FIELD OF THE INVENTION The present invention provides ungulate animals, tissue and organs as well as cells and cell lines derived from such animals, tissue and organs, which lack expression of functional endogenous immunoglobulin loci. The present invention also provides ungulate animals, tissue and organs as well as cells and cell lines derived from such animals, tissue and organs, which express xenogenous, such as human, immunoglobulin loci. The present invention further provides ungulate, such as porcine genomic DNA sequence of porcine heavy and light chain immunogobulins. Such animals, tissues, organs and cells can be used in research and medical therapy. In addition, methods are provided to prepare such animals, organs, tissues, and cells.
BACKGROUND OF THE INVENTION An antigen is an agent or substance that can be recognized by the body as ‘foreign’. Often it is only one relatively small chemical group of a larger foreign substance which acts as the antigen, for example a component of the cell wall of a bacterium. Most antigens are proteins, though carbohydrates can act as weak antigens. Bacteria, viruses and other microorganisms commonly contain many antigens, as do pollens, dust mites, molds, foods, and other substances. The body reacts to antigens by making antibodies. Antibodies (also called immunoglobulins (Igs)) are proteins that are manufactured by cells of the immune system that bind to an antigen or foreign protein. Antibodies circulate in the serum of blood to detect foreign antigens and constitute the gamma globulin part of the blood proteins. These antibodies interact chemically with the antigen in a highly specific manner, like two pieces of a jigsaw puzzle, forming an antigen/antibody complex, or immune complex. This binding neutralizes or brings about the destruction of the antigen.
When a vertebrate first encounters an antigen, it exhibits a primary humoral immune response. If the animal encounters the same antigen after a few days the immune response is more rapid and has a greater magnitude. The initial encounter causes specific immune cell (B-cell) clones to proliferate and differentiate. The progeny lymphocytes include not only effector cells (antibody producing cells) but also clones of memory cells, which retain the capacity to produce both effector and memory cells upon subsequent stimulation by the original antigen. The effector cells live for only a few days. The memory cells live for a lifetime and can be reactivated by a second stimulation with the same antigen. Thus, when an antigen is encountered a second time, its memory cells quickly produce effector cells which rapidly produce massive quantities of antibodies.
By exploiting the unique ability of antibodies to interact with antigens in a highly specific manner, antibodies have been developed as molecules that can be manufactured and used for both diagnostic and therapeutic applications. Because of their unique ability to bind to antigenic epitopes, polyclonal and monoclonal antibodies can be used to identify molecules carrying that epitope or can be directed, by themselves or in conjunction with another moiety, to a specific site for diagnosis or therapy. Polyclonal and monoclonal antibodies can be generated against practically any pathogen or biological target. The term polyclonal antibody refers to immune sera that usually contain pathogen-specific antibodies of various isotypes and specificities. In contrast, monoclonal antibodies consist of a single immunoglobulin type, representing one isotype with one specificity.
In 1890, Shibasaburo Kitazato and Emil Behring conducted the fundamental experiment that demonstrated immunity can be transmitted from one animal to another by transferring the serum from an immune animal to a non-immune animal. This landmark experiment laid the foundation for the introduction of passive immunization into clinical practice. However, wide scale serum therapy was largely abandoned in the 1940s because of the toxicity associated with the administration of heterologous sera and the introduction of effective antimicrobial chemotherapy. Currently, such polyclonal antibody therapy is indicated to treat infectious diseases in relatively few situations, such as replacement therapy in immunoglobulin-deficient patients, post-exposure prophylaxis against several viruses (e.g., rabies, measles, hepatitis A and B, varicella), and toxin neutralization (diphtheria, tetanus, and botulism). Despite the limited use of serum therapy, in the United States, more than 16 metric tons of human antibodies are required each year for intravenous antibody therapy. Comparable levels of use exist in the economies of most highly industrialized countries, and the demand can be expected to grow rapidly in developing countries. Currently, human antibody for passive immunization is obtained from the pooled serum of donors. Thus, there is an inherent limitation in the amount of human antibody available for therapeutic and prophylactic therapies.
The use of antibodies for passive immunization against biological warfare agents represents a very promising defense strategy. The final line of defense against such agents is the immune system of the exposed individual. Current defense strategies against biological weapons include such measures as enhanced epidemiologic surveillance, vaccination, and use of antimicrobial agents. Since the potential threat of biological warfare and bioterrorism is inversely proportional to the number of immune persons in the targeted population, biological agents are potential weapons only against populations with a substantial proportion of susceptible persons.
Vaccination can reduce the susceptibility of a population against specific threats; provided that a safe vaccine exists that can induce a protective response. Unfortunately, inducing a protective response by vaccination may take longer than the time between exposure and onset of disease. Moreover, many vaccines require multiple doses to achieve a protective immune response, which would limit their usefulness in an emergency to provide rapid prophylaxis after an attack. In addition, not all vaccine recipients mount a protective response, even after receiving the recommended immunization schedule.
Drugs can provide protection when administered after exposure to certain agents, but none are available against many potential agents of biological warfare. Currently, no small-molecule drugs are available that prevent disease following exposure to preformed toxins. The only currently available intervention that could provide a state of immediate immunity is passive immunization with protective antibody (Arturo Casadevall “Passive Antibody Administration (Immediate Immunity) as a Specific Defense Against Biological Weapons” from Emerging Infectious Diseases, Posted Sep. 12, 2002).
In addition to providing protective immunity, modern antibody-based therapies constitute a potentially useful option against newly emergent pathogenic bacteria, fungi, virus and parasites (A. Casadevall and M. D. Scharff, Clinical Infectious Diseases 1995; 150). Therapies of patients with malignancies and cancer (C. Botti et al, Leukemia 1997; Suppl 2:S55-59; B. Bodey, S. E. Siegel, and H. E. Kaiser, Anticancer Res 1996; 16(2):661), therapy of steroid resistant rejection of transplanted organs as well as autoimmune diseases can also be achieved through the use of monoclonal or polyclonal antibody preparations (N. Bonnefoy-Berard and J. P. Revillard, J Heart Lung Transplant 1996; 15(5):435-442; C. Colby, et al Ann Pharmacother 1996; 30(10):1164-1174; M. J. Dugan, et al, Ann Hematol 1997; 75(1-2):41 2; W. Cendrowski, Boll Ist Sieroter Milan 1997; 58(4):339-343; L. K. Kastrukoff, et al Can J Neurol Sci 1978; 5(2):175178; J. E. Walker et al J Neurol Sci 1976; 29(2-4):303309).
Recent advances in the technology of antibody production provide the means to generate human antibody reagents, while avoiding the toxicities associated with human serum therapy. The advantages of antibody-based therapies include versatility, low toxicity, pathogen specificity, enhancement of immune function, and favorable pharmacokinetics.
The clinical use of monoclonal antibody therapeutics has just recently emerged. Monoclonal antibodies have now been approved as therapies in transplantation, cancer, infectious disease, cardiovascular disease and inflammation. In many more monoclonal antibodies are in late stage clinical trials to treat a broad range of disease indications. As a result, monoclonal antibodies represent one of the largest classes of drugs currently in development.
Despite the recent popularity of monoclonal antibodies as therapeutics, there are some obstacles for their use. For example, many therapeutic applications for monoclonal antibodies require repeated administrations, especially for chronic diseases such as autoimmunity or cancer. Because mice are convenient for immunization and recognize most human antigens as foreign, monoclonal antibodies against human targets with therapeutic potential have typically been of murine origin. However, murine monoclonal antibodies have inherent disadvantages as human therapeutics. For example, they require more frequent dosing to maintain a therapeutic level of monoclonal antibodies because of a shorter circulating half-life in humans than human antibodies. More critically, repeated administration of murine immunoglobulin creates the likelihood that the human immune system will recognize the mouse protein as foreign, generating a human anti-mouse antibody response, which can cause a severe allergic reaction. This possibility of reduced efficacy and safety has lead to the development of a number of technologies for reducing the immunogenicity of murine monoclonal antibodies.
Polyclonal antibodies are highly potent against multiple antigenic targets. They have the unique ability to target and kill a plurality of “evolving targets” linked with complex diseases. Also, of all drug classes, polyclonals have the highest probability of retaining activity in the event of antigen mutation. In addition, while monoclonals have limited therapeutic activity against infectious agents, polyclonals can both neutralize toxins and direct immune responses to eliminate pathogens, as well as biological warfare agents.
The development of polyclonal and monoclonal antibody production platforms to meet future demand for production capacity represents a promising area that is currently the subject of much research. One especially promising strategy is the introduction of human immunoglobulin genes into mice or large domestic animals. An extension of this technology would include inactivation of their endogenous immunoglobulin genes. Large animals, such as sheep, pigs and cattle, are all currently used in the production of plasma derived products, such as hyperimmune serum and clotting factors, for human use. This would support the use of human polyclonal antibodies from such species on the grounds of safety and ethics. Each of these species naturally produces considerable quantities of antibody in both serum and milk.
Arrangement of Genes Encoding Immunoglobulins
Antibody molecules are assembled from combinations of variable gene elements, and the possibilities resulting from combining the many variable gene elements in the germline enable the host to synthesize antibodies to an extraordinarily large number of antigens. Each antibody molecule consists of two classes of polypeptide chains, light (L) chains (that can be either kappa (κ) L-chain or lambda (λ) L-chain) and heavy (H) chains. The heavy and light chains join together to define a binding region for the epitope. A single antibody molecule has two identical copies of the L chain and two of the H chain. Each of the chains is comprised of a variable region (V) and a constant region (C). The variable region constitutes the antigen-binding site of the molecule. To achieve diverse antigen recognition, the DNA that encodes the variable region undergoes gene rearrangement. The constant region amino acid sequence is specific for a particular isotype of the antibody, as well as the host which produces the antibody, and thus does not undergo rearrangement.
The mechanism of DNA rearrangement is similar for the variable region of both the heavy- and light-chain loci, although only one joining event is needed to generate a light-chain gene whereas two are needed to generate a complete heavy-chain gene. The most common mode of rearrangement involves the looping-out and deletion of the DNA between two gene segments. This occurs when the coding sequences of the two gene segments are in the same orientation in the DNA. A second mode of recombination can occur between two gene segments that have opposite transcriptional orientations. This mode of recombination is less common, although such rearrangements can account for up to half of all Vκ to Jκ joins; the transcriptional orientation of half of the human Vκ gene segments is opposite to that of the Jκ gene segments.
The DNA sequence encoding a complete V region is generated by the somatic recombination of separate gene segments. The V region, or V domain, of an immunoglobulin heavy or light chain is encoded by more than one gene segment. For the light chain, the V domain is encoded by two separate DNA segments. The first segment encodes the first 95-101 amino acids of the light chain and is termed a V gene segment because it encodes most of the V domain. The second segment encodes the remainder of the V domain (up to 13 amino acids) and is termed a joining or J gene segment. The joining of a V and a J gene segment creates a continuous exon that encodes the whole of the light-chain V region. To make a complete immunoglobulin light-chain messenger RNA, the V-region exon is joined to the C-region sequence by RNA splicing after transcription.
A heavy-chain V region is encoded in three gene segments. In addition to the V and J gene segments (denoted VH and JH to distinguish them from the light-chain VL and JL), there is a third gene segment called the diversity or DH gene segment, which lies between the VH and JH gene segments. The process of recombination that generates a complete heavy-chain V region occurs in two separate stages. In the first, a DH gene segment is joined to a JH gene segment; then a VH gene segment rearranges to DJH to make a complete VH-region exon. As with the light-chain genes, RNA splicing joins the assembled V-region sequence to the neighboring C-region gene.
Diversification of the antibody repertoire occurs in two stages: primarily by rearrangement (“V(D)J recombination”) of Ig V, D and J gene segments in precursor B cells resident in the bone marrow, and then by somatic mutation and class switch recombination of these rearranged Ig genes when mature B cells are activated. Immunoglobulin somatic mutation and class switching are central to the maturation of the immune response and the generation of a “memory” response.
The genomic loci of antibodies are very large and they are located on different chromosomes. The immunoglobulin gene segments are organized into three clusters or genetic loci: the κ, λ, and heavy-chain loci. Each is organized slightly differently. For example, in humans, immunoglobulin genes are organized as follows. The λ light-chain locus is located on chromosome 22 and a cluster of Vλ gene segments is followed by four sets of Jλ gene segments each linked to a single Cλ gene. The κ light-chain locus is on chromosome 2 and the cluster of Vκ, gene segments is followed by a cluster of Jκ gene segments, and then by a single Cκ gene. The organization of the heavy-chain locus, on chromosome 14, resembles that of the κ locus, with separate clusters of VH, DH, and JH gene segments and of CH genes. The heavy-chain locus differs in one important way: instead of a single C-region, it contains a series of C regions arrayed one after the other, each of which corresponds to a different isotype. There are five immunoglobulin heavy chain isotypes: IgM, IgG, IgA, IgE and IgD. Generally, a cell expresses only one at a time, beginning with IgM. The expression of other isotypes, such as IgG, can occur through isotype switching.
The joining of various V, D and J genes is an entirely random event that results in approximately 50,000 different possible combinations for VDJ(H) and approximately 1,000 for VJ(L). Subsequent random pairing of H and L chains brings the total number of antibody specificities to about 107 possibilities. Diversity is further increased by the imprecise joining of different genetic segments. Rearrangements occur on both DNA strands, but only one strand is transcribed (due to allelic exclusion). Only one rearrangement occurs in the life of a B cell because of irreversible deletions in DNA. Consequently, each mature B cell maintains one immunologic specificity and is maintained in the progeny or clone. This constitutes the molecular basis of the clonal selection; i.e., each antigenic determinant triggers the response of the pre-existing clone of B lymphocytes bearing the specific receptor molecule. The primary repertoire of B cells, which is established by V(D)J recombination, is primarily controlled by two closely linked genes, recombination activating gene (RAG)-1 and RAG-2.
Over the last decade, considerable diversity among vertebrates in both Ig gene diversity and antibody repertoire development has been revealed. Rodents and humans have five heavy chain classes, IgM, IgD, IgG, IgE and IgA, and each have four subclasses of IgG and one or two subclasses of IgA, while rabbits have a single IgG heavy chain gene but 13 genes for different IgA subclasses (Burnett, R. C et al. EMBO J. 8:4047; Honjo, In Honjo, T, Alt. F. W. T. H. eds, Immunoglobulin Genes p. 123 Academic Press, New York). Swine have at least six IgG subclasses (Kacskovics, I et al. 1994 J Immunol 153:3565), but no IgD (Butler et al. 1996 Inter. Immunol 8:1897-1904). A gene encoding IgD has only been described in rodents and primates. Diversity in the mechanism of repertoire development is exemplified by contrasting the pattern seen in rodents and primates with that reported for chickens, rabbits, swine and the domesticated Bovidae. Whereas the former group have a large number of VH genes belonging to seven to 10 families (Rathbun, G. In Hongo, T. Alt. F. W. and Rabbitts, T. H., eds, Immunoglobulin Genes, p. 63, Academic press New York), the VH genes of each member of the latter group belong to a single VH gene family (Sun, J. et al. 1994 J. Immunol. 1553:56118; Dufour, V et al. 1996, J Immunol. 156:2163). With the exception of the rabbit, this family is composed of less than 25 genes. Whereas rodents and primates can utilize four to six JH segments, only a single JH is available for repertoire development in the chicken (Reynaud et al. 1989 Adv. Immunol. 57:353). Similarly, Butler et al. (1996 Inter. Immunol 8:1897-1904) hypothesized that swine may resemble the chicken in having only a single JH gene. These species generally have fewer V, D and J genes; in the pig and cow a single VH gene family exists, consisting of less than 20 gene segments (Butler et al, Advances in Swine in Biomedical Research, eds: Tumbleson and Schook, 1996; Sinclair et al, J. Immunol. 159: 3883, 1997). Together with lower numbers of J and D gene segments, this results in significantly less diversity being generated by gene rearrangement. However, there does appear to be greater numbers of light chain genes in these species. Similar to humans and mice, these species express a single κ light chain but multiple λ light chain genes. However, these do not seem to affect the restricted diversity that is achieved by rearrangement.
Since combinatorial joining of more than 100 VH, 20-30 DH and four to six JH gene segments is a major mechanism of generating the antibody repertoire in humans, species with fewer VH, DH or JH segments must either generate a smaller repertoire or use alternative mechanisms for repertoire development. Ruminants, pigs, rabbits and chickens, utilize several mechanisms to generate antibody diversity. In these species there appears to be an important secondary repertoire development, which occurs in highly specialized lymphoid tissue such as ileal Peyer's patches (Binns and Licence, Adv. Exp. Med. Biol. 186: 661, 1985). Secondary repertoire development occurs in these species by a process of somatic mutation which is a random and not fully understood process. The mechanism for this repertoire diversification appears to be templated mutation, or gene conversion (Sun et al, J. Immunol. 153: 5618, 1994) and somatic hypermutation.
Gene conversion is important for antibody diversification in some higher vertebrates, such as chickens, rabbits and cows. In mice, however, conversion events appear to be infrequent among endogenous antibody genes. Gene conversion is a distinct diversifying mechanism characterized by transfers of homologous sequences from a donor antibody V gene segment to an acceptor V gene segment. If donor and acceptor segments have numerous sequence differences then gene conversion can introduce a set of sequence changes into a V region by a single event. Depending on the species, gene conversion events can occur before and/or after antigen exposure during B cell differentiation (Tsai et al. International Immunology, Vol. 14, No. 1, 55-64, January 2002).
Somatic hypermutation achieves diversification of antibody genes in all higher vertebrate species. It is typified by the introduction of single point mutations into antibody V(D)J segments. Generally, hypermutation appears to be activated in B cells by antigenic stimulation.
Production of Animals with Humanized Immune Systems
In order to reduce the immunogenicity of antibodies generated in mice for human therapeutics, various attempts have been made to replace murine protein sequences with human protein sequences in a process now known as humanization. Transgenic mice have been constructed which have had their own immunoglobulin genes functionally replaced with human immunoglobulin genes so that they produce human antibodies upon immunization. Elimination of mouse antibody production was achieved by inactivation of mouse Ig genes in embryonic stem (ES) cells by using gene-targeting technology to delete crucial cis-acting sequences involved in the process of mouse Ig gene rearrangement and expression. B cell development in these mutant mice could be restored by the introduction of megabase-sized YACs containing a human germline-configuration H- and κ L-chain minilocus transgene. The expression of fully human antibody in these transgenic mice was predominant, at a level of several 100 μg/l of blood. This level of expression is several hundred-fold higher than that detected in wild-type mice expressing the human Ig gene, indicating the importance of inactivating the endogenous mouse Ig genes in order to enhance human antibody production by mice.
The first humanization attempts utilized molecular biology techniques to construct recombinant antibodies. For example, the complementarity determining regions (CDR) from a mouse antibody specific for a hapten were grafted onto a human antibody framework, effecting a CDR replacement. The new antibody retained the binding specificity conveyed by the CDR sequences (P. T. Jones et al. Nature 321: 522-525 (1986)). The next level of humanization involved combining an entire mouse VH region with a human constant region such as gamma1 (S. L. Morrison et al., Proc. Natl. Acad. Sci., 81, pp. 6851-6855 (1984)). However, these chimeric antibodies, which still contain greater than 30% xenogeneic sequences, are sometimes only marginally less immunogenic than totally xenogeneic antibodies (M. Bruggemann et al., J. Exp. Med., 170, pp. 2153-2157 (1989)).
Subsequently, attempts were carried out to introduce human immunoglobulin genes into the mouse, thus creating transgenic mice capable of responding to antigens with antibodies having human sequences (Bruggemann et al. Proc. Nat'l. Acad. Sci. USA 86:6709-6713 (1989)). Due to the large size of human immunoglobulin genomic loci, these attempts were thought to be limited by the amount of DNA, which could be stably maintained by available cloning vehicles. As a result, many investigators concentrated on producing mini-loci containing limited numbers of V region genes and having altered spatial distances between genes as compared to the natural or germline configuration (See, for example, U.S. Pat. No. 5,569,825). These studies indicated that producing human sequence antibodies in mice was possible, but serious obstacles remained regarding obtaining sufficient diversity of binding specificities and effector functions (isotypes) from these transgenic animals to meet the growing demand for antibody therapeutics.
In order to provide additional diversity, work has been conducted to add large germline fragments of the human Ig locus into transgenic mammals. For example, a majority of the human V, D, and J region genes arranged with the same spacing found in the unrearranged germline of the human genome and the human Cμ and Cδ constant regions was introduced into mice using yeast artificial chromosome (YAC) cloning vectors (See, for example, WO 94/02602). A 22 kb DNA fragment comprising sequences encoding a human gamma-2 constant region and the upstream sequences required for class-switch recombination was latter appended to the foregoing transgene. In addition, a portion of a human kappa locus comprising Vκ, Jκ and Cκ region genes, also arranged with substantially the same spacing found in the unrearranged germline of the human genome, was introduced into mice using YACS. Gene targeting was used to inactivate the murine IgH & kappa light chain immunoglobulin gene loci and such knockout strains were bred with the above transgenic strains to generate a line of mice having the human V, D, J, Cμ, Cδ and Cγ2 constant regions as well as the human Vκ, Jκ and Cκ region genes all on an inactivated murine immunoglobulin background (See, for example, PCT patent application WO 94/02602 to Kucherlapati et al.; see also Mendez et al., Nature Genetics 15:146-156 (1997)).
Yeast artificial chromosomes as cloning vectors in combination with gene targeting of endogenous loci and breeding of transgenic mouse strains provided one solution to the problem of antibody diversity. Several advantages were obtained by this approach. One advantage was that YACs can be used to transfer hundreds of kilobases of DNA into a host cell. Therefore, use of YAC cloning vehicles allows inclusion of substantial portions of the entire human Ig heavy and light chain regions into a transgenic mouse thus approaching the level of potential diversity available in the human. Another advantage of this approach is that the large number of V genes has been shown to restore full B cell development in mice deficient in murine immunoglobulin production. This ensures that these reconstituted mice are provided with the requisite cells for mounting a robust human antibody response to any given immunogen. (See, for example, WO 94/02602; L. Green and A. Jakobovits, J. Exp. Med. 188:483-495 (1998)). A further advantage is that sequences can be deleted or inserted onto the YAC by utilizing high frequency homologous recombination in yeast. This provides for facile engineering of the YAC transgenes.
In addition, Green et al. Nature Genetics 7:13-21 (1994) describe the generation of YACs containing 245 kb and 190 kb-sized germline configuration fragments of the human heavy chain locus and kappa light chain locus, respectively, which contained core variable and constant region sequences. The work of Green et al. was recently extended to the introduction of greater than approximately 80% of the human antibody repertoire through introduction of megabase sized, germline configuration YAC fragments of the human heavy chain loci and kappa light chain loci, respectively, to produce XenoMouse™ mice. See, for example, Mendez et al. Nature Genetics 15:146-156 (1997), Green and Jakobovits J. Exp. Med. 188:483-495 (1998), European Patent No. EP 0 463 151 B1, PCT Publication Nos. WO 94/02602, WO 96/34096 and WO 98/24893.
Several strategies exist for the generation of mammals that produce human antibodies. In particular, there is the “minilocus” approach that is typified by work of GenPharm International, Inc. and the Medical Research Council, YAC introduction of large and substantially germline fragments of the Ig loci that is typified by work of Abgenix, Inc. (formerly Cell Genesys). The introduction of entire or substantially entire loci through the use microcell fusion as typified by work of Kirin Beer Kabushiki Kaisha.
In the minilocus approach, an exogenous Ig locus is mimicked through the inclusion of pieces (individual genes) from the Ig locus. Thus, one or more VH genes, one or more DH genes, one or more JH genes, a mu constant region, and a second constant region (such as a gamma constant region) are formed into a construct for insertion into an animal. See, for example, U.S. Pat. Nos. 5,545,807, 5,545,806, 5,625,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429, 5,789,650, 5,814,318, 5,591,669, 5,612,205, 5,721,367, 5,789,215, 5,643,763; European Patent No. 0 546 073; PCT Publication Nos. WO 92/03918, WO 92/22645, WO 92/22647, WO 92/22670, WO 93/12227, WO 94/00569, WO 94/25585, WO 96/14436, WO 97/13852, and WO 98/24884; Taylor et al. Nucleic Acids Research 20:6287-6295 (1992), Chen et al. International Immunology 5:647-656 (1993), Tuaillon et al. J. Immunol. 154:6453-6465 (1995), Choi et al. Nature Genetics 4:117-123 (1993), Lonberg et al. Nature 368:856-859 (1994), Taylor et al. International Immunology 6:579-591 (1994), Tuaillon et al. J. Immunol. 154:6453-6465 (1995), and Fishwild et al. Nature Biotech. 14:845-851 (1996).
In the microcell fusion approach, portions or whole human chromosomes can be introduced into mice (see, for example, European Patent Application No. EP 0 843 961 A1). Mice generated using this approach and containing the human Ig heavy chain locus will generally possess more than one, and potentially all, of the human constant region genes. Such mice will produce, therefore, antibodies that bind to particular antigens having a number of different constant regions.
While mice remain the most developed animal for the expression of human immunoglobulins in humans, recent technological advances have allowed for progress to begin in applying these techniques to other animals, such as cows. The general approach in mice has been to genetically modify embryonic stem cells of mice to knock-out murine immunoglobulins and then insert YACs containing human immunoglobulins into the ES cells. However, ES cells are not available for cows or other large animals such as sheep and pigs. Thus, several fundamental developments had to occur before even the possibility existed to generate large animals with immunoglobulin genes knocked-out and that express human antibody. The alternative to ES cell manipulation to create genetically modified animals is cloning using somatic cells that have been genetically modified. Cloning using genetically modified somatic cells for nuclear transfer has only recently been accomplished.
Since the announcement of Dolly's (a cloned sheep) birth from an adult somatic cell in 1997 (Wilmut, I., et al (1997) Nature 385: 810-813), ungulates, including cattle (Cibelli, J et al 1998 Science 280: 1266-1258; Kubota, C. et al. 2000 Proc. Nat'l. Acad. Sci. 97: 990-995), goats (Baguisi, A. et al., (1999) Nat. Biotechnology 17: 456-461), and pigs (Polejaeva, I. A., et al. 2000 Nature 407: 86-90; Betthauser, J. et al. 2000 Nat. Biotechnology 18: 1055-1059) have been cloned.
The next technological advance was the development of the technique to genetically modify the cells prior to nuclear transfer to produce genetically modified animals. PCT publication No. WO 00/51424 to PPL Therapeutics describes the targeted genetic modification of somatic cells for nuclear transfer.
Subsequent to these fundamental developments, single and double allele knockouts of genes and the birth of live animals with these modifications have been reported. Between 2002 and 2004, three independent groups, Immerge Biotherapeutics, Inc. in collaboration with the University of Missouri (Lai et al. (Science (2002) 295: 1089-1092) & Kolber-Simonds et al. (PNAS. (2004) 101(19):7335-40)), Alexion Pharmaceuticals (Ramsoondar et al. (Biol Reprod (2003)69: 437-445) and Revivicor, Inc. (Dai et al. (Nature Biotechnology (2002) 20: 251-255) & Phelps et al. (Science (2003) January 17; 299(5605):411-4)) produced pigs that lacked one allele or both alleles of the alpha-1,3-GT gene via nuclear transfer from somatic cells with targeted genetic deletions. In 2003, Sedai et al. (Transplantation (2003) 76:900-902) reported the targeted disruption of one allele of the alpha-1,3-GT gene in cattle, followed by the successful nuclear transfer of the nucleus of the genetically modified cell and production of transgenic fetuses.
Thus, the feasibility of knocking-out immunoglobulin genes in large animals and inserting human immunoglobulin loci into their cells is just now beginning to be explored. However, due to the complexity and species differences of immunoglobulin genes, the genomic sequences and arrangement of Ig kappa, lambda and heavy chains remain poorly understood in most species. For example, in pigs, partial genomic sequence and organization has only been described for heavy chain constant alpha, heavy chain constant mu and heavy chain constant delta (Brown and Butler Mol Immunol. 1994 June; 31(8):633-42, Butler et al Vet Immunol Immunopathol. 1994 October; 43(1-3):5-12, and Zhao et al J Immunol. 2003 Aug. 1; 171(3):1312-8).
In cows, the immunoglobulin heavy chain locus has been mapped (Zhao et al. 2003 J. Biol. Chem. 278:35024-32) and the cDNA sequence for the bovine kappa gene is known (See, for example, U.S. Patent Publication No. 2003/0037347). Further, approximately 4.6 kb of the bovine mu heavy chain locus has been sequenced and transgenic calves with decreased expression of heavy chain immunoglobulins have been created by disrupting one or both alleles of the bovine mu heavy chain. In addition, a mammalian artificial chromosome (MAC) vector containing the entire unarranged sequences of the human Ig H-chain and κ L-chain has been introduced into cows (TC cows) with the technology of microcell-mediated chromosome transfer and nuclear transfer of bovine fetal fibroblast cells (see, for example, Kuroiwa et al. 2002 Nature Biotechnology 20:889, Kuroiwa et al. 2004 Nat Genet. June 6 Epub, U.S. Patent Publication Nos. 2003/0037347, 2003/0056237, 2004/0068760 and PCT Publication No. WO 02/07648).
While significant progress has been made in the production of bovine that express human immunoglobulin, little has been accomplished in other large animals, such as sheep, goats and pigs. Although cDNA sequence information for immunoglobulin genes of sheeps, goats and pigs is readily available in Genbank, the unique nature of immunoglobulin loci, which undergo massive rearrangements, creates the need to characterize beyond sequences known to be present in mRNAs (or cDNAs). Since immunoglobulin loci are modular and the coding regions are redundant, deletion of a known coding region does not ensure altered function of the locus. For example, if one were to delete the coding region of a heavy-chain variable region, the function of the locus would not be significantly altered because hundreds of other function variable genes remain in the locus. Therefore, one must first characterize the locus to identify a potential “Achilles heel”.
Despite some advancements in expressing human antibodies in cattle, greater challenges remain for inactivation of the endogenous bovine Ig genes, increasing expression levels of the human antibodies and creating human antibody expression in other large animals, such as porcine, for which the sequence and arrangement of immunoglobulin genes are largely unknown.
It is therefore an object of the present invention to provide the arrangement of ungulate immunoglobin germline gene sequence.
It is another object of the present invention to provide novel ungulate immunoglobulin genomic sequences.
It is a further object of the present invention to provide cells, tissues and animals lacking at least one allele of a heavy and/or light chain immunoglobulin gene.
It is another object of the present invention to provide ungulates that express human immunoglobulins.
It is a still further object of the present invention to provide methods to generate cells, tissues and animals lacking at least one allele of novel ungulate immunoglobulin gene sequences and/or express human immunoglobulins.
SUMMARY OF THE INVENTION The present invention provides for the first time ungulate immunoglobin germline gene sequence arrangement as well as novel genomic sequences thereof. In addition, novel ungulate cells, tissues and animals that lack at least one allele of a heavy or light chain immunoglobulin gene are provided. Based on this discovery, ungulates can be produced that completely lack at least one allele of a heavy and/or light chain immunoglobulin gene. In addition, these ungulates can be further modified to express xenoogenous, such as human, immunoglobulin loci or fragments thereof.
In one aspect of the present invention, a transgenic ungulate that lacks any expression of functional endogenous immunoglobulins is provided. In one embodiment, the ungulate can lack any expression of endogenous heavy and/or light chain immunoglobulins. The light chain immunoglobulin can be a kappa and/or lambda immunoglobulin. In additional embodiments, transgenic ungulates are provided that lack expression of at least one allele of an endogenous immunoglobulin wherein the immunoglobulin is selected from the group consisting of heavy chain, kappa light chain and lambda light chain or any combination thereof. In one embodiment, the expression of functional endogenous immunoglobulins can be accomplished by genetic targeting of the endogenous immunoglobulin loci to prevent expression of the endogenous immunoglobulin. In one embodiment, the genetic targeting can be accomplished via homologous recombination. In another embodiment, the transgenic ungulate can be produced via nuclear transfer.
In other embodiments, the transgenic ungulate that lacks any expression of functional endogenous immunoglobulins can be further genetically modified to express an xenogenous immunoglobulin loci. In an alternative embodiment, porcine animals are provided that contain an xenogeous immunoglobulin locus. In one embodiment, the xenogeous immunoglobulin loci can be a heavy and/or light chain immunoglobulin or fragment thereof. In another embodiment, the xenogenous immunoglobulin loci can be a kappa chain locus or fragment thereof and/or a lambda chain locus or fragment thereof. In still further embodiments, an artificial chromosome (AC) can contain the xenogenous immunoglobulin. In one embodiment, the AC can be a yeast AC or a mammalian AC. In a further embodiment, the xenogenous locus can be a human immunoglobulin locus or fragment thereof. In one embodiment, the human immunoglobulin locus can be human chromosome 14, human chromosome 2, and human chromosome 22 or fragments thereof. In another embodiment, the human immunoglobulin locus can include any fragment of a human immunoglobulin that can undergo rearrangement. In a further embodiment, the human immunoglobulin loci can include any fragment of a human immunoglobulin heavy chain and a human immunoglobulin light chain that can undergo rearrangement. In still further embodiment, the human immunoglobulin loci can include any human immunoglobulin locus or fragment thereof that can produce an antibody upon exposure to an antigen. In a particular embodiment, the exogenous human immunoglobulin can be expressed in B cells to produce xenogenous immunoglobulin in response to exposure to one or more antigens.
In another aspect of the present invention, transgenic ungulates are provided that expresses a xenogenous immunoglobulin loci or fragment thereof, wherein the immunoglobulin can be expressed from an immunoglobulin locus that is integrated within an endogenous ungulate chromosome. In one embodiment, ungulate cells derived from the transgenic animals are provided. In one embodiment, the xenogenous immunoglobulin locus can be inherited by offspring. In another embodiment, the xenogenous immunoglobulin locus can be inherited through the male germ line by offspring. In still further embodiments, an artificial chromosome (AC) can contain the xenogenous immunoglobulin. In one embodiment, the AC can be a yeast AC or a mammalian AC. In a further embodiment, the xenogenous locus can be a human immunoglobulin locus or fragment thereof. In one embodiment, the human immunoglobulin locus can be human chromosome 14, human chromosome 2, and human chromosome 22 or fragments thereof. In another embodiment, the human immunoglobulin locus can include any fragment of a human immunoglobulin that can undergo rearrangement. In a further embodiment, the human immunoglobulin loci can include any fragment of a human immunoglobulin heavy chain and a human immunoglobulin light chain that can undergo rearrangement. In still further embodiment, the human immunoglobulin loci can include any human immunoglobulin locus or fragment thereof that can produce an antibody upon exposure to an antigen. In a particular embodiment, the exogenous human immunoglobulin can be expressed in B cells to produce xenogenous immunoglobulin in response to exposure to one or more antigens.
In another aspect of the present invention, novel genomic sequences encoding the heavy chain locus of ungulate immunoglobulin are provided. In one embodiment, an isolated nucleotide sequence encoding porcine heavy chain is provided that includes at least one variable region, two diversity regions, at least four joining regions and at least one constant region, such as the mu constant region, for example, as represented in Seq ID No. 29. In another embodiment, an isolated nucleotide sequence is provided that includes at least four joining regions and at least one constant region, such as the mu constant region, of the porcine heavy chain genomic sequence, for example, as represented in Seq ID No. 4. In a further embodiment, nucleotide sequence is provided that includes 5′ flanking sequence to the first joining region of the porcine heavy chain genomic sequence, for example, as represented in Seq ID No 1. Still further, nucleotide sequence is provided that includes 3′ flanking sequence to the first joining region of the porcine heavy chain genomic sequence, for example, as represented in the 3′ region of Seq ID No 4. In further embodiments, isolated nucleotide sequences as depicted in Seq ID Nos 1, 4 or 29 are provided. Nucleic acid sequences at least 80, 85, 90, 95, 98 or 99% homologous to Seq ID Nos 1, 4 or 29 are also provided. In addition, nucleotide sequences that contain at least 10, 15, 17, 20, 25 or 30 contiguous nucleotides of Seq ID Nos 1, 4 or 29 are provided. In one embodiment, the nucleotide sequence contains at least 17, 20, 25 or 30 contiguous nucleotides of Seq ID No 4 or residues 1-9,070 of Seq ID No 29. In another embodiment, the nucleotide sequence contains residues 9,070-11039 of Seq ID No 29. Further provided are nucleotide sequences that hybridize, optionally under stringent conditions, to Seq ID Nos 1, 4 or 29, as well as, nucleotides homologous thereto.
In another embodiment, novel genomic sequences encoding the kappa light chain locus of ungulate immunoglobulin are provided. The present invention provides the first reported genomic sequence of ungulate kappa light chain regions. In one embodiment, nucleic acid sequence is provided that encodes the porcine kappa light chain locus. In another embodiment, the nucleic acid sequence can contain at least one joining region, one constant region and/or one enhancer region of kappa light chain. In a further embodiment, the nucleotide sequence can include at least five joining regions, one constant region and one enhancer region, for example, as represented in Seq ID No. 30. In a further embodiment, an isolated nucleotide sequence is provided that contains at least one, at least two, at least three, at least four or five joining regions and 3′ flanking sequence to the joining region of porcine genomic kappa light chain, for example, as represented in Seq ID No 12. In another embodiment, an isolated nucleotide sequence of porcine genomic kappa light chain is provided that contains 5′ flanking sequence to the first joining region, for example, as represented in Seq ID No 25. In a further embodiment, an isolated nucleotide sequence is provided that contains 3′ flanking sequence to the constant region and, optionally, the 5′ portion of the enhancer region, of porcine genomic kappa light chain, for example, as represented in Seq ID Nos. 15, 16 and/or 19.
In further embodiments, isolated nucleotide sequences as depicted in Seq ID Nos 30, 12, 25, 15, 16 or 19 are provided. Nucleic acid sequences at least 80, 85, 90, 95, 98 or 99% homologous to Seq ID Nos 30, 12, 25, 15, 16 or 19 are also provided. In addition, nucleotide sequences that contain at least 10, 15, 17, 20, 25 or 30 contiguous nucleotides of Seq ID Nos 30, 12, 25, 15, 16 or 19 are provided. Further provided are nucleotide sequences that hybridizes, optionally under stringent conditions, to Seq ID Nos 30, 12, 25, 15, 16 or 19, as well as, nucleotides homologous thereto.
In another embodiment, novel genomic sequences encoding the lambda light chain locus of ungulate immunoglobulin are provided. The present invention provides the first reported genomic sequence of ungulate lambda light chain regions. In one embodiment, the porcine lambda light chain nucleotides include a concatamer of J to C units. In a specific embodiment, an isolated porcine lambda nucleotide sequence is provided, such as that depicted in Seq ID No. 28. In one embodiment, a nucleotide sequence is provided that includes 5′ flanking sequence to the first lambda J/C unit of the porcine lambda light chain genomic sequence, for example, as represented by Seq ID No 32. Still further, nucleotide sequence is provided that includes 3′ flanking sequence to the J/C cluster region of the porcine lambda light chain genomic sequence, for example, approximately 200 base pairs downstream of lambda J/C, such as that represented by Seq ID No 33. Alternatively, nucleotide sequence is provided that includes 3′ flanking sequence to the J/C cluster region of the porcine lambda light chain genomic sequence, for example, as represented by Seq ID No 34, 35, 36, 37, 38, and/or 39.
In a further embodiment, nucleic acid sequences are provided that encode bovine lambda light chain locus, which can include at least one joining region-constant region pair and/or at least one variable region, for example, as represented by Seq ID No. 31. In further embodiments, isolated nucleotide sequences as depicted in Seq ID Nos 28, 31, 32, 33, 34, 35, 36, 37, 38, or 39 are provided. Nucleic acid sequences at least 80, 85, 90, 95, 98 or 99% homologous to Seq ID Nos 28, 31, 32, 33, 34, 35, 36, 37, 38, or 39 are also provided. In addition, nucleotide sequences that contain at least 10, 15, 17, 20, 25 or 30 contiguous nucleotides of Seq ID Nos 28, 31, 32, 33, 34, 35, 36, 37, 38, or 39 are provided. Further provided are nucleotide sequences that hybridizes, optionally under stringent conditions, to Seq ID Nos 28, 31, 32, 33, 34, 35, 36, 37, 38, or 39, as well as, nucleotides homologous thereto.
In another embodiment, nucleic acid targeting vector constructs are also provided. The targeting vectors can be designed to accomplish homologous recombination in cells. These targeting vectors can be transformed into mammalian cells to target the ungulate heavy chain, kappa light chain or lambda light chain genes via homologous recombination. In one embodiment, the targeting vectors can contain a 3′ recombination arm and a 5′ recombination arm (i.e. flanking sequence) that is homologous to the genomic sequence of ungulate heavy chain, kappa light chain or lambda light chain genomic sequence, for example, sequence represented by Seq ID Nos. 1, 4, 29, 30, 12, 25, 15, 16, 19, 28 or 31, as described above. The homologous DNA sequence can include at least 15 bp, 20 bp, 25 bp, 50 bp, 100 bp, 500 bp, 1 kbp, 2 kbp, 4 kbp, 5 kbp, 10 kbp, 15 kbp, 20 kbp, or 50 kbp of sequence homologous to the genomic sequence.
In one embodiment, the 5′ and 3′ recombination arms of the targeting vector can be designed such that they flank the 5′ and 3′ ends of at least one functional variable, joining, diversity, and/or constant region of the genomic sequence. The targeting of a functional region can render it inactive, which results in the inability of the cell to produce functional immunoglobulin molecules. In another embodiment, the homologous DNA sequence can include one or more intron and/or exon sequences. In addition to the nucleic acid sequences, the expression vector can contain selectable marker sequences, such as, for example, enhanced Green Fluorescent Protein (eGFP) gene sequences, initiation and/or enhancer sequences, poly A-tail sequences, and/or nucleic acid sequences that provide for the expression of the construct in prokaryotic and/or eukaryotic host cells. The selectable marker can be located between the 5′ and 3′ recombination arm sequence.
In one particular embodiment, the targeting vector can contain 5′ and 3′ recombination arms that contain homologous sequence to the 3′ and 5′ flanking sequence of the J6 region of the porcine immunoglobulin heavy chain locus. Since the J6 region is the only functional joining region of the porcine immunoglobulin heavy chain locus, this will prevent the expression of a functional porcine heavy chain immunoglobulin. In a specific embodiment, the targeting vector can contain a 5′ recombination arm that contains sequence homologous to genomic sequence 5′ of the J6 region, including J1-4, and a 3′ recombination arm that contains sequence homologous to genomic sequence 3′ of the J6 region, including the mu constant region (a “J6 targeting construct”), see for example, FIG. 1. Further, this J6 targeting construct can also contain a selectable marker gene that is located between the 5′ and 3′ recombination arms, see for example, Seq ID No 5 and FIG. 1. In other embodiments, the targeting vector can contain a 5′ recombination arm that contains sequence homologous to genomic sequence 5′ of the diversity region, and a 3′ recombination arm that contains sequence homologous to genomic sequence 3′ of the diversity region of the porcine heavy chain locus. In a further embodiment, the targeting vector can contain a 5′ recombination arm that contains sequence homologous to genomic sequence 5′ of the mu constant region and a 3′ recombination arm that contains sequence homologous to genomic sequence 3′ of the mu constant region of the porcine heavy chain locus.
In another particular embodiment, the targeting vector can contain 5′ and 3′ recombination arms that contain homologous sequence to the 3′ and 5′ flanking sequence of the constant region of the porcine immunoglobulin kappa light chain locus. Since the present invention discovered that there is only one constant region of the porcine immunoglobulin kappa light chain locus, this will prevent the expression of a functional porcine kappa light chain immunoglobulin. In a specific embodiment, the targeting vector can contain a 5′ recombination arm that contains sequence homologous to genomic sequence 5′ of the constant region, optionally including the joining region, and a 3′ recombination arm that contains sequence homologous to genomic sequence 3′ of the constant region, optionally including at least part of the enhancer region (a “Kappa constant targeting construct”), see for example, FIG. 2. Further, this kappa constant targeting construct can also contain a selectable marker gene that is located between the 5′ and 3′ recombination arms, see for example, Seq ID No 20 and FIG. 2. In other embodiments, the targeting vector can contain a 5′ recombination arm that contains sequence homologous to genomic sequence 5′ of the joining region, and a 3′ recombination arm that contains sequence homologous to genomic sequence 3′ of the joining region of the porcine kappa light chain locus.
In another particular embodiment, the targeting vector can contain 5′ and 3′ recombination arms that contain homologous sequence to the 3′ and 5′ flanking sequence of the J/C region of the porcine lambda light chain. See FIG. 3. Disruption of the J/C region will prevent the expression of a functional porcine kappa light chain immunoglobulin. In one embodiment, the targeting vector can contain a 5′ recombination arm that contains sequence homologous to genomic sequence 5′ of the first J/C unit and a 3′ recombination arm that contains sequence homologous to genomic sequence 3′ of the last J/C unit. Further, this lambda light chain targeting construct can also contain a selectable marker gene that is located between the 5′ and 3′ recombination arms, see for example FIG. 4.
In a further embodiment, more than one targeting vector can be used to target the ungulate heavy chain, kappa light chain or lambda light chain genes via homologous recombination. For example, two targeting vectors can be used to target the gene of interest. A first targeting vector can contain 5′ and 3′ recombination arms that contain homologous sequence to the 5′ flanking sequence of at least one functional variable, joining, diversity, and/or constant region of the genomic sequence. A second targeting vector can contain 5′ and 3′ recombination arms that contain homologous sequence to the 3′ flanking sequence at least one functional variable, joining, diversity, and/or constant region of the genomic sequence.
In a particular embodiment, the first targeting vector can contain 5′ and 3′ recombination arms that contain homologous sequence to the 5′ flanking sequence of the first J/C unit in the J/C cluster region. See FIG. 5. According to this embodiment, a second targeting vector can contain 5′ and 3′ recombination arms that contain homologous sequence to the 3′ flanking sequence of the last J/C unit in the J/C cluster region. See FIG. 6.
In another embodiment, primers are provided to generate 3′ and 5′ sequences of a targeting vector. The oligonucleotide primers can be capable of hybridizing to porcine immunoglobulin genomic sequence, such as Seq ID Nos. 1, 4, 29, 30, 12, 25, 15, 16, 19, 28 or 31, as described above. In a particular embodiment, the primers hybridize under stringent conditions to Seq ID Nos. 1, 4, 29, 30, 12, 25, 15, 16, 19, 28 or 31, as described above. Another embodiment provides oligonucleotide probes capable of hybridizing to porcine heavy chain, kappa light chain or lambda light chain nucleic acid sequences, such as Seq ID Nos. 1, 4, 29, 30, 12, 25, 15, 16, 19, 28 or 31, as described above. The polynucleotide primers or probes can have at least 14 bases, 20 bases, 30 bases, or 50 bases which hybridize to a polynucleotide of the present invention. The probe or primer can be at least 14 nucleotides in length, and in a particular embodiment, are at least 15, 20, 25, 28, or 30 nucleotides in length.
In one embodiment, primers are provided to amplify a fragment of porcine Ig heavy-chain that includes the functional joining region (the J6 region). In one non-limiting embodiment, the amplified fragment of heavy chain can be represented by Seq ID No 4 and the primers used to amplify this fragment can be complementary to a portion of the J-region, such as, but not limited to Seq ID No 2, to produce the 5′ recombination arm and complementary to a portion of Ig heavy-chain mu constant region, such as, but not limited to Seq ID No 3, to produce the 3′ recombination arm. In another embodiment, regions of the porcine Ig heavy chain (such as, but not limited to Seq ID No 4) can be subcloned and assembled into a targeting vector.
In other embodiments, primers are provided to amplify a fragment of porcine Ig kappa light-chain that includes the constant region. In another embodiment, primers are provided to amplify a fragment of porcine Ig kappa light-chain that includes the J region. In one non-limiting embodiment, the primers used to amplify this fragment can be complementary to a portion of the J-region, such as, but not limited to Seq ID No 21 or 10, to produce the 5′ recombination arm and complementary to genomic sequence 3′ of the constant region, such as, but not limited to Seq ID No 14, 24 or 18, to produce the 3′ recombination arm. In another embodiment, regions of the porcine Ig heavy chain (such as, but not limited to Seq ID No 20) can be subcloned and assembled into a targeting vector.
In another aspect of the present invention, ungulate cells lacking at least one allele of a functional region of an ungulate heavy chain, kappa light chain and/or lambda light chain locus produced according to the process, sequences and/or constructs described herein are provided. These cells can be obtained as a result of homologous recombination. Particularly, by inactivating at least one allele of an ungulate heavy chain, kappa light chain or lambda light chain gene, cells can be produced which have reduced capability for expression of ungulate antibodies. In other embodiments, mammalian cells lacking both alleles of an ungulate heavy chain, kappa light chain and/or lambda light chain gene can be produced according to the process, sequences and/or constructs described herein. In a further embodiment, porcine animals are provided in which at least one allele of an ungulate heavy chain, kappa light chain and/or lambda light chain gene is inactivated via a genetic targeting event produced according to the process, sequences and/or constructs described herein. In another aspect of the present invention, porcine animals are provided in which both alleles of an ungulate heavy chain, kappa light chain and/or lambda light chain gene are inactivated via a genetic targeting event. The gene can be targeted via homologous recombination.
In other embodiments, the gene can be disrupted, i.e. a portion of the genetic code can be altered, thereby affecting transcription and/or translation of that segment of the gene. For example, disruption of a gene can occur through substitution, deletion (“knock-out”) or insertion (“knock-in”) techniques. Additional genes for a desired protein or regulatory sequence that modulate transcription of an existing sequence can be inserted. To achieve multiple genetic modifications of ungulate immunoglobulin genes, in one embodiment, cells can be modified sequentially to contain multiple genetic modifications. In other embodiments, animals can be bred together to produce animals that contain multiple genetic modifications of immunoglobulin genes. As an illustrative example, animals that lack expression of at least one allele of an ungulate heavy chain gene can be further genetically modified or bred with animals lacking at least one allele of a kappa light chain gene.
In embodiments of the present invention, alleles of ungulate heavy chain, kappa light chain or lambda light chain gene are rendered inactive according to the process, sequences and/or constructs described herein, such that functional ungulate immunoglobulins can no longer be produced. In one embodiment, the targeted immunoglobulin gene can be transcribed into RNA, but not translated into protein. In another embodiment, the targeted immunoglobulin gene can be transcribed in an inactive truncated form. Such a truncated RNA may either not be translated or can be translated into a nonfunctional protein. In an alternative embodiment, the targeted immunoglobulin gene can be inactivated in such a way that no transcription of the gene occurs. In a further embodiment, the targeted immunoglobulin gene can be transcribed and then translated into a nonfunctional protein.
In a further aspect of the present invention, ungulate, such as porcine or bovine, cells lacking one allele, optionally both alleles of an ungulate heavy chain, kappa light chain and/or lambda light chain gene can be used as donor cells for nuclear transfer into recipient cells to produce cloned, transgenic animals. Alternatively, ungulate heavy chain, kappa light chain and/or lambda light chain gene knockouts can be created in embryonic stem cells, which are then used to produce offspring. Offspring lacking a single allele of a functional ungulate heavy chain, kappa light chain and/or lambda light chain gene produced according to the process, sequences and/or constructs described herein can be breed to further produce offspring lacking functionality in both alleles through mendelian type inheritance.
In one aspect of the present invention, a method is provided to disrupt the expression of an ungulate immunoglobulin gene by (i) analyzing the germline configuration of the ungulate heavy chain, kappa light chain or lambda light chain genomic locus; (ii) determining the location of nucleotide sequences that flank the 5′ end and the 3′ end of at least one functional region of the locus; and (iii) transfecting a targeting construct containing the flanking sequence into a cell wherein, upon successful homologous recombination, at least one functional region of the immunoglobulin locus is disrupted thereby reducing or preventing the expression of the immunoglobulin gene. In one embodiment, the germline configuration of the porcine heavy chain locus is provided. The porcine heavy chain locus contains at least four variable regions, two diversity regions, six joining regions and five constant regions, for example, as illustrated in FIG. 1. In a specific embodiment, only one of the six joining regions, J6, is functional. In another embodiment, the germline configuration of the porcine kappa light chain locus is provided. The porcine kappa light chain locus contains at least six variable regions, six joining regions, one constant region and one enhancer region, for example, as illustrated in FIG. 2. In a further embodiment, the germline configuration of the porcine lambda light chain locus is provided. The porcine lambda light chain locus contains a variable region and the J/C region. See FIG. 3.
In a further aspect of the present invention, a method is provided to disrupt the expression of an ungulate lambda light chain locus by (i) analyzing the germline configuration of the ungulate lambda light chain genomic locus; (ii) determining the location of nucleotide sequences that flank the 5′ end of at least one functional region of the locus; (ii) constructing a 5′ targeting construct; (iv) determining the location of nucleotide sequences that flank the 3′ end of at least one functional region of the locus; (v) constructing a 3′ targeting construct; (vi) transfecting both the 5′ and the 3′ targeting constructs into a cell wherein, upon successful homologous recombination of each targeting construct, at least one functional region of the immunoglobulin locus is disrupted thereby reducing or preventing the expression of the immunoglobulin gene. See FIGS. 5 and 6.
In one embodiment, the germline configuration of the porcine lambda light chain locus is provided. The porcine lambda light chain locus contains a variable region and a J/C region. See FIG. 3.
In further aspects of the present invention provides ungulates and ungulate cells that lack at least one allele of a functional region of an ungulate heavy chain, kappa light chain and/or lambda light chain locus produced according to the processes, sequences and/or constructs described herein, which are further modified to express at least part of a human antibody (i.e. immunoglobulin (Ig)) locus. In additional embodiments, porcine animals are provided that express xenogenous immunoglobulin. This human locus can undergo rearrangement and express a diverse population of human antibody molecules in the ungulate. These cloned, transgenic ungulates provide a replenishable, theoretically infinite supply of human antibodies (such as polyclonal antibodies), which can be used for therapeutic, diagnostic, purification, and other clinically relevant purposes. In one particular embodiment, artificial chromosomes (ACs), such as yeast or mammalian artificial chromosomes (YACS or MACS) can be used to allow expression of human immunoglobulin genes into ungulate cells and animals. All or part of human immunoglobulin genes, such as the Ig heavy chain gene (human chromosome 414), Ig kappa chain gene (human chromosome #2) and/or the Ig lambda chain gene (chromosome #22) can be inserted into the artificial chromosomes, which can then be inserted into ungulate cells. In further embodiments, ungulates and ungulate cells are provided that contain either part or all of at least one human antibody gene locus, which undergoes rearrangement and expresses a diverse population of human antibody molecules.
In additional embodiments, methods of producing xenogenous antibodies are provided, wherein the method can include: (a) administering one or more antigens of interest to an ungulate whose cells comprise one or more artificial chromosomes and lack any expression of functional endogenous immunoglobulin, each artificial chromosome comprising one or more xenogenous immunoglobulin loci that undergo rearrangement, resulting in production of xenogenous antibodies against the one or more antigens; and/or (b) recovering the xenogenous antibodies from the ungulate. In one embodiment, the immunoglobulin loci can undergo rearrangement in a B cell.
In one aspect of the present invention, an ungulate, such as a pig or a cow, can be prepared by a method in accordance with any aspect of the present invention. These cloned, transgenic ungulates (e.g., porcine and bovine animals) provide a replenishable, theoretically infinite supply of human polyclonal antibodies, which can be used as therapeutics, diagnostics and for purification purposes. For example, transgenic animals produced according to the process, sequences and/or constructs described herein that produce polyclonal human antibodies in the bloodstream can be used to produce an array of different antibodies which are specific to a desired antigen. The availability of large quantities of polyclonal antibodies can also be used for treatment and prophylaxis of infectious disease, vaccination against biological warfare agents, modulation of the immune system, removal of undesired human cells such as cancer cells, and modulation of specific human molecules.
In other embodiments, animals or cells lacking expression of functional immunoglobulin, produced according to the process, sequences and/or constructs described herein, can contain additional genetic modifications to eliminate the expression of xenoantigens. Such animals can be modified to eliminate the expression of at least one allele of the alpha-1,3-galactosyltransferase gene, the CMP-Neu5Ac hydroxylase gene (see, for example, U.S. Ser. No. 10/863,116), the iGb3 synthase gene (see, for example, U.S. Patent Application 60/517,524), and/or the Forssman synthase gene (see, for example, U.S. Patent Application 60/568,922). In additional embodiments, the animals discloses herein can also contain genetic modifications to express fucosyltransferase and/or sialyltransferase. To achieve these additional genetic modifications, in one embodiment, cells can be modified to contain multiple genetic modifications. In other embodiments, animals can be bred together to achieve multiple genetic modifications. In one specific embodiment, animals, such as pigs, lacking expression of functional immunoglobulin, produced according to the process, sequences and/or constructs described herein, can be bred with animals, such as pigs, lacking expression of alpha-1,3-galactosyl transferase (for example, as described in WO 04/028243).
BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 illustrates the design of a targeting vector that disrupts the expression of the joining region of the porcine heavy chain immunoglobulin gene.
FIG. 2 illustrates the design of a targeting vector that disrupts the expression of the constant region of the porcine kappa light chain immunoglobulin gene.
FIG. 3 illustrates the genomic organization of the porcine lambda immunoglobulin locus, including a concatamer of J-C sequences or units as well as flanking regions that include the variable region 5′ to the JC cluster region. Bacterial artificial chromosomes (BAC1 and BAC2) represent fragments of the porcine immunoglobulin genome that can be obtained from BAC libraries.
FIG. 4 represents the design of a targeting vector that disrupts the expression of the JC cluster region of the porcine lambda light chain immunoglobulin gene. “SM” stands for a selectable marker gene, which can be used in the targeting vector.
FIG. 5 illustrates a targeting strategy to insert a site specific recombinase target or recognition site into the region 5′ of the JC cluster region of the porcine lambda immunoglobulin locus. “SM” stands for a selectable marker gene, which can be used in the targeting vector. “SSRRS” stands for a specific recombinase target or recognition site.
FIG. 6 illustrates a targeting strategy to insert a site specific recombinase target or recognition site into the region 3′ of the JC cluster region of the porcine lambda immunoglobulin locus. “SM” stands for a selectable marker gene, which can be used in the targeting vector. “SSRRS” stands for a specific recombinase target or recognition site.
FIG. 7 illustrates the site specific recombinase mediated transfer of a YAC into a host genome. “SSRRS” stands for a specific recombinase target or recognition site.
DETAILED DESCRIPTION The present invention provides for the first time ungulate immunoglobin germline gene sequence arrangement as well as novel genomic sequences thereof. In addition, novel ungulate cells, tissues and animals that lack at least one allele of a heavy or light chain immunoglobulin gene are provided. Based on this discovery, ungulates can be produced that completely lack at least one allele of a heavy and/or light chain immunoglobulin gene. In addition, these ungulates can be further modified to express xenoogenous, such as human, immunoglobulin loci or fragments thereof.
In one aspect of the present invention, a transgenic ungulate that lacks any expression of functional endogenous immunoglobulins is provided. In one embodiment, the ungulate can lack any expression of endogenous heavy and/or light chain immunoglobulins. The light chain immunoglobulin can be a kappa and/or lambda immunoglobulin. In additional embodiments, transgenic ungulates are provided that lack expression of at least one allele of an endogenous immunoglobulin wherein the immunoglobulin is selected from the group consisting of heavy chain, kappa light chain and lambda light chain or any combination thereof. In one embodiment, the expression of functional endogenous immunoglobulins can be accomplished by genetic targeting of the endogenous immunoglobulin loci to prevent expression of the endogenous immunoglobulin. In one embodiment, the genetic targeting can be accomplished via homologous recombination. In another embodiment, the transgenic ungulate can be produced via nuclear transfer.
In other embodiments, the transgenic ungulate that lacks any expression of functional endogenous immunoglobulins can be further genetically modified to express an xenogenous immunoglobulin loci. In an alternative embodiment, porcine animals are provided that contain an xenogeous immunoglobulin locus. In one embodiment, the xenogeous immunoglobulin loci can be a heavy and/or light chain immunoglobulin or fragment thereof. In another embodiment, the xenogenous immunoglobulin loci can be a kappa chain locus or fragment thereof and/or a lambda chain locus or fragment thereof. In still further embodiments, an artificial chromosome (AC) can contain the xenogenous immunoglobulin. In one embodiment, the AC can be a yeast AC or a mammalian AC. In a further embodiment, the xenogenous locus can be a human immunoglobulin locus or fragment thereof. In one embodiment, the human immunoglobulin locus can be human chromosome 14, human chromosome 2, and human chromosome 22 or fragments thereof. In another embodiment, the human immunoglobulin locus can include any fragment of a human immunoglobulin that can undergo rearrangement. In a further embodiment, the human immunoglobulin loci can include any fragment of a human immunoglobulin heavy chain and a human immunoglobulin light chain that can undergo rearrangement. In still further embodiment, the human immunoglobulin loci can include any human immunoglobulin locus or fragment thereof that can produce an antibody upon exposure to an antigen. In a particular embodiment, the exogenous human immunoglobulin can be expressed in B cells to produce xenogenous immunoglobulin in response to exposure to one or more antigens.
In another aspect of the present invention, transgenic ungulates are provided that expresses a xenogenous immunoglobulin loci or fragment thereof, wherein the immunoglobulin can be expressed from an immunoglobulin locus that is integrated within an endogenous ungulate chromosome. In one embodiment, ungulate cells derived from the transgenic animals are provided. In one embodiment, the xenogenous immunoglobulin locus can be inherited by offspring. In another embodiment, the xenogenous immunoglobulin locus can be inherited through the male germ line by offspring. In still further embodiments, an artificial chromosome (AC) can contain the xenogenous immunoglobulin. In one embodiment, the AC can be a yeast AC or a mammalian AC. In a further embodiment, the xenogenous locus can be a human immunoglobulin locus or fragment thereof. In one embodiment, the human immunoglobulin locus can be human chromosome 14, human chromosome 2, and human chromosome 22 or fragments thereof. In another embodiment, the human immunoglobulin locus can include any fragment of a human immunoglobulin that can undergo rearrangement. In a further embodiment, the human immunoglobulin loci can include any fragment of a human immunoglobulin heavy chain and a human immunoglobulin light chain that can undergo rearrangement. In still further embodiment, the human immunoglobulin loci can include any human immunoglobulin locus or fragment thereof that can produce an antibody upon exposure to an antigen. In a particular embodiment, the exogenous human immunoglobulin can be expressed in B cells to produce xenogenous immunoglobulin in response to exposure to one or more antigens.
Definitions
The terms “recombinant DNA technology,” “DNA cloning,” “molecular cloning,” or “gene cloning” refer to the process of transferring a DNA sequence into a cell or organism. The transfer of a DNA fragment can be from one organism to a self-replicating genetic element (e.g., bacterial plasmid) that permits a copy of any specific part of a DNA (or RNA) sequence to be selected among many others and produced in an unlimited amount. Plasmids and other types of cloning vectors such as artificial chromosomes can be used to copy genes and other pieces of chromosomes to generate enough identical material for further study. In addition to bacterial plasmids, which can carry up to 20 kb of foreign DNA, other cloning vectors include viruses, cosmids, and artificial chromosomes (e.g., bacteria artificial chromosomes (BACs) or yeast artificial chromosomes (YACs)). When the fragment of chromosomal DNA is ultimately joined with its cloning vector in the lab, it is called a “recombinant DNA molecule.” Shortly after the recombinant plasmid is introduced into suitable host cells, the newly inserted segment will be reproduced along with the host cell DNA.
“Cosmids” are artificially constructed cloning vectors that carry up to 45 kb of foreign DNA. They can be packaged in lambda phage particles for infection into E. coli cells.
As used herein, the term “mammal” (as in “genetically modified (or altered) mammal”) is meant to include any non-human mammal, including but not limited to pigs, sheep, goats, cattle (bovine), deer, mules, horses, monkeys, dogs, cats, rats, mice, birds, chickens, reptiles, fish, and insects. In one embodiment of the invention, genetically altered pigs and methods of production thereof are provided.
The term “ungulate” refers to hoofed mammals. Artiodactyls are even-toed (cloven-hooved) ungulates, including antelopes, camels, cows, deer, goats, pigs, and sheep. Perissodactyls are odd toes ungulates, which include horses, zebras, rhinoceroses, and tapirs. The term ungulate as used herein refers to an adult, embryonic or fetal ungulate animal.
As used herein, the terms “porcine”, “porcine animal”, “pig” and “swine” are generic terms referring to the same type of animal without regard to gender, size, or breed.
A “homologous DNA sequence or homologous DNA” is a DNA sequence that is at least about 80%, 85%, 90%, 95%, 98% or 99% identical with a reference DNA sequence. A homologous sequence hybridizes under stringent conditions to the target sequence, stringent hybridization conditions include those that will allow hybridization occur if there is at least 85, at least 95% or 98% identity between the sequences.
An “isogenic or substantially isogenic DNA sequence” is a DNA sequence that is identical to or nearly identical to a reference DNA sequence. The term “substantially isogenic” refers to DNA that is at least about 97-99% identical with the reference DNA sequence, or at least about 99.5-99.9% identical with the reference DNA sequence, and in certain uses 100% identical with the reference DNA sequence.
“Homologous recombination” refers to the process of DNA recombination based on sequence homology.
“Gene targeting” refers to homologous recombination between two DNA sequences, one of which is located on a chromosome and the other of which is not.
“Non-homologous or random integration” refers to any process by which DNA is integrated into the genome that does not involve homologous recombination.
A “selectable marker gene” is a gene, the expression of which allows cells containing the gene to be identified. A selectable marker can be one that allows a cell to proliferate on a medium that prevents or slows the growth of cells without the gene. Examples include antibiotic resistance genes and genes which allow an organism to grow on a selected metabolite. Alternatively, the gene can facilitate visual screening of transformants by conferring on cells a phenotype that is easily identified. Such an identifiable phenotype can be, for example, the production of luminescence or the production of a colored compound, or the production of a detectable change in the medium surrounding the cell.
The term “contiguous” is used herein in its standard meaning, i.e., without interruption, or uninterrupted.
“Stringent conditions” refers to conditions that (1) employ low ionic strength and high temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium citrate/0.1% SDS at 50° C., or (2) employ during hybridization a denaturing agent such as, for example, formamide. One skilled in the art can determine and vary the stringency conditions appropriately to obtain a clear and detectable hybridization signal. For example, stringency can generally be reduced by increasing the salt content present during hybridization and washing, reducing the temperature, or a combination thereof. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press, Cold Spring Harbour, N.Y., (1989).
I. Immunoglobulin Genes
In one aspect of the present invention, a transgenic ungulate that lacks any expression of functional endogenous immunoglobulins is provided. In one embodiment, the ungulate can lack any expression of endogenous heavy and/or light chain immunoglobulins. The light chain immunoglobulin can be a kappa and/or lambda immunoglobulin. In additional embodiments, transgenic ungulates are provided that lack expression of at least one allele of an endogenous immunoglobulin wherein the immunoglobulin is selected from the group consisting of heavy chain, kappa light chain and lambda light chain or any combination thereof. In one embodiment, the expression of functional endogenous immunoglobulins can be accomplished by genetic targeting of the endogenous immunoglobulin loci to prevent expression of the endogenous immunoglobulin. In one embodiment, the genetic targeting can be accomplished via homologous recombination. In another embodiment, the transgenic ungulate can be produced via nuclear transfer.
In another aspect of the present invention, a method is provided to disrupt the expression of an ungulate immunoglobulin gene by (i) analyzing the germline configuration of the ungulate heavy chain, kappa light chain or lambda light chain genomic locus; (ii) determining the location of nucleotide sequences that flank the 5′ end and the 3′ end of at least one functional region of the locus; and (iii) transfecting a targeting construct containing the flanking sequence into a cell wherein, upon successful homologous recombination, at least one functional region of the immunoglobulin locus is disrupted thereby reducing or preventing the expression of the immunoglobulin gene.
In one embodiment, the germline configuration of the porcine heavy chain locus is provided. The porcine heavy chain locus contains at least four variable regions, two diversity regions, six joining regions and five constant regions, for example, as illustrated in FIG. 1. In a specific embodiment, only one of the six joining regions, J6, is functional.
In another embodiment, the germline configuration of the porcine kappa light chain locus is provided. The porcine kappa light chain locus contains at least six variable regions, six joining regions, one constant region and one enhancer region, for example, as illustrated in FIG. 2.
In a further embodiment, the germline configuration of the porcine lambda light chain locus is provided.
Isolated nucleotide sequences as depicted in Seq ID Nos 1-39 are provided. Nucleic acid sequences at least 80, 85, 90, 95, 98 or 99% homologous to any one of Seq ID Nos 1-39 are also provided. In addition, nucleotide sequences that contain at least 10, 15, 17, 20, 25 or 30 contiguous nucleotides of any one of Seq ID Nos 1-39 are provided. Further provided are nucleotide sequences that hybridize, optionally under stringent conditions, to Seq ID Nos 1-39, as well as, nucleotides homologous thereto.
Homology or identity at the nucleotide or amino acid sequence level can be determined by BLAST (Basic Local Alignment Search Tool) analysis using the algorithm employed by the programs blastp, blastn, blastx, tblastn and tblastx (see, for example, Altschul, S. F. et al (1997) Nucleic Acids Res 25:3389-3402 and Karlin et al, (1900) Proc. Natl. Acad. Sci. USA 87, 2264-2268) which are tailored for sequence similarity searching. The approach used by the BLAST program is to first consider similar segments, with and without gaps, between a query sequence and a database sequence, then to evaluate the statistical significance of all matches that are identified and finally to summarize only those matches which satisfy a preselected threshold of significance. See, for example, Altschul et al., (1994) (Nature Genetics 6, 119-129). The search parameters for histogram, descriptions, alignments, expect (i.e., the statistical significance threshold for reporting matches against database sequences), cutoff, matrix and filter (low co M'plexity) are at the default settings. The default scoring matrix used by blastp, blastx, tblastn, and tblastx is the BLOSUM62 matrix (Henikoff et al., (1992) Proc. Natl. Acad. Sci. USA 89, 10915-10919), which is recommended for query sequences over 85 in length (nucleotide bases or amino acids).
Porcine Heavy Chain
In another aspect of the present invention, novel genomic sequences encoding the heavy chain locus of ungulate immunoglobulin are provided. In one embodiment, an isolated nucleotide sequence encoding porcine heavy chain is provided that includes at least one variable region, two diversity regions, at least four joining regions and at least one constant region, such as the mu constant region, for example, as represented in Seq ID No. 29. In another embodiment, an isolated nucleotide sequence is provided that includes at least four joining regions and at least one constant region, such as the mu constant region, of the porcine heavy chain genomic sequence, for example, as represented in Seq ID No. 4. In a further embodiment, nucleotide sequence is provided that includes 5′ flanking sequence to the first joining region of the porcine heavy chain genomic sequence, for example, as represented in Seq ID No 1. Still further, nucleotide sequence is provided that includes 3′ flanking sequence to the first joining region of the porcine heavy chain genomic sequence, for example, as represented in the 3′ region of Seq ID No 4. In further embodiments, isolated nucleotide sequences as depicted in Seq ID Nos 1, 4 or 29 are provided. Nucleic acid sequences at least 80, 85, 90, 95, 98 or 99% homologous to Seq ID Nos 1, 4 or 29 are also provided. Further provided are nucleotide sequences that hybridize, optionally under stringent conditions, to Seq ID Nos 1, 4 or 29, as well as, nucleotides homologous thereto.
In addition, nucleotide sequences that contain at least 10, 15, 17, 20, 25 or 30 contiguous nucleotides of Seq ID Nos 1, 4 or 29 are provided. In one embodiment, the nucleotide sequence contains at least 17, 20, 25 or 30 contiguous nucleotides of Seq ID No 4 or residues 1-9,070 of Seq ID No 29. In other embodiments, nucleotide sequences that contain at least 50, 100, 1,000, 2,500, 4,000, 4,500, 5,000, 7,000, 8,000, 8,500, 9,000, 10,000 or 15,000 contiguous nucleotides of Seq ID No. 29 are provided. In another embodiment, the nucleotide sequence contains residues 9,070-11039 of Seq ID No 29.
In further embodiments, isolated nucleotide sequences as depicted in Seq ID Nos 1, 4 or 29 are provided. Nucleic acid sequences at least 80, 85, 90, 95, 98 or 99% homologous to Seq ID Nos 1, 4 or 29 are also provided. In addition, nucleotide sequences that contain at least 10, 15, 17, 20, 25 or 30 contiguous nucleotides of Seq ID Nos 1, 4 or 29 are provided. Further provided are nucleotide sequences that hybridize, optionally under stringent conditions, to Seq ID Nos 1, 4 or 29, as well as, nucleotides homologous thereto.
In one embodiment, an isolated nucleotide sequence encoding porcine heavy chain is provided that includes at least one variable region, two diversity regions, at least four joining regions and at least one constant region, such as the mu constant region, for example, as represented in Seq ID No. 29. In Seq ID No. 29, the Diversity region of heavy chain is represented, for example, by residues 1089-1099 (D(pseudo)), the Joining region of heavy chain is represented, for example, by residues 1887-3352 (for example: J(psuedo): 1887-1931, J(psuedo): 2364-2411, J(psuedo): 2756-2804, J (functional J): 3296-3352), the recombination signals are represented, for example, by residues 3001-3261 (Nonamer), 3292-3298 (Heptamer), the Constant Region is represented by the following residues: 3353-9070 (J to C mu intron), 5522-8700 (Switch region), 9071-9388 (Mu Exon 1), 9389-9469 (Mu Intron A), 9470-9802 (Mu Exon 2), 9830-10069 (Mu Intron B), 10070-10387 (Mu Exon 3), 10388-10517 (Mu Intron C), 10815-11052 (Mu Exon 4), 11034-11039 (Poly(A) signal).
Seq ID No. 29
tctagaagacgctggagagaggccagacttcctcggaacagctcaaagag
ctctgtcaaagccagatcccatcacacgtgggcaccaataggccatgcca
gcctccaagggccgaactgggttctccacggcgcacatgaagcctgcagc
ctggcttatcctcttccgtggtgaagaggcaggcccgggactggacgagg
ggctagcagggtgtggtaggcaccttgcgccccccaccccggcaggaacc
agagaccctggggctgagagtgagcctccaaacaggatgccccacccttc
aggccacctttcaatccagctacactccacctgccattctcctctgggca
cagggcccagcccctggatcttggccttggctcgacttgcacccacgcgc
acacacacacttcctaacgtgctgtccgctcacccctccccagcgtggtc
catgggcagcacggcagtgcgcgtccggcggtagtgagtgcagaggtccc
ttcccctcccccaggagccccaggggtgtgtgcagatctgggggctcctg
tcccttacaccttcatgcccctcccctcatacccaccctccaggcgggag
gcagcgagacctttgcccagggactcagccaacgggcacacgggaggcca
gccctcagcagctggctcccaaagaggaggtgggaggtaggtccacagct
gccacagagagaaaccctgacggaccccacaggggccacgccagccggaa
ccagctccctcgtgggtgagcaatggccagggccccgccggccaccacgg
ctggccttgcgccagctgagaactcacgtccagtgcagggagactcaaga
cagcctgtgcacacagcctcggatctgctcccatttcaagcagaaaaagg
aaaccgtgcaggcagccctcagcatttcaaggattgtagcagcggccaac
tattcgtcggcagtggccgattagaatgaccgtggagaagggcggaaggg
tctctcgtgggctctgcggccaacaggccctggctccacctgcccgctgc
cagcccgaggggcttgggccgagccaggaaccacagtgctcaccgggacc
acagtgactgaccaaactcccggccagagcagccccaggccagccgggct
ctcgccctggaggactcaccatcagatgcacaagggggcgagtgtggaag
agacgtgtcgcccgggccatttgggaaggcgaagggaccttccaggtgga
caggaggtgggacgcactccaggcaagggactgggtccccaaggcctggg
gaaggggtactggcttgggggttagcctggccagggaacggggagcgggg
cggggggctgagcagggaggacctgacctcgtgggagcgaggcaagtcag
gcttcaggcagcagccgcacatcccagaccaggaggctgaggcaggaggg
gcttgcagcggggcgggggcctgcctggctccgggggctcctgggggacg
ctggctcttgtttccgtgtcccgcagcacagggccagctcgctgggccta
tgcttaccttgatgtctggggccggggcgtcagggtcgtcgtctcctcag
gggagagtcccctgaggctacgctgggg*ggggactatggcagctccacc
aggggcctggggaccaggggcctggaccaggctgcagcccggaggacggg
cagggctctggctctccagcatctggccctcggaaatggcagaacccctg
gcgggtgagcgagctgagagcgggtcagacagacaggggccggccggaaa
ggagaagttgggggcagagcccgccaggggccaggcccaaggttctgtgt
gccagggcctgggtgggcacattggtgtggccatggctacttagattcgt
ggggccagggcatcctggtcaccgtctcctcaggtgagcctggtgtctga
tgtccagctaggcgctggtgggccgcgggtgggcctgtctcaggctaggg
caggggctgggatgtgtatttgtcaaggaggggcaacagggtgcagactg
tgcccctggaaacttgaccactggggcaggggcgtcctggtcacgtctcc
tcaggtaagacggccctgtgcccctctctcgcgggactggaaaaggaatt
ttccaagattccttggtctgtgtggggccctctggggcccccgggggtgg
ctcccctcctgcccagatggggcctcggcctgtggagcacgggctgggca
cacagctcgagtctagggccacagaggcccgggctcagggctctgtgtgg
cccggcgactggcagggggctcgggtttttggacaccccctaatgggggc
cacagcactgtgaccatcttcacagctggggccgaggagtcgaggtcacc
gtctcctcaggtgagtcctcgtcagccctctctcactctctggggggttt
tgctgcattttgtgggggaaagaggatgcctgggtctcaggtctaaaggt
ctagggccagcgccggggcccaggaaggggccgaggggccaggctcggct
cggccaggagcagagcttccagacatctcgcctcctggcggctgcagtca
ggcctttggccgggggggtctcagcaccaccaggcctcttggctcccgag
gtccccggccccggctgcctcaccaggcaccgtgcgcggtgggcccgggc
tcttggtcggccaccctttcttaactgggatccgggcttagttgtcgcaa
tgtgacaacgggctcgaaagctggggccaggggaccctagtctacgacgc
ctcgggtgggtgtcccgcacccctccccactttcacggcactcggcgaga
cctggggagtcaggtgttggggacactttggaggtcaggaacgggagctg
gggagagggctctgtcagcggggtccagagatgggccgccctccaaggac
gccctgcgcggggacaagggcttcttggcctggcctggccgcttcacttg
ggcgtcagggggggcttcccggggcaggcggtcagtcgaggcgggttgga
attctgagtctgggttcggggttcggggttcggccttcatgaacagacag
cccaggcgggccgttgtttggcccctgggggcctggttggaatgcgaggt
ctcgggaagtcaggagggagcctggccagcagagggttcccagccctgcg
gccgagggacctggagacgggcagggcattggccgtcgcagggccaggcc
acaccccccaGGTTTTTGTggggcgagcctggagattgcacCACTGTGAT
TACTATGCTATGGATCTCTGGGGCCCAGGCGTTGAAGTCGTCGTGTCCTC
AGgtaagaacggccctccagggcctttaatttctgctctcgtctgtgggc
ttttctgactctgatcctcgggaggcgtctgtgccccccccggggatgag
gccggcttgccaggaggggtcagggaccaggagcctgtgggaagttctga
cgggggctgcaggcgggaagggccccaccggggggcgagccccaggccgc
tgggcggcaggagacccgtgagagtgcgccttgaggagggtgtctgcgga
accacgaacgcccgccgggaagggcttgctgcaatgcggtcttcagacgg
gaggcgtcttctgccctcaccgtctttcaagcccttgtgggtctgaaaga
gccatgtcggagagagaagggacaggcctgtcccgacctggccgagagcg
ggcagccccgggggagagcggggcgatcggcctgggctctgtgaggccag
gtccaagggaggacgtgtggtcctcgtgacaggtgcacttgcgaaacctt
agaagacggggtatgttggaagcggctcctgatgtttaagaaaagggaga
ctgtaaagtgagcagagtcctcaagtgtgttaaggttttaaaggtcaaag
tgttttaaacctttgtgactgcagttagcaagcgtgcggggagtgaatgg
ggtgccagggtggccgagaggcagtacgagggccgtgccgtcctctaatt
cagggcttagttttgcagaataaagtcggcctgttttctaaaagcattgg
tggtgctgagctggtggaggaggccgcgggcagccctggccacctgcagc
aggtggcaggaagcaggtcggccaagaggctattttaggaagccagaaaa
cacggtcgatgaatttatagcttctggtttccaggaggtggttgggcatg
gctttgcgcagcgccacagaaccgaaagtgcccactgagaaaaaacaact
cctgcttaatttgcatttttctaaaagaagaaacagaggctgacggaaac
tggaaagttcctgttttaactactcgaattgagttttcggtcttagctta
tcaactgctcacttagattcattttcaaagtaaacgtttaagagccgagg
cattcctatcctcttctaaggcgttattcctggaggctcattcaccgcca
gcacctccgctgcctgcaggcattgctgtcaccgtcaccgtgacggcgcg
cacgattttcagttggcccgcttcccctcgtgattaggacagacgcgggc
actctggcccagccgtcttggctcagtatctgcaggcgtccgtctcggga
cggagctcaggggaagagcgtgactccagttgaacgtgatagtcggtgcg
ttgagaggagacccagtcgggtgtcgagtcagaaggggcccggggcccga
ggccctgggcaggacggcccgtgccctgcatcacgggcccagcgtcctag
aggcaggactctggtggagagtgtgagggtgcctggggcccctccggagc
tggggccgtgcggtgcaggttgggctctcggcgcggtgttggctgtttct
gcgggatttggaggaattcttccagtgatgggagtcgccagtgaccgggc
accaggctggtaagagggaggccgccgtcgtggccagagcagctgggagg
gttcggtaaaaggctcgcccgtttcctttaatgaggacttttcctggagg
gcatttagtctagtcgggaccgttttcgactcgggaagagggatgcggag
gagggcatgtgcccaggagccgaaggcgccgcggggagaagcccagggct
ctcctgtccccacagaggcgacgccactgccgcagacagacagggccttt
ccctctgatgacggcaaaggcgcctcggctcttgcggggtgctggggggg
agtcgccccgaagccgctcacccagaggcctgaggggtgagactgaccga
tgcctcttggccgggcctggggccggaccgagggggactccgtggaggca
gggcgatggtggctgcgggagggaaccgaccctgggccgagcccggcttg
gcgattcccgggcgagggccctcagccgaggcgagtgggtccggcggaac
caccctttctggccagcgccacagggctctcgggactgtccggggcgacg
ctgggctgcccgtggcaggccTGGGCTGACCTGGACTTCACCAGACAGAA
CAGGGCTTTCAGGGCTGAGCTGAGCCAGGTTTAGCGAGGCCAAGTGGGGC
TGAACCAGGCTCAACTGGCCTGAGCTGGGTTGAGCTGGGCTGACCTGGGC
TGAGCTGAGCTGGGCTGGGCTGGGCTGGGCTGGGCTGGGCTGGGCTGGAC
TGGCTGAGCTGAGCTGGGTTGAGCTGAGCTGAGCTGGCCTGGGTTGAGCT
GGGCTGGGTTGAGCTGAGCTGGGTTGAGCTGGGTTGAGCTGGGTTGATCT
GAGCTGAGCTGGGCTGAGCTGAGCTAGGCTGGGGTGAGCTGGGCTGAGCT
GGTTTGAGTTGGGTTGAGCTGAGCTGAGCTGGGCTGTGCTGGCTGAGCTA
GGCTGAGCTAGGCTAGGTTGAGCTGGGCTGGGCTGAGGTGAGCTAGGCTG
GGCTGATTTGGGCTGAGCTGAGCTGAGCTAGGCTGCGTTGAGCTGGCTGG
GCTGGATTGAGCTGGCTGAGCTGGCTGAGCTGGGCTGAGCTGGCCTGGGT
TGAGCTGAGCTGGACTGGTTTGAGCTGGGTCGATCTGGGTTGAGCTGTCC
TGGGTTGAGCTGGGCTGGGTTGAGCTGAGCTGGGTTGAGCTGGGCTCAGC
AGAGCTGGGTTGGGCTGAGCTGGGTTGAGCTGAGCTGGGCTGAGCTGGCC
TGGGTTGAGCTGGGCTGAGCTGAGCTGGGCTGAGCTGGCCTGTGTTGAGC
TGGGCTGGGTTGAGCTGGGCTGAGCTGGATTGAGCTGGGTTGAGCTGAGC
TGGGCTGGGCTGTGCTGACTGAGCTGGGCTGAGCTAGGCTGGGGTGAGCT
GGGCTGAGCTGATCCGAGCTAGGCTGGGCTGGTTTGGGCTGAGCTGAGCT
GAGCTAGGCTGGATTGATCTGGCTGAGCTGGGTTGAGCTGAGCTGGGCTG
AGCTGGTCTGAGCTGGCCTGGGTCGAGCTGAGCTGGACTGGTTTGAGCTG
GGTCGATCTGGGCTGAGCTGGCCTGGGTTGAGCTGGGCTGGGTTGAGCTG
AGCTGGGTTGAGCTGGGCTGAGCTGAGGGCTGGGGTGAGCTGGGCTGAAC
TAGCCTAGCTAGGTTGGGCTGAGCTGGGCTGGTTTGGGCTGAGCTGAGCT
GAGCTAGGCTGCATTGAGCAGGCTGAGCTGGGCTGAGCAGGCCTGGGGTG
AGCTGGGCTAGGTGGAGCTGAGCTGGGTCGAGCTGAGTTGGGCTGAGCTG
GCCTGGGTTGAGGTAGGCTGAGCTGAGCTGAGCTAGGCTGGGTTGAGCTG
GCTGGGCTGGTTTGCGCTGGGTCAAGCTGGGCCGAGCTGGCCTGGGTTGA
GCTGGGCTCGGTTGAGCTGGGCTGAGCTGAGCCGACCTAGGCTGGGATGA
GCTGGGCTGATTTGGGCTGAGCTGAGCTGAGCTAGGCTGCATTGAGCAGG
CTGAGCTGGGCCTGGAGCCTGGCCTGGGGTGAGCTGGGCTGAGCTGCGCT
GAGCTAGGCTGGGTTGAGCTGGCTGGGCTGGTTTGCGCTGGGTCAAGCTG
GGCCGAGCTGGCCTGGGATGAGCTGGGCCGGTTTGGGCTGAGCTGAGCTG
AGCTAGGCTGCATTGAGCAGGCTGAGCTGGGCTGAGCTGGCCTGGGGTGA
GCTGGGCTGAGCTAAGCTGAGCTGGGCTGGTTTGGGCTGAGCTGGCTGAG
CTGGGTCCTGCTGAGCTGGGCTGAGCTGACCAGGGGTGAGCTGGGCTGAG
TTAGGCTGGGCTCAGCTAGGCTGGGTTGATCTGGCAGGGCTGGTTTGCGC
TGGGTCAAGCTCCCGGGAGATGGCCTGGGATGAGCTGGGCTGGTTTGGGC
TGAGCTGAGCTGAGCTGAGCTAGGCTGCATTGAGCAGGCTGAGCTGGGCT
GAGCTGGCCTGGGGTGAGCTGGGCTGGGTGGAGCTGAGCTGGGCTGAACT
GGGCTAAGCTGGCTGAGCTGGATCGAGCTGAGCTGGGCTGAGCTGGCCTG
GGGTTAGCTGGGCTGAGCTGAGCTGAGCTAGGCTGGGTTGAGCTGGCTGG
GCTGGTTTGCGCTGGGTCAAGCTGGGCCGAGCTGGCCTGGGTTGAGCTGG
GCTGGGCTGAGCTGAGCTAGGCTGGGTTGAGCTGGGCTGGGCTGAGCTGA
GCTAGGCTGCATTGAGCTGGCTGGGATGGATTGAGCTGGCTGAGCTGGCT
GAGCTGGCTGAGCTGGGCTGAGCTGGCCTGGGTTGAGCTGGGCTGGGTTG
AGCTGAGCTGGGCTGAGCTGGGCTCAGCAGAGCTGGGTTGAGCTGAGCTG
GGTTGAGCTGGGGTGAGCTGGGCTGAGCAGAGCTGGGTTGAGCTGAGCTG
GGTTGAGCTGGGCTCGAGCAGAGCTGGGTTGAGCTGAGCTGGGTTGAGCT
GGGCTCAGCAGAGCTGGGTTGAGCTGAGCTGGGTTGAGCTGGGCTGAGCT
AGCTGGGCTCAGCTAGGCTGGGTTGAGCTGAGCTGGGCTGAACTGGGCTG
AGCTGGGCTGAACTGGGCTGAGCTGGGCTGAGCTGGGCTGAGCAGAGCTG
GGCTGAGCAGAGCTGGGTTGGTCTGAGCTGGGTTGAGCTGGGCTGAGCTG
GGCTGAGCAGAGTTGGGTTGAGCTGAGCTGGGTTCAGCTGGGCTGAGCTA
GGCTGGGTTGAGCTGGGTTGAGTTGGGCTGAGCTGGGCTGGGTTGAGCGG
AGCTGGGCTGAACTGGGCTGAGCTGGGCTGAGCGGAACTGGGTTGATCTG
AATTGAGCTGGGCTGAGCCGGGCTGAGCCGGGCTGAGCTGGGCTAGGTTG
AGCTTGGGTGAGCTTGCCTCAGCTGGTCTGAGCTAGGTTGGGTGGAGCTA
GGCTGGATTGAGCTGGGCTGAGCTGAGCTGATCTGGCCTCAGCTGGGCTG
AGGTAGGCTGAACTGGGCTGTGCTGGGCTGAGCTGAGCTGAGCCAGTTTG
AGCTGGGTTGAGCTGGGCTGAGCTGGGCTGTGTTGATCTTTCCTGAACTG
GGCTGAGCTGGGCTGAGCTGGCCTAGCTGGATTGAACGGGGGTAAGCTGG
GCCAGGCTGGACTGGGCTGAGCTGAGCTAGGCTGAGCTGAGTTGAATTGG
GTTAAGCTGGGCTGAGATGGGCTGAGCTGGGCTGAGCTGGGTTGAGCCAG
GTCGGACTGGGTTACCCTGGGCCACACTGGGCTGAGCTGGGCGGAGCTCG
attaacctggtcaggctgagtcgggtccagcagacatgcgctggccaggc
tggcttgacctggacacgttcgatgagctgccttgggatggttcacctca
gctgagccaggtggctccagctgggctgagctggtgaccctgggtgacct
cggtgaccaggttgtcctgagtccgggccaagccgaggctgcatcagact
cgccagacccaaggcctgggccccggctggcaagccaggggcggtgaagg
ctgggctggcaggactgtcccggaaggaggtgcacgtggagccgcccgga
ccccgaccggcaggacctggaaagacgcctctcactcccctttctcttct
gtcccctctcgggtcctcagAGAGCCAGTCTGCCCCGAATCTCTACCCCC
TCGTCTCCTGCGTCAGCCCCCCGTCCGATGAGAGCCTGGTGGCCCTGGGC
TGCCTGGCCCGGGACTTCCTGCCCAGCTCCGTCACCTTCTCCTGGAACTA
CAAGAACAGCAGCAAGGTCAGCAGCCAGAACATCCAGGACTTCCCGTCC
GTCCTGAGAGGCGGCAAGTACTTGGCCTCCTCCCGGGTGCTCCTACCCTCT
GTGAGCATCCCCCAGGACCCAGAGGCCTTCCTGGTGTGCGAGGTCCAGCA
CCCCAGTGGCACCAAGTCCGTGTCCATCTCTGGGCCAGgtgagctgggct
ccccctgtggctgtggcgggggcggggccgggtgccgccggcacagtgac
gccccgttcctgcctgcagTCGTAGAGGAGCAGCCCCCCGTCTTGAACATC
TTCGTCCCCACCCGGGAGTCCTTCTCCAGTACTCCCCAGCGCACGTCCAAG
CTCATCTGCCAGGCCTCAGACTTCAGCCCCAAGCAGATCTCCATGGCCTGG
TTCCGTGATGGGAAACGGGTGGTGTCTGGCGTCAGGACAGGCCCCGTGGAG
ACCCTACAGTCCAGTGGGGTGACCTACAGGCTCCACAGCATGCTGACCGTCA
CGGAGTCCGAGTGGGTCAGCCAGAGCGTCTTCACCTGCCAGGTGGAGCACAAA
GGGCTGAACTACGAGAAGAACGCGTCCTCTCTGTGCACCTCCAgtgagtgcag
cccctcgggccgggcggcggggcggcgggagccacacacacaccagctgctcc
ctgagccttggcttccgggagtggccaaggcggggaggggctgtgcagggcagc
tggagggcactgtcagctggggcccagcaccccctcaccccggcagggcccggg
ctccgaggggccccgcagtcgcaggccctgctcttgggggaagccctacttggc
cccttcagggcgctgacgctccccccacccacccccgcctagATCCCAACTCTC
CCATCACCGTCTTCGCCATCGCCCCCTCCTTCGCTGGCATCTTCCTCACCAAGT
CGGCGAAGCTTTCCTGCCTGGTCAGGGGCCTCGTCACCAGGGAGAGCGTCAACA
TCTCCTGGACCCGCCAGGACGGCGAGGTTCTGAAGACCAGTATCGTCTTCTCTG
AGATCTACGCCAACGGCACCTTCGGCGCCAGGGGCGAAGCCTCCGTCTGCGTGG
AGGACTGGGAGTCGGGCGACAGGTTCACGTGCACGGTGACCCACACGGACCTGC
CCTCGCCGCTGAAGCAGAGCGTCTCCAAGCCCAGAGgtaggccctgccctgccc
ctgcctccgcccggcctgtgccttggccgccggggcgggagccgagcctggccg
aggagcgccctcggccccccgcggtcccgacccacacccctcctgctctcctcc
ccagGGATCGCCAGGCACATGCCGTCCGTGTACGTGCTGCCGCCGGCCCCGGAG
GAGCTGAGCCTGCAGGAGTGGGCCTCGGTCACCTGCCTGGTGAAGGGCTTCTCC
CCGGCGGACGTGTTCGTGCAGTGGCTGCAGAAGGGGGAGCCCGTGTCCGCCGAC
AAGTACGTGACCAGCGGGCCGGTGCCCGAGCCCGAGCCCAAGGCCCCCGCCTCC
TACTTCGTGCAGAGCGTCCTGACGGTGAGCGCCAAGGACTGGAGCGACGGGGAG
ACCTACACCTGCGTCGTGGGCCACGAGGCCCTGCCCCACACGGTGACCGAGAGG
ACCGTGGACAAGTCCACCGGTAAACCCACCCTGTACAACGTCTCCCTGGTCCTG
TCCGACACGGCCAGCACCTGCTACTGACCCCCTGGCTGCCCGCCGCGGCCGGGG
CCAGAGCCCCCGGGCGACCATCGCTCTGTGTGGGCCTGTGTGCAACCCGACCC
TGTCGGGGTGAGCGGTCGCATTTCTGAAAATTAGAaataaaAGATCTCGTGC
CG
Seq ID No. 1
TCTAgAAGACGCTGGAGAGAGGCCagACTTCCTGGGAACAGCTCAAAGAG
CTCTGTCAAAGCCAGATCCCATCACACGTGGGCACCAATAGGCCATGCCA
GCCTCCAAGGGCCGAACTGGGTTCTCCACGGCGCACATGAAGCCTGCAGC
CTGGCTTATCCTCTTCCGTGGTGAAGAGGCAGGCCCGGGACTGGACGAGG
GGCTAGCAGGGTGTGGTAGGCACCTTGCGCCCCCCACCCCGGCAGGAACC
AGAGACCCTGGGGCTGAGAGTGAGCCTCCAAACAGGATGCCCCACCCTTC
AGGCCACCTTTCAATCCAGCTACACTCCACCTGCCATTCTCCTCTGGGCA
CAGGGCCCAGCCCCTGGATCTTGGCCTTGGCTCGACTTGCACCGACGCGC
ACACACACACTTCCTAACGTGCTGTCCGCTCACCCCTCCCCAGCGTGGTC
CATGGGCAGCACGGCAGTGCGCGTCCGGCGGTAGTGAGTGCAGAGGTCCC
TTCCCCTCCCCCAGGAGCCCCAGGGGTGTGTGCAGATCTGGGGGCTCCTG
TCCCTTACACCTTCATGCCCCTCCCCTCATACCCACCCTCCAGGCGGGAG
GCAGCGAGACCTTTGCCCAGGGACTCAGCCAACGGGCACACGGGAGGCC
A GCCCTCAGCAGCTGGG
Seq ID No. 4
GGCCAGACTTCCTCGGAACAGCTCAAAGAGCTCTGTCAAAGCCAGATCCC
0
ATCACACGTGGGCACCAATAGGCCATGCCAGCCTCCAAGGGCCGAACTGG
GTTCTCCACGGCGCACATGAAGCCTGCAGCCTGGCTTATCCTCTTCCGTG
GTGAAGAGGCAGGCCCGGGACTGGACGAGGGGCTAGCAGGGTGTGGTAG
GCACCTTGCGCCCCCCACCCCGGCAGGAACCAGAGACCCTGGGGCTGAGA
G
TGAGCCTCCAAACAGGATGCCCCACCCTTCAGGCCACCTTTCAATCCAGC
TACACTCCACCTGCCATTCTCCTCTGGGCACAGGGCCCAGCCCCTGGATC
TTGGCCTTGGCTCGACTTGCACCCACGCGCACACACACACTTCCTAACGT
GCTGTCCGCTCACCCCTCCCCAGCGTGGTCCATGGGCAGCACGGCAGTGC
GCGTCCGGCGGTAGTGAGTGCAGAGGTCCCTTCCCCTCCCCCAGGAGCCC
CAGGGGTGTGTGCAGATCTGGGGGCTCCTGTCCCTTACACCTTCATGCCC
CTCCCCTCATACCCACCCTCCAGGCGGGAGGCAGCGAGACCTTTGCCCAG
GGACTCAGCCAACGGGCACACGGGAGGCCAGCCCTCAGCAGCTGGCTCCC
AAAGAGGAGGTGGGAGGTAGGTCCACAGCTGCCACAGAGAGAAACCCTG
ACGGACCCCACAGGGGCCACGCCAGCCGGAACCAGCTCCCTCGTGGGTGA
GCAATGGCCAGGGCCCCGCCGGCCACCACGGCTGGCCTTGCGCCAGCTGA
G
AACTCACGTCCAGTGCAGGGAGACTCAAGACAGCCTGTGCACACAGCCTC
GGATCTGCTCCCATTTCAAGCAGAAAAAGGAAACCGTGCAGGCAGCCCTC
AGCATTTCAAGGATTGTAGCAGCGGCCAACTATTCGTCGGCAGTGGCCGA
TTAGAATGACCGTGGAGAAGGGCGGAAGGGTCTCTCGTGGGCTCTGCGGC
CAACAGGCCCTGGCTCCACCTGCCCGCTGCCAGCCCGAGGGGCTTGGGCC
GAGCCAGGAACCACAGTGCTCACCGGGACCACAGTGACTGACCAAACTCC
CGGCCAGAGCAGCCCCAGGCCAGCCGGGCTCTCGCCCTGGAGGACTCACC
ATCAGATGCACAAGGGGGCGAGTGTGGAAGAGACGTGTCGCCCGGGCCA
T
TTGGGAAGGCGAAGGGACCTTCCAGGTGGACAGGAGGTGGGACGCACTC
C
AGGCAAGGGACTGGGTCCCCAAGGCCTGGGGAAGGGGTACTGGCTTGGG
G
GTTAGCCTGGCCAGGGAACGGGGAGCGGGGCGGGGGGCTGAGCAGGGAG
G
ACCTGACCTCGTGGGAGCGAGGCAAGTCAGGCTTCAGGCAGCAGCCGCAC
ATCCCAGACCAGGAGGCTGAGGCAGGAGGGGCTTGCAGCGGGGCGGGGG
C
CTGCCTGGCTCCGGGGGCTCCTGGGGGACGCTGGCTCTTGTTTCCGTGTC
CCGCAGCACAGGGCCAGCTCGCTGGGCCTATGCTTACCTTGATGTCTGGG
GCCGGGGCGTCAGGGTCGTCGTCTCCTCAGGGGAGAGTCCCCTGAGGCTA
CGCTGGGG*GGGGACTATGGCAGCTCCACCAGGGGCCTGGGGACCAGGG
G
CCTGGACCAGGCTGCAGCCCGGAGGACGGGCAGGGCTCTGGCTCTCCAGC
ATCTGGCCCTCGGAAATGGCAGAACCCCTGGCGGGTGAGCGAGCTGAGA
G
CGGGTCAGACAGACAGGGGCCGGCCGGAAAGGAGAAGTTGGGGGCAGAG
C
CCGCCAGGGGCCAGGCCCAAGGTTCTGTGTGCCAGGGCCTGGGTGGGCAC
ATTGGTGTGGCCATGGCTACTTAGATTCGTGGGGCCAGGGCATCCTGGTC
ACCGTCTCCTCAGGTGAGCCTGGTGTCTGATGTCCAGCTAGGCGCTGGTG
GGCCGCGGGTGGGCCTGTCTCAGGCTAGGGCAGGGGCTGGGATGTGTATT
TGTCAAGGAGGGGCAACAGGGTGCAGACTGTGCCCCTGGAAACTTGACCA
CTGGGGCAGGGGCGTCCTGGTCACGTCTCCTCAGGTAAGACGGCCCTGTG
CCCCTCTCTCGCGGGACTGGAAAAGGAATTTTCCAAGATTCCTTGGTCTG
TGTGGGGCCCTCTGGGGCCCCCGGGGGTGGCTCCCCTCCTGCCCAGATGG
GGCCTCGGCCTGTGGAGCACGGGCTGGGCACACAGCTCGAGTCTAGGGCC
ACAGAGGCCCGGGCTCAGGGCTCTGTGTGGCCCGGCGACTGGCAGGGGG
C
TCGGGTTTTTGGACACCCCCTAATGGGGGCCACAGCACTGTGACCATCTT
CACAGCTGGGGCCGAGGAGTCGAGGTCACCGTCTCCTCAGGTGAGTCCTC
GTCAGCCCTCTCTCACTCTCTGGGGGGTTTTGCTGCATTTTGTGGGGGAA
AGAGGATGCCTGGGTCTCAGGTCTAAAGGTCTAGGGCCAGCGCCGGGGCC
CAGGAAGGGGCCGAGGGGCCAGGCTCGGCTCGGCCAGGAGCAGAGCTTC
C
AGACATCTCGCCTCCTGGCGGGTGCAGTCAGGCCTTTGGCCGGGGGGGTC
TCAGCACCACGAGGCCTCTTGGCTCCCGAGGTGGCCGGCCCCGGCTGCCT
CACCAGGCACCGTGCGCGGTGGGCCCGGGCTCTTGGTCGGCCACCCTTTC
TTAACTGGGATCCGGGCTTAGTTGTCGCAATGTGACAACGGGCTCGAAAG
CTGGGGCCAGGGGACCCTAGT*TAGGACGCCTCGGGTGGGTGTCCCGCAC
CCCTCCCCACTTTCACGGCACTCGGCGAGACCTGGGGAGTCAGGTGTTGG
GGACACTTTGGAGGTCAGGAACGGGAGCTGGGGAGAGGGCTCTGTCAGC
G
GGGTCCAGAGATGGGCCGCCCTCCAAGGACGCCCTGCGCGGGGACAAGG
G
CTTCTTGGCCTGGCCTGGCCGCTTCACTTGGGCGTCAGGGGGGGCTTCCC
GGGGCAGGCGGTCAGTCGAGGCGGGTTGGAATTCTGAGTCTGGGTTCGGG
GTTCGGGGTTCGGCCTTCATGAACAGACAGCCCAGGCGGGCCGTTGTTTG
GCCCCTGGGGGCCTGGTTGGAATGCGAGGTCTCGGGAAGTCAGGAGGGA
G
CCTGGCCAGCAGAGGGTTCCCAGCCCTGCGGCCGAGGGACCTGGAGACG
G
GCAGGGCATTGGCCGTCGCAGGGCCAGGCCACACCCCCCAGGTTTTTGTG
GGGCGAGCCTGGAGATTGCACCACTGTGATTACTATGCTATGGATCTCTG
GGGCCCAGGCGTTGAAGTCGTCGTGTCCTCAGGTAAGAACGGCCCTCCAG
GGCCTTTAATTTCTGCTCTCGTCTGTGGGCTTTTCTGACTCTGATCCTCG
GGAGGCGTCTGTGCCCCCCCCGGGGATGAGGCCGGCTTGCCAGGAGGGGT
CAGGGACCAGGAGCCTGTGGGAAGTTCTGACGGGGGCTGCAGGCGGGAA
G
GGCCCCACCGGGGGGCGAGCCCCAGGCCGCTGGGCGGCAGGAGACCCGT
G
AGAGTGCGCCTTGAGGAGGGTGTCTGCGGAACCACGAACGCCCGCCGGG
A
AGGGCTTGCTGCAATGCGGTCTTCAGACGGGAGGCGTCTTCTGCCCTCAC
CGTCTTTCAAGCCCTTGTGGGTCTGAAAGAGCCATGTCGGAGAGAGAAGG
GACAGGCCTGTCCCGACCTGGCCGAGAGCGGGCAGCCCCGGGGGAGAGC
G
GGGCGATCGGCCTGGGCTCTGTGAGGCCAGGTCCAAGGGAGGACGTGTG
G
TCCTCGTGACAGGTGCACTTGCGAAACCTTAGAAGACGGGGTATGTTGGA
AGCGGCTCCTGATGTTTAAGAAAAGGGAGACTGTAAAGTGAGCAGAGTCC
TCAAGTGTGTTAAGGTTTTAAAGGTCAAAGTGTTTTAAACCTTTGTGACT
GCAGTTAGCAAGCGTGCGGGGAGTGAATGGGGTGCCAGGGTGGCCGAGA
G
GCAGTACGAGGGCCGTGCCGTCCTCTAATTCAGGGCTTAGTTTTGCAGAA
TAAAGTCGGCCTGTTTTCTAAAAGCATTGGTGGTGCTGAGCTGGTGGAGG
AGGCCGCGGGCAGCCCTGGCCACCTGCAGCAGGTGGCAGGAAGCAGGTC
G
GCCAAGAGGCTATTTTAGGAAGCCAGAAAACACGGTCGATGAATTTATAG
CTTCTGGTTTCCAGGAGGTGGTTGGGCATGGCTTTGCGCAGCGCCACAGA
ACCGAAAGTGCCCACTGAGAAAAAACAACTCCTGCTTAATTTGCATTTTT
CTAAAAGAAGAAACAGAGGCTGACGGAAACTGGAAAGTTCCTGTTTTAAC
TACTCGAATTGAGTTTTCGGTCTTAGCTTATCAACTGCTCACTTAGATTC
ATTTTCAAAGTAAACGTTTAAGAGCCGAGGCATTCCTATCCTCTTCTAAG
GCGTTATTCCTGGAGGCTCATTCACCGCCAGCACCTCCGCTGCCTGCAGG
CATTGCTGTCACCGTCACCGTGACGGCGCGCACGATTTTCAGTTGGCCCG
CTTCCCCTCGTGATTAGGACAGACGCGGGCACTCTGGCCCAGCCGTCTTG
GCTCAGTATCTGCAGGCGTCCGTCTCGGGACGGAGCTCAGGGGAAGAGCG
TGACTCCAGTTGAACGTGATAGTCGGTGCGTTGAGAGGAGACCCAGTCGG
GTGTCGAGTCAGAAGGGGCCCGGGGCCCGAGGCCCTGGGCAGGACGGCC
C
GTGCCCTGCATCACGGGCCCAGCGTCCTAGAGGCAGGACTCTGGTGGAGA
GTGTGAGGGTGCCTGGGGCCCCTCCGGAGCTGGGGCCGTGCGGTGCAGGT
TGGGCTCTCGGCGCGGTGTTGGCTGTTTCTGCGGGATTTGGAGGAATTCT
TCCAGTGATGGGAGTCGCCAGTGACCGGGCACCAGGGTGGTAAGAGGGA
G
GCGGCCGTCGTGGCCAGAGCAGCTGGGAGGGTTCGGTAAAAGGCTCGCCC
GTTTCCTTTAATGAGGACTTTTCCTGGAGGGCATTTAGTCTAGTCGGGAC
CGTTTTCGACTCGGGAAGAGGGATGCGGAGGAGGGCATGTGCCCAGGAG
C
CGAAGGCGCCGCGGGGAGAAGCCCAGGGCTCTCCTGTCCCCACAGAGGC
G
ACGCCACTGCCGCAGACAGACAGGGCCTTTCCCTCTGATGACGGCAAAGG
CGCCTCGGCTCTTGCGGGGTGCTGGGGGGGAGTCGCCCCGAAGCCGCTCA
CCCAGAGGCCTGAGGGGTGAGACTGACCGATGCCTCTTGGCCGGGCCTGG
GGCCGGACCGAGGGGGACTCCGTGGAGGCAGGGCGATGGTGGCTGCGGG
A
GGGAACCGACCCTGGGCCGAGCCCGGCTTGGCGATTCCCGGGCGAGGGCC
CTCAGCCGAGGCGAGTGGGTCCGGCGGAACCACCCTTTCTGGCCAGCGCC
ACAGGGCTCTCGGGACTGTCCGGGGCGACGCTGGGCTGCCCGTGGCAGGC
CTGGGCTGACCTGGACTTCACCAGACAGAACAGGGCTTTCAGGGCTGAGC
TGAGCCAGGTTTAGCGAGGCCAAGTGGGGCTGAACCAGGCTCAACTGGCC
TGAGCTGGGTTGAGCTGGGCTGACCTGGGCTGAGCTGAGCTGGGCTGGGC
TGGGCTGGGGTGGGCTGGGCTGGGCTGGACTGGCTGAGCTGAGCTGGGTT
GAGCTGAGCTGAGCTGGCCTGGGTTGAGCTGGGCTGGGTTGAGCTGAGCT
GGGTTGAGCTGGGTTGAGCTGGGTTGATCTGAGCTGAGCTGGGCTGAGCT
GAGCTAGGCTGGGGTGAGCTGGGCTGAGCTGGTTTGAGTTGGGTTGAGCT
GAGCTGAGCTGGGCTGTGCTGGCTGAGCTAGGCTGAGCTAGGCTAGGTTG
AGCTGGGCTGGGCTGAGCTGAGCTAGGCTGGGCTGATTTGGGCTGAGCTG
AGCTGAGCTAGGCTGCGTTGAGCTGGGTGGGCTGGATTGAGCTGGCTGAG
CTGGCTGAGCTGGGCTGAGCTGGCCTGGGTTGAGCTGAGCTGGACTGGTT
TGAGCTGGGTCGATCTGGGTTGAGCTGTCCTGGGTTGAGCTGGGCTGGGT
TGAGCTGAGCTGGGTTGAGCTGGGCTCAGCAGAGCTGGGTTGGGCTGAGC
TGGGTTGAGCTGAGCTGGGCTGAGCTGGCCTGGGTTGAGCTGGGCTGAGC
TGAGCTGGGCTGAGCTGGCCTGTGTTGAGCTGGGCTGGGTTGAGCTGGGC
TGAGCTGGATTGAGCTGGGTTGAGCTGAGCTGGGCTGGGCTGTGCTGACT
GAGCTGGGCTGAGCTAGGCTGGGGTGAGCTGGGCTGAGCTGATCCGAGCT
AGGCTGGGCTGGTTTGGGCTGAGCTGAGCTGAGCTAGGCTGGATTGATCT
GGCTGAGCTGGGTTGAGCTGAGCTGGGCTGAGCTGGTCTGAGCTGGGCTG
GGTCGAGCTGAGCTGGACTGGTTTGAGCTGGGTCGATCTGGGCTGAGCTG
GCCTGGGTTGAGCTGGGCTGGGTTGAGCTGAGCTGGGTTGAGCTGGGCTG
AGCTGAGGGCTGGGGTGAGCTGGGCTGAACTAGCCTAGCTAGGTTGGGCT
GAGCTGGGCTGGTTTGGGCTGAGCTGAGCTGAGCTAGGCTGCATTGAGCA
GGCTGAGCTGGGCTGAGCAGGCCTGGGGTGAGCTGGGCTAGGTGGAGCT
G
AGCTGGGTCGAGCTGAGTTGGGCTGAGCTGGCCTGGGTTGAGGTAGGCTG
AGCTGAGCTGAGCTAGGCTGGGTTGAGCTGGCTGGGCTGGTTTGCGCTGG
GTCAAGCTGGGCCGAGCTGOCCTGGGTTGAGCTGGGCTCGGTTGAGCTGG
GCTGAGCTGAGCCGACCTAGGCTGGGATGAGCTGGGCTGATTTGGGCTGA
GCTGAGCTGAGCTAGGCTGCATTGAGCAGGCTGAGCTGGGCCTGGAGCCT
GGCCTGGGGTGAGCTGGGCTGAGCTGCGCTGAGCTAGGCTGGGTTGAGCT
GGCTGGGCTGGTTTGCGCTGGGTCAAGCTGGGCCGAGCTGGCCTGGGATG
AGCTGGGCCGGTTTGGGCTGAGCTGAGCTGAGCTAGGCTGCATTGAGCAG
GCTGAGCTGGGCTGAGCTGGCCTGGGGTGAGCTGGGCTGAGCTAAGCTGA
GCTGGGCTGGTTTGGGCTGAGCTGGCTGAGCTGGGTCCTGCTGAGCTGGG
CTGAGCTGACCAGGGGTGAGCTGGGCTGAGTTAGGCTGGGCTCAGCTAGG
CTGGGTTGATCTGGCAGGGCTGGTTTGCGCTGGGTCAAGCTCCCGGGAGA
TGGCCTGGGATGAGCTGGGCTGGTTTGGGCTGAGCTGAGCTGAGCTGAGC
TAGGCTGCATTGAGCAGGCTGAGCTGGGCTGAGCTGGCCTGGGGTGAGCT
GGGCTGGGTGGAGCTGAGCTGGGCTGAACTGGGCTAAGCTGGCTGAGCTG
GATCGAGCTGAGCTGGGCTGAGCTGGCCTGGGGTTAGCTGGGCTGAGCTG
AGCTGAGCTAGGCTGGGTTGAGCTGGCTGGGCTGGTTTGCGCTGGGTCAA
GCTGGGCCGAGCTGGCCTGGGTTGAGCTGGGCTGGGCTGAGCTGAGCTAG
GCTGGGTTGAGCTGGGCTGGGCTGAGCTGAGCTAGGCTGCATTGAGCTGG
CTGGGATGGATTGAGCTGGCTGAGCTGGCTGAGCTGGCTGAGCTGGGCTG
AGCTGGCCTGGGTTGAGCTGGGCTGGGTTGAGCTGAGCTGGGCTGAGCTG
GGCTCAGCAGAGCTGGGTTGAGCTGAGCTGGGTTGAGCTGGGGTGAGCTG
GGCTGAGCAGAGCTGGGTTGAGCTGAGCTGGGTTGAGCTGGGCTCGAGCA
GAGCTGGGTTGAGCTGAGCTGGGTTGAGCTGGGCTCAGCAGAGCTGGGTT
GAGCTGAGCTGGGTTGAGCTGGGCTGAGCTAGCTGGGCTCAGCTAGGCTG
GGTTGAGCTGAGCTGGGCTGAACTGGGCTGAGCTGGGCTGAACTGGGCTG
AGCTGGGGTGAGCTGGGCTGAGCAGAGCTGGGCTGAGCAGAGCTGGGTT
G
GTCTGAGCTGGGTTGAGCTGGGCTGAGCTGGGCTGAGCAGAGTTGGGTTG
AGCTGAGCTGGGTTCAGCTGGGCTGAGCTAGGCTGGGTTGAGCTGGGTTG
AGTTGGGCTGAGCTGGGCTGGGTTGAGCGGAGCTGGGCTGAACTGGGCTG
AGCTGGGCTGAGCGGAACTGGGTTGATCTGAATTGAGCTGGGCTGAGCCG
GGCTGAGCCGGGCTGAGCTGGGCTAGGTTGAGCTTGGGTGAGCTTGCCTC
AGCTGGTCTGAGCTAGGTTGGGTGGAGCTAGGCTGGATTGAGCTGGGCTG
AGCTGAGCTGATCTGGCCTCAGCTGGGCTGAGGTAGGCTGAACTGGGCTG
TGCTGGGCTGAGCTGAGCTGAGCCAGTTTGAGCTGGGTTGAGCTGGGCTG
AGCTGGGCTGTGTTGATCTTTCCTGAACTGGGCTGAGCTGGGCTGAGCTG
GCCTAGCTGGATTGAACGGGGGTAAGCTGGGCCAGGCTGGACTGGGCTGA
GCTGAGCTAGGCTGAGCTGAGTTGAATTGGGTTAAGCTGGGCTGAGATGG
GCTGAGCTGGGCTGAGCTGGGTTGAGCCAGGTCGGACTGGGTTACCCTGG
GCCACACTGGGCTGAGCTGGGCGGAGCTCGATTAACCTGGTCAGGCTGAG
TCGGGTCCAGCAGACATGCGCTGGCCAGGCTGGCTTGACCTGGACACGTT
CGATGAGCTGCCTTGGGATGGTTCACCTCAGCTGAGCCAGGTGGCTCCAG
CTGGGCTGAGCTGGTGACCCTGGGTGACCTCGGTGACCAGGTTGTCCTGA
GTCCGGGCCAAGCCGAGGCTGCATCAGACTCGCCAGACCCAAGGCCTGGG
CCCCGGCTGGCAAGCCAGGGGCGGTGAAGGCTGGGCTGGCAGGACTGTC
CCGGAAGGAGGTGCACGTGGAGCCGCCCGGACCCCGACCGGCAGGACCT
GGAAAGACGCCTCTCACTCCCCTTTCTCTTCTGTCCCCTCTCGGGTCCTCA
GAGAGCCAGTCTGCCCCGAATCTCTACCCCCTCGTCTCCTGCGTCAGCCCC
CCGTCCGATGAGAGCCTGGTGGCCCTGGGCTGCCTGGCCCGGGACTTCCT
GCCCAGCTCCGTCACCTTCTCCTGGAA
Porcine Kappa Light Chain
In another embodiment, novel genomic sequences encoding the kappa light chain locus of ungulate immunoglobulin are provided. The present invention provides the first reported genomic sequence of ungulate kappa light chain regions. In one embodiment, nucleic acid sequence is provided that encodes the porcine kappa light chain locus. In another embodiment, the nucleic acid sequence can contain at least one joining region, one constant region and/or one enhancer region of kappa light chain. In a further embodiment, the nucleotide sequence can include at least five joining regions, one constant region and one enhancer region, for example, as represented in Seq ID No. 30. In a further embodiment, an isolated nucleotide sequence is provided that contains at least one, at least two, at least three, at least four or five joining regions and 3′ flanking sequence to the joining region of porcine genomic kappa light chain, for example, as represented in Seq ID No 12. In another embodiment, an isolated nucleotide sequence of porcine genomic kappa light chain is provided that contains 5′ flanking sequence to the first joining region, for example, as represented in Seq ID No. 25. In a further embodiment, an isolated nucleotide sequence is provided that contains 3′ flanking sequence to the constant region and, optionally, the 5′ portion of the enhancer region, of porcine genomic kappa light chain, for example, as represented in Seq ID Nos. 15, 16 and/or 19.
In further embodiments, isolated nucleotide sequences as depicted in Seq ID Nos 30, 12, 25, 15, 16 or 19 are provided. Nucleic acid sequences at least 80, 85, 90, 95, 98 or 99% homologous to Seq ID Nos 30, 12, 25, 15, 16 or 19 are also provided. In addition, nucleotide sequences that contain at least 10, 15, 17, 20, 25 or 30 contiguous nucleotides of Seq ID Nos 30, 12, 25, 15, 16 or 19 are provided. In addition, nucleotide sequences that contain at least 10, 15, 17, 20, 25 or 30 contiguous nucleotides of Seq ID Nos 1, 4 or 29 are provided. In other embodiments, nucleotide sequences that contain at least 50, 100, 1,000, 2,500, 5,000, 7,000, 8,000, 8,500, 9,000, 10,000 or 15,000 contiguous nucleotides of Seq ID No. 30 are provided. Further provided are nucleotide sequences that hybridizes, optionally under stringent conditions, to Seq ID Nos 30, 12, 25, 15, 16 or 19, as well as, nucleotides homologous thereto.
In one embodiment, an isolated nucleotide sequence encoding kappa light chain is provided that includes at least five joining regions, one constant region and one enhancer region, for example, as represented in Seq ID No. 30. In Seq ID No. 30, the coding region of kappa light chain is represented, for example by residues 1-549 and 10026-10549, whereas the intronic sequence is represented, for example, by residues 550-10025, the Joining region of kappa light chain is represented, for example, by residues 5822-7207 (for example, J1:5822-5859, J2:6180-6218, J3:6486-6523, J4:6826-6863, J5:7170-7207), the Constant Region is represented by the following residues: 10026-10549 (C exon) and 10026-10354 (C coding), 10524-10529 (Poly(A) signal) and 11160-11264 (SINE element).
Seq ID No 30
GCGTCCGAAGTCAAAAATATCTGCAGCCTTCATGTATTCATAGAAACAAG
GAATGTCTACATTTTCCAAAGTGGGACCAGAATCTTGGGTGATGTCTAAG
GCATGTGCATTTGCACATGGTAGGCAAAGGACTTTGCTTCTCCCAGCACA
TCTTTCTGCAGAGATCCATGGAAACAAGACTCAACTCCAAAGCAGCAAAG
AAGCAGCAAGTTCTCAAGTGATCTCCTCTGACTCCCTCCTCCCAGGCTAA
TGAAGCCATGTTGCCCCTGGGGGATTAAGGGCAGGTGTCCATTGTGGCAC
CCAGCCCGAAGACAAGCAATTTGATCAGGTTCTGAGCACTCCTGAATGTG
GACTCTGGAATTTTCTCCTCACCTTGTGGCATATCAGCTTAAGTCAAGTA
CAAGTGACAAACAACATAATCCTAAGAAGAGAGGAATCAAGCTGAAGTC
A
AAGGATCACTGCCTTGGATTCTACTGTGAATGATGACCTGGAAAATATCC
TGAACAACAGCTTCAGGGTGATCATCAGAGACAAAAGTTCCAGAGCCAGg
tagggaaaccctcaagccttgcaaagagcaaaatcatgccattgggttct
taacctgctgagtgatttactatatgttactgtgggaggcaaagcgctca
aatagcctgggtaagtatgtcaaataaaaagcaaaagtggtgtttcttga
aatgttagacctgaggaaggaatattgataacttaccaataattttcaga
atgatttatagatgtgcacttagtcagtgtctctccaccccgcacctgac
aagcagtttagaatttattctaagaatctaggtttgctgggggctacatg
ggaatcagcttcagtgaagagtttgttggaatgattcactaaattttcta
tttccagcataaatccaagaacctctcagactagtttattgacactgctt
ttcctccataatccatctcatctccgtccatcatggacactttgtagaat
gacaggtcctggcagagactcacagatgcttctgaaacatcctttgcctt
caaagaatgaacagcacacatactaaggatctcagtgatccacaaattag
tttttgccacaatggttcttatgataaaagtctttcattaacagcaaatt
gttttataatagttgttctgctttataataattgcatgcttcactttctt
ttcttttctttttttttctttttttgctttttagtgccgcaggtgcagca
tatgaaatttcccaggctaggggtcaaatcagaactacacctactggcct
acgccacagccacagcaactcaggatctaagccatgtcggtgacctacac
tacagctcatggcaatgccagatccttaacccaatgagcgaggccaggga
tcgaacccatgtcctcatggatactagtcaggctcattatccgctgagcc
ataacaggaactcccgagtttgctttttatcaaaattggtacagccttat
tgtttctgaaaaccacaaaatgaatgtattcacataattttaaaaggtta
aataatttatgatatacaagacaatagaaagagaaaacgtcattgcctct
ttcttccacgacaacacgcctccttaattgatttgaagaaataactactg
agcatggtttagtgtacttctttcagcaattagcctgtattcatagccat
acatattcaattaaaatgagatcatgatatcacacaatacataccataca
gcctatagggatttttacaatcatcttccacatgactacataaaaaccta
cctaaaaaaaaaaaaaaccctacttcatcctcctattggctgctttgtgc
tccattaaaaagctctatcataattaggttatgatgaggatttccatttt
ctacctttcaagcaacatttcaatgcacagtcttatatacacatttgagc
ctacttttctttttctttctttttttggtttttttttttttttttttttt
ggtctttttgtcttttctaaggctgcatatggaggttcccaggctagctg
tctaatcagaactatagctgctggcctacgccacatccacagcaatacaa
gatctgagccatgtctgcaacttacaccacagctcacagcaacggtggat
ccttaaaccactgagcaaggccagggatcaaacccataacttcatggctc
ctagttggatttgttaaccactgagccatgatggcaactcctgagcctac
ttttctaatcatttccaaccctaggacacttttttaagtttcatttttct
ccccccaccccctgttttctgaagtgtgtttgcttccactgggtgacttc
actcccaggatctcatctgcaggatactgcagctaagtgtatgagctctg
aatttgaatcccaactctgccactcaaagggataggagtttccgatgtgg
cccaatgggatcagtggcatctctgcagtgccaggacgcaggttccatcc
ctggcccagcacagtgggttaagaatctggcattgctgcagctgaggcat
agatttcaattgtgcctcagatctgatccttggcccaaggactgcatatg
cctcagggcaaccaaaaaagagaaaaggggggtgatagcattagtttcta
gatttgggggataattaaataaagtgatccatgtacaatgtatggcattt
tgtaaatgctcaacaaatttcaactattatggagttcccatcatggctca
gtggaagggaatctgattagcatccatgaggacacaggtccaaccccgac
cttgctcagtgggcattgctgtgagctgtggcatgggttacagacgaagc
tcggatctggcattgctgtggctgtggtgtaagccagcaactacagctct
cattcagcccctagcctgggaacctccatatgcctaaaagacaaaaaata
aaatttaaattaaaaataaagaaatgttaactattatgattggtactgct
tgcattactgcaaagaaagtcactttctatactctttaatatcttagttg
actgtgtgctcagtgaactattttggacacttaatttccactctcttcta
tctccaacttgacaactctctttcctctcttctggtgagatccactgctg
actttgctctttaaggcaactagaaaagtgctcagtgacaaaatcaaaga
aagttaccttaatcttcagaattacaatcttaagttctcttgtaaagctt
actatttcagtggttagtattattccttggtcccttacaacttatcagct
ctgatctattgctgattttcaactatttattgttggagttttttcctttt
ttccctgttcattctgcaaatgtttgctgagcatftgtcaagtgaagata
ctggactgggccttccaaatataagacaatgaaacatcgggagttctcat
tatggtgcagcagaaacgaatccaactaggaaatgtgaggttgcaggttc
gatccctgcccttgctcagtgggttaaggatccagcattaccgtgagctg
tggtgtaggttgcagacgtggctcagatcctgcgttgctgtggctgtggc
ataggctggcagctctagctctgattcgaccgctagcctgggaacctcca
tgcgccccgagtgcagcccttaaaaagcaaaaaaaaaagaaagaaagaaa
aagacaatgaaacatcaaacagctaacaatccagtagggtagaaagaatc
tggcaacagataagagcgattaaatgttctaggtccagtgaccttgcctc
tgtgctctacacagtcgtgccacttgctgagggagaaggtctctcttgag
ttgagtcctgaaagacattagttgttcacaaactaatgccagtgagtgaa
ggtgtttccaagcagagggagagtttggtaaaaagctggaagtcacagaa
agactctaaagagtttaggatggtgggagcaacatacgctgagatggggc
tggaaggttaagagggaaacaactatagtaagtgaagctggactcacagc
aaagtgaggacctcagcatccttgatggggttaccatggaaacaccaagg
cacaccttgatttccaaaacagcaggcacctgattcagcccaatgtgaca
tggtgggtacccctctagctctacctgttctgtgacaactgacaaccaac
gaagttaagtctggattttctactctgctgatccttgtttttgtttcaca
cgtcatctatagcttcatgccaaaatagagttcaaggtaagacgcgggcc
ttggtttgatatacatgtagtctatcttgtttgagacaatatggtggcaa
ggaagaggttcaaacaggaaaatactctctaattatgattaactgagaaa
agctaaagagtcccataatgacactgaatgaagttcatcatttgcaaaag
ccttcccccccccccaggagactataaaaaagtgcaattttttaaatgaa
cttatttacaaaacagaaatagactcacagacataggaaacgaacagatg
gttaccaagggtgaaagggagtaggagggataaataaggagtctggggtt
agcagatacaccccagtgtacacaaaataaacaacagggacctactatat
agcacagggaactatatgcagtagcttacaataacctataatggaaaaga
atgtgaaaaagaatatatgtatgcgtgtgtgtgtaactgaatcactttgc
tgtaacctgaatctaacataacattgtaaatcaactacagtttttttttt
ttttaagtgcagggttttggtgttttttttttttcatttttgtttttgtt
tttgttttttgctttttagggccacacccagacatatgggggttcccagg
ctaggggtctaattagagctacagttgccggcttgcaccacagccacagc
aacatcagatccgagccgcacttgcgacttacaccacagctcatggcaat
accagatccttaacccactgagcaaggcccagggatcgtacccgcaacct
catggttcctagtcagattcatttctgctgcgctacaatgggaactccaa
gtgcagttttttgtaatgtgcttgtctttctttgtaattcatattcatcc
tacttcccaataaataaataaatacataaataataaacataccattgtaa
atcaactacaattttttttaaatgcagggtttttgttttttgttttttgt
tttgtctttttgccttttctagggccgctcccatggcatatggaggttcc
caggctaggggtcgaatcggagctgtagccaccggcctacgccagagcca
cagcaacgcgggatccgagccgcgtctgcaacctacaccacagctcacgg
caacgccggatcgttaacccactgagcaagggcagggatcgaacctgcaa
cctcatggttcctagtcagattcgttaactactgagccacaacggaaact
cctaaagtgcagtttttaaatgtgcttgtctttctttgtaatttacactc
aacctacttcccaataaataaataaataaacaaataaatcatagacatgg
ttgaattctaaaggaagggaccatcaggccttagacagaaatacgtcatc
ttctagtattttaaaacacactaaagaagacaaacatgctctgccagaga
agcccagggcctccacagctgcttgcaaagggagttaggcttcagtagct
gacccaaggctctgttcctcttcagggaaaagggtttttgttcagtgaga
cagcagacagctgtcactgtgGTGGACGTTCGGCCAAGGAACCAAGCTGG
AACTCAAACgtaagtcaatccaaacgttccttccttggctgtctgtgtct
tacggtctctgtggctctgaaatgattcatgtgctgactctctgaaacca
gactgacattctccagggcaaaactaaagcctgtcatcaaactggaaaac
tgagggcacattttctgggcagaactaagagtcaggcactgggtgaggaa
aaacttgttagaatgatagtttcagaaacttactgggaagcaaagcccat
gttctgaacagagctctgctcaagggtcaggaggggaaccagtttttgta
caggagggaagttgagacgaacccctgtgTATATGGTTTCGGCGCGGGGA
CCAAGCTGGAGCTCAAACgtaagtggctttttccgactgattctttgctg
tttctaattgttggttggctttttgtccatttttcagtgttttcatcgaa
ttagttgtcagggaccaaacaaattgccttcccagattaggtaccaggga
ggggacattgctgcatgggagaccagagggtggctaatttttaacgtttc
caagccaaaataactggggaagggggcttgctgtcctgtgagggtaggtt
tttatagaagtggaagttaaggggaaatcgctatgGTTGACTTTTGGCTC
GGGGACCAAAGTGGAGCCCAAAAttgagtacattttccatcaattatttg
tgagatttttgtcctgttgtgtcatttgtgcaagtttttgacattttggt
tgaatgagccattcccagggacccaaaaggatgagaccgaaaagtagaaa
agagccaacttttaagctgagcagacagaccgaattgttgagtttgtgag
gagagtagggtttgtagggagaaaggggaacagatcgctggctttttctc
tgaattagcctttctcatgggactggcttcagagggggtttttgatgagg
gaagtgttctagagccttaactgtgGGTTGTGTTCGGTAGCGGGACCAAG
CTGGAAATCAAACgtaagtgcacttttctactcctttttctttcttatac
gggtgtgaaattggggacttttcatgtttggagtatgagttgaggtcagt
tctgaagagagtgggactcatccaaaaatctgaggagtaagggtcagaac
agagttgtctcatggaagaacaaagacctagttagttgatgaggcagcta
aatgagtcagttgacttgggatccaaatggccagacttcgtctgtaacca
acaatctaatgagatgtagcagcaaaaagagatttccattgaggggaaag
taaaattgttaatattgtgGATCACCTTTGGTGAAGGGACATCCGTGGAG
ATTGAACgtaagtattttttctctactaccttctgaaatttgtctaaatg
ccagtgttgacttttagaggcttaagtgtcagttttgtgaaaaatgggta
aacaagagcatttcatatttattatcagtttcaaaagttaaactcagctc
caaaaatgaatttgtagacaaaaagattaatttaagccaaattgaatgat
tcaaaggaaaaaaaaattagtgtagatgaaaaaggaattcttacagctcc
aaagagcaaaagcgaattaattttctttgaactttgccaaatcttgtaaa
tgatttttgttctttacaatttaaaaaggttagagaaatgtatttcttag
tctgttttctctcttctgtctgataaattattatatgagataaaaatgaa
aattaataggatgtgctaaaaaatcagtaagaagttagaaaaatatatgt
ttatgttaaagttgccacttaattgagaatcagaagcaatgttattttta
aagtctaaaatgagagataaactgtcaatacttaaattctgcagagattc
tatatcttgacagatatctcctttttcaaaaatccaatttctatggtaga
ctaaatttgaaatgatcttcctcataatggagggaaaagatggactgacc
ccaaaagctcagatttaaagaaatctgtttaagtgaaagaaaataaaaga
actgcattttttaaaggcccatgaatttgtagaaaaataggaaatatttt
aataagtgtattcttttattttcctgttattacttgatggtgtttttata
ccgccaaggaggccgtggcaccgtcagtgtgatctgtagaccccatggcg
gccttttttcgcgattgaatgaccttggcggtgggtccccagggctctgg
tggcagcgcaccagccgctaaaagccgctaaaaactgccgctaaaggcca
cagcaaccccgcgaccgcccgttcaactgtgctgacacagtgatacagat
aatgtcgctaacagaggagaatagaaatatgacgggcacacgctaatgtg
gggaaaagagggagaagcctgatttttattttttagagattctagagata
aaattcccagtattatatccttttaataaaaaatttctattaggagatta
taaagaatttaaagctatttttttaagtggggtgtaattctttcagtagt
ctcttgtcaaatggatttaagtaatagaggcttaatccaaatgagagaaa
tagacgcataaccctttcaaggcaaaagctacaagagcaaaaattgaaca
cagcagccagccatctagccactcagattttgatcagttttactgagttt
gaagtaaatatcatgaaggtataattgctgataaaaaaataagatacagg
tgtgacacatctttaagtttcagaaatttaatggcttcagtaggattata
tttcacgtatacaaagtatctaagcagataaaaatgccattaatggaaac
ttaatagaaatatatttttaaattccttcattctgtgacagaaattttct
aatctgggtcttttaatcacctaccctttgaaagagtttagtaatttgct
atttgccatcgctgtttactccagctaatttcaaaagtgatacttgagaa
agattatttttggtttgcaaccacctggcaggactattttagggccattt
taaaactcttttcaaactaagtattftaaactgttctaaaccatttaggg
ccttttaaaaatcttttcatgaatttcaaacttcgttaaaagttattaag
gtgtctggcaagaacttccttatcaaatatgctaatagtttaatctgtta
atgcaggatataaaattaaagtgatcaaggcttgacccaaacaggagtat
cttcatagcatatttcccctcctttttttctagaattcatatgattttgc
tgccaaggctattttatataatctctggaaaaaaaatagtaatgaaggtt
aaaagagaagaaaatatcagaacattaagaattcggtattttactaactg
cttggttaacatgaaggtttttattttattaaggtttctatctttataaa
aatctgttcccttttctgctgatttctccaagcaaaagattcttgatttg
ttttttaactcttactctcccacccaagggcctgaatgcccacaaagggg
acttccaggaggccatctggcagctgctcaccgtcagaagtgaagccagc
cagttcctcctgggcaggtggccaaaattacagttgacccctcctggtct
ggctgaaccttgccccatatggtgacagccatctggccagggcccaggtc
tccctctgaagcctttgggaggagagggagagtggctggcccgatcacag
atgcggaaggggctgactcctcaaccggggtgcagactctgcagggtggg
tctgggcccaacacacccaaagcacgcccaggaaggaaaggcagcttggt
atcactgcccagagctaggagaggcaccgggaaaatgatctgtccaagac
ccgttcttgcttctaaactccgagggggtcagatgaagtggttttgtttc
ttggcctgaagcatcgtgttccctgcaagaagcggggaacacagaggaag
gagagaaaagatgaactgaacaaagcatgcaaggcaaaaaaggccttagg
atggctgcaggaagttagttcttctgcattggctccttactggctcgtcg
atcgcccacaaacaacgcacccagtggagaacttccctgttacttaaaca
ccattctctgtgcttgcttcctcagGGGCTGATGCCAAGCCATCCGTCTT
CATCTTCCCGCCATCGAAGGAGCAGTTAGCGACCCCAACTGTCTCTGTGG
TGTGCTTGATCAATAACTTCTTCCCCAGAGAAATCAGTGTCAAGTGGAAA
GTGGATGGGGTGGTCCAAAGCAGTGGTCATCCGGATAGTGTCACAGAGCA
GGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTCTCGCTGCCCA
CGTCACAGTACCTAAGTCATAATTTATATTCCTGTGAGGTCACCCACAAG
ACCCTGGCCTCCCCTCTGGTCACAAGCTTCAACAGGAACGAGTGTGAGGC
TtagAGGCCCACAGGCCCCTGGCCTGCCCCCAGCCCCAGCCCCCCTCCCC
ACCTGAAGCCTCAGGCCCTTGCCCCAGAGGATCCTTGGCAATCCCCCAGC
CCCTCTTCCCTCCTCATCCCCTCCCCCTCTTTGGCTTTAACCGTGTTAAT
ACTGGGGGGTGGGGGAATGAATAaataaaGTGAACCTTTGCACCTGTGAt
ttctctctcctgtctgattttaaggttgttaaatgttgttttccccatta
tagttaatcttttaaggaactacatactgagttgctaaaaactacaccat
cacttataaaattcacgccttctcagttctcccctcccctcctgtcctcc
gtaagacaggcctccgtgaaacccataagcacttctctttacaccctctc
ctgggccggggtaggagactttttgatgtcccctcttcagcaagcctcag
aaccattttgagggggacagttcttacagtcacat*tcctgtgatctaat
gactttagttaccgaaaagccagtctctcaaaaagaagggaacggctaga
aaccaagtcatagaaatatatatgtataaaatatatatatatccatatat
gtaaaataacaaaataatgataacagcataggtcaacaggcaacagggaa
tgttgaagtccattctggcacttcaatttaagggaataggatgccttcat
tacattttaaatacaatacacatggagagcttcctatctgccaaagacca
tcctgaatgccttccacactcactacaaggttaaaagcattcattacaat
gttgatcgaggagttcccgttgtggctcagcaggttaagaacgtgactgg
tatccaggaggatgcgggtttggtccccagcctcgctcagtggattaagg
atccagtgttgctgcaagatcacgggctcagatcccgtgttctatggcta
tggtgtaggctggtagctgcatgcagccctaatttgacccctagcctggg
aactgccatatgccacatgtgaggcccttaaaacctaaaagaaaaaaaaa
gaaaagaaatatcttacacccaatttatagataagagagaagctaaggtg
gcaggcccaggatcaaagccctacctgcctatcttgacacctgatacaaa
ttctgtcttctagggtttccaacactgcatagaacagagggtcaaacatg
ctaccctcccagggactcctcccttcaaatgacataaattttgttgccca
tctctgggggcaaaactcaacaatcaatggcatctctagtaccaagcaag
gctcttctcatgaagcaaaactctgaagccagatccatcatgacccaagg
aagtaaagacaggtgttactggttgaactgtatccttcaattcaatatgc
tcaatttccaactcccagtccccgtaaatacaaccccctttgggaagaga
gtccttgcagatgtagccacgttaaaaagagattatacagaaaggctagt
gaggatgcagtgaaacgggatctttcatacattgctggtggaaatgtaaa
atgctgcaggcactctagaaaataatttgccagttttttgaaaagctaaa
caaaatagtttagttgcattctgggttatttatcccccagaaattaaaaa
ttatgtccgcacaaaaacgtgtacataatcattcataacagccttgtac
Seq ID No. 12
caaggaaccaagctggaactcaaacgtaagtcaatccaaacgttccttcc
ttggctgtctgtgtcttacggtctctgtggctctgaaatgattcatgtgc
tgactctctgaaaccagactgacattctccagggcaaaactaaagcctgt
catcaaactggaaaactgagggcacattttctgggcagaactaagagtca
ggcactgggtgaggaaaaacttgttagaatgatagtttcagaaacttact
gggaagcaaagcccatgttctgaacagagctctgctcaagggtcaggagg
ggaaccagtttttgtacaggagggaagttgagacgaacccctgtgtatat
ggtttcggcgcggggaccaagctggagctcaaacgtaagtggctttttcc
gactgattctttgctgtttctaattgttggttggctttttgtccattttt
cagtgttttcatcgaattagttgtcagggaccaaacaaattgccttccca
gattaggtaccagggaggggacattgctgcatgggagaccagagggtggc
taatttttaacgtttccaagccaaaataactggggaagggggcttgctgt
cctgtgagggtaggtttttatagaagtggaagttaaggggaaatcgctat
ggttcacttttggctcggggaccaaagtggagcccaaaattgagtacatt
ttccatcaattatttgtgagatttttgtcctgttgtgtcatttgtgcaag
tttttgacattttggttgaatgagccattcccagggacccaaaaggatga
gaccgaaaagtagaaaagagccaacttttaagctgagcagacagaccgaa
ttgttgagtttgtgaggagagtagggtttgtagggagaaaggggaacaga
tcgctggctttttctctgaattagcctttctcatgggactggcttcagag
ggggtttttgatgagggaagtgttctagagccttaactgtgggttgtgtt
cggtagcgggaccaagctggaaatcaaacgtaagtgcacttttctactcc
tttttctttcttatacgggtgtgaaattggggacttttcatgtttggagt
atgagttgaggtcagttctgaagagagtgggactcatccaaaaatctgag
gagtaagggtcagaacagagttgtctcatggaagaacaaagacctagtta
gttgatgaggcagctaaatgagtcagttgacttgggatccaaatggccag
acttcgtctgtaaccaacaatctaatgagatgtagcagcaaaaagagatt
tccattgaggggaaagtaaaattgttaatattgtggatcacctttggtga
agggacatccgtggagattgaacgtaagtattttttctctactaccttct
gaaatttgtctaaatgccagtgttgacttttagaggcttaagtgtcagtt
ttgtgaaaaatgggtaaacaagagcatttcatatttattatcagtttcaa
aagttaaactcagctccaaaaatgaatttgtagacaaaaagattaattta
agccaaattgaatgattcaaaggaaaaaaaaattagtgtagatgaaaaag
gaattcttacagctccaaagagcaaaagcgaattaattttctttgaactt
tgccaaatcttgtaaatgatttttgttctttacaatttaaaaaggttaga
gaaatgtatttcttagtctgttttctctcttctgtctgataaattattat
atgagataaaaatgaaaattaataggatgtgctaaaaaatcagtaagaag
ttagaaaaatatatgtttatgttaaagttgccacttaattgagaatcaga
agcaatgttatttttaaagtctaaaatgagagataaactgtcaatactta
aattctgcagagattctatatcttgacagatatctcctttttcaaaaatc
caatttctatggtagactaaatttgaaatgatcttcctcataatggaggg
aaaagatggactgaccccaaaagctcagattt*aagaaaacctgtttaag
*gaaagaaaataaaagaactgcattttttaaaggcccatgaatttgtaga
aaaataggaaatattttaataagtgtattcttttattttcctgttattac
ttgatggtgtttttataccgccaaggaggccgtggcaccgtcagtgtgat
ctgtagaccccatggcggccttttttcgcgattgaatgaccttggcggtg
ggtccccagggctctggtggcagcgcaccagccgctaaaagccgctaaaa
actgccgctaaaggccacagcaaccccgcgaccgcccgttcaactgtgct
gacacagtgatacagataatgtcgctaacagaggagaatagaaatatgac
gggcacacgctaatgtggggaaaagagggagaagcctgatttttattttt
tagagattctagagataaaattcccagtattatatccttttaataaaaaa
tttctattaggagattataaagaatttaaagctatttttttaagtggggt
gtaattctttcagtagtctcttgtcaaatggatttaagtaatagaggctt
aatccaaatgagagaaatagacgcataaccctttcaaggcaaaagctaca
agagcaaaaattgaacacagcagccagccatctagccactcagattttga
tcagttttactgagtttgaagtaaatatcatgaaggtataattgctgata
aaaaaataagatacaggtgtgacacatctttaagtttcagaaatttaatg
gcttcagtaggattatatttcacgtatacaaagtatctaagcagataaaa
atgccattaatggaaacttaatagaaatatatttttaaattccttcattc
tgtgacagaaattttctaatctgggtcttttaatcacctaccctttgaaa
gagtttagtaatttgctatttgccatcgctgtttactccagctaatttca
aaagtgatacttgagaaagattatttttggtttgcaaccacctggcagga
ctattttagggccattttaaaactcttttcaaactaagtattttaaactg
ttctaaaccatttagggccttttaaaaatcttttcatgaatttcaaactt
cgttaaaagttattaaggtgtctggcaagaacttccttatcaaatatgct
aatagtttaatctgttaatgcaggatataaaattaaagtgatcaaggctt
gacccaaacaggagtatcttcatagcatatttcccctcctttttttctag
aattcatatgattttgctgccaaggctattttatataatctctggaaaaa
aaatagtaatgaaggttaaaagagaagaaaatatcagaacattaagaatt
cggtattttactaactgcttggttaacatgaaggtttttattttattaag
gtttctatctttataaaaatctgttcccttttctgctgatttctccaagc
aaaagattcttgatttgttttttaactcttactctcccacccaagggcct
gaatgcccacaaaggggacttccaggaggccatctggcagctgctcaccg
tcagaagtgaagccagccagttcctcctgggcaggtggccaaaattacag
ttgacccctcctggtctggctgaaccttgccccatatggtgacagccatc
tggccagggcccaggtctccctctgaagcctttgggaggagagggagagt
ggctggcccgatcacagatgcggaaggggctgactcctcaaccggggtgc
agactctgcagggtgggtctgggcccaacacacccaaagcacgcccagga
aggaaaggcagcttggtatcactgcccagagctaggagaggcaccgggaa
aatgatctgtccaagacccgttcttgcttctaaactccgagggggtcaga
tgaagtggttttgtttcttggcctgaagcatcgtgttccctgcaagaagc
ggggaacacagaggaaggagagaaaagatgaactgaacaaagcatgcaag
gcaaaaaaggccttaggatggctgcaggaagttagttcttctgcattggc
tccttactggctcgtcgatcgcccacaaacaacgcacccagtggagaact
tccctgttacttaaacaccattctctgtgcttgcttcctcaggggctgat
gccaagccatccgtcttcatcttcccgccatcgaaggagcagttagcgac
cccaactgtctctgtggtgtgcttgatca
Seq ID No. 15
gatgccaagccatccgtcttcatcttcccgccatcgaaggagcagttagc
gaccccaactgtctctgtggtgtgcttgatcaataacttcttccccagag
aaatcagtgtcaagtggaaagtggatggggtggtccaaagcagtggtcat
ccggatagtgtcacagagcaggacagcaaggacagcacctacagcctcag
cagcaccctctcgctgcccacgtcacagtacctaagtcataatttatatt
cctgtgaggtcacccacaagaccctggcctcccctctggtcacAAGCTTC
AACAGGAACGAGTGTGAGGCTTAGAGGCCCACAGGCCCCTGGCCTGCCCC
CAGCCCCAGCCCCGCTCCCCACCTCAAGCCTCAGGCCCTTGCCCCAGAGG
ATCCTTGGCAATCCCCCAGCCCCTCTTCCCTCCTCATCCCCTCCCCCTCT
TTGGCTTTAACCGTGTTAATACTGGGGGGTGGGGGAATGAATAAATAAAG
TGAACCTTTGCACCTGTGATTTCTCTCTCCTGTCTGATTTTAAGGTTGTT
AAATGTTGTTTTCCCCATTATAGTTAATCTTTTAAGGAACTACATACTGA
GTTGCTAAAAACTACACCATCACTTATAAAATTCAcgCCTTCTCAGTTCT
CCCCTCCCCTCCTGTCCTCCGTAAGACAGGCCTCCGTGAAACCCATAAGC
ACTTCTCTTTACACCCTCTCCTGGGCCGGGGTAGGAGACTTTTTGATGTC
CCCTcTTCAGCAAGCCTCAGAACCATTTTGAGGGGGACAGTTCTTACAGT
CACAT*TCCtGtGATCTAATGACTTTAGTTaCCGAAAAGCCAGTGTCTCA
AAAAGAAGGGAACGGCTAGAAACCAAGTCATAGAAATATATATGTATAAA
ATATATATATATCCATATATGTAAAATAACAAAATAATGATAACAGCATA
GGTCAACAGGCAACAGGGAATGTTGAAGTCCATTCTGGCACTTCAATTTA
AGGGAATAGGATGCCTTCATTACATTTTAAATACAATACACATGGAGAGC
TTCCTATCTGCCAAAGACCATCCTGAATGCCTTCCACACTCACTACAAGG
TTAAAAGCATTCATTACAATGTTGATCGAGGAGTTCCCGTTGTGGCTCAG
CAGGTTAAGAACGTGACTGGTATCCAGGAGGATGCGGGTTTGGTCCCCAG
CCTCGCTCAGTGGATTAAGGATCCAGTGTTGCTGCAAGATCACGGGCTCA
GATCCCGTGTTCTATGGCTATGGTGTAGGCTGGTAGCTGCATGCAGCCCT
AATTTGACCCCTAGCCTGGGAACTGCCATAtGCCACATGTGAGGCCCTTA
AAACCTAAAAGAAAAAaAAAGAAAAGAAATATCTTACACCCAATTTATAG
ATAAGAGAGAAGCTAAGGTGGCAGGCCCAGGATCAAAGCCCTACCTGCCT
ATCTTGACACCTGAtACAAATTCTGTCTTCTAGGGtTTCCAACACTGCAT
AGAACAGAGGGTCAAACATGCTACCCTCCCAGGGACTCCTCCCTTCAAAT
GACATAAATTTTGTTGCCCATCTCTGGGGGCAAAACTCAACAATCAATGG
CATCTCTAGTACCAAGCAAGGCTCTTCTCATGAAGCAAAACTCTGAAGCC
AGATCCATCATGACCCAAGGAAGTAAAGACAGGTGTTACTGGTTGAACTG
TATCCTTCAATTCAATATGCTCAATTTCCAACTCCCAGTCCCCGTAAATA
CAACCCCCTTTGGGAAGAGAGTCCTTGCAGATGTAGCCACGTTAAAAAGA
GATTATACAGAAAGGCTAGTGAGGATGCAGTGAAACGGGATCTTTCATAC
ATTGCTGGTGGAAATGTAAAATGCTGCAGGCACTCTAGAAAATAATTTGC
CAGTTTTTTGAAAAGCTAAACAAAATAGTTTAGTTGCATTCTGGGTTATT
TATCCCCCAGAAATTAAAAATTATGTCCGCACAAAAACGTGTACATAATC
ATTCATAACAGCCTTGTACGAAAAGCTT
Seq ID No. 16
GGATCCTTAACCCACTAATCGAGGATCAAACACGCATCCTCATGGACAAT
ATGTTGGGTTCTTAGCCTGCTGAGACACAACAGGAACTCCCCTGGCACCA
CTTTAGAGGCCAGAGAAACAGCACAGATAAAATTCCCTGCCCTCATGAAG
CTTATAGTCTAGCTGGGGAGATATCATAGGCAAGATAAACACATACAAAT
ACATCATCTTAGGTAATAATATATACTAAGGAGAAAATTACAGGGGAGAA
AGAGGACAGGAATTGCTAGGGTAGGATTATAAGTTCAGATAGTTCATCAG
GAACACTGTfGCTGAGAAGATAACATTTAGGTAAAGACCGAAGTAGTAAG
GAAATGGACCGTGTGCCTAAGTGGGTAAGACCATTCTAGGCAGCAGGAAC
AGCGATGAAAGCACTGAGGTGGGTGTTCACTGCACAGAGTTGTTCACTGC
ACAGAGTTGTGTGGGGAGGGGTAGGTCTTGCAGGGTCTTATGGTCACAGG
AAGAATTGTTTTACTCCCACCGAGATGAAGGTTGGTGGATTTTGAGCAGA
AGAATAATTCTGCCTGGTTTATATATAACAGGATTTCCCTGGGTGCTCTG
ATGAGAATAATCTGTCAGGGGTGGGATAGGGAGAGATATGGCAATAGGAG
CCTTGGCTAGGAGCCCACGACAATAATTCCAAGTGAGAGGTGGTGCTGCA
TTGAAAGCAGGACTAACAAGACCTGCTGACAGTGTGGATGTAGAAAAAGA
TAGAGGAGACGAAGGTGCATCTAGGGTTTTCTGCCTGAGGAATTAGAAAG
ATAAAGCTAAAGCTTATAGAAGATGCAGCGCTCTGGGGAGAAAGACCAGC
AGCTCAGTTTTGATCCATCTGGAATTAATTTTGGCATAAAGTATGAGGTA
TGTGGGTTAACATTATTTGTTTTTTTTTTTTCCATGTAGCTATCCAACTG
TCCCAGCATCATTTATTTTAAAAGACTTTCCTTTCCCCTATTGGATTGTT
TTGGCACCTTCACTGAAGATCAACTGAGCATAAAATTGGGTCTATTTCTA
AGCTCTTGATTCCATTCCATGACCTATTTGTTCATCTTTACCCCAGTAGA
CACTGCCTTGATGATTAAAGCCCCTGTTACCATGTCTGTTTTGGACATGG
TAAATCTGAGATGCCTATTAGCCAACCAAGCAAGCACGGCCCTTAGAGAG
CTAGATATGAGAGCCTGGAATTCAGACGAGAAAGGTCAGTCCTAGAGACA
TACATGTAGTGCCATCACCATGCGGATGGTGTTAAAAGCCATCAGACTGC
AACAGACTGTGAGAGGGTACCAAGCTAGAGAGCATGGATAGAGAAACCCA
AGCACTGAGCTGGGAGGTGCTCCTACATTAAGAGATTAGTGAGATGAAGG
ACTGAGAAGATTGATCAGAGAAGAAGGAaAATCAGGAAAATGGTGCTGTC
cTGAAAATCCAAGGGAAGAGATGTTCCAAAGAGGAGAaAACTGATCAGTT
GTCAGCTAGCGTCAATTGGGATGAAAATGGACCATTGGACAGAGGGATGT
AGTGGGTCATGGGTGAATAGATAAGAGCAGCTTCTATAGAATGGCAGGGG
CAAAATTCTCATCTGATCGGCATGGGTTcTAAAGAAAACGGGAAGAAAAA
ATTGAGTGCATGACCAGTCCCTTCAAGTAGAGAGGTgGAAAAGGGAAGGA
GGAAAATGAGGCCACGACAACATGAGAGAAATGACAGCATTTTTAAAAAT
TTTTTATTTTATTTtATTTATTTATTTTTGCTTTTTAGGGCTGCCCCTGC
AAcatatggaggttcccaggttaggggtctaatcagagctatagctgcca
gcctacaccacagccatagcaatgccagatctacatgacctacaccacag
ctcacagcaacgccggatccttaacccactgagtgaggccagagatcaaa
cccatatccttatggatactagtcaggttcattaccactgagccaaaatg
ggaaATCCTGAGTAATGACAGCATTTTTTAATGTGCCAGGAAGCAAAACT
TGCCACCCCGAAATGTCTCTCAGGCATGTGGATTATTTTGAGCTGAAAAC
GATTAAGGCCCAAAAAACACAAGAAGAAATGTGGACCTTCCCCCAACAGC
CTAAAAAATTTAGATTGAGGGCCTGTTCCCAGAATAGAGCTATTGCCAGA
CTTGTCTACAGAGGCTAAGGGCTAGGTGTGGTGGGGAAACCCTCAGAGAT
CAGAGGGACGTTTATGTACCAAGCATTGACATTTCCATCTCCATGCGAAT
GGCCTTCTTCCCCTCTGTAGCCCCAAACCACCACCCCCAAAATCTTCTTC
TGTCTTTAGCTGAAGATGGTGTTGAAGGTGATAGTTTCAGCCACTTTGGC
GAGTTCCTCAGTTGTTCTGGGTCTTTCCTCCGGATCCACATTATTCGACT
GTGTTTGATTTTCTCCTGTTTATCTGTCTCATTGGCACCCATTTCATTCT
TAGACCAGCCCAAAGAACCTAGAAGAGTGAAGGAAAATTTCTTCCACCCT
GACAAATGCTAAATGAGAATCACCgCAGTAGAGGAAAATGATCTGGTgCT
GCGGGAGATAGAAGAGAAAATcGCTGGAGAGATGTCACTGAGTAGGTGAG
ATGGGAAAGGGGGGGCACAGGTGGAGGTGTTGCCCTCAGCTAGGAAGACA
GACAGTTcacagaagagaagcgggtgtccgtGGACATCTTGCCTCATGGA
TGAGGAAACCGAGGCTAAGAAAGACTGCAAAAGAAAGGTAAGGATTGCAG
AGAGGTCGATCCATGACTAAAATCACAGTAACCAACCCCAAACCACCATG
TTTTCTCCTAGTCTGGCACGTGGCAGGTACTGTGTAGGTTTTCAATATTA
TTGGTTTGTAACAGTACCTATTAGGCCTCCATCcCCTCCTCTAATACTAA
CAAAAGTGTGAGACTGGTCAGTGAAAAATGGTCTTCTTTCTCTATGCAAT
CTTTCTCAAGAAGATACATAACTTTTTATTTTATCATaGGCTTGAAGAGC
AAATGAGAAACAgCCTCCAACCTATGACACCGTAACAAAGTGTTTATGAT
CAGTGAAGGGCAAGAAACAAAACATACACaGTAAAGACCCTCCATAATAT
TGtGGGCTGGCCCAaCACAGGCCAGGTTGTAAAAGCTTTTTATTCTTTGA
TAGAGGAATGGATAGTAATGTTTCAACCTGGACAGAGAT*CATGTTCACT
GAATCCTTCCAAAAATTCATGGGTAGTTTGAAtTATAAGGAAAATAAGAC
TTAGGATAAATACTTTgTCCA*GATCCCAGAGTTAATgCCAAAATCAGTT
TTCAGACTCCAGGCAGCCTGATCAAGAGCCTAAACTTTAAAGACACAGTC
CCTTAATAACTACTATTCACAGTTGCACTTTCAgGGCGCAAAGACTCATT
GAATCCTACAATAGAATGAGTTTAGATATCAAATCTCTCAGTAATAGATG
AGGAGACTAAATAGCGGGCATGACCTGGTCACTTAAAGACAGAATTGAGA
TTCAAGGCTAGTGTTCTTTCTACCTGTTTTGTTTCTACAAGATGTAGCAA
TGCGCTAATTACAGACCTCTCAGGGAAGGAATTCACAACCCTCAGCAAAA
ACCAAAGACAAATCTAAGACAACTAAGAGTGTTGGTTTAATTTGGAAAAA
TAACTCACTAACCAAACGCCCCTCTTAGCACCCCAATGTCTTCCACCATC
ACAGTGCTCAGGCCTCAACCATGCCCCAATCACCCCAGCCCCAGACTGGT
TATTACCAAGTTTCATGATGACTGGCCTGAGAAGATCAAAAAAGCAATGA
CATCTTACAGGGGACTACCCCGAGGACCAAGATAGCAACTGTCATAGCAA
CCGTCACACTGCTTTGGTCA
Seq ID No. 19
ggatcaaacacgcatcctcatggacaatatgttgggttcttagcctgctg
agacacaacaggaactcccctggcaccactttagaggccagagaaacagc
acagataaaattccctgccctcatgaagcttatagtctagctggggagat
atcataggcaagataaacacatacaaatacatcatcttaggtaataatat
atactaaggagaaaattacaggggagaaagaggacaggaattgctagggt
aggattataagttcagatagttcatcaggaacactgttgctgagaagata
acatttaggtaaagaccgaagtagtaaggaaatggaccgtgtgcctaagt
gggtaagaccattctaggcagcaggaacagcgatgaaagcactgaggtgg
gtgttcactgcacagagttgttcactgcacagagttgtgtggggaggggt
aggtcttgcaggctcttatggtcacaggaagaattgttttactcccaccg
agatgaaggttggtggattttgagcagaagaataattctgcctggtttat
atataacaggatttccctgggtgctctgatgagaataatctgtcaggggt
gggatagggagagatatggcaataggagccttggctaggagcccacgaca
ataattccaagtgagaggtggtgctgcattgaaagcaggactaacaagac
ctgctgacagtgtggatgtagaaaaagatagaggagacgaaggtgcatct
agggttttctgcctgaggaattagaaagataaagctaaagcttatagaag
atgcagcgctctggggagaaagaccagcagctcagttttgatccatctgg
aattaattttggcataaagtatgaggtatgtgggttaacattatttgttt
tttttttttccatgtagctatccaactgtcccagcatcatttattttaaa
agactttcctttcccctattggattgttttggcaccttcactgaagatca
actgagcataaaattgggtctatttctaagctcttgattccattccatga
cctatttgttcatctttaccccagtagacactgccttgatgattaaagcc
cctgttaccatgtctgttttggacatggtaaatctgagatgcctattagc
caaccaagcaagcacggcccttagagagctagatatgagagcctggaatt
cagacgagaaaggtcagtcctagagacatacatgtagtgccatcaccatg
cggatggtgttaaaagccatcagactgcaacagactgtgagagggtacca
agctagagagcatggatagagaaacccaagcactgagctgggaggtgctc
ctacattaagagattagtgagatgaaggactgagaagattgatcagagaa
gaaggaaaatcaggaaaatggtgctgtcctgaaaatccaagggaagagat
gttccaaagaggagaaaactgatcagttgtcagctagcgtcaattgggat
gaaaatggaccattggacagagggatgtagtgggtcatgggtgaatagat
aagagcagcttctatagaatggcaggggcaaaattctcatctgatcggca
tgggttctaaagaaaacgggaagaaaaaattgagtgcatgaccagtccct
tcaagtagagaggtggaaaagggaaggaggaaaatgaggccacgacaaca
tgagagaaatgacagcatttttaaaaattttttattttattttatttatt
tatttttgctttttagggctgcccctgcaacatatggaggttcccaggtt
aggggtctaatcagagctatagctgccagcctacaccacagccatagcaa
tgccagatctacatgacctacaccacagctcacagcaacgccggatcctt
aacccactgagtgaggccagagatcaaacccatatccttatggatactag
tcaggttcattaccactgagccaaaatgggaaatcctgagtaatgacagc
attttttaatgtgccaggaagcaaaacttgccaccccgaaatgtctctca
ggcatgtggattattttgagctgaaaacgattaaggcccaaaaaacacaa
gaagaaatgtggaccttcccccaacagcctaaaaaatttagattgagggc
ctgttcccagaatagagctattgccagacttgtctacagaggctaagggc
taggtgtggtggggaaaccctcagagatcagagggacgtttatgtaccaa
gcattgacatttccatctccatgcgaatggccttcttcccctctgtagcc
ccaaaccaccacccccaaaatcttcttctgtctttagctgaagatggtgt
tgaaggtgatagtttcagccactttggcgagttcctcagttgttctgggt
ctttcctccTgatccacattattcgactgtgtttgattttctcctgttta
tctgtctcattggcacccatttcattcttagaccagcccaaagaacctag
aagagtgaaggaaaatttcttccaccctgacaaatgctaaatgagaatca
ccgcagtagaggaaaatgatctggtgctgcgggagatagaagagaaaatc
gctggagagatgtcactgagtaggtgagatgggaaaggggtgacacaggt
ggaggtgttgccctcagctaggaagacagacagttcacagaagagaagcg
ggtgtccgtggacatcttgcctcatggatgaggaaaccgaggctaagaaa
gactgcaaaagaaaggtaaggattgcagagaggtcgatccatgactaaaa
tcacagtaaccaaccccaaaccaccatgttttctcctagtctggcacgtg
gcaggtactgtgtaggttttcaatattattggtttgtaacagtacctatt
aggcctccatcccctcctctaatactaacaaaagtgtgagactggtcagt
gaaaaatggtcttctttctctatgaatctttctcaagaagatacataact
ttttattttatcataggcttgaagagcaaatgagaaacagcctccaacct
atgacaccgtaacaaaatgtttatgatcagtgaagggcaagaaacaaaac
atacacagtaaagaccctccataatattgtgggtggcccaacacaggcca
ggttgtaaaagctttttattctttgatagaggaatggatagtaatgtttc
aacctggacagagatcatgttcactgaatccttccaaaaattcatgggta
gtttgaattataaggaaaataagacttaggataaatactttgtccaagat
cccagagttaatgccaaaatcagttttcagactccaggcagcctgatcaa
gagcctaaactttaaagacacagtcccttaataactactattcacagttg
cactttcagggcgcaaagactcattgaatcctacaatagaatgagtttag
atatcaaatctctcagtaatagatgaggagactaaatagcgggcatgacc
tggtcacttaaagacagaattgagattcaaggctagtgttctttctacct
gttttgtttctacaagatgtagcaatgcgctaattacagacctctcaggg
aaggaattcacaaccctcagcaaaaaccaaagacaaatctaagacaacta
agagtgttggtttaatttggaaaaataactcactaaccaaacgcccctct
tagcaccccaatgtcttccaccatcacagtgctcaggcctcaaccatgcc
ccaatcacc
Seq ID No. 25
GCACATGGTAGGCAAAGGACTTTGCTTCTCCCAGCACATCTTTCTGCAGA
GATCCATGGAAACAAGACTCAACTCCAAAGCAGCAAAGAAGCAGCAAGTT
CTCAAGTGATCTCCTCTGACTCCCTCCTCCCAGGCTAATGAAGCCATGTT
GCCCCTGGGGGATTAAGGGCAGGTGTCCATTGTGGGACCCAGCCCGAAGA
CAAGCAATTTGATCAGGTTCTGAGCACTCCTGAATGTGGACTCTGGAATT
TTCTCCTCACCTTGTGGCATATCAGCTTAAGTCAAGTACAAGTGACAAAC
AACATAATCCTAAGAAGAGAGGAATCAAGCTGAAGTCAAAGGATCACTGC
CTTGGATTCTACTGTGAATGATGACCTGGAAAATATCCTGAACAACAGCT
TCAGGGTGATCATCAGAGACAAAAGTTCCAGAGCCAGGTAGGGAAACCCT
CAAGCCTTGCAAAGAGCAAAATCATGCCATTGGGTTCTTAACCTGCTGAG
TGATTTACTATATGTTACTGTGGGAGGCAAAGCGCTCAAATAGCCTGGGT
AAGTATGTCAAATAAAAAGCAAAAGTGGTGTTTCTTGAAATGTTAGACCT
GAGGAAGGAATATTGATAACTTACCAATAATTTTCAGAATGATTTATAGA
TGTGCACTTAGTCAGTGTCTCTCCACCCCGCACCTGACAAGCAGTTTAGA
ATTTATTCTAAGAATCTAGGTTTGCTGGGGGCTACATGGGAATCAGCTTC
AGTGAAGAGTTTGTTGGAATGATTCACTAAATTTTCTATTTCCAGCATAA
ATCCAAGAACCTCTCAGACTAGTTTATTGACACTGCTTTTCCTCCATAAT
CCATCTCATCTCCGTCCATCATGGACACTTTGTAGAATGACAGGTCCTGG
CAgAGACTCaCAGATGCTTCTGAAACATCCTTTGCCTTCAAAGAATGAAC
AGCACACATACTAAGGATCTCAGTGATCCACAAATTAGTTTTTGCCACAA
TGGTTCTTATGATAAAAGTCTTTCATTAACAGCAAATTGTTTTATAATAG
TTGTTCTGCTTTATAATAATTGCATGCTrCACTTTCTTTTCTTTTCTTTT
TTTTTCTTTTTTTGCTTTTTAGTGCCGCAGGTgcagcatatgaaatttcc
caggctaggggtcaaatcagaactacacctactggcctacgccacagcca
cagcaactcaggatctaagccatgtcggtgacctacactacagctcatgg
caatgccagatccttaacccaatgagcgaggccagggatcgaacccatgt
cctcatggatactagtcaggctcattatccgctgagccataacaggaact
cccGAGTTTGCTTTTTATCAAAATTGGTACAGCCTTATTGTTTCTGAAAA
CCACAAAATGAATGTATTCACATAATTTTAAAAGGTTAAATAATTTATGA
TATACAAGACAATAGAAAGAGAAAACGTCATTGCCTCTTTCTTCCACGAC
AACACGCCTCCTTAATTGATTTGAAGAAATAACTACTGAGCATGGTTTAG
TGTACTTCTTTCAGCAATTAGCCTGTATTCATAGCCATACATATTCAATT
AAAATGAGATCATGATATCACACAATACATACCATACAGCCTATAGGGAT
TTTTACAATCATCTTCCACATGACTACATAAAAACCTACCTAAAAAAAAA
AAAAACCCTACTTCATCCTCCTATTGGCTGCTTTGTGCACCATTAAAAAG
CTCTATCATAATTAGGTTATGATGAGGATTTCCATTTTCTACCTTTCAAG
CAACATTTCAATGCACAGTCTTATATACACATTTGAGCCTACTTTTCTTT
TTCTTTCTTTTTTTGGTTTTTTTTTTTTTTTTTTTTTTGGTCTTTTTGTC
TTTTCTAAGgctgcatatggaggttcccaggctagctgtctaatcagaac
tatagctgctggcctacgccacatccacagcaatacaagatctgagccat
gtctgcaacttacaccacagctcacagcaacggtggatccttaaaccact
gagcaaggccagggatcaaacccatAACTTCATGGCTCCTAGTTGGATTT
GTTAACCACTGAGCCATGATGGCAACTCCTGAGCCTACTTTTCTAATCAT
TTCCAACCCTAGGACACTTTTTTAAGTTTCATTTTTCTCCCCCCACCCCC
TGTTTTCTGAAGtGTGTTTGCTTCCACTGGGTGACTTCACtCCCAGGATC
TCATCTGCAGGATACTGCAGCTAAGTGTATGAGCTCTGAATTTGAATCCC
AACTCTGCCACTCAAAGGGATAGGAGTTTCCGATGTGGCCCAATGGGATC
AGTGGCATCTCTGCAGTGCCAGGACGCaggttccatccctggcccagcac
agtgggttaagaatctggCATTGCTGCAGCTGAGGCATAGATTTCAATTG
TGCCTCAgATCTGATCCTTGGCCCAAGGACTGCATATGCCTCAGGGCAAC
CAAAAAAGAGAAAAGGGGGGTGATAGCATTAGTTTCTAGATTTGGGGGAT
AATTAAATAAAGTGATCCATGTACAATGTATGGCATTTTGTAAATGCTCA
ACAAATTTCAACTATTATggagttcccatcatggctcagtggaagggaat
ctgattagcatccatgaggacacaggtCCAACCCCGACCTTGCTCAGTGG
GCATTGCTGTGAGCTGTGGCATGGGTTACAGACGAAGCTCGGATCTGGCA
TTGCTGTGGCTGTGGTGTAAGCCAgCAActacagctctcattcagcccct
agcctgggaacctccatatgccTAAAAGACAAAAAATAAAATTTAAATTA
AAAATAAAGAAATGTTAACTATTATGATTGgTACTGCTTGCATTACTGCA
AAGAAAGTCACTTTCTATACTCTTTAATATCTTAGTTGACTGTGTGCTCA
GTGAACTATTTTGGACACTTAATTTCCACTCTCTTCTATCTCCAACTTGA
CAACTCTCTTTCCTCTCTTCTGGTGAGATCCACTGCTGACTTTGCTCTTT
AAGGCAACTAGAAAAGTGCTCAGTGACAAAATCAAAGAAAGTTACCTTAA
TCTTCAGAATTACAATCTTAAGTTCTCTTGTAAAGCTTACTATTTCAGTG
GTTAGTATTATTCCTTGGTCCCTTACAACTTATCAGCTCTGATCTATTGC
TGATTTTCAACTATTTATTGTTGGAGTTTTTTCCTTTTTTCCCTGTTCAT
TCTGCAAATGTTTGCTGAGCATTTGTCAAGTGAAGATACTGGACTGGGCC
TTCCAAATATAAGACAATGAAACATCGGGAGTTCTCATTATGGTGCAGCA
GAaacgaatccaactaggaaatgtgaggttgcaggttcgatccctgccct
tgctcagtgggttaaggatccagcattaccgtgagctgtggtgtaggttg
cagacgtggctcagatcctgcgttgctgtggctgtggcataggctggcag
ctctagctctgattcgaccgctagcctgggaacctccatGCGCCCCGAGT
GCAGCCCTTAAAAAGCAAAAAAAAAAGAAAGAAAGAAAAAGACAATGAAA
CATCAAACAGCTAACAATCCAGTAGGGTAGAAAGAATCTGGGAACAGATA
AGAGCGATTAAATGTTCTAGGTCCAGTGACCTTGCCTCTGTGCTCTACAC
AGTCGTGCCACTTGCTGAGGGAGAAGGTCTCTCTTGAGTTGAGTCCTGAA
AGACATTAGTTGTTCACAAACTAATGCCAGTGAGTGAAGGTGTTTCCAAG
CAGAGGGAGAGTTTGGTAAAAAGCTGGAAGTCACAGAAAGACTCTAAAGA
GTTTAGGATGGTGGGAGCAACATACGCTGAGATGGGGCTGGAAGGTTAAG
AGGGAAACAACTATAGTAAGTGAAGCTGGACTCACAGCAAAGTGAGGACC
TCAGCATCCTTGATGGGGTTACCATGGAAACACCAAGGCACACCTTGATT
TCCAAAACAGCAGGCACCTGATTCAGCCCAATGTGACATGGTGGGTACCC
CTCTAGCTCTACCTGTTCTGTGACAACTGACAACCAACGAAGTTAAGTCT
GGATTTTCTACTCTGCTGATCCTTGTTTTTGTTTCACACGTCATCTATAG
CTTCATGCCAAAATAGAGTTCAAGGTAAGACGCGGGCCTTGGTTTGATAT
ACATGTAGTCTATCTTGTTTGAGACAATATGGTGGCAAGGAAGAGGTTCA
AACAGGAAAATACTCTCTAATTATGATTAAGTGAGAAAAGCTAAAGAGTC
CCATAATGACACTGAATGAAGTTCATCATTTGCAAAAGCCTTCCCCCCCC
CCCAGGAGACTATAAAAAAGTGCAATTTTTTAAATGAACTTATTTACAAA
ACAGAAATAGAGTCACAGACATAGGAAACGAACAGATGGTTACCAAGGGT
GAAAGGGAGTAGGAGGGATAAATAAGGAGTCTGGGGTTAGCAGATACACC
CCAGTGTACACAAAATAAACAACAGGGACCTACTATATAGCACAGGGAAC
TATATGCAGTAGCTTACAATAACCTATAATGGAAAAGAATGTGAAAAAGA
ATATATGTATGCGTGTGTGTGTAACTGAATCACTTTGCTGTAACCTGAAT
CTAACATAACATTGTAAATCAACTACAGTTTTTTTTTTTTTTAAGTGCAG
GGTTTTGGTGTTTTTTTTTTTTCATTTTTGTTTTTGTTTTTGTTTTTTGC
TTTTTAGGGCCACACCCAGACATATGGGGGTTCCCAGGctAGGGGTcTAa
TTAGAGcTACAGtTGCCGGCTTGCAccacagccacagcaacatcagatcc
gagccgcacttgcgacttacaccacagctcatggcaataccagatcctta
acccactgagcaaggcccagggatcgtacccgcaacctcatggttcctag
tcagattcattTCTGCTGCGCTACAATGGGAACTCCAAGTGCAGTTTTTT
GTAATGTGCTtGTCTTTCTTTGTAATTCATATTCATCCTACTTCCCAATA
AATAAATAAATACATAAATAATAAACATACCATTGTAAATCAACTACAAT
TTTTTTTAAATGCAGGGTTTTTGTTTTTTGTTTTTTGTTTTGTCTTTTTG
CCTTTTGTAgggccgctcccatggcatatggaggttcccaggctaggggt
cgaatcggagctgtagccaccggcctacgccagagccacagcaacgcggg
atccgagccgcgtctgcaacctacaccacagctcacggcaacgccggatc
gttaacccactgagcaagggcagggatcgaacctgcaacctcatggttcc
tagtcagattcgttaactactgagccacaacggaaacTCCTAAAGTGCAG
TTTTTAAATGTGCTTGTCTTTCTTTGTAATTTACACTCAACCTACTTCCC
AATAAATAAATAAATAAACAAATAAATCATAGACATGGTTGAATTCTAAA
GGAAGGGACCATCAGGCCTTAGACAGAAATACGTCATCTTCTAGTATTTT
AAAACACACTAAAGAAGACAAACATGCTCTGCCAGAGAAGCCCAGGGCCT
CCACAGCTGCTTGCAAAGGGAGTTAGGCTTCAGTAGCTGACCCAAGGCTC
TGTTCCTCTTCAGGGAAAAGGGTTTTTGTTCAGTGAGACAGCAGACAGCT
GTCACTGTGgtggacgttcggccaaggaaccaagctggaactcaaacGTA
AGTCAATCCAAACGTTCCTTCCTTGGCTGTCTGTGTCTTACGGTCTCTGT
GGCTCTGAAATGATTCATGTGCTGACTCTCTGAAACCAGACTGACATTCT
CCAGGGCAAAACTAAAGCCTGTCATGAAACcGGAAAACTGAGGGCACATT
TTCTGGGCAGAACTAAGAGTCAGGCACTGGGTGAGGAAAAACTTGTTAGA
ATGATAGTTTCAGAAACTTACTGGGAAGCAAAGCCCATGTTCTGAACAGA
GCTCTGCTCAAGGGTCAGGAGGGGAACCAGTTTTTGTACAGGAGGGAAGT
TGAGACGAACCCCTGTGTAtatggtttcggcgcggggaccaagctggagc
tcaaacGTAAGTGGCTTTTTCCGACTGATTCTTTGCTGTTTCTAATTGTT
GGTTGGCTTTTTGTCCATTTTTCAGTGTTTTCATCGAATTAGTTGTCAGG
GACCAAACAAATTGCCTTCCCAGATTAGGTACCAGGGAGGGGACATTGCT
GCATGGGAGACCAGAGGGTGGCTAATTTTTAACGTTTCCAAGCCAAAATA
ACTGGGGAAGGGGGCTTGCTGTCCTGTGAGGGTAGGTTTTTATAGAAGTG
GAAGTTAAGGGGAAATCGCTATGGTtcacttttggctcggggaccaaagt
ggagcccaaaattgaGTACATTTTCCATCAATTATTTGTGAGATTTTTGT
CCTGTTGTGTCATTTGTGCAAGTTTTTGACATTTTGGTTGAATGAGCCAT
TCCCAGGGACCCAAAAGGATGAGACCGAAAAGTAGAAAAGAGCCAACTTT
TAAGCTGAGCAGACAGACCGAATTGTTGAGTTTGTGAGGAGAGTAGGGTT
TGTAGGGAGAAAGGGGAACAGATCGCTGGCTTTTTCTCTGAATTAGCCTT
TCTCATGGGACTGGCTTCAGAGGGGGTTTTTGATGAGGGAAGTGTTCTAG
AGCCTTAACTGTGGgttgtgttcggtagcgggaccaagctggaaatcaaa
CGTAAGTGCACTTTTCTACTCC
Porcine Lambda Light Chain
In another embodiment, novel genomic sequences encoding the lambda light chain locus of ungulate immunoglobulin are provided. The present invention provides the first reported genomic sequence of ungulate lambda light chain regions. In one embodiment, the porcine lambda light chain nucleotides include a concatamer of J to C units. In a specific embodiment, an isolated porcine lambda nucleotide sequence is provided, such as that depicted in Seq ID No. 28. See FIG. 3 for a diagram of the organization of the porcine lamba immunoglobulin locus.
In one embodiment, nucleotide sequence is provided that includes 5′ flanking sequence to the first lambda J/C region of the porcine lambda light chain genomic sequence, for example, as represented by Seq ID No 32.
Still further, nucleotide sequence is provided that includes 3′ flanking sequence to the J/C cluster region of the porcine lambda light chain genomic sequence, for example, approximately 200 base pairs downstream of lambda J/C, such as that represented by Seq ID No 33. Alternatively, nucleotide sequence is provided that includes 3′ flanking sequence to the J/C cluster region of the porcine lambda light chain genomic sequence, for example, approximately 11.8 kb downstream of the J/C cluster, near the enhancer (such as that represented by Seq ID No. 34), approximately 12 Kb downstream of lambda, including the enhancer region (such as that represented by Seq ID No. 35), approximately 17.6 Kb downstream of lambda (such as that represented by Seq ID No. 36, approximately 19.1 Kb downstream of lambda (such as that represented by Seq ID No. 37), approximately 21.3 Kb downstream of lambda (such as that represented by Seq ID No. 38), and/or approximately 27 Kb downstream of lambda (such as that represented by Seq ID No. 39).
In still further embodiments, isolated nucleotide sequences as depicted in Seq ID Nos 28, 31, 32, 33, 34, 35, 36, 37, 38, or 39 are provided. Nucleic acid sequences at least 80, 85, 90, 95, 98 or 99% homologous to Seq ID Nos 28, 31, 32, 33, 34, 35, 36, 37, 38, or 39 are also provided. In addition, nucleotide sequences that contain at least 10, 15, 17, 20, 25, 30, 40, 50, 75, 100, 150, 200, 250, 500 or 1,000 contiguous nucleotides of Seq ID Nos 28, 31, 32, 33, 34, 35, 36, 37, 38, or 39 are provided. Further provided are nucleotide sequences that hybridizes, optionally under stringent conditions, to Seq ID Nos 28, 31, 32, 33, 34, 35, 36, 37, 38, or 39, as well as, nucleotides homologous thereto.
Seq ID No. 28
CCTTCCTCCTGCACCTGTCAACTCCCAATAAACCGTCCTCCTTGTCATTC
AGAAATCATGCTCTCCGCTCACTTGTGTCTACCCATTTTCGGGCTTGCAT
GGGGTCATCCTCGAAGGTGGAGAGAGTCCCCCTTGGCCTTGGGGAAGTCG
AGGGGGGCGGGGGGAGGCCTGAGGCATGTGCCAGCGAGGGGGGTCACCTC
CACGCCCCTGAGGACCTTCTAGAACCAGGGGCGTGGGGCCACCGCCTGAG
TGGAAGGCTGTCCACTTTTCCCCCGGGCCCCCAGGCTCCCTCCTCCGTGT
GGACCTTGTCCACCTCTGACTGGCCCAGCCACTCATGCATTGTTTCCCCG
AAACCCCAGGACGATAGCTCAGCACGCGACAGTGTCCCCCTCTGAGGGCC
TCTGTCCATTTCAGGACGACCCGCATGTACAGCGTGACCACTCTGCTCAC
GCCCACTCACCACGTCCTAGAGCCCCACCCCCAGCCCCATCCTTAGGGGC
ACAGCCAGcTCCGACCGCCCCGGGGACACCACCCTCTGCCCCTTcCCCAG
GCCCTCCCTGTCACACGCACCACAGGGCCCTCCGTCCCGAGACCCTGCTC
CCTCATCCCTCGGTCCCCTCAGGTAGCCTTCCACCCGCGTGTGTCCCGAG
GTCCCAGATGCAGCAAGGCCCCTGGGACAACGCCAGATCTCTGCTCTcCC
CGACCCCTCAGAAGCCAGCCCACGCCTGGCCCCACCACCACTGCCTAACg
TCCAAGTGTCCATAGGCCTCGGGACCTCCAAGTCCAGGTTCTGCCTCTGG
GATTCCGCCATGGGTCTGCCTGGGAAATGATGCACTTGGAGGAGCTCAGC
ATGGGATGCGGGACCTTGTCTCTAGGCGCTcCCTCAGGATCCCACAGCTG
CCCTGTGAGACACACACACACACACACACACACACACACACACACACACA
CACACAAACACGCATGCACGCACGCCGGCACACACGCTATTGCAGAGATG
GCCACGGTAGCTGTGCCTCGAGGCCGAGTGGAGTGTCTAGAACTCTCGGG
GGTCCCCTCTGCAGACGACACTGCTCCATCCCCCCCGTGCCCTGAAGGGC
TCCTCACTCTCCCATCAGGATCTCTCCAAGCTGCTGACCTGGAGAGGAAG
GGGCCTGGGACAGGCGGGGACACTCAGACCTCCCTGCTGCCCCTCCTCTG
CCTGGGCTTGGACGGCTCCCCCCTTCCCACGGGTGAAGGTGCAGGTGGGG
AGAGGGCACCCCCCTCAGCCTCCCAGACCCAGACCAGCCCCCGTGGCAGG
GGCAGCCTGTGAGCCTCCAGCCAGATGCAGGTGGCCTGGGGTGGGGGGTG
GAGGGGGCGGGAGGTTTATGTTTGAGGCTGTATCACTGTGTAATATTTTC
GGCGGTGGGACCCATCTGACCGTCCTCGGTGAGTCTCCCCTTTTCTCTCC
TCCTTGGGGATCCGAGTGAAATCTGGGTCGATCTTCTCTCCGTTCTCCTC
CGACTGGGGCTGAGGTCTGAACCTCGGTGGGGTCCGAAGAGGAGGCCCCT
AGGCCAGGCTCCTCAGCCCCTCCAGCCCGACcgGCCCTCTTGACACAGGG
TCCAGCTAAGGGCAGACATGGAGGCTGCTAGTCCAGGGCCAGGCTCTGAG
ACCCAAGGGCGCTGCCCAAGGAACCCTTGCCCCAGGGACCCTGGGAGCAA
AGCTCCTCACTCAGAGCCTGCAGCCCTGGGGTCTGAGGACAAGGAGGGAC
TGAGGACTGGGCGTGGGGAGTTCAGGCGGGGACACCAGGTCCAGGGAGGT
GACAAAGGCGCTGGGAGGGGGCGGACGGTGCCGGGGACTCCTCCTGGGCC
CTGTGGGCTCGGGGTCCTTGTGAGGACCCTGAGGGACTGAGGGGCCCCTG
GGCCTAGGGACTTGCAgTgAGGGAGGCAGGGAGTGTCCCTTGAGAACGTG
GCCTCCGCGGGCTGGGTCCCCCTGCTGCTCCCAGCC*GGGAGGACACCCC
AGAGCAAGCGCCCCAGGTGGGCGGGGAGGGTCTCCTCACAGGGGCAGCTG
ACAGATAGAGGCCCCCGCCAGGCAGATGCTTGATCCTGGCAgTTATACTG
GGTTC**GCACAACTTTCCCTGAACAAGGGGCCCTCCGAACAGACACAGA
CGCAACCCAGTCGACCcaggCTCAGCACAgAAAATGCACTGACACCCAAA
ACCCTCATCTggggGCCTGGCCGGcAtCCCGCCCCAGGACCCAAGGCCCC
TGCCCCCTGGCAGCCCTGGACACGGTCCTCTGTGGGCGGTGGGGTCgGGG
CTGTGGTGACGGTGGCATCGGGGAGCCTGTGCCCCCTCCCTGAAAGGGCG
GAGAGGCTCAAGAGGGGAGAGAAATGTCCTCCCCTAGGAAGACGTCGGAC
GGGGGCGGGGGGGTGGTCTCCGACAGACAGATGCCCGGGACCGACAGACC
TGCCGAGGGAAGAGGGCACCTCGGTCGGGTTAGGCTCCAGGCAGCACGAG
GGAGCGAGGCTGGGAGGGTGAGGACATGGGAGCCTGAGGAGGAGCTGGAG
ACTTCAGCAGGCCCCCAGCTCCGGGCTTCGGGCTCTGAGATGCTCGGACG
CAAGGTGAGTGACCCCACCTGTGGCTGACCTGACCTCAgGGgGACAAGGC
TCAGCCTGGGAGTCTGTGTCCCCATCGCCTGcACAGGGGATTCCCCTGAT
GGACACTGAGCCAACGACCTCCCGTCTGTCCCCGACCCCCAGGTCAGCCC
AAgGCCaCTCCCACGGTCAACCTCTTCCCGCCCTCCTCTGAGGAGCTCGG
CACCAACAAGGCCACCCTGGTGTGTCTAATAAGTGACTTCTACCCGGGCG
CCGTGACGGTGACCTGGAAGGCAGGCGGCACCACCGTCACCCAGGGCGTG
GAGACCACCAAGCCCTCGAAACAGAGCAACAACAAGTACGCGGCCAGCAG
CTACCTGGCCCTGTCCGCCAGTGACTGGAAATCTTCCAGCGGCTTCACCT
GCCAGGTCACCCACGAGGGGACCATTGTGGAGAAGACAGTGACGCCCTCC
GAGTGCGCCTAGGTCCCTGGGCCCCCACCCTCAGGGGCCTGGAGCCACAG
GACCCCCGCGAGGGTCTCCCCGCGACCCTGGTCCAGCCCAGCCCTTCCTC
CTGCACCTGTCAACTCCCAATAAACCGTCCTCCTTGTCATTCAGAAATCA
TGCTCTCCGCTCACTTGTGTCTACCCATTTTCGGGCTTGCATGGGGTCAT
CCTCGAAGGTGGAGAGAGTCCCCCTTGGCCTTGGGgAAATCGAGGGGGGC
GGGGGGAGGCCTGAGGCATGTGCCAGCGAGGGGGGTCACCTCCACGCCCC
TGAGGACCTTCTAGAACCAGGGGCGTGGGGCCACCGCCAGAGTGGAAGGC
TGTCCACTTTTCCCCCGGGCCCCCAGGCTCCCTCCTCCGTGTGGACCTTG
TCCACCTCTGACTGGCCCAGCCACTCATGCATTGTTTCCCCGAAACCCCA
GGACGATAGCTCAGCACGCGACAGTGTCCCCCTCTGAGGGCCTCTGTCCA
TTTCAGGACGACCCGCATGTACAGCGTGACCACTCTGCTCACGCCCACTC
ACCACGTCCTAGAGCCCCACCCCCAGCCCCATCCTTAGGGGCACAGCCAG
CTCCGACCGCCCCGGGGACACCACCCTCTGCCCCTTCCCCAGGCCCTCCC
TGTCACACGCACCACAGGGCCCTCCGTCCCGAGACCCTGCTCCCTCATCC
CTCGGTCCCCTCAGGTAGCCTTCCACCCGCGTGTGTCCCGAGGTCCCAGA
TGCAGCAAGGCCCCTGGGACAACGCCAGATCTCTGCTCTCCCCGACCCTC
AGAAGCCAGCCCACGCCTGGCCCACCACCACTGCCTAACGTCCAAGTGTC
CATAGGCTCGGGAcCTCcAaGTCCAGGTTCTGCCTCTGGGATTCCGCCAT
GGGTCTGCCTGGAATGATGCACTTGGAGgAgCTCAGcATGGGATGcGGAA
CTTGTCTAGcGCTCCTCAGATCCAcAGcTGCCTGtGAgAcacacacacac
acacacacacaccAAAcaCGcATGCACGCACGCCGGCACACACGCTATTA
CAGAGATGGCCACGGTAGCTGTGCCTCGAGGCCGAGTGGAGTGTCTAGAA
CTCTCGGGGGTCCCCTCTGCAGACGACACTGCTCCATCCCCCCCGTGCCC
TGAAGGGCTCCTCACTCTCCCATCAGGATCTCTCCAAGCTGCTGACCTGG
AGAGGAAGGGGCCTGGGACAGGCGGGGACACTCAGACCTCCCTGCTGCCC
CTGCTCTGCCTGGGCTTGGACGGCTCCCCCCTTCCCACGGGTGAAGGTGC
AGGTGGGGAGAGGGCACCCCCCTCACCCTCCCAGACCCAGACCAGCCCCC
GTGGCAGGGGCAGCCTGTGAGCCTCCAGCCAGATGCAGGTGGCCTGGGGT
GGGGGGTGGAGGGGGCGGGAGGTTTATGTTTGAGGCTGTATTCATCTGTG
TAATATttTCGGCGGTGGGACCCATCTGACCGTCCTCGGTGAGTCTCCCC
TtttctttcctccttggggatccgagtgaaATcTGGGTCGATCTTCTCTC
CGTTCTCCTCCGACTGGGGCTGAGGTCTGAACCTCGGTgGGGTCCGAAGA
GGAGGCCCCTAGGCC*GGCTCcTCAGCCCCTCCAGGCCGACCCGCCCTCT
TGACACAGGGTCCAGCTAAGGGCAGACAT***GGCTGCTAGTCCAGGGCC
AGGCTcTGAGACCCAAGGGCGCTGCCCAAGGAACCCTTGCCCCAGGGACC
CTGGGAGCAAAGCTCCTCACTCAGAGCCTGCAGCCCTGGgGTCTGAGGAC
AAGGAGGGACTGAGGACTGGGCGTGGGGAGTTCAGGCgGGGACACCGGGT
CCAGGGAGGTGACAAAGGCGCTGGGAGGGGGCGGACGGTGCCGGAGACTC
CTCCTGGGCCCTGTGGGCTCGTGGTCCTTGTGAGGACCCTGAGGG*CTGA
GGGGCCCCTGGGCCTAGGGACTTGCAGTGAGGGAGGCAGGGAGTGTCCCT
TGAGAACGTGGCCTCCGCGGGCTGGGTCCCCCTCGTGCTCCCAGCAGGGA
GGACACCCCAGAGCAAGCGCCCCAGGTGGGCGGGGAGGGTCTCCTCACAG
GGGCAGCTGACAGATAGAC*GgccCCCGCCAGACAGATGCTTGATCCTGG
TCag***TACTGGGTTCGCcACTTCCCTGAACAGGGGCCCTCCGAACAGA
CACAGACGCAGACCaggCTCAGCACAgAAAATGCACTGACACCCAAAACC
CTCATCTGggGGCCTGGCCGGCATCCCGCCCCAGGACCCAAGGCCCCTGC
CCCCTGGCAGCCCTGGACACGGTCCTCTGTGGGCGGTGGGGTCgGGGCTG
TGGTGACGGTGGCATCGGGGAGCCTGTGCCCCCTCCCTGAAAGGGCGGAG
AGGCTCAAGAGGGGACAGAAATGTCCTCCCCTAGGAAGACCTCGGACGGG
GGCGGGGGGGTGGTCTCCGACAGACAGATGCCCGGGACCGACAGACCTGC
CGAGGGAAGAGGGCACCTCGGTCGGGTTAGGCTCCAGGCAGCACGAGGGA
GCGAGGCTGGGAGGGTGAGGACATGGGAGCCTGAGGAGGAGCTGGAGACT
TCAGCAGGCCCCCAGCTCCGGGCTTCGGGCTCTGAGATGCTCGGACGCAA
GGTGAGTGACCCCACCTGTGGCTGACCTGACCTGACCtCAGGGGGACAAG
GCTGAGCCTGGGACTCTgTGTCCCCATCGCCTGCACAGGGGATTCCCCTG
ATGGACACTGAGCCAACGACCTCCCGTCTCTCCCCGACCCCCAGGTCAGC
CCAAGGCCACTCCCACGGTCAACCTGTTCCCGCCCTCCTCTGAGGAGCTC
GGCACCAACAAGGCCACCCTGGTGTGTCTA
Seq ID No. 32
GCCACGCCCACTCCATCATGCGGGGAGGGGATGGGCAGACCCTCCAGAAA
GAAGCTCCCTGGGGTGCAGGTTAACAGCTTTCCCAGACACAGCCAGTACT
AGAGTGAGGTGAATAAGACATCCTCCTTGCTTGTGAAATTTAGGAAGTGC
CCCCAAACATCAGTCATTAAGATAAATAATATTGAATGCACTTTTTTTTT
TTTATTTTTTTTTTTTGCTTTTTAGGGCCTAATCTGCAGCatatggaagt
tcccaggctacaagtcgaaccagagctgcagctgccagcctacatcacag
ccacagcaacaccagatccgagccacatctgtgactaacactgcagttca
cagcaacgccagatccttaacccattgagtgaggccagggatcaaaccca
catcctcatggatactagtctggttcgtaaaccactgagccaCAAGGGGA
ACTCCTGAATGCAATATTTTTGAAAATTGAAATTAAATCTGTCACTCTTT
CACTTAAGAGTCCCCTTAGATTGGGGAAAATTTAAATATCTGTCATCTTA
GTGCATCTTTGCTCATATGATGTGAATAAAATCCCAAAATCCATATGAAT
GAAGCATCAAAATGTACATGAAGTCAGCCTGACCCTGCACTGCCCTCACT
TGCCTCATGTACCCCCCACCTCAAAGGAAGATGCAGAAAGGAGTCCAGCC
CCTACACCGCCACCTGCCCCCACCACTGGAGCCCCTCAGGTCTCCCACCT
CCTTTTCTGAGCTTCAGTCTTCCTGTGGCATTGCCTACCTCTACAGCTGC
CCCCTACTAGGCCCTCCCCCTGGGGCTGAGCTCCAGGCACTGGACTGGGA
AAGTTAGAGGTTAAAGCATGGAAAATTCCCAAAGCCACCAGTTCCAGGCT
GCCCCCCACCCCACCGCCACGTCCAAAAAGGGGCATCTTCCCAGATCTCT
GGCTGGTATTGGTAGGACCCAGGACATAGTCTTTATACCAATTCTGCTGT
GTGTCTTAGGAAAGAaactctccctctctgtgcttcagtttcctcatcaa
taaaAGGAGCAGGCCAGGTTGGAGGGTCTGTGACGTCTGCTGAAGCAGCA
GGATTCTCTCTCCTTTTGCTGGAGGAGAACTGATCCTTCACCCCCAGGAT
CAACAGAGAAGCCAAGGTCTTCAGCCTTCCTGGGGACCCCTCAGAGGGAA
CTCAGGGCCACAGAGCCAGACCCTGATGCCAGAACCTTTGTCATATGCCC
AGACGGAGACTTCATCCCCCTCCTCCTCAGACCCTCCAGGCCCCAACAGT
GAGATGCTGAAGATATTAAGAGAAGGGCAAGTCAGcTTAAGTTTGGGGGT
AGAGGGGAACAGGGAGTGAGGAGATCTGGCCTGAGAGATAGGAGCCCTGG
TGGCCACAGGAGGACTCTTTGGGTCCTGTCGGATGGACACAGGGCGGCCC
GGGGGCATGTTGGAGCCCGGCTGGTTCTTACCAGAGGCAGGGGGCACCCT
CTGACACGGGAGCAGGGCATGTTCCATACATGACACACCCCTCTGCTCCA
GGGCAGGTGGGTGGCGGCACAGAGGAGCCAGGGACTCTGAGCAAGGGGTC
CACCAGTGGGGCAGTTGGATCCAGACTTCTCTGGGCCAGCGAGAGTCTAG
CCCTCAGCCGTTCTCTGTCCAGGAGGGGGGTGGGGCAGGCCTGGGCGGCC
AGAGCTCATCCCTCAAGGGTTCCCAGGGTCCTGCCAGACCCAGATTTCCG
ACCGCAGCCACCACAAGAGGATGTGGTCTGCTGTGGCAGCTGCCAAGACC
TTGCAGCAGGTGCAGGGTGGGGGGGTGGGGGCACCTGGGGGCAGCTGGGG
TCACTGAGTTCAGGGAAAACCCCTTTTTTCCCCTAAACCTGGGGCCATCC
CTAGGGGAAACCACAACTTCTGAGCCCTGGGCAGTGGCTGCTGGGAGGGA
AGAGCTTCATCCTGGACCCTGGGGGGGAACCCAGCTCCAAAGGTGCAAGG
GGCCCAGGTCCAAGGCTAGAGTGGGCCAAGCACCGCAATGGCCAGGGAGT
GGGGGAGGTGGAGCTGGACTGGATCAGGGCCTCCTTGGGACTCCCTACAC
CCTGTGTGACATGTTAGGGTACCCACACCCCATCACCAGTCAGGGCCTGG
CCCATCTCCAGGGCCAGGGATGTGCATGTAAGTGTGTGTGAGTGTGTGTG
TGTGGTGTAGTACACCCCTTGGCATCCGGTTCCGAGGCCTTGGGTTCCTC
CAAAGTTGCTCTCTGAATTAGGTCAAACTGTGAGGTCCTGATCGCCATCA
TCAACTTCGTTCTCCCCACCTCCCATCATTATCAAGAGCTGGGGAGGGTC
TGGGATTTCTTCCCACCCACAAGCCAAAAGATAAGCCTGCTGGTGATGGC
AGAAGACACAGGATCCTGGGTCAGAGACAAAGGCCAGTGTGTCACAGCGA
GAGAGGCAGCCGGACTATCAGCTGTCACAGAGAGGCCTTAGTCCGCTGAA
CTCAGGCCCCAGTGACTCCTGTTCCACTGGGCACTGGCCCCCCTCCACAG
CGCCCCCAGGCCCCAGGGAGAGGCGTCACAGCTTAGAGATGGCCCTGCTG
AACAGGGAACAAGAACAGGTGTGCCCCATCCAGCGCCCCAGGGGTGGGAC
AGGTGGGCTGGATTTGGTGTGAAGCCCTTGAGCCCTGgAACCCAAcCACA
GCAgGGCAGTTGGTAGATGCCATTTGGGGAGAGGCCCCAGGAGTAAGGGC
CATGGGCCCTTGAGGGGGCCAGGAGCTGAGGACAGGGACAGAGACGGCCC
AGGCAGAGGACAGGGCCATGAGGGGTGCACTGAGATGGCCACTGCCAGCA
GGGGCAGCTGCCAACCCGTCCAGGGAACTTATTCAGCAGTCAGCTGGAGG
TGCCATTGACGCTGAGGGCAGATGAAGCCCAGGCCAGGCTAGGTGGGCTG
TGAAGACCCCAGGGGACAGAGCTCTGTCCCTGGGCAGCACTGGCCTCTCA
TTCTGCAGGGCTTGACGGGATCCCAAGGCCTGCTGCCCCTGATGGTAGTG
GCAGTACCGCCCAGAGCAGGACCCCAGCATGGAAACCCCAACGGGACGCA
GCCTGCGGAGCCCACAAAACCAGTAAGGAGCCGAAGCAGTCATGGCACGG
GGAGTGTGGACTTCCCTTTGATGGGGCCCAGGCATGAAGGACAGAATGGG
ACAGCGGCCATGAGCAGAAAATCAGCCGGAGGGGATGGGCCTAGGCAGAC
GCTGGCTTTATTTGAAGTGTTGGCATTTTGTCTGGTGTGTATTGTTGGTA
TTGATTTTATTTTAGTATGTCAGTGACATACTGACATATTATGTAACGAC
ATATTATTATGTGTTTTAAGAAGCACTCCAAGGGAACAGGCTGTCTGTAA
TGTGTCCAGAGAAGAGAGCAAGAGCTTGGCTCAGTCTCCCCCAAGGAGGT
CAGTTCCTCAACAGGGGTCCTAAATGTTTCCTGGAGCCAGGCCTGAATCA
AGGGGgTCATATCTACACGTGGGGCAGACCCATGGACCATTTTCGGAGCA
ATAAGATGGCAGGGAGGATACCAAGCTGGTCTTACAGATCCAGGGCTTTG
ACCTGTGACGCGGGCGCTCCTCCAGGCAAAGGGAGAAGCCAGCAGGAAGC
TTTCAGAACTGGGGAGAACAGGGTGCAGACCTCCAGGGTCTTGTACAACG
CACCCTTTATCCTGGGGTCCAGGAGGGGTCACTGAGGGATTTAAGTGGGG
GACCATCAGAACCAGGTTTGTGTTTTGGAAAAATGGCTCCAAAGCAGAGA
CCAGTGTGAGGCCAGATTAGATGATGAAGAAGAGGCAGTGGAAAGTCGAT
GGGTGGCCAGGTAGCAAGAGGGCCTATGGAGTTGGCAAGTGAATTTAAAG
TGGTGGCACCAGAGGGCAGATGGGGAGGAGCAGGCACTGTCATGGACTGT
CTATAGAAATCTAAAATGTATACCCTTTTTAGCAATATGCAGTGAGTCAT
AAAAGAACACATATATATTTAAATTGTGTAATTCCACTTCTAAGGATTCA
TCCCAAGGGGGGAAAATAATCAAAGATGTAACCAAAGGTTTACAAACAAG
AACTCATCATTAATCTTCCTTGTTGTTATTTCAACGATATTATTATTATT
ACTATTATTATTATTATTATTttgtctttttgcattttctagggccactc
ccacggcatagagaggttcccaggctaggggtcaaatcggagctacagct
gccggcctacgccagagccacagcaacgcaggatctgagccacagcaatg
caggatctacaccacagctcatggtaacgctggatccttaacccaatgag
tgaggccagggatcgaacctgtaacttcatggttcctagtcggattcatt
aaccactgagccacgacaggaactccAACATTATTAATGATGGGAGAAAA
CTGGAAGTAACCTAAATATCCAGCAGAAAGGGTGTGGCCAAATACAGCAT
GGAGTAGCCATCATAAGGAATCTTACACAAGCCTCCAAAATTGTGTTTCT
GAAATTGGGTTTAAAGTACGTTTGCATTTTAAAAAGCCTGCCAGAAAATA
CAGAAAAATGTCTGTGATATGTCTCTGGCTGATAGGATTTTGCTTAGTTT
TAATTTTGGCTTTATAATTTTCTATAGTTATGAAAATGTTCACAAGAAGA
TATATTTCATTTTAGCTTCTAAAATAATTATAACACAGAAGTAATTTGTG
CTTTAAAAAAATATTCAACACAGAAGTATATAAAGTAAAAATTGaggagt
tcccatcgtggctcagtgattaacaaacccaactagtatccatgaggata
tggatttgatccctggccttgctcagtgggttgaggatccagtgttgctg
tgagctgtggtgtaggttgcagacacagcactctggcgttgctgtgactc
tggcgtaggccggcagctacagctccatttggacccttagcctgggaacc
tccatatgcctgagatacggcccTAAAAAGTCAAAAGCCAAAAAAATAGT
AAAAATTGAGTGTTTCTACTTACCACCCCTGCCCACATCTTATGCTAAAA
CCCGTTCTCCAGAGACAAACATCGTCAGGTGGGTCTATATATTTCCAGCC
CTCCTCCTGTGTGTGTATGTCCGTAAAACACACACACACACACACACACG
CACACACACACACACGTATCTAATTAGCATTGGTATTAGTTTTTCAAAAG
GGAGGTCATGCTCTACCTTTTAGGCGGCAAATAGATTATTTAAACAAATC
TGTTGACATTTTCTATATCAACCCATAAGATCTCCCATGTTCTTGGAAAG
GCTTTGTAAGACATCAACATCTGGGTAAACCAGCATGGTTTTTAGGGGGT
TGTGTGGATTTTTTTCATATTTTTTAGGGCACACCTGCAgcatatggagg
ttcccaggctaggggttgaatcagagctgtagctgccggcctacaccaca
gccacagcaacgccagatccttaacccactgagaaaggccagggattgaa
cctgcatcctcatggATGCTGGTCAGATTTATTTCTGCTGAGCCACAACA
GGAACTCCCTGAACCAGAATGCTTTTAACCATTCCACTTTGCATGGACAT
TTAGATTGTTTCCATTTAAAAATACAAATTACAaggagttcccgtcgtgg
ctcagtggtaacgaattggactaggaaccatgaggtttcgggttcgatcc
ctggccttgctcggtgggttaaggatccagcattgatgtgagatatggtg
taggtcgcagacgtggctcggatcccacgttgctgtggctctggcgtagg
ccggcaacaacagctccgattcgacccctagccTGggaacctccatgtgc
cacaggagcagccctaGAAAAGGCAAAAAGACAAAAAAATAAAAAATTAA
AATGAAAAAATAAAATAAAAATACAAATTACAAGAGACGGCTACAAGGAA
ATCCCCAAGTGTGTGCAAATGCCATATATGTATAAAATGTACTAGTGTCT
CCTCGCGGGAAAGTTGCCTAAAAGTGGGTTGGCTGGACAGAGAGGACAGG
CTTTGACATTCTCATAGGTAGTAGCAATGGGCTTCTCAAAATGCTGTTCC
AGTTTACACTCACCATAGCAAATGACAGTGCCTCTTCCTCTCCACCCTTG
CCAATAATGTGACAGGTGGATCTTTTTCTATTTTGTGTATCTGACAAGCA
AAAAATGAGAACAggagttcctgtcgtggtgcagtggagacaaatctgac
taggaaccatgaaatttcgggttcaatccctggcctcactcagtaggtaa
aggatccagggttgcagtgagctgtggggtaggtcgcagacacagtgcaa
atttggccctgttgtggctgtggtgtaggccggcagctatagctccaatt
ggacccctagcctgggaacctccttatgccgtgggtgaggccctAAAAAA
AAGAGTGCAAAAAAAAAAAATAAGAACAAAAATGATCATCGTTTAATTCT
TTATTTGATCATTGGTGAAACTTATTTTCCTTTTATATTTTTATTGACTG
ATTTTATTTCTCCTATGAATTTACCGGTCATAGTTTTGCCTGGGTGTTTT
TACTCCGGTTTTAGTTTTGGTTGGTTGTATTTTCTTAGAGAGCTATAGAA
ACTCTTCATCTATTTGGAATAGTAATTCCTCATTAAGTATTTGTGCTGCA
AAAAATTTTCCCTGATCTGTTTTATGCTTTTGTTTGTGGGGTCTTTCACG
AGAAAGCCTTTTTAGTTTTTACACCTCAGCTTGGTTGTTTTTCTTGATTG
TGTCTGTAATCTGCGGCCAACATAGGAAACACATTTTTACTTTAGTGTTT
TTTTCCTATTTTCTTCAAGTACGTCCATTGTTTTGGTGTCTGATTTTACT
TTGCCTGGGGTTTGTTTTTGTGTGGCAGGAATATAAACTTATGTATTTTC
CAAATGGAGAGCCAATGGTTGTATATTTGTTGAATTCAAATGCAACTTTA
TCAAACACCAAATCATCGATTTATCACAACTCTTCTCTGGTTTATTGATC
TAATGATCAATTCCTGTTCCACGCTGTTTTAATTATTTTAGCTTTGTGGA
TTTTGGTGCCTGGTAGAGAACAAAGCCTCCATTATTTTCATTCAAAATAG
TCCCGTCTATTATCTGCCATTGTTGTAGTATTAGACTTTAAAATCAATTT
ACTGATTTTCAAAAGTTATTCCTTTGGTGATGTGGAATACTTTATACTTC
ATAAGGTACATGGATTCATTTGTGGGGAATTGATGTCTTTGCTATTGTGG
CCATTTGTCAAGTTGTGTAATATTTTACCCATGCCAACTTTGCATATTGT
ATGTGAGTTTATTCCCAGGGTTTTTAATAGGATGTTTATTGAAGTTGTCA
GTGTTTCCACAATTTCATCGCCTCAGTGCTTACTGTTTGCATAAAAGGAA
ACCTACTCACTTTTGCCTATTGCTCTTGTATTCAATCATTTTAGTTAACT
CTTGTGTTAATTTTGAGAGTTTTTCAGCTGACTGTCTGGGGTTTTCTTTA
ATAGACTAGCCCTTTGTCTGTAAAGAATAATTTTATCGAATTTTTCTTAA
CACTCACACTCTCCCCACCCCCACCCCCGCTGATCTCCTTTCATTGGGTC
AAATCTGTAGAATACAATAAAAGTAAGAGTGGGAACCTTAGCCTTTAAGT
CGATTTTGCCTTTAAATGTGAATGTTGCTATGTTTCGGGACATTCTCTTT
ATCAAGTTGCGGATGTTTCCTTAGATAATTAACTTAATAAAAGACTGGAT
GTTTGCTTTCTTCAAATCAGAATTGTGTTGAATTTATATTGCTATTCTGT
TTAATTTTGTTTCAAAAAATTTACATGCACACCTTAAAGATAACCATGAC
CAAATAGTCCTCCTGCTGAGAGAAAATGTTGGCCCCAATGCCACAGGTTA
CCTCCCGACTCAGATAAACTACAATGGGAGATAAAATCAGATTTGGCAAA
GCCTGTGGATTCTTGCCATAACTCTCAGAGCATGACTTGGGTGTTTTTTC
CTTTTCTAAGTATTTTAATGGTATTTTTGTGTTACAATAGGAAATCTAGG
ACACAGAGAGTGATTCAATGAGGGGAACGCATTCTGGGATGACTCTAGGC
CTCTGGTTTGGGGAGAGCTCTATTGAAGTAAAGACAATGAGAGGAAGCAA
GTTTGCAGGGAACTGTGAGGAATTTAGATGGGGAATGTTGGGTTTGAGGT
TTCTATAGGGCACGCAAGCAGAGATGCACTCAGGAGGAAGAAGGAGCATA
AATCTAGAGGCAAAAAGAGAGGTCAGGACTGGAAATAGAGATGCGAGACA
CCAGGGTGGCAGTCAGAGAGCACAGTGTGGGTCAGAAGACAGTGGAAGAA
CACAAGGGACAGAGAGGGATCTCCAACTTCACTGGGATGAGGGCCTTGTT
GGCCTTGACCTGAGAGATTTCCAGGAGTTGAGGGTGGGAAGGAGAGGGCT
CCTGCACATGTCCTGACATGAAACGGTGCCCAGCATATGGGTGCTTGGAA
GACATTGTTGGACAGATGGATGGATGATGGATGATGGATGAATGGATGGA
TGGAAGATGATGGATAAATGGATGATGGATGGATGGACAGAAGGACAAAG
AGATGGACAGAAAGACAGTGATCTGAGAGAGCAGAGAAGGCTTCATGAAA
GGACAGGAACTGAACTGTCTCAGTGGGTGGAGACAATGGTGTAGGGGGTT
TCCACATGGAGGCACCAGGGGTCAGGAATAATCTAGTGTCCACAGGCCCA
GGAAGGAAGCTGTCTGCAGGAAATTGTGGGGAAGAACCTCAGAGTCCTTA
AATGAGGTCAGGAGTGGTCAGGAGGGTCTGATCAGGTAAGGACTCATGTC
CATCATCACATGGTCACCTAAGGGCATGTAGCTCTCAGCATCTCCATCAG
GACAGTCTCAGAATGGGGGCGGGGTCACACACTGGGTGACTCAAGGCGTG
GGTCATGCCTGCCTCGGACGTGGGCCTGGGCATGGGGACACCTCCAGACC
ATGGGCCCGCCCAGGGCTGCACTGGcctctggtgggctagctacccgtcc
aagcaacacaggacacagccctacctgctgcaaccctgtgcccgaaacgc
ccatctggttcctgctccagcccggccccagggaacaggactcaggtgct
agcccaatggggttttgttcgagcctcagtcagcgtggTATTTCTCCGGC
AGCGAGACTCAGTTCACCGCCTTAGGttaagtggttctcatgaatttcct
agcagtcctgcactctgctatgccgggaaagtcacttttgtcgctggggg
ctgtttccccgtgcccttggagaatcaaggattgcccaactttctctgtg
ggggaggtggctggtcttggggtgaccagcaggaagggccccaaaagcag
gagcagctgcctccagAATACAACTGTCGGCTACAGCTCAAACAGGAGGC
CTGGACTGGGGTTTAACCACCAGGGCGGCACGAAGGAGCGAGGCTGGGAG
GGTGAGGACATGGGAGCCTGAGGAGGAGCTGGAGACTTCAGCAGGCCCCC
AGCTCCGGGCTTCGGGCTCTGAGATGCTCGGACGCAAGGTGAGTGACCCC
ACCTGTGGCTGACCTGACCTCAGGGGGACAAGGCTCAGCCTGAGACTCTG
TGTCCCCATCGCCTGCACAGgggattcccctgatggacactgagccaacg
acctcccgtctctccccgacccccaggtcagcccaaggccgcccccacgg
tcaacctcttcccgccctcctctgaggagctcggcaccaacaaggccacc
ctggtgtgtctaataagtgacttctacccgAAGGGCGAATTCCAGCACAC
TGGCGGCCGTTACTAGTGGATCCGAGCTCGGTACCAAGCTTGATGCATAG
CTTGAGTATCTA
Seq ID No. 33
agatctttaaaccaccgagcaaggccagggatcgaacccgcatcctcatg
aatcctagttgggttcgttaaccgctgaaccacaatgggaactcctGTCT
TTCACATTTAATTCACAACCTCTCCAGGATTCTGGGGGTGGGTGGGGAAT
CCTAGGTACCCACTGGGAAAGTAATCCAAGGGGAGAGGCTCACGGACTcT
AGGGATCGGCGGAGGAGGGAAGGTATCTCCCAGGAAACTGGCCAGGACAC
ATTGGTCCTCCGCCCTCCCCTTCCTCCCACTCCTCCTCCAGACAGGACTG
TGCCCACCCCCTGCCACCTTTCTGGCCAGAACTGTCCATGGCAGGTGACC
TTCACATGAGCCCTTCCTCCCTGCCTGCCCTAGTGGGACCCTCCATACCT
CCCCCTGGACCCCGTTGTCCTTTCTTTCCAGTGTGGCCCTGAGCATAACT
GATGCCATCATGGGCTGCTGACCCACCCGGGACTGTGTTGTGCAGTGAGT
CACTTCTCTGTCATCAGGGCTTTGTAATTGATAGATAGTGTTTCATCATC
ATTAGGACCGGGTGGCCTCTATGCTCTGTTAGTCTCCAAACACTGATGAA
AACCTTCGTTGGCATAGTCCCAGCTTCCTGTTGCCCATCCATAAATCTTG
ACTTAGGGATGCACATCCTGTCTCCAAGCAACCACCCCTCCCCTAGGCTA
ACTATAAAACTGTCCCAATGGCCCTTGTGTGGTGCAGAGTTCATGCTTCC
AGATCATTTCTGTGCTAGATCCATATCTCACCTTGTAAGTCATCCTATAA
TAAACTGATCCATTGATTATTTGCTTCTGTTTTTTCCATCTCAAAACAGC
TTCTCAGTTCAGTTCGAATTTTTTATTCCCTCCATCCACCCATACTTTCC
TCAGCCTGGGGAACCCTTGCCCCCAGTCCCATGCCCTTCCTCCCTCTCTG
CCCAGCTCAGCACCTGCCCACCCTCACCCTTCCTGTGACTCCCTAGGACT
GGACCATCCACTGGGGCCAGGACACTCCAGCAGCCTTGGCTTCATGGGCT
CTGAAATCCATGGCCCATCTCTATTCCTCACTGGATGGCAGGTTCAGAGA
TGTGAAAGGTCTAGGAGGAAGCCAGGAAGGAAACTGTTGCATGAAAGGCC
GGCCTGATGGTTCAGTACTTAAATAATATGAGCTCTGAGCTCCCCAGGAA
CCAAAGCATGGAGGGAGTATGTGCCTCAGAATCTCTCTGAGATTCAGCAA
AGCCTTTGCTAGAGGGAAAATAGTGGCTCAACCTTGAGGGCCAGCATCTT
GCACCACAGTTAAAAGTGGGTATTTGTTTTACCTGAGGCCTCAGCATTAT
GGGAACCGGGCTCTGACACAAACACAGGTGCAGCCCGGCAGCCTCAGAAC
ACAGCAACGACCACAAGCTGGGACAGCTGCCCCTGAACGGGGAGTCCACC
ATGCTTCTGTCTCGGGTACCACCAGGTCACCATCCCTGGGGGAGGTAGTT
CCATAGCAGTAGTCCCCTGATTTCGCCCCTCGGGCGTGTAGCCAGGCAAG
CTCCTGCCTCTGGACCCAGGGTGGACCCTTGCTCCCCACTACCCTGCACA
TGCCAGACAGTCAAGACCACTCCCACCTCTGTCTGAGGCCCCCTTGGGTG
TCCCAGGGCCCCCGAGCTGTCCTCTACTCATGGTTCTTCCACCTGGGTAC
AAAAGAGGCGAGGGACACTTTTCTCAGGTTTGCGGCTCAGAAAGGTACCT
TCCTAGGGTTTGTCCACTGGGAGTCACCTCCCTTGCATCTCAATGTCAGT
GGGGAAAACTGGGTCCCATGGGGGGATTAGTGCCACTGTGAGGCCCCTGA
AGTCTGGGGCCTCTAGACACTATGATGATGAGGGATGTGGTGAAAAACCC
CACCCCAGCCCTTCTTGCCGGGACCCTGGGCTGTGGCTCCCCCATTGCAC
TTGGGGTCAGAGGGGTGGATGGTGGCTATGGTGAGGCATGTTTCCCATGA
GCTGGGGGCACCCTGGGTGACTTTCTCCTGTGAATCCTGAATTAGCAGCT
ATAACAAATTGCCCAAACTCTTAGGCTTAAAACAACACACATTTATTCCT
CTGGGTCCCAGGGTCAGAAGTCCAAAATGAGTCCTATAGGCTAAATTTGA
GGTGTCTCTGGGTTGAGCTCCTCCTGGAAGCCTTTTCCAGCCTCTAGAGT
CCCAAGTCCTTGGCTCTGGGCCCCTCCCTCAAGCTTCAAAGCCACAGAAG
CTTCTAATCTCTCTCCCTTCCCCTCTGACCTCTGCTCCCATCCTCATACC
CTGTCCCCTCACTCTGACCCTCCTGCCTCCCTCTTTCCCTTATAAAGACC
CTGCATGGGGCCACGGAGATAATCCAGGGTAATCGCCCCTCTTCCAGCCC
TTAACTCCATCCCATCTGCAAAATCCCTGTCACCCCATAATGGACCTACT
GATGGTCTGGGGGTTAGGACGTGGACAACTTGGGGCCTTATTCATCTGAT
CACAACTCCAGTTCCCAGACCCCCAGACCCCCGGGCATTAGGGAAACTTC
TCCCAGTTCCTCTCCCTCTGTGTCCTGCCCAGTCTCCAGGATGGGCCACT
CCCGAGGGCCCTTCAGCTCAGGCTCCCCCTCCTTTCTCCCTGGCCTCTTG
TGGCCCCATCTCCTCCTCCGCTCACAGGGAGAGAACTTTGATTTCAGCTT
TGGCTCTGGGGCTTTGCTTCCTTCTGGCCATTGGCTGAAGGGCGGGTTTC
TCCAGGTCTTACCTGTCAGTCATCAAACCGCCCTTGGAGGAAGACCCTAA
TATGATCCTTACCCTACAGATGGAGACTCGAGGCCCAGAGATCCTGAGTG
ACCTGCTCACATTCACAGCAGGGACTGAACCCCAGTCACCTACCCAACTC
CAGGGCTCAGCGCTTTTTTTTTTTTTTTTCTTTTTgccttttcgagggcc
gctcccgcaacatatggagatttccaggctaggggtctaattggagcagt
cgacactggcctaagccaaagccacagcaacaagggcaagccgcttctgc
agcctataccacagctcacggcaatgccggatccttaacccactgagcaa
agccagggattgaacctgcaacctcatgtttcctagtcaaatttgttaac
cactgacccatgacgggaactcccAGGGCTCAGCTCTTGACTCCAGGTTC
GCAGCTGCCCTGAAAGCAATGCAACCCTGGCTGGCCCCGCCTCATGCATC
CGGCCTCCTGCCCAAAGAGCTCTGAGCCCACCTGGGCCTAGGTCCTCCTC
CCTGGGACTCATGGCCTAAGGGTACAGAGTTACTGGGGCTGATGAAGGGA
CCAATGGGGACAGGGGCCTCAAATCAAAGTGGCTGTCTCTCTCATGTCCC
TTCCTCTCCTCAGGGTCCAAAATCAGGGTCAGGGCCCCAGGGCAGGGGCT
GAGAGGGCCTCTTTCTGAAGGCCCTGTCTCAGTGCAGGTTATGGGGGTCT
GGGGGAGGGTCAATGCAGGGCTCACCCTTCAGTGCCCCAAAGCCTAGAGA
GTGAGTGCCTGCCAGTGGCTTCCCAGGCCCAATCCCTTGACTGCCTGGGA
ATGCTCAAATGCAGGAACTGTCACAACACCTTCAGTCAGGGGCTGCTCTG
GGAGGAAAAACACTCAGAATTGGGGGTTCAGGGAAGGCCCAGTGCCAAGC
ATAGCAGGAGCTCAGGTGGCTGCAGATGGTGTGAACCCCAGGAGCAGGAT
GGCCGGCACTCCCCCCAGACCCTCCAGAGCCCCAGGTTGGCTGCCCTCTT
CACTGCCGACACCCCTGGGTCCACTTCTGCCCTTTCCCACCTAAAACCTT
TAGGGCTCCCACTTTCTCCCAAATGTGAGACATCACCACGGCTCCCAGGG
AGTGTCCAGAAGGGCATCTGGCTGAGAGGTCCTGACATCTGGGAGCCTCA
GGCCCCACAATGGACAGACGCCCTGCCAGGATGCTGCTGCAGGGCTGTTA
GCTAGGCGGGGTGGAGATGGGGTACTTTGCCTCTCAGAGGCCCCGGCCCC
ACCATGAAACCTCAGTGACACCCCATTTCCCTGAGTTCACATACCTGTAT
CCTACTCCAGTCACCTTCCCCACGAACCCCTGGGAGCCCAGGATGATGCT
GGGGCTGGAGCCACGACCAGCCCACGAGTGATCCAGCTCTGCCAATCAGC
AGTCATTTCCCAAGTGTTCCAGCCCTGCCAGGTCCCACTACAGCAGTAAT
GGAGGCCCCAGACACCAGTCCAGCAGTTAGAGGGCTGGACTAGCACCAGC
TTTCAAGCCTCAGCATCTCAAGGTGAATGGCCAGTGCCCCTCCCCGTGGC
CATCACAGGATCGCAGATATGACCCTAGGGGAAGAAATATCCTGGGAGTA
AGGAAGTGCCCATACTCAAGGATGGCCCCTCTGTGACCTAACCTGTCCCT
GAGGATTGTACTTCCAGGCGTTAAAACAGTAGAACGCCTGCCTGTGAACC
CCCGCCAAGGGACTGCTTGGGGAGGCCCCCTAAACCAGAACACAGGCACT
CCAGCAGGACCTCTGAACTCTGACCACCCTCAGCAAGTGGCACCCCCCGC
AGCTTCCAAGGCAC
Seq ID No. 34
AACAAGATGCTACCCCACCAACAAAATTCACCGGAGAAGACAAGGACAGG
GGGTTCCTGGGGTCCTGACAGGGTCACCAAAGAGGGTTCTGGGGCAGCAG
CAACTCCAGCCGCCTCAGAACAGAGCCTGGAAGCTGTACCCTCAGAGCAG
AGGCGGAGAGAGAAAGGGCCTCTTGGTGGGTCAGCAGGAGCAGAGGCTCA
GAGGTGGGGGTTGCAGCCCCCCCTTCAACAGGCCAACACAGTGAAGCAGC
TGACCCCTCCACCTTGGAGACCCCAGACTCCTGTCTCCCACGCCACCTTG
GTTTTTAAGGTAATTTTTATTTTATATCAGAGTATGGTTGACTTACAATG
TTGTGTTGGTTTCAGGTGTACAGCAGAGTGATTCACTTCTACATAGACTC
ATATCTATTCTTTCTCAGATTCTTTTCCCATATAGGTTATTACAGAATAT
TGAGTAGATCCCTGCTGATTACCCATTTTTATAATTGTATATGTTAATCC
CAAACTCCTAATTTATCCCTCCCCAGACTATGATTCTTTATATCTCTATC
TGTTTCCTAATCTGTCTCCTCTAAGTCACCCTAGGAGAGCAGAGGGGTCA
CGTCTGTCCTGTCCTGGCCCAGCCACCTCTCTCCACCCAGGAATCCCTTG
CATTTGGTGCCAAGGGCCCGGCCCCGCCCTAAAGAGAAAGGAGAACGGGA
TGTGGACAGGACACCGGGCAGAGAGGGACAAGCAGAGGATGCCAGGGTAG
GGAGGTCTCCAGGGTGGATGGTGGTCTGTCCGGAGGCAGGATGAGGCAGG
AAGGGTGTGGATGTACTCGGTGAGGCTGGCGCATGGCCTGGAGTGTCCTG
AGCCCTGGGAGGCCTCAGCCCTGGATCAGATCTGTGATTCCAAAGGGCCA
CTGCATCCAGAGACCGTTGAGTGGCCCATTGTCCTGAACCATTTATAGAA
CACAGGACAAGCGGTACCTGACTAAGCTGCTCACAGATTCCATGAGGCTG
ATGCCAGGGTTGTCACCCCATCTCACAGGCAGGGAAACTGATGCATATAC
TGCAGAGCCAGGCAGAGGCCCTCCCAGTGCCCCCTCCCAGCCTGTGGCCC
CCCTCCAGTGGCTGGACACTGAGGCCACACTGGGGCACCCTGTGGAGATC
t
Seq ID No. 35
AGATCTGGCCAGGCCAGAGAAGCCCATGTGGTGACCTCCCTCCATCACTC
CACGCCCTGACCTGCCAGGGAGCAGAAAGTAGGCCCAGGGTGGACCCGGT
GGCCACCTGCCACCCCATGGCTGGGAGAAGGGAGGGCCTGGGCAAAGGGC
CTGGGAAGCCTGTGGTGGGACCCCAGACCCCAGGGTGGACAGGGAGGGTC
CCACACCCACAGCCATTTGCTTCCCTCTGTGGGTTCAGTGTCCTCATCTC
ATCTGTGGGGAGGGGGCTGATAATGAATCTCCCCCATTGGGGTGGGCTTG
GGGATTAAAGGGCCAGTGTCTGTGATATGCCTGGACCATAGTGACCCTCA
CCCTCCCCAGCCATTGCTGTCACCTTCCGGGCTCTTGCCCAGGCCTGCCT
GACATGCTGTGTGACCCTGGGCAAGATGATCCCCCTTTCTGGGCCCCAGC
CTTCCTCTCTGCTCCGGAAGTGCTTCCTGGGGAAACCTGTGGGCTGGATC
CTATAGGAAACCTGTCCAATTCCTGGATGCACAGAGGGGCAGGGAGGCCC
TGGGCCTGGAGGGGCAGGGAGGCTCGAGGTGGGAGCAGGGTAGGGGCCAG
TCCAGGGCAAGGAGGTGGGTGGGTAGGGTG
Seq ID No. 36:
GATCTGTGTTCCATCTCAGAGCTATCTTAGCAGAGAGGTGCAGGGGCCTC
CAGGGCCACCAAAGTCCAGGCTCAGCCAGAGGCAATGGGGTATCGATGAG
CTACAGGACACAGGCGTCAGCCCAGTGTCAGGGAGAATCACCTTGTTTGT
TTTCTGAGTTCCTCTTAAAATAGAGTTAATTGGTCTTGGCCTTACGGTTT
ACAATAACAACTGCACCCTGTAAACAACGTGAAGAGTACAGAACAACAAA
TGGGGGAAAACATATTTCACCTGAAAGAGCCACCGCTCATATTTTGATGG
ATTTCCTTCTAGTTTAATCCTGTTTTAATTGTAAACTGTTAAAACAAACA
TAAATAAAGAAAATGCATCTGTAAAGTTTAAAAGTCATATCTATGGTGAT
GGTTGCAAAACACTGTGAATGTTCACTTTGAAATCGTGAACTCTACGTGA
TATGCATGTCCCGTTAATTAACCTCACAGGCTCAGAATGTGGTTCATTAT
TTCTTTAATTTTCCTTTAATTTTATGTCCTCTGTGTGTGCCCTTAAACCA
ACTACTTTTCAGCTCTGCCTGTTTTTGACCTTCACATAGATGACATTTGT
GAGTGTTTTCTTTCTCAACACTGGGTCTGATACCCACCCACGCTGTCTGC
TGTCACTGCGGACGTGGAGGGCCACCACCCAGCTATGGCCCCAGCCAGGC
CAACACTGGATGAATCTGCCCCCAGAGCAGGGCCACCAACACTGGAGGTG
CAGAGAGGGTTTCTTCAGGGCCATCATTATCCAAGGCATTGTTTCTACTG
TAAGCTTTCAAAATGCTTCCCCTGATTATTAAAAGAAATAATAAGATGGG
GGGAAAGTACAAGAAGGGAAGTTTCCAGCCCAGCCTGAAGATCGTGCTGG
TTGTATCTGGAGCCTGTCTTCCTGACAGGCCTCTATTCCCAGAGTTA
Seq ID No. 37:
GGATCCTAGGGAAGGGAGGGCGGGGGCCTGGACAAAGGGGGCCTAAAGGA
CATTCTCACCTATCCCACTGGACCcctgctgtgctctgagggagggagca
gagagggggtctgaggccttttcccagCTCCTCTGAGTCCCTCCTCCGAG
CACCTGGACGGAAGCCCCTCCTCAGGGAGTCCTCAGACCCCTCCCCTCCA
GCCAGGTTGGCCTGTGTGGAGTCCCCAGTAAGAATAGAATGCTCAGGGCT
TCGAGCTGAGCCCTGGCTACTTGGGGGGGTGCTGGGGATTGGGGGTGCTG
GGCGGGGAGCTGGGGTGTCACTAGATGCCAGTAGGCTGTGGGCTCGGGTC
TGGGGGGTCTGCACATGTGCAGCTGTGGGAAGGCCCTATTGGTGGTACCC
TCAGACACATATGGCCCCTCAATTTCTGAGACCAGAGAGCCCAGTCTGGC
CTTCCCAGAACAGCTGCCCCTGGTGGGGGAGATGTAGGGGGGCCTTCAGC
CCAGGACCCCCAACGGCAGGGCCTGAGGCCCCCATCCCCTTGTCCTGGGC
CCAGAGCCTCAGCTATCAGGCCTATCAGAGATCCTGGCTGCCCAGCTCAG
GTTCCCCAGGAGCCAGAGGGAGGCCAGGGGTTACTAGGAAATCCGGAAAG
GGTCTTTGAGGCTGGGCCCCACCCTCTCAGCTTTCACAGGAGAAACAGAG
GCCCACAGGGGGCAAAGGACTTGCCAGACTCACAATGAGCCCAGCAGCTG
GACTCAAGGCCCAGTGTTCGGCCCCACAACAGCACTCACGTGCCCTTGAT
CGTGAGGGGCCCCCTCTCAGCCAGGCATTCAGACCTGTGACCTGCATCTA
AGATTCAGCATCAGCCATTCTGAGCTGAAGAGCCCTCAGGGTCTGCAGTC
AAGGCCAGAGGGCCAGACCTCCAACGGCCAGACATCCCAGCCAGATTCCT
TTCTGGTCAATGGGCCCCAGTCTGGCTTGGCTCCTGCAGGCCCAGTGCCG
CCTTCTTCCCCTGGGCCTGTGGAGTCCAGCCTTTCAGTTTCCCACCCACA
TCCTCAGCCACAATCCAGGCTCAGAGGCAATGTCCGTGGGCAGCCCCTGT
GTGACCCCTCTGTGGGTGATCCTCAGTCCTACCCTTAGCAGACAGCGCAT
GAGGGGCCCTCTTGAACCTGAGGGATACTCCATGTCGGAGGGGAGAAGCT
GGCCTTCCCCACCCCCACTTCCAGGCCTTGGGGAGCAGAGAAAGACCCCA
GACCTGGGTCCCTTCTAACAGGCCAGGCCCCAGCCCAGCTCTCCACCAGC
CCCAGGGGCCTGGGGTCCACGCCTGGGGACTGGAGGGTGGGCCTGTCAGG
CGCTGACCCAGAGGCAGGACAGCCAAGTTCAGGATCCCAGCCAGGTGGTC
CCCGTGCACCATGCAGGGGTGTCACCCACACAGGGGTGTTGCCACCCTCA
CCTGACTGTCCTCATGGGCCACATGGAGGTATCCTGGGTTCATTACTGGT
CAACATACCCGTGTCCCTGCAGTGCCCCCTCTGGcgcacgcgtgcacgcg
cacacgcacacactcatacaGAGGCTCCAGCCAAGAGTGCCCTGTAGTAG
GCACTGCTGTCACTTCTCTAAAAGGTCGCAATCATACTTGTAAAGACCCA
AGATTGTTCAGAAATCCCAGATGGAGAAGTCTGGAAAGATCtTTTTCTCC
TTTCACGGGCTGGGGAAATGTGACCTGGCCAAGGTCACACAGCAAGTGGT
GGAACCCTGGCCCCTGATTCCAGCTCATTCCAGTTCCCAAGGCCCTGCCA
GAGCCCAGAGGCTGGGCCCTCTGGGGCAGAGGAGCTGGGGTCCTCCCCCC
TACACAGAGCACACAGCCCCGCAAGAGAGAAGAGACACCTTGGGGAGAGG
AATCTCCAGACCAGAGATCCCAGTATGGGTCTCCTCTATGCTGACGGGAT
GGGATGTCAAGAGGGGAGGGGGCTGGGCTTTAGGGAAACACACAAAAATC
GCTGAGAACACTGACAGGTGCGACACACCCACCCCTAATGCTAACCTGTG
GCCCATTACTCAgatct
Seq ID No. 38
GATCTTCTCCTAAGACCAAGGAAAACTGGTCATACCAGGTCCACTTGTCC
CCTGTGGCCATTGTCCCTCCTTCCCCAGAAGAAACAAGCACTTTCCACTC
CACAAGTAGCTCCTGATCAGCTTGGAAGCCCGGTGCTGCTCTGGGCCCTG
GGGACACGGCAGGGGCATCAGAGACCAAATCCTGGAACAAAGTTCCAGTG
GGTGAGGCAGGCCGGACAAGCAACACGTTATACCATAATATGAGGCAAAA
TATAATGTGAGTTCTTTATGAAAGGAAGGGGTTGCAGGTGCAACTGTTGG
CTTAGGTGGATGGTCACCCCTGAATGGAGGAGGGGGTTCCCAGGGCATGT
GCCTGGGGAGAAGGGCTCCTGGCAGGAGGGACAGCAAGTGCAAGGGCCCT
GTGATCAAATGTGCCTGGCAAGTTGCAGGAACAGCTAGAAGGCCAGCAAG
GTTGGAACCAAGGAAGGGGTGAGGGGAGGGGCAGGGCCCTCAGGGCCTTG
CCCAGCAGCCTGAGCATCTGGAGATTTGTCCAAAGTTTCAAATGTACCTG
GGCAACCTCATGCCCATATACCATTCCTAACTTCTGCACTTAACATCTCT
AGGACTGGGACCCAGCCAGTCAAGCGGGGGGACCCAGAGAGCTCCGGTGT
GAACACCGAGGTGCTGGTGGGTCTGCGTGTGTGGACATAGGGCAGTCCCG
GTCCTTCCTTCACTAACACGGCCCGGGAAGCCCTGTGCCTCCCTGGTGCG
CGGGTCGGCGCTTCCGGAGGGTACAGGCCCACCTGGAGCCCGGGCACAGT
GCATGCAAGTCGGGTTCACGGCAACCTGAGCTGGCTCTGCAGGGCAGTGG
GACTCACAGCCAGGGGTACAGGGCAGACCGGTCCTGCCTCTGCGCCCCTC
CCTGGCCTGTGGCCCCTGGACGTGATCCCCAACAGTTAGCATGCCCCGCC
GGTGCTGAGAACCTGGACGAGGTCCGCAGGCGTCACTGGGCGGTCACTGA
GCCCGCCCCAGGCCCCCTCTGCCCCTTCCTGGGGTGACCGTGGACTCCTG
GATGACCCTGGACCCTAGACTTCCCAGGGTGTCTCGCGGAGGTTCCTCAG
CCAGGATCTCTGCGTCTCCTCCTTCCATAGAGGGGACGGCGCCCCCTTGT
GGCCAAGGAGGGGACGGTGGGTCCCGGAGCTGGGGCGGAGAACACAGGGA
GCCCCTCCCAGACCCCGCTCTGGGCAGAACCTGGGAAGGGATGTGGCCAT
CGGGGGATCCCTCCAGGCCATCTCCTCAGATGGGGGCTGGTCGACTAGCT
TCTGAGTCCTCCAAGGAACCGGGTCCTTCTAGTCATGACTCTGCCCAGAT
GAAGAAGGAGAGCACTTCTCTCCATCAGGAGGATCTGAGCTTCTCTTAAT
TAGAATCAGCTCCTTGGCTTCTACCCCTTAAAAAAAGGTACAGAAACTTT
GCACCTTGATCCAGTATCAGGGGAATTTATCAATCAATGTGGGAGAAATT
GGCATCTTTACCACACTGAATCTTTCAATCCATGAATATCCTCTCTCTCT
TCCATGCATAGGTTTTAATAATTCTCAATGGAGTTTAATGTAAGTTTTCC
TCATAGACAATTGCCTTTGGACATCTCTTTAGAGTCATCTCTAGTAAACT
GATATTCTTAATGCAATTATAAAATGTATCCTGCTTAATGTTATTTTCTA
TTCATTTGCTGTTATATAGAGATACAATGAGTTTCCACATTTGAAACTGG
ATCTGGTAAATTGGCTACCCTTTTTTTATAGATTCTATTAATTTTTATAC
ATTCTGTGGGACTTGCTACATACTTAATCATGTCACCTGTGAAGAATGAC
AATTTGGTTGCTACCCTCCCAATTCTTATATGTCTCATTTCTTTCCCTCT
GCTGGTACTCTGGCAGCAGCAGGGAAGATAATGGGCCTCCTTATCTTGTC
ACAAAAGGATGTTTTTAAAGATTTCGTTATAAAACATAACGCTTTCTGGT
TTTCTTTAAAGATTCTCTCACCAGCTTAAGAAAATTTTCTTATACTCTGT
ATGATAAATGGGTTTTTGACAATCATTTGTTGCATTTTACCTAGTGTTTT
CTCTGCATCTTTATATGCTTTTTCTCCTTTAATCCTGAAAATTGTTTCGA
TTTTTCTAACATTGAACCAATCTTACATTCCTGGAATGGATGGACCAGAC
TAGTCCACATGTTTATTCTGCCCAATGGCTAGATTTTGTGTTCaatattt
tgttcagaatgtttgcatctatattcttGAGTGAGACAGAGCTGCCCTTG
TTAGGTTTCACAACCGAGGTTGTGTTAGCTTCATAAAATGAGACGTTTAT
TCTCTAAAAGAATTGTTTCGCTTCTCTGGATGAATTTGTGTAAGGTTAGA
ATTGCTTACCAGTGAagatctCGGGgCCAGTTCTTCTTTAGGGGAAGATT
TTCAACAATTAAGCTCAATGCCTTTAGAAGAACTGAGAGTTTCTATTATT
TCTTGAGTTAAATATATGTATTTAATTAGACTTTCTAGGAATAGTCTCAT
TTCATCTCAAATAATTGACATATGCTATTAAAGCAGATTCTCATGAACCA
TTGTAGGTATTCCAGGTCTAGAAAAATGTTCCCCTTTGCATCCCTAATGT
GTTTAATTTTCACCTTCTTTCTTTTGTTCTTGAGAAATTCACCAAATCAT
TTTCAATTTCAGTCATATCCCAAAGCAACCAACTCTCTACCTTCTTGTTT
TATCATCCCTGCTGGATTTTTGTTATCTACTTCTTCAGTATTTGTTCTTC
CCTTTCTTCTATTCCTCATTCCATTTTTCCCTTGTTTTCTAACTTTCTGA
GATATATGCTTAGTTCCTTCATTTGAAGCCTTTTTATTTTCTTTTTTTTT
TTTTGGTCTTTTTGTCTTTtGTTGTTGTTGTTGTGCTATTtCTTGGGCCG
CTCCCGCGGCATATGGAGGTTCCCAGGCTAGGAGTCGAATCGGAGCTGTA
GCCACCGGCCTACGCCAGAGCCACAGCAATGCGGGATCCGAGCCGCGTCT
GCAACCTACACCACAGCTCATGGCAACGCCGGATCGTTAACCCACTGAGC
AAGGGCAGGAACCGAACCCGCAACCTCATGGTTCCTAGTCGGATTCGTAA
CCACTGTGCCACAACAGGAACTCCGCCTTTTTATTTTCTATAAAAATTTC
TATGTACATTTTAAGGTTATAGGTTTCCTTCTATGTACCCCATTGGCTGT
ATCCTCAGGGTTCTGTGGAGTGATTTCATTATTGTTCAAGTTCAATATGT
CTTCTGATTTTCCAATTTGAATACCTCTCTAAATCAGTAGGTGAATATTT
CTTTTTCTTTTTCTTTTCTTTTCTTCTTTTTTTTTTTCTTTCAGCCAGGT
CCATGGCATGCAGAAATTCCCAGGCCAGGAATCAAACTCTCACCATGGCA
GTGACAATGTCGGATCCTTTACCCACTAGGCCACCAGGGAACTCTGGGAG
CATATGTTTTTATTTCCCGACATCTGAGGATGCCTAGTATGTCTTCATTA
TTGATTTCTAGTTTGCCACTGATTTCTAGTATTTTGCTCATAGAGTGTAT
GCTCAATGGTTTTGGTCATTTGAAATGTATTTAGTCCTGCTTTATGACCC
AGTATGTGGTCAGTTTTGTCAATGTTCCTTTTCTGCTTGAAGAGAACCTA
CATGCTGTAACTCTGGGTGCATGTTCTGTATATAAGTCTATAGGCTGAGC
CGGGGGAGCCTTCTAATCTGCCGTTATCTTCTTCGAGTTATTCTAGGTAC
TATTTCTTAGCCATAAACCTTTAAATTCTGATATCAATATAATGACCCCA
GCCCGCTTAGGGTCGGCACTTCATGTTATCTTTTTCCATCCATTTAATGC
CTCCCCACTGTTTTGGCCACACCCGTGGGATATGGGAGTTCCTGGGCCAA
GGATCaGATCTGAGCCGCAGCTGCCACCTATGCCACAGCAgcagcaatga
tggatctttaacccactgcaccacactggggattgaacccaagcctcagc
agcaacccaagctactgcagagacaacaccagatccttaacctgctgtgc
catagcgggaaTTTCCATCCATTTACTTTCAAGCCAGCTGAATAACCTAG
CCCACCATGGCTGGACATGGGTGCTCTGCTTCAAATGATTTTGTTCAGTC
AGCATCCATCTCTGAAATGTGTGCCAAGCATTTATATGCATGCAAGAGTC
ATGTTGGCACTTCTATCATTTCCAACAGTTCAGTAGCCTTTGTATCATGA
CATTTCTTGGCCTTTTCTCTACAATATTTGAGGCTGAGCAGACTGGCCGT
GCCCCTGTCCATGCTTCCAGAGCCTGTGTGCAGACTTCTGCTCTAGACAG
AGACAGCTAACCATCCTGCAGTGCCCAGAAAACCCAACTCAAAGACCCTC
AAGTAAGGAAGGATTTATTGGCTCACGTAATCTGGAATCCAGGCATGGGG
TATTCAGGGCGACCTGAACCAGAGGCCCTGGCCCTGTTCTCTAAGCTTCT
TCCTGCCCTGCCCTCGTTCTGGAAGTGACCCTGAAGGACAGCAATGAAGG
GCAGCTCCCCCAGGGACAGATGACTGAGAGGTCCATTTCAAGTCCAACTT
GGCCTAGATTGAGAGGCAGCAAGAAATATGGACCTACAGTGAGTCACAGG
ATTTACCAGTGGTTTGGCTGGGTTGTCAGTGTTACAGGCTAAACATTTGG
GTCCCTCCAAAATTAACATGTTGCCACTCTAACCACCAAAATCatggtat
ttgggggtggggcccttggaggtaattaggtttagaaAGAATGAAGAGGG
GGCCCTTGTGATGGGACTAGTGCCTTTATAGAGAGAGAAGAGAGAGGG
Seq ID No. 39
CACCTCATCCCCAACCACCTGGATGGTGGCAAGTGGCAGGCTGAGAGGCT
GCATATGAGCTCATCAAGAGGGTCCCCACCCCACAGAGGCTGACCCAGCT
GCCACTGCCACCTAGTGGCTGATCGGCCAAGAGCAGGAGCCCCAGGGGCA
GCTCCATTCCCTGGGGCGGCCAGGGAACCACCTGGTGGTAGGACAATTCC
ATTGCACCTCATCCATCAGGAAAAGGTTTGCCTTCCCTGGCAGTAATGCA
TCTTCCCATAACATGGTCCCTGGCCTCTTGGAATGGCTTGGCCACCGTCA
TGGCCTCACCCACAAAGCCTTGTGTCTCAGCAAGGAACTTATTCCACAGC
AAAGGACTTGCAGCCTGGAATGAACTGGTCTGACTACATACCCCATTGCC
CAGAAGTAGGTGGTCTATTGCAAAGTGGAGTGGCTTACCCAAGACTCAGT
TGTGCCCAAGTTGAGAGATAGCATCCTAAAATATGGGCTTATGTCTCACT
GGCTGAGGTTTATTCTTTGAATCAAAGACAATTATATGGTGTGGTCCCCC
CAGAGATAGAATACATGAGTCTGGGAATCAAGGGATAGAAGTAAGAAGAG
ATTTTGTCACCATTAATCCCAATAACTCGCCCAAAGAATATTTGCTTTCT
GTCCTGGCAGCTCTGCTGCTTTGGCAATAACTTCCTAGAATATAATGTCT
CCACCAGGGGACTCCACAACGGTTCCATTGATTTGAAGCCAATGGGCAGA
GGAGGGGCTGCCTTACTGGTCGGACTGGTCAGCCCTGATTACTAAGGAGA
AATCAGGCAACTTCAACAAAACTAAGGCAGGGGGGACTTTGTCTAGAACC
CAAAGCACTAAGCATCTTAGTACTTTTTAGTTCTCAGAGCCTCCAAGAAC
AAAGATTTAGCCCCTCAGCACCACCAGGTAAAGAACAGGTAAATCCAGCT
GAGGACAAGAGAAATATTGAATGGATAGAGGAAGAAAGAAATTATAGATA
TCAACTATGGCCTCATGACTAGAGTCTCCAGATTAAGCGGAATAAAAATA
CAGATGATTaGATCTGAACATCAGGCCAAACAACGAACAACAGTTTAAGT
GCGACCTAGGCAATATTTGGGACATACTTATACTAAAATTTTTTCGCTAT
TTGAGCATCCTGTATTTTATCTGGCAACTTTATTCATCCCTAGCGAAAAA
GGAACTGTGGTAACTTAGTGTATTTTTACTTTGCTCATTATTGTGTATAT
ACCTACTTGTATTTATCAATCATATTTACTCTGTTCTCAGTATTACTTTA
TATAGCAGTTGGTGGTGATGGTTAGCAACATATTCAGTGGAACTGTGACT
GAATTTGAGGAGAAATTAACAGAGTTGGCTGTGGCTACAATAACCCTTCG
GGACATGTGTCCCCTCATTTTGGGGAGATGGTTagatctCTGGGTAAATG
TTAGGGCATCTGAGCCAGAAACCAAGATTTTGCCAGCTGGTGCAATGTCA
GATTTTACCAGCAGAGGGTGCCAGAGGAATGCGGCAAAACCCGAGTGCCA
GAAAGCACCTCCCTGTTTTCCAGCTTTTCTTCCTTTTTATTTATTTTATT
TACGGCCCAGGAGTCCGTAATAGCGCTGAGGATGGCCCAGGCTCTTCTCA
GCAGCCCTGACTGACTAGTTCAGCAATGCGCTCAGGCCCCATCTGGCCAC
CGGGCAGCCTCTTCTGTGGTAGCTCCAGCCTCAGCCAGTGCAAAAGGCTA
CCCTACACTGGCGCCACTTCTACAATCAGCACTGGCCACACCCTCCACGC
CATCCGGCACGGAGCCAGGTGATCTGCCGGCCAGATTGCAGTTCGTGCTG
CCTGAGTCCAGGTGATTACACTGGCTGCATCTTTTCTTTCTGGACCAtTC
attccattttttt
Bovine Lambda Light Chain
In a further embodiment, nucleic acid sequences are provided that encode bovine lambda light chain locus, which can include at least one joining region-constant region pair and/or at least one variable region, for example, as represented by Seq ID No. 31. In Seq ID No 31, bovine lambda C can be found at residues 993-1333, a J to C pair can be found at the complement of residues 33848-35628 where C is the complement of 33848-34328 and J is the complement of 35599-35628, V regions can be found at (or in the complement of) residues 10676-10728, 11092-11446, 15088-15381, 25239-25528, 29784-30228, and 51718-52357. Seq ID No. 31 can be found in Genbank ACCESSION No. AC117274. Further provided are vectors and/or targeting constructs that contain all or part of Seq ID No. 31, for example at least 100, 250, 500, 1000, 2000, 5000, 10000, 20000, 500000, 75000 or 100000 contiguous nucleotides of Seq ID No. 31, as well as cells and animals that contain a disrupted bovine lambda gene.
Seq ID No 1 tgggttctat gccacccagc ttggtctctg
31 atggtcactt gaggccccca tctcatggca
61 aagagggaac tggattgcag atgagggacc
gtgggcagac atcagaggga cacagaaccc
121 tcaaggctgg ggaccagagt cagagggcca
ggaagggctg gggaccttgg gtctagggat
181 ccgggtcagg gactcggcaa aggtggaggg
ctccccaagg cctccatggg gcggacctgc
241 agatcctggg ccggccaggg acccagggaa
agtgcaaggg gaagacgggg gaggagaagg
301 tgctgaactc agaactgggg aaagagatag
gaggtcagga tgcaggggac acggactcct
361 gagtctgcag gacacactcc tcagaagcag
gagtccctga agaagcagag agacaggtac
421 cagggcagga aacctccaga cccaagaaga
ctcagagagg aacctgagct cagatctgcg
481 gatgggggga ccgaggacag gcagacaggc
tccccctcga ccagcacaga ggctccaagg
541 gacacagact tggagaccaa cggacgcctt
cgggcaaagg ctcgaacaca catgtcagct
601 caaaatatac ctggactgac tcacaggagg
ccagggaggc cacatcatcc actcagggga
661 cagactgcca gccccaggca gaccccatca
accgtcagac gggcaggcaa ggagagtgag
721 ggtcagatgt ctgtgtggga aaccaagaac
cagggagtct caggacagcg ctggcagggg
781 tccaggctca ggctttccca ggaagatggg
gaggtgcctg agaaaacccc acccaccttc
841 cctggcacag gccctctggc tcacagtggt
gcctggactc ggggtcctgc tgggctctca
901 aaggatcctg tgtccccctg tgacacagac
tcaggggctc ccatgacggg caccagacct
961 ctgattgtgg tcttcttccc ctcgcccact
ttgcaggtca gcccaagtcc acaccctcgg
1021 tcaccctgtt cccgccctcc aaggaggagc
tcagcaccaa caaggccacc ctggtgtgtc
1081 tcatcagcga cttctacccg ggtagcgtga
ccgtggtcta gaaggcagac ggcagcacca
1141 tcacccgcaa cgtggagacc acccgggcct
ccaaacagag caacagcaag tacgcggcca
1201 gcagctacct gagcctgatg ggcagcgact
ggaaatcgaa aggcagttac agctgcgagg
1261 tcacgcacga ggggagcacc gtgacgaaga
cagtgaagcc tcagagtgtt cttagggccc
1321 tgggccccca ccccggaaag ttctaccctc
ccaccctggt tccccctagc ccttcctcct
1381 gcacacaatc agctcttaat aaaatgtcct
cattgtcatt cagaaatgaa tgctctctgc
1441 tcatttttgt tgatacattt ggtgccctga
gctcagttat cttcaaagga aacaaatcct
1501 cttagccttt gggaatcagg agagagggtg
gaagcttggg ggtttgggga gggatgattt
1561 cactgtcatc cagaatcccc cagagaacat
tctggaacag gggatggggc cactgcagga
1621 gtggaagtct gtccaccctc cccatcagcc
gccatgcttc ctcctctgtg tggaccgtgt
1681 ccagctctga tggtcacggc aacacactct
ggttgccacg ggcccagggc agtatctcgg
1741 ctccctccac tgggtgctca gcaatcacat
ctggaagctg ctcctgctca agcggccctc
1801 tgtccactta gatgatgacc cccctgaagt
catgcgtgtt ttggctgaaa ccccaccctg
1861 gtgattccca gtcgtcacag ccaagactcc
ccccgactcg acctttccaa gggcactacc
1921 ctctgcccct cccccagggc tccccctcac
agtcttcagg ggaccggcaa gcccccaacc
1981 ctggtcactc atctcacagt tcccccaggt
cgccctcctc ccacttgcat ggcaggaggg
2041 tcccagctga cttcgaggtc tctgaccagc
ccagctctgc tctgcgaccc cttaaaactc
2101 agcccaccac ggagcccagc accatctcag
gtccaagtgg ccgttttggt tgatgggttc
2161 cgtgagctca agcccagaat caggttaggg
aggtcgtggc gtggtcatct ctgaccttgg
2221 gtggtttctt aggagctcag aatgggagct
gatacacgga taggctgtgc taggcactcc
2281 cacgggacca cacgtgagca ccgttagaca
cacacacaca cacacacaca cacacacaca
2341 cacacacgag tcactacaaa cacggccatg
ttggttggac gcatctctag gaccagaggc
2401 gcttccagaa tccgccatgg cctcactctg
cggagaccac agctccatcc cctccgggct
2461 gaaaaccgtc tcctcaccct cccaccgggg
tgacccccaa agctgctcac gaggagcccc
2521 cacctcctcc aggagaagtt ccctgggacc
cggtgtgaca cccagccgtc cctcctgccc
2581 ctcccccgcc tggagatggc cggcgcccca
tttcccaggg gtgaactcac aggacgggag
2641 gggtcgctcc cctcacccgc ccggagggtc
aaccagcccc tttgaccagg aggggggcgg
2701 acctggggct ccgagtgcag ctgcaggcgg
gcccccgggg gtggcggggc tggcggcagg
2761 gtttatgctg gaggctgtgt cactgtgcgt
gtttgctcgg tggagggacc cagctggcca
2821 tccggggtga gtctcccctt tccagctttc
cggagtcagg agtgacaaat gggtagattc
2881 ttgtgttttt cttacccatc tggggctgag
gtctccgtca ccctaggcct gtaaccctcc
2941 cccttttagc ctgttccctc tgggcttctt
cacgtttcct tgagggacag tttcactgtc
3001 acccagcaaa gcccagagaa tatccagatg
gggcaggcaa tatgggacgg caagctagtc
3061 caccctctta ccttgggctc cccgcggcct
ccggataatg tctgagctgc ctccctggat
3121 gcttcacctt ctgagactgt gaggcaagaa
accccctccc caaaagggag gagacccgac
3181 cccagtgcag atgaacgtgc tgtgagggga
ccctgggagt aagtggggtc tggcggggac
3241 cgtgatcatt gcagactgat gccccaggca
gggtgagagg tcatggccgc cgacaccagc
3301 agctgcaggg agcacaggcc gggggcaagt
catgcagaca ggacaggacg tgtgaccctg
3361 aagagtcaga gtgacacgcg gggggggggc
ccggagctcc cgagattagg gcttgggtcc
3421 taacgggatc caggagggtc cacgggccca
ccccagccct ctccctgcac ccaatcaact
3481 tgcaataaaa cgtcctctat tgtcttacaa
aaaccctgct ctctgctcat gtttttcctt
3541 gccccgcatt taatcgtcaa cctctccagg
attctggaac tggggtgggg nnnnnnnnnn
3601 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnmnnnnnnn nnnnnnnnnn
3661 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
agcttatgtg gtgggcaggg gggtagtaag
3721 atcaaaagtg cttaaattaa taaagccggc
atgatatacg agtttggata aaaaatagat
3781 ggaaaagtaa gaaaggacag gaggggggtg
aggcggaaga aagggggaag aaggaaaaaa
3841 aaataagaga gaggaacaaa gaaagggagg
ggggccggtg atgggggtgg gatagaatat
3901 aataattgga gtaaagagta gcgggtggct
gttaattccg ggggggaata gagaaaaaaa
3961 aaaaaaaatg tgcgggtggg cggtaagtat
ggagatttta taaatattat gtgtggaata
4021 atgagcgggg gtggacgggc aaggcgagag
taaaaagggg cgagagaaaa aaattaggat
4081 ggaatatatg gggtaaattt taaatagagg
gtgatatatg ttagattgag caagatataa
4141 atatagatgg tgggggaaaa gagacaaggg
tgagcgccaa aacgccctcc cgtatcattt
4201 gccttccttc ctttaccacc tcgttcaaac
tctttttcga gaaccctgaa gcggtcaggc
4261 ccggggctgg gggtgggata cccggggagg
ggctgcgcct cctcctttgc agagggggtc
4321 gaggagtggg agctgaggca ggagactggc
aggctggaga gatggctgtt gacttcctgc
4381 ctgtttgaac tcacagtcac agtgccagac
ccactgaatt gggctaaata ccatattttt
4441 ctggggagag agtgtagagc gagcgactga
ggcgagctca tgtcatctac agggccgcca
4501 gctgcaggga ctttgtgtgt gtcgtgctcg
ttgctcagtt gtgtccgact ctttatgact
4561 tcatggactg taacctgcca ggctcctctg
tccgtggaat tctccaggca agaatactgg
4621 agtgggtagc cattctcatc tccgggggat
cttcctgacc caagaatcaa acctgagtct
4681 cccgcattgc aggcagcttc tttcttgtct
gagccaccag ggaagcccct taagtggagg
4741 atctaaatag agtgtttagg agtataagag
aaaggaagga cgtctataca agatccttcg
4801 gttcctgtaa ctacgactcg agttaacaag
ccctgtgtga gtgagttgcc agtaattatt
4861 gctaacctgt ttctttcact cactgagcca
ggtatcctgt gagacggcat acttacctcc
4921 tcttctgcat tcctcgggat ggagctgtgc
ggtggcctct aggactacca catcgaccag
4981 gtcagaccca gggacagagg attgctgaga
tgcactgaga agtttgtcag cctaggtctt
5041 cacccacaca gactgtgctg tcgtctacca
cgtaattctt cctgtccaaa gaactggtta
5101 aacgctcctg aagcgtattc tggtctgctt
caaaaagtgc ctctttcctt tataagttcc
5161 gccaatcctg gactttgtcc caggccagtc
tactttattt gtgggaaagg tttttttggt
5221 cttttttgtt ttaaactctg cagaaattgc
ttacactttt ggtgtgcaat ggctcactct
5281 tacggttcta gctgtattca aaggggttgc
ttttctttgt ttttaaagct ttttgaacgt
5341 ggaccatttt taaagtcttt attaaacgtc
taacatcgtt tctggtttat tttctggtgg
5401 tctggccatg aggcctacgg gtcttagctc
ccctaccagg gtccaaccca catcccttgc
5461 actggacggc aaggtcttaa cctttgaacc
accagagagc ttctgaaagg ggctgctttt
5521 ctccaatcct ctttgctccc tgcctgctgg
tagggattca gcacccctgc aatagccctg
5581 tctgttctta ggggctcagt agcctttctg
cctgggtgtg gagctggggt tgtaagagag
5641 cttcatggat ttggacacga cctacgactc
agaggtaaga ctccatctta gcgctgtaat
5701 gacctctttc caacaaccac ccccaccacc
ctggaccact gatcaggaga gatgattctc
5761 tctcttatca tcaacgtggt cagtcccaaa
cttgcacccg gcctgtcata gatgtagcag
5821 gtaagcaata aatatttgtt gaatgttaag
tgaattgaaa taacataagt gaaaaagaaa
5881 acacttaaaa acatgtgttt ttataattac
acagtaaaca tataatcatt gtagaaaaaa
5941 atcgaaagag tggcgggggc caagtgaaaa
ccaccatccc tggtatgtcc acccgcccgg
6001 gtagccccag gtaagaggtg cggacacgga
tggccctgta gacacagaga cacacgctca
6061 tatgctgggt cttgtcttgt gacctcttgg
ggatgatgtt attttcacga tgccattcaa
6121 accttctacc acaccatttt tagagggtcg
ttcatcgtaa atcagttcac tgctttgttt
6181 tctgattttg aaagtgtcac attcttcgag
aaatgagaag gaacaggcgc gcataaggaa
6241 gaaagtaaac acgtggcctt gcttccaggg
ggcactcagc gtgttggtgt gcacgctggc
6301 agtcttttct ctgtgacagt catggccttt
tcccaaaggt gggctcagat aagaccgcct
6361 cccatcccct gtccctgtcc ccgtccccta
cggtggaacc cacccacggc acgtctccga
6421 ggccctttgg ggctgtggac gttaggctgt
gtggacatgc tgctggtggg gacccagggc
6481 tgggcagcac gttgtccctg ggtcccgggc
cagtgaggag ctcccaagga gcagggctgc
6541 tgggccaaag ggcagtgcgt cccgaggcca
tggacaaggg gatacatttc ctgctgaagg
6601 gctggactgc gtctccctgg ggccccttgg
agtcatgggc agtggggagg cctctgctca
6661 ccccgttgcc cacccatggc tcagtctgca
gccaggagcg cctggggctg ggacgccgag
6721 gccggagccc ctccctgctg tgctgacggg
ctcggtgacc ctgccgcccc ctccctgggg
6781 ccctgctgac cgcgggggcc accccggcca
gttctgagat tcccctgggg tccagccctc
6841 caggatccca ggacccagga tggcaaggat
gttgaggagg cagctagggg gcagcatcag
6901 gcccagaccg gggctgggca ggggctgggc
gcaggcgggt gggggggtct gcacnccccc
6961 acctgcnagc tgdncnnncn tttgntnncg
tcctccctgn tcctggtctg tcccgcccgg
7021 ggggcccccc ctggtcttgt ttgttccccc
tccccgtccc ttcccccctt tttccgtcct
7081 cctcccttct tttattcgcc ccttgtggtc
gttttttttc cgtccctctt ttgttttttt
7141 gtctttttct ttttccccct cttctccctt
gctctctttt tcattcgtcg gtttttctgc
7201 tcccttccct ctcccccccg ctttttttcc
ctgtctgctt tttgtgttct ccctctctac
7261 cccccctgca gcctattttt tttatatatc
catttccccc tagtatttgg cccccgctta
7321 cttctcccta atttttattt tcctttcttt
aactaaaatc accgtgtggt tataagtttt
7381 aacctttttt gcaccgccca caatgcaatc
ttcacgcacg ccccccccgt cagcctcctt
7441 aaataccttt gcctactgcc cccctccttg
tataataacg cgtcacgtgg tcaaccatta
7501 tcacctctcc accaccttac cacattttcc
ttcnnnnnnn nnnnnnnnnn nnnnnnnnnn
7561 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
7621 nnnnnnnnnn nnntgaaaaa agaaaaggct
gggcaggttt taatatgggg gggttggagt
7681 ggaatgaaaa tgcattggag tggttgcaac
aaatggaaag gtctcaggag cgctcctccc
7741 ccatcaggag ctggaaagaa gtggaagcaa
agcaaggaat tcgtgtgatg gccagaggtc
7801 aggggcaggg agctgcaaag actgccggct
gtttgtgact gnccgtctcc gggtgcattt
7861 gttagcaggg aggcattaca ctcatgtctt
ggtttgctaa ctaattctta ctattgttta
7921 gttgcaaggt catgtctgac tctttgcaac
ccagggactg cagcccgcca ggctcctctg
7981 tccatgggat ttcgcaggca agaatactgg
aggtggtagc cattttcttc accatgggat
8041 cttcccgagc cagaaatgga acccgagtcg
cctcctgtgc atggggtctg ctgcctaaca
8101 ggcagatatt tgacgtctga gccaacaggg
aggacagacg gtaattatac caaccattga
8161 aagaggaatt acacactaat ctttatcaaa
atctttcaaa cagtagagga gaaaggatac
8221 tctctagttt attccataaa gttggaatta
cgcttatcaa taaagacatt acaagaaaag
8281 aaagtgaagc cccaaatgcc ttataaatat
acaagaaaaa atcttttaag atattagcca
8341 acttaatcaa caaaaaatgt atcaaaagtc
caagtaacat tcaccccagg aatgcaagtg
8401 tggttcagcc taagacaatc agtcatgagt
ataccacgga aacaaattaa agagaaaaga
8461 cattaaatct cacaaatggt gcagaaaaag
atttggcaat atcgaacatc ttttcatgac
8521 caaaggaaaa aaaagaaaca aaacaccaga
aaattctgtg tagaaagaat atatctcaac
8581 ccaatgaagg gcatttatga aaaacccaca
gcatacatca cactccatga gaaagactga
8641 aagctttccc cactgccatt gaactctgtc
ctggaaattc tagtcacagc gacagaacaa
8701 gagaaagaaa taacggccgt ctaaactggt
aggaagaaat caaagcgtct ctattctctg
8761 ggcgcataat acaatataga caaatttcta
aagtccacaa aaattcctag agctcataat
8821 gaatccagaa atgcgtcagg gctcaagatt
cagatgcaaa aatcgtctgg gttttgatgc
8881 accaacaaac aattccatta acaataatac
caaggaatta atttaactta gaagagaaaa
8941 gacctgttta cagagagtta taaaacattt
ggtgatgaaa ttaaataaga gtaaatcata
9001 tagaaacacc gttcgtgttt tggagaccta
atgtcataaa cgtggcaaca cagagacgcc
9061 tcacggggaa ccctgagcct ccttctccaa
acaggcctgc tcatcatttc acaggtaacc
9121 tgagacccta aagcttgact ctgaggcact
ttgagggcat gaagagagca gtagctcctc
9181 ccatgggacc gacagtcaag gcccagggaa
tgaccacctg gacagatgac ttcccggcct
9241 catcagcagt cggtgcagag tggccaccag
ggggcagcag agagtcgctc aacactgcac
9301 ctggagatga ggcaacctgg gcatcaggtg
cccatgcagg ggctggatac ccacacctca
9361 cacctgagga caggggccgg ctttctgtgg
tgtcgccctc tcaggatgca cagactccac
9421 cctcttcgct tgcattgaca gcctctgtcc
ttcctggagg acaagctcca ccttccccat
9481 ctctccccag ggggctgggg ccaacagtgt
tctctcttgt ccactccagg aacacagagc
9541 caagagattt atttgtctta attagaaaaa
ctatttgtat tcctgcattt ccccagtaac
9601 tgaaggcaac tttaaaaaat gtatttcctg
gacttccctg gtgggccagt ggctagactc
9661 tgagctccca gtgcatgggg cctgggttca
atccctgctc aggaaactac atcccacagg
9721 ctgcaaataa gatcctgcat gccacccgat
gcaggcaaag aaacaagtgt tcggtatgca
9781 tgtatttcac gtgaggtgtt tctataattt
acagccagta ttctgtctta cacttagtca
9841 ttcctttgag cacatgatcg gtcgatggcc
cagaccacac acaggaatac tgaggcccag
9901 cacccaccgg ctgcccagaa cctcatggcc
aagggtggac acttacagga cctcagggga
9961 cctttaagaa cgccccgtgc tcttggcagc
ggagcagtgt taagcatggc tctgtccctc
10021 gggagctgtg tctgggctgc gtgcatcacc
tgtggtgtgg gcctggtgag ggtcaccgtc
10081 caggggccct cgagggtcag aagaaccttc
ccttaaaagt tctagaggtg gagctagaac
10141 cagacccaca tgtgaactgc acccaaaaac
agtgaaggat gagacacttc aaagtcctgg
10201 gtgaaattaa gggccttccc ctgaaccagg
atggagcaga ggaaggactt ggcttccagg
10261 aaaccctgac gtctccaccg tgactctggc
cggggtcatg gcagggccca ggatcctttg
10321 gtgcaaagga ctcagggttc ctggaaaata
cagtctccac ctctgagccc tcagtgagaa
10381 gggcttctct cccaggagtg gggcaaggac
ccagattggg gtggagctgt ccccccagac
10441 cctgagacca gcaggtgcag gagcagcccc
gggctgaggg gagtgtgagg gacgttcccc
10501 ccgctctcaa ccgctgtagc cctgggctga
gcctctccga ccacggctgc aggcagcccc
10561 caccccaccc cccgaccctg gctcggactg
atttgtatcc ccagcagcaa ggggataaga
10621 caggcctggg aggagccctg cccagcctgg
gtttggcgag cagactcagg gcgcctccac
10681 catggcctgg accccctcct cctcggcctc
ctggctcact gcacaggtga gccccagggt
10741 ccacccaccc cagcccagaa ctcggggaca
ggcctggccc tgactctgag ctcagtggga
10801 tctgcccgtg agggcaggag gctcctgggg
ctgctgcagg gtgggcagct ggaggggctg
10861 aaatccccct ctgtgctcac tgctaggtca
gccctgaggg ctgtgcctgc cagggaaagg
10921 ggggtctcct ttactcagag actccatcca
ccaggcacat gagccggggg tgctgagact
10981 gacggggagg gtgtccctgg gggccagaga
atctttggca cttaatctgc atcaggcagg
11041 gggcttctgt tcctaggttc ttcacgtcca
gctacctctc ctttcctctc ctgcaggcgc
11101 tgtgtcctcc tacgagctga ctcagtcacc
cccggcatcg atgtccccag gacagacggc
11161 caggatcacg tgttgggggc ccagcgttgg
aggtganaat gttgagtggc accagcagaa
11221 gccaggccag gcctgtgcgc tggtctccta
tggtgacgat aaccgaccca cgggggtccc
11281 tgaccagttc tctggcgcca actcagggaa
catggccacc ctgcccatca gcggggcccg
11341 ggccaaggat gaggccgact attactgtca
gctgtgggac agcagcagta acaatcctca
11401 cagtgacaca ggcagacggg aagggagatg
caaaccccct gcctggcccg cgcggcccag
11461 cctcctcgga gcagctgcag gtcccgctga
ggcccggtgc cctctgtgct cagggcctct
11521 gttcatcttg ctgagcagcg gcaagtgggc
attggttcca agtcctgggg gcatatcagc
11581 acccttgagc cagagggtta ggggttaggg
ttagggttag gctgtcctga gtcctaggac
11641 agccgtgtcc cctgtccatg ctcagcttct
ctcaggactg gtgggaagat tccagaacca
11701 ggcaggaaac cgtcagtcgc ttgtggccgc
tgagtcaggc agccattctg gtcagcctac
11761 cggatcgtcc agcactgaga cccggggcct
ccctggaggg caggaggtgg gactgcagcc
11821 cggcccccac accgtcaccc caaaccctcg
gagaaccgcg ctccccagga cgcctgcccc
11881 tttgcaacct gacatccgaa cattttcatc
agaacttctg caaaatattc acaccgctcc
11941 tttatgcaca ttcctcagaa gctaaaagtt
atcatggctt gctaaccact ctccttaaat
12001 attcttctct aacgtccatc ttccctgctc
cttagacgcg ttttcattcc acatgtctta
12061 ctgcctttgg tctgctcgtg tattttcttt
tttttttttt ttttattgga atatatttgc
12121 gttacaatgt tgaatttgaa ttggtttctg
ttgtacaaca atgtgaatta gttatacatg
12181 tcctgaggag gggcggctgc gtgggtgcag
gagggccgag aggagctact ccacgttcaa
12241 ggtcaggagg ggcggccgtg aggagatacc
cctcgtccaa ggtaagagaa acccaagtaa
12301 gacggtaggt gttgcgagag ggcatcagag
ggcagacaca ctgaaaccat aatcacagaa
12361 actagccaat gtgatcacac ggaccacagc
ctggtctaac tcagtgaaac taagccatgc
12421 ccatggggcc aaccaagatg ggcgggtcat
gtgcccatgg ggccaaccaa gatgggcggg
12481 tcatggtgaa gaggtctgat ggaatgtggt
ccactggaga agggaaaggc aaaccacttc
12541 agtattcttg ccttgagagc cccatgaaca
gtatgaaaag gcaaaatgat aggatactga
12601 aagaggaact ccccaggtca gtaggtgccc
aatatgctac tggagatcag tggagaaata
12661 actccagaaa gaatgaaggg atggagccaa
agcaaaaaca atacccagtt gtggatgtga
12721 ctggtgatag aagcaagggc caatgatgta
aagagcaata ttgcatagga acctggaatg
12781 ttaagtccaa gannnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
12841 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnagaatttt
12901 gagcattact ttactagcgt gtgagacgag
tgcaattgtg cggtagtttg agcattcttt
12961 ggcattgcct ttctttggga ttggaatgaa
aactgacctg ttccaggcct gtggccactg
13021 ctgagttttc caaatttgct ggcgtattga
gtgcatcact ttaacagcat catcttttag
13081 gatttgaaat agctcaactg gaattctatc
actttagcta attccattca ttagctttgt
13141 ttgtagtgat gcttcctaag gcccccctgg
ctttatcttc ctggatgtct ggctctggtg
13201 agtgatcaca ccgctgtgat tatctgggtc
atgaaggtct ttttgtatag ttcttcttag
13261 gaacagatat tatgatctcc atccttgcat
ctcgttatat ctagagaagc actgactccc
13321 ttcatggtga cgtcagatcc tcatgactaa
caaatggcct tttgtaagat gagtgcctca
13381 tggtattgag ctcccccgtc accaagacct
tatgactgac ctcccccact gccccaggtg
13441 cctctcgaag cgtctgagat gccgcctccc
aggctgcact cctcattttg cccccaataa
13501 aacttaactt gcagctctcc agctgtgcat
ctgtgtttag ttgacagtac aaatataatg
13561 gaaaatttaa attaaatata atctatgggg
agaaatccaa acatcttatg agggagagag
13621 agggagagaa aggaaagaag aagaagcagg
aggaggagga gagtagagaa acagggggag
13681 ggcggcaggg agacagaggg gaggacaccg
aggggaaagg gaggaaggcg agtgcagtga
13741 gagagaggcc agagttcatc agagtctgga
ctcgcagccc aatcccacgg gtgtgtcccg
13801 aagcagggga gagcctgagc caggcggaga
cagagctgtg tctccagtcc tcgtggccgt
13861 gacctggagc tgtgtggtca gcccccctga
ccccagcctg gccctgctgg tggtcggagg
13921 cagtgatcct ggacacagtg tctgagcgtc
tgtctgaaat ccctgtggag gcgccactca
13981 ggacggacct cgcctggccc cacctggatc
tgcaggtcca ggcccgagtg gggcttcctg
14041 cctggaactg agcagctgga ggggcgtctg
caccccagca gtggagcggc cccaggggcg
14101 ctcagagctg ccggggggac acagagcttg
tctgagaccc agggctcgtc tccgaggggt
14161 cccctaaggt gtcttctggc cagggtcaga
gccgggatga gcacaggtct gagtcagact
14221 ttcagagctg gtggctgcat ccctggggac
agagggctgg gtcctaacct gggggtcaga
14281 gggcaggacg ggagcccagc tgacccctgg
ggactggcct cctctgtggt ctcccctggg
14341 cagtcacagc ttccccggac gtggactctg
aggaggacag ctggggcctg gctgtcagga
14401 gggggttcga gaggccacac tcagaggagg
agaccctggc ctgcttgggt tgtgactgag
14461 tttttggggt cctctaggag actctggccc
tgcaggccct gcaaggtcat ctctagtgga
14521 gcaggactcc acaagattga tgaactgaat
cctctaggag aggtgtggtt gtgagggggc
14581 agcattctag aaccaacagc gtgtgcaggt
agctggcacc gggtctagtg gcggcgggca
14641 gggcactcag ggccgactag gggtctgggg
gattcaatgg tgcccacagc actgggtctt
14701 ccatcagaat cccagacttc acaaggcagt
ttcggggatt aggtcaggac gtgagggcca
14761 cagagaggtg gtgatggcct agacaagtcc
ttcacagaga gagctccagg ggccatgata
14821 agatggatgg gtctgtattg tcagtttccc
cacatcaaca ccgtggtccc gccagcccat
14881 aatgctctgt ggatgcccct gtgcagagcc
tacctggagg cccgggaggc ggggccgcct
14941 gggggctcag ctccggggta accgggccag
gcctgtccct gctgtgtcca cagtcctccc
15001 ggggttggag gagagtgtga gcaggacagg
agggtttgtg tctcacttcc ctggctgtct
15061 gtgtcactgg gaacattgta actgccactg
gcccacgaca gacagtaata gtcggcttca
15121 tcctcggcac ggaccccact gatggtcaag
atggctgttt tgccggagct ggagccagag
15181 aactggtcag ggatccctga gcgccgctta
ctgtctttat aaatgaccag cttaggggcc
15241 tggcccggct tctgctggta ccactgagta
tattgttcat ccagcagctc ccccgagcag
15301 gtgatcttgg ccgtctgtcc caaggccact
gacactgaag tcaactgtgt cagttcatag
15361 gagaccacgg agcctggaag agaggaggga
gaggggatga gaaggaagga ctccttcccc
15421 aagtgagaag ggcgcctccc ctgaggttgt
gtctgggctg agctctgggt ttgaggcagg
15481 ctcagtcctg agtgctgggg gaccagggcc
ggggtgcagt gctggggggc cgcacctgtg
15541 cagagagtga ggaggggcag caggagaggg
gtccaggcca tggtggacgt gccccgagct
15601 ctgcctctga gcccccagca gtgctgggct
ctctgagacc ctttattccc tctcagagct
15661 ttgcaggggc cagtgagggt ttgggtttat
gcaaattcac cccccggggg cccctcactc
15721 agaggcgggg tcaccacacc atcagccctg
tctgtcccca gcttcctcct cggcttctca
15781 cgtctgcaca tcagacttgt cctcagggac
tgaggtcact gtcaccttcc ctgtgtctga
15841 ccacatgacc actgtcccaa gcccccctgc
ctgtggtcct gggctcccca gtggggcggt
15901 cagcttggca gcgtcctggc cgtggactgc
ggcatggtgt cctggggttc actgtgtatg
15961 tgaccctcag aggtggtcac tagttctgag
gggatggcct gtccagtcct gacttcctgc
16021 caagcgctgc tccctggaca cctgtggacg
cacagggctg gttcccctga agccccgctt
16081 gggcagccca gcctctgacc tgctgctcct
ggccgcgctc tgctgccccc tgctggctac
16141 cccatgtgct gcctctagca gagctgtgat
ttctcagcat aactgattac tgtctccagt
16201 actttcatgt ccctgtgacg ggctgagtta
gcatttctca cactagagaa ccacagtcct
16261 cctgtgtaaa gtgatcacac tcctctctgt
gggacttttg taaaagattc tgcagccagg
16321 agtcatgggt ggtcttagct gagaaatgct
ggatcagaga gacctgataa ccgatgtgaa
16381 gaggggaacc tggaagatct tcagttcagt
tcatttcagt cattcagttg tgtccgactg
16441 tttgggatcc catggactgc cacacgccag
tcctccctgt ccatcaccaa cttctgaagc
16501 ttgttcaaac tcatgtccat caagttggag
atgcctttca accatctcat cctctgtcat
16561 ccccttctcc tcccgccttc aatcttccct
agcattaggg tcttttccgt gagtcagttc
16621 ttcgcatcag gtggccaagt tttggagttt
cagtttcagc atcagtcctt tcaatgaata
16681 gtaaggactg atttccttta ggatggactg
gtttgatatc cttgcagttc aagggactct
16741 caagagtctt ctccaacact gcagttaaaa
gccatcaatt cttcggtgct cagctttctt
16801 tttggtacaa ctctcacatt catacatgac
taccgaaaat acattagtcg tgtagaacca
16861 gtttggggct tcccacgtgg ctctagtggt
aaagaatatg cctgccaact cagaagatgt
16921 aagagatgcg gttcaatctc tgggtcggga
agatcccctg gagaagggca tgacaaccca
16981 ctccagtatt tttgcctgga gaatcccatg
gacagagaag cctggtggac tgcagtccat
17041 ggagtctcac agagtcagac acgactgaag
caacttagct acttggaaaa gagcatgcac
17101 gaagctgtct aaaaaacagg tcaagaagtc
ttgtgttttg aaggtttact gagaaagttg
17161 atgcactgct ccaacacttc ctctcagttg
aaaagatcag aagcgttaga tcaaatggtg
17221 gtcaatacct tggatgcgct ccaacaggtt
atatctgcag atggaaatga aggcagttta
17281 tggggtaact ggaggacaag atgagatcat
acacttggaa cactgtctgg catcaaaggc
17341 gtgtacagta aacattagct gttattagca
aaataaattc agcttgaatc acccaaatca
17401 gatggcattc ttaaagccac tgagtggtaa
aatcaggggt gtgcagccaa aacgtccatt
17461 ttgactcatt atgatttcca tgtcacaaga
ctagaaagtc actttctcct cagcagaaga
17521 gaaggtagaa cattttaacc tttttttgga
gtgtcaaggg aattttgttt acactgtaaa
17581 gtcagtgaaa atattgaagc ttttcatttg
tggaaaatat taaatatgta aaattgaaat
17641 tttaaaattt attcctgggt agttttgttt
ttccagtagt catgcatgga tgtgagagtt
17701 ggactataaa gaaagctgag cgctgaagaa
ttaatgcttt tgaactgtgg cactggagaa
17761 gactcttgag agtcccttgg tctgcaagga
gatcaaacca gtccatccta aaggaaatca
17821 gtcctgaata ttcactggaa ggactgatgc
tgaagctgaa actccaatac tttggccacc
17881 tgatgtgaag aactgactca tatgaaaaga
ctcagatgct gggaaagatt gaaggtggga
17941 ggagaagggg acgacagagg atgagatggc
tgaatggcat caccgactcg atggacatga
18001 gtctgaataa gctctgggag ttgttgatgg
acagggaggc cctggagtgc tgcagtccat
18061 gggattgcaa agagttggac atgactgagt
gactgaactg aactgagttt ggtaacagat
18121 atgagaatta tataatttaa atctaaactc
ttggtatttc tttctttggc ggttccaaaa
18181 gagctgtccc ttctgttaac tatataaatc
ctttttgaga attactaaat tgataatgtt
18241 cacaagttat ccaatttctc attactctta
gttgtcagta taagaaatcc catttgattt
18301 atcatgttat agtatctgca actctaatag
ttcagttctg acaaattttt attttattta
18361 aaaatattgg catacagtaa aatttcaaac
aatatacaat tctccctttc agtttaaaaa
18421 acaaaacaaa acaaaagtaa tattagttaa
aaaaatccgg gaagaatcca agcatttaaa
18481 attgcatcac atttctatgc tagacaagct
gatataaagt tataattaat aaaggattgg
18541 actattaaac tctttacata tgaggtaaca
tggctctcta gcaaaacatt taaaaatatg
18601 ttgtgggtaa attattgttg tccttaaaga
aataaaaaga cataagcgta agcaattggn
18661 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
18721 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnna aaatggataa ggggggagga
18781 catgggtagg ggagcgcgat ggaggaagta
aggtggtcga gggagttggg gggggaataa
18841 gtgggtaaaa gggaagcggg cggaaggagg
gggaagcagg agagaggggt gggcgtcaga
18901 tcggggggag gggtatgagg gagagggaat
ggtagacggg gggtgggaag cataaaggaa
18961 aagatagggg ggggaaaagt tagaagaaga
atgaggggat aggcggaaag ggaagagaaa
19021 tgggagaaga acagaaaaat agggggaggg
ggggcgtaaa gagggggggg gagggcaggt
19081 gtggagatga cagatacggg gaatgccccg
gtataaaaga gtatatggcg tggggcgaga
19141 aggctgtcat cctgtgggag gggggacgcg
gagaaccctt cgggctatag ggaggattcg
19201 gggggatcgt tcgggaaggc agtcagcaca
gcacccacca agggtgcagg gatggatctg
19261 gggtcccaaa gaagaggccc aatcccgcgt
cttggcagca aggagccctg gagactggga
19321 agtgtccagg acactgaccc aggggttcga
ggaacccaga agtgtgtctg tgaagatgtg
19381 ttttgtgggg ggacaggtcc agagctttga
gcagaaaagc ggccatggcc tgtggagggc
19441 caaccacgct gatctttttt aaaaggtttt
tgttttgatg tggaccattt ttaaagtctt
19501 cattgaattt gctacaatat tgtttctggt
ttatgctctg gtttcttcgg ctgcaaggtt
19561 tgtgtgatcg tatctcctca accaggactg
aacccacagc ccctgcactg gaaggcgaag
19621 tcttaaccca gatcgccagg aacgtccctc
ccctcactga tctaatccaa gaccctcatt
19681 aaggaaaaac cgagattcaa agctccccca
ggaggactcg gtggggagga gagagccaag
19741 cactcagcac tcagtccagc acggcgccct
ccctgtccag ggcgagggct cggccgaagg
19801 accaccggag accctgtcgg attcaccagt
aggattgtga ggaatttcaa cttacttttt
19861 aaatctgtct ctcaaggctg ttacaagcgg
actttaccag taacttaaaa gttgaaaggg
19921 acttcccagg cggcacttgc ggtgaagaac
ccgccggctg gttttaggag acataagaga
19981 tgtgggttag atccctggtt caggaggatt
cccctggaga aggaaatggc aacccactcc
20041 agtattcttg cctggaaagc ctcacggaca
gaggaggctg gcgggctaca gtccacgggg
20101 tcgcacacga ctgaatcgac ttagcttcaa
gttgagacag gaagaggcag tgactggtgg
20161 caaaacaccg cacccatgct cccaggggac
ctgcagcgct ctggttcatg agctgtgcta
20221 acaaaaatca acccaacgag aggcccagac
agagggaagc tgagttcatc aaacacgggc
20281 atgatgtgga ggagataatc caggaaggga
cctgccaagc ccatgacaga ccggtgtcct
20341 gtctgagggc cgtcctggca gagcagtgca
gggccctccg agaccgcccg agctccagac
20401 ccggctgggg gctacagggt ggggctgagc
tgcaaggact ctgctgtgag ccccacgtca
20461 gggaggatca ccttgtttgt tttctgagtt
tctcttaaaa tagcctttat gggtcctggt
20521 ctttggtttt aaaataacaa ctgttctccg
taaacaacgt gaaaaaaaac aaacaggagg
20581 aaaacaacgc agcccgggca tttcacccgg
aagagccgcc tctaacactt tgacgggttg
20641 ccttctattt taaccctgtt ttcattgtaa
actgtaaaaa ccacatcata aataaattaa
20701 aggtctctgt gaagtttaaa aagtaagcat
ggcggtggcg atggctgtgc cacaccgtga
20761 acgctcgttt caaaacggta aattctaggg
accccctggt ggtccagtgg gtgagatttt
20821 gcttccattg caggagccgt gggtttgatc
cctggttggg gaactaagat cccacatgct
20881 gtatggagtg gccaaaaaga attttttgta
aatggtgagt tttaggtgac gtgaatttcc
20941 cattgatgca cttcacaggc tcagatgcag
ccaggccctc aggaagcccg agtccaccgg
21001 tcctttactt ttccttagag ttttatggct
tctgtttctg cccttaaacc caccatgttt
21061 caacctcatc tgattttgga ctttataata
aagttaggct gtgtttcagg aaactttgct
21121 cagtattctg taataatcta aatggaaaga
atttgaaaaa agagcagaca cttgtacatg
21181 cataactgaa tcactttggt gtacacctga
aactcgagtg cagccgctca gtcgtgtccg
21241 accctgcgac cccacggact gcagcacgcg
ggcttccctg cccatcacca actcccggag
21301 ttcactcaaa cacatgtccg tcgactcggt
gatgccgtcc aaccgtctca tcctctgtcg
21361 tccccttctc ctcccgcctt caatcttttc
cagcatcagg gtcttttcaa atgagtcagt
21421 tcttcacacc aggtggccag agtattggag
tttcagcttc agcatcagcc cttccaacga
21481 ccccccatac ctgaagctaa cacagtgcta
atccactgtg ctgcaacatg aaagaaaaac
21541 acatttttta agtttaggct gtgtgtgtct
tccttctctc aacactgcgt ctgaccccac
21601 ccacactgcc cagcactgca ttccccgtgg
acaggaggcc ccctgcccca cagctgcgtg
21661 ccggccggtc actgccgagc agacctgccc
gcccagagtg gggcccctgg cactggggac
21721 aaggcagggg cctctccagg gccggtcact
gtccactgtt cctactggtt ttgttttcaa
21781 aagtggaggc agcgtaatat ttccctgatt
ataaaaagaa gtacacaggt tctccacaaa
21841 taaaacaggg gaaaagtata aagaatggaa
gttcccagca cagcctggag atcacgccgg
21901 gtgcacctgg ggtgtccttc caggctggac
ctcacatttc acgcagacat cagaaggctg
21961 cgagatctac ccagaaggct gggtagatgg
gggataggtc agtgacaaac agtagacaga
22021 gagatataca gacagatgat ggatagacag
acgctaagac accgagcgag gggacagacg
22081 gatggaagac accatccttt gtcactgacc
acacacccac atgggtgtgg tgagccggct
22141 gtcatacttg tgaacctgct gctctcacaa
caccagctgg gtccctccag ccccagcgtc
22201 ccacacagca gactcccggc tccatcccca
ggcaggaatc ccaccaccaa ctggggtgga
22261 ccctccccgc aggaaggtcg tgctgtctaa
ggccttgaga gcaagttaca gacctacttc
22321 tgggaagaca gcgcacaacc gcctaccccg
cagagcccag gaggacccct gagtcctagg
22381 gaagggacca cgcggcctgg acggggagcg
gccccaggac gctgccccca acctgtccca
22441 cctcactcct gctctgctct gaggcggggc
gcagagaggg gccctgaggc ctcttcccag
22501 ttcttgggag cacccactgg gcctgaacca
ggccagaagc cccctcctca aggtgtcccc
22561 agaccactcc cctccacctc cggttgctct
gtctcctggc agcagggagc cccagtgaga
22621 agagacagct ccaggctgtg atcttggccc
ctggctgctc tggcagtgtg gggggtgggg
22681 gtcgctggga ggccatgagt gctgggggtc
ggggctgtga aagcacctcg aggtcagtgg
22741 gctgttggtc gggctctgcg aggtccgcac
gggtagagct gtgccaggac acaggaggcc
22801 tggtcagtgg tcccaagagt cagggccaaa
ggaaggggtt cgggcccctc tggttcctca
22861 gcttctgagg ccggggaccc cagtctggcc
ttggtagggg ggcgattgga gggtacaacg
22921 atccaaaaga aaacacacat ctacgaggga
agagtcctga ggaggagaga gctacacaga
22981 gggtctgcac actgcggaca ctgcttggag
tctgagagct cgagtgcggg gcacagtgag
23041 cgaagggagg acggaacctc caaggacacc
ggacgccgat ggccagagac acacgcacgt
23101 cccatgaggg ccggctgctc agacgcaggg
gagctcctca ttaaggcctc tcgctgaata
23161 gtgaggagaa ctggccccgt gtgtggggaa
acttagccca gaagaaacgc tgccctggcc
23221 ccaaggatca nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
23281 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn tgccctttgc
23341 ctccagggag ggaggaagcg tggatcttgg
gtttgccttg ggtttaaagg atccacccac
23401 tcccttttta gccactccct gtgctggcaa
tttcttaaga ctggaggtcg caaagagttg
23461 gacacactga gcgagtgaac tgcactgagc
ctaagaaaag tctttgaatt cctccaaaca
23521 aaacacactt gtcttgggta ctttccttgg
ttttgttaca aatgtctggt ccctctgttc
23581 tcctggccag ctcctgggtg tcattttgac
ctgacgaagt caaagggagc ctggaccctc
23641 aaaatctgta ggacccagca cccctccatt
acacctctgt tcccccgcga acgggcacgt
23701 gtttcgccgt ctggcgtaat gtgtaagcga
cggtgtgata ctcgggagtc ttactctgtt
23761 tctttttctt ctggggtgac accaccatcc
gcacgactct gtctgaatgt gaacatttgg
23821 gtgatttgat gtggcccaga ctcccccaac
gaatgtacct tcaggttggt tttcttcttt
23881 tatattttgc ttttgtgaat agacacagga
tcccatcagt tgtatgtagt gagaaagtaa
23941 aaacccactc agccttagct ggatggagat
ctagtagtaa gatagcacgt tagccggaaa
24001 tggaaatttc agccagaatc tgaaaagcgt
gtcctggaag gagaagaggg actcaggccc
24061 gagcacactg ctccacgctg gagcctcagg
ctctgacagc tgtacctgcc ggggtcttca
24121 tgggacaggc catgcaggcc acgatcccgt
tgagaagttt cttgcctttc catcacattg
24181 gcaattgcac gctttgctct tgcttctaca
tggagtttta cttttatccc agacagtttg
24241 gtttcttctc tgattttcgc caattgtaca
gatcgttaca gtatttctta accacataga
24301 attcggcagg gggggtgggg ggacagggta
gggtggggtg agagtgaggg gagggggctg
24361 caccgagcag catctggggt cgtagctccc
tgacggggat agacctcgtg cccctgcagt
24421 gacagcacag agtcctcctc tctgaactgc
cagggacgct cctgcaattg acttaatgaa
24481 aggcatctaa ttaggaattt tggggtgaca
ttttacattt aagtgtgtga gcagtgatta
24541 tagttcatat cattttatag tttcgtgatt
ttactagctt aaagggtttt tggggtttct
24601 ttttgtttta aaagctaaaa tctgtttttt
aattccatgg aatacaaaaa aaaaaagtct
24661 gtagaatatt ttaaagagtg aaggctttgt
tcggaatgtg agcgctttgc tccactgaac
24721 cgaacggtaa taacatttgt agaagagacg
cagagtgaaa ggtacctctt tttattgagt
24781 gacatgacag cacccatcgc gtgagttatt
ggctggagtt tagagacagg ccatgttggg
24841 ctaaactcct tattgctgtt ctcagccttt
gagtaataat cagaagcttt ctctgaagag
24901 agtggggtca gctgtcagac tcctaggtgt
ctacctgcag cagggctggg attaaatgca
24961 gcagccagta gatacgggat ggggcaagag
gtcaccttgt ccctttgttg ctgctgggag
25021 agaggcttgt cctggtgcca gtggggccaa
agctgtgact ttgtgaccac aggatgtctc
25081 tgaccctgcc ttgggttccc tgagggtgga
gggacagcag ggtctccccg gttccttggc
25141 cggagaagga ccccccaccc cttgctctct
gacatccccc caggacttgc cccggagtag
25201 gttcttcagg atgggcatcc gggccccacc
ctgactcctg gagctggccg gctagagctt
25261 gctgcagaat gaggccttgg ccattgcggc
cctgaaggag ctgcccgtca agctcttccc
25321 gaggctgttt acggcggcct ttgccaggag
gcacacccat gccgtgaagg cgatggtgca
25381 ggcctggccc ttcccctacc tcccgatggg
ggccctgatg aaggactacc agcctcatct
25441 ggagaccttc caggctgtac ttgatggcct
ggacctcctg cttgctgagg aggtccgccg
25501 taggtaaggt cgacctggca gactggtggg
gcctggggtg tgagcaagat gcagccaggc
25561 caggaagatg aggggtcacc tgggaacagg
cgttgggtgt acaggactgg ttgaggctca
25621 gaggggacaa aaggcacgtg ggcctccccc
ccagtgtccc ttaaagtggg aaccaagggg
25681 gccccggaag ccggaggagc tgtggtgtgt
ggagtgcaga gccctcgcgg ggtcctgatg
25741 cccgtcggac tctgcacagc tcagcgtgtg
ccccgcggcc cggtaggcgg tggaagctgc
25801 aggtgctgga cttgcgccgg aacgcccacc
agggacttct ggaccttgtg gtccggcatc
25861 aaggccagcg tgtgctcact gctggagccc
gagtcagccc agcccatgca gaagaggagc
25921 agggtagagg gttccagggg tgggggctga
agcctgtgcc gggccctttg gaggtgctgg
25981 tcgacctgtg cctcaaggag gacacgctgg
acgagaccct ctgctacctg ctgaagaagg
26041 ccaagcagag gaggagcctg ctgcacctgc
gctgccagaa gctgaggatc ttcgccatgc
26101 ccatgcagag catcaggagg atcctgaggc
tggtgcagct ggactccatc caggacctgg
26161 aggtgaactg cacctggaag ctggctgggc
cggatgggca acctgcgcgg ctgctgctgt
26221 cgtgcatgcg cctgttgccg cgcaccgccc
ccgaccggga ggagcactgc gttggccagc
26281 tcaccgccca gttcctgagc ctgccccacc
tgcaggagct ctacctggac tccatctcct
26341 tcctcaaggg cccgctgcac caggtgctca
ggtgaggcgt ggcgccagct ccaaagacca
26401 gagcaggcct ctcttgtttc gtgcccgctg
gggacattgc cagggtgccc ggccactcgg
26461 aagtcctcac gatgccaccg ctctgaccct
gggcatcttg tcaggtcact tccctggtta
26521 gggtcagagg cgtggcctag gttaaatgct
gtcaaagggg actcctttct gggagtccgc
26581 atagtggggg cttggtgtga tgcccttggg
aattctttcc gagagagtga tgtcttagct
26641 gagataatga cagataacta agcgagaagg
acggtccatc aggtgtgagg tttgaagtcc
26701 aaagctctgt ctctccctcc cacctgcccc
ttctgtcctg agctgtttta ggctccaggt
26761 gagctgtggg aagtgggtga ttctggagat
gacaagaagg gatcaggagg ggaaaattgt
26821 ggctcctaag cagtccagag aagagaaaaa
gtcaaataag cattattgtt aaagtggctc
26881 cagtctcttt aagtccaaat tataattata
attttcctct aagacttctg aatacatagg
26941 aaatcctcag taacaggtta ttgctctgcc
ttgaacacag tgataaaagc tgggaggatg
27001 cagcctaatc tgtctgtgtg aatgagttgt
attgattccc tttttggcag ctgcaaactc
27061 caagcattag gaataaatat gttcactgag
aaccccgaag aaagaaagaa agaaaaaaaa
27121 aaagaattgt aggtgttgat ggacggtttg
tggcccctga atatctgggg gatgttcacc
27181 cagggatcac gtgtaactgc tgggaccccc
agccccatgt ccactgcatc cagcctgctg
27241 ttgaattccg cggatcnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
27301 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnncaat
27361 tcgagctcgg taccccaaag gtccgtctag
tcaaggctat ggtttttcca gtggtcatgt
27421 atggatgtga gagttggact gtgaagaaag
ctgagtgcca aagaattatt cttttgtact
27481 gggtgttgga gaagactctt gagagtccct
tgaactgcaa ggagatccaa ccagtccgtt
27541 ctaaaggaga tcagtcctga atgttcattg
gaaggactga tgctgaagct gaaactccaa
27601 tactttggcc acctgacgtg aagagttgac
tcattggaaa agaccatgat gctgagagga
27661 attgggggca ggaggagaag gggacgacag
aggatgagat ggctggatgg catcaccaac
27721 tcgatgngac atgagtttgg ttaaactcca
ggagttggtg atggacttgg aggcctggtg
27781 tgctgggatt catggggtcg cagagtcgga
catgactgag cgactgaact gaactgaact
27841 gagctgaaga gctcacctgt accagagctc
ctcaggtcct cctgcaggcc tggctgtaat
27901 ggcccccagg tcaccgtcct gcctccttca
tcccatcctt tcacgacagg ctgggagtgg
27961 ggtgaggtga gttgtcttgt atctagaatt
tctgcatgcg accctcagag tgcaatttag
28021 ctccagagaa ctgagctcca agagttcatt
ttttcctttt cttctttatg atactaccct
28081 cttctgagca gagacctcat gtcagggaga
aggggactct gccttcctca gccttttgtt
28141 cctccaagac ccacacgggg agggtcgcct
gcttcactga gccggaaggt tcaattgctc
28201 atgtcctcca gaaacacccc cccccccaga
gacccccaga aataagtgga acagcacctt
28261 gtttcccaga caagtgggac acacgttatg
aaccacctca gtgattaaaa tagtaacctc
28321 tgtgtatgtg tatttactgg agaaggaaac
ggcaacctac tccactattc ctgcctagaa
28381 aattccatgg gagagaagcc aggcaggcta
cagtccacgg ggtcacagag actgaacata
28441 cacaagcaca tggaagtgta ttttgcagta
tttttaaatt tgttcagttc aacatggagt
28501 acaagaattc aaatcgtgaa gtcaattgac
caagaaacca gaagaaatca ctgtgttgtg
28561 atctctgtgg aggtaacatg ggtacctgtg
ctctgaccct cacagcctct ggctctctct
28621 ctacatgtac atacacatat atttccatgt
atgtatgtat tcggaagatt tcacatacgt
28681 ctcaccagtc cacagccccc gcgttccctg
atgcccagaa catctgtgat agctgtgagt
28741 attgtcacca gataagatct tccaggttcc
tgcactcaca ttggttatca ggtctctctg
28801 atccagcatt tctcagctaa gattccttgt
gactcctggc tgcagaatct tctgcaaaag
28861 tcccacagag aggagtgtga tcactgtaca
caggagggcc gtggttctct agtgtgagaa
28921 aagctaactc agcccgtcac agggacgtga
atgtacctga gacagtaatc agttatgctg
28981 agaaatcaca gctctgctag aggcagcaca
tggggtagcc agcagggggc agcagagcac
29041 ggccaggagc cgcaggtcag aggctgggct
gcccaagcgg ggcttcaggg gaaccagccc
29101 tgcgggtcca caggtgtcca gggagcagcg
cttggcagga agtcaggacc ggacaggcca
29161 tcccctcagg actagtgacc acctctgagg
gtcacatcca cagtgaaccc cagagcacca
29221 tgcctcagtc cacggccagg acgctgccag
gctgaccgcc ccactgggga gtccagggga
29281 gaccacaggc cggggggctt gggacagtga
tcatgtggtc agacacagag aaggtgacag
29341 tgacctcagt ccctgaggac aagtctgatg
tgcagacgtg agaagccgag gaggaagctg
29401 gggacagaca gggctgatgg tgtggtgacc
ccgcctctca gtgaggggcc cccgggggtg
29461 aatttgcata aacccaagcc ctcactgccc
ccacaaagct ctgagaggga ataaaggggc
29521 tcggagagcc cagcactgct gcgggctcag
aggcagagct cggggcgcgt ccaccatggc
29581 ctgggcccct ctcgtactgc ccctcctcac
tctctgcgca ggtgcggccc cccagcctcg
29641 gtccccaagt gaccaggcct caggctggcc
tgtcagctca gcacaggggc tgctgcaggg
29701 aatcggggcc gctgggagga gacgctcttc
ccacactccc cttcctctcc tctcttctag
29761 gtcacctggc ttcttctcag ctgactcagc
cgcctgcggt gtccgtgtcc ttgggacaga
29821 cggccagcat cacctgccag ggagacgact
tagaaagcta ttatgctcac tggtaccagc
29881 agaagccaag ccaggccccc tgtgctggtc
atttatgagt ctagtgagag accctcaggg
29941 atccctgacc ggttctctgg ctccagctca
gggaacacgg ccaccctgac catcagcggg
30001 gcccagactg aggacgaggc cgactattac
tgtcagtcat atgacagcag cggtgatcct
30061 cacagtgaca cagacagacg gggaagtgag
acacaaacct tccagtcctg ctcacgctct
30121 cctccagccc cgggaggact gtgggcacag
cagggacagg cctggcccgg ttcccccgga
30181 gctgagcccc caggcggccc cgcctcccgg
ccctccaggc aggctctgca caggggcgtt
30241 agcagtggac gatgggctgg caggccctgc
tgtgtcgggg tctgggctgt ggagtgacct
30301 ggagaacgga ggcctggatg aggactaaca
gagggacaga gactcagtgc taatggcccc
30361 tgggtgtcca tgtgatgctg gctggaccct
cagcagccaa aatctcctgg attgacccca
30421 gaacttccca gatccagatc cacgtggctt
tagaaaggct taggaggtga acaagtgggg
30481 tgagggctac catggtgacc tggaccagaa
ctcctgagac ccatggcacc ccactccagt
30541 actcttccct ggaaaatccc atggacggag
gagcctggaa ggcttcagcc catggggtcg
30601 ctaagagtca gacacgactg agcgacgtca
ctttcccttt tcactttcat gcattggaga
30661 aggaaatggc aacccagtcc agtgttcctg
cctggaaaat cccagggaca ggggagcctg
30721 gtgggctgcc atccatgggg ccacacagag
tcagacacga ctgaagcaac ttagcagcag
30781 cagcagcagc ccaataaaac tcagcttaag
taatggcatc taaatggacc ctattgccaa
30841 ataaggtcca ctcgcgtgca ctctgtttag
gacttcagtt cctgattgtg gagggttccc
30901 acaagacgtg tgtgtatatt ggtgttgccg
gaaaacagtg tcaatgtgag catcccagac
30961 tcatcaccct cctactccca ctattccatt
gtctctgcag gtattaagca taaaggttaa
31021 gggtcttatt agatggaaga ggagtgaata
ctcgtctgtg cttaacacat accaagtacc
31081 atcaaggtcc ttcctattta ttaacgtgtg
ttttaatcag aaatatgcta tgtagaagca
31141 tccggacgat agcccatgtt acagacgggg
aagctgaggc atgaagttct cagcaccttg
31201 tttcacgtca gacctgaaac ggggcagagc
cggcagcaaa caaggttcct cttcccaagc
31261 gcccgctctt cacccgcttc ctatggcttc
tcactgtgct tcctaaacta agctctcccc
31321 aaccctgtgg agacaggatt agagacttta
ggagaaaaga ccaggaacat cccacacccg
31381 acccgagtga gccactaaga caaggctttg
taaggacaga accagcaggt gtcctcagcg
31441 agccagggag agacctcgca ccaaaaacaa
tattgtagca tcctgaccct ggacttctga
31501 cctccagaaa tgtgaaaaag aaacgtgtgg
ggtttaatca actcaccggt gttatttggt
31561 tatgactgcc tgagttaaga aggagttggg
aacacttgag tgtaggtgtt tatggaacat
31621 aagtcttgtt tctctgaaat aaattcccaa
gggtataatt cctaggttgt agggtaactg
31681 ccacaaatct aggcagctta ttaaaaaaca
aagatatcac tttgccagca aaggttcata
31741 tagtcaaatt atggttttta tagtagtcat
gtatggatgt aaaagttgga tcataaagaa
31801 ggctgagcac cagagaattg atcccttcaa
atcgtggtgc tggagaagac tcttgagagt
31861 cccttggaca gcaaggagat ccaaccagtc
aatcctaaag gaaatgaact gtgaatattc
31921 actggaagga ctgatgctga agctgaagat
ccaatacttt ggccacctga tgcgaagagt
31981 tgactcattg gaaaagaccc tgatgctgga
aagcttgagg gcaggaggag aagagggcgg
32041 cagaggatga gacggttgga tggcatcact
gactcaatgg acatgagttt gagccaactc
32101 tgggagacag tgaaggatag ggaaggctgg
cgtggtacag tgcatgcggt cacaaagagt
32161 ctgacacatc ttagtgactc aacaacgaca
gcaacacagg catcacacgc ttagtgtgat
32221 aagcggcaga actgttttcc aggggtccgn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
32281 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
32341 nnnnnnnnng tacgattcga gctcggaccc
tgacattgtg agtcacgtca tgagcagctg
32401 ttttccggtc ttcagggatt gtggacgatt
tctgtttggg tttgctcatg ataatttagt
32461 tacagcttag gttctttctt tccaggccac
gagcgacatg ttttcaggtg agatgacgtg
32521 gtgggggatg ggcggccaag cccccactgg
ggggggaggg attctgttgt gggcaggagt
32581 tggcagcatc cctgaactga tgacctgcga
tccaggtgac aagaaccggg ggatattatt
32641 cctctgcctt ctcatgtcat gtcctcggtt
cttcatgatg aaaacatatg acaatacagg
32701 ggagttagat ttgggcgggc acaactctgg
gtgggggacc cggtggcatt gtgcccagca
32761 gggccatcaa gatgagggcg acctgggtgg
tccccttctc ccctggggtc ttagttttcc
32821 cctcatggaa atgggatcag gcagcagcca
tggaacaccg cgaccgtggc ttctctcacc
32881 tcctcgtctg tgattttggg tcgggatacc
aggcatgaag acctggggcg gggggacatc
32941 actcctctgc agcagggagg ccgcagagtc
ctccgtccat gaggacttcg tccctgggct
33001 gaccctgcgg actgctggag gctgaagctg
gaggcacagg cgggctgcga ggccagggtc
33061 ctgaggacga cagagccagt ggggctgcag
ctctgagcag atggcccctc gccccgggcc
33121 ctgagcttgt gtgtccagct gcaggttcgc
tcaggtgagc cactacgtta tgggggaggc
33181 gccctgggca gggatcgggg gtgctgactc
ctccgagatt ccgaccttct gggagcactc
33241 tggccacact ctaagcctgg caagagctgg
gttcatcagt ctaactctcc tcctgaagtc
33301 caatggactc tctccatgcg gcagtcactg
gatggcctct ttatccccga tggtgtcctt
33361 ttccgctgac ctggctctcc tgaccacctc
ccagcccccc accatacagg aagatggcac
33421 ctggtccctg cagagctaag tccacccctg
gcctggcttc agatgcctac agtcctcctg
33481 cgggaggccc cgctccccac taggccccaa
gcctgccgtg tgagtctcag tctcacctgg
33541 aaccctcctc atttctcccc agtcctcagc
tcccaacccc agaggtatcc cctgcccctt
33601 tcaaggccct tgtcccttcc tggggggatg
gggtgtatgg gagggcaagc ctgatccccc
33661 gagcctgtgc cgctgacaat gtccgtctct
ggatcatcgc tcccctggct ctcagagctc
33721 cctggtccct ggggatgggt tgcggtgatg
acaagtggat ggactctcag gtcacacctg
33781 tcccttccct aaggaactga cccttaaccc
cgacactcgg ccagacccag aaagcacttc
33841 agacatgtcg gctgataaat gagaaggtct
ttattcagga gaaacaggaa cagggaggga
33901 ggagaggccc ctggtgtgag gcgacctggg
taggggctca ggggtccatg gagaggtggg
33961 ggagggggtg tgggccagag ggcccccgag
ggtgggggtc cagggcccta agaacacgct
34021 gaggtcttca ctgtcttcgt cacggtgctc
ccctcgtgcg tgacctcgca gctgtaactg
34081 cctttcgatt tccagtcgct gcccgtcagg
ctcagtagct gctggccgcg tatttgctgt
34141 tgctctgttt ggaggcccgg gtggtctcca
cgttgcgggt gatggtgctg ccgtctgcct
34201 tccaggccac ggtcacgcta cccgggtaga
agtcgctgat gagacacacc agggtggcct
34261 tgttggcgct gagctcctcg gtggggggcg
ggaacagggt gaccgagggt gcggacttgg
34321 gctgacccgt gtggacagag gagagggtgt
aagacgccgg ggaggttctg accttgtccc
34381 cacggtagcc ctgtttgcct tctctgtgcc
ctccgaccct tgccctcagc ccctgggcgg
34441 cagacagccc ctcagaagcc attgcaatcc
actctccaag tgaccagcca aacgtggcct
34501 cagagtcccc ggctgcgacc agggctgctc
tcctccgtcc tcctggcccc gggagtctgt
34561 gtctgctctt ggcactgacc ccttgagccc
tcagcccctg ccagacccct ccgtgacctt
34621 ccgctcatgc agcccaggtg cctcctccgt
gaacccgggt ccccccgccc acctgccagg
34681 acggtcctga tgggagatgt ggggacaagc
gtgctagggt catgtgcgga gccgggcccg
34741 ggcctccctc tcctcgccca gcccagcctc
agctctcctg gccaaagccc ggggctcctc
34801 tgaggtcctg cctgtctacc gtccgccctg
cctgagtgca gggcccctcg cctcacctgc
34861 cttcagggga cggtgccccc acacagcacc
tccaaagacc ccgattctgt gggagtcaga
34921 gccctgttca tatctcctaa gtccaatgct
cgcttcgagg ccagcggagg ccgaccctcg
34981 gacaggtgtg acccctgggt cccaggggat
caggtctccc agactgacga gtttctgccc
35041 catgggaccc gctcctttct gaccgctgtc
ctgagatcct ctggtcagct tgccccgtct
35101 cagctgtgtc cacccggccc ctcagcccag
agcgggcgag acccctctct ctctgccctc
35161 cagggccttc cctcaggctg ccctctgtgt
tcctggggcc tggtcatagc ccccgccgag
35221 cccccaagct cctgtctggc ctcccggctg
gggcatggag ctcacagcac agagcccggg
35281 gcttggagat gcccctagtc agcaccagcc
tctggcccgc accccagcgt ctgccctgca
35341 agaggggaac aagtccctgc attcctggac
caaacaccag ccccggcgcc ccgactggcc
35401 ccattggacg gtcggccact ggatgctcct
gctggttacc ccaagaccaa cccgcctccc
35461 ctcccggccc cacggagaaa ggtggggatc
ggcccttaag gccgggggga cagagaggaa
35521 gctgccccca gagcaagaga agtgactttc
ccgagagagc agagggtgag agaggctggg
35581 gtagggtgag agccacttac ccaggacggt
gacccaggtc ccgccgccta agacaaaata
35641 cagagactaa gtctcggacc aaaacccgcc
gggacagcgc ctggggcctg tcccccgggg
35701 gggctgggcc gagcgggaac ctgctgggcg
tgacgggcgc agggctgcag ccggtggggc
35761 tgtgtcctcc gctgaggggt gttgtggagc
cagccttcca gaggccaggg gaccttgtgt
35821 cctggaggtg ccctgtgccc agccccctgg
ccgaggcagc agccacacac gcccttgggg
35881 tcacccagtg ccccctcact cggaggctgt
cctggccacc actgacgcct tagcgctgag
35941 ggagacgtgg agcgccgcgt ctgtgcgggg
cggcagagga gtaccggcct ggcttggacc
36001 tgcccagccg ctcctggcct cactgtaagg
cctctgggtg ttccttcccc acagtcctca
36061 cagtccagcc aggcagcttc cttcctgggg
ctgtggacac cgggctattc ctcaggcccc
36121 aagtggggaa ccctgccctt tttctccacc
cacggagatg cagttcagtt tgttctcttc
36181 aatgaacatt ctctgctgtc agatcactgt
ctttctgtac atctgtttgt ccatccatcg
36241 atccaacatc catccatcca tccatcaccc
agccatccat ctgtcatcca acatccatcc
36301 ttccatccat tgtccatcca tctgtccatc
ttgcatctgt ctgtccaaca gtggccatca
36361 agcacccgtc tgccaagccc tgtgtcacac
gctgggactt ggtgggggga gccctcgccc
36421 tcccaccctc ccatctctcc tgaaacttct
ggggtcaagt ctaacaaggt cccatcccgt
36481 ctagtctgag gtccccccgc agcctcctct
tccactctct ctgcttctga cccacactgt
36541 gcactcggac gaccacccag ggcccttgca
tccctgtttc cttcctgacc tctttttttt
36601 ggctctggat ttatacacat tctgcctcct
ggaggcgtct cagcttgagt gtcccacaga
36661 cgcctcagac tcagcatctt ccatcgaaac
tgctcccagg tccttgcaga cctggtcccc
36721 cacattgttc tcaattcggt agatttctcc
acaagccaga ggcctggact catcccataa
36781 tgcctgcccc tcattgagtc agcctctgtg
tcctaccata accaaacatc cccttaaaaa
36841 tctcagaaga acaaaaaaag cacccagatg
gcactgtcag agtttatgat gacaagaatc
36901 ctcagttcag ttcagtcact cagtcgtgtc
cgactctttg cgaccccatg aatcgcagca
36961 cgccaggcct ccctgtccat caccaactcc
cggagttcac tcagactcac gtccattgag
37021 tcagtgatgc catccagcca tctcatcctc
tctcgtcccc ttctcctcct gcccccaatc
37081 cctcccagca tcagagtttt ttccaatgag
tcaactcttc gcgtgaggtg accaaagtac
37141 tggagtttca gcttcagcat cattccttcc
aaagaaatcc cagggctgat ctccttcaga
37201 atggactggt tggatctcct tacagtccaa
gggactctca agagtcttct ccaacaccac
37261 agttcaaaag cctcaattct ttggcgctca
gccttcttca cagtccaact ctcacatcca
37321 tacatgacca caggaaaaac cataaccttg
actagatgga cctttgttgg caaagtaatg
37381 tctctgcttt ttaatatgct atctaggttg
ctcataactt tccttccaag aagtaagtgt
37441 cttttaattt catggctgca atcaacatct
gcagtgattt tggagcccca aaaaataaag
37501 tctgccactg tttccactgt ttccccatct
atttcccatg aagtgatggg accagatgcc
37561 atgatctttg ttttctgaat gttgagcttt
aagccaactt ttcactctcc actttcactt
37621 tcatcaagag gctttttagt tcctcttcac
tttctgccat aagggtggtg tcatctgcat
37681 atctgaggtt attgatattt ctcctggcaa
tcttgattcc agtttgtgtt tcttccagtc
37741 cagtgtttct catgatgtac tctgcatata
agttaaataa gcagggtgat aatatacagc
37801 cttgacgtac tccttttcct atttggaacc
agtctgttgt tccatgtcca gttctaactg
37861 ttgcttcctg acctgcatac agatttctca
agaggcaggt caggtggtct ggtattccca
37921 tctctttcag aattttccac agttgattgt
gatccacaca gtcaaaggct ttggcatagt
37981 caataaagca gaaatagatg tttttctgaa
actctcttgc tttttccatg atccagcaga
38041 tgttggcaat ttgatctctg gttcctctgc
cttttctaaa accagcttga acatcaggaa
38101 gttcacggtt catgtattgc tgaagcctgg
cttggagaat tttgagcatt cctttgctag
38161 cgtgtgagat gagtgcaatt gtgcggcagt
ttgagcattc tttggcattg cctttctttg
38221 ggattggaat gaaaactgac ctgttccagg
cctgtggcca ctgttgagtt ttcccaattt
38281 gctggcatat tgagtgcagc actttcacag
catcatcttt caggatttga aatcgctcca
38341 ctggaattcc atcacctcca ctagctttgt
ttgtagtgat gctctctaag gcccacttga
38401 cttcacattc caggatgtct ggctctagat
gagtgatcac accatcgtga ttatctgggt
38461 cgtgaagatc ttttttgtac agttcttctg
tgtattcttg ccacctcttc ttaatatctt
38521 ctgcttctgt taggcccata ccgtttctgt
cctcgcctat cgagccctcg cctccctacg
38581 tagagactct aagcaggaag gtgacccgtg
ctgcactggg tccagcatgc ttttaattca
38641 gcagtggaac ttctgggtca tgattgtgtt
taagggatgc gcatacgatt tttgaagcaa
38701 aatttaacag gacagcagtg taaagtcagt
acttatttct gattaaagaa agcaaatatc
38761 cagcctgtta ctaagttaat taactaaaga
aacatcttca acttaataaa cagtatctcc
38821 tgaaacttac agcatgcttc acatttaaag
gcaaaaccat tttagaggcc agggttccca
38881 cgcttacgtt tattatttaa tatatgctac
agattcaagc ccatgacaca aaatgggggg
38941 aagagtgtga gtgttaggaa aaatgagata
aaattggttt ttgcaggtga tgggctagtt
39001 tactttaaaa aaaaaaacaa aacaagctca
agatgaactg aaggactatt agaactggta
39061 caagagttaa cctgtgatcg aatacaagca
ggctgggcaa aactcagcag gttttcttct
39121 atacaggcag taatgattga gaatacgaaa
cggcggaagc gcttacaacc tcgataacag
39181 ttctattaaa agccctagga atgaacttaa
cacggnnnnn nnnnnnnnnn nnnnnnnnnn
39241 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
39301 nnnnnnnnnn nnnnngctcc ccccaccctc
ccctcctccc cccccaccac cagtgcccca
39361 ggtctcgtgc ccagagagct gaagatgcca
gcaggcccgc tgcctgcctc gctcgcgtgg
39421 cccgggctcg ctgccggtct gcctgcccag
cacacagatg cagccccagc tctcgctgcc
39481 acccgcctcc cccaggcagg actctcccac
aacaccaagg gcgtctctgg gttcaggatg
39541 gccctcgttg aggtgtaaag tgcttcccgg
ggctgagacg aatgggccgg agatccaaac
39601 gaggccaagg ccgccacggc gcctggcgca
gggcacccat ggtgcagagc ggcccagctc
39661 cctccctccc tccctccctc cctgcttctt
tatgctcccg gctatgtcta tttttactct
39721 gcaatttaga aatgataccg aaggacaaac
accgttcccc ctgtgtgtct gctctaaacc
39781 ctttatctac ttatctatta gcgtgtccaa
gttttgctgc taagtgaatg aaggaacact
39841 acccacaagc agcaacgtcc ccacgaccct
cgcctgttca actgggaatg taaatgtgct
39901 ttcaaaggac ctaagtttct atgttcaaaa
ccgttgtgtg tttcttttgg gagtgaacct
39961 aggccactcg ttgttctgcc tttcaaagca
ttcttaacaa ctctccagaa cccagggctt
40021 ggcttacgtt tccagaaatt ccaaagacag
acacttggaa acctgatgaa gaaggcctgt
40081 gagcacagca ggggccgggg tacctgaggt
aggtgggggg ctcggtgctg atggacacgg
40141 ccttgtactt ctcatcgttg ccgtccagga
tctcctccac ctcggaggct ttcagcaggg
40201 tcacgctggt ggccagggtc gtgtatccat
gatctgcaac cagagacggg gctgcggtca
40261 gcccgcgggc gggcagcagg caggagcagc
caggagacgc agcacaccga ggtcctcaca
40321 tgcaggaggt gggggaagcg gctgtggacc
tcacgactgc ccgatgtggg cctcttccaa
40381 agggccggcc tggaccctgg ctttctccag
aggccctgct gggccgtccg cacaggctcc
40441 agccacaggg cctcttggga caggagggct
ccagagtgag ccggccggcg ggaagaggtc
40501 tgacaccgct gcagtccaca acacgaagcg
aggtggagat gggatgaggg atgagaaaca
40561 cttttctttt aaaacaagag cccagagagt
tggaaagagc tgctgcacac gcaacatgaa
40621 ctcctggccc cggtgccagc ggcgctggga
gcccgagttc tcggcaatcc gaccacagct
40681 tgcctaggga gccgggtgga gacggagggt
taggggaagg cggctcccca gggagcgcga
40741 ggcccggggt cgccaaggct cgccaggggc
aagcgcagct aggggcgcag ggttagtgac
40801 cggcactgca cccggcgcag gagggccagg
gaggggctga aaggtcacag cagtgtgtgg
40861 acaagaggct ccggctcctg cgttaaaaga
acgcggtgga cagaccacga cagcgccacg
40921 gacacactca taccggacgg actgcggagt
gcacgcgcgc gcacacacac acacacacca
40981 cacacacaca cacacggccc gggacacact
cataccggac ggactgcgga gtgcacgcgc
41041 acacacacac ccaccacaca cacacccacc
acacacacac ccaccacaca cacacacaca
41101 cacacacacc cccacacaca cccacacaca
cccacacaca cccacacaca cacacccaca
41161 cacacacaca cacacacaca cacacacacg
gcccggtggc cccaggcgca cacagcacgg
41221 agcaaacatg cacagagcac agagcgagcg
ctagcggacc ggctgccaga ccaggcgcca
41281 cgcgatggat tgggggcggg gacggggagg
ggcgggagca aacggnnnnn nnnnnnnnnn
41341 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
41401 nnnnnnnnnn nnnnnnnnnn nnnnngtatt
aaagaagccg ggagcgagaa tatgacggca
41461 agaggatgta ggtgggggcg gggcaagagt
aaagagagcg gacggtagag gggatgcgat
41521 tgtgatgcgg aagcgagacg aggagtgatg
ccgtattaga ttgatagcaa gaggaacagt
41581 aggagggggg ggggagagga gggggaggtg
gggggtggtg ggtgggaagg gaactttaaa
41641 aaaaagaggg gagagttgga ggggggaata
aacgggcggt aaaaaagaac aatttgaaat
41701 taccagggtg gggcggccag gggggtgatt
cattcttgga gggggcaaca tatggggggt
41761 ggctgtcgcg gattaggaga aaataaatat
caggggtgat taagtgtttg gcgttgggga
41821 ataatgaagt aagaatcaaa tatgaatcgc
gttggcatcg ttagccatcg ggggaaacat
41881 ttcccatgca aggaacaagg atgtgagaat
gcgtccgtct gaaccaccgt cccggggtcc
41941 cagtaggact cgccgagctg atagttgccg
gagcaacagt taagggagca gaagctgcta
42001 caaaaccacc acctgccaaa gtagggtctc
caattacgga gtgcgcctcc tgggtgtcgg
42061 tccaaacctt tggaaaggac ctggaaataa
gtgctaccca ccagatatta atataaaccc
42121 acctggccag gagaggcagg cgctgctggc
acaggaagtg tccccagact cagtcatcaa
42181 ggtaaataat attttgggac ctccctggaa
atccagtggt taggactctg cggttcaatc
42241 cctggtcggg gaactaagat cccacaagtc
acaagacatg gccaaattta aaaaagaaaa
42301 aaagagagag aaatatttag tgcaataggt
tttagaattg aaattaagct cctgcccacc
42361 cccacccccc aatctggatg aataaagcat
tgaaatagta agtgaagtca ggctctgaca
42421 tgcactgatg tgactcacct taagcaaccc
ccaccctagg actggtcggg gttccaggag
42481 tttcaggggt gccaggaaga tggagtccag
cccctgccct ctccccccac cacgtcctcc
42541 actggagccg cctaccccac ctcccacccc
tccgcaccct gctacccccc acccctgccc
42601 ccaggtctcc cctgtcctgt gtctgagctc
cacactttct gggcagtgtc tccctctaca
42661 gctggtttct gctgcccgct accgggcccg
tcccctctgt tcagttcagt tcagtcgctc
42721 agtcatgtct gactctttgt gaccccatgg
actgcagcac accaggcctc cctggccatc
42781 accaaccccc agaacttact caaactcatg
tccatcgagc cagtgatgcc atccaaccat
42841 ctcatcctct gtcgacccct tctcctggcc
tcaatctttc ccagcatcag ggtcttttcc
42901 aatgagtcag ttctttgcat caggtagcca
aagtattgga gtttcagctt cagcatcatt
42961 tcttccaatg aatattcagg actcatttcc
tttgggatga actggttgga tctccttgca
43021 gtccaaggga ctctcaagag tcttctccaa
caccacagtt caaaagcatc aattcttcag
43081 tgctcagctc tctttatagt ccaactctca
catccatacg tgaccactgg aaaaaccata
43141 gcctcgacta gatggaactt tgtgggcaaa
gtaatgtctc tgcttttgaa tatgctgtct
43201 aggttggtca taacttttct tccaaggagc
aagcgtcttt taatttcatg gctgcagtca
43261 ccatctgcag tgatttttgg agcccaagaa
aataaagtct gtcactgttt ccactgtttc
43321 cccgtctatt taacggaggg aaatttccca
gagcccccag gttccaggct gggccccacc
43381 ccactcccat gtcccagaga gcctggtcct
cccaggctcc cggctggcgc tggtaagtcc
43441 caggatatag tctttacatc aagttgctgt
gtgtcttagg aaagaaactc tccctctctg
43501 tgcctctgtt ccctcatccg cagaagtgac
tgccaggtcg gggagtctgt gacgtctcca
43561 gaagccggag gattttctcc ccatttgctg
aaagagagct cggggtgggg gaagcttctg
43621 cacccctagg atcaccagag gagccagggt
cttcagggtt cccggggacc cctcagtggg
43681 ggctcaggaa ccacagagcc agaccctgat
tccaaaaacc tggtcacacc tccagatgac
43741 cctttgtccc ttggctccgc ctcaaatgct
ccaagcccca acagtgaagc gcttaagaga
43801 aggatccacc aggcttgagt ttggggagga
gggaagtggg gagctggggg agggcctggg
43861 cctgggagac aggaatccac catggcttca
ggcagggtct ctggggcctg cggggtggag
43921 agcgggcagg agcagacaga ggtgactgga
cacgacacac ccctccactc caagggaggt
43981 gggcaggggc ggggcacaga ggaacaagag
accctgagaa ggggtccacc gagcagactg
44041 ctggacccag acatctctga gccagctgga
atccagctct aagccatgct cagcccaggc
44101 agggtatagg gcaggactga gtggagtggc
cagagctgca gctgcatggg ctgggaaggc
44161 cctgcccgtc ccctgagggt cccccagggt
ctagccagac tccaatttcc gaccgcagca
44221 cacacaggag gaagtggtcg gggtggagtt
ggcccagagg tctgggcagg tgcagggtgg
44281 gggaaggggg gcagctggag tcacccgctg
aattcaggga cagtcccttt ttctccctga
44341 aacctggggc tgtcccgggg gccaccgcag
cctccaggca gcggggggac ccagccccca
44401 atatgtgaga agagcaggtc ccaggctgga
gagagcgaag caccatggtg gggagaagtt
44461 agactggatc ggggccccta ggggctcccc
cggacctgca cggcagccgt cagggcaccc
44521 gcaccccatt gctgttcagt gctggccagt
gtccaaggcc agggatgtgt gtgtgtgtgt
44581 gtgcgtgcgt gcgtgcgtgt gtgtgtgcgt
gtgtgcgcgt gcgtgcgtgt gtgtgtgtgt
44641 gcgtgcgtgt gcgtgcgtag acgtgtgcgt
gcgtgcgtgc gtgcgtgcgt gtgtgtgcgc
44701 acgcgcgcag cccagcctca gcactggacc
aggcagcctg ggattcctcc aaaactgcct
44761 tgtgagtttg gtcaaaccgt gaggctctga
tcaccgccat ccattcgccc cctcctgccc
44821 ccctcatcac cgtggttgtt gtcattatcg
agagctgtgg agggtctggg aggtcatccc
44881 acctgccagc taaaccgtga ggctgccgca
atcgcactga tgcgggcaga cccgagacgc
44941 tgtgccggag acgaaggcca gcttgtcacc
ccgccagagc ggcagtcggg ccacaagcat
45001 catccaagca gtggttctct gagcccgacg
gggtgatgca aaggagccag gagacacctg
45061 cgcgtccaag ctgggggacc ccaggtctgt
tatgccggac agtaaacacg ttcagctccg
45121 gagggagagg gttcccctac cttccagggt
ttctcattcc acaaacatcc aaagacaatc
45181 cataccgaag gcgatccgtg cctttgctcc
tgagacgtgc ggaagcacag agatccacag
45241 acactgtctc ccaggatcct atgtatgtaa
aggaaccgaa gtcccaggct gtgtgtctgg
45301 taccacatcc cacggaacag gctggactga
ttttcaccaa atgtagcaga aacgttaagg
45361 agtatcagct tcaaaatatg agggccagac
atgtctgaga agtcccttcc agaaaagtcc
45421 ctttggggtc cttccccaga gttgctgaaa
cagagaaccg gaagggctgc agagctgaac
45481 ttaaacaact ggatcgcaaa ggtccgtctc
atcagagcga tggtttttcc agtggtcatg
45541 tatggatgag agagttggac cataaagaaa
gctgagcgcc gaagaatcga tgcttttgaa
45601 ctctggtgtt ggagaagact cttgagagtc
ccttggactg caaggagatc caaccagtca
45661 atcctaaagg aaatcaatcc tgaatattca
tgggaaggac tgatgctgaa gctgaaactc
45721 caatactttg gccacttgat gcaaagaact
gactcactgg aaaaaccctg atgctgggaa
45781 aggttgaagg caggaggaga aggggtcgac
agaggatgag atggttgggt ggcatcaccc
45841 acccatggac tcaatggaca tgggtttgag
taaactctgg gagttggtga tggacagaga
45901 atcctggcat gctgcggtcc atggggtcat
agagagtcag acacaactga gcgactgaca
45961 gaactgaagc aactggcaag ccggagggta
ggtgccggct gcgatgagcg ggaacgtgca
46021 acctgccacg tggagctctt cctacaccca
gagtcctgac ggcactggga ccctagccct
46081 ccacggcctc tccagggcca cgagacaccc
tcacagagca gagaagcgga acagagctgg
46141 tgtgcagaac caggccccgg gggtggggcg
gggctggtgg gcaggcttta gtgagaagcc
46201 cttgagccct ggaaccagag cagagcagaa
cagttggcag aggcccccct gggagaggcc
46261 ccccgcccag agtaccggcc ctgggccctg
ggggagaggg cggtgctggg ggcagggaca
46321 gaaggcccag gcagaggatg ggccccgtgg
gacggggcgc accaaaacag cccctgccag
46381 caaggggaag ctggggcact ttcgaccccc
tccaaggagg agcccacacc agcgcatctg
46441 cccaaggtgc ccttggccct gggggcacat
gaggcccagg ccaggccagg gggcccatga
46501 ggcccccagg ggtcagtgca gtgtccccag
gcagccctgg cctctcatcc tgctgggcct
46561 ggcctcttat cccgtgggcg cccacggcct
gctgcccccg acagcggcgc ctcagagcac
46621 agccccccgc atggaagccc cgtcaggaaa
gagcccttgg agcctgcagg acaggtaagg
46681 gccgagggag tcatggtgca gggaagtggg
gcttcccttc gatgggaccc aggggtgaat
46741 gaccgcaggg gcggggaacg agaagggaaa
ccagctggag agaaggagcc tgggcagacg
46801 tggctgcacg cacagcgctg accctgggcc
cagtgtgcct ttgtgttggg ttttattttt
46861 aattttgtat tgagatgcta tttatctcgt
ggagcttttg ccgccctgag attttgtacc
46921 cgtggctggt gtccctcttg cctcaccccg
gcctctgtag cagggcagac acggcgcaac
46981 ggggcagggc gtgcccagga ggcactgtca
ttttgggggc agcggcccca caaggcaggt
47041 ctgccttcct cccctcttac aggcagcgac
agaggtccag agaggtgagg caagctgccc
47101 aatgtcacac agcacacggg cgcagtccca
ggactgtaga aatcccggga ctagacaggc
47161 accagagtgt cctgtgtttt taaaaaaacg
gcccaagaga agaggcaagt ctgcaaggcg
47221 tcccgggaag gcagcagggg cttggctcgg
tctcccccaa ggaggccagc tcctcagcga
47281 ggttcctaag tgtctaacgg agccaagcct
gaaccaaggg ggtcacgtgc agctatggga
47341 cactgacctg ggatggggga gctccaggca
aagggagtag ggaggccaag gaggagagag
47401 gggtgcacag gcctgcaggg agcttccaga
gctggggaaa acggggttca gaccacgggg
47461 tcatgtccac ccctccttta tcctgggatc
cggggcaggt attgagggat ttatgtgcgg
47521 ggctgtcagg gtccagttcg tgctgtggaa
aaattgtttc agatcagaga ccagcgtgag
47581 gtcaggttag aggatggaga agaagctgtg
aaaaggtgat ggagagcggg gggacggtcc
47641 tcggtgatca ggcaccgaga tcgcccatgg
aatccgcagg cgaatttaca gtgacgtcgt
47701 cagagggctg tcggggagga acaggcactg
tcatgaactg gctacaaaaa tctaaaatgt
47761 gcaccctttt cggcaatatg cagcaagtca
taaaagaaaa cgcatttctt taaaattgcg
47821 taattccgct tttaggaatt catctggggg
cgggggaaca atcaaaaaga tgtgaccaaa
47881 ggtttacaag ccaggaagtc aactcgttaa
tgatgggaga aaaccggaaa taacctgaat
47941 atccaacaga aagggtgtga tgaagcgcag
catggcacat ccaccgcaag gaatcctaac
48001 acaaacttcc aaaacaatat ttctgacgtt
gggtttttaa agcatgcgtg cactttcaaa
48061 agcttgtcag aaaacataga aatatgccaa
taatgtgtct ctagccaaat tttttaattt
48121 ttgctttata attttataaa gttataattg
tatgaaatat aatgataaaa ttataaacta
48181 taaaaaagtt atgaaaatgt tcacaagaag
atatacatgt aattttatct tctacaatac
48241 tttttaatac cagaataacg tgcttttaaa
aaagattgag cacagaagcg tataaagtaa
48301 aaattgagag tttctgctca ccaaccacac
gtcttacctt aaaacccatt ctccagcgag
48361 agacagtgtc atgtgggtct gtacacttct
ggcctttctc ctaggcatgt atgtccctga
48421 aaactcacac acacggctaa tggtgctggg
attttagttt tcaaaacgga ctcatactct
48481 gcctatgagc ctgcaactat ttattcagtc
tgttgagatt ttctatatca gcccacatgg
48541 atcccgcatg ttctctgaat ggctctgtat
gaattcaaag tttggaagaa gcagcgtgtc
48601 tttaatcatt cgcctattaa tggacgtttg
gggtgtttcc actacaaaan nnnnnnnnnn
48661 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
48721 nnnnnnnnnn nnnnnnnnnn nnnnnnnnng
atacaattcg agctcggtac cctggcttga
48781 actatatgaa cagagaacga tgagaacagt
ttctcaaact tggaacagtt aacattttgg
48841 gctaaatgat tcttttttgt gtggagttgg
cctatgaata gaggatatta gcagcatcat
48901 ttaaccttta ctcactacat acctgtagca
actacatcct ctccatttgt gtcaatcaaa
48961 actgtctccg gacatggaca agtgtgcccc
tgggatgggt ggaatgacct tttgttaaga
49021 accactgggt cagagattca tagatttttg
tcttgttgac tttttaaaaa tacatcttgg
49081 tttttatttt attggtttct gctcttatct
ttatgattac cttcctttta cttggggctt
49141 ccctgataga ttttcccttc tggctcagct
ggtaaagaat ctgcctgcaa tgcaggagac
49201 ctgggttcag tccctgggtt gggaggatcc
cctggagagg agaagggcta cccaccccag
49261 tattctggcc tggaggattc catggagtgt
atagtccatg gggtcgcaga gtcggacatg
49321 actgagtgac tttcacacac acatatgtcc
ctggtagctc agctagtaaa gaatcccacc
49381 cgcaatgcag gagaccccgg tccaattcct
gggtccggaa gattcccttt tgtttactcc
49441 ataagatctt atctggggac aaaactaaca
gctatgccag accttctgga catcagggaa
49501 cgtgaggggt gtggactgga cagatgtgtg
tgttctccca aacacaaaca tacatctgta
49561 tacatgtaca tggagagagg gggagggagg
ctgtgagtct ccaggggacc gtgcaaccat
49621 gtgacattca tggaggcgtt tgcgggtgat
cactacacag tttcttcttc tggtttcttg
49681 gtcaattgac ttcacaattc caattcctat
acttcatttt agactgaggg aattttacac
49741 tattgtaaga catatgtata catgagttat
gttcagcgcc atgagggctc attttgtgtg
49801 tccactttgc ctggaaacaa agttggactg
atttacttct aggggtgcct gggggtgttt
49861 ctggaggaca ggagcatttg aacccaaggg
ctcggtgaag catgagcctc tctgcaggtg
49921 gacccaggag gaacgcaagg ccgaggaagg
cagactctcc tcctccctaa cccgaggtct
49981 ctgctcagaa aagggacaat ataatgacta
gaagaaaaga aagaacatca gctgtgggag
50041 gtttgttctc tggagcagat tcacacgttg
aggctcatgt gcaggaattc taggtgaaac
50101 agagcagtca cccatgtgtg ttggaaaatt
ttaaattaca tttgcagtta cgactttgtt
50161 taagccagac agggtagcac agcaaagtca
ccatgtggtc acctgtgttt tgtaaaggag
50221 agagaacttg ctggcacatt caggaaaggc
cgtgtctcag ctttggaggc acactgagag
50281 gccacaagca gatggtgagg accagggtct
cgggcagagg gatcaattca ctgctcttca
50341 cttttgccac atctgtgtgc tgtccatcct
ggccagagta gttcagtctt cagatgctgg
50401 agttcccatt ggtagaaatc caatctgggt
catttttaaa cctctcttgg ttctacttaa
50461 tggttttaaa atctctttgg ctcaagaaaa
aaaataaaca taattttaaa gggtggtttg
50521 gggccttgac tataaagtac attatctggg
ccatttcaga gcatggttga attaatacat
50581 ttcgtgctta ctatagctcc tattttcttg
attctttaca ggtaattttt gttaggaatc
50641 gggtactgtg aatattttct tgttgaatac
gggatctttg tattttttcc taattttttt
50701 ttttttttca tttttggttt taccttcagg
aaagtcacta ggactcagga aagtcctttg
50761 tccgcctgtt atttcagtct cttacctggg
gccagggcag cgtttcctct gggctaagtt
50821 tccccacaac cggggccagt tctcctcact
cttcaccctg aggccttaat gaggagctcc
50881 cctgcgtctg agcagccggc cctcctgtga
cgtgcgtgtg tctctggcca tcggcgtccg
50941 gtgtccttgg aggttccgtc ctcccttcgc
tcactgtgcc ccgcactcga gctctcaggc
51001 tccaagcagt gtccgcagtg tgcagaccct
ctgtgtagct ctctcctcct caggactctt
51061 ccctctagat gtgtgttttc ttttggctcc
ttggacctcc gctctgaacg caggcctggt
51121 gctgagtgtg atctctggag ggaagcctgg
gaggctggac gggtccgccc tgcggtgtgg
51181 tgacaggtgt gggctcgggg cggggcctgc
acgtcgtcct gacccgagcc gggactgggc
51241 tccgggcctc aggcatcact gactgaatct
ccctcacaga ggggtcaggg cctgggcggg
51301 ggaaccgtct ctgcaatgac agcccctccc
agggagggca cagcggggag ctgccgaggc
51361 tccagcccta gtgggaggtc ggggagccca
ggggagcggc ctgacggccc cacaccggcc
51421 cagggctggt tcgttctgtt tctcgagctc
aacagaagct ccgaggagct gggcagttct
51481 ctgaattcgt cccggagttt tggctgctga
gtgtcctgtc agcaccgtat ggacatccag
51541 agtccattag cagtggtctc tgtccctctg
tctgtccttc atcaggctct ttgtccaggt
51601 caccacacgg ccaacaccag gacagtctgg
tcccgccagc ccatcgtccc tgcggacgcc
51661 cctgtgcagc ctgccgaagg gccgggaggc
cgggggaacc gggccaggcc tgtccctgct
51721 gtgtccacag tcctcccggg gctggaggag
agcgtgagca ggacgggagg gtttgtgtct
51781 cacttccccg tctgtctgtg tcactgtgag
gattatcact gctgtcagct gactgacagt
51841 aatagtcggc ctcgtcctcg gtctgggccc
cgctgatggt cagcgtggct gttttgcctg
51901 agctggagcc agagaaccgg tcagagatcc
ctgagggccg ctcactatct ttataaatga
51961 ccctcacagg gccctggccc ggcttctgct
ggtaccactg agtatattgt tcatccagca
52021 ggtcccccga gcaggtgatc ttggccgtct
gtcccaaggc cactgacact gaagtcggct
52081 gggtcagttc ataggagacc acggagccgg
aagagaggag ggagagggga tgagaaagaa
52141 ggaccccttc cccgggcatc ccaccctgag
gcggtgcctg gagtgcactc tgggttcggg
52201 gcaggcccca gcccagggtc ctgtgtggcc
ggagcctgcg ggcagggccg gggggccgca
52261 cctgtgcaga gagtgaggag gggcagcagg
agaggggtcc aggccatggt ggatgcgccc
52321 cgagctctgc ctctgagccc gcagcagcac
tgggctctct gagacccttt attccctctc
52381 agagctttgc aggggccagt gagggtttgg
gtttatgcaa attcaccccc gggggcccct
52441 cactgagagg cggggtcacc acaccatcag
ccctgtctgt ccccagcttc ctcctcggct
52501 tctcacgtct gcacatcaga cttgtcctca
gggactgagg tcactgtcac cttccccgtc
52561 tctgaccaca tgaccactgt cccaagcccc
ccggcctgtg gtctcccctg gactccccag
52621 tggggcggtc agcctggcag catcctggcc
gtggactgag gcatggtgct ctggggttca
52681 ctgtggatgt gaccctcaga ggtggtcact
agtcctgagg ggatggcctg tccagtcctg
52741 acttcctgcc aagcgctgct ccttggacag
ctgtggaccc gcagggctgc ttcccctgaa
52801 gctccccttg ggcagcccag cctctgacct
gctgctcctg gccacgctct gctgccccct
52861 gctggtggag gacgatcagg gcagcggctc
ccctcccgca ggtcacccca aggcccctgt
52921 cagcagagag ggtgtggacc tgggagtcca
gccctgcctg gcccagcact agaggccgcc
52981 tgcaccggga agttgctgtg ctgtgaccct
gtctcagggc ggagatgacc gcgccgtccc
53041 tttggtttgt tagtggagtg gagggtccgg
gatgactcta gccgtaaact gccaggctcc
53101 gtagcaacct gtgcgatgcc cccggggacc
cagggctcct tgtgctggtg taccaaggtt
53161 ggcactagtc ccaccccagg agggcacttc
gctgatggtg ttcctggcag ttgagtgcat
53221 ttgagaactt acatcatttt catcatcaca
tcttcatcac cagtatcatc accaccatca
53281 ccattccatc atctcttctc tctttttctt
ttatgtcatc tcacaatctc acacccctca
53341 agagtttgca ttggtagcat atttacttta
gcacagtgtg cctcttttta ggaaactggg
53401 ggtctcctgc tgatacccct gggaacccat
ccagaaattg tactgatggc tgaacccctg
53461 cgtttggatt cttgccgagg agaccctagg
gcctcaaagt tctctgaatc actcccatag
53521 ttaacaacac tcattgggcc tttttatact
ttaatttgga aaaatatcct tgaagttagt
53581 acctacctcc acattttaca gcaggtaaag
ctgcttcgca tttgagagca agtccccaga
53641 tcaataaaga gaatgggatg aacccaggat
ggggcccagg ggtcctggat tcagactcca
53701 gccgtttagg acagaacttg actaggtacg
aagtgagcgg ggtggggggg caatctgggg
53761 ggaactgtgg cacccccagg gctcggggcc
atccccacca catcctggct ttcatcagta
53821 gccccctcag cctgcgtgtg gaggaggcca
gggaagctat ggtccaggtc atgctggaga
53881 atatgtgggg ctggggtgct gctgggtcct
aggggtctgg ccaggtcctg ctgcctctgc
53941 tgggcagtga taattggtcc tcatcctcct
gagaagtcac gagtgacagg tgtctcatgg
54001 ccaagctatt ggaggaggca gtgagcactc
ccacccctgc agacatctct ggaggcatca
54061 gtggtcctgt aggtggtcct ggggcttggg
ccgggggacc tgagattcag ccattgactc
54121 tcagaggggc cagctgtggg tgcagcggca
gggctgggcg gtggaggata cctcaccaga
54181 gccaaaataa gagatcaccc aacggataga
aattgactca caccctttgg tctggcacat
54241 tctgtcttga aatttcttgt ggacaggaca
cagtccctgg ataaagggat ttctatcttg
54301 cgtgtgcaat agagctgtcg acacgcttgg
ctgggacatg taatcctttg aacatggtat
54361 taaattctgt tcactaacat ctgaaaggat
ttttgcatca ataaacctaa ggtatattgc
54421 cctgtcattt ccttgtcttg tagtgtctct
gagtaggctg gaaggggtaa ccagcttcac
54481 aaatcgagtt aggaaattcc cttattcttc
cactgtctaa tagactttca taagattagt
54541 gttaattcct ctttaaatcg ctgctataat
catcactgtg gccaccggta ctgaattttt
54601 tgttaggatg atttttaaac aagcatttta
atgatttttc cttttatttt cggctgtgct
54661 gggtctcgtt gctgtgtgcc ggcgttctct
cgctgtggcc agtgggggcg ctgctctcgc
54721 gttgcgaagc tcgggcttct gactgcagtg
gcttctctcg ttgcagagcg cgggctccag
54781 ggcgctcagg ctcgcgtggc tgcggcacgt
gggctcagta gtcctggggc acaggtgcag
54841 cagcctctca ggacgttttg ttcccagatg
gtgggtcggt cgaaccggtg tcccctgcgt
54901 tgcaaggtgg attcttcacc gctggaccac
cagcgacgtt ccctggaggt ttttaattat
54961 ggatttaagc tctcattaga tgtctcctca
catttcctat ttctttttga gtcagtttga
55021 tactttgttt gtgtctgtaa gtttgtccat
tttatccaag tcatctaatg tgttgataga
55081 caattattgg ttagtcatct aattgttggt
ttacaatttt gagagcattg tcctgcaatt
55141 ccttctatct gcaagattgg taataatatc
tcccaagagg agtcacaaac tgaaatgaga
55201 ttanatacag gctttttttt taaaagaatg
aacttatgtt gttgcctttc tcatagatct
55261 tacttcttag catgactgta cttactgact
ggggcgtttt catgtctgtg tggagagcta
55321 ccattagtac ttcttatcgc ccaaagacat
cgggctcctg ggcacagtga aaacactcct
55381 ttctgtggct attttgcaaa atatggccta
gcctagcgtc ataagggatc acagctgaca
55441 actgctggaa cagagggaca tgcgaagcaa
cgtgagggct ggaacctgga gggtcctctc
55501 tggggacagt ttaaccagct ataatggaca
ttccagcatc tgggacatgg agctgtgaac
55561 tggaccaatg actgtcattt ttggaagaga
aatcccagga gagaagggtc caggggaatc
55621 tgaggccgca tgcagtgcct caggacaggg
gacaccttct ccagcagagc aggggggccc
55681 gcccaggccg cctgcagtga ttccaccagg
aggagatgca tccctgcaga cctctgacag
55741 cacggccctc tcctgagaca cagggtcaca
cccggggccc tggaaccctt tgagacccta
55801 aacctttcct ttcctgacca ccctgacagc
agtctagctc agaacagaca tcttcatttt
55861 cagcaggaaa atccttttcc tcgtttgagg
gagcgactgg caccggagga gctgagtctt
55921 ttaaacacag gctgcctgaa cctcagggat
gacctgcagc tgctcagagg aggctggagt
55981 gtgatagctc actctaatgt tactaaaagg
aacatattgg acaccccctc tctgaaaaat
56041 ttccctcctg cctctcatct cttagtccac
tttatcgccg ttttactgct tttctattta
56101 ctactcttaa cgccaaccta tcttatttcc
cctcccagtt taacacggtt ttccctccac
56161 ccgctctctt taatctcaga agattctgcc
tattcctcta ttatcacacg cccctacttt
56221 ttattttttt tcttacccgc cttttattcc
ctcccctcct cactctctat ttaattacat
56281 cttaactaca ccgcctgcgc tatcttcgaa
tgtatccaaa tatttttccc ttatataaca
56341 ctccaggccg agcggctaac ttattataat
ttctttatag cgcctaccta atttcccttt
56401 atttctaatt atctatatat acccatgcaa
tttcgnnnnn nnnnnnnnnn nnnnnnnnnn
56461 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
56521 nnnnnnnnnn nnnnntgggt gtacgttata
gagtaaacgc gcatgaagaa gtgggtcaat
56581 ctatggctgt gagaggcaga aaataatatt
atcatatata atttatgtta taacacactg
56641 aggtggtggg ctcgtagaat agtgcggacg
gggagaaagg tgggaaggag aagacacaag
56701 agagagatgt tcgcctcgcg ggatggatgg
gcggagggat agaagaataa aaagaggaga
56761 ggtatagagg ggggcggggg gcataacgtg
tggtggggta aatagtaggc ggtaattatg
56821 aaaaaaagaa agacgggggg ggcggtaaca
tagaatacgc aaaaaagtca tatactgaac
56881 ggggattagg gagaagaggt ggggggcgtg
gggtgcgggg gaaagaggtg tgtgtataat
56941 tggtatggag tgttatttga atatatatta
atgtaatagg gagtgtaatt agtgaaattg
57001 tgggagtatt atattggggt gtgggggaca
tggcaaagtg atgatcggga taaaaaaagt
57061 aaagcaagag gggaggggaa aataaggggg
gggagaaggt cgaagaaaat aagaggaaga
57121 agaaagaacg ggggtggcgg gcgggggggg
cgccgctctt gtatctggct tttttgttgt
57181 gtcggtggtt gttcgcgtct tgttgggtcc
ggggcgggtg tgcggaaaaa aaaaaaggcg
57241 ggaggcccgg ggcccggtca cgcggcaccc
ccgcgggtcc ctggcttctc cttcggcagc
57301 tccgggggtc ggtgagcctg cgccctccgg
gccgccggcc cgagctgtgt gcgccctgga
57361 gaatcggagc cgctgtggca gcacgcggag
ggcgcgcgca agggccacgg gacggacctt
57421 caaaggccgc ggcggagcgc ggcaagccga
accgagggcg gtctggcgat cggccgagcc
57481 ctgctccccc ctcccgcgtg gccccagggt
cgcgggtgga ctggggcggg tacaaagcac
57541 tcacccccgt cccgccccca gaaagcctcc
caggactctc acagagcacc cgccaggagg
57601 catccggttc ccccctcggc tcagttcagt
tgctcagtcg tgtccaactc tttgcgaccc
57661 catggactgc agcaccccaa gcttccctgt
ccatcaccaa ctcccggagt ttactcaaac
57721 tcatctattg agtcagtgat gccatccaac
cgtctcatcc tctgttgtcc ccttctcctc
57781 ccactttcaa tctttcccag catcagggtc
ttttcttatg agccagttct tcacatcagg
57841 tggtcagagt attggagttt cagcttcagc
atcagtcctt ccaatgaaca ctcaggactg
57901 atttccttta ggatggactg gctggatgca
gcgccagaca ccgaccgcgt ttaccccgtg
57961 tgtcctttcc aatggctgtc ccctgcgggc
ctaggggcat tggtgcgggt ttgaatcctg
58021 tggccttgaa ttttacgcct tagttccagg
tccagggcag ggccatccgg attcaggatg
58081 cttcccagcc cttcaggaat ggcaggtttt
catggtcctt tctgagtgag ttctgagtgg
58141 tcatattggt gcccttggca gggagggctc
ctgactttcc tatcttcaca tcactgtccc
58201 caacccccaa gagaggcctc ttggcccagg
gactgcaggg aggatgaagt caggagcaga
58261 agcatggggt agggggctca ggtgggcaga
ggaggcccct ctgtgaggag gaacggcaag
58321 cgaggaggga acaggggcac cggcagtgcc
tggcaagctg ggtgatgtca cgactacgtc
58381 ccgaccacac agtcctctca gccagcccga
gaagcagggc cctcccctga cccccatctg
58441 ggcctgggct tcagttttct cctccctgca
atggggtgac tgtttgcctc caggagaggg
58501 gagcatgtaa aggtggccac tctcttctgg
cagacatgcc aggcctgggc cagcctccac
58561 ccctttgctc ctgcagcccc tgctgacctg
ctcctgtttg ccacaccggc ccctcctggg
58621 ctgatcaggg cccccctcct gcaggaagcc
ctctgggaca agcccagctt gctgtaactg
58681 tggctttcca ctgtgacctg caacgtggga
ggctgttact taaaactccc atgactggtg
58741 gattgccggt ccccagaaca aggccacgca
tccctggagg ccctcgagac catttaaggt
58801 agttaaacat ttttacttta tgcattttca
tgtgtatcag aaagaaaaaa aatgtatcat
58861 cagttcatca aatccatgat ttcttgacca
atattgctaa gatgaggctg aaataggcat
58921 ttccattttt aaaaaactga atcactctga
agaaacagat ggcaggcttc cctggtggtc
58981 cggtggttaa cagtccatgc ttccagtgct
gggggcatgg gttcgatccc tgaaaatttt
59041 aaaaaggaag aaaaagatgg ctcccccgtc
cctgggattc tccaggcaag aacactggag
59101 tgggttgcca tttccttctc cagtgcatga
aagggaaaag ggaaagtgaa gtcgctcagt
59161 cgtgtgcgac tcttagcaac cccatggact
gcagcctacc agactcctcc gtccatggga
59221 ttttccaggc aagagtactg gagtggggtg
ccattgcctt ctccaggcaa acggcctgct
59281 actgctactg ctgctaaatc gcttcagtcg
tgtccaactc tgtgcgaccc catagacggc
59341 agcccaccag gctcccccgt ccctgggatt
ctccaggcaa gaacactgga gtggggtgcc
59401 attgccttca gcctgctgct gctgctgcta
agtcgcttca gtcgtgtccg actctgtgtg
59461 accgcataga cggcagccca ccaggctccc
ccgtccctgg gattctccag gcaagaacac
59521 tggagtgggt tgccatttcc ttctccaatg
catgaaagtg aaaagttaaa gtgaaattgc
59581 tcagtcgtgt ccgactctta gtgacccaat
ggactgcagc ctaccagggt cctccatcca
59641 tgggattttc caggcaagag tactggagtg
gggtgccatt cggcctaggg agtgagaaat
59701 cacggctgtc ttccctcttc tcgccctcta
ggggtctctg tggagcctcc ctggagaggc
59761 cgcggcggct ccggggactg gagggggagg
gggggttgag tcagccggtg gccctcccct
59821 cgctgcccgt ctcctccctt tttaggcaca
agctgggcgc cctttttagg cgcagcctca
59881 ccctgcgggc cactgcccgt gtttcggctc
cccggagata aaacagattg cctgcacccc
59941 gggtcatcac aaggattgta tgaccgtttc
ccagtgtgct caccaccctc cctctgattc
60001 tcagagacgc gccctcgcct caggaggctg
ctcatcccag gccaaggggc ggcgtggggt
60061 ccccagcgcc ccgcacagac actgccttct
gaccacctcc tcccaacagc ttacctgcca
60121 agaaggcctc ctgacccctc atcctgcccg
gtggtttgga gaaagcctca tctggcccct
60181 ccttctcggg gcctcagttt ccccctctgt
gaactggcgg attctgccaa gctgacgtcc
60241 tggccagccg cctccccgtg gccagtgtcc
cccgggacac agctgaatgt ccctgctcgg
60301 gatgcacctt cccaagttgg cctgtcagga
ggcgggggcg agcagggaaa cccgactcct
60361 ctcagacggc ccatcgcatt ggggacgctg
aggcccggag cagcggcacc ctcctggcca
60421 gggtcattct cccgccccgc cccgtccctc
cgggcctccg agaccgcagc ccggcccgcc
60481 ccgggaagga ccggatccgc gggccgggcc
accccccttc cctggccgcg ggcgcggggc
60541 gagtgcagaa caaaagcggg gggcggggcc
ggggcggggg cggggcggag gatataaggg
60601 gcggcggccg gcggcacccc agcaggccct
gcacccccgg gggggatggc tcgggccgcc
60661 ggcctccgcg gggcggcctc gcgcgccttt
ttgtttttgg tgagggtgat gggggcggtc
60721 gcggggtact attttttcat ttataattgg
gtattagcta gcgagtggaa ccacaccctt
60781 attccactat agccaatttt tgcgggggca
tcttacatta cagactcgcc cgcctcttat
60841 ttcggtacag catatcagat cgtctcttta
ctcagacact agtgattatt gtctatagta
60901 cacaaaaaga acggttgtgt cggcgtaatg
gttgcatttt ccctcctcgt ttctcctgac
60961 cacctcaatt acaccaacac tctactattt
aaatcacgta ttgtacgcca ccctccgccc
61021 gcgaactaaa agaatgtgca gatattctga
agataaaatc gttcattgtt acgccccgcg
61081 cgcttcgcgt atattactct tagaacttct
tattcgcccg agcagttatt caccccccgc
61141 aactagatgt cgccttaata tttgttctaa
ccgttttgga ttctaacgat aggcgggaaa
61201 ggtagacatt cgaccgctac gacaactaaa
atcgacgagc acaggctatt tatatcgcga
61261 ccacacgcgc gcggtataca naccgtaaaa
ttatctaaca tcgagagtaa gggcacagag
61321 cgaaatacaa gcggcgtggt gggaggtgtg
tctgtagtga attcgcacct cgcgccgccg
61381 cctctgtgcg tcgnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
61441 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnngatataa
61501 tattaataaa cagcggatag atgtgtgtaa
gggaggaggt gcataagaga ttaaagagag
61561 gcgggcggag agaaatagag tagaggagga
tgagagaaaa aagaaagcaa gcgtaggtac
61621 aacggcgggt gggtagtatg ataaagtgag
tgtatatatt tgagtaaagg aagggtagat
61681 ggagtataaa gaagtaagga gaggagaggg
cggcggagag agagagtgca aagaaaataa
61741 gtgggcaaag gcggggtggg tgagaagcag
tagaagagaa gatagagaag ggggaaaaag
61801 aggaaaatga ggattagaac aagtaggaca
ggatagatgt gaaaaatgag atcaggtcaa
61861 ggtggagaaa aagtagaaac tggggcgtga
ttgtaaaaaa gggaggccgc gatggggcag
61921 caccataagc gaagagatga attaatgaaa
gcaaggcagg gagaatcaaa tgagttgggt
61981 ggaggaagga ggctgtgact tccttcgctg
ccggaaagag aactagaata gcctcgggct
62041 gtggggggag gtaaagataa agtgacttct
gggccctggg ggaggcccag gagtttctac
62101 cgagctgagc tgggtgcctc tcccaaatgc
ccaaccccct gagagtcgac gggagagcac
62161 agcctggcca aacctgggca gggcacacgt
gtccttcacc ccacagtggt cacgagccca
62221 gcgtggtccc tgcgtctggc gggaaacaca
gaccctcaca ccccacacaa gggtccggcc
62281 gctttcaaat aacagcagcc gtgccctctg
ggccggtgac ccggacacag agagatgaag
62341 tccgcatctc tcagagtgcg ctgtcctccg
cccggtcagg cccgggtccc ctgcttctct
62401 gaggtcacca ggagggattg catgtgggtc
tcagggacac aggttcagtg atgtgacaga
62461 gggtagtggg tcccagcagg gccggtcttt
ggacccgttt ttctgaaaag ccagttggcg
62521 acctggggtc acagcaaagc tgatcctgtt
tggccaggag tctcccagtg acggcctccc
62581 ccagaacatc gggcccagtg ggggctccag
ggggtagact tgcctcccag ctcacgcccg
62641 tgtcttgaca agtccatgat ttggtaaaat
taatttgtgt tggatggagt tgatttagtg
62701 gtgtgtgagt ttctgtggcg cagcaaagtc
aatcagttac gcatacacat gtatccagct
62761 cttcctacga ttctgttccc atataggtca
ttatggggtg tcaggtagag cttcctgtgc
62821 tacgcagtac ggccttattc agttcagctc
agtcgtgtcc gactccttgt gaccccatgg
62881 actgcagcac gccaggctcc cctgtccatc
accaactcct ggagcttatt caaactcatg
62941 tccatcgagc cggtgatgcc atccaaccat
ctcatcctct gtcgttccct ctcctcctgc
63001 cttcagtctt tcccagcacc ccctagagaa
gggaatggca aaccacttcg gtattcttgc
63061 cctgagaacc ccatgaacag tacggaaagt
ccttattagt tttctatttt atatatagca
63121 gtgcacacgt gtcagcccca atctcgcaat
ttatcacccc cctccgccgc cgattggtag
63181 tcatgtttgt tttctacatc tgcgactcta
tttctgtttt gtaaacaagt tcatttacac
63241 cactttttta gattctgcac atacgtggca
agcccacagc aaacatgctc aatggtgaaa
63301 gactgaaagc atttcctcta agatcaaaaa
caagacgagg atgtccactc actccgtttt
63361 tactcaacac agccctgaac gtcctagcca
tggcaatcag agaagagaaa gaaattaagg
63421 aatccaaatt ggaaaagaag aagtaaaact
cactctttgc aaatgacatg acacttatac
63481 ccagaaaatc ctagagatgc taccagataa
ctattagagc tcatcagtga atttgttgca
63541 ggatacaaaa ttaatacaca gaaatctcct
gcattcctat agactgacaa caaaagatct
63601 gagagagaaa ttaaggaaac catcccacgg
catgaaaaag agtaaaatac ctaggaataa
63661 agctacctaa agaggcaaaa gacctgtact
cagaaaacta taaaatactg acaaaggaaa
63721 tcagacgaca cagagagaga gagataccac
gctcttggat gagaagaatc gatagtgtga
63781 caatgactat actacccaga gaaacataca
gattcagtac aacccctatc aaattcccaa
63841 tggcattttt cacagaatca gaattagaac
aaaaagtttt acaagtttca gggaaacaag
63901 aaagatccta aagagccaga gcaatcttga
gaaagaaaaa tggagctgga agagtcaggc
63961 tccctgagtt ctgactgtgt atacaaagct
ggcatgattt ttaacagcag gggtgtaaat
64021 gaacttgttc acaaaacaga tggtggggtg
ggcttccctg gtggctcagc tggtaaagaa
64081 tcctcctgca acgcaggaga cctgggttcg
atccctaggc tgggaagatc ccctggagaa
64141 gggaaaggct acccactcca gtattctggc
ctggaaaatt ccaaggacca tatagtccat
64201 gggtttgcaa agagtcggac acgactgagc
gacttccaat cctggaaacg tcccattgtg
64261 gacggtgaac tggggttgtc caagctcagg
gtaaccgttt gctgagtgac tgacactcct
64321 tctcatgggt taaaatgtgg ggcccaaggc
caggaccaga ccccgcagtc agccaggcag
64381 accctgtgca gccccagcga gtgtgtggcc
gccgtggagt tcctggcccc catgggcctc
64441 gactggagcc cctggagtga gcccattccc
tcccagcccg tgagaggctg ggtgcagccc
64501 taaccatttc ccacccagtg acagatccgc
ctgtgtggaa acctgctctt gtccccaggg
64561 aacctggcag gactcaggga gaatgtctca
gggcggccac agatcagggg ctgggggggc
64621 agggctgggt ccagcagagg ccctgtgccc
actccccgga aagagcagct gatggtcagc
64681 atgacccacc agggcaccga cgcgtgcttg
cacacaggcc gccccctcat ggtgacactc
64741 ttttcctgtg gccacatctc gccccctcag
gtccctcctg ctccccagct cctggcctgg
64801 gaacctcttc cccgccccgg ggacgtcagg
gctggtgtcc actgagcatc ccatgcccgg
64861 gactgtgctg atcaccagca cctgcacccc
ctctcgggtc tcaccaggat gggcaactcc
64921 tgcccatcca gcacccagcc tcctgggtac
acatcggggg aggagggaga agcctgggcc
64981 agacccccag tgggctccct aaggaggaca
gaaaggctgc cgtgggccag ccgagagcag
65041 ctctctgaga gacgtgggac cccagaccac
ctgtgagcca cccgcagtgt ctctgctcac
65101 acgggccacc agcccagcac tagtgtggac
gagggtgagt gggtgaggcc caggtgcacc
65161 agggcaagtg ggtgaggccc gagtggacag
ggtgagtggg tgaggcccag gtagaccagg
65221 gcccatgtgg gtgaggcccg ggtggaccag
agtgagcggg tgaggcccag gtggacaggg
65281 cgagcgggtg aggcccaggt ggacagggcg
agcgggtgag gcccgggtgg acagggcgag
65341 cgggtgaggc ccgggtggac agggcgagcg
ggtgaggccc gggtggacag ggcgagtggg
65401 tgaggcccgg gtggaccagg gcgagtgggt
gaggcccggg tggacagggc gagtgggtga
65461 ggcccgggtg gaccagggcg agtgggtgag
gcccaggtgg acagggtgag tgggtgaggc
65521 ccaggtagac cagggcccag agcaaagccc
cggctcagca gtgatttcct gagcgcccac
65581 tgcttgcagg gacctcagcg atggtaaggc
agccctgttg ggggctcccg actggggaca
65641 gcatgcagag agcgagtggt cccctggaga
aacagccagg gcatggccgg gcgccctgcc
65701 aggctgcccc aggggccaca gctgagcccc
gaggcggcca ggggccggga cagccctgat
65761 tctgggttgg gggctggggg ccagagtgcc
ctctgtgcag ctgggccggt gacagtggcg
65821 cctcgctccc tgggggcccg ggagggacgg
tcaggtggaa aatggacgtt tgcgggtctc
65881 tggggttgac agttgtcgcc attggcactg
ggctgttggg gcccagcagc ctcaggccag
65941 cacccccggg gctccccacg ggccccgcac
cctcacccca cgcagctggc ctggcgaaac
66001 caagaggccc tgacgcccga aatagccagg
aaaccccgac cgaccgccca gccctggcag
66061 caggtgcctc cctctccccg gggtgggggg
aggggttgct ccagttctgg aagcttccac
66121 cagcccagct ggagaaaggc ccacatccca
gcacccaggc cgcccaggcc cctgtgtcca
66181 ggcctggccg cctgagacca cgtccgtcag
aagcggcatc tcttatccca cgatcctgtg
66241 tctgggatcc tggaggtcat ggcccctctc
ggggccccag gagcccatct aagtgccagg
66301 ctcagagctg aggctgccgc gggacacaga
ggagctgggg ctggcctagg gcaccgcggt
66361 cacacttccc ctgccgcccc tcacttggga
ctctttgcgg ggagggactg agccaagtat
66421 ggggatgggg agaaaaatgg ggaccctcac
gatcactgcc ctgggagccc tggtgcgtct
66481 ggagtaacaa tgcggtgact cgaagcacag
ctgttcccca cgaggcctca cagggtcctt
66541 ctccagggga cgggacctca gatggccagt
cactcatcca ttccccacga ggcctcacag
66601 ggtccttctc caggggacgg gacctcagat
ggccagtcac tcatccattc cccatgaggt
66661 ctcacagggt ccttctccag gggacgggac
ctcagatggc cagtcactca tccattcccc
66721 acgaggcctc acagggtcct tctccagggg
acgggacccc agatgggcca gtcactcatc
66781 catccgtctg tgcacccatc cgtccaacca
tcacccttcc ctccatccat ctgaaagctt
66841 ccctgaggcc tccccgggga cccagcctgc
atgcggccct cagctgctca tcccaggcca
66901 gtcaggcccg gcacagtcaa ggccaaagtc
agacctggaa ggtgcctgct tcaccacggg
66961 aggagggggg ctgtggacac agggcgcccc
atgccctgcc cagcctgccc cccgtgctcg
67021 gccgagatgc tgagggcaac gggggggcag
gaggtgggac agacaggcca gcgtgggggg
67081 ccagctgccg cctggctgcg ggtgagcaga
ctgcccccct caccccaggt acaggtctcc
67141 ctgatgtccc ctgccctccc tgcctccctg
tccggctcca atcagagagg tcccggcatt
67201 ccagggctcc gtggtcctca tgggaataaa
aggtggggaa caagtacccg gcacgctctc
67261 ctgagcccac ccccaaacac acacaaaaaa
atccctccac cggtgggact tcaccagctc
67321 gttctcaggg gagctgccag ggggtccccc
agccccagga agccaggggc caggcctgca
67381 agtccacagc cataacacca tgtcagctga
cacagagaga cagtgtctgg tggacaggtg
67441 cccccacctg cgagcctgga gagtgtggcc
ctcgcctgcc ccagccgcgg tcagtcggct
67501 cagcaaccgc tgtccactcc cagcgccctg
gcctcccctg tgggcccagg tcaagtcctg
67561 ggggtgaagc taagtcaggg agcctcatcc
atgcccagcc cggagcccac agcgccatca
67621 agaaatgctt cttccctcca tcaggaaaca
ttagtgggaa agacaagagc tggggggttc
67681 tggggtcctg ggggatcaga tgaaggggtc
tgggagcagc agcagcctca ggcaccccaa
67741 aacaaggccc aggagctgga ctcccagggc
tgaggggcag agggaaggaa ggcctcctgg
67801 ggggttggca tgagcaaagg cacccaggtg
ggggctgagc acccctcggc tggcacacac
67861 aggcccccac tgcagtacct tccccctcgg
agaccctggg ctcccgtctc ccgcctggcc
67921 tgccatcctg ctcaccaccc agaaatccct
gagtgcggtg ccatgtgact gggccctgcc
67981 ctggggagga aggagattca gacagacagg
atgccagggc agagaggggc gagcagagga
68041 tgctgggagg gggcccgggg aggcctgggg
ggcagggggg caggagttct ccagggtgga
68101 cggcgctgtg ctatgctcgg tgagcacaga
ggccccgggt gtcccaggcc tgggaaccca
68161 gcagaggggc agggacgggg ctcaaaggac
ccaaaggccg agccctgacc agacctgtgg
68221 gtccagaagg cagctgcgcc ctgaggccac
tgagtggccc cgtgtcccga accaccgctg
68281 aaacatggga cacacgttcc caggcggagc
cactcctgcc ttccgggagg ctcccagcgg
68341 gctcatcgct ccatcccaca gggagggaaa
ccgaggccca gatgacgaac atcccggcga
68401 gcaggtcaaa gccagcccct ggggtcccct
ctcccggcct ggggcctccc ctctgcaggg
68461 tgggaaaccg aggccacaca ggggctccat
ggggctgccc tctgccaggc cctggacacc
68521 ccgcgggtga cccccgcctc tatcatccca
gccctgccag gccctggaca ccccgtggat
68581 gacccccgcc tctatcatcc cagccctggg
ggacagatgg gaggcccaag cgtggacccc
68641 ctggccaccc cctaccccac agccgggagg
agccgggagc tggtggccaa gggcctagag
68701 gagccagann nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
68761 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnca atatagaggg
68821 ggtgggataa agggtaatat gatgtttagg
tagttagagt taaattagaa gggtttggat
68881 aaagattaat aaaattacaa gcgtacatat
cgtgtgagtg tgggtgataa tatttgtgta
68941 tgtggggaat agaagtgagt gtgagtagta
ttcaagatgt aagtgtgcga atacaggtct
69001 gagcgatttg aatggaagtg aaaaaaagcg
tgtgtgtgga ggaggcggga gaggaagata
69061 gtgtggggga agaaaagaag gctagtgggt
aaagaaatat cagtaggcgg ttgacgaaag
69121 aagaactagg aagaattaat ataaaaataa
agggaggatt aaaaaataaa gagggaggag
69181 gtaacggaaa tagttagtta agaaaagaat
ggagagtgga ggtaagataa ataagggagt
69241 aatgggagtg aggaggaata aataaaaaaa
tggtgaggga aaatagagta gaatgagaac
69301 aagaatgaaa aagggagtga agggggtgaa
aaaaagtgaa gttgaaaaaa gaggaaaaaa
69361 aaggagaaga taaaaaaata aaataaaaaa
aggaaaaaaa agaaaaaaag aaagaagggt
69421 taaaggacga aaagaaggga agagaaaaaa
aatagtttaa gtgggggagg gtaaaaaaga
69481 attaataaag taaatatggt tgtggtcgaa
aaaaaaaaaa aaattgttgt gttgatgaga
69541 agaaaagaaa aaagaagaaa gggaaaagca
aaaagaaagg agagaaaaag acaaccccac
69601 cgcccgggcg catggagggt gaggatggcg
cacgcccgcg gatggcacag catcacagca
69661 atcctaaaac gttttcagac cggtgcatct
tcaccgcgcg cgcgccccgc ccggccctcc
69721 tcccgccctg accgcggacc cccacccgca
ccggggagcc tacccccacc ccggggacgc
69781 tccgccacgc taaggtcagg actgccgtga
agacgcgccg gggtgaaaac gttttatctt
69841 catgacataa gcgagtggtt ttgaaacagg
tttacaaacc ctcgtgaaga cgcaccctta
69901 gcgttaggtt ttgttttttt accatgtgac
gatgcaacta ttttcttcct ctcttccaca
69961 gtggctagtc gcctccagag cgaggggtat
ctcttgtaca gagaccctcg gaacatccgg
70021 aggtagtttc ccacctaggg gtaaagcgag
aaggctcatt acgagggccg gggctcctcg
70081 gggaagggca gggccctggc gcagaggctc
tgccacctca gtgacacgca gaccacgcgc
70141 ggcctgcagg cgccgggctc tgaaagcagg
caaagcccga tctgctgaca tcaggggttc
70201 cgcagcagcg aaggtctggc ccgcacctgg
cccactggca gggggtaagc tctgcctccc
70261 gacgacagca ccaagttcag gaagggccac
gcagacactg gtgagacacg gcccccccgg
70321 agctgcccga gaagctctga ctttgcacta
aagatctctg gcgcggtcca aaaatgtaag
70381 gcctctcttc cttttatctt aagactttga
tatttttacg atgtaataaa taccaagaag
70441 ggcttttaat ttcagacaga tgtaggataa
tttcccccgt agcccttgct gctttgttta
70501 gtaacgaaac tcaaaccaga aataccaaag
gaattttcca aagagtttca aaagcgctta
70561 tcagcaatca ctagactgct gcatacatca
tcactgcccc aaacaatagc ctgcctgtgc
70621 cagttactca aagtactact tacttgacga
aaacaaatct agtcctaacg tttttacaaa
70681 gaaactccac tcttccgcca acttttcaga
aacaaccact cgatcacgtg gcaggggacc
70741 gtggctggac tgggtgctgg ctccttctgt
gaccaggcaa cactgccccc ttctcggcct
70801 ccctacgcct cttgacaaat gttcatcagc
tgtaaagttc accccacgag ggacccactt
70861 ctgctatttc ccacgtacct accccattat
aggagttttc tttgtgacag tttctgcatt
70921 tttcatggat ttagaggttt acataatcag
ggctgctgaa cagcatgaga gacgtggcca
70981 caaggtccct cctgcacctt gccgcagggg
cagggcgagt tatctggctt gagcgtggtt
71041 accatcaggg ggtaaacaca gtttccagga
cgtttttgac aagacactga cccggatgcc
71101 cccactacca ccgtgcaggt cctgcaggcc
tcccagcctc ccaggccctt cccgaggtcc
71161 cttcggaact taggggactc ggtctgcccc
cctgggtttt ccctgcacca gcttttgccc
71221 cctctggacc caggtttccc aaatggaaaa
cgaaggtgtg ggtatggaag ctccctgggc
71281 tcctctcagc tgtgcctctg catggtgatg
acggctgccc atcggggggg gcaggactgg
71341 ggcagctgcg gacaccctcc caaggctgct
acccccgagt ggtgtggggc gctgtgggca
71401 cgctctgctc agcgcacctc ctggaaacca
gcgcctgccg tctgcccggg gcaaccggcc
71461 cgggagccaa gcaccactgc cgtcagagga
gctgctggct gtgagtggac gccagtctag
71521 ctctgaaccc tgcccaggcc tcctgaggtc
tgaacattgt aaaatcaggc cccggacggc
71581 aactgcctct ccctcctgcc gtctggtctc
cataaactgc atctcaggac aaatcttctc
71641 actcaccagg gctgaaacag aagactgcag
ctatctttct caaatctaag gtgtgctaca
71701 gggcaagtcg cagaaactgt ctggcctaag
catctcatca gatgcctgag acaagagctg
71761 tggacgccaa gctggagcca gagctcctcg
cgttctgccc acctggcacc gcgttccacc
71821 cagtaaacgc aggcttgatt ttcaaaagta
ccaccgactc agagccaatg ctaaaccgac
71881 cacttttcct gcccattaga ttgggtgaag
gtttctttaa tcaatctgcc agtcaccaca
71941 tgccgcctct gtgcccacag gctggcgaag
acctttctga gctacggcat gtggcaggca
72001 gcggcacctc tcttcagtac ggccagctgt
caaggggagc gtttctgtga tgatgtgaaa
72061 atacattgca tccggccccg tgtttcatga
acacgggtga ggaaaggaaa cacacaaagt
72121 tctgatgcga ctgacagcac gggtctcata
actcaataca agtcagacaa accacaggga
72181 gtcacaggga atcccaatag cctcatctag
tgtgaccatc atgaggctta atttattcag
72241 tgtattcaat cataaagagg gggaaaaatt
gtaaaaaaaa aaaaaaagaa agagtgaaat
72301 gtgtaatact gaaaactgtt gctaggagaa
gcaagcattg gcgtttgtaa ctgctttgac
72361 tccccaagac ccacactcgc ctcgctacaa
aagggaggca ctgctgctca gtacttgcac
72421 acccgaactg cggatttgta atttaaaaat
gtgtgtgtgg acacagcaca agccagagac
72481 tgccaaaggt tgagggacac tggaagaact
taatatactt ggtgcatgct gccagtgaca
72541 gtcagtcacc agctgattca atagagtgcc
gaaaggtcac cttttaggta aggatgaagg
72601 ggttctgggc tcgtttactt gcactaactc
agagttagtc cgagatatcc gaagtgccag
72661 gtgcctccca tttgctgatg gatctagctc
agggacggct gggccctagc catccaaaaa
72721 tcaagcattg ttctcccaac ctgtcttctc
gctgataatg gaaggtcaga acgcccaccc
72781 gcccacctca aagtcaaaga acaccaagcg
ggtgagtccc cactaagctc ggtgtttcca
72841 atcagcggtt tcaggattcc agctggggca
atgagggagg gagcgtgcga gggatccaac
72901 acctcgcccc gtgcgcagca agggataacc
caacaccccg tttctgtacg tccggctgga
72961 gttgtggaac tcagcgcgga cccggggcca
ccgcgacccc cgggaccctg gccgcgcggc
73021 gcatccccgc tgccgggaca cgggtaagcg
tccccaaact gccggacgcg gggcggggcc
73081 ttctccgcca cgccccgata ggccacgccc
aaggacaagg atggtcgtgc ccagacggcc
73141 ggggcgggnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
73201 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnncg gagggggggg
73261 ggcggggcgg gggctgccgc cgcgcgtata
ggacggtggt cgcccggcct ggggtccggc
73321 cgggaatgac cccgcctctc cccgcatccc
gcagccgccc cgccgcgccc tctgccgcgc
73381 acccgcctgc gcacccgccg ccctcggccg
cggccccggc ccccgccccg tcgggccagc
73441 ccggcctgat ggcgcagatg gcgaccaccg
ccgccggagt ggccgtgggc tcggctgtgg
73501 gccacgtcgt gggcagcgct ctgaccggag
ccttcagtgg ggggagctca gagcccgccc
73561 agcctgcggc ccagcaggtg agcaagggct
caggggaaac tgaggcccga cacagagccg
73621 cagcaagaag gatcctactg gtcactcggc
tgttggcctg gggtcatcac aggcgggctc
73681 tcccaaccca tcccctgagg ccaaggtccc
tagaaccccg tgggcagaca ccaaccagcc
73741 ctttaaatat ggggaaacca aggtgcttag
gggtcagaga tagccctagg tcgcccaacc
73801 ctagtagaag ggagggctgt tggagttcct
gagtgcccgc tctcccaccc cccgggaggc
73861 cccttcctga gcccaagggt gactggtagt
cagtgacttt gggcctgccg acctgtaccc
73921 cactgggcac cccaccagtc ctgagccaca
tttgggctta gtgacggggt cagggatcat
73981 gaggatcaat gtggctgagc caggaaggtg
ttagaacctg tcggcctgga gttcatacca
74041 gcactgccct gggcttttct agacccatgt
cccgcctcct gccccacctg cccctgttcc
74101 cgcaccccac cagcagcggc aggggcttcg
agagggctgt gggctcaccc tatttcaggg
74161 atggagccgc taagacctgg ggcacactgc
ccgctaggga cccctgaggc accagggccg
74221 ggggctctgc ggaggggcag ccgccacccc
cagctttgga gtcctctccc gggtgcccag
74281 cccgagctga tccggctgcc tcccacgctg
tgccccaggg cccggagcgc gccgccccgc
74341 agcccctgca gatggggccc tgtgcctatg
agatcaggca gttcctggac tgctccacca
74401 cccagagcga cctgaccctg tgtgagggct
tcagcgaggc cctgaagcag tgcaagtaca
74461 accacggtga gcggctgctg cccgactggc
gccagggtgg gaagggcggt ccacggctcc
74521 cactccttcg gggtgctccc gctattccca
ggtgctcctg cacttcccat gtgctcccga
74581 ttctccctgg tgctccctct cctcctggct
gctcctttgc ctcccaggtg ctcccacttc
74641 tccctggtgc tcctgctcct cccggcggct
cctgtacctt cggcctgacc tcctccctct
74701 acaggtctga gctccctgcc ctaagagacc
agagcagatt gggtggccag ccctgcaccc
74761 acctgcaccc ccctcccacc gacagccgga
ccatgacgtc agattgtacc caccgagctg
74821 ggacccagag tgaggagggg gtccctcacc
ccacagatga cctgagatga aaacgtgcaa
74881 ttaaaagcct ttattttagc cgaacctgct
gtgtctcctc ttgttggact gtctgcgggg
74941 ggcggggggg agggagatgg aagtcccact
gcggggtggg gtgccacccc ttcagctgct
75001 gccccctgtg gggagggtga ccttgtcatc
ctgcgtaatc cgacgggcag cgcagaccgg
75061 atggtgaggc actaactgct gacctcaagc
ctcaagggcg tccgactccg gccagctgga
75121 gaccctggag gagcgtgccg cctccttctc
gtctctgggg gcccctcggt ggcctcacgc
75181 tctgtcggtc accttgcccc tcttgctgat
gcaatttccc cgtaattgca gattcagcag
75241 gaggaatgct tcgggccttt gcacctgacc
gcatgagcag aggtcacggc cagccccctt
75301 ggatctcagt ccagctcggc cgcttggccg
tgacgttcca ggtcacaggg cctgccggca
75361 cagaggagca ggcccttcag tgccgtcgag
cactcggagc tgctgcctcc gctgagttca
75421 ctcagtgtct acgcacagag cgcccactgt
gtaccaggcc ctattccacg ttccccagtc
75481 accgagcccc cagggctggt ggggacctgc
cctcgggtac actgtgtccc gtcacgtggc
75541 tttacgtgtg tctctgaggg aggctggcat
tgcggtccac ctctcagcac aaacatctgt
75601 cccctgggaa gggggtccca tttctgggtg
cgagcagccc cctggggtcc gtgtctcctc
75661 cttacctggc tcaaggcccc ggctcctggg
tcctggacag cagggagccc acccctcggg
75721 gctgtggagg gggaccttgc ttctggaggc
cacgccgagg gcccaggcgc cgcctccggc
75781 cgtcgccctg agggagcagg cccgacgcca
gcgcggctcc tctgtgaggc ccgggaaacc
75841 ctgcctgagg gtgcgggtgg gcaggtgccc
ctgcccccag gctctcctgt gtgagtgaca
75901 ctcaccagcc agctctggat gccacccatc
cgggttctcc aggaggcact catagcgggt
75961 ggggtcccct ccctcccccc tctgtggagg
gagggagtct gatcactggg aggctggtgg
76021 tccgtacccg cccccccgac tctggacgtg
tttactaccc ccgcctgggc tcaggacagg
76081 gcattggatg ggaaggacag ggctgggtcc
tggccaggct gggggctctg cagggcatgg
76141 gtgcccctgt ctcttcttat attccaacgt
cactgcaggg gggcgcaaat cttggacccc
76201 acttactgat gatctgcatc aggacatagg
tcccccctcc tgcagcgggg ggctggccac
76261 ggagggcgct ggggaaggcc cctcctccag
cccctcggcg aggctcacca ggtgcccatc
76321 ctcagccagc agggcgacgc tcgctgggag
ggcggagagg gaggcagggc agggctggta
76381 cgacccccgc tggggcgggg gggccctcag
ccggtcctcc agcacccttg ctgccccccc
76441 tcaccgtcag ggggcacctg gccgctctgc
ctcaggtggg cggtgagggt cccaaggcca
76501 caccaggtgt tcaccagctc ccagcagctg
gctgtgggag aggggcagag gtgggcgcat
76561 ggcacccgcc ttccccccag accaggatgc
tctgccttcc tcccgcccat ctccccagac
76621 atctgaagga ctcttgcctc caccatgcag
ccccgcctcc accagaagct caggttcccc
76681 gccccccctc cccgaagctg caggacccct
gaccagcgaa gagatgggac agttggaaca
76741 cacgctcccc cagcagcggc acagcagctg
tgtggcccag aagagcccgc ctgtttccct
76801 caagcaactc cccatggatg tcatcccatg
gacaccccct tccccacacc gcctcctcgt
76861 tctccccctc caaggcagag ggaacgcacc
cccacctgtc tgctaggaca ggggacccca
76921 cttacctccg aacatcacct tgataaacat
ggccgtggtg gggacagatc cctccgaccc
76981 ccaacttccg acctggggaa ggagctgggg
tggagctcga ctgcagggtg gggccctgtg
77041 ggaggtgtac gggtggagag ggtgatgggt
gggtgggctc aagcggagct ccttgctcag
77101 tccaggcggt ccctgcagct agtccaggat
cctcagcctt ctccccctca ctggatcagg
77161 gaagactgag gttccctccc ctgccccccc
acccagcttc caagctggtc tctgtggcag
77221 tgggagctgc caagaggtct gagcggccag
tatccgggta acggggtttg tggagggtcc
77281 gggcattccc ggtgcagggc tctagtgggg
gctggagcct cgggcccaga gctgtccaga
77341 gaccagtgcc ctcccaccgc cgccgcccgc
aaggagagac agagctccca ggcggggagt
77401 cggaggttcc tggaggggga gcatcctcaa
ctctgcaggc ccccttccca ggcgcactcc
77461 cggcctcccc gtcttctgtc ccctgctctt
gttgaagtat gattggcata cagttcacag
77521 ccactcttcg gagtgttctc cacactaagg
atacagaaca tgtccctcgt ccccccaaac
77581 tcccagccag gctgtcacga agagggaggc
ggccgacggg gcagggcctt gcactcctgc
77641 gtgtggggtc cacaggggtc gtccccgtgt
cggtggcccc ttcctctcac gccaggaggg
77701 tccccttgcc tggaggtgcc gtggatccgc
tcgctgcctg ctctttgggt tgtttcccgc
77761 atggggtgat gatgaagagg ccagtacaga
cactcgccag caggtctctg ggtgaacagg
77821 catttatttc tctttcctga gggcagatcc
tgggagtggg gtgccggacc gtccggggag
77881 agtatgcttc tgtttctaag aagctgccgt
gttctccagt gtgctgcacc atgtcacggc
77941 ccctctgtgc gtctggactc aggagacctc
cttctcagcg gccctccccc ccaggtggtc
78001 aggccatctg tgcccttctg ggggcagagc
tcagcgccgg aggcgggagg aggcccagat
78061 cccagcgcag cccaccagcg ttgctctgct
tccctcggca ttcatagctg gagaaagggc
78121 aaggagcacc ggctgaagcc ccacctggag
gacgcacttc gatggcagca ggtgctcaga
78181 ggtggccccg ggcagcattc cccagacgca
caggccagtg ctttcttccc aggacaccac
78241 tgtgtctggg gacccgagtc ctgcagcacg
gtcgggagcg gctgtgccca gattccggcc
78301 tgcacccttg gctccagcca ccacccctgt
ttgtcaaggg gtttttgtct ttcgagccgc
78361 cgaggaggga gtcttttgtc tgcagtgtca
cagaagtgcc ataaagaggg gcccacagtg
78421 ggagctttat aacattggtg cggagggctg
taacaggtca gggaggcact tgagggagcc
78481 ttctagggcg atggagatgt tctaaaattt
ggtctgggta caggctacag agatgtgtgg
78541 gtgtgtgtgt gtgtgtgtgt aaaaccctcg
agccacacgt gtgaggtctg tgcatgtgac
78601 cgtacacagg agacctcggt ggaaagcagc
cacctgctct gactgcacct gtggatttcc
78661 agctcctgcc ctcaggcggc cctgcggggc
ccactggctg acggggagac ggcaccgccc
78721 tcccccgctg tcagggtggg ggggctgacg
atttgcatgt cgtgtcaggg tccagcggcc
78781 tcccttgcgt ggaggtcccg aagcacctgg
agcgccgccc gcagaacagc ggactcctgc
78841 ctgcctccct gcctctggcc atggcctgcc
cgcctctggc cctctttctg ctcggggccc
78901 tcctggcagg tgagccctcc caaggcctgg
ctcacctagg ggtgtgtaag acagcacggg
78961 gctctagaag taaatcgcgg ggaagtaaat
cgtagtgggc aggggggatg gtttccgaag
79021 gggccctgag ggggacagga gacctggcct
cagtttcccc actggtgagt gaccagatag
79081 ccagggtacc tttggactct gactctgggg
ggctctcaga gactggtctc ctactcagtt
79141 tttcagaggg gaagctggtg tggccttgtc
actgccctgc agggcctcag ggacaagcta
79201 tccctgagga ggtctccagc agtcagtggc
cggaggctga gccgatggat atagtaacag
79261 cccaggcggc ctcttggggg tggtcagcct
gtagccaggt tttggacgag ccgaagtgac
79321 ctaagtgatg ggggtctgca gagcaaggga
tgagggtggg cagcaggagg acccagagcc
79381 caccagccca ccctctgaat tctggaccct
tagctgcatg tggctccttg ggaagacggg
79441 gcttaagggt tgcccgctct gtggcccaca
cagtgctgat tccacagcac tggctgtgag
79501 cttttgggag cagattctcc cggggagtct
gacccaggct ttgtggggca ggggctggag
79561 ggaaggggcc caggccagac ctgagtgtgt
gtctctcagc ctcccagcca gccctgacca
79621 agccagaagc actgctggtc ttcccaggac
aagtggccca actgtcctgc acgatcagcc
79681 cccattacgc catcgtcggg gacctcggcg
tgtcctggta tcagcagcga gcaggcagcg
79741 ccccccgcct gctcctctac taccgctcag
aggagcacca acaccgggcc cccggcattc
79801 cggaccgctt ctctgcagct gcggatgcag
cccacaacac ctgcatcctg accatcagcc
79861 ccgtgcagcc cgaagatgac gccgattatt
actgctttgt gggtgactta ttctaggggt
79921 gtgggatgag tgtcttccgt ctgcctgcca
cttctactcc tgaccttggg accctctctc
79981 tgagcctcag ttttcctcct ctgtgaaatg
ggttaataac actcaccatg tcaacaataa
80041 ctgctctgag ggttatgaga tccctgtggc
tcggggtgtg ggggtaggga tggtcctggg
80101 gattactgca gaagaggaag cacctgagac
ccttggcgtg gggcccagcc tccccaccag
80161 cccccagggg cccagactgg tggctcttgc
cttcctgtga cgggaggagc tggagtgaga
80221 gaaaaaggaa ccagcctttg ctggtcccgg
ctctgcatgg ctggttgggt tccaacactc
80281 aacgagggga ctggaccggg tcttcgggag
cccctgccta ctcctgggtg gggcaagggg
80341 gcaggtgtga gtgtgtgtgt ggggtgcaga
cactcagagg cacctgaagg caggtgggca
80401 gagggcaggg gaggcatggg cagcagccct
cctggggtag agaggcaggc ttgccaccag
80461 aagcagaact tagccctggg aggggggtgg
gggggttgaa gaacacagct ctcttctctc
80521 ccggttcctc taagaggcgc cacatgaaca
gggggactac ccatcagatg nnnnnnnnnn
80581 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
80641 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
agagggtggg tgggtggaat ttaatatagt
80701 ggtgcgcgtg gagcgtgggc ggcgcattta
aggcggtcat ctaaaatagt ggataggggg
80761 tggtgtgaca ataacgggtg gtggatgtgg
tttacggggg gtgcaatagt tctgagtftg
80821 ttagtgtctt cttgatgggg ttgcggcgtg
tggacctacg ccttgagtat gtgggggggg
80881 aaaagcagtg agggtagtag ggatgggaaa
tattggtgga ggttctttgt tggtgtattt
80941 tttggtatta tgttgggtgg tggagtggtg
ggttgggtgt aatttcgctt gcgttatgtg
81001 ttttttttct ttttcgtgtc gtgggttggg
ttggttggtg ctttgtggtg gtggtgggtt
81061 gtggtataaa aaaaaatgtg tggttgtgct
cagcttagcc ctataacggt cggctttgtt
81121 tcttgtttgt tctgtgggcg tgagcggatg
gctcgggcct ccgtgctccg cggcgcggcc
81181 tcgcgcgccc tcctgctccc gctgctgctg
ctgctgctgc tcccgccgcc gccgctgctg
81241 ctggcccggg ccccgcggcc gccggtgagt
gcccgccgtc ctccagcccc cccgccccgc
81301 cccgccctcc acgccgaggg gcgccggctc
gcagagctgg atccaagggg gtgcccggga
81361 gtggcccggc gcggcccgtt accccgaaac
gctgtctggg tgccccgggg gtgtggtgga
81421 tagtgagctt cccgtccctg gaagtatgca
agtgaagccg gcgccgggat cgctcgggct
81481 ggctggtgag cgggcgggac tcggtcgggc
gctagacgca cgccgccagc cccccagctc
81541 ccagacctgc ccactccgcg cccgcccggc
cgcgatcccg ggtgtgtgtg tgtgttgcag
81601 gggagggaca gcgggagtgg ctacagggct
cccgactcac cgcagggaca aagacccgcg
81661 ggtccccagc tggcgtcagc cgccaggtgt
gtggcctcgg tgagcacacc tccaggcggg
81721 agggttgagg gaagcgctgt ggggagggca
tgcggggtct gagcctggaa gagacggatg
81781 ctaccgcctg ggacctgtga gtggcgggat
tgggaggcta tggaatcagg aggcagccta
81841 agcgtgagag ctccggtgtg gcctggcggg
ggtggtaggg gggggacgcc cctgtgtgtg
81901 ccagcctgcg tgtgccctaa aggctgcgcc
ctcccccact gctggggctt cgggggacca
81961 gtcacagcct aggctactgc aggcgcacag
ctccccggga gcccggccca cgcgggtgtg
82021 ccgctgagcc tccagcctgt cggggcaggg
gtggggggca gggatggggt cgttagcggg
82081 gttgggggca gacgcccagg cagactctct
gggcacagct ccggtgacaa gggaggtctg
82141 gcaagcctgg gccccttctg tccagccacg
ccagctctgc cctggccagt cttgccccct
82201 ggcagtgctg gggatggaag ggggagcggg
tacctcagtc tgggggccct gcctcctccc
82261 cagccccgcc cggcccccta ggcctagggg
cagagtctag gggtcaccct ggggagctgc
82321 tgaatccgcg ggtttaggaa ccggagggac
ctgggctttt gaaccacgtg gccctaggtg
82381 agccctccgg cgcctcggta gccctcaccc
ccagccttgt ccaggtgggc gggtgggagg
82441 cgacagtgcc cactgctggg ctgaacagcg
tctgcaggga ggccaggaga gctgggcaca
82501 cggacacgtt ccatcacctg gagctgccac
tgtgccactt gtgcggggtc aggcggggtc
82561 tgagccgggc tgtcatctgt cacgccacag
atatgcaggg ggcactcggg gtcgcctcgg
82621 acatgcttat ccctggacgg ctgttggcag
ggccgggaag gctctgtaaa tatttatcca
82681 tcccagctca cagctttcag ggttgatgaa
agccccgccg cccgcccact gtgggggacc
82741 ccgccttccc ttctggagcc agcggggtga
gggggtgggg gagatggacc tgcctgccca
82801 ggagcaggcg gtgtgactct ggcaggtcac
ttgacctctc tgagcctcag ggagggcccg
82861 ggatggtgtg cggatgctct ctgccttcct
cccagcctga ccagtgtcct cccctcgggg
82921 tcgcctcctg cccaccgcag agggggtggc
tatggggacc tgggccgatg gcaggcaggc
82981 cggagagggc atgcccggct cagccgtgcc
cagcacttcc cagtccaggg gcccccgcca
83041 ctcccagccg ctggctgcct cccattttcc
cgattgcagg ttggccccga ggctgaccgg
83101 agcctctggc tcagctggga gactgaattc
cccaagcaat tcctcaagga tgtgtgaggc
83161 tgtggtgtgg tgcctatccg ggagaggtgg
ggtgagcgga ctgggcacct ccgcccaggg
83221 caggcccagg gagacgctgg ctgacgagca
ggcaggcctg caaggaggac gagcagccat
83281 ctcaggaatg tgggttttgg agacaagcca
cagctggggg ggtggggggg ccatgggtgg
83341 ggaggcctga tccccaggtc taggtccagc
tctgggctcc ctcgccgtgt gaccctgggc
83401 caagacctgg acctctctgg gccccgtctc
ttcccctggg aggtggggcg atgcctgctc
83461 cccaatcccc cagggctgtg gatgaggcag
acgaggtgtg tgctcatccc cacctcactg
83521 ccttccagca gccccgggcg gggggggtgg
tggggactgg cgcacccagg tgaggatcag
83581 gccttggagc tagggagggc cccccagccc
caggccagaa aggacacggg gagacagaat
83641 gcaggagggc ggcagagcag gggccagcgg
tggggaaact gaggccaaga gcctgtggac
83701 gatgtgctcc aggaaaggac ctcgctgcct
ggggcctgga tcctagagcc tccaggagcg
83761 gtgaccatga cgtgggcagg gaaccggagg
ccccggcttg caggtggacc cggcgcgagt
83821 cactcttcct ctctggccct gagagcttcc
ttccagctgc cgctcctgtg ttctaatgtc
83881 aagtctggag gcctgggggg caggtggggg
ctgactgcca ggtgggggag ggcaggaatt
83941 tggcagagca gcgtcccaga gtgggagaag
ccagcccatg gaggggactc tctccatgcc
84001 tgctgcccca aagggcgtta tagagagagg
tcggttaccc cttcgccatg gccccgttcc
84061 cattgaacag atgggaaagt ggaggctgag
agaaggctgt gacttgccca gggtctccgt
84121 ggcatggaac tgggcctgct gagtctcagg
ccggggatct cgctgctgca ctgagcacgc
84181 caggatgcag gggtctgggc ctggacctag
cgcctcgtgg gggcaagaga ggaaggcacg
84241 ctgggcctgc ctgtcaccct ccaccccacc
gtggcttgtt gctcaggcct tcctgggggc
84301 agaggagagg ggagatttca ctcgctggca
ggctaggccc tgggctctct ggggctccgg
84361 gggaacaatg cagccctggt ctttctgagg
agggtccttg gacctccacc agggttgagg
84421 aaaggatttc tgttcctcct ggaggtcacg
gagccgacat ggggaggagc aggggcaggc
84481 ccggggccca catcctcagt gtgagacctg
gacgtgtgtc ctcccacctg acgctggggg
84541 tggggggtgg gggccggggg ggatccagtg
aaccctgccc ccaaattgtc tggaagacag
84601 cgggtacttg gtcatttccc cttcctcctc
ttcgtttgcc ctggtgggga cagtccctcc
84661 cctggggaag ggggacccca gcctgaagaa
cagagcagag ctggggtcag gggtgtgctg
84721 ggagcgcaga gagcctcctg ctctgcctgc
tggtcattcc tggtggctct ggagtcggca
84781 gctggtgggg agcggctggg gtgctcgtct
gagctctggg gtgcccaggg cctgggagag
84841 ttgccagagg ctgaggccga gggtggggcc
ctggcggccc ggctcctgcc ccaaatatgg
84901 ctcgggaagg ccacagcggc actgagcaga
caggccgggc cagacgggcg ctgaggctcc
84961 cggcctctcc cccagctccg ctgtgaccct
cacctgcggc ccggggtgcc agggcccccg
85021 cttggttctg ccgtgtcttt gcaggctgat
cccacgggct ctccctgcct ctctgagctt
85081 ccgccttttc caggcagggg aaccgcgacc
tccaggctgg gacgcgggga gggtgtatgc
85141 gccaggtcag aatcacccct ccaccgggag
agcgtggtcc aggggccctg gcagggtggg
85201 gaccgagcat ctgggaactg ccagccaccc
ccacccatgc agaggggaca tacagaccac
85261 acggaggctg tgcctccgct gcagcaactg
gagaacaccc agccgcggcc aaacataaat
85321 aactaaataa taaaagtttt aaagatcgtt
acttaaaaaa acaagtgtgc cccagtgatc
85381 ggaccccagt tcccggtgcc ctgagtggtg
ccggccctgt gctgagcatg gcctggttgg
85441 ttcaccccca gatccacact aaagggtggg
atcaccccta ctagtcaggt gagcagatgc
85501 agggggggag ggcggcagcc cctccatgct
ggtgggtggc cgtggtgggt gtcctgggca
85561 ggagccagct cacggagctg gagaggacag
acctgggggg ttgggggcgc ccaggaagaa
85621 acgcaggggg agaggtgtct gccgggggtg
ggggtccctt cgaggctgtg cgtgaagagg
85681 gcaggcgggc ctgcagcccc acctacccgt
ccccggccca aacggcggga gtaagtgacc
85741 ctgggcacct ggggccctcc aggagggggc
gggaggcctt gggatcagca tctggacgcc
85801 agtcagcccg cgccagagcg ccatgctccc
cgacggcctc cgctggagtg aggctgcgct
85861 gacacccaca ccgctgaccc gggcctctct
cccgctcagg atgccccccg ccgccacccc
85921 gtgagcagag ggccacagcc ctggcccgac
gcccctcccg acagtgacgc ccccgccctg
85981 gccacccagg aggccctccc gcttgctggc
cgccccagac ctccccgctg cggcgtgcct
86041 gacctgcccg atgggccgag tgcccgcaac
cgacagaagc ggttcgtgct gtcgggcggg
86101 cgctgggaga agacggacct cacctacagg
tagggccagt ggccacgagc tggcctttga
86161 tctccacctg ctgtctgaga cacgctggag
ctggggggag ggcagatccc tatggccaac
86221 aggctggagt gtcccccaac tcccgtgccc
actgctcaac accccaaacc cacacttaga
86281 tgcactccca tgccctccct tgggagcacg
gtctccacac ccacctggcc accccacaca
86341 cccgtggggc acggccgtta gtcacccacg
caacctctgc gggcaccgtg ctgcgggcca
86401 ggccctggga ctctcagtga gggaggcaga
cacggcccct cctccggggg agcgaggtgc
86461 tccccacgcc cggttcagct ctagcaccgc
actcgggacc ctcacaggga gggacccact
86521 ggggcaggcc aggtgacggc tcgggtgacc
tcggcccctg gcgctgagac tacacttcct
86581 gcagtgggcg gcgaagatgg gtgtggtgtc
ccacgtcgtt gcagcgggga ctcctggggc
86641 ctcggaagtg tcctgggcgg ggagcctggg
gagcaggaag ggcaggtctt ggggtccaag
86701 gcctccccac ggtcaggtct gggagggggc
ctcggggctc ttgggtcctt tccgcccagt
86761 gcagaccctc gcggccacct aagggcacac
agaccacaca aagctgtgcc catgcagtgt
86821 ggggagtggt gcgcaccctc agagcacact
gggcccacat cacgcacgcc tgccccctca
86881 ctgtgcatcc ggggaaactc ctggccccga
cagccagcgg ggctgacgct accccgtgag
86941 ccagacccag gcccccctca ccgcccctgt
cctccccagg atcctccggt tcccatggca
87001 gctgctgcgg gaacaggtgc ggcagacggt
ggcggaggcc ctccaggtgt ggagcgatgt
87061 cacaccgctc accttcaccg aggtgcacga
gggccgcgcc gacatcgtga tcgacttcac
87121 caggtgagcg ggggcctgag ggcaccccca
ccctgggaag gaaacccatc tgccggcagc
87181 cactgactct gcccctaccc accccccgac
aggtactggc acggggacaa tctgcccttt
87241 gatggacctg ggggcatcct ggcccacgcc
ttcttcccca agacccaccg agaaggggat
87301 gtccacttcg actatgatga gacctggacc
atcggggaca accagggtag gggctggggc
87361 cccactttcc ggaggggccc tgtcgaggcc
ccggagccgg gcccgggctc tgcgtccgct
87421 ggggagctcg cgcattgccg ggctgtctcc
ctcttccagg cacggatctc ctgcaggtgg
87481 cggcacacga gtttggccac gtgctcgggc
tgcagcacac gacagctgcg aaggccctga
87541 tgtccccctt ctacaccttc cgctacccac
tgagcctcag cccagacgac cgcaggggca
87601 tccagcagct gtacggccgg cctcagctag
ctcccacgtc caggcctccg gacctgggcc
87661 ctggcaccgg ggcggacacc aacgagatcg
cgccgctgga ggtgaggccc tgctccccct
87721 gcccacggct gcctctgcag ctccaacatg
ggctcctcct aacccttcgc tctcacccca
87781 gccggacgcc ccaccggatg cctgccaggt
ctcctttgac gcagccgcca ccatccgtgg
87841 cgagctcttc ttcttcaagg caggctttgt
gtggcggctg cgcgggggcc ggctgcagcc
87901 tggctaccct gcgctggcct ctcgccactg
gcaggggctg cccagccctg tggatgcagc
87961 cttcgaggac gcccagggcc acatctggtt
cttccaaggt gagtgggagc cgggtcacac
88021 tcaggagact gcagggagcc aggaacgtca
tggccaaggg tagggacaga cagacgtgat
88081 gagcagatgg acagacggag ggggtcccgg
agttttgggg cccaggaaga gcgtgactca
88141 ctcctctggg cacagctggg aggcttcctg
gaggaggcgg ttctcgaagc gggagtagga
88201 taaaaggtat tgcaccccat gaagcacgtg
tgatccttgc ccctagagac aaggctctgg
88261 ggctcagagg tggtgaagtg acccacatga
gggcacagct tggagaatgt cgggagggat
88321 gtgagctcag tgtgccagag atgggagcct
ggagcatgcc aaggggcagg gcctgctgcc
88381 tgagagctgg cactggggtg ggcagccaag
tgcagggatg gagcgggcgc ccaggtggcc
88441 tctttgctgc tcagaacgac ctttcccatg
tatacctccc agcgccgctg gcattgccca
88501 gtgtccttct tgggggcagg agtaccaagc
aggcattatt actggccttt tgtgttttat
88561 ggacaacgaa actgaggctg ggaaggtccg
aggtggtgtt ggtggcggaa ggtggccgct
88621 gggcagccct gttgcagcac acacccccca
cccaccgttt ctccaacagg agctcagtac
88681 tgggtgtatg acggtgagaa gccggtcctg
ggccccgcgc ccctctccga gctgggcctg
88741 caggggtccc cgatccatgc cgccctggtg
tggggctccg agaagaacaa gatctacttc
88801 ttccgaagtg gggactactg gcgcttccag
cccagcgccc gccgcgtgga cagccctgtg
88861 ccgcgccggg tcaccgactg gcgaggggtg
ccctcggaga tcgacgcggc cttccaggat
88921 gctgaaggtg tgcagggggc aggccctctg
cccagccccc tcccattccg cccctcctcc
88981 tgccaaggac tgtgctaact ccctgtgctc
catctttgtg gctgtgggca ccaggcacgg
89041 catggagact gaggcccgtg cccaggtccc
ttggatgtgg ctagtgaaat cagtccgagg
89101 ctccagcctc tgtcaggctg ggtggcagct
cagaccagac cctgagggca ggcagaaggg
89161 ctcgcccaag ggtagaaaga ccctggggct
tccttggtgg ctcagacagt aaagcgtctg
89221 cctgcaatgc gggagacctg gattcgatcc
ctgggtcagg gagatcccct ggagaaggaa
89281 atggcaatgc cctccggtac tgttgcctgg
aaaattccat ggacagagca gcctggaagc
89341 tccatggggt cgcgaagagt cagacacaat
ggagcgactt cactgtctta agggccacct
89401 gaggtcctca ggtttcaagg aacccagcag
tggccaaggc ctgtgcccat ccctctgtcc
89461 acttaccagg ccctgaccct cctgtctcct
caggcttcgc ctacttcctg cgtggccgcc
89521 tctactggaa gtttgacccc gtgaaggtga
aagccctgga gggcttcccc cggctcgtgg
89581 gccccgactt cttcagctgt actgaggctg
ccaacacttt ccgctgatca ccgcctggct
89641 gtcctcaggc cctgacacct ccacacagga
gaccgtggcc gtgcctgtgg ctgtaggtac
89701 caggcagggc acggagtcgc ggctgctatg
ggggcaaggc agggcgctgc caccaggact
89761 gcagggaggg ccacgcgggt cgtggccact
gccagcgact gtctgagact gggcaggggg
89821 gctctggcat ggaggctgag ggtggtcttg
ggctggctcc acgcagcctg tgcaggtcac
89881 atggaaccca gctgcccatg gtctccatcc
acacccctca gggtcgggcc tcagcagggc
89941 tgggggagct ggagccctca ccgtcctcgc
tgtggggtcc catagggggc tggcacgtgg
90001 gtgtcagggt cctgcgcctc ctgcctccca
caggggttgg ctctgcgtag gtgctgcctt
90061 ccagtttggt ggttctggag acctattccc
caagatcctg gccaaaaggc caggtcagct
90121 ggtgggggtg cttcctgcca gagaccctgc
accctggggg ccccagcata cctcagtcct
90181 atcacgggtc agatcctcca aagccatgta
aatgtgtaca gtgtgtataa agctgttttg
90241 tttttcattt tttaaccgac tgtcattaaa
cacggtcgtt ttctacctgc ctgctggggt
90301 gtctctgtga gtgcaaggcc agtatagggt
ggaactggac cagggagttg ggaggcttgg
90361 ctggggaccc gctcagtccc ctggtcctca
gggctgggtg ttggttcagg gctccccctg
90421 ctccatctca tcctgcttga atgcctacag
tggcttcaca gtctgctccc catctcccca
90481 gcggcctctc agaccgtcgt ccaccaagtg
ctgctcacgt tttccgatcc agccactgtc
90541 aggacacaga accgaactca aggttactgt
ggctgactcc tcactctctg gggtctactt
90601 gcctgccacc ctcagagagc caaggatccg
cctgtgatgc aggagtgagt gaagtcgctc
90661 agccgagtcc gactctttgc aaccccatag
gactgtagcc taccaggctc ctctgtctat
90721 gggatttttc aggcaagagt gctggagtgg
gttgccattt ccttctccag gggatcttcc
90781 caaccctggt ctcccgcata gcaggcagac
tctttactgt ctgagccacc aggcaatgca
90841 ggagacctag gttcagtctc tgggtgggga
agatcccctg gagaagggaa tgacaacctg
90901 cttcagtatt cttgattggg gaatcccatg
gacaaaggag cctggaggcc tacagcccat
90961 agggtgcaaa gagacacgac tgagcaagtc
acacacacag agccctacgt ggatgctcat
91021 agcggcacct catagctgcc atgtatcagg
tgttggcatg ggcagccatc agcagggggc
91081 catttctgac ccactgcctt gttccaccgg
atacacgggt gccttcctgt gtgtcgggcc
91141 cactcggctg tcagcgccca agggcagggc
tgtcgggagg cacagggcac agagttaagg
91201 aggggatggg gacgttagct cctccccagc
tctcagcgga tgcagcaggc aaaacaaacg
91261 ctaggaatcc tgccaaaccc ggtagtctct
gcccatgctc gccccatccc cagagccaca
91321 agaacgggag ctggggggtg gcccggagct
gggatactgg tccctgggcc cgcccatgtg
91381 ctcggccgca cagcgtcctc cgggcgggga
aactgaggca cgggcgcctc cggcttcctc
91441 cccgccttcc gggcctcgcc tcgttcctcc
tcaccagggc agtattccag ccccggctgt
91501 gagacggaga agggcgccgt tcgagtcagg
gccgcggctg ttatttctgc cggtgagcgg
91561 ccttccctgg tacctccact tgagaggcgg
ccgggaaggc cgagaaacgg gccgaggctc
91621 ctttaagggg cccgtggggg cgcgcccggc
ccttttgtcc gggtggcggc ggcggcgacg
91681 cgcgcgtcag cgtcaacgcc cgcgcctgcg
cactgagggc ggcctgcttg tcgtctgcgg
91741 cggcggcggc ggcggcggcg gaggaggcga
accccatctg gcttggcaag agactgagnn
91801 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
91861 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnct gcaggtgccg gcggtgacgc
91921 ggacgtacac cgcggcctgc gtcctcacca
ccgccgccgt ggtaaccgcc cccgggggtt
91981 gccaaggtta cgattggacc ctccccgccc
cgaccctgct cccctagggt gggtgggtcg
92041 gggggcagtt tctaagatct cctggttccg
cagcagctgg aactcctcag tcccttccag
92101 ctctacttca acccgcacct cgtgttccgg
aagttccagg tgaggccgcc ccgccccttg
92161 cacttgctgg cccaacccct cccgcccagc
gctggcctga ccgcccccca ccccgcccac
92221 cccacgcagg tttggaggct catcaccaac
ttcctcttct tcgggcccct gggattcagc
92281 ttcttcttca acatgctctt cgtgtatcct
gcgccgtggt ggaagcggga ggagggcggg
92341 gcgggggacc gggcgggagg cagcgggccc
cgggaagctg agaccctcca aggggcacgc
92401 ttcctatacc aaagccgcag gttccgctac
tgccgcatgc tggaggaggg ctccttccgc
92461 ggccgcacgg ccgacttcgt cttcatgttt
ctcttcgggg gcgtcctgat gactgtatcc
92521 ttcccgggct cggggaccta tgggtccggg
cctctgctgg ccctgaggcc ctgcttgagc
92581 gcatgccaca gagggagagt tgcgaccccg
agctgagggt gtttttgagc gtacatcacg
92641 tgctcagctg caggtgcccc tgtcgaactc
cagggctaca cccaaaatac cacagggcag
92701 ggtgcccagg ggctgagtcc tgaatgcagg
tagccaggag gatctagggc tgggcccggg
92761 ggctggggtg aagtggagag gcagggccga
tcagggggcc cctggaggcc accgtttggt
92821 cttagagtgg gaagcgaaac caacctgctt
gagggtttca ggggtttagg aagtcagagg
92881 ggccctgggc agggcacaag accttgactc
tggcccagct actggggctc ctgggtagcc
92941 tcttcttcct gggccaggcc ctcacggcca
tgctggtgta cgtgtggagc cgccgcagcc
93001 ctggggtgag ggtcaacttc tttggcctcc
tcaccttcca ggcgccgttc ctgccctggg
93061 cgctcatggg cttttcaatg ctgctgggca
actccatcct ggtggacctg ctgggtgagc
93121 ctgctgtcca gggagcctgc cccaagctgg
gtgtgctggg ccagagccct ggtcctctcc
93181 ccgcccccac ccctcttccc cactcctggc
gcccccatcc ttccagcccc tccaacaagt
93241 cagcctatag gttttactta ttcgagcctg
acccatttgc tgacgcttgt gtggggcccg
93301 acccggtagg gatgggtggc tcagggtgcc
tgctcacagc tccacttctt ctgacgtcct
93361 caggcctgac ctcctcccag gttctgccta
ctctgggcca agcctggccc cacgctgggc
93421 tggctggccg tgcagggcat cagaccccca
tgctttgggg gcttcagggc tgtggagggt
93481 ggcctcggca ttggcgcctc tcccacaggg
attgcggtgg gccacgtcta ctacttcctg
93541 gaggacgtct tccccaacca gcctggaggc
aagaggctgc tgctgacccc cagcttcctg
93601 tgagtgctga cagccttccc cacccccttc
cccagatggc tctctacccc atgagggggg
93661 gggaccctgc cagctgccgc tcagcgtggg
ctcctcccca caggaaactg ctactggatg
93721 ccccagagga ggaccccaat tacctgcccc
tccccgagga gcagccagga cccctgcagc
93781 agtgaggacg acctcaccca gagccgggtc
ccccaccccc acccctggcc tgcaacgcag
93841 ctccctgtcc tggaggccgg gcctgggccc
agggcccccg ccctgaataa acaagtgacc
93901 tgcagcctgt tcgccacagc actggctctc
ctgccgcggc cagcctctcc acgcggggca
93961 ggtgctgctg gccgagagcc agggccacca
agcctgacgt gctctccgac ccagaacatt
94021 ggcacagctg gaggcccaga gagggtccag
aacctgccca ctcgccagca gaactctgag
94081 cacagagggc agccctgctg gggttctcat
ccctgccctg cctgtgccgt aattcagctt
94141 ccactgatgg ggctcacatc tcaggggcgg
ggctgggact gggatgctgg gttgtgctga
94201 gctttggccg tgggggccct cctgtcccga
actagcaacc cccaagggga cctctgcttc
94261 atttcccagc caggccactg aaggacgggc
caggtgcaga agagggccag gccctttctg
94321 tgactccgaa gcctcaagtg tcagtgtttg
cagagtccag tggctgaggc agaggcctct
94381 gggaagctct gcccctgccg tttgcagctg
aggccggcag gagcctcacc tggtccccag
94441 ctcacgggca ttggaggacc agtccgcacg
gtggtttact cctgggtcgg caccagccgc
94501 cgccggctgt ccctttcaca gaggataaaa
gtactcgctc tggagttgga ctttaatgtt
94561 gtcatgaaac ctctggccca gcagcgggct
ccgcagtggg tggcaggtga aggcccctcc
94621 ccgggcctct ccaggcaggt gccgcctggc
cagcagggaa ggcaggcagt gtcatccccc
94681 actggctctg gggctcaggc tacctcctgc
tgtggccgga acatctcccc cagtggtgga
94741 gcccagtgtc cgtgaggcca gctgggcctg
aaaccttcct ctctgaagcc ccgctgtccc
94801 cttgccctgt atggagggca gaggctggag
cgcaagttcc taggatgtgc ttgcgagacc
94861 cccgagccca ggggcgaggc ccatctcagc
ccacccccga actggaaacc cttggagctc
94921 tgcccctcgt ggtgtgaggc ccctgctatg
cgaccctcag ccctgccagc aacggaaggt
94981 gcagggcccg ggcccacggg cttaacgcaa
ctgggcctgg gtcacctgcg gggcctggtc
95041 ccaggaggaa gacccaggtg ccaccctcct
gggtgccacg tccaggtcac gtggggaccc
95101 gtccatgtca cagaagatgc agggtcaccc
ggtgagctgg cgccgggccc tgccagagca
95161 ccagccgcgg gtggaggtgg gccccagctc
tcctgtcagg cacgtggtgc tgggaggtgc
95221 ggccggagca gtgcccacca gctgcagcag
gacaggtggg cacaggccca ccagcagtgc
95281 ccgcacggga tgggcccctg caagggccag
agaagccacg ctcctggctg ggggctgggc
95341 tgggactgac aggtggccct gccctctgcg
ccccactact tcccagccac ccgggactcc
95401 aaggacttgc tgagctgggc aggtgggacg
ccgaggggag tcaaactgct cgtgggggca
95461 ggaggggcgg tccacagggc tgagccctga
gctgaaccct ggccctgctc gtggttgtgg
95521 gggtgggggg gtccagtggc gccctagccc
tgctgaggcc cagctgggac gtgcgcgccg
95581 gagggcgagg ggccagccca tgccatgctg
tcccccgttc tcagctccat gctaccactt
95641 tgaagaaaca gaacctgttg cctttttatt
tagaaagtgt tgcttgccct gcctggggct
95701 tctatacaaa aaacaaacac agctcaacgt
ggcctctcct gaccagagac gggcggtggg
95761 gactggggct cagcagacgg aatgtgtccc
cggcggcggg agaccaggag gcccctggcc
95821 cgctcctcag gacggctggg ctgtccccac
ctggtcccct ccgagccaga agatggagga
95881 gaggtgggct gatctccaga tgctccctgg
gagccaagcg ccacggggtg gtcaccaggc
95941 cggggccgtg ttggccagac gcctcatccg
cctgtgggag ggggagggca gcaacccccg
96001 gatctctcag gcaaccgagt gaggaggcag
gagcccccag cccctccctc ggccgctctg
96061 ctgcgtgggg ccctgaagtc gtcctctgtc
tcgcccccct ccccagggag agtgagcctg
96121 ttctgggctg tggtcagacc tgcccgaggg
ccagcctcgc ccggggccct gtcctgcctg
96181 gaaggggctg gggcagcacc ttgtgttccg
gtcctggtcc cggatcttct tctccatctc
96241 tgcatccgtc agggtctcca gcagcgggca
ccactggtca gcgtcgcctg tgttccggat
96301 ggcaatctcc accgtgggca gggggttctc
actgtggagg acgagagagg tagacggctc
96361 acagagcagc tgcaggagag gcccctagaa
agcagtgtcc accccgctgc gggcagacag
96421 gacatggagc ctggtttctg cacccggctc
ccgacacagg gcggccgggc acgctgccaa
96481 catggcatct ccgggtctgc atgtggggag
gggtccacag gacagtgctg caggtccagc
96541 cattcccagt ggacttgctg ggaggaggag
ggccgtccgc cccgctcagt gtccaggaga
96601 aaggagagca aaggagtcca tccacccagg
agtggagtcc cagggcccct gccctgacca
96661 gcctgcaggg ggcccctcgg cccacatcac
aggggcccag aatccataag ccctgactgc
96721 tccaccccgg ggcccctcaa agacgcgcct
agactccgtc cgagggccac ctgcacaccc
96781 tctggcgaag tggactcagg gctgggggtc
agcctcggtg aggccgcaaa ggctggggac
96841 tcctggccga gctgctgcct ctgccaggag
ccaggcccag cctgccggcg agcctcagcc
96901 acgccctcac ccaccctgcc cgcggcgcca
cgctggcctc cgggtcctct cctctggcct
96961 cctgctgggc cactggtgct cagccccagc
agtcggcctg ccaggagccc tgcagagtca
97021 gcccccagag ggaggagggg gcccggggga
acagcacagg aacaaacaga cccctggcct
97081 tagttttagc tcctcatctg gaaaatgggg
acagtgtcct tgctgcgagg ggtttcagag
97141 gaccactgcc atgcaacacc cagcacacac
ccactgcgtg ggggctcggg cccgagccgg
97201 tgcccccgag tcccaggctg gtggctgggc
cgccccagcc accctgccga cagctgcttc
97261 ccagccgggc ggtgctgcgg cagtccagaa
gccagcactg cagacccaaa tgtcactcct
97321 cacgttgcgg gctcccagct gccttccttg
ggggcagcag acacgaaagt caccaagccc
97381 acgccgacgg gagcaaacac gtcttcctct
taaacaagtg cgggtcccgg aggccctgtg
97441 tttacctccc tgtggctccg ggaagattgc
atcccagggg gttgttctaa accaagggct
97501 gctcgggcca ggcctggaag gaggggcctg
gagccaggag cccaccctta cgggcattcg
97561 gcttcctggg tctcaaggcc ggctgggacc
ctgcattccc accacccgcc aggtgcaagc
97621 agggaggccg tgtcggagga ggcagagggc
ctggagggtc gtcttcgacg tgacctcact
97681 tttacaacct cacaggtgcg gcaggccagc
tgggaggcat ggctgtgccc tcctggtaga
97741 tgagaacaag actgcaggga gtgatccccc
tgaacttccc caaccaggag gagacaaaac
97801 tcggtgtcgc cctcctgctt aagatcaact
gactctggac aaggggccca gcccacccga
97861 tggggaaagg gcagtccttc caacaagcgg
tgctgggacg ggacccggca ggccatggtt
97921 tctcagctat gacaccagca gcacaagcac
cccgagaaaa acagctaagc tgggcactgt
97981 cacacaagtg aactccaaac ccaagaaaac
cacaaaaagc ctgcggatct tcagatatgt
98041 gggaagggac ctgtatctgg aatgtataac
gaactcctga aaagtgaaag tgttagtcac
98101 tcagtctgtt cagctctttg caaccccatg
gacggtagcc tgccaggctc ctctgcccat
98161 gggattctct aggcaagaat actggagtgg
gttgccatgc cttcctccag gggatcttcc
98221 caacccaggg attgaacctg tgtctctctt
gcactggcag gcgggttctt taccagtagc
98281 gccacctgag tagaaacact ccaggtgccc
tgagtgtcag agcaggaggg actcggccca
98341 ggcctgtgag gggaccctct ccgagtcccc
tgctgcacag cagtgagagg tgcgttctga
98401 gtcagcctcc agggatgagg gacttggtgt
cgacatcact cccaggacct caggatctgc
98461 tctgggaagc gaggctcccc aggctggccc
caggcccgct ggcctcagct cgtgagccgt
98521 gcgtggacag gtgccatgag caggcctccc
acgggactcg gggcgcggcc tggaccccgg
98581 ggctgccagt ggtcgcgggg ggccccgtgt
ggcggctgtt ccctctcttg ctccgagtcc
98641 taggaacatg gtgggcgctg cctcctgggg
tttctggaga agcagctgag atgcaaacag
98701 ccccacgcgc tccctcagct gttccctgtc
acgggtggcc ccttggtgac ggcctccatg
98761 cagggacggt gacagctcga gcagccgcgt
aaaaccacac ggggacggtg gcagctcgag
98821 cagccgcgta aagcctgaca tccaatttgg
aagcctcccg cagtggaaga ggggcccggg
98881 gacggggctg cccggggcga gctccaccgg
gtcgggggtc acgaggagcc cacccgcgtc
98941 cccgccacca gcacctggga ccagataccc
tccccgctct gagggcggcc tgaacgccgc
99001 cccctcccac gggggcgccc accgcctgct
cgtggactga acaagaggcg gcagtggcct
99061 ccagaccccc tcgggggagg gcagacctgt
ccgagactga gcacaagtcc agggaatgag
99121 caagggtctc agtaatgtcc ccaccgggac
gggacgggag gaggcgacag aggccgctga
99181 ggtgcggggc agccctcagt agctggcatc
aaggccccag gcagtcccgg ggcatccccg
99241 cagggggcgg gggcgaccac cggcccgagc
ccaggcagtc ccggggcatc cctgcagcgg
99301 gcgggggcga ccaccggccc gagccctacc
tgaaggcgta ggtcttctga tgccagctca
99361 gctgtccccg gatgctgtag gcgatggtgg
tgacgaactc cccgcccagc cccagctcgg
99421 agcacagctt cagagcgaac ttctcgggcg
agttctcctt ctccgacatg tcccactcga
99481 actggtccac caaggagatg ttccccacgt
ggatgttcag ctggcccggg agcacagaca
99541 tgagccagag cggccccctc tggggccagg
ccgcaccctc accacccctt ctccccggaa
99601 catccccgcc tcgttcttgg ccgcgcccct
gtgctgctac ttggggtaag gaaaacaacc
99661 cccatctctc tgaaaagggt taactagcga
ggaagatgcg ctggtaactg gaaaactccc
99721 tacaaagaaa gcttggatct gatggcttca
ctggtgaatt ccaccaaaca tttcaagcac
99781 taacaccaat ccttatcaaa tcctgccaaa
aaactgaaaa ggaaggaaca catcataact
99841 ccctgccttg ataccaaagc cagacaaaga
tactacgaga aaggaaaggt gcagaccggc
99901 acttactgtg gacattgatg tgaaacctca
gcagacacga gcaaaactac attcaccagc
99961 acgtcagaag aatcacacac cgttataaat
gatgggatga tgacacaacc acattataaa
100021 cggtggggct tactctggtg atgtaaggac
ggctcagtaa gaaaaccggt caatgccatg
100081 aaccacttga acagagtgaa ggacaaaaac
cacacagtca tcttgataat tggaggaaaa
100141 tcattagaca aacttcaacg tgctttcacg
ataaaagcac tcagtaaact aagatcagat
100201 ggaaaccaca tcaacaagat taattcagtc
aaaaaattca ctgcaagtat cacccacaat
100261 ggcagaagac tggtaacttt tcctctaaga
tcaggaacga gccaaagata cccagtcttg
100321 ccacttttgt tcaatatagc gttggaattt
ctactcagtg cagtgcagtc gctcagtcgt
100381 gtccgactct tttcgacccc atggatcaca
gcacgccagg cctccctgtc catcaccaac
100441 tcccggagtt cacccaaact catgtgcact
gagtcagtga tgccatccag ccatctcatc
100501 ctctgtcgtc cccttctcct cctgcctcca
atcccttcca gcagttaggc aagaaaaata
100561 aatcaaaggt atccacctgg aatggaagaa
gtaaaactat ctctggtccg agatgttaca
100621 atcttatatg cagagtttaa gatgctaaca
aaatactatt agaactaatg aatgaattca
100681 gcaaggtacc aggatacaaa gtcaacgtgc
aaaaatcagc cgcatttcta catgctaaca
100741 ctgcacaatc tgaagaagaa aggatgaaca
aattacaata acataaaaaa gaataaaatc
100801 cttagaaatt aacttgatca aagagatgta
caatgaacaa tataaaacat actgaaagaa
100861 attgaagata taaataaatg gaaaaacatc
ctatgtccat ggattggaag acttaaaatt
100921 attaagctgt caaggctatg gtttttccag
tggtcatgta tggatgtgag agttggacta
100981 taaagaaagc tgagcaccga agaagtgatg
cttttgaact gtggtgttgg agaagactct
101041 tgagaggtcc ttggactgca aggagatcca
accagtccat cctaaaggag atcagtcctg
101101 ggtgttcatt ggaaggactg atgttaaagc
tgaaactcca atactttggc cacctgatgc
101161 gaagagctga ctcatttgaa aagaccctga
tgctgggtaa gattgagggc gggaggggaa
101221 ggggacaaca gaggatgaga tggttggatg
gcatcaccga ctcaatggac atgggtttgg
101281 gtggactctg gaagttggtg atggacaggg
aggcctggcg tgctgcggtt catggggttg
101341 tgaggagtcg gacacgactg agcgactgaa
ctgaactgaa catgaatacc caaagcaatc
101401 tacaaagcca aatgtaatcc ctatcaaaat
cccaatagca tttctgcaga aacaggaaaa
101461 aaaatcttaa aattcatatg gaatctaagg
aaaagcaaag gatgtctggt caaaacaatg
101521 acgaaaagaa caacaaagct ggaagactca
cacttcctga tttcagaact tactgcaaag
101581 atacaataat gaaaacactg tgggactaac
gtaaaagcag acacgtgggc caacgggaca
101641 gcccagaaat aaactctcaa ataagcagtc
aaatgatttt caacagagat gccaagacca
101701 ctcagtgaag gaaagtgttt gcaaccaacg
gttttgggaa aaaagaaccc acatgcgaaa
101761 gaatgaagtg ggacccttac ccagccccat
ctacagaaat caactcaaaa cagacagaac
101821 atatggctca agccataaaa cgctcagaaa
aacagagcaa agctttatga tgttggattt
101881 ggcggtgatt tctcagatat gacgtcaaag
gcataggtga taagcgaaaa aataaactgg
101941 acttcaccaa aatacaacac ttctatgcat
ccaaggacac taccgacagc ataacaaggc
102001 agcccaggga aaggaggaaa catccgcaaa
tcacagcatc tgggaacaga ccgctgcctg
102061 tgagatacag ggaaccgata aaaacaagaa
aacagcaaaa cccggactca aaaatgggaa
102121 ggactccagc agacacagga gacagacaag
ccgccagcag gtcactaatc agcaagcaag
102181 gcccgcaaag gcccgtatcc aaggctgtgg
tttttccagt ggtcatgtag gaaagagagc
102241 tggatcgtaa gaaagctgag cgctgaagaa
ttgattgaac tgtggtgttg gagaagactc
102301 ttgagagtcc cttggactgc aagatcaaac
cagtccattc tgaaggagat cagtcccgaa
102361 tagtcactga aggactgatg ctgtagctcc
aatactttgg ccacctgatt cgaagaactg
102421 actcattggc aaagaccctg atgctgggaa
agattgaagg caggaggaga aggggacgac
102481 agaggatgag atggttggat ggcatcactg
actccatgga catgagcttg ggcaagctcc
102541 gggagagagt gaaggacagg gaagcctggc
gtgctgcagc ccgtgggtcc caaatctttg
102601 gaccaagcga ctgaacaata acaaatcaac
agggaaatgc aaatcaaaac cacagtgaga
102661 tactgtccac caccaggcag gcgttcttca
gcggggttcg gggcaggtgg tgccctcttc
102721 tctcgtaacg cccccaggac cgcgggggct
gctgagacag catggggtgt gcttggccta
102781 gcctgcccat gacaagagtg gcagtgtgct
cgcctcactg cgcccttccc tgctctgccc
102841 accagctggg ccacccctgg gaccacccag
cttccgctcc gtggacggca aggccgcagc
102901 agcgcccgga cacgcccaga acgtggtgcc
ctcctcagaa gtcggcctgt gcccttcctg
102961 ggacaagccg cccaagagac agtcttccag
agccctgccc cacaacacgg accccagaca
103021 ggctcctgtg gaggcctcca cgcacctccg
cacctcgcaa gccccgagga caaggcaggc
103081 ccgctgcggg tgaggagccg cctaccttga
taatgacgcg ctggtctgac tggtcttcca
103141 ggatgctgtc cgtggggtag gactcgatct
gctgtctgat ggcagaggca atggctggca
103201 cgaatgtcag tgggttcaga tccaggtcgt
cacagagaat ctctgagaac atctccgggg
103261 tcatcagctt ctctgaaacg atgacggagc
gggggaaccc ccagtggacc acagggccta
103321 cggtcagcgt gctcagcccc ggcctccccc
agccttgcct cctctgccac cgcccccccg
103381 ggtgacgaca ggaccccctg gcagcacgca
gacagagctg agtgcacgcc agccagggcg
103441 gcggacggac cattcatgtt ccaggtaaag
gcatcccgca gcttctgccc gtcaatctcc
103501 atgtccagtc ggatggggac cagcacctcg
ggctgggacg cgttctcgtg gatcacggct
103561 gggtcgtggt cgtcgaagct ggaaggggag
cggccgcgtg ctcagcaaag cgggctgggc
103621 ccctgtgccc agggcctccc tctctgcacc
actggtcgct gagacctgcc cagagaggac
103681 ctgtccacta cgggccgggc cggcagaaac
agggctggcg ggggtccacg cggggcggga
103741 ggggagctgc cgactcggca gcgggacaag
ctcagaggtt ccctgcagga agagaggttt
103801 aagccccaga gcaggcagga ttctcccagc
agctgtgggg aagaaagggt atgtccagaa
103861 gaagaaaccc tggaacaaag gccgaggggc
aggagggttg aggagctgct tggagagcag
103921 tgaagggggg ctgggcggct ggggggtgct
ggggagcctc ggtggccaag cacccagggc
103981 tccccacctg cagcctggac cccgagggag
ccccagagga cggagagcaa ggcagctccg
104041 cactcacacc tgccctttag gatggggaag
agggaagaga cgggggctgc ggggggcaag
104101 gaaaccaggc acgccccgct tagacccggg
ggcgagaacc actttccaag aacgcagggg
104161 cgccaatgat gaacaatggg tagcagcccg
caggcgggag gcccggtggc cgaggcccct
104221 caccagagcg ggaaggtccg cttcttgtcg
cggcccatgc ggttcctgtt gatggtggtg
104281 gagcagggca cggcgtccag gtggtgcgag
ctgttgggca gggtgggcac ccactggctg
104341 ttcctcttgg ccttctgttc cctgggagac
acagacgccc gtccgctcag cctatgggcc
104401 aaaagccgcc ccccagccgc caggttgtgg
ccagtggacg cccgccatgc ccctctgggc
104461 ccaggccccc atggggacct ctgtgcgccc
agctccgcgg tggttattcc ccaggctcca
104521 agcggcacct gctcggggtc accagtttta
ggggaggagg agagggcagg ggccccagcc
104581 cagtctgtga gctgtcaccc ccaggctcca
agcggcacct gctcggggtc accagtttta
104641 ggggaggagg agagggcagg ggccccagcc
cagtctgtga gctgtcaccc ccaggctcca
104701 agcggcacct gctcggggtc accagtttta
ggggaggagg agagggcagg ggccccagcc
104761 cagtctgtga gctgtcaccc ccaggctcca
agcggcacct gctcggggtc accagtttta
104821 ggggaggagg agagggcagg ggccccagcc
cagtctgtga gctgtcaccc gtgctatgtg
104881 ctgggctggg cactcaggaa agagggtcag
ggttcacggg ggggtggcgc gcagatttcc
104941 aggagagccc cgagggcagc agagaggagg
ctcaggtcaa tggttgggca gggggccagg
105001 gctggagaca cagagagggt cccgattcgg
gggggtgccc tcagcaggtg gctgggagtc
105061 cctgggggtt tgcacacttt cgatcaggct
gttatttcag acgcttggtc cagcctgaga
105121 caggtaatgc ctctggcctc cgggccttca
gggatggaaa gatactctag aaagcgggac
105181 tcaaagtaac tcaaggaact cgcgtcccac
agtggggagc ccttctctcc aatttacatg
105241 gggcgtttac tacgaggaaa ataccgaagg
ccgttttgag ctgaggctcc cgggccgggc
105301 tgtccgtttg tgagactgct cgtcacccct
gggccacatc cctggtggcc aagggggcaa
105361 tcagtgcggt gactgcacga cacacctctg
cagccctgcc ccacagctgt caccatcggt
105421 gacgtccacc ccctggagaa cctgaccact
gcccggtttc ccgctaaaac agcgcccttc
105481 caggatgggg ggcagaggga gaggccttgg
ccttttcact cctcttctgc agcgggggcc
105541 cctcgcaccc cagtgcccgg gcccaggagc
gccccttggg gtggggcagg gagggatcca
105601 cacaccaagg ggagccagga cccccccaaa
tctgctgccc tgccctgata cccgagacct
105661 ggggaaacgg gggactgggg ctgatgcggg
caggaccaag aactgaggcg gtgagacggg
105721 gtccccacca caggccatct ggctggcagt
ttctactccg ggcctgcagg ccaagaggga
105781 aaaggtgccc cactcagatc aggcgcctcc
cgtccccagg gagggcctac aaggtcagat
105841 cctttgtaac ttccacgggc aaaactggct
tgctgggcct gtgcgggccg catgggcgtg
105901 gaccaccaca cctttcccca ctgagtctcc
agccggagct gtcacccagg tccccccagg
105961 ccagccccac cccgccacct tgcagtagcc
tctcgtatcc aggccgaggc tgcccggtcg
106021 acccctcctg cctgatggcc tcaagtggac
aatgcgagtc acgttgcagc acgtgagtgg
106081 gacgggcagc gccacgcggg gtccgggcat
ccgagtccca ccactcagcc tcccttccgc
106141 tgcagagagg tctgtccaag agccctgggg
gccatccagc ccctgtccga cctggccggt
106201 gtggaagagg gggtgtgcca cccctcctgg
ggggctggct gggcgctggg caggcccctc
106261 ctaagagtgg agcccactgg tggttttcct
gcagccccac ctccacacag cagttctcac
106321 tgcccagtaa caggaggcta ctggcctagc
tctctccctc gtgtgatgga ctcaaccagg
106381 agcgttcacg gccccacaca gggttctcgg
ctgctgcatg aggatctcaa agccccatcc
106441 acgtgcatgt aatctcctcc ggtaacttct
ctagggaagc ccggctatcc tgccatcctc
106501 accgcaccac cagggcgaga aaagccatct
ccagcgctca catccacaat gggccaggcc
106561 gtgagcacac caccttcttc gggaggttgt
gggggcgggn nnnnnnnnnn nnnnnnnnnn
106621 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
106681 nnnnnnnnnn nnnnnnnnng cgcgcccccc
ccccccgcgg cgccggcacc ccgggcggcg
106741 gcccccggcg ctgggagcag gtgcggggcc
gcggccgctc gtgagcctcc agcccggagg
106801 acgggccccg ggggccggcc cggtgcccag
gccctgggag ccccggaggc cagagtgcca
106861 gagggccgga ggacccggga aggcccgaga
gaggtgggaa gcacggggtt ccagccctag
106921 gccatttcag ccccaaagcc atcggtgaaa
ccattgctgg ccccagataa aagcgtcgcc
106981 aactttttca ccccggcgga gactttagcg
ggtagctgcc ccctaggggg aatggaaaaa
107041 ccaggattta ccaggtgggt ggaggtcaca
actgcccaga tcctgagaaa gaggggtcag
107101 tggggcggga agattagtgg ggagaggagc
tttcagaacc caagggaatg aaacgaggct
107161 tgaggttggt tatccagcag ccgccccctg
ccccgtgagt gagcgaaggc tgggcccctt
107221 attgtcacat cttccagctc ttcgctagaa
aacctagagt tttaaatact gtggcagctg
107281 agtcaaacaa taaggaaaag cccgactctt
tgagagccag gcacaaggcg tctgtgacag
107341 ggtctccagg ctgcccattt gcagtctctg
aaacggaggg tttttcgaga aggaggtctt
107401 ggggtgcctg ccagaattgg aggggggggc
gcgggaagtg aggacccaga agagagggct
107461 tggcccgctg caaggaggtc actggacact
ggagctgaag cgccagccga aactggaaac
107521 tcgaaatctg tctccgtgcc agccacaagg
cctatgattt tccttggcga cgttcagcat
107581 cttaggagga gctggcgggg gaggcgggta
gttcgtgggc ggttgcagca gggcaggaag
107641 gtgaggaacc tgaggctggt cagagagctg
gttggagtga tgcccatcgg tggacccgct
107701 ggagaaggcc tgagtagaga aggtctaagc
ttaacgggga aggggtgggc cagggtggaa
107761 atggggtggg aagtttgagg agggggagca
gtggagatgg gggttgtgag gaatgggagt
107821 gagcttagac gtcttgagga tactgcagtt
ctgtgctttt tttcacacct ggctgaaaat
107881 tcactgaaaa caaaacaacc cttgctctgt
gacagcctag aggggtggga gggaggctta
107941 agagggaggg gacgtgcgtg tgcctatggg
cgattcatgt gggtgtacgg cagaaagcaa
108001 cacagtatgt aattaccctc caattaaaga
tcaagtacaa cttaaaaacc ccaaacacaa
108061 cattgtaagt cagctagact ccagtaaaca
tttcagtaag aagattcaac tgggaatgag
108121 ttccgccgtg actatcctga tgaatttccc
gtgtcttctt gaggccattc ctctttgaac
108181 ttccgtgttt ggggaagcgt gcctttgtat
ggagtcctga ggagtaaatg agacgggctt
108241 gtagaaggcc tagtagtgcc ttgcacgcgg
cagatgctca ataacctcga gttgtcacca
108301 ttatggtacc tcaagagtct ccttggagct
tgcacggttt ctgaatgggg tcctgcgggg
108361 ctcccttggg gctcccacat ggggttgggg
ggctgagtgg ggtgtccccg ctccttgctt
108421 gtcccctgtg gaacaccccc ttccacccga
gcagctctgc ttttgtctct tgtgtttgtt
108481 tatatctcct agattgttgt tcagtcgctc
agtcgtgtcc aactctccga ccccatggac
108541 tgcagcacac caggccttct gccttcacca
tctcccggag cttgctcaaa ctcctgtcca
108601 ttgagttgct gatgccgtcc aaccatctcg
tcctctgtcg tccccttctc cttttgacct
108661 cagtctttcc cagcatcagg gtcttttcca
atgagtcagc tctttgactc aggtggccaa
108721 gtattggagc ttcagcttca ttatcagtcc
ttccaatgaa tattcagggt tgatttcttt
108781 taggattgag tgacttgatc tccttgcagt
ccaagggact ctcaagagtc ttcaacacca
108841 cagttcaaaa gcatcagttc ttcggcactc
agccttcttt atgatccaac gcccacatcg
108901 gtacatgact actggaaaaa ctttggctca
gagataattg acttgattga atacaaagtt
108961 ctttggcaaa aaataaaagt gtggcaagca
gtactgacac aaaagcaagt ggcttttcct
109021 ccgttgagtc atttatttat tcagtgggtg
tgtgcgtgta gagacggagc ggctgtgctg
109081 ggagctgggg cttccacttc agaggagccc
cggacctgcc ctcggggagt tcacaggcag
109141 tgctgcgggg ggtcctgcca ggacgcctgc
cctgcgagtg cccagtgctg tgatggatgc
109201 gtgtcccgca tctgcggcca ctggggccac
gtgcccgaga ttgtccgggt ctgagggtgc
109261 agagaagagg aggcatttgg actgagtctg
gaaaaatgag catgtggcca cgtgagaagc
109321 cagtggtgag gggaccagtc aggcggagga
aagagcggct catacgagtt gtggagctgg
109381 aagcatgagg gtgtgtggaa gcagaggccg
gggacagggc cgcagggccg gccatggagg
109441 gcgtgggctg ctgcaggctc ctgagaaggg
ggacgctgcc atcatgaccg ggtttaggtg
109501 tttgaccctg gtgtccacgt agaggacaga
tgtgtggggg gggagctgga gatgggcatc
109561 catcgggagt cagcctggag agaggcagag
accccgtcag tgggccctca ggacgtggat
109621 ggggcggatg ttgggaagat ctgactcctg
ggttccggct ggggctccgg gctggagggg
109681 tgccgcccac cgagcacagg aggcaaacag
atgccctctc ccagcaagac cccagcccca
109741 gcaccctccg gggccggact ccgcccctct
tccagaatgg ctcccttgct gtcctcgccc
109801 atctttccgg tgccctgagc ctctagagtc
tggacaccag cgtccgcctt gcgcttgttt
109861 ctgggaagtc tctggcttgt ctctgactca
cccaggaccg tcttcgaggg caaggttgtg
109921 tccttggttc catctgcttt ggggtccggc
tcctcgctgc ttgacctgct gatgtgacag
109981 tgtctcttgt tttcttttca gaatccgaga
gcagctgtgt gtgtcccaga cagacccagc
110041 cgctgggatg acgggcccct ctgtggagat
ccccccggcc gccaagctgg gtgaggcttt
110101 cgtgtttgcc ggcgggctgg acatgcaggc
agacctgttc gcggaggagg acctgggggc
110161 cccctttctt caggggaggg ctctggagca
gatggccgtc atctacaagg agatccctct
110221 cggggagcaa ggcagggagc aggacgatta
ccggggggac ttcgatctgt gctccagccc
110281 tgttccgcct cagagcgtcc ccccgggaga
cagggcccag gacgatgagc tgttcggccc
110341 gaccttcctc cagaaaccag acccgactgc
gtaccggatc acgggcagcg gggaagccgc
110401 cgatccgcct gccagggagg cggtgggcag
gggtgacttg gggctgcagg ggccgcccag
110461 gaccgcgcag cccgccaagc cctacgcgtg
tcgggagtgc ggcaaggcct tcagccagag
110521 ctcgcacctg ctccggcacc tggtgattca
caccggggag aagccgtatg agtgcggcga
110581 gtgcggcaag gccttcagcc agagctcgca
cctgctccgg caccaggcca tccacaccgg
110641 ggagaagccg tacgagtgcg gcgagtgcgg
caaggccttc cggcagagct cggccctggc
110701 gcagcacgcg aagacgcaca gcgggaggcg
gccgtacgtc tgccgcgagt gcggcaagga
110761 cttcagccgc agctccagcc tgcgcaagca
cgagcgcatc cacaccgggg agaagcccta
110821 cgcgtgccag gagtgcggca aggccttcaa
ccagagctcg ggcctgagcc agcaccgcaa
110881 gatccactcg ctgcagaggc cgcacgcctg
cgagctgtgc gggaaggcct tctgccaccg
110941 ctcgcacctg ctgcggcacc agcgcgtcca
cacgggcaag aagccgtacg cctgcgcgga
111001 ctgcggcaag gccttcagcc agagctccaa
cctcatcgag caccgcaaga cgcacacggg
111061 cgagaggccc taccggtgcc acaagtgcgg
caaggccttc agccagagct cggcgctcat
111121 cgagcaccag cgcacccaca cgggcgagag
gccttacgag tgcggccagt gcggcaaggc
111181 cttccgccac agctcggcgc tcatccagca
ccagcgcacg cacacgggcc gcaagcccta
111241 cgtgtgcaac gagtgcggca aggccttccg
ccaccgctcg gcgctcatcg agcactacaa
111301 gacgcacacg cgcgagcggc cctacgagtg
caaccgctgc ggcaaggcct tccggggcag
111361 ctcgcacctc ctccgccacc agaaggtcca
cgcggcggac aagctctagg gtccgcccgg
111421 ggcgagggca cgccggccct ggcgcccccg
gcccagcggg tggacctggg gggccagccg
111481 gacggcggaa tcccggccgg ctcttctctg
ccgtgacccc ggggggttgg ttttgccctc
111541 cattcgcttt ttctaaagtg cagacgaata
cacgtcagag ggacgaagtg gggttaagcc
111601 cccgggagac gtccggcgag ctctaacgtc
agacacttga agaagtgaag cggactcgca
111661 gcccgtacag cccggggaag atgagtccaa
agtcgagggt caccttggcc actgcagggt
111721 cgctcggcgg tggggcggag cgggtgcagg
agggctcctc ctgggcttgg ggtggcaggc
111781 gaggaccccg cgcctctcag ccctcggcct
gggttggctg agggcgggcc tggctgtagg
111841 ccctccagcg gaggtggagg cgctgcccgg
ctcagccagg cacaggaccc tgccacgagg
111901 agtagccctc cgccagaccc ggcgtccagg
ctggggcgcc tgcggggcct ccgttctgtg
111961 gctgggcagc ctgcgccctg tccagggatg
aaggggttcc ggtctgaagg gctgggttca
112021 gggtccagct ctggcccctc ctgccttggt
gtcctggagg aagccccaag gctccgtttc
112081 cctctccagg aggtggggac gttgggaatg
ccacattccc ctggggggtg tgtgtgtgtg
112141 ttcaaggctc ccattcagac tgggactggg
cactcacgag ctttggcaac tggcaactga
112201 ggacggagac ccagggtgac accccacctc
ctgctgcggc ccccccggca ggggagacac
112261 aggcccgtct ggttcccaag atggcagggc
ccctccccct ccagcttgtg ccctgggtgt
112321 ggtgcctggg gctacagcga ccctttccgg
ttccccgggc cagttcagct gggcatcctc
112381 agggcggggc tctgagggtg ccatgtttcc
agagctcctc ctcctcccac cagtagcagg
112441 cgggcggcca gctcccaggc agccccctgg
catcgcctag gtgcacacct gcccgctgtg
112501 acccagcaag gcttgaaggt ggccatccca
gttaagtccc ctgcccctgg cccaggaatg
112561 ggctcgggca gggccgcatc tggctgcccc
agaagcgtct gtccctggcc tctgggagtt
112621 ggcggtggtc tctggtactg tccctcgcag
ggccccttag cactgctcgg ggaggaggtg
112681 ggctgaactg attttgaagt tttacatgtc
tgcggccgca gtcctacgag cccgtcaggg
112741 tcatgctggt tatttcagca gatggggctt
ggctcggcag ctaggatggt cctgaataaa
112801 aatgggaagg ccagagctgt tcctccatca
gcaggcttgg cagctgggga cgttgaaagg
112861 acaggtctgc tggtctgggg agaccagctc
tgtgcagccc ctgctgtccg tgggggtact
112921 aaaccagccc ctgtgtgcgc ccatctgagt
ggcagcccgc ctggaggatc gcccatcact
112981 tgtgagaatt gagagaatgc tgacaccccc
gcttggtgca gggggacagg gccccctaag
113041 atctacctcc ttgccccacc cccgggaccc
cctcagcctt ggccaggact gtccttactg
113101 ggcagggcag tcatccactt ccaacctttg
ccgtctcctc cgcgcgctgt gctcccagcc
113161 aaattgtttt atttttttcc aagcatcact
ttgcacacgt caccactctc cttaaaacca
113221 cccttccgga gtctcctgct cgtaaatcgc
cggtttcagc caacctgggt cgccccccaa
113281 gcccagcaag cctgctgagc cccgcgcctc
ccagctactt cacgctcgcc tcaagcttct
113341 aaacgcggac cttctccccc ccacccccat
ccctttcttt tctgatttat gtaacacggc
113401 aggtaagact cctctcctga agggttgaca
gactcacaca aaaccgtggt cagaccaggc
113461 aagtgctttt tttcagaagt gtgagcggaa
cctagtcttc agctcatgct ctttccttgt
113521 tttcttatgt gttctaagtc ctttgacttg
ggctcccaga cagcgacgtt gtaagaggcc
113581 gtcctggtag catttgaatt gtcctcgagt
ttcgttgtcg gattttgttt tattgtctta
113641 gttttccctt cttttagcag acgttgttga
ctgtcgtaaa gctccagttc ttggttctgt
113701 ttactaatca aattgttttg tcaaagtaca
tgtattctgc tcttttcttt atcttttttg
113761 ttgcttaata ttaacacttt acatttctaa
gattaattat ttaggtaatt aataattttt
113821 aacatttcta gtaaacgtgg gtacttgggt
ctgtgtttgt tttcttgtag ttacagcttt
113881 ttctgctcta tactgttgac gtctgggttt
ttttttgctc ttaggaattt ccctttgacc
113941 ccattattat tattttaatt agtatttttt
aataattaaa aattagtgtt tttaaattaa
114001 ccctaatcct aaccccagtg atgactgctt
cagtcattgc tgttacttat tatgtgctgg
114061 tgtcaggatt tttaagtgtc catagacatt
ctctgagcct gaatatatta tcagttttat
114121 acagcatttg tgtactctca agaaacgtgt
tttcactctg tcagttcggt ttgttacctc
114181 agtctttatg ttattttgct ccagtccgca
cttgctctaa cttgtcttcc cttcgaggtg
114241 tgaggacgcc tggcagccgg tgagcatgcc
ggggtccggg gtcgtgggcc caggcgccca
114301 gcaaagccct gtgggtgtgt gcacggctgg
gctgctccgg gaggaagcct gtggccccac
114361 ggtagttagg agcgctggtt tacctggtca
caccacggtc tggttttgtg tgcttttccc
114421 tgacgtgttt ctgttttgcc ttggtttcta
ttctgtttta tgagtgccgt ttacgctttg
114481 ttagtcatgc cgttatctcg atagacaggg
tgtacgtgat caagtgatta ccgtatttgg
114541 agcagatgtc tatttaacag agatgaactg
agaacctgtg cctttgcatg ccctctttgc
114601 ctcttttaat gcttctagct tcaacttctc
ttttccaaac attataatgg aaaccccttg
114661 cttttttttt tttaatttgc atttgcatga
gagtttattt agctcggcat tttattttta
114721 aaatttgtgt atatattttt gctatatatc
tgtaacttat aaacagcaaa ttattggatt
114781 ttgctttctg attctttctg taattcttct
tacataagaa gttctcctat gagtaacatt
114841 gctgtttaga gtgaggcatg atttatttcc
agcttagtat gtattgggtc ggttaacccc
114901 caaaggtcat gctcatcccc gccccatctc
tgtgagttat tgtccgagtg tggagcgccc
114961 tgtctaggcc gacgagagac ccaccatcgg
gcacacctgc ccctcctggt ctggtcagtg
115021 ccgggctctg tcctgagtcc actcctgatg
tcacaggctg gtgcttcagc gacctcggct
115081 gtgacacgga gggtgtgatg gcactgccca
gccccatggg gcttggagga ctaaaggatg
115141 cacacctgcc tggcagactg agggcacagg
tgtttctcac actgtcagcg ttttgaaata
115201 ttcctttgat tttctaccct aactcccaaa
ggccgttcaa cataagctag aatgctacgt
115261 ggtgcttgat tacattttag aaaagtttca
gcaaatacca cgagatgcag caaagaacta
115321 gacctcacag atcaggccgc ctgcataagg
gagcccacac agtcgtggga gacggggacc
115381 ctctcccacg tcctgtctgt cccaggatgg
tcccctcacc cgccccctct ctcccctcgc
115441 cctcctgtgg tgggggccgg ccaccatcac
agctgcagag cctcaagaag ggggtcgccc
115501 tggccactcc cgtggcagga gggacacgag
ggcaggagct taccgcgggt gcagtggtct
115561 cggatcagct cagctggccg ctgcggggtc
ggggggacag ttcagtggga ggcaggagcc
115621 cccactacag ctgccaggac ttctcagagg
tgacaagggg gttcagtcac ctcagcccag
115681 gtggaaacca aatggcctct tgcgcggctc
ctggggccac gcggaggttc gctgggatca
115741 caggtatctg gatgtgtgcg ccatggacat
gcaccacctt cggggggtaa ggggtgggga
115801 aaggcagccc ctttcttttg ggggaccccc
tcttcagtgt ctgataacca ggaaaccaaa
115861 tcagaaggtg gtctgggggt gctgagcagg
gtgtctccta caccacaggc cacacactca
115921 cacagcctcc aggactccag tggggctgag
cgctggagac tcacccacgt ttgctacccc
115981 cccacccaag gccatcccag aacagctgcc
tgcgtcctca cggctggccc ctcccctctg
116041 gtctaaccca gtgtgggtgg gccggcctgg
ggtctccacc tgcctcctgc tgttccctgg
116101 gctgctggct gtctgcagat gcggggccct
ggcccggaga agccccatca gagcccagag
116161 gacgggagtg gagcggggag gtgagccccg
gagtctcgag gggccagagg caaaatactg
116221 ggctgtgtcc ctggaaggca gtttcccatg
aaaccttcaa tataggccgc cccagacgat
116281 cagcctcatc tgctacgtgg attcctcccc
gtagcgaatg gtgattgggt tctacatgga
116341 cccgggactt ctgtttgaat tataatcttt
cccccactgc ccctccaggg atctggaaaa
116401 tggaggcctg ggctagacgg aagcttcctc
caagattctt tattgaaggg attcgaagag
116461 aaacaggtgg tcagtaatct gtgggggatg
gaggggtgag cgctacgtgt aacggtttta
116521 ctgttgctac gggaccagtt ttgatgtctt
tccccttcaa gaagcagacc caaacaccga
116581 gatgctgagg ttagcagcac agagcgggtt
catccacaag gcaaccaggc agggagacca
116641 gagacgctct ggaatctgcc tccctatggg
cacgggctgg gtgctcacgg atgaagacca
116701 agcagcaggt ggcgtggggc gtggggagcc
tgcggaaagc gatggacaag gtgcgggacc
116761 gcggtccgcg cggtggaccc aagctccgcc
tctgcgctgc agcgcgagct gggggcggag
116821 cttccaggga cccgcgaccg cgcccagtgg
gagggtccgc ggtccaccca gtcctaacag
116881 ctcagctcca gctagacgcc gctgagtccg
gctttctaga gagcaacccc ggcgggtatt
116941 ttatggttct ggcttcctga ttggaggaca
cgcgagtctt agaacaccct tgattagtgc
117001 gggcaggcgg aatggatttg actgatcacg
atctgcagtt tcaccatctc aggggccgcc
117061 ctcaccccca cctatcctgc caaagggggg
gcctcggtgc tgagatcggg gccacacgtg
117121 cactagacgg tcggtcagcg ctgctgctga
gcggacccgg ggccatcctc acaccgccac
117181 tggcccctgt gctcaataaa aggaaggaaa
gcgggaaaag cgctttctgg ccgcggtggc
117241 ctcgcgcgtt cctccatcgc catctgctgg
cagagcccgg catggcaccc gctgcacaga
117301 aacctcggtg tccgtttggg tgccccatcc
ttgaccccga gagagcaccc tccgtccaaa
117361 atgaaaaaca gctgctccca agagtcatta
taatcacagc caattgtgtt aattcgtcct
117421 cggatccact cacagttcca cggaacattc
tgctaacctc tgacaactcc tacataaagc
117481 aatactgaga agaaaagaac gtggttgata
aatacaaagg catacaacaa taaggagcaa
117541 agaaaaaaga cagtcctcgc agttctgttt
tgttcatctc tcatgagtag gatggcagat
117601 aaaacacaga atgcccagtg aataatttta
gtctaagtat gtccccaata ctgcctaatc
117661 ttcaaatcta accttatttt taaaatatat
attttttgct ggtcactcat cagttcatgc
117721 accaaagcct ttgtttcttg actcctaact
ttttgacccc tctggggtga ggagcacccc
117781 taacctcgag agcccatcac acagtcccct
tgggactaga cccttctttg cccatcacag
117841 ctgaccggaa gggccagccc atggccagcg
ctcgcgcccc ctggcggaca gactctgcgc
117901 ggcagccccg ggagcccagg tgcgaccccg
cggtctctgg cgccctctag tgtggaaaga
117961 tctcctcctg gtgttcccag tcattgggct
gtattttatt agagaagatg ctcgcgtgac
118021 gatgatgatg gtcctttacc gggaggcacg
tttggggcgc gtcggctcag gggccgagct
118081 attagcctgc atcgcgccca caggcatcgc
gtccccctga gccgggtcag ctgtgggctg
118141 tcctgacacg ggtttccccc agtctctggc
ccgctgtccc tcccaggtca gtgtccagcg
118201 ttgcccttct ggttgtggac ttgtgcagcg
gtctcagcag atggaggggc gaccctaaag
118261 gatgtattga ggcatctcag cactgtcctc
cgcccaggtt tgctggtcag cagtgaagtg
118321 accgggaaaa ggggctgtct tggggtcctt
tcagaggcct gggttagacc aaagttttct
118381 agaagattca ccattgcagg gagtcaaaga
caaaactagg gtggtcagca atctgtgggg
118441 gattcggcgg tgagggaatt ctgaatgcta
catgtaatgg ttttactatt gttagggaac
118501 atttttcccc cctacaaaca gcaggccaaa
atactgagat gtcaggtttg catcaaagag
118561 cgggttcatc cacaaggcaa ccagagaacg
ctctggaatc tgcctccctg cgggcacagg
118621 ctgggtgctc acggatgaag accaagcagc
aggtggcgtg gggagtgggg agcctgggga
118681 aagcgatgga caaggtgcga ggacctccgg
cgcgagctgg aggcggagct tccagggaca
118741 cgcggccacg cccagtggga gggtcagcgg
tccatccagt cctaacagct cagctccaac
118801 tagacgctgc tgagtctggc tttctagaga
acactccggg cgggtatttt attgttttgg
118861 cttcgtgact ggaggacgtt caagtcttaa
aacacccttg attagtgcgg ggaggcggaa
118921 tggatttgac tgatcacgac ccgcagtttc
accatctcag gggccgccct caccccctcc
118981 taccctacca aaggtggggg catcggtgct
gagatctggg gtgacacata aaatcaggtg
119041 aagtcttagg acagggggcc gattccaggt
cctagggtgc agaaaaaacc tacctggccc
119101 cgggctagac agcgtggagg gcgtggcccg
ggctggtgca cagaagtggc ccccaactgg
119161 tcagaaggtg tgggagccca gggctggtct
actgcagaag gggtcgcctg gtggacagag
119221 tggggcctga gtgcctgctg aactggtccg
tcagggctgc tgagcagaca cgggccatca
119281 tcactggctc ctgtgctcga tagaagggag
ggaaaccagg aaagcaaagg cgctttatgg
119341 ccgcttttgt gtttcgcgtt cctctagcac
cgtctgccgg cagaacgcgg cattacatcc
119401 gctggccaaa cctcggggtc cggcttggat
gtccccatcc ttgtctcgga gatctcacct
119461 ctcagcagtt cccctgggga caatgtcgag
aagatgcgac cttgacccgg agctcggtgg
119521 agagggtgcc ctgggttctt tccgcagttg
cttggagtgg aggtgcctca tgttgggctg
119581 ggaacgggag gaaggaaaca ggtcatgatt
gagatgctct agacagactg tccctgctct
119641 tgccaaattt cagaagattg tctttaataa
atattccatt ttttgtatgc ccttaggtct
119701 atttccagac actttaaata tattgaaaga
ctttaaatat ttatataaaa atattattta
119761 tagactgtat aaaaggaaca gttagaactg
gacttggaac aacagactgg ttccaaatag
119821 gaaaaggagt acgtcaaggc tgtatattgt
caccctgctt atttaactta tatgcagagt
119881 acatcatgag aaacgctggg ctggaagaaa
cacaagctgg aatcaagatt gccgggagaa
119941 atatcaataa cctcagatat gcagatgaca
ccacccttat ggcagaaagt gaagaggaac
120001 tcaaaagcct cttgatgaag gtgaaagagg
agagcgaaaa agttggctta aagctcaaca
120061 tttagaaaac gaagatcatg gcatctggtc
ccatcacttc atggaaatag atggggaaac
120121 agttgagaca gtgtcagact ttatttttgg
gggctccaat gaaattaaaa gacgcttact
120181 tcttggaagg aaagttatga ccaacctaga
cagcatatta aaaagcagag acactacttt
120241 gccagcaaag gtccgtctag tcaaggctat
ggtttttcca gtggtcatgt atggatgtga
120301 gagttggact gtgaagaagg ctgagcaccg
aagaagtgat gcttttgaac tgtggtgttg
120361 gagaagactc ttgagaggcc cttggactgc
aaggagatcc aaccagtcca tcgtaaagga
120421 gatcaccccc tgggtggtca ttggaaggac
tgatgttgaa gctgaaactc cagtactttg
120481 gctacctaat gcgaagagct gactcattgg
aaaagaccct gatgctggga aagattgaag
120541 gtgggaggag aaggggacaa cagaggatga
gatggttgga ttgcatcact gactcgatgg
120601 acgtgagtct gagtgaagtc tgggagttgg
tgatggccag ggaggccctg gcgtgctggc
120661 ggttcatggg gtcgcaaaga gtcggccatg
actgagtgac tgaactgaac tgatccagaa
120721 atttaaaatt aatatataaa ccaaatccat
gcagacaatt ataagcatat attataaatg
120781 cataattata agcaagtata tgttatattt
ataatagttt ataatgtatt tataagcaag
120841 tatatattat tataagcata attgtaagta
gaagtaactt tgggctttcc tggtggctca
120901 gacagtaaag aatctgcctg cagtacagga
gaccgggttc gatccctggt ttggggaaat
120961 tccctggaga agggaatggc aaccaactcc
aacatgtttg cctggagaat tccatggaca
121021 gaggagcccg gaaggttgca gtccatgggg
ttgcaaagag ctggatacaa cagagtgact
121081 aacacatgta tataaataaa tttacctata
tattgtatat atatttataa acatattcag
121141 atattataaa taattagaaa catattatac
atgtatttaa atactgttat aaacataaat
121201 ttaaaaaata attttcagcc ctttggcttg
ggggtgtgtt tgtggacgtc tttgtgctac
121261 tgttcctgaa gtggagctct cccctcccaa
accagctttt gaaatgactg ggaaagcaat
121321 ggaatacata agcatcagga agatagcaac
agagctgtca ttcttcacag agggtgtgct
121381 tgagtgtgta gcaagtcccg cagaatgtag
acagattaat atagtctatt aaaaatagtg
121441 tagcaaattt acgaggtgcg atttcaagta
taaagactta ctgggtctct cagttcagtt
121501 cagtcgcttg gttgtgtccg actctttttg
accccatgga ccgcagcacg ccaggcctcc
121561 ctgtccatca ccaactcctg gagttcactc
aaactcatgt ccatcgagtc ggtgatgcca
121621 tccaaccatc tcatcctctg gcgtcccctt
ctcctcccac cttcaatctt tcccagcatc
121681 agggtctttc ccagtgagtc agttctttgc
atcaggtggc cagagtagtg gagtttcagc
121741 ttcagcatcg gtccttccaa tgaatattct
ggactgattt cctttaggat tgactggttg
121801 gatctccttg cagttcaagg gactctcaag
agtcttctcc aacagcacag tctatgaata
121861 gaatagcaaa tgaatagaga ataacattta
cgaggatata ttttaccatt gcataaaata
121921 tatcagcttg tagagaacag acttgttccc
aggggagagg gtgggtaggg atggagtggg
121981 agtttgngat cancagaagc gagctgttat
atagaagatg gataaaaagg atacacaaca
122041 atgtcctact gtgtggcacc gggacctata
ttcagtagct tgtgagaaac cataatcgac
122101 aagactgagg aaaagtatat atatatgtat
gtacttgagt tgctttgctg tacagaagaa
122161 attaacacaa cattgtaaat cgatatttca
atagaatcca cccccccaaa tatataagtt
122221 tcctggagat ggagacggca acccactcca
tttcttgcac ccaatattct tgcctggagg
122281 atcccatgga tagaggatcg caaagactcg
gacataaccc agcgactaac actttccctt
122341 tcaaatgtgt aggtttacta gcgtgaatct
acagagatgc ccaagacatt cgtttatgag
122401 gaaaactcca cacgcagctt cactgagaat
tattaaacct attaaaggga gagagcgcca
122461 ggatattcat ggattgaaag attcgatgtg
gtcaagttgc cagttttccc caaactgatt
122521 ggtaaattcc ccaggagctg gctcaaggcg
caaaattccc tttacctttt tttaagagac
122581 gaagccaagg agccgattct ggttgagaga
cgctcaggtc ctcctgcggg agagcagccc
122641 tcttcctccc ggtcgcctgg gcagtttcga
ggccacgacc agaaggactt ggctccctgt
122701 gtcgcgcact cagaagtctc cctctccgtc
ccaaggactc agaagctggg cgtcctgccc
122761 gcagcagagg aggcagcctg gaggggcccc
gcgggcacag cggtccgggt ttcagccgag
122821 ttgcccgccc cgcccctcta cctgggcgct
gccgcccggc tccggggccg gccgtgccct
122881 ccgtggccgc aaggcgtcgc tgtccccccg
ctggaagtgc tgacccggag gaaggggccc
122941 agacggaggg actcggagcc tccgagtgac
accctgggac tccgagcgct ggagcctggc
123001 gtcaccccag gcaggggcag tgggggcccg
gggcggggtc aggggcctcc cccggttctc
123061 atttgacacc gcgggggtgc gctgggcaca
gtgtccaggg gccacgttcc gagcaggggc
123121 gcgatgcagg cccgggcgcg gcctgtcccg
ggcgcgagtc cagctgcttt gcagaggtgg
123181 cggcaggtcg cagtgaccct cacagagacg
ccccactctg cggctccagg tgggcctgtg
123241 ccccccagaa gtgctgacct gtgcaccggg
aaggcacagg gccccccagc catgtctgcg
123301 atggaagagc cggaaccgcg ccatgcccgt
cctcgctgac cggcaggcac ccgccgtgtg
123361 tccacacgct gagccatctg gctccccttg
cttgacatac acccaggacc tgagtgtgca
123421 ggaagttaga aggggcaggt gtggtgacac
gatgccatcc agcatcacct gagaacctgg
123481 acaaacctca ggggcccagc ctgctctgtg
aggccccgag ggccggcccc tccccggacc
123541 cctgccttga atccggccac actgcccgcc
ttcctgctcc tgcggcttgt cagacacgcc
123601 tgagcccagg gcctgtgcac tcgctgtccc
ttctgccagg actgctcctc cccaggctct
123661 tgctggggct ccccttcttc attcgggggt
ggcctctctt gttcagtggc tcagctgtgc
123721 ccagtctttg caaccccatg gactgcagca
cgccaggctt ccctgtcctt cactagctcc
123781 tggagtttgc tcaaactcat gtccattgag
tcagtgatgc tatccaacca tctcatcctt
123841 tgctgcccac ttcttctcct gctctcaatc
tttcccagca tcagggtctt ttccaatgag
123901 ttagctctct gcatcaggag gccaaagtat
tggagcttca gcatcagtcc ttccagtgaa
123961 tatgcgaggt tgatttccct tagaattgac
tggttggatc tccttcctgt ccagagaact
124021 ctcaagagtc ttctccagca ccacagtcgg
agagcatcag ttcttcagtg atcaggtttc
124081 tttatagccc agctctcaca tcggtacatg
actattggaa aacccatagc tttgattaga
124141 tggaccttca ttggcaaagt gatgggcctt
cattggccct gctttttaat acaccatcta
124201 ggtttgtcgt agctttcctt ccaaagagca
aacatctttt aatttcctgg ctgcagtaac
124261 catccatagt gattttggag cccaagaaaa
taaaatctgc cactgtttcc actttttccc
124321 cttctatttg ctatgaagtg aggggactgg
atgccatgat cttagtttaa accagcagtt
124381 gtcaccccga ccgcttcctt tcctaaagag
ctcatcacac ctcccactgg aatgcaatgt
124441 gttgcctgtc cgcctgcttc acctcctggg
actttgctgc aggtcttggt ctctgaggcc
124501 cctgccgtat ccccagggcc cagagcagtg
ctgggcttcg agtccgatca gggactatgt
124561 gtgtggactg gatggtgctt gcttcttctg
gggaacgaga gacctgggcc tggggaacga
124621 ggggacctgg tgtgaccgga tctcctccct
cgggagagga gccaagcgag tggacacagg
124681 tcagtgtgtc ttgctcctgt gtggcaggtg
tcccgtctgt gtctgtcatc ttggcatttc
124741 ggtgtttctg tgaacccagc ccctcccctc
ctgatacccc atcccatcag cacagaggag
124801 actgggcttg gggactctct ggtcctgaga
ttcctctccg catgtgactc ccccctcctg
124861 gggggagcag gcaccgtgtg tgaggagggt
ggaagctttt caagaccccc agcttttctg
124921 tcccaggggg ctctggcagg gccttgggag
ctggaatgag ctggaatctg ggccagtggg
124981 ggtttccctg gtggtaaaga acccgcctgc
ccatgcacga ggcataagag acgcgggttc
125041 gatcactggg tcgggaagat cccctacagg
agggcatggc aacccactcc agtattcttt
125101 cctgaagaat cccttggaca gaggagcctg
gtgggctaca gtctctgggg tggcaaggag
125161 tcggacacga ctgaagcgac ttaccatgca
cgcacgcggg gtcaggggtc agggccgcgc
125221 tgcttacctg ctgtgtgacc ttagccaggt
cacacccccc aggctgtgaa agagaacagt
125281 cttcccagac tcgggcatcc aggtctttac
agacgtgcct gtgagctttg tgactctggc
125341 tctgtggccg ctagagggcg ctgtccgccg
ggccctatgt gcgtgcacgc atgtgagcat
125401 gttcgcatac gtgtgtgcat ctgtcggggg
cgcacggtgc ggggacacgg gcacgcggtc
125461 aggaacgcag cccggacacc tccacgtggc
ccgcgagtac cgtcaggtgg gggctgtggc
125521 tccgctgtgt gggtgacccg ccctcccccc
gcgaacgtgg tgcatagtga ccgcctggct
125581 gggctcctga gctcagccat cctgcccccc
gggtcagctc ccgacaggcc cagctctagg
125641 ccccaggcgt ggaccgaggc ccccaggccc
cggcctgtga gatgggacct ccgtctgggg
125701 ggctcattct gctcccggag gcctggcagg
cccctcctct ttggcattgc ataccctcgc
125761 attggggtgg gtaagcacag taccccatgc
ctgtggcccc gtgggagcgg cctgctcagg
125821 gaggccggag cctcagctac agggctgtca
caccgggctg cagaggaaga agacgggagc
125881 gaggcctaca ggaacctagc caggccctgg
cccactgagc cgacaggagc ctggccagag
125941 gcctgcacag gacggggtgg cggggggggt
ggggtggggt gctgggcccc gtggccttga
126001 ctgcagaccc cgagggctcc tcagcttaga
acggccaagc ctgagtcttg ggggtgcagg
126061 tcaggggg
Primers
In another embodiment, primers are provided to generate 3′ and 5′ sequences of a targeting vector. The oligonucleotide primers can be capable of hybridizing to porcine immunoglobulin genomic sequence, such as Seq ID Nos. 1, 4, 29, 30, 12, 25, 15, 16, 19, 28 or 31, as described above. In a particular embodiment, the primers hybridize under stringent conditions to Seq ID Nos. 1, 4, 29, 30, 12, 25, 15, 16, 19, 28 or 31, as described above. Another embodiment provides oligonucleotide probes capable of hybridizing to porcine heavy chain, kappa light chain or lambda light chain nucleic acid sequences, such as Seq ID Nos. 1, 4, 29, 30, 12, 25, 15, 16, 19, 28 or 31, as described above. The polynucleotide primers or probes can have at least 14 bases, 20 bases, 30 bases, or 50 bases which hybridize to a polynucleotide of the present invention. The probe or primer can be at least 14 nucleotides in length, and in a particular embodiment, are at least 15, 20, 25, 28, or 30 nucleotides in length.
In one embodiment, primers are provided to amplify a fragment of porcine Ig heavy-chain that includes the functional joining region (the J6 region). In one non-limiting embodiment, the amplified fragment of heavy chain can be represented by Seq ID No 4 and the primers used to amplify this fragment can be complementary to a portion of the J-region, such as, but not limited to Seq ID No 2, to produce the 5′ recombination arm and complementary to a portion of Ig heavy-chain mu constant region, such as, but not limited to Seq ID No 3, to produce the 3′ recombination arm. In another embodiment, regions of the porcine Ig heavy chain (such as, but not limited to Seq ID No 4) can be subcloned and assembled into a targeting vector.
In other embodiments, primers are provided to amplify a fragment of porcine Ig kappa light-chain that includes the constant region. In another embodiment, primers are provided to amplify a fragment of porcine Ig kappa light-chain that includes the J region. In one non-limiting embodiment, the primers used to amplify this fragment can be complementary to a portion of the J-region, such as, but not limited to Seq ID No 21 or 10, to produce the 5′ recombination arm and complementary to genomic sequence 3′ of the constant region, such as, but not limited to Seq ID No 14, 24 or 18, to produce the 3′ recombination arm. In another embodiment, regions of the porcine Ig heavy chain (such as, but not limited to Seq ID No 20) can be subcloned and assembled into a targeting vector.
II. Genetic Targeting of the Immunoglobulin Genes
The present invention provides cells that have been genetically modified to inactivate immunoglobulin genes, for example, immunoglobulin genes described above. Animal cells that can be genetically modified can be obtained from a variety of different organs and tissues such as, but not limited to, skin, mesenchyme, lung, pancreas, heart, intestine, stomach, bladder, blood vessels, kidney, urethra, reproductive organs, and a disaggregated preparation of a whole or part of an embryo, fetus, or adult animal. In one embodiment of the invention, cells can be selected from the group consisting of, but not limited to, epithelial cells, fibroblast cells, neural cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes (B and T), macrophages, monocytes, mononuclear cells, cardiac muscle cells, other muscle cells, granulosa cells, cumulus cells, epidermal cells, endothelial cells, Islets of Langerhans cells, blood cells, blood precursor cells, bone cells, bone precursor cells, neuronal stem cells, primordial stem cells, hepatocytes, keratinocytes, umbilical vein endothelial cells, aortic endothelial cells, microvascular endothelial cells, fibroblasts, liver stellate cells, aortic smooth muscle cells, cardiac myocytes, neurons, Kupffer cells, smooth muscle cells, Schwann cells, and epithelial cells, erythrocytes, platelets, neutrophils, lymphocytes, monocytes, eosinophils, basophils, adipocytes, chondrocytes, pancreatic islet cells, thyroid cells, parathyroid cells, parotid cells, tumor cells, glial cells, astrocytes, red blood cells, white blood cells, macrophages, epithelial cells, somatic cells, pituitary cells, adrenal cells, hair cells, bladder cells, kidney cells, retinal cells, rod cells, cone cells, heart cells, pacemaker cells, spleen cells, antigen presenting cells, memory cells, T cells, B cells, plasma cells, muscle cells, ovarian cells, uterine cells, prostate cells, vaginal epithelial cells, sperm cells, testicular cells, germ cells, egg cells, leydig cells, peritubular cells, sertoli cells, lutein cells, cervical cells, endometrial cells, mammary cells, follicle cells, mucous cells, ciliated cells, nonkeratinized epithelial cells, keratinized epithelial cells, lung cells, goblet cells, columnar epithelial cells, squamous epithelial cells, osteocytes, osteoblasts, and osteoclasts. In one alternative embodiment, embryonic stem cells can be used. An embryonic stem cell line can be employed or embryonic stem cells can be obtained freshly from a host, such as a porcine animal. The cells can be grown on an appropriate fibroblast-feeder layer or grown in the presence of leukemia inhibiting factor (LIF).
In a particular embodiment, the cells can be fibroblasts; in one specific embodiment, the cells can be fetal fibroblasts. Fibroblast cells are a suitable somatic cell type because they can be obtained from developing fetuses and adult animals in large quantities. These cells can be easily propagated in vitro with a rapid doubling time and can be clonally propagated for use in gene targeting procedures.
Targeting Constructs
Homologous Recombination
In one embodiment, immunoglobulin genes can be genetically targeted in cells through homologous recombination. Homologous recombination permits site-specific modifications in endogenous genes and thus novel alterations can be engineered into the genome. In homologous recombination, the incoming DNA interacts with and integrates into a site in the genome that contains a substantially homologous DNA sequence. In non-homologous (“random” or “illicit”) integration, the incoming DNA is not found at a homologous sequence in the genome but integrates elsewhere, at one of a large number of potential locations. In general, studies with higher eukaryotic cells have revealed that the frequency of homologous recombination is far less than the frequency of random integration. The ratio of these frequencies has direct implications for “gene targeting” which depends on integration via homologous recombination (i.e. recombination between the exogenous “targeting DNA” and the corresponding “target DNA” in the genome).
A number of papers describe the use of homologous recombination in mammalian cells. Illustrative of these papers are Kucherlapati et al., Proc. Natl. Acad. Sci. USA 81:3153-3157, 1984; Kucherlapati et al., Mol. Cell. Bio. 5:714-720, 1985; Smithies et al, Nature 317:230-234, 1985; Wake et al., Mol. Cell. Bio. 8:2080-2089, 1985; Ayares et al., Genetics 111:375-388, 1985; Ayares et al., Mol. Cell. Bio. 7:1656-1662, 1986; Song et al., Proc. Natl. Acad. Sci. USA 84:6820-6824, 1987; Thomas et al. Cell 44:419-428, 1986; Thomas and Capecchi, Cell 51:503-512, 1987; Nandi et al., Proc. Natl. Acad. Sci. USA 85:3845-3849, 1988; and Mansour et al., Nature 336:348-352, 1988. Evans and Kaufman, Nature 294:146-154, 1981; Doetschman et al., Nature 330:576-578, 1987; Thoma and Capecchi, Cell 51:503-512, 4987; Thompson et al., Cell 56:316-321, 1989.
The present invention can use homologous recombination to inactivate an immunoglobulin gene in cells, such as the cells described above. The DNA can comprise at least a portion of the gene(s) at the particular locus with introduction of an alteration into at least one, optionally both copies, of the native gene(s), so as to prevent expression of functional immunoglobulin. The alteration can be an insertion, deletion, replacement or combination thereof. When the alteration is introduce into only one copy of the gene being inactivated, the cells having a single unmutated copy of the target gene are amplified and can be subjected to a second targeting step, where the alteration can be the same or different from the first alteration, usually different, and where a deletion, or replacement is involved, can be overlapping at least a portion of the alteration originally introduced. In this second targeting step, a targeting vector with the same arms of homology, but containing a different mammalian selectable markers can be used. The resulting transformants are screened for the absence of a functional target antigen and the DNA of the cell can be further screened to ensure the absence of a wild-type target gene. Alternatively, homozygosity as to a phenotype can be achieved by breeding hosts heterozygous for the mutation.
Targeting Vectors
In another embodiment, nucleic acid targeting vector constructs are also provided. The targeting vectors can be designed to accomplish homologous recombination in cells. These targeting vectors can be transformed into mammalian cells to target the ungulate heavy chain, kappa light chain or lambda light chain genes via homologous recombination. In one embodiment, the targeting vectors can contain a 3′ recombination arm and a 5′ recombination arm (i.e. flanking sequence) that is homologous to the genomic sequence of ungulate heavy chain, kappa light chain or lambda light chain genomic sequence, for example, sequence represented by Seq ID Nos. 1, 4, 29, 30, 12, 25, 15, 16, 19, 28 or 31, as described above. The homologous DNA sequence can include at least 15 bp, 20 bp, 25 bp, 50 bp, 100 bp, 500 bp, 1 kbp, 2 kbp, 4 kbp, 5 kbp, 10 kbp, 15 kbp, 20 kbp, or 50 kbp of sequence, particularly contiguous sequence, homologous to the genomic sequence. The 3′ and 5′ recombination arms can be designed such that they flank the 3′ and 5′ ends of at least one functional variable, joining, diversity, and/or constant region of the genomic sequence. The targeting of a functional region can render it inactive, which results in the inability of the cell to produce functional immunoglobulin molecules. In another embodiment, the homologous DNA sequence can include one or more intron and/or exon sequences. In addition to the nucleic acid sequences, the expression vector can contain selectable marker sequences, such as, for example, enhanced Green Fluorescent Protein (eGFP) gene sequences, initiation and/or enhancer sequences, poly A-tail sequences, and/or nucleic acid sequences that provide for the expression of the construct in prokaryotic and/or eukaryotic host cells. The selectable marker can be located between the 5′ and 3′ recombination arm sequence.
Modification of a targeted locus of a cell can be produced by introducing DNA into the cells, where the DNA has homology to the target locus and includes a marker gene, allowing for selection of cells comprising the integrated construct. The homologous DNA in the target vector will recombine with the chromosomal DNA at the target locus. The marker gene can be flanked on both sides by homologous DNA sequences, a 3′ recombination arm and a 5′ recombination arm. Methods for the construction of targeting vectors have been described in the art, see, for example, Dai et al., Nature Biotechnology 20: 251-255, 2002; WO 00/51424.
Various constructs can be prepared for homologous recombination at a target locus. The construct can include at least 50 bp, 100 bp, 500 bp, 1 kbp, 2 kbp, 4 kbp, 5 kbp, 10 kbp, 15 kbp, 20 kbp, or 50 kbp of sequence homologous with the target locus. The sequence can include any contiguous sequence of an immunoglobulin gene.
Various considerations can be involved in determining the extent of homology of target DNA sequences, such as, for example, the size of the target locus, availability of sequences, relative efficiency of double cross-over events at the target locus and the similarity of the target sequence with other sequences.
The targeting DNA can include a sequence in which DNA substantially isogenic flanks the desired sequence modifications with a corresponding target sequence in the genome to be modified. The substantially isogenic sequence can be at least about 95%, 97-98%, 99.0-99.5%, 99.6-99.9%, or 100% identical to the corresponding target sequence (except for the desired sequence modifications). In a particular embodiment, the targeting DNA and the target DNA can share stretches of DNA at least about 75, 150 or 500 base pairs that are 100% identical. Accordingly, targeting DNA can be derived from cells closely related to the cell line being targeted; or the targeting DNA can be derived from cells of the same cell line or animal as the cells being targeted.
Porcine Heavy Chain Targeting
In particular embodiments of the present invention, targeting vectors are provided to target the porcine heavy chain locus. In one particular embodiment, the targeting vector can contain 5′ and 3′ recombination arms that contain homologous sequence to the 3′ and 5′ flanking sequence of the J6 region of the porcine immunoglobulin heavy chain locus. Since the J6 region is the only functional joining region of the porcine immunoglobulin heavy chain locus, this will prevent the expression of a functional porcine heavy chain immunoglobulin. In a specific embodiment, the targeting vector can contain a 5′ recombination arm that contains sequence homologous to genomic sequence 5′ of the J6 region, optionally including J1-4 and a 3′ recombination arm that contains sequence homologous to genomic sequence 3′ of the J6 region, including the mu constant region (a “J6 targeting construct”), see for example, FIG. 1. Further, this J6 targeting construct can also contain a selectable marker gene that is located between the 5′ and 3′ recombination arms, see for example, Seq ID No S and FIG. 1. In other particular embodiments, the 5′ targeting arm can contain sequence 5′ of J1, such as depicted in Seq ID No. 1 and/or Seq ID No 4. In another embodiments, the 5′ targeting arm can contain sequence 5′ of J1, J2 and/or J3, for example, as depicted in approximately residues 1-300, 1-500, 1-750, 1-1000 and/or 1-1500 Seq ID No 4. In a further embodiment, the 5′ targeting arm can contain sequence 5′ of the constant region, for example, as depicted in approximately residues 1-300, 1-500, 1-750, 1-1000, 1-1500 and/or 1-2000 or any fragment thereof of Seq ID No 4 and/or any contiguous sequence of Seq ID No. 4 or fragment thereof. In another embodiment, the 3′ targeting arm can contain sequence 3′ of the constant region and/or including the constant region, for example, such as resides 7000-8000 and/or 8000-9000 or fragment thereof of Seq ID No 4. In other embodiments, targeting vector can contain any contiguous sequence or fragment thereof of Seq ID No 4. sequence In other embodiments, the targeting vector can contain a 5′ recombination arm that contains sequence homologous to genomic sequence 5′ of the diversity region, and a 3′ recombination arm that contains sequence homologous to genomic sequence 3′ of the diversity region of the porcine heavy chain locus. In a further embodiment, the targeting vector can contain a 5′ recombination arm that contains sequence homologous to genomic sequence 5′ of the mu constant region and a 3′ recombination arm that contains sequence homologous to genomic sequence 3′ of the mu constant region of the porcine heavy chain locus.
In further embodiments, the targeting vector can include, but is not limited to any of the following sequences: the Diversity region of heavy chain is represented, for example, by residues 1089-1099 of Seq ID No 29 (D(pseudo)), the Joining region of heavy chain is represented, for example, by residues 1887-3352 of Seq ID No 29 (for example: J(psuedo): 1887-1931 of Seq ID No 29, J(pseudo): 2364-2411 of Seq ID No 29, J(pseudo): 2756-2804 of Seq ID No 29, J (functional J): 3296-3352 of Seq ID No 29), the recombination signals are represented, for example, by residues 3001-3261 of Seq ID No 29 (Nonamer), 3292-3298 of Seq ID No 29 (Heptamer), the Constant Region is represented by the following residues: 3353-9070 of Seq ID No 29 (J to C mu intron), 5522-8700 of Seq ID No 29 (Switch region), 9071-9388 of Seq ID No 29 (Mu Exon 1), 9389-9469 of Seq ID No 29 (Mu Intron A), 9470-9802 of Seq ID No 29 (Mu Exon 2), 9830-10069 of Seq ID No 29 (Mu Intron B), 10070-10387 of Seq ID No 29 (Mu Exon 3), 10388-10517 of Seq ID No 29 (Mu Intron C), 10815-11052 of Seq ID No 29 (Mu Exon 4), 11034-11039 of Seq ID No 29 (Poly(A) signal) or any fragment or combination thereof. Still further, any contiguous sequence at least about 17, 20, 30, 40, 50, 100, 150, 200 or 300 nucleotides of Seq ID No 29 or fragment and/or combination thereof can be used as targeting sequence for the heavy chain targeting vector. It is understood that in general when designing a targeting construct one targeting arm will be 5′ of the other targeting arm.
In other embodiments, targeting vectors designed to disrupt the expression of porcine heavy chain genes can contain recombination arms, for example, the 3′ or 5′ recombination arm, that target the constant region of heavy chain. In one embodiment, the recombination arm can target the mu constant region, for example, the C mu sequences described above or as disclosed in Sun & Butler Immunogenetics (1997) 46: 452-460. In another embodiment, the recombination arm can target the delta constant region, such as the sequence disclosed in Zhao et al. (2003) J immunol 171: 1312-1318, or the alpha constant region, such as the sequence disclosed in Brown & Butler (1994) Molec Immunol 31: 633-642.
Seq ID No. 5
GGCCAGACTTCCTCGGAACAGCTCAAAGAGCTCTGTCAAAGCCAGATCCC
ATCACACGTGGGCACCAATAGGCCATGCCAGCCTGCAAGGGCCGAACTGG
GTTCTCCACGGCGCACATGAAGCCTGCAGCCTGGCTTATCCTCTTCCGTG
GTGAAGAGGCAGGCCCGGGACTGGACGAGGGGCTAGCAGGGTGTGGTAGG
CACCTTGCGCCCCCCACCCCGGCAGGAACCAGAGACCCTGGGGCTGAGAG
TGAGCCTCCAAACAGGATGCCCCACCCTTCAGGCCACCTTTCAATCCAGC
TACACTCCACCTGCCATTCTCCTCTGGGCACAGGGCCCAGCCCCTGGATC
TTGGCCTTGGCTCGACTTGCACCCACGCGCACACACACACTTCCTAACGT
GCTGTCCGCTCACCCCTCCCCAGCGTGGTCCATGGGCAGCACGGCAGTGC
GCGTCCGGCGGTAGTGAGTGCAGAGGTCCCTTCCCCTCCCCCAGGAGCCC
CAGGGGTGTGTGCAGATCTGGGGGCTCCTGTCCCTTACACCTTCATGCCC
CTCCCCTCATACCCACCCTCCAGGCGGGAGGCAGCGAGACCTTTGCCCAG
GGACTCAGCCAACGGGCACACGGGAGGCCAGCCCTCAGCAGCTGGCTCCC
AAAGAGGAGGTGGGAGGTAGGTCCACAGCTGCCACAGAGAGAAACCCTGA
CGGACCCCACAGGGGCCACGCCAGCCGGAACCAGCTCCCTCGTGGGTGAG
CAATGGCCAGGGCCCCGCCGGCCACCACGGCTGGCCTTGCGCCAGCTGAG
AACTCACGTCCAGTGCAGGGAGACTCAAGACAGCCTGTGCACACAGCCTC
GGATCTGCTCCCATTTCAAGCAGAAAAAGGAAACCGTGCAGGCAGCCCTC
AGCATTTCAAGGATTGTAGCAGCGGCCAACTATTCGTCGGCAGTGGCCGA
TTAGAATGACCGTGGAGAAGGGCGGAAGGGTCTCTCGTGGGCTCTGCGGC
CAACAGGCCCTGGCTCCACCTGCCCGCTGCCAGCCCGAGGGGCTTGGGCC
GAGCCAGGAACCACAGTGCTCACCGGGACCACAGTGACTGACCAAACTCC
CGGCCAGAGCAGCCCCAGGCCAGCCGGGCTCTCGCCCTGGAGGACTCACC
ATCAGATGCACAAGGGGGCGAGTGTGGAAGAGACGTGTCGCCCGGGCCAT
TTGGGAAGGCGAAGGGACCTTCCAGGTGGACAGGAGGTGGGACGCACTCC
AGGCAAGGGACTGGGTCCCCAAGGCCTGGGGAAGGGGTACTGGCTTGGGG
GTTAGCCTGGCCAGGGAACGGGGAGCGGGGCGGGGGGCTGAGCAGGGAGG
ACCTGACCTCGTGGGAGCGAGGCAAGTCAGGCTTCAGGCAGCAGCCGCAC
ATCCCAGACCAGGAGGCTGAGGCAGGAGGGGCTTGCAGCGGGGCGGGGGC
CTGCCTGGCTCCGGGGGCTCCTGGGGGACGCTGGCTCTTGTTTCCGTGTC
CCGCAGCACAGGGCCAGCTCGCTGGGCCTATGCTTACCTTGATGTCTGGG
GCCGGGGCGTCAGGGTCGTCGTCTCCTCAGGGGAGAGTCCCCTGAGGCTA
CGCTGGGG*GGGGACTATGGCAGCTCCACCAGGGGCCTGGGGACCAGGGG
CCTGGACCAGGCTGCAGCCCGGAGGACGGGCAGGGCTCTGGCTCTCCAGC
ATCTGGCCCTCGGAAATGGCAGAACCCCTGGCGGGTGAGCGAGCTGAGAG
CGGGTCAGACAGACAGGGGCCGGCCGGAAAGGAGAAGTTGGGGGCAGAGC
CCGCCAGGGGCCAGGCCCAAGGTTCTGTGTGCCAGGGCCTGGGTGGGCAC
ATTGGTGTGGCCATGGCTACTTAGACGCGTGATCAAGGGCGAATTCCAGC
ACACTGGCGGCCGTTACTAGTggatcccggcgcgccctaccgggtagggg
aggcgcttttcccaaggcagtctggagcatgcgctttagcagccccgctg
ggcacttggcgctacacaagtggcctctggcctcgcacacattccacatc
caccggtaggcgccaaccggctccgttctttggtggccccttcgcgccac
cttctactcctcccctagtcaggaagttcccccccgccccgcagctcgcg
tcgtgcaggacgtgacaaatggaagtagcacgtctcactagtctcgtgca
gatggacagcaccgctgagcaatggaagcgggtaggcctttggggcagcg
gccaatagcagctttggctccttcgctttctgggctcagaggctgggaag
gggtgggtccgggggcgggctcaggggcgggctcaggggcggggcgggcg
cccgaaggtcctccggaagcccggcattctgcacgcttcaaaagcgcacg
tctgccgcgctgttctcctcttcctcatctccgggcctttcgacctgcag
ccaatatgggatcggccattgaacaagatggattgcacgcaggttctccg
gccgcttgggtggagaggctattcggctatgactgggcacaacagacaat
cggctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccgg
ttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcaggac
gaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagc
tgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcg
aagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaa
gtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccggc
tacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgta
ctcggatggaagccggtcttgtcaatcaggatgatctggacgaagagcat
caggggctcgcgccagccgaactgttcgccaggctcaaggcgcgcatgcc
cgacggcgaggatctcgtcgtgacccatggcgatgcctgcttgccgaata
tcatggtggaaaatggccgcttttctggattcatcgactgtggccggctg
ggtgtggcggatcgctatcaggacatagcgttggctacccgtgatattgc
tgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggta
tcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgag
ttcttctgaggggatcaattcTCTAGATGCATGCTCGAGCGGCCGCCAGT
GTGATGGATATCTGCAGAATTCGCCCTtCCAGGCGTTGAAGTCGTCGTGT
CCTCAGGTAAGAACGGCCCTCCAGGGCCTTTAATTTCTGCTCTCGTCTGT
GGGCTTTTCTGACTCTGATCCTCGGGAGGCGTCTGTGCCCCGCCCGGGGA
TGAGGCCGGCTTGCCAGGAGGGGTCAGGGACCAGGAGCCTGTGGGAAGTT
CTGACGGGGGCTGCAGGCGGGAAGGGCCCCACCGGGGGGCGAGCCCCAGG
CCGCTGGGCGGCAGGAGACCCGTGAGAGTGCGCCTTGAGGAGGGTGTCTG
CGGAAGCACGAACGCCGGCCGGGAAGGGCTTGCTGCAATGCGGTCTTCAG
ACGGGAGGCGTCTTCTGCCCTCACCGTCTTTCAAGCCCTTGTGGGTCTGA
AAGAGCCATGTCGGAGAGAGAAGGGACAGGCCTGTCCCGACCTGGCCGAG
AGCGGGCAGCCCCGGGGGAGAGCGGGGCGATCGGCCTGGGCTCTGTGAGG
CCAGGTCCAAGGGAGGACGTGTGGTCCTCGTGACAGGTGCACTTGCGAAA
CCTTAGAAGACGGGGTATGTTGGAAGCGGCTCCTGATGTTTAAGAAAAGG
GAGACTGTAAAGTGAGCAGAGTCCTCAAGTGTGTTAAGGTTTTAAAGGTC
AAAGTGTTTTAAACCTTTGTGACTGCAGTTAGCAAGCGTGCGGGGAGTGA
ATGGGGTGCCAGGGTGGCCGAGAGGCAGTACGAGGGCCGTGCCGTCCTCT
AATTCAGGGCTTAGTTTTGCAGAATAAAGTCGGCCTGTTTTCTAAAAGCA
TTGGTGGTGCTGAGCTGGTGGAGGAGGCCGCGGGCAGCCCTGGCCACCTG
CAGCAGGTGGCAGGAAGCAGGTCGGCCAAGAGGCTATTTTAGGAAGCCAG
AAAACACGGTCGATGAATTTATAGCTTCTGGTTTCCAGGAGGTGGTTGGG
CATGGCTTTGCGCAGCGCCACAGAACCGAAAGTGCCCACTGAGAAAAAAC
AACTCCTGCTTAATTTGCATTTTTCTAAAAGAAGAAACAGAGGCTGACGG
AAACTGGAAAGTTCCTGTTTTAACTACTCGAATTGAGTTTTCGGTCTTAG
CTTATCAACTGCTCACTTAGATTCATTTTCAAAGTAAACGTTTAAGAGCC
GAGGCATTCCTATCCTCTTCTAAGGCGTTATTCCTGGAGGCTCATTCACC
GCCAGCACCTCCGCTGCCTGCAGGCATTGCTGTCACCGTCACCGTGACGG
CGCGCACGATTTTCAGTTGGCCCGCTTCCCCTCGTGATTAGGACAGACGC
GGGCACTCTGGCCCAGCCGTCTTGGCTCAGTATCTGCAGGCGTCCGTCTC
GGGACGGAGCTCAGGGGAAGAGCGTGACTCCAGTTGAACGTGATAGTCGG
TGCGTTGAGAGGAGACCCAGTCGGGTGTCGAGTCAGAAGGGGCCCGGGGC
CCGAGGCCCTGGGCAGGACGGCCCGTGCCCTGCATCACGGGCCCAGCGTC
CTAGAGGCAGGACTCTGGTGGAGAGTGTGAGGGTGCCTGGGGCCCCTCCG
GAGCTGGGGCCGTGCGGTGCAGGTTGGGCTCTCGGCGCGGTGTTGGCTGT
TTCTGCGGGATTTGGAGGAATTCTTCCAGTGATGGGAGTCGCCAGTGACC
GGGCACCAGGCTGGTAAGAGGGAGGCCGCCGTCGTGGCCAGAGCAGCTGG
GAGGGTTCGGTAAAAGGCTCGCCCGTTTCCTTTAATGAGGACTTTTCCTG
GAGGGCATTTAGTCTAGTCGGGACCGTTTTCGACTCGGGAAGAGGGATGC
GGAGGAGGGCATGTGCCCAGGAGCCGAAGGCGCCGCGGGGAGAAGCCCAG
GGCTCTCCTGTCCCCACAGAGGCGACGCCACTGCCGCAGACAGACAGGGC
CTTTCCCTCTGATGACGGCAAAGGCGCCTCGGCTCTTGCGGGGTGCTGGG
GGGGAGTCGCCCCGAAGCCGCTCACCCAGAGGCCTGAGGGGTGAGACTGA
CCGATGCCTCTTGGCCGGGCCTGGGGCCGGACCGAGGGGGACTCCGTGGA
GGCAGGGCGATGGTGGCTGCGGGAGGGAACCGACCCTGGGCCGAGCCCGG
CTTGGCGATTCCCGGGCGAGGGCCCTCAGCCGAGGCGAGTGGGTCCGGCG
GAACCACCCTTTCTGGCCAGCGCCACAGGGCTCTCGGGACTGTCCGGGGC
GACGCTGGGCTGCCCGTGGCAGGCCTGGGCTGACCTGGACTTCACCAGAC
AGAACAGGGCTTTCAGGGCTGAGCTGAGCCAGGTTTAGCGAGGCCAAGTG
GGGCTGAACCAGGCTCAACTGGCCTGAGCTGGGTTGAGCTGGGCTGACCT
GGGCTGAGCTGAGCTGGGCTGGGCTGGGCTGGGCTGGGCTGGGCTGGGCT
GGACTGGCTGAGCTGAGCTGGGTTGAGCTGAGCTGAGCTGGCCTGGGTTG
AGCTGGGCTGGGTTGAGCTGAGCTGGGTTGAGCTGGGTTGAGCTGGGTTG
ATCTGAGCTGAGCTGGGCTGAGCTGAGCTAGGCTGGGGTGAGCTGGGCTG
AGCTGGTTTGAGTTGGGTTGAGCTGAGCTGAGCTGGGCTGTGCTGGCTGA
GCTAGGCTGAGCTAGGCTAGGTTGAGCTGGGCTGGGCTGAGCTGAGCTAG
GCTGGGCTGATTTGGGCTGAGCTGAGCTGAGCTAGGCTGCGTTGAGCTGG
CTGGGCTGGATTGAGCTGGCTGAGCTGGCTGAGCTGGGCTGAGCTGGCCT
GGGTTGAGCTGAGCTGGACTGGTTTGAGCTGGGTCGATCTGGGTTGAGCT
GTCCTGGGTTGAGCTGGGCTGGGTTGAGCTGAGCTGGGTTGAGCTGGGCT
CAGCAGAGCTGGGTTGGGCTGAGCTGGGTTGAGCTGAGCTGGGCTGAGCT
GGCCTGGGTTGAGCTGGGCTGAGCTGAGCTGGGCTGAGCTGGCCTGTGTT
GAGCTGGGCTGGGTTGAGCTGGGCTGAGCTGGATTGAGCTGGGTTGAGCT
GAGCTGGGCTGGGCTGTGCTGACTGAGCTGGGCTGAGCTAGGCTGGGGTG
AGCTGGGCTGAGCTGATCCGAGCTAGGCTGGGCTGGTTTGGGCTGAGCTG
AGCTGAGCTAGGCTGGATTGATCTGGCTGAGCTGGGTTGAGCTGAGCTGG
GCTGAGCTGGTCTGAGCTGGCCTGGGTCGAGCTGAGCTGGACTGGTTTGA
GCTGGGTCGATCTGGGCTGAGCTGGCCTGGGTTGAGCTGGGCTGGGTTGA
GCTGAGCTGGGTTGAGCTGGGCTGAGCTGAGGGCTGGGGTGAGCTGGGCT
GAACTAGCCTAGCTAGGTTGGGCTGAGCTGGGCTGGTTTGGGCTGAGCTG
AGCTGAGCTAGGCTGCATTGAGCAGGCTGAGCTGGGCTGAGCAGGCCTGG
GGTGAGCTGGGCTAGGTGGAGCTGAGCTGGGTCGAGCTGAGTTGGGCTGA
GCTGGCCTGGGTTGAGGTAGGCTGAGCTGAGCTGAGCTAGGCTGGGTTGA
GCTGGCTGGGCTGGTTTGCGCTGGGTCAAGCTGGGCCGAGCTGGCCTGGG
TTGAGCTGGGCTCGGTTGAGCTGGGCTGAGCTGAGCCGACCTAGGCTGGG
ATGAGCTGGGCTGATTTGGGCTGAGCTGAGCTGAGCTAGGCTGCATTGAG
CAGGCTGAGCTGGGCCTGGAGCCTGGCCTGGGGTGAGCTGGGCTGAGCTG
CGCTGAGCTAGGCTGGGTTGAGCTGGCTGGGCTGGTTTGCGCTGGGTCAA
GCTGGGCCGAGCTGGCCTGGGATGAGCTGGGCCGGTTTGGGCTGAGCTGA
GCTGAGCTAGGCTGCATTGAGCAGGCTGAGCTGGGCTGAGCTGGCCTGGG
GTGAGCTGGGCTGAGCTAAGCTGAGCTGGGCTGGTTTGGGCTGAGCTGGC
TGAGCTGGGTCCTGCTGAGCTGGGCTGAGCTGACCAGGGGTGAGCTGGGC
TGAGTTAGGCTGGGCTCAGCTAGGCTGGGTTGATCTGGCAGGGCTGGTTT
GCGCTGGGTCAAGCTCCCGGGAGATGGCCTGGGATGAGCTGGGCTGGTTT
GGGCTGAGCTGAGCTGAGCTGAGCTAGGCTGCATTGAGCAGGCTGAGCTG
GGCTGAGCTGGCCTGGGGTGAGCTGGGCTGGGTGGAGCTGAGCTGGGCTG
AACTGGGCTAAGCTGGCTGAGCTGGATCGAGCTGAGCTGGGCTGAGCTGG
CCTGGGGTTAGCTGGGCTGAGCTGAGCTGAGCTAGGCTGGGTTGAGCTGG
CTGGGCTGGTTTGCGCTGGGTCAAGCTGGGCCGAGCTGGCCTGGGTTGAG
CTGGGCTGGGCTGAGCTGAGCTAGGCTGGGTTGAGCTGGGCTGGGCTGAG
CTGAGCTAGGCTGCATTGAGCTGGCTGGGATGGATTGAGCTGGCTGAGCT
GGCTGAGCTGGCTGAGCTGGGCTGAGCTGGCCTGGGTTGAGCTGGGCTGG
GTTGAGCTGAGCTGGGCTGAGCTGGGCTCAGCAGAGCTGGGTTGAGCTGA
GCTGGGTTGAGCTGGGGTGAGCTGGGCTGAGCAGAGCTGGGTTGAGCTGA
GCTGGGTTGAGCTGGGCTCGAGCAGAGCTGGGTTGAGCTGAGCTGGGTTG
AGCTGGGCTCAGCAGAGCTGGGTTGAGCTGAGCTGGGTTGAGCTGGGCTG
AGCTAGCTGGGCTCAGCTAGGCTGGGTTGAGCTGAGCTGGGCTGAACTGG
GCTGAGCTGGGCTGAACTGGGCTGAGCTGGGCTGAGCTGGGCTGAGCAGA
GCTGGGCTGAGCAGAGCTGGGTTGGTCTGAGCTGGGTTGAGCTGGGCTGA
GCTGGGCTGAGCAGAGTTGGGTTGAGCTGAGCTGGGTTCAGCTGGGCTGA
GCTAGGCTGGGTTGAGCTGGGTTGAGTTGGGCTGAGCTGGGCTGGGTTGA
GCGGAGCTGGGCTGAACTGGGCTGAGCTGGGCTGAGCGGAACTGGGTTGA
TCTGAATTGAGCTGGGCTGAGCCGGGCTGAGCCGGGCTGAGCTGGGCTAG
GTTGAGCTTGGGTGAGCTTGCCTCAGCTGGTCTGAGCTAGGTTGGGTGGA
GCTAGGCTGGATTGAGCTGGGCTGAGGTGAGCTGATCTGGCCTCAGCTGG
GCTGAGGTAGGCTGAACTGGGCTGTGCTGGGCTGAGCTGAGCTGAGCCAG
TTTGAGCTGGGTTGAGCTGGGCTGAGCTGGGCTGTGTTGATCTTTCCTGA
ACTGGGCTGAGCTGGGCTGAGCTGGCCTAGCTGGATTGAACGGGGGTAAG
CTGGGCCAGGCTGGACTGGGCTGAGCTGAGCTAGGCTGAGCTGAGTTGAA
TTGGGTTAAGCTGGGCTGAGATGGGCTGAGCTGGGCTGAGCTGGGTTGAG
CCAGGTCGGACTGGGTTACCCTGGGCCACACTGGGCTGAGCTGGGCGGAG
CTCGATTAACCTGGTCAGGCTGAGTCGGGTCCAGCAGACATGCGCTGGCC
AGGCTGGCTTGACCTGGACACGTTCGATGAGCTGCCTTGGGATGGTTCAC
CTCAGCTGAGCCAGGTGGCTCCAGCTGGGCTGAGCTGGTGACCCTGGGTG
ACCTCGGTGACCAGGTTGTCCTGAGTCCGGGCCAAGCCGAGGCTGCATCA
GACTCGCCAGACCCAAGGCCTGGGCCCCGGCTGGCAAGCCAGGGGCGGTG
AAGGCTGGGCTGGCAGGACTGTCCCGGAAGGAGGTGCACGTGGAGCCGCC
CGGACCCCGACCGGCAGGACCTGGAAAGACGCCTCTCACTCCCCTTTCTC
TTCTGTCCCCTCTCGGGTCCTCAGAGAGCCAGTCTGCCCCGAATCTCTAC
CCCCTCGTCTCCTGCGTCAGCCCCCCGTCCGATGAGAGCCTGGTGGCCCT
GGGCTGCCTGGCCCGGGACTTCCTGCCCAGCTCCGTCACCTTCTCCTGGAA
Porcine Kappa Chain Targeting
In particular embodiments of the present invention, targeting vectors are provided to target the porcine kappa chain locus. In one particular embodiment, the targeting vector can contain 5′ and 3′ recombination arms that contain homologous sequence to the 3′ and 5′ flanking sequence of the constant region of the porcine immunoglobulin kappa chain locus. Since the present invention discovered that there is only one constant region of the porcine immunoglobulin kappa light chain locus, this will prevent the expression of a functional porcine kappa light chain immunoglobulin. In a specific embodiment, the targeting vector can contain a 5′ recombination arm that contains sequence homologous to genomic sequence 5′ of the constant region, optionally including the joining region, and a 3′ recombination arm that contains sequence homologous to genomic sequence 3′ of the constant region, optionally including at least part of the enhancer region (a “Kappa constant targeting construct”), see for example, FIG. 2. Further, this kappa constant targeting construct can also contain a selectable marker gene that is located between the 5′ and 3′ recombination arms, see for example, Seq ID No 20 and FIG. 2. In other embodiments, the targeting vector can contain a 5′ recombination arm that contains sequence homologous to genomic sequence 5′ of the joining region, and a 3′ recombination arm that contains sequence homologous to genomic sequence 3′ of the joining region of the porcine kappa light chain locus. In other embodiments, the 5′ arm of the targeting vector can include Seq ID No 12 and/or Seq ID No 25 or any contiguous sequence or fragment thereof. In another embodiment, the 3′ arm of the targeting vector can include Seq ID No 15, 16 and/or 19 or any contiguous sequence or fragment thereof.
In further embodiments, the targeting vector can include, but is not limited to any of the following sequences: the coding region of kappa light chain is represented, for example by residues 1-549 of Seq ID No 30 and 10026-10549 of Seq ID No 30, whereas the intronic sequence is represented, for example, by residues 550-10025 of Seq ID No 30, the Joining region of kappa light chain is represented, for example, by residues 5822-7207 of Seq ID No 30 (for example, J1:5822-5859 of Seq ID No 30, J2:6180-6218 of Seq ID No 30, J3:6486-6523 of Seq ID No 30, J4:6826-6863 of Seq ID No 30, J5:7170-7207 of Seq ID No 30), the Constant Region is represented by the following residues: 10026-10549 of Seq ID No 30 (C exon) and 10026-10354 of Seq ID No 30 (C coding), 10524-10529 of Seq ID No 30 (Poly(A) signal) and 11160-11264 of Seq ID No 30 (SINE element) or any fragment or combination thereof. Still further, any contiguous sequence at least about 17, 20, 30, 40, 50, 100, 150, 200 or 300 nucleotides of Seq ID No 30 or fragment and/or combination thereof can be used as targeting sequence for the heavy chain targeting vector. It is understood that in general when designing a targeting construct one targeting arm will be 5′ of the other targeting arm.
Seq ID No. 20
ctcaaacgtaagtggctttttccgactgattctttgctgtttctaattgt
tggttggctttttgtccatttttcagtgttttcatcgaattagttgtcag
ggaccaaacaaattgccttcccagattaggtaccagggaggggacattgc
tgcatgggagaccagagggtggctaatttttaacgtttccaagccaaaat
aactggggaagggggcttgctgtcctgtgagggtaggtttttatagaagt
ggaagttaaggggaaatcgctatggttcacttttggctcggggaccaaag
tggagcccaaaattgagtacattttccatcaattatttgtgagatttttg
tcctgttgtgtcatttgtgcaagtttttgacattttggttgaatgagcca
ttcccagggacccaaaaggatgagaccgaaaagtagaaaagagccaactt
ttaagctgagcagacagaccgaattgttgagtttgtgaggagagtagggt
ttgtagggagaaaggggaacagatcgctggctttttctctgaattagcct
ttctcatgggactggcttcagagggggtttttgatgagggaagtgttcta
gagccttaactgtgggttgtgttcggtagcgggaccaagctggaaatcaa
acgtaagtgcacttttctactcctttttctttcttatacgggtgtgaaat
tggggacttttcatgtttggagtatgagttgaggtcagttctgaagagag
tgggactcatccaaaaatctgaggagtaagggtcagaacagagttgtctc
atggaagaacaaagacctagttagttgatgaggcagctaaatgagtcagt
tgacttgggatccaaatggccagacttcgtctgtaaccaacaatctaatg
agatgtagcagcaaaaagagatttccattgaggggaaagtaaaattgtta
atattgtggatcacctttggtgaagggacatccgtggagattgaacgtaa
gtattttttctctactaccttctgaaatttgtctaaatgccagtgttgac
ttttagaggcttaagtgtcagttttgtgaaaaatgggtaaacaagagcat
ttcatatttattatcagtttcaaaagttaaactcagctccaaaaatgaat
ttgtagacaaaaagattaatttaagccaaattgaatgattcaaaggaaaa
aaaaattagtgtagatgaaaaaggaattcttacagctccaaagagcaaaa
gcgaattaattttctttgaactttgccaaatcttgtaaatgatttttgtt
ctttacaatttaaaaaggttagagaaatgtatttcttagtctgttttctc
tcttctgtctgataaattattatatgagataaaaatgaaaattaatagga
tgtgctaaaaaatcagtaagaagttagaaaaatatatgtttatgttaaag
ttgccacttaattgagaatcagaagcaatgttatttttaaagtctaaaat
gagagataaactgtcaatacttaaattctgcagagattctatatcttgac
agatatctcctttttcaaaaatccaatttctatggtagactaaatttgaa
atgatcttcctcataatggagggaaaagatggactgaccccaaaagctca
gattt*aagaaaacctgtttaag*gaaagaaaataaaagaactgcatttt
ttaaaggcccatgaatttgtagaaaaataggaaatattttaataagtgta
ttcttttattttcctgttattacttgatggtgtttttataccgccaagga
ggccgtggcaccgtcagtgtgatctgtagaccccatggcggccttttttc
gcgattgaatgaccttggcggtgggtccccagggctctggtggcagcgca
ccagccgctaaaagccgctaaaaactgccgctaaaggccacagcaacccc
gcgaccgcccgttcaactgtgctgacacagtgatacagataatgtcgcta
acagaggagaatagaaatatgacgggcacacgctaatgtggggaaaagag
ggagaagcctgatttttattttttagagattctagagataaaattcccag
tattatatccttttaataaaaaatttctattaggagattataaagaattt
aaagctatttttttaagtggggtgtaattctttcagtagtctcttgtcaa
atggatttaagtaatagaggcttaatccaaatgagagaaatagacgcata
accctttcaaggcaaaagctacaagagcaaaaattgaacacagcagccag
ccatctagccactcagattttgatcagttttactgagtttgaagtaaata
tcatgaaggtataattgctgataaaaaaataagatacaggtgtgacacat
ctttaagtttcagaaatttaatggcttcagtaggattatatttcacgtat
acaaagtatctaagcagataaaaatgccattaatggaaacttaatagaaa
tatatttttaaattccttcattctgtgacagaaattttctaatctgggtc
ttttaatcacctaccctttgaaagagtttagtaatttgctatttgccatc
gctgtttactccagctaatttcaaaagtgatacttgagaaagattatttt
tggtttgcaaccacctggcaggactattttagggccattttaaaactctt
ttcaaactaagtattttaaactgttctaaaccatttagggccttttaaaa
atcttttcatgaatttcaaacttcgttaaaagttattaaggtgtctggca
agaacttccttatcaaatatgctaatagtttaatctgttaatgcaggata
taaaattaaagtgatcaaggcttgacccaaacaggagtatcttcatagca
tatttcccctcctttttttctagaattcatatgattttgctgccaaggct
attttatataatctctggaaaaaaaatagtaatgaaggttaaaagagaag
aaaatatcagaacattaagaattcggtattttactaactgcttggttaac
atgaaggtttttattttattaaggtttctatctttataaaaatctgttcc
cttttctgctgatttctccaagcaaaagattcttgatttgttttttaact
cttactctcccacccaagggcctgaatgcccacaaaggggacttccagga
ggccatctggcagctgctcaccgtcagaagtgaagccagccagttcctcc
tgggcaggtggccaaaattacagttgacccctcctggtctggctgaacct
tgccccatatggtgacagccatctggccagggcccaggtctccctctgaa
gcctttgggaggagagggagagtggctggcccgatcacagatgcggaagg
ggctgactcctcaaccggggtgcagactctgcagggtgggtctgggccca
acacacccaaagcacgcccaggaaggaaaggcagcttggtatcactgccc
agagctaggagaggcaccgggaaaatgatctgtccaagacccgttcttgc
ttctaaactccgagggggtcagatgaagtggttttgtttcttggcctgaa
gcatcgtgttccctgcaagaagcggggaacacagaggaaggagagaaaag
atgaactgaacaaagcatgcaaggcaaaaaaggGGGTCTAGCCGCGGTCT
AGGAAGCTTTCTAGGGTACCTCTAGGGATCCCGGCGCGCCCTACCGGGTA
GGGGAGGCGCTTTTCCCAAGGCAGTCTGGAGCATGCGCTTTAGCAGCCCC
GCTGGGCACTTGGCGCTACACAAGTGGCCTCTGGCCTCGCACACATTCCA
CATCCACCGGTAGGCGCCAACCGGCTCCGTTCTTTGGTGGCCCCTTCGCG
CCACCTTCTACTCCTCCCCTAGTCAGGAAGTTCCCCCCCGCCCCGCAGCT
CGCGTCGTGCAGGACGTGACAAATGGAAGTAGCACGTCTCACTAGTCTCG
TGCAGATGGACAGCACCGCTGAGCAATGGAAGCGGGTAGGCCTTTGGGGC
AGCGGCCAATAGCAGCTTTGGCTCCTTCGCTTTCTGGGCTCAGAGGCTGG
GAAGGGGTGGGTCCGGGGGCGGGCTCAGGGGCGGGCTCAGGGGCGGGGCG
GGCGCCCGAAGGTCCTCCGGAAGCCCGGCATTCTGCACGCTTCAAAAGCG
CACGTCTGCCGCGCTGTTCTCCTCTTCCTCATCTCCGGGCCTTTCGACCT
GCAGCCAATATGGGATCGGCCATTGAACAAGATGGATTGCACGCAGGTTC
TCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGA
CAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGC
CCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCA
GGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCG
CAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTG
GGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGA
GAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATC
CGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCA
CGTACTCGGATGGAAGCCGGTCTTGTCAATCAGGATGATCTGGACGAAGA
GCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCA
TGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCG
AATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCG
GCTGGGTGTGGCGGATCGCTATCAGGACATAGCGTTGGCTACCCGTGATA
TTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGGTTCCTCGTGCTTTAC
GGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGA
CGAGTTCTTCTGAGGGGATCAATTCTCTAGAGCTCGCTGATCAGCCTCGA
CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCT
TCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGA
GGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTG
GGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCAT
GCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGGGGAAAGAACCAGCTG
GGGGCGCGCCCctcgagcggccgccagtgtgatggatatctgcagaattc
gcccttggatcaaacacgcatcctcatggacaatatgttgggttcttagc
ctgctgagacacaacaggaactcccctggcaccactttagaggccagaga
aacagcacagataaaattccctgccctcatgaagcttatagtctagctgg
ggagatatcataggcaagataaacacatacaaatacatcatcttaggtaa
taatatatactaaggagaaaattacaggggagaaagaggacaggaattgc
tagggtaggattataagttcagatagttcatcaggaacactgttgctgag
aagataacatttaggtaaagaccgaagtagtaaggaaatggaccgtgtgc
ctaagtgggtaagaccattctaggcagcaggaacagcgatgaaagcactg
aggtgggtgttcactgcacagagttgttcactgcacagagttgtgtgggg
aggggtaggtcttgcaggctcttatggtcacaggaagaattgttttactc
ccaccgagatgaaggttggtggattttgagcagaagaataattctgcctg
gtttatatataacaggatttccctgggtgctctgatgagaataatctgtc
aggggtgggatagggagagatatggcaataggagccttggctaggagccc
acgacaataattccaagtgagaggtggtgctgcattgaaagcaggactaa
caagacctgctgacagtgtggatgtagaaaaagatagaggagacgaaggt
gcatctagggttttctgcctgaggaattagaaagataaagctaaagctta
tagaagatgcagcgctctggggagaaagaccagcagctcagttttgatcc
atctggaattaattttggcataaagtatgaggtatgtgggttaacattat
ttgttttttttttttccatgtagctatccaactgtcccagcatcatttat
tttaaaagactttcctttcccctattggattgttttggcaccttcactga
agatcaactgagcataaaattgggtctatttctaagctcttgattccatt
ccatgacctatttgttcatctttaccccagtagacactgccttgatgatt
aaagcccctgttaccatgtctgttttggacatggtaaatctgagatgcct
attagccaaccaagcaagcacggcccttagagagctagatatgagagcct
ggaattcagacgagaaaggtcagtcctagagacatacatgtagtgccatc
accatgcggatggtgttaaaagccatcagactgcaacagactgtgagagg
gtaccaagctagagagcatggatagagaaacccaagcactgagctgggag
gtgctcctacattaagagattagtgagatgaaggactgagaagattgatc
agagaagaaggaaaatcaggaaaatggtgctgtcctgaaaatccaaggga
agagatgttccaaagaggagaaaactgatcagttgtcagctagcgtcaat
tgggatgaaaatggaccattggacagagggatgtagtgggtcatgggtga
atagataagagcagcttctatagaatggcaggggcaaaattctcatctga
tcggcatgggttctaaagaaaacgggaagaaaaaattgagtgcatgacca
gtcccttcaagtagagaggtggaaaagggaaggaggaaaatgaggccacg
acaacatgagagaaatgacagcatttttaaaaattttttattttatttta
tttatttatttttgctttttagggctgcccctgcaacatatggaggttcc
caggttaggggtctaatcagagctatagctgccagcctacaccacagcca
tagcaatgccagatctacatgacctacaccacagctcacagcaacgccgg
atccttaacccactgagtgaggccagagatcaaacccatatccttatgga
tactagtcaggttcattaccactgagccaaaatgggaaatcctgagtaat
gacagcattttttaatgtgccaggaagcaaaacttgccaccccgaaatgt
ctctcaggcatgtggattattttgagctgaaaacgattaaggcccaaaaa
acacaagaagaaatgtggaccttcccccaacagcctaaaaaatttagatt
gagggcctgttcccagaatagagctattgccagacttgtctacagaggct
aagggctaggtgtggtggggaaaccctcagagatcagagggacgtttatg
taccaagcattgacatttccatctccatgcgaatggccttcttcccctct
gtagccccaaaccaccacccccaaaatcttcttctgtctttagctgaaga
tggtgttgaaggtgatagtttcagccactttggcgagttcctcagttgtt
ctgggtctttcctccTgatccacattattcgactgtgtttgattttctcc
tgtttatctgtctcattggcacccatttcattcttagaccagcccaaaga
acctagaagagtgaaggaaaatttcttccaccctgacaaatgctaaatga
gaatcaccgcagtagaggaaaatgatctggtgctgcgggagatagaagag
aaaatcgctggagagatgtcactgagtaggtgagatgggaaaggggtgac
acaggtggaggtgttgccctcagctaggaagacagacagttcacagaaga
gaagcgggtgtccgtggacatcttgcctcatggatgaggaaaccgaggct
aagaaagactgcaaaagaaaggtaaggattgcagagaggtcgatccatga
ctaaaatcacagtaaccaaccccaaaccaccatgttttctcctagtctgg
cacgtggcaggtactgtgtaggttttcaatattattggtttgtaacagta
cctattaggcctccatcccctcctctaatactaacaaaagtgtgagactg
gtcagtgaaaaatggtcttctttctctatgaatctttctcaagaagatac
ataactttttattttatcataggcttgaagagcaaatgagaaacagcctc
caacctatgacaccgtaacaaaatgtttatgatcagtgaagggcaagaaa
caaaacatacacagtaaagaccctccataatattgtgggtggcccaacac
aggccaggttgtaaaagctttttattctttgatagaggaatggatagtaa
tgtttcaacctggacagagatcatgttcactgaatccttccaaaaattca
tgggtagtttgaattataaggaaaataagacttaggataaatactttgtc
caagatcccagagttaatgccaaaatcagttttcagactccaggcagcct
gatcaagagcctaaactttaaagacacagtcccttaataactactattca
cagttgcactttcagggcgcaaagactcattgaatcctacaatagaatga
gtttagatatcaaatctctcagtaatagatgaggagactaaatagcgggc
atgacctggtcacttaaagacagaattgagattcaaggctagtgttcttt
ctacctgttttgtttctacaagatgtagcaatgcgctaattacagacctc
tcagggaaggaa
Porcine Lambda Chain Targeting
In particular embodiments of the present invention, targeting vectors are provided to target the porcine lambda chain locus. In one embodiment, lambda can be targeted by designing a targeting construct that contains a 5′ arm containing sequence located 5′ to the first JC unit and a 3′ arm containing sequence 3′ to the last JC unit of the J/C cluster region, thus preventing functional expression of the lambda locus (see, FIGS. 3-4). In one embodiment, the targeting vector can contain any contiguous sequence (such as about 17, 20, 30, 40, 50, 75, 100, 200, 300 or 5000 nucleotides of contiguous sequence) or fragment thereof. Seq ID No 28. In one embodiment, the 5′ targeting arm can contain Seq ID No. 32, which includes 5′ flanking sequence to the first lambda J/C region of the porcine lambda light chain genomic sequence or any contiguous sequence (such as about 17, 20, 30, 40, 50, 75, 100, 200, 300 or 5000 nucleotides of contiguous sequence) or fragment thereof (see also, for example FIG. 5). In another embodiment, the 3′ targeting arm can contain, but is not limited to one or more of the following: Seq ID No. 33, which includes 3′ flanking sequence to the J/C cluster region of the porcine lambda light chain genomic sequence, from approximately 200 base pairs downstream of lambda J/C; Seq ID No. 34, which includes 3′ flanking sequence to the J/C cluster region of the porcine lambda light chain genomic sequence, approximately 11.8 Kb downstream of the J/C cluster, near the enhancer; Seq ID No. 35, which includes approximately 12 Kb downstream of lambda, including the enhancer region; Seq ID No. 36, which includes approximately 17.6 Kb downstream of lambda; Seq ID No. 37, which includes approximately 19.1 Kb downstream of lambda; Seq ID No. 38, which includes approximately 21.3 Kb downstream of lambda; and Seq ID No. 39, which includes approximately 27 Kb downstream of lambda, or any contiguous sequence (such as about 17, 20, 30, 40, 50, 75, 100, 200, 300 or 5000 nucleotides of contiguous sequence) or fragment thereof of Seq ID Nos 32-39 (see also, for example FIG. 6). It is understood that in general when designing a targeting construct one targeting arm will be 5′ of the other targeting arm.
Seq ID No. 48 (as shown in Example 4) provides a representative, non-limiting example of a targeting construct that contains a 5′ arm containing sequence located 5′ to the first JC unit and a 3′ arm containing sequence 3′ to the last JC unit of the J/C cluster region. Representative 5′ and 3′ arms are shown in Seq ID No. 49 and 50 (also in Example 4).
In another embodiment, lambda is targeted using two targeting vectors. The two lambda targeting vectors, i.e., a vector pair, are utilized in a two step strategy to delete the entire J/C region of porcine lambda. In the first step, a first targeting vector is inserted upstream of the J/C region (or alternatively downstream of the J/C region). If the first targeting vector is inserted upstream of the J/C region, the 5′ and 3′ recombination arms of the first targeted vector contain homologous sequence to the 5′ flanking sequence of the first J/C unit of the J/C cluster region. See FIG. 5, which shows 7 JC units in the J/C cluster region. If the first targeting vector is inserted downstream of the J/C cluster region, the 5′ and 3′ recombination arms of the first targeting vector contain homologous sequence to the 3′ region of the last J/C unit in the JC region.
The first-step vectors are designed with lox sites that flank a fusion gene which can provide both positive and negative selection. Selection of the targeting event utilizes the Tn5 APHII gene commonly described as Neo resistance. Once targeting events are isolated, Cre is provided transiently to facilitate deletion of the selectable marker located between two lox sites. Negative selection is then provided by the Herpes simplex thymidine kinase coding region. This step selects for targeted cells that have deleted the selectable marker and retains a single lox site upstream (alternatively downstream) of the J/C region.
The second step is performed in the same lineage as the first step. The second targeting step also inserts a marker that provides both positive and negative selection. However, the second step inserts the marker on the opposite site of the J/C region in comparison to the first step. That is, if the first vector was inserted upstream of the J/C region, the second targeting vector is inserted downstream, and vice versa. FIG. 6 shows a second targeting vector inserted downstream of the J/C region. In addition, the second targeting vector has a single lox site that is located distally compared to the first vector. In other words, for the first strategy, the second vector has a single lox site located downstream of the marker gene (the alternative vector has the lox site upstream of the marker). After Cre mediated deletion, the region between the first targeting event (which left a lox remnant) and the second targeting event (which has a lox site outside of the marker) is deleted. Cells that have deleted the entire J/C cluster region are thus obtained.
In a representative, non-limiting example, the vector pair is Seq. ID No. 44 (step 1) and Seq. ID No. 45 (step 2).
In a further, non-limiting example, the vector pair is Seq. ID No. 46 (step 1) and Seq. ID No. 47 (step 2).
SEQ. ID taaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtcgctgagcaggccctggcctccctggcc
44 gagggcggtttgcgtattagaggcctaaatggccgaattcagcggataacaatttcacacaggaaacagctatgaccatg
attatctagtaactataacggtcctaaggtagcgagcgatcgcttaattaacctgcagggatatcccatgggggccgccag
tgtgatggatatctgcagaattcgcccttgatattaagagaagggcaagtcagcttaagtttgggggtagaggggaacag
ggagtgaggagatctggcctgagagataggagccctggtggccacaggaggactctttgggtcctgtcggatggacac
agggcggcccgggggcatgttggagcccggctggttcttaccagaggcagggggcaccctctgacacgggagcagg
gcatgttccatacatgacacacccctctgctccagggcaggtgggtggcggcacagaggagccagggactctgagcaa
ggggtccaccagtggggcagttggatccagacttctctgggccagcgagagtctagccctcagccgttctctgtccagg
aggggggtggggcaggcctgggcggccagagctcatccctcaagggttcccagggtcctgccagacccagatttccg
accgcagccaccacaagaggatgtggtctgctgtggcagctgccaagaccttgcagcaggtgcagggtgggggggtg
ggggcacctgggggcagctggggtcactgagttcagggaaaaccccttttttcccctaaacctggggccatccctaggg
gaaaccacaacttctgagccctgggcagtggctgctgggagggaagagcttcatcctggaccctgggggggaaccca
gctccaaaggtgcaaggggcccaggtccaaggctagagtgggccaagcaccgcaatggccagggagtgggggagg
tggagctggactggatcagggcctccttgggactccctacaccctgtgtgacatgttagggtacccacaccccatcacca
gtcagggcctggcccatctccagggccagggatgtgcatgtaagtgtgtgtgagtgtgtgtgtgtggtgtagtacacccct
tggcatccggttccgaggccttgggttcctccaaagttgctctctgaattaggtcaaactgtgaggtcctgatcgccatcatc
aacttcgttctccccacctcccatcattatcaagagctggggagggtctgggatttcttcccacccacaagccaaaagata
agcctgctggtgatggcagaagacacaggatcctgggtcagagacaaaggccagtgtgtcacagcgagagaggcag
ccggactatcagctgtcacagagaggccttagtccgctgaactcaggccccagtgactcctgttccactgggcactggcc
cccctccacagcgcccccaggccccagggagaggcgtcacagcttagagatggccctgctgaacagggaacaagaa
caggtgtgccccatccagcgccccaggggtgggacaggtgggctggatttggtgtgaagcccttgagccctggaaccc
aaccacagcagggcagttggtagatgccatttggggagaggccccaggagtaagggccatgggcccttgagggggc
caggagctgaggacagggacagagacggcccaggcagaggacagggccatgaggggtgcactgagatggccact
gccagcaggggcagctgccaacccgtccagggaacttattcagcagtcagctggaggtgccattgaccctgagggca
gatgaagcccaggccaggctaggtgggctgtgaagaccccaggggacagagctctgtccctgggcagcactggcctc
tcattctgcagggcttgacgggatcccaaggcctgctgcccctgatggtagtggcagtaccgcccagagcaggacccc
agcatggaaaccccaacgggacgcagcctgcggagcccacaaaaccagtaaggagccgaagcagtcatggcacgg
ggagtgtggacttccctttgatggggcccaggcatgaaggacagaatgggacagcggccatgagcagaaaatcagcc
ggaggggatgggcctaggcagacgctggctttatttgaagtgttggcattttgtctggtgtgtattgttggtattgattttatttt
agtatgtcagtgacatactgacatattatgtaacgacatattattatgtgttttaagaagcactccaagggaacaggctgtctg
taatgtgtccagagaagagagcaagagcttggctcagtctcccccaaggaggtcagttcctcaacaggggtcctaaatgt
ttcctggagccaggcctgaatcaagggggtcatatctacacgtggggcagacccatggaccattttcggagcaataagat
ggcagggaggataccaagctggtcttacagatccagggctttgacctgtgacgcgggcgctcctccaggcaaagggag
aagccagcaggaagctttcagaactggggagaacagggtgcagacctccagggtcttgtacaacgcaccctttatcctg
gggtccaggaggggtcactgagggatttaagtgggggaccatcagaaccaggtttgtgttttggaaaaatggctccaaa
gcagagaccagtgtgaggccagattagatgatgaagaagaggcagtggaaagtcgatgggtggccaggtagcaaga
gggcctatggagttggcaagtgaatttaaagtggtggcaccagagggcagatggggaggagcaggcactgtcatgga
ctgtctatagaaatctaaaatgtataccctttttagcaatatgcagtgagtcataaaagaacacatatatatttcctttggccgg
ccggcgcgccacgcgtataacttcgtatagcatacattatacgaagttatcttaagggctatggcagggcctgccgcccc
gacgttggctgcgagccctgggccttcacccgaacttggggggtggggtggggaaaaggaagaaacgcgggcgtatt
ggccccaatggggtctcggtggggtatcgacagagtgccagccctgggaccgaaccccgcgtttatgaacaaacgacc
caacaccgtgcgttttattctgtctttttattgccgtcatagcgcgggttccttccggtatgtctccttccgtgtttcactcgagt
tagaagaactcgtcaagaaggcgatagaaggcgatgcgctgcgaatcgggagcggcgataccgtaaagcacgagga
agcggtcagcccattcgccgccaagctcttcagcaatatcacgggtagccaacgctatgtcctgatagcggtccgccac
acccagccggccacagtcgatgaatccagaaaagcggccattttccaccatgatattcggcaagcaggcatcgccatgg
gtcacgacgagatcctcgccgtcgggcatgcgcgccttgagcctggcgaacagttcggctggcgcgagcccctgatgc
tcttcgtccagatcatcctgatcgacaagaccggcttccatccgagtacgtgctcgctcgatgcgatgtttcgcttggtggtc
gaatgggcaggtagccggatcaagcgtatgcagccgccgcattgcatcagccatgatggatactttctcggcaggagca
aggtgagatgacaggagatcctgccccggcacttcgcccaatagcagccagtcccttcccgcttcagtgacaacgtcga
gcacagctgcgcaaggaacgcccgtcgtggccagccacgatagccgcgctgcctcgtcctgcagttcattcagggcac
cggacaggtcggtcttgacaaaaagaaccgggcgcccctgcgctgacagccggaacacggcggcatcagagcagcc
gattgtctgttgtgcccagtcatagccgaatagcctctccacccaagcggccggagaacctgcgtgcaatccatcttgttc
aatggccgatcccattccagatctgttagcctcccccatctcccgtgcaaacgtgcgcgccaggtcgcagatcgtcggtat
ggagcctggggtggtgacgtgggtctggatcatcccggaggtaagttgcagcagggcgtcccggcagccggcgggc
gattggtcgtaatccaggataaagacgtgcatgggacggaggcgtttggtcaagacgtccaaggcccaggcaaacacg
ttgtacaggtcgccgttgggggccagcaactcgggggcccgaaacagggtaaataacgtgtccccgatatggggtcgt
gggcccgcgttgctctggggctcggcaccctggggcggcacggccgtccccgaaagctgtccccaatcctcccgcca
cgacccgccgccctgcagataccgcaccgtattggcaagcagcccgtaaacgcggcgaatcgcggccagcatagcca
ggtcaagccgctcgccggggcgctggcgtttggccaggcggtcgatgtgtctgtcctccggaagggcccccaacacg
atgtttgtgccgggcaaggtcggcgggatgagggccacgaacgccagcacggcctggggggtcatgctgcccataag
gtatcgcgcggccgggtagcacaggagggcggcgatgggatggcggtcgaagatgagggtgagggccgggggcg
gggcatgtgagctcccagcctcccccccgatatgaggagccagaacggcgtcggtcacggcataaggcatgcccattg
ttatctgggcgcttgtcattaccaccgccgcgtccccggccgatatctcaccctggtcaaggcggtgttgtgtggtgtagat
gttcgcgattgtctcggaagcccccagcacccgccagtaagtcatcggctcgggtacgtagacgatatcgtcgcgcgaa
cccagggccaccagcagttgcgtggtggtggttttccccatcccgtggggaccgtctatataaacccgcagtagcgtgg
gcattttctgctccgggcggacttccgtggcttcttgctgccggcgagggcgcaacgccgtacgtcggttgctatggccg
cgagaacgcgcagcctggtcgaacgcagacgcgtgctgatggccggggtacgaagccatggtggctctagaggtcga
aaggcccggagatgaggaagaggagaacagcgcggcagacgtgcgcttttgaagcgtgcagaatgccgggcttccg
gaggaccttcgggcgcccgccccgcccctgagcccgcccctgagcccgcccccggacccaccccttcccagcctctg
agcccagaaagcgaaggagccaaagctgctattggccgctgccccaaaggcctacccgcttccattgctcagcggtgc
tgtccatctgcacgagactagtgagacgtgctacttccatttgtcacgtcctgcacgacgcgagctgcggggcgggggg
gaacttcctgactaggggaggagtagaaggtggcgcgaaggggccaccaaagaacggagccggttggcgcctaccg
gtggatgtggaatgtgtgcgaggccagaggccacttgtgtagcgccaagtgcccagcggggctgctaaagcgcatgct
ccagactgccttgggaaaagcgcctcccctacccggtagggatccgcgttacataacttacggtaaatggcccgcctgg
ctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattg
acgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctatt
gacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatct
acgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacgggg
atttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacggttaacaagcttataacttcgtat
agcatacattatacgaagttattacgtagcggccgcgtcgacgataaattgtgtaattccacttctaaggattcatcccaagg
ggggaaaataatcaaagatgtaaccaaaggtttacaaacaagaactcatcattaatcttccttgttgttatttcaacgatattat
tattattactattattattattattattttgtctttttgcattttctagggccactcccacggcatagagaggttcccaggctagggg
tcaaatcggagctacagctgccggcctacgccagagccacagcaacgcaggatctgagccacagcaatgcaggatct
acaccacagctcatggtaacgctggatccttaacccaatgagtgaggccagggatcgaacctgtaacttcatggttcctag
tcggattcattaaccactgagccacgacaggaactccaacattattaatgatgggagaaaactggaagtaacctaaatatc
cagcagaaagggtgtggccaaatacagcatggagtagccatcataaggaatcttacacaagcctccaaaattgtgtttctg
aaattgggtttaaagtacgtttgcattttaaaaagcctgccagaaaatacagaaaaatgtctgtgatatgtctctggctgatag
gattttgcttagttttaattttggctttataattttctatagttatgaaaatgttcacaagaagatatatttcattttagcttctaaaata
attataacacagaagtaatttgtgctttaaaaaaatattcaacacagaagtatataaagtaaaaattgaggagttcccatcgtg
gctcagtgattaacaaacccaactagtatccatgaggatatggatttgatccctggccttgctcagtgggttgaggatccag
tgttgctgtgagctgtggtgtaggttgcagacacagcactctggcgttgctgtgactctggcgtaggccggcagctacag
ctccatttggacccttagcctgggaacctccatatgcctgagatacggccctaaaaagtcaaaagccaaaaaaatagtaa
aaattgagtgtttctacttaccacccctgcccacatcttatgctaaaacccgttctccagagacaaacatcgtcaggtgggtc
tatatatttccagccctcctcctgtgtgtgtatgtccgtaaaacacacacacacacacacacacgcacacacacacacacg
tatctaattagcattggtattagtttttcaaaagggaggtcatgctctaccttttaggcggcaaatagattatttaaacaaatctg
ttgacattttctatatcaacccataagatctcccatgttcttggaaaggctttgtaagacatcaacatctgggtaaaccagcat
ggtttttagggggtttgtggatttttttcatattttttagggcacacctgcagcatatggaggttcccaggctaggggttgaat
cagagctgtagctgccggcctacaccacagccacagcaacgccagatccttaacccactgagaaaggccagggattga
acctgcatcctcatggatgctggtcagatttatttctgctgagccacaacaggaactccctgaaccagaatgcttttaaccat
tccactttgcatggacatttagattgtttccatttaaaaatacaaattacaaggagttcccgtcgtggctcagtggtaacgaatt
ggactaggaaccatgaggtttcgggttcgatccctggccttgctcggtgggttaaggatccagcattgatgtgagatatgg
tgtaggtcgcagacgtggctcggatcccacgttgctgtggctctggcgtaggccggcaacaacagctccgattcgaccc
ctagcctgggaacctccatgtgccacaggagcagccctagaaaaggcaaaaagacaaaaaaataaaaaattaaaatga
aaaaataaaataaaaatacaaattacaagagacggctacaaggaaatccccaagtgtgtgcaaatgccatatatgtataaa
atgtactagtgtctcctcgcgggaaagttgcctaaaagtgggttggctggacagagaggacaggctttgacattctcatag
gtagtagcaatgggcttctcaaaatgctgttccagtttacactcaccatagcaaatgacagtgcctcttcctctccacccttg
ccaataatgtgacaggtggatctttttctattttgtgtatctgacaagcaaaaaatgagaacaggagttcctgtcgtggtgca
gtggagacaaatctgactaggaaccatgaaatttcgggttcaatccctggcctcactcagtaggtaaaggatccagggttg
cagtgagctgtggggtaggtcgcagacacagtgcaaatttggccctgttgtggctgtggtgtaggccggcagctatagct
ccaattggacccctagcctgggaacctccttatgccgtgggtgaggccctaaaaaaaagagtgcaaaaaaaaaaaataa
gaacaaaaatgatcatcgtttaattctttatttgatcattggtgaaacttattttccttttatatttttattgactgattttatttctcctat
gaatttaccggtcatagttttgcctgggtgtttttactccggttttagttttggttggttgtattttcttagagagctatagaaactct
tcatctatttggaatagtaattcctcattaagtatttgtgctgcaaaaaattttccctgatctgttttatgcttttgtttgtggggtctt
tcacgagaaagcctttttagtttttacacctcagcttggttgtttttcttgattgtgtctgtaatctgcggccaacataggaaaca
catttttactttagtgtttttttcctattttcttcaagtacgtccattgttttggtgtctgattttactttgcctggggtttgtttttgtgtg
gcaggaatataaacttatgtattttccaaatggagagccaatggttgtatatttgttgaattcaaatgcaactttatcaaacacc
aaatcatcgatttatcacaactcttctctggtttattgatctaatgatcaattcctgttccacgctgttttaattattttagctttgtgg
attttggtgcctggtagagaacaaagcctccattattttcattcaaaatagtcccgtctattatctgccattgttgtagtattaga
ctttaaaatcaatttactgattttcaaaagttattcctttggtgatgtggaatactttatacttcataaggtacatggattcatttgtg
gggaattgatgtctttgctattgtggccatttgtcaagttgtgtaatattttacccatgccaactttgcatattgtatgtgagtttat
tcccagggtttttaataggatgtttattgaagttgtcagtgtttccacaatttcatcgcctcagtgcttactgtttgcataaaagg
aaacctactcacttttgcctattgctcttgtattcaatcattttagttaactcttgtgttaattttgagagtttttcagctgactgtctg
gggttttctttaatagactagccctttgtctgtaaagaataattttatcgaatttttcttaacactcacactctccccacccccacc
cccgctcatctcctttcattgggtcaaatctgtagaatacaataaaagtaagagtgggaaccttagcctttaagtcgattttgc
ctttaaatgtgaatgttgctatgtttcgggacattctctttatcaagttgcggatgtttccttagataattaacttaataaaagact
ggatgtttgctttcttcaaatcagaattgtgttgaatttatattgctattctgtttaattttgtttcaaaaaatttacatgcacacctta
aagataaccatgaccaaatagtcctcctgctgagagaaaatgttggccccaatgccacaggttacctcccgactcagata
aactacaatgggagataaaatcagatttggcaaagcctgtggattcttgccataactctcagagcatgacttgggtgttttttc
cttttctaagtattttaatggtatttttgtgttacaataggaaatctaggacacagagagtgattcaatgaggggaacgcattct
gggatgactctaggcctctggtttggggagagctctattgaagtaaagacaatgagaggaagcaagtttgcagggaact
gtgaggaatttagatggggaatgttgggtttgaggtttctatagggcacgcaagcagagatgcactcaggaggaagaag
gagcataaatctagtggcgctgccggcaagcttgctggaggaggccaattgggagctgctggaatgcatggaggcggc
gctctcgaggctggaggaggccagctgatttaaatcggtccgcgtacgatgcatattaccctgttatccctaccgcggtta
ctggccgtcgttttacaacgtcgtgactgggaaaaccctggcgatgctcttctcccggtgaaaacctctgacacatggctct
tctaaatccggagtttaaacgcttccttcatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgtt
gctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccg
acaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccgga
tacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgtt
cgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtc
caacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcgg
tgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagcca
gttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagc
agcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaa
aactcacgttaagggattttggtcatgcctaggtggcaaacagctattatgggtattatgggtctaccggtgcatgagattat
caaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctga
cagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgt
gtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggct
ccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatcc
agtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggc
atcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccat
gttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggtta
tggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctg
agaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaa
aagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacc
cactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgcc
gcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcaggg
ttattgtctcgggagcggatacatatttgaatgtatttagaaaaa
SEQ ID 45 taaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtcgctgagcaggccctggcctccctggcc
gagggcggtttgcgtattagaggcctaaatggccgaattcagcggataacaatttcacacaggaaacagctatgaccatg
attatctagtaactataacggtcctaaggtagcgagcgatcgcttaattaacctgcagggataaccactgacccatgacgg
gaactcccagggctcagctcttgactccaggttcgcagctgccctcaaagcaatgcaaccctggctggccccgcctcat
gcatccggcctcctccccaaagagctctgagcccacctgggcctaggtcctcctccctgggactcatggcctaagggta
cagagttactggggctgatgaagggaccaatggggacaggggcctcaaatcaaagtggctgtctctctcatgtcccttcc
tctcctcagggtccaaaatcagggtcagggccccagggcaggggctgagagggcctctttctgaaggccctgtctcagt
gcaggttatgggggtctgggggagggtcaatgcagggctcacccttcagtgccccaaagcctagagagtgagtgcctg
ccagtggcttcccaggcccaatcccttgactgcctgggaatgctcaaatgcaggaactgtcacaacaccttcagtcaggg
gctgctctgggaggaaaaacactcagaattgggggttcagggaaggcccagtgccaagcatagcaggagctcaggtg
gctgcagatggtgtgaaccccaggagcaggatggccggcactccccccagaccctccagagccccaggttggctgcc
ctcttcactgccgacacccctgggtccacttctgccctttcccacctaaaacctttagggctcccactttctcccaaatgtga
gacatcaccacggctcccagggagtgtccagaagggcatctggctgagaggtcctgacatctgggagcctcaggcccc
acaatggacagacgccctgccaggatgctgctgcagggctgttagctaggcggggtggagatggggtactttgcctctc
agaggccccggccccaccatgaaacctcagtgacaccccatttccctgagttcacatacctgtatcctactccagtcacct
tccccacgaacccctgggagcccaggatgatgctggggctggagccacgaccagcccacgagtgatccagctctgcc
aatcagcagtcatttcccaagtgttccagccctgccaggtcccactacagcagtaatggaggccccagacaccagtcca
gcagttagagggctggactagcaccagctttcaagcctcagcatctcaaggtgaatggccagtgcccctccccgtggcc
atcacaggatcgcagatatgaccctaggggaagaaatatcctgggagtaaggaagtgcccatactcaaggatggcccct
ctgtgacctaacctgtccctgaggattgtacttccaggcgttaaaacagtagaacgcctgcctgtgaacccccgccaagg
gactgcttggggaggccccctaaaccagaacacaggcactccagcaggacctctgaactctgaccaccctcagcaagt
gggcaccccccgcagcttccaaggcaccccagggctcaccacagcggcccctcctggcagcccctcacccaggccc
agaccctctaagatggcacatctaagccaatccacctccttgtcattcctcctgtccccacccaggacccttctcagatgaa
accttcgctccagccgctgggccctctctcctgcccctctggcagttctccagggactccgcctcccactctctgtctctcc
ctgcactcctaggaacaagcgacctccaggaagcccagtccaattatcccctctgtgtcctccccaatctctgcctctggg
tggatttgagcaccacatcctgttctcttcgacctgaaactccttggccccggtgtccgctctcctgggccctcttttctctcct
cccctcttccgtgccccgtttgtttggtgttacaggcaggccccggggagccgtccctccagctgctcttccttgtctgtctc
aggagccagaaactggcagcatctaaaaagggctcctgtttcttcatctgcccagcctcctagcccaaccagggctctgg
cctcactccagagggtgggctccagagggcaggggttgcaccctcttagtgcctcagaggctcagctgggtgcaggat
gggggggccctcagggagcccctcagtgactgctgatcacttactgcaggactgttcccagctcttcccaatcattggaat
gacaatacctagttctgctccatcatagtgatgcaggaaaaatgttactgaaatcctggttcttgtttagcaatcgaagaatg
aattccgcgaacacacaggcagcaagcaagcgaagcctttattaaaggaaagcagatagctcccagggctgcaggga
gcggggagaagagctccccactctctattgtcctatagggctttttaccccttaaagttggggggatacaaaaaaaataga
agaaaaagggagttcccgtcagggcacagcagaaacaaatccaactaggaaccatgaggttgggggttcgattcctgg
cctctctcagtgggttaaggatgcagcgttgccgtgagctatgatacaggtcacagatgcagctcagatctactagtcaatt
gacaggcgccggagcaggagctaggcctttggccggccggcgcgccagatctcttaagggctatggcagggcctgcc
gccccgacgttggctgcgagccctgggccttcacccgaacttggggggtggggtggggaaaaggaagaaacgcggg
cgtattggccccaatggggtctcggtggggtatcgacagagtgccagccctgggaccgaaccccgcgtttatgaacaaa
cgacccaacaccgtgcgttttattctgtctttttattgccgtcatagcgcgggttccttccggtattgtctccttccgtgtttcact
cgagttagaagaactcgtcaagaaggcgatagaaggcgatgcgctgcgaatcgggagcggcgataccgtaaagcacg
aggaagcggtcagcccattcgccgccaagctcttcagcaatatcacgggtagccaacgctatgtcctgatagcggtccg
ccacacccagccggccacagtcgatgaatccagaaaagcggccattttccaccatgatattcggcaagcaggcatcgcc
atgggtcacgacgagatcctcgccgtcgggcatgcgcgccttgagcctggcgaacagttcggctggcgcgagcccctg
atgctcttcgtccagatcatcctgatcgacaagaccggcttccatccgagtacgtgctcgctcgatgcgatgtttcgcttggt
ggtcgaatgggcaggtagccggatcaagcgtatgcagccgccgcattgcatcagccatgatggatactttctcggcagg
agcaaggtgagatgacaggagatcctgccccggcacttcgcccaatagcagccagtcccttcccgcttcagtgacaacg
tcgagcacagctgcgcaaggaacgcccgtcgtggccagccacgatagccgcgctgcctcgtcctgcagttcattcagg
gcaccggacaggtcggtcttgacaaaaagaaccgggcgcccctgcgctgacagccggaacacggcggcatcagagc
agccgattgtctgttgtgcccagtcatagccgaatagcctctccacccaagcggccggagaacctgcgtgcaatccatctt
gttcaatggccgatcccattccagatctgttagcctcccccatctcccgtgcaaacgtgcgcgccaggtcgcagatcgtcg
gtatggagcctggggtggtgacgtgggtctggatcatcccggaggtaagttgcagcagggcgtcccggcagccggcg
ggcgattggtcgtaatccaggataaagacgtgcatgggacggaggcgtttggtcaagacgtccaaggcccaggcaaac
acgttgtacaggtcgccgttgggggccagcaactcgggggcccgaaacagggtaaataacgtgtccccgatatggggt
cgtgggcccgcgttgctctggggctcggcaccctggggcggcacggccgtccccgaaagctgtccccaatcctcccg
ccacgacccgccgccctgcagataccgcaccgtattggcaagcagcccgtaaacgcggcgaatcgcggccagcatag
ccaggtcaagccgctcgccggggcgctggcgtttggccaggcggtcgatgtgtctgtcctccggaagggcccccaaca
cgatgtttgtgccgggcaaggtcggcgggatgagggccacgaacgccagcacggcctggggggtcatgctgcccata
aggtatcgcgcggccgggtagcacaggagggcggcgatgggatggcggtcgaagatgagggtgagggccggggg
cggggcatgtgagctcccagcctcccccccgatatgaggagccagaacggcgtcggtcacggcataaggcatgccca
ttgttatctgggcgcttgtcattaccaccgccgcgtccccggccgatatctcaccctggtcaaggcggtgttgtgtggtgta
gatgttcgcgattgtctcggaagcccccagcacccgccagtaagtcatcggctcgggtacgtagacgatatcgtcgcgc
gaacccagggccaccagcagttgcgtggtggtggttttccccatcccgtggggaccgtctatataaacccgcagtagcgt
gggcattttctgctccgggcggacttccgtggcttcttgctgccggcgagggcgcaacgccgtacgtcggttgctatggc
cgcgagaacgcgcagcctggtcgaacgcagacgcgtgctgatggccggggtacgaagccatggtggctctagaggtc
gaaaggcccggagatgaggaagaggagaacagcgcggcagacgtgcgcttttgaagcgtgcagaatgccgggcttc
cggaggaccttcgggcgcccgccccgcccctgagcccgcccctgagcccgcccccggacccaccccttcccagcct
ctgagcccagaaagcgaaggagccaaagctgctattggccgctgccccaaaggcctacccgcttccattgctcagcgg
tgctgtccatctgcacgagactagtgagacgtgctacttccatttgtcacgtcctgcacgacgcgagctgcggggcgggg
gggaacttcctgactaggggaggagtagaaggtggcgcgaaggggccaccaaagaacggagccggttggcgcctac
cggtggatgtggaatgtgtgcgaggccagaggccacttgtgtagcgccaagtgcccagcggggctgctaaagcgcatg
ctccagactgccttgggaaaagcgcctcccctacccggtagggatccgcgttacataacttacggtaaatggcccgcctg
gctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccatt
gacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctat
tgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatc
tacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggg
gatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacggttaacaagcttataacttcgta
tagcatacattatacgaagttattacgtagcggccgcgtcgacgatatcgctgccggagcccccggggccgctgccgga
agatctggcattgctgtgactgtggtgtaggccggcagctggagctctgattagacccctcacctgggaatctccatatgc
tgcacgtgcggccctaaaaagacaaaagacaaaaaaaaaaaaaaaaaaaaaaaatcaaaaaaaaacatagggggtta
ccaacgtggggtccagaaagatgtggttttctcccattggccttgcccagttacctatatcagtccttgtccaacaggggttt
taggggggaaatgccccataaattttacggtttctttgcccttctcttcctttagactgagtcaccattgctctcattccttttcta
tcagttgaggagtgggttagagattaaggtccatgtggtggaggtacacttcttatagtaaacaaggcctatggggaattac
tctctggagcccttaaaccacaaatgataatccatgccacatcaaagatgcatcgaagcccatgctcctacactgactacct
gagttagcattctgcctcaacaggactgaccatccccagctctggggcagatatcctctctctgccacaagggcagtgac
ccccatgctgtctgagggtcacgctttaccccccccccacccctgccgtgaccccccagaccaccccaggaggtgggc
actaatatccctcattaccccatagatgaggaaacagaggttcccccggggtcccacaggtgctcagggtcacatgcacc
gtgggcacccaggccccatcccaaggccaccctccctcctcaggaagctgtgctgcgctgggccagaaggtactgcac
acgactcctcagcctccggtggtgggaggcagcctcaagcctctgagtgggggggcacccgggctcctcaatctatact
gactcctgggggtgggagaaggggagggggagctgtggcctctgagtccactaagcaaatcagggtgggcaatgcg
ggcccatttcaaggaggagagaaccgaggctctgacagcaggccgggggtccagggacctgcccagggtcataggc
tgaactgctggctgacctgccttgggttctttccttggctcctcagccctgtgtgatgtgacaggtcattcattcactcactcg
ctcattcattcagcaaaccctcagtgagccctgctgggagcaggtgctaggggcaaggagacaggacctcttgccctgg
aacagctgaagcactgggggacaggcagtggcagggaggtgcgtgatcaccgctgaccccattccatcctccagccc
ccaggtcagtttccacccaccattgaccccaccatgtcctccatccccaaggtcagtttcccgcccaaggagcatctcctt
acacactagggacaaaatttcacggctgtcactgggcatctctccacgctcatcacagccctctagcagccttgaagtcct
gtagagcccttcccatttcacagaagggacaagactatgagggccacaccgtgagccatgagccttaggctgtgagccg
ggacagcccctgcaggactggtggcctcagggcactgggtggggagggtgcacagtgggtgggccccttgtggaata
gagaggagtgtcaggtcaggggagggggcttggcctggccctggcctgcctggtgtgcaaccctaggcagcccctcct
tcccaggcctcctacttcctggaggccaagcctcagggaggtaattgagtcaggtgggggagggggggttgtggctttc
ttcacagcagaaaaacagagcccacaatagtgtccactgagacagaggggtcctgggggaggggaggggtgggagg
tgactgctgagccctgtgggagggagggagcaactactgagctgagctgggtgactctcccatctgccccgccccctgt
ggggccagcagagtcaccgagagaacatgacccagccaggcctggacagggggacacccatgtcctttaccccaca
gggttcactgagcctatctgccccaagcctgtgtctccctgggacggagaccctcactcccaaccacaaaggtctaaact
caagttcccaacagccttgaaaatacagcttccgggggcctccaaggagcagtcagccgtccactgccaggctcgctg
gctcagtgacacaggacacatcctgatgacggtccacctgtctccaagcaggttctcctctgccgatggggcaacgagct
cctcctgtggctccctggctggatgcgtgggaggcggggtgggggggcaggcggtgttcctggccgcacacaaggag
cacccccaccagcatccgaagacgggggcccggtctttccccaaaacactgcttgcgggagactttgtgacgtttccag
gggccatgctcccttcgggcagcttgggggacttctgctcctatgtggtcacctgcagggactccccccaggccttgggg
acaaacaaagtgatgagagggagggttagtgggtcggggcagggccagtctttggaccggtttatctgaaaagccagtt
ggtcaccgggaaccacagcaaacctaaacccatttggccaggcatctcccagggacagtctcccccaggatgcgggg
cccaggggggctccaggggtgacctgcgtcctggatttccctgatgctcccagttcgtgcctctgtccaagcatgattttta
atagtgccccttccactcccagaaatgtccaagtgtgggcaataaattctggtcacctgagctcagtgtaactgtttgctgaa
tgacacttactgtaacaggttaaaatgggaggcccaaggccacgcagagccatcgaaggctctgtgtgtcccagccctg
atagaagcatcaggatggggactgtggcctcaccaggggccacatccaggcggtcaccatggggttcctggtctccgt
gggccttgactggagcccctggtgtgagctcaccccatcccagcctgtgagaggcctggatgtgggcctgacatcatttc
ccacccagtgacagcactgcatgtgatggggcctctgggcagcctttttcccgggggaaactggcaggaatcaggacc
accaggacaggggtcaggggagaggcgatgctgggcaccagagcctggaccaccctcgggttctcagcgatgggca
acccctgccacccagggccccgccttcctggggagacatcggggtttccaggccatcctgggaggagggtgggagcc
tcagctagaccccagctggcttgcccccccatgccccggccaagagagggtcttggagggaagggggaccccagac
cagcctggcgagcccatcctcagggtctctggtcagacaggggctcagctgagctccagggtagaccaaggccctgc
gtggatgaggccagtgtggtcactgcccagagcaaagccacctctcagcagccctttcctgagcaccttctgtgtgcggg
gacatcagcagtggcaacacagccatgctggggactcagggctagagacaggggaccagcctatggagagtgggta
gtgtcctgcagggcaggcttgtgccctggagaaaacaaaccagggtgaggccagggacgctggccgggttcacagg
gtgatggctgagcacagagtgccaggggctggactgtcctgactctgggttggtggctgagggcctgtgtccctctatgc
ctctgggttggtgataatggaaacttgctccctggagagacaggacgaatggttgatgggaaatgaatgtttgcttgtcact
tggttgactgttgttgccgttagcattgggcttcttgggccaggcagcctcaggccagcactgctgggctccccacaggc
ccgacaccctcagccctgtgcagctggcctggcgaaaccaagaggccctgatgcccaaaatagccgggaaaccccaa
ccagcccagccctggcagcaggtgcctcccatttgcctgggctgggggaggggtggctctggttctggaagtttctgcc
agtccagctggagaagggacctgtatcccagcacccaggccgcccaagcccctgcaccagggcctgggccaggcag
agttgacatcaatcaattgggagctgctggaatgcatggaggcggcgctctcgaggctggaggaggccagctgatttaa
atcggtccgcgtacgatgcatattaccctgttatccctaccgcggttactggccgtcgttttacaacgtcgtgactgggaaa
accctggcgatgctcttctcccggtgaaaacctctgacacatggctcttctaaatccggagtttaaacgcttccttcatgtga
gcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgac
gagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccct
ggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtg
gcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaacccc
ccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactgg
cagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaacta
cggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttga
tccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctca
agaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgcctaggt
ggcaaacagctattatgggtattatgggtctaccggtgcatgagattatcaaaaaggatcttcacctagatccttttaaattaa
aaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatct
cagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatct
ggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccgga
agggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagt
agttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttca
ttcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctc
cgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccat
ccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgc
ccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcg
aaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttac
tttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaat
gttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcgggagcggatacatatttgaatgtat
ttagaaaaa
SEQ ID 46 taaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtcgctgagcaggccctggcctccctggcc
gagggcggtttgcgtattagaggcctaaatggccgaattcagcggataacaatttcacacaggaaacagctatgaccatg
attatctagtaactataacggtcctaaggtagcgagcgatcgcttaattaacctgcagggatatcccatgggggccgccag
tgtgatggatatctgcagaattcgcccttgatattaagagaagggcaagtcagcttaagtttgggggtagaggggaacag
ggagtgaggagatctggcctgagagataggagccctggtggccacaggaggactctttgggtcctgtcggatggacac
agggcggcccgggggcatgttggagcccggctggttcttaccagaggcagggggcaccctctgacacgggagcagg
gcatgttccatacatgacacacccctctgctccagggcaggtgggtggcggcacagaggagccagggactctgagcaa
ggggtccaccagtggggcagttggatccagacttctctgggccagcgagagtctagccctcagccgttctctgtccagg
aggggggtggggcaggcctgggcggccagagctcatccctcaagggttcccagggtcctgccagacccagatttccg
accgcagccaccacaagaggatgtggctgctgtggcagctgccaagaccttgcagcaggtgcagggtgggggggtg
ggggcacctgggggcagctggggtcactgagttcagggaaaaccccttttttcccctaaacctggggccatccctaggg
gaaaccacaacttctgagccctgggcagtggctgctgggagggaagagcttcatcctggaccctgggggggaaccca
gctccaaaggtgcaaggggcccaggtccaaggctagagtgggccaagcaccgcaatggccagggagtgggggagg
tggagctggactggatcagggcctccttgggactccctacaccctgtgtgacatgttagggtacccacaccccatcacca
gtcagggcctggcccatctccagggccagggatgtgcatgtaagtgtgtgtgagtgtgtgtgtgtggtgtagtacacccct
tggcatccggttccgaggccttgggttcctccaaagttgctctctgaattaggtcaaactgtgaggtcctgatcgccatcatc
aacttcgttctccccacctcccatcattatcaagagctggggagggtctgggatttcttcccacccacaagccaaaagata
agcctgctggtgatggcagaagacacaggatcctgggtcagagacaaaggccagtgtgtcacagcgagagaggcag
ccggactatcagctgtcacagagaggccttagtccgctgaactcaggccccagtgactcctgttccactgggcactggcc
cccctccacagcgcccccaggccccagggagaggcgtcacagcttagagatggccctgctgaacagggaacaagaa
caggtgtgccccatccagcgccccaggggtgggacaggtgggctggatttggtgtgaagcccttgagccctggaaccc
aaccacagcagggcagttggtagatgccatttggggagaggccccaggagtaagggccatgggcccttgagggggc
caggagctgaggacagggacagagacggcccaggcagaggacagggccatgaggggtgcactgagatggccact
gccagcaggggcagctgccaacccgtccagggaacttattcagcagtcagctggaggtgccattgaccctgagggca
gatgaagcccaggccaggctaggtgggctgtgaagaccccaggggacagagctctgtccctgggcagcactggcctc
tcattctgcagggcttgacgggatcccaaggcctgctgcccctgatggtagtggcagtaccgcccagagcaggacccc
agcatggaaaccccaacgggacgcagcctgcggagcccacaaaaccagtaaggagccgaagcagtcatggcacgg
ggagtgtggacttccctttgatggggcccaggcatgaaggacagaatgggacagcggccatgagcagaaaatcagcc
ggaggggatgggcctaggcagacgctggctttatttgaagtgttggcattttgtctggtgtgtattgttggtattgattttatttt
agtatgtcagtgacatactgacatattatgtaacgacatattattatgtgttttaagaagcactccaagggaacaggctgtctg
taatgtgtccagagaagagagcaagagcttggctcagtctcccccaaggaggtcagttcctcaacaggggtcctaaatgt
ttcctggagccaggcctgaatcaagggggtcatatctacacgtggggcagacccatggaccattttcggagcaataagat
ggcagggaggataccaagctggtcttacagatccagggctttgacctgtgacgcgggcgctcctccaggcaaagggag
aagccagcaggaagctttcagaactggggagaacagggtgcagacctccagggtcttgtacaacgcaccctttatcctg
gggtccaggaggggtcactgagggatttaagtgggggaccatcagaaccaggtttgtgttttggaaaaatggctccaaa
gcagagaccagtgtgaggccagattagatgatgaagaagaggcagtggaaagtcgatgggtggccaggtagcaaga
gggcctatggagttggcaagtgaatttaaagtggtggcaccagagggcagatggggaggagcaggcactgtcatgga
ctgtctatagaaatctaaaatgtataccctttttagcaatatgcagtgagtcataaaagaacacatatatatttcctttggccgg
ccggcgcgccacgcgtataacttcgtatagcatacattatacgaagttatcttaagggctatggcagggcctgccgcccc
gacgttggctgcgagccctgggccttcacccgaacttggggggtggggtggggaaaaggaagaaacgcgggcgtatt
ggccccaatggggtctcggtggggtatcgacagagtgccagccctgggaccgaaccccgcgtttatgaacaaacgacc
caacaccgtgcgttttattctgtctttttattgccgtcatagcgcgggttccttccggtattgtctccttccgtgtttcactcgagt
tagaagaactcgtcaagaaggcgatagaaggcgatgcgctgcgaatcgggagcggcgataccgtaaagcacgagga
agcggtcagcccattcgccgccaagctcttcagcaatatcacgggtagccaacgctatgtcctgatagcggtccgccac
acccagccggccacagtcgatgaatccagaaaagcggccattttccaccatgatattcggcaagcaggcatcgccatgg
gtcacgacgagatcctcgccgtcgggcatgcgcgccttgagcctggcgaacagttcggctggcgcgagcccctgatgc
tcttcgtccagatcatcctgatcgacaagaccggcttccatccgagtacgtgctcgctcgatgcgatgtttcgcttggtggtc
gaatgggcaggtagccggatcaagcgtatgcagccgccgcattgcatcagccatgatggatactttctcggcaggagca
aggtgagatgacaggagatcctgccccggcacttcgcccaatagcagccagtcccttcccgcttcagtgacaacgtcga
gcacagctgcgcaaggaacgcccgtcgtggccagccacgatagccgcgctgcctcgtcctgcagttcattcagggcac
cggacaggtcggtcttgacaaaaagaaccgggcgcccctgcgctgacagccggaacacggcggcatcagagcagcc
gattgtctgttgtgcccagtcatagccgaatagcctctccacccaagcggccggagaacctgcgtgcaatccatcttgttc
aatggccgatcccattccagatctgttagcctcccccatctcccgtgcaaacgtgcgcgccaggtcgcagatcgtcggtat
ggagcctggggtggtgacgtgggtctggatcatcccggaggtaagttgcagcagggcgtcccggcagccggcgggc
gattggtcgtaatccaggataaagacgtgcatgggacggaggcgtttggtcaagacgtccaaggcccaggcaaacacg
ttgtacaggtcgccgttgggggccagcaactcgggggcccgaaacagggtaaataacgtgtccccgatatggggtcgt
gggcccgcgttgctctggggctcggcaccctggggcggcacggccgtccccgaaagctgtccccaatcctcccgcca
cgacccgccgccctgcagataccgcaccgtattggcaagcagcccgtaaacgcggcgaatcgcggccagcatagcca
ggtcaagccgctcgccggggcgctggcgtttggccaggcggtcgatgtgtctgtcctccggaagggcccccaacacg
atgtttgtgccgggcaaggtcggcgggatgagggccacgaacgccagcacggcctggggggtcatgctgcccataag
gtatcgcgcggccgggtagcacaggagggcggcgatgggatggcggtcgaagatgagggtgagggccgggggcg
gggcatgtgagctcccagcctcccccccgatatgaggagccagaacggcgtcggtcacggcataaggcatgcccattg
ttatctgggcgcttgtcattaccaccgccgcgtccccggccgatatctcaccctggtcaaggcggtgttgtgtggtgtagat
gttcgcgattgtctcggaagcccccagcacccgccagtaagtcatcggctcgggtacgtagacgatatcgtcgcgcgaa
cccagggccaccagcagttgcgtggtggtggttttccccatcccgtggggaccgtctatataaacccgcagtagcgtgg
gcattttctgctccgggcggacttccgtggcttcttgctgccggcgagggcgcaacgccgtacgtcggttgctatggccg
cgagaacgcgcagcctggtcgaacgcagacgcgtgctgatggccggggtacgaagccatggtggctctagaggtcga
aaggcccggagatgaggaagaggagaacagcgcggcagacgtgcgcttttgaagcgtgcagaatgccgggcttccg
gaggaccttcgggcgcccgccccgcccctgagcccgcccctgagcccgcccccggacccaccccttcccagcctctg
agcccagaaagcgaaggagccaaagctgctattggccgctgccccaaaggcctacccgcttccattgctcagcggtgc
tgtccatctgcacgagactagtgagacgtgctacttccatttgtcacgtcctgcacgacgcgagctgcggggcgggggg
gaacttcctgactaggggaggagtagaaggtggcgcgaaggggccaccaaagaacggagccggttggcgcctaccg
gtggatgtggaatgtgtgcgaggccagaggccacttgtgtagcgccaagtgcccagcggggctgctaaagcgcatgct
ccagactgccttgggaaaagcgcctcccctacccggtagggatccgcgttacataacttacggtaaatggcccgcctgg
ctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattg
acgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctatt
gacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatct
acgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacgggg
atttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacggttaacaagcttagatctgcggc
cgcgtcgacgataaattgtgtaattccacttctaaggattcatcccaaggggggaaaataatcaaagatgtaaccaaaggt
ttacaaacaagaactcatcattaatcttccttgttgttatttcaacgatattattattattactattattattattattattttgtctttttg
cattttctagggccactcccacggcatagagaggttcccaggctaggggtcaaatcggagctacagctgccggcctacg
ccagagccacagcaacgcaggatctgagccacagcaatgcaggatctacaccacagctcatggtaacgctggatcctt
aacccaatgagtgaggccagggatcgaacctgtaacttcatggttcctagtcggattcattaaccactgagccacgacag
gaactccaacattattaatgatgggagaaaactggaagtaacctaaatatccagcagaaagggtgtggccaaatacagca
tggagtagccatcataaggaatcttacacaagcctccaaaattgtgtttctgaaattgggtttaaagtacgtttgcattttaaaa
agcctgccagaaaatacagaaaaatgtctgtgatatgtctctggctgataggattttgcttagttttaattttggctttataattn
ctatagttatgaaaatgttcacaagaagatatatttcattttagcttctaaaataattataacacagaagtaatttgtgctttaaaa
aatattcaacacagaagtatataaaaaaattgaggagttcccatcgtggctcagtgattaacaaacccaactagtatc
catgaggatatggatttgatccctggccttgctcagtgggttgaggatccagtgttgctgtgagctgtggtgtaggttgcag
acacagcactctggcgttgctgtgactctggcgtaggccggcagctacagctccatttggacccttagcctgggaacctc
catatgcctgagatacggccctaaaaagtcaaaagccaaaaaaatagtaaaaattgagtgtttctacttaccacccctgcc
cacatcttatgctaaaacccgttctccagagacaaacatcgtcaggtgggtctatatatttccagccctcctcctgtgtgtgta
tgtccgtaaaacacacacacacacacacacacgcacacacacacacacgtatctaattagcattggtattagtttttcaaaa
gggaggtcatgctctaccttttaggcggcaaatagattatttaaacaaatctgttgacattttctatatcaacccataagatctc
ccatgttcttggaaaggctttgtaagacatcaacatctgggtaaaccagcatggtttttagggggttgtgtggatttttttcata
ttttttagggcacacctgcagcatatggaggttcccaggctaggggttgaatcagagctgtagctgccggcctacaccac
agccacagcaacgccagatccttaacccactgagaaaggccagggattgaacctgcatcctcatggatgctggtcagat
ttatttctgctgagccacaacaggaactccctgaaccagaatgcttttaaccattccactttgcatggacatttagattgtttcc
atttaaaaatacaaattacaaggagttcccgtcgtggctcagtggtaacgaattggactaggaaccatgaggtttcgggttc
gatccctggccttgctcggtgggttaaggatccagcattgatgtgagatatggtgtaggtcgcagacgtggctcggatccc
acgttgctgtggctctggcgtaggccggcaacaacagctccgattcgacccctagcctgggaacctccatgtgccacag
gagcagccctagaaaaggcaaaaagacaaaaaaataaaaaattaaaatgaaaaaataaaataaaaatacaaauacaag
agacggctacaaggaaatccccaagtgtgtgcaaatgccatatatgtataaaatgtactagtgtctcctcgcgggaaagtt
gcctaaaagtgggttggctggacagagaggacaggctttgacattctcataggtagtagcaatgggcttctcaaaatgctg
ttccagtttacactcaccatagcaaatgacagtgcctcttcctctccacccttgccaataatgtgacaggtggatctttttctatt
ttgtgtatctgacaagcaaaaaatgagaacaggagttcctgtcgtggtgcagtggagacaaatctgactaggaaccatga
aatttcgggttcaatccctggcctcactcagtaggtaaaggatccagggttgcagtgagctgtggggtaggtcgcagaca
cagtgcaaatttggccctgttgtggctgtggtgtaggccggcagctatagctccaattggacccctagcctgggaacctcc
ttatgccgtgggtgaggccctaaaaaaaagagtgcaaaaaaaaaaaataagaacaaaaatgatcatcgtttaattctttattt
gatcattggtgaaacttattttccttttatatttttattgactgattttatttctcctatgaatttaccggtcatagttttgcctgggtgtt
tttactccggttttagttttggttggttgtattttcttagagagctatagaaactcttcatctatttggaatagtaattcctcattaagt
atttgtgctgcaaaaaattttccctgatctgttttatgcttttgtttgtggggtctttcacgagaaagcctttttagtttttacacctc
agcttggttgtttttcttgattgtgtctgtaatctgcggccaacataggaaacacatttttactttagtgtttttttcctattttcttca
agtacgtccattgttttggtgtctgattttactttgcctggggtttgtttttgtgtggcaggaatataaacttatgtattttccaaatg
gagagccaatggttgtatatttgttgaattcaaatgcaactttatcaaacaccaaatcatcgatttatcacaactcttctctggtt
tattgatctaatgatcaattcctgttccacgctgttttaattattttagctttgtggattttggtgcctggtagagaacaaagcctc
cattattttcattcaaaatagtcccgtctattatctgccattgttgtagtattagactttaaaatcaatttactgattttcaaaagttat
tcctttggtgatgtggaatactttatacttcataaggtacatggattcatttgtggggaattgatgtctttgctattgtggccattt
gtcaagttgtgtaatattttacccatgccaactttgcatattgtatgtgagtttattcccagggtttttaataggatgtttattgaag
ttgtcagtgtttccacaatttcatcgcctcagtgcttactgtttgcataaaaggaaacctactcacttttgcctattgctcttgtatt
caatcattttagttaactcttgtgttaattttgagagtttttcagctgactgtctggggttttctttaatagactagccctttgtctgt
aaagaataattttatcgaatttttcttaacactcacactctccccacccccacccccgctcatctcctttcattgggtcaaatct
gtagaatacaataaaagtaagagtgggaaccttagcctttaagtcgattttgcctttaaatgtgaatgttgctatgtttcggga
cattctctttatcaagttgcggatgtttccttagataattaacttaataaaagactggatgtttgctttcttcaaatcagaattgtgt
tgaatttatattgctattctgtttaattttgtttcaaaaaatttacatgcacaccttaaagataaccatgaccaaatagtcctcctg
ctgagagaaaatgttggccccaatgccacaggttacctcccgactcagataaactacaatgggagataaaatcagatttg
gcaaagcctgtggattcttgccataactctcagagcatgacttgggtgttttttccttttctaagtattttaatggtatttttgtgtta
caataggaaatctaggacacagagagtgattcaatgaggggaacgcattctgggatgactctaggcctctggtttgggga
gagctctattgaagtaaagacaatgagaggaagcaagtttgcagggaactgtgaggaatttagatggggaatgttgggttt
gaggtttctatagggcacgcaagcagagatgcactcaggaggaagaaggagcataaatctagtggcgctgccggcaa
gcttgctggaggaggccaattgggagctgctggaatgcatggaggcggcgctctcgaggctggaggaggccagctga
tttaaatcggtccgcgtacgatgcatattaccctgttatccctaccgcggttactggccgtcgttttacaacgtcgtgactgg
gaaaaccctggcgatgctcttctcccggtgaaaacctctgacacatggctcttctaaatccggagtttaaacgcttccttcat
gtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccc
tgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttcc
ccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagc
gtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaac
cccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccac
tggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaa
ctacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctct
tgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatct
caagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgcctag
gtggcaaacagctattatgggtattatgggtctaccggtgcatgagattatcaaaaaggatcttcacctagatccttttaaatt
aaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcaccta
tctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccat
ctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccg
gaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaa
gtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggctt
cattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtc
ctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgc
catccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctct
tgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggg
gcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatctt
ttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacgga
aatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcgggagcggatacatatttgaat
gtatttagaaaaa
SEQ ID 47 taaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtcgctgagcaggccctggcctccctggcc
gagggcggtttgcgtattagaggcctaaatggccgaattcagcggataacaatttcacacaggaaacagctatgaccatg
attatctagtaactataacggtcctaaggtagcgagcgatcgcttaattaacctgcagggataaccactgacccatgacgg
gaactcccagggctcagctcttgactccaggttcgcagctgccctcaaagcaatgcaaccctggctggccccgcctcat
gcatccggcctcctccccaaagagctctgagcccacctgggcctaggtcctcctccctgggactcatggcctaagggta
cagagttactggggctgatgaagggaccaatggggacaggggcctcaaatcaaagtggctgtctctctcatgtcccttcc
tctcctcagggtccaaaatcagggtcagggccccagggcaggggctgagagggcctctttctgaaggccctgtctcagt
gcaggttatgggggtctgggggagggtcaatgcagggctcacccttcagtgccccaaagcctagagagtgagtgcctg
ccagtggcttcccaggcccaatcccttgactgcctgggaatgctcaaatgcaggaactgtcacaacaccttcagtcaggg
gctgctctgggaggaaaaacactcagaattgggggttcagggaaggcccagtgccaagcatagcaggagctcaggtg
gctgcagatggtgtgaaccccaggagcaggatggccggcactccccccagaccctccagagccccaggttggctgcc
ctcttcactgccgacacccctgggtccacttctgccctttcccacctaaaacctttagggctcccactttctcccaaatgtga
gacatcaccacggctcccagggagtgtccagaagggcatctggctgagaggtcctgacatctgggagcctcaggcccc
acaatggacagacgccctgccaggatgctgctgcagggctgttagctaggcggggtggagatggggtactttgcctctc
agaggccccggccccaccatgaaacctcagtgacaccccatttccctgagttcacatacctgtatcctactccagtcacct
tccccacgaacccctgggagcccaggatgatgctggggctggagccacgaccagcccacgagtgatccagctctgcc
aatcagcagtcatttcccaagtgttccagccctgccaggtcccactacagcagtaatggaggccccagacaccagtcca
gcagttagagggctggactagcaccagctttcaagcctcagcatctcaaggtgaatggccagtgcccctccccgtggcc
atcacaggatcgcagatatgaccctaggggaagaaatatcctgggagtaaggaagtgcccatactcaaggatggcccct
ctgtgacctaacctgtccctgaggattgtacttccaggcgttaaaacagtagaacgcctgcctgtgaacccccgccaagg
gactgcttggggaggccccctaaaccagaacacaggcactccagcaggacctctgaactctgaccaccctcagcaagt
gggcaccccccgcagcttccaaggcaccccagggctcaccacagcggcccctcctggcagcccctcacccaggccc
agaccctctaagatggcacatctaagccaatccacctccttgtcattcctcctgtccccacccaggacccttctcagatgaa
accttcgctccagccgctgggccctctctcctgcccctctggcagttctccagggactccgcctcccactctctgtctctcc
ctgcactcctaggaacaagcgacctccaggaagcccagtccaattatcccctctgtgtcctccccaatctctgcctctggg
tggatttgagcaccacatcctgttctcttcgacctgaaactccttggccccggtgtccgctctcctgggccctcttttctctcct
cccctcttccgtgccccgtttgtttggtgttacaggcaggccccggggagccgtccctccagctgctcttccttgtctgtctc
aggagccagaaactggcagcatctaaaaagggctcctgtttcttcatctgcccagcctcctagcccaaccagggctctgg
cctcactccagagggtgggctccagagggcaggggttgcaccctcttagtgcctcagaggctcagctgggtgcaggat
gggggggccctcagggagcccctcagtgactgctgatcacttactgcaggactgttcccagctcttcccaatcattggaat
gacaatacctagttctgctccatcatagtgatgcaggaaaaatgttactgaaatcctggttcttgtttagcaatcgaagaatg
aattccgcgaacacacaggcagcaagcaagcgaagcctttattaaaggaaagcagatagctcccagggctgcaggga
gcggggagaagagctccccactctctattgtcctatagggctttttaccccttaaagttggggggatacaaaaaaaataga
agaaaaagggagttcccgtcagggcacagcagaaacaaatccaactaggaaccatgaggttgggggttcgattcctgg
cctctctcagtgggttaaggatgcagcgttgccgtgagctatgatacaggtcacagatgcagctcagatctactagtcaatt
gacaggcgccggagcaggagctaggcctttggccggccggcgcgccacgcgtataacttcgtatagcatacattatac
gaagttatcttaagggctatggcagggcctgccgccccgacgttggctgcgagccctgggccttcacccgaacttgggg
ggtggggtggggaaaaggaagaaacgcgggcgtattggccccaatggggtctcggtggggtatcgacagagtgcca
gccctgggaccgaaccccgcgtttatgaacaaacgacccaacaccgtgcgttttattctgtctttttattgccgtcatagcgc
gggttccttccggtattgtctccttccgtgtttcactcgagttagaagaactcgtcaagaaggcgatagaaggcgatgcgct
gcgaatcgggagcggcgataccgtaaagcacgaggaagcggtcagcccattcgccgccaagctcttcagcaatatcac
gggtagccaacgctatgtcctgatagcggtccgccacacccagccggccacagtcgatgaatccagaaaagcggccat
tttccaccatgatattcggcaagcaggcatcgccatgggtcacgacgagatcctcgccgtcgggcatgcgcgccttgag
cctggcgaacagttcggctggcgcgagcccctgatgctcttcgtccagatcatcctgatcgacaagaccggcttccatcc
gagtacgtgctcgctcgatgcgatgtttcgcttggtggtcgaatgggcaggtagccggatcaagcgtatgcagccgccg
cattgcatcagccatgatggatactttctcggcaggagcaaggtgagatgacaggagatcctgccccggcacttcgccc
aatagcagccagtcccttcccgcttcagtgacaacgtcgagcacagctgcgcaaggaacgcccgtcgtggccagccac
gatagccgcgctgcctcgtcctgcagttcattcagggcaccggacaggtcggtcttgacaaaaagaaccgggcgcccct
gcgctgacagccggaacacggcggcatcagagcagccgattgtctgttgtgcccagtcatagccgaatagcctctccac
ccaagcggccggagaacctgcgtgcaatccatcttgttcaatggccgatcccattccagatctgttagcctcccccatctc
ccgtgcaaacgtgcgcgccaggtcgcagatcgtcggtatggagcctggggtggtgacgtgggtctggatcatcccgga
ggtaagttgcagcagggcgtcccggcagccggcgggcgattggtcgtaatccaggataaagacgtgcatgggacgga
ggcgtttggtcaagacgtccaaggcccaggcaaacacgttgtacaggtcgccgttgggggccagcaactcgggggcc
cgaaacagggtaaataacgtgtccccgatatggggtcgtgggcccgcgttgctctggggctcggcaccctggggcggc
acggccgtccccgaaagctgtccccaatcctcccgccacgacccgccgccctgcagataccgcaccgtattggcaagc
agcccgtaaacgcggcgaatcgcggccagcatagccaggtcaagccgctcgccggggcgctggcgtttggccaggc
ggtcgatgtgtctgtcctccggaagggcccccaacacgatgtttgtgccgggcaaggtcggcgggatgagggccacga
acgccagcacggcctggggggtcatgctgcccataaggtatcgcgcggccgggtagcacaggagggcggcgatgg
gatggcggtcgaagatgagggtgagggccgggggcggggcatgtgagctcccagcctcccccccgatatgaggagc
cagaacggcgtcggtcacggcataaggcatgcccattgttatctgggcgcttgtcattaccaccgccgcgtccccggcc
gatatctcaccctggtcaaggcggtgttgtgtggtgtagatgttcgcgattgtctcggaagcccccagcacccgccagtaa
gtcatcggctcgggtacgtagacgatatcgtcgcgcgaacccagggccaccagcagttgcgtggtggtggttttccccat
cccgtggggaccgtctatataaacccgcagtagcgtgggcattttctgctccgggcggacttccgtggcttcttgctgccg
gcgagggcgcaacgccgtacgtcggttgctatggccgcgagaacgcgcagcctggtcgaacgcagacgcgtgctgat
ggccggggtacgaagccatggtggctctagaggtcgaaaggcccggagatgaggaagaggagaacagcgcggcag
acgtgcgcttttgaagcgtgcagaatgccgggcttccggaggaccttcgggcgcccgccccgcccctgagcccgcccc
tgagcccgcccccggacccaccccttcccagcctctgagcccagaaagcgaaggagccaaagctgctattggccgct
gccccaaaggcctacccgcttccattgctcagcggtgctgtccatctgcacgagactagtgagacgtgctacttccatttgt
cacgtcctgcacgacgcgagctgcggggcgggggggaacttcctgactaggggaggagtagaaggtggcgcgaag
gggccaccaaagaacggagccggttggcgcctaccggtggatgtggaatgtgtgcgaggccagaggccacttgtgta
gcgccaagtgcccagcggggctgctaaagcgcatgctccagactgccttgggaaaagcgcctcccctacccggtagg
gatccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatga
cgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggc
agtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgccca
gtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcag
tacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttg
gcaccaaaatcaacggttaacaagcttataacttcgtatagcatacattatacgaagttattacgtagcggccgcgtcgacg
atatcgctgccggagcccccggggccgctgccggaagatctggcattgctgtgactgtggtgtaggccggcagctgga
gctctgattagacccctcacctgggaatctccatatgctgcacgtgcggccctaaaaagacaaaagacaaaaaaaaaaa
aaaaaaaaaaaaatcaaaaaaaaacatagggggttaccaacgtggggtccagaaagatgtggttttctcccattggcctt
gcccagttacctatatcagtccttgtccaacaggggttttaggggtggaaatgccccataaattttacggtttctttgcccttct
cttcctttagactgagtcaccattgctctcattccttttctatcagttgaggagtgggttagagattaaggtccatgtggtggag
gtacacttcttatagtaaacaaggcctatggggaattactctctggagcccttaaaccacaaatgataatccatgccacatc
aaagatgcatcgaagcccatgctcctacactgactacctgagttagcattctgcctcaacaggactgaccatccccagctc
tggggcagatatcctctctctgccacaagggcagtgacccccatgctgtctgagggtcacgctttaccccccccccaccc
ctgccgtgaccccccagaccaccccaggaggtgggcactaatatccctcattaccccatagatgaggaaacagaggttc
ccccggggtcccacaggtgctcagggtcacatgcaccgtgggcacccaggccccatcccaaggccaccctccctcctc
aggaagctgtgctgcgctgggccagaaggtactgcacacgactcctcagcctccggtggtgggaggcagcctcaagc
ctctgagtgggggggcacccgggctcctcaatctatactgactcctgggggtgggagaaggggagggggagctgtgg
cctctgagtccactaagcaaatcagggtgggcaatgcgggcccatttcaaggaggagagaaccgaggctctgacagca
ggccgggggtccagggacctgcccagggtcataggctgaactgctggctgacctgccttgggttctttccttggctcctc
agccctgtgtgatgtgacaggtcattcattcactcactcgctcattcattcagcaaaccctcagtgagccctgctgggagca
ggtgctaggggcaaggagacaggacctcttgccctggaacagctgaagcactgggggacaggcagtggcagggag
gtgcgtgatcaccgctgaccccattccatcctccagcccccaggtcagtttccacccaccattgaccccaccatgtcctcc
atccccaaggtcagtttcccgcccaaggagcatctccttacacactagggacaaaatttcacggctgtcactgggcatctc
tccacgctcatcacagccctctagcagccttgaagtcctgtagagcccttcccatttcacagaagggacaagactatgag
ggccacaccgtgagccatgagccttaggctgtgagccgggacagcccctgcaggactggtggcctcagggcactggg
tggggagggtgcacagtgggtgggccccttgtggaatagagaggagtgtcaggtcaggggagggggcttggcctggc
cctggcctgcctggtgtgcaaccctaggcagcccctccttcccaggcctcctacttcctggaggccaagcctcagggag
gtaattgagtcaggtgggggagggggggttgtggctttcttcacagcagaaaaacagagcccacaatagtgtccactga
gacagaggggtcctgggggaggggaggggtgggaggtgactgctgagccctgtgggagggagggagcaactactg
agctgagctgggtgactctcccatctgccccgccccctgtggggccagcagagtcaccgagagaacatgacccagcca
ggcctggacagggggacacccatgtcctttaccccacagggttcactgagcctatctgccccaagcctgtgtctccctgg
gacggagaccctcactcccaaccacaaaggtctaaactcaagttcccaacagccttgaaaatacagcttccgggggcct
ccaaggagcagtcagccgtccactgccaggctcgctggctcagtgacacaggacacatcctgatgacggtccacctgt
ctccaagcaggttctcctctgccgatggggcaacgagctcctcctgtggctccctggctggatgcgtgggaggcggggt
gggggggcaggcggtgttcctggccgcacacaaggagcacccccaccagcatccgaagacgggggcccggtctttc
cccaaaacactgcttgcgggagactttgtgacgtttccaggggccatgctcccttcgggcagcttgggggacttctgctcc
tatgtggtcacctgcagggactccccccaggccttggggacaaacaaagtgatgagagggagggttagtgggtcgggg
cagggccagtctttggaccggtttatctgaaaagccagttggtcaccgggaaccacagcaaacctaaacccatttggcca
ggcatctcccagggacagtctcccccaggatgcggggcccaggggggctccaggggtgacctgcgtcctggatttccc
tgatgctcccagttcgtgcctctgtccaagcatgatttttaatagtgccccttccactcccagaaatgtccaagtgtgggcaa
taaattctggtcacctgagctcagtgtaactgtttgctgaatgacacttactgtaacaggttaaaatgggaggcccaaggcc
acgcagagccatcgaaggctctgtgtgtcccagccctgatagaagcatcaggatggggactgtggcctcaccaggggc
cacatccaggcggtcaccatggggttcctggtctccgtgggccttgactggagcccctggtgtgagctcaccccatccca
gcctgtgagaggcctggatgtgggcctgacatcatttcccacccagtgacagcactgcatgtgatggggcctctgggca
gcctttttcccgggggaaactggcaggaatcaggaccaccaggacaggggtcaggggagaggcgatgctgggcacc
agagcctggaccaccctcgggttctcagcgatgggcaacccctgccacccagggccccgccttcctggggagacatc
ggggtttccaggccatcctgggaggagggtgggagcctcagctagaccccagctggcttgcccccccatgccccggc
caagagagggtcttggagggaagggggaccccagaccagcctggcgagcccatcctcagggtctctggtcagacag
gggctcagctgagctccagggtagaccaaggccctgcgtggatgaggccagtgtggtcactgcccagagcaaagcca
cctctcagcagccctttcctgagcaccttctgtgtgcggggacatcagcagtggcaacacagccatgctggggactcag
ggctagagacaggggaccagcctatggagagtgggtagtgtcctgcagggcaggcttgtgccctggagaaaacaaac
cagggtgaggccagggacgctggccgggttcacagggtgatggctgagcacagagtgccaggggctggactgtcct
gactctgggttggtggctgagggcctgtgtccctctatgcctctgggttggtgataatggaaacttgctccctggagagac
aggacgaatggttgatgggaaatgaatgtttgcttgtcacttggttgactgttgttgccgttagcattgggcttcttgggccag
gcagcctcaggccagcactgctgggctccccacaggcccgacaccctcagccctgtgcagctggcctggcgaaacca
agaggccctgatgcccaaaatagccgggaaaccccaaccagcccagccctggcagcaggtgcctcccatttgcctgg
gctgggggaggggtggctctggttctggaagtttctgccagtccagctggagaagggacctgtatcccagcacccagg
ccgcccaagcccctgcaccagggcctgggccaggcagagttgacatcaatcaattgggagctgctggaatgcatggag
gcggcgctctcgaggctggaggaggccagctgatttaaatcggtccgcgtacgatgcatattaccctgttatccctaccg
cggttactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgatgctcttctcccggtgaaaacctctgacacat
ggctcttctaaatccggagtttaaacgcttccttcatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggc
cgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaa
acccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgctta
ccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtag
gtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtctt
gagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgta
ggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctg
aagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgttt
gcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtgg
aacgaaaactcacgttaagggattttggtcatgcctaggtggcaaacagctattatgggtattatgggtctaccggtgcatg
agattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttg
gtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccc
cgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctca
ccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcct
ccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgcta
caggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatc
ccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactc
atggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaag
tcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcag
aactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcga
tgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggca
aaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcattt
atcagggttattgtctcgggagcggatacatatttgaatgtatttagaaaaa
The two-step strategy outline above, utilizing a vector pair, can be used to delete the entire J/C cluster region (i.e., all J/C units), multiple J/C units or an individual J/C unit.
Selectable Marker Genes
The DNA constructs can be designed to modify the endogenous, target immunoglobulin gene. The homologous sequence for targeting the construct can have one or more deletions, insertions, substitutions or combinations thereof. The alteration can be the insertion of a selectable marker gene fused in reading frame with the upstream sequence of the target gene.
Suitable selectable marker genes include, but are not limited to: genes conferring the ability to grow on certain media substrates, such as the tk gene (thymidine kinase) or the hprt gene (hypoxanthine phosphoribosyltransferase) which confer the ability to grow on HAT medium (hypoxanthine, aminopterin and thymidine); the bacterial gpt gene (guanine/xanthine phosphoribosyltransferase) which allows growth on MAX medium (mycophenolic acid, adenine, and xanthine). See, for example, Song, K-Y., et al. Proc. Nat'l Acad. Sci. U.S.A. 84:6820-6824 (1987); Sambrook, J., et al., Molecular Cloning—A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989), Chapter 16. Other examples of selectable markers include: genes conferring resistance to compounds such as antibiotics, genes conferring the ability to grow on selected substrates, genes encoding proteins that produce detectable signals such as luminescence, such as green fluorescent protein, enhanced green fluorescent protein (eGFP). A wide variety of such markers are known and available, including, for example, antibiotic resistance genes such as the neomycin resistance gene (neo) (Southern, P., and P. Berg, J. Mol. Appl. Genet. 1:327-341 (1982)); and the hygromycin resistance gene (hyg) (Nucleic Acids Research 11:6895-6911 (1983), and Te Riele, H., et al., Nature 348:649-651 (1990)). Other selectable marker genes include: acetohydroxyacid synthase (AHAS), alkaline phosphatase (AP), beta galactosidase (LacZ), beta glucoronidase (GUS), chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase (NOS), octopine synthase (OCS), and derivatives thereof. Multiple selectable markers are available that confer resistance to ampicillin, bleomycin, chloramphenicol, gentamycin, hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin, and tetracycline.
Methods for the incorporation of antibiotic resistance genes and negative selection factors will be familiar to those of ordinary skill in the art (see, e.g., WO 99/15650; U.S. Pat. No. 6,080,576; U.S. Pat. No. 6,136,566; Niwa et al., J. Biochem. 113:343-349 (1993); and Yoshida et al., Transgenic Research 4:277-287 (1995)).
Combinations of selectable markers can also be used. For example, to target an immunoglobulin gene, a neo gene (with or without its own promoter, as discussed above) can be cloned into a DNA sequence which is homologous to the immunoglobulin gene. To use a combination of markers, the HSV-tk gene can be cloned such that it is outside of the targeting DNA (another selectable marker could be placed on the opposite flank, if desired). After introducing the DNA construct into the cells to be targeted, the cells can be selected on the appropriate antibiotics. In this particular example, those cells which are resistant to G418 and gancyclovir are most likely to have arisen by homologous recombination in which the neo gene has been recombined into the immunoglobulin gene but the tk gene has been lost because it was located outside the region of the double crossover.
Deletions can be at least about 50 bp, more usually at least about 100 bp, and generally not more than about 20 kbp, where the deletion can normally include at least a portion of the coding region including a portion of or one or more exons, a portion of or one or more introns, and can or can not include a portion of the flanking non-coding regions, particularly the 5′-non-coding region (transcriptional regulatory region). Thus, the homologous region can extend beyond the coding region into the 5′-non-coding region or alternatively into the 3′-non-coding region. Insertions can generally not exceed 10 kbp, usually not exceed 5 kbp, generally being at least 50 bp, more usually at least 200 bp.
The region(s) of homology can include mutations, where mutations can further inactivate the target gene, in providing for a frame shift, or changing a key amino acid, or the mutation can correct a dysfunctional allele, etc. The mutation can be a subtle change, not exceeding about 5% of the homologous flanking sequences. Where mutation of a gene is desired, the marker gene can be inserted into an intron or an exon.
The construct can be prepared in accordance with methods known in the art, various fragments can be brought together, introduced into appropriate vectors, cloned, analyzed and then manipulated further until the desired construct has been achieved. Various modifications can be made to the sequence, to allow for restriction analysis, excision, identification of probes, etc. Silent mutations can be introduced, as desired. At various stages, restriction analysis, sequencing, amplification with the polymerase chain reaction, primer repair, in vitro mutagenesis, etc. can be employed.
The construct can be prepared using a bacterial vector, including a prokaryotic replication system, e.g. an origin recognizable by E. coli, at each stage the construct can be cloned and analyzed. A marker, the same as or different from the marker to be used for insertion, can be employed, which can be removed prior to introduction into the target cell. Once the vector containing the construct has been completed, it can be further manipulated, such as by deletion of the bacterial sequences, linearization, introducing a short deletion in the homologous sequence. After final manipulation, the construct can be introduced into the cell.
The present invention further includes recombinant constructs containing sequences of immunoglobulin genes. The constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation. The construct can also include regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available. The following vectors are provided by way of example. Bacterial: pBs, pQE-9 (Qiagen), phagescript, PsiX174, pBluescript SK, pBsKS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLneo, pSv2cat, pOG44, pXT1, pSG (Stratagene) pSVK3, pBPv, pMSG, pSVL (Pharmiacia), viral origin vectors (M13 vectors, bacterial phage 1 vectors, adenovirus vectors, and retrovirus vectors), high, low and adjustable copy number vectors, vectors which have compatible replicons for use in combination in a single host (pACYC184 and pBR322) and eukaryotic episomal replication vectors (pCDM8). Other vectors include prokaryotic expression vectors such as pcDNA II, pSL301, pSE280, pSE380, pSE420, pTrcHisA, B, and C, pRSET A, B, and C (Invitrogen, Corp.), pGEMEX-1, and pGEMEX-2 (Promega, Inc.), the pET vectors (Novagen, Inc.), pTrc99A, pKK223-3, the pGEX vectors, pEZZ18, pRIT2T, and pMC1871 (Pharmacia, Inc.), pKK233-2 and pKK388-1 (Clontech, Inc.), and pProEx-HT (Invitrogen, Corp.) and variants and derivatives thereof. Other vectors include eukaryotic expression vectors such as pFastBac, pFastBacHT, pFastBacDUAL, pSFV, and pTet-Splice (Invitrogen), pEUK-C1, pPUR, pMAM, pMAMneo, pBI101, pBI121, pDR2, pCMVEBNA, and pYACneo (Clontech), pSVK3, pSVL, pMSG, pCH110, and pKK232-8 (Pharmacia, Inc.), p3′SS, pXT1, pSG5, pPbac, pMbac, pMC1neo, and pOG44 (Stratagene, Inc.), and pYES2, pAC360, pBlueBacHis A, B, and C, pVL1392, pBlueBacIII, pCDM8, pcDNA1, pZeoSV, pcDNA3 pREP4, pCEP4, and pEBVHis (Invitrogen, Corp.) and variants or derivatives thereof. Additional vectors that can be used include: pUC18, pUC19, pBlueScript, pSPORT, cosmids, phagemids, YAC's (yeast artificial chromosomes), BAC's (bacterial artificial chromosomes), P1 (Escherichia coli phage), pQE70, pQE60, pQE9 (quagan), pBS vectors, PhageScript vectors, BlueScript vectors, pNH8A, pNH116A, pNH18A, pNH46A (Stratagene), pcDNA3 (Invitrogen), pGEX, pTrsfus, pTrc99A, pET-5, pET-9, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia), pSPORT1, pSPORT2, pCMVSPORT2.0 and pSV-SPORT1 (Invitrogen), pTrxFus, pThioHis, pLEX, pTrcHis, pTrcHis2, pRSET, pBlueBacHis2, pcDNA3.1/His, pcDNA3.1(−)/Myc-His, pSecTag, pEBVHis, pPIC9K, pPIC3.5K, pAO815, pPICZ, pPICZ□, pGAPZ, pGAPZ□, pBlueBac4.5, pBlueBacHis2, pMelBac, pSinRep5, pSinHis, pIND, pIND(SP1), pVgRXR, pcDNA2.1, pYES2, pZErO1.1, pZErO-2.1, pCR-Blunt, pSE280, pSE380, pSE420, pVL1392, pVL1393, pCDM8, pcDNA1.1, pcDNA1.1/Amp, pcDNA3.1, pcDNA3.1/Zeo, pSe, SV2, pRc/CMV2, pRc/RSV, pREP4, pREP7, pREP8, pREP9, pREP 10, pCEP4, pEBVHis, pCR3.1, pCR2.1, pCR3.1-Uni, and pCRBac from Invitrogen; □ ExCell, □ gt11, pTrc99A, pKK223-3, pGEX-1□T, pGEX-2T, pGEX-2TK, pGEX-4T-1, pGEX-4T-2, pGEX-4T-3, pGEX-3X, pGEX-5X-1, pGEX-5X-2, pGEX-5X-3, pEZZ18, pRIT2T, pMC1871, pSVK3, pSVL, pMSG, pCH110, pKK232-8, pSL1180, pNEO, and pUC4K from Pharmacia; pSCREEN-1b(+), pT7Blue(R), pT7Blue-2, pCITE-4abc(+), pOCUS-2, pTAg, pET-32LIC, pET-30LIC, pBAC-2 cp LIC, pBACgus-2 cp LIC, pT7Blue-2 LIC, pT7Blue-2, □SCREEN-1, □BlueSTAR, pET-3abcd, pET-7abc, pET9abcd, pET11abcd, pET12abc, pET-14b, pET-15b, pET-16b, pET-17b-pET-17xb, pET-19b, pET-20b(+), pET-21abcd(+), pET-22b(+), pET-23abcd(+), pET-24abcd(+), pET-25b(+), pET-26b(+), pET-27b(+), pET-28abc(+), pET-29abc(+), pET-30abc(+), pET-31b(+), pET-32abc(+), pET-33b(+), pBAC-1, pBACgus-1, pBAC4x-1, pBACgus4x-1, pBAC-3 cp, pBACgus-2 cp, pBACsurf-1, plg, Signal plg, pYX, Selecta Vecta-Neo, Selecta Vecta-Hyg, and Selecta Vecta-Gpt from Novagen; pLexA, pB42AD, pGBT9, pAS2-1, pGAD424, pACT2, pGAD GL, pGAD GH, pGAD10, pGilda, pEZM3, pEGFP, pEGFP-1, pEGFP-N, pEGFP-C, pEBFP, pGFPuv, pGFP, p6xHis-GFP, pSEAP2-Basic, pSEAP2-Contral, pSEAP2-Promoter, pSEAP2-Enhancer, p□gal-Basic, p□gal-Control, p□gal-Promoter, p□gal-Enhancer, pCMV□, pTet-Off, pTet-On, pTK-Hyg, pRetro-Off, pRetro-On, pIRES1neo, pIRES1hyg, pLXSN, pLNCX, pLAPSN, pMAMneo, pMAMneo-CAT, pMAMneo-LUC, pPUR, pSV2neo, pYEX4T-1/2/3, pYEX-S1, pBacPAK-His, pBacPAK8/9, pAcUW31, BacPAK6, pTrip1Ex, □gt10, □gt11, pWE15, and □Trip1Ex from Clontech; Lambda ZAP II, pBK-CMV, pBK-RSV, pBluescript II KS +/−, pBluescript II SK +/−, pAD-GAL4, pBD-GAL4 Cam, pSurfscript, Lambda FIX II, Lambda DASH, Lambda EMBL3, Lambda EMBL4, SuperCos, pCR-Scrigt Amp, pCR-Script Cam, pCR-Script Direct, pBS +/−, pBC KS +/−, pBC SK +/−, Phagescript, pCAL-n-EK, pCAL-n, pCAL-c, pCAL-kc, pET-3abcd, pET-11abcd, pSPUTK, pESP-1, pCMVLacI, pOPRSVI/MCS, pOPI3 CAT, pXT1, pSG5, pPbac, pMbac, pMC1neo, pMC1neo Poly A, pOG44, pOG45, pFRT□GAL, pNEO□GAL, pRS403, pRS404, pRS405, pRS406, pRS413, pRS414, pRS415, and pRS416 from Stratagene and variants or derivatives thereof. Two-hybrid and reverse two-hybrid vectors can also be used, for example, pPC86, pDBLeu, pDBTrp, pPC97, p2.5, pGAD1-3, pGAD10, pACt, pACT2, pGADGL, pGADGH, pAS2-1, pGAD424, pGBT8, pGBT9, pGAD-GAL4, pLexA, pBD-GAL4, pHISi, pHISi-1, placZi, pB42AD, pDG202, pJK202, pJG4-5, pNLexA, pYESTrp and variants or derivatives thereof. Any other plasmids and vectors may be used as long as they are replicable and viable in the host.
Techniques which can be used to allow the DNA construct entry into the host cell include, for example, calcium phosphate/DNA co precipitation, microinjection of DNA into the nucleus, electroporation, bacterial protoplast fusion with intact cells, transfection, or any other technique known by one skilled in the art. The DNA can be single or double stranded, linear or circular, relaxed or supercoiled DNA. For various techniques for transfecting mammalian cells, see, for example, Keown et al., Methods in Enzymology Vol. 185, pp. 527-537 (1990).
In one specific embodiment, heterozygous or homozygous knockout cells can be produced by transfection of primary fetal fibroblasts with a knockout vector containing immunoglobulin gene sequence isolated from isogenic DNA. In another embodiment, the vector can incorporate a promoter trap strategy, using, for example, IRES (internal ribosome entry site) to initiate translation of the Neor gene.
Site Specific Recombinases
In additional embodiments, the targeting constructs can contain site specific recombinase sites, such as, for example, lox. In one embodiment, the targeting arms can insert the site specific recombinase target sites into the targeted region such that one site specific recombinase target site is located 5′ to the second site specific recombinase target site. Then, the site specific recombinase can be activated and/or applied to the cell such that the intervening nucleotide sequence between the two site specific recombinase sites is excised.
Site-specific recombinases include enzymes or recombinases that recognize and bind to a short nucleic acid site or sequence-specific recombinase target site, i.e., a recombinase recognition site, and catalyze the recombination of nucleic acid in relation to these sites. These enzymes include recombinases, transposases and integrases. Examples of sequence-specific recombinase target sites include, but are not limited to, lox sites, att sites, dif sites and frt sites. Non-limiting examples of site-specific recombinases include, but are not limited to, bacteriophage P1 Cre recombinase, yeast FLP recombinase, Inti integrase, bacteriophage λ, phi 80, P22, P2, 186, and P4 recombinase, Tn3 resolvase, the Hin recombinase, and the Cin recombinase, E. coli xerC and xerD recombinases, Bacillus thuringiensis recombinase, TpnI and the β-lactamase transposons, and the immunoglobulin recombinases.
In one embodiment, the recombination site can be a lox site that is recognized by the Cre recombinase of bacteriophage P1. Lox sites refer to a nucleotide sequence at which the product of the cre gene of bacteriophage P1, the Cre recombinase, can catalyze a site-specific recombination event. A variety of lox sites are known in the art, including the naturally occurring loxP, loxB, loxL and loxR, as well as a number of mutant, or variant, lox sites, such as loxP511, loxP514, lox.DELTA.86, lox.DELTA.117, loxC2, loxP2, loxP3 and lox P23. Additional example of lox sites include, but are not limited to, loxB, loxL, loxR, loxP, loxP3, loxP23, loxΔ86, loxΔ117, loxP511, and loxC2.
In another embodiment, the recombination site is a recombination site that is recognized by a recombinases other than Cre. In one embodiment, the recombinase site can be the FRT sites recognized by FLP recombinase of the 2 pi plasmid of Saccharomyces cerevisiae. FRT sites refer to a nucleotide sequence at which the product of the FLP gene of the yeast 2 micron plasmid, FLP recombinase, can catalyze site-specific recombination. Additional examples of the non-Cre recombinases include, but are not limited to, site-specific recombinases include: att sites recognized by the Int recombinase of bacteriophage λ (e.g. att1, att2, att3, attP, attB, attL, and attR), the recombination sites recognized by the resolvase family, and the recombination site recognized by transposase of Bacillus thruingiensis.
In particular embodiments of the present invention, the targeting constructs can contain: sequence homologous to a porcine immunoglobulin gene as described herein, a selectable marker gene and/or a site specific recombinase target site.
Selection of Homologously Recombined Cells
The cells can then be grown in appropriately-selected medium to identify cells providing the appropriate integration. The presence of the selectable marker gene inserted into the immunoglobulin gene establishes the integration of the target construct into the host genome. Those cells which show the desired phenotype can then be further analyzed by restriction analysis, electrophoresis, Southern analysis, polymerase chain reaction, etc to analyze the DNA in order to establish whether homologous or non-homologous recombination occurred. This can be determined by employing probes for the insert and then sequencing the 5′ and 3′ regions flanking the insert for the presence of the immunoglobulin gene extending beyond the flanking regions of the construct or identifying the presence of a deletion, when such deletion is introduced. Primers can also be used which are complementary to a sequence within the construct and complementary to a sequence outside the construct and at the target locus. In this way, one can only obtain DNA duplexes having both of the primers present in the complementary chains if homologous recombination has occurred. By demonstrating the presence of the primer sequences or the expected size sequence, the occurrence of homologous recombination is supported.
The polymerase chain reaction used for screening homologous recombination events is known in the art, see, for example, Kim and Smithies, Nucleic Acids Res. 16:8887-8903, 1988; and Joyner et al., Nature 338:153-156, 1989. The specific combination of a mutant polyoma enhancer and a thymidine kinase promoter to drive the neomycin gene has been shown to be active in both embryonic stem cells and EC cells by Thomas and Capecchi, supra, 1987; Nicholas and Berg (1983) in Teratocarcinoma Stem Cell, eds. Siver, Martin and Strikland (Cold Spring Harbor Lab., Cold Spring Harbor, N.Y. (pp. 469-497); and Linney and Donerly, Cell 35:693-699, 1983.
The cell lines obtained from the first round of targeting are likely to be heterozygous for the targeted allele. Homozygosity, in which both alleles are modified, can be achieved in a number of ways. One approach is to grow up a number of cells in which one copy has been modified and then to subject these cells to another round of targeting using a different selectable marker. Alternatively, homozygotes can be obtained by breeding animals heterozygous for the modified allele, according to traditional Mendelian genetics. In some situations, it can be desirable to have two different modified alleles. This can be achieved by successive rounds of gene targeting or by breeding heterozygotes, each of which carries one of the desired modified alleles.
Identification of Cells that have Undergone Homologous Recombination
In one embodiment, the selection method can detect the depletion of the immunoglobulin gene directly, whether due to targeted knockout of the immunoglobulin gene by homologous recombination, or a mutation in the gene that results in a nonfunctioning or nonexpressed immunoglobulin. Selection via antibiotic resistance has been used most commonly for screening (see above). This method can detect the presence of the resistance gene on the targeting vector, but does not directly indicate whether integration was a targeted recombination event or a random integration. Certain technology, such as Poly A and promoter trap technology, increase the probability of targeted events, but again, do not give direct evidence that the desired phenotype, a cell deficient in immunoglobulin gene expression, has been achieved. In addition, negative forms of selection can be used to select for targeted integration; in these cases, the gene for a factor lethal to the cells is inserted in such a way that only targeted events allow the cell to avoid death. Cells selected by these methods can then be assayed for gene disruption, vector integration and, finally, immunoglobulin gene depletion. In these cases, since the selection is based on detection of targeting vector integration and not at the altered phenotype, only targeted knockouts, not point mutations, gene rearrangements or truncations or other such modifications can be detected.
Animal cells believed to lacking expression of functional immunoglobulin genes can be further characterized. Such characterization can be accomplished by the following techniques, including, but not limited to: PCR analysis, Southern blot analysis, Northern blot analysis, specific lectin binding assays, and/or sequencing analysis.
PCR analysis as described in the art can be used to determine the integration of targeting vectors. In one embodiment, amplimers can originate in the antibiotic resistance gene and extend into a region outside the vector sequence. Southern analysis can also be used to characterize gross modifications in the locus, such as the integration of a targeting vector into the immunoglobulin locus. Whereas, Northern analysis can be used to characterize the transcript produced from each of the alleles.
Further, sequencing analysis of the cDNA produced from the RNA transcript can also be used to determine the precise location of any mutations in the immunoglobulin allele.
In another aspect of the present invention, ungulate cells lacking at least one allele of a functional region of an ungulate heavy chain, kappa light chain and/or lambda light chain locus produced according to the process, sequences and/or constructs described herein are provided. These cells can be obtained as a result of homologous recombination. Particularly, by inactivating at least one allele of an ungulate heavy chain, kappa light chain or lambda light chain gene, cells can be produced which have reduced capability for expression of porcine antibodies. In other embodiments, mammalian cells lacking both alleles of an ungulate heavy chain, kappa light chain and/or lambda light chain gene can be produced according to the process, sequences and/or constructs described herein. In a further embodiment, porcine animals are provided in which at least one allele of an ungulate heavy chain, kappa light chain and/or lambda light chain gene is inactivated via a genetic targeting event produced according to the process, sequences and/or constructs described herein. In another aspect of the present invention, porcine animals are provided in which both alleles of an ungulate heavy chain, kappa light chain and/or lambda light chain gene are inactivated via a genetic targeting event. The gene can be targeted via homologous recombination. In other embodiments, the gene can be disrupted, i.e. a portion of the genetic code can be altered, thereby affecting transcription and/or translation of that segment of the gene. For example, disruption of a gene can occur through substitution, deletion (“knock-out”) or insertion (“knock-in”) techniques. Additional genes for a desired protein or regulatory sequence that modulate transcription of an existing sequence can be inserted.
In embodiments of the present invention, alleles of ungulate heavy chain, kappa light chain or lambda light chain gene are rendered inactive according to the process, sequences and/or constructs described herein, such that functional ungulate immunoglobulins can no longer be produced. In one embodiment, the targeted immunoglobulin gene can be transcribed into RNA, but not translated into protein. In another embodiment, the targeted immunoglobulin gene can be transcribed in an inactive truncated form. Such a truncated RNA may either not be translated or can be translated into a nonfunctional protein. In an alternative embodiment, the targeted immunoglobulin gene can be inactivated in such a way that no transcription of the gene occurs. In a further embodiment, the targeted immunoglobulin gene can be transcribed and then translated into a nonfunctional protein.
III. Insertion of Artificial Chromosomes Containing Human Immunoglobulin Genes
Artificial Chromosomes
One aspect of the present invention provides ungulates and ungulate cells that lack at least one allele of a functional region of an ungulate heavy chain, kappa light chain and/or lambda light chain locus produced according to the processes, sequences and/or constructs described herein, which are further modified to express at least part of a human antibody (i.e. immunoglobulin (Ig)) locus. This human locus can undergo rearrangement and express a diverse population of human antibody molecules in the ungulate. These cloned, transgenic ungulates provide a replenishable, theoretically infinite supply of human antibodies (such as polyclonal antibodies), which can be used for therapeutic, diagnostic, purification, and other clinically relevant purposes.
In one particular embodiment, artificial chromosome (ACs) can be used to accomplish the transfer of human immunoglobulin genes into ungulate cells and animals. ACs permit targeted integration of megabase size DNA fragments that contain single or multiple genes. The ACs, therefore, can introduce heterologous DNA into selected cells for production of the gene product encoded by the heterologous DNA. In a one embodiment, one or more ACs with integrated human immunoglobulin DNA can be used as a vector for introduction of human Ig genes into ungulates (such as pigs).
First constructed in yeast in 1983, ACs are man-made linear DNA molecules constructed from essential cis-acting DNA sequence elements that are responsible for the proper replication and partitioning of natural chromosomes (Murray et al. (1983), Nature 301:189-193). A chromosome requires at least three elements to function. Specifically, the elements of an artificial chromosome include at least: (1) autonomous replication sequences (ARS) (having properties of replication origins—which are the sites for initiation of DNA replication), (2) centromeres (site of kinetochore assembly that is responsible for proper distribution of replicated chromosomes at mitosis and meiosis), and (3) telomeres (specialized structures at the ends of linear chromosomes that function to both stabilize the ends and facilitate the complete replication of the extreme termini of the DNA molecule).
In one embodiment, the human Ig can be maintained as an independent unit (an episome) apart from the ungulate chromosomal DNA. For example, episomal vectors contain the necessary DNA sequence elements required for DNA replication and maintenance of the vector within the cell. Episomal vectors are available commercially (see, for example, Maniatis, T. et al., Molecular Cloning, A Laboratory Manual (1982) pp. 368-369). The AC can stably replicate and segregate along side endogenous chromosomes. In an alternative embodiment, the human IgG DNA sequences can be integrated into the ungulate cell's chromosomes thereby permitting the new information to be replicated and partitioned to the cell's progeny as a part of the natural chromosomes (see, for example, Wigler et al. (1977), Cell 11:223). The AC can be translocated to, or inserted into, the endogenous chromosome of the ungulate cell. Two or more ACs can be introduced to the host cell simultaneously or sequentially.
ACs, furthermore, can provide an extra-genomic locus for targeted integration of megabase size DNA fragments that contain single or multiple genes, including multiple copies of a single gene operatively linked to one promoter or each copy or several copies linked to separate promoters. ACs can permit the targeted integration of megabase size DNA fragments that contain single or multiple human immunoglobulin genes. The ACs can be generated by culturing the cells with dicentric chromosomes (i.e., chromosomes with two centromeres) under such conditions known to one skilled in the art whereby the chromosome breaks to form a minichromosome and formerly dicentric chromosome.
ACs can be constructed from humans (human artificial chromosomes: “HACs”), yeast (yeast artificial chromosomes: “YACs”), bacteria (bacterial artificial chromosomes: “BACs”), bacteriophage P1-derived artificial chromosomes: “PACs”) and other mammals (mammalian artificial chromosomes: “MACs”). The ACs derive their name (e.g., YAC, BAC, PAC, MAC, HAC) based on the origin of the centromere. A YAC, for example, can derive its centromere from S. cerevisiae. MACs, on the other hand, include an active mammalian centromere while HACs refer to chromosomes that include human centromeres. Furthermore, plant artificial chromosomes (“PLACs”) and insect artificial chromosomes can also be constructed. The ACs can include elements derived from chromosomes that are responsible for both replication and maintenance. ACs, therefore, are capable of stably maintaining large genomic DNA fragments such as human Ig DNA.
In one embodiment, ungulates containing YACs are provided. YACs are genetically engineered circular chromosomes that contain elements from yeast chromosomes, such as S. cerevisiae, and segments of foreign DNAs that can be much larger than those accepted by conventional cloning vectors (e.g., plasmids, cosmids). YACs allow the propagation of very large segments of exogenous DNA (Schlessinger, D. (1990), Trends in Genetics 6:248-253) into mammalian cells and animals (Choi et al. (1993), Nature Gen 4:117-123). YAC transgenic approaches are very powerful and are greatly enhanced by the ability to efficiently manipulate the cloned DNA. A major technical advantage of yeast is the ease with which specific genome modifications can be made via DNA-mediated transformation and homologous recombination (Ramsay, M. (1994), Mol Biotech 1:181-201). In one embodiment, one or more YACs with integrated human Ig DNA can be used as a vector for introduction of human Ig genes into ungulates (such as pigs).
The YAC vectors contain specific structural components for replication in yeast, including: a centromere, telomeres, autonomous replication sequence (ARS), yeast selectable markers (e.g., TRP1, URA3, and SUP4), and a cloning site for insertion of large segments of greater than 50 kb of exogenous DNA. The marker genes can allow selection of the cells carrying the YAC and serve as sites for the synthesis of specific restriction endonucleases. For example, the TRP1 and URA3 genes can be used as dual selectable markers to ensure that only complete artificial chromosomes are maintained. Yeast selectable markers can be carried on both sides of the centromere, and two sequences that seed telomere formation in vivo are separated. Only a fraction of one percent of a yeast cell's total DNA is necessary for replication, however, including the center of the chromosome (the centromere, which serves as the site of attachment between sister chromatids and the sites of spindle fiber attachment during mitosis), the ends of the chromosome (telomeres, which serve as necessary sequences to maintain the ends of eukaryotic chromosomes), and another short stretch of DNA called the ARS which serves as DNA segments where the double helix can unwind and begin to copy itself.
In one embodiment, YACs can be used to clone up to about 1, 2, or 3 Mb of immunoglobulin DNA. In another embodiment, at least 25, 30, 40, 50, 60, 70, 75, 80, 85, 90, or 95 kilobases.
Yeast integrating plasmids, replicating vectors (which are fragments of YACs), can also be used to express human Ig. The yeast integrating plasmid can contain bacterial plasmid sequences that provide a replication origin and a drug-resistance gene for growth in bacteria (e.g., E. coli), a yeast marker gene for selection of transformants in yeast, and restriction sites for inserting Ig sequences. Host cells can stably acquire this plasmid by integrating it directly into a chromosome. Yeast replicating vectors can also be used to express human Ig as free plasmid circles in yeast. Yeast or ARS-containing vectors can be stabilized by the addition of a centromere sequence. YACs have both centromeric and telomeric regions, and can be used for cloning very large pieces of DNA because the recombinant is maintained essentially as a yeast chromosome.
YACs are provided, for example, as disclosed in U.S. Pat. Nos. 6,692,954, 6,495,318, 6,391,642, 6,287,853, 6,221,588, 6,166,288, 6,096,878, 6,015,708, 5,981,175, 5,939,255, 5,843,671, 5,783,385, 5,776,745, 5,578,461, and 4,889,806; European Patent Nos. 1 356 062 and 0 648 265; PCT Publication Nos. WO 03/025222, WO 02/057437, WO 02/101044, WO 02/057437, WO 98/36082, WO 98/12335, WO 98/01573, WO 96/01276, WO 95/14769, WO 95/05847, WO 94/23049, and WO 94/00569.
In another embodiment, ungulates containing BACs are provided. BACs are F-based plasmids found in bacteria, such as E. Coli, that can transfer approximately 300 kb of foreign DNA into a host cell. Once the Ig DNA has been cloned into the host cell, the newly inserted segment can be replicated along with the rest of the plasmid. As a result, billions of copies of the foreign DNA can be made in a very short time. In a particular embodiment, one or more BACs with integrated human Ig DNA are used as a vector for introduction of human Ig genes into ungulates (such as pigs).
The BAC cloning system is based on the E. coli F-factor, whose replication is strictly controlled and thus ensures stable maintenance of large constructs (Willets, N., and R. Skurray (1987), Structure and function of the F-factor and mechanism of conjugation. In Escherichia coli and Salmonella Typhimurium: Cellular and Molecular Biology (F. C. Neidhardt, Ed) Vol. 2 pp 1110-1133, Am. Soc. Microbiol., Washington, D.C.). BACs have been widely used for cloning of DNA from various eukaryotic species (Cai et al. (1995), Genomics 29:413-425; Kim et al. (1996), Genomics 34:213-218; Misumi et al. (1997), Genomics 40:147-150; Woo et al. (1994), Nucleic Acids Res 22:4922-4931; Zimmer, R. and Gibbins, A.M.V. (1997), Genomics 42:217-226). The low occurrence of the F-plasmid can reduce the potential for recombination between DNA fragments and can avoid the lethal overexpression of cloned bacterial genes. BACs can stably maintain the human immunoglobulin genes in a single copy vector in the host cells, even after 100 or more generations of serial growth.
BAC (or pBAC) vectors can accommodate inserts in the range of approximately 30 to 300 kb pairs. One specific type of BAC vector, pBeloBac11, uses a complementation of the lacZ gene to distinguish insert-containing recombinant molecules from colonies carrying the BAC vector, by color. When a DNA fragment is cloned into the lacZ gene of pBeloBac11, insertional activation results in a white colony on X-Gal/IPTG plates after transformation (Kim et al. (1996), Genomics 34:213-218) to easily identify positive clones.
For example, BACs can be provided such as disclosed in U.S. Pat. Nos. 6,713,281, 6,703,198, 6,649,347, 6,638,722, 6,586,184, 6,573,090, 6,548,256, 6,534,262, 6,492,577, 6,492,506, 6,485,912, 6,472,177, 6,455,254, 6,383,756, 6,277,621, 6,183,957, 6,156,574, 6,127,171, 5,874,259, 5,707,811, and 5,597,694; European Patent Nos. 0 805 851; PCT Publication Nos. WO 03/087330, WO 02/00916, WO 01/39797, WO 01/04302, WO 00/79001, WO 99/54487, WO 99/27118, and WO 96/21725.
In another embodiment, ungulates containing bacteriophage PACs are provided. In a particular embodiment, one or more bacteriophage PACs with integrated human Ig DNA are used as a vector for introduction of human Ig genes into ungulates (such as pigs). For example, PACs can be provided such as disclosed in U.S. Pat. Nos. 6,743,906, 6,730,500, 6,689,606, 6,673,909, 6,642,207, 6,632,934, 6,573,090, 6,544,768, 6,489,458, 6,485,912, 6,469,144, 6,462,176, 6,413,776, 6,399,312, 6,340,595, 6,287,854, 6,284,882, 6,277,621, 6,271,008, 6,187,533, 6,156,574, 6,153,740, 6,143,949, 6,017,755, and 5,973,133; European Patent Nos. 0 814 156; PCT Publication Nos. WO 03/091426, WO 03/076573, WO 03/020898, WO 02/101022, WO 02/070696, WO 02/061073, WO 02/31202, WO 01/44486, WO 01/07478, WO 01/05962, and WO 99/63103.
In a further embodiment, ungulates containing MACs are provided. MACs possess high mitotic stability, consistent and regulated gene expression, high cloning capacity, and non-immunogenicity. Mammalian chromosomes can be comprised of a continuous linear strand of DNA ranging in size from approximately 50 to 250 Mb. The DNA construct can further contain one or more sequences necessary for the DNA construct to multiply in yeast cells. The DNA construct can also contain a sequence encoding a selectable marker gene. The DNA construct can be capable of being maintained as a chromosome in a transformed cell with the DNA construct. MACs provide extra-genomic specific integration sites for introduction of genes encoding proteins of interest and permit megabase size DNA integration so that, for example, genes encoding an entire metabolic pathway, a very large gene [e.g., such as the cystic fibrosis (CF) gene (−600 kb)], or several genes [e.g., a series of antigens for preparation of a multivalent vaccine] can be stably introduced into a cell.
Mammalian artificial chromosomes [MACs] are provided. Also provided are artificial chromosomes for other higher eukaryotic species, such as insects and fish, produced using the MACS are provided herein. Methods for generating and isolating such chromosomes. Methods using the MACs to construct artificial chromosomes from other species, such as insect and fish species are also provided. The artificial chromosomes are fully functional stable chromosomes. Two types of artificial chromosomes are provided. One type, herein referred to as SATACs [satellite artificial chromosomes] are stable heterochromatic chromosomes, and the another type are minichromosomes based on amplification of euchromatin. As used herein, a formerly dicentric chromosome is a chromosome that is produced when a dicentric chromosome fragments and acquires new telomeres so that two chromosomes, each having one of the centromeres, are produced. Each of the fragments can be replicable chromosomes.
Also provided are artificial chromosomes for other higher eukaryotic species, such as insects and fish, produced using the MACS are provided herein. In one embodiment, SATACs [satellite artificial chromosomes] are provided. SATACs are stable heterochromatic chromosomes. In another embodiment, minichromosomes are provided wherein the minichromosomes are based on amplification of euchromatin.
In one embodiment, artificial chromosomes can be generated by culturing the cells with the dicentric chromosomes under conditions whereby the chromosome breaks to form a minichromosome and formerly dicentric chromosome. In one embodiment, the SATACs can be generated from the minichromosome fragment, see, for example, in U.S. Pat. No. 5,288,625. In another embodiment, the SATACs can be generated from the fragment of the formerly dicentric chromosome. The SATACs can be made up of repeating units of short satellite DNA and can be fully heterochromatic. In one embodiment, absent insertion of heterologous or foreign DNA, the SATACs do not contain genetic information. In other embodiments, SATACs of various sizes are provided that are formed by repeated culturing under selective conditions and subcloning of cells that contain chromosomes produced from the formerly dicentric chromosomes. These chromosomes can be based on repeating units 7.5 to 10 Mb in size, or megareplicons. These megareplicaonscan be tandem blocks of satellite DNA flanked by heterologous non-satellite DNA. Amplification can produce a tandem array of identical chromosome segments [each called an amplicon] that contain two inverted megareplicons bordered by heterologous [“foreign”] DNA. Repeated cell fusion, growth on selective medium and/or BrdU [5-bromodeoxyuridine] treatment or other genome destabilizing reagent or agent, such as ionizing radiation, including X-rays, and subcloning can result in cell lines that carry stable heterochromatic or partially heterochromatic chromosomes, including a 150-200 Mb “sausage” chromosome, a 500-1000 Mb gigachromosome, a stable 250-400 Mb megachromosome and various smaller stable chromosomes derived therefrom. These chromosomes are based on these repeating units and can include human immunoglobulin DNA that is expressed. (See also U.S. Pat. No. 6,743,967
In other embodiments, MACs can be provided, for example, as disclosed in U.S. Pat. Nos. 6,743,967, 6,682,729, 6,569,643, 6,558,902, 6,548,287, 6,410,722, 6,348,353, 6,297,029, 6,265,211, 6,207,648, 6,150,170, 6,150,160, 6,133,503, 6,077,697, 6,025,155, 5,997,881, 5,985,846, 5,981,225, 5,877,159, 5,851,760, and 5,721,118; PCT Publication Nos. WO 04/066945, WO 04/044129, WO 04/035729, WO 04/033668, WO 04/027075, WO 04/016791, WO 04/009788, WO 04/007750, WO 03/083054, WO 03/068910, WO 03/068909, WO 03/064613, WO 03/052050, WO 03/027315, WO 03/023029, WO 03/012126, WO 03/006610, WO 03/000921, WO 02/103032, WO 02/097059, WO 02/096923, WO 02/095003, WO 02/092615, WO 02/081710, WO 02/059330, WO 02/059296, WO 00/18941, WO 97/16533, and WO 96/40965.
In another aspect of the present invention, ungulates and ungulate cells containing HACs are provided. In a particular embodiment, one or more HACs with integrated human Ig DNA are used as a vector for introduction of human Ig genes into ungulates (such as pigs). In a particular embodiment, one or more HACs with integrated human Ig DNA are used to generate ungulates (for example, pigs) by nuclear transfer which express human Igs in response to immunization and which undergo affinity maturation.
Various approaches may be used to produce ungulates that express human antibodies (“human Ig”). These approaches include, for example, the insertion of a HAC containing both heavy and light chain Ig genes into an ungulate or the insertion of human B-cells or B-cell precursors into an ungulate during its fetal stage or after it is born (e.g., an immune deficient or immune suppressed ungulate) (see, for example, WO 01/35735, filed Nov. 17, 2000, US 02/08645, filed Mar. 20, 2002). In either case, both human antibody producing cells and ungulate antibody-producing B-cells may be present in the ungulate. In an ungulate containing a HAC, a single B-cell may produce an antibody that contains a combination of ungulate and human heavy and light chain proteins. In still other embodiments, the total size of the HAC is at least to approximately 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 Mb.
For example, HACs can be provided such as disclosed in U.S. Pat. Nos. 6,642,207, 6,590,089, 6,566,066, 6,524,799, 6,500,642, 6,485,910, 6,475,752, 6,458,561, 6,455,026, 6,448,041, 6,410,722, 6,358,523, 6,277,621, 6,265,211, 6,146,827, 6,143,566, 6,077,697, 6,025,155, 6,020,142, and 5,972,649; U.S. Pat. Application No. 2003/0037347; PCT Publication Nos. WO 04/050704, WO 04/044156, WO 04/031385, WO 04/016791, WO 03/101396, WO 03/097812, WO 03/093469, WO 03/091426, WO 03/057923, WO 03/057849, WO 03/027638, WO 03/020898, WO 02/092812, and WO 98/27200.
Additional examples of ACs into which human immunoglobulin sequences can be inserted for use in the invention include, for example, BACs (e.g., pBeloBAC11 or pBAC108L; see, e.g., Shizuya et al. (1992), Proc Natl Acad Sci USA 89(18):8794-8797; Wang et al. (1997), Biotechniques 23(6):992-994), bacteriophage PACs, YACs (see, e.g., Burke (1990), Genet Anal Tech Appl 7(5):94-99), and MACs (see, e.g., Vos (1997), Nat. Biotechnol. 15(12):1257-1259; Ascenzioni et al. (1997), Cancer Lett 118(2):135-142), such as HACs, see also, U.S. Pat. Nos. 6,743,967, 6,716,608, 6,692,954, 6,670,154, 6,642,207, 6,638,722, 6,573,090, 6,492,506, 6,348,353, 6,287,853, 6,277,621, 6,183,957, 6,156,953, 6,133,503, 6,090,584, 6,077,697, 6,025,155, 6,015,708, 5,981,175, 5,874,259, 5,721,118, and 5,270,201; European Patent Nos. 1 437 400, 1 234 024, 1 356 062, 0 959 134, 1 056 878, 0 986 648, 0 648 265, and 0 338 266; PCT Publication Nos. WO 04/013299, WO 01/07478, WO 00/06715, WO 99/43842, WO 99/27118, WO 98/55637, WO 94/00569, and WO 89/09219. Additional examples includes those AC provided in, for example, PCT Publication No. WO 02/076508, WO 03/093469, WO 02/097059; WO 02/096923; US Publication Nos US 2003/0113917 and US 2003/003435; and U.S. Pat. No. 6,025,155.
In other embodiments of the present invention, ACs transmitted through male gametogenesis in each generation. The AC can be integrating or non-integrating. In one embodiment, the AC can be transmitted through mitosis in substantially all dividing cells. In another embodiment, the AC can provide for position independent expression of a human immunogloulin nucleic acid sequence. In a particular embodiment, the AC can have a transmittal efficiency of at least 10% through each male and female gametogenesis. In one particular embodiment, the AC can be circular. In another particular embodiment, the non-integrating AC can be that deposited with the Belgian Coordinated Collections of Microorganisms—BCCM on Mar. 27, 2000 under accession number LMBP 5473 CB. In additional embodiments, methods for producing an AC are provided wherein a mitotically stable unit containing an exogenous nucleic acid transmitted through male gametogenesis is identified; and an entry site in the mitotically stable unit allows for the integration of human immunoglobulin genes into the unit.
In other embodiments, ACs are provided that include: a functional centromere, a selectable marker and/or a unique cloning site. Tin other embodiments, the AC can exhibit one or more of the following properties: it can segregate stably as an independent chromosome, immunoglobulin sequences can be inserted in a controlled way and can expressed from the AC, it can be efficiently transmitted through the male and female germline and/or the transgenic animals can bear the chromosome in greater than about 30, 40, 50, 60, 70, 80 or 90% of its cells.
In particular embodiments, the AC can be isolated from fibroblasts (such as any mammalian or human fibroblast) in which it was mitotically stable. After transfer of the AC into hamster cells, a lox (such as loxP) site and a selectable marker site can be inserted. In other embodiments, the AC can maintain mitotic stability, for example, showing a loss of less than about 5, 2, 1, 0.5 or 0.25 percent per mitosis in the absence of selection. See also, US 2003/0064509 and WO 01/77357.
Xenogenous Immunoglobulin Genes
In another aspect of the present invention, transgenic ungulates are provided that expresses a xenogenous immunoglobulin loci or fragment thereof, wherein the immunoglobulin can be expressed from an immunoglobulin locus that is integrated within an endogenous ungulate chromosome. In one embodiment, ungulate cells derived from the transgenic animals are provided. In one embodiment, the xenogenous immunoglobulin locus can be inherited by offspring. In another embodiment, the xenogenous immunoglobulin locus can be inherited through the male germ line by offspring. In still further embodiments, an artificial chromosome (AC) can contain the xenogenous immunoglobulin. In one embodiment, the AC can be a yeast AC or a mammalian AC. In a further embodiment, the xenogenous locus can be a human immunoglobulin locus or fragment thereof. In one embodiment, the human immunoglobulin locus can be human chromosome 14, human chromosome 2, and human chromosome 22 or fragments thereof. In another embodiment, the human immunoglobulin locus can include any fragment of a human immunoglobulin that can undergo rearrangement. In a further embodiment, the human immunoglobulin loci can include any fragment of a human immunoglobulin heavy chain and a human immunoglobulin light chain that can undergo rearrangement. In still further embodiment, the human immunoglobulin loci can include any human immunoglobulin locus or fragment thereof that can produce an antibody upon exposure to an antigen. In a particular embodiment, the exogenous human immunoglobulin can be expressed in B cells to produce xenogenous immunoglobulin in response to exposure to one or more antigens.
In other embodiments, the transgenic ungulate that lacks any expression of functional endogenous immunoglobulins can be further genetically modified to express an xenogenous immunoglobulin loci. In an alternative embodiment, porcine animals are provided that contain an xenogeous immunoglobulin locus. In one embodiment, the xenogeous immunoglobulin loci can be a heavy and/or light chain immunoglobulin or fragment thereof. In another embodiment, the xenogenous immunoglobulin loci can be a kappa chain locus or fragment thereof and/or a lambda chain locus or fragment thereof. In still further embodiments, an artificial chromosome (AC) can contain the xenogenous immunoglobulin. In one embodiment, the AC can be a yeast AC or a mammalian AC. In a further embodiment, the xenogenous locus can be a human immunoglobulin locus or fragment thereof. In one embodiment, the human immunoglobulin locus can be human chromosome 14, human chromosome 2, and human chromosome 22 or fragments thereof. In another embodiment, the human immunoglobulin locus can include any fragment of a human immunoglobulin that can undergo rearrangement. In a further embodiment, the human immunoglobulin loci can include any fragment of a human immunoglobulin heavy chain and a human immunoglobulin light chain that can undergo rearrangement. In still further embodiment, the human immunoglobulin loci can include any human immunoglobulin locus or fragment thereof that can produce an antibody upon exposure to an antigen. In a particular embodiment, the exogenous human immunoglobulin can be expressed in B cells to produce xenogenous immunoglobulin in response to exposure to one or more antigens.
In other embodiments, the transgenic ungulate that lacks any expression of functional endogenous immunoglobulins can be further genetically modified to express an xenogenous immunoglobulin loci. In an alternative embodiment, porcine animals are provided that contain an xenogeous immunoglobulin locus. In one embodiment, the xenogeous immunoglobulin loci can be a heavy and/or light chain immunoglobulin or fragment thereof. In another embodiment, the xenogenous immunoglobulin loci can be a kappa chain locus or fragment thereof and/or a lambda chain locus or fragment thereof. In still further embodiments, an artificial chromosome (AC) can contain the xenogenous immunoglobulin. In one embodiment, the AC can be a yeast AC or a mammalian AC. In a further embodiment, the xenogenous locus can be a human immunoglobulin locus or fragment thereof. In one embodiment, the human immunoglobulin locus can be human chromosome 14, human chromosome 2, and human chromosome 22 or fragments thereof. In another embodiment, the human immunoglobulin locus can include any fragment of a human immunoglobulin that can undergo rearrangement. In a further embodiment, the human immunoglobulin loci can include any fragment of a human immunoglobulin heavy chain and a human immunoglobulin light chain that can undergo rearrangement. In still further embodiment, the human immunoglobulin loci can include any human immunoglobulin locus or fragment thereof that can produce an antibody upon exposure to an antigen. In a particular embodiment, the exogenous human immunoglobulin can be expressed in B cells to produce xenogenous immunoglobulin in response to exposure to one or more antigens.
In another embodiment, porcine animals are provided that contain an xenogeous immunoglobulin locus. In one embodiment, the xenogeous immunoglobulin loci can be a heavy and/or light chain immunoglobulin or fragment thereof. In another embodiment, the xenogenous immunoglobulin loci can be a kappa chain locus or fragment thereof and/or a lambda chain locus or fragment thereof. In still further embodiments, an artificial chromosome (AC) can contain the xenogenous immunoglobulin. In one embodiment, the AC can be a yeast AC or a mammalian AC. In a further embodiment, the xenogenous locus can be a human immunoglobulin locus or fragment thereof. In one embodiment, the human immunoglobulin locus can be human chromosome 14, human chromosome 2, and human chromosome 22 or fragments thereof. In another embodiment, the human immunoglobulin locus can include any fragment of a human immunoglobulin that can undergo rearrangement. In a further embodiment, the human immunoglobulin loci can include any fragment of a human immunoglobulin heavy chain and a human immunoglobulin light chain that can undergo rearrangement. In still further embodiment, the human immunoglobulin loci can include any human immunoglobulin locus or fragment thereof that can produce an antibody upon exposure to an antigen. In a particular embodiment, the exogenous human immunoglobulin can be expressed in B cells to produce xenogenous immunoglobulin in response to exposure to one or more antigens.
Human immunoglobulin genes, such as the Ig heavy chain gene (human chromosome 414), Ig kappa chain gene (human chromosome #2) and/or the Ig lambda chain gene (chromosome #22) can be inserted into Acs, as described above. In a particular embodiment, any portion of the human heavy, kappa and/or lambda Ig genes can be inserted into ACs. In one embodiment, the nucleic acid can be at least 70, 80, 90, 95, or 99% identical to the corresponding region of a naturally-occurring nucleic acid from a human. In other embodiments, more than one class of human antibody is produced by the ungulate. In various embodiments, more than one different human Ig or antibody is produced by the ungulate. In one embodiment, an AC containing both a human Ig heavy chain gene and Ig light chain gene, such as an automatic human artificial chromosome (“AHAC,” a circular recombinant nucleic acid molecule that is converted to a linear human chromosome in vivo by an endogenously expressed restriction endonuclease) can be introduced. In one embodiment, the human heavy chain loci and the light chain loci are on different chromosome arms (i.e., on different side of the centromere). In one embodiments, the heavy chain can include the mu heavy chain, and the light chain can be a lambda or kappa light chain. The Ig genes can be introduced simultaneously or sequentially in one or more than one ACs.
In particular embodiments, the ungulate or ungulate cell expresses one or more nucleic acids encoding all or part of a human Ig gene which undergoes rearrangement and expresses more than one human Ig molecule, such as a human antibody protein. Thus, the nucleic acid encoding the human Ig chain or antibody is in its unrearranged form (that is, the nucleic acid has not undergone V(D)J recombination). In particular embodiments, all of the nucleic acid segments encoding a V gene segment of an antibody light chain can be separated from all of the nucleic acid segments encoding a J gene segment by one or more nucleotides. In a particular embodiment, all of the nucleic acid segments encoding a V gene segment of an antibody heavy chain can be separated from all of the nucleic acid segments encoding a D gene segment by one or more nucleotides, and/or all of the nucleic acid segments encoding a D gene segment of an antibody heavy chain are separated from all of the nucleic acid segments encoding a J gene segment by one or more nucleotides. Administration of an antigen to a transgenic ungulate containing an unrearranged human Ig gene is followed by the rearrangement of the nucleic acid segments in the human Ig gene locus and the production of human antibodies reactive with the antigen.
In one embodiment, the AC can express a portion or fragment of a human chromosome that contains an immunoglobulin gene. In one embodiment, the AC can express at least 300 or 1300 kb of the human light chain locus, such as described in Davies et al. 1993 Biotechnology 11:911-914.
In another embodiment, the AC can express a portion of human chromosome 22 that contains at least the λ light-chain locus, including Vλ gene segments, Jλ gene segments, and the single Cλ gene. In another embodiment, the AC can express at least one Vλ gene segment, at least one Jλ gene segment, and the Cλ gene. In other embodiment, ACs can contain portions of the lambda locus, such as described in Popov et al. J Exp Med. 1999 May 17; 189(10):1611-20.
In another embodiment, the AC can express a portion of human chromosome 2 that contains at least the κ light-chain locus, including Vκ gene segments, Jκ gene segments and the single Cκ gene. In another embodiment, the AC can express at least one Vκ gene segment, at least one Jκ gene segment and the Cκ gene. In other embodiments, AC containing portions of the kappa light chain locus can be those describe, for example, in Li et al. 2000 J Immunol 164: 812-824 and Li S Proc Natl Acad Sci USA. 1987 June; 84(12):4229-33. In another embodiment, AC containing approximately 1.3 Mb of human kappa locus are provided, such as described in Zou et al FASEB J. 1996 August; 10(10):1227-32.
In further embodiments, the AC can express a portion of human chromosome 14 that contains at least the human heavy-chain locus, including VH, DH, JH and CH gene segments. In another embodiment, the AC can express at least one VH gene segment, at least one DH gene segment, at least one JH gene segment and at least one at least one CH gene segment. In other embodiments, the AC can express at least 85 kb of the human heavy chain locus, such as described in Choi et al. 1993 Nat Gen 4:117-123 and/or Zou et al. 1996 PNAS 96: 14100-14105.
In other embodiments, the AC can express portions of both heavy and light chain loci, such as, at least 220, 170, 800 or 1020 kb, for example, as disclosed in Green et al. 1994 Nat Gen 7:13-22; Mendez et al 1995 Genomics 26: 294-307; Mendez et al. 1997 Nat Gen 15: 146-156; Green et al. 1998 J Exp Med 188: 483-495 and/or Fishwild et al. 1996 Nat Biotech 14: 845-851. In another embodiment, the AC can express megabase amounts of human immunoglobulin, such as described in Nicholson J Immunol. 1999 Dec. 15; 163(12):6898-906 and Popov Gene. 1996 Oct. 24; 177(1-2):195-201. In addition, in one particular embodiment, MACs derived from human chromosome #14 (comprising the Ig heavy chain gene), human chromosome #2 comprising the Ig kappa chain gene) and human chromosome #22 (comprising the Ig lambda chain gene) can be introduced simultaneously or successively, such as described in US Patent Publication No. 2004/0068760 to Robl et al. In another embodiments, the total size of the MAC is less than or equal to approximately 10, 9, 8, or 7 megabases.
In a particular embodiment, human Vh, human Dh, human Jh segments and human mu segments of human immunoglobulins in germline configuration can be inserted into an AC, such as a YAC, such that the Vh, Dh, Jh and mu DNA segments form a repertoire of immunoglobulins containing portions which correspond to the human DNA segments, for example, as described in U.S. Pat. No. 5,545,807 to the Babraham Instititute. Such ACs, after insertion into ungulate cells and generation of ungulates can produce heavy chain immunoglobulins. In one embodiment, these immunoglobulins can form functional heavy chain-light chain immunoglobulins. In another embodiment, these immunoglobulins can be expressed in an amount allowing for recovery from suitable cells or body fluids of the ungulate. Such immunoglobulins can be inserted into yeast artificial chromosome vectors, such as described by Burke, D T, Carle, G F and Olson, M V (1987) “Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors” Science, 236, 806-812, or by introduction of chromosome fragments (such as described by Richer, J and Lo, C W (1989) “Introduction of human DNA into mouse eggs by injection of dissected human chromosome fragments” Science 245, 175-177).
Additional information on specific ACs containing human immunoglobulin genes can be found in, for example, recent reviews by Giraldo & Montoliu (2001) Transgenic Research 10: 83-103 and Peterson (2003) Expert Reviews in Molecular Medicine 5: 1-25.
AC Transfer Methods
The human immunoglobulin genes can be first inserted into ACs and then the human-immunoglobulin-containing ACs can be inserted into the ungulate cells. Alternatively, the ACs can be transferred to an intermediary mammalian cell, such as a CHO cell, prior to insertion into the ungulate call. In one embodiment, the intermediary mammalian cell can also contain and AC and the first AC can be inserted into the AC of the mammalian cell. In particular, a YAC containing human immunoglobulin genes or fragments thereof in a yeast cell can be transferred to a mammalian cell that harbors an MAC. The YAC can be inserted into the MAC. The MAC can then be transferred to an ungulate cell. The human Ig genes can be inserted into ACs by homologous recombination. The resulting AC containing human Ig genes, can then be introduced into ungulate cells. One or more ungulate cells can be selected by techniques described herein or those known in the art, which contain an AC containing a human Ig.
Suitable hosts for introduction of the ACs are provided herein, which include but are not limited to any animal or plant, cell or tissue thereof, including, but not limited to: mammals, birds, reptiles, amphibians, insects, fish, arachnids, tobacco, tomato, wheat, monocots, dicots and algae. In one embodiment, the ACs can be condensed (Marschall et al Gene Ther. 1999 Sep.; 6(9):1634-7) by any reagent known in the art, including, but not limited to, spermine, spermidine, polyethylenimine, and/or polylysine prior to introduction into cells. The ACs can be introduced by cell fusion or microcell fusion or subsequent to isolation by any method known to those of skill in this art, including but not limited to: direct DNA transfer, electroporation, nuclear transfer, microcell fusion, cell fusion, spheroplast fusion, lipid-mediated transfer, lipofection, liposomes, microprojectile bombardment, microinjection, calcium phosphate precipitation and/or any other suitable method. Other methods for introducing DNA into cells, include nuclear microinjection, electroporation, bacterial protoplast fusion with intact cells. Polycations, such as polybrene and polyornithine, may also be used. For various techniques for transforming mammalian cells, see e.g., Keown et al. Methods in Enzymology (1990) Vol. 185, pp. 527-537; and Mansour et al. (1988) Nature 336:348-352.
The ACs can be introduced by direct DNA transformation; microinjection in cells or embryos, protoplast regeneration for plants, electroporation, microprojectile gun and other such methods known to one skilled in the art (see, e.g., Weissbach et al. (1988) Methods for Plant Molecular Biology, Academic Press, N.Y., Section VIII, pp. 421-463; Grierson et al. (1988) Plant Molecular Biology, 2d Ed., Blackie, London, Ch. 7-9; see, also U.S. Pat. Nos. 5,491,075; 5,482,928; and 5,424,409; see, also, e.g., U.S. Pat. No. 5,470,708,).
In particular embodiments, one or more isolated YACs can be used that harbor human Ig genes. The isolated YACs can be condensed (Marschall et al Gene Ther. 1999 September; 6(9):1634-7) by any reagent known in the art, including, but not limited to spermine, spermidine, polyethylenimine, and/or polylysine. The condensed YACs can then be transferred to porcine cells by any method known in the art (for example, microinjection, electroporation, lipid mediated transfection, etc). Alternatively, the condensed YAC can be transferred to oocytes via sperm-mediated gene transfer or intracytoplasmic sperm injection (ICSI) mediated gene transfer. In one embodiment, spheroplast fusion can be used to transfer YACs that harbor human Ig genes to porcine cells.
In other embodiments of the invention, the AC containing the human Ig can be inserted into an adult, fetal, or embryonic ungulate cell. Additional examples of ungulate cells include undifferentiated cells, such as embryonic cells (e.g., embryonic stem cells), differentiated or somatic cells, such as epithelial cells, neural cells epidermal cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, B-lymphocytes, T-lymphocytes, erythrocytes, macrophages, monocytes, fibroblasts, muscle cells, cells from the female reproductive system, such as a mammary gland, ovarian cumulus, granulosa, or oviductal cell, germ cells, placental cell, or cells derived from any organ, such as the bladder, brain, esophagus, fallopian tube, heart, intestines, gallbladder, kidney, liver, lung, ovaries, pancreas, prostate, spinal cord, spleen, stomach, testes, thymus, thyroid, trachea, ureter, urethra, and uterus or any other cell type described herein.
Site Specific Recombinase Mediated Transfer
In particular embodiments of the present invention, the transfer of ACs containing human immunoglobulin genes to porcine cells, such as those described herein or known in the art, can be accomplished via site specific recombinase mediated transfer. In one particular embodiment, the ACs can be transferred into porcine fibroblast cells. In another particular embodiment, the ACs can be YACs.
In other embodiments of the present invention, the circularized DNA, such as an AC, that contain the site specific recombinase target site can be transferred into a cell line that has a site specific recombinase target site within its genome. In one embodiment, the cell's site specific recombinase target site can be located within an exogenous chromosome. The exogenous chromosome can be an artificial chromosome that does not integrate into the host's endogenous genome. In one embodiment, the AC can be transferred via germ line transmission to offspring. In one particular embodiment, a YAC containing a human immunoglobulin gene or fragment thereof can be circularized via a site specific recombinase and then transferred into a host cell that contains a MAC, wherein the MAC contains a site specific recombinase site. This MAC that now contains human immunoglobulin loci or fragments thereof can then be fused with a porcine cell, such as, but not limited to, a fibroblast. The porcine cell can then be used for nuclear transfer.
In certain embodiments of the present invention, the ACs that contain human immunoglobulin genes or fragments thereof can be transferred to a mammalian cell, such as a CHO cell, prior to insertion into the ungulate call. In one embodiment, the intermediary mammalian cell can also contain and AC and the first AC can be inserted into the AC of the mammalian cell. In particular, a YAC containing human immunoglobulin genes or fragments thereof in a yeast cell can be transferred to a mammalian cell that harbors a MAC. The YAC can be inserted in the MAC. The MAC can then be transferred to an ungulate cell. In particular embodiments, the YAC harboring the human Ig genes or fragments thereof can contain site specific recombinase target sites. The YAC can first be circularized via application of the appropriate site specific recombinase and then inserted into a mammalian cell that contains its own site specific recombinase target site. Then, the site specific recombinase can be applied to integrate the YAC into the MAC in the intermediary mammalian cell. The site specific recombinase can be applied in cis or trans. In particular, the site specific recombinase can be applied in trans. In one embodiment, the site specific recombinase can be expressed via transfection of a site specific recombinase expression plasmid, such as a Cre expression plasmid. In addition, one telomere region of the YAC can also be retrofitted with a selectable marker, such as a selectable marker described herein or known in the art. The human Ig genes or fragments thereof within the MAC of the intermediary mammalian cell can then be transferred to an ungulate cell, such as a fibroblast.
Alternatively, the AC, such as a YAC, harboring the human Ig genes or fragments thereof can contain site specific recombinase target sites optionally located near each telomere. The YAC can first be circularized via application of the appropriate site specific recombinase and then inserted into an ungulate cell directly that contains its own site specific recombinase target site within it genome. Alternatively, the ungulate cell can harbor its own MAC, which contains a site specific recombinase target site. In this embodiment, the YAC can be inserted directly into the endogenous genome of the ungulate cell. In particular embodiments, the ungulate cell can be a fibroblast cell or any other suitable cell that can be used for nuclear transfer. See, for example, FIG. 7; Call et al., Hum Mol Genet. 2000 Jul. 22; 9(12):1745-51.
In other embodiments, methods to circularize at least 100 kb of DNA are provided wherein the DNA can then be integrated into a host genome via a site specific recombinase. In one embodiment, at least 100, 200, 300, 400, 500, 1000, 2000, 5000, 10,000 kb of DNA can be circularized. In another embodiment, at least 1000, 2000, 5000, 10,000, or 20,000 megabases of DNA can be circularized. In one embodiment, the circularization of the DNA can be accomplished by attaching site specific recombinase target sites at each end of the DNA sequence and then applying the site specific recombinase to result in circularization of the DNA. In one embodiment, the site specific recombinase target site can be lox. In another embodiment, the site specific recombinase target site can be Flt. In certain embodiments, the DNA can be an artificial chromosome, such as a YAC or any AC described herein or known in the art. In another embodiment, the AC can contain human immunoglobulin loci or fragments thereof.
In another preferred embodiment, the YAC can be converted to, or integrated within, an artificial mammalian chromosome. The mammalian artificial chromosome is either transferred to or harbored within a porcine cell. The artificial chromosome can be introduced within the porcine genome through any method known in the art including but not limited to direct injection of metaphase chromosomes, lipid mediated gene transfer, or microcell fusion.
Site-specific recombinases include enzymes or recombinases that recognize and bind to a short nucleic acid site or sequence-specific recombinase target site, i.e., a recombinase recognition site, and catalyze the recombination of nucleic acid in relation to these sites. These enzymes include recombinases, transposases and integrases. Examples of sequence-specific recombinase target sites include, but are not limited to, lox sites, att sites, dif sites and frt sites. Non-limiting examples of site-specific recombinases include, but are not limited to, bacteriophage P1 Cre recombinase, yeast FLP recombinase, Inti integrase, bacteriophage λ, phi 80, P22, P2, 186, and P4 recombinase, Tn3 resolvase, the Hin recombinase, and the Cin recombinase, E. coli xerC and xerD recombinases, Bacillus thuringiensis recombinase, TpnI and the β-lactamase transposons, and the immunoglobulin recombinases.
In one embodiment, the recombination site can be a lox site that is recognized by the Cre recombinase of bacteriophage P1. Lox sites refer to a nucleotide sequence at which the product of the cre gene of bacteriophage P1, the Cre recombinase, can catalyze a site-specific recombination event. A variety of lox sites are known in the art, including the naturally occurring loxP, loxB, loxL and loxR, as well as a number of mutant, or variant, lox sites, such as loxP511, loxP514, lox.DELTA.86, lox.DELTA.117, loxC2, loxP2, loxP3 and lox P23. Additional example of lox sites include, but are not limited to, loxB, loxL, loxR, loxP, loxP3, loxP23, loxΔ86, loxΔ117, loxP511, and loxC2.
In another embodiment, the recombination site is a recombination site that is recognized by a recombinases other than Cre. In one embodiment, the recombinase site can be the FRT sites recognized by FLP recombinase of the 2 pi plasmid of Saccharomyces cerevisiae. FRT sites refer to a nucleotide sequence at which the product of the FLP gene of the yeast 2 micron plasmid, FLP recombinase, can catalyze site-specific recombination. Additional examples of the non-Cre recombinases include, but are not limited to, site-specific recombinases include: att sites recognized by the Int recombinase of bacteriophage λ (e.g. att1, att2, att3, attP, attB, attL, and attR), the recombination sites recognized by the resolvase family, and the recombination site recognized by transposase of Bacillus thruingiensis.
IV. Production of Genetically Modified Animals
In additional aspects of the present invention, ungulates that contain the genetic modifications described herein can be produced by any method known to one skilled in the art. Such methods include, but are not limited to: nuclear transfer, intracytoplasmic sperm injection, modification of zygotes directly and sperm mediated gene transfer.
In another embodiment, a method to clone such animals, for example, pigs, includes: enucleating an oocyte, fusing the oocyte with a donor nucleus from a cell in which at least one allele of at least one immunoglobulin gene has been inactivated, and implanting the nuclear transfer-derived embryo into a surrogate mother.
Alternatively, a method is provided for producing viable animals that lack any expression of functional immunoglobulin by inactivating both alleles of the immunoglobulin gene in embryonic stem cells, which can then be used to produce offspring.
In another aspect, the present invention provides a method for producing viable animals, such as pigs, in which both alleles of the immunoglobulin gene have been rendered inactive. In one embodiment, the animals are produced by cloning using a donor nucleus from a cell in which both alleles of the immunoglobulin gene have been inactivated. In one embodiment, both alleles of the immunoglobulin gene are inactivated via a genetic targeting event.
Genetically altered animals that can be created by modifying zygotes directly. For mammals, the modified zygotes can be then introduced into the uterus of a pseudopregnant female capable of carrying the animal to term. For example, if whole animals lacking an immunoglobulin gene are desired, then embryonic stem cells derived from that animal can be targeted and later introduced into blastocysts for growing the modified cells into chimeric animals. For embryonic stem cells, either an embryonic stem cell line or freshly obtained stem cells can be used.
In a suitable embodiment of the invention, the totipotent cells are embryonic stem (ES) cells. The isolation of ES cells from blastocysts, the establishing of ES cell lines and their subsequent cultivation are carried out by conventional methods as described, for example, by Doetchmann et al., J. Embryol. Exp. Morph. 87:27-45 (1985); Li et al., Cell 69:915-926 (1992); Robertson, E. J. “Tetracarcinomas and Embryonic Stem Cells: A Practical Approach,” ed. E. J. Robertson, IRL Press, Oxford, England (1987); Wurst and Joyner, “Gene Targeting: A Practical Approach,” ed. A. L. Joyner, IRL Press, Oxford, England (1993); Hogen et al., “Manipulating the Mouse Embryo: A Laboratory Manual,” eds. Hogan, Beddington, Costantini and Lacy, Cold Spring Harbor Laboratory Press, New York (1994); and Wang et al., Nature 336:741-744 (1992). In another suitable embodiment of the invention, the totipotent cells are embryonic germ (EG) cells. Embryonic Germ cells are undifferentiated cells functionally equivalent to ES cells, that is they can be cultured and transfected in vitro, then contribute to somatic and germ cell lineages of a chimera (Stewart et al., Dev. Biol. 161:626-628 (1994)). EG cells are derived by culture of primordial germ cells, the progenitors of the gametes, with a combination of growth factors: leukemia inhibitory factor, steel factor and basic fibroblast growth factor (Matsui et al., Cell 70:841-847 (1992); Resnick et al., Nature 359:550-551 (1992)). The cultivation of EG cells can be carried out using methods described in the article by Donovan et al., “Transgenic Animals, Generation and Use,” Ed. L. M. Houdebine, Harwood Academic Publishers (1997), and in the original literature cited therein.
Tetraploid blastocysts for use in the invention may be obtained by natural zygote production and development, or by known methods by electrofusion of two-cell embryos and subsequently cultured as described, for example, by James et al., Genet. Res. Camb. 60:185-194 (1992); Nagy and Rossant, “Gene Targeting: A Practical Approach,” ed. A. L. Joyner, IRL Press, Oxford, England (1993); or by Kubiak and Tarkowski, Exp. Cell Res. 157:561-566 (1985).
The introduction of the ES cells or EG cells into the blastocysts can be carried out by any method known in the art. A suitable method for the purposes of the present invention is the microinjection method as described by Wang et al., EMBO J. 10:2437-2450 (1991).
Alternatively, by modified embryonic stem cells transgenic animals can be produced. The genetically modified embryonic stem cells can be injected into a blastocyst and then brought to term in a female host mammal in accordance with conventional techniques. Heterozygous progeny can then be screened for the presence of the alteration at the site of the target locus, using techniques such as PCR or Southern blotting. After mating with a wild-type host of the same species, the resulting chimeric progeny can then be cross-mated to achieve homozygous hosts.
After transforming embryonic stem cells with the targeting vector to alter the immunoglobulin gene, the cells can be plated onto a feeder layer in an appropriate medium, e.g., fetal bovine serum enhanced DMEM. Cells containing the construct can be detected by employing a selective medium, and after sufficient time for colonies to grow, colonies can be picked and analyzed for the occurrence of homologous recombination. Polymerase chain reaction can be used, with primers within and without the construct sequence but at the target locus. Those colonies which show homologous recombination can then be used for embryo manipulating and blastocyst injection. Blastocysts can be obtained from superovulated females. The embryonic stem cells can then be trypsinized and the modified cells added to a droplet containing the blastocysts. At least one of the modified embryonic stem cells can be injected into the blastocoel of the blastocyst. After injection, at least one of the blastocysts can be returned to each uterine horn of pseudopregnant females. Females are then allowed to go to term and the resulting litters screened for mutant cells having the construct. The blastocysts are selected for different parentage from the transformed ES cells. By providing for a different phenotype of the blastocyst and the ES cells, chimeric progeny can be readily detected, and then genotyping can be conducted to probe for the presence of the modified immunoglobulin gene.
In other embodiments, sperm mediated gene transfer can be used to produce the genetically modified ungulates described herein. The methods and compositions described herein to either eliminate expression of endogenous immunoglobulin genes or insert xenogenous immunoglobulin genes can be used to genetically modify the sperm cells via any technique described herein or known in the art. The genetically modified sperm can then be used to impregnate a female recipient via artificial insemination, intracytoplasmic sperm injection or any other known technique. In one embodiment, the sperm and/or sperm head can be incubated with the exogenous nucleic acid for a sufficient time period. Sufficient time periods include, for example, about 30 seconds to about 5 minutes, typically about 45 seconds to about 3 minutes, more typically about 1 minute to about 2 minutes. In particular embodiments, the expression of xenogenous, such as human, immunoglobulin genes in ungulates as described herein, can be accomplished via intracytoplasmic sperm injection.
The potential use of sperm cells as vectors for gene transfer was first suggested by Brackett et al., Proc., Natl. Acad. Sci. USA 68:353-357 (1971). This was followed by reports of the production of transgenic mice and pigs after in vitro fertilization of oocytes with sperm that had been incubated by naked DNA (see, for example, Lavitrano et al., Cell 57:717-723 (1989) and Gandolfi et al. Journal of Reproduction and Fertility Abstract Series 4, 10 (1989)), although other laboratories were not able to repeat these experiments (see, for example, Brinster et al. Cell 59:239-241 (1989) and Gavora et al., Canadian Journal of Animal Science 71:287-291 (1991)). Since then, there have been several reports of successful sperm mediated gene transfer in chicken (see, for example, Nakanishi and Iritani, Mol. Reprod. Dev. 36:258-261 (1993)); mice (see, for example, Maione, Mol. Reprod. Dev. 59:406 (1998)); and pigs (see, for example, Lavitrano et al. Transplant. Proc. 29:3508-3509 (1997); Lavitrano et al., Proc. Natl. Acad. Sci. USA 99:14230-5 (2002); Lavitrano et al., Mol. Reprod. Dev. 64-284-91 (2003)). Similar techniques are also described in U.S. Pat. No. 6,376,743; issued Apr. 23, 2002; U.S. Patent Publication Nos. 20010044937, published Nov. 22, 2001, and 20020108132, published Aug. 8, 2002.
In other embodiments, intracytoplasmic sperm injection can be used to produce the genetically modified ungulates described herein. This can be accomplished by co-inserting an exogenous nucleic acid and a sperm into the cytoplasm of an unfertilized oocyte to form a transgenic fertilized oocyte, and allowing the transgenic fertilized oocyte to develop into a transgenic embryo and, if desired, into a live offspring. The sperm can be a membrane-disrupted sperm head or a demembranated sperm head. The co-insertion step can include the substep of preincubating the sperm with the exogenous nucleic acid for a sufficient time period, for example, about 30 seconds to about 5 minutes, typically about 45 seconds to about 3 minutes, more typically about 1 minute to about 2 minutes. The co-insertion of the sperm and exogenous nucleic acid into the oocyte can be via microinjection. The exogenous nucleic acid mixed with the sperm can contain more than one transgene, to produce an embryo that is transgenic for more than one transgene as described herein. The intracytoplasmic sperm injection can be accomplished by any technique known in the art, see, for example, U.S. Pat. No. 6,376,743. In particular embodiments, the expression of xenogenous, such as human, immunoglobulin genes in ungulates as described herein, can be accomplished via intracytoplasmic sperm injection.
Any additional technique known in the art may be used to introduce the transgene into animals. Such techniques include, but are not limited to pronuclear microinjection (see, for example, Hoppe, P. C. and Wagner, T. E., 1989, U.S. Pat. No. 4,873,191); retrovirus mediated gene transfer into germ lines (see, for example, Van der Putten et al., 1985, Proc. Natl. Acad. Sci., USA 82:6148-6152); gene targeting in embryonic stem cells (see, for example, Thompson et al., 1989, Cell 56:313-321; Wheeler, M. B., 1994, WO 94/26884); electroporation of embryos (see, for example, Lo, 1983, Mol Cell. Biol. 3:1803-1814); cell gun; transfection; transduction; retroviral infection; adenoviral infection; adenoviral-associated infection; liposome-mediated gene transfer; naked DNA transfer; and sperm-mediated gene transfer (see, for example, Lavitrano et al., 1989, Cell 57:717-723); etc. For a review of such techniques, see, for example, Gordon, 1989, Transgenic Animals, Intl. Rev. Cytol. 115:171-229. In particular embodiments, the expression of xenogenous, such as human, immunoglobulin genes in ungulates as described herein, can be accomplished via these techniques.
Somatic Cell Nuclear Transfer to Produce Cloned, Transgenic Offspring
In a further aspect of the present invention, ungulate, such as porcine or bovine, cells lacking one allele, optionally both alleles of an ungulate heavy chain, kappa light chain and/or lambda light chain gene can be used as donor cells for nuclear transfer into recipient cells to produce cloned, transgenic animals. Alternatively, ungulate heavy chain, kappa light chain and/or lambda light chain gene knockouts can be created in embryonic stem cells, which are then used to produce offspring. Offspring lacking a single allele of a functional ungulate heavy chain, kappa light chain and/or lambda light chain gene produced according to the process, sequences and/or constructs described herein can be breed to further produce offspring lacking functionality in both alleles through mendelian type inheritance.
In another embodiment, the present invention provides a method for producing viable pigs that lack any expression of functional alpha-1,3-GT by breeding a male pig heterozygous for the alpha-1,3-GT gene with a female pig heterozygous for the alpha-1,3-GT gene. In one embodiment, the pigs are heterozygous due to the genetic modification of one allele of the alpha-1,3-GT gene to prevent expression of that allele. In another embodiment, the pigs are heterozygous due to the presence of a point mutation in one allele of the alpha-1,3-GT gene. In another embodiment, the point mutation can be a T-to-G point mutation at the second base of exon 9 of the alpha-1,3-GT gene. In one specific embodiment, a method to produce a porcine animal that lacks any expression of functional alpha-1,3-GT is provided wherein a male pig that contains a T-to-G point mutation at the second base of exon 9 of the alpha-1,3-GT gene is bred with a female pig that contains a T-to-G point mutation at the second base of exon 9 of the alpha-1,3-GT gene, or vise versa.
The present invention provides a method for cloning an animal, such as a pig, lacking a functional immunoglobulin gene via somatic cell nuclear transfer. In general, the animal can be produced by a nuclear transfer process comprising the following steps: obtaining desired differentiated cells to be used as a source of donor nuclei; obtaining oocytes from the animal; enucleating said oocytes; transferring the desired differentiated cell or cell nucleus into the enucleated oocyte, e.g., by fusion or injection, to form NT units; activating the resultant NT unit; and transferring said cultured NT unit to a host animal such that the NT unit develops into a fetus.
Nuclear transfer techniques or nuclear transplantation techniques are known in the art(Dai et al. Nature Biotechnology 20:251-255; Polejaeva et al Nature 407:86-90 (2000); Campbell et al, Theriogenology, 43:181 (1995); Collas et al, Mol. Report Dev., 38:264-267 (1994); Keefer et al, Biol. Reprod., 50:935-939 (1994); Sims et al, Proc. Natl. Acad. Sci., USA, 90:6143-6147 (1993); WO 94/26884; WO 94/24274, and WO 90/03432, U.S. Pat. Nos. 4,944,384 and 5,057,420).
A donor cell nucleus, which has been modified to alter the immunoglobulin gene, is transferred to a recipient oocyte. The use of this method is not restricted to a particular donor cell type. The donor cell can be as described herein, see also, for example, Wilmut et al Nature 385 810 (1997); Campbell et al Nature 380 64-66 (1996); Dai et al., Nature Biotechnology 20:251-255, 2002 or Cibelli et al Science 280 1256-1258 (1998). All cells of normal karyotype, including embryonic, fetal and adult somatic cells which can be used successfully in nuclear transfer can be employed. Fetal fibroblasts are a particularly useful class of donor cells. Generally suitable methods of nuclear transfer are described in Campbell et al Theriogenology 43 181 (1995), Dai et al. Nature Biotechnology 20:251-255, Polejaeva et al Nature 407:86-90 (2000), Collas et al Mol. Reprod. Dev. 38 264-267 (1994), Keefer et al Biol. Reprod. 50 935-939 (1994), Sims et al Proc. Nat'l. Acad. Sci. USA 90 6143-6147 (1993), WO-A-9426884, WO-A-9424274, WO-A-9807841, WO-A-9003432, U.S. Pat. No. 4,994,384 and U.S. Pat. No. 5,057,420. Differentiated or at least partially differentiated donor cells can also be used. Donor cells can also be, but do not have to be, in culture and can be quiescent. Nuclear donor cells which are quiescent are cells which can be induced to enter quiescence or exist in a quiescent state in vivo. Prior art methods have also used embryonic cell types in cloning procedures (Campbell et al (Nature, 380:64-68, 1996) and Stice et al (Biol. Reprod., 20 54:100-110, 1996).
Somatic nuclear donor cells may be obtained from a variety of different organs and tissues such as, but not limited to, skin, mesenchyme, lung, pancreas, heart, intestine, stomach, bladder, blood vessels, kidney, urethra, reproductive organs, and a disaggregated preparation of a whole or part of an embryo, fetus, or adult animal. In a suitable embodiment of the invention, nuclear donor cells are selected from the group consisting of epithelial cells, fibroblast cells, neural cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes (B and T), macrophages, monocytes, mononuclear cells, cardiac muscle cells, other muscle cells, extended cells, cumulus cells, epidermal cells or endothelial cells. In another embodiment, the nuclear donor cell is an embryonic stem cell. In a particular embodiment, fibroblast cells can be used as donor cells.
In another embodiment of the invention, the nuclear donor cells of the invention are germ cells of an animal. Any germ cell of an animal species in the embryonic, fetal, or adult stage may be used as a nuclear donor cell. In a suitable embodiment, the nuclear donor cell is an embryonic germ cell.
Nuclear donor cells may be arrested in any phase of the cell cycle (G0, G1, G2, S, M) so as to ensure coordination with the acceptor cell. Any method known in the art may be used to manipulate the cell cycle phase. Methods to control the cell cycle phase include, but are not limited to, G0 quiescence induced by contact inhibition of cultured cells, G0 quiescence induced by removal of serum or other essential nutrient, G0 quiescence induced by senescence, G0 quiescence induced by addition of a specific growth factor; G0 or G1 quiescence induced by physical or chemical means such as heat shock, hyperbaric pressure or other treatment with a chemical, hormone, growth factor or other substance; S-phase control via treatment with a chemical agent which interferes with any point of the replication procedure; M-phase control via selection using fluorescence activated cell sorting, mitotic shake off, treatment with microtubule disrupting agents or any chemical which disrupts progression in mitosis (see also Freshney, R. I., “Culture of Animal Cells: A Manual of Basic Technique,” Alan R. Liss, Inc, New York (1983).
Methods for isolation of oocytes are well known in the art. Essentially, this can comprise isolating oocytes from the ovaries or reproductive tract of an animal. A readily available source of oocytes is slaughterhouse materials. For the combination of techniques such as genetic engineering, nuclear transfer and cloning, oocytes must generally be matured in vitro before these cells can be used as recipient cells for nuclear transfer, and before they can be fertilized by the sperm cell to develop into an embryo. This process generally requires collecting immature (prophase I) oocytes from mammalian ovaries, e.g., bovine ovaries obtained at a slaughterhouse, and maturing the oocytes in a maturation medium prior to fertilization or enucleation until the oocyte attains the metaphase II stage, which in the case of bovine oocytes generally occurs about 18-24 hours post-aspiration. This period of time is known as the “maturation period”. In certain embodiments, the oocyte is obtained from a gilt. A “gilt” is a female pig that has never had offspring. In other embodiments, the oocyte is obtained from a sow. A “sow” is a female pig that has previously produced offspring.
A metaphase II stage oocyte can be the recipient oocyte, at this stage it is believed that the oocyte can be or is sufficiently “activated” to treat the introduced nucleus as it does a fertilizing sperm. Metaphase II stage oocytes, which have been matured in vivo have been successfully used in nuclear transfer techniques. Essentially, mature metaphase II oocytes can be collected surgically from either non-superovulated or superovulated animal 35 to 48, or 39-41, hours past the onset of estrus or past the injection of human chorionic gonadotropin (hCG) or similar hormone. The oocyte can be placed in an appropriate medium, such as a hyaluronidase solution.
After a fixed time maturation period, which ranges from about 10 to 40 hours, about 16-18 hours, about 40-42 hours or about 39-41 hours, the oocytes can be enucleated. Prior to enucleation the oocytes can be removed and placed in appropriate medium, such as HECM containing 1 milligram per milliliter of hyaluronidase prior to removal of cumulus cells. The stripped oocytes can then be screened for polar bodies, and the selected metaphase II oocytes, as determined by the presence of polar bodies, are then used for nuclear transfer. Enucleation follows.
Enucleation can be performed by known methods, such as described in U.S. Pat. No. 4,994,384. For example, metaphase II oocytes can be placed in either HECM, optionally containing 7.5 micrograms per milliliter cytochalasin B, for immediate enucleation, or can be placed in a suitable medium, for example an embryo culture medium such as CR1aa, plus 10% estrus cow serum, and then enucleated later, such as not more than 24 hours later, or not more than 16-18 hours later.
Enucleation can be accomplished microsurgically using a micropipette to remove the polar body and the adjacent cytoplasm. The oocytes can then be screened to identify those of which have been successfully enucleated. One way to screen the oocytes is to stain the oocytes with 1 microgram per milliliter 33342 Hoechst dye in HECM, and then view the oocytes under ultraviolet irradiation for less than 10 seconds. The oocytes that have been successfully enucleated can then be placed in a suitable culture medium, for example, CR1aa plus 10% serum.
A single mammalian cell of the same species as the enucleated oocyte can then be transferred into the perivitelline space of the enucleated oocyte used to produce the NT unit. The mammalian cell and the enucleated oocyte can be used to produce NT units according to methods known in the art. For example, the cells can be fused by electrofusion. Electrofusion is accomplished by providing a pulse of electricity that is sufficient to cause a transient breakdown of the plasma membrane. This breakdown of the plasma membrane is very short because the membrane reforms rapidly. Thus, if two adjacent membranes are induced to breakdown and upon reformation the lipid bilayers intermingle, small channels can open between the two cells. Due to the thermodynamic instability of such a small opening, it enlarges until the two cells become one. See, for example, U.S. Pat. No. 4,997,384 by Prather et al. A variety of electrofusion media can be used including, for example, sucrose, mannitol, sorbitol and phosphate buffered solution. Fusion can also be accomplished using Sendai virus as a fusogenic agent (Graham, Wister Inot. Symp. Monogr., 9, 19, 1969). Also, the nucleus can be injected directly into the oocyte rather than using electroporation fusion. See, for example, Collas and Barnes, Mol. Reprod. Dev., 38:264-267 (1994). After fusion, the resultant fused NT units are then placed in a suitable medium until activation, for example, CR1aa medium. Typically activation can be effected shortly thereafter, for example less than 24 hours later, or about 4-9 hours later, or optimally 1-2 hours after fusion. In a particular embodiment, activation occurs at least one hour post fusion and at 40-41 hours post maturation.
The NT unit can be activated by known methods. Such methods include, for example, culturing the NT unit at sub-physiological temperature, in essence by applying a cold, or actually cool temperature shock to the NT unit. This can be most conveniently done by culturing the NT unit at room temperature, which is cold relative to the physiological temperature conditions to which embryos are normally exposed. Alternatively, activation can be achieved by application of known activation agents. For example, penetration of oocytes by sperm during fertilization has been shown to activate prefusion oocytes to yield greater numbers of viable pregnancies and multiple genetically identical calves after nuclear transfer. Also, treatments such as electrical and chemical shock can be used to activate NT embryos after fusion. See, for example, U.S. Pat. No. 5,496,720, to Susko-Parrish et al. Fusion and activation can be induced by application of an AC pulse of 5 V for 5 s followed by two DC pulses of 1.5 kV/cm for 60 μs each using an ECM2001 Electrocell Manipulator (BTX Inc., San Diego, Calif.). Additionally, activation can be effected by simultaneously or sequentially by increasing levels of divalent cations in the oocyte, and reducing phosphorylation of cellular proteins in the oocyte. This can generally be effected by introducing divalent cations into the oocyte cytoplasm, e.g., magnesium, strontium, barium or calcium, e.g., in the form of an ionophore. Other methods of increasing divalent cation levels include the use of electric shock, treatment with ethanol and treatment with caged chelators. Phosphorylation can be reduced by known methods, for example, by the addition of kinase inhibitors, e.g., serine-threonine kinase inhibitors, such as 6-dimethyl-aminopurine, staurosporine, 2-aminopurine, and sphingosine. Alternatively, phosphorylation of cellular proteins can be inhibited by introduction of a phosphatase into the oocyte, e.g., phosphatase 2A and phosphatase 2B.
The activated NT units, or “fused embryos”, can then be cultured in a suitable in vitro culture medium until the generation of cell colonies. Culture media suitable for culturing and maturation of embryos are well known in the art. Examples of known media, which can be used for embryo culture and maintenance, include Ham's F-10+10% fetal calf serum (FCS), Tissue Culture Medium-199 (TCM-199)+10% fetal calf serum, Tyrodes-Albumin-Lactate-Pyruvate (TALP), Dulbecco's Phosphate Buffered Saline (PBS), Eagle's and Whitten's media, and, in one specific example, the activated NT units can be cultured in NCSU-23 medium for about 1-4 h at approximately 38.6° C. in a humidified atmosphere of 5% CO2.
Afterward, the cultured NT unit or units can be washed and then placed in a suitable media contained in well plates which can contain a suitable confluent feeder layer. Suitable feeder layers include, by way of example, fibroblasts and epithelial cells. The NT units are cultured on the feeder layer until the NT units reach a size suitable for transferring to a recipient female, or for obtaining cells which can be used to produce cell colonies. These NT units can be cultured until at least about 2 to 400 cells, about 4 to 128 cells, or at least about 50 cells.
Activated NT units can then be transferred (embryo transfers), zero(0)-144 hours post activation, to the oviduct of an female pigs. In one embodiment, the female pigs can be an estrus-synchronized recipient gilt. Crossbred gilts (large white/Duroc/Landrace) (280-400 lbs) can be used. The gilts can be synchronized as recipient animals by oral administration of 18-20 mg Regu-Mate (Altrenogest, Hoechst, Warren, N.J.) mixed into the feed. Regu-Mate can be fed for 14 consecutive days. One thousand units of Human Chorionic Gonadotropin (hCG, Intervet America, Millsboro, Del.) can then be administered i.m. about 105 h after the last Regu-Mate treatment. Embryo transfers can then be performed about 22-26 h after the hCG injection. In one embodiment, the pregnancy can be brought to term and result in the birth of live offspring. In another embodiment, the pregnancy can be terminated early and embryonic cells can be harvested.
Breeding for Desired Homozygous Knockout Animals
In another aspect, the present invention provides a method for producing viable animals that lack any expression of a functional immunoglobulin gene is provided by breeding a male heterozygous for the immunoglobulin gene with a female heterozygous for the immunoglobulin gene. In one embodiment, the animals are heterozygous due to the genetic modification of one allele of the immunoglobulin gene to prevent expression of that allele. In another embodiment, the animals are heterozygous due to the presence of a point mutation in one allele of the alpha-immunoglobulin gene. In further embodiments, such heterozygous knockouts can be bred with an ungulate that expresses xenogenous immunoglobulin, such as human. In one embodiment, a animal can be obtained by breeding a transgenic ungulate that lacks expression of at least one allele of an endogenous immunoglobulin wherein the immunoglobulin is selected from the group consisting of heavy chain, kappa light chain and lambda light chain or any combination thereof with an ungulate that expresses an xenogenous immunoglobulin. In another embodiment, a animal can be obtained by breeding a transgenic ungulate that lacks expression of one allele of heavy chain, kappa light chain and lambda light chain with an ungulate that expresses an xenogenous, such as human, immunoglobulin. In a further embodiment, an animal can be obtained by breeding a transgenic ungulate that lacks expression of one allele of heavy chain, kappa light chain and lambda light chain and expresses an xenogenous, such as human, immunoglobulin with another transgenic ungulate that lacks expression of one allele of heavy chain, kappa light chain and lambda light chain with an ungulate and expresses an xenogenous, such as human, immunoglobulin to produce a homozygous transgenic ungulate that lacks expression of both alleles of heavy chain, kappa light chain and lambda light chain and expresses an xenogenous, such as human, immunoglobulin. Methods to produce such animals are also provided.
In one embodiment, sexually mature animals produced from nuclear transfer from donor cells that carrying a homozygous knockout in the immunoglobulin gene, can be bred and their offspring tested for the homozygous knockout. These homozygous knockout animals can then be bred to produce more animals.
In another embodiment, oocytes from a sexually mature homozygous knockout animal can be in vitro fertilized using wild type sperm from two genetically diverse pig lines and the embryos implanted into suitable surrogates. Offspring from these matings can be tested for the presence of the knockout, for example, they can be tested by cDNA sequencing, and/or PCR. Then, at sexual maturity, animals from each of these litters can be mated. In certain methods according to this aspect of the invention, pregnancies can be terminated early so that fetal fibroblasts can be isolated and further characterized phenotypically and/or genotypically. Fibroblasts that lack expression of the immunoglobulin gene can then be used for nuclear transfer according to the methods described herein to produce multiple pregnancies and offspring carrying the desired homozygous knockout.
Additional Genetic Modifications
In other embodiments, animals or cells lacking expression of functional immunoglobulin, produced according to the process, sequences and/or constructs described herein, can contain additional genetic modifications to eliminate the expression of xenoantigens. The additional genetic modifications can be made by further genetically modifying cells obtained from the transgenic cells and animals described herein or by breeding the animals described herein with animals that have been further genetically modified. Such animals can be modified to eliminate the expression of at least one allele of the alpha-1,3-galactosyltransferase gene, the CMP-Neu5Ac hydroxylase gene (see, for example, U.S. Ser. No. 10/863,116), the iGb3 synthase gene (see, for example, U.S. Patent Application 60/517,524), and/or the Forssman synthase gene (see, for example, U.S. Patent Application 60/568,922). In additional embodiments, the animals discloses herein can also contain genetic modifications to express fucosyltransferase, sialyltransferase and/or any member of the family of glucosyltransferases. To achieve these additional genetic modifications, in one embodiment, cells can be modified to contain multiple genetic modifications. In other embodiments, animals can be bred together to achieve multiple genetic modifications. In one specific embodiment, animals, such as pigs, lacking expression of functional immunoglobulin, produced according to the process, sequences and/or constructs described herein, can be bred with animals, such as pigs, lacking expression of alpha-1,3-galactosyl transferase (for example, as described in WO 04/028243).
In another embodiment, the expression of additional genes responsible for xenograft rejection can be eliminated or reduced. Such genes include, but are not limited to the CMP-NEUAc Hydroxylase Gene, the isoGloboside 3 Synthase gene, and the Forssman synthase gene. In addition, genes or cDNA encoding complement related proteins, which are responsible for the suppression of complement mediated lysis can also be expressed in the animals and tissues of the present invention. Such genes include, but are not limited to CD59, DAF, MCP and CD46 (see, for example, WO 99/53042; Chen et al. Xenotransplantation, Volume 6 Issue 3 Page 194-August 1999, which describes pigs that express CD59/DAF transgenes; Costa C et al, Xenotransplantation. 2002 January; 9(1):45-57, which describes transgenic pigs that express human CD59 and H-transferase; Zhao L et al.; Diamond L E et al. Transplantation. 2001 Jan. 15; 71(1):132-42, which describes a human CD46 transgenic pigs.
Additional modifications can include expression of tissue factor pathway inhibitor (TFPI), heparin, antithrombin, hirudin, TFPI, tick anticoagulant peptide, or a snake venom factor, such as described in WO 98/42850 and U.S. Pat. No. 6,423,316, entitled “Anticoagulant fusion protein anchored to cell membrane”; or compounds, such as antibodies, which down-regulate the expression of a cell adhesion molecule by the cells, such as described in WO 00/31126, entitled “Suppression of xenograft rejection by down regulation of a cell adhesion molecules” and compounds in which co-stimulation by signal 2 is prevented, such as by administration to the organ recipient of a soluble form of CTLA-4 from the xenogeneic donor organism, for example as described in WO 99/57266, entitled “Immunosuppression by blocking T cell co-stimulation signal 2 (B7/CD28 interaction)”.
In one embodiment, the animals or cells lacking expression of functional immunoglobulin, produced according to the present invention, can be further modified to transgenically express a cytoxic T-lymphocyte associated protein 4-immunoglobin (CTLA4). The animals or cells can be modified to express CTLA4 peptide or a biologically active fragment (e.g., extracellular domain, truncated form of the peptide in which at least the transmembrane domain has been removed) or derivative thereof. The peptide may be, e.g., human or porcine. The CTLA4 peptide can be mutated. Mutated peptides may have higher affinity than wildtype for porcine and/or human B7 molecules. In one specific embodiment, the mutated CTLA4 can be CTLA4 (Glu104, Tyr29). The CTLA4 peptide can be modified such that it is expressed intracellularly. Other modifications of the CTLA4 peptide include addition of a golgi retention signal to the N or C terminus. The golgi retention signal may be, e.g., the sequence KDEL. The CTLA4 peptide can be fused to a peptide dimerization domain or an immunoglobulin (Ig) molecule. The CTLA4 fusion peptides can include a linker sequence that can join the two peptides.
Certain aspects of the invention are described in greater detail in the non-limiting Examples that follow.
EXAMPLES Example 1 Porcine Heavy Chain Targeting and Generation of Porcine Animals that Lack Expression of Heavy Chain A portion of the porcine Ig heavy-chain locus was isolated from a 3× redundant porcine BAC library. In general, BAC libraries can be generated by fragmenting pig total genomic DNA, which can then be used to derive a BAC library representing at least three times the genome of the whole animal. BACs that contain porcine heavy chain immunoglobulin can then be selected through hybridization of probes selective for porcine heavy chain immunoglobulin as described herein.
Sequence from a clone (Seq ID 1) was used to generate a primer complementary to a portion of the J-region (the primer is represented by Seq ID No. 2). Separately, a primer was designed that was complementary to a portion of Ig heavy-chain mu constant region (the primer is represented by Seq ID No. 3). These primers were used to amplify a fragment of porcine Ig heavy-chain (represented by Seq ID No. 4) that led the functional joining region (J-region) and sufficient flanking region to design and build a targeting vector. To maintain this fragment and subclones of this fragment in a native state, the E. coli (Stable 2, Invitrogen cat #1026-019) that harbored these fragments was maintained at 30° C. Regions of Seq. ID No. 4 were subcloned and used to assemble a targeting vector as shown in Seq. ID No. 5. This vector was transfected into porcine fetal fibroblasts that were subsequently subjected to selection with G418. Resulting colonies were screened by PCR to detect potential targeting events (Seq ID No. 6 and Seq ID No. 7, 5′ screen primers; and Seq ID No. 8 and Seq ID No. 9, 3′ screen primers). See FIG. 1 for a schematic illustrating the targeting. Targeting was confirmed by southern blotting. Piglets were generated by nuclear transfer using the targeted fetal fibroblasts as nuclear donors.
Nuclear Transfer.
The targeted fetal fibroblasts were used as nuclear donor cells. Nuclear transfer was performed by methods that are well known in the art (see, e.g., Dai et al., Nature Biotechnology 20: 251-255, 2002; and Polejaeva et al., Nature 407:86-90, 2000).
Enucleation of in vitro-matured oocytes (BoMed, Madison, Wis.; TransOva Genetics, Sioux City, Iowa) was begun between 40 and 42 hours post-maturation as described in Polejaeva, I. A., et al. (Nature 407, 86-90 (2000)). For enucleation, we incubated the oocytes in calcium-free phosphate-buffered NCSU-23 medium containing 5 μg ml−1 cytochalasin B (Sigma) and 7.5 μg ml−1 Hoechst 33342 (Sigma) at 38° C. for 20 min. A small amount of cytoplasm from directly beneath the first polar body was then aspirated using an 18 μM glass pipette (Humagen, Charlottesville, Va.). We exposed the aspirated karyoplast to ultraviolet light to confirm the presence of a metaphase plate.
For nuclear transfer, a single fibroblast cell was placed under the zona pellucida in contact with each enucleated oocyte. Fusion and activation were induced by application of an AC pulse of 5 V for 5 s followed by two DC pulses of 1.5 kV/cm for 60 μs each using an ECM2001 Electrocell Manipulator (BTX Inc., San Diego, Calif.). Fused embryos were cultured in NCSU-23 medium for 1-4 h at 38.6° C. in a humidified atmosphere of 5% CO2, and then transferred to the oviduct of an estrus-synchronized recipient gilt. Crossbred gilts (large white/Duroc/landrace) (280-400 lbs) were synchronized as recipients by oral administration of 18-20 mg Regu-Mate (Altrenogest, Hoechst, Warren, N.J.) mixed into their feed. Regu-Mate was fed for 14 consecutive days. Human chorionic gonadotropin (hCG, 1,000 units; Intervet America, Millsboro, Del.) was administered intra-muscularly 105 h after the last Regu-Mate treatment. Embryo transfers were done 22-26 h after the hCG injection.
Nuclear transfer produced 18 healthy piglets from four litters. These animals have one functional wild-type Ig heavy-chain locus and one disrupted Ig heavy chain locus.
Seq ID 2: primer from ggccagacttcctcggaacagctca
Butler subclone to am-
plify J to C heavychain
(637Xba5′)
Seq ID 3: primer for C ttccaggagaaggtgacggagct
to amplify J to C heavy-
chain (JM1L)
Seq ID 6: heavychain 5′ tctagaagacgctggagagaggccag
primer for 5′ screen
(HCKOXba5′2)
Seq ID 7: heavychain 3′ taaagcgcatgctccagactgcctt
primer for 5′ screen
(5′arm5′)
Seq ID 8: heavychain 5′ catcgccttctatcgccttctt
primer for 3′ screen
(NEO4425)
Seq ID 9: heavychain 3′ Aagtacttgccgcctctcagga
primer for 3′ screen
(650 + CA)
Southern blot analysis of cell and pig tissue samples. Cells or tissue samples were lysed overnight at 60° C. in lysis buffer (10mM Tris, pH 7.5, 10 mM EDTA, 10 mM NaCl, 0.5% (w/v) Sarcosyl, 1 mg/ml proteinase K) and the DNA precipitated with ethanol. The DNA was then digested with NcoI or XbaI, depending on the probe to be used, and separated on a 1% agarose gel. After electrophoresis, the DNA was transferred to a nylon membrane and probed with digoxigenin-labeled probe (SEQ ID No 41 for NcoI digest, SEQ ID No 40 for XbaI digest). Bands were detected using a chemiluminescent substrate system (Roche Molecular Biochemicals).
Probes for Heavy Chain Southern:
HC J Probe (used with Xba I digest)
(Seq ID No 40)
CTCTGCACTCACTACCGCCGGACGCGCACTGCCGTGCTGCCCATGGACCA
CGCTGGGGAGGGGTGAGCGGACAGCACGTTAGGAAGTGTGTGTGTGCGCG
TGGGTGCAAGTCGAGCCAAGGCCAAGATCCAGGGGCTGGGCCCTGTGCCC
AGAGGAGAATGGCAGGTGGAGTGTAGCTGGATTGAAAGGTGGCCTGAAGG
GTGGGGCATCCTGTTTGGAGGCTCACTCTCAGCCCCAGGGTCTCTGGTTC
CTGCCGGGGTGGGGGGCGCAAGGTGCCTACCACACCCTGCTAGCCCCTCG
TCCAGTCCCGGGCCTGCCTCTTCACCACGGAAGAGGATAAGCCAGGCTGC
AGGCTTCATGTGCGCCGTGGAGAACCCAGTTCGGCCCTTGGAGG
HC Mu Probe (used with NcoI digest)
(Seq ID No 41)
GGCTGAAGTCTGAGGCCTGGCAGATGAGCTTGGACGTGCGCTGGGGAGTA
CTGGAGAAGGACTCCCGGGTGGGGACGAAGATGTTCAAGACGGGGGGCTG
CTCCTCTACGACTGCAGGCAGGAACGGGGCGTCACTGTGCCGGCGGCACC
CGGCCCCGCCCCCGCCACAGCCACAGGGGGAGCCCAGCTCACCTGGCCCA
GAGATGGACACGGACTTGGTGCCACTGGGGTGCTGGACCTCGCACACCAG
GAAGGCCTCTGGGTCCTGGGGGATGCTCACAGAGGGTAGGAGCACCCGGG
AGGAGGCCAAGTACTTGCCGCCTCTCAGGACGG
Example 2 Porcine Kappa Light Chain Targeting and Generation of Porcine Lacking Expression of Kappa Light Chain A portion of the porcine Ig kappa-chain locus was isolated from a 3× redundant porcine BAC library. In general, BAC libraries can be generated by fragmenting pig total genomic DNA, which can then be used to derive a BAC library representing at least three times the genome of the whole animal. BACs that contain porcine kappa chain immunoglobulin can then be selected through hybridization of probes selective for porcine kappa chain immunoglobulin as described herein.
A fragment of porcine Ig light-chain kappa was amplified using a primer complementary to a portion of the J-region (the primer is represented by Seq ID No. 10) and a primer complementary to a region of kappa C-region (represented by Seq ID No.11). The resulting amplimer was cloned into a plasmid vector and maintained in Stable2 cells at 30° C. (Seq ID No. 12). See FIG. 2 for a schematic illustration.
Separately, a fragment of porcine Ig light-chain kappa was amplified using a primer complementary to a portion of the C-region (Seq ID No. 13) and a primer complementary to a region of the kappa enhancer region (Seq ID No. 14). The resulting amplimer was fragmented by restriction enzymes and DNA fragments that were produced were cloned, maintained in Stable2 cells at 30 degrees C. and sequenced. As a result of this sequencing, two non-overlapping contigs were assembled (Seq ID No. 15, 5′ portion of amplimer; and Seq ID No. 16, 3′ portion of amplimer). Sequence from the downstream contig (Seq ID No. 16) was used to design a set of primers (Seq ID No. 17 and Seq ID No. 18) that were used to amplify a contiguous fragment near the enhancer (Seq ID No. 19). A subclone of each Seq ID No. 12 and Seq ID No. 19 were used to build a targeting vector (Seq ID No. 20). This vector was transfected into porcine fetal fibroblasts that were subsequently subjected to selection with G418. Resulting colonies were screened by PCR to detect potential targeting events (Seq ID No. 21 and Seq ID No. 22, 5′ screen primers; and Seq ID No. 23 and Seq Id No 43, 3′ screen primers, and Seq ID No. 24 and Seq Id No 24, endogenous screen primers). Targeting was confirmed by southern blotting. Southern blot strategy design was facilitated by cloning additional kappa sequence, it corresponds to the template for germline kappa transcript (Seq ID No. 25). Fetal pigs were generated by nuclear transfer.
Nuclear Transfer.
The targeted fetal fibroblasts were used as nuclear donor cells. Nuclear transfer was performed by methods that are well known in the art (see, e.g., Dai et al., Nature Biotechnology 20: 251-255, 2002; and Polejaeva et al., Nature 407:86-90, 2000).
Oocytes were collected 46-54 h after the hCG injection by reverse flush of the oviducts using pre-warmed Dulbecco's phosphate buffered saline (PBS) containing bovine serum albumin (BSA; 4 g−1) (as described in Polejaeva, I. A., et al. (Nature 407, 86-90 (2000)). Enucleation of in vitro-matured oocytes (BoMed, Madison, Wis.) was begun between 40 and 42 hours post-maturation as described in Polejaeva, I. A., et al. (Nature 407, 86-90 (2000)). Recovered oocytes were washed in PBS containing 4 gl−1 BSA at 38° C., and transferred to calcium-free phosphate-buffered NCSU-23 medium at 38° C. for transport to the laboratory. For enucleation, we incubated the oocytes in calcium-free phosphate-buffered NCSU-23 medium containing 5 μg ml−1 cytochalasin B (Sigma) and 7.5 μg ml−1 Hoechst 33342 (Sigma) at 38° C. for 20 min. A small amount of cytoplasm from directly beneath the first polar body was then aspirated using an 18 μM glass pipette (Humagen, Charlottesville, Va.). We exposed the aspirated karyoplast to ultraviolet light to confirm the presence of a metaphase plate.
For nuclear transfer, a single fibroblast cell was placed under the zona pellucida in contact with each enucleated oocyte. Fusion and activation were induced by application of an AC pulse of 5 V for 5 s followed by two DC pulses of 1.5 kV/cm for 60 μs each using an ECM2001 Electrocell Manipulator (BTX Inc., San Diego, Calif.). Fused embryos were cultured in NCSU-23 medium for 1-4 h at 38.6° C. in a humidified atmosphere of 5% CO2, and then transferred to the oviduct of an estrus-synchronized recipient gilt. Crossbred gilts (large white/Duroc/landrace) (280-400 lbs) were synchronized as recipients by oral administration of 18-20 mg Regu-Mate (Altrenogest, Hoechst, Warren, N.J.) mixed into their feed. Regu-Mate was fed for 14 consecutive days. Human chorionic gonadotropin (hCG, 1,000 units; Intervet America, Millsboro, Del.) was administered intra-muscularly 105 h after the last Regu-Mate treatment. Embryo transfers were done 22-26 h after the hCG injection.
Nuclear transfer using kappa targeted cells produced 33 healthy pigs from 5 litters. These pigs have one functional wild-type allele of porcine Ig light-chain kappa and one disrupted Ig light-chain kappa allele.
Seq ID 10: kappa J to C caaggaqaccaagctggaactc
5′ primer (kjc5′1)
Seq ID 11: kappa J to C tgatcaagcacaccacagagacag
3′ primer (kjc3′2)
Seq ID 13: 5′ primer for gatgccaagccatccgtcttcatc
Kappa C to E (porKCS1)
Seq ID 14: 3′ primer for tgaccaaagcagtgtgacggttgc
Kappa C to E (porKCA1)
Seq ID 17: kappa 5′ ggatcaaacacgcatcctcatggac
primer for amplification
of enhancer region
(K3′arm1S)
Seq ID 18: kappa 3′ ggtgattggggcatggttgagg
primer for amplification
of enhancer region
(K3′arm1A)
Seq ID 21: kappa screen, cgaacccctgtgtatatagtt
5′ primer, 5′
(kappa5armS)
Seq ID 22: kappa screen, gagatgaggaagaggagaaca
3′ primer, 5′
(kappaNeoA)
Seq ID 23: kappa screen, gcattgtctgagtaggtgtcatt
5′ primer, 3′
(kappaNeoS)
Seq ID 24: kappa screen, cgcttcttgcagggaacacgat
3′ primer, 5′
(kappa5armProbe3′)
Seq ID No 43, Kappa GTCTTTGGTTTTTGCTGAGGGTT
screen, 3′ primer
(kappa3armA2)
Southern blot analysis of cell and pig tissue samples. Cells or tissue samples were lysed overnight at 60° C. in lysis buffer (10mM Tris, pH 7.5, 10 mM EDTA, 10 mM NaCl, 0.5% (w/v) Sarcosyl, 1 mg/ml proteinase K) and the DNA precipitated with ethanol. The DNA was then digested with SacI and separated on a 1% agarose gel. After electrophoresis, the DNA was transferred to a nylon membrane and probed with digoxigenin-labeled probe (SEQ ID No 42). Bands were detected using a chemiluminescent substrate system (Roche Molecular Biochemicals).
Probe for Kappa Southern:
Kappa5ArmProbe 5′/3′
(SEQ ID No 42)
gaagtgaagccagccagttcctcctgggcaggtggccaaaattacagttg
acccctcctggtctggctgaaccttgccccatatggtgacagccatctgg
ccagggcccaggtctccctctgaagcctttgggaggagagggagagtggc
tggcccgatcacagatgcggaaggggctgactcctcaaccggggtgcaga
ctctgcagggtgggtctgggcccaacacacccaaagcacgcccaggaagg
aaaggcagcttggtatcactgcccagagctaggagaggcaccgggaaaat
gatctgtccaagacccgttcttgcttctaaactccgagggggtcagatga
agtggttttgtttcttggcctgaagcatcgtgttccctgcaagaagcgg
Example 3 Characterization of the Porcine Lambda Gene Locus To disrupt or disable porcine lambda, a targeting strategy has been devised that allows for the removal or disruption of the region of the lambda locus that includes a concatamer of J to C expression cassettes. BAC clones that contain portions of the porcine genome can be generated. A portion of the porcine Ig lambda-chain locus was isolated from a 3× redundant porcine BAC library. In general, BAC libraries can be generated by fragmenting pig total genomic DNA, which can then be used to derive a BAC library representing at least three times the genome of the whole animal. BACs that contain porcine lambda chain immunoglobulin can then be selected through hybridization of probes selective for porcine lambda chain immunoglobulin as described herein.
BAC clones containing a lambda J-C flanking region (see FIG. 3), can be independently fragmented and subcloned into a plasmid vector. Individual subdlones have been screened by PCR for the presence of a portion of the J to C intron. We have cloned several of these cassettes by amplifying from one C region to the next C region. This amplification was accomplished by using primers that are oriented to allow divergent extension within any one C region (Seq ID 26 and Seq ID 27). To obtain successful amplification, the extended products converge with extended products originated from adjacent C regions (as opposed to the same C region). This strategy produces primarily amplimers that extend from one C to the adjacent C. However, some amplimers are the result of amplification across the adjacent C and into the next C which lies beyond the adjacent C. These multi-gene amplimers contain a portion of a C, both the J and C region of the next J-C unit, the J region of the third J-C unit, and a portion of the C region of the third J-C unit. Seq ID 28 is one such amplimer and represents sequence that must be removed or disrupted.
Other porcine lambda sequences that have been cloned include: Seq ID No. 32, which includes 5′ flanking sequence to the first lambda J/C unit of the porcine lambda light chain genomic sequence; Seq ID No. 33, which includes 3′ flanking sequence to the J/C cluster region of the porcine lambda light chain genomic sequence, from approximately 200 base pairs downstream of lambda J/C; Seq ID No. 34, which includes 3′ flanking sequence to the J/C cluster region of the porcine lambda light chain genomic sequence, approximately 11.8 Kb downstream of the J/C cluster region, near the enhancer; Seq ID No. 35, which includes approximately 12 Kb downstream of lambda, including the enhancer region; Seq ID No. 36, which includes approximately 17.6 Kb downstream of lambda; Seq ID No. 37, which includes approximately 19.1 Kb downstream of lambda; Seq ID No. 38, which includes approximately 21.3 Kb downstream of lambda; and Seq ID No. 39, which includes approximately 27 Kb downstream of lambda.
Seq ID 26: 5′ primer for ccttcctcctgcacctgtcaac
lambda C to C amplimer
(lamC5′)
Seq ID 27: 3′ primer for tagacacaccagggtggccttg
lambda C to C amplimer
(lamC3′)
Example 4 Production of Targeting Vectors for the Lambda Gene Following a first targeting strategy, shown in FIG. 4, a vector is designed and built with one targeting arm that is homologous to a region upstream of J1 (i.e., the first J/C unit or sequence) and the other arm homologous to a region that is downstream of the last C (i.e., the last J/C unit or sequence) This targeting vector utilizes a selectable marker (SM).
Seq ID No. 48 represents one example of a vector used in the first targeting strategy. Seq ID No. 48 is a lambda light chain knockout vector which includes both 5′ and 3′ homology arms and Neo resistance factor.
Seq ID GCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTT
No. 48 TCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGC
TCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCA
GGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGA
CCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGA
AGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTC
GGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCC
CCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTT
GAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGC
CACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTA
CAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGG
ACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGG
AAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTG
GTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGA
AAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTC
TGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCA
TGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAA
AAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTG
GTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAG
CGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTC
GTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAG
TGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATT
TATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGT
GGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTG
CCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCA
ACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCG
TTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCG
AGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCT
TCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTA
TCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCAT
GCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCA
AGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGC
CCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTT
AAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCT
CAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACT
CGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGT
TTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGG
GAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTT
TTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAG
CGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGG
TTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCAAACAG
CTATGACCATGGCGGCCGCgtcgacAGGGTGTGGCCAAATACAG
CATGGAGTAGCCATCATAAGGAATCTTACACAAGCCTCCAAAAT
TGTGTTTCTGAAATTGGGTTTAAAGTACGTTTGCATTTTAAAAA
GCCTGCCAGAAAATACAGAAAAATGTCTGTGATATGTCTCTGGC
TGATAGGATTTTGCTTAGTTTTAATTTTGGCTTTATAATTTTCT
ATAGTTATGAAAATGTTCACAAGAAGATATATTTCATTTTAGCT
TCTAAAATAATTATAACACAGAAGTAATTTGTGCTTTAAAAAAA
TATTCAACACAGAAGTATATAAAGTAAAAATTGAGGAGTTCCCA
TCGTGGCTCAGTGATTAACAAACCCAACTAGTATCCATGAGGAT
ATGGATTTGATCCCTGGCCTTGCTCAGTGGGTTGAGGATCCAGT
GTTGCTGTGAGCTGTGGTGTAGGTTGCAGACACAGCACTCTGGC
GTTGCTGTGACTCTGGCGTAGGCCGGCAGCTACAGCTCCATTTG
GACCCTTAGCCTGGGAACCTCCATATGCCTGAGATACGGCCCTA
AAAAGTCAAAAGCCAAAAAAATAGTAAAAATTGAGTGTTTCTAC
TTACCACCCCTGCCCACATCTTATGCTAAAACCCGTTCTCCAGA
GACAAACATCGTCAGGTGGGTCTATATATTTCCAGCCCTCCTCC
TGTGTGTGTATGTCCGTAAAACACACACACACACACACACACGC
ACACACACACACACGTATCTAATTAGCATTGGTATTAGTTTTTC
AAAAGGGAGGTCATGCTCTACCTTTTAGGCGGCAAATAGATTAT
TTAAACAAATCTGTTGACATTTTCTATATCAACCCATAAGATCT
CCCATGTTCTTGGAAAGGCTTTGTAAGACATCAACATCTGGGTA
AACCAGCATGGTTTTTAGGGGGTTGTGTGGATTTTTTTCATATT
TTTTAGGGCACACCTGCAGCATATGGAGGTTCCCAGGCTAGGGG
TTGAATCAGAGCTGTAGCTGCCGGCCTACACCACAGCCACAGCA
ACGCCAGATCCTTAACCCACTGAGAAAGGCCAGGGATTGAACCT
GCATCCTCATGGATGCTGGTCAGATTTATTTCTGCTGAGCCACA
ACAGGAACTCCCTGAACCAGAATGCTTTTAACCATTCCACTTTG
CATGGACATTTAGATTGTTTCCATTTAAAAATACAAATTACAAG
GAGTTCCCGTCGTGGCTCAGTGGTAACGAATTGGACTAGGAACC
ATGAGGTTTCGGGTTCGATCCCTGGCCTTGCTCGGTGGGTTAAG
GATCCAGCATTGATGTGAGATATGGTGTAGGTCGCAGACGTGGC
TCGGATCCCACGTTGCTGTGGCTCTGGCGTAGGCCGGCAACAAC
AGCTCCGATTCGACCCCTAGCCTGGGAACCTCCATGTGCCACAG
GAGCAGCCCTAGAAAAGGCAAAAAGACAAAAAAATAAAAAATTA
AAATGAAAAAATAAAATAAAAATACAAATTACAAGAGACGGCTA
CAAGGAAATCCCCAAGTGTGTGCAAATGCCATATATGTATAAAA
TGTACTAGTGTCTCCTCGCGGGAAAGTTGCCTAAAAGTGGGTTG
GCTGGACAGAGAGGACAGGCTTTGACATTCTCATAGGTAGTAGC
AATGGGCTTCTCAAAATGCTGTTCCAGTTTACACTCACCATAGC
AAATGACAGTGCCTCTTCCTCTCCACCCTTGCCAATAATGTGAC
AGGTGGATCTTTTTCTATTTTGTGTATCTGACAAGCAAAAAATG
AGAACAGGAGTTCCTGTCGTGGTGCAGTGGAGACAAATCTGACT
AGGAACCATGAAATTTCGGGTTCAATCCCTGGCCTCACTCAGTA
GGTAAAGGATCCAGGGTTGCAGTGAGCTGTGGGGTAGGTCGCAG
ACACAGTGCAAATTTGGCCCTGTTGTGGCTGTGGTGTAGGCCGG
CAGCTATAGCTCCAATTGGACCCCTAGCCTGGGAACCTCCTTAT
GCCGTGGGTGAGGCCCTAAAAAAAAGAGTGCAAAAAAAAAAAAT
AAGAACAAAAATGATCATCGTTTAATTCTTTATTTGATCATTGG
TGAAACTTATTTTCCTTTTATATTTTTATTGACTGATTTTATTT
CTCCTATGAATTTACCGGTCATAGTTTTGCCTGGGTGTTTTTAC
TCCGGTTTTAGTTTTGGTTGGTTGTATTTTCTTAGAGAGCTATA
GAAACTCTTCATCTATTTGGAATAGTAATTCCTCATTAAGTATT
TGTGCTGCAAAAAATTTTCCCTGATCTGTTTTATGCTTTTGTTT
GTGGGGTCTTTCACGAGAAAGCCTTTTTAGTTTTTACACCTCAG
CTTGGTTGTTTTTCTTGATTGTGTCTGTAATCTGCGGCCAACAT
AGGAAACACATTTTTACTTTAGTGTTTTTTTCCTATTTTCTTCA
AGTACGTCCATTGTTTTGGTGTCTGATTTTACTTTGCCTGGGGT
TTGTTTTTGTGTGGCAGGAATATAAACTTATGTATTTTCCAAAT
GGAGAGCCAATGGTTGTATATTTGTTGAATTCAAATGCAACTTT
ATCAAACACCAAATCATCGATTTATCACAACTCTTCTCTGGTTT
ATTGATCTAATGATCAATTCCTGTTCCACGCTGTTTTAATTATT
TTAGCTTTGTGGATTTTGGTGCCTGGTAGAGAACAAAGCCTCCA
TTATTTTCATTCAAAATAGTCCCGTCTATTATCTGCCATTGTTG
TAGTATTAGACTTTAAAATCAATTTACTGATTTTCAAAAGTTAT
TCCTTTGGTGATGTGGAATACTTTATACTTCATAAGGTACATGG
ATTCATTTGTGGGGAATTGATGTCTTTGCTATTGTGGCCATTTG
TCAAGTTGTGTAATATTTTACCCATGCCAACTTTGCATATTGTA
TGTGAGTTTATTCCCAGGGTTTTTAATAGGATGTTTATTGAAGT
TGTCAGTGTTTCCACAATTTCATCGCCTCAGTGCTTACTGTTTG
CATAAAAGGAAACCTACTCACTTTTGCCTATTGCTCTTGTATTC
AATCATTTTAGTTAACTCTTGTGTTAATTTTGAGAGTTTTTCAG
CTGACTGTCTGGGGTTTTCTTTAATAGACTAGCCCTTTGTCTGT
AAAGAATAATTTTATCGAATTTTTCTTAACACTCACACTCTCCC
CACCCCCACCCCCGCTCATCTCCTTTCATTGGGTCAAATCTGTA
GAATACAATAAAAGTAAGAGTGGGAACCTTAGCCTTTAAGTCGA
TTTTGCCTTTAAATGTGAATGTTGCTATGTTTCGGGACATTCTC
TTTATCAAGTTGCGGATGTTTCCTTAGATAATTAACTTAATAAA
AGACTGGATGTTTGCTTTCTTCAAATCAGAATTGTGTTGAATTT
ATATTGCTATTCTGTTTAATTTTGTTTCAAAAAATTTACATGCA
CACCTTAAAGATAACCATGACCAAATAGTCCTCCTGCTGAGAGA
AAATGTTGGCCCCAATGCCACAGGTTACCTCCCGACTCAGATAA
ACTACAATGGGAGATAAAATCAGATTTGGCAAAGCCTGTGGATT
CTTGCCATAACTCTCAGAGCATGACTTGGGTGTTTTTTCCTTTT
CTAAGTATTTTAATGGTATTTTTGTGTTACAATAGGAAATCTAG
GACACAGAGAGTGATTCAATGAGGGGAACGCATTCTGGGATGAC
TCTAGGCCTCTGGTTTGGGGAGAGCTCTATTGAAGTAAAGACAA
TGAGAGGAAGCAAGTTTGCAGGGAACTGTGAGGAATTTAGATGG
GGAATGTTGGGTTTGAGGTTTCTATAGGGCACGCAAGCAGAGAT
GCACTCAGGAGGAAGAAGGAGCATAAATCTAGAGGCAAAAAGAG
AGGTCAGGACTGGAAATAGAGATGCGAGACACCAGGGTGGCAGT
CAGAGAGCACAGTGTGGGTCAGAAGACAGTGGAAGAACACAAGG
GACAGAGAGGGATCTCCAACTTCACTGGGATGAGGGCCTTGTTG
GCCTTGACCTGAGAGATTTCCAGGAGTTGAGGGTGGGAAGGAGc
cgcggTCTAGGAAGCTTTCTAGGGTACCTCTAGGGATCCGAACA
ATGGAAGTCCGAGCTCATCGCTAATAACTTCGTATAGCATACAT
TATACGAAGTTATATTCGATGCGGCCGCAAGGGGTTCGCGTCAG
CGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCA
GAGCAGagatccCGGCGCGCCCTACCGGGTAGGGGAGGCGCTTT
TCCCAAGGCAGTCTGGAGCATGCGCTTTAGCAGCCCCGCTGGGC
ACTTGGCGCTACACAAGTGGCCTCTGGCCTCGCACACATTCCAC
ATCCACCGGTAGGCGCCAACCGGCTCCGTTCTTTGGTGGCCCCT
TCGCGCCACCTTCTACTCCTCCCCTAGTCAGGAAGTTCCCCCCC
GCCCCGCAGCTCGCGTCGTGCAGGACGTGACAAATGGAAGTAGC
ACGTCTCACTAGTCTCGTGCAGATGGACAGCACCGCTGAGCAAT
GGAAGCGGGTAGGCCTTTGGGGCAGCGGCCAATAGCAGCTTTGG
CTCCTTCGCTTTCTGGGCTCAGAGGCTGGGAAGGGGTGGGTCCG
GGGGCGGGCTCAGGGGCGGGCTCAGGGGCGGGGCGGGCGCCCGA
AGGTCCTCCGGAAGCCCGGCATTCTGCACGCTTCAAAAGCGCAC
GTCTGCCGCGCTGTTCTCCTCTTCCTCATCTCCGGGCCTTTCGA
CCTGCAGCCAATATGGGATCGGCCATTGAACAAGATGGATTGCA
CGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATG
ACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTC
CGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGA
CCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGC
TATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTC
GACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGA
AGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCG
AGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACG
CTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCG
CATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCAATC
AGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAA
CTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATCT
CGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGG
AAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGT
GTGGCGGATCGCTATCAGGACATAGCGTTGGCTACCCGTGATAT
TGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGC
TTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTAT
CGCCTTCTTGACGAGTTCTTCTGAGGGGATCAATTCtctagtGA
ACAATGGAAGTCCGAGCTCATCGCTAATAACTTCGTATAGCATA
CATTATACGAAGTTATATTCGATGCGGCCGCAAGGGGTTCGCGT
CAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCA
TCAGAGCAGtctagaGCTCGCTGATCAGCCTCGACTGTGCCTTC
TAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCT
TGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAAT
GAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCT
GGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAG
ACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCT
GAGGCGGAAAGAACCAGCTGGGGGCGCGCCCctcgagGGGAAGG
TATCTCCCAGGAAACTGGCCAGGACACATTGGTCCTCCGCCCTC
CCCTTCCTCCCACTCCTCCTCCAGACAGGACTGTGCCCACCCCC
TGCCACCTTTCTGGCCAGAACTGTCCATGGCAGGTGACCTTCAC
ATGAGCCCTTCCTCCCTGCCTGCCCTAGTGGGACCCTCCATACC
TCCCCCTGGACCCCGTTGTCCTTTCTTTCCAGTGTGGCCCTGAG
CATAACTGATGCCATCATGGGCTGCTGACCCACCCGGGACTGTG
TTGTGCAGTGAGTCACTTCTCTGTCATCAGGGCTTTGTAATTGA
TAGATAGTGTTTCATCATCATTAGGACCGGGTGGCCTCTATGCT
CTGTTAGTCTCCAAACACTGATGAAAACCTTCGTTGGCATAGTC
CCAGCTTCCTGTTGCCCATCCATAAATCTTGACTTAGGGATGCA
CATCCTGTCTCCAAGCAACCACCCCTCCCCTAGGCTAACTATAA
AACTGTCCCAATGGCCCTTGTGTGGTGCAGAGTTCATGCTTCCA
GATCATTTCTCTGCTAGATCCATATCTCACCTTGTAAGTCATCC
TATAATAAACTGATCCATTGATTATTTGCTTCTGTTTTTTCCAT
CTCAAAACAGCTTCTCAGTTCAGTTCGAATTTTTTATTCCCTCC
ATCCACCCATACTTTCCTCAGCCTGGGGAACCCTTGCCCCCAGT
CCCATGCCCTTCCTCCCTCTCTGCCCAGCTCAGCACCTGCCCAC
CCTCACCCTTCCTGTCACTCCCTAGGACTGGACCATCCACTGGG
GCCAGGACACTCCAGCAGCCTTGGCTTCATGGGCTCTGAAATCC
ATGGCCCATCTCTATTCCTCACTGGATGGCAGGTTCAGAGATGT
GAAAGGTCTAGGAGGAAGCCAGGAAGGAAACTGTTGCATGAAAG
GCCGGCCTGATGGTTCAGTACTTAAATAATATGAGCTCTGAGCT
CCCCAGGAACCAAAGCATGGAGGGAGTATGTGCCTCAGAATCTC
TCTGAGATTCAGCAAAGCCTTTGCTAGAGGGAAAATAGTGGCTC
AACCTTGAGGGCCAGCATCTTGCACCACAGTTAAAAGTGGGTAT
TTGTTTTACCTGAGGCCTCAGCATTATGGGAACCGGGCTCTGAC
ACAAACACAGGTGCAGCCCGGCAGCCTCAGAACACAGCAACGAC
CACAAGCTGGGACAGCTGCCCCTGAACGGGGAGTCCACCATGCT
TCTGTCTCGGGTACCACCAGGTCACCATCCCTGGGGGAGGTAGT
TCCATAGCAGTAGTCCCCTGATTTCGCCCCTCGGGCGTGTAGCC
AGGCAAGCTCCTGCCTCTGGACCCAGGGTGGACCCTTGCTCCCC
ACTACCCTGCACATGCCAGACAGTCAAGACCACTCCCACCTCTG
TCTGAGGCCCCCTTGGGTGTCCCAGGGCCCCCGAGCTGTCCTCT
ACTCATGGTTCTTCCACCTGGGTACAAAAGAGGCGAGGGACACT
TTTCTCAGGTTTGCGGCTCAGAAAGGTACCTTCCTAGGGTTTGT
CCACTGGGAGTCACCTCCCTTGCATCTCAATGTCAGTGGGGAAA
ACTGGGTCCCATGGGGGGATTAGTGCCACTGTGAGGCCCCTGAA
GTCTGGGGCCTCTAGACACTATGATGATGAGGGATGTGGTGAAA
AACCCCACCCCAGCCCTTCTTGCCGGGACCCTGGGCTGTGGCTC
CCCCATTGCACTTGGGGTCAGAGGGGTGGATGGTGGCTATGGTC
AGGCATGTTTCCCATGAGCTGGGGGCACCCTGGGTGACTTTCTC
CTGTGAATCCTGAATTAGCAGCTATAACAAATTGCCCAAACTCT
TAGGCTTAAAACAACACACATTTATTCCTCTGGGTCCCAGGGTC
AGAAGTCCAAAATGAGTCCTATAGGCTAAATTTGAGGTGTCTCT
GGGTTGAGCTCCTCCTGGAAGCCTTTTCCAGCCTCTAGAGTCCC
AAGTCCTTGGCTCTGGGCCCCTCCCTCAAGCTTCAAAGCCACAG
AAGCTTCTAATCTCTCTCCCTTCCCCTCTGACCTCTGCTCCCAT
CCTCATACCCTGTCCCCTCACTCTGACCCTCCTGCCTCCCTCTT
TCCCTTATAAAGACCCTGCATGGGGCCACGGAGATAATCCAGGG
TAATCGCCCCTCTTCCAGCCCTTAACTCCATCCCATCTGCAAAA
TCCCTGTCACCCCATAATGGACCTACagatctCCTAGAGTTAAC
ACTGGCCGTCGTTTTACCGGTCCGTAGTCAGGTTTAGTTCGTCC
GGCGGCGCCAGAAATCCGCGCGGTGGTTTTTGGGGGTCGGGGGT
GTTTGGCAGCCACAGACGCCCGGTGTTCGTGTCGCGCCAGTACA
TGCGGTCCATGCCCAGGCCATCCAAAAACCATGGGTCTGTCTGC
TCAGTCCAGTCGTGGACTGACCCCACGCAACGCCCAAAATAATA
ACCCCCACGAACCATAAACCATTCCCCATGGGGGACCCCGTCCC
TAACCCACGGGGCCCGTGGCTATGGCAGGCCTGCCGCCCGACGT
TGGCTGCGAGCCCTGGGCCTTCACCCGAACTTGGGGGGTGGGGT
GGGGAAAAGGAAGAAACGCGGGCGTATTGGCCCCAATGGGGTCT
CGGTGGGGTATCGACAGAGTGCCAGCCCTGGGACCGAACCCCGC
GTTTATGAACAAACGACCCAACACCCGTGCGTTTTATTCTGTCT
TTTTATTGCCGACATAGCGCGGGTTCCTTCCGGTATTGTCTCCT
TCCGTGTTTCAGTTAGCCTCCCCCATCTCCCGTGCAAACGTGCG
CGCCAGGTCGCAGATCGTCGGTATGGAGCCTGGGGTGGTGACGT
GGGTCTGGATCATCCCGGAGGTAAGTTGCAGCAGGGCGTCCCGG
CAGCCGGCGGGCGATTGGTCGTAATCCAGGATAAAGACGTGCAT
GGGACGGAGGCGTTTGGCCAAGACGTCCAAGGCCCAGGCAAACA
CGTTGTACAGGTCGCCGTTGGGGGCCAGCAACTCGGGGGCCCGA
AACAGGGTAAATAACGTGTCCCCGATATGGGGTCGTGGGCCCGC
GTTGCTCTGGGGCTCGGCACCCTGGGGCGGCACGGCCGTCCCCG
AAAGCTGTCCCCAATCCTCCCGCCACGACCCGCCGCCCTGCAGA
TACCGCACCGTATTGGCAAGCAGCCCGTAAACGCGGCGAATCGC
GGTCAGCATAGCCAGGTCAAGCCGCTCGCCGGGGCGCTGGCGTT
TGGCCAGGCGGTCGATGTGTCTGTCCTCCGGAAGGGCCCCCAAC
ACGATGTTTGTGCCGGGCAAGGTCGGCGGGATGAGGGCCACGAA
CGCCAGCACGGCCTGGGGGGTCATGCTGCCCATAAGGTATCGCG
CGGCCGGGTAGCACAGGAGGGCGGCGATGGGATGGCGGTCGAAG
ATGAGGGTGAGGGCCGGGGGCGGGGCATGTGAGCTCCCAGCCTC
CCCCCCGATATGAGGAGCCAGAACGGCGTCGGTCACGGTATAAG
GCATGCCCATTGTTATCTGGGCGCTTGTCATTACCACCGCCGCG
TCCCCGGCCGATATCTCACCCTGGTCAAGGCGGTGTTGTGTGGT
GTAGATGTTCGCGATTGTCTCGGAAGCCCCCAGCACCCGCCAGT
AAGTCATCGGCTCGGGTACGTAGACGATATCGTCGCGCGAACCC
AGGGCCACCAGCAGTTGCGTGGTGGTGGTTTTCCCCATCCCGTG
GGGACCGTCTATATAAACCCGCAGTAGCGTGGGCATTTTCTGCT
CCGGGCGGACTTCCGTGGCTTCTTGCTGCCGGCGAGGGCGCAAC
GCCGTACGTCGGTTGCTATGGCCGCGAGAACGCGCAGCCTGGTC
GAACGCAGACGCGTGCTGATGGCCGGGGTACGAAGCCATACGCG
CTTCTACAAGGCGCTGGCCGAAGAGGTGCGGGAGTTTCACGCCA
CCAAGATGTGCGGCACGCTGTTGACGCTGTTAAGCGGGTCGCTG
CAGGGTCGCTCGGTGTTCGAGGCCACACGCGTCACCTTAATATG
CGAAGTGGACCTGGGACCGCGCCGCCCCGACTGCATCTGCGTGT
TCCAATTCGCCAATGACAAGACGCTGGGCGGGGTTTGCTCGACA
TTGGGTGGAAACATTCCAGGCCTGGGTGGAGAGGCTTTTTGCTT
CCTCTTGCAAAACCACACTGCTCGACATTGGGTGGAAACATTCC
AGGCCTGGGTGGAGAGGCTTTTTGCTTCCTCTTGAAAACCACAC
TGCTCGACTCTACGGTCCG
Seq ID No. 49 is a lambda light chain 5′ arm sequence
Seq ID AGGGTGTGGCCAAATACAGCATGGAGTAGCCATCATAAGGAATC
No. 49 TTACACAAGCCTCCAAAATTGTGTTTCTGAAATTGGGTTTAAAG
TACGTTTGCATTTTAAAAAGCCTGCCAGAAAATACAGAAAAATG
TCTGTGATATGTCTCTGGCTGATAGGATTTTGCTTAGTTTTAAT
TTTGGCTTTATAATTTTCTATAGTTATGAAAATGTTCACAAGAA
GATATATTTCATTTTAGCTTCTAAAATAATTATAACACAGAAGT
AATTTGTGCTTTAAAAAAATATTCAACACAGAAGTATATAAAGT
AAAAATTGAGGAGTTCCCATCGTGGCTCAGTGATTAACAAACCC
AACTAGTATCCATGAGGATATGGATTTGATCCCTGGCCTTGCTC
AGTGGGTTGAGGATCCAGTGTTGCTGTGAGCTGTGGTGTAGGTT
GCAGACACAGCACTCTGGCGTTGCTGTGACTCTGGCGTAGGCCG
GCAGCTACAGCTCCATTTGGACCCTTAGCCTGGGAACCTCCATA
TGCCTGAGATACGGCCCTAAAAAGTCAAAAGCCAAAAAAATAGT
AAAAATTGAGTGTTTCTACTTACCACCCCTGCCCACATCTTATG
CTAAAACCCGTTCTCCAGAGACAAACATCGTCAGGTGGGTCTAT
ATATTTCCAGCCCTCCTCCTGTGTGTGTATGTCCGTAAAACACA
CACACACACACACACACGCACACACACACACACGTATCTAATTA
GCATTGGTATTAGTTTTTCAAAAGGGAGGTCATGCTCTACCTTT
TAGGCGGCAAATAGATTATTTAAACAAATCTGTTGACATTTTCT
ATATCAACCCATAAGATCTCCCATGTTCTTGGAAAGGCTTTGTA
AGACATCAACATCTGGGTAAACCAGCATGGTTTTTAGGGGGTTG
TGTGGATTTTTTTCATATTTTTTAGGGCACACCTGCAGCATATG
GAGGTTCCCAGGCTAGGGGTTGAATCAGAGCTGTAGCTGCCGGC
CTACACCACAGCCACAGCAACGCCAGATCCTTAACCCACTGAGA
AAGGCCAGGGATTGAACCTGCATCCTCATGGATGCTGGTCAGAT
TTATTTCTGCTGAGCCACAACAGGAACTCCCTGAACCAGAATGC
TTTTAACCATTCCACTTTGCATGGACATTTAGATTGTTTCCATT
TAAAAATACAAATTACAAGGAGTTCCCGTCGTGGCTCAGTGGTA
ACGAATTGGACTAGGAACCATGAGGTTTCGGGTTCGATCCCTGG
CCTTGCTCGGTGGGTTAAGGATCCAGCATTGATGTGAGATATGG
TGTAGGTCGCAGACGTGGCTCGGATCCCACGTTGCTGTGGCTCT
GGCGTAGGCCGGCAACAACAGCTCCGATTCGACCCCTAGCCTGG
GAACCTCCATGTGCCACAGGAGCAGCCCTAGAAAAGGCAAAAAG
ACAAAAAAATAAAAAATTAAAATGAAAAAATAAAATAAAAATAC
AAATTACAAGAGACGGCTACAAGGAAATCCCCAAGTGTGTGCAA
ATGCCATATATGTATAAAATGTACTAGTGTCTCCTCGCGGGAAA
GTTGCCTAAAAGTGGGTTGGCTGGACAGAGAGGACAGGCTTTGA
CATTCTCATAGGTAGTAGCAATGGGCTTCTCAAAATGCTGTTCC
AGTTTACACTCACCATAGCAAATGACAGTGCCTCTTCCTCTCCA
CCCTTGCCAATAATGTGACAGGTGGATCTTTTTCTATTTTGTGT
ATCTGACAAGCAAAAAATGAGAACAGGAGTTCCTGTCGTGGTGC
AGTGGAGACAAATCTGACTAGGAACCATGAAATTTCGGGTTCAA
TCCCTGGCCTCACTCAGTAGGTAAAGGATCCAGGGTTGCAGTGA
GCTGTGGGGTAGGTCGCAGACACAGTGCAAATTTGGCCCTGTTG
TGGCTGTGGTGTAGGCCGGCAGCTATAGCTCCAATTGGACCCCT
AGCCTGGGAACCTCCTTATGCCGTGGGTGAGGCCCTAAAAAAAA
GAGTGCAAAAAAAAAAAATAAGAACAAAAATGATCATCGTTTAA
TTCTTTATTTGATCATTGGTGAAACTTATTTTCCTTTTATATTT
TTATTGACTGATTTTATTTCTCCTATGAATTTACCGGTCATAGT
TTTGCCTGGGTGTTTTTACTCCGGTTTTAGTTTTGGTTGGTTGT
ATTTTCTTAGAGAGCTATAGAAACTCTTCATCTATTTGGAATAG
TAATTCCTCATTAAGTATTTGTGCTGCAAAAAATTTTCCCTGAT
CTGTTTTATGCTTTTGTTTGTGGGGTCTTTCACGAGAAAGCCTT
TTTAGTTTTTACACCTCAGCTTGGTTGTTTTTCTTGATTGTGTC
TGTAATCTGCGGCCAACATAGGAAACACATTTTTACTTTAGTGT
TTTTTTCCTATTTTCTTCAAGTACGTCCATTGTTTTGGTGTCTG
ATTTTACTTTGCCTGGGGTTTGTTTTTGTGTGGCAGGAATATAA
ACTTATGTATTTTCCAAATGGAGAGCCAATGGTTGTATATTTGT
TGAATTCAAATGCAACTTTATCAAACACCAAATCATCGATTTAT
CACAACTCTTCTCTGGTTTATTGATCTAATGATCAATTCCTGTT
CCACGCTGTTTTAATTATTTTAGCTTTGTGGATTTTGGTGCCTG
GTAGAGAACAAAGCCTCCATTATTTTCATTCAAAATAGTCCCGT
CTATTATCTGCCATTGTTGTAGTATTAGACTTTAAAATCAATTT
ACTGATTTTCAAAAGTTATTCCTTTGGTGATGTGGAATACTTTA
TACTTCATAAGGTACATGGATTCATTTGTGGGGAATTGATGTCT
TTGCTATTGTGGCCATTTGTCAAGTTGTGTAATATTTTACCCAT
GCCAACTTTGCATATTGTATGTGAGTTTATTCCCAGGGTTTTTA
ATAGGATGTTTATTGAAGTTGTCAGTGTTTCCACAATTTCATCG
CCTCAGTGCTTACTGTTTGCATAAAAGGAAACCTACTCACTTTT
GCCTATTGCTCTTGTATTCAATCATTTTAGTTAACTCTTGTGTT
AATTTTGAGAGTTTTTCAGCTGACTGTCTGGGGTTTTCTTTAAT
AGACTAGCCCTTTGTCTGTAAAGAATAATTTTATCGAATTTTTC
TTAACACTCACACTCTCCCCACCCCCACCCCCGCTCATCTCCTT
TCATTGGGTCAAATCTGTAGAATACAATAAAAGTAAGAGTGGGA
ACCTTAGCCTTTAAGTCGATTTTGCCTTTAAATGTGAATGTTGC
TATGTTTCGGGACATTCTCTTTATCAAGTTGCGGATGTTTCCTT
AGATAATTAACTTAATAAAAGACTGGATGTTTGCTTTCTTCAAA
TCAGAATTGTGTTGAATTTATATTGCTATTCTGTTTAATTTTGT
TTCAAAAAATTTACATGCACACCTTAAAGATAACCATGACCAAA
TAGTCCTCCTGCTGAGAGAAAATGTTGGCCCCAATGCCACAGGT
TACCTCCCGACTCAGATAAACTACAATGGGAGATAAAATCAGAT
TTGGCAAAGCCTGTGGATTCTTGCCATAACTCTCAGAGCATGAC
TTGGGTGTTTTTTCCTTTTCTAAGTATTTTAATGGTATTTTTGT
GTTACAATAGGAAATCTAGGACACAGAGAGTGATTCAATGAGGG
GAACGCATTCTGGGATGACTCTAGGCCTCTGGTTTGGGGAGAGC
TCTATTGAAGTAAAGACAATGAGAGGAAGCAAGTTTGCAGGGAA
CTGTGAGGAATTTAGATGGGGAATGTTGGGTTTGAGGTTTCTAT
AGGGCACGCAAGCAGAGATGCACTCAGGAGGAAGAAGGAGCATA
AATCTAGAGGCAAAAAGAGAGGTCAGGACTGGAAATAGAGATGC
GAGACACCAGGGTGGCAGTCAGAGAGCACAGTGTGGGTCAGAAG
ACAGTGGAAGAACACAAGGGACAGAGAGGGATCTCCAACTTCAC
TGGGATGAGGGCCTTGTTGGCCTTGACCTGAGAGATTTCCAGGA
GTTGAGGGTGGGAAGGAG
Seq. ID No. 50 is a lambda 3′ arm sequence
Seq. ID GGGAAGGTATCTCCCAGGAAACTGGCCAGGACACATTGGTCC
No. 50 TCCGCCCTCCCCTTCCTCCCACTCCTCCTCCAGACAGGACTG
TGCCCACCCCCTGCCACCTTTCTGGCCAGAACTGTCCATGGC
AGGTGACCTTCACATGAGCCCTTCCTCCCTGCCTGCCCTAGT
GGGACCCTCCATACCTCCCCCTGGACCCCGTTGTCCTTTCTT
TCCAGTGTGGCCCTGAGCATAACTGATGCCATCATGGGCTGC
TGACCCACCCGGGACTGTGTTGTGCAGTGAGTCACTTCTCTG
TCATCAGGGCTTTGTAATTGATAGATAGTGTTTCATCATCAT
TAGGACCGGGTGGCCTCTATGCTCTGTTAGTCTCCAAACACT
GATGAAAACCTTCGTTGGCATAGTCCCAGCTTCCTGTTGCCC
ATCCATAAATCTTGACTTAGGGATGCACATCCTGTCTCCAAG
CAACCACCCCTCCCCTAGGCTAACTATAAAACTGTCCCAATG
GCCCTTGTGTGGTGCAGAGTTCATGCTTCCAGATCATTTCTC
TGCTAGATCCATATCTCACCTTGTAAGTCATCCTATAATAAA
CTGATCCATTGATTATTTGCTTCTGTTTTTTCCATCTCAAAA
CAGCTTCTCAGTTCAGTTCGAATTTTTTATTCCCTCCATCCA
CCCATACTTTCCTCAGCCTGGGGAACCCTTGCCCCCAGTCCC
ATGCCCTTCCTCCCTCTCTGCCCAGCTCAGCACCTGCCCACC
CTCACCCTTCCTGTCACTCCCTAGGACTGGACCATCCACTGG
GGCCAGGACACTCCAGCAGCCTTGGCTTCATGGGCTCTGAAA
TCCATGGCCCATCTCTATTCCTCACTGGATGGCAGGTTCAGA
GATGTGAAAGGTCTAGGAGGAAGCCAGGAAGGAAACTGTTGC
ATGAAAGGCCGGCCTGATGGTTCAGTACTTAAATAATATGAG
CTCTGAGCTCCCCAGGAACCAAAGCATGGAGGGAGTATGTGC
CTCAGAATCTCTCTGAGATTCAGCAAAGCCTTTGCTAGAGGG
AAAATAGTGGCTCAACCTTGAGGGCCAGCATCTTGCACCACA
GTTAAAAGTGGGTATTTGTTTTACCTGAGGCCTCAGCATTAT
GGGAACCGGGCTCTGACACAAACACAGGTGCAGCCCGGCAGC
CTCAGAACACAGCAACGACCACAAGCTGGGACAGCTGCCCCT
GAACGGGGAGTCCACCATGCTTCTGTCTCGGGTACCACCAGG
TCACCATCCCTGGGGGAGGTAGTTCCATAGCAGTAGTCCCCT
GATTTCGCCCCTCGGGCGTGTAGCCAGGCAAGCTCCTGCCTC
TGGACCCAGGGTGGACCCTTGCTCCCCACTACCCTGCACATG
CCAGACAGTCAAGACCACTCCCACCTCTGTCTGAGGCCCCCT
TGGGTGTCCCAGGGCCCCCGAGCTGTCCTCTACTCATGGTTC
TTCCACCTGGGTACAAAAGAGGCGAGGGACACTTTTCTCAGG
TTTGCGGCTCAGAAAGGTACCTTCCTAGGGTTTGTCCACTGG
GAGTCACCTCCCTTGCATCTCAATGTCAGTGGGGAAAACTGG
GTCCCATGGGGGGATTAGTGCCACTGTGAGGCCCCTGAAGTC
TGGGGCCTCTAGACACTATGATGATGAGGGATGTGGTGAAAA
ACCCCACCCCAGCCCTTCTTGCCGGGACCCTGGGCTGTGGCT
CCCCCATTGCACTTGGGGTCAGAGGGGTGGATGGTGGCTATG
GTCAGGCATGTTTCCCATGAGCTGGGGGCACCCTGGGTGACT
TTCTCCTGTGAATCCTGAATTAGCAGCTATAACAAATTGCCC
AAACTCTTAGGCTTAAAACAACACACATTTATTCCTCTGGGT
CCCAGGGTCAGAAGTCCAAAATGAGTCCTATAGGCTAAATTT
GAGGTGTCTCTGGGTTGAGCTCCTCCTGGAAGCCTTTTCCAG
CCTCTAGAGTCCCAAGTCCTTGGCTCTGGGCCCCTCCCTCAA
GCTTCAAAGCCACAGAAGCTTCTAATCTCTCTCCCTTCCCCT
CTGACCTCTGCTCCCATCCTCATACCCTGTCCCCTCACTCTG
ACCCTCCTGCCTCCCTCTTTCCCTTATAAAGACCCTGCATGG
GGCCACGGAGATAATCCAGGGTAATCGCCCCTCTTCCAGCCC
TTAACTCCATCCCATCTGCAAAATCCCTGTCACCCCATAATG
GACCTAC
In a second strategy, the targeting strategy utilizes a vector pair. One targeting vector is designed to target upstream of J1. See FIG. 5. This targeting vector utilizes a selectable marker that can be selected for or against. Any combination of positive and negative selectable markers described herein or known in the art can be used. A fusion gene composed of the coding region of Herpes simplex thymidine kinase (TK) and the Tn5 aminoglycoside phosphotransferase (Neo resistance) genes is used. This fusion gene is flanked by recognition sites for any site specific recombinase (SSRRS) described herein or known in the art, such as lox sites. Upon isolation of targeted cells through the use of G418 selection, Cre is supplied in trans to delete the marker gene (See FIG. 5). Cells that have deleted the marker gene are selected by addition of any drug known in the art that can be metabolized by TK into a toxic product, such as ganciclovir. The resulting genotype is then targeted with a second vector. The second targeting vector (FIG. 6) is designed to target downstream of last C and uses a positive/negative selection system that is flanked on only one side by a specific recombination site (lox). The recombination site is placed distally in relation to the first targeting event. Upon isolation of the targeted genotype, Cre is again supplied in trans to mediate deletion from recombination site provided in the first targeting event to the recombination site delivered in the second targeting event. The entire J to C cluster region will be removed. The appropriate genotype is again selected by administration of ganciclovir.
Two vector pairs, i.e., lambda targeting constructs, were designed and built to target the first and last J/C regions and to include site-specific recombination sites. The first vector pair was composed of Seq ID No. 44 (step 1 vector) and Seq ID No. 45 (step 2 vector). The second vector pair was composed of Seq ID No. 46 (step 2 vector) and Seq ID No. 47 (step 1 vector).
Overview of Seq ID No. 44 (upstream vector, step 1, double lox):
Feature Map
CDS (3 total)
-
- NEO (+STOP) CDS
- Start: 3311 End: 4114 (Complementary)
- TK CDS (from VEC1198)
- Start: 4118 End: 5251 (Complementary)
- AP(R)
- Start: 11732 End: 12589 (Complementary)
- bla gene-Ap(r) determinant
Enhancer (1 total)
-
- CMV Enhancer
- Start: 5779 End: 6199 (Complementary)
Misc. Binding Site (2 total)
-
- Left Homology Arm
- Right Homology Arm
Misc. Feature (5 total)
-
- loxP-1
- HSVTK-polyA
- Start: 3046 End: 3304 (Complementary)
- loxP-2
Promoter Eukaryotic (1 total)
-
- Mus-PGK Promoter (correct)
- Start: 5264 End: 5772 (Complementary)
Replication Origin (2 total)
-
- Replication Origin
- Start: 10921 End: 11509 (Complementary)
Overview of Seq ID No. 45 (Downstream vector, step 2, single lox
Feature Map
CDS (3 total)
-
- NEO (+STOP) CDS
- Start: 3115 End: 3918 (Complementary)
- TK CDS (from VEC1198)
- Start: 3922 End: 5055 (Complementary)
- AP(R)
- Start: 11322 End: 12179 (Complementary)
- bla gene-Ap(r) determinant
Enhancer (1 total)
-
- CMV Enhancer
- Start: 5583 End: 6003 (Complementary)
Misc. Binding Site (2 total)
-
- Left Homology Arm
- Right Homology Arm
Misc. Feature (4 total)
-
- HSVTK-polyA
- Start: 2850 End: 3108 (Complementary)
- loxP-2
Promoter Eukaryotic (1 total)
-
- Mus-PGK Promoter (correct)
- Start: 5068 End: 5576 (Complementary)
Replication Origin (2 total)
-
- ORI
- Start: 10511 End: 10511
- RNaseH cleavage point
- Replication Origin
- Start: 10511 End: 11099 (Complementary)
Overview of Seq ID No. 46 (upstream vector alternative, step 2, single lox)
Feature Map
CDS (3 total)
-
- NEO (+STOP) CDS
- Start: 3311 End: 4114 (Complementary)
- TK CDS (from VEC1198)
- Start: 4118 End: 5251 (Complementary)
- AP(R)
- Start: 11698 End: 12555 (Complementary)
- bla gene-Ap(r) determinant
Enhancer (1 total)
-
- CMV Enhancer
- Start: 5779 End: 6199 (Complementary)
Misc. Binding Site (2 total)
-
- Left Homology Arm
- Right Homology Arm
Misc. Feature (4 total)
-
- loxP-1
- HSVTK-polyA
- Start: 3046 End: 3304 (Complementary)
Promoter Eukaryotic (1 total)
-
- Mus-PGK Promoter (correct)
- Start: 5264 End: 5772 (Complementary)
Replication Origin (2 total)
-
- ORI
- Start: 10887 End: 10887
- RNaseH cleavage point
- Replication Origin
- Start: 10887 End: 11475 (Complementary)
Overview of Seq ID No. 47 (Downstream vector alternative, step 1, double lox)
Feature Map
CDS (3 total)
-
- NEO (+STOP) CDS
- Start: 3149 End: 3952 (Complementary)
- TK CDS (from VEC1198)
- Start: 3956 End: 5089 (Complementary)
- AP(R)
- Start: 11356 End: 12213 (Complementary)
- bla gene-Ap(r) determinant
Enhancer (1 total)
-
- CMV Enhancer
- Start: 5617 End: 6037 (Complementary)
Misc. Binding Site (2 total)
-
- Left Homology Arm
- Right Homology Arm
Misc. Feature (5 total)
-
- loxP-1
- HSVTK-polyA
- Start: 2884 End: 3142 (Complementary)
- loxP-2
Promoter Eukaryotic (1 total)
-
- Mus-PGK Promoter (correct)
- Start: 5102 End: 5610 (Complementary)
Replication Origin (2 total)
-
- Replication Origin
- Start: 10545 End: 11133 (Complementary)
The first vector pair is used to produce cells in which the entire J/cluster region is deleted.
The second vector pair is used to produce cells in which the entire J/C cluster region is deleted.
Example 5 Crossbreeding of Heavy Chain Single Knockout with Kappa Single Knockout Pigs To produce pigs that have both one disrupted Ig heavy chain locus and one disrupted Ig light-chain kappa allele, single knockout animals were crossbred. The first pregnancy yielded four fetuses, two of which screened positive by both PCR and Southern for both heavy-chain and kappa targeting events (see examples 1 and 2 for primers). Fetal fibroblasts were isolated, expanded and frozen. A second pregnancy resulting from the mating of a kappa single knockout with a heavy chain single knockout produced four healthy piglets.
Fetal fibroblast cells that contain a heavy chain single knockout and a kappa chain single knockout will be used for further targeting. Such cells will be used to target the lambda locus via the methods and compositions described herein. The resulting offspring will be heterozygous knockouts for heavy chain, kappa chain and lambda chain. These animals will be further crossed with animals containing the human Ig genes as described herein and then crossbred with other single Ig knockout animals to produce porcine Ig double knockout animals with human Ig replacement genes.
This invention has been described with reference to its preferred embodiments. Variations and modifications of the invention, will be obvious to those skilled in the art from the foregoing detailed description of the invention.