcagctc
gtgttgtgaaatgttgggttaagtcccgcaacgagcgcaacccttatcctttgttgccagcggtccggccgcgaactcaaaggagactgccagtgataa
actggaggaaggtggggatgacgtcaagtcatcatggcccttacgaccagggctacacacgtgctacaatggcgcatacaaagagaagcgacctcg
cgagagtaagcggacctcataaagtgcgtcgtagtccggattggagtctgcaactcgactccatgaagtcggaatcgctagtaatcgtggatcagaat
gccacggtgaatacgttcccgggccttgtacacaccgcccgtcacaccatgggagtgggttgcaaaagaagtaggtagcttaaccttcgggagggc
gcttaccactttgtgattcatgactggggtgaagtcgtaacaaggtaaccgtaggggaacctgcggttggatcacctcctt
63 ttgaagagtttgatcatggctcagattgaacgctggcggcaggcctaacacatgcaagtcgaacggtagcacagagagcttgctctcgggtgacgagt
ggcggacgggtgagtaatgtcgggaaactgcctgatggagggggataactactggaaacggtagctaataccgcataacgtcgcaagaccaaaga
gggggaccttcgggcctcttgccatcagatgtgcccagatgggattagctagtaggtggggtaacggctcacctaggcgacgatccctagctggtctg
agaggatgaccagccacactggaactgagacacggtccagactcctacgggaggcagcagtggggaatattgcacaatgggcgcaagcctgatgc
agccatgccgcgtgtgtgaagaaggccttcgggttgtaaagcactttcagcggggaggaggnantanggttaataacctgtgttnattgacgttacc
cgcagaagaagcaccggctaactccgtgccagcagccgcggtaatacggagggtgcaagcgttaatcggaattactgggcgtaaagcgcacgcag
gcggtctgtcaagtcggatgtgaaatccccgggctcaacctgggactgcatccgaaactggcaggcttgagtctcgtagagggaggtagaattcca
ggtgtagcggtgaaatgcgtagagatctggaggaataccggtggcgaaggcggcctcctggacgaagactgacgctcaggtgcgaaagcgtggg
gagcaaacaggattagataccctggtagtccacgccgtaaacgatgtctatttggaggttgtgcccttgaggcgtggcttccggagctaacgcgttaaa
tagaccgcctggggagtacggccgcaaggttaaaactcaaatgaattgacgggggcccgcacaagcggtggagcatgtggtttaattcgatgcaac
gcgaagaaccttacctggtcttgacatccacagaacttagcagagatgctttggtgccttcgggaactgtgagacaggtgctgcatggctgtcgtcagc
tcgtgttgtgaaatgttgggttaagtcccgcaacgagcgcaacccttatcctttgttgccagcggttaggccgggaactcaaaggagactgccagtgat
aaactggaggaaggtggggatgacgtcaagtcatcatggcccttacgaccacggctacacacgtgctacaatggcgcatacaaagagaagcgacct
cgcgagagtaagcggacctcataaagtgcgtcgtagtccggattggagtctgcaactcgactccatgaagtcggaatcgctagtaatcgtggatcaga
atgccacggtgaatacgttcccgggccttgtacacaccgcccgtcacaccatgggagtgggttgcaaaagaagtaggtagcttaaccttcgggaggg
cgcttaccactttgtgattcatgactggggtgaagtcgtaacaaggtaaccgtaggggaacctgcggttggatcacctcctt
64 attgaagagtttgatcatggctcagattgaacgctggcggtcaggcctaacacatgcaagtcgagcggcancgggaagtagcttgctactttgccggc
gagcggcggacgggtgagtaatgtctgggaaactgcctgatggagggggataactactggaaacggtagctaataccgcatgacctcgaaagagc
aaagtggggatcttcggacctcacgccatcggatgtgcccagatgggattagctagtaggtgaggtaatggctnacctaggcgacgatccctagct
ggtctgagaggatgaccagccacactggaactgagacacggtccagactcctacgggaggcagcagtggggaatattgcacaatgggcgcaagc
ctgatgcagccatgccgcgtgtgtgaagaaggccttagggttgtaaagcactttcagcgaggaggaggcancanacttaatacgtgtgntgattgac
gttactcgcagaagaagcaccggctaactccgtgccagcagccgcggtaatacggagggtgcaagcgttaatcggaattactgggcgtaaagcgca
cgcaggcggtttgttaagtcagatgtgaaatccccgagcttaacttcgggaactgcatttgaaactggcaagctagagtcttgtagaggggggtagaatt
ccaggtgtagcggtgaaatgcgtagagatctggaggaataccggtggcgaaggcggccccctggacaaagactgacgctcaggtgcgaaagcgt
ggggagcaattcaggattagataccctggtagtccacgctgtaaacgatgtcgacttggaggttgtgcccttgaggcgtggcttccggagctaacgcgt
taagtcgaccgcctggggagtacggccgcaaggttaaaactcaaatgaattgacgggggcccgcacaagcggtggagcatgtggtttaattcgatgc
aacgcgaagaaccttacctactcttgacatccagagaatttgccagagatggcgaagtgccttcgggaactctgagacaggtgctgcatggctgcgt
cagctcgtgttgtgaaatgttgggttaagtcccgcaacgagcgcaacccttatcctttgttgccagcacgtnatggtgggaactcaaaggagactgccg
gtgataaaccggaggaaggtggggatgacgtcaagtcatcatggcccttacgagtagggctacacacgtgctacaatggcatatacaaagagaagc
gaactcgcgagagcaagcggacctcataaagtatgtcgtagtccggattggagtagcaactcgactccatgaagtcggaatcgctagtaacgtaga
tcagaatgctacggtgaatacgttccgggccttgtacacaccgcccgtcacaccatgggagtgggttgcaaaagaagtaggtagcttaaccttcggg
agggcgcttaccactttgtgattcatgactggggtgaagtcgtaacaaggtaaccgtaggggaacctgcggttggatcacctcctt
65 atgaccatgcgtcaatgcgccatttacggcaaaggtgggatcggcaaatcgaccaccacacagaacctggtcgccgcgctggcggagatgggtaaa
aaagtcatgattgtcggctgtgacccgaaagccgattccacgcgtttgatcctgcatgcgaaagcgcagaacaccattatggagatggctgctgaagt
cggctccgtggaagacctggagttagaagacgtgctgcaaatcggttacggcggcgtgcgctgcgcagagtccggcggcccggagccaggcgtg
ggctgtgccggtcgcggggtgatcaccgcgattaacttcctcgaagaagaaggcgcttacgtgccggatctcgattttgttttctacgacgtgctgggc
gacgtggtatgcggtggtttcgccatgccgattcgtgaaaacaaagcgcaggagatctacatcgtttgctctggcgaaatgatggcgatgtacgccgc
caacaacatctccaaaggcatcgtgaaatacgccaaatccggtaaagtgcgcctcggcgggctgatttgtaactcgcgccagaccgaccgtgaagat
gaactgatcattgcgctggcagaaaaactcggcacgcagatgatccactttgttcccgcgacaacattgtgcagcgtgcggaaatccgccgtatgac
ggttatcgaatatgacccgacctgcaatcaggcgaacgaatatcgcagccttgccagcaaaatcgtcaacaacaccaaaatggtggtgcccaccccc
tgcaccatggatgaactggaagaactgctgatggagttcggcattatggatgtggaagacaccagcatcattggtaaaaccgccgccgaagaaaacg
ccgtctga
66 atgagcaatgcaacaggcgaacgcaacctggagataatcgagcaggtgctcgaggttttcccggagaagacgcgcaaagaacgcagaaaacacat
gatggtgacggacccggagcaggaaagcgtcggtaagtgcatcatctctaaccgcaaatcgcagccaggcgtgatgaccgtgcgcggctgctcgta
tgccggttcgaaaggggtggtatttgggccaatcaaggatatggcgcatatctcgcatggcccaatcggctgcggccaatactcccgcgccgggcgg
cggactactacaccggcgtcagcggcgtggacagcttcggcacgctcaacttcacctccgattttcaggagcgcgacatcgtgtttggcggcgataa
aaagctcgccaaactgattgaagagctggaagagctgttcccgctgaccaaaggcatttcgattcagtcggaatgcccggtcggcctgattggcgatg
acattgaggccgtcgcgaacgccagccgcaaagccatcaacaaaccggttattccggtgcgttgcgaaggctttcgcggcgtgtcgcaatccctcgg
tcaccatattgccaacgatgtgatccgcgactgggtgctggataaccgcgaaggcaaaccgttcgaatccaccccttacgatgtggcgatcatcggcg
attacaacatcggcggcgatgcctgggcttcgagcattttgctcgaagagatgggcttgcgggtggtggcacagtggtctggcgacggtacgctggt
ggagatggaaaacacgccgttcgtcaaactgaacctggtgcattgttaccgctcaatgaactacatctcgcgccatatggaggagaagcacggtattc
cgtggatggaatacaacttcttggtccgacgaaaatcgcggaatcgctgcgcaaaatcgccgaccagtttgacgacaccattcgcgccaacgccga
agcggtgatcgccagataccaggcgcaaaacgacgccattatcgccaaatatcgcccgcgtctggaggggcgcaaagtgctgctttatatgggcgg
gctgcgtccgcgccatgtgattggcgcctatgaagacctgggaatggagatcatcgctgccggttatgagttcggtcataacgatgattacgaccgca
ccttgccggatctgaaagagggcacgctgctgtttgatgatgccagcagttatgagaggaggcgttcgtcaacgcgctgaaaccggatctcatcggtt
ccggcatcaaagagaagtacatctttcagaaaatgggcgtgccgtttcgccagatgcactcctgggattactccggcccgtaccacggctatgacggc
ttcgccatcttcgcccgcgatatggatatgacgctcaacaaccccgcgtggggccagttgaccgcgccgtggctgaaatccgcctga
67 atgagccagactgctgagaaaatacagaattgccatcccctgtttgaacaggatgcttaccagacgctgtttgccggtaaacgggcactcgaagaggc
gcactcgccggagcgggtgcaggagtgtttcaatggaccactaccccggaatatgaagcgctgaactttaaacgcgaagcgctgactatccaccc
ggcaaaagcctgccagccgctgggcgcggtgctctgttcgctggggtttgccaataccctaccgtatgtgcacgcttcacaggttgcgtggcctattt
ccgcacgtactttaaccgccactttaaagaaccggtggcctgcgtgtcggattcaatgacggaagacgcggcggtgttcggcgggaataacaacctc
aacaccggcttacaaaacgccagcgcgctgtataaaccggagattatcgccgtctctaccacctgtatggcggaagtgatcggtgatgatttgcaggc
ctttatcgccaacgccaaaaaagatggttttctcgatgccgccatccccgtgccctacgcgcacacccccagttttatcggcagccatatcaccggctg
ggataacatgtttgaaggttttgcccggacctttacggagaccatgaagctcagcccggcaaactttcacgcatcaacctggtgaccgggtttgaaac
ctatctcggcaatttccgcgtgctgaaacgcatgatggaacaaatggaggtgccggcgagtggctctccgatccgtcggaagtgctgcatactcccg
ccaacgggcattaccagatgtacgcgggcgggacgacgcagcaagagatgcgcgaggcgccggatgctatcgacaccctgttcctgcagccctg
gcaactggtgaaaagcaaaaaagtggtgcaggagatgtggaatcagcccgccaccgaggtttctgttcccgttgggctggcaggaacacacgaact
gttgatggcgattagccagttaaccggcaaggccattcccgattcactggcgctggagcgccggcggctggtcgatatgatgctcgattcccacacct
ggttgcacggtaaaaaattcggcctgaggcgatccggattttgtcatgggattgacccgtttcctgctggagctgggctgcgaaccgaccgttatcctc
tgccacaacggtaacaaacgctggcagaaagcaatgaagaaaatgcttgacgcctcgccgtacggccaggagagcgaagtttatcaactgcgatt
tgtggcatttccgctcgctgatgtttacccgccagccggattttatgattggcaactcgtacggcaagttcattcagcgcgacaccttagccaaaggcga
gcagtttgaagttccgctgatccgcctcggttttccccttcgaccgccaccatctgcaccgccagaccacctggggctacgagggcgccatgagca
ttctcactacccttgtgaatgcggtactggagaaagtggacaaagagaccatcaagctcggcaaaaccgactacagcttcgatcttatccgttaa
68 atgaccctgaatatgatgatggatgccggcgcgcccgaggcaatccccggtgcgctttcgcgacaccatcctgggctgttttttaccatcgttgaagaa
gcgcccgtcgccatttcgctgactgatgccgacgcacgcattgtctatgccaacccggctttctgccgccagaccggctatgaactagaagcgttgttg
cagcaaaatccccgcctgatgcaagtcgccaaaccccacgggaaatctatcaggatatgtggcacaccttgttacaacgccgaccgtggcgcgggc
aattgattaaccgccaccgcgacggcagcctgatctggtcgagatcgatatcaccccggtgattaacccgtttggcgaactggaacactacctggca
atgcagcgcgatatcagcgccagttatgcgctggagcagcggttgcccaatcacatgacgctgaccgaagcggtgctgaataacattccggcggcg
gtggttgtagtggatgaacgcgatcatgtggttatggataaccttgcctacaaaacgttctgtgccgactgcggcggaaaagagctcctgagcgaactc
aatttttcagcccgaaaagcggagctggcaaacggccagctcttaccggtggtgctgcgcggtgaggtgcgctggttgtcggtgacctgctgggcgc
tgccgcgcgtcagcgaagaagccagcgctactttattgataacaggctgacgcgcacgctggggtgatcaccgacgacacccaacaacgccagc
agcaggaacagggccgacttgaccgccttaaacagcagatgaccaacggcaaactactggcagcgatccgcgaagcgcttgacgccgcgctgatc
cagcttaactgccccatcaatatgctggcggcggcgcgacgtttaaacggcagtgataacaacaatgtggcgctcgacgccgcgtggcgcgaaggt
gaagaggcgatggcgcggctgaaacgttgccgcccgtcgctggaactggaaaggcggccgtctggccgctgcaacccattttgacgatctgcgc
gcgctttatcttcacccgctacgagcaggggaaaaatttgcaggtcacgctggattcccatcatctggtgggatttggtcagcgtacgcaactgttagcct
gcctgagtctgtggctcgatcgcacgctggatattgccgccgggctgggtgatttcaccgcgcaaacgcagatttacgcccgcgaagaagagggctg
gctactttgtatatcactgacaatgtgccgagatcccgctgcgccacacccactcgccggatgcgcttaacgctccgggaaaaggcatggagctgc
gcctgatccagacgctggtggcacaccaccacggcgcaatagaactcacttcacaccccgaagggggaagttgcctgaccctacgattcccgctatt
tcattcactgaccgcaggttcaaaatga
69 atgacccagcgaaccgagtcgggtaataccgtctggcgatcgatttgtcccagcagttcactgcgatgcagcgcataaggtggtactcagccggg
cgaccgaggtcgatcagacgctccagcaagtgctgtgcgtattgcacaatgacgcctttttgcagcacggcatgatctgctgtacgacagccagcag
gcgattttgaatattgaagcgttgcaggaagccgatcagcagttaatccccggcagctcgcaaatccgctatcgtccgggcgaagggctggtcggga
cggtgctttcgcagggccaatcattaggctggcgcgcgttgctgacgatcagcgctttcttgaccggctcgggttgtatgattacaacctgccgtttatc
gccgtgccgctgatagggccagatgcgcagactttcggtgtgagacggcacaacccatggcgcgttacgaagagcgattacccgcctgcacccgc
tttctggaaacggtcgctaacctggtcgcgcaaaccgtgcgtttgatggcaccaccggcagtgcgcccttccccgcgcgccgccataacacaggccg
ccagcccgattatcctgcacggcctcacgcgcatttggttttgaaaatatggtcggtaacagtccggcgatgcgccagaccatggagattatccgcag
gtttcgcgctgggacaccaccgttctggtacgcggcgagagtggcaccggcaaggagctgattgccaacgccatccaccaccattcgccgcgtgcc
ggtgcgccatttgtgaaattcaactgtgcggcgctgccggacacactgctggaaagcgaattgttcggtcacgagaaaggggcatttaccggcgcgg
tacgccagcgtaaaggccgttttgagctggccgatggcggcacgctgtttcttgacgagatcggcgagagtagcgcctcgtttcaggctaagctgctg
cgcattttgcaggaaggcgaagggaacgcgtcggcggcgacgagacattgcaagtgatttgtgcgcattattgccgcgacgaaccgcaatcttgaa
gatgaagtccggctggggcactttcgcgaagatctctattatcgcctgaatgtgatgcccatcgccctgccgccactacgcgaacgccaggaggacat
tgccgagctggcgcactactggtgcgtaaaatcgcccataaccagagccgtacgctgcgcattagcgagggcgctatccgcctgctgatgagctaca
actggcccggtaatgtgcgcgaactggaaaactgcatgagcgctcagcggtgatgtcggagaacggtctgatcgatcgggatgtgattttgtttaatc
atcgcgaccagccagccaaaccgccagttatcaggtctcgcatgatgataactggctcgataacaaccttgacgagcgccagcggctgattgcggc
gctggaaaaagcgggatgggtacaagccaaagccgcgcgcttgctggggatgacgccgcgccaggtcgcctatcgtattcagacgatggatataac
cctgccaaggctataa
70 atggcaatgcgtcaatgtgcaatctacgggaaagggggtattggtaaatccaccactacccaaaaccttgtagcggctctggccgaaatgaataagaa
ggtcatgatcgtcggctgtgaccctaaggctgattcaacccgactcattctgcatgcgaaagcacagaacaccatcatggaaatggccgctgaagtgg
gctccgtggtagatctggagctggaagatgtgatgcaaatcggctatggcggcgtgcgctggcggaatcaggcggccctgagcctggtgtgggtt
gtgccggacgcgggggtgatcaccgccatcaacttcctcgaagaagaaggcgcgtatgtgccggatctggatttcgtgttttacgacgtattgggcgat
gtggtctgtggcggtttcgcgatgccaattcgcgaaaacaaagcgcaggaaatctacatcgtatgctccggtgaaatgatggcgatgtatgccgccaa
caacatttccaaaggcatcgtgaaatacgcgaaatcgggcaaagttcgcctggccgggctgatctgtaactcccgccagacggatcgcgaagatgaa
ctgatcatcgcgaggctgaaaaacttggcacgcaaatgatccacttcgtgccgcgtgacaacattgtgcaacgcgctgaaatccgccgcatgacggt
catcgaatacgacccgacttgtgcgcaggcagatcagtcgtgcactggcgaacaaaatcgtcaacaacaccaaaatggtggtgccgacaccggtc
accatggatgagctggaagccctgttaatggaatttggcattatggaagaagaagacctgaccatcgtcggtcgtaccgccgccgaagaggcgtga
71 atgaccagtgaaacacgcgaacgtaacgaggcattgatccaggaagtgctggagatcttccccgagaaggcgcttaaagatcgtaagaaacacatg
atgaccaccgacccggcgatggaatctgtcggcaaggtattgtctcaaaccgcaaatcacagccgggcgtgatgaccgtgcgaggctgcgcttacg
ccggttccaaaggcgtggtctttggcccgatcaaagacatggcgcatatctcccacggcccggttggttgcggccagtattctcgtgccggacgccgt
aactattacaccggctggagggcgtgaacagctttggcaccctcaacttcaccagtgattttcaggaacgggacatcgtatttggcggcgataaaaag
ctcgacaaactgatcgacgaactggagatgttgtcccgctgaccaaaggcatttcggtacagtcggaatgtccggtcggtctgatcggcgatgacatt
tctgccgtcgccaaagccagcagcgccaaaatcggtaagccggtcgtgccggtacgctgcgaggggttccgcggtgtgtcgcaatcgctcggccat
cacattgctaacgatgtcatccgcgactgggtgctggataaccgcgaaggcaatgaatttgaaaccacgccttacgacgtggcgattatcggcgacta
caacatcggcggtgacgcctgggcctcacgtattctgctcgaagaaatggggctgcgtgtggtggcgcagtggtccggcgacggcacgctggtgga
gatggaaaacaccccgaaagtcgcactcaatctggtgcactgctaccgctcgatgaactacatctcccgcatatggaagaaaaacacggcattccgt
ggatggaatacaacttctttggcccgtccaaaattgcggaatctctgcgcgaaatcgcggcgcgttttgacgataccatccggaaaaacgccgaagc
ggtgattgaaaaatatcaggcgcaaacgcaggcggtgatcgacaaataccgtccgcgtaggaaggcaaaaaggtgctgttgtatctcggcggtttac
gtccgcgccacatcatcggggcgtatgaagatctgggaatggaaatcatcggtaccggctatgaattcggtcataacgatgattacgaccgcaccttac
cgatgctcaaagaaggcacgttgctgttcgatgacctgtgcagttatgagctggaagcgttcgttaaagcgctgaaaccggatcttgtcgggtcaggc
atcaaagaaaaatacattttccacaaaatgggcgtgccgttccgccagatgcactcctgggattattccggcccttatcacggctacgacggtttcggca
tttttgcccgtgacatggacatgacgctgaacaatccgggctggagtcagctgaccgccccctggttgaaatcggcctga
72 atgagtcaagatcttggcaccccaaaatcctgtttcccgctgacgagcaggatgaataccagagtatgtttacccacaaacgcgcgaggaagaagca
cacggcgaggcgatagtgcgggaagtgtttgaatggaccaccacgcaggaatatcaggatctgaacttctcgcgtgaagcgctgaccgtcgacccg
gcgaaagcctgccagccgttaggcgcggtactttgcgcgctgggttttgccaacacgttgccgtatgtccacggttcacaaggctgtgtggcgtatttc
cgtacctattttaatcctcatttcaaagagccggtggcctgtgtttccgactcaatgaccgaagatgccgccgtttttggcggaaataacaacatgaatgtc
ggtctggaaaacgccagcgcgctgtacaagccgganattattgctgtctccaccacctgtatggcggaagtgatcggtgatgacctgcaggcttttatc
gccaacgccaaaaaagacggatttgtggatgccgggatgccaatcccgtatgcccatacaccgagtttctgggcagtcatgtcaccggctgggacaa
catgtttgaaggcttcgcccgtacctttaccaccgacgccacgcgggaatatcagccgggcaaacttgccaaactgaacgtggtgaccggttttgaaa
cttatctcggcaactaccgggttattcaccgcatgatgagccagatgggggtcgaatgcagcgtcttgtccgatccgtctgaagtgctcgacaccccgg
ctgacggccaataccgcatgtatgccggcggcaccacgcaaaccgaaatgcgtgatgcaccggatgccatcgacaccttgctgctgcaaccgtggc
aattgcagaaaaccaaaaaagtggtgcagggcgactggaatcagccgggcaccgaagtcagtgtaccgattggcctggcggcgaccgatgccttg
ctgatgacggtangcgaactgaccggcaaaccgatagctgacacgctggcgactgaacgtggccgtctggtggacatgatgctcgattcccacacct
ggctgcatggcaagcgtttcggtctctacggtgacccggattttgtgatgggcatgaccgcattcctgctggaactgggctgtgaaccgaccaccattct
cagccataacggcaacaaacgctggcagaaagccatgaagaaaatgctggctgattcgccttacgggcaggacagcgaagtgtattgtgaactgcga
tctgtggcatttccgctcgctgatgtttacccgtaaaccggactttatgatcggcaactcaacggaaaattcattcagcgtgacacgctggccaaaggcg
aacagttcgaagtgccgctgatccgcatcggttttccgatttttgaccggcaccatttgcaccgtcagaccacctggggatacgaaggggcgatgagc
atactgacgcaactggtgaatgccgtacttgaacaactggatcgcgaaaccatgaagctcggcaaaaccgactacaacttcgtcctgttccgctaa
73 atgagcatcacggcgttatcagcatcatttcctgaggggaatatcgccagccgcttgtcgctgcaacatccttcactgttttataccgtggttgaacaatct
tcggtggcgatttcgctgaccgatccgcaggcgcgcatttgttatgccaatccggcanctgccgccagacgggttttgcacttgagacacttttgggcg
agaaccaccgtctgctggccagccagcagacgccgaaacatatctatgacgaaatgtggcgcactttgagcagggcaaatcctggaacggccaact
gatcaaccggcgtaataaccgttcgctttatctggcggatgtcactatcacgcctgttttaggcgggacgggcaggtggagcattacctcggcatgca
caaagatatcagcgagaaatacgcgctggaacagcggttgcgcaaccacatcaccttgttcacggaggtgctgaacaatattcccgccgccgtggtg
gtggtggatgagcaggacaatgtggtgatggacaatctggcctacaaaaccctttgcgcggactgcggcggcaaagagctgctggctgaaatgggc
tatccgcaactcaaagagatgctcancagtggcgaaccggtgccggtttccatgcgcggcaacgtacgctggttttctttcggtcaatggttattcagg
gcgttaatgattgaggccagccgcttttttaccggcattaccgcgccgggaaaactgattgttctgaccgactgcaccgatcagcatcaccggcagcag
cagggttatcttgaccggcttaagcaaaaactcaccaacggcaaattattggcggccatccgtgagtcgctcgatgccgcgcttatccagctcaacgg
gccaatcaatatgctggcggctccgcgtcgtcttaacggcgaagaaggcaacaacatggcgctggaattcgcctggcgcgaaggcgagcaggcgg
tgagtcgcttacaggcctgccgtccgtcgctggattttgagccgcaggcagaatggccggtcagtgaattctttgacgatctgagcgcgctgtacgaca
gccattttctcagtgacggtgaattgcgttacgtggtcatgccatctgatctgcacgctgtcgggcaacgaacgcaaatccttaccgcgctgagcttatg
gattgatcacacgctgtcacaggcgcaggccatgccgtctctgaagctctcggtgaacattgcgcgaggcaggatgcgagctggttgtgttttgacatt
accgataatgtgccgcgtgaacgggtgcgttatgcccgcccggaagcggcgttttcccgtccggggaatggcatggagctgcgccttatccagacgc
tgatcgcccatcatcgcggttctttagatctctcggtccgccctgatggcggcaccttgctgacgttacgcctgccggtacagcaggttatcaccggag
gcttaaatga
74 atgacccagttcctaccgcgggcccggttatccggcgctttgatatgtctgcccagtttacggcgctttatcgcatcagcgtggcgctgagtcaggaa
agcaacaccgggcgcgcactggcggcgatcctcgaagtgcttcacgatcatgcatttatgcaatacggcatggtgtgtctgtttgataaagaacgcaat
gcactctttgtggaatccctgcatggcatcgacggcgaaaggaaaaaagagacccgccatgtccgttaccgcatgggggaaggcgtgatcggcgcg
gtgatgagccagcgtcaggcgctcgtgttaccgcgcatttcagacgatcagcgttttctcgaccgcctgaatatttacgattacagccttccgttgattgg
cgtgcccgatccccggtgcggataatcagccatcgggcgtgctggtggcacagccgatggcgttgcacgaagaccggctgactgccagtacgcggtt
tttagaaatggtcgccaatctcatcagccagccactgcgttctgccacgcccccggaatcattgcctgctcaaacgccggtccggtgcagtgttccgcg
ccagtttggtttcgagcagatggtcgggaaaagtcaggcgatgcgccagacgatggacattttacggcaggtttccaaatgggataccacggttctggt
gcgtggtgaaagcggcaccggcaaggaacttatcgccaatgccattcattacaactcaccccgtgcggccgcgccatttgtgaaattcaactgcgccg
cgctgccggataacctgctggaaagcgaactgttcggtcatgaaaaaggggccttcaccggcgctatccgtacccgtaaaggccgctttgaactggc
ggacgggggcacgttattcctcgatgaaatcggcgaatcgagcgcgtcgtttcaggccaaattgctgcgcattttgcaggaaggtgaaatggaacgg
gtcggcggcgataccacgctgaaagttgatgtgcgcattattgctgccaccaaccgtaatcttgaagaggaagtgcgtgccgggaattttcgcgaaga
cctgtattatcgcctgaacgtgatgccggtttcgctgcctgcactgcgtgaaaggctggatgatatcgccgatctggcgccgtttaggtcaattaagatt
gcgctgcgtcaggggcgggaactgcgcatcagcgacggtgcggtgcgtctgctgatgacctacagctggccaggcaacgtgcgtgaactggaaaa
ctgtctcgaacgggcgtcggtaatgaccgatgaagggctgatcgaccgcgacgtgatcctgttcaatcaccatgaatccccggcgctgtccgtcaaac
ccggcctgccgctcgcgacagatgaaagctggctggatcaggaactcgacgaacgccagcgggtgattgccgcactggagaaaaccggctgggt
gcaggccaaagcggcccgactgctgggcatgacaccgcgccagattgcctaccgtatccagattatggacatcaacatgcaccgtatctga
75 aaaactaccgccgcaattaatgaacccaacgctactgttgccgggccatgctcttccccggcgcgctgcccggaaaggatatagattgcccagcacg
cgccagcaccaagcgcgaacgccgcgccagtgagatcaacatgtgaaacattttcgcccagcggcagcagatacaagaggccaagtaccgccag
gatcacccagatgaaatccaccgggcggcgtgaggcaaaaagcgccaccgccagcgggccggtaaattccagcgccaccgcaacgccgagcgg
tatcgtctggatcgataaatagaacatatagttcatggcgccgagcgacaggccataaaacagcagtggcaggcgttgttcacgggtaaaatgtaaac
gccagggcttgaacactacgaccaaaataagggtgccaagtgcgagacgcagcgcggtgacgccgggtgcgccaacaatcggaaacagtgatttc
gccagcgacgcgcctccctgaatggacatcatcgcgacaaacaatattaataccggcaaccacaccggcaatttacgagactgcgcaggcatccttt
ctcccgtcaatttctgtcaaataaagtaaaagaggcagtctacttgaattacccccggctggttgagcgttgttgaaaaaaagtaactgaaaaatccgta
gaatagcgccactctgatggttaattaacctattcaattaagaattatctggatgaatgtgccattaaatgcgcagcataatggtgcgttgtgcgggaaaa
ctgcttttttttgaaagggttggtcagtagcggaaactttctgttacatcaaatggcgctttagaccccaattcccgcaaagagtttcttaactaattttgatat
atttaaacgcgtaggacgtaggatttacttgaagcacatttgaggtggattatgaaaaaaattgcatgtctttcagcactggccgcacttctggcggtttct
gcaggttccgcagtagcagcaacttcaaccgtaactggcggctacgctcagagcgacgctcagggtattgctaacaaaactaacggtttcaacctgaa
atatcgctacgagcaggacaacaacccgctgggtgttatcggttcctttacttacactgaaaaagatcgcaccgaaagcagcgtttataacaaagcgca
gtactacggcatcaccgcaggcccggcttaccgcatcaacgactgggcgagcatctacggtgttgtgggtgtaggttacggtaaattccagcagattg
tagacaccgctaaagtgtctgacaccagcgactacg
76 aacacacgctcctgttganaaagagatcccgccgggaaatgcggtgancgtgtctgatattgcgaagagtgtgccagttttggcgcgggcaaaacct
gcaccagtttggtattaatgcaccagtctggcgctttttttcgcgagtttctcctcgctaatgcccgccaggcgcggctttggcgctgatagcgcgctg
aataccgatctggatcaaggttttgtcgggttatcagccaaaaggtgcactctttgcatggttatacgtgcctgacatgttgtccgggcgacaaacggcct
ggtggcacaaattgtcagaactacgacacgactaactgaccgcaggagtgtgcgatgaccctgaatatgatgatggatgccggcgcgcccgaggca
atcgccggtgcgctttcgcgacaccatcctgggctgttttttaccatcgttgaagaagcgcccgtcgccatttcgctgactgatgccgacgcacgcattg
tctatgccaacccggctttctgccgccagaccggctatgaactagaagcgttgttgcagcaaaatccccgcctgcttgcaagtcgccaaaccccacgg
gaaatctatcaggatatgtggcacaccattgttacaacgccgaccgtggcgcgggcaattgattaaccgccaccgcgacggcagcctgtatctggtcga
gatcgatatcaccccggtgattaacccgtttggcgaactggaacactacctggcaatgcagcgcgatatcagcgccagttatgcgctggagcagggt
tgcgcaatcacatgacgctgaccgaagcggtgctgaataacauccggcggcggtggngtagtggatgaacgcgatcatgtggttatggataaccttg
cctacaaaacgttctgtgccgactgcggcggaaaagagctcctgaggaactcaatttttcagcccgaaaagggagctggcaaacggccaggtctt
accggtggtgctgcgcggtgaggtgcgctggttgtcggtgacctgctgggcgctgccgggcgtcagcgaagaagccagttcgctactttattgataac
aggctgacgcgcacgctggtggtgatcaccgacgacacccaacaacgccagcagcaggaacagggccgacttgaccgccttaaacagcagatga
ccaacggcaaactactggcagcgatccgcgaagcgcttgacgccgcgctgatccagataactgccccatcaatatgctggcggcggcgcgacgttt
aaacggcagtgataacaacaatgtggcgctcgacgccgcgtggcgcgaaggtgaagtggcgatggcgcggctgaaacgttgccgcccgtcgctg
gaactggaaagtgcggccgtctggccgctgcaaccctttttgacgatctgcgcgcgctttatcacacccgctacgagcaggggaaaaatttgcaggt
cacgctggattcccatcatctggtgggatttggtcagcgtacgcaactgttagcctgcctgagtctgtggctcgatcgcacgctggatattgccgccgg
gctgggtgatttcaccgcgcaaacgcagatttacgcccgcgaagaagagggctggctctctttgtatatcactgacaatgtgccgctgatcccgctgcg
ccacacccactcgccggatgcgcttaacgctccgggaaaaggcatgcacctgcgcctgatccagacgctggtggcacaccaccacggcgcaatag
aactcacttcacaccccgaagggggaagttgcctgaccctacgattcccgctattcattcactgaccggaggttcaaatgacccagcgaaccgagt
cgggtaataccgtctggcgcttcgatttgtcccagcagttcactgcgatgcagcgcataagcgtggtactcagccgggcgaccgaggtcgatcagac
gctccagcaagtgctgtgcgtattgcacaatgacgcctttttgcagcacggcatgatctgtctgtacgacagccagcaggcgattttgaatattgaagcg
ttgcaggaagccgatcagcagttaatccccggcagctcgcaaatccgctatcgtccgggcgaagggctggtcgggacggtgctttcgcagggccaa
tcattagtgctggcgcgcgttgctgacgatcaggctttcttgaccggctcgggttgtatgattacaacctgccgtttatcgccgtgccgctgatagggcc
agatgcgcagactttcggtgtgctgacggcacaacccatggcgcgttacgaagagcgattacccgcctgcacccgctttctggaaacggtcgctaac
ctggtcgcgcaaaccgtgcgtttgatggcaccaccggcagtgcgcccttccccgcgcgccgccataacacaggccgccagcccgaaatcctgcac
ggcctcacgcgcatttggttttgaaaatatggtcggtaacagtccggcgatgcgccagaccatggagattatccgtcaggtttcgcgctgggacaccac
cgttctggtacgcggcgagagtggcaccggcaaggagctgattgccaacgccatccaccaccattcgccgcgtgccggtgcgccatttgtgaaattc
aactgtgcggcgctgccggacacactgctggaaagcgaattgttcggtcacgagaaaggggcatttaccggcgcggtacgccagcgtaaaggccg
ttttgagctggccgatggcggcacgctgtttcttgacgagatcggcgagagtagcgcctcgtttcaggctaagctgctgcgcattttgcaggaaggcga
aatggaacgcgtcggcggcgacgagacattgcaagtgaatgtgcgcattattgccgcgacgaaccgcaatcttgaagatgaagtccggctggcgca
ctttcgcgaagatctctattatcgcctgaatgtgatgcccatcgccctgccgccactacgcgaacgccaggaggacattgccgagctggcgcactttct
ggtgcgtaaaatcgcccataaccagagccgtacgctgcgcattagcgagggcgctatccgcctgctgatgagctacaactggcccggtaatgtgcgc
gaactggaaaactgccttgagcgctcagcggtgatgtcggagaacggtctgatcgatcgggatgtgattttgtttaatcatcgcgaccagccagccaaa
ccgccagttatcagcgtctcgcatgatgataactggctcgataacaaccttgacgagcgccagggctgattgcggcgctggaaaaaggggatgg
gtacaagccaaagccgcgcgcttgctggggatgacgccgcgccaggtcgcctatcgtattcagacgatggatataaccctgccaaggctataa
77 MTLNMMMDAGAPEAIAGALSRHHPGLFFTIVEEAPVAISLTDADARIVYANPAFcRQTGYELE
ALLQQNPRLLASRQTPREIYQDMWHTLLQRRPWRGQLINRHRDGSLYLVEIDITPVINPFGELE
HYLAMQRDISASYALEQRLRNHMTLTEAVLNNIPAAVVVVDERDHVVMDNLAYKTFcADcG
GKELLSELNFSARKAELANGQVLPVVLRGEVRWLSVTcWALPGVSEEASRYFIDNRLTRTLVV
ITDDTQDRQQQEQGRLDRLKQQMTNGKLLAAIREALDAALIQLNcPINMLAAARRLNGSDNN
NVALDAAWREGEEAMARLKRcRPSLELESAAVWPLQPFFDDLRALYHTRYEQGKNLQVTLD
SHHLVGFGQRTQLLAcLSLWLDRTLDIAAGLGDFTAQTQIYAREEEGWLSLYITDNVPLIPLRH
THSPDALNAPGKGMELRLIQTLVAHHHGAIELTSHPEGGScLTLRFPLFHSLTGGSK
78 MTQRTESGNTVWRFDLSQQFTAMQRISVVLSRATEVDQTLQQVLcVLHNDAFLQHGMIcLYD
SQQAILNIEALQEADQQLIPGSSQIRYRPGEGLVGTVLSQGQSLVLARVADDQRFLDRLGLYDY
NLPFIAVPLIGPDAQTFGVLTAQPMARYEERLPAcTRFLETVANLVAQTVRLMAPPAVRPSPRA
AITQAASPKScTASRAFGFENMVGNSPAMRQTMEIIRQVSRWDTTVLVRGESGTGKELIANAIH
HHSPRAGAPFVKFNcAALPDTLLESELFGHEKGAFTGAVRQRKGRFELADGGTLFLDEIGESSA
SFQAKLLRILQEGEMERVGGDETLQVNVRIIAATNRNLEDEVRLGHFREDLYYRLNVMPIALPP
LRERQEDIAELAHFLVRKIAHNQSRTLRISEGAIRLLMSYNWPGNVRELENcLERSAVMSENGL
IDRDVILFNHRDQPAKPPVISVSHDDNWLDNNLDERQRLIAALEKAGWVQAKAARLLGMTPR
QVAYRIQTMDITLPRL
79 atgccgcaccacgcagcattgtcgcagcactggcaaacggtattttctcgtctgccggaatcgctcaccgcgcagccattgagcgcgcaggcgcagt
cagtgctcacttttagtgattttgttcaggacagcatcatcgcgcatcctgagtggctggcagagcttgaaagcgcgccgccgcctgcgaacgaatggc
aacactatgcgcaatggctgcaagcggcgctggatggcgtcaccgatgaagcctcgctgatgcgcgcgctgcggctgtttcgccgtcgcatcatggt
gcgcatcgcctggagccaggcgttacagttggtggcggaagaagatatcctgcaacagcttagcgtgctggcggaaaccctgatcgtcgccgcgcg
cgactggctttatgaggcctgctgccgtgagtggggaacgccgagcaatccacaaggcgtggcgcagccgatgctggtactcggcatgggcaaact
gggtggcggcgaactcaatttctcatccgatatcgatttgattttcgcctggccggaaaatggcgcaacgcgcggtggacgccgtgagctggataacg
cgcaatttttcactcgccttggtcaacggctgattaaagtcctcgaccagccaacgcaggatggctttgtctaccgcgtcgatatgcgcttgcgcccgttt
ggcgacagcggcccgctggtgctgagctttgccgcgctggaagattactaccaggagcaggggcgcgattgggaacgctacgcgatggtgaaagc
gcgcattatgggcgataacgacggcgaccatgcgcgggagttgcgcgcaatgctgcgcccgtttgttttccgccgtatatcgacttcagcgtgaaca
gtccctgcgtaacatgaaaggcatgattgcccgcgaaggtgcgtcgccgtggcctgaaggacaacattaagctcggcgcgggcgggatccgcgaaat
agaatttatcgtccaggttttccagagattcgcggcggtcgcgagcctgcactgcaatcgcgacactgttgccgacgcttgctgccatagatcaactg
catctgctgccggatggcgacgcaacccggctgcgcgaggcgtatttgtggctgcgacggctggagaacctgctgcaaagcatcaatgacgaacag
acacagacgctgccgggcgatgaactgaatcgcgcgcgcctcgcctggggaatgggcaaagatagctgggaagcgctctgcgaaacgctggaag
cgcatatgtcggcggtgcgtcagatatttaacgatctgattggcgatgatgaaacggattcgccggaagatgcgctttctgagagctggcgcgaattgt
ggcaggatgcgttgcaggaggaggattccacgcccgtgctggcgcatctctcagaggacgatcgccgccgcgtggtggcgctgattgccgattttcg
caaagagttggataaacgcaccattggcccgcgagggcggcaggtactcgatcacttaatgccgcatctgacagcgatgtatgctcgcgcgacgat
gcgccagtaccgctgtcacgcctgacgccgctgacaccggaattattacccgcaccacttaccttgagctgctaagtgaatttcccggcgcactgaaa
cacctcatttccctgtgtgccgcgtcgccgatggttgccagtcagctggcgcgctacccgatcctgcttgatgaattgctcgacccgaatacgctctatc
aaccgacggcgatgaatgcctatcgcgatgagagcgccaatacctgctgcgcgtgccggaagatgatgaagagcaacagcttgaggcgctgcgg
cagtttaagcaggcgcagttgctgcgcgtggcggcggcggatattgccggtacgttgccagtaatgaaagtgagcgatcacttaacctggctggcgg
aagcgattattgatgcggtggtgcagcaagcctgggggcagatggtggcgcgttatggccagccaacgcatctgcacgatcgcgaagggcgcggtt
ttgcggtggtcggttatggcaagctgggcggctgggagctgggttacagctccgatctggatctggtattcctgcacgactgcccgatggatgtgatga
ccgatggcgagcgtgaaatcgatggtcgccagttctatttgcgtctcgcgcagcgcgtgatgcacctgtttagcacgcgcacgtcgtccggcatccttt
atgaagttgatgcgcgtctgcgtccatctggcgctgcggggatgctggtcactactacggaatcgttcgccgattaccagcaaaacgaagcctggacg
tgggaacatcaggcgctggcccgtgcgcgcgtggtgtacggcgatccgcaactgaccgccgaatttgacgccattcgccgcgatattctgatcacgc
ctcgcgacggcgcaacgctgcaaaccgacgtgcgagaaatgcgcgagaaaatgcgtgcccatcttggcaacaagcataaagaccgcttcgatctga
aagccgatgaaggcgftatcaccgacatcgagtttatcgcccaatataggtgaggctttgcccatgacaagccgaaactgacgcgctggtcggat
aatgtgcgcattctcgaagggctggcgcaaaacggcatcatggaggagcaggaagcgcaggcattgacgctggcgtacaccacattgcgtgatga
gctgcaccacctggcgctgcaagagttgccgggacatgtggcgctctcctgttttgtcgccgagcgtgcgcttattaaaaccagctgggacaagtggc
tggtggaaccgtgcgccccggcgtaa
80 atgtttaacgatctgattggcgatgatgaaacggattcgccggaagatgcgctttctgagagctggcgcgaattgtggcaggatgcgttgcaggagga
ggattccacgcccgtgctggcgcatctctcagaggacgatcgccgccgcgtggtggcgctgattgccgattttcgcaaagagttggataaacgcacc
attggcccgcgagggcggcaggtactcgatcacttaatgccgcatctgctcagcgatgtatgctcgcgcgacgatgcgccagtaccgctgtcacgcc
tgacgccgctgctcaccggaattattacccgcaccacttaccttgagctgctaagtgaatttcccggcgcactgaaacacctcatttccctgtgtgccgc
gtcgccgaggttgccagtcagctggcgcgctacccgatcctgcttgatgaattgctcgacccgaatacgctctatcaaccgacggcgatgaatgccta
tcgcgatgagctgcgccaatacctgctgcgcgtgccggaagatgatgaagagcaacagcttgaggcgctgcggcagtttaagcaggcgcagttgct
gcgcgtggcggcggcggatattgccggtacgttgccagtaatgaaagtgagcgatcacttaacctggctggcggaagcgattattgatgcggtggtg
cagcaagcctgggggcagatggtggcgcgttatggccagccaacgcatctgcacgatcgcgaagggcgcggttttgcggtggtcggttatggcaag
ctgggcggctgggagctgggttacagctccgatctggatctggattcctgcacgactgcccgatggatgtgatgaccgatggcgagcgtgaaatcg
atggtcgccagttctatttgcgtctcgcgcagcgcgtgatgcacctgtttagcacgcgcacgtcgtccggcatcctttatgaagttgatgcgcgtctgcgt
ccatctggcgctgcggggatgctggtcactactacggaatcgttcgccgattaccagcaaaacgaagcctggacgtgggaacatcaggcgctggcc
cgtgcgcgcgtggtgtacggcgaccgcaactgaccgccgaatttgacgccattcgccgcgatattctgatgacgcctcgcgacggcgcaacgctgc
aaaccgacgtgcgagaaatgcgcgagaaaatgcgtgcccatcttggcaacaagcataaagaccgcttcgatctganagccgatgaaggcggtatca
ccgacatcgagtttatcgcccaatatctggtgctgcgctttgcccatgacaagccgaaactgacgcgctggtcggataatgtgcgcattctcgaagggc
tggcgcaaaacggcatcatggaggagcaggaagcgcaggcattcacgctggcgtacaccacattgcgtgatgagctgcaccacctggcgctgcaa
gagttgccgggacatgtggcgctctcctgttttgtcgccgagcgtgcgcttattaaaaccagctgggacaagtgcctggtggaaccgtgcgccccggc
gtaa
81 MPHHAGLSQHWQTVFSRLPESLTAQPLSAQAQSVLTFSDFVQDSIIAHPEWLAELESAPPPANE
WQHYAQWLQAALDGVTDEASLMRALRLFRRRIMVRIAWSQALQLVAEEDILQQLSVLAETLI
VAARDWLYEAccREWGTPSNPQGVAQPMLVLGMGKLGGGELNFSSDIDLIFAWPENGATRG
GTRELDNAQFFTRLGQRLIKVLDQPTQDGFVYRVDMRLRPFGDSGPLVLSFAALEDYYQEQGR
DWERYAMVKARIMGDNDGDHARELRAMLRPFVFRRYIDFSVIQSLRNMKGMIAREVRRRGL
KDNIKLGAGGIREIEFIVQVFQLIRGGREPALQSRSLLPTLAAIDQLHLLPDGDATRLREAYLWL
RRLENLLQSINDEQTQTLPGDELNRARLAWGMGKDSWEALcETLEAHMSAVRQIFNDLIGDD
ETDSPEDALSESWRELWQDALQEEDSTPVLAHLSEDDRRRVVALIADFRKELDKRTIGPRGRQ
VLDHLMPHLLSDVcSRDDAPVPLSRLTPLLTGIITRTTYLELLSEFPGALKHLISLcAASPMVAS
QLARYPILLDELLDPNTLYQPTAMNAYRDELRQYLLRVPEDDEEQQLEALRQFKQAQLLRVA
AADIAGTLPVMKVSDHLTWLAEAIIDAVVQQAWGQMVARYGQPTHLHDREGRGFAVVGYG
KLGGWELGYSSDLDLVFLHDcPMDVMTDGEREIDGRQFYLRLAQRVMHLFSTRTSSGILYEV
DARLRPSGAAGMLVTTTESFADYQQNEAWTWEHQALARARVVYGDPQLTAEFDAIRRDILM
TPRDGATLQTDVREMREKMRAHLGNKHKDRFDLKADEGGITTDIEFIAQYLVLRFAHDKPKLT
RWSDNVRILEOLAQNGIMEEQEAQALTLAYTTLRDELHHLALQELPGHVALScFVAERALIKT
SWDKWLVEPcAPA
82 MFNDLIGDDETDSPEDALSESWRELWQDALQEEDSTPVLAHLSEDDRRRVVALIADFRKELDK
RTIGPRGRQVLDHLMPHLLSDVcSRDDAPVPLSRLTPLLTGIITRTTYLELLSEFPGALKHLISLc
AASPMVASQLARYPILLDELLDPNTLYQPTAMNAYRDELRQYLLRVPEDDEEQQLEALRQFKQ
AQLLRVAAADIAGTLPVMKVSDHLTWLAEAIIDAVVQQAWGQMVARYGQPTHLHDREGRGF
AVVGYGKLGGWELGYSSDLDLVFLHDcPMDVMTDGEREIDGRQFYLRLAQRVMHLFSTRTSS
GILYEVDARLRPSGAAGMLVTTTESFADYQQNEAWTWEHQALARARVVYGDYQLTAEFDAIR
RDILMTPRDGATLQTDVREMEREKMRAHLGNKHKDRFDLKADEGGITDIEFIAQYLVLRFAHD
KPKLTRWSDNVRILEGLAQNGIMEEQEAQALTLAYTTLRDELHHLALQELPGHVALScFVAER
ALIKTSWDKWLVEPcAPA
83 EEQQLEALRQFKQAQLLRVAAADIAGTLPVMKVSDHLTWLAEAIIDAVVQQAWGQMVARYG
QPTHLHDREGRGFAVVGYGKLGGWELGYSSDLDLVFLHDcPMDVMTDGEREIDGRQFYLRL
AQRVMHLFSTRTSSGILYEVDARLRPSGAAGMLVTTTESFADYQQNEAWTWEHQALARARV
VYGDPQLTAEFDAIRRDILMTPRDGATLQTDVREMREKMRAHLGNKHKDRFDLKADEGGITD
IEFIAQYLVLRFAHDKPKLTRWSDNVRILEGLAQNGIMEEQEAQALTLAYTTLRDELHHLALQE
LPGHVALScFVAERALIKTSWDKWLVEPcAPA
84 ccgagcgtcggggtgcctaatatcagcaccggatacgagagaaaagtgtctacatcggttcggttgatattgaccggcgcatccgccagcccgccca
gtttctggtggatctgtttggcgattttgcgggtcttgccggtgtcggtgccgaaaaaaataccaatatttgccataacacacgctcctgttgaaaaagag
atcccgccgggaaatgcggtgaacgtgtctgatattgcgaagaggtgccagttttatcgcgggcaaaacctgcaccagtttggttattaatgcacca
gtctggcgctttttttcgccgagtttctcctcgctaatgcccgccaggcgcggctttggcgctgatagcgcgctgaataccgatctggatcaaggttttgtc
gggttatcagccaaaaggtgcactctttgcatggttatacgtgcctgacatgttgtccgggcgacaaacggcctggtggcacaaattattgtcagaactacg
acacgactaactgaccgcaggagtgtgcgatgaccctgaatatgatgatggatgccggcggacatcatcgcgacaaacaatattaataccggcaacc
acaccggcaatttacgagactgcgcaggcatcctttctcccgtcaatttctgtcaaataaagtaaaagaggcagtctacttgaattacccccggctggttg
agcgtttgttgaaaaaaagtaactgaaaaatccgtagaatagcgccactctgatggttaattaacctattcaattaagaattatctggatgaatgtgccatta
aatgcgcagcataatggtgcgttgtgcgggaaaactgcttttttttgaaagggttggtcagtagcggaaacaactcacttcacaccccgaagggggaa
gttgcctgaccctacgattcccgctatttcattcactgaccggaggttcaaaatgacccagcgaaccgagtcgggtaataccgtctggcgcttcgatttgt
cccagcagttcactgcgatgcagcgcataagcgtggtactcagccgggcgaccgaggtcgatcagacgctccagcaagtgctgtgcgtattgcaca
atgacgcctttttgcagcacggcatgatctgtctgtacgacagccagcaggcgattttgaatattgaagcgttgcaggaagccgatcagcagttaatccc
cggcagctcgcaaatccgctatcgtccgggcgaagggctggtcgggacggtgctttcgcagggccaatcattagtgctggcgcgcgttgctgacgat
cagcgctttcttgaccggctcgggttgtatgattacaacctgccgtttatc
85 attgaagagtttgatcatggctcagattgaacgctggcggcaggcctaacacatgcaagtcgagcggcagcgggaagtagatgctactttgccggc
gagcggcggacgggtgagtaatgtctgggtatctgcctgatggagggggataactactggaaacggtagctaataccgcatgacctcgaaagagc
aaagtgggggatcttcggacctcacgccatcggatgtgcccagatgggattagctagtaggtgaggtaatggctcacctaggcgacgatccctagct
ggtctgagaggatgaccagccacactggaactgagacacggtccagactcctacgggaggcagcagtggggaatattgcacaatgggcgcaagc
ctgatgcagccatgccgcgtgtgtgaagaaggccttagggttgtaaagcactttcagcgaggaggaaggcatcanacttaatacgtcggtgattgac
gttactcgcagaagaagcaccggctaactccgtgccagcagccgcggtaatacggagggtgcaagcgttaatcggaattactgggcgtaaagcgca
cgcaggcggtttgttaagtcagatgtgaaatccccgcgcttaacgtgggaactgcatttgaaactggcaagctagagtcttgagaggggggtagaatt
ccaggtgtagcggtgaaatgcgtagagatctggaggaataccggtggcgaaggcggccccctggacaaagactgacgctcaggtgcgaaagcgt
ggggagcaaacaggattagataccctggtagtccacgctgtaaacgatgtcgacttggaggttgtgcccttgaggcgtggcttccggagctaacgcgt
taagtcgaccgcctggggagtacggccgcaaggttaaaactcaaatgaattgacgggggcccgcacaagcggtggagcatgtggtttaattcgatgc
aacgcgaagaaccttacctactcttgacatccacgcaattcgccagagatggcttagtgccttcgggaaccgtganacaggtgctgcatggctgtcgt
cagctcgtgttgtgaaatgttgggttaagtcccgcaacgagcgcaacccttatcctttgttgccagcnngtnatgnngggaactcaaaggagactgcc
ggtgataaaccggaggaaggtggggatgacgtcaagtcatcatggcccttacgagtagggctacacacgtgctacaatggcatatacaaacagaag
cgaactcgcgagg
86 atggcaatgcgtcaatgcgcaatctacgggaaagggggtattgggaaatccaccactacccaaaaccttgtagcggctctggccgaaatgaataaga
aggtcatgatcgtcggctgtgaccctaaggctgattcaacccgcctcattctgcatgcgaaagcacagaacaccatcatggaaatggccgctgaagtg
ggctccgtggaagatctggagctggaagatgtgatgcaaatcggctatggcggcgtgcgctgtgcggaatcaggcggccctgagcctggtgtgggt
tgtgccggacgcggggtgatcaccgccatcaacttcctcgaagaagaaggcgcgtatgtgccggatctggattttgtgttttacgacgtattgggcgat
gtggtctgtggcggtttcgcgatgccaattcgcgaaaacaaagcgcaggaaatctacatcgtgtgctccggtgaaatgatggcgatgtatgccgccaa
caacatttccaaaggcatcgtgaaatacgcgaaatcgggcaaagttcgcctggccgggctgatctgtaactcccgccagacggatcgcgaagatgaa
ctgatcatcgcgctggctgaaaaacttacacgcaatttgatccacttcgtgccgcgtgacaacattgtgcaacgcgctgaaatccgccgcatgacggt
catcgaatacgacccgacttgtgcgcaggcagatcagtatcgtgcactggcgaacaaaatcgtcaacaacaccaaaatggtggtgccgacaccggtc
accatggatgagctggaagccctgttaatggaatttggcattatggaagaagaagacctcgccatcgtcggtcgtaccgccgccgaagaggcgtga
87 atgaaggcaaaagagattctggcgctgattgatgagccagcctgtgagcataaccacaagcagaagtcgggttgcagcctgccgaaaccgggcgc
gacggcaggcggttgtgcgtttgatggcgcgcagattgcgctgctgccggtcgcggacgtcgcgcatctggtgcacggcccgattggctgtaccgg
cagttcatgggacaaccgtggcagccgcagttccgggccttccatcaaccgcatgggcttcaccaccgacatgagcgagcaggatgtgattatgggg
cgcggcgagcgacgcttatttcacgccgtgcagcacatcgtcagccattaccatccggtggcggtctttatttacaacacctgcgtacccgcgatggaa
ggggatgacgttgaagccgtgtgtcgcgccgcatcggccgctgccggtgtgccggttatttcagtcgatgccgccggtttctacggcagcaaaaatct
cggtaaccgcattgccggggacgtgatggtcaaaaaggtgatcggccagcgcgaacccgcgccgtggccggaaaactcaccgatccccgccgga
caccgccacagcatcagcctgattggcgaattcaatattgccggcgagttctggcacgttctgccgctgctcgatgagctcgggatccgcgtgctgtgc
agcctttccggggattcccgttttgctgaaatccagactatgaaccgtggcgaagccaacatgctggtgtgctcgcgggcgctgatcaacgtcgcccg
aaaaatggaagagcgttaccagatcccatggtttgaaggcagtttttatggcctgcgttccatggctgattccctgcgcacgatcgccgtgctgctcaaa
gacccggatttacaggcgcgcacagaacgtctgattgagcgcgaggaggcggcgacacatcttgcgcttgcgccttaccgtgcgcggctcagcgg
gcgcaaggcgctgctgtataccggtggcgtgaaatcctggtcggtggtctcggcgttacaggatttaggcatcacggtggtggcgaccggcacccga
aaatcaaccgaagaagacaagcagcgtattcgcgaactgatgggtgaagacgtgctgatgctcgacgaaggcaatgccagaaccttgctcgacacc
ctctatcgtttcggcggcgacatcatgatcgccgggggccgcaacatgtataccgcgtacaaagccgcctgccgttcctggatatcaatcaggagcg
cgagcatgcgtttgccggatatcacgggctggtaaatctggccgaacagttgtgtatcaccctggaaagcccggtctgggcgcaggtcaaccgtctg
gcgccgtggcgctaa
88 atgaccagtgaaacacgcgaacgtaacgaggcattgatccaggaagtgctggagatcttccccgagaaggcgcttaaagatcgtaagaaacacatg
atgaccaccgacccggcgatggaatctgtcggcaagtgtattgtctcaaaccgcaaatcacagccgggcgtgatgaccgtgcgaggctgcgcttacg
ccggttccaaaggcgtggtctttggcccgatcaaagacatggcgcatatctcccacggcccggttggttgcggccagtattcccgtgccggacgccgt
aactattacaccggctggagcggcgtgaacagctttggcaccctcaacttcaccagtgattttcaggaacgggacatcgtatttggcggcgataaaaag
ctcgacaaattgatcgatgaactggagatgttgttcccgctgagcaaaggcatttcggtgcagtcggaatgtccggtcggtctgatcggcgatgacattt
ctgccgtcgccaaagccagcagcgccaaaatcggtaagccggtcgtgccggtacgctgcgaggggttccgcggtgtgtcgcaatcgctcggccatc
acattgctaacgatgtcatccgcgactgggtgctggataaccgcgaaggcaatgaatttgaaaccacgccttacgacgtggcgattatcggcgactac
aacatcggcggtgacgcctggccctcacgtattctgctcgaagaaatggggctgcgcgtggtggcgcagtggtccggcgacggcacgctggtgga
gatggaaaacaccccgaaagtcgcgctcaatctggtgcactgctaccgctcgatgaactacatctcccgtcatatggaagaaaaacacggcattccgt
ggatggaatacaacttctttggcccgaccaaaattgcggaatctctgcgcgaaatcgcggcggttttgacgataccatccggaaaaacgccgaagc
ggtgattgaaaaatatcaggcgcaaacgcaggcggtgatcgacaaataccgtccgcgtctggaaggcaaaaaggtgctgttgtatctcggcggtttac
gtccgcgccacatcatcggggcgtatgaagatctgggaatggaaatcatcggtaccggctatgaattcggtcataacgatgattacgaccgcaccttac
cgatgctcaaagaaggcacgttgctgttcgatgacctgagcagttatgagctggaagcgttcgttaaagcgctgaaaccggatcttgtcgggtcaggta
tcaaagaaaaatacattttccagaaaatgggcgtgccgttccgccagatgcactcctgggattattccggcccttatcacggctacgacggtttcggcat
ttttgcccgtgacatggacatgacgctgaacaatccgggctggagtcagctgaccgccccctggttgaaaagcctga
89 atggctcaaattctgcgtaatgccaagccgcttgccaccacgcctgtcaaaagcgggcaaccgctcgcggcgatcctggccagtcaggggctggaa
aattgcatcccgctggttcacggcggcaaggttgtagcgcgttcgccaaagttttcttcatccagcattttcacgatccgatcccgttgcagtccacggc
gatggaatcgaccacgactatcatgggctcggatggcaacgtcagtactgcgttgaccacgttgtgtcagcgcagtaatccaaaagccattgtgattttg
agcaccggactgtcagaagcgcagggcagtgatttgtcgatggcgctgcgtgagtttcgcgacaaagaaccgcgctttaatgccatcgctattctgac
cgttaacacgccggatttttacggctcgctggaaaacggctacagcgcgctgatggaaagcgtgatcactcagtgggtgccggaaaagccgccgac
cggcatgcgtaacaagcgcgtgaacctgctggtgagccatctgctgacgccgggggatctggaattactgcgcagctatgtcgaagcctttggcctg
caaccggtgatcctgccggatttatcacagtcgctggacggacatctggcgaatggcgatttcaatccggtcacgcagggcggcacgtcgcaacgcc
agattgaacaaatggggcagagcctgaccaccattaccattggcagttcgctcaactgcgccgccagtctgatggcgatgcgcagccgtggcatggc
gctgaacctgccgcacctgatgacgctggaaaacatggacagtctgatccgccatctgcatcaggtgtcaggccgcgaggtaccggcatggattgag
cgccagcgcgggcaactgaggacgccatgatcgactgccatacctggctgcagtcacagcgtattgcgctggcggcagaagcggatttgctggtg
gcgtggtgcgatttcgctcagagccagggaatgcgcgtcgggccggtgattgcgccggttaatcagcagtcactggccgggctgccggtcgaacag
gtggtgatcggcgatctggaagatttacaaacccggctcgacagctacccggtttcactgctggtggcgaactcccacgctgcaccactggcggaaa
aaaacggtatcgcgctggtacgtgccggtttcccgctttacgaccgtctcggggaatttcgccgcgtgcggcagggctatgcgggtattcgcgacacc
ttgttcgaactcgcgaacctgatgaggcgcgccatcacatgctgacggcgtatcactcaccgcttaggcaggtgttcggcctgagcccggtaccgg
aggccagtcatgaggcggctaa
90 atgagtcaagatcttggcaccccaaaatcctgtttcccgctcttccagcaggatgaataccagaatatgtttacccacaaacgcgcgctggaagaagca
cacggcgaggcgaaagtgcgggaagtgtttgaatggaccaccacgcaggaatatcaggatctgaacttctcgcgtgaagcgctgaccgtcgacccg
gcgaaagcctgccagccgttaggcgccgtactttgcgcgctgggttttaccaacacgttgccgtatgtccatggttcacaaggctgtgtggcctatttcc
gtacctattttaatcgtcatttcaaagagccggtggcctgtgtttccgactcaatgaccgaagatgccgccgtttttggcggaaataacaacatgaatgtc
ggtctggaaaacgccagcgcgctgtacaagccggaaattattgcggtctccaccacctgtatggcggaagtgatcggtgatgacctgcaggcttttatc
gccaacgccaaaaaagacggatttgtggatgcggtatgccaatcccgtatgcccatacaccgagttttctgggcagtcatgtcaccggctgggacaa
catgtttgaaggcttcgcccgtacctttaccaccgacgccacgcgggaatatcagccgggcaaacttgccaaactgaacgtggtgaccggttttgaaa
cttatctcggcaactaccgggttattcaccgcatgatgagccagatgggggtcgaatgcagcgtcttgtccgatccgtctgaagtgctcgacaccccgg
ctgacggccaataccgcatgtatgccgacggcaccacgcaaaccgaaatgcgtgatgaccggatgccatcgacaccttgctgctgcaaccgtggc
aattacagaaaaccaaaaaggtggtgcagggcgactggaatcagccgggcaccgaagtcagtgtaccgattggcctggcggcgaccgatgccttg
ctgatgacggtaaggaactgaccggcaaaccgatagctgacgcgctggcgactgaacgtggccgtctggtggacatgatgctcgattctcacacct
ggctgcacggcaagcgtttcggtctctacggtgacccggattttgtgatgggcatgaccgcattcctgctggaactgggctgtgaaccgaccaccattc
tcagccataacggcaacaaacgctggcagaaagccatgaagaaaatgctggctgattcgccttacggacaggacagcgaagtgtatgtgaactgcg
atctgtggcatttccgctcgctgatgtttacccgtaaaccggactttatgatcggcaactatacggaaaattcattcagcgtgacacgctggccaaaggc
gaacagttcgaagtgccgctgatccgtatcggattcccgatttttgaccggcaccatttgcaccgtcagaccacctggggatacgagggcgcgatgag
catcctgacgcaactggtgaatgcggtgctcgaacagctggatcgcgaaaccatgaagctcggcaaaaccgactacaacttcgatctgatccgctaa
91 atgagcatcacggcgttatcagcatcatttcctgaggggaatatcgccagcgcttgtcgctgcaacatccttcactgttttataccgtggttgaacaatca
tcggtggcgatttcgctgaccgatccgcaggcgcgcatttgttatgccaatccggcattctgccgccagacgggttttgcacttgagacacttttgggcg
agaaccaccgtctgctggccagccagcagacgccgaaacatatctatgacgaaatgtggcgcactttgttgcagggctaatcctggaacggccaact
gatcaaccggcgtaataaccgttcgctttatctggcggatgtcactatcacgcctgttttaggcgcggacgggcaggtggagcattacctcggcatgca
caaagatatcagcgagaaatacgcgctgcagcagggttgcgcaaccacatcaccttgttcacggaggtgctgaacatttattcccgccgccgtggtg
gtggtggatgagaggacaatgtggtgatggacaatctggcctacaaaaccctgtgcgctgactgcggcgcaaaagagctgttggccgaaatgggc
tatccgcaactcaaagagatgctcaacagtggcgaaccggtgccggtttccatgcgcggcaacgtacgctggttttctttcggtcagtggtcattgcag
ggcgttaatgaagaggccagccgcttttttaccggcattaccgcgccgggaaaactgattgttctcaccgactgaccgatcagcatcaccggcagca
gcagggttatcttgaccggctcaagcaaaaacttaccaacggcaaattgctggcagccatccgcgagtcgcttgatgccgcgctgattcagctcaacg
ggccattttaatatgctggcggctgcgcgtcgtcttaacggcgaagaaggcaacaacatggcgctggaattcgcctggcgcgaaggcgagcaggcg
gtgagtcgcttacaggcctgccgtccgtcgctggattttgagccgcaggcagaatggccggtcagtgaattcttcgacgatctgagcgcgctgtacga
cagccattttctcagtgacggtgaattgcgtacgtggtcatgccatctgatctgcacgctgtcgggcaacgaacgcaaatccttaccgcgctgagcttat
ggattgatcacacgctgtcacaggcgcaggccatgccgtctctgaagctctcggtgaacattgttgcgaagcaggatgcgagctggttgtgttttgaca
ttaccgataatgtgccgcgtgaacgggtgcgttatgcccgcccggaagcggcgttttcccgtccgggcaatggcatggagctgcgccttatccagac
gctgatcgcccatcatcgcggttctttagatctctcggtccgccctgatggcggcaccttgagacgttacgcctgccggtacagcaggttatcaccgga
ggcttaaaatga
92 atgacccagttacctaccgcgggcccggttatccggcgctttgatatgtntgcccagtttacggcgctttatcgcatcagcgtggcgctgagtcaggaa
agcaataccgcgcgcgcactggcggcgatcctcgaagtgcttcacgatcatgcatttatgcaatacggcatggtgtgtctgttcgataaagaacgcaat
gcactgtttgtggaatccctgcatggcatcgacggcgaaaggaaaaaagagacccgccatgtccgttaccgcatgggggaaggcgtgatcggcgc
ggtgatgagccagcgtcaggcgctggtgtaccgcgcatttcagacgatcagcgttttctcgaccgcctgaatatttacgattacagcctgccgatgatt
ggtgtgccgatccccggtgcggataatcagcctgcgggtggctggtggcacagccgatggcgttgcacgaagaccggctggctgccagtacgcg
gtttttagaaatggcgccaatctcatcagccagccactgcgttctgccacgcccccggaatcattgcctgctcaaacgccggtccggtgcagtgttccg
cgccagtttggttttgagcagatggtcgggaaagtcaggcgatgcgccagacgatggacattttacggcaggtttccaaatgggataccacggttctg
gtgcgtggtgaaagcggcaccggcaaggaacttatcgccaatgccattcattacaactcaccccgtgcggccgcgccatttgtgaaattcaactgcgc
cgcgctgccggataacctgctggaaagcgaactgttcggtcatgaaaaaggggccttcaccggcgctatacgcacccgaaaaggccgctttgaact
ggcggacgggggcacgttattcctcgatgaaatcggcgaatcgagcgcgtcgtttcaggccaaattgctgcgcattttgcaggaaggtgaaatggaa
cgggtcggcggcgataccacgctgaaagttgatgtgcgcattattgctgccaccaaccgtaatcttgaggaggaagtgcgtgccgggaattttcgcga
agacctgtattatcgcctcaacgtgatgccggtttcgctgcctgcactgcgtgaaaggctggatgatatcgccgatctggcgccgtttctggtcaaaaag
attgcgctgcgtcaggggcgggaactgcgcatcagtgatggtgcggtgcgtctgctgatgacctacagctggccaggcaacgtgcgtgaactggaa
aactgcctcgaacgggcgtcggtaatgaccgatgaagggctgatcgaccgcgacgtgatcctgttcaatcaccatgagtccccggcgctgtccgtca
aacccggtctgccgctcgcgacagatgaaagctggctggatcaggaactcgacgaacgccagcgggtgattgctgcactggagaaaaccggctgg
gtgcaggccaaagcggcccgactgagggcatgacaccgcgccagattgcctaccgtatccagattatggacatcaacatgcaccgtatctga
93 atgttgccactttcttctgttttgcaaagccacgcgcagagtttgcctgaacgctggcatgaacatcctgaaaacctgcccctccccgatgatgaacagct
ggctgtgctgagcagcagtgaattcatgacggacagtttgctggcttttccgcagtggtggcatgaaattgtccaaaatccccctcaggcgcaggagtg
gcaactttaccgtcagtggctggatgaatcactgacgcaggtgactgacgaagccgggttaatgaaagctttgcgtctgttccgccgccgtattctgac
ccgcattgcgtggtcacagtccgcgcaaaccagcgaagcaaaagatacgcttcaccagctgagtgaactggcggaattattgattgtcagcgcccgt
gactggctgtatgccgcttgctgtcgcgagttcggtacgccggtcaatgccgcaggggaaccgcagagaatgctgatcctcgggatgggcaaactc
ggcggtggcgagctgaatttcatcggacatcgacctgacctgatttttgcttatccggaaaatggccagacacgcggcggtcggcgtgaactggataacgc
acaattttcacccggctcggccagcgtctgatcaaagcgctggatcagcccactatcgacggttttgtctatcgcgtggacatgcgtttgcgtccgttcg
gcgacagtggcccgctggtgatgagcttcccggcactggaagattattatcaggaacaggggcgcgactgggaacgctacgcaatggtgaaagcg
cgtctgatgggcggcgcggaggacatcagcagtcaggaattgcgtaaaatgctgatgcttttgtcttccgccgttatatcgatttcagtgtgatccagtc
cctgcgtaacatgaaaggcatgatcgcccgcgaagtacgccgccgtggtctgaaagacaacatcaaactcggcgcaggcggtattcgtgaaattgaa
tttatcgtgcaggtatttcagctgatccgtggcggtcgtgaaccggcattgcagcagcgtgcgttgttgccaacgcttcaggcgctggaaaatctgggg
ctgctgccggtagagcaggtgttgcagttgcgtaacagctatctgttcctgcgacgtctggaaaacctgttgcaggccattgctgacgagcaaacgcaa
accttaccgtccgatgagctgaatcaggcgcgtctggcgtgggggatgaattacgctggctggccgcagcttaggatgcagtgaatgctcacatgca
ggccgtacgcgcggtatttaacgatctgattggcgatgacacgccagatgccgaagatgacgtgcaactctcccggttcagcagtttatggattgatac
gcttgagcctgacgagctggctccgctggtgccgcaacttgacgaaaatgcgcaacggcatgttttacatcagattgctgattttcgccgtgacgtggat
aaacgcacgatagggccacgtgggcgtgatcagttggatttgctgatgccgcgtttactggcccaggtctgcacctataaaaatgcggatgtgacgtta
cagcgcctgatgcagttgctgctcaatatcgtcacgcgcacgacgtatatcgagctgctggtggaatatcccggtgcgctcaaacagttaatacgtctgt
gcgctgcctcgccgatggtggcgacgcaacttgcgcgtcatcctttattgctcgacgaactgctcgacccgcgcacgctttaccagccgattgagccg
ggcgcgtaccgtgatgaactgcggcaatatctgatgcgggtgccaaccgaagacgaagaacaacagcttgaagccgtgcgccagttcaaacaggc
acagcatttacgtattgcggccggggatatttccggtgcgttgccggtgatgaaagtcagtgaccatttaacctaccttgcggaggccattctgacgtt
gtggttgcaacaggcgtgggaacaaatggtcgtaaaatacggtcagccaacccatcttcagcaccgtaaagggcgcggttttgccgtggtgggttacg
gaaaactcggtggctgggagctgggttacagctcggatctggatctggtcttcctgctcgattgcgcgccggaagtcatgaccgacggcgaacgcag
cattgacgggcgtcagttttatctgcggctggcgcagcgcatcatgcatttattagcacccgtacgtcgtcaggcattctttatgaggttgacccgcgtc
tgcggccttccggtgcttccggcatgctggtcagcaccatcgaagcttttgcggattatcaggccaacgaagcctggacatgggagcatcaggcgctg
gttcgcgcgcgtgtggtttatggtgatccgcaactgacgcagcaatttaatgccacgcgtcgcgacattctttgccgccagcgcgatgccgacggcttg
cgtaaggaagtccgtgaaatgcgcgagaaaatgtatgcccatctgggcagcaaaagagccgacgagtttgatctgaaagccgatccgggtggcata
acggatattgaattcatcgcacaatatctggttctgcgtttcgcgcatgatgagccgaagagacccgctggtagataacgtgcggattttcgaactgat
ggcgcgacatgacatcatgccggaagaggaagcacgccatctgacgcaggcttacgtgacattgcgcgatgaaattcatcatctggcgtgcaggaa
cacagcgggaaagtggccgcagacagctttgccactgagcgcgcccaaatccgcgccagctgggcaaactggcttggctga
94 atgaaaaaacttttatccatgatggggcttggtgcagtggctttgctaccttcgcttgccatggcagcaccaccagcagcggcaaacggtgctgataac
gcctttatgatgatttgtaccgcgctggtattgttcatgaccgtacccggtgtggcgttgttctacggcggcttactgcgttctaaaaacgttttgtccatgct
gactcaggttattgttacctttgctctggtctgcgtcctgtggatcctctacggttacagccttgccttcagtgaaggtaacgcgttcttcggtggtttcagca
acgtaataatgaaaggcattggcctggattctgtgactggcaccttctcgcagatgatccacgttgcattccagtgttcatttgcctgcatcactgtagcgc
tgatcgtaggtggtattgctgaacgtgtgcgtttctcagcagttctgattttcactgtgatctggctgactttctcttatattccgatggctcacatggtatggg
caggcggtttcctggctgctgacggtgcgctggactttgccggtggtaccgttgttcatatcaatgccgcaattgctggcctggtaggggcttatctgctg
ggtaaacgcgccggttttggcaaagaagctttcaaaccacacaacctgccaatggtcttcactggcgcctcaatcctgtatgtgggctggttcggcttca
atgcgggttcagcaagtgccgcaagctctgttgccgcgctggctttcctgaacactgtcattgctactgctggcgcaatcctgtcctggacgctggttga
gtggatggtgcgcggtaagccctcactgctgggcgcaagctccggtgctatcgcaggtctggtggctatcacgcctgcatgtggtacggtcggcgta
ggtggtgctctgattatcggtctggtaggcggtatcactggtctgtggggggttgttaccctgaaaaaatggctgcgtgttgatgacacctgtgatgtgtt
cggtgttcatggcgtgtgcggtatcgtaggttgtctgctgacgggtgtattcacgtccagttcacttggcggcgtgggctacgcagaaggcgtgaccat
gggccatcaggtttgggtgcagttcttcagcgtgtgcgtaacattggtctggtcaggcgttgttgccttcatcggttacaaagtggctgacatgatcgtag
gtctgcgtgttcctgaagaacaagaacgcgaaggtctggacgttaacagccacggcgaaaacgcttacaaccaataa
95 tgaatatcactgactcacaagctacctatgtcgaagaattaactaaaaaactgcaagatgcaggcattcgcgttaaagccgacttgagaaatgagaaga
ttggctttaaaattcgcgaacacacgctacgccgtgttccttatatgttagtttgtggcgataaagaggtcgaagcaggcaaagttgctgttcgtactcgtc
gcggcaaagacttaggaagcatggatgttagcgaagtcgttgacaaactgctggcggaaatccgcagcagaagtcatcatcaactggaggaataaa
gtattaaaggcggaaaacgagttcaaccggcgtcctaatcgcattaacaaagagattcgcgcgcaagaagttcgcctcaccggcgtcgatggcga
gcagattggtattgtcagtctgaatgaagctcttgaaaaagctgaggaagcgggcgtcgatttagtagaaatcagtccgaatgccgagccgccagtttg
tcgaatc
96 attgaagagtttgatcatggctcagattgaacgctggcggcaggcctaacacatgatagtcgagcggtagcacagagagcttgctctcgggtgacga
gcggcggaccggtgagtaatgtctgggaaactgcctgatggagggggataactactggaaacggtagctaataccgcataacgtcgcaagaccaaa
gtgggggaccttcgggcctcatgccatcagatgtgcccagatgggattagctagtaggtggggtaacggctcacctaggcgacgatccctagctggt
ctgagaggatgaccagccacactggaactgagacacggtccagactcctacgggaggcagcagtggggaatattgcacaatgggcgcaagcctga
tgcagccatgccgcgtgtgtgaagaaggccttcgggttgtaaagcactttcagcggggaggaaggcggtgaggttaataacctcaccgattgacgtta
cccgcagaagaagcaccggctaactccgtgccagcagccgcggtaatacggagggtgcaagcgttaatcggaattactgggcgtaaagcgcacgc
acgcggtctgtcaagtcggatgtgaaatccccgggctcaacctgggaactgcattcgaaactggcaggctagagtcttgtagaggggggtagaattc
caggtgtagggtgaaatgcgtagagatctggaggaataccggtggcgaaggcggccccctggacaaagactgacgctcaggtgcgaaagcgtg
gggagcaaacaggattagataccctggtagtccacgctgtaaacgatgtcgatttggaggttgtgcccttgaggcgtggcttccggagctaacgcgtta
aatcgaccgcctggggagtacggccgcaaggttaaaactcaaatgaattgacgggggcccgcacaagcggtggagcatgtggtttaattcgatgca
acgcgaagaaccttacctggtcttgacatccacagaactttccagagatggattggtgccttcgggaactgtgagacaggtgctgcatggctgtcgtca
gctcgtgttgtgaaatgttggg
97 atgaccatgcgtcaatgcgctatctacggtaaaggcggtatcggtaaatccaccaccacccagaatctcgtcgcggccctcgccgagatgggtaaga
aagtgatgatcgtcggctgcgatccgaaagcggattccacccgtctgatcctccacgctaaagcccagaacaccatcatggagatggcggcggaagt
gggctcggtcgaggatctggagctcgaagacgttctgcaaatcggctatggcgatgtccgttgcgccgaatccggcggcccggagccaggcgtcg
gctgcgccggacgcggggtgatcaccgccatcaacttcctcgaggaagaaggcgcctatgaagaagatttggatttcgtcttctatgacgtcctcggc
gacgtcgtctgcggcggcttcgctatgccgatccgcgaaaacaaagcccaggagatctacatcgtctgctccggcgagatgatggcgatgtatgccg
ccaacaatatctccaaagggatcgtgaagtacgccaaatccggcaaggtgcgcctcggcggcctgatctgtaactcgcaaaaccgaccgggaag
acgaactgatcatcgccctggcggagaagcttggcacgcagatgatccacttcgttccccgcgacaacattgtgcagcgcgcggagatccgccgga
tgacggtgatcgagtacgacccgacctgtcagcaggcgaatgaatatcgtcaactggcgcagaagatcgtcaataacaccaaaaaagtggtgccga
cgccgtgcaccatggacgagctggaatcgctgctgatggagttccgcatcatggaagaagaagacaccagcatcattggtaaaaccgccgctgaag
aaaacgcggcctga
98 atggttaggaaaagtagaagtaaaaatacaaatatagaactaactgaacatgaccatttattaataagtcaaataaaaaagcttaaaacacaaaccacttg
cttttttaataataaaggaggggttgggaagactacattagtagcaaatttaggagcagagctatcaataaactttagtgcaaaagttcttattgtggatgcc
gaccctcaatgtaatctcacgcagtatgtatttagtgatgaagaaactcaggacttatatgggcaagaaaatccagatagtatttatacagtaataagacc
actatcctttggtaaaggatatgaaagtgacctccctataaggcatgtagagaatttcggttttgacataattgtcggtgaccctagacttgctttacaggaa
gaccttttagctggagactggcgagatgccaaaggcggtgggatgcgaggaattaggacaacttttgtatttgcagagttaattaagaaagctcgtgag
ctaaattatgattttgttttctttgacatgggaccatcattaggcgcaatcaacagggcagtattactggcaatggaattctttgtcgtcccaatgtcaatcga
tgtattttcactatgggctattaaaaatattggctccacggtttcaatatggaaaaaagaattagacacagggattcggctctcagaggaacctagcgaatt
atcacaattatcacctcaaggaaaactaaagtttctcggttacgtcacccaacaacataaagaacgctctggatacgatacaattcagcttgagaatactg
aggaagaaataaaatcgaaacgtcgggtaaaggcgtatgaagacattggagaggtgtttccttctaaaattactgagcatctttctaaactttatgcatca
aaagatatgaacccacaccttggagatatacgtcatttaggtagtttagctccgaaatcacaatcacaacacgttccgatgatatcagtgtctggtacagg
aaattacaccagacttagaaaaagcgcgcgtgaactttatcgagatattgcaagaagatacttagagaacattcagactgctaatggcgagaaatag
99 atgaagggaaaggaaattctggcgctgctggacgaacccgcctgcgagcacaaccagaagcaaaaatccggctgcagcgcccctaagcccggcg
ctaccgccggcggttgcgccttcgacggcgcgcagataacgctcctgcccatcgccgacgtcgcgcacctggtgcacggccccatcggctgcgcg
ggcagctcgtgggataaccgcggcagcgtcagcgccggcccggccctcaaccggctcggctttaccaccgatcttaacgaacaggttgtgattatg
ggccgcggcgaacgccgcctgttccacgccgtgcgtcacatcgtcgaccgctatcatccggcggcggtctttatctacaacacctgcgtaccggcga
tggagggcgatgacatcgaggcggtctgccaggccgcacagaccgccaccggcgtcccggtcatcgctattgacgccgccggtttctacggcagta
aaaatcttggcaaccgaatggcgggcgacgtgatgctcaggcaggtgattggccagcgcgaaccggccccgtggccagacaacacgccctttgcc
ccggcccagcgccacgatatcggcctgattggcgaattcaatatcgccggcgagttctggcaggtccagccgctgctcgacgagctggggatccgc
gtcctcggcagcctctccggcgacggccgctttgccgagatccagaccctgcaccgggcgcaggccaatatgctggtgtgctcgcgcgcgctgatc
aacgtcgcccgggggctggagctgcgctacggcacgccgtggtttgaaggatgcttctacgggatccgcgccacctccgacgccttgcgccagct
ggcgacgctgctgggggatgacgacctgcgccgccgcaccgaggcgctgatcgcccgcgaagagcaggcggcggagcaggctcttgcgccgt
ggcgtgagcagctccgcgggcgcaaagtgctgctctataccggcggcgtgaaatcctggtcggtggtatcggccctgcaggatctcggcatgaccg
tggtggccaccggcacgcgcaaatccaccgaggaggacaaacagcggatccgtgagctgatgggcgacgaggcggtgatgcttgaggagggca
atgcccgcaccctgctcgacgtggtgtaccgctatcaggccgacctgatgatcgccggcggacgcaatatgtacaccgcctggaaagcccggctgc
cgttctcgatatcaatcaggagcgcgagcacgcctacgccggctatcagggcatcatcaccctcgcccgccagctctgtctgaccctcgccagcccc
gtctggccgcaaacgcatacccgcgccccgtggcgctag
100 atgaccaacgcaacaggcgaacgtaaccttgcgctcatccaggaagtcctggaggtgtttcccgaaaccgcgcgcaaagagcgcagaaagcacat
gatgatcagcgatccgcagatggagagcgtcggcaagtgcattatctcgaaccgtaaatcgcagcccggggtgatgaccgtgcgaggctgcgccta
tgcgggctcgaaaggggtggtgtttgggccaatcaaagacatggcccatatctcgcacggccccatcggctgcggccagtattcccgcgccggacg
gcgcaactactataccggcgtcagcggtgtcgacagcttcggcaccctgaacttcacctctgattttcaggagcgcgatattgttttcggcggcgataaa
aagctgaccaaactgatcgaagagatggagctgctgttcccgctgaccaaagggatcaccatccagtcggagtgcccggtgggcctgatcggcgat
gacatcagcgccgtagccaacgccagcagcaaggcgctggataaaccggtgatcccggtgcgctgcgaaggctttcgcggcgtatcgcaatcgct
gggccaccatatcgccaacgacgtggtgcgcgactgggtgctgaacaatcgcgaagggcagccgtttgccagcaccccgtacgatgttgccatcatt
ggcgattacaacatcggcggcgacgcctgggcctcgcgcattctgctggaagagatgggcctgcgcgtagtggcgcagtggtccggcgacggca
ccctggtggagatggagaacaccccattcgttaagcttaacctcgtccactgctaccgttcgatgaactatatcgcccgccatatggaggagaaacatc
agatcccatggatggaatataacttcttcggcccgaccaaaatcgccgaatcgctgcgcaagatcgccgatcaatttgatgacaccattcgcgccaatg
cggaagcggtgatcgcataatatgaggggcagatggcggccatcatcgccaaatatcgcccgcggctggaggggcgcaaagtgctgctgtacatg
ggggggctgcggccgcgccacgtcatcggcgcctatgaggatctcgggatggagatcatcgccgccggctacgagtttgcccataacgatgattac
gaccgcaccctgccggacctaaagagggcaccctgctgtttgacgatgccagcagctatgagctgtgaggccttcgtcaaagcgctgaaacctgac
ctcatcggctccgggatcaaagagaaatatatcttccagaaaatgggggtgccgttccgccagatgcactcctgggactattccggcccctatcacgg
ctatgacggcttcgccatctttgcccgcgatatggatatgaccctgaacaatccggcgtggaacgaactgactgccccgtggctgaagtctgcgtga
101 atggcagatattatccgcagtgaaaaaccgctggcatgagcccgattaaaaccgggcaaccgctcggggcgatcctcgccagcctcgggctggc
ccaggccatcccgctggtccacggcgcccagggctgcagcgccttcgccaaagttttctttattcagcatttccatgacccggtgccgctgcagtcgac
ggccatggatccgaccgccacgatcatgggggccgacggcaatatcttcaccgcgctcgacaccctctgccagcgccacagcccgcaggccatcg
tgctgctcagcaccggtctggcggaagcgcagggcagcgatatcgcccgggtggtgcgccagtttcgcgaggcgcatccgcgccataacggcgtg
gcgatcctcaccgtcaataccccggatttttttggctctatggaaaacggctacagcgcggtgatcgagagcgtgatcgagcagtgggtcgcgccgac
gccgcgtccggggcagcggccccggcgggtcaacctgctggtcagccacctctgttcgccaggggatatcgaatggctgggccgctgcgtggag
gcctttggcctgcagcccgtgatcctgccggacctctcgcagtcaatggatggccacctcggtgaaggggattttacgcccctgacccagggcggcg
cctcgctgcgccagattgcccagatgggccagagtctgggcagcttcgccattggcgtgtcgctccagcgggcggcatcgctcctgacccaacgca
gccgaggcgacgtgatcgccctgccgcatctgatgaccctcgaccattgcgatacctttatccatcagctggcgaagatgtccggacgccgcgtacc
ggcctggattgagcgccagcgtggccagctgcaggatgcgatgatcgactgccatatgtggcttcagggccagcgcatggcgatggcggcggagg
gcgacctgctggcggcgtggtgtgatttcgcccgcagccaggggatgcagcccggcccgctggtcgcccccaccagccaccccagcctgcgcca
gctgccggtcgagcaagtcgtgccgggggatcttgaggatctgcagcagctgctgagccaccaacccgccgatctgctggtggctaactctcacgc
ccgcgatctggcggagcagtttgccctgccgctgatccgcgtcggttttcccctcttcgaccggctcggtgagtttcgtcgcgtccgccaggggtacgc
cggtatgcgagatacgctgtttgaactggccaatctgctgcgcgaccgccatcaccacaccgccctctaccgctcgccgcttcgccagggcgccgac
ccccagccggcttcaggagacgcttatgccgcccattaa
102 atgagccaaacgatcgataaaattcacagctgttatccgctgtttgaacaggatgaataccagaccctgttccagaataaaaagacccttgaagaggcg
cacgacgcgcagcgtgtgcaggaggtttttgcctggaccaccaccgccgagtatgaagcgctgaaatccagcgcgaggcgctgaccgtcgaccc
ggccaaagcctgccagccgctcggcgccgtactctgcgcgctggggttcgccggcaccctgccctacgtgcacggctcccagggctgcgtcgcct
attttcgcacctactttaaccgccattttaaagagccggtcgcctgcgtctccgactccatgaccgaggacgcggcggtgttcggcggcaacaacaac
atgaatctgggcctgcagaatgccagcgcgctgtataaacccgagattatcgccgtctccaccacctgtatggccgaggtgatcggcgacgatctgca
ggcgtttatcgccaacgccaaaaaagagggatttgttgacgaccgcatcgccattccttacgcccatacccccagctttatcggcagccatgtcaccgg
ctgggacaatatgttcgaagggttcgcgaagacctttaccgctgactacgccgggcagccgggcaaacagcaaaagctcaatctggtgaccggattt
gagacctatctcggcaacttccgcgtgctgaagggatgatggcgcagatggatgtcccgtgcagcctgctctccgacccatcagaggtgctcgaca
cccccgccgacggccattaccggatgtacgccggcggcaccagccagcaggagatcaaaaccgcgccggacgccattgacaccctgctgctgca
gccgtggcagctggtgaaaagcaaaaaggtggttcaggagatgtggaaccagcccgccaccgaggtggccgttccgctgggcctggccgccacc
gacgcgctgctgatgaccgtcagtcagctgaccggcaaaccgatcgccgacgactgaccaggagcgcggccggctggtcgacatgatgctggat
tcccacacctggctgcatggcaaaaaattcggcctctacggcgatccggatttcgtgatggggctgacgcgcttcctgctggagctgggctgcgagcc
gacggtgatcctcagtcataacgccaataaacgctggcaaaaagcgatgaagaaaatgctcgatgcctcgccgtacggtcaggaaagcgaagtgttc
atcaactgcgacctgtggcacttccggtcgctgatgttcacccgtcagccggactttatgatcggtaactcctacggcaagtttatccaggcgataccc
tggcaaagggcaaagccttcgaagtgccgctgatccgtctgggctttccgctgttcgaccgccatcatctgcaccgccagaccacctggggctatgaa
ggcgcaatgaacatcgtcacgacgctggtgaacgccgtgctggaaaaactggaccacgacaccagccagttgggcaaaaccgattacagcttcgac
ctcgttcgttaa
103 atgaccctgaatatgatgctcgataacgccgcgccggaggccatcgccggcgcgctgactcaacaacatccggggctgttttttaccatggtggaaca
ggcctcggtggccatctccctcaccgatgccagcgccaggatcatttacgccaacccggcgttttgccgccagaccggctattcgctggcgcaattgtt
aaaccagaacccgcgcctgctggccagcagccagacgccgcgcgagatctatcaggagatgtggcataccctgctccagcgtcagccctggcgcg
gtcagctgattaatcagcgtcgggacggcggcctgtacctggtggagattgacatcaccccggtgcttagcccgcaaggggaactggagcattatct
ggcgatgcagcgggatatcagcgtcagctacaccctcgaacagcggctgcgcaaccatatgaccctgatggaggcggtgctgaataatatccccgc
cgccgtggtagtggtggacgagcaggatcgggtggtgatggacaacctcgcctacaaaaccttctgcgctgactgcggcggccgggagctgctcac
cgagctgcaggtctcccctggccggatgacgcccggcgtggaggcgatcctgccggtggcgctgcgcggggccgcgcgctggctgtcggtaacc
tgctggccgttacccgccgtcagtgaagaggccagccgctactttatcgacagcgcgctggcgcggaccctggtggtgatcgccgactgtacccag
cagcgtcagcagcaggagcaagggcgccttgaccggctgaagcagcaaatgaccgccggcanctgctggcggcgatccgcgagtcgctggac
gccgcgctgatccagctgaactgcccgattaatatgctggcggcagcccgtcggctgaacggcgagggaagcgggaatgtggcgctggaggccg
cctggcgtgaaggggaagaggcgatggcgcggctccagcgctgtcgcccatcgctggaactcgaaaaccccgccgtctggccgctgcagccctttt
tcgacgatctgtgcgccctctaccgtacacgcttcgatcccgacgggctgcaggtcgacatggcctcaccgcatctgatcggctttggccagcgcacc
ccactgctggcgtgcttaagcctgtggctcgatcgcaccctggccctcgccgccgaactcccctccgtgccgctggcgatgcagctctacgccgagg
agaacgacggaggctgtcgctgtatctgactgacaacgtaccgctgctgcaggtgcgctacgctcactcccccgacgcgctganctcgccgggca
aaggcatggagctgcggctgatccagaccctggtggcgcaccatcgcgagccattgagctggcttcccgaccgcagggcggcacctgcctgacc
ctgcgtttcccgctgtttaacaccctgaccggaggtgaagcatga
104 atgatccctgaatccgacccggacaccaccgtcagacgcttcgacctctctcagcagttcaccgccatgagcggataagcgtggtgctgagccggg
ccaccgaggccagcaaaacgctgcaggaggtgctcagcgtattacacaacgatgcctttatgcagcacgggatgatctgcctgtacgacagcgagc
aggagatcctcagtatcgaagcgctgcagcaaaccggccagcagcccctccccggcagcacgcagatccgctatcgccccggcgagggactggt
ggggaccgtgctggcccaggggcagtcgctggtgctgccccgggtcgccgacgatcagcgttttctcgaccgcctgagcactacgattacgatctg
ccgtttatcgccgtaccgttgatggggcccaacgcccggccaataggggtgctggcggcccagccgatggcgcgccaggaagagcggctgccgg
cctgcacccgttttctcgaaaccgtcgccaacctcgtcgcccagaccatccggctgatgatccttccggcctcacccgccctgtcgagccgccagccg
ccgaaggtggaacggccgccggcctgctcgtcgtcgcgcggcgtgggccttgacaatatggtcggcaagagcccggcgatgcgccagatcgtgg
aggtgatccgtcaggtttcgcgctgggacaccaccgtgctggtacgcggcgaaagcggcaccgggaaagagctgatcgccaacgccatccatcac
cattcgccacgggctggcgccgccttcgtcaaatttaaactgcgcggcgctgccggacaccctgctggaaagcgaactgttcggccatgagaaaggc
gcctttaccgagcggtgcgtcagcgtaaaggacgttttgagctggcggatggcggcaccctgttcctcgatgagattggtganagcagcgcctcg
ccaggccaagctgctgcgatcctccaggagggggagatggagcgggtcggcggcgatgagaccctgcgggtgaatgtccgcatcatcgccgcc
accaaccgtcacctggaggaggaggtccggctgggccatttccgcgaggatctctactatcgctgaacgtgatgccatcgccctgcccccgagc
gcgagcgtcaggaggacatcgccgagctggcgcacttcctggtgcgcaaaatcggccagcatcaggggcgcacgctgcggatcagcgagggcg
cgatccgcctgctgatggagtacagctggccgggtaacgttcgcgaactggagaactgcctcgaacgatcggcggtgatgcggagagtggcctga
tcgatcgcgacgtgatcctcttcactcaccaggatcgtcccgccaaagccctgcctgccctgcgggccagcggaagacagctggctggacaacagcc
tggacgaacgtcagcgactgatcgccgcgctggaaaaagccggctgggtgcaggccaagccggcacggctgctggggatgacgccgcgccagg
tcgcttatcggatccagatcatggatatcaccctgccgcgtctgtag
105 atgatgccgctttctccgcaattacagcagcactggcagacggtcgctgaccgtctgccagcggattttcccattgccgaactgagcccacaggccag
gtccgtcatggcgttcagcgattttgtcgaacagagtgtgatcgcccagccgggctggctgaatgagcttgcggactcctcgccggaggcggaagag
tggcggcattacgaggcctggctgcaggatcgcctgcaggccgtcactgacgaagcggggttgatgcgagagctgcgtctcttccgccgccagatg
atggtccgcatcgcctgggcgcaggcgctgtcgctggtgagcgaagaagagactctgcagcagctgagcgtcctggcggagaccctgattgtcgcc
gcccgcgactggctgtacgccgcctgctgtaaggagtggggaacgccatgcaatgccgagggccagccgcagccgctgctgatcctcgggatgg
gaaaagctgggcggcggcgagctgaacttctcttccgatatcgatctgatctttgcctggcctgagcatggcgccacccgcggcggccgccgcgagct
ggataacgcccaggctttacccgtctggggcagcggctgatcaaggcccttgaccagccgacgcaggacggctttgtctatcgggttgacatgcgcc
tgcggccgtttggcgacagtgggccgctggtactcagttttgcggcgctggaagattattaccaggagcagggtcgggactgggaacgctatgcgat
ggtgaaagcgcggatcatgggcgataacgacggcgtgtacgccagcgagttgcgcgcgatgctccgtcctttcgtcttccgccgttatatcgacttca
gcgtgatccagtcgctgcgtaacatgaaaggcatgatcgcccgcgaagtgcggcgtcgcgggctgaaagacaacatcaagctcggcgccggcgg
gatccgtgaaattgagtttatcgttcaggtctttcaactgatccgcggtggtcgcgaacctgcactgcagcaggcgccctgctgccgacgctggcgg
cgattgatgagctacatctgctgccggaaggcgacgcggcgctgctgcgcgaggcctatctgttcctgcgccggctggaaaacctgctgcaaagcat
caacgatgagcagacccagaccctgccgcaggatgaacttaaccgcgccaggctggcgtgggggatgcataccgaagactgggagacgctgagc
gcgcagctggcgagccagatggccaacgtgcggcgagtgtttaatgaactgatcggcgatgatgaggatcagtccccggatgagcaactggccga
gtactggcgcgagctgtggcaggatgcgctggaagaagatgacgccagcccggcgctggcgcatttaaacgataccgaccgccgtagcgtgctgg
cgctgattgccgattttcgtaaagagctggatcggcgcaccatcggcccgcgggccgccaggtgctggatcagctgatgccgcatctgctgagcga
aatctgctcgcgcgccgatgcgccgctgcctctggcggatcacgccgctgttgaccgggatcgtcacccgtaccacctatcttgagctgctgagc
gaattccccggcgcgctgaagcacctgatcacgctctgcgcggcgtcgccgatggtcgccagccagctggcgcgccacccgctgctgctggatga
gctcgctggatcccaacaccctctatcagccgacggcgaccgatgcctatcgcgacgagctgcgccagtacctgctgcgcgtgccggaagaggatga
agagcagcagctggaggcgttgcgccagtttaagcaggcgcagcagagcatatcgcggcggcggatatcgctggtaccctgccggtgatgaagg
tcagcgatcatttaacctggcttgccgaagcgatcctcgacgcggtggtgcagcaggcatgggggcagatggtcgctcgctacggccagccgaccc
acctgcacgatcgccagggtcgcggcttcgccgtcgtcggctacggtaagcttggcggctgggagctgggctacagctccgatctcgatctggtgttc
ctccatgactgcccggcggaggtgatgaccgacggcgagcgggagattgacggccgtcagttctacctgcggctggcccagcggatcatgcacct
gttcagcacccgcacctcgccggtattctctacgaagtggacgcccggctgcgtccttctggcgcggcggggatgctggtcaccaccgccgacgc
gtttgctgactatcagcagaacgaagcctggacgtgggaacatcaggcgctggtgcgcgcccgcgtggtctatggcgacccggcgctgcaggcgc
gctttgacgccattcgtcgcgatatcctgaccaccccgcgggaggggatgaccctgcagaccgaggttcgcgagatgcgcgagaagatgcgcgcc
caccttggcaacaaacatcccgatcgtttgatatcaaagccgatgccggcgggatcaccgatattgaatttattactcagtatctggtcctacgctatgcc
agtgacaagccgaagctgacccgctggtctgacaacgtgcgtattcttgagctgctggcgcagaagacatcatggacgaggaggaggcgcgcgc
cttaacgcatgcgtacaccaccttgcgtgatgcgctccatcacctggccctgcaggagcagccgggacacgtggcgccagaggccttcagccggga
gcgtcagcaggtcagcgccagctggcagaagtggctgatggcttaa
106 agcgtcaggtaccggtcatgattcaccgtgcgattctcggttccctggagcgcttcattggcatcctgaccgaagagttcgctggcttcttcccaacctg
gattgcaccagtgcaggtagtggtcatgaatattaccgattctcaggctgaatacgttaacgaattgacgcgtaaactacaaaatgcgggcattcgtgta
aaagcagacttgagaaatgagaagattggctttaaaatccgcgagcacactttacgtcgtgcccgtatatgttggtctgtggcgacaaagaagtcgaa
gccggcaaagtggccgtgcgcacccgtcgcgggaaagacctcggcagcatggacgtaagtgaagtgattgagaagctgcaacaagagattcgca
gccgcagtcttcaacaactggaggaataaggtattaaaggcggaaaacgagttcaaacggcacgtccgaatcgtatcaatggcgagattcgcgccct
ggaagttcgc
107 atgaaaatggcaacaatgaaatcgggtctgggggcattagcccttcttccgggactggcaatggccgcgcccgcagtggcggacaaagccgataac
gcgtttatgatgatttgcaccgcgctggttctgtttatgaccatcccggggatcgcgctgttttacggcggcctgatccgcggcaaaaacgtcctttccat
gctgactcaggtgattgtgacctttggcctcgtctgcgtactgtgggtgatttatggctataccctggccttcggcaccggcggcagcttcttcgctagttt
tgactgggtgatgctgaaaaatattgaactgaaagcgctgatgggcaccttctatcagtacatccacgtggcatccagggctcgttcgcctgtatcacc
gtcgggctgatcgtgcgggcgctggctgagcgtattcgtttctccgccgtgctgatttttgtggtggtgtggatgacgctctcttatgttccgattgcgcac
atggtctggggcggcggtctgctggcgacccacggcgcgctggacttcgcgggcggcaccgttgtacacatcaacgccgcggttgccgggctggt
gggtgcgtacatgatgggcaaacgtggggcttcggcaaagaagcgttcaaaccgcacaatctgccgatggtgttcaccggaaccgccatcctctac
gtgggctggttcggcttcaacgccggctccgccagcgcagcgaacgaaattgccgcattggctttcgtcaacaccgtcgtcgccacagcggctgcca
tcctggcgtggacctttggcgaatgggccctgcgcggtaaaccttcactgctgggcgcctgctccggggcgattgccggtctggttggcgtcacacca
gcctgtgggtatatcggtgtcggtgggcgttgattgtgggtatcgcatctggtctggcgggcatctggggcgtaacggcgctgaaacgctggctgcg
ggttgatgacccttgcgacgtcttcggcgtccacggcgtctgcggcatcgtcggctgtatcctgaccggtatcttcgcggccacctctctgggcggcgt
gggttatgcagaaggcgtcaccatgggccatcagctgctggtgcaactcgagagtatcgcgattaccatcgtctggtcgggcgttgtcgctttcattgg
ctacaaagtggcggacatgaccgtggggctgcgcgtaccagaagagcaggagcgcgaaggactggacgtcaacagccatggcgaaaacgccta
caacgcctga
108 cgccgtcctcgcagtaccattgcaaccgactttacagcaagaagtgattctggcacgcatggaacaaattcttgccagtcgggctttatccgatgacga
acgcgcacagcttttatatgagcgcggagtgttgtatgatagtctcggtctgagggcattagcgcgaaatgatttttcacaagcgctggcaatccgaccc
gatatgcctgaagtattcaattacttaggcatttacttaacgcaggcaggcaattttgatgctgcctatgaagcgtttgattctgtacttgagcttgatc
109 gctaaagttctcggctaatcgctgataacatttgacgcaatgcgcaataaaagggcatcatttgatgccctttttgcacgctttcataccagaacctggctc
atcagtgattttttttgtcataatcattgctgagacaggctctgaagagggcgtttatacaccaaaccattcgagcggtagcgcgacggcaagtcagcgtt
ctcctttgcaatagcagggaagaggcgccagaaccgccagcgttgaagcagtttgaacgcgttcagtgtataatccgaaacttaatttcggtttgga
110 gcccgctgaccgaccagaacttccaccttggactcggctatacccttggcgtgacggcgcgcgataactgggactacatccccattccggtgatctta
ccattggcgtcaataggttacggtccggcgactttccagatgacctatattcccggcacctacaataacggtaacgtttacttcgcctgggctcgataca
gttttaattcgctaagtcttagcaataaatgagataagcggtgtgtcttgtggaaaaacaaggactaaagcgttacccactaaaaaagatagcgacttttat
cactttttagcaaagttgcactggacaaaaggtaccacaattggtgtactgatactcgacacagcattagtgtcgatttttcatataaaggtaattttg
111 ttgaagagtttgatcatggctcagattgaacgctggcggcaggcctaacacatgcaagtcgagcggtagcacagagagcttgctctcgggtgacgag
cggcggacgggtgagtaatgtagggaaactgcctgatggagggggataactactggaaacggtagctaataccgcataacgtcgcaagaccaaag
tgggggaccttcgggcctcatgccatcagatgtgcccagatgggattagctagtaggtggggtaacggctcacctaggcgacgatccctagctggtct
gagaggatgaccagccacactggaactgagacacggtccagactcctacgggaggcagcagtggggaatattgcacaatgggcgcaagcctgat
gcagccatgccgcgtgtgtgaagaaggccttcgggttgtaaagcactttcagcggggaggaaggcgntnaggttaataaccttgtcgattgacgttac
ccgcagaagaagcaccggctaactccggccagcagccgcggtaatacggagggtgcaagcgttaatcggaattactgggcgtaaagcgcacgca
ggcggtagtcaagtcggatgtgaaatccccgggctcaacctgggaactgcattcgaaactggcaggctagagtcttgtagaggggggtagaattcc
agggtagcggtgaaatgcgtagagatctggaggaataccggtggcgaaggcggccccctggacaaagactgacgctcaggtgcgaaagcgtgg
ggagcaaacaggattagaccctggtagtccacgctgtaaacgatgtcgatttggaggttgtgcccttgaggcgtggcttccggagctagcgcgttaa
atcgaccgcctggggagtacggccgcaaggttaaaactcaaatgaattgacgggggcccgcacaagcggtggagcatgtggtttaattcgatgcaa
cgcgaagaaccttacctggtcttgacatccacagaactttccagagatggattggtgccttcgggaactgtgagacaggtgctgcatggctgtcgtcag
ctcgtgttgtgaaatgttgggttaagtcccgcaacgagcgcaacccttatcctttgttgccagcggtnnggccgggaactcaaaggagactgccagtg
ataaactggaggaaggtggggatgacgtcaagtcatcatggcccttacgaccagggctacacacgtgctacaatggcatatacaaagagaagcgac
ctcgcgagagcaagcggacctcataaagtatgtcgtagtccggattggagtctgcaactcgactccatgaagtcggaatcgctagtaatcgtagatca
gaatgctacggtgaatacgttcccgggccttgtacacaccgcccgtacaccatgggagtgggttgcaaaagaagtaggtagcttaaccttcgggag
ggcgcttaccactttgtgattcatgactggggtgaagtcgtaacaaggtaaccgtaggggaacctgcggttggatcacctcctt
112 atgaccatgcgtcaatgcgctatctacggtaaaggcggtatcggtaaatccaccaccacccagaatctcgtcgcggcctcgccgagatgggtaaga
aagtgatgatcgtcggctgcgatccgaaagcggactccacccgtctgatccttcacgctaaagcccagaacaccatcatggagatggcggcggaagt
gggctcggtcgaggatctggagctcgaagacgttctgcaaatcggctatggcgatgtccgttgcgccgaatccggcggcccggagccaggcgtcg
gctgcgccggacgcggggtgatcaccgccatcaacttcctcgaggaagaaggcgcctatgaggaagatttggatttcgtcttctatgacgtcctcggc
gacgtagtctgcggcggcttcgccatgccgatccgcgaaaacaaagcccaggagatctacatcgtctgctccggcgagatgatggcgatgtatgccg
ccaacaatatctccaaggggatcgtgaagtacgcgaaatctggcaaggtgcgcctcggcggcctgatctgtaactcgcgcaaaaccgaccgggaag
acgaactgatcatcgccctggcggagaagcttggcacgcagatgatccacttcgttccccgcgacaacattgtgcagcgcgcggagatccgccgga
tgacggtgatcgagtacgacccgacctgtcagcaggcgaatgaatatcgtcaactggcgcagaagatcgtcaataacaccaaaaaagtggtgccaa
cgccgtgcaccatggacgagctggaatcgctgctgatggagttcggcatcatggaagaagaagacaccagcatcattggtaaaaccgccgctgaag
aaaacgcggcctga
113 atgaccaacgcaacaggcgaacgtaaccttgcgctcatccaggaagtcctggaggtgtttcccgaaaccgcgcgcaaagagcgcagaaagcacat
gatgatcagcgatccgcagatggagagcgtcggcaagtgcattatctcgaaccgtaaatcgcagcccggggtgatgaccgtgcgtggctgcgcctat
gcgggcttcgaaaggggtggtgtttgggccaatcaaagacatggcccatatctcgcacggccccatcggctgcggccagtactgcgcgccggacg
gcgcaactactataccggcgtcagcggtgtcgacagcttcggcaccctgaacttcacctctgattttcaggagcgcgatattgttttcggcggcgataaa
aagctgaccaaactgatcgaagagatggagctgcttcccgctgctccaaagggatcaccatccaatcggagtgcccggtgggcctgatcggcgat
gacatcagcgccgtggccaacgccagcagcaaggcgctggataaaccggtgatcccggtgcgctgcgaaggctttcgcggcgtatcgcaatcgct
gggccaccatatcgccaacgacgtggtgcgcgactgggtgctgaacaatcgcgaagggcagccgtttgccagcaccccgtatgatgttgccatcatt
ggcgattacaacatcggcggcgacgcctgggcctcgcgcattctgctggaagagatggggctgcgcgtagtggcgcagtggtccggcgacggca
ccctggttgagatggagaacaccccattcgttaagcttaacctcgccactgctaccgacgatgaactatatcgcccgccatatggaggagaaacatc
agatcccgtggatggaatataacttcttcggcccgaccaaaatcgccgaatcgctgcgcaagatcgccgatcaatttgatgacaccattcgcgccaatg
cggaagcggtgatcgccaaatatgaggggcagatggcggccatcatcgccaaatatcgcccgcggctggaggggcgcaaagtgctgctgtacatg
gagggctgcggccgcgccacgtcatcggcgcctatgaggatctcgggatggagatcatcgccgccggctacgagtttgcccataacgatgattac
gaccgcaccctgccggacctgaaagagggcaccctgctgtttgacgatgccagcagctatgagctggaggccttcgtcaaagcgctgaaacctgac
ctcatcggctccgggatcaaagagaaatatatcttccagaaaatgggggtgccgttccgccagatgcactcctgggactattccggcccctatcacgg
ctatgacggcttcgccatctttgcccgccatatggatatgaccctgaacaatccggcgtggaacgaactgactgccccgtggctgaagtctgcgtga
114 atgaagggaaaggaaattaggcgctgctggacgaacccgcctgcgagcacaaccagaagcaaaaatccggctgcagcgctcctaagcccggcg
caaccgccggcggctgcgccttcgacggcgcgcagataacgctcctgcccatcgccgacgtcgcgcacctggtgcacggccccatcggctgcgc
gggcagctcgtgggataaccgcggcagcgtcagcgccggcccggccctcaaccggctcggctttaccaccgatcttaacgaacaggatgtgattat
gggccgcggcgaacgccgcctgttccacgccgtccgtcacatcgtcgaccgctatcatccggcggcggtctttatctacaacacctgcgtaccggcg
atggagggggatgacctggaggccgtctgccaggccgcacagaccgccaccggcgtcccggtcatcgccattgacgccgccggtttctacggcag
taaaaatcttggcaaccgaatggcgggcgacgtgatgctcaggcaggtgattggccagcgcgaaccggccccgtggccagacaacacgccctttg
ccccggcccagcgccacgatatcggcctgattggcgaattcaatatcgccggcgagttctggcaggtccagccgctgctcgacgagctggggatcc
gcgtcctcggcagcctctccggcgacggccgctttgccgagatccagaccctgcaccgggcgcaggccaatatgctggtgtgctcgcgcgcgctga
tcaacgtcgcccgggggctggagctgcgctacggcacgccgtggtttgaaggcagcttctacgggatccgcgccacctccgacgccttgcgccagc
tggcggcgctgctgggaatgacgacctgtgccgccgcaccgaggcgctgatcgcccgcgaagagcaggcggcggagcaggcgctggcgccg
tggcgcgagcagctccgtgggcgcaaagtgttgctctacaccggcggcgtgaaatcctggtcggtggtatcagccctgcaggatctcggcatgacc
gtggtggccaccggcacgcggaaatccaccgaggaggacaaacagggatccgtgagctgatgggcgacgaggcggtgatgcttgaggagggc
aatgctcgcaccctgctcgacgtggtgaccgctatcaggccgacctgatgatcgccggcggacgcaatatgtacaccgcctggaaagcccggctg
ccgtttctcgatatcaatcaggagcgcgagcacgcctacgccggctatcagggcatcatcaccctcgcccgccagactgtctgaccctcgccagtcc
cgtctggccgcaaacgcatacccgcgccccgtggcgctag
115 atggcagacattatccgcagtgaaaaaccgctggcggtgagcccgattaaaaccgggcaaccgctcggggcgatcctcgccagcctcgggctggc
ccaggccatcccgctggtccacggcgcccagggctgcagcgccttcgccaaagttttctttattcagcatttccatgacccggtgccgctgcagtcgac
ggccatggatccgaccgccacgatcatgggggccgacggcaatatcttcaccgcgctcgacaccctctgccagcgccacagcccgcaggccatcg
tgctgctcagcaccggtctggcggaagcgcagggcagcgatatcgcccgggtggtgcgccagtttcgtgaggcgcatccgcgccataacggcgtg
gcgatcctcaccgtcaataccccggatttttttggctcgatggaaaacggctacagcgcggtgatcgagagcgtgatcgagcagtgggcgcgccga
cgccgcgtccggggcagcggccccggcgggtcaacctgctggtcagccacctctgttcgccaggggatatcgaatggctgggccgctgcgtggag
gcctttggcctgcagccggtgatcctgccggacctctcgcagtcaatggatggccacctcggtgaaggggattttacgcccctgacccagggcggcg
cctcgctgcgccagattgcccagatgggccagagtctgggcagcttcgccattggcgtgtcgctccagcgggcggcatcgctcctgacccaacgca
gccgcggcgacgtgatcgccctgccgcatctgatgaccctcgaccattgcgatacctttatccatcagctggcgaagatgtccggacgccgcgtacc
ggcctggattgagcgccagcgcggccagctgcaggatgcgatgatcgactgccatatgtggcttcagggccagcgcatggcgatggcggcggag
ggcgacctgaggcggcgtggtgtgatttcgcccgcagccaggggatgcagcccggcccgctggtcgcccccaccagccaccccagcctgagcc
agctgccggtcgatcaggtcgtgccgggggatcttgaggatctgcagcagctgctgagccaccaacccgccgatctgctggtggctaactctcacgc
ccgctgatctggcggagcagtttgccctgccgctgatccgcgtcggttttcccctatcgaccggctcggtgagatcgtcgcgtccgccaggcgtacgc
cggtatgcgagatacgctgtttgagctggccaatctgctgcgcgaccgccatcaccacaccgccctctaccgctcgccgcttcgccagggcgccgac
cccctgccggcttcaggagacgcttatgccgcccattaa
116 gtgccgctgatccgtctgggctttccgctgttcgaccgccatcatctgcaccgccagaccacctggggctatgaaggcgcaatgaacatcgtcacgac
gctggtgaacgccgtgctggaaaaactggaccacgacaccagttgggcaaaaccgattacagcttcgacctcgttcgttaa
117 atgaccctgaatatgatgctcgataacgccgcaccggaggccatcgccggcgcgctgactcaacaacatccggggctgttttttaccatggtggaaca
ggcctcggtggccatatccctcaccgatgccagcgccaggatcatttacgccaacccagcgtttgccgccagaccggctattcgctggcgcaattgt
aaaccagaacccgcgcctgctggccagcagccagacgccgcgcgcgatctatcaggagatgtggcataccctgctccagcgtcagccctggcgcg
gtcagctgattaatcagcgtcgggacggcggcctgtgcctggtggagattgacatcaccccggtgcttagcccgcaaggggaactggagcattatct
ggcgatgcagcgggatatcagcgtcagctacaccctcgaacaacggctgcgcaaccatatgaccctgatggaggcggtgctgaataatatccccgc
cgccgtggtggtggtggacgagcaggatcgggtggtgatggacaacctcgcctacataaccttctgcgctgactgcggcggccgggagctgctca
ccgagctgcaggtctcccctggccggatgacgcccggcgtggaggcgatcctgccggtagcgctgcgcggggccgcgcgctggctgtcggtaac
ctgctggccgttgcccggcgtcagtgaagaggccagccgctactttatcgacagcgcgctggcgcggaccctggtggtgatcgccgactgtaccca
gcagcgtcagcagcaggagcaaggacgccttgaccggctgaagcagcaaatgaccgccggcaagctgctggcggcgatccgcgagtcgctgga
cgccgcgctgatccagctgaactgcccgattaatatgaggcggcagcccgtcggctgaacggcgagggaagcgggaatgtggcgctggaggcc
gcctggcgtgaagggcaagaggcgatggcgcggctccagcgctgtcgcccatcgctggaactcgaaaaccccgccgtctggccgctgcagccctt
tttcgacgatctgtgcgccctctaccgtacccgcttcgatcccgacgcgctgcaggtcgacatggcctcaccgcatctgatcggctttggccagcgcac
cccgctgctgccgtgcttaagcctgtggctcgaccgcaccctggccctcgccgccgaattgccctccatgcgctggcgatgcagctctatgccgag
gagaacgacggctggctgtcgctgtacctgactgataacgtaccgctgttgcagggcgctacgcccactcccccgacgcgctgaactcgccgggta
aaggcatggagctgcgcctgatccagaccctggtggcgcaccatcgcggggccattgagctggcttcccgaccgcagggcggcacctgcctgacc
ctgcgtttcccgctgtttaacaccctgaccggaggtgaagcatga
118 atgatccctgaatccgacccggacaccaccgtcagacgcttcgacctctacagcagttcaccgccatgcagcggataagcgtggtgctgagccggg
ccaccgaggccagcaaaacgctgcaggaggtactcactgtattgcacaacgatgccatatgcagcacgggatgatctgcctgtacgacagcgagca
ggagatcctcagtatcgaagcgctgcagcaaaccggccagcagcccctccccggcagcacgcagatccgctatcgccccggcgagggactggtg
gggaccgtgctggcccaggggcagtcgctggtgctgccccgggtcgccgacgatcagcgttttctcgaccgcctgagcctctacgattacgatctgc
cgtttatcgccgtaccgttgatggggcccaacgcccggccaataggggtgctggcggcccagccgatggcgcgccaggaagagcggctgccggc
ctgcacccgttttacgaaaccgtcgccaacacgtcgcccagaccatccggctgatgatccttccggcctcacccgccagtcgagccgccagccgc
cgaaggtggaacggccgccggcctgctcgtcgtcgcgcggcgtgggccttgacaatatggtcggcaagagcccggcgatgcgccagatcgtgga
ggtgatccgtcaggtttcgcgctgggacaccaccgtgctggtgcgcggtgaaagcggcaccgggaaagagctgatcgccaacgccatccatcacc
attcgccacgggctggcgccgccttcgtcaaatttaactgcgcggcgctgccggacaccctgctggaaagcgaactgttcggccatgagaaaggcg
cctttaccggggcggtgcgtcagcgtaaaggacgttttgagctggccgatcgcggcaccctgttcctcgatgagattggtgaaagcagcgcctcgttc
caggccaagctgctgcgtatcctccaggagggggagatggagcgggtcggcggcgatgagaccctgcgggtgaatgtccgcatcatcgccgcca
ccaaccgtcacctggaggaggaggtccggctgggccatttccgcgaggatctctattatcgtctgaacgtgatgcccatcgccctgcccccgctgcgc
gagcgtcaggaggacatcgccgagctggcgcacttcctggtgcgcaaaatcggccagcatcaggggcgcacgctgcggatcagcgagggcgcg
atccgcctgctgatggagtacagctggccgggtaacgttcgcgaactggagaactgcctcgaacgatcggcggtgatgtcggagagtggcctgatc
gatcgcgacgtgatcctcttcactcaccaggatcgtcccgccaaagccctgcctgccaggggccagcggaagacagctggctggacaacagcct
ggacgaacgtcagcgactgatcgccgcgctggaaaaagccggctgggtgcaggccaaggcgccacggctgctggggatgacgccgcgccaggt
cgcttaccggatccagatcatggatatcaccctgccgcgtctgtag
119 atgatgccgctttctccgcaattacagcagcactggcagacggtcgctgaccgtctgccagcggattttcccattgcagaactgagcccacaggccag
gtcggtcatggcgttcagcgattttgtcgaacagagtgtgatcgcccagccgggctggctgaatgagatgcggactcctcgccggaggcggaagag
tggcggcattacgaggcctggctgcaggatcgcctgcaggccgtcactgacgaagcggggttgatgcgagagctgcgtctatccgccgccagatg
atggtccgcatcgcagggcgcaggcgctgtcgctggtgagcgaagaagagaccctgagcagctgagcgccctggcggagaccctgattgtcgc
cgcccgcgactggctctacgccgcctgctgtaaggagtggggaacgccatgcaatgccgagggccagccgcagccgctgctgatcctcgggatgg
gaaagctgggcggcggcgagctgaacttctatccgatatcgatagatctttgcctggcctgagcatggcgccacccgcggcggccgccgcgagct
ggataacgcccagttctttacccgtctggggcagcggctgatcaaggcccttgaccagccgacgcaggacggctttgtctatcgggttgacatgcgcc
tgcggccgtttggcgacagtgggccgctggtactcagctttgcggcactggaagattattaccaggagcagggtcgggactgggaacgctatgcgat
ggtgtaagcgcggatcatgggcgataacgacggcgtgtacgccagcgagttgcgcgcgatgctccgtcctttcgtcttccgccgttatatcgacttca
gcgtgatccagtcgctgcgtaacatgaaaggcatgatogcccgcgaagtgcggcgtcgcgggctgaaagacaacatcaagctcggcgccggcgg
gatccgtgaaattgagtttatcgttcaggtctttcagctgatccgcggtggtcgcgaacctgcactgcagcagcgcgccctgctgccgacgctggcgg
cgattgatgagctacatctgctgccggaaggcgacgcggcgctgctgcgcgaggcctatctgttcctgcgccggctggaaaacctgctgcaaagcat
caacgatgattcagacccagaccctgccgcaggatgaacttaaccgcgccaggctggcgtgggggatgcataccgaagactgggagacgctgagc
gcgcagctggcgagccagatggccaacgtgcggcgagtgtttaatgaactgatcggcgatgatgaggatcagtccccggatgagcaactggccga
gtactggcgcgagctgtggcaggatgcgctggaagaagatgacgccagcccggcgctggcgcatttaaacgataccgaccgccgtagcgtgctgg
cgctgattgccgattttcgtaaagagctggatcggcgcaccatcggcccgcgcggccgccaggtgctggatcagctgatgccgcatctgagagcga
aatctgctcgcgtgccgatgcgccgagcctctggcgcggatcacgccgctgttgaccgggatcgtcacccgtaccacctatcttgagctgctgagcg
aattccccggcgcgctgaagcacctgatcacgctctgcgcggcgtcgccgatggtcgccagccagctggcgcgccacccgctgctgctggatgag
ctgctggatcccaacaccctctatcagccgacggcgaccgatgcctatcgcgacgagctgcgccagtacctgctgcgcgtgccggaagaggatcaa
gagcagcagctggaggcgtgcgccagtttaagcaggcgcagagctgcatatcgcggcggcggatatcgctggtaccctgccggtgatgaaggt
caggatcacttaacctggcttgccgaagcgatcctcgacgcggtggtgcagcaggcatgggggcagatggtcgctcgctacggtcagccgaccca
cctgcacgatcgccagggtcgcggatcgccgttgtcggctacggtaagctcggcgcctgggagctggactacagctccgatctcgatctggtgttcc
tccatgactgcccggcggaggtgatgaccgacggcgaggggagattgacggccgtcagttctacctgcggctggcccagcggatcatgcacctgt
tcagcacccgcacctcgtccggtattactacgaagtggacgcccggctgcgtcatctggcgcggcaggttgctggtcaccaccgccgacgcgtt
tgctgactatcagagaacgaagcctggacgtgggaacatcaggcgctggtgcgcgcccgcgtggtctatggcgacccggcgctgcaggcgcgct
ttgacgccattcgtcgcgatatcctgaccaccccgcgggaggggacgaccctgcagaccgaggttcgcaagatgcgcgagaagatgcgcgcccac
cttggcaacaaacatcccgatcgattgatatcaaagccgatgccggcgggatcaccgatattgaatttattactcagtatctggtcctacgctatgccagt
gacaagccgaagctgacccgctggtctgacaacgtgcgtattcttgagctgctggcgcaggacgacatcatggacgaggaggaggcgcgcgcctta
acgcatgcatacaccaccttgcgtgatgcgctccatcacctggccctgcaggagcagccgggacacgtggcgccagaggccttcagccgggagg
tcagcaggtcagcgccagctggcagaagtggctgatggcttaa
120 atgaaaatggcaacaatgaaatcgggtctgggggcattagcccttcttccgggactggcaatggccgcgcccgcagtggcggacaaagccgataac
gcgtttatgatgatttgcaccgcgaggttctgtttatgaccatcccggggatcgcgctgattacggcggcctgatccgcggcaaaaacgtcctttccat
gctgactcaggtgattgtgacctttggcctggtntgcgtactgtgggtgatttatggctataccctggccttcggaaccggcggcagcttcttcggtagctt
tgactgggtgatgctgaaaaatattgaactgaaagcgctgatgggcaccttctatcagtacatccacgtggccttccagggctcgttcgcctgtatcacc
gtcgggctgatcgtgggggcgctggctgagcgtattcgtttctccgccgtgctgattttcgtggtggtgtggatgacgctacttatgttccgattgcgca
catggtctggggcggcggtctgctggcgacccacggcgcgctggacttcgcgggcggcaccgttgtacacatcaacgccgcggttgccgggctgg
tgggtgcgtatatgatgggcaaacgtgtgggcttcggcaaagaagcgttcaaaccgcacaatctgccgatggtgttcaccggaaccgccatcctctac
gtgggctggttcggcttcaacgccggctccgccagcgcagcgaacgaaattgccgcactggctttcgtcaacaccgtcgtcgccacagcggcagcc
atcctggcctggacctttggcgaatgggctctgcgcggcaaaccttcactgctgggcgcctgctccggggcgattgccggtctggttggcgtcacacc
agcctgtgggtatatcggtgtcggtggggcgttgattgtgggtatcgcatctggtctggcgggcatctggggcgtaacggcgctgaaacgctggctgc
gggttgatgacccttgcgacgtcttcggcgtccacggcgtctgcggtcgtcggctgtatcctgaccggtatcttcgcggccacctctctgggcggc
gtgggttatgcagaaggcgtcaccatgggccatcagctgctggtgcaactcgagagtatcgcgattaccatcgtctggtcgggcgttgtcgattcattg
gctacaaagtggcggacatgaccgtggggctgcgcgtaccagaagagaggagcgcgaaggactggacgtcaacagccatggcgaaaacgcct
acaacgcctga
121 ctgaagagtttgatcctggctcagattgaacgctagcgggatgccttacacatgcaagtcgaacggcagcacggacttcggtctggtggcgagtggc
gaacgggtgagtaatgtatcggaacgtgcctagtagcgggggataactacgcgaaagcgtagctaataccgcatacgccctacgggggaaagcag
gggatcgcaagaccttgcactattagagcggccgatatcggattagatagttggtggggtaanggatcaccaaggcgacgatccgtagctggattgag
aggacgaccagccacactgggactgagacacggcccagactcctacgggaggcagcagtggggaattttggacaatgggggaaaccctgatcca
gccatcccgcgtgtgcgatgaaggccttcgggatgtaaagcacttttggcaggaaagaaacgtcatgggntaataccccgtgaaactgacggtacctg
cagaataagcaccggctaactacgtgccagcagccgcggtaatacgtagggtgcaagcgttaatcggaattactgggcgtaaagcgtgcgcaggcg
gttcggaaagaaagatgtgaaatcccagagcttaactttggaactgcatttttaactaccgggctagagtgtgtcagagggaggtggaattccgcgtgta
gcagtgaaatgcgtagatatgcggaggaacaccgatggcgaaggcagcctcctgggataacactgacgctcatgcacgaaagcgtggggagcaa
acaggattagataccctgctagtccacgccctaaacgatgtcaactagctgttggggccttcgggccttagtagcgcagataacgcgtgaagttgacc
gcctggggagtacggtcgcaagattaaaactcaaaggaattgacggggacccgcacaagcggtggatgatgtggattaattcgatgcaacgcgaaa
aaccttacctacccttgacatgtctggaattctgaagagattcggaagtgctcgcaagagaaccggaacacaggtgctgcatggctgtcgtatgatcgt
gtcgtgagatgttgggttaagtcccgcaacgagcgcaacccttgtcattagttgctacgaaagggcactctaatgagactgccggtgacaaaccggag
gaaggtggggatgacgtcaagtcctcatggcccttatgggtagggcttcacacgtcatacaatggtcgggacagagggtcgccaacccgcgagggg
gagccaatcccagaaacccgatcgtagtccggatcgcagtctgcaactcgactgcgtgaagtcggaatcgctagtaatcgcggatcagcatgtcgcg
gtgaatacgttcccgggtcttgtacacaccgcccgtcacaccatgggagtgggttttaccagaagtagttagcctaacc
122 ctgaagagtttgatcctcgctcagattgaacgctaggggatgccttacacatgcaagtcgaacggcagcacggacttcggtctggtggcgagtggc
gaacgggtgagtaatgtatcggaacgtgcctagtagcgggggataactacgcgaaagcgtagctaataccgcatacgccctacgggggaaagcag
gggatcgcaagaccttgcactattagagcggccgatatcggattagatagttggtggggtaaaggctcaccaaggcgacgatccgtagatggtttgag
aggacgaccagccacactgggactgagacacggcccagactcctacgggaggcagcagtggggaattttggacaatgggcgaaaccctgatcca
gccatcccgcgtgtgcgatgaaggccttcgggttgtaaagcacttttggcaggaaagaaacgtcatgggttaataccccgtgaaactgacggtacctg
cagaataagcaccggctaactacgtgccagcagccgcggtaatacgtagggtgcaagcgttaatcggaattactgggcgtaaagcgtgcgcaggcg
gttcggaaagaaagatgtgaaatcccagagcttaactttggaactgcatttttaactaccgggctagagtgtgtcagagggaggtggaattccgcgtgta
gcagtgaaatgcgtagatatgcggaggaacaccgatggcgaaggcagcctcctgagataacactgacgctcatgcacgaaagcgtggggagcaa
acaggattagataccctggtagtccacgccctaaacgatgtcaactagctgttggggcatcgggccttagtagcgcagctaacgcgtgaagttgacc
gcctggggagtacggtcgcaagattaaaactcaaaggaattgacggggacccgcacaagcggtggatgatgtggattaattcgatgcaacgcgaaa
aaccttacctacccttgacatgtctggaattcngaagagattnggaagtgctcgcaagagaaccggaacacaggtgctgcatggctgtcgtcagctcg
tgtcgtgagatgttgggttaagtcccgcaacgagcgcaacccttgtcattagttgatacgattagggcactctaatgagactgccggtgacaaaccgga
ggaaggtgggcatgacgtcaagtcctcatggcccttatgggtagggcttcacacgtcatacaatggtcgggacagagggtcgccaacccgcgaggg
ggagccaatcccagaaacccgatcgtagtccggatcgcagtctgcaactcgactgcgtgaagtcggaatcgctagtaatcgcggatcagcatgtcgc
ggtgaatacgttcccgggtcttgtacacaccgcccgtcacaccatgggagtgggttttaccagaagtagttagcctaaccgnaaggggggcgattacc
acggtaggattcatgactggggtgaagtcgtaacaaggtagccgtatcggaaggtgaggctggatcacctccttt
123 tacggagagtttgatcctggctcaggatgaacgctcgcggcctgcttaacacatgcaagtcgaacggttgaacacggagcttgctctctgggatcagtg
gcgaacgggtgagtaacacgtcagcaacctgcccctgactctgggataagcgctggaaacggcgtctaatactggatatgtgacgtggccgcatgga
ctgcgtctggaaagaatttcggttggggatgggatcgcggcctatatgcttgttggtgaggtaatggctcaccaaggcgtcgacgggtagccggcctg
agagggtgaccggccacactgggactgagacacggcccagactcctacgggaggcagcagtggggaatattgcacaatgggcgcaagcctgatg
cagcaacgccgcgtgagggatgacggccttcgggagtaaacctcttttagcagggaagaaggaaagtgacggtacctgcagaaaaagcgccgg
ctaactacgtgccagcagccgcggtaatacgtagggcgcaagcgttatccggaattattgggcggaaagagctcgtaggcggtttgtcgcgtctgctgt
gaaatccggaggctcaacctccggcctgcagtgggtacgggcagactagagtgcggtaggggagattggaattcctggtgtagcggtggaatgcgc
agatatcaggaggaacaccgatggcgaaggcagatctctgggccgtaactgacgctgaggagcgaaagggtggggagcaaacaggcttagatac
cctggtagtccaccccgtaaacgttgggaactagttgtggggtccattccacggattccgtgacgcagataacgcattaagttccccgcctggggagta
cggccgcaaggctaaaactcaaaggaattgacggggacccgcacaagcgacggagcatgcggattaattcgatgcaacgcgaagaaccttaccaa
ggcttgacatatacgagaacgggccagaaatggtcaactctttggacactcgtaaacaggtggtgcatggttgtcgtcagatcgtgtcgtgagatgttg
ggttaagtcccgcaacgagcgcaaccctcgttctatgttgccagcacctaatggtgggaactcatgggatactgccggggtcaactcggaggaaggt
ggggatgacgtcaaatcatcatcccccttatgtcttgggcttcacgcatgctacaatggccggtacaaagggctgcaataccgcgaggtggaccgaat
cccaaaaagccggtcccagttcggattgaggtctgcaactcgacctcatgaagtcggagtcgctagtaatcgcagatcagcaacgctgcggtgaata
cgttcccgggtcttgtacacaccgcccgtcaagtcatgaaagtcggtaacacctgaagccggtggcctaacccttgtggagggagccgtcgaaggtg
ggatcggtaattaggactaagtcgtaacaaggtagccgtaccggaaggtgcggctggatcacctccttt
124 attgaagagtttgatcatggctcagattgaacgctggcggcagccctaacacatgcaagtcgaacggtagcacagagagcttgctctcgggtgacga
gtggcggacgggtgagtaatgtctgggaaactgcccgatggagggggataactactggaaacggtagctaataccgcataatgtcgcaagaccaaa
gagggggaccttcggccctcttgccatcggatgtgcccagatgggattagattgatggtgaggtaatggatcaccaaggcgacgatccctagatggtc
tgagaggatgaccagccacactggaactgagacacggtccagactcctacgggaggcagcagtggggaatattgcacaatgggcgcaagcctgat
gcagccatgccgcgtgtgtgaagaaggccttcgggttgtaaagcactttcagcggggaggaaggcgatncggttaataaccgtgttgattgacgttac
ccgcagaagaagcaccggctaactccgtgccagcagccccggtaatacggagggtgcaagagataatcggaattactgggcgtaaagcgcacgca
ggcggtctgtcaagtcggatgtgaaatccccgggctcaacctgggaactgcattcgaaactggcaggcttgagtcttgtagaggggggtagaattcca
ggtgtagcggtgaaatgcgtagagatctggaggaataccggtggcgaaggcggccccctggacaaagactgacgctcaggtgcgaaagcgtggg
gagcaaacaggattagataccctggtagtccacgccgtaaacgatgtcgacttggaggttgtgcccttgaggcgtggcttccggagctaacgcgttaa
gtcgaccgcctggggagtacggccgcaaggttaaaactcaaatgaattgacgggggcccgcacaagcggtggagcatgtggtttaattcgatgcaa
cgcgaagaaccttacctggtcttgacatccacggaattnggcagagatgccttagtgccttcgggaaccgtgagacaggtgctgcatggctgtcgtca
gctcgtgttgtgaaatgttgggttaagtcccgcaacgagcgcaacccttatcctttgttgccagcggtccggccgggaactcaaaggagactgccagt
gataaactggaggaaggtggggatgacgtcaagtcatcatggcccttacgaccagggctacacacgtgctacaatggcatatacaaagagaagcga
cctcgcgagagcaagcggacctcataaagtatgtcgtagtccggattggagtctgcaactcgactccatgaagtcggaatcgctagtaatcgtggatc
agaatgccacggtgaatacgttcccgggccttgtacacaccgcccgtcacaccatgggagtgggttgcaaaagaagtaggtagcttaaccttcggga
gggcgcttaccactttgtgattcatgactggggtgaagtcgtaacaaggtaaccgtaggggaacctgcggttggatcacctcctt
125 atgaccatgcgtcaatgcgccatttatggcaaaggtgggatcggcaaatccaccaccacgcaaaacctcgtcgccgctctcgcggaaatgggtaaaa
aagtgatgatcgtcggctgcgacccgaaagggactccacccgtctgatcctgcatgcgaaagcacagaacaccattatggagatggccgccgaag
tgggttcagtggaagaccttgaactggaagatgtgctgcaaatcggttacggcggcgtgcgttgtgcagaatccggcggcccggagccaggcgtgg
gttgtgcaggccgcggcgttattaccgccattaacttccttgaagaagaaggcgcctatgtcagcgacctcgactttgtcttctatgacgtcctcggtgac
gtggtctgcggcgggttcgccatgccgattcgtgaaaacaaagcgcaagagatctatatcgtctgctccggggaaatgatggcgatgtatgccgctaa
caacataccaaaggcatcgtgaaatacgctaaatccggcaaggtgcgcctgggcgggctgatttgtaactcccgtcagaccgaccgcgaagatgaa
ctgatcatcgcgctggcagaaaaactgggcacccagattgattcactttgtgccacgcgacaacatcgtccagcgcgcggaaattcgccgtatgacgg
ttatcgaatatgacccgaaatgcaaccaggccgacgaataccgcgcgctggcgaacaagatcgtcaacaacaccctgatggtcgtcccgaccccttg
caccatggatgaactggaagagctgctgatggaattcggcattatggatgtggaagacgccagcatcatcggtaaaaccgccgccgaagaaaacgc
ggcctga
126 atgaacgataacgatgtccttttctggcgcatgctgccgctatttcagtgtctgccggaactgcaacccgcgcagatcctggcctggctgacaggagaa
cgcgacgacgccttaaccccggcgtacctcgataagcttaacgtccgcgaactggaagcgaccttcccgtctgaaacggcgatgatgtcgcccgca
cgctggagccgcgttaacgcgtgccttcacggtacgctgcccgcacacctgcaggtaaaaagcaccactcgtcaggggcaattacgggtagcattt
gttcacaggatggattgctgatcaatggtcattttggtcaggggcggctgttttttatctacgcctttgatgaacagggcggatggctacacgcgttacgc
cgtcttccctcggccccgcaaacccaggagccgaatgaagttcgcgcgcagctcctgagtgattgccacctgctgttttgtgaagccattggcggccc
tgcggcggcccggctgattcgtcacaatatccacccgatgaaagtgtcgccagggatgtccattgccgcccagtgtgatgccattaccgcactgctga
gcggacgtctgccaccgtggctggcaaaacgtcttgagaaagccaacccgctggagaagggtgttttaa
127 atgaagggaaatgacattctcgcgctgctggatgaacccgcctgcgaacacaatcacaaacagaaatccggctgtagcgcccctaaacccggtgcc
acggcgggcggttgcgcgttcgacggcgcgcaaatcaccctgttgccgctgtcggtagtggcgcacctggtccacggaccgattggctgcacggg
aagctcctgggataaccggggcagtatgagctccggccccagtctcaaccggctcggctttaccaccgacctgaacgagcaggatgtcaaatgggg
cgcggcgaacggcggcttttccacgcggtgcgtcatatcgtcaaccgttatcaccctgccgccgtgtttatctataacacctgcgttccggcgatggag
ggtgatgatattgacgccgtctgtcaggcggcggaaaccgccaccggcgtgccagtgattgccgttcatgccgccgggttctatggcagcaaaaacc
ttggcaaccgtctcgcgggtgaagtgatggttaacaaggtcattggacggcgcccgcccgccccctggccggacgatacccccttcgcgccggaac
accgccacgatatcggcctgattggcgaatttaatatcgccggggagttctggcacgttcagccgctgctcgatgagctgggtattcgcgtgctgggca
gcctttccggggatggccgttttagtgaaatccagaccctgcaccacgcgcaggtcaatatgctggtctgctcaagagcgctgatcaatgttgcccgca
ccctggaacagcgctatggcaccccctggtttgagggcagtttttacggcgtgcgcgctacctccgatgccctgcgtcaactggcatccctgcttggcg
acagcgatctgattgcccccaccgaagccgttattgcccgcgaagaagccacggcaaatcagccgctcgccccgtggcgcgaacggctacagggt
cgcaaagtgctgctctataccggtggcgtgaaatcaggtcggtgatctccgcattgcaggatttagggatgaccctcgtgccgactggcacccgcaa
atctaccgaagaagataagcagcgtattcgcgaattaatgggcgatgacgcgctaatgctggaagaaggcaacgcccgcaccctgctggatgtggtg
taccgctatcaggcggatttgatgatcgctggggggcgtaacatgtataccgcgtacaaagcgggctgccgtttctggatatcaaccaggagcgtga
acacgcctttgcgggttatcgcggcatcgtcaccctcgcccaacagctttgccagactattgaaagccccgtctggccgcaaacacacgcccgcgcg
ccgtgccaataa
128 atgagcaatgcaacaggcgaacgtaatctggaaattatccaggaagtgctggagatctttcccgaaaaaacgcgcaaagaacgcagaaagcacatg
atggttaccgacccggagatggaaagcgtcgggaaatgcatcatctctaaccgcaaatcgcagccgggtgtgatgactgtccgcggctgacctacg
ccgggtcgaaaggcgtggtttttgggccgattaaagatatggcccacatctcccacggcccgatcggctgtgggcagtactcccgtgccgggcggc
gcaactactacaccggggtcagcggcgttgattccttcgggacgctgaactttacctctgattttcaggagcgcgatatcgtcttcggcggcgataaaaa
gctcaccaaactgattgaggagatggaggaactgttcccgctgaccaaaggcatctccattcagtcggagtgcccggtaggtttaatcggtgacgatat
cgaagcggtggcgaatgccagtaaaaaagcgctcaacaagccggtgatcccggtgcgttgcgaaggctttcgcggcgtgtcgcagtcgctcggtca
ccatatcgccaacgacgttatccgcgactgggtgctggataaccgcgaagggaagcccttcgaatctaccccctatgacgtggccatcatcggcgatt
acaacatcgggggggatgcctgggcgtcgcgcattctgcttgaagagatggggttacgcgtggtggcgcagtggtccggtgacggcacgctggtag
agatggtaaacaccccgttcgtcaagctgaacaggtgcactgctaccgctctatgaactacatctctcgccatatggaagagaaacacggtatcccgt
ggatggagtacaacttcttcggcccgaccaattatcgccgaatcgagcgtaagatcgccgatcaatttgacgacaccatccgcgccaatgcggaagc
ggtgatcgccaaatatcaggcgcaaattcgatgcgattatcgccaaataccgcccgcgtctcgaaggccgcaaggtgctgctctatatgggtggcctg
cgtcctcgccacgtgattggcgcgtatgaggatttgggcatggagattgtcgccgccgggtatgaatttgcccataacgacgattacgaccgcaccct
gccggacctcaaagagggcacgctgttgttcgacgatgccagcagttatgaactggaagccttcgtgaaggcgattaagccggacctcattggctca
ggcatcaaggaaaaatacattttccagaaaatgggggtaccgtttcgccagatgcactcctgggattactccggcccgtatcacggctatgacggcttt
gccatctttgcccgcaatatggacatgacgctcaacaatcccgcctggggcgagttgaccgcaccctggctgaaatcagcctga
129 atggcagatatcatccgtaatcagaaaccgctggcggtaagcccggtaaaaagcggccagccgttaggcgccattctggcgagcctcggctttgtgc
acagattccactggtgcacggtgcgcagggatgcagcgcgttcgccaaagtgttttttatccaacattttcatgaccctattccgctgcaatccacggcg
atggaccccacctcaacggtcatgggggcggacggcaatatccttgccgcgctcaatacgctgtgccagcgcattcaccccgaaagctatcgtcctgt
tgagtaccggcctgtctgaggcgcagggcagcgatatcagccgcgtggtacgtcagtttcgtgaggattttccccgccacaaaaatatcgccctcctg
acggtcaacaccccggatttttacggcacgctggagaacggctttagtgcggtggtggaaagcgtcatcgaacagtgggtgccggaaagcctcag
catggcctgcgtaaccggcgggtcaacttgttgttaagtcacctgctgacgcccggtgatgttgagttgctgcgcagctacgtcgaggcttttggcctgc
aaccggtgatcgtgccggatctttcacagtcgctggatggtcacctggcaagcggtgatttttcgccggtcactcaggggggaacgcccctgcgcatt
atcgaacagatgggacagagcctgtgcacgtttgctattggcgtgtcgctgtcccgtgcggcatcgagctggcacagcgtagccgtggcgangtga
tcgtgcttccccatctgatgaccatggaacattgcgaccgttttattcatcaactgaagatcatttccgggcgcgaggttcccgcctggattgagcgccag
cgcggacaattgcaggatgcgatgatcgattgtcatatgtggttgcaggatacccggctcgcgctggccgccgagggcgatctgctggcgggctggt
gtgatttcgcccgtagccagggcatgctccccggccccgttgtggcgccggtcagccagccgggcctgcgacagcttcccgtggagaaagtggtca
ttggcgatctggaagatatgcaggatttactctgcgctatacctgctgacctgctggtcgccaactcccatgccgcagacctggccgaacaattctccat
cccgctgatccgcgccgggttccctatcttcgacaggcttggcgaatttcgtcgcgtgcgtcagggataccccggcattcgcgacacgctgtttgagct
ggcgaacctgatgcgcgaacgtcatcaccacctgcccgtctaccgctcccocctgcgccagcaatttgcccaggacgctgacggaggccgctatgc
aacatgttaa
130 atgagccaaactgctgagaaaattgtcacctgtcatccgctgtttgaacaggacgaataccagacgctgtttcgcaataagcgcggtctggaagaggc
gcacgacccgcagcgcgtgaagaggtttttgaatggaccaccacggcggagtatgaagcgctgaactttaagcgtgaagcgttaaccgtcgatccg
gcaaaggcctgccagcctttaggatcggtactctgctcgctgggttttgccaatacgctgccttagtgcacggttcccagggctgtgtggcctatttccg
cacctattttaaccgtcatttcaaagagccgatcgcttgcgttccgactctatgacagaggatgcggtcttcggcgccggcaacaacaacctaacacc
gggttgcaaaatgccagcgccctgacaaaccggaaattgtcgctgtgctccactacctgtatggcggaggtcatcggcgatgacctgcaggcctttatc
gccaacgccaaaaaggacgggtttattgatgccgccattccggtgccctacgcccatacgccaagttttatcggtagccacatcaccggctgggacaa
catgtttgaaggtttcgcccgggcatttaccgccgatcacgtggcgcaaccgggcaaactggcgaagctaaacctggtgaccggttttgaaacctatct
tggcaattaccgcgtgctcaaacgcatgatggcccagatggaggtgccctgtagcctgctgtctgacccgtctgagggttagatacgccagccgacg
gccactatcgcatgtatgcgggcggcacaacgcaacaagagatgcgcgacgcccccgatgctatcgacacccagctgctgctgcaaccctggcatctgg
tgaagagtaaaaaagtggtgcaggagtcctggggccagcccgccacagaagtgtccatcccaatgggactgaccgggaccgacgaactgctgatg
gcagtcagtcagttaaccggcaaaccggtggccgatgaactgacgctggagcgtgggcgcctggtggatatgattctcgattcacacacctggctgc
acggtaagaaattcggtctctacggcgatccggatttgtgatggggctgacgcgtttcctgctggaactgggctgcgagccgacggttatcctctgtca
taacggtagcaagcgctggcagaaagcgatgaagaaaatgcttgaggcatcgccctacggtcaggagagcgaagtgttcatcaactgcgatctgtg
gcatttccgctcgctgatgtttacccgcaaaccggactttatgatcggcaactcgtacgccaaattcatccagcgtgacacgctggcgaaaggcgaaca
gtttgaagttccgctgatccgtcttggcttcccgttgttcgaccgccaccacctgcatcgccagaccacatggggttatgaaggggcgatgaatatcgtc
accaccctggtcaacgccgtgctggaaaaagtcgaccgcgataccatcaaactgggcaaaacggactacagcttccgaccttgtccgctaa
131 atgacctttaatatgatgctggagaccagcgcaccgcagcacattgcgggcaacctctcacttcaacatcccggactgttttccacgatggttgaacag
gctccgatcgcgatttcgctgaccgacccggacgcgaggattctgtacgctaatccggccttttgtcgccagaccggttatagcctggaagagctgctc
aaccagaaccatcgcatactggcaagccancagacgccgcgcagcatttatcaggaactgtggcaaacgctgctgcaacagatgccctggcgcggt
cagctcatcaatcgccgtcgggatggcagcctttatctggctgaggtcgatatcaccccggtcgtcaacaaacagggcgaactggaacactacctcgc
catgcaacgtgatatcagcgccagctatgcgctcgaacagcgattgcgcaatcacaccaccatgagcgaggcggtgctgaacaacattcctgccgcc
gtggtggtggtcaacgagcaggaccaggtagtcatggacaacctcgcctacaaaaccttctgtgccgactgcggtggcaaggagctgctcaccgaa
ctggatttctcccggcgcaaaagcgatctctatgccgggcaaatactgcctgtggtgctgcgcggcgccgtgcgctggactctgtcacctgaggac
cttgccgggggtgagcgaagaagccagccgctactttattgataccgcgagccccgcaccctggtggtgatcaccgactgcacccagcaacaaca
acaggcccgaacagggccgtctcgatcgtctcaaacaggagatgaccaccgggaagctgctggccgcgatccgtgaatcgttggatgccgcgctggt
tcagctaaactgccccatcaatatgctggcggcggcgcgacgtctcaacggtgaagataaccataacgtggcgctggatgccgcgtggcgcgaggg
ggaagaggcgctggcccgcctgcaacgctgccgcccttctctcgatctggaagagagcgcgctgtggcctctgcaaccgctgtttgacgacctgcg
cgccctttaccatacccgctataacaatggcgaaaatctgcacgttgaaatggcctctccgcatctggcggggtttggtcagcgcacgcagatccttgc
ctgctcagtttgtggctcgaccgtacgctggccctcgccgccgcgctaccggacagaacgctgcatacccagctttacgcccgtgaagaagatggct
ggctgtgtccatttggctgacagataatggccgctcatccatgtgcgatacgcccactcccccgatgccctgaacgcccccggcaaagggatggagct
gcgattgattcaaaccctggttgcccatcatcgcggcgcaatagaactaactacccgccagatggcggtacctgcctgaccctgcgattcccgttattt
cattcactgacccgaggcccacgatga
132 atgacccagcgacccgagtcgggcaccaccgtctggcgttttgatctctcacagcaatttaccgccatgcagcgcatcagcgtggtgttgagtcgcgc
aaccgagataagccagacgctgcaggaggtgctgtgtgttctgcataatgacgcatttatgcaacacggcatgctgtgtctgtatgacaaccagcagg
aaattctgagtattgaagccttgcaggaggcagaccaacatctgatccccggcagctcgcaaattcgctatcgccctggcgaagggctggtaggagc
cgtactgtcccagggacaatctcttgtgctgccgcgtgtcgccgacgatcaacgctttctcgacaggcttggcatctatgattacacctgccgtttatcg
ccgtccccttaatggggccaggcgcgcagacgattggcgtgctcgccgcgcagccgatggcgcgtctggaggagcggcttccttctgtacgcgct
ttctggaaaccgtcgccaatctggtcgcacagacagtccggctgatgaccccgcctgccgccgccacaccgcgcgccgcgattgcccagaccgaa
cgccagcgcaactgtggcactcctcgccccttcggctttgagaatatggtgggcaaaagcccggccatgcagcagacaatggacattatccgccagg
tttcgcgctgggataccacggtactggtgcgcggcgaaagcggcaccggtaaagaacttatcgccaatgctattcatcacaactcccctcgcgccgcc
gcgccctttgtgaaatttaactgcgcggcgctaccggatacgctggagagagcgaattgttcggccatgaaaaaggggcgttcaccggcgcggttc
gccagcgtaaaggacgtttgaactggccgatggcggcacactgtttcttgatgaaattggcgaaagcagcgcctcgttccaggccaaactgctgcgt
attttgcaggagggtgaaatggagcgcgttggcggcgacgaaaccctgcgcgtcaatgtgcgtatcatcgccgccaccaaccggaatctggaagaa
gaggtgcggatgggcaatttccgcgaggatctctattatcgcctcaacgtaatgcccatctccctgcccccgctgcgtgaacgtcaggaggacattgc
cgagctggcgcactttctggtgcgcaaaatcgcccataaccaggggcgtacgctgcgcatcagtgatggcgccatccgtctgctgatgggttacaact
ggcccggtaacgtgctgtgagctggaaaattgcctggaacgttcggcagtgatgtcagaaaacggcctgatcgaccgcgatgtggtgctctttaaccac
cgtgagaacacgccaaaactcgctatcgccgccgcgccaaaagaggatagctggcttgatcaaacgctggatgaacgtcaacggctgattgccgcg
ctggaaaaagccgggtgggtgcaggccaaagcggcgcgtctgctgggtatgacgccccgtcaggtcgcctatcggatacaaattatggatatcagc
atgcccaggatgtga
133 atgatgccgcactctccacagctacagcagcactggcaaactgtactggcccgcttgcctgagtcattcagtgaaacaccgcttagtgaacaagcgca
gttagtgcttactttcagtgattttgtgcaggatagccttgccgcgcatcctgactggctggctgagctggaaagcgcaccgccacaggcggacgagtg
gaagcagtatgcgcaaacccttcgcgaatcgctggaaaggtgtgggagatgaggcatcattaatgcgtgcgctgcgcctgttccgtcgccatatgatgg
tgcgcattgcctgggcgcagtcgctggcgctggtggcagaagatgagacgttgcagcagttgagcgtactggcggagaccctgatcgtcgctgcac
gcgactggctttacgatgcctgctgtcgcgagtggggaacgccgtgcaatcagcagggggaaccgcagccgttgctgatcctgggcatgggcaag
ctgggtggcggggagcttaacttttcgtccgatatcgatctgatttttgcctggccggaaaacggttcaacgcgcggtgggcgacgcgaacttgataac
gcccagttttttactcgcttgggacagcgcctgatcaaagtgctcgaccagccgacgcaggatggctttgtctatcgcgtggatatgcggctgcgcccg
tttggcgacagcggtccgctggtgctgagttttgccgcgctggaagattattatcaggagcaggggcgcgactgggaacgttatgcgatggtgaaagc
ccgcattatgggcgataaggacgatgtttacgctggcgaattacgggccatgctgcggccgttcgtcttccgtcgctatatcgatttcagcgttattcagt
ctctgcgtaacatgaaagggatgattgcccgcgaagtgcgccgccgtggtctgaaagataacattaagctgggcgcgggcggcatccgtgagattga
gtttatcgttcaggtgttccagttgatacgcggtgggcgcgagccgtcgttgcagtcccgttcactgttaccgacgctggacgctatcgataagctgggt
ttgctgccgcctggcgatgcaccggcgttacgccaggcctatttgtatctgcgccgtctggaaaaacctgctgcaaagcattaacgacgaacaaacgca
gacgctgccgacagatgaactcaatcgcgcgcgtctggcctgggggatgcgggtcgcagactgggaaaccctgaccgctgagcttgaaaagcaga
tgtctgccgtacgagggatattcaacaccctgattggcgatgacgaagccgaagagcagggggatgcgctctgcgggcaatggagtgagttgtggc
aggatgcgtttcaggaagatgacagcacgcctgtgctggcgcacctttctgacgatgatcgccgccgcgtggtcgcgatgattgctgattttcgcaaag
agctggataaacgcaccattggcccacgcggccgccaggtgctcgaccatctgatgccgcatctgttgagtgatgtctgctcccgtgaggatgcccct
gtaccgttgtctcgcgtgacgccgctgttaacgggaattgtcacgcgtacgacgtatcttgagctgctcagcgagtttcctggtgcgcgtaagcatctga
tttcactctgtgccgcctcgccgatggtggccagtaagctggcgcgctatccgttattgctggatgagttgctcgatccgaataccctttatcagcccacg
gcgatgaatgcctaccgggatgagctacgtcagtatctgctgcgtgtgccggatgacgatgaagagcagcaactggaggcgttacgccagtttaaac
aggctcaattgttgcgtgtggcggcagcagatctggcaggcacactccccgtgatgaaagtgagcgatcacttaacatggcttgccgaagccatcatt
gaagccgtggtacaacaggcgtggagcctgatggtatcgcgttatgggcagccgaaacacttacgcgaccgtgaaggccgtgggtttgcagtggtc
ggttacggcaaactgggcggttgggagctgggctatagttccgatctggatttgattttccttcatgactgtccggtggacgtgatgactgacggcgagc
gggaaatcgatggccgccaattttatctgcgccttgcccagcgcgtgatgcacctgttcagtacgcgcacctcatccgggatcctgtatgaggtagacg
cgcgcttgcgcccgtccggtgcggcgggaatgctggtgacctcaaccgaatcctttgccgactaccagcgcaccgaagcctggacctgggaacatc
aggcgctggttcgcgcccgcgttgtctatggcgatccacaattaaacgcgcaatttgatgccatccgccgcgatatcaccatgaccgtgcgtaatggtg
caacgttacaaaccgaggtgcgcgagatgcgcgaaaaaatgcgcgcccacttgagcaataagcacaaggatcgctttgatattaaagccgatgagg
gtggaattaccgatatcgaatttatcacccagtatctggtgctgcgttatgcccatgccaaaccgaaactgacgcgctggtcggacaatgtccgcattct
ggaagggctggcgcaaaacggcattatggaagagcaggaagcgcaggcacttaccaccgcctatacaacgttgcgtgatgagctgcatcacctgg
cgctacaggagctgccaggacatgttccggaggcatgttttgtcgctgaacgcgcgatggtgcgagcctgctggaacaagtggttggtggagccgtg
cgaggacgcgtaa
134 atgaagaaagcactattaaaagcgggtctggcctcgctggcattactgccgtgtctggctatggcagccgatccggttgtcgtcgataaagccgacaat
gcctttatgatgatttgcaccgcgctggtgctgtttatgtcaattccgggcatcgccctgttctatggtggtttaatccgcggtaaaaacgtcctttctatgct
gacacaggttgcggttacgttcgcactggtgtgcgtgctgtgggtggtttacggctactctctggcctttggcactggcggcagcttcttcggtagcttcg
actgggtgatgctgaaaaatattgagctgaaagcgctgatgggcaccatctatcagtacattcacgttgcgttccagggctcgtttgcctgtattaccgtc
ggcctgattgtcggtgcgctggcagaacgtatccgtttctccgcagtactgattttcgtcgtggtatggctgacgctgtcctacgtgccgatcgcacacat
ggtctggggcggcggtctgctggcaacccatggcgccatggattttgcgggcggtacagtcgttcacatcaacgcagccgttgcaggcctggtgggt
gcttacctgattggcaaacgtgtcggtttcggtaaagaagcgtttaaaccgcacaacctgccgatggtgtttaccggtacggcaatcctctactttggctg
gttcggattcaacgcgggttctgcaagcgcggcgaacgaaattgcgggtctggcttttgttaacaccgtcgtggcaacagcgggtgcaatcctctcctg
ggtcttcggtgagtgggcgctgcgcggcaaaccgtctctgttgggtgcctgttctggtgcgattgctggcctcgtgggtatcaccccggcgtgtggtta
cgttggtgtgggtggcgcgctgatcgtgggcatcgttgcaggcctggcgggtctgtggggcgttaccgcgctgaaacgctggctgcgtgttgacgac
ccgtgtgatgtcttcggtgttcacggcgtgtgcggtatcgtaggttgtatcatgacaggtatcttcgcagccacttcactgggcggcgtgggttatgccga
aggcgtgaccatgggccatcaggttctggtacaactggaaagtatcgccattactatcgtatggtctggtatcgtcgcctttatcggttacaaactggctg
atatgacagtgggtctgcgtgttccggaagatcaggaacgcgaagggctggacgtcaacagccacggcgagaacgcctacaacgcctga
135 ctggggtcactggagcgctttatcggcatcctgaccgaagaatttgccggtttcttcccgacctggctggcccctgttcaggttgtggtgatgaatatcac
tgattctcaagctgaatatgtcaacgaattgacccgtaaattgcaaaatgcgggcattcgtgtaaaagcggacttgagaaacgagaagattggctttaaa
atccgcgagcacactttacgtcgtgtcccttatatgttggtctgtggtgataaagaggtggaagcaggcaaagtggccgttcgcacccgccgcggtaa
agacctgggcagcctggacgtaagtgaagtgattgagaagctgcaacaagagattcgcagccgcagtcttcaacaactggaggaataaggtattaaa
ggcggaaaacgagttcaaacggcacgtccgaatcgtatcaatggcgagattcgcgcccaggaagacgcttaactggtctggaaggtgagcagctg
ggtatt
136 attgaagagtttcatcatggctcagattgaacgctggcggcaggcctaacacatgcaagtcgaacggtagcacagagagcttgctctcgggtgacga
gtggcggacgggtgagtaatgtctgggaaactgcctgatggagggggataactactggaaacggtagctaataccgcataacgtcgcaagaccaaa
gagggggaccttcgggcctcttgccatcagatgtgcccagatgggattagctagtaggtgggctaacggctcacctaggcgacgatccctagctggt
ctgagaggatgaccagccacactggaactgagacacggtccagactcctacgggaggcagcagtggggaatattgcacaatgggcgcaagcctga
tgcagccatgccgcgtgtatgaagaaggccttcgggttgtaaagtactttcagcggggaggaaggcganacggttaataaccgtgttgattgacgtta
cccgcagaagaagcaccggctaactccgtgccagcagccgcggtaatacggagggtgaagcgttaatcggaattactgggcgtaaagcgcacgc
aggcggtctgtcaagtcggatgtgaaatccccgggctcaacctgggaactgcatccgaaactggcaggcttgagtacgtagagggaggtagaattc
caggtgtagcggtgaaatgcgtagagatctggaggaataccggtggcgaaggcggcctcctggacgaagactgacgctcaggtgcgaaagcgtg
gggagcaaacaggattagataccctggtagtccacgccgtaaacgatgtctatttggaggttgtgcccttgaggcgtggcttccggagctaacgcgtta
aatagaccgcctggggagtacggccgcaggttaaaactcaaatgaattgacgggggcccgcacaaccggtggagcatgtggtttaattcgatgca
acgcgaagaaccttacctggtcttgacatccacagaacttgccagagatggcttggtgccttcgggaactgtgagacaggtgctgcatggctgtcgtca
gctcgtgttgtgaaatgttgggttaagtcccgcaacgagcgcaacccttatcctttgttgccagcggtccggccgggaactcaaaggagactgccagt
gataaactggaggaaggtggggatgacgtcaagtcatcatggcccttacgaccagggctacacacgtgctacaatggcgcatacaaagaagcga
cctcgcgagagcaagcggacctcataaagtgcgtcgtagtcccgattggagtctgcaactcgactccatgaagtcggaatcgctagtaatcgtggatc
agaatgccacggtgaatacgttcccgggccttgtacacaccgcccgtcacaccatgggagtgggttgcaaaagaagtaggtagcttaaccttcggga
gggcgcttaccactttgtgattcatgactggggtgaagtcgtaacaaggtaaccgtaggggaacctgcggttggatcacctcctt
137 atgaccatgcgtcaatgcgccatttacggcaaaggtgggatcggtaaatcgaccaccacacagaacctggtcgccgcgctggcggagatgggtaag
aaagtcatgatcgtcggctgcgatccgaaagccgactccacgcgtttgatcctgcatgcgaaagcgcagaacaccattatggagatggccgccgaag
tcggctccgtcgaagacctggaattagaagacgtgctgcaaatcggttacggcggcgtgcgctgcgcggaatccggtggcccggagccaggtgtg
ggttgtgccggtcgtggcgtgatcaccgcgattaacttcctcgaagaagaaggcgcttacgtgccggatctggattttgttttctacgacgtgctgggcg
acgtggtatgcggtggtttcgccatgccgattcgtgaaaacaaagcgcaggagatctacatcgtttgctctggcgaaatgatggcgatgtacgccgcc
aataacatctccaaaggcatcgtgaaatatgccaaatccggtaaagtgcgcctcggcgggctgatttgtaactcgcgccagaccgaccgcgaagatg
aactcatcattgcgctggcggaaaaactcggcacgcaaatgatccactttgttccccgcgacaacattgtgcagcgtgcggaaatccgccgtatgacg
gttatcgaatatgacccgacctgcaatcaggccaacgaatatcgcagccttgccagcaaaatcgtcaacaacaccaaaatggtggtaccaaccccctg
caccatggatgaactggaagaactgctgatggagttcggcattatggatgtggaagacgccagcatcattggtaaaaccgccgccgaagaaaacgc
cgtctga
138 atgagcaatgcaacaggcgaacgtaacctggaaatcatcgagcaggtgctggaggttttcccggaaaagacgcgcaaagagcgcagaaaacacat
gatggtgacggacccggagcaggagagcgtcggcaagtgcatcatctctaaccgcaaatcgcagccgggcgtgatgaccgtgcgtggctgctcgt
atgccggatcaaaaggggtggtatttgggccaatcaaagatatggcgcatatctcccacggcccgatcggctgcgggcagtactcccgcgccgggc
ggcgtaactactataccggcgtcagcggcgtggacagtttcggcacgctcaacttcacctccgatttccaggagcgcgacatcgtgtttggcggcgac
aaaaagctcgccaaactgattgaagagctggaagaactgtttccgctgaccaaaggcatttcgattcagtcggaatgcccggtcggcctgattggcga
tgatattgaagccgtggcgaacgccagccgcaaagcgatcaacaaaccggttattccggtgcgttgcgaaggctttcgcggcgtgtcgcaatccctc
ggtcaccatattgccaacgatgtgatccgcgactgggtactggataaccgcgaaggcaaaccgtttgaatccaccccttacgatgtggcgatcatcgg
cgattacaacatcggtggcgacgcctgggcctcgcgcattttgctcgaagagatggggttgcgggtggtcgcgcagtggtccggcgacatacgct
ggtgcagatggaaaacacgccgttcgtcaaactgaacctggtgcactgctaccgctcgatgaactacatctcgcgccatatggaggagaagcacggt
attccgtggatggaatacaacttctttggcccgacgaaaatcgcggaatcgctgcgcaaaatcgccgacctgttcgacgacaccattcgcgccaacgc
cgaagcggtgatcgcccgataccaggcgcagaacgacgccattatcgccaaatatcgcccacgtctggagggtcgcaaagtgttgctctatatgggc
gggctgcgtccgcgccatgtgattggcgcctatgaagatctgggaatggagatcatcgccgccggttatgagtttggtcataacgacgattacgaccg
caccctgccggatctgaaagagggcacgctgctgtttgatgacgccagcagctatgagctggaggcgtttgtcaacgcgctgaaaccggatctcatc
ggttccggcatcaaagagaagtacatctttcagaaaatgggcgtgccgtttcgccagatgcactcctgggattactccggcccgtaccacggctatgac
ggcttcgccatcttcgcccgcgatatggatatgacgctcaacaaccccgcctggggtcagttgaccgcgccgtggcttaaatccgcctga
139 atgaaggggaacgacatcctggctctgctcgatgaaccagcctgcgagcataaccataaacagaaaaccggctgtagcgcgccaaacccggcgc
caccgccggaggctgcgccttcgacggcgcacagatcaccctgctgccactttccgatgtggcgcatctggtacatggcccgattggctgcgccggc
agctcatgggataaccgtggcagcctgagttctggcccgctgattaaccgactcggattcaccactgatttgaacgaacaggatgtcatcatggggcg
cggcgagcggcggttgtttcacgcggtgcgccatattgtcgagcgctatcacccggcggcggtatttatttacaacacctgcgttccggctatggaagg
cgatgacattgacgcggtctgccaggccgccgcgaccgccaccggtgtgcccgtgattgccgtagatgtggccggtttttacggtagcaaaaacctg
ggtaaccgcctcgcgggcgaggtgatggtgaaaaaagttatcggcgggcgcgaacccgcgccgtggccggacaatacaccttttgccccggcgca
ccgccatgacataggcctgattggcgaatttaacatcgccggcgagttctggcatatccagccgctgcttgatgagctgggtattcgcgtccttggctcc
ctttccggcgacgggcgctttgccgagatccagacgttgcaccgcgcgcaggtcaatatgctggtgtgctccagggcgctgattaatgtcgccagatc
gcttgaacaacgttatggcacaccctggtttgaaggcagtttttatggcgttcgcgccacctccgatgccctgcgccagctggcaacactcaccggcga
tagcgatttaatggcgcgaaccgaacggctgatcgcacgtgaagagcaagccacagaacaggcgctagcaccgctgcgtgaacggttacacggcc
ggaaagtgctgctctataccggtggcgtgaaatcctggtcggtggtttcggcgctgcaggatctcggcatgacggtcgttgctaccggaacgcgcaaa
tccaccgaagaggataaacaacgcatccgtgaactgatgggcgatgacgccatcatgctggatgaaggcaatgcccgcgccttgctggatgtggtct
atcgctacaaagccgacatgatgatcgcgggcgggcgcaacatgtacaccgcctataaagcgcgtctgccctttctggatatcaaccaggagcgtga
acacgcgtttgccggttatcgcggcatcatcacgcttgccgaacaactttgtcagacgctggaaagcccggtctggccgcaaacacatgcccgcgcc
ccgtggcaataa
140 atgagccagactgctgagaaaatacagaattgccatcccctgtttgaacaggacgcctaccagacactatttgccggtaaacgggcactcgaagagg
ctcactcgccggagcgggtgcaggaagtgtttcaatggaccaccaccccggaatacgaagcgctgaacttcaaacgcgaagcgctgactatcgacc
cggcaaaagcctgccagccgctgggggcggtgctagttcgctggggtttgccaacaccctgccgtatgtgcacggttcacagggttgtgtggcctat
ttccgtacgtactttaaccgccacttcaaagaaccggtggcctgcgtgtcggattcgatgacggaagacgcggccgtgttcggcgggaataacaacct
caacaccgattacaaaacgccagcgcactgtataaaccggtgattatcgccgtctctaccacctgtatggcggaagtgatcggtgatgatttacagg
cgtttatcgccaacgccaaaaaagatggttttctcgatgccgccatccccgtgccctacgcccacacccccagttttatcggtagccatatcaccggctg
ggacaacatgtttgaaggttttgcccgtacctttaccgcaaaccatcagccacagcccggtaaactttcacgcctgaacctggtgaccgggtttgaaac
ctatctcggcaatttccgcgtgagaaacgcatgatggaacaaatggaggtgcaggcgagtgtgctctccgatccgtcggaggtgctggacaccccc
gccaatggccattaccagatgtacgcgggcggtacgacgcagcaagagatgcgcgaggcaccggatgccatcgacaccctgctgctgcaaccgtg
gcagctggtgaaaagcaaaaaagtggtgcaggagatgtggaatcagcccgccaccgaggttgccattcccgtcgggctggcaggcacagacgaa
ctgttgatggcgattagccagttaaccggcaaagccattcccgattcgctggcgctggagcgcgggcggctggtcgatatgatgctcgactcccacac
ctggttacacggtaaaaaattcggtctgtttggcgatccggattttgtcatgggattgacccgcttcctgctggaactgggctgtgaacctgccgtcatcct
ctgccataacggtaacaaacgctggcaaaaagcgatgaagaaaatgctcgatgcttcaccgtacggccaggagagcgaagtgtttatcaactgcgac
ttgtggcatttccgctcgctgatgttcacccgccagccggattttatgattggcaactcgtacgccaagtttattcagcgcgacaccttagccaagggcga
acagtttgaagtcccgctgatccgcctcggttttccgctgttcgaccgtcaccatctgcaccgccagaccacctggggctacgagggcgcgatgagca
ttctcacgacgctggtgaatgcggtactggagaaagtggacaaagagaccatcaagctcggcaaaaccgactacagcttcgatcttatccgttaa
141 atggctgatattgttcgtagtaaaaaaccgctggcggtgagcccgataaaaagcggccagccgctgggggcgatcctggcaagcctgggtttcgaac
agtgcataccgctggtacacggcgctcaggggtgcagcgcgttcgcgaaagtgttctttattcaacattttcacgacccgatcccgctgcaatcgacgg
cgatggacccgacttccaccattatgggcgccgatgaaaacatttttaccgcgctcaatgttctctgccagcgcaacgccgcgaaagccatcgtgctgc
tcagcaccgggctgtcagaagcccagggcagcgatatttcacgagtggtgcgccagtttcgtgatgactttccgcggcataaaaacgtggcgctgctc
accgtcaacaccccggatttctacggctcgctggaaaacggctacagcgccgtgctggaaagcatgattgaacagtgggtgcccgcgcagcccgcc
gccagcctgcgcaaccgtcgcgtcaacctgctggtcagccatttactgacgccgggcgatatcgaactgttacgcagttatgtggaagcattcggtctg
caaccggtgattgtgccggatctatcgcagtcgctggacggacatctggccaacggtgatttttcgcccgtcacccaggggggaacaccgctgcgca
tgattgaacagatgggccaaaacctggccacttttgtgattggccactcgctggggcgggcggcggcgttactggcgcagcgcagccgtggcgagg
tgatcgccctgccgcatctgatgacgcttgatgcgtgcgacacctttatccatcgcctgaaaaccctctccgggcgcgacgtgcccgcgtggattgag
cgccagcgcgggcaagtgcaggatgcgatgatcgattgccatatgtggttgcagggcgcggctatcgccatggccgcagaaggcgatcacctggc
ggcatggtgcgatttcgcccgcagccagggcatgatccccggcccggttgtcgcgccggtcagccagccggggttgcaaaatctgccggttgaaat
ggtggttcatcggcgatctggaagatatgcaggcttcggctttgcgcgacgcccgccgcgttactggtggccaattctcatgccgccgatctcgccacgc
agtttgatatgtcgcttatccgcgccggttttccggtgtatgaccggctgggggaatttcgtcggctgcgccaggggtatagcggcattcgtgacacgc
tgtttgagctggcgaatgtgatgcgcgaacgccattgcccgcttgcaacctaccgctcgccgctgcgtcagcgcttcggcgacaacgttacgccagg
agatcggtatgccgcatgttaa
142 atgaccctgaatatgatgatggatgccagcgcgcccgaggccatcgccggtgcgctttcgcaacaacatcctgggctgttttttaccatcgttgaagaa
gccccgtcgctatttcactaaccgatgccgaggcacgtattgtctatgccaacccggcattctgccgccagaccggctatgagcttgaggagttgttg
cagcaaaatccccgcctgcttgccagtcagcagaccccacgggaaatctaccaggatatgtggcacaccctgttacaacgtcgaccatggcgcggg
caattgatcaaccgccaccgtgacggcagcctttttctggttgagatcgatatcaccccggtgattaacccgtttggcgaactggaacactacctggcca
tgcagcgcgatatcagcgccggttatgcgctggagcagcggttgcgtaatcacatggcgctgaccgaagcggtgctgaataacattccggcggcgg
tggtcgtggtcgatgaacgcgatcgtgtggttatggataacctcgcctataaaactttctgtgctgattgcggcggaaaagagctactgagcgaactcca
tttttcagcccgtaaagcggagctggcaaacggccaggtcttaccggtggtgctgcgcggcgcggtgcgctggttgtcggtcacctgctgggcgctg
ccaggcgtcagcgaagaagccagtcgctactttattgataataccttgacgcgcacgctggtggtcatcaccgacgacacccagcagcgccagcag
caagagcaaggacggcttgaccgccttaaacagcagatgaccagcggcaaactgctggcggcgatccgcgaagcgcttgacgccgcgctgatcc
agcttaactgccccatcaatatgctggcggcggcgcggcgtttaaacggcagtgataacagcaacgtagcgctggacgccgcgtggcgcgaaggt
gaagaagcgatggcgcggctgaaacggtgccgcccgtcgctggagctggaaagtgccgccgtctggccgctgcaacccttttttgacgacttgcgc
gcgctttatcacacccgctacgagcagggtaaaaatttgcaggtcacgctggattcgacgcatctggtgggatttggtcagcgaacccaactgctggc
ctgcctgagtctgtggctcgatcgcacgctggatattgccgtcgggctgcgtgatttcaccgcccaaacgcagatttacgcccgggaagaagcgggct
ggctctcgttgtatatcactgacaatgtgccgttgattccgctgcgccatacccattcgccggatgcgcttaacgcaccgggaaaaggtatggagttgc
ggctgatccagacgctggtagcgcatcacaacggcgcgatagaactcacttcacgccccgaagggggaagctgcctgaccctacgattcccgctatt
tcattcactgaccggaggttcaaaatga
143 atgacccagcgaaccgagtcgggtaataccgtctggcgcttcgatttatcccagcagttcaccgcgatgagcggataagcgtggttctcagccggg
cgaccgaggttgaacagacactccagcaggtgtgtgcgtattgcacaatgacgcctttttgcagcacggcatgatctgtctgtacgacagccagcag
gcgattttgactattgaagcgttgcaggaagccgatcagcagttgatccccggcagctcgcaaattcgctaccgtccgggtgaagggctggtcggga
cggtgctttcgcaggggcaatcgttagtgctggcgcgtgtggctgacgatcagcgctttcttgaccgcctgggactgtatgattacaacctgccgtttatc
gccgtgccgctgatagggccggatgcgcagacttttggcgtgctgacggcgcaaccgatggcgcgttacgaagagcggttacccgcctgcacccg
ctttctggaaacggtcgcgaatctggtggcgcagaccgtgcgtttgatgacgccgccggctgcacgcccttccccacgcgctgccatcacgccaacc
gccagcccgaaatcgtgcagtacttcacgcgcgttcggcttcgaaaatatggtcggcaacagcccggcaatgcgccagaccatggagattatccgtc
aggtttcgcgctgggataccaccgttctggtgcgcggcgagagcggcaccggcaaggaactgattgccaacgccatccatcacaattcgccgcgcg
ccagtgcgccatttgtgaaattcaactgtgcggcgctgccggacacattgcttgaaagcgaattatttggtcatgaaaaaggcgcctttaccggcgcgg
tacgccagcgtaaaggccgttttgagctggccgatggcggcacgctgtttcttgacgaaattggggaaagcagcgcctcgtttcaggctaagctgctg
cgtattttgcaggagggcgaaatggaacgcgtcggtggtgacgagacattgcaagtgaatgtgcgcatcattgccgcgacgaaccgcaaccttgaag
atgaagtacgcctgggacattttcgcgaagatctctattaccgcctgaatgtgatgcccatcgccctgccgccgctgcgcgaacgccaggaccacatc
gccgaactggcacattttctggtgcgtaaaatcgcccacaaccagaaccgcacgctgcgcattagcgagggcgctatccgcctgctgatgagctaca
gctggcccggcaatgtgcgcgaactggaaaactgccttgagcgctctgcggtgatgtoggaaaacggtctgatcgatcgggacgtgattttatttaatc
atcgcgaccagccagccaaaccgccggttatcagcgtcacgcccgacgataactggctcgataacacccttgacgagcgccagcggctgattgccg
cgctgaaaaaagcgggatgggtacaagccaaagccgcccgcttgctggggatgacgccgcgccaggtcgcttatcgtattcagaccatggatatca
ccctgccaaggctataa
144 atgccgcaccacgcaggattgtcgcagcactggcaaacggttttttctcgtctgccggaagcgctcaccgcgcaaccattgagcgcgcaggcgcagt
cagtgctcacttttagtgattttgttcaggacagcatcatcgtgcatcctgagtggctggcagagcttgaaagcgcaccgccgccagcgaacgagtggc
aacactacgcgcaatggctgcaagcggcgctggagggcgtcaccgatgaaacctcgctgatgcgcacgctgcggctgtttcgccgtcgcattatggt
gcgcatcgcctggagtcaggcgctacagttggtggcggaagaggatatcctgcaacagctcagcgtgctggcggaaactctgatcgtcgccgcgcg
cgactggctctatgacgcctgctgccgtgagtggggaacgccgtgcaatccgcaaggcgtcgcgcagccgatgctggtgctcggcatgggcaaact
tggcggcggcgaactcaatttctcatccgatatcgatttgatttttgcctggccggaaaatggcaccacgcgcggcggacgccgtgaactggataacg
cgcagttttttacccgccttggtcaacggctaattaaagtcctcgaccagcccacgcaggatggctttgtctaccgcgtcgatatgcgcttgcgtcccttt
ggcgacagcggcccgctggtgctgagttttgccgcgctggaagattactaccaggagcaggggcgcgactgggaacgatacgcgatggtgaaagc
gcgcattatgggggacaacgacggcgaccatgcgcgagagttgcgcgccatgctgcgcccgttcgttttccgccgctatatcgacttcagcgtgatcc
agtctctgcgcaacatgaaaggcatgattgcccgcgaagtgcggcgtcgcggcctgaaggacaacataaaactcggcgcgggcggtattcgcgaa
atagagtttatcgtgcaggttttccagttgattcgcggcggtcgcgagcctgcgctgcaatcgcgttcgctgttgccgacgcttgctgccattgatcaact
acatctgctgccagatggtgatgcaccccggctgcgcgaggcgtatttgtggctgcgacggctggaaaacttgctgaaagcattaatgacgaacag
acacagacgctgccggccgatgatttgaatcgcgcgcgcctcgcctggggaatgggcaaagagagctgggaagcgctctgcgaaacgctggaag
cgcatatgtcggcggtgggcagattttcaacgatctgattggcgatgatgaaacggattcgccggaagatgcgctttctgagggctggcgcgaattgt
ggcaggatgcgttgcaggaagaggactctacgcccgtgctggcgcatctttccgaggacgatcgccgccgcgtggtggcgctgattgctgattttcg
caaagagctggataaacgcaccattggcccgcgcgggcgacaggtactcgatcacttaatgccgcatctgctcagcgatgtatgctcgcgtgacgat
gcgccagtgccgctgtcgcgtctgacgccgctgctcaccggtattattacgcgcaccacttaccttgagctgctgagtgaattccccggtgcgctgaaa
cacctcatttccctgtgcgccgcgtcgccgatggtggccagccaactggcgcgctacccgatcctgctcgatgaactgctcgacccgaacacgctcta
tcaaccgacggcgatgaacgcctatcgcgatgaactgcgacaatacctgttgcgcgtgccggaagaggatgaagagcagcaactggaggcgctac
ggcagtttaagcacgcgcagttgttgcgcgtagcggcggcggatatcgccggtacgttacccgtcatgaaagtgagcgatcacttaacctggctggc
ggaagcgattatcgatgcggtggtgcagcaagcctggaaccagatggtggcgcgttacggccagccgacgcatctgcacgatcgcgaagggcgc
ggtttcgccgtggtcggttacggcaaacttggcggctgggaattaggttacagctccgatctggatctggtgttcctgcacgactgccccatggatgtga
tgaccgatggcgagcgtgaaatcgatggccgccagttctatttgcgcctcgcgcagcgcgtcatgcacctgttcagcacgcgcacgtcgtccggcatt
ctttatgaagtcgatgcgcgtttgcgcccgtccggcgcggccggaatgctggtgaccactgcggaagcgttcgccgattatcaaaaaaatgaagcctg
gacatgggagcatcaggcgctggcgcgtgcgcgcgtggtgtacggcgatccgcaactgaccgccgaatttgacgccattcgccgcgatatcctgat
gacctcccgcgatgccgctaccctgcaaaccgaagtgcgggaaatgcgtgagaaaatgcgcgcccatcttagtaacaagcacaaagaccgtttcga
tctgaaagccgatgaaggcggtatcaccgatattgagtttatcgctcagtatctggtgctgcgctttgcccatgagaagccgaaactgacgcgctggtc
ggataatgtgcgcatcctcgaagggctggcgcaaaacggcatcatggatgagcaggaagagcaggcattgacgctggcgtacaccacgttgcgtg
atgagctgcaccacctggcgctgcaagagctgccaggacatgtggcgctctcctgttttgtcgccgagcgtgcgcttatcaaaaccagctgggacaa
gtggctggtggaaccgtgcgccccggcgtaa
145 atgaaaaacacaacattaaaaacggctcttgctttcgctggcgttgctgccaggcctggcgatggcggctcccgctgtggcggataaagccgacaacg
gctttatgatgatttgcaccgcgctggtgctgtttatgaccattccgggcattgcgagttctacggcggtttgatccgcggtaaaaacgtgctgtcgatgc
tgacgcaggttgccgtcaccttcgctctggtgtgcatcctgtgggtggtttacggctactctctggcatttggcgagggcaacagcttcttcggcagtttc
aactgggcgatgttgaaaaacatcgaattgaaagccgtgatgggcagcatttatcagtacatccacgtggcgttccagggctcctttgcttgtatcaccg
ttggcctgattgtcggtgcgctggctgagcgtattcgcttctctgcggtgctgatttttgtggtggtatggctgacgctttcttatgtgccgattgcgcacat
ggtctggggtggcggtctgctggcaacccacggcgcgctggatttcgcgcgcggtacggttgttcacatcaacgccgcgatcgcaggtctggtggg
ggcttacctgattggcaaacgcgtgggctttggcaaagaagcgttcaaaccgcataacctgccgatggtcttcaccggcaccgcgatcctctatgttgg
ctggtttggcttcaacgccggctctgcaagctcggcgaacgaaatcgctgcgctggctttcgtgaacacggttgttgccactgcggccgctattctggc
gtgggtatttggcgagtgggcaatgcgcggtaagccgtctctgctcggtgcctgttctggtgccatcgcgggtctggttggtatcaccctcggcgtgcg
gttatgtgggtgtcggcggcgcgctgattgtgggtctgattgccggtctggcagggctgtggggcgttactgcactgaaacgtatgttggtgttgatga
cccatgcgatgtcttcggtgtgcacggcgtgtgcggcatcgtgggttgtatcctgaccggtatcttcgcgtctacgtcgctgggcggtgtcggtttcgct
gaaggggtgaccatgggccatcaggtactggtacagctggaaagcgttgccatcactatcgtgtggtctggcgtggtggcctttatcggttacaaactg
gcggatatgacggtaggcctgcgcgtaccggaagagcaagagcgtgaagggctggatgtgaacagccacggcgaaaatgcgtataacgcctga
146 ttcttggttctctggagcgctttatcggcatcctgactgaagaatttgcaggcttcttcccaacctggcttcacccgtgcaggtagttgtgatgaacatca
ctgattcgcaggctgaatacgttaacgaattgacccgtaaactgcaaaatgcgggcattcgtgtaaaagcagacttgagaaacgagaagattggcttta
aaatccgcgagcacactttacgtcgtgtcccttatatgctggtttgtggtgacaaagaggtcgaagccggcaaagttgctgtgcgtacccgtcgcggta
aagacctgggtagcctggacgtaaatgatgttatcgagaagctgcaacaagagattcgcagccgcagtcttcaacaactggaggaataaggtattaaa
ggcggaaaacgagttcaaacggcgcgtcccaatcgtattaatggcgagattcgcgccacggaagttcgcttaacaggtaggaaggcgagcagctt
ggtatt
147 cgttctgtaataataaccggacaattcggactgattaaaaaagcgccctcgcggcgctttttttatattctcgactccatttaaaataaaaaatccaatcgga
tttcactatttaaactggccattatctaagatgaatccgatggaagctcgctgttttaacacgcgttttttaaccattattgaaagtcggtgcttctttgagcga
acgatcaaatttaagtcgattcccatcaaaaaaatattctcaacctaaaaaagtttgtgtaatacttgtaacgctacatggagattaactcaatctagagggt
attaataatgaatcgtactaaactggtactgggcgc
148 cgcgtcaggttgaacgtaaaaaagtcggtctgcgcaaagcacgtcgtcgtccgcagttctccaaacgttaattggtttctgcttcggcagaacgattggc
gaaaaaacccggtgcgaaccgggtttttttatggataaagatcgtgttatccacagcaatccattgattatctcttctttttcagcatttccagaatcccctca
ccacaaagcccgcaaaatctggtaaactatcatccaattttctgcccaaatggctgggattgttcattttttgtttgccttacaacgagagtgacagtacgc
gcgggtagttaactcaacatctgaccggtcgat
149 ttgaagagtttgatcatggctcagattgaacgctggcggcaggcctaacacatgcaagtcgngcggaagcacaggagagcttgctctctgggtgacg
agcggcggacgggtgagtaatgtctgggaaactgcctgatggagggggataactactggaaacggtagctaataccgcataacgtcgcaagaccaa
agagggggaccttcgggcctcttgccatcagatgtgcccagatgggattagctagtaggtggggtaacggcncacctaggcgacgatccctagctg
gtctgagaggatgaccagccacactggaactgagacacggtccagactcctacgggaggcagcagtggggaatattgcacaatgggcgcaagcct
gatgcagccatgccgcgtgtatgaagaaggccttcgggttgtttaagtactttcagcggggaggaaggtgttgnggttaataaccncagcaattgacgt
tacccgcagaagaagcaccggctaactccgtgccagcagccgcggtaatacggaggntgcaagcgttanncggaatnantgggcgtaaagcgtn
cgcaggcggtntgtnaagtcggatgtgaaatccccgggctcaacctgggaactgcattcgaaactggcaggctagagtnnngtagaggngggtag
aattccnggtgtagcggtgaaatgcgtagagatcnggangaanaccngtggcgaaggcggcccnctggacaaagactgacgctnaggngcgaa
agcgggggagcaaacaggattagataccctngtagtccacgccgtaaacgatgtcgacttggaggttgtgcccttgaggcgtggcttccggagcta
acgcgttaagtcgaccgcctggggagtacggccgcaaggttaaaactcaaatgaattgacgggggcccgcacaagcggtggagcatgtggtttaatt
cgatgcaacgcgaagaaccttacctactcttgacatccagagaacttnncagagatgnnttggtgccttcgggaactctgagacaggtgctgcatggc
tgtcgtcagctcgtgttgtgaaatgttgggttaagtcccgcaacgagcgcaacccttatcctttgttgccagcggtncggccgggaactcaaaggagac
tgccagtgataaactggaggaaggtggggatgacgtcaagtcatcatggcccttacgagtagggctacacacgtgctacaatggcgcatacaaagag
aagcgacctcgcgagagcaagcggacctcataaagtgcgtcgtagtccggattggagtctgcaactcgactccatgaagtcggaatcgctagtaatc
gtagatcagaatgctacggtgaatacgttcccgggccttgtacacaccgcccgtcacaccatgggagtgggttgcaaaagaagtaggtagcttaacct
tcgggagggcgcttaccactttgtgattcatgactggggtgaagtcgtaacaaggtaaccgtaggggaacctgcggttggatcacctcctt
150 atgaccatgcgtcaatgtgccatttacggcaaaggtggtatcggtaaatccactaccacgcaaaacctggtcgccgcgctggcggagatgggcaaga
aagtaatgatcgtcggctgcgacccgaaagcagactccactcgtctgatcctgcatgcgaaagcgcagaacaccattatggagatggcggctgaagt
cggctccgtggaagaccttgaactggaagatgtgctgcaaatcggttacggcgacgtacgctgcgcagaatccggcggcccggaaccaggcgttg
gctgtgctggtcgcggggtaattaccgccatcaacttcctggaagaagaaggcgcctatgttcccgacctcgatttcgtcttttacgacgtgttgggcga
cgtggtgtgcggggggttcgccatgccgattcgcgaaaacaaagcgcaggagatctacatcgtctgctccggcgaaatgatggcgatgtacgccgc
caacaacatctctaaaggcatcgtgaaatacgccaaatccggcaaaggcgccttggcgggctgatctgtaactcccgtcagaccgaccgcgaagat
gagctgatcatagcgctggcggaaaaactcggcacccagatgatccacttcgtgccgcgcgacaacatcgtgcaacgcgctgaaatccgccgtatg
acggtgattgagtacgatccgaaatgcaaccaggccaatgaataccgcacgctggcgaacaagatcgtcaacttacaccaaaatggtcgtgccaacg
cccatcaccatggacgaactggaagagctgttgatggaattcggcattatggatgtggaagacaccagcattatcggtaaaaccgccgcagaagaaa
acgcggtttga
151 atgagcaatgcaacaggcgaacgtaataggagatcatccaggaagtgctggagatctttccggaaaaaacgcgcaaagaacgcagaaagcacatg
atggtgagcgacccggagatggaaagcgtcgggaaatgcatcatctccaaccgtaagtcgcagcccggcgtaatgaccgtgcgcggttgctcttac
gccggttctaaaggggtggtattcgggccgatcaaagatatggcccatatttcccacggcccggtcggctgcggtcagtactcccgcgccgggcggc
gtaactactacaccggcgtcagcggtgtggatagcttcggtacgctcaactttacctccgattttcaggagcgcgatatcgtgtttggcggcgataaaaa
gctgaccaaactgattgaagagatggagacgctgttcccgctgaccaaagggatctccattcagtccgaatgcccggtcggcctgattggcgacgac
attgaagccgttgccaacgccagccgcaaagccatcaataaaccggtcattccggtgcgctgcgaaggttttcgcggcgtttcccagtcactcggtca
ccacattgccaacgacgtgatccgcgactgggtactggataaccgcgaaggcaagccgtttgaggccggtccttatgacgtggcgatcatcgccgat
tacaacatcggcggcgatgcctgggcgtcgcgcattttgctcgaagagatgggcctgcgcgtggtggcgcagtggtccggcgacggcacgctggtt
gagatggagaccacgccgttcgtcaaactcaaccttgtgcactgctaccgctcaatgaactatatctcccgccatatggaggagaaacacggtattccg
tggatggagtacaacttcttcggtccgaccaaagtcgccgaatcgttgcgcaaaatcgccgatatgtttgatgacaccattcgcgccaacgccgaagc
ggtgatcgccaaatatcaggcgcagaacgacgccatcatcgccaaataccgtccgcgtctggaaggccgcaaagtgctgctgtatatgggcggttta
cgtcctcgccatgtgattggcgcttatgaagatctgggaatggaaattatcgctgcgggttatgaattcgcccacaacgatgactacgaccgcaccctg
ccggatctgaaagaaggcaccttgctgttcgacgatgccagcagttatgaactggaagcctttgtcaaagcgctgaagccggatctgatcggctccgg
cattaaagagaagtacatatccagaaaatgggcgtgccgtttcgccagatgcactcctgggattactccggcccctatcacggttatgacggctttgcc
atcttcgcccgcgatatggatatgacgatcaacaaccccgcgtggggccagttgaccgcgccgtggctgaaatccgcctga
152 atgggacgcggcgagcgccgcctgttccatgccgtgcgccacatcgtcaaccgctaccacccggccgccgtctttatctataacacctgcgttcccgc
gatggagggcgacgatatcgaagccgtctgccaggcggcagaaaccgccatccgcgtaccggtgattgccgttgatgtcgccgggttttacggcag
caaaaatctcggcaaccggttggccggtgaagtgatggtgaaaaaaggtgattggcgggcgtgaacccgcgccgtggccggaagataccccttttgc
cccggcgcaccgccacgatatcgggctgattggcgaattcaatattgccggagagttctggcatattcagccgctgctcgatgagctgggtattcgcgt
gctcggcagcctctccggcgacgggcgcttcagtgaaatccagacgctgcaccgggcgcaggtcaatatgctggctcctccagggcgctgatcaa
cgtcgcccgctcgctggagcagcgctacggcacgccgtggtttgaaggcagtttttatggtgttcgcgccacctctgacgccctgcgccaactggcg
gcgctgaccggagaccgcgatctgatgcagcgcaccgattcagctcattgcccgcgaagagcagcaaacagagcaggcgctggccccgctgcgc
gagcgcctgcgcgggcgcaaagcgctgctctatnccggcggcgtgaaatcctggacggtggtttcggcgcttcaggatctgggcatggaagtggtg
gcgaccggcacgcgcaaatccaccgaagaggataaacagcgcatccgcgaactgatgggcgccgacgcgctgatgcttgatgaaggtaacgccc
gctcgctgctggacgggtttaccgctacaaggcggacatgatgatcgccgggggacgcaatatgtcaccgcctacaaagcgcggctgccgttcct
cgatatcaatcaggagcgcgagcacgcctttgccggctaccgcggcattgtcaccctggccgaacagctctgcctgaccatggaaagcccggtctg
gccgcaaacccattcccgcgcaccgtggcaataa
153 atgatggagcaaatggacgtgccgtccagcctgctttccgatccctccgaagtgctggataccccggctgacgggcattaccacatgtatgcggggcg
gtacgacccagcaggagatgcgcgaagcgcctgacgctatcgacaccctgctgctgcaaccctggcaactggtgtaaaccaaaaaagtggtgcag
gaaagctggaaccagcccgctaccgaggtgcaaatcccaatggggctggccggaaccgacgagctgctgatgacggtaagccagttaaccggca
aagccattccggatagcttagcgctggaacgcggtcggctggtggatatgatgctcgactcccacacctggctgcacggcaagaaattcggcctgttc
ggtgacccggattttgtcatggggctgacccgcttcctgctggaactgggctgcgaaccgacggtgattctgtgccataacggcagcaagcgctggc
agaaagcgatgaagaaaatgcttgaagcctcgccgtacgcgaaagagagcgaagtctttatcaactgcgatttgtggcatttccgctcgctgatttac
ccgtcagccggactttatgatcggcaactcctacgccaagtttatccaccgcgatacgctggcgaagggtgagcagtttgaagtgccgctgatccgcc
tggggttcccgctgttcgatcgccaccatctgcaccgccagaccacctggggttacgaaggggccatgagtatcctcaccacgctggttaatgcggtg
ctggagaaagtcgacagagagaccatcaagctcggcaaaaccgactacagcttcgatcttatccgttaa
154 atgcagcgcgacatcagcaccagctacgcgctggaacaacggctgcgcaatcatatgacgctgaccgaagccgtcttgaataacattccggcggcg
gttgtagtggtggatgaacgcgatcgggtggtgatggataacctcgcctacaaaaccttttgcgccgattgcggcggtaaagaactactcaccgaaatc
aacttttccgcccataaggcggagctggcgcagggcctggtactgccggtagtgctgcgcggcaccgtgcgctggttgtccgttacctgttgggcgct
gccgggcgtcagcgaagaagcaggccgctactttattgatagcgccgtgccgcgcacgctggtggtgatcaccgataatactcagcagcagcaaca
acaggagcaggggcgtcttgatcgtctgaagcagcagataaccagcggtaaattgctggcggcgatccgcgaatcgctggacgccgcgctggtac
aactcaattgcccaattaatatgctggccgccgcacgccgcttaaatggcgacgagcatagcaatctggcgctggatgccgcatggcgtgaaggcga
agaagcgatggcgcggttgcagcgctgccgcccgtcgctggaactggaaagcccggcagtctggccgctccagccgttccttgacgatctgcgtgc
cctgtatcacacccgatataaccagggcgaaaacctgcaaattgagctggaatcccccgacctggtgggctttggccagcgaacacaactgcttgcct
gcctgagcctgtggctcgacagaaccctggatattgccgcggagctacgtgatttcacggtacagactcaactttacgcccgcgaagagagcggctg
gctgtcgttctatttaaacgacaatgtgccgctgattcaggtgcgctacacccattcacccgatgcactnaatgcgcccggtaaaggcatggagctgcg
gctgatccagacgctggtcgcccaccatcgaggcgcaatagaactgacctcacgccctcagggaggcacctgtctgatcctgcgtttcccattatttta
ctcgctgacaggaggctcactatga
155 atgactcagcgaaccgagtcgggtacaaccgtctggcgctttgacctctcccaacagtttacagccatgcagcgtatcagtgtggtgttaagccgcgc
gacggagatcgggcagacgctacaggaagtgctgtgcgtgctgcacaacgatgcctttatgcagcacgggatgatctgtccgtacgcgcgggtgcg
cgtcttcgcgagcgtatggctttga
156 atgcgcgtggaagactggtcaacgctgaccgaacggctcgatgcccatatggcaggcgtgcgccgaatctttaacgaactgatcggtgatgacgaaa
gtgagtcgcaggacgatgcgctctccgagcactggcgcgagctgtggcaggacgcgcttcaggaagatgacaccacgccggtgagacgcactta
accgacgacgcgcgccatcgcgtggtggcgctgatcgctganttccgtcttgagagaacaaacgcgccatcggcccgcgtngcgccaggtgctg
gatcacctgatgccgcacctgagagcgaagtctgctcgcgtgccgatgcgccggtgccgctgtcgcggatgatgcccctgagaggggattatca
cccgtactacctaccttgaactcctgagcgagttccctggcgcgcttaagcacctgatttcactctgcgccgcgtcgccgatggtggccaacaagctgg
cgcgttacccgctgctgctggatgagctgctcgatccgaataccctttatcaaccgacggcgaccgacgcctaccgggacgaactgcgtcagtatctg
ctgcgcgtgccggaagaagacgaagagcaacagctcggaggcgctgcgtcagtttaagcaggcccagatgctgcgcgtggcggccgcagatattg
ccggaacgctgccggtgatgaaagtgagcgatcacttaacctggcttgcggaagcgattatcgacgcggtggtgcatcaggcctgggtgcagatggt
ggcgcgctatggccagccgaaacatctggctgaccgtgatggtcgcggcttcgctggtggtgggttacggtaagctcggcggttgggagctgggcta
tagctccgatctggatttaatcttcctccacgactgcccggttgatgtgatgaccgacggcgagcgcgagattgacgggcgtcagttctacctgcgcct
ggcgcagcgcatcatgcacctgttcagcacccgcacctcgtcgggcattttgtatgaagtggatgcccgtagcgcccgtccggcgcggcgggcatg
ctggtcacctcgacggagtccttcgctgattaccagaagaatgaagcctggacgtgggagcatcaggcgctggtgcgcgcccggtggtgtatggcg
atccgctgctgaaaacgcagtttgacgtgattcgtaaggaagtcatgaccaccgtgcgcgatggcagcacgctgcaaacggaagtgcgcgaaatgc
gcgagaaaatgcgcgcgcacttaggcaataaacatcgcgatcgctttgatattaaagccgatgagggcggtattaccgatattgagtttattacccagta
tctggtgttgctgcacgcgcatgacaagccgaagctgacgcgctggtcggataacgtgcgcattaggaactgctggcgcaaaacgacattatggac
gagcaggaggcgcaggccttaacccgtgcctatacaacgcttcgcgatgagctccatcatctggcgttgcaggagcagcngggncacgtggcgct
ggactgtttcaccgctgaacgcgctcaggtaacggccagctggcagaagtggctggtggaaccgtgcgtaacaaatcaagtgtga
157 agatgtgcccagatgggattagctagtaggtggggtaacggcncacctaggcgacgatccctagctggtctgagaggatgaccagccacactggaa
ctgagacacggtccagactcctacgggaggcagcagtggggaatattgcacaatgggcgcaagcctgatgcagccatgccgcgtgtatgaagaag
gccttcgggttgtaaagtactttcagcggggaggaaggtgttgtggttaataaccncagcaattgacgttacccgcagaagaagcaccggctaactcc
gtgccagcagccgcggtaatacggagggtgcaagcgttaatcggaattactgggcgtaaagcgcacgcaggcggtctgtcaagtcggatgtgaaat
ccccgggctcaacctgggaactgcattcgaaactggcaggctagagtcttgtagaggggggtagaattccaggtgtagcggtgaaatgcgtagagat
ctggaggaataccggtggcgaaggcggccccctggacaaagactgacgctcaggtgcgaaagcgtggggagcaaacaggattagataccctggt
agtccacgccgtaaacgatgtcgacttggaggttgtgcccttgaggcgtggcttccggagctaacgcgttaagtcgaccgcctggggagtacggccg
caaggttaaacctcaaatgaattgacgggggcccgcacaagcggtggagcatgtggtttaattcgatgcaacgcgaagaaccttacctactcttgacat
ccagagaacttnncagagatgnnttggtgccttcgggaacctgagacaggtctgcatggctgtcgtcagctcgtgttgtgaaatgttgggttaagtc
ccgcaacgagcgcaacccttatcctttgttgccagggtccggccgggaactcaaaggagactgccagtgataaactggaggaaggtggggatgac
gtcaagtcatcatggcccttacgagtagggctacacacgtgctacaatggcgcatacaaagagaagcgacctcgcgagagcaagggacctcataa
agtgcgtcgtagtccggattggagtctgcaactcgactccatgaagtcggaatcgctagtaatcgtagatcagaatgctacggtgaatacgttcccggg
ccttgtacacaccgcccgtcacaccatgggagtgggttgcaaaagaagtaggtagcttaaccttcgggagggcgcttaccactttgtgattcatgactg
gggtgaagtcgtaacaaggtaaccgtaggggaacctgcggttggatcacctcctt
158 atgaccatgcgcaatgtgccatttacggcaaaggtggtatcggtaaatccactaccacgcaaaacctggtcgccgcgctggcggagatgggcaaga
aagtaatgatcgtcggctgcgacccgaaagcagactccactcgtctgatcctgcatgcgaaagcgcagaacaccattatggagatggcggctgaagt
cggctccgtggaagaccttgaactggaagatgtgctgcaaatcggttacggcgacgtacgctgcgcagaatccggcggcccggaaccaggcgttg
gagtgctggtcgcggggtaattaccgccatcaacttcctggaagaagaaggcgcctatgttcccgacctcgatttcgtcttttacgacgtgttgggcga
cgtggtgtgcggggggttcgccatgccgattcgcgaaaacaaagcgcaggagatctacatcgtctgctccggcgaaatgatggcgatgtacgccgc
caacaacatctctaaaggcatcgtgaaatacgccaaatccggcaaagtgcgccttggcgggctgatctgtaactcccgtcagaccgaccgcgaagat
gagctgatcatagcgctggcggaaaaactcggcacccagatgatccacttcgtgccgcgcgacaacatcgtgcaacgcgctgaaatccgccgtatg
acggtgattgagtacgatccgaaatgcaaccaggccaatgaataccgcacgctggcgaacaagatcgtcaacaacaccaaaatggtcgtgccaacg
cccatcaccatggacgaactggaagagctgttgatggaattcggcattatggatgtggaagacaccagcattatcggtaaaaccgccgcagaagaaa
acgcggtttga
159 atgagcaatgcaacaggcgaacgtaatctggagatcatccaggaagtgctggagatctttccggaaaaaacgcgcaaagaacgcagaaagcacatg
atggtgagcgacccggagatggaaagcgtcgggaaatgcatcatctccaaccgtaagtcgcagcccggcgtaatgaccgtgcgcggttgctcttac
gccggttctaaaggggtggtattcgggccgatcaaagatatggcccatatttcccacggcccggtcggctgcggcagtactcccgcgccgggcggc
gtaactactacaccggcgtcagcggtgtggatagcttcggtacgctcaactttacctccgattttcaggagcgcgatatcgtgtttggcggcgataaaaa
gctgaccaaactgattgaagtgatggagacgctgttcccgctgaccaaagggatctccattcagtccgaatgcccggtcggcctgattggcgacgac
attgaagccgttgccaacgccagccgcaaagccatcaataaaccggtcattccggtgcgctgcgaaggttttcgcggcgtttcccagtcactcggtca
ccacattgccaacgacgtgatccgcgactgggtactggataaccgcgaaggcaagccgtttgaggccggtccttatgacgtggcgatcatcggcgat
tacaacatcggcggcgatgcctgggcgtcgcgcattttgctcgaagagatgggcctgcgcgtggtggcgcagtggtccggcgacggcacgctggtt
gagatggagaacacgccgttcgtcaaactcaaccttgtgcactgctaccgctcaatgaactatatctcccgccatatggaggagaaacacggtattccg
tggatggagtacaacttcttcggtccgaccaaagtcgccgaatcgttgcgcaaaatcgccgatatgtttgatgacaccattcgcgccaacgccgaagc
ggtgatcgccaaatatcaggcgcagaacgacgccatcatcgccaaataccgtccgcgtctggaaggccgcaaagtgctgctgtatatgggcggttta
cgtcctcgccatgtgattggcgcttatgaagatctggggatggaaattatcgctgcgggttatgaattcgcccacaacgatgactacgaccgcaccctg
ccggatctgaaagaaggcaccttgctgttcgacgatgccagcagttatgaactggaagcctttgtcaaaggctgaagccggatctgatcggctccgg
cattaaagagaagtacatcttccagaaaatgggcgtgccgtttcgccagatgcactcctgggattactccggcccctatcacggttatgacggctttgcc
atcttcgcccgcgatatggatatgacgatcaacaaccccgcgtggggccagttgaccgcgccgtggctgaaatccgcctga
160 atgaaggggaacgagatcctggctttgctcgatgaacctgcctgcgagcacaaccataaacagaaatccggctgcagcgcgccgaaacccggcgc
gacagcgggcggctgcgcctttgacggtgcgcagatcaccctgctgccactctccgatgttgcccacctggtacacggccccattggttgtaccggta
gctcatgggataaccgtggcagcttcagttccggcccgacgatcaaccggctgggttaccaccgatctgagcgaacaggatgtgatcatgggacg
cggcgagcgccgcctgttccatgccgtgcgccacatcgtcaaccgctaccacccggccgccgtctttatctataacacctgcgttcccgcgatggagg
gcgacgatatcgaagccgtctgccaggcggcagaaaccgccatcggcgtaccggtgattgccgttgatgtcgccgggttttacggcagcaaaaatct
cggcaccggttggccggtgaagtgatggtgaaaaaggtgattggcgggcgtgaacccgcgccgtggccggaagataccccttttgccccggcgc
accgccacgatatcgggctgattggcgaattcaatattgccggagagttctggcatattcagccgctgctcgatgagctgggtattcgcgtgctcggca
gcctctccggcgacgggcgcttcagtgaaatccagacgctgcaccgggcgcaggtcaatatgctggtctgctccagggcgctgatcaacgtcgccc
gctcgctggagcagcgctacggcacgccgtggtttgaaggcagtttttatggtgttcgcgccacctctgacgccctgcgccaactggcggcgctgac
cggagaccgcgatctgatgcagcgcaccgaacagctcattgcccgcgaagagcagcaaacagagcaggcgctggccccgctgcgcgagcgcct
gcggggcgcaaagcgctgctctataccggcggcgtgaaatcaggtcggggtttcggcgcttcaggatctgggcatggaagtggtggcgaccgg
cacgcgcaaatccaccgaagaggataaacagcgcatccgcgaactgatgggcgccgacgcgctgatgcttgatgaaggtaacgcccgctcgctgc
tggacgtggtttaccgctacaaggcggacatgatgatcgccgggggacgcaatatgtacaccgcctacaaagcgcggctgccgtcctcgatatcaat
caggagcgcgagcacgcctttgccggctaccgcggcattgtcaccctggccgaacagctctgcctgaccatggaaagcccggtctcgccgcaaac
ccattcccgcgcaccgtggcaataa
161 atgagccaaagtgctgagaaaattcaaaactgtcatccgctgtttgaacaggatgcgtaccagatgctgataaagataaacggcaactggaagaggc
ccacgatccggcgcgcgtgcaggaggtctttcaatggaccaccaccgccgagtatgaagcgcttaactttcaacgcgaagcgctgactatcgatccg
gccaaagcctgccagcgctgggtgcggtactgtgctcgctgggctttgccaataccctgccctatgttcacgactcccaggggtgcgtggcctatttc
cgcacctattttaaccgtcactttaaagagccgattgcctgtgtttctgactcgatgacggaagatgcggcagtattcggcggcaacaacaacctgaaca
ccgggttgcagaacgccaggccctctacaagccggaaatcattgccgtctccaccacctgtatggcggaggtcatcggcgacgacctgcaggcgt
ttattgctaacgccaaagaagacggctttatcgacgcggcgatcccggtgccttacgcgcacacgccaagctttatcggcagccatatcaccggctgg
gacaatatgtttgagggcttcgcccgtacctttaccgccgattacagcggacaaccgggcaaattaccgcgtatcaatctggtcagcggatttgaaacct
atctcggtaatttccgcgtgctgaaacgcatgatggagcaaatggacgtgccgtgcagcctgctttccgatccctccgaagtgctggataccccggctg
acgggcattaccacatgtatgcgggcggtacgacccagcaggagatgcgcgaagcgcctgacgctatcgacaccctgctgctgcaaccctggcaa
ctggttgaaaaccaaaaaaagtggtgcaggaaagctggaaccagcccgctaccgaggtgataatcccaatggggctggccggaaccgacgagagc
tgatgacggtaagccagttaaccggcaaagccattccggatagcttagcgaggaacgcggtcggctggtggatatgatgctcgactcccacacctg
gctgcacggcaagaaattcggcctgttcggtgacccggattttgtcatggggctgacccgcttcctgctggaactgggctgcgaaccgacggtgattct
gtgccatagcggcagcaagcgaggcagaaagcgatgaagaaaatgcttgaagcctcgccgtacgggaaagagagcgaagtctttatcaaagcga
tttgtggcatttccgctcgctgatgtttacccgtcagccggactttatgatcggcaactcctacgccaagtttatccagcgcgatacgctggcgaagggtg
agcagtttgaagtgccgctgatccgcctggggttcccgctgttcgatcgccaccatctgcaccgccagaccacctggggttacgaaggggccatgag
tatcctcaccacgctggttaatgcggtgctggagaaagtcgacagagagaccatcaagctcggcaaaaccgactacagcttcgatcttatccgttaa
162 atgccagaaattatccgtagttaaaaagccgctggccgtcagcccggtaaaaagtggccagccgctgggcgcgattctggcgagcatgggctttgaa
cagagcattccgctggttcatggcgctcacgggtgcagcgccttcgcgaaggtcttttttatccagcattttcacgatccgatcccgctgcaatcgacgg
caatggacccgacatcgaccattatgggtgccgatgagaacatctttaccgcgctgaatgtgctgtgttcacgcaacaacccgaaagcgattgttctgct
gagcactggcctttccgaggcgcagggaagcgatatttcgcgcgtggtgcgccagttccgcgatgaatatccgcgccataaaggggtggcgctgct
gaccgtcaacacgccggatttttacggcagcctggaaaacggctacagcgcggtgctggagagcatggttgaacagtgggtgccggaaaaaccgc
agccgggcgtgcgcaatcgccgcgtgaacctgctgctcagccatttgcttacgccgggcgacattgagctgctgcgaagttatgtcgaggcatttggc
ctgcagccggtgatggtccggatctttcccagtcgctggatggccatctcgccagcggggatttctcgccaattacccagggcggcagcagcctgc
ggctgattgaacagatgggacagagtcttggcacgttcgccattggcgtatccctctcccgcgccgcgcaattgctggcgcagcgcagccatgcgga
agtggtcaccctgccgcatctgatgaccatgagccagtgcgatacgtttattcatcaactgaagcgcctctccgggcgcgatgttccggcgtggatcga
acgccagcgcgggcaactgcaggatgcgatgatcgattgtcatatgtggttgcagggcgcgcctgtcgcgctggccgccgagggcgatctgctcgc
cgcctggtgcgatttcgcctgcgatatgggcatggtgcccggcccggtggtggcgccggtgagccagaaagggttgcaggatctgccggtcgaaaa
agtcattatcggcgatctggaggatatgcaggatctgttgtgtgaaacgcctgcatcgctgctcgtctctaattctcacgccgctgatttggccgggcagt
tcgacattccgctggtgcgcgccggtttccccctgttcgaccgtctgggcgagtttcgccgcgtgcgccagggttacgccgggatgcgcgacaccttg
tttgagctggcgaatgccctgcgcgatcgccatcatcatcttgccgcttatcactcgccgctgcgccagcgtttttacgaacccgcatcttcgggaggtg
actatgcaacatgttaa
163 atgaccctgaatatgatgatggacgccaccgcgcccgccgagatcgccggagcgctctcacaacagcatcccggattgtttttcaccatggttgaaca
ggcgcccgtcgcgatttcactgaccgatgccgatgcccacattctctacgccaaccccgcgttttgtcgccagtcggggtatgaactggaagagttgtt
gcagcaaaacccgcgcctgcttgccagtaagaagacgccgcgtgaaatctaccaggaaatgtggcacaccctgctgcaacaccgtccgtggcgcg
gacaactgatcaaccgtcgccgcgacggcagcctgtttctggtggaaatcgacatcaccccactgtttgatgcgttcggcaaactcgaacattacctgg
ccatgagcgcgacatcagcaccagctacgcgctggaacaacggctgcgcaatcatatgacgctgaccgaagccgtcttgaataacattccggcgg
cggttgtagtggtggatgaacgcgatcgggtggtgatggataacctcgcctacaaaaccttttgcgccgattgcggcggtaaagaactactcaccgaa
atcaacttttccgcccataaggcggagctggcgcagggcctggtactgccggtagtgctgcgggcaccgtgcgctggttgtccgttacctgttgggc
gctgccgggcgtcagcgaagaagaggccgctactttattgatagcgccgtgccgcgcacgctggtggtgatcaccgataatactcagcagcagca
acaacaggagcaggggcgtcttgatcgtctgaagcagagataaccagcggtaaattgctggcggcgatccgcgaatcgctggacgccgcgctgg
tacaactcaattgcccaattaatatgctggccgccgcacgccgcttaaatggcgacgagcatagcaatctggcgctggatgccgcatggcgtgaagg
cgaagaagcgatggcgcggttgcagcgctgccgcccgtcgctggaactggaaagcccggcagtctggccgctccagccgttccttgacgatctgc
gtgccctgtatcacacccgatataaccagggcgaaaacctgcaaattgagctggaatcccccgacctggtgggctttggccagcgaacacaactgctt
gcctgcctgagcctgtggctcgacagaaccctggatattgccgcggagctacgtgatttcacggtacagactcaactttacgcccgcgaagagagcg
gctggctgtcgttctatttaaacgacaatgtgccgctgattcaggtgcgctacacccattcacccgatgcactcaatgcgcccggtaaaggcatggagc
tgcggctgatccagacgctggtcgcccaccatcgaggcgcaatagaactgacctcacgccctcagggaggcacctgtctgatcctgcgtttcccatta
ttttactcgctgacaggaggctcactatga
164 atgactcagcgaaccgagtcggggacaaccgtctggcgctttgacctctcccaacagtttacagccatgcagcgtatcagtgtggtgttaaaccgcgc
gacggagatcgggcagacgctacaggaagtgctgtgcgtgctgcacaacgatgcctttatgcagcacgggatgatctgtctgtacgacagtaagcaa
gcgatcctttccattgaagccttgcatgaggccgatcagagttaattcccggcagttcacagattcgctaccgtccgggcgaagggctggtaggcac
ggtgctttcacagggacagtcgctggtactgccctgtgtctccgacgatcggcgttttctcgatcgcctgggattgtatgattacagcttgccgtttatcgc
cgtgccgctgatggggccaaactcgcagcctatcggcgtgctggccgcccagcctatggcgcgttacgaggagggctgcccgcctgcacgcgttt
tcttgaaaccgtcgccaatctggtggcgcaaaccgttcgcctgatgacaccgcccagcgtcgcgtctccaccccgtgctgctgccgcgcagattgcca
gccagcgcgggtgcgcgtcttcgcgagcgtatggctttgaaaacatggtcggtaaaagcgcggctatgcgtcagacgctggaaattattcgccaggt
atcacgctgggacaccaccgtgctggtgcgtggcgaaagcggaaccggtaaagagttgatagccaacgctatccaccacaattcaccgcgcgccg
ccgcgccgtttgtcaaattcaactgcgcggcgctgcccgatacgctgaggagagtgaactcttcggtcatgaaaaaggcgcgtttaccggcgcggtg
cgccagcgcaaaggccgtttcgaactggcggatggcggtacgctgtttcttgatgagatcggcgaaagtagcgcctcgtttcaggcgaaattgctgcg
tatcttgcaggaaggcgaaatggaacgcgtcggcggcgacgaaacgctgcgggtgaatgtacggatcattgccgccaccaaccgcaatctggaag
aggaagtgcggctgggtaattttcgcgaagatctctactatcgccttaatgtgatgccgatctccctgcccccgctccgcgagcgtcaggaggacatcg
tcgagctggcgcattttctggtgcgcaaaatcgcgcaaaaccagaaccgcacgctgcgcatcagcgatggcgcgatccgtttgttgatgagctatagc
tggcctggaaacgtgcgtgagctggaaaactgccttgagcgatcggcggtgatgtcggaaaacgggctgatcgatcgcgacgtgattttgtttcacca
cagggaaaatctgcaaaaacgccacagaccagtgcgccgcgcgaagagagctggctcgatcagaacctcgatgaggacaaagattgatcgcc
gcgctggagaaagccggttgggtacaggcaaaagccgcgcgcctgagggaatgaccccgcgccaggtggcctatcgtattcagacgatggacat
tgccatgccgagattgtag
165 atgccgctttcttcgcaggttacagcagcagtggcagaccgtttgcgaacgtctgcctgagtcattaccggcgtcatcgttaagcgagcaggcaaagag
cgtgctcgtcttcagtgattttgtgcaggaaagtatcaccgccaacccgaactggctggcggaacttgagaacgcaccaccgcaggcagaagagtgg
cggcactatgctggctggctgcaaactgtactcgaagacgttacggatgaggccacgctgatgcgcgtcctgcgccagttccgtcgtcggctgatggt
ggcattgcctgggctcaggcgctggaactggtgagcgaagagagtacgctgcagcagttaagcgagctggcgcaaacgttgattgtcgccgcgc
gagactggctctatgcctgctgtaaagagtggggcacgccgtgcagcgaggaaggggttcctcagccgctgttgattctgggcatgggaaagc
tgggcggctgcgagctgaacttctcctctgatatcgacctgatttttgcctggccggagaacggctccacgcgcggaggccgccgcgagctgaacaa
cgcgcagttctttacccgtctcggccagcgcctgattaaagcgctggatcagcccacgcaggacggttttgtttaccgcgtggacatgcgcctgcgtcc
gtttggcgacagcgggccgctggtgctgagctttgcggcgctggaagattattaccaggagcaaggtcgcgactgggagcgttacgcgatggtcaa
agcgcggatcatgggcaacagcgacgacgcttatgccaacgagctgcgcgccatgctgcgtccgttcgtgttccgtcgctatatcgacttcagcgtca
tccagtccctgcgaaatatgaaagggatgattgcccgcgaggtgcgccgccgtgggctgaaagacaatatcaagctcggtgcgggcggcatccgc
gaaatcgaatttatcgtccaggtcttccagcttattcgcggcggacgcgagccgtcgctgcagtcccgttccttattaccgacgctgagcgccattgcgc
agctgcatctcctgccggacggcgacgcgcaaaccctgcgcgaggcctatcttttcctgcgtcgtctggaaaacctgctgcaaagcattaatgacgaa
cagacccaaaccctgccgggcgacgaccttaaccgggcgcgtctggcctggggaatgcgcgtggaagactggtcaacgctgaccgaacggctcg
atgcccatatggcaggcgtgcgccgaatctttaacgaactgatcggtgatgacgaaagtgagtcgcaggacgatgcgctctccgagcactggcgcg
agctgtggcaggacgcgcttcaggaagatgacaccacgccggtgctgacgcacttaaccgacgacgcgcgccatcgcgtggtggcgctgatcgct
gatttccgtcttgagctgaacaaacgcgccatcggcccgcgtggtcgccaggtgctggatcacctgatgccgcacctgctgagcgaagtctgctcgc
gtgccgatgcgccggtgccgctgtcgcggatgatgcccctgctgaggggattatcacccgtactacctaccttgaactcctgagcgagttccctggc
gcgcttaagcacctgatttcactctgcgccgcgtcgccgatggtggccaacaagctggcgcgttacccgctgctgctggatgagctgctcgatccgaa
taccctttatcaaccgacggcgaccgacgcctaccgggacgaactgcgtcagtatctgctgcgcgtgccggaagaagacgaagagcaacagctgg
aggcgctgcgtcagtttaagcaggcccagatgctgcgcgtggcggccgcagatattgccggaacgctgccggtgatgaaagtgagcgatcacttaa
cctggcttgcggaagcgattatcgacgcggtggtgcatcaggcctgggtgcagatggtggcgcgctatggccagccgaaacatctggctgaccgtg
atggtcgcggcttcgcggtggtgggttacggtaagctcggcggttgggagctgggctatagctccgatctggatttaatcttcctccacgactgcccgg
ttgatgtgatgaccgacggcgagcgcgagattgacgggcgtcagttctacctgcgcctggcgcagcgcatcatgcacctgttcagcacccgcacctc
gtcgggcattgtatgaagtggatgcccgtctgcgcccgtccggcgcggcgggcatgctggtcacctcgacggagtccttcgctgattaccagaaga
atgaagcctggacgtgggtgcatcaggcgctggtgcgcgcccgtgtggtgtatggcgatccgctgctgaaacgcagtttgacgtgattcgtaagga
agtcatgaccaccgtgcgcgatggcagcacgctgaaacggaaggcgcgaaatgcgcgagaaaatgcgcgcgcacttaggcaataaacatcgc
gatcgctttgatattaaagccgatgagggcggtattaccgatattgagtttattacccagtatctggtgttgctgcacgcgcatgacaagccgaagctgac
gcgctggtcggataacgtgcgcattctggaactgctggcgcaaaacgacattatggacgagcaggaggcgcaggccttaacccgtgcctatacaac
gcttcgcgatgagctccatcatctggcgttgcaggagcagccgggacacgtggcgctggactgtttcaccgctgaacgcgctcaggtaacggccag
ctggcagaagtggctggtggaaccgtgcgtaacaaatcaagtgtga
166 atgaagatagcaacacttaaaacgggtctgggttcgctggcactgctgccgggcctggcgctggctgctgcacctgcggtggcagacaaagccgat
aacgcctttatgatgatcagcaccgcgctggtgctgttcatgtccattccgggcattgcgctgttctatggcggcctgatccgtggcaaaaacgttctctc
catgctgacgcaggttgccgtaacgttcgcgctggtctgcgtactgtgggtggtttacggttactcgctggctttcggcacgggcaacgcgttctttggta
acttcgactgggtgatgctgaaaaatattgaactgaccgcgctgatgggcagtttctaccagtatattcacgttgctttccagggctcgttcgcctgcatta
ccgtcgggctgattgtaggcgcgcttgccgagcgtattcgtttctctgcggtcctgatcttcgtggtggtctggctgacgctctcctatgtgccgattgcg
cacatggtctggggtggcggctgctggcgacgcatggcgcgctggacttcgcgggcggtaccgttgtgcacattaacgccgcatagcgggtctg
gttggcgcatacctgattggcaaacgcgtgggcttcggtaaagaagcgttcaaaccgcacaacctgccgatggtcttcaccggtaccgcgatcctcta
ctttggctggtttggtttcaacgccggctcagcaagtgccgcgaacgaaatcgccgcgctggccttcgtgaataccgttgtggccacggcaggtgcaa
tcctctcctgggtctttggcgagtgcgctgtgcgcggtaaaccttctctgctgggtgcctgttcgggggcgattgctggtctggtcggtatcaccccagc
atgtggttatgtcggtgtgggtggcgcgctgctggtcggcctggtgtcaggtctggcgggtctgtggggcgtgacggcgctgaaacgtattctgcgcg
ttgatgacccttgcgatgtgtttggcgtgcacggcgtgtgcggcatcgtcggctgtatcatgaccggtatctttgcagcgaaatcgctgggtggcgtgg
gctacgcagaaggcgtcaccatggcccatcaggtgctggtgcagctggaaagtattgctgtcaccgtggtgtggtctgccgttgtcgctttcattggcta
caaactggcggacatgacggttggtctgcgcgtgccggaagagcaggaacgcgaaggtctggacgtcaacagccacggcgagaatgcgtataac
gcatga
167 gccgagagaggggcccgcgtcggattaggtagttggtgaggtaatggctcaccaagccttcgatccgtagctggtctgagaggatgatcagccaca
ctgggactgagacacggcccagactcctacgggaggcagcagtggggaatattggacaatgggcgcaagcctgatccagcaatgccgcgtgagtg
atgaaggccttagggttgtaaagctctttcgcacgcgacgatgatgacggtagcgtgagaagaagccccggctaacttcgtgccagcagccgcggta
atacgnagggagcnagcgttnntcggaattactgggcgtaaagngcgcgtaggcggcntgttnagtcagaagtgaaagccccgggctcaacctgg
gaatagcttttgatactggcaggcttgagttccggagaggatggtggaattcccngtgtagnggtgaaatncgtagatattgggangaacaccngtgg
cgaaggcggcnatctggacgganactgacgctgaggcgcgaaagcgtggggagcaaacaggattagataccctngtagtccacgccgtaaacga
tgaatgctagacgtcggggtgcatgacttcggtgtcgccgctaacgcattaagcattccgcctggggagtacggccgcaaggttaaaactcaaagg
aattgacgggggcccgcacaagcggtggagcatgtggtttaattcgaagcaacgcgcagaaccttaccaacccttgacatgtccactttgggctcgag
agatngggtccttcagttcggctgggtggaacacaggtgctgcatggctgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgc
aacccctaccgtcagttgccatcattcagttgggcactctggtggaaccgccggtgacaagccggaggaaggcggggatgacgtcaagtcctcatg
gcccttatgggttgggctacacacgtgctacaatggcggtgacagtgggaagcgaagtcgcgagatggagcaaatccccaaaagccgtctcagttc
ggatcgcactctgcaactcgagtgcgtgaagttggcaatcgctagtaatcgcggatcagcacgccgcggtgaatacgttcccgggccttgtacacacc
gcccgtcacaccatgggagttggttttacccgaaggtggtgcgctaaccgcaaggaggcagccaaccacggtaaggtcagcgactggggtgaagt
cgtaacaaggtagccgtagggcaacctgcggctggatcacctccttt
168 atggccaaagcgcctctgcgtcagatcgccttttacggcaagggcggtatcggcaagtccaccacctctcagaacacgctggccgcgctggtcgag
ctggatcagaggatcctgatcgtcggctgcgacccgaaggccgactcgacccgcctgatcctgcacgcaaaggcccaggacaccgtcctgcatctg
gccgccgaggccggctcggtcgaggatctggagctcgaggacgttctcaagatcggctacaagaacatcaagtgcgtcgagtccggcggtccgga
gccgggggtcggctgcgccggccgcggcgtcatcacctcgatcaacttcctggaagagaacggcgcctacgacgacgtggactatgtgtcctacga
cgtgctgggcgacgtggtctgcggcggcttctccatgccgatccgcgagaacaaggcccaggaaatctacatcgtcatgccggcgagatgatggc
gctgtacgccgccaacaacatcgccaagggcatcctgaagtacgcgcacagcggcggcgtccgtctcggcggcctgatagcaacgagcgccag
accgacaaggaatgggatctggccgacgcgctggccaaccgcctgggctccaagctgatccacttcgtgccgcgcgacaacatcgtccagcacgc
cgagagcgccgcatgacgctcatcgagtacgccccggacagcaagcaggccggcgaataccgcgcgctcgccaacaagatccatgcgaactcc
ggccagggttgcatcccgaccccgatcaccatggaagagctggaagagatgctgatggacttcggcatcatgaagaccgaggagcagcagctcgc
cgagctcgccgccaaggaagcggcgaaggccggcgcctga
169 atgagcctgtccgagaacaccacggtcgacgtcaagaacctcgtcaacgaagtcctcgaagcctatcccgaaaaatcccgcaagcgccgcgccaa
gcacctgaacgtgctggaggccgaggccaaggaagggcgtcaagtcgaacgtcaagtccatccccggcgtcatgaccatccgcggctgcgcct
atgccggctccaagggcgtggtgtggggtccgatcaaggacatgatccacatctcccacggtccggtcggctgcggctactactcaggtccggccg
ccgcaactactacatcggcgacaccggtgtggacagctggggcacgatgcacttcacctccgacttccaggagaaggacatcgtcttcggcggcga
caagaagctgcacaaggtcatcgaggaaatcaacgagctgttcccgctggtgaacggcatctcgatccagtcggaatgcccgatcggcctgatcgg
cgacgacatcgaggctgtcgcccgcgccaagtcggcggaaatcggcaagccggtcatccccgtgcgctgcgaaggcttccgcggcgtgtcccagt
cgctgggccaccacatcgccaacgacgccatccgagactgggtgttcgagaagacggaacccaaggccggcttcgtctccaccccctatgacgtca
ccatcatcggcgactacaacatcggcggcgacgcaggtcgtcccgcatcctgctggaggagatcggcctgcgcgtgatcgcccagtggtcgggc
gacggcacgctcgccgaactggagaacacgccgaaggccaaggtcaacctgatccactgctaccgctcgatgaactacatcgcgcgccacatgga
agagaagttcaacattccttggatggaatacaacttcttcggcccgagccagatcgccgaatccctgcgcaagatcgccgctctcttcgacgacaagat
caaggagaacgccgagaaggtcatcgcccgctaccagccgatggtcgatgcggtcatcgccaagtacaagccgcggctcgaaggcaagaaggtc
atgatctacgtcggcggcctgcgtccccgccacgtcgtcgatgcctaccatgacctcggcatggagatcaccggcaccggctacgagttcgcccaca
acgacgactatcagcgcacgcagcactacgtgaaggaaggcacgctgatctacgacgacgtcaccgcgttcgaactggagaagttcgtcgaggcg
atgcgtcccgacctcgtcgcgtcgggcatcaaggaaaagtacgtgttccagaagatgggcctgccgttccgccagatgcacagctgggactactccg
gcccgtaccacggctatgacggcttcgcgatcttcgcccgcgacatggacctggccatcaacaaccccgtctggggcgtgatgaaggccccgttctg
a
170 atgctccaggacaagatccaggatgtcttcaacgaaccgggctgcgcgaccaaccaagccaaatcggccaaggagaagaagaagggctgcacca
agtcgctgaaaccgggggcggcagccggcggctgagcctatgacggggcgatgatcgtgctccagccgatcgccgacgccgcccatctggtccat
ggccccatcgcctgcctcggaaacagttgggacaaccgcggctccaaatcctccggctcgcagctctaccgcaccggcttcaccaccgatctgtcgg
aactggacgtcatcggcggcggcgagaagaagctctaccgcgccatcaaggagatcgttcagcaatacgacccgccggccgtcttcgtctatcaga
cctgcgtgcccgccatgaccggcgacgacatcgccgcattgcaagttcgccacgcagaagctgagcaagccggtgatcacggtggactcgcc
gggcttcgtcgggtcgaagaatctcggcaacaagctggccggcgaagccctgctggagcatgtcatcggcacggtcgaaccggactacaccaccc
cgaccgacgtctgcatcatcggcgaatacaaccttgccggcgagctgtggctggtcaagccgctgctggacgagatcggcatccgcctcctgtcctg
catttccggcgacggccgctaccgggaggtggcgcaggcccaccgcgcccgcgtcaccatgatggtgtgcagccaggcgctggtgaatgtcggg
cgcaagatggaggagcgctacggcatcctctatttcgaggggtccttctacggcgtgtccgacatgtcggacaccctgcgcaccatgacccgcatgc
tggtggagcgggcgccgacaagggcctgatcgaccgggcggagggcgtgatcgcgcgggaggaaagccgggtctggcgccggctggaaccc
tacaagccgcgcttcgacggcaagcgcgtccttctcttcaccggcggcgtcaagagctggtcgatggtcagcgcgctggagggtgcggggctgacc
atcctcggcacctccaccaagaaatcgaccagggaggacaaggagcgcatcaagaagatgaagggcgaagagttccaccagtgggacgatttgaa
gccgcgcgacatctacaggatgctggccgacgatcaggccgacatcatgatgtccggcggccgctcgcagttcatctcgctgaaggccaaggttcc
ctggctcgacatcaaccaggagcgccaccacgcctatgccggctatgacggcatcgtcaatctctgcgaggagatcgacaaaacgctgtcgaatcc
gatctggcgtcaggtgcgtcagccggcaccgtgggagtccggcgcgtcctccacccttctggcttcctcgatggcggcggagtga
171 atgtcccacatccagcgcttcccctccgccgccaaggccgcctccaccaacccgctgaagatgagccagccgctgggtgcggctctggcctatctcg
gcgtcgaccgctgcctgccgctgttccatggctcgcagggctgcaccgccttcgggctggtcctgctggtgcgccatttccgcgaggcgatcccgct
ccagaccacggcgatggatcaggtcgccaccatcctcggcggctacgacaatctggagcaggcgatccgcaccatcgtcgagcgcaaccagccc
gccatgatcgccgtcgccaccaccggcgtcaccgagaccaagggcgaggatatggccggacagtacacgctgttccgccagcgcaaccccgactt
ggccgacacggccctggtcttcgccaacacccccgacttcgccggcggcttcgaggacggcttcgccgccgcggtcaccgcgatggtcgagcggt
tggtcgaaccgtcgccggtgcgcatcccgacccaggtcaacgtgctggccggctgccatctgtcccccggcgacgtggaggaactgcgcgacatc
atcgaaggcttcggcctgtcgccgatcttcctgaccgacctgtcgctgtcgatggcgggccgccagccggccgacttcaccgccacctcgctgggcg
gcgtgaccgtcgatcagatccgcgccatgggcgcttcggccctcaccatcgtggtcggtgagcatatgcgggtggccggtaacgcgctggagctga
agaccgacgtgcccagccatttatcaaccgcctgaccgggctggaggcgacggacaagctggtccggctgctgatggagttgtcgggcaagccg
gcgcccgcccggctgcggcgccagcgcgaaagcctggtcgatgccatgctcgacgggcatttcttctacagccgcaagcgcatcgccgtcgcgct
ggagcccgacctgctctatgccgtcaccggcttcctcgccgacatgggggccgaggtgatcgccgcggtgtccccgacgcagagcccggtgctgg
agcggttgaaggccgccaccatcatggtcggcgatcattccgacgtggagacgctggcccgcgacgccgacctgatcgctccaactcgcacggg
cggcagggagccgcgcggatcggcgtggctctgcaccgcatgggcctgccgctgttcgaccggctgggggccggcctgcgcgtccaggtcggct
accgaggcacgcgggaactgctgtgcgacatcggcaacctgttcctcgcccgcgagatggaccacgagcacgggcacgagagccacgaccacg
gggaatcccacggctgccgaggcggatcatgcggatgcaacgccgtctga
172 atgaccgacaagctttcgcagagcgccgacaaggtcctcgaccactacaccctcttccggcagcccgaatacgcggcgatgttcgagaagaagaag
accgagttcgagtacggccattcggacgaggaagtcgcccgcgtgtccgaatggaccaagtccgaggactacaaggcgaagaacttcgcccgtga
agcggtcgtcatcaacccgaccaaggcctgccagccgatcggcgcaatgttcgccgcccagggcttcgaaggcaccctgcccttcgtccacggctc
ccagggctgcgtcgcctattaccgcacccacctgacccgtcacttcaaggagccgaacagcgcggtctcctcgtcgagacggaggacgcggcggt
gttcggcggcctgaacaacatgatcgacggcctggcgaacgcctatgcgctctacaagccgaagatgatcgaggtgatgaccacctgcatggccga
agtcatcggcgacgatttgcagggcttcatcgccaatgcgaagaccaaggacaggtcccggccgacttcccggtcccctacgcccacaccccggc
cttcgtcggcagccacatcgtcggctacgacaacatgatcaaggggatcctgaccaacttctggggtacgtcggagaatttcgacacacccaagacc
gagcagatcaacctgatcccgggattcgacggcttcgccgtcggcaacaaccgcgaactgaagcgcatcgccggcgaattcggcgtgaagctgca
aatcctgtccgacgtgtccgacaatttcgacacgccgatgaatggcgagtaccgcatgtatgacggcggcaccaccatcgaggagaccaaggaggc
cctgcacgccaaggccaccatctccatgcaggagtacaacacgacccagaccctgcaattctgcaaggagaagggtcaggaagtcgccaagttcaa
ctacccgatgggcgtcaccggcaccgacgagctgctgctgaagctcgccgaactgtcgggcaagccggtcccggccagcctgaagctggagcgc
ggccgtctggtcgacgccatcgccgacagccacacccacatgcacggcaagcgcttcgccgtctatggcgacccggacttctgcctgggcatgtcc
aagttcctgctggagctgggtgcggagccggtgcacatcctgtcgacgtcgggctccaagaaggggagaagcaggtccagaaggtgctggacgg
ctcgcccttcggcgcctcgggcaaggcccatggcggcaaggatctgtggcacctgcgttcgctgatcttcaccgacaaggtggactacatcatcggc
aacagctacggcaagtatctggagcgcgacaccaaggttccgctgatccgcctgacctacccgatcttcgaccgccaccaccaccaccgctacccg
acctggggctaccagggcgcgctgaacgtgctggtacggatcctaaccggatcttcgaggacatcgacgccaacaccaacatcgtcggccagac
cgactactcgttcgacctgatccgctga
173 atgttgacctctgatattgttggcaaattgcgctgcatcgcagcagaccccaaagcgggcatcgcaaggggcctcgacaccgggacgacgaagatc
ggtcccgtttgggagggtgacgtgggcgacaccgtggatttcgaagcgctgcgccagcgggcggtccactccctgttcgaacatctggaatccatgt
gcgtcagcgccgtcgccgtcgaccacaccggccgcatcgcctggatggacgagaagtacaaggctctgctgggcgttcccgacgacccgcgcgg
ccggcaggtggaggacgtcatccccaacagccagctgcgccgggtgatcgacagcggccagccgcagccgatggacatcatggagttcgacgac
cggtccttcgtggtgacgcgcatgccgctgttcggcaccgacggttcgatcatcggcgccatcggcttcgtgctgttcgaccgcgccgaatatctccg
cccgctggtccgcaaatacgagaagatgcaggaggagctggcccgcacccagcaggagctggcgcatgagcgccgcgccaaatactccttctcg
cagttcctgggcgccagcgaatcgatccgcgagatcaagcggctggggcgccgcgccgcccagatggattcgaccgtcctgctgctgggcgaaac
cgggaccggcaaggagctgctggcccaggccatccattccgccagcccgcaggcgtccaagcccttcgtcggcgtcaatgtcgccgccattccgg
aaaccctgctggaggcggagttcttcggcgtcgoccccggcgccttcaccggcgccgaccgccgccaccgcgacggcaagttccagctcgccaac
ggcggcaccctgttcctcgacgagatcggcgacatgccgctgccggtgcaggccaagcttctgcgcgtgctgcaggagcgggagatcgagccgct
cggctccaacaaggtggtgcgggtcgatgtccgcatcatcgccgccaccagccgtgacctgcacgccctggtgcgtgagaagcagttccgcgccg
acctctattaccggctgaatgtggtgccgatcaccctgccgccgctgcgcgaccggccggaggacatcgagagcatcgccgaccgcatcctggaac
agctggcgatccagcagggcacgccgcogcgcgagctgctggaatcggcggtgcaggtgctgcgcgactatgactggcccggcaatgtgcgcga
gctttacaacacgctggaacgggtggtggcgctgaccgatgcgccgatcctgaccgcgccgcacatccgcagcgtgctgcccggccagcatccgg
ccggcgcgtcggccctgccgctggcggccggcgcgcggccgttgcaggaggtgctgacgccgccgagcgccacgccatcgccgcggcgcttg
aggaggcgaacggcgtcaaggcgcgggcggcgaagctgctgggcatttcgcgcgcgtcgctgtacgaacgcatggtgacgctggggttggggg
cgacgcagtag
174 atgccgagtcccatcgcgttctcaagccccttgccgaagcctttcgacagcgcgcaggggcgctggggatggaggctggcgccagcaggccgc
cgcggcggagccggagacccgcgcctgggcggaagccttcgccgattcggagaccggccgggcgctgatcggggcggtgtgcggcaacagcc
cgtatctcggccacagcctgacgcgggagttgcccttcgtcgcccgtacagtgcaggacggcttcgacgacaccttcgccgcgctgatcgccgctct
ccatgccgagcatggcgaggagaaatcgatggaccggctgatggccggcctgcgggtggcgaagcggcgggcggcgagctgatcgcgctggc
cgacatcgccgggcgtggccgctgttccgcgtcaccggcgccctgtcggagctggcggagacgggggtgcagctggccgcgaatttcctgctgc
gccgcgccagggaggccgggacgctgacgctgccggatccgcagcgaccgtgggtcggttcgggcctgatcgttttgggcatgggtaagcttggc
gggcgcgaactcaactattccagcgacatcgacctgatcgtcctgtatgacgacgctgttgtgcagacgccccagccggacaacctcgcgcgaacct
tcatcaggctcgcacgcgatcttgtccgcattatggatgaacggaccaaggacggctacgtcttccgcaccgaccttcggcttaggcccgatcccggc
gccacgccgctggcggtttccgtctccgcagccgaaatttattacggcagcgtcggtcagaactgggaacgcgcggcgatgatcaaggcccgtccc
atcgccggcgatctggaggcgggcgcgcctcctttgtccgcttcctggagcccttcgtctggcgccgcaacctggatttcgccgccatccaggacatcca
ttcgatcaaacgccagatcaacgcccacaagggccaccgcgaggtgacggtcaacggccacgacatcaaggtcggccgcggcggcatccgcga
gatcgagacttcgcccagacccagcagctgatcttcggcgggcgcgacccgcgcgtgcgaatcgctccgaccctgatggcgaacgaggcgctgc
gcgacgtcggccgcgtgccgccgcagacggtggaagagcttgccggggcctatcatttcctgcgccgtgtcgaacatcgcatccagatgatcgacg
accagcagacccatcgtattcccgccgacgatgccggggtggcgcatttggccaccttcctcggctatgacgaccccgccgccttccgggcggaact
gctggcgacgctggggcaggtggaggaccgctattccgagctgttcgaggaggcgccgtcgctttccggccccggcaatctggtcttcaccggca
ccgaccccgatccgggcacgatggagacgctgaagggcatgggcttcgccgatccggccgcgtcatcagcgtggtgtcggctggcatcgcgg
ccgctaccgcgccacccggtcgggccgggcgcgggagctgctgacggagctgacgccggccctgctgagtgcgctgaccaagaccccggcccc
cgattcggcgctgatgaacttcgacgatttcctcggcaagctgccggccggcgtcggtctgttctcgctgttcgtcgccaatccctggctcctggagctg
gtggcggagatcatgggcatcgcgccgcagatggcgcagacgctgtcgcgcaacccgtcgctgctcgacgccgtgctgtcgccggacttcttcgac
ccgctgccgggcaaggaggacgggctggccgacgaccacgcccgcgtgatggcgccggcccgcgatttcgaggatgcgctgaccctgtcgcgg
cgctggaccaacgaccagcgcttccgcgccggggtgcatatcctgcgcggcatcaccgatggcgaccgctgcggcgccttcctggccgatctggc
cgacatcgtcgtccccgaccttgcccgccgggtggaggaggagttcgcccagcgccacggccatattcccggcggcgcctgggtggtggtggcga
tgggcaagtcggcagccggcagctgaccatcacgtccgacatcgacctgatcgtcatctacgatgtggcgcccggccaagggggcgggggcgg
tccccgcttgtcggatggtgccaagccgctgtcgcccaacgagtattacatcaagtgactcagcgtctgaccaacgccattaccgcgccgatgcgc
gacggccggctctacgaggtcgacatgcggctgcgcccgtcgggcaacgccgggccgctcgccacctcgctggacgctttcctgaaatatcaggc
gaccgatgcctggacctgggagcatatggccctgacccgcgcccgggtgatcggcggtgatgcggagctggccgggcgggtgtcggcagcgatc
cgctcggtgctgacggcgccgcgcgatgccgaccggctgctgtgggacgtggccgacatgcggcggcggatcgagaaggagttcgggacgacc
aatgtctggaacgtcaaatacgcccgcggcggcctgatcgacatcgagttcatcgcccagtacctgaactgcgccatggtcacgagcggccggac
atcctgcacatcggcaccgccaaggcgctgggctgcgccgcccggacgggcgcgctggcgccggaggtggcggaggatctggagacgacgct
gcggctgtggcggcgggtgcagggctttctgcggttgaccaccgccggggtgctcgatcccaatcaggtgtcgcccagcctgctggccgggctggt
ccgcgccgcctttcctgctgactttcagggcgagcgtgagcctcgactgttgacttccccgaactggaccacaaaatccgtgccgtcgccgcccgc
gcccatggtcatttcaagaccctggtcgaggaaccggcgggccgtctggccccacccgccaccacgcctccagcctga
175 atgaaccgtctgttccaatggccgcaccgatgatggcggttgctctgggcgcggtcggcatgccggccgcagcccttgcccaggatccggcggctg
ccgccgctgccgcggctgcggctgcggagccgccgctgctgccgcaccggcggctccggcgctgaatggcggcgacaccgcctggatgctcat
ctccaccgcgctggtgctgatgatgaccatccccggcctggcgctgttctacggcggcatggtccgcaagatgaacgtgctgtcgacggtgatgcag
agcttcgccatcacctgcctgatcagcgtcctgtggtacgtcatcggctacagcctggccttcaccggcaccggtgcctatgtcggcggtctcgaccg
gctgttcctcaacgggctcgacttcacgaaggccttcgtgctgggcgaggcgaccgggtcgggcgtcccgacgaccatccccgagccggtcttcat
gatgttccagatgacctttgcgatcatcaccccggccctgatcaccggcgccttcgccgaccgatgaagttctcctccctgctggtcttcaccgcgctg
tggtcgatcgtggtctatgcgccgatcgcccactgggtctggtacccgtcgggcttcctgttcggcctagcgtgaggacttcgccggcggcacggt
cgtgcacatcaaccgccggcgtcgccggcctggtcgccgcgctggtgatcggcaagcgcaagggctacccgaaggaagccttcatgccgcacaac
ctggtgctgtcgctgatcggcgcctcgctgctgtgggtcggctggttcggcttcaacgccggttcggccctgaccgccggtccgcgtgccggcatgg
cgctggccgccacgcacatcgccaccgccggtgccgccatgggctggctgttcgcggagtggatcgtcaagggcaagccgtcgatcctcggcatc
atctccggcgccgtcgccggcctggtcgggtgaccccggccgccggcttcgtcgacccgacgggcgccatcgtcatcggcatcgtcgcccgcgt
ggtctgcttctggtcggccaccagcctcaagcacatgctgggctatgacgacagcctggacgccttcggcgtgcacggcgtcggcggcctgatcgg
cgccatcctgaccggcgtcttcgccaagatgtcggtgtccaacagcgaaggcggcttcgcctccgtcctgcaggccgacccgaaggccacgctgg
gcctgctggaaggcaacgccgccgccgtctggatccaggtccagggcgtcctctacaccatggtctggtgcgccatcgccaccttcgtcctgctgaa
gatcgtcgatgtggtcatgggcctgcgcgtcgaagaggatgtggagcgcgacggtctcgacctcgccctgcatggcgagagcatccactaa
176 atggatgcggcaaagacgggtggcgacgtccttttcgtgctgatgggcgcggtgatggtgctggcgatgcattgcggcttcgccctgctggaggtcg
ggacggtccggcgcaagaatcaggtcaacgcgctggtgaagatcctgtcggacttcgccatgtcgaccatcgcctattttttcgtcggttatgccgtgg
cctacggcatcgacttcttcgccgacgcccacacgctggtcggcaagggaagcggcgggttcgcggcctatggctacgatctggtgaagttcttcttc
ctggcgaccttcgccgccgcggtgccggccatcgtctcgggcggcatcgccgagcgtgctaggttctggccgcaggccgccgccacgctggcgct
gatcgcgctgttctatccattgctggaaggcacggtctggggcacccgcttcggcctgcaaagctggatggccgcgaccttcggccagcctttccacg
acttcgccggatctgtggtggtgcatgccttcggcggctgggtggcgctgggtgccgtgctgaacctcggcaaccgccgcggccgctaccgtccga
acggctcgctgatcgccattccgccgtcgaacatccccttcctggcgctgggcgcctgggtgctgtgcgtggggtggttcggcttcaacgtgatgagc
gcccaggtgctggatggcgtgacgggtctggtggcgctgaactcgctgatggcgatggtcggcggcatcgtcacctcgctggtgatcagccgcacc
gatcccggcttcgtccacaacggcgcgctggccggtctggtggcggtctgcgccgggtccgacgtgatgcacccgctgggcgcgctggtcaccgg
cggcatcgccgggctgctgttcgtctgggccttcaacaaatgccagatcgactggaagatcgacgacgtgctgggcgtctggccgctgcacggctg
tgcggcctgaccggcggcctgctggccggcgtcttcgggcaggaggcactgggcggccttggcggcgtgtcgatcctcagccagatcgtcggcac
ggcaagcggcgccagcttcggattcgctacgggtctggcggtctaccgcctgctgcgcgtcaccgtccgcatccgcctcgatcccgaccaggagta
caagggcgccgacttgtcgttgcaccatatcaccgcgtacccggaagaggacgcgccgaccctgtaa
177 atgaccctgaatatgatgatggattcttggttctctggagcgctttatcggcatcctgactgaagatttgcaggcttcttcccaacctggcttgcacccgt
gcaggtagttgtgatgaacatcactgattcgcaggctgaatacgttaacgaattgacccgtaaactgcaaaatgcgggcattcgtgtaaaagcagactt
gagaaacgagaagattggctttaaaatccgcgagcacactttacgtcgtgtcccttatatgctggtttgtggtgacaaagaggtcgaagccggcaaagt
tgctgtgcgtacccgtcgcggtaaagacctgggtagcctggacgtaaatgatgatcgagaagctgcaacaagagattcgcagccgcagtcttcaac
aactggaggaataaggtattaaaggcggaaaacgagttcaaacggcgcgtcccaacgtattaatggcgagattcgcgccacggaagttcgcttaac
aggtctggaaggcgagcagcttggtattgcgatagaactcacttcacgccccgaagggggaagctgcctgaccctacgattcccgctatttcattcact
gaccggaggttcaaaatga
178 accggataagagagaaaagtgcgacgtcggtccggttgatattgaccggcgcatccgccagctcgcccagtttttggtggatctgtttggcgattttgc
gggtcttgccggtgtcggtgccganaaaaataccaatatttgccataacacacgctcctgttgaaaaagagatcccgcggggaaaatgcggtgaacat
gtcagctattgcgaagagtgtgccagttttgctcacgggcaaaagctgcaccagaatgggattaatgcaccagcctggcgctttttttcgcggcacgtc
ccctcgctaatgcccgtctggcgcggctttgacgctgataaggcgctgaataccgatctggatcaaggtttgtcgggttatcgtccaaaaggtgcactc
ttgcatggttataagtgcctgacatggtgtccgggcgaacgtcgccaggtggcacaaattgtcagaactacgacacgactaaccgaccgcaggagt
gtgcgatgaccctgaatatgatgatggattatggttctctggagcgctttatcggcatcctgactgaagaatttgcaggcttcttcccaacctggcttgca
cccgtgcaggtagttgtgatgaacatcactgattcgcaggctgaatacgttacgaattgacccgtaaactgcaaaatgcgggcattcgtgtaaaagca
gacttgagaaacgagaagattggctttaaaatccgcgagcacactttacgtcgtgtcccttatatgctggtttgtggtgacaaagaggtcgaagccggc
aaagttgctgtgcgtacccgtcgcggtaaagacctgggtagcctggacgtaaatgatgttatcgagaagctgcaacaagagattcgcagccgcagtct
tcaacaactggaggaataaggtattaaaggcggaaaacgagttcaaacggcgcgtcccaatcgtattaatggcgagattcgcgccacggaagttcgc
ttaacaggtctggaaggcgagcagcttggtattgcgatagaactcacttcacgccccgaagggggaagctgcctgaccctacgattcccgctatttcat
tcactgaccggaggttcaaaatgacccagcgaaccgagtcgggtaataccgtctggcgcttcgatttatcccagcagttcaccgcgatgcagcggata
agcgtggttctcagcgggcgaccgaggttgaacagacactccagcaggtgctgtgcgtattgcacaatgacgcctttttgcagcacggcatgatctg
tctgtacgacagccagcaggcgattttgactattgaagcgttgcaggaagccgatcagcagttgatccccggcagctcgcaaattcgctaccgtccgg
gtgaagggaggtcgggacggtgctttcgcaggggcaatcgttagtgctggcgcgtgtggctgacgatcagcgctttcttgaccgcctgggactgtat
gattacaacctgccgtttatcgccgtgccgctgatagggccggatgcgcagacttttggcgtgctgacggcgcaaccgatggcgcgttacgaagagc
ggttacccgcctgcacccgctttctggaaacggtc
179 tccctgtgcgccgcgtcgccgatggtgaccagcaactggcgcgctacccgatcctgctcgagaactgctcgacccgaacacgctctatcaaccga
cggcgatgaacgcctatcgcgatgaactgcgacaatacctgttgcgcgtgccggaagaggatgaagagcagcaactggaggcgctacggcagttta
agcaggcgcagttgttgcgcgtagcggcggcggatatcgccggtacgttacccgtcatgaaagtgagcgatcacttaacctggctggcggaagcga
ttatcgatgcggtggtgcagcaagcctggaaccagatggtggcgcgttacggccagccgacgcatctgcacgatcgcgaagggcgcggtttcgccg
tggtcggttacggcaaacttacggctgggaattaggttacagctccgatctggatctggtgttcctgcacgactcccccatggatgtgatgaccgatg
gcgagcgtgaaatcgatggcccccagttctatttgcgcctcgcgcagcgcgtgatgcacctgttcagcacgcgcacgtcgtccggcattctttatgaag
tcgatgcgcgtttgcgcccgtccgggcggccggaatgctggtgaccactgcggaagcgttcgccgattatcaaaaaaatgaagcctggacatggg
agcatcaggcgctggcgcgtgcgcgcgtggtgtacggcgatccgcaactgaccgccgaatttgacgccattcgccgcgatatcctgatgacctcccg
cgatgccgctaccctgcaaaccgaagtgcgggaaatgcgtgagaaaatgcgcgcccatcttggtaacaagcacaaagaccgtttcgatctgaaagc
cgatgaaggcggatcaccgatattgagtttatcgctcagtatctggtgctgcgctttgcccatgagaagccgaaactgacgcgctggtcggataatgg
cgcatcctcgaagggctggcgcaaaacggcatcatggatgagcaggaagcgcaggcattgacgctggcgtacaccacgttgcgtgatgagctgca
ccacctggcgctgcaagagctgccaggacatgtggcgctctcctgttttgtcgccgagcgtgcgcttatcaaaaccagctgggacaagtggctggtg
gaaccgtgcgccccggcgtaa
180 taaagcgagcgctcacttacgtgatctgttgacgcagtccgaagcgaccattacttcagccgtttcagcagatacggcggtgtggagtgcgcaatcag
ccctggcgaaactggtgctcaccgagtggttagtgacgcagggctggcgaaccttccttgatgaaaaagcgcaggctaagtttgccgactcctttaaa
cgctttgctgacgttcatctgtcacgcagcgccgccgagctgaaaaaagcctttgcccagccgctgggcgacagctatcgcgaccagttaccgcggc
tggcgcgtgatatcgacagcgcgttattgctggccggacattacgatcgcgcgcgcgccgtggagtggctggaaaactggcaggggcttcagcacg
ctattgaaacgcgccagagagttgaaatcgaacatttccgtaataccgccattacccaggagccgttctggttgcacagggaaaacgttaacgaaag
gatatttcgcatgtccctgtgcgccgcgtcgccgatggtggccagccaactggcgcgctacccgatcagctcgatgaactgctcgacccgaacacg
ctctatcaaccgacggcgatgaacgcctatcgcgatgaactgcgacaatacctgttgcgcgtgccggaagaggatgaagagcagcaactcgaggc
gctacggcagtttaagcaggcgcagttgttgcgcgtagcggcggcggatatcgccggtacgttacccgtcatgaaagtgagcgatcacttaacctgg
ctggcggaagcgattatcgatgcggtggtgcagcaagcctggaaccagatggtggcgcgttacggccagccgacgcatctgcacgatcgcgaagg
gcgcggtttcgccgtggtcggttacgccaaacttggcggctgggaattaggttacagctccgatctggatctggtgttcctgcacgactgccccatgga
tgtgatgaccgatggcgagcgtgaaatcgatggccgccagttctatttgcgcctcgcgcagcgcgtgatgcacctgttcagcacgcgcacgtcgtccg
gcattctttatgaagtcgatgcgcgtttgcgcccgtccggcgcggccggaatgctggtgaccactgcggaagcgttcgccgattatcaaaaaaatgaa
gcctggacatgggagcatcaggcgctggcgcgtgcgcgcgtggtgtacggcgatccgcaactgaccgccgaatttgacgccattcgccgcgatatc
ctgatgacctcccgcgatgccgctaccctgcaaaccgaagtgcgggaaatgcgtgagaaaatgcgcgcccatcttggtaacaagcacaaagaccgt
ttcgatctgaaagccgatgaaggcggtatcaccgatattgagtttatcgctcagatctggtgctgcgctttgcccatgagaagccgaaactgacgcgct
ggtcggataatgtgcgcatcctcgaagggctggcgcaaaacggcatcatggatgagcaggaagcgcaggcattgacgctggcgtacaccacgttg
cgtgatgagctgcaccacctggcgctgcaagagctgccaggacatgtggcgactctcctgttttgtcgccgagcgtgcgcttatcaaaaccagctggga
caagtggctggtggaaccgtgcgccccggcgtaagtgtggtatcatcgcgcgcaaattttgtatctctcaggagacaggaatgaaagttacgctgcca
gagttcaatcaagccggtgtcatggtggtgggtgatgtgatgctggatcgctactggtacggcccaaccagccgcatttctccggaagcgccagttcc
ggttgttaaagtcgatactattgaagagcgaccgggcggtgcggcaaacgtggcgatgaacattgcctcgctgggcgcaacggcgcgtctggttggc
ctgactggcattgatgatgcggcgcgcgcgctgagcaaagcgctggcggatgttaatgttaaatgtgacttcgtttctgttccgactcaccccaccatca
ctaagctgcgcgtgctgtcgcgtaaccagcaactgattcgc
181 atgaccctgaatatgatgatggatgccagccgttctgtaataataaccggacaattcggactgattaaaaaagcgccctcgcggcgctttttttatattctc
gactccatttaaaataaaaaatccaatcggatttcactatttaaactggccattatctaagatgaatccgatggaagctcgctgttttaacacgcgtttttaa
ccttttattgaaagtcggtgcttctttgagcgaacgatcaaatttaagtggattcccatcaaaaaaatattctcaacctaaaaaagtttgtgtaatacttgtaac
gctacatggagattaactcaatctagagggtattaataatgaatcgtactaaactggtactgggcgcaactcacttcacgccccgaagggagaagctgc
ctgaccctacgattcccgctatttcattcactgaccggaggttcacaatga
182 accggataagagagaaaagtgtcgacgtcggtccggttgatattgaccggcgcatccgccagctcgcccagtttttggtggatctgtttggcgattttgc
gggtcttgccggtgtcggtgccgaaaaaaataccaatatttgccataacacacgctcctgttgaaaaagagatcccgcggggaaaatgcggtgaacat
gtcagctattgcgaagactgtgccagttttgctcacgggcaaaagctgcaccagaatgggtattaatgcaccagcctggcgctttttttcgcggcacgtc
ccctcgctaatgcccgtctggcgcggctttgacgctgataaggcgctgaataccgatctggatcaaggttttgtcgggttatcgtccaaaaggtgcactc
tttgcatggttataagtgcctgacatggtgtccgggcgaacgtcgccaggtgccacaaattgtcagaactacgacacgactaaccgaccgcaggagt
gtgcgatgaccctgaatatgatgatggatgccagccgttctgtaataatcaccggacaattcggactgattaaaaaagcgccctcgcggcgctttttttat
attctcgactccatttaaaataaaaaatccaatcggatttcactatttaaactggccattatctaagatgaatccgatggaagctcgctgttttaacacgcgtt
ttttaaccttttattgaaagtcgggcttctttgagcgaacgatcaaatttaagtggattcccatcaaaaaaatattctcaacctaaaaaagtttgtgtaatactt
gtaacgctacatggagattaactcaatctagagggtattaataatgaatcgtactaaactggtactgggcgcaactcacttcacgccccgaagggggaa
gctgcctgaccctacgattcccgctatttcattcactgaccggaggttcaaaatgacccagcgaaccgagtcgggtaataccgtctggcgcttcgattta
tcccagcagttcaccgcgatgcagcggataagcgtggttctcagccggccgaccgaggttgaacagacactccagcaggtgctgtgcgtattgcaca
atgacgcctttttgcagcacggcatgatctgtctgtacgacagccagcaggcgattttgactattgaagcgttgcaggaagccgatcagcagttgatccc
cggcagctcgcaaattcgctaccgtccgggtgaagggctggtcgggacggtgctttcgcaggggcaatcgttagtgctggcgcgtgtggctgacgat
cagcgctttcttgaccgcctgggactgtatgattacaacctgccgtttatcgccgtgccgctgatagggccggatgcgcagacttttggcgtgctgacg
gcgcaaccgatggcgcgttacgaagagcggttacccgcctgcacccgctttctggaaacggtc
183 tccctgtgcgccgcgtcgccgatggtggccagccaactggcgcgctacccgatcctgctcgatgaactgctcgacccgaacacgctctatcaaccga
cggcgatgaacgcctatcgcgatgaactgcgacaatacctgttgcgcgtgccggaagaggatgaagagcagcaactggaggcgctacggcagttta
agcaggcgcagttgttgcgcgtagcggcggcggatatcgccggtacgttacccgtcatgaaagtgagcgatcacttaacctggctggcggaagcga
ttatcgatgcggtggtgcagcaagcctggaaccagatggtggcgcgttacggccagccgacgcatctgcacgatcgcgaagggcgcggtttcgccg
tggtcggttacggcaaacttggcggctgggaattaggttacagctccgatctggatctggtgttcctgcacgactgccccatggatgtgatgaccgatg
gcgagcgtgaaatcgatggcccccagttctatttgcgcctcgcgcagcgcgtgatgcacctgttcagcacgcgcacgtcgtccggcattctttatgaag
tcgatgcgcgtttgcgcccgtccggcgggccggaatgctggtgaccactgggaagcgttcgccgattatcaaaaaaatgaagcctggacatggg
agcatcaggcgctggcgcgtgcgcgcgtggtgtacggcgatccgcaactgaccgccgaatttgacgccattcgccgcgatatcctgatgacctcccg
cgatgccgctaccctgcaaaccgaagtgcgggaaatgcgtgagaaaatgcgcgcccatcttggtaacaagcacaaagaccgtttcgatctgaaagc
cgatgaaggcggtatcaccgatattgagtttatcgctcagtatctgggctgcgctttgcccatgagaagccgaaactgacgcgctggtcggataatgtg
cgcatcctcgaagggctggcgcaaaacggcatcatggatgagcagcaagcgcaggcattgacgctggcgtacaccacgttgcgtgatgagctgca
ccacctggcgctgcaagagctgccaggacatgtggcgctctcctgttttgtcgccgagcgtgcgcttatcaaaaccagctgggacaagtggctggtg
gaaccgtgcgccccggcgtaa
184 taaagcgagcgctcacttacgtgatctgttgacgcagtccgaagcgaccattacttcagccgtttcagcagatacggcggtgtggagtgcgcaatcag
ccctggcgaaactggtgctcaccgagtggttagtgacgcagggctggcgaaccttccttgatgaaaaagcgcaggctaagtttgccgactcctttaaa
cgctttgctgacgttcatctgtcactcagcgccgccgagctgtaaaaagcctttgcccagccgctgggcgacagctattgcgaccagttaccgcggc
tggcgcgtgatatcgacagcgcgttattgctggccggacattacgatcgcgcgcgcgccgtggagtggctggaaaactggcaggggcttcagcacg
ctattgaaacgcgccagagagttgaaatcgaacatttccgtaataccgccattacccaggagccgttctggttgcacagcggaaaacgttacgaaag
gatatttcgcatgtccctgtgcgccgcgtcgccgatggtggccagccaactggcgcgctacccgatcctgctcgatgaactgctcgacccgaacacg
ctctatcsaccgacggcgatgaacgcctatcgcgatgaactgcgacaatacctgttgcgcgtgccggaagaggatgaagagcagcaactggaggc
gctacggcagtttaagcaggcccagttgttgcgcgtagcggcggcggatatcgccggtacgttacccgtcatgaaagtgagcgatcacttaacctgg
ctggcggaagcgattatcgatgcggtggtgcagcaagcctggaaccagatggtggcgcgttacggccagccgacgcatctgcacgatcgcgaagg
gcgcggtttcgccgtggtcggttacggcaaacttggcggctgggaattaggttacagctccgataggatctggtgttcctgcacgactgccccatgga
tgtgatgaccgatggccagcctgaaatcgatggccgccagttctatttgcgcctcgcgcagcgcgtgatgcacctgttcagcacgcgcacgtcgtccg
gcattctttatgaagtcgatgcgcgtttgcgcccgtccggcgcggccggaatgctggtgaccactgcggaagcgttcgccgattatcaaaaaaatgaa
gcctggacatgggagcatcagccgtagcgcgtgcgcgcgtggtgtaccgcgatccgcaactgaccgccgaatttgacgccattcgccgcagatatc
ctgatgacctcccgcgatgccgctaccctgcaaaccgaagtgcgggaaatgcgtgagaaaatgcgcgcccatcttggtaacaagcacaaagaccgt
ttcgatctgaaagccgatgaaggcggtatcaccgatattgagtttatcgctcagtatctggtgctgcgctttgcccatgagaagccgaaactgacgcgct
ggtcggataatgtgcgcatcctcgaagggctggcgcaaaacggcatggatggatgagcaggaagcgcaggcattgacgctggcgtacaccacgttg
cgtgatgagctgcaccacctggcgctgcaagagctgccaggacatgtggcgctctcctgttttgtcgccgagcgtgcgatatcaaaaccagctggga
caagtggctggtggaaccgtgcgccccggcgtaagtgtggtatcatcgcgcgcaaattttgtatctctcaggagacaggaatgaaagttacgctgcca
gagttcaatcaagccggtgtcatggtggtgggtgatgtgatgctggatcgctactatacggcccaaccagocgcatttctccggaagcgccagcc
ggttgttaaagtcgatactattgaagagcgaccgggcggtgcggcaaacgtggcgatgaacattgcctcgctgggcgcaacggcgcgtctggttggc
ctgactggcattgatgatgcggcgcgcgcgctgagcaaagcgctggcggagttaatgttaaatgtgacttcgtttctgttccgactcaccccaccatca
ctaagctgcgcgtgctgtcgcgtaaccagcaactgattcgc
185 atgaccctgaatatgatgatggatgccagccgttctgaataataaccggacaattcggactgattaaaaaagcgccctcgcggcgctttttttatattctc
gactccatttaaaataaaaaatccaatcggatttcactatttaaactggccattatctaagatgaatccgatggaagacgctgattttaacacgcgttttttaa
ccttttattgaaagtcggtgcttctttgagcgaacgatcaaatttattgtggattcccatcaaaaaaatattctcaacctaaaaaagtttgtgtaatacttgtaac
gctacatggagattaactcaatctagagggtattaataatgaatcgtactaaactggtactgggcgcaactcacttcacgccccgaagggggaagctgc
ctgaccctacgattcccgctatttcattcactgaccggaggttcaaaatga
186 accggataagagagaaaagtgtcgacgtcggtccggttgatattgaccggcgcatccgccagctcgcccagtttttggtggatctgtttggcgattttgc
gggtcttgccggtgtcgctgccgaaaaaaataccaatatttgccataacacacgctcctgttgaaaaagagatcccgcggggaaaatgcggtgaacat
gtcagctattgcgaagagtgtgccagttttgctcacgggcaaaagctgcaccagaatgggtattaatgcaccagcctggcgctttttttcgcggcacgtc
ccctcgctaatgcccgtctggcgcggctttgacgctgataaggcgctgaataccgatctggatcaaggttttgtcgggttatcgtccaaaaggtgcactc
tttgcatggttataagtgcctgacatggtgtccgggcgaacgtcgccaggtggcacaaattgtcagaactacgacacgactaaccgaccgcaggagt
gtgcgatgaccctgaatatgatgatggatgccagccgttctgtaataataaccggacaattcggactgattaaaaaagcgccctcgcggcgctttttat
attctcgactccatttaaaataaaaaatccaatcggatttcactatttaaactggccattatctaagatgaatccgatggaagctcgctgtttaacacgcgtt
ttttaaccttttattgaaagtcggtgcttctttgagcgaacgatcaaatttaagtggattcccatcagaaaaatattctcaacctaaaaaagtttgtgtaatactt
gtaacgctacatggagattaactcaatctagagggtattaataatgaatcgtactaaactggtactgggcgcaactcacttcacgccccgaagggggaa
gctgcctgaccctacgattcccgctatttcattcactgaccggaggttcaaaatgacccagcgaaccgagtcgggtaataccgtctggcgcttcgattta
tcccagcagtcaccgcgatgcagcggataagcgtggttctcagccgggcgaccgaggttgaacagacactccagcaggtgctgtgcgtattgcaca
atgacgcctttttgcagcacggcatgatctgtctgtacgacagccagcaggcgattttgactattgaagcgttgcaggaagccgatcagcagttgatccc
cggcagctcgcaaattcgctaccgtccgggtgaagggctggtcgggacggtgctttcgcaggggcaatcgttagtgaggcgcgtgtggctgacgat
cagcgctttcttgaccgcctgggactgtatgattacaacctgccgtttatcgccgtgccgctgatagggccggatgcgcagacttttggcgtgctgacg
gcgcaaccgatggcgcgttacgaagagcggttacccgcctgcacccgctttctggaaacggtc
187 tccctgtgcgccgcgtcgccgatggtggccagccaactggcgcgctacccgatcctgctcgatgaactgctcgacccgaacacgctctatcaaccga
cggcgatgaacgcctatcgcgatgaactgcgacaatacctgttgcgcgtgccggaagaggatgaagagcagcaactggaggcgctacggcagttta
agcaggcgcagttgttgcgcgtagcggcggcggatatcgccggtacgttacccgtcatgaaagtgagcgatcacttaacctggctggcggaagcga
ttatcgatgcggtggtgcagcaagcctggaaccagatggtggcgcgttacggccagccgacgcatctgcacgatcgcgaagggcgcggtttcgccg
tggtcggttacggcaaacttggcggctgggaattaggttacagctccgatctggatctggtgttcctgcacgactgccccatggatggatgaccgatg
gcgagcgtgaaatcgatggcccccagttctatttgcgcctcgcgcagcgcgtgatgcacctgttcagcacgcgcacgtcgtccggcattctttatgaag
tcgatgcgcgtttgcgcccgtccggcgcggccggaatgctggtgaccactgcggaagcgttcgccgattatcaaaaaaatgaagcctggacatggg
agcatcaggcgctggcgcgtcgcggtggtgtacggcgatccgcaactgaccgccgaatttgacgccattcgccgcgatatcctgatgacctcccg
cgatgccgctaccctgcaaaccgaagtgcgggaaatgcgtgagaaaatgcgcgcccatcttggtaacaagcacaaagaccgtttcgatctgaaagc
cgatgaaggcggtatcaccgatattgagtttatcgctcagtatctggtgctgcgctttgcccatgagaagccgaaactgacgcgctggtcggataatgtg
cgcatcctcgaagggctggcgcaaaacggcatcatggatgagcaggaagcgcaggcattgacgctggcgtacaccacgttgcgtgatgagctgca
ccacctggcgctgcaagagagccaggacatgtggcgctctcctgttttgtcgccgagcgtgcgcttatcaaaaccagctgggacaagtggctggtg
gaaccgtgcgccccggcgtaa
188 taaagcgagcgctcacttacgtgatctgttgacgcagtccgaagcgaccattacttcagccgtttcagcagatacggcggtgtggagtgcgcaatcag
ccctggcgaaactggtgctcaccgagtggttagtgacgcagggctggcgaaccttccttgatgaanagcgcaggctaagtttgccgactcctttaaa
cgctttgctgacgttcatctgtcacgcagcgccgccgagctgaaaaaagcctttgcccagccgctgggcgacagctatcgcgaccagttaccgcggc
tggcgcgtgatatcgacagcgcgttattgctggccggacattacgatcgcgcgcgcgccgtggagtggctggaaaactggcaggggcttcagcacg
ctattgaaacgcgccagagagttgaaatcgaacatttccgtaataccgccattacccaggagccgttctggttgcacagcggaaaacgttaacgaaag
gatatttcgcatgtccctgtgcgccgcgtcgccgatggtggccagccaactggcgcgctacccgatcctgctcgatgaactgctcgacccgaacacg
ctctatcaaccgacggcgatgaacgcctatcgcgatgaactgcgacaatacctgttgcgcgtgccggaagaggatgaagagcagcaactggaggc
gctacggcagtttaagcaggcccagttgttgcgcgtagcggcggcggatatcgccggtacgttacccgtcatgaaagtgagcgatcacttaacctgg
ctggcggaagcgattatcgatgcggtggtgcagcaagcctggaaccagatggtggcgcgttacggccagccgacgcatctgcacgatcgcgaagg
gcgcggtttcgccgtggtcggttacggaaacttggcggctgggaattaggttacagctccgatctggatctggtgttcctgcacgactgccccatgga
tgtgatgaccgatggcgagcgtgaaatcgatggccgccagttctatttgcgcctcgcgcagcgcgtgatgcacctgttcagcacgcgcacgtcgtccg
gcattctttatgaagtcgatgcgcgtttgcgcccgtccggcgcgccggaatgctggtgaccactgcggaagcgttcgccgattatcaaaaaaatgaa
gcctggacatgggagcatcaggctggcgcgtgcgcgcgtggtgtacggcgatccgcaactgaccgccgaatttgacgccattcgccgcgatatc
ctgatgacctcccgcgatgccgctaccctgcaaaccgaagtgcgggaaatgcgtgagaaaatgcgcgcccatcttggtaacaagcacaaagaccgt
ttcgatctgaaagccgatgaaggcggtatcaccgatattgagtttatcgctcagtatctggtgctgcgctttgcccatgagaagccgaaactgacgcgct
ggtcggataagtgcgcatcctcgaagggctggcgcaaaacggcatcatggatgagcacgaagcgcaggcattgacgctggcgacaccacgttg
cgtgatgagagcaccacctggcgctgcaagagctgccaggacatgtggcgactcctgttttgtcgccgagcggcgcttatcaaaaccagctggga
caagtggctggtggaaccgtgcgccccggcgtaagtgtggtatcatcgcgcgcaaattttgtatctctcaggagacaggaatgaaagttacgctgcca
gagttcaatcaagccggtgtcatggtggtgggtgatgtgatgctggatcgctactggtacggcccaaccagccgcatttctccggaagcgccagttcc
ggttgttaaagtcgatactattgaagagcgaccgggcggtgcggcaaacgtggcgatgaacattgcctcgctgggcgcaacggcgcgtctggttggc
ctgactggcattgatgatgcggcgcgcgcgctgagcaaagcgctggcggatgttaatgttaaatgtgacttcgtttctgttccgactcaccccaccatca
ctaagctgcgcgtgctgtcgcgtaaccagcaactgattcgc
189 atgaccctgaatatgatgatggatgccagccgcgtcaggttgaacgtaaaaaagtcggtctgcgcaaagcacgtcgtcgtccgcagttctccaaacgtt
aattggtttctgcttcggcagaacgattggcgaaaaaacccggtgcgaaccgggtttttttatggataaagatcgtgttatccacagcaatccattgattat
ctcttctttttcagcatttccagaatcccctcaccacaaagcccgcaaaatctggtaaactatcatccaattttctgcccaaatggctgggattgttcattttt
gtttgcatacaacgagagtgacagtacgcgcgggtagttaactcaacatctgaccggtcgataactcacttcacgccccgaagggggaagctgcctg
accctacgattcccgctatttcattcactgaccggaggttcaaaatga
190 accggataagagagaaaagtgtcgacgtcggtccggttgatattgaccggcgcatccgccagctcgcccagtttttggtggatctgtttggcgattttgc
gggtcttgccggtgtcggtgccgaaaaaaataccaatatttgccataacacacgctcctgttgaaaaagagatcccgcggggaaaatgcgggaacat
gtcagctattgcgaagagtgtgccagttttgctcacgggcaaaagctgcaccagaatgggtattaatgcaccagcctggcgctttttttcgcggcacgtc
ccctcgctaatgcccgtctggcgcggctttgacgctgataaggcgctgaataccgatctggatcaaggttttgtcgggttatcgtccaaaaggtgcactc
tttgcatggttataagtgcctgacatggtgtccgggcgaacgtcgccaggtggcacaaattgtcagaactacgacacgactaaccgaccgcaggagt
gtgcgatgaccctgaatatgatgatggatgccagccgcgtcaggttgaacgtaaaaaagtcggtctgcgcaaagcacgtcgtcgtccgcagttctcca
aacgttaattggtttctgcttcggcagaacgattggcgaaaaaacccggtgcgaaccgggtttttttatggataaagatcgtgttatccacagcaatccatt
gattatctcttctttttcagcatttccagaatcccctcaccacaaagcccgcaaaatctggtaaactatcatccaattttctgcccaaatggctgggattgttc
attttttgtttgccttacaacgagagtgacagtacgcgcgggtagttaactcaacatctgaccggcgataactcacttcacgccccgaagggggaagct
gcctgaccctacgattcccgctatttcattcactgaccggaggttcaaaatgacccagcgaaccgagtcgggtaataccgtctggcgcttcgatttatcc
cagcagttcaccgcgatgcagcggataagcgtggttctcagccgggcgaccgaggttgaacagacactccagcaggtgctgtgcgtattgcacaat
gacgcctttttgcagcacggcatgatctgtctgtacgacagccagcaggcgattttgactattgaagcgttgcaggaagccgatcagcagttgatcccc
ggcagctcgcaaattcgctaccgtccgggtgaagggctggtcgggacggtgctttcgcaggggcaatcgttagtgctggcgcgtgtggctgacgatc
agcgctttcttgaccgcctgggactgtatgattacaacctgccgtttatcgccgtgccgctgatagggccggatgcgcagacttttggcgtgctgacgg
cgcaaccgatggcgcgttacgaagagcggttacccgcctgcacccgctttctggaaacggt
191 atggcgctgaagcacctgatcacgctctgcgcggcgtcgccgatggtcgccagccagctggcgcgccacccgctgctgctggatgagctgctggat
cccaacaccctctatcagccgacggcgaccgatgcctatcgcgacgagctgcgccagtacctgctgcgcgtgccggaagaggatgaagagcagca
gctggaggcgttgcgccagtttaagcaggcgcagcagctgcatatcgcggcggcggatatcgctggtaccctgccggtgatgaaggtcagcgatca
cttaacctggcttgccgaagcgatcctcgacgcggtggtgcagcaggcatgggggcagatggtcgctcgctacggccagccgacccacctgcacg
atcgccagggtcgcggcttcgccgtcgtcggctacggtaagcttggcggctgggagctgggctacagctccgatctcgatctggtgttcctccatgac
tgcccggcggaggtgatgaccgacggcgagcgggagattgacgtccgtcagttctacctgcggctggcccagcggatcatgcacctgttgcagcac
ccgcacctcgtccggtattctctacgaagtggacgcccggctgcgtccttctggcgcggcggggatgctgtggtcaccaccgccgacgcgtttgctgact
atcagcagaacgaagcctggacgtgggaacatcaggcgctggtgcgcgcccgcgtggtctatggcgacccggcgctgcaggcgcgctttgacgc
cattcgtcgcgatatcctgaccaccccgcgggaggggatgaccctgcagaccgaggttcgcgagatgcgcgagaagatgcgcgcccaccttggca
acaaacatcccgatcgttttgatatcaaagccgatgccggcgggatcaccgatattgaatttattactcagtatctggtcctacgctatgccagtgacaag
ccgaagctgacccgctggtctgacaacgtgcgtattcttgagctgctggcgcagaacgacatcatggacgaggaggaggcgcgcgccttaacgcat
gcgtacaccaccttgcgtgatgcgctccatcacctggccctgcaggaccagccgggacacgtggcgccagaggccttcagccgggagcgtcagca
ggtcagcgccagctggcagaagtggctgatggcttaa
192 cgtaaggcgaccacccagctccgcgcgttgctgaacgacgctgaagccgttctgctggccgcggacaccgccgacgaggcgttattccgcaccga
ggtcgtcggcgccaaactggccctgactgaatggctggtccagcgcggctggcgtccgttcctcaacgaggcaggagagaaaaaatagccggat
cgttcaaacggtttgccgatattaacctctcgcgggtggcggccgagctgcgcagcgccgtgcagcatctggcggttgaagatgccgccgaccagtt
gccgaagctgcccgcgacatcgacagcgtccagctgctggcgggcgcctatggcgacgccgtcgcgccgtggctggagaactggcaggagctt
caccgtgcaatagcacatgacgatcgcagcgtctttgaatatttccgtcgccaggcgctggctgccgagccgttctggctgcatagtggaaaacgata
atttcaggccagggagcccttatggcgctgaagcacctgatcacgctctgcgcggcgtcgccgatggtcgccagccagctggcgcgccacccgctg
ctgctggatgagctgctggatcccaacaccctctatcagccgacggcgaccgatgcctatcgcgacgagctgcgccagtacctgctgcgcgtgccg
gaagaggatgaagagcagcagctggaggcgttgcgccagtttaagcaggcgcagcagctgcatatcgcggcggcggatatcgctggtaccctgcc
ggtgatgaaggtcagcgatcacttaacctggcttgccgaagcgatcctcgacgcggtggtgcagcaggcatgggggcagatggtcgctcgctacgg
ccagccgacccacctgcacgatcgccagggtcgcggcttcgccgtcgtcggctacggtaagcttggcggctgggagctgggctacagctccgatct
cgatctggtgttcaccatgactgcccggcggaggtgatgaccgacggcgagcgggagattgacggccgcagttctacctgcggctggcccagcg
gatcatgcacctgttcagcacccgcacctcgtccggtattctctacgaagtggacgcccggctgcgtccttctggcgcggcggggatgctggtcacca
ccgccgacgcgtttgctgactatcagcagaacgaagcctggacgtgggaacatcaggcgctggtgcgcgcccgcgtggtctatggcgacccggcg
ctgcaggcgcgctttgacgccattcgtcgcgatatcctgaccaccccccaaggggatgaccctgcagaccgaggttcgcgagatgcgcgagaa
gatgcgcgcccaccttggcaacaaacatcccgatcgttttgatatcaaagccgatgccggcgggatcaccgatattgaatttattactcagtatctggtcc
tacgctatgccagtgacaagccgaagctgacccgctggtctgacagcgtgcgtattcttgagctgctggcgcagaqacgacatcatgcacgaggagga
ggcgcgcgccttaacgcatgcgtacaccaccttgcgtgatgcgctccatcacctggccctgcaggagcagccgggacacgtggcgccagaggcct
tcagccgggagcgtcagcaggtcagcgccagctggcagaagtggctgatggcttaactataaaatcgggtgtgctatatcgcgcgcaaagtttgcgt
ctcgcaggagagagtcatgaaagtaacgctgccggagtttgaacgtgcaggagtgttggtggtgggtgatgtgatgctggaccgctactggtacggc
cccaccagtcgtatttcccggaagccccggtgccggtggtgaaggtggaaaatatcgaagaacgtcctggcggcgcggcaaacgtagcgatgaa
catcgcctccctgggggcaacgtcgcgcctggtgggattgaccgggattgatgacgctgcccgcgctgagccaggcgctggccaatgtgaatgt
gaagtgcgacttcgtctccgtcccgactcacccgaccatcaccaagctgcgggtgctgtcgcgcaatcagcagctgatccgcctcgactttgaagag
ggcttctccggcgtggatccgcagccgatgcatgagcgcattcagcaggcgctgggagccattggcgcactgg
193 atgaccctgaatatgatgctagaagcgtcaggtaccggtcatgattcaccgtgcgattctcggttccctggagcgcttcattggcatcctgaccgaagag
ttcgctggcttcttcccaacctggattgcaccagtgcaggtagtggtcatgaatattaccgattctcaggctgaatacgttaacgaattgacgcgtaaacta
caaaatgcgggcattcgtgtaaaagcagacttgagaaatgagaagattggctttaaaatccgcgagcacactttacgtcgtgtcccgtatatgttggtct
gtggcgacaaagaagtcgaagccggcaaagtggccgtgcgcacccgtcgcgggaaagacctcggcagcatggacgtaagtgaagtgattgagaa
gctgcaacaagagattcgcagccgcagtcttcaacaactggaggaataaggtattaaaggcggaaaacgagttcaaacggcacgtccgaatcgtatc
aatggcgagattcgcgccctggaagttcgcgccattgagctggcttcccgaccgcagggcggcacagcctgaccctgcgtttcccgctgtttaacac
cctgaccggaggtgaagcatga
194 ggccgtcgcccagcgtcggcgtccccaacagcagggcgggtaggccagcaggtccgccagcgtggcgcggttaatattgaccggggcggcgg
cggcctcccccagctgcttgtggatcattttcgcgatcttgcgggtttttaccggtatcggtaccaaagaaaatgccaatgttcgccatagtacgctcctgt
cggnatggtgttgaaaaaaggaatgacgacagaggtattgcgaaggctgtgccaggttgccctgcaccgcgacggcccatccctgccccatcagga
tcgcttcgcatcacgatgccgcgcgccaaaggcgcacccggcggggcgaaaggtaaaaatccgtgaattttccccctgtcggatcaatgtttcgcgtg
gtcgttccgataagggcgcacactttgcatggttatccgggttcggcttaccccgccgagttttgcgcacggtgtcggacaatttgtcataactgcgaca
caggagtttgcgatgaccctgaatatgatgctagaagcgcaggtaccggtcatgattcaccgtgcgattctcggttccctggagcgcttcattggcatc
ctgaccgaagagttcgctggcttcttcccaacctggattgcaccagtgcaggtagtggtcatgaatattaccgattctcaggctgaatacgttaacgaatt
gacgcgtaaactacaaaatgcgggcattcgtgtaaaagcagacttgagaaatcagaagattggctttaaaatccgcgagcacactttacgtcgtgtccc
gtatatgttggtctgtgccgacaaagaagtcgaagccggcnaagtggccgtgcgcacccgtcgcgggaaagacctcggcagcatggacgtaagtg
aagtgattgagaagctgcaaccagagattcgcagccgcagtcttcaacaactggaggaataaggtattaaaggcggaaaacgagttcaaacggcac
gtccgaatcgtatcaatggcgagattcgcgccctggaagttcgcgccattgagctggcttcccgaccgcagggcggcacctgcctgaccctgcgtttc
ccgctgtttaacaccctgaccggaggtgaagcatgatccctgaatccgacccggacaccaccgtcagacgcttcgacctctctcagcagttcaccgcc
atgcagcggataaggtggtgagagccgggccaccgaggccagcaaaacgctgcaggaggtgctcagcgtattacacaacgatgcctttatgcag
cacgggatgatctgcctctacgacagcgagcaggagatcctcagtatcgaagcgctgcagcaaaccggccagcagcccctccccggcagcacgc
agatccgctatcgccccggcgagggactggtggggaccgtgctggcccaggggcagtcgctggtgctgccccgggtcgccgacgatcagcgtttt
ctcgaccgcctgagcctctacgattacgatctgccgtttatcgccgtaccgttgatggggcccaacgcccggccaataggggtgctggcggcccagc
cgatggcgcgccaggaagagcggctgccggcctgcacccgttttctcgaaaccgtc
195 atggcgctgaagcacctgatcacgctctgcgcggcgtcgccgatggtcgccagccagctggcgcgccacccgctgctgaggatgagctgctggat
cccaacaccctctatcagccgacggcgaccgatgcctatcgcgacgagctgcgccagtacctgctgcgcgtgccggaagaggatgaagagcagca
gctgcatatcgcggcggcggatatcgctggtaccctgccggtgatgaaggtcagcgatcacttaacctggcttgccgaagcgatcctcgacgcggtg
gtgcagcaggcatgggggcagatggtcgctcgctacggccagccgacccacctgcacgatcgccagggtcgcggcttcgccgtcgtcggctacg
gtaagcttggcggctgggagctgggctacagctccgatctcgatctggtgttcctccatgactgcccggcggaggtgatgaccgacggcgagcggg
agattgacggccgtcagttctacctgcggctggcccagcggatcatgcacctgttcagcacccgcacctcgtccggtattctctacgaagtggacgcc
cggctgcgtccttaggcgcggcggggatgctggtcaccaccgccgacgcgtttgctgactatcagcagaacgaagcctggacgtgggaacatcag
gcgctggtgcgcgcccgcgtggtctatggcgacccggcgctgcaggcgcgctttgacgccattcgtcgcgatatcctgaccaccccgcgggaggg
gatgaccctgcagaccgaggttcgcgagatgcgcgagaagatgcgcgcccaccttggcaacaaacatcccgatcgttttgatatcaaagccgatgcc
ggcgggatcaccgatattgaatttattactcagtatctggtcctacgctatgccagtgacaagccgaagctgacccgctggtctgacancgtgcgtattct
tgagctgctggcgcagaacgacatcatggacgaggaggaggcgcgcgccttaacgcatgcgtacaccaccttgcgtgatgcgctccatcacctggc
cctgcaggagcagccgggacacgtggcgccagaggccttcagccgggagcgtcagcaggtcagcgccagctggcagaagtggctgatggcttaa
196 cgtaaggcgaccacccagctccgcgcgttgctgaacgacgctgaagccgttctgctaccgcggacaccgccgacgaggcgttattccgcaccga
ggtcgtcggcgccaaactggccctgactgaatggctggtccagcgcggctggcgtccgttcctcaacgaggcaggagagaaaaaaatagccggat
cgttcaaacggtttgccgatattaacctctcgcgggtggcggccgagctgcgcagcgccgtgcagcatctggcggttgaagatgccgccgaccagtt
gccgaagctgtcccgcgacatcgacagcgtccagctgctggcgggcgcctatggcgacgccgtcgcgccgtggctggagaactggcaggagctt
caccgtgcaatagcacatgaccatcgcagcgtctttgaatatttccgtcgccaggcgctggctgccgagccgttctggctgcatagtggaaaacgata
atttcaggccagggagcccttatggcgctgaagcacctgatcacgctctgcgcggcgtcgccgatggtcgccagccagctggcgcgccacccgctg
ctgctggatgagctgctggatcccaacaccctctatcagccgacggcgaccgatgcctatcgcgacgagagcgccagtacctgctgcgcgtgccg
gaagaggatgaagagcaggctgcatatcgcggcggcggatatcgctggtaccctgccggtgatgaaggtcagcgatcacttaacctggcttgcc
gaagcgatcctcgacgcggtggtgcagcaggcatgggggcagatggtcgctcgctacggccagccgacccacctgcacgatcgccagggtcgcg
gcttcgccgtcgtcggctacggtaagcttggcggctgggagctgggctacagctccgatctcgatctggtgttcctccatgactgcccggcggaggtg
atgaccgtcggcgagcgggagattgacggccgtcagttctacctgcggctggcccagcggatcatgcacctgttcagcacccgcacctcgtccggt
attctctacgaagtggacgcccggctgcgtccttctggcgcggcggggatgctggtcaccaccgccgacgcgtttgagactatcagcagaacgaag
cctggacgtgggattcatcaggcgctggtgcgcgcccgcgtggtctatggcgacccggcgctgcaggcgcgctttgacgccattcgtcgcgatatcc
tgaccaccccgcgggaggggatgaccctgcagaccgaggttcgcgagatgcgcgagaagatgcgcgcccaccttggcaacaaacatcccgatcg
ttttgatatcaaagccgatgccggcgggatcaccgatattgaatttattactcagtatctggtcctacgctatgccagtgacaagccgaagctgacccgct
ggtctgacaacgtgcgtattcttgagctgctggcgcagaacgacatcatggacgaggaggaggcgcgcgccttaacgcatgcgtacaccaccttgc
gtgatgcgctccatcacctggccctgcaggagcagccgggacacgtggccccagaggccttcagccgggagcgtcagcaggtcagcgccagctg
gcagaagtggctgatggcttaactataaaatcgggtgtgctattatcgcgcgcaaagtttgcgtctcgcaggagagagtcatgaaagtaacgctgccg
gagtttgaacgtgcaggagtgttggtggtgggtgatgtgatgctggaccgctactggtacggccccaccagtcgtatttccccggaagcccoggtgcc
ggtggtgaaggtggtaaatatcgaagaacgtcctggcggcgcggcaaacgtagcgatgaacatcgcctccctgggggcaacgtcgcgcctggtgg
gattgaccgggattgatgacgctgcccgcgcgctgagccaggcgctggccaatgtgaatgtgaagtgcgacttcgtctccgtcccgactcacccgac
catcaccaagctgcgggtgctgtcgcgcaatcagcagctgatccgcctcgactttgaagagggcttctccggcgtggatccgcagccgatgcatgag
cgcattcagcaggcgctgggagccattggcgcactgg
197 atgaccctgaatataagctcgacgccgtcctcgcagtaccattgcaaccgactttacagcaagaagtgattctggcacgcatggaacaaattcttgcca
gtcgggctttatccgatgacgaacgcgcacagcttttatatgagcgcggagtgttgtatgatagtctcggtctgagggcattagcgcgaaatgatttttca
caagcgctggcaatccgacccgatatgcctgaagtattcaattacttaggcatttacttaacgcaggcaggcaattttgatgctgcctatgaagcgtttgat
tctgtacttgagcttgatcgccattgagctggcttcccgaccgcagggcggcacctgcctgaccctgcgtttcccgctgtttaacaccctgaccggaggt
gaagcatga
198 cccaacagcagggccgggtaggccagcaggtccgccagcgtggcgcggttaatattgaccggggcggcggcggcctcccccagctgcttgtgga
tcattttcgcgatcttgcgggttttaccggtatcggtaccaaagaaaatgccaatgttcgccatagtacgctcctgtcggaatggtgttgaaaaaaggaat
gacgacagaggtattgcgaaggctgtgccaggttgccctgcaccgcgacggcccatccctgccccatcaggatcgcttcgcatcacgatgccgcgc
gccaaaggcgcacccggcggggcgaaaggtaaaaatccgtgaattttccccctgtcggatcaatgatcgcgtggtcgttccgataagggcgcacac
tttgcatggttatccgggttcggcttaccccgccgcgttttgcgcacggtgtcggacaatttgtcataactgcgacacaggagtttgcgatgaccctgaat
atgatgctcgacgccgtcctcgcagtaccattgcaaccgactttacagcaagaagtgattctggcacgcatggaacaaattcttgccagtcgggctttat
ccgatgacgaacgcgcacagcattatatgagcgcggagtgttgtatgatagtctcggtctgagggcattagcgcgaaatgatttttcacaagcgctggc
aatccgacccgatatgcctgaagtattcaattacttaggcatttacttaacgcaggaggcaattttgatgctgcctatgaagcgtttgattctgtacttgag
cttgatcgccattgagaggcttcccgaccgcagggcggcacctgcctgaccctgcgtttcccgctgtttaacaccctgaccggaggtgaagcatgat
ccctgaatccgacccggacaccaccgtcagacgcttcgacctctctcagcagttcaccgccatgcagcggataagcgtggtgctgagccgggccac
cgaggccagcaaaacgctgcaggaggtgctcagcgtattacacaacgatgcctttatgcagcacgggatgatctgcctgtacgacagcgagcagga
gatcctcagtatcgaagcgctgcagcaaaccggccagcagcccctccccggcagcacgcagatccgctatcgccccggcgagggactggtgggg
accgtgctggcccaggggcagtcgctggtgctgccccgggtcgccgacgatcagcgttttctcgaccgcctgagcctctacgattacgatctgccgtt
tatcgccgtaccgttgatggggcccaacgcccggccaataggggtgctggcggcccagccgatggcgcgccaggaagagcggctgccggcctgc
acccgttttctcgaaaccgtc
199 atggcgctgaagcacctgatcacgctctgcgcggcgtcgccgatggtcgccagccagctggcgcgccacccgctgctgctggatgagctgctggat
cccaacaccctctatcagccgacggcgaccgatgcctatcgcgacgagctgcgccagtacctgctccgcgtgccggaagaggatgaagagcagca
gctgcatatcgcggcggcggatatcgctggtaccctgccggtgatgaaggtcagcgatcacttaacctggcttgccgaagcgatcctcgacgcggtg
gtgcagcaggcatgggggcagatggtcgctcgctacggccagccgacccacctgcacgatcgccagggtcgcggcttcgccgtcgtcggctacg
gtaagcttggcggctgggagctgggctacagctccgatctcgatctggtgttcctccatgactgcccggcggaggtgatgaccgacggcgagcggg
agattgacggccgtcagttctacctgcggctggcccagcggatcatgcacctgttcagcacccgcacctcgtccggtattctctacgaagtggacgcc
cggctgcgtccttctggcgcggcggggatgctggtcaccaccgccgacgcgtttgctgactatcagcagaacgaagcctggacgtgggaacatcag
gcgctggtgcgcgcccgcgtggtctatggcgacccggcgctgcaggcgcgctttgacgccattcgtcgcgatatcctgaccaccccgcgggaggg
gatgaccctgcagaccgaggttcgcgagatgcgcgagaagatgcgcgcccaccttggcaacaaacatcccgatcgttttgatatcaaagccgatgcc
ggcgggatcaccgatattgaatttattactcagtatctggtcctacgctatgccagtgacaagccgaagctgacccgctggtctgacaacgtgcgtattct
tgagctgctggcgcagaacgacatcatggacgaggaggaggcgcgcgccttaacgcatgcgtacaccaccttgcgtgatgcgctccatcacctggc
cctgcaggagcagccgggacacgtggcgccagaggccttcagccgggagcgtcagcaggtcagcgccagctggcagaagtggctgatggcttaa
200 cgtaaggcgaccacccagctccgcgcgttgctgaacgacgctgaagccgttctgctggccgcggacaccgccgacgaggcgttattccgcaccga
ggtcgtcggcgccaaactggccctgactgaatggctggtccagcgcggctggcgtccgttcctcaacgaggcaggagagaaaaaaatagccggat
cgttcaaacggtttgccgatattaacctctcgcgggtggcggccgagctgcgcagcgccgtgcagcatctggcggttgaagatgccgccgaccagtt
gccgaagctgtcccgcgacatcgacagcgtccagctgctggcgggcgcctatggcgacgccgtcgcgccgtggctggagaactggcaggagctt
caccgtgcaatagcacatgacgatcgcagcgtctttgaatatttccgtcgccaggcgctggctgccgagccgttctggctgcatagtgggaaaacgata
atttcaggccagggagcccttatggcgctgaagcacctgatcacgctctgcgcggcgtcgccgatggtcgccagccagctggcgcgccacccgctg
ctgctggatgagctgctggatcccaacaccctctatcagccgacggcgaccgatgcctatcgcgacgagctgcgccagtacctgctgcgcgtgccg
gaagaggatgaagagcagcagctgcatatcgcggcggcggatatcgctggtaccctgccggtgatgaaggtcagcgacacttaacctggcttgcc
gaagcgatcctcgacgcggtggtgcagcaggcatgggggcagatggtcgctcgctacggccagccgacccacctgcacgatcgccagggtcgcg
gcttcgccgtcgtcggctacggtaagcttggcggctgagagctgggctacagctccgatctcgatctggtgttcctccatgactgcccggcggaggtg
atgaccgacggcgagcgggagattgacggccgtcagttctacctccggctggcccagcggatcatgcacctgttcagcacccgcacctcgtccggt
attctctacgaagtggacgcccggctgcgtccttctggcgcggcggggatgctggtcaccaccgccgacgcgtttgctgactatcagcagaacgaag
cctggacgtgggaacatcaggcgctggtgcgcgcccgcgtggtctatggcgacccggcgctgcaggcgcgctttgacgccattcgtcgcgatatcc
tgaccaccccgcgggaggggatgaccctgcagaccgaggttcgcgagatgcgcgagaagatgcgcgcccaccttggcaacaaacatcccgatcg
ttttgatatcaaagccgatgccggcgggatcaccgatattgaatttattactcagtatctggtcctacgctatgccagtgacaagccgaagctgacccgct
ggtctgacaacgtgcgtattcttgagctgctggcgcagaacgacatcatggacgaggaggaggcgcgcgccttaacgcatgcgtacaccaccttgc
gtgatgcgctccatcacctggccctgcaggagcagccgggacacgtggcgccagaggccttcagccgggagcgtcagcaggtcagcgccagctg
gcagaagtggctgatggcttaactataaaatcgggtgtgctattatcgcgcgcaaagtttgcgtctcgcaggagagagtcatgaaactaacgctgccg
gagtttgaacgtgcaggagtgttggtggtgggtgatgtgatgctggaccgctactggtacggccccaccagtcgtatttccccggaagccccggtgcc
ggtggtgaaggtggaaaatatcgaagtacgtcctggcggcgcggcaaacgtagcgatgaacatcgcctccctgggggcaacgtcgcgcctggtgg
gattgaccgggattgatgacgctgcccgcgcgctgagccaggcgctggccaatgtgaatgtgaagtgcgacttcgtctccgtcccgactcacccgac
catcaccaagctgcgggtgctgtcgcgcaatcagcagctgatccgcctcgactttgaagagggcttctccggcgtggatccgcagccgatgcatgag
cgcattcagcaggcgctgggagccattggcgcactgg
201 atgaccctgaatatgatgctcgagctaaagttctcggctaatcgctgattacatttgacgcaatgcgcaataaaagggcatcatttgatgccctttttgcac
gctttcataccagaacctggctcatcagtgattttttttgtcataatcattgctgagacaggctctgaagagggcgtttatacaccaaaccattcgagcggt
agcgcgacggcaagtcagcgttctcctttgcaatagcagggaagaggcgccagaaccgccagcgttgaagcagtttgaacgcgttcagtgtataatc
cgaaacttaatttcggtttggagccattgagctggcttcccgaccgcagggcggcacctgcctgaccctgcgtttcccgctgtttaacaccctgaccgg
aggtgaagcatga
202 ggccgtcgcccagcgtcggcgtccccaacagcagggccgggtaggccagcaggtccgccagcgtggcgcggttaatattgaccggggcggcgg
cggcctcccccagctgcttgtggatcattttcgcgatcttgcgggttttaccggtatcggtaccaaagaaaatgccaatgttcgccatagtacgctcctgt
cggaatggtgttgaaaaaaggaatgacgacagtggtattgcgaaggctgtgccaggttgccctgcaccgcgacggcccatccctgccccatcagga
tcgcttcgcatcacgatgccgcgcgccaaaggcgcacccggcggggcgaaaggtaaaaatccgtgaattttccccctgtcggatcaatgtttcgcgtg
gtcgttccgataagggcgcacactttgcatggttatccgggttcggcttaccccgccgcgttttgcgcacggtgtcggacaatttgtcataactgcgaca
caggagtttgcgatgaccctgaatatgatgctcgagctaaagttctcggctaatcgctgataacatttgacgcaatgcgcaataaaagggcatcatttgat
gcccttttgcacgctttcataccagaacctggctcatcagtgattttttttgtcataatcattgctgagacaggctctgaagagggcgtttatacaccaaac
cattcgagcggtagcgcgacggcaagtcagcgttctcctttgcaatagcagggaagaggcgccagaaccgccagcgttgaagcagtttgaacgcgt
tcagtgtataatccgaaacttaatttcggtttggagccattgagctggcttcccgaccgcagggcggcacctgcctgaccctgcgtttcccgctgtttaac
accctgaccggaggtgtagcatgatccctgaatccgacccagacaccaccgtcagacgcttcgacctctctcagcagttcaccgccatgcagcggat
aagcgtggtgctgagccgggccaccgaggccagcaaaacgctgcaggaggtgctcagcgtattacacaacgatgcctttatgcagcacgggatgat
ctgcctgtacgacagcgagcaggagatcctcagtatcgaagcgctgcagcaaaccggccagcagcccctccccggcagcacgcagatccgctatc
gccccggcgagggactggtggggaccgtgctggcccaggggcagtcgctggtgctgccccgggtcgccgacgatcagcgttttctccaccgcctg
agcctctacgattacgatctgccgtttatcgccgtaccgttgatggggcccaacgcccggccaataggggtgctggcggcccagccgatggcgcgc
caggaagagcggctgccggcctgcacccgttttctcgaaaccgtc
203 atgaccctgaatatgatgctcgagcccgctgaccgaccagaacttccaccttggactcggctatacccttggcgtgacggcgcgcgataactgggact
acatccccattccggtgatcttaccattggcgtcaataggttacggtccggcgactttccagatgacctatattcccggcacctacaataacggtaacgttt
acttcgcctgggctcgtatacagttttaattcgctaagtcttagcaataaatgagataagcggtgtgtcttgtggaaaaacaaggactaaagcgttaccca
ctaaaaaagatagcgacttttatcactttttagcaaagttgcactggacaaaaggtaccacaattggtgtactgatactcgacacagcattagtgtcgatttt
tcatataaaggtaattttggccattgagctggcttcccgaccgcagggcggcacctgcctgaccctgcgtttcccgctgtttaacaccctgaccggaggt
gaagcatga
204 ggccgtcgcccagcgtcggcgtccccaacagcagggccgggtaggccagcaggtccgccagcgtggcgcgcttaatattgaccggggcggcgg
cggcctcccccagctgcttgtggatcattttcgcgatcttgcgggttttaccggtatcggtaccaaagaaaatgccaatgttcgccatagtacgctcctgt
cggaatggtgttgaaaaaaggaatgacgacagaggattgcgaaggctgtgccaggttgccctgcaccgcgacggcccatccctgccccatcagga
tcgcttcgcatcacgatgccgcgcgccaaaggcgcacccggcggggcgaaaggtaaaaatccgtgaattttccccctgtcggatcaatgtttcgcgtg
gtcgttccgataagggcgcacactttgcatggttatccgggttcggcttaccccgccgcgttttgcgcacggtgtcggacaatttgtcataactgcgaca
caggagtttgcgatgaccctgaatatgatgctcgagcccgctgaccgaccagaacttccaccttggactcggctatacccttggcgtgacggcgcgcg
ataactgggactacatccccattccggtgatcttaccattggcgtcaataggttacggtccggcgactttccagatgacctatattcccggcacctacaat
aacggtaacgtttacttcgcctgggctcgtatacagttttaattcgctaagtcttagcaataaatgagataagcggtgtgtcttgtggaaaaacaaggacta
aagcgttacccactaaaaaagatagcgacttttatcactttttagcaaagttgcactggacaaaaggtaccacaattggtgtactgatactcgacacagca
ttagtgtcgatttttcatataaaggtaattttggccattgagctggcttcccgaccgcagggcggcacctgcctgaccctgcgtttcccgctgtttaacacc
ctgaccggaggtgaagcatgatccctgaatccgacccggacaccaccgtcagacgatcgacctctctcagcagttcaccgccatgcagcggataag
cgtggtgctgagccgggccaccgaggccagcaaaacgctgcaggaggtgctcagcgtattacacaacgatgcctttatgcagcacgggatgatctg
cctgtacgacagcgagcaggagatcctcagtatcgaagcgctgcagcaaaccggccagcagcccctccccggcagcacgcagatccgctatcgcc
ccggcgagggactggtggggaccgtgctggcccaggggcagtcgctggtgctgccccgggtcgccgacgatcagcgttttctcgaccgcctgagc
ctctacgattacgatctgccgtttatcgccgtaccgttgatggggcccaacgcccggccaataggggtgctggcggcccagccgatggcgcgccag
gaagagcggctgccggcctgcacccgttttctcgaaaccgtc
205 atggcgctgaagcacctgatcacgctctgcgcggcgtcgccgatggtcgccagccagctggcgcgccacccgctgctgctggatgagctgctggat
cccaacaccctctatcagccgacggcgaccgatgcctatcgcgacgagctgcgccagtacctgctgcgcgtgccggaagaggatgaagagcagca
gctgcatatcgcggcggcggatatcgctggtaccctgccggtgatgaaggtcagcgatcacttaacctggcttgccgaagcgatcctcgacgcggtg
gtgcagcaggcatgggggcagatggtcgctcgctacggccagccgacccacctgcacgatcgccagggtcgcgccttcgccgtcgtcggctacg
gtaagcttggcggctgggagagggctacagctccgatctcgatctggtgttcctccatgactgcccggcggaggtgatgaccgacggcgagggg
agattgacggccgtcagttctacctgcggctggcccagcggatcatgcacctgttcagcacccgcacctcgtccggtattctctacgaagtggacgcc
cggctgcgtccttctggcgcggcggggatgctggtcaccaccgccgacgcgtttgctgactatcagcagaacgaagcctggacgtgggaacatcag
gcgctggtgcgcgcccgcgtggtctatggcgacccggcgctgcaggcgcgctttgacgccattcgtcgcgatatcctgaccaccccgcgggaggg
gatgaccctgcagaccgaggttcgcgagatgcgcgagaagatgcgcgcccaccttggcaacaaacatcccgatcgttttgatatcaaagccgatgcc
ggcgggatcaccgatattgaatttattactcagatctggtcctacgctatgccagtgacaagccgaagctgacccgctggtctgacaacgtgcgtattct
tgagctgctggcgcagaacgacatcatggacgaggaggaggcgcgcgccttaacgcatgcgtacaccaccttgcgtgatgcgctccatcacctggc
cctgcaggagcagccgcgacacgtggcgccagaggccttcagccgggagcgtcagcaggtcagcgccagctggcagaagtggctgatggcttaa
206 cgtaaggcgaccacccagctccgcgcgttgctgaacgacgctgaagccgttctgctggccgcggacaccgccgacgaggcgttattccgcaccga
ggtcgtcggcgccaaactggccctgactgaatggctggtccagcgcggctggcgtccgttcctcaacgaggcaggagagaaaaaaatagccggat
cgttcaacggtttgccgatattaacctctcgcaggtggcggccgagctgcgcagcgccgtgcagcatctggcggttgaagatgccgccgaccagtt
gccgaagctgtcccgcgacatcgacagcgtccagctgctggcgggcgcctatggcgacgccgtcgcgccgtggctggagaactggcaggagctt
caccgtgcaatagcacatgacgatcgcagcgtctttgaatatttccgtcgccaggcgctggctgccgagccgttctggctgcatagggaaaacgata
atttcaggccagggagcccttatggcgctgaagcacctgatcacgctctgcgcggcgtcgccgatggtcgccagccagctggcgcgccacccgctg
ctgctggatgagctgctggatcccaacaccctctatcagccgacggcgaccgatgcctatcgcgacgagctgcgccagacctgctgcgcgtgccg
gaagaggatgaagagcagcagctgcatatcgcggcggcggatatcgctggtaccctgccggtgatgaaggtcagcgatcacttaacctggcttgcc
gaagcgatcctcgacgcggtggtgcagcaggcatgggggcagatggtcgctcgctacggccagccgacccacctgcacgatcgccagggtcgcg
gcttcgccgtcgtcggctacggtaagcttggcggctgggagctgggctacagctccgatctcgatctggtgttcctccatgactgcccggcggaggtg
atgaccgacggcgaggggagattgacggccgcagttctacctgcggctggcccagcggatcatgcacctgttcagcacccgcacctcgccggt
attctctacgaagtggacgcccggctgcgtccttctggcgcggcgcggatgctggtcaccaccgccgacgcgtttgctgactatcagcagaacgaag
cctggacgtgggaacatcaggcgctggtgcgcgcccgcgtggtctattgcgacccggcgctgcaggcgcgctttgacgccattcgtcgcgatatcc
tgaccaccccgcgggaggggatgaccctgcagaccgaggttcgcgagatgcgcgagaagatgcgcgcccaccttggcaacaaacatcccgatcg
ttttgatatcaaagccgatgccggcgggatcaccgatattgaatttattactcagtatctggtcctacgctatgccagtgacaagccgaagctgacccgct
ggtctgacaacgtgcgtattcttgagctgctggcgcagaacgacatcatggacgaggaggaggcgcgcgccttaacgcatgcgtacaccaccttgc
gtgatgcgctccatcacctggccctgcaggagcagccgggacacgtggcgccagaggccttcagccgagagcgtcagcaggtcagcgccagctg
gcagaagtggctgatggcttaactataaaatcgggtgtgctattatcgcgcgcaaagtttgcgtctcgcaggagagagtcatgaaagtaacgctgccg
gagtttgaacgtgcaggagttggtggtgggtgatgtgatgctggaccgctactggtacggccccaccagtcgtatttccccggaagccccggtgcc
ggtggtgaaggtggaaaatatcgaagaacgtcctggcggcgcggcaaacgtagcgatgaacatcgcctccctgggggcaacgtcgcgcctggtgg
gattgaccgggattgatgacgctgcccgcgcgctgagccaggcgctggccaatgtgaatgtgaagtgcgacttcgtctccgtcccgactcacccgac
catcaccaagctgcgggtgctgtcgcgcaatcagcagctgatccgcctcgactttgaagagggcttctccggcgtggatccgcagccgatgcatgag
cgcattcagcaggcgctgggagccattggcgcactgg
207 atgaccctgaatatgatgctagaagcgtcaggtaccggtcatgattcaccgtgcgattctcggttccctggagcgcttcattggcatcctgaccgaagag
ttcgctggcttcttcccaacctggattgcaccagtgcaggtagtggtcatgaatattaccgattctcaggctgaatacgttaccgaangacgcgtaaacta
caaaatgcgggcattcgtgtaaaagcagacttgagaaatgagaagattggctttaaaatccgcgagcacactttacgtcgtgtcccgtatatgtggtct
gtggcgacaaagaagtcgaagccggcaaagtggccgtgcgcacccgtcgcgggaaagacctcggcagcatggacgtaagtgaagtgattgagaa
gctgcaacaagagattcgcagccgcagtcttcaacaactggaggaataaggtattaaaggcggaaaacgagttcaaacggcacgtccgaatcgtatc
aatgccgagattcgcgccctcgaagttcgcgccattgagctggcttcccgaccgcagggcgccacctgcctgaccctgcgtttcccgctgtttaacac
cctgaccggaggtgaagcatga
208 ggccgtcgcccagcgtcggcgtccccaacagcagggccgggtaggccagcaggccgccagcgtggcgcggttaatattgaccggggcggcgg
cggcctcccccagagcttgtggatcattttcgcgatcttgcgggttttaccggtatcgaaccaattgaaaatgccaatgttcgccatagtacgctcctgt
cggaatggtgttgaaaaaaggaatgacgacagaggtattgcgaaggctggccaggttgccctgcaccgcgacggcccatccctgccccatcagga
tcgcttcgcatcacgatgccgcgcgccaaaggcgcacccggcggggcgaaaggtaaaaatccgtgaattttccccctgtcggatcaatgtttcgcgtg
gtcgttccgataagggcgcacactttgcatggttatccgggttcggcttaccccgccgcgttttgcgcacggtgtcggacaatttgtcataactgcgaca
caggagtttgcgatgaccctgaatatgatgctagaagcgtcaggtaccggtcatgattcaccgtgcgattctcggttccctggagcgatcattggcatc
ctgaccgaagagttcgctggcttcttcccaacctggattgcaccagtgcaggtagtggtcatgaatattaccgattctcaggctgaatacgttaacgaatt
gacgcgtaaactacaaaatgcggcattcgtgtaaaagcagacttgagaaatgagaagattggctttaaaatccgcgagcacactttacgtcgtgtccc
gtatatgttggtctgtggcgacaaagaagtcgaagccggcaaagtggccgtgcgcacccgtcgcgggaaagacctcggcagcatggacgtaagtg
aagtgattgagaagctgcaacaagagattcgcagccgcagtcttcaacaactggaggaataaggtattaaaggcggaaancgagttcaaacggcac
gtccgaatcgtatcaatggcgagattcgcgccctggaagttcgcgccattgagctggcttcccgaccgcagggcggcacctgcctgaccctgcgtttc
ccgctgtttaacaccctgaccggaggtgaagcatgatccctgaatccgacccggacaccaccgtcagacgcttcgacctctctcagcagttcaccgcc
atgcagcggataagcgtggtgctgagccgggccaccgaggccagcaaaacgctgcaggtggtgctcagcgtattacacaacgatgcctttatgcag
cacgggatgatctgcctgtacgacagcgagcaggagatcctcagtatcgaagcgctgcagcaaaccggccagcagcccctccccggcagcacgc
agatccgctatcgccccggcgagggactggtggggaccgtgctggcccaggggcagtcgctggtgctgccccgggtcgccgacgatcagcgtttt
ctcgaccgcctgagcctctacgattacgatctgccgtttatcgccgtaccgttgatggggcccaacgcccggccaataggggtgctggcggcccagc
cgatggcgcgccaggaagagcggctgccggcctgcacccgttttctcgaaaccgtc
209 atggcgctgaagcacctgatcacgactgcgcggcgtcgccgatggtcgccagccagctcgcgcgccacccgctgctgctggatgagctgctggat
cccaacaccctctatcagccgacggcgaccgatgcctatcgcgacgagctgcgccagtacctgctgcgcgtgccggaagaggatgaagagcagca
gctggaggcgttgcgccagtttaagcaggcgcagcagctgcatatcgcggcggcggatatcgctggtaccctgccggtgatgaaggtcagcgatca
cttaacctggcttgccgaagcgatcctcgacgcggtggtgcagcaggcatgggggcagatggtcgctcgctacggccagccgacccacctgcacg
atcgccagggtcgcggcttcgccgtcgtcggctacggtaagcttacggctgggagctgggctacagctccgatctcgatctggtgttcctccatgac
tgcccggcggaggtgatgaccgacggcgagcggcagattgacggccgtcagttctacctgcggctggcccagcggatcatgcacctgttcagcac
ccgcacctcgtccggtattctctacgaagtggacgcccggctgcgtccttctggcgcggcggggatgctggtcaccaccgccgacgcgtttgctgact
atcagcagaacgaagcctggacgtgggaacatcaggcgctggtgcgcgcccgcggtctatggcgacccggcgctgcaggcgcgctttgacgc
cattcgtcgcgatatcctgaccaccccgcgggaggggatgaccctgcagaccgaggttcgcgagatgcgcgagaagatgcgcgcccaccttggca
acaaacatcccgatcgttttgatatcaaagccgatgccggcgggatcaccgatattgaatttattactcagtatctggtcctacgctatgccagtgacang
ccgaagctgacccgctggtagacaacgtgcgtattcttgagctgctggcgcagaacgacatcatggacgaggaggaggcgcgcgccttaacgcat
gcgtacaccaccttgcgtcatgcgctccatcacctggccctgcaggagcagccgggacacgtggcgccagaggccttcagccgggagcgtcagca
ggtcagcgccagctggcagaagtggctgatggcttaa
210 cgtaaggcgaccacccagctccgcgcgttgctgaacgacgctgaagccgttctgctggccgcggacaccgccgacgaggcgttattccgcaccga
ggtcgtcggcgccaaactggccctgactgaatggctggtccagcgcggctggcgtccgttcctcaacgaggcaggagagaaaaaaatagccggat
cgttcaaacggtttgccgatattaacctctcgcgggtggcggccgagctgcgcagcgccgtgcagcatctggcggttgaagatgcgccgaccagtt
gccgaagctgtcccgcgacatcgacagcgtccagctgctggcgggcgcctatggcgacgccgtcgcgccgtggctggagaactggcaggagctt
caccgtgcaatagcacatgacgatcgcagcgtctttgaatatttccgcgccaggcgctggctgccgagccgttctggcttcatagtggaaaacgata
atttcaggccagggagcccttatggcgctgaagcacctgatcacgctagcgcggcgtcgccgatggtcgccagccagctggcgcgccacccgctg
ctgctggatgagctgctggatcccaacaccctctatcagccgacggcgaccgatgcctatcgcgacgagctgcgccagtacctgctgcgcgtgccg
gaagaggatgaagagcagcagctggaggcgttgcgccagtttagcaggcgcagcagctgcatatcgcggcggcggatatcgctggtaccctgcc
ggtgatgaaggtcagcgatcacttaacctggcttgccgaagcgatcctcgacgcggtggtgcagcaggcatgggggcagatggtcgctcgctacgg
ccagccgacccacctgcacgatcgccagcgtcgcggcttcgccgtcgtcggctacggtaagcttggcggctgggagctgggctacagctccgatct
cgatctggtgttcctccatgtctgcccggcggaggtgatgaccgacggcgagcgggagattgacggccgtcagttctacctgcggctggcccagcg
gatcatgcacctgttcagcacccgcacctcgtccggtattctctacgaagtggacgcccggctgcgtccttctggcgcggcggggatgctggtcacca
ccgccgacgcgtttgctgactatcagcagaacgaagcctggacgtgggaacatcaggcgctggtgcgcgcccgcgtggtctatgccgacccggcg
ctgcaggcgcgctttgacgccattcgtcgcgatatcctgaccaccccgcgggaggggatgaccctgcagaccgaggttcgcgagatgcgcgagaa
gatgcgcgcccaccttggcaacaaacatcccgatcgttttgatatcaaagccgatgccggcgggatcaccgatattgaatttattactcagtatctggtcc
tacgctatgccagtgacaagccgaagctgacccgctggtctgacaacgtgcgtattcttgagctgctggcgcagaacgacatcatggacgaggagga
ggcgcgcgccttaacgcatgcgtacaccaccttgcgtgatgcgctccatcacctggccctgcaggagcagccgggacacgtggcgccagaggcct
tcagccgggagcgtcagcaggtcagcgccagctggcagaagtggctgatcgcttaactataaaatcgggtgtgctattatcgcgcgcaaagtttgcgt
ctcgcaggagagagtcatgaaagtaacgctgccggagttgaacgtgcaggagtgttggtggtggggatgtgatgctggaccgctactggtacggc
cccaccagtcgtatttccccggaagccccggtgccggtggtgaaggtggaaaatatcgaagaacgtcctggcggcgcggcaaacgtagcgatgaa
catcgcctccctgggggcaacgtcgcgcctggtgggattgaccgggattgatgacgctgcccgcgcgctgagccaggcgctggccaatggaatgt
gaagtgcgacttcgtctccgtcccgactcacccgaccatcaccaagctgcgggtgctgtcgcgcaatcagcagctgatccgcctcgactttgaagag
ggcttctccggcgtggatccgcagccgatgcatgagcgcattcagcaggcgctgggagccattggcgcactgg
211 atggcgctcaaacagttaatccgtctgtgtgccgcctcgccgatggtcgcgacaacttgcacgtcatcctttattgctcgatgaactgctcgacccgc
gcacgctttaccagccgattgagccgggcgcttaccgcgacgaactgcgtcagtatagatgcgggtgccaacagaagacgaagaacagcagcttg
aagccgtgcgccagttcaaacaggcccagcatttgcgtatcgcagccggggatatttccggggcattgccggtgatgaaagtcagtgaccatttaacc
taccttgccgaggccattctcgatgtcgtggtgcagcatgcgtgggaacaaatggtcgtaaaatacgggcagcccgcgcatcttcagcaccgtgagg
ggcgcggttttgccgtggtcggttacgggaaactcggtggctgggagctgggttatagctcagatctggatctggtcttcctgctcgattgcgcgccgg
aggtgatgacggacggcgaacgcagcatcgacggacgtcagttttatatcggctggcgcagcgcattatgcacttattcagcacccggacatcgtca
ggcattctttacgaggttgatccgcgtagcgaccttccggcgcatccggcatgctggtcagtaccattgaagcgtttgcagattatcaggccaatgaag
cctggacgtgggagcatcaggcgctggttcgcgcgcgcgtggtttacggggatccgcaactgacacagcaatttaacgccacgcgtcgcgacattct
ttgccgccagcgcgatggcgacggcctgcgtaaggaggtccgtgaaatgcgcgagaaaatgtatgcccatctggggagtaaaaaagcccacgagtt
tgatctgaaagccgatccgggtggcatcacggatattgaattcattgcaccatacctggttctgcgtttcgcgcntgatgagccgaagctgacgcgctg
gtctgataacgtgcggatttttgaactgatggcacgatatgacatcatgccggaagaggaagcgcgccatctgacgcaggcttatgtgacgctgcgcg
atgaaattcatcatctggcgttgcaggaacacagcgggaagtggccgcggacagctttgctactgagcgcgcgcagatccgtgccagctgggcaa
agtggctcggctga
212 cggtactggaacagaaatcggcggatgcgtaggagatttgttatgacacggcctgtctgaagtgcaagtagtgcttacttcctggctggcaacctcag
gctggacgccgtttattgatgataaatctgcgaagaaactggacgcttccttcaaacgttttgctgacatcatgctcggtcgtaccgcagcggatctgaaa
gaagcctttgcgcagccactgacggaagaaggttatcgcgatcagctggcgcgcctgaaacgccagatcattaccttccatttgcttgccggtgcttac
cctgaaaaagacgtcgatgcgtatattgccggctgggtggacctgcaacaggccatcgttcagcagcaacacgcctgggaggattcggcccgttctc
acgcggtgatgatggatgattctggttaaacgggcaacctcgtaactgactgactagcctgggcaaactgcccgggcttttttttgcaaggaatctgat
ttcatggcgctcaaacagttaatccgctgtggccgcctcgccgatggtcgcgacacaacttgcacgtcatcctttattgctcgatgaactgctcgaccc
gcgcacgctttaccagccgattgagccgggcgcttaccgcgacgaactgcgtcagtatctgatgcgggtgccaacagaagacgaagaacagcagct
tgaagccgtgcgccagttcaaacaggcccagcatttgcgtatcgcagccggggatatttccggggcattgccggtgatgaaagtcagtgaccatttaa
cctaccttgccgaggccattctcgatgtcgtggtgcagcatgcgtgggaacaaatggtcgtaaaatacgggcagcccgcgcatcttcagcaccgtga
ggggcgcggttttgccgtggtcggttacgggaaactcggtggctgggagctgggttatagctcagatctggatctggtcttcctgctcgattgcgcgcc
ggaggtgatgacggacggcgaacgcagcatcgacggacgtcagttttatcttcggctggcgcagcgcattatgcacttattcagcacccggacatcgt
caggcattctttacgaggttgatccgcgctgcgaccttccggcgcatccggcatgaggtcagtaccattgaagcgtttgcagattatcaggccaatga
agcctggacgtgggagcatcaggcgaggttcgcgcgcgcgtggtttacggggatccgcaactgacacagcaatttaacgccacgcgtcgcgacat
tctttgccgccagcgcgatggcgacggcctgcgtaaggaggtccgtgaaatgcgcgagaaaatgtatgcccatctggggagtaaaaaagcccacga
gtttgatctgaaagccgatccgggtggcatcacggatattgaattcattgcacaatacctggttctgcgtttcgcgcatgatgagccgaagctgacgcgc
tggtctgataacgtgcggatttttgaactgatggcacgatatgacatcatgcccgaagaggaagcgcgccatctgacgcaggcttatgtgacgctgcg
cgatgaaattcatcatctggcgttgcaggaacacagcgggaaagtggccgcggacagctttgctactgagcgcgcgcagatccgtgccagctgggc
aaagtggctcggctgagggtttttattcggctaacaggcgcttgtgatattatccggcgcattgtatttacccgatttgatttatctgttttggagtcttgggat
gaaagtgactttgcctgattttcaccgcgcaggtgtgctggttgtcggtgacgtaatgttagaccgttactggtatggcccgaccaatcgtatttctccgga
agctccggtgccggtggtgaaggtcagtaccattgaagagcggcctggcggtgagctaacgtggcgatgaacatttcatctctgggcgcctcttcct
gtctgatcggcctgaccggcgtagacgacgctgcgtgccctcagtgagcgtctggcagaagtgaaagaaactgcgatttcgtcgcactatccaca
catcctaccatcaccaaactgcgaattttgtcccgtaaccagcaactgatccgcctcgactttgaggaaggttttgaaggcgttgatctcgagccgatgct
gaccaaaataga
213 atgagcatcacggcgttatcagcatcatttcctgaggggaatatcgccagccgcttgtcgctgcaacatccttcactgttttataccgtggttgaacaatct
tcggtggcgatttcgctgaccgatccgcaggcgcgcatttgttatgccaatccggcattctgccgccagacgggttttgcacttgagacacttttgggcg
agaaccaccgtctgctggaattttttttcacaaaggtagcgttattgaatcgcacattttaaactgttggccgctgtggaaggaatattggtgaaaggtg
cggttttaaggcctttttctttgactctctgtcgttacaaagttaatatgcgcgccctccgtctctgaagctctcggtgaacattgttgcgaggcaggatgcg
agctggttgtgttttgacattaccgataatgtgccgcgtgaacgggtgcgttatgcccgcccggaagcggcgttttcccgtccggggaatggcatggag
ctgcgccttatccagacgctgatcgcccatcatcgcggttctttagatctctcggtccgccctgatggcggcaccttgctgacgttacgcctgccggtac
agcaggttatcaccggaggcttaaaatga
214 tgtttcgtctcgaggccgggcaactgagcggccccgttgaaaccgacctgggctggcatctgttgttgtgcgaacaaattcgcctgccgcaacccttgc
cgaaagccgaagccttaacgcgggtgcgtcagcaactgattgcccggcaacagaaacattatcagcgccagtggctgcaacaactgatcaacgcct
gagcctgttctccttcttgttgatgcagacgggttaatgcccgttttgacgaaaaatgcacataaattgcctgcgttgccttataacagcgcagggaaat
cctgcctccggccttgtgccacaccgcgctttgcctggtttgtggtaaaaactggcccgctttgcatcctgatgcttaaaacaccccgttcagatcaacct
ttgggcagataagcccgcgaaaggcctgcaaattgcacggttattccgggtgagtatatgtgtgatttgggttccggcattgcgcaataaaggggaga
aagacatgagcatcacggcgttatcagcatcatttcctgaggggaatatcgccagccgcttgtcgctgcaacatccttcactgttttataccgtggttgaa
caatcttcggtggcgatttcgctgaccgatccgcaggcgcgcatttgttatgccaatccggcattctgccgccagacgggttttgcacttgagacactttt
gggcgagaaccaccgtctgctggaattttttttcacaaagcgtagcgttattgaatcgcacattttaaactgttggccgctgtggaagcgaatattggtga
aaggtgcggttttaaggcctttttctttgactctctgtcgttacaaagttaatatgcgcgccctccgtctctgaagctctcggtgaacattgttgcgaggcag
gatgcgagctggttgtgttttgacattaccgataatgtgccgcgtgaacgggtgcgttatgcccgcccggaagcggcgttttcccgtccggggaatggc
atggagctgcgccttatccagacgctgatcgcccatcatcgcggttctttagatctctcggtccgccctgatggcggcaccttgctgacgttacgcctgc
cggtacagcaggttatcaccggaggcttaaaatgacccagttacctaccgcgggcccggttatccggcgctttgatatgtctgcccagtttacggcgctt
tatcgcatcagcgtggcgctgagtcaggaaagcaacaccgggcgcgcactggcggcgatcctcgaagtgcttcacgatcatgcatttatgcaatacg
gcatggtgtgtctgtttgataaagaacgcaatgcactctttgtggaatccctgcatggcatcgacggcgaaaggaaaaaagagacccgccatgtccgtt
accgcatgggggaaggcgtgatcggcgcggtgatgagccagcgtcaggcgctggtgttaccgcgcatttcagacgatcagcgttttctcgaccgcct
gaatatttacgattacagcctgccgttgattggcgtgccgatccccggtgcggataatcagccatcgggcgtgctggtggcacagccgatggcgttgc
acgaagaccggctgactgccagtacgcggtttttagaaatggtc
215 atgagcatcacggcgttatcagcatcatttcctgaggggaatatcgccagccgcttgtcgctgcaacatccttcactgttttataccgtggttgaacaatct
tcggtggcgatttcgctgaccgatccgcaggcgcgcatttgttatgccaatccggcattctgccgccagacgggttttgcacttgagacacttttgggcg
agaaccaccgtctgctggaattttttttcacaaagcgtagcgttattgaatcgcacattttgatgttggccgctgtggaagcgaatattggtgaaaggtg
cggttttaaggcctttttctttgactctctgtcgttacaaagttaatatgcgcgccctccgtctctgaagctctcggtgaacattgttgcgaggcaggatgcg
agctggttgtgttttgacattaccgataatgtgccgcgtgaacgggtgcgttatgcccgcccggttgcggcgttttcccgtccggggaatggcatggag
ctgcgccttatccagacgctgatcgcccatcatcgcggttctttagatctctcggtccgccctgatggcggcaccttgctgacgttacgcctgccggtac
agcaggttatcaccggaggcttaaaatga
216 tgtttcgtctcgaggccgggcaactgagcggccccgttgaaaccgacctgggctggcatctgttgttgtgcgaacaaattcgcctgccgcaacccttgc
cgaaagccgaagccttaacgcgggtgcgtcagcaactgattgcccggcaacagaaacattatcagcgccagtggctgcaacaactgatcaacgcct
gagcctgttctccttcttgttgatgcagacgggttaatgcccgttttgcacgaaaaatgcacataaattgcctgcgttgccttataacagcgcagggaaat
cctgcctccggccttgtgccacaccgcgctttgcctggtttgtggtaaaaactggcccgctttgcatcctgatgcttaaaacaccccgttcagatcaacct
ttgggcagataagcccgcgaaaggcctgcaaattgcacggttattccgggtgagtatatgtgtgatttgggttccggcattgcgcaataaaggggaga
aagacatgagcatcacggcgttatcagcatcatttcctgaggggaatatcgccagccgcttgtcgctgcaacatccttcactgttttataccgtggttgaa
caatcttcggtggcgatttcgctgaccgatccgcaggcgcgcatttgttatgccaatccggcattctgccgccagacgggttttgcacttgagacactttt
gggcgagaaccaccgtctgctggaattttttttcacaaagcgtagcgttattgaatcgcacattttaaactgttggccgctgtggaagcgaatattggtga
aaggtgcggttttaaggcctttttctttgactctctgtcgttacaaagttaatatgcgcgccctccgtctctgaagctctcggtgagcattgttgcgaggcag
gatgcgagctggttgtgttttgacattaccgataatgtgccgcgtgaacgggtgcgttatgcccgcccggaagcggcgttttcccgtccggggaatggc
atggagctgcgccttatccagacgctgatcgcccatcatcgcggttctttagatctctcggtccgccctgatggcggcaccttgctgacgttacgcctgc
cggtacagcaggttatcaccggaggcttaaaatgacccagttacctaccgcgggcccggttatcccgcgctttgatatgtctgcccagtttacggcgctt
tatcgcatcagcgtggcgctgagtcaggaaagcaacaccgggcgcgcactggcggcgatcctcgaagtgcttcacgatcatgcatttatgcaatacg
gcatggtgtgtctgtttgataaagaacgcaatgcactctttgtggaatccctgcatggcatcgacggcgaaaggaaaaaagagacccgccatgtccgtt
accgcatgggggaaggcgtgatcggcgcggtgatgagccagcgtcaggcgctggtgttaccgcgcatttcagacgatcagcgttttctcgaccgcct
gaatatttacgattacagcctgccgttgtttggcgtgccgatccccggtgcggataatcagccatcgggcgtgctggtggcacagccgatggcgttgc
acgaagaccggctgactgccagtacgcggtttttagaaatggtc
217 atgagcatcacggcgttatcagcatcatttcctgaggggaatatcgccagccgcttgtcgctgcaacatccttcactgttttataccgtggttgaacaatct
tcggtggcgatttcgctgaccgatccgcaggcgcgcatttgttatgccaatccggcattctgccgccagacgggttttgcacttgagacacttttgggcg
agaaccaccgtctgctggttaaaaacgtgaccacgagcattaataaacgccacgaaatgtggcgtttatttattcaaaaagtatcttctttcataaaaagtg
ctaaatgcagtagcagcaaaattgggataagtcccatggaatacggctgttttcgctgcaatttttaactttttcgtaaaaaaagatgtttctttgagcgaac
gatcaaaatataggttaaccggcaaaaaattattctcattagaaaatagtttgtgtaatacttgtaacgctacatggagattaacttaatctagagggtttta
taccgtctctgaagctacggtgaacattgttgcgaggcaggatgcgagctggttgtgttttgacattaccgataatgtgccgcgtgaacgggtgcgttat
gcccgcccggaagcggcgttttcccgtccggggaatggcatggagagcgccttatccagacgctgatcgcccatcatcgcggttctttagatctctcg
gtccgccctgatggcggcaccttgctgacgttacgcctgccggtacagcaggttatcaccggaggcttaaaatga
218 gtttcgtctcgaggccgggcaactgagcggccccgttgaaaccgacctgggctggcatctgttgttgtgcgaacaaattcgcctgccgcaacccttgc
cgaaagccgaagccttaacgcgggtgcgtcagcaactgattgcccggcaacagaaacattatcagcgccagtggctgcaacaactgatcaacgcct
gagcctgttctccttatgttgatgcagacgggttaatgcccgttttgcacgaaaaatgcacataaattgcctgcgttgccttataacagcgcagggaaat
cctgcctccggccttgtgccacaccgcgctttgcctggtttgtggtaaaaactggcccgctttgcatcctgatgcttaaaacaccccgttcagatcaacct
ttgggcagataagcccgcgaaaggcctgcaaattgcacggttattccgggtgagtatatgtgtgatttgggttccggcattgcgcaataaaggggaga
aagacatgagcatcacggcgttatcagcatcatttcctgaggggaatatcgccagccgcttgtcgctgcaacatccttcactgttttataccgtggttgaa
caatcttcggtggcgatttcgctgaccgatccgcaggcgcgcatttgttatgccaatccggcattctgccgccagacgggttttgcacttgagacactttt
gggcgagaaccaccgtagctggttaaaaacgtgaccacgagcattaataaacgccacgaaatgtggcgtttatttattcaaaaagtatatctttcataa
aaagtgctaaggcagtagcagcaaaattgggataagtcccatggaatacggctgttttcgctgcaatttttaactttttcgtaaaaaaagatgtttctttgag
cgaacgatcaaaatatagcgttaaccggcaaaaaattattacattagaaaatagtttgtgtaatacttgtaacgctacatggagattaacttaatctagagg
gttttataccgtctctgaagctctcggtgaacattgttgcgaggcaggatgcgagaggttgtgttttgacattaccgataatgtgccgcgtgaacgcgtg
cgttatgcccgcccggaagcggcgttttcccgtccggggaatggcatggagctgcgccttatccagacgctgatcgcccatcatcgcggttctttagat
ctctcggtccgccctgatggcggcaccttgctgacgttacgcctgccggtacagcaggttatcaccggaggcttaaaatgacccagttacctaccgcg
ggcccggttatccggcgctttgatatgtctgcccagtttacggcgctttatcgcatcagcgtggcgctgagtcaggaaagcaacaccgggcgcgcact
ggcggcgatcctcgaagtgcttcacgatcatgcatttatgcaatacggcatggtgtgtctgtttgataaagaacgcaatgcactctttgtggaatccctgc
atggcatcgacggcgaaaggaaaaaagagacccgccatgtccgttaccgcatgggggaaggcgtgatcggcgcggtgatgagccagcgtcaggc
gctggtgttaccgcgcatttcagacgatcagcgttttctcgaccgcctgaatatttacgattacagcctgccgttgattggcgtgccgatccccggtgcgg
ataatcagccatcgggcgtgctggtggcacagccgatggcgttgcacgaagaccggctgactgccagtacgcgctttttagaaatggtcg
219 atgagcatcacggcgttatcagactcatttcctgagggaatatcgccagccgcttgtcgctgcaacatccttcactgttttataccgtggttgaacaatct
tcggtggcgatttcgctgaccgatccgcaggcgcgcatttgttatgccaatccggcattctgccgccagacgggttttgcacttgagacacttttgggcg
agaaccaccgtctgctggtgaacatcactgatgcacaagctacctatgtcgaagaattaactaaaaaactgaagatgcaggcattcgcgttaaagcc
gacttgagaaatgagaagattggctttaaaattcgcgaacacacgctacgccgtgttccttatatgttagtttgtggcgataaagaggtcgaagcaggca
aagttgctgttcgtacccgccgcggcaaagacttaggaagcatggatgttagcgaagtcgttgacaaactgctggcggaaatccgcagcagaagtctt
catcaactggaggaataaagtattaaaggcggaaaacgagttcaaccggcgcgtcctaatcgcattaacaaagagattcgcgcgcaagaagttcgcc
tcacaggcgtcgatggcgagcagattggtattgtcagtctgaatgaagctcttgaaaaagctgaggaagcgggcgtcgatttagtagaaatcagtccg
aatgccgagccgccagtttgtcgaatcccgtctagaagctctcggtgaacattgttgcgaggcaggatgcgagctggttgtgttttgacattaccgata
atggccgcgtgaacgggtgcgttatgcccgcccggaagcggcgttttcccgtccggggaatggcatggagctgcgccttatccagacgctgatcgc
ccatcatcgcggttctttagatctctcggtccgccctgatggcggcaccttgctgacgttacgcctgccggtacagcaggttatcaccggaggcttaaaa
tga
220 tgtttcgtctcgaggccgggcaactgagcggccccgttgaaaccgacctgggctggcatctgttggtgcgaacaaattcgcctgccgcaacccttgc
cgaaagccgaagccttaacgcgggtgcgtcagcaactgattgcccggcaacagaaacattatcagcgccagtggctgcaacaactgatcaacgcct
gagcctgttctccttcttgttgatgcagacgggttaatgcccgttttgcacgaaaaatgcacataaattgcctgcgttgccttataacagcgcagggaaat
cctgcctccggccttgtgccacaccgcgctttgcctggtttgtggtaaaaactggcccgctttgcatcctgatgcttaaaacaccccgttcagatcaacct
ttgggcagataagcccgcgaaaggcctgcaaattgcacggttattccgggtgagtatatgtgtgatttgggttccggcattgcgcaataaaggggaga
aagacatgagcatcacggcgttatcagcatcatttcctgaggggaatatcgccagccgcttgtcgctgcaacatccttcactgttttataccgtggttgaa
caatcttcggtggcgatttcgctgaccgatccgcaggcgcgcatttgttatgccaatccggcattctgccgccagacgggttttgcacttgagacactttt
gggcgagaaccaccgtctgctggtgaacatcactgatgcacaagctacctatgtcgaagaattaactaaaaaactgcaagatgcaggcattcgcgtta
aagccgacttgagaaatgagaagattggctttaaaattcgcgaacacacgctacgccgtgttccttatatgttagtttgtggcgataaagaggtcgaagc
aggcaaagttgctgttcgtacccgccgcggcattagacttaggaagcatggatgttagcgaagtcgttgacaaactgctggcggaaatccgcagcaga
agtcttcatcaactggaggaataaagtattaaaggcggaaaacgagttcaaccggcgcgtcctaatcgcattaacaaagagattcgcgcgcaagaagt
tcgcctcacaggcgtcgatggcgagcagattggtattgtcagtctgaatgaagctcttgaaaaagctgaggaagcgggcgtcgatttagtagaaatca
gtccgaatgccgacccgccagtttgtcgaatcccgtctctgaagctctcggtgaacattgttgcgaggcaggatgcgagctggttgtgttttgacattac
cgataatgtgccgcgtgaacgggtgcgttatgcccgcccggagggcgttttcccgtccggggaatggcatggagctgcgccttatccagacgctg
atcgcccatcatcgcggttctttagatctctcggtccgccctgatggcggcaccttgctgacgttacgcctgccggtacagcaggttatcaccggaggct
taaaatgacccagttacctaccgcgggcccggttatccggcgctttgatatgtctgcccagtttacggcgctttatcgcatcagcgtggcgctgagtcag
gaaagcaacaccgggcgcgcactggcggcgatcctcgaagtgcttcacgatcatgcatttatgcaatacggcatggtgtgtctgtttgataaagaacg
caatgcactctttgtggaatccctgcatggcatcgacggcgaaaggaaaaaagagacccgccatgtccgttaccgcatgggggaaggcgtgatcgg
cgcggtgatgagccagcgtcaggcgctggtgttaccgcgcatttcagacgatcagcgttttctcgaccgcctgaatatttacgattacagcctgccgttg
attggcgtgccgatccccggtgcggataatcagccatcgggcgtgctggtggcacagccgatggcgttgcacgaagaccggctgactgccagtacg
cggtttttagaaatggtc
221 atgagcatcaccgcgttatcagcatcatttcctgaggggaatatcgccagccgcttgtcgctgcaacatccttcactgttttataccgtggttgaacaatct
tcggtggcgatttcgctgaccgatccgcaggcgcgcatttgttatgccaatccggcattctgccgccagacgggttttgcacttgagacacttttgggcg
agaaccaccgtctgctggtgaacatcactgatgcacaagctacctatgtcgaagaattaactaaaaaactgcaagatgcaggcattcgcgttaaagcc
gacttgagaaatgagaagattggctttaaaattcgcgaacacacgctacgccgtgttccttatatgttagtggtggcgataaagaggtcgaagcaggca
aagttgctgttcgtacccgccgcggcaaagacttaggaagcatggatgttagcgaagtcgttgacaaactgctggcggaaatccgcagcagaagtctt
catcaactggaggaataaagtattaaaggcggaaaacgagttcaaccggcgcgtcctaatcgcattaacaaagagattcgcgcgcaagaagttcgcc
tcacaggcgtcgatggcgagcagattggtattgtcagtctgaatgaagctcttgaaaaagctgaggaagcgggcgtcgatttagtagaaatcagtccg
aatgccgagccgccagtttgtcgaatcccgtctctgaagctctcggtgaacattgttgcgaggcaggatgcgagctggagtgttttgacattaccgata
atgtgccgcgtgaacgggtgcgttatgcccccccggaagcgccgttttcccgtccggggaatggcatggagctgcgccttatccagacgctgatcgc
ccatcatcgcggttctttagatctctcggtccgccctgatggcggcaccttgctgacgttacgcctgcggtacagcaggttatcaccggacgcttaaaa
tga
222 tgtttcgtctcgaggccgggcaactgagcggccccgttgaaaccgacctgggctggcatctgttgttgtgcgaacaaattcgcctgccgcaacccttgc
cgaaagccgaagccttaacgcgggtgcgtcagcaactgattgcccggcaacagaaacattatcagcgccagtggctgcaacaactgatcaacgcct
gagcctgttctccttcttgttgatgcagacgggttaatgccgttttgcacgaaaaatgcacataaattgcctgcgttgccttataacagcgcagggaaat
cctgcctccggccttgtgccacaccgcgctttgcctggtttgtggtaaaaactggcccgctttgcatcctgatgcttaaaacaccccgttcagatcaacct
ttgggcagataagcccgcgaaaggcctgcaaattgcacggttattccgggtgagtatatgtgtgatttgggttccggcattgcgcaataaaggggaga
aagacatgagcatcacggcgttatcagcatcatttcctgaggggaatatcgccagccgcttgtcgctgcaacatccttcactgttttataccgtggttgaa
caatcttcggtggcgatttcgctgaccgatccgcaggcgcgcatttgttatgccaatccggcattctgccgccagacgggttttgcacttgagacactttt
gggcgagaaccaccgtctgctggtgaacatcactgatgcacaagctacctatgtcgaagaattaactaaaaaactgcaagatgcaggcattcgcgtta
aagccgacttgagaaatgagaagattggctttaaaattcgcgaacacacgctacgccgtgttccttatatgttagtttgtggcgataaagaggtcgaagc
aggcaaagttgctgttcgtacccgccgcggcaaagacttaggaagcatggatgttagcgaagtcgttgacaaactgctggcggaaatccgcagcaga
agtcttcatcaactggaggaataaagtattaaaggcggaaaacgagttcaaccggcgcgtcctaatcgcattaacaaagagattcgcgcgcaagaagt
tcgcctcacaggcgtcgatggcgagcagattggtattgtcagtctgaatgaagctcttgaaaaagctgaggaaggggcgtcgatttagtagaaatca
gtccgaatgccgagccgccagtttgtcgaatcccgtctctgaagctctcggtgaacattgttgcgaggcaggatgcgagctggttgtgttttgacattac
cgataatgtgccgcgtgaacgggtgcgttatgcccgcccggaagggcgttttcccgtccggggaatggcatggagctgcgccttatccagacgctg
atcgcccatcatcgcggttctttagatctctcggtccgccctgatggcggcaccttgctgacgttacgcctgccggtacagcaggttatcaccggaggct
taaaatgacccagttacctaccgcgggcccggttatccggcgctttgatatgtctgcccagtttacggcgctttatcgcatcagcgtggcgctgagtcag
gaaagcaacaccgggcgcgcactggcggcgatcctcgaagtgcttcacgatcatgcatttatgcaatacggcatggtgtgtctgtttgataaagaacg
caatgcactctttgtggaatccctgcatggcatcgacggcgaaaggaaaaaagagacccgccatgtccgttaccgcatgggggaaggcgtgatcgg
cgcggtgatgagccaggtcaggcgctggtgttaccgcgcatttcagacgatcagcgttttctcgaccgcctgaatatttacgattacagcctgccgttg
attggcgtgccgatccccggtgcggataatcagccatcgggcgtgctggtggcacagccgatggcgttgcacgaagaccggctgactgccagtacg
cggtttttagaaatggtc
223 atggcgctcaaacagttaatccgtctgtgtgccgcctcgccgatggtcgcgacacaacttgcacgtcatcctttattgctcgatgaactgctcgacccgc
gcacgctttaccagccgattgagccgggcgcttaccgcgacgaactccgtcagtatctgatgcgggtgccaacagaagacgaagaacagcagcttg
aagccgtgcgccagttcaaacaggcccagcatttgcgtatcgcagccggggatatttccggggcattgccggtgatgaaagtcagtgaccatttaacc
taccttcccgaggccattctcgatgtcgtggtgcagcatgcgtgggaacaaatggtcgtaaaatacgggcagcccgcgcatcttcagcaccgtgagg
ggcgcggttttgccgtggtcggttacgggaaactcggtggctgggagctgggttatagctcagatctggatctggtcttcctgctcgattgcgcgccgg
acgtgatgacggacggcgaacgcagcatcgacggacgtcagttttatcttcggctggcgcagcgcattatgcacttattcagcacccggacatcgca
ggcattctttacgaggttgatccgcgtctgcgaccttccggcgcatccggcatgctggtcagtaccattgaagcgtttgcagattatcaggccaatgaag
cctggacgtgggagcatcaggcgctggttcgcgcgcgcgtggtttacggggatccgcaactgacacagcaatttaacgccacgcgtcgcgacattct
ttgccgccagcgcgatggcgacggcctgcgtaaggaggtccgtgaaatgcgcgagaaaatgtatgcccataggggagtaaaaaagcccacgagtt
tgatctgaaagccgatccgggtggcatcacggatattgaattcattgcacaatacctggttctgcgtttcgcgcatgatgagccgaagctgacgcgctg
gtctgataacgtgcggatttttgaactgatggcacgatatgacatcatgccggaagaggaagcgcgccatctgacgcaggcttatgtgacgctgcgcg
atgaaattcatcatctggcgttgcaggaacacagcgggaaagtggcgcggacagctttgctactgagcgcgcgcagatccgtgccagctgggcaa
agtggctcggctga
224 cggtactggaacagaaatcggcggatgcgcaggaaatttgttatgacacggcctgtctgaagtgcaagttagtgcttacttcctggctggcaacctcag
gctggacgccgtttattgatgataaatctgcgaagaaactggacgcttccttcaaacgttttgctgacatcatgctcggtcgtaccgcagggatctgaaa
gaagcctttgcgcagccactgacggaagaaggttatcgcgatcagctggcgcgcctgaaacgccagatcattaccttccatttgcttgccggtgcttac
cctgaaaaagacgtcgatgcgtatattgccggctgggtggacctgcaacaggccatcgttcagcagcaacacgcctgggaggattcggcccgttctc
acgcggtgatgatggatgctttctggttaaacgggcaacctcgttaactgactgactagcctgggcaaactgcccgggcttttttttgcaaggaatctgat
ttcatggcgctcaaacagttaatccgtctgtgtgccgcctcgccgatggtcgcgacacaacttgcacgtcatcctttattgctcgatgaactgctcgaccc
gcgcacgctttaccagccgattgagccgggcgcttaccgcgacgaactgcgtcagtatctgatgcgcgggtgccaacagaagacgaagaacagcagct
tgaagccgtgcgccagttcaaacaggcccagcatttgcgtatcgcagccggggatatttccggggcattgccggtgatgaaagtcagtgaccatttaa
cctaccttgccgaggccattctcgatgtcgtggtgcagcatgcgtgggaacaaatggtcgtaaaatacgggcagcccgcgcatcttcagcaccgtga
ggggcgcggttttgccgtggtcggttacgggaaactcggtggctgggagctgggttatagctcagatctggatctggtcttcctgctcgattgcgcgcc
ggaggtgatgacggacggccaacgcagcatcgacggtcgtcagttttatcttcggctggcgcaggcattatgcacttattcagcacccggacatcgt
caggcattctttacgaggttgatccgcgtctccgaccttccggcgcatccggcatgctggtcagtaccattgaaggtttgcagattatcaggccaatga
agcctggacgtgggagcatcaggcgctggttcgcgcgcgcgtggtttacggggatccgcaactgacacagcaatttaacgccacgcgtcgcgacat
tctttgccgccagcgcgatggcgacggcctgcgtaaggaggtccgtgaaatgcgcgagaaaatgtatgcccatctggggagtaaaaaagcccacga
gtttgatctgaaagccgatccgggtggcatcacggatattgaattcattgacaatacctggttctgcgtttcgcgcatgatgagccgaagctgacgcgc
tggtctgataacgtgcggatttttgaactgatggcacgatatgacatcatgccggaagaggaagagcgccatctgacgcaggcttatgtgacgctgcg
cgatgaaattcatcatctggcgttgcaggaacacagcgggaaagtggccgcggacagctttgctactgagcgcgcgcagatccgtgccagctgggc
aaagtggctcggctgagggtttttattcggctaacaggcgcttgtgatattatccggcgcattgtatttacccgatttgatttatctgttttggagtcttgggat
gaaagtgactttgcctgattttcaccgcgcaggtgtgctggttgtcggtgacgtaatgttagaccgttactggtatggcccgaccaatcgtatttctccgga
agctccggtgccggtggtgaagctcagtaccattgaagagcggcctggcggtgcagctaacgtggcgatgaacatttcatctctgggcgcctcttcct
gtctgatcggcctgaccggcgtagacgacgctgcgcgtgccctcagtgagcgtctggcagaagtgattagttaactgcgatttcgtcgcactatccaca
catcctaccatcaccaaactgcgaattttgtcccgtaaccagcaactgatccgcctcgactttgaggaaggttttgaaggcgttgatctcgagccgatgct
gaccaaaataga
225 atgagcatcacggcgttatcagcatcatttcctgaggggaatatcgccagccgcttgtcgagcaacatccttcactgttttataccgtattgaacaatct
tcggtggcgatttcgctgaccgatccgcaggcgcgcatttgttatgccaatccggcattctgccgccagacgggttttgcacttgagacacttttgggcg
agaaccaccgtctgctggtacagtaggcctctcaaaaatagataaacggctcatgtacgtgggccgtttattttttctacccataatcgggaaccggtgt
tataatgccgcgccctcatattgtggggatttcttaatgacctatcctgggcctaaagagtagttgacattagcggagcactaacccgtctagaagctct
cggtgaacattgttgcgaggcaggatgcgagctggttgtgttttgacattaccgataatgtgccgcgtgaacgggtgcgttatgcccgcccggaagcg
gcgttttcccgtccggggaatggcatggagctgcgccttatccagacgctgatcgcccatcatcgcggttctttagatctctcggtccgccctgatggcg
gcaccttgctgacgttacgcctgccggtacagcaggttatcaccggaggcttaaaatga
226 tgtttcgtctcgaggccgggcaactgagcggccccgttaaaccgacctgggctggcatctgttgttgtgcgaacaaattcgcctgccgcaacccttgc
cgaaagccgaagccttaacgcgggtgcgtcagcaactgattgcccggcaacaggaatcattatcagcgccagtggctgcaacaactgatcaacgcct
gagcctgttctccttcttgttgatgcagacgggttaatgcccgttttgcacgaaaaatgcacataaattgcctgcgttgccttataacagcgcagggaaat
cctgcctccggccttgtgccacaccgcgctttgcctggtttgtggtaaaaactggcccgctttgcatcctgatgataaaacaccccgtttcagatcaacct
ttgggcagataagcccgcgaaaggcctgcaaattgcacggttattccgggtgagtatatgtgtgatttgggttcaggcattgcgcaataaaggggaga
aagacatgagcatcacggcgttatcagcatcatttcctgaggcgaatatcgccagccgcttgtcgctgcaacatccttcactgttttataccgtgcttgaa
caatcttcggtggcgatttcgctgaccgatccgcaggcgcgcatttgttatgccaatccggcattctgccgccagacgggttttgcacttgagacactttt
gggcgagaaccaccgtctgctggtacagtagcgcctctcaaaaatagataaacggctcatgtacgtgggccgtttattttttctacccataatcgggaac
cggtgttataatgccgcgccctcatattgtggggatttcttaatgacctatcctgggtcctaaagttgtagttgacattagcggagcactaacccgtctctg
aagctctcggtgaacattgttgcgaggcaggatgcgagctggttgtgttttgacattaccgataatgtgccgcgtgaacgggtgcgttatgcccgcccg
gaagcggcgttttcccgtccggggaatggcatggagctgcgccttatccagacgctgatcgcccatcatcgcggttctttagatctctcggtccgccct
gatggcggcaccttgctgacgttacgcctgccggtacagcaggttatcaccggaggcttaaaatgacccagttacctaccgcgggcccggttatccg
gcgctttgatatgtctgcccagtttacggcgctttatcgcatcagcgtggcgctgagtcaggaaagcaacaccgggcgcgcactggcggcgatcctcg
aagtgcttcacgatcatgcatttatgcaatacggcatggtgtgtctgtttgataaagaacgcaatgcactctttgtggaatccctgcatggcatcgacggc
gaaaggaaaaaagagacccgccatgtccgttaccgcatgggggaaggcgtgatcggcgcggtgatgagccagcgtcaggcgctggtgttaccgc
gcattttagacgatcagcgttttctcgaccgcctgaatatttacgattacagcctgccgttgattggcgtgccgatccccggtgcggataatcagccatcg
ggcgtgctggtggcacagccgatggcgttgcacgaagaccggctgactgccagtacgcggtttttagaaatggtc
227 atggcgctcaaacagttaatccgtctgtgtgccgcctcgccgatggtagcgacacaacttgcacgtcatcctttattgctcgatgaactgctcgacccgc
gcacgctttaccagccgattgagccgggcgcttaccgcgacgaactgcgtcagtatctgatgcgggtgccaacagaagacgaagaacagcagcttg
aagccgtgcgccagttcaaacaggcccagcatttgcgtatagcagccggggatatttccggggcattgccggtgatgaaagtcagtgaccatttaacc
taccttgccgaggccattctcgatgtcgtggtgcagcatgcgtgggaacaaatggtcgtaaaatacgggcagcccgcgcatcttcagcaccgtgagg
ggcgcggttttgccgtggtcggttacgggaaactcggtggctgggagctgggttatagctcagatctggatctggtcttcagctcgattgcgcgccgg
aggtgatgacggacggcgaacgcagcatcgacggacgtcagttttatcttcggctggcgcagcgcattatgcacttattcagcacccggacatcgtca
ggcattctttacgaggttgatccgcgtctgcgaccttccggcgcatccggcatgctggtcagtaccattgaagcgtttgcagattatcaggccaatgaag
cctggacgtgggagcatcaggcgctggttcgcgcgcgcgtggtttacggggatccgcaactgacacagcaatttaacgccacgcgtcgcgacattct
ttgccgccagcgcgatggcgacggcctgcgtaaggaggtccgtgaaatgcgcgagaaaatgtatgcccatctggggagtaaaaaagcccacgagtt
tgatctgaaagccgatcccggtggcatcacggatattgaattcattgacaatacctggttctgcgtttcgcgcatcatgagccgaagctgacgcgctg
gtctgataacgtgcggatttttgtactgatggcacgatatgacatcatgcggaagaggaagcgcgccatctgacgcaggcttatgtgacgctgcgcg
atgaaattcatcatctggcgttgcacgaacacagcgggaaagtggccgcggacagctttgctactgagcgcgcgcagatccgtgccagctgggcaa
agtggctcggctga
228 cggtactggaacagaaatcggcggatgcgcaggaaatttgttatgacacggcctgtctgaagtgcaagttagtgcttacttcctggctggcaacctcag
gctggacgccgtttattgatgataaatctgcgaagaaactggacgcttccttcaaacgttttgctgacatcatgctcggtcgtaccgcagcggatctgaaa
gaagcctttgcgcagccactgacggaagaaggttatcgcgatcagctggcgcgcctgaaacgccagatcattaccttccatttgcttgccggtgcttac
cctgaaaaagacgtcgatgcgtatattgccggctgggtggacctgcaacaggccatcgttcagcagcaacacgcctgggaggattcggcccgttctc
acgcggtgatgatggatgctttctggttacgggcaacctcgttaactgactgactagcctgggcaaactgcccgggcttttttttgcaaggaatctgat
ttcatggcgctcaaacagttaatccgtctgtgtgccgcctcgccgatggtcgcgacacaacttgcacgtcatcctttattgctcgatgaactgctcgaccc
gcgcacgctttaccagccgattgagccgggcgcttaccgcgacgaactgcgtcagtatctgatgcgggtgccaacagaagacgaagaacagcagct
tgaagccgtgcgccagttcaaacaggcccagcatttgcgtatcgcagccggggatatttccggggcattgccggtgatgaaagtcagtgaccatttaa
cctaccttgccgaggccattctcgatgtcgtggtgcagaatgcgtgggaacaaatggtcgtaaaatacgggcagcccgcgcatcttcagcaccgtga
ggggcgcggttttgccgtggtcggttacgggaaactcggtggctgggagctgggttatagctcagatctggatctggtcttcctgctcgattgcgcgcc
ggaggtgatgacggacggcgaacgcagcatcgacggacgtcagttttcttcggaggcgcagcgcattatgcacttattcagcacccggacatcgt
caggcattctttacgaggttgatccgcgtctgcgaccttccggcgcatccggcatgctggtcagtaccattgaagcgtttgcagattatcaggccaatga
agcctggacgtgggagcatcaggcgctgtgtcgcgcgcgcgtggtttacggggatccgcaactgacacagcaatttaacgccacgcgtagcgacat
tctttgccgccagcgcgatggcgacggcctgcgtaaggaggtccgtgaaatgcgcgagaaaatgtatgcccatctggggagtaaaaaagcccacga
gtttgatctgaaagccgatccgggtggcatcacggatattgaattcattgcacaatacctggttctgcgtttcgcgcatgatgagccgaagctgacgcgc
tggtctgataacgtgcggatttttgaactgatggcacgatatgacatcatgccggaagaggaaggcgccatctgacgcaggcttatgtgacgctgcg
cgatgaaattcatcatctggcgttgcaggaacacagcgggaaagtggccgcggacagctttgctactgagcgcgcgcagatccgtgccagctgggc
aaagtggctcggctgagggtttttattcggctaacaggcgcttgtgatattatccggcgcattgtatttacccgatttgatttatctgttttggagtcttgggat
gaaagtgactttgcctgattttcaccgcgcaggtgtgctggttgtcggtgacgtaatgttagaccgttactggtatggcccgaccaatcgtatttctccgga
agctccggtgccggtggtgaaggtcagtaccattgaagagcggcctggcggtgcagctaacgtggcgatgaacattcatctctggacgcctcttcct
gtctgatcggcctgaccggcgtagacgacgctgcgcgtgccctcagtgagcgtctggcagaagtgaaagttaactgcgatttcgtcgcactatccaca
catcctaccatcaccaaactgcgaattttgtcccgtaaccagcaactgatccgcctcgactttgaggaaggttttgaaggcgttgatctcgagccgatgct
gaccaaaataga
229 atgagcatcacggcgttatcagctgaatatcactgactcacaagctacctatgtcgaagaattaactaaaaaactgcaagatgcaggcattcgcgttaaa
gcgacttgagaaatgagaagattggctttaaaattcgcgaacacacgctacgccgtgtccttatatgttagtttgtggcgataaagaggtcgaagcag
gcaaagttgctgttcgtactcgtcgcggcaaagacttaggaagcatggatgttagcgaagtcgttgacaaactgctggcggaaatccgcagcagaagt
catcatcaactggaggaataaagtattaaaggcggaaaacgagttcaaccggcgcgtcctaatcgcattaacaaagagattcgcgcgcaagaagttc
gcctcaccggcgtcgatggcgagcagattggtattgcagtctgaatgaagctcttgaaaaagctgaggaagcgggcgtcgatttagtagaaatcagt
ccgaatgccgagccgccagtttgtcgaatctctttagatctctcggtccgccctgatggcggcaccttgctgacgttacgcctgccggtacagcaggtta
tcaccggaggcttaaaatga
230 tgtttcgtcgaagccgggcaactgagcagccccgttgaaaccgaactgggctggcatctgttgttgtgcgaacaaattcgcctgcgcaacccttgc
cgaaagccgaggccttaacgcgggtgcgtcagcaactgattgcccggcaacagaatcattatcagcgccagtggctgcaacaactgatcaacgcct
gagcctgttctccttcttgttggtgcagacgggttaatgcccgttttgcacgaaaaatgcacataaactgccttcgctgccttataacagcgcatggaaatc
ctgcctcctgccttgtgccacgccgcgctttgcctggtttgtggtaaaaactggcccgctttgcatcctgatgtttaaaacaccccgttcagatcaaccttt
gggcagataagcccgcgaaaggcctgcaaattgcacggttattccgggtgagtatatatgtgatttgggttccggcattgcgcaataaaggggagaaa
gacatgagcatcacggcgttatcagctgaatatcactgactcacaagctacctatgtcgaagaattaactaaaaaactgcaggatgcaggcattcgcgtt
aaagccgacttgagaaatgagaagattggctttaaaattcgcgaacacacgctacgccgtgttccttatatgttagtttgtggcgataaagaggtcgaag
caggcaaagtgctgttcgtactcgtcgcggcaaagacttaggaagcatggatgttagcgaagtcgttgacaaactgctggcggagatccgcagcag
aagtcatcatcaactggaggaataaagtattaaaggcggaaaacgagttcaaccggcgcgtcctaatcgcattaacaaagagattcgcgcgcaagaa
gttcgcctcaccggcgtcgatggcgagcagattggtattgtcagtctgaatgaagacttgaaaaagctgaggaagcgggcgtcgatttagtagaaatc
agtccgaatgccgagccgccagtttgtcgaatctctttagatctctcggtccgccctgatggcggcaccttgctgacgttacgcctgccggtacagcag
gttatcaccggaggcttaaaatgacccagttacctaccgcgggcccggttatccggcgctttgatatgtctgcccagtttacggcgctttatcgcatcagc
gtggcgctgagtcaggagagcaataccgcgcgcgcactggcggcgatcctcgaagtgcttcacgatcatgcatttatgcaatacggcatggtgtgtct
gttcgataaagaacgcaatgcactgtttgtgcaatccctgcatggcatcgacggcgaaaggaaaaaagaaacccgccatgtccgttaccgcatgggg
gaaggcgtgatcggcgcggtgatgagccagcgtcaggcgtggtgttaccgcgcatttcagacgatcagcgttttctcgaccgcctgaatatttacgat
tacagcctgccgctgattggtgtgccgatccccggtgcggataatcagcctgcgggtgtgctggtggcacagccgatggcgttgcacgaagaccgg
ctggctgccactacgcgctttttagaaatggtc
231 atgaccctgaatatgatgatggatgccggcggacatcatcgcgacaaacaatattaataccggcaaccacaccggcaatttacgagactgcgcaggc
atcctttacccgtcaatttctgtcaaataaagtaaaagaggcagtctacttgaattacccccggctggttgagcgtttgttgaaaaaaagtaactgaaaa
tccgtagaatagcgccactctgatggttaattaacctattcaattaagaattatctggatgaatgtgccattaaatgcgcagcataatggtgcgttgtgcgg
gaaaactgcttttttttgaaagggttggtcagtagggaaacaactcacttcacaccccgaagggggaagttgcctgaccctacgattcccgctatttcat
tcactgaccggaggttcaaaatga
232 accggatacgagagaaaagtgtctacatttggttcggttgatattgaccggcgcatccgccagcccgcccagtttctggggatctgtttggcgattttgc
gggtcttgccggtgtcggtgccgaaaaaaataccaatatttgccataacacacgctcctgttgaaaaagagatcccgccgggaaatgcggtgaacgtg
tctgatattgcgaagagtgtgccagttttggtcgcgggcaaaacctgcaccagtttggttattaatgcaccagtctggcgctttttttcgccgagtttctcct
cgctaatgcccgccaggcgcggctttggcgctgatagcgcgctgaataccgatctggatcaaggttttgtcgggttatcagccaaaaggtgcactcttt
gcatggttatacgtgcctgacatgttgtccgggcgacaaacggcctggtggcacaaattgtcagaactacgacacgactaactgaccgcaggagtgt
gcgatgaccctgaatatgatgatggatgcccgcggacatcatcgcgacaaacaatattaataccggcaaccacaccggcaatttacgagactgcgca
ggcatcctttctcccgtcaatttctgtcaaataaagtaaaagaggcagtctacttgaattacccccggctggttgagcgtttgttgaaaaaaagtaactgaa
aaatccgtagaatagcgccactctgatggttaattaacctattcaattaagaattatctggatgaatgtgccattaaatgcgcagattaatggtgcgttgtg
cgggaaaactgctttttttgaaagggttggtcagtagcggaaacaactcacttcacaccccgaagggggaagttgcctgaccctacgattcccgctatt
tcattcactgaccggaggttcaaaatgacccagcgaaccgagtcgggtaataccgtctggcgcttcgatttgtcccagcagttcactgcgatgcagcgc
ataagcgtggtactcagccgggcgaccgaggtcgatcagacgctccagcaagtgctgtgcgtattgcacaatgacgcctttttgcagcacggcatgat
ctgtctgtacgacagccagcaggcgattttgaatattgaagcgttgcaggaagccgatcagcagttaatccccggcagctcgcaaatccgctatcgtcc
gggcgaagggctggtcgggacggtgctttcgcagggccaatcattagtgcttgcgcgcgttgctgacgatcagcgctttcttgaccggctcgggttgt
atgattacaacctgccgtttatcgccgtgccgctgatagggccagatgcgcagactttcggtggctgacggcacaacccatggcgcgttacgaagag
cgattacccgcctgcacccgctttctggaaacggtc
233 atgaccctgaatatgatgatggatgccggccgtcctgtaataataaccggacaattcggactgattaaaaaagcgcccttgtggcgctttttttatattccc
gcctccatttaaaataaaaaatccaatcggatttcactatttaaactggccattatctaagatgaatccgatggaagctcgctgttttaacacgcgttttttaa
ccttttattgaaagtcggtgcttctttgagcgaacgatcaaatttaagtggattcccatcaaaaaaatattctcaacctaaaaaagtttgtgtaatacttgtaac
gctacatggagattaactcaatctagagggtattaataatgaatcgtactaaactggactgggcgcaactcacttcacaccccgaagggggaagttgc
ctgaccctacgattcccgctatttcattcactgaccggaggttcaaaatga
234 accggatacgagagaaaagtgtctacatcggttcggttgatattgaccggcgcatccgccagcccgcccagtttctggtggatctgtttggcgattttgc
gggtcttgccggtgcggtgccgaaaaaaataccaatatttgccataacacacgctcctgttgaaaaagagatcccgccgggaaatgcggtgaacgtg
tctgatattgcgaagagtgtgccagttttggtcgcgggcaaaacctgcaccagtttggttattaatgcaccagtctggcgctttttttcgccgagtttctcct
cgctaatgcccgccaggcgcggctttggcgctgatagcgcgctgaataccgatctggatcaaggttttgtcgggttatcagccaaaaggtgcactcttt
gcatggttatacgtgcctgacatgttgtccgggcgacaaacggcctggtggcacaaattgtcagaactacgacacgactaactgaccgcaggagtgt
gcgatgaccctgaatatgatgatggatgccggccgtcctgtaataataaccgcacaattcggactgattaaaaaagcgcccttgtggcgctttttttatatt
cccgcctccatttaaaataaaaaatccaatcggatttcactatttaaactggccattatctaagatgaatccgatggaagctcgctgttttaacacgcgttttt
taaccttttattgaaagtcggtgcttctttgagcgaacgatcaaatttaagtggattcccatcaaaaaaatattctcaacctaaaaaagtttgtgtaatacttgt
aacgctacatggagattaactcaatctagagggtattaataatgaatcgtactaaactggtactgggcgcaactcacttcacaccccgaagggggaagt
tgcctgaccctacgattcccgctatttcattcactgaccggaggttcataatgacccagcgaaccgagtcgggtaataccgtctggcgcttcgatttgtc
ccagcagttcactgcgatgcagcgcataagcgtggtactcagccgggcgaccgaggtcgatcagacgctccagcaagtgctgtgcgtattgcacaat
gacgcctttttgcagcacggcatgatctgtctgtacgacagccagcaggcgattttgaatattgaagcgttgcaggaagccgatcagcagttaatcccc
ggcagctcgcaaatccgctatcgtccgggcgaagggctggtcgggacggtgtttcgcagggcaatcattagtgctggcgcgcgttgctgacgatc
agcgctttcttgaccggctcgggttgtatgattacaacctgccgtttatcgccgtgccgctgatagggccagatgcgcagactttcggtgtgctgacggc
acaacccatggcgcgttacgaagagcgattacccgcctgcacccgctttctggaaacggtc
235 atgaccctgaatatgatgatggatgccggccgtcctgtaataataaccggacaattcggactgattaaaaaagcgcccttgtggcgcatttttttattccc
gcctccatttaaaataaaaaatccaatcggatttcactatttaaactggccattatctaagatgaatccgatggaagctcgctgttttaacacgcgtttttaa
ccttttattgaaagtcggtgcttctttgagcgaacgatcaaatttaagtggattcccatcaaaaaaatattctcaacctaaaaaagtttgtgtaatacttgtaac
gctacatggagattaactcaatctagagggtattaataatgaatcgtactaaactggtactgggcgcaactcacttcacaccccgaagggggaagttgc
ctgaccctacgattcccgctatttcattcactgaccggaggttcaaaatga
236 accggatacgagagaaaagtgtctacatcggttcggttgatattgaccggcgcatccgccagcccgcccagtttctggtggatctgtttggcgattttgc
gggtcttgccggtgtcggtgccgaaaaaaataccaatatttgccataacacacgctcctgttgaaaaagagatcccgccgggaaatgcggtgaacgtg
tctgatattgcgaagagtgtgccagttttggtcgcgggcaaaacctgcaccagtttggttattaatgcaccagtctggcgctttttttcgccgagtttctcct
cgctaatgcccgccaggcgcggctttggcgctgatagcgcgctgaataccgatctggatcaaggttttgtcgggttatcagccaaaaggtgcactcttt
gcatggttatacgtgcctgacatgttgtccgggcgacaaacggcctggtggcacaaattgtcagaactacgacacgactaactgaccgcaggagtgt
gcgatgaccctgaatatgatgatggatgccggccgtcctgtaataataaccggacaattcggactgattaaaaaagcgcccttgtggcgctttttttatatt
cccgcctccatttaaaataaaaaatccaatcggatttcactatttaaactggccattatctaagatgaatccgatggaagctcgctgttttaacacgcgttttt
taaccttttattgaaagtcggtgcttctttgagcgaacgatcaaatttaagtggattcccatcaaaaaaatattctcaacctaaaaaagtttgtgtaatacttgt
aacgctacatggagattaactcaatctagagggtattaataatgaatcgtactaaactggtactgggcgcaactcacttcacaccccgaagggggaagt
tgcctgaccctacgattcccgctatttcattcactgaccggaggttcaaaatgacccagcgaaccgagtcgggtaataccgtctggcgatcgatttgtc
ccagcagttcactgcgatgcagcgcataagcgtggtactcagccgggcgaccgaggtcgatcagacgctccagcaagtgctgtgcgtattgcacaat
gacgcctttttgcagcacggcatgatctgtctgtacgacagccagcaggcgattttgaatattgaagcgttgcaggaagccgatcagcagttaatcccc
ggcagctcgcaaatccgctatcgtccgggcgaagggctggtcgggacggtgctttcgcagggccaatcattagtgctggcgcgcgttgctgacgatc
agcgctttcttgaccggctcgggttgtatgattacaacctgccgtttatcgccgtgccgctgatagggccagatgcgcagactttcggtgtgctgacggc
acaacccatggcgcgttacgaagagcgattacccgcctgcacccgctttctggaaacggtc
237 atggcactgaaacacctcatttccctgtgtgccgcgtcgccgatggttgccagtcagctggcgcgctacccgatcctgcttgatgaattgctcgacccg
aatacgctctatcaaccgacggcgatgaatgcctatcgcgatgagctgcgccaatacctgctgcgcgtgccggaagatgatgaagagcaacagcttg
aggcgctgcggcagtttaagcaggcgcagttgctgcgcgtggcggcggcggatattgccggtacgttgccagtaatgaaagtgagcgatcacttaac
ctggctggcggaagcgattattgatgcggtggtgcagcaagcctgggggcagatggtggcgcgttatggccagccaacgcatctgcacgatcgcga
agggcgggttttgcggtggtcggttatggcaagctgggcggctgggagctgggttacagctccgatctggatctggtattcctgcacgactgcccga
tggatgtgatgaccgatggcgagcgtgaaatcgatggtcgccagttctatttgcgtctcgcgcagcgcgtgatgcacctgtttagcacgcgcacgtcgt
ccggcatcctttatgaagttgatgcgcgtctgcgtccatctggcgctgcggggatgctggtcactactacggaatcgttcgccgattaccagcaaaacg
aagcctggacgtgggaacatcaggcgctggcccgtgcgcgcgtggtgtacggcgatccgcaactgaccgccgaatttgacgccattcgccgcgata
ttctgatgacgcctcgcgacggcgcaacgctgcaaaccgacgtgcgagaaatgcgcgagaaaatgcgtgcccatcttggcaacaagcataaagacc
gcttcgatctgaaagccgatgaaggcggtatcaccgacatcgagtttatcgcccaatatctggtgctgcgctttgcccatgacaagccgaaactgacgc
gctggtcggataatgtgcgcattctcgaagggctggcgcaaaacggcatcatggaggagcaggaagcgcaggcattgacgctggcgtacaccaca
ttgcgtgatgagctgcaccacctggcgctgcaagagttgccgggacatgtggcgctctcctgttttgtcgccgagcgtgcgcttattaaaaccagctgg
gacaagtggctggtggaa
238 gcgcaaagcgagtgctcacttacgtgatctgttgacacaatctgaagcgaccataacttctgccgtttcagcgaatacggcggtgtggagcgcacaatc
agccctggcgaagctggtgctcaccgagtggctagtgacgcagggctggcgaaccttccttgatgaaaaagcgcaggccaaattcgccgactccttt
aaacgctttgctgacatccatctgtcacgcagcgccgccgagctgaaaaaaggcctttgcccaaccgctgggcgacagctatcgcgaccagttgccgc
gcctggcgcgtgatatcgactgcgcgttactgctggccgggcattacgatcgcgcgcgcgccgtggaatggctggaaaactggcaggggcttcagc
acgccattgaaacgcgccagagagtcgaaatcgaacatttccgtaataccgcgattacccaggagccgttctggttgcacagcggaaaacgttaacg
aaaggatatttcgcatggcactgaaacacctcatttccctgtgtgccgcgtcgccgatggttgccagtcagctggcgcgctacccgatcctgcttgatga
attgctcgacccgaatacgctctatataccgacggcgatgaatgcctatcgcgatgagctgcgcatatacctgctgcgcgtgccggaagatgatgaa
gagcaacagcttgaggcgctgcggcagtttaagcaggcgcagttgctgcgcgtggcggcggcggatattgccggtacgttgccagtaatgaaagtg
agcgatcacttaacctggctggcggaagcgattattgatgcggtggtgcagcaagcctgggggcagatggtggcgcgttatggccagccaacgcat
ctgcacgatcgcgaagggcgcggttttgcggtggtcggttatggcaagctgggcggctgggagctgggttacagctccgatctggatctggtattcct
gcacgactgcccgatggatgtgatgaccgatggcgagcgtgaaatcgatggtcgccagttctatttgcgtctcgcgcagcgcgtgatgcacctgtttag
cacgcgcacgtcgtccggcatcctttatgaagttgatgcgcgtctgcgtccatctggcgctgcggggatgctggtcactactacggaatcgttcgccga
ttaccagcaaaacgaagcctgcacgtgggaacatcaggcgctggcccgtgcgcgcgtggtgtacggcgatccgcaactgaccgccgaatttgacg
ccattcgccgcgatattctgatgacgcctcgcgacggcgcaacgctgcaaaccgacgtgcgagaaatgcgcgagaaaatgcgtgcccatcttggca
acaagcataaagaccgcttcgatctgaaagccgatgaaggcggtatcaccgacatcgagtttatcgcccaatatctggtgctgcgctttgcccatgaca
aaccgaaactgacgcgctggtcggataatgtgcgcattctcgaagggctggcgcaaaacggcatcatggaggagcaggaagcgcaggcattgacg
ctggcgtacaccacattgcgtgatgagagcaccacctggcgctgcaagagttgccgggacatgtggcgctctcctgttttgtcgccgagcgtgcgctt
attaaaaccagctgggacaagtggctggtggaaccgtgcgccccggcgtaagtgtggtatcatcgcgcgcaaattttgtatctctcaggagacaggaa
tgaaagtgacgagccagagtttaagcaagccggtgtaatggtggtgggtgatgtgatgctggatcgttactggtatggcccaaccagccgtatctctc
cggaagcgccagtcccggttgttaaagtcgataccattgaagagcgtcctggcggcgcggcaaacgtggcgatgaatatcgcctcactgggcgcca
cggcgcgtctggttggcctgactggcattgacgatgcggcgcgcgcgctgagcaaagcgctggccgatgttaacgttaaatgtgacttcgtttctgttc
cgacgcatcccaccatcactaagctgcgcgtgctgtcgcgtaaccagcagctgattcgcctggactttgaagagggttttgaaggagtcgatccgcaa
ccgatgcatcgaacgcatcagccaggcgcttggtaatattggcgcgctggtgctgtcggatt
239 atgtttaacgatctgattggcgatgatgaaacggattcgccggaagatgcgctttctgagagctggcgcgaattgtggcaggatgcgttgcaggagga
ggattccacgcccgtgctggcgcatctctcagaggacgatcgccgccgcgtggtggcgctgattgccgattttcgcaaagagttggataaacgcacc
attggcccgcgagggcggcaggtactcgatcacttaatgccgcatctgctcagcgatgtatgctcgcgcgacgatgcgccagtaccgctgtcacgcc
tgacgccgctgctcaccggaattattacccgcaccacttaccttgagctgctaagtgaatttcccggcgcactgaaacacctcatttccctgtgtgccgc
gtcgccgatggttgccagtcagctggcgcgctacccgatcctgcttgatgaattgctcgacccgaatacgctctatcaaccgacggcgatgaatgccta
tcgcgatgagctgcgccaatacctgctgcgcgtgccggaagatgatgaagagcaacagcttgaggcgctgcggcagtttaagcaggcgcagttgct
gcgcgtggcggcggcggatattgccggtacgttgccagtaatgaaagtgagcgatcacttaacctggctggcggaagcgattattgatgcggtggtg
cagcaagcctgggggcagatggtggcgcgttatggccagccaacgcatctgcacgatcgcgaagggcgcggttttgcggtggtcggttatggcaag
ctgggcggctgggagctgggttacagctccgatctggatctggtattcctgcacgactgcccgatggatgtgatgaccgatggcgagcgtgaaatcg
atggtcgccagttctatttgcgtctcgcgcagcgcgtgatgcacctgtttagcacgcgcacgtcgtccggcatcctttatgaagttgatacgcgtctgcgt
ccatctggcgctgcggggatgctggtcactactacggaatcgttcgccgattaccagcaaaacgaagcctggacgtgggaacatcaggcgctggcc
cgtgcacgcgtgggtacggcgatccgcaactgaccgccgaatttgacgccattcgccgcgatattctgatgacgcctcgcgacggcgcaacgctgc
aaaccgacgtgcgagaaatgcgcgagaaaatgcgtgcccatcttggcaacaagcataaagaccgcttcgatctgaaagccgatgaaggcggtatca
ccgacatcgagtttatcgcccaatatctggtgctgcgctttgcccatgacaagccgaaactgacgcgctggtcggataatgtgcgcattctcgaagggc
tggcgcaaanggcatcatggaggagcaggaagcgcaggcattgacgctggcgtacaccacattgcgtgatgagctgcaccacctggcgctgcaa
gagttgccgggacatgtggcgctctcctgttttgtcgccgagcgtgcgcttattaaaaccagctgggacaagtggctggtggaaccgtgcgccccggc
gtaa
240 atgaccctgaatatgatgatggatgccggccgtcctgtaataataaccggacaattcggactgattaaaaaagcgcccttgtggcgctttttttatattccc
gcctccatttaaaataaaaaatccaatcggatttcactatttaaactggccattatctaagatgaatccgatggaagctcgctgtttaacacgcgttttttaa
ccttttattgaaagtcggtgcttctttgagcgaacgatcaaatttaagtggattcccatcaaaaaaatattctcaacctaaaaaagtttgtgtaatacttgtaac
gctacatggagattaactcaatctagagggtattaataatgaatcgtactaaactggtactgggcgcaactcacttcacaccccgaagggggaagttgc
ctgaccctacgattcccgctatttcattcactgaccggaggttcaaaatga
241 accggatacgagagaaaagtgtctacatcgatcggttgatattgaccggcgcatccgccagcccgcccagtttctggtggatctgtttggcgattttgc
gggtcttgccggtgtcggtgccgaaaaaaataccaatatttgccataacacacgctcctgttgaaaaagagatcccgccgggaaatgcggtgaacgtg
tctgatattgcgaagagtgtgccagttttggtcgcgggcaaaacctgcaccagtttggttattaatgcaccagtctggcgctttttttcgccgagtttctcct
cgctaatgcccgccaggcgcggctttggcgctgatagcgcgctgaataccgatctggatcaaggttttgtcgggttatcagccaaaaggtgcactcttt
gcatggttatacgtgcctgacatgttgtccggccgacaaacggcctggtggcacaaattgtcagaactacgacacgactaactgaccgcaggagtgt
gcgatgaccctgaatatgatgatggatgccggccgtcctgtaataataaccggacaattcggactgattaaaaaagcgcccttgtggcgctttttttatatt
cccgcctccatttaaaataaaaaatccaatcggatttcactatttaaactggccattatctaagatgaatccgatggaagctcgctgttttaacacgcgttttt
taaccttttattgaaagtcggtgcttctttgagcgaacgatcaaatttaagtggattcccatcaaaaaaatattctcaacctaaaaaagtttgtgtaatacttgt
aacgctacatggagattaactcaatctagagggtattaataatgaatcgtactaaactggtactgggcgcaactcacttcacaccccgaaggggcaagt
tgcctgaccctacgattcccgctatttcattcactgaccggaggttcaaaatgacccagcgaaccgagtcgggtaataccgtctggcgcttcgatttgtc
ccagcagttcactgcgatgcagcgcataagcgtggtactcagccgggcgaccgaggtcgatcagacgctccagcaagtgctgtgcgtattgcacaat
gacgcctttttgcagcacggcatgatctgtctgtacgacagccagcagccgattttgaatattgaagcgttgcaggaagccgatcagcagttaatcccc
ggcagctcgcaaatccgtatcgtccgggcgaagggctggtcgggacggtgctttcgcagggccaatcattagtgctggcgcgcgttgctgacgatc
agcgctttcttgaccggctcgggttgtatgattacaacctgccgtttatcgccgtgccgctgatagggccagatgcgcagactttcggtgtgctgacggc
acaacccatggcgcgttacgaagagcgattacccgcctgcacccgctttctggaaacggtc
242 gcgcaaagcgagtgctcacttacgtgatctgttgacacaatctgaagcgaccataacttctgccgtttcagcgaatacggcggtgtggagcgcacaatc
agccctggcgaagctggtgctcaccgagtggctagtgacgcagggctggcgaaccttccttgatgaaaaagcgcaggccaaattcgccgactccttt
aaacgctttgctgacatccatctgtcacgcagcgccgccgagctgaaaaaagcctttgcccaaccgctgggcgacagctatcgcgaccagttgccgc
gcctggcgcgtgatatcgactgcgcgttactgctggccgggcattacgatcgcgcgcgcgccgtggaatggctggaaaactggcaggggcttcagc
acgccattgaaacgcgccagagagtcgaaatcgaacatttccgtaataccgcgattacccaggagccgttctggttgcacagcggaaaacgttaacg
aaaggatatttcgcatgtttaacgatctgattggcgatgatgaaacggattcgccggaagatgcgctttctgagagctggcgcgaattgtggcaggatg
cgttgcaggaggaggattccacgcccgtgaggcgcatctctcagaggacgatcgccgccgcgtggtggcgctgattgccgattttcgcaaagagtt
ggataaacgcaccattggcccgcgagggcggcaggtactcgatcacttaatgccgcatctgctcagcgatgtatgacgcgcgacgatgcgccagta
ccgctgtcacgcctgacgccgctgacaccggaattattacccgcaccacttaccttgagctgctaagtgaatttcccggcgcactgaaacacctcattt
ccctgtgtgccgcgtcgccgatggttgccagtcagctggcgcgctacccgatcctgcttgatgaattgctcgacccgaatacgctctatcaaccgacg
gcgatgaatgcctatcgcgatgagctgcgccaatacctgctgcgcgtgccggaagatgatgaagagcaacagcttgaggcgctgcggcagtttaag
caggcgcagttgctgcgcgtcgcggcggcggatattgccggtacgttgccagtaatgaaagtgagcgatcacttaacctggctggcggaagcgatta
ttgatgcggtggtgcagcaagcctgggggcagatggtggcgcgttatggccagccaacgcatctgcacgatcgcgaagggcgcggttttgcggtgg
tcggttatggcaagctgggcggctgggagctgggttacagctccgatctggatctggtattcctgcacgactgcccgatggatgtgatgaccgatggc
gagcgtgaaatcgatggtcgccagttctatttgcgtctcgcgcagcgcgtgatgcacctgtttagcacgcgcacgtcgccggcatcctttatgaagttg
atgcgcgctgcgtccatctggcgctgcggggatgctggtcactactacggaatcgttcgccgattaccagcaaaacgaagcctggacgtgggaaca
tcaggcgctggcccgtgcgcgcgggtgtacggcgatccgcaactgaccgccgaatttgacgccattcgccgcgatattctgatgacgcctcgcgac
ggcgcaacgctgcaaaccgacgtgcgagaaatgcgcgagaaaatgcgtgcccatcttggcaacaagcataaagaccgcttcgatctgaaagccgat
gaaggcggtatcaccgacatcgagtttatcgcccaatatctggtgctgcgctttgcccatgacaagccgaaactgacgcgctggtcggataatgtgcg
cattctcgaagggctggcgcaaaacggcatcatggaggagcaggaagcgcaggcattgacgctggcgtacaccacattgcgtgatgagctgcacc
acctggcgctgcaagagttgccgggacatgtggcgctctcctgttttgtcgccgagcgtgcgcttattaaaaccagctgggacaagtggctggtggaa
ccgtgcgccccggcgtaagtgtggtatcatcgcgcgcaaattttgtatctctcaggagacaggaatgaaagtgacgctgccagagtttaagcaagccg
gtgtaatggtggtgggtgatgtgatgctggatcgttactggtatggcccaaccagccgtatctctccggaagcgccagtcccggttgttaaagtcgatac
cattgaagagcgtcctggcgccgcggcaaacgtggcgatgaatatcgcctcactgggcgccacggcgcgtctggttggcctgactggcattgacga
tgcggcgcgcgcgctgagcaaagcgctggccgatgttaacgttaaatgtgacttcgtttctgttccgacgcatcccaccatcactaagctgcgcgtgct
gtcgcgtaaccagcagctgattcgcctggactttgaagagggttttgaaggagtcgatccgcaaccgatgcatgaacgcatcagccaggcgcttggta
atattggcgcgctggtgctgtcggatt
243 atgtttaacgatctgattggcgatgatgaaacggattcgccggaagatgcgctttctgagagctggcgcgaattgtggcaggatgcgttgcaggagga
ggattccacgcccgtgctggcgcatctctcagaggacgatcgccgccgcgtggtggcgctgattgccgattttcgcaaagagttggataaacgcacc
attggcccgcgagggcggcaggtactcgatcacttaatgccgcatctgctcagcgatgtatgctcgcgcgacgatgcgccagtaccgctgcacgcc
tgacgccgctgctcaccggaattattacccgcaccacttaccttgagctgctaagtgaatttcccggcgcactgattacacctcatttccctgtgtgccgc
gtcgccgatggttgccagtcagctggcgcgctacccgatcctgcttgatgaattgctcgacccgaatacgctctatcaaccgacggcgatgaatgccta
tcggatgagctgcgccaatacctgctgcgcgtgccggaagatgatgaagagcaacagcttgaggcgctgcggcagtttaagcaggcgcagttgct
gcgcgtggcggcggcggatattgccggtacgttgccagtaatgaaagtgagcgatcacttaacctggctggcggaagcgattattgatgcggtggtg
cagcaagcctgggggcagatggtggcgcgttatggccagccaacgcatctgacgatcgcgaagggcgcggtttgcggggtcggttatggcaag
ctgggcggctgggagctgggtacagctccgatctggatctggtattcctgcacgactgcccgatggatgtgatgaccgatggcgagcgtgaaatcg
atggtcgccagtctatttgcgtctcgcgcagcgcgtgatgcacctgtttagcacgcgacgtcgtccggcatcctttatgaagttgatgcgcgtctgcgt
ccatctggcgctgcggggatgctggtcactactacggaatcgttcgccgattaccagcaaaacgaagcctggacgtgggaacatcaggcgctggcc
cgtgcgcgcgtggtgtacggcgatccgcaactgaccgccgaatttgacgccattcgccgcgatattctgatgacgcctcgcgacggcgcaacgctgc
aaaccgacgtgcgagaaatgcgcgagaaaatgcgtgcccatcttggcaacaagcataaagaccgcttcgatctgaaagccgatgaaggcggtatca
ccgacatcgagtttatcgcccaatatctggtgctgcgctttgcccatgacaagccgaaactgacgcgctggtcggataatgtgcgcattctcgaagggc
tggcgcaaaacggcatcatggaggagcaggaagcgcaggcattgacgctggcgtacaccacattgcgtgatgagctgcaccacctgggctgcaa
gagttgccgggacatgtggcgctctcctgtttgtcgccgagcgtgcgcttattaaaaccagctgggacaagtggctggtggaaccgtgcgccccggc
gtaa
244 gcgcaaagcgagtgctcacttacgtgatctgttgacacaatctgaagcgaccataacttctgccgtttcagcgaatacggcggtgtggagcgcacaatc
agccctggcgaagctggtgctcaccgagtggctagtgacgcagggctggcgaaccttccttgatgaaaaagcgcaggccaaattcgccgactccttt
aaacgctttgctgacatccatctgtcacgcagcgccgccgagctgaaaaaagcctttgcccaaccgctgggcgacagctatcgcgaccagttgccgc
gcctggcgcgtgatatcgactgcgcgttactgctggccgggcattacgatcgcgcgcgcgccgtggaatggctggaaaactggcaggggcttcagc
acgccattgaaacgcgccagagagtcgaaatcgaacatttccgtaataccgcgattacccaggagccgttctggttgcacagcggaaaacgttaacg
aaaggatatttcgcatgtttaacgatctgattggcgatgatgaaacggattcgccggaagatgcgctttctgagagctggcgcgaattgtggcaggatg
cgttgcaggaggaggattccacgcccgtgctggcgcatctctcagaggacgatcgccgccgcgtggtggcgctgattgccgattttcgcaaagagtt
ggataaacgcaccattggcccgcgagggcggaaggtactcgatcacttaatgccgcatctgctcagcgatgtatgctcgcgcgacgatgcgccagta
ccgctgtcacgcctgacgccgctgctcaccggaattattacccgcaccacttaccttgagctgctaagtgaatttcccggcgcactgaaacacctcattt
ccctgtgtgccgcgtcgccgatggttgccagtcagctggcgcgctacccgatcctgcttgatgaattgctcgacccgaatacgctctatcaaccgacg
gcgatgaatgcctatcgcgatgagctgcgccaatacctgctgcgcgtgccggaagatgatgaagagcaacagcttgaggcgctgcggcagtttaag
caggcgcagttgctgcgcgtggcggcggcggatattgccggtacgttgccagtaatgaaagtgagcgatcacttaacctggctggcggaagcgatta
ttgatgcggtgggcagcaagcctgggggcagatggtggcgcgttatggccagccaacgcatagcacgatcgcgaagggcgcggttttgcggtgg
tcggttatggcaagctgggcggctgggagctgggttacagctccgatctggatctggtattcctgacgactgcccgatggatgtgatgaccgatggc
gagcgtgaaatcgatggtcgccagttctatttgcgtctcgcgcagcgcgtgatgcacctgtttagcacgcgcacgtcgtccggcatcctttatgaagttg
atgcgcgtctgcgtccatctggcgctgcggggatgctggtcactactacggaatcgttcgccgattaccagcaaaacgaagcctggacgtgggaaca
tcagccgctggcccgtgcgcgcgtggtgtacggcgatccgcaactgaccgccgaatttgacgccattcgccgcgatattctgatgacgcctcgcgac
ggcgcaacgctgcaaaccgacgtgcgagaaatgcgcgagagaaaatgcgtgcccatcttggcaacaagcataaagaccgcttcgatctgaaagccgat
gaaggcggtatcaccgacatcgagtttatcgcccaatatctgggctgcgctttgcccatgacaagccgaaactgacgcgctggcggataatgtgcg
cattctcgaagggctggcgcaaaacggcatcatggaggagcaggaagcgcaggcattgacgctggcgtacaccacattgcgtgatgagctgcacc
acctggcgctgcaagagttgccgggacatgtggcgctctcctgttttgtcgccgagcgtgcgcttattaaaaccagctgggacaagtggctggtggaa
ccgtgcgccccggcgtaagtgtggtatcatcgcgcgcaaattttgtatctctcaggagacagcaatgaaagtgacgctgccagagtttaagcaagccg
gtgtaatggtggtgggtgatgtgatgctggatcgttactggtatggcccaaccagccgtatctctccggaagcgccagtcccggttgttaaagtcgatac
cattgaagagcgtcctggcggcgcggcaaacgtggcgatgaatatcgcctcactgggcgccacggcgcgtctggttggcctgactggcattgacga
tgcggcgcgcgcgctgagcaaagcgctggccgatgttaacgtaaatgtgacttcgtttctgttccgacgcatcccaccatcactaagctgcgcgtgct
gtcgcgtaaccagcagctgattcgcctggactttgaagagggttttgaaggagtcgatccgcaaccgatgcatgaacgcatcagccaggcgcttggta
atattggcgcgctggtgctgcggatt
245 atgaccctgaatatgatgatggatgccggcggacatcatcgcgacaaacaatattaataccggcaaccacaccggcaatttacgagactgcgcaggc
atcctttctcccgtcaatttctgtcaaataaagtaaaagaggcagtctacttgaattacccccggctggttcagcgtttgttgaaaaaaagtaactgaaaaa
tccgtagaatagcgccactctgatggtaattacctattcaattaagaattatctggatgaatgtgccattaaatgcgcagcataatggtggttgtgcgg
gaaaactgcttttttttgaaagggttggtcagtagcggaaacaactcacttcacaccccgaagggggaagttgcctgaccctacgattcccgctatttcat
tcactgaccggaggttcaaaatga
246 accggatacgagagaaaagtgtctacatcggttcggttgatattgaccggcgcatccgccagcccgcccagtttctggtggatctgtttggcgattttgc
gggtcttgccggtgtcggtgccgaaaaanataccaatatttgccataacacacgctcctgttgaaaaagagatcccgccgggaaatgcggtgaacgtg
tctgatattgcgaagagtgtgccagttttggtcgcgggcaaacctgcaccagtttggttattaatgcaccagtctggcgctttttttcgccgagtttctcct
cgctaatgcccgccaggcgcggctttggcgctgatagcgcgctgaataccgatctggatcaaggttttgtcgggttatcagccaaaggtgcactcttt
gcatggtatacgtgcctgacatgttgtccgggcgacaaacggcctggtggcacaaattgtcagaactacgacacgactaactgaccgcaggagtgt
gcgatgaccctgaatatgatgatggatgccggcggacatcatcgcgacaaacaatattaataccggcaaccacaccggcaatttacgagactgcgca
ggcatcctttctcccgtcaatttctgcaaataaagtaaaagaggcagtctacttgaattacccccggctggttgagcgtttgttgaaaaaaagtaactgaa
aaatccgtagaatagcgccactctgatggttaattaacctattcaattaagaattatctggatgaatgtgccattaaatgcgcagcataatggtgcgttgtg
cgggaaaactgcttttttttgaaagggttggtcagtagcggaaacaactcacttcacaccccgaagggggaagttgcctgaccctacgattcccgctatt
tcattcactgaccggaggttcaaaatgacccagcgaaccgagtcgggtaataccgtaggcgcttcgatttgtcccagcagttcactgcgatgcagcgc
ataagcgtggtactcagccgggcgaccgaggtcgatcagacgctccagcaagtgctgtgcgtattgacaatgacgcctttttgcagcacggcatgat
ctgctgtacgacagccaccaggcgattttgaatattgaagcgttgcaggaagccgatcagcagttaatccccggcagctcgcaaatccgctatcgtcc
gggcgaagggctggtcgggacggtgctttcgcagggccaatcattagtgctggcgcgcgttgctgacgatcagcgctttcttgaccggctcggttgt
atgattacaacctgccgtttatcgccgtgccgctgatagggccagatgcgcagactttcggtgtgctgacggcacaacccatggcgcgttacgaagag
cgattacccgcctgcacccgctttctggaaacggtc
247 atgaccctgaatatgatgatggatgccggccgtcctgtaataataaccggacaattcggactgattaaaaaagcgcccttgtggcgctttttttatattccc
gcctccatttaaaataaaaaatccaatcggattcactatttaaactggccattatctaagatgaatccgatggaagctcgctgttttaacacgcgttttttaa
ccttttattgaaagtcggtgcttctttgagcgaacgatcaaatttaagtggattcccatcaaaaaaatattctcaacctaaaaaagtttgtgtaatacttgtaac
gctacatggagattaactcaatctagagggtattaataatgaatcgtactaaactggtactgggcgcaactcacttcacaccccgaagggggaagttgc
ctgaccctacgattcccgctatttcattcactgaccggaggttcaaaatga
248 accggatacgagagaaaagtgctacatcggttcggttgatattgaccggcgcatccgccagcccgcccagtttctggtggatctgtttggcgattttgc
gggtcttgccggtgtcggtgccgaaaaaaataccaatatttgccataacacacgctcctgttgaaaaagagatcccgccgggaaatgcggtgaacgtg
tctgatattgcgaagagtgtgccagttttggtcgcgggcaaaacctgcaccagtttggttattaatgcaccagtctggcgctttttttcgccgagtttctcct
cgctaatgcccgccaggcgcggctttggcgctgatagcgcgctgaataccgatctggatcaaggtttgtcgggttatcagccaaaaggtgcactcttt
gcatggttatacgtgcctgacatgttgtccgggcgacaaacggcctggtggcacaaattgtcagaactacgacacgactaactgaccgcaggagtgt
gcgatgaccctgaatatgatgatggatgccggccgtcctgtaataataaccggacaattcggactgattaaaaaagcgcccttgtggcgctttttttatatt
cccgcctccatttaaaataaaaaatccaatcggatttcactatttaaactggccattatctaagatgaatccgatggaagctcgctgttttaacacgcttttt
taaccttttattgaaagtcggtgcttctttgagcgaacgatcaaatttaagtggattcccatcaaaaaaatattctcaacctaaaaaagtttgtgtaatacttgt
aacgctacatggagattaactcaatctagagggtattaataatgaatcgtactaaactggtactgggcgcaactcacttcacaccccgaagggggaagt
tgcctgaccctacgattcccgctatttcattcactgaccggaggttcaaaatgacccagcgaaccgagtcggggtaataccgtctggcgcttcgatttgtc
ccagcagttcactgcgatgcagcgcataagcgtggtactcagccgggcgaccgaggtcgatcagacgctccagcaagtgctgtgcgtattgcacaat
gacgccttttgcagcacggcatgatctgtctgtacgacagccagcaggcgattttgaatattgaagcgttgcaggaagccgatcagcagttaatcccc
ggcagctcgcaaatccgctatcgtccgggcgaagggctggtcgggacggtgctttcgcagggccaatcattagtgctggcgcgcgttgctgacgatc
agcgctttcttgaccggctcgggttgatgattacaacctgccgtttatcgccgtgccgctgatagggccagatgcgcagactttcggtgtgctgacggc
acaacccatggcgcgttacgaagagcgattcccgcctgcacccgctttctggaaacggtc
249 atggcactgaaacacctcattccctgtgtgccgcgtcgccgatggttgccagtcagctggcgcgctacccgatcctgcttgatgaattgctcgacccg
aatacgctctatcaaccgacggcgatgaatgcctatcgcgatgagagcgccaatacctgctgcgcgtgccggaagatgatgaagagcaacagcttg
aggcgctgcggcagtttaagcaggcgcagttgctgcgcgtggcggcggcggatattgccggtacgttgccagtaatgaaagtgagcgatcacttaac
ctggctggcggaggcgattattgatgcggtggtgcagcaagcctgggggcagatggtggcgcgttatggccagccaacgcatctgcacgatcgcga
agggcgcggttttgcggtggtcggttatggcaagctgggcggctgggagctgggttacagctccgatctggatctggtattcctgcacgactgcccga
tggatgtgatgaccgatggcgagcgtgaaatcgatggtcaccagttctatttgcgtctcgcgcagcgcgtgatgcacctgtttagcacgcgcacgtcgt
ccggcatcctttatgaagttgatgcgcgtctgtccatctggcgctgcggatgctggtcactactacggaatcgttcgccgattaccagcaaaacg
aagcctggacgtgggaacatcaggcgctggcccgtgcgcgcgtggtgtacggcgatccgcaactgaccgccgaattgacgccattcgccgcgata
ttctgatgacgcctcgcgacggcgcaacgctgcaaaccgacgtgcgagaaatgcgcgagaaaatgcgtgcccatcttggcaacaagcataaagacc
gcttcgatctgaaagccgatgaaggcggtatcaccgacatcgagtttatcgcccaatatctggtgctgcgctttgcccatgacaagccgaaactgacgc
gctggtcggataatgtgcgcattctcgaagggctggcgcaaaacggcatcatggaggagcaggaagcgcaggcattgacgctggcgtacaccaca
ttgcgtgatgagctgcaccacctggcgctgcaagagttgccgggacatgtggcgctctcctgttttgtcgccgagcgtgcgcttattaaaaccagctgg
gacaagtggctggtggaa
250 gcgcaaagcgagtgctcacttacgtgatctgttgacacaatctgaagcgaccataacttctgccgtttcagcgaatacggcggtgtggagcgcacaatc
agccctggcgaagctggtgctcaccgagtggctagtgacgcagggctggcgaaccttccttgatgaaaaagcgcaggccaaatcgccgactccttt
aaacgctttgctgacatccatctgtcacgcagcgccgccgagctgaaaaaagcctttgcccaaccgctgggcgacagctatcgcgaccagttgccgc
gcctggcgcgtgatatcgactgcgcgttactgctggccgggcattacgatcgcgcgcgcgccgtggaatggctggaaaactggcaggggcttcagc
acgccattgaaacgcgccagagagtcgaaatcgaacatttccgtaataccgcgattacccaggagccgttctggttgcacagcggaaaacgtttacg
aaaggatatttcgcatggcactgaaacacctcatttccctgtgtgccgcgtcgccgatggttgccagtcagctggcgcgctacccgatcctgcttgatga
attgctcgacccgaatacgctctatcaaccgacggcgatgaatgcctatcgcgatgagctgcgccaatacctgctgcgcgtgccggaagatgatgaa
gagcaacagcttgaggcgctgcggcagtttaagcaggcgcagttgctgcgcgtggcggcgggatattgccggtacgttgccagtaatgaaagtg
agcgatcacttaacctggctggcggaagcgattattgatgcggtggtgcagcaagcctgggggcagatggtggcgcgttatggccagccaacgcat
ctgcacgatcgcgaagggcgcggttttgcggtggtcggttatggcaagagggcggctgggagctgggttacagctccgatctggatctggtattcct
gcacgactgcccgatggatgtgatgaccgatggcgagcgtgaaatcgatggtcgcagttctatttgcgtctcgcgcagcgcgtgatgcacctgttag
cacgcgcacgtcgtccggcatcctttatgaagttgatgcgcgtctgcgtccatctagcgctgcggggatgctggtcactactacggaatcgttcgccga
ttaccagcaaaacgaagcctggacgtgggaacatcaggcgctggcccgtgcgcgtggtgtacggcgatccgcaactgaccgccgaatttgacg
ccattcgccgcgatattctgatgacgcctcgcgacggcgcaacgctgcaaaccgacgtgcgagaaatgcgcgagaaaatgcgtgcccatcttggca
acaagcataaagaccgcttcgatctgaaagccgatgaaggcggtatcaccgacatcgagtttatcgcccaatatctggtgctgcgctttgcccatgaca
agccgaaactgacgcgctggtcggataatgtgcgcattctcgaacggctggcgcaaaacggcatcatggaggagcaggaagcgcaggcattcacg
ctggcgtacaccacattgcgtgatgagctgcaccacctggcgctgcaagagttgccgggacatgtggcgctctcctgttttgtcgccgagcgtgcgctt
attaaaaccagctgggacaagtggctggtggaaccgtgcgccccggcgtaagtgtggtatcatcgcgcgcaaattttgtatctctcaggagacaggaa
tgaaagtgacgctgccagagtttaagcaagccggtgtaatggtggtgggtgatgtgatgctggatcgttactggtatggcccaaccagccgtatctctc
cggaaggccagtcccggttgttaaagtcgataccattgaagagcgtcctggcggcgcggcaaacgtggcgatgaatatcgcctcactgggcgcca
cggcgcgctggttggcctgactggcattgacgatgcggcgcgcgcgctgagcaaagcgctggccgatgttaacgttaaatgtgacttcgtttctgttc
cgacgcatcccaccatcactaagctgcgcgtgctgtcggtaaccagcagctgattcgcctggactttgaagagggttttgaaggagtcgatccgcaa
ccgatgcatgaacgcatcagccaggcgcttggtaatattggcgcgctggtgctgtcggatt
251 tttcgctgaaggtgtgaccatgggccatcaggtgctggtgcagctggaaagtgttgccatcactatcgtgtggtctggcgtggtggcctttattggttaca
aactggcggacatgacggtaggcctgcgcgtaccggaagaacaagaacgtgaagggctggatgaaacagccacggcgaaaacgcctataacgc
ctga
252 tttcctttctgactctgcccgtccgggcgcactaacggcctgaaatactccctcttttcattcctggcacaacgattaaatgtagttgcgtgttagctgcg
gccattatcgaattcgactggagggggatctatgaagctggttaccgtggtgattaagccattcaaacttgaagacgtgcgtgaagcgctttcttctattg
gtattcaagggttgaccgtaactgaagtgaaaggctttggccgtcagaagggtcacgctgagctgtaccgcggtgcggaatatagcgttaatttcctgc
cgaaagtgaaaattgatgtggcgatcgctgacgatcaactcgatgaagtaatcgatgtgatcagcaaagcggcctacaccggaaaaattggcgacgg
caaaattttcgttgctgagctgcaacgcgtcattcgtattcgtaccggcgaagccgacgaagcggcactgtaatacaagacacacagtgatggggatc
ggtttcgctgaaggtgtgaccatgggccatcaggtgctggtgcagctggaaagtgttgccatcactatcgtgtggtctggcgtggtggcctttattggtta
caaactggcggacatgacggtaggcctgcgcgtaccggaagaacaagaacgtgaagggctggatgtaaacagccacggcgaaaacgcctataac
gcctgattgcgttgagttatctcctgagcataaaaaagcctccattcggaggcttttctttttttaagtttaaaggcggttagttgcgattgcgcatgacgcc
ttcctgcacgctggacgcgaccagcacaccctcttgcgtatagaactcgccgcgcacattaaccgcgagcgctggaggctgacgtgctttccacactg
tagagcagccattcgttcatattaaacgggcgatggaaccacatggagtggtcaatggtggcaacctgcataccgcgctcaaggaagcccacgccgt
gcggctgaagtgcaaccggcaggaagttaaagtctgaggcatatccaagcagatattgatgtacgcgaaaatcgtccacaccgtgccgtttgcgcg
gatccatacctggcgggtgggatcggcaacgtggcctttcagcgggttatgaaactcaaccgggcggatctccagtggtttatcactaagaaacttctc
tttggcctgcgg
253 atgtttaacgatctgattggcgatgatgaaacggattcgccggaagatgcgctttctgagagctggcgcgaattgtggcaggatgcgttgcaggagga
ggattccacgcccgtgctggcgcatctctcagaggacgatcgccgccgcgtggtggcgctgattgccgattttcgcaaagagttggataaacgcacc
attggcccgcgagggcggcaggtactcgatcacttaatgccgcatctgctcagcgatgtatgctcgcgcgacgatgcgccagtaccgctgtcacgcc
tgacgccgctgctcaccggaattattacccgcaccacttaccttgagctgctaagtgaatttcccggcgcactgaaacacctcatttccctgtgtgccgc
gtcgccgatggttgccagtcagctggcgcgctacccgatcctgcttgatgaattgctcgacccgaatacgctctatcaaccgacggcgatgaatgccta
tcgcgatgagctgcgccaatacctgctgcgcgtgccggaagatgatgaagagcaacagcttgaggcgctgcggcagtttaagcaggcgcagttgct
gcgcgggcggcggcggatattgccggtacgttgccagtaatgaaagtgagcgatcacttaacctggctggcggaagcgattattgatgcggtggtg
cagcaagcctgggggcagatggtggcgcgttatggccagccaacgcatctgcacgatcgcgaagggcgcggttttgcggtggtcggttatggcaag
ctgggcggctgggagctgggttacagatccgatctggatctggtattcctgcacgactgcccgatggatgtgatgaccgatggcgagcgtgaaatcg
atggtcgccagttctatttgcgtctcccgcagcgcgtgatgcacctgtttagcacgcgcacgtcgtccggcatcctttatgaagttgatgcgcgtctgcgt
ccatctggcgctgcggggatgctggtcactactacggaatcgttcgccgattaccagcaaaacgaagcctggacgtgggaacatcaggcgctggcc
cgtgcgcgcgtggtgtacggcgatccgcaactgaccgccgaatttgacgccattcgccgcgatattctgatgacgcctcgcgacggcgcaacgctgc
aaaccgacgtgcgagaaatgcgcgagaaaatgcgtgcccatcttggcaacaagcataaagaccgcttcgatctgaaagccgatgaaggcggtatca
ccgacatcgagtttatcgcccaatatctggtgctgcgctttgcccatgacaagccgaaactgacgcgctggtcggataatgtgcgcattctcgaagggc
tggcgcaaaacggcatcatggacgagcaggaagcgcaggcattgacgctggcgtacaccacattgcgtgatgagctgaccacctggcgctgcaa
gagttgccgggacatgggcgctctcctgttttgtcgccgagcgtgcgcttattaaaaccagctgggacaagtggctggtggaaccgtgcgccccggc
gtaa
254 gcgcaaagcgagtgctcacttacgtgatctgttgacacaatctgaaggaccataacttctgccgtttcagcgaatacggcggtgtggaggcacaatc
agccctggcgaagctggtgctcaccgagtggctagtgacgcagggctggcgaaccttccttgatgaaaaagcgcaggccaaattcgccgactccttt
aaacgctttgctgacatccatctgtcacgcagcgccgccgagctgaaaaaagcctttgcccaaccgctgggcgacagctatcgcgaccagttgccgc
gcctgccgcgtgatatcgactgcgcgttactgctggccgggcattacgatcgcgcgcgcgccgtggaatggctggaaaactggcaggggcttcagc
acgccattgaaacgcgccagagagtcgaaatcgaacatttccgtaataccgcgattacccaggagccgttctggttgcacagcggaaaacgttaacg
aaaggatatttcgcatgtttaacgatctgattggcgatgatgaaacggattcgccggaagatgcgctttctgagagctggcgcgaattgtggcaggatg
cgttgcaggaggaggattccacgcccgtgctggcgcatctctcagaggacgatcgccgccgcgtggtggcgctgattgccgattttcgcaaagagtt
ggataaacgcaccattcgcccgcgagggcggcaggtactcgatcacttaatgccgcatctgctcagcgatgtatgctcgcgcgacgatgcgccagta
ccgctgtcacgcctgacgccgctgctcaccggaattattacccgcaccacttaccttgagctgctaagtgaatttcccggcgcactgaaacacctcattt
ccctgtgtgccgcgtcgccgatggttgccagtcagctggcgcgctacccgatcctgcttgatgaattgctcgacccgaatacgctctatcaaccgacg
gcgatgaatgcctatcgcgatgagctgcgccaatacctgctgcgcgtgccggaagatgatgaagagcaacagcttgaggcgtgggcagtttaag
caggcgcagttgctgcgcgtggcggcggcggatattgccggtacgttgccagtaatgaaagtgagcgatcacttaacctggctggcggaagcgatta
ttgatgcggtggtgcagcaagcctgggggcagatggtggcgcgttatggccagccaacgcatctgcacgatcgcgaagggcgcggttttgcggtgg
tcggttatggcaagctgggcggctgggagctgggttacagctccgatctggatctggtattcctgcacgactgcccgatggatgtgatgaccgatggc
gagcgtgaaatcgatggtcgccagttctatttgcgtctcgcgcagcgcgtgatgcacctgtttagcacgcgcacgtcgtccggcatcctttatgaagttg
atgcgcgtctgcgtccatctggcgctgcggggatgctggtcactactacggaatcgttcgccgattaccagcaaaacgaagcctggacgtgggaaca
tcaggcgctggcccgtgcgcgcgtggtgtacggcgatccgcaactgaccgccgaatttgacgccattcgccgcgatattctgatgacgcctcgcgac
ggcgcaacgctgcaaaccgacgtgcgagaaatgcgcgagaaaatgcgtgcccatcttggcaacaagcataaagaccgcttcgatctgaaagccgat
gaaggcggtatcaccgacatcgagtttatcgcccaatatctggtgctgcgctttgcccatgacaagccgaaactgacgcgctggtcggataatgtgcg
cattctcgaagggctggcgcaaaacggcatcatggaggagcaggaagcgcaggcattgacgctggcgtacaccacattgcgtgatgagctgcacc
acctggcgctgcaagagttgccgggacatgtggcgctctcctgttttgtcgccgagcgtgcgcttattaaaaccagctgggacaagtggctggtggaa
ccgtgcgccccggcgtaagtgtggtatcatcgcgcgcaaattttgtatctctcaggagacaggaatgaaagtgacgctgccagagtttaagcaagccg
gtgtaatggtggtgggtgatgtgatgctggatcgttactggtatggcccaaccagccgtatctctccggaagcgccagtcccggttgttaaagtcgatac
cattgaagagcgtcctggcggcgcggcaaacgtggcgatgaatatcgcctcactgggcgccacggcgcgtctggttggcctgactggcattgacga
tgcggcgcgcgcgctgagcaaagcgctggccgatgttaacgttaaatgtgacttcgtttctgttccgacgcatcccaccatcactaagctgcgcgtgct
gtcgcgtaaccagcagctgattcgcctggactttgaagagggttttgaaggagtcgatccgcaaccgatgcatgaacgcatcagccaggcgcttggta
atattggcgcgctggtgctgtcggatt
255 atgaccctgaatatgatgatggatgccggccgtcctgtaataataaccggacaattggactgattaaaaaagcgcccttgtggcgattttttatattccc
gcctccatttaaaataaaaaatccaatcggatttcactatttaaactggccattatctaagatgaatccgatggaagctcgctgttttaacacgcgttttttaa
ccttttattgaaagtcggtgcttctttgagcgaacgatcaaatttaagtggattcccatcaaaaaaatattctcaacctaaaaaagtttgtgtaatacttgtaac
gcttcatggagattaactcaatctagagggtattaataatgaatcgtactaaactggtactgggcgcaactcacttcacaccccgaaggcgaagttgc
ctgaccctacgattcccgctatttcattcactgaccggaggttcaaaatga
256 accggatacgagagaaaagtgtctacatcggttcggttgatattgaccggcgcatccgccagcccgcccagtttctggtggatctgtttggcgattttgc
gggtcttgccggtgtcggtgccgaaaaaaataccaatatttgccataacacacgctcctgttgaaaaagagatcccgccgggaaatgcggtgaacgtg
tctgatattgcgaagagtgtgccagttttggtcgcgggcaaaacctgcaccagtttggttattaatgcaccagtctggcgctttttttcgccgagtttctcct
cgctaatgcccgccaggcgcggctttggcgctgatagcgcgctgaataccgatctggatcaaggttttgtcgggttatcagccaaaaggtgcactcttt
gcatggttatacgtgcctgacatgttgtccgggcgacaaacggcctggtggcacaaattgtcagaactacgacacgactaactgaccgcaggagtgt
gcgatgaccctgaatatgatgatggatgccggccgtcctgtaataataaccggacaattcggactgattaaaaaagcgcccttgtggcgctttttttatatt
cccgcctccatttaaaataaaaaatccaatcggatttcactatttaaactggccattatctaagatgaatccgatggaagctcgctgttttancacgcgttttt
taaccttttattgaaagtcggtgcttctttgagcgaacgatcaaatttaagtggattcccatcaaaaaaatattcttcaacctaaaaaagtttgtgtaatacttgt
aacgctacatggagattaactcaatctagagggtattaataatgaatcgtactaaactggtactgggcgcaactcacttcacaccccgaagggggaagt
tgcctgaccctacgattcccgctatttcattcactgaccggaggttcaaaatgacccagcgaaccgagtcgggtaataccgtctggcgcttcgatttgtc
ccagcagttcactgcgatgcagcgcataagcgtggtactcagccgggcgaccgaggtcgatcagacgctccagcaagtgctgtgcgtattgcacaat
gacgcctttttgcagcacggcatgatctgtctgtacgacagccagcaggcgattttgaatattgaagcgttgcaggaagccgatcagcagttaatcccc
ggcagctcgcaaatccgctatcgtccgggcgaagggctggtcgggacggtgctttcgcagggccaatcattagtgctggcgcgcgttgctgacgatc
agcgctttcttgaccggctcgggttgtatgattacaacctgccgtttatcgccgtgccgctgatagggccagatgcgcagactttcggtgtgctgacggc
acaacccatggcgcgttacgaagagcgattacccgcctgcacccgctttctggaaacggtc
257 tttcgctgaaggtgtgaccatgggccatcaggtgctggtgcagctggaaagtgttgccatcactatcgtgtggtctggcgtggtggcctttattggttaca
aactggcggacatgacggtaggcctgcgcgtaccggaagaacaagaacgtgaagggctggatgtaaacagccacggcgaaaacgcctataacgc
ctga
258 tttcctttctgactctgcccgtccgggcgcactaacggcctgaaatactccctcttttcattcctggcacaacgattgcaatgtctgttgcgtgttagctgcg
gccattatcgaattcgactggagggggatctatgaagctggttaccgtggtgattaagccattcaaacttgaagacgtgcgtgaagcgctttcttctattg
gtattcaagggttgaccgtaactgaagtgaaaggctttggccgtcagaagggtcacgctgagctgtaccgcggtgcggaatatagcgttaatttcctgc
cgaaagtgaaaattgatgtggcgatcgctgacgatcaactcgatgaagtaatcgatgtgatcagcaaagcggcctacaccggaaaaattggcgacgg
caaaattttcgttgctgagctgcaacgcgtcattcgtattcgtaccggcgaagccgacgaagggcactgtaatacaagacacacagtgatggggatc
ggtttcgctgaaggtgtgaccatgggccatcaggtgctggtgcagctggaaagtgttgccatcactatcgtgtggtctggcgtggtggcctttattggtta
caaactggcggacatgacggtaggcctgcgcgtaccggaagaacaagaacgtgaagggctggatgtaaacagccacgacgaaacgcctataac
gcctgattgcgttgagttatctcctgagcataaaaaagcctccattcggaggcttttctttttttaagtttaaagcgcggttagttgcgattgcgcatgacgcc
ttcctgcacgctggacgcgaccagcacaccctcttgcgtatagaactcgccgcgcacaaaaccgcgagcgctggaggctgacgtgctttccacactg
tagagcagccattcgttcatattaaacgggcgatggaaccacatggagtggtcaatggtggcaacctgcataccgcgctcaaggaagcccacgccgt
gcggctgaagtgcaaccggcaggaagttaaagtctgaggcatatccaagcagatattgatgtacgcgaaaatcgtccggcaccgtgccgtttgcgcg
gatccatacctggcgggtggcatcggcaacgtggcctttcagcgggttatgaaactcaaccgggcggatctccagtggtttatcactaagaaacttctc
tttggcctgcgg
259 atgacctttaatatgatgcctggggtcactggagcgcttttatcggcatcctgaccgaagaatttgccggtttcttcccgacctggctggcccctgttcagg
ttgtggtgatgaatatcactgattctcaagctgaatatgtcaacgaattgacccgtaaattgataaatgcgggcattcgtgtaaaagcggacttgagaaac
gagaagattggctttaaaatccgcgagcacactttacgtcgtgtcccttatatgttggtctgtggtgataaagaggtggaagcaggcaaagtggccgttc
gcacccgccgcggtaaagacctgggcagcctggacgtaaggaagtgattgagaagctgcaacaacagattcgcagccgcagtcttcaacaactgg
aggaataaggtattaaaggcggaatacgagttcaaacggcacgtccgaatcgtatcaatggcgagattcgcgcccaggaagttcgcttaactggtctg
gaaggtgagcagctgggtattgcaatagaactaactacccgccctgaaggcggtacctgcctgaccctgcgattcccgttatttcattcactgaccgga
ggcccacgatga
260 ggtacgacaaaaacgtctccagcgacgtgcggttaatattgactggcgcatccgccacatcccccagtttttgctggatcagtttggcgattttgcgggtt
tttcccgtgtcactgccaaaaaaaataccaatgttagccatgtcgcgctcctgttgagaaagaataaggccgcctgcaaacggcggatatcccttctcct
gttgcgaaggctgtgccaggtttttttaaggccttctgtgcactgaaatgcgtgaaaaaatgactcttttttgtgcaggcaccgtcctctctccgctatccag
acctgctttgaaggcctctgagggccaaatcagggccaaaacacgaatcacgatcaatgtttcggcgcgttacctgttcgaaaggtgcactctttgcat
ggttaatcacacccaatcaggcctgcggatgtcgggcgtttcacaacacaaaatgttgtaaatgcgacacagccgggcctgaaaccaggagcgtgtg
atgacctttaatatgatgcctggggtcactggagcgctttatcggcatcctgaccgaagaatttgccggtttcttcccgacctggctggcccctgttcagg
ttgtggtgatgaatatcactgattctcaagctgaatatgtcaacgaattgacccgtaaattgcaaaatgcgggcattcgtgtaaaagcggacttgagaaac
gagaagattggctttaaaatccgcgagcacactttacgtcgtgtcccttatatgttggtctgtggtgataaagaggtggaagcaggcaaagtggccgttc
gcacccgccgcggtaaagacctgggcagcctggacgtaagtgaagtgattgagaagctgcaacaagagattcgcagccgcagtcttcaacaactgg
aggaataaggtattaaaggcggaaaacgagttcaaacggcacgtccgaatcgtatcaatggcgagattcgcgcccaggaagttcgcttaactggtctg
gaaggtgagcagctgggtattgcaatagaactaactacccgccctgaaggcggtacctgcctgaccctgcgattcccgttatttcattcactgaccgga
ggcccacgatgacccaccgacccgagtcgggcaccaccgtctggcgttttgatctctcacagcaatttaccgccatgcagcgcatcagcgtggtgttg
agtcgcgcaaccgagataagccagacgctgcaggaggtgctgtgtgttctgcataatgacgcatttatgcaacacggcatgctgtgtctgtatgacaac
cagcaggaaattctgagtattgaagccttgcaggaggcagaccaacatctgatccccggcagctcgcaaattcgctatcgccctggcgaagggctgg
taggagccgtactgtcccagggacaatctcttgtgctgccgcgtgtcgccgacgatcaacgctttctcgacaggcttggcatctatgattacaacctgcc
gtttatcgccgtccccttaatggggccaggcgcgcagacgattggcgtgctcgccgcgcagccgatggcgcgtctggaggagcggcttccttcctgt
acgcgctttctggaaaccgtc
261 ttgaagagtttgatcatggctcagattgaacgctggcggcaggcctaacacatgcaagtcgaacggtagcacagagagcttgctctcgggtgacgagt
ggcggacgggtgagtaatgtctgggaaactgcctgatggagggggataactactggaaacggtagctaataccgcataacgtcgcaagaccaaaga
ggcggaccttcgggcctcttgccatcagatgtgcccagatgggattagctagtaggtggggtaacggctcacctaggcgacgatccctagctggtctg
agaggatgaccagccacactggaactgagacacggtccagactcctacgggaggcagcagtggggaatattgcacaatgggcgcaagcctgatgc
agccatgccgcgtgtgtgaagaaggccttcgggttgtaaagcactttcagcggggaggaagggagtaaggttaataaccttattcattgacgttacccg
cagaagaagcaccggctaactccgtgccagcagccgcggtaatacggagggtgcaagcgttaatcggaattactgggcgtaaaccgcacgcaggc
ggtctgtcaagtcggatgtgaaatccccgggctcaacctgggaactgcatccgaaactggcaggcttgagtctcgtagagggaggtagaattccagg
tgtagcggtgaaatgcgtagagatctggaggaataccggtggcgaaggcggcctcctggacgaagactgacgctcaggtgcgaaagcgtgggga
gcaaacaggattagataccctggtagtccacgccgtaaacgatgtctatttggaggttgtgcccttgaggcgtggcttccggagctaacgcgttaaata
gaccgcctggggagtacggccgcaaggttaaaactcaaatgaattgacgggggcccgcacaagcggtggagcatgtggtttaattcgatgcaacgc
gaagaaccttacctggtcttgacatccacagaactttccagagatggattggtgccttcgggaactgtgagacaggtgctgcatggctgtcgtcagctc
gtgttgtgaaatgttgggttaagtcccgcaacgagcgcaacccttatcctttgttgccagcggtccggccgggaactcaaaggagactgccagtgataa
actggaggaaggtggggatgacgtcaagtcatcatggcccttacgaccagggctacacacgtgctacaatggcgcatacaaagagaagcgacctcg
cgagagtaagcggacctcataaagtgcgtcgtagtccggattggagtctgcaactcgactccatgaagtcggaatcgctagtaatcgtggatcagaat
gccacggtgaatacgttcccgggccttgtacacaccgcccgtcacaccatgggagtgggttgcaaaagaagtaggtagcttaaccttcgggagggc
gcttaccactttgtgattcatgactggggtgaagtcgtaacaaggtaaccgtaggggaacctgcggttggatcacctcctt
262 ttgagagtttgatcatggctcagattgaacgctggcggcaggcctaacacatgcaagtcgaacggtagcacagagagcttgctctcgggtgacgagt
ggcggacgggtgagtaatgtctgggaaactgcctgatggagggggataactactggaaacggtagctaataccgcataacgtcgcaagaccaaaga
gggggaccttcgggcctcttgccatcagatgtgcccagatgggattagctagtaggtggggtaacggctcacctaggcgacgatccctagctggtctg
agaggatgaccagccacactggaactgagacacggtccagactcctacgggaggcagcagtggggaatattccacaatgggcgcaagcctgatgc
agccatgccgcgtgtgtgaagaaggccttcgggttgtaaagcactttcagcggggaggaaggnantanggttaataacctgtgttnattgacgttacc
cgcagaagaagcaccggctaactccgtgccagcagccgcggtaatacggagggtgcaagcgttaatcggaattactgggcgtaaagcgcacgcag
gcggtctgtcaagtcggatgtgaaatccccgggctcaacctgggaactgcatccgaaactggcaggcttgagtctcgtagagggaggtagaattcca
ggtgtagcggtgaaatgcgtagagatctggaggaataccggtggcgaaggcggcctcctggacgaagactgacgctcaggtgcgaaagcgtggg
gagcaaacaggattagataccctggtagtccacgccgtaaacgatgtctatttggaggttgtgcccttgaggcgtggcttccggagctaacgcgttaaa
tagaccgcctggggagtacggccgcaaggttaaaactcaaatgaattgacgggggcccgcacaagcggtggagcatgtggtttaattcgatgcaac
gcgaagaaccttacctggtcttgacatccacagaacttagcagagatgctttggtgccttcgggaactgtgagacaggtgctgcatggctgtcgtcagc
tcgtgttgtgaaatgttgggttaagtcccgcaacgagcgcaaccatatcctttgttgccagcggttaggccgggaactcaaaggagactgccagtgat
aaactggaggaaggtggggatgacgtcaagtcatcatggcccttacgaccagggctacacacgtgctacaatggcgcatacaaagagaagcgacct
cgcgagagtaagcggacctcataaagtccgtcgtagtccggattggagtctgcaactcgactccatgaagtcggaatcgctagtaatcgtggatcaga
atgccacggtgaatacgttcccgggccttgtacacaccgcccgtcacaccatgggagtgggttgcaaaagaagtaggtagcttaaccttcgggaggg
cgcttaccactttgtgattcatgactggggtgaagtcgtaacaaggtaaccgtaggggaacctgcggttggatcacctcctt
263 atgaccatgcgtcaatgcgccatttacggcaaaggtgggatcggcaaatcgaccaccacacagaacctggtcgccgcgctggcggagatgggtaaa
aaagtcatgattgtcggctgtgacccgaaagccgattccacgcgtttgatcctgcatgcgaaagcgcagaacaccattatggagatggctgagaagt
cggctccgtggaagacctggagttagaagacgtgctgcaaatcggttacggcggcgtgcgctgcgcagagtccggcggcccggagccaggcgtg
ggctgtgccggtcgccgggtgatcaccgcgattaacttcctcgaagaagaaggcgcttacgtgccggatctcgattttgttttctacgacgtgctgggc
gacgtggtatgcggtggtttcgccatcccgattcgtgaaaacaaagcgcaggagatctacatcgtttgctctcgcgaaatgatggcgatgtacgccgc
caacaacatctccaaaggcatcgtgaaatacgccaaatccggtaaagtgcgcctcggcgggctgatttgtaactcgcgccagaccgaccgtgaagat
gaactgatcattgcgctggcagaaaaactcggcacgcagatgatccactttgttccccgcgacaacattgtgcagcgtgcggaaatccgccgtatgac
ggttatcgaatatgacccgacctgcaatcaggcgaacgaatatcgcagccttgccagcaaaatcgtcaacaacaccaaaatggtggtgcccaccccc
tgcaccatggatgaactggaagaactgctgatggagttcggcattatggatgtggaagacaccagcatcattggtaaaaccgccgccgaagaaaacg
ccgtctga
264 atgagcaatgcaacaggcgaacgcaacctggagataatcgagcaggtgctcgaggttttcccggagaagacgcgcaaagaacgcagaaaacacat
gatggtgacggacccggagcaggaaagcgtcggtaagtgcatcatctctaaccgcaaatcgcagccaggcgtgatgaccgtgcgcggctgctcgta
tgccggttcgaaaggggtggtatttgggccaatcaaggatatggcgcatatctcgcatggcccaatcggctgcggccaatactcccgcgccgggcgg
cggaactactacaccggcgtcagcggcgtggacagcttcggcacgctcaacttcacctccgattttcaggagcgcgacatcgtgtttggcggcgataa
aaagctcgccaaactgattgaagagctggaagagctgttcccgctgaccaaaggcatttcgattcagtcggaatgcccggtcggcctgattggcgatg
acattgaggccgtcgcgaacgccagccgcaaagccatcaacaaaccggttattccggtgcgttgcgaaggctttcgcggcgtgtcgcaatccctcgg
tcaccatattgccaacgatgtgatccgcgactgggtgctggataaccgcgaaggcaaaccgttcgaatccaccccttacgatgtggcgatcatcggcg
attacaacatcggcggcgatgcctgggcttcgcgcattttgctcgaagagatgggcttgcgggggtggcacagtggtctggcgacggtacgctggt
ggagatggaaaacacgccgttcgtcaaactgaacctggtgcattgttaccgctcaatgaactacatctcgcgccatatggaggagaagcacggtattc
cgtggatggaatacaacttctttggtccgacgaaaatcgcggaatcgctgcgcaaaatcgccgaccagtttgacgacaccattcgcgccaacgccga
agcggtgatcgccagataccagccgcaaaacgacgccattatcgccaaatatcgcccgcgtctggaggggcgcaaagtgctgctttatatgggcgg
gctgcgtccgcgccatgtgattgccgcctatgaagacctgggaatggagatcatcgctgccgctatgagttcggtcataacgatgattacgaccgca
ccttgccggatctgaaagagggcacgctgctgtttgagatgccagcagttatgagctggaggcgttcgtcaacgcgctgaaaccggatctcatcggtt
ccggcatcaaagagaagtacatctttcagaaaatgggcgtgccgtttcgccagatgcactcctgggattactccggcccgtaccacggctatgacggc
ttcgccatcttcgcccgcgatatggatatgacgctcaacaaccccgcgtggggccagttgaccgcgccgtggctgaaatccgcctga
265 atgagccagactgctgagaaaatacagaattgccatcccctgtttgaacaggatgcttaccagacgctgtttgccggtaaacgggcactcgaagaggc
gcactcgccggagcgggtgcaggaagtgtttcaatggaccactaccccggaatatgaagcgctgaactttaaacgcgaagcgctgactatcgaccc
ggcaaaagcctgccagccgctgggcgcggtgctctgttcgctggggtttgccaataccctaccgtatgtgcacggttcacagggttgcgtggcctattt
ccgcacgtactttaaccgccactttaaagaaccggtggcctgcgtgtcggattcaatgacggaagacgcggccgtgttcggcgggaataacaacctc
aacaccggcttacaaaacgccagcgcgctgtataaaccggagattatcgccgtctctaccacctgtatggcggaagtgatcggtgatgatttgcaggc
ctttatcgccaacgccaaaaaagatggttttctcgatgccgccatccccgtgccctacgcgcacacccccagttttatcggcagccatatcaccggctg
ggataacatgtttgaaggttttgcccggacctttacggcagaccatgaagctcagcccggcaaactttcacgcatcaacctggtgaccgggtttgaaac
ctatctcggcaatttccgcgtgctgaaacgcatgatggaacaaatggaggtgccggcgagtgtgctctccgatccgtcggaagtgctggatactcccg
ccaacgggcattaccagatgtacgcgggcgggacgacgcagcaagagatgcgcgaggcgccggatgctatcgacaccctgttgctgcagccctg
gcaactggtgaaaagcaaaaaagtggtgcaggagatgtggaatcagcccgccaccgaggtttctgttcccgttgggctggcaggaacagacgaact
gttgatggcgattagccagttaaccggcaaggccattcccgattcactggcgctggagcgcgggcggctggtcgatatgatgctcgattcccacacct
ggttgcacggtaaaaaattcggcctgtttggcgatccggattttgtcatgggattgacccgtttcctgctggagctgggctgcgaaccgaccgttatcctc
tgccacaacggtaacaaacgctggcagaaagcaatgaagaaaatgcttgacgcctcgccgtacggccaggagagcgaagtgtttatcaactgcgatt
tgtggcatttccgctcgctgatgtttacccgccagccggattttatgattggcaactcgtacggcaagttcattcagcgcgacaccttagccaaaggcga
gcagtttgaagttccgctgatccgcctcggttttcccctgttcgaccgccaccatctgcaccgccagaccacctggggctacgagggcgccatgagca
ttctcactacccttgtgaatgcggtactggagagagtggacaaagagaccatcaagctcggcaaaaccgactacagcttcgatatcttatccgttaa
266 atgaccctgaatatgatgatggatgccggcgcgcccgaggcaatcgccggtgcgctttcgcgacaccatcctgggctgttttttaccatcgtgaagaa
gcgcccgtcgccatttcgctgactgatgccgacgcacgcattgtctatgccaacccggctttctgccgccagaccggctatgaactagaagcgttgttg
cagcaaaatccccgcctgcttgcaagtcgccaaaccccacgggaaatctatcaggatatgtggcacaccttgttacaacgccgaccgtggcgcgggc
aattgattaaccgccaccgcgacggcagcctgtatctggtcgagatcgatatcaccccggtgattaacccgtttggcgaactggaacactacctggca
atgcagcgcgatatcagcgccagttatgcgctggagcagcggttgcgcaatcacatgacgctgaccgaagcggtgctgaataacattccgccggcg
gtggttgtagtggatgaacgcgatcatgtggttatggataaccttgcctacaaaacgttctgtgccgactgcggcggaaaagagctcctgagcgaactc
aatttttcagcccgaaaagcggagctggcaaacggccaggtcttaccggtggtgctgcgcggtgaggtgcgctggttgtcggtgacctgctgggcgc
tgccgcgcgtcagcgaagaagccagtcgctactttattgataacaggctgacgcgcacgctggtggtgatcaccgacgacacccaacaacgccagc
agcaggaacagggccgacttgaccgccttaaacagcagatgaccaacggcaaactactggcagcgatccgcgaaggcttgacgccgcgctgatc
cagcttaactgccccatcaatatgctggcggcggcgcgacgtttaaacggcagtgataacaacaatgtggcgctcgacgccgcgtacgcgaaggt
gaagaggcgatggcgcggctgaaacgttgccgcccgtcgctggaactggaaagtgcggccgtctggccgctgcaacccttttttgacgatctgcgc
gcgctttatcacacccgctacgagcaggggaaaaatttgcaggtcacgctggattcccatcatctggtgggatttggtcagcgtacgcaactgttagcct
gcctgagtctgtggctcgatcgcacgctggatattgccgccgggctgggtgatttcaccgcgcaaacgcagatttacgcccgcgaagaagagggctg
gctctctttgtatatcactgacaatgtgccgctgatcccgctgcgccacacccactcgccggatgcgcttaacgctccgggaaaaggcatggagctgc
gcctgatccagacgctggtggcacaccaccacggcgcaatagaactcacttcacaccccgaagggggaagttgcctgaccctacgattcccgctatt
tcattcactgaccggaggttcaaaatga
267 atgacccagcgaaccgagtcgggtaataccgtctggcgatcgatttggcccagcagttcactgcgatgcagcgcataagcgtggtactcagccggg
cgaccgaggtcgatcagacgctccagcaagtgctggcgtattgcacaatgacgcctttttgcagcacggcatgatctgtctgtacgacagccagcag
gcgattttgaatattgaagcgttgcaggaagccgatcagcagttaatccccggcagctcgcaaatccgctatcgtccgggcgaagggctggtcggga
cggtgctttcgcagggccaatcattagtgctggcgcgcgttgctgacgatcagcgctttcttgaccggctcgggttgtatgattacaacctgccgtttatc
gccgtgccgctgatagggccagatgcgcagactttcggtgtgctgacggcacaacccatggcgcgttacgaagagcgattacccgcctgcacccgc
tttctggaaacggtcgctaacctggtcgcgcaaaccgtgcgtttgatggcaccaccggcagtgcgcccttccccgcgcgccgccataacacaggccg
ccagcccgaaatcctgcacggcctcacgcgcatttggttttgaaaatatggtcggtaacagtccggcgatgcgccagaccatggagattatccgtcag
gtttcgcgctgggacaccaccgttctggtacgcggcgagagtggcaccggcaaggagctgattgccaacgccatccaccaccattcgccgcgtgcc
ggtgcgccatttgtgaaattcaactgtgcggcgctgccggacacactgctggaaagcgaattgttcggtcacgagaaaggggcatttaccggcgcgg
tacgccagcgtaaaggccgttttgagctggccgatggcggcacgctgtttcttgacgagatcggcgagtgtagcgcctcgtttcaggctaagctgctg
cgcattttgcaggaaggcgaaatggaacgcgtcggcggcgacgagacattgcaagtgaatgtgcgcattattgccgcgacgaaccgcaatcttgaa
gatgaagtccggctggggcactttcgcgaagatctctattatcgcctgaatgtgatgcccatcgccctgccgccactacgcgaacgccaggaggacat
tgccgagctggcgcactttctggtgcgtaaaatcgcccataaccagagccgtacgctgcgcattagcgagggcgctatccgcctgctgatgagctaca
actggcccggtaatgtgcgcgaactggaaaactgccttgagcgctcagcggtgatgtcggagaacggtctgatcgatcgggatgtgattttgtttaatc
atcgcgaccagccagccaaaccgccagttatcagcgtctcgcatgatgataactggctcgataacaaccttgacgagcgccagcggctgattgcggc
gctggaaaaagcgggatgggtacaagccaaagccgcgcgcttgctggggatgacgccgcgccaggtcgcctatcgtattcagacgatggatataac
cctgccaaggctataa
268 atgccgcaccacgcaggattgtcgcagcactggcaaacggtattttctcgtctgccggaatcgctcaccgcgcagccattgagcgcgcaggcgcagt
cagtgctcacttttagtgattttgttcaggacagcatcatcgcgcatcctgagtggctggcagagcttgaaagcgcgccgccgcctgcgaacgaatggc
aacactatgcgcaatggctgcaagcggcgctggatggcgtcaccgatgaagcctcgagatgcgcgcgctgcggctgtttcgccgtcgcatcatggt
gcgcatcgcctggagccaggcgttacagttggtggcggaagaagatatcctgcaacagcttagcgtgctggcggaaaccctgatcgtcgccgcgcg
cgactggctttatgaggcctgctgccgtgagtggggaacgccgagcaatccacaaggcgtggcgcagccgatgctggtactcggcatgggcaaact
gggtggcggcgaactcaatttctcatccgatatcgatttgattttcgcctgggacggaaaatggcgcaacgcgcggtggacgccgtgagctggataacg
cgcaatttttcactcgccttggtcaacggctgattaaagtcctcgaccagccaacgcaggatggctttgtctaccgcgtcgatatgcgcttgcgcccgttt
ggcgacagcggcccgctggtgctgagctttgccgcgctggaagattactaccaggagcaggggcgcgattgggaacgctacgcgatggtgaaagc
gcgcattatgggcgataacgacggcgaccatgcgcgggagttgcgcgcaatgctgcgcccgtttgttttccgccgttatatcgacttcagcgtgattca
gtccctgcgtaacatgaaaggcatgattgcccgcgaagtgcgtcgccgtggcctgaagacaacattaagctcggcgcgggcgggatccgcgaaat
agaatttatcgtccaggttttccagctgattcgcggcggtcgcgagcctgcactgcaatcgcgttcactgttgccgacgcttgctgccatagatcaactg
catctgctgccggatggcgacgcaacccggctgcgcgaggcgtatttgtggctgcgacggctggagaacctgctgcaaagcatcaatgacgaacag
acacagacgctgccgggcgatgaactgaatcgcgcgcgcctcgcctggggaatgggcaaagatagctgggaagcgctctgcgaaacgctggaag
cgcatatgtcggcggtgcgtcagatatttaacgatctgattggcgatgatgaaacggattcgccggaagatgcgctttctgagagctggcgcgaattgt
ggcaggatgcgagcaggaggaggattccacgcccgtgctggcgcatctctcagaggacgatcgccgccgcgtggtggcgctgattgccgattttcg
caaagagttggataaacgcaccattggcccgcgagggoggcaggtactcgatcacttaatgccgcatctgctcagcgatgtatgctcgcgcgacgat
gcgccagtaccgctgtcacgcctgacgccgctgctcaccggaattattacccgcaccacttaccttgagctgctaagtgaatttcccggcgcactgaaa
cacctcatttccctgtgtgccgcgtcgccgatggttgccagtcagaggcgcgctacccgatcctgcttgatgaattgctcgacccgaatacgctctatc
aaccgacggcgatgaatgcctatcgcgatgagctgcgccaatacctgctgcgcgtgccggaagatgatgaagagcaacagcttgaggcgctgcgg
cagtttaagcaggcgcagttgctgcgcgtggcggcggcggatattgccggtacgttgccagtaatgaaagtgagcgatcacttaacctggctggcgg
aagcgattattgatgcggtggtgcagcaagcctgggggcagatggtggcgcgttatggccagccaacgcatctgcacgatcgcgaagggcgcggtt
ttgcggtggtcggttatggcaagctgggcggctgggagctgggttacagctccgatctggatctggtattcctgcacgactgcccgatggatgtgatga
ccgatggcgagcgtgaaatcgatggtcgccagttctatttgcgtctcgcgcagcgcgtgatgcacctgtttagcacgcgcacgtcgtccggcatccttt
atgaagttgatgcgcgtctgcgtccatctggcgctgcggggatgctggtcactactacggaatcgttcgccgattaccagcaaaacgaagcctggacg
tgggaacatcaggcgctggcccggcggcgtggtgtacggcgatccgcaactgaccgccgaatttgacgccattcgccgcgatattctgatgacgc
ctcgcgacggcgcaacgctgcaaaccgacgtgcgagaaatgcgcgagaaaatgcgtgcccatcttggcaacaagcataaagaccgcttcgatctga
aagccgatgaaggcggtatcaccgacatcgagtttatcgcccaatatctggtgctgcgctttgcccatgacaagccgaaactgacgcgctggtcggat
aatgtgcgcattctcgaagggctggcgcaaaacggcatcatggaggagcaggaagcgcaggcattgacgctggcgtacaccacattgcgtgatga
gctgcaccacctggcgctgcaagagttgccgggacatgtggcgctctcctgttttgtcgccgagcgtgcgcttattaaaaccagctgggacaagtggc
tggtggaaccgtgcgccccggcgtaa
269 attgaagagtttgatcatggctcagattgaacgctggcggcaggcctaacacatgcaagtcgaacggtagcacagagagcttgactctcgggttgacga
gtggcggacgggtgagtaatgtctgggaaactgcctgatggagggggataactactggaaacggtagctaataccgcataacgtcgcaagaccaaa
gagggggaccttcgggcctcttgccatcagatgtgcccagatgggattagctagtaggtggggtaacggctcacctaggcgacgatccctagctggt
ctgagaggatgaccagccacactggaactgagacacggtccagactcctacgggaggcagcagtggggaatattgcacaatgggcgcaagcctga
tgcagccatgccgcgtgtgtgaagaaggccttcgggttgtaaagcactttcagcggggaggaagggagtaaggttaataaccttgctcattgacgttat
ccgcagaagaagcaccggctaactccgtgccagcagccgcggtaatacggagggtgcaagcgttaatcggaattactgggcgtaaagcgcacgca
ggcggtctgtcaagtcggatgtgaaatccccgggctcaacctgggaactgcatccgaaactggcaggcttgagtctcgtagagggaggtagaattcc
aggtgtagcggtgaaatgcgtagagatctggaggaataccggtggcgaaggcggcctcctggacgaagactgacgctcaggtgcgaaagnnnnn
nnnnnnaacaggattagataccctggtagtccatgccgtaaacgatgtctactagccgttggggcctttgaggctttagtggcgcagctaacgcgata
agtagaccgcctggggagtacggtcgcaagactaaanctcaaatgaattgacgggggcccgcacaagcggtggagcatgtggtttaattcgatgca
acgcgaagaaccttacctggccttgacatagtaagaattttccagagatggattggtgccttcgggaacttacatacaggtgctgcatggctgtcgtcag
ctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaacccttgtcattagttgctacatttagttgggcactctaatgagactgccggtgacaaa
ccggaggaaggtggggatgacgtcaagtcctcatggcccttataggtggggctacacacgtcatacaatggctggtacaaagggttgccaacccgc
gagggggagctaatcccataaaaccagtcgtagtccggatcgcagtctgcaactcgactgcgtgaagtcggaatcgctagtaatcgtggatcagaat
gtcacggtgaatacgttcccgggtcttgtacacaccgcccgtcacaccatgggagcgggttctgccagaagtagttagcttaaccgcaaggagggcg
attaccacggcagggttcgtgactggggtgaagtcgtaacaaggtagccgtatcggaaggtgcggctgcatcacctccttt
270 atgaccatgcgtcaatgcgccatttacggcaaaggtgggatcggcaaatcgaccaccacacagaacctggtcgccgcgctggcggagatgggtaaa
aaagtcatgattgtcggctgtgacccgaaagccgattccacgcgtttgatcctgcatgcgaaagcgcagaacaccattatggagatggctgctgaagt
cggctccgtggaagacctggagttagaagacgtgctgcaaatcggttacggcggcgtgcgctgcgcagagtccggcggcccggagccaggcgtg
ggctgtgccggtcgcggggtgatcaccgcgattaacttcctcgaagaagaaggcgcttacgtgccggatctcgattttgttttctacgacgtgctgggc
gacgtggtatgcggtggtttcgccatgccgattcgtgaaaacaaagcgcaggagatctacatcgtttgctctggcgaaatgatggcgatgtacgccgc
caacaacatctccaaaggcatcgtgaaatacgccaaatccggtaaagtgcgcctcggcgggctgatttgtaactcgcgccagaccgaccgtgaagat
gaactgatcattgcgctggcagaaaaactcggcacgcagatgatccactttgttccccgcgacaacattgtgcagcgtgcggaaatccgccgtatgac
ggttatcgaatatgacccgacctgcaatcaggcgaacgaatatcgcagccttgccagcaaaatcgtcaacaacaccatnatggtggtgcccaccccc
tgcaccatggatgaactggaagaactgctgatggagttcggcattatggatgtggaagacaccagcatcattggtaaaaccgccgccgaagaaaacg
ccgtctga
271 atggccgaaattctgcgcagtaaaaaaccgctggcggtcagcccgataaaaagggccagccgctgggggcgatcctcgcaagcctgggtgtcga
acagtgcataccgctggtacacggcgcacagggatgtagcgcgttcgcgaaggtgttctttattctacattttcacgatccgatcccgctgcaatcgac
ggcgatggatccgacttccaccattatgggcgccgatgaaaacatttttaccgcgctcaatgtgctctgccagcgcaacgccgcgaaagccattgtgct
gctcagcaccgggctttcagaagcccagggcagcgacatttcgcgggtggtgcgccagtttcgtgatgattttccccggcataaaggcgttgcgctgc
tcaccgtcaacacacccgatttctacggctcgctggaaaacggctacagcgccgtgctggaaagcatgattgaacagtgggtacccgcacagcccg
ccgccagcctgcgcaaccgccgtgtcaacctgctggtcagccatttactgacaccaggcgatatcgaactgttgcgcagttatgttgaagccttcggcc
tgcaaccggtgattgtgccggatctgtcgctgtcgctggacgggcatctggcagacggtgatttttcgcctgttacccaagggggaacatcgctgcgc
atgattgaacagatggggcaaaacctggccacctttgtgattggcgcctcgctgggccgtgcggcggcgttactggcgcagcgcagccgtggcgag
gtgatcgccctgccgcatctgatgacgcttgcagcctgcgacacgtttattcatcgactgaaaaccctctccgggcgcgatgtccccgcgtggattgag
cgccagcgcggccaagttcaggatgcgatgatcgattgccatatgtggctgcagggtgcggctatcgccatggcagcagaaggcgatcacctggcg
gcatggtgcgatttcgcccgcagccagggcatgatccccggcccgattgtcgcaccggtcagccagccggggttgcaaaatctgccggttgaaacc
gtggttatcggcgatctggaagatatgcaggatcggctttgcgcgacgcccgccgcgttactggtggccaattctcatgccgccgatctcgccacgca
gtttgatttgtcacttatccgcgccgggttcccggtgtatgaccggctgggggaatttcgtcgcctgcgccaggggtacagcggcattcgtgacacgct
gtttgagctgggaatgtgatgcgcgagcgccatcacocgcttgcaacctaccgctcgccgctgcgccagcacgccgacgacaacgttacgcctgg
agatctgtatgccgcatgttaa
272 atgaaaaacacaacattaaaaacagcgcttgcttcgctggcgttactgcctggcctggcgatggcggctcccgctgtggcggataaagccgacaacg
gctttatgatgatttgcaccgcgctggtgctgtttatgaccattccgggcattgcgctgttctacggcggtttgatccgcggtaaaaacgtgctgtcgatgc
tgacgcaggttgccgtcaccttcgcactggtgtgcattctgtgggtggtgtatggctactcgctggcatttggcgagggcaacagcttcttcgggagtttt
aactgggcgatgttgaaaaacatcgaactgaaagccgtgatgggcagcatttatcagtatatccacgtggcgttccagggttccttcgcctgtatcaccg
ttggcctgattgtcggtgcactggctgagcgtattcgcttactgcggtgctgatttttgtggtggtatggctgacgctttcttacgtgccgattgcacacat
ggtggggggcggcggtctgctggcaacccacggtgcgctggatttcgcaggcggtacggttgttcacatcaacgctgcgattgcaggtctggtgggg
gcttacctgattggcaaacgcgtgggctttggcaaagaagcattcaaaccgcataacctgccgatggtcttcactggcaccgctatcctgatgttggct
ggtttggtttcaacgccggctccgcaagctcggcgaacgaaattgctgcgctggccttcgtgaacactgtcgttgccactgctgccgctattctggcgtg
ggtatttggcgaatgggcaatgcgcggcaagccgtctctgctcggtgcctgttctggtgccatcgcgggtctggttggtatcacccccgcctgtggttat
gtgggtgtcggcggtgcgctgattgtgggtctgattgccggtctggctgggctgtggggcgttactgcgctgaaacgtatgttgcgtgtcgatgacccg
tgtgacgtattcggtgtgcacggcgtgtgcggcatcgtgggctgtatcctgacgggtatcttcgcctctacgtcgctgggtggtgtcggtttcgctgaag
gtgtgaccatgggccatcaggtgctggtgcagctggaaagtgttgccatcactatcgtgtggtctggcgtggtggcctttattggttacaaactggcgga
catgacggtaggcctgcgcgtaccggaagaacaagaacgtgaagggctggatgtaaacagccacggcgaaaacgcctataacgcctga
273 cgtcctgtaataataaccggacaattcggactgattaaaaaagcgcccttgtggcgctttttttatattcccgcctccatttaaaataaaaaatccaatcgga
tttcactatttaaactggccattatctaagatgaatccgatggaagacgctgttttaacacgcgttttttaaccttttattgaaagtcggtgcttctttgagcga
acgatcaaatttaaatggattcccatcaaaaaaatattctcaacctaaaaaagtttgtgtaatacttgtaacgctacatggagattaactcaatctagagggt
attaataatgaatcgtactaaactggtactgggcgc
274 ggacatcatcgcgacaaacaatattaataccggcaaccacaccggcaatttacgagactgcgcaggcatcctttctcccgtcaatttctgtcaaataaag
taaaagaggcagtctacttgaattacccccggctggttgagcgtttgttgaaaaaaagtaactgaaaaatccgtagaatagcgccactctgatggttaatt
aacctattcaattaagaattatctggatgaatgtgccattaaatgcgcagcataatggtgcgttgtgcgggaatactgcttttttttgaaagggttagtcagt
agcggaaac
275 atgaccctgaatatgatgctcgataacgccgcgccggacgccatcgccggcgcgctgactcaacaacatccggggagttttttaccatggtggaaca
ggcctcggtggccatctccctcaccgatgccagcgccaggatcatttacgccaacccggcgttttgccgccagaccggctattcgctggcgcaattgtt
aaaccagaacccgcgcctgctggccagcagccagacgccgcgcgagatctatcaggagatgtggcataccctgaccagcgtcagccctggcgcg
gtcagctgattaatcagcgtcgggacggcggcctgtacctggtggagattgacatcaccccggtgcttagcccgcaaggggaactggagcattatct
ggcgatgcagcgggatatcagcgtcagctacaccctcgaacagcggctgcgcaaccatatgaccctgatggaggcggtgctgaataatatccccgc
cgccgtggtagtggtggacgagcaggatcgggtggtgatggacaacctcgcctacaaaaccttctgcgctgactgcggcggccgggagctgctcac
cgagctgcaggtctcccctggccggatgacgcccggcgtggaggcgatcctgccggtggcgctgcgcggggccgcgcgctggctgtcggtaacc
tgctggccgttgcccggcgtcagtgaagaggccagccgctactttatcgacagcgcgctggcgcggaccctggtggtgatcgccgactgtacccag
cagcgtcagcagcaggagcaagggcgccttgaccggctgaagcagcaaatgaccgccggcaagctgctggcggcgatccgcgagtcgctggac
gccgcgctgatccagctgaactgcccgattaatatgctggcggcagcccgtcggctgaacggcgagggaagcgggaatgtggcgctggaggccg
cctggcgtgaaggggaagaggcgatggcgcggctccagcgctgtcgcccatcgctggaactcgaaaaccccgccgtctggccgctgcagccctttt
tcgacgatctgtgcgccctctaccgtacacgcttcgatcccgacgggctgcaggtcgacatggcctcaccgcatctgatcggctttggccagcgcacc
ccactgctggcgtgcttaagcctgtggctcgatcgcaccctggccctcgccgccgaactcccctccgtgccgctggcgatgcagctctacgccgagg
agaacgacggctggctgtcgctgtatctgactgacaacgtaccgctgctgcagggcgctacgctcactcccccgacgcgctgaactcgccgggca
aaggcatggagctgaggctgatccagaccctggtggcgcaccatcgcggggccattgagctggcttcccgaccgcagggcggcacctgcctgacc
ctgcgtttcccgctgtttaacaccctgaccggaggtgaagcatga
276 atgatccctgaatccgacccggacaccaccgtcagacgcttcgacctctctcagcagttcaccgccatgcagcggataagcgtggtgctgagccggg
ccaccgaggccagcaaaacgctgcaggaggtgctcagcgtattacacaacgatgcctttatgcagcacgggatgatctgcctgtacgacagcgagc
aggagatcctcagtatcgaagcgctgcagcaaaccggccagcagcccctccccggcagcacgcagatccgctatcgccccggcgagcgactggt
ggggaccgtgctggcccaggggcagtcgctggtgctgccccgggtcgccgacgatcagcgttttctcgaccgcctgagcctctacgattacgatctg
ccgtttatcgccgaccgttgatgcggcccaacgcccggccaataggggtgctggcggcccagccgatggcgcgccaggaagagcggctgccgg
cctgcacccgttttctcgaaaccgtcgccaacctcgtcgcccagaccatccggctgatgatccttccggcctcacccgccctgtcgagccgccagccg
ccgaaggtggaacggccgccggcctgctcgtcgtcgcgcggcgtgggccttgacaatatggtcggcaagagcccggcgatgcgccagatcgtgg
aggtgatccgtcaggtttcgcgctgggacaccaccgtgctggtacgcggcgaaagcggcaccgggaaagagctgatcgccaacgccatccatcac
cattcgccacgggctggcgccgccttcgtcaaatttaactgcgcggcgctgccggacaccctgctggaaagcgaactgttcggccatgagaaaggc
gcctttaccggggcggtgcgtcagcgtaaaggacgttttgagctggcggatggcggcaccctgttcctcgatgagattggtgaaagcagcgcctcgtt
ccaggccaagagctgcgtatcctccaggagggggagatggagcgggtcggcggcgatgagaccctgcgggtgaatgtccgcatcatcgccgcc
accaaccgtcacctggaggaggaggtccggctgggccatttccgcgaggatctctactatcgtctgaacgtgatgcccatcgccctgcccccgctgc
gcgagcgtcaggaggacatcgccgagctggcgcacttcctggtgcgcaaaatcggccagcatcaggggcgcacgctgcggatcagcgagggcg
cgatccgcctgctgatggagtacagctggccgggtaacgttcgcgaactggagaactgcctcgaacgatcggcggtgatgtcggagagtggcctga
tcgatcgcgacgtgatcctcttcactcaccaggatcgtcccgcaaagccctgcctgccagcgggccagcggaagacagctggctggacaacagcc
tggacgaacgtcagcgactgatcgccgcgctggaaaaagccggctgggtgcaggccaaggcggcacggctgctggggatgacgccgcgccagg
tcgcttatcggatccagatcatggatatcaccctgccgcgtctgtag
277 attgaagagtttgatcatggctcagattgaacgctggcggcaggcctaacacatgcaagtcgagcggcagcgggaagtagcttgctactttgccggc
gagcggcggacgggtgagtaatgtctgggaaactgcctgatggagggggataactactggaaacggtagctaataccgcatgacctcgaaagagc
aaagtgggggatcttcggacctcacgccatcggatgtgcccagatgggattagctagtaggtgaggtaatggctcacctaggcgacgatccctagct
ggtctgagaggatgaccagccacactggaactgagacacggtccagactcctacgggaggcagcagtggggaatattgcacaatgggcgcaagc
ctgatgcagccatgccgcggtgtgaagaaggccttagggtttgtaaagcactttcagcgaggaggaaggcatcatacttaatacgtgtggtgattgtcg
ttactcgcagaagaagcaccggctaactccgtgccagcagccgcggtaatacggagggtgcaagcgttaatcggaattactgggcgtaaagcgcac
gcaggcggtttgttaagtcagatgtgaaatccccgagcttaacttgggaactgcatttgaaactggcaagctagagtcttgtagaggggggtagaattc
caggtgtagcggtgaaatgcgtagagatctggaggaataccggtggcgaaggcggccccctggacaaagactgacgctcaggtgcgaaagcgtg
gggagcaaacaggattagataccctggtagtccacgctgtaaacgatgtcgacttggaggttgtgcccttgaggcgtggcttcggagctaacgcgtt
aagtcgaccgcctggggagtacggccgcaagtttaaaactcaaatgaattgacgggggcccgcacaaagcggtggagcatgtggtttaattcgatgc
aacgcgaagaaccttacctactcttgacatccagagaatttgccagagatggcgaagtgccttcgggaactctgagacaggtgctgcatggctgtcgt
cagctcgtgttgtgaaatgttgggttaagtcccgcaacgagcgcaacccttatcctttgttgccagcacgtaatggtgggaactcaaaggagactgccg
gtgataaaccggaggaaggtggggatgacgtcaagtcatcattgcccttacgagtagggctacacacgtgctacaatggcatatacaaagagaagc
gaactcgcgagagcaagcggacctcataaagtatgtcgtagtccggattggagtctgcaactcgactccatgaagtcggaatcgctagtaatcgtaga
tcagaatgctacggtgaatacgttccgggccttgtacacaccgcccgtcacaccatgggagtgggttgcaaaagaagtaggtagcttaaccttcggg
agggcgcttaccactttgtgattcatgactggggtgaagtcgtaacaaggtaaccgtaggggaacctgcggttggatcacctcctt
278 attgaagagtttgatcatggctcagattgaacgctggcggcaggcctaacacatgcaagtcgagcggcatcgggaagtagcttgctactttgccggcg
agcggcggacgggtgagtaatgtctgggaaactgcctgatggagggggataactactggaaacggtagctaataccgcatgacctcgaaagagca
aagtgggggatcttcggacctcacgccatcggatgtgcccagatgggattagctagtaggtgaggtaatggctnacctaggcgacgatccctagctg
gtctgagaggatgaccagccacactggaactgagacacggtccagactcctacgggaggcagcagtggggaatattgcacaatcggcgcaagcct
gatgcagccatgccgcgtgtgtgaagaaggccttaggggttaaagcactttcagcgaggaggaaggcatcatacttaatacgtgtggtgattgacgtt
actcgcagaagaagcaccggctaactccgtgccagcagacgggtaatacggagggtgcaagcgttaatcggaattactgggcgtaaagcgcacg
caggcggtttgttaagtcagatgtgaaatccccgagcttaacttgggaactgcatttgaaactggcaagctagagtcttgtagaggggggtagaattcca
ggtgtagggtgaaatgcgtagagatctggaggaataccggtggcgaaggcggccccctggacaaagtctgacgctcaggtgcgaaagcgtggg
gagcaaacaggattagataccctggtagtccacgctgtaaacgatgtcgacttggaggttgtgcccttgaggcgtggcttccggagctaacgcgttaa
gtcgaccgcctggggagtacggccgcaaggttaaaactcaaatgaattgacgggggcccgcacaagcggtggagcatgtggtttaattcgatgcaa
cgcgaagaaccttacctactcttgacatccagagaatttgccagagatggcgaagtgccttcgggaactctgagacaggtgctgcatggctgtcgtca
gctcgtgttgtgaaatgttgggttaagtcccgcaacgagcgcaacccttatcctttgttgccagcacgtgatggtgggaactcaaaggagactgccggt
gataaaccggaggaaggtggggatgacgtcaagtcatcatggcccttacgagtagggctacacacgtgctacaatggcatatacaaagagaagcga
actcgcgagagcaagggacctcataaagtatgtcgtagtccggattggagtctgcaactcgactccatgaagtcggaatcgctagtaatcgtagatca
gaatgctacggtgaatacgttcccgggccttgtacacaccgcccgtcacacattgggagtgggttgcaaaagaagtaggtagcttaaccttcgggag
ggcgcttaccactttgtgattcatgactggggtgaagtcgtaacaaggtaaccgtaggggaacctgcggttggatcacctcctt
279 attgaagagtttgatcatggctcagattgaacgctggcggcaggcctaacacatgcaagtcgagcggcaggggaagtagcttgctactttgccggc
gagcggcggacgggtgagtaatgtctgggaaactgcctgatggagggggataactactggaaacggtagctaataccgcatgacctcgaaagagc
aaagttgggggatcttcggacctcacgccatcggatgtgcccagatgggattagctagtaggtgaggttaatggctcacctaggcgacgatccctagct
ggtctgagaggatgaccagccacactggaactgagacacggtccagactcctacgggaggcagcagtggggaatattgcacaatgggcgcaagc
ctgatgcagccatgccgcgtgtgtgaagaaggccttagggttgtaaagcactttcagcgaggaggaaggcancatacttaatacgtgtggtgattgac
gttactcgcagaagaagcaccggctaactccgtgccagcagccgcggtaatacggagggtgcaagcgttaatcggaattactgggcgtaaagcgca
cgcaggcggtttgttaagtcagatgtgaaatccccgagcttaacttgggaactgcatttgaaactggcaagctagagtcttgtagaggggggtagaatt
ccaggtgtagcggtgaaatgcgtagagatctggaggaataccggtggcgaaggcggccccctggacaaagactgacgctcaggtgcgaaaggcgt
ggggagcaaacaggattagataccctggtagtccacgctgtaaacgatgtcgacttggaggttgtgcccttgaggcgtggcttccggagctaacgcgt
taagtcgaccgcctggggagtacggccgcaaggttaaactcaaatgaattgacgggggcccgcacaagcggtggagcatgtggtttaattcgatgc
aacgcgaagaaccttacctactcttgacatccagagaatttgccagagatggcgaagtgccttcgggaactctgagacaggtgctgcatggagtcgt
cagctcgtgttgtgaaatgttgggttaagtcccgcaacgagcgcaacccttatcctttgttgccagcacgtgatggtgggaactcaaaggagactgccg
gtgataaaccggaggaaggtggggatgacgtcaagtcatcatggcccttacgagtagggctacacacgtgctacaatggcatatacaaagagaagc
gaactcgcgagagcaagcggacctcataaagtatgtcgtagtccggattggagtagcaactcgactccatgaagtcggaatcgctagtaatcgtaga
tcagaatgctacggtgaatacgttcccgggccttgtacacaccgcccgtcacaccatgggagtgggttgcaaaagaagtaggtagcttaaccttcggg
agggcgcttaccactttgtgattcatgactggggtgaagtcgtaacaaggtaaccgtaggggaacctgcggttggatcacctcctt
280 attgaagagtttgatcatggctcagattgaacgctggcggcaggcctaacacatgcaagtcgagcgacancgggaagtagcttgctactttgccggc
gagcggcggacgggtgagtaatgtctgggaaactgcctgatggagggggataactactggaaacggtagctaataccgcatgacctcgaaagagc
aaagtgggggatcttcggacctcacgccatcggatgtgcctagatgggattagctagtaggtgaggtaatggcttacctaggcgacgatccctagctg
gctgagaggatgaccagccacactggaactgagacacggtccagactcctacgggaggcagcagggggaatattgcacaatgggcgcaagcct
gatgcagccatgccgcgtgtgtgaagaaggccttagggttgtaaagcactttcagcgaggaggaaggcatcacacttaatacgtgggtgattgacgtt
actcgcagaagaagcaccggctaactccgtgccagcagccgcggtaatacggagggtgcaagcgttaatcggaattactgggcgtaaagcgcacg
caggcggtttgttaagtcagatgtgaaatccccgagcttaacttgggaactgcatttgaaactggcaagctagagtcttgtagagggggtgagaattcca
ggtgtagcggtgaaatgcgtagagatctggaggaataccggtggcgaaggcggccccctggacaaagactgacgctcaggtgcgaaagcgtggg
gagcaaacaggattagataccctggtagtccacgctgtaaacgatgtcgacttggaggttgtgcccttgaggcgtggcttccggagctaacgcgttaa
gtcgaccgactggggagtacggccgaaaggtttaaaactcaaatgaattgacgggggcccgcacaagcggtggagcatgtggtttaattcgatgcaa
cgcgaagaaccttacctactcttgacatccagagaatttgccagagatggcgaaggccttcgggaactctgagacaggtgctgcatggctgtcgtca
gctcgtgttgtgaaatgttgggttaagtcccgcaacgagcgcaacccttatcctttgttgccagcacgtgatggtgggaactcaaaggagactgccggt
gataaaccggaggaaggtggggatgacgtcaagtcatcatggcccttacgagtagggctacacacggctacaatggcatatacaaagagaagcga
actcgcgagagcaagcggacctcataaagtatgtcgtagtccggattggagtctgcaactcgactccatgaagtcggaatcgctagtaatcgtagatca
gaatgctactgtgaatacgttcccgggccttgtacacaccgcccgtcacaccatgggagtgggttgcaaaagaagtaggtagcttaaccttcgggag
ggcgcttaccactttgtgattcatgactggggtgaagtcgtaacaaggtaaccgtaggggaacctgcggttggatcacctcctt
281 attgaagagtttgatcatggctcagattgaacgctggcggcaggcctaacacatgcaagtcgagcggcagcgggaagaagcttgctactttgccggc
gagcggcggacgggtgagtaatgtctgggaaactgcctgatggagggggataactactggaaacggtagctaataccgcatgacctcgaaagagc
aaagtgggggatcttcggacctcacgccatcggatgtgcccagatgggattagctagaggtgaggtaatggctcacctaggcgacgatccctagct
ggtctgagaggatgaccagccacactggaactgagacacggtccagactcctacgggaggcagcagtggggaatattgcacaatgggcgcaagc
ctgatgcagccatgccgcgtgtgtgaagaaggccttagggttgtaaagcactttcagcgaggaggaaggcatcacacttaatacgtgtgttgattgacg
ttactcgcagaagaagcaccggctaactccgtgccagcagccgcgattaatacggagggtgcaagcgttaatcggaattactgggcgtaaagcgcac
gcaggcggtttgttaagtcagatgtgaaatccccgagcttaacttgggaactgcatttgaaactggcaagctagagtcttgtagaggggggtagaattc
caggtgtagcggtgaaatgcgtagagatctggaggaataccggtggcgaaggcggccccctggacaaagactgacgctcaggtgctgaaagcgtg
gggagcaaacaggattagataccaggtagtccacgctgtaaacgatgtcgacttggaggttgtgcccttgaggcgtggcttccggagctaacgcgtt
aagtcgaccgcctggggagtacggccgcaaggttaaaactcaaatgaattgacgggggcccgcacaagcggtggagcatgtggtttaattcgatgc
aacgcgaagaaccttacctactcttgacatccagagaatttgccagagatggcgaagtgccttcgggaactctgagacaggtgctgcatggctgtcgt
cagctcgtgttgtgaaatgttgggttaagtacgcaacgagcgcaacccttatcctttgttgccagcacgtgatggtgggaactcaaaggagactgccg
gtgataaaccggaggaaggtggggatgacgtcaagtcatcatggcccttacgagtagggctacacacgtgctacaatggcatatacaaagagaagc
gaactcgcgagagcaagcggacctcataaagtatgtcgtagtccggattggagtctgcaactcgactccatgaagtcggaatcgctagtaatcgtaga
tcagaatgctacggtgaatacgttcccgggccttgtacacaccgcccgtcacaccatgggagtgggttgcaaaagaagtaggtagcttaaccttcggg
agggcgcttaccactttgtgattcatgactggggtgaagtcgtaacaaggtaaccgtaggggaacctgcggttggatcacctcctt
282 gtagctaataccgcatgacctcgaaagagcaaagtgggggatcttcggacctcacgccatcggatgtgcccagatgggattagctagtaggtgaggt
aatggctcacctaggcgacgatccctagctggtctgagaggatgaccagccacactggaactgagacacggtccagactcctacgggaggcagca
gtggggaatattgcacaatgggcgcaagcctgatgcagccatgccgcgtgtgtgaagaaggccttagggttgtaaagcactttcagcgaggaggaa
ggcatcanacttaatacgtgtgntgattgacgttactcgcagaagaagcaccggctaactccgtgccagcagccgcggtaatacggagggtgcaagc
gttaatcggaattactgcgcgtaaagcgcacgcaggcggtttgttaagtcagatgtgaaatccccgagcttaacttgggaactgcatttgaaactggca
agctagagtcttgtagaggggggtagaattccaggtgtagcggtgaaatgcgtagagatctggaggaataccggtggcgaaggcggccccctggac
aaagactgacgctcaggtgcgaaagcgtgggagcaaacaggattagataccctggtagtccacgctaaacgatgtcgacttggaggttgtgccc
ttgaggcgtggcttccggagctaacgcgttaagtcgaccgcctggggagtacggccgcaaggttaaaactcaaatgaattgacgggggcccgcaca
agcggtggagcatgtggtttaattcgatgcaacgcgaagaaccttacctactcttgacatccagagaatttgccagagatggcgaagtgccttcgggaa
ctctgagacaggtgctgcatggctgtcgtcagctcgtgttgtgaaatgttgggttaagtcccgcaacgagcgcaacccttatcctttgttgccagcacgt
natggtgggaactcaaaggagactgccggtgataaac
283 attgaagagtttgatcatggctcagattgaacgctggcggcaggcctaacacatgaagtcgagcggcagcgggaagtagcttgctactttgccggc
gagcggcggacgggtgagtaatgtcctgatggaggggataactactggaacggtagctaataccgcacctcgaaagagcaaagtgggggatcttcg
gacctcacgccatcggatgtgcccagatgggattagctagtaggtgaggtaatggctcacctaggcgacgatccctagctggtctgagaggatgacc
agccacactggaactgagacacggtccagactcctacgggaggcagcagtggggaatattgcacaatgggcgcaagcctgatgcagccatgccgc
gtgtgtgaagaaggccttagggttctaaagcactttcagcgagcaggaaggcatcatacttaatacgtgtggtgattgacgttactcgcagaagaagca
ccggctaactccgtgccagcagccgcggtaatacggagggtgcaagcttsatcggaattactgggcgtaaagcgcacgcaggcggttgttaagtca
gatgtgaaatccccgagcttaacttgggaactgcatttgaaactggcaagctagagtcttgtttgaggggggtagaattccaggtgtagcggtgaaatgc
gtagagatctggaggaataccggtggcgaaggcggccccctggacaaagactgacgctcaggtgcgaaagcgggggagcaaacaggattaata
ccctggtagtccacgctgtaacgatgtcgacttggaggttgtgccctgaggcgtggcttccggagctaacgcgttaagtcgaccgcctggggagtacg
gccgcaaggtaaaactaaatgaattgacgggggcccgcacaagcggtggagcatgtggtttaattcgatgcaacgcgaagaaccttacctactctt
gacatccagagaatttgccagagatggcgaagtgccttcgggaactctgagacaggtgctgcatggctgtcgtcagctcgtgttgtgaaatgttgggtt
aagtcccgcaacgagcgcaacccttatcctttgttgccagcacgtaatggtgggaactcaaaggagactgccggtgataaaccggaggaaggtggg
gatgacgtcaagtcatcatggcccttacgagtagggctacacacgtgctacaatggcatatacaaagagaagcgaactcgcgagagcaagcggacc
tcataaagtatgtcgtagtccggattggagtctgcgactcgactccatgaagtcggaatcgctagaatcgtagatcagaatgctacggtgaatacgttcc
cgggccttgtacacaccgcccgtcacaccatgggagtgggttgcaaaagaagtaggtagcttaacatcgggagggcgcttaccactttgtgattcatg
actggggtgaagtcgtaacaaggtaaccgtaggggaacctgcggttggatcacctcctt
284 atgaccatgcgtcaatgcgctatctacggtaaaggcggtatcggtaaatccaccaccacccagaatctcgtcgcggccctcgccgagatgggtaaga
aagtgatgatcgtcggctgcgatccgaaagcggattccacccgtctgatcctccacgctaaagcccagaacaccatcatggagatggcggcggaagt
gggctcggtcgaggatctggagctcgaagacgttctgcaaatcggctatggcgatgtccgttgcgccgaatccggcggcccggagccaggcgtcg
gctgcgccggacgcggggtgatcaccgccatcaacttcctcgaggaaggcgcctatgaagaagatttggatttcgtcttctatgacgtcctcggc
gacgtggtctgcggcggcttcgctatgccgatccgcgaaaacaaagcccaggagatctacatcgtctgctccggcgagatgatggcgatgtatgccg
ccaacaatatctccaaagggatcgtgaagtacgccaaatccggcaaggtgcgcctcggcggcctgatctgtaactcgcgcaaaaccgaccgggaag
acgaactgatcatcgccctggcggagaagcttggcacgcagatgatccacttcgttccccgcgacaacattgtgcagcgcgcggagatccgccgga
tgacggtgatcgagtacgacccgacctgtcagcaggcgaggaatatcgtcaactggcgcagaagatcgtcaataacaccaaaaaagtggtgccga
cgccgtgcaccatggacgagctggaatcgctgctgatggagttcggcatcatggaagaagaagacaccagcatcattggtaaaaccgccgctgaag
aaaacgcggcctga
285 atggttaggaaaagtagaagtaaaaatacaaatatagaactaactgaacatgaccatttattaataagtcaaataaaaaagcttaaaacacaaaccacttg
cttttttaataataaaggaggggttgggaagactacattagtagcaaatttaggagcagagctatcaataaactttagtgcaaaagttcttattgtggatgcc
gaccctcaatgtaatctcacgcagtatgtattaagtgatgaagaaactcaggacttatatgggcaagaaaatccagatagtatttatacagtaataagacc
actatcctttggtaaaggatatgaaagtgacctccctataaggcatgtagagaatttcggttttgacataattgtcggtgaccctagacttgctttacaggaa
gaccttttagctggagactggcgagatgccaaaggcggtgggatgcgaggaattaggacaacttttgtatttgcagagttaattaagaaagctcgtgag
ctaaattatgattttgttttctttgacatgggaccatcattaggcgcaatcaacagggcagtattactggcaatggaattctttgtcgtcccaatttcaatcga
tgtattttcactatgggctattaaaaatattggctccacggtttcaatatggaaaaaagaattagacacagggattcggctctcagaggaacctagcgaatt
atcacaattatcttcctcaaggaaaactaaagtttctcggttacgtcacccaacaacataaagaacgctctggatacgatacaattcagcttgagaatactg
aggaagaaataaaatcgaaacgtcgggtaaaggcgtatgaagacattggagaggtgtttccttctaaaattactgagcatctttctaaactttatgatca
aaagatatgaacccacaccttggagatatacgtcatttaggtagtttagctccgaaatcacaatcacaacacgttccgatgatatcagtgtctggtacagg
aaattacaccagacttagaaaaagcgcgcgtgaactttatcgagatattgcaagaagatacttagagaacattcagactgctaatggcgagaaatag
286 atgaagggaaaggaaattctggcgctgctggacgaacccgcctgcgagcacaaccagaagcaaaaatccggctgcagcgcccctaagcccggcg
ctaccgccggcggttgcgccttcgacggcgcgcagataacgctcctgcccatcgccgacgtcgcgcacctggtgcacggccccatcggctgcgcg
ggcagctcgtgggataaccgcggcagcgtcagcgccggcccggccctcaaccggctcggctttaccaccgatcttaacgaacaggatgtgattatg
ggccgcggcgaacgccgcctgttccacgccgtgcgtcacatcgtcgaccgctatcatccggcggcggtctttatctacaacacctgcgtaccggcga
tggagggcgatgacatcgaggcggtctgccaggccgcacagaccgccaccggcgtcccggtcatcgctattgacgccgccggtttctacggcagta
aaaatcttggcaaccgaatggcgggcgacgtgatgctcaggcaggtgattggccagcgcgaaccggccccgtggccagacaacacgccctttgcc
ccggcccagcgccacgatatcggcctgattggcgaattcaatatcgccggcgagttctggcaggtccagccgctgacgacgagctggagatccgc
gtcctcggcaccctctccggcgacggccgctttgccgagatccagaccctgcaccgggcgcaggccaatatgctggtgtgctcgcgcgcgctgatc
aacgtcgcccgggggctggagctgcgctacggcacgccgtggtttgaaggcagcttctacgggatccgcgccacctccgacgccttgcgccagct
ggcgacgctgctgggggatgacgacctgcgccgccgcaccgaggcgctgatcgcccgcgaagagcaggcggcggagcaggctcttgcgccgt
ggcgtgagcagctccgcgggcgcaaatgctgctctataccggcggcgtgaaatcctggtcggtggtatcggccctgcaggatctcggcatgaccg
tggtggccaccggcacgcgcaaatccaccgaggaggacaaacagcggatccgtgagctgatgggcgacgaggcggtgatgcttgaggagggca
atgcccgcaccctgctcgacgtggtgtaccgctatcaggccgacctgatgatcgccggcggacgcaatatgtacaccgcctggaaagcccggctgc
cgtttctcgatatcaatcaggagcgcgagcacgcctacgccggctatcagggcatcatcaccctcgcccgccagctctgtctgaccctcgccagcccc
gtctggccgcaaacgcatacccgcgccccgtggcgctag
287 atgaccaacgcaacaggcgaacgtaaccttgcgctcatccaggaagtcctggaggtgtttcccgaaaccgcgcgcaaagagcgcagaaagcacat
gatgatcagcgatccgagatggagagcgtcggcaagtgcattatctcgaaccgtaaatcgcagcccggggtgatgaccgtgcgcggctgcgccta
tgcgggctcgaaaggggtggtgtttgggccaatcaaagacatggcccatatctcgcacggccccatcggctgcggccagtattcccgcgccggacg
gcgcaactactataccggcgtcagcggtgtcgacagcttcggcaccctgaacttcacctctgattttcaggagcgcgatattgttttcggcggcgataaa
aagctgaccaaactgatcgaagagatggagagctgttcccgctgaccaaagggatcaccatccagtcggagtgcccggtgggcctgatcggcgat
gacatcagcgccgtagccaacgccagcagcaaggcgctggataaaccggtgatcccggtgcgctgcgaaggctttcgcggcgtatcgcaatcgct
gggccaccatatcgccaacgacgtggtgcgcgactgggtgctgaacaatcgcgaagggcagccgtttgccagcaccccgtacgatgttgccatcatt
ggcgattacaacatcggcggcgacgcctgggcctcgcgcattctgctggaagagatggggctgcgcgtagtggcgcagtggtccggcgacggca
ccctggtggagatggagagcaccccattcgttaagcttaacctcgtccactgctaccgttcgatgaactatatcgcccgccatatggaggagaaacatc
agatcccatggatggaatataacttcttcggcccgaccaaaatcgccgaatcgctgcgcaagatcgccgatcaatttgatgacaccattcgcgccaatg
cggaagcggtgatcgccaaatatgaggggcagatggcggccatcatcgccaaatatcgcccgcggctggaggggcgcaaagtgctgctgtacatg
ggggggctgaggccgcgccacgtcatcggcgcctatgaggatctcgggatggagatcatcgccgccggctacgagtttgcccataacgatgattac
gaccgcaccctgacggacctgaaagagggcaccctgctgtttgacgatgccagcagatatgagctggaggccttcgtcaaagcgctgaaacctgac
ctcatcggctccgggatcaaagagaaatatatcttccagaaaatgggggtgcgttccgccagatgcactcctgggactattccggcccctatcacgg
ctatgacggcttcgccatctttgcccgcgatatggatatgaccctgaacaatccggcgtggaacgaactgactgccccgtggctgaagtctgcgtga
288 atggcagatattatccgcagtgaaaaaccgctggcggtgagcccgattaaaaccgggcaaccgctcggggcgatcctcgccagcctcgggctggc
ccaggccatcccgctggccacggcgcccagggctgcagcgccttcgccaaagttttctttattcagcatttccatgacccggtgccgctgcagtcgac
ggccatggatccgaccgccacgatcatgggggccgacggcaatatcttcaccgcgctcgacaccctctgccagcgccacagcccgcaggccatcg
tgctgctcagcaccggtctggcggaagcgcagggcagcgatatcgcccgggtggtgcgccagtttcgcgaggcgcatccgcgccataacggcgtg
gcgatcctcaccgtcaataccccggatttttttggctctatggaaaacggctacagcgcggtgatcgagagcgtgatcgagaagtgggtcgcgccgac
gccgcgtccgggcagcggccccggcgggtcaacctgctggtcagccacctctgttcgccaggggatatcgaatggctgggccgctgcgtggag
gcctttggcctgcagccggtgatcctgccggacctctcgcagtcaatggatggccacctcggtgaaggggattttacgcccctgacccagggcggcg
cctcgctgcgccagattgcccagatgggccagagtagggcagcttcgccattggcgtgtcgctccagagggcggcatcgatcctgacccaacgca
gccgcggcgacgtgatcgccctgccgatctgatgaccctcgaccattgcgatacctttatccatcagctggcgaagatgtccggacgccgcgtacc
ggcctggattgagcgccagcgtggccagctgcaggatgcgatgatcgactgccatatgtggcttcagggccagcgcatggcgatggcggcggagg
gcgacctgctggcggcgtggtgtgatttcgcccgcagccaggggatgcagcccggcccgctggtcgcccccaccagccaccccagcctgcgcca
gctgccggcgagcaagtcgtgccgggggatcttgaggatctgcagcagctgctgagccaccaacccgccgatctgaggtggctaactctcacgc
ccgcgatctggcggagcagtttgccctgccgctgatccgcgtcggttttcccctcttcgaccggctcggtgagtttcgtcgagtccgccaggggtacgc
cggtatgcgagatacgctgtttgaactggccaatctgctgcgcgaccgccatcaccacaccgccctctaccgctcgccgcttcgccagggcgccgac
ccccagccggcttcaggagacgcttatgccgcccattaa
289 atgagccaaacgatcgataaaattcacagctgttatccgatatttgaacaggatgaataccagaccctgttccagaataaaaagacccttgaagaggcg
cacgacgcgcagcgtgtgcaggaggtttttgcctggaccaccaccgccgagtatgaagcgctgaacttccagcgcgaggcgctgaccgtcgaccc
ggccaaagcctgccagccgctcggcgccgtactctgcgcgctggggttcgccggcaccctgccctacgtgcacggctcccagggctgcgtcgcct
atttcgcacctactttaaccgccattttaaagagccggtcgcctgcgtctccgactccatgaccgaggacgcggcggtgttcggcggcaacaacaac
atgaatctgggcctgcacaatgccagcgcgctgtataaacccgagattatcgccgtctccaccacctgtatggccgaggtgatcggcgacgatctgca
ggcgtttatcgccaacgccaaaaaagagggatttgttgacgaccgcatcgccattccttacgcccatacccccagctttatcggcagccatgtcaccgg
ctgggacaatatgttcgaagggttcgcgaagacctttaccgctgactacgccgggaagccgggcaaacagaaaaagctcaatctggtgaccggattt
gagacctatctcggcaacttccgcgtgctgaagcggatgatggcgcagatggatgtcccgtgcagcctgctctccgacccatcagaggtgctcgaca
cccccgccgacggccattaccggatgtacgccggcggcaccagccagcaggagatcaaaaccgcgccggacgccattgacaccctgctgctgca
gccgtggcagatggtgaaaagcaaaaaggtggttcaggagatgtggaaccagcccgccaccgaggtggccgttccgctgggcctggacgccacc
gacgcgctgctgatgaccgtcagtcagctgaccggcaaaccgatcgccgacgctctgaccctggagagaggccggctggtcgacatgatgctggat
tcccacacctggctgcatggcaaaaaattcggcctctacggcgatccggatttcgtgatggggctgacgcgcttcctgctggagctgggctgcgagcc
gacggtgatcctcagtcataacgccaataaacgctggcaaaaagcgatgaagaaaatgctcgatgcctcgccgtacggtcaggaaagcgaagtgttc
atcaactgcgacctgtggcacttccggtcgctgatgttcacccgtcagccggactttatgatcggtaactcctacggcaagtttatccagcgcgataccc
tggcaaagggcaaagccttcgaagtgccgatgatccgtctgggctttccgctgttcgaccgccatcatctgcaccgccagaccacctggggctatgaa
ggcgcaatgaacatcgtcacgacgctggtgaacgccgtgctggaaaaactggaccacgacaccagccagttgggcaaaaccgattacagcttcgac
ctcgttcgttaa
290 atgatgccgctttctccgcaattacagcagcactggcagacggtcgctgaccgtctgccagaggattttcccattgccgaactgagcccacaggccag
gtcggtcatggcgttcagcgattttgtcgaacagagtgtgatcgcccagccgggctggctgaatgagcttgaggactcctcgccggaggcggaagag
tggcggcattacgaggcctggctgcaggatcgcctgcaggccgtcactgacgaagggggttgatgcgagagctgcgtctcttccgccgccagatg
atggtccgcatcgcctgggcgcaggcgctgtcgctggtgagcgaagaagagactctgcagcagctgagcgtcctggcggagaccctgattgtcgcc
gcccgcgactggctgtacgccgcctgctgtaaggagtggggaacgccatgcaatgccgagggccagccgcagccgctgctgatcctcgggatgg
gaaagctgggcggcggcgagctgaacttctcttccgatatcgatctgatctttgcctggcctgagcatggcgccacccgcggcggccgccgcgagct
ggataacgcccagttctttacccgtctggggcagcggctgatcaaggcccttgaccagccgacgcaggacggctttgtctatcgggttgacatgcgcc
tgcggccgtttggcgacagtgggccgctggtactcagttttgcggcgctggaagattattaccaggagaagggtcgggactgggaacgctatgcgat
ggtgaaagcgcggatcatggccgataacgacggcgtgtacgccagcgacttgcgcgcgatgctccgtcctttcgtcttccgccgttatatcgacttca
gcgtgatccagtcgctgcgtaacatgaaaggcatgatcgcccgcgaagtgcggcgtcgcgggctgaaagacaacatcaagctcggcgccggcgg
gatccgtgaaattgagtttatcgttcaggctttcaactgatccgcggtggtcgcgaacctgcactgcagcagcgcgccctgctgccgacgctggcgg
cgattgatgagctacatctgctgccggaaggcgacgcggcgctgctgcgcgaggcctatctgttcctgcgccggctggaaaacctgctgcaaagcat
caacgatgagcagacccagaccctgccgcaggatgaacttaaccgcgccaggctggcgtgggggatgcataccgaagactgggagacgctgagc
gcgcagctggcgagccagatggccaacgtgcggcgagtgtttaatgaactgatcggcgatgatgaggatcagtccccggatgagcaactggccga
gtactggcgcgagctgtggcagcatgcgctggaagaagatgacgccagcccggcgctggcgcatttaaacgataccgaccgccgtagcgtgctgg
cgctgattgccgattttcgtaaagagctggatcggcgcaccatcggcccgcgcggccgccaggtgctggatcagcttgatgccgcatctgctgagcga
aatctgctcgcgcgccgatgcgccgctgcctctggcgcggatcacgccgctgttgaccgggatcgtcacccgtaccacctatcttgagctgctgagc
gaattccccggcgcgctgaagcacctgatcacgctctgcgcggcgtcgccgatggtcgccagccagctggcgcgccacccgctgctgctggatga
gctgctggatcccaacaccctctatcagccgacggcgaccgatgcctatcgcgacgagctgcgccagtacctgctgcgcgtgccggaagaggatga
agagcagcagatggaggcgttgcgccagtttaagcaggcgcagcagctgcatatcgcggcggcggatatcgctggtaccctgccggtgatgaagg
tcagcgatcacttaacctggcttgccgaagcgatcctcgacgcggtggtgcagcaggcatgggggcagatggtcgctcgctacggccagccgaccc
acctgcacgatcgccagggtcgcggcttcgccgtcgtcggctacgctaagcttggcggctgggagctgggctacagctccgatctcgatctggtgttc
ctccatgactgcccggcggaggtgatgaccgacggcgagcgggagattgacggccgtcagttctacctgcggctggcccagcggatcatgcacct
gttcagcacccgcacctcgtccggtattctctacgaagttgacgcccggagcgccttctggcgcggcggggatgaggtcaccaccgccgacgc
gtttgctgactatcagcagaacgaagcaggacgtgggaacatcaggcgctggtgcgcgcccgcgtggtctatggcgacccggcgctgcaggcgc
gctttgacgccattcgtcgcgatatcctgaccaccccgcgggaggggatgaccctgcagaccgaggttcgcgagatgcgcgagaagatgcgcgcc
caccttggcaacaaacatcccgatcgttttgatatcaaagccgatgccggcgggatcaccgatattgaatttattactcagtatctggtcctacgctatgcc
agtgacaagccgaagctgacccgctggtctgacaacgtgcgtattcttgagctgctggcgcagaacgacatcatggaggaggaggaggcgcgcgc
cttaacgcatgcgtacaccaccttgcgtgatgcgctccatcacctggccctgcaggagcagccgggacacgtggcgccagaggccttcagccggga
gcgtcagcaggtcagcgccagctggcagaagtggctgatggcttaa
291 agtctgaactcatcctgcggcagtcggtgagacgtatttttgaccaaagagtgatctacatcacggaattttgtggttgttgctgcttaaaagggcaaatct
acccttagaatcaactgttatatcagggggattcagagagatattaggaatttgcacaagcgcacaatttaaccacatcatgataacgccatgtaaaaca
aagataaaaaaacaaaatgcagtgacttacatcgcaagcaaggcattttcttatccaattgctcaaagtttggcctttcatatcgcaacgaaaatgcgtaat
atacgcgcccttgcggacatcagtatggtcattcctagttcatgcgcatcggacaccaccagcttacaaattgcctgattgcggccccgatggccggtat
cactgaccgaccatttcgtgccttatgtcatgcgatgggggctgg
292 tgaacatcactgatgcacaagctacctatgacgaagaattaactaaaaaactgcaagatgaggcattcgcgttaaagccgacttgagaaatgagaag
attggctttaaaattcgcgaacacacgctacgccgtgttcatatatgttagtttgtggcgataaagaggtcgaagcaggcaaagttgctgttcgtacccg
ccgcggcaaagacttaggaagcatggatgttagcgaagtcgttgacaaactgctggcggaaatccgcagcagaagtcttcatcaactggaggaataa
agtattaaaggcggaaaacgagttcaaccggcgcgtcctaatcgcattaacaaagagattcgcgcgcaagaagttcgcctcacaggcgtcgatggcg
agcagattggtattgtcagtagaatgaagctcttgaaaaagctgaggaaggggcgtcgatttagtagaaatcagtccgaatgccgagccgccagttt
gtcgaatc
293 tacagtagcgcctctcaaaaatagataaacggctcatgtacgtgggccgtttattttttctacctataatcgggaaccggtgttataatgccgcgccctcat
attgtggggattcttaatgacctatcctgggtcctaaagttgtagttgacattagcggagcactaac
294 aattttttttcacaaagcgtagcgttattgaatcgcacattttaaactgttggccgctgtggaagcgaatattcgtgaaagtgcggttttaaggcctttttctt
tgactctctgtcgttacaaagttaatatgcgcgccct
295 ttaaaaacgtgaccacgagcattaataaacgccacgaaatgtggcgtttatttattcaaaaagtatctctttcataaaaagtgctaaatgcagtagcagca
aaattgggataagtcccatggaatacggctgttttcgctgcaatttttaactttttcgtaaaaaaagatgtttctttgagcgaacgatcaaaatatagcgttaa
ccggcaaaaaattattctcattagaaaatagtttgtgtaatacttgtaacgctacatggagattaacttaatctagagggttttata
296 atggcgctcaaacagttaatccgtctgtgtgccgcctcgccgatggtcgcgacacaacttgcacgtcatcctttattgctcgatgaactgctcgacccgc
gcacgctttaccagccgattgagccgggcgcttaccgcgacgaactgcgtcagtatctgatgcgggtgccaacagnagacgaagaacagcagcttg
aagccgtgcgccagttcaaacaggcccagcatttgcgtatcgcagccggggatatttccggggcattgccggtgatgaaagtcagtgaccatttaacc
taccttgccgaggccattctcgatgtcgtggtgcagcatgcgtgggaacaaatggtcgtaaaatacgggcagcccgcgcatcttcagcaccgtgagg
ggcgcggttttgccgtggtcggttacgggaaactcggtggctgggagctggttatagctcagatctggatctggtcttcctgctcgattgcgcgccgg
aggtgatgacggacggcgaacgcagcatcgacggacgtcagttttatcttcggctggcgcagcgcattatgcacttattcagcacccggacatcgtca
ggcattctttacgaggttgatccgcgtctgcgaccttccggcgcatccggcatgctggtcagtaccattgaagcgtttgcagattatcaggccaatgaag
cctggacgtgggagcatcaggcgctggttcgcgcgcgcgtggtttacggggatccgcaactgacacagcaatttaacgccacgcgtcgcgacattct
ttgccgccagcgcgatggcgacggcctgcgtaaggaggtccgtgaaatgcgcgagaaaatgtatgcccatctggggagtaaaaaagcccacgagtt
tgactgaaagccgatccgggtggcatcacggatattgaattcattgcacaatacctggttctgcgtttcgcgcatgatgagccgagctgacgcgctg
gtctgataacgtgcggatttttgaactgatggcacgatatgacatcatgccggaagaggaagcgcgccatctgacgcaggcttatgtgacgctgcgog
atgaaattcatcatctggcgttgcaggaacacagcgggaaagtggccgcggacagctttgctactgagcgcgcgcagatccgtgccagctgggcga
agtggctcggctga
297 cggtactggaacagaaatcggcggatgcgcaggaaatttgttatgacacggcctgtctgaagtgcaagttagtgcttacttcctggctggcaacctcag
gctggacgccgtttattgatgataaatctgcgaagaaactggacgcttccttcaaacgttttgctgacatcatgctcggtcgtaccgcagcggatctgaaa
gaggcctttgcgcagccactgacggaagaaggttatcgcgatcagctggcgcgcctgaaacgccagatcattaccttccatttgatgccggtgcttac
cctgaaaaagacgtcgatgcgtatattgccggctgggtggacctgcaacaggccatcgttcagcagcaacacgcctgggaggattcggcccgtctc
acgcggtgatgatggatgctttctggttaaacgggcaacctcgttaactgactgactagcctgggcaaactgcccgggcttttttttgcaaggaatctgat
ttcatggcgctcaaacagttaatccgtctgtgtgccgcctcgccgatggtcgcgacacaacttgcacgtcatcctttattgctcgatgaactgctcgaccc
gcgcacgctttaccagccgattgagccgggcgcttaccgcgacgaactgcgtcagtatctgatgcgggtgccaacagaagacgaagaacagcagct
tgaagccgtgcgccagttcaaacaggcccagcatttgcgtatcgcagccggggatatttccggggcattgccggtgatgaangtcagtgaccatttaa
cctaccttgccgaggccattctcgatgtcgggtgcagcatgctgtgggaacaaatggtcgtaaaatacgggcagcccgcgcatcttcagcaccgtga
ggggcgcggttttgccgtggtcggttacgggaaactcggtggctgggagctgggttatagctcagatctggatctggtcttcctgctcgattgcgcgcc
ggaggtgatgacggacggcgaacgcagcatcgacggacgtcagttttatcttcggctggcgcagcgcattatgcacttattcagcacccggacatcgt
caggcattctttacgaggttgatccgcgtctgcgaccttccggcgcatccggcatgctggtcagtaccattgaagcgtttgcagattatcaggccaatga
agcctggacgtgggagcatcaggcgctggttcgcgcgcgcgtggtttacggggatccgcaactgacacagcaatttaacgccacgcgtcgcgacat
tctttgccgccagcgcgatggcgacggcctgcgtaaggaggtccgtgaaatgcgcgagaaaatgtatgcccatctggggagtaaaaaagcccacga
gtttgatagaaagccgatccgggtggcatcacggatattgaattcattgcacaatacctggttctgcgtttcgcgcatgatgagccgaagctgacgcgc
tggtctgataacgtgcggatttttgaactgatggcacgatatgacatcatgccggaagaggaagcgcgccatctgacgcaggcttatgtgacgctgcg
cgatgaaattcatcatctggcgttgcaggaacacagcgggaaagtggccgcggacagctttgctactgagcgcgcgcagatccgtgccagctgggc
aaagtggctcggctgagggtttttattcggctaacaggcgcttgtgatattatccggcgcattgtatttacccgatttgatttatctgttttggagtcttgggat
gaaagtgactttgcagattttcaccgcgcaggtgtgctggttgtcggtgacgtaatgttagaccgttactggtatggcccgaccaatcgtatttctccgga
agctccggtgccggtggtgaaggtcagtaccattgaagagcggcctggcggtgcagctaacgtggcgatgaacatttcatctctgggcgcctcttcct
gtctgatcggcctgaccggcgtagacgacgctgcgcgtgccctcagtgagcgtctggcagaagtgaaagttaactgcgttttcgtcgcactatccaca
catcctaccatcaccaaactgcgaattttgtcccgtaaccagcaactgatccgcctcgactttgaggaaggttttgaaggcgttgatctcgagccgatgct
gaccaaaataga
298 atgagcatcacggcgttatcagcatcatttcctgaggggaatatcgccagccgcttgtcgctgcaacatccttcactgttttataccgtggttgaacaatct
tcggtggcgatttcgctgaccgatccgcaggcgcgcatttgttatgccaatccggcattctgccgccagacgggttttgcacttgagacacttttgggcg
agaaccaccgtctgctggagtctgaactcatcctgcgatgggggctgggccgtctctgaagctctcggtgaacattgttgcgaggcaggatgcgagct
ggttgtgttttgacattaccgataatgtgccgcgtgaacgggtgcgttatgcccgcccggaagggcgttttcccgtccggggaatggcatggagctgc
gccttatccagacgctgatcgcccatcatcgcggttctttagatctctcggtccgccctgatggcggcaccttgctgacgttacgcctgccggtacagca
ggttatcaccggaggcttaaaatga
299 tgtttcgtctcgaggccgggcaactgagggccccgttgaaaccgacctgggctggcatctgttgttgtgcgaacaaattcgcctgccgcaaccttgc
cgaaagcccaagccttaacgcgggtgcgtcagcaactgattgcccggcaacagaaacattatcagcgccagtggctgcaacaactgatcaacgcct
gagcctgttctccttcttgttgatgcagacgggttaatgcccgttttgcacgaaaaatgcacataaattgcctgcgttgccttataacaggcagggaaat
cctgcctccggccttgtgccacaccgcgctttgcctggtttgtggtaaaaactggcccgctttgcatcctgatgcttaaaacaccccgttcagatcaacct
ttgggcagataagcccgcgaaacgcctgcaaattgcacggttattccgggtgagtatatgtgtgatttgggttccggcattgcgcaataaaggggaga
aagacatgagcatcacggcgttatcagcatcatttcctgacgggaatatcgccagccgcttgtcgctgcaacatccttcactgttttataccgtggttgaa
caatcttcggtggcgatttcgctgaccgatccgcaggcgcgcatttgttatgccaatccggcattctgccgccagacgggttttgcacttgagacactttt
gggcgagaaccaccgtctgctggagtctgaactcatcctgcgatgggggctgggccgtctctgaagctctcggtgaacattgttgcgaggcaggatg
cgagctggttgtgttttcacattaccgataatgtgccgcgtgaacgggtgcgttatgcccgcccggaagcggcgttttcccgtccggggaatggcatcg
agctgcgccttatccagacgctgatcgcccatcatcgcggttctttagatctctcggtccgccctgatggcggcaccttgctgacgttacgcctgccggt
acagcaggttatcaccggaggcttaaaatgacccagttacctaccgcgcgcccggttatccggcgctttgatatgtctgcccagtttacggcgctttatc
gcatcagcgtggcgctgagtcaggaaagcaacaccgggcgcgcactggcggcgatcctcgaagtgcttcacgatcatgcatttatgcaatacggcat
ggtgtgtctgtttgataaagaacgcaatgcactctttgtggaatccctgcatggcatcgacggcgaaaggaaaaaagagacccgccatgtccgttacc
gcatgggggaaggcgtgatcggcgcggtgatgagccagcgtcaggcgctggtgttaccgcgcatttcagacgatcagcgttttctcgaccccctgaa
tatttacgattacagcctgccgttgattggcgtgccgatccccggtgcggataatcagccatcgggcgtgctggtggcacagccgatggcgttgcacg
aagaccggctgactgccagtacgcggtttttagaaatggtc
300 atgagcatcacggcgttatcagcatcatttcctgaggggaatatcgccagccgcttgtcgctgcaacatccttcactgttttataccgtggttgaacaatct
tcggtggcgatttcgctgaccgatccgcaggcgcgcatttgttatgccaatccggcattctgccgccagacgggttttgcacttgagacacttttgggcg
agaaccaccgtctgctggttaaagcctgccggtacagcaggttatcaccggaggcttaaatga
301 tgtttcgtctcgaggccgggcaactgagcggccccgttgaaaccgacctgggctggcatctgttgttgtgcgaacaaattcgcctgccgcaacccttgc
cgaaagccgaagccttaacgcgggtgcgtcagcaactgattgcccggcaacagaaacattatcagcgccagtggagcaacaactgatcaacgcct
gagcctgttctccttcttgttgatgcagacgggttaatgcccgttttgcacgaaaaatgcacataaatttgcctgcgttgccttataacagcgcagggaaat
cctgcctccggccttgtgccacaccgcgctttgcctggtttgtggtaaaaactggcccgctttgcatcctgatgcttaaaacaccccgttcagatcaacct
ttgggcagataagcccgcgaaaggcctgcaaattgcacggttattccgggtgagtatctgtgtgatttgggttccggcattgcgcaataaaggggaga
aagacatgagcatcacggcgttatcagcatcatttcctgaggggaatatcgccagccgcttgtcgctgcaacatccttcactgttttataccgtggttgaa
caattcttcggtggcgatttcgctgaccgatccgcaggcgcgcatttgttatgccaatccggcattctgccgccagacgggttttgcacttgagacactttt
gggcgagaaccaccgtctgctggttaaagcctgccggtacagcaggttatcaccggaggcttaaaatgacccagttacctaccgcgggcccggttat
ccggcgctttgatatgtctgcccagtttacggcgctttatcgcatcagcgtggcgctgagtcaggaaagcaacaccgggcgcgcactggcggcgatc
ctcgaagtgcttcacgatcatgcatttatgcaatacggcatggtgtgtctgtttgataaagaacgcaatgcactctttgtggaatccctgcatggcatcgac
ggcgaaaggaaaaaagagacccgccatgtccgttaccgcatgggggaaggcgtgatcggcgcggtgatgagccagcgtcaggcgctggtgttac
cgcgcatttcagacgatcagcgttttctcgaccgcctgaatatttacgattacagcctgccgttgattggcgtgccgatccccggtgcggataatcagcc
atcgggcgtgctggtggcacagccgatggcgttgcacgaagaccggctgactgccagtacgcggtttttagaaatggtc
302 atgagcatcacggcgttatcagcatcatttcctgaggggaatatcgccagccgcttgtcgctgcaacatccttcactgttttataccgtggttgaacaatct
tcggtggcgatttcgctgaccgatccgcaggcgcgcatttgttatgccaatccggcattctgccgccagacgggttttgcacttgagacacttttgggcg
agaaccaccgtctgctggagtctgaactcatcctgcggcagtcggtgagacgtatttttgaccaaagagtgatctacatcacggaattttgtggttgttgc
tgcttaaaagggcaaatctacccttagaatcaactgttatatcagggggattcagagagatattaggaatttgcacaagcgcacaatttaaccacatcatg
ataacgccatgtaaaacaaagataaaaaaacaaaatgcagtgacttacatcgcaagcaaggcattttcttatccaattgctcaaagtttggcccttcatatc
gcaacgaaaatgcgtaatatacgcgcccttgcggacatcagtatggtcattcctagttcatgcgcatcggacaccaccagcttacaaattgcctgattgc
ggccccgatggccggtatcactgaccgaccatttcgtgccttatgtcatgcgatgggggctggcccgtctctgaagctctcggtgaacattgttgcgag
gcaggatgcgagctggttgtgttttgacattaccgataatgtgccgcgtgaacgggtgcgttatgcccgcccggaagcggcgttttcccgtccgggga
atggcatggagagcgccttatccagacgctgatcgcccatcatcgcggttctttagatctctcggtccgccctgatggcggcaccttgctgacgttacg
cctgccggtacagcaggttatcaccggaggcttaaaatga
303 tgtttcgtctcgaggccgggcaactgagcggccccgttgaaaccgacctgggctggcatctgttgttgtgcgaacaaattcgcctgccgcaacccttgc
cgaaagccgaagccttaacgcgggtgcgtcagcaactgattgcccggcaacagaaacattatcagcgccagtggctgcaacaactgatcaacgcct
gagcctgttctccttcttgttgatgagacgggttaatgcccgttttgcacggaaaatgcacataaattgcctgcgttgccttataacagcgcagggaaat
cctgcctccggccttgtgccacaccgcgctttgcctggtttgtggtaaaaactggcccgctttgcatcctgatgcttaaaacaccccgttcagatcaacct
ttgggcagataagcccgcgaaaggcctgcaaattgcacggttattccgggtgagtatatgtgtcatttgggttccgccattgcgcaataaaggggaga
aagacatgagcatcacggcgttatcagcatcatttcctgaggggaatatcgccagccgcttgtcgctgcaacatccttcactgttttataccgtggttgaa
caatcttcggtggcgatttcgctgaccgatccgcaggcgcgcatttgttatgccaatccggcattctgcccccagacgggttttgcacttgagacactttt
gggcgagaaccaccgtctgctggagtctgaactcatcctgcggcagtcggtgagacgtatttttgaccaaagagtgatctacatcacggaattttgtggt
tgttgctgcttaaaagggcaaatctacccttagaatcaactgttatatcagggggattcagagagatattaggaatttgcacaagcgcacaatttaaccac
atcatgataacgccatgtaaaacaaagataaaaaaacaaaatgcagtgacttacatcgcaagcaaggcattttcttatccaattgctcaaagtttggccttt
catatcgcaacgaaaatgcgtaatatacgcgcccttgcggacatcagtatggtcattcctagttcatgcgcatcggacaccaccagcttacaaattgcct
gattgcggccccgatggccggtatcactgaccgaccatttcgtgccttatgtcatgcgatgggggctgggccgtctctgaagctctcggtgaacattgtt
gcgaggcaggatgcgagctggttgtgttttgacattaccgataatgtgccgcgtgaacgggtgcgttatgcccgcccggaagcggcgttttcccgtcc
ggggaatggcatggagctgcgccttatccagacgctgatcgcccatcatcgcggttctttagatctctcggtccgccctgatggcggcaccttgctgac
gttacgcctgccggtacagcaggttatcaccggaggcttaaaatgacccagttacctaccgcgggcccggttatccggcgctttgatatgtctgcccag
tttacggcgctttatcgcatcagcgtggcgctgagtcacgaaagcaacaccgggcgcgcactggcgccgatcctcgaagtgcttcacgatcatgcatt
tatgcaatacggcatggtgtgctgtttgataaagaacgcaatgcactctttgtggaatccctgcatggcatcgacggcgaaaggaaaaaagagaccc
gccatgtccgttaccgcatgggggaaggcgtgatcggcgcggtgatgagccagcgtcaggcgctggtgttaccgcgcatttcagacgatcagcgttt
tctcgaccgcctgaatatatttacgattacagcctgccgttgattggcgtgccgatccccggtgcggataatcagccatcgggcgtgctggtggcacagcc
gatggcgttgcacgaagaccggctgactgccagtacgcggtttttagaaatggtc
The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if the range 10-15 is disclosed, then 11, 12, 13, and 14 are also disclosed. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
CLAUSES
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- 1. A method of increasing nitrogen fixation in a non-leguminous plant, comprising:
- a. applying to the plant a plurality of non-intergeneric bacteria, said plurality comprising non-intergeneric bacteria that:
- i. have an average colonization ability per unit of plant root tissue of at least about 1.0×104 bacterial cells per gram of fresh weight of plant root tissue; and/or
- ii. produce fixed N of at least about 1×104′ mmol N per bacterial cell per hour, and
- wherein the plurality of non-intergeneric bacteria, in planta, produce 1% or more of the fixed nitrogen in the plant, and
- wherein each member of the plurality of non-intergeneric bacteria comprises at least one genetic variation introduced into at least one gene, or non-coding polynucleotide, of the nitrogen fixation or assimilation genetic regulatory network, such that the non-intergeneric bacteria are capable of fixing atmospheric nitrogen in the presence of exogenous nitrogen.
- 2. The method according to clause 1, wherein the plurality of non-intergeneric bacteria comprise bacteria that: have an average colonization ability per unit of plant root tissue of at least about 1.0×104 bacterial cells per gram of fresh weight of plant root tissue.
- 3. The method according to clause 1, wherein the plurality of non-intergeneric bacteria comprise bacteria that: produce fixed N of at least about 1×10−17 mmol N per bacterial cell per hour.
- 4. The method according to clause 1, wherein the plurality of non-intergeneric bacteria comprise bacteria that: have an average colonization ability per unit of plant root tissue of at least about 1.0×104 bacterial cells per gram of fresh weight of plant root tissue and produce fixed N of at least about 1×10−17 mmol N per bacterial cell per hour.
- 5. The method according to clause 1, wherein the at least one genetic variation comprises an introduced control sequence operably linked to the at least one gene of the nitrogen fixation or assimilation genetic regulatory network.
- 6. The method according to clause 1, wherein the at least one genetic variation comprises a promoter operably linked to the at least one gene of the nitrogen fixation or assimilation genetic regulatory network.
- 7. The method according to clause 1, wherein the at least one genetic variation comprises an inducible promoter operably linked to the at least one gene of the nitrogen fixation or assimilation genetic regulatory network.
- 8. The method according to clause 1, wherein the plurality of non-intergeneric bacteria do not comprise a constitutive promoter operably linked to a gene of the nitrogen fixation or assimilation genetic regulatory network.
- 9. The method according to clause 1, wherein the plurality of non-intergeneric bacteria do not comprise a constitutive promoter operably linked to a gene in the nif gene cluster.
- 10. The method according to clause 1, wherein the plurality of non-intergeneric bacteria, in planta, excrete nitrogen-containing products of nitrogen fixation.
- 11. The method according to clause 1, wherein the plurality of non-intergeneric bacteria applied to the plant do not stimulate an increase in the uptake of exogenous non-atmospheric nitrogen.
- 12. The method according to clause 1, wherein the plant is grown in soil from a field which has been administered at least about 50 lbs of nitrogen-containing fertilizer per acre, and wherein the nitrogen-containing fertilizer comprises at least about 5% nitrogen by weight.
- 13. The method according to clause 1, wherein the plant is grown in soil from a field which has been administered at least about 50 lbs of nitrogen-containing fertilizer per acre, and wherein the nitrogen-containing fertilizer comprises at least about 5% nitrogen by weight, and wherein the nitrogen-containing fertilizer comprises ammonium or an ammonium containing molecule.
- 14. The method according to clause 1, wherein the exogenous nitrogen is selected from fertilizer comprising one or more of: glutamine, ammonia, ammonium, urea, nitrate, nitrite, ammonium-containing molecules, nitrate-containing molecules, and nitrite-containing molecules.
- 15. The method according to clause 1, wherein the plurality of non-intergeneric bacteria, in plan/a, produce 5% or more of the fixed nitrogen in the plant.
- 16. The method according to clause 1, wherein the plurality of non-intergeneric bacteria, in planta, produce 10% or more of the fixed nitrogen in the plant.
- 17. The method according to clause 1, wherein the plurality of non-intergeneric bacteria, in planta, fix atmospheric nitrogen in non-nitrogen-limiting conditions.
- 18. The method according to clause 1, wherein the plurality of non-intergeneric bacteria, in planta, excrete nitrogen-containing products of nitrogen fixation.
- 19. The method according to clause 1, wherein the fixed nitrogen produced by the plurality of non-intergeneric bacteria is measured through dilution of enriched fertilizer by atmospheric N2 gas in plant tissue.
- 20. The method according to clause 1, wherein the at least one gene, or non-coding polynucleotide, of the nitrogen fixation or assimilation genetic regulatory network are selected from the group consisting of: nifA, nifL, ntrB, ntrC, polynucleotide encoding glutamine synthetase, glnA, glnB, glnK, drat, amtB, polynucleotide encoding glutaminase, glnD, glnE, nifJ, nifD, nifK, nifY, nifE, nifL, nifV, nifW, nifZ, nifM, nifF, nifB, nifQ, and a gene associated with biosynthesis of a nitrogenase enzyme.
- 21. The method according to clause 1, wherein the at least one genetic variation is a mutation that results in one or more of: increased expression or activity of NifA or glutaminase; decreased expression or activity of NifL, NtrB, glutamine synthetase, GlnB, GlnK, DraT, AmtB; decreased adenylyl-removing activity of GlnE; or decreased uridylyl-removing activity of GlnD.
- 22. The method according to clause 1, wherein the at least one genetic variation is selected from: (A) a knock-out mutation; (B) alters or abolishes a regulatory sequence of a target gene; (C) comprises the insertion of a heterologous regulatory sequence; or (D) a domain deletion.
- 23. The method according to clause 1, wherein the at least one genetic variation is a mutated nifL gene that has been altered to comprise a heterologous promoter inserted into said nifL gene.
- 24. The method according to clause 1, wherein the at least one genetic variation is a mutated glnE gene that results in a truncated GlnE protein lacking an adenylyl-removing (AR) domain.
- 25. The method according to clause 1, wherein the at least one genetic variation is a mutated amtB gene that results in the lack of expression of said amtB gene.
- 26. The method according to clause 1, wherein the at least one genetic variation is selected from: a mutated nifL gene that has been altered to comprise a heterologous promoter inserted into said nifL gene; a mutated glnE gene that results in a truncated GlnE protein lacking an adenylyl-removing (AR) domain; a mutated amtB gene that results in the lack of expression of said amtB gene; and combinations thereof.
- 27. The method according to clause 1, wherein the at least one genetic variation is a mutated nifL gene that has been altered to comprise a heterologous promoter inserted into said nifL gene and a mutated glnE gene that results in a truncated GlnE protein lacking an adenylyl-removing (AR) domain.
- 28. The method according to clause 1, wherein the at least one genetic variation is a mutated nifL gene that has been altered to comprise a heterologous promoter inserted into said nifL gene and a mutated glnE gene that results in a truncated GlnE protein lacking an adenylyl-removing (AR) domain and a mutated amtB gene that results in the lack of expression of said amtB gene.
- 29. The method according to clause 1, wherein the plant comprises the seed, stalk, flower, fruit, leaves, or rhizome.
- 30. The method according to clause 1, wherein the plurality of non-intergeneric bacteria are formulated into a composition.
- 31. The method according to clause 1, wherein the plurality of non-intergeneric bacteria are formulated into a composition comprising an agriculturally acceptable carrier.
- 32. The method according to clause 1, wherein the plurality of non-intergeneric bacteria are applied into furrows in which seeds of said plant are planted.
- 33. The method according to clause 1, wherein the plurality of non-intergeneric bacteria are formulated into a liquid in-furrow composition comprising bacteria at a concentration of about 1×107 to about 1×1010 du per milliliter.
- 34. The method according to clause 1, wherein the plurality of non-intergeneric bacteria are applied onto a seed of said plant.
- 35. The method according to clause 1, wherein the plurality of non-intergeneric bacteria are formulated into a seed coating and are applied onto a seed of said plant.
- 36. The method according to clause 1, wherein the plurality of non-intergeneric bacteria are formulated into a seed coating and are applied onto a seed of said plant, at a concentration of about 1×105 to about 1×107 cfu per seed.
- 37. The method according to clause 1, wherein the plant is a cereal crop.
- 38. The method according to clause 1, wherein the plant is selected from the group consisting of: corn, rice, wheat, barley, sorghum, millet, oat, rye, and triticale.
- 39. The method according to clause 1, wherein the plant is corn.
- 40. The method according to clause 1, wherein the plant is an agricultural crop plant.
- 41. The method according to clause 1, wherein the plant is a genetically modified organism.
- 42. The method according to clause 1, wherein the plant is not a genetically modified organism.
- 43. The method according to clause 1, wherein the plant has been genetically engineered or bred for efficient nitrogen use.
- 44. The method according to clause 1, wherein the plurality of non-intergeneric bacteria comprise at least two different species of bacteria.
- 45. The method according to clause 1, wherein the plurality of non-intergeneric bacteria comprise at least two different strains of the same species of bacteria.
- 46. The method according to clause 1, wherein the plurality of non-intergeneric bacteria comprise bacteria selected from: Rahnella aquatilis, Klebsiella variicola, Achromobacter spiritinus, Achromobacter marplatensis, Microbacterium murale, Kluyvera intermedia, Kosakonia pseudosacchari, Enterobacter sp., Azospirillum lipoferum, Kosakonia sacchari, and combinations thereof.
- 47. The method according to clause 1, wherein the plurality of non-intergeneric bacteria are endophytic, epiphytic, or rhizospheric.
- 48. The method according to clause 1, wherein the plurality of non-intergeneric bacteria comprise bacteria selected from: a bacteria deposited as PTA-122293, a bacteria deposited as PTA-122294, a bacteria deposited as NCMA 201701002, a bacteria deposited as NCMA 201708004, a bacteria deposited as NCMA 201708003, a bacteria deposited as NCMA 201708002, a bacteria deposited as NCMA 201712001, a bacteria deposited as NCMA 201712002, and combinations thereof.
- 49. A bacterial composition, comprising:
- at least one genetically engineered bacterial strain that fixes atmospheric nitrogen in an agricultural system that has been fertilized with more than 20 lbs of Nitrogen per acre.
- 50. A bacterial composition, comprising:
- at least one bacterial strain that has been bred to fix atmospheric nitrogen in an agricultural system that has been fertilized with more than 20 lbs of Nitrogen per acre.
- 51. The bacterial composition of clause 49 or clause 50, wherein said fertilizer is a chemical fertilizer selected from the group consisting of anhydrous ammonia, ammonia sulfate, urea, diammonium phosphate, urea-form, UAN (urea ammonium nitrate) monoammonium phosphate, ammonium nitrate, nitrogen solutions, calcium nitrate, potassium nitrate, and sodium nitrate.
- 52. A bacterial composition, comprising:
- at least one genetically engineered bacterial strain that fixes atmospheric nitrogen, the at least one genetically engineered bacterial strain comprising exogenously added DNA wherein said exogenously added DNA shares at least 80% identity to a corresponding native bacterial strain.
- 53. The bacterial composition of clause 52, wherein said exogenously added DNA shares at least 85% identity to a corresponding native bacterial strain.
- 54. The bacterial composition of clause 52, wherein said exogenously added DNA shares at least 90% identity to a corresponding native bacterial strain.
- 55. The bacterial composition of clause 52, wherein said exogenously added DNA shares at least 95% identity to a corresponding native bacterial strain.
- 56. The bacterial composition of clause 52, wherein said exogenously added DNA shares at least 99% identity to a corresponding native bacterial strain.
- 57. The bacterial composition of clause 52, wherein said exogenously added DNA is derived from a same bacterial strain as said corresponding native bacterial strain.
- 58. The bacterial composition of any of the preceding clauses, wherein said bacterial composition is a fertilizing composition.
- 59. The bacterial composition of any of the preceding clauses, wherein said at least one genetically engineered bacterial strain comprises at least one variation in a gene or intergenic region within 10,000 bp of a gene selected from the group consisting of: nifA, nifL, ntrB, ntrC, glnA, glnB, glnK, draT, amtB, glnD, glnE, nifJ, nifH, nifD, nifK, nifY, nifE, nifN, nifU, nifS, nifV, nifW, nifZ, nifM, nifF, nifB, and nifQ.
- 60. The bacterial composition of any of the preceding clauses, further comprising at least one additional component selected from the group consisting of a tackifier, a microbial stabilizer, a fungicide, an antibacterial agent, a preservative, a stabilizer, a surfactant, an anti-complex agent, an herbicide, a nematicide, an insecticide, a plant growth regulator, a fertilizer, a rodenticide, a dessicant, a bactericide, and a nutrient.
- 61. The bacterial composition of any of the preceding clauses, further comprising at least one additional component selected from the group consisting of a colorant, primer, pellet, disinfectant, plant growth regulator, safener, and a nematicide.
- 62. The bacterial composition of any of the preceding clauses, wherein said bacterial composition is formulated to be applied to a field.
- 63. The bacterial composition of clause 62, wherein said bacterial composition is formulated to be applied in-furrow.
- 64. The bacterial composition of clause 62, wherein said bacterial composition is formulated to be applied as a seed coating, seed dressing, or seed treatment.
- 65. The bacterial composition of clause 61 or clause 62, wherein said bacterial composition is formulated to be applied at, prior to, or post planting of the seed.
- 66. A seed composition comprising a seed of a plant that is inoculated with a bacterial composition of any of the preceding clauses.
- 67. A method of growing a crop using a plurality of seeds having a seed composition of clause 66.
- 68. The method of clause 67, further comprising harvesting said crop.
- 69. A method of applying a bacterial composition of any of the preceding clauses to a field.
- 70. The method of clause 69, wherein said bacterial composition is applied to said field in a form selected from the group consisting of a liquid form, a dry form, a granule, a powder, and a pellet.
- 71. The method of clause 69, wherein said bacterial composition is applied to said field as a seed coating, seed dressing, or seed treatment.
- 72. The method of clause 69, wherein said bacterial composition is applied to said field as an in-furrow treatment.
- 73. The method of clause 69, wherein said bacterial composition is applied to said field at, prior to, or post planting of the seed.
- 74. A fertilizer composition comprising a bacterial composition of any of the preceding clauses.
- 75. The fertilizer composition of clause 74, wherein said fertilizer composition is a seed coating, seed dressing, or seed treatment composition.
- 76. The fertilizer composition of clause 74, wherein said fertilizer composition is an in-furrow composition.
- 77. The fertilizer composition of clause 74, wherein said fertilizer composition is provided to a crop at, prior to, or post planting.
- 78. The fertilizer composition of clause 74, further comprising a porous carrier.
- 79. The fertilizer composition of clause 74, further comprising an additional synergistic component that, when combined with said bacterial composition, increases a fertilizing benefit of said fertilizer composition to a crop that is beyond a cumulative benefit of its individual components.
- 80. A method of maintaining soil nitrogen levels, comprising:
- planting, in soil of a field, a crop inoculated by a genetically engineered bacterium that fixes atmospheric nitrogen; and
- harvesting said crop, wherein no more than 90% of a nitrogen dose required for producing said crop is administered to said soil of said field between planting and harvesting.
- 81. The method of clause 80, wherein no more than 80% of a nitrogen dose required for producing said crop is administered to said soil of said field between planting and harvesting.
- 82. The method of clause 80, wherein no more than 70% of a nitrogen dose required for producing said crop is administered to said soil of said field between planting and harvesting.
- 83. The method of clause 80, wherein no more than 60% of a nitrogen dose required for producing said crop is administered to said soil of said field between planting and harvesting.
- 84. The method of clause 80, wherein no more than 50% of a nitrogen dose required for producing said crop is administered to said soil of said field between planting and harvesting.
- 85. The method of clause 80, wherein no more than 40% of a nitrogen dose required for producing said crop is administered to said soil of said field between planting and harvesting.
- 86. The method of clause 80, wherein said genetically engineered bacterium comprises a bacterial composition of any of the preceding clauses.
- 87. The method of clause 80, wherein said genetically engineered bacterium consists of a bacterial composition of any of the preceding clauses.
- 88. A method of delivering a probiotic supplement to a crop plant, comprising:
- coating a crop seed with a seed coating, seed treatment, or seed dressing, wherein said seed coating, seed dressing, or seed treatment comprises living representatives of said probiotic; and
- applying, in soil of a field, said crop seeds.
- 89. The method of clause 88, wherein said seed coating, seed dressing, or seed treatment is applied in a single layer to said crop seed.
- 90. The method of clause 88, wherein said seed coating is applied in multiple layers to said crop seed.
- 91. The method of clause 88, wherein said seed coating is applied in a blend to said crop seed.
- 92. The method of clause 88, wherein said crop seed is non-nodulating.
- 93. The method of clause 88, wherein said seed coating comprises a bacterial composition of any of the preceding clauses.
- 94. The method of any of the proceeding clauses, wherein the genetically engineered bacterial strain is a genetically engineered Gram-positive bacterial strain.
- 95. The method of clause 94, wherein the genetically engineered Gram-positive bacterial strain has an altered expression level of a regulator of a Nif cluster.
- 96. The method of clause 94, wherein the genetically engineered Gram-positive bacterial strain expresses a decreased amount of a negative regulator of a Nif cluster.
- 97. The method of clause 94, wherein the genetically engineered bacterial strain expresses a decreased amount of GlnR.
- 98. The method of any one of the proceeding clauses, wherein the genome of the genetically engineered bacterial strain encodes a polypeptide with at least 75% identity to a sequence from the Zehr lab NifH database.
- 99. The method of any one of the proceeding clauses, wherein the genome of the genetically engineered bacterial strain encodes a polypeptide with at least 85% identity to a sequence from the Zehr lab NifH database.
- 100. The method of any one of the proceeding clauses, wherein the genome of the genetically engineered bacterial strain encodes a polypeptide with at least 75% identity to a sequence from the Buckley lab NifH database.
- 101. The method of any one of the proceeding clauses, wherein the genome of the genetically engineered bacterial strain encodes a polypeptide with at least 85% identity to a sequence from the Buckley lab NifH database.
- 102. A method of breeding microbial strains to improve specific traits of agronomic relevance, comprising:
- providing a plurality of microbial strains that have the ability to colonize a desired crop;
- improving regulatory networks influencing the trait through intragenomic rearrangement;
- assessing microbial strains within the plurality of microbial strains to determine a measure of the trait; and
- selecting one or more microbial strains of the plurality of microbial strains that generate an improvement in the trait in the presence of the desired crop.
- 103. The method of clause 102, wherein the specific trait which is improved is the ability of the microbial strain to fix nitrogen.
- 104. The method of clause 103, wherein the specific trait which is improved is the ability of the microbial strain to fix atmospheric nitrogen in the presence of N-fertilized growing conditions.
- 105. A method of breeding microbial strains to improve specific traits of agronomic relevance, comprising:
- providing a plurality of microbial strains that have the ability to colonize a desired crop;
- introducing genetic diversity into the plurality of microbial strains;
- assessing microbial strains within the plurality of microbial strains to determine a measure of the trait; and
- selecting one or more microbial strains of the plurality of microbial strains that generate an improvement in the trait in the presence of the desired crop.
- 106. The method of clause 105, wherein the specific trait which is improved is the ability of the microbial strain to fix nitrogen.
- 107. The method of clause 106, wherein the specific trait which is improved is the ability of the microbial strain to fix atmospheric nitrogen in the presence of N-fertilized growing conditions.
- 108. A method of increasing the amount of atmospheric derived nitrogen in a non-leguminous plant, comprising:
- exposing said non-leguminous plant to engineered non-intergeneric microbes, said engineered non-intergeneric microbes comprising at least one genetic variation introduced into a nitrogen fixation genetic regulatory network or at least one genetic variation introduced into a nitrogen assimilation genetic regulatory network.
- 109. The method of clause 108, wherein said engineered non-intergeneric microbes comprise at least one genetic variation introduced into said nitrogen fixation genetic regulatory network.
- 110. The method of clause 108, wherein said engineered non-intergeneric microbes comprise at least one genetic variation introduced into said nitrogen assimilation genetic regulatory network.
- 111. The method of clause 108, wherein said engineered non-intergeneric microbes comprise at least one genetic variation introduced into said nitrogen fixation genetic regulatory network and at least one genetic variation introduced into said nitrogen assimilation genetic regulatory network.
- 112. The method of clause 108, wherein said engineered non-intergeneric microbes are applied into furrows in which seeds of said non-leguminous plant are planted.
- 113. The method of clause 108, wherein said engineered non-intergeneric microbes are coated onto a seed of said non-leguminous plant.
- 114. The method of clause 108, wherein said non-leguminous plant is a non-leguminous agricultural crop plant selected from the group consisting of sorghum, canola, tomato, strawberry, barley, rice, corn, wheat, potato, millet, cereals, grains, and maize.
- 115. The method of clause 108, wherein said engineered non-intergeneric microbes colonize at least a root of said non-leguminous plant such that said engineered non-intergeneric microbes are present in said non-leguminous plant in an amount of at least 105 colony forming units per gram fresh weight of tissue.
- 116. The method of clause 108, wherein said engineered non-intergeneric microbes are capable of fixing atmospheric nitrogen in non-nitrogen-limiting conditions.
- 117. The method of clause 108, wherein said engineered non-intergeneric microbes, in planta, excrete nitrogen-containing products of nitrogen fixation.
- 118. The method of clause 108, wherein said at least one genetic variation is introduced into a gene selected from the group consisting of nifA, nut, ntrB, ntrC, polynucleotide encoding glutamine synthetase, glnA, glnB, glnK, drat, amtB, polynucleotide encoding glutaminase, glnD, glnE, nifJ, nifH, nit)), nifK, nifY, nifE, nifN, nifU, nifS, nifV, nifW, nifZ, nifM, nifF, nifB, nifQ, and a gene associated with biosynthesis of a nitrogenase enzyme.
- 119. The method of clause 108, wherein said engineered non-intergeneric microbes, in planta, produce at least 1% of fixed nitrogen in said non-leguminous plant.
- 120. The method of clause 119, wherein said fixed nitrogen in said non-leguminous plant produced by said engineered non-intergeneric microbes is measured by dilution of 15N in crops grown in fields treated with fertilizer containing 1.2% 15N.
- 121. The method of clause 119, wherein said engineered non-intergeneric microbes, in planta, produce 5% or more of the fixed nitrogen in said non-leguminous plant.
- 122. The method of clause 108, wherein said non-intergeneric microbes are engineered using at least one type of engineering selected from the group consisting of directed mutagenesis, random mutagenesis, and directed evolution.
- 123. A method of increasing an amount of atmospheric derived nitrogen in a corn plant, comprising
- exposing said corn plant to engineered non-intergeneric microbes comprising engineered genetic variations within at least two genes selected from the group consisting of nifL, glnB, glnE, and amtB.
- 124. The method of clause 123, wherein said engineered non-intergeneric microbes, in planta, excrete nitrogen-containing products of nitrogen fixation.
- 125. The method of clause 123, wherein said engineered non-intergeneric microbes are applied into furrows in which seeds of said corn plant are planted.
- 126. The method of clause 123, wherein said engineered non-intergeneric microbes are coated onto a seed of said corn plant.
- 127. A method of increasing an amount of atmospheric derived nitrogen in a corn plant, comprising:
- exposing said corn plant to engineered non-intergeneric microbes comprising at least one genetic variation introduced into a nitrogen fixation genetic regulatory network and at least one genetic variation introduced into a nitrogen assimilation genetic regulatory network, wherein said engineered non-intergeneric microbes, in planta, produce at least 5% of fixed nitrogen in said corn plant as measured by dilution of 15N in crops grown in fields treated with fertilizer containing 1.2% 15N.
- 128. A method of increasing nitrogen fixation in a non-leguminous plant, comprising:
- a. applying to the plant a plurality of non-intergeneric bacteria, said plurality comprising non-intergeneric bacteria that:
- i. have an average colonization ability per unit of plant root tissue of at least about 1.0×104 bacterial cells per gram of fresh weight of plant root tissue; and
- ii. produce fixed N of at least about 1×10−17 mmol N per bacterial cell per hour, and
- wherein the product of (i) the average colonization ability per unit of plant root tissue and (ii) produced fixed N per bacterial cell per hour, is at least about 2.5×104 mmol N per gram of fresh weight of plant root tissue per hour, and
- wherein the plurality of non-intergeneric bacteria, in planta, produce 1% or more of the fixed nitrogen in the plant, and wherein each member of the plurality of non-intergeneric bacteria comprises at least one genetic variation introduced into at least one gene, or non-coding polynucleotide, of the nitrogen fixation or assimilation genetic regulatory network, such that the non-intergeneric bacteria are capable of fixing atmospheric nitrogen in the presence of exogenous nitrogen.
- 129. The method according to clause 128, wherein the at least one genetic variation comprises an introduced control sequence operably linked to the at least one gene of the nitrogen fixation or assimilation genetic regulatory network.
- 130. The method according to clause 128, wherein the at least one genetic variation comprises a promoter operably linked to the at least one gene of the nitrogen fixation or assimilation genetic regulatory network.
- 131. The method according to clause 128, wherein the at least one genetic variation comprises an inducible promoter operably linked to the at least one gene of the nitrogen fixation or assimilation genetic regulatory network.
- 132. The method according to clause 128, wherein the plurality of non-intergeneric bacteria do not comprise a constitutive promoter operably linked to a gene of the nitrogen fixation or assimilation genetic regulatory network.
- 133. The method according to clause 128, wherein the plurality of non-intergeneric bacteria do not comprise a constitutive promoter operably linked to a gene in the nif gene cluster.
- 134. The method according to clause 128, wherein the plurality of non-intergeneric bacteria, in planta, excrete the nitrogen-containing products of nitrogen fixation.
- 135. The method according to clause 128, wherein the plurality of non-intergeneric bacteria applied to the plant do not stimulate an increase in the uptake of exogenous non-atmospheric nitrogen.
- 136. The method according to clause 128, wherein the plant is grown in soil from a field which has been administered at least about 50 lbs of nitrogen-containing fertilizer per acre, and wherein the nitrogen-containing fertilizer comprises at least about 5% nitrogen by weight.
- 137. The method according to clause 128, wherein the plant is grown in soil from a field which has been administered at least about 50 lbs of nitrogen-containing fertilizer per acre, and wherein the nitrogen-containing fertilizer comprises at least about 5% nitrogen by weight, and wherein the nitrogen-containing fertilizer comprises ammonium or an ammonium containing molecule.
- 138. The method according to clause 128, wherein the exogenous nitrogen is selected from fertilizer comprising one or more of: glutamine, ammonia, ammonium, urea, nitrate, nitrite, ammonium-containing molecules, nitrate-containing molecules, and nitrite-containing molecules.
- 139. The method according to clause 128, wherein the plurality of non-intergeneric bacteria, in planta, produce 5% or more of the fixed nitrogen in the plant.
- 140. The method according to clause 128, wherein the plurality of non-intergeneric bacteria, in planta, produce 10% or more of the fixed nitrogen in the plant.
- 141. The method according to clause 128, wherein the plurality of non-intergeneric bacteria, in planta, fix atmospheric nitrogen in non-nitrogen-limiting conditions.
- 142. The method according to clause 128, wherein the plurality of non-intergeneric bacteria, in planta, excrete nitrogen-containing products of nitrogen fixation.
- 143. The method according to clause 128, wherein the fixed nitrogen produced by the plurality of non-intergeneric bacteria is measured through dilution of enriched fertilizer by atmospheric N2 gas in plant tissue.
- 144. The method according to clause 128, wherein the at least one gene, or non-coding polynucleotide, of the nitrogen fixation or assimilation genetic regulatory network are selected from the group consisting of: nifA, nifL, ntrB, ntrC, polynucleotide encoding glutamine synthetase, glnA, glnB, glnK, drat, amtB, polynucleotide encoding glutaminase, glnD, glnE, nifJ, nifH, nifD, nifK, nifY, nifE, nifN, nifU, nifS, nifV, nifW, nifZ, nifM, nifF, nifQ, nifA, and a gene associated with biosynthesis of a nitrogenase enzyme.
- 145. The method according to clause 128, wherein the at least one genetic variation is a mutation that results in one or more of: increased expression or activity of NifA or glutaminase; decreased expression or activity of NifL, NtrB, glutamine synthetase, GlnB, GlnK, DraT, AmtB; decreased adenylyl-removing activity of GlnE; or decreased uridylyl-removing activity of GlnD.
- 146. The method according to clause 128, wherein the at least one genetic variation is selected from: (A) a knock-out mutation; (B) alters or abolishes a regulatory sequence of a target gene; (C) comprises the insertion of a heterologous regulatory sequence; or (D) a domain deletion.
- 147. The method according to clause 128, wherein the at least one genetic variation is a mutated nifL gene that has been altered to comprise a heterologous promoter inserted into said nifL gene.
- 148. The method according to clause 128, wherein the at least one genetic variation is a mutated glnE gene that results in a truncated GlnE protein lacking an adenylyl-removing (AR) domain.
- 149. The method according to clause 128, wherein the at least one genetic variation is a mutated amtB gene that results in the lack of expression of said amtB gene.
- 150. The method according to clause 128, wherein the at least one genetic variation is selected from: a mutated nifL gene that has been altered to comprise a heterologous promoter inserted into said n/IL gene; a mutated glnE gene that results in a truncated GlnE protein lacking an adenylyl-removing (AR) domain; a mutated amtB gene that results in the lack of expression of said amtB gene; and combinations thereof.
- 151. The method according to clause 128, wherein the at least one genetic variation is a mutated nifL gene that has been altered to comprise a heterologous promoter inserted into said nifL gene and a mutated glnE gene that results in a truncated GlnE protein lacking an adenylyl-removing (AR) domain.
- 152. The method according to clause 128, wherein the at least one genetic variation is a mutated nifL gene that has been altered to comprise a heterologous promoter inserted into said nifL gene and a mutated glnE gene that results in a truncated GlnE protein lacking an adenylyl-removing (AR) domain and a mutated amtB gene that results in the lack of expression of said amtB gene.
- 153. The method according to clause 128, wherein the plant comprises the seed, stalk, flower, fruit, leaves, or rhizome.
- 154. The method according to clause 128, wherein the plurality of non-intergeneric bacteria are formulated into a composition.
- 155. The method according to clause 128, wherein the plurality of non-intergeneric bacteria are formulated into a composition comprising an agriculturally acceptable carrier.
- 156. The method according to clause 128, wherein the plurality of non-intergeneric bacteria are applied into furrows in which seeds of said plant are planted.
- 157. The method according to clause 128, wherein the plurality of non-intergeneric bacteria are formulated into a liquid in-furrow composition comprising bacteria at a concentration of about 1×107 to about 1×1010 cfu per milliliter.
- 158. The method according to clause 128, wherein the plurality of non-intergeneric bacteria are applied onto a seed of said plant.
- 159. The method according to clause 128, wherein the plurality of non-intergeneric bacteria are formulated into a seed coating and are applied onto a seed of said plant.
- 160. The method according to clause 128, wherein the plurality of non-intergeneric bacteria are formulated into a seed coating and are applied onto a seed of said plant, at a concentration of about 1×105 to about 1×107 cfu per seed.
- 161. The method according to clause 128, wherein the plant is a cereal crop.
- 162. The method according to clause 128, wherein the plant is selected from the group consisting of: corn, rice, wheat, barley, sorghum, millet, oat, rye, and triticale.
- 163. The method according to clause 128, wherein the plant is corn.
- 164. The method according to clause 128, wherein the plant is an agricultural crop plant.
- 165. The method according to clause 128, wherein the plant is a genetically modified organism.
- 166. The method according to clause 128, wherein the plant is not a genetically modified organism.
- 167. The method according to clause 128, wherein the plant has been genetically engineered or bred for efficient nitrogen use.
- 168. The method according to clause 128, wherein the plurality of non-intergeneric bacteria comprise at least two different species of bacteria.
- 169. The method according to clause 128, wherein the plurality of non-intergeneric bacteria comprise at least two different strains of the same species of bacteria.
- 170. The method according to clause 128, wherein the plurality of non-intergeneric bacteria comprise bacteria selected from: Rahnella aquatilis, Klebsiella variicola, Achromobacter spiritinus, Achromobacter marplatensis, Microbacterium murale, Kluyvera intermedia, Kosakonia pseudosacchari, Enterobacter sp., Azospirillum lipoferum, Kosakonia sacchari, and combinations thereof.
- 171. The method according to clause 128, wherein the plurality of non-intergeneric bacteria are endophytic, epiphytic, or rhizospheric.
- 172. The method according to clause 128, wherein the plurality of non-intergeneric bacteria comprise bacteria selected from: a bacteria deposited as PTA-122293, a bacteria deposited as PTA-122294, a bacteria deposited as NCMA 201701002, a bacteria deposited as NCMA 201708004, a bacteria deposited as NCMA 201708003, a bacteria deposited as NCMA 201708002, a bacteria deposited as NCMA 201712001, a bacteria deposited as NCMA 201712002, and combinations thereof.
- 173. A method of increasing nitrogen fixation in a non-leguminous plant, comprising:
- a. applying to the plant a plurality of bacteria, said plurality comprising bacteria that:
- i. have an average colonization ability per unit of plant root tissue of at least about 1.0×104 bacterial cells per gram of fresh weight of plant root tissue; and/or
- ii. produce fixed N of at least about 1×10−17 mmol N per bacterial cell per hour, and
- wherein the plurality of bacteria, in planta, produce 1% or more of the fixed nitrogen in the plant.
- 174. The method according to clause 173, wherein the plurality of bacteria comprise bacteria that: have an average colonization ability per unit of plant root tissue of at least about 1.0×104 bacterial cells per gram of fresh weight of plant root tissue.
- 175. The method according to clause 173, wherein the plurality of bacteria comprise bacteria that: produce fixed N of at least about 173×10−17 mmol N per bacterial cell per hour.
- 176. The method according to clause 173, wherein the plurality of bacteria comprise bacteria that: have an average colonization ability per unit of plant root tissue of at least about 1.0×104 bacterial cells per gram of fresh weight of plant root tissue and produce fixed N of at least about 1×10−17 mmol N per bacterial cell per hour.
- 177. The method according to clause 173, wherein the plurality of bacteria comprise bacteria that: have an average colonization ability per unit of plant root tissue of at least about 1.0×104 bacterial cells per gram of fresh weight of plant root tissue and produce fixed N of at least about 1×10−17 mmol N per bacterial cell per hour, and wherein the product of (i) the average colonization ability per unit of plant root tissue and (ii) produced fixed N per bacterial cell per hour, is at least about 2.5×10−8 mmol N per gram of fresh weight of plant root tissue per hour.
- 178. The method according to clause 173, wherein the plurality of bacteria, in planta, excrete the nitrogen-containing products of nitrogen fixation.
- 179. The method according to clause 173, wherein the plurality of bacteria applied to the plant do not stimulate an increase in the uptake of exogenous non-atmospheric nitrogen.
- 180. The method according to clause 173, wherein the plant comprises the seed, stalk, flower, fruit, leaves, or rhizome.
- 181. The method according to clause 173, wherein the plurality of bacteria are formulated into a composition.
- 182. The method according to clause 173, wherein the plurality of bacteria are formulated into a composition comprising an agriculturally acceptable carrier.
- 183. The method according to clause 173, wherein the plurality of bacteria are applied into furrows in which seeds of said plant are planted.
- 184. The method according to clause 173, wherein the plurality of bacteria are formulated into a liquid in-furrow composition comprising bacteria at a concentration of about 1×107 to about 1×1010 cfu per milliliter.
- 185. The method according to clause 173, wherein the plurality of bacteria are applied onto a seed of said plant.
- 186. The method according to clause 173, wherein the plurality of bacteria are formulated into a seed coating and are applied onto a seed of said plant.
- 187. The method according to clause 173, wherein the plurality of bacteria are formulated into a seed coating and are applied onto a seed of said plant, at a concentration of about 1×105 to about 1×107 cfu per seed.
- 188. The method according to clause 173, wherein the plant is a cereal crop.
- 189. The method according to clause 173, wherein the plant is selected from the group consisting of: corn, rice, wheat, barley, sorghum, millet, oat, rye, and triticale.
- 190. The method according to clause 173, wherein the plant is corn.
- 191. The method according to clause 173, wherein the plant is an agricultural crop plant.
- 192. The method according to clause 173, wherein the plant is a genetically modified organism.
- 193. The method according to clause 173, wherein the plant is not a genetically modified organism.
- 194. The method according to clause 173, wherein the plant has been genetically engineered or bred for efficient nitrogen use.
- 195. The method according to clause 173, wherein the plurality of bacteria comprise at least two different species of bacteria.
- 196. The method according to clause 173, wherein the plurality of bacteria comprise at least two different strains of the same species of bacteria.
- 197. The method according to clause 173, wherein the plurality of bacteria comprise bacteria selected from: Rahnella aquatilis, Klebsiella variicola, Achromobacter spiritinus, Achromobacter marplatensis, Microbacterium murale, Kluyvera intermedia, Kosakonia pseudosacchari, Enterobacter sp., Azospirillum lipoferum, Kosakonia sacchari, and combinations thereof.
- 198. The method according to clause 173, wherein the plurality of bacteria are endophytic, epiphytic, or rhizospheric.
- 199. The method according to clause 173, wherein the plurality of bacteria are selected from: a bacteria deposited as NCMA 201701003, a bacteria deposited as NCMA 201701001, and a bacteria deposited as NCMA 201708001.
- 200. A non-intergeneric bacterial population capable of increasing nitrogen fixation in a non-leguminous plant, comprising:
- a. a plurality of non-intergeneric bacteria, that:
- i. have an average colonization ability per unit of plant root tissue of at least about 1.0×104 bacterial cells per gram of fresh weight of plant root tissue; and/or
- ii. produce fixed N of at least about 1×10−17 mmol N per bacterial cell per hour, and
- wherein the plurality of non-intergeneric bacteria, in planta, produce 1% or more of the fixed nitrogen in a plant grown in the presence of the plurality of non-intergeneric bacteria, and
- wherein each member of the plurality of non-intergeneric bacteria comprises at least one genetic variation introduced into at least one gene, or non-coding polynucleotide, of the nitrogen fixation or assimilation genetic regulatory network, such that the non-intergeneric bacteria are capable of fixing atmospheric nitrogen in the presence of exogenous nitrogen.
- 201. The non-intergeneric bacterial population according to clause 200, wherein the plurality of non-intergeneric bacteria comprise bacteria that: have an average colonization ability per unit of plant root tissue of at least about 1.0×104 bacterial cells per gram of fresh weight of plant root tissue.
- 202. The non-intergeneric bacterial population according to clause 200, wherein the plurality of non-intergeneric bacteria comprise bacteria that: produce fixed N of at least about 1×10−17 mmol N per bacterial cell per hour.
- 203. The non-intergeneric bacterial population according to clause 200, wherein the plurality of non-intergeneric bacteria comprise bacteria that: have an average colonization ability per unit of plant root tissue of at least about 1.0×104 bacterial cells per gam of fresh weight of plant root tissue and produce fixed N of at least about 1×10−17 mmol N per bacterial cell per hour.
- 204. The non-intergeneric bacterial population according to clause 200, wherein the at least one genetic variation comprises an introduced control sequence operably linked to the at least one gene of the nitrogen fixation or assimilation genetic regulatory network.
- 205. The non-intergeneric bacterial population according to clause 200, wherein the at least one genetic variation comprises a promoter operably linked to the at least one gene of the nitrogen fixation or assimilation genetic regulatory network.
- 206. The non-intergeneric bacterial population according to clause 200, wherein the at least one genetic variation comprises an inducible promoter operably linked to the at least one gene of the nitrogen fixation or assimilation genetic regulatory network.
- 207. The non-intergeneric bacterial population according to clause 200, wherein the plurality of non-intergeneric bacteria do not comprise a constitutive promoter operably linked to a gene of the nitrogen fixation or assimilation genetic regulatory network.
- 208. The non-intergeneric bacterial population according to clause 200, wherein the plurality of non-intergeneric bacteria do not comprise a constitutive promoter operably linked to a gene in the nif gene cluster.
- 209. The non-intergeneric bacterial population according to clause 200, wherein the plurality of non-intergeneric bacteria, in planta, excrete nitrogen-containing products of nitrogen fixation.
- 210. The non-intergeneric bacterial population according to clause 200, wherein the plurality of non-intergeneric bacteria applied to the plant do not stimulate an increase in the uptake of exogenous non-atmospheric nitrogen.
- 211. The non-intergeneric bacterial population according to clause 200, wherein the plant is grown in soil from a field which has been administered at least about 50 lbs of nitrogen-containing fertilizer per acre, and wherein the nitrogen-containing fertilizer comprises at least about 5% nitrogen by weight.
- 212. The non-intergeneric bacterial population according to clause 200, wherein the plant is grown in soil from a field which has been administered at least about 50 lbs of nitrogen-containing fertilizer per acre, and wherein the nitrogen-containing fertilizer comprises at least about 5% nitrogen by weight, and wherein the nitrogen-containing fertilizer comprises ammonium or an ammonium containing molecule.
- 213. The non-intergeneric bacterial population according to clause 200, wherein the exogenous nitrogen is selected from fertilizer comprising one or more of: glutamine, ammonia, ammonium, urea, nitrate, nitrite, ammonium-containing molecules, nitrate-containing molecules, and nitrite-containing molecules.
- 214. The non-intergeneric bacterial population according to clause 200, wherein the plurality of non-intergeneric bacteria, in planta, produce 5% or more of the fixed nitrogen in the plant.
- 215. The non-intergeneric bacterial population according to clause 200, wherein the plurality of non-intergeneric bacteria, in planta, produce 10% or more of the fixed nitrogen in the plant.
- 216. The non-intergeneric bacterial population according to clause 200, wherein the plurality of non-intergeneric bacteria, in planta, fix atmospheric nitrogen in non-nitrogen-limiting conditions.
- 217. The non-intergeneric bacterial population according to clause 200, wherein the plurality of non-intergeneric bacteria, in planta, excrete nitrogen-containing products of nitrogen fixation.
- 218. The non-intergeneric bacterial population according to clause 200, wherein the fixed nitrogen produced by the plurality of non-intergeneric bacteria is measured through dilution of enriched fertilizer by atmospheric N2 gas in plant tissue.
- 219. The non-intergeneric bacterial population according to clause 200, wherein the at least one gene, or non-coding polynucleotide, of the nitrogen fixation or assimilation genetic regulatory network are selected from the group consisting of: nifA, nifL, ntrB, ntrC, polynucleotide encoding glutamine synthetase, glnA, glnB, glnK, drat, amtB, polynucleotide encoding glutaminase, glnD, glnE, nifJ, nifH, nifD, nifK, nifY, nifE, nifN, nifU, nifS, nifV, nifW, nifZ, nifM, nifF, nifB, nifQ, and a gene associated with biosynthesis of a nitrogenase enzyme.
- 220. The non-intergeneric bacterial population according to clause 200, wherein the at least one genetic variation is a mutation that results in one or more of: increased expression or activity of NifA or glutaminase; decreased expression or activity of NifL, NtrB, glutamine synthetase, GlnB, GlnK, DraT, AmtB; decreased adenylyl-removing activity of GlnE; or decreased uridylyl-removing activity of GlnD.
- 221. The non-intergeneric bacterial population according to clause 200, wherein the at least one genetic variation is selected from: (A) a knock-out mutation; (B) alters or abolishes a regulatory sequence of a target gene; (C) comprises the insertion of a heterologous regulatory sequence; or (D) a domain deletion.
- 222. The non-intergeneric bacterial population according to clause 200, wherein the at least one genetic variation is a mutated/tiff, gene that has been altered to comprise a heterologous promoter inserted into said nifL gene.
- 223. The non-intergeneric bacterial population according to clause 200, wherein the at least one genetic variation is a mutated glnE gene that results in a truncated GlnE protein lacking an adenylyl-removing (AR) domain.
- 224. The non-intergeneric bacterial population according to clause 200, wherein the at least one genetic variation is a mutated amtB gene that results in the lack of expression of said amtB gene.
- 225. The non-intergeneric bacterial population according to clause 200, wherein the at least one genetic variation is selected from: a mutated nifL gene that has been altered to comprise a heterologous promoter inserted into said nifL gene; a mutated glnE gene that results in a truncated GlnE protein lacking an adenylyl-removing (AR) domain; a mutated amtB gene that results in the lack of expression of said amtB gene; and combinations thereof.
- 226. The non-intergeneric bacterial population according to clause 200, wherein the at least one genetic variation is a mutated nifL gene that has been altered to comprise a heterologous promoter inserted into said nifL gene and a mutated glnE gene that results in a truncated GlnE protein lacking an adenylyl-removing (AR) domain.
- 227. The non-intergeneric bacterial population according to clause 200, wherein the at least one genetic variation is a mutated nifL gene that has been altered to comprise a heterologous promoter inserted into said nifL gene and a mutated glnE gene that results in a truncated GlnE protein lacking an adenylyl-removing (AR) domain and a mutated amtB gene that results in the lack of expression of said amtB gene.
- 228. The non-intergeneric bacterial population according to clause 200, wherein the plant comprises the seed, stalk, flower, fruit, leaves, or rhizome.
- 229. The non-intergeneric bacterial population according to clause 200, wherein the plurality of non-intergeneric bacteria are formulated into a composition.
- 230. The non-intergeneric bacterial population according to clause 200, wherein the plurality of non-intergeneric bacteria are formulated into a composition comprising an agriculturally acceptable carrier.
- 231. The non-intergeneric bacterial population according to clause 200, wherein the plurality of non-intergeneric bacteria are formulated into a liquid in-furrow composition comprising bacteria at a concentration of about 1×107 to about 1×1010 cfu per milliliter.
- 232. The non-intergeneric bacterial population according to clause 200, wherein the plurality of non-intergeneric bacteria are formulated into a seed coating.
- 233. The non-intergeneric bacterial population according to clause 200, wherein the plant is a cereal crop.
- 234. The non-intergeneric bacterial population according to clause 200, wherein the plant is selected from the group consisting of: corn, rice, wheat, barley, sorghum, millet, oat, rye, and triticale.
- 235. The non-intergeneric bacterial population according to clause 200, wherein the plant is corn.
- 236. The non-intergeneric bacterial population according to clause 200, wherein the plant is an agricultural crop plant.
- 237. The non-intergeneric bacterial population according to clause 200, wherein the plant is a genetically modified organism.
- 238. The non-intergeneric bacterial population according to clause 200, wherein the plant is not a genetically modified organism.
- 239. The non-intergeneric bacterial population according to clause 200, wherein the plant has been genetically engineered or bred for efficient nitrogen use.
- 240. The non-intergeneric bacterial population according to clause 200, wherein the plurality of non-intergeneric bacteria comprise at least two different species of bacteria.
- 241. The non-intergeneric bacterial population according to clause 200, wherein the plurality of non-intergeneric bacteria comprise at least two different strains of the same species of bacteria.
- 242. The non-intergeneric bacterial population according to clause 200, wherein the plurality of non-intergeneric bacteria comprise bacteria selected from: Rahnella aquatilis, Klebsiella variicola, Achromobacter spiritinus, Achromobacter marplatensis, Microbacterium morale, Kluyvera intermedia, Kosakonia pseudosacchari, Enterobacter sp., Azospirillum lipoferum, Kosakonia sacchari, and combinations thereof.
- 243. The non-intergeneric bacterial population according to clause 200, wherein the plurality of non-intergeneric bacteria are endophytic, epiphytic, or rhizospheric.
- 244. The non-intergeneric bacterial population according to clause 200, wherein the plurality of non-intergeneric bacteria comprise bacteria selected from: a bacteria deposited as PTA-122293, a bacteria deposited as PTA-122294, a bacteria deposited as NCMA 201701002, a bacteria deposited as NCMA 201708004, a bacteria deposited as NCMA 201708003, a bacteria deposited as NCMA 201708002, a bacteria deposited as NCMA 201712001, a bacteria deposited as NCMA 201712002, and combinations thereof
- 245. A bacterial population capable of increasing nitrogen fixation in a non-leguminous plant, comprising:
- a. a plurality of bacteria, that:
- i. have an average colonization ability per unit of plant root tissue of at least about 1.0×104 bacterial cells per gram of fresh weight of plant root tissue; and/or
- ii. produce fixed N of at least about 1×10−17 mmol N per bacterial cell per hour, and
- wherein the plurality of bacteria, in planta, produce 1% or more of the fixed nitrogen in die plant.
- 246. The bacterial population according to clause 245, wherein the plurality of bacteria comprise bacteria that: have an average colonization ability per unit of plant root tissue of at least about 1.0×104 bacterial cells per gram of fresh weight of plant root tissue.
- 247. The bacterial population according to clause 245, wherein the plurality of bacteria comprise bacteria that: produce fixed N of at least about 245×10−17 mmol N per bacterial cell per hour.
- 248. The bacterial population according to clause 245, wherein the plurality of bacteria comprise bacteria that: have an average colonization ability per unit of plant root tissue of at least about 1.0×104 bacterial cells per gram of fresh weight of plant root tissue and produce fixed N of at least about 1×10−17 mmol N per bacterial cell per hour.
- 249. The bacterial population according to clause 245, wherein the plurality of bacteria comprise bacteria that: have an average colonization ability per unit of plant root tissue of at least about 1.0×104 bacterial cells per gram of fresh weight of plant root tissue and produce fixed N of at least about 1×10−17 mmol N per bacterial cell per hour, and wherein the product of (i) the average colonization ability per unit of plant root tissue and (ii) produced fixed N per bacterial cell per hour, is at least about 2.5×mmol N per gram of fresh weight of plant root tissue per hour.
- 250. The bacterial population according to clause 245, wherein the plurality of bacteria, in planta, excrete the nitrogen-containing products of nitrogen fixation.
- 251. The bacterial population according to clause 245, wherein the plurality of bacteria applied to the plant do not stimulate an increase in the uptake of exogenous non-atmospheric nitrogen.
- 252. The bacterial population according to clause 245, wherein the plant comprises the seed, stalk, flower, fruit, leaves, or rhizome.
- 253. The bacterial population according to clause 245, wherein the plurality of bacteria are formulated into a composition.
- 254. The bacterial population according to clause 245, wherein the plurality of bacteria are formulated into a composition comprising an agriculturally acceptable carrier.
- 255. The bacterial population according to clause 245, wherein the plurality of bacteria are applied into furrows in which seeds of said plant are planted.
- 256. The bacterial population according to clause 245, wherein the plurality of bacteria are formulated into a liquid in-furrow composition comprising bacteria at a concentration of about 1×107 to about 1×1010 cfu per milliliter.
- 257. The bacterial population according to clause 245, wherein the plurality of bacteria are applied onto a seed of said plant.
- 258. The bacterial population according to clause 245, wherein the plurality of bacteria are formulated into a seed coating and are applied onto a seed of said plant.
- 259. The bacterial population according to clause 245, wherein the plurality of bacteria are formulated into a seed coating and are applied onto a seed of said plant, at a concentration of about 1×105 to about 1×107 cfu per seed.
- 260. The bacterial population according to clause 245, wherein the plant is a cereal crop.
- 261. The bacterial population according to clause 245, wherein the plant is selected from the group consisting of: corn, rice, wheat, barley, sorghum, millet, oat, rye, and triticale.
- 262. The bacterial population according to clause 245, wherein the plant is corn.
- 263. The bacterial population according to clause 245, wherein the plant is an agricultural crop plant.
- 264. The bacterial population according to clause 245, wherein the plant is a genetically modified organism.
- 265. The bacterial population according to clause 245, wherein the plant is not a genetically modified organism.
- 266. The bacterial population according to clause 245, wherein the plant has been genetically engineered or bred for efficient nitrogen use.
- 267. The bacterial population according to clause 245, wherein the plurality of bacteria comprise at least two different species of bacteria.
- 268. The bacterial population according to clause 245, wherein the plurality of bacteria comprise at least two different strains of the same species of bacteria.
- 269. The bacterial population according to clause 245, wherein the plurality of bacteria comprise bacteria selected from: Rahnella aquatilis, Klebsiella variicola, Achromobacter spiritinus, Achromobacter marplatensis, Microbacterium murale, Kluyvera intermedia, Kosakonia pseudosacchari, Enterobacter sp., Azospirillum lipoferum, Kosakonia sacchari, and combinations thereof.
- 270. The bacterial population according to clause 245, wherein the plurality of bacteria are endophytic, epiphytic, or rhizospheric.
- 271. The bacterial population according to clause 245, wherein the plurality of bacteria are selected from: a bacteria deposited as NCMA 201701003, a bacteria deposited as NCMA 201701001, and a bacteria deposited as NCMA 201708001.
- 272. A bacterium that:
- i. has an average colonization ability per unit of plant root tissue of at least about 1.0×104 bacterial cells per gram of fresh weight of plant root tissue; and/or
- ii. produces fixed N of at least about 1×10−17 mmol N per bacterial cell per hour.
- 273. A non-intergeneric bacterium, comprising: at least one genetic variation introduced into at least one gene, or non-coding polynucleotide, of the nitrogen fixation or assimilation genetic regulatory network, such that the non-intergeneric bacterium is capable of fixing atmospheric nitrogen in the presence of exogenous nitrogen, and wherein said bacterium:
- i. has an average colonization ability per unit of plant root tissue of at least about 1.0×104 bacterial cells per gram of fresh weight of plant root tissue; and/or
- ii. produces fixed N of at least about 1×10−17 mmol N per bacterial cell per hour.
- 274. The non-intergeneric bacterium according to clause 273, wherein the bacterium has an average colonization ability per unit of plant root tissue of at least about 1.0×104 bacterial cells per gram of fresh weight of plant root tissue.
- 275. The non-intergeneric bacterium according to clause 273, wherein the bacterium produces fixed N of at least about 1×10−17 mmol N per bacterial cell per hour.
- 276. The non-intergeneric bacterium according to clause 273, wherein the bacterium has an average colonization ability per unit of plant root tissue of at least about 1.0×104 bacterial cells per gram of fresh weight of plant root tissue and produces fixed N of at least about 1×10−17 mmol N per bacterial cell per hour.
- 277. The non-intergeneric bacterium according to clause 273, wherein the bacterium has an average colonization ability per unit of plant root tissue of at least about 1.0×104 bacterial cells per grain of fresh weight of plant root tissue and produces fixed N of at least about 1×10−17 mmol N per bacterial cell per hour, and wherein the product of (i) the average colonization ability per unit of plant root tissue and (ii) produced fixed N per bacterial cell per hour, is at least about 2.5×10−8 mmol N per gram of fresh weight of plant root tissue per hour.
- 278. The non-intergeneric bacterium according to clause 273, wherein the at least one genetic variation comprises an introduced control sequence operably linked to the at least one gene of the nitrogen fixation or assimilation genetic regulatory network.
- 279. The non-intergeneric bacterium according to clause 273, wherein the at least one genetic variation comprises a promoter operably linked to the at least one gene of the nitrogen fixation or assimilation genetic regulatory network.
- 280. The non-intergeneric bacterium according to clause 273, wherein the at least one genetic variation comprises an inducible promoter operably linked to the at least one gene of the nitrogen fixation or assimilation genetic regulatory network.
- 281. The non-intergeneric bacterium according to clause 273, wherein the bacterium does not comprise a constitutive promoter operably linked to a gene of the nitrogen fixation or assimilation genetic regulatory network.
- 282. The non-intergeneric bacterium according to clause 273, wherein the bacterium does not comprise a constitutive promoter operably linked to a gene in the nif gene cluster.
- 283. The non-intergeneric bacterium according to clause 273, wherein the bacterium, in planta, excretes the nitrogen-containing products of nitrogen fixation.
- 284. The non-intergeneric bacterium according to clause 273, wherein the at least one gene, or non-coding polynucleotide, of the nitrogen fixation or assimilation genetic regulatory network are selected from the group consisting of: nifA, nifL, ntrB, ntrC, polynucleotide encoding glutamine synthetase, glnA, glnB, glnK, drat, amtB, polynucleotide encoding glutaminase, glnD, glnE, nifJ, nifH, nifD, nifK, nifY, nifE, nifN, nifU, nifS, nifV, nifW, nifZ, nifM, nifF, nifB, nifQ, and a gene associated with biosynthesis of a nitrogenase enzyme.
- 285. The non-intergeneric bacterium according to clause 273, wherein the at least one genetic variation is a mutation that results in one or more of: increased expression or activity of NifA or glutaminase; decreased expression or activity of NifL, NtrB, glutamine synthetase, GlnB, GlnK, DraT, AmtB; decreased adenylyl-removing activity of GlnE; or decreased uridylyl-removing activity of GlnD.
- 286. The non-intergeneric bacterium according to clause 273, wherein the at least one genetic variation is selected from: (A) a knock-out mutation; (B) alters or abolishes a regulatory sequence of a target gene; (C) comprises the insertion of a heterologous regulatory sequence; or (D) a domain deletion.
- 287. The non-intergeneric bacterium according to clause 273, wherein the at least one genetic variation is a mutated MIL gene that has been altered to comprise a heterologous promoter inserted into said nifL gene.
- 288. The non-intergeneric bacterium according to clause 273, wherein the at least one genetic variation is a mutated glnE gene that results in a truncated GlnE protein lacking an adenylyl-removing (AR) domain.
- 289. The non-intergeneric bacterium according to clause 273, wherein the at least one genetic variation is a mutated amtB gene that results in the lack of expression of said amtB gene.
- 290. The non-intergeneric bacterium according to clause 273, wherein the at least one genetic variation is selected from: a mutated nifL gene that has been altered to comprise a heterologous promoter inserted into said nifL gene; a mutated glnE gene that results in a truncated GlnE protein lacking an adenylyl-removing (AR) domain; a mutated amtB gene that results in the lack of expression of said amtB gene; and combinations thereof.
- 291. The non-intergeneric bacterium according to clause 273, wherein the at least one genetic variation is a mutated nifL gene that has been altered to comprise a heterologous promoter inserted into said nifL gene and a mutated glnE gene that results in a truncated GlnE protein lacking an adenylyl-removing (AR) domain.
- 292. The non-intergeneric bacterium according to clause 273, wherein the at least one genetic variation is a mutated nifL gene that has been altered to comprise a heterologous promoter inserted into said tuft gene and a mutated glnE gene that results in a truncated GlnE protein lacking an adenylyl-removing (AR) domain and a mutated amtB gene that results in the lack of expression of said amtB gene.
- 293. The non-intergeneric bacterium according to clause 273, formulated into a composition.
- 294. The non-intergeneric bacterium according to clause 273, formulated into a composition comprising an agriculturally acceptable carrier.
- 295. The non-intergeneric bacterium according to clause 273, formulated into a liquid in-furrow composition.
- 296. The non-intergeneric bacterium according to clause 273, formulated into a seed coating.
- 297. The non-intergeneric bacterium according to clause 273, wherein said bacterium is selected from: Rahnella aquatilis, Klebsiella variicola, Achromobacter spiritinus, Achromobacter marplatensis, Microbacterium murale, Kluyvera intermedia, Kosakonia pseudosacchari, Enterobacter sp., Azospirillum lipoferum, Kosakonia sacchari, and combinations thereof.
- 298. The non-intergeneric bacterium according to clause 273, wherein said bacterium is endophytic, epiphytic, or rhizospheric.
- 299. The non-intergeneric bacterium according to clause 273, wherein said bacterium is selected from: a bacteria deposited as PTA-122293, a bacteria deposited as PTA-122294, a bacteria deposited as NCMA 201701002, a bacteria deposited as NCMA 201708004, a bacteria deposited as NCMA 201708003, a bacteria deposited as NCMA 201708002, a bacteria deposited as NCMA 201712001, a bacteria deposited as NCMA 201712002, and combinations thereof.
- 300. A method of increasing nitrogen fixation in a plant, comprising administering to the plant an effective amount of a composition comprising:
- i. a purified population of bacteria that comprises bacteria with a 16S nucleic acid sequence that is at least about 97% identical to a nucleic acid sequence selected from SEQ ID NOs: 85, 96, 111, 121, 122, 123, 124, 136, 149, 157, 167, 261, 262, 269, 277-283;
- ii. a purified population of bacteria that comprises bacteria with a nucleic acid sequence that is at least about 90% identical to a nucleic acid sequence selected from SEQ ID NOs: 86-95, 97-110, 112-120, 125-135, 137-148, 150-156, 158-166, 168-176, 263-268, 270-274, 275, 276, 284-295; and/or
- iii. a purified population of bacteria that comprises bacteria with a nucleic acid sequence that is at least about 90% identical to a nucleic acid sequence selected from SEQ ID NOs: 177-260, 296-303; and
- wherein the plant administered the effective amount of the composition exhibits an increase in nitrogen fixation, as compared to a plant not having been administered the composition.
- 301. The method of clause 300, wherein the composition comprises: a purified population of bacteria that comprises bacteria with a 16S nucleic acid sequence that is at least about 97% identical to a nucleic acid sequence selected from SEQ ID NOs: 85, 96, 111, 121, 122, 123, 124, 136, 149, 157, 167, 261, 262, 269, and 277-283.
- 302. The method of clause 300, wherein the composition comprises: a purified population of bacteria that comprises bacteria with a 16S nucleic acid sequence that is at least about 99% identical to a nucleic acid sequence selected from SEQ ID NOs: 85, 96, 111, 121, 122, 123, 124, 136, 149, 157, 167, 261, 262, 269, and 277-283.
- 303. The method of clause 300, wherein the composition comprises: a purified population of bacteria that comprises bacteria with a 165 nucleic acid sequence selected from SEQ ID NOs: 85, 96, 111, 121, 122, 123, 124, 136, 149, 157, 167, 261, 262, 269, and 277-283.
- 304. The method of clause 300, wherein the composition comprises: a purified population of bacteria that comprises bacteria with a nucleic acid sequence that is at least about 90% identical to a nucleic acid sequence selected from SEQ ID NOs: 86-95, 97-110, 112-120, 125-135, 137-148, 150-156, 158-166, 168-176, 263-268, 270-274, 275, 276, and 284-295.
- 305. The method of clause 300, wherein the composition comprises: a purified population of bacteria that comprises bacteria with a nucleic acid sequence that is at least about 95% identical to a nucleic acid sequence selected from SEQ ID NOs: 86-95, 97-110, 112-120, 125-135, 137-148, 150-156, 158-166, 168-176, 263-268, 270-274, 275, 276, and 284-295.
- 306. The method of clause 300, wherein the composition comprises: a purified population of bacteria that comprises bacteria with a nucleic acid sequence that is at least about 99% identical to a nucleic acid sequence selected from SEQ ID NOs: 86-95, 97-110, 112-120, 125-135, 137-148, 150-156, 158-166, 168-176, 263-268, 270-274, 275, 276, and 284-295.
- 307. The method of clause 300, wherein the composition comprises: a purified population of bacteria that comprises bacteria with a nucleic acid sequence selected from SEQ ID NOs: 86-95, 97-110, 112-120, 125-135, 137-148, 150-156, 158-166, 168-176, 263-268, 270-274, 275, 276, and 284-295.
- 308. The method of clause 300, wherein the composition comprises: a purified population of bacteria that comprises bacteria with a nucleic acid sequence that is at least about 90% identical to a nucleic acid sequence selected from SEQ ID NOs: 177-260 and 296-303.
- 309. The method of clause 300, wherein the composition comprises: a purified population of bacteria that comprises bacteria with a nucleic acid sequence that is at least about 95% identical to a nucleic acid sequence selected from SEQ ID NOs: 177-260 and 296-303.
- 310. The method of clause 300, wherein the composition comprises: a purified population of bacteria that comprises bacteria with a nucleic acid sequence that is at least about 99% identical to a nucleic acid sequence selected from SEQ ID NOs: 177-260 and 296-303.
- 311. The method of clause 300, wherein the composition comprises: a purified population of bacteria that comprises bacteria with a nucleic acid sequence selected from SEQ ID NOs: 177-260 and 296-303.
- 312. The method of clause 300, wherein the composition comprises: a purified population of bacteria that comprise bacteria with at least one genetic variation introduced into a gene selected from the group consisting of nifA, nifL, ntrB, ntrC, polynucleotide encoding glutamine synthetase, glnA, glnB, glnK, drat, amtB, polynucleotide encoding glutaminase, glnD, glnE, nifJ, nifH, nifH, nifD, nifY, nifE, nifN, nifU, nifS, nifV, nifW, nifZ, nifM, nifB, nifQ, and a gene associated with biosynthesis of a nitrogenase enzyme.
- 313. The method of clause 300, wherein the composition comprises: a purified population of bacteria that comprise bacteria with at least one genetic variation introduced into a gene selected from the group consisting of nifA, nifL, ntrB, ntrC, polynucleotide encoding glutamine synthetase, glnA, glnB, glnK, drat, amtB, polynucleotide encoding glutaminase, glnD, glnE, nifJ, nifH, nifD, nifK, nifE, nifN, nifU, nifS, nifV, nifW, nifZ, nifM, nifF, nifB, nifQ, and a gene associated with biosynthesis of a nitrogenase enzyme, wherein the genetic variation (A) is a knock-out mutation; (B) alters or abolishes a regulatory sequence of a target gene; (C) comprises the insertion of a heterologous regulatory sequence; or (D) a domain deletion.
- 314. The method of clause 300, wherein the composition comprises: a purified population of bacteria that comprise bacteria with at least one mutation that results in one or more of: increased expression or activity of NifA or glutaminase; decreased expression or activity of NifL, NtrB, glutamine synthetase, GlnB, GlnK, DraT, or AmtB; decreased adenylyl-removing activity of GlnE; or decreased uridylyl-removing activity of GlnD.
- 315. The method of clause 300, wherein the composition comprises: a purified population of bacteria that comprise bacteria with a mutated MIL gene that has been altered to comprise a heterologous promoter inserted into said nifL gene.
- 316. The method of clause 300, wherein the composition comprises: a purified population of bacteria that comprise bacteria with a mutated glnE gene that results in a truncated GlnE protein lacking an adenylyl-removing (AR) domain.
- 317. The method of clause 300, wherein the composition comprises: a purified population of bacteria that comprise bacteria with a mutated amtB gene that results in the lack of expression of said amtB gene.
- 318. The method of clause 300, wherein the composition comprises: a purified population of bacteria that comprise bacteria with at least one of the following genetic alterations: a mutated nifL gene that has been altered to comprise a heterologous promoter inserted into said nifL gene; a mutated glnE gene that results in a truncated GlnE protein lacking an adenylyl-removing (AR) domain; a mutated amtB gene that results in the lack of expression of said amtB gene; and combinations thereof.
- 319. The method of clause 300, wherein the composition comprises: a purified population of bacteria that comprise bacteria with a mutated nifL gene that has been altered to comprise a heterologous promoter inserted into said nifL gene and a mutated glnE gene that results in a truncated GlnE protein lacking an adenylyl-removing (AR) domain.
- 320. The method of clause 300, wherein the composition comprises: a purified population of bacteria that comprise bacteria with a mutated nifL gene that has been altered to comprise a heterologous promoter inserted into said nifL gene and a mutated glnE gene that results in a truncated GlnE protein lacking an adenylyl-removing (AR) domain and a mutated am/B gene that results in the lack of expression of said amtB gene.
- 321. The method of clause 300, wherein the composition comprises: a purified population of bacteria that comprise bacteria with a nifL modification that expresses a Nil protein at less than about 50% of a bacteria lacking the nifL modification.
- 322. The method of clause 300, wherein the composition comprises: a purified population of bacteria that comprise bacteria with a nifL modification that expresses a Nil protein at less than about 50% of a bacteria lacking the nifL modification, wherein the modification is a disruption, knockout, or truncation.
- 323. The method of clause 300, wherein the composition comprises: a purified population of bacteria that comprise bacteria with a raft, modification that expresses a Nil protein at less than about 50% of a bacteria lacking the nifL modification, and wherein the bacteria further comprise a promoter operably linked to a nifA sequence.
- 324. The method of clause 300, wherein the composition comprises: a purified population of bacteria that comprise bacteria with a nifL modification that expresses a Nil protein at less than about 50% of a bacteria lacking the nifL modification, and wherein the bacteria lack a nifL homolog.
- 325. The method of clause 300, wherein the composition comprises: a purified population of bacteria that comprise bacteria with a glnE modification that expresses a GlnE protein at less than about 50% of a bacteria lacking the glnE modification.
- 326. The method of clause 300, wherein the composition comprises: a purified population of bacteria that comprise bacteria with a glnE modification that expresses a GlnE protein at less than about 50% of a bacteria lacking the glnE modification, wherein the modification is a disruption, knockout, or a truncation.
- 327. The method of clause 300, wherein the composition comprises: a purified population of bacteria that comprise bacteria with a glnE modification that expresses a GlnE protein at less than about 50% of a bacteria lacking the glnE modification, and wherein the GlnE protein sequence lacks an adenylyl removing (AR) domain.
- 328. The method of clause 300, wherein the composition comprises: a purified population of bacteria that comprise bacteria with a glnE modification that expresses a GlnE protein at less than about 50% of a bacteria lacking the glnE modification, and wherein the GlnE protein sequence lacks adenylyl removing (AR) activity.
- 329. The method of clause 300, wherein the purified population of bacteria, in planta, provide at least 1% of fixed nitrogen to the plant.
- 330. The method of clause 300, wherein the purified population of bacteria, in planta, provide at least 5% of fixed nitrogen to the plant.
- 331. The method of clause 300, wherein the purified population of bacteria, in planta, provide at least 10% of fixed nitrogen to the plant.
- 332. The method of clause 300, wherein the purified population of bacteria are capable of fixing atmospheric nitrogen in non-nitrogen-limiting conditions.
- 333. The method of clause 300, wherein the purified population of bacteria, in planta, excrete nitrogen-containing products of nitrogen fixation.
- 334. The method of clause 300, wherein the purified population of bacteria, in planta, provide at least 1% of fixed nitrogen to the plant, and wherein said fixed nitrogen is measured through dilution of enriched fertilizer by atmospheric N2 gas in plant tissue.
- 335. The method of clause 300, wherein the purified population of bacteria colonize a root of said plant and are present in an amount of at least about 1.0×104 bacterial cells per gram of fresh weight of plant root tissue.
- 336. The method of clause 300, wherein the plant comprises the seed, stalk, flower, fruit, leaves, or rhizome.
- 337. The method of clause 300, wherein the composition comprises: an agriculturally acceptable carrier.
- 338. The method of clause 300, wherein the composition comprising the purified population of bacteria is administered into furrows in which seeds of said plant are planted.
- 339. The method of clause 300, wherein the composition comprising the purified population of bacteria is formulated as a liquid in-furrow composition comprising bacteria at a concentration of about 1×107 to about 1×1010 du per milliliter.
- 340. The method of clause 300, wherein the composition comprising the purified population of bacteria is administered onto a seed of said plant.
- 341. The method of clause 300, wherein the composition comprising the purified population of bacteria is formulated as a seed coating and is administered onto a seed of said plant.
- 342. The method of clause 300, wherein the composition comprising the purified population of bacteria is formulated as a seed coating and is administered onto a seed of said plant, at a concentration of about 1×105 to about 1×107 cfu per seed.
- 343. The method of clause 300, wherein the plant is non-leguminous.
- 344. The method of clause 300, wherein the plant is a cereal crop.
- 345. The method of clause 300, wherein the plant is selected from the group consisting of: corn, rice, wheat, barley, sorghum, millet, oat, rye, and triticale.
- 346. The method of clause 300, wherein the plant is corn.
- 347. The method of clause 300, wherein the plant is a legume.
- 348. The method of clause 300, wherein the plant is a grain crop.
- 349. The method of clause 300, wherein the purified population of bacteria comprise bacteria selected from: Rahnella aquatilis, Klebsiella variicola, Achromobacter spiritinus, Achromobacter marplatensis, Microbacterium murale, Kluyvera intermedia, Kosakonia pseudosacchari, Enterobacter sp., Azospirillum lipoferum, Kosakonia sacchari, and combinations thereof.
- 350. The method of clause 300, wherein the purified population of bacteria comprise a bacteria of genus Rahnella.
- 351. The method of clause 300, wherein the purified population of bacteria comprise a bacteria of species Rahnella aquatilis.
- 352. The method of clause 300, wherein the purified population of bacteria comprise a bacteria deposited as PTA-122293.
- 353. The method of clause 300, wherein the purified population of bacteria comprise a bacteria deposited as NCMA 201701003.
- 354. The method of clause 300, wherein the purified population of bacteria comprise a bacteria of genus Kosakonia.
- 355. The method of clause 300, wherein the purified population of bacteria comprise a bacteria of species Kosakonia sacchari.
- 356. The method of clause 300, wherein the purified population of bacteria comprise a bacteria deposited as PTA-122294.
- 357. The method of clause 300, wherein the purified population of bacteria comprise a bacteria deposited as NCMA 201701001.
- 358. The method of clause 300, wherein the purified population of bacteria comprise a bacteria deposited as NCMA 201701002.
- 359. The method of clause 300, wherein the purified population of bacteria comprise a bacteria deposited as NCMA 201708004.
- 360. The method of clause 300, wherein the purified population of bacteria comprise a bacteria deposited as NCMA 201708003.
- 361. The method of clause 300, wherein the purified population of bacteria comprise a bacteria deposited as NCMA 201708002.
- 362. The method of clause 300, wherein the purified population of bacteria comprise a bacteria of genus Klebsiella
- 363. The method of clause 300, wherein the purified population of bacteria comprise a bacteria of species Klebsiella variicola.
- 364. The method of clause 300, wherein the purified population of bacteria comprise a bacteria deposited as NCMA 201708001.
- 365. The method of clause 300, wherein the purified population of bacteria comprise a bacteria deposited as NCMA 201712001.
- 366. The method of clause 300, wherein the purified population of bacteria comprise a bacteria deposited as NCMA 201712002.
- 367. An isolated bacteria, comprising:
- i. a 16S nucleic acid sequence that is at least about 97% identical to a nucleic acid sequence selected from SEQ ID NOs: 85, 96, 111, 121, 122, 123, 124, 136, 149, 157, 167, 261, 262, 269, 277-283;
- ii. a nucleic acid sequence that is at least about 90% identical to a nucleic acid sequence selected from SEQ ID NOs: 86-95, 97-110, 112-120, 125-135, 137-148, 150-156, 158-166, 168-176, 263-268, 270-274, 275, 276, 284-295; and/or
- iii. a nucleic acid sequence that is at least about 90% identical to a nucleic acid sequence selected from SEQ ID NOs: 177-260, 296-303.
- 368. The isolated bacteria of clause 367, comprising: a 1.65 nucleic acid sequence that is at least about 97% identical to a nucleic acid sequence selected from SEQ ID NOs: 85, 96, 111, 121, 122, 123, 124, 136, 149, 157, 167, 261, 262, 269, and 277-283.
- 369. The isolated bacteria of clause 367, comprising: a 16S nucleic acid sequence that is at least about 99% identical to a nucleic acid sequence selected from SEQ ID NOs: 85, 96, 111, 121, 122, 123, 124, 136, 149, 157, 167, 261, 262, 269, and 277-283.
- 370. The isolated bacteria of clause 367, comprising: a 16S nucleic acid sequence selected from SEQ ID NOs: 85, 96, 111, 121, 122, 123, 124, 136, 149, 157, 167, 261, 262, 269, and 277-283.
- 371. The isolated bacteria of clause 367, comprising: a nucleic acid sequence that is at least about 90% identical to a nucleic acid sequence selected from SEQ ID NOs: 86-95, 97-110, 112-120, 125-135, 137-148, 150-156, 158-166, 168-176, 263-268, 270-274, 275, 276, and 284-295.
- 372. The isolated bacteria of clause 367, comprising: a nucleic acid sequence that is at least about 95% identical to a nucleic acid sequence selected from SEQ ID NOs: 86-95, 97-110, 112-120, 125-135, 137-148, 150-156, 158-166, 168-176, 263-268, 270-274, 275, 276, and 284-295.
- 373. The isolated bacteria of clause 367, comprising: a nucleic acid sequence that is at least about 99% identical to a nucleic acid sequence selected from SEQ ID NOs: 86-95, 97-110, 112-120, 125-135, 137-148, 150-156, 158-166, 168-176, 263-268, 270-274, 275, 276, and 284-295.
- 374. The isolated bacteria of clause 367, comprising: a nucleic acid sequence selected from SEQ ID NOs: 86-95, 97-110, 112-120, 125-135, 137-148, 150-156, 158-166, 168-176, 263-268, 270-274, 275, 276, and 284-295.
- 375. The isolated bacteria of clause 367, comprising: a nucleic acid sequence that is at least about 90% identical to a nucleic acid sequence selected from SEQ ID NOs: 177-260 and 296-303.
- 376. The isolated bacteria of clause 367, comprising: a nucleic acid sequence that is at least about 95% identical to a nucleic acid sequence selected from SEQ ID NOs: 177-260 and 296-303.
- 377. The isolated bacteria of clause 367, comprising: a nucleic acid sequence that is at least about 99% identical to a nucleic acid sequence selected from SEQ ID NOs: 177-260 and 296-303.
- 378. The isolated bacteria of clause 367, comprising: a nucleic acid sequence selected from SEQ ID NOs: 177-260 and 296-303.
- 379. The isolated bacteria of clause 367, comprising: at least one genetic variation introduced into a gene selected from the group consisting of nifA, nifL, ntrB, ntrC, polynucleotide encoding glutamine synthetase, glnA, glnB, glnK, drat, amtB, polynucleotide encoding glutaminase, glnD, glnE, nifJ, nifH, nifD, nifK, nifY, nifE, nifN, nifU, nifS, nifV, nifW, nifW, nifZ, nifM, nifF, nifB, nifQ, and a gene associated with biosynthesis of a nitrogenase enzyme.
- 380. The isolated bacteria of clause 367, comprising: at least one genetic variation introduced into a gene selected from the group consisting of nifA, nifL, ntrB, polynucleotide encoding glutamine synthetase, glnA, glnB, glnK, drat, amtB, polynucleotide encoding glutaminase, glnD, glnE, nifJ, nifH, nifD, nifK, nifY, nifE, nifN, nifU, nifS, nifV, nifW, nifZ, nifM, nifF, nifB, nifQ, and a gene associated with biosynthesis of a nitrogenase enzyme, wherein the genetic variation (A) is a knock-out mutation; (B) alters or abolishes a regulatory sequence of a target gene; (C) comprises the insertion of a heterologous regulatory sequence; or (D) a domain deletion.
- 381. The isolated bacteria of clause 367, comprising: at least one mutation that results in one or more of: increased expression or activity of NifA or glutaminase; decreased expression or activity of NifL, NtrB, glutamine synthetase, GlnB, GlnK, DraT, or AmtB; decreased adenylyl-removing activity of GlnE; or decreased uridylyl-removing activity of GlnD.
- 382. The isolated bacteria of clause 367, comprising: a mutated nifL gene that has been altered to comprise a heterologous promoter inserted into said nifL gene.
- 383. The isolated bacteria of clause 367, comprising: a mutated glnE gene that results in a truncated GlnE protein lacking an adenylyl-removing (AR) domain.
- 384. The isolated bacteria of clause 367, comprising: a mutated amtB gene that results in the lack of expression of said amtB gene.
- 385. The isolated bacteria of clause 367, comprising: at least one of the following genetic alterations: a mutated nifL gene that has been altered to comprise a heterologous promoter inserted into said MIL gene; a mutated glnE gene that results in a truncated GlnE protein lacking an adenylyl-removing (AR) domain; a mutated amtB gene that results in the lack of expression of said amtB gene; and combinations thereof.
- 386. The isolated bacteria of clause 367, comprising: a mutated nifL gene that has been altered to comprise a heterologous promoter inserted into said nifL gene and a mutated glnE gene that results in a truncated GlnE protein lacking an adenylyl-removing (AR) domain.
- 387. The isolated bacteria of clause 367, comprising: a mutated nifL gene that has been altered to comprise a heterologous promoter inserted into said nifL gene and a mutated glnE gene that results in a truncated GlnE protein lacking an adenylyl-removing (AR) domain and a mutated amtB gene that results in the lack of expression of said amtB gene.
- 388. The isolated bacteria of clause 367, comprising: a nifL modification that expresses a NifL protein at less than about 50% of a bacteria lacking the nifL modification.
- 389. The isolated bacteria of clause 367, comprising: a MIL modification that expresses a nifL protein at less than about 50% of a bacteria lacking the nifL modification, wherein the modification is a disruption, knockout, or truncation.
- 390. The isolated bacteria of clause 367, comprising: a nifL modification that expresses a NifL protein at less than about 50% of a bacteria lacking the nifL modification, and wherein the bacteria further comprise a promoter operably linked to a nifA sequence.
- 391. The isolated bacteria of clause 367, comprising: a nifL modification that expresses a NifL protein at less than about 50% of a bacteria lacking the nifL modification, and wherein the bacteria lack a nifL homolog.
- 392. The isolated bacteria of clause 367, comprising: a glnE modification that expresses a GlnE protein at less than about 50% of a bacteria lacking the glnE modification.
- 393. The isolated bacteria of clause 367, comprising: a glnE modification that expresses a GlnE protein at less than about 50% of a bacteria lacking the glnE modification, wherein the modification is a disruption, knockout, or a truncation.
- 394. The isolated bacteria of clause 367, comprising: a glnE modification that expresses a GlnE protein at less than about 50% of a bacteria lacking the glnE modification, and wherein the GlnE protein sequence lacks an adenylyl removing (AR) domain.
- 395. The isolated bacteria of clause 367, comprising: a glnE modification that expresses a GlnE protein at less than about 50% of a bacteria lacking the glnE modification, and wherein the GlnE protein sequence lacks adenylyl removing (AR) activity.
- 396. The isolated bacteria of clause 367, wherein the bacteria, in planta, provide at least 1% of fixed nitrogen to a plant exposed to said bacteria.
- 397. The isolated bacteria of clause 367, wherein the bacteria, in planta, provide at least 5% of fixed nitrogen to a plant exposed to said bacteria.
- 398. The isolated bacteria of clause 367, wherein the bacteria, in planta, provide at least 10% of fixed nitrogen to a plant exposed to said bacteria.
- 399. The isolated bacteria of clause 367, wherein the bacteria is capable of fixing atmospheric nitrogen in non-nitrogen-limiting conditions.
- 400. The isolated bacteria of clause 367, wherein the bacteria, in planta, excretes nitrogen-containing products of nitrogen fixation.
- 401. The isolated bacteria of clause 367, wherein the bacteria, in planta, provide at least 1% of fixed nitrogen to a plant exposed to said bacteria, and wherein said fixed nitrogen is measured through dilution of enriched fertilizer by atmospheric N2 gas in plant tissue.
- 402. The isolated bacteria of clause 367, wherein the bacteria colonize a root of a plant exposed to said bacteria to a concentration of at least about 1.0×104 bacterial cells per gram of fresh weight of plant root tissue.
- 403. The isolated bacteria of clause 367, formulated into an agricultural composition.
- 404. The isolated bacteria of clause 367, formulated into an in-furrow composition.
- 405. The isolated bacteria of clause 367, formulated as a liquid in-furrow composition that comprises bacteria at a concentration of about 1×107 to about 1×1010 cfu per milliliter.
- 406. The isolated bacteria of clause 367, formulated as a seed treatment or seed coating.
- 407. The isolated bacteria of clause 367, formulated as a seed treatment or seed coating that comprises bacteria at a concentration of about 1×105 to about 1×107 cfu per seed.
- 408. The isolated bacteria of clause 367, wherein the bacteria is in contact with a plant and increases nitrogen fixation in the plant.
- 409. The isolated bacteria of clause 367, disposed on a non-leguminous plant.
- 410. The isolated bacteria of clause 367, disposed on a cereal crop.
- 411. The isolated bacteria of clause 367, disposed on a plant selected from the group consisting of: corn, rice, wheat, barley, sorghum, millet, oat, rye, and triticale.
- 412. The isolated bacteria of clause 367, disposed on corn.
- 413. The isolated bacteria of clause 367, disposed on a legume.
- 414. The isolated bacteria of clause 367, disposed on a grain crop.
- 415. The isolated bacteria of clause 367, selected from: Rahnella aquatilis, Klebsiella variicola, Achromobacter spiritinus, Achromobacter marplatensis, Microbacterium murale, Kluyvera intermedia, Kosakonia pseudosacchari, Enterobacter sp., Azospirillum lipoferum, Kosakonia sacchari, and combinations thereof.
- 416. The isolated bacteria of clause 367, wherein the bacteria is of the genus Rahnella.
- 417. The isolated bacteria of clause 367, wherein the bacteria is of the species Rahnella aquatilis.
- 418. The isolated bacteria of clause 367, deposited as PTA-122293.
- 419. The isolated bacteria of clause 367, deposited as NCMA 201701003.
- 420. The isolated bacteria of clause 367, wherein the bacteria is of the genus Kosakonia.
- 421. The isolated bacteria of clause 367, wherein the bacteria is of the species Kosakonia sacchari.
- 422. The isolated bacteria of clause 367, deposited as PTA-122294.
- 423. The isolated bacteria of clause 367, deposited as NCMA 201701001.
- 424. The isolated bacteria of clause 367, deposited as NCMA 201701002.
- 425. The isolated bacteria of clause 367, deposited as NCMA 201708004.
- 426. The isolated bacteria of clause 367, deposited as NCMA 201708003.
- 427. The isolated bacteria of clause 367, deposited as NCMA 201708002.
- 428. The isolated bacteria of clause 367, wherein the bacteria is of the genus Klebsiella.
- 429. The isolated bacteria of clause 367, wherein the bacteria is of the species Klebsiella variicola.
- 430. The isolated bacteria of clause 367, deposited as NCMA 201708001.
- 431. The isolated bacteria of clause 367, deposited as NCMA 201712001.
- 432. The isolated bacteria of clause 367, deposited as NCMA 201712002.
- 433. A composition comprising any one or more bacteria of clauses 415 to 432.
- 434. A method of detecting a non-native junction sequence, comprising: amplifying a nucleotide sequence that shares at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ED NOs: 372-405.
- 435. The method according to clause 434, wherein said amplifying is by conducting a polymerase chain reaction.
- 436. The method according to clause 434, wherein said amplifying is by conducting a quantitative polymerase chain reaction.
- 437. A method of detecting a non-native junction sequence, comprising: amplifying a nucleotide sequence that shares at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least a 10 contiguous base pair fragment contained in SEQ ID NOs: 372-405, said contiguous base pair fragment being comprised of nucleotides at the intersection of: an upstream sequence comprising SEQ ID NOs: 304-337 and downstream sequence comprising SEQ ID NOs: 338-371.
- 438. The method according to clause 437, wherein said amplifying is by conducting a polymerase chain reaction.
- 439. The method according to clause 437, wherein said amplifying is by conducting a quantitative polymerase chain reaction.
- 440. A non-native junction sequence, comprising: a nucleotide sequence that shares at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 372-405.
- 441. A non-native junction sequence, comprising: a nucleotide sequence that shares at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 1.00% sequence identity to at least a 10 contiguous base pair fragment contained in SEQ ID NOs: 372-405, said contiguous base pair fragment being comprised of nucleotides at the intersection of: an upstream sequence comprising SEQ ID NOs: 304-337 and downstream sequence comprising SEQ ID NOs: 338-371.
- 442. A bacterial composition, comprising: at least one remodeled bacterial strain that fixes atmospheric nitrogen, the at least one remodeled bacterial strain comprising exogenously added DNA wherein said exogenously added DNA shares at least 80% identity to a corresponding native bacterial strain.
- 443. A method of maintaining soil nitrogen levels, comprising:
- planting, in soil of a field, a crop inoculated by a remodeled bacterium that fixes atmospheric nitrogen; and
- harvesting said crop, wherein no more than 90% of a nitrogen dose required for producing said crop is administered to said soil of said field between planting and harvesting.
- 444. A method of delivering a probiotic supplement to a crop plant, comprising:
- coating a crop seed with a seed coating, seed treatment, or seed dressing, wherein said seed coating, seed dressing, or seed treatment comprising living representatives of said probiotic; and
- applying in soil of a field, said crop seeds.
- 445. A method of increasing the amount of atmospheric derived nitrogen in a non-leguminous plant, comprising:
- exposing said non-leguminous plant to remodeled non-intergeneric microbes, said remodeled non-intergeneric microbes comprising at least one genetic variation introduced into a nitrogen fixation genetic regulatory network or at least one genetic variation introduced into a nitrogen assimilation genetic regulatory network.
- 446. A method of increasing an amount of atmospheric derived nitrogen in a corn plant, comprising:
- exposing said corn plant to remodeled non-intergeneric microbes comprising remodeled genetic variations within at least two genes selected from the group consisting of nifL, glnB, glnE, and amtB.
- 447. A method of increasing an amount of atmospheric derived nitrogen in a corn plant, comprising:
- exposing said corn plant to remodeled non-intergeneric microbes comprising at least one genetic variation introduced into a nitrogen fixation genetic regulatory network and at least one genetic variation introduced into a nitrogen assimilation genetic regulatory network, wherein said remodeled non-intergeneric microbes, in planta, produce at least 5% of fixed nitrogen in said corn plant as measured by dilution of 15N in crops grown in fields treated with fertilizer containing 1.2% 15N.
- 448. The method of any of the previous clauses, wherein the bacterium produces fixed N of at least about 1×10−17 mmol N per bacterial cell per hour when in the presence of exogenous nitrogen.
- 449. The bacterium of any of the previous clauses, wherein the bacterium produces fixed N of at least about 1×10−17 mmol N per bacterial cell per hour when in the presence of exogenous nitrogen.
- 450. The bacterial population of any of the previous clauses, wherein the bacterium produces fixed N of at least about 1×10−17 mmol N per bacterial cell per hour when in the presence of exogenous nitrogen.
- 451. The isolated bacteria of any of the previous clauses, wherein the bacterium produces fixed N of at least about 1×10−17 mmol N per bacterial cell per hour when in the presence of exogenous nitrogen.
- 452. The non-intergeneric bacterial population of any of the previous clauses, wherein the bacterium produces fixed N of at least about 1×10−17 mmol N per bacterial cell per hour when in the presence of exogenous nitrogen.
- 453. The non-intergeneric bacterium of any of the previous clauses, wherein the bacterium produces fixed N of at least about 1×10−17 mmol N per bacterial cell per hour when in the presence of exogenous nitrogen.
- 454. The method of any of the previous clauses, wherein the plurality of bacteria, in planta, produce 5% or more of the fixed nitrogen in the plant.
- 455. The method of any of the previous clauses, wherein the plurality of non-intergeneric bacteria, in planta, produce 5% or more of the fixed nitrogen in the plant.
- 456. The bacterium of any of the previous clauses, wherein the plurality of bacteria, in plank; produce 5% or more of the fixed nitrogen in the plant.
- 457. The bacterial population of any of the previous clauses, wherein the plurality of bacteria, in planta, produce 5% or more of the fixed nitrogen in the plant.
- 458. The isolated bacteria of any of the previous clauses, wherein the plurality of bacteria, in planta, produce 5% or more of the fixed nitrogen in the plant.
- 459. The non-intergeneric bacterial population of any of the previous clauses, wherein the plurality of non-intergeneric bacteria, in planta, produce 5% or more of the fixed nitrogen in the plant.
- 460. The non-intergeneric bacterium of any of the previous clauses, wherein the plurality of non-intergeneric bacteria, in planta, produce 5% or more of the fixed nitrogen in the plant.
- 461. The method of any of the previous clauses, wherein the plurality of bacteria, in planta, produce 10% or more of the fixed nitrogen in the plant.
- 462. The method of any of the previous clauses, wherein the plurality of non-intergeneric bacteria, in planta, produce 10% or more of the fixed nitrogen in the plant.
- 463. The bacterium of any of the previous clauses, wherein the plurality of bacteria, in planta, produce 10% or more of the fixed nitrogen in the plant.
- 464. The bacterial population of any of the previous clauses, wherein the plurality of bacteria, in planta, produce 10% or more of the fixed nitrogen in the plant.
- 465. The isolated bacteria of any of the previous clauses, wherein the plurality of bacteria, in planta, produce 10% or more of the fixed nitrogen in the plant.
- 466. The non-intergeneric bacterial population of any of the previous clauses, wherein the plurality of non-intergeneric bacteria, in planta, produce 10% or more of the fixed nitrogen in the plant.
- 467. The non-intergeneric bacterium of any of the previous clauses, wherein the plurality of non-intergeneric bacteria, in planta, produce 10% or more of the fixed nitrogen in the plant.
- 468. The method of any of the previous clauses, wherein the plurality of bacteria, in planta, produce 15% or more of the fixed nitrogen in the plant.
- 469. The method of any of the previous clauses, wherein the plurality of non-intergeneric bacteria, in planta, produce 15% or more of the fixed nitrogen in the plant.
- 470. The bacterium of any of the previous clauses, wherein the plurality of bacteria, in planta, produce 15% or more of the fixed nitrogen in the plant.
- 471. The bacterial population of any of the previous clauses, wherein the plurality of bacteria, in planta, produce 15% or more of the fixed nitrogen in the plant.
- 472. The isolated bacteria of any of the previous clauses, wherein the plurality of bacteria, in planta, produce 15% or more of the fixed nitrogen in the plant.
- 473. The non-intergeneric bacterial population of any of the previous clauses, wherein the plurality of non-intergeneric bacteria, in planta, produce 15% or more of the fixed nitrogen in the plant.
- 474. The non-intergeneric bacterium of any of the previous clauses, wherein the plurality of non-intergeneric bacteria, in planta, produce 15% or more of the fixed nitrogen in the plant.
- 475. The method of any of the previous clauses, wherein the plurality of bacteria, in planta, produce 20% or more of the fixed nitrogen in the plant.
- 476. The method of any of the previous clauses, wherein the plurality of non-intergeneric bacteria, in planta, produce 20% or more of the fixed nitrogen in the plant.
- 477. The bacterium of any of the previous clauses, wherein the plurality of bacteria, in planta, produce 20% or more of the fixed nitrogen in the plant.
- 478. The bacterial population of any of the previous clauses, wherein the plurality of bacteria, in planta, produce 20% or more of the fixed nitrogen in the plant.
- 479. The isolated bacteria of any of the previous clauses, wherein the plurality of bacteria, in planta, produce 20% or more of the fixed nitrogen in the plant.
- 480. The non-intergeneric bacterial population of any of the previous clauses, wherein the plurality of non-intergeneric bacteria, in planta, produce 20% or more of the fixed nitrogen in the plant.
- 481. The non-intergeneric bacterium of any of the previous clauses, wherein the plurality of non-intergeneric bacteria, in planta, produce 20% or more of the fixed nitrogen in the plant.
- 482. The method of any of the previous clauses, wherein the product of (i) the average colonization ability per unit of plant root tissue and (ii) produced fixed N per bacterial cell per hour, is at least about 2.0×10−7 mmol N per gram of fresh weight of plant root tissue per hour.
- 483. The bacterium of any of the previous clauses, wherein the product of (i) the average colonization ability per unit of plant root tissue and (ii) produced fixed N per bacterial cell per hour, is at least about 2.0×10−7 mmol N per gram of fresh weight of plant root tissue per hour.
- 484. The bacterial population of any of the previous clauses, wherein the product of (i) the average colonization ability per unit of plant root tissue and (ii) produced fixed N per bacterial cell per hour, is at least about 2.0×10−7 mmol N per grain of fresh weight of plant root tissue per hour.
- 485. The isolated bacteria of any of the previous clauses, wherein the product of (i) the average colonization ability per unit of plant root tissue and (ii) produced fixed N per bacterial cell per hour, is at least about 2.0×10−7 mmol N per gram of fresh weight of plant root tissue per hour.
- 486. The non-intergeneric bacterial population of any of the previous clauses, wherein the product of (i) the average colonization ability per unit of plant root tissue and (ii) produced fixed N per bacterial cell per hour, is at least about 2.0×10−7 mmol N per gram of fresh weight of plant root tissue per hour.
- 487. The non-intergeneric bacterium of any of the previous clauses, wherein the product of (i) the average colonization ability per unit of plant root tissue and (ii) produced fixed N per bacterial cell per hour, is at least about 2.0×10−7 mmol N per gram of fresh weight of plant root tissue per hour.
- 488. The method of any of the previous clauses, wherein the product of (i) the average colonization ability per unit of plant root tissue and (ii) produced fixed N per bacterial cell per hour, is at least about 2.0×10−6 mmol N per gram of fresh weight of plant root tissue per hour.
- 489. The bacterium of any of the previous clauses, wherein the product of (i) the average colonization ability per unit of plant root tissue and (ii) produced fixed N per bacterial cell per hour, is at least about 2.0×10−6 mmol N per gram of fresh weight of plant root tissue per hour.
- 490. The bacterial population of any of the previous clauses, wherein the product of (i) the average colonization ability per unit of plant root tissue and (ii) produced fixed N per bacterial cell per hour, is at least about 2.0×10−6 mmol N per gram of fresh weight of plant root tissue per hour.
- 491. The isolated bacteria of any of the previous clauses, wherein the product of (i) the average colonization ability per unit of plant root tissue and (ii) produced fixed N per bacterial cell per hour, is at least about 2.0×10−6 mmol N per gram of fresh weight of plant root tissue per hour.
- 492. The non-intergeneric bacterial population of any of the previous clauses, wherein the product of (i) the average colonization ability per unit of plant root tissue and (ii) produced fixed N per bacterial cell per hour, is at least about 2.0×1016 mmol N per gram of fresh weight of plant root tissue per hour.
- 493. The non-intergeneric bacterium of any of the previous clauses, wherein the product of (i) the average colonization ability per unit of plant root tissue and (ii) produced fixed N per bacterial cell per hour, is at least about 2.0×100.6 mmol N per grain of fresh weight of plant root tissue per hour.
- 494. The method of any of the previous clauses, wherein the plant has been remodeled or bred for efficient nitrogen use.
- 495. The bacterial composition of any of the previous clauses, wherein said at least one remodeled bacterial strain comprises at least one variation in a gene or intergenic region within 10,000 bp of a gene selected from the group consisting of: nifA, ntrB, ntrC, glnA, glnB, glnK, draT, amtB, glnD, glnE, nifJ, nifH, nifD, nifK, nifY, nifE, nifN, nifU, nifS, nifV, nifW, nifZ, nifM, nifF, nifB, and nifQ.
- 496. The method of any of the previous clauses, wherein said remodeled bacterium comprises a bacterial composition of any of the preceding clauses.
- 497. The method of any of the previous clauses, wherein said remodeled bacterium consists of a bacterial composition of any of the preceding clauses.
- 498. The method of any of the previous clauses, wherein the remodeled bacterial strain is a remodeled Gram-positive bacterial strain.
- 499. The method of any of the previous clauses, wherein the remodeled Gram-positive bacterial strain has an altered expression level of a regulator of a Nif cluster.
- 500. The method of any of the previous clauses, wherein the remodeled Gram-positive bacterial strain expresses a decreased amount of a negative regulator of a Nif cluster.
- 501. The method of any of the previous clauses, wherein the remodeled bacterial strain expresses a decreased amount of GlnR.
- 502. The method of any of the previous clauses, wherein the genome of the remodeled bacterial strain encodes a polypeptide with at least 75% identity to a sequence from the Zehr lab NifH database.
- 503. The method of any of the previous clauses, wherein the genome of the remodeled bacterial strain encodes a polypeptide with at least 85% identity to a sequence from the Zehr lab NifH database.
- 504. The method of any of the previous clauses, wherein the genome of the remodeled bacterial strain encodes a polypeptide with at least 75% identity to a sequence from the Buckley lab NifH database.
- 505. The method of any of the previous clauses, wherein the genome of the remodeled bacterial strain encodes a polypeptide with at least 85% identity to a sequence from the Buckley lab NifH database.
- 506. The method of any of the previous clauses, wherein said remodeled non-intergeneric microbes comprise at least one genetic variation introduced into said nitrogen fixation genetic regulatory network.
- 507. The method of any of the previous clauses, wherein said remodeled non-intergeneric microbes comprise at least one genetic variation introduced into said nitrogen assimilation genetic regulatory network.
- 508. The method of any of the previous clauses, wherein said remodeled non-intergeneric microbes comprise at least one genetic variation introduced into said nitrogen fixation genetic regulatory network and at least one genetic variation introduced into said nitrogen assimilation genetic regulatory network.
- 509. The method of any of the previous clauses, wherein said remodeled non-intergeneric microbes are applied into furrows in which seeds of said non-leguminous plant are planted.
- 510. The method of any of the previous clauses, wherein said remodeled non-intergeneric microbes are coated onto a seed of said non-leguminous plant.
- 511. The method of any of the previous clauses, wherein said remodeled non-intergeneric microbes colonize at least a root of said non-leguminous plant such that said remodeled non-intergeneric microbes are present in said non-leguminous plant in an amount of at least 105 colony forming units per gram fresh weight of tissue.
- 512. The method of any of the previous clauses, wherein said remodeled non-intergeneric microbes are capable of fixing atmospheric nitrogen in non-nitrogen-limiting conditions.
- 513. The method of any of the previous clauses, wherein said remodeled non-intergeneric microbes, in planta, excrete nitrogen-containing products of nitrogen fixation. 514. The method of any of the previous clauses, wherein said remodeled non-intergeneric microbes, in planta, produce at least 1% of fixed nitrogen in said non-leguminous plant.
- 515. The method of any of the previous clauses, wherein said fixed nitrogen in said non-leguminous plant produced by said remodeled non-intergeneric microbes is measured by dilution of 15N in crops grown in fields treated with fertilizer containing 1.2% 15N.
- 516. The method of any of the previous clauses, wherein said remodeled non-intergeneric microbes, in planta, produce 5% or more of the fixed nitrogen in said non-leguminous plant.
- 517. The method of any of the previous clauses, wherein said non-intergeneric microbes are remodeled using at least one type of engineering selected from the group consisting of directed mutagenesis, random mutagenesis, and directed evolution.
- 518. The method of any of the previous clauses, wherein said remodeled non-intergeneric microbes, in planta, excrete nitrogen-containing products of nitrogen fixation.
- 519. The method of any of the previous clauses, wherein said remodeled non-intergeneric microbes are applied into furrows in which seeds of said corn plant are planted.
- 520. The method of any of the previous clauses, wherein said remodeled non-intergeneric microbes are coated onto a seed of said corn plant.
- 521. The method of any of the previous clauses, wherein said remodeled non-intergeneric microbes comprise at least one genetic variation introduced into a nitrogen fixation genetic regulatory network and at least one genetic variation introduced into a nitrogen assimilation gentic regulatory network.
- 522. The method of any of the previous clauses, wherein said non-intergeneric microbes comprise at least one genetic variation introduced into a nitrogen fixation genetic regulatory network and at least one genetic variation introduced into a nitrogen assimilation gentic regulatory network.
- 523. The method of any of the previous clauses, wherein said genetically engineered non-intergeneric microbes comprise at least one genetic variation introduced into a nitrogen fixation genetic regulatory network and at least one genetic variation introduced into a nitrogen assimilation gentic regulatory network.
- 524. The method of any of the previous clauses, wherein said remodeled non-intergeneric bacteria comprise at least one genetic variation introduced into a nitrogen fixation genetic regulatory network and at least one genetic variation introduced into a nitrogen assimilation gentic regulatory network.
- 525. The method of any of the previous clauses, wherein said non-intergeneric bacteria comprise at least one genetic variation introduced into a nitrogen fixation genetic regulatory network and at least one genetic variation introduced into a nitrogen assimilation gentic regulatory network.
- 526. The method of any of the previous clauses, wherein said genetically engineered non-intergeneric bacteria comprise at least one genetic variation introduced into a nitrogen fixation genetic regulatory network and at least one genetic variation introduced into a nitrogen assimilation gentic regulatory network.
- 527. The bacterium of any of the previous clauses, wherein said bacterium comprise at least one genetic variation introduced into a nitrogen fixation genetic regulatory network and at least one genetic variation introduced into a nitrogen assimilation gentic regulatory network.
- 528. The bacterial population of any of the previous clauses, wherein bacteria within said bacterial population comprise at least one genetic variation introduced into a nitrogen fixation genetic regulatory network and at least one genetic variation introduced into a nitrogen assimilation gentic regulatory network.
- 529. The isolated bacteria of any of the previous clauses, wherein said isolated bacteria comprise at least one genetic variation introduced into a nitrogen fixation genetic regulatory network and at least one genetic variation introduced into a nitrogen assimilation gentic regulatory network.
- 530. The non-intergeneric bacterial population of any of the previous clauses, wherein non-intergeneric bacteria within said non-intergeneric bacterial population comprise at least one genetic variation introduced into a nitrogen fixation genetic regulatory network and at least one genetic variation introduced into a nitrogen assimilation gentic regulatory network.
- 531. The non-intergeneric bacterium of any of the previous clauses, wherein said non-intergeneric bacterium comprises at least one genetic variation introduced into a nitrogen fixation genetic regulatory network and at least one genetic variation introduced into a nitrogen assimilation gentic regulatory network.
- 532. A composition comprising any one or more bacteria of any of the previous clauses.
While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.
