Patent Application: US-6006502-A

Abstract:
selection of clones having the kinase and / or phosphatase - like structure from clones which had been isolated and the structures thereof had been determined in the helix research institute was conducted . two novel genes were provided by carrying out homology search for all the helix clones by using the amino acid sequences of known kinases and phosphatases as queries . the genes are expected to be involved in intracellular signal transduction . the physiological functions of the inventive genes can be tested by using reporter gene assay systems capable of detecting signal transduction . the proteins of the present invention are useful as target molecules in drug discovery and in the development of new pharmaceuticals .

Description:
the invention is illustrated more specifically with reference to the following examples , but is not to be construed as being limited thereto . the nt - 2 neuron progenitor cells ( stratagene ), teratocarcinoma cells from human fetal testis , which can be differentiated into neurons by the treatment with retinoic acid were cultured for two weeks after induction treatment by the addition of retinoic acid according to the manufacturer &# 39 ; s instructions . after the culture , the respective cells were collected , and mrna was extracted according to the method described in the literature ( sambrook et al ., molecular cloning 2nd edition , cold spring harbor laboratory press , 1989 ). then , poly ( a ) + rna was purified by using oligo dt cellulose . similarly , human ovary cancer tissue ( ovarc1 ) was used to extract mrna by the method described in the literature ( sambrook et al ., molecular cloning 2nd edition , cold spring harbor laboratory press , 1989 ). furthermore , poly ( a ) + rna was purified from the mrna using oligo - dt cellulose . this poly ( a ) + rna was used to construct a cdna library by the oligo - capping method ( maruyama et al ., gene 138 : 171 - 174 , 1994 ). using the oligo - cap linker ( agcaucgagu cggccuuguu ggccuacugg / seq id no : 5 ) and the oligo - dt primer ( gcggctgaag acggcctatg tggccttttt tttttttt tt / seq id no : 6 ), bacterial alkaline phosphatase ( bap ) treatment , tobacco acid phosphatase ( tap ) treatment , rna ligation , the first strand cdna synthesis , and removal of rna were performed according to the references ( suzuki et al ., protein , nucleic acid and enzyme , 41 : 197 - 201 , 1996 ; suzuki et al ., gene 200 : 149 - 156 , 1997 ). then , 5 ′- and 3 ′- pcr primers ( agcatcgagt cggccttgtt g / seq id no : 7 , and gcggctgaag acggcctatg t / seq id no : 8 , respectively ) were used for performing pcr to convert the cdna into double stranded cdna , which was then digested with sfii . then , the draiii - cleaved vector puc19fl3 or pme18sfl3 ( genbank ab009864 , expression vector ) ( nt2rp3 , ovarc1 ) was used for cloning the cdna in a unidirectional manner , and cdna libraries were obtained . the nucleotide sequence of the 5 ′- and 3 ′- ends of the cdna clones was analyzed with a dna sequencer ( abi prism 377 , pe biosystems ) after sequencing reactions performed with the dna sequencing reagents ( dye terminator cycle sequencing fs ready reaction kit , drhodamine terminator cycle sequencing fs ready reaction kit , or bigdye terminator cycle sequencing fs ready reaction kit , pe biosystems ), according to the instructions . the obtained data were used for a database . oligo - cap high full - length ratio cdna library of nt2rp3 was prepared by using an expression vector , pme18sfl3 , which can be expressed in eukaryotic cells . pme18sfl3 vector contains the srα promoter and sv40 small t intron in the upstream , as well as the sv40 polya addition signal sequence downstream of the cloning site , respectively . as the cloning site of pme18sfl3 has asymmetrical draiii sites , and the ends of cdna fragments contain sfii sites complementary to the draiii sites , the cloned cdna fragments can be unidirectionally inserted downstream of the srα promoter . therefore , clones containing full - length cdna can be expressed transiently by introducing the obtained plasmid directly into cos cells . thus , the clones can be analyzed very easily in terms of the proteins that are the gene products of the clones , or in terms of the biological activities of the proteins . estimation of the completeness at the 5 ′- ends of the clones contained in the cdna libraries constructed by the oligo - capping method the full - length ratio at the 5 ′- end sequence of respective clones in the human cdna libraries constructed by the oligo - capping method was determined as follows . the clones whose 5 ′- end sequences were consistent with those of known human mrna in the public database were judged to be “ full - length ” if they had a longer 5 ′- end sequence than that of the known human mrna ; or even though the 5 ′- end sequence was shorter , if it contained the translation initiation codon it was judged to have the “ full - length ” sequence . clones which did not contain the translation initiation codon were judged to be “ not - full - length ”. the full - length ratio (( the number of full - length clones )/( the number of full - length and not - full - length clones )) at the 5 ′- end of the cdna clones from each library was determined by comparing with known human mrna . as a result , the full - length ratio of the 5 ′- ends was 63 . 5 %. the result indicates that the full - length ratio at the 5 ′- end sequence was extremely high in the human cdna clones obtained by the oligo - capping method . assessment of the full - length ratio of the 5 ′- end of the cdna by the atgpr and the estimatefl the atgpr , developed by salamov a . a ., nishikawa t ., and swindells m . b . in the helix research institute , is a program for prediction of the translation initiation codon based on the characteristics of the sequences in the vicinity of the atg codon ( salamov et al ., bioinformatics 14 : 384 - 390 , 1998 ; http :// www . hri . cojp / atgpr /). the results are shown with expectations ( also mentioned as atgpr1 below ) whether the atg is a true initiation codon ( 0 . 05 - 0 . 94 ). when it was not considered that the sequence was the 5 ′- end of the cdna or not , both of the sensitivity and specificity of analytical results by this program were estimated as 66 %. when the program was applied to the 5 ′- end sequences of the clones from the cdna library that was obtained by the oligo - capping method having 65 % full - length ratio , the sensitivity and specificity of the estimation of the full - length clone ( clone containing the n - terminus of the orf ) were improved to 82 to 83 % by selecting only clones having an atgpr1 score 0 . 6 or higher . the maximum atgpr1 scores for 5 ′- end sequences of nt2rp3001938 and ovarc1000945 were 0 . 32 and 0 . 74 , respectively . next , the estimatefl was used for the assessment of the clones . the estimatefl , developed by nishikawa and ota in the helix research institute , is a method for selecting clones expected to have a full - length cdna by comparing with the 5 ′- end or 3 ′- end sequences of ests in the public database . by this method , a cdna clone is judged to be most likely not to be full - length if there exist any ests which have longer 5 ′- end or 3 ′- end sequences than the clone . the method is systematized for high throughput analysis . a clone is judged to be full - length if the clone has a longer 5 ′- end sequence than the ests in the public database corresponding thereto . even if a clone has a shorter 5 ′- end , the clone is judged to be full - length if the difference in length is within 50 bases , and otherwise judged not to be full - length , for convenience . those clones whose 5 ′- end sequence is matching with the known mrna , about 80 % of the clones judged to be full - length by the comparison with ests were also judged to be full - length by the assessment of the 5 ′- end sequence by comparing with known mrna . also , about 80 % of the clones judged to be not full - length in the 5 ′- end sequence by comparing with ests were also judged to be not full - length in the 5 ′- end sequence by comparison with known mrna . the precision of the estimation by comparing with ests is improved with increasing numbers of ests to be compared . however , in case with limited numbers of ests , the reliability becomes low . thus , the method is effective in excluding clones with high probability of being not - full - length from the cdna clones that is synthesized by the oligo - capping method having a 5 ′- end sequence full - length ratio of about 60 %. in particular , the estimatefl is efficiently used in estimating the full - length ratio at the 3 ′- end sequence of cdna of a human unknown mrna , a significant number of which are deposited in the public database as est deposits . results of the above assessment for the full - length ratio showed that the clone “ c - ovarc1000945 ” was a novel clone with a high probability of being full - length and also which shares no sequence identity with any of human est sequences at least either at the 5 ′- end sequence or 3 ′- end sequence , or both ends . furthermore , “ c - nt2rp3001938 ” is also a full - length clone ; the number of human est sequences that shared a common sequence to each of these clones at the 5 ′- end was 20 or less ( clones which do not share sequences with certain human est sequences at least either at the 5 ′- end or at 3 ′- end , or at both ends of the clone ; excluding clones in which the number of human est sequences that shared a common sequence to each of the clones at both of the 5 ′- and 3 - end was 1 or more and 5 or less ). accordingly , they were concluded to be novel clones . clones having a kinase / phosphatase - like sequence were selected from the helix clones . all the helix clones were searched for homology by ncbi tblastn2 . 0 by using the following 31 amino acid sequences of known kinases and phosphatases ( also including phospholipid kinases ) as queries . clones with a expectation value ( expect ) 1 . 0e - 05 or lower were selected . the query sequences used in the homology search as well as their seq id nos and genbank accession numbers are as follows . query sequence no . seq id no : genbank accession no . hlkb1 9 gi | 3024670 hvrk1 10 gi | 4507903 hcdc2 11 gi | 4502709 haurorak1 12 gb | aac12708 . 1 haurorak2 13 gi | 4759178 hikka 14 gb | aac51662 . 1 hmkk3 15 gb | aab40653 . 1 herk1 16 pir | a48082 hraf1 17 gi | 4506401 hakt 18 gi | 4885061 hpikp85 19 sp | p27986 hatm 20 gi | 4502267 hc - src 21 gi | 4758078 hjak1 22 ref | np_002218 . 1 hflt1 23 gb | aac16449 . 1 hpp2a 24 gi | 4506017 hmkp2 25 gb | aac50452 . 1 hvhr 26 gi | 4758208 hptp - sl 27 gi | 4506325 hstep 28 sp | p54829 hpten 29 gi | 4506249 cdc14b1 30 gb | aad15415 . 1 dusp12 31 gi | 6005956 ak000449 32 gi | 8923413 dus7 33 sp | q16829 calcineurin a alpha 34 gi | 6715568 pnp1 35 emb | caa56124 . 1 tpte 36 gi | 7019559 ppp1cc 37 gi | 4506007 pp - 1 gamma 38 gb | aaa19823 . 1 pp2a 39 gi | 4506017 based on the result , non - overlapping 2 clones , c - nt2rp3001938 and c - ovarc 1000945 , were selected as clones having kinase / phosphatase - like structure ( kp clones ). the clones encode novel human proteins , and each of the proteins was deduced to function as a protein kinase and / or a protein phosphatase . dna for spotting onto the nylon membranes was prepared according to the following procedure . e . coli was cultured in each well of a 96 - well plate ( in a lb medium at 37 ° c . for 16 hours ). a part of each culture was suspended in 10 μl of sterile water in the well of a 96 - well plate . the plate was heated at 100 ° c . for 10 minutes . then the samples were analyzed by pcr . pcr was performed in a 20 μl solution per one reaction by using takara pcr amplification kit ( takara ) according to the supplier &# 39 ; s protocol . a pair of sequencing primers , me761fw ( 5 ′ tacggaagtgttacttctgc 3 ′/ seq id no : 40 ) and me1250rv ( 5 ′ tgtgggaggffttttctcta 3 ′/ seq id no : 41 ), or a pair of primers , m13m4 ( 5 ′ gttttcccagtcacgac 3 ′/ seq id no : 42 ) and m13rv ( 5 ′ caggaaacagctatgac 3 ′/ seq id no : 43 ) were used for the amplification of the insert cdna in the plasmid . pcr was performed in a thermal cycler , geneamp system 9600 ( pe biosystems ). the cycling profile consisted of pre - heating at 95 ° c . for 5 minutes ; 10 cycles of denaturation at 95 ° c . for 10 seconds , and annealing / extension at 68 ° c . for 1 minute ; 20 cycles of denaturation at 98 ° c . for 20 seconds and annealing / extension at 60 ° c . for 3 minutes ; and final extension at 72 ° c . for 10 minutes . after the pcr , 2 μl of the reaction solution was electrophoresed on a 1 % agarose gel . dna on the gel was stained with ethidium bromide to confirm the amplification of cdna . when cdnas were not amplified by pcr , plasmids containing the corresponding insert cdnas were prepared by the alkali - extraction method ( sambrook et al ., molecular cloning , a laboratory manual , 2nd edition , cold spring harbor laboratory press , 1989 ). dna array was prepared by the following procedure . an aliquot of the dna solution was added to each well of a 384 - well plate . dna was spotted onto a nylon membrane ( boehringer ) by using a 384 - pin tool of biomek 2000 laboratory automation system ( beckman - coulter ). more specifically , the 384 - well plate containing the dna was placed under the 384 - pin tool . the independent 384 needles of the pin tool were simultaneously dipped into the dna solution to fix the dna on the needles . the needles were gently pressed onto a nylon membrane , and the dna fixed on the needles was spotted onto the membrane . denaturation of the spotted dna and immobilization of the dna on the nylon membrane were carried out according to conventional methods ( sambrook et al ., molecular cloning , a laboratory manual , 2nd edition , cold spring harbor laboratory press , 1989 ). 1 st strand cdna labeled with radioisotope was used as the hybridization probe . the 1 st strand cdna was synthesized by using thermoscript ™ rt - pcr system ( gibco ). more specifically , the 1st strand cdna was synthesized by using 1 . 5 μg mrnas from various human tissues ( clontech ), 1 μl 50 μm oligo ( dt ) 20 , and 50 μci [ α 33 p ] datp according to the attached protocol . purification of the probe was carried out by using probequant ™ g - 50 micro column ( amersham - pharmacia biotech ) according to the attached protocol . in the next step , 2 units of e . coli rnaseh were added to the reaction mixture . the mixture was incubated at room temperature for 10 minutes , and then 100 μg of human cot - 1 dna ( gibco ) was added thereto . the mixture was incubated at 97 ° c . for 10 minutes , and then was allowed to stand on ice to give the hybridization probe . hybridization of the radioisotope - labeled probe to the dna array was performed in a usual manner ( sambrook et al ., molecular cloning , a laboratory manual , 2nd edition , cold spring harbor laboratory press , 1989 ). the membrane was washed as follows : the nylon membrane was washed three times by incubating in the washing solution 1 ( 2 × ssc , 1 % sds ) at room temperature ( about 26 ° c .) for 20 minutes ; then the membrane was washed 3 times by incubating it in the washing solution 2 ( 0 . 1 × ssc , 1 % sds ) at 65 ° c . for 20 minutes . autoradiography was performed by using an image plate for bas2000 ( fuji photo film co ., ltd .). specifically , the nylon membrane used for the hybridization was wrapped with a piece of saran wrap , and was contacted with the light - sensitive surface of the image plate . the membrane with the image plate was placed in an imaging cassette for radioisotope and was allowed to stand in dark for 4 hours . the radioactivity recorded on the image plate was analyzed by bas2000 ( fuji photo film co ., ltd .) and was recorded as an image file of the autoradiogram by electronic conversion . the signal intensity of each dna spot was analyzed by using visage high density grid analysis systems ( genomic solutions inc .). the signal intensity was converted into numerical data . the data were taken by duplicated measurements . the reproducibility was assessed by comparing the signal intensities of the corresponding spots on the duplicated dna filters that were hybridized to a single dna probe . the ratio between the corresponding spots falls within a range of 2 - folds or less in 95 % of entire spots , and the correlation coefficient was r = 0 . 97 . thus , the reproducibility was assumed to be satisfactory . the detection sensitivity in gene expression analysis was estimated by examining increases in the signal intensity of the probe concentration - dependent spot of the hybridization using a probe complementary to the dna spotted on the nylon membrane . place1008092 ( the same dna as that deposited in genbank accession no . af107253 ) was used as the dna . the dna array with the dna of place1008092 was prepared according to the above - mentioned method . the probe was prepared as follows : mrna was synthesized in vitro from the clone , place1008092 ; using this mrna as the template , radioisotope - labeled 1st strand cdna was synthesized in the same manner as the probe preparation method described above ; and the cdna was used as the probe . the cdna place1008092 was inserted into pbluescript sk (−), so that the 5 ′- end of the place1008092 is ligated to the t7 promoter of the pbluescript sk (−) to give a recombinant plasmid for in vitro synthesis of the mrna from place1008092 . specifically , the place1008092 inserted at the draiii site of the pme18sfl3 was cut out by xhoi digestion . the resulting place1008092 fragment was ligated to xhoi - predigested pbluescript sk (−) by using the dna ligation kit ver . 2 ( takara ). the in - vitro mrna synthesis from place1008092 inserted in pbluescript sk (−) was carried out by using the ampliscribe ™ t7 high yield transcription kit ( epicentre technologies ). the hybridization and analysis of signal intensity of each dna spot were conducted using the same methods described above . when the probe concentration was 1 × 10 7 μg / ml or less , there was no increase of signal intensity proportional to the probe concentration . therefore it was assumed to be difficult to compare the signals with one another in this concentration range . thus , spots with a intensity of 40 or less were indiscriminately taken as low - level signals . within a concentration of the probe ranging from 1 × 10 7 μg / ml to 0 . 1 μg / ml , signals were found to increase in a probe concentration - dependent manner . the detection sensitivity is 1 : 100 , 000 in a ratio of mrna expression level in a sample . table 2 shows the expression of each cdna in human normal tissues ( heart , lung , pituitary gland , thymus , brain , kidney , liver and spleen ). the expression levels are indicated by numerical values of 0 to 10 , 000 . each of the “ c - nt2rp3001938 ” and “ c - ovarc1000945 ” was expressed in at least one tissue . non - enzymic protein glycation reaction is believed to be a cause of a variety of chronic diabetic complications . accordingly , genes of which expression is elevated or decreased in a glycated protein - specific manner are associated with diabetic complications caused by glycated proteins . vascular endothelial cells are affected with glycated proteins present in blood . reaction products of non - enzymic protein glycation include amadori compound ( glycated protein ) as a mildly glycated protein and advanced glycation endproduct as a heavily glycated protein . hence , whether or not the expression of the kp genes of this invention was varied depending on the presence of these proteins in endothelial cells was examined . the mrnas were extracted from endothelial cells that were cultured in the presence or absence of glycated protein . the mrnas were converted into radiolabeled first strand cdnas for preparing probes . the probes were hybridized to the above - mentioned dna array . signal of each dna spot was detected by bas2000 and analyzed by arraygauge ( fuji photo film co ., ltd .). advanced glycation endproduct of bovine serum albumin was prepared as follows : bovine serum albumin ( bsa ; sigma ) was incubated in a phosphate buffer solution containing 50 mm glucose at 37 ° c . for 8 weeks ; and the resulting brownish bsa was dialyzed against a phosphate buffer solution . human normal pulmonary arterial endothelial cells ( cell applications ) were cultured in an endothelial cell growth medium ( cell applications ). the culture dish ( falcon ) with the cells was incubated in a co 2 incubator ( 37 ° c ., 5 % co 2 , in a humid atmosphere ). when the cells were grown to be confluent in the dish , 250 μg / ml of bovine serum albumin ( sigma ), glycated bovine serum albumin ( sigma ) or advanced glycation endproduct of serum albumin was added thereto and the cells were incubated for 33 hours . the mrna was extracted from the cells by using a fasttrack ™ 2 . 0 kit ( invitrogen ). the labeling of hybridization probe was carried out by using the mrna according to the same procedure as described above . table 3 shows the expression level of each cdna in human pulmonary arterial endothelial cells cultured in a medium containing bovine serum albumin , glycated bovine serum albumin or advanced glycation endproduct of bovine serum albumin . the expression of “ c - nt2rp3001938 ” was detected in the endothelial cell . it is known that ultraviolet rays give considerably adverse influence on health . in recent years , the risks of tissue damage by ultraviolet rays has been increased due to the destruction of the ozone layer , and ultraviolet radiation has been recognized as a risk factor for diseases such as skin cancers ( united states environmental protection agency : ozone depletion home page , http :// www . epa . gov / ozone /). genes whose expression levels change with exposure of the skin epidermal cells to ultraviolet rays are considered to be associated with skin damage caused by ultraviolet radiation . culturing primary cultured skin fibroblast cells irradiated with ultraviolet ray , it was examined whether the expression of kp genes of this invention varies depending on the irradiation of ultraviolet ray . first , after culturing to confluence in a culture dish , the primary cultured skin fibroblast cells ( cell applications ) were exposed to 10 , 000 pj / cm 2 of 254 - nm ultraviolet light . thereafter , messenger rnas were extracted by using a fasttrack ™ 2 . 0 mrna isolation kit ( invitrogen ) from the unexposed cells and from the cells that were exposed to the ultraviolet light and then cultured for 4 or 24 hours . the labeling of the hybridization probe was carried out by using 1 . 5 μg of each mrna in the same manner as described above . the data were obtained in triplicate ( n = 3 ). the hybridization signals were compared between the cells exposed to the ultraviolet light and the unexposed cells . the comparison was preformed by statistical treatment with two - sample t - test . clones with significant differences in the signal distribution were selected under the condition of p & lt ; 0 . 05 . according to the analysis , the difference in the signal values can be also detected statistically even when the signal values are low . accordingly , clones with signal value of 40 or lower were also assessed . table 4 shows the expression of each cdna in skin - derived fibroblast cells exposed and unexposed to ultraviolet light . averaged signal values ( m 1 , m 2 ) and sample variances ( s 1 2 , s 2 2 ) were calculated for each gene in each of the cells , and then , pooled sample variances s were obtained from the sample variances of the two types of cells to be compared . the t values were determined according to the following formula : t =( m 1 − m 2 )/ s /( 1 / 3 + 1 / 3 ) 1 / 2 . when the determined t - value was greater than a t - value at p , probability of significance level , of 0 . 05 or 0 . 01 in the t - distribution table with 4 degrees of freedom , it was judged that a difference exists in the expression level of the gene between the two types of cells at p & lt ; 0 . 05 or p & lt ; 0 . 01 , respectively . the table also includes the information of an increase (+) or decrease (−) in the average expression level of a signal in the clones compared with that of undifferentiated cells . the results showed that the expression level of “ c - ovarc1000945 ” was reduced 4 hours or 24 hours after ultraviolet ray irradiation , suggesting that it is a clone associated with ultraviolet ray disorders . the present invention provides novel human protein kinase and protein phosphatase proteins , as well as genes encoding the proteins . the regulation of the phosphorylation state of proteins by kinase and / or phosphatase plays central roles in normal differentiation and / or proliferation of cells , as well as in physiological functions at the cellular level . the novel kinases and phosphatases of the present invention can be assumed to be closely associated with intracellular physiological functions , and thus , the inventive proteins are useful as target molecules of agents in the development of pharmaceuticals . furthermore , agents acting on the inventive proteins are expected to be effective pharmaceuticals which can control intracellular physiological functions more precisely than agents represented by previous receptor agonists and antagonists . attca atg tct ctt ttg gat tgc ttc tgc act tca aga aca caa gtt gaa 410 met ser leu leu asp cys phe cys thr ser arg thr gln val glu tca ctc aga cct gaa aaa cag tct gaa acc agt atc cat caa tac ttg 458 ser leu arg pro glu lys gln ser glu thr ser ile his gln tyr leu gtt gat gag cca acc ctt tcc tgg tca cgt cca tcc act aga gcc agt 506 gaa gta cta tgt tcc acc aac gtt tct cac tat gag ctc caa gta gaa 554 ata gga aga gga ttt gac aac ttg act tct gtc cat ctt gca cgg cat 602 ile gly arg gly phe asp asn leu thr ser val his leu ala arg his act ccc aca gga aca ctg gta act ata aaa att aca aat ctg gaa aac 650 tgc aat gaa gaa cgc ctg aaa gct tta cag aaa gcc gtg att cta tcc 698 cys asn glu glu arg leu lys ala leu gln lys ala val ile leu ser cac ttt ttc cgg cat ccc aat att aca act tat tgg aca gtt ttc act 746 gtt ggc agc tgg ctt tgg gtt att tct cca ttt atg gcc tat ggt tca 794 val gly ser trp leu trp val ile ser pro phe met ala tyr gly ser gca agt caa ctc ttg agg acc tat ttt cct gaa gga atg agt gaa act 842 ala ser gln leu leu arg thr tyr phe pro glu gly met ser glu thr tta ata aga aac att ctc ttt gga gcc gtg aga ggg ttg aac tat ctg 890 cac caa aat ggc tgt att cac agg agt att aaa gcc agc cat atc ctc 938 his gln asn gly cys ile his arg ser ile lys ala ser his ile leu att tct ggt gat ggc cta gtg acc ctc tct ggc ctg tcc cat ctg cat 986 agt ttg gtt aag cat gga cag agg cat agg gct gtg tat gat ttc cca 1034 ser leu val lys his gly gln arg his arg ala val tyr asp phe pro cag ttc agc aca tca gtg cag ccg tgg ctg agt cca gaa cta ctg aga 1082 cag gat tta cat ggg tat aat gtg aag tca gat att tac agt gtt ggg 1130 gln asp leu his gly tyr asn val lys ser asp ile tyr ser val gly att aca gca tgt gaa tta gcc agt ggg cag gtg cct ttc cag gac atg 1178 ile thr ala cys glu leu ala ser gly gln val pro phe gln asp met cat aga act cag atg ctg tta cag aaa ctg aaa ggt cct cct tat agc 1226 his arg thr gln met leu leu gln lys leu lys gly pro pro tyr ser cca ttg gat atc agt att ttc cct caa tca gaa tcc aga atg aaa aat 1274 pro leu asp ile ser ile phe pro gln ser glu ser arg met lys asn tcc cag tca ggt gta gac tct ggg att gga gaa agt gtg ctt gtc tcc 1322 agt gga act cac aca gta aat agt gac cga tta cac aca cca tcc tca 1370 aaa act ttc tct cct gcc ttc ttt agc ttg gta cag ctc tgt ttg caa 1418 caa gat cct gag aaa agg cca tca gca agc agt tta ttg tcc cat gtt 1466 gln asp pro glu lys arg pro ser ala ser ser leu leu ser his val ttc ttc aaa cag atg aaa gaa gaa agc cag gat tca ata ctt tca ctg 1514 ttg cct cct gct tat aac aag cca tca ata tca ttg cct cca gtg tta 1562 cct tgg act gag cca gaa tgt gat ttt cct gat gaa aaa gac tca tac 1610 met ser leu leu asp cys phe cys thr ser arg thr gln val glu ser leu arg pro glu lys gln ser glu thr ser ile his gln tyr leu val val leu cys ser thr asn val ser his tyr glu leu gln val glu ile gly arg gly phe asp asn leu thr ser val his leu ala arg his thr asn glu glu arg leu lys ala leu gln lys ala val ile leu ser his gly ser trp leu trp val ile ser pro phe met ala tyr gly ser ala ser gln leu leu arg thr tyr phe pro glu gly met ser glu thr leu gln asn gly cys ile his arg ser ile lys ala ser his ile leu ile leu val lys his gly gln arg his arg ala val tyr asp phe pro gln thr ala cys glu leu ala ser gly gln val pro phe gln asp met his leu asp ile ser ile phe pro gln ser glu ser arg met lys asn ser gly thr his thr val asn ser asp arg leu his thr pro ser ser lys asp pro glu lys arg pro ser ala ser ser leu leu ser his val phe ttgaggtcac accttcagtc cttcgagcaa at atg cct ctt cat gtt cga cgc 53 agt agt gac cca gct cta att ggc ctc tcc act tct gtc agt gat agt 101 aat ttt tcc tct gaa gag cct tca agg aaa aat ccc aca cgc tgg tca 149 aca aca gct ggc ttc ctc aag cag aac act gct ggg agt cct aaa gcc 197 tgc gac agg aag aaa gat gaa aac tac aga agc ctc ccg cgg gat act 245 cys asp arg lys lys asp glu asn tyr arg ser leu pro arg asp thr agt aac tgg tct aac caa ttt cag aga gac aat gct cgc tcg tct ctg 293 agt gcc agt cac cca atg gtg ggc aag tgg cag gag aaa caa gaa cag 341 ser ala ser his pro met val gly lys trp gln glu lys gln glu gln gat gag gat ggg aca gaa gag gat aac agt cgt gtt gaa cct gtt gga 389 cat gct gac acg ggt ttg gag cat ata ccc aac ttt tct ctg gat gat 437 his ala asp thr gly leu glu his ile pro asn phe ser leu asp asp atg gta aag ctc gta gaa gtc ccc aac gat gga ggg cct ctg gga atc 485 cat gta gtg cct ttc agt gct cga ggc ggc aga acc ctg ggg tta tta 533 gta aaa cga ttg gag aaa ggt ggt aaa gct gaa cat gaa aat ctt ttt 581 cgt gag aat gat tgc att gtc agg att aat gat ggc gac ctt cga aat 629 aga aga ttt gaa caa gca caa cat atg ttt cgc caa gcc atg cgt aca 677 ccc atc att tgg ttc cat gtg gtt cct gca gca aat aaa gag cag tat 725 pro ile ile trp phe his val val pro ala ala asn lys glu gln tyr gaa caa cta tcc caa agt gag aag aac aat tac tat tca agc cgt ttt 773 agc cct gac agc cag tat att gac aac agg agt gtg aac agt gca ggg 821 ser pro asp ser gln tyr ile asp asn arg ser val asn ser ala gly ctt cac acg gtg cag aga gca ccc cga ctg aac cac ccg cct gag cag 869 ata gac tct cac tca aga cta cct cat agc gca cac ccc tcg gga aaa 917 cca cca tcc gct cca gcc tcg gca cct cag aat gta ttt agt acg act 965 gta agc agt ggt tat aac acc aaa aaa ata ggc aag agg ctt aat atc 1013 cag ctt aag aaa ggt aca gaa ggt ttg gga ttc agc atc act tcc aga 1061 gat gta aca ata ggt ggc tca gct cca atc tat gtg aaa aac att ctc 1109 asp val thr ile gly gly ser ala pro ile tyr val lys asn ile leu ccc cgg ggg gcg gcc att cag gat ggc cga ctt aag gca gga gac aga 1157 ctt ata gag gta aat gga gta gat tta gtg ggc aaa tcc caa gag gaa 1205 gtt gtt tcg ctg ttg aga agc acc aag atg gaa gga act gtg agc ctt 1253 ctg gtc ttt cgc cag gaa gac gcc ttc cac cca agg gaa ctg aat gca 1301 leu val phe arg gln glu asp ala phe his pro arg glu leu asn ala gag cca agc cag atg cag att cca aaa gaa acg aaa gca gaa gat gag 1349 gat att gtt ctt aca cct gat ggc acc agg gaa ttt ctg aca ttt gaa 1397 gtc cca ctt agt gat tca gga tct gca ggc ctt ggt gtc agt gtc aaa 1445 ggt aac cgg tca aaa gag aac cac gca gat ttg gga atc ttt gtc aag 1493 gly asn arg ser lys glu asn his ala asp leu gly ile phe val lys tcc att att aat gga gga gca gca tct aaa gat gga agg ctt cgg gtg 1541 aat gat caa ctg ata gca gta aat gga gaa tcc ctg ttg ggc aag aca 1589 asn asp gln leu ile ala val asn gly glu ser leu leu gly lys thr aac caa gat gcc atg gaa acc cta aga agg tct atg tct act gaa ggc 1637 asn gln asp ala met glu thr leu arg arg ser met ser thr glu gly aat aaa cga gga atg atc cag ctt att gtt gca agg aga ata agc aag 1685 asn lys arg gly met ile gln leu ile val ala arg arg ile ser lys tgc aat gag ctg aag tca cct ggg agc ccc cct gga cct gag ctg ccc 1733 att gaa aca gcg ttg gat gat aga gaa cga aga att tcc cat tcc ctc 1781 tac agt ggg att gag ggg ctt gat gaa tcg ccc agc aga aat gct gcc 1829 tyr ser gly ile glu gly leu asp glu ser pro ser arg asn ala ala ctc agt agg ata atg ggt aaa tac cag ctg tcc cct aca gtg aat atg 1877 leu ser arg ile met gly lys tyr gln leu ser pro thr val asn met ccc caa gat gac act gtc att ata gaa gat gac agg ttg cca gtg ctt 1925 cct cca cat ctc tct gac cag tcc tct tcc agc tcc cat gat gat gtg 1973 ggg ttt gtg acg gca gat gct ggt act tgg gcc aag gct gca atc agt 2021 gat tca gcc gac tgc tct ttg agt cca gat gtt gat cca gtt ctt gct 2069 ttt caa cga gaa gga ttt gga cgt cag act gac gag act aaa ctc aat 2117 aca gtg gat gac cag aaa gca ggt tct ccc agc aga gat gtg ggt cct 2165 tcc ctg ggt ctg aag aag tca agc tca ttg gag agt ctg cag acc gca 2213 gtt gcc gag gtg act ttg aat ggg gat att cct ttc cat cgt cca cgg 2261 val ala glu val thr leu asn gly asp ile pro phe his arg pro arg ccg cgg ata atc aga ggc agg gga tgc aat gag agc ttc aga gct gcc 2309 atc gac aaa tct tat gat aaa ccc gcg gta gat gat gat gat gaa ggc 2357 atg gag acc ttg gaa gaa gac aca gaa gaa agt tca aga tca ggg aga 2405 gag tct gta tcc aca gcc agt gat cag cct tcc cac tct ctg gag aga 2453 glu ser val ser thr ala ser asp gln pro ser his ser leu glu arg caa atg aat gga aac caa gag aaa ggt gat aag act gat aga aaa aag 2501 aaa atg aaa gcc aag aag gga atg ctg aag ggc ttg gga gac atg ttc 2597 agc ctt gcc aaa ctg aag ccc gag aag aga tgaacaacaa agcgattcaa 2647 met pro leu his val arg arg ser ser asp pro ala leu ile gly leu lys asn pro thr arg trp ser thr thr ala gly phe leu lys gln asn thr ala gly ser pro lys ala cys asp arg lys lys asp glu asn tyr asp asn ala arg ser ser leu ser ala ser his pro met val gly lys ser arg val glu pro val gly his ala asp thr gly leu glu his ile asp gly gly pro leu gly ile his val val pro phe ser ala arg gly ala glu his glu asn leu phe arg glu asn asp cys ile val arg ile asn asp gly asp leu arg asn arg arg phe glu gln ala gln his met phe arg gln ala met arg thr pro ile ile trp phe his val val pro arg ser val asn ser ala gly leu his thr val gln arg ala pro arg ile tyr val lys asn ile leu pro arg gly ala ala ile gln asp gly met glu gly thr val ser leu leu val phe arg gln glu asp ala phe his pro arg glu leu asn ala glu pro ser gln met gln ile pro lys arg glu phe leu thr phe glu val pro leu ser asp ser gly ser ala glu ser leu leu gly lys thr asn gln asp ala met glu thr leu arg arg ser met ser thr glu gly asn lys arg gly met ile gln leu ile val ala arg arg ile ser lys cys asn glu leu lys ser pro gly ser ser pro ser arg asn ala ala leu ser arg ile met gly lys tyr gln leu ser pro thr val asn met pro gln asp asp thr val ile ile glu thr asp glu thr lys leu asn thr val asp asp gln lys ala gly ser asn glu ser phe arg ala ala ile asp lys ser tyr asp lys pro ala pro ser his ser leu glu arg gln met asn gly asn gln glu lys gly leu met ser val gly met asp thr phe ile his arg ile asp ser thr glu val ile tyr gln pro arg arg lys arg ala lys leu ile gly lys glu val leu asp ser glu thr leu cys arg arg ala val lys ile leu met glu tyr cys val cys gly met gln glu met leu asp ser val pro glu lys arg phe pro val cys gln ala his gly tyr phe cys gln leu ile asp gly leu glu tyr leu his ser gln gly ile val his lys asp thr cys arg thr ser gln gly ser pro ala phe gln pro pro glu ile ala asn gly leu asp thr phe ser gly phe lys val asp ile trp ser ala gly val thr leu tyr asn ile thr thr gly leu tyr pro phe glu met leu glu tyr glu pro ala lys arg phe ser ile arg gln ile arg gln his ser trp phe arg lys lys his pro pro ala glu ala pro val gly leu pro lys ala val cys met asn gly thr glu ala ala gln leu arg his leu ala glu gln phe ala val gly glu ile ile thr asp met ala lys lys glu trp lys val gly leu pro ile gly gln gly gly phe gly cys ile tyr leu ala asp met asn ser ser glu ser val gly ser asp ala pro cys val val lys val glu pro ser asp asn gly pro leu phe thr glu leu lys phe tyr gln arg ala ala lys pro glu gln ile gln lys trp ile arg thr arg lys leu lys tyr leu gly val pro lys tyr trp gly ser gly leu his asp lys asn gly lys ser tyr arg phe met ile met asp arg phe gly ser asp leu gln lys ile tyr glu ala val his lys glu tyr lys glu asp pro lys arg cys his asp gly thr ile glu phe thr ser ile asp ala his asn gly val ala pro ser arg arg gly asp leu glu ile leu gly tyr cys met ile gln trp leu thr gly his leu pro trp glu asp asn leu lys asp pro lys tyr val arg asp ser lys ile arg tyr arg glu asn ile ala ser leu met asp lys cys phe pro glu lys asn lys pro gly glu ile ala lys tyr met glu gly val glu asp thr glu trp ser asn thr gln thr glu glu ala ile lys lys ile arg leu glu ser glu glu glu gly val pro ser thr ala leu gln gly ile val phe cys his ser arg arg val leu his arg asp gly ser ala arg tyr ser thr pro val asp ile trp ser ile gly thr ile phe ala glu leu ala thr lys lys pro leu phe his gly asp ser glu ile asp gln leu phe arg ile phe arg ala leu gly thr pro asn asn glu val trp pro glu val glu ser leu gln asp tyr lys asn thr phe pro lys trp lys pro gly ser leu ala ser his val lys asn leu asp glu asn gly leu asp leu leu ser lys met leu ile tyr asp pro ala lys arg ile ser gly lys met ala leu asn his pro tyr phe asn met asp arg ser lys glu asn cys ile ser gly pro val lys ala thr gly asn val tyr leu ala arg glu lys gln ser lys phe ile leu ala ile leu arg leu tyr gly tyr phe his asp ala thr arg val tyr leu ile leu glu tyr ala pro leu gly thr val tyr arg glu leu gln lys leu ser lys phe asp glu gln arg thr ala thr tyr ile thr glu leu ala asn ala leu ser tyr cys his ser lys arg val ile his arg asp ala asp phe gly trp ser val his ala pro ser ser arg arg thr thr leu cys gly thr leu asp tyr leu pro pro glu met ile glu gly arg met his asp glu lys val asp leu trp ser leu gly val leu cys tyr glu phe leu val gly lys pro pro phe glu ala asn thr tyr gln glu thr tyr lys arg ile ser arg val glu phe thr phe pro asp phe val thr glu gly ala arg asp leu ile ser arg leu leu lys his asn pro ser gln arg pro met leu arg glu val leu glu his pro trp ile thr met ala gln lys glu asn ser tyr pro trp pro tyr gly arg gln thr ala pro ser gly leu ser thr leu pro gln arg val leu arg lys glu pro thr ala ala pro gly gln lys val met glu asn ser ser gly thr arg pro leu gly lys gly lys phe gly asn val tyr leu ala arg glu glu leu tyr lys glu leu gln lys ser cys thr phe asp glu gln arg gly lys lys val ile his arg asp ile lys pro glu asn leu leu leu gly leu lys gly glu leu lys ile ala asp phe gly trp ser val his ala pro ser leu arg arg lys thr met cys gly thr leu asp tyr leu pro pro glu met ile glu gly arg met his asn glu lys val asp leu phe glu ser ala ser his asn glu thr tyr arg arg ile val lys val asp leu lys phe pro ala ser val pro thr gly ala gln asp leu ile met arg glu arg leu gly thr gly gly phe gly asn val cys leu tyr gln his arg glu leu asp leu lys ile ala ile lys ser cys arg leu glu leu ser thr lys asn arg glu arg trp cys his glu ile gln ile tyr cys ser gly gly asp leu arg lys leu leu asn lys pro glu asn gly ser gly ile arg tyr leu his glu asn lys ile ile his arg asp his lys ile ile asp leu gly tyr ala lys asp val asp gln gly ser leu cys thr ser phe val gly thr leu gln tyr leu ala pro glu leu phe glu asn lys pro tyr thr ala thr val asp tyr trp ser phe gly thr met val phe glu cys ile ala gly tyr arg pro phe leu his his leu gln pro phe thr trp his glu lys ile lys lys lys asp pro lys his leu pro gln pro asn ser leu cys ser leu ile val glu pro met gly pro val asp leu thr leu lys gln pro arg cys phe val leu met glu leu leu ser glu thr gly ile ser leu asp pro arg lys pro ala tyr leu phe asp lys ser lys thr val tyr glu gly pro phe ala ser arg ser leu ser asp cys val asn tyr ile val gln asp ser lys ile gln leu pro ile ile gln leu arg lys val trp ala glu ala val his tyr val ser gly leu lys glu asp tyr ser arg leu phe gln gly gln glu phe phe his lys ser ile gln leu asp leu glu arg tyr ser glu gln met thr tyr gly ile ser ser glu lys met leu lys ala trp lys gln lys ser pro tyr gly arg arg gln gly asp leu met glu ser leu glu gln arg ala ile asp leu tyr lys gln leu lys his arg pro ser thr val gln ser gln asp arg val leu lys glu leu phe gly his leu val glu val ala leu ser asn ile lys glu ala asp asn thr val met phe met gln gly lys arg gln lys glu ile trp his leu leu lys ile glu his asp his ser leu ser cys val val thr pro gln asp gly glu thr ser ala gln met ile glu glu asn leu asn cys leu gly his leu ser arg thr phe ile thr ile gly asp arg asn phe glu val glu ala asp asp leu val thr ile ser glu leu gly arg gly ala tyr gly val val glu lys val arg his ala gln ser gly thr ile met ala val lys arg ile arg ala thr val asn ser gln glu gln lys arg leu leu met asp leu asp ile asn met arg thr val asp cys phe tyr thr val thr phe tyr gly ala leu phe arg glu gly asp val trp ile cys met glu asn met thr ile pro glu asp ile leu gly glu ile ala val ser ile asp val lys pro ser asn val leu ile asn lys glu gly his val lys met cys asp phe gly ile ser gly tyr leu val asp ser val ala lys thr met asp ala gly cys lys pro tyr met ala pro glu arg ile asn pro glu leu asn gln lys gly tyr asn val lys ser asp val trp ser leu gly ile thr met ile glu met ala ile leu arg phe pro tyr glu ser trp gly thr pro phe gln gln leu lys gln val val glu glu pro thr ala gln cys leu arg lys asn pro ala glu arg met ser tyr leu gly gln pro phe asp val gly pro arg tyr thr gln leu gln tyr ile lys thr arg val ala ile lys lys ile ser pro phe glu his gln thr his glu asn val ile gly ile arg asp ile leu arg ala ser thr leu glu ala met arg asp val tyr ile val gln asp leu met glu thr asp tyr phe leu tyr gln ile leu arg gly leu lys tyr ile his ser ala pro glu his asp his thr gly phe leu thr glu tyr val ala thr arg trp tyr arg ala pro glu ile met leu asn ser lys gly tyr thr lys ser ile asp ile trp ser val gly cys ile leu ala glu met leu ser asn arg pro ile phe pro gly lys his tyr leu asp gln leu asn his ile asn met lys ala arg asn tyr leu gln ser leu pro ser lys thr met glu his ile gln gly ala trp lys thr ile ser asn gly phe gly phe lys asp ala val phe asp gly ser ser cys ile ser pro thr ile val gln gln phe gly tyr gln arg arg ala ser asp asp gly lys leu thr asp pro ser lys thr ser asn thr ile arg val phe leu pro asn lys gln arg thr val val asn val arg asn gly met ser leu his asp cys leu met lys ala leu lys val arg gly leu gln pro glu cys cys asp trp asn thr asp ala ala ser leu ile gly glu glu leu gln val asp phe leu asp his val pro leu thr thr his asn phe ala arg lys asn gly phe arg cys gln thr cys gly tyr lys phe his glu his cys ser thr lys val pro thr met cys val asp trp ser asn ile arg gln leu leu leu phe pro asn ser thr ile gly asp ser gly val pro ala val ser gly thr gln glu lys asn lys ile arg pro arg gly gln arg arg his val asn ile leu leu phe met gly tyr met thr lys asp asn leu ala ile val thr gln trp cys glu gly ser ser leu tyr lys his leu his val gln glu thr lys phe gln met phe gln leu ile asp ile ala arg gln thr ala gln gly met asp tyr leu his ala lys asn ile ile his arg asp met lys ser asn asn ile phe leu his glu gly leu thr val lys ile gly asp phe gly leu ala thr val lys ser arg trp ala pro glu val ile arg met gln asp asn asn pro phe ser phe gln glu leu pro tyr ser his ile asn asn arg asp gln ile ile phe met val gly arg gly tyr ala ser pro asp leu ser lys leu tyr lys asn leu gln his ser leu pro lys ile asn arg ser ala ser glu pro ser met ser asp val ala ile val lys glu gly trp leu his lys arg gly glu tyr ile lys thr trp arg pro arg tyr phe leu leu lys asn asp gly thr phe ile gly tyr lys glu arg pro gln asp val asp gln arg glu ala pro leu asn asn phe ser val ala gln cys gln leu met lys thr glu arg pro arg pro asn thr phe ile ile arg cys leu gln trp glu glu trp thr thr ala ile gln thr val ala asp gly leu lys lys ser gly ala glu glu met glu val ser leu ala lys pro lys his arg phe gly lys val ile leu val lys glu lys ala thr gly arg tyr tyr ala his thr leu thr glu asn arg val leu gln asn ser arg his pro phe leu thr ala leu lys tyr ser phe gln thr his asp arg leu cys phe val met glu tyr ala asn gly gly glu leu phe phe his leu ser ile val ser ala leu asp tyr leu his ser glu lys asn val val tyr thr met lys thr phe cys gly thr pro glu tyr leu ala pro glu val val val met tyr glu met met cys gly arg leu pro phe tyr asn gln glu ile met gln his arg phe phe ala gly ile val trp gln his val met ser ala glu gly tyr gln tyr arg ala leu tyr asp tyr lys lys asn lys gly ser leu val ala leu gly phe ser asp gly gln glu ala glu arg gly asp phe pro gly thr tyr val glu tyr ile gly arg lys leu arg gln leu leu asp cys asp thr pro ser val asp leu glu met lys leu ile arg ser pro ser ile pro his gln tyr trp leu thr leu asn leu leu asn ala arg val leu ser glu ile phe ser pro met leu phe arg phe ser ala ala ser ser asp asn thr glu asn leu ile lys val ile glu ile leu ile ser thr glu trp asn glu arg gln pro ala asp ile ser arg glu glu val asn glu lys leu arg asp thr ala asp gly thr phe leu val arg asp ala ser thr lys met his gly asp tyr his arg asp gly lys tyr gly phe ser asp pro leu thr phe ser ser val val glu leu ile asn his tyr arg asn glu ser leu ala gln tyr leu his glu tyr asn thr gln phe gln glu lys ser arg glu tyr asp lys arg met asn ser ile lys pro asp leu ile gln leu arg lys thr arg asp gln tyr leu met trp leu thr gln lys gly val arg gln lys lys leu asn glu trp leu gly asn glu asn thr glu asp gln tyr ser lys arg asp gly thr phe leu val arg glu ser ser lys gln gly cys tyr ala cys ser val val val asp gly glu val lys his cys val ile gln his asn asp ser leu asn val thr leu ala tyr pro val tyr ala met ser leu val leu asn asp leu leu ile cys cys arg gln leu glu ser lys gln gly lys tyr leu asn trp asp ala val phe arg phe leu gln lys tyr ile gln lys glu thr glu cys leu arg ile ala lys pro ile ser ser leu val lys tyr phe ile lys cys ala asn arg arg ala pro arg leu lys cys gln glu leu leu asn tyr ile met asp thr val leu leu lys asp ile leu ser val arg lys tyr trp cys glu ile ser lys pro ser gln asp val his arg val leu val ala arg ile ile his ala val thr lys gly cys cys ser gln thr asp gly leu asn ser lys phe leu asp phe phe ser lys ala ile gln cys ala arg gln glu lys lys thr leu ala val asn phe arg ile arg val cys glu leu gly asp glu ile leu pro thr leu leu tyr ile trp thr gln his arg leu asn ser thr lys trp arg ser ile leu tyr asn leu tyr asp leu leu val cys his gln val phe asn glu asp thr arg ser leu glu ile ser gln gln lys ser gln asn asp phe asp leu val pro trp leu gln ile ala thr gln leu ile ser lys tyr pro ala ser leu pro asn cys glu leu leu lys leu trp asn lys ile trp cys ile thr phe arg gly ile ser gln gly ser leu val glu val asp arg glu phe trp lys leu phe thr ala leu thr thr ser ile val pro gly ala val lys met gly ile glu gln asn met cys glu val asn arg ser phe ser leu lys glu ser ile met lys trp leu leu phe tyr gln leu glu gly asp leu glu asn ser glu lys ile leu val ser leu thr met lys asn cys lys ala ala met asn phe phe gln ser val pro glu cys glu his his gln lys asp lys phe asp lys met asp phe leu thr ile val arg glu cys gly ile glu lys his gln ser ser ile gly phe ser val his gln asn leu lys glu met gln cys ala gly glu ser ile thr leu phe lys asn lys thr asn glu glu phe arg ile gly ser leu arg asn met met gln leu cys thr gly phe phe leu arg leu leu thr ser lys leu met asn asp ile ala val glu asp gln ser ser met asn leu phe asn asp tyr pro asp ser ser val ser asp ala asn glu pro gly glu ser gln ser thr ile gly ala ile asn pro leu ala glu glu tyr leu ser lys gln asp leu leu thr asn thr val ser phe arg ala ala asp ile arg arg lys leu leu cys ser leu tyr arg arg asp gln asp val cys lys thr ile leu asn glu asn thr arg asp ala gln gly gln phe leu thr val ile gly ala phe trp his leu thr lys glu arg lys tyr ile phe ser val arg met ala leu val asn cys leu lys thr leu leu glu ala asp pro tyr ser lys trp ala ile leu asn val met gly lys asp phe pro val asn glu val phe thr gln phe leu ala asp asn his his gln val arg met leu ala ala glu ser ile asn arg leu phe gln asp thr lys gly asp ser glu asn ala tyr leu lys ala gln glu gly met arg glu met ser his ser ala glu asn pro glu thr leu asp glu ile tyr asn arg lys ser glu lys gln ala leu phe ala leu cys lys ser val lys glu asn gly phe gly tyr arg arg leu glu asp phe met ala ser his leu asp tyr phe asp glu val lys ser ile ala asn gln ile gln glu asp trp lys ser leu leu thr asp cys phe pro lys ile leu val asn ile leu pro tyr phe ala tyr glu gly thr arg asp ser gly met ala gln gln arg glu thr ala thr lys val tyr asp met leu lys ser glu asn leu leu gly lys gln ile asp his leu phe ile ser asn leu pro glu ile val asn pro pro his phe pro ser his val ile lys ala thr phe ala tyr ser lys ser pro asp ser tyr gln lys ile leu leu ala ile cys glu gln ala ala glu thr asn asn val tyr lys lys his arg ile leu lys leu gly gly ala trp ala phe val leu arg asp val ile tyr thr leu ile his tyr ile asn gln arg pro ser cys ile met asp val ser leu ala val thr tyr cys lys asp ala leu glu asn his leu his val ile glu asn leu tyr ile thr ile lys leu leu asp pro phe pro asp his val val phe lys asp leu arg ile thr gln gln lys ile lys tyr ser arg gly pro phe ser leu leu glu glu ile asn his phe leu ser val ser val tyr asp ala leu pro leu thr arg leu glu gly leu lys asp arg ala ser gln asp asn pro gln asp gly ile met val lys leu val val asn leu leu gln leu ser lys met ala ile asn his thr gly glu ile met leu thr tyr leu asn asn thr leu val glu asp cys val lys thr gly his ser phe trp glu ile tyr lys met thr thr asp pro met leu ala tyr leu gln pro phe arg thr ser arg lys lys phe leu glu val pro arg phe asp lys glu asn pro phe glu gly leu asp asp ile asn leu trp ile pro leu ser glu asn his asp ile trp ile lys thr leu thr cys ala phe leu asp ser gly gly thr lys cys glu ile leu gln leu leu lys pro met cys glu val lys thr asp phe cys gln thr val leu pro tyr leu ile his asp ile leu leu gln asp thr asn glu ser trp arg asn leu leu ser thr his val gln gly phe phe thr ser cys leu arg his phe ser gln thr ser arg ser thr thr pro ala asn ser gln arg thr met leu ala val val asp tyr met arg arg gln lys arg pro ser ser gly thr ile phe asn asp ala phe trp leu asp leu asn tyr leu glu val ala lys val ala gln ser cys ala ala his phe asp gln glu lys arg ser leu ala phe glu glu gly ser gln ser thr ser leu tyr gly cys gly gly gly lys met leu gln pro ile thr arg leu arg thr tyr glu his glu ala met trp gly lys ala leu val thr tyr asp leu glu thr ala ile pro ser ser thr arg gln ala gly ile ile gln ala leu gln asn leu gly leu cys his ile leu ser val tyr glu leu his tyr gln ala ala trp arg asn met gln trp asp his cys tyr asn ala leu gln ser leu arg asp arg glu phe ser thr phe tyr val thr his arg gln leu ser glu val tyr ile lys trp gln lys his asn ser gln arg glu cys ile lys asp ile leu thr lys his leu val glu leu ser ile leu ala arg thr phe lys asn thr gln leu pro glu arg ala ile phe gln ile lys gln tyr asn ser val ser cys gly val pro ala val ile met gln thr tyr leu glu lys ala val glu val ala ile glu asn tyr met lys ser ser glu phe glu asn lys gln ala leu lys ala val glu asn tyr ile asn cys leu leu ser gly glu glu his asp met trp val phe arg leu cys ser leu trp leu glu asn ser gly val ser glu val asn gly met met lys arg asp gly met lys ile pro thr tyr lys phe leu pro leu met tyr gln leu ala ala arg met gly ile arg ser arg arg pro gln met val arg ser val glu ala leu cys gln arg lys gly ile asn ile pro ala asp gln pro ile thr lys leu his thr gly glu tyr gly asn leu val thr ile gln ser phe lys ala glu phe arg leu ala gly gly val asn leu pro lys ile ile asp cys cys thr tyr lys val val 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pro gly asp tyr thr leu cys val ser cys asp gly lys val glu his tyr arg ile met tyr his ala ser lys leu ser ile asp glu glu val tyr phe glu asn leu met gln leu val glu his tyr thr ser asp ala asp gly leu cys thr arg leu ile lys pro lys val met glu gly thr val ala ala gln asp glu phe tyr arg ser gly trp ala leu asn met val met leu gly asp tyr arg gly asn lys val ala val lys cys ile lys asn asp ala thr ala gln ala phe leu ala glu ala ser val met asp cys leu leu lys phe ser leu asp val cys glu ala met glu tyr leu val ser glu asp asn val ala lys val ser asp phe gly leu thr lys glu ala ser ser thr gln asp thr gly lys leu pro val lys trp glu val met lys asn cys trp his leu asp ala ala met arg pro ser met ala phe cys ala lys met arg ser ser lys lys thr glu val asn leu glu ala pro glu pro gly val glu val ile phe tyr leu ser asp leu phe ala leu tyr asp glu asn thr lys leu trp tyr ala pro asn arg thr ile thr val asp asp lys met ser leu arg leu his tyr arg met arg phe tyr phe thr asn trp his gly 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ser lys leu leu ser gly val asp pro tyr ser asp asp asp glu ile asn gly val thr gln gly asp arg ser ala ser arg val ser cys ala gly gln met leu glu val gln pro arg glu ala gly ile thr ala val leu thr val asp ser glu glu pro ser phe lys ala gly pro gly val glu asp leu trp arg leu phe val phe leu met lys thr asp gln leu pro phe glu lys ala tyr glu lys leu gln ile leu lys pro glu ala lys met asn glu gly phe glu trp gln leu lys leu tyr gln ala met gly tyr glu val asp thr ser ser ala ile tyr lys gln tyr arg leu gln lys val thr glu lys tyr pro val ser gln gly leu lys asp glu val leu tyr lys cys arg lys cys ser gly pro ile ala phe ala his lys arg met thr pro ser ser met leu thr thr gly arg gln ala gln cys thr ser tyr phe ile glu pro leu cys pro lys cys ser ala lys leu gly ser phe asn trp tyr gly glu gln cys ser cys gly arg trp ile thr pro ala phe gln ile his lys asn arg val asp glu met lys ile leu pro val leu gly ser gln met gly val gln pro pro asn phe ser trp val leu pro gly arg leu leu gly 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