專利名稱：：硫鏈絲菌素的生物合成基因簇的制作方法
技術(shù)領(lǐng)域：
：本發(fā)明屬于微生物基因資源和基因工程領(lǐng)域，具體涉及抗革蘭氏陽性菌抗生素硫鏈絲菌素(Thiostrepton)的生物合成基因簇的克隆、分析、功能研究及其應(yīng)用。
背景技術(shù)：
：硫鏈絲菌素(Thiostrepton)是硫肽類抗生素家族的一個(gè)著名成員，可由鏈霉菌S/r，o附戸s/a,"",ATCC31255、S/re聲w拜sATCC14921、S/r印towj;c^/awa//era/;yATCC12236所產(chǎn)生。人們首次發(fā)現(xiàn)硫鏈絲菌素是在1954年，但是由于它結(jié)構(gòu)十分復(fù)雜，直到1989年借助了X射線晶體衍射技術(shù)才最終闡明了其分子結(jié)構(gòu)[J^"幼Z饑1989,^,1649]。它含有兩個(gè)大環(huán)結(jié)構(gòu)(分別為26員環(huán)和27員環(huán))，4個(gè)噻唑環(huán)，l個(gè)噻唑啉，一個(gè)四取代的哌啶和一個(gè)喹萘啶酸結(jié)構(gòu)，整個(gè)分子共有17個(gè)手性中心。前體標(biāo)記實(shí)驗(yàn)表明其中噻唑、噻唑啉雜環(huán)來自于半胱氨酸，哌啶環(huán)由兩個(gè)絲氨酸經(jīng)酶催化后形成，喹萘啶酸由色氨酸衍生而來。硫鏈絲菌素具有良好的生物活性。它對(duì)革蘭氏陽性菌有廣譜而且強(qiáng)有力的抑制作用，特別是對(duì)多種耐藥性的條件致病菌具有極強(qiáng)的殺傷作用。硫鏈絲菌素抑制革蘭氏陽性菌的機(jī)制是因?yàn)樗芘c50S核糖體亞單元上的23SrRNA-Lll蛋白復(fù)合物結(jié)合，抑制延長(zhǎng)因子的GTPase活性,從而抑制蛋白質(zhì)的合成[/.她/.歷0/1998,276,391-401]。近年來還發(fā)現(xiàn)硫鏈絲菌素有抗瘧疾的活性，對(duì)人類瘧原蟲(P/oywo必ww々/">7^/附)的IC5o值達(dá)到了3.2nM[尸ra"w，1999，150,189]，Jonghee等人還在2002年報(bào)道了硫鏈絲菌肽的抗癌活性[PC7VW.^p/.WO2002066046，2002.]但是，由于硫鏈絲菌素水溶性不好，生物利用度較低，大大限制了它的醫(yī)藥用途，目前只作為畜用抗生素。人們希望能獲得一些它的結(jié)構(gòu)類似物，從中篩選出活性更好，更有利于實(shí)際使用的分子。由于硫鏈絲菌素極其復(fù)雜的分子結(jié)構(gòu)，即使是在有機(jī)合成化學(xué)髙度發(fā)達(dá)的今天，通過化學(xué)全合成來獲得產(chǎn)品也是一項(xiàng)艱巨的挑戰(zhàn)。直到2004年才首次報(bào)道了硫鏈絲菌素A的化學(xué)合成[J"gewC/zem五"g/.2003Jul28;42(29):3418-24]，整個(gè)合成過程操作繁瑣，產(chǎn)率低下。通過化學(xué)合成所獲得的結(jié)構(gòu)類似物即使在活性和水溶性方面的性質(zhì)得到改善，也可能因?yàn)楹铣傻膹?fù)雜性而使實(shí)際生產(chǎn)成本過高。我們以微生物來源的硫鏈絲菌素為目標(biāo)分子，從克隆生物合成基因簇出發(fā)，采用微生物學(xué)、分子生物學(xué)、生物化學(xué)及有機(jī)化學(xué)相結(jié)合的方法研究其生物合成，通過對(duì)其生物合成機(jī)制的研究揭示了其經(jīng)由核糖體合成的生物合成機(jī)制，并揭示了包括喹萘啶酸在內(nèi)的獨(dú)特化學(xué)結(jié)構(gòu)形成的酶學(xué)機(jī)理，在此基礎(chǔ)上運(yùn)用代謝工程的原理，合理修飾硫鏈絲菌素的生物合成途徑，探索水溶性好、生物利用度高、活性更好、并能通過微生物發(fā)酵大量生產(chǎn)的新型藥物。
發(fā)明內(nèi)容本發(fā)明涉及一種由鏈霉菌5^印tow;;c^/"wew衍ATCC31255產(chǎn)生的具有抗革蘭氏陽性菌活性的抗生素一一硫鏈絲菌素(Thiostrepton)的生物合成基因簇的克隆、測(cè)序、分析、功能研究及其應(yīng)用。本發(fā)明中整個(gè)基因簇共包含22個(gè)基因的核苷酸序列或互補(bǔ)序列(序列1)，其中一個(gè)基因(TsrH)負(fù)責(zé)編碼硫鏈絲菌素的前體肽，九個(gè)基因(加0、加M、加丄to《、加S、加Z、加/、加C、加及)負(fù)責(zé)對(duì)加/Z編碼的前體肽進(jìn)行后修飾，形成整個(gè)大環(huán)骨架；七個(gè)基因(加F、加丄"W、加B、加￡>、加f/、加尸)負(fù)責(zé)以色氨酸為前體，合成喹萘啶酸結(jié)構(gòu)單元；一個(gè)基因負(fù)責(zé)對(duì)形成的喹萘啶酸進(jìn)行活化，在其他酶的催化作用下連接到大環(huán)骨架上，并最終形成分子中的第二個(gè)大環(huán)。本發(fā)明還提供了一個(gè)編碼氨基轉(zhuǎn)移酶的核苷酸序列，由序列2中的氨基酸序列組成，命名為feM，其基因的核苷酸序列位于序列l(wèi)中第3774-2686堿基處。,本發(fā)明還提供了一個(gè)脂肪水解酶的核苷酸序列，由序列3中的氨基酸序列組成，命名為加W，其基因的核苷酸序列位于序列l(wèi)中第4626-3796堿基處。本發(fā)明還提供了一個(gè)編碼天門冬酰胺合成酶的核苷酸序列，由序列4中的氨基酸序列組成，命名為&rC，其基因的核苷酸序列位于序列l(wèi)中第6541-4685堿基處。本發(fā)明還提供了一個(gè)編碼酯環(huán)化酶的核苷酸序列，由序列5中的氨基酸序列組成，命名為"rZ)，其基因的核苷酸序列位于序列l(wèi)中第7088-6681堿基處。本發(fā)明還提供了一個(gè)編碼酰基-CoA脫水酶的核苷酸序列，由序列6中的氨基酸序列組成，命名為to^，其基因的核苷酸序列位于序列1中第8449-7163堿基處。本發(fā)明還提供了一個(gè)編碼甲基轉(zhuǎn)移酶的核苷酸序列，由序列7中的氨基酸序列組成，命名為tsrF，其基因的核苷酸序列位于序列l(wèi)中第9529-8594堿基處。本發(fā)明還提供了一個(gè)編碼未知蛋白的核苷酸序列，由序列8中的氨基酸序列組成，命名為"K，其基因的核苷酸序列位于序列l(wèi)中第10241-9600堿基處。本發(fā)明還提供了一個(gè)編碼硫鏈絲菌素前體肽的核苷酸序列，由序列9中的氨基酸序列組成，命名為"rH，其基因的核苷酸序列位于序列1中第10570-10746堿基處。本發(fā)明還提供了一個(gè)編碼水解酶的核苷酸序列，由序列10中的氨基酸序列組成，命名為&r/，其基因的核苷酸序列位于序列l(wèi)中第11461-12054堿基處。本發(fā)明還提供了一個(gè)編碼脫水酶的核苷酸序列，由序列11中的氨基酸序列組成，命名為ten/，其基因的核苷酸序列位于序列l(wèi)中第12047-14848堿基處。本發(fā)明還提供了一個(gè)編碼脫水酶的核苷酸序列，由序列12中的氨基酸序列組成，命名為/^《，其基因的核苷酸序列位于序列l(wèi)中第14845-15885堿基處。本發(fā)明還提供了一個(gè)編碼未知蛋白(可能負(fù)責(zé)催化D-A反應(yīng))的核苷酸序列，由序列13中的氨基酸序列組成，命名為"M，其基因的核苷酸序列位于序列1中第15872-16978堿基處。本發(fā)明還提供了一個(gè)編碼NADH依賴的氧化酶的核苷酸序列，由序列14中的氨基酸序列組成，命名為"rM，其基因的核苷酸序列位于序列1中第16975-18744堿基處。本發(fā)明還提供了一個(gè)編碼未知蛋白的核苷酸序列，由序列15中的氨基酸序列組成，命名為/^7V，其基因的核苷酸序列位于序列1中第18741-20579堿基處。本發(fā)明還提供了一個(gè)編碼氮雜五元環(huán)環(huán)化酶的核苷酸序列，由序列16中的氨基酸序列組成，命名為欲O，其基因的核苷酸序列位于序列1中第20576-22621堿基處。本發(fā)明還提供了一個(gè)編碼細(xì)胞色素P450氧化酶的核苷酸序列，由序列17中的氨基酸序列組成，命名為tof，其基因的核苷酸序列位于序列1中第22599-24023堿基處。本發(fā)明還提供了一個(gè)編碼?；?CoA合成酶的核苷酸序列，由序列18中的氨基酸序列組成，命名為加2，其基因的核苷酸序列位于序列l(wèi)中第23999-25312堿基處o本發(fā)明還提供了一個(gè)編碼細(xì)胞色素P450氧化酶的核苷酸序列，由序列19中的氨基酸序列組成，命名為其基因的核苷酸序列位于序列1中第25422-26612堿基處。本發(fā)明還提供了一個(gè)編碼脫水酶的核苷酸序列，由序列20中的氨基酸序列組成，命名為"nS，其基因的核苷酸序列位于序列1中第26603-29413堿基處。本發(fā)明還提供了一個(gè)編碼硫腺苷甲硫氨酸轉(zhuǎn)移酶的核苷酸序列，由序列21中的氨基酸序列組成，命名為toT，其基因的核苷酸序列位于序列1中第29430-31229堿基處。本發(fā)明還提供了一個(gè)編碼脫氫酶的核苷酸序列，由序列22中的氨基酸序列組成，命名為加仏其基因的核苷酸序列位于序列l(wèi)中第31226-32044堿基處。本發(fā)明還提供了一個(gè)未知蛋白的核苷酸序列，由序列23中的氨基酸序列組成，命名為加r，其基因的核苷酸序列位于序列1中第32391-33113堿基處。序列1的互補(bǔ)序列可根據(jù)DNA堿基互補(bǔ)原則隨時(shí)得到。序列1的核苷酸序列或部分核苷酸序列可以通過聚合酶鏈?zhǔn)椒磻?yīng)(PCR)或用合適的限制性內(nèi)切酶酶切相應(yīng)的DNA或使用其他合適的技術(shù)得到。本發(fā)明提供了得到至少包含部分序列1中DNA序列的重組DNA質(zhì)粒的途徑。本發(fā)明還提供了產(chǎn)生硫鏈絲菌素生物合成基因被中斷、被置換或同框缺失的微生物體的途徑，至少其中之一的基因包含有序列l(wèi)中的核苷酸序列。本發(fā)明所提供的核苷酸序列或部分核苷酸序列，可利用聚合酶鏈?zhǔn)椒磻?yīng)(PCR)的方法或包含本發(fā)明序列的DNA作為探針以Southern雜交等方法從其他生物體中得到與硫鏈絲菌素生物合成基因相似的基因。包含本發(fā)明所提供的核苷酸序列或至少部分核苷酸序列的克隆DNA可用于從鏈霉菌Sr印ro附y(tǒng)c^/m^e油7ATCC31255基因組文庫中定位更多的文庫質(zhì)粒。這些文庫質(zhì)粒至少包含本發(fā)明中的部分序列，也包含有5^印tom;;cM/aww"招ATCC31255基因組中鄰近區(qū)域以前未克隆的DNA序列。包含本發(fā)明所提供的核苷酸序列或至少部分核苷酸序列可以被修飾或突變。這些途徑包括插入、置換或缺失，位點(diǎn)特異性突變，特別是可以通過定點(diǎn)突變編碼前體肽的基因序來實(shí)現(xiàn)thiostrepton大環(huán)骨架的改造，獲得一系列的thiostrepton類似物。還可以通過不同序列的重新連接，序列的不同部分或與其他來源的同源序列進(jìn)行定向進(jìn)化(DNAshuffling),或通過紫外線或化學(xué)試劑誘變等。包含本發(fā)明所提供的核苷酸序列或至少部分核苷酸序列的克隆基因可以通過合適的表達(dá)體系在外源宿主中表達(dá)以得到相應(yīng)的酶或其他更高的生物活性或產(chǎn)量。這些外源宿主包括鏈霉菌、假單孢菌、大腸桿菌、芽孢桿菌、酵母、植物和動(dòng)物等。本發(fā)明所提供的氨基酸序列可以用來分離所需要的蛋白并可用于抗體的制備。包含本發(fā)明所提供的氨基酸序列或至少部分序列的多肽可能在去除或替代某些氨基酸之后仍有生物活性甚至有新的生物學(xué)活性，或者提高了產(chǎn)量或優(yōu)化了蛋白動(dòng)力學(xué)特征或其他致力于得到的性質(zhì)。包含本發(fā)明所提供的核苷酸序列或至少部分核苷酸序列的基因或基因簇可以在異源宿主中表達(dá)并通過DNA芯片技術(shù)了解它們?cè)谒拗鞔x鏈中的功能。包含本發(fā)明所提供的核苷酸序列或至少部分核苷酸序列的基因或基因簇可以通過遺傳重組來構(gòu)建重組質(zhì)粒以獲得新型生物合成途徑，也可以通過插入、置換、缺失或失活進(jìn)而獲得新型生物合成途徑。包含本發(fā)明所提供的核苷酸序列或至少部分核苷酸序列的克隆基因或DNA片段可以通過缺失硫鏈絲菌素生物合成中的一個(gè)或幾個(gè)步驟而得到新的硫鏈絲菌素結(jié)構(gòu)類似物或前體。包含DNA片段或基因可以用來提高硫鏈絲菌素或其衍生物的產(chǎn)量，本發(fā)明提供了在基因工程微生物中提高產(chǎn)量的途徑。本發(fā)明所提供的硫鏈絲菌素骨架的后修飾基因提供了通過遺傳修飾得到類似物的途徑，所包含的氧化還原反應(yīng)也可有其他應(yīng)用?？傊景l(fā)明所提供的包含硫鏈絲菌素生物合成相關(guān)的所有基因和蛋白信息可以幫助人們理解硫鏈絲菌素類天然產(chǎn)物的生物合成機(jī)制，為進(jìn)一步遺傳改造提供了材料和知識(shí)。本發(fā)明所提供的基因及其蛋白質(zhì)也可以用來尋找和發(fā)現(xiàn)可用于醫(yī)藥、工業(yè)或農(nóng)業(yè)的化合物或基因、蛋白。圖l:硫鏈絲菌素的化學(xué)結(jié)構(gòu)圖2:硫鏈絲菌素生物合成基因簇的限制性內(nèi)切酶譜。6個(gè)交疊的粘粒代表了抗生鏈霉菌基因組60kb的DNA區(qū)域，B代表限制性內(nèi)切酶5amHI;圖3:硫鏈絲菌素生物合成基因簇的基因組成。圖4:提出的硫鏈絲菌素的生物合成途徑。圖5:喹萘啶酸單元的合成途徑圖6:基因中斷與基因缺失突變菌株發(fā)酵產(chǎn)物的高效液相色譜(HPLC)分析。從上至下依次為標(biāo)準(zhǔn)品、野生型、環(huán)化酶基因中斷的突變體(AwO)、脫水酶基因同框缺失的突變體(Jtov/)、脫水酶基因同框缺失的突變體(Jto《)、脫水酶基因同框缺失的突變體(zfoW)的發(fā)酵產(chǎn)物。符號(hào)說明圖lThiostrepton:硫鏈絲菌素。圖2peptideskeleton:大環(huán)骨架；quinaldicacidmoiety:喹萘卩定酸單元；tailoring:后修飾；propeptide:前體肽；unknown:未知功能。圖4Leadingpeptide:信號(hào)肽；Structuralpeptide:結(jié)構(gòu)肽；NAD(P)H:煙酰胺腺嘌呤二核苷酸(磷酸)；LP:信號(hào)肽具體實(shí)施例方式以下結(jié)合圖1-圖6對(duì)本發(fā)明進(jìn)一步詳細(xì)說明。1.克隆硫鏈絲菌素的生物合成基因片斷盡管在硫鏈絲菌素的化學(xué)合成和作用機(jī)制方面的研究很多，但有關(guān)其生物合成起源方面的認(rèn)識(shí)卻非常少。之前，人們依據(jù)非核糖體聚肽合成酶的保守序列設(shè)計(jì)簡(jiǎn)并引物，希望用這種簡(jiǎn)并引物能擴(kuò)增獲得與硫鏈絲菌素生物合成相關(guān)的基因，總是不能成功。本專利依據(jù)硫鏈絲菌素分子中富含噻唑環(huán)這一特點(diǎn)，以噻唑環(huán)合成酶的保守序列設(shè)計(jì)簡(jiǎn)并引物，以5^ptomycM/awe"招的總DNA為模板，成功克隆得到了負(fù)責(zé)催化噻唑環(huán)合成的環(huán)化酶基因。在S/rQ^omycM/"wrew份體內(nèi)失活該基因，得到的突變體無法再產(chǎn)生硫鏈絲菌素，證實(shí)了克隆到的基因與硫鏈絲菌素生物合成相關(guān)。2.硫鏈絲菌素生物合成基因簇的克隆，序列分析及功能分析-將上述雜環(huán)合成酶基因片段用地高辛標(biāo)記為探針，從S/r印r(W^CM/aww"衍的基因組文庫約2000個(gè)克隆中篩選，分離得到的粘粒涵蓋了染色體約60kb的區(qū)域。(圖2)。DNA順序分析了39，556bp的染色體區(qū)域，GC含量74.19%。生物信息學(xué)分析包含了29個(gè)開放讀碼框(圖3)。開放讀碼閱讀框詳細(xì)的分析結(jié)果列于表l。表1Thiostr印ton的生物合成基因簇中各基因及編碼蛋白的功能分析￥i~~氨基酸數(shù)相似蛋白同一性/推測(cè)功能目相似性％193SSDG一05344(YP—002196146)94/98208SGR—3卯3(YP—001825415)41/53177SSCG—02932(YP一002192918)68/84142SSDG一05347(YP—002196149)80/87362Acel一0035(YP一871797)47/63276SSEG—03719(YP—002206063)50/65618STIAU—7558(ZP—01461966)56/69135GeobDRAFT—1690(ZP_01388898)32/54428STH2357(YP—076186)35/51311Sare—0495(YP—001535415)41/57213Mext—1123(YP一00腿598)28/4658----------—陽197Bml—20485(XP—001895551)29/39933BC5084(NP—834752)19/38346BC5083(NP—834751)25/47368BC5082(NP—834750)21/41589BAT3535(ZP03054696)24/40尿嘧啶磷酸核糖基轉(zhuǎn)移酶未知蛋白未知蛋白脫氨酶氨基轉(zhuǎn)移酶脂肪水解酶天門冬酰胺合成酶酯環(huán)化酶?；?CoA脫氫酶甲基轉(zhuǎn)移酶未知蛋白硫鏈絲菌素前體肽未知蛋白NADH依賴的氧化酶幼§612BL02837(YP—078248)25/39未知蛋白681GWCH70DRAFT—037932/43雜環(huán)合成酶(ZP—02912149)474SACE—0651(YP—001102921)29/44細(xì)胞色素P450氧化酶424TM1040—2789(YP—614783)27/42?；?CoA合成酶加及396RoseRS—0765(YP一OO1275131)31/48細(xì)胞色素P450氧化酶ten936BC5084(NP—834752)20/34脫水酶加r599Gura一3608(YP—001232335)27/39硫腺苷甲硫氨酸轉(zhuǎn)移酶272Krad—3162(YP—001362890)43/58脫氣,247orf—L7(CAN89623)39/60未知蛋白1447SsviAJ)1010001707338/51WD-40重復(fù)蛋白(ZP—03195299)99SCO4036(NP—628218)82/91未知蛋白29336SclaA—01010001372579/81RNA聚合酶o因子(ZP—03183088)3.Thiostrepton的生物合成基因簇邊界的確定根據(jù)基因編碼蛋白的功能分析以及和結(jié)構(gòu)類似物Siomycin生物合成基因簇對(duì)比，硫鏈絲菌素的生物合成基因簇被確定為從基因ton4到to^(圖3)，包含22個(gè)開放讀碼框。本發(fā)明中整個(gè)基因簇共包含22個(gè)基因的核苷酸序列或互補(bǔ)序列(序列i),其中一個(gè)基因(to"/0負(fù)責(zé)編碼硫鏈絲菌素的前體肽，十個(gè)基因(加CK加M、"n/、加《、n加Z、加/、加C、欲及、加戶)負(fù)責(zé)X寸加i7編碼的前體肽進(jìn)行后修飾，形成整個(gè)大環(huán)骨架；七個(gè)基因(加f、to"、/^￡、&M、加￡>、欲仏加，P)負(fù)責(zé)以色氨酸為前體，合成喹萘啶酸結(jié)構(gòu)單元；一個(gè)基因欲g負(fù)責(zé)對(duì)形成的喹萘啶酸進(jìn)行活化，在其他酶的催化作用下連接到大環(huán)骨架上，并最終形成分子中的第二個(gè)大環(huán)。其中l(wèi)個(gè)基因("rC0中斷及基因同框缺失加v/,^^,和"W完全抑制了硫鏈絲菌素的產(chǎn)生(圖6)，進(jìn)一步證明了克隆到基因簇為硫鏈絲菌素的生物合成基因簇。4.喹萘啶酸結(jié)構(gòu)單元的生物合成途徑喹萘啶酸結(jié)構(gòu)單元的生物合成如圖5所示。to^編碼的重復(fù)使用的聚酮合成酶甲基轉(zhuǎn)移酶催化色氨酸的2位連上一個(gè)甲基，"M編碼的氨基轉(zhuǎn)移酶使得色氨酸的氨基轉(zhuǎn)化為羰基，^"五編碼的酰基-CoA脫氫酶脫去一份子氫，^^編碼的脂肪水解酶催化使氮碳雙鍵斷裂，形成氨基和羰基，to"編碼的酯環(huán)化酶催化氨基和另一羰基縮合，形成喹啉酸，^"[/編碼的脫氫酶將羰基還原為羥基，to^編碼的P450氧化酶氧化喹啉8，9位上的雙鍵為環(huán)氧，最后^"g編碼的?；?CoA合成酶負(fù)責(zé)活化喹萘啶酸的羧基，將其轉(zhuǎn)變成CoA-硫酯鍵。5.硫鏈絲菌素骨架合成硫鏈絲菌素生物合成基因簇中有一個(gè)基因to^編碼硫鏈絲菌素的前體肽，經(jīng)核糖體將該前體肽合成之后，由z^O編碼的環(huán)化酶負(fù)責(zé)催化前體肽中的巰基親核進(jìn)攻臨位的羰基，然后脫去一份子水，形成噻唑啉，W*M負(fù)責(zé)編碼的脫氫酶將噻唑啉轉(zhuǎn)變?yōu)猷邕?，然后?^丄加《、toW編碼的脫水酶負(fù)責(zé)催化使得絲氨酸殘基的羥基脫水，編碼蛋白的催化作用下，兩個(gè)脫水絲氨酸殘基發(fā)生D-A反應(yīng)，形成第一個(gè)大環(huán)；然后在"W編碼的水解酶作用下，信號(hào)肽被切除，經(jīng)活化的喹萘啶酸單元連接到肽鏈的氮端；最后在^rC的脂肪水解酶作用下，喹萘啶酸的羧基與大環(huán)骨架上的蘇氨酸殘基的羥基發(fā)生成酯反應(yīng)，形成第二個(gè)大環(huán)。(或"rP)負(fù)責(zé)編碼的P450氧化酶負(fù)責(zé)羥化異亮氨酸殘基上的仲丁基，最終形成完整的硫鏈絲菌素分子。整個(gè)過程如圖4所示。以下進(jìn)一步提供實(shí)施實(shí)例，這些實(shí)施實(shí)例有助于理解本發(fā)明，僅用作說明而不限制本發(fā)明的應(yīng)用范圍。實(shí)施例l硫鏈絲菌素產(chǎn)生菌鏈霉菌5^印tom少ce;y/awren衍ATCC31255總DNA的提取將100nLlxl08S./aww油7孢子懸液接種到3mLTSB液體培養(yǎng)基中，3(TC，230rpm培養(yǎng)約24hr后達(dá)到對(duì)數(shù)生長(zhǎng)期后期，取2mL接種到50mLTSB(含10mM氯化鎂，0.1%甘氨酸)，3(TC，250rpm培養(yǎng)約23hr后達(dá)到穩(wěn)定生長(zhǎng)期前期，呈乳黃色渾濁，將菌液4。C，3500rpm，離心15min收集菌絲，用裂解液洗滌，收集淡乳黃色菌絲0.5mL。向1mL菌絲中加入10mL裂解液(含溶菌酶5mg/mL)共四管，渦旋至均一，37。C水浴15mim。加入0.1mL蛋白酶K(10mg/mL，用裂解液新鮮配制)，lmL10%SDS，混勻后迅速放入70。C水浴15mim，呈澄清。置冰上冷卻，加入2.5mL5MKAc，冰上冷卻15min。加入10mL飽和酚，混勻，lOmL氯仿，混勻，12000rpm,4。C離心20min。用破口的槍頭將水相吸出置于新的離心管，加等量的CHClr異戊醇(24:1)抽提，12000rpm，4'C離心10min。用破口的槍頭將水相吸出置于新的離心管，加2倍的無水乙醇，混勻，有大團(tuán)的DNA出現(xiàn)。將其鉤出置于新的離心管，加5mL70。/。乙醇洗滌，將液體傾出，用槍吸凈，加5mLTE溶解，加RNaseA使終濃度為50pg/mL，37'C溫育0.5小時(shí)。依次用等體積的飽和酚抽提兩次，CHCb-異戊醇抽提兩次，向水相中加入0.1體積的3MNaAc，2體積的無水乙醇，輕輕的混合充分，有絮狀DNA出現(xiàn)。將四管DNA合并到兩管(每管中有1mL70%乙醇用于洗滌)，將液體吸出，再加1mL無水乙醇洗滌，吸出乙醇，超凈臺(tái)中吹干，溶于適當(dāng)體積的TE(pH8.0)中。實(shí)施例2硫鏈絲菌素產(chǎn)生菌鏈霉菌S&卬/om;;c^/awe"招ATCC31255遺傳轉(zhuǎn)移系統(tǒng)的建培養(yǎng)含有適當(dāng)質(zhì)粒的ET12567至0D6(K)0.3-0.4，30mLLB培養(yǎng)液中的細(xì)菌細(xì)胞離心收集，用等體積的LB洗兩次，重懸于2mLLB中，作為大腸桿菌供體細(xì)胞。取適量?jī)龃嬗谝?0。C的5"/r印towj^;y/aww"招ATCC31255的20%甘油孢子懸液500pL，用等體積的TES緩沖液(50mMTESNa,pH8.0)洗兩次，重懸于等體積的TES緩沖液，5(TC熱激10min使孢子萌發(fā)。再加等體積的TSB培養(yǎng)基，37。C溫育2—5hr。離心重懸于0.5—lmLLB中作為鏈霉菌受體細(xì)胞。將不同濃度的受體細(xì)胞100pL與等體積的供體細(xì)胞混合直接涂布在含有l(wèi)OmMMgCl2的平板上，3(TC溫浴20hr后，采用無菌水輕輕洗滌平板表面以洗去大部分大腸桿菌，在每一平板的表面覆蓋lmL含萘啶酮酸(終濃度為50ng爾L)和相應(yīng)抗生素的無菌水。3(TC培養(yǎng)5天以上挑取接合子。由于S/reptomyc^/awe"衍ATCC31255對(duì)阿泊拉霉素和紅霉素都敏感，最后確定遺傳轉(zhuǎn)移所用抗生素的濃度阿泊拉霉素25pg/mL，紅霉素100昭/mL。MS(甘露醇2.0g,黃豆粉2.0g,瓊脂粉2.0g用自來水稀釋至100mL，并調(diào)節(jié)pH=7)培養(yǎng)基最好，接合轉(zhuǎn)移效率最高。在MS培養(yǎng)基上，無論是在鏈霉菌中可以自主復(fù)制的質(zhì)粒pKC1139、自殺型質(zhì)粒pOJ260都可以得到相應(yīng)的接合子。實(shí)施例3硫鏈絲菌素產(chǎn)生菌鏈霉菌5^印to附y(tǒng)c^/flt^"招ATCC31255基因組文庫的構(gòu)建首先用lml注射器吹吸S/rgpto附;;ce;y/awe油'z'ATCC31255總DNA數(shù)次，使得總DNA隨機(jī)斷裂，使用蔗糖梯度離心對(duì)DNA進(jìn)行梯度離心，收集略大于40kb的DNA片段，脫磷，待用。pJTU2554先用EcoRV從兩個(gè)cos序列中間切開，與制備好的的40kb的DNA片段連接過夜。于冰上融化存于一80。C的PromegaPackageneexract，立即加入10ul連接產(chǎn)物，輕彈混勻，于室溫(約22。C)放置3hr。加入445ulPhagebuffer,倒轉(zhuǎn)混合；加入25ul氯仿終止反應(yīng)，離心使氯仿沉于底部，4"C保存。將凍存于-80。C的菌株E.coliLE392涂布在LB培養(yǎng)基上復(fù)蘇。調(diào)取單菌落接種于3MLLB培養(yǎng)基中(0.2%111&08610和mMMgS04)，37°C，220rpm振蕩培養(yǎng)過夜，轉(zhuǎn)接1%到50MLLB培養(yǎng)基中(0.2%maltose10和mMMgS04)，37°C，220rpm振蕩培養(yǎng)至OD600=0.6—0.8。取5ul包裝液，加入95ulphagebuffer,稀釋，再加入100ul￡.Co//LE392(OD600=0.67)混勻，37°C，30min，涂于LB(含100ug/mlApramycin)平板上。37。C倒置培養(yǎng)過夜，測(cè)定噬菌斑形成單位(pfo)以估算文庫的效價(jià)。取lOOul包裝液加入3.9mlphagebuffer混勻，再加入4mlE.ColiLE392(OD600=0.72)菌液，室溫，30min，加入4mlLB，37°C，75min;2500rpm，10min，去上清，剩余1一3ml涂于LB(含100ug/mlApramycin)平板上。37'C倒置培養(yǎng)過夜。用LB刮下平板上的菌株，加入甘油(終濃度18%)和Apramycin(終濃度50ug/ml)，分裝，于一8(TC保存。隨機(jī)從平板中挑取10個(gè)克隆，接種于LB培養(yǎng)基中培養(yǎng)，按大腸桿菌的質(zhì)粒的堿法小量制備的方法抽提重組黏粒。用^"附M鑒定，于0.8%的瓊脂糖凝膠上電泳，根據(jù)限制酶切的電泳分析圖譜，我們判斷出黏粒被酶切產(chǎn)生的片斷大小，加和這些片斷，從而推算出整個(gè)黏粒的大小，實(shí)驗(yàn)表明每個(gè)黏粒的插入片斷約為40Kb左右。對(duì)于鏈霉菌而言，其染色體DNA的大小約為8Mb，如果插入片斷為20kb的文庫效價(jià)是2000—5000cfo，就足以代表它的整個(gè)基因組。根據(jù)以上實(shí)驗(yàn)，我們建立的文庫效價(jià)超過為10000pfU/iigDNA，插入片斷約為40kb左右，這表明我們建立的文庫具有良好的質(zhì)量，能夠滿足文庫篩選的需要。實(shí)施例4硫鏈絲菌素產(chǎn)生菌鏈霉菌S加;7tow;;cfty/awm7衍ATCC31255的發(fā)酵、產(chǎn)物分離純化與鑒定取100ulS加/tow;;ce;y/做w"衍ATCC31255孢子接入S./做re油7—級(jí)發(fā)酵培養(yǎng)基(TSB15g,可溶性淀粉15g;蔗糖50g，蒸餾水定容到1L)28°C、250轉(zhuǎn)每分鐘培養(yǎng)48小時(shí)，取10ml種子培養(yǎng)液接入S./m^ew冶'二級(jí)發(fā)酵培養(yǎng)基(TSB15g，CaS0415g，酵母提取物llg,葡萄糖50g，2x微量元素lml，蒸餾水定容到1L)28°C、250轉(zhuǎn)每分鐘培養(yǎng)72小時(shí).發(fā)酵液以3800rpm的轉(zhuǎn)速，離心15分鐘；上清用等量的乙酸乙酯萃取兩次，萃取后的乙酸乙酯用適量無水硫酸鎂3(TC蒸干，所得固體用3ml甲醇溶解。發(fā)酵產(chǎn)物分析取20ial進(jìn)樣，流動(dòng)相為乙腈和水(含0.1XTFA)。HPLC洗脫條件為(紫外254nm處檢測(cè)，柱子為NUCLEOSIL100-5C18)。<table>tableseeoriginaldocumentpage16</column></row><table>實(shí)施例5PCR克隆硫鏈絲菌素的生物合成基因PCR體系包含DMSO(8%,v/v)，MgCl2(25mM)，dNTP(2.5mM)，簡(jiǎn)并性引物(40mM)，TaqDNA聚合酶(2.5u)及適量模板5^e^o附y(tǒng)ces/flwe"招ATCC31255總DNA。首先95。C，3min,1輪；然后94。C，lmin,68°C，lmin，72°C，2min，5輪;94°C，lmin，65。C,lmin，72°C，2min，30輪；最后72。C，10min,1輪。PCR結(jié)束后，1%瓊脂糖電泳檢査結(jié)果。低熔點(diǎn)膠回收預(yù)期大小的DNA片段，與pMD19-Tvector連接，轉(zhuǎn)化大腸桿菌DH5a感受態(tài)細(xì)胞，涂布在含有氨芐青霉素，IPTG(異丙基硫代—p—D-半乳糖苷)和X-gal(5—溴—4—氯_3—吲哚一p—D-半乳糖苷)的LB平板上進(jìn)行藍(lán)白斑篩選。挑取白色菌落過夜培養(yǎng)，抽提質(zhì)粒，五coRl酶切鑒定是否含有預(yù)期大小的DNA插入片段。插入有預(yù)期大小DNA片段的質(zhì)粒測(cè)序。實(shí)施例6核酸分子雜交1)DIGDNA標(biāo)記將待標(biāo)記的DNA用無菌水稀釋至總體積15pL，沸水浴中加熱變性10分鐘，立即置于冰鹽浴中冷卻。接著加入2^L引物混合物，2pLdNTP混合物，lpL酶，混合均勻后，37。C水浴約16小時(shí)。加入O.一L0.8MEDTA(pH8.0)以終止反應(yīng)，加入2.5pL4MLiCl混合均勻，再加入75pL預(yù)冷的無水乙醇沉淀標(biāo)記后的DNA，置于一80。C沉降40分鐘。4'C，12000rpm離心20分鐘收集DNA，用預(yù)冷的70。/Q乙醇洗滌DNA沉淀，真空干燥后重新溶于50pLTE((pH8.0)中。2)DIGDNA探針標(biāo)記后的質(zhì)量檢測(cè)稀釋標(biāo)記的DNA探針，至以下六個(gè)梯度，1、10"、l(T2、l(T3、l(T4、l(T5。稀釋標(biāo)記的對(duì)照DNA至濃度分別為以下濃度l昭/mL，100ng/mL，10ng/mL，lng/mL，0.1ng/mL，0.01ng/mL。分別取lpiL上述梯度的DNA樣品點(diǎn)在雜交用的尼龍膜上，根據(jù)7)所述步驟進(jìn)行顯色反應(yīng)，對(duì)比標(biāo)記的DNA探針和DIG標(biāo)記的對(duì)照DNA的顯色強(qiáng)度以決定標(biāo)記的DNA探針濃度。3)菌落雜交(文庫篩選)的膜轉(zhuǎn)移將保存于一8(TC的基因文庫稍融，取50pL,用450pLLB稀釋得到l(T1的稀釋倍數(shù)，倍比稀釋得到l(T2，l(T3，IO"4，l(T5，l(T6。300pL涂平板(15cmxl5cm，平板為L(zhǎng)B/50昭/mL卡那霉素)。選取合適的比例，使每塊平板約1200—1500個(gè)克隆。照選定的比例均勻涂布四塊平板，37。C培養(yǎng)過夜。根據(jù)平板的大小剪取尼龍膜，小心地覆蓋于平板表面不要產(chǎn)生氣泡，做好位置標(biāo)記，1分鐘后取下尼龍膜置于干燥濾紙上，干燥10分鐘直至菌落結(jié)合在尼龍膜上。原始的平板置于培養(yǎng)箱中4一5hr，使克隆重新生長(zhǎng)作為原平板。將尼龍膜置于變性液(0.25MNaOH，1.5MNaCl)飽和的濾紙上15分鐘(不要浸過膜)，轉(zhuǎn)移至中和液(1.0MTris.HCl，1.5MNaCl，pH7.5)飽和的濾紙上5分鐘。轉(zhuǎn)移至2xSSC(20xSSC儲(chǔ)備液(L"):NaCl，175.3g，擰檬酸鈉，88.2g，pH=7.0)飽和的濾紙上自然風(fēng)干。取下尼龍膜置于烘箱中，12(TC固定45分鐘。常溫下于3xSSC/0.1%SDS溶液中振蕩洗滌3小時(shí)，以除去細(xì)胞碎片。4)Southera雜交的膜轉(zhuǎn)移:DNA樣品在適當(dāng)濃度的瓊脂糖凝膠上電泳至適當(dāng)距離，做好標(biāo)記。浸泡于400mL0.25MHCl中脫嘌呤20分鐘，使溴酚藍(lán)變黃，用去離子水洗數(shù)次。室溫下浸入堿性緩沖液(0.5MNaOH，1MNaCl)15分鐘并輕輕振蕩。換液一次繼續(xù)浸泡凝膠20分鐘，并輕輕振蕩，去離子水洗三次。取一張每邊都比凝膠大lmm的尼龍膜，用去離子水完全浸濕，做好標(biāo)記。采用向上毛細(xì)管轉(zhuǎn)移方法，用10xSSC轉(zhuǎn)移緩沖液轉(zhuǎn)移8—24hr。用2xSSC略微洗膜，12(TC烘烤30分鐘。5)預(yù)雜交和雜交預(yù)熱雜交液(20mL/100cm2)至雜交溫度68'C，放入雜交尼龍膜，輕輕振蕩并保溫30分鐘。將DIG標(biāo)記的DNA探針在沸水浴中變性5分鐘，立即置于冰鹽浴中冷卻。冷卻后，將DNA探針與合適體積的DIG雜交液(2.5mL/100cm"混合均勾。去除預(yù)雜交液并立即把DNA探針/DIG雜交液加入，輕輕振蕩保持雜交溫度64'C或68""C約16小時(shí)。6)雜交后嚴(yán)緊洗脫室溫下用2xSSC/0.1%SDS漂洗兩次，每次5分鐘。68'C，用0.1xSSC/0.1%SDS振蕩漂洗兩次，每次15分鐘。7)顯色反應(yīng)和檢測(cè):嚴(yán)緊洗脫后的尼龍膜在洗滌緩沖液(0.1M馬來酸，0.15MNaCl，pH=7.5,0.3%(v/v)Tween20)中平衡1-5分鐘，接著在封閉緩沖液(封閉試劑以10%的濃度溶于O.IM馬來酸，0.15MNaCl，pH=7.5)中封閉30分鐘，然后在抗體中浸泡30分鐘。用洗滌緩沖液漂洗尼龍膜兩次后，用檢測(cè)緩沖液(0.1MTris-HCl，0.1MNaCl,pI^9.5)中平衡2-5分鐘，最后將尼龍膜置于10mL新配制的顯色溶液[NBT(nitrobluetetrazoliumchloride)溶于70%DMF，濃度為70mg/mL，BCIP(5-bromo-4-chloro-3-indolyl-phosphate)溶于水，濃度為50mg/mL。用時(shí)10mL顯色溶液中加45^LNBT，35pLBCIP]中，置于黑暗中顯色。顯色合適后用去離子水漂洗以終止反應(yīng)。實(shí)施例7基因中斷突變菌株的獲得將獲得的轉(zhuǎn)化子接種到TSB液體培養(yǎng)基(Am25pg/ml)中，3(TC振蕩約28hr。取出200nl涂布在ISP-2(Am50嗎/ml)平板，3(TC培養(yǎng)6-8天，收孢子，保存于37'C整合生長(zhǎng)的單菌落，接種至液體培養(yǎng)基ISP-2(Am25^ig/ml)，37'C，振蕩2—3天。取出涂布在ISP-2平板(Am50pg/ml)，37。C整合2-3天，收孢子，保存于-80。C。基因中斷或基因置換所用載體為pOJ260或pKC1139，基因置換用紅霉素抗性基因替代目標(biāo)基因中間的DNA片段。構(gòu)建好的質(zhì)粒通過實(shí)施例2所述屬間接合轉(zhuǎn)移的方式導(dǎo)入5^//omyc^ATCC31255中得到雙交換突變體，所得突變體通過Southern雜交在基因型上加以證明。以下根據(jù)本
發(fā)明內(nèi)容提供的基因和蛋白序列氨基酸/核苷酸序列表:SEQUENCELISTING<iio中國科學(xué)院上海有機(jī)化學(xué)研究所<120>硫鏈絲菌素的生物合成基因簇<130>說明書、權(quán)利要求書<160>1<170>Patentlnversion3,3<210>1<211>39556<212>DNA<213>鏈霉菌/m/"""7ATCC31255<400>1ATGTAGCCGTTCTCGTTGAGCCGCTCGTCGCCTGCCAGCTCGCGCTCCATGACCGCGACGGTGACATCGTCCGCGCCGCGCCGGATCAGTGTCGCCAGCATCGGGTCCAGGACGTACACCGTCGCGTAGGTCGACGCCTCCAGCGTCTCCTCGGCGGTCGGGAGCAGCCGCACCATGCCGGGGACCACGAGCGGGCGGGGGTACGAGAGCTCGATGTCGACCCGCTCGGTGCGCACGTCCAGCTCGTCGGCGAGCCGGCGGAAGGTCGGGAGCTTGTGGGCGACCAGCGGGTGATCGACGGGCCGGTGACGGGCGCGGCACAGCGGCCGGCGGCGGGGGAAGGTGGGGACACAGGGACCCCCGAAGAAGACGCCCGGAACGCCCGGAATGGACCCTCCGCCGCTCCCGAGGGAGTACCCAACCGAGGCCGTCACGACGGTCACCGGCGTA60CCCTCGGGGGCGGCGAGGAGCACGACCGCG120TCCTTGATCGCCGCGACGAGCGTGCCGCCG180TGGCGGCCGGACAGGTCCTCCGGCATCCGC240TCGTTGCGGATCATCCCGAGGAAGCCGACC300TCGAGCATGCCGAGACCGGCGCGCAGGATC360TTCACGCCGGCCGTCTTCGTGACGGGGGTC420CTGGTCGCCTCGTAGGCGAGCAGGGTGACC480GAGTCCGTGCGCTTGTCGCGCAGAGTGGTG540ACGTGGAGACGCATGCCTCAACAGTAGCCC600GGTGACCCGCGGGGTGGTGCCGGCCCGGGG660AGCCGTGGGGTGGTGTGGCCGATGCCCGAC720CCTGGTGAGGCGGCCGGAGCGGAGGCTTTC780CCGGCTTCGGGCCCTTCCCGGGCCTCCGGG840TCGAACCCCGGGCCCAGCCCCGAGCTCGGGCCGGACACGCCGGACCCGGACGCGAACCCGGAGCGCCGGCGGCGGCGCGCGCAGTTCCTGGAACGCGTCCAGCCCCGGCGCGCCCGGGCGTCGTTCCGGTGGTGAAGGGGCCGATCGAGGCGCGAGGCCGTGCGCCCCTGCGTGCGCCGGTCAGACTGATGCACGACGCAAGAGCCGAAGATGCCGATTGGGCGGGGCATCAGGAGCGCGAGAGTCAGGTGTACTTCGCCGCACTGCTCGATACAGAGCTCGACGACGTGGAAACGCTGGCCGCCGACGACGGAGAGACCGTGATCGTCTTCGTCCGGGTCGACGGCGAGGACGACCCACCCCGCTCCTCGTACGGGGAGATCCTCACCTCACTCGACGAACTGACCGACCTGGACGGCAAGGACGACGAGGCGGCGGACAGCGCCGCGGCGCTCATCCTGGCCGACCTCGGCATGGACGACGCCCTCGGGGAGATCGCGGACGCCATCGAGTGCCCCGCGCAGCACACGGCGAGGACACGGAGCCGATCCGGGAGCCGGTCCCGGAACCGGGCCCCGCGATGGACCCCGTAGGGGATCCGTACGCGCTCGCCGAGGCCGACCTGGCCGCCGTGCTCGCCCCGGACGGCTCCGTCCTCGCCGACCCGACCGCCCACGCCGAGGTGCTCGCGTGGCGGCTGACCGGCTGCACGCTCGTGGTGGCCCTCGTCCAGGCGCGGGTCGAACGGGTCGCGACCGGCTCGCTCTGGGACCTGGTCCGGATCCACGGCGTACTGGAGGCGGAGTGCTCCTGACACCCGGCCGACGACCCCGCCGACCTCCTTTGGGCTAAGCTCTCTCTCGGTAGCGTAGCGAGTGTGGGGAAACTCACCGAGGGTTCGCCGGGCTCGGGGCCGGGCCCGGCCCCGAG900GAGGCGGACCCGGAGGCGGACCCGGAGAGC960CGCGACCTGCACGAGGCCAAGGCGCTGCGC1020GCCCGGATGCGGCAGGCGATGCGGATGCGT1080TAGTGCACCCGGGTGACCTGCGGGAAGTCC1140GGTCGCGCGCCCGCCCGATCGCGGTGTCGT1200ACCTCTCCTGACGGCGAGGTTTCTGTCACG1260CCGCCGGTCATCACACCTCTGGACAGTGGG1320CGCGCACCGAAGACGGGTGGGAAGCGAGCG1380CGGACCTCGCCGAACTGGCCCGTGAGGCCG1440TCATCGAGCAGGAGGACGCCTGGTTCGGCG1500GCGTGTTCGTCTCGAACGGCGCCGCCGCCG1560CGGAGATCCTCGGCGACGAACCGGACGACG1620CGGAGGACGGCGAGCCGGAGGCCGCGGACG1680CCGTCGCCCCGCCCGGGCCGCTCGGCGAGA1740AGCAGGCGCTGCTCGGTCTGGAGACCGGCG1,GCGCCGCGGACATCCTGGAGGCCGTCCGCT1860CGGCGCCGCCCCTCGCCCCGAACCCGTACG1920GGTCCATGAACCCGCCGCGGGATCCGGACC1980CGTACGGGACCCCTGGCGTGCCCCGATGCG2040GCTGGCCGGTGACGTACCGGTCGGCGCGGT2100CACCGGCCACAACGAACGCGAGGCGACCGG2160CCTGCGCCGGGCCGCGGCGGCGACGGGCGA2220GACCCTGGAGCCCTGCGTGATGTGCGCGGG2280GGTCTTCGGCGCCTTCGACGAGAAGGCCGG2340CGACCGCCGCCTCAACCACCGCCCCGAAGT2400GGCCCAGCTGACCGCCTTCTTCCGCACCCG2460GCGCGGGGATACGGATTTCAGTCCTGGGCG2520GTCCGAGCGGCCGAAGGAGCTCGCCTCGAA2580AAATCCCTCCGCTACCGCTCTCGAACCGCC2640CCTGATCTGCGACAGCAGGTCAGGGGCGGTCTCCCCCGGCTCCGTCGGCCCCTCCACTGACTTGGCCAGACGGGCGGTCAGCCGGAGGACGACGCGGACGCCCTCGCCCGCGACGGCGCGGACGAAGGCGGCCGAGCGGGAGCCCAGCGGCACCTCCCAGCCCTGCCCGCGCAGTTCCGCGTCCACGCGCGCGTACACCTCGGACCGCCGGACGGTGCCGACGGCGAAGGGCAGCAGCCGGGCGAGCGCGAAGCCGACGCGCAGGCCGGCGACCACCACGTTCGGCAGGCCGCGGTAGACGGCGAACTCCCGGTACGCCTCGTCCAGGACGAAGGCGAGCAGGGCGTCGGCGCCGAGCGCGACGAGCGCGCGGGTGTCGGGGGTGACGGCGTCCCGCAGCGGGATCTCGACGGCGGTGGCGTCGAACGACGGCCGGGCGTAGACGACCTCGTGCTGGAGGATCACGGCGGAGCCGGGGCCGGTGGCCGCGATCGCCGCCGTCAGCTCCCGGCGCAGCGCGTCCCGGAGCGCCTCGTCCAGCAGCGAAAGCCGGTGCTCCGGGCCGGACGCGCACCTCCTCGCGGGTCAGGGACGCGTGGGGAGGTCCGCGGTCGCGCGGTCGCGCCCCTCATAGACGCGTTCGGCCAGGTGGAAGCCGCGGATCAGGGCCAGCGGATCGGCGTCGCCGTGCTCGCCCCCGTTGATCCGTTCGAGGCAGGTCGCGCCGTGCCAGATCAGCGGGTCCGCGTTCATCACCGAGCCGACGTCCACCGGCATCTCCGCCGTGTGCCAGGAGCCGAGCACGGGCGCGTGGCGCTGGGCGTACCGGACGGCCACCATGGGGAGCCCCGGGTGGTCCGCGCGGGCCCGCTCGTCGTGGTCCGTCACGAGCGCGCGTGCTCTGCGTGTGCCCGCTCAGTCTCCCGGCGC2700TGCCGCCGGCCCCACCGGCTCCTCGCGCTC2760GGCGTCGTTGGCCGCCGGGTCGCCGACGGT2820GACGAGGATGCCCTCGCGGGCCGCCGCCGC2880CAGCCAGCAGAAGTTGGCGGCCGACGGCGC2940GGTGAGCCGGTCCCGTTCGGCGACGACGCG3000GGCGAGAACGGTCCGCGCGGCCGCCTCGGC3060GGTGCGCAGGACGTCCGCCACCTCGCGGCG3120CAGCCCGTACGCCTTCGAGAAGGACCGGAG3180GTCCGGCCCGTCGAGGGTGTCCTTGTCCCC3240GACGACCACGTGCTCCGGGACCGCGCGGAG3300GGTGCCCGTGGGGTTGTGGGGGTTGCACAC3360CGCCAGCATGCGCGGCAGATCGTGCCGGTG3420CCCGGCGGCGGCCACGGCCAGCGGGTACGC3480GCCCCGCCGCGGGGCCGCCCACTGGATGAC3540CGTGACCACGTCGTGCTCGGGTACGGAGTG3600GGCGAACGGGTCGGGGTAGCGGTGCGCCCC3660GCCGGTCACGGGGACGGCCGGCATCTCGTT3720TCCCGGTTCGGTGACTCCCGTCATGTCGTC3780AACGGTGAGGGCGTCGTCGAGGAACGCCGT3840GTCGTGGAACAGGCCGTGCAGCGCTCCCGG3900GATCTTCTCCACGCCGGCCCGGGTCTCGTC3960CAGCCAGAGCGTCGGCAGGAAGGCCAGGCT4020GACGACGGACTCCAGGGTGTCCCGGACGAA4080GTAGCGGGCCGCCTCGGCGGGGTCGCGGGA4140GGGGATCTCCGCGAAGGCGAGGAGCGAGGT4200GGCGAGCACGAGCGCGGCGAGGTCCTCGGG4260CCCGCCGACCGAGTGCCCGATCATCACCAC4320CTCGACGACCGTGGCGAGGTCGGCGACCTG4380GCCGCCCGAGCGGCCGCTGCCGGCGTGGTC4440CGGCGCGTACACGGCGGCGCCGCGTGGTCGGCGTACCCGTCCAGACGTGCAGATGGATCGGGTCATCGTTCCCTCTTCCCGGCGTCACTCCGCCAGCTCCACTCGAATCCCACCCGCAGGACGCCTCGGCCGCGCTCTTCTGAGTGCGCCGAGCCGGCCCACGGCTCCTTCTTCCGGGTCGCAGGCTCTTCTCCCGGCCGCCACCAGGGCGTGGTCGAGGGGTCCTTGCGTTCCAGGAGCGCATCCGCCGGTCCTCCTCGCGTACCGGTCGGCCCGGTACACGCCGGGCCGCCGGCGGCGGGACGGCGTTCTCGTCGTGGCGGACAGCGCGACCGTGGAACCATGTCGGCGAGGCCCTGGGGTCGGACAGCAGCTTCGCGCCAGTTCGGCGGCGTACGCGCGGAGCCGGCGGCGGAGAAGGCAGCGCCCCGACGGCGAGCAGCAGAGCGGCACGTCCGCGCCCGGCCGATCGTGGTGTCGCGCGCTCCGTGATCCCGCCGTCCCGTCGAAGATCGCGTGCCCGAGGTCAGCCGGGCGCGGAGAGGACACCGTCCGGGGTCTCAGCAGGAGTTCCTGGCCGGCCGGGTGACGTCCTCGCCCCGTGCCGGGTCAGCGCGTCGGCCAGCCGGCCGTAGCGGCC4500GCACGAACACCGCGAGATAGCGGGGCGCCGTCTCCGGCGG4560GGTGCCGGACGCCGGCGAACCGCCGTGCCGTCGTCTCCTC4620TTCGTCGCCGGTCCACCGAGCCGCGCGCGCCGTCGCGCCC4680ACCCGGTAGTCCCGCATCCAGTCGTTGATCTTCAGCAGGG4740AACCGCAGTTCCTCCGGGGACGCCTTCCGCACCGCGGCGT權(quán)OTCGTCCATCAGCGCGCGGACCGGTGCCCCGGTGTCGTCCA4860TTGAGCAGCTGGTCGTAGGCGGGATCGGCGATCGTCGGGT4920AGTACGGAGTCCGGGAGCTCGGGGGCCATGGCCGCGCGCA4980TCGAAGGTCTTCATCGACCACGGCACGTTGAAGACGTACT5040AACGGCACCCGCACCTCCAGGCCCACGGCCATGCTCATCC5100ATCGGCAGGAAGCGGGTGAGGAAGAGGTGGCCGACCTTCC5160GCCTCCCCGGGCAGGACGGGGACGGCGGCGACCGCCTCGG5220GCGTCCAGGGAGAGCCGCTCGGGCAGGGCCGGGTCGAGGA5280GCCACGCCCGCCATGTACATCCAGGGGAAGGTCTCCGCGC5340AACCAGGGGTAGCCGCCGAAGAGTTCGTCGGCGCCCTCGC5400CGCTGCCGGATCGCCTTGAACAGCAGGTAGAGGGACGTGT5460GGCAGGTCGCGGGCCCGGAGCACGGCCGCCCGCGCCGCCG5520TCGAGGGTCAGGTCCTCGTGGCGGGCGCCGATGTGGCGGG5580CTGTCGTCGCCACCGCGCGGGCCGGTGACGAAGCCCGGAC5640CCGACCGAGAAACAGGTGAGCGGGTCCTCGCCGCGGTCCC5700GCCGCCACGGAGCTGGAGTCCAGACCGCCCGAGAGCAGCG5760ACGGTCTGGCCGGCGACGGACGACCCCAGCAGGTCACGGA5820AGATCGTCCTCGTGCGGGCGCGCGGGGAGTTCCCAGTACG5880CGCGCGACGCGGACGACGTGCCCGGGGCGCACTTGGCGCA5940CCGGGGGTGTTCACCCAGGTCATCAGCTCGCGCAGACCGT6000GCCTCGGGGTGGGCCAGGATCGCCTTCGGCTCGGAGCCGA6060GGGTAGTAGTAGAGCGGCTTGACGCCCAACCGGTCCCGGA6120CGGGCGTCCCAGAGCGCGAACGCGAACATGCCGGTGAGGT6180CACTCCAGGCAGGCGCGCAGGACGACCTCGGTGTCGCTCC6240GGGTGCGGAAGGTGTGGCCGGCGGAGACGATCTCCCCGCTGTAGGAGAGGGCGGCCGTGAGCGCGCCGCCGGCGATGTCGATGACGGCGACGTACCAGGTGCCCCCGGCGTCGGGGCCGCCCGCGGAACGCTCGGCGCCCAGGTCCCGCCTCCGTCCTCCTCCATTTCCTGCCGCTTTCCCCCTGCCGTTGCCCTGCCGTTGCCCTGCCGACGTGGGCGCGCCCGCCGGGTCAGTCCGGCAGGTCCATCTCGTCCCAGTGCTGGGCGAACAGGCCGACCGAGAAGCGCCGTCCGGAGGCCCCGCTGAAGGTCATCCGCGCGACGGCCTTGAGGCTGAGGTCCTCCACCGTCGAGGCCCAGCCGCGCTCGACGGCCGGGTTGGGGTGCCGGCGCGGGTCTCCTTCGTTGTACGCCGCCAGGCGGTCCACGGGGATCGCCTTCCGGTCCGGTGGGGCGGGGGAGGCCGGGGGTGTCAGGAGGTGGCCCCGGCCTCCACCTCGGCCTCGGCCTGGGGCGGATGCACGGGAGCCGTCTCCTCGCCGTCGGCGTTGCTGTACGGGTGCATCGTCCACACCGAGCCGGGCCGCAGGGCCGGGGTGCCCACGGCGTTGTTGGTGCCCACGTTCTTGGTGGTGAGCGCGTTCCACAAGCCGTTGCCCACGCGCGAGGTCTCGATCAGCTCCTCGCAGGCCTCCCAGCCGCGGCTCCGGACGTCCCGTACGAGATAGACCGCGGCGAAGGTGGGCATCGCGTCCAGACTCCCGACGGGGTAGGAGGTGAGGAGTTCGATGTTCTCGCTGCGGGTGGCCCGGACCCGGACGCCGGGACTGTCGGCGGGGAGGTGTTCGCGGTACTCGGCCATCGAGGTCAAGGTCTTGTCGCCGGTGACCAGGTAACCGCGCCGGGCGCGCAGCTCGTCGAAGCCGTAGA6300CCTGCCCGTTCTCCTCGGCGAGCATGGGCT6360GCCGCCGGTGGCCCAGGGCCACCGGGCCGC6420GACAGGCCATCGTCCGTGTCATCGCCTCCA6480CGAAGGCCACCCAGCCCGCTATGCCGCACA6540TGCCGCTTTCCTGCCGCTTTCCTGCCGCTG6600TTGCCCTGCCGTTCCGACGACTCGCGAGGT6660AGCGCGCCGAGCTGCCGGTGCAGCCCGAGC6720AGACCGTCCTCGATGAGGAAGATGTCGATG6780GGGATGCCGAGGATCGGGCCGACCTGCCGG6840TCCCCCTCCACGACGAGGTCGTCGAGGGTG6900CGGCGGATCGCGGCCCGCATGCCCTCGATG6960TACTCGGGGTGCAGACAGTGGTCCAGGTGG7020AAATGCTCCATGAGCTGCTGTACGGATGCT7080GGGTGTGCGGGGCAGGACGGGTTACGGGGG7140CGGCAGGGCGTACGGCCGTGTCGCTGGCCT7200CGGGCGCCGGCTCGGCGAGCCGGTCCAGCC7260CGAGCGTCGTCGCCGCGATGAGGTCGCACG7320CGGCCCGTACGTCGCGGTGGGCCCGCTCGT7380CGATGATCCGGATCACCCGGTCGACGATCT7440TCAACACGGCACGGGCCATGCCCTGTTGGA7500TCACCTCGCCGGCGGTCCGCTCGATCAGGG7560CGGCGATCTCCGCGCGGATGTACGGCCGGT7620CGCAGGTCTCGAACGCGCCGACGGCCACGC7680CCCAGCCCCACACGGTCTGGAGCAGCGAAC7740GGACGTTCTCCTCCGGCACCATGACGTCGC7800GCAGTCCCGAGGTGTTCCAGGTGCCTTCGA7860TACGGAAGACCAGGACGTGCGGGCCGTCGG7920TGTGGGTCATGACGGCCGAGCCGGTGCCGA7980CTTCCACCTTGCGGGCCTTGGCGCCGGTGG8040TGCGCACGAGCGCGTGTCCCGGCTCGGCCGTGGCGCCCATGAGGATCACGCGGCCCTCGA8100CGATCTCCGTCAGCGTCTTCCGGGCGCGCTCGTCGCCCGTGGTGCGCCAGGCGCGGGCCA8160TCTGGCCGGCCATGGAGAGGTGCATGTTGACGGCGAGGGCGGTGGAGGCGCAGCCGGCGG8220CCAGCCGTTCCTGGGCCACGGCCATGTCGGCGAGGGTGCCGCCCAGCCCGCCGAGTTCGC8280GCGGCACGGTCAGCGCCAGATAGCCGGCGGCGCGGAGTTCGTCGATGTTGCGGAACGGGA8340ACGCGGCCGTCCGGTCGTGCTCGGCGGCCCGCTCGCGGAAGACCGCGGCCATGTCCTCCG8400CCAGCTCGACGTACCGCTGCCGGGCCGGCCGGGTGATCAGGAGGTGCATCAGGACTCCAG8460CTGTCGGAGGAGGGGTGTCACGAGGAGAAGGGCCTTCGCCGGGCGCCGGTCACACCGGTC8520GCTCCGGAGGCGCCGGTCACACGGACGCCGGTCACAACGAGGCGCAAACGGAGGCGGGTT8580CGGAGGCCGCACGTCAGGCGACGGTGGGTGAAGCGGAGGCGGATTCGGAGACGGAATCGG8640AGACGGAATCGGGGACGGAATCGGGGACGGAATCGGGGACGGAATCGATGACGGCCGGGC8700CGGTGACCAGCTCGGCGGCGACGAGCCACGTGCGGGCGGCCCCGGGCGTCCCGCGCGGCG8760GGGTCCGCGGCACCATCCGCCCGGCCGCCTCGTCCAGGTCGCAGGGTTCGCGCACCTCGG8820CCCGCCAGCCGCGGCCGGCGAGCCACGCCTCGGGCTCGGCCACCCCGGAACGCCAGGGGC8880GCCCCGCGTCCCGCATCTGCCGCAGCCAGTCCCGCATCCAAGGGGCCCGGTACAGATCGG8940TGTTGACCTGCTCCAGGCAGAGCACGCTGCCGGCGGCGGAGAGCTCGGTGACCCGGTCGA9000CGACCGCGTCCACCTGCGCCTCGGTCAGGTAATAGAGGACTCCTTCGACCAGCCATACGG9060TCGGCAGACCGGGGCGGAAGCCCGCCTCGGCGAGCGCGGCGGGCCAGTCGCCGGTCAGGT9120CGGCGGCGACCGGGACGCGCCGACAGCGGTCGGCGACCAGGCCGGAGCGGGACAGGAAGG9180CGCTCTTCTCCTCCACTCCCTCGGGCAGGTCCACCTCGTAGAGGGTGACGCCCGGGGGCC9240AGGGGAGCCGGAAGGCCCGGCCGTCCATGCCGGCGGCGAGCACGACGACCTGCCGGGCCG9300GGCCGCCGGTCACGGCGCGGATCAGACGGTCGTCGAAGAACGCGGTCCGCACGGCGAGTA9360CGTCGAAGCGGGAACTCGGTTCGGTGCGGAGGCGGTCGTAGAGGTCCGGGTCGGTGCGCC9420GGACGAACTCCACCGCGAGCGGGTCCTCGAACAGCCGGCCGTCGGGACGGAGCGACTCGG9480CTGCCCGGGCGCAGGCGATCCAGTGGGCGGTCTCGATCAAACTCGACATGGGCTCTTCTC9540GCAGAGAGCCGGGCGCCGGCGCGCCCGGGTGAGGGTCACCACGGATCCGCGGCCGCGCGT9600CAGCGGCCTTCGGTCGCCTCGCCGACGGCGGGCGCGGCAGACGCGGGAGGTGCGGCGGGT9660GCGGCAGGCGCGGCAGGCGCGGTGGGCGTACCCGTCGCCGCCGGCTCGTCGTCGATCCCG9720AGCGCGCGGGACATCCCGCGGCGGAGCGACCAGTGCAGGATGTCCGTCTGGTTGTGCCGG9780AACTGGGCCCAGTAGGCGTCGCCCGCCTTGTTCCTGGTCCGCAGGACGACACCGATGTAC9840GCCTTGCCGTAGGAGCACTTCGGGGCAGCGATGACGACGCAGGCTGGGACGGGATCGCCAAGAGCCGAGTTGGCTTCGCCTGGCGCATCACATTTCCGTACTTGACTTGCTTTCCCGTGGGTGAACGGCAGTCGACACCCACCGTCACGATCCTGAGGTACAGTCGACCAGCAGGTCCGGGGGCCCGACCCCGGCCTCCGGGTACGTCTTCCTCCCATGGGCCCGGACGGGCGGCCCTTAGTGCACGGTCCGCCCTGTCACCCGCCGCCGGTGGTCGTGGCCCGCCACCGCTGAACCGCTCGGCGACGATCTCGCTCGCTGCTGTTCCCGCCTGGACCCGGGTCCATGGTCCTCCGCCGCCGTTCGGAATACGCTCAGGGCTGCACCGAGTGTGATCTACCTATGGTCGACCGGCCCACGTGAGCAATGCTGGAGATCAGTGATCGCGTCACACCCGGCGGGTCCGCGATGGCCCGACCCCGGAGTCATCGACGAGCACCCGGCATGTCCCCGCGGAAACGCTTCCCGTCTTCTGTTTACTCCTGGGCACCGGAAATACGCGGCGATAGAGGCTTTCCTATGGCCGGAATGGCTGGCGGGGAAGGACATCCGCCTCGAAGCTTGCTGCGCGATCCACAGGCAGCCGGGCGCTCCGCGTGGTGAACAGCTGCCTCGGGACGCCTATGCAGAGTCACAGCCAGATCAGCCGGTGCGGCGCGACGCCCTGGAGCGACTACATGCGCCTCCTGCCGGAGGACTGCTAACCGGGAGGAGTACCCCTCCGGCCGTGCTCACTATGCTATTGCACCGGGCCCCATACGGGTGCGCCGGGCAACAGAAGGAAGACGCGTCTTCCAGGCCCGCGTCCGCTCTGGGTGCCCGTCATGTCGCTTCACCAGACGGTGTTCGAACCTCGTACGATCGTGTTGGTCGACCAGCCAGCTCCCTGGGTGTGCTCTCGCTATATGCATCATAGGTGTGATCGATTCTTTTATCCGGAGCGACGGAGTTCGACGCGACATCGGTGTCGGACGAGACGCACCACCTGCATTCCTCCCCGGTGGGACCCGGGACCTGCCCCTCGCTCGGACCGATGACGGCGGACGGCTTGGCCGAGAGAGGCCCCGAATAGCTGATATCCATTACGCGGCACGGAACCGGAGGCCATCCGCCGTGGCCTGGCTACTCGTATGGCCTCCCGCAGCTTGTCGACCGAGGCGGCTTGACGCCAGAACGCCCGCGTCGATCCTCACTGTCACGTTTCGAGATTCCAGAATTCGCGACTCAGCCGGTGCCCTTGCCGGAAGCCGCGCAGACTTGAGGGACTCACTGCTCGACGGTCTGCACCTGCCCGCCGGACGACCTGCCCGGTTGCAGTCGGGCTGTCGGCCGTGGACAACTCGCACGTCCACGGAGTGTCGACGCTCCGCACGTGCGACCGGTGCCGGAAGCGGCGAAATGCGAACAGCCTCCGGGCCGCTCCTGGTGTCATCAGCCCGC26CATGTCGCCG9900CTCCCCGACC9960GAAGGGGCGC10020GTCGGGCGTG10080CCGATGGTCC10140GAAACGCGCG10200CCCCATGGGT10260ACGGCCGAAG10320GGAGAGTAAA10380AGCGCTTCAA10440GCTACAGCAG10500CCGAAAGGTT10560GGGTCTGGAC10620TGAGGACCTG10680CAGCTGCAGC10740CTGACCGTTG10800TTGCAGTCGA10860AGCAGGTCCG10920GGACCCGCCG10980TAGTTCCGGC11040GTTCAAACGG11100GGTCCCGCAT11160CGGGTATTGA11220CGTAATTCTG11280ATGGTGGACC11340CCCACCCCAT11400GGAACCTCCC11460GGCCGGTTTT11520GCCGGACACC11580GGCCCAGCGC11640CATTTCGCCGAACTGCACGGCCCCACCTGGCTGTCCGACGGCTGCCTGCGGCTGCCCGGCGTCCAGCTCGTCAACGGCTCACTGCTCCAGGCCGCCGGTGCCGACGTCGCGGTGATCGCCCCGCGCGTCTTCGCCGCCACCGTCGAGGACGCTTTCCACGAGCGGCGGCGCGCGGCCGAAGAGGCCGGCACCGGAACCGAGCCCGTCGCCGATCACGTACGCCGGATACGGCGTCGTCCGCCAGTTGTCCTCGCGCGAGGCCGCCCGCGTCTGGCAGGACCTCGCCCCCCGGGTCGCCGACGACCGGGACGAGCGGCGGACCGTTCTCGCCGTCCCGGCCGCCGACCTCGACGTCCTGAACGGGGCGGCCGGGACCGCGGTGGCCGGGCCCCTCGCCGAGCGCCGCGACGACCTCGCGGCGAACGCGGAACGGAAGGTGCTCGCCTCCGCCCAGCTGAGCGCGGACGGTCTGCTGCGCAAGGGCACCGCCCCCGGCAAGCGGGAGCGGATCCGCAGCACCCTCAAGCCTTCCCCCTTCGGGCCGAAGCGCGCGACGGACGCCACGCCCGTCGAGGCGGCCGAGCGGCCGGTTTCCGTCTGGGAGCGTTCCCTCGCCGGCCGGCCCGCCTTGGTCGTCGGCTCCTCGAAGGAGGGTGTCGTCGAGCCGGCCGCCAAGGAACGGTTCGTCCGCCTGGCCCTCGCGGCGGACGCCGCCGTACCCCTGCCCGGCACGGACCCTGCCGCCGCCGGCGTCCTCGCCCGCGACCTGGGCGTCGGCGACGCCCTCCTCCCCCCGGACTGCGACGCCGGCGCCGAGTCCGCGTTCGAGACCGCCGGCCTCAAGGAGCACGTGGCCCGGGTCGCCGAACTCGCCCGCACCCTCGTCTACGAGGACGCGGTGAGAATCTGCTCACCGCGACCTTCGCCGAG11700GTCGCGGACCTGGAGCACCTCCCGGTACCC11760AACGAGCGGAACGCGATCGGTCCGGCGGCC11820GGGGCCGGCCATGTCGTACCGATGGACGCG11880TTCGCCGACCGGGTCACGGCCGGGCGCACC11940AGGGCCGCGGCCGAACCGGCGGTACCGCCC12000CGGAAAGCGGCCACCCATGGATAGTCCGGA12060CGTCTGTTCCCAACCCGCCGACATCATCGC12120CTGCGACGAGCTCAGGGACGTACGGCGTAC12180GGAACTCACCGAGGTGGTCCCCCTCCTCAC12240GCTGCGCCGCCGGCTGCACCGCGCCGCGGA12300CCTCGCGTACGGAACCTCGG(^GGACGGGCC12360CCTGGCGCCCGGGGCCGCCGCGCTGCGGGC12420CCGCCTGGAGGGCGCGTACGAGGAAGGGCT12480CGCCCTCGACCCCGCCCTGCGTGCCGGAGC12540CATGGACCGCTACGCCGCCGCCGTCGCCGA12600GACCGAGTCCACCGTCCTCCATCTGCTGAC12660GCAGTTGGTGCACACCCGGCCGGTCCTCCT12720CATCGCTCCCGTCACCGCTCCCGGCACGGA12780CCGGCTCCCCCGCCAGCTCGTGAGCTGGGT12840CCGGCATGTCGCCGTCCTGCGCCGCGCACC12900CTTCCTGCTGCGGGGCCGGGACGGCACCCA12960CGTCCCGCCCGGCGTGGTGGTCACGGCGGT13020CGAGCGGGAGCTGCGGGAACGGTGCGCCGC13080TGCCGCCCTCGACGCGCTGATCGGCCAGGG13140CCAGCACCCGCACCCCCTCGCCCGCCTGGC13200CCTGCGCGCGGCCGTCAGCGGGCTGGCCGG13260CGAACGGCGCGAGGAACTCGTCTCCCGTAT13320CACCGGCGTCGCCACCCCGCCGATCGACGT13380CGTGGCCGTCCCGCGCGCCGAGGCGCCCGG13440CCGGTGGGGGCGGCATCTGCCCGCGCTGGCCGACGCCGCGCACGTCCGGGCGATCGTCGCGCCGCACCGGCTCCTTCCCCTGTTCGCGGCCACCGAGCGGCTCACCGACGTGTCGGCGCCCGCGGTGTTCGCGCTCGCCGATCCCGACGGGCGGGCCGCGCGGCTCGCCGACGACCTGCCCTGGCACGTCCAGCACCACGCCGCCCCCGAGATCGGCTTCGGCCGCGCGATCTCCCGCTTCGGTTTCGGCGACCTGGTCACGGCCGAGATCACGGACCTCGCGGCCGTGCTCGGCATCAAGCACCTCCGCTACCCCTGCGCGACCCCGGAGGAGGACTGCTGGGTCGTGTACGACGCGGAGGACGGTCCCGTCCTGCGGCTGGTCATGCTTTTCTACCAGTTCCTCAACTTCTTCAGCTCCCGGGTCGACCAGCGCCGCGAAGCCGGTGCTGGTGCCGGTGCCGGTGGTGGTGCCGGCTCGGTCCGCTCCGGCAGCCTCGTCCTGGCCCGCCCCGGCCTCGCCGGCGCGACCGGTGTCGCGTCGCTGGGCCTCCCCGACCAGGTCTTCTACCCGATGTCCCGGGAGGAGCAGCGGACCTGCGCCGAACGCAAGCCCACCTTCCTGGACTTGCGGGCGCTGCGCCGGTGCCAGGGCGGCCTCGAGGACGAAGGGGCGGCGGGCGGCCTCCGGACCACCGAGACCGAGGAGGAAGGGTGAGCCATGTCTTCCACCACGGCGACACCGATCCGGACGACCTGCGCCGGGCGGGACGCGTGGAAGGCCCGCACGTCCGGCTCCGGATGCGGGTGGAGACCGAGCGGCGCGTCCTCGACTACCTCCCGGCACGGCTGCGGGACGCGCTGCGCCGGGATGTCGCCGTGCGCACCCCGGAGCCGCCCCCCGTGGCACCGGCTGCTGCCGCTCTTCGA13500CGACGTGGTCAGGACCCACTTCGGGCCCGG13560GCTGTCCTCGGCCAAGGTGCAGGCCGTCCT13620GCTGCCGACCCGGTTGCGCACCCTTCAGGA13680CGGCGCGGACACGGTGACGGTCGACCCGGA13740GTCCTGGATCCCGCGCTGGTCCCGCGCGAG13800CGGCGGTCTGCTCGTCGTGAACGGCGGATC13860CCTGCCCGCCTACGCCGCCGCCGGGGACGC13920CGCCCGGGACGACGACCCGTCCGCGCCCCT13980CGCCAACGTGCACTCCCCGCTGCTCGGCCG14040GGAGTGGGGCGGGAACACCGGCGTCTCCCT14100CACGGGCCGGCTGCTGCCGCGCCTGGGCAA14160GAACTTCCTGCTCAACGAACTCGCCCCGCG14220GGGCAGCCTCGCCAACCTCGCCTGGTGGGA14280CGGTGCCGACGGTGGTGCCGGTGCCGACGG14340GGGTACGGACTCCGTACGCCACTACCCGCG14400CCGCACCTGGAAGGTCCCCGACGCCCGGCT14460GGCCTTCGCGGAGGTACGGCGCTGGCAGGA14520CCGCGGCTTCACCCTCCCCGACCCGCTCGT14580GGCGAGGACCCTCGCCCGGTTCCCCACCTC14640CACCAGCGTCACCAGCGTGACCGCCTGGCA14700GACCTTCCAGGAGTGCCTGCCCGCCGTGGG14760GGAGCGGTTCACCGAGGAATTCATCGTCGA14820ACCGCCCCGGCACACCCCTGGCTGTCCTGT14謹(jǐn)CTGATCGCGGAGTGCCTGCTCCCCCTGGTC14940CGCCACTTCTATCTGCGGCACTGGGAACGC15000CCGGCGGAGCACCACGACGCGGTACGCGCG15060GGGCGGCGCCCCGGCCCCGTCGACACCGAT15120CTCAGCGAACTGGAGCACGGCGACGCCTCC15180AACTCCCTTCGCTGGATCGACTATCAGCCC15240GAGCTGGCCAAGTACGGCGGGGCTCGGTGCTGACCGGCCAMCGCGCTCCAGCTGCTGCTGCCGTCTTCCTCCGTACCTACTCGCCTCCTGGGACCGGCAGGCCTCGACGCCGGCGACCCGTCCAGGCCCTGGAGCGGCTACCGACCCCGCGGCACCGCCACCACCAACAACCGGATGAATCGCGCGCGGCCGAGGACCAGCCCAGACGGCACACGAGAGCGTCAGCGTCCCGTGGTCGGCGACGAGTCGGTGGAACTCGCGGGCGGCCGTTCCTGCGGCGAAATCACGGATCCTGGCGCCCAGCCGTACAACCACGAACGAACTTCTGGACCGAGACCACCGGGCCCTGCGGCTCGCCGGCCCCATCTGCCGCCGTCCAGGCCACGTTCGAGGGCTACACGCCCAACGGTACGAGAAGAGCAGATGAGCGAGCCGGACGGGTCCGGCTGCGCGACTACGCCTCTACGCCACGCCGCGCACTACCGCAGGCCCGACGTGGCCACCGGGCGATCGCCGACACCTGGCCTCGCGGCTGGCGCGCTCGCCGACCGGTACACGCCCTCGACGCGGCGGCGCTGAGCCCCGGGGACCGCCGAGGCGCCCGAAGGCGTGGCGATCGCGGAGGACGTCTTCCACGCC15300GGTCCTCCGGCTGGTCGACAACGACCGCGCCCGGATGGGC15360GCTCGCCTCGCGCGCGCTGGGCTTCGGTCCCGACGAACGC15420CCAGGAACGCTGGTCCGGATACGTCACGGACCCCCGCGTC15480GTACCAGCTGCAGCGGGAGCAGTACGCGGCCCTGCTCGAC15540CGTCGGGAAGGGGCTCGGCGCGTACTGGGAACGGACCCTC15600GCGCCCGCTCGTCGGGACGGGCCGGGTGTGGCCGGCGGAG15660GGTGGTCGCGCTCGCGGCCCTCGTCAGCCAGTTCCTGCAC15720CGTCCGCCCGCAAGGCGAGTGCTTCGTCGCCTACCTGGCC15780GCGCGCCGCCCGGTCCGGCACCGCCCCGACGGCCGACCCC15840GGATGAGCGTCATGTCCACCAGTGAACAGAAGGATCTCAC15900TGCAGGAGGACCTCCTTCTCGACGTGGCCGCGCCCCTGCT15960GGGAGACGGACTCGTGGTTCTACCTCCGCGAGAACCACGG16020TGCGCTTCGCGAGCCGCAGCCCGTCGGTCGAACGCCGCCT16080ACGTCGGGCCGACGATCGACGCCGGCGACGTCTTCACGTA16140ACGACTGGCTGGGCGGCACCGCCGGGCTCGGGCTCGCCGA16200CCCCGCTGGCGCTGGACACCCTGCGCGCCACCCGCGGCAA16260TGGCCTTCGACTTCCTCGTGTGCACCGGCGTCMGCTCGC16320TCGCCAAGTTCGGCTACAAGGCCGGCTATCTCTCCTACCT16380TGCTGCTGATCCGGGACCCCGAGGGCACCCGGGCCAAGCA16440ACCGGGAGTTGCTGCGGCCACGCCTTCGCACGCTCGTCGA16500GCGAGCTGACCGACGTGCCCGAGCTGGCCCGCGAGTGGCT16560TCCCCGCCCTCCAGAAGGGCTTCGACGAGGGCCGCTTCTA16620AGGCCGAGACGGCCAAGCTCACGCCGTCGCCCGACGGCCT16680AGTGGCTGTCCGACCTGCCGGAGCCCCCGGTGGCCGGCAT16740ACACGTACTACCAGGGCATGATCCGCGAGGACCGCCGCTT16800AGGCGTACACCAACTGGCATCTGTACCGGCTGGGCTTCCT16860TCTTCTACCTGATCGCCCGCGCCTTCGAGGAGGAGTACGA16920TCCGGTCGGTACGGCCGGAAGCAGAGGTCGCCGGATGACG16980CACCGCTCCGGTCGAGGCCGCCGCCCCCGCCGAGGCCGCC17040GGAACCGGGCCCTTCGACGCCGCCGGAGTGGACGGCTTCGTTCGACGAAGTCCACCCGGACCAGTGGCAGATCGACTGGCCGCTGGTACGTCGACCTGCCCCGGCGCGATCTGCCCGCGCGGCGGGCCCGCCGCGGCGGCCGCGTTCGCCGCGGCCGGGCCTGCTCCAGGGCGCCTACGGACTGACCGGCGTGCGGTGGTAGCAGCCCCGCCGAGCCCACCCCCGTCCACCGCAACCCGCGTGCCCTCGGGAGGCGTGACGTACCCCGCCGAGTGCTACGGGCACGGAGGGCACGGAGGAAGAGGACGGTCTCGACGCCGGTACGCCACGCACTGGCCGTGCTGTCCGCCGGCCGGCCGCGAGGGCGGTGTCCGGATCGTCGTCACCCAGCCGCTGTGGAGACTTCTCCTACCGGCTCGGCGCCATGGACACCGGTGCCGGCCGCCCGGTCCTGGGAACTGCGCTGCCGGGTGACGTTCGGCGGACCTGCTCGGGCTCGATCCGGAACACGAGGCCGTCCCTGGGAGACCCGGTGGGACCGGCGGCATCCGCGGCCGAGACCCCGCTGGCTGGGCGAGTCCGTCCCGCCCGGCCCCGCCAGCCGCGTGCCTGGCCGCCGGCCGCCCCGGCCGGCCCGTCGCCCGACCTGGCCGGGCAGGCGGACGTGGAGCTGCCCCGGCGTCGCGGACACCTGGTCCCGGTCGGCGCTCGGCGAGCAGTGCCGAGGTCCTGGCGGACTGGCTGACCACGGTGCTGGCCCGGAGAGCAAGCCGGCCGCGCCGACACCGACACCGGAGAGCGGGACCGTCACCGGCGAGGCCGCCCCGACGCACGCCCGCTGTGCCCGGCGTGCCTCCGGGCGCCTATCTGCCACACCCGGCTCGCCGCCGGGGACTTCGGGCCCGAGGTCCAGCGCAGCAGTCGGGCAGGCGCCGCTGACCGCCGTCATGATCGGCGCCGGCCGGCCCGATCGGCTACCGCACCCAGCATCTCCTCGCGGGCCGCGTGCGCGGCGCGCGACGGCAGATCGGCGCACCCGGGTCCTCGACGACGCGTTCGGGCTGCGGGCCCGGGGCCTGAGTGGGCGGTTACCGCCGGGGCCTCTATCTCGAGAACAGTCGCGGGCGACGGGAGTGGAATATGACAGCGCCGACCGACACCGGCCGGGACCAGCGAGGCGGCCGGAACGAGCGACGCGACTCCGCCGGCTCCGGGGCGAC17100GCAGCATCCCCAGCCCCTTC17160TGTCGCGCGCGGCCGCGGCC17220CGCTCGGCCGGCTCGGCATG17280ACCCCGAGGGCATCGCCAAG17340ACTACCAGCTGCGCCGCGCA17400TCGTCACGGCGGGCACGGCG17460GGGTGTACCACTACGACGCG17520CTGCGGAGCTGACGCCGCCG17580AGAACTACTTCAAGTACGGC17640TCGTCGGCCAGTTCGCGCTC17700GGGTGGACGAGCGGCGGATC17760CGGCCGTGCTGGACGTCTCC17820CCGCGCCCCCGGTCGGGCGG17880CCGGCACCCGCCGGATGCAC17940ACCTCCCGCCGGTCGTCCTG18000CGGCCGGGCGGCCGGTGTCG18060TCCGGGGCACGCCGCTGCCC18120CGCTGGACTGCGACGTCACC18180GGGCCGGCCGCCCCGGCACC18240GCCTGCTGATCGCGCGGTCC18300ACGGGCGCCGGCTCACCCGG18360TGTTCGCCGACTACATGAAC18420CCGATCCGCATCGCGGTCCG18480GCGTCCTCGCCCAGCGTCTC18540TGCTCGGCTTCGTCAGCGGC18600CGTCCCTGCTCCTCGTCCCC18660TGAGTCCGCTCGCCACGCGG18720GACGACCGGGACGAGCGAGG18780CGGGACGAGCGACGCGACCG1884030GGACGAGCGACGCGACCGGCACCGTTCTGCGGCTGCGCGCCGACGCCTACTACGTACCCG18900TGCCGAACGGGGTGTGGATCCGGACGACGGACGGCTCGTTCACCCTCAACGGCAAGGCGG18960TCGCGACCTGGGTCGAGCGGCTCGCGCCCCTCCTGGACGAGGGGGTGGTACCCGCCCGGC19020TCCTCGGGGCGCTCCGGCCCGAGCAGGCCGGGTTCGTGGAACAGCTCCTGACCCTGCTGG19080AGAAGCGGGGAGTGGTACGCCGGGAGGCCGCCGCCGACGGATTCGGCCCGGACCTGGCCC19140GGCTCTTCCCCCAACAGGCCGCGTTCCTCGGCCACTTCGCCTCGCGGCCCGCCGAGGCGT19200TCGACCGCGTACGGCGGCACCGGGTGGCGGTCGCGGGACCCGCAGGACGCGCGGACGTCC19260TCGTCACGACGCTCATGGAGGCCGGGTTCGGCGACTTCGCCCTCCTGGACGCCGAGCCGA19320GCACCGAACTCCGGGACCTGGCCGCCGACTTCGAGCGGCGCGGCGCACCCGTACGCCTGT19380CCGGGCCGGACGGTGATCCCCACGACCGCACGCTCATCGGGGTGTTCGACCCCGGGACGG19440ACGGCGAGGCCGACGCCCGGCGGTTCCTCGACCGGGCCGGCGCGCTCGGCGGCGGCGCCT19500GGGCGGGCCTGGTCAGCGGCCAGGCGATGCTCCTCAAGGGACAGGTGCCGGGCTCCGGCC19560GGGCCTGCGTACGGTGTGCCTGGCGGCGGCTGGCCCACAAGGCCGTCGCGCTGCCGCACT19620CCGACACGCTGGGCCATGTACCGGTGTCGATGGCGGCGACCGTGCTCGCCCAGGAGCTGT19680TCCAGTACGTCGCCGGGGCCGACGACTCCGTACTCGGCGAGGGCGTCGTGGTGGACCTGA19740CCCGGCTCAGCGTCTGGCGTACGGACGTCGACCCGGACCCGGACTGCCCGGCCTGCTCCG19800CGGCCCGCCCGTCCGGCGTACCGGCGCCGGCGCACCGCCGGACGTCCGCGTACCCCGACG19860CCGTCTTCGGCGCCCGCTGCTTCGGCCCGCTGTTCTCGTGCACCCCCGAGGAACTGCCCC19920AGTTCCCGCTCACCGCGCTGCGGGTGCGGGTCAACCCGCCGGGGCGCACCGCGCCGACGG19980GGGCGCTCGACGGCCCGGTCGTCGTGGCGCGGTCCATGGTCGACGCGCGCGCCGAGGCGG20040CCCTGACCGCCGTCGAGGCGACGCTCCCCGCGGCGGACGAGGCAGCCGTCGCGGGCGTGG20100GCCGGGACCACGCCGAGGCGCTGGCGCGCGCCCTGCTCCGGTGGGCCGACCGGACGCCGG20160AGGACGCCCGCGCGGAAGGCGGCAGGGAACCCGTCCGCACCGAGGGGACGGGCACGGCGC20220CCGCCCTCGCGGAGCTGGCCGCCCTCGCCGACACGGACGTCGCCGTCGGCGTCACGCACC20280TGCCGAGCGGCTTGTGGCGGGCCCGGGCCGACGACGGCACGGGTGCCGTCGTCCGGACCG20340GGTTCGACCCCGACCAGGCGGCGGAGCGCGCGCTGACCGCCCTGCTGGCCCGCCGGCAGC20400TGGGACCGCGGCCCGATGGAGCACCGGTCGTGGCGGCGTGGCCGGGCCCGGTCCCGTCCC20460GGGAGCGGGTCGGCGAGGTCGCGTCCGCGCTCGGCCTGACCTGGCGCGAGGCCGTCCTGC20520CGCCGCTGGTCGCCGCCGATCTGACCGCGATCGTCCTGACCGGGGAGTCCACCGGATGAG20580CGCGCCGCCGACGACGGCACCCCACGCGCCCGAGGGCGTCGACGACACGGCGGCCGAGCT20640GCGCCGTCTGCTCGACGACGTCGCCCGGGTCGTCGACACCCCGGCCGCGATCACCGCCGC20700CGCCCCTCTCCACGCCGGCCTCCCCGCCGAGGGGGACGGCCGCCCGGTCGTCCTGGTGAA20760CCTCGGCTGGGACGCCGACCTGTCCGGCCTGCCCGCCGCGGGCACCACCGTGCTGCCCGT20820GTACGGGCTCCGCGGCCGCGTCGTCGTCGGCCCGGTGACCCGACCCGGACTGCCCGGCTG20880CCCCCACTGTCTGGGGCTGCGCGTGCGCGCCACGGACCGCCCCGAGGGCGTCCCCCGGGG20940CGAGCCGGCCGTCCCCCGCCGGCTGTCCGCGCGGCCCGACGCGTGGCTGCCGCCGGGGAC21000CGCGCGCCTGGTGGCGGCCGTCGTCCGCGCCGAACTGGCCCGGCTCGCCGCCGGTGAGCC21060GCCGCTCGGCACCGAGGCCGCCTACACGGTCGGGCCCGCCCGGCTGGCCGGCGACTGGCA21120TCCGGCGCTGCCGCACACCCTGTGCGACACGCCGCACTGCCGGGGCGCCCGCCGCCCGCT21180GCCGCCCCCGCTCCCCGACCTGGCCACGCCGCTGCCCGCGACCGGTGTGGGCTCGGCCCG21240CCGCTTCCCGGCCGCCCGGTTCGCCGAACGGCTGGAGCGGGAGTACCTCGACGGCTGGTC21300GGGCGTCACCCGCTCCGCCGCCGTCGGCGACCGGGCCGTCCTGCCGTCGACGCAGGTGCG21360GGTGCCCACGGTCTGGGGGTTCGACGAGATCGCGATCGGCCGCGCCGAGGACTACGCGGG21420CGCGCGCCCGGCGGCGATCCTGGAGGGGCTGGAACGGTACGCCGGCTGGCACTGCGGCGG21480CCGCGACCCCGTCCGCTTCGCCTCCTACGCCGAACTCGCCTCCGCCGACGCCGCTGAGCC21540CTCCGGCGGACCCGGCGGGCCGGACCGACCCGCCGCCTCCGACGCCGTCGTCGATCCCCG21600CTCCCTGCTCCTGCACCCCGAAGAGGCGTATGGACAACCGGGGTTCGAGTACACGCCGTA21660CGCCCCGGAGATCCCGACCGGATGGGCGGAGGCGTACTCCGCGCTCACGGGCCGGCGGAC21720CCTCGTGCCGTTCCACGTCGCGTACTACGGCGCGACCCGCCGCCCCGAGACCGGCCCGCG21780CTTCGTCTACGAGAACTCCAACGGCTGCGCCCTCGGCTCCGGCACCGAAGAGGCGCTCCT21840GGCCGCCCTGTTGGAGGTGTCCGAGCGCGACGCCTTCCTGTGCGCGTGGCTCTCCGGGAC21900GCCGCTGCCGGAGATCGACCTCGGCCGGCTGACCGGCGAGGCGGCGCGGGCGGTACGAAC21960GGTGCGCCACCGCACCGGCCGCGAGCTGCGGGCCTTCCGGGCGCTGGGCGCCTTCGACGT22020CCCCGTGGCCCTCCTCGTGTCGACAGCGGAGGATCCGGACCTGCCGGCGACCCTGGTCAC22080CGCCGGCTCGGGCCTCACGGTGGAACGGGCGCTGCTCGGCGCCGTCCACGAGATGGCCGC22140CTCCGCACCCGTGAACACGGTCGAATTCCAGCGCCGCCGGGCCGAGTTGGAGGCGGCACT22200GGACGATCCCGGGCTGGTCCGCCGGATGGAGGACCACGCGCTGGTGGGCGCCCTGCCCGA22260GGCGCGCCCGTGGTTCTCCTTCCTCCTCGACGGCACCCCGCCGGGCGTGCCCGGCCCGGC22320CGCCCCGGACGGCCTCACGCCGTCCGGCGACGTCGCCGCCGATCTCGCCGCGATGCTCGC22380GGCCGCGCGGCGCGACGGGCAGGACGTCGTCGTCGTGGACCACACCACCAGCGAGCTGGA2244032CCGCCTGGGCCTGCGCTGTGTGAAGGCGATCGTGCCCGGCACGGTGCCGATGACCTTCGG22500CCACCGGCACCGCCGGCTGCCGCCGCCCGCGACGCTCCGCGCGTTCCGGGCCCGGCACAC22560CGACGGCCCCGTCGAGTTCTCCCCCGAGGAAGTACGGCATGAGCCACACCCGTTCCCCTG22620AGACGACGACTCCCGCTCCCGCGACGCCCGTCACCGCGGACGACGGCCTGACCGCCCTGC22680CCCACGGCATGGGCTGGGTCGTCGGCAGCCGCGCCCTGGCGGTCCGGCTGCTCGGTGACC22740AGCGCCTCGGCGTGCTGGAACCGCGCCCGGTCATGGAGCACCTCGCCACCAAGGTCGATC22800CGGGCCTGCGCCCGCTGATCGACGAGATCTCCTCGTTCGCCGAACGCGTCATGCTCCAGA22860CCGGCCACGACCGGCACCAGGAGCTGCGCAGGGCGTTCGGCGGATTCTTCGACGCCGACG22920CGGTCGAGGCCCGCGCTCCGCGGCTGCGGCAGGCGGCCGAGGAGATCGTGCGGCCCTTCG22980CCGCGGCCGGGGGCGGCGAGTTCATGACGGCGGTCGCCTTCCCGTACGCGGTGCGCACCG23040GCGCGCGGCTCATCGGCTTCACCGACGCGGAGTACCGGCTGCTGCACAAGCTGAGCCAGC23100AGGTCGCGCTCGTCGCCTACGCCGCCCGGGTCGAGGACCCGGCGGAGGCGGTCCGGGCGG23160GCCACGCCGCGCTGGTCCGGGTCCGGGAGATCCTCGCGGACGCCCGGGCGGTCGCGCCCC23220GCGACTCGGTGCTCGGCGCCTGGCACGCCGGGGAGATCGGCCTGCCGGACGCGGACGTCG23280AGGCCAACGCCGTCATGCTGGTGCAGGCGTCGCTGGAGACCGTCGCCGGCATGCTCGGGA23340ACACCGCCGTACGGGTCCTGGACACCCCGGGGGCGCTCGCCGACCCCGTACTCCTGGAGG23400AGCGGATCGACGCGGCCCTGCGCGCGGAGCCGCCGCTCAAGACGCTGGAGCGGGTGGTCA23460GGGAGCCGTTCCGGCTGGCCGGGCGGGAGTTCCGGGCCGGCCAGGTCGTCTCCGTCCGGG23520TCGCGGACGCCAACCGGCTGGACGACGCGCCGGTGGCCGGCCGCGCGCTGTTCTCCTTCG23580GCTGGGGCGCGTACCGCTGTCTCGGCGCGCGGCTCGCCCGCCACCAGGCGCGGGAACTGT23640TCCAGGCGCTGCACCGCGCCGCGCCGGACGCCACGTACGCCGACGCCGGCGCCGCGGTCG23700AGCGGGTCCGGCACATCCGCTTCGACATGCCGCGCCGCCTCCCCGTGCGGTGCGCCCCGC23760CCGTCGCCGAGGCCGCCCCGCCGCCATCCGGTGTGCTCCCCGACGCGCACACCGAGGTAC23820CCACCGAGGTACCCACCGACGTGCTCACCGACGTCCTGACGGAAGCACTCGAAGAGGACG23880ACGTGGACCGGCCGTTGACCTCGCTGGAGCGCGAGGTGACGTACGTCGTCCTGACCGAGA23940ACGGGGTGACCGTCCCCGAGGACCTGCGCGGTCCCACGACGATCCGGGAGTGGGTGACAT24000GGGCGGCCTCGGCGCGTGGCTGAAGCAGGCCGAACCGGTGCTGCGGCGTTCGCGCGCCGG24060GGTGGTGACCGAGCGCGGACGTCATCCGTGGCCGTCGCTCCTCGCCCGGGCGGAGCGGCT24120GGCGGCGGGGCTGCCCGACGCCCCGTACGGCTGGGTCGTCCCGGCGGACGGCGGCGAGCG24180GTCGATCGTGGGGCTGCTCGCGCTCGGGCTGCTCGGCCCCGCCCCCCGCTGGGTGCTCGG24240CGACCCCGCGGGCCGGGCCCCTCGGGCACCGGTACGGCTGCTCCGCCATCCCGCGACCTCCCGCCCCGGCCGGACGGCACGCGGGCGGGCCACCGAGACGGCCCCTGCCAGTGGACCAGGGGACGGCGCCGCTGGACGGCGATCACGGCCTTCCCGGACCCCGGGTCCGCCAGACCGGCCAGCCGCCCAGCCGAGCCCGCCCCGCGCGGCGCCGCCAGGGAGCTCGCCCGCGCAGTGGTCAGGGAGAGCGTCCGCGCCCGAGCGCTCCTCGCGACCTCGTTCAGCAGGGGTGACCCACGTGCCTGGGCCGCAGGCGGCGCACCGGGCAGCTACGCCGACGGACTTCGCGGGTCCTGTTCCGTGTGCCTGGGACTTCCGCGCGGATCCGCGGGACTCGTCAGGCAAACCCGGGGCCCGACAGTGACCACCGCGCGCGGACGGCCGTCCGGGGGCGACCGCCGACGCCTGCGGACGAGACCGGCGCCGAGACCGAGCCCGTTGGTCACCGAGCCCCCTCGTAGCTGGTGCTGGGCCGACGGCGCACGCGCGCCACCGTGCGGCCTGGACGGGCGGCATCCCCCGGCGGCGTGGGACGCGCTGCGGACGGCCACTCCGCCCGACGAAACTGCTGCATGCCGGACGGCCTTCTAGGCCCGGCTCCGCCGCCCGCGCACCTCCTTCGGGGTTCCCCGGTCGGGCGTCTGGCTGGACCCGGTTCGCGCGATCTCGCACCTGGTCAACGCTGCCCGGTCACCGTGATCCCGCCTGCCCCTACGACGCGCCGAGACGCGAGAAAGAGCGCACTGCTCATGGAGCCGTCCGCGACCCCCAGCCACGCCGCTGCGCCGGTATCGAGGCCGGTCGCGGAGCGACGCCGACCGCGTCCGGGAGGCAGGCTCGCAAGCCGCTGGGTCGCCGGCCTTCGGCCGGGTACGCGGCGCATGGTGGCCCTGGGCGACGGCTCGCCGCGCGTCCCCGCCCGGCTGCGACTACGGTGCGCGGACGTCGGGGGTGACCGGGCCACCGGGCCGGTCGACGGCTGTCCGGGACCGTCATGGGGCGTCCCGGTCCCGCGGCAAGCGAGGTCACCGTGATGACGAAGTGCCCGGCCCGACCGCACGCTTCAGCCTGGTTCTTCCATCCTCGGACGCGATGGCGGCTCGCCGCCCGGCGACGAGGTAGCCCGCCGATCGACCGCTGGGCGACGGGGCCCACGTACGCCGCACGCGAGCCGAGGTGTGCCCCGGCGAGCCGTCCCCGCTGGGCGCCCGGCGGCGGCCTTCCTGACCAGCCGACGGCCCCGGACGGCGGGCCGGCTCAGACGGCGGCGGTCAGCGTCTGCCTCGGTGTACCGACGCCGACCCCGCACCGTGCTGCGCTCAGCCGTCGAGATGACGACGCCCCCTCTACCTGGGTGGTGGCCCGGCCCGGAGCATGCTGGCTGTTCACCGCTCCTGGACGCTGCCCGTGCAGCGAACTGGGACGACGTCGACGCGGACTAGTTGACGGCCCACCGCCTCTCCCCTCGGCTCGCCACCTGTGACGCTCGGAGCTGGCCGGGTACGGCGGCTCACCCCCGATGCTCGGCTCATGTCGGCGCTCGGCACCCTCCACCGGGCCGGGGGTTCGTACCCGCGCGACCGTCGACCGTGACCGAGGAGGGTCCTGTGGAGCGCGTCGAGCCGACGAGACCGGACCCTGAGCGGATGCACCGGCTACGCCGCCGGGATCTGGCCTGCGCCGCGGGCGAGAGGCGGAGCAGCGTCATCGTGCCGGGTGAGACCGCGCGCCCGGAACTGC34243002436024420244802454024600246602472024780248402490024960250202508025140252002526025320253802544025500255602562025680257402580025860259202598026040TCGTGCCCGACTCCGTGCTGGCGCTCGCCGACGAGGCGCGCGACACCCCGGACGCGCTGA26100CCCGTACCGAGGTCCTCGCCAACGTCTTCAGCCTTTACATCGCCGGGCACGATACCTCCC26160GCAACATGCTCAGCGGCCTACTGCTGCGCCTCGCCACCCACCCCGGACTCCTCGCGGGCC26220TGGCCGACGGCACGGTGGACGCCCAGGCGGAGGTCGACCTGCTGCTGCTCGGCGAGTCCC26280CGCTCACGTTCACCGTCCGCGTGGCGCGGGAGGAGTGCGAGCTGGCGTCGCGGCGCATCC26340AGCCCGGCGACCGCATCCGGGTGATGCTCGGCGCCGCCAACCACGAGCTGCTCACCAGCG26400GCGACGCCAAGGACGGCAACACCGGCGTCAGTTTCGGCGAGGGCCGCCATGTGTGCCTCG26460GCGCGCACCTGGCGCGCACCGAGGGCCGGGTGATGCTCGACGCCGTGAGCCGGCGATGGT26520CCGGGCTGCGGCTCGCGGACGACCCGGCGTGGACGCCGCACTTCCTGCACCGGGGCCTGG26580ACCGCCTGCCGCTGGAGATCGCATGGCGGTGACGAGCGGGCCCGTACACGAGACGGCCCC26640CGTGGCTGCCGACCACGTCGTCCCGGGGCCGGTGGTGGTACGCCGCCGGTGCCGCGTCCC26700GGGCTCCGTCCTGGCGGACCTGGGCAGCGACCGGGTGTGGGGCGCGGCCGCGGCCTGGGC26760GTACGCGGAGGACGCCGTCCAGCGCACCGGCACCCGACTCGCCGACCAGGTCGGGGAGTC26820GGTGCCGACCGTACGGCAGGAGGAGCGCCGCGATCTGCTGCGGGTACGCCGGGCCGCGTT26880CAACTCCCGCCCGCTGCCCGCCGATTCCGGCGCCGTGCTCGGGGGCCCGGCCGCCGCCCT26940CGCGGAGGAGTACACGCGCGCCCTGACCGCGCGCGACACGAACCGTCACGAACTAGACCT27000GGCCCTCGCGGACTTGGCCCGTACGTCCGGCGCGACCGTCGACGCCCTGCTGCGCGACCC27060GCACCTCGCCCGGTCCGTCGAGCTCTCCGTACCCGGCCTCCTGGCCGCCGGCCCCGTCGG27120CGCACTGGACCCGGCCGCCGCGAAGAAGACCCGGCGCCGGGCCCTCACCCTGCTCCGCCT27180CATGCAGCGCGCCGCGTCGAAGCCGACCCCGTTCGCCGGCTTCGCCACCACGCACGTGCT27240CTTCGAGAACACGGCACCGGCCGCTTCGGACCTCCCCTCGCGCAGCCATGTGCGGATCGA27300CCGCGCGGTGATCGAGTGGGTACGGCAGTGGATCGCCGCGGGCGGCCACCGGAGCCTGCG27360GCCGGAGCGGCTGTGGCTCACCGCCAACCCCACCGCGGCCGTCCGCCAGGACGCCTCCGG27420GACGCGCGTGACCTGGCTCCTGAGCAGGGCGGACGGCACCGAACGGGTGCTGTCCGCGCC27480ATGCGGCGCCGCGCTCGCCGGCACCCTCGCCCGGCTGCGGGAACCGGTCCGCCTGGACAC27540CGTCGCGCCGGGCGGCACCCTGCCCGACGGCCTCGCGCGCCTCGTCGCGCGCGGGCTCCT27600GGAGATCGGTCCACGTCTTCCCTCCCATGGCCGGCGGACGCTGCACGCGGCGGCCGACGC27660GACGGCGCCGGCACCGGACCTGGACCTGGACCTGGACCCGGACGCGACCGAGGCCCGGCA27720GGCCCGTCAACTCCACGACGCGCTGGGCGTGTTGCGTGACGCCGAGGACGCCCTCGCCGC27780CGATCCGGCACACGCGGGCGCCCGGCGCGACGCGAAGGACGGCATCGCCGCCGTCGCCAC27840CGCGCTGGGGCGTGGGCGTCCTTCGGCCCCCTTCGAGCTGTCCGGTGCCCCGCGTTGCTCGCTCTTCGACCGATCCCGGCCTGCGCGGCCCGGCTACGGCGCTGCGCGACCGCCGCGTCGCTATCCGGGCCTCCTGCGCGCGCCCCGGGCCGGCCGCTTCGGCCCCCGGCCCTGTGGGATTCCGCGGCTGGGCGCTGCCCCCTGGCCTGGCACGACGCTCCGCCGCCCGCCCAGCCGCTGACCGCTGCCGGTACGTCCTGCACACGGCCGACCCCAACCATGGAGGCCTCGGCAGACCCTATCGGCGTCCATGGGCGTCCGAGGTCCTCGCCCTTCCAGCGTCCTGACCGGCCGACTGTCGGGCTCCGCGCTCCTCGACGTGGCCGGTCCCGCTTCGCCGTGGGCGGCGACTCGGCGCCACGCGCCCTGGGTGCGCGTGGACCGGCCCTGGCGGGACGCCCTGTACCGGTCACGTGGACGACCCTGGGGCGCGGAAGCCCTACCGCTGGTCCGGACCCCTTCCACCTCCGTGCTGGCCGGGATCCCGCCGGAGACCTCGCTAAGCAGGAAGATCCACCCCCGGCTTCGAGCCGGGACTACGGGCGGGCGGGTGCCGACGACCGACCTCGGTGGCCACGGCCCTACGCGTGAGGCCGAGGACGGCGGCGGACGGTGGCGGCAACTCGCCGGCCCGCGTCGCCCGCCGCCACCCGTCAACGAAGTGCGCCCCCGGGCGGGCGGCCCCGGTGCCGCTGTTCCCGGCTGAGCCGCCCTCGCGGCGGCTCGTGCTTCCCGGGCGCGCGCGCGTACGGGACGTCGTGCGGCGCGGCCCCTGGCGCCGCGGCACCACAGCCTCCGCGGACGAGATCACCGATCGCGCGCGCACGTCCTTCCGGGCGGCGGCGACXGTCGACGTTCCGCCGCGTCCAGCCTCGCGTTCGTTCCTCGACACTCGCCACGCCGGAACCCGCGATCTGCCGGGACCGCGTGCGCCGGCCTGCCCTCACGAGGCGGCCCCTGCGAGACCCCCGCTGCTGCTCCGGCGCCGGCCGTACCGCACCCTCGGGCCGGCCCCGGGACGAGCCGGCGCGGCGGGAGCCGGGCGTGCCCCCGCGGCCTGCCTCCGCAAGCGCGCCCGGGCGCCGCCGCACGCACGAGCGTGCTCCGTAACGTACGCGCTTGGACCTCACAGCGCCCGCTTCGGCGGTCGCGCGCCGACGCGCGTCGTGCCGGCGCCGCTGAGGGCCGCCGTCCTCGCCCGGCGCGCCGGGACGCGGTCAGTGGTGAACGGACCCGGCCCCACGGGGTCCCTGCTGCCCGGGGGCCCGGGTTCGGCGGCGGCCGGCACCGAACGCGACCCGGATCCGGGGCCGGAGGCCGACCTGGAAGGGGCGGAACCGCGCCGCTCGTGCCGACAGACCCCCTCTACGGACACGCTGACCCGGCAGCCACCGAGGGCTAGGCACCGTCGGACGTGCTCCTTCCACCTGGCTCGTCGGCGCGAGGACGTGCACGGGCCCCGCTGCTCGGTCGGCACCGAGGGAATCCGATGCGCCACCGAGGCCGCGTGTGGACCTCTCGGGTCGGCACGGCTCGCCGTCGGTCGACATCCGCGCTCGGTCGACCTGTCAGGGCGACGAGCACCGGCACTGCCGACCGTCGACGCTCGCTACGGCACTATGCCCGCGGCCGCGGGAGGCTCCTGCGCGGGCCCGGTGGATCGACTTCTCCCTTCACCGAGCGTCACCGAGCGCCGCCGCGCTCTGATCAACCACCGCGCGACGACTGGCGTGACCTGCC362790027960280202808028140282002826028320283802844028500285602862028680287402,028860289202固02904029100291602922029280293402940029460295202958029640GCAACACCGACACGGTGTACGCGCTTGAGCAACGCCCCTTCGTGGACGGGTACAAGGCGG29700TCATCGACGAGGTACGGCGGCTCACCGCCGCGCCGGTCGTCGCGGGCGGAGTGGGCTTCT29760CCACCATGCCCTTCGCCCTGGTCGACTACTTCGGCATCGAGTACGGGGTGAAGGGGCCGG29820GCGAGAAGATCATCTGCGATCTGGCGCGGGCGCTCGCGGAGGGACGGTCCGCCGACCGCA29880TCCACATCCCTGGACTGCTGGTCAACCGCGGCCCCGGGAACGTCACCAGAGTGGCCCCGC29940CCGCGCTGGATCCCCGCGCGGCCCCCGCGCCTTCTTCCTCGCCCTCACCCTCGCCCGCGC30000CCTCTTCCTCGTCCGCCCCGGTCCCCGTCCCGCTGTCGTTCGCGGCCGTCGGCCACCACG30060AGAGCCGGGCCTGGCAGGCGGAGACCGAACTCCCCTACACCCGGCGGTCGGGCGAGCCGT30120ACAAGGTCGACAACCTGCGCTACTACCGCGAGGGCGGCCTGGGCAGCATCCTGACCAAGA30180ACGGCTGCGTCTACAAATGCAGCTTCTGCGTCGAACCGGACGCCAAGGGAACGCAGTTCG30240CGCGGCGCGGCATCACGGCGGTGGTGGACGAGATGGAGGCGCTGACCGCCCAGGGCATCC30300ATGATCTGCACACCACGGACAGCGAGTTCAACCTCTCGATCGCGCACAGCAAGAACCTGC30360TGCGGGAGATCGTGCGGCGCCGCGACCACGACGCGACCAGCCCGCTGCGCGACCTCCGGC30420TCTGGGTCTACTGTCAACCTTCCCCGTTCGACGAGGAGTTCGCGGAGCTGCTGGCCGCCG30480CCGGCTGCGCGGGCGTGAACATCGGCGCCGACCACACCCGTCCCGAGATGCTCGACGGGT30540GGAAGGTGACCGCGAAGGGCACCCGGTACTACGACTTCGCCGACACCGAGCGGCTGGTGC30600AGCTGTGCCACCGCAACGGCATGCTGACGATGGTCGAGGCGCTCTTCGGCATGCCGGGGG30660AGACCCTGGAGACCATGCGCGACTGCGTCGACCGGATGATGGAACTCGACGCCACGGTCA30720CCGGGTTCAGCCTCGGCCTCCGGCTCCTCCCCTACATGGGCCTGGCCAAGTCCCTGGCCG30780AGCAGTGCGACGGGGTACGCACGGTCCGCGGGCTCCAGTCCAACAACGCGTCGGGGCCGA30840TCGTCCTGAAGCAGCTGCACCAGTGCGACGGACCCATCGAGTACGAACGGCAGTTCATGT30900TCGACGAGTCCGGCGACTTCCGGCTCGTCTGTTACTTCTCCCCCGACCTCCCCGAGGCTC30960CGGGGACGGCGGACAGCCCCGACGGGATCTGGCGGGCCTCGGTCGACTTCCTCTGGGACC31020GCATCCCGAAGTCCGAGCAGTACCGGGTGATGCTGCCGACCCTCTCCGGCAGCAGCGAGA31080ACGACAACAACTACGCCGACAACCCCTTCCTCACCAGCCTGAACAGGAAGGGATACACGG31140GCGCTTTCTGGGCCCATTGGCGCGACCGCGAGGCGATCATGAGCGGCGCCACGCTCCCGC31200TCGGGGAGCTCGCGGAGGCCGTCCGGTGACCGCCCCCGCGCTCCCGCTCGCCGGGCAGGT31260CGCGCTGGTCACCGGGGCCGGCCGCGGCATCGGGCGGATGGTCGCGGTCGCCCTCGCCGA31320GGCGGGCATGACCGTCGGACTGGTCGGCCGGAACCGGCGGCTCCTCGACGTCACCGCGCG31380CGCCTGCGCCGCCGCCCGCGAAGCCGCCTCCGGGGACGGCTCCGGGGACGGCTCCGGAGG31440CGGTCCGGAAGGAGGCCGCCCGGCCTGGTGGGTCTTCGAGGACCCGCCGCCCCCCGCTACGGCGGAACCGCACCGACATGCCTGTTCCTCCTTCCTCCACCTGACGGCACCCGGGACCCGGGTCCTCGGGCTCTTCGGGGAGACGGCAGACGCCGGCCCGACATCCGTCTGGTGCGGCATCCTCGGTCCGGGCCCCTGTCACCGGGTCGACGGTCGGGTAGCACGGTCAAAGMGACCGGCGGACGTCAATGGCCGCGGACGGGCACGGGCCGACGACCCTCAAGAACTCCGTAGTCCTGGCCGGTGTCGGAGGCGGTACGACCAGGGCGACGAACGTCCGGATCGGGCCTCCGCCTACTCTCCGGGACCACCCTGTCGAGCGGCGATACGTGGGCGCCGCACCTGCGAGGGCCGGCCTCCCGGTCCTCGGTTTCCCTGACGTCGGGCACGCCCGGGGCCACTCGGCGTCGGTGGTGAACGCTGGAGAACGCTGCGCCGCGAACGGGGTGCAGCGTCGACGCTGACGAAGGGTGAACGGCGGGCGGCGGGCCTGGACGTCCTACCGGATAGGCGGGCCCGCTGACCGGCCCCCGGGGACGCCGTGGCGGTGGGCCGCACTAACTCCCCTTGCGGCACCGTGCGTCGTCAAGCGGCTCCAAGCGGGGTCCATGGCCGTCTGTCCGCGTGGCACGACTTCGAGACGAGTGCGGGGCCGGACCGGCCGGAACCTGTCGGCGTGCACGGGGGTCGGGCGACCGTTCCGTGGCGGGAGATGTACGGGCTCGGGCCACGGTCTCGTCTGATCCTCGGTCCCCGCCGGTGGGCGCGGGCGGTCGACGATCAAGGCCCCAGGCGACGAGACGCGCAGACACCGGCTCACGGTCTCGTCTACTGCGCCCGGCGGATGTCCGGCCCCGTCCTGGGAAGCCCAACACCTGCCGTCAACTCCGGCGACGCTGCCTGTTCGACCGCGGGCCGCCCACGGCGACCGCGTTACTGGAGCGGACCGTCGGCCGGTCCTGATCGAACCCGGCGGGGCTGGTCGTGGCTCGCAACAGGCGGCCGGACTAGGACAGCAGGCGTCACCGTACCAGGACAAGCGACTGCGGGCATCCCGGTTCCAGGTGACTGAGGACCACGGACGTCCAACGACCAACGGCGGCCAACGGCGCTGAAGACCGGCTCACGGACAGGGCCGGCGCGCGCCCGGACCTCCTGGGATCCGGAGCCGGGCCGTCCCGCCTCGCCGCTCGCCCTGGATCAGTCCGGGACGACTGGACTCGACCCCCAGCGAACGGCCGGGGCGGACCTCGGGCCGGCCTGGGGCAACGTTAGCGTCCCGGCGTCGCGGGATCTCGCGGCCGTCGGCTTCGCTGCCCCAACGGCACCGAAGCCCGACTGACGACATCGAGCGGCAAGCACCACGTCCCAACGGCCGCCGGGGGCATCCACCATCGCCCGACAAGCGGCAGCAACGGGGGCGTCCCGGGGTGAGGACGCCGAGATCCGTGAACAACGCGGTGGTGGGATGCCGGAGATTCCAGGGAGAACGCGGTCGCGCATGGTGCGCGGACCCCGCTCGCCGGCCGGGCTCGCCGTCGGAATCCGGTCCTCGGGCCTCCAGGGGTTGTAGAGCGTCCGTCGGGCCAATGCGCAAGAGCGCTCTCCGGAGAAGATCAAAGGCCGGCGGGCCCCACCCGACTTCTGCTGTCACCAACGGGCCGGATCGATCACCGGATGAGCTGAACCCTGAAGGTGCATCGGTCTGAAGCATCAGCGCACGGCGGGTTTCCCGGTGG38315003156031620316803174031,318603192031980320403210032160322203228032340324003246032520325803264032700327603282032謂329403300033060331203318033240CCGCGCAGACGGTGCGGGCCGCGGCCTCCGCCTCCGCGGTGCTCGCCAGAGTGCGCCCGTGCGCGCTGCCGGCGATGGCGCGGCTGAGGATGCCGCCTTCGCCGCTGCCCACCGCCACCATGGTCGTGCCGCGCGGGTCCGAGGAGCCGCCCCAGAGGGTCAGTTCGCCGTCCTGGTCGGGCGCCAGCGCCTGGAGTTTGTCCTGTTTGGACGCCGCCCGGTCCCACAGCTCGGCGCTCCCGTCCGCACTGACCGTCAGCCCCTCGATGCCGGCGGGCCGGTCCGGCGGGGTGAGACCCCCCAGCGTCCGGCCGTCCCCGCTCAGCCTCGGCGTACGGGAGAGTTCCCGGGGTCGGGCCGCGTCGCCGCCCGTGGCGAGCGTCCGGCCGTTCGTCGTCCCGGTGTTCAGCGTGCCGAGCGTCCACAGCCGGACCGTCCCGTCGGACCCGCAGGCGAGCGACAACGCGCCGTACGCGGCGGGCTGGAGCTGTCCGGCCTGGGTACCGCCGAGGCCCCGGACGTTGGAGGGAGGGGCGGGGACGGTGACGTTCCACAGCCGCACGGTCCGGCCGGCGCTGAAGGCCAGGGAGAGCACATCGCCGGGGCGGGCCGGGTCGGCGAGGTCCCACAGGGTCCGCCCGTCGGGGCTGTAGGCGATCCCCGGTTCCACGGGTGCCCGGTCCGGATGGGGCCAGGCCCCGGTGAGCGTGCGGCCGTCGGAGGCGCCGGCGAGCCGGCGGGGCGGGGCGGGGGTGCCGAGGTCCACGAGGACCATCCGGCCGTCCGGACCGAACGCCACGACGTAGCTGGCGGGCTTGGCGGGGTCGGTGACGTCCCACATGGCCAGGAGCCGGCCGTTCGGGCTGAAGACCGGTGCGCCGAGCGGCCGGAGGCGGGCCGGGTCGGTGGTCCAGATACGTACGGGCTGGT33300CCGGGGCGAAGGCGAGAGCGACCATCTTCT33360AGGACGCACGGTCGGCACCCGTGAAGCGGA33420GGTCTCCGCGCGCGGTCGCCGCCAGGGCGG33480GTGCGACCGGCCGCGCCGGGTCCGCGAGGT33540CCCGGGCCAGCGTCCGGCCGTCGCCGCTGA33600AAGGCCCGGGCAGCGTGGCCGTACGGTGCG33660CGCCGTGGCGCCCGACGCCGATCATCCGCC33720CGTCCTCCTTGCCCGGCAGGCTTCCGGTCG33780AGACCCGCACGGTGGCCCCGTCCTCGAAGA33840CGTCGTCACCGGAGCCCTGGCTCGCCCCGG33900GATCGGCGAGGTCCCACAGCACGAGAGGGC33960CCCGGCCGAAGCCGAGGACGGTCACTTGCT34020GGCGGGGGGCGCCGGCCGGCGAGGCGAGGT34080CGGTGGCGAGAGTCCGGCCGTCCGGGGCGA34140CGGGAAGCACCCGCCCGGGCAGGCTCCACA34200CGAGGGTGCGGCCGTCGGGGCTGAAGGCCA34260CGGCGGTACCGGCCGGGGACTGGGCCGGGT34320CCCGGTCCGAGACGGCCAGGGTCTGGCCGT34380CCGCCGCCGGCAGATAGAACCGGAGCCGGA34440GCCAGACCCGGGCGTCGTTGCCCATCGCGA34500GGTAACCGCTCGCGCTGGGCAACGGCGGGA34560CCAGATCCCACACCCGTACGGGGCCGTTGC34620CCCCGAAGCGCAGGCTCTCCGGGAAGCCGT34680GCCGGGCGGGACCGTCCGGGCCGGCGGCCG34740GGTCCGGGCCGACGCCCGCCAGGGTGCGGC3權(quán)0TCGCGACAGCCGAGGGCAGCGGAAGCACGA34860GCCGGGCCGGGGTGTTGAAGCTCCGGCCGC34920CCATGTTGGTGCTCAAGGCGGAGTCGGCGA34980GATCGGAGGCGTCCCACAGCCGCAGCTTGC3504039CGTCCTCACCCGTACTGGCGAGGACGCGTCCGTCGGGGCGGTAGACGGTCTCGTGGACCA35100TGTTCTGCCGATGGGCCGCCCTCGTGCGCGGAAGGATCGAGTTGCCGTCGGCGAGCACGT35160TCAGGGTGGTCTCGGGGCGCGGTGCGAGGCGGTGGGCCACCAGGTCCAGCTGGGCCGCGA35220GCGCCGGGTCCGTCGCGCGCACCCCGGCGGCCTGCGAGACGATCCGGGCGGCCAGCGCCT35280GGCGCTGCTCGGCGCGGGCCGAGCCACTCTGCCGCAGGGCCGCGAACGAGGCCGCGGCGG35340CGACGACCACGAGCACGACGAGGACGGCCGTGAGCCGGTGCCGGATCCGTACCGCCCGCT35400TGGCGCGCCGCACGGACGTGTCGAGGAAGGCGCGCCGCAGCGGGTCGTCCTCGTCCGGGT35460CGTCGGGCGGGGGCGCCGCGTCGAGGGCGCGGGCGAGCGGGGCTCCCCGGTAGAGGGCGT35520CGTGCTCGTGTCCGGCGGCGGCCCAGGCGCGGGCGGCGGCGTCCAGGTCCTGGAGGATCA35580GGTTGCGGGGCCGGTCGTCGTCGATCCAGCCGCGCAGCCGGGGCCAGGCGGTCAGCAGCG35640CCTCGTGCGTGATGGCGACGGTGTCGCGGTCCTGGACGAGCAGGCGCTTGGTGGTGAACG35700CGGCGAGCACCGCCTCGGCGCGCGCCCGGTCGGGGATGGCGTCGAGAAGCTGTTCGCGGC35760GGACCCGGCGGCGGCTGTCGGCGGTGTCCTCGCCGACCGTCACCAGGCCGAGGAACAGCC35820GCCGGGCGATCCGCCGTTCGGCGGCGTCGAGGCGGTCGAAGACCCGTTCTGCGGTGTTAC35880CGACGGCGTGGGCGACACCACCCGTGCCCGTGTAGCCGTCGACGGTGAGCAGGCGGCCCT35940GCCGGTTCTGCCAGGTGGCGCGCAGGGCGTGGGCGAGCAGCGGCAGCGCTCCCGTCTCGG36000GGTGGTCGCCGCTCCGGTCGCCGCCGGAGCCCGCTCCGCGGCCTTCCGGGCCGGCCGCGG36060GCTCGGTGGCGGAGAAGTCGGCGAGGAGGAGTTCGACGAGCCCCGGTTCGAGGCGCAGTC36120CGGCGGCGCGGGCGGGCCGGCGTATCGCGTCGGCGAGTTCGGCGCGGGTCAGGGCGGGGA36180GGAGCAGCTGGCCGTTCTGTACGGCGGTCCGCAGCCAGGGAAGTTCGGCGCAGCGGGCGT36240AGAAGTCGGCGCGCAGGGCGAGGACGACGACGGCGAGCGGGGCGTCGCCGCTGCCCGCGC36300CCGGGTCCGCCAGCCAGGCCAGCAGTTCGGTGAAGCCCTGCCGTTCGGCGGGGTCGGCGC36360AGAGGGTGAAGAGTTCCTCGGCCTGGTCGACCACCACGAGCGTCCGTGCGGGCACGGCCT36420CGCGCAGCCGCCGCGCGAAGCGCGTCCGGTCGGCGGTCCACCAGTGCGCCATCCGGTCCG36480GCTCCTCGTCGGCGAGCCGGGCGAGACGGGCGGTCAGTTCGGCGAGGGGGCGGGCCGTCG36540GGGTGAGCAGCAGCCGCGGCCAGTGCCGCGATCCGGGCACCGGCACCCGGGCCTGGTCGA36600GGTCCTGCCAGAGCCCGGCCTGGAGCAGCGAGGTCTTGCCCGCGCCGGACGGGGCGACGA36660CGACCAGCGGGGTGCGGCCGGCATGGCAGTCGGCGAGCCGTTCGCCGAGTTCGGTGAGGA36720GCCGGCGGCGGCCGAAGAAGCGGTCGGCCTGGTCGGCGGTGAAGGCGGAGAGACCGGGAT36780AGGGGCAGTCGTCGGTGACCGGGCCGACGGTGTGGTGCTCGCGGCCGTCGGCGGTCTCGG36840TGTAGTCGATGTGGAGATGGACGTCGCCCTGCTCGGCGACGTACATGTCCCGCCCCGAGG36900CGAACGCGTGCGAAGACGCCGGCCCCGGGATGTCGGACAACGGGTCGGTGGGCCCCTCGT36960CCGGCCGGTCGCCTGGCCGCTCGCGCGGCCGGCCGGGCCGACGTTCCTCGTCGGGAGTGC37020TCACCGCTCGGCCCCCTCGGGACTCGCTGCCCGCCGTGCACATGACGTGCCGCCAGGCTA37080GCGGCGCCCCGGACGGCCGGGGAAGGCGGAAGCGAAGGGACAAGCGGGGAACAAGCGGGA37140AGGGAGAGCGGAGGAAGCGTGAAGGCGCGCGGGTGACGTGCGGTGGCCGGGCCGGGAAGG37200GCTGGGGCTGCCGGGAGACCCGGGCCGGGAAAACGGCCCGCCAGGGGAAGGGCCGCCAAG37260GGAAGGGCCGGCCGGGGAAAGGCTTGGCCGGGGGGAACGGCATCGGGGGGACAGCCGTTC37320CCCCCGATGAGAACCGGACCTGCCAGTCCAGCGCCGTGATCAGGGGACGCACTCGGCGCG37380GACCCCGCAGTGAGGGCCGGTTCCAAAGCCCTGCGGATTCCTGCCAGACCCGCTGGGCTC37440GAGACCCATCCTGCTCGTTCGCCCGCCGCTCCGGGGGTCGGCGAAAGGCCCCGTTCACTG37500GGCCATTGGTCCATAAAGACCCGGCCGGAAGGCGCTCGGTTAGACTCGGCGACCGCGTAA37560ACGCGGCGCCGGGAGCGGTGACGGCGGCGGCAGCGGTGGTGACAGGGGTTTCAGGGGCGA37620CTGGGGAGAGCGGACGAATGGCGCAGGTGAAGAAGGTCGCGCTCTATGCGGTCGGGGTTT37680TCGTGCTGTACACGATCATCACGTCCCCGGCCCGGGCGGCGGACCTCGTCCAGGTAGGGT37740TCGAGGGCATATCGAGCGCCGCTCAGAGCGTCGGCCAGTTCATGACGGAGCTCGTCAACT37800GAGTCGACCGACTCGCACCGGACTCGCCCACGGGAGACGGCAGTGATCCGCCATCTGGTC37860CTCTTCAAGCTCGACGACGGCGTCCGGCGCGACGACCCCCGCGTGATCGCGGGCGCCGAG37920GCGTTCCGCGCCCTCGGTTCGCGGATCCCCGAGCTGACGTTCTGGGAATGCGCCTGGAAC37980ATCAGCGACCGGCCGATCGCGTACGACTTCGCCATCAACTCCGCCGTCGCCGACCGGGAC3謝0GCGCTCCAGCGCTATCTGGACCACCCGGCGCACCAGGCGGGCGTCGCGCTCTGGCGCGAG38100TTCTCCACCTGGGTGATCGCCGACTACCCGTTCGAGGCCTGATCCCCCGCCTCGTCCTCC38160GCGGCGCGCACGCGTCACCACCCTTCCGGGCCCTTCGCCGTCGACGGCGGAGGGCCCGGA38220TTCTTACCCGCCCGACGCTCACTCAACACGTGGTTATGCGGTGCTTGCACACAGTGGACA38280TGTCTTGTGATGCTATGACCGCTTTTGACGAATGAGTCGACCGAAGTTGACGTGAAGGGG38340TGGCGTGAACGTGCCGGCCAGTGCAGCGCCTCAGGTCCCTCCCCAGAACGAGCCCGGAAC38400CCCGACCACGCCCGGCGCCCCCCGTACGCCCGGCACCGCCGGCGCCCCCGACGCCGACGC38460CGCCGCCGACATCCCGAGCGCTTCCGACGACTCCGGAGACACCGAAGAGTCGGGCGACTC38520CGGCGGTCCCGGCGACTCCGACACGCCCGAGACCGCCGACGGCACCCCCGGCGCGCGCAC38580CGGCCACCGCCCCGGCACCCGCGGCGCCGACACCCGCGCCCTCACCCAGGTGCTCTTCGC38640GCGGCTCAAGAACCTGGAGCCGGGCACCCCCGAGGCGAACCTCCCCCTCGTCCGCTACGCGATGGAGGACGTGGTCCAGGTCGGCACCATCCCGGACCGGGGCGTGCAGTTCCCGACCTTGCGCTACTTCCGCGACAACGTCCGCACGGTGGTCCAGGTCAACGGCGCCACCGAGGACCTCGCCGAGATCGCCGAACGCCTCAAGATCGGCGGCCGCTCGTACCACGCCACCTCCCTGGACCTGCTCGACCGCCTCGGCTACGAGGACCCCGTCCGGCATCTGCTCGTCCAGCTGCCCGACTACAGCAACCTGACGCAGTCCCAGATCAGGTCAAGGCTCCTCGCCCGGAGCTTCGCCCGACCGGATCGGGTAGACCCTTCCGGAGCACCTGTTTCCTGCGGATACTCGACTCCGCTGTGTGTGACATTCTGCAGAAACCGCGTTTGCCATGCGTTCCTCAGATGG39556GGAACACCAGCACGTCCGCGGCGCCCTGAT38700CGCCGCCCGCTTCCGCAGCCGCAACGAGCC38760CGGCCTGATCAACGCCATCGACCGCTTCGA38820CGCCATGCCGACCGTCGTCGGCGAGATCAA38880CCACGTCCCCCGCCGCCTCCACGAACTGTG38940CACCACCGCCCACGGCCGCTCCCCGACCAC39000CGAGGACGAGGTGCTCGCCTGCATCGAGGC39060GGCCGCCCAGGAGGGCGACGGCCTGCCCGG39120CGCGCTCGCCGGCGTCGAACACCGCGACCT39180GCGCGAGCAGCGCATCCTGATGCTCCGCTA39240TCAGGAGCTCGGGGTCTCGCAGATGCACGT39300CCTGAGATCCGCAAATCGGATCGAGGCGTA39360TTTAAGGCACCTCAAAAGCCGCACACCCCT39420TCGACAAGTCACTACAGCGCGTTGCCGACT39480CGGCCTCACCTCCGGTATTCAGGTGGAGGC39540<210>2<211>362<212〉PRT<213>iSf邵^ww，s/a騰"riz'ATCC31255<400>1MetThrGlyValThrGluProGlyAlaSerGlyProGluHisArgLeu151015SerLeuAsnGluMetProAlaValProValThrGlyLeuAspGluAla202530LeuArgAspAlaLeuArgGlyAlaHisArgTyrProAspProPheAla354045ArgGluLeuThrAlaAlalieAlaAlaThrHisSerValProGluHis5055AspValValThrGlyProGlySerAlaVal6570GinTrpAlaAlaProArgArgGly60LeuGinHisPheAspAlaTyr100LeuGluliePro115ValThrProAsp130ThrPro85ProLeuAlaValArgGlulie75ValTyrAlaArgVal90AlaAlaGlyAlaAlaPro145AlaValProGluAspHisArg120ThrArgAla135GlyThrAlaLeuGlyAla150ValValValAla105HisAspLeuVallie80ProSer95ThrAlaVal110MetLeuAlaProArg125LeuValValCysAsnProHisAsn140LeuAlaPheAlaGlyAsp180ValLeuProAsn195LeuHis165LysAspThrLeuValAspAlaLeu155AspValLeu170GlyProAspValAsp185SerPheSerPheLeuArg160GluAlaTyrArgGlu175ArgGlyAlaGly210LeuArgVal225AlaAlaAlaValAspArgGlyGinGly275ProLeuGlyValLeuArg200ArgValGlyPheAlaLeuAlaArg215ThrArgLeuLeuProPheAlaVal230235ArgThrValLeuAlaArgArgSer245250ValValAlaGluArgAspArgLeu260265TrpGluValAlaProSerAlaAla280SerArgSerAlaAlaPheValAlaTyr190LysAlaTyrGlyLeu205ArgGluValAlaAsp220GlyThrValAlaGlu240GluValTyrAlaArg255ThrAlaGluLeuArg270AsnPheCysTrpLeu285AlaAlaAlaArgGlu43Gly305GlyAlaLysGlu295LeuValArgAlaVal310AlaAsnAsp325GluGluPro290lieAspProAlaProThrGlu355Arg340ProGlyGluAlaAlaGlyGluGly315AlaValLeuArg330ValGlyProAla345ProGlyAsp360300ValArgValThrVal320LeuThrAlaArgLeu335AlaSerValGluGly350<210><211><212><213><400>MetThr1lieHisValPheAlaAsp50AlaGly65AspGlu3276PRTS/rejf^o附y(tǒng)cesATCC31255GluLeuVal35AlaSerGinAspHisProGluThrThrAla5HisValTrpPro20HisGlyTyrAlaLeuThrArgHis55GlyArgSerGly70ValAIeiAspLeu85GlyLeuProVal100ArgArgPheAla10ProAsp40GlyGlu25HisThrAlaAlaGlyAlaAlaValGlyAlaArgAlaThrValMet105Val90lieGlyValArgHisPro15ProArgTyrLeuAla30ArgTyrGlyArgLeu45TyrAlaProAspHis60AlaLeuValThrAspHis7580ValGluArgAlaArgAla95GlyHisSerValGlyGly11044MetValAlaValArgTyrAlaGinArgHisProGluAspLeuAlaAla120125LeuGlySerTrpHisThrAlaThrSerLeuAla115LeuAlaAlaProVal130LeuLeuAlaPheAla145ValMetSerArgVal135Glulie150ProAlaGluAlaTrpHis140ProGluMetProValAspValGly155ArgTyrAsnAlaAspProAlaGluAlaAla165170LeulieTrpHisGlyAlaPheValArgAspThrLeu180185GluArglieAsnGlyGlyAsp175ValSer160ProValThrCysLeuGluArglieAsnGly195200TrpLeuHisGlyAspAlaAspProThrLeu210ThrArgAlaGlyVal225ValTyrProGlyAsp215GluLyslieArgGly230LeuHisGlyLeuGluSer190GluSerLeuAlaPheLeuPro205LeulieAspGluArgAlaThrAla260ProThrArgPro275Ala245AspLeuThrAlaLeuAla220PheHisLeuAlaGlu235HisAspGluGlyPhe265Phe250LeuAspAspAlaLeu270Arg255ThrArg240AspVal<210>4<211>618<212>PRT<213>iS/re/^o附少cey/a訓(xùn)加7ATCC31255<400>1MetCysGlylieAlaGlyTrpValAlaPheGlyArgAspLeuGlyAla45<formula>formulaseeoriginaldocumentpage46</formula>ValProLeuAlaAlaThrVal305AlaCys290ThrLeu275PheCys260Ala245SerLeuLeuSerValGlyAlaSerValGlyGlyProArgArgHislieLeuLeuSerLeuProLysAsp385ArgAla370GluGin355lieAsp340GlyGly325ProGly310AlaPhe295GlyLeu280SerGly265Arg250GlyLeuAspSerAspArgGlyAlaAlaGlyAspAspSerArgHisGluAlaAlaArgLeuAlaAspArgGinArgLeuPheGlyAlaGluThrGlyGlyProAspAlaValHis465ArgLeu450LeuTyr435ProAla420ArgPhe405PheGly390ProSer375TyrMet360ThrAla345AspAsp330AlaAla315LeuSer300TyrGlu285GlySer270Asp255SerValProLeuProGlyPheAlaAlaGluThrLeuAspValLeuArgThrSerLeuValAlaLeuProTrpPheTrpMetTyrLeuAspAlaAspGlyGluAlaPheLeuThr470ProAla425ArgTyrAla440GluGluAsp455PheLeuProMet410LeuHis395AlaSer380AspTyr365GlyAla350LeuAla335ArgLeu320LysAspLeuPheGluGlyAlaGluAsnAlaGlyValAlaProGluGluAlaValArgArgMetLeu475Met460LeuArgLeu430AlaAla445ArgLysAla415SerVal400AlaLeuValProValGlyGluArgLysMetSerMetAlaValGlyLeuGluValArgValProPheLeuAsp480Asp47485490495HisAlaLeuValGluTyrValPheAsnValProTrpSerMetLysThr500505510PheAspGlyArgGluLysSerLeuLeuArgAlaAlaMetAlaProGlu515520525LeuProAspSerValLeuThrArgLysLysGluProTyrProThrlie530535540AlaAspProAlaTyrAspGinLeuLeuLysGlyArgLeuGlyAlaLeu545550555560MetAspAspThrGlyAlaProValArgAlaLeuMetAspGluLysSer565570575AlaAlaGluAlaTyrAlaAlaValArgLysAlaSerProGluGluLeu580585590ArgPheLeuArgValGlyPheGluSerLeuLeuLyslieAsnAspTrp595600605MetArgAspTyrArgValGluLeuAlaGlu610615<210>5<211>135<212>PRT<213>Sfr^o考ces/awre礎(chǔ)ATCC31255<400>1ValAspArgAlaSerValGinGinLeuMetGluHisPheLeuAlaAla151015TyrAsnGluGlyAspProArgHisLeuAspHisCysLeuHisProGlu202530TyrArgHisProAsnProAlaValGluArgGlylieGluGlyMetArg354045AlaAla50LeuAsp65PheSerArgArgLeuPheGinLeu130<210><211><212><213><400>MetHis1AlaGluArgThrGlyTyr50ThrLeu65AlaSerlieArgArgTrpAlaSerThrValGlu55GluAspLeuValValGluGly70GlyArgGinValGlyPro85ValGlyLeulieAspLeuSerLeuThr60ValAlaArgPheSer100AlaGinHisTrpAsp115GlyAlaLeuProAsp135Glu120AspLysAla75lieLeuGlylieProAla90AspliePheLeulieGlu105110MetAspLeuLeuGlyLeuHisArg125MetThr80SerGly95AspGly6428PRTSfre/f歸戸51/aATCC31255LeuLeulieThrArgProAlaArgGinArgTyrVal510AlaAlaValPheArgGluArgAlaAlaAspMetAlaAlaValPheArg2025AlaPheProPheArgAsnlieAspGluLeu15HisAspAlaAlaPheProPheArg3540LeuAlaLeuThrValProArgGluLeuGlyGlyLeuGlyGly55AlaAspMetAlaValAlaGinGluGluLeu45GlyGly60LeuAlaGlu30ArgAlaAlaAlaAlaValAlaGinGluArg7075ThrAlaLeuAlaValAsnMetHisLeuSerMetAlaGlyCys80GlyGin4985MetAlaArgAlaTrpArgThrThr100lieValGluGlyLeuThrGlu115GluProGlyHisAla130ValGluGlyGlyTyr145SerAlaValMet90GlyAspGluArgAla105VallieLeuMetThr165ProAspGlyProHisValLeuVal180ValLeuGluGlyArg120LeuValArgThrThrGly135LeuValThrGlyAspLys150155HisMetThrSerMetAla95ArgLysThr110AlaThrAlaMet170ArglieProAlaGlyValArg195SerGluAsnPhe185TrpAsnThrSerGly125AlaLysAlaArgLys140ThrPheGlyThrGly160GluTyrArgGluHis175SerProThr200LeuArgAspValThrArgSerGluAsnlieGlu210215AsnValLeuThrSerTyrProValGlySer225230GinThrValTrpGlyTrpAlaMetProValTrpGly245GlyValAlaValGlyAlaPheGlu260ArgGlyTrpGluAspArgProTyr275280LeulieGluThrSerArgAlaLeuMet220LeuAspGlySer235PheAlaAlaValAsp190GlyLeuArgAla205ValProGluGluThr250CysValArgAspThr265lieArgAlaGluGluGluLeulieGlu290GluValMetGlyAsn305MetAlaArgAlaValThrSer295GlyLeuTrpAsnAla310LeuThrLysAsnVallieGlu300LeuThrlieGin315GlyThrAsnAsnLeuLeu240TyrLeu255ValArgSer270lieAlaAlaCys285ArgThrAlaGlyGinGly320AlaVal50325GlulieValAspArgVallieArg340ValTyrGluArgProGlySer355MetHisProTyrSerAsn370ThrLeuGlyGluGlu385LeuAlaGluProAla360AlaAspAlaCysAspLeu375380ThrAlaProValHisProPro390395ProGluAlaGluAlaGluVal330335lieGlyThrProAlaLeuArg350AlaGlyThrlie345HisArgAspValLysAlaSerAsp420Ala405ThrAlaValArgPro425Arg365lieAlaAlaThrAla410AlaAlaSerArgGluLeuAspArg400AlaGlyAla415<210>7<211>311<212>PRT<213>Sfre/^owyces/"wre礎(chǔ)'ATCC31255<400>1MetSerSerLeulieGluThrAlaHisTrplieAlaCysAlaArgAla151015AlaGluSerLeuArgProAspGlyArgLeuPheGluAspProLeuAla202530ValGluPheValArgArgThrAspProAspLeuTyrAspArgLeuArg354045ThrGluProSerSerArgPheAspValLeuAlaValArgThrAlaPhe505560PheAspAspArgLeulieArgAlaValThrGlyGlyProAlaArgGin65ValValVal707580AlaGlyMetAspGlyArgAlaPheArgLeuProLeuAlaAlaGlyMetAspGly8590TrpProProGlyValThrLeuTyrGluValAspLeuProGluGlyValGluGluLys115ArgGly100SerAlaPheLeuLeu95GlyGluValAspLeuProGlu105110ArgSerGlyLeuValAlaAspArgSerArgSerGlyLeuVal120125ValProValAlaAlaAspLeuThrGlyAspTrpProAlaAla135AlaGluAlaGlyPheArgProGlyLeuProThrValTrpGly140ProCysArg130AlaLeuAlaGluAlaGlyPheArgProGlyLeu145150155ValGluGlyValLeuTyrTyrLeuThrGluAlaGinValAsp165170ValAspArgValThrGluLeuSerAlaAlaGlySerValAla175CysLeu160ValLeuGluGinVal195GinValThrGluLeuSerAlaAlaGlySerValLeu180185190AsnThrAspLeuTyrArgAlaProTrpMetArgAspTrp200205MetArgAspAlaGlyArgProTrpArgSerGlyValAlaAlaGlyArgProTrpArg215220GluAlaTrpLeuAlaGlyArgGlyTrpArgAlaGluValLeuArg210GluProGluAlaTrpLeu225230GluProCysAspLeuAspLeu245ProProArgGlyThrProGlyTrp235GluAlaAlaGlyArgMetValProGly260ThrGly250ArgThrTrpLeuAlaAla265GluLeuValThrGlyProAlaVallieAspSerValProAspSerVal280285ValSerAspSerValSerGluSerAlaVal275SerValProAspProAspSerValProAspSer290295SerAlaSerProThrValAlaVal300Val270AspArg240ArgThr255AlaAla52<210><211><212><213><400>MetHis8213PRTSfre/^o附y(tǒng)ces/a訓(xùn)礎(chǔ)ATCC31255lieAlaSerCysSerlieProAsnAspSerAlaLeuAlaArg1PheArgAsplieProMetLeu65ProAsp50TrpAsp35ArgSer20His5GlyGluHisThrArgArglieProSerLeulieArgValAsnThrMetGluArgLeuLeuLeuValLys145lieAla130AlaGly115AspGly100AspArg85ProGin70SerThr55GlyAsp40ProHis25Ala10AlaGluAlaAlaArgGluLeuAspArgValValMetLysArgGluTyrGluValMetAspLysGlyLysAlaGlyAspAlaLeuHisTrpSer165Tyr150Leu■Tyr135TrpLeu120liePhe105LeuGin90GluAlaPhe60ArgPro75ProLeuAla45TrpGlu30Arg15ValAlaLeuThrLeuValAspPheGlyValProArgAlaSerGluGluHisArgGlyValAlaGinPheArgArgGlyMet170Met125ValLeuArg140ArgHis155SerArgAlaArg110SerVal95GluLys80GlyValPhelieThrArgAsnAsnGinThrLeuGly175Asp160lie53AspAspAlaAlaAlaThr210<210><211><212><213><400>MetSer1AspValAspGlyThrCys50<210><211><212><213><400>ValVal1GluProAlaAlaThrGlyThrProThrAlaProAlaAlaPro180185190ProAlaAlaProProAlaSerAlaAlaProAlaValGlyGlu195200205GluGlyArg958PRT浙e/^譜jces/做re"衍ATCC31255AsnAlaAlaLeuGlulieGlyValGluGlyLeuThrGlyLeu51015AspThrLeuGlulieSerAspTyrMetAspGluThrLeuLeu202530GluAspLeuThrValThrMetlieAlaSerAlaSerCysThr354045lieCysThrCysSerCysSerSer5510197PRT5!f邵fo附y(tǒng)ce51/owe""!'ATCC31255ValGlyLeuSerTyrLeuGlyAlaAlaValAlaPheArgAla51015AlaAlaGlyPheProAlaThrValAlaGlylieValMetSerGlyTyr2025ValSerProAspThrLeuAsnArgTrpLeuAlaGlyPheSerPheLeuValSerProAspThr3540ThrArgMetAlaSerHisGinProAlaAla5055LeuHisGlyProThrTrpGluAsnLeuLeu6570LeuSerAspGlyCysLeuArgLeuProCysLeuArgLeuProGly8590LeuProValProValGluLeuValAsnGlySerLeuMetSer30LeuAla45GinArgHisPheAlaGlu60ThrAlaThrPheAla75ValAlaAspLeuPro100lielieAlaGlyAlaGlyHisAla115GlyAla120ValAsn105AlaGlu95AsnGlu80HisGluLeuGin110ArgAsnAlalieGlyProAlaAlaAlaAlaGlyAlaAspValAlaVal125ProArgValPheProMetAlaGlyAlaGlyHisVal130135AlaAlaThrValGluA印PheAlaAspArgValThrAlaGlyArg145150AlaPheHisGluArgArgArgAlaAlaAlaValProPro180AlaAlaThrHis195Arg165GluGlyAspAla140ValThr155ArgAlaGlu170AlaGlyThrGlyThrGluPro185AlaAlaGlu175ValAlaArg190Thr160ProLys<210>11<211>933<212>PRT<213>Sfreptomyces/fl訓(xùn)油7ATCC31255<400>MetAsp1CysSerAlaArgLeuAla50ThrAsp65HisArgAlaTyrAlaGlyArgArg130LeuAsn145LeuArgAspArgGluArgLeuLys210LeuPro225AlaProLeuProProAlaSerSer290HisGlu305ValValArgGluSerGinVal35ProArgAlaGlyPro115AspAlaAlaTyrMet195ProLysGlyArgPhe275LysProThrLeuProPro20CysArgAspAlaThr100LeuAspGluGlyAla180ThrSerArgThrGin260ArgGluAlaAlaArgGlu5AlaAspValGluAsp85SerAlaLeuArgAla165AlaGluProAlaAsp245LeuHisGlyAlaVal325GlulieAspGluAlaArg70ValAlaProAlaLys150GinAlaSerPheThr230GluValValValLys310LeuArgThrlieLeuGlu55ArgProAspGlyAla135ValLeuValThrGly215AspAlaSerAlaVal295GluAlaCysTyrlieArg40GluThrAlaGlyAla120ArgLeuSerAlaVal200GinAlaAlaTrpVal280PheArgLeuAlaAlaAla25AspLeuValAlaPro105AlaLeuAlaAlaGlu185LeuLeuThrGluVal265LeuLeuPheAlaAlaGly10GinValThrLeuAsp90GlyAlaGluSerAsp170GlyHisValProArg250GluArgLeuValAla330LeuTyrLeuArgGluAla75LeuAlaLeuGlyAla155GlyThrLeuHisVal235ProArgArgArgArg315AspProGlySerArgVal60LeuAspAlaArgAla140AlaLeuAlaLeuThr220lieValSerAlaGly300ValAlaGlyValSerThr45ValArgValGlyAla125TyrLeuLeuProThr205ArgAlaSerLeuPro285ArgProAlaThrValArg30TrpProArgLeuThr110LeuGluAspArgGly190ArgProProValAla270ValAspProValAspArg15GluGinILeuArgAsn95AlaAlaGluProAsn175LysSerValValCys255GlyValGlyGlyPro335ProValAlaAspLeuLeu80LeuValGluGlyAla160MetArgThrLeuThr240ArgArgGlyThrVal320GluAla56340345350AlaAlaGlyAlaAlaLeuAspAlaLeulieGlyGinGlyValLeuAla355360365ArgAspLeuGlyValGlyAspGinHisProHisProLeuAlaArgLeu370375380AlaAlaLeuLeuProProAspCysAspAlaGlyLeuArgAlaAlaVal385390395400SerGlyLeuAlaGlyAlaGluSerAlaPheGluThrAlaGlyLeuGlu405410415ArgArgGluGluLeuValSerArglieLysGluHisValAlaArgVal420425430AlaGluLeuThrGlyValAlaThrProProlieAspValAlaArgThr435440445LeuValTyrGluAspAlaValValAlaValProArgAlaGluAlaPro450455460GlyArgTrpGlyArgHisLeuProAlaLeuAlaProTrpHisArgLeu465470475480LeuProLeuPheAspAspAlaAlaHisValArgAlalieValAlaAsp485490495ValValArgThrHisPheGlyProGlyProHisArgLeuLeuProLeu500505510PheAlaAlaLeuSerSerAlaLysValGinAlaValLeuThrGluArg515520525LeuThrAspValSerAlaProLeuProThrArgLeuArgThrLeuGin530535540AspAlaValPheAlaLeuAlaAspProAspGlyGlyAlaAspThrVal545550555560ThrValAspProGluArgAlaAlaArgLeuAlaAspAspLeuProSer565570575TrplieProArgTrpSerArgAlaSerTrpHisValGinHisHisAla580585590AlaProAspGlyGlyLeuLeuValValAsnGlyGlySerlieGlyPhe595600605GlyArgAlalieSerArgPheLeuProAlaTyrAlaAlaAlaGlyAsp610615620AlaGlyPheGlyAspLeuValThrAlaGlulieAlaArgAspAspAsp625630635640ProSerAlaProLeuThrAspLeuAlaAlaValLeuGlylieAsnAla645650655AsnValHisSerProLeuLeuGlyArgHisLeuArgTyrProCysAla660665670ThrProGluGluTrpGlyGlyAsnThrGlyValSerLeuGluAspCys675680685TrpValValTyrAspAlaAspThrGlyArgLeuLeuProArgLeuGly690LysAsp705GluLeuSerLeuAlaGlyAlaGly770ArgVal785ProAspPheAlaValPheArgGlu850SerAla865ValThrPheGinGlyLeuThrGlu930GlyAlaAlaAla755GlyArgAlaGluTyr835GluGluAlaGluArg915GluProProAsn740GlyGlySerArgVal820ArgGinArgTrpCys900GluGluValArg725LeuAlaAlaGlyLeu805ArgGlyArgLysGin885LeuArgGly695LeuArg710PheTyrAlaTrpAspGlyGlySer775LeuValGinPheTrpAsp745GlyAla760GlyThrSer790ProArgPheThrPro870ArgProPheLeuGlyTrpThrTrp855ThrAlaAlaThrValLeuGinLeu840AlaPheLeuValGlu920LeuAlaGlu825ProArgLeuArgGly905GluMetLeu730ArgGlyAspAlaGly810SerAspThrAspArg890GluPheLeu715AsnValAlaSerArg795AlaLeuProLeuPhe875CysAsplie12346PRTS/reptow戸s/awe加'/ATCC31255700AsnPhePhePheAspGinAspGly765ValArg780ArgThrThrGlyGlyLeuLeuVal845AlaArg860ThrSerGinGlyGluGlyValGlu925<210><211><212><213><權(quán)>1ValSerThrAlaProAlaHisProTrpLeuSer1510LeuSerArg750GlyHisTrpValPro830ProPheValGlyAla910ThrLeuSer735ArgAlaTyrLysAla815AspMetProThrLeu895AlaThrAsn720GlyGluGlyProVal800AlaGinSerThrSer880ThrGlyGluCysHisValPheHis1558HisGlyAspThrAspProLeulieAlaGluCysLeu2025AspAspLeuArgArgAlaGlyArgValGluArgHis3540HisTrpGluArgGlyProHisValArgLeuArgMet505560GluHisHisAspAlaValArgAlaGluThrGluArg657075TyrLeuGlyArgArgProGlyProValAspThrAsp8590ArgAspAlaLeuArgArgLeuSerGluLeuGluHis100105AspValAlaValArgThrProGluProProAsnSer115120AspTyrGinProGluLeuAlaLysTyrGlyGlyPro130135140lieAlaGluAspValPheHisAlaGlySerValLeu145150155LeuArgLeuValAspAsnAspArgAlaArgMetGly165170LeuLeuLeuLeuAlaSerArgAlaLeuGlyPheGly180185AlaValPheLeuArgThrTyrGinGluArgTrpSer195200AspProArgValLeuAlaSerTrpAspArgGinTyr210215220GluGinTyrAlaAlaLeuLeuAspGlyLeuAspAla225230235GlyLysGlyLeuGlyAlaTyrTrpGluArgThrLeu245250GluArgLeuArgProLeuValGlyThrGlyArgVal260265ThrAspProAlaAlaProProValValAlaLeuAla275280GinPheLeuHisThrThrAsnAsnArgMetAsnVal290295300GluCysPheValAlaTyrLeuAlaSerArgAlaAla305310315AlaAlaArgSerGlyThrAlaProThrAlaAspPro325330HisGluArgAspGluArgHisValHisGin340345<210>13LeuProLeu30PheTyrLeu45ArgValProArgValLeuProAlaArg95GlyAspAla110LeuArgTrp125GluGlyValThrGlyGinlieAlaLeu175ProAspGlu190GlyTyrVal205GinLeuGinGlyAspProValGinAla255TrpProAla270AlaLeuVal285ArgProGinGluAspGinAlaGinThr335ValArgAlaAsp80LeuSerlieAlaVal160GinArgThrArgVal240LeuGluSerGlyArg320Ala59<211><212><213><糊>MetSer1ValGinSerValHisGly50SerVal65ThrlieHisAspTrpThrGlyAsn130ThrGly145GlyTyrMetLeuArgTyrValGlu210LeuAla225GinLysLysAlaArgProGlylie368PRTThrSerGluGinLysAspLeuThrValSerValProTrpSer51015GluAspLeuLeuLeuAspValAlaAlaProLeuLeuAspGlu202530GluLeuGlyGluThrAspSerTrpPheTyrLeuArgGluAsn354045GlyArgProPheLeuArgLeuArgPheAlaSerArgSerPro5560GluArgArgLeuLysSerArglie乙euAlaHisValGlyPro707580AspAlaGlyAspValPheThrTyrGinProTyrAsnHisGlu859095TrpLeuGlyGlyThrAlaGlyLeuGlyLeuAlaGluAsnPhe100105110GluThrThrProLeuAlaLeuAspThrLeuArgAlaThrArg115120125ArgAlaLeuArgLeuAlaValAlaPheAspPheLeuValCys135140ValMetLeuAlaProHisLeuProProSerlieAlaLysPhe150155160LysAlaGlyTyrLeuSerTyrLeuAlaThrPheGluGlyTyr165170175LeulieArgAspProGluGlyThrArgAlaLysHisAlaGin180185190GluLysAsnArgGluLeuLeuArgProArgLeuArgThrLeu195200205GinMetSerGluProAspGlyGluLeuThrAspValProGlu215220ArgGluTrpLeuValArgLeuArgAspTyrValProAlaLeu230235240GlyPheAspGluGlyArgPheTyrLeuTyrAlaThrProArg245250255GluThrAlaLysLeuThrProSerProAspGlyLeuTyrArg260265270AspValGluTrpLeuSerAspLeuProGluProProValAla275280285HisArgAlalieAlaAspAsnThrTyrTyrGinGlyMetlie60290ArgGlu305AsnTrpLeuPheAlaAla<210><211><212><213><400>MetThrAlaProAspGlyAspGin50TyrVal65AlaAlaLeuGlyValArg295300AspArgArgPheLeuAlaSerArgLeuAlaGinAlaTyrThr310315320HisLeuTyrArgLeuGlyPheLeuLeuAlaAspArgTyrThr325330335TyrLeulieAlaArgAlaPheGluGluGluTyrAspLeuAsp340345350AlaLeulieArgSerValArgProGluAlaGluValAlaGly35536036514589PRTS/re/^o/wyces/owe油7ATCC31255AlaProGlyThrAlaGluAlaThrAlaProValGluAlaAla51015AlaGluAlaAlaGlyThrGlyProPheAspAlaAlaGlyVal202530PheValArgArgLeuArgGlyAspPheAspGluValHisPro354045TrpGinlieAspTrpArgSerlieProSerProPheArgTrp5560AspLeuProArgArgAspLeuProAlaLeuSerArgAlaAla707580GlyGlyProAlaAlaAlaAlaAlaPheAlaAlaAlaGlyPro859095ArgLeuGlyMetLeuLeuGinGlyAlaTyrGlyLeuThrGly100105110TrpTyrProGluGlylieAlaLysSerSerProAlaGluPro115120125ThrProValHisArgAsnProHisTyrGinLeuArgArgAlaValPro130135140Ser145ThrGlyGlyValThrAlaGlyThrValTyrHisGlyArgValSer225AlaVal210TyrPro195ThrTyr180ProGlu165AspTyr150GlyProAlaGluCysThrGluGluAlaValArgAlaGluLeuGinProLeuArgLeuGlyLeuAlaAlaValAspGluGluAlaProTrp305MetAla290LeuArg260ValPro275AlaSerArg245ArgAla230SerTrp215MetThr200LysHis185ProGlu170AlaTyr155AspValValThrAlaGlyLeuAspLeuAlaValProGluGlyAsnTyrPheAspThrGlyTrpGluLeulieAlaAspAlaValLeuAlaAlaGlyGluSerHisAlaAlaLeuProLeuProProVal340ArgPro355Cys325ValVal310LeuGlu295ProAsp280ThrLeu265ValArg250LeuAla235CysLys220ValGly205TyrLeu190ValAla175SerGly160GlyAlaArglieGlyAspPheValGlyGinArgValThrGlyLeuAspSerLeuGlyAlaProProProGlyProAlaAlaGlyLeuProAspAlaGlyArgPro360Leu345ValArg330AlaAla315ProVal300ThrAsp285GlyPro270ProPhe255GluPhe240GlyHisValGlyArgProArgGlyThrArgGlyArgProGlyGinAlaSerValAlaAsp365Asp350ThrVal335ValArg320AspGluTrpSerArgSer370VslLeu385ValThrAlaLeuGlyGluGinPheArgGlyThrProLeuProAlaGlu375380AlaAspTrpGlyGluAlaGlyArgHisAlaGlyAla450AlaGly465MetAsnArg435TyrPro420CysGin405GlyLeu390AlaThrThrValLeuGlyArgAlaThrGlyThrLeuLeulieLeuProHisAspPheGlyValGlyAspProHisLeuLeuAspGly530AspAsp545ValProAla515ArgAlaVal500GlyGin485GlyPro470AlaPro455GluAla440AspVal425ArgAsp410ThrAla395Thr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Asn320ValArgGlyArg<210>20<211>936<212>PRT<213>iSfre/^o/wyces/flwre"riz'ATCC31255<400>1MetAlaValThrSerGlyProValHisGluThrAlaProValAlaAla151015AspHisValValProGlyProValValValArgArgArgCysArgVal202530ProAlaArg65GluProLeuHisThr145LeuProLeuThrPro225ArgLeuGlyValLeu305ProProAlaThrGlyAla50LeuArgLeuAlaGlu130ValSerAlaMetThr210SerGinTrpThrLeu290ArgAspArgThrGlu370Ser35AlaAlaArgProGlu115LeuAspValAlaGin195HisArgTrpLeuArg275SerGluGlyLeuAla355AlaValTrpAspAspAla100GluAspAlaProAla180ArgValSerlieThr260ValAlaProLeuPro340ProArgLeuAlaGinLeu85AspTyrLeuLeuGly165LysAlaLeuHisAla245AlaThrProValAlaTyrVal70LeuSerThrAlaLeu150LeuLysAlaPheVal230AlaAsnTrpCysArg310AlaArg325SerHisAlaProGinAlaAspAla55GlyArgGlyArgLeu135ArgLeuThrSerGlu215ArgGlyProLeuGly295LeuLeuGlyAspArg375Leu40GluGluValAlaAla120AlaAspAlaArgLys200AsnlieGlyThrLeu280AlaAspValArgLeu360GinGlyAspSerArgVal105LeuAspProAlaArg185ProThrAspHisAla265SerAlaThrAlaArg345AspLeuSerAlaValArg90LeuThrLeuHisGly170ArgThrAlaArgArg250AlaArgLeuValArg330ThrLeuHisAspValPro75AlaGlyAlaAlaLeu155ProAlaProProAla235SerValAlaAlaAla315GlyLeuAspAspArgGin60ThrAlaGlyArgArg140AlaValLeuPheAla220ValLeuArgAspGly300ProLeuHisLeuAla380Val45ArgValPheProAsp125ThrArgGlyThrAla205AlalieArgGinGly285ThrGlyLeuAlaAsp365LeuTrpThrArgAsnAla110ThrSerSerAlaLeu190GlySerGluProAsp270ThrLeuGlyGluAla350ProGlyGlyGlyGinSer95AlaAsnGlyValLeu175LeuPheAspTrpGlu255AlaGluAlaThrlie335AlaAspValAlaThrGlu80ArgAlaArgAlaGlu160AspArgAlaLeuVal240ArgSerArgArgLeu320GlyAspAlaLeu73Arg385ArglieValAspVal465AlaValAspAlaAsn545ValTrpThrAlaHis625ValArgSerGlyAla705LeuAspArgGlyValGly450GinThrLeuAlaLeu530ProAlaProGlyArg610GluAsnAlaSerGlu690GlyPheAlaAspValGly435ThrArgAlaThrLeu515ArgGlyAlaValTyr595LeuHisGluLeuCys675GlyAlaProGluAlaArg420ValGlyValLeuAla500LeuHisAlaAspGin580GlyAlaGlyArgGlu660AlaAspGlyValAspLys405AlaMetProValAla485TyrAlaArgProLeu565LeuArgValValPro645CysValAspThrPro725Ala390AspGlyGlyPhePro470PheAlaAspLeuGlu550ProAlaPheLeuPro630LeuAlaArgAlaGly710HisLeuGlyGlyValGly455LeuArgTrpCysPhe535AlaAspGlyAlaArg615ValLeuProValPro695ThrAsnAlalieGlyArg440ProLeuArgPheGin520AspAlaAlaAspAla600AspAspLeuGluAla680GlyGlyAlaAlaAlaAsp425AlaGluGlyArgLeu505AlaGlyCysValArg585ArgTrplieProThr665GlyThrThrThrAspAla410ArgAspValPhePhe490AspGluLeuAspMet570ValValAlaAlaAla650ProGlyGlyGlyLeu730Pro395ValSerAspLeuGlu475GlyGluGluArgPro555AspValAlaAlaAla635AlaGlyArgProArg715ProAlaAlaAlaAspAla460LeuThrGlyLeuAla540GlyLeuValAlaThr620SerLeuAlaLeuGly700PheThrHisThrValVal445AspProAspArgAla525AlaLeuSerAsnGly605AlaLeuGlyArgLeu685ProAlaValAlaAlaGly430ProLeuPheProArg510ThrAlaLeuCysGly590LeuAlaGlyTyrVal670LeuGlyGlyAlaGlyLeu415GluArgGlyGinVal495GluValAspAspAla575ValAspThrAlaPro655AspAla400GlyAspAlaArgLeu480ProSerLeuArgAla560AlaGlyProProThr640GlyLeuPheGlyAlaGlyArgPro720ProGly735LeuAlaAlaArg785ProTyrGlyArgArg865LeuAspGluGlyTyrLeuVal770ArgGlyArgThrArg850LysAlaProTyrCys930ArgTrp740TrpAsp755ArgHisThrTrpAlaAlaLeuSerArgHisValAspTyrProArg775LysValPro790GlyAlaGly805AlaArgThrTrpCys820ThrLeuProAspPro835ProValProLeuProAlaAspAlaAla855TyrValAsp870ProGlyAsp885GlyThrThrAlaGly900ValLeuGluThrSer915AlaAlaAlaThrArg935LeuAla760LeuAlaGlyGlyTrp840ArgPheThrAlaGin920ProGly745LeuThrAlaThrVal825AspSerSerLeuAla905ProProAlaLeuAlaAla810ProValThrGinThr890AlaProGlySerAlaProGlyArg780LeuPro795ArgGluArgArgValArgSerGly860ProLeu875PheThrProGlyArgAlaGlyArg765LeuAlaAlaSerGly845GlyCysGluSerArg925Ala750AspValGluLeuPhe830LeuAlaTrpProThrLeuProGluLeuSerAlaLeu800AlaTrp815LeuArgAlaAspAlaLeuProAla880LeuPro895ValThrArg910AlaThrGlu<210>21<211>599<212>PRT<213>S/rep/omyces/ai/rew"'!'ATCC31255<400>1MetLeuArgLysGlyThrValAlaLeulieAsnProAsnGinlieHis151015ProProlieAlaProTyrAla.LeuAspValLeuThrThrAlaLeuGlu202530AlaSerGlyPheGluAlaHisValLeuAspLeuThrPheHisLeuAsp354045AspTrpArgGinThrLeuArgAspTyrPheArgAlaGluArgProLeu50556075LeuValGlyValThrCysArgAsnThrAspThrValTyrAlaLeuGlu65707580GinArgProPheValAspGlyTyrLysAlaVallieAspGluValArg859095ArgLeuThrAlaAlaProValValAlaGlyGlyValGlyPheSerThr100105110MetProPheAlaLeuValAspTyrPheGlylieGluTyrGlyValLys115120125GlyProGlyGluLyslielieCysAspLeuAlaArgAlaLeuAlaGlu130135140GlyArgSerAlaAspArglieHislieProGlyLeuLeuValAsnArg145150155160GlyProGlyAsnValThrArgValAlaProProAlaLeuAspProArg165170175AlaAlaProAlaProSerSerSerProSerProSerProAlaProSer180185190SerSerSerAlaProValProValProLeuSerPheAlaAlaValGly195200205His.HisGluSerArgAlaTrpGinAlaGluThrGluLeuProTyrThr210215220ArgArgSerGlyGluProTyrLysValAspAsnLeuArgTyrTyrArg225230235240GluGlyGlyLeuGlySerlieLeuThrLysAsnGlyCysValTyrLys245250255CysSerPheCysValGluProAspAlaLysGlyThrGinPheAlaArg260265270ArgGlylieThrAlaValValAspGluMetGluAlaLeuThrAlaGin275280285GlylieHisAspLeuHisThrThrAspSerGluPheAsnLeuSerlie290295300AlaHisSerLysAsnLeuLeuArgGlulieValArgArgArgAspHis305310315320AspAlaThrSerProLeuArgAspLeuArgLeuTrpValTyrCysGin325330335ProSerProPheAspGluGluPheAlaGluLeuLeuAlaAlaAlaGly340345350CysAlaGlyValAsnlieGlyAlaAspHisThrArgProGluMetLeu355360365AspGlyTrpLysValThrAlaLysGlyThrArgTyrTyrAspPheAla370375380AspThrGluArgLeuValGinLeuCysHisArgAsnGlyMetLeuThr385390395400MetValGluAlaLeuPheGlyMetProGlyGluThrLeuGluThrMet40541041576ArgAspCysValAspArgMetMetGluLeuAspAlaThrValThrGly420425430PheSerLeuGlyLeuArgLeuLeuProTyrMetGlyLeuAlaLysSer435440445LeuAlaGluGinCysAspGlyValArgThrValArgGlyLeuGinSer450455柳AsnAsnAlaSerGlyProlieValLeuLysGinLeuHisGinCysAsp465470475480GlyProlieGluTyrGluArgGinPheMetPheAspGluSerGlyAsp485490495PheArgLeuValCysTyrPheSerProAspLeuProGluAlaProGly500505510ThrAlaAspSerProAspGlylieTrpArgAlaSerValAspPheLeu515520525TrpAspArglieProLysSerGluGinTyrArgValMetLeuProThr530535540LeuSerGlySerSerGluAsnAspAsnAsnTyrAlaAspAsnProPhe545550555560LeuThrSerLeuAsnArgLysGlyTyrThrGlyAlaPheTrpAlaHis565570575TrpArgAspArgGluAlalieMetSerGlyAlaThrLeuProLeuGly580585590GluLeuAlaGluAlaValArg595<210>22<211>272<212>PRT<213>Sfre;7to附y(tǒng)ces/aww油7ATCC31255<■>1ValThrAlaProAlaLeuProLeuAlaGlyGinValAlaLeuValThr151015GlyAlaGlyArgGlylieGlyArgMetValAlaValAlaLeuAlaGlu202530AlaGlyMetThrValGlyLeuValGlyArgAsnArgArgLeuLeuAsp354045ValThrAlaArgAlaCysAlaAlaAlaArgGluAlaAlaSerGlyAsp505560GlySerGlyAspGlySerGlyGlyGlyProGluAlaGlyValAlaVal7765AlaValAlaValGlyLeuArgTrp130CysArg145ValAsnAlsTyrAlaGluGlyMet210ArgAsp225ValAlaGlyAla<210><211><212><213><權(quán)>MetArg1LeuAlaTyrGluGluAsn50AlaArgVal115TrpAlaValCysPro195ValAspHisAspAspAla100AspGluValAsnGly180LeuArgTrpGlyAsp260Val85AlaGinValLeuSer165SerArgThrThrAsp245Phe70ArgLeuGlyPhePro150ArgLysAspAspAsp230LeuAspAlaGlyGluGlu135GluLeuAlaArgMet215ProAspAlaProProLeu120ThrMetAlaThrGly200ThrAlaProLeuAlaVal105ProAsnThrValLeu185ValLeuLeuLeuLeu265Glu90AspPheValArgGin170LeuHisSerPheAla250Ser75lieLeuTrpArgArgGluAlaAla95AsnLeuValAsn110GluAlaAsp125GlyThrVal140GlySerGlyArg155GlyAspProArgAlaLeuMetLeuAspAla190PheAsplie205AlaValCys220AlaAlalieLeu235GlyArgPheLeuGluArgArgLeu270ProAsnArgTyr175ValSerAlaLeuHis255Ala80GluAlaGluThrVal160SerAlaProGlyArg240ValVal23247PRTS/repfowycey/a訓(xùn)""7ATCC31255LysAsnlieArgLeuLeuGlyValSerValAlaAlaAlaGly51015ValGlyAlaLeuSerGlyCysGlyMetValValAsnGluMet202530AspSerSerSerLeuProGluLyslieThrSerValArgLeu354045GlySerGlyArgValThrValAsnGlyThrLysAlaGlyGly5560ProLeuSerLeuArgArgThrValSer6570GlyProThrHisArgValGluAsnGly85GlyAspAsplieAspPheCysSerVal100105AlaGlylieProValSerGlyLysVal115120ThrLysValGlyAlaValGinValThr130135MetThrGlyValAsnGlyAlaValAsp145150lieThrGlySerAspValLysGlyGly165AsnGlyGlylieGluLeuAsnLeuAla180185ThrThrThrAsnGlyThrlieAlaLeu195200ArglieAspAlaGinThrAlaAsnGly210215AspAspProAlaGlyProHisArgLeu225230SerlieSerValLysAsnSer245TyrGinAspLysLysProAsp7580ValLeulieLeuGlyAspCys9095GlyTyrSerValAspValPro110ThrAsnGlyThrValLysLeu125ThrThrSerGlyArglieGlu140ValArgThrThrAsnGlyArg155160GlylieLysAlaArgThrSer170175AlaAspLeuAspValGinAla190LysValProGlyThrGlyTyr205AspLysArglieGlyLeuThr220ThrLeuLysThrSerAsnGly23524079權(quán)利要求1.一種抗革蘭氏陽性菌活性的抗生素—硫鏈絲菌素的生物合成基因簇，其特征在于編碼硫鏈絲菌素生物合成所涉及的22個(gè)基因，具體為1)1個(gè)氨基轉(zhuǎn)移酶基因，即tsrAtsrA位于基因簇核苷酸序列第3774-2686個(gè)堿基處，長(zhǎng)度為1089個(gè)堿基對(duì)，氨基轉(zhuǎn)移酶，362個(gè)氨基酸；2)脂肪酶基因，即tsrBtsrB位于基因簇核苷酸序列第4626-3796個(gè)堿基處，長(zhǎng)度為831個(gè)堿基對(duì)，編碼脂肪酶，276個(gè)氨基酸；3)天門冬酰胺合成酶基因，即tsrCtsrC位于基因簇核苷酸序列第6541-4685個(gè)堿基處，長(zhǎng)度為1857個(gè)堿基對(duì)，編碼天門冬酰胺合成酶，618個(gè)氨基酸；4)酯環(huán)化酶基因，即基因tsrDtsrD位于基因簇核苷酸序列第個(gè)7088-6681堿基處，長(zhǎng)度為408個(gè)堿基對(duì)，編碼酯環(huán)化酶，135個(gè)氨基酸；5)?；?CoA脫氫酶基因，即tsrEtsrE位于基因簇核苷酸序列第8449-7163個(gè)堿基處，長(zhǎng)度為1287個(gè)堿基對(duì)，編碼酰基-CoA脫氫酶，428個(gè)氨基酸；6)甲基轉(zhuǎn)移酶基因，即tsrFtsrF位于基因簇核苷酸序列第9529-8594個(gè)堿基處，長(zhǎng)度為936個(gè)堿基對(duì)，編碼甲基基轉(zhuǎn)移酶，311個(gè)氨基酸；7)與thiostrepton側(cè)鏈生物合成相關(guān)但具體功能未知的基因，即tsrGtsrG位于基因簇核苷酸序列第10241-9600個(gè)堿基處，長(zhǎng)度為642個(gè)堿基對(duì)，編碼一個(gè)未知功能蛋白，213個(gè)氨基酸；8)前體肽基因，即tsrHtsrH位于基因簇核苷酸序列第10570-10746個(gè)堿基處，長(zhǎng)度為177個(gè)堿基對(duì)，編碼Thiostrepton前體肽，58個(gè)氨基酸；9)水解酶基因，即tsrItsrI位于基因簇核苷酸序列第11461-12054個(gè)堿基處，長(zhǎng)度為594個(gè)堿基對(duì)，編碼水解酶，197個(gè)氨基酸；10)脫水酶基因，即tsrJtsrJ位于基因簇核苷酸序列第12047-14848個(gè)堿基處，長(zhǎng)度為2802個(gè)堿基對(duì)，編碼脫水酶，933個(gè)氨基酸；11)脫水酶基因，即tsrKtsrK位于基因簇核苷酸序列第14845-15885個(gè)堿基處，長(zhǎng)度為1041個(gè)堿基對(duì)，編碼脫水酶，346個(gè)氨基酸；12)與thiostrepton大環(huán)合成相關(guān)具體功能未知基因，即tsrLtsrL位于基因簇核苷酸序列第15872-16978個(gè)堿基處，長(zhǎng)度為1107個(gè)堿基對(duì)，編碼一個(gè)具體功能未知的蛋白，368個(gè)氨基酸；13)NADH依賴的氧化酶基因，即tsrMtsrM位于基因簇核苷酸序列第16975-18744個(gè)堿基處，長(zhǎng)度為1770個(gè)堿基對(duì)，編碼NADH依賴的氧化酶，589個(gè)氨基酸；14)與thiostrepton大環(huán)合成相關(guān)具體功能未知基因，即tsrNtsrN位于基因簇核苷酸序列第18741-20579個(gè)堿基處，長(zhǎng)度為1839個(gè)堿基對(duì)，編碼一個(gè)具體功能未知的蛋白，612個(gè)氨基酸；15)與thiostrepton大環(huán)合成相關(guān)具體功能未知基因，即tsrOtsrO位于基因簇核苷酸序列第20576-22621個(gè)堿基處，長(zhǎng)度為2046個(gè)堿基對(duì)，編碼一個(gè)具體功能未知的蛋白，681個(gè)氨基酸；16)細(xì)胞色素P450氧化酶基因，即tsrPtsrN位于基因簇核苷酸序列第22599-24023個(gè)堿基處，長(zhǎng)度為1425個(gè)堿基對(duì)，編碼細(xì)胞色素P450氧化酶，474個(gè)氨基酸；17)?；?CoA合成酶，即tsrQtsrQ位于基因簇核苷酸序列第23999-25312個(gè)堿基處，長(zhǎng)度為1314個(gè)堿基對(duì)，編碼酰基-CoA合成酶，437個(gè)氨基酸；18)細(xì)胞色素P450氧化酶基因，即tsrRtsrR位于基因簇核苷酸序列第25422-26612個(gè)堿基處，長(zhǎng)度為1191個(gè)堿基對(duì)，編碼細(xì)胞色素氧化酶，396個(gè)氨基酸；19)脫水酶基因，即tsrStsrS位于基因簇核苷酸序列第26603-29413個(gè)堿基處，長(zhǎng)度為2811個(gè)堿基對(duì)，編碼脫水酶，936個(gè)氨基酸；20)鐵硫簇氧化還原酶基因，即tsrTtsrT位于基因簇核苷酸序列第29430-31229個(gè)堿基處，長(zhǎng)度為1800個(gè)堿基對(duì)，編碼一個(gè)具體功能未知的蛋白，599個(gè)氨基酸；21)短鏈脫氫酶基因，即tsrUtsrU位于基因簇核苷酸序列第31226-32044個(gè)堿基處，長(zhǎng)度為819個(gè)堿基對(duì)，編碼一個(gè)具體功能未知的蛋白，272個(gè)氨基酸；22)與thiostrepton大環(huán)合成相關(guān)具體功能未知基因，即tsrVtsrV位于基因簇核苷酸序列第32391-33113個(gè)堿基處，長(zhǎng)度為744個(gè)堿基對(duì)，編碼一個(gè)具體功能未知的蛋白，247個(gè)氨基酸。2.根據(jù)權(quán)利要求1所述的硫鏈絲菌素的生物合成基因簇的用途，其編碼蛋白用于催化合成抗生素硫鏈絲菌素及其類似物、或者催化合成含噻唑、噻唑啉、吡啶雜環(huán)或喹萘啶酸結(jié)構(gòu)單元的大環(huán)骨架。3.根據(jù)權(quán)利要求2所述的硫鏈絲菌素的生物合成基因簇的用途，其特征在于由核糖體依據(jù)所述的硫鏈絲菌素的生物合成基因簇中編碼硫鏈絲菌素前體肽的基因合成出的相應(yīng)的聚肽是硫鏈絲菌素的前體肽；該前體肽經(jīng)由后修飾酶的催化反應(yīng)生成硫鏈絲菌素。全文摘要本發(fā)明是一種由鏈霉菌產(chǎn)生的具有抗革蘭氏陽性菌抗生素-硫鏈絲菌素(Thiostrepton)的生物合成基因簇的克隆、測(cè)序、分析、功能研究及其應(yīng)用。整個(gè)基因簇共包含22個(gè)基因10個(gè)與Thiostrepton大環(huán)生物合成相關(guān)基因，8個(gè)與側(cè)鏈生物合成相關(guān)基因，還有4個(gè)基因目前功能不明。通過對(duì)上述生物合成基因的遺傳操作可阻斷硫鏈絲菌素的合成。本發(fā)明所提供的基因及其蛋白也可以用來尋找和發(fā)現(xiàn)可用于醫(yī)藥、工業(yè)或農(nóng)業(yè)的化合物或基因、蛋白。文檔編號(hào)C12N15/54GK101445803SQ20081020241公開日2009年6月3日申請(qǐng)日期2008年11月7日優(yōu)先權(quán)日2008年11月7日發(fā)明者瑩丁,文劉,廖日晶,煉段,沂虞,春雷申請(qǐng)人:中國科學(xué)院上海有機(jī)化學(xué)研究所