專利名稱：組胺n－甲基轉(zhuǎn)移酶基因與原發(fā)性高血壓的相關(guān)性的制作方法
技術(shù)領(lǐng)域：
本發(fā)明涉及分子生物學(xué)和醫(yī)學(xué)領(lǐng)域。更具體地涉及人組胺N-甲基轉(zhuǎn)移酶基因(histamine N-methyltransferase，HNMT)的單核苷酸多態(tài)性(singlenucleotide polymorphism，SNP)及其組成的單倍型(haplotype)與原發(fā)性高血壓的相關(guān)性。本發(fā)明還涉及檢測這些SNP和單倍型的方法和試劑盒。
背景技術(shù)：
原發(fā)性高血壓(essential hypertension，EH)是最常見的心血管疾病，其發(fā)病率逐年上升，并可引起嚴(yán)重的心、腦、腎并發(fā)癥，是腦卒中和冠心病的主要危險(xiǎn)因素。EH是一種由遺傳因素和環(huán)境因素相互作用而發(fā)病的多基因病，尋找EH相關(guān)基因進(jìn)而闡明高血壓發(fā)病的遺傳機(jī)制已經(jīng)成為目前研究的熱點(diǎn)(HarrapSB.Hypertensiongenes versus environment.Lancet，1994，344169-171)。
定位克隆法是研究EH遺傳機(jī)制的主要研究之一，即利用一定數(shù)量的覆蓋整個基因組的微衛(wèi)星引物，對大樣本家系或同胞對進(jìn)行連鎖分析，而將EH易感基因位點(diǎn)定位到某一染色體區(qū)域。上海市高血壓研究所已完成對上海地區(qū)漢族高血壓家系的全基因掃描，研究提示2q14-q23區(qū)域與高血壓存在緊密連鎖關(guān)系(Zhu DL，Wang HY，Xiong MM，et al.Linkage of hypertension to chromosome2q14-q23 in Chinese families.J Hypertens 2001；1955-61)，然而并沒有確定該區(qū)域內(nèi)的眾多基因中哪個基因?yàn)镋H易感基因。
HNMT基因是一種定位于該區(qū)域內(nèi)的基因，但目前沒有證實(shí)HNMT基因與原發(fā)性高血壓相關(guān)性的報(bào)道，更沒有證實(shí)HNMT基因的SNP與原發(fā)性高血壓相關(guān)性的報(bào)道。
綜上所述，為了盡早治療高血壓，本領(lǐng)域迫切需要尋找原發(fā)性高血壓易感基因，并開發(fā)檢測原發(fā)性高血壓的方法、試劑盒，以及相關(guān)的治療藥物。
發(fā)明內(nèi)容
本發(fā)明的目的就是提供一種輔助性診斷(尤其是早期診斷)原發(fā)性高血壓等高血壓的方法及檢測試劑盒。
本發(fā)明的另一目的是提供一種新的治療高血壓的方法。
在本發(fā)明的第一方面，提供了一種體外對個體的高血壓易感性進(jìn)行輔助性診斷的方法，它包括步驟檢測該個體的HNMT基因的基因組序列并與正常的HNMT基因的基因組序列相比較，存在差異就表明該個體患高血壓的可能性高于正常人群，其中所述的差異是在HNMT的基因組序列的第616、7798、40000、51951、和52109位，存在選自下組的單倍型(i)TAGAA(ii)TGAGT，其中，核苷酸位置編號基于SEQ ID NO1。
在另一優(yōu)選例中，檢測的HNMT基因組序列來自于個體細(xì)胞的DNA抽提物。
在另一優(yōu)選例中，所述的細(xì)胞是外周血白細(xì)胞。
在本發(fā)明的第二方面，提供了一種檢測樣品是否存在HNMT基因的單倍型的方法，包括步驟(a)用HNMT基因特異性引物擴(kuò)增樣品的HNMT基因，得到擴(kuò)增產(chǎn)物；和(b)檢測擴(kuò)增產(chǎn)物中對應(yīng)于HNMT的基因組序列的第616、7798、40000、51951、和52109位的核苷酸，并判斷是否存在選自下組的單倍型(i)TAGAA；(ii)TGAGT；(iii)TGAAA；(iv)CAAAA其中，核苷酸位置編號基于SEQ ID NO1。
在另一優(yōu)選例中，所述的單倍型是(i)TAGAA；或(ii)TGAGT。
在本發(fā)明的第三方面，提供了一種用于輔助性檢測高血壓的試劑盒，它包括特異性擴(kuò)增HNMT基因的引物和說明書，所述的引物所擴(kuò)增出的擴(kuò)增產(chǎn)物含有對應(yīng)于HNMT的基因組序列的第616、7798、40000、51951、和52109位的核苷酸，其中，核苷酸位置編號基于SEQ ID NO1。
在另一優(yōu)選例中，它還含有與第616、7798、40000、51951、和52109位的部位結(jié)合的探針。
在本發(fā)明的第四方面，提供了一種組胺N-甲基轉(zhuǎn)移酶的cDNA序列或基因組序列的用途，它們被用于制備檢測原發(fā)性高血壓的試劑。
具體實(shí)施例方式
本發(fā)明人經(jīng)過多年研究，首次發(fā)現(xiàn)和證明了HNMT基因與高血壓密切相關(guān)，而且發(fā)現(xiàn)了它的新功能HNMT的改變將導(dǎo)致高血壓，其中關(guān)聯(lián)研究結(jié)果顯示，在HNMT第616位的SNP01(C/T)、第7798位的SNP05(A/G)、第40000位的SNP07(A/G)、第51951位的SNP08(A/G)和第52109位的SNP09(A/T)組成的單倍型在病例和對照組中的分布存在顯著性差異?；诖税l(fā)現(xiàn)，本發(fā)明提供了利用檢測HNMT基因或轉(zhuǎn)錄本是否存在所述單倍型，從而為診斷高血壓提供輔助性或早期參考指標(biāo)的方法和試劑盒。
HNMT基因的總長度為52427bp，其詳細(xì)序列可參見網(wǎng)址http://genome.ucsc.edu/或http://www.ncbi.nlm.nih.gov/或參見SEQ IDNO1。
基于本發(fā)明的新發(fā)現(xiàn)，HNMT蛋白或多肽有多方面的新用途。這些用途包括(但不限于)直接做為藥物治療HNMT蛋白功能低下或喪失所致的疾病(如高血壓)，和用于篩選促進(jìn)HNMT蛋白功能的物質(zhì)，如抗體、多肽或其它配體。用表達(dá)的重組人HNMT蛋白篩選多肽庫可用于尋找有治療價值的能刺激人HNMT蛋白功能的多肽分子。
另一方面，本發(fā)明還包括對人HNMT DNA或是其片段編碼的多肽具有特異性的多克隆抗體和單克隆抗體，尤其是單克隆抗體。這里，“特異性”是指抗體能結(jié)合于人HNMT基因產(chǎn)物或片段。較佳地，指那些能與人HNMT基因產(chǎn)物或片段結(jié)合但不識別和結(jié)合于其它非相關(guān)抗原分子的抗體。本發(fā)明中抗體包括那些能夠結(jié)合并抑制人HNMT蛋白的分子，也包括那些并不影響人HNMT蛋白功能的抗體。
本發(fā)明不僅包括完整的單克隆或多克隆抗體，而且還包括具有免疫活性的抗體片段，如Fab’或(Fab)2片段；抗體重鏈；抗體輕鏈；遺傳工程改造的單鏈Fv分子；或嵌合抗體。
本發(fā)明的抗體可以通過本領(lǐng)域內(nèi)技術(shù)人員已知的各種技術(shù)進(jìn)行制備。例如，純化的人HNMT基因產(chǎn)物或者其具有抗原性的片段，可被施用于動物以誘導(dǎo)多克隆抗體的產(chǎn)生。與之相似的，表達(dá)人HNMT蛋白或其具有抗原性的片段的細(xì)胞可用來免疫動物來生產(chǎn)抗體。多種佐劑可用于增強(qiáng)免疫反應(yīng)，包括但不限于弗氏佐劑等。
本發(fā)明的抗體也可以是單克隆抗體。此類單克隆抗體可以利用雜交瘤技術(shù)來制備。本發(fā)明的抗體包括能阻斷人HNMT蛋白功能的抗體以及不影響人HNMT蛋白功能的抗體。本發(fā)明的各類抗體可以利用人HNMT基因產(chǎn)物的片段或功能區(qū)，通過常規(guī)免疫技術(shù)獲得。這些片段或功能區(qū)可以利用重組方法制備或利用多肽合成儀合成。與人HNMT基因產(chǎn)物的未修飾形式結(jié)合的抗體可以用原核細(xì)胞(例如E.Coli)中生產(chǎn)的基因產(chǎn)物來免疫動物而產(chǎn)生；與翻譯后修飾形式結(jié)合的抗體(如糖基化或磷酸化的蛋白或多肽)，可以用真核細(xì)胞(例如酵母或昆蟲細(xì)胞)中產(chǎn)生的基因產(chǎn)物來免疫動物而獲得。
抗人HNMT蛋白的抗體可用于免疫組織化學(xué)技術(shù)中，檢測活檢標(biāo)本中的人HNMT蛋白。一種優(yōu)選的抗HNMT抗體是不識別正常HNMT但識別突變HNMT的抗體，或者識別正常HNMT但不識別突變HNMT的抗體。利用該抗體對正常和異常HNMT蛋白的特異性差異，可以方便地進(jìn)行蛋白質(zhì)水平的高血壓易感性檢測。
利用本發(fā)明HNMT蛋白，通過各種常規(guī)篩選方法，可篩選出與HNMT蛋白發(fā)生相互作用的物質(zhì)，如抑制劑、激動劑或拮抗劑等。
本發(fā)明HNMT蛋白及其抗體、抑制劑、激動劑、拮抗劑等，當(dāng)在治療上進(jìn)行施用(給藥)時，可提供不同的效果。通常，可將這些物質(zhì)配制于無毒的、惰性的和藥學(xué)上可接受的水性載體介質(zhì)中，其中pH通常約為5-8，較佳地pH約為6-8，盡管pH值可隨被配制物質(zhì)的性質(zhì)以及待治療的病癥而有所變化。配制好的藥物組合物可以通過常規(guī)途徑進(jìn)行給藥，其中包括(但并不限于)肌內(nèi)、靜脈內(nèi)、或皮下給藥。
正常的HNMT多肽有望用于疾病治療，例如，用于高血壓治療。在使用本發(fā)明HNMT蛋白時，還可同時使用其他治療高血壓的藥劑。
本發(fā)明還提供了一種藥物組合物，它含有安全有效量的本發(fā)明HNMT蛋白以及藥學(xué)上可接受的載體或賦形劑。這類載體包括(但并不限于)鹽水、緩沖液、葡萄糖、水、甘油、乙醇、及其組合。藥物制劑應(yīng)與給藥方式相匹配。本發(fā)明的藥物組合物可以被制成針劑形式，例如用生理鹽水或含有葡萄糖和其他輔劑的水溶液通過常規(guī)方法進(jìn)行制備。諸如片劑和膠囊之類的藥物組合物，可通過常規(guī)方法進(jìn)行制備。藥物組合物如針劑、溶液、片劑和膠囊宜在無菌條件下制造?；钚猿煞值慕o藥量是治療有效量，例如每天約0.1微克/千克體重-約10毫克/千克體重。此外，本發(fā)明的多肽還可與其他治療劑一起使用。
使用藥物組合物時，是將安全有效量的HNMT蛋白或其拮抗劑、激動劑施用于哺乳動物，其中該安全有效量通常至少約0.1微克/千克體重，而且在大多數(shù)情況下不超過約10毫克/千克體重，較佳地該劑量是約0.1微克/千克體重-約100微克/千克體重。當(dāng)然，具體劑量還應(yīng)考慮給藥途徑、病人健康狀況等因素，這些都是熟練醫(yī)師技能范圍之內(nèi)的。
人HNMT蛋白的多聚核苷酸也可用于多種治療目的?；蛑委熂夹g(shù)可用于治療由于HNMT蛋白的無表達(dá)或異常/無活性的HNMT蛋白的表達(dá)所致的細(xì)胞增殖、發(fā)育或代謝異常。構(gòu)建攜帶HNMT基因的重組病毒載體的方法可見于已有文獻(xiàn)(Sambrook，et al.)。另外重組人HNMT基因可包裝到脂質(zhì)體中，然后再轉(zhuǎn)移至細(xì)胞內(nèi)。
檢測HNMT基因的突變也可用于診斷高血壓。檢測可以針對cDNA，也可針對基因組DNA。HNMT蛋白突變的形式包括與正常野生型HNMT DNA序列相比的點(diǎn)突變、易位、缺失、重組和其它任何異常等。一類優(yōu)選的突變是由SNP構(gòu)成的單倍型?？捎靡延械募夹g(shù)如Southern印跡法、DNA序列分析、PCR和原位雜交檢測突變。
本發(fā)明的主要優(yōu)點(diǎn)在于首次用實(shí)驗(yàn)證實(shí)了HNMT的改變與高血壓的密切相關(guān)性，進(jìn)而提供了輔助性診斷高血壓(或易感性)的方法和試劑盒。本發(fā)明可以為臨床診斷(尤其是早期診斷)高血壓提供極為有價值的輔助性參考指標(biāo)，從而有利于實(shí)現(xiàn)早診斷、早預(yù)防、早得益。
下面結(jié)合具體實(shí)施例，進(jìn)一步闡述本發(fā)明。應(yīng)理解，這些實(shí)施例僅用于說明本發(fā)明而不用于限制本發(fā)明的范圍。下列實(shí)施例中未注明具體條件的實(shí)驗(yàn)方法，通常按照常規(guī)條件如Sambrook等人，分子克隆實(shí)驗(yàn)室手冊(New YorkCold Spring Harbor Laboratory Press，1989)中所述的條件，或按照制造廠商所建議的條件。
實(shí)施例11對象與方法1.1研究對象1.1.1中國人SNP發(fā)現(xiàn)和確認(rèn)樣本樣本為來自24個核心漢族高血壓家系、無親緣關(guān)系的24例原發(fā)性高血壓病人。
1.1.2SNP分型和關(guān)聯(lián)分析研究樣本所有受試者均為漢族。原發(fā)性高血壓組(EH組)和正常血壓組(NT組)各96例，均來自中國上海地區(qū)，彼此無親緣關(guān)系。EH組為上海市瑞金醫(yī)院高血壓科住院病人，均為漢族，無親緣關(guān)系。平均年齡為57.32±11.75歲。入選標(biāo)準(zhǔn)收縮壓140mmHg和/或舒張壓90mmHg，或正在接受抗高血壓藥物治療至少一年以上；在30歲之前或60歲以后發(fā)病者除外；臨床上除外繼發(fā)性高血壓，如原發(fā)性醛固酮增多癥、嗜鉻細(xì)胞瘤、腎性高血壓等。
NT組為上海地區(qū)40歲以上居民，均為漢族，無親緣關(guān)系。平均年齡56.39±6.45歲。收縮壓＜140mmHg且舒張壓＜90mmHg，無服用抗高血壓藥物史，排除肝、腎功能障礙、甲狀腺疾病、糖尿病、冠心病史。
1.2實(shí)驗(yàn)方法1.2.1DNA提取常規(guī)酚-氯仿法提取外周血白細(xì)胞DNA，并標(biāo)化DNA濃度值至20ng/μl。
1.2.2PCR及測序引物的設(shè)計(jì)根據(jù)GenBank中HNMT的基因組序列，應(yīng)用prime3軟件(美國麻省理工生物醫(yī)學(xué)白頭研究所，http://www-gonome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi)設(shè)計(jì)引物。具體引物如下表所示。
表1 引物序列表
1.2.3HNMT基因的調(diào)控區(qū)和編碼區(qū)分段體外擴(kuò)增以提取的DNA為模板，用Taq酶，在GeneAmp 9700 PCR儀上以Touchdown4～16倍。
表4 冬氨酸阿奇霉素粉針對75株實(shí)驗(yàn)菌的MIC于MBC比較
(3)殺菌曲線的檢測選取金黃色葡萄球菌和大腸埃希菌各一株，以MH肉湯配成2×105CFU/ml的菌懸液，再用MH肉湯分別將門冬氨酸阿奇霉素粉針劑或乳糖酸紅霉素藥液均配成8個MIC的濃度，加入到等體積的上述菌懸液中，設(shè)置無藥對照(在菌懸液中加入等體積的MH肉湯)，于加入藥液后的0，2，4，6，8，12和24小時分別取該菌藥混和物1∶100稀釋后傾皿培養(yǎng)(每皿0.1ml)，計(jì)數(shù)各皿活菌數(shù)，繪制時間-菌濃度曲線即殺菌曲線。殺菌曲線的實(shí)驗(yàn)結(jié)果見表5，表明門冬氨酸阿奇霉素粉針劑對所選金黃色葡萄球菌和大腸埃希菌的殺菌時間為12小時左右，比乳糖酸紅霉素效果明顯。
表5 門冬氨酸阿奇霉素粉針劑藥物對實(shí)驗(yàn)菌的殺菌曲線
∞示菌落太多，無法統(tǒng)計(jì)數(shù)。
(4)實(shí)驗(yàn)條件對MIC的影響選取金黃色葡萄球菌和大腸埃希菌各一株，按常規(guī)以試管肉湯稀釋法測各藥液的MIC，改變菌液濃度(103，105，107CFU/ml)，MH肉<p>說明(1)表中第一列為SNP的名稱；第二列為SNP在HNMT基因序列中的位置；第三列為SNP所屬基因區(qū)域，Promoter為啟動子區(qū)，Exon為外顯子區(qū)，Intron為內(nèi)含子區(qū)，3′UTR為3′末端非翻譯序列；第四列為在等位基因中SNP的兩個堿基；第五列為測序所用引物。
(2)SNP02，SNP04，SNP05，SNP06，SNP08，SNP09為新發(fā)現(xiàn)的SNP。
2.2SNP位點(diǎn)的基因分型在96例EH和96例NT中，對SNP01、SNP05、SNP07、SNP08和SNP09進(jìn)行基因分型、關(guān)聯(lián)分析和單倍型分析。
關(guān)聯(lián)分析結(jié)果提示，各SNP等位基因分布在兩組中無顯著差異(P均＞0.05)。
5個SNP位點(diǎn)，單倍型分析估計(jì)得9種單倍型(頻率均＞1％)，解釋96.74％的EH和94.51％的NT個體，單倍型分布在EH組和NT組間存在顯著差異(見表3)。其中，EH組TAGAA和TGAGT單倍型頻率顯著高于NT組，TGAAA和CAAAA單倍型頻率顯著低于NT組(P均＜0.05)。
具體地，對于TAGAA而言，在EH和NT的出現(xiàn)頻率之比為0.2011∶0.0659(約3∶1)，故每4個具有該單倍型的個體中有3個是高血壓患者(或尚未患病的高血壓易感者)。
具體地，對于TGAGT而言，在EH和NT的出現(xiàn)頻率之比為0.0598∶0.0165(約3.6∶1)，故每4.6個具有該單倍型的個體中有3.6個是高血壓患者(或尚未患病的高血壓易感者)。
具體地，對于TGAAA和CAAAA而言，具有該單倍型的個體中大多數(shù)不高血壓患者，也不易患高血壓。
表3 HNMT基因單倍型在EH組和NT組分布比較
EH，原發(fā)性高血壓NT，正常血壓對照
3.討論高血壓是遺傳因素與多種環(huán)境因素相互作用而導(dǎo)致的多基因疾病，其遺傳機(jī)制的研究已經(jīng)成為目前的研究熱點(diǎn)。迄今研究者已經(jīng)成功地確定了幾種單基因遺傳性高血壓的候選基因，但占高血壓約95％的原發(fā)性高血壓的遺傳方式和易感基因仍未確認(rèn)。定位克隆法是研究EH遺傳機(jī)制的主要研究之一，上海市高血壓研究所已完成對上海地區(qū)漢族高血壓家系的全基因掃描，研究提示2q14-q23區(qū)域與高血壓存在緊密連鎖關(guān)系，HNMT即為該定位區(qū)域內(nèi)的眾多候選基因之一。
由于多基因疾病表現(xiàn)型的復(fù)雜性、相關(guān)基因的微效性和人群的遺傳異質(zhì)性，其相關(guān)基因定位克隆研究一直是一個很大的挑戰(zhàn)。在原發(fā)性高血壓候選基因中篩選多態(tài)性標(biāo)記，然后在病例-對照中進(jìn)行相關(guān)分析是目前尋找疾病相關(guān)基因的重要方法之一。SNP數(shù)量多、分布廣、密度高，是進(jìn)行遺傳連鎖分析和關(guān)聯(lián)研究最理想的遺傳標(biāo)記。
人的HNMT基因位于2號染色體上(2q22.1)，編碼組胺甲基轉(zhuǎn)移酶，有6個外顯子，廣泛表達(dá)于肝、腎、肺、脾等組織(Aksoy S，Raftogianis R，Weinshilboum R.Human histamine N-methyltransferase genestructuralcharacterization and chromosomal location.Biochem Biophys Res Commun 1996；219(2)548-54)。已發(fā)現(xiàn)該基因變異可能減少組胺的代謝，進(jìn)而導(dǎo)致支氣管收縮。由于組胺的擴(kuò)血管作用，這個能夠調(diào)節(jié)組胺降解的基因可能與高血壓有一定關(guān)系。
本發(fā)明首次對中國漢族人群進(jìn)行了HNMT基因的SNP檢測，發(fā)現(xiàn)了位于啟動子區(qū)的4個SNP、編碼區(qū)的1個SNP、內(nèi)含子區(qū)的2個SNP和3′UTR區(qū)的2個SNP。其中6個SNP尚未被收錄于NCBI的SNP數(shù)據(jù)庫(http://www.ncbi.nlm.nih.gov/SNP)中。本發(fā)明對其中的5個SNP在各為96例的高血壓病例和正常對照人群中進(jìn)行了基因分型，雖然沒有發(fā)現(xiàn)單個位點(diǎn)的等位基因頻率和基因型在兩組之間有統(tǒng)計(jì)學(xué)差異，但發(fā)現(xiàn)4種單倍型分布在EH組和NT組間存在顯著差異，EH組中TAGAA和TGAGT單倍型頻率升高，NT組中TGAAA和CAAAA單倍型頻率升高?；谶@些發(fā)現(xiàn)，可以開發(fā)輔助性診斷(尤其是早期輔助性診斷)原發(fā)性高血壓的試劑和試劑盒。
實(shí)施例2原發(fā)性高血壓易感性檢測試劑盒制備一試劑盒，它含有
抽取待檢測高血壓病人的血液3ml，使用常規(guī)方法(或使用特定的試劑盒)從血液中提取DNA。將高血壓檢測試劑盒中的PCR引物稀釋到1μmol/μl，以所提取的DNA為模板與所提供的引物進(jìn)行PCR反應(yīng)。PCR產(chǎn)物純化后，用ABI-PRISMTM377 DNA測序儀進(jìn)行熒光標(biāo)記末端終止法雙向測序，用Polyphred軟件進(jìn)行序列的判讀和SNP確認(rèn)。
用本實(shí)施例的試劑盒對近百例待測對象進(jìn)行了檢測，發(fā)現(xiàn)26位具有(i)TAGAA或(ii)TGAGT單倍型的個體，再對其進(jìn)行血壓測量，結(jié)果證實(shí)其中19位的血壓高于正常人。
在本發(fā)明提及的所有文獻(xiàn)都在本申請中引用作為參考，就如同每一篇文獻(xiàn)被單獨(dú)引用作為參考那樣。此外應(yīng)理解，在閱讀了本發(fā)明的上述講授內(nèi)容之后，本領(lǐng)域技術(shù)人員可以對本發(fā)明作各種改動或修改，這些等價形式同樣落于本申請所附權(quán)利要求書所限定的范圍。
序列表<110>上海市高血壓研究所國家人類基因組南方研究中心<120>組胺N-甲基轉(zhuǎn)移酶基因與原發(fā)性高血壓的相關(guān)性<130>055215<160>23<170>PatentIn version 3.1<210>1<211>52427<212>DNA<213>智人(Homo sapiens)<400>1tcctcactgg gatccacatg aagaactggc tctgttggag aatatttgac agcaggcaag 60ggaggtgatg gtctcagaag accaagtttc agttaaggga ggccttaaca gcagctttcc120aagagaaagt catgtcagga gtattcctca gcagaatagc aaacagagac tcttactaaa180cttttattca atagatgaat acattattgg ccatttttgg ttttggttaa agtgaaacaa240aaacagatag atgttacaca tgaatgaatg aatgatttgg gttgagctct aaagagaaaa300atggaagatg gtaatagaag caaaagtttg tcagagccca tctgaggaaa gggaaggctg360gttggataga gatgtgagta aagaaccagc aaagacctta tctaaggatg agtgaagggc420acaggagcta aatcagggct tggccaaata aggcctgtgg ccaaacccaa ccctaccaac480ctgatttgtg cagcccatga gctaccaata acttttatgt ttttatatgg ttaaagtaaa540ttcaaagaag gataatattt catgacatgt gaaaatgata tgaaactcaa atttcaatgt600ccgtgaataa agtgacgact gtttttgtca ttttatatat atatattttt tttgcagagg660gactggcttg cactttttaa acattagtta tattattctc agtacaaccc tataagataa720aattcattcc cattttactg atgagcaaac tgcagcttat gtttcagcca aagtcagaca780gttcattaag ggcagagtca tccatggcaa tctgcctgtc cagatattac actttccccc840ggggtaaaag cactggctct aagaatagtg agaacaaaac atacttataa aagggagttg900ggggctggga atggtggctc acgcctgtaa tcccagcact ttgggaggcc gaggcgggtg960gatcacttga gatcaggagt tcgagatcag cctggccaac atggtgaaac cctgtctcta 1020ctaaaagtac aaaaattagc caggcgtggt ggcgggtgcc tgtaatccca gttattcggg 1080aggctaagac aggagaatca cttgagtcca ggaagcggag gttgcagtga gtggagatcg 1140caccattgca ctccaggctg ggcgacagag cgatactctg tctcaaaaaa aaaaagagag 1200ttgggaagtg ttttgaatgc ttagagggca gcttgggaac tcaattagaa gaagggacgg 1260tttaagcaga catatttcta agagacttat atgagttaac tcatttgatc ctcagcactc 1320ctccatgagt tggatgctat tattaacaac ccaatattag ataaaggatg accttcttaa 1380agacctcttc ccaggaaaaa gactgtgtgt gggtactctg aagctttggc tcttctggtg 1440gggacaccac tgggtttcca aaggctcatc cagccaagcg aagtatctat gacctcgtcg 1500tgttcagcgc gcttagggaa ggattctatg gactgcactt ttaccctggt gtcctgcatt 1560cttgctccaa gacttctctt accactctca ttcccctaaa tgcagcttct gacaccttct 1620agctacgagt tcttgggctc ctccttcgtg acttcctttt aagataattt ctgagttaca 1680acaaatcagc atttgaagaa atgcagcaga taagttgaaa attcaagtgc tgaggaatgt 1740aaaggcccaa aacaatcccg tagaaagcac agaggcagat gacagtcttc gttaaagatt 1800tcactgctgt agatttctga gagtgctgtg tagctgtcct cttatcctgg tgtttgccac 1860ttcgaacagt cactttccca cttggaactt acttgtggct ttgctgacaa aacaggaagt 1920gatttgaata cagtttgttt ttatgacctt tcaacctcag gaatttgaaa agttttagca 1980gtggatttac agccttgctt aatacttcta tttcttacaa aacaaccgta taggggttaa 2040agtatcagtg caaagtcccg ttcaaatatt gactcagtaa tgagctctac aagccagcct 2100ttgtggcatg ttaaattcaa aacaagctta gcattttttt tttttttttt ggatagaaac 2160tggtgggggt ggggaaagaa agagggaact gggaagagaa ggaggggaac ttaaaccttg 2220cttcctgctc tgtctttctc agaaaaccaa atatggcatc ttccatgagg agcttgtttt 2280
ctgaccacgg gaaatatgtt gaatctttcc ggaggtttct caaccattcc acggaacacc2340agtgcatgca ggaattcatg gacaagaagc tgccaggcat aataggaagg taacaaaagg2400gacgttgttg tcaaagggac aagcctcaga ctggctgtgc gtcagtgata gatgggtctc2460gctcagcctc gcaggctggg cacagggata agggcgaatt actgatagca gctccccgtc2520gtcccctcct cgctgccctg ccctgccttt ctcctgcagt gctttggttc cacgtttgct2580ttgaacactc aaaggctccc atttgtgttg tatttccagc tgcatagaga taagcgttaa2640ccatttgaag gcacaaatcc atgttcattt gggaaaagat aagatgctaa tcttcgaact2700cagatttttc agccctcctc caaattttac atcccaaaaa agtcccaaag ggagtctggt2760atgcttttca ttagaatcaa atataaataa ttgccaaagt acgatttgcc ctgaggattg2820agtgcctcag ttctcataat atccatttaa attaattttc cttatccatt gttatagtta2880tcactctctt cctcaaaatt gtctttttct ttcagcttaa tattagcaat gtgttcctta2940aaaaaagaac cattttccca ttatcagtga aactcactgg ttcctatcct tcttgcagag3000ttcttttttt tgggtgatta atgtgttcaa tttgtgccag agctgataag cattatgaga3060gtaacagatt ccacatgcta aagataatca aatcacctat ctgggggtga tttgctgtaa3120ctcattctac ttcaggaaat aataatggaa aactgggaag aaatgactat ggatccaatt3180tgcaaacagc aaaggaaagt ggtattttat cttttgctga atttctctat tgcaggcctg3240aaattctttc accagggcag tacctatgtc tgaccagagc aggcattatt agatgtacat3300aaaattttat ttccttttct atttaacatg attcagattt taaatgttag ctttcaaaaa3360ataggatatt atatattaaa aattaatatg taataggata aggatgcaaa catcaaagca3420aatagcaaat atagagaact tcttacctag gaactaatta tagttaacct ctttgggttt3480ctttgctcat ttctaaagtt aaaggtaatg agttaatttc tcaattcttt ttcagtacta3540aaagtatttg ctcatgtctg tagcttacca gatccttggt tttcttcagc catacacagt3600ttggtttgaa gaggtaccgt ggatggagat aacataatcg atgatgaatg accccttcct3660tatcttcaaa gtttaaatct aactggaaat attataaaat ctgtgaaaga ctgaagaatt3720tttaagggca cactgtttgt cttataacag tagtatatag ttaggaatga tcagattcag3780tgtcatttat ttttttcctg aaggtaatgt ttgaaataag atttagctga aggactttta3840aaattgttac ttagagttgt ctataagtta tgaagtacca aaatgtgatt aggatactct3900ttgtgtatgt tagggcaggg tttgttgagt ttgtggctca ctgtgctaat tgagtgtgga3960gcgagcctat taaattttta ttaaagcaag acatgccata ttttaaaaca tcaaatggtt4020ctaaagtgtg tggggactat aacaaacccc acccactcca ttcttgattc ccactctccg4080gaagcaacca ctttcagcac tttgaaatct tttagttatt tatggaattt tctttcataa4140ttcaaaataa catgttattg ttctatttct tcatttcgct gtattaaaat agctaagtaa4200gcatgcatat ttcccaccct atcatgtcac tatatgtagt caaaattttg gttaaataat4260ttgagtgttt atgttatttt gcctatgtac atattcttaa taattgagca atatattcga4320ctatgcttat atttcctttt acgtacaact ttttgttctc cctagagttg actattgcct4380tgattttggt ttcttttctt agttctccat gtacctatta ctattttgat ccccaagcag4440atatattaat ctcctctcaa tatatgcaaa catatcaagg aatctgttta ttttacccca4500agggaacaac ttttctgaag ccctctgttc tttttttcag tttaggaaga tctgctttgg4560cctgctatac aactattaag gagggaattt atttcacttt ctgagaatca ttttctttct4620ccattatttg attccctgat tctatatctc atgcctttct tttctttgtt tcctcggtcc4680tttgtttcct gaacaattct ttaagaactt taagagtgaa cgagaggtaa actttttgag4740actttgtatg tttaaaaatc ccttggttct atgttaatcg tttgactgga tatagaactc4800tatactgaaa atggttttgt ataatagatc taaaggtact tttccactgt cttttagata4860ccagaattgc tgttaacaaa taaaatactg gccagatgtg ttggttcacg cctgtaatcc4920tagcacttcg ggaggctgag gcgggaggat tacttgagcc taggagtttg agaccagcct4980gggcaacata gcaagatccc atctctacaa aaaagtgaaa aagttagctg aacaaggcgg5040catgcacatg ctactccaga cgctgaagtg ggaagatcac ttaagtccga gagatcgagg5100cttcagtgag atatggctga gacactgctc tcagcctgga tgacagagtg agaacctgtc5160tcaaacaaga gaaaaaaata aatcaaatgc tactcaaaat tctcatcctt ggcatgtgga5220ataactttta cattgctatg tgttttgttt tccctatttt gaacttttta gaatcttctt5280acgtctagta ttctgaaatt ttaaaatact gagctgtgaa atggttcctt tttacccatt5340ttatgggtac tccctgggct atttcaattg agattcagat ccttcagtta tgcggaactt5400tggtaatcat tcttcaccac tgttttcttt ttctggaact tttattcatc aaatacttcc5460tggattgatt acctaatttt cttatatttc cagtttgcta caactttcct ctttcagccc5520atgtattaac tttttactat tatattttta atttccaaga atgttttcta tagcattctt5580ttcatcattc ccagatgtaa taccttctta ttcattacaa aatgttcctc tgctctctct5640gcctctttcc atctctgttt cttatgttag acagctcccc aaccacctaa agagccttgg5700ttttctattc aatcaaataa tgaggcacta aaaggtagtc agattctctt tgtgtaagtc5760
agggcagggt ttgttgagtg agtagctcac tgaataattg agtgtggact cagctctttc5820ctgggagatc ctcagatata aaatcatgtt aactttatca ctgagatgat ttgtttccct5880atagaggaat tcttcaatcc cctatttgag ggtagatggt ggggcagggc ttgggggcag5940gaaaaattat agtaaataac tataaatagt aaagttggtt attgttgttg catataaaac6000atttaaatcc ttggctagaa aatagtaagt tttcaataat tatttgatgt cattataata6060tgtgaaatgg gatgaataat agtatctaac tcatagagga atttcaagga ttcaaagagt6120taattcacat aaatcactta catgtaccta gaatcataat aattgctaat atatgtggag6180tgcttgtcat gtgcagatac atttctaaga gatttgcatg aattaaatca ttgaatcctc6240acaacttctc catgaattag aaacagttat taataatcct acaagagaga tgatattatt6300atcttatttc acagagatgt caaataactt gctcaaggta acataacagt aacctgggat6360ttcaacttgg gtgctctgct tcatagcctc cctatgctgt catagatgta agcattttaa6420agaatgaaca aaaggtgtag atagaaatgg atgtgttagg actcaggatt atgagttgga6480ggataattag aattgaagaa ggcaaatcat ttataggtgt tctcgaccct agactttgca6540agattccttt agaaccttac ttcaattttt gttcccatcc tgtgactgag caggcttttt6600gcactgtaga caaaacacag ttttgtttgc catcccaaag tccaaagaaa caaacaccat6660ccttttccct tcatggttct cctacagcag tcatctcttt tccttctctc tttcaccctg6720tttcaacaga ctctagatct gcttcctcat ttttgagctc agttttaatc ccattgccat6780ctgacttcag tcttcaatct tcttttacaa ctgccattac taaagacact cgtattgcca6840aattcgatgg agatttttcc ttttttaagt agtacactct gctgcttttg ccatgacgac6900gggctttctc tgcctccaat tactctggta cctctctgtg ctttatttct tttcttctct6960gtcttctttg tatattcttc acaatccagt gatctttaga agttggtatt gcatcaccat7020ccactactac tataactctg caatatcttt gggtgatgga atctactttt ctagcactga7080ccattacttt atgatgatga ttctccagaa tacatcattg accctgattt ctctcctccc7140agcttaaatc ttgcaaatcc aaatgtctgc tgaattcgta tttatttttt ttaaaagccc7200tgacaaaaca taagcttgct gccaaactgt atttctcagc ttcctttttg gcatcaccct7260ttgcccagtc atccaagcct gaaacaggga agtcactgtg cattcatatt tttttctgac7320ctcttcacat caatctaaca gcaagtccta ctggcttatt tcccaatctg tccactccac7380tctatttcta gtacctgagt cataattcgg gctttattat ctcacatttc attttgagag7440caaagtgata tctgtttttt taatgcctag gatagaatac acataatgca tgtttaataa7500acacttgttc attaaaagga aggttaaaaa aagggggagg aaagaaggca aacattatag7560atagtgggaa ccagctgagc ctttccttta tctacgatga gtggcttatt atatgactag7620actcaaaact ccactttttc tgttcttgtt ctccaagcca taaccaaaca aatggcattt7680tgtacctctg atatgtacat actgccattt ccaagtctcc cagttaaaga ttgttaatga7740ataaaaccta tattttgaaa tatactctaa agatggcaat ataactgata taattgggac7800atttcatgtt ggcctagttt tcattcattg tatttttagt ctgttctctt caactagact7860agataatcag atttcacaaa gcacctaaca catttttcta aaactacata atttttttct7920ttcaggattg gagacacaaa atcagaaatt aagattctaa gcataggcgg aggtgcaggt7980atgagtaata tatttttaaa gttcatattt cactttaacc attatgctgt gtgatgacaa8040tattgttcat tttttaggaa gacttttttt ttttgctttt ggaagagatc ggcttattaa8100aactagcaat attacttcaa ctaacattaa tgcccccata gatttacagg gtttaggaca8160ttagccttca ttactatctc tattgtgaat aagtgatgtg acttgttctt ttgatagatg8220aaaaatcaaa taggataact ttaaggataa ctcttatcca aaatgattaa aatactcaca8280gaaaaggaaa atctcataat aagcttctta agtttgtgcc atttaaaacc ttgttcatgg8340actggttact gttttcaaac tgttattggt ccatgtgaag atctgaaatg tgcacaggag8400gctgggcgcg gtggctcacg cctgtaatcc cagcactttg ggaggctgag gggggcggat8460cacgaggtca ggagatcgag accatcctgg ctaacgcggt gaaaccctgt ctctattaaa8520aatacaaaaa attagccaag cgtggtggca gacgcctgta gtcccagcta ctcgggaggc8580tgaggcagga gaatggcgtg aatcctggag gcggagcttg cagtgagccg agatcgcgcc8640actgcactcc agcctgggtg acagagcgag actacgtctc aaaaaaaaaa aaaaaaagaa8700agaaagaaag aaagaaagaa agaaagaaag aaaaaagaaa tgtgcactgg aatataaact8760gatgcacagc ttttttcatt gagaaagtct caccatgggg aaaaatgtgc tcagtgacag8820agttaattta cattcttgtg aactcattct ttgagtggca tgggcctaat ctgttccctt8880cctcttactc agttccaaaa ctgaatgctt agaattacct ctagcattca gataactcct8940aagcaccttt ttttttttga cggattctca ctctgtcgcc caggctgaaa tgtgcagtgg9000tgcaatcttg gctcactgca acctccacct cccgggttca agcaattctc ctccctcagc9060atcctgagta gctgggatta caggcaccaa ccaccgcacc tggctaattt ttgtattttt9120aatagagacg gggtttcacc atcttgtcca ggctggtctc aaactcctga ccttgtgatc9180cactcacctt ggcctcccaa agtgctggga ttacaggcat gagtcacatg cccagcccta9240
agcatacttt tataggcatt ttatagtgtc attactttta gatccagtgc tttcattcta 9300ggggaaaatt taaagtctgg aaaggtagat ggacacaggt aactcagcct ccagtgaaat 9360ttatgatctt ggctaaaaca tcagtaaaat gggaacaatg aattttatct cataaggttg 9420tagtgagagt gatataggta acatttacaa agtgcttagg acattcttat tgctcctgga 9480gagtgaaggc tattcccttc tgcaaaagta aaataagttt catctcaaga cttttcctaa 9540tatcaactcc tgaacagtgt ttgccttatt tttaagtcat ctggaagttt gggttaatgc 9600attaatatat aaaatatgta gatatattat ttatatattt atctttagtt attttctttt 9660gtctgagtga tagttcagta atggttacaa ctcgatatca agtggacact attgtcactt 9720gtaacttaat gccaagtggt aatcccttga acctcttaag tataagtgtt ctagtgcccc 9780atgaatgagt tgaactgtgg ctcatattaa cacaaagata actaagagct ctctaatgaa 9840atgagatcac tgattttttg tttttgtttt ttgtttgttt tttgagatgg agtctcgctc 9900tgttgcccag gctagagtac agtggcatga tcttggctca ctgcaacctc tgcctcctgg 9960gttcaagcaa ttctccctgc ctcagtctcc cgagtagctg ggattacagg aaactgccac 10020cacacccgtc taatttttgt attttttagt agagaagggt ttcaccatgt tggccaggct 10080agtctcgaac tccttacctc aggtgatttg tctgcctcgg cctcccaaag tgctgagatt 10140acaggcatga gccaccatgc ccgacagaga tcactaattg ttaatgtcat tatttgttaa 10200tttttcatga aatacactgt gatatagtgt aataagaact aatcattaaa atcattttac 10260tttattatca tctttcacca agaaaataga aaccaacaaa gacagacaat gaagggaatt 10320gtcatgaaca ataccataga gggtatttaa gctaaaatac tttattattg gttatacttt 10380aagtccccaa aattataata gttatgaatt gtctaaagaa tgtcttatta attcatttaa 10440ccaagacagt tatttcctgt gtgttttgga tatgagtttc aggcaaccaa caggcaatga 10500gactatcatt tgatagtgtc acttgtgctt ggacaagaag agaaggccta caagaaaaag 10560taattaaaaa caatctctat catggccaag ttaattgacc ttttaatata aagttaattg 10620aatttctaat gcaaatttat ccagatgctt ataagctatc tagagaaagg aacgtttggc 10680ctgcacagtg agtccttaca taaaggtgca catggaagct aatacagaga acttaacaaa 10740ataaagggaa caactcttgg agttcttctg actgagccct aataatgtat taataacaaa 10800ctccaccttg gggagaatgt gggtgccctt taaggtcact tttcataaga aatcaatcag 10860aaatgtaaga aaaactccaa gaaaaattta agttatatat acttagcttt cactacccaa 10920ctaagttata tgaaatcaaa agcaatgcat ttaaaatccc taggtctagg tgtctgttgc 10980ttgaaagaac agtgttttga ggggaaagga cactctactt cagttagttt gagtgctcat 11040gtagcaactg ggagatggtt atctaatcac ctattatggc ctgactacaa gcatcaggca 11100gaacaatgca ttactataca gcaaggaact ttctatcctt gccttagaga aatggtctcc 11160ccacatattc taagatctac agggtgcctg agagttttgt attgtcccat ggcttgagta 11220tccttccaaa tcattcatta aactccagtc cctagcaaat gaggtcattg atcctgttca 11280ctcctttgtg ctcagctcct gtaatgtatc tcatagggtc ccatttctgt cttagtttac 11340aggttttatt acctcaagtt tctttccttg tttgcacaga tagataggca catggctttg 11400gtgcaaatat tctctaagca ttcataatgg tgctaccact tctcccaccg tgcttcaaaa 11460acacacaaaa gaaaaccagc taaaagcctt cctagatgaa actgctactt ttataccact 11520taattctcat ggtttttttt gggggggggt ggtctatttg atttttttca gaaacatgag 11580cagggagggg atgcacatat gccctatcga ttagcctctc ctcttgagtc tggtgtttgt 11640gtggaaagag ttttcatagt ttaagagaga ttgtgaggtt tttgcaaagg tgcttggcac 11700ttcagtttag atgcgaagca attattaggg ctctgagaag ttcaaaagaa aggtagatga 11760ctctgagtca tctaccatat caataaatta taactggtaa ataacactgt tatccataga 11820ttacacattc taatgttagg aaaatgtatg aaataaatat tatatgaacc caataggaag 11880aaagagagga aaacaaacca tgaacagaaa aacagaccat gtagctttta cagagaccaa 11940acttggaagt gtaaagttat gcatgtatgt tcatgtatat gagtttgtgt gctttaatga 12000gacaaaatga gtggccagaa ttgctttatt cagggtcttg aagtattcat gttgttaatt 12060tctgcaattg tttttaggtt ttgagagtaa tgcatatatt ctaaatctca tagaacatgc 12120ttctgggtcc atcaacattg gtgcttcctt aaattaaacc tcttgatggg ggttttcatt 12180attattcaac ctctaatgag tgttgtcttc atgctagatg gtacgtggga aaaaagaaac 12240ccatattctt tttggtaaga gagaatgcta ttaatttaca gtgcatatat ttatacctga 12300aaacatgttc cagaaagcag tcacggccaa tgtgttctcc ttgctctggg ctctgcttaa 12360aggataatca ttttatctgt cccagtgttt ggttcttatg ttcaaatgcc agaaagtttt 12420ttaagagtta gacagagctg taactcaaat aatcacattt aatccagctt cctacttcta 12480ttttcattaa cctattgtat attaactcaa ccactatacc attgctatac caataattgt 12540atatacattt atagataaat tttttaaccc actcaagcaa ctcatatatg aaatacaggt 12600gttatcaacc ctcataatat tttttgtaat aatttttctg acatatgttg agaccagttt 12660gatgggtctc ctgttctgca gcatatagac accagcattt ctacagaggt taaaacctag 12720
aagaaaattt acagaccatc aacactctca cttcacaaat aagaaacctg gggaccatca12780aggcagaaga aaacccagat ctcttgactt tagtccagtc caacaaattg tactaaacag12840atttaatagt taattcttaa cctggtaagg gagaaagtgt ctgagacagc tctcaatcaa12900tttagaggtt tattttacca aggttgagga tgcaactgag aaaaagagac ctaggttgca12960gtagcatctg tgatctgtgc tttttccaaa gaggtgtttg aaggcttcaa tatttaaagg13020gggaagagca gcaggagaga aggaggaaag aaaaaaatag ggagggtagg gagtaaggta13080agtggtcact ttcttgtgag ggtttgatca gtactcactg aatccacatg ttatatgtaa13140aaggaggggg gccacatgga gaaatagtgg ccttctgtca ttgcacctat ctgtttaggt13200acaaaaggga aggcagtttt tgtgtgaccc agttctcaag tgtaactctt ccttttaaca13260tagtgagttt ggagtcccag gattttattt tcctttcaca ggagagatta ggtacttagc13320aatgactagc tatttgaaaa tctaacctta tcagattatt aaccttgtct cctttcataa13380ataacatgtt aaggtaacac agaagcttaa tactttaagc tgattggatg gcagtagaat13440gaatgtaatt caagtaggaa ctggaagatc cagtgtgtca ggtttagaaa ctaggaggtg13500gcaggaagca aaggttaatg tttaccaatg atccatagta agccaacagt acagatcaac13560aaacttaaaa tgtgtgagaa aatacaggga attgaggtat tttttgcttg gaaaaaagaa13620tttgattgtg cttattgcca ttttcgtata ttttctgatg ggcaaacaat agaatatttg13680ccctcaaagc gccaatggtt gaaagcttca gagagtgatt tcttggctca gtgtaagagc13740ctgttggctg ggcctggtgg ctcacgcctg taatcccagt actttgggag gccaactggg13800gccgatcacc tgaggtcagg agtttgagac cagcctggcc aacatagtga aacccggctt13860ttctaaaaac acaaaaatta gctgggcatg gtggcacaca cctgcaatcc cagctactcg13920ggaggctgat gcaggagaat ctcttgaacc tgggaggagg aggttgcagt gagccgagat13980cgggccattg cactccagcc tgggggacag agcaagactc catctcaaaa aaacaaaaaa14040caaaaaaaaa aaaacctgtc tataaataga aaataacttc cactccttgg gagataaaaa14100gtcattctag tggtgtcagc acaggttgga gaaatacttt acagatatgt tatggaaggg14160acttaggcac tgattttctt gtgtgtttgg gccatcaaac gtgctttcag tcttgaaatc14220ttgttttatg agtcaccttc tagaaaagag gtaggaatat ttttcggagt agtgtggcta14280ttaccatgtt tgctgtattg aggaagcggt attctaatgg ggtttctaga cctgcagaca14340actttgggaa tggattttaa ttgcttataa ctaaaaatga gtaaaaacta gtacttatat14400gcagcatcat agcagttaac tcctttacca gaaggatata gattataatc ataaactaat14460ctacaagatt tagaactact ttacataaac acagttatag tttggggcat cattttatta14520aaggcatttg ttaaatatat gattaaaaaa taaagtattc ttcataaata attttaccaa14580aatagcttac cccattgtat tgtatttaaa actttcagtg agcattgagc tattgttaaa14640gacagataag ttacagattc aagagaaggg agcatagtgt tagaacactg gagtgattgt14700cattaaaagc agagtaaaca agattgtttt ggctaattca ataaggttag aaacagcaaa14760acaagggata atgaaccaag tgaggacaaa actatcattg cagattaaat gactgtatac14820ttacaaaagc aaataaactt agttctaaag ccttataatt aataagaatg cagcaatggg14880cttaaacagg taaaatagaa aaaaagcagt atgttaggtt cttattttgt tttgttgttg14940ttaacgtttg agtgttttcc attcccaatt ttgattattt acaatctaca taaaagtcca15000tggcatgtgt ttatctgtaa ttttgctgaa gggcaaattt gtatcttgcc agtttttgtg15060ccattttggt ctgcaatctc ttaatcagtg aataagtttc ctcctgacag tcatgctgta15120taaaactttg caatttaata tacacagtca tgcaactctg gtgtaggtga cttgttgtta15180tcttcatttt atagatagtg aacctaataa tatatgcctg aaggggattg ggggggttgc15240agggattgag atcaattcct tgggtctcct tcagcttgat ctctattaac aataacaatc15300tgagagaaac gacgaaaaaa tatttggtta attagctaat aatatatttt attcaaaata15360taaaaaattt aaagaaaagt tcaaatgttt gctaagaata attcaaaaag acttaaaaag15420ctaataacat ataatactga tgaggttaca gtgatatgga agttttcatg tgcaaaaggt15480aaaggtatga aattaaaatt tccctgaaag taatttggca ttatatagca agaacagttc15540tgattttcat ctaatatgcc ctggcctggc cctgctggtt gttaaaaata ttgaaattct15600tgataaacag ctactcttct tccatattat cacagcccca gatactgctt cagtgctttc15660tcctggatat cccaccttgc ctccccaccc cccatctcca gggctccagc aattggcctg15720gccttgccag tccaggaggg ctgctaggac tgtgctacag ttcctcagcc tgcatccttt15780ctgagcacct gccacccttg gacttgagct ttttgtggtg gttgttattg ttgtttatca15840ctgtttttaa aaatatcatc agagatcaag agtcttattc caatcattaa ttcccattat15900ggaaattaac ttaaaaaagt aagatttttg cacaaaaatt tatggctcat tttcagtctc15960ctcacaatac caaaatatct aaaactatat atgagaccaa cgatagtgaa ttttaagtat16020atttatatac gtatatatta actatactac agttatgtat gtatatatta actgtaatat16080aatattacat aattatataa tatatgatta tataatatag tattatacaa tacatatgat16140tatataatat agtattatac aatacatatg attatataat atagtattat atacaataca16200
tatgattaga taatatagta ttatacaata catatgatta gataatatag tattatacaa16260tacatatgat tatataatat agtattatac aatacatatg attagataat atagtattat16320acaatacata tgactagata atatagtatt atacaataca tatgatataa tatagtatta16380tacaatacat atgattagat aatatagtat tatacaatac atatgattag ataatatagt16440attatacaat acatatgatt agataatata gtattataca atacatatga ttatataata16500tagtattata caatacatat gattatataa tatagtatta tataatactt tataattatg16560taatatagta ttatataata ctttataatt atgtaatata gtataattat ataatttagt16620attatataat agtcttatat aataaatagt ataaagtata ctccatatta cataattata16680ataggtagta tacataaaat aggtaatcta ttatattaca tataataagt aatatataat16740atatataatt ggtaatacat tatataatat tcttatataa tatactatat aggcatacta16800taatagtaaa ttgtataata tatagcatag ttcatatatg tttatagtat agttatatat16860attatgtgtt atatatagta cataatatat aaagtataat ttatgttata taagacaaac16920tatatgtaaa attatatatt cacatataaa atatatccat atatatttcc atatataaag16980tatatataaa tacaaatata aatgaatttt acataaatgt atacttttat ttatttattt17040atttattttg agacggagtc ttgctctgtc gcccaggctg gagtgcagtg gtgtgatctc17100ggctcactgc aagctccgcc tcccgggttc acgccattct cctgcctcag cctcccgcgt17160agctgggact acaggcgccc atcaccacgt ttttagtaga gacggggttt caccctgtta17220gccaagatgg tctcgatctc ctgacctcgt gatccaccca ccttggcctc ccaaagtgct17280gggattatag gcgtgagcca ccacgcccgg ccatactttt attttattta tattatatat17340gaaagattaa taattaaatg aattatttca catgtccatt taagagaata tagtatagcc17400actacaaata acacattaaa ataaattttg atatattcat agtttaacac tcaagagaaa17460ggcaaaataa tttatataca taataatctc acctagatag actaagaaaa aaattgattt17520gatcaatgga gaaaaataaa agtttaggaa gcagtgagat gtttcttttc tactttttac17580ctttctctat gtcctgagtt ttctgcatta ttaattttta gccagagaga gaaaaaaaca17640aaactataat tttatatacg agagatgatg gtaaggacta gacctccggc ctctccttca17700tctccttcag ctaaacagct gaagcccaaa gcactgctcc cacaagtgtg ttttcctgac17760agtttttctt cagaagattt tgattcacct gtaacaggtt tttttttttt ttttgagaca17820gagtctccct ctgtcaccca ggctggagtg cagtggcacg atctcagctc actgcaagct17880ccaccaccca ggttcatgcc attctcctgc ctcgccctcc ccagtagctg ggactacagg17940cgtcagccac cacacccagc taatttttaa atatttttta gtagagacgg ggtttcactg18000tgttagccag gatggtctcg atctcctgac ctcatgatcc tcccatctcg gcctcccaaa18060gtgctgggat tacaggcgtg agccaccatg cccggtcctg taacagtttt atacctgtgc18120aaatatttaa agagcaatcc cagctatgtt gtagattagc agagcttttc ttcagagcct18180atagattctc agtgcctagc accgagtata cttcagctct gaggacagtg aggatgatgc18240tgatcaagag aatggtggct atcctctgtt tcttgaggta tttgttgtat cttttctgag18300ttttccatca aatatctgta catttcagtc ttcagcaccc tttgaagcat tttctctaag18360tttgtgtttt gtagaacatt ttggttgcat gacttttctt agacttcctg atgttgcaaa18420accctcaggc agtagatttc ctgggccaag aaccagaaga cacatgaaaa caaatggcaa18480cacccttttg actaagaatt tatatttata acccccttct tgaaactcta cagctatttt18540aatattttat actcatattt tcatatagac agaactgctt taatactgag gtacccaggg18600tgttggaggg agatagagag agaaaaagag aagtaaagaa aagaaaggat cttgtcagga18660atcttctgtc tttcatattg cagttcacaa tcagattgtt ctttctctgg ctgcaaaccc18720acacctgaaa ttatttttca tctccaagca cggagttata tgagcaatcg ctcttcctag18780aaaccatttt ttttctgcct gatattctag tagagggaac aaaaatgata ataatcttaa18840tgcaacatgt aatagaatta aatttttcct gctgataata tatattttga tatgagaaaa18900gaagcaggga taggctgaaa tcgaatggca ggagatatgg ccacagtctt taatccccta18960ttttcttgac ctgcagattg tctcattcgg ggaagctcca gggttctcaa gcggaattcg19020tgtttcattt tgtgtagcac ccgtcagaaa gacaagccag gcatgaggat ccatgatgag19080cgctcttctg agttgccatt tggagctgcc cgtttagaaa gcaaatctgc atttccctca19140ttcctagttt ccttcatcct cttttaagtg tcatcttttc catgaagcct actagatcac19200acagttgaaa attgcaaagt cttcacaccg cagcttcccc tttctttgtt gctttggttt19260tctccgtggg acttcccacc atccgtaaaa ttaggtgttt ttgcctgttt tattcactgc19320tacgtctcca atggttagaa cctgacattt agtgggtgct caataatatt tgttgaagta19380atgtataata acttcacttt cttgagtaat tactatatat catgaactgt ggtaaattat19440ctccatataa tgcctgtaat ctttacaaca cctctaaccc tataacataa accatgttat19500acccatttta cacatatgca atttaaagga cagagagacc cacacatttg cataggataa19560ttctagtgaa taagaaattt gcgttctttt gtctctctac aggcttcttc ttattattat19620tatcactatt ttgaaacaga gtctttctct gtcacccagg ttggagtgca gtgccgcgat19680
ctaggctcac tgcaacctcc gtctctcagg ctcatgtgat tcttgtgcct cagccacccg19740agtagctgag attacaggct cacgccacca cgcccagata atttttgtat ttttagtaga19800gatttcacca tgttggccag gctggtcacg aactcctggc ctcaagtgat ccaccagcct19860aggcctcccg aagtgctggg attacaggca ggagccactg cgcccagcct taattttttc19920ttttcacgat ttttttccat tttaaattaa atgcctttgg attttaaaaa atgcaacatt19980cctctgctat cttattaaac gtatccttga acttcaggga gtggagaata ggtgtctgga20040aaatgagaca ctgcttttct ttgtttttga acttaacaag ttccacattc agagatctca20100attacatgcc cttcattgaa tgtaagtgtg atgcatttaa catacagctt gtgtgaaagc20160aggatattat aaccacaaac atatctgact ttaaaattaa gtaaaatact caacatagcc20220cagaatggaa acatgggaat taattctata tctgtggcag atctgaggga cacggtggta20280ccaaaaattg agatttacag agaaggagat ttttaataat tcagttttca ttaacttccc20340aaatcagaaa gccagcatat tctgtttggt aggaaattac acaggagtga agaaaaaaat20400ccacaattca gctgattaga aaacaagttt tcattaaact aaaatttatt gaatttttaa20460gcaatgtata gccagctagc ttccaaaaat ttgctcatcg atattgctta ataaaaagtg20520ttttctttcc ctttatatgc tgtaataaca taaactgcct aagtgctatt ttggaagctg20580aatcatagag gggctaagtg ttatgaaggc ttattatttt ccagacactt tgctagccac20640ttgacatatg taatcacatt ttattctaat ggaaactctg tggggacttc ctttaaaaat20700gaaagagctg aggttcagat attttgaggc tcagatggtt taagatgcat ttcatcaagt20760aatagacggt ggagccgatc tccaagccca ggttggattt ttcctgaagc gcatgatttt20820ttcctattat cccaaagcct catccataaa tatcatgcag aacctatgta gttgctcatt20880atagagaaag ttctagagcc taaagtagtc caaagtaagt gacccaaaac caggaagaca20940gttaacagta gaatacacag acttgcccaa ggggtatatc ctccacatcc tccacaaagt21000atttcctgct cttcttcttg gactcagcct cccctgtcct cactacagtc actaactctg21060cttttgtatt ttcactgttt gccctgaaga attccattat accattgttg taatttattg21120cacttaatgt gttttatgtt tccgattacc aatctgtaca tttttattct cctattgtat21180atttttatac aatatacata tagtgtatac aatatatatt gtagggaaca aaaaaaaatt21240ccccctcaac cctcatgaat tcttagctgg gatgggtccc tataacaaaa gatagattaa21300taagagggaa acaaacacgt ttattaacgt gcagtgctca tcatacagga tacactccaa21360ggaaaagtaa ctcgaagtag tggtttggaa ctctggctca tagagcatct tcaacaaaaa21420acaataaatt tgtggggaaa tgacaggaca aaggaaaatt attttaggct tccaagggag21480ggaaactgtg ggaaggtaaa tatatgggtg gaaaatatga gtaagtttgt ttgcagattc21540ctctggtgct gtctctggga cggtaagagt ttagagttgt ctccagtaaa ggagaattta21600taacctgccc ttaggcagaa gagtaggagg atagggaaaa ttttccttcc atttgctgct21660tcttaattgc ctttagctca aaaacaattt tcttgtcaaa gaggcagatg tcgaggtgac21720ctattctggt ttccctcaat atattactct ttcttgcatt gtcatggagt actataccct21780gaagattacc gtattgctaa gtattgatct gatctatgga ttctgactgt tacagaatac21840tgagtgttgt gcatccacca caatttttct gccttataac tttccatcta actcccttct21900atcccaagca atgctgtagt gaacgtggtt cagtatctcc ttatccaccc atgtgagaat21960ttctttggat tatgcatttt tattacattc tcaattcaga actcctgggt cgtaggttat22020gcatatattt aatacgaata agtattacca aattagtccc catagtggct gctccagtct22080atattctcag tggcctggat gaagtttctc tatctgaata tccctgacat cacttagcat22140tctttagctt tctgatgttt tgcaagtcca acaagcatca ggtgatattg tattgccatt22200ttaattttca tctctttgat tattaatgag tttgagcaat gtcaatgtgc ctattaactt22260tttaagaaat tatctctata agcccaacag tgggcatttc ctggcatata atggatcctt22320agtggatgta tgattgaaag aaagacatta tagcatgcaa aattcccaaa ttagtctcag22380gctttgattt ttgatacttg ttggttatct cgaaatctga acactaagta aatattggaa22440ttaagttttt gaggtaatta actatattaa tcagttgtcc tgattttaca gtcgaagatt22500gttggtaagt ttgacatagg gaaattaaac ttaaaaaaat cataaaaata acatttttta22560aaagttacca tatgatgtaa ggagtgactg tgagttatat tttcccattt tgtagatgag22620caaatgagtt caggagagat ttaaataccc tatgaaaagc atgcagaatt ctggtagcac22680tataaggata aataaaaaga ttgggccatt tggctagggc tggcatgagt taattataat22740cattatgtga ttaataaaac atttgacata ccagcaatca tgtcatgaag ttctattctc22800tgcctttgaa tagtagcaaa ctaggattat aattttataa tgacagacca gtttaagata22860gaagagacat gacacgtaag actctcagct tctcactatc tgcctaaaac gtgcatggac22920aaaattggtg tgtgctaaag gcagtgccac aggaaccgaa actaaaaaaa aaaaaaaaga22980ttaagaatgt ataaaactgc tctaactcca tcccttgact gccttgggat ctcaaatagc23040agaaatgtat gtccatcaac aatgcaagtg gaagtgtatg tctaatttaa agccgtgtcc23100taagaatagg atttcacata atgtgctagc taaagcgtag agggttggta gagcttttgt23160
tagggatttc cttgcagcct atcttcacat cctacccttt aaacatgctt ttttaaatgt23220aacagcagag cacctagaaa acctattttt ccaagttgtg acttctctct ttcctacata23280cttttattct tctagtcatc caagtaacta tcttactagt agcagtctgt gagtctgtct23340atgcattcat gcatccgtct aaccagctgt ctacctactt atccatattt tttaagcaat23400aaaaatttca ttttgtagcc tcttttacta gtgcacttgc ccagaacaga cagtctccca23460ctcagaagac cagttctttg agtcaccaga taaagttcat atatgatata aggtatagtc23520acatcttcag tatactaaca tttactataa tacagcaatg ccttttaaaa tcaaatacac23580gtgattttac aaaaccaaat gtaagcctcc aggatgtaag agaacaactt gcattataaa23640gtatccttga acccaggagg cagagcttgc agtgagccaa aatcacacca ctgcactcca23700gcctgggcga cagagcgaga ctccatctca aaaaataaaa taaaattaaa attaaataaa23760aatgtttaat tagatacata atggtaaatg tggtactagt gttagtggta aatatggtaa23820ttagcgtaga gaggttaaat gattacagta agctaaaggc caaattgttg ttaatttgag23880ttgttttata tatgttgata aatgccttgg aatagtcact tgcatacaaa ataataaaag23940aaaaaattta gacctgtagt aattagtcag ggttctctag agaaacagaa ccaacgagat24000tatatatata taatataaat acaatttata tatatatacc ttaatatgta aaataaggaa24060ttagcttata tgattatgaa ggctgacaag tcccaagatc tgcagtcggt aggctggaga24120cccaggagag cccatggtgt aagttctagt ctcaaagcca gcaggcctga aggcttgaga24180ctcaacaaag gctgatattt cagtctgagt ctgaaggcca gaaatgacca gttccagctc24240aagctgttct cttctggtct tggattgatt ggatgaggga gggcagtctg ctttactcag24300tctaccaatt caaacattaa tctcatccaa aaatgccctg acagacacac ccagaatcat24360gtttatccaa atgtctgagc actccatggc ccagtagaga tgacacataa aagtaaccat24420cacagaaagg ttttctaatt ggaagataaa cacttcgaaa ataattaatt tgaataaatt24480tttatagaaa catttcaaat tatagataaa aaaagagaat taaaaataag ctataattct24540atcgccatta gataactctt gatttgaatt tggtgcatat ctttctaaac attgtatatt24600gaaatgaagt gctgtgtatc ccattgattt atgctgatac atacttggat ttgcccagct24660ttgctaaaat tacacacaag ggatcaattg agaaaatcag ttacatcatc atcacaagca24720cagaatgaaa aatcattaat ttgccttgct attcacagaa ggggagaaaa gaaaaaaaga24780aagaaaatac tatgatatgt ttgaatagag tttgtttaat catcaaaatt atttctctat24840ttgtcacaga aaatgctgta gcctttgttt ctttagggag aaacatttaa tgtcatttct24900taatagggct tttgttaacc caaacagctg ttggtgtgta tgtatctaaa ggccttaggg24960ggtcaaaaat aagtagcctt caatcatcag agttatgacc aagcaatggt catgagaggt25020cacatgtggg aagagactga ctataaactg agcatgtatt gccctactcc ccttaaatgc25080cattttctgg atctcaagtt taagattcct agaaagtttt gcaatgtaca ataaaagtag25140attgctgctt ccacattttt ttcacttacc cctgaatttg ttgagaattt ctgaattatt25200tgactcaagt ctagtttact ttctgccatt attttgacac attagtttgg cttgtgttct25260ctctgaaagc atttgtaagg cattccatac ctgattctgg aacatagtca taacattgtg25320ggtcctgctc tgaaggattc agttagaaag gacaatggcc actttttttt tttttttttt25380tttttttttt actttgaatc tcttatactg atggcttatg gttctatagg gtatagtaac25440ccctcatatg cctgcaaaga ttaatgatca aatcagctca gttattcaat ccagcattta25500agtatcctta gttcttttta acaaacttgt tggaagcaag cctttgctag tacatcatat25560aaatgaagaa gagcaataat ttagatcagg aaagacaagt gtgttttatc tgtgttgcca25620actccagtca attgatgggt agttagtgtc tggaagattc tcatgctgct tctgggctct25680gtgggaaaga ccactaagat taccagctat gagtggtaga gttttagatg tgtaggagtt25740agccagctga agtgaagaag tgagggagac ctttttaggc agaggagaaa gcatgggaga25800ggatggtacg taaaggaaag agggtagaat gcaagcaatt cagggctcct ggagccgcaa25860aattcagaat ataaaactgt gagtgatgag aataaggacc aggccagata gtctatgttt25920agtggagaag cttgaccttc atccatttta tagtcactag aagattttaa gcagggaagt25980gataggtttg cttagcaggg tttaaaatgg ttatcatggt aagtgctttg aggaatacaa26040gaccagaggt aggctgacct tttaggatat tattgcagta ttccaaatgt gaataatctt26100gacctgaata agagcacagc tagtagggac agaattgaga aagtaaactt cttgatggcc26160aggaaaggaa gataaaggaa gaagaaactt tgagtgatga tggatagcaa acagcatggg26220tgaatgtttc aagaaaagat aatactttaa aactaaatgt taaatggtta aacaagacta26280taatatgaca gatctgaact attgaccccc ctttttttta aacaggcaag aggattatat26340acatcttttt gttggccgga cgtggttact cacgcctgta atcccagcac tttgggaggc26400caaggtaggc agatcacttg aggtcaagag ttcgagacca gcctggccaa tatggtgaaa26460ccccgtctct actaaaaata tgaaaattag cctggcatgg tggtacgtgt ctgtaatccc26520agctacttgg gagggtgagg caggagaatc gcttgaacct gggaggcaga ggttgtagtg26580agccgagatt gcaccactgc actccagcct gggcaacaga gcaagactgt ttcaaaaaaa26640
taaaaataaa tatttttgtc attgttgttt tattttaaag aaaggttttt taaaggaagt26700ttagggtttg cagataaatt gacagcaagg tacagagatt ttccatacaa cccctgctct26760caagcatgca ttgtctcccc attatatata tattccctcc cagaattaca atctgctaca26820atttataaac atccattgac atataattac tagaagctca tagtttactt tagggtttcc26880ttttggtgtt gtattccacg ggtttaaaca aatgtataaa gacatgtatc caccattaca26940gtattataca gagtagtttt actgtccccg aaatcatcta tgctccaccc cttcacatcc27000acccctcagc ccctggcaac cactgatctt ttcactgttt ccataatttt gtcttttcca27060gaatgtcatc tagttgaaat cacacagcat gtaacctttt catattggct ccgttcagat27120agtaatatgc atttaagttt cttccatgtc ttttcatggc ttagtaactc atttctttct27180tgtactgaat gatattctat tgtctgcatg taccacagat tctttgtttg tttgttcttt27240tacttatcag tttatatgct gaaggacaca tcttggttac ttccaagctt tggcaattat27300gaataaagct aataaacatc tatgtgcaga tttttgtgtg actgtaagtt ttcaactcct27360ttgggtagaa accaaggagc ttgattactg aatcatatgg taaggttatg cttcgtcttt27420taagaaactg ccaaactgta ttccaaagtg gacccaaagt tacctttccc accagcagcg27480aatgagagtt cctgttgctc catatctttc ccagcatttg ttgtcattag tgctctggat27540tttggccctt ccaggtgtgt aatggtattt tgttaatgtt ttaatttgca tttcattgat27600ggcatatgat atgaaatatc tctttatata tttatctgtc atctgtatat cttctttggt27660gaggtgttta ggtctttggc ccaattgtta attgggctgg ttgttttctt atcgttgagc27720tttaagagtt ctttgtatat tttggatagt aatcctttat cagatatgct cttttcaatt27780attttctttc atgcttgtca tctcattctc ttgacatttt cttttgtaga gcagaagttt27840ttaattttaa taaagttcag cttattaatt acttatttca tagattatgc ctttggtttc27900tatttaaaaa gtcatcgccg tacccaaggt catctaggtt ttcctctgtg ttatatttta27960ggaattttac agttttatgt tttacacata gttctatgat tcattttgag ttaatttttg28020tgaaggatgt aaggtccttg tctagattca tttttttgca ggtgggtatc cagttgttgc28080agcaccattt gttgaaaaga ctatctttgc tccattgtat tgcctttgct cccttgtcaa28140agatcaattc actatattta tgtgggtcta tttctggtct ctctattctg ttccactgat28200ctatctgtct attcttttgc cattaccagg ctgttttgat tcatatagct ttatagtaag28260tcttgaagtc agatagtgtc ggttctccaa ctttattctt ctctttccat attatattgc28320agattatggg tataaattat tttttaaaaa tacatgtctg ctatttctga gggatactga28380aaagcaaatc atgtagaata caatgtcaag gaatatttta ttttatgatt ttatatctta28440tggatatgta tatataccca cacatattta tatgtacata catatgtata tgttctgagg28500gaaaaactca aactttttcc tctgctctca caccacaaca atcaacacag aagacttctg28560tgaccaaatg catgagtttt ttttttctcc caacaccaag cagcagatac ttccaggtat28620cttccaattc aattcaattc tgatgctgtc tccctgaaaa taatgtcaga tcccacaggt28680tgagggctca gtcccacaaa actgtcccct ttcagacacc agttgcaagt ttgggcctcc28740agaatttctc agtagccagc ttcaagttga ggtttccaca acccctcttt gggtttgatt28800aatttactaa agtggttcac agaactcagg gaaactggct taatttgctg gtttatgata28860aaggatgtta caaaggctac agatgaagag atgcataggg taaggtatga gagaaggtac28920acagagcttt catgctctgc ctggatatgc cacctgccag gatcctacac atgttcacta28980tctggaagct cttcaaatcc tgttctcttg ggccttttat gaaaacttca ttggataggc29040atgactgaag catggacaac catgtagtaa tgtgattgga tgaaaaggtt ataatctaat29100gctgataggc tgagtgggaa aacccagtga ggcctatctg ttcagattct tcttgatctc29160tctgtgcagc attaattcct ctaggatatg ggacaggacc tcctctgaaa tgaggtctta29220tgaccctcaa tcacaaaggt ggaaacacgt tatagtcctg ccttgtatag gtgaaaggtt29280ttctgaggtc tgctcctgag gccaaaagca ccataaaatt ataataaaag actattaaca29340agggctatgg gaatcataag ccaggagcca tagataaaaa catatagata tataaaataa29400tatcacacac atatatcgat ctatgcatat acacacattt ctataaagat acatatatgt29460atgtaaatag atagaagtag atatttatat agctagatat agaagtagat aattgtatta29520tttattacat atatatcaca ataagaaata ctatacttat tataaaaata acacagggcg29580gggccaagat ggccgactag aaacagcagc gtttggaggc ccccattgaa aaaaaacata29640agcttgtgaa tacttcacca gcaaccaagg catccaggtt ctctcatcag aactgactag29700gaggttggtg tgacccacgg agagaaagaa gaacagtgtg gtgtggcagc ccacatgaga29760gccacatgga gtgggggagc cccctccccc ccagccaagg gaggcaatgg gtgagcgtgc29820tttttccaca gaattgtgca acccacagat tggaagatcc cactcgtgaa cccacaccac29880cagggactag cgtcccaacc caggaatgca cagactctta gcttctcggt tggaaggaat29940ctgcttaagc ctacggaact cctggaggga ggggtgacca gaactgggtg tggctgcctg30000ctgtcaaagc tatttgagct ccttgtggga ggagcagcag ccagtactgg gatttgcatc30060tgcctaacac aaagctccct gggcagggga agggaggcat ccatctctat agccccaggc30120
tgtgcttttc ctctgttgga gacagggagg ctggacggct tggtcccaag acttgtccct30180cacagtccaa cacatctgct tgcagtctgt ggccagagtg cctcttcagg cctgaccctg30240acccatcctt cctcattggg tgaggcttcc cccaccaact ccagtaacac cagccagagg30300ctcagggaca gaacctgcat ctccctggtc ctgtgtccct agggggaggg atggctgcag30360tctctgtgga ccagcagact tagcctttcc tcctggtagt tctgaggaat ccaggcagcc30420cagacaagtg ggttccccgc aagtgaggca ctgtgttaaa caggttctgt tccccatgcc30480acccaactgg gtgagaccct ccaatagagg ttgtcagaca ccctatacag gagtgctcct30540actggcatca ggctggtgcc cctcaaggtc agaagtccca gaagaagaag caggcactca30600tctttgctgt tctccagcct ctttgagtga catctccagg cacagcagcg aatcagataa30660ataggtcctg aagtgaaccc ccagcaaact gcagcagccc tacagaagag ggacctaacc30720attgaaagaa aaaaaacaag cagaaagcaa caacaacagc atcaacaaca agaacaacaa30780aaagtctcca caaaaactcc atccaagggt cagcagcctc aaagaccaaa actagacaaa30840ctcacgaagt caagaaagaa tcaatgaaac aatgctgaaa acccaaaagg tcagagtgcc30900ttttgtcctc caaatgattt caacctttct ccgtcggggt aacagaactg gagggaggat30960cagatggaca aattgacaga agtaggcttc aaaatatggg taataaaaac ctacactgat31020ctaaaggagc atgttctaac ccaatgcaaa gaagctaaga accttgataa aaggttagag31080gaattgctaa ctagaataac cagtttagag aggagcataa acaacatgtt ggagctgaaa31140aacacagcac gagaacttcc tgaagcatac acaagtatca actgatgaat caaccaagtg31200gaagaaagaa tatcagagtc tgaagacaag actagagaaa aaagaatgaa aaggaatgaa31260caaagcttcc aagaaatatg agacttcata agaagaccaa acctacaatt gattggagtc31320caagaaggag atggagagaa tggaaacaag ctggaaaaca cacttcagga tattatccag31380gagaacttcc tcaatctagc aagactgcca acatgcaaac tgaggaaaaa cagagaacat31440cattaagata ctccacaaga aattcaaccc caagacacat aatcatcaga ttgaagattg31500aaatgaaaga aaattgttaa gggcagccaa agagaaaagc caggtcacct acaaagggaa31560acccatcaga ctgacagacc tctcagcaga aactctacaa gccagaaaag attgggggcc31620aatattcaac attcttaaag aaaaaaaaaa ttcaacctag aatttcatat ccagccaaac31680tagcttcata agcaaaggag aaataaaatc ctttccagac aagcaaatgc tgaggaattt31740tgttgttacc aggcctgccc tgcaagagct cctgaaggaa gcactaaaga tggaaaggaa31800aagccaggag cagccactgc aaaaacacat caaaatataa agaccaatga cactatgaag31860aaactgcaac aactagtgta caaaataaac aaatagcagc atgatggtag ggtcaaattc31920acacataatg atactaacct taaatgtaaa tgagctaaat gtcccaatta aaagacacag31980tctgacaaat cggataaaga gtcaagaccc atcacccatc agtgtgctgt attcaggaga32040cccatctcat gtgcaaagac atgcataggc tcaaaataag gggatggagg aaaatttacc32100aagcaaatgg aaagcaaaaa ataagcaggg gttgaaatcc tagtctctga caaaacagac32160tttaaaccaa caaagatcaa aaaagacaag ggcattacat aaggtaaagg gaacaattca32220acaagaagag ctaactattc taaatatata tgcaccctat acaagagcac ccagattcat32280aaaacaagtt tttagagacc tacaaagaga ggtatactcc cagacaataa tagtgggaga32340ttttaacacc ccactgtcag tattagatca atgagacaga aaattaataa ggatattcaa32400gacttgaact cagatctgga tcaagtggac ctagtagaca tctacaaacc tctgtacccc32460aaatcaacag aatatacatt cttcgcaatg ccacttggca cttattctaa aatctacgac32520ataattggaa ataaaacact ccttagcaaa tgcaaaagaa ctgaaatcat aacagtctct32580cagaccacag tgcaatcaaa ttagaactca ggattaagaa actcactcaa aaccacataa32640ttacatggaa attgaacaac ctgctcctga atgactcctg ggtaaataat gaaattaaag32700cagagatcaa gaagttcttt gaaaccaaca agaacaaaga gacaacatac cagaatctct32760gggacacaac taaagcagtg ttaagaggga aatttatagc acttaaatgc ccccatcaga32820aagcctgaag atctcaagtt gacaccctaa catcacaatt aaaagagtta tagaggcaag32880aacaaatgaa tccaaaagct ggcagaagac tagaaataac tatgatcaga gaagaattaa32940aggagataga gacccaaaaa ccctccaaaa aaaaaatcaa tgagtccagg agctggtttt33000ttgaaaaaat taacaaaata gattgctagc tagactaata aaggagaaaa gagagaagga33060tcaaatagac acaataaaaa atgataaagg ggatatcacc actgacccca cagaaataca33120aactaccatc agagaatact ataaacacct ctacacaaat aaactagaaa ctctagaaga33180aatggttaaa ttcctgggca catacaccct actaagacta gaccaagaag aagttgaatc33240cctgaataga ccaataacaa gttctgaaat taaggcagta attaatagcc taccaaccaa33300agaaagccca ggaccagatg gattcacagc tgagtcctac cagaaataca aagaggagct33360ggtaccattc cttcagatac tattacaaat gattgaaaag gtgggactct tccctaactc33420attttatgaa gccagcatct tcctgataac gaaatcaaga agagacacaa caaaaaaaag33480gaaacttcag gccagtatct ctgatgaata tcgatgggaa aatcctcaat aaaatactgg33540caaaccaaat tcagcagcac attaaaaaaa attatccacc acaatcaagt cagcttcatc33600
cctgggatgc aaggctggtt caacatacac aaatcaaaaa acataatcta tcacataaac33660agaccgaaag acaaaaacca cattattatc tcaatagatg cagaaaaggc ctttgataaa33720attaaactac tcttcttgtt aaaaactctc aataaactag atattaatgg aacatatctc33780aaaataatga aagctatgta tgacaaactt acccaaccaa tatcatattg aataggcaaa33840agctggaagc attcctgttg aaaaccagta caagacaagg atgtcctctc tcaccactcc33900tattcaacat agtattggaa gttcttgcca gggcaatcaa gcaatagaaa caaataaaag33960gtattcaaat aggaagagag gaagtcaaat tgtctctgtt tgtagacaac atgattgtat34020atttagaaaa ccccatcatc tcagcctaaa aacttcttaa actgataagc aacttcagca34080aagtcttagg atacaaaatc aatgtgcaaa aatcacaagc attcctttac accaacaata34140gacaaaaaca gagccaaatc atgaataaac tccaaatcac agttgctaca aagagaataa34200aatacctagg aatacagcta acaagggatg caaaggacct cttcaaggag atctacaaag34260catttttcaa ggatataaga gaggatgcaa tcaaatggaa aaacattcca tcctcatgga34320taagaagaat caatattatg aaaatgacca tactgcccaa attgatttat agattcaatg34380ctattcccat caaactacca tcgacattct tcacagaatt agaaaaagtt attttaaatt34440tcatatgtaa tcaaagaaga ccacatatag ccaagacaat cctaagcaaa aggagcaaag34500ctggaggcat catgccacct gacttcaaac tatactaaaa gtctacagta accaaaacag34560catggtactg gtaccaaaac agatatatag accaatggaa cagaacagag acctcagaaa34620taacaccaca cacctacaac catctgatct tcaacaaacc tgacaaaaac aagcaacggg34680gaaaggatct cctattcagt aaatggtgct gggaaaactg gctagccata tgcagaaaac34740tgaaactcga ccccttcctt acaccttata acaaaaatta acccaagatg gattaaagac34800ttaaatgtaa aacccaaaac cataaaaacc ctagaagaca acctaggcaa taccattcag34860gacataggca tgggcaaaga cttcatgaca aaaatgccaa aagcaattgc aacaaaagcc34920aacatttaca aatgggatct aattaaacta aagaacttct gcacagcaaa agaaactatc34980atcagagtga acaggcaacc tacagaatgg gataaaattt ctgtgctcta cccatctgac35040aaagatctaa tatccagaat ttacaaggaa cttaaatgaa tttataagaa aaaaaaagcc35100accccatcaa aaactggaca aaagatatga acagacactt ctcaaaagaa gacatttatg35160tggccaacaa acatatgaaa aaaagctcaa tatcgctgat catcagagaa atgcaaatca35220aaaccaccat gagataccat ctcatgccag tcagaatggc aattattaaa acatcaggaa35280acaatagatg ctggcaaagc cgtggagaga taggagcact tttacattgt tggtgggaat35340gtcaattagt tcaaccattg tggaagacag tatggagatt cctcaagaat ttagaaccag35400aaataccatt tgacccagca atgggattgc tgggtatata cccaaaggaa tataaatcat35460tctactataa agacacatgc atatgtatgc tcactgcagc actatttaca atagcaaagt35520catggaacca acccaaatgc ccatcggtga tagactggat aaagaaaatg tgcatataca35580ccatgttata ctgtaaagcc ataaaaaaga atgagttcat gtccttcaca gggacatgga35640tgatgctggg agccatcatc ctcagcaaac taacaggaac agagaaccaa ataccgcatg35700ttctcattca taagtggcag ttgaacgatg agagcacatg gacacagagc ggggagcaac35760agacaccagg gcctgttggg ggttggggtt gaggggaggg aacacagaag acaggtcaat35820aggtgcagca aaccaccatg gtgcatgtat acctatgtaa caaacctgta cattctgcat35880atatattcca ttctttctca gaagaaataa aaaaaagcac agaatgtaat cgcctatgag35940cacagtggta ttatttattg tccaatgtag ggtactttag agaattagtt ggaaagagta36000ggtaaaataa ttaaaattat tataattttt gaaatttgta tgcattgaac aaatttctca36060tattagtaaa tttatgaaat gttctttttt aaaaactact tccaaaacta aaattctgct36120aatgatattt tattaaaaat gaacaagtta ggaccaaaaa acgaaagcag atggacacta36180agtcaacagg tataaaatgg aaatgtccct gacaaacaag tatggtcacc ctacttatga36240ggtcagaaat agattttacg ttctaattga ctcttaagga aatagttgca ttatcacctc36300ttcttaaact taatttattt ttgtcaggaa tccagcacac atgagcttat actctagagg36360ctgttatgca aaccaatggt ttccatatgt caattgtgat tctagcattg tgtcctctat36420cagtgcccca gggagaggga ctgcctggct ctgaacatgt cccagactgt gctagatata36480ttttgccaac caggccaacc tgaaatgtca aggtccatct ttggaggaga gctcatcaag36540ccctgagggg atataattgc atttcagtta tctttttcaa aatgtcgaat ttgagatcct36600cataattgat aatttttcat ttaaaatgta tggctaggtt aatatgaggc aagaacagaa36660aattttggtc tctagtcttt tgggggcaag ctggaatcta tttcccatta gcccacagct36720attcttacca gccttttgtc ttagttgtct ttttttccct tcagaatagt tgctgaacag36780aagaataaaa ggcttttcat gtctttttag catttgtgat ctttctactt cttagttgaa36840tgagttagaa gaataaacat tgtagattta gacaaatgtg acttaaactt caggtatatc36900aatatccctt gaatgtcaac atactcaaat cacctaatcc agttgggaat attaataata36960tcatggaact tgctgtcagg attaatgaga tgttccaggt aaaaagccta gcacagtgtt37020tggcaaatag taggtggcca ataataaatg gcagctatag tcatcagtga agaaatacat37080
gatttcaagt aggtagggct ggctgtgaag aaaattgtca cataagtgac ttcatactct37140atttcagctt ccttcccact ctccctcctt ccctccacac acaccatgta cgccatctcc37200tattgtcagt acttgggact ctcatatgca cctggaccac tgtcttttcc cagtctagca37260tcttctgttc ctggtttatc ttcctcatct ctttccttcc agacctccca gtcttatgac37320ctctgctttt gcttcctgct atgtgcagcc ttggaaagtt tctgctaatg tcatttatgc37380caaatgttaa agactaacat cattttccta tgctaatctt gccaggaggt atttccattt37440tagtgaaggt caacatcata acctaactga ataaaagtgc aaaacataag ttggaggcat37500cagcaagagg gagagaagag atattttcaa atagatgtac tcaagataac aataaaaata37560atgaacagct aacatttatt cactgccaac tttattttgc actgtgcatt atatgcatta37620cttcatttaa ccctcacaac taataaatta agtactatta ttatttttat ttttctaata37680aaaaaaaaaa agaagcttgg agaggtaaag caaattgccc aaagtcgtat acaccgagta37740agtgaagaag acaggtttaa aactcaagtc catctgactt attgtcatct ctcttacact37800aacccttaca ggctgaattt tcaccagggc agggattctt gcttggaagt cctgccttgc37860tcaatattct aagaaatcta gactgctagt agttccaaat atactctttt attttctgtg37920tgtgcatctg tcatcttgtc ccctgtacct gcaatccctc acttccccac agtttctggg37980ctaactccca cttaccctgg gagtgacaga gttcatgagt gatcttcccc catgaagcat38040catcactctg gcatcatctc tcgtcctctc cccatatgtt ctcttccctc ttcctacctt38100gattcaactg agccagggct atttctgtgt ctgtgaacag gtaatagtgt ttatgagggc38160agatatctga catgtttttc ttctttattt tacctgacat atgcttgtac cctttattta38220atctgacatt tttttgtact cttctggcct agcctactct gccagttctg aggtgcattt38280ttttttcgta ttttaatacc tctgatattg ttatgctctc cagttgataa aaatcttaaa38340attttaattg acagtggctt ttttctaact tggtgaaaca aaaataaggg ttaatttcct38400tctacaatca acagcatctt tgatttgatg aaatatagtg atagatgtta aagatcatgt38460aaacgaatgg atggcactca cagccctcct tgagtcacat tactatgcct acttagaacc38520tagctgccct gcatcatggc agggcagcag ttgaacatta ttctttattt atgttaggct38580ttcctagtaa aggtagggca gataataaat cagctaaaat tgtttttaat catttcttgc38640tggaatgatg tgacctgtcc catatgttta tcttctaggt gaaattgatc ttcaaattct38700ctccaaagtt caggctcaat acccaggagt ttgtatcaac aatgaagttg ttgagccaag38760tgctgaacaa attgccaaat acaaaggtac ctgtaactcc tggtcctcta caccagatcc38820tatcccaaaa gacttaactc aaattgttcc cttgaatgat taaaaatata gttactgtgg38880tatgcttttc acaagcttat tgggagaaga actgaattag ttcttggcag gcatgactaa38940acatctcaaa atgtgaacag tgaataataa actccctttt ctattaacac ttcatccatt39000ccccagttgt catcaatgat taccttttga tgtttatgct taagtacaga ttcattcatt39060gatccagaat tatttaaacc aaccttaaag agcttatgtt tagtgggtta cagtaaaagt39120tcacaaactt ttcctggcaa aggccaaata gtaaatattt tagattcaga aggtcaattc39180agtctctgcc ataactaatc aacctgccat gtgccacaaa ggcagccaaa aagaaaaata39240ggtaaactaa caagcatggg tgtgtttgaa taaaacttta tttgtaaaca ctaaaatttg39300aatttcatat cattttcaca tatgaattag catccttctt ttgttttttt aaatcattca39360aaaatataat gacgtgaaac atgaatggca gtgacacaaa gtcaggaagt agaacgatat39420atgtttttag cttgcggttc atacaaaaat aagtagcagg ctagatatag tccctggact39480gtagtttgtc ttctcctgaa ttatacaaac atatctgttt ccaattcact ttccattgat39540aaaattcacc agttgattct tcattaatac taagacaata taacctattc tgcccagggt39600tcttgtaatt atttcttcct gactctttct ttcacctcta atttcattaa atactagaaa39660ccaaaaaaag cagtatgaac tctctggtct cccagacata agaaagaaaa acgttctttc39720tatctgtttg tatataacat tgattgcatt aaaattctaa atggaaatgg tttccacatt39780tgcaattaaa attgatggtg tgtcacctct tctctgtatt ttagagcttg tagccaagac39840atcgaacctc gagaacgtaa agtttgcttg gcataaggag acatcatctg aataccaaag39900tagaatgttg gagaaaaagg agcttcaaaa gtgggacttt attcatatga ttcaagtaag39960aaatatgtat tataatatat actcagaaag aagacttttc agaatatata tataatgaat40020atctactatt ctaattttta aaaggtaaat gaaaatcttc tgatttctaa ttttacaagt40080gaccatgatc tgcccaaata aaaggtaaag atgtttatta tactaagatt ctctttaaca40140ttccaaaaac cagttttctc taatcaaaat acctctacaa gttatccatt tttaaaaatg40200ttggcttaat tcttatttct tttttgagat gggagtctcg ctgtgtcatc caggctggag40260tgcggtggca tgatcttagc tcactctagc ctcaaaatcc tgagcttaag caattctccc40320actgcagctt cctggttagt tgagaccaca ggtgtgcacc accacaccca actaattttt40380tattttttgt agagatgggg tcatactatg ttgcccaggc tggtcttgaa atcctgggct40440caagtgatct tccctcttca acctccaaaa gtgttggaat tacaggtgtg agccactgca40500cccagtcttt aatttatttt tatatcattg gatgtttata tagagatttg agaatgatag40560
catcataaca tattctctag tggctctaat ggtttgggtg gaccaatgtc ccagagtgtt40620tctgtagtgg acatatggtc gaaggggcag gttaaatgat gcctcagttc aataaatttt40680tacttccatt atccagatcc gacaaactga ctcatacatg ttatagaaag ggagtgacgc40740aagaacaaaa aaccaaacac cgcatattct cactcatagg tgggaattga acaatgagaa40800cacatggaca caggaaaggg aacatcacac tctggggtct gttgtggggt ggggggaggg40860gggagggata gcatcgggag atatacctaa tgctagatga cgagttagtg ggtgcagcgc40920accagcatgg cacatgtata catatgtaac taacctgcac attgtgcaca tgtaccctaa40980aacttaaagt ataataataa taataataaa agaaagggag tgactattga gtagatagca41040tggggctgga tttccagaga taactaacat ttctgaatag cataagggat gtatgggttt41100tggaagttgg ttaccaacgg gaaatgtacg tattttggca ataccacttg attaacgaca41160aaacactatt tttcttcctg gtctacaatg ataatcaccc caaaagaata aacttacaag41220aagtcaaagg aagaaatact ccaattgtgt catatacagg caattctatg tctctgcttc41280tttagctgat gaaatctttt gcctgtcagg tagtacttat cactgtccag gatgccaaga41340tatttctcta ccagacttgc ttttgttccc acttcctcac ataactattt cataactccc41400ctgtctcctt accactcctg caatctctcc cagaccattc gttctgagcc aatgacctca41460cttcataatg tatagagaaa atagaaactg ccagatactc attttttacc ctctaacttt41520gcaaacataa ctgcatttgt gcatcatctc cttcttccat cttattgcaa aaacctcctc41580ttatcaaaaa ccactccctc tgcttaaccc tggatcccat tcttttctct gttataaata41640ttgtgatttc aggaatcagc ttctctttcc tatttctctt aaaacttcca tgtttgttcc41700ttctttctta taacaactct gtttccctct gaatctctat cctgttcctc tctctccatc41760tctgtctatg ccccattttt ctgtgcagcg tctctgcctc tctattactc attatagtcc41820tcacactctt atgatacttt tacaacctca ctgtcaattc agaaaatttc actgtctttg41880agaaaaattt aaggtatctt ctatggccca cttccacatt tctagccagg tcatatcgta41940gattgttagc catcatattg attcactata tttgggtacc tgcctatggt ccctgtggtg42000gaaaatgtgg agtgtggccc ctaaggcacc atgagccatg cacaggtcag gtccatttta42060tttcattcat tcatatgaaa gaggcacagc taaagtactt ttcctttgtg caatgaattc42120tctaaggaca ggctatatgg aatattgaat tcttgtccaa caaagatgaa gatgagatat42180aagaagtgtc tatgggggtg gggtccaaag atgcaagcca tggggaaaaa tttcccacgt42240caatgcctaa gtccaagttc tgtgttgggt ggaaaggcct gtacttcact agagattgat42300ccagaattat taaaaatggg tgtcctgacc cctaaacacc ctgtgaccaa gcagataacc42360tcatgcttta tttagtccaa cttcctacag aatctgtcat catcagaggc tgtcacattt42420tctgtgattt taaaaaagca tctgtgagca tatggagaag ctagtgcaaa gggcttaatt42480gtaaagtaac gttccatgaa atggggtctg tttttatggt gccagaacca ttttctccca42540gaagaactgc aattcacgaa ttatagaata ggcataggat tcatgtcccc atgtggaatg42600gtgactaagt gtctccagct ataaatgtct gtgctatgga agccagaaag agtaaattta42660aaaccatttg actctagtcg aacaaatacc atacttttcc atttccccaa atattttctc42720acattattgt tctatgtctt catgcaacgt ctttaatctt taaatttctt aagtgttttc42780ttgggtaaaa attcactcaa ctagaaaatc aaactttcta ggagtatcta gcccaagcaa42840taaagacttc aacatacaga agcagctcat ttctcttttt cctcctttct agatgctgta42900ttatgtaaaa gacatcccag ctaccctgaa attcttccat agtctcttag gtaccaatgc42960taagatgctc attattgttg tgtcaggtaa gttattttca ttcagcctga attttaaaac43020agcaataata cacgtatgca tggattcctg tgtttgaaag aagcttatat attttgtctt43080cattatgaaa ttcttgcctt ggcctctaat catagccaat taattttcac agtattaggg43140aagtcattca caacagttct caatttagaa ggtatagtgg aattaaacat ctgtctactt43200ctttcttttg ggaaaatatg tctttgtctt ctatatatga atccttttta tatagcttaa43260attcaactga caaaagattt taaatgagac taaaaatagt aaacatttat tttacctact43320tcgtgtcatt tgagtgtttc ttttccctct atgatgttta ttaataaata aaggttaaac43380tatttcagat gtcatcattt ttaattgact tgcattctta gagccatgat caacactgct43440tgctataggt tttttcaaat gcctatcaaa aacagttcat ttgttttgtt ttgtgttggt43500gttttatttt ccaaggaggc atgtgagcat gtccagttgc tgccagggat ttagtaagta43560tcacacagag aacccaagta aacattttta gataatgaaa cattgacatt gttgaggaca43620gtatttatta cttctttcat tcagcaatct ttatggattt tctaccagta cttacaggtt43680gattttagat acacagcaga ctctgtcttc ctaaggttac accagacccc atactttaac43740accgtatgtc acaaaattga cttgttctta aatatgaaga attgctttaa agatggcaag43800catggttata gcttgtaatc ctgggtttga ccttatgtga aagtttggca atagagagct43860cctgtacaca acctctcctg caagggaaca tttagatata ctatttttat ctctatttag43920ttaaaaatac tcatttatgt gggagaggtc tcatttccca tttagaaggg cttatggaaa43980ctggactcat cctactaacc tctctctgag ctagtaaagc actataaact gcatttagaa44040
ttgcaaagta aacttagtta tcaacattgt aggcagccta ttcttgatta tattcatcaa44100tcatacagaa aaagtattgt agacggaaat aacaattcat tgagaagtca tgtatttttt44160atcttagaat atatttcatc tcaatagtag gtctaatctc ttatttatat ttggcttatt44220ctaacatatt ttcagaaatt aagtatctga gcacccagaa gttagctttc tataagtata44280tattaccaat aagacaaagc cagcctgaag tttagaaggg agcatgaata gtccacaagt44340aactgagaac tgattaagtg cccttatggc tacagtattc tgctggctgt atcatgaatg44400aattaattga tgattattgg catcaagaaa tttgaagacc aaaataaacc caatgaaatg44460tgttgtatgt taatacatgg tgcaagaata ctgaggcact tttaactatg tgaatttggt44520ctctgcatct cagtttattc aagggctatt tctggacaac cttatctgca ttatattaca44580tcttttcata tatgtggctc ataagtacca tctgtaaata agcattcatc ataaaaaaaa44640tcaaaagaaa gggagtaaag actcaacaat cagttagctt tttttgaaaa gtatttttag44700tcaaaaatta tttaaccaaa gtatttgatg atggaaatgt tttactcact ttctcaatat44760gcagttaaca attcttcatt actgcttata gcacaggaga cagctgttcc aaagaggaaa44820acattgttaa tagaatgcaa cgaataacat ttgacagatg attaaagaga ttctgatgat44880ctccttcctt taccatatca cttcttctga gaagagattt acacagctat agccatctat44940ttcttcctaa aagaaaaacc acttcgcaaa agtagaagaa agatttcgat attttatttg45000gggctttcta cgtctctctc tcctaagcct aagaatgtct tggctttttt tgcctctatt45060tgtcagaatg ctaagaaaat aaataaatta aatataagtt aatttttttt aaaacctact45120tttcccaata atgaatactt tgcagtgaaa aataattcta cctgaaaaga gaaatagaat45180tgtgaaatat gttgatcatt gtcatcaatt tagtagaact ggaatgtccc cagattctcc45240aatcctatcg gcatgtcata cttaactctt aaagatccag agtaaatgat ggagttctca45300tttcaagtaa cttgggtaat ccatcccctc atcatcactc ctttactcct gagggttcct45360gggggtcaga cttaaatgtt cctggtgatt gccatccact gtgtgctcca agttcaactt45420ttgatttctt ctccccaaat cttgcatgtg ctgcagccgc cctttctcat cttgtttcac45480attgccttaa acatggacat agtcctacct ccttccttct ccaaaacacc attcttgctt45540ttgacagatt ttgggacctg tgggggagaa agaggaacaa agaacagaat ttagcattaa45600ggtcccaccg tgactctttt tctcaccttt ctcttgctca atctggacac acttccgttg45660aacctgctct cataacttct ctcagtcttc cctcatatcc tcttagttca gtgctaagat45720gccacctcct cattttctat tgcaaccctc ttttttaact tttattttaa gttcaggggt45780acatctgaag gtttgtcaca taggtaaacg tgtccggggg gttgttaaac agattatttc45840atcacccagg tattaaaccc actacccaat aataatcttt tctgctcctc tttctccccc45900caccctccat cctcaagtag acctcaatgt ctgctgtttc cttctttgtg ttcatcagtt45960ctcatcattt agctcccact tataattgag aacatgcggt atttggtttt ctgtttctgc46020attagttttt taggccaata gcttctggct ccatccatgt ccccagaaaa gacatgatct46080ctttattttt tatggctgca tagtattcca tagtgtatat gtatcacatt ttctttaacc46140actctgtcac tgaagggttt ttaggttgat tccatgtctt tgctattgtg aataatcctg46200caatgaacat ttatgtgcat gtgtctttat gacagaatga ttaatattcc tctgggtata46260tacccagtaa taggtttcca aaccatacta taggggggta caataaccaa acagcatggt46320actggtacaa aaacaagcac atagaccaat gaaacagaat agagagacca gaaataaggc46380cgcacaccta cgaccatctg atctttgaca aagctgacaa aaacaagcaa tggggaaaag46440acttcctatt caataaatgg tggtgggata actggctagc catatgcaga agactgaagc46500cagtctcctt ccttatacta tatacaaaaa tcaactcaat gtggcttaaa gactacctaa46560aactataaaa acctggaaga caacctaggc aataccatcc tgcacatagg aacaggcaaa46620gattctgtga caaagacact aaaatcaata aaaacattgg caaatggcat ctcattaaac46680ttaagagctt ctgtacagca aaagaaacta tcaatagaat aaacagacaa cctacggaat46740ggtagaaaat atttgcatag tatgcatttg acaaaggtct aatatccatc atctataagg46800aacttaaatt tacaagagag aaacaacccc attaaaacgt gggcaaagga catgaataaa46860caattctcaa aagaagacag agatgtggcc aagaagcatt tgaaaaaaag ctcaacatca46920ctgatcatta gtgaaatgaa aatcaaaacc acaatgagat accatctcat accagtcaga46980atgactattt attaaaaaga caaaaaataa cagatgctga tgaggttgtg gagtaaaagg47040aacacataca ttgttggtga gaatgtaaag tagttcaacc actgtggaaa gcagtatggc47100aatttcttag agagctaaaa gcagaactac cagttgaccc agcaaccctc ttatagagta47160gtttaaatcc cttttgttct atttctatca cctagataaa cctgtgcaca catgaacttg47220tcttaacaac aacacatttc cctgagataa attgttttgc ccccctcagt gtatgatgag47280ggtgatggta gtttcctatt ttaaaaaaat agcaatagtt gctttgttgt attaacatac47340ccaatagaaa tatattggtc agcttttgga aaaatagatc aagtctagtt tccaacattg47400gaaacaaatt tgatcacaag aaatttccaa tcagacattt tcatattgat gtcagtgaac47460acacatagga atgatccaca aaattactac tcattgtttt ctttcaatga tgtagcttca47520
aatccctatc tgctagacta gtcagtggaa agaaaacaag tttgagaaac ctcatcagcc47580gaggtgttcc atctacatcc ctccttaccc ctgtttagga accattgcct agcttgcctt47640ctccaccaga gctataattc tttaaacccc atttttcatt tactcaacag attgaaacat47700ttcctaaagt tgaatattga cccagaactt ggaatgtcta taccacattg gtttacacta47760agttctgttt ttattaagat tctttgggtc actttcttaa tggaatttgc ttcataatta47820cctaaaaggg aagatgcttc tgtgtatgtt catttgtttc agggtaatat tgtcatccat47880agctgctctc caatgcacaa aactgagtac tcatggtata tagggaggca tatagggaca47940ttcatctctg ttcctacatc tttgtaaaga ctttggcaca gagacaatac ttagtaaata48000tgttttaatt gaactgaata attgtttatt atttctgcct tatgatatca gaactacaat48060aagctctgaa tatacatcca aaaaaagaga atatatttca gctaaaactg acttgacgaa48120atcagagaac acaggaagtt ctggatattt gcagatgaag tttaataaat aggaataaaa48180ggctcaataa aaaagacaca cgcacacaca cacacacaca cacacacaca cacacacaca48240cacagcaaat ggaagcagcc agacaaaagc aataaagaac aaacaactaa aactatttgt48300gtatttttag ggaaagaaaa agaaattgaa atcagaatta aatgacaaca tttactggtc48360agcttatgga aatatatgtt gcttgatgga aatcaacctt tcctctgtgt ttctaaatat48420ccattacaga attaccctaa ctcaagagac tgtcatttag tgataactag tagaagttcc48480tgataatgga gatgaaagaa atttgatttg gggataaaaa agaagagata taagaacaag48540agagttggat ttctttaaaa aatgtggagt atcccacact aaaatggtat caaagaaaag48600tttttttctg caaatgtgat agccaagcat caattgggag ggttgctgtt taaaaggcag48660cctgtctggt taagggagat gagccagaag gagatcactg aagctttctg tctgtcctct48720ccttacctga gtaaggaagg aggaaaatca ttgtgtgtta tttttagtgc agtgagaaaa48780agaaaatctg gcttacatca tgttgaaaac aaaagtaaca ccatatgttg tgcccaccga48840gagccattat gataatgcat tagtcgggaa gtgcataatt gctcagctca gaaatggcac48900cgagtcacag cactgacacc agcaaatttt ttgcaattag tgtttggttc ttttttctct48960gccaggttaa aaataataat aatgatgata acagagcctc tcttcaacca ctgaaatgct49020gattttcttg taatgtcttg tttgccggca aactcttatt taggaagaca atccactgtc49080tttggaataa aagattttta aataaatact cacaaccctc atttattttc attgaacaga49140ggttttattc tttctgcttc attccaattc caccttctcc tagagcatat cctgcagtta49200cctctccctc ttctgaaata taagtagaac atcaattatc ctaggctatt tattatttag49260cctattattt gtcttggttc atctcttgag ttaaattaga tctgggaact ttagcttaat49320ccatgcatga gcgccaaggg tctttgagca ttctataatc ttatgcagaa ctgtgtgggt49380atgtattgtt tgtgtatttt tctggagagt cagttactct cactagatca taaaggggac49440ttgggaacca aaagtatctc aagacattta atcctagaag cacaaagaaa gtataagttg49500tctcttatat tgcttttgcc aaatccagtt taacacttca gtaacgttaa ctattgtgtc49560atggttctaa gggtctttac tttattaact cagtgaatcc ttgtagcagt ctataaatgg49620attatattat cacccctgtt ttacagatga taaaactgag acacaggttc ataaagttgg49680taagaaggaa agccaggctt catactcagg cagttgggtt ttagagacca tgctctttat49740tactgccctg tactgactgt gtatgtaata tgcatgtatt agaaggccca taaatagaaa49800ctcattatta ttattataga tttctgaata gcaggcttgc ttttttagtc tcaagaatgc49860aatcagtgga aatacactag caaactaatt catgtatttt ataagcactt attacacatc49920tttcatgtgc gtaccattac gcaataattc acttaaatta tctcatgtaa tgcttgtagt49980tgcattcaag gtaagtttga taatccccat tttacagatg aggaaattta agttctgaga50040ggctagataa caagcctcat ctcattcata aagcatcaga agaagagttc agctcatgtc50100tgtctccctc agataaggta ttccgtagct tctctgcagg gttagaattt cagtttagta50160ctacaaacat tgctaaaagg tcttctaaac ttggggacaa gatttataga aatccaccta50220gttattgtca gtccaaggca gcatcttttt ttatatagag atgagagtaa aaaatcattg50280atttgagtaa atcaccattt actgtgctct ttctttccat tttaattttc actgttaaag50340attcttgtct gaattccaga ctaacaacta ccttcctcat atcaccacct gaatgtccag50400taggtatctc aaatttatat tcctgaaacc agaggcctga tgatacaaaa ctgctcgtct50460tcattcttct ctccaacaat ggcaattctg tcttctcagg cgccctcaca gaaattactg50520gggtcatcac tgattctttc tttttaccca tatcctacat ccaaattgtt ggcgagccct50580gttggatctc ggagtataac acataaaaac tcccaccact tctcttcacc tcccagctca50640ctggtctgtg ggacccaccc ccacctatcc cccaggtgat tacagtagcc ttctcgctgt50700tctccccacc tctgccctta tgcgccagta ctctcttccc atcggtgagg ccagggtgac50760cttattaaat gtatattgag tcattctaat gtgctgttcc atacagaaac catgttttag50820tagtgaagtt gaaactgaaa ttaatttaaa tgaatgaaac ttaaaattca ggtcttcagt50880aacacttgaa actcaagtcc taagccacct ttcaagtgct ccatagcaac aagtggtgag50940tgacaatcct gctggacaat gcagatatcg aacgtgtcca ttggttcaga aaattctatc51000
agacagcatt acagtggttt cccattgggc cccaagtgat ctgggccctt catccactcc51060ccacccctaa tctctccaac ctcatctctt ttccctctgc cctcactcat tcagctccaa51120tttcactggc ttcctggcta gttctttaat gggccaagta tcatcctgta ttcaggtcct51180ttgcagctgc tcatctctct cctggagtca tcttccccct gatatctcca tgtcttgctg51240tcaggtctct gctcaaatgt caggttatta gacagactct gcgaccaccc tagttaaaac51300agcagcccat caaagtcttt aaccctctta gcatttactc agattacttt ttcatttagc51360atttatcacc atatatttac ttatttttct ctctcctcct gtactccttg aaatatgagc51420tgcacaaagg acaagattat tattttgttc tattctaagc atctagtgta ctttctggca51480gactgcaaat gaaagaaaga ataaatgaaa gcatgacttg tgttttattg cacaggaagc51540agtggctggg acaagctgtg gaaaaagtac ggatcacgct ttccccagga tgacctctgc51600cagtatatca catcagatga cctcactcag atgctggaca acctagggct taagtatgag51660tgctatgacc ttttgtccac catggatata tctgactgct ttattgatgg taatgaaaat51720ggagacctgc tttgggattt tttgactgaa acctgcaact ttaatgccac agcaccacct51780gatctcagag cagagcttgg gaaagatcta caagagcctg aatttagtgc taagaaagag51840gggaaggttc tttttaataa tactctgagt ttcatagtga ttgaggcata actatcaatc51900acaaaagtat attcaaaaat tatattttga acaactcgaa tcactcattt atttccatat51960taaaatcaca aactcatcca ttaatgtaga taaagcactg tttggatatg agatgtagca52020aattccaata cattattgga cttccatttg gaatcatatg ggatactgct ggtcttatcc52080tgtccctcct ccaggtagag agaccacaag caggctcaac ataacataag ctagaaaaat52140tagatgactg aatttctatg gcatattgat aataaaattc attccatttg ctgattgtct52200gaaattttct agaatactaa taaaatacat actatagatt ctttattagt gaagtatgca52260ctaatcaata ctttgaacac aaagcctgtg ttactgattt ggccgttttg tgaagaaaca52320tttatctttg tacgttcttc tattgtgctt tctatctaat ttttattaat ttgtaagagt52380aagcaccttt agaatattaa aaattaattc tttatcacat gttgtca 52427<210>2<211>20<212>DNA<213>智人(Homo sapiens)<400>2atatttgaca gcaggcaagg20<210>3<211>20<212>DNA<213>智人(Homo sapiens)<400>3ccgggggaaa gtgtaatatc20<210>4<211>20<212>DNA<213>智人(Homo sapiens)<400>4gtttcagcca aagtcagaca20<210>5<211>20<212>DNA<213>智人(Homo sapiens)<400>5ctgccctcta agcattcaaa20<210>6
<211>20<212>DNA<213>智人(Homo sapiens)<400>6aggttgcagt gagtggagat20<210>7<211>20<212>DNA<213>智人(Homo sapiens)<400>7gaagctgcat ttaggggaat20<210>8<211>20<212>DNA<213>智人(Homo sapiens)<400>8tgcactttta ccctggtgtc20<210>9<211>20<212>DNA<213>智人(Homo sapiens)<400>9gcaaggctgt aaatccactg20<210>10<211>20<212>DNA<213>智人(Homo sapiens)<400>10tgcaaagtcc cgttcaaata20<210>11<211>20<212>DNA<213>智人(Homo sapiens)<400>11taattcgccc ttatccctgt20<210>12<211>20<212>DNA<213>智人(Homo sapiens)<400>12actgccattt ccaagtctcc20<210>13<211>20<212>DNA
<213>智人(Homo sapiens)<400>13cgatctcttc caaaagcaaa20<210>14<211>20<212>DNA<213>智人(Homo sapiens)<400>14agggcagcag ttgaacatta20<210>15<211>20<212>DNA<213>智人(Homo sapiens)<400>15gtttagtcat gcctgccaag20<210>16<211>21<212>DNA<213>智人(Homo sapiens)<400>16tctggtctcc cagacataag a 21<210>17<211>20<212>DNA<213>智人(Homo sapiens)<400>17ggcagatcat ggtcacttgt20<210>18<211>20<212>DNA<213>智人(Homo sapiens)<400>18tgctatggaa gccagaaaga20<210>19<211>20<212>DNA<213>智人(Homo sapiens)<400>19gccaaggcaa gaatttcata20<210>20<211>20<212>DNA<213>智人(Homo sapiens)
<400>20tatgagctgc acaaaggaca20<210>21<211>20<212>DNA<213>智人(Homo sapiens)<400>21ccttcccctc tttcttagca20<210>22<211>20<212>DNA<213>智人(Homo sapiens)<400>22actttaatgc cacagcacca20<210>23<211>20<212>DNA<213>智人(Homo sapiens)<400>23tcagacaatc agcaaatgga20
權(quán)利要求
1.一種體外對個體的高血壓易感性進(jìn)行輔助性診斷的方法，其特征在于，它包括步驟檢測該個體的組胺N-甲基轉(zhuǎn)移酶HNMT基因的基因組序列并與正常的HNMT基因的基因組序列相比較，存在差異就表明該個體患高血壓的可能性高于正常人群，其中所述的差異是在HNMT的基因組序列的第616、7798、40000、51951、和52109位，存在選自下組的單倍型(i)TAGAA(ii)TGAGT，其中，核苷酸位置編號基于SEQ ID NO1。
2.如權(quán)利要求1所述的方法，其特征在于，檢測的HNMT基因組序列來自于個體細(xì)胞的DNA抽提物。
3.如權(quán)利要求1所述的方法，其特征在于，所述的細(xì)胞是外周血白細(xì)胞。
4.一種檢測樣品是否存在HNMT基因的單倍型的方法，其特征在于，包括步驟(a)用HNMT基因特異性引物擴(kuò)增樣品的HNMT基因，得到擴(kuò)增產(chǎn)物；和(b)檢測擴(kuò)增產(chǎn)物中對應(yīng)于HNMT的基因組序列的第616、7798、40000、51951、和52109位的核苷酸，并判斷是否存在選自下組的單倍型(i)TAGAA；(ii)TGAGT；(iii)TGAAA；(iv)CAAAA其中，核苷酸位置編號基于SEQ ID NO1。
5.如權(quán)利要求4所述的方法，其特征在于，所述的單倍型是(i)TAGAA；或(ii)TGAGT。
6.一種用于輔助性檢測高血壓的試劑盒，其特征在于，它包括特異性擴(kuò)增HNMT基因的引物和說明書，所述的引物所擴(kuò)增出的擴(kuò)增產(chǎn)物含有對應(yīng)于HNMT的基因組序列的第616、7798、40000、51951、和52109位的核苷酸，其中，核苷酸位置編號基于SEQ ID NO1。
7.如權(quán)利要求6所述的試劑盒，其特征在于，它還含有與第616、7798、40000、51951、和52109位的部位結(jié)合的探針。
8.一種組胺N-甲基轉(zhuǎn)移酶的cDNA序列或基因組序列的用途，其特征在于，用于制備檢測原發(fā)性高血壓的試劑。
全文摘要
本發(fā)明公開了一種檢測原發(fā)性高血壓易感性的方法，它包括檢測個體的組胺N－甲基轉(zhuǎn)移酶基因HNMT的基因組序列，并與正常對照相比是否存在變異，存在變異就表明該個體患原發(fā)性高血壓的可能性大于正常人群。其中，所述的變異是HNMT的單核苷酸多態(tài)性(SNP)及其組成的單倍型(haplotype)。本發(fā)明還公開了相應(yīng)的檢測試劑盒。
文檔編號C12Q1/68GK1861803SQ20051002568
公開日2006年11月15日 申請日期2005年5月9日 優(yōu)先權(quán)日2005年5月9日
發(fā)明者張怡, 張奎星, 朱鼎良, 高平進(jìn), 王谷亮, 黃薇 申請人:上海市高血壓研究所, 上海人類基因組研究中心