GAB1 INVOLVEMENT IN GLUCOSE HOMEOSTASIS REGULATION BY HEPATOCYTES

Abstract

to the regulation of glucose homeostasis by modulating the activity of Grb2-associated binder 1 (Gab1) in hepatocytes. This invention also provides for a method for identifying compounds capable of modulating the glucose homeostasis regulatory activity of Gab1. In one aspect, the invention provides a method for identifying a compound that can effectively modulate glucose homeostasis wherein Gab1 mediated MapK activity indicates that the candidate compound is an effective compound that modulates glucose homeostasis. In another aspect, the invention provides a method for identifying a compound that can effectively modulate the glucose homeostasis regulating activity of Gab1 wherein MAPK is activated to phosphorylate Serine residue 612 of IRS-1, indicating that the candidate compound is an effective compound that modulates glucose homeostasis. In another aspect of the invention is provided a method for diagnosing Gab1 related disorders.

Description

RELATED APPLICATIONS

[0001]This application is a continuation of U.S. patent application Ser. No. 10/987,384, filed on Nov. 12, 2004, which claims benefit of priority under 35 U.S.C. 119(e) of U.S. Provisional Application No. 60/519,358, filed Nov. 12, 2003. The disclosures of the above referenced applications are herein incorporated by reference in their entireties.

REFERENCE TO SEQUENCE LISTING

[0003]The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled BURNHAM031C1.TXT, created Sep. 10, 2010, which is 147 KB in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0004]This invention relates to the field of molecular biology and molecular medicine, and more specifically to proteins involved in blood glucose homeostasis and diabetes.

BACKGROUND

[0005]In humans, free glucose is present in the plasma and interstitial fluid at a concentration of approximately 80 mg per 100 ml. Blood glucose levels; however, are in a dynamic flux. Organs of the body remove the glucose from the blood for metabolic energy, while food intake loads glucose into the blood system. Under normal conditions, the body maintains a balance of blood glucose by absorbing excess glucose into many tissues.

[0006]A rise in blood glucose is normally followed by a rise in blood insulin. Insulin secretion is stimulated by many events associated with glucose intake. Primarily, insulin is secreted by the pancreas. High glucose concentration in the vicinity of the βcells of the pancreas is sensed by the glucose transporter GLUT-2 and is carried into the cells. The glucose is modified and begins a signal transduction cascade that results in insulin exocytosis.

[0007]Rising concentrations of insulin in the blood have an effect on three main tissues--liver, muscle and adipose tissue. Liver tissues play a central role in glucose homeostasis primarily orchestrated by insulin (Saltiel, A. R. & Kahn, C. R. Nature 414: 799-806 (2001), Michael, M. D. et al. Mol Cell 6: 87-97 (2000)), although it is not well understood how the insulin-elicited signals are tightly controlled in hepatocytes.

[0008]In general, insulin activates a four-subunit transmembrane receptor (insulin receptor) expressed on the surface of these tissue types. The activated insulin receptor phosphorylates and recruits different substrate adaptors such as the Insulin Receptor Substrate (IRS) family of proteins. IRS-1 and IRS-2 proteins are known to be positively required for relay of signals emanating from insulin receptor (Araki, E. et al. Nature 372: 186-90 (1994), Tamemoto, H. et al. Nature 372: 182-6 (1994), Withers, D. J. et al. Nature 391: 900-4 (1998)).

[0009]Tyrosine phosphorylated IRS displays binding sites for numerous signaling partners. Among them, PI3K has a role in insulin function mainly characterized by the activation of the Akt/PKB and the PKCζ cascades. These cascades are associated with glucagon synthesis and with glucose uptake. Glucose uptake is mediated by the translocation of glucose transport vesicles to the plasma membrane, which is regulated by numerous signal cascades including those discussed.

[0010]Grb2-associated binder 1 (Gab1) is part of a family of adaptor proteins recruited by a wide variety of receptor tyrosine Kinases, such as the insulin receptor. Upon stimulation of the receptor by its cognate ligand, Gab is recruited to the plasma membrane, undergoes phosphorylation and functions as a multi protein assembly center Gab1 shares structural and functional homology with the IRS family of proteins, possessing a PH domain at the N-terminus, multiple tyrosine phosphorylation sites and proline-rich motifs for entertaining SH2- and SH3-containing proteins (Holgado-Madruga, M., et al. Nature 379: 560-4 (1996), Gu, H. & Neel, B. G Trends Cell Biol 13: 122-30 (2003)). Homozygous Gab1 mutant mice are embryonic-lethal with the phenotype revealing an essential role of Gab1 in promoting signals from epidermal growth factor (EGF) and hepatocyte growth factor (HGF), etc (Sachs, M. et al. J Cell Biol 150: 1375-84. (2000), Itoh, M. et al. Mol Cell Biol 20: 3695-704 (2000)).

[0011]The insulin signalling pathway is critical for the regulation of intracellular and blood glucose levels (glucose homeostasis), and dysregulation of glucose homeostasis is indicated in numerous disorders. For example, diabetes mellitus is a group of diseases characterized by high levels of blood glucose resulting from defects in insulin production, insulin action, or both. There are two main forms of diabetes; type 1 and type 2. Type 1 patients are unable to produce insulin, and thus must receive exogenous insulin to survive. On the other hand, type 2 patients have at least partially preserved insulin production, but often are insulin resistant. Insulin resistance is caused by defects that may arise at the insulin receptor or post-insulin receptor levels. Post-insulin receptor defects often involve signal transduction proteins.

[0012]For the year 2003, the Center for Disease Control estimates that 17 million Americans have some type of diabetes, with an increasing prevalence during the last decade resulting in an incidence of about 1 million new cases per year. The prevalence and incidence of diabetes is even higher world wide, making diabetes a global health problem. Diabetes is the sixth leading cause of death in the United States.

[0013]The primary treatment for diabetes is the delivery of exogenous insulin via pumps and/or injection. Total annual costs for treating diabetes is $132 billion, with $92 billion attributed to direct medical costs.

[0014]Thus, there exists in the art a need to develop novel treatments for managing glucose homeostasis and blood glucose levels. There also exists in the art, a need to prevent the dysregulation of glucose homeostasis and blood glucose levels. Thus, there is a need in the art to better understand the insulin signaling pathway and Gab1.

SUMMARY OF INVENTION

[0015]In one embodiment, a method of identifying a modulator of Gab1 includes providing a first group comprising cells that express Gab1 protein and a second group comprising cells modified to lack Gab1 protein expression; contacting the first group and second group with a candidate agent; comparing the extracellular glucose concentration of the first and second groups after contact with the candidate agent to a baseline value indicated by the difference in extracellular glucose concentration between the first and second groups without contact with the candidate agent; and identifying the candidate agent as a modulator of Gab1 if a change in glucose concentration between the first and second groups is found after contact with the candidate agent compared to the baseline value.

[0016]In one aspect, the cells of the first and second groups comprise mammalian cells. In the same aspect, the cells of the first and second groups comprise murine cells. In the same aspect, the cells modified to lack Gab1 protein expression are from a transgenic mouse whose genome comprises a homozygous disruption of the endogenous Gab1 gene. In another aspect, the cells of the first and second groups are from the same organ type. In the same aspect, the organ type is liver.

[0017]In a further aspect, the providing and the contacting are performed in vitro. In an additional aspect, the providing and the contacting are performed in vivo. In the same aspect, the first and second groups are mice. In the same aspect, the second group is a transgenic mouse whose genome comprises a homozygous disruption of the endogenous Gab1 gene. In the same aspect, the method further includes administering a known amount of glucose to the mice. In the same aspect, the extracellular glucose concentration is measured as plasma glucose levels.

[0018]In a further aspect, the candidate agent is identified as a positive modulator of Gab1 if the change in glucose concentration is an increase. In yet an additional aspect, the candidate agent is identified as a negative modulator of Gab1 if the change in glucose concentration is a decrease.

[0019]In another embodiment, a transgenic mouse is provided whose genome includes a homozygous disruption of the endogenous Gab1 gene, wherein the disruption results in the functional inactivation of the Gab1 gene, and wherein the mouse exhibits hypoglycemia relative to a mouse whose genome comprises the functional endogenous Gab1 gene. In one aspect, the homozygous disruption is specifically in the hepatic cells of the mouse. In another aspect, the disruption in the Gab1 gene is a deletion of SEQ ID NO: 3. In the same aspect, the Gab1 gene is replaced with a replacement gene. In the same aspect, the replacement gene includes a neomycin resistance gene In the same aspect, the replacement gene further includes a thymidine kinase gene.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020]FIG. 1a: Generation of liver-specific Gab1 knockout mice: the targeting strategy. Genomic DNA fragments were cloned into the targeting vector as left, central and right arms. Three loxP sequences were marked by a black triangle (negative: HSV-TK; positive: PGK-NEO). Another negative selection marker, PGK-DT, was put outside of the right arm. The numbered boxes represent exons in the Gab1 gene. TK: thymidine kinase; NEO: neomycin; DT: diphtheria toxin.

[0021]FIG. 1b: Southern blot analysis. In the upper panel, genomic DNA was digested with BamHI and hybridized to the 32P-labelled 5' probe. Three properly targeted ES cell clones flox/+ (F/+) showed a 6 kb band for the Gab1flox allele and a 10 kb band for the wild-type allele. The lower panel shows a 24 kb band for the wt allele and a 12 kb band for Gab1 fox allele, detected by the 3' probe upon ApaI digestion of genomic DNA.

[0022]FIG. 1c: PCR with A and B primers on DNA extracted from a Gab1flox/flox mouse (+/+; F/F) and a LGKO mouse. The Gab1flox allele produces a 630 by fragment and the Gab1-allele produces a 150 by band. Deletion of the loxP-floxed sequences was detected only in the liver (L), but not in the tail (T), skeletal muscle (M), pancreas (P), brown adipose tissue (Ba), white adipose tissue (Wa), hypothalamus (H), pituitary gland (Pi), and kidney (K).

[0023]FIG. 1d: Immunoblot analysis of Gab1 protein expression in the liver, skeletal muscle, brain and white adipose tissue (Wa) isolated from mice of different genotypes: wild-type (WT), Albumin-Cre/+ (Cre/+), Gab1flox/flox (F/F), Alb-cre/+: Gab1flox/+ (Cre/+, F/+), and LGKO. Gab1 protein was barely detectable in the liver of LGKO mice.

[0024]FIG. 1e: Immunoblot analysis of Gab1 expression in liver and skeletal muscle of control (Ctl, Gab1flox/flox) and LGKO mice at the age of 2-month and 1-year, respectively.

[0025]FIGS. 2a and 2b: Body weights of LGKO mice. Solid bars are for the control and open bars for LGKO mice. No statistically significant difference was observed between the two groups using the Student's t test analysis. Values are the means±SEM. (FIG. 2a) Fed body weight of control and LGKO mice at the indicated ages and sex, N=19-24 for 2-month-old groups, N=6-25 for 6-month-old groups, N=7 to 19 for 1-year-old groups. (FIG. 2b) Fasting body weight of control and LGKO mice at the indicated ages, N=9-14 for 2-month-old groups, N=5-22 for 6-month-old groups, N=7 for each 1-year-old group. M: Male; F: Female.

[0026]FIG. 3a. Metabolic changes of LGKO mice. Blood glucose levels were measured on randomly-fed (Fed) or 16-hr fasted (Fast) male (M) or female (F) mice; N=8 to 39 for each group. Serum insulin levels were measured on fed and fasted male mice; N=10 to 13 for each group. *P<0.05; **P<0.01; ***P<0.001 for control versus LGKO. Values are the means±SEMs.

[0027]FIG. 3b. Metabolic changes of LGKO mice. Blood glucose and serum insulin during glucose tolerance test performed on 16-hr-fasted male mice; N=5 to 8 for each group. *P<0.05; **P<0.01; ***P<0.001 for control versus LGKO. Values are the means±SEMs.

[0028]FIG. 3c. Metabolic changes of LGKO mice. Blood glucose level during insulin tolerance test performed on randomly-fed male animals; N=6 to 10 per group. *P<0.05; **P<0.01; ***P<0.001 for control versus LGKO. Values are the means±SEMs.

[0029]FIG. 3d: In vivo insulin sensitivity measured by Glucose infusion rate. One-year-old male mice were used, N=8. *P<0.05; **P<0.01 for control versus LGKO. Values are the means±SEMs.

[0030]FIG. 3e: In vivo insulin sensitivity measured by IS-GDR at the infusion rate of 12 mU/kg/min. One-year-old male mice were used, N=8. *P<0.05; **P<0.01 for control versus LGKO. Values are the means±SEMs.

[0031]FIG. 3f: In vivo insulin sensitivity measured by basal (bas.) and clamped (cl.) hepatic glucose productions (HGP). One-year-old male mice were used, N=8. *P<0.05; **P<0.01 for control versus LGKO. Values are the means±SEMs.

[0032]FIG. 3g: In vivo insulin sensitivity measured by insulin suppression of HGP, as determined by euglycemic, hyperinsulinemic clamp analyses. One-year-old male mice were used, N=8. *P<0.05; **P<0.01 for control versus LGKO. Values are the means±SEMs.

[0033]FIG. 3h: Serum triglyceride levels were measured for 16 hr-fasted mice (N=8, for 2-month-old mice; N=12-13 for 1-year-old animals). Solid bars represent the control mice and open bars and squares represent the LGKO mice. *P<0.05; **P<0.01; ***P<0.001 for control versus LGKO, Student's t tests. Values are the mean±SEM.

[0034]FIG. 4a: Biochemical analysis of insulin signalling in LGKO mice. The phosphorylation levels of Akt at Ser473 (p-Akt) were quantified against Akt proteins amounts at different time-points in the liver and muscle. Shown are statistical data collected from 3-4 mice, by setting the value of the control at 2 min to 100, as well as a representative immunoblot. *P<0.05; **P<0.01;***P<0.001 for control versus LGKO. Values are the means±SEMs.

[0035]FIG. 4b: Insulin-induced tyrosine phosphorylation of IRβ in LGKO liver. Left panel is the statistical data of 3-4 experiments using different mice each time, by setting the control value at 2 min to 100. Right panel is a representative immunoblot result. Tyrosine phosphorylation of IRβ (PY-IR), normalized against IRβ level (IR), in the liver at 0, 2 or 5 min after insulin treatment (N=4). Relative tyrosine phosphorylation of IRβ (PY-IR), in skeletal muscle at 0, 4, 8 min of insulin treatment (N=3). Solid bars represent the Control (Ctl) mice and open bars LGKO mice. No statistical significances were found between the two groups using the Student's t test. Values are the means±SEMs.

[0036]FIG. 4c: Biochemical analysis of insulin signalling in LGKO mice. Upper graph: relative PY levels of IRS-1, -2, in the liver at 0 and 2 min for insulin treatment (N=3). Lower graph: relative amounts of p85α binding to IRS-1 & -2, in the liver at 0, 2 min of insulin treatment (N=2 to 3). *P<0.05; **P<0.01; ***P<0.001 for control versus LGKO. Values are the means±SEMs.

[0037]FIG. 4d: Deletion of Gab1 leads to enhanced insulin signalling through IRS-1, -2 in hepatocytes. Mice were injected with 5 U insulin (Ins) (+) or saline (-) as a control via vena cava and liver extracts were prepared 2 min after injection. IRS-1 (left panel) or IRS-2 (right panel) were immunoprecipitated from liver lysates with specific antibodies and immunoblotted with antibodies against p85α subunit of PI3K, phosphotyrosine or against IRS-1, IRS-2 as a loading control.

[0038]FIG. 4e: Biochemical analysis of insulin signalling in LGKO mice. Tyrosine phosphorylation of Gab1 in the liver were measured 2 min after vena cava injection with 5 U insulin (Ins) (+) or saline (-). Two mice were included in each group. *P<0.05; **P<0.01; ***P<0.001 for control versus LGKO. Values are the means±SEMs.

[0039]FIG. 4f: High levels of Gab1 tyrosine phosphorylation in hepatocytes induced by EGF or peroxovanadate. Tyrosine phosphorylation of Gab1 in the liver was measured 2 min after vena cava injection with EGF or peroxovanadate. Gab1 was immunoprecipitated from liver lysates with Gab1 antibody and immunoblotted with antibodies against phosphotyrosine or Gab1 as a loading control.

[0040]FIG. 4g: Biochemical analysis of insulin signalling in LGKO mice. Gab1 phosphorylation on the p-YXXM motifs and its association with p85α in response to saline solution, insulin (ins), EGF, or peroxovanadate. *P<0.05; **P<0.01; *P<0.001 for control versus LGKO. Values are the means±SEMs.

[0041]FIG. 4h: Biochemical analysis of insulin signalling in LGKO mice. Insulin-induced p-Erk1/2 and p-IRS-1S612 were shown together with Erk2 and IRS-1 blots as loading controls. *P<0.05; **P<0.01; ***P<0.001 for control versus LGKO. Values are the means±SEMs.

[0042]FIG. 4i: Biochemical analysis of insulin signalling in LGKO mice. Inhibition of insulin-induced p-Erk1/2 and p-IRS-1S612 by PD98059 MEK inhibitor (left) or U0126 MEK inhibitor (right). *P<0.05; **P<0.01; ***P<0.001 for control versus LGKO. Values are the means±SEMs.

[0043]FIG. 5: Normal expression levels of proteins involved in insulin signaling. Immunoblot analysis was performed with lysates from liver or skeletal muscle of control and LGKO mice. Liver and muscle extracts were prepared after saline (-) or insulin (Ins) stimulation for 2 and 4 min, respectively (+). Deletion of Gab1 in the liver did not affect the hepatic expression of IRS-1, IRS-2, p85α, Shp-2 or IRβ

[0044]FIG. 6: Inventors model for Gab1 activity (a negative feedback mechanism) in insulin signaling in the liver through the insulin mediated Gab1 protein signaling pathway. Gab1 promotes activation of MapK (Erk), leading to the phosphorylation of the Ser612 residue on IRS-1 and suppressing tyrosine phosphorylation on IRS-1. This, in turn, attenuates insulin-initiated signals flowing through IRS-1/PI3K/Akt pathway.

DETAILED DESCRIPTION OF THE INVENTION

[0045]Definitions Used Herein:

[0046]The term "site-specific recombination," refers to DNA transfer from a donor DNA or vector to an acceptor DNA or vector.

[0047]The term "lox sequence" refers to a nucleotide sequence which undergoes recombination (e.g., DNA cross-over and exchange) when catalyzed by a recombinase, such as Cre, Flp or another member of the Int family of recombinases (Argos et al. (1986) EMBO J. 5: 433). Suitable lox sequences include, for example, the lox sequences recognized by Cre recombinase, and the frt sequences recognized by Flp recombinase.

[0048]The term "recombinase" refers to any recombinase capable of catalyzing a site-specific recombination at a lox site. Suitable recombinases include, for example, Cre recombinase (Sauer et al. (1993) Methods in Enzymology 225: 898) and Flp recombinase (Buchholz et al. (1996) Nucl. Acids Res. 24:4256-4262; Buchholz et al. (1998) Nat. Biotechnol. 16:657-662).

[0049]The terms "acceptor DNA" and "acceptor vector," are used interchangeably herein and refer to any DNA or vector which, preferably, is capable of integrating into the genome of a cell. For example, the acceptor DNA or vector can be of viral origin, such as a retroviral vector or adeno-associated vector. Generally, the acceptor DNA or vector contains an exchange cassette (i.e., DNA which is replaced by DNA from the donor vector) and can also, optionally, contain a selectable (e.g., negative) marker gene.

[0050]The terms "donor DNA" and "donor vector" are used interchangeably herein and refer to any DNA or vector (e.g., circular plasmid DNA) containing DNA which is transferred to the acceptor DNA or vector via a recombinase-mediated exchange reaction. Generally, the donor DNA or vector comprises plasmid DNA and, optionally, also can contain a selectable (e.g., positive) marker gene.

[0051]Methods for preparing large libraries of compounds, including simple or complex organic molecules, metal-containing compounds, carbohydrates, peptides, proteins, peptidomimetics, glycoproteins, lipoproteins, nucleic acids, antibodies, and the like, are well known in the art and are described, for example, in Huse, U.S. Pat. No. 5,264,563; Francis et al., Curr. Opin. Chem. Biol. 2:422-428 (1998); Tietze et al., Curr. Biol., 2:363-371 (1998); Sofia, Mol. Divers. 3:75-94 (1998); Eichler et al., Med. Res. Rev. 15:481-496 (1995); and the like. Libraries containing large numbers of natural and synthetic compounds also can be obtained from commercial sources.

[0052]The number of different candidate compounds to test in the methods of the invention will depend on the application of the method. For example, one or a small number of candidate compounds can be advantageous in manual screening procedures, or when it is desired to compare efficacy among several predicted ligands, agonists or antagonists. However, it is generally understood that the larger the number of candidate compounds, the greater the likelihood of identifying a compound having the desired activity in a screening assay. Additionally, large numbers of compounds can be processed in high-throughput automated screening assays. Therefore, "one or more candidate compounds" can be, for example, 2 or more, such as 5, 10, 15, 20, 50 or 100 or more different compounds, such as greater than about 103, 105 or 107 different compounds, which can be assayed simultaneously or sequentially.

[0053]The present invention relates to the discovery that Gab1 protein is a negative regulator of the glucose homeostasis pathway. In the current discovery, Inventors have shown that when Gab1 is knocked-out in the liver in mammals, the mammal will display hypoglycemia and enhanced glucose tolerance. Inventors' showing leads to the discovery that Gab1 promotes action in the MapK (Erk) pathway, which in turn downregulates the IRS pathway, leading to hypoglycemia and enhanced glucose tolerance.

[0054]To determine how Gab1 functions in glucose homeostasis, Inventors generated a mouse model in which the Gab1 gene was specifically disrupted in the liver. Methods for generating a knock out mouse are generally well known in the art. (Brown, T. A., Gene Cloning and DNA Analysis, Ed. 4, Blackwell Science Press (2001)). Liver-specific Gab1 knockout (LGKO) mice were created to possess a conditional Gab1 knockout allele, a loxp-foxed allele of Gab1 (Gab1^flox). Two loxP sites were inserted into introns flanking exon 3 of the Gab1 gene using homologous recombination in embryonic stem (ES) cells (FIG. 1a). Methods and techniques for achieving efficient and stable site-specific DNA recombination using a recombinase/lox system, such as the Cre/lox system or the Flp/frt system, are well known in the art.

[0055]Genomic DNA fragments of Gab1 were isolated by screening a λDASHII mouse genomic library, (Stratagene, La Jolla Calif.), with a Gab1 cDNA fragment as a probe. The sequence of Gab1 is known and presented in a variety of databases, including Project Ensembl from The Wellcome Trust Sanger Institute. The Mouse Gab1 gene is listed herein as SEQ ID No.: 1. Similarly, the cDNA probe is listed herein as SEQ ID No.: 2, and is generated from a partial sequence of SEQ ID No.: 1. Based on the restriction map, (determined using the Flash Non-Radioactive Gene Mapping Kit, Stratagene, La Jolla Calif.), and partial sequencing of Gab1 genomic DNA fragments, a targeting construct was engineered using a triple-loxP construction system, wherein the genomic DNA fragments were cloned into the targeting vector as left, central and right arms. (See e.g., Jr-Wen Shui and Tse-Hua Tan, Genesis, 39:3, p217. See also, Wang, Xinhe, et al., PNAS, 99:26, 16881; and Zhu, Y. J. et al. Journal of Bio. Chem. 278 (3): 1986-1990; and Gainetdinov, R. R. et al., Neuron, 24, 1029 (1999); and Gainetdinov, R. R. et al., Neuron, 38, 291 (2003)). The Gab1 gene and the target construct are homologously recombined, resulting in three loxP sequences flanking the central arm and the negative/positive selection marker genes (negative: HSV-TK; positive: PGK-NEO), allowing for cell selection. (Dr. R. T. Premont (Duke University, Durham, N.C.) provided the triple-loxp vectors. See also Gainetdinov, R. R. et al., Neuron, 24, 1029 (1999); and Gainetdinov, R. R. et al., Neuron, 38, 291 (2003)). Another negative selection marker, PGK-DT, was put outside of the right arm. Exon 3 codes for amino acids 124-198 of the Gab1 protein and deletion of this exon leads to a frame-shift mutation and to the introduction of a new stop codon immediately. Exon 3 nucleic acids are listed at SEQ ID No.: 3. The recombinant construct DNA is then linearized using NotI (NEB, Inc., Beverly Mass., Catalog No.: R0189S) and introduced into R1 ES cells using electroporation.

[0056]ES cell colonies resistant to the Geneticin Antibiotic G418 (Sigma, St. Louis, Mo., Cat No.: G-9516) were screened for homologous recombination by PCR using a primer in the neo cassette and a primer outside of the right arm. The Gab1^flox allele and the Gab1 knockout (Gab1-) allele can be distinguished by PCR analysis using primers A (SEQ ID No.: 4) and B (SEQ ID No.: 5) that produces DNA fragments at 670 by and 200 bp, respectively. Results were confirmed using Southern Blot analysis with a 5' probe (Southern Blot techniques are well known in the art. See for example, Maniatis et al., Molecular Cloning: A Laboratory Manual (1989), 2nd Ed., Cold Spring Harbor, N.Y.) (FIG. 1b). Briefly, genomic DNA was digested with ApaI or BamHI and hybridized to the ³²P-labelled 3' probe or 5' probe. Three properly targeted clones (F/+) show one 6 kb band for the Gab1^flox allele and another 10 kb band for the wild-type allele. The lower panel of FIG. 1b shows a 24 kb band for the wild-type allele and 12 kb for Gab1^flox allele, detected by the 3' probe upon ApaI digestion of genomic DNA. Gene Targeting techniques are well known to those of ordinary skill in the art, and are described in the literature. (See e.g., Joyner, A., Gene Targeting: A Practical Approach, The Practical Approach Series, edited by B. D. Hames, Oxford University Press 2^nd Ed., Oxford 1999.)

[0057]ES cells were then transiently transfected with a CMV-Cre construct (pBS185; Invitrogen Corp., Carlsbad, Calif.). Cells containing the CMV-cre construct were selected in fialuridine (FIAU)-containing medium against the TK-neo cassette. (FIAU, Moravek Biochemicals, Brea Calif., Cat. No. M-251.) Surviving clones were further screened by PCR analysis using primers A and B (SEQ ID No.: 4 and SEQ ID No.: 5, respectively), described above, thereby allowing for isolation of ES cell clones with a loxP-floxed Gab1 allele (Gab1_flox). The engineered ES cells were injected into C57BL/6 blastocysts and chimeric animals were obtained. Germline transmission of the Gab1_flox allele was obtained from two independent ES cell clones isolated from the screen.

[0058]Generation of liver-specific Gab1 knockout (LGKO) mice:

[0059]The Gab1^flox allele was generated in a 129/Sv background and mutant mice were crossed with wild-type C57BL/6 animals for 4 generations to acquire the C57BL/6 background. To generate liver-specific Gab1 knockout (LGKO) mice, Gab1^flox/+ mice were crossed with Albumin-Cre transgenic mice (C57BL/6-TgN (Alb-cre)21Mgn). (See e.g., Michael, et al. Mol Cell 6: 87-97 (2000); and Postic et al., J Biol Chem 274, 305-15 (1999)). LGKO mice (Gab1^flox/flox:Alb-cre/+) were born with the expected Mendelian frequency, morphologically indistinguishable from their wild-type (WT) littermates. All animals were housed in virus-free facility on a 12 hr light/dark cycle and were fed with a standard mouse food. All protocols for animal use and euthanasia were approved by the institutional animal committee.

[0060]To determine the efficiency of exon 3 deletion in the bred mice, PCR analysis was performed on genomic DNA extracted from the tails and liver of weaned mice. Genotyping PCR used the following primers: Cre primers GCC TGC ATT ACC GGT CGA TGC AAC GA, (SEQ ID No.: 6) GTG GCA GAT GGC GCG GCA ACA CCA TT (SEQ ID No. 7) and Gab1.sup.Flox/wild-type primers: GGT GAA TCG ACG GGT GCT TGT GA, (SEQ ID No.: 8) CAG ATT GGC CTT GAA CTG GTA AG (SEQ ID No.: 9). The PCR program used was: 94° C. for 5 min; 39 cycles at 94° C. for 30 s, 58° C. for 30 s, 72° C. for 45 s; 72° C. for 5 min. PCR analysis of the LGKO mice showed an efficient deletion of exon 3 in the liver but not other organs such as skeletal muscles, pancreas, brown and white adipose tissue, hypothalamus, pituitary and kidney. (FIG. 1c).

[0061]The efficiency of Gab1 deletion in LGKO hepatocytes was further demonstrated by immunoblot analysis.

[0062]Male mice at age of 2 month were fasted for 16 hr and were anaesthetized intraperitoneally with Avertin (0.015 ml/g) (2,2,2 tribromoethanol, purchased at Aldrich Cat. No.: T4,840.2). Either 5 U of human insulin (Humalin, Eli Lilly & Company) or 20 microliters saline solution was injected into the inferior vena cava. Liver and muscle tissue were harvested as indicated above, and quickly frozen in liquid nitrogen. Frozen liver and muscle samples were homogenized in a Dounce apparatus in Protein lysis buffer (50 mM Hepes pH7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM EGTA, 100 mM NaF, 10 mM sodium pyrophosphate, 10 mM Na3VO4, 10 micrograms/ml leupeptin, 10 ug/ml aprotinin, 2 mM PMSF). After 30 min incubation on ice, tissue lysates were clarified by centrifugation at 37,000 g for 1 hr at 4° C. Protein concentration was determined using a kit from Bio-Rad (Bio-Rad Protein Assay, Hercules, Calif., Catalogue No.: 500-0001).

[0063]For immunoprecipitation, 1.5 mg of total protein was incubated with 4-5 μl antibody for 2-4 hr at 4° C. and the mixture was further incubated for 2-4 hr at 4° C. with protein A/G plus agarose (Santa-Cruz Biotechnology, Santa Cruz, Calif., Cat No.: sc-2003). The bead-bound complex was washed three times with cold HNTG buffer (20 mM Hepes, pH7.5, 150 mM NaCl, 10% glycerol, 0.1% Triton X-100) and resuspended in 10 μl SDS sample buffer. For immunoblot analysis, lysates were separated on SDS-polyacrylamide gels, transferred to a nitrocellulose membrane, and blotted with primary antibodies as indicated. Specific signals were detected by enhanced chemiluminescence (ECL analysis kit; Amersham Corp.) following blotting with horseradish peroxidase-conjugated secondary antibodies. Antibody against Gab1 was produced by injection of rabbits with purified glutathione S-transferase fusion protein of Gab1 following standard procedures. Antibodies to phospho-Akt (serine 473), Akt, phospho-p44/42 Erk, Phospho-(Tyr) p85 of PI3K, phospho-IRS-1 (Ser612) were obtained from Cell Signaling Technologies (Beverly Mass.). Antibodies to p85α, p110α, IRβ, Erk1/2 were obtained from Santa Cruz Biotechnology (Santa Cruz, Calif.). Anti-phosphotyrosine antibody was purchased from Upstate Biotechnology Inc. (Charlottesville, Va.). IRS-1 and IRS-2 antibodies were kindly provided by Joslin Diabetes Center, Boston, Mass., and are further available from Upstate--Cell Signaling Solutions (Charlottesville, Va., Catalog Nos.: 06-248 and 06-506). Those of skill in the art will readily prepare antibodies to an antigen using well known techniques (See e.g., Harlow, Ed, Lane, David, Antibodies: A Laboratory Manual, 1988, Cold Springs Harbor Press). Blots were scanned and signals were quantified using IMAGEQUANT.sup.®. software. Statistical analysis of the data was performed using a two-tailed unpaired t-test, expressing values as mean±SEM.

[0064]As seen in FIG. 1d, the efficiency of Gab1 deletion was higher than 90% in LGKO liver, but Gab1 expression was not changed in skeletal muscle, brain or white adipose tissue. Gab1 protein was barely detectable in the liver of LGKO mice (Gab1^flox/flox Alb-Cre/+), but Gab1 expression was not changed in the liver of Gab1.sup.+/+:+/+ (wt); Gab1.sup.+/+:Alb-Cre/+ (Cre/+); Gab1^flox/flox:+/+ (F/F); Gab1. sup.flox/+:Alb-cre/+ (Cre/+, F/+) mice. Stable deletion of Gab1 was detected in young adult mice (2 months) as well as in aged 1-year old mice (FIG. 1e). Deletion of Gab1; however, did not affect normal development and morphology of the liver. The ratio of liver versus body weight of mutant mice was normal (WT: 5.21±0.22%, N=6; LGKO: 5.23±0.13%, N=9). The body weight for mutant mice was also similar to the wild-type over a one-year period in both fed and fasted mice. (FIGS. 2a and 2b).

[0065]Glucose Homeostasis and LGKO Mice:

[0066]To determine the effects of the Gab1 knockout on glucose homeostasis a series of metabolic and biochemical studies were performed.

[0067]Blood glucose levels of both WT and LGKO mice were measured using whole venous blood and an automatic glucose monitor (e.g., One Touch Basic, Lifescan, Milpitas, Calif.). Surprisingly, the results of these studies revealed that LGKO animals are hypoglycaemic under both fed and fasted status when blood glucose levels are measured on sex- and age-matched mice (FIG. 3a). Selective deletion of hepatic Gab1 led to reduced blood glucose levels, ranged from 9.1 to 28.9%, in comparison between both fed and fasted states for gender- and age-matched mice (FIG. 3a). For example, in comparison to control mice, the blood glucose concentrations in fasted male LGKO mice at the age of 2-month and 1-year were reduced by 19% (P=0.006) and 28.9% (P=0.009), respectively. Significant decrease of blood glucose levels was also detected in fed female LGKO animals (FIG. 3a). Consistently, serum insulin levels in fed and fasted LGKO male mice were significantly lower than control littermates when measured at 2-month (Fed: P=0.0192; Fasted: P=0.0275) or 1-year (Fed: P=0.0356; Fasted: P=0.0171) (FIG. 3a). This result strongly suggests a negative regulatory role of Gab1 in insulin-controlled glucose homeostasis.

[0068]To further determine the role of Gab1 in glucose homeostasis, Inventors performed a glucose tolerance test (GTT). Glucose tolerance tests were performed intravenously (IGTT), although the test can be performed orally (OGTT). In the current discovery, mice were fasted for 16 hours, and then given a solution containing a known amount of glucose via intra-peritoneal (IP) injection (2 g glucose per 1 kg body weight). Blood was obtained before IP injection of the glucose solution (time point 0), and was drawn again at 15, 30, 60 and 120 min (time point 15, 30, 60 and 120, respectively). Following intraperitoneal glucose injection, LGKO mice exhibited significantly reduced plasma glucose levels for each of the sampling time-points during the 120-minute glucose tolerance test, GTT (FIG. 3b); and thus the area under the glucose curve was significantly decreased (72% of control at 2-month; 59% at 1 year) for LGKO animals as compared to controls. In addition, the insulin response to the glucose load, measured at 60 and 120 min during the GTT, was significantly diminished in LGKO animals, with a decrease in the area under the curve by 44% (FIG. 3b). Thus, selective deletion of hepatic Gab1 significantly improved glucose tolerance. In addition, the lower glucose response curve, in spite of significantly diminished insulin levels, also implicates enhanced insulin sensitivity.

[0069]To assess the potential difference in peripheral glucose disposal between the genotypes of mice, we first performed in vivo insulin tolerance tests. Insulin tolerance tests (ITT) were performed on randomly fed animals via IP delivery of a bolus of insulin (1 U insulin per kg of body weight), (Humulin, Eli Lilly and Company, Indianapolis, Ind. 46285). Blood glucose levels were measured at time point 0, 15, 30 and 60 minutes after intra-peritoneal injection of human insulin using Lifescan's One Touch system. Serum insulin levels were measured by ELISA, using rat insulin as a standard (Cat#: INSKR 020, Crystal Chem, Inc. Downers Grove, Ill. 60515). Serum triglyceride levels were measured by Animal Care Program, Diagnostic Laboratory, University of California, San Diego. As seen in FIG. 3c, insulin's ability to reduce circulating glucose levels was similar between the genotypes at both ages. Since the insulin tolerance test is a relatively crude technique for assessment of insulin stimulated glucose disposal, the more accurate glucose clamp technique was used to quantitatively assess overall in vivo insulin action, quantify the rates of glucose disposal and hepatic glucose production.

[0070]Briefly, at 1 year of age each mouse was implanted with two catheters in the right jugular vein which were tunneled subcutaneously, exteriorized at the back of the neck, and encased in silastic tubing. Four days after surgery, animals were fasted for 6 hours and glucose turnover was measured in the basal state and during a hyperinsulinemic-euglycemic clamp. Following basal sampling (approximately 90 min), a constant infusion (5.micro.Ci/h) of [3-3H] D-glucose (NEN Life Science Products Inc.) was infused into one of the jugular cannulas. One hour after the start of the tracer infusion, a second basal blood sample was taken for measurement of glucose concentration and tracer specific activity as previously described 28. Following this, regular human insulin (12 mUkg-1min-1, Novolin R; Novo Nordisk Pharmaceutical Industries Inc.) combined with [3-3H] D-glucose (5.micro.Ci/h) was infused. Steady state was achieved by 80 minutes and held for the duration of the 120 minutes clamp. Following the steady state period (minimal of 30 minutes) at the end of the clamp, a final blood sample was taken for measurement of glucose turnover. Plasma glucose concentration was measured with a HemoCue glucose analyzer (Hemocue Inc., Lake Forrest, Calif. 92630). Circulating plasma human insulin following the clamp was measured using a radioimmunoassay kit (Linco Research, St. Charles, Mo. 63304, RIA Kit, Catalog No.: MENDO-75K). Plasma glucose specific activity was measured after deproteinization with barium hydroxide and zinc sulfate as previously described by our laboratory. Hepatic glucose production (HGP) and glucose disposal rate (GDR) were calculated at basal and during the 30-minute steady state portion of the glucose clamp. Tracer determined rates were quantified using the Steele equation for steady state conditions. Comparisons between the two groups were conducted using analysis variance (ANOVA), and values were presented as means±SEMs. Clamp techniques are well know in the art. (Miles, P. D., Barak, Y., He, W., Evans, R. M. & Olefsky, J. M. Improved insulin-sensitivity in mice heterozygous for PPAR-gamma deficiency. J Clin Invest 105, 287-92 (2000); Revers, R. R., Fink, R., Griffin, J., Olefsky, J. M. & Kolterman, O. G. Influence of hyperglycemia on insulin's in vivo effects in type II diabetes. J Clin Invest 73, 664-72 (1984); Steele, R. Influences of glucose loading and of injected insulin on hepatic glucose output. Ann N.Y. Acad Sci 82, 420-30 (1959).)

[0071]Both groups of animals were clamped at the 6-h fasting blood glucose values, 125±2.4 mg/dl. The exogenous glucose infusion rate (GIR) required to maintain euglycemia during the glucose clamp was increased by ˜30% (P=0.0016) in LGKO mice (FIG. 3d). Similar to results for the insulin tolerance test (FIG. 3c), no significant difference was observed between the genotypes with respect to insulin's ability to stimulate glucose disposal into skeletal muscle (IS-GDR, FIG. 3e). However, we did detect more marked suppression of hepatic glucose production (HGP) from basal during the clamp in LGKO mice (FIGS. 3f and 3g), revealing enhanced hepatic insulin sensitivity.

[0072]Glucose is converted to fat in the liver, and triglycerides are released in blood by the liver. The amount of fasting triglycerides was slightly lower for both the 2-month-old (14.8% reduction) and 1-year-old (22.7% reduction) knockout mice (FIG. 3h). At 2-months-old, the difference was not significant (P=0.41), whereas at one-year-old of age, the difference was statistically significant (P<0.05). Thus, Gab1 deletion in the liver also changed hepatic lipid metabolism.

[0073]Inventors' have discovered that hypoglycemia and enhanced glucose tolerance in LGKO is related to the Gab1 actions in the liver. Gab1 promotes the Erk pathway in attenuating insulin-elicited signals through the IRS signalling pathway. Thus, Gab1 deficiency in hepatocytes removes Gab1 mediated attenuation of IRS, resulting in increased basal and insulin-stimulated Akt/PKB activity.

[0074]To investigate the molecular basis for hypoglycemia and improved glucose tolerance, we examined the activity of Akt/PKB kinase, a critical enzyme in insulin signaling, by measuring levels of phospho-Akt (p-Akt) in both liver and muscle of wild-type and LGKO mice. Tissue lysates were made from the liver at 0, 2 and 5 min after insulin administration, and muscle lysates were prepared at 0, 4 and 8 min following insulin injection (FIG. 4a), and lysates were detected using immunoblot techniques as described hereinabove. Both basal and insulin-stimulated levels of p-Akt were higher in LGKO liver than that in wild-type, without a change in protein expression levels of Akt. Notably, elevated Akt activity was detected in the LGKO liver only, with no difference observed in skeletal muscle. Immunoblot and other techniques for measuring the level of protein in tissue samples are well known in the art.

[0075]Inventors measured the tyrosine phosphorylation status of the beta subunit of insulin receptor (IRβ) and observed its normal activation in LGKO liver as well as in muscle (FIGS. 4b and 4c). The IRβ expression level was not changed either (FIG. 5). Therefore, the increased Akt activity can not be attributed to a change in IRβ activation or expression but is rather caused by a downstream event.

[0076]Inventors then examined the protein amounts of IRS-1, IRS-2, Shp-2, and the p85α subunit of PI3-kinase in both liver and muscle, and found no change in their expression in LGKO mice (FIG. 5). However, using antibodies specific for the phosphorylated form of IRS-1 or IRS-2, Inventors discovered that enhanced tyrosine phosphorylation levels of both IRS-1 and IRS-2 were detected in the LGKO liver with or without insulin stimulation (FIGS. 4c and 4d). IRS-1 tyrosine phosphorylation was the most affected, with a 2.0 fold increase at the basal level and a 1.4 fold increase after insulin stimulation, whereas it was about 1.4 fold improved at both conditions for IRS-2. Consistently, higher amounts of p85 were detected in complex with IRS-1 and IRS-2 in Gab1-deficient hepatocytes, under both control and insulin-stimulated status (FIGS. 4c and 4d). Thus, Gab1 acts to attenuate insulin-triggered signals going through both IRS-1 and IRS-2, and deletion of Gab1 in the liver leads to augmented activation of the IRS proteins, which results in promotion of the PI3K/Akt pathway. The binding of IRS-1, -2 to IRβ is not affected by Gab1 deletion (FIG. 4d), excluding the possibility of a competition between IRS and Gab1 for binding IRβ Unlike the IRS proteins, Gab1 is weakly tyrosine-phosphorylated following insulin stimulation (FIGS. 4e, 4f and 4g), despite high levels of Gab1 phosphorylation being detected following induction by injection of EGF or peroxo-vanadate (an agent known to induce tyrosine phosphorylation). Neither phosphorylation of the p85 binding motif on Gab1 (p-YXXM), nor association of Gab1 with p85 was detected in insulin treated liver (FIG. 4g), suggesting that Gab1 may not be found directly involved in insulin stimulated pI3K/Akt pathway. Instead it appears that Gab1 has a negative effect on insulin stimulated IRS activation and thus deletion of Gab1 in the liver leads to augmented IRS tyrosine phosphorylation. This observation is contrary to what would be expected if Gab1 was directly involved in modulation of PI3K/Akt pathway in response to insulin.

[0077]Those of ordinary skill in the art are familiar with a variety of techniques for detecting phosphorylation of an amino acid residue within a protein or peptide fragment. Briefly, and by way of example only, wild-type mice were injected via vena cava with a saline solution (control), insulin (5 U), EGF (100 μg), or peroxo-vanadate. Liver extracts were prepared 2 min after injection; immunoprecipitated with Gab1 antibody; and immunoblotted with antibodies against either phosphotyrosine (PY), Gab1, phospho-(Tyr) p85 PI3K (p-YXXM), or p85. Peroxo-vanadate solution was prepared following a procedure published previously (S. J. Ruff, K. Chen, S. Cohen, J Biol Chem 272, 1263-7 (Jan. 10, 1997)).

[0078]Vena cava injection of insulin dramatically induced the activation of extracellular signal-regulated kinase (Erk) in the liver in vivo (FIG. 4h). However, the insulin-stimulated Erk activity was abolished in Gab1 deficient hepatocytes (FIG. 4h). This result suggests that Gab1, rather than IRS protein, plays a critical role in mediating insulin-stimulated activation of the Erk pathway in hepatocytes. It was previously reported that serine phosphorylation of IRS-1 by Erk down regulated its tyrosine phosphorylation and association with p85 ((Mothe, I. & Van Obberghen, E. J Biol Chem 271: 11222-7 (1996), De Fea, K. & Roth, R. A. Biochemistry 36: 12939-47 (1997), De Fea, K. & Roth, R. A. J Biol Chem 272: 31400-6 (1997)). Using an antibody specific for phospho-serine 612 (p-IRS-1.sup.S612), Inventor detected insulin-induced phosphorylation of IRS-1 on this amino acid residue, a known negative regulatory site on IRS-1 for insulin signaling (De Fea, K. & Roth, R. A. Biochemistry 36, 12939-47 (1997)). Notably, this serine-phosphorylation event was abolished in Gab1-deficient hepatocytes (FIG. 4h). Consistently, injection of MEK inhibitors (PD98059 or U0126. Cell Signaling Technologies, Beverly, Mass., Cat. Nos.: 9900S and 9903, respectively) also attenuated Erk activation as well as phosphorylation of IRS-1 on Ser612 (FIG. 4i).

[0079]Inventors have discovered and herein disclosed that Gab1 is the primary mediator for insulin-stimulated Erk activation, which leads to serine phosphorylation of IRS-1 and attenuation of its tyrosine phosphorylation. This, in turn, results in down-regulation of the IRS/PI3K/Akt pathway. This negative feedback loop in hepatocytes is a critical molecular mechanism for glucose homeostasis, as controlled by insulin (FIG. 6). Interestingly, the negative regulatory role of Gab1 in glucose metabolism is connected with its positive effect in mediating cell mitogenesis via the Erk pathway. Thus, pharmaceutical interference of the Gab1 activity in insulin signaling not only reduces blood glucose levels but also may suppress unwanted mitogenic activities in the liver.

[0080]Dysregulation of the glucose homeostasis pathway can lead to numerous disorders ranging from altered blood pressure to diabetes. Diabetes mellitus is the most common public health problem worldwide, affecting over 5% of the population in western countries. Ninety-five percent (95%) of the cases are classified as type II or non-insulin-dependent diabetes mellitus, which results from resistance to insulin activity (Kahn, B. B. & Rossetti, L. Nat Genet 20, 223-5 (1998), Taylor, S. I. Cell 97, 9-12 (1999)). To this end, search for negative regulators of the glucose homeostasis pathway is of particular interests in development of therapeutic strategies for related diseases.

[0081]Through Inventor's novel discovery of the Gab1 signalling pathway, Gab1 presents as an attractive target for addressing these goals. The negative regulatory role of Gab1 in glucose homeostasis is connected with its positive effect in mediating cell mitogenesis via the Erk pathway. Disruption of the Gab1 activity in insulin signalling may not only reduce blood glucose level but also may suppress unwanted mitogenic activities in the liver, thereby preventing hepatic carcinogenesis.

[0082]Thus, Inventors have uncovered a novel balancing mechanism for control of insulin signal strength in liver via the actions of Gab1. Inventors' discovery leads to a novel method for discovering new therapeutic modulators for treating type II diabetes mellitus, and other regulatory disorders of the glucose homeostasis pathway. Inventors' discovery also leads to a novel method for diagnosing the origin of disorders related to glucose homeostasis dysregulation, and developing specific treatments therefore.

EXAMPLES

[0083]While the present invention has been described in some detail for purposes of clarity and understanding, one skilled in the art will appreciate that various changes in form and detail can be made without departing from the true scope of the invention.

[0084]High Throughput Screening techniques are well known in the art and applicable to methods using Inventors' discovery. The number of different candidate compounds used to test in the methods of the invention will depend on the application of the method For example, one or a small number of candidate compounds can be advantageous in manual screening procedures, or when it is desired to compare efficacy among several predicted ligands, agonists or antagonists. However, it is generally understood that the larger the number of candidate compounds, the greater the likelihood of identifying a compound having the desired activity in a screening assay. Additionally, large numbers of compounds can be processed in high-throughput automated screening assays. Therefore, "one or more candidate compounds" can be, for example, 2 or more, such as 5, 10, 15, 20, 50 or 100 or more different compounds, such as greater than about 103, 105 or 107 different compounds, which can be assayed simultaneously or sequentially

[0085]The following non-limiting examples are useful in describing Inventor's discovery, and are in no way meant to limit the current invention. Those of ordinary skill in the art will readily adopt the underlying principles of Inventor's discovery to design a variety of screening assays without departing from the spirit of the current invention.

Screening Assay One

[0086]A first screening assay takes advantage of the methods and procedures described above. In this example, LGKO and WT mice are generated using the previously described procedures. Blood glucose levels, Glucose tolerance tests and Insulin tolerance tests are performed at time point 0, as described. The mice are then given a bolus of glucose. The control group comprises both WT mice and the LGKO mice, which receive the bolus of glucose alone. Similarly, the test group comprises WT and the LGKO mice; however, the test group receives a bolus of glucose and one or more candidate compounds. A candidate compound can be a naturally occurring macromolecule, such as a polypeptide, amino acid, nucleic acid, carbohydrate, lipid, or any combination thereof. A candidate compound also can be a partially or completely synthetic derivative, analog or mimetic of such a macromolecule, or a small organic molecule prepared by combinatorial chemistry methods. Candidate compounds can be given concurrently with the bolus of glucose or at a time point before or after the administration of said bolus of glucose. The Blood glucose levels, Glucose tolerance tests and Insulin tolerance tests are again performed, this time at time points 15, 30, 60 and 120, as described.

[0087]Measurements received from the control group establish baseline glucose levels, glucose tolerance and baseline insulin tolerance. As described above, there is a difference in the blood glucose levels and in glucose tolerance, but not insulin tolerance, between the WT and LGKO mice, thus the control group will establish data parameters for mice with functional Gab1 and with a knockout Gab1.

[0088]The test group will receive a glucose bolus and one or more candidate compounds. Each candidate compound will be tested in WT and LGKO mice. Candidate compounds that modulate the glucose homeostasis pathway are useful in treating the conditions associated with dysregulation of said glucose homeostasis pathway. For example, LGKO mice receiving a candidate compound that increases blood glucose levels reveal an ideal candidate modulator for treating hypoglycemia. Similarly, WT mice receiving a candidate compound that decreases blood glucose levels reveal an ideal candidate modulator for treating hyperglycemia. Glucose tolerance modulators are also discovered using this screening method.

[0089]Those of skill in the art will readily use Inventor's disclosure and will design numerous animal models to screen candidate compounds for modulators of the glucose homeostasis pathway. Such uses are all within the spirit of the current invention.

Screening Assay Two

[0090]In a second example, test agents are screened to see if said test agent is capable of modulating Gab1 mediated Erk1/2 activity. Those of ordinary skill in the art will readily uncover this same information using a variety of methods; however, in this example hepatic cell lines containing the Gab1 mediated glucose homeostasis pathway are used.

[0091]Briefly, hepatic cells are plated in a multi-well cell culture plate containing 96 wells, 384 wells, 1594 or other commercially available well numbers, and are incubated according to well known procedure. Otherwise identical wells are then either exposed to one or more candidate compounds in a glucose solution (test wells) or are exposed to glucose solution alone (control wells). A candidate compound can be a naturally occurring macromolecule, such as a polypeptide, amino acid, nucleic acid, carbohydrate, lipid, or any combination thereof. A candidate compound also can be a partially or completely synthetic derivative, analog or mimetic of such a macromolecule, or a small organic molecule prepared by combinatorial chemistry methods.

[0092]The wells are immediately and incrementally assayed for glucose concentration. In this situation, the response of the test cell to a candidate compound is compared to the response (or lack of response) of the control cell to the same compound under substantially the same reaction conditions. Candidate compounds that reduce the level of glucose in the test well as compared to the control are considered to negatively modulate Gab1 mediated Erk1/2 activity, thereby leading to a hypoglycemic environment. Conversely, candidate compounds that result in a higher level of glucose in the test well as compared to the control are considered to positively modulate Gab1 mediated Erk1/2 activity, thereby leading to a hyperglycemic environment.

[0093]Candidate compounds shown to modulate Gab1 mediated Erk1/2 activity are useful in treating the conditions associated with dysregulation of the glucose homeostasis pathway.

Screening Assay Three

[0094]In an alternative embodiment of Screening Assay Two, hepatic cell lines comprise either a wild type Gab1 or a Gab1 knockout. In this embodiment, the assay is performed as is Screening Assay Two: cells are plated in wells; the wells are exposed to glucose alone (control well) or glucose and one or more candidate compounds (test wells); and subsequent glucose concentration data is acquired.

[0095]In this embodiment, the control wells present data for the Gab1 mediated glucose homeostasis pathway wherein either Gab1 is functional or wherein Gab1 is knocked out. Candidate compounds that cause a decrease in glucose concentration in the wild type wells function to modulate the Gab1 mediated glucose homeostasis pathway in a manner similar to when Gab1 is knocked out. These modulators are useful for treating hyperglycemia. Conversely, candidate compounds that cause an increase in glucose concentration in the LGKO wells function to modulate the Gab1 mediated glucose homeostasis pathway in a manner similar to when Gab1 is functionally present.

Screening Assay Four

[0096]In a fourth example, candidate compounds are screened to determine whether said candidate compound is capable of modulating Gab1 wherein said screening method includes (a) contacting Gab1 under conditions suitable to promote MapK activation by insulin; (b) measuring the activity of insulin-stimulated MapK; (c) contacting Gab1 with a candidate compound; and (d) determining the ability of the candidate compound to modulate glucose homeostasis, where modulation of insulin-stimulated MapK activity indicates that the candidate compound is an effective compound that modulates glucose homeostasis.

[0097]For example, such a screening method can include a hepatic cell line comprising the Gab1 signaling pathway. The hepatocytes are plated in a cell culture well under suitable conditions. Such cell culture wells may be part of a multi-well plate having 96-wells, 384-wells or any number of wells commercially available. Those of skill in the art will readily adapt the current example to high throughput screening. Such applications are anticipated by the current disclosure.

[0098]The wells are designated as either negative control (cell media only); positive control (cell media and insulin) or test wells type A (cell media, insulin and candidate compound) and test wells type B (cell media and candidate compound). The cells are incubated under suitable conditions along with negative control media, positive control media, type A media or type B media. Following incubation, the cells are lysed at the optimal time and the extracts are assayed for MapK (Erk1/2) activity using immunoblot techniques. An antibody specific for phospho serine 612 of IRS-1 is used to detect insulin mediated activity of Erk1/2 t. In short, wells are stained for IRS-1 phosphorylation using p-IRS-1.sup.Ser612 antibodies followed by Cy3 conjugated anti-goat IgG (Cell Signaling Technologies, Beverly, Mass.). Fluorescent images can be collected and analyzed using, for example, MRC-1024 MP laser-scanning confocal microscope, and the images then compared.

[0099]The phosphorylation of IRS-1 in the presence of a candidate compound is compared to the control wells. Data acquired from the control wells establishes the degree of Gab1 mediated glucose homeostasis pathway activity in the presence and absence of insulin. Data from the test wells can be compared to the control wells. Candidate compounds causing an increase or decrease in insulin mediated activity of Erk1/2, as compared to control wells, determined by relative levels of phosphorylation at Ser612 residue of IRS-1, are useful as modulators of the Gab1 mediated glucose homeostasis pathway. Furthermore, the action of these modulators is further elucidated in that those that activate the Gab1 pathway in the absence of insulin are useful for treating insulin production and/or release mediated disorders, while those that act in the presence of insulin are useful for disorder occurring despite the presence of insulin. Modulators that increase IRS-1 phosphorylation are useful for treating hypoglycemia, while those that reduce IRS-1 phosphorylation are useful for treating hyperglycemia.

Diagnostic Assay

[0100]The current invention is additionally useful in diagnosing whether a dysregulation of the glucose homeostasis pathway is related to the Gab1 signaling pathway. For example, using the technique described in Screening Assay Two, hepatic cells obtained from the liver biopsy of a patient suffering from a disorder related to glucose homeostasis dysregulation can be assayed.

[0101]Biopsied hepatic cells are plated in a cell culture plate, which may be multi well containing 96 wells, 384 wells, 1594 wells or other commercially available well numbers, and are incubated according to well known procedure. Otherwise identical wells are then either exposed to Gab1 modulators in a glucose solution (test wells) or are exposed to glucose solution alone (control wells). Gab1 modulators, as used in this Diagnostic Assay, are those that have been identified to restore glucose homeostasis in LGKO mice, or other Gab1 deficient systems.

[0102]The wells are immediately and incrementally assayed for glucose concentration. In this situation, the response of the test cell to the modulator is compared to the response in the control cell under substantially the same reaction conditions. If the Gab1 modulator causes a restoration of glucose homeostasis in said test cell, then the diagnosis and subsequent treatments can be tailored to treating a Gab1 deficiency.

Pharmaceutical Compositions

[0103]Methods of using the compounds and pharmaceutical compositions of the invention are also provided herein. The methods involve both in vitro and in vivo uses of the compounds and pharmaceutical compositions for altering preferred nuclear receptor activity, in a cell type specific fashion.

[0104]In certain embodiments, the claimed methods involve the discovery and use of modulating compounds including agonists, antagonists, ligands, small molecules, peptides and nucleic acid molecules.

[0105]Once identified as a modulator using a method of the current invention, an agent can be put in a pharmaceutically acceptable formulation, such as those described in Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co., Easton, Pa. (1990), incorporated by reference herein, to generate a pharmaceutical composition useful for specific treatment of diseases and pathological conditions.

[0106]Agents identified by the methods taught herein can be administered to a patient either by themselves, or in pharmaceutical compositions where it is mixed with suitable carriers or excipient(s). In treating a patient exhibiting a disorder of interest, a therapeutically effective amount of agent or agents such as these is administered. A therapeutically effective dose refers to that amount of the agent resulting in amelioration of symptoms or a prolongation of survival in a patient.

[0107]The agents also can be prepared as pharmaceutically acceptable salts. Examples of pharmaceutically acceptable salts include, but are not limited to acid addition salts such as those containing hydrochloride, sulfate, phosphate, sulfamate, acetate, citrate, lactate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, cyclohexylsulfamate and quinate. Such salts can be derived using acids such as hydrochloric acid, sulfuric acid, phosphoric acid, sulfamic acid, acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclohexylsulfamic acid, and quinic acid.

[0108]Pharmaceutically acceptable salts can be prepared by standard techniques. For example, the free base form of the agent is first dissolved in a suitable solvent such as an aqueous or aqueous-alcohol solution, containing the appropriate acid. The salt is then isolated by evaporating the solution. In another example, the salt is prepared by reacting the free base and acid in an organic solvent.

[0109]Carriers or excipients can be used to facilitate administration of the agent, for example, to increase the solubility of the agent. Examples of carriers and excipients include calcium carbonate, calcium phosphate, various sugars or types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols and physiologically compatible solvents.

[0110]For applications that require the compounds and compositions to cross the blood-brain barrier, or to cross the cell membrane, formulations that increase the lipophilicity of the compound are particularly desirable. For example, the compounds of the invention can be incorporated into liposomes (Gregoriadis, Liposome Technology, Vols. I to III, 2nd ed. (CRC Press, Boca Raton Fla. (1993)). Liposomes, which consist of phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer. Additionally, the therapeutic compound can be conjugated to a peptide that facilitates cell entry, such as penetratin (also known as Antennapedia peptide), other homeodomain sequences, or the HIV protein Tat.

[0111]Toxicity and therapeutic efficacy of such agents can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Agents which exhibit large therapeutic indices are preferred The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such agents lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.

[0112]For any agent identified by the methods taught herein, the therapeutically effective dose can be estimated initially from cell culture assays. For example, a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 as determined in cell culture (i.e., the concentration of the test agent which achieves a half-maximal disruption of the protein complex, or a half-maximal inhibition of the cellular level and/or activity of a complex component). Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by HPLC.

[0113]The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g. Fingl et al., in The Pharmacological Basis of Therapeutics, Ch. 1 p. 1 (1975)). It should be noted that the attending physician would know how to and when to terminate, interrupt, or adjust administration due to toxicity, or to organ dysfunctions. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity). The magnitude of an administrated dose in the management of the disorder of interest will vary with the severity of the condition to be treated and to the route of administration. The severity of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods. Further, the dose and perhaps dose frequency, will also vary according to the age, body weight, and response of the individual patient. A program comparable to that discussed above may be used in veterinary medicine.

[0114]Depending on the specific conditions being treated, such agents may be formulated and administered systemically or locally. Techniques for formulation and administration may be found in Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co., Easton, Pa. (1990). Suitable routes may include oral, rectal, transdermal, vaginal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections, just to name a few.

[0115]For injection, the agents may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. For such transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0116]Use of pharmaceutically acceptable carriers to formulate the agents herein disclosed into dosages suitable for systemic administration is contemplated. With proper choice of carrier and suitable manufacturing practice, these agents, in particular, those formulated as solutions, may be administered parenterally, such as by intravenous injection. The agents can be formulated readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administration. Such carriers enable the agents of the invention to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.

[0117]Agents intended to be administered intracellularly may be administered using techniques well known to those of ordinary skill in the art. For example, such agents may be encapsulated into liposomes, then administered as described above. Liposomes are spherical lipid bilayers with aqueous interiors. All molecules present in an aqueous solution at the time of liposome formation are incorporated into the aqueous interior. The liposomal contents are both protected from the external microenvironment and, because liposomes fuse with cell membranes, are efficiently delivered into the cell cytoplasm. Additionally, due to their hydrophobicity, small organic molecules may be directly administered intracellularly.

[0118]Pharmaceutical compositions suitable for use in the context of the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. Determination of the effective amounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active agents into preparations which can be used pharmaceutically. The preparations formulated for oral administration may be in the form of tablets, dragees, capsules, or solutions. The pharmaceutical compositions contemplated by the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levitating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0119]Pharmaceutical formulations for parenteral administration include aqueous solutions of the active agents in water-soluble form. Additionally, suspensions of the active agents may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the agents to allow for the preparation of highly concentrated solutions.

[0120]Pharmaceutical preparations for oral use can be obtained by combining the active agents with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0121]Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active agent doses.

[0122]Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active agents may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added.

[0123]Some methods of delivery that may be used include: [0124]a. encapsulation in liposomes, [0125]b. transduction by retroviral vectors, [0126]c. localization to nuclear compartment utilizing nuclear targeting site found on most nuclear proteins, [0127]d. transfection of cells ex vivo with subsequent reimplantation or administration of the transfected cells, [0128]e. a DNA transporter system.

Sequence CWU 1

91117329DNAMus musculusmisc_feature(4762)...(4861)n is a, c, g, or t 1agtggaagca gtgtgagact ttatcctgtc ttagagatac acgcagctga acgctttgtg 60gaggctaagc ggtagcatca ctcggtagaa cagctcctgg gtcaggccct aggcacccag 120gaggtccccc acccccttgg tctctgggtg cacagtgagg gtcctagcgc gctcctggtc 180cgggagacgc agtccttcag tctctcgagc agccaggacc cgcttgagtg agttctcctc 240ctggctgggt tattttttca gactgtcaag tagggaaatc cttccgaaaa ctctggatcg 300cgaaggcaca gccgagtaca aactcctgac cctgggggtt gtcaagatgg caaactcagt 360cggagctggc gagagtgccc ggcactcccg ctctcacact ggggtggcgc tgcgttcccg 420gagaccgccg ccttccatag cgcgcgggcc cggggcggcg gggcggcgag gaccccagcg 480cgcctgctga ctacagatcc cagggtgcct ctgccatctc gctccctccc agcgccggcc 540ccgcctcccc agcccttgtt gttttggctg gaagcgctcc ggaggagttt cagagaaagt 600ctgggctgaa gctagaggcg accgatcgtg ggaaggggag gcagaaaggc taggaggagg 660agggccgggg cccaggccgc gcctccccga acccgccgcg cgcccccggg gtgggggagg 720agcgcagggg acaggacaga ggggtccctc cccgaagcag agccgccctg cgcctcgtcc 780ctgtgctgat ccccgccctc atccgaggct ggagcgcaga cccaccctcc caccgcggac 840ccgcgacctc cccgacgccc ggcgacgccc tgaccctcgc tgctggtccg cgaactccct 900aggctgatca ggacctgccc ctgtgccggc tgccacccgg acgccgcacg ccttcccagg 960cgcccttttc cgagcagagg gaaagagaag atcgagcccc tctcagtgtg aatgcgccaa 1020cgggcggagc ggagcggagc ggacaccgcg cgcgggcatt gtgtgtgcgc gtgcagcgtg 1080gggtccgcag cggggagcac ccgcgggagg tcccgtttcc aaggggcgga gcgcagggct 1140tccagttctg ggctccctgt ccggacagag tcccagcgga gcccgaccgc tgcctaggcg 1200gcgggacggc gcgcctggcg gccaggaggg cgcactgaaa gaaggtcggc gagccctggt 1260ccccgcggtt cccgatcgag ttcctcttca gtccgcgaat ctgcgggaga ggttcgatcg 1320ccgacacagg gcgcggggag ccgggccgcc ccgtcggggg aatctgagac gtcctctggg 1380ctgcgtttga ctgccgtgcc cgccgtgcac ggagcgcgtc cactgtgtcc accgacccct 1440ttggtgtctg gtcctcgagt cctcacggcg tgcaccatga gcggcggcga agtggtttgc 1500tcgggatggc tccgcaagtc gcccccggag aagaagttga agcgttatgt aagtacaggt 1560caggtcaccg tctgccccgc tgcagcagcc tgcgcgcccg gttggctctt ccacgaatcc 1620cgccctcctt ttcctcgatc cgtctctcgg ccggcgggct ccggaccggg tgcggtccac 1680tcccaccctg acgtccactc ggcggccaaa ctttctccct ccatattgca gactgcctcc 1740cttgtaggcc tcgttgcctg ttagagaagc ccagttttcc agctcaaaac tggctgcata 1800ctttggttgt tgttatttcc atttagcaag ggtggagtga aacagggaga aagagccttc 1860ttttattatc tgtatcagat tctcatggag ctgttaaatc tcccctcccc gagaaaccgc 1920ggaggtagta agtactcatg ggttctcgat tgggtccgcg cccgctctcc tttgcgcctc 1980caggaggtgg agggaagtga actgtggacg caagaagtgg ataatatagg caaacttctc 2040aaaatattcg ctcaagggaa aggggaaaaa agttcttatg aaccctgccg tctgcttagc 2100attttcatgg ttttgctttg aggacactgg attttggcct gagaaccctg gcggtttgat 2160tcggcaggag ggcgctgggg ggagcctggt ggctggtgtt tgggcgcctt agaagcgctg 2220tcaagccccg caggagggga cagctgaagc caacagggat ttcttgatgc tggcgtttct 2280tttcccgggc accagaacgg cttaactggt atttgcgtaa ttgattcaag atgctgattg 2340tgagatgact gtgttcacgg ttaggaaaac tgtatcttga aggagcctat aatacttgag 2400tggaactata tcctcatcgg gaatctaggg tcctaagtct tccttcagat tggctaattt 2460tgcgatgtac cctgtcagta aagaaatgac actttgtacc ggcatttgaa acaagtattt 2520ttagggtgaa atgtgtttcc caaaagcaaa acgtgtaccc acttaattag agtttactac 2580ctgttttgat gaaacctgtt tctggtggag gggcgtctac accaagaatc atcggactac 2640tcgcttgctt gtgtgaaacc aggcaatatt tattttcacg gagctcacca gcgagtttcg 2700ggctcacctt ctggtataat ttaatacagc caagtaattt cattgaaaga tgtgatttga 2760caaattgtca gaggttcccc aaagatgtct ctccatctgt cacagtttgc ttggatttct 2820tggtaaagat ggcaacttga ttccagaaat ttgagagtgg cagtgacatc ttcagaatta 2880gttctaaaga gacgattgtg gtgaggcgat gaggggtgag gagatagagg taaggcacta 2940tttaggaaag acggttttag gattcttgag gaaactaaag tacaatatat ttgctgatca 3000tgcagtccgg tgcattcttt cattacttat ggcctgccag acatactgag ttctgtacta 3060ggcaccagcc acaaggtaag gagaaaagcc ttgcagggaa ggaaaatgac aactaataag 3120ggacagatat ggtggtatta tatgcccaat accacccagc actccagagg cagaaatggg 3180aggctccgtg gacaaactgg tctccatagc agattccagg ctagccagga ccgtgtctca 3240aaaatagaaa acacaagcac aaaaaccaaa tacagctgat tatttataaa gatattaata 3300ataatatgat tatgtttact tatatgtatc attatatgac cacagcaatg ttctcccaca 3360aaagtcccaa tcatgccaaa cctgttagct tctgaatcag ggaagagtga ttgaattcag 3420gggggtctgc gaaacgtgaa gctcagatta agtctaaggt cttttcagtc tagactttct 3480gtgaattagc tgagaactca aggaagaaga accaatcgga tacacagtgc tccaaccttt 3540gctcctaagt agtttttaaa gtttggggaa caacaaagtc ttaaagtaaa tgcagagaaa 3600gtcagccaga atgcaaagtg gcttttatct gcatgtctac attgagtggt ggtgttttta 3660taaatgcctt caggagaaaa ctcagtccct aaattgtgtt tcagtaagca ggaggtgggg 3720gaagccaagg gttttcttgc ttggtagaaa actagtggtt tgtgtgtgtg tgtgtgtgtg 3780tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tttgtgtgtg ggtatgcaca taggcatgta 3840tgcacatgtg cgtttgtggt ggccagaggt cagcctcagg catgtcattc cagtggagct 3900acccaccttg ctttgctcaa agggtctcac tgggatctgg ggcttatgga ttagagtcgg 3960ctgtgggctg gttagtgaga tcaggcatcc tcctgtctct gcgtcaccag tgttggggtt 4020ataaactgca gcagcatgct tggcttgtta caaaggagct ggggttgggc tgagtttctt 4080gggcttctgt ggaaagcatt ttatcacgtg agccatgtct ccagctgact agtgtttctt 4140tacagtttag tggagtaggt gaggatggcc caggatggta atagacaggt gacagccaag 4200aggatgactg aggtctgcta agtgacactt atttgtcagg tttagtcctc agatttgaca 4260tcctcgggat gttgaacctt tgacttgggc atccatccag tttgattttg ttaaagggat 4320ggctacgtac gatggctatg tgtatttttc tattcttttc cttttttctt ttttttcttt 4380ttggtgaaag gaatcctgca aagtaggcca aagaatgatt gataattttt tttgtttgtt 4440tttgttttag tttttcgaga cagggtttct ctgtaaagcc ctggctgttc tggaactcac 4500tttgtagacc aggctagcct cgaactcaga aatccgcctg cctctgcctt ccgagtgctg 4560gaattaaagg cgtgcgccac cacacccggc tgataatggt ttttattttt caaattttta 4620tgtgtgtgcc tgtgggagta gagcccaagg ccttgagccc aaggcctcac acataattcc 4680aaatgttctg caattgagct gttttcacac ccttgacagg gcttctctct ctctctctct 4740ctctctctct ctctctctct cnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4800nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4860ntctgcttct gcctgccaag tgctgggata aaagacatgt gccaccttgc ctggcaatat 4920ttttaaatca atatttttaa atgtcttctc ctctcctctc ctctcctctc ctctcctctc 4980ctctcctctc ctctcctctc ctctcctctc ctctcctctc ctctcctctc ctctcctctc 5040ctctcctctc ctctcctctc ctttcctttc ctttcctttc atgtgtgcat gtttcttatg 5100tgtgtacatg agagtgagtg tgtgctatgg aggtcagcgg acaacttgtg ctccagagtt 5160tgttctttcc ttcccccacg tgggtcctgc gtgagtatcc aactgtggtt gacagaagta 5220ggggcaagtg tcttaaccct ctgggccatc tttctggcct gataatgctt tttaatgagc 5280atattcacta tgaaaagctt atcttggatg cttgcactgt aattattaaa tgagacttca 5340gggagtttga gtatcatcgt cctgctggga aatgctgccc gtgggacgtt gactcttgtc 5400ttggtatttg tttgcagcat ttgtagagtg gtccaaatga ttgttaaggt ctgtaagtga 5460caaacacaca cacacaaaca aacaaatttt tggctcaggg agatggctta atgaataaat 5520cattttgtct catgtttgag aatgtgagaa cttgagttct gtctcccgaa ctcatgcgag 5580taccagggca ggcatggtgg cagcctagaa tgccaactgt agggaactgt gtatactcag 5640ggccagctgg acaattagat tagcaaactt ctatattgaa gagtgttccg gcttgctgag 5700atggctcagt ggttaagagc attgactgct cttccaaagg tcttgagttg aaatcccagc 5760aactacatgg tggctcacaa ccatctgtaa tggggatctg atgccctctt ctggtgtgtc 5820taaaagacag ctacagtgta cttacatata ataatcaata aatcttaaaa aaaagaagag 5880agtttctcca ataattgaaa tagccaatga gaaagacatc tgatgttatt gatgggcctg 5940cgcacatatt ataaatgttc ctgcccatat gtgtgtgtcc atatacatac agacatggac 6000atcacatgca tatgcaagaa agtattgatt tcatctttaa aactatctca agggctgggg 6060agatggctta gcagttaaga ctgctcttcc agaggtcctg agttcaaatc ccagcaacca 6120catgctggct cacaaccatt tgtaacaata tctgactccc tcttctagag tgtctgaaga 6180aagctacagt gtacctagat ataagaaata aatcttaaaa agaaaaaaat ctcaaattgt 6240ggcccctgat gagttctgta cagcccctgt tgcggtaaat ctcccaccca aataagcctg 6300gggaatgtaa acacaatgaa tacatgctgt gcgcctagat tgggcagatc tacctctacg 6360ctatcatctt ccccatataa gagacccctt agaacttgca gtttctccag gccaggtgct 6420ctgctccact ttccttcttt tcctcctcct ccattttgtt tgtttggttg gctggttttt 6480tttttgtttt gttttgtttg tttgtttttg tattggtttc atctttaaaa ccagctcaaa 6540ttgtagcccc gatgagtcct gtacagcccc aacgtctgtg tccgtatgtc cgcgtgactg 6600ctctgtctgg tcactgtcac agcctgtttt cttccttgtc tttgtgctca tctaatagag 6660gaactttctc tcagagaaag tgacagggga taaaggaaga gactccccag tctcagagtc 6720acttctttgc ggacctttcc catgcaaaag ggtaagaact ttttacatgc gcagtcctgc 6780agtctgtcct ctggcggctt tgctgagggt tatgcacagt ggggatgaac atcctcacgc 6840tttctctcag tgttgctgag tgacctccag cgctcagtca ccagggtcat agagttgagg 6900gaactgctat tctgtactca gctcagctcg gatttcttat taaaatttaa atttgcaggg 6960ctggggatgg agctcagcct tgcctagtgt acaggaagtc gtgtgtgtgt gtgtgtgtgt 7020gnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 7080nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 7140nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 7200nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnncctctg acctcaaccc 7260aagctcctta gcacaaatac cacacacaca cacacacaca cacacaccac acaccacaca 7320ccatacacta aaaattaaat tagtaagatc attctttttt ttgtttgttt gtttccgaga 7380cagggtttct ctgtgtagcc ctggctgtcc tggaactctc tctgtagacc aggctggcct 7440cgaactcaga aatccgcctg cctctggtgc tgggattaaa ggtgtgcgcc accatgcctg 7500gctagtaaga tcattcttac tagtagagtt gaggcttgcc aaggcaacat ttttttttta 7560aagtcttcag tgctgtctaa gcatttgggg cgacagtgtg gcgattcagt accagtacac 7620aaaaatgtaa tggtcaggtc tgggtactta gacttctcct tacacatttt atctgtggtt 7680tggcagccat tgaacatctc tcttgatttg taaaatacac attgtatttt cctaaggact 7740tattttagaa agtagtggtg ggggttagca tatccagaat gcacatgaca catccatgaa 7800aacatcaaag aacaaatgca atgaaaggtt ttgttttttt ttttaaagca atggtggact 7860ttcccatcag gatgtgaaat tttcacagaa ataaatgtaa gtggataaat aatagcttgc 7920ttcaatccag ctggccctca ctgctactct gcaggggtag tttccatgtg gcccgcctcc 7980agcctgacgc ccagggaggt tcctccacag cctgctgtct gtcagctctg ggcaggtgtc 8040ctggaggagc aggtgttcct cctcggtagg agaacctgcc tgggaagcca gggcaaggtg 8100gcctgagagc agacagccct ggagcaaggc ctcagccagt tcttgggtgg ataatctgtc 8160acccagcttg agctctgtct gctgcagctg tcaattatca gagcaggttt tgtctggact 8220ttgggagcag cctctagttc aggagaggag tctctcactg ggtgggcatg cgacatctga 8280tgtggtggag gggaacctga gcagcatctc tgtggctcaa ggatgcccta gggtgtgtgc 8340tgatcctgaa gaccgtggtc ccactcgcct ggcctgtctc acctgtttca cattgagacc 8400ctccagctgc cctgttgaag gacaattttc ctggttgcct tcatgttagt gaacattcac 8460gtcagattcc tgccatctcc attccaccct ttgaagtccg gcaggaccat tgtctgttaa 8520cacaggtctt taactagcaa tcacattaag ccttcttact tagttaatga cagttaattc 8580ttctgttgtg cctgggcaga atggtaagtt gtataatgct ctccattaag ctcgctgaat 8640ccctgaatac agttggtacc ttttggggat ttagaattcc tatttggaag ttaatcattt 8700gggtggtaat ttcttaccac tttttaaccc tctaactgca ctcttctttt ctttctaaat 8760ttgtagaacc cgaaacatgt actcactcag ctttgccaag aacagaagtt ggaagggaat 8820ttataagctt ttcaattttt tctttctttt ctctctttgt ctctctctct ctccttcctt 8880tctttctttc tttctttctt tctttctttc tttctttctt tctttctttc tttctttctt 8940ccttccttcc ttccttcctt ccttccttcc ttccttcctt cctttagggt ttctctgtgt 9000agccctggct gtcctggatg gaactcaccc tgtagaccag gctggcctcg aactcagaaa 9060ttcacctgcc tctgcctcct gagtgctggg attaaaggcg tgaaccacca ctgcccggcc 9120ttcaactttt ccactcttgg ggtatataca agcaaaataa ataaatgaat gaatgaatga 9180gaggagggat aggccagtga aatggaggaa tgttcgtgcc ttttctggga ataagagatt 9240gcttgaagaa ttcaggtgcc agttttcttt gttcgtttaa tggttctttc cagtatcgac 9300gtggcaacta taaacggtac tgacattggg agctgtgtca tttgccgtgt agatgggatt 9360atagaaaagc tagaggccat ctggtggtct ctctcagcca atttagatcc aaccagtttc 9420catcttggcc agcccacctg aagaggaact gctggcattt aaaaatcctg tccttgaagg 9480tagacaagat taggggtaga agttctagta actcaacttg gcatctgtca ggattacaga 9540tttagaattc tgctggttta gaacaaatga ctgtcacttt cggtctgggg ctgaggctta 9600gttgagtgag tgtctagcat gacagatgaa gctcaggctc tgtcctcaac actgggcatg 9660gtggagaaca ccagtaatcc ctgcctttgg gaggtggagg cagaaggatc gccatattca 9720aggtcaccag agctatacgc tttgaggtta gcattttaaa aaatatcttg tccttttaaa 9780gaaaatagtt atgagcagga tattgtggct cagacttgta gtcttagcag ctgggaggta 9840gaagcaggag gactgagttt gagactacat agcaagaccc ggtctcagaa tagtgaaggg 9900tggaagtgct aaggatttca gcatgcattc tgcttgggct gtgggaggga acgcaggctg 9960agggtcgggc tgtgcctctg gcctcccacc tgatccttga ctggtctgat cctgtttcct 10020tatctggagg ggaggggaca gaagggcagt agagctccag ctgtggtaga gagtggggtg 10080gggggagcat cagagggtag accgagagct actcacttgc taggctctat tgctcttctc 10140tggtcgttgc gtgaacgtgg taggaattct gtgggctcca cagtgttact cagtgcagga 10200gaacacagac acacagttgg cttcagtgtt cgggtgctgg gtgcacccac aagcagtacc 10260tggctcagga tggaaggttc cacagcttat ctgctggctg agtaaataaa caggtatttc 10320gggagctttg gtcctgggcc ttatgtggaa tgcagtgcag acacctgtcc actccctgcc 10380tgtaaggagt ttctgatctg tgaaaaccag ctgtagcttc atgaacactt agctttcctc 10440agagattggc tggggagctc tctgtgttta gttgccaaat gagggctcca ggttgtagtt 10500gcctaagagt cgagactgag agagggaggg aagaggatga ccccagaaaa cttcccatca 10560gagtctcaag ctgttccagg aaggattgag gtgcagcccc ccccctctcg gtagcaaaat 10620cctaagggaa agatgtttta gatgaacaaa gagtccttag cagtgacacg aagggcctca 10680ggatgctgga gctcaggcct ggggtttgca tgtcagctgt aactttctca ttggcaaagt 10740ttttgtttct gctatgactt aataaggttc tcatgggcac ctagccacag tgtgttttcc 10800gcaaccaaga agacagaaac acagcaaccc gtgcggtcac ctgggcttat taaaattcca 10860agggacctgc gttagttaag atttctttat gtctgttttg agctaagtaa ttcattctgg 10920caagccttca gtttacaaca gagagggcgc atgttcgtgt gggaaggaga ataaaggagt 10980ctttgattca tttgcttctg agctgctagt gcctgaactt ggactttgga ggccatctta 11040gcccagctgt gtgagggcca gccagtgttg cagggcgaga ataacgccat tggctcgctt 11100gaaagcgttc tgactgtcga tggggtgttg gaggtcctct cattcacatt cctggcatta 11160acatggtggc ccgtctccat ccatccctag ttctctctga agctgccatt tgaacttgaa 11220gtcacatttc tgtgactctt tatctcagcc cgtgtgtcca ctaagtatag aaagcttagt 11280gtctcaaggc tctttttctc tccccttgga ttagagcaca gataaaatta gtattcgtta 11340gattactgag gagaaagtgg tccattgtga tgtggatttc cacatttgtt acattcctcc 11400actctttgcc tactaaccca gggtggggtg gtttaggaac tttcaggggt tgtaggcaat 11460gcctgtattg ctgggaaaag tcatttcagg agccagaggt ttctgataga tcattgcaga 11520ctccttcaca tagtactgta aataatacaa ggactcatga actggaagaa tctttatatt 11580ttggacaatt cttttattca tgggggggtg gggtgggaaa tctccctatt tgaaaagttc 11640tttccaaaat catagcgatg tggtaacctg ggctttggcc gtcctgcagc ctcttgagag 11700gagaggcagg gcctagtaat gcatatttgg gccttatctc tgttacttag ctcttgacgc 11760ttgctgatta caacttgtag ccgccaagtc ccgtcctgca ccattatctg gaacaaaaac 11820tttccatgtg gtttgcctca gctctgcctc ggtaggcaca gcactcggtg gaggtggcag 11880aggtggcgga ggtggcggga gtagtggggg tggaggtgga agtttgtaaa gagcttctgg 11940aaggggtcac gtgagtaatt taatattttc ctactgttta agttatcctt tccctgcctt 12000ggcatgtctt ccagaaatgt ttgtcattct tctaaactga gttatctttc acttttctgc 12060tctggataga atgccgcatt tactaacggt tcttttctca cgttctttcc ctctttgctc 12120tcacactctt tgtcttagct tcttgctttg aattcctcct tgccctagat aggtgtctat 12180tcttaaaaca aaaaacaaaa tccccaacat cctaaaaagg aggtgaggac caaattgcca 12240cattgttttt tgaatttaaa acaatatact aaagtctaaa catttctttt caatttaaaa 12300atatcccccc cccgtttttt tttatttaaa ggaagaaaaa tactaacctt agctttaact 12360atattattgt gtttccttcc tccctttttg tttattttct tttgctttga ctcagctcaa 12420gtccagctcg atgcagtctt ctggtcgtct ccagagtatg gagcacatgg agactaacaa 12480tggcggcccc actggctgtc aggagagagg cctgacctct gaagagaagg gggatgatat 12540cacaaagcaa agaaggcttt attgtgtcct ctcgtgacac taaattgaat tatcttagca 12600ttattagaac actacaagaa gtaagcagat aaatggaatt gtattccttt aaaaatactt 12660tagatttatt gatgtagttt tacatggatg gatgtttgct ggagctgttt gtctgtgcca 12720ccagtacatg tgtgcccagt gtgcagcagt ggcgtgtaga agaggcaatc gggtcatctg 12780gtctggcgtc cttgatagct gtgaagcacc tgtcgtgcag gtgctgggaa cggaacctgg 12840gacccctgaa gagcagtaag tggtcctaag tgttgtacca ccgctctggg taaccgtaat 12900tttggttagg aggtctgctt tctctgatag atactgactg ttgcactagg attttttttt 12960ttttgatagc ttgacttatt tttgttgtcg tttctgttgt atttaatgct ttggcttgcc 13020tgttgctgtc aaatggtttt taccagagaa aagtaagtct gtccctgttg ctggagtcct 13080cagccctctg ctcttaactt gtgctactac tgtaaggtag ccaagactgg agtagtggcg 13140gtaactttac tcagtaggca gcttgcttca tccagtctgc tacatggcct tagttaccag 13200tgcattggga aatgtggccg ggctacttct gactccgccc agaggtgact accacattaa 13260agaattagca gccctagtcc ctagctcagc ctccccattg gtgctgtagc cagagtctca 13320tagccaagat ttaacttctg ctgcttggca aaatgccact tgattcccag gccacaagca 13380ctttagcatt tcagtttggg atgagaaata cttgggaagc tgccaccttc cataatttga 13440gttctgatgg aggtatgggg cttagtgagg aaccatggcc tcgtttagtg aggggggtat 13500tatttgaaag ttataatgga cctgacaaat tctccctgat aatttctggt ttaagtgtgt 13560tgaagcattg agaaaaagtg acagggagca ctgagtactg gcgaaagcag tacaatctag 13620tgctactgtt acctaaagcc tcctcatggg gtatgtaggc catttaccac ccagaaaagg 13680gccagaatca actagatttt tgggagtagt atcctcattt tttgttctgt acgaacttgt 13740caagggtcaa acttctcagc gtacagtgct ctagacaaga gcagcgagcc atgtggttcg 13800ggcagcagca gcaagctacg tagtgtcatt ttcaagttga ttttcataat ggcccacaga 13860aagcgcagaa aacgagagtg tgtacttatt ccatccattg tgacatttag tcgtcctttg 13920tgtttgtggc agggtttgat gtccctgaag gtatctatgc tttgtctaaa gcatagtgtg 13980ggaccttgtg cgatcgcttg aacagtataa ccaagtagct gtagactttt gggcccagaa 14040ttctaaatca ccttgtccag tttctcctta gctttttgat tccttcctct tggtgtgttc 14100aatgccctct ggcttttcat ttactgtgcc tttgatttgc acagcaaaaa catttactat 14160tgaatattct tttttttttc agttgttaca tctctgtcct ggtggagaag agtccgaccc 14220cccctttttt ttttacgggg aatgaaaatg agcctcaggg agactaatcg atttgtccaa 14280ggtcacacaa cataaccagc tgtcccacca gggtcagatg tgtcatggct cagggcaaat 14340cctttagtta cccagtgaga gctgagggta agcaaggcac tgtggaaaac tataaagaag 14400aggccaaggg ggcattgctg tcaccttcaa gaggttatat gttcatagag gacctgggat 14460gtgctgctca cactcagaat cctatggtga ctcatcctga gatgagtatg agatgcactc 14520acagaattca gcaaaaaggg attgattcag aagttcagga agacttcgta agaggaacat 14580ttttttcaga gcttaattag ggacactttc agaagtagga gcagggctac agtactcagt 14640tacatggttc ccttttctag ccacccttga attggttatg taggtgtagc tgctggctct 14700ctcgacagtc agagccgttg ccaccaactc ctaccatgga gcctcgtctt atcagtgtgg 14760ggataactat agttcttcca tgaaattctt gggtgtgttg agggacgtag gttgtagtct 14820gcttaagtcc tggaatccct aagcactcca gtcctgtttt gtttttatcc attgtgagac 14880tgtatactcc ttgaaagttt aataaaagcc ctgtgctcag gtggtctgcc acatcagctt 14940taaagaaaga cttctgccac taagcgtgat gatccacact cgatccccag aacccacata

15000gctgaagaag aaacggtctc tggccttctt ctcatgccca cagtgtggca tacaacatag 15060taatgttaaa acaaaacaaa aggccctgag cgctcagttt catcagaact tttagaactt 15120ttagaaactt acaagtgtta taacttgtag gatttctgac ctgtgaaacc tgtctgtgag 15180tctctctgcg tagttacagg ccttccagtt ttatgctgat aatctgcggc ttctctccac 15240ccagtcttat gggtccagct gcttagatgg tctacagaag tgccaagggc ccatgccagc 15300attttaccct tcagacttgg tctccctgtt gcgccctcaa gtggacagtg ctgagctacc 15360cttcttcagt ggcttgagaa tactggtttc cttattctgc acacgcagta accaaaacaa 15420accctgtcag tttaccacct cagtggctct cgactgttgg ccctctcctg cctttgtttg 15480gaatgctgga gctgttaact ggtctctcag tacagccttg caaagatctc tttgtgaaca 15540gagccaggca gcttttctga actgaaaagc tctggactga cgttgttggt tctgctgtgt 15600cgctgtgctc ttagtagcgc ctaggccttt ccccttggct cagcagggtt agtggtcatg 15660tggcattggc acatctgtcc aggacacatg ctgccacatc cctcattcag ccactgcctc 15720agctttgccg gacttttgtg tccccacttg tccccaagac ctgttattct gggctagccc 15780cctccaggaa gtcttggcct tctttttcac tctacaccga gactaacttc tgctgcttct 15840cctctctgcc agcttcccct ggtctccatc accagcacag acacagtgtg caagactgtt 15900gtgtcgttag cataaactgg ccgctcttgg ttgtactgac ttctgcaact gctcatctca 15960aactgtgtat ggtatggtgg gtagcaggca ctgcgacttc tgtgagtcca gctactcctc 16020ctcgcgtggg atcgccttgt gtatgaaatt taagactagc tgtagtagca cctgttccta 16080atctcaacac ttgggaagcc gaggcaggat gacttcacgt tggagagcag tgggggctac 16140agaacaagtt ccaggctggc catggatgca gagtgaaagc ctttgccaac aaaccaaaaa 16200caagcaaaca gaaaatggaa aattaggtaa aggtgtttgc ctaaaggctg aagacctcag 16260tgtgataggt tgggagaaaa attgtatgat cgttacacaa acagatgtaa cgggagaaag 16320caaaaatcat attttgaaat ataaatataa tctgagctca attttgttaa agtatcatgg 16380tcaccaaaca ctgtttctta tgtgtgtggt gggtacatcg atgttgaatg aacagaaata 16440ctttgcagat aaagtccaca gcatatgtca gctgatggat tgggacaaag gcaagacaac 16500tactcagctc ttcagttacc tgacactttt gtaatcaccc aggtggctct caaagaataa 16560tcctaaaatg tcattgtgta gtccctggga atagaaccag tctcaggaaa ttagacacac 16620ttaacacaca tatactcact accttataat atatatttaa gagtaagaat cggggctggt 16680gagatggctc agtgggtaag agcacccgac tgctcttcca aaggtccaga gttcaaatcc 16740cagcaaccac atggtggctc acaaccatcc gtaacgagat ctgactccct cttctggagt 16800gtctgaagac agctacaatg tacttacata taataaataa ataaatcttt aaaaaaaaaa 16860aaaaaaaaag agtaagaatc attgagaagt ttctgatttc aaaacgattc cctgagttgg 16920cttactaaca ggcatatttc ttgagacgtt ttgacataga tttgtgtagg gctgggatgc 16980ctgttttgct gtgtctgaag acagatttct cttgcagcat ttagtagagt caggactgaa 17040acgcctgggt aaaccattgc ctccctgcac ccacccccac ccaccgcgag acgtggtgag 17100ctgccttcta tagagaagct ctctctaggc ctcgtcctta cactcctgct ctctgttggc 17160cttacttccc ctgtgcttca gctacaagct gcagttcaga tgcagagtcc tgttgtatgc 17220ctggagtcaa agcattgttt ttaccctggt aaagttgaga agaacagaca tttgagaggc 17280tgaccagaag ctctctccag tgaagaatca aaacccaata ctcattttgc ctcactctcc 17340tagctagagc gcagcctgtt gtttgggggt ggggtgctga gtcatgcttt tattcatgag 17400tttatccatg cttttttatt acctctgctg gcagatcacc atccattgtt catgagccgc 17460ccagcttgtt gacttttata agttacatgt aatcaagccc tcttgcaggt ctggctacca 17520taactggcag tcccaccaag ccgggctgac cagtctgcat ttgtgctgac tcccatgttg 17580ggtatctgtt ctgctcgcca agtgcccttg cctcctccac ctgtactgcc gatggaagag 17640aagacacctg cttgggggtt gggggggggg ggagcttgca gatgtaatgg gacacctcca 17700cagaacttgg tggagagctc attgtccttt agtttaggat aaatgaaggg tttgagggag 17760ttcgtgatgt cacctgggtc acttctaaag gaaggcttgc tgttttcttc attccctttt 17820cctagctgat gacttagcaa gtataggtaa agtccagaaa ctttgaaaaa caaaaggtaa 17880gcgctgtgtt agagcgatgg ctgggcagta aggagtactt gctgcttttc tagatgaggg 17940tttttgtttc cccacagttt gtttcggtgg ctcatggctg cctgtggctc tcagctccag 18000gggatctgcc catcacacat gtggcatata catacacagg caaaaaaacc acatgcacat 18060agatagaata acttttaaaa agcaaatgtt tatatatttt atgtatatct gtgctgtttg 18120tggatataca tatttgtgta tgtacatatg tgtgtaccat ggaggagaaa ggtctatgcc 18180aatatcttca gttgctctcc actctgtctg tctgtctgtc tgtctgtctg tctatctatc 18240tgaggcagta tctctcacta acctggaaca cactaattgt ctagaccaga tggtcagtag 18300gtcccaggaa tccccctgcc tctgattttt cttcattaaa gttactgatg tacactcaca 18360tacctggctt tttaaaatgt ggctactggg gggtagctat ggattactca ggtactccag 18420agcaaacatc ttacaactga gtccctgccc cagccctgga atcccacagc aaaaatctga 18480acaaccctta tggtctgatg taggtgggcc tcagattata gttggggaaa taatgatctt 18540ggtgcaaagg gaaaaagaag aaagaaaaat ctagtagaag caaatattat ttacattgta 18600ataagaagac taaaccaatg gccagaaagt tcctctttga ttttcttttt ccttccttcc 18660ttccttcctt ccttccttcc ttctttcttt ctttctttcc cttttttttt ttgttccttt 18720gagatgagag cggactagag aatccttggg aatacaggtg tttagttttg cctctttagt 18780ttcattttgt tttgctgttt tctcattgtg attgacatca gcttatcctt ccaaaaggat 18840gtgcttaaga tgacaaaaat aagagaatat tcagaatggt tagaactaaa aagtacctaa 18900gtattgctaa gacctcaaaa gatgtcgagt caaagaagga attaagttga cactcattag 18960acaagaatgt gaatccattc caacgaggat gcagactttg tgaatggctt agaggtgaag 19020agcagagagg attagttctt cttcatgagt tctggttatt ctggttatag gatctgaaag 19080catattatta aagtcatatt tcctggaata gttcaaaatt caaatacctt tcccctgcct 19140gttgaagaca ggacaactgg cttgtacttc tttttcgttt tcagattgat tggttcagtt 19200aggagcacca ggcgggttgt acctaacaac agaagtcaga cctcctattt ttcaactttt 19260aaaaaaaata tatgtgtaga ctccaaagtc cacactcaag aatgagagct gagagaccca 19320gtctgttata ggaagtcatc taagaagtag accaggcagg aatggctgtt taagcctagc 19380agaggagaga aagcaattcc tcctgtgtcc taatgaattc ccttttcatg tggaaagctg 19440gcttttgttg ttgctaataa tacaggattt ccttcagaga atttttaatg tcttgttcag 19500ataccacaca gagagatatt gaccacaaat attaatactg gtttgggaga tatgttgatg 19560tgatgttgtc cctaccttta ttaaactgac catcttaata actccatcca tcttggcacc 19620gacacctctg cttttctact tactgtgttc cttacaactc ctaggcctta gagcggtgct 19680gactacatta cccgtcctct tccctctctt ctatcttcca ccccgccctg cccccctttc 19740tccagggctt acttccacag gagcactcca tcctcccaag gaccctgccc atctgcctac 19800aagatctgcg agcatcttcg tatctgcctt gtaagtgttg aggatcatta ctgtcattat 19860ggggagcaga ggccacgccc gtgtgctgct cagagcccaa gtgagttggg tcagtagatc 19920cttagctgct gaagcaaaca ggagggctag tgctgtgtgc tggtggtgac agagccagct 19980tctctcactg acgctggcaa cccaggaagg tgctcttcat ggctgccact ccgctctaag 20040cactgaagag caggcgcagg agtgtttctt tttccaggtg acttcctatc tttgctgttt 20100ggtgctgcca tactcatatg cttgtaggtg cttcttactg gccttaatat tcctggttta 20160gcttttggag cctactctat tccatatata tttaaccaag aggtggaggt ttccagtgat 20220ccccttattt tgacaggaaa cgggtcttag tgtttttgtc aggcttgtgt gtgtgtgtgt 20280gtgtgtggtc taatcaggaa gtcatccaag ttcaacttta aaatgcccat aaactatcaa 20340tgcaagtgtt gcactcagag tattcttttg gggatgtagc acatttttga aaaaaaaaaa 20400aacaaaacga ttgtaagaca ttaagttagt gcatttagtg acaatgacat gataagctac 20460taaagggaaa taccttgacc ctcaggccta ccctatcgcc ctaagtccct ctccaaaccc 20520tcccaccctc ccttcagaca tggtcttatg tagcccatgg gggaatgttt ctttattaga 20580cttattgttt aactatcctg tatttattat atatttatgt gttataacta cctatttaag 20640tagaatgtgt acataaactg atatactatg ttggtattta attttaggta atactgttat 20700taattttgag acagagccat tatctcactg tgtagccttg gctggcctgg gactctgtgt 20760agaccaggct ggtcttgaat tcaacaaaga tttgtttctg tctccctagt gctgggatta 20820aaggtctgct acactgtgta tgtcttaggt cataaatctt aataatccca gtatatttaa 20880atctttaaaa cgctatatat aacctagaaa ctttttattt tgcttcaagg gtatttcatt 20940tggttgtttt tctatctgtc catttgcatc agagtttaag tgaaatcagt ttgttttgtt 21000gctggaccat tttttttttt ttagagcaaa tgtactttgt tccaatttgg aaagctagtt 21060ggtgagaacc ctgagctcct atcaagggca gcggaagctg gctctttaaa cactttgcat 21120ctttgaagta agctcttgtc ttaacacagt cctcttttca cacatgagtt cttggttttt 21180tttttttttt ttttttttga actgcatgct ttctaactct gagctgatta ttgattattg 21240aagcatttaa gtaaagagat gggagaaaga tagaagtgca cggaccaggt gaacatgttg 21300tcaggcaaag gaccgtccgc gcaagaagac atcttcagga cccgagttta taggccacct 21360acgctggtgc atcttgtatt cccaacatgg aggtggcgga gactcatggc tccctagcca 21420ttttgcccgc tgtctcagaa acaaaatgga ggacggcgga gggctgacac ctgcgcgcat 21480gcgtgcatgc gcacaaatgc acagagcacg gccgtttctc ttattttaat gaacggagaa 21540aaaatttata ttgtctttac ttacccattt ttgttctctt ttgagacagg gtctccttag 21600gtagccctgg cgctcacgga actctcgatg tagatcaagt tgaccaatga tgttgagaga 21660acatcttggg tctgtatgga tgtgacagct gtcagtaata tctgatggcc tatagcttag 21720gcaggaaata ggaggtggga catctggcag gcagaaagga ttctgggata acgacaggcg 21780ggagagtcgc cgggaccatt cgaggtggca gacgcatgac acctgagtac aggtaaccag 21840ccacatggca gaatatagag tagaataaat gggttacttt ataagctagt gagagaagag 21900cctagttata tgtccaaggt atttgtaatt tgtaatattg taaatgtgtt ttgagtctga 21960gtcttactcc tgggagcatg gggctgggag gaaaaaccat caaactaata tactcagact 22020tgcgggaaac cttctgtagg tgcttctagg tatggtggta gacatggacc actatgtccg 22080gcaacttttt tctgagataa acgtttaaac tgttgtgaga ttgggctcca actccctgcg 22140gcatctcatt gacacctggt agaccctcca aattgctggg gctgtagttg tgggccgcca 22200tagccagcat tctccccaca ctctttgctg ggcctgcatt tcaggccaag cgcctttcca 22260ctgagcccac ccggccccac cccacctccc cacccccaac cctctttcta ttcttgatga 22320gaaaaatgaa acttattgga aacggaagcc taagaaatac tctgaaaaca agaccacttg 22380ttcaagcagt cagagtcgga ctagaattca aatttggact agaattcgaa ttcactttcc 22440caacaggctg cttcttggac agcctcaggc tcctcccagc gagcaaagtc ttcccgccta 22500agaaactgga cactgctaat ctttacacac caaaagagag agcctttagg ggtccccaaa 22560gcagtggcaa tatcatacat tcatctaagt tggctcctaa tttgttaaat gtggttagaa 22620accattttct gttttatttt gatacagggc ctcctgtagc ccaggcctca agtctctgta 22680gttgagatag ccttggtcta ctgagcttgc actttccccc accccacccc caccccaggc 22740tgggcttgca agcatgagtc accattgctc ttcggccact cagtagtttg tagagttagt 22800gctcctcgtt gttctacagc tcatccctgg tatctccaga cagtctctgg aggcagtgca 22860cagcacatcg gaggggctca gtacccacgc ctcccacgaa ggcgtgctcc tctgtggtat 22920tcccaccctt accgcaggaa gaaggtaatt tcatatgcga tttgctcagc tgacctacgc 22980agatgatctt actgtctcgt atacttctgg aaatcccaca cacctttcct gcccctgagg 23040aaaaatttct tcctgatgct gaattcagag ctgtatttaa tcctgagcat atgagagtga 23100gtcaccttgg agtggtgagc aatcccactc ataccttcca ccttcacgtc agcgtttgaa 23160actgaagacc ttggtgcatg aataaagtct gacatggaga ctgctttcaa acctgagaac 23220agtagtgaaa cttctttcca tcccctccct ctcttccctt cttcccttct ttctttcttc 23280cctccttcct tcctctcttc ctttctttct ttctttcttt ctttctttct ttctttcttt 23340ctttttttct ttctttcttt ctttcttccc tccttccttc cttcctttct ttctttcttt 23400ctttctttct ttctttcttc cctccttcct tccttccttt ctttctttct ttctttcttt 23460ctttctttct ttctttcctt ccttccttcc ttccttcctt ccttccttcc ttccttcttt 23520ccttccttcc ttccttcctc tctctctctc tctctctctc tctctctctc tctctctctc 23580tctctctctc tttctctctc tctctctcac cagtgttttt tagttcagct gcaaatgaaa 23640ctgcataact tcagctctga aatgcgggct atgactggaa tgtttgttca gacaagaaag 23700caaatgtttt tctgaggagg cagggaaaag gaggattgaa aattttgggt agtctgagtt 23760ttcaaattca gactaattga gctgaatgac taccgttttt gtgatgtcac tgtgtcccca 23820aaaagacaga ctttcgaggt tcaggagata gtgttgaaca tgtgtgtcat tgttagggtg 23880atatttctgt cattacctgc acagtgggta acctttacct ctatttctct tatttgtaca 23940gtgtcactgg aagttatttt aaaagcttac agtccttgtc aggggccttg agatttctag 24000gataatttca aagaatgtaa atcattaatg atccttttta taccaataga acaaaatatg 24060aaagccaatg actgcttccc caaaaggccc tgcgctgtcc cctcctaaga gcttgtctta 24120aactggcagt tctccagcta gcctcagcct tctctcttag caagtgttgg gcacgcctgt 24180tttaacctgc gcaggttcac ttcccacaga tcgaaggagg gggccctgtg tgatgcgggc 24240cctgggctag ccttattaac atattttgaa taactgtatt ggaatgtgtg ccagctttgc 24300caggcttttg gaaacttgcc aggcagcagt aggagttttt gagaggactg tggagatgtg 24360agcctgcaat cttttttctt tttgttcttg gaagagaaag attttagaac acgccattag 24420gcccacctca tgaaatgaga accgggcacc tgtatctcat caccccaact ttcccaacga 24480cctctgtctg ctttagtttt gtttcgtttt aattttgtgt aaatttggat cttctctatc 24540actttttggt tttttgagac agcgtttctc gctgtagccc tggctatcct ggcactcact 24600ctgtagacca ggctggcctc gaactcagaa acccacctgc ctctgcctcc caagtgctgg 24660gactaaaggc gtgcgacacc acgcctggct tctatcactt ttttttttct tcgaaatatg 24720tgctgtcact ctgtagctgg ggctggcttg gaattccttc tgtagatcag gccagacttg 24780aacttttgag agatcgacct gctctgcctc ctcccaagtg ctgggattac aggtgtgaaa 24840taccacaccc agatttttaa ctttaatttt ttaaaagtat cttaggagat atttggtaag 24900ttgacctttg tccagcaggt ttgaagcttt ttctgggagg cagactttct tctgggaggt 24960tttgtgtgtg tgtgtgttgt gcgggtgcct atgtaaaccc ataaatgctt ctaccaggac 25020agacagggta aactatggcc cctgtagagt gatgctcaca ttataaagat gccatatctg 25080tagcctggga tctgtgtctg tccttgacac gtgatcaaga ccaaaacttt gggttctaat 25140attagaattg aaagagaagg cttctagatg tgttcatttg tactgttttg tttgtttgtt 25200tttgtttgtg tgtgtgtgtg tggggggggg gtggtgatga gatacaggtg tccagttctc 25260atttcatgag gcgggtttga ggagcactgc tcatcggagg gcctctcagt gtgaagggtg 25320tgtgggatgg atggctctgt ggtgaggcgc ctttataatc tgtacccagt atgatgcttt 25380cttactacgt accctctcag ttcattttgc ctccttcttc tgtccagtgc tagggattga 25440acccagggtt tcacacatgt gagacaggcg ttgtggctgc actcccagct catgcctctt 25500tgggatggag tgtttccggt tagagacagt agctctgacc tatcaaagac tatgaattga 25560agctgggtgc ctgtaatcct agcccttagg cgggcgaggc aggaggatcg gtggggcttt 25620gagggtagcc tggaatatct agacagacct tgtcaataat aataataata attattatta 25680ttattattat tattattatt cagcaatgac ggtacattta ctaggctcac caaggggcta 25740tagaggtgtc tcagcggtta agagcacttg ttgctcttgc agaggacctg ggttcagttc 25800ccagcaccca catggtggtt cacaaccata tcatccagga aatccaatgc ccttttcggg 25860gctctgtaac actgtgtgtt ccatatacac gcaggcagaa acactcacac acagaaaaca 25920aaacacatct aaaacataat tgaaaaagta gactcaccag aaatggggtt ccccttccca 25980gttgagggct ttactttttc ttttgtctct tcaaagcctg ttcttaatac tttcaatata 26040gggtctcatg gtgcccagga tggttttgaa ctcctgattc tcctgcctta gccttctcat 26100ttgctttgta tgtatcacca caagacttac ttaatttttt taaatcatgt aatatggggt 26160atcagaaata ggaaagaaca catttgatta ttgttactaa aaaggctttt aaagttggaa 26220tttagtttat gggactatct ggcagaaaac cctgagttcc aattctaacc ccctataggt 26280tgaacatggt ggtacctaga tgtgatccta gcactcagga tgttgaggtg taaagaacca 26340aaagtttggg gttatcctca gctatatcgg gagtttaaag ttagccttgg ctacatgaaa 26400ccacatcacc tgccctccaa aaaaattcct tttagaattc agtgtctaga gaagggttct 26460ggccttataa gcgaaactgt acctctactt atttacttta aaaatatgtg tccaaggctg 26520acatagtact cttagtagcc aggctagcca cccactatag cgttcctcct cccacttcac 26580tttctcaagt gctggcatga tagatgtcag ccactgtgcc cggaagccca ccgtttttaa 26640tagatatttt aatgagccac atgcactcgc atttcaagcc tttttttatc cagaacgctt 26700tcaagtttct agattggaat tattgtgtcg ggttcttttg ttgaacctca gtgtctgtct 26760gacttgtagt tttgtcaata ctgcctgtga ttcataggtt tatatggtca ccatttacaa 26820ttaagagggg aaatataact ttgttttttt ccaaaatttt tcggagttga tatataacag 26880actgttaaaa acaaacaaac aaacaaacaa ataaaaaaaa accattgtga gtctttatga 26940aacaatgtgt gtttatgctg aacacctttg aatatcattt cagaaaatct ttcttctctt 27000ggtatagagt gttattttct tcgcggtgct tcttcacctt ggcttgcaca gggtgcaagt 27060ttatgaagcg ctgcaaattt gactgcttgg ctgtcctctc cgacacccgt gagggtggta 27120gatatgaaat ctcactcttc aacaagtaca tgcatgagag taaaacagtt tattcaggtc 27180ataaaacagt gctggctccc agcccaagtt taagctgata gcgtgctggg ggcgcagcgg 27240cgatgactaa ttactgaaaa tctcttcaga atgcgttacc tttaccagac ccaaggaaga 27300cttatttagg ggtacgtcag atatcatagg cgaagctcaa atgctttttt tgttgttgct 27360ttcttatgtg tgtgttgtca atcacagcag ctcggttggt atttgtgggg acacatgcag 27420cctttttagg ggtggttggg ctctccctgt tttgggggta aaaccatttc cacacaagtg 27480aatctgcaga ggctgccttc aggtgtatgt agtgagcttt aactctttca gcccttgtcc 27540tgacttgtct gacccaggag cagctggctc cctggtatat tgtataatgt agactggctc 27600gtcaattcag actagctcgt cctgtttggt gggtttgtta gaaactgcag agctaagtaa 27660atatgcgata acagtttgaa tggtatttga agttaaagag cgcttgcaga ctgactgact 27720ctgagggaca ctgtggccag ggaccccggg gctgcctctt gggtcagttt ccatttttat 27780ctgagtttta ggaaggcacc gtagtaatga aggcctggtc agatgacagc accttccaca 27840tgaggcacgc agtatgtcat gtctgatact acggatgcca ggggttaact ctgtcacctc 27900atgagccagt gcccactcag tgcccactca gtgcccactg agaaaacagg gagggctgaa 27960tggggcctgc tgtgtgggag agtgtttgct gatgactatt tctgtgtggc actgtgttcc 28020ctccgctgta gggttatttc ctggtttctc agaacaacac tgtctccctg ttaacttcat 28080gagggacgtg tcttgactga tgggcgagcc tctaagttct ctgaagggct ttgtgccttg 28140tcagtaccgt ccccctacac acacacacac acacacacac acacacacac acacccatct 28200ccttcataag atcacctctc taggatgcag agtggaagtc ggatgctgac acatacacac 28260acacacacac acacacacac acacacacac acacacacac acacaggttc actttctgat 28320cagagacaag tgaagtctta gaagatctca atttagcaac aacctgtgtc cccaccccca 28380cccccagtga gtcactctta gtttgaaata ccctaaaaga gtctctcatt tctgtaggtc 28440agacaggttg taacctgccc gactgctcag cggcattggt gttggatgat tggagagaga 28500agaggccaga actgtgtggc cttgtactga caacctaact gtaggtctgc ttgcagacag 28560agcagtcctt tgtgcaatga gtttccccat tccattctac tcgtaatagt gaggacatga 28620tagctctatg tgtgctatat ctctttttta aaaaaaatta gttctgaaat tggaatgctt 28680ttcaagctct ttgtttttct gattctgttt tttaaaaaca gttttctgtt ttaaatttgc 28740ttttattgag attgagaaac tactttttct ttcttccttc cttctttcct ttcttctttc 28800tttcctttct tcctcccttt ctttctttcc ttgagacacg gtttctctgt gtagtcctgg 28860ctgtcttgga actctctata gaccaggctg gtctcaactc acagagatct ggaattaaag 28920gcataaacca ctctacctgg ttacttttta ttactctttg aaagtttcct atgtatatat 28980aattttgcac ctatttttgt gtgtgtgtgt gtgtatgtgt gtgttggctg gcatgcatgt 29040ggaggtcagg ataatttgtg ggagtcggtc ttttccttcc accatgtggg taccaaagat 29100agaactccgt tgttcaggcc tagtggtaag cagctttatg cactgcggtg tccttcctgg 29160ccccactgtg gtctttttct gttcatgaac tgaggattgg agaacctctg ttcattacag 29220tggccgcttg tgtgtgggta accgtcagag gtcaggagaa gataagatga gggttagtgt 29280aaggcgtcct ccttggacat ttataaagtg gtgtcagcat caagagtgtg aaggatagtt 29340ggcaatggga acttcttttc ctgtaagtcc cttccccaaa ccctctccct ccctcgtgaa 29400aaaaagtttg catgcaagtt gatgcttctc agaatcctga tgcttgcttt cagaggaatt 29460ttttttcctg tgccaaagtt ttgtttttca tgtgacttct tgtagcaact tgggtcttcc 29520tgaaaagact ttttaaagtg cattggtaaa tgtactggta ataaacccaa aggcaaattt 29580actgtagacg tttgtacagt ttagttaaag ttactaggga aggcaagcac tacccacagt 29640gattgaggtg aaagtgactg tcacttgtag gtaaggcaaa gtcgcttccc actggagagt 29700gagtgtgagc tccagagaag cttccagttg ttcctgtgtc ccaccaggac accacggctt 29760ctgctcctcg ctgacccagc taggtggagt agcatcatgg cgcgttaacc aggaaaagac 29820gcttcagctt tcaggctgga cgtggaactc tgtggtaaac ttccatgtgt aaacccctgg 29880atcaagtctt agtctgtctg tctgtctgtc tgtctctcta tctctctctt taggtgtgtg 29940tgataaactg tcaaacacac aagaatgctt tttctttttc tttttctttt tttttttttt 30000tttggttttt caagacaggg tttctctgta tagctctttg tagaccaggc tggcctcgaa

30060ctcagaaatc cgcctgcctc tgcctcccga gtgctgggat taaaggcgtg cgtcaccacg 30120cccggcttac aagaatgttt ttttaaaaca gattttttaa tgtgaatctg tatatccatg 30180tgactatatg acacatgtat gctgaggtca agagagggca tcggtcttcc cagaacccgg 30240gttgcaggta gatgtgggtc ctgggaactg aactcagtaa gaatagcaag cgttcaaact 30300catactgagc cacctcttgg aagatgtttt ttttgaggaa agagtggtat tacatcttga 30360cctgattggt aaaaacaaat cttgtttttc tagttttggg ttcttttgct acagggtctt 30420gtctttgtag cccaggctag ctatgaattt gcagtttagt ccagtctagc ctccaactct 30480gcatgcatcc tcctgtctca gcctgaccca tgtcatagat gatcaccatt aaaacactac 30540aataaggccg ggcagtggtg gcacacgcct ttcatcccaa cactcaggag gcagaggtag 30600gtgaatctct tgagttcaag gctaccttgg tctacagagc tgagctccaa gacagccagg 30660gctacacaga gaaactgtct ccaaaaacaa aaacaacaaa caaacaaaca aacaaacaca 30720aacccaaaat gagagcgaca gaacacgatg gtaatatact catccttcca ttaatttgaa 30780gaaatgactc aggaatgcca ataagtgctt gctcagcaac ttacagatgc tgactaagat 30840gattcgttgc atgtgtttgg tctgaaaact taacagtggc tttgaccaaa atagtaagct 30900gttacctgat gtgccatatc agttaagagt tgctgttgta agcaacagaa atctctgctt 30960taaccggaag gggaggtttt ctccctctct ccctctcttt cccacccctc ctctccttcc 31020tccctcctcc cctcccccct caagtcctcc ctttcttgcg cgcgcggggg gggggggggg 31080gcttgcaggt attcgggagc tcatagactt tctgggactc agcaacccat gtctggggcc 31140tcattcttgc tccttgagat gattcaaaag ctggggtgga tttgctgagt gaagccagtg 31200tagtgaatct tgtgcctaca ttttaggtta gagcagtggt tcacagcctg tggattgtga 31260cccctttggc aaacctctgt ctgcaaaaat atttacctta tgattcataa cagtagcaaa 31320tttactgcca tgaggaagca atgaaaataa tttggggggg ggaggcagtc gctacaactt 31380gaggagccgt aataaagagt cacagcatta ggaaggtttg agacccactg ggttagaggg 31440aacacagaaa atgattatct aagattttca gttttgtggg gttaagggtt ttctgacaca 31500ggaaggggag accatacatt gggtaacaga aaagaatcag ttttcacatg tgccatgttg 31560gtggcatgtg ttggacaaca ggaaggctta tgtctccatt acatgtgaca tgtggtgaca 31620agtggaaaaa ataaagatga gctccactct tacaaatacc ttaactagac agtggtctct 31680gggaccaggg aggttgtcca ggaaggaaag gaacttgtga agtcaaggat ccgaagtcgg 31740tcccttgaac tcccgtggca gcaggagagg actgcctcct ccccagcccg tacacaaccc 31800ctcacaggca attcacagga aaaatataaa atgtctgtta accgccatga gaaaaatgta 31860gtaagaccaa tagttttcag tattcacaca gtagcatcca caaatactaa gtagatattt 31920ggtgtagtga agggctgttt tgtggttggc agctccagga cttgggggcg gggtggggtc 31980tcagacaaag gttaattcaa gttggggtgg gagtgcagtg tgcctcgtgg ttttatgagg 32040tggctctgct gtgtttctcg gtcccctcgc actttctctt ggtggaatgt ttgtcctcac 32100acacactcct gtgcaacccc tgagctccag tggaccccta ttttgggggt gccaaagtgt 32160atggcgccca gactgtacct gccagaactc agaaggttcg gtgatgcacc tgaacagata 32220ctttcagcac cagctgcacc ttggcctgtt tattttctca gtcactgtgc ggctggcctt 32280tgaggcctat agctctaggg tagtgcagag ctgtagtggc tcaatgattt ccccaccggc 32340aagccctcca gagaggccag gtggtggtgg tggtggcggg ggcggcgggg gcggcgggga 32400cttggctcca ctgctccctt ctttatgcag aaggaaactg atgctcagac tggctcccca 32460acctatgcag ctggaatggg gtgacagatt caaagacaag agactgttca aaaccctttc 32520cctggtcttt gaaccagttt cctcctcaca gacatctttt agaggagaac agagagcctc 32580cctgacctgg agagagtttc ccagcgcttc tgcatcacgt cccttcgccc acgatttaac 32640ttttttcctg agttgaaaga tgtccccaga aagcgacaga ggccagcacc acaggttctg 32700gagtcacagg gtgaaagata gcctgtggtt tgggtctaga ggggcgttca cagaatgaag 32760agagaacagg ctcggaagca ccttagagga ggagggacta tcaaaagatt gtttcggact 32820tggcaggagg caagccagaa gagccagaat gggaacggct gccggagcca gaacacactt 32880ttaaaaggaa aatggatccc ggccaggtgc tctggagagg gtggggtgaa ctggccaagc 32940aggaagttac ggcgacgttg tgtaatttgc agttgtgttc tctgcctacc ttctctggct 33000aaatcagcct agaggtgcag tgatcttaaa gagagaaagg aaaaaaaagc ctagcttgtt 33060ctgggacagg tgcagttttg tgcttctggg ttttagttgt ttttcagtgt ggccactttg 33120gccttggggc ccaaatatct tgactactct tggtgttttc ccgtgccgca tgctgcagct 33180gcatcgttgc tttaggcagt cctcctgtaa gagtaactgg gctcttacat tcagaggcta 33240ctctgccacc tcccttctga ctcgccagtg gtaacttcca tccagtctga cctctgcttc 33300gtcccctttc ctgatgttac cctgcaggat cattgcaatc cacctcccaa aggctgagga 33360tttaaatgtg caggcttctg ggctggagcc atgcatggtc cccagcatcc acacagcaac 33420tcaccatcac ctgtaactcc cgacctctgc aggcttccag ggccaccagc cacgtacatg 33480gtatacatac agacatgaag gtgaaacacc cataaataaa aaactaaaca aacaaacaaa 33540tacaaatgtg gctagctaga ttattctgat cagccttccg gagagaacta tacagtgctt 33600tggtgtaggt aagaatacgt gtccccatgn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 33660nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 33720nnnnnnnnnc tgctgtattt ctcagatttt aatagtcatt ggtaaaaatc ttggaaagca 33780ttccgtgtgt gctatatatg cacaggctct agtactttat ccagtattta cacgtcactg 33840gttactagga tgaacacgaa gaggctttag gcaggatgag agaatgtggg taggcttttt 33900gcaaatactc cggcacttgt ggaagggact tgaatctaga accagctccc aacccacaca 33960tacacacacc cagggaaatg ccaaggtacg atcatagcgc agaaaagagt gagcatcttc 34020ttgaaggcca cgggtgcggt tggatcatcc aagtgaagcg cacttggatg accggaaggt 34080tgagtatcca gagtggcgtc tttgtcagct gagctgagga ggtgtgctac tttcttctgg 34140ctttttggta cagcctcggc ctgtgattcc agcttctctc cattgtgcta gggtctcgaa 34200gagcccttcc tcaaatccgg cgagcagctc cagtgtttgc ttgtccccca ccccgcccca 34260cgtctgtgca gatgagtgtt tctccaggct ccttgttgtg gagatctgtt caaagaggtg 34320ggagaagggg aaggactcaa aattaaacct ccccatgtct acttttggtg atctgttgct 34380gatctccata tcccttcacc ccagtggagg gacctgttaa ctggcttttc tgagtgcctt 34440tttctttcta ctctgcatta cccctctggt ctcacccctc tttttctaac ccttaagggt 34500tagttggttt gtttttgttt tgattttgtt tttgtatttt tccctctgtt agaatccatg 34560tcttggtctg gagagatagc tcagtgctta agagcactag atattcttcc agaggtcttg 34620agttcaattc ccagcaacaa tgtggtagct tacaaccatc tgtaatgtgt tctgatgccc 34680tcctctggca tgcagatgta catgtagata gtacactcac acattaaaca aacaaacaaa 34740caaataagta aatctttaaa aagagaaaaa gagaaaccaa gtctagcaga ggatgtgaca 34800tataggaggc actcagaatg tggggctcgt agctagcttt gtgatagaac ctgtgtggac 34860catgtgaggc cctaggctcc tccttcccca acaccaccaa taaataaaca agtaaatctg 34920cctgcctgcc tgcctatcat ctatctatct aacatctata tatctatctt gaatgaacaa 34980gtatcagaag gagagatgga accagctctc tgtcctcgcc cttgcatccc ttaggctctg 35040acttgaagga gtctttcatc tggagagcat ttgaggtggt tcccactctg aagctctgag 35100tagtttctac atggaacatt tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtggtgttt 35160agtgtctctc agctgcaata aaccataaca gacatcgttt ctatgggaac atgcattctg 35220agccctgatg aatccattgc atccaagccc gtgagagaac ttgtcagtca gtggggacct 35280taatcaatca catgtctcct gttcggcttt ttaaccactt ttcgtgtaaa tgaacaacgg 35340gctgtttctt tgctgaagga ccagaatgag agaagccttg tcttctgaga atataaatta 35400ttacatttta aagtgaatct ccttcatcca acacacaggg ctagtttatg ccaagtatat 35460ttgtagtgtg cctagcacag gctgcatgat ggtataggag tgcttcacac atgtataagt 35520aagaccgtcc cccgcagagg tcaggattag ctttcactta aagaagactg gaactcagcc 35580tcgaaggtcg tttccagatg tgacgcggcg attctgcgcc tttggaactg ctttatccat 35640ttgcctgtct tccttgactc acgcggtcct gttaccaaat ttatttttag acacagtgga 35700caaaagcaca atgacatttt caagtagcca atagcagtgt caccctttca agacccgaaa 35760atgtccctgc tatcacgacg caaaacctag aagcaaacag tatggtgctg ttttatttcc 35820tctcactgta ttttggggca taaccgatta ttgacttcgg agtgggttag ccttcctaat 35880ttattttatt gtattttact tactctgtgt gagtgtttgt gtgttatatg tgtgtgcgcg 35940catgtgtgtg gtgtgtgagt gtgtgtatgt acatctgtgt gtgtgaatgt gtatgagtgt 36000gtgcatgcat gtgagtgtgt gtatgtacat ctgtgtgtgt tttgtgtgtg tgtcagtgtg 36060tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtacatg gatgcttgca gacacacagg 36120tcagaagtca gttttgaggt tcagttctgt ctgcccgtgg gccctggtag tcagacgtgg 36180ttgaggtttg ggggcttttg ctcactgagc catcttgttg acccacacag tagattttta 36240ttctaacttt aaaaaaaaat ccttaaaaaa agaaaagaat aatggcacat caagaaaata 36300gcataaaagt agcatgttaa agacttaaag tccagatact ttcactttaa aaatctatgt 36360gatgccaagc agtagtggta cacaccatta atccctgggg aagcagaggc agttggatct 36420ttgagtctga ggccatcctg gtctataaag tgagttccag tacagccagg gctactcaga 36480caaaccatgt cttgaaaatc caaaaataaa taaaataaaa taataataat acaaataaat 36540aagcaaaggg agaaaaagtg tatgtgatat tccataaaga tgatctatat gtgttaagaa 36600tattcacttt tggctggtgt ctgtttgttc ggttttttga gacagggttt ctctgtataa 36660cagctctgac tgtcctggaa ctctccttgt agaccaggct gacctctgtc ttacaaagat 36720tctcctgcct ctgcctcctg agtgctggga ttgaaggtag ctgccacccc tccaccatgt 36780cctttctttc tgttttcttt cagtctgtgt actttctgca acagttgtgt tcttctttcc 36840tgtccacatc acatgcgtac actcactcat gcacacagca cacagcagag cgtctctaaa 36900gtctttctgt ggctcagact tctcaaggtc agctccgtag tagagcaatt tcacttctct 36960acgccagcta ctgataagta tttgtagtga tatgtatttg ttgccgtgtg gatttctcat 37020ctaacatcag agcagattct tttaatatga aacttgatct ttggaatctg gttcccataa 37080ttatgagaaa ttgaaacctg gctcatgaga gattagttgt gttggaggga gagaacaatc 37140catccgttgt atgcagtaac cttccttggt gaggctgaaa gcacctgagt cactggggtt 37200gaaggcagac actaccacag taggcttgtc tgtcacacgg gtgctggcga tggaactcag 37260gcctgcatgc ttgtgctgca agcttgtgct ccactgctgt aaaagcattt tttcataact 37320ggcaagtaat agctaagtaa tagtttcatg atgggaagga aaatcaacag ccggatttag 37380aaaattaaga ttatttaaca gacactgaca tctgcaaata actggtcttt ttcctatggc 37440aacagaggca tgtatggtgg aacaccgcct ctggtgctac tagcctggtt cttgggtggc 37500ggggggtggg ggtggggtgt tagtcttaca ctgaaaactg cattcacatg ttgatcttcc 37560tactaacttt gttttttttt aaagatttat ttattattat atatgagtac actgtagctg 37620tcttcagaca catcacaaga gggcatcaga tctcattatg gatggttgtg agccaccatg 37680tggttgctgg gatttgaact cgggaccttt ggaagagcag tcagtgctct taacccctga 37740gccatctctc cagccctgtt tttatttttt aatatccaac atgatccaac atgatctagg 37800gtcttaaaag acaacaaatt catttttctt tctgggtgtt tttttttaaa ctttttattg 37860attctctttg caccccagtc ccactaatct ccccaccccc tcatatctac ccttctcctt 37920tgcaacgtcc tcccaaaaca tacacacaca cacattgtgg aagctgtagt gtgtcacagt 37980gtgtcccaca gtgtagccct ctgtccacac atcttcactt gcaaatgtgc attgacatga 38040gtcactggtc tatgttcctg gtctctggct tctgggacac catcaatact ggatcctcac 38100cagaactccc agttattctg ttgttgccct gtgccatgga gaccctgcag ctttggaaca 38160gcaggactgg cccttccatg catccaacct ttcacagatg acacagattt gggggtaagg 38220cagttcagag ccctgggtgt gggcctgggg gatagctgag ctagtcagcc caccggctct 38280ctgttatcca caccaccaga gcaagctctg gctaggttac cactcccagc acccacccct 38340tcagagccag ctctgatgct gcccagttca ggttcagggc cctctccaag tactgtaacc 38400ttgaggggct gggccagctc tcctgttctc acacccttag ggctggctca ccagtgcccc 38460cttaaccagg tccatctgtg ctggtgtagg gctcactcat ctgagtgttg gagctggtga 38520tgggcagcac taactctccc aaggtcatga ccctgtgggc agctttcttg agtgctggag 38580gtgacgagga acaggggccg tcacctctgc atcagtccca tcccaaggca gatgagtaac 38640ggggtcagct ctcccatatt cctgccctta gggctgacac acacacacac acacactgcc 38700aggaccagct ctactcttct gtctaggcga agcacagggc ctgctttaca gagtggtgca 38760cccaatgaga ggcagggcat cccctggata tccgcgtgat cccctcaacc tgatccagcc 38820ctactgcgct gcccaggcaa attgtcgagc ctgctctcct gagtgctgca actggcgagg 38880gggcaggtac agtgactgcc ttgaccagag acatctccaa gttctctagt ggtaatacaa 38940accacagaca ttgatagtga cccctgtact acataggcat ggactcacac gtggtcttca 39000gtggcagctc aggctgggac ctcgccatgg ccccaagtga caggcagggc tggccactca 39060caacaagcta ctcctctcca tcctccagtc tctcgttcca tctccttcat catgcccaag 39120ctgctccact tctttctctc tcatctgacc accacatact tgcatatcgt ggtggctcgt 39180gctgcacact ggacacacag ctggtgggcc cctggacaag tcctccatcc atgttgcatg 39240gtgtcgtggc aagtgtatgt ctgtggcctg cctgtgctgt gtgctggagg gcaggtctat 39300ggatgccatg gtggtccaca ggtctctttc tgtgctgccc ggatgtgatt tggtttggtt 39360tggtttttat gagtcctagg cgtaagacag ctttggccac caagccaggc tccaagttag 39420aatgaataga gaggcctgcc atctgctctg ccccttggcc gggttggggt gggcaaatca 39480atgttaaact cttaaaggtt tctggcgttt gtattctaca ctctgtgcac gttgtagtct 39540tcactcgtaa ttttcaagtg caagggagga gcccctgctc ctgtgccagc tgtagtttgt 39600agttatccac agggttttgt tttgttttgg agaaaaaata agatgatttg ctctagcttt 39660gaagaaacga aaaaaactat gggctcctat cagcatggca aaacatttgt tgtgttttat 39720ggctaaattg ctttctgaga agagaaattg atttcaagaa gaggaacccg tgagcaggac 39780cacacagctc atttgagaag aggggagaaa cttgactcct ggggccttag tttttgtttt 39840ccctttagct gatgtcaagt gtcttggact ctgctgttct tggggtagat gttggttgtg 39900gttgttcaaa gcttggacag ggaggtggct gtagaaaggg ctgctttgca ggagcgaggt 39960ctgccaggaa ccatgcccct caccacagcc tccattctca cctccaggcc gatccatata 40020atgggtgcct tgtcaggaag cagggcagga ctcaatgcag tgtactctgg tgggaaggtg 40080gacactgtct agcacaaatg tcttccacgt tgacatcagg gaactctgtg ggaggatttg 40140ttaactcaaa gatgtgtgaa gttaccctgt caagtatcct aaagttgtat ttactctgca 40200tccaacttag tatattcagc taaggggaac aaatttctgc tccaatgggc ctacctacat 40260cactgaacca tccctctaga aagagttgtc tctgcattga catctgtttc ctgtaacttc 40320ttgttttcct aaacttgttg tttgcttgtt tgtttgtttt tggatgtagg ggagggtggc 40380atgtactata gtgtgcagtc caccgcactc ctgttgagtt caaaaaatat cttgtgggag 40440tcagttcttc cttctaccac atagcatctg gggatccaat tcaggttgtt ggccttgtct 40500cacctggcca cctgtttctc tctccctttt ttgaaactct acaagacaaa gctactttct 40560tgcctttgtc ttttctatcc agacagtttc attgttattt ctttaacagt cctccattgg 40620ctcagctcac acttactgca cagctagtgt gggcatgatt tgttcttggt gcccagtgtc 40680agacagaggc atcttgaagg gtaacgactg atatggagcc agtgaatata aaacatgctg 40740tctgccagcc ctaacagtgg agacaaagag cagttaaccc cggtcacgag aaaactgtgg 40800tttcaataag gtttcaattc cttatgcctc aaccaggtga tgcagttgcc aagaagccca 40860gccggactgc ctcatcaccc tgtggatgag tgatgagagc caagaagtcc cacgtacttt 40920cttcctgtca agcaagcctt agagtgcttc ggacagacag cagcaaacta ggaagtgttt 40980ataaggtctc tccattgcca ccttgaagaa agccaccatg tccctggttc tgggctcagc 41040ctttccagtt agatcaaatt gatcacacag catgatttca gtcccttaac tgtgcgaatc 41100tttctgtgta taattaatat cgcctcttaa actgctgctc tcagaaggca cctggtgtct 41160ccaggtgtgg tttcttctaa tgccttttga ctttctttcc ttgagctagg ttttgttttt 41220tgttttttgt tttgttttgt ttttacagga tttatgtatt tattatatgt aagtacactg 41280tagctttctt cagacacccc agaagagggc atcagatctc attacggatg gttgtgagcc 41340accacgtggt tgctggggtt tgaacctagg accttcggaa gagcaatcag tgctcttaac 41400cgctgagcca tctctccagc ccttgagcta ggtttttgtt tgtttgtttt gttgttttgt 41460ttaattaggc agttgacgat tgaggtaccc gtaaaagaat aaaagtagaa gaatgctctg 41520gttggttctc agtgtcaaag tattgctagg ccgcttccct tgttcagttt attcctgtgt 41580acagtatagt aacttagatt tctctctctt aaaattataa gttctttgct ttccacactt 41640tattttctgt caggatctta tttttttaat ttgtttattt ttatgtgtgc atgtttggct 41700ttttatgtgt ttttgtgctt tgtgtgtgtg cttgggaccg gtggaggaca gaagagggtg 41760tcacatctcc tggaactgga gtttcatgtg gttatggcag ttaaataagt taactagtta 41820atagttaata gtgagctgtt gagtaggtgc tgggaattga accggggtcc ttggggagaa 41880ctgtcagtgt tcttaactgc tgagccattt ttccagcctg agatcctttt gaatattgtc 41940tctcaccctt actttgccat ttgcagaact gataagcaca tttgcagtct tcatctaggt 42000gattaattca attgtttcct taaatgatga aatagtacca ggctgttccc agtctcttaa 42060gtgacttttc ttttaaaatg gcattgcagg gagctggaga ggttggagag gtggctctgt 42120ggctaagggc atttgctgcc cttgttatat ggtggcacac agaaacaatt ataacaactg 42180cttcagggga gccaacaccc tcttctggct gctgtgggca gcaagtatac atgtggtaca 42240cagacatata tgcatacata catgtataaa tgcatgcatg caagtatgca tgtgatacac 42300agacatacat tcaggcaaac acacacataa aataaaaata agtaaatcca ttttagaaac 42360cacaaacaac aactgaaatg attttttttc caagatggca tggcagttaa cagtgagcac 42420tcttgactct gtcatccaca gtctcatttt atccaagcat tgactgcagt ggcttcttgg 42480tccttaaaga tgagacatga ccacaattta aagtagtcac ttttagctct tctctaactc 42540attcattatc tggaatggtt aggtagaaat ggaagctaaa tctcttgtct ttgcacgcca 42600ccctttcttc tggtattttc ctcttgcttc tggtaaacct taaaagaaag tgggaggaga 42660ctaggctctg ccctgtggtc cgtgctgctg tgtcgactgc tgttgggctc tgtccagttc 42720atctggcctc cgttcgggag ccacattcct tcttgtattc agtcttagtt atctgccaaa 42780gcactaggga atgtgtttgg cttagttttt aacttatgat ctcaggtcag ttcaatccct 42840ggggcctaca tggtaggaag agagaactga ttcctgtgag ctgtccttgg acttctgcac 42900gagggctaca gcatctgtgc cccccccccc acacacacac aaaaataaat aagtaaatgt 42960gattgacatg tttgctttct ttatgttttt caagacaagg tttctctctg cagccctgtc 43020aatcctggaa ctttgtctgt agaccaggct gacctggaac tcacagagat ccacctgcct 43080ctgtcctctg agtgctggga ttaaaggcat gcactaccac tttgtggctt atatcacata 43140ttaactctgc ttaaatgact gtggctgatt gtgactttct tttgtttgag agggaggccc 43200atatagctca ggctatcctt aagctcactc agttgctaag gctggccttg aactacctca 43260cttacagatg tgcctcacca tgagtgactg acaatgtgac tttcaaagga gaattattaa 43320agccatttct tgcctaataa agaagtccac tgagagtggt tcttaggctg agcagagggc 43380caggtttttg tttttgtttt tgtttttgtt taagctacat ttaaagtaaa gaagctactt 43440aatgtttatg tgtgtgtcct gaagaaaagg caccaacagt gtgtgaagtc tgtggacttt 43500actccagggg tcctgtgtct gtcctttact ccaggggtcc tgtgtctgtc ctttacttgt 43560aaatgatttt cagctgacaa gaagatgtag cagtcacgct tatagttgtt cagcatggct 43620gacacagagt acatgtgggc accattgtcc acatgggctg atggtgaaat gccaccattg 43680gggtggtggc tttgatggac tctgctcttg ccccacctgg acagactcat tttcagaagt 43740gtctgagttt gcagggaccc cacacaatgc agaatgtcct cggaagcagg cagatccccc 43800cccctccctt taagcaagac ttccgcttca ggtgatttcc ttgaactaac actcagcggc 43860cattacagta ctgttatttc tgctcatggg attttgggtc atagccttaa aaggtgactc 43920aggaacaact ggcatgattt tgttagattg gaaacatttg aatatgacca agctttttgg 43980ttgaatgtat ctactttaaa ttcatcaatg agcactggag agatagctta gagattaaga 44040tcactggctg ctcttccaaa ggacctgggt tcagttccta gcacctacat ggcactgcac 44100aattatctgt gacttctggc ctctctgggc accaggcaca ctggaacaca aatatacatg 44160cagacaaaac tcatgcatat agaatcaatt aattaaaaag ataaaattca tcgatgaaag 44220caccttaata atagctgaag attaaattcc atttagattc cctctaaaga tcctgtgggt 44280ttctgtaagg ctcctgccta cttcataccc tgctgttctg gtggctttgt gtcctttgct 44340agttggtggt ccctgcaagg caagaatcac cacttactgt cttggctccc agcactggtc 44400accgagggat ggagattgag aggaggatct gactttgagc tctcttgcct cagaatagga 44460gatgctggga atggccttca actcttgact gtctagtttc ccctacttta gcctgcagtg 44520ggtcttagcc ttggcatggc tgccattttg gcctgtgaga ctgttttgat gcctgtgcta 44580attaattgtg tatttggtct ctaatcattg aatgctgtgc tctccacccc tcaaaaaatc 44640tgacatccaa aatgtctcca gccatagcca aattcccctg ggagcaagga gccttaagct 44700ctctggtaaa tgctgagcag ggccatcagc aaattataga cgttcagctt tggaaaggac 44760tgaaaactat tgccctctgg ggcactcctc cctaccccag ctctgaatta gtgaatattt 44820gaagaagaag aagaagaaga gggagaggag gaggaggagg aagaggaagg agaaggagaa 44880ggagaaggag aagaagagag gaggagaaga ctgtatgtgt gttaagactg ggttggaatt 44940gtatgattat ttggttcaag tttatttaca aaaggtgaac ttgttttacg gtaaacgttt 45000ttacacttat ttatttactg tggcatgagc ggatgcatgg gaatcctggc atgtatgtag 45060agaggtcaga ggacaacttg caggggctga ttttctcctt ttaccatgtg ggccgcaagg

45120gtccaagtca ggtcagcagg cttgttggta aagcatcctt acctgctggc ctgtctcacc 45180agccctaaga aagtacagac ttatatggcc ttattaacat cccaggaaga gtagtgtcta 45240gacccactct tctttaaatg taagatacat agtaagcatt tctccagttc aaacgaactg 45300agcaaactct atgagtcacc ggaacctgcc ttctggcgta gccctggagt actgtgtact 45360gtagcttctt ggagagattt aagtactcat gtcaagaaag agacaatgat ttgattcccc 45420ttcacctgtg ccaaaaaacc aacttggttt ttcctctctt gagaattgcg acgaggctca 45480ttgtgtgact cctactgtgg gttgttgttt tataaggggc gtttgggtct tactaggttt 45540cctcacatga aggtaagaac accaaattaa atattagctg cttgtggtta ttttaagact 45600catctcaata acctagagaa tttggttctc acactttgta ctttctgatt acaaacagct 45660tttaaatggg tttatgttgt ttttccatca ggacttacag agggtgggca gcggagggac 45720gcttttggga ccaacatgag gcaattcaga gtctctcttg cgtgtcagct ctttctgctt 45780tgatctctat ctattattct tactctgtct tttcaaagat tctgtttgga gaacttctct 45840ttagcgagat cacaagagaa aaatggcagg acgctgtgct ctggagtcat ccacctattt 45900ctgaggtagg gtaaaatctc actgacatgc gctttcttgg cctttggtgt cctgtgctct 45960gtttagatgg ctcttttttc ttttatttgg tttcaggtag accctagaca gtctcaccta 46020gatgtaacac aaatgtctct aaaagttgtg gatgcatgtt tctgtgtaac tcggaaggta 46080gcaggaggct tcaggagctg ttttcttcat gttttcttca tgttttcctt gcttcatcgg 46140cagatcaggc tttcaatttt ctctgttctc tctttttgta acctctttta aatattaaac 46200ttgatataat ctctctgaaa atttttttga ctatgtatgt tggtgattta cttgttcaaa 46260attaataact gcaaagcctg cctgcctgcc tctctgcact ttgtatttga aatctcttag 46320cagggagaca tttttctcag aacaaatctt ttggcagaca tcaagaagtt aattattcct 46380acatgaaatg ctacttggaa ctttttgaaa catttcaata tctatgctga tttaaccaaa 46440agaaaagaaa atgggttctg ggttgaataa ctggggagtc ctggcagaca tgaccgtgtc 46500agatgccttg gcacggcctc agggcagagg ggactttccc acttctcatt gtaatctccc 46560atcttcgtat gtgggagttt tgtgaaggtc ctgtgtttca ctgaaatgct cagctttccg 46620cactatgtat actgctctgt gagcacaggc tctgtggatg tctacacatg tttaaaagaa 46680aatgcaaagg aatggtggag aaaggcgtca ctgtggttat tattagctca tataataacg 46740tgatccttaa ctttgcagtc ggtcggtcag aatgtaaagt ggaacgctct gtagaaacag 46800gacatcatgt gtggtggtga gggtggcagg ctctcagcat ttatttatcc catcatctct 46860taatagatgt tgttgtgcag gccagagatg ttttcatcca agcagggtca gtggtacctt 46920ggcatcaact tatgttcagg agtcaaagtg aaaacacagc taaaacacta atgaagtcta 46980tagcttggaa aaggtgtctt ttaagtctca tcgtccccag tccctgcatg ctttctccat 47040cacccccagt ttcaggtctc acaggcccag ctctggctac cctctgatgt cagggagctt 47100cacgtcacca gacccctggt gccaaggtgg aggtgagaag aggagttaca ggcagttgtg 47160agccaccata tgggttgtgg gagcagaacc tgggttctct gccagggcag accactgagc 47220tacctagaag tcgttaaggt atcagtgttt gtgtcaccat tcaaggagca ggcagagtaa 47280cagtgttggt gattagaagg tacccttttt tcatttttga gtaaaaggat ctcccacctg 47340ccaaccttga gtcatttcag aggacatcaa cgtcttgtca cactttgtag ccactgtaag 47400tttgtcacca actttacaga agtaggtgtt gagatcctag actagattga agccagaggt 47460agtctcctct caccctctct tctgggcatg gggtgtttac tgagcaggac aatgaagaag 47520tgaggtgtca ggaaggacag gttccttttc ctccgtgggt tccattagtg tggtgttagc 47580attgcagggc ttgtgttacg atgggcccag tggttctttc actgagtctc gtaaagactt 47640ggagaacatc atttacttaa ctcctggact tcaattccca agtccccatc tatcttgatc 47700cctacatatt gatgtatcat actctgtcta ctgtctgtct gtctggcagt cgtttgtctg 47760tctgtctgtc atctacctat cttgaacata cttcccctct tagccccctc tgcctctttt 47820ctcacccctc attcttacta ttagtccctg gccttcctct ccacaacatc ggaagattac 47880atctcgaagc cctcttacct gtgagtcaca gtctcgctat gcttctataa ttataatcat 47940caggcttttg atgcctactc tttttaattc aagatagcct tagccaagct gttaactgca 48000gattaattca tatgctgtca tgataggcaa tttagcaatc catagagatg cagacagaca 48060cagaggggga ctgactgtag agtggcccat tcttctagtt taaggccaac ccacctagca 48120agctcatgtc aaataatgtg ggcgactata tattatacct taagagatct gtgtaaaatt 48180gactatcgct gcttctgaaa aggagtaaga aagttagatt attataaaaa gaactgtagc 48240ctttacaaaa gaatttaatc tcaggattct tcatagtagc aggatctcca gtttggttaa 48300actgtaaaaa tgaatcactt cttagcattt tggcagaggt ctagtgtaaa atgcaccatg 48360acttatagag gcttggcttc cctatgccag aaatgcagtc tttgaacagc actgctcagc 48420atgagccctc tggggacatg ctatagggct tgagaatttg tgctgtactg gagtttgcag 48480gcatacattt tccctttcct gtgtccctgc ccacacccaa agaagtgtcc attaaaatat 48540ggatgattga cctcagctgt tgactgaggg agccacctgt agagttaacc tttctgtggg 48600gataggaaca tggagggttt cgatgccagc actacctctc aatgatttta aacaaggtac 48660ccaactcccc agagtcttat atttgacccg ttacatggga agagggaacc tcaactgaga 48720aaatgccttt attagattgg cccataggca agtctatggg gtgtttttct tgattaatga 48780ttgatatgga agagtccatc ccactgtggg tggtgttgcc cctgagcaag tggccatggg 48840ttgtataaat atgcaagctg agcaaactag taagcagagc tcctccatga agtcttgcct 48900tgccttctct aggatggact gcgattagat gtgtaagcca tgcagtccca taggaggaac 48960aacaatatga accaaccagt accccaagag ctccctggga ctaaaccacc taccaaagag 49020tacacatggt aggacccatg gttccagctg tatatgtagc aatggcctag tcggtcatca 49080atgggaggag aggcctttgg ccctgagaag gctcgatgcc ccagtgtagg ggggtgccag 49140gtccaggaag tgggagtggg tagattggtg agcaggggga ggggggaggg gggtttcaga 49200ggggaaacca aaaaagggga taacatttga aatgtaaata aagaaaatat ctagaaaagg 49260ccaagcgtgg tggtgcatgc ctttaatccc agctctctgg aggcagagac aggtggattt 49320ctgagttcga ggccagcctg gtctacaaaa tgagttccag gatagccagg actatacaga 49380gaaaccctgt ctcgaacccc ccgcccccca aaaaaagaaa gaaaagaaaa gatataataa 49440taataataat aataataata ataataataa taaaagaaaa catctaagtc agaaataaaa 49500aataaaaagt aaaagatgtg taagccaatt aaatgttttc cttccccaaa ttgctttggg 49560tcatggtcct tatgacagca atagaaagca gaataagaca ggatctgaca caaagtgggc 49620gctcagcaaa ggtcagcatc ttgccctctt tctgtgtggg cagctcacct gcagtgtctc 49680tagccccata gggttgagct gtgagcctgg agcactcaca gtatgagagt gaagtacagt 49740ggctttaacc atagcctctg gagccagaca gcctacatgt gcatcagctc actgtgctct 49800tgaagagtgt cttcctatca gtacctcatt tgcctgctct gcaaacggag gctaatggca 49860aatgttcgat tggactattg aagtccagca gcctctgagt cgtgtagagt gcagagaaca 49920cgcctacagt gttgtgcagt gaagctcttt gtgttgttag agctcagtgt ggaccaggct 49980ttaaaatctc atgggccgac agcaggcttc ttagcagcta atagctcatt ggaggaaact 50040cttgcttccg aacactcagg ccttggccgc ctctttaaca agaggtagct gttctcttag 50100aagctgccct ggtctttctt ctcttccctg ctccctgcat ccactctggc tggcttttgt 50160tattaatgtg gagcagttga agtcattgac ttgggtgaag gccaggcagc attactgaag 50220tcatatatta ctttcctgga ctggccatga gcaggatgat aggagtcggg ggccagggaa 50280gccgcctctc taggcagccc acatgaaagg tgtgtttgtt ttctccatca tatttgtgta 50340agtagatctg aagcccattc actgtctcta gttttcccca cgggctgctc ttctctgctt 50400aggctgacta ctcctgtgtg tgtcgcatct ttctttgtcc tttgtgacgt cattgcacaa 50460actcctgtct actaattgca ggtagaattg ttgagtgagc ctggctgtat ctgtaggcac 50520tttataatat gctaggtcag gtctgtgctt tggtgttggc ttttgttttt ctcatgttag 50580tggaatactg ctgggagaga gtccttagtg acagtcagtg aaagtcttcc tcctcggagt 50640gtggttgaag cacacacttg tttgttctgc tctggtattt ggggcctggg aaacggcagc 50700ggaatgttat taggcatttt cctgaacttc ctacctgctc tttgctggaa tactccttgt 50760ttggctctgg aaaggttcca tgctttgatg agcacagacg ctcgggggat cggaggctgt 50820tttgatggtc tgttggcatc atgaaaggag atattgtttg ccaggggaga tacagtctgg 50880gaaagagtgc tcaaggatcg tgtgacctca gagtgagaca gaacagtggt ctttgcctgt 50940gccattgagc ttttaaaaaa ttgtttgtaa ctaataataa ctgaatatac tggtgtagcc 51000tcgggttctg ttttgggttt ggtgggaatg gggaagatgg cattgtccag acttaaagtt 51060tcttttaact gtgttcgtaa atgattaggg gtggttttcc actgtgaagt aaaaactgat 51120tgctactggc attagccact gttaaattgt gatgcgcatt cacttcatta ttatttcaac 51180agtttcggtg gggctttctt ctattttctc aattcaagaa cagtaattgt gaatcataat 51240tcctaggaaa attctacatc ataatgacca cagatacatg agaactacac ccaccccctc 51300tgtttttgct gtcagtttaa taaaatctgg acatcactca ggcgtgttgt ggtgtttaga 51360ccctgtattt tgacgtattg tggtagctgc tgatttgtcc tgagagtacc actgttgcat 51420cctgttgcgt ccctgagaca ctgttgcatc cctgagagat ccactgttgc gtccctggga 51480cactgttgca tccctgagag atccactgtt gcgtccctga gacactgttg catccctgag 51540agatccactg ttgcgtccct gagacactgt tgcatccctg agagatccac tgttgcgtcc 51600ctgagacatt gttgcatccc tgagagatcc actgttttgc ccctgagcct tctcttgctg 51660tttatttgtg agctttctag agtccaatga ttcagagaca agagcttgtt ctatccctca 51720gactactttt tttttatcct ctcaatacaa tgaaaggaga agtatattct tttagtgctg 51780tggtattttt atacctagtt ctcttacaat atatatatgg atgaatgatg gacccagtat 51840agtggtatgt ctttaattcc agcaattgga aggctgaggc aggaaagtct agagttcaga 51900taaaaattaa atacacagtg agacttcatc tttaaaaaaa tctgtgtgtg tgtgtgtgtg 51960tttgtgtgtt cactcatgca tgcgcatgta tgtacatgtt tctgagtgtg tccatgtgtg 52020cgtgtgtgta agctgggtgt gtaactcggt agacacttgc ctagcatatg tgagaccctg 52080agttaaactc tataaacatc ctaaataaat aataaaacca tatttgtaca atgttttcta 52140gaaattcaaa ttcttacgtt ataaagaaga aaacaattat taaaccaatc atagaaaacc 52200tgttaaaatg ttgagatagt tactttcata tacatatatc ttttttccct tgcttttaaa 52260gatagaatct caggctagcc ctgaactctc taattctccc gtgtctcttt ctgtgggcac 52320cactgcatcc aacagccctg atcatttgct ttggaaaaat actttaaatg gtttggaaac 52380ctaggtttat aaagcataat atgtgagcat atttcccaca tcccagacca ttcttggcaa 52440atattattta aatagctgca tagttctgta tctgatatgc tttttcttat aaagagttag 52500agaaaaatat gcactaaaat taaaaagcag ggtttgaaaa cgtcgccttc tcttcctttt 52560taaagaccat ttctgtttgt ttgtgtgccc atgtgcgtgc atttatatgt cagaggctga 52620catgggtacc ttcctcgttg ctccccgtct tactctctga gagactctct cattgaactc 52680acagatttcg ttaaactgat tgggcaagga tcccaggtcc tgcctgtgcc tcctcagagg 52740taggattact gctattactt ggtgctggga attaaactca gatcttcata cttgtatggt 52800aaacatttta ctgactgtac catctcccaa acacccctgt gccttttaaa taaaaaaatt 52860aaggcgttgt ctcatggaat acaggctgcc cttggactcc tgatctccct ccccatgctt 52920ccctaagaaa tgggtatgcg gctgtctttg tctccttgac acaagctata gtcagtgtgg 52980aagagggagc ctcagttgag taaatgtcct cactacattg gccattgatg tgggagggtc 53040cagctcactg tggatggtca cagcctgggc tgatggtcct gtgtgctgta aaagaaagca 53100gactgagcaa gccatgagtg agaagcagca gtgaacagca gtccctcatg gtctccacat 53160actttcctac ctccaggttc ctgccctgtt tgagttcctg tcctatctct tccttcaaca 53220atgagctatg atgtagtagt gtaagcgaag taaacccttt cctttccagg ctgcttttgg 53280ttgaggtgtt tgagcacagc catagaaagc ctaagatgct gggattcctc acctggaaac 53340ccacgctcac ctcgtggttc tgttctaact ttaaaagtaa ttagtttcaa agtttgagac 53400tatgcaaact ttatgtttat atacaaaaga tttacccttt gtaaaagtga caatatataa 53460aacgtatgaa catatataat agaagtcttt tgattggcaa caattaatta gaataattat 53520tttaattggc tattattcat agctattgaa tcagtacaat tttcaaataa tcaactttta 53580tccccactta aaaaaaaaaa ccaaaccaac ctctgaaaca gtcttctctt tttatttgaa 53640gtgaggtcaa ctatttttgc aactaacttg agtttatcta cttgtgatat agattgaggg 53700tgtacctgat tgtccttgat ctcccaggcc gcataggaca agaaaggctg tggcagtgaa 53760ggaaacggag ccagatggcc cccatcaagg gtgccaaacc tccagcaggg atggatgcca 53820caccagacag acagggcagg aaaatcactt cttcaagtag attttgccac tgctgcacct 53880ggtgcgggga gtaagcgtgt agattgttaa ggtccactgg gcaagcgagg aagaggtaag 53940gacaccggcc acgaggccag gactgggggt tggcagtcct tgggctctct gatgaaatcc 54000acttggcatt ctcatagttt ttcaaaggaa tctgggtaag gcatttaatc ttgtgttatt 54060ggtttagttg gctcttaaag gagagtcata attaaaccag agccatctgc cagtgaatct 54120tagaggtgct tttcagtttg aaagttgaaa cccagagtca caaagccatc tcttaagcat 54180cggcctacag ttgactgcat gttgctgtgg tttgtttttt ccaccaaggg agttgatttg 54240ggagaaagaa tgtggttaag aattctatgt ccccaaagac atctgtacaa ctgctcccaa 54300acttcgtttc taaaagtgac aatagtaaca cttattttaa aggtaagagg gaggttgcgt 54360gttaaagcat agcactgact tggcctctcc gagatattag agactttgct attaataaat 54420catggcgagt cacgagattc cacggtgtgg aactgtgctt ggatcactgg ggttcgttaa 54480ataaagttac ttttcctttc tgagtcaatt acagtctcct gcccagaact acgcacggaa 54540atgcaagtct ttatgaaaga tctccaaagc ccattgaact aaacaacaaa cccactttgg 54600tttatttgct ctccccctcc ctctctccat ccttccctac gtctctctgt ccctcctgct 54660ccttcctcca gcccaggtga ttccttccag cttgtttctc ttgtcattct gaaagctggc 54720ctgaccttcg cagtcagata attctaattt cagctcaaga aaagggagac ggtcttgatt 54780tgtgtggccg tctgcattca tgcccaatta tacacaacag tcatttgtcc atggctgcag 54840gggaggctgg gagatacaca cgtgttctgt tagcgtagag tagggatgag ggatcggtgg 54900tgccaatcgt gtcacctgta cttgcaaata aatcatgttg agacatagcc atgcccttgg 54960cctgtggagg gtctgtgtct cctttcatat agcacagtga atgagctcag ttccctgaga 55020ccatatagac ttcaaagcac accacattta ctgtctcgcc cttttcagac gcttgctgac 55080atttgctagg gaaaatggag aaatgaaatt ataagagtca ccaagagttt gtgccacact 55140ttgcagctta gtaaactgtg agccattgcc atagctggga ctttgtgctt ctgaagagtg 55200gtagccaaca gaaagcacgg ttgatttgaa gtaacgcttg aagaagcact gttcagagat 55260ggatttttgg agcaaagcag gaagttattg gtgagggtga agagagtata aaacgttgtt 55320tagaggagct agagaaatga acacttgcca ctcttccaga ggatctgagt tcagttccca 55380gcacccacat caggcagctt acaactgatt ggactttatc tcctagggat ccaatgcctt 55440tctgtagcca ctgtgggtaa acacacacac acacacagga ttttttaaaa gctttcatgt 55500gaatttctgc ttttaaggcg acaggccata tttcaagcgt atcctcatct cctgcatccc 55560acagattgtt ctgtgttttt tctttgcttt cttcatgact ggggcagaag agggatacat 55620ccataggagg gcatagacgg tatctccact ctgcagctgg gttcatttcc ccaacttcag 55680ggtgtgccag ggagcagagc ggcataagga ggatcataaa agctgagctc cctgtgagtg 55740acagagtgct gcccaggcca cttggaagga caggaccttg cccacagctg gtgtcttgtg 55800tgtccacagc cacagtcaag ggaagggggg ttttgatttg gagagacatg attggccctg 55860tgagctgcct cttctttcct tagtctgtat aaccacgcat acaatgagga gactaataga 55920taatgagaga cttgctcctg ctgtcaaaga cctcccgtcc ttcactcagc tcatattgat 55980taaaacctgg tgagcttggc cacagcattg gaaagacagg tggacgacca ggaacttcta 56040aaaggatcag caggagagag agaccccgag atggggtgct ggggtttccg ctgagtccac 56100tgtgctgctt gaagcataaa tacaaggggg cttccagacc cctgtgacag agggacacct 56160gtctgataac ggaggaaagc agaggtctgg ttccttatgc tttggtgact ggagctgttg 56220aagaacaaaa gcagattaat gtagaatttt tctcaagctt gacttaggat aacaacaaga 56280tggacttttg ctaatgtgca tccagcccct ggcagaacac tctggagaca gtcagtgctc 56340agtacatgtt tgttgagtga ataaattagg tggattttgg cacttaatta tcagagcttt 56400ctagaagatg aaaggattcc ctagcagaat gatgttttta cagagaaaaa ggcaaaaggc 56460tcttatcctc caggagcccc tccttcactc tctcatcttc cacatgttca tgtctcactg 56520gcccctcctg ctttcaggac tttgggagag taatggcgtg tagccgcctt ttatcaggag 56580agcctgtgtt atttaaccac gcttcatggc ccctttgtag ccgacacaga tggcagaatg 56640agttcgtttt taataatcta ggttttatag gaatgaactg tggtgtgaac tgcttggcct 56700tccatgagac gaagtagaat cttgtctgtc tgactatcta ttgtttttgt gtgtgtgtgt 56760ggtttttcga gacagggttt ctctgtgtag ctctggctgt cctgggaatc gctctgtaca 56820ccaggctggc ctcgaactca gatatctacc tgcctctgtc tcctgagtgc tgggatcaaa 56880ggcgtgcgcc accaccactc agcaacctcc acagaatttc tgaacagtaa aaaacgtttc 56940cttctgaagc tgcttccttc agagtttttg agttttacat aaatctgctg taatagttag 57000attacggaac tggtgaaatg gcttagcagg taaaggtgct taccagtgcg tgcagcaagg 57060cgtgcgtgca gcaaggcgtg cgtgcacgtg cacaggagtg gacacacata gccttataat 57120gtaataaaaa gtaagcagta cattaagtgt atttgtgaac aagtcaaata taaatccctt 57180aattctaagc taatgtttct ggcccagatc catgagagag tccacagagg aaaaatgtca 57240gagagaaact ctgtgtaaca aggaactttc cctgaggaca cagcatctgc agtcactggc 57300tgtttgtgct ctgcaccacg caggcctgca gttgatcatt gtaccagact gcactactat 57360tgaggggaca ataagacatc cagcccatca tgtccctgtc tttatttcaa agcagggaac 57420aatagcagct cttgtgccca aaactcacgc atgagatagt ccatgggaca cacctgggac 57480agcctcacat gtagtaaggt actgctccca gccacgacgg ctgaacagag acttcaaaat 57540ctgacccaaa gcagctaaga ctgctttgcc tgctgcccca atggggagga tcttgctggc 57600atgcactgta gcgtgccgct tgcaggcatt ctagttcctc ccacccgttg ctccagctcc 57660cagcctctgg tagagccatc ttcatggtag gagctagggt gtgctatgcc tgtgtctcag 57720ctggaggcag agggtggcct gggaagtcaa actgctgaca gtgttcatgg cacaacttgt 57780gtatttttag gaatttgtgt gtctctgatg aggcaagctg tttacttcct acttaatgtc 57840taacctgaat tctaggcctg aatctagaaa aggtacattt ttgccatgct gtgtctgttg 57900agagactcgg gttatcggtc cacaattgac tgccagagag actgggaagt atccctgcca 57960cctggatttt tgtatgtcca gttactgcac attctcagtg taggaaatat ttattaaaac 58020agcatttcgg gtaaggattt agatgtcagg acaaggaatc gcagagctgt gccacaggcc 58080ctgtatctcc catgagataa taaatccttc gaatattgaa tttaaacttg gggaaatttt 58140tggattacag tgactctcag catgtctata atcctgggaa ttggaggtga gatgcaaaat 58200ttgagggcac cctcaactgc ataggggata caaggctagc ttgggctaca tgagaccctg 58260cttccgaaag gatagcccaa aaggactaag gatggctgtg agcacttgag tctttctaaa 58320gtcatgtcta aaatattgct taggtggtat agcctgtatc tcctccattg tgctgttgag 58380ttttagaatg attttattct tcctgactgt gattggacta ctagttgttc cccaatgccc 58440ttagcctctt aacgattact ggtgtatgtg gctgtatgtg ttgtgaactc tttggcatcc 58500ccatgtaaga gaggacatgc gacacttacc ttcccatgct agactaacta gacataacat 58560taactaatta gttaacttaa ttaacttcca gctccatcag tttcagtgcc tatcataggc 58620tatcatttta gtaactgaat aatattccat tgcatacatc ctgaaattga tatgtcattt 58680ttccatatcc atcacaccaa tggtcatcta aattgattac acatcttgtg aattgtgctg 58740ccacatacac aaagtaacaa caaggatgga gacattgatg accctgactg tgtcagtaca 58800catcgtatac atcgtatgca tgcgtcgaaa cattgcgctg taccctacaa acacatagga 58860ttattacatt ttttttcttt tttttcagga ttattacatt aattgaaaga aatgtaatgc 58920ttaaggagta catattttac tactatgatt tgatcattat tggctgtctc cagctatcaa 58980actgtagctc tgaacatgta caattaacgt aataactagg agagtggaaa caccttggaa 59040ggcctgctct ggaataagcc taacccagta tctgacctgg caagggcaaa gtacatagaa 59100gcagggtagt ttacttcaga gtactggagg ggaggcgggg accgggcaag gcatggggct 59160ttcgagactt gacttttctt gttcagatca ctgctgtgta tgtgaaggag acaatcacac 59220gtgcatttac tgtagttttt cttccccatg tcactgtccc ccaggagaca aggggttgaa 59280gacacaggtt tctgcatcta gaagaaagga attcaggtta gacattaggt aggaagtaaa 59340cagcctgcct catcagagac acacaaattc ttaaaaagca gacaccttgt gccgtgagcg 59400ctgtcaacag atactgtcac gtggcagtaa aagcctcagg ttcctgcctc gtccatgctt 59460agaaactgct gaaaacggaa ggactgggaa agatgactct tgacctgctg tgtagggttt 59520tagaaccgtc tggaatagtg ggtagtaaca ccctgggagc tttgtgtgaa cgtgaagccc 59580tgcactggct aagaagagaa gaggtgactg tttctggttt ttataagtac cctcttgtct 59640aactgctaaa aataactgct gttactttaa acagatggcc tcaatttaat atgaaagaaa 59700gtaaaagtga gacagtttct tagcatctaa gaactaccac ccccactggc taaaatagcc 59760aaattttata taaatactta tttacaggga aaacttctga gggtggggtt tgtagatgga 59820gctatgtatc ttggtagatt gagagccagt ataaatgact taattcaaaa cagaacggtg 59880gatccttgcg tttaaaacaa ttattatcat tatgaggtgg tgcacaggtg tggagatcag 59940aggacactct gacatgggtc cctggatcaa gatgagatca ccagccttgt gtggcaagtg 60000tgtttcttta cccaatgagc cctctcccca gtctaggact ttggattttt aaaagacatt 60060taagttgtta taaaatatat gtaacctaag aaagttaatg tcagggatcg taacacactg 60120agagggtgag agagcacgcc gaggttattg ttttaggtcc tctccaatgg ccaaagagaa

60180catgagctga tagggttcaa ttccaggagc acggagatag tagttaatgt acgactttac 60240gtcatttctg acatcataga taatattttg ataaggaaac agtgagccag catttatgag 60300actttgacta aagactgatt cttaagttca cagacactgt ctatgtcatc gagtaatgcc 60360aaaatactat taagttcaac aggtagagaa gttttattgt gaaactcggt cattgatttt 60420ttttaaaaga tttatttatt catttaatgt atatgagtac actgttgctg tcttcagaca 60480caccagaaga ggccatcaga tcccattaca gatgtttgtg agcctccatg tggctgctgg 60540gaattgaact caggacctct ggacgactag tcagtcagtg ctcttaaccg ctgagccatc 60600tctccagccc agtcattgat ttttattcct acttttgctg ttcatttgtg attattgcac 60660ctggttcttg tgcggggagg ggtgctgtct gtcctgtgtg catccatgga ccacagaccg 60720tgcttggatt tagatgtttg catttcctca cctggcctgt acctgcacag ttgtttgtgt 60780tgggtcctgg gttgcgcctg gaatcttact tgtttccttc tgaacttgtc tctgtaaaag 60840aagtagactg gctcatagga aatatgaggc tggggaggcg gcactctgta agatgtcagg 60900ggcacaagtg tgaggacctg aatttgcatc cctagaatgc ttgtaaaaag accaggcttt 60960gctgagggag ccagggaccc tggggaggaa ggtgggtagg gtatggagtt aggtcggatc 61020cctgtaccta gttggccagc tcttaccttt ccaacttcat gggcctcagt tttagtgact 61080gatacaccct gtctcaaaac caaagaagaa cactggagga aagcccccgg cttctggtct 61140ctgtgtgcac tggcatgtgt gtgcacatgc accgacatgt tcacattaac aagtgcctcc 61200cctaccacac cactaccctt acacacgact accacacacg tacatgtata tacaaacaca 61260cacatgtaga ccagagatca gcaaagtgta ctgctgggct atataaaaaa ataacatgag 61320acagatattt gcagtagtta gcctttacaa aatgtttgct acctcttgaa gacaagatgc 61380tcgtgggccg acgctggttc agatcttggt tttaccattt ccagttcgac ttgggcaaat 61440tacttaatct ctctgtgact agttttccaa tctgtaattg taattatagc ctgcctcatt 61500gggttgtggt gaggactaac ttaatgtaca aaatgctcag aacaatgtct gggattaatg 61560ggtgctcaat gaatgttagt cagtgctcct gtgcgggaga gagccccgaa ggagcttctg 61620aaccattctt ggtttcagct ggtacacacc caaattatcc agcccactcg ctctgtggtt 61680cctgtattga gaagggaagg ctgtcttgtt gttgttttgt tttgtttttt atttttaaat 61740agtgaatgcg ttcctgaaaa acaaagtcaa gttgaatcta cactgaaatg tgaaaaataa 61800tcttgaaaaa aaaaaaggag acataagcag aaagcagaga tgctttgtaa gttaagggaa 61860gtcaaaggtc gcttgtgctg ggtgtgttgg cacacaggtg gcttatcatc tcaggactct 61920gaggctgagg agagagatga atgcattcca ggcctgcctt gcctatgcag taaagacctt 61980gcctcagaaa gagcaaacac aaaagtcctt tgctgtactt tggtaaagca acttgtgatc 62040gtgagagatg gagaggagac agtggcgggg gcgaggggga gtatctcagg ggaagctggg 62100gccttctcaa gggtgtccag tgtcagcatc cacgaggcta tgcagagtgc cgtgtgacca 62160ctctgcttct tttctatgga agtgatacat tttcagcatc aaatcaacca taggtggtaa 62220aggtcacctc agacccccat gtatacataa ttgtcatagt ttgcatcttt agtttttaag 62280acacctggct agtcactcag ttgctgtcgt catcaaacat ttgatcagcg tctcaatgcc 62340tttgtcctct ctgatacctg tctcctcgct tgggctttcc agtagggaaa ggacaagtaa 62400ttacttagaa ttttttaaat ttaataaagt aatattccct ccctctcact ctccttcttt 62460ccactctagc tgaacacgct gttgatggat attggagtgg aatgatgggt ggccagagat 62520gggtgtttga cgatgtcaga ggactggagt aatggccttg cgtgaggagt acagtgaatg 62580acctcatctt cttagctgat ggaggcattc aagaggacga ggttgcagag cttttaagcc 62640tatgcagctc ccaggctctg agcctaagag ctagctaact gtcatctccc agagggacca 62700tggcaagggt tggggctgtt gaaatacgtt cagggaccct gttagggcct ttctctttga 62760cattaggatg atttagggaa ggttgggact tacagaccag cttgagtttt cttttgtggc 62820aattatagac ttaacttaaa agatcagttg ttttttgtta gtgtcctctg agtttgggtt 62880tgctgacgca gatacacact gcttcattag agcacctccc tcccaaccat gacaacagtg 62940gcactcgtca gtactcaata tgttatttct ggagtgagtg agcggatgaa tggagtaaac 63000aaatataacc cattattccc aataaaatac ctctcccaca tggaggtaag ttgtcagttg 63060gcattatttt agatcaattt ttgctattaa aaaaaaaaga gtgttgtaaa ttttgagtat 63120gccagaggga agctcaactc gggctttcat ggtttcccag ggcaaatggt gacttgatga 63180gatagttaaa acacagcaac ccaggcagca aaggctttgg tcagaactct taacctctaa 63240gactatctga gagcacttgg aaaagcttgc tgttctcttt gttctaagat gatagtgcag 63300caagaactaa aacaactttg agacatttct ctttcagaca aagcaaatta taaaacatga 63360ttaaccatta catcatcata cgtcctttaa ggcctgactt ccacaccctt cataggccct 63420ggacagaaca agatgaacat ccaaggtgga aggcatgctg tagggatctg ctggctcatg 63480ggtcaggctt cctgagtgga caagaggaca aagggtagat tccagcagaa cctacctgca 63540tagaattgct aagtatgtgc atgcattaga ggggatgcat attagtttag tcttgctgtt 63600ataaaatacc ctgaccaatg aaacagctta caggaaaaca agtttattta ggtttataat 63660tctgaagggt cagagtccat catggtggaa aagagatggt tcctggggtc aacaacaaga 63720aactggtgat cacacagcat ctgcacttag gaggtgtgga gagagaggac aggaagtgag 63780gccagactat aacattgcaa agctcaactc ctgtgaccca cttcctccaa caaggctttg 63840cctcctgggt tccatagcct caccaaacag tgccaccagc tggggaccaa gtgttaagcc 63900acagaagcct gtggaggacc cttcgtattc aaaccctagc agcctgtctc tgagagcatt 63960cacccttgat tgacatgggg aacacgacaa ggacactagg ctagcagaaa agcaaatata 64020attctagacc ttcttggggc ctagggaaag tgcaccctgt gactgcactg ttgattgaaa 64080actgatgcaa actttagaaa taaaagggaa aatgtcgggg gctggagaga tggccagtgg 64140ttaagagcac tgactgctct tctgggggtc ctgagttcaa atcccagcaa ccacatggtg 64200gctcacaacc atctgtaatg agatctgaag ataactacag tgtacttaca tataattaat 64260aaataaaatc tttaaaagtt ggggggagga aatgtccagc tagtgctaca caagtttttt 64320tttttggggg gggggggcca aggggttgac ttaaataaag ctaatggtgc cagagaaata 64380aagagcaggt taagagtggc tccactccac cttgccactt ttagaagaag ggcaaggcgg 64440gttacaaagc ctcaaaacaa gtctggcatg attgactgca ggtgagtgag catgttgttc 64500aacttgtaaa tacnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 64560nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnttttttt 64620ctccctccct cccttctttc ttttctccct ccctcccttc tttccttcct agcgtattca 64680ccataaaccg aattaactcc agatatttca actactcctt tggtttaggt gacttgagag 64740acacaggaaa gaaaaataca aacattaaat ggcattttga aaacctgttc ttaagtccag 64800tgggcatcag agcagttgaa cagcaggtac ctgggagcac tctggtgaca caggttgaca 64860cctttggaag gaaaggaatt agtctgccca ctcctcccca caaacagatg ggcagagatt 64920ggcaggtgag gtggagagaa gaggggtaaa cccaggctaa ccgaaagggg agatggctat 64980ccacaaagag aaatcaaagc ctaggcacac ttgtgtagaa ctgggaacca ggatgcctgc 65040gacagagatt acggtattgc cccagaagcc cttttatact acttgatgtt gtgtgtatgt 65100atagatttta tagaacaact taaggtaaac atttacattc ggtacatgtt atttaatatt 65160aatatttaat agtttgtgcc ctcagtttgc ttgcaaatta tgattgataa aagaagttta 65220attcagaatc tataaaaata ctaacactct atgttaaaaa aattaattcc tggaggtcag 65280agaaatgatg acctaggttt aattcctgga actcatataa aaaagctaga cgtgttagtg 65340aatatttata attccagccc tcctggtgtg aggtaggaga ggcaggagac tctctaggct 65400ctggagccag cgatcctgga ctacagcatc ttggcagaag caaaacaaag accaaggtga 65460gaagggactc cacacatgtg cacaaacaca tttataaaaa tgggttcttg aatgaatgat 65520ttgcatggtt atttttttgc agtataacta cctaggaata agaatggctt tcccgcgtag 65580cccagtttgc tgtttaacta taaatacacc acttgtggca aatcggacgt ccaaaagcaa 65640cagctgctgc ttctgtgttt tgttctttgt tattcatttt aatgaataat ttgaaccgac 65700tccagaacat gaagttagac atagaagcat gctctaagac ttatatcatg tgtttcaaac 65760cgtgtggtga catcattctc taaatatagt gaattttaag aaagaattat ttgagctgtt 65820cccccccccc cccataaata atttggctgg aagacagaga gagagagaga caagggataa 65880aaatcaactt gggagcttta tccaagaata gagagctggg gaaattctcc cacatccttc 65940tcctgacacg tgaacctctt ttactaaaac aaatgggggg gatgtgtctc cgctgagatt 66000tgcttttcta gtgaagatcc caagtgatgt taggcgtatc cccccctgcc ttccgtagtg 66060tgtcctgtag ctgaaggatt taggttcagg tggaatagag aggaagatgg attcagagct 66120tcgtgattgt gattcctgtc tgtctctcgt ggcattctca acagttgaaa agcaagaaaa 66180tgcagccttt atcttcctat gtcctgggca tttgcttggc gtatatatgg gctttgtgtg 66240ctccatctgc accgatttca gcccttcctt caggaaagct ggtaggactc attcaggtta 66300acactgcatc ggtgcaaggt gtggtcgcag atcagcaagg ctgatttcct ttaaaacctg 66360ctaaactgat tggtccagta tacttcagta gaaggtgata caactgttaa ttaagtaagc 66420agttttgtac tttgattaga atattgttgt atgccttttc tcactggact gctgtacagt 66480gttgagaagt ggaattccta ctaatgcaga aggagtttta tgtgccctta tatatgggga 66540gaaatttaga attgcctctg gagagtggtt gcttaggaac gaagaaattg aaccacccat 66600cacttaaggc cagaaactct atcctgtact tgtgtcctac cttaggcatc atttttctgt 66660gtttcgttct gttcaagcct cttatatagc tgtggctggc tgtcttggat ggaaacccat 66720gtcgcagtcc tcaggggtgg agtctccaga tgggctgtgt acgccggccg gccagcagcc 66780ctgtctgtca tcctgtctgt caggaactga gtgtgtattc gtgaggaacg ctaatataaa 66840aggcagctgt gtttaaggat gaggtcatag gcaaaccatg caccccctct ccacagaaca 66900cagaaggatc ccaacggaga taaatatgcg tctggaggag ccacaccacc ttgcctgctg 66960ccatgcctgg cttcattcac tttgcctgca cctgcggaaa gcaaacccga gagccctcta 67020tgcaaatgtt attttaatta ttcatgttta ggacatatga aaaacaaacc tagaagggcc 67080atgtctagat gctggcatgc cagtgacttg ttatcttatc tgtagagaaa tccacatcgt 67140atctagatac aatctagaga tgaggggttt tccttacaag gtaatgatca ctgtccaaag 67200gtgcgagagt aagtggcttg cccaaggtgt agacatgtta agtctctggg tcaaagtgct 67260taccacagtc acctcccttt tcttggatgg tggatctgta gaagatggaa gcttcccctg 67320gttttatgtc tccttttctg ccactgtcct gtatgttttt cagattcgtt ttgttgttgt 67380cgtcgttgtt gttttgtttt gtttttgttt tttgtttttt tccagtgcaa gcctattgta 67440agcagtccct gtttatgggc agtccactag ggggcagaca tgtcctcatt cactatgtcc 67500cagacacttg gggcaagtct tcttacatag ttatgcttca tataatggtt tccagtttgc 67560ttttcctgta tttgaggtat attcctatct aaaagttcag tggtaattcc tcagttcgtt 67620gacagtgtct cttgtttttc tgttctacgc atcaccctaa aggtcattta tttattggtg 67680actgtcttca tgcttagcat tcttctgtgc ctggggatat taaattctct ccttcacact 67740ccattttgtt gggttattgt gtgttgggct gaataaaatt aatctgggag tagactgggc 67800tgtgagtgag gaccagaacc tgagtcacac acagtaggta cagtggcctg gaggccatct 67860gctgagagga ccaagctgtg agggggtgaa aaggggaatt catgataagg acagtgctgt 67920ggacttttag ctctggttat tctttataag atgcttagaa gaacgggcct tgctgagaca 67980ggggggttgg aggttcaact ccagcctaca ctgcttagta gaacccagtc tccaaagact 68040gagggctggg gggtgtggct cactgtgggg gacccaccta acatctgtga agctctgtct 68100ctagtagctc agtgtaggtg accctcctag catcacaaaa ctccgtctgc agccgaaaac 68160tccagcagtt aggtacttga aagcatgtgt gtgtgcatga ataaaaacca gatcagaata 68220tggaaaatgt tatttattgt aatattatct atattcacct cataacttat cagttttact 68280attaatttct tcaaagtaaa cttgataaag aaaacaatac tccctgcaga acagtaactc 68340agaatgtttt cccaaagccc tgggcaactt tcagaaagga aaagtacacc acagtaattt 68400aagagttgca ggtatttgca gtttatcatt tggaagtata cagctgagct gtgacaaaag 68460attctctctc tctctctctc tctctctctc tctcactcac acacacacac acacacacac 68520acacgttaaa attttaaggt ttcttgaaat gacagaggga tacaatataa gtcaacagtg 68580tctgagcggg tatctcgcag tagctcactc actgatgaaa tgctgagttg gctctgcctt 68640agccaggctc cctttgagat cccgctgaga acagtgttgg gcttttattg ggaatacttg 68700ccctgattca cctgagaaca ccctgctcct caggaacaga gctagaagct tgatgtggtg 68760gtgcacgcct ttaatcccag cacttaggag gcagaggcaa gcagatctct gagtttgagg 68820ccagcctggt ctacataggg aattccaggc caaccagggc tatgtagtga gaccctgtat 68880agaaaaacaa aaggaaacaa acaaaattac aggattaaga gttcaaatta ctgtctttca 68940catgtgtcag atggtgtgtg gggacttgca tatgtccatt ctccatggct tatctgtgtg 69000tctctctgaa gcttctgtgt ggggaacgaa gacagggcag ggggcaggga gcacagggat 69060agagagtgga aaactacaag caggacactc cacagaggtc gggtcctcca tcaagtacgg 69120ggcctgagat ttatgtaatg ttagttaaag ggaggcaaag tggcattctt cctatttgct 69180gcccccttcc cttgtcccaa gtccactcct ccttcattct agttaaacat caacgatgac 69240atatttaaag taaaacagca aaatgtgaga atctctcgtg tcctgtggga tgtctggaga 69300catgtcacaa acccaaagcc agccgctgca gtctgaactc aggggctata tttagatgac 69360tgtgtggatc ctgtattttt tcagtgctgt aaacatgttg atgtggttat ttttagcttt 69420cacagacctt agtgaacttc tgagttggcc agcagacaga ggcagttgtg gctgttgccc 69480gcacctctgt caatggctct gattgtctct cagggtttcc agtgcattca gagcttgtca 69540gtgttcaggc tgtctagcat taactgccca gtgtggttgt gtgtaggtga ctctactaca 69600gtctggtggg cttcattttg ttttgatgtg tgtctctggc catagttttg attccagttg 69660aagatatatc aagcccactg gtcctctcat gccctgaccc tccagcattg atgacagatt 69720cttgctgttg tcattacctt cagtctgttg tactcttttc ctgctgggca aaggttataa 69780ggcggcagtg tggtaaaggg ggcaatggaa agctgacagg aaatgggaag gcagggctca 69840gtttggtccc cagcatagat tggaccagct agtgtgaaaa gaagtaatta agtagtcaac 69900agcttttctt gatgaccaaa tcatgctgtc tctgctgctt acatgagtgg aacattttcc 69960tcgaagagcc atgcctggat ccctgacatc gattagtcac ttgagctggg ccttccaagt 70020ttgtgttaga gaattcactt ccttctttgt gaagaggctg ctgttctgag ctctgctttg 70080tacctggtgg ctggctttgg ttggataagg tcaattactt tgtatggtgc aggaagcagt 70140tcctgagcgg gtgactcgag agagcatgat ttatctcttt ggaactactt ttcccaagga 70200aactcataac tgcctcatac cttttgcctg gaagccttaa actgagttgt acattcagaa 70260gagagtggta ttgagcgtca tccagactta gttcatatta ataccattat ggcaatttgt 70320tttgaaatgt tttcagtgat tcatatgaaa gtgatgtttt tgaactgtgt cccaattttt 70380aaaattattt gtttttgttt gtttgtttgt ttctatgggt atgtttgtgt gcccagtgca 70440tgcatgccca ccacatgtgc aggacccctc agagtttaga agatggcttt cccctggagc 70500tggagttaca ggcgattatg agctaccatc tgggtgctaa gaactgaccc caggtcctct 70560gctagaacgg tgtgtgccct tgactgacga ctcacatctc tggcacccat gacctctggt 70620tcttactgta gcctttacac attgtaaaca cgccacaaaa caaaacaaaa caaaacactt 70680gacaggaagc atgctttttg tattttgctg aaaactcaga aacaaacata cacattttac 70740cacagaaata tatattttgg atttggtagg tgaacaagaa atggaattga gaaatccaag 70800ggttttagca aaagataggg ttttttcaaa tgtttttatt atgtgtaatt aatttgtatg 70860tgtgtttttt ttttttaaag atttatttat ttattatatg taagtacact gtagctgtcc 70920tcagatactc cagaagaggg catcagatct cattacggat ggttgtgagc caccatgtgg 70980ttgctgggat ttgaactagg gaccttcgga agagcagtcg gcactcttaa ccactgagcc 71040atctcgccag cccctgtatg tgtgttttta tgtggagcct tgtgcacgtg tgtgcaggtc 71100cctatggagg ctgggagaga atgttgtgac gtctgaagca ggaattacat gtgagcggtc 71160tgacctgggt gctgagaact cagctctggt cctctggaag atcaatactc actcttgact 71220cctcaacatc tttccagcca gaagttgaaa ttaaaacaaa acaaaacaaa aagaatcaag 71280attgctgtta atgttttgat gagctttttg ttgttgttgt tcattttggg cttggtttag 71340ttttgagaca gggccctgta tagcccacgc tggtcttaga tttactctgt agctttgtgt 71400gaacttttct ggggggaaaa ggggtctatt caccccagaa aagtaccaac catgaactga 71460agatgtgacc ctcccaagtc taacagggtg aaccagtgtg cttaattagg gttacttaaa 71520ggcgtacaga cactttgcaa gcaggcaagt gattatacca ttgaagaaaa cgtctctttc 71580tttccagcag ttttagctgt cagtcagtct ttgatgatga ctgggtcctc aggagccctt 71640gccgtcctct gtggctccat gttataggct caggcctgtg tagaagccta tagacagtca 71700cagctgccaa gaattataga gggtaatggc catgccacag ctaggagaca gccttgtaag 71760catagagttt gaccttgaat gagatgcttc tatttccatt tcccaaatgc tgagatctca 71820tgcgtgagcc aacatcccag gtgctgggtt catgctttct aggcatccac cctgacaact 71880aagttacagc ccagcccagg acagacaggg gttgggatat attgctactg gtcactatgt 71940agaccgggct gaccttgaat tcagagatct gtctgcctct gtttccaatt ctattggcat 72000taaatgtgtg tgccaccaca tcagcaccat ttttttaaaa tccccgttta tatagacaag 72060tccattgttt tcccaatagc agtggcagta acccaggtgg catgcatttg tggtgcatag 72120taggcacccc agtttacagt cgagcatagt ttaaacctcg atcattgtag gtctcatcct 72180ttggagccgg gctttgtaac aagatgctgt ctcgaacatg agttacagaa ttgtcatttc 72240aagctgtaat gtccacatga cagcttcagg ccacagcagc caaatctaag tgaagtcaga 72300aaatcagctc tctgtttgtg acagtcacta atgttctggt tctgtcgctc agtgcctcct 72360ctggcctgtt tgggactcat gatgatgctt gtaccctgtg gctctgttca gccagattgc 72420ctgggtctct acctgggcgt tcaacccacc tctgcctttg aagccgtttg actttgtggt 72480agttggtggt ctttggtggc catcgttaga atccctcacc ttccccacac ttggctagcc 72540tgcttccatc caggccgagc atgtatgtaa actctgatac ttcagaagtt caccggacag 72600ttgggagcca ggagcctgga gccaggcatt ggtctgcagt tttttccagt gtattcgtct 72660gtttgtatta ctaagctaaa gttagcacaa atgggacaag gctagaaaat ggtgcgcctg 72720gtttgaaaac aggaagtttt aggcttctgt gtgtgtggaa cagacggctg taatgataga 72780gctggagcgt ggaaattccc acgaagcctg ctttttcctg attttgctcg aggagagagg 72840gtaatactgc ttcctggaaa acagtcgagc acacgtttta aagcacacgg ctcgcttttg 72900tccttgttca cggataattt gtttttgtcc atttcattta cacttggagc agtattttgg 72960aaatgtagta gttttaatgg agaaagcaaa tccctaggca cgtacttgat gttttcagac 73020atttagacat ctgagtgtat gccctttaac tggcatagcc tttgaagaca atgagagcat 73080tatggatgtt ggatggggga ggggagcgat aggcttgtct attttaagtg tccccagctt 73140tgctggcttg tttgttttta acccctgtat ccagtcttca aaaccaagtc tctgtctaag 73200ccatttcctg actatgctgc atccagatgt atcctggaat tctatatctg agagaaacat 73260taaattcatt cactaattgc agaggaacaa aatggcattt ccaggcggag gcagcacaaa 73320gttctcactt tatgtacacc atgcctagat atttttttaa aaatgtattt tctttttcaa 73380agccttttta gacataaact caaaatatat aacattttcc attataaagg caataaatgg 73440aagatctatg ggagaaaaaa tgaaaggcct gtataccttg tctgaatatc tcatcccagt 73500tacagactcg tcatggttag cattgggttt tacatgcttt cagatctttt ctctaaactt 73560gttgacgtgc acacaaacat aattatttca gacaaatgga gccatacctt ctatacaact 73620ctgaaacttt ggttttatcg ttctgtagtt gagacaaggt ctcagcagtc taggctggcc 73680tataattcat ttgataacag cccaatctaa ctgaattcct gcaatcctcc tgcctcagct 73740tctcaggtaa tgggatcaca gatagacaag aaccatgttc ccccctcccc cttttttcat 73800ttaacagtta tcttgcaggg attatcatat atattttcca tgtttttaaa atggtttgca 73860tagtacacgt attgcataat ttaaccattt tcctgtcctt gaacgttagt ctaactcctg 73920tcattaaaaa atccagtgtt caggttggag agcactgact gctcttccag aggtcctgag 73980ttcaactccc agcaaccaca tggtggctta caaccatctg taatagtatc tgatgtcctc 74040ttctggtgtg tctgaagaca gctacagtat actcatatac ataaaataaa taaannnnnn 74100nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 74160nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnaaataa ataaataaat ctttaaaaaa 74220aatccagtgt tcttagtcag acatggatac acattaatca tagcactcag aaggtagagg 74280ctggcaggat tacacaaaga aacactcttg aaaaaacaaa taaacaaaca aagataattc 74340agtgttctca ctagccttgg tggccaacac tgcagtccta tcacttggga ggcagatact 74400attagaggct agcctgagct acatgaaact gcctcaaaag gctaacccaa acaacaaaca 74460ccctatcatc tttatgacag tttacagact tctgagatga tatgtaaagt ttgcattgaa 74520tccatcagag aactctcagt tcattgtttt tccctacaga atatcaggtg tgtagtacct 74580tttagtagac ttcaaaacaa aacaagcctt cctgcactct gtcctcaaca ccttctcagg 74640gtgcagacta atgagagatc tatcaccatt tgcatcttgt cccctgaaac ttggttaggc 74700tctggcaacc agtgagggtc ccagagaatt ctccctagct tctgtaagca agtggagccg 74760tggacaggtt ctggagttgc tgtctaccaa gacacaaacg agcaggaaac agcaagtgcc 74820tttacaagga cgggacttgg gggcttgctt tattttgttt tgtttgttgt ttgtttgttt 74880ttgagacagg gtttctctat gttcccttga ctgttctgga acttcctctg tagaccagac 74940tcgtctccag ttcacagaga tccacctgtc tctgcccccc ccccccacca gggccgccac 75000ctcagggctg agttgggggc ttaagtgaag gtcttgacct ggaatttgaa ggaagttgat 75060cattggaaga ggaatgaagt gtgagagtgt ggaggggaat gctcagaaga caacctggtt 75120tggaagatgc aaggtcatgt gccgtcctgg aactgacact tggactgcac taacttccta 75180tctctgattt ctttcccaga gatcttctaa tcaagttcac tggctctctg tggtgatttt

75240gatgggcaga aaacactaga ttgtctatat caaggctttc aagaccttag taatttgtta 75300attgttgttg ctgattcaaa ataattggaa aggaaaggaa aggaaaggaa aggaaaggaa 75360aggaaaggaa aggaaagggg aaaggaaaga ggaaaggaaa gaggaaagga aaggaaagaa 75420aggcccctcg tgcttcgagt tggagaagca gttccgaggc gtttggaccc agtgcagagt 75480ttggaggtca gctaccccac gtccaccgag cacagcacat gtatacagca tgatgcagtt 75540gctgctctga aggaatcact tccctcctgt aaaacagaat cctctggtta tttgtaacca 75600cagaaggctc ctagaacaag tgtatggcag agacagtggt tttctttaaa gctgaacata 75660gcatcacttg gggtaattgg gcacgttcta taaacttgct aattgcatat agcaagtgat 75720ggtgaggggc tagtccaggt gggaattctt atgacagggc tccatctgtc ctttgtgaca 75780gtgtgttcca gactgctacc ctagtagctg agaagcctgt tttcagatct tgccaaagtg 75840tgataggcct gccttgagct tagcatagcc taatttgctt aagtgtagat gtgcttatac 75900ggaattcttt tcaactgaag ttcatagaca cctcccaagt gtgtagaggt tgaaagcaag 75960ttagttgaaa atttatggag cgctatcttc tgacagttct tgagtaggcc aggcaggtta 76020tgctcagacc agtgtccctg tgatgtgtag gtcatatgag gatgtccata ttgtcctaac 76080ttcaatgggc atacctgctt cttcatcaat gggtgataca agctccctgg ggagcagggg 76140gatgggagcg gaggaaaggt acagagacaa ggcttcccaa aacccccaag taatttggat 76200taagagtacc aaacagcatg cgactgggaa ggacgtgagg atggagggtc ccaagctagc 76260ttggggcaag ctagttaggg gatggttcag agaggaggac tgagctgagt tcggggtggc 76320ttatgtgctc agaatagcat ctgttacagg gccaaaggcg gaaaagaaga cacaccgaag 76380aggatggcaa acctttgagg agatacaggt tgggttatta tgaatttgag ctctctccca 76440tgtagacagc agacagataa ccagtaagca gagggcaccg atatcagagg atggtacatc 76500taaagattca ctaaagttgg tactacattg attattgggc ttgaaactcg agtcattgtt 76560gtaagacatg atggccacac cgtaagttgt tatgtaattt tctcagctaa ccttatggat 76620actgtttcat tatcatggaa gtctaatagt taaaatggtt tttcatgaga tttttattta 76680aagttgattt gagcatttgc tttgaaggtt tcaagaaaac tcatctgctt tgactgttac 76740tcaagcaaga ctgggaggca gtgtacagct gtctctgggg ctcccgagac agcagggtaa 76800aaataggaca aactttaaga atcccagata ttctcagata acaaagactg gtttattggg 76860gccttttttg tgcttggaca aaacagagaa gcctttgtgc tgtgattagc aagggtgctg 76920atgacagatc tcctgtcaag catcaggctt tattctatga ctaaatggtg ctttgtcccg 76980ccatccagag aacctttgta tgtgccttgt taaagtgtga gtttatgtaa ttgcttttgt 77040ttttcccgtt tcattggtgc cagattcaat gagagtgact cattttgtga ccgagacaaa 77100cattcacatt tagaccatgt taaaaataaa caaacaagag gcaggtgctg cctgcctcgg 77160ttctgcatct ccgggctgct gtttcctgtc tcaggagttc aggtctcagg tagtggctac 77220tactcagctc ccgtggtcac tggtccactg tgccccaggg cagtttctaa aggccttttg 77280gatgttggtg acaggaaggt ccaaattagg aaatctggtt agatgggaag tagaaagtga 77340tgtgtacagt gtctcaaagg cacacaggaa gacgacagtc ccagtctctt ctggctctgt 77400agttttccca ccacacagca gtggtttgga gcagttactt tacatgtgtt tttcacacat 77460atgcctcatt caggggctct ttttctcaca gagaagacgt ttggaaagca ctgatcaaat 77520tttaatgaga ccatggagtg catttaatag tccccaaact gtacaacttt atttcccttg 77580ggaactctga aggtggccca ggaaggaaag gcctagaagg ttgggaaaga aaacaatggg 77640tttttggaaa gagaacatac gaagagtttg gcctcccaag acagacgtga tagttgatga 77700aaatgtaagg gaggacgggt agaaagttcg cttaaaggat tgtaggcaga gtgggagaga 77760tgttcgcttg agaaagtact tagcagccaa gtgtcgggcc ttgagttaga ttccccagag 77820cacatgcaaa agagctgggc atagaggggt gtgcttataa acccagtcct ggaaagaaag 77880gttagcagag gtagatcatt ggggctcaca gaccagacag cctaccctcc ttagtaagcg 77940ccaggccagt gagggaccaa actaaaaagc aaaacaagat caaggttgag catgtatgtg 78000tgcatgggtg cgcacactct ctcacacaca cacacaccca cacacacaca cacacctata 78060atggttaggt gaactgggca cacccagcag tgggctggtt cggtatgggg ctgatgaccc 78120tacacagctc agcagccttg gtggccctcg atgacttgca gtctagagat tgacacctgc 78180accactgacc cgagccactg gggaatggat gaattatgtt caatgaatta tgtagtaagg 78240catttcagaa agatttcctt gcgaggcatt aaagttttag aaatcatttc tgtatttaaa 78300gacagaacct cttcaatctg gggggtggag ggcactaacc agagaaaggc tcctctgcat 78360aaataatact gttttctgac tttctgcttc tccgttgctg tatcaggctt aatacttatg 78420gatgccttta taggtctcag ggcagagtct tatgtgtgct accacacctt gcaaaaggtg 78480ataaattttc tacaatggct gttgttcatt tgagttagat gaatggactt ttgtcatgca 78540gtatcatgtg tatctataat cttgtgactt cccagtacat ggaacatcca cagagctcca 78600tgcctttctt cttggccacc ttcgttactt cctttgagca ggactaggac agaggttgct 78660ggagcctccg ttgccacccc agaccctttg gcacatccct agcaccacca tccaaatcta 78720cataaaaaat gcagttagtg tggaggctaa acccaaagta cgtgctagag aaatggcaag 78780tctgtggtgc tcggcagctc tggggcactg ccatgttgcc tctgctggct ttaaacagta 78840ggtgctttct gatgagcaca caactatcta tgggtgctga tgaagacaca ggaggtcttg 78900agttagggtc ggctcaggct tgaagcacaa gcatgaagtt cactcttggt ctgatgagac 78960catagaagac caaactccta ttggatgatg gacagactct gaaaacactg taaacccaag 79020ctgactctat ccccactcgt tactgaggga caagcacttt gtataggtac agttttctaa 79080accaaatcat tgatgcaatt tagtgtggcg ctaggtgaca tgaactgtgg ccatgaataa 79140tgcccccgag agctacggaa actcgtatct ctgtcctccc agctcacagg gcgccttctt 79200tgcatcacct catctctggt gttcattctg gagcgtcagt gtgtcttaga aggtgaccaa 79260gacttactgc ctcttaatat ttagctgtct atgccagaaa aaatcactca aagagcagtg 79320acgcttttgg gggatccagg gccatatttg ggggtataaa aggtgactag actgtcagcc 79380acctagacct gctttaccta atgaatatag ggtttaggaa aggtggctgt aagcaatgtt 79440acagcctcct ctaaaacatt ctttttaggt ttaggtattt aagtttatgt gttacatgtt 79500ttgccagcat gtatgtatgt atgtatgtgc atcatatata tacactggtg cccttggagg 79560caacaagagc ggttggatca cctggaagtg gagttacggg tggttgtgag ctatcatgtg 79620ggtgctggga actgaacctg ggtcctctgt aagagcagcc agtattgtta actactgagt 79680tacctttagg ataactcagt cctcagccct ctttaggaat ggtattaaag atctagatga 79740aggaactatt gagtatagat cccgattgca tgcatgcgtg tgtgtgtgtg tgtgtgtgtg 79800tgtgtgtgtg tgtgtgtgtg tgtgtgcaca tgctaggaag agacgcatga caattcagga 79860atacagatct ctgtgatttc acaacagcag actggccatt agagtgatag ctcattgcta 79920gcatctcagg gaagcaacca ggaaaacatc caagcccgtc tctccctccc ccatcgtgtc 79980actcctgaga aaacagaatt ctacgtttta atgtctgtca gtcttatgaa gaagcaccat 80040aagtgtattc ttttgtacct aggggtgggg gacagtgtgt gcgtgtatgt gtgtgtgcgt 80100gtgtgtgcac tcacacacgc tcatggaggc tcaaagagga catcaggtga cctgctctat 80160cttatccctt taatacagag gactgaacac agagctacac tggtggccag aaagcctcag 80220cagcccttct tgtctctgct ctgtgtggca ggagaaagag aagcacatat agccatgttc 80280agctttttta tgtgggtgtt ggggaccgga tggagctcaa gtcctcgtgc tctcatggca 80340gcctcttcct actgagccac ccctccagcc cttctttgac atgttaaact atggaaaacc 80400attttttaaa gtgaaaaata acctgcagct taaaattttc cactaatttt cagtagccag 80460ttaagtgctc atacattcac attgttatat actttgaccc cttctctaga actagtccac 80520agtctgtctc tggatttagc tactgtaaac ggttcatata acaagacggc agtatctgcc 80580ctttgtggat ggcttctttg tcttagcatg atatctgtcc ttgtcctttg gctgataccg 80640tttccccaac gttactcatt aaaatgattt tatatttggt tccatttgct gtgtcataag 80700agaactgaga caagatacta caactttttt tttcagacag tacattcata aagttagctt 80760aacaattgtg ctccaccaca gaatgttttg acctgacagc cagtggctat ttcacttgtt 80820tctaggcgtg gaagagaagg tggtttgtgt tgcgcagtgg ccgtttgact ggagacccgg 80880atgtcctgga gtattacaaa aacgatcatg ccaagaagcc tattcggatt attgatttaa 80940atttatgtca gcaagttgat gctgggttga cattcaacaa aaaggagttt gaaaacagct 81000atatctttga tatcaacacc atcgaccgga ttttctactt ggtggcagat agtgaggaag 81060acatgaacaa gtgggtccgt tgtatctgtg acatctgtgg attcaatccc acagaagaag 81120gtacgtccaa ggttttgttt tgtttacatt tttaaaaatt tattatatgt gtgtgggtat 81180tttgcctgtg tgtatgtcta tgcaccacaa gcattgcagt ggctgtgaag actagaggtg 81240gccaatgaat cccctgggac tgaagttaca gatgttctga gtggtcatgt gggagctggg 81300aattgaacca gggtcctaga ccttttcagc actaatccat ctctccagac ccagagttta 81360aggttttgtt atccagttag tgcaatcatt tttctctttt tttggggggg ggggcggggc 81420gggaggttgt agttttagaa atggtacaaa attgttattt cagttatatg catcaatatt 81480tttcttttac aatcggaatt tgacattttg agcaggcact gaatgcttca ggcagtttag 81540agagaaatgc tacaggtttt gagagactta actgatatgg accagtctaa ataactgtct 81600tcgtgaagaa tttggggagg ggagtggcgt gtgtgtgtca caatgtgagt gtgggggtct 81660gtctgggtgg caggtgcttt acctgcagag caccttgtgc cataggctgt gacatacaca 81720ggctgtgctc tcactgctcc ccacagccca cccctcccca gcttgaaagc agcaggaaca 81780tttttaatag cagaactcca tagtccttaa aaatgggtga ccaatatatt tgggtacttt 81840gtgttttcgt aatcaagtat gaagggtaga agacagtgag ctcatttata aaccattatt 81900caaatatgga attgtggttt gtgagctgga caccagaaca ttgctaatca caaacccttt 81960gtatttggtc cctaaacaac aaaatccctc gtaatttcaa tctccccata cttcctacag 82020cttataaact atatcaaagt aactatatct aagaactata tgtaagtaat ggctttaaaa 82080tcagccattc ctcatgcatt tgcagtacat accttcactg tatctcaaga cagatgtgga 82140ctctaaacag cagagcagag ctttgtagca gtaatcccca ccattccatg tgctagagtg 82200aaaaccggga tgtttgtgca ctatgtgggt gcagtgccag cagtggtcag aagagggcat 82260cagatcccgt ggaactggtt tacagacagg atttttgttg gtttttttaa tgtgtggggg 82320tattttgcct gcatgtatta gtgtgtgtca catgtatgcc tggtgcccga ggaggccaga 82380ataggatgct gggatcccta ggactagagt tacagacagt tgtgagctag gtcttctgga 82440agaacaacca gttctcttga ccgctgggcc atctctccag cccctctatc agcaattttc 82500tattctcttg tctccaacca tccctcataa ctactcccga aagagcaatg gcttccttgg 82560cctttgttag gtctgtctct caggcatgtg ctctcccttt ccaagactga atatgcagtg 82620ttcttgtttc aacagttggc tgataccttg tgacactttt tttactattt gaatttcaag 82680ccagatgtgg gaagaaatag actctgcttg gacagcactg tctgagaaaa tcagtctccc 82740gatccctctg agaataacta attttccctc aaaacaaaag gaaaggggaa aattctgacc 82800tgtgttttga ttccaggaac ctctcaaaat atcattacca tgttatttcc gctgccccag 82860gtgaccactg ctattattga atactcggaa cgctggcagg ccctcctgtt tttaaattct 82920agctcactga actgtgtgcg gttcctgcta aggtttttct ttagtggatt tgggggtaga 82980aactggggtt cttttgtttt tcttggaaat gtgcagattg actttttatt ttggttcggg 83040ctcaccaagg tggaagctgt taaatttgct aaacaagcac taggaagccc agccagggct 83100gcaggaagct cactgacatc tagcaaaacc tctgggctaa gtttagggta tgagcttaaa 83160ttgcttttct cagagtgtcc agatcatggt agtttaaatt aaaagaaaat gcaattcccg 83220atttgaggtt cttgccttta gtaagataaa ctgatactta aggaaaaaat atatagactg 83280agaatatctc agcagtgtgt ttgtctagcg tgtacaaagc tctgcgtttg atcctcagaa 83340cagcatagat gtgacataat ggtatatgcc tgtaaatgtc agcacctaag aggtggaggc 83400aggaggatca gaagttcaag gtcttacaaa catagaagac cgtttctcag aggtggaaag 83460aaagtgtagg gggttgggtg ggtacaagga tttcatcttt tctataacac agtagtcacc 83520aagggcttct aaatttcctt tgactgagtc tggagagatg gctcagacat taagatcact 83580ggctgctctt ccagaggacc ccgggttcca ttcccagcaa tcacgtaaca acttaaaact 83640gtctgtaact ccagttctgg ggactcaata cctccttctg acttctgcag gcaccaggca 83700ggtacacaga cacacataca aacaaggaaa aagacaaaac ggaaaccaaa taaacaagca 83760gaaacccgcc cttcacataa actaataagt aaaatttact ttgagatttt tttttttact 83820tttttttttt tttaatgaga caggattcaa acagaggttc tcttgcctct gcctcctggg 83880tgctggaact taaactgtgc gtcgccacgc ccaactcttt ttacgttttt attaagacat 83940ccactattac tattgtgtag gcatagatga ccagtgcatt gcttagaggt tttctatgtc 84000tacatttgta catggatata cgtgagtgtg ctttaaaggc caagctcaga tcaagaattt 84060caggattcac attccagcaa agaatccttt tcctagagaa atcctagaga aatttcctag 84120agtagatttg ggataatttt gttttgtttc attttaagat gtatgtatgt atgtatatga 84180gtacaccgtc tcgctctctt cttgcacacc agaagaaggt atcagatccc cttacagatg 84240attgtgagcc accaggttgc tggaaattga actcaggagc agtcagtact cttaacatct 84300gagccatctc tccagcggga tagttttaac ttctgtcccc agtcatgaat tttgcctgtt 84360ttaaatcctc taaaaaaaaa aaaaaaaaaa aagaaccatg gagtctggct tacctcttcc 84420ataaaagtca ccatgttgaa gtatgtggtg ggtttttttc cccatacagc actccctcac 84480tcctattact ctcatacaat atgggtctat ttgaatatcg gtaaatggac atttcagttg 84540ttctacaaag gtctgaccat tcttacgtgt ctttttacaa caaacaaaca aacaaacaaa 84600caaacaaaaa gatccttttt tagtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt 84660gtatacaaag gtgctaggga ggccttgaat ctaggattcc tactaggtag ttggaagctg 84720cctagtgtgg atgtcaggaa ttgaactcag ctcctcccaa gagccgtctc tctagtctgt 84780tttgtctttg gatagacatt agctcttagt tcttttggat acatgccaat gagtagaact 84840gccacgtcat agcgtatatg cattttagca tttgtaaaac ataaatgtct tcaaagccat 84900tgtaccagct gctgtcctaa cagtgtatgg caaatagagt tagcccatat tgttagtgac 84960atttggcatt agtcagtggt tcatcttggc tttcccagta gctgtgtact ggcaagttca 85020tgatttattt tgtgtgggtt tttttttttt tttttttttt tttgaaaagc agttcaacta 85080gaaagattat ctcttctgtt tggctttctt gcaaagtttt agttgcgatt cagtgggacc 85140cctgtggtgg ggtccaagct catagaaccc caggatttcc atgccataga gctctggata 85200aaaacagtgc tgtttgtcag catccccgaa ggattagaaa ggtatttgaa atgtgtgagg 85260gacaagaagc ttggaaaatt gagacgtttg tacttgaaac ccagtaaaca agttgggaag 85320tgcaggcatt cagggtcttg cctggctgat taacagtgga ggcggcggca gggtgcctca 85380ctgacagcag ctactactgc agtgtctatt tattatccat tcattagact gattgacctt 85440ttaaatttca tatatgtatg gagagagaca gacagagaca gagagacaga gagagagaga 85500gggagaaagg gagagagaga cagagaatgc gcgcgcaaga gagtgcccac ttgtgtgggg 85560gtcccaggat accctgcaga agttatttct ctccttccac caggtagctc ctggggactg 85620atctaagata gccaagctgg caacaaatat ctttacccgc tgagccatct cactggctca 85680ttaaaggttt cttatcataa gttataggtt tgacatgacg tttccattca cgtacgtaat 85740gtattttgaa cgtgttcacc accacacaag acacctctct tctcttgccc cctattcttg 85800tggagccctt atacatacct ttgtatatca cacgaagcgt gacataccta agtaggagaa 85860gccaatagta gggcgagggc tcctgtgagc cagttcactt gtggatgtgc tcatcacctc 85920cccctgcact gaaggcttgc ctgctggaag gcctcgcatt agcttattag ctttctaagt 85980aagtttcaat taggagtttg ctccagtggc ttattaggcc tttaatggat gctaattata 86040ctctcggtgc attctgagtt aagaattggg aaatgatttt acatgtgtaa gcaaaccttg 86100agccagtgtt tgtttcttag aagaaactta gagagagcag taagtcagaa atcaaaacag 86160gactgagacc agaatgatct ataccactgg gcttgtcgcc cagaattggc tgaagtccca 86220tgcctgtaag gccaagcagg agtctacttt gggccagtat tgactctcgc aggaatctta 86280aatttgagat tatttagaca caattcaaca aatttttaaa tttacattat ccacgtgtgt 86340gagcgtccgt gcttgtgttc atgtgtgcca cggggcatgt cagaacgata tatacttctc 86400tccaccctgt ggttccaggg aatgaactca ggtcactggg gctgtggcat gtacccttac 86460ctgcggagcc atctcactag ctctgcacca tccaattttt atatccacat actcagtacc 86520aggtagatgg tcggctcaag acagaatgct gaagatggtg cattttccag gttaacacgc 86580tagcatgatc tctgtactct taaagctact gcctgccact gtcacttggg tgtttggggg 86640tacctaccgc ttatgtctcc acctttatga acttgcttgg ggttgtggtg gaattttaat 86700ttcagctctt gcctcttgaa gacataggtg agtcctgaca tgctgttaag cacagcattt 86760tttttttttt tttgaaacag gcccttctga gtcccaggcc tgttttcagg agattccact 86820gtctttctca ctcctgggta ggaaatctga aaggtcagag gtgaagctgc tctatctgtg 86880acagtagaac aagaccgggg agtgcgagtc tctgccagga caattttctt tttcccacga 86940gagttcctca atttattatt ttatggctgt cagatgtaac aaaagaaaca aattaaaaaa 87000aaaaaaaaaa acaaacccta caaaacaaaa cccgccactg aaggaaagtg tatctgtgca 87060aaccgtcatc actaggcgag tcagaaggag gtcctgggtg ggctgcagtt cacggacctt 87120cactgaggga actgtgcgtc tgcagcgttt ggttttcttt ccaactgagc gctgagcttg 87180gttggtggta ccatgcatgt agctgaagag ctaacagaaa gcaggggcca cgcactgggg 87240agagagcttg gagatgtggt agctgcttcc ccatttccct gctagtgctg gctggggctc 87300ttgagctcat ctttgaatag taaatatttt ttcatggttc tcttaaggcc agaaggatat 87360agtctgaatc attatttaat ctgcagcatt tgaaatcctc tgatgctaaa tccctgtatt 87420agcaactgga tctcgagttg aacgcgttgt gggggcagct tacctcactt tataatcaac 87480ctctcctaga cacagcaaca ggaacttaag aggacccact gaaattcgga caggatgtag 87540taggagtggt cccaactgta ttttctgctt ctgtcttcct cctggcaaag ggtgttaaat 87600catattagag aaatcgattt tttaatagcc atttcatgaa aattcataaa ttaaaagtta 87660gaactcctct tggtctaaac caaggagatt cacagacctc attagcatag agcaggactc 87720caagagatct caagcaacca gtccaaggct acacatgtag gcaaatgggc gtcgaggatt 87780ttcaggtgcc tcctgctctt taaaaccctc ttccagtctg acaaagtgag cctggatgtg 87840ctgcccagta gttgaaatgt ctccctgccc gtcccccaac ctcagctaca cgtaagagag 87900acagttatga acacagcttc ctgatcatcc ctgaggcctg tgggcagcac gatactgttc 87960tcctcagaca tcacaattaa gatgtgccct ggatactcct atctgagggg aagcattgta 88020aagttggccg ttgtgaacat atgaatgagg taaacacaca aaaccatcaa aagtgtgcag 88080attgctctag gaatccagag gagggtcagt tagaaggcca aatggctggt gttcagtcag 88140agttgatgga ccatccaccc catcatacct tttagcatgt tttacgtatt ttattgttta 88200aaatctgctg ttcaccagcc ttctctgttc actcttatga ccttatattt ctaaccagaa 88260taacagatca tagtctatca gggccgttaa aattattctt tgtcatgtca gaaactacag 88320agggacttta tttctagctg tacaacttat tttctagctg taagctgcac agtttatttg 88380ttttccttca tctttggaac atgctcaagg gatttgtggg atttttggac aggtgtagtt 88440acaagctcgg ggaagggtct gacgctacag atgagcaccg gattcccctt ggctcagagt 88500tagagaggtt ttcccaataa cccggtaggc aaacctgaag agcagatcca ggcactctta 88560gcagcattgt ggccatcaag tctgtcggtg taagtgtgtc tccttgctag gttggcgtaa 88620cctagggaaa tgggggaggg gggaatccaa aacaacaaaa caactgtctc ttaggactgg 88680agagatggca tagtggttca gagtactagc tgttcttgca gaagacccag gctccactcc 88740cagcccctac ataataacgc atgaccatag taactccaat tccaggggac tctaatgccc 88800ccttctggcc tccaagagca ccaagtacac atatagtgag cagacttatg tgcaggcgaa 88860gtacctatgc acattaaatt acgttaacat taaaaatggt gcctactaag atcgtaggac 88920agtcttattt taatttaagt agtttcaaaa ctcagtattt ttcagggttg cattagtgtg 88980gcctcactgg gtgtagatct gggtctgggt gtttgcacat gagtgtgtat gcttgtggag 89040gctggaggtt ggggtcaggt gtgattctct atctctgtcc atgtgagtca aagtcattgt 89100atttggagct tgttaagagg gttagagtag gtggccagtg agctttcggg gtctgcttgg 89160gtttcatgta agtttgggga tcttagctcg agtcttcctg cttatgtgac aggcgcttat 89220ctcctcaggc cccatcttga tttctctcag gcctctggac taaaggaaag gctgaccttc 89280ttcagatgtc tttggcaaga gttaattaac atataaccat tgcgatttag gtagtatttc 89340tttcttagtt caaagctatc atctcgttct ttttgcccag attggccttg aactggtaag 89400gcgagcattt cagtcttcag aatagctggc agagcaggtg aataccactg cccatggcct 89460caggtagtgt ttgtttcagc taacactgcc atcattgatt ctggttatgt gcattaaaac 89520aatctatctc cagactttca aagtagctta gtctaagaac aacagaatct tcaactgtga 89580tagactaaaa cttaacttga ttacagatcc tgtgaagccg ctgactggct cctcacaagc 89640acccgtcgat tcacctttcg ctataagtac agcaccagcc tccagtcaga tggaagcttc 89700ttcagtcgcg ctacctcctc cttaccaggt catcagcctt ccgccacacc cagacaccct 89760cggcctccag gacgatccac aagactacct cttgctgatc aactgtcaaa gcaagaagcc 89820tgaacctaac aggtacatgg tacatgctag cgcatgtgcg cacactcaca cttgtgtgtg 89880tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtttgtgtg tgtgtgtgtg 89940agtgagtgtg tgtgtgtatg tatgtgtgtg tgtgtttgtg tatgtgtgtg tgtgtgagtg 90000agtgtgtgtg tgtgtgtgtg tgtgattcag atttgtgttg caggtttata atcctctgtt 90060aatgatttct gttatgtggg aggtattttg ttgttggcac tttctcccat catggctgat 90120catagttgtg cctacagggc tgacttggaa agagtctcga agtctttgtt agggactcac 90180agcgttactt gcttgacaga tgtcactgat ttgtaaatgg tgaccttgct ccatttctta 90240tgtgattgta aagcagccaa gggtcatctt ttgtttctga agaaggagag gagtacctac

90300tactagaaga ttttgaaagc aaagcgattc cattgcagtg agtgtgctgt agttgtggaa 90360ctgtgtgaga aactaatcat acaggtaaaa tcccattatg gcaaatccta gaggctaggc 90420agaccaggag gcaatgacat ttcatgaaat caaatagact gctacaagct gtgaaccttt 90480caaaccttcc tccccagcca tggagcagtg ctgactccaa gctgtgactt acaactttta 90540aaatagatat atgtggggtg tatgcctgac ccagagggtc caaagagcct tttccagatg 90600cttggtagag ggtcatcacc acacacacac acattcctgt gctaagcttg aagctatttt 90660aacttggcca tctttggcat ttgatagtga aaagtgaaaa ggcataagta agtggcagag 90720gatattgaag gacagttact ttatcatttg aagaagcaac aggaaagtga agatcaatat 90780acaagctagt gtctgctgct tacaactcgt gaccattggt gacagcacca acaactggaa 90840tatttttcac tgtgcaaaaa tggcaatgac tcccaaggaa gatttataaa atatggtgtt 90900aaggagttaa gctttatttt taataacaaa agtgtttctt ttctttggtt aaaaaatgat 90960tattagttag aataagatta tagagtagtt gtttaaaaaa tgtatgtaga acttaaaact 91020cccagaaatg gtttctatga ccccttccaa ggggcagtga gtgaaaggag agagacgaca 91080ttccatggat ggttttgtct gtttgttata atggtatttt acctgtaaga gcaagaaaat 91140tgtgtaagtc aacactagtt aatactatca aattctaact caactgtgcc tgtttgctgt 91200gagcattgag gcagcacact tgtgggcttg cgccctgtgt gactaactga ttctttcctc 91260tgtggaaact cactgttact tgctggtgtg gttgtttttg ttttgtttta ttttgtcttt 91320tagaatccaa ctgatcagtg acaatgtttg gtgtattttc ttcttatgac agtgcctttc 91380attgattttg ctggtacaac accattactg ccacaggaga aattgttctc caagttactg 91440agtagatact ctgctgcagt gacagttaaa taacaaatat ttgtaaagtc cctaccacat 91500gcccactagt atgttaggtg ctatgaagat acaaatggat tacagtcccc tgagattcag 91560tcaccctggg agctctcaaa gtaaacctgc tcaagaagcc agataaacta accatttata 91620tatgttatat ttatatatga gagcacaggg gacggttgct cacacatcct tctccaccat 91680gactcagctc atatacatgc cccttcagga agcctgtcta caggcttaca ttgaaatgtc 91740cctctgggtt gatgaggttt tgtttttgtt ttagtgtgtg tgtgtgtgtg tgtgtgtgtg 91800tgtgtgtgcg cgcgcgaatg tgtgtgtctg tgtctctgtg tgtgtctgtg tgtgtctgtg 91860tgtgtgtgtg tgtgtttcct tggaggaact cggcatgggc aaatacagga caggagttac 91920tgagaacgct ctggcagttt ggtggtggta gttctgctgg tttataatta taaacagcta 91980aagaacattt gtgctttttg aatcttaatg gaccacaaac aaacaatctt aacaaagcaa 92040gttttgattg ctgagctgca gtgtatatgt tgccaaaact gactcgactg tggtttggtt 92100tttgattttg gttttttgtc attcagaacc ctctttgact ctgccaagcc caccttttct 92160gagacagact gcaatgacaa cgtcccttcc caccagactc ctgcttcctc ccagagcaaa 92220cacggaatga atggcttttt ccagcaacaa atgatgtatg actgcccacc gtcccggctg 92280acatctgtct cgggagagtc cagcctctat aacctgccca ggagctattc ccatgacgtg 92340ttgccaaagg aatccccatc aagcacggag gccgacgggg agctgtacac ctttaacacc 92400ccatctggga ctgcaggtgt agaaacgcag atgagacatg tatccatcag ttacgacatt 92460ccgccaacac ctggcaacac ttaccagatc ccacggacat ttccagaaag cacactggga 92520cagtcatcaa agctggacac cattcctgat atccccccac ctcggccacc aaagccacat 92580ccaactcatg accggtctcc tgtggaaacg tgtggagtcc cacgcacggc ctcggacact 92640gacagcagtt actgtatccc tcctccagca ggcatgacgc cctcccggag taataccatt 92700tccaccgtgg atttgaacaa gttgcggaaa ggtaaagctc tggccggttc tgctctttag 92760ggggtcagtg atagaaagtc tgtttccaca aggcttaatg agtaagagcc ttttctgccc 92820aagtatagca cctacactta gatcctagca tcctctcagg tttctgatcc tagtgaaatg 92880tatgggacgg agacaggggg gttattggta cttgtgtgcc agcctacctc tcggttcaga 92940gaaagatcct gtctcaggag aataaggcaa agtgtgatat aggaggacac ccaatattct 93000cccctggtgt ccactcactg tgaaggcaca caagcaagta aacccacata catcacgcac 93060atacatacac atgcaagaaa agcaattttc tcagtaattg ctctcttcag catagaatgg 93120tggtgaagtg tatttatcct ttcttcacct ctgcagcccc cacacgtgta gccatgatac 93180cagaatcagt agatccctgt agggtgtgtg tgtgtgatca ggggggctgg tgggtgtctg 93240tgtgtgtgtc tgtctctgtc catttgtctg tgtttctgtg tgtatgtctg tatatctgtc 93300tatgtccatt tgtctgggtt tctatgtgta tgtctgtata tctgtctgta tctgtgtgtg 93360tctttgtgtg ggagtctggg tctatgtgtc tgtgtctgtg tgtgtgtgtc tgcctgtgtc 93420catgtccgtc tgtctgcctg cctgcctgtg agcatacacg tctgtgtgtc tgctctgtta 93480gaagaaaaag ctggatcctc tcatgcctga aaacgtgtgg tcttacggct tcctacctcg 93540gttctctccc atttcctaga cttctgtaga ggtgggctgt ctgaacactc ctagtgatgt 93600ccttgcctgt gtctgtgtgc caccatgact atttctatgt catatctcca gggttttccc 93660cttccttcct tgaccagcag cttttacatt tttgtcttca taggcagcga tgctgttcaa 93720gtcgagccat tgtactttga ctttttcaat ttttagagca ctgcgtctct cctctctctc 93780tcttcagctt agctgacagt gggtatgcct gtcttgtccc ataaagcctg caaacacacg 93840ctaaagacct cgggagtgtg ggcttggtct agcttaatcc tcccaagatt ctggaatttt 93900tgtgcagtat aaatactgtg tgttcagcat attgtcccat ctcctctgcc ttctgtttcc 93960tccatgctcc ctgttcctct tagttccctt catctcctag agagttttgt gtctactttc 94020atattaaata tgattttatg tatcaatata aaatctagtt accataaaat atctttgtct 94080ctatgagatt agcttaatta tcttaattat ctcatgttct accttctagc aagtggtatg 94140acttcattct tcgttttgtt ttgcttttca tctttattta ttagtttatt atgtgtagga 94200ggtcagagga caacattcaa gagttgcttc tccccttcca ccatgtgggt cccagagaac 94260aaagtcaggc catcagactt agcagcaact gcctttattt gctgagccat caggccagcc 94320caaattcatt gttctttaag ggtgaaagaa tccactgcta taggtgggac cacactttcc 94380ttaccctgtc ttctgctgct ggaccctctg ttggccgctg tctagtttag ctgttgtgag 94440ttgtgctgaa gcaaagactg ctgggcacgt ctctgtgatc tgtcgctctc aagtactcag 94500ggtggtagct gggttgtatg gtagatgtgg tttcacttat ttaattttta ggaaacttcc 94560ataatgattt tcctaatagt ttgtctagtt tatgttccca cctgcgatgt ctgaaggtcc 94620tcgttctctt ttatccttcg cccttgccag cacttgttag gtttttgctg ctgttttatg 94680tgagcgaatg tttgtgaggg gcagatttgg tgtggttttg gttttctggt tttttgtttg 94740tttgtttgtt tgtttgtttt gtttaatgac caacattatg actgggataa ggagaaatct 94800cagtgtggtt ttggtttgca tttctctgac agctattgag gttattaaac tgtgtgtgtg 94860tgtgtctgtg tgtgtgtgtg tctgtgtgtg tgtgtgtctg tgtgtgtgtg tgtctgtgtg 94920ttaatggcca tttgtattcc tttctttggg tgctatatac tcctttagtt agcccattta 94980ctggctaggt agttttctta tttagttttt aagttcattc ttgctatgag cgccatgttg 95040ggtgtacagc tggcaaaggc tctctcccac tctgtaggct gcttttactt ggtcactgct 95100ccctgcactg tcatttgtaa tttcatgagg tcttgtttgt cagctgttgg ttccatgtgc 95160tggatggccg aggtcaactg agcgtgtgca ttgcctggcc tgtgcttctt tcttccccag 95220ccacttctga gtttcaggtc acatccctct gtaaggtctt tgagccactg gagttggttt 95280ttggaggatg aactgctggg atctagtatc attgttacac agatggctat ccagattctc 95340actactacct gttggagnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 95400nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnttc 95460cttccttctt tccttcctgt ctttcccctc cctccctgtc cttctcccct ccccactccc 95520ttcctttcac cttgccacag gatctcatgt agccctgtct cactgtgtcc ctgagggtga 95580ccttgaactt cacacacaca cacacacaca cacacacaca cactgctcgt tgctgcgatt 95640tcaaagaggc taaataacct agttataagt tagtggctca aaatggatat catttctttt 95700ttccatcttt agataatcct tttcctgcca ggcttggaca ttcggttgct gtcatagtca 95760gatgatgaca ttggtaaaca cgtaggtgtg tgcttaacat gccagttaga gaccctgctt 95820gatgagtgtg ctttcagtgt taacagatat gtgttcatgg acaagtgtga agagagagta 95880ccttcgagct ggtaacacgg gaacaaaagc tgactttttt tttcttaatt ccagatgcta 95940gttctcaaga ttgctatgat attccacgga cctttccgag cgatagatct agttccctgg 96000aaggcttcca tagccagtat gtaagtacaa ctgaccactg gccctgccac gttctgactc 96060tttagactta gtgccccgta ctctatatgc gaccatgaaa tgaaagtaac tacgggttct 96120gtagatacag gcgtgatact tgtgtgtgac agtggctttg cttattaaga gaaatgtatg 96180tgtgttttag aaaatcaaaa gcgtgttgac agcgggaggt gtctcgggtg aagagctgga 96240tgagaactac gttcccatga accccaactc gccacctcga caacattccg gcagctttac 96300cgagccaatc caggagccaa actatgtgcc aatgacccca gggacctttg acttttcttc 96360ctttggaatg caagtccctc ctcctgctca tatgggcttc aggtccagcc caaagacccc 96420tcccaggagg ccagttcctg ttgctgactg tgaaccaccc ccggtggata ggaacctcaa 96480gccagacaga aaaggtaagg gggtgtggcc agtaaaggat aaggtgggga catgcagtta 96540gggctctatt aaccatacca aaacatgaaa ctgtttaggc tggcatggga ctatgctgaa 96600gcactttata tagcctgggt ccttaactta agttcagaag tcctactatt cgtaaatgaa 96660tatgttgatg tccaggtggg atttccaggt ctcctcagac ctacagtaga gcttacagac 96720taagaatgcc cagagcaatg gcagcagcag gcaagcacta ggagaatctg gaatgttctt 96780cccagaggat ataggtagag ctcccgaccc ctgcctgact gtcccctccc ctgatgtatg 96840acactcacac atgtcaagag agacctgaaa tctcaggttg tatgtgtgtc agtgtcttga 96900ttgttttgtt gttgttaatt tctgccaact gttttgcttt tgtaaagcat agcaaaccaa 96960gcttaacact agatatgacc ttgctttgct actttgtata ttatcaaatt agattccaga 97020taataatgca cttgatgtgg ttatttaaga atagtgaaga tgtctattat agcctttctc 97080tttccccaca tcatgacttt aaaaacacct gccttggctc caggctaagt ggaagcttct 97140atggctctaa ttctggatga tgtaaggaaa gttgacttct gaagcagggg agctagtgag 97200gcctggttct ctttgtcaaa ggaagccttc agccactttg ggtaatgtct cctcaagttt 97260taaaaactta ggttcaagcc aggcatggtg gccttagctt ttcattctgg gatctgggat 97320acagaggcag gtaaatttct gtggttcaag gccagcctgg tctacacagt tctgggtcag 97380ccagagctgc acccatctcc aaaacaaaac aaatttaggc ttaagtcttt taaatgcaac 97440acactgtgct cagcacacca ctagtgtgtg gcttagtctt gataaccaac taaagttaga 97500gttgtatgat agaatgatgg atttctggct tcttgcagag aaattaaggt catttgaaaa 97560gccacgtgtc atttatagct tttttcaggc catgtaatgc ttatgcaaac ttattaacct 97620gggcttaagt cattaacact aattatgttg tagttgtggg agactcaggt ttggttttat 97680tcttacgtaa gaaagggtgt atttctgctg tactcaatgg taatttgtgg tctttgccct 97740gtgcacttgt cttgtgggac gtctctgcat cttctctgtc cgctcccttt cttttctcct 97800tccctccctg agttctctcc cttgactggg ttctgccccc tttggcctgc agcacttctc 97860tgtggcctca gtcctcccgc ctggatccct ccttggccct tgcccgtcaa catctgcctc 97920taatgcctcc tccgtcattt tcaggcttca gattcttaga gatcacttga gtggtccagg 97980ttatcccttc acacagatgc ctggctcctg ttgggacaga cgcccacctt tggtctcaca 98040cagttgagaa tgaccccatg tggtagccgt agactcccag acacccatgc ttcatggcca 98100ccatctggaa ttgtgttaga aagggtgtga gtgccacagg ctttttgtcc cttttgtttg 98160tctttgtctc taactcaatt agaaatctca ctaattgttt tcttcttgac ataaaatgta 98220agtgaacatg aagagatctg taattggtcc aagggtctct ggaagatcaa tacacatctt 98280gtagacacaa agtattgaaa tcttttttat tatttttttt ttattttatt tgtttctctg 98340tgtagctctg gctgacctgg aacttgtctg tcagaccagg ctggcctcca tctcagaaat 98400ccacctgcct ctacctccca agtgctggtc ccactacctg gctttgagat ccccttttca 98460aaagttaaag cggcatgtta tttccataga atagaacaaa gtaagcttgg atcgcgtttg 98520gagacagtta ttcttccaag aactaggcaa acttgaaatg ggcaggacat ccatccccag 98580caccacactg tgatttcatc ggccagacac tttatttggc ctatctgctt agatacatga 98640tgaacggaca aagagcaccc aggggactac atccagggac agttttagcc aaaggttaaa 98700gaggccattg agatttagcg ttagtaaagg ggttttactt ttctttcgta cctttaattt 98760aaatcaatag gattcatggg aagaagcctg tgaaggtgtt tgctgcatcc agaatactgt 98820gcctgggaaa gtataactgt taagaaaagc agtttagggc tggagagatg gctcagcggt 98880taggagcact gactggtctt ctagaggtcc tgggttcaaa tcccagcaac cacatggtgg 98940ctcacaacca tctgtaatgg gatctgatgc cttcttcnnn nnnnnnnnnn nnnnnnnnnn 99000nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 99060nnnnnnnnnn nnnnnnnagg gaagaaggga gggaggaggg aggggaggga gggagggagg 99120aaggaaggaa ggaaggaagg aaaggaagga aagggaagga aggaaggaag gaaagcagtt 99180taaaacccag attccagctg gacagggtta gcactccaga aggcagaggc agaggcaggc 99240agatctctgt gagttctaag ccagctaggg ctatgcagtg agaccttgtc tcaaagcact 99300aaaaagaaaa tctaaaagct gagattaaag tctcacaact aaacttatcc ttgagtctgt 99360gtgagctggg agcatgtgtc ccattcgggc cacgagattg ttttccctcg gtgtggcaag 99420agatgagagt tgtacctgca aattcagtga gtctgggtaa attacttgag ccgtcctttc 99480agtgtcttat taactaattc ataaggaaat gcgattgtgc caggtgtgcc tggctcctct 99540cagagcctca gccagctggg tgttggtttg tttgagctgc tctctttgag agagtccatg 99600acaaatagtt ggcagcctcc tctcggttct cccgcagttt tatgtgccct gccacagtat 99660acatgtggtg ctggccagtt ttcagacgct tcccacatac tgaggccaca gaagtacaga 99720ggtgctgtgt gtgcagacgc ctggcgtgtc cctgctgtcg gctgtcacag cttgatcgct 99780agctcagagt tcacgatgct aaccctgtag tgtttgtttc aaacaagatt atgtgtgcat 99840ttaataagga gagctttaag cgtattgtaa gagataccag aagagtagag cagtcgctct 99900ctagagccag agtggaacag tttgtagctg cttgtgtttc taatggaaaa ggaattttgg 99960aagagttgga gatttttata aaatatctgt tgggtttcag taaaagtaag ccgtgacaca 100020gagggccctg cttaaccact gggctcatct ggattctgct gcttccatct tccaggtcat 100080gccagaggca gaagccacca acgatggtgt tgttttcctc tgtttgaaga gatattatca 100140ttcttctttt aaagtttgtt tttgttttgt tttgtttttt gttttttttt tgagacaggg 100200tttctctgta tagctctggc tgtcctggaa ctcattctgt agaccaggct ggccttgaac 100260taagaaatcc acctgcctct gcctcgcaag tgctgggatt aaaggtgtgc tccaccactg 100320cctggctcat tctaactttt taaaaatcga gaccttgaag tttttgaata tcattaaaaa 100380tgaccgtgct ttctttaaaa ttatgcttat aagataataa atatgtaaag accagatgag 100440tgatactagt accttaacta attttttaaa agtttttttc taaattttta tattcaccaa 100500gtagatgcct agcggctgag gaaacccaaa aagggtatca gatccagtgt gcaactggag 100560ttacagacag ttgtgaacca ccatgtcggt gctgagactc accccaatcc tctacaagag 100620cagcccgtgc actgacccac gtctctacct tccttcacta aatttccata aacaggagtt 100680aatgtttaat tcctaaatca gactgtggat acacttgtta gagcttaaag gaaggaagta 100740tggaatgtgt tcctacagca tagccttacc agcctgagac agtttcccca tgtaatactt 100800accattaggt actacagtgt gtattatatt cctcctctca tagttaaaac aaggacaaat 100860ggtttttctt tcacaaccct gcctcaaatc acctgcttta cttttcttcc accttggttt 100920tggggtgccc actggaaaaa gtcctgcaac tttagaagag tatgcctgca tgatttgaag 100980ccaattcaga aagttctagt gtagcactga gtttatgtag gcattacatg acacgtagga 101040gaccattcat atgcagcgct ttccaaaaag gcttgcccct gtgtatttga ggtgggactt 101100tcatgtgatg attttgactg tactcaagct gaacacttct gaaaggactt cagtataaat 101160gaggtttgct tatcagagag ctaagggctt accctcagta aatccccttt gctaggcatg 101220gcgtctgtga acccaaggaa agatagagtc acttccctaa gacactcacc atctaggttt 101280gacagaggag gtggacgggt gagcacacac acacactgtg tgtgtgtgtg tgtgtgtgtg 101340tgtgtgtgtg tgtgtgaatg cccagggaca ttcacaatgc tggttagctt tgtcactcta 101400agggcagctc ttctaacgca agcctgataa agggggaggg gcaatgaggg ctggagcaag 101460ggagaggagg cagagctggg gagctgtgag tggcaggaag acctttccca acatgggagg 101520ggattgctca gtcattgatg ggtgagcacc gccaagacag tagggagagc aagtagggag 101580agcacgggaa agaaagaaga atctcacatc ccgaggagac accctcatcc aagctaacgt 101640gctaatgagt gctttattct gagcactctg ggtgaagagc tttatgcaga cagtgacaat 101700cacttggcac taaacgtttc attctgattt aaaataaaat aattatcctt tgctataaat 101760gatacaaaaa tcacatttgt ttattatctc ccctagtgag aacttttccc agtttcccaa 101820acccttcttg gtcgttggtt gggatgatgg agctatactg aggtcctttt tgcttggaga 101880tcattgttga gaaggttgtt ttcttcatga cacatgagga ttggaataaa actaacaaga 101940gttatcctaa ttgtgaaggc gatatctatg gaacacttta agtacacttg ggaagacttg 102000ctatgcaaag catgaccttg atggggattc caggcaggag caaacacatg gagataagaa 102060agtcacacct cctcctagac tcaaaggttc caattctatt agtaccttag tcattgccct 102120tgggactctc tgcagcttca ctaatcagtg accacttgag tatttgttct ggtcatactt 102180tcttccgaaa gcttggggag gagggttgga tgagttgaga caatacctgg ccatcagaag 102240ttaagggcca cagtgtctat agagccagtc ttacaaaaca taacttagca agcttcgtgt 102300agagaggatg gccctggatt caagaagcca tggtgggctg ataattctga gaaagagcag 102360ggagcagtct ggccaatgca gagcgtgtat acttcctgat aagaatagaa ggagaaaggc 102420cggtgttgtg aaaggcaaca ccgtccacac caccaaggtc tcccatattg tctgccaaaa 102480ccaactcatg tttggagtaa ttcattgaaa gtgactggat gctctgttgt gctgtctttc 102540ttatttattt atagatttat ttatttatgt atgtgagtgc actgtagctg tacagatggt 102600tgtgagcctt tatatggttg ttgagaactg aatttttttt tagatctggt cgcctcccct 102660ccttcagtcc tgctcgttct ggcccaaaga ttcatttatt attataaata agtacactgt 102720aactgacttc agatgcacca gaagagggcg tcagatccta ttacaggtgg ttgtgagcca 102780ccatgtggtt gctgggattt gaactcagga ctttctgaag agcagtcggt gctcttcccc 102840gctgagccat ctcaccagcc cgagttgtgc tgtcttacac ggcaggcccc tgatgcctct 102900tgggttcttc tgtctttcag tttgaagttt ctcctagtta gagaagacga aaacaacatt 102960gaccttcgag gtcccaacag tccctgccaa gattacttcc tcaggcaagg actgacaccc 103020aaaggatgtt gtctgaccgc cacacttaac acacacttac accttcacac aaacagacac 103080actcacatta cacactctct cgcgcgcgca cgcgcacaca cacacagagc ttcctccaca 103140aatagtaaat ttgctcttac ttttcttcta agcatagata gattttgttt tgttttacta 103200cattaagatt tagtaaaaga aacttaaaga aacatggata ggaactaatt tgaaacagtc 103260ccaccgctga aaaaaaaaaa tacaacaaaa caaaatcatt tcctagatag ctaagcattt 103320aacaagtcta gtacaatgag accccagaga gttaggactg tgaactctgt tatcagattc 103380ttgatgatcc tgatttgtct gtggacactc cagccccttt aggttttaaa agagcttact 103440ggttttaaga acctcactat tttttgtttg tttttctttg tttgtttttt gagacagggt 103500tcctcgatgt agccctggct gtcctggaac tcgatctgta gagagtccgg cttcacactc 103560agagatccac ctgcctctgc ctcccaagtg ctgagatcaa aagctgtgcc cctcctaccc 103620agcaagcttc acggtttctc gtagatgagg tcattgtgca cgggagttgt cagggctgtg 103680ctcaaaggct ctcgtctccc cagataacct gctccttcta ttttactctc agttaaaagt 103740tctttcttca ttgacatgtt cagactagat gcaactgcgc agaggagccg ggctcttcct 103800ataccaccct gaggattgaa cctcaggccc cctgggctga ccctgctagt agtgcagctg 103860gtttggacat gtggttcatt aagcactgga cagcaattgc aggcaggaat atgaagctaa 103920agaaatgtga gaaaccacac caagagagca gccattactt aacaccaaac ataaagataa 103980aatttggtga ctacagcccg acatagtggc tcactgcttt aattaactct ggcactgagg 104040aggcagaggc aggaagatct ttgagtctca ggccagccag gcctgtgtag ggagaccctg 104100tctcaacaaa caaacaaaca aacaaacaaa agatttggag aatactaagg agaaaaaatc 104160aggagggatg tgaagccatt gtgggccctt ctgaaaaagt ttgtgggaga ggcaatttgc 104220aaactgataa ccccaggaac agtttgtgat ggttgtgtac tagacactcc atgtgccagt 104280gctctgtggt gttcccacgg gtcacgtccc agccacagcc gtgtccacag agcagcagca 104340gcagcagcag cagcagcttc acatctaaga acagacgcgc taagaagcct ctttctgctc 104400gccctggttt gactgactca gtgtgtgacc actgaggtct aaggtcagag gctgacctgg 104460agaactcgga actgagctcc ggaggacaag ccacctggct tccctgcagg accatgttag 104520agccagggga cgcctcacct gcccctccca ttctgccttt cccattgtct tgttgtctga 104580catgttagaa gacacactaa catggcccat gtgcttcctt cctttcctca aggaaggaaa 104640agcaagcagc tgcttgtccc tttagccagc cctctctcgt cccctgtgcc tgtgacgctg 104700tagcatgcca ttccctgcat tctggtgcac tgagcatgaa ttgtgtctag cgagactgga 104760tgtctttcgc taacaagttt ttgtttatgc ttttaatatg tcacatttct gcacacgact 104820ctgtttcttt tattgtttgt ttgtgtaaca gaatccattt tccaatttaa aaataacatt 104880tttaataaga gagtcttctt tttaccaatt aggtcagagt cctaaaattt taagacccaa 104940accccatggt ttagagcgaa ctgattcaca aaccataggt gactttgcta caagaagaaa 105000gggtatgtac ctatgtgggg cactgggatt ttatttattt ttatggtttt tttttttctt 105060cagataaaaa gaaggagagc ttatgtaggg ctgctattgc cagccgtccc tgtgtgatag 105120ttataagaga gcacatctgg agatggaggt gcgcagcagg cctgcgtcct cactgcacgg 105180ggattatctg atagtgtgtg ccctcagtca ggcttggctc cctccctcca tcctccttac 105240ctttccaggc agtaactcat tgtttccctt ctgcttctgg gctgctgtga gcccaggttg 105300tctcctgtct ttgtatatgt atatgttgtt ttctgttgtg aaatagaata agaaataatt

105360tcagtgtttt tactatggca tggcttagaa aatgacagct aaactcttat tcaccttttt 105420aagtttttgt tttgtttgtt tgtttgtttg tttttaaaaa gggaacatga agaaaagtcc 105480cctttcttca gcccaggatg ctgtatgtgt ctgttttaag gatacagatg ggaaagctct 105540taactcaaga tttctaactc tatctttact ctgcttgagc tgtgagccta tgtgatctgc 105600agctttgaaa cgagaacttt gtgcccctgt gctgttgata agatagttga ttatgattac 105660atatttttga acccaaagta agaaaaccta ggaggttttg ttttgttgtt ttttatgatt 105720cttttggttt ggggtgtagc aacgatttta gtgctttcta cacatcattt tacatatgaa 105780aaactatact gaggcttcaa tagtggggac catgtctgac acagatgaag atgagattca 105840taaaacaatt cattaatgcg agcagtgccc agaggagctt taaaggggca tactttacgt 105900tgtcgtttaa tttatattta caggtagagg ttagaccttg agctgagtca agcagtatcg 105960ttgtgttgaa gccatatata gtaggagcag tagtagaggt tagacctttc gttgctgagc 106020gttagactct gtgtaagacg atgtctgcct accaagtgtg aattgcagca ttgagggggc 106080cttgtttatt ttgtgaagtt ctgtccactc acaccgtccc tccctttctt tgcagtcaag 106140ccggcacctt tagacataaa acctctgtca gaatgggaag agctgcaagc cccagtcaga 106200tctcccatca ccaggagctt cgctcgggag taagtgttct actgaaaacg cacagttgga 106260gtggcaagac aatgaaaatt gtttatttca agagagatgg gttgaaaaag aatctagaga 106320aagcccaaag tcaagagtat cttcatttgt cataataaaa atgcccgata tgtaactgta 106380aactaatgac cattgcagaa agagcatgtt ggagattgct tttatatggg tgggtatgtg 106440gggtgatttg acacctgttc cgttaggttc atgatgtcac tttctctagc tcctctaggt 106500ttcccatgtc ccctcggcct gattctgtgc acagtacgac atcgagcagc gactctcatg 106560acagtgaaga gaactatgtc cccatgaatc caaatctgtc tggcgaagac ccggtatgtg 106620caggcctcgt gttcatttct ctacctgagc tgccctttga agcgagttca gcttaaagtg 106680ttctatctgt acacatttac cctcccgtct ttcttcctat ccttgaaaaa acaatgaaat 106740aatttttaag tgatgctttc ttacgtactg tcaaagtctc agaccagctg ggatcaaact 106800gttctgtttg tacagttgga aatcagtgaa aacatgaagg ttttcattac agctttctgt 106860agcttgtagc aagttcccga ggcaatcaac ttataaagac agagaaggtg tccgtgactt 106920ggagtgttag gagtgtctgt gattgctcag tgttgtcact ttgggccagt ggtgacacat 106980ggttgtggct agtggcacag caaagctatt ggccttatga cctatagtga agtgagacaa 107040ggagcttgct tgatatgata tgttcaaaga aatatgaggt gataaatttt caaaattcca 107100cataagcaaa ctggccgaca ctggggccag agagcacgtg ctgctcttac tggagttcag 107160cttccagtac actgcagatg gttcactgca gcacctctac ccagggcatc gggtaacgtc 107220tgctgtgtgc caggtacagc tgaaaagtca actgtcccct ctgcagcttc ccgtgccacc 107280acgactctga ccctcccagc cctgcattag attgtcaagc aagcttcatc agagctcttc 107340actcttggaa cacggtgtgt tgatggaatg ccatggaagt tcacagggct aacatgctct 107400tggacttatt aaatcagtgt aaacaaaatt aatagtctta gaaaagctag gacagactca 107460ttaccacatc tgatttgtgt gtaaaggtca agtcacacgt ccaccaagtg aactctaact 107520agatggccta aattttagcc atggtccagc acttctgcca taactgtccc tagggaataa 107580cagaagcatc actcagtttt ctatatgtaa gaattttaga aaacctagca ctattgtttg 107640tttttttcct tgatatacag aatatctaga acagtctctg aatcttaaaa gtttctctaa 107700aaactgtgtg gaaaacactg ggtgacaggc tactgtagag tctaacagct gggtttcagc 107760cctactttaa gatgaaaaag aagtccagtt ctgctgataa ttagacagag ttcagtgaga 107820aatgctgaca gattctcttg gggaacagct ggctgcagtt gactgtgcca ggtcctaggg 107880atttatgcct tgcttttatg actttataat attgtaaaaa ttgtcccagc cagcgcaagg 107940tagggcatgg gtgtgtttgc agttgtcacc tttagacaca aagtgttacc ctcaccccaa 108000gctgagagtg aaagatggtg tccttcagag gacaaaaaga ttgttattgc acatttcagg 108060gttcctttct cataacctag gaaaggggcg aggggtgcca gccattttaa ctgggcatcc 108120agatgttcct gaattcttgt gagctgcgcc attggtctga aagttcttgc caaagtctgt 108180ttctgtttct ctacatcaag cttgtttgtt cttacttgtt agtgctgccc tctatcttcc 108240tggttgtgct acatgattct agagagtttt atcatatgct tcttagagga agaatggcat 108300cagactaaga gaaaacagaa aagttactgg aaggaccgca gtcatgatcc acatccttta 108360gttaatgtac tccaaattca ttcttctcta aatacctaaa agttaaatgt ggatctttaa 108420ttctttatta atttttttaa gatttattta ttattgtatg taagtacact gtagctggta 108480tcagatttca ttatggatgg ttgtgagcca ccatgtggtt gctgggattt gaactcagga 108540ccttcagaaa aacagtcagt gctcttaacc actgagccat ctctccagac cctttattaa 108600atttttaatg caatatgttt tggccgggca gtggtggcac acatgtttga acccaaaact 108660tgggaggcag aggcaggtgg atttctgagt tcgaggccag cctggtctac agagtgagtt 108720ccaggacagc cagggctaca cagagaaacc ctgtctcgaa aaaaccaaag aaatacatac 108780atacatatat atatatatat atatatatat atatatatat atatatatat atatatgtca 108840tattctttcc cctcccctcc ctttgaaagc aagaaatatg aggatccttc actataataa 108900tgtaactggg tacatacata tatggttttc ttttgttttg ctttgttttg agatatgaag 108960ttgcagttgt taacctagga cagtcctttt tcactgttca aggtttattt ggtgttttca 109020ttggcatgac atttaagtgg ttggttccat cacccatatg cagatcttac atttcacata 109080atattgcagt gcacactaca gacacatata atacataaaa tatcctgtag aatatttatg 109140caaagttata cataatggac gtacacactg gagccaggtt atgactaact gggaactcgt 109200tggcctaggc atgcttggtg agggtgcaca gggcagcaat gcagaaggct cgcagggtaa 109260tcaaagcctg cacagttggc agtgtgtctc ggagacagtg ggactgtcac actaaccagg 109320ttaccactac acaagtacca cttggcagca gggcagagct gtgggaactg agatgtttcc 109380ccacaccacc tcaacatcat tgtactaacc acagctcaga tgagaagggt gtggtctcca 109440tagaatccct gctgcagcct ggacagtgaa gtgcgaggac actgggaaga ctttagaact 109500gcagctcctt ctcgattccc cagagggtgt ctgcactctt cttcaggcgg gtctcttcct 109560caggagtcaa tgtcaccttc acaacatctg agattccatt ttgtcccaag atacttggga 109620cactgaggaa aacgtcatca ttgattccat agagaccttt aatcatggtg gaaatgggat 109680gcacccgcct aaggttcttc gttatgctct tggccaagtc tgccacagag aggccaatgg 109740tccaggatgt gtaacctctc agcttgatcg tcgtatatgc actgtccacc acctgctttt 109800gagcctcctt ccactgctcc ttaaaacagt ctgtatcagt gcccagttct gggttcagag 109860acttcaggga gacgccagca acgttcacac cactccacac agggacactg gagttgccat 109920gttctcccag gacccagctg tgacagctca gagggtgaac ccccagcctt tctcccatca 109980ggtaacggaa ccgaactgaa cccagatggc aaccacttcc aataactcga cttttgagac 110040agccactgat tttccgagcc acttaggtca aaatatccaa aggatttgag acaatcagca 110100gcgtgcagtg tggactgtac ttgacaatgt tgggaaggat gaacttgaag atgttcacgt 110160ttcgctggac cagattaagt cagctctctc cttcttgctg acggcccctg ctgtgatgac 110220cagcttggag tttgcagtca cacagttgtc tttgctggag accgtttttg gtgttttaag 110280gaagaggttg ccacgctgga gatccatcat ctctcccttt agcttgtctt ccatgacatc 110340cgccaagtcc ttcattaaga tacggatggc acaagccatg ccaacagcac caaccccaac 110400aactctaatc ttgttctggg ggacctgttc ttccttaaga tccacagtca gctggtcttt 110460gagggttgcc atcttggact ttgaatcttt tgagaccgct agagcacgat ggggggtgat 110520gggcaagcag cagcgtggcc tgggctcagc gacagtggct ccagcaccag cctaggacag 110580tcttgaactt gtaattctcc ttcatcatcg gcttccggat atcggaatta caagtgcgct 110640ttcacaccca gacctgcatg gttttaagaa tgagagcttt tgcagctgtc tgtgacttga 110700tgtctaagac ttttttttat tattactctt tacataattc tgagtgtgtg tgtgtgtgtg 110760tgtgtgtgtg tgtgtgtgtg tgtgtgtgca tcttggttag ggtttctatt gctctgaaga 110820gacaccatga ccacggcaac tcatataaag gaaaacattc cattggggct ggtttatagt 110880tcagaggttc aatccattat caccttggtg ggaagcatgg cagcatccag gaaggcctgg 110940tgcctgagaa ggagctgagc attctgcttc tgagagggca ggcaacagaa gtgaatgtct 111000gagccggagg cagacttgag cttctgagac ctgaaagaga cctcaagtga cacacccatt 111060ccaacaaggc ctcacctcct aatggcgcca ctctcaatgt gccaatgggg ggaccatttt 111120ctttcagagc ctcgcagttt gtttctgcga agggttactt acctgtgcaa acatgagtaa 111180agttatttac ttgagtaagg gacagttacc agtggctaca gcactcaggc tgagtattag 111240tttaactaaa tttaacttca gagcccagga gttcaagacc atttttgaac aaggtagtga 111300ccctccccat cccccagggg gaacaagaga gggtgggaga agagggcatc tcctgagtgt 111360ctgcccactg ttcgctgttc tgttttcaga atctctttgc cagcaacagc cttgatgggg 111420gaagcagccc gatgaataaa cccaaaggag acaaacaagt cgaatacctg gatttagacc 111480tagattctgg gaagtccacg ccaccacgga aggtaagtga gcccgtgttc tctagatagg 111540tcaagcacac tttgagggac catgttcttt ggggaaagtt gggggcctga ttatttaaat 111600ttagtgactg ttgtttccta agtcacaagc aacagaatca tgaagtctga tagatttgtt 111660tctccctttg tagaggtcgg tcagttgtgc agtttctact cgtcatggtc agtgcttttg 111720ctgtctttag aatgaagaaa atgtggcttg gatgagtggg gtagccacat ctttaatccc 111780agcacaggga agacagaatt tatctgtaaa aaggaagaaa aggagaacat ggctcatggc 111840tcaagtcata tgctgttttg tcttaaattt taatttctat taaacctacc aattacacag 111900tttctttata attctttggt tttggtgggt tttttaaata tttaacatat atgtgggtat 111960atgcacatgg gtgcagtgtc cctcagagac tggaagaggg cattagctcc caggagctag 112020agtgacaacg ggttcagttc ctccgaaaga gcagtgtaca gcctccccca cccccagccc 112080tccttccagc ccgtgcatca ctggacttac tttgttattt aagtcctatt ttcttacatt 112140gtttttggct tatcattctc tcttagactc agattttcaa cagtgtcctg ttgaagtccc 112200tgtaatctga aggtcttttg gtagtgaatt cttctcaggt tgtgtataac tgaagtctct 112260ttattttaga aacaagtttt gttgagatca cagttcttgt tttttttcct gtgacataat 112320tacataattt tcaataattt gaattctgaa aataaggttg aaaaaaattg aaagatttgc 112380ttacagaaga tgccattatt tgcacagaga aaagctcacg gcagtcatgt taatgtgtgt 112440tggaaaaggc cgctgtcata ctgtcagggt aactgagcta ctgagtcagt tcttaactga 112500gcctctttac tgcatggggg ccctctgttg ctgtccagtc tcttgttctg tcaaatgtgc 112560cctttggaca cttcctatct gtttttctgg ctgcttttca gatgtttcct gctgaattta 112620cagttttatg acgtagatgt cttttcacta atctgcttgg aagttgtgct attcacacac 112680gtgtacgcat atacacatac tcatacgcac caacacatac acatacttac acatataaac 112740atatatacac acatacacac agacacacca cacagataca tacacacaca tacacacata 112800cttatacaca catgcacaca tatgcacaca tatgcataca catgtacgtg tacacgccaa 112860cacacatgcc atggctgtac acatgacttt tctgcttctt ttgattacct ttttgtcacc 112920tgccaggaac acatacagta tacctactat ttctatatct ttgtagtttt ctgttctatg 112980atctatgtat ctctgttcta tgtatctgtt taattttgga tagtttttag ctctatcacc 113040agatttccta attctccttt ctttttgttg aggtgttttg gtttggttgt tttgttttgt 113100cttttgttgt tgttgttatt ttgtatttat ctctggggtg aaatgctgtt tgatttactt 113160taactgacat taatctcttt gtgttacttg tatgtttaag gccttttctg gcttatcttt 113220taacactgtt cacacacttc ataagtcctt ggaggccgga atttgcatgt actgtttctg 113280ttagcccaca ctctttatgg ctgtctcctt gtgtgtttat acatttttat tacaaattca 113340tggttgaact tggaggacct gaacttcctc cacagagcat ctctaattcc ccctggaggc 113400ccactgagat tcctgctctg ggctcacgtc agcttccttc aagcttgtca ggagccttgt 113460tatcagcatc ccaccttccc tgcctccttc cccacccacc gagctgagtc cctccccatc 113520ctccctgaag atcttagaat gtccttgcta tctaggagcc ctacagagca ttgcccaccc 113580agctggctca gctactgcat atctagctgc accttaagaa aagagggcct ggttgtggtg 113640gcacaggcca gtacaatttg ctgaaggagg cagaggcagg aggatcacaa gttcaaggcc 113700agtctgggct acacattttg ggctagaaag atggctcagt gtttaagagc acagactgct 113760cttccagaga tccagagttc aattcccaga aaccacatgg tggctcacaa ccatctttaa 113820tgggatccaa tgccctcttc tggtgtgtat gaagatgagc tacagtgtat taatatacat 113880aaaataaata aatcttaaaa aaatagagaa gaggaggcag ctttcagtta ctgtgtatct 113940agttgcacgt taagacaaga ggggacatct ttgtagttta ctttcctgca aatataactt 114000tgccttcaga cacaccagaa aaggacatca tatcccatta tagatggttg tgagccagta 114060tggttgctgg gaattgaact taggacatca ggaagagtaa ccagtgctct taaactctga 114120gctatctctc cagcccccaa aagtactttt cttgcacaaa gagtaaagag ctgtttggtg 114180ctgatccggg cgagaggaaa aaacactcaa acataagcta aagagagtct agttctgtgt 114240gttagatgag ctagattttc tatgaatgcc atctgtgata gcagtattat tagtttgaat 114300agttctatat aattttaaaa ttaagagaaa tcggtgtcaa gttagagagt ggacagacaa 114360taccattaat ctgaggaaat gccagtttgt gtgtgtaatt ccaagattac atatgtctat 114420ctatctacgt ttgtttgttt gtttgtttgt ttgctttgag acaagatttc attatgcaac 114480ccttgctgat tcggaactct ccatagagca ggctggcctt gaactctaat atagagatcc 114540accagcctac ctatgcctcc caagtgctgg catgatacca ccataccctg catatgtatc 114600aatatcaaat cttgggtttc actcagaacc accctcacaa atggcccctt gggggtattt 114660ccttgtgttt tttagcaaaa gagcagtggt tctggcagca gcatggcaga cgagagggtg 114720gattacgttg tggtggacca acagaagact ctggccctga agagtaccag agaagcttgg 114780acggatggga ggcagtccac agagtccgag acacccacca agaatgtgaa gtgaagacat 114840gccgtcgcct ctgccggcag acgagatctg agtggaaaga gagatgccaa gtgaagatgt 114900tcccactctc agtggagcct cgagccagca ggggcagaga gaaggatctc tcacacatgt 114960tcaagcaaat taggttgtga atggtgctgt gtggtattgg atttataacg tgtaaataac 115020ccggggaaat agtgttttta gttcacagag aagcttctgt ccctaattaa cacacctgta 115080gtattactat actgatgcac ttttcattta aaaccttggt ttgggtcttc ccgatctacc 115140ttaacagact ttccttggga ggtcttttgg cctcctcaca ctactctata taacaatact 115200aagtgaactg agctacttgt aattctggaa attccagttg aagctacagg gctaacacca 115260ttaaaacaag aagtaagttg acacattcgc ttttctcttg aaggtggtag ccattagctt 115320aagctgtaga acatagttgg acttgtcctt cgttgttttc caaaaattcc ggggatattg 115380tatatagcag gtcaagacct agctctctga ctcatgtaca cttaggtttt aactgtagga 115440ctttgttatt attatttttt ttgttaatga cagtgttggg ttcatcgtgt gaaggttctg 115500ctgggtagga tcttgcacct ttcaaagact gcctcttagt tacactagta agcccccaaa 115560tcatccacag catggactgc tggcctgctc ttactcctgt ttatgtgtta aacattatct 115620gcaaaaggca gattatacga ctgaccaatc aggtacgtac aaggcactga tgtgctaata 115680cagtgattgg gtcagacaaa gtgcttcagt tagtgtgcgt tcgtcctaat cttggtttag 115740aattaatgaa acagttggcg ttcactgtca gcagcatagt gtgattttga atgaattagg 115800caggaattca agattactac tcttagctcg ctcgctctct ctctctctcc accgtagtgc 115860tcttcctagg gttttctttc ttctacttaa tatcttcttg gccttatatt taaatcccta 115920tgcaattaat gttttatatc tgcgttttta aaaaagaaat gtcattttaa gtgattcttg 115980tatgtagcaa gcacctattg cttttgtgag taaatgaatt aagacttttg tactgtgatt 116040tgtactcact gccccagttc cccaactgtt ggagccttgc tgctgtgaaa cgctgtagtc 116100accatagttg tgtccaccac ccagccgggt ctgtgagtct cacctgtcac gtgacatcgt 116160ctggtgtgga tgttggctct gaattagtgt cactgcagtt acacgtgtct gtctaggctt 116220tgcaagatgc tgtagtcacc atagtcgtga ccaccaccta gccaggtctg tgagtctcac 116280ctgtcacgtg ccatcatctg gtgtcactgc agttacacgt gtctgtctag gctttgccgc 116340aaccttgaga agcagctagc attttccgtt gttcacacag taaggacaat gtctctgcat 116400tgatctgagg ctcactggtg gcctggggaa gggacacaga gaacagaatg tctgcagctg 116460aggcttgtct ctcttcgttc agacctctta cctgttgcct gagtacacaa tgcaccccgc 116520tttctggccc actggccacg gtgctgtgcc taatctgagt tctcccctgg cttttccagt 116580cagtttgaaa gctgtgttca taactagatg aagtgtagaa tagtaatatt agatgctttt 116640aaatgttcgc ttctttttaa acaaaaacta aaacccagaa ctgaattttg aggtggattt 116700ttaaataaaa aaagattgag tttgctgtgt gagctgtgtt acttttttct tacttttaat 116760atactttcta tttttttgag caagcaacaa gtgtcaagaa aacacatact ggggttggag 116820agatgattca gtgattaaaa gcactggctg ctcgtaagaa aagactggat ttcagttcga 116880gcttgacata gcctctcaca ggcaatcata attctggttc cgggagtctg gctcctctcc 116940tggcctctgg acactcactg cagacgcgtg cacagacatg catgcaggtg aaacacccac 117000acacatacta aaaatgttgt tatgtatgta ttctcttggg aacatgacac gtagagagcc 117060agagagaggg cccagtgctt acagcacgtg ttgcttctgc tgagaccagg gattcatggt 117120agatctcagc catccatttc tccaggtcta gggacccaga tgccttcttc tgacttcctt 117180tggcactagg catgcatggt gatgcaaaag aagttaggag acacagggcc catcctattt 117240ctgggtatct gaatataatt atacatatta ctggtagccc agagacaatc acagccttga 117300atgttctgta ataaaactac caaagaaag 11732924859DNAMus musculus 2ctgggctgaa gctagaggcg accgatcgtg ggaaggggag gcagaaaggc taggaggagg 60agggccgggg cccaggccgc gcctccccga acccgccgcg cgcccccggg gtgggggagg 120agcgcagggg acaggacaga ggggtccctc cccgaagcag agccgccctg cgcctcgtcc 180ctgtgctgat ccccgccctc atccgaggct ggagcgcaga cccaccctcc caccgcggac 240ccgcgacctc cccgacgccc ggcgacgccc tgaccctcgc tgctggtccg cgaactccct 300aggctgatca ggacctgccc ctgtgccggc tgccacccgg acgccgcacg ccttcccagg 360cgcccttttc cgagcagagg gaaagagaag atcgagcccc tctcagtgtg aatgcgccaa 420cgggcggagc ggagcggagc ggacaccgcg cgcgggcatt gtgtgtgcgc gtgcagcgtg 480gggtccgcag cggggagcac ccgcgggagg tcccgtttcc aaggggcgga gcgcagggct 540tccagttctg ggctccctgt ccggacagag tcccagcgga gcccgaccgc tgcctaggcg 600gcgggacggc gcgcctggcg gccaggaggg cgcactgaaa gaaggtcggc gagccctggt 660ccccgcggtt cccgatcgag ttcctcttca gtccgcgaat ctgcgggaga ggttcgatcg 720ccgacacagg gcgcggggag ccgggccgcc ccgtcggggg aatctgagac gtcctctggg 780ctgcgtttga ctgccgtgcc cgccgtgcac ggagcgcgtc cactgtgtcc accgacccct 840ttggtgtctg gtcctcgagt cctcacggcg tgcaccatga gcggcggcga agtggtttgc 900tcgggatggc tccgcaagtc gcccccggag aagaagttga agcgttatgc gtggaagaga 960aggtggtttg tgttgcgcag tggccgtttg actggagacc cggatgtcct ggagtattac 1020aaaaacgatc atgccaagaa gcctattcgg attattgatt taaatttatg tcagcaagtt 1080gatgctgggt tgacattcaa caaaaaggag tttgaaaaca gctatatctt tgatatcaac 1140accatcgacc ggattttcta cttggtggca gatagtgagg aagacatgaa caagtgggtc 1200cgttgtatct gtgacatctg tggattcaat cccacagaag aagatcctgt gaagccgctg 1260actggctcct cacaagcacc cgtcgattca cctttcgcta taagtacagc accagcctcc 1320agtcagatgg aagcttcttc agtcgcgcta cctcctcctt accaggtcat cagccttccg 1380ccacacccag acaccctcgg cctccaggac gatccacaag actacctctt gctgatcaac 1440tgtcaaagca agaagcctga acctaacaga accctctttg actctgccaa gcccaccttt 1500tctgagacag actgcaatga caacgtccct tcccaccaga ctcctgcttc ctcccagagc 1560aaacacggaa tgaatggctt tttccagcaa caaatgatgt atgactgccc accgtcccgg 1620ctgacatctg tctcgggaga gtccagcctc tataacctgc ccaggagcta ttcccatgac 1680gtgttgccaa aggaatcccc atcaagcacg gaggccgacg gggagctgta cacctttaac 1740accccatctg ggactgcagg tgtagaaacg cagatgagac atgtatccat cagttacgac 1800attccgccaa cacctggcaa cacttaccag atcccacgga catttccaga aagcacactg 1860ggacagtcat caaagctgga caccattcct gatatccccc cacctcggcc accaaagcca 1920catccaactc atgaccggtc tcctgtggaa acgtgtggag tcccacgcac ggcctcggac 1980actgacagca gttactgtat ccctcctcca gcaggcatga cgccctcccg gagtaatacc 2040atttccaccg tggatttgaa caagttgcgg aaagatgcta gttctcaaga ttgctatgat 2100attccacgga cctttccgag cgatagatct agttccctgg aaggcttcca tagccagtat 2160aaaatcaaaa gcgtgttgac agcgggaggt gtctcgggtg aagagctgga tgagaactac 2220gttcccatga accccaactc gccacctcga caacattccg gcagctttac cgagccaatc 2280caggagccaa actatgtgcc aatgacccca gggacctttg acttttcttc ctttggaatg 2340caagtccctc ctcctgctca tatgggcttc aggtccagcc caaagacccc tcccaggagg 2400ccagttcctg ttgctgactg tgaaccaccc ccggtggata ggaacctcaa gccagacaga 2460aaagtcaagc cggcaccttt agacataaaa cctctgtcag aatgggaaga gctgcaagcc 2520ccagtcagat ctcccatcac caggagcttc gctcgggact cctctaggtt tcccatgtcc 2580cctcggcctg attctgtgca cagtacgaca tcgagcagcg actctcatga cagtgaagag 2640aactatgtcc ccatgaatcc aaatctgtct ggcgaagacc cgaatctctt tgccagcaac 2700agccttgatg ggggaagcag cccgatgaat aaacccaaag gagacaaaca agtcgaatac 2760ctggatttag acctagattc tgggaagtcc acgccaccac ggaagcaaaa gagcagtggt 2820tctggcagca gcatggcaga cgagagggtg gattacgttg tggtggacca acagaagact 2880ctggccctga agagtaccag agaagcttgg acggatggga ggcagtccac agagtccgag 2940acacccacca agaatgtgaa gtgaagacat gccgtcgcct ctgccggcag acgagatctg 3000agtggaaaga gagatgccaa gtgaagatgt tcccactctc

agtggagcct cgagccagca 3060ggggcagaga gaaggatctc tcacacatgt tcaagcaaat taggttgtga atggtgctgt 3120gtggtattgg atttataacg tgtaaataac ccggggaaat agtgttttta gttcacagag 3180aagcttctgt ccctaattaa cacacctgta gtattactat actgatgcac ttttcattta 3240aaaccttggt ttgggtcttc ccgatctacc ttaacagact ttccttggga ggtcttttgg 3300cctcctcaca ctactctata taacaatact aagtgaactg agctacttgt aattctggaa 3360attccagttg aagctacagg gctaacacca ttaaaacaag aagtaagttg acacattcgc 3420ttttctcttg aaggtggtag ccattagctt aagctgtaga acatagttgg acttgtcctt 3480cgttgttttc caaaaattcc ggggatattg tatatagcag gtcaagacct agctctctga 3540ctcatgtaca cttaggtttt aactgtagga ctttgttatt attatttttt ttgttaatga 3600cagtgttggg ttcatcgtgt gaaggttctg ctgggtagga tcttgcacct ttcaaagact 3660gcctcttagt tacactagta agcccccaaa tcatccacag catggactgc tggcctgctc 3720ttactcctgt ttatgtgtta aacattatct gcaaaaggca gattatacga ctgaccaatc 3780aggtacgtac aaggcactga tgtgctaata cagtgattgg gtcagacaaa gtgcttcagt 3840tagtgtgcgt tcgtcctaat cttggtttag aattaatgaa acagttggcg ttcactgtca 3900gcagcatagt gtgattttga atgaattagg caggaattca agattactac tcttagctcg 3960ctcgctctct ctctctctcc accgtagtgc tcttcctagg gttttctttc ttctacttaa 4020tatcttcttg gccttatatt taaatcccta tgcaattaat gttttatatc tgcgttttta 4080aaaaagaaat gtcattttaa gtgattcttg tatgtagcaa gcacctattg cttttgtgag 4140taaatgaatt aagacttttg tactgtgatt tgtactcact gccccagttc cccaactgtt 4200ggagccttgc tgctgtgaaa cgctgtagtc accatagttg tgtccaccac ccagccgggt 4260ctgtgagtct cacctgtcac gtgacatcgt ctggtgtgga tgttggctct gaattagtgt 4320cactgcagtt acacgtgtct gtctaggctt tgcaagatgc tgtagtcacc atagtcgtga 4380ccaccaccta gccaggtctg tgagtctcac ctgtcacgtg ccatcatctg gtgtcactgc 4440agttacacgt gtctgtctag gctttgccgc aaccttgaga agcagctagc attttccgtt 4500gttcacacag taaggacaat gtctctgcat tgatctgagg ctcactggtg gcctggggaa 4560gggacacaga gaacagaatg tctgcagctg aggcttgtct ctcttcgttc agacctctta 4620cctgttgcct gagtacacaa tgcaccccgc tttctggccc actggccacg gtgctgtgcc 4680taatctgagt tctcccctgg cttttccagt cagtttgaaa gctgtgttca taactagatg 4740aagtgtagaa tagtaatatt agatgctttt aaatgttcgc ttctttttaa acaaaaacta 4800aaacccagaa ctgaattttg aggtggattt ttaaataaaa aaagattgag tttgctgtg 48593226DNAMus musculus 3atcctgtgaa gccgctgact ggctcctcac aagcacccgt cgattcacct ttcgctataa 60gtacagcacc agcctccagt cagatggaag cttcttcagt cgcgctacct cctccttacc 120aggtcatcag ccttccgcca cacccagaca ccctcggcct ccaggacgat ccacaagact 180acctcttgct gatcaactgt caaagcaaga agcctgaacc taacag 226423DNAMus musculus 4acaagtgtga gtgtgcgcac atg 23523DNAMus musculus 5cagattggcc ttgaactggt aag 23626DNAMus musculus 6gcctgcatta ccggtcgatg caacga 26726DNAMus musculus 7gtggcagatg gcgcggcaac accatt 26823DNAMus musculus 8ggtgaatcga cgggtgcttg tga 23923DNAMus musculus 9cagattggcc ttgaactggt aag 23

Claims

1. A method of identifying a modulator of Gab1 comprising:providing a first group comprising cells that express Gab1 protein and a second group comprising cells modified to lack Gab1 protein expression;contacting said first group and second group with a candidate agent;comparing the extracellular glucose concentration of said first and second groups after contact with said candidate agent to a baseline value indicated by the difference in extracellular glucose concentration between the first and second groups without contact with said candidate agent; andidentifying the candidate agent as a modulator of Gab1 if a change in glucose concentration between the first and second groups is found after contact with the candidate agent compared to the baseline value.

2. The method of claim 1, wherein the cells of the first and second groups comprise mammalian cells.

3. The method of claim 2, wherein the cells of the first and second groups comprise murine cells.

4. The method of claim 3, wherein the cells modified to lack Gab1 protein expression are from a transgenic mouse whose genome comprises a homozygous disruption of the endogenous Gab1 gene.

5. The method of claim 1, wherein the cells of said first and second groups are from the same organ type.

6. The method of claim 4, wherein the organ type is liver.

7. The method of claim 1, wherein said providing and said contacting are performed in vitro.

8. The method of claim 1, wherein said providing and said contacting are performed in vivo.

9. The method of claim 8, wherein said first and second groups are mice.

10. The method of claim 9, wherein said second group is a transgenic mouse whose genome comprises a homozygous disruption of the endogenous Gab1 gene.

11. The method of claim 10, further comprising administering a known amount of glucose to the mice.

12. The method of claim 11, wherein the extracellular glucose concentration is measured as plasma glucose levels.

13. The method of claim 1, wherein the candidate agent is identified as a positive modulator of Gab1 if said change in glucose concentration is an increase.

14. The method of claim 1, wherein the candidate agent is identified as a negative modulator of Gab1 if said change in glucose concentration is a decrease.

15. A transgenic mouse whose genome comprises a homozygous disruption of the endogenous Gab1 gene, wherein said disruption results in the functional inactivation of the Gab1 gene, and wherein said mouse exhibits hypoglycemia relative to a mouse whose genome comprises the functional endogenous Gab1 gene.

16. The transgenic mouse of claim 15, wherein the homozygous disruption is specifically in the hepatic cells of the mouse.

17. The transgenic mouse of claim 15, wherein the disruption in the Gab1 gene is a deletion of SEQ ID NO: 3.

18. The transgenic mouse of claim 17, wherein the Gab1 gene is replaced with a replacement gene.

19. The transgenic mouse of claim 18, wherein the replacement gene comprises a neomycin resistance gene.

20. The transgenic mouse of claim 17, wherein the replacement gene further comprises a thymidine kinase gene.

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