硫链丝菌素的生物合成基因簇的制作方法

专利名称：：硫链丝菌素的生物合成基因簇的制作方法
技术领域：
：本发明属于微生物基因资源和基因工程领域，具体涉及抗革兰氏阳性菌抗生素硫链丝菌素(Thiostrepton)的生物合成基因簇的克隆、分析、功能研究及其应用。
背景技术：
：硫链丝菌素(Thiostrepton)是硫肽类抗生素家族的一个著名成员，可由链霉菌S/r，o附戸s/a,"",ATCC31255、S/re声w拜sATCC14921、S/r印towj;c^/awa//era/;yATCC12236所产生。人们首次发现硫链丝菌素是在1954年，但是由于它结构十分复杂，直到1989年借助了X射线晶体衍射技术才最终阐明了其分子结构[J^"幼Z饥1989,^,1649]。它含有两个大环结构(分别为26员环和27员环)，4个噻唑环，l个噻唑啉，一个四取代的哌啶和一个喹萘啶酸结构，整个分子共有17个手性中心。前体标记实验表明其中噻唑、噻唑啉杂环来自于半胱氨酸，哌啶环由两个丝氨酸经酶催化后形成，喹萘啶酸由色氨酸衍生而来。硫链丝菌素具有良好的生物活性。它对革兰氏阳性菌有广谱而且强有力的抑制作用，特别是对多种耐药性的条件致病菌具有极强的杀伤作用。硫链丝菌素抑制革兰氏阳性菌的机制是因为它能与50S核糖体亚单元上的23SrRNA-Lll蛋白复合物结合，抑制延长因子的GTPase活性,从而抑制蛋白质的合成[/.她/.历0/1998,276,391-401]。近年来还发现硫链丝菌素有抗疟疾的活性，对人类疟原虫(P/oywo必ww々/">7^/附)的IC5o值达到了3.2nM[尸ra"w，1999，150,189]，Jonghee等人还在2002年报道了硫链丝菌肽的抗癌活性[PC7VW.^p/.WO2002066046，2002.]但是，由于硫链丝菌素水溶性不好，生物利用度较低，大大限制了它的医药用途，目前只作为畜用抗生素。人们希望能获得一些它的结构类似物，从中筛选出活性更好，更有利于实际使用的分子。由于硫链丝菌素极其复杂的分子结构，即使是在有机合成化学髙度发达的今天，通过化学全合成来获得产品也是一项艰巨的挑战。直到2004年才首次报道了硫链丝菌素A的化学合成[J"gewC/zem五"g/.2003Jul28;42(29):3418-24]，整个合成过程操作繁琐，产率低下。通过化学合成所获得的结构类似物即使在活性和水溶性方面的性质得到改善，也可能因为合成的复杂性而使实际生产成本过高。我们以微生物来源的硫链丝菌素为目标分子，从克隆生物合成基因簇出发，采用微生物学、分子生物学、生物化学及有机化学相结合的方法研究其生物合成，通过对其生物合成机制的研究揭示了其经由核糖体合成的生物合成机制，并揭示了包括喹萘啶酸在内的独特化学结构形成的酶学机理，在此基础上运用代谢工程的原理，合理修饰硫链丝菌素的生物合成途径，探索水溶性好、生物利用度高、活性更好、并能通过微生物发酵大量生产的新型药物。
发明内容本发明涉及一种由链霉菌5^印tow;;c^/"wew衍ATCC31255产生的具有抗革兰氏阳性菌活性的抗生素一一硫链丝菌素(Thiostrepton)的生物合成基因簇的克隆、测序、分析、功能研究及其应用。本发明中整个基因簇共包含22个基因的核苷酸序列或互补序列(序列1)，其中一个基因(TsrH)负责编码硫链丝菌素的前体肽，九个基因(加0、加M、加丄to《、加S、加Z、加/、加C、加及)负责对加/Z编码的前体肽进行后修饰，形成整个大环骨架；七个基因(加F、加丄"W、加B、加￡>、加f/、加尸)负责以色氨酸为前体，合成喹萘啶酸结构单元；一个基因负责对形成的喹萘啶酸进行活化，在其他酶的催化作用下连接到大环骨架上，并最终形成分子中的第二个大环。本发明还提供了一个编码氨基转移酶的核苷酸序列，由序列2中的氨基酸序列组成，命名为feM，其基因的核苷酸序列位于序列l中第3774-2686碱基处。,本发明还提供了一个脂肪水解酶的核苷酸序列，由序列3中的氨基酸序列组成，命名为加W，其基因的核苷酸序列位于序列l中第4626-3796碱基处。本发明还提供了一个编码天门冬酰胺合成酶的核苷酸序列，由序列4中的氨基酸序列组成，命名为&rC，其基因的核苷酸序列位于序列l中第6541-4685碱基处。本发明还提供了一个编码酯环化酶的核苷酸序列，由序列5中的氨基酸序列组成，命名为"rZ)，其基因的核苷酸序列位于序列l中第7088-6681碱基处。本发明还提供了一个编码酰基-CoA脱水酶的核苷酸序列，由序列6中的氨基酸序列组成，命名为to^，其基因的核苷酸序列位于序列1中第8449-7163碱基处。本发明还提供了一个编码甲基转移酶的核苷酸序列，由序列7中的氨基酸序列组成，命名为tsrF，其基因的核苷酸序列位于序列l中第9529-8594碱基处。本发明还提供了一个编码未知蛋白的核苷酸序列，由序列8中的氨基酸序列组成，命名为"K，其基因的核苷酸序列位于序列l中第10241-9600碱基处。本发明还提供了一个编码硫链丝菌素前体肽的核苷酸序列，由序列9中的氨基酸序列组成，命名为"rH，其基因的核苷酸序列位于序列1中第10570-10746碱基处。本发明还提供了一个编码水解酶的核苷酸序列，由序列10中的氨基酸序列组成，命名为&r/，其基因的核苷酸序列位于序列l中第11461-12054碱基处。本发明还提供了一个编码脱水酶的核苷酸序列，由序列11中的氨基酸序列组成，命名为ten/，其基因的核苷酸序列位于序列l中第12047-14848碱基处。本发明还提供了一个编码脱水酶的核苷酸序列，由序列12中的氨基酸序列组成，命名为/^《，其基因的核苷酸序列位于序列l中第14845-15885碱基处。本发明还提供了一个编码未知蛋白(可能负责催化D-A反应)的核苷酸序列，由序列13中的氨基酸序列组成，命名为"M，其基因的核苷酸序列位于序列1中第15872-16978碱基处。本发明还提供了一个编码NADH依赖的氧化酶的核苷酸序列，由序列14中的氨基酸序列组成，命名为"rM，其基因的核苷酸序列位于序列1中第16975-18744碱基处。本发明还提供了一个编码未知蛋白的核苷酸序列，由序列15中的氨基酸序列组成，命名为/^7V，其基因的核苷酸序列位于序列1中第18741-20579碱基处。本发明还提供了一个编码氮杂五元环环化酶的核苷酸序列，由序列16中的氨基酸序列组成，命名为欲O，其基因的核苷酸序列位于序列1中第20576-22621碱基处。本发明还提供了一个编码细胞色素P450氧化酶的核苷酸序列，由序列17中的氨基酸序列组成，命名为tof，其基因的核苷酸序列位于序列1中第22599-24023碱基处。本发明还提供了一个编码酰基-CoA合成酶的核苷酸序列，由序列18中的氨基酸序列组成，命名为加2，其基因的核苷酸序列位于序列l中第23999-25312碱基处o本发明还提供了一个编码细胞色素P450氧化酶的核苷酸序列，由序列19中的氨基酸序列组成，命名为其基因的核苷酸序列位于序列1中第25422-26612碱基处。本发明还提供了一个编码脱水酶的核苷酸序列，由序列20中的氨基酸序列组成，命名为"nS，其基因的核苷酸序列位于序列1中第26603-29413碱基处。本发明还提供了一个编码硫腺苷甲硫氨酸转移酶的核苷酸序列，由序列21中的氨基酸序列组成，命名为toT，其基因的核苷酸序列位于序列1中第29430-31229碱基处。本发明还提供了一个编码脱氢酶的核苷酸序列，由序列22中的氨基酸序列组成，命名为加仏其基因的核苷酸序列位于序列l中第31226-32044碱基处。本发明还提供了一个未知蛋白的核苷酸序列，由序列23中的氨基酸序列组成，命名为加r，其基因的核苷酸序列位于序列1中第32391-33113碱基处。序列1的互补序列可根据DNA碱基互补原则随时得到。序列1的核苷酸序列或部分核苷酸序列可以通过聚合酶链式反应(PCR)或用合适的限制性内切酶酶切相应的DNA或使用其他合适的技术得到。本发明提供了得到至少包含部分序列1中DNA序列的重组DNA质粒的途径。本发明还提供了产生硫链丝菌素生物合成基因被中断、被置换或同框缺失的微生物体的途径，至少其中之一的基因包含有序列l中的核苷酸序列。本发明所提供的核苷酸序列或部分核苷酸序列，可利用聚合酶链式反应(PCR)的方法或包含本发明序列的DNA作为探针以Southern杂交等方法从其他生物体中得到与硫链丝菌素生物合成基因相似的基因。包含本发明所提供的核苷酸序列或至少部分核苷酸序列的克隆DNA可用于从链霉菌Sr印ro附yc^/m^e油7ATCC31255基因组文库中定位更多的文库质粒。这些文库质粒至少包含本发明中的部分序列，也包含有5^印tom;;cM/aww"招ATCC31255基因组中邻近区域以前未克隆的DNA序列。包含本发明所提供的核苷酸序列或至少部分核苷酸序列可以被修饰或突变。这些途径包括插入、置换或缺失，位点特异性突变，特别是可以通过定点突变编码前体肽的基因序来实现thiostrepton大环骨架的改造，获得一系列的thiostrepton类似物。还可以通过不同序列的重新连接，序列的不同部分或与其他来源的同源序列进行定向进化(DNAshuffling),或通过紫外线或化学试剂诱变等。包含本发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过合适的表达体系在外源宿主中表达以得到相应的酶或其他更高的生物活性或产量。这些外源宿主包括链霉菌、假单孢菌、大肠杆菌、芽孢杆菌、酵母、植物和动物等。本发明所提供的氨基酸序列可以用来分离所需要的蛋白并可用于抗体的制备。包含本发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学活性，或者提高了产量或优化了蛋白动力学特征或其他致力于得到的性质。包含本发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异源宿主中表达并通过DNA芯片技术了解它们在宿主代谢链中的功能。包含本发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过遗传重组来构建重组质粒以获得新型生物合成途径，也可以通过插入、置换、缺失或失活进而获得新型生物合成途径。包含本发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片段可以通过缺失硫链丝菌素生物合成中的一个或几个步骤而得到新的硫链丝菌素结构类似物或前体。包含DNA片段或基因可以用来提高硫链丝菌素或其衍生物的产量，本发明提供了在基因工程微生物中提高产量的途径。本发明所提供的硫链丝菌素骨架的后修饰基因提供了通过遗传修饰得到类似物的途径，所包含的氧化还原反应也可有其他应用。总之，本发明所提供的包含硫链丝菌素生物合成相关的所有基因和蛋白信息可以帮助人们理解硫链丝菌素类天然产物的生物合成机制，为进一步遗传改造提供了材料和知识。本发明所提供的基因及其蛋白质也可以用来寻找和发现可用于医药、工业或农业的化合物或基因、蛋白。图l:硫链丝菌素的化学结构图2:硫链丝菌素生物合成基因簇的限制性内切酶谱。6个交叠的粘粒代表了抗生链霉菌基因组60kb的DNA区域，B代表限制性内切酶5amHI;图3:硫链丝菌素生物合成基因簇的基因组成。图4:提出的硫链丝菌素的生物合成途径。图5:喹萘啶酸单元的合成途径图6:基因中断与基因缺失突变菌株发酵产物的高效液相色谱(HPLC)分析。从上至下依次为标准品、野生型、环化酶基因中断的突变体(AwO)、脱水酶基因同框缺失的突变体(Jtov/)、脱水酶基因同框缺失的突变体(Jto《)、脱水酶基因同框缺失的突变体(zfoW)的发酵产物。符号说明图lThiostrepton:硫链丝菌素。图2peptideskeleton:大环骨架；quinaldicacidmoiety:喹萘卩定酸单元；tailoring:后修饰；propeptide:前体肽；unknown:未知功能。图4Leadingpeptide:信号肽；Structuralpeptide:结构肽；NAD(P)H:烟酰胺腺嘌呤二核苷酸(磷酸)；LP:信号肽具体实施例方式以下结合图1-图6对本发明进一步详细说明。1.克隆硫链丝菌素的生物合成基因片断尽管在硫链丝菌素的化学合成和作用机制方面的研究很多，但有关其生物合成起源方面的认识却非常少。之前，人们依据非核糖体聚肽合成酶的保守序列设计简并引物，希望用这种简并引物能扩增获得与硫链丝菌素生物合成相关的基因，总是不能成功。本专利依据硫链丝菌素分子中富含噻唑环这一特点，以噻唑环合成酶的保守序列设计简并引物，以5^ptomycM/awe"招的总DNA为模板，成功克隆得到了负责催化噻唑环合成的环化酶基因。在S/rQ^omycM/"wrew份体内失活该基因，得到的突变体无法再产生硫链丝菌素，证实了克隆到的基因与硫链丝菌素生物合成相关。2.硫链丝菌素生物合成基因簇的克隆，序列分析及功能分析-将上述杂环合成酶基因片段用地高辛标记为探针，从S/r印r(W^CM/aww"衍的基因组文库约2000个克隆中筛选，分离得到的粘粒涵盖了染色体约60kb的区域。(图2)。DNA顺序分析了39，556bp的染色体区域，GC含量74.19%。生物信息学分析包含了29个开放读码框(图3)。开放读码阅读框详细的分析结果列于表l。表1Thiostr印ton的生物合成基因簇中各基因及编码蛋白的功能分析￥i~~氨基酸数相似蛋白同一性/推测功能目相似性％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尿嘧啶磷酸核糖基转移酶未知蛋白未知蛋白脱氨酶氨基转移酶脂肪水解酶天门冬酰胺合成酶酯环化酶酰基-CoA脱氢酶甲基转移酶未知蛋白硫链丝菌素前体肽未知蛋白NADH依赖的氧化酶幼§612BL02837(YP—078248)25/39未知蛋白681GWCH70DRAFT—037932/43杂环合成酶(ZP—02912149)474SACE—0651(YP—001102921)29/44细胞色素P450氧化酶424TM1040—2789(YP—614783)27/42酰基-CoA合成酶加及396RoseRS—0765(YP一OO1275131)31/48细胞色素P450氧化酶ten936BC5084(NP—834752)20/34脱水酶加r599Gura一3608(YP—001232335)27/39硫腺苷甲硫氨酸转移酶272Krad—3162(YP—001362890)43/58脱氣,247orf—L7(CAN89623)39/60未知蛋白1447SsviAJ)1010001707338/51WD-40重复蛋白(ZP—03195299)99SCO4036(NP—628218)82/91未知蛋白29336SclaA—01010001372579/81RNA聚合酶o因子(ZP—03183088)3.Thiostrepton的生物合成基因簇边界的确定根据基因编码蛋白的功能分析以及和结构类似物Siomycin生物合成基因簇对比，硫链丝菌素的生物合成基因簇被确定为从基因ton4到to^(图3)，包含22个开放读码框。本发明中整个基因簇共包含22个基因的核苷酸序列或互补序列(序列i),其中一个基因(to"/0负责编码硫链丝菌素的前体肽，十个基因(加CK加M、"n/、加《、n加Z、加/、加C、欲及、加户)负责X寸加i7编码的前体肽进行后修饰，形成整个大环骨架；七个基因(加f、to"、/^￡、&M、加￡>、欲仏加，P)负责以色氨酸为前体，合成喹萘啶酸结构单元；一个基因欲g负责对形成的喹萘啶酸进行活化，在其他酶的催化作用下连接到大环骨架上，并最终形成分子中的第二个大环。其中l个基因("rC0中断及基因同框缺失加v/,^^,和"W完全抑制了硫链丝菌素的产生(图6)，进一步证明了克隆到基因簇为硫链丝菌素的生物合成基因簇。4.喹萘啶酸结构单元的生物合成途径喹萘啶酸结构单元的生物合成如图5所示。to^编码的重复使用的聚酮合成酶甲基转移酶催化色氨酸的2位连上一个甲基，"M编码的氨基转移酶使得色氨酸的氨基转化为羰基，^"五编码的酰基-CoA脱氢酶脱去一份子氢，^^编码的脂肪水解酶催化使氮碳双键断裂，形成氨基和羰基，to"编码的酯环化酶催化氨基和另一羰基縮合，形成喹啉酸，^"[/编码的脱氢酶将羰基还原为羟基，to^编码的P450氧化酶氧化喹啉8，9位上的双键为环氧，最后^"g编码的酰基-CoA合成酶负责活化喹萘啶酸的羧基，将其转变成CoA-硫酯键。5.硫链丝菌素骨架合成硫链丝菌素生物合成基因簇中有一个基因to^编码硫链丝菌素的前体肽，经核糖体将该前体肽合成之后，由z^O编码的环化酶负责催化前体肽中的巯基亲核进攻临位的羰基，然后脱去一份子水，形成噻唑啉，W*M负责编码的脱氢酶将噻唑啉转变为噻唑，然后由/^丄加《、toW编码的脱水酶负责催化使得丝氨酸残基的羟基脱水，编码蛋白的催化作用下，两个脱水丝氨酸残基发生D-A反应，形成第一个大环；然后在"W编码的水解酶作用下，信号肽被切除，经活化的喹萘啶酸单元连接到肽链的氮端；最后在^rC的脂肪水解酶作用下，喹萘啶酸的羧基与大环骨架上的苏氨酸残基的羟基发生成酯反应，形成第二个大环。(或"rP)负责编码的P450氧化酶负责羟化异亮氨酸残基上的仲丁基，最终形成完整的硫链丝菌素分子。整个过程如图4所示。以下进一步提供实施实例，这些实施实例有助于理解本发明，仅用作说明而不限制本发明的应用范围。实施例l硫链丝菌素产生菌链霉菌5^印tom少ce;y/awren衍ATCC31255总DNA的提取将100nLlxl08S./aww油7孢子悬液接种到3mLTSB液体培养基中，3(TC，230rpm培养约24hr后达到对数生长期后期，取2mL接种到50mLTSB(含10mM氯化镁，0.1%甘氨酸)，3(TC，250rpm培养约23hr后达到稳定生长期前期，呈乳黄色浑浊，将菌液4。C，3500rpm，离心15min收集菌丝，用裂解液洗涤，收集淡乳黄色菌丝0.5mL。向1mL菌丝中加入10mL裂解液(含溶菌酶5mg/mL)共四管，涡旋至均一，37。C水浴15mim。加入0.1mL蛋白酶K(10mg/mL，用裂解液新鲜配制)，lmL10%SDS，混匀后迅速放入70。C水浴15mim，呈澄清。置冰上冷却，加入2.5mL5MKAc，冰上冷却15min。加入10mL饱和酚，混匀，lOmL氯仿，混匀，12000rpm,4。C离心20min。用破口的枪头将水相吸出置于新的离心管，加等量的CHClr异戊醇(24:1)抽提，12000rpm，4'C离心10min。用破口的枪头将水相吸出置于新的离心管，加2倍的无水乙醇，混匀，有大团的DNA出现。将其钩出置于新的离心管，加5mL70。/。乙醇洗涤，将液体倾出，用枪吸净，加5mLTE溶解，加RNaseA使终浓度为50pg/mL，37'C温育0.5小时。依次用等体积的饱和酚抽提两次，CHCb-异戊醇抽提两次，向水相中加入0.1体积的3MNaAc，2体积的无水乙醇，轻轻的混合充分，有絮状DNA出现。将四管DNA合并到两管(每管中有1mL70%乙醇用于洗涤)，将液体吸出，再加1mL无水乙醇洗涤，吸出乙醇，超净台中吹干，溶于适当体积的TE(pH8.0)中。实施例2硫链丝菌素产生菌链霉菌S&卬/om;;c^/awe"招ATCC31255遗传转移系统的建培养含有适当质粒的ET12567至0D6(K)0.3-0.4，30mLLB培养液中的细菌细胞离心收集，用等体积的LB洗两次，重悬于2mLLB中，作为大肠杆菌供体细胞。取适量冻存于一80。C的5"/r印towj^;y/aww"招ATCC31255的20%甘油孢子悬液500pL，用等体积的TES缓冲液(50mMTESNa,pH8.0)洗两次，重悬于等体积的TES缓冲液，5(TC热激10min使孢子萌发。再加等体积的TSB培养基，37。C温育2—5hr。离心重悬于0.5—lmLLB中作为链霉菌受体细胞。将不同浓度的受体细胞100pL与等体积的供体细胞混合直接涂布在含有lOmMMgCl2的平板上，3(TC温浴20hr后，采用无菌水轻轻洗涤平板表面以洗去大部分大肠杆菌，在每一平板的表面覆盖lmL含萘啶酮酸(终浓度为50ng尔L)和相应抗生素的无菌水。3(TC培养5天以上挑取接合子。由于S/reptomyc^/awe"衍ATCC31255对阿泊拉霉素和红霉素都敏感，最后确定遗传转移所用抗生素的浓度阿泊拉霉素25pg/mL，红霉素100昭/mL。MS(甘露醇2.0g,黄豆粉2.0g,琼脂粉2.0g用自来水稀释至100mL，并调节pH=7)培养基最好，接合转移效率最高。在MS培养基上，无论是在链霉菌中可以自主复制的质粒pKC1139、自杀型质粒pOJ260都可以得到相应的接合子。实施例3硫链丝菌素产生菌链霉菌5^印to附yc^/flt^"招ATCC31255基因组文库的构建首先用lml注射器吹吸S/rgpto附;;ce;y/awe油'z'ATCC31255总DNA数次，使得总DNA随机断裂，使用蔗糖梯度离心对DNA进行梯度离心，收集略大于40kb的DNA片段，脱磷，待用。pJTU2554先用EcoRV从两个cos序列中间切开，与制备好的的40kb的DNA片段连接过夜。于冰上融化存于一80。C的PromegaPackageneexract，立即加入10ul连接产物，轻弹混匀，于室温(约22。C)放置3hr。加入445ulPhagebuffer,倒转混合；加入25ul氯仿终止反应，离心使氯仿沉于底部，4"C保存。将冻存于-80。C的菌株E.coliLE392涂布在LB培养基上复苏。调取单菌落接种于3MLLB培养基中(0.2%111&08610和mMMgS04)，37°C，220rpm振荡培养过夜，转接1%到50MLLB培养基中(0.2%maltose10和mMMgS04)，37°C，220rpm振荡培养至OD600=0.6—0.8。取5ul包装液，加入95ulphagebuffer,稀释，再加入100ul￡.Co//LE392(OD600=0.67)混匀，37°C，30min，涂于LB(含100ug/mlApramycin)平板上。37。C倒置培养过夜，测定噬菌斑形成单位(pfo)以估算文库的效价。取lOOul包装液加入3.9mlphagebuffer混匀，再加入4mlE.ColiLE392(OD600=0.72)菌液，室温，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倒置培养过夜。用LB刮下平板上的菌株，加入甘油(终浓度18%)和Apramycin(终浓度50ug/ml)，分装，于一8(TC保存。随机从平板中挑取10个克隆，接种于LB培养基中培养，按大肠杆菌的质粒的碱法小量制备的方法抽提重组黏粒。用^"附M鉴定，于0.8%的琼脂糖凝胶上电泳，根据限制酶切的电泳分析图谱，我们判断出黏粒被酶切产生的片断大小，加和这些片断，从而推算出整个黏粒的大小，实验表明每个黏粒的插入片断约为40Kb左右。对于链霉菌而言，其染色体DNA的大小约为8Mb，如果插入片断为20kb的文库效价是2000—5000cfo，就足以代表它的整个基因组。根据以上实验，我们建立的文库效价超过为10000pfU/iigDNA，插入片断约为40kb左右，这表明我们建立的文库具有良好的质量，能够满足文库筛选的需要。实施例4硫链丝菌素产生菌链霉菌S加;7tow;;cfty/awm7衍ATCC31255的发酵、产物分离纯化与鉴定取100ulS加/tow;;ce;y/做w"衍ATCC31255孢子接入S./做re油7—级发酵培养基(TSB15g,可溶性淀粉15g;蔗糖50g，蒸馏水定容到1L)28°C、250转每分钟培养48小时，取10ml种子培养液接入S./m^ew冶'二级发酵培养基(TSB15g，CaS0415g，酵母提取物llg,葡萄糖50g，2x微量元素lml，蒸馏水定容到1L)28°C、250转每分钟培养72小时.发酵液以3800rpm的转速，离心15分钟；上清用等量的乙酸乙酯萃取两次，萃取后的乙酸乙酯用适量无水硫酸镁3(TC蒸干，所得固体用3ml甲醇溶解。发酵产物分析取20ial进样，流动相为乙腈和水(含0.1XTFA)。HPLC洗脱条件为(紫外254nm处检测，柱子为NUCLEOSIL100-5C18)。<table>tableseeoriginaldocumentpage16</column></row><table>实施例5PCR克隆硫链丝菌素的生物合成基因PCR体系包含DMSO(8%,v/v)，MgCl2(25mM)，dNTP(2.5mM)，简并性引物(40mM)，TaqDNA聚合酶(2.5u)及适量模板5^e^o附yces/flwe"招ATCC31255总DNA。首先95。C，3min,1轮；然后94。C，lmin,68°C，lmin，72°C，2min，5轮;94°C，lmin，65。C,lmin，72°C，2min，30轮；最后72。C，10min,1轮。PCR结束后，1%琼脂糖电泳检査结果。低熔点胶回收预期大小的DNA片段，与pMD19-Tvector连接，转化大肠杆菌DH5a感受态细胞，涂布在含有氨苄青霉素，IPTG(异丙基硫代—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平板上进行蓝白斑筛选。挑取白色菌落过夜培养，抽提质粒，五coRl酶切鉴定是否含有预期大小的DNA插入片段。插入有预期大小DNA片段的质粒测序。实施例6核酸分子杂交1)DIGDNA标记将待标记的DNA用无菌水稀释至总体积15pL，沸水浴中加热变性10分钟，立即置于冰盐浴中冷却。接着加入2^L引物混合物，2pLdNTP混合物，lpL酶，混合均匀后，37。C水浴约16小时。加入O.一L0.8MEDTA(pH8.0)以终止反应，加入2.5pL4MLiCl混合均匀，再加入75pL预冷的无水乙醇沉淀标记后的DNA，置于一80。C沉降40分钟。4'C，12000rpm离心20分钟收集DNA，用预冷的70。/Q乙醇洗涤DNA沉淀，真空干燥后重新溶于50pLTE((pH8.0)中。2)DIGDNA探针标记后的质量检测稀释标记的DNA探针，至以下六个梯度，1、10"、l(T2、l(T3、l(T4、l(T5。稀释标记的对照DNA至浓度分别为以下浓度l昭/mL，100ng/mL，10ng/mL，lng/mL，0.1ng/mL，0.01ng/mL。分别取lpiL上述梯度的DNA样品点在杂交用的尼龙膜上，根据7)所述步骤进行显色反应，对比标记的DNA探针和DIG标记的对照DNA的显色强度以决定标记的DNA探针浓度。3)菌落杂交(文库筛选)的膜转移将保存于一8(TC的基因文库稍融，取50pL,用450pLLB稀释得到l(T1的稀释倍数，倍比稀释得到l(T2，l(T3，IO"4，l(T5，l(T6。300pL涂平板(15cmxl5cm，平板为LB/50昭/mL卡那霉素)。选取合适的比例，使每块平板约1200—1500个克隆。照选定的比例均匀涂布四块平板，37。C培养过夜。根据平板的大小剪取尼龙膜，小心地覆盖于平板表面不要产生气泡，做好位置标记，1分钟后取下尼龙膜置于干燥滤纸上，干燥10分钟直至菌落结合在尼龙膜上。原始的平板置于培养箱中4一5hr，使克隆重新生长作为原平板。将尼龙膜置于变性液(0.25MNaOH，1.5MNaCl)饱和的滤纸上15分钟(不要浸过膜)，转移至中和液(1.0MTris.HCl，1.5MNaCl，pH7.5)饱和的滤纸上5分钟。转移至2xSSC(20xSSC储备液(L"):NaCl，175.3g，拧檬酸钠，88.2g，pH=7.0)饱和的滤纸上自然风干。取下尼龙膜置于烘箱中，12(TC固定45分钟。常温下于3xSSC/0.1%SDS溶液中振荡洗涤3小时，以除去细胞碎片。4)Southera杂交的膜转移:DNA样品在适当浓度的琼脂糖凝胶上电泳至适当距离，做好标记。浸泡于400mL0.25MHCl中脱嘌呤20分钟，使溴酚蓝变黄，用去离子水洗数次。室温下浸入碱性缓冲液(0.5MNaOH，1MNaCl)15分钟并轻轻振荡。换液一次继续浸泡凝胶20分钟，并轻轻振荡，去离子水洗三次。取一张每边都比凝胶大lmm的尼龙膜，用去离子水完全浸湿，做好标记。采用向上毛细管转移方法，用10xSSC转移缓冲液转移8—24hr。用2xSSC略微洗膜，12(TC烘烤30分钟。5)预杂交和杂交预热杂交液(20mL/100cm2)至杂交温度68'C，放入杂交尼龙膜，轻轻振荡并保温30分钟。将DIG标记的DNA探针在沸水浴中变性5分钟，立即置于冰盐浴中冷却。冷却后，将DNA探针与合适体积的DIG杂交液(2.5mL/100cm"混合均勾。去除预杂交液并立即把DNA探针/DIG杂交液加入，轻轻振荡保持杂交温度64'C或68""C约16小时。6)杂交后严紧洗脱室温下用2xSSC/0.1%SDS漂洗两次，每次5分钟。68'C，用0.1xSSC/0.1%SDS振荡漂洗两次，每次15分钟。7)显色反应和检测:严紧洗脱后的尼龙膜在洗涤缓冲液(0.1M马来酸，0.15MNaCl，pH=7.5,0.3%(v/v)Tween20)中平衡1-5分钟，接着在封闭缓冲液(封闭试剂以10%的浓度溶于O.IM马来酸，0.15MNaCl，pH=7.5)中封闭30分钟，然后在抗体中浸泡30分钟。用洗涤缓冲液漂洗尼龙膜两次后，用检测缓冲液(0.1MTris-HCl，0.1MNaCl,pI^9.5)中平衡2-5分钟，最后将尼龙膜置于10mL新配制的显色溶液[NBT(nitrobluetetrazoliumchloride)溶于70%DMF，浓度为70mg/mL，BCIP(5-bromo-4-chloro-3-indolyl-phosphate)溶于水，浓度为50mg/mL。用时10mL显色溶液中加45^LNBT，35pLBCIP]中，置于黑暗中显色。显色合适后用去离子水漂洗以终止反应。实施例7基因中断突变菌株的获得将获得的转化子接种到TSB液体培养基(Am25pg/ml)中，3(TC振荡约28hr。取出200nl涂布在ISP-2(Am50吗/ml)平板，3(TC培养6-8天，收孢子，保存于37'C整合生长的单菌落，接种至液体培养基ISP-2(Am25^ig/ml)，37'C，振荡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。基因中断或基因置换所用载体为pOJ260或pKC1139，基因置换用红霉素抗性基因替代目标基因中间的DNA片段。构建好的质粒通过实施例2所述属间接合转移的方式导入5^//omyc^ATCC31255中得到双交换突变体，所得突变体通过Southern杂交在基因型上加以证明。以下根据本
发明内容提供的基因和蛋白序列氨基酸/核苷酸序列表:SEQUENCELISTING<iio中国科学院上海有机化学研究所<120>硫链丝菌素的生物合成基因簇<130>说明书、权利要求书<160>1<170>Patentlnversion3,3<210>1<211>39556<212>DNA<213>链霉菌/m/"""7ATCC31255<400>1ATGTAGCCGTTCTCGTTGAGCCGCTCGTCGCCTGCCAGCTCGCGCTCCATGACCGCGACGGTGACATCGTCCGCGCCGCGCCGGATCAGTGTCGCCAGCATCGGGTCCAGGACGTACACCGTCGCGTAGGTCGACGCCTCCAGCGTCTCCTCGGCGGTCGGGAGCAGCCGCACCATGCCGGGGACCACGAGCGGGCGGGGGTACGAGAGCTCGATGTCGACCCGCTCGGTGCGCACGTCCAGCTCGTCGGCGAGCCGGCGGAAGGTCGGGAGCTTGTGGGCGACCAGCGGGTGATCGACGGGCCGGTGACGGGCGCGGCACAGCGGCCGGCGGCGGGGGAAGGTGGGGACACAGGGACCCCCGAAGAAGACGCCCGGAACGCCCGGAATGGACCCTCCGCCGCTCCCGAGGGAGTACCCAACCGAGGCCGTCACGACGGTCACCGGCGTA60CCCTCGGGGGCGGCGAGGAGCACGACCGCG120TCCTTGATCGCCGCGACGAGCGTGCCGCCG180TGGCGGCCGGACAGGTCCTCCGGCATCCGC240TCGTTGCGGATCATCCCGAGGAAGCCGACC300TCGAGCATGCCGAGACCGGCGCGCAGGATC360TTCACGCCGGCCGTCTTCGTGACGGGGGTC420CTGGTCGCCTCGTAGGCGAGCAGGGTGACC480GAGTCCGTGCGCTTGTCGCGCAGAGTGGTG540ACGTGGAGACGCATGCCTCAACAGTAGCCC600GGTGACCCGCGGGGTGGTGCCGGCCCGGGG660AGCCGTGGGGTGGTGTGGCCGATGCCCGAC720CCTGGTGAGGCGGCCGGAGCGGAGGCTTTC780CCGGCTTCGGGCCCTTCCCGGGCCTCCGGG840TCGAACCCCGGGCCCAGCCCCGAGCTCGGGCCGGACACGCCGGACCCGGACGCGAACCCGGAGCGCCGGCGGCGGCGCGCGCAGTTCCTGGAACGCGTCCAGCCCCGGCGCGCCCGGGCGTCGTTCCGGTGGTGAAGGGGCCGATCGAGGCGCGAGGCCGTGCGCCCCTGCGTGCGCCGGTCAGACTGATGCACGACGCAAGAGCCGAAGATGCCGATTGGGCGGGGCATCAGGAGCGCGAGAGTCAGGTGTACTTCGCCGCACTGCTCGATACAGAGCTCGACGACGTGGAAACGCTGGCCGCCGACGACGGAGAGACCGTGATCGTCTTCGTCCGGGTCGACGGCGAGGACGACCCACCCCGCTCCTCGTACGGGGAGATCCTCACCTCACTCGACGAACTGACCGACCTGGACGGCAAGGACGACGAGGCGGCGGACAGCGCCGCGGCGCTCATCCTGGCCGACCTCGGCATGGACGACGCCCTCGGGGAGATCGCGGACGCCATCGAGTGCCCCGCGCAGCACACGGCGAGGACACGGAGCCGATCCGGGAGCCGGTCCCGGAACCGGGCCCCGCGATGGACCCCGTAGGGGATCCGTACGCGCTCGCCGAGGCCGACCTGGCCGCCGTGCTCGCCCCGGACGGCTCCGTCCTCGCCGACCCGACCGCCCACGCCGAGGTGCTCGCGTGGCGGCTGACCGGCTGCACGCTCGTGGTGGCCCTCGTCCAGGCGCGGGTCGAACGGGTCGCGACCGGCTCGCTCTGGGACCTGGTCCGGATCCACGGCGTACTGGAGGCGGAGTGCTCCTGACACCCGGCCGACGACCCCGCCGACCTCCTTTGGGCTAAGCTCTCTCTCGGTAGCGTAGCGAGTGTGGGGAAACTCACCGAGGGTTCGCCGGGCTCGGGGCCGGGCCCGGCCCCGAG900GAGGCGGACCCGGAGGCGGACCCGGAGAGC960CGCGACCTGCACGAGGCCAAGGCGCTGCGC1020GCCCGGATGCGGCAGGCGATGCGGATGCGT1080TAGTGCACCCGGGTGACCTGCGGGAAGTCC1140GGTCGCGCGCCCGCCCGATCGCGGTGTCGT1200ACCTCTCCTGACGGCGAGGTTTCTGTCACG1260CCGCCGGTCATCACACCTCTGGACAGTGGG1320CGCGCACCGAAGACGGGTGGGAAGCGAGCG1380CGGACCTCGCCGAACTGGCCCGTGAGGCCG1440TCATCGAGCAGGAGGACGCCTGGTTCGGCG1500GCGTGTTCGTCTCGAACGGCGCCGCCGCCG1560CGGAGATCCTCGGCGACGAACCGGACGACG1620CGGAGGACGGCGAGCCGGAGGCCGCGGACG1680CCGTCGCCCCGCCCGGGCCGCTCGGCGAGA1740AGCAGGCGCTGCTCGGTCTGGAGACCGGCG1,GCGCCGCGGACATCCTGGAGGCCGTCCGCT1860CGGCGCCGCCCCTCGCCCCGAACCCGTACG1920GGTCCATGAACCCGCCGCGGGATCCGGACC1980CGTACGGGACCCCTGGCGTGCCCCGATGCG2040GCTGGCCGGTGACGTACCGGTCGGCGCGGT2100CACCGGCCACAACGAACGCGAGGCGACCGG2160CCTGCGCCGGGCCGCGGCGGCGACGGGCGA2220GACCCTGGAGCCCTGCGTGATGTGCGCGGG2280GGTCTTCGGCGCCTTCGACGAGAAGGCCGG2340CGACCGCCGCCTCAACCACCGCCCCGAAGT2400GGCCCAGCTGACCGCCTTCTTCCGCACCCG2460GCGCGGGGATACGGATTTCAGTCCTGGGCG2520GTCCGAGCGGCCGAAGGAGCTCGCCTCGAA2580AAATCCCTCCGCTACCGCTCTCGAACCGCC2640CCTGATCTGCGACAGCAGGTCAGGGGCGGTCTCCCCCGGCTCCGTCGGCCCCTCCACTGACTTGGCCAGACGGGCGGTCAGCCGGAGGACGACGCGGACGCCCTCGCCCGCGACGGCGCGGACGAAGGCGGCCGAGCGGGAGCCCAGCGGCACCTCCCAGCCCTGCCCGCGCAGTTCCGCGTCCACGCGCGCGTACACCTCGGACCGCCGGACGGTGCCGACGGCGAAGGGCAGCAGCCGGGCGAGCGCGAAGCCGACGCGCAGGCCGGCGACCACCACGTTCGGCAGGCCGCGGTAGACGGCGAACTCCCGGTACGCCTCGTCCAGGACGAAGGCGAGCAGGGCGTCGGCGCCGAGCGCGACGAGCGCGCGGGTGTCGGGGGTGACGGCGTCCCGCAGCGGGATCTCGACGGCGGTGGCGTCGAACGACGGCCGGGCGTAGACGACCTCGTGCTGGAGGATCACGGCGGAGCCGGGGCCGGTGGCCGCGATCGCCGCCGTCAGCTCCCGGCGCAGCGCGTCCCGGAGCGCCTCGTCCAGCAGCGAAAGCCGGTGCTCCGGGCCGGACGCGCACCTCCTCGCGGGTCAGGGACGCGTGGGGAGGTCCGCGGTCGCGCGGTCGCGCCCCTCATAGACGCGTTCGGCCAGGTGGAAGCCGCGGATCAGGGCCAGCGGATCGGCGTCGCCGTGCTCGCCCCCGTTGATCCGTTCGAGGCAGGTCGCGCCGTGCCAGATCAGCGGGTCCGCGTTCATCACCGAGCCGACGTCCACCGGCATCTCCGCCGTGTGCCAGGAGCCGAGCACGGGCGCGTGGCGCTGGGCGTACCGGACGGCCACCATGGGGAGCCCCGGGTGGTCCGCGCGGGCCCGCTCGTCGTGGTCCGTCACGAGCGCGCGTGCTCTGCGTGTGCCCGCTCAGTCTCCCGGCGC2700TGCCGCCGGCCCCACCGGCTCCTCGCGCTC2760GGCGTCGTTGGCCGCCGGGTCGCCGACGGT2820GACGAGGATGCCCTCGCGGGCCGCCGCCGC2880CAGCCAGCAGAAGTTGGCGGCCGACGGCGC2940GGTGAGCCGGTCCCGTTCGGCGACGACGCG3000GGCGAGAACGGTCCGCGCGGCCGCCTCGGC3060GGTGCGCAGGACGTCCGCCACCTCGCGGCG3120CAGCCCGTACGCCTTCGAGAAGGACCGGAG3180GTCCGGCCCGTCGAGGGTGTCCTTGTCCCC3240GACGACCACGTGCTCCGGGACCGCGCGGAG3300GGTGCCCGTGGGGTTGTGGGGGTTGCACAC3360CGCCAGCATGCGCGGCAGATCGTGCCGGTG3420CCCGGCGGCGGCCACGGCCAGCGGGTACGC3480GCCCCGCCGCGGGGCCGCCCACTGGATGAC3540CGTGACCACGTCGTGCTCGGGTACGGAGTG3600GGCGAACGGGTCGGGGTAGCGGTGCGCCCC3660GCCGGTCACGGGGACGGCCGGCATCTCGTT3720TCCCGGTTCGGTGACTCCCGTCATGTCGTC3780AACGGTGAGGGCGTCGTCGAGGAACGCCGT3840GTCGTGGAACAGGCCGTGCAGCGCTCCCGG3900GATCTTCTCCACGCCGGCCCGGGTCTCGTC3960CAGCCAGAGCGTCGGCAGGAAGGCCAGGCT4020GACGACGGACTCCAGGGTGTCCCGGACGAA4080GTAGCGGGCCGCCTCGGCGGGGTCGCGGGA4140GGGGATCTCCGCGAAGGCGAGGAGCGAGGT4200GGCGAGCACGAGCGCGGCGAGGTCCTCGGG4260CCCGCCGACCGAGTGCCCGATCATCACCAC4320CTCGACGACCGTGGCGAGGTCGGCGACCTG4380GCCGCCCGAGCGGCCGCTGCCGGCGTGGTC4440CGGCGCGTACACGGCGGCGCCGCGTGGTCGGCGTACCCGTCCAGACGTGCAGATGGATCGGGTCATCGTTCCCTCTTCCCGGCGTCACTCCGCCAGCTCCACTCGAATCCCACCCGCAGGACGCCTCGGCCGCGCTCTTCTGAGTGCGCCGAGCCGGCCCACGGCTCCTTCTTCCGGGTCGCAGGCTCTTCTCCCGGCCGCCACCAGGGCGTGGTCGAGGGGTCCTTGCGTTCCAGGAGCGCATCCGCCGGTCCTCCTCGCGTACCGGTCGGCCCGGTACACGCCGGGCCGCCGGCGGCGGGACGGCGTTCTCGTCGTGGCGGACAGCGCGACCGTGGAACCATGTCGGCGAGGCCCTGGGGTCGGACAGCAGCTTCGCGCCAGTTCGGCGGCGTACGCGCGGAGCCGGCGGCGGAGAAGGCAGCGCCCCGACGGCGAGCAGCAGAGCGGCACGTCCGCGCCCGGCCGATCGTGGTGTCGCGCGCTCCGTGATCCCGCCGTCCCGTCGAAGATCGCGTGCCCGAGGTCAGCCGGGCGCGGAGAGGACACCGTCCGGGGTCTCAGCAGGAGTTCCTGGCCGGCCGGGTGACGTCCTCGCCCCGTGCCGGGTCAGCGCGTCGGCCAGCCGGCCGTAGCGGCC4500GCACGAACACCGCGAGATAGCGGGGCGCCGTCTCCGGCGG4560GGTGCCGGACGCCGGCGAACCGCCGTGCCGTCGTCTCCTC4620TTCGTCGCCGGTCCACCGAGCCGCGCGCGCCGTCGCGCCC4680ACCCGGTAGTCCCGCATCCAGTCGTTGATCTTCAGCAGGG4740AACCGCAGTTCCTCCGGGGACGCCTTCCGCACCGCGGCGT權OTCGTCCATCAGCGCGCGGACCGGTGCCCCGGTGTCGTCCA4860TTGAGCAGCTGGTCGTAGGCGGGATCGGCGATCGTCGGGT4920AGTACGGAGTCCGGGAGCTCGGGGGCCATGGCCGCGCGCA4980TCGAAGGTCTTCATCGACCACGGCACGTTGAAGACGTACT5040AACGGCACCCGCACCTCCAGGCCCACGGCCATGCTCATCC5100ATCGGCAGGAAGCGGGTGAGGAAGAGGTGGCCGACCTTCC5160GCCTCCCCGGGCAGGACGGGGACGGCGGCGACCGCCTCGG5220GCGTCCAGGGAGAGCCGCTCGGGCAGGGCCGGGTCGAGGA5280GCCACGCCCGCCATGTACATCCAGGGGAAGGTCTCCGCGC5340AACCAGGGGTAGCCGCCGAAGAGTTCGTCGGCGCCCTCGC5400CGCTGCCGGATCGCCTTGAACAGCAGGTAGAGGGACGTGT5460GGCAGGTCGCGGGCCCGGAGCACGGCCGCCCGCGCCGCCG5520TCGAGGGTCAGGTCCTCGTGGCGGGCGCCGATGTGGCGGG5580CTGTCGTCGCCACCGCGCGGGCCGGTGACGAAGCCCGGAC5640CCGACCGAGAAACAGGTGAGCGGGTCCTCGCCGCGGTCCC5700GCCGCCACGGAGCTGGAGTCCAGACCGCCCGAGAGCAGCG5760ACGGTCTGGCCGGCGACGGACGACCCCAGCAGGTCACGGA5820AGATCGTCCTCGTGCGGGCGCGCGGGGAGTTCCCAGTACG5880CGCGCGACGCGGACGACGTGCCCGGGGCGCACTTGGCGCA5940CCGGGGGTGTTCACCCAGGTCATCAGCTCGCGCAGACCGT6000GCCTCGGGGTGGGCCAGGATCGCCTTCGGCTCGGAGCCGA6060GGGTAGTAGTAGAGCGGCTTGACGCCCAACCGGTCCCGGA6120CGGGCGTCCCAGAGCGCGAACGCGAACATGCCGGTGAGGT6180CACTCCAGGCAGGCGCGCAGGACGACCTCGGTGTCGCTCC6240GGGTGCGGAAGGTGTGGCCGGCGGAGACGATCTCCCCGCTGTAGGAGAGGGCGGCCGTGAGCGCGCCGCCGGCGATGTCGATGACGGCGACGTACCAGGTGCCCCCGGCGTCGGGGCCGCCCGCGGAACGCTCGGCGCCCAGGTCCCGCCTCCGTCCTCCTCCATTTCCTGCCGCTTTCCCCCTGCCGTTGCCCTGCCGTTGCCCTGCCGACGTGGGCGCGCCCGCCGGGTCAGTCCGGCAGGTCCATCTCGTCCCAGTGCTGGGCGAACAGGCCGACCGAGAAGCGCCGTCCGGAGGCCCCGCTGAAGGTCATCCGCGCGACGGCCTTGAGGCTGAGGTCCTCCACCGTCGAGGCCCAGCCGCGCTCGACGGCCGGGTTGGGGTGCCGGCGCGGGTCTCCTTCGTTGTACGCCGCCAGGCGGTCCACGGGGATCGCCTTCCGGTCCGGTGGGGCGGGGGAGGCCGGGGGTGTCAGGAGGTGGCCCCGGCCTCCACCTCGGCCTCGGCCTGGGGCGGATGCACGGGAGCCGTCTCCTCGCCGTCGGCGTTGCTGTACGGGTGCATCGTCCACACCGAGCCGGGCCGCAGGGCCGGGGTGCCCACGGCGTTGTTGGTGCCCACGTTCTTGGTGGTGAGCGCGTTCCACAAGCCGTTGCCCACGCGCGAGGTCTCGATCAGCTCCTCGCAGGCCTCCCAGCCGCGGCTCCGGACGTCCCGTACGAGATAGACCGCGGCGAAGGTGGGCATCGCGTCCAGACTCCCGACGGGGTAGGAGGTGAGGAGTTCGATGTTCTCGCTGCGGGTGGCCCGGACCCGGACGCCGGGACTGTCGGCGGGGAGGTGTTCGCGGTACTCGGCCATCGAGGTCAAGGTCTTGTCGCCGGTGACCAGGTAACCGCGCCGGGCGCGCAGCTCGTCGAAGCCGTAGA6300CCTGCCCGTTCTCCTCGGCGAGCATGGGCT6360GCCGCCGGTGGCCCAGGGCCACCGGGCCGC6420GACAGGCCATCGTCCGTGTCATCGCCTCCA6480CGAAGGCCACCCAGCCCGCTATGCCGCACA6540TGCCGCTTTCCTGCCGCTTTCCTGCCGCTG6600TTGCCCTGCCGTTCCGACGACTCGCGAGGT6660AGCGCGCCGAGCTGCCGGTGCAGCCCGAGC6720AGACCGTCCTCGATGAGGAAGATGTCGATG6780GGGATGCCGAGGATCGGGCCGACCTGCCGG6840TCCCCCTCCACGACGAGGTCGTCGAGGGTG6900CGGCGGATCGCGGCCCGCATGCCCTCGATG6960TACTCGGGGTGCAGACAGTGGTCCAGGTGG7020AAATGCTCCATGAGCTGCTGTACGGATGCT7080GGGTGTGCGGGGCAGGACGGGTTACGGGGG7140CGGCAGGGCGTACGGCCGTGTCGCTGGCCT7200CGGGCGCCGGCTCGGCGAGCCGGTCCAGCC7260CGAGCGTCGTCGCCGCGATGAGGTCGCACG7320CGGCCCGTACGTCGCGGTGGGCCCGCTCGT7380CGATGATCCGGATCACCCGGTCGACGATCT7440TCAACACGGCACGGGCCATGCCCTGTTGGA7500TCACCTCGCCGGCGGTCCGCTCGATCAGGG7560CGGCGATCTCCGCGCGGATGTACGGCCGGT7620CGCAGGTCTCGAACGCGCCGACGGCCACGC7680CCCAGCCCCACACGGTCTGGAGCAGCGAAC7740GGACGTTCTCCTCCGGCACCATGACGTCGC7800GCAGTCCCGAGGTGTTCCAGGTGCCTTCGA7860TACGGAAGACCAGGACGTGCGGGCCGTCGG7920TGTGGGTCATGACGGCCGAGCCGGTGCCGA7980CTTCCACCTTGCGGGCCTTGGCGCCGGTGG8040TGCGCACGAGCGCGTGTCCCGGCTCGGCCGTGGCGCCCATGAGGATCACGCGGCCCTCGA8100CGATCTCCGTCAGCGTCTTCCGGGCGCGCTCGTCGCCCGTGGTGCGCCAGGCGCGGGCCA8160TCTGGCCGGCCATGGAGAGGTGCATGTTGACGGCGAGGGCGGTGGAGGCGCAGCCGGCGG8220CCAGCCGTTCCTGGGCCACGGCCATGTCGGCGAGGGTGCCGCCCAGCCCGCCGAGTTCGC8280GCGGCACGGTCAGCGCCAGATAGCCGGCGGCGCGGAGTTCGTCGATGTTGCGGAACGGGA8340ACGCGGCCGTCCGGTCGTGCTCGGCGGCCCGCTCGCGGAAGACCGCGGCCATGTCCTCCG8400CCAGCTCGACGTACCGCTGCCGGGCCGGCCGGGTGATCAGGAGGTGCATCAGGACTCCAG8460CTGTCGGAGGAGGGGTGTCACGAGGAGAAGGGCCTTCGCCGGGCGCCGGTCACACCGGTC8520GCTCCGGAGGCGCCGGTCACACGGACGCCGGTCACAACGAGGCGCAAACGGAGGCGGGTT8580CGGAGGCCGCACGTCAGGCGACGGTGGGTGAAGCGGAGGCGGATTCGGAGACGGAATCGG8640AGACGGAATCGGGGACGGAATCGGGGACGGAATCGGGGACGGAATCGATGACGGCCGGGC8700CGGTGACCAGCTCGGCGGCGACGAGCCACGTGCGGGCGGCCCCGGGCGTCCCGCGCGGCG8760GGGTCCGCGGCACCATCCGCCCGGCCGCCTCGTCCAGGTCGCAGGGTTCGCGCACCTCGG8820CCCGCCAGCCGCGGCCGGCGAGCCACGCCTCGGGCTCGGCCACCCCGGAACGCCAGGGGC8880GCCCCGCGTCCCGCATCTGCCGCAGCCAGTCCCGCATCCAAGGGGCCCGGTACAGATCGG8940TGTTGACCTGCTCCAGGCAGAGCACGCTGCCGGCGGCGGAGAGCTCGGTGACCCGGTCGA9000CGACCGCGTCCACCTGCGCCTCGGTCAGGTAATAGAGGACTCCTTCGACCAGCCATACGG9060TCGGCAGACCGGGGCGGAAGCCCGCCTCGGCGAGCGCGGCGGGCCAGTCGCCGGTCAGGT9120CGGCGGCGACCGGGACGCGCCGACAGCGGTCGGCGACCAGGCCGGAGCGGGACAGGAAGG9180CGCTCTTCTCCTCCACTCCCTCGGGCAGGTCCACCTCGTAGAGGGTGACGCCCGGGGGCC9240AGGGGAGCCGGAAGGCCCGGCCGTCCATGCCGGCGGCGAGCACGACGACCTGCCGGGCCG9300GGCCGCCGGTCACGGCGCGGATCAGACGGTCGTCGAAGAACGCGGTCCGCACGGCGAGTA9360CGTCGAAGCGGGAACTCGGTTCGGTGCGGAGGCGGTCGTAGAGGTCCGGGTCGGTGCGCC9420GGACGAACTCCACCGCGAGCGGGTCCTCGAACAGCCGGCCGTCGGGACGGAGCGACTCGG9480CTGCCCGGGCGCAGGCGATCCAGTGGGCGGTCTCGATCAAACTCGACATGGGCTCTTCTC9540GCAGAGAGCCGGGCGCCGGCGCGCCCGGGTGAGGGTCACCACGGATCCGCGGCCGCGCGT9600CAGCGGCCTTCGGTCGCCTCGCCGACGGCGGGCGCGGCAGACGCGGGAGGTGCGGCGGGT9660GCGGCAGGCGCGGCAGGCGCGGTGGGCGTACCCGTCGCCGCCGGCTCGTCGTCGATCCCG9720AGCGCGCGGGACATCCCGCGGCGGAGCGACCAGTGCAGGATGTCCGTCTGGTTGTGCCGG9780AACTGGGCCCAGTAGGCGTCGCCCGCCTTGTTCCTGGTCCGCAGGACGACACCGATGTAC9840GCCTTGCCGTAGGAGCACTTCGGGGCAGCGATGACGACGCAGGCTGGGACGGGATCGCCAAGAGCCGAGTTGGCTTCGCCTGGCGCATCACATTTCCGTACTTGACTTGCTTTCCCGTGGGTGAACGGCAGTCGACACCCACCGTCACGATCCTGAGGTACAGTCGACCAGCAGGTCCGGGGGCCCGACCCCGGCCTCCGGGTACGTCTTCCTCCCATGGGCCCGGACGGGCGGCCCTTAGTGCACGGTCCGCCCTGTCACCCGCCGCCGGTGGTCGTGGCCCGCCACCGCTGAACCGCTCGGCGACGATCTCGCTCGCTGCTGTTCCCGCCTGGACCCGGGTCCATGGTCCTCCGCCGCCGTTCGGAATACGCTCAGGGCTGCACCGAGTGTGATCTACCTATGGTCGACCGGCCCACGTGAGCAATGCTGGAGATCAGTGATCGCGTCACACCCGGCGGGTCCGCGATGGCCCGACCCCGGAGTCATCGACGAGCACCCGGCATGTCCCCGCGGAAACGCTTCCCGTCTTCTGTTTACTCCTGGGCACCGGAAATACGCGGCGATAGAGGCTTTCCTATGGCCGGAATGGCTGGCGGGGAAGGACATCCGCCTCGAAGCTTGCTGCGCGATCCACAGGCAGCCGGGCGCTCCGCGTGGTGAACAGCTGCCTCGGGACGCCTATGCAGAGTCACAGCCAGATCAGCCGGTGCGGCGCGACGCCCTGGAGCGACTACATGCGCCTCCTGCCGGAGGACTGCTAACCGGGAGGAGTACCCCTCCGGCCGTGCTCACTATGCTATTGCACCGGGCCCCATACGGGTGCGCCGGGCAACAGAAGGAAGACGCGTCTTCCAGGCCCGCGTCCGCTCTGGGTGCCCGTCATGTCGCTTCACCAGACGGTGTTCGAACCTCGTACGATCGTGTTGGTCGACCAGCCAGCTCCCTGGGTGTGCTCTCGCTATATGCATCATAGGTGTGATCGATTCTTTTATCCGGAGCGACGGAGTTCGACGCGACATCGGTGTCGGACGAGACGCACCACCTGCATTCCTCCCCGGTGGGACCCGGGACCTGCCCCTCGCTCGGACCGATGACGGCGGACGGCTTGGCCGAGAGAGGCCCCGAATAGCTGATATCCATTACGCGGCACGGAACCGGAGGCCATCCGCCGTGGCCTGGCTACTCGTATGGCCTCCCGCAGCTTGTCGACCGAGGCGGCTTGACGCCAGAACGCCCGCGTCGATCCTCACTGTCACGTTTCGAGATTCCAGAATTCGCGACTCAGCCGGTGCCCTTGCCGGAAGCCGCGCAGACTTGAGGGACTCACTGCTCGACGGTCTGCACCTGCCCGCCGGACGACCTGCCCGGTTGCAGTCGGGCTGTCGGCCGTGGACAACTCGCACGTCCACGGAGTGTCGACGCTCCGCACGTGCGACCGGTGCCGGAAGCGGCGAAATGCGAACAGCCTCCGGGCCGCTCCTGGTGTCATCAGCCCGC26CATGTCGCCG9900CTCCCCGACC9960GAAGGGGCGC10020GTCGGGCGTG10080CCGATGGTCC10140GAAACGCGCG10200CCCCATGGGT10260ACGGCCGAAG10320GGAGAGTAAA10380AGCGCTTCAA10440GCTACAGCAG10500CCGAAAGGTT10560GGGTCTGGAC10620TGAGGACCTG10680CAGCTGCAGC10740CTGACCGTTG10800TTGCAGTCGA10860AGCAGGTCCG10920GGACCCGCCG10980TAGTTCCGGC11040GTTCAAACGG11100GGTCCCGCAT11160CGGGTATTGA11220CGTAATTCTG11280ATGGTGGACC11340CCCACCCCAT11400GGAACCTCCC11460GGCCGGTTTT11520GCCGGACACC11580GGCCCAGCGC11640CATTTCGCCGAACTGCACGGCCCCACCTGGCTGTCCGACGGCTGCCTGCGGCTGCCCGGCGTCCAGCTCGTCAACGGCTCACTGCTCCAGGCCGCCGGTGCCGACGTCGCGGTGATCGCCCCGCGCGTCTTCGCCGCCACCGTCGAGGACGCTTTCCACGAGCGGCGGCGCGCGGCCGAAGAGGCCGGCACCGGAACCGAGCCCGTCGCCGATCACGTACGCCGGATACGGCGTCGTCCGCCAGTTGTCCTCGCGCGAGGCCGCCCGCGTCTGGCAGGACCTCGCCCCCCGGGTCGCCGACGACCGGGACGAGCGGCGGACCGTTCTCGCCGTCCCGGCCGCCGACCTCGACGTCCTGAACGGGGCGGCCGGGACCGCGGTGGCCGGGCCCCTCGCCGAGCGCCGCGACGACCTCGCGGCGAACGCGGAACGGAAGGTGCTCGCCTCCGCCCAGCTGAGCGCGGACGGTCTGCTGCGCAAGGGCACCGCCCCCGGCAAGCGGGAGCGGATCCGCAGCACCCTCAAGCCTTCCCCCTTCGGGCCGAAGCGCGCGACGGACGCCACGCCCGTCGAGGCGGCCGAGCGGCCGGTTTCCGTCTGGGAGCGTTCCCTCGCCGGCCGGCCCGCCTTGGTCGTCGGCTCCTCGAAGGAGGGTGTCGTCGAGCCGGCCGCCAAGGAACGGTTCGTCCGCCTGGCCCTCGCGGCGGACGCCGCCGTACCCCTGCCCGGCACGGACCCTGCCGCCGCCGGCGTCCTCGCCCGCGACCTGGGCGTCGGCGACGCCCTCCTCCCCCCGGACTGCGACGCCGGCGCCGAGTCCGCGTTCGAGACCGCCGGCCTCAAGGAGCACGTGGCCCGGGTCGCCGAACTCGCCCGCACCCTCGTCTACGAGGACGCGGTGAGAATCTGCTCACCGCGACCTTCGCCGAG11700GTCGCGGACCTGGAGCACCTCCCGGTACCC11760AACGAGCGGAACGCGATCGGTCCGGCGGCC11820GGGGCCGGCCATGTCGTACCGATGGACGCG11880TTCGCCGACCGGGTCACGGCCGGGCGCACC11940AGGGCCGCGGCCGAACCGGCGGTACCGCCC12000CGGAAAGCGGCCACCCATGGATAGTCCGGA12060CGTCTGTTCCCAACCCGCCGACATCATCGC12120CTGCGACGAGCTCAGGGACGTACGGCGTAC12180GGAACTCACCGAGGTGGTCCCCCTCCTCAC12240GCTGCGCCGCCGGCTGCACCGCGCCGCGGA12300CCTCGCGTACGGAACCTCGG(^GGACGGGCC12360CCTGGCGCCCGGGGCCGCCGCGCTGCGGGC12420CCGCCTGGAGGGCGCGTACGAGGAAGGGCT12480CGCCCTCGACCCCGCCCTGCGTGCCGGAGC12540CATGGACCGCTACGCCGCCGCCGTCGCCGA12600GACCGAGTCCACCGTCCTCCATCTGCTGAC12660GCAGTTGGTGCACACCCGGCCGGTCCTCCT12720CATCGCTCCCGTCACCGCTCCCGGCACGGA12780CCGGCTCCCCCGCCAGCTCGTGAGCTGGGT12840CCGGCATGTCGCCGTCCTGCGCCGCGCACC12900CTTCCTGCTGCGGGGCCGGGACGGCACCCA12960CGTCCCGCCCGGCGTGGTGGTCACGGCGGT13020CGAGCGGGAGCTGCGGGAACGGTGCGCCGC13080TGCCGCCCTCGACGCGCTGATCGGCCAGGG13140CCAGCACCCGCACCCCCTCGCCCGCCTGGC13200CCTGCGCGCGGCCGTCAGCGGGCTGGCCGG13260CGAACGGCGCGAGGAACTCGTCTCCCGTAT13320CACCGGCGTCGCCACCCCGCCGATCGACGT13380CGTGGCCGTCCCGCGCGCCGAGGCGCCCGG13440CCGGTGGGGGCGGCATCTGCCCGCGCTGGCCGACGCCGCGCACGTCCGGGCGATCGTCGCGCCGCACCGGCTCCTTCCCCTGTTCGCGGCCACCGAGCGGCTCACCGACGTGTCGGCGCCCGCGGTGTTCGCGCTCGCCGATCCCGACGGGCGGGCCGCGCGGCTCGCCGACGACCTGCCCTGGCACGTCCAGCACCACGCCGCCCCCGAGATCGGCTTCGGCCGCGCGATCTCCCGCTTCGGTTTCGGCGACCTGGTCACGGCCGAGATCACGGACCTCGCGGCCGTGCTCGGCATCAAGCACCTCCGCTACCCCTGCGCGACCCCGGAGGAGGACTGCTGGGTCGTGTACGACGCGGAGGACGGTCCCGTCCTGCGGCTGGTCATGCTTTTCTACCAGTTCCTCAACTTCTTCAGCTCCCGGGTCGACCAGCGCCGCGAAGCCGGTGCTGGTGCCGGTGCCGGTGGTGGTGCCGGCTCGGTCCGCTCCGGCAGCCTCGTCCTGGCCCGCCCCGGCCTCGCCGGCGCGACCGGTGTCGCGTCGCTGGGCCTCCCCGACCAGGTCTTCTACCCGATGTCCCGGGAGGAGCAGCGGACCTGCGCCGAACGCAAGCCCACCTTCCTGGACTTGCGGGCGCTGCGCCGGTGCCAGGGCGGCCTCGAGGACGAAGGGGCGGCGGGCGGCCTCCGGACCACCGAGACCGAGGAGGAAGGGTGAGCCATGTCTTCCACCACGGCGACACCGATCCGGACGACCTGCGCCGGGCGGGACGCGTGGAAGGCCCGCACGTCCGGCTCCGGATGCGGGTGGAGACCGAGCGGCGCGTCCTCGACTACCTCCCGGCACGGCTGCGGGACGCGCTGCGCCGGGATGTCGCCGTGCGCACCCCGGAGCCGCCCCCCGTGGCACCGGCTGCTGCCGCTCTTCGA13500CGACGTGGTCAGGACCCACTTCGGGCCCGG13560GCTGTCCTCGGCCAAGGTGCAGGCCGTCCT13620GCTGCCGACCCGGTTGCGCACCCTTCAGGA13680CGGCGCGGACACGGTGACGGTCGACCCGGA13740GTCCTGGATCCCGCGCTGGTCCCGCGCGAG13800CGGCGGTCTGCTCGTCGTGAACGGCGGATC13860CCTGCCCGCCTACGCCGCCGCCGGGGACGC13920CGCCCGGGACGACGACCCGTCCGCGCCCCT13980CGCCAACGTGCACTCCCCGCTGCTCGGCCG14040GGAGTGGGGCGGGAACACCGGCGTCTCCCT14100CACGGGCCGGCTGCTGCCGCGCCTGGGCAA14160GAACTTCCTGCTCAACGAACTCGCCCCGCG14220GGGCAGCCTCGCCAACCTCGCCTGGTGGGA14280CGGTGCCGACGGTGGTGCCGGTGCCGACGG14340GGGTACGGACTCCGTACGCCACTACCCGCG14400CCGCACCTGGAAGGTCCCCGACGCCCGGCT14460GGCCTTCGCGGAGGTACGGCGCTGGCAGGA14520CCGCGGCTTCACCCTCCCCGACCCGCTCGT14580GGCGAGGACCCTCGCCCGGTTCCCCACCTC14640CACCAGCGTCACCAGCGTGACCGCCTGGCA14700GACCTTCCAGGAGTGCCTGCCCGCCGTGGG14760GGAGCGGTTCACCGAGGAATTCATCGTCGA14820ACCGCCCCGGCACACCCCTGGCTGTCCTGT14謹CTGATCGCGGAGTGCCTGCTCCCCCTGGTC14940CGCCACTTCTATCTGCGGCACTGGGAACGC15000CCGGCGGAGCACCACGACGCGGTACGCGCG15060GGGCGGCGCCCCGGCCCCGTCGACACCGAT15120CTCAGCGAACTGGAGCACGGCGACGCCTCC15180AACTCCCTTCGCTGGATCGACTATCAGCCC15240GAGCTGGCCAAGTACGGCGGGGCTCGGTGCTGACCGGCCAMCGCGCTCCAGCTGCTGCTGCCGTCTTCCTCCGTACCTACTCGCCTCCTGGGACCGGCAGGCCTCGACGCCGGCGACCCGTCCAGGCCCTGGAGCGGCTACCGACCCCGCGGCACCGCCACCACCAACAACCGGATGAATCGCGCGCGGCCGAGGACCAGCCCAGACGGCACACGAGAGCGTCAGCGTCCCGTGGTCGGCGACGAGTCGGTGGAACTCGCGGGCGGCCGTTCCTGCGGCGAAATCACGGATCCTGGCGCCCAGCCGTACAACCACGAACGAACTTCTGGACCGAGACCACCGGGCCCTGCGGCTCGCCGGCCCCATCTGCCGCCGTCCAGGCCACGTTCGAGGGCTACACGCCCAACGGTACGAGAAGAGCAGATGAGCGAGCCGGACGGGTCCGGCTGCGCGACTACGCCTCTACGCCACGCCGCGCACTACCGCAGGCCCGACGTGGCCACCGGGCGATCGCCGACACCTGGCCTCGCGGCTGGCGCGCTCGCCGACCGGTACACGCCCTCGACGCGGCGGCGCTGAGCCCCGGGGACCGCCGAGGCGCCCGAAGGCGTGGCGATCGCGGAGGACGTCTTCCACGCC15300GGTCCTCCGGCTGGTCGACAACGACCGCGCCCGGATGGGC15360GCTCGCCTCGCGCGCGCTGGGCTTCGGTCCCGACGAACGC15420CCAGGAACGCTGGTCCGGATACGTCACGGACCCCCGCGTC15480GTACCAGCTGCAGCGGGAGCAGTACGCGGCCCTGCTCGAC15540CGTCGGGAAGGGGCTCGGCGCGTACTGGGAACGGACCCTC15600GCGCCCGCTCGTCGGGACGGGCCGGGTGTGGCCGGCGGAG15660GGTGGTCGCGCTCGCGGCCCTCGTCAGCCAGTTCCTGCAC15720CGTCCGCCCGCAAGGCGAGTGCTTCGTCGCCTACCTGGCC15780GCGCGCCGCCCGGTCCGGCACCGCCCCGACGGCCGACCCC15840GGATGAGCGTCATGTCCACCAGTGAACAGAAGGATCTCAC15900TGCAGGAGGACCTCCTTCTCGACGTGGCCGCGCCCCTGCT15960GGGAGACGGACTCGTGGTTCTACCTCCGCGAGAACCACGG16020TGCGCTTCGCGAGCCGCAGCCCGTCGGTCGAACGCCGCCT16080ACGTCGGGCCGACGATCGACGCCGGCGACGTCTTCACGTA16140ACGACTGGCTGGGCGGCACCGCCGGGCTCGGGCTCGCCGA16200CCCCGCTGGCGCTGGACACCCTGCGCGCCACCCGCGGCAA16260TGGCCTTCGACTTCCTCGTGTGCACCGGCGTCMGCTCGC16320TCGCCAAGTTCGGCTACAAGGCCGGCTATCTCTCCTACCT16380TGCTGCTGATCCGGGACCCCGAGGGCACCCGGGCCAAGCA16440ACCGGGAGTTGCTGCGGCCACGCCTTCGCACGCTCGTCGA16500GCGAGCTGACCGACGTGCCCGAGCTGGCCCGCGAGTGGCT16560TCCCCGCCCTCCAGAAGGGCTTCGACGAGGGCCGCTTCTA16620AGGCCGAGACGGCCAAGCTCACGCCGTCGCCCGACGGCCT16680AGTGGCTGTCCGACCTGCCGGAGCCCCCGGTGGCCGGCAT16740ACACGTACTACCAGGGCATGATCCGCGAGGACCGCCGCTT16800AGGCGTACACCAACTGGCATCTGTACCGGCTGGGCTTCCT16860TCTTCTACCTGATCGCCCGCGCCTTCGAGGAGGAGTACGA16920TCCGGTCGGTACGGCCGGAAGCAGAGGTCGCCGGATGACG16980CACCGCTCCGGTCGAGGCCGCCGCCCCCGCCGAGGCCGCC17040GGAACCGGGCCCTTCGACGCCGCCGGAGTGGACGGCTTCGTTCGACGAAGTCCACCCGGACCAGTGGCAGATCGACTGGCCGCTGGTACGTCGACCTGCCCCGGCGCGATCTGCCCGCGCGGCGGGCCCGCCGCGGCGGCCGCGTTCGCCGCGGCCGGGCCTGCTCCAGGGCGCCTACGGACTGACCGGCGTGCGGTGGTAGCAGCCCCGCCGAGCCCACCCCCGTCCACCGCAACCCGCGTGCCCTCGGGAGGCGTGACGTACCCCGCCGAGTGCTACGGGCACGGAGGGCACGGAGGAAGAGGACGGTCTCGACGCCGGTACGCCACGCACTGGCCGTGCTGTCCGCCGGCCGGCCGCGAGGGCGGTGTCCGGATCGTCGTCACCCAGCCGCTGTGGAGACTTCTCCTACCGGCTCGGCGCCATGGACACCGGTGCCGGCCGCCCGGTCCTGGGAACTGCGCTGCCGGGTGACGTTCGGCGGACCTGCTCGGGCTCGATCCGGAACACGAGGCCGTCCCTGGGAGACCCGGTGGGACCGGCGGCATCCGCGGCCGAGACCCCGCTGGCTGGGCGAGTCCGTCCCGCCCGGCCCCGCCAGCCGCGTGCCTGGCCGCCGGCCGCCCCGGCCGGCCCGTCGCCCGACCTGGCCGGGCAGGCGGACGTGGAGCTGCCCCGGCGTCGCGGACACCTGGTCCCGGTCGGCGCTCGGCGAGCAGTGCCGAGGTCCTGGCGGACTGGCTGACCACGGTGCTGGCCCGGAGAGCAAGCCGGCCGCGCCGACACCGACACCGGAGAGCGGGACCGTCACCGGCGAGGCCGCCCCGACGCACGCCCGCTGTGCCCGGCGTGCCTCCGGGCGCCTATCTGCCACACCCGGCTCGCCGCCGGGGACTTCGGGCCCGAGGTCCAGCGCAGCAGTCGGGCAGGCGCCGCTGACCGCCGTCATGATCGGCGCCGGCCGGCCCGATCGGCTACCGCACCCAGCATCTCCTCGCGGGCCGCGTGCGCGGCGCGCGACGGCAGATCGGCGCACCCGGGTCCTCGACGACGCGTTCGGGCTGCGGGCCCGGGGCCTGAGTGGGCGGTTACCGCCGGGGCCTCTATCTCGAGAACAGTCGCGGGCGACGGGAGTGGAATATGACAGCGCCGACCGACACCGGCCGGGACCAGCGAGGCGGCCGGAACGAGCGACGCGACTCCGCCGGCTCCGGGGCGAC17100GCAGCATCCCCAGCCCCTTC17160TGTCGCGCGCGGCCGCGGCC17220CGCTCGGCCGGCTCGGCATG17280ACCCCGAGGGCATCGCCAAG17340ACTACCAGCTGCGCCGCGCA17400TCGTCACGGCGGGCACGGCG17460GGGTGTACCACTACGACGCG17520CTGCGGAGCTGACGCCGCCG17580AGAACTACTTCAAGTACGGC17640TCGTCGGCCAGTTCGCGCTC17700GGGTGGACGAGCGGCGGATC17760CGGCCGTGCTGGACGTCTCC17820CCGCGCCCCCGGTCGGGCGG17880CCGGCACCCGCCGGATGCAC17940ACCTCCCGCCGGTCGTCCTG18000CGGCCGGGCGGCCGGTGTCG18060TCCGGGGCACGCCGCTGCCC18120CGCTGGACTGCGACGTCACC18180GGGCCGGCCGCCCCGGCACC18240GCCTGCTGATCGCGCGGTCC18300ACGGGCGCCGGCTCACCCGG18360TGTTCGCCGACTACATGAAC18420CCGATCCGCATCGCGGTCCG18480GCGTCCTCGCCCAGCGTCTC18540TGCTCGGCTTCGTCAGCGGC18600CGTCCCTGCTCCTCGTCCCC18660TGAGTCCGCTCGCCACGCGG18720GACGACCGGGACGAGCGAGG18780CGGGACGAGCGACGCGACCG1884030GGACGAGCGACGCGACCGGCACCGTTCTGCGGCTGCGCGCCGACGCCTACTACGTACCCG18900TGCCGAACGGGGTGTGGATCCGGACGACGGACGGCTCGTTCACCCTCAACGGCAAGGCGG18960TCGCGACCTGGGTCGAGCGGCTCGCGCCCCTCCTGGACGAGGGGGTGGTACCCGCCCGGC19020TCCTCGGGGCGCTCCGGCCCGAGCAGGCCGGGTTCGTGGAACAGCTCCTGACCCTGCTGG19080AGAAGCGGGGAGTGGTACGCCGGGAGGCCGCCGCCGACGGATTCGGCCCGGACCTGGCCC19140GGCTCTTCCCCCAACAGGCCGCGTTCCTCGGCCACTTCGCCTCGCGGCCCGCCGAGGCGT19200TCGACCGCGTACGGCGGCACCGGGTGGCGGTCGCGGGACCCGCAGGACGCGCGGACGTCC19260TCGTCACGACGCTCATGGAGGCCGGGTTCGGCGACTTCGCCCTCCTGGACGCCGAGCCGA19320GCACCGAACTCCGGGACCTGGCCGCCGACTTCGAGCGGCGCGGCGCACCCGTACGCCTGT19380CCGGGCCGGACGGTGATCCCCACGACCGCACGCTCATCGGGGTGTTCGACCCCGGGACGG19440ACGGCGAGGCCGACGCCCGGCGGTTCCTCGACCGGGCCGGCGCGCTCGGCGGCGGCGCCT19500GGGCGGGCCTGGTCAGCGGCCAGGCGATGCTCCTCAAGGGACAGGTGCCGGGCTCCGGCC19560GGGCCTGCGTACGGTGTGCCTGGCGGCGGCTGGCCCACAAGGCCGTCGCGCTGCCGCACT19620CCGACACGCTGGGCCATGTACCGGTGTCGATGGCGGCGACCGTGCTCGCCCAGGAGCTGT19680TCCAGTACGTCGCCGGGGCCGACGACTCCGTACTCGGCGAGGGCGTCGTGGTGGACCTGA19740CCCGGCTCAGCGTCTGGCGTACGGACGTCGACCCGGACCCGGACTGCCCGGCCTGCTCCG19800CGGCCCGCCCGTCCGGCGTACCGGCGCCGGCGCACCGCCGGACGTCCGCGTACCCCGACG19860CCGTCTTCGGCGCCCGCTGCTTCGGCCCGCTGTTCTCGTGCACCCCCGAGGAACTGCCCC19920AGTTCCCGCTCACCGCGCTGCGGGTGCGGGTCAACCCGCCGGGGCGCACCGCGCCGACGG19980GGGCGCTCGACGGCCCGGTCGTCGTGGCGCGGTCCATGGTCGACGCGCGCGCCGAGGCGG20040CCCTGACCGCCGTCGAGGCGACGCTCCCCGCGGCGGACGAGGCAGCCGTCGCGGGCGTGG20100GCCGGGACCACGCCGAGGCGCTGGCGCGCGCCCTGCTCCGGTGGGCCGACCGGACGCCGG20160AGGACGCCCGCGCGGAAGGCGGCAGGGAACCCGTCCGCACCGAGGGGACGGGCACGGCGC20220CCGCCCTCGCGGAGCTGGCCGCCCTCGCCGACACGGACGTCGCCGTCGGCGTCACGCACC20280TGCCGAGCGGCTTGTGGCGGGCCCGGGCCGACGACGGCACGGGTGCCGTCGTCCGGACCG20340GGTTCGACCCCGACCAGGCGGCGGAGCGCGCGCTGACCGCCCTGCTGGCCCGCCGGCAGC20400TGGGACCGCGGCCCGATGGAGCACCGGTCGTGGCGGCGTGGCCGGGCCCGGTCCCGTCCC20460GGGAGCGGGTCGGCGAGGTCGCGTCCGCGCTCGGCCTGACCTGGCGCGAGGCCGTCCTGC20520CGCCGCTGGTCGCCGCCGATCTGACCGCGATCGTCCTGACCGGGGAGTCCACCGGATGAG20580CGCGCCGCCGACGACGGCACCCCACGCGCCCGAGGGCGTCGACGACACGGCGGCCGAGCT20640GCGCCGTCTGCTCGACGACGTCGCCCGGGTCGTCGACACCCCGGCCGCGATCACCGCCGC20700CGCCCCTCTCCACGCCGGCCTCCCCGCCGAGGGGGACGGCCGCCCGGTCGTCCTGGTGAA20760CCTCGGCTGGGACGCCGACCTGTCCGGCCTGCCCGCCGCGGGCACCACCGTGCTGCCCGT20820GTACGGGCTCCGCGGCCGCGTCGTCGTCGGCCCGGTGACCCGACCCGGACTGCCCGGCTG20880CCCCCACTGTCTGGGGCTGCGCGTGCGCGCCACGGACCGCCCCGAGGGCGTCCCCCGGGG20940CGAGCCGGCCGTCCCCCGCCGGCTGTCCGCGCGGCCCGACGCGTGGCTGCCGCCGGGGAC21000CGCGCGCCTGGTGGCGGCCGTCGTCCGCGCCGAACTGGCCCGGCTCGCCGCCGGTGAGCC21060GCCGCTCGGCACCGAGGCCGCCTACACGGTCGGGCCCGCCCGGCTGGCCGGCGACTGGCA21120TCCGGCGCTGCCGCACACCCTGTGCGACACGCCGCACTGCCGGGGCGCCCGCCGCCCGCT21180GCCGCCCCCGCTCCCCGACCTGGCCACGCCGCTGCCCGCGACCGGTGTGGGCTCGGCCCG21240CCGCTTCCCGGCCGCCCGGTTCGCCGAACGGCTGGAGCGGGAGTACCTCGACGGCTGGTC21300GGGCGTCACCCGCTCCGCCGCCGTCGGCGACCGGGCCGTCCTGCCGTCGACGCAGGTGCG21360GGTGCCCACGGTCTGGGGGTTCGACGAGATCGCGATCGGCCGCGCCGAGGACTACGCGGG21420CGCGCGCCCGGCGGCGATCCTGGAGGGGCTGGAACGGTACGCCGGCTGGCACTGCGGCGG21480CCGCGACCCCGTCCGCTTCGCCTCCTACGCCGAACTCGCCTCCGCCGACGCCGCTGAGCC21540CTCCGGCGGACCCGGCGGGCCGGACCGACCCGCCGCCTCCGACGCCGTCGTCGATCCCCG21600CTCCCTGCTCCTGCACCCCGAAGAGGCGTATGGACAACCGGGGTTCGAGTACACGCCGTA21660CGCCCCGGAGATCCCGACCGGATGGGCGGAGGCGTACTCCGCGCTCACGGGCCGGCGGAC21720CCTCGTGCCGTTCCACGTCGCGTACTACGGCGCGACCCGCCGCCCCGAGACCGGCCCGCG21780CTTCGTCTACGAGAACTCCAACGGCTGCGCCCTCGGCTCCGGCACCGAAGAGGCGCTCCT21840GGCCGCCCTGTTGGAGGTGTCCGAGCGCGACGCCTTCCTGTGCGCGTGGCTCTCCGGGAC21900GCCGCTGCCGGAGATCGACCTCGGCCGGCTGACCGGCGAGGCGGCGCGGGCGGTACGAAC21960GGTGCGCCACCGCACCGGCCGCGAGCTGCGGGCCTTCCGGGCGCTGGGCGCCTTCGACGT22020CCCCGTGGCCCTCCTCGTGTCGACAGCGGAGGATCCGGACCTGCCGGCGACCCTGGTCAC22080CGCCGGCTCGGGCCTCACGGTGGAACGGGCGCTGCTCGGCGCCGTCCACGAGATGGCCGC22140CTCCGCACCCGTGAACACGGTCGAATTCCAGCGCCGCCGGGCCGAGTTGGAGGCGGCACT22200GGACGATCCCGGGCTGGTCCGCCGGATGGAGGACCACGCGCTGGTGGGCGCCCTGCCCGA22260GGCGCGCCCGTGGTTCTCCTTCCTCCTCGACGGCACCCCGCCGGGCGTGCCCGGCCCGGC22320CGCCCCGGACGGCCTCACGCCGTCCGGCGACGTCGCCGCCGATCTCGCCGCGATGCTCGC22380GGCCGCGCGGCGCGACGGGCAGGACGTCGTCGTCGTGGACCACACCACCAGCGAGCTGGA2244032CCGCCTGGGCCTGCGCTGTGTGAAGGCGATCGTGCCCGGCACGGTGCCGATGACCTTCGG22500CCACCGGCACCGCCGGCTGCCGCCGCCCGCGACGCTCCGCGCGTTCCGGGCCCGGCACAC22560CGACGGCCCCGTCGAGTTCTCCCCCGAGGAAGTACGGCATGAGCCACACCCGTTCCCCTG22620AGACGACGACTCCCGCTCCCGCGACGCCCGTCACCGCGGACGACGGCCTGACCGCCCTGC22680CCCACGGCATGGGCTGGGTCGTCGGCAGCCGCGCCCTGGCGGTCCGGCTGCTCGGTGACC22740AGCGCCTCGGCGTGCTGGAACCGCGCCCGGTCATGGAGCACCTCGCCACCAAGGTCGATC22800CGGGCCTGCGCCCGCTGATCGACGAGATCTCCTCGTTCGCCGAACGCGTCATGCTCCAGA22860CCGGCCACGACCGGCACCAGGAGCTGCGCAGGGCGTTCGGCGGATTCTTCGACGCCGACG22920CGGTCGAGGCCCGCGCTCCGCGGCTGCGGCAGGCGGCCGAGGAGATCGTGCGGCCCTTCG22980CCGCGGCCGGGGGCGGCGAGTTCATGACGGCGGTCGCCTTCCCGTACGCGGTGCGCACCG23040GCGCGCGGCTCATCGGCTTCACCGACGCGGAGTACCGGCTGCTGCACAAGCTGAGCCAGC23100AGGTCGCGCTCGTCGCCTACGCCGCCCGGGTCGAGGACCCGGCGGAGGCGGTCCGGGCGG23160GCCACGCCGCGCTGGTCCGGGTCCGGGAGATCCTCGCGGACGCCCGGGCGGTCGCGCCCC23220GCGACTCGGTGCTCGGCGCCTGGCACGCCGGGGAGATCGGCCTGCCGGACGCGGACGTCG23280AGGCCAACGCCGTCATGCTGGTGCAGGCGTCGCTGGAGACCGTCGCCGGCATGCTCGGGA23340ACACCGCCGTACGGGTCCTGGACACCCCGGGGGCGCTCGCCGACCCCGTACTCCTGGAGG23400AGCGGATCGACGCGGCCCTGCGCGCGGAGCCGCCGCTCAAGACGCTGGAGCGGGTGGTCA23460GGGAGCCGTTCCGGCTGGCCGGGCGGGAGTTCCGGGCCGGCCAGGTCGTCTCCGTCCGGG23520TCGCGGACGCCAACCGGCTGGACGACGCGCCGGTGGCCGGCCGCGCGCTGTTCTCCTTCG23580GCTGGGGCGCGTACCGCTGTCTCGGCGCGCGGCTCGCCCGCCACCAGGCGCGGGAACTGT23640TCCAGGCGCTGCACCGCGCCGCGCCGGACGCCACGTACGCCGACGCCGGCGCCGCGGTCG23700AGCGGGTCCGGCACATCCGCTTCGACATGCCGCGCCGCCTCCCCGTGCGGTGCGCCCCGC23760CCGTCGCCGAGGCCGCCCCGCCGCCATCCGGTGTGCTCCCCGACGCGCACACCGAGGTAC23820CCACCGAGGTACCCACCGACGTGCTCACCGACGTCCTGACGGAAGCACTCGAAGAGGACG23880ACGTGGACCGGCCGTTGACCTCGCTGGAGCGCGAGGTGACGTACGTCGTCCTGACCGAGA23940ACGGGGTGACCGTCCCCGAGGACCTGCGCGGTCCCACGACGATCCGGGAGTGGGTGACAT24000GGGCGGCCTCGGCGCGTGGCTGAAGCAGGCCGAACCGGTGCTGCGGCGTTCGCGCGCCGG24060GGTGGTGACCGAGCGCGGACGTCATCCGTGGCCGTCGCTCCTCGCCCGGGCGGAGCGGCT24120GGCGGCGGGGCTGCCCGACGCCCCGTACGGCTGGGTCGTCCCGGCGGACGGCGGCGAGCG24180GTCGATCGTGGGGCTGCTCGCGCTCGGGCTGCTCGGCCCCGCCCCCCGCTGGGTGCTCGG24240CGACCCCGCGGGCCGGGCCCCTCGGGCACCGGTACGGCTGCTCCGCCATCCCGCGACCTCCCGCCCCGGCCGGACGGCACGCGGGCGGGCCACCGAGACGGCCCCTGCCAGTGGACCAGGGGACGGCGCCGCTGGACGGCGATCACGGCCTTCCCGGACCCCGGGTCCGCCAGACCGGCCAGCCGCCCAGCCGAGCCCGCCCCGCGCGGCGCCGCCAGGGAGCTCGCCCGCGCAGTGGTCAGGGAGAGCGTCCGCGCCCGAGCGCTCCTCGCGACCTCGTTCAGCAGGGGTGACCCACGTGCCTGGGCCGCAGGCGGCGCACCGGGCAGCTACGCCGACGGACTTCGCGGGTCCTGTTCCGTGTGCCTGGGACTTCCGCGCGGATCCGCGGGACTCGTCAGGCAAACCCGGGGCCCGACAGTGACCACCGCGCGCGGACGGCCGTCCGGGGGCGACCGCCGACGCCTGCGGACGAGACCGGCGCCGAGACCGAGCCCGTTGGTCACCGAGCCCCCTCGTAGCTGGTGCTGGGCCGACGGCGCACGCGCGCCACCGTGCGGCCTGGACGGGCGGCATCCCCCGGCGGCGTGGGACGCGCTGCGGACGGCCACTCCGCCCGACGAAACTGCTGCATGCCGGACGGCCTTCTAGGCCCGGCTCCGCCGCCCGCGCACCTCCTTCGGGGTTCCCCGGTCGGGCGTCTGGCTGGACCCGGTTCGCGCGATCTCGCACCTGGTCAACGCTGCCCGGTCACCGTGATCCCGCCTGCCCCTACGACGCGCCGAGACGCGAGAAAGAGCGCACTGCTCATGGAGCCGTCCGCGACCCCCAGCCACGCCGCTGCGCCGGTATCGAGGCCGGTCGCGGAGCGACGCCGACCGCGTCCGGGAGGCAGGCTCGCAAGCCGCTGGGTCGCCGGCCTTCGGCCGGGTACGCGGCGCATGGTGGCCCTGGGCGACGGCTCGCCGCGCGTCCCCGCCCGGCTGCGACTACGGTGCGCGGACGTCGGGGGTGACCGGGCCACCGGGCCGGTCGACGGCTGTCCGGGACCGTCATGGGGCGTCCCGGTCCCGCGGCAAGCGAGGTCACCGTGATGACGAAGTGCCCGGCCCGACCGCACGCTTCAGCCTGGTTCTTCCATCCTCGGACGCGATGGCGGCTCGCCGCCCGGCGACGAGGTAGCCCGCCGATCGACCGCTGGGCGACGGGGCCCACGTACGCCGCACGCGAGCCGAGGTGTGCCCCGGCGAGCCGTCCCCGCTGGGCGCCCGGCGGCGGCCTTCCTGACCAGCCGACGGCCCCGGACGGCGGGCCGGCTCAGACGGCGGCGGTCAGCGTCTGCCTCGGTGTACCGACGCCGACCCCGCACCGTGCTGCGCTCAGCCGTCGAGATGACGACGCCCCCTCTACCTGGGTGGTGGCCCGGCCCGGAGCATGCTGGCTGTTCACCGCTCCTGGACGCTGCCCGTGCAGCGAACTGGGACGACGTCGACGCGGACTAGTTGACGGCCCACCGCCTCTCCCCTCGGCTCGCCACCTGTGACGCTCGGAGCTGGCCGGGTACGGCGGCTCACCCCCGATGCTCGGCTCATGTCGGCGCTCGGCACCCTCCACCGGGCCGGGGGTTCGTACCCGCGCGACCGTCGACCGTGACCGAGGAGGGTCCTGTGGAGCGCGTCGAGCCGACGAGACCGGACCCTGAGCGGATGCACCGGCTACGCCGCCGGGATCTGGCCTGCGCCGCGGGCGAGAGGCGGAGCAGCGTCATCGTGCCGGGTGAGACCGCGCGCCCGGAACTGC34243002436024420244802454024600246602472024780248402490024960250202508025140252002526025320253802544025500255602562025680257402580025860259202598026040TCGTGCCCGACTCCGTGCTGGCGCTCGCCGACGAGGCGCGCGACACCCCGGACGCGCTGA26100CCCGTACCGAGGTCCTCGCCAACGTCTTCAGCCTTTACATCGCCGGGCACGATACCTCCC26160GCAACATGCTCAGCGGCCTACTGCTGCGCCTCGCCACCCACCCCGGACTCCTCGCGGGCC26220TGGCCGACGGCACGGTGGACGCCCAGGCGGAGGTCGACCTGCTGCTGCTCGGCGAGTCCC26280CGCTCACGTTCACCGTCCGCGTGGCGCGGGAGGAGTGCGAGCTGGCGTCGCGGCGCATCC26340AGCCCGGCGACCGCATCCGGGTGATGCTCGGCGCCGCCAACCACGAGCTGCTCACCAGCG26400GCGACGCCAAGGACGGCAACACCGGCGTCAGTTTCGGCGAGGGCCGCCATGTGTGCCTCG26460GCGCGCACCTGGCGCGCACCGAGGGCCGGGTGATGCTCGACGCCGTGAGCCGGCGATGGT26520CCGGGCTGCGGCTCGCGGACGACCCGGCGTGGACGCCGCACTTCCTGCACCGGGGCCTGG26580ACCGCCTGCCGCTGGAGATCGCATGGCGGTGACGAGCGGGCCCGTACACGAGACGGCCCC26640CGTGGCTGCCGACCACGTCGTCCCGGGGCCGGTGGTGGTACGCCGCCGGTGCCGCGTCCC26700GGGCTCCGTCCTGGCGGACCTGGGCAGCGACCGGGTGTGGGGCGCGGCCGCGGCCTGGGC26760GTACGCGGAGGACGCCGTCCAGCGCACCGGCACCCGACTCGCCGACCAGGTCGGGGAGTC26820GGTGCCGACCGTACGGCAGGAGGAGCGCCGCGATCTGCTGCGGGTACGCCGGGCCGCGTT26880CAACTCCCGCCCGCTGCCCGCCGATTCCGGCGCCGTGCTCGGGGGCCCGGCCGCCGCCCT26940CGCGGAGGAGTACACGCGCGCCCTGACCGCGCGCGACACGAACCGTCACGAACTAGACCT27000GGCCCTCGCGGACTTGGCCCGTACGTCCGGCGCGACCGTCGACGCCCTGCTGCGCGACCC27060GCACCTCGCCCGGTCCGTCGAGCTCTCCGTACCCGGCCTCCTGGCCGCCGGCCCCGTCGG27120CGCACTGGACCCGGCCGCCGCGAAGAAGACCCGGCGCCGGGCCCTCACCCTGCTCCGCCT27180CATGCAGCGCGCCGCGTCGAAGCCGACCCCGTTCGCCGGCTTCGCCACCACGCACGTGCT27240CTTCGAGAACACGGCACCGGCCGCTTCGGACCTCCCCTCGCGCAGCCATGTGCGGATCGA27300CCGCGCGGTGATCGAGTGGGTACGGCAGTGGATCGCCGCGGGCGGCCACCGGAGCCTGCG27360GCCGGAGCGGCTGTGGCTCACCGCCAACCCCACCGCGGCCGTCCGCCAGGACGCCTCCGG27420GACGCGCGTGACCTGGCTCCTGAGCAGGGCGGACGGCACCGAACGGGTGCTGTCCGCGCC27480ATGCGGCGCCGCGCTCGCCGGCACCCTCGCCCGGCTGCGGGAACCGGTCCGCCTGGACAC27540CGTCGCGCCGGGCGGCACCCTGCCCGACGGCCTCGCGCGCCTCGTCGCGCGCGGGCTCCT27600GGAGATCGGTCCACGTCTTCCCTCCCATGGCCGGCGGACGCTGCACGCGGCGGCCGACGC27660GACGGCGCCGGCACCGGACCTGGACCTGGACCTGGACCCGGACGCGACCGAGGCCCGGCA27720GGCCCGTCAACTCCACGACGCGCTGGGCGTGTTGCGTGACGCCGAGGACGCCCTCGCCGC27780CGATCCGGCACACGCGGGCGCCCGGCGCGACGCGAAGGACGGCATCGCCGCCGTCGCCAC27840CGCGCTGGGGCGTGGGCGTCCTTCGGCCCCCTTCGAGCTGTCCGGTGCCCCGCGTTGCTCGCTCTTCGACCGATCCCGGCCTGCGCGGCCCGGCTACGGCGCTGCGCGACCGCCGCGTCGCTATCCGGGCCTCCTGCGCGCGCCCCGGGCCGGCCGCTTCGGCCCCCGGCCCTGTGGGATTCCGCGGCTGGGCGCTGCCCCCTGGCCTGGCACGACGCTCCGCCGCCCGCCCAGCCGCTGACCGCTGCCGGTACGTCCTGCACACGGCCGACCCCAACCATGGAGGCCTCGGCAGACCCTATCGGCGTCCATGGGCGTCCGAGGTCCTCGCCCTTCCAGCGTCCTGACCGGCCGACTGTCGGGCTCCGCGCTCCTCGACGTGGCCGGTCCCGCTTCGCCGTGGGCGGCGACTCGGCGCCACGCGCCCTGGGTGCGCGTGGACCGGCCCTGGCGGGACGCCCTGTACCGGTCACGTGGACGACCCTGGGGCGCGGAAGCCCTACCGCTGGTCCGGACCCCTTCCACCTCCGTGCTGGCCGGGATCCCGCCGGAGACCTCGCTAAGCAGGAAGATCCACCCCCGGCTTCGAGCCGGGACTACGGGCGGGCGGGTGCCGACGACCGACCTCGGTGGCCACGGCCCTACGCGTGAGGCCGAGGACGGCGGCGGACGGTGGCGGCAACTCGCCGGCCCGCGTCGCCCGCCGCCACCCGTCAACGAAGTGCGCCCCCGGGCGGGCGGCCCCGGTGCCGCTGTTCCCGGCTGAGCCGCCCTCGCGGCGGCTCGTGCTTCCCGGGCGCGCGCGCGTACGGGACGTCGTGCGGCGCGGCCCCTGGCGCCGCGGCACCACAGCCTCCGCGGACGAGATCACCGATCGCGCGCGCACGTCCTTCCGGGCGGCGGCGACXGTCGACGTTCCGCCGCGTCCAGCCTCGCGTTCGTTCCTCGACACTCGCCACGCCGGAACCCGCGATCTGCCGGGACCGCGTGCGCCGGCCTGCCCTCACGAGGCGGCCCCTGCGAGACCCCCGCTGCTGCTCCGGCGCCGGCCGTACCGCACCCTCGGGCCGGCCCCGGGACGAGCCGGCGCGGCGGGAGCCGGGCGTGCCCCCGCGGCCTGCCTCCGCAAGCGCGCCCGGGCGCCGCCGCACGCACGAGCGTGCTCCGTAACGTACGCGCTTGGACCTCACAGCGCCCGCTTCGGCGGTCGCGCGCCGACGCGCGTCGTGCCGGCGCCGCTGAGGGCCGCCGTCCTCGCCCGGCGCGCCGGGACGCGGTCAGTGGTGAACGGACCCGGCCCCACGGGGTCCCTGCTGCCCGGGGGCCCGGGTTCGGCGGCGGCCGGCACCGAACGCGACCCGGATCCGGGGCCGGAGGCCGACCTGGAAGGGGCGGAACCGCGCCGCTCGTGCCGACAGACCCCCTCTACGGACACGCTGACCCGGCAGCCACCGAGGGCTAGGCACCGTCGGACGTGCTCCTTCCACCTGGCTCGTCGGCGCGAGGACGTGCACGGGCCCCGCTGCTCGGTCGGCACCGAGGGAATCCGATGCGCCACCGAGGCCGCGTGTGGACCTCTCGGGTCGGCACGGCTCGCCGTCGGTCGACATCCGCGCTCGGTCGACCTGTCAGGGCGACGAGCACCGGCACTGCCGACCGTCGACGCTCGCTACGGCACTATGCCCGCGGCCGCGGGAGGCTCCTGCGCGGGCCCGGTGGATCGACTTCTCCCTTCACCGAGCGTCACCGAGCGCCGCCGCGCTCTGATCAACCACCGCGCGACGACTGGCGTGACCTGCC362790027960280202808028140282002826028320283802844028500285602862028680287402,028860289202固02904029100291602922029280293402940029460295202958029640GCAACACCGACACGGTGTACGCGCTTGAGCAACGCCCCTTCGTGGACGGGTACAAGGCGG29700TCATCGACGAGGTACGGCGGCTCACCGCCGCGCCGGTCGTCGCGGGCGGAGTGGGCTTCT29760CCACCATGCCCTTCGCCCTGGTCGACTACTTCGGCATCGAGTACGGGGTGAAGGGGCCGG29820GCGAGAAGATCATCTGCGATCTGGCGCGGGCGCTCGCGGAGGGACGGTCCGCCGACCGCA29880TCCACATCCCTGGACTGCTGGTCAACCGCGGCCCCGGGAACGTCACCAGAGTGGCCCCGC29940CCGCGCTGGATCCCCGCGCGGCCCCCGCGCCTTCTTCCTCGCCCTCACCCTCGCCCGCGC30000CCTCTTCCTCGTCCGCCCCGGTCCCCGTCCCGCTGTCGTTCGCGGCCGTCGGCCACCACG30060AGAGCCGGGCCTGGCAGGCGGAGACCGAACTCCCCTACACCCGGCGGTCGGGCGAGCCGT30120ACAAGGTCGACAACCTGCGCTACTACCGCGAGGGCGGCCTGGGCAGCATCCTGACCAAGA30180ACGGCTGCGTCTACAAATGCAGCTTCTGCGTCGAACCGGACGCCAAGGGAACGCAGTTCG30240CGCGGCGCGGCATCACGGCGGTGGTGGACGAGATGGAGGCGCTGACCGCCCAGGGCATCC30300ATGATCTGCACACCACGGACAGCGAGTTCAACCTCTCGATCGCGCACAGCAAGAACCTGC30360TGCGGGAGATCGTGCGGCGCCGCGACCACGACGCGACCAGCCCGCTGCGCGACCTCCGGC30420TCTGGGTCTACTGTCAACCTTCCCCGTTCGACGAGGAGTTCGCGGAGCTGCTGGCCGCCG30480CCGGCTGCGCGGGCGTGAACATCGGCGCCGACCACACCCGTCCCGAGATGCTCGACGGGT30540GGAAGGTGACCGCGAAGGGCACCCGGTACTACGACTTCGCCGACACCGAGCGGCTGGTGC30600AGCTGTGCCACCGCAACGGCATGCTGACGATGGTCGAGGCGCTCTTCGGCATGCCGGGGG30660AGACCCTGGAGACCATGCGCGACTGCGTCGACCGGATGATGGAACTCGACGCCACGGTCA30720CCGGGTTCAGCCTCGGCCTCCGGCTCCTCCCCTACATGGGCCTGGCCAAGTCCCTGGCCG30780AGCAGTGCGACGGGGTACGCACGGTCCGCGGGCTCCAGTCCAACAACGCGTCGGGGCCGA30840TCGTCCTGAAGCAGCTGCACCAGTGCGACGGACCCATCGAGTACGAACGGCAGTTCATGT30900TCGACGAGTCCGGCGACTTCCGGCTCGTCTGTTACTTCTCCCCCGACCTCCCCGAGGCTC30960CGGGGACGGCGGACAGCCCCGACGGGATCTGGCGGGCCTCGGTCGACTTCCTCTGGGACC31020GCATCCCGAAGTCCGAGCAGTACCGGGTGATGCTGCCGACCCTCTCCGGCAGCAGCGAGA31080ACGACAACAACTACGCCGACAACCCCTTCCTCACCAGCCTGAACAGGAAGGGATACACGG31140GCGCTTTCTGGGCCCATTGGCGCGACCGCGAGGCGATCATGAGCGGCGCCACGCTCCCGC31200TCGGGGAGCTCGCGGAGGCCGTCCGGTGACCGCCCCCGCGCTCCCGCTCGCCGGGCAGGT31260CGCGCTGGTCACCGGGGCCGGCCGCGGCATCGGGCGGATGGTCGCGGTCGCCCTCGCCGA31320GGCGGGCATGACCGTCGGACTGGTCGGCCGGAACCGGCGGCTCCTCGACGTCACCGCGCG31380CGCCTGCGCCGCCGCCCGCGAAGCCGCCTCCGGGGACGGCTCCGGGGACGGCTCCGGAGG31440CGGTCCGGAAGGAGGCCGCCCGGCCTGGTGGGTCTTCGAGGACCCGCCGCCCCCCGCTACGGCGGAACCGCACCGACATGCCTGTTCCTCCTTCCTCCACCTGACGGCACCCGGGACCCGGGTCCTCGGGCTCTTCGGGGAGACGGCAGACGCCGGCCCGACATCCGTCTGGTGCGGCATCCTCGGTCCGGGCCCCTGTCACCGGGTCGACGGTCGGGTAGCACGGTCAAAGMGACCGGCGGACGTCAATGGCCGCGGACGGGCACGGGCCGACGACCCTCAAGAACTCCGTAGTCCTGGCCGGTGTCGGAGGCGGTACGACCAGGGCGACGAACGTCCGGATCGGGCCTCCGCCTACTCTCCGGGACCACCCTGTCGAGCGGCGATACGTGGGCGCCGCACCTGCGAGGGCCGGCCTCCCGGTCCTCGGTTTCCCTGACGTCGGGCACGCCCGGGGCCACTCGGCGTCGGTGGTGAACGCTGGAGAACGCTGCGCCGCGAACGGGGTGCAGCGTCGACGCTGACGAAGGGTGAACGGCGGGCGGCGGGCCTGGACGTCCTACCGGATAGGCGGGCCCGCTGACCGGCCCCCGGGGACGCCGTGGCGGTGGGCCGCACTAACTCCCCTTGCGGCACCGTGCGTCGTCAAGCGGCTCCAAGCGGGGTCCATGGCCGTCTGTCCGCGTGGCACGACTTCGAGACGAGTGCGGGGCCGGACCGGCCGGAACCTGTCGGCGTGCACGGGGGTCGGGCGACCGTTCCGTGGCGGGAGATGTACGGGCTCGGGCCACGGTCTCGTCTGATCCTCGGTCCCCGCCGGTGGGCGCGGGCGGTCGACGATCAAGGCCCCAGGCGACGAGACGCGCAGACACCGGCTCACGGTCTCGTCTACTGCGCCCGGCGGATGTCCGGCCCCGTCCTGGGAAGCCCAACACCTGCCGTCAACTCCGGCGACGCTGCCTGTTCGACCGCGGGCCGCCCACGGCGACCGCGTTACTGGAGCGGACCGTCGGCCGGTCCTGATCGAACCCGGCGGGGCTGGTCGTGGCTCGCAACAGGCGGCCGGACTAGGACAGCAGGCGTCACCGTACCAGGACAAGCGACTGCGGGCATCCCGGTTCCAGGTGACTGAGGACCACGGACGTCCAACGACCAACGGCGGCCAACGGCGCTGAAGACCGGCTCACGGACAGGGCCGGCGCGCGCCCGGACCTCCTGGGATCCGGAGCCGGGCCGTCCCGCCTCGCCGCTCGCCCTGGATCAGTCCGGGACGACTGGACTCGACCCCCAGCGAACGGCCGGGGCGGACCTCGGGCCGGCCTGGGGCAACGTTAGCGTCCCGGCGTCGCGGGATCTCGCGGCCGTCGGCTTCGCTGCCCCAACGGCACCGAAGCCCGACTGACGACATCGAGCGGCAAGCACCACGTCCCAACGGCCGCCGGGGGCATCCACCATCGCCCGACAAGCGGCAGCAACGGGGGCGTCCCGGGGTGAGGACGCCGAGATCCGTGAACAACGCGGTGGTGGGATGCCGGAGATTCCAGGGAGAACGCGGTCGCGCATGGTGCGCGGACCCCGCTCGCCGGCCGGGCTCGCCGTCGGAATCCGGTCCTCGGGCCTCCAGGGGTTGTAGAGCGTCCGTCGGGCCAATGCGCAAGAGCGCTCTCCGGAGAAGATCAAAGGCCGGCGGGCCCCACCCGACTTCTGCTGTCACCAACGGGCCGGATCGATCACCGGATGAGCTGAACCCTGAAGGTGCATCGGTCTGAAGCATCAGCGCACGGCGGGTTTCCCGGTGG38315003156031620316803174031,318603192031980320403210032160322203228032340324003246032520325803264032700327603282032謂329403300033060331203318033240CCGCGCAGACGGTGCGGGCCGCGGCCTCCGCCTCCGCGGTGCTCGCCAGAGTGCGCCCGTGCGCGCTGCCGGCGATGGCGCGGCTGAGGATGCCGCCTTCGCCGCTGCCCACCGCCACCATGGTCGTGCCGCGCGGGTCCGAGGAGCCGCCCCAGAGGGTCAGTTCGCCGTCCTGGTCGGGCGCCAGCGCCTGGAGTTTGTCCTGTTTGGACGCCGCCCGGTCCCACAGCTCGGCGCTCCCGTCCGCACTGACCGTCAGCCCCTCGATGCCGGCGGGCCGGTCCGGCGGGGTGAGACCCCCCAGCGTCCGGCCGTCCCCGCTCAGCCTCGGCGTACGGGAGAGTTCCCGGGGTCGGGCCGCGTCGCCGCCCGTGGCGAGCGTCCGGCCGTTCGTCGTCCCGGTGTTCAGCGTGCCGAGCGTCCACAGCCGGACCGTCCCGTCGGACCCGCAGGCGAGCGACAACGCGCCGTACGCGGCGGGCTGGAGCTGTCCGGCCTGGGTACCGCCGAGGCCCCGGACGTTGGAGGGAGGGGCGGGGACGGTGACGTTCCACAGCCGCACGGTCCGGCCGGCGCTGAAGGCCAGGGAGAGCACATCGCCGGGGCGGGCCGGGTCGGCGAGGTCCCACAGGGTCCGCCCGTCGGGGCTGTAGGCGATCCCCGGTTCCACGGGTGCCCGGTCCGGATGGGGCCAGGCCCCGGTGAGCGTGCGGCCGTCGGAGGCGCCGGCGAGCCGGCGGGGCGGGGCGGGGGTGCCGAGGTCCACGAGGACCATCCGGCCGTCCGGACCGAACGCCACGACGTAGCTGGCGGGCTTGGCGGGGTCGGTGACGTCCCACATGGCCAGGAGCCGGCCGTTCGGGCTGAAGACCGGTGCGCCGAGCGGCCGGAGGCGGGCCGGGTCGGTGGTCCAGATACGTACGGGCTGGT33300CCGGGGCGAAGGCGAGAGCGACCATCTTCT33360AGGACGCACGGTCGGCACCCGTGAAGCGGA33420GGTCTCCGCGCGCGGTCGCCGCCAGGGCGG33480GTGCGACCGGCCGCGCCGGGTCCGCGAGGT33540CCCGGGCCAGCGTCCGGCCGTCGCCGCTGA33600AAGGCCCGGGCAGCGTGGCCGTACGGTGCG33660CGCCGTGGCGCCCGACGCCGATCATCCGCC33720CGTCCTCCTTGCCCGGCAGGCTTCCGGTCG33780AGACCCGCACGGTGGCCCCGTCCTCGAAGA33840CGTCGTCACCGGAGCCCTGGCTCGCCCCGG33900GATCGGCGAGGTCCCACAGCACGAGAGGGC33960CCCGGCCGAAGCCGAGGACGGTCACTTGCT34020GGCGGGGGGCGCCGGCCGGCGAGGCGAGGT34080CGGTGGCGAGAGTCCGGCCGTCCGGGGCGA34140CGGGAAGCACCCGCCCGGGCAGGCTCCACA34200CGAGGGTGCGGCCGTCGGGGCTGAAGGCCA34260CGGCGGTACCGGCCGGGGACTGGGCCGGGT34320CCCGGTCCGAGACGGCCAGGGTCTGGCCGT34380CCGCCGCCGGCAGATAGAACCGGAGCCGGA34440GCCAGACCCGGGCGTCGTTGCCCATCGCGA34500GGTAACCGCTCGCGCTGGGCAACGGCGGGA34560CCAGATCCCACACCCGTACGGGGCCGTTGC34620CCCCGAAGCGCAGGCTCTCCGGGAAGCCGT34680GCCGGGCGGGACCGTCCGGGCCGGCGGCCG34740GGTCCGGGCCGACGCCCGCCAGGGTGCGGC3權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ycesATCC31255GluLeuVal35AlaSerGinAspHisProGluThrThrAla5HisValTrpPro20HisGlyTyrAlaLeuThrArgHis55GlyArgSerGly70ValAIeiAspLeu85GlyLeuProVal100ArgArgPheAla10ProAsp40GlyGlu25HisThrAlaAlaGlyAlaAlaValGlyAlaArgAlaThrValMet105Val90lieGlyValArgHisPro15ProArgTyrLeuAla30ArgTyrGlyArgLeu45TyrAlaProAspHis60AlaLeuValThrAspHis7580ValGluArgAlaArgAla95GlyHisSerValGlyGly11044MetValAlaValArgTyrAlaGinArgHisProGluAspLeuAlaAla120125LeuGlySerTrpHisThrAlaThrSerLeuAla115LeuAlaAlaProVal130LeuLeuAlaPheAla145ValMetSerArgVal135Glulie150ProAlaGluAlaTrpHis140ProGluMetProValAspValGly155ArgTyrAsnAlaAspProAlaGluAlaAla165170LeulieTrpHisGlyAlaPheValArgAspThrLeu180185GluArglieAsnGlyGlyAsp175ValSer160ProValThrCysLeuGluArglieAsnGly195200TrpLeuHisGlyAspAlaAspProThrLeu210ThrArgAlaGlyVal225ValTyrProGlyAsp215GluLyslieArgGly230LeuHisGlyLeuGluSer190GluSerLeuAlaPheLeuPro205LeulieAspGluArgAlaThrAla260ProThrArgPro275Ala245AspLeuThrAlaLeuAla220PheHisLeuAlaGlu235HisAspGluGlyPhe265Phe250LeuAspAspAlaLeu270Arg255ThrArg240AspVal<210>4<211>618<212>PRT<213>iS/re/^o附少cey/a訓加7ATCC31255<400>1MetCysGlylieAlaGlyTrpValAlaPheGlyArgAspLeuGlyAla45<formula>formulaseeoriginaldocumentpage46</formula>ValProLeuAlaAlaThrVal305AlaCys290ThrLeu275PheCys260Ala245SerLeuLeuSerValGlyAlaSerValGlyGlyProArgArgHislieLeuLeuSerLeuProLysAsp385ArgAla370GluGin355lieAsp340GlyGly325ProGly310AlaPhe295GlyLeu280SerGly265Arg250GlyLeuAspSerAspArgGlyAlaAlaGlyAspAspSerArgHisGluAlaAlaArgLeuAlaAspArgGinArgLeuPheGlyAlaGluThrGlyGlyProAspAlaValHis465ArgLeu450LeuTyr435ProAla420ArgPhe405PheGly390ProSer375TyrMet360ThrAla345AspAsp330AlaAla315LeuSer300TyrGlu285GlySer270Asp255SerValProLeuProGlyPheAlaAlaGluThrLeuAspValLeuArgThrSerLeuValAlaLeuProTrpPheTrpMetTyrLeuAspAlaAspGlyGluAlaPheLeuThr470ProAla425ArgTyrAla440GluGluAsp455PheLeuProMet410LeuHis395AlaSer380AspTyr365GlyAla350LeuAla335ArgLeu320LysAspLeuPheGluGlyAlaGluAsnAlaGlyValAlaProGluGluAlaValArgArgMetLeu475Met460LeuArgLeu430AlaAla445ArgLysAla415SerVal400AlaLeuValProValGlyGluArgLysMetSerMetAlaValGlyLeuGluValArgValProPheLeuAsp480Asp47485490495HisAlaLeuValGluTyrValPheAsnValProTrpSerMetLysThr500505510PheAspGlyArgGluLysSerLeuLeuArgAlaAlaMetAlaProGlu515520525LeuProAspSerValLeuThrArgLysLysGluProTyrProThrlie530535540AlaAspProAlaTyrAspGinLeuLeuLysGlyArgLeuGlyAlaLeu545550555560MetAspAspThrGlyAlaProValArgAlaLeuMetAspGluLysSer565570575AlaAlaGluAlaTyrAlaAlaValArgLysAlaSerProGluGluLeu580585590ArgPheLeuArgValGlyPheGluSerLeuLeuLyslieAsnAspTrp595600605MetArgAspTyrArgValGluLeuAlaGlu610615<210>5<211>135<212>PRT<213>Sfr^o考ces/awre础ATCC31255<400>1ValAspArgAlaSerValGinGinLeuMetGluHisPheLeuAlaAla151015TyrAsnGluGlyAspProArgHisLeuAspHisCysLeuHisProGlu202530TyrArgHisProAsnProAlaValGluArgGlylieGluGlyMetArg354045AlaAla50LeuAsp65PheSerArgArgLeuPheGinLeu130<210><211><212><213><400>MetHis1AlaGluArgThrGlyTyr50ThrLeu65AlaSerlieArgArgTrpAlaSerThrValGlu55GluAspLeuValValGluGly70GlyArgGinValGlyPro85ValGlyLeulieAspLeuSerLeuThr60ValAlaArgPheSer100AlaGinHisTrpAsp115GlyAlaLeuProAsp135Glu120AspLysAla75lieLeuGlylieProAla90AspliePheLeulieGlu105110MetAspLeuLeuGlyLeuHisArg125MetThr80SerGly95AspGly6428PRTSfre/f歸戸51/aATCC31255LeuLeulieThrArgProAlaArgGinArgTyrVal510AlaAlaValPheArgGluArgAlaAlaAspMetAlaAlaValPheArg2025AlaPheProPheArgAsnlieAspGluLeu15HisAspAlaAlaPheProPheArg3540LeuAlaLeuThrValProArgGluLeuGlyGlyLeuGlyGly55AlaAspMetAlaValAlaGinGluGluLeu45GlyGly60LeuAlaGlu30ArgAlaAlaAlaAlaValAlaGinGluArg7075ThrAlaLeuAlaValAsnMetHisLeuSerMetAlaGlyCys80GlyGin4985MetAlaArgAlaTrpArgThrThr100lieValGluGlyLeuThrGlu115GluProGlyHisAla130ValGluGlyGlyTyr145SerAlaValMet90GlyAspGluArgAla105VallieLeuMetThr165ProAspGlyProHisValLeuVal180ValLeuGluGlyArg120LeuValArgThrThrGly135LeuValThrGlyAspLys150155HisMetThrSerMetAla95ArgLysThr110AlaThrAlaMet170ArglieProAlaGlyValArg195SerGluAsnPhe185TrpAsnThrSerGly125AlaLysAlaArgLys140ThrPheGlyThrGly160GluTyrArgGluHis175SerProThr200LeuArgAspValThrArgSerGluAsnlieGlu210215AsnValLeuThrSerTyrProValGlySer225230GinThrValTrpGlyTrpAlaMetProValTrpGly245GlyValAlaValGlyAlaPheGlu260ArgGlyTrpGluAspArgProTyr275280LeulieGluThrSerArgAlaLeuMet220LeuAspGlySer235PheAlaAlaValAsp190GlyLeuArgAla205ValProGluGluThr250CysValArgAspThr265lieArgAlaGluGluGluLeulieGlu290GluValMetGlyAsn305MetAlaArgAlaValThrSer295GlyLeuTrpAsnAla310LeuThrLysAsnVallieGlu300LeuThrlieGin315GlyThrAsnAsnLeuLeu240TyrLeu255ValArgSer270lieAlaAlaCys285ArgThrAlaGlyGinGly320AlaVal50325GlulieValAspArgVallieArg340ValTyrGluArgProGlySer355MetHisProTyrSerAsn370ThrLeuGlyGluGlu385LeuAlaGluProAla360AlaAspAlaCysAspLeu375380ThrAlaProValHisProPro390395ProGluAlaGluAlaGluVal330335lieGlyThrProAlaLeuArg350AlaGlyThrlie345HisArgAspValLysAlaSerAsp420Ala405ThrAlaValArgPro425Arg365lieAlaAlaThrAla410AlaAlaSerArgGluLeuAspArg400AlaGlyAla415<210>7<211>311<212>PRT<213>Sfre/^owyces/"wre础'ATCC31255<400>1MetSerSerLeulieGluThrAlaHisTrplieAlaCysAlaArgAla151015AlaGluSerLeuArgProAspGlyArgLeuPheGluAspProLeuAla202530ValGluPheValArgArgThrAspProAspLeuTyrAspArgLeuArg354045ThrGluProSerSerArgPheAspValLeuAlaValArgThrAlaPhe505560PheAspAspArgLeulieArgAlaValThrGlyGlyProAlaArgGin65ValValVal707580AlaGlyMetAspGlyArgAlaPheArgLeuProLeuAlaAlaGlyMetAspGly8590TrpProProGlyValThrLeuTyrGluValAspLeuProGluGlyValGluGluLys115ArgGly100SerAlaPheLeuLeu95GlyGluValAspLeuProGlu105110ArgSerGlyLeuValAlaAspArgSerArgSerGlyLeuVal120125ValProValAlaAlaAspLeuThrGlyAspTrpProAlaAla135AlaGluAlaGlyPheArgProGlyLeuProThrValTrpGly140ProCysArg130AlaLeuAlaGluAlaGlyPheArgProGlyLeu145150155ValGluGlyValLeuTyrTyrLeuThrGluAlaGinValAsp165170ValAspArgValThrGluLeuSerAlaAlaGlySerValAla175CysLeu160ValLeuGluGinVal195GinValThrGluLeuSerAlaAlaGlySerValLeu180185190AsnThrAspLeuTyrArgAlaProTrpMetArgAspTrp200205MetArgAspAlaGlyArgProTrpArgSerGlyValAlaAlaGlyArgProTrpArg215220GluAlaTrpLeuAlaGlyArgGlyTrpArgAlaGluValLeuArg210GluProGluAlaTrpLeu225230GluProCysAspLeuAspLeu245ProProArgGlyThrProGlyTrp235GluAlaAlaGlyArgMetValProGly260ThrGly250ArgThrTrpLeuAlaAla265GluLeuValThrGlyProAlaVallieAspSerValProAspSerVal280285ValSerAspSerValSerGluSerAlaVal275SerValProAspProAspSerValProAspSer290295SerAlaSerProThrValAlaVal300Val270AspArg240ArgThr255AlaAla52<210><211><212><213><400>MetHis8213PRTSfre/^o附yces/a訓础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yce51/owe""!'ATCC31255ValGlyLeuSerTyrLeuGlyAlaAlaValAlaPheArgAla51015AlaAlaGlyPheProAlaThrValAlaGlylieValMetSerGlyTyr2025ValSerProAspThrLeuAsnArgTrpLeuAlaGlyPheSerPheLeuValSerProAspThr3540ThrArgMetAlaSerHisGinProAlaAla5055LeuHisGlyProThrTrpGluAsnLeuLeu6570LeuSerAspGlyCysLeuArgLeuProCysLeuArgLeuProGly8590LeuProValProValGluLeuValAsnGlySerLeuMetSer30LeuAla45GinArgHisPheAlaGlu60ThrAlaThrPheAla75ValAlaAspLeuPro100lielieAlaGlyAlaGlyHisAla115GlyAla120ValAsn105AlaGlu95AsnGlu80HisGluLeuGin110ArgAsnAlalieGlyProAlaAlaAlaAlaGlyAlaAspValAlaVal125ProArgValPheProMetAlaGlyAlaGlyHisVal130135AlaAlaThrValGluA印PheAlaAspArgValThrAlaGlyArg145150AlaPheHisGluArgArgArgAlaAlaAlaValProPro180AlaAlaThrHis195Arg165GluGlyAspAla140ValThr155ArgAlaGlu170AlaGlyThrGlyThrGluPro185AlaAlaGlu175ValAlaArg190Thr160ProLys<210>11<211>933<212>PRT<213>Sfreptomyces/fl訓油7ATCC31255<400>MetAsp1CysSerAlaArgLeuAla50ThrAsp65HisArgAlaTyrAlaGlyArgArg130LeuAsn145LeuArgAspArgGluArgLeuLys210LeuPro225AlaProLeuProProAlaSerSer290HisGlu305ValValArgGluSerGinVal35ProArgAlaGlyPro115AspAlaAlaTyrMet195ProLysGlyArgPhe275LysProThrLeuProPro20CysArgAspAlaThr100LeuAspGluGlyAla180ThrSerArgThrGin260ArgGluAlaAlaArgGlu5AlaAspValGluAsp85SerAlaLeuArgAla165AlaGluProAlaAsp245LeuHisGlyAlaVal325GlulieAspGluAlaArg70ValAlaProAlaLys150GinAlaSerPheThr230GluValValValLys310LeuArgThrlieLeuGlu55ArgProAspGlyAla135ValLeuValThrGly215AspAlaSerAlaVal295GluAlaCysTyrlieArg40GluThrAlaGlyAla120ArgLeuSerAlaVal200GinAlaAlaTrpVal280PheArgLeuAlaAlaAla25AspLeuValAlaPro105AlaLeuAlaAlaGlu185LeuLeuThrGluVal265LeuLeuPheAlaAlaGly10GinValThrLeuAsp90GlyAlaGluSerAsp170GlyHisValProArg250GluArgLeuValAla330LeuTyrLeuArgGluAla75LeuAlaLeuGlyAla155GlyThrLeuHisVal235ProArgArgArgArg315AspProGlySerArgVal60LeuAspAlaArgAla140AlaLeuAlaLeuThr220lieValSerAlaGly300ValAlaGlyValSerThr45ValArgValGlyAla125TyrLeuLeuProThr205ArgAlaSerLeuPro285ArgProAlaThrValArg30TrpProArgLeuThr110LeuGluAspArgGly190ArgProProValAla270ValAspProValAspArg15GluGinILeuArgAsn95AlaAlaGluProAsn175LysSerValValCys255GlyValGlyGlyPro335ProValAlaAspLeuLeu80LeuValGluGlyAla160MetArgThrLeuThr240ArgArgGlyThrVal320GluAla56340345350AlaAlaGlyAlaAlaLeuAspAlaLeulieGlyGinGlyValLeuAla355360365ArgAspLeuGlyValGlyAspGinHisProHisProLeuAlaArgLeu370375380AlaAlaLeuLeuProProAspCysAspAlaGlyLeuArgAlaAlaVal385390395400SerGlyLeuAlaGlyAlaGluSerAlaPheGluThrAlaGlyLeuGlu405410415ArgArgGluGluLeuValSerArglieLysGluHisValAlaArgVal420425430AlaGluLeuThrGlyValAlaThrProProlieAspValAlaArgThr435440445LeuValTyrGluAspAlaValValAlaValProArgAlaGluAlaPro450455460GlyArgTrpGlyArgHisLeuProAlaLeuAlaProTrpHisArgLeu465470475480LeuProLeuPheAspAspAlaAlaHisValArgAlalieValAlaAsp485490495ValValArgThrHisPheGlyProGlyProHisArgLeuLeuProLeu500505510PheAlaAlaLeuSerSerAlaLysValGinAlaValLeuThrGluArg515520525LeuThrAspValSerAlaProLeuProThrArgLeuArgThrLeuGin530535540AspAlaValPheAlaLeuAlaAspProAspGlyGlyAlaAspThrVal545550555560ThrValAspProGluArgAlaAlaArgLeuAlaAspAspLeuProSer565570575TrplieProArgTrpSerArgAlaSerTrpHisValGinHisHisAla580585590AlaProAspGlyGlyLeuLeuValValAsnGlyGlySerlieGlyPhe595600605GlyArgAlalieSerArgPheLeuProAlaTyrAlaAlaAlaGlyAsp610615620AlaGlyPheGlyAspLeuValThrAlaGlulieAlaArgAspAspAsp625630635640ProSerAlaProLeuThrAspLeuAlaAlaValLeuGlylieAsnAla645650655AsnValHisSerProLeuLeuGlyArgHisLeuArgTyrProCysAla660665670ThrProGluGluTrpGlyGlyAsnThrGlyValSerLeuGluAspCys675680685TrpValValTyrAspAlaAspThrGlyArgLeuLeuProArgLeuGly690LysAsp705GluLeuSerLeuAlaGlyAlaGly770ArgVal785ProAspPheAlaValPheArgGlu850SerAla865ValThrPheGinGlyLeuThrGlu930GlyAlaAlaAla755GlyArgAlaGluTyr835GluGluAlaGluArg915GluProProAsn740GlyGlySerArgVal820ArgGinArgTrpCys900GluGluValArg725LeuAlaAlaGlyLeu805ArgGlyArgLysGin885LeuArgG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inCysAspGlyValArgThrValArgGlyLeuGinSer450455柳AsnAsnAlaSerGlyProlieValLeuLysGinLeuHisGinCysAsp465470475480GlyProlieGluTyrGluArgGinPheMetPheAspGluSerGlyAsp485490495PheArgLeuValCysTyrPheSerProAspLeuProGluAlaProGly500505510ThrAlaAspSerProAspGlylieTrpArgAlaSerValAspPheLeu515520525TrpAspArglieProLysSerGluGinTyrArgValMetLeuProThr530535540LeuSerGlySerSerGluAsnAspAsnAsnTyrAlaAspAsnProPhe545550555560LeuThrSerLeuAsnArgLysGlyTyrThrGlyAlaPheTrpAlaHis565570575TrpArgAspArgGluAlalieMetSerGlyAlaThrLeuProLeuGly580585590GluLeuAlaGluAlaValArg595<210>22<211>272<212>PRT<213>Sfre;7to附yces/aww油7ATCC31255<■>1ValThrAlaProAlaLeuProLeuAlaGlyGinValAlaLeuValThr151015GlyAlaGlyArgGlylieGlyArgMetValAlaValAlaLeuAlaGlu202530AlaGlyMetThrValGlyLeuValGlyArgAsnArgArgLeuLeuAsp354045ValThrAlaArgAlaCysAlaAlaAlaArgGluAlaAlaSerGlyAsp505560GlySerGlyAspGlySerGlyGlyGlyProGluAlaGlyValAlaVal7765AlaValAlaValGlyLeuArgTrp130CysArg145ValAsnAlsTyrAlaGluGlyMet210ArgAsp225ValAlaGlyAla<210><211><212><213><權>MetArg1LeuAlaTyrGluGluAsn50AlaArgVal115TrpAlaValCysPro195ValAspHisAspAspAla100AspGluValAsnGly180LeuArgTrpGlyAsp260Val85AlaGinValLeuSer165SerArgThrThrAsp245Phe70ArgLeuGlyPhePro150ArgLysAspAspAsp230LeuAspAlaGlyGluGlu135GluLeuAlaArgMet215ProAspAlaProProLeu120ThrMetAlaThrGly200ThrAlaProLeuAlaVal105ProAsnThrValLeu185ValLeuLeuLeuLeu265Glu90AspPheValArgGin170LeuHisSerPheAla250Ser75lieLeuTrpArgArgGluAlaAla95AsnLeuValAsn110GluAlaAsp125GlyThrVal140GlySerGlyArg155GlyAspProArgAlaLeuMetLeuAspAla190PheAsplie205AlaValCys220AlaAlalieLeu235GlyArgPheLeuGluArgArgLeu270ProAsnArgTyr175ValSerAlaLeuHis255Ala80GluAlaGluThrVal160SerAlaProGlyArg240ValVal23247PRTS/repfowycey/a訓""7ATCC31255LysAsnlieArgLeuLeuGlyValSerValAlaAlaAlaGly51015ValGlyAlaLeuSerGlyCysGlyMetValValAsnGluMet202530AspSerSerSerLeuProGluLyslieThrSerValArgLeu354045GlySerGlyArgValThrValAsnGlyThrLysAlaGlyGly5560ProLeuSerLeuArgArgThrValSer6570GlyProThrHisArgValGluAsnGly85GlyAspAsplieAspPheCysSerVal100105AlaGlylieProValSerGlyLysVal115120ThrLysValGlyAlaValGinValThr130135MetThrGlyValAsnGlyAlaValAsp145150lieThrGlySerAspValLysGlyGly165AsnGlyGlylieGluLeuAsnLeuAla180185ThrThrThrAsnGlyThrlieAlaLeu195200ArglieAspAlaGinThrAlaAsnGly210215AspAspProAlaGlyProHisArgLeu225230SerlieSerValLysAsnSer245TyrGinAspLysLysProAsp7580ValLeulieLeuGlyAspCys9095GlyTyrSerValAspValPro110ThrAsnGlyThrValLysLeu125ThrThrSerGlyArglieGlu140ValArgThrThrAsnGlyArg155160GlylieLysAlaArgThrSer170175AlaAspLeuAspValGinAla190LysValProGlyThrGlyTyr205AspLysArglieGlyLeuThr220ThrLeuLysThrSerAsnGly23524079权利要求1.一种抗革兰氏阳性菌活性的抗生素—硫链丝菌素的生物合成基因簇，其特征在于编码硫链丝菌素生物合成所涉及的22个基因，具体为1)1个氨基转移酶基因，即tsrAtsrA位于基因簇核苷酸序列第3774-2686个碱基处，长度为1089个碱基对，氨基转移酶，362个氨基酸；2)脂肪酶基因，即tsrBtsrB位于基因簇核苷酸序列第4626-3796个碱基处，长度为831个碱基对，编码脂肪酶，276个氨基酸；3)天门冬酰胺合成酶基因，即tsrCtsrC位于基因簇核苷酸序列第6541-4685个碱基处，长度为1857个碱基对，编码天门冬酰胺合成酶，618个氨基酸；4)酯环化酶基因，即基因tsrDtsrD位于基因簇核苷酸序列第个7088-6681碱基处，长度为408个碱基对，编码酯环化酶，135个氨基酸；5)酰基-CoA脱氢酶基因，即tsrEtsrE位于基因簇核苷酸序列第8449-7163个碱基处，长度为1287个碱基对，编码酰基-CoA脱氢酶，428个氨基酸；6)甲基转移酶基因，即tsrFtsrF位于基因簇核苷酸序列第9529-8594个碱基处，长度为936个碱基对，编码甲基基转移酶，311个氨基酸；7)与thiostrepton侧链生物合成相关但具体功能未知的基因，即tsrGtsrG位于基因簇核苷酸序列第10241-9600个碱基处，长度为642个碱基对，编码一个未知功能蛋白，213个氨基酸；8)前体肽基因，即tsrHtsrH位于基因簇核苷酸序列第10570-10746个碱基处，长度为177个碱基对，编码Thiostrepton前体肽，58个氨基酸；9)水解酶基因，即tsrItsrI位于基因簇核苷酸序列第11461-12054个碱基处，长度为594个碱基对，编码水解酶，197个氨基酸；10)脱水酶基因，即tsrJtsrJ位于基因簇核苷酸序列第12047-14848个碱基处，长度为2802个碱基对，编码脱水酶，933个氨基酸；11)脱水酶基因，即tsrKtsrK位于基因簇核苷酸序列第14845-15885个碱基处，长度为1041个碱基对，编码脱水酶，346个氨基酸；12)与thiostrepton大环合成相关具体功能未知基因，即tsrLtsrL位于基因簇核苷酸序列第15872-16978个碱基处，长度为1107个碱基对，编码一个具体功能未知的蛋白，368个氨基酸；13)NADH依赖的氧化酶基因，即tsrMtsrM位于基因簇核苷酸序列第16975-18744个碱基处，长度为1770个碱基对，编码NADH依赖的氧化酶，589个氨基酸；14)与thiostrepton大环合成相关具体功能未知基因，即tsrNtsrN位于基因簇核苷酸序列第18741-20579个碱基处，长度为1839个碱基对，编码一个具体功能未知的蛋白，612个氨基酸；15)与thiostrepton大环合成相关具体功能未知基因，即tsrOtsrO位于基因簇核苷酸序列第20576-22621个碱基处，长度为2046个碱基对，编码一个具体功能未知的蛋白，681个氨基酸；16)细胞色素P450氧化酶基因，即tsrPtsrN位于基因簇核苷酸序列第22599-24023个碱基处，长度为1425个碱基对，编码细胞色素P450氧化酶，474个氨基酸；17)酰基-CoA合成酶，即tsrQtsrQ位于基因簇核苷酸序列第23999-25312个碱基处，长度为1314个碱基对，编码酰基-CoA合成酶，437个氨基酸；18)细胞色素P450氧化酶基因，即tsrRtsrR位于基因簇核苷酸序列第25422-26612个碱基处，长度为1191个碱基对，编码细胞色素氧化酶，396个氨基酸；19)脱水酶基因，即tsrStsrS位于基因簇核苷酸序列第26603-29413个碱基处，长度为2811个碱基对，编码脱水酶，936个氨基酸；20)铁硫簇氧化还原酶基因，即tsrTtsrT位于基因簇核苷酸序列第29430-31229个碱基处，长度为1800个碱基对，编码一个具体功能未知的蛋白，599个氨基酸；21)短链脱氢酶基因，即tsrUtsrU位于基因簇核苷酸序列第31226-32044个碱基处，长度为819个碱基对，编码一个具体功能未知的蛋白，272个氨基酸；22)与thiostrepton大环合成相关具体功能未知基因，即tsrVtsrV位于基因簇核苷酸序列第32391-33113个碱基处，长度为744个碱基对，编码一个具体功能未知的蛋白，247个氨基酸。2.根据权利要求1所述的硫链丝菌素的生物合成基因簇的用途，其编码蛋白用于催化合成抗生素硫链丝菌素及其类似物、或者催化合成含噻唑、噻唑啉、吡啶杂环或喹萘啶酸结构单元的大环骨架。3.根据权利要求2所述的硫链丝菌素的生物合成基因簇的用途，其特征在于由核糖体依据所述的硫链丝菌素的生物合成基因簇中编码硫链丝菌素前体肽的基因合成出的相应的聚肽是硫链丝菌素的前体肽；该前体肽经由后修饰酶的催化反应生成硫链丝菌素。全文摘要本发明是一种由链霉菌产生的具有抗革兰氏阳性菌抗生素-硫链丝菌素(Thiostrepton)的生物合成基因簇的克隆、测序、分析、功能研究及其应用。整个基因簇共包含22个基因10个与Thiostrepton大环生物合成相关基因，8个与侧链生物合成相关基因，还有4个基因目前功能不明。通过对上述生物合成基因的遗传操作可阻断硫链丝菌素的合成。本发明所提供的基因及其蛋白也可以用来寻找和发现可用于医药、工业或农业的化合物或基因、蛋白。文档编号C12N15/54GK101445803SQ20081020241公开日2009年6月3日申请日期2008年11月7日优先权日2008年11月7日发明者莹丁,文刘,廖日晶,炼段,沂虞,春雷申请人:中国科学院上海有机化学研究所