CROSS REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. patent application Ser. No. 15/783,243, filed Oct. 13, 2017, which is a continuation of U.S. patent application Ser. No. 14/806,175, filed Jul. 22, 2015, which is a continuation of U.S. patent application Ser. No. 14/091,572, filed Nov. 27, 2013, which is a continuation of International Application No. PCT/US2012/040599, filed Jun. 1, 2012, which claims priority to U.S. Provisional application No. 61/492,174 filed Jun. 1, 2011, the disclosures of all of which are hereby incorporated by reference in their entireties for all purposes.
FIELD OF THE INVENTION The invention relates to the field of molecular biology and recombinant nucleic acid technology. In particular, the invention relates to methods of inserting genes into defined locations in the chromosomal DNA of cultured mammalian cell lines which are subject to gene amplification. The invention also relates to meganucleases, vectors and engineered cell lines necessary for performing the methods, cell lines resulting from the application of the methods, and use of the cell lines to produce protein products of interest.
BACKGROUND OF THE INVENTION Therapeutic proteins are the primary growth driver in the global pharmaceutical market (Kresse, Eur J Pharm Biopharm 72, 479 (2009)). In 2001, biopharmaceuticals accounted for $24.3 billion in sales. By 2007, this number had more than doubled to $54.5 billion. The market is currently estimated to reach $78 billion by 2012 (Pickering, Spectrum Pharmaceutical Industry Dynamics Report, Decision Resources, Inc., 5 (2008)). This includes sales of “blockbuster” drugs such as erythropoietin, tissue plasminogen activator, and interferon, as well as numerous “niche” drugs such as enzyme replacement therapies for lysosomal storage disorders. The unparalleled growth in market size, however, is driven primarily by skyrocketing demand for fully human and humanized monoclonal antibodies (Reichert, Curr Pharm Biotechnol 9, 423 (2008)). Because they have the ability to confer a virtually unlimited spectrum of biological activities, monoclonal antibodies are quickly becoming the most powerful class of therapeutics available to physicians. Not surprisingly, more than 25% of the molecules currently undergoing clinical trials in the United States and Europe are monoclonal antibodies (Reichert, Curr Pharm Biotechnol 9, 423 (2008)).
Unlike more traditional pharmaceuticals, therapeutic proteins are produced in living cells. This greatly complicates the manufacturing process and introduces significant heterogeneity into product formulations (Field, Recombinant Human IgG Production from Myeloma and Chinese Hamster Ovary Cells, in Cell Culture and Upstream Processing, Butler, ed., (Taylor and Francis Group, New York, 2007)). In addition, protein drugs are typically required at unusually high doses, which necessitates highly scalable manufacturing processes and makes manufacturing input costs a major price determinant. For these reasons, treatment with a typical therapeutic antibody (e.g., the anti-HER2-neu monoclonal Herceptin®) costs $60,000-$80,000 for a full course of treatment (Fleck, Hastings Center Report 36, 12 (2006)). Further complicating the economics of biopharmaceutical production is the fact that many of the early blockbuster biopharmaceuticals are off-patent (or will be off-patent soon) and the US and EU governments are expected to greatly streamline the regulatory approval process for “biogeneric” and “biosimilar” therapeutics (Kresse, Eur J Pharm Biopharm 72, 479 (2009)). These factors should lead to a significant increase in competition for sales of many prominent biopharmaceuticals (Pickering, Spectrum Pharmaceutical Industry Dynamics Report, Decision Resources, Inc., 5 (2008)). Therefore, there is enormous interest in technologies which reduce manufacturing costs of protein therapeutics (Seth et al., Curr Opin Biotechnol 18, 557 (2007)).
Many of the protein pharmaceuticals on the market are glycoproteins that cannot readily be produced in easy-to-manipulate biological systems such as bacteria or yeast. For this reason, recombinant therapeutic proteins are produced almost exclusively in mammalian cell lines, primarily Chinese hamster ovary (e.g., CHO-K1), mouse myeloma (e.g., NS0), baby hamster kidney (BHK), murine C127, human embryonic kidney (e.g., HEK-293), or human retina-derived (e.g., PER-C6) cells (Andersen and Krummen, Curr Opin Biotechnol 13, 117 (2002)). Of these, CHO cells are, by far, the most common platform for bioproduction because they offer the best combination of high protein expression levels, short doubling time, tolerance to a wide range of media conditions, established transfection and amplification protocols, an inability to propagate most human pathogens, a paucity of blocking intellectual property, and the longest track record of FDA approval (Field, Recombinant Human IgG Production from Myeloma and Chinese Hamster Ovary Cells, in Cell Culture and Upstream Processing, Butler, ed. (Taylor and Francis Group, New York, 2007)).
Large-market biopharmaceuticals are typically produced in enormous stirred-tank bioreactors containing hundreds of liters of CHO cells stably expressing the protein product of interest (Chu and Robinson, Curr Opin Biotechnol 12, 180 (2001), Coco-Martin and Harmsen, Bioprocess International 6, 28 (2008)). Under optimized industrial conditions, such manufacturing processes can yield in excess of 5 g of protein per liter of cells per day (Coco-Martin and Harmsen, Bioprocess International 6, 28 (2008)). Because of the large number of cells involved (˜50 billion cells per liter), the level of protein expression per cell has a very dramatic effect on yield. For this reason, all of the cells involved in the production of a particular biopharmaceutical must be derived from a single “high-producer” clone, the production of which constitutes one of the most time- and resource-intensive steps in the manufacturing process (Clarke and Compton, Bioprocess International 6, 24 (2008)).
The first step in the large-scale manufacture of a biopharmaceutical is the transfection of mammalian cells with plasmid DNA encoding the protein product of interest under the control of a strong constitutive promoter. Stable transfectants are selected by using a selectable marker gene also carried on the plasmid. Most frequently, this marker is a dihydrofolate reductase (DHFR) gene which, when transfected into a DHFR deficient cell line such as DG44, allows for the selection of stable transfectants using media deficient in hypoxanthine. The primary reason for using DHFR as a selectable marker is that it enables a process called “gene amplification”. By growing stable transfectants in gradually increasing concentrations of methotrexate (MTX), a DHFR inhibitor, it is possible to amplify the number of copies of the DHFR gene present in the genome. Because the gene encoding the protein product of interest is physically coupled to the DHFR gene, this results in amplification of both genes with a concomitant increase in the expression level of the therapeutic protein (Butler, Cell Line Development for Culture Strategies: Future Prospects to Improve Yields, in Cell Culture and Upstream Processing, Butler, ed., (Taylor and Francis Group, New York, 2007)). Related systems for the creation of stable bioproduction lines use the glutamine synthetase (GS) or hypoxanthine phosphoribosyltransferase (HPRT) genes as selectable markers and require the use of GS- or HPRT-deficient cell lines as hosts for transfection (Clarke and Compton, Bioprocess International 6, 24 (2008)). In the case of the GS system, gene amplification is accomplished by growing cells in the presence of methionine sulphoximine (MSX) (Clarke and Compton, Bioprocess International 6, 24 (2008)). In the case of the HPRT system, gene amplification is accomplished by growing cells in HAT medium, which contains aminopterin, hypoxanthine, and thymidine (Kellems, ed. Gene amplification in mammalian cells: a comprehensive guide, Marcel Dekker, New York, 1993).
In all of these systems, the initial plasmid DNA comprising a biotherapeutic gene expression cassette and a selectable marker integrates into a random location in the genome, resulting in extreme variability in therapeutic protein expression from one stable transfectant to another (Collingwood and Urnov, Targeted Gene Insertion to Enhance Protein Production from Cell Lines, in Cell Culture and Upstream Processing, Butler, ed. (Taylor and Francis Group, New York, 2007)). For this reason, it is necessary to screen hundreds to thousands of initial transfectants to identify cells which express acceptably high levels of gene product both before and after gene amplification (Butler, Cell Line Development for Culture Strategies: Future Prospects to Improve Yields, in Cell Culture and Upstream Processing, Butler, ed. (Taylor and Francis Group, New York, 2007)). A second and more problematic consequence of random gene integration is the phenomenon of transgene silencing, in which recombinant protein expression slows or ceases entirely over time (Collingwood and Urnov, Targeted Gene Insertion to Enhance Protein Production from Cell Lines, in Cell Culture and Upstream Processing, Butler, ed. (Taylor and Francis Group, New York, 2007)). Because these effects often do not manifest themselves for weeks to months following the initial transfection and screening process, it is generally necessary to carry and expand dozens of independent clonal lines to identify one that expresses the protein of interest consistently over time (Butler, Cell Line Development for Culture Strategies: Future Prospects to Improve Yields, in Cell Culture and Upstream Processing, Butler, ed. (Taylor and Francis Group, New York, 2007)).
This large number of screening and expansion steps results in a very lengthy and expensive process to simply generate the cell line that will, ultimately, produce the therapeutic of interest. Indeed, using conventional methods, a minimum of 10 months (with an average of 18 months) and an upfront investment of tens of millions of dollars in labor and material is required to produce an initial pool of protein-expressing cells suitable for industrial manufacturing (Butler, Cell Line Development for Culture Strategies: Future Prospects to Improve Yields, in Cell Culture and Upstream Processing, Butler, ed. (Taylor and Francis Group, New York, 2007)). If one takes into account lost time on market for a blockbuster protein therapeutic, inefficiencies in cell line production can cost biopharmaceutical manufacturers hundreds of millions of dollars (Seth et al., Curr Opin Biotechnol 18, 557 (2007)).
Much of the time and expense of bioproduction cell line creation can be attributed to random genomic integration of the bioproduct gene resulting in clone-to-clone variability in genotype and, hence, variability in gene expression. One way to overcome this is to target gene integration to a defined location that is known to support a high level of gene expression. To this end, a number of systems have been described which use the Cre, Flp, or ΦC31 recombinases to target the insertion of a bioproduct gene (reviewed in Collingwood and Urnov, Targeted Gene Insertion to Enhance Protein Production from Cell Lines, in Cell Culture and Upstream Processing, Butler, ed. (Taylor and Francis Group, New York, 2007)). Recent embodiments of these systems, most notably the Flp-In® system marketed by Invitrogen Corp. (Carlsbad, Calif.), couple bioproduct gene integration with the reconstitution of a split selectable marker so that cells with correctly targeted genes can be selected. As expected, these systems result in greatly reduced heterogeneity in gene expression and, in some cases, individual stable transfectants can be pooled, obviating the time and expense associated with expanding a single clone.
The principal drawback to recombinase-based gene targeting systems is that the recombinase recognition sites (loxP, FRT, or attB/attP sites) do not naturally occur in mammalian genomes. Therefore, cells must be pre-engineered to incorporate a recognition site for the recombinase before that site can be subsequently targeted for gene insertion. Because the recombinase site itself integrates randomly into the genome, it is still necessary to undertake extensive screening and evaluation to identify clones which carry the site at a location that is suitable for high level, long-term gene expression (Collingwood and Urnov, Targeted Gene Insertion to Enhance Protein Production from Cell Lines, in Cell Culture and Upstream Processing, Butler, ed. (Taylor and Francis Group, New York, 2007)). In addition, the biomanufacturing industry is notoriously hesitant to adopt “new” cell lines, such as those that have been engineered to carry a recombinase site, that do not have a track record of FDA approval. For these reasons, recombinase-based cell engineering systems may not readily be adopted by the industry and an approach that allows biomanufacturers to utilize their existing cell lines is preferable.
SUMMARY OF THE INVENTION The present invention depends, in part, upon the development of mammalian cell lines in which sequences of interest (e.g., exogenous, actively transcribed transgenes) are inserted proximal to an endogenous selectable gene in an amplifiable locus, and the discovery that (a) the insertion of such exogenous sequences of interest does not inhibit amplification of the endogenous selectable gene, (b) the exogenous sequence of interest can be co-amplified with the endogenous selectable gene, and (c) the resultant cell lines, with an amplified region comprising multiple copies of the endogenous selectable gene and the exogenous sequence of interest, are stable for extended periods even in the absence of the selection regime which was employed to induce amplification. Thus, in one aspect, the invention provides a method for producing cell lines which can be used for biomanufacturing of a protein product of interest by specifically targeting the insertion of an exogenous sequence of interest capable of actively expressing the protein product of interest proximal to an endogenous selectable gene. In another aspect, the invention provides engineered cell lines that can be used to produce protein products of interest (e.g., therapeutic proteins such as monoclonal antibodies) at high levels.
It is understood that any of the embodiments described below can be combined in any desired way, and any embodiment or combination of embodiments can be applied to each of the aspects described below, unless the context indicates otherwise.
In one aspect, the invention provides a recombinant mammalian cell comprising an engineered target site stably integrated within selectable gene within an amplifiable locus, wherein the engineered target site disrupts the function of the selectable gene and wherein the engineered target site comprises a recognition sequence for a site specific endonuclease.
In some embodiments, the selectable gene is glutamine synthetase (GS) and the locus is methionine sulphoximine (MSX) amplifiable. In some embodiments, the selectable gene is dihydrofolate reductase (DHFR) and the locus is Methotrexate (MTX) amplifiable.
In some embodiments, the selectable gene is selected from the group consisting of Dihydrofolate Reductase, Glutamine Synthetase, Hypoxanthine Phosphoribosyltransferase, Threonyl tRNA Synthetase, Na,K-ATPase, Asparagine Synthetase, Ornithine Decarboxylase, Inosine-5′-monophosphate dehydrogenase, Adenosine Deaminase, Thymidylate Synthetase, Aspartate Transcarbamylase, Metallothionein, Adenylate Deaminase (1,2), UMP-Synthetase and Ribonucleotide Reductase.
In some embodiments, the selectable gene is amplifiable by selection with a selection agent selected from the group consisting of Methotrexate (MTX), Methionine sulphoximine (MSX), Aminopterin, hypoxanthine, thymidine, Borrelidin, Ouabain, Albizziin, Beta-aspartyl hydroxamate, alpha-difluoromethylornithine (DFMO), Mycophenolic Acid, Adenosine, Alanosine, 2′deoxycoformycin, Fluorouracil, N-Phosphonacetyl-L-Aspartate (PALA), Cadmium, Adenine, Azaserine, Coformycin, 6-azauridine, pyrazofuran, hydroxyurea, motexafin gadolinium, fludarabine, cladribine, gemcitabine, tezacitabine and triapine.
In some embodiments, the engineered target site is inserted into an exon of the selectable gene. In some embodiments, the site specific endonuclease is a meganuclease, a zinc finger nuclease or TAL effector nuclease. In some embodiment, the recombinant cell further comprises the site specific endonuclease.
In one aspect, the invention provides a recombinant mammalian cell comprising an engineered target site stably integrated proximal to a selectable gene within an amplifiable locus, wherein the engineered target site comprises a recognition sequence for a site specific endonuclease.
In some embodiments, the engineered target site is downstream from the 3′ regulatory region of the selectable gene. In some embodiments, the engineered target site is 0 to 100,000 base pairs downstream from the 3′ regulatory region of the selectable gene. In other embodiments, the engineered target site is upstream from the 5′ regulatory region of the selectable gene. In some embodiments, the engineered target site is 0 to 100,000 base pairs upstream from the 5′ regulatory region of the selectable gene.
In another aspect, the invention provides a method for inserting an exogenous sequence into an amplifiable locus of a mammalian cell comprising: (a) providing a mammalian cell having an endogenous target site proximal to a selectable gene within the amplifiable locus, wherein the endogenous target site comprises: (i) a recognition sequence for an engineered meganuclease; (ii) a 5′ flanking region 5′ to the recognition sequence; and (iii) a 3′ flanking region 3′ to the recognition sequence; and (b) introducing a double-stranded break between the 5′ and 3′ flanking regions of the endogenous target site; (c) contacting the cell with a donor vector comprising from 5′ to 3′: (i) a donor 5′ flanking region homologous to the 5′ flanking region of the endogenous target site; (ii) an exogenous sequence; and (iii) a donor 3′ flanking region homologous to the 3′ flanking region of the endogenous target site; whereby the donor 5′ flanking region, the exogenous sequence and the donor 3′ flanking region are inserted between the 5′ and 3′ flanking regions of the endogenous target site by homologous recombination to provide a modified cell.
In some embodiments, the method further comprises growing the modified cell in the presence of a compound that inhibits the function of the selectable gene to amplify the copy number of the selectable gene. In some embodiments, the exogenous sequence comprises a gene of interest.
In some embodiments endogenous target site is downstream from the 3′ regulatory region of the selectable gene. In some embodiments, the endogenous target site is 0 to 100,000 base pairs downstream from the 3′ regulatory region of the selectable gene. In other embodiments, the endogenous target site is upstream from the 5′ regulatory region of the selectable gene. In some embodiments, the endogenous target site is 0 to 100,000 base pairs upstream from the 5′ regulatory region of the selectable gene.
In one aspect, the invention provides a method for inserting an exogenous sequence into an amplifiable locus of a mammalian cell comprising: (a) providing a mammalian cell having an endogenous target site proximal to a selectable gene within the amplifiable locus, wherein the endogenous target site comprises: (i) a recognition sequence for an engineered meganuclease; (ii) a 5′ flanking region 5′ to the recognition sequence; and (iii) a 3′ flanking region 3′ to the recognition sequence; and (b) introducing a double-stranded break between the 5′ and 3′ flanking regions of the endogenous target site; (c) contacting the cell with an engineered target site donor vector comprising from 5′ to 3′: (i) a donor 5′ flanking region homologous to the 5′ flanking region of the endogenous target site; (ii) an exogenous sequence comprising an engineered target site; and (iii) a donor 3′ flanking region homologous to the 3′ flanking region of the endogenous target site; whereby the donor 5′ flanking region, the exogenous sequence and the donor 3′ flanking region are inserted between the 5′ and 3′ flanking regions of the endogenous target site by homologous recombination to provide a mammalian cell comprising the engineered target site; (d) introducing a double-stranded break between the 5′ and 3′ flanking regions of the engineered target site; (e) contacting the cell comprising the engineered target site with a sequence of interest donor vector comprising from 5′ to 3′: (i) a donor 5′ flanking region homologous to the 5′ flanking region of the engineered target site; (ii) an exogenous sequence comprising a sequence of interest; and (iii) a donor 3′ flanking region homologous to the 3′ flanking region of the engineered target site; whereby the donor 5′ flanking region, the exogenous sequence comprising the sequence of interest and the donor 3′ flanking region are inserted between the 5′ and 3′ flanking regions of the engineered target site by homologous recombination to provide an engineered mammalian cell comprising the sequence of interest.
In some embodiments, the method further comprises growing the engineered mammalian cell in the presence of a compound that inhibits the function of the selectable gene to amplify the copy number of the selectable gene. In some embodiments, the sequence of interest comprises a gene.
In another aspect, the invention provides a method for inserting an exogenous sequence into an amplifiable locus of a mammalian cell comprising: (a) providing a mammalian cell having an endogenous target site within a selectable gene within the amplifiable locus, wherein the endogenous target site comprises: (i) a recognition sequence for an engineered meganuclease; (ii) a 5′ flanking region 5′ to the recognition sequence; and (iii) a 3′ flanking region 3′ to the recognition sequence; and (b) introducing a double-stranded break between the 5′ and 3′ flanking regions of the endogenous target site; (c) contacting the cell with an engineered target site donor vector comprising from 5′ to 3′: (i) a donor 5′ flanking region homologous to the 5′ flanking region of the endogenous target site; (ii) an exogenous sequence comprising an engineered target site; and (iii) a donor 3′ flanking region homologous to the 3′ flanking region of the endogenous target site; whereby the donor 5′ flanking region, the exogenous sequence and the donor 3′ flanking region are inserted between the 5′ and 3′ flanking regions of the endogenous target site by homologous recombination to provide a mammalian cell comprising the engineered target site; (d) introducing a double-stranded break between the 5′ and 3′ flanking regions of the engineered target site; (e) contacting the cell comprising the engineered target site with a sequence of interest donor vector comprising from 5′ to 3′: (i) a donor 5′ flanking region homologous to the 5′ flanking region of the engineered target site; (ii) an exogenous sequence comprising a sequence of interest; and (iii) a donor 3′ flanking region homologous to the 3′ flanking region of the engineered target site; whereby the donor 5′ flanking region, the exogenous sequence comprising the sequence of interest and the donor 3′ flanking region are inserted between the 5′ and 3′ flanking regions of the engineered target site by homologous recombination to provide a engineered mammalian cell comprising the sequence of interest.
In some embodiments, the method further comprises growing the engineered mammalian cell in the presence of a compound that inhibits the function of the selectable gene to amplify the copy number of the selectable gene.
In some embodiments, the sequence of interest comprises a gene.
In some embodiments, the endogenous target site is within an intron of the selectable gene. In other embodiments, the endogenous target site is within an exon of the selectable gene.
In one aspect, the invention provides a recombinant meganuclease comprising a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO: 15.
In another aspect, the invention provides a recombinant meganuclease comprising the amino acid sequence of SEQ ID NO: 15.
In another aspect, the invention provides a recombinant meganuclease which recognizes and cleaves a recognition site having 75%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO: 14. In one embodiment, the meganuclease recognizes and cleaves a recognition site of SEQ ID NO: 14.
In another aspect, the invention provides a recombinant meganuclease comprising a polypeptide having at least 75%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO: 9. In one embodiment, the recombinant meganuclease has the sequence of the meganuclease of SEQ ID NO: 9.
In another aspect, the invention provides a recombinant meganuclease which recognizes and cleaves a recognition site having at least 75%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO: 7. In one embodiment, the meganuclease recognizes and cleaves a recognition site of SEQ ID NO: 7.
In another aspect, the invention provides a recombinant meganuclease comprising a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO: 10. In one embodiment, the recombinant meganuclease comprises the polypeptide of SEQ ID NO: 10.
In another aspect, the invention provides a recombinant meganuclease which recognizes and cleaves a recognition site having at least 75%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO: 8. In one embodiment, the meganuclease recognizes and cleaves a recognition site of SEQ ID NO: 8.
In another aspect, the invention provides a recombinant meganuclease comprising a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO: 13. In one embodiment, rhe recombinant meganuclease comprises the polypeptide of SEQ ID NO: 13.
In another aspect, the invention provides a recombinant meganuclease which recognizes and cleaves a recognition site having at least 75%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO: 12. In one embodiment, the meganuclease recognizes and cleaves a recognition site of SEQ ID NO: 12.
In another aspect, the invention provides a recombinant meganuclease comprising a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO: 29. In one embodiment, the recombinant meganuclease comprises the polypeptide of SEQ ID NO: 29.
In another aspect, the invention provides a recombinant meganuclease which recognizes and cleaves a recognition site having at least 75%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to SEQ ID NO: 30. In one embodiment, the meganuclease recognizes and cleaves a recognition site of SEQ ID NO: 30.
In another aspect, the invention provides recombinant mammalian cell lines which continue to express a protein product of interest from an exogenous sequence of interest present in an amplified region of the genome (i.e., present in 2-1,000 copies, co-amplified with a selectable gene in an amplifiable locus) for a period of at least 8, 9, 10, 11, 12, 13, or 14 weeks after removal of the amplification selection agent, and with a reduction of expression levels and/or copy number of less than 20, 25, 30, 35 or 40%.
In another aspect, the invention provides methods of producing recombinant cells with amplified regions including a sequence of interest and a selectable gene by subjecting the above-described recombinant cells to selection with a selection agent which causes co-amplification of the sequence of interest and the selectable gene.
In another aspect, the invention provides methods of producing a protein product of interest by culturing the above-described recombinant cells, or the above-described recombinant cells with amplified regions, and obtaining the protein product of interest from the culture medium or a cell lysate.
BRIEF DESCRIPTION OF THE FIGURES FIG. 1. A general strategy for targeting a sequence of interest to an amplifiable locus.
FIGS. 2A and 2B. (A) Schematic of the CHO DHFR locus showing a preferred region for targeting a sequence of interest 5,000-60,000 base pairs downstream of the DHFR gene. (B) Schematic of the CHO GS locus showing a preferred region for targeting a sequence of interest 5,000-55,000 base pairs downstream of the GS gene.
FIG. 3. Strategy for inserting a sequence of interest into an amplifiable locus in a two-step process involving a pre-integrated engineered target sequence.
FIG. 4. Strategy for inserting an engineered target sequence into an amplifiable locus with concomitant removal of a portion of the selectable gene, followed by insertion of a sequence of interest and reconstitution of the selectable gene.
FIG. 5. Strategy for inserting an engineered target sequence into an amplifiable locus with concomitant disruption of the coding sequence of a selectable gene, followed by insertion of a sequence of interest and reconstitution of the selectable gene.
FIG. 6. Strategy for inserting an engineered target sequence into an amplifiable locus with concomitant disruption of the mRNA processing, followed by insertion of a sequence of interest and reconstitution of the selectable gene.
FIGS. 7A through 7D. (A) A direct-repeat recombination assay for site-specific endonuclease activity. (B) Results of the assay in (A) applied to the CHO-23/24 and CHO-51/52 meganucleases. (C) Alignment of sequences obtained from CHO cells transfected with mRNA encoding the CHO-23/24 meganuclease (SEQ ID NOS 37-39, 38, 40, 38, and 38, respectively, in order of appearance). (D) Alignment of sequences obtained from CHO cells transfected with mRNA encoding the CHO-51/52 meganuclease (SEQ ID NOS 41-51, respectively, in order of appearance).
FIGS. 8A and 8B. (A) Strategy for inserting an exogenous DNA sequence into the CHO DHFR locus using the CHO-51/52 meganuclease. (B) PCR products demonstrating insertion of an engineered target sequence.
FIGS. 9A through 9C. (A) Strategy for inserting an engineered target sequence into the CHO DHFR locus using the CHO-23/24 meganuclease, followed by Flp recombinase-mediated insertion of a sequence of interest. (B) PCR products from hygromycin-resistant clones produced in (A). (C) GFP expression by the 24 clones produced in (B).
FIGS. 10A through 10C. Results of experiments with a GFP-expressing CHO line produced by integrating a GFP gene expression cassette into the DHFR locus using a target sequence strategy as shown in FIG. 9.
FIGS. 11A through 11C. (A) A direct-repeat recombination assay, as in FIG. 5A. (B) The assay in (A) applied to the CHO-13/14 and CGS-5/6 meganucleases. (C) Alignment of sequences obtained from CHO cells transfected with mRNA encoding the CGS-5/6 meganuclease (SEQ ID NOS 52-56, 56, 56-63, 63, 63, and 63-64, respectively, in order of appearance).
DETAILED DESCRIPTION OF THE INVENTION 1.1 Introduction The present invention depends, in part, upon the development of mammalian cell lines in which exogenous actively transcribed transgenes have been inserted proximal to an endogenous amplifiable locus, and the discovery that (a) the insertion of such exogenous actively transcribed transgenes does not prevent or substantially inhibit amplification of the endogenous amplifiable locus, (b) the exogenous actively transcribed transgene can be co-amplified with the endogenous amplifiable locus, and (c) the resultant cell line, with an amplified region comprising multiple copies of the endogenous amplifiable locus and the exogenous actively transcribed transgene is stable for extended periods even in the absence of the selection regime which was employed to induce amplification. Thus, in one aspect, the invention provides a method for producing cell lines which can be used for biomanufacturing of a protein product of interest by specifically targeting the insertion of an exogenous gene capable of actively expressing the protein product of interest proximal to an endogenous amplifiable locus. In another aspect, the invention provides engineered cell lines that can be used to produce protein products of interest (e.g., therapeutic proteins such as monoclonal antibodies) at high levels.
1.2 References and Definitions The patent and scientific literature referred to herein establishes knowledge that is available to those of skill in the art. The entire disclosures of the issued U.S. patents, pending applications, published foreign applications, and scientific and technical references cited herein, including protein and nucleic acid database sequences, are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference.
As used herein, the term “meganuclease” refers to naturally-occurring homing endonucleases (also referred to as Group I intron encoded endonucleases) or non-naturally-occurring (e.g., rationally designed or engineered) endonucleases based upon the amino acid sequence of a naturally-occurring homing endonuclease. Examples of naturally-occurring meganucleases include I-SceI, I-CreI, I-CeuI, I-DmoI, I-MsoI, I-AniI, etc. Rationally designed meganucleases are disclosed in, for example, WO 2007/047859 and WO 2009/059195, and can be engineered to have modified DNA-binding specificity, DNA cleavage activity, DNA-binding affinity, or dimerization properties relative to a naturally occurring meganuclease. A meganuclease may bind to double-stranded DNA as a homodimer (e.g., wild-type I-CreI), or it may bind to DNA as a heterodimer (e.g., engineered meganucleases disclosed in WO 2007/047859). An engineered meganuclease may also be a “single-chain meganuclease” in which a pair of DNA-binding domains derived from a natural meganuclease are joined into a single polypeptide using a peptide linker (e.g., single-chain meganucleases disclosed in WO 2009/059195).
As used herein, the term “single-chain meganuclease” refers to a polypeptide comprising a pair of meganuclease subunits joined by a linker. A single-chain meganuclease has the organization: N-terminal subunit-Linker-C-terminal subunit. The two meganuclease subunits will generally be non-identical in amino acid sequence and will recognize non-identical DNA sequences. Thus, single-chain meganucleases typically cleave pseudo-palindromic or non-palindromic recognition sequences. Methods of producing single-chain meganucleases are disclosed in WO 2009/059195.
As used herein, the term “site specific endonuclease” means a meganuclease, zinc-finger nuclease or TAL effector nuclease.
As used herein, with respect to a protein, the term “recombinant” means having an altered amino acid sequence as a result of the application of genetic engineering techniques to nucleic acids which encode the protein, and cells or organisms which express the protein. With respect to a nucleic acid, the term “recombinant” means having an altered nucleic acid sequence as a result of the application of genetic engineering techniques. Genetic engineering techniques include, but are not limited to, PCR and DNA cloning technologies; transfection, transformation and other gene transfer technologies; homologous recombination; site-directed mutagenesis; and gene fusion. In accordance with this definition, a protein having an amino acid sequence identical to a naturally-occurring protein, but produced by cloning and expression in a heterologous host, is not considered recombinant. As used herein, the term “engineered” is synonymous with the term “recombinant.”
As used herein, with respect to a meganuclease, the term “wild-type” refers to any naturally-occurring form of a meganuclease. The term “wild-type” is not intended to mean the most common allelic variant of the enzyme in nature but, rather, any allelic variant found in nature. Wild-type homing endonucleases are distinguished from recombinant or non-naturally-occurring meganucleases.
As used herein, the term “recognition sequence” refers to a DNA sequence that is bound and cleaved by a meganuclease. A recognition sequence comprises a pair of inverted, 9 base pair “half sites” which are separated by four base pairs. In the case of a homo- or heterodimeric meganucleases, each of the two monomers makes base-specific contacts with one half-site. In the case of a single-chain heterodimer meganuclease, the N-terminal domain of the protein contacts a first half-site and the C-terminal domain of the protein contacts a second half-site. In the case if I-CreI, for example, the recognition sequence is 22 base pairs and comprises a pair of inverted, 9 base pair “half sites” which are separated by four base pairs.
As used herein, the term “target site” refers to a region of the chromosomal DNA of a cell comprising a target sequence into which a sequence of interest can be inserted. As used herein, the term “engineered target site” refers to an exogenous sequence of DNA integrated into the chromosomal DNA of a cell comprising an engineered target sequence into which a sequence of interest can be inserted.
As used herein, the term “target sequence” means a DNA sequence within a target site which includes one or more recognition sequences for a nuclease, integrase, transposase, and/or recombinase. For example, a target sequence can include a recognition sequence for a meganuclease. As used herein, an “engineered target sequence” means an exogenous target sequence which is introduced into a chromosome to serve as the insertion point for another sequence.
As used herein, the term “flanking region” or “flanking sequence” refers to a sequence of >3 or, preferably, >50 or, more preferably, >200 or, most preferably, >400 base pairs of DNA which is immediately 5′ or 3′ to a reference sequence (e.g., a target sequence or sequence of interest).
As used herein, the terms “amplifiable locus” refers to a region of the chromosomal DNA of a cell which can be amplified by selection with one or more compounds (e.g., drugs) in the growth media. An amplifiable locus will typically comprise a gene encoding a protein which, under the appropriate conditions, is necessary for cell survival. By inhibiting the function of such an essential protein, for example with a small molecule drug, the amplifiable locus is duplicated many times over as a means of increasing the copy number of the essential gene. A gene of interest, if integrated into an amplifiable locus, will also become duplicated with the essential gene. Examples of amplifiable loci include the chromosomal regions comprising the DHFR, GS, and HPRT genes.
As used herein, the term “amplified locus” or “amplified gene” or “amplified sequence” refers to a locus, gene or sequence which is present in 2-1,000 copies as a result of gene amplification in response to selection of a selectable gene. An amplified gene or sequence can be a gene or sequence which is co-amplified due to selection of a selectable gene in the same amplifiable locus. In preferred embodiments, a sequence of interest is amplified to at least 3, 4, 5, 6, 7, 8, 9 or 10 copies.
As used herein, the term “selectable gene” refers to an endogenous gene that is essential for cell survival under some specific culture conditions (e.g., presence or absence of a nutrient, toxin or drug). Selectable genes are endogenous to the cell and are distinguished from exogenous “selectable markers” such as antibiotic resistance genes. Selectable genes exist in their natural context in the chromosomal DNA of the cell. For example, DHFR is a selectable gene which is necessary for cell survival in the presence of MTX in the culture medium. The gene is essential for growth in the absence of hypoxanthine and thymidine. If the endogenous DHFR selectable gene is eliminated, cells are able to grow in the absence of hypoxanthine and thymidine if they are given an exogenous copy of the DHFR gene. This exogenous copy of the DHFR gene is a selectable marker but is not a selectable gene. An amplifiable locus comprises a selectable gene and a target site. A target site is found outside of a selectable gene such that a selectable gene does not comprise a target site. Examples of selectable genes are given in Table 1.
As used herein, when used in connection with the position of a target site, recognition sequence, or inserted sequence of interest relative to the position of a selectable gene, the term “proximal” means that the target site, recognition sequence, or inserted sequence of interest is within the same amplifiable locus as the selectable gene, either upstream (5′) or downstream (3′) of the selectable gene, and preferably between the selectable gene and the next gene in the region (whether upstream (5′) or downstream (3′)). Typically, a “proximal” target site, recognition sequence, or inserted sequence of interest will be within <100,000 base pairs of the selectable gene, as measured from the first or last nucleotide of the first or last regulatory element of the selectable gene.
As used herein, the term “homologous recombination” refers to the natural, cellular process in which a double-stranded DNA-break is repaired using a homologous DNA sequence as the repair template (see, e.g. Cahill et al. (2006), Front. Biosci. 11:1958-1976). The homologous DNA sequence may be an endogenous chromosomal sequence or an exogenous nucleic acid that was delivered to the cell. Thus, for some applications of engineered meganucleases, a meganuclease is used to cleave a recognition sequence within a target sequence in a genome and an exogenous nucleic acid with homology to or substantial sequence similarity with the target sequence is delivered into the cell and used as a template for repair by homologous recombination. The DNA sequence of the exogenous nucleic acid, which may differ significantly from the target sequence, is thereby inserted or incorporated into the chromosomal sequence. The process of homologous recombination occurs primarily in eukaryotic organisms. The term “homology” is used herein as equivalent to “sequence similarity” and is not intended to require identity by descent or phylogenetic relatedness.
As used herein, the term “stably integrated” means that an exogenous or heterologous DNA sequence has been covalently inserted into a chromosome (e.g., by homologous recombination, non-homologous end joining, transposition, etc.) and has remained in the chromosome for a period of at least 8 weeks.&&
As used herein, the term “non-homologous end-joining” or “NHEJ” refers to the natural, cellular process in which a double-stranded DNA-break is repaired by the direct joining of two non-homologous DNA segments (see, e.g. Cahill et al. (2006), Front. Biosci. 11:1958-1976). DNA repair by non-homologous end-joining is error-prone and frequently results in the untemplated addition or deletion of DNA sequences at the site of repair. Thus, for certain applications, an engineered meganuclease can be used to produce a double-stranded break at a meganuclease recognition sequence within an amplifiable locus and an exogenous nucleic acid molecule, such as a PCR product, can be captured at the site of the DNA break by NHEJ (see, e.g. Salomon et al. (1998), EMBO J. 17:6086-6095). In such cases, the exogenous nucleic acid may or may not have homology to the target sequence. The process of non-homologous end-joining occurs in both eukaryotes and prokaryotes such as bacteria.
As used herein, the term “sequence of interest” means any nucleic acid sequence, whether it codes for a protein, RNA, or regulatory element (e.g., an enhancer, silencer, or promoter sequence), that can be inserted into a genome or used to replace a genomic DNA sequence. Sequences of interest can have heterologous DNA sequences that allow for tagging a protein or RNA that is expressed from the sequence of interest. For instance, a protein can be tagged with tags including, but not limited to, an epitope (e.g., c-myc, FLAG) or other ligand (e.g., poly-His). Furthermore, a sequence of interest can encode a fusion protein, according to techniques known in the art (see, e.g., Ausubel et al., Current Protocols in Molecular Biology, Wiley 1999). In preferred embodiments, a sequence of interest comprises a promoter operably linked to a gene encoding a protein of medicinal value such as an antibody, antibody fragment, cytokine, growth factor, hormone, or enzyme. For some applications, the sequence of interest is flanked by a DNA sequence that is recognized by the engineered meganuclease for cleavage. Thus, the flanking sequences are cleaved allowing for proper insertion of the sequence of interest into genomic recognition sequences cleaved by an engineered meganuclease. For some applications, the sequence of interest is flanked by DNA sequences with homology to or substantial sequence similarity with the target site such that homologous recombination inserts the sequence of interest within the genome at the locus of the target sequence.
As used herein, the term “donor DNA” refers to a DNA molecule comprising a sequence of interest flanked by DNA sequences homologous to a target site. Donor DNA can serve as a template for DNA repair by homologous recombination if it is delivered to a cell with a site-specific nuclease such as a meganuclease, zinc-finger nuclease, or TAL-effector nuclease. The result of such DNA repair is the insertion of the sequence of interest into the chromosomal DNA of the cell. Donor DNA can be linear, such as a PCR product, or circular, such as a plasmid. In cases where a donor DNA is a circular plasmid, it may be referred to as a “donor plasmid.”
As used herein, unless specifically indicated otherwise, the word “or” is used in the inclusive sense of “and/or” and not the exclusive sense of “either/or.”
2.1 Transgene Targeting to Amplifiable Loci The present invention provides methods for generating transgenic mammalian cell lines expressing a desired protein product of interest, including “high-producer” cell lines, by targeting the insertion of a gene encoding the protein product of interest (e.g., a therapeutic protein gene expression cassette) to regions of the genome that are amplifiable. Such regions in mammalian cells include the DHFR, GS, and HPRT genes, as well as others shown in Table 1.
The precise mechanism of gene amplification is not known. Indeed, it is very likely that there is no single mechanism by which gene amplification occurs but that a variety of different random chromosomal aberrations, in combination with strong selection for amplification, results in increased gene copy number (reviewed in Omasa (2002), J. Biosci. Bioeng. 94:600-605). It is clear that chromosomal location plays a major role in amplification and the stable maintenance of amplified genes (Brinton and Heintz (1995), Chromosoma 104:143-51). It has been found that transgenes integrated into chromosomal locations adjacent to telomeres are more easily amplified and, once amplified, tend to be stable at high copy numbers after the selection agent is removed (Yoshikawa et al. (2000), Cytotechnology 33:37-46; Yoshikawa et al. (2000), Biotechnol Prog. 16:710-715). This is significant because selection agents such as MTX and MSX are toxic and cannot be included in the growth media in a commercial biomanufacturing process. In contrast, transgenes integrated into regions in the CHO genome that are not adjacent to telomeres amplify inefficiently and rapidly lose copy number following the removal of selection agents from the media. For example, Yoshikawa et al. found that randomly-integrated transgenes linked to a DHFR selectable marker amplified to greater than 10-fold higher copy numbers when the integration site was adjacent to a telomere (Yoshikawa et al. (2000), Biotechnol Prog. 16:710-715). These researchers also found that an amplified transgene integrated into a non-telomeric region will lose >50% of its copies in only 20 days following the removal of MTX from the growth media. None of the selectable genes identified in Table 1 is adjacent to a telomere in the mouse genome (www.ensembl.com) and the similarity in genome organization between mouse and CHO makes it likely that these genes are in non-telomeric regions in CHO as well (Xu et al. (2011), Nat. Biotechnol. 29:735-741). Thus, the prior art instructs that the loci identified in Table 1, including the DHFR and GS loci, are not preferred locations to target transgene insertion if the goal is efficient and stable gene amplification.
In addition, in the case of endogenous gene amplification, it is clear that chromosomal sequences outside of the selectable gene sequence play an important role in facilitating amplification and in defining the length of DNA sequence that is co-amplified with the gene under selection (Looney and Hamlin (1987), Mol. and Cell. Biol. 7:569-577). In particular, it has been shown that the sequence and location of the DNA replication origin in relation to the selectable gene plays a major role in amplification. For example, it has been shown that amplification of the endogenous CHO DHFR locus is dependent upon a pair of replication origins found in the region 5,000-60,000 base pairs downstream of the DHFR gene coding sequence (Anachkova and Hamlin (1989), Mol. and Cell. Biol. 9:532-540; Milbrandt et al. (1981), Proc. Natl. Acad. Sci. USA 78:6042-6047). Further, Brinton and Heintz have shown that these same replication origins fail to promote gene amplification when incorporated randomly into the genome with a transgenic DHFR sequence (Brinton and Heintz (1995), Chromosoma. 104:143-51). This clearly demonstrates the importance of maintaining both the sequence and proper chromosomal context of these replication origins to promote DHFR gene amplification. Thus the art instructs that the region downstream of DHFR is critical to gene amplification and should not be disrupted by, for example, inserting a transgenic gene expression cassette as described in the present invention.
Surprisingly, we have discovered that DNA sequences, including exogenous transcriptionally active sequences, which are inserted proximal to (e.g., within <100,000 base pairs) selectable genes in mammalian cell lines (e.g., CHO-K1) will co-amplify in the presence of appropriate compounds which select for amplification. Thus, the present invention provides methods for reliably and reproducibly producing isogenic cell lines in which transgenes encoding protein products of interest (e.g., biotherapeutic gene expression cassettes) can be amplified but in which it is not necessary to screen a large number of randomly generated cell lines to identify those which express high levels of the protein product of interest and are resistant to gene silencing.
In addition, we have surprisingly found that the mammalian cell lines of the invention, in which a sequence of interest is co-amplified with a selectable gene in an amplifiable locus, are stable with respect to expression of the sequence of interest and/or copy number of the sequence of interest even in the absence of continued selection. That is, whereas the art teaches that amplified sequences will be reduced in copy number over time if selection is not maintained (see, e.g., Yoshikawa et al. (2000), Biotechnol Prog. 16:710-715), we have found that cell lines produced according to the methods of the invention continue to produce the protein products of interest (encoded by the sequences of interest) at levels within 20%-25% of the initial levels, even 14 weeks after removal of the selection agent. This is significant, as noted above, because selection agents such as MTX and MSX are toxic, and it would be highly desirable to produce biotherapeutic proteins in cell lines which do not require continued exposure to such selection agents. Therefore, in some embodiments, the invention provides recombinant mammalian cell lines which continue to express a protein product of interest from an exogenous sequence of interest present in an amplified region of the genome (i.e., present in 2-1,000 copies, co-amplified with a selectable gene in an amplifiable locus) for a period of at least 8, 9, 10, 11, 12, 13, or 14 weeks after removal of the amplification selection agent, and with a reduction of expression levels and/or copy number of less than 20, 25, 30, 35 or 40%.
The present invention also provides the products necessary to practice the methods, and to target insertion of sequences of interest into amplifiable loci in mammalian cell lines. A common method for inserting or modifying a DNA sequence involves introducing a transgenic DNA sequence flanked by sequences homologous to the genomic target and selecting or screening for a successful homologous recombination event. Recombination with the transgenic DNA occurs rarely but can be stimulated by a double-stranded break in the genomic DNA at the target site (Porteus et al. (2005), Nat. Biotechnol. 23: 967-73; Tzfira et al. (2005), Trends Biotechnol. 23: 567-9; McDaniel et al. (2005), Curr. Opin. Biotechnol. 16: 476-83). Numerous methods have been employed to create DNA double-stranded breaks, including irradiation and chemical treatments. Although these methods efficiently stimulate recombination, the double-stranded breaks are randomly dispersed in the genome, which can be highly mutagenic and toxic. At present, the inability to target gene modifications to unique sites within a chromosomal background is a major impediment to routine genome engineering.
One approach to achieving this goal is stimulating homologous recombination at a double-stranded break in a target locus using a nuclease with specificity for a sequence that is sufficiently large to be present at only a single site within the genome (see, e.g., Porteus et al. (2005), Nat. Biotechnol. 23: 967-73). The effectiveness of this strategy has been demonstrated in a variety of organisms using ZFNs (Porteus (2006), Mol Ther 13: 438-46; Wright et al. (2005), Plant J. 44: 693-705; Urnov et al. (2005), Nature 435: 646-51). Homing endonucleases are a group of naturally-occurring nucleases which recognize 15-40 base-pair cleavage sites commonly found in the genomes of plants and fungi. They are frequently associated with parasitic DNA elements, such as Group I self-splicing introns and inteins. They naturally promote homologous recombination or gene insertion at specific locations in the host genome by producing a double-stranded break in the chromosome, which recruits the cellular DNA-repair machinery (Stoddard (2006), Q. Rev. Biophys. 38: 49-95). Homing endonucleases are commonly grouped into four families: the LAGLIDADG (SEQ ID NO: 65) family, the GIY-YIG family, the His-Cys box family and the HNH family. These families are characterized by structural motifs, which affect catalytic activity and recognition sequence. For instance, members of the LAGLIDADG (SEQ ID NO: 65) family are characterized by having either one or two copies of the conserved LAGLIDADG (SEQ ID NO: 65) motif (see Chevalier et al. (2001), Nucleic Acids Res. 29(18): 3757-3774). The LAGLIDADG (SEQ ID NO: 65) homing endonucleases with a single copy of the LAGLIDADG (SEQ ID NO: 65) motif form homodimers, whereas members with two copies of the LAGLIDADG (SEQ ID NO: 65) motif are found as monomers.
Natural homing endonucleases, primarily from the LAGLIDADG (SEQ ID NO: 65) family, have been used to effectively promote site-specific genome modification in plants, yeast, Drosophila, mammalian cells and mice, but this approach has been limited to the modification of either homologous genes that conserve the endonuclease recognition sequence (Monnat et al. (1999), Biochem. Biophys. Res. Commun. 255: 88-93) or to pre-engineered genomes into which a recognition sequence has been introduced (Rouet et al. (1994), Mol. Cell. Biol. 14: 8096-106; Chilton et al. (2003), Plant Physiol. 133: 956-65; Puchta et al. (1996), Proc. Natl. Acad. Sci. USA 93: 5055-60; Rong et al. (2002), Genes Dev. 16: 1568-81; Gouble et al. (2006), J. Gene Med. 8(5):616-622).
Systematic implementation of nuclease-stimulated gene modification requires the use of engineered enzymes with customized specificities to target DNA breaks to existing sites in a genome and, therefore, there has been great interest in adapting homing endonucleases to promote gene modifications at medically or biotechnologically relevant sites (Porteus et al. (2005), Nat. Biotechnol. 23: 967-73; Sussman et al. (2004), J. Mol. Biol. 342: 31-41; Epinat et al. (2003), Nucleic Acids Res. 31: 2952-62).
I-CreI (SEQ ID NO: 1) is a member of the LAGLIDADG (SEQ ID NO: 65) family of homing endonucleases which recognizes and cuts a 22 base pair recognition sequence in the chloroplast chromosome of the algae Chlamydomonas reinhardtii. Genetic selection techniques have been used to modify the wild-type I-CreI cleavage site preference (Sussman et al. (2004), J. Mol. Biol. 342: 31-41; Chames et al. (2005), Nucleic Acids Res. 33: e178; Seligman et al. (2002), Nucleic Acids Res. 30: 3870-9, Arnould et al. (2006), J. Mol. Biol. 355: 443-58). More recently, a method of rationally-designing mono-LAGLIDADG (SEQ ID NO: 65) homing endonucleases was described which is capable of comprehensively redesigning I-CreI and other homing endonucleases to target widely-divergent DNA sites, including sites in mammalian, yeast, plant, bacterial, and viral genomes (WO 2007/047859).
Thus, in one embodiment, the invention provides engineered meganucleases derived from the amino acid sequence of I-CreI that recognize and cut DNA sites in amplifiable regions of mammalian genomes. These engineered meganucleases can be used in accordance with the invention to target the insertion of gene expression cassettes into defined locations in the chromosomal DNA of cell lines such as CHO cells. This invention will greatly streamline the production of desired cell lines by reducing the number of lines that must be screened to identify a “high-producer” clone suitable for commercial-scale production of a therapeutic glycoprotein.
The present invention involves targeting transgenic DNA “sequences of interest” to amplifiable loci. The amplifiable loci are regions of the chromosomal DNA that contain selectable genes that become amplified in the presence of selection agents (e.g., drugs). For example, the Chinese Hamster Ovary (CHO) cell DHFR locus can be amplified to ˜1,000 copies by growing the cells in the presence of methotrexate (MTX), a DHFR inhibitor. Table 1 lists additional examples of selectable genes that can be amplified using small molecule drugs (Kellems, ed. Gene amplification in mammalian cells: a comprehensive guide. Marcel Dekker, New York, 1993; Omasa (2002), J. Biosci. Bioeng. 94:6 600-605).
TABLE 1
Amplifiable Genes
Selectable Gene Name Amplified With
Dihydrofolate Reductase Methotrexate (MTX)
Glutamine Synthetase Methionine sulphoximine (MSX)
Hypoxanthine Phosphoribosyl- Aminopterin, hypoxanthine, and thymidine
transferase
Threonyl tRNA Synthetase Borrelidin
Na,K-ATPase Ouabain
Asparagine Synthetase Albizziin or Beta-aspartyl hydroxamate
Ornithine Decarboxylase alpha-difluoromethylornithine (DFMO)
Inosine-5′-monophosphate Mycophenolic Acid
dehydrogenase
Adenosine Deaminase Adenosine, Alanosine, 2′deoxycoformycin
Thymidylate Synthetase Fluorouracil
Aspartate Transcarbamylase N-Phosphonacetyl-L-Aspartate (PALA)
Metallothionein Cadmium
Adenylate Deaminase (1, 2) Adenine, Azaserine, Coformycin
UMP-Synthetase 6-azauridine, pyrazofuran
Ribonucleotide Reductase hydroxyurea, motexafin gadolinium,
fludarabine, cladribine, gemcitabine,
tezacitabine, triapine.
Several considerations must be taken into account when selecting a specific target site for the insertion of a sequence of interest within an amplifiable locus. First, the selected insertion site must be co-amplified with the gene under selection. In many cases, experimental data already exists in the art which delimits the amount of flanking chromosomal sequence that co-amplifies with a selectable gene of interest. This data, which precisely defines the extent of the amplifiable locus, exists for CHO DHFR (Ma et al. (1988), Mol Cell Biol. 8(6):2316-27), human DHFR (Morales et al. (2009), Mol Cancer Ther. 8(2):424-432), and CHO GS (Sanders et al. (1987), Dev Biol Stand. 66:55-63). Where such data does not already exist in the art, we predict that chromosomal DNA sequences <100,000 base pairs upstream or downstream of the selectable gene coding sequence are likely to co-amplify. Hence, these regions could be suitable sites for targeting the insertion of a sequence of interest.
Second, target sites should be selected which will not greatly impact the function of the selectable gene (e.g., the endogenous DHFR, GS, or HPRT gene). Because amplification requires a functional copy of the selectable gene, insertion sites within the promoter, exons, introns, polyadenylation signals, or other regulatory sequences that, if disrupted, would greatly impact transcription or translation of the selectable gene, should be avoided. For example, WO 2008/059317 discloses meganucleases which cleave DNA target sites within the HPRT gene. To the extent WO 2008/059317 discloses the insertion of genes into the HPRT locus, it teaches that the HPRT gene coding sequence should be disrupted in the process of transgene insertion to facilitate selection for proper targeting using 6-thioguanine. 6-thioguanine is a toxic nucleotide analog that kills cells having functional HPRT activity. Because cells produced in accordance with WO 2008/059317 will not have HPRT activity, they will not amplify an inserted transgene in response to treatment with an HPRT inhibitor and, so, cannot be used in the present invention. For the present invention, unless the precise limits of all regulatory sequences are already known for a particular selectable gene, insertion sites >1,000 base pairs, >2,000 base pairs, >3,000 base pairs, >4,000 base pairs, or, preferably, >5,000 base pairs, upstream or downstream of the gene coding sequence should be selected. However, if the location of the regulatory sequences are known, the sequence of interest can be inserted immediately adjacent to the either the most 5′ or 3′ regulatory sequence (e.g., immediately 3′ to the polyadenylation signal).
Lastly, target sites should be selected which do not disrupt other chromosomal genes which may be important for normal cell physiology. In general, gene insertion sites should be >1,000 base pairs, >2,000 base pairs, >3,000 base pairs, >4,000 base pairs, or, preferably, >5,000 base pairs, away from any gene coding sequence.
Various methods of the invention are described schematically in the figures as follows:
FIG. 1 depicts a general strategy for targeting a sequence of interest to an amplifiable locus. In the first step, a site-specific endonuclease introduces a double-stranded break in the chromosomal DNA of a cell at a site that is proximal to an endogenous selectable gene. The cleaved chromosomal DNA then undergoes homologous recombination with a donor DNA molecule comprising a sequence of interest flanked by DNA sequences homologous to sequences flanking the endonuclease recognition sequence in the target site. As a result, the sequence of interest is inserted into the chromosomal DNA of the cell adjacent to the endogenous selectable gene. The modified cell is then grown in the presence of one or more compounds that inhibit the function of the selectable gene to induce an increase in the copy number (i.e., amplification) of the selectable gene. The sequence of interest, which is genetically linked to the selectable gene, will co-amplify with the selectable gene. The result is a stable transgenic cell line comprising multiple copies of the sequence of interest.
FIG. 2(A) depicts a schematic of the CHO DHFR locus showing a preferred region for targeting a sequence of interest 5,000-60,000 base pairs downstream of the DHFR gene. FIG. 2(B) depicts a schematic of the CHO GS locus showing a preferred region for targeting a sequence of interest 5,000-55,000 base pairs downstream of the GS gene. Promoters are shown as arrows. Exons are shown as rectangles, with non-coding exons in white and protein coding exons in gray.
FIG. 3 depicts a strategy for inserting a sequence of interest into an amplifiable locus in a two-step process involving a pre-integrated target sequence. In the first step, the chromosomal DNA of a cell is cleaved by a site-specific endonuclease at a site that is proximal to a selectable gene. The cleaved chromosomal DNA then undergoes homologous recombination with a donor DNA molecule comprising an exogenous target sequence flanked by DNA sequences homologous to the sequences flanking the endogenous target site. This results in the insertion of the new engineered target sequence into the chromosomal DNA of the cell proximal to the selectable gene. A sequence of interest can subsequently be targeted proximal to the same selectable gene using a nuclease, integrase, transposase, or recombinase that specifically recognizes the pre-integrated engineered target sequence. The modified cell is then grown in the presence of one or more compounds that co-amplify the selectable gene and the sequence of interest.
FIG. 4 depicts a strategy for inserting an engineered target sequence into a selectable gene (e.g., DHFR) with concomitant removal of a portion of the selectable gene. A site-specific endonuclease is first used to cleave the chromosomal DNA of the cell proximal to or within the selectable gene sequence. As shown in the figure, the endogenous target site is between exons 2 and 3 of the CHO DHFR gene (although the target site could be within any intron or exon, and the selectable gene could be any gene subject to amplification). The chromosomal DNA then undergoes homologous recombination with a first donor DNA (“donor DNA #1”) such that the sequence of the first donor DNA is inserted into the chromosomal DNA of the cell. As shown in the figure, this results in the replacement of the promoter and first two exons of DHFR by the new engineered target sequence (although the first donor DNA could replace more or less of the chromosomal DNA, such as only a portion of one exon). If such a replacement is made to all DHFR alleles in a cell, the resultant cell line is DHFR (−/−). A sequence of interest can subsequently be targeted proximal to the selectable gene in the cell line using an endonuclease, integrase, transposase, or recombinase that recognizes the engineered target sequence. As shown in the figure, the second donor DNA (“donor DNA #2”) comprises a sequence of interest as well as a promoter and the first two exons of DHFR. Proper targeting of this second donor DNA molecule results in the insertion of the sequence of interest at the engineered target sequence while simultaneously reconstituting a functional DHFR gene. Thus, properly targeted cell lines will be DHFR+ and can be selected using media deficient in hypoxanthine/thymidine. In addition, the sequence of interest can be co-amplified with the DHFR gene using MTX selection. The strategy diagrammed here for DHFR can be applied to any selectable gene in an amplifiable locus.
FIG. 5 depicts a strategy for inserting an engineered target sequence into an amplifiable locus with concomitant disruption of the coding sequence of a selectable gene. A site-specific endonuclease is first used to cleave the chromosomal DNA of the cell within the selectable gene coding sequence. As shown in the figure, the endogenous target site is in the third exon of the CHO GS gene. The chromosomal DNA then undergoes homologous recombination with a first donor DNA (“donor DNA #1”) such that the sequence of the first donor DNA is inserted into the chromosomal DNA of the cell. This results in the insertion of a new engineered target sequence into the GS coding sequence. If such an insertion occurs in both alleles of the GS gene and results in a frameshift mutation or otherwise disrupts the function of the GS gene, the resultant cell line will be GS (−/−). A sequence of interest can subsequently be targeted proximal to the amplifiable locus in the cell line using an endonuclease, integrase, transposase, or recombinase that recognizes the engineered target sequence. As shown in the figure, a second donor DNA (“donor DNA #2”) comprises a sequence of interest operably linked to a promoter as well as the 3′ portion of the GS coding sequence comprising exons 3, 4, 5, and 6. (The figure shows exons 3, 4, 5, and 6 joined into a single nucleotide sequence (i.e., with introns removed), but a sequence including either the naturally-occurring introns or one or more artificial introns could also be employed). Proper targeting of the second donor DNA molecule results in the insertion of the sequence of interest at the engineered target sequence while simultaneously reconstituting a functional GS gene. Thus, properly targeted cell lines will be GS+ and can be selected using media deficient in L-glutamine. In addition, the sequence of interest can be co-amplified with the GS gene using MSX selection. The strategy diagrammed here for GS can be applied to any selectable gene in an amplifiable locus.
FIG. 6 depicts a strategy for inserting an engineered target sequence into an amplifiable locus with concomitant disruption of the mRNA processing of a selectable gene. A site-specific endonuclease is first used to cleave the chromosomal DNA of the cell within an intron in the selectable gene. As drawn, the endogenous target site is in the intron between the third and fourth coding exons of the CHO GS gene. The chromosomal DNA then undergoes homologous recombination with a donor DNA #1 such that the sequence of the donor DNA is inserted in the chromosomal DNA of the cell. This results in the insertion of a new engineered target sequence into the GS coding sequence with an additional sequence that causes the GS mRNA to be processed incorrectly. As drawn, this additional sequence comprises a strong splice acceptor. If such an insertion occurs in both alleles of the GS gene, the artificial splice acceptor will cause the GS mRNA to splice incorrectly, resulting in a loss of GS expression and a requirement for growth in media containing L-glutamine. A sequence of interest can subsequently be targeted to the amplifiable locus in the cell line using an endonuclease, integrase, transposase, or recombinase that recognizes the engineered target sequence. As diagrammed, donor DNA #2 comprises a sequence of interest operably linked to a promoter as well as the 3′ portion of the GS coding sequence comprising exons 4, 5, and 6 joined into a single nucleotide sequence. (The figure shows exons 4, 5, and 6 joined into a single nucleotide sequence (i.e., with introns removed), but a sequence including either the naturally-occurring introns or one or more artificial introns could also be employed). Proper targeting of this donor DNA #2 molecule results in the insertion of the sequence of interest at the engineered target sequence while simultaneously reconstituting a functional GS gene. Thus, properly targeted cell lines will be GS+ and can be selected using media deficient in L-glutamine and the sequence of interest can be co-amplified with the GS gene using MSX selection. The strategy diagrammed here for GS can be applied to any selectable gene in an amplifiable locus.
FIG. 7(A) depicts a direct-repeat recombination assay for site-specific endonuclease activity. A reporter plasmid is produced comprising the 5′ two-thirds of the GFP gene (“GF”), followed by an endonuclease recognition sequence, followed by the 3′ two-thirds of the GFP gene (“FP”). Mammalian cells are transfected with this reporter plasmid as well as a gene encoding an endonuclease. Cleavage of the recognition sequence by the endonuclease stimulates homologous recombination between direct repeats of the GFP gene to restore GFP function. GFP+ cells can then be counted and/or sorted on a flow cytometer.
FIG. 7(B) depicts the results of the assay of FIG. 7(A) as applied to the CHO-23/24 and CHO-51/52 meganucleases. Light bars indicate the percentage of GFP+ cells when cells are transfected with the reporter plasmid alone (−endonuclease). Dark bars indicate the percentage of GFP+ cells when cells are co-transfected with a reporter plasmid and the corresponding meganuclease gene (+endonuclease). The assay was performed in triplicate and the standard deviation is shown.
FIG. 7(C) depicts alignment of sequences obtained from CHO cells transfected with mRNA encoding the CHO-23/24 meganuclease. The top sequence is from a wild-type (WT) CHO cell with the recognition sequence for CHO-23/24 underlined.
FIG. 7(D) depicts alignment of sequences obtained from CHO cells transfected with mRNA encoding the CHO-51/52 meganuclease. The top sequence is from a wild-type (WT) CHO cell with the recognition sequence for CHO-51/52 underlined.
FIG. 8(A) depicts a strategy for inserting an exogenous DNA sequence into the CHO DHFR locus using the CHO-51/52 meganuclease. CHO cells were co-transfected with mRNA encoding CHO-51/52 and a donor plasmid comprising an EcoRI site flanked by 543 base pairs of DNA sequence homologous to the region upstream of the CHO-51/52 recognition site and 461 base pairs of DNA sequence homologous to the region downstream of the CHO-51/52 recognition site. 48 hours post-transfection, genomic DNA was isolated and subjected to PCR using primers specific for the downstream region of the DHFR locus (dashed arrows).
FIG. 8(B) depicts PCR products that were cloned into pUC-19 and 48 individual plasmid clones and were digested with EcoRI and visualized on an agarose gel. 10 plasmids (numbered lanes) yielded a 647 base pair restriction fragment, consistent with cleavage of a first EcoRI site within the pUC-19 vector and a second EcoRI site in the cloned PCR fragment. These 10 plasmids were sequenced to confirm that they harbor a PCR fragment comprising a portion of the downstream DHFR locus with an EcoRI restriction site inserted into the CHO-51/52 recognition sequence. This restriction pattern was not observed when CHO cells were transfected with the donor plasmid alone.
FIG. 9(A) depicts a strategy for inserting an engineered target sequence into the CHO DHFR locus using the CHO-23/24 meganuclease. CHO cells were co-transfected with mRNA encoding CHO-23/24 and a donor plasmid comprising, in 5′ to 3′ orientation, an SV40 promoter, an ATG start codon, an FRT site, and a Zeocin-resistance (Zeo) gene. Zeocin-resistant cells were cloned by limiting dilution and screened by PCR to identify a clonal cell line in which the donor plasmid sequence integrated into the CHO-23/24 recognition site. After expansion, this cell line was co-transfected with a first plasmid encoding Flp recombinase operably linked to a promoter and second plasmid (donor plasmid #2) comprising a GFP gene under the control of a CMV promoter, an FRT site, and a hygromycin-resistance (Hyg) gene lacking a start codon. Flp-mediated recombination between FRT sites resulted in the integration of the donor plasmid #2 sequence into the engineered target sequence (i.e., the FRT site) such that a functional Hyg gene expression cassette was produced. FIG. 9(B) depicts PCR products from hygromycin-resistant clones produced as in (A) that were cloned by limiting dilution. Genomic DNA was extracted from 24 individual clones and PCR amplified using a first primer in the DHFR locus and a second primer in the Hyg gene (dashed lines). All 24 clones yielded a PCR product consistent with Hyg gene insertion into the engineered target sequence. FIG. 9(C) depicts GFP expression by the 24 clones produced in (B) using flow cytometry. All clones were found to express high levels of GFP with relatively little clone-to-clone variability.
FIG. 10. A GFP-expressing CHO line was produced by integrating a GFP gene expression cassette into the DHFR locus using an engineered target sequence strategy as shown in FIG. 9. This cell line was then grown in MTX as described in Example 2 to amplify the integrated GFP gene. (A) Flow cytometry plots showing GFP intensity on the Y-axis. In the pre-MTX cell line, GFP intensity averages approximately 2×103 whereas in the cell line grown in 250 nM MTX, a distinct sub-population is visible (circled) in which GFP intensity approaches 104. (B) MTX treated cell lines were sorted by FACS to identify individual cells expressing higher amounts of GFP. Five such high-expression cells were expanded and GFP intensity was determined by flow cytometry. All five clones were found to have significantly increased GFP expression relative to the pre-MTX cell line. (C) Genomic DNA was isolated from the five clonal cell lines produced in (B) and subjected to quantitative PCR using a primer pair specific for the GFP gene. It was found that the five high-expression clones had significantly more copies of the GFP gene than the pre-MTX cell line. These results demonstrate that the copy number and expression level a transgene integrated downstream of CHO DHFR can amplify in response to MTX treatment.
FIG. 11. (A) A direct-repeat recombination assay, as in FIG. 5A. (B) The assay in (A) applied to the CHO-13/14 and CGS-5/6 meganucleases. Light bars indicate the percentage of GFP+ cells when cells are transfected with the reporter plasmid alone (−endonuclease). Dark bars indicate the percentage of GFP+ cells when cells are co-transfected with a reporter plasmid and the corresponding meganuclease gene (+endonuclease). The assay was performed in triplicate and standard deviation is shown. (C) Alignment of sequences obtained from CHO cells transfected with mRNA encoding the CGS-5/6 meganuclease. The top sequence is from a wild-type (WT) CHO cell with the recognition sequence for CGS-5/6 underlined. Dashes indicate deleted bases. Bases that are italicized and in bold are point mutations or insertions relative to the wild-type sequence. Note that the mutations observed in at least clones 6d4, 6g5, 3b7, 3d11, 3e5, 6f10, 6hH8, 6d10, 6d7, 3g8, and 3a9 are expected to knockout GS gene function.
2.1.1 Gene Targeting to the CHO DHFR Locus The CHO DHFR locus is diagrammed in FIG. 2A. The locus comprises the DHFR gene coding sequence in 6 exons spanning ˜24,500 base pairs. The Msh3 gene is located immediately upstream of DHFR and is transcribed divergently from the same promoter as DHFR. A hypothetical gene, 2BE2121, can be found ˜65,000 base pairs downstream of the DHFR coding sequence. Thus, there is a ˜65,000 base pair region downstream of the DHFR gene that does not harbor any known genes and is a suitable location for targeting the insertion of sequences of interest. Target sites for insertion of a sequence of interest generally should not be selected which are <1,000 base pairs, and preferably not <5,000 base pairs from either the DHFR or 2BE2121 genes. This limits the window of preferred target sites to the region 1,000-60,000 base pairs, or 5,000-60,000 base pairs downstream of the DHFR coding sequence. The sequence of this region is provided as SEQ ID NO: 2.
The human and mouse DHFR loci have an organization similar to CHO locus. In both cases, the Msh3 gene is immediately upstream of DHFR but there is a large area devoid of coding sequences downstream of DHFR. In humans, the ANKRD34B gene is ˜55,000 base pairs downstream of DHFR while the ANKRD34B gene is ˜37,000 base pairs downstream of DHFR in mouse. Therefore, the genomic region downstream of DHFR is an appropriate location to insert genes of interest in CHO, human, and mouse cells and cell lines. Further, gene expression cassettes inserted into this region will be expressed at a high level, resistant to gene silencing, and capable of being amplified by treatment with MTX. Methods for amplifying the CHO cell DHFR locus are known in the art (see, e.g., Kellems, ed., Gene amplification in mammalian cells: a comprehensive guide. Marcel Dekker, New York, 1993) and typically involve gradually increasing the concentration of MTX in the growth media from 0 to as high as 0.8 mM over a period of several weeks.
2.1.2 Gene Targeting to the GS Locus The CHO, human, and mouse glutamine synthetase (also known as “glutamate-ammonia ligase” or “GluL”) loci share a common organization (FIG. 2B). The TEDDM1 gene is immediately upstream of GS in all species (˜5,000 bp upstream in the case of human, ˜7,000 bp upstream in the case of mouse and CHO). The closest downstream gene, however, is ˜46,000 away in the case of human and ˜117,000 bp away in the case of mouse and CHO. Therefore, we predict that the chromosomal region 1,000-41,000 bp, or 5,000-41,000 bp downstream of GS in human cells and 1,000-100,000 bp, or 5,000-100,000 bp downstream of GS in mouse and CHO cells are appropriate locations to target the insertion of sequences of interest. Because DNA sites distal to the GS coding sequence are more likely to be susceptible to gene silencing, the chromosomal region 5,000-60,000 bp downstream of GS is a preferred location to target the insertion of a sequence of interest even in mouse or CHO cells. The sequence of this region from the CHO genome is provided as SEQ ID NO: 3. Gene expression cassettes inserted into this region will be expressed at a high level, resistant to gene silencing, and capable of being amplified by treatment with MSX. Less-preferred regions include the chromosomal region between the TEDDM1 and GS genes or the region <10,000 bp downstream of TEDDM1 (see FIG. 2B). Methods for amplifying the GS locus are known in the art (Bebbington et al. (1992), Biotechnology (N Y). 10(2):169-75).
2.2 Engineered Endonucleases for Gene Targeting A sequence of interest may be inserted into an amplifiable locus using an engineered site-specific endonuclease. Methods for generating site-specific endonucleases which can target DNA breaks to pre-determined loci in a genome are known in the art. These include zinc-finger nucleases (Le Provost et al. (2010), Trends Biotechnol. 28(3):134-41), TAL-effector nucleases (Li et al. (2011), Nucleic Acids Res. 39(1):359-72), and engineered meganucleases (WO 2007/047859; WO 2007/049156; WO 2009/059195). In one embodiment, the invention provides engineered meganucleases derived from I-CreI that can be used to target the insertion of a gene of interest to an amplifiable locus. Methods to produce such engineered meganucleases are known in the art (see, e.g., WO 2007/047859; WO 2007/049156; WO 2009/059195). In preferred embodiments, a “single-chain” meganuclease is used to target gene insertion to an amplifiable region of the genome. Methods for producing such “single-chain” meganucleases are known in the art (see, e.g., WO 2009/059195 and WO 2009/095742). In some embodiments, the engineered nuclease is fused to a nuclear localization signal (NLS) to facilitate nuclear uptake. Examples of nuclear localization signals include the SV40 NLS (amino acid sequence MAPKKKRKV (SEQ ID NO: 36)) which can be fused to the C- or, preferably, the N-terminus of the protein. In addition, an engineered nuclease may be tagged with a peptide epitope (e.g., an HA, FLAG, or Myc epitope) to monitor expression levels or localization or to facilitate purification.
2.3 Engineered Cell Lines with Sequences of Interest Targeted to Amplifiable Loci In some embodiments, the invention provides methods for using engineered nucleases to target the insertion of transgenes into amplifiable loci in cultured mammalian cells. This method has two primary components: (1) an engineered nuclease; and (2) a donor DNA molecule comprising a sequence of interest. The method comprises contacting the DNA of the cell with the engineered nuclease to create a double strand DNA break in an endogenous recognition sequence in an amplifiable locus followed by the insertion of the donor DNA molecule at the site of the DNA break. Such insertion of the donor DNA is facilitated by the cellular DNA-repair machinery and can occur by either the non-homologous end-joining pathway or by homologous recombination (FIG. 1).
The engineered nuclease can be delivered to the cell in the form protein or, preferably, as a nucleic acid encoding the engineered nuclease. Such nucleic acid can be DNA (e.g., circular or linearized plasmid DNA or PCR products) or RNA. For embodiments in which the engineered nuclease coding sequence is delivered in DNA form, it should be operably linked to a promoter to facilitate transcription of the engineered nuclease gene. Mammalian promoters suitable for the invention include constitutive promoters such as the cytomegalovirus early (CMV) promoter (Thomsen et al. (1984), Proc Natl Acad Sci USA. 81(3):659-63) or the SV40 early promoter (Benoist and Chambon (1981), Nature. 290(5804):304-10) as well as inducible promoters such as the tetracycline-inducible promoter (Dingermann et al. (1992), Mol Cell Biol. 12(9):4038-45).
In some embodiments, mRNA encoding the engineered nuclease is delivered to the cell because this reduces the likelihood that the gene encoding the engineered nuclease will integrate into the genome of the cell. Such mRNA encoding an engineered nuclease can be produced using methods known in the art such as in vitro transcription. In some embodiments, the mRNA is capped using 7-methyl-guanosine. In some embodiments, the mRNA may be polyadenylated.
Purified engineered nuclease proteins can be delivered into cells to cleave genomic DNA, which allows for homologous recombination or non-homologous end-joining at the cleavage site with a sequence of interest, by a variety of different mechanisms known in the art. For example, the recombinant nuclease protein can be introduced into a cell by techniques including, but not limited to, microinjection or liposome transfections (see, e.g., Lipofectamine™, Invitrogen Corp., Carlsbad, Calif.). The liposome formulation can be used to facilitate lipid bilayer fusion with a target cell, thereby allowing the contents of the liposome or proteins associated with its surface to be brought into the cell. Alternatively, the enzyme can be fused to an appropriate uptake peptide such as that from the HIV TAT protein to direct cellular uptake (see, e.g., Hudecz et al. (2005), Med. Res. Rev. 25: 679-736).
Alternatively, gene sequences encoding the engineered nuclease protein are inserted into a vector and transfected into a eukaryotic cell using techniques known in the art (see, e.g., Ausubel et al., Current Protocols in Molecular Biology, Wiley 1999). The sequence of interest can be introduced in the same vector, a different vector, or by other means known in the art. Non-limiting examples of vectors for DNA transfection include virus vectors, plasmids, cosmids, and YAC vectors. Transfection of DNA sequences can be accomplished by a variety of methods known to those of skill in the art. For instance, liposomes and immunoliposomes are used to deliver DNA sequences to cells (see, e.g., Lasic et al. (1995), Science 267: 1275-76). In addition, viruses can be utilized to introduce vectors into cells (see, e.g., U.S. Pat. No. 7,037,492). Alternatively, transfection strategies can be utilized such that the vectors are introduced as naked DNA (see, e.g., Rui et al. (2002), Life Sci. 71(15): 1771-8).
General methods for delivering nucleic acids into cells include: (1) chemical methods (Graham et al. (1973), Virology 54(2):536-539; Zatloukal et al. (1992), Ann. N.Y. Acad. Sci., 660:136-153; (2) physical methods such as microinjection (Capecchi (1980), Cell 22(2):479-488, electroporation (Wong et al. (1982), Biochim. Biophys. Res. Commun. 107(2):584-587; Fromm et al. (1985), Proc. Nat'l Acad. Sci. USA 82(17):5824-5828; U.S. Pat. No. 5,384,253) and ballistic injection (Johnston et al. (1994), Methods Cell. Biol. 43(A): 353-365; Fynan et al. (1993), Proc. Nat'l Acad. Sci. USA 90(24): 11478-11482); (3) viral vectors (Clapp (1993), Clin. Perinatol. 20(1): 155-168; Lu et al. (1993), J. Exp. Med. 178(6):2089-2096; Eglitis et al. (1988), Avd. Exp. Med. Biol. 241:19-27; Eglitis et al. (1988), Biotechniques 6(7):608-614); and (4) receptor-mediated mechanisms (Curiel et al. (1991), Proc. Nat'l Acad. Sci. USA 88(19):8850-8854; Curiel et al. (1992), Hum. Gen. Ther. 3(2):147-154; Wagner et al. (1992), Proc. Nat'l Acad. Sci. USA 89 (13):6099-6103). In some preferred embodiments, 7-methyl-guanosine capped mRNA encoding the engineered nuclease is delivered to cells using electroporation.
The donor DNA molecule comprises a gene of interest operably linked to a promoter. In many cases, a donor molecule may comprise multiple genes operably linked to the same or different promoters. For example, donor molecules comprising monoclonal antibody expression cassettes may comprise a gene encoding the antibody heavy chain and a second gene encoding the antibody light chain. Both genes may be under the control of different promoters or they may be under the control of the same promoter by using, for example, an internal-ribosome entry site (IRES). Donor molecules may also comprise a selectable marker gene operably linked to a promoter to facilitate the identification of transgenic cells. Such selectable markers are known in the art and include neomycin phosphotransferase (NEO), hypoxanthine phosphoribosyltransferase (HPRT), glutamine synthetase (GS), dihydrofolate reductase (DHFR), and hygromycin phosphotransferase (HYG) genes.
In some embodiments, donor DNA molecules will additionally comprise flanking sequences homologous to the target sequences in the DNA of the cell. Such homologous flanking sequences comprise >3 or, preferably, >50 or, more preferably, >200 or, most preferably, >400 base pairs of DNA that are identical or nearly identical in sequence to the chromosomal locus recognized by the engineered nuclease (FIG. 1). Such homologous DNA sequences facilitate the integration of the donor DNA sequence into the amplifiable locus by homologous recombination.
The “donor” DNA molecule can be circular (e.g., plasmid DNA) or linear (e.g., linearized plasmid or PCR products). Methods for delivering DNA molecules are known in the art, as discussed above.
In some embodiments, the engineered nuclease gene and donor DNA are carried on separate nucleic acid molecules which are co-transfected into cells or cell lines. For example, the engineered nuclease gene operably linked to a promoter can be transfected in plasmid form simultaneously with a separate donor DNA molecule in plasmid or PCR product form. In an alternative embodiment, the engineered nuclease can be delivered in mRNA form with a separate donor DNA molecule in plasmid or PCR product form. In a third embodiment, the engineered nuclease gene and donor DNA are carried on the same DNA molecule, such as a plasmid. In a fourth embodiment, cells are co-transfected with purified engineered nuclease protein and a donor DNA molecule in plasmid or PCR product form.
Following transfection with the engineered nuclease and donor DNA, cells are typically allowed to recover from transfection (24-72 hours) before being cloned using methods known in the art. Common methods for cloning a genetically engineered cell line include “limiting dilution” in which transfected cells are transferred to tissue culture plates (e.g., 48 well, 96 well plates) at a concentration of <1 cell per well and expanded into clonal populations. Other cloning strategies include robotic clone identification/isolation systems such as ClonePix™ (Genetix, Molecular Devices, Inc., Sunnyvale, Calif.). Clonal cell lines can then be screened to identify cell lines in which the sequence of interest is integrated into the intended target site. Cell lines can easily be screened using molecular analyses known in the art such as PCR or Southern Blot. For example, genomic DNA can be isolated from a clonal cell line and subjected to PCR amplification using a first (sense-strand) primer that anneals to a DNA sequence in the sequence of interest and a second (anti-sense strand) primer that anneals to a sequence in the amplifiable locus. If the donor DNA molecule comprises a DNA sequence homologous to the target site, it is important that the second primer is designed to anneal to a sequence in the amplifiable locus that is beyond the limits of homology carried on the donor molecule to avoid false positive results. Alternatively, cell lines can be screened for expression of the sequence of interest. For example, if the sequence of interest encodes a secreted protein such as an antibody, the growth media can be sampled from isolated clonal cell lines and assayed for the presence of antibody protein using methods known in the art such as Western Blot or Enzyme-Linked Immunosorbant Assay (ELISA). This type of functional screen can be used to identify clonal cell lines which carry at least one copy of the sequence of interest integrated into the genome. Additional molecular analyses such as PCR or Southern blot can then be used to determine which of these transgenic cell lines carry the sequence of interest targeted to the amplifiable locus of interest, as described above.
The method of the invention can be used on any culturable and transfectable cell type such as immortalized cell lines and stem cells. In preferred embodiments, the method of the invention is used to genetically modify immortalized cell lines that are commonly used for biomanufacturing. This includes:
-
- 1. Hamster cell lines such as baby hamster kidney (BHK) cells and all variants of Chinese Hamster Ovary (CHO) cells, e.g., CHO-K1, CHO-S (Invitrogen Corp., Carlsbad, Calif.), DG44, or Potelligent™ (Lonza Group Ltd., Basel, Switzerland). Because the genome sequences of different hamster cell lines are very nearly identical, an engineered meganuclease which can be used to practice the invention in one hamster cell type (e.g., BHK cells) can generally be used to practice the invention in another hamster cell type (e.g., CHO-K1).
- 2. Mouse cell lines such as mouse hybridoma or mouse myeloma (e.g., NS0) cells. Because the genome sequences of different mouse cell lines are very nearly identical, an engineered meganuclease which can be used to practice the invention in one mouse cell type (e.g., mouse hybridoma cells) can generally be used to practice the invention in another mouse cell type (e.g., NS0).
- 3. Human cell lines such as human embryonic kidney cells (e.g., HEK-293 or 293S) and human retinal cells (e.g., PER.C6). Because the genome sequences of different human cell lines are very nearly identical, an engineered meganuclease which can be used to practice the invention in one human cell type (e.g., HEK-293 cells) can generally be used to practice the invention in another human cell type (e.g., PER.C6).
2.6 Pre-Engineered Cell Lines with Engineered Target Sequences in Amplifiable Loci In one embodiment, the invention provides cell lines which are pre-engineered to comprise a targetable “engineered target sequence” for gene insertion in an amplifiable locus in a mammalian cell line (FIG. 3). An engineered target sequence comprises a recognition sequence for an enzyme which is useful for inserting transgenic nucleic acids into chromosomal DNA sequences. Such engineered target sequences can include recognition sequences for engineered meganucleases derived from I-CreI (e.g., SEQ ID NO 37-87 from WO 2009/076292), recognition sequences for zinc-finger nucleases, recognition sequences for TAL effector nucleases (TALENs), the LoxP site (SEQ ID NO 4) which is recognized by Cre recombinase, the FRT site (SEQ ID NO: 5) which is recognized by FLP recombinase, the attB site (SEQ ID NO: 6) which is recognized by lambda recombinase, or any other DNA sequence known in the art that is recognized by a site specific endonuclease, recombinase, integrase, or transpose that is useful for targeting the insertion of nucleic acids into a genome. Thus, the invention allows one skilled in the art to use an engineered nuclease (e.g., a meganuclease, zinc-finger nuclease, or TAL effector nuclease) to insert an engineered target sequence into an amplifiable locus in a mammalian cell line. The resulting cell line comprising such an engineered target sequence at an amplifiable locus can then be contacted with the appropriate enzyme (e.g., a second engineered meganuclease, a second zinc-finger nuclease, a second TAL effector nuclease, a recombinase, an integrase, or a transposase) to target the insertion of a gene of interest into the amplifiable locus at the engineered target sequence. This two-step approach can be advantageous because the efficiency of gene insertion that can be achieved using an optimal meganuclease, zinc-finger nuclease, recombinase, integrase, or transposase might be higher than what can be achieved using the initial endonuclease (e.g., meganuclease or zinc-finger nuclease) that cleaves the endogenous target site to promote insertion of the engineered target sequence.
In an alternative embodiment, a cell line is produced by inserting an engineered target sequence into an amplifiable locus with the concomitant removal of all or a portion of the adjacent endogenous marker gene (FIG. 4). For example, an engineered meganuclease, zinc-finger nuclease, or TAL-effector nuclease can be used to remove the first two exons of both alleles of the CHO DHFR gene and replace them with an engineered target sequence for a different engineered meganuclease, ZFN, TALEN, recombinase, integrase, or transposase. The resulting cell line will be DHFR deficient and unable to grow in the absence of hypoxanthine/thymidine. Alternatively, for example, an engineered meganuclease, ZFN or TALEN can be used to remove the first exon of both alleles of the CHO GS gene and replace it with an engineered target sequence for a different engineered meganuclease, ZFN, TALEN, recombinase, integrase, or transposase (FIG. 4). The resulting cell line will be GS deficient and unable to grow in the absence of L-glutamine. Such a cell line is useful because a gene of interest can be inserted into the engineered target sequence in the pre-engineered cell line while simultaneously reconstituting the selectable gene (e.g., DHFR or GS). Thus, it is possible to select for transfectants harboring the gene of interest at the amplifiable locus using media conditions that select for DHFR+ or GS+ cells.
In an alternative embodiment, a cell line is produced in which an engineered target sequence is inserted into an amplifiable locus with disruption of the selectable gene (FIGS. 5, 6). This can be accomplished, for example, using a meganuclease which recognizes a DNA site in the coding sequence of the selectable gene. Such a meganuclease can be used to target the insertion of an engineered target sequence into the selectable gene coding sequence resulting in disruption of gene function by, for example, introducing a frameshift (FIG. 5). Alternatively, for example, an engineered target sequence can be inserted into an intron in the selectable gene sequence with an additional sequence that promotes improper processing of the selectable gene transcript (FIG. 6). Such sequences that promote improper processing include, for example, artificial splice acceptors or polyadenylation signals. Splice acceptor sequences are known in the art (Clancy (2008), “RNA Splicing: Introns, Exons and Spliceosome,” Nature Education 1:1) and typically comprise a 20-50 base pair pyrimidine-rich sequence followed by a sequence (C/T)AG(A/G). SEQ ID NO: 33 is an example of a splice acceptor sequence. Likewise, polyadenylation signals are known in the art and include, for example, the SV40 polyadenylation signal (SEQ ID NO: 34) and the BGH polyadenylation signal (SEQ ID NO: 35). In some embodiments, the resulting cell line harboring the new engineered target sequence in all alleles of the selectable gene will be deficient in the function of the gene due to mis-transcription or mis-translation and will be able to grow only under permissive conditions. For example, an engineered target sequence can be inserted into the GS gene sequence using a meganuclease resulting in a cell line that is GS−/− that can grow only in the presence of L-glutamine in the growth media. In a subsequent step, a gene of interest can be inserted into the engineered target sequence while simultaneously reconstituting the selectable gene (e.g., DHFR or GS). Thus, it is possible to select for transfectants harboring the gene of interest at the amplifiable locus using media conditions that select for DHFR+ or GS+ cells.
2.5 Transgenic Cell Lines for Biomanufacturing In some embodiments, the invention provides transgenic cell lines suitable for the production of protein pharmaceuticals. Such transgenic cell lines comprise a population of cells in which a gene of interest, operably linked to a promoter, is inserted into the genome of the cell at an amplifiable locus wherein the gene of interest encodes a protein therapeutic. Examples of protein therapeutics include: monoclonal antibodies, antibody fragments, erythropoietin, tissue-type plasminogen activator, Factor VIII, Factor IX, insulin, colony stimulating factors, interferons (e.g., interferon-α, interferon-β, and interferon-γ), interleukins (e.g., interleukin-2), vaccines, tumor necrosis factor, and glucocerebrosidase. Protein therapeutics are also referred to as “biologics” or “biopharmaceuticals.”
To be used for biomanufacturing, a transgenic cell line of the invention should undergo: (1) adaptation to serum-free growth in suspension; and (2) amplification of the gene of interest. In some embodiments, the invention is practiced on adherent cell lines which can be adapted to growth in suspension to facilitate their maintenance in shaker-flasks or stirred-tank bioreactors as is typical of industrial biomanufacturing. Methods for adapting adherent cells to growth in suspension are known in the art (Cell Culture and Upstream Processing, Butler, ed. (Taylor and Francis Group, New York, 2007)). For regulatory reasons, it is generally necessary to further adapt biomanufacturing cell lines to chemically-defined media lacking animal-derived components (i.e., “serum-free” media). Methods for preparing such media and adapting cell lines to it are known in the art (Cell Culture and Upstream Processing, Butler, ed. (Taylor and Francis Group, New York, 2007)). Such media can also be purchased commercially (e.g., CD-3 media for maintenance of CHO cells, available from Sigma-Aldrich, St. Louis, Mo.) and cells can be adapted to it by following the manufacturers' instructions. In some embodiments, the cell line is adapted to growth in suspension and/or serum-free media prior to being transfected with the engineered nuclease.
Lastly, methods for gene amplification are known in the art (Cell Culture and Upstream Processing, Butler, ed. (Taylor and Francis Group, New York, 2007)). In general, the process involves adding an inhibitor of a selectable gene product to the growth media to select for cells that express abnormally high amounts of the gene product due to gene-duplication events. In general, the concentration of inhibitor added to the growth media is increased slowly over a period of weeks until the desired level of gene amplification is achieved. Inhibitor is then generally removed from the media prior to initiating a bioproduction run to avoid the possibility of the inhibitor contaminating the protein therapeutic formulation. For example, the CHO DHFR locus can be amplified by slowly increasing the concentration of MTX in the growth media from 0 mM to as high as 0.8 mM over a period of several weeks. The GS locus can, likewise, be amplified by slowly increasing the concentration of MSX in the media from 0 μM to as high as 100 μM over a period of several weeks. Methods for evaluating gene amplification are known in the art and include Southern Blot and quantitative real-time PCR (rtPCR). In addition, or as an alternative, expression levels of the sequence of interest, which are generally correlated to gene copy number, can be evaluated by determining the concentration of protein therapeutic in the growth media using conventional methods such as Western Blot or ELISA.
Following cell line production, adaptation, and amplification, protein therapeutics can be produced and purified using methods that are standard in the biopharmaceutical industry.
EXAMPLES This invention is further illustrated by the following examples, which should not be construed as limiting. Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein. Such equivalents are intended to be encompassed in the scope of the claims that follow the examples below. Example 1 refers to engineered meganucleases that can be used to target the insertion of a gene of interest downstream of the DHFR gene in CHO cells. Example 2 refers to engineered meganucleases that can be used to target the insertion of an engineered target sequence into the CHO DHFR gene with concomitant removal of DHFR exons 1 and 2. Example 2 also refers to engineered meganucleases that can be used to target the insertion of an engineered target sequence into the CHO GS gene. Example 3 refers to meganucleases that can be used to target the insertion of a gene of interest downstream of the GS gene in CHO cells.
Example 1 Targeted Gene Insertion into the CHO DHFR Locus Using Engineered Meganucleases
The CHO genomic DNA sequence 10,000-55,000 base pairs downstream of the DHFR gene was searched to identify DNA sites amenable to targeting with engineered meganucleases. Two sites (SEQ ID NO: 7 and SEQ ID NO: 8) were selected which are, respectively, 35,699 and 15,898 base pairs downstream of the DHFR coding sequence (Table 2).
TABLE 2
Example Recognition Sites For Engineered Meganucleases in
the CHO DHFR Locus.
SEQ ID Location Relative to CHO
NO: Target Site Sequences DHFR Coding Sequence
7 5′-TAAGGCCTCATATGAAAATATA-3′ 35,699 bp downstream
8 5′-ATAGATGTCTTGCATACTCTAG-3′ 15,898 bp downstream
1. Meganucleases that Recognize SEQ ID NO: 7 and SEQ ID NO: 8
An engineered meganuclease (SEQ ID NO: 9) was produced which recognizes and cleaves SEQ ID NO: 7. This meganuclease is called “CHO-23/24”. A second engineered meganuclease (SEQ ID NO: 10) was produced which recognizes and cleaves SEQ ID NO: 8. This meganuclease is called “CHO-51/52.” Each meganuclease comprises an N-terminal nuclease-localization signal derived from SV40, a first meganuclease subunit, a linker sequence, and a second meganuclease subunit.
2. Site-Specific Cleavage of Plasmid DNA by Meganucleases CHO-23/24 and CHO-51/52 CHO-23/24 and CHO-51/52 were evaluated using a direct-repeat recombination assay as described previously (Gao et al. (2010), Plant J. 61(1):176-87, FIG. 7A). A defective GFP reporter cassette was generated by first cloning a 5′ 480 bp fragment of the GFP gene into NheI/HindIII-digested pcDNA5/FRT (Invitrogen Corp., Carlsbad, Calif.) resulting in the plasmid pGF. Next, a 3′ 480 bp fragment of the GFP gene (including a 240 bp sequence duplicated in the 5′ 480 bp fragment) was cloned into BamHI/XhoI-digested pGF. The resulting plasmid, pGFFP, consists of the 5′ two-thirds of the GFP gene followed by the 3′ two-thirds of the GFP gene, interrupted by 24 bp of the pcDNA5/FRT polylinker. To insert the meganuclease recognition sites, complementary oligonucleotides comprising the sense and anti-sense sequence of each recognition site were annealed and ligated into HindIII/BamHI-digested pGFFP.
The coding sequences of the engineered meganucleases were inserted into the mammalian expression vector pCP under the control of a constitutive (CMV) promoter. Chinese hamster ovary (CHO) cells at approximately 90% confluence were transfected in 96-well plates with 150 ng pGFFP reporter plasmid and 50 ng of meganuclease expression vector or, to determine background, 50 ng of empty pCP, using Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen Corp., Carlsbad, Calif.). To determine transfection efficiency, CHO cells were transfected with 200 ng pCP GFP. Cells were washed in PBS 24 h post-transfection, trypsinized and resuspended in PBS supplemented with 3% fetal bovine serum. Cells were assayed for GFP activity using a Cell Lab Quanta SC MPL flow cytometer and the accompanying Cell Lab Quanta analysis software (Beckman Coulter, Brea, Calif.).
Results are shown in FIG. 7B. It was found that both of the engineered meganucleases were able to cleave their intended recognition sites significantly above background within the context of a plasmid-based reporter assay.
3. Site-Specific Cleavage of CHO DHFR Locus by Meganucleases CHO-23/24 and CHO-51/52 To determine whether or not CHO-23/24 and CHO-51/52 are capable of cleaving their intended target sites in the CHO DHFR locus, we screened genomic DNA from CHO cells expressing either CHO-23/24 or CHO-51/52 to identify evidence of chromosome cleavage at the intended target site. This assay relies on the fact that chromosomal DNA breaks are frequently repaired by NHEJ in a manner that introduces mutations at the site of the DNA break. These mutations, typically small deletions or insertions (collectively known as “indels”) leave a telltale scar that can be detected by DNA sequencing (Gao et al. (2010), Plant J. 61(1):176-87).
CHO cells were transfected with mRNA encoding CHO-23/24 or CHO-51/52. mRNA was prepared by first producing a PCR template for an in vitro transcription reaction (SEQ ID NO: 20 and SEQ ID NO: 21). Each PCR product included a T7 promoter and 609 bp of vector sequence downstream of the meganuclease gene. The PCR product was gel purified to ensure a single template. Capped (m7G) RNA was generated using the RiboMAX T7 kit (Promega Corp., Fitchburg, Wis.) according to the manufacturer's instructions and. Ribo m7G cap analog (Promega Corp., Fitchburg, Wis.) was included in the reaction and 0.5 μg of the purified meganuclease PCR product served as the DNA template. Capped RNA was purified using the SV Total RNA Isolation System (Promega Corp., Fitchburg, Wis.) according to the manufacturer's instructions.
1.5×106CHO-K1 cells were nucleofected with 3×1012 copies of CHO-23/24 or CHO-51/52 mRNA (2×106 copies/cell) using an Amaxa Nucleofector II device (Lonza Group Ltd., Basel, Switzerland) and the U-23 program according to the manufacturer's instructions. 48 hours post-transfection, genomic DNA was isolated from the cells using a FlexiGene kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The genomic DNA was then subjected to PCR to amplify the corresponding target site. In the case of cells transfected with mRNA encoding CHO-23/24, the forward and reverse PCR primers were SEQ ID NO: 16 and SEQ ID NO: 17. In the case of cells transfected with mRNA encoding CHO-51/52, the forward and reverse PCR primers were SEQ ID NO: 18 and SEQ ID NO: 19. PCR products were gel purified and cloned into pUC-19. 40 plasmids harboring PCR products derived from cells transfected with CHO-23/24 mRNA were sequenced, 13 of which were found to have mutations in the CHO-23/24 target site (FIG. 7C). 44 plasmids harboring PCR products derived from cells transfected with CHO-51/52 mRNA were sequenced, 10 of which were found to have mutations in the CHO-51/52 target site (FIG. 7D). These results indicate that CHO-23/24 and CHO-51/52 are able to cut their intended target sites downstream of the CHO DHFR gene.
4. Site-Specific Integration into the CHO DHFR Locus Using an Engineered Meganuclease
To evaluate the efficiency of DNA insertion into the CHO DHFR locus using an engineered meganuclease, we prepared a donor plasmid (SEQ ID NO: 11) comprising an EcoRI restriction enzyme site flanked by DNA sequence homologous to the CHO-51/52 recognition site (FIG. 8A). Specifically, the donor plasmid of SEQ ID NO: 11 comprises a pUC-19 vector harboring a homologous recombination cassette inserted between the KpnI and HindIII restriction sites. The homologous recombination cassette comprises, in 5′- to 3′-order: (i) 543 base pairs of DNA identical to the sequence immediately upstream of the CHO-51/52 cut site, including the upstream half-site of the CHO-51/52 recognition sequence and the four base pair “center sequence” separating the two half-sites comprising the CHO-51/52 recognition sequence; (ii) an EcoRI restriction enzyme site (5′-GAATTC-3′); and iii) 461 base pairs of DNA identical to the sequence immediately downstream of the CHO-51/52 cut site, including the downstream half-site of the CHO-51/52 recognition sequence and the four base pair “center sequence” separating the two half-sites comprising the CHO-51/52 recognition sequence. Note that this results in a duplication of the four base pair “center sequence” (5′-TTGC-3′) to maximize the likelihood of strand invasion by the 3′ overhangs generated by CHO-51/52 cleavage. We have discovered that donor plasmids comprising such a duplication of the center sequence are optimal substrates for gene targeting by homologous recombination.
mRNA encoding CHO-51/52 was prepared as described above. 1.5×106 CHO-K1 cells were nucleofected with 3×1012 copies of CHO 51-52 mRNA (2×106 copies/cell) and 1.5 μg of the donor plasmid (SEQ ID NO: 11). Nucleofection was performed using an Amaxa Nucleofector II device (Lonza Group Ltd., Basel, Switzerland) and the U-23 program according to the manufacturer's instructions. 48 hours post-transfection, genomic DNA was isolated from the cells using a FlexiGene kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The DNA was subjected to PCR using primers flanking the CHO-51/52 recognition site (SEQ ID NO: 18 and SEQ ID NO: 19). Importantly, these primers are beyond the limits of homologous sequence carried in the donor plasmid and, therefore, will amplify only the chromosomal DNA sequence and not the donor plasmid. PCR products were cloned into a pUC-19 plasmid and 48 clones were purified and digested with EcoRI (FIG. 8B). 10 plasmids yielded a restriction pattern consistent with the insertion of an EcoRI site into the CHO-51/52 recognition sequence. These data demonstrate that it is possible to use CHO-51/52 to precisely insert DNA downstream of the CHO DHFR gene at SEQ ID NO: 8.
5. Site-Specific Integration of an Engineered Target Sequence into the CHO DHFR Locus
A donor plasmid (SEQ ID NO: 25) was produced comprising an FRT sequence (SEQ ID NO: 5) adjacent to a zeocin resistance gene under the control of an SV40 early promoter (FIG. 9A). This cassette was flanked by DNA sequence homologous to the CHO DHFR locus immediately upstream or downstream of the CHO-23/24 recognition sequence. CHO cells were co-transfected with this donor plasmid and mRNA encoding CHO-23/24 as described above. 72 hours post-transfection, zeocin-resistant cells were cloned by limiting dilution and expanded for approximately 3 weeks. Clonal populations were then screened by PCR using a first primer in the SV40 promoter (SEQ ID NO: 26) and a second primer in the DHFR locus (SEQ ID NO: 16) to identify cell lines carrying the FRT/Zeocin sequence downstream of the DHFR gene. One such cell line carrying the integrated FRT Insertion target sequence was subsequently co-transfected with a second donor plasmid (SEQ ID NO: 27) and a plasmid encoding Flp recombinase. SEQ ID NO: 27 comprises a GFP gene under the control of a CMV promoter, a FRT sequence, and a non-functional hygromycin resistance gene lacking an ATG start codon. Flp-stimulated recombination between FRT sites in the genome and the plasmid resulted in the incorporation of the entire plasmid sequence into the CHO genome at the site of the engineered target sequence. Such recombination restored function to the hygromycin-resistance gene by orientating it downstream of an ATG start codon integrated as part of the engineered target sequence. As such, successful integrations could be selected using hygromycin.
Hygromycin-resistant cells were cloned by limiting dilution and 24 individual clonal lines were assayed by PCR using a first primer in the hygromycin-resistance gene (SEQ ID NO: 28). All 24 clones yielded the expected PCR product (FIG. 9B), indicating that the GFP gene expression cassette was successfully inserted into the DHFR engineered target sequence in all cases. The 24 cell lines were then evaluated by flow cytometry and were found to express consistent levels of GFP (FIG. 9C).
6. Transgene Amplification A GFP-expressing CHO line produced as described above was seeded at a density of 3×105 cells/mL in 30 mL of media containing 50 nM MTX. Cells were cultured for 14 days before being re-seeded at the same density in media containing 100 nM MTX. Cells were cultured for another 14 days before being re-seeded in media containing 250 nM MTX. Following 14 days in culture, GFP expression in the treated cells was evaluated by flow cytometry and compared to GFP expression in the parental (pre-MTX) cell population (FIG. 10A). It was found that the MTX-treated cells had a distinct sub-population in which GFP expression was significantly increased. Individual high-expression cells from the MTX-treated population were then isolated using a cell sorter and 5 clones were expanded for 14 days in the absence of MTX. GFP expression in the 5 clonal cell populations was then evaluated by flow cytometry and compared with the parental (pre-MTX) cell population. It was found that the MTX-treated clones had approximately 4-6 times the GFP intensity as the pre-MTX cells. Quantitative PCR was then performed using a primer set specific for the GFP gene and it was found that the MTX-treated clones all had approximately 5-9 times as many copies of the GFP gene as the pre-MTX population. These data provide conclusive evidence that a transgene inserted downstream of the CHO DHFR gene can be amplified by treatment with MTX.
7. Stability of Gene Amplification The five clonal cell lines expressing high levels of GFP that were produced in (6) above were then passaged for a period of 14 weeks in media with or without 250 nM MTX to evaluate the stability of gene amplification. GFP intensity was determined on a weekly basis and the quantitative PCR assay used to determine GFP gene copy number described above was repeated at the end of the 14 week evaluation period. As expected, the clones passaged in media with MTX maintained a high level of GFP expression with no clone deviating more than 20% from the GFP intensity determined in week 1. Quantitative PCR revealed that gene copy number likewise deviated by less than 20% for all clones. Surprisingly, gene amplification was equally stable in cell lines grown in media lacking MTX. Contrary to what would have been predicted based on the existing art, GFP gene expression was not reduced by more than 18% in any of the five cell lines over the 14 week evaluation period. Gene copy number determined by quantitative PCR was also stable with less than 24% deviation over time for all of the cell lines. These results indicate that a transgene amplified in the CHO DHFR locus is stable for an extended period of time, obviating the need to grow the cells in toxic selection agents that that could contaminate bioproduct formulations.
Example 2 Insertion of an Engineered Target Sequence into the CHO DHFR or GS Gene Coding Regions As diagrammed in FIG. 4, an alternative method for targeting a sequence of interest to an amplifiable locus involves the production of a cell line in which a portion of a selectable gene is replaced by an engineered target sequence. The advantage of this approach is that the subsequent insertion of a sequence of interest can be coupled with reconstitution of the selectable gene so that cell lines harboring the properly targeted sequence of interest can be selected using the appropriate media conditions. A cell line harboring such an engineered target sequence can be produced using nuclease-induced homologous recombination. In this case, a site-specific endonuclease which cuts a recognition sequence near or within the selectable gene sequence is preferred.
1. Engineered Meganucleases that Cut within the DHFR or GS Genes.
A meganuclease called “CHO-13/14” (SEQ ID NO: 12) was produced which cuts a recognition sequence in the CHO DHFR gene (SEQ ID NO: 13). The recognition sequence is in an intron between Exon 2 and Exon 3 of CHO DHFR. A meganuclease called “CGS-5/6” (SEQ ID NO: 14) was produced which cuts a recognition sequence in the CHO GS gene (SEQ ID NO: 15). Each meganuclease comprises an N-terminal nuclease-localization signal derived from SV40, a first meganuclease subunit, a linker sequence, and a second meganuclease subunit.
2. Site-Specific Cleavage of Plasmid DNA by Meganucleases CHO-13/14 and CGS-5/6 CHO-13/14 and CGS-5/6 were evaluated using a direct-repeat recombination assay as described in Example 1 (FIG. 7A). Both meganucleases were found to efficiently cleave their intended recognition sequences within the context of a plasmid-based reporter assay (FIG. 7B).
3. Site-Specific Cleavage of the CHO GS Gene by CGS-5/6 CHO cells were transfected with mRNA encoding CGS-5/6. mRNA was prepared by first producing a PCR template for an in vitro transcription reaction (SEQ ID NO: 22). Each PCR product included a T7 promoter and 609 bp of vector sequence downstream of the meganuclease gene. The PCR product was gel purified to ensure a single template. Capped (m7G) RNA was generated using the RiboMAX T7 kit (Promega Corp., Fitchburg, Wis.) according to the manufacturer's instructions and. Ribo m7G cap analog (Promega Corp., Fitchburg, Wis.) was included in the reaction and 0.5 μg of the purified meganuclease PCR product served as the DNA template. Capped RNA was purified using the SV Total RNA Isolation System (Promega Corp., Fitchburg, Wis.) according to the manufacturer's instructions.
1.5×106 CHO-K1 cells were nucleofected with 3×1012 copies of CGS-5/6 using an Amaxa Nucleofector II device (Lonza Group Ltd., Basel, Switzerland) and the U-23 program according to the manufacturer's instructions. 48 hours post-transfection, genomic DNA was isolated from the cells using a FlexiGene kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The genomic DNA was then subjected to PCR to amplify the CGS-5/6 target site using the primers of SEQ ID NO: 23 and SEQ ID NO: 24. The PCR products were cloned into a pUC-19 plasmid and 94 plasmids harboring PCR products were digested with the BssSI restriction enzyme, which recognized and cuts the sequence 5′-CTCGTG-3′ found within the CGS-5/6 recognition sequence. 17 plasmids were found to be resistant to BssSI, suggesting that the CGS-5/6 recognition site was mutated. These 17 plasmids were sequenced to confirm the existence of indels or point mutations within the CGS-5/6 recognition sequence (FIG. 7C). These results indicate that CGS-5/6 is able to cut its intended target site within the CHO GS gene. Because the CGS-5/6 recognition sequence is within an exon in the GS coding sequence, many of the mutations introduced by CGS-5/6 are expected to frameshift the GS gene. Therefore, CGS-5/6 is useful for knocking-out CHO GS to produce GS (−/−) cell lines. Such cell lines are useful because they are amenable to GS selection and amplification for producing biomanufacturing cell lines.
Example 3 Meganucleases for Targeting Gene Insertion to the CHO GS Locus 1. Engineered Meganucleases that Cut Downstream of the CHO GS Gene.
An engineered meganuclease called “CHOX-45/46” (SEQ ID NO: 29) was produced which recognizes a DNA sequence (SEQ ID NO: 30) approximately 7700 base pairs downstream of the CHO GS coding sequence. CHO cells were transfected with mRNA encoding CHOX-45/46 as described in Example 2. 72 hours post transfection, genomic DNA was extracted from the transfected cell pool and the region downstream of the CHO GS gene was PCR amplified using a pair of primers (SEQ ID NO: 31 and SEQ ID NO: 32) flanking the CHOX-45/46 recognition sequence. PCR products were then cloned and 24 cloned products were sequenced. It was found that 14 of the 24 clones PCR products (58.3%) had large mutations in the sequence consistent with meganuclease-induced genome cleavage followed by mutagenic repair by non-homologous end-joining. From these data, we conclude that the CHOX-45/46 meganuclease is able to specifically cleave a DNA site downstream of the CHO GS gene coding sequence and will likely be able to target the insertion of transgenes to this amplifiable locus in the genome.
SEQUENCE LISTING
SEQ ID NO: 1 (wild-typeI-CreI, Genbank Accession #P05725)
1 MNTKYNKEFL LYLAGFVDGD GSIIAQIKPN QSYKFKHQLS LTFQVTQKTQ RRWFLDKLVD
61 EIGVGYVRDR GSVSDYILSE IKPLHNFLTQ LQPFLKLKQK QANLVLKIIE QLPSAKESPD
121 KFLEVCTWVD QIAALNDSKT RKTTSETVRA VLDSLSEKKK SSP
SEQ ID NO: 2 (Chromosomal region 5,000-55,000 base pairs downstream of CHO
DHFR gene coding sequence)
1 taaaactcaa gatgccagct ttgtagctag cttaggaaac aaagtagtaa aaaataataa
61 tgggtgggtg aaggtctgaa gcatttacag agttctctca agacaaagca cagaggctgg
121 tggccacata acttggcaac tgatttgggg gaacagaata caagaaagga aatttaaata
181 ctgtttttct caatgttgaa ctatatgggc atagtcacag ctgcctaacc tatagagact
241 ggaagctgga acctcggcta tctaagatag aataatcaag aaatgtcaat tatttgagaa
301 aaacatcagg aataaatagc tgctaagtta caagttggtg ctttagacat ttggagagga
361 taggatgggg gctcccagac ctggggctcc ctaataaagc tgtgctggcc tacaagttcc
421 agggatcctc cagtccatgc ctcccactgt tgggactgcg ggcgatggtt tctgacgtgg
481 gtactgaggg cctgaactgt ccacacactt aagccacacg ccttttactg agtcatctcc
541 tcatctcaga acattttcct ttaatctttc ttaatgaaaa ggtcgcattt cttccgaggg
601 ctagcctcct gttactctct atacatgtca cataaaacta catgaaaact ttgaaggcac
661 tatatgtcca tactcagatg aaaagccatt agctgtggtc atacaaaacc ccacagacca
721 actgttggga aacatcagac ttttttcctg cagcgcctgc cctgatcttc cacagagaat
781 tcagtctcac tttttccagg atgacttctg aactatcacc gtaagatgag aatttgaaac
841 aaagatgtaa gtaatgaact tcatgtgttc tgaacacaca gcttagtgca ttgaaattac
901 gtaacacccg cttccttata agccatttct caaaatgttc ccattacacc tgcatcgggg
961 atgggtccca gaatcttcct tttaaataaa caccccagag gattctgaag ctagaacacc
1021 aaggactgac agagagaagc atgcctgtgg gcgactccag acacctggga gctgcctgct
1081 ttcttgctac tgatttagaa ggcatttgcc cccgaatggg gctgggggac tgtcactatt
1141 tctcattctc gggactttga aaggaagcaa aacagaaaac catgcaaagt ataagccacc
1201 atggaataat ggcagacgat ccggttgtgc agattagatt ttacatattg ctgattttga
1261 agctaaagac ctttcacttc ttaaatatat aataaaattc atacaagagt attttgtgta
1321 ggtaactcag tcagatacaa ggtaagcaaa gtaaatgata ggtgcccctt aacaaaatgc
1381 attctcatag ttcatttatc aattatagaa atggtggact ggagggaagg cttgaggtca
1441 ggagaatgtg ctgctcttcc agacagcccg ggttcttttc cccagcaatc tgggactcac
1501 gtctgcctgt agctccaggc ccaggggatc tggcaccttc ttctggcctc tgcaggcacc
1561 catacacaca tggcatacac acacatacac aaattctaaa attaaatagt aggttgtagg
1621 cctacacaaa aacatgcata cattaactaa ataattaata gttaataaat aaaaatcaac
1681 caaacacata cactgattaa gtaacatgac tctgtaaggt caaaggcggc tgaccagctg
1741 tgggaagggt taaataataa caatcacctt tgaaagactg gacctggtga ttaaggatgt
1801 tccagctgtg tcgtggatga gaaatcaaat gcataattga atgagtgcca ggaatagaac
1861 tggagacttt ctggtgagaa tgcttttact ggcagtagag tccctgtcta aacaggagag
1921 agacctgcag tagccctgtg gcggccctgc agtggccctg tgatggctct gcagttgtac
1981 tcttcctgag ataggagaca cactagagag tgtttctaat gagcagctcc tgtactttct
2041 gttcccctgg agaccgcacg tgtttctccg ataatacatt gacatttctg ttaaaccatt
2101 ttcttcttgg aacaaaaatg gagaacaaat cagattggtg tgtggtcttt taaataactt
2161 ggtacttaat aacacaaaac aaaattatca gaggctggat tttaggtgct ctcagcatct
2221 gccacccctg agccatcagt caggtcttgg aggaacaatc tccaaggaga aaacagttct
2281 gtcctcagaa aagctggagg aatatgagat tttctacagc actcatagca aaatcattta
2341 cggaagggat cctgagtaag atggcctctt cttcatcaca tggtcatagt ctgcttcaat
2401 ggggagaata gttcaatcta gcatcgagaa atcgaaggtt cccttttgac tggcaatgcc
2461 ccatagatag atagatatag attatgtata tattgtgtaa aacacacgta tgtatatata
2521 atacacatac atgtatgtgt atacatacat acatacatac atacatacat acatacatac
2581 atacatagat acgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt
2641 ttgagactga gtttctctac tatgtagctc tggctgtcct gaaagttgct aagtagacca
2701 gactggccag accagatcca ccctcctctg cctcctaagt gctgagatta aaggcctgca
2761 cccaccccca cccagcccat cttatatttt gcttcatttc aaagtaagct ctatgcatca
2821 tttattcctg catattatta gccatggttc agtcttgttt gtgttttgga atatttactt
2881 aacaaaactt gaaaaacatt tttcaagatt tgtttgtttt taagatttat ttatttatta
2941 tgtataataa taaatattat tatgaaaaac ggtgttctgc ctgcagggca gaagagggca
3001 ccagattgaa ttacagatgg ttgtgagcca ccatgtggtt gctgggactt gaactcagga
3061 cctctggaag agcagccagt acttttaact gctgagccat ctccccaggc ccaaaataca
3121 catcttaagt gtattgccac aagcatacat cttcatggcc caatcttctg tccatcactt
3181 cagacagctc tccttctttc cctggccagt cacaacaccc tcagctatca ggaaaggccc
3241 tatgggggtt gttttgtttt cccactccag ttcccttgcc tgctctgacc tcatgagtag
3301 actcatacag gatgtgctca cttcacttgg gatgatttct ttttcaccca ttgttgctct
3361 gcccagaatt tgttcctttt tattgtctta gtgttaatca actatcaaag ccagcaacaa
3421 aaaatagtag ggaaactttt ttgatagggt aaacctgatt gattgcaggc tttggttgcc
3481 ttgtttggtc tatccccttg agagtccctt acaatgtgag ttagttagtg gctgctaact
3541 agttgaatct caacttcctt tttctttaat gtgggtattt gtaaggaata gcccccttaa
3601 atctagattc tgttctcaaa tcaagcaagc tcaaggctgt aagcatggat tcaccaactt
3661 tcctgctcaa ggaatttaaa tgtctggtct ccatcatatt actttaatag taatagttta
3721 ttatacacat gtgccagctg tatatccctt ttcttcttga tggacctatg aactctgttg
3781 aggtgagatt tgaacccctt agaaggtgct agagaagagg tacctgatgg tcaaggcaag
3841 gctgatactt attcatgggt cccacatctg ctaatgtaag caataacaga taatatgctt
3901 tgtgtttaga cccacagtgg ttgcatgtac actaagtatg tatcatcatt gtcttatcgt
3961 tcctttagaa tacagctaat aattatgacc gctattctca tagcatttat attatatgag
4021 cattgtaaat tattttgaaa tgctttaaga tatacttgag aactatgcat atcatgcgta
4081 tgttgttcta ccagctggga ccttgaaatg agatcccttg aggccagcat aaagagaaag
4141 ttttcatctc aaacaaacaa aagatacact tgataataga tgagggataa atgtcatact
4201 ttttatatag tgattgagaa tctacagatt tgggtatcct ggtcacttag gagaccaagg
4261 gaggactatt agctctagag ctatgaactt tatctccaga ttccaaagcc aatacaaact
4321 ctagccaagt tggggtgctg ttacctgtat ccctctgtca aattccaagt gttttcacca
4381 cctttactgt atctttccaa ctgttctctt ttataaccac acatagttca tggtctttcc
4441 ttctctcact tgactgtgga gtaacctaac ttgcgtgttt ccagttttcg atctcttcct
4501 taaatctaca ctagttaacc acaaagaccc tcttttctga gctgtgtcta ttctatcact
4561 gtcaccattc cttaatgctc tcccagatgc agccaaactt cactttgggc ttgagagtct
4621 tctccaggtg acagtgacta atgtctccag attgagcatc taccatctac cctgtgtatt
4681 acacatgaat agccttagct tttcagcaat agacagatag atccatagtt agccatgtca
4741 acacccttct tcatgctgtt ctcacagtaa taagtcctaa ttcctgtttt ctcccatcta
4801 aactcaaccc tgtcctaaat accttactca aatcctaatt gtatctcttc cacaaacatt
4861 tcccccttct ctccattaca aggtggaaac tcagagatcc aggtgtcttg catgttgttg
4921 attctgtcct caacaaggaa ttccccaggt tcctgcacga aggaaagcat ggaggaccat
4981 acttgaggct actggtgtag tgggaagaca ggcccaaacc atgtcacaga aacccatcac
5041 cagaaagttg ggggaggcag cccagttgtg gagcaggaga aggagaaaac aggcttgggg
5101 aactgctagc tatgctttgt cacagtcaca agaaaaaagg gccctagcct ggcctacata
5161 ttctacaact tcctgaatct ttgctctgaa atgaagaggt ttggatggct gtctgggaat
5221 tcatcttgct tgcagtgaag ctccttgggg tatttgaaac caggaagttt gaaggagttg
5281 atgctaattg ttttctaaag tgtgtgagga gtactggcag agttcaggcc ttgtgaggaa
5341 agaatcctat atctagtctg cactcctggg cacatgagac attcagctat ctcccttata
5401 aagcatagaa agtactcttg tacttgacac agaaataatt tcagtatgta gagcattaaa
5461 aaaaagtatg aatgacttag agagatggct catcagttaa aagcacatac tgctcttcca
5521 gaggtcctga gttcaattcc caacaaccac aaaaactcac acatatgcat gtgattaaaa
5581 ataaaatctc tctctctctc tctctctgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgag
5641 tgtgtgtgtg tgtgtgtgag tgtgtgagtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg
5701 tgtgtgtgtg tgtgtgtgtg tgtgtgatgg tgggcttgtg tttgcaagcc cagcactagg
5761 gagttaaggc ctcactcaca gtgccaggcc agtctaggtt acagtgagtt ctagacagcc
5821 caagctacag agtaaggtac tgacaaagaa agaaagaaag aaaaaaagaa agaaagaaag
5881 aaagaaagaa agaaagaaag aaagaaagaa agaaagaaag aaagaaagga gagaggtgag
5941 agggagggaa ggaactggaa gggggaagga gggaaagaaa agaaaaagaa acaaccaaag
6001 gaacaaacca ctgtatgcca ttatacatta gctttgggct ttacaggtta tacactctat
6061 attgtcatag ccaatgtctc aatattccat aagaggtgtc tagttgtggg tatgttcttt
6121 cttagtcctt ttatttagac tacatgacct gtttttgcct aataggccat tagtaatact
6181 gacttctcca catgctgccc tcaaaactta ctcctggaag atctttattt aagctatgaa
6241 cgaaaatctt aaccctgtga cctgccaccc agaatgcctc tgggaacaac ctcaggcaac
6301 ctatcaagcc gcttttccaa catttggggc aacagggatt aaaattatga ttgttgtctg
6361 cctgctgagt tcaaactcac agagggacca gaagctgact cactgatatc aagcagttct
6421 aaattttcag tttaaaactc taattattaa acaggggatg tcctcagacc agcactcaag
6481 agaaggagat aggcagagct ctatgagttg agttataggc cagcctggtt ttcatagtga
6541 gttttagctc tccagagagt taccagcaag accctgtcac aaacaaataa aaacaaacaa
6601 acaattaggg gatatacata taactaaatg ataaagcctt acctagcaca ttcaagtccc
6661 caggttcaat tgctagccct gggtggggat ttggacaaat ttaaaaagac cttttttgta
6721 tcacacataa atatgactgc actggttgtt gttttccatg gaaacagaat caatgtggca
6781 tgtattttac ggcattagct catatagttg tgcaggctgg caagtgtgga atgtataggg
6841 caggccagga atcagaaatt gatacaaaat tcaggaaaga cctctgggtg caatggtgca
6901 cacctttaat tcaagcactt gaaaggcaga ggcaggtgat ctttgtgagt tccaggccag
6961 cctggtctac atagtgaatt ccgggacagc cagggcttca tagaaagaac ctgtctcaaa
7021 acacacaaac aatcagaggg aagggcttat tttgtttttg agacagggtc ttctatgtag
7081 cccaggctgg cctcaaactc atgctcttga tatgcccacc tcacaagtgc atgttaagat
7141 tacaggtgcc tgacacacac cacttttgtg aagtgctgaa gagtaagccc agggcttcat
7201 ggacgctggg caagcactgt gccagctgag ccacactccc cagtgtgcac gatactttgc
7261 aaagatagat ccatatggat gctgtgcttc tatctaaaca gaatgacaac cacactctgg
7321 caggttctgg ttcataactg agtcttattg gtcacctcct tctccatttt tcgctggtat
7381 ttctcaagga gagaccacaa atgagaagtg aagcctaact tttaatgcgg tctctcctat
7441 gtcacctaaa ttctagctca aacagggttt ctggctctta ccttttcctc gggtttctgg
7501 atacttgaag tgttaacggg catttctctt aaagaccaaa tctggccaga ttcaaatggc
7561 tggccttcaa ctcggcaaac taggaacaat aatgtccgct gcatgtggct tgtagcactc
7621 tgtttctatt catggacttg tgagtgattt ctgggaaaca cgaattataa gataagtcct
7681 tttcagtgga cttcacaagt tcaccctcag gtagtatact gtcaggtaga aacgtctttc
7741 agagaagcga gaggtgacaa gccctctggg ctggccattg tccctgctgg cattgaacag
7801 cctgttcagc acatgaaagc atcgcctgat gctcccaaag ctggagcact ggcagccccc
7861 tgcagtcagg tgtgtagggt gggttagcag gggtgcttag gcgggttttg tagttacctt
7921 ttcaacacaa atgcaaaagc cagagagaga gagagagaga gagagagaga gagagagaga
7981 gagagggaga gagagagaga gagagagaga gagagagaga gagagagaga gcaggaaagc
8041 atccaggctt tgaagcaagc cagccttcag ctctgtcctt gagccattct gagtggaatg
8101 gagtaattgt ctgcttggag aactgaagaa tagcacatgg caaagaacaa tttgtacctg
8161 gaatatattc attagcttgc atgtcaaaag gccacatgca gatagaaacc attatcttgg
8221 cattctttaa aaccttgcag ccttgagact tgaggtgcag aaacccacat gcccatgtga
8281 ctgactacct gtcgatctct ccagccctgc ctggctaaca gggacaatat agggggatgg
8341 tgggagggga cagcttagac tcctgtggac ttggattgaa agaagaacag ggaagacagg
8401 ggactgtgca aataagcact ctattaggac ctatttttgg tgtcttggga ccctcctact
8461 ggtttagctt aaattgagag gggatttggt ttgcctcact agctgtttct tcccactcaa
8521 ttcacaatta cagctttctt cattgtcatt aaaatacatt aaatgtgtac ttgttggggt
8581 aaggctttct gttgaaatct gcataaagac aatgtccaca gcccccagtc agtggaaaga
8641 gcagtaggac cagaaggcat gtgtttccat cccgagtcta tattggaatg tttgttaaaa
8701 cctgcacttg taagagacaa acactagaac catcagcttg caggtctaca ggccagtgtt
8761 gccagtgcag ataatgccca aactggaacc taaagatgaa ggcctttggg agctgaggtg
8821 gaagagtcag ctgtgatctc ccagatgtcc tcctcatgcc ccattgccac tctagcctcc
8881 cacctccaag cacatttggg atccaactgc taacccctgg tgttcttttc ttagttgaaa
8941 ttctcaggga ataacctaag agtctctgtc actcagtcta tggcatccta tgataacagc
9001 caaggctaaa tagccatcat tgttcttttt ccagatgctc agcaatgagg atgcagaggt
9061 gaacaaaggt ggttcagggc tgccctgatg atgaatttga caagccagaa tctaacaaga
9121 tcagtcggta aacagaatcc tccttcctat ccagagatgt tggcttgttc tgtcactgga
9181 tgggcatcat ttactataag tcatacaggc accagacact cagagataaa taacatgaag
9241 tttccagtct tatgcagtcc tgtctagttg acttgccagt attctcaagg aagttccacc
9301 ccagcccctg gcatccatag accaaggact ctggaatgtt ctgggaaagc tccacctgag
9361 ctcctagcac ccatatatcc aaagagtctg gaacgttatg gtggaagccc cacctctctc
9421 tccccagacc tcgccccctc aaaaagtcca ccaaagactc cccacccccc acacaccccc
9481 agatgctcaa gaccacttcc atagagtatt taaactgcct cccagaaaac agaattcatt
9541 ttttcagtct ctcttcccca tgtcctctca gggtgggggg caggggtatt agtattcaag
9601 cacctatact ggcctgtcct tggggttctg acaagatatg acctcagcta cagccactaa
9661 gatcaccacc tgtgtatatc cactatgctc ccttttaaaa gggccctgtc cacctcccat
9721 tctctctgtc tctctctctg tctctgtctc tgtgtgtgtg tgtctctgtc tctctctctc
9781 tttctctctc tctctgtctc tctctctctc tccttctctg cctgactctc cctccctccc
9841 ctgctctctt ctttcctgct gcttttgtcc ctagaggcta gtctcctctc tccccttccc
9901 ccttttccca ttcactttcc cccaataaaa aactctccac ccaagctcta tcacatggca
9961 tcattctctt gctccatgat tttaaaatca caatgaggag gggagcatgg aaaaattatc
10021 caggaagact ttatccatta aacctgggtg ctttttcttt cttccttcct tcctttcttt
10081 ccttctttct ttcttccttt cttttttcct ttcttccttt cttttttcct tttttccttt
10141 ctttttgttt tgttttgttt tgagacagcg tttctctgta gctttggaga ctgccctgaa
10201 actcaatctg tagagcaggc tggccttgag ctcacagaga tccacctgcc tctgcctccc
10261 atgtgcttga attaaaggtg tgcaccacca ctgcctggct taaaactggg ctttttctaa
10321 gtcagtttga tttggattgc tgcattggca gagaggttta ttggggtgca gaaacctttc
10381 aaccagcttt tgagctaatg atagagagaa gctcaaggaa ttggagcaat gcttgactag
10441 ggatgtcaga gggaggctat ccagaggagc ttacaactga ggtaaactta aaagttaggg
10501 agtttgtcaa cttcaaccca cagaatagag cagagccagg aggagctgag gcttctgagt
10561 gttatggtgg aagcatcacc ccaacccttg acatccatat gcctgaagag tctggaatgt
10621 tatggtggaa gttccaccca agcctccctt cccggtcgcc ctccaaaccc tgctacatct
10681 cagaaatccc accaaatgat gactccctcc cccagagata ttcaagacca ctcccacagg
10741 gtatttaaac tgccccccaa cccccagaaa atagatgtgt ggttttccaa tctctctttc
10801 ctatcacgtc tctggggagc tggcaggcca tttgggagca ttgtatccat taaacgactt
10861 ctcagtggag actctgaaag ccagaagagc ctagacagat agatgtcttg catactctag
10921 agactacaga tgccggccca gactattata tccagcaaaa gtttcaaaca ccatacaaag
10981 tcaaatttaa acagtatcta tctacaaatc caatattaca gaaggtgcta gtaggaaaac
11041 tccaaactaa gattaactat acctgtgaag acacaggaaa taatctcaca ctggcaaaag
11101 aagaaaaacc tctctctctc tctcctctct ctctctctct ctctctctct ctctctctct
11161 ctctctctct ctctctcaca cacacacaca cacacacaca cacacaccaa caccaatacc
11221 atgaacaaca aaataacagg aattaacaat aattgatgtg tgtgtatgtc cctgtgtgtg
11281 tgtccttgtg tgtgtctgtt tgtgtgtctg tgtatatgtt tgtcacctga ggggtggctc
11341 ttccttggtt tgtgaggttt ctacccaatc tataactccc ttttcttcat tcacttcctc
11401 atgtccttac tagtctctat tgtggattaa ggaaactgtg tggagaacag ttttcttcta
11461 gaaaagaaca ctagccatct catgtaatca aattggtgac tatcctaatt attatgagag
11521 agcttccgtc cagtaagtgc tagaagtaga tgcagagatc cacagacaag cactgagcca
11581 agctccagga gtcctgttga aaagagagag gaaggattgt aggagccaaa gagtcaagag
11641 catgacaggg aaacccacag agacagctga cctgggcttg tgggtgggag ctcatggact
11701 cttgaccaac aattagggaa cctgcatgag gccaacctag gaactctgca tgtgtgtgac
11761 agttgtatag catggtctgt ttgtgaggct tctagcagtg ggatcagggc ctgtccttgg
11821 cgcttgagct ggcttttggg aacctgttcc gcatgctgga ttaccacacc cagccttgat
11881 gctgggggaa gcacttggtc ctgcctcaac ttgatgcgcc ttgcattgtt ggattctcat
11941 gggaggactg cccctttctg aaaaagaaca aggagaagtg aataggggag gggattggga
12001 ggagaggaag gagaggaaac tgtgataggg atgtaaaata aattaaaaaa ttaattaatt
12061 aaaaaagaac acttgtactg gtagattggc taaaatgaaa caaagataaa agtacacagg
12121 aaaaagagag gagaaacctg gggagggggg ctccaaagag aggtgagggg gggatgggaa
12181 tggcagctta gtggaggaag gaagacatga cctacacgaa tcgagctgta gtttttatct
12241 ggagcatagg gtaaagatgt ttgaggagaa ggaggaacac atgcttgtaa aacatggtct
12301 tcagaaccag caacaatcat acagagtgtc cagggtccat gggcacatga aggacagacc
12361 aacacatatt taacagtaaa gtgtccatat ttggtatgaa agtgatgggt aaattgtcct
12421 gggactgtaa tttagttgta aaggacttgt ctggcatgtg ggtattcttg ggttccctcc
12481 ttagcactga aaaaaaaaaa aaacacacac acacacacac atatattcta gtgttttgta
12541 gaaaaggatt caaagaaagc catgatttct cttttgataa atccagaata atgtaataag
12601 aacacacagt ggtgtgattt cagcaatcaa gtacaggttg cttgtctgtt tgttgtatgg
12661 gatggttggg tggttgtttg cttggtttgt aagatgggtg ggtgggttgg tgggtggttg
12721 cttggttggg tagttggttg ggtgattggg tgggtgggta tttggttggg tgggtggtgg
12781 gttggttggt cgtttggttg ggtggggtgg gttttgtttt gagacaggga tttactctat
12841 atctcagttt gtctcaaact cactatgtgc acatgagtat gtgatgagat tatctaagac
12901 catagtgtct gtgttcatgg aatgtctctc tagcttagag aatttaaaaa atggccatgt
12961 agggaaaccc ctcagaaaag gagtttctat ggcctccaag aataagaatg gatcctccta
13021 gctcggagtc agcaaggaac tgaagccctt aattttatag acacaaagga atccattgtg
13081 tggctccttc ccagccaagt ctcagatgag tcacagacct gcatggcacc ttatgcagtc
13141 ttttgaggtc ccaagaatag gatgcagata agccatgcca gaatcccaac acacaaagcc
13201 ttagtgatat agtaaatatg tattgtgtct aggctgctgc atttctggtt atgctactgt
13261 gcagtaatac acaactaata cagatgtgat ggttaatatt atgtgacaac ttgagtgggg
13321 cacagaggta cagacacttg gtaaaccatt ctgggtgcac gtaaggatag ttttggatga
13381 cataaacatt tagattagta tgctgggtaa aatacattgt ccatcccaat gggcatgggc
13441 tttgtccaac tagatgacag ctggaataga aaagtctgcc tctctcatag ttctcaggcc
13501 tttgagctca gactagacag aactcacagg ttctctgagc tttccagctt gatgaatgtc
13561 catggcagtc ttcacactta acacctgaca gacttaatga tcatatgaac caattcaaat
13621 ctgaccatca ctcgggtcat tcttttgatt ctgtcacttt ggagaactaa taccgaggac
13681 ataaaatgcc atcacatcgt tattttcttc ctgtctgtga atatttttct tttttttctt
13741 ggtttttttt tttttttttt tttttttttt tttgtttttc tctgtgtagc tttggagcct
13801 atcctggcac ttgctctgga gaccaggctg accttgaact ctcagagatc cgcctgcctc
13861 tgcctcccga gtgctgggat taaaggcgtg taccaccaac gctcggcctg tctgtgaata
13921 tttaaaatga aaactttgga aatgttctga aaccagctgg tgtcagatag tcagagaact
13981 ttcgtaaggt aggtgtgggt tatagcataa tcccacacaa gaggctgaag caggaggatt
14041 ttgtgtttga gggcagctag agccacatgg tgagtccctg cctcaaaaca caaaagcaag
14101 acaaaaacaa gctccaaata agattcactg ggccctttct ttccttcctt ctcagtgagt
14161 ccacttgctt taaaatcagg tcttaaagac gcactagatg ctgaacttaa cagtaataat
14221 aaatatcttc tcttacagta cagattatgc tctataaaca ctgcactgat aaagttcagc
14281 cttaaccttt gttctgtaaa tgtttcctag tttttctact gccgtattat aagacaaatg
14341 tcagcatgaa ggcaggtttt tcagaaaaca cagcagctcc acagatggcc tctaatccat
14401 aatcattaaa gacaagactg caactttttc aactggaaat cattcaagat gtttttctga
14461 agtccctacc aggacacaag ccaccctggt tgctgtgtga catcagttag gtagactctg
14521 aactggcttc ccaagaaatt atacaaaagc aaggtgtcac ctagtattag cataacttct
14581 gataactact gtcttagctg gggtttctat tgctgtgaag agacaccatg accacagaaa
14641 ctcttataaa ggaaagcaat tattgggtcc agcttacagt tcagaggttt aatccattgt
14701 catgattgca ggaagtatgg tggcccacag gcagacatgg tgctggagaa gtagatgaga
14761 gttctatatc agattgacac acttcttcca acaaggccac acctccactc actctgagcc
14821 tatggggcca ttttcattca aaccaccaaa gctacaaggt agcttatacc ccagcttgct
14881 atttctgatg agacttagta aatagtctta aaagcccata aaatgactca aaactagttt
14941 ttttattatt attattagtt caaattagga agaagcttgc tttacatgtc aatcccttct
15001 ccctctccct catcaaaact agttttttgt tttttaggtt ttttttcaag acagggtttc
15061 tctgtgtagc tttggagcct atcctggcac tcgctctgga gaccaggctg gcctcgaact
15121 cacagagatc tgcctgcctt tgcctcccga gtgctgggat taaaggcatg caccaccaac
15181 acctggccaa aattagtttt aagtccagtt ctaggagctc caatgccctc ttttggcttc
15241 catgggaacc aggaacacta tatatatata tatatatata tatatatata tatatatata
15301 tatatattca ggcaaatatt tatgcatata aaaataaaat aaatcttttt tccttttttt
15361 tttaaagaag tgacattgtc ttggaatttt tgtggctgct ctgcccttat gtgtaactgg
15421 acactaccag catctaaaca ctggcctgaa accagccaaa gaaaaccttt gtgccaggtc
15481 ctgtgtcaaa gtattatgtt ccttttagga tatcctatat cctaaaggat ttattttact
15541 gatagcatct taacttcctt tgaaaggttg gtcttctcaa gcagtcctcg tggagctggc
15601 tcctcagcta atgccagggg acaataatga tcccctccca aaaccaaaca gaaaaccatg
15661 gcaactctgg tttccttggg cagcacctgc tttaagaatg agcaaatgac caatcagctc
15721 atgaaactaa atactctatt attactaaaa tatttttttg agacagggca tggaattcat
15781 cacatagttc aggttggcct tgaactcaga gagactcact tacctttgcc tcccacgtgc
15841 tggaattaaa ggcatgaacc accacaccaa acataacact tgaattttgg aagagtcctt
15901 cttccaatag atttgaggtt ttgaaaatgt ggcacagaaa atatgaattc aaatataatg
15961 aaaacaagag ataactttca actaagtttc tataggttct tgctaggaat cctaagcttg
16021 tctgaaactc tagagcttct gtttctagct tctgagtgtt agtattgtag gtatgtgccc
16081 tgcctcagtg tgatgttttt gataatctta aagaaatcaa agaaatttta taaaagacta
16141 gactgtgcta cacaaaaaga atattcagat gccaagaaag agttcttaga aattaagaaa
16201 tatgctacta gtataaatcc tttataaagt ggaatgacaa atctgatgaa atcttactaa
16261 aagtagaaaa acataaacat caaagacatg aataataaga aaatcatatt gtgcatatga
16321 ttaacctaaa acattaactt gcaaaaatag aatagtccca aaaagtaaac aaaataaata
16381 aatcaccaag aacatgatac aaggacaatt cctaggatga taaaacaaga atattcatta
16441 taaaaggccc tatcactaaa gcacaacaga aacagactca aaagataaat cttcattgtc
16501 actggagaga agtccatact atcatagcac tcagaaggaa ataaaaatca aaatgtcaaa
16561 aaggacctca gcctctgaaa cacaaataca aaatatgtcc cgccttcttg acacgcatta
16621 ctcttcaatt aacattttaa gaaaactata aactgttaaa gagagcttag tattttaaga
16681 aatctgtagc tatttctttt ataagcatga caactaagtt tccctgattt aaacagacct
16741 aaaaaaccgg tgaagtgagt ggagaaaggg gatacgaaga cagcatccca catgactgct
16801 cccagtaaag gcaaggtctt catccatttt atcctgaact ctgggaaatt tataaagaac
16861 agaaatgtat ttctctcagt tctggagcct cagtccagga cactaagtct aggtactaca
16921 ctctcacatg gtggaaagta gaaagcaagc tcacttgtca ctcactacct gatgcctctt
16981 tcatcaatcc cattgataag gaagagacct ggcatctcag tttcctaagg actcagctct
17041 tactaacatt agctgtcatt tctgggtcac tgtaacagaa agcctgacag aagcaaccca
17101 ggggaagaag gatgtatttt ggctcactgt ctctgaggat ttcaacttat cccagcaata
17161 aagggataaa ggcattgcag caggaatatg tgtggcagaa gctgtttatg tcacaataaa
17221 caaataaaca cacgctagcg cgcgcgcaca cacacacaca cacacacaca cacacacaca
17281 cacagagaga gagagagaga gagagagaga gagagagaga gagagggggg ggggcagaca
17341 gacagacaga gagggagaga ggcagagagg gagagagaga gagagagaga gagagagaga
17401 gagagagaga gagagagaga gagagaaatc aaaggcccac ctccatcaga ctggtcccat
17461 atcccaaatt tctagaacct cctaaaacaa caccatcaac tgagggagac atttttggat
17521 tgaaagcata atgccattac ccaggcagaa tctgcctgtc tgggggagtc acatttaagc
17581 catggtatca attgacctca tgtaatttca gaatactaca taaaactatc agatattttt
17641 catgatgaat ttctaaagct tgaaattccc tttgaataaa ggaccaacta cagaattttg
17701 ctgagtctac aattacatac atgaaaatgt aactacgaag tggccagcca caatgaaaat
17761 taaagtgttt gggtggtctg tctctattga tgctcttctt tgccctgttt ttttttaata
17821 ttgttgatgg tttgtttttc ttttaagata cttggcccca agaaaaaaaa tgacagcctt
17881 aattaatttt gtttactctc ctgacatttt aaaagacaaa tttatgaaga cctgactgtt
17941 ccatgtagta ttagaaagat gtaaaattaa gggttgctta agctgtgtag aattgaagag
18001 cacagcattt gagtgacagg gtacaattag agatcatcag ggatgtggca caaagtgtac
18061 tcaacctcac cttttcctgc ttagcagaga acagggtgcc tcggtgagat aggaaattaa
18121 tcaaatagaa gaagaaatag taattttaga aggatcaaat tttcctggtt agaatgatca
18181 aaactacaag acttgtaact aaaatatagt caaacccatt tcaactggaa tctgtgctat
18241 tcatgtatag attaactaga atctaatttt taaattttca tcttacttcc aaaaatattt
18301 gtccaaatac tctgtgaatg cattagtttc ttatgggaaa acatcatatc ttttgtacaa
18361 tgtgtttctt agcttgaggt tctctccaaa caggaccaag acgaggccag gaccatgtga
18421 tacaacccat agtcctcaag aaatagttgt cattttctta ttccaattgc atcccaaggt
18481 ctcatctcat tttgcgtgtg cctttgacac cccataccca cataaactaa ggtggtgtta
18541 ttttttgagg ccctgaaggt atcttcagga atccataagt gagccttaag ctgcatctgg
18601 atataggaat ctgaaagtgt cccttctctg catgatctct tctttcagtt tttcaagtca
18661 gtgtgccaca ggaatcagga acgataaatg gagaggggaa gtgcagttgc ttggtataga
18721 caccccagag ggctatttgc atcctgtcct tcaaaatctc tctgagcctt cctgcctaag
18781 ctgttttgag ttgggtttgt ggtaccagaa cccctgcccc cgccccattc tgactaatga
18841 gagagagaga gagagagaga gagagagaga gagagagaga gcagcagagc atagaatgaa
18901 agtaggttag aagggcaggt aaaagcactt tagacaagag caggtataag ggccttggac
18961 tccctcccca gaacacacac atgaaggtaa acgatggtta aaggatacag ataggatgtc
19021 gaagctggac gatcacttgc ttttgtgtgc ttgaagtgac aggctgtggc tttcgggttc
19081 atggggtctg ttgttgagtt cacagtctca ccatgttagc aagcatgtca ctattaagct
19141 ctatccccgc cccccttttt tgagacatgg tcttgctaac atacccagac cggcctagga
19201 agcactttgc agtctcagct cccctgagtg ctatgatcac tcgtgtgagc tacagtaccc
19261 aaaccagaat atgtgtgttg ggtgttatga gagtttacac attgctgcct tgaatgctgc
19321 tctgcttgag ttcctgtagg aagctgagct gggaacctaa gcttcctcct cccagatagc
19381 agtaaccctg cagagacctc ccaccaagac tagctaaccc ctccttcttg tgctgtactt
19441 agcaagaacc ccaaggttct gggtccttgt gctacagttc cagaagagta tgaacaatct
19501 tagcttttct gtatatgtgt ctgtgtctgt cctgtcagat caagtcccag cctcactgta
19561 tgcaacatga aaggctgtga aaactgtgca ttttgagaat gaacatcatt agtctccagt
19621 aagttcaaaa acaaatgaag gcagccactc ataagggtct ttaatgaggc aagggggcaa
19681 aagggtggtt tctgtttgtt caaagaagcc tgtcatacat tttcagaaaa tttagaaaca
19741 cgtatcatgt catttcacgt tagtatgaag tccttataat tcatttcata ttaaatgatt
19801 tcctttggtt agaagcaaaa ttatgcataa aatgtgttcc tttgtgtttg gagcaaaatt
19861 acaagttaca ttattagtta atattctagt tcttattttt cccaatctcc aagaagcaaa
19921 atattcccct aaaccctaaa gcatcaaatt atcctatcac acagtgacca gtcatcgtaa
19981 cctaaatatt aaagcatcag attatcctgt ctatggtgac cagtcattgt aacctaaata
20041 ttattgtaat gtggattaga gttaactata ccttttcatc acactataat gtaaacactc
20101 tccaaatctt tcaaagtctt gaaaacacaa tttataaata ctgtgttctg tttgttttga
20161 gacctgatcc ggttaggaat ttcaggctgt cctcaaactc atcatcttcc tgcctcactc
20221 aggtcctaag tgctgagatt aaaggtctat gctaccacag ccatacgaat gccatgtctc
20281 catcagctta tcacttctta acttttttct tttcttcttc tacatactgc tgagtaggag
20341 catcgatgac ctcagcctag taggaatggt tcccatgtga acccttaatc tgtaggaaga
20401 tgctggactt cttccattaa gactgatctc catttgaact tgacttgtct ctctcttgtg
20461 tggagctacc atcccatata taatcttctg gtttataaac agattgcttt accctcaaga
20521 tcctttgcta gcgcagcaat gtaagtttta atacaaacag taaggtctct gattggagtg
20581 tcatggtttg gttaagtgcc ctttccaagg gcccatatag ttaagggctc aaccaccaag
20641 tgatgcttgt ggataggagg cagggcctag tggacagtct ttaggtcatg gagctatgct
20701 gttgaggggg actgtggggt cctggtcttt ttcccactcc tttttaggtc ctagctatga
20761 ggtgagtggt tttgtcctat caagcacctc tgtcctgcca tggtgtaatt gattataact
20821 acaacctctg aaactaagcc agtataacct atttatctca agatgtaact tacaggtaat
20881 ggtaagataa agctaacaaa agacaaattg ttataatcca ggcaagcctg gccccatccc
20941 ttgggggcat ggcacagagt gtgtcaccca tctgtgcatg gcaagcagta ccctgactct
21001 gtatgctgat tcaaaggtcc cttaaagcaa actcctccca cttcctctct ttttctgcca
21061 tttctctgag gagggaggcc actgtctctc tgtctctctc tgtgtctctt tttctatctt
21121 cctctccctc tcttcccttt ccccaataaa ctttccacat taagttttgt ctgaaggtat
21181 ctgtttgtct ctcacccgcc ttttaggccc cacctaccat gggatctgcc aaaggtctca
21241 cctcgagctg tattcataac acaaatgaca gacaaagatc aaccctgaag actagtagga
21301 tgtagaaggc ctggagctga cctgaagaac actgctgact tcaacattgc ccatccgtca
21361 gttatgtagc attaaagtta tagtggttcc tcagaaagca gtctcctttg aaaacttctc
21421 gttttgtgtc taaatggaat taaatacctt gttcccgaat aattgtttta gttctcttga
21481 aagatcccgt atacttacta ttaagatgta tataaacctc aagctgaaag aatgacttcc
21541 cctatggcca gatcacaaga ctctccactg atgtgcccgt tgcaacctga ttagaggaag
21601 agggtcaaag ttccccaaga ttcagctgag ttcatgcaag ttttagaaaa aaaacaagat
21661 gttcctccac agttagaaag gagtggggct ggagggatga ctcactgaga aaggttattg
21721 tcgtacaagc atgaagacct gagctcgaag cctggcaccc atgtaaaaag aaaccatgca
21781 tggtagtgtg catcttcaat cccagcattg gggagacaga gaaagagaaa gggacatccc
21841 tagagcttcc tggtcagcca gccttggcaa gccagtgaac tccaggttca gtgagagacc
21901 tgtctgggga ggaaaaaggg agggagggag ggagagagag agagacacac acacacacac
21961 acacacacag agagagagag agagagagag agagagagag agagagagag agagattgag
22021 gaagatacct gatatcaacc tcacacactc atgtacccat gtatgtaggt accttcacac
22081 acacacacac acacacacac acacacacac acacacacac acacacacac acacacacac
22141 acggatggtg ttgaattcta aggctcttat ccacacatat atggagacaa atagaagaat
22201 tacagtcgtc cctgcctttg acgctactct gtttctccaa ccctgcttcc cagatatttt
22261 tcaacatcta ctcagccttg agtggttgca ctctgacccc aggacctctt tctgtgactt
22321 ccttggcctc ctgttttgtt tttctgatgc taaaaactga atctggggcc tcatgcacac
22381 aggaagatgc tataccaatg agctacaatt ttgttgccct ttttaatttt tgagatggtc
22441 tcactaaatt gttcaggatg gcccacttgt aattctcctg ccttagcttc ccaagtagct
22501 gggcttttat acagatctgt gcttccacac ctggctgagc agacactcat gatttcattt
22561 ctgctaatca ggtagttttc ttgcccctcg ctgccatttc ctacctgcct ttccttgcca
22621 actaaactgg ttcccacaag cgacaggcta tcatttctca gctcttccac aggttagctg
22681 tgcaatttgg tatgaatcat ttagcaagcc cagttctcct ctttgtaaaa cagatgattt
22741 agatgaaatt ttttcaaagt tctctttgaa ttaaaactat cactgccttg cttgctctct
22801 gactcttgga gaccatggcc tatccctgat tagtccttgg tccacagaag gatgggtggc
22861 attggatgtg ctgaacaatc aggtactttc atgtcacttg gagtcttaca gtaactgcat
22921 gtttcaaatg aatcctttct ggctctatta gtttcttttt tgtcactgtg aaaaaaacac
22981 ctgaaagaaa caaggcacgg tttgttctga ctctcggttc agaggatata gttcaccatg
23041 gaggcaggag cttctcacag ctgtaacagc catggagtca ggtggctagt tacagtcagc
23101 tggccttagc agtcagagag ccaagagagc tcagttgagg agagtccagc caggctgtag
23161 cccttaggac ctgctcccca gagatccact ttctacagta tcttctaaac agtgtcacta
23221 gatggtgacc aggtagtcaa gcacatgagc ctgagggata atatcattca aaccatagga
23281 ttagtctaga actgaaccag atcaagaacc aggttttctt ctcacataat agataccaca
23341 catcatgttc tcatatagag tgtgatctag gtattgtttc tccaaatgga gaagccaaca
23401 ctggatgact tacatagaaa gaaagagagg gaggaaacaa gcaagggagg gggaagagtg
23461 agaattattg gaacagtacc agtgcctcaa aatccttggt ggactagaga attagcctca
23521 ggaagaagcg actaggcttc ttacagcata gacatacagt tcttaccaga ggcacagcca
23581 tcatgggtgc catggggagc atgaagttca gctccatcca gccattccta gcgatttctg
23641 gcaacctctg tcctttgaga cacttcctga agatataaga gtccagggag agacatctga
23701 ttgctttgat cccaggatct tgggatggaa ttggtgttgt ctctgctcca gctccagggt
23761 caggaaggtg aaactggaaa cacaagctag cttttcttac ttagcaaaaa cccacaggtg
23821 acataaaaga cagattgaca cgagaacagc atggcagatt tatttagtca aagttttacc
23881 agacacaagc accttcagaa aggtaaagtc agagacctta ggggaatttt cttgccagaa
23941 tttttccaga agaatcaaca gccgtgtaac aataggacta gataaacaag taagactgga
24001 cctgcagcac aaatgtgaca ataggagttg gaatccccag gactcacata aagccatggg
24061 agccgaatgt aatggtcact tgtagtttca gcctcagatg ggggtgggga ttctccagaa
24121 taagcaggct agcaagacta gccatgttgc caagctctgg gttatattga gacactctgc
24181 ctcaatgagt aagtggaaga atgatggagg ccaacttcaa ccttggactt ccacatgaac
24241 acacatacac aatgcaacca tgcatccaca gtgtatgtac acacacacac acacacacac
24301 acacacacac acacacacac acgcaaatgg acaaagaaag aggtaaaacc tacaaggaat
24361 caactgaaca gaagccaact ggtctgcctg ttcagatcct ttttggcctc tctgtgtgct
24421 tccctttctc ctgggcatgg ggcaggcagg atctgtatgg ggtgagggtc ttcagagaag
24481 cgaacagcct tcctaggttt tatggctcag tttggtggag aggggatcta gtttctctta
24541 atcatctttt taaaaattta ttaatttatt ttttatattc caatcccagt tttccctccc
24601 tcctctcttc ccctccccca cctcccatct gttccttaga gagggtaaga cctcctctag
24661 gaagtctact aagtctgccc catcatctca ttgaggcagg accaaggcac ctctccaccc
24721 ctacactctg gtgtctaggc agaacaaggt atctctccat atagaatggg ctccactaag
24781 tcagtttgtg cattagtgtt agatcttgga cccacttcca gtggcctcat atattgtccc
24841 agtcacatcg ttgtcaccta tattaaggga gtctagttcg gtcttatgca ggttccccat
24901 ttgtcagact ggagtcagtg atctctcact agctctggtc agctgattct gtggtttccc
24961 catcatgatc ttgactcctt tgttcatatt gtcactcttg cctcacttca attgtactcc
25021 aggagcttgc ccattggtta gttgtggatt tctgcatctg cttccatcta tttctggaag
25081 agggttctat cttctctggg gttgtgaatt gtagactggg tatcttttgc tttatgtctg
25141 gtatatgctt atgagtgagt acatacaaca tttgtccttc tgggtctggg ttaccccact
25201 caggatgttt tttttctagt tctgtccatt tgcctgcaaa ttttagaatg tcattgtttc
25261 ttactgctga gtagtactgc attgtgtaaa tgtaccacat tttctttatc cattcttcag
25321 ttgaggggca tctaggttgt ttccaagttc tggttattac aaataatgtt cctatgaata
25381 tagttgagca aatgtccttg tggtatgaat gtgcctcctt tgggtatatg cacaaaagtg
25441 atatttcagg gtcttgaggt aggttgattc ctaattttct gagaaatcga catactaatt
25501 tccatggagg ctgtacaagt ttgcactccc accagcaatg gaggagtgtt ctctttactc
25561 cacatcctct ccaccataag ctgtcatcag tgtttttgat cttagccttt ctgatcagct
25621 taaaatggta tctcagggtt gttttgttaa tcatcttgag aaaaaggaat tctattttct
25681 gtgactggct ctgagagaga gagaagaggg aaaggtggga ggaatgtgtg ctttcaagac
25741 cttgtgttct cccttagctc aaagtactca ccatgaaaaa ccaccagcct ttggaggagc
25801 atgctcttgc agaggcaaga tcctggcttc ctcccatctt gaatttgcca aaatagcaaa
25861 gatgtttggg tgctggacag ccaaaaatga cagctgctca cttcacagct tcctcacgta
25921 tgattacaac tccactcatc atcaagcttt aattacatca tgagcaggct tatggctgag
25981 ccgttatcct cgcatccctt cgtctcatca ctgattcaca caaatcacta ggtgctccgg
26041 ttaatgaaaa catattcatc agtacagtga ctaattcatc aggccaacat ttacatggct
26101 cctctgcatg acaaaaatga atgtttagaa tgaataatga gtcaccagag gtgggggaca
26161 tcttctgagc acaggttgcc cttgtctttc ctggtactca atcccggctg aagagctgaa
26221 caaagctgag gttatttttc ccatgacagt gcattgtggt ttagagatct gtaagcggct
26281 tatcttgatt ggcagtttga ttggttctgg gatgtactaa gagacgtgcc tcatgggcat
26341 ttccagaaag aattaactga gggggaagct cctcgccccg agaatgggta ggagcatctg
26401 gtggggtaca gatgtaaagt ggtccaaggg agaagccgca tggcctgcct gccttcactc
26461 cttgctgctg agtgtgttta tcccatctat cccgttgttg cttctgttgc agttgcaatc
26521 ctgcttctcc aggccccagc gtagactgaa cagtggctgc ccagaaattc ccaattgaag
26581 cagccgaatg gtggactgag cacctctcag tcttcagtct ctctagtttg taggcaacca
26641 ttgttggacc caactcttag tagtaagcca atctactaaa tacagaaagg ccagtgagat
26701 ggctcagtat aggtgcttac caccaagctt ggtgacccga gttcaatccc caagactcat
26761 aaggaaagaa ctaactaccg agagttgttc tctgagctcc acacatgctg aaacatgggc
26821 ctccacatgt catgaacatg ttcacacaat acatatttat ctctatatat tcatttctta
26881 taatttttag aaaatttcat tttatgtata tgagtgtttt atctgtttgt atgtctgtgt
26941 accacatgca tgcctggtgc ctgaagaagt cataagaacg tatcagattc cctctaactg
27001 gagctaaaag aagattgaga ggtacctacc atctgagtgc taggaaccaa acctgtgtct
27061 tctggaagat cagtaagcat gcttaaccac tgagccatca tgccacttat ttgtaacaca
27121 tatccatcct attggttaca gtcctgactc atacagttag atagctgagg aacctagaat
27181 tcttctgctt ttttattaca aaacaaagaa ttttatctga cttacagttc tggccttagt
27241 cagggagctg cattgggaga tggcttctct actgtcagag tccagaggtg gccgtaaagt
27301 atcatatgac atgaggcaga aagtctaact tacttgagag ttaacttgga aatgtccaaa
27361 gagacagggg gctaagtccc tcttattgaa gagaccttcc atagaagtta gcctgacaga
27421 tggccttgcc tgaactgcat tgacagtctt acttggaagg cctgttttgg ttcctaagaa
27481 attcaaggat ccaccagaga agtgtgcagc cagcaagctg gactccctat cccaagcccc
27541 agctcctcct cagggacctc agcagtcctg tgtctagctt acctcagcga tggggggaaa
27601 gatgctgttt tcctgctaag agcacactat tttatattat tgttgacaca ggttggactg
27661 catgtaacag actctccaac aacacagtga agatacaagt gtgttttgct gcatttaaat
27721 gtctccccat ctgtccctgc taagacacct actgtccttc acatgtcact gaaaactcca
27781 ccccttatga gaagtcttcc ctgatgccat ctagacaagc taagagtgct ctgctctgca
27841 ctgagcagct tctcaactct ggggttatca ttgctctgca tcacaattag cacacgtggt
27901 agtggctgtg tttgtgtttt tccacaccat gagtccagac agcatccctc tcaccagcac
27961 gccataggca caagtgctca agagtagcag gacttgaaca tgtgtggttt atcatacaga
28021 cagctgctgc tcagagacca gatcaaattc aaagcaaaat agagagatga tggttcctgc
28081 catgagcgta ctgaacaagg acaaacatca ccatcataag gaactcagct gacagggagc
28141 ggtcaccaaa cttttttttc tgtaaagtga caaaaatagt taagtatttt gccctagaca
28201 tagtgggtgg tacacatgta atctcagcat ttgtcagagt gaggcagaga gttgaatgct
28261 gggctacgta gatagtctca aaaaataaat aaataagtaa ataaataaat aaataaataa
28321 aaggaagaaa taaaaaaaag aatttgttac tcaactctgc acaatggtgc aaaagaaaca
28381 ataagcatta tgtaacctag tgggtattgg ctgtttcact ttactaacag gcattgaaat
28441 ttcaattttg caaaattttc atgttccata ttacccttat ttttattctc ccctataaat
28501 ggtgactcac caatacgcaa ctggataaga ttagggtatt tttattaggg aatatgcctt
28561 acttacagag cacctaacca gccagcagga aacatagtaa agtagcgcat gccgatgaaa
28621 caaggaaaaa gaagaactac catgtgtgac ccctaaccct taaaacctct cccacatcac
28681 cctgaccatg cccattaggc gtggtcacct agccagcccc taggaggcat ggttacggtg
28741 tccccctaca ctcccctaat catttaaaga tgcaaatgca tgcttggtga tgggctaacc
28801 ttggctcatg ggctaatctt ggctcatggg ctaaccttag ctcatgggct aataatcaag
28861 gtttactaat ctctgtcaga cagccatttt ttttttgcag agaagaatcc ccatctttgg
28921 atcatttatt tattcctttt gtatatttga tgcaatttat aaccacaaga acctactatg
28981 tgactgcact gtgccagatg gcagagaaag ctaagccccg attcttgtgg catggactca
29041 cacaactcca gtacaggact gttagtgaca atctccttaa ggcataagca tactgcagtg
29101 gcagcctctg ggttaggaga caaggataca gtttatgaca cctggtatct ggaaggcatg
29161 aaacatgtca aatgctggct acacctaaga atcagcaaca tctagtctgg ccatagccta
29221 ggatgaatgt cacagggtct taggccagaa atgtatggcc gagctgtagc agggtcctct
29281 ctagggccag aattaattcc agtgtgatgg acagccaaga ccacagggat aacaaatgag
29341 cagtgccaat gacacgtgct tctccttatt attgctgcac agtgtttgtt acacatagca
29401 ttttcgcaca gtaatataat gtgcttgggt catcttgctt catatcccat cactccctcc
29461 atctccctag tgcctcccct gttacctttg cttctcagtt ttgtttctgc tttgatgtca
29521 acagcacata caagatttta tgcaatacat cacttcctga atggctctat ttggaaatca
29581 ctaaaaggta atttatggaa catttggggt ctttttgatt ttctaattta ccaaaaaatc
29641 cacctgggga aagacaatgg agttcaagga cttctaagag gggaatgtac catggtatgc
29701 tccagccagg ggaaccagtg cttcccagga gctatggctt acaaagtggg ttatcacatg
29761 aaagcaagac taaaataatc atctcaaata ttcattagat gtgggactcc taaccatctc
29821 acaatgcctc cctcggtcta cattaaataa gaaacctcca ttttgtgctt tgcgagaaaa
29881 tgactgaaga ttatacattt ggccttgaag tggaagtatt tttgaaaatc atgaatagga
29941 aaataataaa tctctcattt caacataaaa tataagggac aaggacatct actcatgctc
30001 caaggacgga cactgaattt tccatcaggt agttgcagaa cgctgtgtcg ctcaatcaaa
30061 aattcaggat gcattgctca gagtgcatta tattaaaaga tagcatcttg gaacacagga
30121 tgctcaggaa atgggaggga cattaatctg catgcagtga tcatctcctg caaagcgggc
30181 atgagagcct gatgggagac aagccatcca gatgcccata cccaggggag ctgtactggg
30241 ctgcagccct gcgccattca gccatgcacc aggctactcc ctcctcttcc agctttctcc
30301 ttctgatggc cataggatta gaagataagg gactctagtg caggtcaact gctgaccagt
30361 gtgaaaatgc acagactaca tgctggtaga tcagcacttc aaactactgt tcaccatcat
30421 ctctggaata agcactacat ttacagggtt caaacctcaa tgaatataaa caaacaaaac
30481 acacctccct tccttcactg tctcccattt ctttggttcc catctccaca tagaatttat
30541 aattaaaatt tctaagtatc tttccagaaa tacttcacac atgttataag caaatgtgct
30601 tttaaagata ctattttaaa ttatgaaaat ggttatatta gttgagataa aagaatagaa
30661 tgggaagttc cagaatttaa ggcctcatat gaaaatataa agcgctttct cttttaagtc
30721 tagggtaggt gtactagatc agcgctcagc tccataccat gaagccatcc aggagtcaga
30781 cctctctgac agccctgcca ttgtcacaga gaagtttctg tcaccagtgc tcatgctgtc
30841 agaggagcga aggagaaaag atgtgagacc tcccaagtca aagtcatcta tggataaaac
30901 cttagttgca tggcacacca gtgttaggga gtcggggaaa cacagccata gcccagcttc
30961 ctctctgttc ttgctcttat taccaccaga aagaggttgc ttagacaacc caaaccaaga
31021 cacagggctc tgtgggaggg aatcagtccc aggcttctgg cacatgctat gtcaccggaa
31081 agccccagcc ctactccgaa tccccacaag tacagcaaat atcagattat agcatttaaa
31141 ggggcactct tgccaaagag aagcaccatt ggaatagcca tgcttgagaa ctggtcctac
31201 ttactgcaga accatggata caggctccct tttgtagatg ggcttaataa atacttctat
31261 aagtgatact ctgctttgtg aaaatgacct cgtcaatatt caaagtaatc ctctggttta
31321 ggactactat gaacctgtgg ggttcattgt tcatgtggtt aaacagcaaa gagtagttag
31381 acagttgtcc tacgtcacag agggggacat atgctatgct tggttaaata gctgtcctgg
31441 tcagagggga ggcatgctat tctgcccttt ctgacagacc ctgattgcat agacatttca
31501 gtgagataaa ggaaggaagg gaagaaggag gaaagacaac attttttgct tctgttaagg
31561 tagagactat ctgtgatcca gttcagcaca gtgcctgtga gtagaagcta caggtcaggc
31621 aggagccaag gaaatgtatt gcttttctaa ttgaacaaag gacacacagc tgccatttat
31681 tttcttcatt ttgacccttc agccctgcac tgtggatatg acatcaagaa actaagcagc
31741 cattttgtga aaatgagatc taagttagta aatgtggctg aaaaagaagc cagctgcatc
31801 ctccctggat ttacgagggg gaaatgtagg catactaaat taaaacacta aaattgaccc
31861 aaagctattt tgactgatat ttaaatatag attctgctcc tggacattcc agagttcata
31921 ggacagttgc ttctgttcag aggattcctc ttcggggttg cctctccttc cttaggcctg
31981 cttgtcctgc ccaaagctgc ccaagtgcat caggccccaa accaacttct ccatcctgac
32041 gcacagcaga ctaaatatgc aactttgtgt ctcttcatcc caggacaaaa ctttcaccca
32101 gcccctgaca tctgagactc tactacaggt tatctattaa atcttttata aagaccaaga
32161 aacaaagtgt tggcatccaa actttggtaa atcatagcct tttaataaag tcaaatggac
32221 caatgtactc taacaaaaaa atatgggtct ctcatttctg aatggcagat ttcaagccct
32281 aagaaccaca atgctcacct actgggcaac actgagttac agagacccag ctcccccacc
32341 cctcaccaag ccagagaaac actctatctg aacaatcctt ggtccatgga gcaagaatta
32401 gacatagaat ttgtatctca ttgtttttta ggaaaacccc aaaggctatt atgaagtcag
32461 tttttctggg caccttttct ttcccatgac aacgagttgt gggcagtctc agcagaatac
32521 tgaagctgtg gcttggggag acagagcata tactggattg gagttcatgg gtgggtgcat
32581 ggaatcaatg ccgggcatgg gattcaagac cttatgcatg tgggtagatg ctttgttact
32641 gggataaatc ccccacctgg gatctgactt caagcacaat ctttggaagg cggcattggc
32701 tctctgctaa tttttctagc acttttattc cacttatttt ctgcttgttt gctttgggag
32761 ttttgttcgt tataagacag tcttgctgtg tatcctaggc tgatcacaaa cctgtggcag
32821 tccttttgtc agcaggccaa aattcccact ttatctctga agacagaaag tagattgagg
32881 aatatatgat aaagacactc atcaaagcca ggcatctatc tttacttttc ttaaagcatg
32941 tttttgaatg gcataaaacc atgtagacaa ggagtcttat gttgtacatg gtcctacttt
33001 gtcacttaca atataggata ctttcaataa gcttggtagc ccttgcccta ttctacttat
33061 tctgttctct cttcctcggg tcttggggag ccttcttacc aggtggggtg gcataaaggg
33121 aaaagtcaca aagctcttcc tattcctggt tcccctccta agtgtacctt gctggtggcc
33181 ttgctagcaa atgtagtata acatctgact tatctcctct cagatatggt tgttgtactt
33241 agataaattt aatctagaaa ctcaagctgt atgtctttgg ggaccagcat tacagagctc
33301 ttcccttcct gtccttacct caccttggct actgtagtaa gttaatcctg atgattcctc
33361 catgagtcct gaaactgatt agttccaaga gctggaggat gagaagggat atagcctggt
33421 gcagggacac tttccaatga ccacaagacc ttgcacaagg tacacatgga atgtgttaga
33481 ctgtctcctt tctgtcccta gcctcagttg ccccagtgtt tatcaatgtt tattaacatt
33541 gccctagcaa aaatactaca gactaggaag cttgggtaca attgaaaaga gcttctcagg
33601 gttctggata ccgggaagtg caaaggttca gcatctggac agggctgcta ttgtagtttc
33661 aaatggttct gctgcaacac ccctttgaga gaatgaacac tgcttttcac atggtggaga
33721 gtgcacagac accaacccaa ctcctgaagg ccctttctcg agggctctaa tccatcatga
33781 gggccatact ctcaggactc attacctccc caacatcccc tctctaaata gtaccacact
33841 gcatttgcat ttcaatatat cactggagat atataaatct ccagaccaca gcataccata
33901 aatcagataa ggcaggcctg ccttctatag cctttcactc agcaaaggtg tttctagccc
33961 aaagcagtct ggactctcac tctgaaacct cttgggagtg gtggccagaa atgacttccc
34021 atcatccctc tctcctgacc tggtccagca ccaggtcacc aggaaatcct ccaagtttca
34081 ttatccccac ccccaattgt ctcttgtctc tagcaaacct cttccaatac ttccttcctt
34141 ggtgggtgta gcaagccaga tgatagcctg ccaaagaagt tcacagcctc atttctggag
34201 cctatgaata tgttacattg tgtggtaaaa ggaactttgt aggtgtgatt aaattatgaa
34261 tcttgaagtg ggcagattat ccaagtgagt ccagtgaaat tgcaaaggta catcaccaac
34321 agtgaggcag gaaggccaga gggggagaag gaagcagaga ggcagaggga ggaaaagaca
34381 agccagggga ggggagtggg gggaaagaaa ggagagagag agagagagag agagagagag
34441 agagagagag agagagagag aaatatcaca cacacacaca cacacacaca cacacacaca
34501 cacacacaca cacacctgaa cctgattgtg gaggaagaaa ccactaacca aggcattcga
34561 ggcagccttt gaaagtcaca agagacaggg aaaacagatt ctctccctcg gcccttcaga
34621 atcaacacag ccccacaact gctgatttta gtcatgttaa agccaagttg gacttctgac
34681 tgccaaaact ttagacgagc aaataaatct gcactatttt aagataccaa tgtgatttgt
34741 tcatgaaaac aatcaataag gaactaataa agtagaagtg aaaattggat cacttctgaa
34801 gtttggtaat atccacagaa actggacaca tgctgacttt gtgagccata gctccacacc
34861 caggtatgcc ccctacagaa atgtgtatat aggtgggcag gagatgtcac ctgctgtgtt
34921 catagtcgca cctttagact ttcccaagcc tgagaatagc ccaaacacct accaggagca
34981 aaataaattg agatatacag acgcagtggg atactacact tctaaaagaa tgagaaaacc
35041 acgctataca ctgtatatcg tcggaacagt aacacagggg tgacaatcag gcaataggac
35101 atattctcta tggctttaga aaacataaaa atagcataac agttctgtta gtggcaatgt
35161 gttctgtttt gtgatctgta tgatgcttcg gtttgtgcaa aagctctgga cttacctttt
35221 aaatgtatgg tggtctatac cttttaaatg tatgctagat atacatgagt aaaaatgatt
35281 aaaagagatg gaggggagga gactcatgcc ttcataaaag tttgttctgt cctttctggc
35341 actgtccaag tgaatgtgtg taaacaaaga gtgacccacc ccaggtagtc caccttctta
35401 gaacctactt ctgctacaac atgtcctgtg aatgtgcacc aaatgtttac taagggatca
35461 tgccacaggg ttttgtttaa ataaagtatg tctacctagg ggtatattga ttgtctttcc
35521 ttttgagggg gggtctcaaa actacaaact agtttgtttt gagacaagta tgtagcccag
35581 gatggccttg aactcacacc ttctgtcctg cctctttccc agcactagga tggcaggtga
35641 gactatcagc ctggccccag gaaactatct ttgattgaca ttatctggtc agaaaagatc
35701 taccttttcc tccaccaggt cctccaaata catgaagagc tgaaacagtt ctgtctaccg
35761 aatttccttt tttcttgatg tttctgtgga atttaataca taaattttaa tttgcatttt
35821 tagcttttct attaagcctt aattagagta taatgaagtt atgaatttat aaaaataaaa
35881 acaaaacggt tgctcccaca atcactcagt cttgaagtga ggttctgact ttacctgaag
35941 tgggggaaga gagtgaggaa agggacctgc ggaagctgaa tctcagaccc acaagatgga
36001 tctgagatcc atccaagcga acgtggacgc agacccggag tagggacatc caggggtcat
36061 cttcatctgt cctcgctgtg cttctgcccc tttgctcctc taccagtctc agctgtcaaa
36121 gctcagtggc ctggagggga gatggggcgg ggcttaggat cgaaggcgga gcctcggaga
36181 gcatcttctg gcccccgggg cctggactgg cccgccgccc ccacctgcag cgcggcggag
36241 cgcgggcgcg tcactcccag cggaagcgcc agcctcgcgt ctggcgaggt gcgcgcttcg
36301 cggctcccgc tccagagctt cgtggcccgc ctgtgtctgc agagcagggg cgggggcccg
36361 gcggcaccga ctgggcactg agatccaagt agccactgaa tcgtagacag tcacccagct
36421 cggacagcgc gtcggggcgg gagcagatcg ggaaggtgaa ggaccactgc ggatccgaca
36481 gcgcgtccca ggtcagtcct cccgctgcac ttggggaaac tttgggatgc ggtgacggct
36541 gcgagatgag gacactgagg gtcgcgaggc cgcgtggccc ctgtgaaccc cgcgaacccg
36601 tacctgccgc gcacctgaca ccgcagctgc cagggcgggg accgaNaccc tgctgccgcg
36661 gaccactgcg ggccaccaag ggctagcggg cttcaggggc ctctcgggag cctccggctt
36721 gcccgcgccc agccgcgcgc ctccggtcct cgcgggtccc cagctccttt tggcggctcg
36781 cgcccggacc ccgcggggct gcggattccg ccgtcttcgg gcctcgtggc gctggaggag
36841 cggcccgggg gcccatggct gcagggtggc ggccccgcgg cgggagcggc gcgtgctcgg
36901 ccggtggagc gcgcgggtcg cggggttcgg ctggagcgcg tggccgcagg tgcctgtggc
36961 cgctgggcag cggaggtgag agcgcgggct ggggacgcgg agcggattgc aacctctggc
37021 tgcaggaacc agggtcgctg ggtgagcagt cctgtccccg cggcttccgg gcgtgcacat
37081 ccctggcacc cggcatccag accccatcag ctggaggcgg gctgcagagc ggcgcctgcc
37141 cgggccgagg accagtgcct cctgctctga cacgccatct caccaacgag ggcggggtgc
37201 tagattggcg ggctgcgcgg ggaccactgg ccagggcctt ctggcacaag cccttttcgt
37261 ggacagctgc ctgctctggc ttggagtgga ggagacgaaa tgagtacccc gcccccatca
37321 gcgccccaac actgtcgccc cagtcacctt cctttgccct tctccgacag caccttggac
37381 ttgctccctc ccgaattggg gaaaatctga ggaaaccagg cagggacctt ggagataccg
37441 cagcctgcat actcaacagc ctggaaatcc agtcaccttg gtacctcgct gcttcccaga
37501 cactttggag gagcaggttt gccatttcta ccccacatcc gtaccccatc ccccgtccgt
37561 ctctgctgag gaagggactc ttatgagaga agttgggatc taggtacccc ttaaggtagc
37621 cccagagtct gtggtaacta ggctcatagg taactaaaag gcatcctagc tctgtagctt
37681 tgtgagggaa acaaacctta ccaactaatt ccttcccttt ctgaatattt cttagaagac
37741 tggagaccaa cggaagccga ctgttctggc cagtctttgc accctttgct tggctctgac
37801 tctccttcct aggcagagaa acattttgct tatgacctct ggctggcctc cttccaatcg
37861 ctgcctggcc ttggactgcc catcaggact gtgatttttt ttttttttta agacctgatt
37921 aggaaaggct gcaagcctcc ggttctagaa ggctcaaact caggggtata ctcttctctg
37981 atacccatgt gctccctaat tccactgtgg caacacctct gcccttcact cccacaagaa
38041 aattggttgt caaacctctt ggggaagatg atggaggcat ccctgtggga gcagatgcag
38101 gatttggaag caaccaggaa acaaccagga gtgaggaatc ttttttaaag gctcacatga
38161 ttctggaact aagaaaagat ggagatgcca ccagtgtatg aagcttggcc tctcctcggc
38221 ccatcccacc caactcaggg aactggcata tgcaggacct gtattgggtg atgcatattt
38281 ggaacctagt acttattgaa ttcctaagca gtaaacacat tccgaatttg aaattcctca
38341 caatcatcta ctgNaatgta gatattaaac ccccaactta tgaatgatag ccccaaaatt
38401 gttaacattg agagagccca ggttccctgc cacctcttcc acaacaggac aggaactagg
38461 acaatgaata ggaccatttg agctttaggg tcatgtgccc actttacagc tccatagcca
38521 gacaactgtt ttataagaga gggcacaaag gaaaatcact gtcctgtcca aatgaataga
38581 aagctgggga tggtggcagg acaaaggcaa caggaaaaat catctccaac aaggctttcc
38641 aagcatatca gtcttatact actgccatgt tgggtaccac acaaatcagg tatctcaaac
38701 tggacgctgc ctagggaggt ctgtcatcta aaaaggcagg gagatattga gataaaatac
38761 acagaagcta gtatttaact ccaggctggc agataatagg aatgaccttg ggagggtgtg
38821 cttacctttc cttctctctt gaacaaaatg tggactggac cagatgagca ccaaggctcc
38881 accaactcta acagaccttg tgtggtgggc ttgcctgcaa acagacttga gctaggttgc
38941 tgtgcgtggg atccattcca gactcattta caaactcgta gtcagtgaaa tgtgataaac
39001 cgaacactgt agggatttct aaacaaggaa ttaaaaaact cgactccaaa tgggagagat
39061 gcaggcaaca aatcgacagt gtttatgtgc ctctgaatag ctttgatttc cttcggtagg
39121 agctgacagc tggctgacag aaagctcacc cagggagaga agagagaaaa atcaagtatg
39181 agattaggaa taatgttttc aggtaacttt ctattcccat tcggagtggg tgtctggaag
39241 ggcgagtgta gttatggctt gaattgctcc atttatccac agatattttc ttcccaaggg
39301 ctcctgattc taagatgctg ggctttgctt ctgtctccta gtttcctggt agcagggtag
39361 agagctgggg gtcccagcat tcagcctgca tattcttcct ctatcctcac tatctgctgc
39421 ctccattatt tgtggtcttt tggatctatt tggtcagaga gtcagtcttt ggtttcttgc
39481 cctggaaact gcttgttgct acttgtggtg ggggcagcat ttggaagtcc aggtgctctg
39541 cccacaaact ttcaacccat catttgtttt tcatcccttt ctcattgcca ctttgtgtgg
39601 tgcctgggac ttctgggacc tatagttcaa gggtcatata taccaatggc tcacatgaca
39661 gcactgatca ctctgccagc tctcctctct ttgcaaaact tatttcagat ttttcatttg
39721 acaatacctt tcctccagtt gtctttattc ttggcagcat atgccttgta acctttaaaa
39781 aggaaggtaa ataatttgag aaaaaatgta ccaagtcctc agtgatacat tcttactaaa
39841 gactcccagt tttaacaagg agttgggctg gagccatggc tcaacagtta agagcactac
39901 ctgctcttcc aaaggacaca aattccattc ccagaaccca catggcccct tccaaacatt
39961 gataactctc gttccagggc acctcatgcc ctttcctggc atctgagaga accagcataa
40021 acatacatgc aggtgaacat tcatacacat aaaatgaaca ttaaaaaaga aatgaaatag
40081 agaaagggtt tacataacta tttaataact aagactgcct aataatgtag ggacccataa
40141 agaaaatcta gtaagttttt acaagattcc actcaatcag accaaacatt actgttactg
40201 acagagtaaa aagtcacttc caatagtcca agaacaactt tgtttcattt ctcaggcact
40261 gtctgttttg tggcatatgt gcatggtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg
40321 tgtgtacagg tgaatgctgc tcgtgtatga gcacatgcag gtgtgtgttt gcatggtgtg
40381 tagacagagt ttctgacctg cctggtccca cagctgtttg gccacaaata aacatacaga
40441 ggcttatatt aattagaaac tgtttggcct atggcttagg cttctcactg gctatctctg
40501 tcttaattat taacccataa ctactaatct atgtatttct acgtggcgtt atcttaccgg
40561 agaatacttg gtgtcctatc ttctcagcaa ctacatggcg tcttctctct gcgtcttctc
40621 cccagaattc tcctcgtctg gttgccccgc ctatactttc tacctggcta ctggccaatc
40681 agtgttttat tcatcagcca ataagagaaa catatgtgaa gaaggacatt tccctatcaa
40741 tggtgtgtgt gtgtgtgttt gtgtgtgtgt gtgtgtgtgt gtgtgtatgt gtgtacatgg
40801 gtatgtgagc acatgtgggt atatgggtgc atgtgcacct gtgtgtgtgc atggtggcta
40861 gagttgaggt tagatgtctt ccttggctgc tctccacctt ttttttattg aagctctcac
40921 tgaacttaga gctcactgat tcagctagtc tagctacccg gcctgctctg ggggtcccct
40981 gccttcactt tccatgtggc taccatatct actttacatt tatgtgggta atggggatct
41041 gaactatggg gtcctcatgc ttgcatggca agtgctttat ggactaagac atctttctag
41101 cctttacctt tttttttttt gaaagagttt ttttttgcta actgggaact caacaccaga
41161 tagctagtct actggtcact gaggcccagg gatctactat ttctgcttct cttcccaagt
41221 gctgggacta cagactgtac caccatatcc atatttcttt tagcatgagc tctggaagtc
41281 aaactcaggt cctcacgctc acaaagtaag tgttttatct accaagccat cttcccatct
41341 ctgttgtttt aaaaggcttt gaatatggga tgtgatgaag ggaggtgaaa ttctgagata
41401 aatttcttga aaagaagaat gaatcaagta ggagaacctc ctcctggtgc tgtctttcag
41461 ttccatgtcc acacagcata aacattatga ttatcattcc acagattgta attagtcttt
41521 ctctgttttg ccagtctgct cccaaaaaat gacacagaga gacttcttat taatgatgaa
41581 agctttgcct tagcttaggc ttgtttctaa ctaactcttg taacttaaat taacccattt
41641 ctattcatct acctgctgcc acgtgattca tgacttttac ctctctctca ttctgcatat
41701 cctgcttcct ctgcttctgg ctcatgatcc cgcttttctt cctctccgag tgctctgtcc
41761 ccagaagtcc cgcctaacct cttcctgcct agcaattgcc catttggctc tttactaaac
41821 caatcacagt gacacatctt cacgcagtgt aaaggagtat tctgcaacaa caggtgatga
41881 agccaacatt ccaagaggcc agggcttgcc tagggcacat agctaactta agaaaattag
41941 gatcgcattc tacatctgtc tgactctgaa ttggatctga actgtgactt gcatggaaga
42001 cccaaagacc ctgagaaagt acaatgacaa aggggctgac tctgtccaca tggtgttagc
42061 ccaggtttcc cacaggagga aaacccatcc taggcaagag aagtggtctt catcaaacac
42121 tctatgaaaa gcaaatcaga ctcaaatgtc aggatttgtg ctttacagat cgatccggta
42181 agatgaaaga acttcctgaa agtgtgtgaa ggcctaaagt cagggctgtt catggaaggc
42241 actgactaca gaatgaggtg ccagaagcct agtcagagcc tctagggaat aaagtgtcag
42301 atgatcttct aaaaaagttg aagtttcacc agtaacagaa tggccccact attaaaatgt
42361 gagcaaactc agaagtcatt gtagcatata gaagcacaga cctatggatt gctggatgga
42421 gcccaggtat tcactccatc ctgaatagcc agctggggag ctagctcagt cagttaagta
42481 tttgctatgc aaatctgagg accagacttt ggtctcctgc atccacagaa atggtgcaca
42541 cttgtaatct cagcactggg gaagcagtca gccagatcca acagctgcct agccagcgga
42601 aacagcctta tcagaaactc atgggtcctg gtgaaagata ttatctcaaa taacaaggtg
42661 ggaagctcct gaaggacact ggaggttaac ttctggataa acataggctc gccccaccac
42721 cagtgagcat gtgcctaaat ccgtacataa caatgatgta aagatggaat tcattccagt
42781 gaaaagtaag cctcctggac tctttttttt tttttgttgc tagatattct cgagacctca
42841 ggagagaagg tttgccatca tctatataac atggtactca acttccctgt agtccacaac
42901 attcctattt ctatatgatg gagaagaggc cactgcccct cccagacatc tcagtctcaa
42961 atttgttacc agttccctct cctaataagt gcttagggtt agtgttgtag agaagggctt
43021 tacatgaagt gtgtgtgtgt gtgtgtggtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg
43081 tgtgtgtgtg tgtgtgtaac ctaaaggctt tccatgtttc cacactgaaa ggttcttaag
43141 actgagaaca accagataag agtccaaatt ctagaaacca tgggaaagtg taatattgaa
43201 agtcagaaca aggcatggtg gtgctcacct tgaaacccac cacttggggc agaggcagtc
43261 agatctctgt gagttcaagg cccagcctgg tctacagact gtacatagtg agttccaggg
43321 ccagaactac atagtgagat cttgtctggc caaaaatata taagtaaata aaataaatca
43381 gtacatggta acttgttctt atttcagtgt ctgtttctca agcatgactt tggcttaagg
43441 atttttccca acttgttttt gtgattgcca ctgtatcatt tctttgtgtg aagttactaa
43501 gtggtttctg tatttgatat tatgttctga cctagtttct tttcatatta aacccatttg
43561 tatatgaaaa ctgcaaagaa gtgggttttt tgttttttgg gttttttttt gtttgtttgt
43621 ttgtttgttt tttcttggtg ttctcatgtg acctttccaa tgtttgcttc cagaatagac
43681 ctgcaagttg ggatccacac tgccatctga agtcctgcac cccaagtttc aggtatgttt
43741 tgatggcaga atagcttttc tagactgtga caataggggc ataaagccac aaagcattcg
43801 ctttcctaca ggttatgcac ccactctctg agtgattggc tgtgcatcat gaatattatc
43861 aaaatggagg cagttcagtt tggagtgctg tcttttatgc gcttattcat ggcaatgcca
43921 atggaacatt cggcaacata tactactaat catgcatggt aactgaactg tgttgtgcaa
43981 ggaagacctc atatgaccta cctttgcata tgctgacctt ttctgtgaca gactcctata
44041 atactgagag tggtactgta tggaagagtg tgtgaaaatg tattgtttaa ataacagaca
44101 gatgcctcta aatacaacac ccaagcagag aaatggagca tcactggcac tttggaggcc
44161 tctgggtaac ctttccagat cacactgttt tccttcctcc accaataacc actttccctt
44221 tggatgctac tcatagttaa catctttact tttgttgttg tcccactgat gctaagaaaa
44281 ataacttcaa ctagcaagca caacactaga tgaattaaga gtgatattga ctgtgtgtgg
44341 tgagtctcag aagactagct gcctcaggat tcatgaatgc ttacaggaac cctttagcaa
44401 ggtcaggaat gagtcttagg atccatgtgg ctcatagtct ccagcctgga catggagtag
44461 cacagtgtct gagtgcccca agggaatggg cttgttcagg ctcccctccc cgtccccagt
44521 tccaacaggt ctcagatcca ggacatcaga gctgagtgaa gagcagagct aaaaggagca
44581 ccatcggagc cctagaagca gaataggggg ggacacagca cacagagaca agaactgagg
44641 ccaggctgct gtgtgctttg ggcctaagtt gacagatgaa acatggtagg gtgaccacat
44701 ggaggatgtc tgtgcacatc catcaaactg gcaggtcccc ccagcatttt ctgggagctt
44761 ggggtcctct tttccatgat cttcagcttc tgtattctat gtgcgctgtt accatttcat
44821 cttggtagag tctatccttc tgttatttct tgagagtatg tcccaattct tgcctggagg
44881 tttggctaaa tatagaattc taagcagagg gtcatttctc cttcagatat ttaaagacac
44941 tttctgtatt gtgcctcatt gccattgttg atatacctga atctaaattg atcccttggt
45001 gcgtgactta tccccacagc caagggcccc ttcccttctg gtctgtgctc tggaagtctg
45061 caggcacatg gtatgggtag ccactgtttc attcatagtt caatgctccg ataggccctt
45121 ttgatttgat aactctatcc ctttccccca ttcccgttga tgatttcttc ttttgttccc
45181 cttttgatat agtttccttg ctgatgctgt gctaaaatat tcctaccaaa aacaacctgg
45241 ggaggagagg cttcatttgg cttacaattc cagctcacag tcattgaggg aagtcagggc
45301 aggaactcaa ggcagggagc atggaggaat tgcctgctgg cttcctctct gacttactca
45361 caggttcttg taggctagct ttctgataac atctcaggac cacctgctta gcaatagtgt
45421 ggtccacagc aggtttgaac cttctgcatc agttactaat caagacattt gcccaaagac
45481 atgcccacag gccagattga tgtaggcagt tcttaaatca agtctttttt gtcaagtgac
45541 tctagactgt caagtcgaca gttgatgcta actaggacac tattctacca cttttcttgg
45601 tagaaatatt attcggatat tggagttctt ggactagttt ttctggttct ccttttcttt
45661 cttttcctgt tatttatatt tgttttatga gatagggtct ctctgtgaag ttgtcctaga
45721 ccttctggcc ctcctgctta taattcctaa gaactgatat tacaggcagg tgccatgagc
45781 ccaacgtttt ttcttttctt ttcactgcac tctgtttgag agtctcatcg tcacagtcat
45841 tcacatcttc tattgtcttg tttttctttt taaatgtgca ttggtgtttt gcctgtatgt
45901 atgtctgtgt gagggtgtca gatcttggaa ttacagttcc aaataatatt tctaccaaga
45961 aaaagtggta gttgtatcct agttggcatc aaatgtcacc ttgacagcct tgagtcacct
46021 gagaagaaag acttgattta ggagctacca tgtggttgct ggtaattgaa cccaggacct
46081 ctggaagagc acccagtgct cttaactgct gagccatctc tctggcttcc ttctattgac
46141 ttttgcaggc ttctttcttg ttcttttgca atttcatggt ctctgactgt tcttcacaga
46201 ctcttacctc atgcttaaga tgtctcttac tccttcaagg atactgagtt tttgaagttt
46261 taattctcct gactactgtc ttttccctcc tgtttgtcat tctctgtttg ccctggcctc
46321 tgtctttcat gcaggaagac ttttcatttg cttttaggtt tttattttaa ctattggttc
46381 atgactaaag ggctagatga aaaggccagt gagaaggctg gagcatatgg gtgatacttg
46441 tcaaccggga gcctcactgt ggaatgcttc agtggcatgt gaaatcctgt ggtatttgct
46501 caggcaagtg cagctgttga atgcagacca gagcagcttc cttcgaagga gtcagatgtt
46561 gctgactgtc tttctgcagc tggtcaggaa ggtgggatag acttcagctc ttttcaaaca
46621 gtggtcacca aacaaccact tgcccagaga ctttgtgctt taccattctc agagaacaga
46681 cctctggatg gccccatggt ggaagcagcg cacctgtcta tcacaggtgc tctgaaggag
46741 ttggaagaac tacccattgt ccacatttcc cacattttca catgccagct tcactctggg
46801 atctgggtga cagtggggct gacataatgg caggggttgc agtttcagac tcagagtatg
46861 tggtaggaat gctgctgtct gagggaagac tcatctgagc agtggaggct ttgcctgttc
46921 cctggcatca tttgacctgc ccctccttag aactgggaac cccagttcta aagctccctg
46981 ctttaaagat tctgtgttgg ggtaagttct tagctttctc aggctaggtc ctctgctctt
47041 gggtttccac ggcactgttg ttttccctct ggctttgtga gtggttgtct tttgaaaaac
47101 tagttagttt ggaaaatttt gggagggagt caaataagat gtatgcattt tgccatgtaa
47161 gtcctaacca agccatctgc tgtggtattt tcctgagttt ggttctgccc ctataggcag
47221 agtctgtcat cacagataat tgcattttga acttgagcat ctcccttcct tctttgtctg
47281 cctgaaaaag tctctttata aaaaaatgta atgttaattt aaaaagtatt cattattctt
47341 gtgttgtgat acatgagtat atatatgcta tgatgcatat gtgcaggttg gaggacaact
47401 ttctgtagtt ggttctctct ttctcccttc atgtaggttc tggggatcga acccaagtca
47461 tcaagcttgc acaacagcac ctttaccttc taagccttct catcagccct ttttttattg
47521 attgattggt tgattgattg attgattgat gctagggata gagcctaggg tcttttacat
47581 gctaagaaaa tgctctacca ctgaactgca ctcctagccc aacctgctaa attcttacac
47641 tgtcttcaaa aagaagctct gatgctggat tctgcaaagt ccatttttat ccctaaattc
47701 ctaaagctgt ttaaatctcg tgagtcttac tgtacagacc agctctgtgc accatcttcc
47761 acaatctcca tgacctcctc aggatgggct ggtatctctg cagctctgcc cagtgcctac
47821 caggaactta caggtgtcac caatgaattt attggtgcat gctcacttca tcttgtccct
47881 atccactttc tgctttgact ccttctggta agagacaagt gtgttaacta cttgtgctat
47941 caccacacag aaatccatat cccataatct tagtcctttt tatttactta tttttgagac
48001 agggtcacac tctgtagctc ccacactggc cttaaacact gacctcgaac tcatggtgat
48061 tctcctgcct aaacttctca aataccatga ttacaagagt gacacaccat gctgggagtc
48121 ataatcttaa gtttaaaagt gagggactgg tcagtttact gtgctaggtt gacattgtat
48181 agaaatgaac agccatgttg gtctggaaat gttcctagtt ttcatttgta caaggatatg
48241 cagtgtgtga aatagggaga gtcttaccta tgtgggtttg atcacagcaa ttaataaaat
48301 atgctctaaa taatgaaaaa agccagtaac tagtagtgtt tctgaatcct cactaaagct
48361 ttaatacatc ataaataata tatcactgca gattatgtct acatgttata catatcacat
48421 ttatagtaca atctgatctt tgtcacctac tgtaagcaca actgaaaaac aaattttctc
48481 atagctcaat attaagtcat tattatcccc ataataagta attattatcc ccataatgaa
48541 actatctatt gagggagtca gaatctgaga tagttaaata aatttaagca tgtattttta
48601 gtgtcaatgg taaaaattaa atgttcataa agcctgtatg actcctttta aagtagtttt
48661 aattttatgt gtatacatat atgcatgttt tgccttcttg tatgtctgag taccacttgt
48721 atgtctggtg cctgaggagg ccagaacgta tcagatcccc tgaaactggt attacagttt
48781 tgagctacta tgtggctgtt gggaattgaa cctggatgct ctgaaagagc agccagtgct
48841 cttaatgact aggccatctc tccattttct taaaaaaaaa tttaaaacat ttactctaag
48901 atttactttt atgtaggtgc gtgtgtgaat gtgtatggtt tatgcattgg ggtggggagg
48961 atggattagc acagtcacag aagactagag gagggtctct actattgctt tctgtcttct
49021 acccttgaga cagggtctct cactaaacct gaaactcacc tttgcagctg gggtagctgg
49081 tcagaaagat cctggaatct gtctttctcc ctggccctaa tgcttgagtt acaggcccat
49141 gtgaccatac ctgtcgtttt actggggttc tacagagtca aacccaagtc ctcacgcttg
49201 catagccagc gattttaccg actgagacat ttatctgccc caattcataa ttcttctctg
49261 cttccattaa taatcccatc tatgtcccct tcatacatat ttctgaaata gacaaaatga
49321 atacaagtta gacatcgagt ctgattaatc ttcaacttct ttgataacca ggtattgatt
49381 tctgactttt gaagatggat gaaggcacag aagtctccac tgatggaaat tccctgatca
49441 aagctgtcca tcagagccgg cttcgcctca caagactttt gctcgaaggt ggtgcttaca
49501 tcaacgagag caatgaccgt ggcgaaacac ctttaatgat tgcttgtaag accaaacaca
49561 ttgaccagca gagcgttggt agagccaaga tggttaaata ccttctagag aacagtgctg
49621 accccaacat ccaggacaaa tctgggaaaa gcgctctgat gcacgcatgc ttggaaagag
49681 cgggcccgga agtggtttcc ttgctgctca agagtggggc tgacctcagc ttgcaggacc
49741 attctggcta ctcagctctg gtgtatgcta taaatgcaga agacagagat accctcaaag
49801 tcctccttag tgcttgccag gcgaaaggaa aagaggtcat tatcataacc acagcaaagt
49861 caccctctgg gaggcatacc acccagcagt acctcaacat gcctcccgca gacatggatg
49921 agagccatcc gccagccacg ccttcagaaa ttgacatcaa gacagcctcc ttgccactct
49981 catgttcttc agagacggac c
SEQ ID NO: 3 (Chromosomal region 5,000-55,000 basepairs downstream of CHO GS
gene coding sequence)
1 GGGCTCAGGC ATTTATCGTT CAGAGATTGA CTGAGCTGTA AAGATGGAAA GACAAACTTT
61 TTTTTTTTTT GATTGAGTCG GGGTTTCTCT ATGTAACAGC CCTGGCTGTC CAGGAACTCA
121 CTCTGTAGAC CAGGCTGGCC TTGAACTCAC AGAGATCTGC CTGCCCCTGC CTGTCGAATG
181 TTGGGATTAA AGGTGTGAGC CACCACCGCC CCGCTGACAA ACTAGACTTT TAGAATGTAT
241 TATGAGATAA GGTTTTGTTA TGTTGCCCAG GCTGGACTCA GATCTGTAGC AATCTATCTG
301 CTCCAGACTC CTGAGTGCTG GGATATACAG ACCTGAGTTA CCTGTACAGC TTTCTAATCA
361 TCCCCCGCTC CCCCAGAGAC AGGGTTTCTC TTTATTGTTT TGGAGCCTGT CCTGGCACTG
421 GCACTCACTC TGTAGACCAG GTTGGCCTCG AACTCACAGA GATCCACCTG TCTCTGCCTC
481 CTGAGTGCCG AGATTAAAGG TGTGCACCAC CAACACCCTA CTTTCTAATT CTTAAAGCAA
541 GGCTCCCAAC TCCTCCCTTG TGTGTAATCA ACAAGGTTCT TAGACCCTGT CTGCAGTGTG
601 GATTCCCACT AATAAGACAG TGGCGGCACA GTGCTGTGTG GCAGAGCAAG CGTCCATCTA
661 GTTCCTATTG TCATTCTATG ATTTGCTCTT CTGGGAGCCT TGTCATTCAG CAAGTTCCTG
721 GGCTTGTCTT GGGATTGCAA TGTGCCTCAG CTTGGCTAGT TCCTCTGCGG CAGAAGCAGT
781 GTTTGAACTC AGTGGGCACT CAGTCACTAC ATCTAACTTG TTTGAGGGCT CTCTGCATTT
841 GCTTTCCAAT TAAGGTTTAG GATGACTCCT CCCTGTGACT CTTATCATCC TGCCTATTAA
901 TGCTAAATTA GAGAGGCATT CAAGATAACT GCCGAAGATC TAATAAATAA ATGGGGTGGG
961 TGGGTAGGAC TATAAACCAG TTTATAGCAT GCAAGAAAGC TCTGAGCACC ACATTCAAAA
1021 ATAAAGTGCT GTGAGCCTGG TGGTGGTGGC TCACACCCTG ATCCCAGAAC TCAAGAAGTA
1081 GACAGAAGGC TCAGATTCAA GATTCAAGTT CTTCCACTAT ACAGCCAATT TGAAGTCAGC
1141 CCAGACTACA TGAGACCCTG TCTCAACTAA GCAAATGAAA GCAAACTGGG GTCCAAATAG
1201 GCACTATTCG ATGTTTTGAT GCAAGTTTGT GACTGAGGAG TGGAGGTGGC AAATGAAGAC
1261 TTTTTTCTTC CTCTTCTTCT TCCTCCTGGG TCCCGTTTTT TTTAGGGTGT TCTTAGGATA
1321 TGTATGTCTC ATTGGCACTA CTAAGAAGTG TGGGGTCTAG GGAACTTCCT GTTATGTATA
1381 CAAGCTAATC TTCAAACAAT TGTGTGGGCT GTTTTGGTAA CTACTCAAAT AATGCTATAG
1441 AAAATTGTAC AATATATTGG GGAAGGAAGG GAGTTTTACA CAGGAGTCAA CATGACTCTT
1501 GTCTCTGGAA AGCAACTTGT GATCCAATGA GGAGCTAAAT TTAGAGACAC AATTCAGGAA
1561 GAGAATCCAA TCAGAGCTTC CTTGTAAAAC AACTCACCTT CACAAACAAG TTCATTCCTA
1621 ATCGAATTTA AGGTCTAGAA ACTGCCAACC TATTAATGTT TCTATAAATA CACTTGGGGT
1681 CAACTACGTA GCCAAGGAAA TCTTTAATAA ATTGAACACA AATTGTCAGG GGAAGGTTAT
1741 TGCTGGGACT CCTGGAAGCA TGTATAAGCA GGGTAGGGGT GACATAGGGG TGGGGGGCAG
1801 TTAACTCACA GATATTAGTC TCAGATATTA ATGGCTTGTG TGTGAGCTGT CTGCCACACT
1861 TAATGTCAGT CACCTTGCCC GGAACTATTT TTCTCTCTGA TTCCAAATGT AGCTATTGGT
1921 CTATTAAATG ATTAACTTCC ACAGAAACTG ATAATATCCT TATGGAATCT GACTGTGGTA
1981 AGCCTGTACA CCCCCGCCCC AATTTCCTTC TAGATTTAGA ATTCCATTCC ATGAGCCATC
2041 ACACCCACGC TGAAAAAAGA AAACCTGTTG AATCAAATTT GTGTTTTGGA GGGTAAGAGC
2101 CACCCTTCCA ATTTATAAGG CTGTCTATTT CTTTGGGGGG GGGGAAATGA ACCAGTATCT
2161 TCTATTAGTA AAAGGAGTGT TTGAGCATGG GCACTACAAC CCACTTCTTT CAGGGAGATT
2221 CATTTTTCTC TGAGAACTCA GCCTCTCTGT GCTGGTGCCA CAGGAATTCT TAAACTCTTT
2281 CAACTCTCCA ATTAACCAGA GAGCAAACCC AGCACTTTCC ATCTATGAGA AATCTACACC
2341 ACTCATGGAA TCATTGTGTG CCCTCTCTCA CTGCCTAACA GGGGTACCCT TGCCAAAGAA
2401 AAGCAACTTA ATGCCAAAAA GGTGCATCAC CTGGCACTGC TTCCGAGGAT GGGCAATGTG
2461 CAAGCACTTT GTTCAGTGGC TCTGCCTTGG GGTCTCTTGA GGGGCGGCAG GTTACCTGGG
2521 GTGGGGGCGC ACACTCTCTG AAGGTGGGCT GCGTTCAGTT TCCTGCTTCA GGGGCTCCTT
2581 CATAGTACCG CCCCCTGATG AGTTTCTGCT CAGACTGGAA GGTGTCAGGT CCCAAAGAAA
2641 CCTGGGACAA GGCTCACTCA GTACCTGTCG CTTCTCCCAG CACGTCTCAC CCCACCCCTA
2701 CCCTAAACTT CTCTAGCCCA GAGGCTGGGC TCCCCCTTTC TCTTTCCTAC ATAACCCTGC
2761 CATTTTAGCT GTGAGCTCTC TCCGTCTTTA GCTCCTCTAC TGTTCTTTTA TCCTCTCTTT
2821 TCTCTCTCCT CTTCTTCTCT CACCCCCACC CCCACCCCCA TCTCTCCCCC CATGGTCTGG
2881 TTCAGTCTGG ACCCTTTCAG ATGCCTCTGT CTGAACTCTC CCTCATATCT CAATAAAACC
2941 CTTCTCTTCA GCCACGCCTT GGAGAGGTCA TAGGCTCATT TTCGTTCAGA AGGCCTATCA
3001 AAGAATCTGT GGGCTTATCT TTACATTCAC AATAGGCAGC TTGGCCCTGA GACCACAGTC
3061 CAGGTTAAAG TGTTACCTTG GAAAGAAAGT CTTTTATTCA AGGTGTCTGG TTTCTTTTCT
3121 TGTTTTTGTT TTTGTTTTTG GAGACAGGGT TTCTCTGTAT TATTTTGGAG GCTGTCCTGG
3181 AACTCGCTCT GTAGACCAGG CTGGCCTTGA ACTCACAGAG ATCCGCCTGC CTCTACCTCC
3241 TGAGTGCTGG GATTAAAGGC GTGAGCCACC AACGCCCGGC TCAAGTGTCT GGTTTCTTTT
3301 GATGTCTTTA GTTTCTTTAA TCCCATAATT CCTTTAATTA TACCCTCTTG TCTGTCGGAG
3361 AATGACATCA AGGATATCCA GTTCAAGGTT TCCTATGTAG TTCAGTCATA GAGTGCTTGC
3421 CCAGCTGCCA GACTCTGTCA GATGCCCAGC ACCACACACA TACAAAGCAT TTCCAGCTCT
3481 GTGTCTGTGT CAATTACTCC TGTCTGCTTC TCCATCCCCA GACACCAGGA GGGCCCACAA
3541 GAAGCTTGGA GCAGGGAAGA ATAAAGAGAC AATATCCATA GACACACAAA ACCTCCAAAG
3601 TACTTATGCA TTGAGGAATT ACAGCTTACA AATCCAGTCA CAGTATCTAT ATTCATGTTA
3661 GCCTGATTTC AATCCCCCAG CTACATATTC TTCCATGAGC TAGCTCCTTT CCTATTCAAG
3721 ACTCCCTTGA TAATAGTTGT TATCAGACTT TACCCCTATT AAAATATTTG GACCGTTTGA
3781 GAGCAATAGC TCACCTCTAT AATCTAGAAC CCAGGAAGTT AAAACAAGAT GTTTGCTGCA
3841 AGTTTGATGC CAGCCTGGGC TACATAGCAA TTTCCAGAAC ATCCTGAGCT ACAGGGCAAA
3901 ATTCTATCTT AAAAAACAAA AAGTAGACAG ATCAGGTGTT TCACCTTGTT TCAAAAAATG
3961 CAAAAAATAT TTTTTAATTG TAGAAATATA TACGCTAATT CCTTTGGTAC CCTAGGCCAA
4021 GTGACTAGAT GGGTTAGTCT TCCTTCTGGT CCTCACAGAA GAAAGTTAAG TTCTCAGCAG
4081 GAATAATAAA AAATATTAAA AAAAAAAACA AGCTGCAAAA TTCTGTTGTG GTTCTGCCAA
4141 AGTGTTCTCA GGAGTGAGGG CATACTGGGA TTTAGTCAAG CAGATATTTC TGTTTGAATA
4201 ACTAGGATCT GGGAGCCATG GGACACCACC CCCACCCATA AGGGCTACTG AAAACCACCC
4261 CTGGAAATCT GTAAATATTG CTAAGGCTCT ACCCTTTTGC TCAGAGAACA ACCACCCACA
4321 AGGATAGGGG ATAAGTTAGT TCTGTAGTAG AGTGCTTGCT TAGCACACAG AAAGTCTTTC
4381 TCTCTCTGTC TTTCTCTCTG TCTCTGTCTC TGTCTCTCTC TCTCTCTCTC TCTCACACAC
4441 ACACACACAC ACAAACAAAC ACATGAGTGC ACAAGAAACT TCTAGGTGCT ACTAAACTAA
4501 TGTAAAATCA TGCAAAGTTC ATAGAGAATT CAACAGCTAG TGACAGGATG ACCCGAACAC
4561 AAGATTCTGC CCTAGTCCTT GTATTCTGTA GTCCCCAGTT TCTCTTTACT GCCACAGTCT
4621 CCTATCTCTG ACAGCCTCCC TCTTTGCAGA TCTGGCAGTT TCTGGGCCTG GAACTGCTTT
4681 GGTAGAATGT CTGTACAGCA TGCACTAGGC ACTGGGTTTG ATCCCCAGCA CTGCATAAAT
4741 CAACTTTGAT GTCACACCTA TAATTTCAGC ACTTGGCAGG GATCGAAGCA GGAGGATCAG
4801 AGGTGAATCA AGGCCAGCCT GGGCTACTTG AAACCCTGGG GAGAGGGATA GAAGAAGGGG
4861 GAGGGGGGAG GGAGAAGAAA GGAAGGAGGG GGAGGGAAGA GGAGAGGAAG AGAGGAGGGA
4921 GAGGGAGGGA AACAGGGAGG GAGGAAGAGA AGGAGGGAGA GAGGGAGGAG GGAGGGAGAG
4981 ACTAGTGTAA GCAGAACCTG TAAGTTCTCT CCTCAGCCTC AACACACCCC AGCTCCCTGC
5041 TGTCTCCCGG TCCAGGGCTT CAGGGCCTGG CAGGACAGGC AGCAGGTTGT TTTGCTCTCA
5101 TAAAGCCATG TTACATAACT AACTAATGTT TTGAGCAGTG GAGCTGAGCC AATCTAGGTC
5161 ACATCAAGAG GGAATGGGGA AAGAGGATGA TCACGGAAGT GGTGAGAGGA AGGGAAACAA
5221 GAAGGGAGGA ATAAAAAAAA GAGGCGAGAG TGGAAATGGG GTGCGATTAT TTAATATCTG
5281 CTGCCTGTTC ATAGTTCCTG GTCCTTAGGG ACAGCATATA TTATCCTGAA AAGTCCTCTC
5341 TCTATTTTAT CTAGGCATTC TGTCATCCTA TAGCCCCCAC TCTGGATGGC TGAACTCTGT
5401 GCCAGCAGCC TGCAGGTATC ACCCCTTATT GGAGTGAGGT CTATTCCTTA TTGGAAGCAG
5461 TGGCAGGCTG GTAGGAAACA AACAGGCCTG GTGTTGTGGA ATGCTGTCCT CCCAGCATGA
5521 CCATCATTAG ACCTTATGGA AGCAGAGCGA GGGGGGCATT GTCCTCCTCC CCAGGCTCCT
5581 GCAAGCCTAC TCAGCTCAAC TGGTTCCCCG GGCCAGACTT AGGTGCAAGA GTTGCTTTGG
5641 TTTGTTATTG GTGGCCTGTG TAGCTGAGTA GACACATGCT CACCTACATG ATATATGATG
5701 GCTTGCAACC TTCTAAAAGT TCAGTTTCAG GAGATCCAGA ACCCTCTTTT GCCCTCCAAG
5761 GACACCAGAC ACCCATGTGG TACCCATACG TACATGCGGG CAAAACACTT GTGCATATAA
5821 AATAAAAAGA GATGGCTCCG TGGCTAAGAA TGCTCCCTAC CTCCAGCTCA CCCACATCTT
5881 CACAACTGAC TGTGAATCCA TCCATGGTTC TCTTCTGACC TCGGAGGGCA CCTGTGCCCA
5941 TGGGGCATAC ACATACACAT ACACAAAACA AGTATGTAAA TAAATAAATA TTTAAAATTG
6001 GGGCTGGAGA TGGCTTAGTG GTTGAGAGCA CTGGCTGATC CTCCAGAGGT CCAGAGTTCA
6061 ATTCCCAGCA CCTACATGGT GGCTCCCAAT CACCTAAAGT GGGACCTGAT GTCCTCTTCT
6121 GACATAAGGT CATACATGCA GATAGAGGAC TCAAATGCAT AAAATAAATA AATAAATCTT
6181 TAGAAAATAA GTACATAATA AATAAATATT TAAAATGACC CAAATTAAGA AAAAAATGAA
6241 GCCAGGCAGT GGTGGTACAC TCAGAAGGCA GAGGCAGGCA GATCTCTGAG TTTGAGACCA
6301 GCAGTTCCAG GACAGCCAGA GTTACACAGA GAAACTCTGT CTCAAAAAAA AAAAAGAAAA
6361 AAAAACAGAG AAAGAAGAGA GGAGAAAAAC AAGAACAAAA AATAACAAAA CAAAAACATG
6421 GCTTTCCCTT CATGGCATCT GCTTCATCTG CCTATTTGGT AATGATCAGG GCACTACACA
6481 CCCAGTGCTT CATACCCTGG CCATGTTTCT GTTCTTGGTG TCACCACCAA GTTTACTAAA
6541 GATGGTTCCA GAGTGACATT AGCAGCCCCA CACCCCAATT GCAGCTAGCA GTTGAGGAGA
6601 TTTCTGGCTT TTTGTCTAAG AGGAAGGTTC TTTGGCTAGG AGATATACTG AGAAGGACTA
6661 GGAAAAGGGG TGTCTAAGAA ACTTGGAGAG CACATTTTTC AAGTCAGAAA GAACATAGAC
6721 ATATTCTGGG GGTGGGGGTA GTAAGATAAT GGACCCTCCT AAGGGAAGGA TTGTGGGGTT
6781 TGCCTGAAGG GGCTGAAGCA GACCACTGAG CAGGCCAGAC CACCAGCAGC TTTTGAGAGG
6841 TGGGAACACT GCAGCTGAAG TCACTTGTCA CCTTCCCAGG TAGTTCTTAC TTCCAGCTCT
6901 GGCAGGGCTA GATAGCCTAG GAACTCCCAG ATAGGAGTTC TAGTTCTTCT TCTCCCAAGC
6961 TGACAGAACG TGAGCTCAGA GTCTAGGGAC ACTCCAGGTT AAGGACGGGG CCATTCTTGA
7021 TTGTCAGCAC AGATAGATTT TAATTAGAGA GCAATGACAT GACAGATAAA CAGCCCCTTA
7081 TCTAAAGGGG TACATCCCAA GACCCTGGAG GACTCTTGAA AACCCAGATA GGAGCCAGCC
7141 ACGGAAGCAT ATACCTTTAA TCCTAAGATT TGGGAGGCTG AGGTAGGAGG ATCTCTGTGA
7201 GTTTGAGGCC AGTCTTGTCT ACAAAGTGAA TTTTGGGACA GCTACACAGA GAAACCCTGT
7261 AAGAAAAAAA AAAAAAAGAA AGAAAGGAAG GAAGGAAGGA AGGAAGGAAG GAAGGAAGGG
7321 AAAGGAAGAA AAAGATAAAG GAAGAAAATC CAAATAGGAA AGAATCCCAT ATATACCATA
7381 TTTTTCTTAA ACATACATAG GTTTATTCAT TCTCTCTGTG TCTGTGTGTC TGTGTGTCTG
7441 TGTGTCTGTG TGTCTGTGTC TGTCTGTCTG TCTGTCTGTC TGTCTCTCTC TCTCTCTCTC
7501 TCTTTCTCTC CCTCTCTCTC TCTTTCTTGT CTCATAAATC TCAACACTCA GGGACCCAGA
7561 AGATATCCCA GTGGTTAAGA ATACACACTG CTCTTGCAGA CCTAAACTCA GTTCCTTGTC
7621 CCTACTTGGG GCAGCTCACA ACCACACCTG TAAGTCTAGC TCCAGGGAAT CCACACCTTC
7681 TGGCCTGTGC AGGCACCTGT GTGAAGGAGC ACATATCCTT CCCCATAATT AAAAAACAAT
7741 CATTGAAAAA TAAAACTCAA CCCCCTCCCC CGGGACTCAA ACCAGAGGTA GTCTCCCTGC
7801 CGTAGGCGCT CAAAAACTGG ACTTTCAGGT GTGAGCCTCT AGGCCAGGCT GCTTTTCTTA
7861 ACTGGCTACC GTGCTCTTGC CTGAAACTTC CAGCTTGAGA CCTCATAGTA AAAAGAACAT
7921 ACACGTCTTC TGTCTGTACT ATTTTACAGA CGGCTGACAT GTTCATACCA CGTATTTTAG
7981 CAATTTCAGC ACTTGGTATA TTTTCTGTCA TTCTCAAATA ACTTTCACCT TGCCACTTAG
8041 GGCAGTCCAA GGCTCCTCTT AGATATATCC AAATTATCAG CCACCACTTC TGCCTTTACT
8101 AAGTAAGACA GGGTACTTAA CATGGAGTAC TTAACACAAG CACTGTGATC TGAAGGTGGA
8161 GACTGCTTGC TACTCAGTCA CAGCTTAGCA TTGCTAGAAC AAATCCTGAA CAAAGGGTAA
8221 TTCATGACCC AGGCAGGGCA GAGGCGGATG GCTGTTCTTG CTCCTCAGAA ACCCCTGTGT
8281 ATAATTTCAA GCTTAGGAGT TGTTTGTCTT TGGATGGAGA GGGTCAGACC TAGGGCTTCA
8341 CTCACACTAG GCAAGCACCG CAGGTCTACC TTCGAAGAGA AGAATTTTCA CTTAGCGTTT
8401 TCAGATATAG GTCAACCTCA GCTGGCTGAA ACTTTGACTA AGTGAGCAAC TGTGAGGGTG
8461 GGGAACACAT GCATGCATTT CTTCATGTTA TAACATCTAT TTATACATAA ACATATCATA
8521 TAAATATATT CTATTGCATA TAAATATACA TAAATGCACA CTCATGTATA GATATCAATC
8581 ACATAATTTA TGCTTTTATT CATAGATTAT CTCTGGGAGG TGTACAATTA CTGACAATAC
8641 CTGCACATGA TAGTACACGT TGTTCTAGTT AGGTTTCTTT TGCTGTGACA AACACCACAA
8701 CCAAAAGCAA CTTGCAGAGG GAAGGGTTTA TTTCAGCTTA CAGTTGTATT CATTATGAAG
8761 AGTTGGGAAG TCAGGACAGG AACCTGGAGG CAGGAACTGA AGCAGAAACC ATGGAATAAT
8821 GCTGCTTACT GGTTTACCCA CCATGACTCA ACCTGCTTTC TTATATCACC AGGACTGCTT
8881 GCCCAGGGAT AGAACCACAC ATGGGGACTG TACCTCCCAC AACAATCATT GATCAAGAAA
8941 TGCCCTAGAG TCAGGGATGG TGGCAAATGC TTTTAATCCC AGCACTCGGG AGGCAGAACC
9001 AGGCCTTGAC TGTGAGGTCA AGGCCAGGCT GGTCTACAGA TTGAGTTCCA GGACAGCCAG
9061 GGCTACTCAG AGAAACCATG TCTCATGGAA AAGAAAAGGA GGAGGAGGAG AAAGGAGAAG
9121 GAAAAAGAGG AGGAGGAGGA GGAGGAGGAG GAGGAGGAGG AGGAGGAAAG AAGAAGAAGA
9181 AGAAGAAGAA GAAGAAGAAG AAGAAGAAGA AGAAGTAGAA GAAGAAGTGT CCACTGGACA
9241 ATCTGATGGT GGCGTTTCCC AATTGAAGTT CCCCTTCCAA GATAACTCCA GGATGTGTCA
9301 AGCAGACAAA AACAAGAACC AAGACACATG TTTATAATCC CAACACTGGG GAAGTGGAAT
9361 AAGAGGTTTG GCAGTTTAAG GCCATTTTCA GCTACATAGG GAGTTCCAGA CTATCCTGGC
9421 TACATGAGAC CCTGTCTCAA AACACCAAAA TGCAAGGGAA AAACAAAAAG CAAAATAATG
9481 AGTACAAATA GCAGTGACAT TCTGGGGAGA CAGCCTGGAG GGGGGGATTG CTTATTATCT
9541 CTCCCTACCG TTTGGAGTTT TTAAAATCAT GAATCTAACC CCAGAAAAAA AAGCATTGAG
9601 ATTCTGGGAC ACTCGGGTGG TAGAGAAGAT CATCTGATCC TGTCACCTTT CGGGTACGTC
9661 ACTTTATTAA TCTCTCTGAG ATTCAGTTTC ATCACCTCTG AAGTGGTTTG TGTCGACGTA
9721 CAGTCCTCAG GACTAAGTAA GGCCACTTGG TGGCTGTGCC AAAGCACTGT GTCAGGGACA
9781 CGGCAGATGT CTGACACATC TTGTTAGATT CCTTTTCTGT CCTCCGCTCC CCTACCCCAG
9841 AGGTGGGTAC AGCCCCATGG CACCTCATCT TTAATGGCTT GGGTTTCTTT TCTCCAGCCA
9901 GGAAAGTTGT CGCTTTGGTG ACAGCTATTT TAAGTCAACT GACCTTTCCT GCAAATGATC
9961 CAGATGCCTC TATCTTAGGC TGGTGATGAC GAAGATGGCC TATGACGGGG TTCCTGGGGG
10021 TGTGTTGGGA GGTGGGGCAG GGGTGGGGCC CGGCATTTGT CAGACCCATA TGATCTTCTG
10081 GCTCCCGGGC TCTGCAGATT TCTCCTGCTG GAGATGCCTA CCTGCCAGCA ATCTTGGAGA
10141 AGACAGAAAT AGCAGCTTTG GGTTCCAGGT CCCCTCCTCC CTTTGGCCCA ATGTAGCTAG
10201 AGCTTTGGTT TCCTGCTGCT GTCTTGGTGC CTGGAGCCCT CTCTGGATGG TCATGGAGTC
10261 TTGTCAGAGA AGCAACTTTG GGCTGGCAGA CAGTCATTCC AGAAGACATG ATCTGGAAAA
10321 ACTGCTTCAT CGTTTCCTTC AGAGGCACTG TCCCGAGCCC ATTTCCTTGT CTGGTTCCTG
10381 AAATCTCAGG GATGCCATCA GAAGAAGGTG TTCTTGTGTT TACTTTGGAC ATGGTTTTCT
10441 GTAGTGCAGA CTGCCCTTAA ACTCTACGTA GCTGAAAATG ACCTTGGTCT CCAGACCTCT
10501 TGATCTGTCA GCATCCCTGG GAAATCCAGG GTTCTGTAAT CCTCCCCTCT CACCTTGACT
10561 TACTGTACCA GCATCAAACA TCCTAAACAA ATCCAGTGTT TAGCCAAATA CAGCGGTGCA
10621 TGTCTGTAAT CCCAGCCACC TGGGAAGCCG AGGCAGAAGG ATTAAGGGAG CTGGAGGCCA
10681 GTCTGTGCAA TTTAGCAGGA CTGTCTCAAA ACAAAATTTA ATGGTTAGGG GTGGGCATGT
10741 CATTTATTTG ACTCTTATCA CATGAACACA CCTGTAATCT CATCACGAAA CGACAAGGCA
10801 GGAAAATCAA AAGTTCAAAG TCATCTTTGG CTACATAGCA AGTTCTAACC TGACCTAGGG
10861 TATGTAAGAC CTTGTCTCAA AAGCAAACAA ACAAACCCCA AATAACAACA ACAACAAAAC
10921 AAAAAGCAAA CAAGGAGAGG GTGTGCAGCT AGGGATATAA TTCAATGGGT GAGGGCTTAC
10981 CTCACATGCA CGAGGCCTTG GTTTCAACTT CCAGTTGAAA TGAAGTTTAG TGGTAGAGTT
11041 CTGTGCAAGG CTGTAGTTTC AGCTCTCCAT ACTGCAAACT GGAAAGAACA ACAGTGACAA
11101 ACAGAAACAA AAAACCCCCA CAAACAATGT GCTTTCTCAC TCAATAAAAC CACCTCTTTA
11161 CATACAACTA CAACTGCTAA GAAAGTTCTT CAGTGTTCTA GAGCCTGAGC ACCTCAAATG
11221 GTTTCCATAA AGCTGTATGC AAACACTGAT AAGCCACGAG AAGCAACTGT ACAAAGCACC
11281 CTTTGATTTT CATAGTTTAT CTACACAAGG ATTCTAGGAA AGTGTGCTAG GAAAATTTTA
11341 TGTATCAGCC TTGCGGGTTT GTCCAATAGT TTTAGATTTT GCCAGTGAAG ATTTTCCTTT
11401 CTTTATTTTT TACATGGGAA GGAAGTTTAA TTGGGGGAAG GGACGGGAGT GGGCTTTATT
11461 TTTATTTTTT AATGAGACTA GCATTTGCAT TGGTGGACAT TGAAGGAAAC AGTTTCCCCT
11521 CCCTAATGTG TGTGGGCCTC ACCTAACTCA TTGAAAGTCT TAGATAAAAC TAAGCTGAGT
11581 GAGTGAGTTG GCCCATACCT GTAGATGGAA GGAAAAGGGT CTTGAGTTTT GGTTTATCCT
11641 AGAGAGAACT TGATCCCCCA AACACCAAAC TTTCAAACCA AACCCCAGCC TCCTCAGTGT
11701 GAAGGGATGC TGTTACATGA CCACCTATGG ACTCAGACAA CCTCTCTTCC CTGAGTCTGC
11761 TGGCTTACTC ATCAGAGTCT GGGCTCACGA AGCCGCCACA CATATATGAG CCTCGTTCTC
11821 CCCACTCTTC TCTTGTGGCA CTGAGGTTCA AACCAAGGAC CTCGCACATG ATAGCAAATA
11881 CTGTACTGAA CCATAGAGCC AGCCCTTGTC AGTTTCTTAA CACAAACATA TAGATGTATA
11941 TGTATATGAA TATTTCCATG CTACCAATTC CATTTTCTCA GAGAACCAAA GAATACACCA
12001 AGTAGTCACA CTTGAAATTC TGTTCTGAGA TTGAATAAAA CCTGATCAAA TGTGAATTCG
12061 GTCCCTTCTC CCCCATCCCT GACGCCACCA CGTTGCTATA CAGACCAGGC ACAAACTCTT
12121 CTCCTTGTGA ATGTGTGTAA CACATGTTAC CACTGTGCTT GGCTTTTGTA GTTAGAAGGT
12181 TGGTTGATAT TTAAAAAAAA ACTTTAATAT TTAGTCATTA CTTTTTAGTA AAGATTTGCC
12241 TTGCTTTTAT TTTATTCATG TGCATGTGTG TGTATCTGTG TGAGTGTATG CCACGTGTGT
12301 TTGGGTGCCT CTGGAGATTG GAAAAGAATG TCAAAATCCC AGGACCTGGA GTTCCAGGCA
12361 GTTGTAAACT TCCCAATGTG GGTAATTATA ATGAACTTGG ATCCTCTAAA AGAGCAGAAC
12421 TCACTCTTAA CTGATGAGTT ATCCTTCTAC CCCCAAATTT ATTTGTTTTG TTTATTTGTT
12481 TATTTATTTG AGAGGGTCTC ACTGTGTAGC TCTGACAGTA TTAGAATTTA CTATGTAGAC
12541 CAGACTTGAT AAATGTCTAA CCCTAGAAAA AAATAGTTTT GTTTTGATTT TATGTCTGTG
12601 CCATCCACTC CTTGAACATA TATTTGGTAT CTGTGAAGCC AGTGAAGGCT GTTGGTTCCC
12661 TTAGGACTGG AGTTACAGAT GGCTCTGAGC TACCATGTGC ATGCTGGGAA ACAAACTCAG
12721 GTCCTTTGGA AGAGCAAAAA ATGTCCTTTG ATGGTGGTGG TTTGAATGAG AATTGCCCTA
12781 TCGAGCATAA AAACTTGGCA GCTTTGGCTA CATGGTTCTG GATTAAGAGT CAAGAAGGAT
12841 ACAAGAAAGC GGTTGTGGAA TCATCCCCCA TGGTTAAGGA AAACCACCAA AGCCAGGCTT
12901 GTGGCAGGGG AGTTCCTGCA TGGAGGCCAA GAGAAGCCAC TATGTCAAGC TGTGAAGGTG
12961 AAGCCTGGAT TGTGTTGGAG ACCCAAGCTA CTGGAGATGT AAGAGATGTG AGATAATGCC
13021 CAGGAGAGCT GCAGACAGGG CATGGAATCA GGCCAAGCGA GAGAAGTGTG TTGCAGTCAG
13081 CAGAACTGGG AGGGAAGAGT CATCTAAGTC CTTTGTCATC AGACATAGAG ATACAGGATC
13141 TGAAATTTGC TCTGCTGGGT TTTGGTCTTG ATTTGGCCCA GTACTTCCTA ACTATGTCCC
13201 CTTTTCTCCC TTTTAGAATA CTAATTTATA TTCTGTGCCA TTGCCGGTGG ATCAGGATGG
13261 TTCTCAGATA CTGTTTTAGT TCCATGCCTG TCTACTTCCC GTCATGACAG TCATGCACTA
13321 ACACTCTAAA ACTGTAAGCA AGCTCCCAAT GAAATGTTTT CATTTATAGA GGTGCCTTGA
13381 TCATGCTGTC TCTTCACAGC AATACAACAG TGATTAAGTC AGCTGCTGAG CAATCTCTCT
13441 GGCCCCAGAA GTATGCATGT GTGCAATTGT GTGTGTGTGT GTGTGTGTGT GTGTGTGTGT
13501 GTGTGTGNNN NNNNNNNNNN NNNNNNNNNN NAGGAAATGT CATTCTGTAA ATATGTTTAT
13561 CTTATTGGTT GATGAATAAA ACACTGTTGG CCAATAGGGC AACAAAATAG GTGGGGCCAG
13621 GATATAAGGA GGATTTTGGG AAGTGTAGGC AGAGGGGAAT TGTCATATGA TCCCAGGAAG
13681 AGACATAGAT GGGCAGAAAC TGCCTCTAGC TAACCATAGA GGTCTGGAGG TCTGTACAGA
13741 CAGGCAGGAA GTGATGTAGC TGGAAGAATC AGAATATAAG CAGGAACAAA CAGGAAATCG
13801 AGCTCTTCTT CTCTCTCCAC TTCAGAGATG CTGAACAGTT GAGATGCAGG ATGCCAGAAG
13861 AGTAAGAGGT CCCTGGACCT TTCTCCAGTA AGATAAGACC ATGTGGAAAT AGATTGATAG
13921 AAATGGGTTA GAGATTAAGT CAGAGCTAGC CAATAAGAAG CCGTAGATAT TGGCCAACCG
13981 TTTCATAATT AATATAGCAT CTGTGTATTT ATTTGGGGGA CCTGGTAGAC CAGAAAACTC
14041 GTGTTAGAGA CATCTTATCA AAGTTGAAAA AAGAAAAAAT GTGATAAAGT TAGGAAAAAA
14101 TATAGTAAAT GTTAAAAGCT AAATTCTAAA ACTACAACTT ATTTATCATT TCCTAAATGT
14161 TTAAAAATAT TATTTTATAA TGAAGATACT TAAAATTCAT TTCTCTGTCT TTTGAGACAG
14221 GGTCTCAGTG TCCTGGAACT CATTATATAC AGCAGGCTGG CTTGGAACTC ACAGAGATCC
14281 ACCTGCCTCT GTCTCCTAAA TGCTGGGATT AAAGGTGTGT GCCACCAAGC CTCAATTAAA
14341 ATGCGTTTCT TTTTCTTTCT TTCTTCCTGT CTTTCATTTT TTTGTTTGTT TAGATTTTTT
14401 TTTTTAGACA GGGTTTCTCT GTTAGCATTA GTTGTACTGG AACTCACTCT GTAGACCAGG
14461 CTGGCCATGA ACTGAGAGAT CTGCCTGCCT CTGCCTTCTG AGTGCTAGGA TTAAAGGCAT
14521 GCACCACCAC TGCCAGGCTT AAAATGTATT TCTTTTTTTA ATTTAGAAAT TTATTCTGTT
14581 TAATCCACAC GCTTTATATA GCTTTAGTTA AGAAATAAAA TAAAATGAAA CAGTGAAACC
14641 AAGAGACTAT GTCCAAGTCC AGGTCCTCCC AGCCTGCCAA TGCCAAGAGC TCTTTAGTTC
14701 TGTGTACCAA TTGGAAGAGT AAGAAAAAAA TATGGATGGG AACCACACAG TTTCATAAAA
14761 CAGATTTATG GAACTGAAGG GTCCTTGCTG AGTCTAGCAA ATTGCCTTTA CAAAAGAGAA
14821 AGAAAAAAGG GGGAGGTAGA AAAACAAAAC AAATCAACCC AAAGAGGACA AAATCCCAGA
14881 GTTCTAAATT GACTTAGGAA CCTGTCACAC TGGGACAGAA GCTTCAGCAT CCATGAGCTG
14941 TGCCTCCCCT GCTCTCTAGA GCTGGGATCT CGAGGTGTCA GCAGAGACCC CACAGGTAAC
15001 AGGAGCAAAA ACACTCACTC AGACCTTTGT GGTACTTCAA CAGTGGTCTC ACTTCTGGGC
15061 AAGCTTACAA ACCTATACAA AGTTGAAGGT GTACTTTACA TGAGTGCTAA ACTTCAAGAG
15121 GAAGGAAGAA AAAAAGGGAG GTGGAGGGGA CAGAGAGAGA GAGAAAAAAA CAAAACAAAA
15181 CAAAAACAAC CACCTCAGGA GAGGCAAGGG CATTTAAAGG AACCACAAGA ATGCCAACGA
15241 TATTAAAATG TATTTCTTAA TAGTAAATTT TATGGGAAAA GAGAGTCTCC TCTTCCTCCA
15301 AGTAGGCTAG GTAAGTACCT TGCCACTGAG CTCTATCTAT ACCCTTCAAA GTGGACAAAA
15361 TGACAAAGAT AGTTCATCTC CCCCAAAGGC CCTGTTGGGG TGCTGATTGT CACATCTGGT
15421 GAGATTTCTG TTTTTGTTTT TATTTCAAGA CAGGGCCTCT CTACATAGAT AGTCCTGGCT
15481 GCCCTGGAAC TCACTCTGTA GACCAGGCTG GCCTGGAACT CATAGACCCA CTTGCTTCTG
15541 TCTCCCAAGT GCTGGTGCTA AAGGTGTGCA CTGCCACTCT TTTTAAGTAA CTATGAGTTT
15601 CAAAACAAAT TAAAGAGCAC TGTTAAAGTG GCTTGTTGTG TAAGCCTAGC TTCAAGTCAA
15661 AGGCCCGAGG CTCCCCTACC AACCAGCTGC TATCACCTAG ACACTGTCTG TAGATCTTGC
15721 ACTGACTCAA AACTGTGGCC TAAGGTCAAA ATAATGGTCT TCCTGGATTC TGATGTGAGT
15781 GAGATTGTGT AGGAGGGCTG GCCGCTGGCC TGGCTTGAGT CACTCTCAGC TGGTTTCATC
15841 CCATTCCTGC AACTCTGTGT AAGAGGTGGA TGATCCTTGC TTAACTGATG AAGAAACCAA
15901 AGCTGTAGAA AGGATCATTT GCTTAACTCT TCACAGATGG CAAGAGGCAG AGTCAGGATT
15961 GGCAGAGTCA CTTCTGCCAA CTTCACCCTC CTGCTAACTC CACCCTCCTG CTAACTCCAC
16021 CCTCTTGCTT ATACTTGACA GTGGAGGAAA AGCCACTGAG GGAATTAAAA GTTGTTACTG
16081 GTAATGGTCA GGAAAAAAGC TGAACAAAGG AGATTAGATT CAGGGATCTT TTTCTGAAAA
16141 GAAAGAAAGA AAGGGGGACT ATAGTCTAGA AATGCTGAGA TAAAAGGGTG GATTATCATA
16201 TCTACTCTCA AACTAAAGAA GCAACTACTA GTCTCAAATA CTTTATATTG GTATGGATTT
16261 TTGTGTATTG GTACAAATTT AAGGTTATTT TTGTTATACT GTATATATGT TTTTCTTTCT
16321 TGTTTAAGGT ATTGTACCTG TATAGCTTAT TTAAAAATGC AATGTAAACA TATAGTCCTT
16381 GAAAACTATT TAAGATAATA AAGAAATACA GGTTAATAGT CATCTATAGC AATCAAACTT
16441 ATAGTCATGT TAGGTATGTT TTCAAGGGCA TACAGAAATA AATTTGAGAT AGATAGGTCA
16501 TCTTCAAACA CTCCAGAGAT CTACAGAAAA TGGCATTTAT AAAATGTTTT AATGACATAA
16561 GATTTTTCAT GATAGTGAGA AATGTCTACT CTTGGCAGCA CCAATTTACT TCAAAAATGG
16621 ACAATGGGCA TTGAAGAAAC TCCATGTGGA TTTTGCTTTC TTTGTGGCAA AAATCTAGCT
16681 ATCTGGGCAA GAAACTTCCC TTACCTTGAC TGCTGTCCTA ACTGGACAAG CAGGACATAA
16741 AAGAAATTGA CTGCTGAACT TTGCCAAGAT AGTATACATT AGTCTTTCAA AAATCCCTGC
16801 TTTACAAAAA AGTCTATCAG ATATTCTAAG CTTCTAGGCC AAAGATGGAT GCTTCAATGT
16861 TAACAGAGGA ATCTTCTGTG ACTGATGTTT CTGTCATTTC TATAGTTTTG AAAATTGCTT
16921 GCTCTGTTCT TCCCTGTTTG CTCAGGTAGT ATTATTTCCT TCTTGAGTGT CTAATGGAGT
16981 TAAAGACTAG ATAGTTATAG CTACAGTTTT CCTTGTAACC AAATTCAGAA AAGAAACTCC
17041 CAAAAGAGGT GTAAAAGTAT GAGGCTGAGA AATATAAAAA CTTAAATTTA TCTAAGAAAA
17101 TGTTTTGTTA TCTAAAAAAA AATAATTTTG GGTTAGTAAT ACAAGTTAGG ATAGAAAATG
17161 AATTAGGTAC AAAACTTTGG ACTCATCAAG AAAAAATAGA TAATGGAGTA TTTTCTCTGA
17221 ATTTGCCAAA TACAAATAGA CTGGGTATTG TAAATGTAAT TCTTACTTGA TAATTGTTCT
17281 TATTGTTTAT AGTTTATTAT GTTAGAGTCA AAACCTTTCT TTTTTATTTA GACAAAAAGG
17341 GGGAATGTAG AATATTTCTT TACACTGTGT GAAGATGTAT CACTGTGATT GGTTTAATAA
17401 AGAGCTGAAT AGCCAATAGT TAGGCAGGAA GAGGTTAGGT GAGACTTCTG GGAACAGAAG
17461 TCTCAGGGAA GGAAACAGGC TAGGTCACCA GCTAAATGAA GAGGAAATAG GACACTCAGG
17521 AGGAGAGGTA ACAGCCACAA GCCAAGTGGT GGAATATAGA TGAATGGAAA TGGGTTAATT
17581 TAAGTCATAG GAGCTAGTTA GAAACAAGCC TGAGCTAAAG CTGAGCTGTC ATAACTAAAA
17641 GTGGAGCTTT CATAATTAGT AAGTCTCTGT GTCATGATTT GGGGGCTGAC GGCCCAAAAA
17701 AGCCTGCTAC CCAAGTTCTT TTCAATTTTC AAGTTCTAGG ATTCTGGCCT TTTATTGGAA
17761 AACACTGTCA AGTTTCTATA GAGGTCTGAC TCCACAGTGT TGCCTGTGCA ATGAAATTTA
17821 TTTAATTTAT TCCGAGGCCT TGTGCACTCT GGATAATCAC TGTACCACTT AATCTATATT
17881 CCCATCCTTC ATTATAATTT AAAATGGTCT TATTAATCTG GTCACTTGGC TTTTTTTTTT
17941 TTTTTTTTCT GAGACAGGAT TTCTCTGTGT AGCCTTGGCC ATCCTAGAAC TTGCTCTGTA
18001 GACCAGCCTG GCCTGGAACT CACAGAGATC CACCTGCCTC CCCTCCAGAG TTCTGGGATT
18061 AAAGGCGTGT GCCACCACCT CCCAGTGAGT TTATGTCTTT GCAAATTATA CATGGTTTCA
18121 GTTTTTTTTT CTGTTTGTAA GTCACTTTAT TTCAAATGTA AAGTTTAAAA CAAGAAGCAA
18181 ATTACTATGA ATTTTTGTTA ACAGTCATTT TCCTTAACTA ATAAGTTTTA AATTTTCATT
18241 AATATGTTTT GATCATATTT TTTCCATGCC CCAACACCTC CAAAATCTCC CCACTCATTC
18301 AGTTCTTTCT CTATCTCAAA AAATGAAAAA TCCAAGCAAA CAACCATTAG ACAAAAAATA
18361 ACAAAACAAA ACAAAGCAAA GCAAAATAAA AGCACACGGG CTGGAGAGAT GGCTCAGAGG
18421 TTAAGAGCAC CGACTGCTCT TCCAGAGGTC CTGAGTTCAA TTCCCAGCAA CCACATGGTG
18481 GCTCACAACC ATCTGTAATG AGATCTGGTG CCCTCTTCTG GTGTACAGAT ATACATGGAA
18541 GCAGAATGTT GTATACATAA TAAATAAATA AAATCTAAAA AAAAAAAAGA AAAAAGCACA
18601 CAAAAAACCC AGAGAGTGTG TATTGAGTTG GTTAACCCCT ACTCCTCTGG AGTGTGATTG
18661 ATACAGCCAG TGCCGCTATT GGAGAACACT GATTGTCCCT GTCCTTACAG GTATCAATTG
18721 TGTGTAGCTC CTTGGTTAGG AATGGGGCTT TGTGTGCACT TCCCCTTTCA GCTTTGTAAA
18781 GGGTGTCCGA TTGAAGTTCG TATCTTCTGG GAGAGCATAA AATCAAAAAA AGATAAATGG
18841 ACTCCAGTGA AAAAGGAGCA AGCGGCACCT ATCTTTAAGG TAGAGAGGCA GAGGAGTGTG
18901 GTGTGGCCTG TCACAAACAC CCAATTCCCA ATCAGCTGGC GTCTACCAGG CTGCTTTCAC
18961 TTAGATGAAC CCTGACCTCC ATGTCTCCTT AACATTGCCA TTGTTTAACT GTTAGTGAGT
19021 CTGCCCTCTG TTCACTGAAA GACTTTCAGA AGGTGGTGTC GCCTGCCTTT AATCCTAGCA
19081 CTCGGGAGTC AGAAGCAGGT AGATAGAGCT CTGTGAGTTT GAGGCCAGGC TGGTCTGCAG
19141 AGTTCCAGGA CAGGCTACAG AGTGAAACCC AGTCTCACAA ACACCGCCTC CACCACAAAA
19201 AAAAAAGGAA ACAAGATAGA GTGAACAAAC CCAGCTACCT AGACATCTAT CTGGTAAACT
19261 GACTCATCCC AATCCTCCCT GCCCTCCCAA AGAGCTTGGC TGGCTCACTT CCCCAAATGC
19321 TCTTCCCCTT TAACATTTAA CTAGTTCTTG TCTCTTGTAT GGTTTCCTTT TAACTGTATC
19381 CACCACCCCT ACCTTGACTT TTGTCCTGGT TGGTTTTTAA TTGTAAACTT GACACACAAA
19441 GTCACCTGGG AAAAGGGAAC CTTAATTGAA GAATTGTCTT AGATTGGCCT GTGGGTGTAT
19501 TTATAGGGCA TTGTCTTGAT TGCCAATTGA TTCGGGGTGG GGAGTGGGAG GGTAGGGTGG
19561 GGGTGGGAGC AGCCCACTAT GGGACTCACT TTCCCTAGGC AGATGGCTAT ATTAGAAAGG
19621 TAGCTGAGCC TAAGCCAGCG GGTGAGCCGA GCCAGCAAGT AGCATTCTTC TATGGTTTCT
19681 TTCTTTCTTT TTCTTTTTCT TTTTCTTTTT CTTTTTCTTT TTCTTTTTCT CTTTCTTTTC
19741 TTTTCTTTTT TTTTTTTTCT TCCCGAGACA GGGTTTCTTT GTGTAGCTTT GGAGCCTATC
19801 CTGGCACTCG CTCTGGAGAC CAGGCTGGCC TCAAACTCAC AGAGATCCTC CTGCCTCTGC
19861 CTCCCGAGTG CTGGGATTAA AGGCATGCGT CACCAACGCC CAGCTCTTCT GTGGTTTCTG
19921 CTTCAGATTT CTGCTTTGAG TTCCTGTCTG ACTTCCCTCA ATAATTGTTT GTAACCTAGG
19981 AGTGTAAGAC AAATGAACCC TTTCATCCCC AAGTAGCTAT GGATTTAGAG TGGTTTATCA
20041 CAGCCACAGA GTGAAACCAG AACAACTTTC TAGTAGCCTC TTGTTCTACT CCAGCTGCTC
20101 CTCTGACTAT TCCTAAAAGG TAGTTGGGCT CAGGGAACCA CATCCCGAGA GATTCAGCCC
20161 ATATGAAAAT AGCTCCATTG TGTTGAAGAA ATGTGACCCT CCAGGATTTC AGGCATCAGG
20221 ATTCCATGTT GAAAATGAAA ACAATTATTT TCCTCTCTCT CAAGATTCCT TTAGTCACCT
20281 TCCCTTACCC CAGTTCCTGG CTTTCCTTCT AAACAAATGT TCAGGGAGGT TCAAACAAAC
20341 AGCTGTGAAG AGCAGCATCC CATACCCCCA CCTTCCGACC CAACACTTGC CAGTGCTATA
20401 AGTAGACTGG GATCATCCCT GGACACTGTG TTAAATTACC CATGACCAAC CTTCTAGCAA
20461 GCTCTCCTTT TCAGGATTTT GTTGTTTGTT TGGGTTTGTT TGTTTGTGAC TTGATCTCAT
20521 GTAAGCTGAC CTGGAATTTG CTTAATAGCC AAGGATAGAC TTACAACCTG TGATGCTCCA
20581 GCCTCTGACT CCTGAGTACC AGGGATTACA CATGTGTGGC ATCACAATGA AAGATTTTAG
20641 TTTGCTGAGA GAAAAAGTTT TTAAAGATTT TAGTTCACAG AGAGAATAAG TTTCCCACAG
20701 GCCTTGGTCC AGGACAAGGA AGTTGGTCCC AACCCGAGGG CAGACAAACA ATCCTTTTTG
20761 GGTCACACCT GGCTGGCCAA CAGACAATAA AGGACTTCTC AGGGTACATT CTATGGTTGA
20821 CCACTCTAAC ATGAGATCAT ACTTTGTAAT CAATCACTTT GTGCCCCTTG CCTGTATGCT
20881 GATCTGCGGT TTTTTACAGG CTCCTATATA AGGAGTCTGT AACCCTTGCT GGGGTGTGCA
20941 GCTTCCCCGA TATTGCTGAC ACCCGAATGA GCATTCGTTC AATAAACCCT CTTGCTTTTG
21001 CAGCTCTTGG TCTGGTTTCT GAGTCTTGGG GCCTCCTTGG GATCCTGAGA CCCTTAAGGG
21061 TCTGGGGGTC TTTCAACACT TAACTTTCCT GTTTTTAAGT AGGAAGATCT GAAATCCCAG
21121 ATTCCTGACT CCATTGCACA TTTTCTGTAT TAGAGGCTGT AGCTCTGTAT AGTGGGTTGT
21181 GTGGCTTACA CATGCTCTGA GCTGGAGATT CTAGGGACAC TTAGGGTAAA GTGGAGTGTC
21241 AGCCCCTTTC CCTGCTAGAC TGAGGCCTTT CTGTTCTTTC CTAACTGGGA GGCTGTATAG
21301 CACCCAATGT GTTCATTAAA CTCCATATGT TAGCACTGCA TGGAATCTGA CACACACACA
21361 CACACACACA CACCCTCTAC CACCACCATC ATCAGCACCA CCCCCATCAG CACCACCCTC
21421 ATCCCCCCAC CCCCCACCCT GCCCCNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNC
21481 AACTGGAGGG TAGCATTAGC ACCCAGATGC CATTAATGTG CCAAATATTT GCTTGCTTGC
21541 TTGCTTGTTT GTTCCAGCAT CCTTAGTGAA TGCTCCTGCC CTCCTGGTTA AAGATGGCTT
21601 TGGCATCTCT TGGCATCTTT CTTGTATTCT AGGCCTGAAA TAGGGATGAA TGGTGAAGGG
21661 CAAGGAGCTC AAGTGTCACT TACCACCTGC ACTTGTCCCT TTAAGGGGTT TCCCTAGAAG
21721 CAGTCTACAT TTCATTAGCC AGAGCTTTGT CACCTGGCTA CTTGTGAAGG AGGTGGTGAA
21781 GAAGCCTTAC CTTTGACTCT GCCACTTGGA GCCAAGTCAG GATTCTCTCC CTGGAAAGGA
21841 AATGGAAGAT TAATACCTTG TTGGTTGTTA GACCTAGCCC ATTATGCGCC ATGAGGAAAG
21901 AGAGACAACA GTGGGTCACT GATTGATCAG GGTTACAGGA CAAGGAGCCT TGTTTCTCCT
21961 AACAGCTCTG AGCGGAGACA GAAGTGGAGT ATATAGGCAT AAAATTCACA AACATTTGCT
22021 GCCACGTTAC AGGTACATTT TTTCACCAGT CAGAAATCAA AGATTAGGGA CTTTGCTTGT
22081 GTGTTCCATC ACTGTCAACT GACATACACG GCAAGCCTTT TAGTCCAACC AATCAGAATC
22141 ATTTGTTCCT TCTGTTGTTA GGAGCAGCCA TAATGATTCT AAAGAACTAA CAATGCATAA
22201 TGACTATTTT TGTAGTTTAG GGATGAGGTA TGTCAGCCAT TGGACAGTTC TCAGCTCCCC
22261 TAGGGCTTGG GAACTTGAAC TTTATTTCAT CCTGCATGTA ATGGAGTCTG AAGTCAAAAT
22321 GGCAGTACTT AGGTCAAGGT GCTCGTGCCT GCTGCCTTCA AGGTGGTTTC CCATTCCCAC
22381 CATACCAGAG ACTTCCTACT GCATCTCCAG TCAAGGACAC AAACACTTTT AAGTCCTGAC
22441 TGTTGATTCA ATCTATATAG TTACCAGCAT AGAGGCTAAG AGTCACACTG GCTTGCAGGG
22501 GACTTCTCTA GCATATGTGA AGCCCCGTTT GAATCCTAAA CACAAGAGTC TAAGCTTTGG
22561 AGTCAGAGAC AAGCATGTTC AAATCTGTAC GTCACCACCC TATAGACATA GACAAGTCCC
22621 TTGGGCTCAG TTTTTTCACT ACAGAGAGTA ATTGTTATTT CAGATTCCTA GGGTTGTGGT
22681 AATTAAATAG TTGAAAGATA TAGCCCATGG AACATAAAAA AAACTCAAAA CCAGGCACAG
22741 TGGCACATGT CTTTAATTTC AGCACTCAAG AGACAGAGGC AAGTGGATCT CTGTGAGTTT
22801 GAGGCCAGGC TGGTCTATAT AGAGAGTTCC AGGTCTACAC AGAGAAACAG GCTCAAAACC
22861 AAAGCAAAAG CAAAACCTCA ACTAATGTTC ATAAAATTAT GAAATTGCTG GTACCAGTGA
22921 CATGACTCAT TGGTAAAGAC ACTTGCTAGC AAGTTTAATG ATCTGAGTTT TATCTCCGGG
22981 ATCTACAATG TAGAAGAAGA AAAACAACTC TCAAGAGTTG TCCTCTGATT TCCACTTATG
23041 CAAAATAGCA TGGGAACACA CTTAAGCAGG TAGGTAGGTA GGTAGATAGA TAGATAGATA
23101 GATAGATAGA TAGATAGATA ATAGACATAA TTAAGAACGT TCAGTTGCAG CACAGTTCAT
23161 ACTGAACTGC ATTTGGACAC CTCTGTGAAA AGTCAGGAGC TCTCCTGTCC TCCTGGTGAC
23221 ATTTAAACAT TGAAGGCAAC TATTTTAACT GTCAGTTATA TACAAATCCA CTGGCCTTGT
23281 AAAATTTTAA AACATAACAG AGGAGGCTAA AGTCCTGTTT AACAACCCTC TCCTTTTACC
23341 ATCCCAGGAA GCCAAAATTG TTCACAATTT GTTCTCTTCC CTCAGGCCTT CCATATTTCA
23401 AATACCACAT AAAACACCTA TGGAAAAACA TGAGGTATTA AAAATGTCAC TTGGAAATCC
23461 TTCTTCAAAC AAGCTTGTTC TTTCTTTTTT CTTTTATGTA CAGTGAATGG AATCCAGGAC
23521 CTTTGCAGAT GCTAGGCGAG TCCTTTACCT CATTCCTCTT TCGATTTAAA ACTTTTTCTT
23581 GTTTTGTGGA GACAGGGTTT CTCTGTGTAG CCATAGATGT CCTAGAACTA GCTCTGTAGA
23641 CTAGGCTGGT CTCAAATTCA GAAGCCAGTC TGCCTCTGCC TCGGGAGCGC TAGGATTAAA
23701 GGTGTGGGCA GAGTGCTAGG ATGAAAGGTA TGCACACCAC CACTCCTGGT TGATTTTAAA
23761 AAGATGCTTT TTAAAAAAAA TGATGTGTAG GTAGTGGGGG GAGAGACGGT TTCATGCCTA
23821 AGAGCACTGA CAGCTCTTCT AGAGGACTCA GGTTCAATTC CCAGCACCCA CATGGCAGCT
23881 CATAACCATC TGTAACCCCG GTCCCAGGGA ATCCAACACC CTCTTCTGGT CTCTGTGAAT
23941 GACAGATATG CATGGGATAT ACAAACATAT ACGCAGACAA AACACTGTAT ACATTAAATA
24001 AGTACAAATT TAAAATATGT GTAGGCATGT ATGTCTGCAT GTGGGTATGT GTACACTGAA
24061 TGCAAGTTCA CTTGGAGGCC AGAGATATAT AGATCCCCTG GAGTTGCAGT TACAGATACT
24121 TGCGAGCTGC TGTGAGTGTG CTGGGAACCA AATCCTCTGG AACAGCAGCA AGTGCTCTCA
24181 CCTGCTGAGC CATTTCTTCA CCCGCTTCTT TCTACTTTTT ATTTTGAGAC AAGGTCTTAC
24241 TAAGTTATAT ATTCACTTGG GGCTTGAATT CATTTTGTCA GCAGGCAGAC CATAAACTTG
24301 CCTTCCTCTT GCCTCGGGCT CCTGAGTAGC TGAGACTTCA CCATGAGGTC TGGCTTTGAT
24361 TACATTTTTC TTTGTTTTCT TTTTGGGGGT GGGGCTGATC ATGAACTCTA AATAGCCAAG
24421 GATTGATAGT GAAGTCCAGA TTCCCCCACC TATCACCGGG TGGAATTACA GGTGTGCACT
24481 ACCACACCCA ATTTGGTTTG ATTTTTTTTT TTTTTTTCAG GACAAGCTCT CCTTTTATAG
24541 CTCTGACTGG GTTGGAATTT ACTATGTAGA CTAGGCTAGT GTCAAAATCA CAGAGATCTT
24601 CCTGTCCCTG CTTCCTGAGT ACTGGGATTA AAGGCATGTA CCACCACACC TTCGGGTGTG
24661 GTGATGCACA GCTTTAATCC CAGCACTCAG GCAGGCGAAT CTCTCTGAGT TTGAGGCTAG
24721 CCTAGTCTTC AGAGTGAGTT CCAGAACAGC CAAGGCTACA CAGAGACACT TTGTTTCGAA
24781 AAACAAACAA AAACAAAAGA GGCTAGCCTG AAACTCCTGA TTCTACCAGC ACCTCCCAAG
24841 GGCTGGGATG ACAGGTTGTG GCCCCATGCT CTCTGCCGGG GCCTCTCTTT TCTTTCTTCT
24901 GTTTGAGGTA GAGGCTTACT AGGTTGGCTG GGTGAGTTGT GAACTCACTC TGCAGCCCAC
24961 ACAGGAACTG ATCTTGTGAT CCTCCTGCCT CAGTCTCCCT AGCAGCTAGG ATTGCAGGCC
25021 TGCACCATCA GGCCCATCGT ACACTGTTTT CTGAGTTTGA AAATTGCCTC TGTTGTTGAC
25081 TATAAGGCAT GCTCTCCTCC TAACATTGTC CTTGGTGCCT CTGCCACCCT TTGGGACTAG
25141 AGAGAACAGA TCTTATTCCT ATTTCACATG CTGTGCCAAC CCAGTAACAA ACTCAGATTC
25201 CTGCTTCCGC CCCCACCACC CCCATCTAAT TGTTCAGTGT TTCTGTGAAG ATAAACACGA
25261 TCATCTTTGT GAAAGCCACT TAAGTTCCTT TCAAGGTTGG GATATAAGTT AGAGTGATAG
25321 CTTGTTCCCA GGGTGGGGAG AGCATGTGAA TTCCCCTCTC GCTCAAGTAG GCTATACTAA
25381 TTTTCATTTA GATATTTCTG AGGCAAAGTC TCATGCTGGC CATCCACCTG CCTTAGCTTC
25441 TCAAGTGCTT GGATTACAGG CATGAGCTAC AATATCTGGC TTAGTTTCAA GGTTGTGAAA
25501 ATTATACTGT GTTCTGATGA CCTGAGTTCA ATTCCCTGGA CCTGGGTGAT GGACGGAGAG
25561 GACAGACCCC TGCAGATTGT CCTTTGACCT CCCTGTCACT ATGTGAACAC TCGTGTACAC
25621 ACACACACAC ACACACACAC ACACACACTA AATGAATGTA ATAAAATATA AAAAGGTGTT
25681 CACTAGTTAA TAAGACATGA GAGAAAAAGC TTACCATCCC TAATCAATGG GGAAGCATTG
25741 AATATAAGTG ACTGTGGTCA TGGAAAGCAG TATAGAGGTT CCTCAATAAA CTGGAATATA
25801 GCAGCATATA CTTGTAAGCC TCCCACAACA GGAGAAAGGT AAAGAGGGGC GGCCACTCTG
25861 GAATATTATT AATATCCTGT TTCATAAACA AGTAAATAGA ACAAACCCCT CAACAACAAG
25921 AACCGGTGTG CTGGCACACA CCTGCAATCC CAGCATTTGG GACTTGGAGG CAGCACAATT
25981 GAAGTTCGTT CTTGGTCATC CTCAGCTATG TATGAAATCT GAAGCCTGCC TGGCCTACAG
26041 GAGACCCTGT CTCAAAAAAA TAAACTAAAT AGATTAAAAT GAAAATTAGA AGCAGGTAGT
26101 GTGGAAGTTG AATAAGAATA GCCGCCATGG GCTCATGTAT TTGAATGTTT AGTGGCACAA
26161 CTTGAGTGAG TTAGGAGGTG TGGCCTGTTG GAGTTGTGTG TCACTGGGAG TGAGCTTTGG
26221 GATTTTAGAA GCCCAAGCCA GGCCCAGGGA CTTGCTCTCT TCCTGCGATC TGAGGAACTG
26281 GATGTAGAAC GCTTAGCTAC TTCTTCAGCA CCATGTCTGC CTGCATGCTG CCATGTTCCC
26341 TGTCAAAATG ATAATGGACT GACCCTCTGA AACTTGGTCT CTTTTGGCTG AGGAGTTAGC
26401 AAGGTAAGAG GTGGCTGTGG CTTGCTCTTG TTTCTCTCTC TCTGATCTTT CATCATTTTC
26461 TCCCGTATCT GGCTGTGGGT TTTTATTATT AAGAGTAATT AGAACTCATG TTACAGTGGT
26521 ACATGCATGC CACAGACCCA GTGTGGATGC CAGAGGACAA CATGTGTAAA TTTTTTCTTT
26581 CCTTGTATGT GCGTCCAGGC TAGTTTCAGA CTTGTGGGCT TCTGCTTCAG CCTCCCAAAG
26641 GTGGGGACCA CAGGCTTATA TACCTACACT CACCTCTTTA TTCCCAGTGG ATGTGTGTGT
26701 GTGTGTGTGT GTGTGTGTGT GTGTGTGTGT GTGTTTGTGT GTTTTACACA GACCTGTACC
26761 ACATTCATTT GGTTACTTTT TTTTCCTGCA TTTTGTTTTT AGGTAGGGTC TCACTATGTA
26821 ACCCTGACTG TCCTGGAACA TGCTATTTAG ATTAGACTGA CCTGCTGGTC CCTACCTTCC
26881 GAGTGCTGGG ATTAAAGGTG TGTACTACCA TACCTGGTGA TTAGTTTGTC TTTTGAGACT
26941 GGGTCTCTTG TAGCCCAGGT TGGTCTTGAA CTCCTGGTTT TCCAGACTCT ACCTTCCAAA
27001 TATTGATATT GCAGGTGGTC ACTACCATGT GTGGAATTTA TTTTTGAGCA GTGTTCTGTG
27061 GGTGGATGAT AAGGTCATGT CTATGGTAAA ATTGTTTCTA ATAATGATGA ATAGCTTCAT
27121 GTGTGTATGC ATCTATCAGG TTTGTTCAAC CTGAAGTGTA GGCCTAATAT TTGGATTTAT
27181 TTAGCCAGTG ATAGCTATGA ATTGAGCCCA GAAAAAATCA TAAACTTGAC TAAAACATCT
27241 TAAGAATTTT GTAACTTCTT TTGTAACTCA ACTGTATTGT TTCTGAGCAT GAATGTTGTA
27301 AATGACAATG TCAGCTGCCA TGTCAAAAGG TTGAACATTA CTTGGCAGTG GTGGCACACA
27361 CCTTTAACTC CAACACTCAG GAGGCAGAGG CAGGCAGATC TCTGAGTTAG AGGCCAGCCT
27421 GGTCCACATA GGGAGTTCCA CACCAGCTAA GGTGACAGAG TGAGACCTTG TCTAATTTTT
27481 TTTTAAGGTT GGACATGTAT AATTCCAGAG AATAATTTTT CACTAATCGG AAAAGAGGCA
27541 GTTTCAACTT GGAGTTCACA AGATTTAATC TTTCTTTGAA GATTTATTTA TTTTTAGTTA
27601 TGTGTGTGTA TATATGTATG TATGTATGTA TGTATTGGTG TGTTAAACCC CTGGGGCTGG
27661 AATTACAGGT GGTTGTGAAC CTGATGTTGT AATAAGCTCC CAGACCGTAG CACAAATGAC
27721 TCTATGAAGA AAGTACCATT CAGGCTGTAA AATCCACATA GACAGCACCA CCTGGAAAAA
27781 CTAAAACAAA AATCCAATCC ATCAAACTCC ACAGATCTGG GAAAGTATCT AAATGCACTA
27841 ACCTTGATTT TTGGCTTCTG TAGTTCTGCT TCTGGCTAAC TATTCTTGTT AACTGAAGTA
27901 TGTGAACCCA CAACATGGTT TTTGTGCTTA AAAGTTCTCT GTTCTACAGA ATGAATTCCA
27961 GGACAGCCAG AGCTGCATGG AGAAAATCTG CCTCAAAACA AAACAAACAA ATAAAAACCT
28021 TGAGAAAGGC TCAGGGCTAT ACTGGTATCC CATACACTCA GTGTAGTCGC CAACTGTCAA
28081 AGACTTTTTG TTGACTTAAA CCCATTTCTA AGCAGTATTC TCTTATGGAT ACCCCTTACA
28141 AGTGGGTGCT GGGACTTGAA CTCAGGTCCT CTGGAAAAGC AGAGGATTTC TCACCTGCTG
28201 AGCACCTCTC CAGGCCCATA AGATCTATCT TAAGACAAGA CCTGAGCAGC CTTATGGAGA
28261 TGGCAGTCTG GGGAACCACT GGTGCGCCTT TTCTTCTGCT GGTCACAAAC TGCTGTGGGA
28321 ATTTCCATCT GAAGTTCCTG CCTCTTCTCA CATTCCATGA TATGAGAAAG CTATCAATGT
28381 TCTAAATCTG TTTGCTTTCT GCTTTGCAAG ACCTTTCTCT TTCCTAGGTC ACCCTCCAAG
28441 AGTTCTTGAC CTCAGCCCCG ACTGGTGTCT TGGGATGGGT GACTGGGTTC TGGGGGCTTC
28501 CCTGTGCCTT GGAATATGGT AAAAGAGCAT CTCAGGTATT CACTCAGTAG ATGCTAGTAG
28561 CACTCCCTCC CTCCATTTCT GTCTACAGAT GTTGCTAGCT GGCCCCTATG AGGTAGTCTT
28621 TGCCCCTTTG TTATTGCTGC AGACTCAGAA AAAAGAGGAA ATATAGAACT CCTCGTGGTC
28681 TTCTACTCAA TATCCAAGCA AGGGGGAACA ACTGAGCATC CATACACTGC TGTTTTGGCT
28741 TCTCAATTGC TTGCTTGTAC ATCACCAAGA AGCTTTCATT GGTCAGTGTA AACAAGATCT
28801 GGGAGTTGAT GGTAGAGCAG TTGGATGAGT GACTCTGTCT TTCACCTTTG TTGAGTCATT
28861 TGGTGTGTGC ACATTGTGGG TCCCTGCCTC GCTTCCCATT AAATGTCAAG GTGAACTTTA
28921 TGAGGTTGAA ACTTTTATAT GTAGTGCAAC TGTACTCCTT CCTCTCTATC TCTTCCTTCA
28981 TTTTTCTTCC TTCACCTTCT CTTCCTTTAA AAAAAGAAAA ACTTTAAAAA ATGTGAATCT
29041 GATGTATCCC AGGATGGCCT CAAACTGTTT GCTTTCTCAG AAGATGACCT TGAACTTTCA
29101 ATCCTCCTGC CTCCACCTCC CAAATGCTGG GCTTACAGGA ATTCATCACC ATGCCTGGTT
29161 TTCCTCTCTC CTGGTGAGTG AATCCAGGGC TTCATGCTTG CCAGGCAAGT GTTCTGCTGA
29221 CTGAGTTACA TGCTTAGCCT GTATCCACAT CTTGACTGAG TAATTTCTGC ACCAAAACTT
29281 TAGGTTTCAT CTCAGTGACT CTGCCAATGT GTTTCCATTT TAGAGTGACG ACTGGCCTTA
29341 GAGGAGAGTG TAAGAGAAAT AGAGTCTCTT TCCTTGGTCT GCTTTTTAAA TTTTAATTTC
29401 TTTTTAGACA TCTTATATTT ATTCATGCAT GTGTGTGTAT AACTAGCAGA ACTCAGCTGT
29461 CTCTTTCTAC CACTCAGGTC ACCAGGCTTG GTGGCAGGGA CTCTTACCTG CCTTCGAGCA
29521 GGCTCTGCCC TCCTTTTGGA GAAACTGGTT TGCAGAAGGA AGAGACAGCA CAGCTCAGAA
29581 GACAGCCGTG CTTTCAGATG CCTGAGAATC CTGCCAAGGA CACTGCTGCA TTCTCCTATT
29641 CTTTTGTAAG GGTCCCATCT CTGCTGAGCT AAACTGGGCT TTCTCAGCCC TTCTCCTCTG
29701 ACAGTATTTT AAAACCCTAC CTAAAGGGGG ATGGAGAGAT GGCTCAGCAA TTAGGAGCAT
29761 ATCCTACTCT TCCGGAGACC CCTACTTCTG TTCCCAGCAC CAATGCTGGT CAATTTACAA
29821 CTGTAACTCT GCTCCAGGTC ATCGGATGCT GCTATCCTCC TCAGGCAACT TCACTCATGT
29881 GCACATACAC ATACTTAAAA ACAAAATAAG TCTTTAAAAA TCACCTAAGA AATATAAAGG
29941 CACATATCAT AATTCAGCCT GCTGTGACGT ATAGCTATAG TCCCAGAATT CTGAAGGCAG
30001 AGGCAAGAGG ATCACCTCAA GCTTGGGGCC AGCGTGGTCT ACAGTGAGAC CCTGGAGACT
30061 TTAATCTCAA AATATGTAAC AAAACAAATA TGTAAATAGA CATATATCAC AATTTATATT
30121 TAAGTAAAAT GGGGGGCATT GGAGAGATAG CTTTGTGGTT AAGAGCATGT ACTGTTCTTG
30181 TCAAGGACCC AAGTTTGATT CCCAGTGTCT ACACTGGTTG GTCTCCAACC CAATTCCAAG
30241 AGATCTGCTG CCTTCTTCTC CTCTCTACTG GAACTGCATT CATGTGCAAA TGTCCATATG
30301 CACACACATA CCCACATGCA TACACACAAA CACATACATA CTCATTTTGC CTGACATCGT
30361 GGTAAAGTGG GAAGACTTGT TGCCCTATTA CTTGGTCTTC ATTTGCCTAT GAGCACCATG
30421 TTGGCATGAA CTCATTCATT AATATCTTTC CTGTACAACT CCCCAATAAC CAAGATGACA
30481 CTTGGCACAC ATTAATTGCT AAGTATAATG AAAATTTAGT TTAAATTAGC TAAATAATTT
30541 AAAGTTCCCC CTCAAGCCTC ATGCCTGATT TAAAGTAGTA CTTATTAATG CTGGGCCTGG
30601 TGGCATACAT TTCTAATTCT AACACTTAGG AGGCTGAGGC AGGAGGATGG CCAATTCAAG
30661 GCCAGCTTAG CCAGCTTAGT AAGACCTTGT CTCCAAGCAA ATTACAGCAA AGTCTGAGAT
30721 ATAGTTCAGT AATTAGGGTG TTTGTCTACC ATGTGTGAAG ACCTGAGTTC AGTTTCTAAC
30781 AACAAAACAA AACTAAACAA ACCAGAACCT AGAGGTTATC ATTTATTTTT TTATTTTTAT
30841 TTTTTTTTGG AGTTTATGCC TTTGGATTAT CCATTCTATG TCCAGACATC AGTACTGCCA
30901 TGTTACAGTC AATAAAAGTC TTCCTTCATC ACCCTTAATC TTATCACCAC TAAAGTCTCT
30961 ACTTGACAGA CATGCCATAC ATAATTATAG CTGTTACCTT CTATCATAAA GTAGACATTT
31021 TATTTTATTT GTGTATTCAT TTTCATTTAT TTTGTTGTTG TTGTTGTTTT ATGAGACAGA
31081 GTTTCTCTGT GCAGCCCTGG TTATCCTGGA ACTCACTCTG CAGACCAGGC TGGCTTCAAA
31141 CACACAGAGA TCCACCTGCC TCTGCCTCCT GAGTGCTAAG ATTAAAGGAG TGTGCTGCCA
31201 TCTTCCCAGC AACATTCTAA ATTATTTTTT GTTTATGTTT TGAAATGGTC TAATGTAGCT
31261 GAGGTGGGCC TCAAGCTTGT TATATAGCTG GGGAACCTTG AACTTGTGTT CTTCCTACCT
31321 CTAGAACTCT GGAGTGCTGG AATTACAGGT ATGAACCATC ACATTCCAGT TTTAATCAAA
31381 TCCAGACTTC ATGGGTACTA GGAAAGCACT CTACAAATTA AACTTCACCC CTAGTTCATA
31441 TATATATATG TGTGTGTGTG TCCATGTATG TATGCCTACA TGATTTTATG TGTGCCACAT
31501 GTGTGCAGGT GCTCTTGGAG GTCAGAGGGT GTCAAATCCC CTGGCACCTG AGTTATAGGT
31561 GGTTGTGAGC CACCTGATGT GGATTCTGGG AACTGAACTT TGGTCCTCTG CAGGAGAAGT
31621 CACTGTTCCT CTGAGTGAAC GTTTCTACTT TTTAATATAC TTCCCATTCG AATTAGAAAG
31681 TAGAAGCTCT CGGAGGTTGA GACCTTACCT AAAGTCACCC AACTAGTAAG AAAACTAAAA
31741 TATCAACTTG GTTTTCTGAG TTTTAAATAT TTTTTCCCAA TGTGTAATTA CACAGGAGAA
31801 TTAATGGGGA CACTTCAAGG TAAAACAGAA GCTTTAGACA TAGCAAGGCA TGGTGGCACA
31861 CATCCCATTG AGAGGCAGGA GGATCAGGAG GCCAGCTTTG GCTGCATACT TAAGAGGCAT
31921 CCAGGGCTAC ATGAGGCGCT ACCTAAAAAA ATTAAATTAG GCAGGGCGTT GGTGGCGCAC
31981 GCCTTTAATC CCAGCACTCG GGAGGCAGAG GCAGGCGGAT CTCTGTGAGT TCAAGGCTAG
32041 CCTGGTCTTC AGAGCGAGTG CCAGGATAGG CTCCAAAGCT ACACAGAGAA ACCCTGTCTT
32101 GAAAAACCAA AAAAGCACTG GTCATTGTCA TTTTCTTTCC TAACAGGGCA CTGGAACCCT
32161 GATGTTGGTT GGCTCCTAGA TTTCTTCTCC ACAGCAGAGA GTTCTTGCCC TGTTAGAGCC
32221 AGAAGGATGC TCTGGAGAGT CAGTATATAG CAAAGCAGGG TCATCTGGAG TAGTAAAAAC
32281 CCTCTGGCAC AGTCAGACCT CATTTCCTCT TGTCCTGTGC TCGTGGCTCT AGCATTATGC
32341 AAGGAGAGGC GCAAACAGCA AACAATTTGG AAGGGCTAGC ACTTGAGCAA CTCTTTGTAG
32401 CTTCCTCTTC TCTACTCTTT TGCCCCTGGC TTCTACTGGA ACAGGTGACT TTCCATTGCA
32461 TTGCATTCTC CAAACTCAGA TGATTTTGAG AATGTGGCAC TACTAAAAGT CACATGGACA
32521 TACAAGGTAC AACTAGAACT ATCCCGGGAA ACAGTGATAC ACGATCTAGT TTGAGGCCTT
32581 GAGCCATAGC TTGTCAGAAG CTCAGAAATG ATTGAGTCTC TGGGAGCCCT CACCTCAGCA
32641 TCCCTGCTTG CAAAAGGCTT CTTGAAGTAG TAAAAACTGC TGGGACCTTG TCTAGGCTGG
32701 GTAACCTTGC ATAATTACTC AACCTTACTG AGCTCAGTCC CCTCCTCTAT AAAATAAGTG
32761 CAACAGTATT TACCTTAGTG GCCCACCTGA AAACATCACA GCTGCCATAG CTAGCTCTTG
32821 GCTTTTGTTC TATCTCCTCC TCCCCCTACT TTCTCTTCCC TCCCTCCCTC CCTCCCTCAT
32881 TTTTCTTTAT TCCTTTCTTT GTATTTTTTT CTTTTTTCTT CCTCACACCT CTCCTTATTC
32941 CCCACCCTCC TCTCTCTCTC TCCCTTCCCA CTTCTCTTTC TTTCATGGCA GGATATCATG
33001 TATCCTAGCT ATACTTGAAT TCACTATATA GCTGAAGAGG AGCTTCCAGC CCTTTTGCCT
33061 CTGCCTCCCA AGTGCTGAGA TTATAGGTGT CCACCTCCAC GTCTACTTAT GCTTTGCTAA
33121 GGATCAAACC AGGGCTTTGT ATGTGCATGC TAGGCAAGAG CCAACTACAT CGCCAGACCT
33181 ATATAATACC CCTTTCTCAG CGAAACTGGG GTTGCTGATG GCTGGTGTTG GGGGAAGGCA
33241 CTAAATATTT AGCAGAAGTA TAGGAAAACT CTAGAAGTCT AGAGATCCTC AAAGTAAGTT
33301 TGGAGAGCCT TGGCCTTTTC TTAGTTGAAA GTCATGGTGC CTACTCACTT TGACTGCTCA
33361 AGGAATATCC ATTCACCACC TGGAAATAAG AAAGGAGGGA GAACCAGCTA GGGATGTGAC
33421 TTAGTAGTAG AGCACTTGTC TAGCATGAGC GTGGTCCTGG GTTCAAGCTC CAGTACAAAG
33481 GCTGGGTGGG GGGGTGGAGA AAGGCTTCTT TCCCATGGCG TTCTAGAGAT GGCGGGGAGA
33541 AACCACCAAT CCACATCTAT CTACAACAGT TCAAGTAGAA CTAATCTTGG TGGTATGGCT
33601 ATAGTAGTCC TAATCCCATC TCAGGGATGC TTCTCTTTGC AATTGATACA AAACACATTA
33661 CAGAAAACCA CAGTGAATCA AAATGCAGAG TTGTGGTGCC TAGTTCCAAT GGATGCATCT
33721 ACAGTACAAC TCCCATGCCT AAGGCTCAGG GATCATTGTG GAAGACAAAG ATCCTCCCAG
33781 GAGATCAGGG AGTTTGCTGT CTCCTAGGAA TTTCAGAAAA TACATCTGTA AAGGCTCACC
33841 AACGTGAATT CCTAAACATG AGCTGAACAA GGATGACAAT AGACATGCTA ACAAGGATGG
33901 GAAAAAGCCC TTGAAGCCTC AGACCTACAC AAAGAGCCGC AGTTGATTAA GGAATGCTGA
33961 TTGTGGGAGA AACCATCTTC CCAAATTGTT ATCTAATACC ACATAGTCAG CCCTGAAAAC
34021 ACACATGCAA ATAAGATTAT ACAAAACAAG GGGGTTGTAC ATATGTATTT AGGAATATAT
34081 ATATATATAT ATATATATAT ATATATATAT GTAACAATAA TTAATAGAAA AAGAGACCAT
34141 GAATTTGAAA AAGAACAAGG AGGGGTACAT GGAAGGGTTT AGGATGCTTT GACCCTTTAA
34201 TATAGTTTCT TGTGTTGTGG TGACCCCAAT CATAAAATTA TTTTTGTTGC TAGTTCACAA
34261 CTGTAATTTT GCTGCTGTTA TGAATTGTAA AGTAAATACC TATGGTTTTT GATGATCTTA
34321 GGCAATCCCT GTTAAACTGT CATTCAGTCC CCAAAGGGGT CAAGACCCAC AGGTTGAGAA
34381 CTGCTGATTT AGAGAGAGGA AAGGGAAGGG GGGGTGAAAT GCTGTAATTA TAATTCCAAA
34441 AAAAAATTTT TAAAAATTTC TTAAAGGAAC TGAAGAAAAG AGCTGAACAT TCTAAGCTTA
34501 AGGGGGGAAA GGTTCTGGAA TGTTACATTT TTCTGGTTTC CTTAGTCTCA GCAACAGGCT
34561 CCCAGCCTTC TGTTTGGACA GTGGTTTACA GGCATGTGAG CTCAGGGAAC ACTCTTCCAA
34621 GTGAATCAGA CTTCAGGAGA AGACATTCAG TTCAGGGCCC TGGGGAAAGT AAGGACAGAA
34681 CTCCATTCCT GAGAATTACC AGGTTTGCTC AGAAGATAAA ACTGGTGAGC CCAATGGCTG
34741 TGTGCACAAC CCTGACCTCA GTGTCTAGGA TAGCTGGACT CTAGCTGCTA GAAGATAGTC
34801 AGAGGGCCAT CCTTTCCCTG AGGCTAATCT GTGAATCAAG TAAACTACAG TCAGGAAGGG
34861 AGCTGGAGAT GGGGGCCCAG CAAACAGGTC CCCCTTAAAG CCCAGCACAT AGGTGGGGAA
34921 CCCAACCTCC CATTTTGTCT TCACCCCACC ACCAGGCCTT TACCAAGGCC CGAGGTTGCC
34981 ACTATTTTCA GCTTGCCAGG CTCTTTGCAG TTTTAGGGGG ATGAGGAGGA GATGCTCTGA
35041 GGTGCTGGGA GGCACATGGC GGGTGCTATT TATGGCTTGG GCTGAACTCC GATGTCCTAG
35101 AAAGAGTGTT TCTGACACTT TCTGCCTTCT GGGAATCAGG AGACTCATGA CAAACACTGC
35161 CTGGCAGTGT TTCTTTCTTG TTCACAGCAA GAAGTGTGCA GTCCATGGCA CGAAAGAGGC
35221 CTGAGCAGGG CAAGATGGAC ACGATGACAT CACTGAAGGA GCTTCCCAGG GGCTGTCTTG
35281 ACTGCTTCAT TAACTCATTC ATGCAGTTTA TTCAGCAGCT ATGCCTGTCA GACCCCATTC
35341 TGTCTGCACA AGACACATGG CAACAAAGGA GACTTACTAT TCCCATCTTC ATGGGTTTTA
35401 TGTTCTGGCA AGAGGAAGAT AGTAATAATT TTTAAAAAGT AACCAGTCTT GAGAGCATGA
35461 TAAATATGGT TGATAACAAT ATGCTATATT TTAAAAGTTG TGAGATAGTA TACTTTAAGT
35521 GTTCTCAAAA CAAAATGATG AATATGGGTG ATATAACATG TTAATTGGTT TAATTTAGCC
35581 ATGCCTTTGT GAACATACTG TATCGTGTAT CATAATTGTG CATGACTTTA TTTATGAGCT
35641 AAATAAATGA ATGGAAAAAA AAGTAACCAG TCTTGATGCT TACCTGCCAT CCTGGAAGGA
35701 AATGGAAATA GGATCTGCCG CCGCAGCATT GCCCTATGCT CTTATTTCTT CTCTTGAAGA
35761 GGTAGGGGTG TGTGTGTGTG TGTGTGTGTG TGTGTGTGTG TGTTACTAGA GACTGAGCTA
35821 CCGGCCTCAC ACATTCTAGG CAAATGCTCT ACTTTATATT AAACACTTTA TAAAACATTA
35881 AGCCTTTCAG GGTCAGCAAG GTAGCTCAGA GAGTCCGGGC ATTTGCTACC AAGCCTGACA
35941 ACCTGAGTTC GATTGATGAT CCCCCAGACT CACGTGATAG GAGGAAGCTG ACACCTGTGG
36001 GTTGTCCTCT GACTATAGGC ATGCACACAC ATCCCATGAA TAAATATTTA TACATTTTCA
36061 AATCATACTT ATTTTACAAT GATTTTTATT TGTTTGCCTG TCTTTCTGTC TGTGTAGAGA
36121 CAAGGTTTCA TGCAGCTCAG GTTGGCCTCA AACTCACTCT GTGGCAAGGA TGCCTTAACT
36181 TCAGGTCTTC CAGGTCCAGG TAACAAAATG TTCAGGAGGA ACCTGGTACC TCATCATAAC
36241 CGGTTCTAGA TGGTCTTCCC AGGGCTGCTG TAAGAAAGTG CTACACGACG AGTTATTTCA
36301 AACATTCTCA CAGTTCTGGG GATTAGAAGT TTGAAACTAA GGTGCTGAAG AGATTAGTTC
36361 CTTCTGGAAG CTCAGAAGAG CCATCTGGTC CATACTTTTC TCCAGGTTTC TCTTAGTTTT
36421 TGGCAATCCT TGGAATCCCT TGGTTTGTAG ATGCAGCTTC CAAAGCTCAA GATCTCTCTC
36481 CAATGCTGTG TGGCATTTCC CCGTGTTTAT GAGTGTCTAA ATGGCTTTTA AAAACATTTT
36541 TGAGATGTGA AATTCTGGCT GACCCAGAAT ATATAAACCA GGCTGACCTT TGTCTCCCAG
36601 AGATCTCCCT GCCTCTGCTT CCCAAACCTT TTGATTAAAG GTGTGTGTCA AGTGCCCAGA
36661 CCAAATGCCC TTCTTGTAAG GACAACGGTC ATATTGGATT TAGTGTCTAA GTGAGTCCCC
36721 TATGAACTCA TCTCGAACTC AGTTTGCATA GAACACTGTA CCATGCAAAA TAAATGACAC
36781 AGAGACTGAT ATTGGGGTTC ACACTTCAAG CTGAAGGTCA GAAAAGCAAA GCATTGGGCC
36841 ACTAGCTCTT ACCACTACCT CAGGCTGAAC GGGCTGATCC TGCTGCCTCT CCTCAGCATG
36901 GCTGGAGAAT ATCTTCATAT CCTCATTGTG GCTGGAAAAT GAATGCCTGA TATGGAGAAC
36961 TTGCTCCTGT TTTATATAAC TCCCTAATGC TGGGATTAAA GATGTGTGAT CCCAGGTGCT
37021 GAGATCATCT TTGTGTGAGC TGTTTCTCTT TAGGACTGGA TCAATTTTGT GTAGATCTGG
37081 ATGGCTTTGG GCTCACTGAG ATCTATCTAC CTCTTAATCC CTGGTCCTAG GATTAAAGGT
37141 ATGTACCACC ACATCCTAGC TTCTGGCTGC TGGGATTAAA GGTGTATGCC TGGCTTCGAT
37201 GGCTTGTGGC TGACTTTGCT TTCTGAATCC GCAGGCAAGC TTAAAAAAAT CATAAATAAT
37261 ATATCACCAT AGACCACACT TCCAAATAGG CTTCCATTTA GAGGCGCCAG TGGGTGATAA
37321 TGTAGGCGGT TTTACTCAGT TTTGTGCAGA TGGCTGGCGT CCTGTCTGGT GAGTTCAGAT
37381 TTTTTTTTTT TTTTTTTTTA AGTTCAGAAT CTTACCCAGC TCAGCTTTTC AGGCTGCATT
37441 CAGTGTCCGG CTTTTTTCTC ACCGTCTTGA CTTCCTGTCC TGCATCCCAT TTCTCAGCCT
37501 GGACCCTGCC AGTCTATCAG ATAGATAACA TAAACAAAAT TGTACTGGAT TAATGGGAGC
37561 TGTTTGGACA TTTCCTACTT TTGCCTTTTC ACCAATGATT TGCATACTTA AGCCTGCAAC
37621 TACAGCCCCG ATGCAGTAAG CTCAGTCTCT GGCAAGCAAA GGTCTCTCTG GGGTCTTGTT
37681 TAAGAACCAG CTCAGGCTGC TGGCTCTGTT GGCAGTGGAG GTATTTCCTA TAATGGGATG
37741 ATGGGATGGG TTATTCACAC ACATCTCAGT TACTGGGCTA CATGGATCCA AATCAGCCAC
37801 CCAAGGGTTT GCAGTCACAT GTGAGTCACT TAGCACAGAG AAAGAAGCCT GGAGGAGGAG
37861 GGGTCCTCCC AGCTTCAGGA GGGTTTTCCA GGATATAGGC TTCTAGTCTC GTTTTGGATC
37921 AATTTATCAG TTTTGGATTG GGTCTAATAA CTCTTTCCTG AGCCTGGACT GGGCTCAAAG
37981 GCATGAGTAT GTGAGGGGAA TTTACTAGAA TTCACCTGTA GTTTCTGTAT CATTCCTAGA
38041 GAAGGGGAAG TAGAGACACT GGTGATGGGA AATAAAAACA AAACAAAACC TAAATATTGG
38101 GAGCACAGAG GTCCTTGTTC CACAGCTCTT GATAGAAGTC AGGAATGTTA TGTATGTACA
38161 ATTGCCCTTG AAAAGGAAAG GATGTATGAC CTGTTTTTCT GTCCCGAAGG CTGGGAACTG
38221 GGGATGATTA ACAGCCTGTT GATCTGCATT ATCTGAAGGG CTAGGCCATA TCAAGCTCCC
38281 ACAGCTAGCA CTGAAGGAGA ATAGGGCCTT ACAAAGGGAA TTCCCTCTTT GGATCGAACC
38341 TAGGAACATC TTCTGTTTTA CCGCTCTCTC CTTGTTTCAT CTGCAAAGGG AGGAGCTTGG
38401 TAGTGATGTT GAGGCAGGCA CCACTTGTAT TTTTCTAAGC CACAGAGACT GTTTCCCTAC
38461 CTTACAAACA TCCCTGTGCA TCACTGCAGC TCTGTCTCTT ATGGCAGTGT CTCAGTTAGG
38521 GCTTCTATTG CTGCGACTAA ACACCATGAC CAAAAAAGCT CACACTTCCA TACTCCTGTT
38581 CATTATTGAA GAATGTCAGG ACTGGAGCGC AAACAGGGCA GGGTCCTGGA GGCAGGAGCT
38641 GATGCAGAGG TCATGGAGGA AGGCTGCTTA CTGGCTTGCT CTCCATGGCT TGCTCAGCCT
38701 GCTTTCTTAT AGAACCCAGG ACCACCTGCC CAGGGATGAC ACCACCTACA ATGGGCTGGG
38761 CGCTAATATG AGGGATCAAA GAGATGGAGT TGTGGGAGGG ACAGAGGGGG AGAGCAATGA
38821 AAGAGATAAT CTTGATAGAG GGAGCCGTTA TGGGGTTAGG GAGAAACCTG GTGCTAGAGA
38881 AATTCCCAGG AATCCACAAG GAAGACCCCA GCTAAGACTC CTAGCAATAA TGAAGAGGAT
38941 GTCTGAACGG GTCTTCCCCT TTAATCAGAT TAGTGACTAC CCTAATTGTC ATCACAGAAC
39001 CTACATCCAG TAACTGATGG AAGCAGATGC AGTGATCCAC AGCCAAGCAC TGGGCTGAGC
39061 TTCGGGAGTT CAGTTGAAGA GAGAAGGGAT CATGTGAGCA AGGGGGTGGG GGAAGTCAAG
39121 ATCATGATGG GGAAAACCAC AGAGACAGCT GACCCGAGCT AGTGGGAGCT CATGGACTAT
39181 GAAACGCCAG ACGTTGTAGA CTCCCTAAGG AAGGCCTTAC CCCCTCTGAA GAGTGGATGG
39241 GGGGTGGGAA GTGGGGACGC TGGGGGACAG GAGAAAGGGA GGGAGGGGGA ACTGGGTTGG
39301 TTTGTAAAAT GAAAAAATAG ATTTTTTTTA AATAAAAAAA GAAAGTGCTT TACATCTGGA
39361 TTTCATGGAG GCATTTTCTT AACTGAAGCT CCTTCCTCTC TGGCGACTCT AGTTTGTGTC
39421 AAGTTAACAC AGAACCAGCC AGTACAGGCA GCAGAAATAC CTTGCAGAAA TATCTTAGTT
39481 CAGGAGTCCA CGGTGGTCTC AGTCACTTCC TCATGTGCCA CCTGAGTTTA ACATTCCCCA
39541 AAACTTGGAA CACAGGCCAC CACATCATGG AGCCCTGGCT TAAAGCTCAA GTTTTATGGT
39601 ATTTTCTTTT ATCACTGTCT ATAATTCCTA AACATGCTAC AATGTTGTGA GCCCTCACCG
39661 TCTCCTAGGT CCATAGTGAC TTCCTGGCAT TAATAGACTG TGCCCCAAGA GCTCTATGGC
39721 CACGACCACC ACCTGCCATT CCCCTCCCCC TCCATGGTCC CAGCCTCACT TCTTCACTTC
39781 CTGGTCCTTC CGAGCCCAAT GTGCAAACCC ACAGAATCTG TCTGCTTATG TAAGTTTCCT
39841 GGTCACTGAG TGGGGTGACT CAGCACCAAG GTGGTGCCCT GCGATTTCCC AGCCCCAGGC
39901 AGCAGAACAA CTGAAATGGA AAACAAGTCC CGTTAATAGG GTCCAGCTGA GAGCCTCCCT
39961 TTCTCAGGGA GTCTGGCAAA TCTACTCCTC GGGGAACTGC CCTGGGCAGT GGAATTCTCC
40021 AGCTCCCTGC TCATTTCCTA GTTCCTCTTC CCTCTTCTCA CCTTTGGCTG AGGATCAGAA
40081 AGGTTCCCAC TGAGGTCTGC TTTGCCCTGG GCCTGCTCTT TTCAGAGTCC CATTTTTGGA
40141 ATGAATTTTT TTTGTCTCCT ACTTTCAAGT TCACATATTG AAGCCATTAT TGCCAAGGTG
40201 ATGGTATCAG AAGGAGGGAC CTTTGGGAGA TGAATGGATG GATTCCAAGA GGTTATGTGG
40261 GCAGAGCACC CATGATGGGG TTGGTGCCTT CATAGGAAGA AGACACAGTA GAAGGGAAAG
40321 AGATGCCGAC TGAAAAACAG GAAGTCTCCT GGAGTAGGCC ACTCAGCCTA TGACACGCCA
40381 GCACTCAGAT CTCGGACTTC CCATCTCCCA AATGGTGATA AACAAATGCT GTTGTCCAGG
40441 CTGCACAGTC TACGGCATTT TGTTGCAAGG GCCTGGACCA ACCAGGCTCA GGCAGGAAGT
40501 GAATCTAGTG TGGGAGGATG TACAGACTGC CACTCAGTCT GGACACAAAC TGTCCTCAGG
40561 GATCACCTGA GCCACATCTA CCTAAGAATG GCTATTCTTT CCATTTGTTA ACATCAAATG
40621 CCAAGCCCCT ACTGTATGTA GGCTCTTGCT AGCAGTGGAT ATGATGCTAT GTGAGATGGG
40681 AGCAATCCTC TCTGCACAGA ACTATACATA GAACTATGCA TAGAAGACCA ACAGGGAGAC
40741 ATCAGATAAC TATTAACTGT GATAGCTCTG TGGGAGACAA ACAGAATGAG GGAATGGACA
40801 ATGACTTTGA GGAAAAACTA TGATTGAAAA TACTCTATCT GGCTGGGCGG TGGTGGCGCA
40861 TGCCTTTAAT CCCAGCACTT GGGAGGCAGA GGCAGGTAGA TCTCTGTGAG TTCGAGACCA
40921 GCCTGGTCTA TAAGAGCTAG TTCCAGGACA GCCTCCAAAG CCACAGAGAA ACCCTGTCTC
40981 AAAAAAAACA AAACAAACAC ACAAAAAAGA AAATATTCTG TGAGGTAAAC AAGCATCTGG
41041 AAGGGTTGGG AGATAATGCA GGCAAAAATG CATTAGACAG CACACAGTAC AACACAGCAA
41101 TCAAACTTAA TATAAACACA GCAAATGTCA TCTTTGGGCT TTGCCCCATT TCCTGATCTG
41161 ACCATAACAG CCTAGTGTCT GGAAAGCACA CTAAAGCCAT TTACGTCACA CAGGAGTTCA
41221 ATGTTGAGTT CAGAGGGAGG GGGTGGAGGG CAGATTAGCG AGGTACAAGT TCTGGTCCCT
41281 TTGATGAAGT GTTGATGTAC CCATCGACAC CACACAAATA TACCATCATG CTCCATGTTA
41341 GGGTCAGTGA AGGATTGCAT ATGTGACGGT GGCCCACTGG GCTGAGAAAG CCCTATTGCT
41401 TAGTGACATC TGTGATAATG ACATGCGAGC CCTATTGCTT AGTGACATCA CTCTTCTCAT
41461 AGTGTGGGAT CCAATGTGTT TCTTGTACAC TTGTGATAAT GACATGCAAA CAAGTCTATT
41521 GTGCGGCCAG TCACACAAAA AATATATTAT GTGCAGTCAG GAACAGTCCA TAGTACTTGA
41581 TTGGGACAGC ACAAGTCTGT GTTGCTGGTT CACACATTAA TCATTACCAC TGTTTTAGTG
41641 TGCTCCTATA TATATATATT TAAAAATTAC TATAAAATGA TACACCGTGC TGAGCAATAG
41701 CACCTCTTAT ACCTTGTGTT TACTGGATGT ACTCAAGCTA TTTTCTCTTG TGCTTGATTT
41761 ATTTGTATTT GTATTTTTGA GAGAACCTCA TCTAGTCCAT GCTGGCTTCA AACTTGTTAT
41821 AAAGCTGAGG ATGGCTTCGA ACTCCTGATC CCCCAGCCTC TGCCTCCCAA ATGATGAGAT
41881 TACAGGCATA TGCTACCAAA CATGACTTTT ATTTATTTTT ATTACTTAGG TGGTATGGGT
41941 GGTTTGAATG AGACTGTCCC CTTTGGCTTA TATATTTGTA GGTGGACCTT TGGAAAGGTT
42001 TAACAGGTAT GACCATAGTG GAGGCAGTGT GTCAGTAGGG GAGGTCTTTG GGGAACCCAA
42061 TACTCAATCA ATTCCAAGTT AGGGCTGTCT GTCTGTCTGT CCCCTGATTG TGTCACAAGG
42121 CAGAAACTCT CAGCTACTGC TCTAGTTCTA TGCCTACCCA CCTGTTGCCA TGGTCCCTGC
42181 CATGATGGTC ATGTACTTCA ACCCTTTGGA TAGGTGGCCC CCAAATTAAA TGGTTTCTTT
42241 TATAAGTTGC CTTGGTCATG GTGTTTTGTC ATGGCGATAA GAAAGTGACT GAGACAGGTT
42301 TGTTGCTGTT GTTACAAGGT TTAGTCCAGG CATCTGGCAC CACCTCTGGC CTGTGCTTGA
42361 TTCAATCATG TTACCTTTAG AAATAGCAGG CTAAAGGACA TATACCTGTG TACGTATATG
42421 TGTACGTATA TATTAGCTGT ATAGTCTAAG TGTGCACCTG ACTCTAATAT CTAGGTTTGT
42481 GTAAGTAGAC TCCACCAAGC TCACTAAGCA ATGGTATCAC AGTTTTCAGA TAGTGTTCAG
42541 CGATGCTTGG CTGAGTGTTA GTTCTTTTTT TAATATTTTA TTTATTTATT ATGTATACAA
42601 CATTCTGCTT CCATGTATCT CTGCACACCA GAAGAGGACA CCAAATCTCA TAACGGATGG
42661 TTTTGAGCCA CCATGTGGTT GCTGGGAATT GAACTCAGGA CCTCTGGAAG AGCAGTCGGT
42721 GCTCTTAACC TCTGAGCCAT CTCTCCAGCC CCTGAGTGTT TTTAAATCAA GGAAAAAAGC
42781 CTGAGGGAAG GGAGCTCAGG CTGAAGGGGA GGAGTCAAGA CAGTCTGACC CCAAGGCATT
42841 GTGGGACGTA AAGAGTTCTG GGACAAGACT GAGGTCTCTT CCTTCTCAGA GACTGTGGGC
42901 TTCAGTTTCC TTGGTAGCCG GAAGCAAAGC TAATCCATGG CTTAAAATAT AATACTCAGT
42961 GTAACCTTGT GTTGTAGAAG TGACTTGCTT GTCTTCTTCC ATAATTCTAA AACATCTTTA
43021 AGAGCAGGAT CCAGGAAGGG AAAAGGAGAG ATTCTCATCT TCTTCAAAAG GCAGCTTTCC
43081 CTAAAGCATT TTCTGATGAA ATTTAAGTTC TAAAACCAGC AGTGGTATAA TCCCATCATG
43141 AATGGGGATC TCTGAGTTTA AGGCCAGCCT GGTCTACAGA GCAAGTTCCA GGACAGCCAC
43201 GGTTACACAA AGAAATCCTG TCTTAAAACA AAACAAAACC CAAAACAAAC ATAAACAAAA
43261 ACTATCCAAA ACCAACCAAC CCCCCCAACT CAGAAAGAAA GAAAGAAAGA AATCAAGAAA
43321 GAACTGCCCA CCGGGTGTTG GTGGTGCAAG CCTTTAATCC CAGCACTCGG GAGGCAGAGG
43381 CAGGCAGATC TCTGTGAGTT TGAGGCCAAC CTGTTCTCCA GAAAGAGTGC CAGGATAGGC
43441 TCCAAAGCTA CACAGAGAAA CCCTGTCTTG AAAAAAGAAA AGAAAGAACT ACCCATGACC
43501 AAACAGTTCC ATGGCCAGGT AGAGAATGAG GACGCTGAAA GTCACACCTT CTCAGAGTCT
43561 CAAACTGCAC ATCTGGCCTC AAAGTCCAGA AATGAGTGCA AGACCATTAA TGACAGTCTT
43621 TGGAAACAAA CCAGACCAAA GAACATTTGG CTCCTGATAC ATATTCTGAG GGTCACATAG
43681 AAAGAAAGAT CTGCCTTTGG CCACCTCCTT TTGAAGTGGG GAATTTTATT TTCTTCTGCA
43741 TGGAAACTTC ATGTAGGTAT TTGAGAATAC ATACAGACAT GCAGGTGCAC ATGCACGGAC
43801 ATGAACACAC ACATACACCC CGGGTAGGCA GGCAAGAAAG TGTGTGGAAT AACACTTGAA
43861 CTTCCCTTCC AGAACAGAAG CCCTCTGAAG TGTGACATTC ATGCTGGCTG CATGGGGTCT
43921 GATCAGTACT AGTGAGTGGA GGTGGAGGGG TAGGAAACAT GGGGATGATA ATAGGTTGTC
43981 AGGAAAGTGG TGCCCCAGGT AGCACAGAGT AGAAATTTGT CCCCCAAAAT CCTTTTGAAC
44041 CCAGTTGATT TGAATGCCGT GCCCCTGCCA CCCAGGCTTC AGAGCTAAGT GACTTATGTC
44101 TTCAGGTCAG TGATGATTAC CACGGTTGCA GTGCTAACAC AGATGCTTTA TCTACCAGGA
44161 CAGAAACAAG AAAGATGCTC CTTCCCAGGC CCCTTAGCAC TCTCTGGGTG GGGAGGATTG
44221 CCCCACCTTC CAAAAATAGA ATACTGTTTT GGTAAACAGC CACTTTGAGC CCATGAGGAT
44281 ATCTTCATTA GCTATGGAGA CAGGTTTTAG TAAGAAAGCA AGATGAGAGG CTAAAAAACC
44341 CTTGGGGAGC AGGAACTGGG AAGACTGTGG TACCTTGTTC CCAGATCCAC CAGAAACCTT
44401 GCCACCAGAC GATGTGTCCA GGCCCCACAT ATTTCACAAA AAGTTGGATC TGATAACAAT
44461 GAGGATGGAA TCCCGGTCTT AAGGTGGGTT TGGGGTGGGA AGAGGCGGGA TAATGGGTGA
44521 GAGGGTCGGT GGGGACAGGT GAGATGGGGT ATGGTGGGGA GAGGTGGAAT GGGGTGGGGT
44581 GGGTTGAGAT GGAGTATGGT ACAGCGGGGA GGGATAGAAT TGTCTTTTCC CTGTACCACA
44641 GAGAAGTTTG ACTGCTACCC TTGGCAATTA ATCAATTATA GAAAATGCAA CTTTGCTTTT
44701 AAAATGTGTC TATTTCCAAA GGCTTCTTCC CCTCCCCTAC CTAGGGAGAA GGAAAGAATG
44761 GATAATGCTA CTGTAGAGGA GGGTAGCATC ACTATAGAGG CCTCAGTATC TGCCCCAGGG
44821 AGCTGGGAGA GAGTTCTATC ACACAAACAC AGCCCGAGTC ACATACTCAA CAAACCCCAC
44881 AAAACAAAAC AACAATAATG AAGATACAAA ATCTCATTAT GTAGCCCAGG CTAGTCCTAG
44941 ATTTCTGTTT TCTTTTTTTG TTTTTCGAGA CAGGGTTTCT CTGTGTAGCT TTGGAGCCTA
45001 TCCTGGCACT TGCTCTGAAG CCCAGGCTGC CCTCACTCAC AGAGATCCGC CTGCCTCTGT
45061 CTCCAGAGTG CTGGGATTAA AGGCGTGCAC CACTAATGCC TGGCTAGTCC TAGATTTTTT
45121 TATCCTCCTG CCTCAGGCTC CCAACTGTTG GGTTTACTTT TGGGAGTCCA TTTTCTTCCA
45181 GCATGGATTC TTTGAATTGA AATTCAGATT ATCAGGTTTC TGTAGCAATC CCACCAGCCC
45241 ATTTTTTTGT CTGACACTGC TTGTTTTGAG ACACAGTCTC CCACTGCTGT AGCCCAGGCT
45301 GCCCTAGATT TTCTATGTAG CCCAGGCTGG CCTTGAACTC CCAGGAGTCC TCTGGCCTCT
45361 CCCTTTTGAT TACTGGAACT AGAAGAAGTC ACTATGCTTG ACTTGGAACT AATATTAGAA
45421 CAAAATATAT TTTTCATTGA GATTCAACTT TGAAATCCTG ATGCTCCTGC CTCACTCAGG
45481 TCATCAGGGT TGGCAGCAAG AGCCTTTATC CACTGAGTCA TATTGGGCCC TGACCTGCTT
45541 TTAAATTTTG CCTTTAGGGC TGGAGATGTA GCTCGGCTGG TTCAGTGCTT GCCTGGTACC
45601 CACGAAGCCC TGGGTTTGAT CTACAACACA GTATAAGCCA GGCCTGATGG CGTATACATG
45661 TAATCCTAAC ACTTGGGGAG CAAGAGGGAG GCCAAAGCCA TCCTCTGCTA CTTGGTGAGC
45721 TTGAGGCCAG CCTGGGATCC TTGAGACCCT GTTTCAAAAC AATAACAACA AACACAGACT
45781 ACTAAAAAAA ATTAATAAGG GCCAGACTGG GTGGTGTATT CCTTTAATCC AAGCAATGAG
45841 GAGGCAGAGG CAGGCAAATG TCTGTGAGTC TGGGGACAGC CTGGTCTACT GAGCAGCAGG
45901 CCAACTAAGG CTACATAGTG AGACTATCTC AAAAAAAGCA AAATAACAAT AAACAGACCA
45961 GTTCCCCATC TCCTATTTTG CCTTTACCTC CTATTCCCTG CTCAGCAGGT TATTTTTTGT
46021 TCCTGCATCT TGGTTCACTG ATCTGTAAAC TTGTCTGAAT AAGTAGGTAC AGGGTTGTTT
46081 TAAAATTAGA TAATATATTC AATGAGAAGG GCTACCAAGT GCTCAACCAA TGTATGCATA
46141 TGTATGTATG TATGTATGTA TGTATGTATT TATTTTTGTT TTGTTTTTCA AGATAAGGTT
46201 TCTCTGTGTA GTTTTAGAGT CTGTCCTAGA ACTTGCTCTG AAGAGCAGGC TGGTCTTGAA
46261 CTCACAAAGA TCCACCTGTC TCTGCCTCCC AAGTGCTGGG ATTAAAGGCA TGTGACACCA
46321 CCCCCAAAGC CAATGTTCTT ATAGGCATCT TTGATTTTTT TTCTCTTTCT TTGAGTGGAG
46381 TCTGACTAAG TAGCCCATAC TAGCTCTGCA TTTACAATCT GAACACATGG ATAAGAGTGG
46441 TGAAAATTAT CAAGATCATG TTATGCTATG CCTCCTGAGT CACCATGCCC TGCTTCAGAC
46501 TTCTTTGTAT TAAAGAACTG TGTAAAAAAA AAAAAAAAGA CATTTGAAGG CACATAATCA
46561 GAGGAATTTG TCAGTGATTT TTCACATACT GTCTTATTTG TGGCCAAGGT AAGCCTAGAG
46621 AGTATTTCTT AAAATTAAAA ATAGTGGGCA GATTTTGGAG GCGATCTGAT ATGAAAATCC
46681 CTTCCCACCC CAGGTAGTCA TGGGCTGACT ATCAAGGATA CATTCTGAGA CATATATCCT
46741 CAAGCAGTTT CTGCCTTACG CAAATATCAT AGGTCATAGC ACACTGAGAC TATGTGGCAG
46801 TCTATGTGTC TATATACACA TGGTGTGGCC TATTGTTCCC ATGGTCACAA AGAACAAAAC
46861 AACTTTTTCA CAAGGCTTTA CCCCTAGAGG AAGAGCTACA GGCAATCAAT GGTTGCTGAG
46921 AGGAGTATCA GTCTTCTCCA GGGACTTAGC CAATCCCAAG AGGTCAGCCA CGCATAGGAA
46981 CGCTTAGCCA CGCTTGTATA GAACATCTCA AACAACAACC ACCTCAGTGT AAAGCAAGCA
47041 CACAAGGAAC TGATGCAACT AAGAGACAAA GGGCCCGGTG TGTGTGGCCC GTAGCTGTCA
47101 TCCCAGCACT TGAGACTAAG GAAGGAAGGT TGAGAATTTG AGGCCAGCAT GGACTCCACA
47161 GAAAGACCGT TTTCTTTCTC AGAAAAAAGA AGCAAAAACC AAGAACAAGG TGTATGGGAA
47221 TGCTACTGTC TTGGCATATT GTTTATAGAA AACTTTTTTA TATATAAAAG GAATGCACTA
47281 CAAAAATTAT AAACTACTGT AATATTAACT GCATAGATCT ATAACATGGT CATTTATTAT
47341 TGAGTATGAT TATCTATCTA CCCACGCTGC AGGTTTAGAC AGTTGCACTA CAGTAGATCT
47401 GTTTGCAGTA GCATCATTAT TAGACATTTT GGACAAAGCC AAGTGGTAAT GGCACATGCC
47461 TTTAATCCCA GCACTTGGGA AGCAGAGGTA GGCGGATCTC TGTGAGTCAG AGACCAGCCT
47521 GGTCTACAAA GAACTAGTTC CAGGAGAGTC TCCAAGGCCA CAGAGAAACC CTGTCTCGAA
47581 AAACCAAAAG AAAAAAAGAA AACAAAAAAC TAAAAAATAA ATAAATTTGG GGCAATATCT
47641 TGTCCTATGA TGTTACTGGG TAATGGGATT TCCTCCTCTT GTATTATTTT TTCTTTGGGG
47701 GTTTTACTTA TTATTTACTT GAGACAGAGT CTCATTTATG ACAGGCTGGC CTCAAACAGG
47761 AAATGAAGCC AAGGAAGACC TTGAAGACCT AATCCTTCTG TTTCTTCCTC CTATATGGTG
47821 AGTTAAAGGC ATACAGTACC ATGCCCAGTC TATTCACTGC CCAGGGCTTC ATGCATGCTA
47881 GCAAAGCACC AACTGAGCTG CATCCCCACC CCTCCTCCTG GCTTCCATCT CCTTATGTAG
47941 CTAGAAATGA GCCTGTCTGT CTCAAATACT GGGATTATGG GTGTGTGCCA CCACACCTGG
48001 CTTCCTATTA TAGCCTTGTG GGATCACTGT TGTTTACTGA AGCATTGTGA CACACTGCAG
48061 ATTGCTGGAA CAGCGTCTGC CATCATCATG ACACAACTTC AGAGAAAGAG AGAGTTCCCA
48121 ACCAGCCACA CACTTAACTC AATGCCTGTA GCCCTTATTC TGTTAAGACG ATTTCCTGCC
48181 ATCTTACTCA AAGACCCTCT TTAACTCGGT AGGAACATCT GTTACACTGA AAGTCCTGCC
48241 TGTTGCTCCA CTGACCTCCT TCACAAATTA TTATATTTTG GAGCCAATTC TGAACCCAGG
48301 TTTTCTGAGT GACACATTTT AGTATTTTTT TTTTCTTTCT ATTTTCTTTC ATGGAAAGTC
48361 TCTTGTTACT GTTCACATGA CCAAGGATCA CTGCATCATC TTCCAAGGCC AATTTTGGAT
48421 GTTTCAGCAA GGGAGACTGA AGATCCTGAG TCTCAGTGTT GATCTCCTTT AGAATGTCCT
48481 CTGGAGAAGG TAGTGACAAC ACTGCAAGGA TAATAGGTGA ATAAAGGGAA GCCAGAGTGT
48541 CCTCTGGGAT GTGCGGCACT TACATGAAGG ATTCATTTAT AAATTTTAAG TTATGGAGTA
48601 TAATAATAAG ACTAAATATG TAGTGTCGTA ATTTTATAAC TATACATATG TATATAGTAA
48661 ATATAAATTT ATATGTAATG TATTTATAGT AAGTGTACAT AGAATTGAAC ATATGTTACA
48721 TAAATGGCAG AAAGGAATGA TTCTCAATTG CTTTTTTTCT AATTATAATT TCTATTGCTC
48781 TTTGTGGATT TCACACCATG CATTCTGATC CCACTTATCT CCTTGTCTCC TTGCATTTGC
48841 CCTCTGCCCT TGCAACCTCA CCCCCAAATC AAAGCCAAAT TTAAAAAAAA AACCAAAATC
48901 CAAACAAAAC AGAGACAAAA CAAAAATAAA AGCAACAACA AAAAAAGGAG AATCTTGTCA
48961 TGGTAGCTGT AGTGTGGCCT GTTGAATCAC ACAGTATACC CTTTAGTCCA TTCATCTTTT
49021 CTTCCAAGTG TTCATTGATA CAAGTCACGG TCTGGCTCGA GGATTCTGGT TTCTGCTATA
49081 TTACTAATAA TGGGCTCTCA CTGGGGCTCC CCTTGGATAT CCTATTGTCC TGTGTTATGG
49141 AGAGCCTGCT GTTTTGGATA TGTAGGTTTG TCCCCTTCAC ATGCTATAAC AATTCATAAA
49201 TTCAGTGAAT GTTGGGGTGG GCCAACTCAT AGCCCTGGTT CTGGGCTTGG GTGGTATTAT
49261 TAAACCCACT GATGGAGAAT AAGACCACTA CCATAATTTA AAAGCCAAAT TGAAGCAAGT
49321 TTTAATTCAA TACTGCCCAG GTGGACAGGC TCTGGCTAGG TCCATCTCTG AGTTTCCAGG
49381 AGGTGGCCCT GACTCACGGT TTACAGTGGC TTGAGTATTT TCCATAAGGT CCAATCAGGG
49441 GCAAGCATAC ATCCTGATGT ACCTCCAGTC TATATCCAAT CGGGGGCAAG TGTACATCTT
49501 GATGTATTTC CTGCCTGTGA ACCTACTGCC CACATGTGAT CAAGCACATC CGGTGCAGTT
49561 GGGTCAAACA GACTTGTTTA GGGCAATGAA AAACACATGG CTTTTTATCT CCCATAAACA
49621 ATAGCCTCCA GCGGTTCAGG GACTATTTGT CCTTGGGCAA GGAATTTACA GATCCTATAG
49681 GTGAGTCAGG GTCAGCATCC TGCTCTCATG CCCTCAGGGC TGGCTCACTT GTTACCTCCC
49741 CGACCCTCTC TCAACAGGGT CAGCTCTGAG GTGCTGCCCA GGTGGGGTGC AGGGCCTACT
49801 CTTCCGCATG TTGCAGCTGG TCAGGGTTAG TTCTCTCATA TGCCACAGGT GGCAATGGGT
49861 GAAGGGGGAG GGCATGTTTC CCTCATCAAC GCCATTACAT GGGGGGATGG GGTCAGCTCT
49921 CATGCCCTTA GGGTTGGCTC ACCTGCATCC TTGACCATAG GGTCAGCTCT AGTATGCTGC
49981 TCAAGTGAGG CGCACACCTA
SEQ ID NO: 4 (LoxP sequence from bacteriaphage P1)
1 ATAACTTCGT ATAGCATACA TTATACGAAG TTAT
SEQ ID NO: 5 (FRT sequence from the 2 μm plasmid of the baker′s yeast
Saccharomyces cerevisiae)
1 GAAGTTCCTA TTCtctagaa aGTATAGGAA CTTC
SEQ ID NO: 6 (attB sequence from E. coli)
1 cCTGCTTt t TtatAc tAA CTTGa
SEQ ID NO: 7 (Recognition site for the CHO-23/24 meganuclease, 35,699
basepairs downstream of CHO DHFR)
1 TAAGGCCTCA TATGAAAATA TA
SEQ ID NO: 8 (Recognition site for the CHO-51/52 meganuclease, 15,898
basepairs downstream of CHO DHFR)
1 ATAGATGTCT TGCATACTCT AG
SEQ ID NO: 9 (CHO-23/24 meganuclease)
1 MAPKKKRKVH MNTKYNKEFL LYLAGFVDGD GSIKAQIFPN QCYKFKHQLR LRFQVTQKTQ
61 RRWFLDKLVD EIGVGYVTDR GSVSDYMLSQ IKPLHNFLTQ LQPFLKLKQK QANLVLKIIE
121 QLPSAKESPD KFLEVCTWVD QIAALNDSKT RKTTSETVRA VLDSLPGSVG GLSPSQASSA
181 ASSASSSPGS GISEALRAGA GSGTGYNKEF LLYLAGFVDG DGSIIAQIKP GQSYKFKHTL
241 QLVFQVTQKT QRRWFLDKLV DEIGVGYVID RGSASDYRLS EIKPLHNFLT QLQPFLKLKQ
301 KQANLVLKII EQLPSAKESP DKFLEVCTWV DQIAALNDSK TRKTTSETVR AVLDSLSEKK
361 KSSP
SEQ ID NO: 10 (CHO-51/52 meganuclease)
1 MAPKKKRKVH MNTKYNKEFL LYLAGFVDGD GSIIAQIPPN QSCKFKHQLR LTFQVTQKTQ
61 RRWFLDKLVD EIGVGYVRDR GSVSDYILSE IKPLHNFLTQ LQPFLKLKQK QANLVLKIIE
121 QLPSAKESPD KFLEVCTWVD QIAALNDSKT RKTTSETVRA VLDSLPGSVG GLSPSQASSA
181 ASSASSSPGS GISEALRAGA GSGTGYNKEF LLYLAGFVDG DGSIYAGIAP NQSCKFKHQL
241 RLWFVVSQKT QRRWFLDKLV DEIGVGYVID NGSVSHYRLS EIKPLHNFLT QLQPFLKLKQ
301 KQANLVLKII EQLPSAKESP DKFLEVCTWV DQIAALNDSK TRKTTSETVR AVLDSLSEKK
361 KSSP
SEQ ID NO: 11 (CHO-51/52 donor plasmid with EcoRI site)
1 TCGCGCGTTT CGGTGATGAC GGTGAAAACC TCTGACACAT GCAGCTCCCG GAGACGGTCA
61 CAGCTTGTCT GTAAGCGGAT GCCGGGAGCA GACAAGCCCG TCAGGGCGCG TCAGCGGGTG
121 TTGGCGGGTG TCGGGGCTGG CTTAACTATG CGGCATCAGA GCAGATTGTA CTGAGAGTGC
181 ACCATATGCG GTGTGAAATA CCGCACAGAT GCGTAAGGAG AAAATACCGC ATCAGGCGCC
241 ATTCGCCATT CAGGCTGCGC AACTGTTGGG AAGGGCGATC GGTGCGGGCC TCTTCGCTAT
301 TACGCCAGCT GGCGAAAGGG GGATGTGCTG CAAGGCGATT AAGTTGGGTA ACGCCAGGGT
361 TTTCCCAGTC ACGACGTTGT AAAACGACGG CCAGTGAATT CGAGCTCGGT ACCCAGAAAC
421 CTTTCAACCA GCTTTTGAGC TAATGATAGA GAGAAGCTCA AGGAATTGGA GCAATGCTTG
481 ACTAGGGATG TCAGAGGGAG GCTATCCAGA GGAGCTTACA ACTGAGGTAA ACTTAAAAGT
541 TAGGGAGTTT GTCAACTTCA ACCCACAGAA TAGAGCAGAG CCAGGAGGAG CTGAGGCTTC
601 TGAGTGTTAT GGTGGAAGCA TCACCCCAAC CCTTGACATC CATATGCCTG AAGAGTCTGG
661 AATGTTATGG TGGAAGTTCC ACCCAAGCCT CCCTTCCCGG TCGCCCTCCA AACCCTGCTA
721 CATCTCAGAA ATCCCACCAA ATGATGACTC CCTCCCCCAG AGATATTCAA GACCACTCCC
781 ACAGGGTATT TAAACTGCCC CCCAACCCCC AGAAAATAGA TGTGTGGTTT TCCAATCTCT
841 CTTTCCTATC ACGTCTCTGG GGAGCTGGCA GGCCATTTGG GAGCATTGTA TCCATTAAAC
901 GACTTCTCAG TGGAGACTCT GAAAGCCAGA AGAGCCTAGA CAGATAGATG TCTTGCGAAT
961 TCTTGCATAC TCTAGAGACT ACAGATGCCG GCCCAGACTA TTATATCCAG CAAAAGTTTC
1021 AAACACCATA CAAAGTCAAA TTTAAACAGT ATCTATCTAC AAATCCAATA TTACAGAAGG
1081 TGCTAGTAGG AAAACTCCAA ACTAAGATTA ACTATACCTG TGAAGACACA GGAAATAATC
1141 TCACACTGGC AAAAGAAGAA AAACCTCTCT CTCTCTCTCC TCTCTCTCTC TCTCTCTCTC
1201 TCTCTCTCTC TCTCTCTCTC TCTCTCTCTC TCACACACAC ACACACACAC ACACACACAC
1261 ACCAACACCA ATACCATGAA CAACAAAATA ACAGGAATTA ACAATAATTG ATGTGTGTGT
1321 ATGTCCCTGT GTGTGTGTCC TTGTGTGTGT CTGTTTGTGT GTCTGTGTAT ATGTTTGTCA
1381 CCTGAGGGGT GGCTCTTCCT TGGTTTGTGA GGTTTCTACC CAAAAGCTTG GCGTAATCAT
1441 GGTCATAGCT GTTTCCTGTG TGAAATTGTT ATCCGCTCAC AATTCCACAC AACATACGAG
1501 CCGGAAGCAT AAAGTGTAAA GCCTGGGGTG CCTAATGAGT GAGCTAACTC ACATTAATTG
1561 CGTTGCGCTC ACTGCCCGCT TTCCAGTCGG GAAACCTGTC GTGCCAGCTG CATTAATGAA
1621 TCGGCCAACG CGCGGGGAGA GGCGGTTTGC GTATTGGGCG CTCTTCCGCT TCCTCGCTCA
1681 CTGACTCGCT GCGCTCGGTC GTTCGGCTGC GGCGAGCGGT ATCAGCTCAC TCAAAGGCGG
1741 TAATACGGTT ATCCACAGAA TCAGGGGATA ACGCAGGAAA GAACATGTGA GCAAAAGGCC
1801 AGCAAAAGGC CAGGAACCGT AAAAAGGCCG CGTTGCTGGC GTTTTTCCAT AGGCTCCGCC
1861 CCCCTGACGA GCATCACAAA AATCGACGCT CAAGTCAGAG GTGGCGAAAC CCGACAGGAC
1921 TATAAAGATA CCAGGCGTTT CCCCCTGGAA GCTCCCTCGT GCGCTCTCCT GTTCCGACCC
1981 TGCCGCTTAC CGGATACCTG TCCGCCTTTC TCCCTTCGGG AAGCGTGGCG CTTTCTCATA
2041 GCTCACGCTG TAGGTATCTC AGTTCGGTGT AGGTCGTTCG CTCCAAGCTG GGCTGTGTGC
2101 ACGAACCCCC CGTTCAGCCC GACCGCTGCG CCTTATCCGG TAACTATCGT CTTGAGTCCA
2161 ACCCGGTAAG ACACGACTTA TCGCCACTGG CAGCAGCCAC TGGTAACAGG ATTAGCAGAG
2221 CGAGGTATGT AGGCGGTGCT ACAGAGTTCT TGAAGTGGTG GCCTAACTAC GGCTACACTA
2281 GAAGAACAGT ATTTGGTATC TGCGCTCTGC TGAAGCCAGT TACCTTCGGA AAAAGAGTTG
2341 GTAGCTCTTG ATCCGGCAAA CAAACCACCG CTGGTAGCGG TGGTTTTTTT GTTTGCAAGC
2401 AGCAGATTAC GCGCAGAAAA AAAGGATCTC AAGAAGATCC TTTGATCTTT TCTACGGGGT
2461 CTGACGCTCA GTGGAACGAA AACTCACGTT AAGGGATTTT GGTCATGAGA TTATCAAAAA
2521 GGATCTTCAC CTAGATCCTT TTAAATTAAA AATGAAGTTT TAAATCAATC TAAAGTATAT
2581 ATGAGTAAAC TTGGTCTGAC AGTTACCAAT GCTTAATCAG TGAGGCACCT ATCTCAGCGA
2641 TCTGTCTATT TCGTTCATCC ATAGTTGCCT GACTCCCCGT CGTGTAGATA ACTACGATAC
2701 GGGAGGGCTT ACCATCTGGC CCCAGTGCTG CAATGATACC GCGAGACCCA CGCTCACCGG
2761 CTCCAGATTT ATCAGCAATA AACCAGCCAG CCGGAAGGGC CGAGCGCAGA AGTGGTCCTG
2821 CAACTTTATC CGCCTCCATC CAGTCTATTA ATTGTTGCCG GGAAGCTAGA GTAAGTAGTT
2881 CGCCAGTTAA TAGTTTGCGC AACGTTGTTG CCATTGCTAC AGGCATCGTG GTGTCACGCT
2941 CGTCGTTTGG TATGGCTTCA TTCAGCTCCG GTTCCCAACG ATCAAGGCGA GTTACATGAT
3001 CCCCCATGTT GTGCAAAAAA GCGGTTAGCT CCTTCGGTCC TCCGATCGTT GTCAGAAGTA
3061 AGTTGGCCGC AGTGTTATCA CTCATGGTTA TGGCAGCACT GCATAATTCT CTTACTGTCA
3121 TGCCATCCGT AAGATGCTTT TCTGTGACTG GTGAGTACTC AACCAAGTCA TTCTGAGAAT
3181 AGTGTATGCG GCGACCGAGT TGCTCTTGCC CGGCGTCAAT ACGGGATAAT ACCGCGCCAC
3241 ATAGCAGAAC TTTAAAAGTG CTCATCATTG GAAAACGTTC TTCGGGGCGA AAACTCTCAA
3301 GGATCTTACC GCTGTTGAGA TCCAGTTCGA TGTAACCCAC TCGTGCACCC AACTGATCTT
3361 CAGCATCTTT TACTTTCACC AGCGTTTCTG GGTGAGCAAA AACAGGAAGG CAAAATGCCG
3421 CAAAAAAGGG AATAAGGGCG ACACGGAAAT GTTGAATACT CATACTCTTC CTTTTTCAAT
3481 ATTATTGAAG CATTTATCAG GGTTATTGTC TCATGAGCGG ATACATATTT GAATGTATTT
3541 AGAAAAATAA ACAAATAGGG GTTCCGCGCA CATTTCCCCG AAAAGTGCCA CCTGACGTCT
3601 AAGAAACCAT TATTATCATG ACATTAACCT ATAAAAATAG GCGTATCACG AGGCCCTTTC
3661 GTC
SEQ ID NO: 12 (Recognition site for the CHO-13/14 meganuclease, in Intron 2 of
CHO DHFR)
1 TACATGTATG TACAAAATAT AT
SEQ ID NO: 13 (CHO-13/14 meganuclease)
1 MAPKKKRKVH MNTKYNKEFL LYLAGFVDGD GSIFASITPR QCYKFKHELQ LTFVVTQKTQ
61 RRWFLDKLVD EIGVGYVIDQ GSVSHYRLSE IKPLHNFLTQ LQPFLKLKQK QANLVLKIIE
121 QLPSAKESPD KFLEVCTWVD QIAALNDSKT RKTTSETVRA VLDSLPGSVG GLSPSQASSA
181 ASSASSSPGS GISEALRAGA GSGTGYNKEF LLYLAGFVDG DGSIIAQIKP NQSCKFKHQL
241 MLTFTVAQKT QRRWFLDKLV DEIGVGYVID IGSVSEYRLS QIKPLHNFLT QLQPFLKLKQ
301 KQANLVLKII EQLPSAKESP DKFLEVCTWV DQIAALNDSK TRKTTSETVR AVLDSLSEKK
361 KSSP
SEQ ID NO: 14 (Recognition site for the CGS-5/6 meganuclease, in Exon 4 of
CHO GS)
1 AAGGCACTCG TGTAAACGGA TA
SEQ ID NO: 15 (CGS-5/6 meganuclease)
1 MAPKKKRKVH MNTKYNKEFL LYLAGFVDGD GSIKAIIRPE QSYKFKHRLR LVFQVTQKTQ
61 RRWFLDKLVD EIGVGYVYDR GSVSDYYLSE IKPLHNFLTQ LQPFLKLKQK QANLVLKIIE
121 QLPSAKESPD KFLEVCTWVD QIAALNDSKT RKTTSETVRA VLDSLPGSVG GLSPSQASSA
181 ASSASSSPGS GISEALRAGA GSGTGYNKEF LLYLAGFVDG DGSIWARIKP GQSYKFKHTL
241 ELVFQVTQKT QRRWILDKLV DEIGVGYVTD AGSASVYRLS EIKPLHNFLT QLQPFLKLKQ
301 KQANLVLKII EQLPSAKESP DKFLEVCTWV DQIAALNDSK TRKTTSETVR AVLDSLSEKK
361 KSSP
SEQ ID NO: 16 (Forward PCR primer for evaluating CHO-23/24 target site)
1 ggagggacat taatctgcat gcagtgatc
SEQ ID NO: 17 (Reverse PCR primer for evaluating CHO-23/24 target site)
1 gtcttggttt gggttgtcta agcaacctc
SEQ ID NO: 18 (Forward PCR primer for evaluating CHO-51/52 target site)
1 CACAGGTGTC CACTCCCAGT TCAATTACAG CTCTTAAGG
SEQ ID NO: 19 (Reverse PCR primer for evaluating CHO-51/52 target site)
1 CGATGGCCCA CTACGTGAAC CATCACC
SEQ ID NO: 20 (PCR template for mRNA encoding CHO-23/24)
1 CACAGGTGTC CACTCCCAGT TCAATTACAG CTCTTAAGGC TAGAGTACTT AATACGACTC
61 ACTATAGGCT AGCCTCGAGC CGCCACCATG GCACCGAAGA AGAAGCGCAA GGTGCATATG
121 GCACCGAAGA AGAAGCGCAA GGTGCATATG AACACCAAGT ACAACAAGGA GTTCCTGCTC
181 TACCTGGCGG GCTTCGTCGA CGGGGACGGC TCCATCAAGG CCCAGATCTT TCCGAACCAG
241 TGCTACAAGT TCAAGCATCA GCTGAGGCTC CGTTTCCAGG TCACCCAGAA GACACAGCGC
301 CGTTGGTTCC TCGACAAGCT GGTGGACGAG ATCGGGGTGG GCTACGTGAC TGACCGCGGC
361 AGCGTCTCCG ACTACATGCT GAGCCAGATC AAGCCTCTGC ACAACTTCCT GACCCAGCTC
421 CAGCCCTTCC TGAAGCTCAA GCAGAAGCAG GCCAACCTCG TGCTGAAGAT CATCGAGCAG
481 CTGCCCTCCG CCAAGGAATC CCCGGACAAG TTCCTGGAGG TGTGCACGTG GGTGGACCAG
541 ATCGCGGCCC TCAACGACAG CAAGACCCGC AAGACGACCT CGGAGACGGT GCGGGCGGTC
601 CTGGACTCCC TCCCAGGATC CGTGGGAGGT CTATCGCCAT CTCAGGCATC CAGCGCCGCA
661 TCCTCGGCTT CCTCAAGCCC GGGTTCAGGG ATCTCCGAAG CACTCAGAGC TGGAGCAGGT
721 TCCGGCACTG GATACAACAA GGAATTCCTG CTCTACCTGG CGGGCTTCGT GGACGGGGAC
781 GGCTCCATCA TCGCCCAGAT CAAGCCGGGT CAGTCCTACA AGTTCAAGCA TACCCTGCAG
841 CTCGTTTTCC AGGTCACGCA GAAGACACAG CGCCGTTGGA TCCTCGACAA GCTGGTGGAC
901 GAGATCGGGG TGGGCTATGT GATCGACCGC GGCAGCGCCT CCGACTACCG CCTGAGCGAG
961 ATCAAGCCTC TGCACAACTT CCTGACCCAG CTCCAGCCCT TCCTGAAGCT CAAGCAGAAG
1021 CAGGCCAACC TCGTGCTGAA GATCATCGAG CAGCTGCCCT CCGCCAAGGA ATCCCCGGAC
1081 AAGTTCCTGG AGGTGTGCAC CTGGGTGGAC CAGATCGCCG CTCTGAACGA CTCCAAGACC
1141 CGCAAGACCA CTTCCGAGAC CGTCCGCGCC GTTCTAGACA GTCTCTCCGA GAAGAAGAAG
1201 TCGTCCCCCT AGACAGTCTC TCCGAGAAGA AGAAGTCGTC CCCCTAGCGG CCGCTTCGAG
1261 CAGACATGAT AAGATACATT GATGAGTTTG GACAAACCAC AACTAGAATG CAGTGAAAAA
1321 AATGCTTTAT TTGTGAAATT TGTGATGCTA TTGCTTTATT TGTAACCATT ATAAGCTGCA
1381 ATAAACAAGT TAACAACAAC AATTGCATTC ATTTTATGTT TCAGGTTCAG GGGGAGATGT
1441 GGGAGGTTTT TTAAAGCAAG TAAAACCTCT ACAAATGTGG TAAAATCGAT AAGATCTTGA
1501 TCCGGGCTGG CGTAATAGCG AAGAGGCCCG CACCGATCGC CCTTCCCAAC AGTTGCGCAG
1561 CCTGAATGGC GAATGGACGC GCCCTGTAGC GGCGCATTAA GCGCGGCGGG TGTGGTGGTT
1621 ACGCGCAGCG TGACCGCTAC ACTTGCCAGC GCCCTAGCGC CCGCTCCTTT CGCTTTCTTC
1681 CCTTCCTTTC TCGCCACGTT CGCCGGCTTT CCCCGTCAAG CTCTAAATCG GGGGCTCCCT
1741 TTAGGGTTCC GATTTAGTGC TTTACGGCAC CTCGACCCCA AAAAACTTGA TTAGGGTGAT
1801 GGTTCACGTA GTGGGCCATC G
SEQ ID NO: 21 (PCR template for mRNA encoding CHO-51/52)
1 CACAGGTGTC CACTCCCAGT TCAATTACAG CTCTTAAGGC TAGAGTACTT AATACGACTC
61 ACTATAGGCT AGCCTCGAGC CGCCACCATG GCACCGAAGA AGAAGCGCAA GGTGCATatg
121 gCACCGAAGA AGAAGCGCAA GGTGCATATG AACACCAAGT ACAACAAGGA GTTCCTGCTC
181 TACCTGGCGG GCTTCGTGGA CGGGGACGGC TCCATCATCG CCCAGATCCC GCCGAACCAG
241 TCCTGCAAGT TCAAGCATCA GCTGCGCCTC ACCTTCCAGG TCACGCAGAA GACACAGCGC
301 CGTTGGTTCC TCGACAAGCT GGTGGACGAG ATCGGGGTGG GCTACGTGCG CGACCGCGGC
361 AGCGTCTCCG ACTACATCCT GAGCGAGATC AAGCCTCTGC ACAACTTCCT GACCCAGCTC
421 CAGCCCTTCC TGAAGCTCAA GCAGAAGCAG GCCAACCTCG TGCTGAAGAT CATCGAGCAG
481 CTGCCCTCCG CCAAGGAATC CCCGGACAAG TTCCTGGAGG TGTGCACCTG GGTGGACCAG
541 ATCGCCGCTC TGAACGACTC CAAGACCCGC AAGACCACTT CCGAGACTGT CCGCGCCGTT
601 CTAGACAGTC TCCCAGGATC CGTGGGAGGT CTATCGCCAT CTCAGGCATC CAGCGCCGCA
661 TCCTCGGCTT CCTCAAGCCC GGGTTCAGGG ATCTCCGAAG CACTCAGAGC TGGAGCAGGT
721 TCCGGCACTG GATACAACAA GGAATTCCTG CTCTACCTGG CGGGCTTCGT GGACGGGGAC
781 GGCTCCATCT ACGCCGGGAT CGCGCCGAAC CAGTCCTGCA AGTTCAAGCA TCAGCTGCGC
841 CTCTGGTTCG TGGTCAGCCA GAAGACACAG CGCCGTTGGT TCCTCGACAA GCTGGTGGAC
901 GAGATCGGGG TGGGCTACGT GATTGACAAT GGCAGCGTCT CCCATTACCG CCTGAGCGAG
961 ATCAAGCCTC TGCACAACTT CCTGACCCAG CTCCAGCCCT TCCTGAAGCT CAAGCAGAAG
1021 CAGGCCAACC TCGTGCTGAA GATCATCGAG CAGCTGCCCT CCGCCAAGGA ATCCCCGGAC
1081 AAGTTCCTGG AGGTGTGCAC CTGGGTGGAC CAGATCGCCG CTTTGAACGA CTCCAAGACC
1141 CGCAAGACCA CTTCCGAGAC TGTCCGCGCC GTTCTAGACA GTCTCTCCGA GAAGAAGAAG
1201 TCGTCCCCCT AGACAGTCTC TCCGAGAAGA AGAAGTCGTC CCCCTAGCGG CCGCTTCGAG
1261 CAGACATGAT AAGATACATT GATGAGTTTG GACAAACCAC AACTAGAATG CAGTGAAAAA
1321 AATGCTTTAT TTGTGAAATT TGTGATGCTA TTGCTTTATT TGTAACCATT ATAAGCTGCA
1381 ATAAACAAGT TAACAACAAC AATTGCATTC ATTTTATGTT TCAGGTTCAG GGGGAGATGT
1441 GGGAGGTTTT TTAAAGCAAG TAAAACCTCT ACAAATGTGG TAAAATCGAT AAGATCTTGA
1501 TCCGGGCTGG CGTAATAGCG AAGAGGCCCG CACCGATCGC CCTTCCCAAC AGTTGCGCAG
1561 CCTGAATGGC GAATGGACGC GCCCTGTAGC GGCGCATTAA GCGCGGCGGG TGTGGTGGTT
1621 ACGCGCAGCG TGACCGCTAC ACTTGCCAGC GCCCTAGCGC CCGCTCCTTT CGCTTTCTTC
1681 CCTTCCTTTC TCGCCACGTT CGCCGGCTTT CCCCGTCAAG CTCTAAATCG GGGGCTCCCT
1741 TTAGGGTTCC GATTTAGTGC TTTACGGCAC CTCGACCCCA AAAAACTTGA TTAGGGTGAT
1801 GGTTCACGTA GTGGGCCATC G
SEQ ID NO: 22 (PCR template for mRNA encoding CGS-5/6)
1 CACAGGTGTC CACTCCCAGT TCAATTACAG CTCTTAAGGC TAGAGTACTT AATACGACTC
61 ACTATAGGCT AGCCTCGAGC CGCCACCATG GCACCGAAGA AGAAGCGCAA GGTGCATATG
121 GCACCGAAGA AGAAGCGCAA GGTGCATATG AACACCAAGT ACAACAAGGA GTTCCTGCTC
181 TACCTGGCGG GCTTCGTCGA CGGGGACGGC TCCATCAAGG CCATTATCCG GCCAGAGCAG
241 TCCTACAAGT TCAAGCATCG CCTGCGGCTC GTTTTCCAGG TCACGCAGAA GACACAGCGC
301 CGTTGGTTCC TCGACAAGCT GGTGGACGAG ATCGGGGTGG GCTACGTGTA CGACCGCGGC
361 AGCGTCTCCG ACTACTATCT GAGCGAGATC AAGCCTCTGC ACAACTTCCT GACCCAGCTC
421 CAGCCCTTCC TGAAGCTCAA GCAGAAGCAG GCCAACCTCG TGCTGAAGAT CATCGAGCAG
481 CTGCCCTCCG CCAAGGAATC CCCGGACAAG TTCCTGGAGG TGTGCACGTG GGTGGACCAG
541 ATCGCGGCCC TCAACGACAG CAAGACCCGC AAGACGACCT CGGAGACGGT GCGAGCGGTC
601 CTGGACTCCC TCCCAGGATC CGTGGGAGGT CTATCGCCAT CTCAGGCATC CAGCGCCGCA
661 TCCTCGGCTT CCTCAAGCCC GGGTTCAGGG ATCTCCGAAG CACTCAGAGC TGGAGCAGGT
721 TCCGGCACTG GATACAACAA GGAATTCCTG CTCTACCTGG CGGGCTTCGT GGACGGGGAC
781 GGCTCCATCT GGGCCCGGAT CAAGCCGGGG CAGTCCTACA AGTTCAAGCA TACCCTGGAG
841 CTCGTGTTCC AGGTCACCCA GAAGACACAG CGCCGTTGGA TCCTCGACAA GCTGGTGGAC
901 GAGATCGGGG TGGGCTACGT GACCGACGCC GGCAGCGCCT CCGTCTACCG CCTGAGCGAG
961 ATCAAGCCTC TGCACAACTT CCTGACCCAG CTCCAGCCCT TCCTGAAGCT CAAGCAGAAG
1021 CAGGCCAACC TCGTGCTGAA GATCATCGAG CAGCTGCCCT CCGCCAAGGA ATCCCCGGAC
1081 AAGTTCCTGG AGGTGTGCAC CTGGGTGGAC CAGATCGCCG CTCTGAACGA CTCCAAGACC
1141 CGCAAGACCA CTTCCGAGAC CGTCCGCGCC GTTCTAGACA GTCTCTCCGA GAAGAAGAAG
1201 TCGTCCCCCT AGACAGTCTC TCCGAGAAGA AGAAGTCGTC CCCCTAGCGG CCGCTTCGAG
1261 CAGACATGAT AAGATACATT GATGAGTTTG GACAAACCAC AACTAGAATG CAGTGAAAAA
1321 AATGCTTTAT TTGTGAAATT TGTGATGCTA TTGCTTTATT TGTAACCATT ATAAGCTGCA
1381 ATAAACAAGT TAACAACAAC AATTGCATTC ATTTTATGTT TCAGGTTCAG GGGGAGATGT
1441 GGGAGGTTTT TTAAAGCAAG TAAAACCTCT ACAAATGTGG TAAAATCGAT AAGATCTTGA
1501 TCCGGGCTGG CGTAATAGCG AAGAGGCCCG CACCGATCGC CCTTCCCAAC AGTTGCGCAG
1561 CCTGAATGGC GAATGGACGC GCCCTGTAGC GGCGCATTAA GCGCGGCGGG TGTGGTGGTT
1621 ACGCGCAGCG TGACCGCTAC ACTTGCCAGC GCCCTAGCGC CCGCTCCTTT CGCTTTCTTC
1681 CCTTCCTTTC TCGCCACGTT CGCCGGCTTT CCCCGTCAAG CTCTAAATCG GGGGCTCCCT
1741 TTAGGGTTCC GATTTAGTGC TTTACGGCAC CTCGACCCCA AAAAACTTGA TTAGGGTGAT
1801 GGTTCACGTA GTGGGCCATC G
SEQ ID NO: 23 (Forward PCR primer for evaluating CGS-5/6 target site)
1 tgacagctct ggccttaagt gcctacgaaa ctag
SEQ ID NO: 24 (Reverse PCR primer for evaluating CGS-5/6 target site)
1 gtctttcctc tttgctgtag ccttggtaga actactgcc
SEQ ID NO: 25 (CHO-23/24 Insertion target sequence donor plasmid)
1 TCGCGCGTTT CGGTGATGAC GGTGAAAACC TCTGACACAT GCAGCTCCCG GAGACGGTCA
61 CAGCTTGTCT GTAAGCGGAT GCCGGGAGCA GACAAGCCCG TCAGGGCGCG TCAGCGGGTG
121 TTGGCGGGTG TCGGGGCTGG CTTAACTATG CGGCATCAGA GCAGATTGTA CTGAGAGTGC
181 ACCATATGCG GTGTGAAATA CCGCACAGAT GCGTAAGGAG AAAATACCGC ATCAGGCGCC
241 ATTCGCCATT CAGGCTGCGC AACTGTTGGG AAGGGCGATC GGTGCGGGCC TCTTCGCTAT
301 TACGCCAGCT GGCGAAAGGG GGATGTGCTG CAAGGCGATT AAGTTGGGTA ACGCCAGGGT
361 TTTCCCAGTC ACGACGTTGT AAAACGACGG CCAGTGAATT CCATACCCAG GGGAGCTGTA
421 CTGGGCTGCA GCCCTGCGCC ATTCAGCCAT GCACCAGGCT ACTCCCTCCT CTTCCAGCTT
481 TCTCCTTCTG ATGGCCATAG GATTAGAAGA TAAGGGACTC TAGTGCAGGT CAACTGCTGA
541 CCAGTGTGAA AATGCACAGA CTACATGCTG GTAGATCAGC ACTTCAAACT ACTGTTCACC
601 ATCATCTCTG GAATAAGCAC TACATTTACA GGGTTCAAAC CTCAATGAAT ATAAACAAAC
661 AAAACACACC TCCCTTCCTT CACTGTCTCC CATTTCTTTG GTTCCCATCT CCACATAGAA
721 TTTATAATTA AAATTTCTAA GTATCTTTCC AGAAATACTT CACACATGTT ATAAGCAAAT
781 GTGCTTTTAA AGATACTATT TTAAATTATG AAAATGGTTA TATTAGTTGA GATAAAAGAA
841 TAGAATGGGA AGTTCCAGAA TTTAAGGCCT CATATGGATC CCAGCTGTGG AATGTGTGTC
901 AGTTAGGGTG TGGAAAGTCC CCAGGCTCCC CAGCAGGCAG AAGTATGCAA AGCATGCATC
961 TCAATTAGTC AGCAACCAGG TGTGGAAAGT CCCCAGGCTC CCCAGCAGGC AGAAGTATGC
1021 AAAGCATGCA TCTCAATTAG TCAGCAACCA TAGTCCCGCC CCTAACTCCG CCCATCCCGC
1081 CCCTAACTCC GCCCAGTTCC GCCCATTCTC CGCCCCATGG CTGACTAATT TTTTTTATTT
1141 ATGCAGAGGC CGAGGCCGCC TCGGCCTCTG AGCTATTCCA GAAGTAGTGA GGAGGCTTTT
1201 TTGGAGGCTA CCATGGAGAA GTTACTATTC CGAAGTTCCT ATTCTCTAGA AAGTATAGGA
1261 ACTTCAAGCT TGGCACTGGG TACCGCCAAG TTGACCAGTG CCGTTCCGGT GCTCACCGCG
1321 CGCGACGTCG CCGGAGCGGT CGAGTTCTGG ACCGACCGGC TCGGGTTCTC CCGGGACTTC
1381 GTGGAGGACG ACTTCGCCGG TGTGGTCCGG GACGACGTGA CCCTGTTCAT CAGCGCGGTC
1441 CAGGACCAGG TGGTGCCGGA CAACACCCTG GCCTGGGTGT GGGTGCGCGG CCTGGACGAG
1501 CTGTACGCCG AGTGGTCGGA GGTCGTGTCC ACGAACTTCC GGGACGCCTC CGGGCCGGCC
1561 ATGACCGAGA TCGGCGAGCA GCCGTGGGGG CGGGAGTTCG CCCTGCGCGA CCCGGCCGGC
1621 AACTGCGTGC ACTTCGTGGC CGAGGAGCAG GACTGACACC CGAGCGAAAA CGGTCTGCGC
1681 TGCGGGACGC GCGAATTGAA TTATGGCCCA CACCAGTGGC GCGGCGACTT CCAGTTCAAC
1741 ATCAGCCGCT ACAGTCAACA GCAACTGATG GAAACCAGCC ATCGCCATCT GCTGCACGCG
1801 GAAGAAGGCA CATGGCTGAA TATCGACGGT TTCCATATGG GGATTGGTGG CGACGACTCC
1861 TGGAGCCCGT CAGTATCGGC GGAATTCCAG CTGAGCGCCG GTCGCTACCA TTACCAGTTG
1921 GTCTGGTGTC AAAAATAATA ATAACCGGGC AGGGGGGATC TGCATGGATC TTTGTGAAGG
1981 AACCTTACTT CTGTGGTGTG ACATAATTGG ACAAACTACC TACAGAGATT TAAAGCTCTA
2041 AGGTAAATAT AAAATTTTTA AGTGTATAAT GTGTTAAACT ACTGATTCTA ATTGTTTGTG
2101 TATTTTAGAT TCCAACCTAT GGAACTGATG AATGGGAGCA GTGGTGGAAT GCCTTTAATG
2161 AGGAAAACCT GTTTTGCTCA GAAGAAATGC CATCTAGTGA TGATGAGGCT ACTGCTGACT
2221 CTCAACATTC TACTCCTCCA AAAAAGAAGA GAAAGGTAGA AGACCCCAAG GACTTTCCTT
2281 CAGAATTGCT AAGTTTTTTG AGTCATGCTG TGTTTAGTAA TAGAACTCTT GCTTGCTTTG
2341 CTATTTACAC CACAAAGGAA AAAGCTGCAC TGCTATACAA GAAAATTATG GAAAAATATT
2401 CTGTAACCTT TATAAGTAGG CATAACAGTT ATAATCATAA CATACTGTTT TTTCTTACTC
2461 CACACAGGCA TAGAGTGTCT GCTATTAATA ACTATGCTCA AAAATTGTGT ACCTTTAGCT
2521 TTTTAATTTG TAAAGGGGTT AATAAGGAAT ATTTGATGTA TAGTGCCTTG ACTAGAGATC
2581 ATAATCAGCC ATACCACATT TGTAGAGGTT TTACTTGCTT TAAAAAACCT CCCACACCTC
2641 CCCCTGAACC TGAAACATAA AATGAATGCA ATTGTTGTTG TTAACTTGTT TATTGCAGCT
2701 TATAATGGTT ACAAATAAAG CAATAGCATC ACAAATTTCA CAAATAAAGC ATTTTTTTCA
2761 CTGCATTCTA GTTGTGGTTT GTCCAAACTC ATCAATGTAT CTTATCATGT CTGGATCCCC
2821 AGGAAGCTCC TCTGTGTCCT CATAAACCCT AACCTCCTCT ACTTGAGAGG ACATTCCAAT
2881 CATAGGCTGC CCATCCACCC TACTAGTATA TGAAAATATA AAGCGCTTTC TCTTTTAAGT
2941 CTAGGGTAGG TGTACTAGAT CAGCGCTCAG CTCCATACCA TGAAGCCATC CAGGAGTCAG
3001 ACCTCTCTGA CAGCCCTGCC ATTGTCACAG AGAAGTTTCT GTCACCAGTG CTCATGCTGT
3061 CAGAGGAGCG AAGGAGAAAA GATGTGAGAC CTCCCAAGTC AAAGTCATCT ATGGATAAAA
3121 CCTTAGTTGC ATGGCACACC AGTGTTAGGG AGTCGGGGAA ACACAGCCAT AGCCCAGCTT
3181 CCTCTCTGTT CTTGCTCTTA TTACCACCAG AAAGAGGTTG CTTAGACAAC CCAAACCAAG
3241 ACACAGGGCT CTGTGGGAGG GAATCAGTCC CAGGCTTCTG GCACATGCTA TGTCACCGGA
3301 AAGCCCCAGC CCTACTCCGA ATCCCCACAA GTACAGCAAA TATCAGATTA TAGCATTTAA
3361 AGGGGCACTC TTGCCAAAGA GAAGCACCAT TGGAATAGCC ATGCTTGAGA ACTAAGCTTG
3421 GCGTAATCAT GGTCATAGCT GTTTCCTGTG TGAAATTGTT ATCCGCTCAC AATTCCACAC
3481 AACATACGAG CCGGAAGCAT AAAGTGTAAA GCCTGGGGTG CCTAATGAGT GAGCTAACTC
3541 ACATTAATTG CGTTGCGCTC ACTGCCCGCT TTCCAGTCGG GAAACCTGTC GTGCCAGCTG
3601 CATTAATGAA TCGGCCAACG CGCGGGGAGA GGCGGTTTGC GTATTGGGCG CTCTTCCGCT
3661 TCCTCGCTCA CTGACTCGCT GCGCTCGGTC GTTCGGCTGC GGCGAGCGGT ATCAGCTCAC
3721 TCAAAGGCGG TAATACGGTT ATCCACAGAA TCAGGGGATA ACGCAGGAAA GAACATGTGA
3781 GCAAAAGGCC AGCAAAAGGC CAGGAACCGT AAAAAGGCCG CGTTGCTGGC GTTTTTCCAT
3841 AGGCTCCGCC CCCCTGACGA GCATCACAAA AATCGACGCT CAAGTCAGAG GTGGCGAAAC
3901 CCGACAGGAC TATAAAGATA CCAGGCGTTT CCCCCTGGAA GCTCCCTCGT GCGCTCTCCT
3961 GTTCCGACCC TGCCGCTTAC CGGATACCTG TCCGCCTTTC TCCCTTCGGG AAGCGTGGCG
4021 CTTTCTCATA GCTCACGCTG TAGGTATCTC AGTTCGGTGT AGGTCGTTCG CTCCAAGCTG
4081 GGCTGTGTGC ACGAACCCCC CGTTCAGCCC GACCGCTGCG CCTTATCCGG TAACTATCGT
4141 CTTGAGTCCA ACCCGGTAAG ACACGACTTA TCGCCACTGG CAGCAGCCAC TGGTAACAGG
4201 ATTAGCAGAG CGAGGTATGT AGGCGGTGCT ACAGAGTTCT TGAAGTGGTG GCCTAACTAC
4261 GGCTACACTA GAAGAACAGT ATTTGGTATC TGCGCTCTGC TGAAGCCAGT TACCTTCGGA
4321 AAAAGAGTTG GTAGCTCTTG ATCCGGCAAA CAAACCACCG CTGGTAGCGG TGGTTTTTTT
4381 GTTTGCAAGC AGCAGATTAC GCGCAGAAAA AAAGGATCTC AAGAAGATCC TTTGATCTTT
4441 TCTACGGGGT CTGACGCTCA GTGGAACGAA AACTCACGTT AAGGGATTTT GGTCATGAGA
4501 TTATCAAAAA GGATCTTCAC CTAGATCCTT TTAAATTAAA AATGAAGTTT TAAATCAATC
4561 TAAAGTATAT ATGAGTAAAC TTGGTCTGAC AGTTACCAAT GCTTAATCAG TGAGGCACCT
4621 ATCTCAGCGA TCTGTCTATT TCGTTCATCC ATAGTTGCCT GACTCCCCGT CGTGTAGATA
4681 ACTACGATAC GGGAGGGCTT ACCATCTGGC CCCAGTGCTG CAATGATACC GCGAGACCCA
4741 CGCTCACCGG CTCCAGATTT ATCAGCAATA AACCAGCCAG CCGGAAGGGC CGAGCGCAGA
4801 AGTGGTCCTG CAACTTTATC CGCCTCCATC CAGTCTATTA ATTGTTGCCG GGAAGCTAGA
4861 GTAAGTAGTT CGCCAGTTAA TAGTTTGCGC AACGTTGTTG CCATTGCTAC AGGCATCGTG
4921 GTGTCACGCT CGTCGTTTGG TATGGCTTCA TTCAGCTCCG GTTCCCAACG ATCAAGGCGA
4981 GTTACATGAT CCCCCATGTT GTGCAAAAAA GCGGTTAGCT CCTTCGGTCC TCCGATCGTT
5041 GTCAGAAGTA AGTTGGCCGC AGTGTTATCA CTCATGGTTA TGGCAGCACT GCATAATTCT
5101 CTTACTGTCA TGCCATCCGT AAGATGCTTT TCTGTGACTG GTGAGTACTC AACCAAGTCA
5161 TTCTGAGAAT AGTGTATGCG GCGACCGAGT TGCTCTTGCC CGGCGTCAAT ACGGGATAAT
5221 ACCGCGCCAC ATAGCAGAAC TTTAAAAGTG CTCATCATTG GAAAACGTTC TTCGGGGCGA
5281 AAACTCTCAA GGATCTTACC GCTGTTGAGA TCCAGTTCGA TGTAACCCAC TCGTGCACCC
5341 AACTGATCTT CAGCATCTTT TACTTTCACC AGCGTTTCTG GGTGAGCAAA AACAGGAAGG
5401 CAAAATGCCG CAAAAAAGGG AATAAGGGCG ACACGGAAAT GTTGAATACT CATACTCTTC
5461 CTTTTTCAAT ATTATTGAAG CATTTATCAG GGTTATTGTC TCATGAGCGG ATACATATTT
5521 GAATGTATTT AGAAAAATAA ACAAATAGGG GTTCCGCGCA CATTTCCCCG AAAAGTGCCA
5581 CCTGACGTCT AAGAAACCAT TATTATCATG ACATTAACCT ATAAAAATAG GCGTATCACG
5641 AGGCCCTTTC GTC
SEQ ID NO: 26 (reverse PCR primer in the SV40 early promoter)
1 AGATGCATGC TTTGCATACT TCTGCCTGC
SEQ ID NO: 27 (donor plasmid for inserting GFP into FRT Insertion target
sequence)
1 GACGGATCGG GAGATCTCCC GATCCCCTAT GGTGCACTCT CAGTACAATC TGCTCTGATG
61 CCGCATAGTT AAGCCAGTAT CTGCTCCCTG CTTGTGTGTT GGAGGTCGCT GAGTAGTGCG
121 CGAGCAAAAT TTAAGCTACA ACAAGGCAAG GCTTGACCGA CAATTGCATG AAGAATCTGC
181 TTAGGGTTAG GCGTTTTGCG CTGCTTCGCG ATGTACGGGC CAGATATACG CGTTGACATT
241 GATTATTGAC TAGTTATTAA TAGTAATCAA TTACGGGGTC ATTAGTTCAT AGCCCATATA
301 TGGAGTTCCG CGTTACATAA CTTACGGTAA ATGGCCCGCC TGGCTGACCG CCCAACGACC
361 CCCGCCCATT GACGTCAATA ATGACGTATG TTCCCATAGT AACGCCAATA GGGACTTTCC
421 ATTGACGTCA ATGGGTGGAG TATTTACGGT AAACTGCCCA CTTGGCAGTA CATCAAGTGT
481 ATCATATGCC AAGTACGCCC CCTATTGACG TCAATGACGG TAAATGGCCC GCCTGGCATT
541 ATGCCCAGTA CATGACCTTA TGGGACTTTC CTACTTGGCA GTACATCTAC GTATTAGTCA
601 TCGCTATTAC CATGGTGATG CGGTTTTGGC AGTACATCAA TGGGCGTGGA TAGCGGTTTG
661 ACTCACGGGG ATTTCCAAGT CTCCACCCCA TTGACGTCAA TGGGAGTTTG TTTTGGCACC
721 AAAATCAACG GGACTTTCCA AAATGTCGTA ACAACTCCGC CCCATTGACG CAAATGGGCG
781 GTAGGCGTGT ACGGTGGGAG GTCTATATAA GCAGAGCTCT CTGGCTAACT AGAGAACCCA
841 CTGCTTACTG GCTTATCGAA ATTAATACGA CTCACTATAG GGAGACCCAA GCTGGCTAGC
901 GTTTAAACTT AAGCTTAGCC ACCaTGGTGA GCAAGGGCGA GGAGCTGTTC ACCGGGGTGG
961 TGCCCATCCT GGTCGAGCTG GACGGCGACG TAAACGGCCA CAAGTTCAGC GTGTCCGGCG
1021 AGGGCGAGGG CGATGCCACC TACGGCAAGC TGACCCTGAA GTTCATCTGC ACCACCGGCA
1081 AGCTGCCCGT GCCCTGGCCC ACCCTCGTGA CCACCCTGAC CTACGGAGTG CAGTGCTTCA
1141 GCCGCTACCC CGACCACATG AAGCAGCACG ACTTCTTCAA GTCCGCCATG CCCGAAGGCT
1201 ACGTCCAGGA GCGCACCATC TTCTTCAAGG ACGACGGCAA CTACAAGACC CGCGCCGAGG
1261 TGAAGTTCGA GGGCGACACC CTGGTGAACC GCATCGAGCT GAAGGGCATC GACTTCAAGG
1321 AGGACGGCAA CATCCTGGGG CACAAGCTGG AGTACAACTA CAACAGCCAC AACGTCTATA
1381 TCATGGCCGA CAAGCAGAAG AACGGCATCA AGGTGAACTT CAAGATCCGC CACAACATCG
1441 AGGACGGCAG CGTGCAGCTC GCCGACCACT ACCAGCAGAA CACCCCCATC GGCGACGGCC
1501 CCGTGCTGCT GCCCGACAAC CACTACCTGA GCACCCAGTC CGCCCTGAGC AAAGACCCCA
1561 ACGAGAAGCG CGATCACATG GTCCTGCTGG AGTTCGTGAC CGCCGCCGGG ATCACTCTCG
1621 GCATGGACGA GCTGTACAAG TAAGGATCCA CTAGTCCAGT GTGGTGGAAT TCTGCAGATA
1681 TCCAGCACAG TGGCGGCCGC TCGAGTCTAG AGGGCCCGTT TAAACCCGCT GATCAGCCTC
1741 GACTGTGCCT TCTAGTTGCC AGCCATCTGT TGTTTGCCCC TCCCCCGTGC CTTCCTTGAC
1801 CCTGGAAGGT GCCACTCCCA CTGTCCTTTC CTAATAAAAT GAGGAAATTG CATCGCATTG
1861 TCTGAGTAGG TGTCATTCTA TTCTGGGGGG TGGGGTGGGG CAGGACAGCA AGGGGGAGGA
1921 TTGGGAAGAC AATAGCAGGC ATGCTGGGGA TGCGGTGGGC TCTATGGCTT CTGAGGCGGA
1981 AAGAACCAGC TGGGGCTCTA GGGGGTATCC CCACGCGCCC TGTAGCGGCG CATTAAGCGC
2041 GGCGGGTGTG GTGGTTACGC GCAGCGTGAC CGCTACACTT GCCAGCGCCC TAGCGCCCGC
2101 TCCTTTCGCT TTCTTCCCTT CCTTTCTCGC CACGTTCGCC GGCTTTCCCC GTCAAGCTCT
2161 AAATCGGGGG CTCCCTTTAG GGTTCCGATT TAGTGCTTTA CGGCACCTCG ACCCCAAAAA
2221 ACTTGATTAG GGTGATGGTT CACGTACCTA GAAGTTCCTA TTCCGAAGTT CCTATTCTCT
2281 AGAAAGTATA GGAACTTCCT TGGCCAAAAA GCCTGAACTC ACCGCGACGT CTGTCGAGAA
2341 GTTTCTGATC GAAAAGTTCG ACAGCGTCTC CGACCTGATG CAGCTCTCGG AGGGCGAAGA
2401 ATCTCGTGCT TTCAGCTTCG ATGTAGGAGG GCGTGGATAT GTCCTGCGGG TAAATAGCTG
2461 CGCCGATGGT TTCTACAAAG ATCGTTATGT TTATCGGCAC TTTGCATCGG CCGCGCTCCC
2521 GATTCCGGAA GTGCTTGACA TTGGGGAATT CAGCGAGAGC CTGACCTATT GCATCTCCCG
2581 CCGTGCACAG GGTGTCACGT TGCAAGACCT GCCTGAAACC GAACTGCCCG CTGTTCTGCA
2641 GCCGGTCGCG GAGGCCATGG ATGCGATCGC TGCGGCCGAT CTTAGCCAGA CGAGCGGGTT
2701 CGGCCCATTC GGACCGCAAG GAATCGGTCA ATACACTACA TGGCGTGATT TCATATGCGC
2761 GATTGCTGAT CCCCATGTGT ATCACTGGCA AACTGTGATG GACGACACCG TCAGTGCGTC
2821 CGTCGCGCAG GCTCTCGATG AGCTGATGCT TTGGGCCGAG GACTGCCCCG AAGTCCGGCA
2881 CCTCGTGCAC GCGGATTTCG GCTCCAACAA TGTCCTGACG GACAATGGCC GCATAACAGC
2941 GGTCATTGAC TGGAGCGAGG CGATGTTCGG GGATTCCCAA TACGAGGTCG CCAACATCTT
3001 CTTCTGGAGG CCGTGGTTGG CTTGTATGGA GCAGCAGACG CGCTACTTCG AGCGGAGGCA
3061 TCCGGAGCTT GCAGGATCGC CGCGGCTCCG GGCGTATATG CTCCGCATTG GTCTTGACCA
3121 ACTCTATCAG AGCTTGGTTG ACGGCAATTT CGATGATGCA GCTTGGGCGC AGGGTCGATG
3181 CGACGCAATC GTCCGATCCG GAGCCGGGAC TGTCGGGCGT ACACAAATCG CCCGCAGAAG
3241 CGCGGCCGTC TGGACCGATG GCTGTGTAGA AGTACTCGCC GATAGTGGAA ACCGACGCCC
3301 CAGCACTCGT CCGAGGGCAA AGGAATAGCA CGTACTACGA GATTTCGATT CCACCGCCGC
3361 CTTCTATGAA AGGTTGGGCT TCGGAATCGT TTTCCGGGAC GCCGGCTGGA TGATCCTCCA
3421 GCGCGGGGAT CTCATGCTGG AGTTCTTCGC CCACCCCAAC TTGTTTATTG CAGCTTATAA
3481 TGGTTACAAA TAAAGCAATA GCATCACAAA TTTCACAAAT AAAGCATTTT TTTCACTGCA
3541 TTCTAGTTGT GGTTTGTCCA AACTCATCAA TGTATCTTAT CATGTCTGTA TACCGTCGAC
3601 CTCTAGCTAG AGCTTGGCGT AATCATGGTC ATAGCTGTTT CCTGTGTGAA ATTGTTATCC
3661 GCTCACAATT CCACACAACA TACGAGCCGG AAGCATAAAG TGTAAAGCCT GGGGTGCCTA
3721 ATGAGTGAGC TAACTCACAT TAATTGCGTT GCGCTCACTG CCCGCTTTCC AGTCGGGAAA
3781 CCTGTCGTGC CAGCTGCATT AATGAATCGG CCAACGCGCG GGGAGAGGCG GTTTGCGTAT
3841 TGGGCGCTCT TCCGCTTCCT CGCTCACTGA CTCGCTGCGC TCGGTCGTTC GGCTGCGGCG
3901 AGCGGTATCA GCTCACTCAA AGGCGGTAAT ACGGTTATCC ACAGAATCAG GGGATAACGC
3961 AGGAAAGAAC ATGTGAGCAA AAGGCCAGCA AAAGGCCAGG AACCGTAAAA AGGCCGCGTT
4021 GCTGGCGTTT TTCCATAGGC TCCGCCCCCC TGACGAGCAT CACAAAAATC GACGCTCAAG
4081 TCAGAGGTGG CGAAACCCGA CAGGACTATA AAGATACCAG GCGTTTCCCC CTGGAAGCTC
4141 CCTCGTGCGC TCTCCTGTTC CGACCCTGCC GCTTACCGGA TACCTGTCCG CCTTTCTCCC
4201 TTCGGGAAGC GTGGCGCTTT CTCATAGCTC ACGCTGTAGG TATCTCAGTT CGGTGTAGGT
4261 CGTTCGCTCC AAGCTGGGCT GTGTGCACGA ACCCCCCGTT CAGCCCGACC GCTGCGCCTT
4321 ATCCGGTAAC TATCGTCTTG AGTCCAACCC GGTAAGACAC GACTTATCGC CACTGGCAGC
4381 AGCCACTGGT AACAGGATTA GCAGAGCGAG GTATGTAGGC GGTGCTACAG AGTTCTTGAA
4441 GTGGTGGCCT AACTACGGCT ACACTAGAAG GACAGTATTT GGTATCTGCG CTCTGCTGAA
4501 GCCAGTTACC TTCGGAAAAA GAGTTGGTAG CTCTTGATCC GGCAAACAAA CCACCGCTGG
4561 TAGCGGTGGT TTTTTTGTTT GCAAGCAGCA GATTACGCGC AGAAAAAAAG GATCTCAAGA
4621 AGATCCTTTG ATCTTTTCTA CGGGGTCTGA CGCTCAGTGG AACGAAAACT CACGTTAAGG
4681 GATTTTGGTC ATGAGATTAT CAAAAAGGAT CTTCACCTAG ATCCTTTTAA ATTAAAAATG
4741 AAGTTTTAAA TCAATCTAAA GTATATATGA GTAAACTTGG TCTGACAGTT ACCAATGCTT
4801 AATCAGTGAG GCACCTATCT CAGCGATCTG TCTATTTCGT TCATCCATAG TTGCCTGACT
4861 CCCCGTCGTG TAGATAACTA CGATACGGGA GGGCTTACCA TCTGGCCCCA GTGCTGCAAT
4921 GATACCGCGA GACCCACGCT CACCGGCTCC AGATTTATCA GCAATAAACC AGCCAGCCGG
4981 AAGGGCCGAG CGCAGAAGTG GTCCTGCAAC TTTATCCGCC TCCATCCAGT CTATTAATTG
5041 TTGCCGGGAA GCTAGAGTAA GTAGTTCGCC AGTTAATAGT TTGCGCAACG TTGTTGCCAT
5101 TGCTACAGGC ATCGTGGTGT CACGCTCGTC GTTTGGTATG GCTTCATTCA GCTCCGGTTC
5161 CCAACGATCA AGGCGAGTTA CATGATCCCC CATGTTGTGC AAAAAAGCGG TTAGCTCCTT
5221 CGGTCCTCCG ATCGTTGTCA GAAGTAAGTT GGCCGCAGTG TTATCACTCA TGGTTATGGC
5281 AGCACTGCAT AATTCTCTTA CTGTCATGCC ATCCGTAAGA TGCTTTTCTG TGACTGGTGA
5341 GTACTCAACC AAGTCATTCT GAGAATAGTG TATGCGGCGA CCGAGTTGCT CTTGCCCGGC
5401 GTCAATACGG GATAATACCG CGCCACATAG CAGAACTTTA AAAGTGCTCA TCATTGGAAA
5461 ACGTTCTTCG GGGCGAAAAC TCTCAAGGAT CTTACCGCTG TTGAGATCCA GTTCGATGTA
5521 ACCCACTCGT GCACCCAACT GATCTTCAGC ATCTTTTACT TTCACCAGCG TTTCTGGGTG
5581 AGCAAAAACA GGAAGGCAAA ATGCCGCAAA AAAGGGAATA AGGGCGACAC GGAAATGTTG
5641 AATACTCATA CTCTTCCTTT TTCAATATTA TTGAAGCATT TATCAGGGTT ATTGTCTCAT
5701 GAGCGGATAC ATATTTGAAT GTATTTAGAA AAATAAACAA ATAGGGGTTC CGCGCACATT
5761 TCCCCGAAAA GTGCCACCTG ACGTC
SEQ ID NO: 28 (reverse PCR primer in the hygromycin-resistance gene)
1 CAGAAACTTC TCGACAGACG TCGCGGTGAG
SEQ ID NO: 29 (CHOX-45/46 amino acid sequence)
1 MAPKKKRKVH MNTKYNKEFL LYLAGFVDGD GSICASIRPE QERKFKHRLV LRFEVTQKTQ
61 RRWFLDKLVD EIGVGYVYDS GSVSRYYLSQ IKPLHNFLTQ LQPFLKLKQK QANLVLKIIE
121 QLPSAKESPD KFLEVCTWVD QIAALNDSKT RKTTSETVRA VLDSLPGSVG GLSPSQASSA
181 ASSASSSPGS GISEALRAGA GSGTGYNKEF LLYLAGFVDG DGSIFATICP RQQYKFKHQL
241 RLRFEVDQKT QRRWFLDKLV DEIGVGYVYD LGSVSRYGLS EIKPLHNFLT QLQPFLKLKQ
301 KQANLVLKII EQLPSAKESP DKFLEVCTWV DQIAALNDSK TRKTTSETVR AVLDSLSEKK
361 KSSP
SEQ ID NO: 30 (CHOX-45/46 recognition site sequence)
1 CAGCACGTCT CACCCCACCC CT
SEQ ID NO: 31 (CHOX-45/46 forward screening primer)
1 GGAATCTGAC TGTGGTAAGC CTGTACAC
SEQ ID NO: 32 (CHOX-45/46 reverse screening primer)
1 CAGCACTCAG GAGGTAGAGG CAGG
SEQ ID NO: 33 (artificial splice acceptor)
1 TCTTACTGAC ATCCACTTTG CCTTTCTCTC CACAGG
SEQ ID NO: 34 (SV40 polyadenylation signal)
1 ACTTGTTTAT TGCAGCTTAT AATGGTTACA AATAAAGCAA TAGCATCACA AATTTCACAA
61 ATAAAGCATT TTTTTCACTG CATTCTAGTT GTGGTTTGTC CAAACTCATC AATGTATCTT
121 ATCATGTCTG
SEQ ID NO: 35 (BGH polyadenylation signal)
1 CTGTGCCTTC TAGTTGCCAG CCATCTGTTG TTTGCCCCTC CCCCGTGCCT TCCTTGACCC
61 TGGAAGGTGC CACTCCCACT GTCCTTTCCT AATAAAATGA GGAAATTGCA TCGCATTGTC
121 TGAGTAGGTG TCATTCTATT CTGGGGGGTG GGGTGGGGCA GGACAGCAAG GGGGAGGATT
181 GGGAAGACAA TAGCAGGCAT GCTGGGGATG CGGTGGGCTC TATGG
