Isolated human phosphodiesterase proteins, nucleic acid molecules encoding human phosphodiesterase proteins, and uses thereof

Abstract

vides amino acid sequences of peptides that are encoded by genes within the human genome, the phosphodiesterase peptides of the present invention. The present invention specifically provides isolated peptide and nucleic acid molecules, methods of identifying orthologs and paralogs of the phosphodiesterase peptides, and methods of identifying modulators of the phosphodiesterase peptides.

Description

FIELD OF THE INVENTION

[0001]The present invention is in the field of phosphodiesterase proteins that are related to cGMP-stimulated 3',5'-cyclic nucleotide phosphodiesterase 2A (PDE2A), recombinant DNA molecules, and protein production. The present invention specifically provides novel peptides and proteins that effect protein phosphorylation and nucleic acid molecules encoding such peptide and protein molecules, all of which are useful in the development of human therapeutics and diagnostic compositions and methods. In particular, the phosphodiesterase protein provided by the present invention is a novel alternative splice form of PDE2A.

BACKGROUND OF THE INVENTION

Phosphodiesterases

[0002]In general, phosphodiesterases ("PDEs") catalyze the hydrolysis of a phosphodiester bond. Specific classes of phosphodiesterases include those catalyzing the degradation of cyclilc monophosphates.

[0003]The signaling pathways regulated by PDEs include the transduction of photon capture in the outer segment of a photoreceptor as well as changes in neurotransmitter release from its inner segment. PDEs also regulate the aldosterone production by atrial natriuretic peptide and platelet aggregation by endothelial relaxation factor.

[0004]Experimental data have demonstrated the role of phosphodiesterases in a range of diseases, including inflammatory diseases such as asthma, chronic obstructive pulmonary disease, rheumatoid arthritis and atopy. Drugs that selectively inhibit individual PDE isozymes have a wide variety of different effects on an animals, suggesting specific roles for most of the different PDEs.

[0005]Experimental evidence indicates the existence of several related gene families coding for different phosphodiesterases, and that each of these families contain more than one gene. Furthermore, each gene product is differentially spliced in different tissues to yield different isozymes. Isolation of cDNAs for many of the isozymes has allowed a series of structure/function studies to be initiated. Several of these isozymes are regulated by phosphorylation/dephosphorylation mechanisms.

[0006]Over 30 phosphodiesterases have been identified. Categories of phosphodiesterases include seven major classes. Class I phosphodiesterases include calmodulin-dependent phosphodiesterases which are expressed in tissues such as the brain, testes, sperm, coronary artery, lung, heart, and pancreas. Class II phosphodiesterases include cGMP-stimulated phosphodiesterases which are expressed in tissues such as the brain, adrenal gland, and the heart. Class III phosphodiesterases include cGMP-inhibited phosphodiesterases expressed in tissues such as T-lymphocytes, macrophages, platelets, smooth muscle, heart, and adipose tissue. Class IV phosphodiesterases include cAMP-specific phosphodiesterases which are expressed in tissues such as monocytes, leukocytes, and the central nervous system. Class V phosphodiesterases include cGMP-specific phosphodiesterases which are expressed in tissues such as lung, smooth muscle, platelets, and the aorta. Class VI phosphodiesterases include photoreceptor-specific phosphodiesterases expressed in the retina. Class VII phosphodiesterases include high affinity cAMP-specific phosphodiesterases.

[0007]Cyclic Nucleotide Phosphodiesterases

[0008]As is well-known in the art, a myriad of physiological processes are controlling by causing changes in the steady state levels of the second messengers cAMP and cGMP. One of the major mechanisms by which these levels are controlled is via the cyclic nucleotide PDEs that control their degradation by catalyzing the hydrolysis of a phosphodiester bond, yielding 5'-AMP and 5'-GMP, respectively.

[0009]Experimental data have demonstrated the role of cyclic nucleotide phosphodiesterases in a range of diseases, including inflammatory diseases such as asthma, chronic obstructive pulmonary disease, rheumatoid arthritis and atopy.

[0010]In mammals, four genes are known to code for cAMP-specific PDEs. These genes are known as PDE4A, PDE4B, PDE4C and PDE4D. This was first demonstrated in rats and later in humans and in mice. The four human and four rat genes show a one to one correspondence, in that each of the four human PDE4 genes is more closely related to its homologous rat gene than to any other human gene. The PDE4 genes are located on three different human chromosomes: PDE4B on chromosome 1, PDE4D on chromosome 5; PDE4A on p13.2 of chromosome 19 and PDE4C on p13.1 of chromosome 19. Their four murine homologues are each located in correspondingly conserved regions of the mouse genome. The mammalian PDE4 genes thus comprise a well-conserved multigene family.

[0011]The existence of a large number of mRNA transcripts from many of the mammalian PDE4 genes suggests that the genomic structure of these genes is likely to be complex. Partial genomic sequences have been published for the rat PDE4B and PDE4D genes. However, the published data indicate that sequences at the 5' end of the genes, which would include a number of upstream exons and promoter sites, were not included.

[0012]cGMP-stimulated 3',5'-cyclic nucleotide phosphodiesterase 2A (PDE2A)

[0013]The novel human protein, and encoding gene, provided by the present invention is an alternative splice form of cGMP-stimulated 3',5'-cyclic nucleotide phosphodiesterase 2A (PDE2A). Specifically, the phosphodiesterase provided by the present invention differs from known phosphodiesterases, particularly bovine PDE2A1 (gi116569) and human PDE2A3 (gi4505657), in exon 1. These difference are illustrated in the Figures, particularly in the amino acid sequence alignments shown in FIGS. 2 and 3.

[0014]For a further review of PDE2A and related proteins, see Rosman et al., Gene 1997 May 20; 191(1):89-95; Sonnenburg et al., J Biol Chem 1991 Sep. 15; 266(26):17655-61; Trong et al., Biochemistry 1990 Nov. 6; 29(44):10280-8; and Charbonneau et al., Proc Natl Acad Sci USA 1986 December; 83(24):9308-12.

[0015]Phosphodiesterase proteins, particularly alternative splice forms of PDE2A, are a major target for drug action and development. Accordingly, it is valuable to the field of pharmaceutical development to identify and characterize previously unknown splice forms of phosphodiesterase proteins. The present invention advances the state of the art by providing a previously unidentified human PDE2A alternative splice form.

SUMMARY OF THE INVENTION

[0016]The present invention is based in part on the identification of amino acid sequences of human phosphodiesterase peptides and proteins that are related to PDE2A, as well as allelic variants and other mammalian orthologs thereof. Specifically, the phosphodiesterase protein provided by the present invention is a novel alternative splice form of PDE2A. These unique peptide sequences, and nucleic acid sequences that encode these peptides, can be used as models for the development of human therapeutic targets, aid in the identification of therapeutic proteins, and serve as targets for the development of human therapeutic agents that modulate phosphodiesterase activity in cells and tissues that express the phosphodiesterase. Experimental data as provided in FIG. 1 indicates expression in humans in the amygdala, brain (including infant brain), uterus, testis, placenta choriocarcinomas, Hela cells, and a pooled melanocyte/fetal heart/pregnant uterus sample.

DESCRIPTION OF THE FIGURE SHEETS

[0017]FIG. 1 provides the nucleotide sequence of a cDNA molecule that encodes the phosphodiesterase protein of the present invention. (SEQ ID NO: 1) In addition, structure and functional information is provided, such as ATG start, stop and tissue distribution, where available, that allows one to readily determine specific uses of inventions based on this molecular sequence. Experimental data as provided in FIG. 1 indicates expression in humans in the amygdala, brain (including infant brain), uterus, testis, placenta choriocarcinomas, Hela cells, and a pooled melanocyte/fetal heart/pregnant uterus sample.

[0018]FIG. 2 provides the predicted amino acid sequence of the phosphodiesterase of the present invention. (SEQ ID NO:2) In addition structure and functional information such as protein family, function, and modification sites is provided where available, allowing one to readily determine specific uses of inventions based on this molecular sequence.

[0019]FIG. 3 provides genomic sequences that span the gene encoding the phosphodiesterase protein of the present invention (SEQ ID NO:3), allowing one to readily determine specific uses of inventions based on this molecular sequence. As illustrated in FIG. 3, SNPs were identified at 231 different nucleotide positions. FIG. 3 also provides a multiple alignment of isoform 1 (11000567083206_pep), human PDE2A3 (gi4505657_pep) and bovine PDE2A1 (gi116569_pep) amino acid sequences, illustrating the differences in exon 1 between the phosphodiesterase of the present invention and known phosphodiesterases.

DETAILED DESCRIPTION OF THE INVENTION

General Description

[0020]The present invention is based on the sequencing of the human genome. During the sequencing and assembly of the human genome, analysis of the sequence information revealed previously unidentified fragments of the human genome that encode peptides that share structural and/or sequence homology to protein/peptide/domains identified and characterized within the art as being a phosphodiesterase protein or part of a phosphodiesterase protein and are related to PDE2A. Specifically, the phosphodiesterase protein provided by the present invention is a novel alternative splice form of PDE2A. Utilizing these sequences, additional genomic sequences were assembled and transcript and/or cDNA sequences were isolated and characterized. Based on this analysis, the present invention provides amino acid sequences of human phosphodiesterase peptides and proteins that are related to the PDE2A subfamily, nucleic acid sequences in the form of transcript sequences, cDNA sequences and/or genomic sequences that encode these phosphodiesterase peptides and proteins, nucleic acid variation (allelic information), tissue distribution of expression, and information about the closest art known protein/peptide/domain that has structural or sequence homology to the phosphodiesterase of the present invention.

[0021]In addition to being previously unknown, the peptides that are provided in the present invention are selected based on their ability to be used for the development of commercially important products and services. Specifically, the present peptides are selected based on homology and/or structural relatedness to known phosphodiesterase proteins of the PDE2A subfamily and the expression pattern observed. Experimental data as provided in FIG. 1 indicates expression in humans in the amygdala, brain (including infant brain), uterus, testis, placenta choriocarcinomas, Hela cells, and a pooled melanocyte/fetal heart/pregnant uterus sample. The art has clearly established the commercial importance of members of this family of proteins and proteins that have expression patterns similar to that of the present gene. Some of the more specific features of the peptides of the present invention, and the uses thereof, are described herein, particularly in the Background of the Invention and in the annotation provided in the Figures, and/or are known within the art for each of the known PDE2A family or subfamily of phosphodiesterase proteins.

SPECIFIC EMBODIMENTS

Peptide Molecules

[0022]The present invention provides nucleic acid sequences that encode protein molecules that have been identified as being members of the phosphodiesterase family of proteins and are related to PDE2A (protein sequences are provided in FIG. 2, transcript/cDNA sequences are provided in FIG. 1 and genomic sequences are provided in FIG. 3). Specifically, the phosphodiesterase protein provided by the present invention is a novel alternative splice form of PDE2A. The peptide sequences provided in FIG. 2, as well as the obvious variants described herein, particularly allelic variants as identified herein and using the information in FIG. 3, will be referred herein as the phosphodiesterase peptides of the present invention, phosphodiesterase peptides, or peptides/proteins of the present invention.

[0023]The present invention provides isolated peptide and protein molecules that consist of, consist essentially of, or comprise the amino acid sequences of the phosphodiesterase peptides disclosed in the FIG. 2, (encoded by the nucleic acid molecule shown in FIG. 1, transcript/cDNA or FIG. 3, genomic sequence), as well as all obvious variants of these peptides that are within the art to make and use. Some of these variants are described in detail below.

[0024]As used herein, a peptide is said to be "isolated" or "purified" when it is substantially free of cellular material or free of chemical precursors or other chemicals. The peptides of the present invention can be purified to homogeneity or other degrees of purity. The level of purification will be based on the intended use. The critical feature is that the preparation allows for the desired function of the peptide, even if in the presence of considerable amounts of other components (the features of an isolated nucleic acid molecule is discussed below).

[0025]In some uses, "substantially free of cellular material" includes preparations of the peptide having less than about 30% (by dry weight) other proteins (i.e., contaminating protein), less than about 20% other proteins, less than about 10% other proteins, or less than about 5% other proteins. When the peptide is recombinantly produced, it can also be substantially free of culture medium, i.e., culture medium represents less than about 20% of the volume of the protein preparation.

[0026]The language "substantially free of chemical precursors or other chemicals" includes preparations of the peptide in which it is separated from chemical precursors or other chemicals that are involved in its synthesis. In one embodiment, the language "substantially free of chemical precursors or other chemicals" includes preparations of the phosphodiesterase peptide having less than about 30% (by dry weight) chemical precursors or other chemicals, less than about 20% chemical precursors or other chemicals, less than about 10% chemical precursors or other chemicals, or less than about 5% chemical precursors or other chemicals.

[0027]The isolated phosphodiesterase peptide can be purified from cells that naturally express it, purified from cells that have been altered to express it (recombinant), or synthesized using known protein synthesis methods. Experimental data as provided in FIG. 1 indicates expression in humans in the amygdala, brain (including infant brain), uterus, testis, placenta choriocarcinomas, Hela cells, and a pooled melanocyte/fetal heart/pregnant uterus sample. For example, a nucleic acid molecule encoding the phosphodiesterase peptide is cloned into an expression vector, the expression vector introduced into a host cell and the protein expressed in the host cell. The protein can then be isolated from the cells by an appropriate purification scheme using standard protein purification techniques. Many of these techniques are described in detail below.

[0028]Accordingly, the present invention provides proteins that consist of the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example, proteins encoded by the transcript/cDNA nucleic acid sequences shown in FIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG. 3 (SEQ ID NO:3). The amino acid sequence of such a protein is provided in FIG. 2. A protein consists of an amino acid sequence when the amino acid sequence is the final amino acid sequence of the protein.

[0029]The present invention further provides proteins that consist essentially of the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example, proteins encoded by the transcript/cDNA nucleic acid sequences shown in FIG. 1 (SEQ ID NO: 1) and the genomic sequences provided in FIG. 3 (SEQ ID NO:3). A protein consists essentially of an amino acid sequence when such an amino acid sequence is present with only a few additional amino acid residues, for example from about 1 to about 100 or so additional residues, typically from 1 to about 20 additional residues in the final protein.

[0030]The present invention further provides proteins that comprise the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example, proteins encoded by the transcript/cDNA nucleic acid sequences shown in FIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG. 3 (SEQ ID NO:3). A protein comprises an amino acid sequence when the amino acid sequence is at least part of the final amino acid sequence of the protein. In such a fashion, the protein can be only the peptide or have additional amino acid molecules, such as amino acid residues (contiguous encoded sequence) that are naturally associated with it or heterologous amino acid residues/peptide sequences. Such a protein can have a few additional amino acid residues or can comprise several hundred or more additional amino acids. The preferred classes of proteins that are comprised of the phosphodiesterase peptides of the present invention are the naturally occurring mature proteins. A brief description of how various types of these proteins can be made/isolated is provided below.

[0031]The phosphodiesterase peptides of the present invention can be attached to heterologous sequences to form chimeric or fusion proteins. Such chimeric and fusion proteins comprise a phosphodiesterase peptide operatively linked to a heterologous protein having an amino acid sequence not substantially homologous to the phosphodiesterase peptide. "Operatively linked" indicates that the phosphodiesterase peptide and the heterologous protein are fused in-frame. The heterologous protein can be fused to the N-terminus or C-terminus of the phosphodiesterase peptide.

[0032]In some uses, the fusion protein does not affect the activity of the phosphodiesterase peptide per se. For example, the fusion protein can include, but is not limited to, enzymatic fusion proteins, for example beta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-His fusions, MYC-tagged, HI-tagged and Ig fusions. Such fusion proteins, particularly poly-His fusions, can facilitate the purification of recombinant phosphodiesterase peptide. In certain host cells (e.g., mammalian host cells), expression and/or secretion of a protein can be increased by using a heterologous signal sequence.

[0033]A chimeric or fusion protein can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different protein sequences are ligated together in-frame in accordance with conventional techniques. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and re-amplified to generate a chimeric gene sequence (see Ausubel et al., Current Protocols in Molecular Biology, 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST protein). A phosphodiesterase peptide-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the phosphodiesterase peptide.

[0034]As mentioned above, the present invention also provides and enables obvious variants of the amino acid sequence of the proteins of the present invention, such as naturally occurring mature forms of the peptide, allelic/sequence variants of the peptides, non-naturally occurring recombinantly derived variants of the peptides, and orthologs and paralogs of the peptides. Such variants can readily be generated using art-known techniques in the fields of recombinant nucleic acid technology and protein biochemistry. It is understood, however, that variants exclude any amino acid sequences disclosed prior to the invention.

[0035]Such variants can readily be identified/made using molecular techniques and the sequence information disclosed herein. Further, such variants can readily be distinguished from other peptides based on sequence and/or structural homology to the phosphodiesterase peptides of the present invention. The degree of homology/identity present will be based primarily on whether the peptide is a functional variant or non-functional variant, the amount of divergence present in the paralog family and the evolutionary distance between the orthologs.

[0036]To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of the length of a reference sequence is aligned for comparison purposes. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[0037]The comparison of sequences and determination of percent identity and similarity between two sequences can be accomplished using a mathematical algorithm. (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (Devereux, J., et al., Nucleic Acids Res. 12(1):387 (1984)) (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of E. Myers and W. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

[0038]The nucleic acid and protein sequences of the present invention can further be used as a "query sequence" to perform a search against sequence databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (J. Mol. Biol. 215:403-10 (1990)). BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the proteins of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (Nucleic Acids Res. 25(17):3389-3402 (1997)). When utilizing BLAST and gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0039]Full-length pre-processed forms, as well as mature processed forms, of proteins that comprise one of the peptides of the present invention can readily be identified as having complete sequence identity to one of the phosphodiesterase peptides of the present invention as well as being encoded by the same genetic locus as the phosphodiesterase peptide provided herein. The gene encoding the novel phosphodiesterase protein of the present invention is located on a genome component that has been mapped to human chromosome 11 (as indicated in FIG. 3), which is supported by multiple lines of evidence, such as STS and BAC map data.

[0040]Allelic variants of a phosphodiesterase peptide can readily be identified as being a human protein having a high degree (significant) of sequence homology/identity to at least a portion of the phosphodiesterase peptide as well as being encoded by the same genetic locus as the phosphodiesterase peptide provided herein. Genetic locus can readily be determined based on the genomic information provided in FIG. 3, such as the genomic sequence mapped to the reference human. The gene encoding the novel phosphodiesterase protein of the present invention is located on a genome component that has been mapped to human chromosome 11 (as indicated in FIG. 3), which is supported by multiple lines of evidence, such as STS and BAC map data. As used herein, two proteins (or a region of the proteins) have significant homology when the amino acid sequences are typically at least about 70-80%, 80-90%, and more typically at least about 90-95% or more homologous. A significantly homologous amino acid sequence, according to the present invention, will be encoded by a nucleic acid sequence that will hybridize to a phosphodiesterase peptide encoding nucleic acid molecule under stringent conditions as more fully described below.

[0041]FIG. 3 provides information on SNPs that have been found in the gene encoding the phosphodiesterase protein of the present invention. SNPs were identified at 231 different nucleotide positions. Changes in the amino acid sequence caused by these SNPs can readily be determined using the universal genetic code and the protein sequence provided in FIG. 2 as a reference. These SNPs may also affect control/regulatory elements.

[0042]Paralogs of a phosphodiesterase peptide can readily be identified as having some degree of significant sequence homology/identity to at least a portion of the phosphodiesterase peptide, as being encoded by a gene from humans, and as having similar activity or function. Two proteins will typically be considered paralogs when the amino acid sequences are typically at least about 60% or greater, and more typically at least about 70% or greater homology through a given region or domain. Such paralogs will be encoded by a nucleic acid sequence that will hybridize to a phosphodiesterase peptide encoding nucleic acid molecule under moderate to stringent conditions as more fully described below.

[0043]Orthologs of a phosphodiesterase peptide can readily be identified as having some degree of significant sequence homology/identity to at least a portion of the phosphodiesterase peptide as well as being encoded by a gene from another organism. Preferred orthologs will be isolated from mammals, preferably primates, for the development of human therapeutic targets and agents. Such orthologs will be encoded by a nucleic acid sequence that will hybridize to a phosphodiesterase peptide encoding nucleic acid molecule under moderate to stringent conditions, as more fully described below, depending on the degree of relatedness of the two organisms yielding the proteins.

[0044]Non-naturally occurring variants of the phosphodiesterase peptides of the present invention can readily be generated using recombinant techniques. Such variants include, but are not limited to deletions, additions and substitutions in the amino acid sequence of the phosphodiesterase peptide. For example, one class of substitutions are conserved amino acid substitution. Such substitutions are those that substitute a given amino acid in a phosphodiesterase peptide by another amino acid of like characteristics. Typically seen as conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu, and Ile; interchange of the hydroxyl residues Ser and Thr; exchange of the acidic residues Asp and Glu; substitution between the amide residues Asn and Gln; exchange of the basic residues Lys and Arg; and replacements among the aromatic residues Phe and Tyr. Guidance concerning which amino acid changes are likely to be phenotypically silent are found in Bowie et al., Science 247:1306-1310 (1990).

[0045]Variant phosphodiesterase peptides can be fully functional or can lack function in one or more activities, e.g. ability to bind substrate, ability to phosphorylate substrate, ability to mediate signaling, etc. Fully functional variants typically contain only conservative variation or variation in non-critical residues or in non-critical regions. FIG. 2 provides the result of protein analysis and can be used to identify critical domains/regions. Functional variants can also contain substitution of similar amino acids that result in no change or an insignificant change in function. Alternatively, such substitutions may positively or negatively affect function to some degree.

[0046]Non-functional variants typically contain one or more non-conservative amino acid substitutions, deletions, insertions, inversions, or truncation or a substitution, insertion, inversion, or deletion in a critical residue or critical region.

[0047]Amino acids that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham et al., Science 244:1081-1085 (1989)), particularly using the results provided in FIG. 2. The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity such as phosphodiesterase activity or in assays such as an in vitro proliferative activity. Sites that are critical for binding partner/substrate binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al., J. Mol. Biol. 224:899-904 (1992); de Vos et al. Science 255:306-312 (1992)).

[0048]The present invention further provides fragments of the phosphodiesterase peptides, in addition to proteins and peptides that comprise and consist of such fragments, particularly those comprising the residues identified in FIG. 2. The fragments to which the invention pertains, however, are not to be construed as encompassing fragments that may be disclosed publicly prior to the present invention.

[0049]As used herein, a fragment comprises at least 8, 10, 12, 14, 16, or more contiguous amino acid residues from a phosphodiesterase peptide. Such fragments can be chosen based on the ability to retain one or more of the biological activities of the phosphodiesterase peptide or could be chosen for the ability to perform a function, e.g. bind a substrate or act as an immunogen. Particularly important fragments are biologically active fragments, peptides that are, for example, about 8 or more amino acids in length. Such fragments will typically comprise a domain or motif of the phosphodiesterase peptide, e.g., active site, a transmembrane domain or a substrate-binding domain. Further, possible fragments include, but are not limited to, domain or motif containing fragments, soluble peptide fragments, and fragments containing immunogenic structures. Predicted domains and functional sites are readily identifiable by computer programs well known and readily available to those of skill in the art (e.g., PROSITE analysis). The results of one such analysis are provided in FIG. 2.

[0050]Polypeptides often contain amino acids other than the 20 amino acids commonly referred to as the 20 naturally occurring amino acids. Further, many amino acids, including the terminal amino acids, may be modified by natural processes, such as processing and other post-translational modifications, or by chemical modification techniques well known in the art. Common modifications that occur naturally in phosphodiesterase peptides are described in basic texts, detailed monographs, and the research literature, and they are well known to those of skill in the art (some of these features are identified in FIG. 2).

[0051]Known modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.

[0052]Such modifications are well known to those of skill in the art and have been described in great detail in the scientific literature. Several particularly common modifications, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, for instance, are described in most basic texts, such as Proteins--Structure and Molecular Properties, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993). Many detailed reviews are available on this subject, such as by Wold, F., Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York 1-12 (1983); Seifter et al. (Meth. Enzymol. 182: 626-646 (1990)) and Rattan et al. (Ann. N. Y. Acad. Sci. 663:48-62 (1992)).

[0053]Accordingly, the phosphodiesterase peptides of the present invention also encompass derivatives or analogs in which a substituted amino acid residue is not one encoded by the genetic code, in which a substituent group is included, in which the mature phosphodiesterase peptide is fused with another compound, such as a compound to increase the half-life of the phosphodiesterase peptide (for example, polyethylene glycol), or in which the additional amino acids are fused to the mature phosphodiesterase peptide, such as a leader or secretory sequence or a sequence for purification of the mature phosphodiesterase peptide or a pro-protein sequence.

[0054]Protein/Peptide Uses

[0055]The proteins of the present invention can be used in substantial and specific assays related to the functional information provided in the Figures; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its binding partner or ligand) in biological fluids; and as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state). Where the protein binds or potentially binds to another protein or ligand (such as, for example, in a phosphodiesterase-effector protein interaction or phosphodiesterase-ligand interaction), the protein can be used to identify the binding partner/ligand so as to develop a system to identify inhibitors of the binding interaction. Any or all of these uses are capable of being developed into reagent grade or kit format for commercialization as commercial products.

[0056]Methods for performing the uses listed above are well known to those skilled in the art. References disclosing such methods include "Molecular Cloning: A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds., 1989, and "Methods in Enzymology: Guide to Molecular Cloning Techniques", Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

[0057]The potential uses of the peptides of the present invention are based primarily on the source of the protein as well as the class/action of the protein. For example, phosphodiesterases isolated from humans and their human/mammalian orthologs serve as targets for identifying agents for use in mammalian therapeutic applications, e.g. a human drug, particularly in modulating a biological or pathological response in a cell or tissue that expresses the phosphodiesterase. Experimental data as provided in FIG. 1 indicates that phosphodiesterase proteins of the present invention are expressed in humans in the amygdala, brain (including infant brain), uterus, testis, placenta choriocarcinomas, Hela cells, and a pooled melanocyte/fetal heart/pregnant uterus sample, as indicated by virtual northern blot analysis. PCR-based tissue screening panels also indicate expression in the brain. A large percentage of pharmaceutical agents are being developed that modulate the activity of phosphodiesterase proteins, particularly members of the PDE2A subfamily (see Background of the Invention). The structural and functional information provided in the Background and Figures provide specific and substantial uses for the molecules of the present invention, particularly in combination with the expression information provided in FIG. 1. Experimental data as provided in FIG. 1 indicates expression in humans in the amygdala, brain (including infant brain), uterus, testis, placenta choriocarcinomas, Hela cells, and a pooled melanocyte/fetal heart/pregnant uterus sample. Such uses can readily be determined using the information provided herein, that which is known in the art, and routine experimentation.

[0058]The proteins of the present invention (including variants and fragments that may have been disclosed prior to the present invention) are useful for biological assays related to phosphodiesterases that are related to members of the PDE2A subfamily. Such assays involve any of the known phosphodiesterase functions or activities or properties useful for diagnosis and treatment of phosphodiesterase-related conditions that are specific for the subfamily of phosphodiesterases that the one of the present invention belongs to, particularly in cells and tissues that express the phosphodiesterase. Experimental data as provided in FIG. 1 indicates that phosphodiesterase proteins of the present invention are expressed in humans in the amygdala, brain (including infant brain), uterus, testis, placenta choriocarcinomas, Hela cells, and a pooled melanocyte/fetal heart/pregnant uterus sample, as indicated by virtual northern blot analysis. PCR-based tissue screening panels also indicate expression in the brain.

[0059]The proteins of the present invention are also useful in drug screening assays, in cell-based or cell-free systems. Cell-based systems can be native, i.e., cells that normally express the phosphodiesterase, as a biopsy or expanded in cell culture. Experimental data as provided in FIG. 1 indicates expression in humans in the amygdala, brain (including infant brain), uterus, testis, placenta choriocarcinomas, Hela cells, and a pooled melanocyte/fetal heart/pregnant uterus sample. In an alternate embodiment, cell-based assays involve recombinant host cells expressing the phosphodiesterase protein.

[0060]The polypeptides can be used to identify compounds that modulate phosphodiesterase activity of the protein in its natural state or an altered form that causes a specific disease or pathology associated with the phosphodiesterase. Both the phosphodiesterases of the present invention and appropriate variants and fragments can be used in high-throughput screens to assay candidate compounds for the ability to bind to the phosphodiesterase. These compounds can be further screened against a functional phosphodiesterase to determine the effect of the compound on the phosphodiesterase activity. Further, these compounds can be tested in animal or invertebrate systems to determine activity/effectiveness. Compounds can be identified that activate (agonist) or inactivate (antagonist) the phosphodiesterase to a desired degree.

[0061]Further, the proteins of the present invention can be used to screen a compound for the ability to stimulate or inhibit interaction between the phosphodiesterase protein and a molecule that normally interacts with the phosphodiesterase protein, e.g. a substrate or a component of the signal pathway that the phosphodiesterase protein normally interacts (for example, another phosphodiesterase). Such assays typically include the steps of combining the phosphodiesterase protein with a candidate compound under conditions that allow the phosphodiesterase protein, or fragment, to interact with the target molecule, and to detect the formation of a complex between the protein and the target or to detect the biochemical consequence of the interaction with the phosphodiesterase protein and the target, such as any of the associated effects of signal transduction such as protein phosphorylation, cAMP turnover, and adenylate cyclase activation, etc.

[0062]Candidate compounds include, for example, 1) peptides such as soluble peptides, including Ig-tailed fusion peptides and members of random peptide libraries (see, e.g., Lam et al., Nature 354:82-84 (1991); Houghten et al., Nature 354:84-86 (1991)) and combinatorial chemistry-derived molecular libraries made of D- and/or L-configuration amino acids; 2) phosphopeptides (e.g., members of random and partially degenerate, directed phosphopeptide libraries, see, e.g., Songyang et al., Cell 72:767-778 (1993)); 3) antibodies (e.g., polyclonal, monoclonal, humanized, anti-idiotypic, chimeric, and single chain antibodies as well as Fab, F(ab')₂, Fab expression library fragments, and epitope-binding fragments of antibodies); and 4) small organic and inorganic molecules (e.g., molecules obtained from combinatorial and natural product libraries).

[0063]One candidate compound is a soluble fragment of the receptor that competes for substrate binding. Other candidate compounds include mutant phosphodiesterases or appropriate fragments containing mutations that affect phosphodiesterase function and thus compete for substrate. Accordingly, a fragment that competes for substrate, for example with a higher affinity, or a fragment that binds substrate but does not allow release, is encompassed by the invention.

[0064]The invention further includes other end point assays to identify compounds that modulate (stimulate or inhibit) phosphodiesterase activity. The assays typically involve an assay of events in the signal transduction pathway that indicate phosphodiesterase activity. Thus, the phosphorylation of a substrate, activation of a protein, a change in the expression of genes that are up- or down-regulated in response to the phosphodiesterase protein dependent signal cascade can be assayed.

[0065]Any of the biological or biochemical functions mediated by the phosphodiesterase can be used as an endpoint assay. These include all of the biochemical or biochemical/biological events described herein, in the references cited herein, incorporated by reference for these endpoint assay targets, and other functions known to those of ordinary skill in the art or that can be readily identified using the information provided in the Figures, particularly FIG. 2. Specifically, a biological function of a cell or tissues that expresses the phosphodiesterase can be assayed. Experimental data as provided in FIG. 1 indicates that phosphodiesterase proteins of the present invention are expressed in humans in the amygdala, brain (including infant brain), uterus, testis, placenta choriocarcinomas, Hela cells, and a pooled melanocyte/fetal heart/pregnant uterus sample, as indicated by virtual northern blot analysis. PCR-based tissue screening panels also indicate expression in the brain.

[0066]Binding and/or activating compounds can also be screened by using chimeric phosphodiesterase proteins in which the amino terminal extracellular domain, or parts thereof, the entire transmembrane domain or subregions, such as any of the seven transmembrane segments or any of the intracellular or extracellular loops and the carboxy terminal intracellular domain, or parts thereof, can be replaced by heterologous domains or subregions. For example, a substrate-binding region can be used that interacts with a different substrate then that which is recognized by the native phosphodiesterase. Accordingly, a different set of signal transduction components is available as an end-point assay for activation. This allows for assays to be performed in other than the specific host cell from which the phosphodiesterase is derived.

[0067]The proteins of the present invention are also useful in competition binding assays in methods designed to discover compounds that interact with the phosphodiesterase (e.g. binding partners and/or ligands). Thus, a compound is exposed to a phosphodiesterase polypeptide under conditions that allow the compound to bind or to otherwise interact with the polypeptide. Soluble phosphodiesterase polypeptide is also added to the mixture. If the test compound interacts with the soluble phosphodiesterase polypeptide, it decreases the amount of complex formed or activity from the phosphodiesterase target. This type of assay is particularly useful in cases in which compounds are sought that interact with specific regions of the phosphodiesterase. Thus, the soluble polypeptide that competes with the target phosphodiesterase region is designed to contain peptide sequences corresponding to the region of interest.

[0068]To perform cell free drug screening assays, it is sometimes desirable to immobilize either the phosphodiesterase protein, or fragment, or its target molecule to facilitate separation of complexes from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay.

[0069]Techniques for immobilizing proteins on matrices can be used in the drug screening assays. In one embodiment, a fusion protein can be provided which adds a domain that allows the protein to be bound to a matrix. For example, glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtitre plates, which are then combined with the cell lysates (e.g., ³⁵S-labeled) and the candidate compound, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads are washed to remove any unbound label, and the matrix immobilized and radiolabel determined directly, or in the supernatant after the complexes are dissociated. Alternatively, the complexes can be dissociated from the matrix, separated by SDS-PAGE, and the level of phosphodiesterase-binding protein found in the bead fraction quantitated from the gel using standard electrophoretic techniques. For example, either the polypeptide or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin using techniques well known in the art. Alternatively, antibodies reactive with the protein but which do not interfere with binding of the protein to its target molecule can be derivatized to the wells of the plate, and the protein trapped in the wells by antibody conjugation. Preparations of a phosphodiesterase-binding protein and a candidate compound are incubated in the phosphodiesterase protein-presenting wells and the amount of complex trapped in the well can be quantitated. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the phosphodiesterase protein target molecule, or which are reactive with phosphodiesterase protein and compete with the target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the target molecule.

[0070]Agents that modulate one of the phosphodiesterases of the present invention can be identified using one or more of the above assays, alone or in combination. It is generally preferable to use a cell-based or cell free system first and then confirm activity in an animal or other model system. Such model systems are well known in the art and can readily be employed in this context.

[0071]Modulators of phosphodiesterase protein activity identified according to these drug screening assays can be used to treat a subject with a disorder mediated by the phosphodiesterase pathway, by treating cells or tissues that express the phosphodiesterase. Experimental data as provided in FIG. 1 indicates expression in humans in the amygdala, brain (including infant brain), uterus, testis, placenta choriocarcinomas, Hela cells, and a pooled melanocyte/fetal heart/pregnant uterus sample. These methods of treatment include the steps of administering a modulator of phosphodiesterase activity in a pharmaceutical composition to a subject in need of such treatment, the modulator being identified as described herein.

[0072]In yet another aspect of the invention, the phosphodiesterase proteins can be used as "bait proteins" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300), to identify other proteins, which bind to or interact with the phosphodiesterase and are involved in phosphodiesterase activity. Such phosphodiesterase-binding proteins are also likely to be involved in the propagation of signals by the phosphodiesterase proteins or phosphodiesterase targets as, for example, downstream elements of a phosphodiesterase-mediated signaling pathway. Alternatively, such phosphodiesterase-binding proteins are likely to be phosphodiesterase inhibitors.

[0073]The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for a phosphodiesterase protein is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein ("prey" or "sample") is fused to a gene that codes for the activation domain of the known transcription factor. If the "bait" and the "prey" proteins are able to interact, in vivo, forming a phosphodiesterase-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the phosphodiesterase protein.

[0074]This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model. For example, an agent identified as described herein (e.g., a phosphodiesterase-modulating agent, an antisense phosphodiesterase nucleic acid molecule, a phosphodiesterase-specific antibody, or a phosphodiesterase-binding partner) can be used in an animal or other model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent identified as described herein can be used in an animal or other model to determine the mechanism of action of such an agent. Furthermore, this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.

[0075]The phosphodiesterase proteins of the present invention are also useful to provide a target for diagnosing a disease or predisposition to disease mediated by the peptide. Accordingly, the invention provides methods for detecting the presence, or levels of, the protein (or encoding mRNA) in a cell, tissue, or organism. Experimental data as provided in FIG. 1 indicates expression in humans in the amygdala, brain (including infant brain), uterus, testis, placenta choriocarcinomas, Hela cells, and a pooled melanocyte/fetal heart/pregnant uterus sample. The method involves contacting a biological sample with a compound capable of interacting with the phosphodiesterase protein such that the interaction can be detected. Such an assay can be provided in a single detection format or a multi-detection format such as an antibody chip array.

[0076]One agent for detecting a protein in a sample is an antibody capable of selectively binding to protein. A biological sample includes tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject.

[0077]The peptides of the present invention also provide targets for diagnosing active protein activity, disease, or predisposition to disease, in a patient having a variant peptide, particularly activities and conditions that are known for other members of the family of proteins to which the present one belongs. Thus, the peptide can be isolated from a biological sample and assayed for the presence of a genetic mutation that results in aberrant peptide. This includes amino acid substitution, deletion, insertion, rearrangement, (as the result of aberrant splicing events), and inappropriate post-translational modification. Analytic methods include altered electrophoretic mobility, altered tryptic peptide digest, altered phosphodiesterase activity in cell-based or cell-free assay, alteration in substrate or antibody-binding pattern, altered isoelectric point, direct amino acid sequencing, and any other of the known assay techniques useful for detecting mutations in a protein. Such an assay can be provided in a single detection format or a multi-detection format such as an antibody chip array.

[0078]In vitro techniques for detection of peptide include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence using a detection reagent, such as an antibody or protein binding agent. Alternatively, the peptide can be detected in vivo in a subject by introducing into the subject a labeled anti-peptide antibody or other types of detection agent. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. Particularly useful are methods that detect the allelic variant of a peptide expressed in a subject and methods which detect fragments of a peptide in a sample.

[0079]The peptides are also useful in pharmacogenomic analysis. Pharmacogenomics deal with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, e.g., Eichelbaum, M. (Clin. Exp. Pharmacol. Physiol. 23(10-11):983-985 (1996)), and Linder, M. W. (Clin. Chem. 43(2):254-266 (1997)). The clinical outcomes of these variations result in severe toxicity of therapeutic drugs in certain individuals or therapeutic failure of drugs in certain individuals as a result of individual variation in metabolism. Thus, the genotype of the individual can determine the way a therapeutic compound acts on the body or the way the body metabolizes the compound. Further, the activity of drug metabolizing enzymes effects both the intensity and duration of drug action. Thus, the pharmacogenomics of the individual permit the selection of effective compounds and effective dosages of such compounds for prophylactic or therapeutic treatment based on the individual's genotype. The discovery of genetic polymorphisms in some drug metabolizing enzymes has explained why some patients do not obtain the expected drug effects, show an exaggerated drug effect, or experience serious toxicity from standard drug dosages. Polymorphisms can be expressed in the phenotype of the extensive metabolizer and the phenotype of the poor metabolizer. Accordingly, genetic polymorphism may lead to allelic protein variants of the phosphodiesterase protein in which one or more of the phosphodiesterase functions in one population is different from those in another population. The peptides thus allow a target to ascertain a genetic predisposition that can affect treatment modality. Thus, in a ligand-based treatment, polymorphism may give rise to amino terminal extracellular domains and/or other substrate-binding regions that are more or less active in substrate binding, and phosphodiesterase activation. Accordingly, substrate dosage would necessarily be modified to maximize the therapeutic effect within a given population containing a polymorphism. As an alternative to genotyping, specific polymorphic peptides could be identified.

[0080]The peptides are also useful for treating a disorder characterized by an absence of, inappropriate, or unwanted expression of the protein. Experimental data as provided in FIG. 1 indicates expression in humans in the amygdala, brain (including infant brain), uterus, testis, placenta choriocarcinomas, Hela cells, and a pooled melanocyte/fetal heart/pregnant uterus sample. Accordingly, methods for treatment include the use of the phosphodiesterase protein or fragments.

[0081]Antibodies

[0082]The invention also provides antibodies that selectively bind to one of the peptides of the present invention, a protein comprising such a peptide, as well as variants and fragments thereof. As used herein, an antibody selectively binds a target peptide when it binds the target peptide and does not significantly bind to unrelated proteins. An antibody is still considered to selectively bind a peptide even if it also binds to other proteins that are not substantially homologous with the target peptide so long as such proteins share homology with a fragment or domain of the peptide target of the antibody. In this case, it would be understood that antibody binding to the peptide is still selective despite some degree of cross-reactivity.

[0083]As used herein, an antibody is defined in terms consistent with that recognized within the art: they are multi-subunit proteins produced by a mammalian organism in response to an antigen challenge. The antibodies of the present invention include polyclonal antibodies and monoclonal antibodies, as well as fragments of such antibodies, including, but not limited to, Fab or F(ab')₂, and Fv fragments.

[0084]Many methods are known for generating and/or identifying antibodies to a given target peptide. Several such methods are described by Harlow, Antibodies, Cold Spring Harbor Press, (1989).

[0085]In general, to generate antibodies, an isolated peptide is used as an immunogen and is administered to a mammalian organism, such as a rat, rabbit or mouse. The full-length protein, an antigenic peptide fragment or a fusion protein can be used. Particularly important fragments are those covering functional domains, such as the domains identified in FIG. 2, and domain of sequence homology or divergence amongst the family, such as those that can readily be identified using protein alignment methods and as presented in the Figures.

[0086]Antibodies are preferably prepared from regions or discrete fragments of the phosphodiesterase proteins. Antibodies can be prepared from any region of the peptide as described herein. However, preferred regions will include those involved in function/activity and/or phosphodiesterase/binding partner interaction. FIG. 2 can be used to identify particularly important regions while sequence alignment can be used to identify conserved and unique sequence fragments.

[0087]An antigenic fragment will typically comprise at least 8 contiguous amino acid residues. The antigenic peptide can comprise, however, at least 10, 12, 14, 16 or more amino acid residues. Such fragments can be selected on a physical property, such as fragments correspond to regions that are located on the surface of the protein, e.g., hydrophilic regions or can be selected based on sequence uniqueness (see FIG. 2).

[0088]Detection on an antibody of the present invention can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or ³H.

[0089]Antibody Uses

[0090]The antibodies can be used to isolate one of the proteins of the present invention by standard techniques, such as affinity chromatography or immunoprecipitation. The antibodies can facilitate the purification of the natural protein from cells and recombinantly produced protein expressed in host cells. In addition, such antibodies are useful to detect the presence of one of the proteins of the present invention in cells or tissues to determine the pattern of expression of the protein among various tissues in an organism and over the course of normal development. Experimental data as provided in FIG. 1 indicates that phosphodiesterase proteins of the present invention are expressed in humans in the amygdala, brain (including infant brain), uterus, testis, placenta choriocarcinomas, Hela cells, and a pooled melanocyte/fetal heart/pregnant uterus sample, as indicated by virtual northern blot analysis. PCR-based tissue screening panels also indicate expression in the brain. Further, such antibodies can be used to detect protein in situ, in vitro, or in a cell lysate or supernatant in order to evaluate the abundance and pattern of expression. Also, such antibodies can be used to assess abnormal tissue distribution or abnormal expression during development or progression of a biological condition. Antibody detection of circulating fragments of the full length protein can be used to identify turnover.

[0091]Further, the antibodies can be used to assess expression in disease states such as in active stages of the disease or in an individual with a predisposition toward disease related to the protein's function. When a disorder is caused by an inappropriate tissue distribution, developmental expression, level of expression of the protein, or expressed/processed form, the antibody can be prepared against the normal protein. Experimental data as provided in FIG. 1 indicates expression in humans in the amygdala, brain (including infant brain), uterus, testis, placenta choriocarcinomas, Hela cells, and a pooled melanocyte/fetal heart/pregnant uterus sample. If a disorder is characterized by a specific mutation in the protein, antibodies specific for this mutant protein can be used to assay for the presence of the specific mutant protein.

[0092]The antibodies can also be used to assess normal and aberrant subcellular localization of cells in the various tissues in an organism. Experimental data as provided in FIG. 1 indicates expression in humans in the amygdala, brain (including infant brain), uterus, testis, placenta choriocarcinomas, Hela cells, and a pooled melanocyte/fetal heart/pregnant uterus sample. The diagnostic uses can be applied, not only in genetic testing, but also in monitoring a treatment modality. Accordingly, where treatment is ultimately aimed at correcting expression level or the presence of aberrant sequence and aberrant tissue distribution or developmental expression, antibodies directed against the protein or relevant fragments can be used to monitor therapeutic efficacy.

[0093]Additionally, antibodies are useful in pharmacogenomic analysis. Thus, antibodies prepared against polymorphic proteins can be used to identify individuals that require modified treatment modalities. The antibodies are also useful as diagnostic tools as an immunological marker for aberrant protein analyzed by electrophoretic mobility, isoelectric point, tryptic peptide digest, and other physical assays known to those in the art.

[0094]The antibodies are also useful for tissue typing. Experimental data as provided in FIG. 1 indicates expression in humans in the amygdala, brain (including infant brain), uterus, testis, placenta choriocarcinomas, Hela cells, and a pooled melanocyte/fetal heart/pregnant uterus sample. Thus, where a specific protein has been correlated with expression in a specific tissue, antibodies that are specific for this protein can be used to identify a tissue type.

[0095]The antibodies are also useful for inhibiting protein function, for example, blocking the binding of the phosphodiesterase peptide to a binding partner such as a substrate. These uses can also be applied in a therapeutic context in which treatment involves inhibiting the protein's function. An antibody can be used, for example, to block binding, thus modulating (agonizing or antagonizing) the peptides activity. Antibodies can be prepared against specific fragments containing sites required for function or against intact protein that is associated with a cell or cell membrane. See FIG. 2 for structural information relating to the proteins of the present invention.

[0096]The invention also encompasses kits for using antibodies to detect the presence of a protein in a biological sample. The kit can comprise antibodies such as a labeled or labelable antibody and a compound or agent for detecting protein in a biological sample; means for determining the amount of protein in the sample; means for comparing the amount of protein in the sample with a standard; and instructions for use. Such a kit can be supplied to detect a single protein or epitope or can be configured to detect one of a multitude of epitopes, such as in an antibody detection array. Arrays are described in detail below for nucleic acid arrays and similar methods have been developed for antibody arrays.

[0097]Nucleic Acid Molecules

[0098]The present invention further provides isolated nucleic acid molecules that encode a phosphodiesterase peptide or protein of the present invention (cDNA, transcript and genomic sequence). Such nucleic acid molecules will consist of, consist essentially of, or comprise a nucleotide sequence that encodes one of the phosphodiesterase peptides of the present invention, an allelic variant thereof, or an ortholog or paralog thereof.

[0099]As used herein, an "isolated" nucleic acid molecule is one that is separated from other nucleic acid present in the natural source of the nucleic acid. Preferably, an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. However, there can be some flanking nucleotide sequences, for example up to about 5 KB, 4 KB, 3 KB, 2 KB, or 1 KB or less, particularly contiguous peptide encoding sequences and peptide encoding sequences within the same gene but separated by introns in the genomic sequence. The important point is that the nucleic acid is isolated from remote and unimportant flanking sequences such that it can be subjected to the specific manipulations described herein such as recombinant expression, preparation of probes and primers, and other uses specific to the nucleic acid sequences.

[0100]Moreover, an "isolated" nucleic acid molecule, such as a transcript/cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. However, the nucleic acid molecule can be fused to other coding or regulatory sequences and still be considered isolated.

[0101]For example, recombinant DNA molecules contained in a vector are considered isolated. Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the isolated DNA molecules of the present invention. Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.

[0102]Accordingly, the present invention provides nucleic acid molecules that consist of the nucleotide sequence shown in FIG. 1 or 3 (SEQ ID NO: 1, transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleic acid molecule that encodes the protein provided in FIG. 2, SEQ ID NO:2. A nucleic acid molecule consists of a nucleotide sequence when the nucleotide sequence is the complete nucleotide sequence of the nucleic acid molecule.

[0103]The present invention further provides nucleic acid molecules that consist essentially of the nucleotide sequence shown in FIG. 1 or 3 (SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleic acid molecule that encodes the protein provided in FIG. 2, SEQ ID NO:2. A nucleic acid molecule consists essentially of a nucleotide sequence when such a nucleotide sequence is present with only a few additional nucleic acid residues in the final nucleic acid molecule.

[0104]The present invention further provides nucleic acid molecules that comprise the nucleotide sequences shown in FIG. 1 or 3 (SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleic acid molecule that encodes the protein provided in FIG. 2, SEQ ID NO:2. A nucleic acid molecule comprises a nucleotide sequence when the nucleotide sequence is at least part of the final nucleotide sequence of the nucleic acid molecule. In such a fashion, the nucleic acid molecule can be only the nucleotide sequence or have additional nucleic acid residues, such as nucleic acid residues that are naturally associated with it or heterologous nucleotide sequences. Such a nucleic acid molecule can have a few additional nucleotides or can comprises several hundred or more additional nucleotides. A brief description of how various types of these nucleic acid molecules can be readily made/isolated is provided below.

[0105]In FIGS. 1 and 3, both coding and non-coding sequences are provided. Because of the source of the present invention, humans genomic sequence (FIG. 3) and cDNA/transcript sequences (FIG. 1), the nucleic acid molecules in the Figures will contain genomic intronic sequences, 5' and 3' non-coding sequences, gene regulatory regions and non-coding intergenic sequences. In general such sequence features are either noted in FIGS. 1 and 3 or can readily be identified using computational tools known in the art. As discussed below, some of the non-coding regions, particularly gene regulatory elements such as promoters, are useful for a variety of purposes, e.g. control of heterologous gene expression, target for identifying gene activity modulating compounds, and are particularly claimed as fragments of the genomic sequence provided herein.

[0106]The isolated nucleic acid molecules can encode the mature protein plus additional amino or carboxyl-terminal amino acids, or amino acids interior to the mature peptide (when the mature form has more than one peptide chain, for instance). Such sequences may play a role in processing of a protein from precursor to a mature form, facilitate protein trafficking, prolong or shorten protein half-life or facilitate manipulation of a protein for assay or production, among other things. As generally is the case in situ, the additional amino acids may be processed away from the mature protein by cellular enzymes.

[0107]As mentioned above, the isolated nucleic acid molecules include, but are not limited to, the sequence encoding the phosphodiesterase peptide alone, the sequence encoding the mature peptide and additional coding sequences, such as a leader or secretory sequence (e.g., a pre-pro or pro-protein sequence), the sequence encoding the mature peptide, with or without the additional coding sequences, plus additional non-coding sequences, for example introns and non-coding 5' and 3' sequences such as transcribed but non-translated sequences that play a role in transcription, mRNA processing (including splicing and polyadenylation signals), ribosome binding and stability of mRNA. In addition, the nucleic acid molecule may be fused to a marker sequence encoding, for example, a peptide that facilitates purification.

[0108]Isolated nucleic acid molecules can be in the form of RNA, such as mRNA, or in the form DNA, including cDNA and genomic DNA obtained by cloning or produced by chemical synthetic techniques or by a combination thereof. The nucleic acid, especially DNA, can be double-stranded or single-stranded. Single-stranded nucleic acid can be the coding strand (sense strand) or the non-coding strand (anti-sense strand).

[0109]The invention further provides nucleic acid molecules that encode fragments of the peptides of the present invention as well as nucleic acid molecules that encode obvious variants of the phosphodiesterase proteins of the present invention that are described above. Such nucleic acid molecules may be naturally occurring, such as allelic variants (same locus), paralogs (different locus), and orthologs (different organism), or may be constructed by recombinant DNA methods or by chemical synthesis. Such non-naturally occurring variants may be made by mutagenesis techniques, including those applied to nucleic acid molecules, cells, or organisms. Accordingly, as discussed above, the variants can contain nucleotide substitutions, deletions, inversions and insertions. Variation can occur in either or both the coding and non-coding regions. The variations can produce both conservative and non-conservative amino acid substitutions.

[0110]The present invention further provides non-coding fragments of the nucleic acid molecules provided in FIGS. 1 and 3. Preferred non-coding fragments include, but are not limited to, promoter sequences, enhancer sequences, gene modulating sequences and gene termination sequences. Such fragments are useful in controlling heterologous gene expression and in developing screens to identify gene-modulating agents. A promoter can readily be identified as being 5' to the ATG start site in the genomic sequence provided in FIG. 3.

[0111]A fragment comprises a contiguous nucleotide sequence greater than 12 or more nucleotides. Further, a fragment could at least 30, 40, 50, 100, 250 or 500 nucleotides in length. The length of the fragment will be based on its intended use. For example, the fragment can encode epitope bearing regions of the peptide, or can be useful as DNA probes and primers. Such fragments can be isolated using the known nucleotide sequence to synthesize an oligonucleotide probe. A labeled probe can then be used to screen a cDNA library, genomic DNA library, or mRNA to isolate nucleic acid corresponding to the coding region. Further, primers can be used in PCR reactions to clone specific regions of gene.

[0112]A probe/primer typically comprises substantially a purified oligonucleotide or oligonucleotide pair. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, 20, 25, 40, 50 or more consecutive nucleotides.

[0113]Orthologs, homologs, and allelic variants can be identified using methods well known in the art. As described in the Peptide Section, these variants comprise a nucleotide sequence encoding a peptide that is typically 60-70%, 70-80%, 80-90%, and more typically at least about 90-95% or more homologous to the nucleotide sequence shown in the Figure sheets or a fragment of this sequence. Such nucleic acid molecules can readily be identified as being able to hybridize under moderate to stringent conditions, to the nucleotide sequence shown in the Figure sheets or a fragment of the sequence. Allelic variants can readily be determined by genetic locus of the encoding gene. The gene encoding the novel phosphodiesterase protein of the present invention is located on a genome component that has been mapped to human chromosome 11 (as indicated in FIG. 3), which is supported by multiple lines of evidence, such as STS and BAC map data.

[0114]FIG. 3 provides information on SNPs that have been found in the gene encoding the phosphodiesterase protein of the present invention. SNPs were identified at 231 different nucleotide positions. Changes in the amino acid sequence caused by these SNPs can readily be determined using the universal genetic code and the protein sequence provided in FIG. 2 as a reference. These SNPs may also affect control/regulatory elements.

[0115]As used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences encoding a peptide at least 60-70% homologous to each other typically remain hybridized to each other. The conditions can be such that sequences at least about 60%, at least about 70%, or at least about 80% or more homologous to each other typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. One example of stringent hybridization conditions are hybridization in 6× sodium chloride/sodium citrate (SSC) at about 45 C, followed by one or more washes in 0.2×SSC, 0.1% SDS at 50-65 C. Examples of moderate to low stringency hybridization conditions are well known in the art.

[0116]Nucleic Acid Molecule Uses

[0117]The nucleic acid molecules of the present invention are useful for probes, primers, chemical intermediates, and in biological assays. The nucleic acid molecules are useful as a hybridization probe for messenger RNA, transcript/cDNA and genomic DNA to isolate full-length cDNA and genomic clones encoding the peptide described in FIG. 2 and to isolate cDNA and genomic clones that correspond to variants (alleles, orthologs, etc.) producing the same or related peptides shown in FIG. 2. As illustrated in FIG. 3, SNPs were identified at 231 different nucleotide positions.

[0118]The probe can correspond to any sequence along the entire length of the nucleic acid molecules provided in the Figures. Accordingly, it could be derived from 5' noncoding regions, the coding region, and 3' noncoding regions. However, as discussed, fragments are not to be construed as encompassing fragments disclosed prior to the present invention.

[0119]The nucleic acid molecules are also useful as primers for PCR to amplify any given region of a nucleic acid molecule and are useful to synthesize antisense molecules of desired length and sequence.

[0120]The nucleic acid molecules are also useful for constructing recombinant vectors. Such vectors include expression vectors that express a portion of, or all of, the peptide sequences. Vectors also include insertion vectors, used to integrate into another nucleic acid molecule sequence, such as into the cellular genome, to alter in situ expression of a gene and/or gene product. For example, an endogenous coding sequence can be replaced via homologous recombination with all or part of the coding region containing one or more specifically introduced mutations.

[0121]The nucleic acid molecules are also useful for expressing antigenic portions of the proteins.

[0122]The nucleic acid molecules are also useful as probes for determining the chromosomal positions of the nucleic acid molecules by means of in situ hybridization methods. The gene encoding the novel phosphodiesterase protein of the present invention is located on a genome component that has been mapped to human chromosome 11 (as indicated in FIG. 3), which is supported by multiple lines of evidence, such as STS and BAC map data.

[0123]The nucleic acid molecules are also useful in making vectors containing the gene regulatory regions of the nucleic acid molecules of the present invention.

[0124]The nucleic acid molecules are also useful for designing ribozymes corresponding to all, or a part, of the mRNA produced from the nucleic acid molecules described herein.

[0125]The nucleic acid molecules are also useful for making vectors that express part, or all, of the peptides.

[0126]The nucleic acid molecules are also useful for constructing host cells expressing a part, or all, of the nucleic acid molecules and peptides.

[0127]The nucleic acid molecules are also useful for constructing transgenic animals expressing all, or a part, of the nucleic acid molecules and peptides.

[0128]The nucleic acid molecules are also useful as hybridization probes for determining the presence, level, form and distribution of nucleic acid expression. Experimental data as provided in FIG. 1 indicates that phosphodiesterase proteins of the present invention are expressed in humans in the amygdala, brain (including infant brain), uterus, testis, placenta choriocarcinomas, Hela cells, and a pooled melanocyte/fetal heart/pregnant uterus sample, as indicated by virtual northern blot analysis. PCR-based tissue screening panels also indicate expression in the brain. Accordingly, the probes can be used to detect the presence of, or to determine levels of, a specific nucleic acid molecule in cells, tissues, and in organisms. The nucleic acid whose level is determined can be DNA or RNA. Accordingly, probes corresponding to the peptides described herein can be used to assess expression and/or gene copy number in a given cell, tissue, or organism. These uses are relevant for diagnosis of disorders involving an increase or decrease in phosphodiesterase protein expression relative to normal results.

[0129]In vitro techniques for detection of mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detecting DNA includes Southern hybridizations and in situ hybridization.

[0130]Probes can be used as a part of a diagnostic test kit for identifying cells or tissues that express a phosphodiesterase protein, such as by measuring a level of a phosphodiesterase-encoding nucleic acid in a sample of cells from a subject e.g., mRNA or genomic DNA, or determining if a phosphodiesterase gene has been mutated. Experimental data as provided in FIG. 1 indicates that phosphodiesterase proteins of the present invention are expressed in humans in the amygdala, brain (including infant brain), uterus, testis, placenta choriocarcinomas, Hela cells, and a pooled melanocyte/fetal heart/pregnant uterus sample, as indicated by virtual northern blot analysis. PCR-based tissue screening panels also indicate expression in the brain.

[0131]Nucleic acid expression assays are useful for drug screening to identify compounds that modulate phosphodiesterase nucleic acid expression.

[0132]The invention thus provides a method for identifying a compound that can be used to treat a disorder associated with nucleic acid expression of the phosphodiesterase gene, particularly biological and pathological processes that are mediated by the phosphodiesterase in cells and tissues that express it. Experimental data as provided in FIG. 1 indicates expression in humans in the amygdala, brain (including infant brain), uterus, testis, placenta choriocarcinomas, Hela cells, and a pooled melanocyte/fetal heart/pregnant uterus sample. The method typically includes assaying the ability of the compound to modulate the expression of the phosphodiesterase nucleic acid and thus identifying a compound that can be used to treat a disorder characterized by undesired phosphodiesterase nucleic acid expression. The assays can be performed in cell-based and cell-free systems. Cell-based assays include cells naturally expressing the phosphodiesterase nucleic acid or recombinant cells genetically engineered to express specific nucleic acid sequences.

[0133]The assay for phosphodiesterase nucleic acid expression can involve direct assay of nucleic acid levels, such as mRNA levels, or on collateral compounds involved in the signal pathway. Further, the expression of genes that are up- or down-regulated in response to the phosphodiesterase protein signal pathway can also be assayed. In this embodiment the regulatory regions of these genes can be operably linked to a reporter gene such as luciferase.

[0134]Thus, modulators of phosphodiesterase gene expression can be identified in a method wherein a cell is contacted with a candidate compound and the expression of mRNA determined. The level of expression of phosphodiesterase mRNA in the presence of the candidate compound is compared to the level of expression of phosphodiesterase mRNA in the absence of the candidate compound. The candidate compound can then be identified as a modulator of nucleic acid expression based on this comparison and be used, for example to treat a disorder characterized by aberrant nucleic acid expression. When expression of mRNA is statistically significantly greater in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of nucleic acid expression. When nucleic acid expression is statistically significantly less in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of nucleic acid expression.

[0135]The invention further provides methods of treatment, with the nucleic acid as a target, using a compound identified through drug screening as a gene modulator to modulate phosphodiesterase nucleic acid expression in cells and tissues that express the phosphodiesterase. Experimental data as provided in FIG. 1 indicates that phosphodiesterase proteins of the present invention are expressed in humans in the amygdala, brain (including infant brain), uterus, testis, placenta choriocarcinomas, Hela cells, and a pooled melanocyte/fetal heart/pregnant uterus sample, as indicated by virtual northern blot analysis. PCR-based tissue screening panels also indicate expression in the brain. Modulation includes both up-regulation (i.e. activation or agonization) or down-regulation (suppression or antagonization) or nucleic acid expression.

[0136]Alternatively, a modulator for phosphodiesterase nucleic acid expression can be a small molecule or drug identified using the screening assays described herein as long as the drug or small molecule inhibits the phosphodiesterase nucleic acid expression in the cells and tissues that express the protein. Experimental data as provided in FIG. 1 indicates expression in humans in the amygdala, brain (including infant brain), uterus, testis, placenta choriocarcinomas, Hela cells, and a pooled melanocyte/fetal heart/pregnant uterus sample.

[0137]The nucleic acid molecules are also useful for monitoring the effectiveness of modulating compounds on the expression or activity of the phosphodiesterase gene in clinical trials or in a treatment regimen. Thus, the gene expression pattern can serve as a barometer for the continuing effectiveness of treatment with the compound, particularly with compounds to which a patient can develop resistance. The gene expression pattern can also serve as a marker indicative of a physiological response of the affected cells to the compound. Accordingly, such monitoring would allow either increased administration of the compound or the administration of alternative compounds to which the patient has not become resistant. Similarly, if the level of nucleic acid expression falls below a desirable level, administration of the compound could be commensurately decreased.

[0138]The nucleic acid molecules are also useful in diagnostic assays for qualitative changes in phosphodiesterase nucleic acid expression, and particularly in qualitative changes that lead to pathology. The nucleic acid molecules can be used to detect mutations in phosphodiesterase genes and gene expression products such as mRNA. The nucleic acid molecules can be used as hybridization probes to detect naturally occurring genetic mutations in the phosphodiesterase gene and thereby to determine whether a subject with the mutation is at risk for a disorder caused by the mutation. Mutations include deletion, addition, or substitution of one or more nucleotides in the gene, chromosomal rearrangement, such as inversion or transposition, modification of genomic DNA, such as aberrant methylation patterns or changes in gene copy number, such as amplification. Detection of a mutated form of the phosphodiesterase gene associated with a dysfunction provides a diagnostic tool for an active disease or susceptibility to disease when the disease results from overexpression, underexpression, or altered expression of a phosphodiesterase protein.

[0139]Individuals carrying mutations in the phosphodiesterase gene can be detected at the nucleic acid level by a variety of techniques. FIG. 3 provides information on SNPs that have been found in the gene encoding the phosphodiesterase protein of the present invention. SNPs were identified at 231 different nucleotide positions. Changes in the amino acid sequence caused by these SNPs can readily be determined using the universal genetic code and the protein sequence provided in FIG. 2 as a reference. These SNPs may also affect control/regulatory elements. The gene encoding the novel phosphodiesterase protein of the present invention is located on a genome component that has been mapped to human chromosome 11 (as indicated in FIG. 3), which is supported by multiple lines of evidence, such as STS and BAC map data. Genomic DNA can be analyzed directly or can be amplified by using PCR prior to analysis. RNA or cDNA can be used in the same way. In some uses, detection of the mutation involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g. U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al., Science 241:1077-1080 (1988); and Nakazawa et al., PNAS 91:360-364 (1994)), the latter of which can be particularly useful for detecting point mutations in the gene (see Abravaya et al., Nucleic Acids Res. 23:675-682 (1995)). This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a gene under conditions such that hybridization and amplification of the gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. Deletions and insertions can be detected by a change in size of the amplified product compared to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to normal RNA or antisense DNA sequences.

[0140]Alternatively, mutations in a phosphodiesterase gene can be directly identified, for example, by alterations in restriction enzyme digestion patterns determined by gel electrophoresis.

[0141]Further, sequence-specific ribozymes (U.S. Pat. No. 5,498,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site. Perfectly matched sequences can be distinguished from mismatched sequences by nuclease cleavage digestion assays or by differences in melting temperature.

[0142]Sequence changes at specific locations can also be assessed by nuclease protection assays such as RNase and SI protection or the chemical cleavage method. Furthermore, sequence differences between a mutant phosphodiesterase gene and a wild-type gene can be determined by direct DNA sequencing. A variety of automated sequencing procedures can be utilized when performing the diagnostic assays (Naeve, C. W., (1995) Biotechniques 19:448), including sequencing by mass spectrometry (see, e.g., PCT International Publication No. WO 94/16101; Cohen et al., Adv. Chromatogr. 36:127-162 (1996); and Griffin et al., Appl. Biochem. Biotechnol. 38:147-159 (1993)).

[0143]Other methods for detecting mutations in the gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA duplexes (Myers et al., Science 230:1242 (1985)); Cotton et al., PNAS 85:4397 (1988); Saleeba et al., Meth. Enzymol. 217:286-295 (1992)), electrophoretic mobility of mutant and wild type nucleic acid is compared (Orita et al., PNAS 86:2766 (1989); Cotton et al., Mutat. Res. 285:125-144 (1993); and Hayashi et al., Genet. Anal. Tech. Appl. 9:73-79 (1992)), and movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (Myers et al., Nature 313:495 (1985)). Examples of other techniques for detecting point mutations include selective oligonucleotide hybridization, selective amplification, and selective primer extension.

[0144]The nucleic acid molecules are also useful for testing an individual for a genotype that while not necessarily causing the disease, nevertheless affects the treatment modality. Thus, the nucleic acid molecules can be used to study the relationship between an individual's genotype and the individual's response to a compound used for treatment (pharmacogenomic relationship). Accordingly, the nucleic acid molecules described herein can be used to assess the mutation content of the phosphodiesterase gene in an individual in order to select an appropriate compound or dosage regimen for treatment. FIG. 3 provides information on SNPs that have been found in the gene encoding the phosphodiesterase protein of the present invention. SNPs were identified at 231 different nucleotide positions. Changes in the amino acid sequence caused by these SNPs can readily be determined using the universal genetic code and the protein sequence provided in FIG. 2 as a reference. These SNPs may also affect control/regulatory elements.

[0145]Thus nucleic acid molecules displaying genetic variations that affect treatment provide a diagnostic target that can be used to tailor treatment in an individual. Accordingly, the production of recombinant cells and animals containing these polymorphisms allow effective clinical design of treatment compounds and dosage regimens.

[0146]The nucleic acid molecules are thus useful as antisense constructs to control phosphodiesterase gene expression in cells, tissues, and organisms. A DNA antisense nucleic acid molecule is designed to be complementary to a region of the gene involved in transcription, preventing transcription and hence production of phosphodiesterase protein. An antisense RNA or DNA nucleic acid molecule would hybridize to the mRNA and thus block translation of mRNA into phosphodiesterase protein.

[0147]Alternatively, a class of antisense molecules can be used to inactivate mRNA in order to decrease expression of phosphodiesterase nucleic acid. Accordingly, these molecules can treat a disorder characterized by abnormal or undesired phosphodiesterase nucleic acid expression. This technique involves cleavage by means of ribozymes containing nucleotide sequences complementary to one or more regions in the mRNA that attenuate the ability of the mRNA to be translated. Possible regions include coding regions and particularly coding regions corresponding to the catalytic and other functional activities of the phosphodiesterase protein, such as substrate binding.

[0148]The nucleic acid molecules also provide vectors for gene therapy in patients containing cells that are aberrant in phosphodiesterase gene expression. Thus, recombinant cells, which include the patient's cells that have been engineered ex vivo and returned to the patient, are introduced into an individual where the cells produce the desired phosphodiesterase protein to treat the individual.

[0149]The invention also encompasses kits for detecting the presence of a phosphodiesterase nucleic acid in a biological sample. Experimental data as provided in FIG. 1 indicates that phosphodiesterase proteins of the present invention are expressed in humans in the amygdala, brain (including infant brain), uterus, testis, placenta choriocarcinomas, Hela cells, and a pooled melanocyte/fetal heart/pregnant uterus sample, as indicated by virtual northern blot analysis. PCR-based tissue screening panels also indicate expression in the brain. For example, the kit can comprise reagents such as a labeled or labelable nucleic acid or agent capable of detecting phosphodiesterase nucleic acid in a biological sample; means for determining the amount of phosphodiesterase nucleic acid in the sample; and means for comparing the amount of phosphodiesterase nucleic acid in the sample with a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect phosphodiesterase protein mRNA or DNA.

[0150]Nucleic Acid Arrays

[0151]The present invention further provides nucleic acid detection kits, such as arrays or microarrays of nucleic acid molecules that are based on the sequence information provided in FIGS. 1 and 3 (SEQ ID NOS:1 and 3).

[0152]As used herein "Arrays" or "Microarrays" refers to an array of distinct polynucleotides or oligonucleotides synthesized on a substrate, such as paper, nylon or other type of membrane, filter, chip, glass slide, or any other suitable solid support. In one embodiment, the microarray is prepared and used according to the methods described in U.S. Pat. No. 5,837,832, Chee et al., PCT application WO95/11995 (Chee et al.), Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) and Schena, M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all of which are incorporated herein in their entirety by reference. In other embodiments, such arrays are produced by the methods described by Brown et al., U.S. Pat. No. 5,807,522.

[0153]The microarray or detection kit is preferably composed of a large number of unique, single-stranded nucleic acid sequences, usually either synthetic antisense oligonucleotides or fragments of cDNAs, fixed to a solid support. The oligonucleotides are preferably about 6-60 nucleotides in length, more preferably 15-30 nucleotides in length, and most preferably about 20-25 nucleotides in length. For a certain type of microarray or detection kit, it may be preferable to use oligonucleotides that are only 7-20 nucleotides in length. The microarray or detection kit may contain oligonucleotides that cover the known 5', or 3', sequence, sequential oligonucleotides which cover the full length sequence; or unique oligonucleotides selected from particular areas along the length of the sequence. Polynucleotides used in the microarray or detection kit may be oligonucleotides that are specific to a gene or genes of interest.

[0154]In order to produce oligonucleotides to a known sequence for a microarray or detection kit, the gene(s) of interest (or an ORF identified from the contigs of the present invention) is typically examined using a computer algorithm which starts at the 5' or at the 3' end of the nucleotide sequence. Typical algorithms will then identify oligomers of defined length that are unique to the gene, have a GC content within a range suitable for hybridization, and lack predicted secondary structure that may interfere with hybridization. In certain situations it may be appropriate to use pairs of oligonucleotides on a microarray or detection kit. The "pairs" will be identical, except for one nucleotide that preferably is located in the center of the sequence. The second oligonucleotide in the pair (mismatched by one) serves as a control. The number of oligonucleotide pairs may range from two to one million. The oligomers are synthesized at designated areas on a substrate using a light-directed chemical process. The substrate may be paper, nylon or other type of membrane, filter, chip, glass slide or any other suitable solid support.

[0155]In another aspect, an oligonucleotide may be synthesized on the surface of the substrate by using a chemical coupling procedure and an ink jet application apparatus, as described in PCT application WO95/251116 (Baldeschweiler et al.) which is incorporated herein in its entirety by reference. In another aspect, a "gridded" array analogous to a dot (or slot) blot may be used to arrange and link cDNA fragments or oligonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical or chemical bonding procedures. An array, such as those described above, may be produced by hand or by using available devices (slot blot or dot blot apparatus), materials (any suitable solid support), and machines (including robotic instruments), and may contain 8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or any other number between two and one million which lends itself to the efficient use of commercially available instrumentation.

[0156]In order to conduct sample analysis using a microarray or detection kit, the RNA or DNA from a biological sample is made into hybridization probes. The mRNA is isolated, and cDNA is produced and used as a template to make antisense RNA (aRNA). The aRNA is amplified in the presence of fluorescent nucleotides, and labeled probes are incubated with the microarray or detection kit so that the probe sequences hybridize to complementary oligonucleotides of the microarray or detection kit. Incubation conditions are adjusted so that hybridization occurs with precise complementary matches or with various degrees of less complementarity. After removal of nonhybridized probes, a scanner is used to determine the levels and patterns of fluorescence. The scanned images are examined to determine degree of complementarity and the relative abundance of each oligonucleotide sequence on the microarray or detection kit. The biological samples may be obtained from any bodily fluids (such as blood, urine, saliva, phlegm, gastric juices, etc.), cultured cells, biopsies, or other tissue preparations. A detection system may be used to measure the absence, presence, and amount of hybridization for all of the distinct sequences simultaneously. This data may be used for large-scale correlation studies on the sequences, expression patterns, mutations, variants, or polymorphisms among samples.

[0157]Using such arrays, the present invention provides methods to identify the expression of the phosphodiesterase proteins/peptides of the present invention. In detail, such methods comprise incubating a test sample with one or more nucleic acid molecules and assaying for binding of the nucleic acid molecule with components within the test sample. Such assays will typically involve arrays comprising many genes, at least one of which is a gene of the present invention and or alleles of the phosphodiesterase gene of the present invention. FIG. 3 provides information on SNPs that have been found in the gene encoding the phosphodiesterase protein of the present invention. SNPs were identified at 231 different nucleotide positions. Changes in the amino acid sequence caused by these SNPs can readily be determined using the universal genetic code and the protein sequence provided in FIG. 2 as a reference. These SNPs may also affect control/regulatory elements.

[0158]Conditions for incubating a nucleic acid molecule with a test sample vary. Incubation conditions depend on the format employed in the assay, the detection methods employed, and the type and nature of the nucleic acid molecule used in the assay. One skilled in the art will recognize that any one of the commonly available hybridization, amplification or array assay formats can readily be adapted to employ the novel fragments of the Human genome disclosed herein. Examples of such assays can be found in Chard, T, An Introduction to Radioimmunoassay and Related Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques in Immunocytochemistry, Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2 (1983), Vol. 3 (1985); Tijssen, P., Practice and Theory of Enzyme Immunoassays: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985).

[0159]The test samples of the present invention include cells, protein or membrane extracts of cells. The test sample used in the above-described method will vary based on the assay format, nature of the detection method and the tissues, cells or extracts used as the sample to be assayed. Methods for preparing nucleic acid extracts or of cells are well known in the art and can be readily be adapted in order to obtain a sample that is compatible with the system utilized.

[0160]In another embodiment of the present invention, kits are provided which contain the necessary reagents to carry out the assays of the present invention.

[0161]Specifically, the invention provides a compartmentalized kit to receive, in close confinement, one or more containers which comprises: (a) a first container comprising one of the nucleic acid molecules that can bind to a fragment of the Human genome disclosed herein; and (b) one or more other containers comprising one or more of the following: wash reagents, reagents capable of detecting presence of a bound nucleic acid.

[0162]In detail, a compartmentalized kit includes any kit in which reagents are contained in separate containers. Such containers include small glass containers, plastic containers, strips of plastic, glass or paper, or arraying material such as silica. Such containers allows one to efficiently transfer reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated, and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another. Such containers will include a container which will accept the test sample, a container which contains the nucleic acid probe, containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, etc.), and containers which contain the reagents used to detect the bound probe. One skilled in the art will readily recognize that the previously unidentified phosphodiesterase gene of the present invention can be routinely identified using the sequence information disclosed herein can be readily incorporated into one of the established kit formats which are well known in the art, particularly expression arrays.

[0163]Vectors/Host Cells

[0164]The invention also provides vectors containing the nucleic acid molecules described herein. The term "vector" refers to a vehicle, preferably a nucleic acid molecule, which can transport the nucleic acid molecules. When the vector is a nucleic acid molecule, the nucleic acid molecules are covalently linked to the vector nucleic acid. With this aspect of the invention, the vector includes a plasmid, single or double stranded phage, a single or double stranded RNA or DNA viral vector, or artificial chromosome, such as a BAC, PAC, YAC, OR MAC.

[0165]A vector can be maintained in the host cell as an extrachromosomal element where it replicates and produces additional copies of the nucleic acid molecules. Alternatively, the vector may integrate into the host cell genome and produce additional copies of the nucleic acid molecules when the host cell replicates.

[0166]The invention provides vectors for the maintenance (cloning vectors) or vectors for expression (expression vectors) of the nucleic acid molecules. The vectors can function in prokaryotic or eukaryotic cells or in both (shuttle vectors).

[0167]Expression vectors contain cis-acting regulatory regions that are operably linked in the vector to the nucleic acid molecules such that transcription of the nucleic acid molecules is allowed in a host cell. The nucleic acid molecules can be introduced into the host cell with a separate nucleic acid molecule capable of affecting transcription. Thus, the second nucleic acid molecule may provide a trans-acting factor interacting with the cis-regulatory control region to allow transcription of the nucleic acid molecules from the vector. Alternatively, a trans-acting factor may be supplied by the host cell. Finally, a trans-acting factor can be produced from the vector itself. It is understood, however, that in some embodiments, transcription and/or translation of the nucleic acid molecules can occur in a cell-free system.

[0168]The regulatory sequence to which the nucleic acid molecules described herein can be operably linked include promoters for directing mRNA transcription. These include, but are not limited to, the left promoter from bacteriophage λ, the lac, TRP, and TAC promoters from E. coli, the early and late promoters from SV40, the CMV immediate early promoter, the adenovirus early and late promoters, and retrovirus long-terminal repeats.

[0169]In addition to control regions that promote transcription, expression vectors may also include regions that modulate transcription, such as repressor binding sites and enhancers. Examples include the SV40 enhancer, the cytomegalovirus immediate early enhancer, polyoma enhancer, adenovirus enhancers, and retrovirus LTR enhancers.

[0170]In addition to containing sites for transcription initiation and control, expression vectors can also contain sequences necessary for transcription termination and, in the transcribed region a ribosome binding site for translation. Other regulatory control elements for expression include initiation and termination codons as well as polyadenylation signals. The person of ordinary skill in the art would be aware of the numerous regulatory sequences that are useful in expression vectors. Such regulatory sequences are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1989).

[0171]A variety of expression vectors can be used to express a nucleic acid molecule. Such vectors include chromosomal, episomal, and virus-derived vectors, for example vectors derived from bacterial plasmids, from bacteriophage, from yeast episomes, from yeast chromosomal elements, including yeast artificial chromosomes, from viruses such as baculoviruses, papovaviruses such as SV40, Vaccinia viruses, adenoviruses, poxviruses, pseudorabies viruses, and retroviruses. Vectors may also be derived from combinations of these sources such as those derived from plasmid and bacteriophage genetic elements, e.g. cosmids and phagemids. Appropriate cloning and expression vectors for prokaryotic and eukaryotic hosts are described in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1989).

[0172]The regulatory sequence may provide constitutive expression in one or more host cells (i.e. tissue specific) or may provide for inducible expression in one or more cell types such as by temperature, nutrient additive, or exogenous factor such as a hormone or other ligand. A variety of vectors providing for constitutive and inducible expression in prokaryotic and eukaryotic hosts are well known to those of ordinary skill in the art.

[0173]The nucleic acid molecules can be inserted into the vector nucleic acid by well-known methodology. Generally, the DNA sequence that will ultimately be expressed is joined to an expression vector by cleaving the DNA sequence and the expression vector with one or more restriction enzymes and then ligating the fragments together. Procedures for restriction enzyme digestion and ligation are well known to those of ordinary skill in the art.

[0174]The vector containing the appropriate nucleic acid molecule can be introduced into an appropriate host cell for propagation or expression using well-known techniques. Bacterial cells include, but are not limited to, E. coli, Streptomyces, and Salmonella typhimurium. Eukaryotic cells include, but are not limited to, yeast, insect cells such as Drosophila, animal cells such as COS and CHO cells, and plant cells.

[0175]As described herein, it may be desirable to express the peptide as a fusion protein. Accordingly, the invention provides fusion vectors that allow for the production of the peptides. Fusion vectors can increase the expression of a recombinant protein, increase the solubility of the recombinant protein, and aid in the purification of the protein by acting for example as a ligand for affinity purification. A proteolytic cleavage site may be introduced at the junction of the fusion moiety so that the desired peptide can ultimately be separated from the fusion moiety. Proteolytic enzymes include, but are not limited to, factor Xa, thrombin, and enterokinase. Typical fusion expression vectors include pGEX (Smith et al., Gene 67:31-40 (1988)), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al., Gene 69:301-315 (1988)) and pET 11d (Studier et al., Gene Expression Technology: Methods in Enzymology 185:60-89 (1990)).

[0176]Recombinant protein expression can be maximized in host bacteria by providing a genetic background wherein the host cell has an impaired capacity to proteolytically cleave the recombinant protein. (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 119-128). Alternatively, the sequence of the nucleic acid molecule of interest can be altered to provide preferential codon usage for a specific host cell, for example E. coli. (Wada et al., Nucleic Acids Res. 20:2111-2118 (1992)).

[0177]The nucleic acid molecules can also be expressed by expression vectors that are operative in yeast. Examples of vectors for expression in yeast e.g., S. cerevisiae include pYepSec1 (Baldari, et al., EMBO J. 6:229-234 (1987)), pMFa (Kujan et al., Cell 30:933-943 (1982)), pJRY88 (Schultz et al., Gene 54:113-123 (1987)), and pYES2 (Invitrogen Corporation, San Diego, Calif.).

[0178]The nucleic acid molecules can also be expressed in insect cells using, for example, baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al., Mol. Cell. Biol. 3:2156-2165 (1983)) and the pVL series (Lucklow et al., Virology 170:31-39 (1989)).

[0179]In certain embodiments of the invention, the nucleic acid molecules described herein are expressed in mammalian cells using mammalian expression vectors. Examples of mammalian expression vectors include pCDM8 (Seed, B. Nature 329:840 (1987)) and pMT2PC (Kaufman et al., EMBO J. 6:187-195 (1987)).

[0180]The expression vectors listed herein are provided by way of example only of the well-known vectors available to those of ordinary skill in the art that would be useful to express the nucleic acid molecules. The person of ordinary skill in the art would be aware of other vectors suitable for maintenance propagation or expression of the nucleic acid molecules described herein. These are found for example in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

[0181]The invention also encompasses vectors in which the nucleic acid sequences described herein are cloned into the vector in reverse orientation, but operably linked to a regulatory sequence that permits transcription of antisense RNA. Thus, an antisense transcript can be produced to all, or to a portion, of the nucleic acid molecule sequences described herein, including both coding and non-coding regions. Expression of this antisense RNA is subject to each of the parameters described above in relation to expression of the sense RNA (regulatory sequences, constitutive or inducible expression, tissue-specific expression).

[0182]The invention also relates to recombinant host cells containing the vectors described herein. Host cells therefore include prokaryotic cells, lower eukaryotic cells such as yeast, other eukaryotic cells such as insect cells, and higher eukaryotic cells such as mammalian cells.

[0183]The recombinant host cells are prepared by introducing the vector constructs described herein into the cells by techniques readily available to the person of ordinary skill in the art. These include, but are not limited to, calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, lipofection, and other techniques such as those found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

[0184]Host cells can contain more than one vector. Thus, different nucleotide sequences can be introduced on different vectors of the same cell. Similarly, the nucleic acid molecules can be introduced either alone or with other nucleic acid molecules that are not related to the nucleic acid molecules such as those providing trans-acting factors for expression vectors. When more than one vector is introduced into a cell, the vectors can be introduced independently, co-introduced or joined to the nucleic acid molecule vector.

[0185]In the case of bacteriophage and viral vectors, these can be introduced into cells as packaged or encapsulated virus by standard procedures for infection and transduction. Viral vectors can be replication-competent or replication-defective. In the case in which viral replication is defective, replication will occur in host cells providing functions that complement the defects.

[0186]Vectors generally include selectable markers that enable the selection of the subpopulation of cells that contain the recombinant vector constructs. The marker can be contained in the same vector that contains the nucleic acid molecules described herein or may be on a separate vector. Markers include tetracycline or ampicillin-resistance genes for prokaryotic host cells and dihydrofolate reductase or neomycin resistance for eukaryotic host cells. However, any marker that provides selection for a phenotypic trait will be effective.

[0187]While the mature proteins can be produced in bacteria, yeast, mammalian cells, and other cells under the control of the appropriate regulatory sequences, cell-free transcription and translation systems can also be used to produce these proteins using RNA derived from the DNA constructs described herein.

[0188]Where secretion of the peptide is desired, which is difficult to achieve with multi-transmembrane domain containing proteins such as phosphodiesterases, appropriate secretion signals are incorporated into the vector. The signal sequence can be endogenous to the peptides or heterologous to these peptides.

[0189]Where the peptide is not secreted into the medium, which is typically the case with phosphodiesterases, the protein can be isolated from the host cell by standard disruption procedures, including freeze thaw, sonication, mechanical disruption, use of lysing agents and the like. The peptide can then be recovered and purified by well-known purification methods including ammonium sulfate precipitation, acid extraction, anion or cationic exchange chromatography, phosphocellulose chromatography, hydrophobic-interaction chromatography, affinity chromatography, hydroxylapatite chromatography, lectin chromatography, or high performance liquid chromatography.

[0190]It is also understood that depending upon the host cell in recombinant production of the peptides described herein, the peptides can have various glycosylation patterns, depending upon the cell, or maybe non-glycosylated as when produced in bacteria. In addition, the peptides may include an initial modified methionine in some cases as a result of a host-mediated process.

[0191]Uses of Vectors and Host Cells

[0192]The recombinant host cells expressing the peptides described herein have a variety of uses. First, the cells are useful for producing a phosphodiesterase protein or peptide that can be further purified to produce desired amounts of phosphodiesterase protein or fragments. Thus, host cells containing expression vectors are useful for peptide production.

[0193]Host cells are also useful for conducting cell-based assays involving the phosphodiesterase protein or phosphodiesterase protein fragments, such as those described above as well as other formats known in the art. Thus, a recombinant host cell expressing a native phosphodiesterase protein is useful for assaying compounds that stimulate or inhibit phosphodiesterase protein function.

[0194]Host cells are also useful for identifying phosphodiesterase protein mutants in which these functions are affected. If the mutants naturally occur and give rise to a pathology, host cells containing the mutations are useful to assay compounds that have a desired effect on the mutant phosphodiesterase protein (for example, stimulating or inhibiting function) which may not be indicated by their effect on the native phosphodiesterase protein.

[0195]Genetically engineered host cells can be further used to produce non-human transgenic animals. A transgenic animal is preferably a mammal, for example a rodent, such as a rat or mouse, in which one or more of the cells of the animal include a transgene. A transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal in one or more cell types or tissues of the transgenic animal. These animals are useful for studying the function of a phosphodiesterase protein and identifying and evaluating modulators of phosphodiesterase protein activity. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, and amphibians.

[0196]A transgenic animal can be produced by introducing nucleic acid into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal. Any of the phosphodiesterase protein nucleotide sequences can be introduced as a transgene into the genome of a non-human animal, such as a mouse.

[0197]Any of the regulatory or other sequences useful in expression vectors can form part of the transgenic sequence. This includes intronic sequences and polyadenylation signals, if not already included. A tissue-specific regulatory sequence(s) can be operably linked to the transgene to direct expression of the phosphodiesterase protein to particular cells.

[0198]Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No. 4,873,191 by Wagner et al. and in Hogan, B., Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence of the transgene in its genome and/or expression of transgenic mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene can further be bred to other transgenic animals carrying other transgenes. A transgenic animal also includes animals in which the entire animal or tissues in the animal have been produced using the homologously recombinant host cells described herein.

[0199]In another embodiment, transgenic non-human animals can be produced which contain selected systems that allow for regulated expression of the transgene. One example of such a system is the cre/loxP recombinase system of bacteriophage P1. For a description of the cre/loxP recombinase system, see, e.g., Lakso et al. PNAS 89:6232-6236 (1992). Another example of a recombinase system is the FLP recombinase system of S. cerevisiae (O'Gorman et al. Science 251:1351-1355 (1991). If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein is required. Such animals can be provided through the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.

[0200]Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, I. et al. Nature 385:810-813 (1997) and PCT International Publication Nos. WO 97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell, from the transgenic animal can be isolated and induced to exit the growth cycle and enter G_o phase. The quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated. The reconstructed oocyte is then cultured such that it develops to morula or blastocyst and then transferred to pseudopregnant female foster animal. The offspring born of this female foster animal will be a clone of the animal from which the cell, e.g., the somatic cell, is isolated.

[0201]Transgenic animals containing recombinant cells that express the peptides described herein are useful to conduct the assays described herein in an in vivo context. Accordingly, the various physiological factors that are present in vivo and that could effect substrate binding, phosphodiesterase protein activation, and signal transduction, may not be evident from in vitro cell-free or cell-based assays. Accordingly, it is useful to provide non-human transgenic animals to assay in vivo phosphodiesterase protein function, including substrate interaction, the effect of specific mutant phosphodiesterase proteins on phosphodiesterase protein function and substrate interaction, and the effect of chimeric phosphodiesterase proteins. It is also possible to assess the effect of null mutations, that is mutations that substantially or completely eliminate one or more phosphodiesterase protein functions.

[0202]All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described modes for carrying out the invention which are obvious to those skilled in the field of molecular biology or related fields are intended to be within the scope of the following claims.

Sequence CWU 1

514171DNAHomo sapien 1gggccggcgg gcgggcgggc ggctgcgagc atggtcctgg tgctgcacca catcctcatc 60gctgttgtcc aattcctcag gcggggccag caggtcttcc tcaagccgga cgagccgccg 120ccgccgccgc agccatgcgc cgacagcctg cagccagcct ggaccccctt gcaaaggagc 180caggaccccc agggagtaga gacgaccgac tggaggacgc cttgctgagt ctgggctctg 240tcatcgacat ttcaggcctg caacgtgctg tcaaggaggc cctgtcagct gtgctccccc 300gagtggaaac tgtctacacc tacctactgg atggtgagtc ccagctggtg tgtgaggacc 360ccccacatga gctgccccag gaggggaaag tccgggaggc tatcatctcc cagaagcggc 420tgggctgcaa tgggctgggc ttctcagacc tgccagggaa gcccttggcc aggctggtgg 480ctccactggc tcctgatacc caagtgctgg tcatgccgct agcggacaag gaggctgggg 540ccgtggcagc tgtcatcttg gtgcactgtg gccagctgag tgataatgag gaatggagcc 600tgcaggcggt ggagaagcat accctggtcg ccctgcggag ggtgcaggtc ctgcagcagc 660gcgggcccag ggaggctccc cgagccgtcc agaacccccc ggaggggacg gcggaagacc 720agaagggcgg ggcggcgtac atcgaccgcg accgcaagat cctccaactg tgcggggaac 780tctacgacct ggatgcctct tccctgcagc tcaaagtgct ccaatacctg cagcaggaga 840cccgggcatc ccgctgctgc ctcctgctgg tgtcggagga caatctccag ctttcttgca 900aggtcatcgg agacaaagtg ctcggggaag aggtcagctt tcccttgaca ggatgcctgg 960gccaggtggt ggaagacaag aagtccatcc agctgaagga cctcacctcc gaggatgtac 1020aacagctgca gagcatgttg ggctgtgagc tgcaggccat gctctgtgtc cctgtcatca 1080gccgggccac tgaccaggtg gtggccttgg cctgcgcctt caacaagcta gaaggagact 1140tgttcaccga cgaggacgag catgtgatcc agcactgctt ccactacacc agcaccgtgc 1200tcaccagcac cctggccttc cagaaggaac agaaactcaa gtgtgagtgc caggctcttc 1260tccaagtggc aaagaacctc ttcacccacc tggatgacgt ctctgtcctg ctccaggaga 1320tcatcacgga ggccagaaac ctcagcaacg cagagatctg ctctgtgttc ctgctggatc 1380agaatgagct ggtggccaag gtgttcgacg ggggcgtggt ggatgatgag agctatgaga 1440tccgcatccc ggccgatcag ggcatcgcgg gacacgtggc gaccacgggc cagatcctga 1500acatccctga cgcatatgcc catccgcttt tctaccgcgg cgtggacgac agcaccggct 1560tccgcacgcg caacatcctc tgcttcccca tcaagaacga gaaccaggag gtcatcggtg 1620tggccgagct ggtgaacaag atcaatgggc catggttcag caagttcgac gaggacctgg 1680cgacggcctt ctccatctac tgcggcatca gcatcgccca ttctctccta tacaaaaaag 1740tgaatgaggc tcagtatcgc agccacctgg ccaatgagat gatgatgtac cacatgaagg 1800tctccgacga tgagtatacc aaacttctcc atgatgggat ccagcctgtg gctgccattg 1860actccaattt tgcaagtttc acctataccc ctcgttccct gcccgaggat gacacgtcca 1920tggccatcct gagcatgctg caggacatga atttcatcaa caactacaaa attgactgcc 1980cgaccctggc ccggttctgt ttgatggtga agaagggcta ccgggatccc ccctaccaca 2040actggatgca cgccttttct gtctcccact tctgctacct gctctacaag aacctggagc 2100tcaccaacta cctcgaggac atcgagatct ttgccttgtt tatttcctgc atgtgtcatg 2160acctggacca cagaggcaca aacaactctt tccaggtggc ctcgaaatct gtgctggctg 2220cgctctacag ctctgagggc tccgtcatgg agaggcacca ctttgctcag gccattgcca 2280tcctcaacac ccacggctgc aacatctttg atcatttctc ccggaaggac tatcagcgca 2340tgctggatct gatgcgggac atcatcttgg ccacagacct ggcccaccat ctccgcatct 2400tcaaggacct ccagaagatg gctgaggtgg gctacgaccg aaacaacaag cagcaccaca 2460gacttctcct ctgcctcctc atgacctcct gtgacctctc tgaccagacc aagggctgga 2520agactacgag aaagatcgcg gagctgatct acaaagaatt cttctcccag ggagacctgg 2580agaaggccat gggcaacagg ccgatggaga tgatggaccg ggagaaggcc tatatccctg 2640agctgcaaat cagcttcatg gagcacattg caatgcccat ctacaagctg ttgcaggacc 2700tgttccccaa agcggcagag ctgtatgagc gcgtggcctc caaccgtgag cactggacca 2760aggtgtccca caagttcacc atccgcggcc tcccaagtaa caactcgctg gacttcctgg 2820atgaggagta cgaggtgcct gatctggatg gcactagggc ccccatcaat ggctgctgca 2880gccttgatgc tgagtgatcc cctccaggga cacttccctg cccaggccac ctcccacagc 2940cctccactgg tctggccaga tgcactggga acagagccac gggtcctggg tcctagacca 3000ggacttcctg tgtgaccctg gacaagtact accttcctgg gcctcagctt tctcgtctgt 3060ataatggaag caagacttcc aacctcacgg agactttgta atttgttctc tgagagcaca 3120ggggtgacca atgagcagtg ggccctactc tgcacctctg accacacctt ggcaagtctt 3180tcccaagcca ttctttgtct gagcagcttg atggtttctc cttgccccat ttctgcccca 3240ccagatcttt gctcctttcc ctttgaggac tcccaccctt tggggtctcc aggatcctca 3300tggaagggga aggtgagaca tctgagtgag cagagtgtgg catcttggaa acagtcctta 3360gttctgtggg aggactagaa acagccgcgg ggcgaaggcc ccctgaggac cactactata 3420ctgatggtgg gattgggacc tgggggatac aggggcccca ggaagaagct gccagagggg 3480cagctcagtg ctctgcagag aggggccctg gggagaagca ggatgggatt gatgggcagg 3540agggatcccc gcactgggag acaggcccag gtatgaatga gccagccatg cttcctcctg 3600cctgtgtgac gctgggcgag tctcttcccc tgtctgggcc aaacagggag cgggtaagac 3660aatccatgct ctaagatcca ttttagatca atgtctaaaa tagctctatc gctctgcgga 3720gtcccagcag aggctatgga atgtttctgc aaccctaagg cacagagagc ccaaccctga 3780gtgtctcaga ggccccctga gtgttcccct tggcctgagc cccttaccca ttcctgcagc 3840cagtgagaga cctggcctca gccctggcag ggctctctct tcaaggccat atccacctgt 3900gccctggggc ttgggagacc ccatagggcc gggactcttg ggtcagcccg gccactggct 3960tctctctttt tctccgtttc attctgtgtg cgttgtgggg tgggggaggg ggtccacctg 4020ccttaccttt ctgagttgcc tttagagaga tgcgtttttc taggactctg tgcaactgtc 4080gtatatggtc ccgtgggctg accgctttgt acatgagaat aaatctattt ctttctacca 4140gaaaaaaaaa aaaaaaaaaa aaaaaaaaaa a 41712920PRTHomo sapien 2Met Arg Arg Gln Pro Ala Ala Ser Leu Asp Pro Leu Ala Lys Glu Pro1 5 10 15Gly Pro Pro Gly Ser Arg Asp Asp Arg Leu Glu Asp Ala Leu Leu Ser20 25 30Leu Gly Ser Val Ile Asp Ile Ser Gly Leu Gln Arg Ala Val Lys Glu35 40 45Ala Leu Ser Ala Val Leu Pro Arg Val Glu Thr Val Tyr Thr Tyr Leu50 55 60Leu Asp Gly Glu Ser Gln Leu Val Cys Glu Asp Pro Pro His Glu Leu65 70 75 80Pro Gln Glu Gly Lys Val Arg Glu Ala Ile Ile Ser Gln Lys Arg Leu85 90 95Gly Cys Asn Gly Leu Gly Phe Ser Asp Leu Pro Gly Lys Pro Leu Ala100 105 110Arg Leu Val Ala Pro Leu Ala Pro Asp Thr Gln Val Leu Val Met Pro115 120 125Leu Ala Asp Lys Glu Ala Gly Ala Val Ala Ala Val Ile Leu Val His130 135 140Cys Gly Gln Leu Ser Asp Asn Glu Glu Trp Ser Leu Gln Ala Val Glu145 150 155 160Lys His Thr Leu Val Ala Leu Arg Arg Val Gln Val Leu Gln Gln Arg165 170 175Gly Pro Arg Glu Ala Pro Arg Ala Val Gln Asn Pro Pro Glu Gly Thr180 185 190Ala Glu Asp Gln Lys Gly Gly Ala Ala Tyr Ile Asp Arg Asp Arg Lys195 200 205Ile Leu Gln Leu Cys Gly Glu Leu Tyr Asp Leu Asp Ala Ser Ser Leu210 215 220Gln Leu Lys Val Leu Gln Tyr Leu Gln Gln Glu Thr Arg Ala Ser Arg225 230 235 240Cys Cys Leu Leu Leu Val Ser Glu Asp Asn Leu Gln Leu Ser Cys Lys245 250 255Val Ile Gly Asp Lys Val Leu Gly Glu Glu Val Ser Phe Pro Leu Thr260 265 270Gly Cys Leu Gly Gln Val Val Glu Asp Lys Lys Ser Ile Gln Leu Lys275 280 285Asp Leu Thr Ser Glu Asp Val Gln Gln Leu Gln Ser Met Leu Gly Cys290 295 300Glu Leu Gln Ala Met Leu Cys Val Pro Val Ile Ser Arg Ala Thr Asp305 310 315 320Gln Val Val Ala Leu Ala Cys Ala Phe Asn Lys Leu Glu Gly Asp Leu325 330 335Phe Thr Asp Glu Asp Glu His Val Ile Gln His Cys Phe His Tyr Thr340 345 350Ser Thr Val Leu Thr Ser Thr Leu Ala Phe Gln Lys Glu Gln Lys Leu355 360 365Lys Cys Glu Cys Gln Ala Leu Leu Gln Val Ala Lys Asn Leu Phe Thr370 375 380His Leu Asp Asp Val Ser Val Leu Leu Gln Glu Ile Ile Thr Glu Ala385 390 395 400Arg Asn Leu Ser Asn Ala Glu Ile Cys Ser Val Phe Leu Leu Asp Gln405 410 415Asn Glu Leu Val Ala Lys Val Phe Asp Gly Gly Val Val Asp Asp Glu420 425 430Ser Tyr Glu Ile Arg Ile Pro Ala Asp Gln Gly Ile Ala Gly His Val435 440 445Ala Thr Thr Gly Gln Ile Leu Asn Ile Pro Asp Ala Tyr Ala His Pro450 455 460Leu Phe Tyr Arg Gly Val Asp Asp Ser Thr Gly Phe Arg Thr Arg Asn465 470 475 480Ile Leu Cys Phe Pro Ile Lys Asn Glu Asn Gln Glu Val Ile Gly Val485 490 495Ala Glu Leu Val Asn Lys Ile Asn Gly Pro Trp Phe Ser Lys Phe Asp500 505 510Glu Asp Leu Ala Thr Ala Phe Ser Ile Tyr Cys Gly Ile Ser Ile Ala515 520 525His Ser Leu Leu Tyr Lys Lys Val Asn Glu Ala Gln Tyr Arg Ser His530 535 540Leu Ala Asn Glu Met Met Met Tyr His Met Lys Val Ser Asp Asp Glu545 550 555 560Tyr Thr Lys Leu Leu His Asp Gly Ile Gln Pro Val Ala Ala Ile Asp565 570 575Ser Asn Phe Ala Ser Phe Thr Tyr Thr Pro Arg Ser Leu Pro Glu Asp580 585 590Asp Thr Ser Met Ala Ile Leu Ser Met Leu Gln Asp Met Asn Phe Ile595 600 605Asn Asn Tyr Lys Ile Asp Cys Pro Thr Leu Ala Arg Phe Cys Leu Met610 615 620Val Lys Lys Gly Tyr Arg Asp Pro Pro Tyr His Asn Trp Met His Ala625 630 635 640Phe Ser Val Ser His Phe Cys Tyr Leu Leu Tyr Lys Asn Leu Glu Leu645 650 655Thr Asn Tyr Leu Glu Asp Ile Glu Ile Phe Ala Leu Phe Ile Ser Cys660 665 670Met Cys His Asp Leu Asp His Arg Gly Thr Asn Asn Ser Phe Gln Val675 680 685Ala Ser Lys Ser Val Leu Ala Ala Leu Tyr Ser Ser Glu Gly Ser Val690 695 700Met Glu Arg His His Phe Ala Gln Ala Ile Ala Ile Leu Asn Thr His705 710 715 720Gly Cys Asn Ile Phe Asp His Phe Ser Arg Lys Asp Tyr Gln Arg Met725 730 735Leu Asp Leu Met Arg Asp Ile Ile Leu Ala Thr Asp Leu Ala His His740 745 750Leu Arg Ile Phe Lys Asp Leu Gln Lys Met Ala Glu Val Gly Tyr Asp755 760 765Arg Asn Asn Lys Gln His His Arg Leu Leu Leu Cys Leu Leu Met Thr770 775 780Ser Cys Asp Leu Ser Asp Gln Thr Lys Gly Trp Lys Thr Thr Arg Lys785 790 795 800Ile Ala Glu Leu Ile Tyr Lys Glu Phe Phe Ser Gln Gly Asp Leu Glu805 810 815Lys Ala Met Gly Asn Arg Pro Met Glu Met Met Asp Arg Glu Lys Ala820 825 830Tyr Ile Pro Glu Leu Gln Ile Ser Phe Met Glu His Ile Ala Met Pro835 840 845Ile Tyr Lys Leu Leu Gln Asp Leu Phe Pro Lys Ala Ala Glu Leu Tyr850 855 860Glu Arg Val Ala Ser Asn Arg Glu His Trp Thr Lys Val Ser His Lys865 870 875 880Phe Thr Ile Arg Gly Leu Pro Ser Asn Asn Ser Leu Asp Phe Leu Asp885 890 895Glu Glu Tyr Glu Val Pro Asp Leu Asp Gly Thr Arg Ala Pro Ile Asn900 905 910Gly Cys Cys Ser Leu Asp Ala Glu915 9203111282DNAHomo sapienmisc_feature(1)...(111282)n = A,T,C or G 3acgtggatga acacccaccc acacacagct ctctaggaaa attgctcccc ttccctcctg 60ctcctcctcc accctgtcct cccaccacca cccacttcca aatgctgaga ccaaagagat 120gggctggacg gtgcctctca ccacttgtca gcctgggacg ccctcctccc tttgtgacta 180gcatgccctc ctccccctgc ccgtctgcct ccccagctct ctctgcctcc ctgtcgccct 240gccacctccc tgcgttcctg tgtatctgcc ctccacacaa gtcactctga ggcctctctt 300tgttactctt gactctgaag tggaaactgc tcctcccagc tctcctgaga ggctcaggat 360ggggacctga cctcataggg ctgatggagg catagggaca agtgaaaggg accccaggtc 420ccgaatctcc tcagctcctg tcaccttcag tccctcctat tgggttaggg gagggctgtg 480tgcctggcac catggagacc agtgtcatgg caacacagtt cgggtgggca cagcttcctc 540ctcctggggt gtggggtcca taagaggagg tgccgaggag gtgggccttg tgctggtgcc 600caccatggct gcctccagct caccattccc aggacagccc acccccatcc ccccagccaa 660ctttgcttgc cactgcagct tccagtgcca caagtcactg atcccatttg ggaaatcctc 720tctcaaacac cagctccaga gctgggcgcc agagagggca ggggcttgcc cagggtcaca 780cagcaagtct ggccaagctc ctgactctca gacctgtttt ctcctccggt ctcccacctt 840ccacccagaa aggggactgg gggcagaggg gtcagtccaa cctcagttcc caccacgatc 900ttcagccagc ccttagagtt ggcagtggga gtgaagatgc aagtgatagt gccgagaaac 960catcatgggg cgcccaccac ctgctgtgcc aaggctttgc atgtgtcatc ccattttatt 1020ccaagaccca ggaggaagat gactggtaag tggagtagct gggacaggaa cacaggtccc 1080tctcagatgg ggcaggtgag tcaaggctcg tgtgtattgc tgtctccatc aggcgctctt 1140ttaaaagaat ggcaaagctt ttaatcccat ctttattacc ggtaagagtg tagggggagg 1200atctggggca tagcctgggt ctggccttag ggtttctaga aaccagggga tatttttcta 1260agaagataga gaatagagct ttcctagtgt ggttagacct agggaagacc tttcttgcag 1320tgcagcaatg cagaccgact tccaatccct aggtcaagct ggagtctagg gacagagggg 1380aggagacccc tgcctcctgt gcccagcctc aacttgtctc ctgaccttca tggagtcacg 1440ttgcagctgc ctccctcctg gcttatgtaa taattcaaat atagcagctg cctttatccc 1500actgagtcac accccctgca tcccccctca ggtgcgggga gttatggggg aagaggtggt 1560tcagggctga gtgggaggtt cggggcctcc tggcaaggaa gatccctagt gtgctggatt 1620ggagggtggt ggtggtgagg gggctggtgc tgaggcccca agaagagcag agccttcgcc 1680agaatatgaa gccacagggg ccacttctgc cctgacccat ccctgctgga attccacatt 1740cctgggggcc ctccccagag tcacaagcta tatgtacagc cttctcttgt gggctgctgt 1800ctcagttgga ggaggaagga gaggtggaag agtatgaaga gggggaagta gtccggtggg 1860gcaatggcca ccgtctttgg tcctaggctc agcctcgccc ttcactcact gggttacctg 1920ggcacccctc tgacctcagt tttcccatct gcacagtgaa ggattagatt aactggctct 1980agcgtctcat tctctccgat ttataaccct ggagatgatc tcaacctgag gctgaaggca 2040cttccgagtg tctggcccag ccgcgttcca ggctgacttc cctccctctt ttctctgcca 2100tccctcctag accaatgcag ccacccccac ccacaagaca aaagaggcag gagagggccc 2160tggactcagc tggggctggg cggcttctcc cttccctgaa ctcgccatct gttccagccc 2220cccagccccc tgcctagcag ccatgggtag gtcactgccc tcacctgggg tcaccccttc 2280ctccccggag agctctgaca gatatcctgg aacctgaagt ggatccttca tgccccatcc 2340tgaatcccaa agccaccttc ctgaggtgtt gaagaagctg ctccaccttg gaactactat 2400aggggctgtg gtggcctttc attcctttat cagcaaaagc ttttgtcact tgtgtggtgg 2460gggacatgct tagtgtgaga atgcagagac ccatgccagg ccctacccaa ggacatggtg 2520ctccttcagc cattgtcatc agagccacag aggggagctt cctggcagag gaggagtggg 2580gagaagctgt ggaatggctc cttgagctcc ccactccacc ccttccccat gcctgggctc 2640ccattgcaaa gacccagatg tgggcttatc ctgtccccca gccagaggga gtcacccagg 2700ggtgttcagg ccaacccttt gtgaaatcca tgttccacca gttaccagcc tttctccgga 2760gagctgaggg ctgtctcaca ctgggtagtc tcagcctgcc ctggggttgg gggggtgctc 2820acagagcagt aagcgtcact gcctgcatcc ccacacacct gcattatctt gtctgcaaga 2880cacgtgtgcc cctgagctga gctctgttgt gcaccacccg atttccgtcg gcctcctttc 2940tgacttttct ccatcaacat ttcctgcttg ggcctgttgc gggctgccca aaggctgtgg 3000actggggccg aggtacatag gactttggct tgtcttttga gctaacagga tcctgtagaa 3060gaaatgagat gagcctgaga gggggtcggg gggtgagaca ttagggaagg gagaggccac 3120caagggtctc tagcccagaa tccaatgccc cttcctgcct acctgtcctt gtgggtggga 3180ggcagggtgt gtgctgactg gcccagcaat ggtgggctag gatttgggat aggcagagaa 3240aaggaagagg agggggaagt cggcctggga ggagaaacac tgtacaaagt cgaggaggag 3300agaaccagag tgtgcttagg gaccagacct ggccccacct ggagcagagg atggtgaggt 3360cagtcagggc tggatcacaa gggacctcaa atgccaggct gaggagcttg gcctttatcc 3420tgagggcact ggggagccct gcaaaggttt tgagagggaa ttccattacc agatagatgt 3480ctttggaagc cgcctctagg tgcaaggagg aggtggagta gagaggttga cctggggtaa 3540gggttggagc atgaccaggg gagggggaag gaagcagggg gtggggatgg agggagtgga 3600tggatctaag agaatctact gtcctttgga acaaacgata caggaagtgt aggagaggga 3660tggggcaagg cgactttgaa gtgtccagct cagagattgg aggtttgctg atgcctttgg 3720gaggccaagg caggcagatc acgaggtcag gagttgaaga ccagcctggc caatatggtg 3780aaaccccgtc tctactaaaa atacaaaaat tagccgggcg tggtgcgggt gcctgtagtc 3840ccagctactt aggaggctga ggcaggagaa ttgcttgaac ccgggaggca gaggttgcag 3900tgagcccaga tcgcaccact gcactcccca ctccacccct tccccatgcc tgggtgacag 3960agcgagactc cgtctcaaaa acaaaacaaa aacccaaaaa acaaaaaact aagaagtttg 4020ctgatgcctt taatagtaac aaaaggtgta ttggatgttc aatatttgag ggacctacgg 4080gttgttccca gaggagatgt ccaagagaca gcctggacac ctggagctcc agggagaggg 4140atgggcagca ggggacaccc ggagttgttg gtatgctggg agaaggctgc atgctccgtg 4200ggagtagggt ggagaatgag gagagacagg gccgccgtcc tgcaaggagc atccatattg 4260agggggcgaa gatagggtgc accagtgagg gagacagagg aggggccgtc tggaaggtgg 4320gagggaaaca gccgcgcagg acggggcggg ggcgggcgct gagaagaagc cgccttcttc 4380ggcaaagagg tagctgaagc ctgtggagcc tgcagtcctc tcaaggctat gggggcagcg 4440cggaggccgg attccagaac tgaatcttcc catcgctttg ggcagccacc ctacctccca 4500ggagcatcct tcctgccatc ccacctccag ttccccagct aacaaaaaac ggtgtttctt 4560gactcccggc agggcggcgg ggcgggcagg tcttgtgaac acggctcgca gggttcagca 4620ccctggagag aggcctgtgg ccggggcggg gcctgcggcg ggggtagggg cgcgcagtca 4680gagcagtcgg gcctttggct ccgtctggga gcggtcttgc aggcaggcaa ttggtggagg 4740agggaaaaac aatcttggat tttctccagc tctctcccct ttatgcacct cccccatccc 4800ggcactggcc tacaggagcc cctatcccag catttggggc tattactctc ctgacgactt 4860caggaaatga gatgggagga gaggggcaac tatttactgg gaacttttca gacattccca 4920aaacctcaca accttttgag cttggaattc gtgaccccat atttcagatg aggaaactaa 4980attgaagttc aggaaggtga aataccttgc ctaggcactt ggcagagctg ggatttgaat 5040tccacctgcc gggctctaag tcctgagtgc ccattagccc ttctgagtcc tgaatcttgc 5100agtttgttcc tgcagactct ccacttctgg gtggctgtgg agtctggtgt ggcagtggga 5160tggggaggag accttccctt ccacctgctt gcttgagtgt attcccagga gatttctgaa 5220gatgaggcca ccaccattgt ttctgaagtg ggagggcaga aaggaggctg agggccaggt 5280gagacctcgt cacacctgca cccatgcatg cccaggagga accctccttt gaactcttct 5340gactcagctt cttgctgcca ggttcctccg accagtgagc aggttcccag gacatgaagg 5400ggagctgtga gggagcagga cgccatggtc cagggctgca gcttcctgag cccagagaat 5460gccttcctag ctgtcaggaa tggagcagcg aggccccagt gataggtgag gtggagaagc 5520aagacatgag ttctgggctg

gctcagctgc tttacaacca gcctgggcct cgttcccttt 5580gagaaaatgg tttgcccaga gttcagagat ctaaaattct atgatgcctt ctggggccac 5640agtgggaaac aaagactcct catattttct ttcctgacac ttcccaggcc acaagacaac 5700tgctttctgc agcacccagc ctgggcaggc catctacaca agctcagtca tttctgacct 5760tgccccctcc accgtgcacc cccatgttct tcaacatggg tcaggtttct attcagcctc 5820agggacttct ctgcttgaag cctgttgtgt ggcggggagg tattctcccc acagctcaga 5880gagatggggt tgctgtggag ggtttgctgt agctcctcta ccctggaata taccctcttc 5940tgccttaaaa gacccaactt ggaccctctc ttccagaaat gcttgctaac cgccccccca 6000ccacccaaac taggtcaggg gtccctctgg gcttcacaga ccctgtgctt ctttctgtca 6060cagcctgcaa gtctcccctc cccactcccc agcccgagtg cttctctgag acaagggata 6120gtgtgagcca tgagctcagc cactggtagg ccaatgaata agtaagttaa tggtgaagcc 6180aggatccaaa tccccatttc ctgcctcaag gtgtggagct gtttctcctg catacaatag 6240tagctctgct gtgacaactc tctatctgtc ctagggccta aaatgcctct atttcactag 6300gttatagctt tatcctaggg agtcctcttt ggaagcaggg tgggggtgca acaggccttc 6360ccccatgcct gtagtctgtg agcagcgaag gccatgtggg gcaggctgtg gcctaggtct 6420ccacagatcc tggtagaagt ccatgctcac gcatcagctc caagtcccag ctaaaccaag 6480ccaccaagag gtgggccctg tgacaaggct ctgagtccaa aggccatcag taaagccccc 6540taagtcttcc gtggacccag ctccaggctg ggatgcacgc taggagatga tacacaccgg 6600gtgagggagc ccagaggaga gggcagctag ctgtgcatgg aggcctgatc tctcagactt 6660gagggcacaa gcgtgtcccc tcatcctgaa ggcttctgcg atggggcagc agagggtctg 6720ggtctgctgc ccctcaagtc cccagcccca tcctagccca tgaggattgt aaatccctcg 6780tcctctcccc tctctcctct gtcagccact cccctttccc cctaccccac tctctttcta 6840tttctgcctc tgattttttt tccttttctg cctttgttcc tctgtgtgtg tgtttctcta 6900tgcctctctg atctctttgt acttccatct tgatctcgct aaggctctga tccctctctc 6960ctctccctct tcatgtgtta ctgtccccct tcctgtctct gtttatctct cagtctctct 7020gtctgtgagt cttttttcct ctctcccagt cagactctct ctctacccct ccctctctcc 7080ctctctccct ctctgtctgg gcctctctct gttcctcctc cctcctccct cccccttctg 7140cattatcaga cctgctccaa cctcctccca gagccagccg agcagcagag gcagtggcag 7200cgggagaggc gggagcagcg gggcagcaga gctggattgg ggtgttgagt ccaggctgag 7260tagggggcag cccactgctc ttggtccctg tgcctgctgg gggtgccctg ccctgaactc 7320caggcagcgg ggacagggcg aggtgccacc ttagtctggc tggggaggcg gacgatgagg 7380agtgatgggg caggcatgcg gccactccat cctctgcagg agccagcagt acccggcagc 7440gcgaccggct gagccgtgag tatagtgagg ggctggggtg gtgagcggct gtgagaggtg 7500ccacagacag ggtcctggga gtccctccaa ggagctgggg ctggcatgga gctgagccac 7560gtggaaggat cgatcctgtt cctgggcacc cctcctcccc gcgttgccag actgcagcct 7620ggggtggggg caggttacct ctgagcagaa tgagggtgtc taacgtcaac ctagtaggtg 7680atgaggctgg ggtcccatgg aaggggctgc tggttggagg aggggctgat aatgaacctg 7740aaccgcttct tcaagggctg agggtgtatg tggggagggg gaggtctgcc aagtagttgg 7800gaggagctct cggggctgca ataggctggt tcaggaccct ggagagggag agtgtcttgg 7860cccaccaagg ctatgtgtgt gtgaaggagg tggggagggg gaaagatgga gaaaatatga 7920ataagagtgg ccctggagca agagagggtt agaggtaacc accttccatg gaattgggaa 7980ttggggttca gggacaccac tttatgaaac tttaccccaa agcgtctgtc ccaggatagg 8040gttctacgga gccagatgga atatggtgcc agcctcgtgt gtgtccacgt gcaggggggt 8100gcatgtgcaa gtgagtgggg ggcgccgtgg cgacacccct ctactaaggg ctgccgaggt 8160ggtaggcagg gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt atacatgtgg aatgtaaggg 8220acatgttggg tgtagagggg cctgtagagc tctagggtcc ttggtggttg gatgtaaagc 8280agcctgtcag agtttgtgat catccctgtg tgagtgagag tttattcgca tgtgtctgag 8340tgtgagtgca ggttggtctg catatgtatg taggtgtgtc tattaggttg agtttgtata 8400ttatgtgtgt tgtgtctgca aaatagagtg aatcagtgtg cattttttat ctgttccatg 8460tgcatttatg tgtgtgtatt tgttagtgtg tgaataatag cattgctgtg tgtggaggtg 8520gatgtggctg tgtgcgtata agtattctgg tgtgggtgtg tgatcatggt gctagtgtgt 8580atatcggtgc ttctgtggct ggtgtgtgtg tgtatctata tgtgtgtatt catctgagtg 8640tgtgtgggtg gctgtttcct tcccctggca attgaggata cagctgggac accatggccc 8700actgatgcag ggcagggagg ggctgaatgt atgaccgcct ctttgaactc aggacaattc 8760attctacacc ctgtgggaaa gatgcagaaa agaaataggc aataatgact ctgccctctg 8820gggcttccta agcttcttag acataaaata gcttgagaat aattaagcag tagagatcaa 8880cgtcatgcta acaggtgggg gtggggtggg aactgcataa gcaaaggccc tgggctgggc 8940atgtcctgga gcagtgaaga cactgtatag agtggggggc aggcaggacc cacattcaat 9000agaactttaa gatccaggac tcttaggctt tatccagaga gccctgggga gcccagaaag 9060gttttatata gcggagagac atgatcagat ttgggttcta gaaacctgcc ctgggccagg 9120catggtggct catgcctgta atcccatcac tttgggaggc agaagcaggt ggatcacttg 9180aggccaggag tttgagacga gactggccaa catggtgaaa cccagtgtct attagaaata 9240caataaaatt agctgggtgt ggtggcacac gcctgtagtc ccagctactt agaaggctga 9300ggcatgagaa tatgagaatc gcttgaactt gggaggtgga ggttgcagtg agctgagatt 9360gccttactgc actttagcct gggggtgaca aagtgagact ctgtctcaaa aaaagaaaaa 9420aaaaagaaga agaaaaataa agaaacctgc ctcggtggca ttgtctgggt tgaactggaa 9480gagagaggtg gggccaggag gctagagtgg aggccaagcc aatacagggg tcagtgagtt 9540ctggagcttt ttgagaactt gggaaaggct ggatagatga gaacagggaa gggaatgtct 9600aggtggctca ggcttggact ggggtcaggg gtgtagtgca gacatctcag taagtcagga 9660tctcatgagg gaaaaggctc atggaaggct caggaaagct gggcgtgggt gggctgaggt 9720agtgggagag atctttgtag tgtttctagc taggatgcag agggtcagag atcatggagc 9780catctcttgc cagacaggga aactgagact atggcttcat cactatcctt tggctgcaag 9840gctggggctc aacctcttca tcagacctga ccctcaatat cattctcctt caggccctgc 9900ccggaacctc ttggttgctg agcttggtca gctcagtgag ggttaattgt ctttatgctc 9960cctgcacccc caccccccgc agtcattccc cctgcccacc aagcagctcc tgccactctt 10020cctgcttccc actccagcct cctgtcccca gggactgctg atggcttggc tgggatctag 10080ccaaatggtg gggggtgggg gcgggggtgg ggggaagagc tcccagcagt cctttacccc 10140ttggtcttaa tggactggga gtctcaccct cagccatgct gctgtcaggc caggcctgcg 10200ctccccgggc ttctgctgct tgggcctatg aaatctcccg actcagcatg attccattgc 10260tgcattcatt cattcaacca ctcaacagga acttctcagt agctgcttgg tgcccacttg 10320gcttgtcacc ggggacacag agcagacact gactgagtcc ctgttctcag ggagtgccca 10380gtctgatgaa ggagaaagaa atggaaagct gcaaccctac agggtgagca gtgctgtgta 10440ggaggtgggg ggcccacagc aagcctgggc ttcagaggaa gagacatttg agccggacct 10500tgaaggatgg gtaggaatca cccaggcagg gaagagcaga gggaacagtt tgtgaaggtg 10560ggtaggaaag gcacagggct aggcacctga ctcagtgcag cctctgggtg ggagaagaca 10620gtaagggcgt ttgggtcatt ttctagcagt tgttttagta ctctctacaa cttgccctgc 10680agatctatcc agcctgctgt ttgcataccc ccggacatag gatgttcatc tcttccctcc 10740tgggcagccc ttcccttgtg gtggttatat ctgtcctggg tcttctccgc agggcccagc 10800aactccaggc tacccagcct ggccttatgt cctttctccg tcctgtgtca ctgtcccctg 10860aagtagggcc aggctggggc acaatgatcc aggagtggca agaacacatc taggcagaga 10920gtgggagaaa tgcgcagcct ttattaacaa aaatctgaga tgggtgcagg ccctgactcc 10980tctccaaaaa taatgataaa gaagcaggca tggccaaata agggagtgag gacagacagc 11040aggaagaact tcctaccaat gcagaagggc tgtgagtctc ttggttttat gagagtgggc 11100tgtacgtgtg aaagggaggg tctcagagga caagaggggg aattggaggc agaggcactg 11160tcagcctctg actctcccat aggtgagtga gtgaagtcat ccagggagag ggaacagagg 11220agggagatca ggactcatca ttcattcatt cagcagccgt tcactggccc taccaaacat 11280gacacccctg ggggcagatg gacagagcca gtgaccacgt ggatggaagc tccgagtctt 11340tcctacctgt gttaatgtcg caggaaggta tttaggagga ggggccattg gggctggcct 11400tataaggaag agccacttca ggctgagttg agggacagca ctaggaagat ggaagagcat 11460ttgcaaaggc ctcaaggtaa gggcaagcag gattttgttc acttagcact ataggagttc 11520agagtggcct aggcatgaag tgccaggctg gggggaagcc ctgggccgtg gtggagcagg 11580agaggagtgg ggaattgagc ctagactgta ggaagcactt tcttccgtga aggtgtctcc 11640aacaggcttg atgtgtaggc attattgtaa gtttgcaact tcttggtctc tcctggtgct 11700cgtgaccaga gcttgctgag ggacccagcc ttgcttgaga aaggggtgtt cagtgaacaa 11760aagagaccct ggaaatgaga gagaagcagt ggctgaagaa tgtgggcccc ttccagaaag 11820tggcgtgcaa acaaatacaa agcaatatgc aaatcagctg gctagggctt ggcagctttg 11880gttggaagaa atgagccatc accccttatt atgccggcct cctaccccct ctgccccagc 11940ctccaggaca gccggaacag ccttgtctgc tccttggagc gccccagctt ttctgagaca 12000caggattgtg gcctccaggg tggtggccgt gggctccctg tcagcaccct cgtcctcctg 12060ggaagtcgat atatttagta acagaaatgt tttcacacat ttatctccta ttgttcagct 12120gcttgctccc tgggaaaggc caggtcccca gtgatgtgac ccacttcttg aagtccctga 12180agtcaccctt ctcactgccc ccccaccccg aaaaacagga ggcaactggg gcttggtgca 12240gcagaacaga tttgagtcaa atatctggga ggacttccca acagtgtggt tgctgagatg 12300tgtggaccct ggatttctgg gctttcattc tttggatggt tgccttgggc gcagaggagg 12360ctttgaagat agagcagaga aggtggcagg caggcttatg ctcaaatttc agcatactga 12420aagatgtact gttactctgt agctgtgtgg tcctgggcaa gttacttaac ttctctgaac 12480cttgtgtgaa tagtggggtg gagataatta tcctttcttg gcaggatgat tctgaagaat 12540ctggaagtgc agagcttagc ccctggcatg cggcaggtgc tcacaaaggt tagctactgt 12600cattatgaac cacccacgat cagccacact ttcagaaaga tttagcgggg cctggagagg 12660gagagaccag agctaggagc tcagggctgt catcgtgtgg gagggaccag gaggcctgaa 12720acagagctgt ggttgtggct acggtgagaa gcacaaagct ctgtgggagg gaccgaggtt 12780tctcagagaa gtgtggccac ctcattaagt tgttctgact ggtctgagac caatccccag 12840ataatacaat ggaagaaagg gcttggtgaa gaaggggtta agtctgtggc cacacccatg 12900cagtctgtga gccattctgg gagctgtagt ctgttgtgaa tttgcagtaa gcatagtttg 12960tactgcctct tttgatccaa atccacaccc tgctgccaag gctggccgag ggccggccct 13020ggtgggtgct gggctgtgtg gagcccaaag gtgaagcagc atcgacctct tccctcaggg 13080accccctggc ttgctatgtg ttggggggtg caggtaggag cagggataga agtattaagc 13140cataattacg acttctcaca tgttcacaca gaagtttaca gcttcctgag cactgtttcc 13200acacctgtga tctcatttaa tcctcaccac aaacccaaga gactgctgtt ttctggatga 13260agaaacagag gatccaggag gggaaatcgc ttgcccacag gtattcagcc agtggagcca 13320gacctggggc acaaatctgt ctgcttccag agctcctgct ctttccatac attactgttc 13380cagatggcag acaggcaaga tgtggacaac taaagttgga tgtgagacat ctcggcagag 13440gaacagctga gcagagagct gctgattcca ggctgagagt ttggactttg tgttgtggcc 13500caccaggatc cacccaaggg ttttctgatt agagctgagc tttgagagaa ttggtcttgc 13560agcttaggct gaatggattg aactggagaa accaaagtca gactgaggct tctaaatccc 13620atccttggtg cacccagcac tttgctgctg tccctcctcc atgcttcttc tcagtttctt 13680ccttctcctc tccttcatct tcttccctca cccttttttt tttttttttt aatagagaca 13740gtgtcttgct ggctggagta cagtggtgcc ataatagctc actgcagcct caaattcctg 13800ggctgaagct atcctcctgc ctgggcctcc caaagtgctg ggattacagg tgtgagccac 13860tgcacccagc tcatcttcct ctttctctcc tactcctctc tgcctcaggc tgaggagtga 13920tgacttttat accatagagc tgtgctgtaa tatcacatgt ctccagaagg gggtgctgtc 13980acatacagtc cattccagcc tgaatcttcg ttgtgtttga agggccagta gaagtgttgg 14040acaagtggca gagatgaagg atggagagaa ggatagccca ttgttctcca cctccattga 14100gcccaggaca tgagggccct gctgaaatgg cactgggagg aatgaaggct gaggagaggt 14160tggaccccaa ccagaaggga cagacatact gagttaagcc agaggaaatt ttctcctcat 14220ggttctggga caggctaaga tttggaaatg catctagaat gacattgcag ttggggtctg 14280ggtttctttt gggtcatgac ttgcttgata ctgaggtgct ggggatattg cttgtgtctc 14340agtgtgtgta tgtgtacctg aatgtgagct tccagttgtg catatgtgta tgctgatctg 14400agagggtgag aatgtgtggg tcagtgttcg tataaaagtg tgaacatact cacatgtgtg 14460agcatgtgag tgtccttttt tttagtttag ttttgagaca gggtctcaca ctctcaccca 14520gactggagtg cagtggcgtg atctcggctc accgcaacct ccgcatccca ggctcaagct 14580attctcctgc ctcagcctcc tgagtagctg ggactacagg catgcaccac cacacctgca 14640taatttttgt atttttagta gagatggggt ttcaccatgt tggccaggct ggtcttgaac 14700tcctgacctc aaatgatcca cccaccttgg cctcccaaag tactgggatt acaggcatga 14760gccactgcac ccggctgtga ctgtccatct ttatgtctga ttttggtaaa cagttatatg 14820catgtgactg tggcttgtgt gtgtgtacat gtatgtagag tgccatatac atatgttcta 14880gtgaaaccgt atgtgtgttc cctgtgtata cagatgcctg tgtctcaatg tgagcacagg 14940gatgagggga tatgtgtgtg tgaaggccca gacacctgct gtgctaacct ttaaggccgc 15000gcctaatgtc tggctattca atactttttc tcctgggtcg cgctttcctg taggtagaga 15060cccttgaagg gctgggcttc cttcagggga ctctgggcca gagtcaggct ttgtgttcag 15120tctcaggttg ggccagccag ggtcctagtc tatcggattg ggcagctaga catggctggg 15180aagtgtctag gttccattct ccccaggaac tcttaatggt cacacttaaa gagtttcagg 15240gactcccagc acggtcctct tgtactgatg caactactga agttcagaga ggtgcagtga 15300ttaacccaag gtcacccagc aggacccagg atgagatgat agggcttgca gcagagaggg 15360gagtgtctga cctggaaggc tgccctccct ccagccccta gagcaggtgg ggagctcaga 15420ggagagccaa gtctgtggtg tgaagccacc tcctgcacct ggctatttcc atgcctcctg 15480ggcctcagag gctgcctttg aagtttttac cagagcttct gcatgctgtg agattcctcc 15540tggggacgtg tgaagtcgac tgttccatgg agcatggaga ctcgatggag aggagcccag 15600tggtgaagtg aggccagagg aggggcttcc tctggaagcc tcaatttctt ctttgcagta 15660gttgcttttt ttttcgtgtt tttttttgtt gttgtttttt aggttttcac cgttctaaca 15720ttcaaggctt tctctgttat ctctctttga gctcttagta ctgagacagt gctggggttt 15780ggggcagtcc tggaggccta tctgggctca aagtgagggt ggcagggcag tcccttaggg 15840aaagggctgc gtgggagaca gggatgagct tcctgcccat agtggggagg catgagcagg 15900ggctggacag cctggttagc aaggctgtat acaaggtacc taccctagtg aggaagttgg 15960ttgcagatta tcttgagtcc cttcaagctg tagctgccat ggggggccag agaagaacgt 16020gcctcagctc tcttgggcct ggggaggatt gagtccacag agtgctcctg gtgtcctggg 16080cagtggaagg tgcaaggtta gactgtgcac ctggaagcag agagatccca ttccctggag 16140aactgaaggg aaatttgtct tcctggaggt ttggggctgg aggcaggggc tggatgggag 16200gacactctgg ggtggagtgg gggtgggatg gggaggactg ggcaagtccg aggcggctct 16260gctgttcagc acccgcagga aggagcaggg aggcatatcc tgaatcatgc agggctctag 16320ggtgggaggc ccatggttgt ggggctcaaa catgggctct ggttggggca gaggagaggc 16380ttcctggggt tggggtctgg gcaggaattg gggtagaaaa ggagagaagc agcaattggg 16440taccacctcc ttcccaggtc aggtaattcg gagttgtctt aaaactctca gtgggccagg 16500catagtggct cgcgcctgta acccaagcac tttgggaggc tgaggtgggt ggatcacctg 16560aggttgggag ttcaagacca gcctggccaa cctggagaaa ctctgtcttt actaaaaata 16620cagaattagc tgggcgtggt ggtggatgcc tgtaatccca gctactcggg gggctgaggc 16680aggagaattg cttgaaccca ggaggcggag gttgcagtga gcctagattg tgccattgca 16740ttccagccta ggcaacaaga gcaaaactct gcctcaaaca aagaaacaaa caaaacctct 16800cagtgagggg ggatctgggg tccagatgga gagaactaat gtttacagag tgacctttaa 16860gttttaaaaa tgattattta aggaggcgat taaacaaatc gcctccttaa ataatccttc 16920cagggaggcc gggcacggtg gctcacacct gtaatcccag tactttggga ggctgaggtg 16980ggcggatcac gaggtcnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17040nnnnnnggaa aagagatatt agntggagta ttagtaagag ataaaagaga aaaacaacga 17100aaaaaaagca gagtgataga agggaaatag aataaggaag aatagattga tagtagcggc 17160ggacgaagaa aaagacgaaa aacagcgagt acggaggcgg gggcggtata atgagaaaat 17220agaagatgaa cgcgatacga aggatgaggg cggagggaaa gtacaatggt ggtggggtat 17280ggaggcgaga gtgaaaggga ggtaaatgac gcacaaaaac aaaagacgga aggggaacag 17340gaggaggggg tggtaggggg gnatcgcctc cttaaataat ccttccaggg aggccgggca 17400cggtggctca cacctgtaat cccagtactt tgggaggctg aggtgggcgg atcacgaggt 17460caggagatcg agaccatcct ggctaacacg gtgaaacccc gtctctacta aaaatacaaa 17520aaattaaccg ggcgtggtgg ggggcgcctg tagtcccagc tactcgggag gctgaggcag 17580gataatggca tgaactcagg aggcggagct tgcagtgagc cgagattgtg ccactgcact 17640ccaagcctga gggacagagc aagactccgt ctcaaaaaaa aaaaaaaaaa aaaaaaaaat 17700ccttcaaggg gctagcccta ttttgtagag gggaaacaga tgacaaactt aaatggttta 17760actgaagaca gttagtgaag caagtattat ggagatgggg aaggcttaag ggaaccagca 17820ggacatgtca agttactcag gactggcatc caaggggcca ggggcgttgg cagagggggg 17880ccgaggagag tgccccagct ccatgagcca gagccctgga gatggggctg ccctgcagga 17940gctgtggctg cagccactgc ttgtcgaagg aggcaggtgg gtgagggggt gaatacccac 18000catgagcctg catgcttctc accctttgct ctcctgccag taccctgacc ctcactggca 18060gaatttctct ggatgccagg gggcaaggga gccctggatg aagctgccac ttagaagtcg 18120gcctctgggg cacacaaccc agcagcaaaa gttagagatt ggatgtggag ggacaaagag 18180atgatgggaa accgaagaac agagagggca tggacttgcc caaggtcaca cagcctgttg 18240atatcagaat tggagtcaga agccaggctc tgcctctgaa cactcacttt tttgtttgtt 18300tggttttctt ttttttcttt cttttttttt ttttttgaga cagtcttgct ctgtcgccca 18360ggctggagtg cagtggtgcg atcttgtctc actgcaacct ccacctcctg ggttcaagtg 18420attgtcctgc ctcagcctcc caagtagctg ggatcacagg cacctgccag catgcccggc 18480taatttttgt acttttggta gagacggggt ttcaccatat tggccaggct ggtctcgaac 18540tcctgccctc aggtgatctg cccgccttga cctcccaaag tgctgggatt acaggcgtga 18600gccactgcac ctggcctgaa cactcacttt gtcacattca ctgaggtctc ctgagtggac 18660tcatatgcgc attatctact ctctggctga gagctgcttc ctgccgtgat caccgcgctc 18720tgtatctggg cagcacaggg gctgctgaag aatgtcattc tcagaacgca gtgtgccctg 18780gagcccccca agccacctgt tcattcatcc caactggcct tgagggtgcc ctggtgtgcc 18840ctgcctgtgc ttgtcaccct ggccatggag atggacccaa aagcccttgc tctccgcttc 18900attagagaca ggcacaccca gacgcaggca atcaattttg tcgggtgagt gctgggaccg 18960ctgatgagga cccttcctga ggaggcgatg ctgggtctta gccttaaaga acaactgaga 19020gttttccagg tggaggagaa aaggaagggt attccaggca aaaatcccca taagagcaaa 19080ggtgtgagca gcaagaaatc aagggtggca ggttcagggc tcctgggctg gaggaagggc 19140ctggcggtgg agaggaaggg agtgaaggcc cagctcacaa agggaagcag aggaaagttt 19200aagcagggtc aggccatggt tagctttggg gttaggaagc tccaaatgat gggtgaagta 19260ggggggctag acccaggtga gaggcagtat tgcggttggc caaggacacg tgagttgcat 19320aaatgggcca gaggaggggt gacagccgct acttcccggc tcacctgcct gagctaaggc 19380cctagttcct cagtgtctgc ccaccaatgc aggtgtgtgg cagctctaga ccctcctcta 19440gggacatccc tccctgcctc atgctgccta tggctttcac tctctggagc actcatccat 19500ggcacccata agccaccccc tcagacaatg gcccctaaag caaaactgtg tcaccgttgc 19560atatctcttg ataacactct gacccctcca ctgccaaatc tgataaaaga cctccctttg 19620aagaccttcc tcctggagtc ggatctcagt ccttcttgct gtccagagcc tgggccttgg 19680gcctccctgg gaggcgagtc agtgagggca gcccccttat ggtgctggga gttgagggac 19740cttggcccag ccaactcatc cctgttgtgt cagcctctct gggcctgggc agccaactca 19800ttttcagtgc taattagcat ctcccctgca gctttctgcc ccactctaag tgcttgacaa 19860tcattaggtg ttactgtgtg caactggatc ccagctccgg cacctccctg ccccagcttc 19920tcctccagac cccagctgcc tgagataagg gacctggcca caaacataca acacaccgaa 19980acccggacac aatctaggca tagggacttg aacaccaaca taaatataca aagaggaaaa 20040acccaataac acagaagaac tccccatacc aggaagcaga ccatagcaca gacagagacc 20100cacagtacac acacaacaca gacaccaaca gatgctgaaa agcagacaca ggatcattcc 20160aaaaagtgac ccagaaacga aaacagaaca aatgggaaca tcaatgcaca tgacacaggt 20220atacacatct agatatgcaa cacaggtacg attcagcaca tgtgtggcgc atcgcaggga 20280agcacttgca cttgaagtat acacagatgc caagatagtc agagggagcc gcctgtggtt 20340ccccacctgt gcagcgtctc tcgcctctgg gctgccgcac atgctgttgc ccggaattcc 20400cttccccaag gccctcctct ttttacctgg ctaattcctg tcattcttca gatcttctag 20460gaagacttct gcctccttga taggggcctt tccatactcc ccagccttgg agtgcttcct 20520gccacatggc atcactgact gttttccaat gagtttctgt caagttttgg gatgaaggat 20580tttgcctgtg ctcgttgagg

tggtgactgt gggtgtgagt gggtgattag ggccaaaaaa 20640accccccaaa aaactggaca gaggcaaatt tggggggaaa tgagttagga atagctgtga 20700ggagccccag ctactcaggg cctcagaaga tatttatttc tgtatttatt tatttattga 20760gacagagtct tgctctgtca cccaggctgg agggcagtgg cgctatcctg gcccactgca 20820acctccacct cccaggttca ggcgattctc cttcctcagc ctcccgagta gctgggatta 20880caggtgcgca ccaccatgcc tggctaattt ttctattttt agcagagacg gggtttcacc 20940atgttggcca ggctggtctt caacttctga gctcaggtga tcctcctgcc tcggcctccc 21000aaagtgctga gattacaggt gtgagccact gcacccgacc tcagaagaca ttgaaaccca 21060cagagaggac acagccagat gccctctgcc tcattttctc agaccctgcc tgatttctct 21120tatgtttctt ctaggcttgc tccctgaccc agttccctcc ttcccagagc tggccttgcc 21180ccttgccacc tctcggagct cacacatact cactcacctt ctctgcttgg ctgtgcccta 21240cccctacttc tacgtgcagt gaaatccttg ttattcaagg cctgaggtca gtgggcacat 21300catccatgcc tggcgtccta acccgtgcca ctgagtatcg tgaagggagg tagtggaggg 21360acgtgcttgg gagcacaagc cttgaggact gtctcttggt tcagatctct gctcctctac 21420ttcttagctg caggattgtg caagttctgc cacctttgtt ccctcatctg tagaaaggag 21480aggataatag agcccacctc attagggcag ccatgaggat taaatgagac acagtgtgta 21540atgtacctgg ctccctctcc aggttgcgtg agagcaggga gaaagctaat gagatcaagg 21600atgtgcaaat gcactcagaa ggtgcctagt gagtccttgc taactggcac ttagtgaaac 21660aaacacctcc tgtgtgagca cctaatatgt gcctctgtag tgggctctgt gacccgcccc 21720tccttagttt ctgcatggct gccagttctg cacagctgtt actgctgtgg gggcttagaa 21780ggtgggggta tgactacttt ttctgaattt atttttaatt ttttacatct gttttatgga 21840ggcataattt acatacagta aaatcaccaa tttaaagtgt ataatgagtt ttgataaata 21900tatatggtca taaccaccat gacaattaag aaaagaatat ttttatcctg gcaaacttcc 21960cttgtgccct ttgtagtcag tccctttgag ggggacttct tataggagtg tgagaagtac 22020tgggttttcc ttgggctgca aacctgggca catggagtgg gggtgcctcc aacatgctgg 22080aagttgccag ggaactgctg accctctctg ggccttggtt cctggcagag gcagtgcagc 22140caggcagggg aagggatgct taggccttgg tctcctgagg gcaagccttg gatgtgaggg 22200ttggatcagc tggaagtggt ggcttcagaa acccatagag tgggtgacag ggtagggact 22260tggtgtttcc acaaacccgc ccctcctttg accaggtgtg ccctgtggtc ctggtggaaa 22320tggctatata ttgtccagac tgtagcaggg gctggccaag atggtccact cctctcccca 22380tcctcctcca accagaggcc ataaacccca ctctatagat taacaattcc ctgaaaagaa 22440gggggtcact tttgttcccc agttctagaa ctaaatatta aagcaattat gtaactagca 22500ataaattact taaagtagtg actcactcag cttaattaga gcgcaagcaa ggagggatta 22560aggtattttt agagcacaca cctcactctc tcctgtgggg gaggcctctg tgcaaggtgg 22620gggtggaaaa aaggctggga actcatggga gcaccccagg tgtctgcaag gagatgaaag 22680ctgatcctcc gccccactga ggtcctaagg aagaaaggcc gagtcagagc tgcagcagga 22740gggattcgga tcagactcaa gaacacttcc cagtggtgct tatttgagaa ctgggacggc 22800aacactagat tgtaaactct gtgagggcag ggattaggtc tgtgaccgcc tcctcaccca 22860gcgggagacc aagaatgaga cttgggagtc agacacaact gggtgtgact cctgcctttg 22920cgggttgcca gcacgtgggc ttgggcaggt tcctttatca ccagaagctt tgccgtctcc 22980tccactataa agtgggcaca ataacatcca cctgcatgca tattataagg attgagtggg 23040ttaaaatgtg caaagcaaga ctttgtgctc agctgggcac agcggctcac acctgtaatc 23100ccagtacttt gggaggctga gacagagtgc ttcagcccag tagttttgag accagcctgg 23160gaaacatagg gagaccctgt ctcttaaaag aaaaaaaaaa ttagaagact cggtgctgac 23220tctgctagac caaaagccca caaaggcagg gattaggttt ggtttgtgtt gttcattgtt 23280gtatctcaag cttcattcat aggactgcac aaagtaggtg ttcagtaaat gctttgttgt 23340gtgactgcgt gttaattttg ttcccattct cctgctccaa aaaaaagttc attttcctga 23400ggttgtgagt gaagaaaata ggcagtgtgg gctgggtgtg gtgtctcatg cctgtaatcc 23460ccagcacttt gggaagctga ggcgggagga tcacttgagg ccaggagttc aagaccagcc 23520tgggtaactt agcgagatcc catctctact tcaaaaaaat ttaaaacaga aaaaatctag 23580ggtgtgtggg ggggcaggtg gggaggttgc aggggtgcct cacaggtggg agtctggcat 23640ttctcctcca ggctgaggag gtggtgactt ccagggaaag tcctgggagg gatcagaacc 23700acagctccag cctgcttgga taagggtggt cttctggctg ccaggagggt agctaggtgg 23760gaagatctgc ccttgtttcc tccataacct ggggtgggag gaggaggagc tcccagccca 23820atctgatggg ggagaccaga accctcaccc accattgctg gcagttcaga gaaggcagcg 23880ataagtcggg gtggggcatc ctgaaaggct tcccagagga ttggatggga ggattagctg 23940agaagacatc cggcatccgt aaaatggagt aatgattctg accctgcagg ttttctggga 24000ggattaaatg agttacattt taaagatgcc tggtacatgc ctgccaggag aaggcacaac 24060atatgaactc cctccctctt ccctccaccc ctcctcagct cctgtgacat caggagggac 24120atgccctgcc ctgctcacag aggctgggtg ggaggctccc atcatggcct tcactgaggc 24180tgcctctgca gttggaccaa gctggacaca cagtaggtgc acataacaga tgggggcagg 24240tctgtgcttg ttttaccagg gtgttgggag gctgagggaa gggcacagct ggattggggt 24300gatggagttc aatccctgct cctcccccag atccaagatc ctaagacgcc tatgtccagt 24360ggctgctctg atcagctctg accagctctc ctcacacctc ataggccttc cagggttcag 24420gtgatgaatt agtgatgaca gcatccagca tcgctatgac aaccacatgg cactcttagc 24480ctccagtcag ggctcagccg cagaggccag agaccccttt ggctctgggc ctttgtactg 24540gcgtgtgtga gcggggctgg ggcctgaggg agatggagga gtgggagggg caggggccgg 24600ggcatggggc tgcatctggc atggactgga gttcattcag attgttccat ccagagggac 24660cttggggaca gttgtttctc tccttccttc cccctttctt ttcattcctc catccctcct 24720ctttcccctc ctcccacttc ttctgagcct tgttcctgtt tgaggccctg ggctgccaac 24780ccttttcccc tcctctggga ataaagccag gctcagccct caccccgggg agctgagtga 24840ggtgggggac agccaccttc tggtctaggc ctcagggaag gtgtgtgggg accactgatg 24900gcttggtgag agggcctgac ccagctgggc caggggctgt gcaagtggct gctgaccctg 24960atgagtgggg aggaggtttt cagtagagag gcagggtcag agatgaagca gcgtgggatg 25020ggggagcgac agatgttcag agtggcctaa gtgtgagatg cggagcagag aacgtgggag 25080gaatcgaggc tcgagaagga ctgggagaga gtggatgcag tgaggagttt gaagtttgtc 25140cctgggggaa gaggagccct gaagattttt gttgttgctt ctttgatttt taaatgggag 25200ggttcatttt agagatgggg aaacaggccc agggtgggaa agtgacttgc tcaagcttaa 25260gtcactagag acagactgag agtacaggct ctgcttgggt cctgctggac tctagctggg 25320acctcttgcc ccagacttgc tggccaggat tttcccaggt aatcactacc tccgagaaag 25380gcgaggagag cccatgggtg actttgccct cagtttgaat gaaatttgca tcagcaaggg 25440ctatgccgat agtcctttct gctcgtgtct ggcctgtttg ggggtgggag tggggtggag 25500gtgagcatcc agggaaggat ctgggaagtc aggggcttgc cagggccagc aaggcattag 25560ggtcagagat ggattcaaac ttgggtcttt ggagacccag cccagactct gtgctccatc 25620tccttcctcc gtctctcagg agcctttggc tgagttaggc acctacagga ggcaagggcc 25680cccccgagcc cctcacattc tcctcagggc tccttctggc cctggggcct gatattgggc 25740ctgctgtgct ggaacttatc caggcagaat aaacctttag ccccattgtc ctgatgaaga 25800aactgaggtc ccgaggtaac agtgactcat tcagggttac aacaggtcag tggctgggct 25860gggcctagcg tctggccctc agcttgtcta catggccccc ctcgtggctc tccccttgcc 25920tctcgcaccc cactgtgcag catggttggg cctgccagcc ttgatggatg gctctgcagc 25980tcaacctccc tcccattcct ctccagatgc cgggccgtga gcctcctaat caccagtcct 26040gcctggtggc cgccaagcca tccatctccc cacacagcct tgcccagcac aggtgatttt 26100gtttggggag aaggggggca cagcaggtct tcctctgagg ctgagccaag agtttggctg 26160cagcccccac tctggggtgc ccgagggtta gggaatagcc tgcactccct tgctggagtg 26220tcagaaatcc ctcctgaatc tccctagggc acgtgcacat gcacacacag gcacacacac 26280tcacagtaac actaataaaa gctctcgtgt agcaaaagaa tattgtatgg caagtattgt 26340tgcagagcca tatgtatcat ctcattcatc actccactgt agagatacag aaactcaggc 26400tcagagaggt taagtgactt gcataggctc catatccagg aaatggagga gctgggattt 26460gaacccacat ccttatggct cacatcttgc attcacaact cctgctctac tgactcacct 26520gtgcacacac acacacatgc acacacacac acgtgcgtgc acacacacac acaggcactc 26580acttgcatgc atgagcacga gccaccattt tggctcttgt accatccatc tacctgggcc 26640aggttcttga ggagtgagga gaatgctggg ctgcagaggg catgaggggt cactgctcat 26700tgtccccagg ctgccccaag ctggctgtgg cactggctgg ctggggagct gcagggaggc 26760agcagcctcc aggcagtgga aaggggaggc tgggagacag tcgatcgatc atccctgcag 26820tgcctccttc caggaactgg ggcccagggg agtgtggcgc cacgggtcga tgttctgggc 26880agcagcacag tctctgagtg cgtacagggt gtgtgtgggg cgaggctggt gtgcagctgc 26940ccgccttccc ctggctccct tcccctgctc ctgccttcct cctgccattc acctgccagc 27000cccacacctt cccctgattc ccccactgtc cccaacctgg gcactacaga ggctgagaat 27060caaactccca gttcccaggc acctgtgtgc ctgctgctac catcccgccc tgccctagag 27120gcaggtctcg ggtgggtgct gcaagagtca ccctatggtg gttggggatg ggtgggtagg 27180gggaccgggg gctggagctg tggggatgtg aggcaagccc acctcagagc ctttggagac 27240ctcgacagac aatacgatga gttaagaaat gtaaaggggc acatagtggg tgctgaattc 27300atcttgtctc gttcctccag taagagtctg gagaaaccaa gagcagctgg gtgcctctga 27360gggcacagga gctcccaggg ctggctggca ggtgcagcta acagtgttag caatcccaag 27420gacaggtagc ttggggcgga ggacagcatg ctgtcaccca tcctgatgag gggagagatg 27480tctggtgcta ggagcagtgg tggccggagg agggctgggg accctcccca ggccacccca 27540cactctccct ctgggagggg ctcctgagca ggcctggtca ccttgcttct tggctgcttc 27600ttccccggcg gaggagcctc ccccaggctc tcccacctgc actggcctca agagagctgg 27660gattgagccc cagttcaggc acctgctggc tggcggaggt tagggcaaat cactttcctc 27720agccctctca tccgtgacag gctctggtga gggttaaatg agatgttgcc cgtcaagtgc 27780ctgccacttc cctgacgccg agcagctagg ctgctctggg ttctctagca cctgcctccc 27840ctggtcccag cactgggtgg gcggctgtgt tctaccggtc actggtgggt cctcagggcc 27900ccgacacagg gcctgctatt gggaaagagg gaagtaaaca tcccagggct ggagctctgc 27960ccactatgga ggtgttccat cttaggctct gtaatctcct cattcactct ggtatgggga 28020caaatgtgcc tctctgcact aactgagccc ccatgggcaa ctaggagtgg tgtcacttgg 28080ggtggaggtg ggcaaggatc tctggactgg gatttccaag ccctgacttc ctgttatttc 28140aggcactacc tcattgttcc atcttgggca agacctgtcc ccttgagggt aagagacaca 28200tgtgacctct gacctccaga gtctctcttc tgagcttctg tgcccagatg attctgtgtt 28260ctaggggaca ggcgaggctg gggggtgacc cccatgccac tgatgggcag actaaggagc 28320aggggcccag gactggggcc agctcaggac tctggtggcc tcggtgccct tgacctggta 28380ttgctgccgt tttgccccac tgctgtctgt ctccgcgtcc gagtcaccac ctgtccctct 28440ccagtcctct cctctcttcc tttattacta tctctatatt gcctcctgcc tcaggcttat 28500ctcctcctgt catgcctcta tccacctctg tcactcccct gcgactctgc ctcactccct 28560ggcacaccct ctccctccct gggagtcggg agtggagcct cgctgggaat caggaccccc 28620ctgcctctgg tctctgtcta agcagtctct gcgattctgg ccagctctta tctttttcca 28680ccttcccgaa tctctcttgc tgtctgatgg tgtctctgcc tttcactgtc tctgaactcc 28740ctttgttttt ctctatatgc ttctctctgc tcttatctct gggcctctgt ctctcagggc 28800ctgactggtc ttgacctctt tgcctccttc ttcccctcga gagcccagcc aggcagcagg 28860tccagccctc cagcccagag aacagatgga gtccaccctc cctctctctt gctggctgcc 28920tcggaagccc caaacaatgg cctccgccct gcaccgtgcc ttgttgctag gccttgggct 28980ggcagcacct ggcttccata gcgacgggtg cttagaaaca gaatgccaca tctcccagtc 29040ccaccacagg agcctttgcc gattgagcga gtgccttttg atcaatcagg aagtgtggcc 29100aggctctagg ttgcctccaa cttgaggagg caagagagga ggggactgtg gtctctgcct 29160tctggagctg gggggactgc tgggctggga ggagttgctc aagtacagcc ctgaagccaa 29220ggaaggactg ggggaggccc tgggctcttt tccccaagtc agcctgctgc aagaggcaca 29280agcttgggag ctggaagggg ctgtgttgaa attgctgttc catcatttct agctgcatga 29340ctttggatga atgacctcag gtcccagggc ctcagtttca tcaactgtaa aattgggcta 29400ataatatcat gaagattaaa tgagagaata gatctggcac ttagtaggtg gtcatcaatg 29460gccattcccc tcccttcccc tttaaagttg tttaaaattt aattgacaga gaggagaagg 29520agggttcttc aggcctgtgg aatggtgtaa gcaaaggggt ggaggctggc atgcacctca 29580catatgctgg agtatttagg gaggaccagg ggccatatct ggaaatggtt ctgccagaag 29640cagccaggcc aagctgggtg ccatgtcatg cacctgtaat tccagctact agggaggctg 29700aggcaggagg atcacttgag ccctggagtt ccagatcagc ctgggcaaca tagtgagacc 29760ccatctcaaa aaaacaaaac acaacaggca ggctgatggg cccatggaga agggactctg 29820tctcctggga ggtatattct tgccaggtgc aaagggatgg gcttgactaa tttctcctct 29880agcatttggg gctgctgggt agggagctac attggggtcc ccttgcttat tctcatgctg 29940ctccctactt ctgccctgtc acttggtccc aggagagggg ctcccactgg ttccttttcc 30000ctgccaggcc tgcccaccaa ggccaccatg gccacacagc ctgaatcctg gggccagcaa 30060gtgtccatgg aaggccccac tctgtcatcg tagagatcag gaaacaggct cagaagtagg 30120agggcttcct ggtcctaggg cccagctctt ccctcttttc aggcctgtct tctgcactaa 30180ggacttcagg ccaccaggga aggtggggag ggaggaaagg agatgagata gacttgggcg 30240ggggcctgag gacagagttt catgtcactt gggcagccag gaaagggtta aagatccctt 30300atcccaagcc atgggcactg gcactgccag aggatgctga ggcctgctgg ggcataagga 30360caacaagcaa catccttttc tgagctgttg ggagtgccaa gctctctgtt aaatactttt 30420gagcctcttc tcatgtattc acagccacct ttcaaggaag gccagttgat ccccagttta 30480gaagtgagaa aacggggtct ccaggaggca cttgtctaag gtgacacagc tggagagttg 30540gagatggtgg ttagaccgag tcaccccccc agaccctggc ctctccctgc gtgccccttc 30600caggacaccc atcactccct tgacacccct tgggagtggg tgttcatttc cttgggctct 30660cccaatccca gtccttggta tccccaactg caggcagaca caggtgcttg ctgctgtgcc 30720ctccccttta cctggcatca cagagactca agcccactga ccattaggct ctcaggggca 30780tagaaaccag gtgctggagt cttagagtcc tgcaatcagg catctcaggc agtcaggaca 30840ttagaatgtt agaatcttgg gcttctacat tctcaagacc ccaggttctc gcattcacag 30900aatgtaagaa aaacagactt tttgaatgat ggggtgttat aacagaagct ttgattttct 30960aagaacatga agctctggga gttcttggag ccttgaagcc atagactggg gcctccctgt 31020gtgatggttt ctgagttagc agggagtgtt cagagtatgg ggccttggtc cctgttgctt 31080agaccttctt gccttggtat ctctgatggg ctcagctctt agtagccttt gtgtatgtgt 31140gtgtgtatgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt agtggggact 31200ggggtcaggg gtcagggact gactctaacc tgaggcaccc ctggagtggg gccagcccag 31260gaatagcagg tggaggaaag ccgggcagcc tcagggctgc agctgtctgg tggtacaggg 31320cagggctctg ggtggctgcc tttggcagag gaccagcctg cctccttcgt cccctaccca 31380gcctgctacc aggatcagga ggaggcatct ccatgggact cctagggctg gagtcagagc 31440agcccctcca ggttctgcag cctggacggt aggaggtgcc actaagggga ggagattggg 31500gaaggattgg gacctttatc tgcggtgagg tggggcacgg ggggatgaga gatatagtgg 31560gagtctttga agggtgtggg atcagtgaag gggctgggga tttagtgatg ggctggggct 31620taggatggag ccaagggctc tgtgggtggg agaccttttg agagggtgga gactcagaga 31680gaaggatggg ggctcagcaa ggggatgtgg ctcagtggag gttgctgaag agtttcttgg 31740ggttggctac acgcggtggc tcacgcctgt aatcccagca ctttgggagg ccaaggcgga 31800tggatcactt gaggtcagga cttcaagacc agcctggcca acatggtgaa accctgcctc 31860taccaaaaaa tacaaatatt agccgggcgt aatggcaggc gcctgtaatc tcagctactc 31920gggaggctga ggcaggagaa ttgcttgaac ctgagaggcg gaggttgcag tgagtcgaga 31980ttgtaccact gcattccagc cctgggcgac agagcaagac tccatctaaa aaaaaaaaaa 32040aaaaaaagtc tcagggctgt ctctgcactg ctccaggttc ctgaggacgg cggttggggc 32100tgggggagtc ttctgtccct ggggtaggct gagaagcaag agctcctttt cccaactctg 32160cccaaagctg gaaaggttgt tagagctgct aagaaagctg gcatctgcct ctccttttgc 32220tcatcttcct ttctggtttc catgggaatc tgtggctcag gatgatcagg ggttgacagg 32280atggcgctgt ggaaggagtc tgtgtcaggc acagccatcc cacatgggaa ggagccggct 32340ggtaagaaag tgagttccct gtccctggga gtgtgcaagc agggtagggg ctgaatggct 32400agagtgactc cagaaagggg ttcagatggg gcagaggaag cagtctggag gccacttccc 32460tgagacaatc atgttttgtg tgattggctc tgggggcccc accagcccca ccttccagac 32520gtccctgggc ctcacaaagg gggttgctgc accctaggca ctgcctctga tccagcccca 32580actcctgtgc tctgtgcctg gcctatgctg aacacggaca tgtgcagctg aatcagattc 32640agtctctgcc tagaggagcc ccagtctgat gggggaggca cacagggaca caaatatagc 32700tgggtaagtc ctacaaaagg gggcatacct ggctgggagg cagttccatc actgattcct 32760gtagtctgta gatgtctttt tgagcaattc ttctgggtca agacttgttc ttatttgctg 32820ggataaaaca gcagtgagca aaacagagct gacagcatgg tgggaaggtt gagctcttcc 32880agaccgtgat gagaagtatt ggtgagtggt ggggagagtg gccagaaggc agagtgtggg 32940cgcagcatga gaggaggctt tgtccagact taaggacctg gaaggccttg aaggccagga 33000ccagggctcc aattgtcctg ctggcaatag gaagccatat gggtgggggt gaggcagaat 33060cagatttagg tgtggaaaag atgactccag ccagtgtggg catcgaagag gaggcacaga 33120agcaggcgtg gccacctgtg cctctgtgta ggagctgtgt gagcatgtgc ttgaggatgt 33180gtgtctgtgt agaggactgg ggtgtaggcg tgataggaac atggacgtgt atctatggaa 33240agactccaat tgtgcatagg ggtgtatgtg tgtaagattc tgtggcccag ggcagcctgt 33300gaaaaggaag gatcttgggg tctctggatg atggggagca gagactaagg cctaaggtat 33360gctggggctc gagccccctg gactttatcc cctgtgagct ggcaggtctt agactagtcc 33420tggactagaa tcctatgggt tcccttcccc cagagggtca tggggccagc catctgctgc 33480agacaagaca aacatgcatg caaatcacat gaaaatggat gaggcctgtg gctgacccac 33540cctacagccc ccatcccctg ggcctgagtt cactcagcct gtacccttcc tgacccagag 33600ctgctgccag ggctctggga acaggccttg cccactagga gctgaaattc acattgtccc 33660cagcacctgc ccgtggccac atcctctctc tgtgagggct acccccacat ctggagccat 33720agccagcgga cacagagctg gatctggact ggtggccatg ggcagcacct ctggcaggtg 33780ctgaggtgga ggaggcagta tccaggcagg catccctggg cagaaggtac ctctcctgag 33840cagacaggcc tacccaggca ccaggcccaa agataggggc aagggctaga tcctggtatt 33900ggaggaccct caggagaggc tgtgtgtgac ttgctctctc tctgacctgg gctagagcat 33960aaacacgtgt cacatacttg cacacacatt cacacgtgaa agcacgcaca tgctattcct 34020ggacacttgt gtacacacac cactgcacac atatacctgc atgtgtgaat atacactcac 34080ttctgcacac agacacatgc ctatctgcat agacacaccc gtgccaaccc ctatagatac 34140acagacatat ctgtgtatac acatataagt tcagctatac cactgcagta tcacacaccc 34200tcacaaggat acaaacctgt gctcacactc tcttccaccc tcacacacat catgcttaca 34260agcctgtgtg cagccttaca cacatgcaca cacgtacaga gcagcctaag ggtggctcac 34320ccctgcccag gtgaacacct gtgcccactc cagggctgga gtgttgagga aagggtctgg 34380atggaggcag aacctgcaga gatgtcagtt tcttccagga agcatcttgg attgtccctt 34440cacagagccc ttggaagtgg ggccctcttt tagtccatgg gctctagccc aggtcacaga 34500gagagcaagt cacacacagc ctctcctgag ggtcctccaa taccaggatc cagcccttgt 34560ccatatcttt gggcctggtg cctgcgtagg accatctgct caggagaggt accttctgcc 34620cagggaggcc tgcctggata ctgcctcctc cacctcagct tcctgagcac tcaaagagaa 34680gcaggccaag cttcacggct gctgagaagt ctgagaccag ggagggccaa agccttgcct 34740gaggtcaccc agcatgtcag ggaagggcta gggtttgaac ctgggcttcc aggtgggggt 34800gtaaccatgg tccatggcaa caggatagat gcatgtcagg cagcagacag gcccttggaa 34860gcaagacatg tggtcatggg ggataggaaa agacttacag tctatggaga tctgccagga 34920ccaagtgtgt gagatggaga gatggtgctt cttcaccaga gctcactggg caccacaggg 34980ctcccagctt ggctggacca tggggactca gggaagaatc agacaggccc tgctcttgag 35040ggagggctgg ggataggtga agaaggaaga gggcattata gactggggag atggtggggg 35100ctacttctcg ttggatggca gttttcttcc tgcatcttga aagatctaac tttcaaattt 35160ctttaccctc aaaactcggc atggagtaca ttctcagtaa atatttatgg catgaatgaa 35220ttaatgaaag tatgatattg gcaggcagat atgcctttgg aagggtattc aaaatgggag 35280ggcaacaggt tgggcaaagg caaagaggtg gaagaaaagc cagaggttca gggtacagct 35340gagtcaggca tggctggacg ggaagtggta ggagaagcag caggaaaaag tcacgtgggg 35400atgagccttg catcttatac tgagtttgga tgttgccttg gaggccatgg ggagcccagt 35460gaagattatg agcagagggt gaacatggtc agagtgaacc tgccctggct ttggggggtc 35520ctgggctaca tagtagctgc ttatccttgg tgcaaagagc actgggtttg gagtctatag 35580gccagggttc acattcctat agtaaccagc tgtgccatct caggtaagca tctacatttc 35640tctgagcctc actttcctta

tttgtaaaat ggggctaatg ccgtgcctcc tgaggctgtt 35700ggatctggcc tgggtgagga aatgctttgc cagcacaagg ccctaccaat gagaggtgtc 35760atttttatta ggaacaaggc agggctggtt cctagacagg gcctgaggtt gagtgggccc 35820aggacccagg ctgacagctg agtcaccttt tccaggccaa gtggcctcta aggtgggaag 35880acaaaaagag ttggctagag gggctgggct atgcattcct aagctggagc tgggaggaaa 35940gctggggctg ggactgggct tcctggtgtc cgagatgggc agagggtgca gacaccggga 36000tagtaggacc ctcagccact gcattcttgg ggacaaaaga ggagctggga aatctgattt 36060ccttacctgg ctttgctcaa gaagcaagga atgtatttaa ggcacagact ggagtgagat 36120ggcctgggtt tgaattttga ctacttacaa gctatgtgac tgtgggcagt ttactttgtg 36180cctgagtttt ccttatctgt gaagtgtgac taataataga tcccacccta taacattgtt 36240gagaagatga aatgtgaggc acacagtatg tgctcaataa atgcgaaagc ctcccagccc 36300cagatgtata cactcggcca gtaggggcca gccctggccc tcacctccat gggacagagg 36360tcagccaggg aggagatgca tctactccag ggttctctga cctggcagca aattagaatc 36420accgggggac attcacaaac atctgggatg ggggttccag atatcagtat ttaaaatgct 36480cccaggcaat tctaacatga gtcagggtga gaacccagaa caggatcaca gattgtgcag 36540ttggagtgag gtagggatct gcgtgtgagt ggaggagtcc ttggagtggg gtcactccta 36600gctataagag ctcggcaagg cctttaaatg tgccaactca aggagccttg gttgccccct 36660caggaagggt gctggttggg gaatttcaag gattgtgtga gagggttttt ctgaaagggc 36720tctgcactct accaagcact ggaagaaagc agtgcacttg tttattgagt ctagtgtaat 36780aacatttcac agatggggaa atagaggcct agagaggtgc tgtggcctgc tcagaatccc 36840acagcaagtc tatggcacag ttaggactca aaccctctga ggaatgcttg gatctgaaag 36900gttgacacag aaagactctt tgagctgagg gacacataga gcacacacca gggaccccag 36960tcattgagct gtagtttgag agattcaagt aagactgaag aaataacttc ttggctgggt 37020gcagtggctc acacctgtaa tcccaacact ttgggaggct gaggtgggtg gatcatgagg 37080tcaagagatc gagaccatcc tggccaacat ggcgaaatcc catctgtact aaaaatataa 37140aaattagctg ggcatggtgg tgcatgcctg tagtcccatc tactcgggag gctgaggcag 37200gagaattgct tgaacccggg aggcggaggt tgcagtgagc tgagatcgcg ccactgcgct 37260ccagcctggt gacagagcga gactccgtct caaaaaaata aaataaaata aaataaaata 37320aaataaaata aaataaaata aaataaataa aataacttct caagaggtga gtgccatgga 37380ggtggtgcct ggagttggga gcccaagaga tggtggcggt gccaggccag ggtcggctgt 37440tgaccatggt ctgaggtggc ctcccctgaa gaacaagtaa ctctggccag tggctgtaac 37500agatacctcc cgggcacctg tatctcaccc agccttgtcc agagcccagg actgagccag 37560tgacacatgc tcagaattta ccaagagact tgtgcactga gctcagactc agacctagtc 37620cttccaacag cccttacatg ggtcatcccc ttttacggaa gagaaaactg aggccaaaaa 37680taggaaggga ggccctgtgg gggccagaac ctttacacat cttagcccag gtaatttttt 37740ctacagtgtt aataagtagg atgaattgcc cctgtttgga agattcagta aaatacattg 37800acttggccca gatcacttac tctacacctc tcctaagtcc ccagatgtga ctcccaggaa 37860agacacaaaa aagggctacc cagagggata agatagtaac cagggaagcc ctcccagagg 37920aggtgggcct tcaaatggcc cctaaatgac aggcaggagg gaaggatctg ggagggtatt 37980gggggtgggg tggcatgggc aaaggcctgg aggtgagagt cagtcagtca ttgatgtgag 38040aagagcaaga agtagaaatg taaggaatgg tggggagggg agtcagagct ggatgaccaa 38100gcaagggttc agctgtagag ggtctggccc gccaggctca gggctcgggc tttattgtgc 38160tggtggtagg gagccactga gggtgagtgg gggagagcat gccagagcat gcctcagaaa 38220gaaaggtggg agaaacgctg gcatggaggg ccgccccctg agttggtggg gtggccgggc 38280tctgccaagg ctatgtgcca gctgcctgga ctgtgtccag gaatgggcac aatgactcaa 38340cattgagaaa atcactcccc agggagaaag ggccctgatg aatcacccag ctgaggtggg 38400gaggctggga ggctgggagg ctgggaggct gggagctcac tgagtcaccg tccaagagtt 38460ggtgaggagg ggagctgcag agagaggggc cggcagtgca gttgacgggg ggattcaggt 38520cagaccacat tgagggctgt cgggggactc taccttcccg ccattcccgg gtttggtcct 38580cctggccgtc ctgtgaggga gatgagaaaa ctgaggccca ggaagtgggg ggaggggatc 38640cgagcaaggt catgcggcaa gtcgctggca aaggcctagc gagacccaag cgcaccctcc 38700agtccagaca cgtcctgccg ccccagccgc tttcatgcca agcagaggcc taagaaccgg 38760gtcggtccgg gcagggagct gaccccggtg acccgctgaa tccccggacg cggcccctcc 38820gggcagccgg caactgaggc cggattgcgc cgccgcgatg ggacggcagg gggcgcagga 38880gcgtcgcggc tgccgcaggc tcctgaaccc agaagccgct ctgcggagaa acgcgctccc 38940ggagcgcggg tcccaccgcg gaactgcgga ccgtgtggcc ctggggcctg caccctctcc 39000ggctccgggg acggcgacag agacctgccc acccaggcct gggggcccca gtcagtggcg 39060gccgccgtgt gtgcgctcgg tgtctgttcg cacgtgtctc cctcgcagat gggcgactgc 39120tccagggcct gtccgtctca cagcgacctc caacattctc ccgacttccc cctgcctcct 39180aggctgaggg agaggagcaa gcccgaggct cctgcggtgt ccgcggcccc tgcccccctt 39240ccccttccct ccccacccca ccccactgcg ccggtctctg cctggggctc tggccgggcc 39300ccggacccca gagtggtggc ggggaaacag ggtgcgatca gacagggtgg aggctctgag 39360agcggcccct gcgagatgcg agagaagtgg cgacggggcg aggggcagcg agcgcaggct 39420gacagcaggc cagctggaag ggccgaggga acccagggcg agacagaagc ggggtgacag 39480cggccgggtg tccggtgggg tcggaggatc cgacgggccg agaggtgcgg tccgcggtgg 39540cggggacata ggcggggccg gggcgggccg ggggcgggcg gggggcgggc cggggcgggg 39600ccggggcgga cactcgggcg gaccaggcga agctgtcgcg gacgcgctga ccgagcgcag 39660cggccgggcc ggcgggcggg cgggcggctg cgagcatggt cctggtgctg caccacatcc 39720tcatcgctgt tgtccaattc ctcaggcggg gccagcaggt cttcctcaag ccggacgagc 39780cgccgccgcc gccgcagcca tgcgccgaca gcctgcaggt aggggggccc ccgcgctggg 39840caccaggaga acggggtgtc cggcgagcgc cgggccgggt ctgcccgccc ccgtaaccct 39900tctcagggta ggagacccct cctctagttc tgaattctac tcctgtgctg ggacggcagc 39960gcagaccaag agcccttgaa gccccagctc tcagtccaca cgtcacccca gactctgaac 40020tcctttcgga tccggggctc caccccaagc actgagcttc cagtccacgg tgggaccgca 40080gtgcacactg agagctgtgc ccaagcctcg aattcccttt ccttagatta gtggggaccc 40140tgcccacgcc tcggaacctt caccatatat gtggggctcc cggcacacct ggagcacctg 40200aacccccagc tgtcatccag gactccacct cagagccggc ctcacccaaa gccccaaacc 40260tccattccaa gcctcaactg gacacccgct cagattccca cccaaacatc tggacttcag 40320tcctcagcct ggaacctacc ccagagtcca aatctctcct tccatcaggg cttcactggc 40380tttctctgtg ggacccactc cccgatccct tccctccctc ctgtgttgga gatctccgag 40440tctttcctcg tggggggccc ctcctcttgt tcctctccag gtacagtggt cccactttat 40500tctctgggct tctcctctgg tttctcttca agtatttctg ggctctctaa tttggtctgt 40560tgccccatgt gcccacctct cttggtctat cttggtctct ctcctgtttc tctaggtctc 40620catcttcgtt ttggggtctc tttctgcagc caccccttcc tcttgtatct acctctgctt 40680tgtggtgagg agggggcagg ctcagagagc agggctagtg tccctgggac acccccgccc 40740cccatgttct caggcatggc atggtgtggg ctcaggtgga agggcctaaa tgtggagtgt 40800gctgccctca ggcgatgccc agggatctga ggtggtgggg ggatgatgtg gtgggcactg 40860gcttttgtaa cttataaagc ccctcatccc agctgccctg gtcttgacgg gggcggctag 40920ggcttgagat agggaagagt aaactgcaat ctggtgtcaa cctgcggtgg gatgtgtcca 40980ggctgggtgg gtctatagtg tatgtgtttg tgtgagtgtt cctgtctgtg tgtagcacgg 41040gctgggttgc atgtgttggg tgtgtcttgt gtgtaatcat gtgtgttgtg ccgtgtatga 41100atgtgtcatc gagagtggga ttatttgtgg ggagattatg ggaattatgg gtctatggca 41160ttgtgtgcta tgtgtggctg gggaggcagt gttgtggctg tggagtggta gctgggtgtg 41220tggctgttgt gtgtgtagag aacttgtgtg tatgtggctg tatgtctgtt attgtacagt 41280ggagttgttg tagggacagc ttgcatacag gattctatat gtagttgtgt gtgttactgg 41340ctgttgtgtg tggccaggaa gggccactgc aggggcctga tggtttccac tgggtgtctt 41400gtcagagagg agttggggca gggggtgccg tgtgtgccaa tgtgtttgca gcctaggtgg 41460ctggcttaga gtcactatgg cacatcctgg gattgcttgg gtaatatatc tattaggacc 41520tgagtgctgg tgtttgaatg tcatgtgtct gtgtggtggc tgctcccgcg attctggaca 41580ggaaagggtt gcagccaggg ctgaggggtc tgaggtgagg agccagttga caagtgtgtg 41640agtgtgtgag tgtgtgtgtg tgcgtgcatg tacacgtgca tatgggaatg gggtggggtg 41700ggaggaggca gtgggccagc agcgctgtct atgctgaggg gctgtgtgtg cccacaaacg 41760tgtgacatta ggtgtgcaca ttatctatgc aggttgtgtc tgcatgtgtc tctgtgtcta 41820ggtggcgtgc gtattgaatt taattggatg catacacctg tggctgggga ggtgagaggt 41880gtgtgaggtg cgtggtggga gacggtgtga gtgtggtgtg aagtgagggt gtgtgagctg 41940ggtgactttt tggtgtgacg tgtgaattat gtgatctttt ctccccatga gctgtgtgtg 42000cctgtggtga ggagtgagtg gaggatggcc agtgagctgg cggtgtgtgt gttgggggtg 42060ttgaggactg tagaatgtgc tgcggtggca gtgtgtgcat gaggtgtgtg tgaggaatga 42120ggtctgtaac atttggggcg tgtggaatat agtgggtgtc cccataaatg tctgtggagt 42180gacgcatgtg tgcaaaaggg cttggctgcc atcctgttct tgctcccctc ctgatcaggt 42240ccctagagat gccctggaat gttctccatg cccccccaac cccagctgcc cctacccttt 42300gcccttcatc ctccttgcct tgaccaagcc ctttgttttg ggtttccggc ggagcaggcg 42360ctggacaggc gggcggcagg caatgtcgtg gtctgagaac ctttgttctc ttagtttgac 42420tggtgtttgg ggccttggtt tggaggaggg tgtggagagg atgcacgtgg cagcaaggtc 42480actgtgttta ctacaccact tcgtgctccg cagaggggag gcgtacggcg caggcagtga 42540ggcctgggtg gtgtctttgg tggcgcctgt tggtgtaaga acagcttagg ctgggcttgg 42600agtttgccag ccatgcagtc ttagtccata gtggcccagc gcccttcctg gctcatgtca 42660gcggggctga gcagccgagc agccaagcac tcacttctcc aagttcacct gccctcgccc 42720cttctctgtg tggctgcagc ccctggagac aaccaggaag acctcgattt agttctattt 42780gtgttcactc caggtcagat ggaggagaaa gagtccccat cctcacagag acacttatct 42840gaaaggagag agctggtcac acctttgggg accctctaga ctgacgcagt ctgtaggggg 42900atcgaggtca taccttccag agagagctgt gggaaaaccc tactgggctg cctcccagca 42960ggtgcttgag agaagaaaca tccaaggttc cttgagattg gaaggcttag agaagtctga 43020gtcagtcagg gaaggggctg gggtcgatgc cgcagtgtca cataccagaa ggttctctga 43080aatgaatagg cttgaactgg accttgaagg gggtgttggg gtgggcagag aaatgcagcc 43140tggggctgag gaaggttctg gcctgactgg caaaagggat cttgctggcc attccccagg 43200caacactgtc tggctttggg tagccatccc tgggcctcca gccttctcaa gctttcacgg 43260tacctttttt atcccattgt ctctggctgg aattatcttc attgtcgttg acattgtcat 43320cttcatcatc ttttaggcag ttatttccaa attccagggt cctttcacaa atgtctcatt 43380tagcaggtta actcatgcaa ttgtccaaaa gtctttatgg aacactgctg tgtaccaggc 43440aggcacagtt ttaagtgccg gggtcatggt ggtggccaaa ctggcctcat ggagctccta 43500ccttctgtgt ccagccatgc tgtcagttgc ccacccttct gtgtttcccc cagtctgggg 43560cgcctggttc tgtggggctc cgcatgtgca ccctctggtg ctggggtctg gctcctacca 43620gaatgtgagc tctgcagagg ctgggcccgg gtctctcctc tacccaccgt gtgtgtcctg 43680agctgggtct ggcagagtcc agatgctcac acctatcatc aagtgactgg aactgccatg 43740tagggttggc agtccagctc tgtctaggga aactggggtc catcggatga ggggactctc 43800atctcatcag gcagcatctc atcaggccct tcttttacca gtagctccag agaccaagaa 43860gggtcaggtg actggtgcag gtctcacagc agggtggcgc ggctggtgtc agaagacagc 43920acttcttgct gccaggttgg gctctggtcc tagcaccatg ctgctctctg gctggcctct 43980gtgctgcctg cggcgggtaa acgattatta atgacccccc tggcaaggag acaggaaatg 44040tttcccagcc acagctgggg acctgctccc tgccagcccc agctatccat acccgtcctg 44100accatggcat cggtgctgat gttatcttca ttctgcctca gtcttcttta ctccttctgc 44160ccatcccccg acctccctga tcttgacatc ctaagggtaa atgacgagaa gctacagagc 44220tttcttttcc atatccctgt ccctcaccac tttctccaac ctgactcatc tctaccttct 44280tccttgtccc atgccagcca gaagtagctc ttcctccaag aaggcttctc tgaatgcaca 44340gccagctcct ccagtgtcca ctaccctgag cccgaggcac tgagtccccc tcattgcaga 44400ctctagtcct ccactgatgg tttgctctga cagccctagg gctggcctgg gcacttcccg 44460cagactgtcc ctaattgctg ccttaggact gacatatgaa gggtcctccc agatgctacc 44520tccaggaagt ctccagttcc actcagccca gggattctta cctcctttga gcacagtgcc 44580cttcctgtct gagtcacaca tgtgtacttc caggacttcc taggtggaca ttagtgaaca 44640cctgctatgt gcccagcaca gagggtggga agagatgagc aggatgcagg ccttaaaatc 44700cccacacctt cctccaagcc tgagcaatgt tgcatcagcc ccttggcagg tggcacagac 44760ctaggtacta gggctggggg aggggaggcg gagaaacagg gagtgatgtt ggtagagtgt 44820gtggggaggg caacgaggga gataaactca ggggccatgg tgatataaag cagggacccc 44880tatttcagcc cagagtggga gggaggggca tgttggggag cttccaggag gaaacaaatc 44940tgaactgaga gctaaggtca agccaggaga aagttctgga cagaggggag agaatggtta 45000ctgtgaaggt tcgctggtgg cagacagagg gaggagagcc tgtggcagca ccactccatg 45060gagcaggccc ctgggtgcca gccggctggg tccgggggga tggggactgg taaagctggc 45120ccagccagat ggtgcaggac ttgtaagcca tgttaaggac tgcggactta ttctggaggg 45180aaattgaccc tggggaagag ttgagagaac ggatatgaca gatcagatct gcatgttcaa 45240tagctccctg gaccatggtg tggagactga aggggaggct ggtgtggtcc aggtaagcgg 45300gggtgatgag gcctggacag ggaaatggct gaagaatgga ggggagggga cggagtggcc 45360agggctggtg gagggagctg gacagctgta gacgtgaagg gcaagggagg agaatgctgc 45420cccacccagg tgtctggatg ggttttgtgc agtctctgag atgtatagga gggaagacag 45480gggttagtgg cagatgcctg ggcctgtgtc agggcccttt aaggaccaaa aggtcttgga 45540aaagcctcag aggagatcat gagctttgag attaaaggga gacctgaagc cggcccaggg 45600ctgctacagc ctcacctgta acatgggaac ttgagatctg ccctgggcaa agggtgttca 45660gaattcaata atcaaaacaa tctgtgaaat gtaatactta ataaaattca aatccaaaaa 45720tgtctgagta cattccaaaa tgagtaaaaa tgtaaattta tgaaaatgct aaacatgcgt 45780gattgttcta atgtaaattg taagcctcag ctgcttccca gaactttgga tctggctccc 45840ttgaagctgc tgcctctgat gtggctgccc cctgcagctc caggaccttc ctgttcagct 45900cccttgagag tagccggcag ggcccctcct ctgcagagcc tgtactctgg ctggtggctt 45960cagggggcag gcattctgcc tttcctgtct cccaccctaa gggagttggc cttgcatgcc 46020tcccatccac ggttgcctct actgggggct gccactggga gacaggaagg gcatgggagt 46080ttcgggagct cagggtaaga ggggctgaga tctcgtggtg tggaggggga gcgggaaggt 46140cgggtggccg aaagaatgga gagggccggg agtgagagca aagggagaca ggcagagctg 46200aagagcagta tcgccccaac atcaatactg gtatttcaga atgggaaagc tgttccattt 46260cccgaaatat cagaatgctg aggtccgatc ttgcagtctc tgagctgggc attccttggc 46320ccccactctc gggtattctt gcacaagacc atttttctgg gctgcatttt ctcacttgta 46380aaaggaggaa gttgggggtc aatatctcca agcgatatat gagctctagc tctaggagta 46440taggattttg agaatctgga attgttagtc tgtggggttc taactgggac aattctagca 46500ttccttgact ctcagctccc agccagggct gtgtggatgc gtggttgtgt gattccgaca 46560ttctgagact ttaagatgct gaggctctag gagctagaga tacggacatt ctgtgaatct 46620aggattctag gatttgatgg tttgatgatt caatgattct aaatggggct gctgggaaga 46680gctgcaacca cctgccttgt taatgtcaat gttcagttat taaaaacata acaagaagca 46740atggagacag atagctcaga atggtgggcg ctccctccac tcccagtgag ggaggacaga 46800agaggctggg ctggccttag agaatagaga ccttttcaac ctgggtcaca caggttgttt 46860ctcctgtcac aacagaactg gtgtgtgtac attcgagaga gcttccactc ccaaagcttg 46920cagggtaagg ggctcatttc cttcagcact ggcctctatt ccttaaccat ttcagactgg 46980gcagagagag gggtaactac cctttcctcc cagccctcga agtctctggg cagaaatggc 47040agcagtggag gaaggagagg tctgctcacc cccgcccctt ccctgacagc ctgaggggga 47100aaacaggaca tgaatacttc ctggacacag acatggaaat gcatgaaccc ctgccttcga 47160gggccccgcg tccaaaggct cagacaaggg cagaggccag gacagccagt ggggtcccat 47220cagcaccctc tcagtatagg ctgaggaggg aagaccctgt tcttgcccca agggtgacag 47280tgagaagggg tcaaggaaag gagtcccagg tcagggactg gaagtgctga caggtcctcc 47340cctgtgtgca aggccacagt ccagcctggc agaaggccag cccaattgtc cagtgtttca 47400ctgcctcctg agtccttctt atgccttggc acccaggcca gagttgggga ggggtccagg 47460ctgcagggga gggtttcctt ccagagtgcc catccctgat ggatccttag aagcccagta 47520cagctgcaca gttccaaggg cttccgctgc ctggtaggtt cacagaccaa agctggccct 47580ggtcacacag cacaacgggg cctgaaatca ggcttcctga ttcccagtcc tgggtgttcc 47640tttttgccca cagcctcccc cacttcccct gggacacctg aggggcagga gtggaggtgg 47700ggctcaggtt agggagcaga gcctctgtcc atcatccctc cgtcttcctc ttcccacagg 47760ccagaagcag gtgtggtggt gacagctgcc cccagtcctc cacaaggctc cattgtcccc 47820ggcagggagc ccctccccag ctgcaggcca gaagtgtgcc tccccgggcc ctcctgtcgt 47880gactctgcca cccgcttcct cctgctgccc cttccctctt ctcatctccg cttgccctca 47940ggccctcccc atccccgtga ggtctcgtct ctggcgctct ctgggtttaa gcctctctcc 48000agtgaaagtt agatttggaa gggccctggg agatcaccaa gtccaaccct tttattcttc 48060ggataaggag gccaggtcag agaggggaag gtcctgtcca aagctgcaca gtaggctgag 48120gcagagccca gtgctgtgct cccttcagcg ctgggtcatg ggtgcacact gcccttggca 48180tcaggcgtcc agggtttgag aactgactgt gatgatcagc gctaagcaca caggcaccta 48240cagaaatgcg gtagggggct tctctcctca gcccttcttc acagccctga gctgccctcc 48300cttcctcttc tttgcccagc tcctctctcc ttcactatcc ctgctgtctg ctgactcctg 48360cctctggcag acactgtcct tgggacacag actagagctc aggcctccag gactgggatg 48420cacacccatg cacccagaca cagacacata aacatgtgca agcgtgtcac ggggtccata 48480aatcccagct gaaaactggt cagaccatca ggaggccacc ctggaaccca gtgtcctcct 48540cttcctgtca ggcctcacac acctcctcca ggaagcccct taggacccct gaagaccatc 48600ttcatccaac tagccccttt gtgacaactg aactctgtga gcctaggttc ctcctgtgac 48660tcgaagggca aggctgagtc cccccttcag tcctggggcc actccttcag tgtcttcagg 48720aggggctcag cttcctgttg ctgggtgggg agagccctga ggtccccaca ggacgtggga 48780caatggggag gcggtgacag atgagaggct gagtcttccc taaagcagac tccaccctcc 48840cctgacctcc ctggctggtg gcttggacac agccctggcc tggactaggg tcctggtctg 48900accccacaat gcagaggtct gggaatcaga agccctggtt ctccagcagc agttctctaa 48960ctggcggcta tggagtccag gcctccaggg cactggtagg ttattggcgg gttggtgcag 49020attccagtgt ccaggagggg tgagctggcc tggggggcct atgtacagga gataggaggg 49080tgataaacac aggctaggtg ggattacagg gagctgggaa tacctagcta agaatcccct 49140catcctaggc actttcccca cacttgaaat tggctggagg gggaaccaga agttaggtgg 49200ggttggggag ggacaggagc cagcaccctg cctccacctc cgggcagtgc ctctgctggg 49260gggagggaac ctgtcctggg ggtggtggga ggtgtgaggg gggagctgga ttctccagtg 49320aaactggccc tccctcctct caggggaggg gagggggctg tccctggctg ctcagcaggt 49380agcccatctg gctgtgggtg gaaaagaaga ctcaggcttt gtggataaaa gggacagccc 49440tgggtcaggc acttatctca accctcgtca tttcctctgc cggacatgac tgggtgagtg 49500gggtcattgc acagagggaa ggaacaggcc agggccagtg cataccaggc cctacaggag 49560agtcaggcac atgggtgacc ctgccacacc ctgggctgca gtcagcccct catagaggcc 49620cagacacaca ccacagtcac tgccggagat ggccacacct agaccatcac accacacaca 49680gacccagtct ctccaggtga cactcaggcc cagctgcagg cgcagctaag agggaagacc 49740ctgcagggca cagggacacg tgggacaacc agacgccctg cttcggccac accacaagcc 49800tccacacacc aggtgcagct cctgtcaccc ctacggtcaa cccaaggaga gccagagatt 49860ccagtagtcg tgggcaggta tccagtgccc aggcgagaag agggggacac cagcagggaa 49920cccagaacct cctccatgcc agactgtgcc ctccccccag ctcacagaag gagtgcctca 49980ggctgtttat ttcctagcag ggactagcag ggatgggtgt ctcatccccc tccccctccc 50040agtccccacc acacgattct gaagctgcca aatcaaatca gcccctgcac ccgcgccagg 50100ctggcatggc ggccagcagc tgacgggaac gaagccaggc tcagaatatc ccaccgcctg 50160tccgatgcct gagtaggctt gttgggtggg ggtggggagg ggcaggagcc tggcagccag 50220gccctgggca gtgcccctca gagaggctgg gggtttggaa tgctgcaggg tggtgggctt 50280ctggagaatg agtgagcagg tctctgttgt gtctccaggc tgctgtggca gtgtctccac 50340cgctagcatt ccgggaactg tggaagtggt gctggtagga tacaggtcgg gggtctgatc 50400ccagtccaga tgactgggcg ccaggctggg gtaggggggc tcccacatgg tctcacattc 50460atttgagact cacagcaccc aggttggaag ccccttggtt gtctgtcagt aaaggcccaa 50520ctcactgtgg aggcccagtg actgtgtgag gtggacatta cggatcccat tttacagaca 50580gagaaactga ggcttagaga gggctagtag agctccctgg agagaagcag aagtggagga 50640ggcctcagaa agagtaaaga ggtggtcatt tccactcctt aggagcccta ggtggaaaga 50700aggaatatgg ctctgttctc

agagtcaagg aacagagaat atggcagagc cagaggtgcc 50760catgggaagc agagaacaag gagggagtct tgggagagag cagggtgcaa gcaggcaagg 50820ctccctggag gagggggcca tccgtgggct tgctgggggc taatgggagg acagtctggg 50880gagaagggga gaaggcctgg ccggcctcag cccctgacct tcttgtctct gcagccagcc 50940tggaccccct tgcaaaggag ccaggacccc cagggagtag agacgaccga ctggaggtga 51000gagctcagtg gagggagaag tgggtgggct tgagggggtg gggcgcagac tgaagatcag 51060tctgagtggt gccctccccc ttgggaggac ggggaggctg gagtcacatc ccagccccag 51120ccctccagac taggaccacc cctatatcaa gaccatctcc cctcacccta tatatcccca 51180gcctggaagt cctcccatga ggattcctcc tcccaactca cctggggagt cactacagac 51240tcctcccttg tcctccccac cctcacccaa caattcccgt tgattctctg ccctgagtat 51300ttcccgagtt cctctcctct ccattctgcc gcctgcttgg gtccaggctc cctcaactct 51360cccctgggcc actcactggc tgcttgcttt cagtttcccc catcatccac gtggccacca 51420ggaggatctt tctaatgcac agacctgaac ttgtcactct cttgccccag aatcctccat 51480gctccccacc cccatgcccc ctccacaacc ccagcctggc aatcgttccc cttcatccat 51540tcctgcctcc cccaaactgc tcctgctggc ctcttcccca ctcagcttcc taaatccttc 51600ggggatcagc tctagcctcc tttcctctgg gaagtcctct ctaccccttg accatgggac 51660caagctcact cctgctccct cctctgagct ctcctgccct ggcagtcaga tgccagggcg 51720ctctgctgtc tgtcgcccac tgtgctgtgc cacgagcacc ctgttttctc catcatgtga 51780ctctgtatgt gtgtctgcct tgtcttctct gcactgtgag ctctttgagc ctcgggactg 51840tgctttcttc attcctgaac cttctaccac ccttggatgg gtaccggtgc agggctcagc 51900cagcgcattt cctgccctgc gaggggtgcc atccccaccc cccgaccatg ccttccttcc 51960ctgtgagggg tgcctcatag gactcttcag tgctcaaagg ggccttgacg agcaaacaag 52020gtgggctgct gatgttgaag atcggcacag aggagggtgt gtgtgtgtgt gagagagaga 52080gagagaactg gacccacagc cagaacagag tctgcccagg cctggctgag agggagagga 52140agatgatgct tgtatcagcc ctcctgtgtg ccaggagcct ttgacaccca ccttgtttaa 52200ttattacagc acccccatga ggtaggggct gctattattc ctatttcaca tttggggaag 52260ctgaggccca gagggatcat tcagcaagtg agttgggaca gagctaagat tggagcctag 52320atgtgtctca ggctcgaggc tcactctttc ccggcccctg agtaagatgg gaaagaaggt 52380gcccacacag ggcctggtgc acaggagggg ctcagcacag gttccctgct gggacacagg 52440gccaagacct gagaatgtgc ctccaagtgg ggctgggccc tgctgctggg agctggcaaa 52500gggagctggg aggggagggc ctggaaagcc acattattaa tttatttact gccatggcat 52560tccccatggg gcggggctcc ccccagagct gggacagatg gtgttcctgg gagcctgcag 52620tgtctcagca gcctcggcca cccgccagga aagactggat ttgtcatcca cccagggagc 52680cacaagaaga gggggctttg gcaaagctga gaccctcctg ggcaacgggg actgtgccct 52740gagggaagga gtatggctcc aggcaccctg ctatgcctct ggggcagccc ccgctgccta 52800ggccatctgc ctgccctctg caggttcaag ttctgctctt tgtccagctc caccggcctc 52860gtccttccca tgaggcttcc ctgggtcggc cccacctgct cctatccctg tattttctct 52920gcctttcctt gagctgggtc ctgctgcctc ttccctctga ccgaggatct ggagccatga 52980gctcctcagc cctcagctct gtcctgaccc catccccaca ctcatcccca aaacagttag 53040tgtctgcctg gactcttggc agggcctgct ggatttctgg gtcctgccag caccccaccc 53100gagtgcccag gcctatactc agcactgctg ggaagagatg ggctgcctga ggggacgctg 53160ccaacatgga gagggcaaga ctggagagag tggggacccg agggcattgc tcagaccaca 53220ggggcagctg gagggaaaag ggactgggag cctgaggggc cctcctgtca gggtggatct 53280gggaagccaa gatggcctca tatagtggac aagccacagg gtcagatgag cacgggttca 53340agtcccaact cccttgcttc ctaggtgtgt ggccttgtgc ctgtcactta accagcctga 53400gcatcagtct cctcacctgc caggcgggat aagaacgtct atcactgccg ggagcggtgg 53460ctcacgcttg taatctcagc actttgggag gccaaggcag gtggatcaca aggtcaggag 53520atcgagacca ttctggttaa cagggtgaaa cctgtctcta ctaaaaatac aaaaaattag 53580ccggttgtgg tggtgggcgc ctgtagtccc agctacttgg gaggctgagg caggagaatg 53640gtgtgaaccc gggaggcaga gcttgcagtg agccgagatc gcgccactgc acttcagcct 53700gggtgacaga gtgagactcc atctcaaaaa aaaaaaaaaa gaacctctat cattcttgga 53760tgtaatcact gttattcaac attaccacaa tagagctgtt gggagaagtt acaaagactg 53820tatgtgtggg gtgcccggcg caggcctggc acatggcaga tccttgggga gagttagcct 53880cctctctgtt tccctcaagg atgacatcct tagagccagg actaggctgt acccctgtga 53940gacaggatgc tctgcagagc tgggctgagg cttatggaag ttctatgggc atggcacact 54000ctcctggcac tggctgggca gcagccaaga aagcagagct gccagcaccc atccccaccc 54060agcaggcgtg tgttcagcac accctcctgg gatggttacc tagcccctgt gccagcagct 54120gacttggagg aggggctctt ccagctcagc ctggcatcct ccttcagggc caggcctctg 54180catcattact gtctctctga aagtcaggtc tggggcagtt caagttggtg aattgagcat 54240gctgagtcaa tgccctcttt gtgatggctc tcagggccca gatggcggct tgggagcctt 54300agctgggatg ggggcatggg gagaggcgga cgtggatgag ggcactgaca tccacaataa 54360gtactgaaat gcactgccca acaccggctc ctctattgct gcccttggga caaagaccac 54420accccttggc agggcattgc tggccttgcc tgctgggtcc cctcatgtcc ccttgtgtcc 54480ccttatgccc tgagacagcc agcgctacag ccacattgtt gtgttcactc ccagcacaca 54540gcagctcccc ctgcctccct gcctttgctc acactgacca cctgtctgga atacctttcc 54600tttctttctc cacctactct cttttcaagg cccagatgaa atgtcacctc ctttgtgacg 54660ttcctcagac tggtccctct acctcaggcc gagccagtct cctcccttcc ctgggcactc 54720acagtcccca tttccctgag cccacagttg ggaaacctgt taccccacgg ggtgctgtgg 54780gtagtgtatc cttccccatg gggttgtaaa cacccaggag gcagaggctg agactgagtc 54840tcctttgtct ctcttgggcc catgtggtgc ttggtatagg cctggtatat ggtaggtgct 54900caataaatac ttcttgaatg aacaagagtg gctgtgagta gggctggagt agttccaaga 54960aggggcacag ttgggttggg cggtcttgga gacttggagg aggcaacctt agaactttga 55020aggatggaga gggtcaaggg caccaaccga agaagccagg gaccagctag gcagtcagag 55080aggtccatga ggtcagcttc tgacagcagc agctaaggac aaccaggacc agaacaggac 55140tgggaaaaag cagatagagg aggctggagc aaggactcag ccccagagga ggctgcagga 55200ggttggctca tgctcagaac ccggctccaa aacactctgc ccatgagtgc tgggctgagg 55260aaggcttggt gccagagtca gggtgaggct gaggccacca gtgaatatgt gggcccagct 55320gcgggggtag cactaggcag gggcgggagc caggttggag ggggtattgc cattgccgct 55380gcaggtggag tagggcttcg ctggggaagg agcagcttgt gcgagagtgt gggcaggagt 55440gggaggggag aaggctccga gtatacgagc atagcttacc agcaagtcct ggggtgaggc 55500tggaggggcc gcgctgtagg cagcactttt caggccctta tctaacattc tcaagtgagt 55560gctcctagct gccagatgtg ctacttcctc ctggattctg cacatcagga gccagtggcc 55620tctacaatgc cccatggccc caagggagtg gctgccaaca agttggcctt agcatctggc 55680atccatgggg gtcctgaggc cctgccatct gtctgtgccc ctgttgggct gcacaggccc 55740ggggcgtgca gggacctggg accagggagg cggtctcagc tgccactcta gcctgtctct 55800ctgcctgccc atccactgtc cacacccctg gctgactgag taaagagaga gatgggcatc 55860gcaggtcctg ccatcaaaga agcctagtct aaaggaggag gcataaagca ccggggactt 55920atacccagag aagacacatg ctgagaccac gccaggctcg cgggcaaggc ctaggcccag 55980ggagggccag cctcgtcaag ggcctggagt tgagactcag ggaaaggcag gagctggctt 56040agaggcgcag gcaggtccaa ggcagtgccc aggccagatg cggcggcccc gggctgaggt 56100tgctccagcc ggccccaccc cccaccgtcc tgcctggcct ttggctgtaa acactgagag 56160aacaagttcc gtttcccggg aaatatttat ctcaggctgt gtgaagagcg tgtgcactgg 56220cctccgtgtg tccttcctgc agaccggctg gggcaggagg agagggagct tggcagcgcc 56280cttgctgggg ggagtctgtg gggctaggag ggaagggtgt gccagaggcc cctgcctaga 56340gcctgaattt gagtgctggc tgagggagag gtgggagcag atgggagaga agcctgtttt 56400ctccaaaccc cacaaatgcc ctccgcctct ctcatgttcc tttcttcttc ctggtccatc 56460ctgtctcctc caggttccgg cctccagcct ggtgtcccct cctcaggctg ccttttcctc 56520ctcctcctcc ctgtttcctg gctcttagcc gctccatctg ggaagtcttc ctcaacttta 56580aaccctcgaa cccttgtcct ctgccctcca tctcccactc ctcaggcttt cagcagcttc 56640acgtggagca ttgggctggt cctgtccaca gttgttcagt tgctgtaaca gcttgtgcag 56700gctgccctgg agccctgttc tgggaagcac aggtctgggc accctggggc tggggcgagg 56760cccggagctg atctcctctg tccatcccag tagagccagc accagtgcag acacatgggg 56820gatccaggtt ggtggaccag gggaggatgg aaagtcccat ggatccagcc ggaatgttgg 56880agtggggagg cagagggccc agggttcctg ctggccagcc tctgggctta ggggtgtgta 56940tcccagacag gccaggcctg ccaggggccc tgacaacagg aaatccttga aggaacaagc 57000agaggctgag gactctgagc acaacaacag gaaacagccg tgacatgggg caacagccct 57060ggcgactgtg cccagttggg gtggggacga ggggccaagc ttgtgggacc cagggtgatg 57120ccaagaggga cactgagaca ctgtgggaca gggggcgttc tgcacatgtg acacggagct 57180tatgacgtgt aatatcaagt acgtgaccat gatcataggg tactgtgtgg agtgtgggtg 57240agtcactgag tatgtgacac tggctgtgag gcactccatg atagcagatg tgtacagtgg 57300ctgtgccacc aagtgtgtaa cactgtgtga tattgattgt gtgatgctga caccgagtgt 57360gtgacattgc acattgcatg ctaccacgtg tgtgacactg aaagtgacag tgagcacatg 57420gagggtgtgt ctccatgaga atcaaataca gaaacgtgag caaatgacgc tgcagtagca 57480ggtatggtcc tgagtctgtg gctcgagtgt ctgacactga attgtgacat tgagtgtgtc 57540ccaagcatat gatctagtga ggctgagtgt gtaaacaaag gcatgacatg gagtgatagc 57600aagtgtgtgg aagtgggtgt gtgatgctgt gtgatcttgg gcctgacatt acatgtgtga 57660tgctctgtaa tggttgtaac agtatgcaat gtgcacatac agtgctgtgt aggacactgt 57720catgggaagg caccgatggg ttcaggcggg aaagtaacac cgtccaaagg atggttttaa 57780aagattgctc tggccggatg cagtggctca cacctataat cccagcactt tgggaggctg 57840agctgggtgg atcacctgag gtcaggagtt caagaccagt ctggtgaaac cccatctcta 57900ctaaaaatac aaaaattagc caggcatggt gacaggcgcc tgtaatctca gctgctcggg 57960aggttgagac aggagaatca cttgaaccca gggggcagag gttgcagtga gccaagattg 58020agccattgca ctccagcctg ggtgacgagt gaaataccat ctcaaaaaaa aaaaaaagaa 58080aaagattgct caggttgcag aatatgtatg tgtgcgagtg tgcatggtgc gtggcagggg 58140aggggagata agttaggggg aggcagagag aaggtgggta gagcaactgg aggctcctgc 58200agctgcccag gcaggagatg gtggtgcctg tgttaatgga atggcagaag agttagagat 58260atggagcaac tttggagata tttgaaaaca gaaatgacag aacttgctga taaatgagaa 58320gatgagcaag agggaaaacc agagaacaat ttccagggtt ctggcttgaa gaaccaagcg 58380atggatggtg aagatgtttc tgagatgggc aaaggcaagg gggagggtca gcactagtgg 58440ggtgggagga caaggaggca gaaaccgagt gagctgtttt ggatgtgtta agggaagcat 58500ccaggtgaag gtgtgcagtg ggcagcgggg ccaggctagg gatacatctg ggagtcgaca 58560ggcatggggg gtttgttaag gtcgtggacc tggctgggat aatggagaga gggagcttgg 58620caacagaaga ggtggggact gaggaccgag ccttaaactc tgaatattcc attgtctaga 58680ggccggggag gtgagaagga gcagcaacga gacagaggag gagggccagg gaggcagagg 58740agaccaggag tgtgaagcca gaagccaagg gaggaaagag gctcaagtgg gagggagggt 58800cggtgtgtgg atggtgctgg cccacaggta agatgggaac cggaagattg tgctgtgctg 58860ggcactgtgg gtgagtcagg ctaatgggag ccatttcagt gatgggctgg agccagaagt 58920cagactggcc tgtgtaggat ggtgagggag gtgaagacgt tagcctggag agccctttgg 58980agacgttggg ctgtgagggc tgcagagaag gacatgatcg ctggaaaggg agattacatt 59040tttttattat gggtgattct aagcagacac aataccagag agaagcatat aagaaactgc 59100catatactca tcaccccagt tcaacagttg ctgggatttg gcctcatttc ttcctctctt 59160gccccctatc tgttctttca ttttcctttg cttaagctta aaatttttta aattgtggta 59220aaatatacat aacttaaact ttaccatcat aaccatttct aagtgtacag ttcagttgtg 59280gtaggtacat tcacactgtt ttgcaaccaa tctctggaac tctttcatct tctcaaactg 59340aaactctgca cctattaaac gacagccccc atcctcctct gtctccagct cctggcaccc 59400accattctac tttctgtctc tatgacttgg actactctag atacctcaag taattggaat 59460aatgtagtat ctgtcttttt gtgactggtt tttaagttta cttagcataa cgtcttcaag 59520ttttacccat gttgtagcat gtgacaggat ttccttcctt tttatggcca cataatattc 59580cagtgtatgg acagaccaca tccatccaac accagacact tgggttgctt tcacatttta 59640gctattgtga gtaatgctgc tatgaacata agtgtacaaa tatctcttca agatcctgct 59700tccaattctt tcagatgtat acctagaagt acgcttgctg gatcacacag tcattctatt 59760ttttggtttt tgaggaactg ccatactgtt ttctgtatct ttttacattc ccacggacag 59820tgtacagggg tttcagtttc tccacatcct tgccaacatg tgttattttc tgttcttttt 59880tttcttttat ttttttaatg gtagccatcc taatgggtgt ggggtgacat ttcattgtgg 59940ttttgatttg catttcccta atgattagtg aagttgagca tcttttcatg tgctggttgg 60000ccacttgtat atcttctttg ggaaaatgtt gattcaagtc ctttgcccat ttaaaacatt 60060gggttgtttg cttttttgtt gttattgaat tgcaggggtt ctttatatat tccagatatt 60120acctctttat cagataaaag ctttgcaaat atttttctcc catttcatag gttgcttcgc 60180tgaaatattt taaagcaaat cccagacatg atgtcatttc accaaaggta gacttttttt 60240ttggtggggg gagctttccg gtgaagactg aaaaacctgc tagacaaatt ctaaaataga 60300tgtgactttg gatttttgtt ttttaaggct aggaggtcct ggatgatgct gaaatgtaac 60360agtgacacag agccagtgtg gaactgtgtc tgatgctgtg tgagggtgac atggtggctt 60420tgggaacatg ggtgcaacac tgaagatatg ggagactcca agtgagggtg acagtgagag 60480atcactgtgt gtgtggccct gtgacaccca gtgacatggg acagtgggac gctgtggacc 60540ctgaaatgac tgtgtgtcac cgagcaggtg ggacctgctg tgtgaaggcc acaggtgtca 60600tgtcttcttg tgtcatcctg gttgatgagt gtgacacagt gcaggactct gcatgggagt 60660aagagggact gaagctgtgc tataggtgac cgggctgcat gtgattcaag tgggctcagc 60720cccagcttca gctgctgagt atgggaggga gcatggacat tgtagggtag atgaggagaa 60780acactgaatg ggaacagaaa tggtgtctgt gcccagatgc gagctcctcc cttctctgaa 60840tacccaggaa ggcttcctgg aggcaggatg tgggcacttc agcaggatgt tgtaggtgct 60900gattaagagc agggcctgtg gtgtcagaca gccctgtcta ggctctgaca ttcagcaggt 60960cattttatct cttgagcctc aatttcctca agtataaaat gggagctctt aggaggattg 61020catgaagcag tgctccaatg catgcagtct ctggcacttg gtaaatactc tatggtctct 61080tggggagcag caacctcaac acctgcaccc caggtcccca aataacagga gcaccagtag 61140gagcacagtg aaggtgcgct gagtgaggtg tcctcttaca cccacagccc tcctctctcc 61200ctctccccca acttctgtcc cctgcttggt gttgtcagcg ataccccctc ctgcccactc 61260actcctgccc cctcctctcc cctgccgtcc ttaccactgt cagcctccag cccaggctcc 61320tgcagcctca tccaattagg ccaatgcaat ttgctcaaga aaaagcccca taatttggtt 61380aatcacacca gtaggggatc tggtcccggt cgggagggtg ggggtggata ggagtccata 61440cccgcagctg aggcacaggt gtcaaagtgc ctgtcttttg ggacctttac ccacttcctt 61500gggctccttt caggagccaa cagagtccca aagcttgggt cttctcaaac cccaactaca 61560gaggccttga aacaggagtc tggacttcct gggttcgctt gtgttcctgg gagggtccct 61620gctactctct gggcctcagt ctccctttcc aaaaatggga gtggaactgg ggagtctcag 61680aggccccagt tggcctagct ctgcatccca gctctggtca gtcccccttg tggcttctga 61740ggggccttct cctgggcctt ggggagggag cactgagggg taggtggaga gcacagggcc 61800ccagggaagt gaggaggggt aagtgtcctc tgagtctcat ctggaatgtg tctaccccag 61860tcctataatc agagaccctc tagttccagg ctgcacacct gaaggtgggg caggaagaaa 61920ggaagctgcc ctttcttggt cacctgcaag gccaaagtct cttaaccgtg caggctatac 61980cttgcacagg agctccagca gaggtggggt ggtgctgaaa ctgagcccac tctccctcac 62040caagcctttc ccctcaggcc cgcatctgcc cagagaattg gggtccctcc tttctaatgt 62100gcacacaggt ggccccagcc ccctgctggg agtcagctta ggcaaggttt gatggctcag 62160cttaatcttc tcagcagctc tgggggaaga gaccatttta cggatgagga actgagccca 62220ggaaggtcca aagacttgtc cagtacatgt ggtgtgtggc agggcaggca gatgagcccg 62280catctgaggg aggcgatggg agaagtgaca ggggtgcgca gaggaggaga attagaccct 62340ctcagattcc accactctca gccacacgtt cactcactca tttggagaca agactaacca 62400ccagcgcatt cacagccccc cagacagcca catactgact ataccactgt cacatggaca 62460tcaatgacct gaatcacata tgcatagatg caggcccaca tggtcactcc cacgtgcaga 62520tggccagtgc acacacatag acacagggta ctcacacatg tttacactct cacgacccat 62580gtgggttaca gattcctaca gagacacaga cctacatact ttcacaagga aattctccca 62640gtgacccagg gaacatagtc tgccatgatg atgtgatggt ccgtaggggc tcgccactat 62700ggaccattaa tgggcaggct gcacacatgc ttaggtcccc agcaaagcgg gagttctgca 62760cagagtgaga ggagaggtca gttctgatga gtgtatccag aattttgcaa tcagaaaaac 62820cacacaaaaa ctattttaat tttcatttcc aagataaaat ttagtttgaa ttgtatagag 62880ggtccgaggg tctggtggga gggcatcatc atcttttcaa ggctttgggg ttctaaggca 62940cccacagatt cacaacagtc ccacaagata tcccaggctg acatatttac ccagcccagt 63000gtgtgcgtgt gtgtgtgtgt gtgtgcgcac gctgtgtgca tgctcatgct ggctcccaga 63060tcctcgggat gtgaggaagg aaagtaggag agattccaga gactccggat gtttgttctc 63120tggcttcctg ggcccttcaa aggaaaataa ctctggatgt cagcctgcct gcctggcggg 63180ctgggtggag aggtgggctg ttttgggagg tgggctgtat gacagcctgc ctcagcccct 63240gtggccccac tgaccgggac cctgtgtaat gaggcagagt gaccaaggcc catggccagc 63300gtcccatggg ctcgtaggcc catcgcctcc cctctctggg gcttggctct ctcatctgaa 63360aaatggaggt gggaaggaga tgagactgga tgggctttct cctggagact gattagagag 63420acagagactc aggcccgggg tccagaaaag acaaccaaag ctggggaggg cacatgaagg 63480ggggcaaaga aggtctgggt tcaggggagt gcgtggggcc ccagagcctg ccatgtctcc 63540gccaactctc tccctcactg gaggagggct ctgtgccttg gtgccccacc tgcccagggc 63600cctgtggctc agccccttgc ttgctctgtg agggggacgg gagaaggatg agagtcccag 63660tgataggggg aggacaagac caggggagag ggctgggggt ttctggaggg ccagagcagg 63720aagagcagga gagaagagag gacaccacag tgcaggaaac ggaggagcaa aggctgggag 63780tggggaggct ggaggggtgc agggaatcag actggggcgc tgcgaagagg cctgaggcca 63840gagcaggcag tgcctggatg gagggagcga gcagctcctc accctcagct ccttgatgag 63900gtaaggtgac cacgagccct gctccaggct gtgtgctgag cactttgctc ggagcctgtc 63960actctggagg aggggagggg gtgttcccag gagctatgac agtcttgtgc aagggaggga 64020cagggtcaca tttatgttta acaaagcact gcgctgggag agaggagctg agagaccccg 64080gccctgggga gcatggtggc tgggaccccg gagggcaggc gtgccccaga cggaccccac 64140tcagaagatt gcttatccca accccccaaa gagaaaggct atttttagga acaataaaag 64200tgctcacaca ttcctgcagg ggcagagaga gggaaagggg gcaggagtca gtgcagagga 64260agagggtgga ccccgctctt ctcccaactc tgccttggtc ttcagggact tctcctcagg 64320ggcttcccca gccagccctg cctctccagc ctccgcctgt ccctggggtt ccctaccggc 64380tcttatgtct atccctctgc ttctgaattg gtacttgttc tgtccctgtc tctctttctc 64440atacttccac tttccccctc cccctggggt ttggggaaca gctgggatgg gccaagctct 64500gttgagagag ccaaatacag tcataggaca aagcagcggg aggctgtggg atacacacat 64560gccgcagagc acagacagag agaggtggcc aggcacagag agagcgccca gggaggctga 64620gaggcaggga gaaaacacgc tgggacagtc agggagagcc ccagggcagg catcaccggg 64680cagccagcct ctgtgccctg ctctctatct tgtccctaag aagaccagca tggctgggct 64740tgcctcccgc catccacccc accagcccta ccccaggctg gcccttcctc cccgccctct 64800gcaggcccac actaacccta ggccaggccg cctccttcag catttacctc ccacacacaa 64860tgggcacagt gaggacataa gagacccagt ctctggcctg gaggcagata ctcagcctta 64920cccgacatct gagagggctc agcccatccc ctggccaagg caggtattag aggggcccca 64980aagacaagca ggactctggg acaaggtgtc ctagtgtggc ccaaagggct gggctgaagc 65040atgggtctcc tggctccaga tgagagcctg ggtgaatcct tccctgcctc ctctggcctt 65100agtctacccc atcaagcttg ggattggact acatgaggcc tgaggccctg tagcccctgg 65160tccctgggaa ttctcagaag gcctgggagg gggacaggtg accacgcagg aaggcttcct 65220ggaggaggtg tcctcactca tgaaagaagg tgatagtgac agtgctcctc ttggggaaga 65280gccctccatc ctgacctgct gcccccaccc ggtctgcacg tggagatgat cctgaagcac 65340aaagggcctc ccggcctgca gaggtgcctg ggagaggttg ccaaaggctc tcagtaggag 65400acaccccatt cctcaggctc cttctctgag actgtaactg tgccagactg gggaggcttt 65460gagaggtctc agctatctcc cctgcctaga tccttcctcc acacccctct tctccctgat 65520ggcatgtagc cctcacagta cagtagtcct gggcacacag gagtttaccc agtcatttac 65580agctcagcaa acacctacca acacctatga ggggctgggt aatgctggag acccggagag 65640gggcaggaca caatctctgc cctccaaaag ctcccagtct gttgtgggag ccagacggga 65700aagggtggca ctgcattgat gcacacagtg catgccatgg tgggggaaag gggggcagtg 65760ggagccccag gtgggagggt

cagacttgcc tggagagaga acaacaacag actctccctg 65820gaggggatcc agagaaggga gatcacttca ttcattcatt cgtcattcat ccatccaccc 65880attcaattat tcctttggcc atcatttcct gagggatgta aactctcttc tgacactgac 65940ccagcgggac actcagcgtc ctcctcctct cctgcttgag ccaccatgcc tgcctcttgg 66000aggctcctgg acttgctttg ctcagctccc aacccaccct gagggggtga ggctgaggag 66060ggtgtacaga cattcagggt caccaaactc agagctggag gcctgccacc tcaccagggg 66120cctttctcag ggcacaggct ccctggtggc agggccttgg cccttgcttg cacacccttg 66180gggactagga gccccctcat ccatcctgct caggctctct tttgtggcgc gactctgatt 66240cacagtgtgc ccaaatctgc ctccttgtga ctgccgcgag ctgcctcgtg ggccccaggc 66300cagaggacaa ggatagctag aatgccaggt gaccaggatg actgtgatgg catggagagg 66360gggatgctgt gatgtgtttg ggaggaagtt tgtggtgtcc aggagaatgt gggcagcaga 66420aatgggacca ctctcggttc ttccctgtag atgaagcagc tgaaggtggg agggggtggg 66480aggagacctg agctggctct gccccgcttg atctgatgtc tgccttgcag ggccatcctc 66540cccctcccca cactcagctc ctgcctccct ccctctaccc actctgactg ttccctcctt 66600tcctgactcc agactctggg tgagggactg aggtgattcc agtgagtcag gccctcaggg 66660aactgatcgt gcaggcaact cttgcctgcc ttctcctgct ctttccctct tcccattcct 66720tcatccaccc ccaaacctag ctcctgatgg atccaagggt gcgggggaca accgggaggt 66780cattttggag gaggcaggag ctggaataga agctgggact ggcttgggaa gggcgagagg 66840ccggggcgga gctggttgtg ggcgctggaa gggaggagcc aacagtgtgg ggtcaggctc 66900ctgtggacgg ggacaccctt gggaggcact gggactggct caggtgtatt ctacagtgca 66960cgtgtctcca gtgtggctcg gaggctggag acgcggccct gttggagtaa caactgaagc 67020cggagtctgc gaagggtggg caggagggtg gagggatggg ggcatggagc gggagggggt 67080aagtagagga gggaggggag gaagagaaag agggaggagg aaaggtctct ggcaggtccc 67140tcctttaaga ctgggctcct gcgctgcgag tggccccgtc catactgcct tgttatccat 67200atctccccac cactagtctc cctctgtcct tccaccccca gcctctcccc tccattggga 67260ccttccctgg ggcgtcccct cattggctgt tctcacctga gcaaggcccc tcccctccag 67320tccttagcct cttcacctgt acaatgggat gacccaaaca ggcacctctt gggcttgtag 67380gaggatccaa gatagtgtca gtgggtctcg aggtgtggtc ccccgaccag cagcatcagt 67440gtcatctagg aatgtttgga aacgcaagtt cttggacctc gtcccagacc tactgtatca 67500gaaaccctgg gggtggggcc agcaatctgc actttaacaa gcactctggg tgggttctgg 67560tgcacatgaa aattggggaa cggctggtgg aaacctctag ccacaggagg tgcttgggaa 67620aggtaccttc ccctccccaa agcctgatgc ctcactcaag catgacactg acagttgggc 67680tagttcagct gcgttctggg tctctgtctt gcctcctcct tcagactaag cctcccaagg 67740gttgccaagc ctctttcctc tattctcctc accctgatcc agctcagcct cattgagaga 67800agtctggggc tgcaagatct tcgcactcac aggcagttcc tctttgcaca tccaaggcac 67860cagtgtcttt gagaggcgtc tccttggcca ggtggcaggc gtgggtgtgt ggggaggaag 67920gaggaggaac cgccttgttc tgctttcttg tctctgactc tgcaggctgg gggtgctgta 67980aggctgcgag gaggcataga gtcagcttgg gtgctgggct gaggccaggg gccgaggctc 68040agctgaagcg ggcttctctg gtctgagcct acaggatgcc tcctttgggg cagttctgcc 68100agtcaccctg actgggcggc tgtgcttgct agtgccagac ccatgctagg cacagaggtc 68160gatacgttct cctgtgctct tgaagggccc tgtcctctgg gaagataaga ggctgtgtat 68220attgcccacc ggaacaggag gcaggaagca aaagaggcgt agatgacact tgcctggcac 68280cccctgtttc ccctctagct gccttcctgg gtttcccatt ctgtgggcgc ttctcttgag 68340ttaggtgctt tctcccagtg ttctcaaggt gactatttgg aggtttgtgg gaggagtggg 68400ctggagacac aggagtaggt gggggcagga agtatgcagg agagagatgg agagtgggag 68460gagaagctat gagaggaaga gaggacgcgg aggtgggaaa agacgtcaag actcctggag 68520aggaacagga gtgcagcctg ggacagaggt ggacgtcggc cgggggaggc agggaggaag 68580gcagggaggt ccacccgaaa ggaagggaag ggatgatgga cagagaatga gagggctccg 68640aggtcctggg ggatctagaa ggacccttcc ctttacagaa ggggacacca aggcccagag 68700agagaggagg gcctcacaga ggacctaaca caagcagagt tgcatgaatc agtgtgaacg 68760gacagtccca agagcacagc cggaccttgg gaggtacttg actcttgagt ttgatgttat 68820tgccttcctg taggccagtg tgaggggcac tgtgaggctt ccttccagag aaggaggcat 68880ggagccagtg ccaggcagtg gggtgagcca taggaggacc tgtggagatg gggaaaggca 68940tagagactca tgaagatgaa acaggaaaga tcttatggca gcgaccccaa ccctcaggaa 69000gggcgttggt cttgtgcttg tggctccaaa ggggataaga ccaaggtctc tggtttcata 69060gaatcttagg ctttaagaac gagttagaag taatttagtc cagaccctct cctctcccca 69120gataagtgca gaaatgcaga tctagcccac ggctgagccc caaccctggc ttcagaggag 69180gcctgactca gaacaggctc ccctttcttg gtacctgggg tgaatgaaag ataagtctgt 69240ggtaatggtg ctgtctgtgg tgctgactgg ccttaccttg gactacagag ctgcaggtgg 69300agctggagag agcagaaagg ctccatctat ccatctaccc acccacccag ccacccatct 69360acctatccac ccaccatcca cccacccatc catccaccat ccctccccca acccatcctg 69420cacccattca tctatccacc tacccactca tccatccagc ctcattgaat taaaccatag 69480aactatatgc tgcagagcta gaaagatcca ttttttagta atgacaaaac tgaggctcag 69540aagaggaaag gtgttgcgta aggccacaca gaacttctgt agtcagtctg gtacaggatt 69600ggaaattgcg gctcttttct acacaccaca agttctcctc tgtggtctgg gaaattgcct 69660ggtttttatg ctgatatcta tactgatatt tgttccaaaa agctgtgaag gcaggaaatg 69720tgacctcctt caccccatcc cgagcctgag ttctgtgtgt gtgtgtgtgt gtgtgtgtgt 69780gtgtgtgtgt gtatgtgatg tgcatgtcta agtgcaacct tgtatatgca ttgaatatat 69840gattgccttt tgatctgtct gtgtgcgtgt ttgtgtgaga gcctgtgcat atacgtatga 69900gtagaggagt gcgtagcaat atgtatttgt gtggcatgtg tagatgggca tgtgagcagg 69960taaagctgtg tctgtatttt tcctttcctc ttccttttaa gatcgaagcc ccctgacttg 70020agccttgctc cccatctgtg cctccaattc aggaatctcc ctgcttccca ttagcagctg 70080ctccccactg attctctcct tccttcactg aagcagcaac tcttccctct gagcccacac 70140ctcatgggct ttgcaatttg agctatttcc tcccctgagt tggtgcaatg ggggtgaagt 70200tgctttgaga tctgaggaag attcatggag gagatggcat ttgagcaagc cttgaaggcc 70260cctttgagtg ccagatctga agtggccctt cccagctgca gttcctgcac ccaacaccct 70320ccattcctgg ggcatgctgg gcaggaccag gaggtggatt gacagaagga tgcccacaaa 70380gagccctggg cttcatcagt cacattacca tccagtccgc tctagcacag atgggaagcc 70440cttccctgct gctgccccaa ctctccccaa ctttcctttc ctgctctcct tattgctact 70500atcctgcact tggcctgaaa agtcacagaa aactgaacaa tcagagcaaa ggtcaggcag 70560gcacccacca attccagtaa aggacagttg agggcattcc ccaattgaag caaagggcag 70620gttgaggagt ccaccaatca gaataaagga cagactgttc tttctgagca ccctagggtg 70680ggagctgggg atcgggtgct gagcaggaac cagacagggc tagagatcca gaggtttggg 70740ttctggacct ggctctgctc tgactggctg tctgaccaca ggttgatcat tgcttctcat 70800tgaacctcag cttcctcatc ggtcaaatgg ggagacttag ctctctgaag gctgtggctt 70860tgaagaattt ctccccctgt atcaggctca ctccgtcacc tgggtctctc ttccccaagt 70920ccacatcaca tacatcagac tccaccaagg gcagggcctc tcaggagtca gcttgtgggc 70980tcctctgcct ccaagaagga atagacacaa accaacacca ccttctgtgc tgtctttaga 71040gcccccgtct ggggagcgtg catctggaag actttatctt gggagtactg ggggcatcag 71100ctcttcctcc cctttttagt cttcagaatt gaccttggaa ggccataata gcctgcgtgt 71160attgtgcaca ggtatcactc gagctcttgc cctgtgaatc tttaaggaac tgtaccagtg 71220agaacgtgtg tgtgtgtgcg cacatggatg gtgtctgaag gcctgctggg atgtctgcga 71280ggacgtggga tctgtggctg tgtggtgctg aagttgtctg tgctgtgatg aggagtgcct 71340aagggtcaaa agacaagtga tccaatttgg gtattgtgtt gtctggaatc agtagcttct 71400gatgtctgag ggtagacatc ttcccatgac caagatatgt gtcttcatcc ttgagcagtg 71460ggagggacca aggaagcctg ggggttgggg aaagcgatgc tgagtaagca tctggggaga 71520aggcccacta ctgccctcct cctgggaaca ctggattggg tgggggaagg ggaggaaact 71580gcagccaaga agacccagga gtgaaatttg gagctgaagc ctggatgcaa gtcttcattg 71640agagcccagc gtggaacttt ctggcaaata ggcattcagc ccactcttgt gcacccttga 71700ggatgggaag ctcacttcct ccctctctcc tggtgacctg tggcatgcct ttgtagcatg 71760gccctacctg gaagaaggtc cttcagccca ctagaccaag gccagcctcc tgtgaaatcc 71820tatgggtccc caggctgtcc atggggccac agagttcaga tcccccatct aggagggtct 71880gagagattgg agttggagac tgataaccct gggtctcctc tgctttagat gaggcatccc 71940tgggttatcc agtcttagtc acatgcaaaa cttggtttcc aattccctcg tttcataggt 72000cgcctcctct ggatgagtgt catcttgtca gcccctggga cacaatgaac aggggatggt 72060ctaactagac tataaaagtg ggggaactgt catcttccca attgggttaa cagacctcta 72120ttaatatggc ctgcagtttg agcattttta tttcttgcca gtcatgctta cactgtgggc 72180tcatgctgaa ctgtggtctt ttaagaccct caacctcata tcatgttcac atgaatgggg 72240acccagccat gtctccttca tcttgcagtt aatcactttg ctttctgaac acagacccaa 72300ccttccactg ggaagacatc tgaaaggact tccaagggct tgcgggaggg catggctggt 72360ggctggtatg agtcacgatc ttgccttggc cctcgtttcc tttgttctgt tacctttctc 72420tttgatcccc atggctctgg ccaagttaat agagcgagaa gcagggactt ttgtctccgt 72480tccggctctg caaggacgag ttctgttcct gggatgggaa ggctgtgaga cagtcaaggc 72540tgacgtctcc ttctcctcct atagttgcca ggggtggccc agctgttctc ccaccttatg 72600ggttatgcac cccataggct cttgctactc tcaacccagc ccctcactag gctggaaaat 72660gagactaggt gagaccacct tccttctggg gaaagtgagc gggacccagc ttcagcgaat 72720attcagctga gcatctactc tgtgttgggc attctgtgag gcacttttag gactctgatt 72780tttattttca tttttaaggg ctcaatttca ttttatcttc atgtcagcct gtagggggca 72840atagccccag ctgcttccaa cttacagata ggagactgag gctcagtgac tgaaccaaga 72900cactcactgc tcatacacag cggagctagg attcaaattt gggtgttttt ttgtttgctt 72960gttttgtttt aatttggagc cttgtggttt ccctactgtg ccagaattgt cctcgactag 73020agaacaagag acctggggtc taggccaggc ttgacctgtt gactcactat gaggcctttg 73080ctaagtccct ggcccttctc tgcgcctcag tttccccacc tgtaagatga gggtacttgg 73140acattctgtg gccttaagac tgtttgattt tgagatccta agatcctggg attcctgtgc 73200ctgaaagact cgggctctgg actaagctgg ggggttttgc tcacagtcct ttgggcagat 73260ggggctgccc tggcctgcct ggcaaagcct ctcactgccc tctcctctct tccaggacgc 73320cttgctgagt ctgggctctg tcatcgacat ttcaggcctg caacgtgctg tcaaggaggc 73380cctgtcagct gtgctccccc gagtggtagg tgcccgccct tgccccacgc ttcccacccc 73440acccccaaat cctttgacca gctctatgct gtacctcact cagggccaag gaggaaggaa 73500gaggcagggt ccctgcccag aggactttca tggggaagtg aagggtctgg atgggtgttc 73560tgagacagct ttctggagga ggaagcctta ggctaagcat caaggaatga acttgcatag 73620gaatcctgca atggctgagc cagaaggggc cttagaggtt aagtggaaaa gctgtgtctc 73680agataatgaa agggattcac ctaggataac aggacgtggt ggagccagct gagttttgga 73740atacatgcag caggagaagt tgagggtaga catgtagaag aacttcctgg aagccaggtc 73800tgggaggtac tagaataggg ctcagctttg atgaatagac atgcattggg ttaaagtgcc 73860ctgcctggag atgggaggct ggaaaaatgg cctctagcag ccttttagca gctttctttc 73920tgtcccatcc caataccatg gatgagttgc aggtttgggg caggtttggg gtgatcatgg 73980ttgcctgagc ccagagtgcc ttactgggga gattgtgccc ctcatcatct gttccaggcc 74040actcccctac ctggcttcaa tggccactgt tcatccctta ggcaggagga tgggtaaacc 74100agcccttgag gcccaaagta gcagggtgtt agttgcacca gaaagaggga agcaggggac 74160gtttgaagcc tggagaaggg agtctgatcc agcctaaggg gcatggaaga cttcctggag 74220gaggagattc cctaactgag tcctgatagc cttgaatgtc ctcttcccta ctctaaaccc 74280ggccaagggc agcctctgct ccaggaaata tggccaactc agaatgtgac cttcccatcc 74340ctccagagcc cattgtccct gaatctgctt gatggatgaa ccaccggagg cccagagaga 74400gagggcactt gtcccaaggt cacacagcat gacagggata aatgggactt ggtatctaag 74460cagccccatt ccctctcttc agctctgcct tccccaaacc tcctagaagt tcagagccca 74520ggaggagggc taatgagtga gctttattga gtgtgaaatt ggtaggaagt gggtggtgtg 74580ttggcgccca aaaataaatc ctcctggaga aggacgggac taaggcaaca tctggcctgg 74640ggtgaaggca catctggaaa gggagggtgg tggaaactgg caggtcggtt tctgtagggc 74700tgccccgaga gcctctgtgg ccactgaggc tgccgtaggg tgggaggagg aagtgactgg 74760ctctgtttca caggcagggt gccctggcgg ctgtgccagc ctagatgctc tgcaacagat 74820taattgtctc cccaaagctg ggggctggga tgacagctgt ggtccaggtt cctgggacag 74880tgggaaatgt cagccctggc ccacccaaga gccctatagg agctagggaa gccctgactt 74940tcgggagtcc tggcttgatt gcacggaggg gctcagcccc cagtgaggta agggagctga 75000ggtctgctct gctgccccca gggagggaag cagagatggg gaggggaccc ccgcccaggg 75060aggagagctg ctggcacctg gcttcctcat cagcacccat tgtggcaggc agccccgaat 75120gcagatggtg ctgatgtgtc tgaaatggtt ccctccttct ctccaataga ctcagctaat 75180tttaacccag agggctgaga gtaagggggt gggagacata cggacatgcg gaagtgaagc 75240gagaatctgt ccccctctgc ccccatggac tacccacccc tccctctgcc tgggcaggac 75300tttctgtata accccggctg gtctcttaac ctctttgggc caaataactc aggcccctcc 75360caggctgctg gaagagatgg atgacaagga ggctagatat agccgaagag tgggcggcct 75420ccttcccact gaattcttta tccctgaaca tcccacttag gtttccttcc agccaaacaa 75480gagggtgtct gcccctctca ctcccttcag gccttatcat tcccacccca tgccacaccc 75540accacggaac ctggctcagt gtctctggaa gtagtggcca ggcatctcct gtggtggggg 75600ctggctggcg acagctgatg acaagaagag tggctggcag gattgtggac gctctcagag 75660tcatggaagg caactgcttc ttctgggaag gattccacac ttactgaggg tgggccttca 75720acacgtagct ccactgtcag ctcctcccaa agccctccag gataccctca gctgggaggc 75780aagcccttct ccatcctcct gcggagaaaa cagcagagtt gtggacaagg ctgcgttgca 75840tgggggttgg tcagggatcc cgaagggttg ccagttctgc ttggaaggaa tgtggatttt 75900tgcctgtagg tcagtgaggg caactacttc tgccaagaca tggcctggaa ctgaggccag 75960agctgctctg ggcccttggg gagggaggat taaagagcaa gagctttgat ctccctctga 76020ggagtaatcg gtccaaaata caaatctgct cacgtctccc tgtgcacgtc ctgccctgcc 76080ccagttctgt tcgtaagccc atcccactca gccctactga ccttgggccc agcccctgtg 76140ccccttccct cactgtctgt tcctaaatgc tccatgcttt atacgcctct ggacctacct 76200gtgtacctgc tataaggcct gggagcccat tctgcaccct gcccactccc tgaatgtgtc 76260taattcccac tcagtgacag ctgaaaggtc acttcctcca ggaagccctc tccagcccca 76320ccggaggatg gcgcagtgcc ctgctctgtg ttcctcccct ggctggggtt atgggtgtgt 76380ggtttcttgt agaggtgaag gagggatgct tcctagaaca ttctgagccc catccctggt 76440acagctcaga gtggatgctc agttattgtt tgctgaatgc ctgaggctgg agtcaggcag 76500ggaaatatcc caggtgggag gtgatttgtc tgcaccctca gtccttgaaa ctctttacct 76560ggcacattgg gttttgggtg gtaaaaaagg tcataggttc atgaatcatt gcctgcttag 76620aattccttcc aagaggagag gacgaggtgc ttagttcacc gggtgttttg ctgccctggc 76680tgcatcttag aatcacctgg agagaaaaac aaacagatca ttgccagagc tccactccca 76740caggttccat gaccttgccc cacagacccc tgtgtacagg ctgggactgg gcagctggga 76800gggcctctcc acagggtctc ataagtgcct tctgtcctag gaaactgtct acacctacct 76860actggatggt gagtcccagc tggtgtgtga ggacccccca catgagctgc cccaggaggg 76920gaaagtccgg tgagccattc tctgcacccc cattgccctc ttgcatggcc aaggattctc 76980agggctgagg caccatccaa ggtcatctgg tctgaccctc cccttccaac attgatcccc 77040gcctccctgc caggtgggat tccttggcca ggttgctgac tccagcacag aagggcagaa 77100gcaatgtctt ctcttccttg gggaaatgga taggcacaga gaaaatacca attgatggta 77160aattttctcc ttctaattgc ttctaaatgg ctgcagcctc ctcagagcag agtctcagaa 77220cattggggct atggggtgta tcagttagaa caccggcatg ctgtgagaac tactgcgagg 77280ctggacctgg aatcccagca tgctgggcct gcaggagctc acagtgccaa ctccttgcat 77340ctgagaacag ggagatcaca ggcagcgtcc tgctgagggt tctggagccc cactgcctgg 77400gttcaaatct cagctccctg tttactagct gtgtaacctt gggcaaatga cacaacctct 77460ctgtgcctca gttttgttta tgaaatggtg ataataatgg tgcttatagg attgtgggga 77520ggattaaatg tgtcacacat gtaaagcatt taaatcaggc ctgatccatg gtgagggctg 77580tctgttgggg attaccattg tgagagaatg ctggaatcac tgacttcagg atcatgggat 77640cagggcactt ggccccctga taccttgatg cccatttaat tcagcctcct catcttccag 77700atgggtggat atcatgagac atgaccaagg ccacatgcca ggtatgaggc agagccaggc 77760ctaggactcg ggtcttctga ctcctggctg tttaggggaa agtgagagga agtggaactc 77820atcagatgag aaaaccttgg gggcaggcat gctgctggga ggaggcaggc tctgaaggat 77880gtggccattg cctgctaagc actgaatgca gggccattgt ggggcccagg gagcactggg 77940caggagctga gggcagagtg ggcaccagtg gggatgtccc aagaaggcag ctctctaccc 78000ctgtgaggag ggcttttcca gcaggccagg tggtccaggg atgtggcttt ttcaggtagc 78060agctgagcct ggcaagccac tcacctttca cagggaccat ggaaagaatt cctgtttgag 78120gatgctggac tcatggtcct gaggcccctc cttgtgctgg aaaccctggt ttctaggatg 78180ctggtctctc ctcagccctt tcccgtggaa ggagttggtt ctgctctgat agccaccttc 78240ccatttccta ttctcccact gagctccttt caccttcccc taacaacttc tccgtcaagg 78300agcatgggaa caaagccatt accacctctc tctagccttt gtgtcccgtc tgtaagagga 78360tggtctgaaa ggtctttaga accttaaggg gaaaaatgtg gtcatgtccc cctttctcct 78420ctaattccaa agaacttcgc tctcctccag catcccccac ctctaattct aaagaacttt 78480gcttcatata agctccactc ctccaggaag gctcctcgga gcagcctggg aggccttcct 78540gggagggatg caggaaaaca ggctcaggag gcagcgggga gcagcctgca ggtttgcttc 78600actccctagg acccacacat gctcccctca gctgtctggg catgtagagt gggtgcgtat 78660ctgcggtcca ggcatttttg agagggctca gatccttggc atcagctgcc ctttcaacat 78720cctccttcca accacttcag actcagtaag gcctttggaa aaaataccaa aaaaaaagca 78780attaaaagtg aatattcaaa tccaattatc ccagagctca gtggagatgg ggaggtgagt 78840gcctgctggt agacaggggc tgaagattcc aggaggaggg ccaggggatg agaaggcaag 78900agagtgagga cagcaaggac ctcccagggg acatacccat catcaggaca cacccgtcat 78960catccccaaa caggaattct ttccatggcc cctgtgaaag gtgagtggct tgccaggctc 79020agctgctacc tgaaaaagga ttgggggaag gcccaggccc agtgctctct ctggtatctg 79080agctctgctt gcccaccttt gtgcctggtg tctggtggtg agcccatctc cacaattagg 79140gcggagaggc cccagggttg gctgggccct gctctcagga gctcccagca ggatggggac 79200ttgagaccca ggtgtatgga cgagggaaga gcactggaat gggattcaga caggtctgga 79260ttctagctca gccccctccc tgtctctctg ctttcctacc tgaggcccgg tctattggct 79320taatggggta acaggggcca agtgcttggc acagtgccca gcacacagta ggagctcagt 79380gattgctact tgcactccca agtcccaacc aatgattagc cttgagtgac cttgagaaaa 79440cgacttctct tctggccttt tttctgtgaa atgggtgggg ttgggtacag ggtccttccg 79500atggtgacct ttgtggctct ggtcccccca ggagggagag ggactgacct acaggctgcc 79560gtggagcctg aggctctagc agtgcccgag gaggtggggg tgtggggagg gtgctactcc 79620aggaaaccct ggactgtggg caaacagcag caggtgtggc gtggaggctg gatcatagag 79680acagataagg aggcccgagg caatgggcag ggaatgggat cagggcagtg tggggagaga 79740cagggtggaa aagggtcaag gcgggagtga ggaggccccc gccagctccc agccccacct 79800gtccctgttc ctgccgctgt ttgggctctc agatgcccag ctgcatcccc ccagtgtgtt 79860tggctttcct gtcttcttgt gcttgtaagg gctgcttgct cccttgcaaa gaccgtccct 79920gctccacttt catctcagcc aatcccattg taattatctt tcatggcctg accagaagct 79980gtcttgggga agcctgctcc acagttccct gacactgaga aggaaccaag tttcagaaaa 80040ggggtctggg ccatattggc ctcccttagg gttcttccac aggaagaacc ttgggctggg 80100agtcagagac ctgggatcca ggacaacatg gctgcaatca caatccgatg ccctcttcct 80160gggcctccat atgcccttct gtaaaatgat acgctgaaca ttctgatatt gagggctggt 80220gaggctctga attgtaaggg ctgcaaacga ccttggggct ggagaggaga gaatcctgga 80280aggctgcctg ggccagggtc ttcctgaaag gaggcttcac ttccctcttg ttggtgcccc 80340acctccatct cccagactgt ttcaggcccc agctctgccg ccttcctctt cttgtgtctc 80400ctgctatctt aaagcctctg attacctgat gctgagtgca gcaaaaatct caggcctttc 80460agctgcaact gaagcaccca ccgcccacct cggcccaggc tggctgtctc cctctgctac 80520cattttgggg tccccagggc ccatccctaa gaaatttctt cccctaagct gaccaggtct 80580tctttcattg cagaatctga ccatccctag gggttgtctc agaggacacc gggaacggtc 80640tgctcccatc tcgggatcct cacatgctgg gggaaggagg gcaagaagag ggtccaggtc 80700ctgggggctc agtgagagtg gggggcttag tgaggggatg ggggcccagt gacagtgggc 80760agcctcagtg aggtgatggg ggcccagtga ggatatgagg gctcagtgag agtggggtgg 80820cccagtgagg ggattggggc

acagtgagag tgaggggctc tgtgaggggg tagggactta 80880agtgagggga tggaggctga gtgagtgtgt gggggctcat tgagagggtg ggggctaagt 80940ggggaatggg ggctcagtga ggggatggag gctcagtgag aggatgaggg ctcagtgagg 81000ggatgggggc tcggtgaggg gatgggggtt caatgagggg atgggggctg agtgagggga 81060tgggggctga gtgaggggat gggggctgag tgagaggatg ggggctgagt gaggggatgg 81120ggctcaatga gaggatgagg gctaggtgag aggatgaggg ttcagtgagg ggatggggct 81180cagtgagggg ataggggctc agtgagaggt tgggggctca gagaggggat ggggactcag 81240tgggggatga gggctcaata aggggatggg ggctgagtga gaggatgggg gctgagtgag 81300gggatggggg ctgagtgaga agatgggggc tgagtgagag gatgggggct gagtgagggg 81360atgggggctc agtgggggat gagggttcag tgagaggatg ggggctcact cgaggggatg 81420ggggctcagt gaggggatgg gggctcagtg agaagttggg ggctcagtga ggggatgggg 81480gctcagtgag aggaagaggg ctaagtaaga ggatgagggc tcaatgaggg gatgggggct 81540gagtgagggg atggggctca gtgagaggat gagggctagg tgagaggatg agggtttggt 81600gatgggatgg gggttagtga ggggataggg gttcagtgag aggatggggg ctcagtgagg 81660tgatgggggc tcagtggggg attagggctc agtgagagga tgggggctca gtgagaggat 81720gagggttagt gaggggatgg ggctcagtga gaggatgggg gcttagtgaa atgatgggag 81780ctcagtgaga ggatgggggc tcagtgaggg gatgaggccg agtgagaggt tgcggctcag 81840tgaggggatg gggacttagt gagaggatag gggctcagtg agggaatggg ggctcagtga 81900gaaggtgggg gctcagtgcg ggattgggtc tcagtgagaa ggtgggggct cagtgagagg 81960gtgagggctt agtgagggta ttcgggctca gtgaggggat gggggctcag tgagaggatg 82020ggggcttggt gaggagatgg gggctcagtg ggggatgggg gctgagtgag gggatggggg 82080ctcagtgaga ggatgagacc tcggtgaggg gatgggggct cagtggggga tgagggctaa 82140gtggtagatg ggggctgagt ggggggatgg gggctcagtg acagggtggg gctcagtgag 82200aggatggggg ctcagtgagg tgatggggct cagtgagagg gtgagggctt agtgagggga 82260ttgggtctca gtgaggggat gggggctcag tgggggatgg gggctcagtg gtagataggg 82320gctgagtggg gggatggggg ctcagtgaga gggtgagggc ctggcgaagg gattggggct 82380cagtgagggg gtggggagtc agcgggggat aggagctcag tgggggatgg agggtcagtg 82440ggggatgggg gctgagtggt agatgggggc tgagtggggg gatggaggct cagtgagagg 82500atgggggctc agtgagggga tggggctcag tgaaagggtg agggcttagt gaggggattg 82560gggctcagtg gtagatgggg gctcaattgg gggatggggg ctcagtgagg gggtggagac 82620ttagtgagag tcggggggct cagtgagggt gggggttccc ctggggggat ggggttccgt 82680gggaggatgg gctcagcaac aggcttggct gcttaatgat gcctgggacc tagtgggtgt 82740tggagggggg cttctccaaa gtagagaacg cgagaaggac acacacaggg gctcagagaa 82800gtgcagggga cccagctctt tccaggctgt tggccctacc agcagagaac ctttccctcg 82860attctttttc cattaaacaa atagttgtta aagggacgga actgccataa agtccacgcc 82920tgttcctctc tccactctgt gcccatctgt ccttatcttc agtggggcag gccatgacca 82980cccaggcacc cagtgctgtc attagccttc gcctgggcag ctggccctgg gttgtggagt 83040tccccacaac ccccagcatg agcctggaag gcagggtggg ggtggggtag tagtaaggga 83100ggaactggag aggagcaggg agcggctctg agttgagcaa ggagctatcg ggggtctgag 83160cagtggacga agctcccgct cccatgtggg tgggggagac tcagccttgg cacattcccc 83220ctcgcagtct gtgggcatct ttggagactt caggaggaca gcagttctgg gagggctatg 83280gcagaggaaa ggggctccca tgggggtagg ttgaggtgag tgtgggctat ggggtcccgc 83340aaagccgggg gagggcaggc tgcagagcaa ggtgccgagg ctgcctaaga attgagggtc 83400cttggaagcc ccagtgcttg ggggcatctc ggcttatcaa gattggtcta tcccagctca 83460gcctctgtct tgtccagggc cactaagatg ataggaccct cactgagacc aggtttccag 83520tgtcacagtc tccttatgtg gagagtttta cccaggcagc atgatcgttc tgaaatcata 83580cctgaccatt accgtccctg ctcaaatccc tcccagggca ccccctgccc tcaggctcaa 83640gcccagctcc atagggccct ggcccctgtc tagccttgct ctcggctgtc cagtcacacc 83700aacctccttg tggccatacc tttcagcagg cacacaatct tctcgcctcc aagccttcac 83760aattgcaatt ccctggacat cctttcctgt ctgcctcgat aacctctgcc tgtcctttag 83820gactcaactc aggtgtctcc ctctacagga agccttctct gactccatca caccctgcac 83880ctgagtgggc tggggcctgc tcttcctgcc tttggcagag ctctcatctc ccgactgaag 83940cgtgggtctg tacgttgatc tctgcgtgtt cttggcctcc tcaagtgagg catatgtctg 84000acccctctgc tcatctcagc cctcagcact gaacctgacc cagaaggacc cagtgaaatg 84060agagacttta agtagaatgc tccccgaggt ttttcatcta gaacacttat tcttgctctg 84120ccatggagaa tggattgaag agacccagct aggaggctag aggcttgggg agaggctgct 84180tcagggttca gggaaaaggt gtctccatgt gagctgggca gtggcttggg catagagagc 84240agaggacagt tgtgagagac aactgggagg tgactcactg atcggatggg ggaggtgagg 84300aaagaaggca ggtttttgga caagccgtga aggacctggt ggatggttgt gctgctttgt 84360tgtgaggtgg agggagtgga gataataatt cagatggtat gggggtccct gggccacctc 84420agggacgtgg tggggaggct ccaggtggcc tttgggtatc tggggtctgg agctcatgag 84480tgagggctgg agagtcatga ggccgtgagc acagaggagg ggttttgtgc aaaagagaag 84540aaaggctgag gacagattcc ttcatcaggg tcctgggaaa gagaggccaa gcagctccag 84600tccaggggtg ggaggggaaa tagttgggag tcggcaggat gaggctgcag tgcgcactga 84660ccagcaacgc aaggaccagt gccaccttgt ggcctccggt taaccagatt gtctgaggcc 84720aaggagctgg gcagggtttg gccaggggtc accccctgcc tccgtgaagc ctcagccttc 84780atcagtttaa tcatcaggaa acgtggctcc cgttgccctc ctgccaccct acgtccctct 84840ccttcccggg gtgactggca atgtggacag ccgggaactg gagcccagca cttcaggaac 84900cttaaaggtc ctgggtgtag gggctggaag gtgggagaca ccaccggttc ctgtagatcc 84960tggattactt aaagtggcca ggaaggaatg ggtttggttc agaatgctgc gtgagcttga 85020acgagatgct caacctcttt ggtcctcgat ttgtctagag tctctgacct agtgatctcg 85080tgacttgcag gccaccccct ccttttcctc atgtgacctt tgctgggctt cccttagtga 85140ccctgtatgc acacagttcc ccaagtttct cttctgtcca ggccaggcag ttcctacaag 85200cacaattaag tggaggcagc atgagggatg aagaacccag gacaattaat catcaaggag 85260tgacatttgg tgcaaacntc aggtgcttaa ttaagcggga tgagccagag gctggggggt 85320agaggaggtg ggttgtgtgg tgggacagag agaaactcat tcttcccata ccaacctccc 85380ctgccttggt tcccaccacc cctctgccac tgtcataccc tgccactcac acctgccccc 85440tgttcaaagc tcacacctcc acaggtattt gggaaggttc cagcatagtg gttagaccta 85500gccctggtgc cacctacctg ggttcaaatc ctggctctac cgcttattca ctgtgtaacc 85560ctgggcaagt gaattagcct cttggtgcca tagcttctcc atctgaaaat gaagatatct 85620aattcataga attgctggga attctgagtt catctatgtg agttgcttgg gctgtgcacg 85680ggacatagga aatggccaat aaactttagt tatgatgatt acctcctgtg cttagcacta 85740aaagctgatc aacaattgtt ttctgaggat ggtgacaggg agggttcttc tctctccacc 85800ctagttctcc ttgggaagat cagagaggtc aggtcatgtg cctaaggtca gattgtagca 85860ggcagcctag ctttgagccc ctgcattcac ttcctctgct ctcccactgc ctggaagatc 85920tgcactgggc cccacccgag cctttaccag caaggggcac cagaggccaa actgtggctg 85980cctgtttctc cacatagggt ccagggtccc ctactttttt acttgtgctg tcatcgtgtc 86040caacctgagg caggtcagct tgcccagatc cttgcacatg tgcagggtcc aaactgtcct 86100gtgttcccag gccaggcctc gttcctccct gagtcggggg ctctcaaggt ggcatcatgt 86160cctcttttca gggaggctat catctcccag aagcggctgg gctgcaatgg gctgggcttc 86220tcagacctgc caggggaagc ccttggccag gctggtggct ccactggctc ctgataccca 86280aggtaagggc taggggctgg gcaggggcag gggcagggag ggactgtggc ccctgcactc 86340caggtcatgt gtgtcttcta ttcctcttca tctctggctc cttnnnnnnn nnnnnnnnnn 86400nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 86460nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 86520nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 86580nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 86640nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 86700nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 86760nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 86820nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 86880nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 86940nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 87000nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 87060nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 87120nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 87180nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 87240nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 87300nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 87360nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 87420nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 87480nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 87540nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 87600nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 87660nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 87720nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 87780nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 87840nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnntctg 87900ccccacctcg aataggtagg atgaagccat tggcccagtc agcagctttc tgaggggtgt 87960gcaagcccct gctggactct cagcccccat aaggttagaa ggcccagcct ttggcgtacg 88020cagcactaac cttggtttct ctttgggatc ctccagatgg ggtggggata atgtccagga 88080gggcatgaac caagtgaggt gtgaagagag atgctgcgga gggacagatg gacataaggg 88140cctgaccaat agagatagtg tggaaaacta gccagagatt tggagtggct ggagctgggg 88200gccttgagga gagaaaggcc atggtcaggc cagccagacc cagggctgag gctgggcgga 88260agacaggcag ctaggggggc tttctggagt ggctgaggaa ggagtgttag tgggaagtgg 88320tggatgctct gccagagccc cagaacccag acaagcctcc ctctgcagcc cctcgaggag 88380ttcccagggt ttctgctccg tggaggggat tgtggaagtg cagagcctgt ggggacggga 88440ttctgccgct ctttccgttt cttctcctgt atggaggtgg gggttcaaac tcgttctatc 88500ctagtaacca aaggatgagt tccccaaatt gctattattg ggggtctgct tctgtcgagg 88560gggctgccat tccagtaaag ggcaggcttg ggggcatgga cagataaatg agcaggtgtt 88620ggttccccgg ggactcctct ccagctcatt aaaatggaat taaacgcttc ccaggcgcct 88680gacctttctg agctgctggc aggagagggc agtgggagtc agggatggct gagaggggag 88740gagagggggg aggggaggga ggggtacttg gctgggtctc ctctgcaccc atcttcctga 88800ctgagggagc caaggccgct gcttggtttc tccgcaagct gctcccccca cccccggttt 88860ctggaacgga gctgctgttg cacggagttc tcagggggtc gcctccccct attctttgct 88920cttggtctgc cccagggctg gtcagctttg aggggcactg aggtgagggg ctgtgtgcag 88980aggtggggat tgggaggtgg tggctgggtc acgcacctcc ttctctgctg agggcaaagg 89040gcctggggtg ccaggtgcct gagaaggact tccttagatt gaggctatga ggactgggtc 89100aggagggaag taaggggaag acatttggaa ggttgcttcc cttgtgggcg gaatgcttgc 89160catggccgct gctcaccttg gctcaggctg ggccagaggc caatgtgtgt gtgtgtgtgt 89220gtgtgtgtgt gtctcaggga cgctggtggg atcagatcac ttacacttaa ctcacccagt 89280agaactctgg tctggtcctg cggcaggctg ggtgctggag ccgactctgc cttcagggag 89340ctctcagtct gggggaaggc agggacaccc tccccaccac aggccagatg aaatactgtt 89400gccagcaggt gttctggtgg caaagcctgg agcagcaagt ggtacctcat cacagagaaa 89460gcatttgtgt ggggccttgg ggcacaggta ggaggtccac aggcagggaa gaagggaaag 89520gggattctag gcagaggagg aatgtgagca aggcaggaag gaggccgggg gaaggggtgg 89580tgtgctgagg ggagagtgtg gtcctgggga agctgaagta ggggtgcatg ctgttgagca 89640ggatgctagc cagctgggct ctggctgcca ggagttttca acaaccgtcc cttcacctcc 89700tcaaataccc tgggcaggga agaactcttg aaaggtgttt agattattca catctgcctc 89760tcctctaagc ctgcaccttc ccactcatgt gtaaatgcac ttccctagga tgccccctcc 89820ccaaggcata catgtctgac agatgcaccc tggctcacag gatctcactg cctcccagag 89880gtagcacagg cagccccaga cttcccagct gctgccagat cctgagggca gagggtgcag 89940aaggaagatg gagagccaca gggtcggttg atcttttggg aaggccgagt caccagctcc 90000ttcctggaca agccccattc ccctctctca gtctctgctc ttagtattca tccactgatc 90060cacctaccca tccaccccct catccatcag ttgaacaact atttcttgag tacccactgt 90120atgtcaggca tggagtgcta ggtgctggga acacagaagt gaccaagaca gattagacag 90180gccctgcctt cacagaccat atggtctgga gggttggtgg tgagaagcac taaagtgagc 90240ataaccccag ctacttggga gtattccttg agctcaggag tttgaatctg gcctggagaa 90300tagagtgcga ctccatctct aaagacaata aaaataacaa aaatactgca aaacaagtga 90360gcaaaataag gataggatca cacgtgatgg caagtgctag caaggaaacc cctcgaggga 90420gtcctgcaaa ggaatgctgg ggtccagtgg cgacgccatc agcgagggct ctctgaggaa 90480tatgctggcc aggtgcaggg ctgtgtggct gacttctggg cctttacagc tcactgctca 90540aagcaccttg attgcaaatt ttttgtgtgg aaagtctttg gctctgttcc ttaatcctgg 90600gataataagt ccctttgagg agtggcagcc cttggtctct ggcatttgat gcctgatttg 90660tgcccactct tcccccagct gcaccagcca caccagctcc ctgcacgggg atggggtgtt 90720catgccatta accattttga acttggttag ggtggggacc tgggggctgt gctgggtttt 90780aaccctctct tacagccaca gtgcccaatg ttgagtgttc ccactgggtc ccagacactt 90840gccctgatta gcaagagcag gtgtaagtgt gtgttcctgt tggcgaacaa aaagccatga 90900gtgtgtgggg gtgattgtgt gtgtttgtgt atgggggcgc caggtgcatg tgcatggctt 90960ttgggtaagt gaactatttc ctgtgtacag gcatgaatgt gcctgtgggg atgctgtgga 91020cactgtaagg gtgggtgtgt ggatgtcttt gtgtctgtga ctgcaccgtg tgtgtgtgtg 91080tgtgtgtgtg tgtaccacct ccatgggaga ttgagtgtaa gtgcatgtgt gtgagggcct 91140gacgttcttc atgagagtgt aggtgtgtgt gtttgtgcac atgctgggtg caagtaggcc 91200aaggcagccc gagaactggt tgcccccaca gccttagagg ggtcccagcc ttctctattc 91260ttgagagatg ggaccaggtg gaaggaacaa gaaccacgtc ctcctccatg tgctaacagt 91320aaaatgccaa catatttata taagccatat gcaaatgaac catagcccca gcttctcctc 91380cctcccgccc ccgctgtcct gtaggagtca cagattgagc ctcatccaga gcttaagttt 91440aaacaccatc cttgacagcc caggtctctt ccaggtcttc ctgcctccaa tctccctccc 91500tcaaattcac cctacacccc acagacagtg gccctcagat gctaaagtct gccatggctc 91560cccagtactc tcaggctaaa gtctaacttc ttagcctggc actcaaggcc cttccttctg 91620tggtaccatg gaccacaccc ctacctggat cctcatctcc ccgcaccacc taccgccagt 91680gtcctggtct cttcaaggtc tacttgtcct ccccatgcca attcaggaag ccttccaaat 91740gtcctcccct ctgagctacc cacctagttt tctctctcct tcctcaggta tgaggctcca 91800cacctcctcc cagtcatgtc tccccactcg tcccacccca gacagactgt gagcttcccg 91860agaacctgct ggtctcctcc tccccatctg ctcctggcag gagacccaga gctgaggcag 91920gcattggctg ctgactgggg agggaggagg aaggaggagc ccccagtgca ggcgctgtgg 91980ggagctctgc aggtggtgag cagctttgag taagctccgg aagctagtga cgcaggtggg 92040gagccttgct ggcagggcct gtagtgggtc cctaggctgc caccctccct ccccaccgtc 92100tctcattttc ctcgacaagc acccaagtag gagtggggga agggacttca cagagtatga 92160aagatgggct gagttccctg gtgactggca cagggagctt ggagagggac aggatgatgg 92220gggtggtgga gagagtggat ctagaggggg gaaggtgtgg gcagagaacg ggagggagtg 92280ggtggtgctg tcttcactct gccactttct gctatgtgat tttgggcaag tcactccact 92340tttctggcca tggcttgcct gccaggtgga ggcttgcatc acagtggtga gaatcacatg 92400taaggcaaag cacttcacga acccatttat ccatttattc attcacccgt ccatcctcac 92460catggcaggt gcatcatact gagcccattc ctttatccaa gacttagggg cagctcaaac 92520cctaactcag acaggataga gccgggaggc tgaggctaat accaccaatt ctaaaatcag 92580gacctgcagc gctccataat ctgtgagcta aaaaggggtc taaggtccct ccatcccctg 92640aaccatacac ccggactgtg ctcttgatga gacagtgatt gaaaaggcag agacaaaaaa 92700gtttcacgtc cttaggttag tcctaggaga cttcctacag gagggatttt cctggagctg 92760tctgagtggt cagaaagatg gggttatcac tgaggcccac agactggagt gtgtatgtgt 92820tggtggggac cagtgtgtgc tgcgcatggg gagggcactc tggcagagac agacactgag 92880agaggtcaca gctagttccc ttctcccatc cctccaggtg cactgtggcc agctgagtga 92940taatgaggaa tggagcctgc aggcggtgga gaagcatgtg agtgggagtg gggccatgtg 93000caatgaggct gaagaccctt atcacagctg gtgggaagat ggcctggcca gggagctgga 93060cagacctggg tttcagcttc ggctttgctg cttttgagct gtgtgacctt gagcaagtca 93120ctaaacctct ctgggcctca gtttcacacc tgaaaatggg gataatgata gcaccgactg 93180acctagggca gtggtgaaac aaaactggtc aaatatcttg taaatacaca cggttgccaa 93240cttacaattt tcgacttttt gatggattta tggggaagca accctatcct aagtccagga 93300gcatctgtac ttagaaagaa ccctccacat aatgatactg aggctctttc atgcctgaga 93360ctttatgatt ttgtgacttt aacaaggact tacgacttcc tgagggggct ggagacagaa 93420atctgacatc ttgtcttgga agaatctagg ggctagggat ggagatagac cctgtaccct 93480cctgttcctg gaccgccgga cgctccaggg gctgtgggag cccccggggg agccctcagg 93540aaggtagagt ccagggatga ggtgtttggg acggcggcgg ggtccctggg cccggcaggc 93600agagggaacg gcgggagcaa aggcaggaat cccgctgcag caagcgcagc gagcttgggg 93660cgagcggcgc gctaaccgct cggcctgccc cagaccctgg tcgccctgcg gagggtgcag 93720gtcctgcagc agcgcgggcc cagggaggct ccccgagccg tccagaaccc cccggagggg 93780acggcggaag accagaaggg cggggcggcg tacaccgacc gcgaccgcaa gatcctccaa 93840ctgtgcggtg agggcccggc ctggacaggt cacgagggcg gggccgggca gaacttggag 93900gggaggtggg cgggttaggc gatcccggga gccggcggcg ggcccggcgc ggagctgagc 93960ggcgcctgag ggacccggac acggaggtgc ggaggggccc tctctctgac cggcgcctgg 94020cccttgcagg ggaactctac gacctggatg cctcttccct gcagctcaaa gtgctccaat 94080acgtgagtcc ctgcgcccct gccggccacc tccccgtcct gtctccctcc ggggaccaac 94140ttccccttga gccctccatc tcagttccaa ttacgatgtc cttccttcct ctctcctcca 94200cccgcctgaa gagccccgga gaggggagca ggtggggagt ggggtgaccc ggatccgcgg 94260tcaccccctc gccctgcctg tccctctctc agctgcagca ggagacccgg gcatcccgct 94320gctgcctcct gctggtgtcg gaggacaatc tccagctttc ttgcaaggtg agggcccagg 94380tccactgtag agcgggggcg gggctgggcg aggaactcgg gtctccgagg gggaaatcca 94440ttgcctttcc tttaaccagc cccctgcact ccgttcctca ggtcatcgga gacaaagtgc 94500tcggggaaga ggtcagcttt cccgtgagtc ccgcgtctgt cttctcgttg gagtctgcag 94560gggcggttga ggccgggtag acactcctgg gatctgcctg gagtatttgg atcttctaga 94620ctcttggaat ccgatggaat tatctggatc ttgggactac ttagaaatgc tgcagggatc 94680acaacctgtg atcagcaggc tctattagga agaatctctt agcatctaca gaaaggctta 94740cctgggacct gttcacttct gttggagtat ttctggatat ggatctgcta gaatctgttg 94800acgagaatcc ttggagtctg cttatctctc tcgtagttag ggaagactct agagtccttt 94860atagcgaatt ctgccagact cccctccatc tctgctcatc aatgctgacc ctgtccacca 94920tttggactga ctgaagggtt ctttgaaact tccagatttg agggtgggga caggttgaga 94980tcccctgacc tggggagtac tgggccctga ctcagtctct cctcaccccc tagttgacag 95040gatgcctggg ccaggtggtg gaagacaaga agtccatcca gctgaaggac ctcacctccg 95100taagtcatgg cctggctgac ccagagggga aagaggagac cccactgcca gcccctagag 95160ccagggtctc tgttacagag cagcctagga atggggcaga taagacctgg ggactttcta 95220ctgtcccatc tccatgacac agagcttcca gccttgcatg agtcccttag aactgcctgt 95280tgcaaaatgt gatggagggc tggaggaggg aagcatactg cgccctgctt ccctgccctg 95340acttgcctcg cctttgcagg aggatgtaca acagctgcag agcatgttgg gctgtgagct 95400gcaggccatg ctctgtgtcc ctgtcatcag ccgggccact gaccaggtgg tggccttggc 95460ctgcgccttc aacaagctag aaggagactt gtgagtcttt gtgggatgat gcagatcagg 95520agatgtcact gagaggctgg ctagggctcc acgagggtaa caatgtggga tgggtactgg 95580gcaggggcta ctgtctcagc agcagtgggt tgaacagtgt gttagtgcaa gagaatgaaa 95640gtcatgttga ggtccaagct agttcctctt ctcttctcct gcttcctgaa gtttgggtaa 95700tctgctcttg gggtattggg ttccctccct tgccatccct gtttttgcat tactgctata 95760aactgctaga tggaggggtg ggtgtgctct gggttggatg aaccctctgg gacccacaaa 95820gcatcatcaa cacagtggac agtggctaaa gggaatatgc ttggggactg ggaaaagctg 95880tggatctttt gagcccctga

cagggcagct ataaaaatga tacacaaaaa tctctttttt 95940tgtgggcagg gcacagtgga caggaaagca ggcttggagg cttagttgga aaggatatct 96000cgagaactga ggacaaacct ggggtctaga aatggtgtca taaataaatt tcatatccta 96060caccaactca taaacaggca gtaggtgcct gaattttatt gcaaatggat cttagttcag 96120ggagaaacag tgctgcgtct gatgagccat ttctgtcctg ggtgcaggtt cacacttggg 96180ctggcaggat gagcagtttg tgctgtgtca cataggtggg gagaagtaga cagatgaggg 96240gctgagtcct gatgcaaaga gatgctgata ggatgctggt ctctggagtc caagcaaaca 96300ggctgggttt cagggcctgg agctcctgca ggaggtggac actagagagc ctgggactag 96360gtaggtgtca gagcccgggc ctgaggtctg ctggggtagg gtggagatcc aggagtccta 96420ggtctgagct gcagaaccta ccagcatgga actgtgttga cagttgggtg ggcctggaga 96480aacaaagata ggggcaaggc agaatcagct gaggcaggga gaatgtggga ttggtggcat 96540ttggaacttg tgggcatcct aatggtggga gaatttatgc cattcagcaa acaaatattg 96600agcacttaat gttgccatcc cagtgctgac cagatggcct tgggaaggcc tttggggaag 96660ggaaggtaga gtgaatgggg gtccagcagg ggccatgact tcttgctgct ggctgtgaga 96720ttgggttcta ggatggcccc agagctggag aagaggtggt atcagcagga aataaggatg 96780gggccttggt ggcagctttg aggcccaggg caggggcagg gctatctctg ggtcccacgc 96840atttcaggga gtgagtgttg aatgactgca tgagccaggg tggggctcag ctcagtgcag 96900tgactacaga gaagcttcct gaaacacagc taagtagcca gagaacaggg gctccagaag 96960cccttcagct gtgagtggga tggggctggt ggcaaggcca gggataggat acactgacga 97020cattagcaaa gacctccgaa gtgtttcctc tgtaccaggc tctgcactgg gcatgggtga 97080tatagtcatg gccccatttc ataagactca aagctcattt tcagggcata gagggaagag 97140agtgagaagg gtattctagg ccgagggaac agtgtagaaa aaaaagcatg aaggtgtgaa 97200agagcccaag gttttctcag aatgatgagg atctttgtgt ggctgaagct gagagatgtt 97260ctgggttgag gggtgacagg tgggtggggc tagctgaggg accacaaatg taagaaaggt 97320gtgcagacag acccaggatg gtggggatgg gatctagatc cgaatcactg gatggcaagc 97380atgaatgggg gatgccccac cagggtggag caccaaggcc agccaaaaag tggggaaggg 97440cttaggcagg gacacctcag ggcagcgtga tgtgggctaa ggcaggctct tcccatgacc 97500cacaccattg gtccacccag ccccatgcag ctccccagtg acaaatcatt tggtggccag 97560attgaatgac gtgagcagga tttggggctt atcttgtctc accagagcta gctccatgag 97620cagggcaagc agtcctctcc acaccaccac cctaagattt ctggaggcac cgaatcaggg 97680ccagcggagt ccagggagag tggggtagtg acaggagctg cacaagatag ggcagtgcca 97740ccgcccctcc ccaaggctgg aggtgtgcct ggggaagagc agaacaccag cttgagccca 97800ggcaatctct agtctgaggg aggagaccca gctttgggct gggtaaatcc caaatcagag 97860acgggaggta tggctctggt ttcaagcatc taaggaggac tggagccctc cccttgggca 97920gcccccagtc tgcagggtca tgggggtggg aagctgttcc aagggcctgt gcagtggtta 97980tatagttggc aggtgggtac ccctgtgggc ttctgatgga acagaagtaa ggagagtggg 98040gagagaagcc agtcttccct tccctcctga gtgagcccac cccctcctcc aggttcaccg 98100acgaggacga gcatgtgatc cagcactgct tccactacac cagcaccgtg ctcaccagca 98160ccctggcctt ccagaaggaa cagaaactca agtgtgagtg ccaggtgagt gacctgcctt 98220cagcctctct cgggcaccga ctcgctcagt tttcagcccc gagagccatt cagaagggaa 98280atgcccatgt ctttctggac tggtggcagc ccttccccag gtggctccat aacctcataa 98340cttgaaggct tgcagttgtt caggacccgc gccactgccc gcaggcactg tatgtgatcg 98400ccctctagtg ttcaatatgt gcactacagc aacacctagg cagctagagc tggcgtgaag 98460gcggctgaga cactcaggag actcctcacc tgcaccgggg ctattccctc actccttcac 98520ttagtagcca aatgatataa ttagacactg acagtttctg gcttgtccag tgagccctag 98580ggaaggaagg agaagacccg ggtgctgttg gaggcagaag gttggatagg gtgaccccta 98640caccccgacc cccctatgat ctccatttcc ttcattccag gctcttctcc aagtggcaaa 98700gaacctcttc acccacctgg gtgagtgcac tgttctctct gcctggctgt gtgtgggcat 98760gggggctggc atttgcagag gagaggcggg aggtcttggc agcctggtct caccctgcct 98820ggtcttctcc cttccccaga tgacgtctct gtcctgctcc aggagatcat cacggaggcc 98880agaaacctca gcaacgcaga gatgtgagtg actctaccca ggggacaggg cgagagaggc 98940tgtggccttc agtccccatc atctcctttc ctgccccacc cacttccctt tctctgcctt 99000ctgcgggact tcatcacctt ttgagggatc ctttatttca tgcctgtctc cctcgctaga 99060ctgtaggctc caatacagca gggacagggc tggctttgga tcctcagctc ctatcacagt 99120gcctggcaca tagtaggtgc ttccaaaaaa aaaaaaaaca aaacacttga atggacacgt 99180ttctggagcc agccagccct gagcagagtg tcttaccttg gagcactcct cccaggcctc 99240ggaaatccgg cctttgcctc cttatgggac gtgagggcga tcagaggggg ttgtcaggcc 99300ccagaggacc aaacccctcc ctccacagct gctctgtgtt cctgctggat cagaatgagc 99360tggtggccaa ggtgttcgac gggggcgtgg tggatgatga ggtgagaggg cgtggaggga 99420gtatgtggcc ctaggggtgt ccgggagtcc gccggcggcg ctggggagcg gcccgaggtt 99480taacagtccc ctctgtggcc gggtcactaa cttcttcctc tcgactccat ctctgctccg 99540gcagagctat gagatccgca tcccggccga tcagggcatc gcgggacacg tggcgaccac 99600gggccagatc ctgaacatcc ctgacgcata tgcccatccg cttttctacc gcggcgtgga 99660cgacagcacc ggcttccgca cgcgcaacat cctctgcttc cccatcaaga acgagaacca 99720gggtgcgcgt ggcggcccgg gcggaggggc ggggcctgcg ccgggcgggg cgggtccgag 99780cgagcggggg tggcaacact tccccaccgc ctccggcgtc ccggagcata agggagtcgg 99840gttccatgcc tgggacgtac gtaacctgcg gaaactgcga gggcaggtcc cggccggatc 99900cctccctcca accgatccct ccctccaccg gtggttcctt gcccctctcc cttccccaga 99960ggtcatcggt gtggccgagc tggtgaacaa gatcaatggg ccatggttca gcaagttcga 100020cgaggacctg gcgacggcct tctccatcta ctgcggcatc agcatcgccc atgtgagggc 100080ggggttggga gtggggtgtg gggtgatagg gggcggggcc cacgaaggac cctcggttct 100140cctcctccga ctgactctcc ttgtggattg atcccttggt ctggcactca gagtcccgcc 100200gctggggtgc agccttcagg acacgctggc cacctctggg ctcagtttcc catctaaaaa 100260ttgggcatac gatttcctgc cctgtccact cagcctcctg ggaccatgag aaactcccgt 100320tgtcaaaacc tcctctcttc cctggaagca gtctcaaccc aagccgagtg cttttttgga 100380agtgctgggt ctcggtgtcc aggcctactg gcgctctggc ctgggaatcc agccccaagg 100440tccctgacat gatcccctcc ttgcttctcc ttccctgcca tgggccttgg gctccatcac 100500tgaagcctgg atcaggtgtg ggggagtgca aagggccaga ccaaatgctg ggagaacttg 100560atgaggagga accggcgcgg gggtctggat gaaagtgggg gtgaggtctt tactgtggac 100620tggagcttga aggttttgac tggggccaga atgggacagg aagtggggtg tctttttgac 100680cccttcatcc cagtcctggg cattgctaaa ttttcacagc caccttcctt gagccccatc 100740tttccctctt tcccctagtc tctcctatac aaaaaagtga atgaggctca gtatcgcagc 100800cacctggcca atgagatgat gatgtaccac atgaaggtga ggcttgcaga gacctctggt 100860cctcctccca gattccccgg ggacccaggg ccaggcaggg cttcctgatc aatctctact 100920gaggatgaga ggataggccc agagccacag caggcctcct gccctcctta ggggcagctc 100980ccacccctgc ttagagacct ctcctccaag ctgcttctga gctcagtccc aaggctggaa 101040gtagccagag gaaccagccc agggagtaat tggttcagcc aggtattccc catgttcagg 101100gaataattcc catcttggga attactgagg gctaggaagc tcacccagga cccgtcccca 101160tggcttccct aggtacaatg cccatgcagc cctgggcagt cttaattgct gataatctat 101220cccattccct accctgggtc acaaaagctg gcttagttcc atgtatatgg tagtcgctgt 101280tcatttggac atttcctctc acctgtgtcc aaaccagaga ggcccagacc ttgtgagttg 101340gatcaaaact gtagtaggaa gagttaaggt tagagagtag aaaggtctcc acaaaaggag 101400gactgctaca gttactgtgt atgaaatgct gccatggttt gggggtgtca tgaaggggtg 101460ttgtcgatct ttgccaaggt tatgctgtta cagataaagg gtggtcacct gcaggaaggc 101520gcgcggggtg ggctgcaggg ctgtgagggg agggtggtga tttcctgccc agttacagtc 101580cacagcgtgg tggcccaact gtggtacatt ctgggtgacg gatcccccac ctgccatggg 101640aatttgaggg tgaagacacc agatggggtg aaggctgtct tctaatgctc tggctggtct 101700cctctaggtc tccgacgatg agtataccaa acttctccat gatgggatcc agcctgtggc 101760tgccattgac tccaattttg caagtttcac ctatacccct cgttccctgc ccgaggatga 101820cacgtccatg gtgagttgct ctcctccact tgactggcca ggccgaaggt atgtagccag 101880aggcttaagt taaatgcgca tcaagaactt cctgggaaga cagagtcatc aaggaaggct 101940gtggagggtc cctcagagat ggaggggctt gtagtctgcc atcaggaagc catggggcct 102000gcccaggggc tagaggctgg actggatgat cccaagggct gctcttggac caaccatgcc 102060cagggcatgt gacctcaggg tttgcatccc tcccaaccct gtttttctaa cattttgtgt 102120gggcttggtt tcaagagttc ttagttctta gatctctaaa aatgcatagc tctgagaacg 102180gttgcttcaa ctattttgtg gttctctagt ttagatgtaa gtttctaaga ctccagatct 102240tgagtgtgga gcttgaagaa ggacccaggc aagggccctg tcttgatact ggcagcccct 102300ctgatacctc cctctgccct ctccaggcca tcctgagcat gctgcaggac atgaatttca 102360tcaacaacta caaaattgac tgcccgaccc tggcccggtt cgtgcgccca cagacagccc 102420cagtcttcgc ctccctcttt cctctactgt cacatccatt gcccccggca ttctggagag 102480gatctctcta aggatgactg gggagaccca gtcttatggg ggtggggagg atccatgaat 102540gagaagcaat tcctagacac tgaactgtca ataaaggcaa gaaatgaggc aaggcaaagc 102600ctggaggcaa ggccgagagt gtgtagccag aggtttaagt tagatgtgca taggaacttc 102660ctgctaagac agagtcatca aggaaggctg tggagggtcc ctcagggatg gaggggacat 102720gtagtttgcc atcatggggc cgtgatggag gaggagaggc tgaggcccct cttctgccct 102780cttccctccc ccaggttctg tttgatggtg aagaagggct accgggatcc cccctaccac 102840aactggatgc acgccttttc tgtctcccac ttctgctacc tgctctacaa gaacctggag 102900ctcaccaact acctcgagtg agtggctgca tctcccccac atctggcagc cactggggtc 102960cccttccctg ggacagggaa gcaccccctg tgtgtcaggc actttacacg cactgcctca 103020tgggatcttc ttagccccag gggactagag gggaaggctg tgagccccat cttccaggag 103080gggcttgctc acagccaagc agctagtgaa gactgagcct gatttaaacc cgggtctgct 103140ggactccaaa ccagtgcttc tttccaggaa gggaacccag gtgttccaac ctcctgtccc 103200agtggctcct gggcatgtca tctcctgtct gtcctcttgg ggatttaggg agggaactgt 103260gggctgacct cttttttttc tcctttctgc ctctcaacca gggacatcga gatctttgcc 103320ttgtttattt cctgcatgtg tcatgacctg gaccacagag gcacaaacaa ctctttccag 103380gtggcctcgg tgagaccctg ccctgctcac agtggggacc ctccatgggg tgtcttggat 103440ctcatcctct cccagcctga atagggtggg agcgagtgag accaggagcc aggtttagac 103500acaggaggag gttcccccag ggtttgcccc tggctctgag atagggagga ggggagaaag 103560gtggaagggc aggacactgc tcagcctaaa gcagtggcac ttggatccgg atgtgaggag 103620tgaccacagt tttcctgggc ttttccagaa atctgtgctg gctgcgctct acagctctga 103680gggctccgtc atggaggtat cactcttctg tcccaccccg tccttcttcc cctttaaggc 103740cagtgacttg caaagttatg acccagctcc tcctattccc aaaccatgct ctccagacag 103800gctgcgagag ctgcagccac acctaggaca tgtctggctc attttcctgg agtgggcttg 103860gaagggtgca ggtgcggatg atagcaagga tttgtgttca gcgtgtttcc ctttggctgc 103920ctgggaacac cccattcagc cccctcctgc caaacttggg atgggctcca ctcccatcac 103980ttagcgtcac cttagattgt ttggtttggg tctgcctacc tcctcgtgca caaggtctga 104040gccatttctg agttccctgc acttggcaca gggcttggca cagagtagga gacacatttc 104100caaggtcacc ttgcctcatg ctacttccca caacacctct ccagaggctg cccctgcttg 104160cacaccccca gagacgaggt tctctgtctc tctcccagga ggcctggtgg cagtgctggt 104220tctgccctct gcccccctga gataagctgc tccttttctg agtgacagcc cttcagcatc 104280cggaaatggg ggccttgccc ttgcctcatc actgcctctc cttgtcagca aacaaatgtg 104340ttctgcatga tttggtgtct aggactccaa aggatcattt caaaaatgtt ccagctttca 104400gggaccccag agcttacctt gttgggtccc tgcatgtgac agctgaggag tctgaggctc 104460agagtggtct agggactcac cctgggtcac acagagggtt gaaacagagc tcagaaaggg 104520aactggggcc cctgactccc cctttctgac tgctctgctt acctgggggc tggagctgga 104580cgaggcccct gcttcctctc ttggggtcaa tggtaaggga gcccatctgc cccagctggg 104640cccccatcac tcctctcccc ccagaggcac cactttgctc aggccatcgc catcctcaac 104700acccacggct gcaacatctt tgatcatttc tcccggaagg tgatggggtt gggggtgggg 104760tggggattga gggggagctg ggagctggct ggaggtggga taaggagcca aggagtggag 104820gctcactggg atgggcaaat gggtgggggt gtccagtagg agggcatgac acccctgccc 104880tcgcctcagg actatcagcg catgctggat ctgatgcggg acatcatctt ggccacagac 104940ctggcccacc atctccgcat cttcaaggac ctccagaaga tggctgaggg tgactgctgt 105000tagccccagt ccttggggct ggggaggaac aaccagggga aggatttgcc aggggagcat 105060tcccagggtg cagacccatc ccctgcaaca tcaacccttc tctggctgca cggccccccc 105120caggcagacc cagcactggc cccttggctc ccatcaaggg tgcccaattc cctggaccgc 105180tctgggttgg gccctgggag ccttgtcctc agaagggcaa agaggctggg ccccgctcct 105240tgaccccatc ctcccctcaa cagtgggcta cgaccgaaac aacaagcagc accacagact 105300tctcctctgc ctcctcatga cctcctgtga cctctctgac cagaccaagg gctggaagac 105360tacgagaaag atcgcggtag gtgtagtcct ccctgggaag gcacaggctg cccaccctgc 105420ccagctttgg gtgccccctg tgcctgaata ccctctctct gctcagctca gcctggctgt 105480gttctgggga gacagaaacc tagaccatct cagggtgaca aatggagact cagagagggg 105540aacagaccta gcaagtcagt ggctggtgga aggtgggccc caacccagcc actccctgcc 105600tcaggccatc ccactgccaa gctggggctg gtggggacgg ctcctgagct gggactgaat 105660ccctgggcct cagttttctc tcctgggaac gggctgtcag aggagcttgg gtggatgtat 105720cctacataga ggatgtgatg agagtgttgg cctttcagga gctgatctac aaagaattct 105780tctcccaggg agacctggta tgtgtggagt gaccccagga tgtccaggat gggggagggt 105840tcctggcctg ggacagggag ggcttgaact agcctgaccc tggtacccga tggaggaatg 105900agagggacag gcctgacgac tcgatgcctg caggagaagg ccatgggcaa caggccgatg 105960gagatgatgg accgggagaa ggcctatatc cctgagctgc aaatcagctt catggagcac 106020attgcaatgc ccatctacaa gtgagtgagc tcatggggac aagctgcacc ctgcacagag 106080agggtaggct ggagtgggga catcacagga aacacaggtg ctgagattgg cctggcccag 106140ctccaactga ttcatcccct tgcctctggg cataactgtc tcccgctgtg cccctcagtg 106200ggtccttcac ttcatccttg gtcctcagtg gaaagagacc atcatgcttt cctaggtgtc 106260ctcctctgtc tcacattctt gtggaagttc ttgttttttt tgagatggag tctcactctg 106320ttgcccaggc tggagtgcaa tggcacgatc ttggctcact gcaacctccc cctcctgggt 106380tcaagcgatt ctcctgcctc agcctcccaa gtagctggga ttacaggcat gcaccaccac 106440gcccagctaa ttttgtattt ttagtagaga tggggcttca ccattttggt caggctggtc 106500ttgaactcct gacttcaggt gatccacaca cctcggcatc tctgagtgtt gggattacag 106560gcgtgagcta ccgtacctgg cccttgtgga aattctattt gttgtgtagc cctagtcttt 106620cttgctgccc atggtctgat ttctggcctc tcaccctctg cccccatgca cccgcaggct 106680gttgcaggac ctgttcccca aagcggcaga gctgtacgag cgcgtggcct ccaaccgtga 106740gcactggacc aaggtgtccc acaagttcac catccgcggc ctcccaagta acaactcgct 106800ggacttcctg gatgaggagt acgaggtgcc tgatctggat ggcactaggg cccccatcaa 106860tggctgctgc agccttgatg ctgagtgatc ccctccaggg acacttccct gcccaggcca 106920cctcccacag ccctccactg gtctggccag atgcactggg aacagagcca cgggtcctgg 106980gtcctagacc aggacttcct gtgtgaccct ggacaagtac taccttcctg ggcctcagct 107040ttctcgtctg tataatggaa gcaagacttc caacctcacg gagactttgt aatttgttct 107100ctgagagcac aggggtgacc aatgagcagt gggccctact ctgcacctct gaccacacct 107160tggcaagtct ttcccaagcc attctttgtc tgagcagctt gatggtttct ccttgcccca 107220tttctgcccc accagatctt tgctcctttc cctttgagga ctcccaccct ttggggtctc 107280caggatcctc atggaagggg aaggtgagac atctgagtga gcagagtgtg gcatcttgga 107340aacagtcctt agttctgtgg gaggactaga aacagccgcg gggcgaaggc cccctgagga 107400ccactactat actgatggtg ggattgggac ctgggggata caggggcccc aggaagaagc 107460tgccagaggg gcagctcagt gctctgcaga gaggggccct ggggagaagc aggatgggat 107520tgatgggcag gagggatccc cgcactggga gacaggccca ggtatgaatg agccagccat 107580gcttcctcct gcctgtgtga cgctgggcga gtctcttccc ctgtctgggc caaacaggga 107640gcgggtaaga caatccatgc tctaagatcc attttagatc aatgtctaaa atagctctat 107700cgctctgcgg agtcccagca gaggctatgg aatgtttctg caaccctaag gcacagagag 107760cccaaccctg agtgtctcag aggccccctg agtgttcccc ttggcctgag ccccttaccc 107820attcctgcag ccagtgagag acctggcctc agccctggca gggctctctc ttcaaggcca 107880tatccacctg tgccctgggg cttgggagac cccatagggc cgggactctt gggtcagccc 107940ggccactggc ttctctcttt ttctccgttt cattctgtgt gcgttgtggg gtgggggagg 108000gggtccacct gccttacctt tctgagttgc ctttagagag atgcgttttt ctaggactct 108060gtgcaactgt cgtatatggt cccgtgggct gaccgctttg tacatgagaa taaatctatt 108120tctttctacc agtcctcccc catggggctg tttgcagact ttgtgcttgg ggtgggtgga 108180gggggggaat agaactggga gaggcaaacg ccctttggaa ctccatggct tccagggtcc 108240tccacccttg gtgcctagcc ccccttctgg ggaagtcata gacctgttgg ggtactccct 108300aggccagatc gtggaggcta aggggtgggt ggcagatgag aaggcctggc catggagcag 108360tgatgggaca tgttggctgg cagagattgt agaatagagg aaaaacaaag gttgaggcaa 108420gcaggcaggc tgcctggagg aggtagcctg gagcttgtcc tagaccctcc cagcgctggc 108480ctgccctggt catgagtgcc catacggcga gggcctaggc ctctgaactc tgtttctagc 108540tgcagtgatg cctggctgtg tcccaggaag tcccacatcc cagttactct gagtcctgcc 108600gaaggtgcac gcctgagtca gactccacac cagatccagc cccgggttgt gtctgaggag 108660ttgcgtctgt tcctctgcat gagagtgttt acttccgccc agtccaagat gggcagactg 108720caggttgggg ctacgcggag gctctgcctg gcacagtctc cagaccctgt ccccgacttg 108780cctacccccc tctgagctcc tctccgtgtt catctcttcc tggtcagtaa aggttgatgt 108840gttaagaggg tgggcactgg ggtctccttt cttggtggga gcaggaagga gatggacagg 108900gccatcctgt gaccatcagc cattgccagc tttgcctttg ggaccacaga gcccatctgc 108960ttcctctgca gctccccctg ccccactagc ctgtctgggt ttggaatctg ctcctctggc 109020tgaatggtct ccaggtttcc agcttccctt agcgtcatgg ggctccaggc tcctcccatt 109080cccagctcct gctgtgggct ccccaagtcc gtctctatcc tctcacagca caggacccag 109140gcttggccag tgggtccccg ggtgggggtg ggagtggtca gtttgtggcc cacggccaat 109200aagagatggc tattctaatg gtgcctggct gaccccaggg tcactgtggg ctgatgtagc 109260tgctcttctg cctgacccct gaccctgagt gtgtgtgcgt gttcctcttc cacaactctt 109320caggcaaaga gaaccttgac cctgcatctg tctgtcccca gcccagccct cctttgaggc 109380tcatgctgtg acacatccct gtttttcacc aaatggaggg aacaaccaca gatatttcct 109440tgtgcacgca ggaccctgtg ctagggctga gggctttgtc tttgtcctgc tctggaaagt 109500ctcacagttt gattggagag ctagatctaa actcagatgc aggccatgac aacgctgtgg 109560ggtgcccggc catggggctc caggcaggat cataaccctg agaacaacaa tgaggtttga 109620aagatgagca gatgttgttt ataggcaaaa ggggacaggc actcctggta gaagaaactg 109680cttttgcaaa ggcctcgaga acagaaggga ctggcaggtg gaggagccga gagatggagg 109740aggaggcaag gccagatcct gaagggcctt aaatgccagg ttgtggagtt tggctttatt 109800ctgtgggcag tggagaacca gagaaaggtt ttcagtagga gagtgactca gaagtgcatt 109860ttagaaagat ccccctggag agcagggaag tgactgcaag gggagagggt gggcagggat 109920tattctatgg gtgatgtgct gtgccctggg ctgggcgagg agaggaattc ggagatgcta 109980ggttggcaga acatggtgac cagtgggtcg ggggatgcag agggaggact tggaggggcc 110040ctgggaggtg gggtctatgc cactccatga agagctgtgg gggctctgtt cagcatcacc 110100ctcacccaca acaggtattg ggtggagcct ctggcagggg tgagctccct gcaaaggtga 110160gcaaaacagc tatctgagga tgcccaggga ggagaggtgg gaggaaggga gagaggacag 110220atgggaggag gctctgcaca gagcctgagg acagccctca ccaggttaca gaacacaagg 110280cttgacccca ttggcttcct gtagctgtcc tgctctccca acttaatggt ttcattttgc 110340attttattta aatttcacaa tgattctagc agataccatt agtctattct gcagccaagt 110400tgtctaaggt ttggagaggt taagtaatgc accaaggtta ggatttgagc cctacctgtc 110460tgattcccct ccgagagctg tctgattcct ttctcctcct ctgggatagg ggaaggagac 110520tcagaaggac ggggtctcca tcttcagtct ttgcaagact attgtagggc attgggatgg 110580tgagcacaaa gtgggttgaa gccccagaga aagagctgag agctgggatc aactgtgtgt 110640gtgcatgtgt gtgtctgtgt gtgtgtgagt tggagtaggg ggcagggaga aaagagtggg 110700gtggtggtgg cttgtagtgc agctcagggc caccaggtgg tgtccagccc tcgctgtcct 110760cacctcccca gaggtcagag aaggatatgg gagggggtgg ggtggggtga gggggacgcg 110820gcggggacgg gggggacggt ggttggtagt ctcactcctg tccattcacc tacaggttga 110880gtatccctta tccaaaatgc ttggggccag aagtgtctca gatttaagat ttttttcgga 110940ttttggaata tttgcatata

cataatgaga tatcttggga atgagacccc aggctaaaca 111000ggaaattcat ttatgtttta tatacacaca gcctgaagca gttttatata atattttgaa 111060taattttatg catgaaacaa agtttgtgca cattgaagca agtgtggaat tttccacttg 111120tggcattatg tcggtgctaa aaaatgtttt agattttgga gcattttgga tctcagaact 111180ttgcattagg aattgaggac taagtctgat attctgtctt acccagattc ctacctaaga 111240ggtctaggaa gtcatgccct acaaaccata cattctcatc ag 1112824905PRTHomo sapien 4Pro Pro Pro Pro Gln Pro Cys Ala Asp Ser Leu Gln Asp Ala Leu Leu1 5 10 15Ser Leu Gly Ser Val Ile Asp Ile Ser Gly Leu Gln Arg Ala Val Lys20 25 30Glu Ala Leu Ser Ala Val Leu Pro Arg Val Glu Thr Val Tyr Thr Tyr35 40 45Leu Leu Asp Gly Glu Ser Gln Leu Val Cys Glu Asp Pro Pro His Glu50 55 60Leu Pro Gln Glu Gly Lys Val Arg Glu Ala Ile Ile Ser Gln Lys Arg65 70 75 80Leu Gly Cys Asn Gly Leu Gly Phe Ser Asp Leu Pro Gly Lys Pro Leu85 90 95Ala Arg Leu Val Ala Pro Leu Ala Pro Asp Thr Gln Val Leu Val Met100 105 110Pro Leu Ala Asp Lys Glu Ala Gly Ala Val Ala Ala Val Ile Leu Val115 120 125His Cys Gly Gln Leu Ser Asp Asn Glu Glu Trp Ser Leu Gln Ala Val130 135 140Glu Lys His Thr Leu Val Ala Leu Arg Arg Val Gln Val Leu Gln Gln145 150 155 160Arg Gly Pro Arg Glu Ala Pro Arg Ala Val Gln Asn Pro Pro Glu Gly165 170 175Thr Ala Glu Asp Gln Lys Gly Gly Ala Ala Tyr Thr Asp Arg Asp Arg180 185 190Lys Ile Leu Gln Leu Cys Gly Glu Leu Tyr Asp Leu Asp Ala Ser Ser195 200 205Leu Gln Leu Lys Val Leu Gln Tyr Leu Gln Gln Glu Thr Arg Ala Ser210 215 220Arg Cys Cys Leu Leu Leu Val Ser Glu Asp Asn Leu Gln Leu Ser Cys225 230 235 240Lys Val Ile Gly Asp Lys Val Leu Gly Glu Glu Val Ser Phe Pro Leu245 250 255Thr Gly Cys Leu Gly Gln Val Val Glu Asp Lys Lys Ser Ile Gln Leu260 265 270Lys Asp Leu Thr Ser Glu Asp Val Gln Gln Leu Gln Ser Met Leu Gly275 280 285Cys Glu Leu Gln Ala Met Leu Cys Val Pro Val Ile Ser Arg Ala Thr290 295 300Asp Gln Val Val Ala Leu Ala Cys Ala Phe Asn Lys Leu Glu Gly Asp305 310 315 320Leu Phe Thr Asp Glu Asp Glu His Val Ile Gln His Cys Phe His Tyr325 330 335Thr Ser Thr Val Leu Thr Ser Thr Leu Ala Phe Gln Lys Glu Gln Lys340 345 350Leu Lys Cys Glu Cys Gln Ala Leu Leu Gln Val Ala Lys Asn Leu Phe355 360 365Thr His Leu Asp Asp Val Ser Val Leu Leu Gln Glu Ile Ile Thr Glu370 375 380Ala Arg Asn Leu Ser Asn Ala Glu Ile Cys Ser Val Phe Leu Leu Asp385 390 395 400Gln Asn Glu Leu Val Ala Lys Val Phe Asp Gly Gly Val Val Asp Asp405 410 415Glu Ser Tyr Glu Ile Arg Ile Pro Ala Asp Gln Gly Ile Ala Gly His420 425 430Val Ala Thr Thr Gly Gln Ile Leu Asn Ile Pro Asp Ala Tyr Ala His435 440 445Pro Leu Phe Tyr Arg Gly Val Asp Asp Ser Thr Gly Phe Arg Thr Arg450 455 460Asn Ile Leu Cys Phe Pro Ile Lys Asn Glu Asn Gln Glu Val Ile Gly465 470 475 480Val Ala Glu Leu Val Asn Lys Ile Asn Gly Pro Trp Phe Ser Lys Phe485 490 495Asp Glu Asp Leu Ala Thr Ala Phe Ser Ile Tyr Cys Gly Ile Ser Ile500 505 510Ala His Ser Leu Leu Tyr Lys Lys Val Asn Glu Ala Gln Tyr Arg Ser515 520 525His Leu Ala Asn Glu Met Met Met Tyr His Met Lys Val Ser Asp Asp530 535 540Glu Tyr Thr Lys Leu Leu His Asp Gly Ile Gln Pro Val Ala Ala Ile545 550 555 560Asp Ser Asn Phe Ala Ser Phe Thr Tyr Thr Pro Arg Ser Leu Pro Glu565 570 575Asp Asp Thr Ser Met Ala Ile Leu Ser Met Leu Gln Asp Met Asn Phe580 585 590Ile Asn Asn Tyr Lys Ile Asp Cys Pro Thr Leu Ala Arg Phe Cys Leu595 600 605Met Val Lys Lys Gly Tyr Arg Asp Pro Pro Tyr His Asn Trp Met His610 615 620Ala Phe Ser Val Ser His Phe Cys Tyr Leu Leu Tyr Lys Asn Leu Glu625 630 635 640Leu Thr Asn Tyr Leu Glu Asp Ile Glu Ile Phe Ala Leu Phe Ile Ser645 650 655Cys Met Cys His Asp Leu Asp His Arg Gly Thr Asn Asn Ser Phe Gln660 665 670Val Ala Ser Lys Ser Val Leu Ala Ala Leu Tyr Ser Ser Glu Gly Ser675 680 685Val Met Glu Arg His His Phe Ala Gln Ala Ile Ala Ile Leu Asn Thr690 695 700His Gly Cys Asn Ile Phe Asp His Phe Ser Arg Lys Asp Tyr Gln Arg705 710 715 720Met Leu Asp Leu Met Arg Asp Ile Ile Leu Ala Thr Asp Leu Ala His725 730 735His Leu Arg Ile Phe Lys Asp Leu Gln Lys Met Ala Glu Val Gly Tyr740 745 750Asp Arg Asn Asn Lys Gln His His Arg Leu Leu Leu Cys Leu Leu Met755 760 765Thr Ser Cys Asp Leu Ser Asp Gln Thr Lys Gly Trp Lys Thr Thr Arg770 775 780Lys Ile Ala Glu Leu Ile Tyr Lys Glu Phe Phe Ser Gln Gly Asp Leu785 790 795 800Glu Lys Ala Met Gly Asn Arg Pro Met Glu Met Met Asp Arg Glu Lys805 810 815Ala Tyr Ile Pro Glu Leu Gln Ile Ser Phe Met Glu His Ile Ala Met820 825 830Pro Ile Tyr Lys Leu Leu Gln Asp Leu Phe Pro Lys Ala Ala Glu Leu835 840 845Tyr Glu Arg Val Ala Ser Asn Arg Glu His Trp Thr Lys Val Ser His850 855 860Lys Phe Thr Ile Arg Gly Leu Pro Ser Asn Asn Ser Leu Asp Phe Leu865 870 875 880Asp Glu Glu Tyr Glu Val Pro Asp Leu Asp Gly Thr Arg Ala Pro Ile885 890 895Asn Gly Cys Cys Ser Leu Asp Ala Glu900 9055921PRTBos taurus 5Met Arg Arg Gln Pro Ala Ala Ser Arg Asp Leu Phe Ala Gln Glu Pro1 5 10 15Val Pro Pro Gly Ser Gly Asp Gly Ala Leu Gln Asp Ala Leu Leu Ser20 25 30Leu Gly Ser Val Ile Asp Val Ala Gly Leu Gln Gln Ala Val Lys Glu35 40 45Ala Leu Ser Ala Val Leu Pro Lys Val Glu Thr Val Tyr Thr Tyr Leu50 55 60Leu Asp Gly Glu Ser Arg Leu Val Cys Glu Glu Pro Pro His Glu Leu65 70 75 80Pro Gln Glu Gly Lys Val Arg Glu Ala Val Ile Ser Arg Lys Arg Leu85 90 95Gly Cys Asn Gly Leu Gly Pro Ser Asp Leu Pro Gly Lys Pro Leu Ala100 105 110Arg Leu Val Ala Pro Leu Ala Pro Asp Thr Gln Val Leu Val Ile Pro115 120 125Leu Val Asp Lys Glu Ala Gly Ala Val Ala Ala Val Ile Leu Val His130 135 140Cys Gly Gln Leu Ser Asp Asn Glu Glu Trp Ser Leu Gln Ala Val Glu145 150 155 160Lys His Thr Leu Val Ala Leu Lys Arg Val Gln Ala Leu Gln Gln Arg165 170 175Glu Ser Ser Val Ala Pro Glu Ala Thr Gln Asn Pro Pro Glu Glu Ala180 185 190Ala Gly Asp Gln Lys Gly Gly Val Ala Tyr Thr Asn Gln Asp Arg Lys195 200 205Ile Leu Gln Leu Cys Gly Glu Leu Tyr Asp Leu Asp Ala Ser Ser Leu210 215 220Gln Leu Lys Val Leu Gln Tyr Leu Gln Gln Glu Thr Gln Ala Ser Arg225 230 235 240Cys Cys Leu Leu Leu Val Ser Glu Asp Asn Leu Gln Leu Ser Cys Lys245 250 255Val Ile Gly Asp Lys Val Leu Glu Glu Glu Ile Ser Phe Pro Leu Thr260 265 270Thr Gly Arg Leu Gly Gln Val Val Glu Asp Lys Lys Ser Ile Gln Leu275 280 285Lys Asp Leu Thr Ser Glu Asp Met Gln Gln Leu Gln Ser Met Leu Gly290 295 300Cys Glu Val Gln Ala Met Leu Cys Val Pro Val Ile Ser Arg Ala Thr305 310 315 320Asp Gln Val Val Ala Leu Ala Cys Ala Phe Asn Lys Leu Gly Gly Asp325 330 335Leu Phe Thr Asp Gln Asp Glu His Val Ile Gln His Cys Phe His Tyr340 345 350Thr Ser Thr Val Leu Thr Ser Thr Leu Ala Phe Gln Lys Glu Gln Lys355 360 365Leu Lys Cys Glu Cys Gln Ala Leu Leu Gln Val Ala Lys Asn Leu Phe370 375 380Thr His Leu Asp Asp Val Ser Val Leu Leu Gln Glu Ile Ile Thr Glu385 390 395 400Ala Arg Asn Leu Ser Asn Ala Glu Ile Cys Ser Val Phe Leu Leu Asp405 410 415Gln Asn Glu Leu Val Ala Lys Val Phe Asp Gly Gly Val Val Glu Asp420 425 430Glu Ser Tyr Glu Ile Arg Ile Pro Ala Asp Gln Gly Ile Ala Gly His435 440 445Val Ala Thr Thr Gly Gln Ile Leu Asn Ile Pro Asp Ala Tyr Ala His450 455 460Pro Leu Phe Tyr Arg Gly Val Asp Asp Ser Thr Gly Phe Arg Thr Arg465 470 475 480Asn Ile Leu Cys Phe Pro Ile Lys Asn Glu Asn Gln Glu Val Ile Gly485 490 495Val Ala Glu Leu Val Asn Lys Ile Asn Gly Pro Trp Phe Ser Lys Phe500 505 510Asp Glu Asp Leu Ala Thr Ala Phe Ser Ile Tyr Cys Gly Ile Ser Ile515 520 525Ala His Ser Leu Leu Tyr Lys Lys Val Asn Glu Ala Gln Tyr Arg Ser530 535 540His Leu Ala Asn Glu Met Met Met Tyr His Met Lys Val Ser Asp Asp545 550 555 560Glu Tyr Thr Lys Leu Leu His Asp Gly Ile Gln Pro Val Ala Ala Ile565 570 575Asp Ser Asn Phe Ala Ser Phe Thr Tyr Thr Pro Arg Ser Leu Pro Glu580 585 590Asp Asp Thr Ser Met Ala Ile Leu Ser Met Leu Gln Asp Met Asn Phe595 600 605Ile Asn Asn Tyr Lys Ile Asp Cys Pro Thr Leu Ala Arg Phe Cys Leu610 615 620Met Val Lys Lys Gly Tyr Arg Asp Pro Pro Tyr His Asn Trp Met His625 630 635 640Ala Phe Ser Val Ser His Phe Cys Tyr Leu Leu Tyr Lys Asn Leu Glu645 650 655Leu Thr Asn Tyr Leu Glu Asp Met Glu Ile Phe Ala Leu Phe Ile Ser660 665 670Cys Met Cys His Asp Leu Asp His Arg Gly Thr Asn Asn Ser Phe Gln675 680 685Val Ala Ser Lys Ser Val Leu Ala Ala Leu Tyr Ser Ser Glu Gly Ser690 695 700Val Met Glu Arg His His Phe Ala Gln Ala Ile Ala Ile Leu Asn Thr705 710 715 720His Gly Cys Asn Ile Phe Asp His Phe Ser Arg Lys Asp Tyr Gln Arg725 730 735Met Leu Asp Leu Met Arg Asp Ile Ile Leu Ala Thr Asp Leu Ala His740 745 750His Leu Arg Ile Phe Lys Asp Leu Gln Lys Met Ala Glu Val Gly Tyr755 760 765Asp Arg Thr Asn Lys Gln His His Ser Leu Leu Leu Cys Leu Leu Met770 775 780Thr Ser Cys Asp Leu Ser Asp Gln Thr Lys Gly Trp Lys Thr Thr Arg785 790 795 800Lys Ile Ala Glu Leu Ile Tyr Lys Glu Phe Phe Ser Gln Gly Asp Leu805 810 815Glu Lys Ala Met Gly Asn Arg Pro Met Glu Met Met Asp Arg Glu Lys820 825 830Ala Tyr Ile Pro Glu Leu Gln Ile Ser Phe Met Glu His Ile Ala Met835 840 845Pro Ile Tyr Lys Leu Leu Gln Asp Leu Phe Pro Lys Ala Ala Glu Leu850 855 860Tyr Glu Arg Val Ala Ser Asn Arg Glu His Trp Thr Lys Val Ser His865 870 875 880Lys Phe Thr Ile Arg Gly Leu Pro Ser Asn Asn Ser Leu Asp Phe Leu885 890 895Asp Glu Glu Tyr Glu Val Pro Asp Leu Asp Gly Ala Arg Ala Pro Ile900 905 910Asn Gly Cys Cys Ser Leu Asp Ala Glu915 920

Claims

1. An isolated peptide comprising an amino acid sequence selected from the group consisting of:(a) an amino acid sequence shown in SEQ ID NO:2;(b) an amino acid sequence of an allelic variant of an amino acid sequence shown in SEQ ID NO:2, wherein said allelic variant is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3;(c) an amino acid sequence of an ortholog of an amino acid sequence shown in SEQ ID NO:2, wherein said ortholog is encoded by a nucleic acid molecule that hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3; and(d) a fragment of an amino acid sequence shown in SEQ ID NO:2, wherein said fragment comprises at least 10 contiguous amino acids.

2. An isolated antibody that selectively binds to a peptide of claim 1.

3. An isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of:(a) a nucleotide sequence that encodes an amino acid sequence shown in SEQ ID NO:2;(b) a nucleotide sequence that encodes of an allelic variant of an amino acid sequence shown in SEQ ID NO:2, wherein said nucleotide sequence hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3;(c) a nucleotide sequence that encodes an ortholog of an amino acid sequence shown in SEQ ID NO:2, wherein said nucleotide sequence hybridizes under stringent conditions to the opposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3;(d) a nucleotide sequence that encodes a fragment of an amino acid sequence shown in SEQ ID NO:2, wherein said fragment comprises at least 10 contiguous amino acids; and(e) a nucleotide sequence that is the complement of a nucleotide sequence of (a)-(d).

4. A nucleic acid vector comprising a nucleic acid molecule of claim 3.

5. A host cell containing the vector of claim 4.

6. A method for producing any of the peptides of claim 1 comprising introducing a nucleotide sequence encoding any of the amino acid sequences in (a)-(d) into a host cell, and culturing the host cell under conditions in which the peptides are expressed from the nucleotide sequence.

7. A method for detecting the presence of any of the peptides of claim 1 in a sample, said method comprising contacting said sample with a detection agent that specifically allows detection of the presence of the peptide in the sample and then detecting the presence of the peptide.

8. A method for detecting the presence of a nucleic acid molecule of claim 3 in a sample, said method comprising contacting the sample with an oligonucleotide that hybridizes to said nucleic acid molecule under stringent conditions and determining whether the oligonucleotide binds to said nucleic acid molecule in the sample.

9. A method for identifying a modulator of a peptide of claim 1, said method comprising contacting said peptide with an agent and determining if said agent has modulated the function or activity of said peptide.

10. The method of claim 9, wherein said agent is administered to a host cell comprising an expression vector that expresses said peptide.

11. A method for identifying an agent that binds to any of the peptides of claim 1, said method comprising contacting the peptide with an agent and assaying the contacted mixture to determine whether a complex is formed with the agent bound to the peptide.

12. A pharmaceutical composition comprising an agent identified by the method of claim 11 and a pharmaceutically acceptable carrier therefor.

13. A method for treating a disease or condition mediated by a human phosphodiesterase protein, said method comprising administering to a patient a pharmaceutically effective amount of an agent identified by the method of claim 11.

14. A method for identifying a modulator of the expression of a peptide of claim 1, said method comprising contacting a cell expressing said peptide with an agent, and determining if said agent has modulated the expression of said peptide.

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