CN101586112B - 诺丝七肽的生物合成基因簇 - Google Patents
诺丝七肽的生物合成基因簇 Download PDFInfo
- Publication number
- CN101586112B CN101586112B CN2009100534277A CN200910053427A CN101586112B CN 101586112 B CN101586112 B CN 101586112B CN 2009100534277 A CN2009100534277 A CN 2009100534277A CN 200910053427 A CN200910053427 A CN 200910053427A CN 101586112 B CN101586112 B CN 101586112B
- Authority
- CN
- China
- Prior art keywords
- ala
- leu
- arg
- gly
- pro
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- OQAOHXRUMXWDLQ-WRHRRJCOSA-N C[C@H]([C@@H](C(N/C(/c1nc(C(N[C@@H](CC(C(OCc2c(c(C)c(C(SC[C@@H](c3nc(-c(c(-c4nc5c[s]4)c4)nc(-c6nc(C(NC(C(N)=O)=C)=O)c[s]6)c4O)c[s]3)NC3=O)=O)[nH]4)c4ccc2)=O)O)c2nc3c[s]2)=O)c[s]1)=C\C)=O)NC5=O)O Chemical compound C[C@H]([C@@H](C(N/C(/c1nc(C(N[C@@H](CC(C(OCc2c(c(C)c(C(SC[C@@H](c3nc(-c(c(-c4nc5c[s]4)c4)nc(-c6nc(C(NC(C(N)=O)=C)=O)c[s]6)c4O)c[s]3)NC3=O)=O)[nH]4)c4ccc2)=O)O)c2nc3c[s]2)=O)c[s]1)=C\C)=O)NC5=O)O OQAOHXRUMXWDLQ-WRHRRJCOSA-N 0.000 description 1
- RKSWXPBYDCMSLZ-ZSEOAQADSA-N C[C@H]([C@@H](C(N/C(/c1nc(C(N[C@@H](CC(C(OCc2cccc3c2c(C)c(C(SC[C@@H](c2nc(C(N=C([C@H](C4)O)c5nc(C(NC(C(NC(C(O)=O)=C)=O)=C)=O)c[s]5)=C4c4nc5c[s]4)c[s]2)NC2=O)=O)[nH]3)=O)O)c3nc2c[s]3)=O)c[s]1)=C\C)=O)NC5=O)O Chemical compound C[C@H]([C@@H](C(N/C(/c1nc(C(N[C@@H](CC(C(OCc2cccc3c2c(C)c(C(SC[C@@H](c2nc(C(N=C([C@H](C4)O)c5nc(C(NC(C(NC(C(O)=O)=C)=O)=C)=O)c[s]5)=C4c4nc5c[s]4)c[s]2)NC2=O)=O)[nH]3)=O)O)c3nc2c[s]3)=O)c[s]1)=C\C)=O)NC5=O)O RKSWXPBYDCMSLZ-ZSEOAQADSA-N 0.000 description 1
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明涉及诺丝七肽的生物合成基因簇,具体说是一种由链霉菌产生的具有抗革兰氏阳性菌抗生素-诺丝七肽(Nosiheptide)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16个基因:6个与Nosiheptide大环生物合成相关基因,5个与侧链生物合成相关基因,4个与翻译后修饰相关基因,1个调控基因。通过对上述生物合成基因的遗传操作可阻断诺丝七肽的合成。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。
Description
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诺丝七肽(Nosiheptide)的生物合成基因簇的克隆、分析、功能研究及其应用。
技术背景:
诺丝七肽(Nosiheptide)是硫肽类抗生素家族的一个著名成员,可由链霉菌Streptomyces actuosus ATCC25421、Streptomyces azureus ATCC14921、Streptomyceshawaiiensis ATCC12236所产生。其分子结构式最先由Depaire.H等人分析鉴定[Depaire,H.;Thomas,J.-P.;Brun,A.;Olesker,A.;Lukacs,G.Tetrahedron Lett.1977,1397.]。结构上,Nosiheptide由五个噻唑环,一个吡啶环组成大环骨架结果,侧链是一个3-甲基-5-甲氧基-2-吲哚酸与主体大环结构通过硫酯键相连。
诺丝七肽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。诺丝七肽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23S rRNA-L11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[J、Mol.Biol 1998,276,391-401]。但是,由于诺丝七肽水溶性不好,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。由于诺丝七肽极其复杂的分子结构,即使是在有机合成化学高度发达的今天,通过化学全合成来获得产品也是一项艰巨的挑战。直到2008年才首次报道了诺丝七肽的部分化学合成[Marc C.Kimber andChristopher J.Moody.Construction of macrocyclic thiodepsipeptides:synthesis of a nosiheptide‘southern hemisphere’model system.Chem.Commun.,2008,591-593.],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诺丝七肽为目标分子,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,合理修饰诺丝七肽的生物合成途径,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Streptomyces actuosus ATCC25421产生的具有抗革兰氏阳性菌活性的抗生素——诺丝七肽(Nosiheptide)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16个基因的核苷酸序列或互补序列(序列1),其中一个基因(nosM)负责编码诺丝七肽的前体肽,八个基因(nosA、nosB、nosC、nosD、nosE、nosF、nosG、nosH)负责对NosH编码的前体肽进行后修饰,形成整个大环骨架;六个基因(nosI、nosJ、nosK、nosL、nosN、nosO)负责以色氨酸为前体,合成吲哚酸结构单元;一个基因nosP负责对诺斯七肽的生物合成进行调控。
本发明还提供了一个编码未知蛋白的核苷酸序列,由序列2中的氨基酸序列组成,命名为nosA,其基因的核苷酸序列位于序列1中第2704-2249碱基处。
本发明还提供了一个编码P450氧化酶的核苷酸序列,由序列3中的氨基酸序列组成,命名为nosB,其基因的核苷酸序列位于序列1中第4068-2701碱基处。
本发明还提供了一个编码P450氧化酶的核苷酸序列,由序列4中的氨基酸序列组成,命名为nosC,其基因的核苷酸序列位于序列1中第5309-4083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5中的氨基酸序列组成,命名为nosD,其基因的核苷酸序列位于序列1中第6365-5328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6中的氨基酸序列组成,命名为nosE,其基因的核苷酸序列位于序列1中第9114-6352碱基处。
本发明还提供了一个编码NADH依赖的脱氢酶的核苷酸序列,由序列7中的氨基酸序列组成,命名为nosF,其基因的核苷酸序列位于序列1中第10778-9129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nosG,其基因的核苷酸序列位于序列1中第12800-10830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9中的氨基酸序列组成,命名为nosH,其基因的核苷酸序列位于序列1中第14751-12797碱基处。
本发明还提供了一个编码乙酰辅酶A合成酶的核苷酸序列,由序列10中的氨基酸序列组成,命名为nos I,其基因的核苷酸序列位于序列1中第16097-14763碱基处。
本发明还提供了一个编码未知功能酶的核苷酸序列,由序列11中的氨基酸序列组成,命名为nosJ,其基因的核苷酸序列位于序列1中第16297-16058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12中的氨基酸序列组成,命名为nosK,其基因的核苷酸序列位于序列1中第17106-16294碱基处。
本发明还提供了一个编码SAM自由基蛋白的核苷酸序列,由序列13中的氨基酸序列组成,命名为nosL,其基因的核苷酸序列位于序列1中第18316-17114碱基处。
本发明还提供了一个编码Nosiheptide前体肽的核苷酸序列,由序列14中的氨基酸序列组成,命名为nosM,其基因的核苷酸序列位于序列1中第18668-18820碱基处。
本发明还提供了一个编码SAM依赖的甲基转移酶的核苷酸序列,由序列15中的氨基酸序列组成,命名为nosN,其基因的核苷酸序列位于序列1中第18891-20078碱基处。
本发明还提供了一个编码与后修饰相关的未知蛋白的核苷酸序列,由序列16中的氨基酸序列组成,命名为nosO,其基因的核苷酸序列位于序列1中第20075-21187碱基处。
本发明还提供了一个编码SARP家族调控基因的核苷酸序列,由序列17中的氨基酸序列组成,命名为nosP,其基因的核苷酸序列位于序列1中第21497-22468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诺丝七肽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1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诺丝七肽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Streptomyces actuosusATCC25421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Streptomyces actuosusATCC25421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Nosiheptide大环骨架的改造,获得一系列的Nosiheptide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(DNA shuffling),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片段可以通过缺失诺丝七肽生物合成中的一个或几个步骤而得到新的诺丝七肽结构类似物或前体。包含DNA片段或基因可以用来提高诺丝七肽或其衍生物的产量,本发明提供了在基因工程微生物中提高产量的途径。
本发明所提供的诺丝七肽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诺丝七肽生物合成相关的所有基因和蛋白信息可以帮助人们理解诺丝七肽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。
附图说明:
图1:诺丝七肽的化学结构
图2:诺丝七肽生物合成基因簇的限制性内切酶谱。3个交叠的粘粒代表了抗生素产生菌基因组40kb的DNA区域,B代表限制性内切酶BamHI
图3:诺丝七肽生物合成基因簇的基因组成。
图4:提出的诺丝七肽的大环骨架部分的生物合成途径。
图5:提出的诺丝七肽的后修饰部分的生物合成途径。
图6:诺丝七肽侧链甲基吲哚酸单元的合成途径。
图7:与Nosiheptide生物合成相关的15个基因的同框缺失突变菌株发酵液的高效液相色谱(HPLC)分析。
图8:基因缺失突变菌株产生的4个新化合物的可能结构与质谱分析。
符号说明:
图1
Nosiheptide:诺丝七肽。
图3
cyclopeptide core biosynthesis:大环骨架生物合成;indolic acid moietybiosynthesis:吲哚酸单元生物合成;tailoring:后修饰;regulation:调控;unrelated:不相关基因。
图4
propeptide:前导肽;complex B:nosO基因同框缺失突变株的发酵液中分离出的化合物。
图5
complex B:nosO基因同框缺失突变株的发酵液中分离出的化合物;complex C:nosC基因同框缺失突变株的发酵液中分离出的化合物;complex D:nosB基因同框缺失突变株的发酵液中分离出的化合物;complex E:nosA基因同框缺失突变株的发酵液中分离出的化合物。
图6
Ado:由SAM蛋白所产生的自由基。
图7
complex E:nosA基因同框缺失突变株的发酵液中分离出的化合物;complex I:nosN基因同框缺失突变株的发酵液中分离出的化合物;complex B,complex F:nosO基因同框缺失突变株的发酵液中分离出的化合物;complex H:nosB基因同框缺失突变株的发酵液中分离出的化合物;complex G:nosC基因同框缺失突变株的发酵液中分离出的化合物。
图8
complex E:nosA基因同框缺失突变株的发酵液中分离出的化合物;complex I:nosN基因同框缺失突变株的发酵液中分离出的化合物;complex B,complex F:nosO基因同框缺失突变株的发酵液中分离出的化合物;complex H:nosB基因同框缺失突变株的发酵液中分离出的化合物;complex G:nosC基因同框缺失突变株的发酵液中分离出的化合物。
具体实施方式:
以下结合图1-图8对本发明进一步详细说明。
1.克隆诺丝七肽的生物合成基因片断:
尽管在诺丝七肽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诺丝七肽生物合成相关的基因,总是不能成功。本专利依据诺丝七肽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Streptomyces actuosus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Streptomyces actuosus体内失活该基因,得到的突变体无法再产生诺丝七肽,证实了克隆到的基因与诺丝七肽生物合成相关。
2.诺丝七肽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Streptomyces actuosus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40kb的区域。(图2)。DNA顺序分析了34,713bp的染色体区域,GC含量71.6%。生物信息学分析包含了26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1。
表1Nosiheptide的生物合成基因簇中各基因及编码蛋白的功能分析
基因 | 氨基酸数目 | 相似及同源蛋白 | 同一性/相似性% | 推测功能 |
orf1 | 324 | SCO4661(NP_628821),fromStreptomyces coelicolorA3 (2) | 91/96 | elongation factor G |
orf2 | 156 | SCO4660(NP_628820),fromStreptomyces coelicolor A3(2) | 99/100 | 30S ribosomalprotein S7 |
orf3 | 123 | SCO4659(NP_628819),fromStreptomyces coelicolor A3(2) | 99/100 | 30S ribosomalprotein S12 |
nosA | 151 | Francci3_4114(YP_483191),fromFrankia sp.CcI3 | 55/67 | hypothetical protein |
nosB | 455 | Sare_3149(YP_001537948),fromSalinispora arenicola CNS-205 | 32/47 | cytochrome P450 |
nosC | 408 | SACE_1426(YP_001103673),FromSaccharopolyspora erythraea NRRL2338 | 39/55 | cytochromeP450-like enzyme |
nosD | 345 | BC5083(NP_834751),from Bacilluscereus ATCC 14579 | 25/44 | lantibioticbiosynthesis protein |
nosE | 920 | PPA0859(YP_055572),fromPropionibacterium acnesKPA17I202 | 28/43 | putative lanthioninebiosynthesis protein |
nosF | 549 | BCE G9241_1259(ZP_00239875),from Bacillus cereus G9241 | 25/38 | NADH oxidase |
nosG | 656 | BC5085(NP_834753),from Bacilluscereus ATCC 14579 | 31/49 | putative cytoplasmicprotein |
nosH | 654 | BC1250(NP_831034),from Bacilluscereus ATCC 14579 | 22/37 | hypothetical protein |
nosI | 444 | SAMR0921(CAJ88630),fromStreptomyces ambofaciens ATCC23877 | 29/39 | putative acyl-CoAsynthetase |
nosJ | 79 | BOV_1126(YP_001259087),fromBrucella ovis ATCC 25840 | 38/58 | ATP-dependent ClpproteaseATP-binding subunitClpA |
nosK | 270 | BCE_2449(NP_978758),fromBacillus cereus ATCC 10987 | 29/43 | alpha/beta foldfamily hydrolase |
nosL | 400 | HoreDRAFT_1581(ZP_01188841),from Halothermothrix orenii H168 | 30/47 | Radical SAM:Biotinand thiaminsynthesis associated |
nosM | 50 | ---- | --/-- | propeptide |
nosN | 395 | Tlm Orf11(ABL74954),fromStreptoalloteichus hindustanus | 32/48 | SAM-dependentoxidase or methyltransferase |
nosO | 370 | BC5082(NP_834750),from Bacilluscereus ATCC 14579 | 23/43 | hypothetical protein |
nosP | 323 | pSLA2-L_p071(NP_851493),fromStreptomyces rochei | 51/66 | SARP familypathway specificregulatory protein |
orf4 | 1299 | SAV_4915(NP_826092),fromStreptomyces avermitilis MA-4680 | 96/98 | DNA-directed RNApolymerase subunitbeta′ |
orf5 | 1181 | SAV_4914(NP_826091),fromStreptomyces avermitilis MA-4680 | 95/98 | DNA-directed RNApolymerase subunitbeta |
orf6 | 129 | SCO4653(NP_628814),fromStreptomyces coelicolor A3(2) | 81/86 | 50S ribosomalprotein L7/L12 |
orf7 | 176 | SSEG_01531(YP_002208236),fromStreptomyces sviceus ATCC 29083 | 89/96 | 50S ribosomalprotein L10 |
orf8 | 272 | SCO4651(NP_628812),fromStreptomyces coelicolor A3(2) | 65/78 | lipoprotein |
orf9 | 297 | SSEG_01533(YP_002208238),fromStreptomyces sviceus ATCC 29083 | 55/70 | lipoprotein |
orf10 | 241 | SCO4649(NP_628810),fromStreptomyces coelicolor A3(2) | 92/97 | 50S ribosomalprotein L1 |
3.Nosiheptide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,诺丝七肽的生物合成基因簇被确定为从基因nosA到nosP(图3),包含16个开放读码框。本发明中整个基因簇共包含16个基因的核苷酸序列或互补序列(序列1),其中一个基因(nosM)负责编码诺丝七肽的前体肽,五个基因(nosD、nosE、nosF、nosG、nosH、nosL)负责对nosM编码的前体肽进行修饰,形成整个大环骨架;五个基因(nosI、nosJ、nosK、nosL、nosN)负责以色氨酸为前体,合成甲基吲哚酸结构单元;一个基因nosP负责对整个生物合成进行正调控。其中基因nosM的同框缺失完全抑制了诺丝七肽的产生(图7),进一步证明了克隆到基因簇为诺丝七肽的生物合成基因簇。
4.甲基吲哚酸结构单元的生物合成途径:
甲基吲哚酸结构单元的生物合成如图6所示。nosL编码的自由基蛋白催化色氨酸发生自由基重排,nosK编码的水解酶使得色氨酸重排产物的亚氨基转化为羟基,然后在nosN所编码的蛋白作用下发生羟甲基化以形成侧链的最终结果,最后nosI编码的酰基-CoA合成酶负责活化吲哚酸的羧基,将其转变成CoA-硫酯键。
5.诺丝七肽骨架合成
诺丝七肽生物合成基因簇中有一个基因nosM编码诺丝七肽的前体肽,经核糖体将该前体肽合成之后,由nosG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,nosF负责编码的脱氢酶将噻唑啉转变为噻唑,然后由nosD、nosE编码的脱水酶负责催化使得丝氨酸残基的羟基脱水,然后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nosO编码的未知蛋白作用下,信号肽被切除,并形成完整的吡啶环中心。整个过程如图5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1
诺丝七肽产生菌链霉菌Streptomyces actuosus ATCC25421总DNA的提取:
将100μL 1×108 S.actuosus孢子悬液接种到3mL TSB液体培养基中,30℃,230rpm培养约24hr后达到对数生长期后期,取2mL接种到50mL TSB(含10mM氯化镁,0.1%甘氨酸),30℃,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℃,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℃水浴15mim。加入0.1mL蛋白酶K(10mg/mL,用裂解液新鲜配制),1mL 10%SDS,混匀后迅速放入70℃水浴15mim,呈澄清。置冰上冷却,加入2.5mL 5M KAc,冰上冷却15min。加入10mL饱和酚,混匀,10mL氯仿,混匀,12000rpm,4℃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CHCl3-异戊醇(24∶1)抽提,12000rpm,4℃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5mL TE溶解,加RNase A使终浓度为50μg/mL,37℃温育0.5小时。依次用等体积的饱和酚抽提两次,CHCl3-异戊醇抽提两次,向水相中加入0.1体积的3M NaAc,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1mL 70%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(pH 8.0)中。
实施例2
诺丝七肽产生菌链霉菌Streptomyces actuosus ATCC25421遗传转移系统的建立:
培养含有适当质粒的E.coli ET12567至OD6000.3-0.4,30mL LB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2mL LB中,作为大肠杆菌供体细胞。取适量冻存于-80℃的Streptomyces actuosus ATCC25421的20%甘油孢子悬液500μL,用等体积的TES缓冲液(50mM TES Na,pH 8.0)洗两次,重悬于等体积的TES缓冲液,50℃热激10min使孢子萌发。再加等体积的TSB培养基,37℃温育2-5hr。离心重悬于0.5-1mL LB中作为链霉菌受体细胞。将不同浓度的受体细胞100μL与等体积的供体细胞混合直接涂布在含有10mMMgCl2的平板上,30℃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1mL含萘啶酮酸(终浓度为50ng/μL)和相应抗生素的无菌水。30℃培养5天以上挑取接合子。
由于Streptomyces actuosus ATCC25421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25μg/mL,红霉素100μg/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
诺丝七肽产生菌链霉菌Streptomyces actuosus ATCC25421基因组文库的构建:
首先用1ml注射器吹吸Streptomyces actuosus ATCC25421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-80℃的PromegaPackagene exract,立即加入10ul连接产物,轻弹混匀,于室温(约22℃)放置3hr。加入445ul Phage buffer,倒转混合;加入25ul氯仿终止反应,离心使氯仿沉于底部,4℃保存。将冻存于-80℃的菌株E.coli LE392涂布在LB培养基上复苏。调取单菌落接种于3ML LB培养基中(0.2%maltose10和mMMgSO4),37℃,220rpm振荡培养过夜,转接1%到50ML LB培养基中(0.2%maltose10和mMMgSO4),37℃,220rpm振荡培养至OD600=0.6-0.8。取5ul包装液,加入95ul phage buffer,稀释,再加入100ul E.Coli LE392(OD600=0.67)混匀,37℃,30min,涂于LB(含100ug/ml Apramycin)平板上。37℃倒置培养过夜,测定噬菌斑形成单位(pfu)以估算文库的效价。取100ul包装液加入3.9ml phage buffer混匀,再加入4ml E.Coli LE392(OD600=0.72)菌液,室温,30min,加入4ml LB,37℃,75min;2500rpm,10min,去上清,剩余1-3ml涂于LB(含100ug/ml Apramycin)平板上。37℃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-80℃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Bam HI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2000-5000cfu,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/μgDNA,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
诺丝七肽产生菌链霉菌Streptomyces actuosus ATCC25421的发酵、产物分离纯化与鉴定:
取100ul Streptomyces actuosus ATCC25421孢子接入S.actuosus一级发酵培养基(TSB 15g,可溶性淀粉15g;蔗糖50g,蒸馏水定容到1L)28℃、250转每分钟培养48小时,取10ml种子培养液接入S.actuosus二级发酵培养基(TSB15g,CaSO415g,酵母提取物11g,葡萄糖50g,2×微量元素1ml,蒸馏水定容到1L)28℃、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30℃蒸干,所得固体用3ml甲醇溶解。
发酵产物分析:
取20μl进样,流动相为乙腈和水(含0.1%TFA)。HPLC洗脱条件为:(紫外254nm处检测,柱子为NUCLEOSIL 100-5 C18)。
时间/分钟 | CH3CN% |
0 | 15 |
3 | 15 |
6 | 40 |
12 | 40 |
19 | 55 |
22 | 85 |
28 | 85 |
32 | 15 |
实施例5
PCR克隆诺丝七肽的生物合成基因:
PCR体系包含:DMSO(8%,v/v),MgCl2(25mM),dNTP(2.5mM),简并性引物(40mM),Taq DNA聚合酶(2.5u)及适量模板Streptomyces actuosusATCC25421总DNA。首先95℃,3min,1轮;然后94℃,1min,68℃,1min,72℃,2min,5轮;94℃,1min,65℃,1min,72℃,2min,30轮;最后72℃,10min,1轮。PCR结束后,1%琼脂糖电泳检查结果。低熔点胶回收预期大小的DNA片段,与pMD19-T vector连接,转化大肠杆菌DH5α感受态细胞,涂布在含有氨苄青霉素,IPTG(异丙基硫代-β-D-半乳糖苷)和X-gal(5-溴-4-氯-3-吲哚-β-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,EcoR I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)DIG DNA标记:将待标记的DNA用无菌水稀释至总体积15μL,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2μL引物混合物,2μLdNTP混合物,1μL酶,混合均匀后,37℃水浴约16小时。加入0.8μL 0.8MEDTA(pH8.0)以终止反应,加入2.5μL 4M LiCl混合均匀,再加入75μL预冷的无水乙醇沉淀标记后的DNA,置于-80℃沉降40分钟。4℃,12000rpm离心20分钟收集DNA,用预冷的70%乙醇洗涤DNA沉淀,真空干燥后重新溶于50μLTE((pH 8.0)中。
2)DIG DNA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、10-1、10-2、10-3、10-4、10-5。稀释标记的对照DNA至浓度分别为以下浓度1μg/mL,100ng/mL,10ng/mL,1ng/mL,0.1ng/mL,0.01ng/mL。分别取1μL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-80℃的基因文库稍融,取50μL,用450μL LB稀释得到10-1的稀释倍数,倍比稀释得到10-2,10-3,10-4,10-5,10-6。300μL涂平板(15cm×15cm,平板为LB/50μg/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℃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4-5hr,使克隆重新生长作为原平板。将尼龙膜置于变性液(0.25M NaOH,1.5M NaCl)饱和的滤纸上15分钟(不要浸过膜),转移至中和液(1.0M Tris.HCl,1.5M NaCl,pH 7.5)饱和的滤纸上5分钟。转移至2×SSC(20xSSC储备液(L-1):NaCl,175.3g,柠檬酸钠,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,120℃固定45分钟。常温下于3×SSC/0.1%SDS溶液中振荡洗涤3小时,以除去细胞碎片。
4)Southern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400mL 0.25M HCl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(0.5M NaOH,1M NaCl)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1mm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10×SSC转移缓冲液转移8-24hr。用2×SSC略微洗膜,120℃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℃,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/100cm2)混合均匀。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℃或68℃约16小时。
6)杂交后严紧洗脱:室温下用2×SSC/0.1%SDS漂洗两次,每次5分钟。68℃,用0.1×SSC/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)Tween 20)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0.1M马来酸,0.15M NaCl,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(0.1M Tris-HCl,0.1M NaCl,pH=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(nitroblue tetrazolium chloride)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45μL NBT,35μL BCIP]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(Am 25μg/ml)中,30℃振荡约28hr。取出200μl涂布在ISP-2(Am 50μg/ml)平板,30℃培养6-8天,收孢子,保存于-80℃;取出10μl在ISP-2(Am 50μg/ml)平板画线,37℃培养,放置2-3天。挑37℃整合生长的单菌落,接种至液体培养基ISP-2(Am 25μg/ml),37℃,振荡2-3天。取出涂布在ISP-2平板(Am 50μg/ml),37℃整合2-3天,收孢子,保存于-80℃。
基因中断或基因置换所用载体为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Streptomyces actuosus ATCC25421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:
SEQUENCE LISTING
<110>中国科学院上海有机化学研究所
<120>诺丝七肽的生物合成基因簇
<130>说明书、权利要求书
<160>1
<170>Patent In version 3.3
<210>1
<211>34713
<212>DNA
<213>链霉菌Streptomyces actuosus ATCC25421
<400>1
CGCCAGCGCC GCGAGGGGCT CCTCGTCGGA CGGCTTGCGC TTGATGACCT CTTCGGCGTT 60
GTCGACCGCG TGGCCCTCGA TGGCCTCGAT GTCGACCGGG GAGGGCAGGT AGCGCACGAC 120
CGCGTCGAGC AGGGGCTGGA CGCCCTTGTT CTTGAACGCG GTGCCGCAGA AGACCGGGGT 180
CACCGTGGTG CCCTGGCCCT TGCCGGAAGC GATCGTGATA CGGCGGATGG CCGCGTACAG 240
CTGCTCCTCG GAGGGCTCCT CGCCCTCCAG GTACAGCTCC ATGATCTCTT CGTCGTTCTC 300
GGCGACGGCC TCGAGCAGCT TGCCGCGCCA CTCCTCGGCG GCCTCGGTGT GCGTGGCCGG 360
GATGTCGACG ACGTCGTACA TCTCGCCCTT GGTCGCCTCG GCGGACCACA CGAGCGCCTT 420
CATGCGGACC AGGTCCACGA CGCCCTTGAA GTCGGCCTCA GCGCCGATCG GCAGCTGCAT 480
CACGATCGGC TGGGCACCCA GCCGGTCGCT GATCATGTCG ACACAGCGGT GGAACTCGGC 540
ACCGGTGCGG TCGAGCTTGT TGACGAAGCA GATACGCGGA ACGCCGTAGC GGTCCGCCTG 600
ACGCCACACC GTCTCGGACT GCGGCTCGAC GCCGGCGACG CCGTCGAACA CCGTCACGGC 660
ACCGTCGAGC ACGCGCAGGG AACGCTCCAC CTCGACGGTG AAGTCGACGT GACCCGGCGT 720
GTCGATGATG TTGATGGTGT GGTCGACGTC GTCCAGCGAC CAGTGACAGG TGGTGGCAGC 780
AGAGGTGATC GTGATGCCAC GCTCCTGCTC CTGCTCCATC CAGTCCATGG TGGCAGCGCC 840
GTCGTGGACC TCACCGATCT TGTAGGACAC ACCGGTGTAG AACAGGATCC GCTCGGTGGT 900
GGTCGTCTTG CCCGCGTCGA TGTGGGCCAT GATGCCGATG TTGCGCACCT TGGCCAGGTC 960
AAGCGAAGTG GTAGCCATAA GGCTTCAGTC TTCTCTCGGT TCTCGAAGGG GTTCCGACTA 1020
CCAGCGGTAG TGCGCGAAGG CCTTGTTGGA CTCGGCCATC TTGTGCGTGT CCTCGCGCTT 1080
CTTCACCGCG GCGCCGAGGC CGTTGGAGGC GTCGAGGAGC TCGTTGAGCA GACGCTCGGT 1140
CATGGTCTTC TCGCGACGGG CGCGGGAGTA ACCGACGAGC CAGCGCAGCG CCAGGGTGTT 1200
GGCACGGCCG GGCTTGACCT CGACCGGGAC CTGGTAGGTG GCGCCACCGA CACGGCGGGA 1260
CTTGACCTCG AGGGTCGGCT TGATGTTCTC GAGAGCGCGC TTGAGCGTGA TGACCGGGTC 1320
GTTGCCGGTC TTCTCGCGCA GGCCCTCCAT GGCGCCGTAG ACGATGCGCT CGGCGGTGGA 1380
GCGCTTGCCG TTCAGCAGCA CCTTGTTGAT CAGGGAGGTC ACCAGAGGAG AACCGTAGAC 1440
CGGGTCGATG ATGACCGGGC GCTTCGGGGC GGGGCCCTTA CGAGGCATTC TTACTTCTCC 1500
TTCTTGGCGC CGTAGCGGCT GCGGGCCTGC TTGCGGTTCT TGACACCCTG GGTGTCGAGC 1560
GAGCCACGGA TGATCTTGTA GCGAACACCC GGCAGGTCCT TCACACGGCC GCCGCGCACG 1620
AGCACGATGG AGTGCTCCTG CAGGTTGTGT CCCTCACCCG GAATGTAAGC GGTGACCTCG 1680
ATCCCGCTGG TCAGACGCAC ACGCGCGACC TTACGCAGGG CCGAGTTCGG CTTCTTCGGG 1740
GTGGTCGTGA ACACACGCGT GCAGACGCCA CGACGCTGAG GGGAACCCTC GAGTGCGGGC 1800
GTCTTGTTCT TCTCGACCTT GTCCTGCCGG CCCTTGCGGA CCAGCTGCTG GATCGTAGGC 1860
ACTACTTCTC CGGTTTCTGT GTGCCGAATG GTGAAGCTAA CCTGGAACGT CGCCGACCCA 1920
CGCGGTCGGG TGTGTCGAAT CGTGCGGACT CCCGCCGCAA GGCAGAAAGA GGCGCAGATT 1980
GCGGTGGCCG GTGACGGCTC TCTGTGCGGT TGAAGGCACG CACGAGAGCC AGGGCACACC 2040
CCAGGCACAA GGTCTGAGCG TACCTAGCCC GTCAGCTCCG GTCAAAACAA ATGGAACCCG 2100
CCCCGGTGGA CCGGGGCCGC GAGCGCACCG CGTACCGATT CATCCCGGTA CGTACGGTAC 2160
CCGGGCATGG CCGGAGCATG CCCGGGTACC GGGGGGCGCG CGAGGGGCGC GCGGAAGGCG 2220
CTGATTTTTG GTGGTGTCTC CGCTCGCGCT ACGCCGGCGG CCGGGAGGGG ATGAAGGCGT 2280
CGGCGCCCGG CGGTACGAAC TCCTGCGGGC GGGTCAGCGG CTCGGGCGAG GCCGGGTCGA 2340
AGGCCAGGCC CAGCCCCTTG CCGAAGGGTG CGGCGGCAGC GGCGGGGCCC TCGGTGGCGG 2400
CCTCGACGCT GTAGTAGCGG CGCTCGGTCG GGCGGTAGCC GATCAGCTCC ACGACCTTGG 2460
ACACGCCGGG CGGGCGGCCG TAGGCGGCCT CGGGCGAGTC CCAGAGGTAG ACCGCGCCCC 2520
ACTGCTCGCC CTCGGGGCCG GTGGAGGAGA TCCACGTCTT CTGCCGCAGG CCGGGCACGG 2580
TCGTGTACGC GTCCACGGCG TGGTCGCGCA GGTAGGCGCG CAGGGAGGCG ACGGTCGCCG 2640
CGGAGCGGGA CAGGTCCCAC AGGACGACCG TGCAGTACAG CTGCTGTGCG GGGTGTTCGG 2700
TCATCGCAGG ACCGCCTTCA GTTCGCGCGG GTACCGGTGG AAGAGGTTCG CGGTGAACTC 2760
CACGGCGGCC GGGTCGGCGG CCGGGGTGAG GGCGGGGAAG CGGCGCAGCA GCCGGCCGAA 2820
GCCCTCCTGG AGTTCCACGC GGGCGAGGGA CGAGCCGAGG CAGTAGTGCG CGCCGGCGCC 2880
GAAGGTGAGG TGGGTGCGCG CGGCGCCCCG GGCCAGGTCG AAGGTGTCGG GGCGGTCGAC 2940
GTGCCGCTCG TCGCGGGCGG CGGAGTCGAG GGAGATGAAG ACCGTGGCGC CCTCGGGGAG 3000
GGTGATGCCG CCGAGGGTCA CCTCCTCGGT GGTGACGGTC GTCCCGGCGA AGCCGGTGCC 3060
GGCCTGGGCG TACCGCAGGA TCTCCTCGAC GGCGGCGGCC TCGGTGACGG TGCCGTCGCG 3120
CAGCCGGGCG AGCTGGTCGG GGTGCTGGAA GAGGGCGAGG AGTCCGTTGG CGAACTGGAC 3180
GGCGGTGGAC TCGTAGCCGG CGGTGGCCAG CAGGACGACG GTGGAGACGA GTTCGTCGGC 3240
GGAGGGGCGG GCGTCCTCGT CGAGGCCCGC GATCAGGCTG CCGATCAGCC CGTCGCCGGG 3300
AGCGGCCAGC TCCTTCTCCA CCAGCTGCCG GGCCACGCCG AACAGCGCGC CCATGGCCGC 3360
GCCGAAGGCG GCGGTCGACG GGGCGGCGGC CCGCACCTGG GCCGCCCACC CCCACAGCCC 3420
GGGGCGCAGC TCTTCGGGCA GGTCGAACAG CTCGCACAGC ACGCGGACGG GCACCTGGTA 3480
GGCGAAGCCG GAGACCAGGT CGACGGGGTC CGGCCCCGCC TCGGCGAGCC CGTCCAGCAC 3540
CTCGTCCACC GCCTGCCCGA CCGCCTTGCG CAGCGACTCG GCGTGCCGGG GGGTGAGGAC 3600
GGTGGCGAGG GTACGGCGCC ACCGGGCGTG CCGCTCGGGG TCGGCCAGCG CCATCCCGAA 3660
GTCGAACCGC TGCCCGGGCC CCTTACCGGG GGCACCCGGG CCCACACTGG CTCCGCCCGG 3720
GCCGCCAGGC CCGGGCACCC CACCCGGCCA ACCACCCTGC GGTGCACCCC CGGCGGAGCC 3780
GCCGGGGCCG CCCGGCCAAC CGCCCTGCGG GGCACCCTCG GCGGAGCCGC CCGGGCCGCC 3840
CGGGCCGGGC GGGCCGCCCC GGAGGGCTCC CGCTCGGATC GCCGCGCGGC TGAAGCGGGG 3900
GTCGGTCAGG ACCGTGCGGA CGTCGTCGTG GCGGGTGACC AGCCAGGCCG TGGCGCCGTT 3960
CGCGAGGCGG ATCTGTGAGA CGGGGGCCTC GTCGCGGCGG CGGACCAGTT CATCGGGTGG 4020
GGCGAAGGGG CACTTCTGCT CGAAGGGGTA CGTCGGGCTC TCGTCCATGG GGGCCTCCAC 4080
GCTCAGATCC GCACCGGCAG CTCGGCCGGA TAGAAGGTGA ACAGGTTGCT GGCGAGCGGC 4140
ACGCCGTCCA GGTCGGGCCC GTCGAACCGC AGGTCCGGGA AGCGGTTCAG CATGCGCGCG 4200
ATGCCCTCCT GCATCTCGAC GCGGGCCAGC GCGGCGCCCA GGCAGTAGTG GGGGCCGTAG 4260
CCGAACGCGG TCTGCCGGTG GGCGCAGCCG CGGCCGACGT CGAAGCGGTC GGCGTCCTCG 4320
CCGAAGACCT TCTCGTCCCG GTTGGCCGAC GCGAGGGAGA CGAAGACGGT CGAGCCGGCC 4380
GGGACGGTCC CGCCGGACAG CTCGACGTCC TCGGTGGTGA ACTTGGCGAT GGCGTACCCG 4440
GTGCCCATCT GGGCGTACCG CAGGATCTCC TCGACGGCCT GGCCGATCAG CGCGGGCTCC 4500
TCGCGCAGCC GGCGCATCTG GGAGGGGTCG CGGAACAGGG CGTAGAAGGC GTTGCCGAGC 4560
TGGACGGCGG TGCTCTCGTA GCCGGCCATC AGCAGCAGGA TGACCGTGGA GACCAGTTCC 4620
TCGTTGCTGA GCCGGCCGTC CTCCTCGTCG GTGAGGCCGA TCAGCGTGCT GAGCAGGTCG 4680
TCGCCCAGGT TCTGCCGCTT GCGGACGATC AGCCCGATGG CGTACCGGCC GATCGCCTGG 4740
AGGGCCTGGC CGAACTCGAC GAACGACGAG GTGAAGTCGC GGCGGGTGAT CTGCGCGGCG 4800
AGCGCGGAGA ACTCGGGGCG GGCGGTCTGC TCCAGTCCGA GCAGGTCGCA GATGACCCGG 4860
ATCGGCAGCT GGTAGGCGTA GTCGGCCATG AGGTCGAAGC GACCGCCGGC GCTTTCGAGG 4920
TCGTCGAGGA GGGCGTCGGT GTGGGCGGCG ACGGCGGGCC GCATCGACTC GGCGTGGCGG 4980
GCGGTGAACG CCTGGTTGAC CAGGCGGCGC CAGCGGGTGT GGCCGGGCGG TTCGGAGATG 5040
GAGCCGCCGA AGTTGAAGAC GGGGCTGTCG GCGTCGGCCT GGAAGCCGGT GTCCACCCGG 5100
GCCGCGCCCT CCCGGTTGAT GGTGCGGGAG AACCGCGGGT CGGTGAGGGC GGTGCGGACG 5160
TCCTCGTAGC GGGTGATCAG CCAGGCGGTG TCGCCGGTGG CGAGGCGGAC CTGGGCGATG 5220
GGCCGGTTCT CGCGCAGCCA GGCGAACTCC TCCGGCATCT CGAACGGGGT GTGGCGGGTG 5280
AAGGGGTAGG TGCGCGCTTC GATGTCCACG GCAGGGGCTC CTGTCGGTCA GTGCTGGCCG 5340
GAGGGCAGAT GGGCGGCGAG GCGTTCCCGG CGGTGGTCGC GGACCCGGTC GAGCAGGCCG 5400
GCGTCCGGGG CGAGGCCCGC GCAGTCGGCG ACGACGTGGT GGCCGAGGTA GCCGAGGAAG 5460
GCCTCCTGCT CCGGGATGAG GCCCAGCCGG TTGTTGTGGG TGTGCAGATA GCTGAGGAGC 5520
ACGATCTGGC GGCGCCGCTC CGCCGGCGCG TCGGGGCCGC CGAGCGTGAC CGCGGGCAGC 5580
ACGGTGTCCG CGGCCGTGTG CAGCGCCTGC CAGGCCTCGC CGACGGCCCG GCCGTAGGGG 5640
TCGTCGGGCG GCTGCGCGGC GGCGAGCAGC GGCCGCAGGT GGGCGGCGGC CGGGCCGCGG 5700
CGCTGGTCCA GCAGCTCGGG CCAGGTGTCG AGGAAGCTGT CGAAGACGTA CGGCGACCAC 5760
ACCGCGCAGT AGTGGGCGAG GAACCCGGCC ATGGCGGGCG GCGAGAGCCG GGCCGCGCAC 5820
AGGCCGCGCA GCGTCATGGT GAACGCGGCG CCCAGCCGGC GGGCCGGGCG GTCGCCGATG 5880
GCGCGCAGCG CGGACAGGGC GACGGCGCTG CTGCGGTCGA AGAACCGCTC GGCGACGGCG 5940
ACACCCTCCG GGCCGCCGTA CTTGCCGTAC TCGGGCTCGT AGCGGGCGGG GTGCACGGTG 6000
TCCGGCGGGA GCAGTTCGCC GGTCGCCTCC GACTCCAGTG CGGCCATGGT CAGCTGGGCC 6060
TCGCGGAACT CCTCGGGGTC GAGGTCTCCG TAGCCGCCGG GATGGGCGGC GAGGTGGCCG 6120
GCGAGGCGGT CGGCGACCTC GCGCACGGTG CGGGCGGCCT GCTCGGGGTC GTGGGCGGCG 6180
GGGCGCAGGC GGAGCCGGAT GTGGTGGCCG CCCTGCCAGT ACCGCAGGAA GAAGGAGCTC 6240
TCCAGCGCGC CGGAGGCGTG CAGCGGGGCG AGCACGGGGG CCAGCCCGTC CACGAGGAAC 6300
GCGTCCTGCC CGGCGTACCG GTGCAGGTGC AGGCTGTGCC AGGGGCCGGT CTCAGGGGCG 6360
GGCATGGGCG TCCTCCGTGG CCTGGGTTCC GGCGGGGGCG GGGGTGCCGA GGGTGTACTC 6420
GACGAAGAAC TCCTCCGCGC CGGAGGTCCG GCCGTCCAGG TCGCCGGCGT GCGGCAGGCA 6480
CTCCTGGAAG GTGACGGTGG CGTCCGGGCC GGCCTCGGCG AGCCGGCGGG CGAAGACCTG 6540
GAGGAGGAAG GGGTTGCGGG CGTCGAGGTA GTGCGGTTTG TGCAGCCGGG CGGTGCGCGC 6600
CTCCAGCGCC CACTGCCGGG TCTCCGCCAG CAGGTCGCGC TCGCCGGCGG GCACGGGCGG 6660
GGGCGTGGTC AGCCGGAAGA AGGTGTGCCG GGGGATGCCG CGCTGCCCGA GCCAGCGGTC 6720
GAACGAGGCG AGGGCCGCCG CCTCCTGCCG TTCCAGGCCG TCGGTGTCCG GGACGTCCGC 6780
CGCGGGCAGC CGCCAGGAGG CCCGGTCGAG GACGAGGTCG CCGAGCAGTA CCCGGGGCAG 6840
CGACCGGTGG GTGGCGGGGA CGCCGGTGCG GCCGGCGGCC CGGCCCGCTT CCGCGTCGGC 6900
CCGGTCGAGC TGGTCCCACA GTCCGCCGCG GTAGGTGCGG GTGGGGGCGA AGGCGCACAG 6960
CAGCCGGTAC AGGCCGGGTG CGGCGGCCGG GTAGAGGAAG TTGAGCGGTA CGAGGTCCAG 7020
GGGCTGCCCG TCGCGGGTGC TGACCAGGGC CAGGGTGCGG CGGGCCGGGT CGGCGCGGAC 7080
GGCCAGGTCG GCGAGGGTCA GGGTGTGCTC GTCGGCGGCG CGCGGCACCG AGCCGGGGTA 7140
GACCAGCTCG TACGGGCTGA GCCGGGGGTG CAGGTTGAAG TTGAGGCCGA GGACGGCCGT 7200
GATGTCGCAC TGGCGGGGCG TCGTGCGGGC GATGTGGCCG CGCAGCGCCT CGGTGAGGCT 7260
CCACTCGTGC GGCCCGTCCG GCTCCAGCAG GTCGCAGAAC CGGGAGAAGA ACACGCCGTG 7320
CCCGGTGGTG ACGCCGTTGA CGACGGCGTA CGGCCGCTCG GGTTCCGCGG TGAACTGCAT 7380
CCGGTAGGCG GTGGACCGCC AGGGGGTCAC CTCGGCGGGC AGCCGGTCGG CGAAGGCGCG 7440
CAGCCGCTCG GCGTCCAGGC GCAGCGCGCC GGTGTGTGCG CTGTCATGGG CGCGCAGTTC 7500
GGTGCGGAGG AGGTCGGCGA AGTCGGCGCG CAGCCGGCGG ATGCGGTCGC TGTGCGGGTC 7560
GCCGACGCCG GAGGCCACGG CACTGGCCTC GGCGGGCGGC AGCGCGGCGA ACCGGCGGTA 7620
GAACTCGATG AACGGGACGT CGCCGTCGCC GTCCGCCGCG CCGAAGTGCC GGGCGAAGAA 7680
GGCATACAGG CCCGCCTTCT CCACGCTCGC GTCGTCCAGC ACCGGGACGA TCCGCTGGAA 7740
CAGCTCCAGG GCCCAGCGGT TGGCGTGCAG CAGGTCCGGG TGCCAGCTGT GGGCGGGTTC 7800
GCGGGTGCCG ACGTCCTCGT AGACGACGGA GCGCATGGCC TCGGGGGCGG GGGCGCGGCA 7860
GCCGACGGCC TCGACGAACA CGGCGACCTG TTCGCGGATC TCGGCCAGCA GCGCGGCCCG 7920
CCGCCGCACG GGGGCGGCCG GGAAGGCGTC CTCGGCGGCC TGGAGCCGTT CGAAGACGAC 7980
GGCGCAGCGG CCGGCCTGGT CGGTGTCGAG GGTGCGCAGC CGCTGGGCGA CGGCGAGCGC 8040
GGGGCGCGGG TGCTGGTCGG GGATGCCGAG CCCGCGTTCG CAGATCCCGG CGCGGACCAG 8100
CTTGTCCAGG GCGGTCGCCG CGGCCTGCGG GGAGAGTCCG GCGGCGGCCA GCCTCTCGCG 8160
CAGCTCGCGC TCGGGCAGCC CGCCGTCGGC GAGCAGGGAC CGCAGCAGGC GCAGCAGGCC 8220
GGTGTCGCGG GCGGTCACCA CCTGGGCGGT GCCGTAGGCC GCCTCGGGGG CGCCGTCCGG 8280
GGCCCGCCGT ACGCACAGGG CCTGTTCCTC CCGGACGAGC AGGGAGTGGT TGAGCCGGAT 8340
CCGCATCAGC TCGTCGGCGC GGTCCAGGCG GTGCAGTTCG TACGTCATCC AGGACAGCAG 8400
TCCCGCGCTG AGCCGGGCCT GGACGGGGCG GGCCGGAGGG CGGTCGGCCG GTGCCGTGCC 8460
GTCGCCGGCG GGCGTCCAGG GGGCGGCGCC GATCTCGGTG AAGGAGGCGA ACGGCGACGG 8520
CTTGAGGGCG ACCCGGTAGG CGAAGCTGGT CAGCGTGTTC TCGGCGCGGC GCCGCTTGCT 8580
GGGCTTGCGG CGCGCGGAGA ACGGGTCGGC GGCCCACTCG GTGACGGTCC GCCAGGTCCG 8640
CTCGTCGGAT AGCTGGAGGC CGCGCTGGAA GAACTCCCCC GTCGCGATGG CCGCCAACGC 8700
CCTTCGACCC GCGTCGAGTT CGGCGGCGAG GGCCGCTTCG GCCTCCTTGT GCAGCCGGTT 8760
GCCCTCGGCG CGGTGCCGCA GCCAGTCCTC CAGCAGTTCC CGGCTCGCCG GGTCGAGCAG 8820
GCGGGCGGCG TCGGGGACGC CGGGGACGCG GTCGTTGTGG ATGTCGCGCC GCAGCCGCAG 8880
CAGTTCGTGG CGCCGGTCGG CGGGCAGCTC CGGCACGGCC GCGTGCAGCG CGTCCTCCAG 8940
GCGCCCGCGC AGCTCCTCCC GCCGCTGCCG GGTCTCCTCG GCCTCCGCGA GCAGCGCCCA 9000
GGTGCGCGCC GGGGTCAGCG CGGCCAGCTC GGACGGGCCG AGCGTGCCGC GGCGGTAGAG 9060
GTAGTAGGGG CTGAACCCGT CGGGCGCGCC GGCAGCGTCG GGCGCGCCGG GCACCCGGGT 9120
CGTGTCGTTC ACCGTGGCAT CACCGCAATC TCCCAGTTCG GGTCGGGGCG TACGGCGCCG 9180
ACGCAGATCT GCGCCTGGGT CCCGCCGTCC GTGTCCGCGC CGCCGTCGAG GCCGTACGCC 9240
GTGTCGATCC GGGTCACGTC GAACCCGAGG ACGGCGTGGC CGCTGAGGCC CTCGGCGGCC 9300
GCGGCCAGCG TCACCGCCTC GATCGCCGCG CCCACGGCGA GCTGCTGCGC GCGGTAGGCG 9360
CGCGGGCCGC CCGCCGGCCG CCAGTCGGCC GGGGTCACCG GGTGGACGGT GAAGGCGGCC 9420
AGCTCGGCGT TGAAAGAGGC GGCGAACAGC GCCTCCTGCA CCCGGCGCGC GCTGCCGGGC 9480
CCGGTCCCCT CCCCCACGGG CAGCAGCGCG CCCAGCTCGT GCGCGTACCG GTACCAGCCC 9540
GGCGGCACGC CGCGCACCCG GTGGACGGCG CAGTACAGGC GGGTCCGCGC GGCCCACCCG 9600
GCCGGCCCGT CCGCCCCGGC TCCGGCCAGC GCCCGCAGCG CCTCGGCCGC CTGCCCCAGC 9660
ACCCCCGCGA GGGCCTCCCC GTCGGCCTCG GCCCCGGTGA ACAAGCGCCC CCGCGAGAAC 9720
CGCCGCGCGG CGGTCTCGGC GGCGAGGAGA TCGAGGGCGC GGGGGGCGGG CAGCGGCACC 9780
GGGCCGACCG GCACGGCGTC CCCGGGCACG GGGCGGGGCG CGGCGGCCGA CACGGCGTCC 9840
TCGTGCACGG GGCCGGGCGC GGCGGCCGGT CCGGCGTCCC CGGGCACGGG GCCGGGCGCG 9900
GTGGCCGGTA CGGCGTCCTC GGGCGGCCGG GCGGGCGCGG CGGTGCTCGT GTCCTCGCGG 9960
GCCGCGCGCT GGAAGGCGGT GAAGCGGTCG GAGCGCCGTA CCGTCCGGGA GCGCTCCAGC 10020
AGGGCGGGGC GGGGCGGGGC GTCGGGTCCG CCGGTGTCCG CCCTGTACGG GAGGTACGGT 10080
CCGAGCGTCA CCGCCGCCCA CGCGTTCTCC TCCTCCCCGT CGAGGCCGAG GCAGGCGTGC 10140
ACGGCCTCGT CGTCGAAGAC CGTCCGCACC TCCGCGTGCT CCCACTCGGC CCGCGCGAGG 10200
CGGGCGGCGC GGCCGAGGGC GACCCCGGCG TCGACCAGGC CGAGGCGGGT CGCGAAGTCG 10260
CCGTACTTGT AGAAGTTCTT CCAGAAGCGG CTGGTCAGGA CGAGCACCGC CGCGGGCGCC 10320
GCCTCCGTCG GCAGGTGCAG CGCGGCGCGC AGCCGGGCCG CCGCGTCGCC GCCGGCCAGC 10380
ACGGTCAGCT CGTGCCGGTA GGGGTCGTAG TGGCAGACCT GCCCGGTCGC GGTGAGGGCG 10440
GCGTACACCT CGGTCGGGTA CATCGCCCCG CCCGACGGGA CGGGCCGGCG CAACTGCACC 10500
TCGGGCCCGT GGTGCGGACG CGGGTCGGCC GGGGTGGCGG CGATGCCGCC GGACGGGTCG 10560
GTGCGGATCC GGCTGACGGC GAAGCCGCCG TGCAGGAGCC GGTGCAGGGC CGCGAGCGGC 10620
GGCGGTCCGT CGGGCCGCAG CGGCAGCCGC CGCGCTCCGC TGTAGACCTT CACCGGCCAG 10680
GGCCCGTCGG CCCAGTCCAC CCGCCAGCCG TCCGGGTTGG CGGAGCGCGG GTCGAGCCGC 10740
AGCCCGGTGG TGTAGGTGTA CGCCTCGTCG GTCGTCATGG TCATGGTCGT GCGGGTTCTC 10800
CTTGCCGGCT GGCCATCGGA CGGCATGTCC TACGGGAAGG GGTGCGGGAC GGCGCCGATC 10860
TCCCGGCCGG GGGCCAGCAG GCTGCGGTAG GGCAGGGTGG TGCCCTCCGT GAGCCGGGGC 10920
AGGCCGCAGG TACGGCGGTT GCGGTGGCCG AAGGTCATCG GGATCAGGCC CGGCACCAGC 10980
ACCCGGACGC AGTGCAGGCC GTTGCGGCGC AGCTCGGGCA TGGTCTGGTC GACGACCAGG 11040
ACGTCGAGGC CGCAGTCGAG GACGCCGGAG ACGGCCGCGT ACAGGTCGGC CCGCAGGTCG 11100
GCGTCCTGCG TGCGGAGCGT CGAGCGGACC CGGCCGAGCG GGACGGGGGC GCCGGTGCGG 11160
GGCCGGTCGA GGAGGAAGGA GAAGCGGTCG CGGGCCTCGG GCAGCGCGCC GACCGTGGAG 11220
TGGTCCTCCA TGCGGCGGAT GAGGAACGGG TCGGCCAGCA TGCGCAGCGC GTCGGGGCGG 11280
CGGCGCTCGT AGGCGTCCCG GGTGGCGAGG ACCGTGCCCA CCAGCTCGTG CAGGGCGCCG 11340
GTGACGGCCT GGACGGGGTC GGGGTGCGCG CCGCAGCCCG CGAAGAGCCG GGGCCCGTCG 11400
CCGCTGTCGT TCTCGGCGGT GAGCAGCAGC GCCGGGACGC CGTACTCCAT CGTGGCGTCG 11460
AAGCAGCGGA AGTCGAAGCC GGTGAAGAGG CGGGACTTGG CCAGCAGGTG GTCCAGGGCG 11520
GGCGAGGTGC CGTCGAGGGC GACCTCTGGG ACGTCGAGTT CGCGGTACCA GGTGAGCAGG 11580
AAGGCGTCCC GCTCGGCGAG TTCGCGCAGG CCGTGCAGGA CGGCCTCCTC GACGCTGTTG 11640
CCGAGGGCGC AGCCGTTCGA GGTGTCGTAG AAGAAGGAGA TCTCGTCGTC GTGGCGCGGT 11700
CCCCAGAACG CGGCGCGCTC CGGGACGAGG ACGCGTCCGT CGCGGCGGAA GGAGTACGCG 11760
CCCACCCAGT CGATCTCGGT GCCGGGGTCG AAGGGCCGGT ACCGGAAGCC TTCGGAGGCG 11820
TAGCTCTCCT CCGGGTGGGT GCCCAGGTCC GGCGGGTACA GGGCCTGGTC GGCCACGTCC 11880
GCGTAGCGGG CCCGCACGGG GGGCAGGGTG CCGCCGCGGT GCAGGCCGGC GTAGCGCTCC 11940
AGGCCCTCCA GGACGGCGAC GGTGCGGCTG GTGGCGTAGT CGGCGGCGCG GCCGATGGCG 12000
GGCTCGCGGC GGCCCCAGCG CGGCGGCAGC TCCACCGAGC AGGCCCCGAA GGGGCTCTGC 12060
AGGTCCTGCC GCAGCTCCTT GAACAGGCCG AGGCCGGCGT AGAGGTAGTC GCCGCGGACG 12120
GCGTCCGCGG GCAGCCGTCC GGTGCGCAGG GTGGCCGGGG TGAGCTTGGT GAGCGGGACG 12180
GCGGCGGGGG TCAGCGCGGG CACGGTGTCC TCGCGGAGGC TGCCGCAGTT CGGGCAGCCG 12240
GAGTCCGGCA GCAGGGTCTG CTGCTCGACC GTGCCGGCGG CGCCGTCGAG GACGAGGACG 12300
CGGCCGTCGG CCGGGCCGGC CGGGCCCTTG CAGGACGCGA GCCGGGCGCG GACGTACTCC 12360
TCGACCAGCC GCAGCACGCC GGGTCCCCAG ATGCGGACGT CCGCGCCGTG CGGCCAGGAC 12420
TCGGCGAGGC TGTCGCCCTC CAGGCCGGGC CCGAAGGGCG AGTTGGCGGT CCGGGTCACC 12480
AGGCAGCGCG GGCAGCCCTG GGTGCCGGGG CGCCAGACGG GGCCGATGTA GACGAGCGAG 12540
CGCCAGCTGC CGACGAAGAG CAGGGAGCGT CCGGTGGCCA GGCAGTCGAC GACGGTGTCC 12600
TGGAACTCGC CGAGCCCGTC GAGCCCGGCG ACGAGCACGG TCGCCCCGGA GGACCGGCCG 12660
AAGCGTGCAC CGCCGTCCGG GTCGGGGGTG TCGTCGCGGC CGAGGCGCCG GACCAGATGC 12720
TCGGCGAGCA CGCCCCGCCC GACGACGACC GGCGGCCCGG CCGGCCCGGT GCGGGGCTGC 12780
GGGGTGGTCG CCGTACTCAT GACGCCTCCT CGGACACGGG CAGGGCCACG ACGCGGACGC 12840
GGGCACCGGC CTCTCCCCCG CTGCCGGTGC CGGTGCCGGT GCCGGTGCCG GTGTCGGTGT 12900
CGGTGAGGAA GGGCAGCAGG TCGGTGATCT CGCGTTCGCC TTCGGCGGTG GGCGCGGCGG 12960
TCCAGGTCGC GGTGGCCGGG GCCAGGAAGG CGGTGGCCTC CTGGGCGTTC CCGGGCAGCG 13020
GCAGGACGCG CGCCACCGCG TTGAGGAGGG CGTGTCCCAC CGCCCGTTCC CGGTCGGTGC 13080
CCACGCCCGC GGTGACCGCG CCGTCGCGGG TGCGCACATG GGCGCGGACG ACGCCGTCGG 13140
GCAGCTCCTC GGCGGCGGTG GCGGTCCAGG GCGTGCCCTC GGCGGCCAGC ACCCCGGCGA 13200
GGTAGCCTGC CGCGGTGTCG CCGGGCGGCG GCTCCTCCGG CGCCGGAGCC CAGTGGAGGT 13260
CCGGCGTGGG CCGGCCGAGC GCCGCGTGCA GCCGGGCGCG CAGCCGGGCC TCGCCCAGGG 13320
ACCAGCCGGC GCCGACCGCG CCGTAGCGCG CGTCCCCTGC GGTGAGGCGG CCGGCCAGCG 13380
ACTCCACCGC GCACAGCACG ACCTGGTTGC GGGCCTCGCG CGGGGACAGC GCCCGGCACA 13440
CCACGCCGAG GGCGGCGGGC GCGTCGGCGC CGCCGTCCGG GTCGGCGACC TGGCAGGCAC 13500
TGGCCGACAG CGGCAGCTGG GCCAGGTCCT CCTCGCCGAG CGCGAGCAGC GGGCCGGTGA 13560
CCGCGTCCGT CAGGGCGGCG CAGGCCGCCA CGATCCGGTC GGAGATCCGC ACCCGCTCGG 13620
GCGGGTCCTC GGAGCGCGGG ATGTCGGGCC GCACGGCGTC CTCGGGTGCG GCGGCCCCGG 13680
CGGGCGGCAG CCGCCGCGGT ACGGCGGGCT CGTGGTGCGG GCAGCGCGGG TGCCGCCGGG 13740
CGGCGTGGCC GCGCACCGCG GGGACGAGGG GTTCGACGGT GGTCACCGGC TTGTCCCCGG 13800
GCGGCCGGAC CCCGGCCAGC CGGGCGAAGG TGTGCTGCGC GACCCGCAGC GCGCCGACCG 13860
CGGCCGGGGC GACCGCCGGG GCGAGGCCCG CGGTGTCGCC GGTGGCCCGG ACGGCGATCG 13920
AGCGGTGCAC GCACTCCCAG CAGTACGGGG TGCCCAGCCC GGGTTCGAGC GCGGTGACGA 13980
AGTCGCCGCA CCGGGCGAGG ACCCCGACGG TCACACCGTC GCGCCACAGC GCGTGCTGGG 14040
AGCGGGCCAG CGCGGCGGCG TCGGCGCTGT CGTAGGCGAG CAGGACGGCG TCGGCGCCCT 14100
CGACGTCGGG GTGTCCGGCC AGCGCGGCCG GTCCGTCCGC CGCGAGCGGG TCGCGCAGCC 14160
GCCAGCGCGC CCCGGGATCC GCCGCGGCGG TCTCCTTCAG CACCTGCTGT ACGGCGGTCA 14220
GGTCGTCGTC GCCGTCCGGG ACCACGTCGA GCCGGGCGAT GCCGAACTCG CGCAGCGCGT 14280
CCAGCAGGGC GTGCAGCGCG GTGCCGCGGC CCGCGCACAC CACGCGTGCC GTGCGGACCT 14340
GCTGGAGGCG GGTGACCGGG CGGTCGGCGT GGTGTTCGAG GAACGCCAGA TGGGTGGCGT 14400
ACCGCTCGCG CATCCAGCCG GGCACCGGCT CGACGGGGTG GGCGACCTCC TTGATGAAGC 14460
CGTTGCGCAG CATGGCCCGC ACCAGCCCCA GCACGGACCG GCGGGCCCGG TCGGGCAGGT 14520
CGCCGTAGAG GTCCTGGAGG GTGCGTTCGC CGTCCAGCCC GGCGAAGAGG GAGCTGACCA 14580
GCTGGTAGGC GCCCTGGCCG CGGACGGAGA AGGAGCCGGT GTTGTTGCCG AGCCAGACGC 14640
CGTCCTGCTG GCGGACGTAG AAGGCGTCGG CGCGGAGCCG GTAGGTGCGG GTGAGGGTGT 14700
CGGTCAGCGG GTCGGCGGCC GGGCCGGCCG GCTGTGCCCT GTCTGCTGCC ACGTCTGTCA 14760
TGTCAGAGCT TCCCGTGCGC GCTGTAGACG GTTGCGGCCT CCTGCTCGGA GGCGCAGCTG 14820
ATCCGGCTGG GCCGGTGCTG TTCGGGGAGG GCGGCGCAGT GCGCGCGGAC GGTGTCCGCG 14880
TCGACGCTGC CGACCACCGT GGCGGCCAGG AACTCCAGGC CGGTCGGGGC GGTCTCGACG 14940
GTGACCCGTA CGTCGCTGAC CCCGTCGAGC GCGAGGACGT GGCGCTCGAT CTCCCGGGGC 15000
CGTACGAGCA CTCCGGCGAG CTTCTCGGCG CCGTCGAGGC GGCCTTCGAG GCAGAGGTTG 15060
CCGTCGGCGT CGAAGCGGCC GTAGTCGGTG CCGGTCACGT GGTCGGCGGG GGCGGAGGGC 15120
GGGCTGCCGG GCCGCCAGGT GCGCCGGCAG CAGCCGGGGC CGGCGACGGC GACCGTGCCG 15180
ATGCCGCGTT CGTCGGTGCC GGTCAGCCAC ACGGACTTGC CGGGGATCGG CCGGCCGACG 15240
TGCCGGTTGT GGCCGGGGTC GTGGTCGAGG GCGATGGCGC CGGTCTCGGC GGTGCCGTAC 15300
AGGTTGCTGA CGGCGGCGCC GGTGAAGTGG CGGCGGATCA GCTCGACCCG CGGGGCGCTG 15360
AGGTGGCCGC CGCCGAGGAA CAGGGCGACG GAGCCGAGGT CGGTGGCGGC GGGCGCGGAC 15420
AGGCAGGCCG CCGCGGCCAG GGCCGGGACG CCGAGGGCCA GCACGCGCCG GTCGCGGGCG 15480
AGGTCGTACA GCGGGCTCCA GTCGTGCGGC GGGTGCACGA CCAGGTCCCG TTTCAGGTAG 15540
AGGGCGGGCA GGACGCACAT GAAGAACGCG GCGGAGAACC CCAGCGGCAG ACAGGTGGCC 15600
AGCGCCTCGG GCGGGCCCGC GGGGAACGCG TCCGCGTACA TCGCCGCCTC GGCGTGCAGG 15660
GCGATCTGCT CCCACTCGTC GGCGGGGCCG ACGGCCACCT TGGGCGCGCC GGAGCTGCCG 15720
GAGGTGCACA CGCCCCACAG CGGGCGCTCC TGTGCCGCGA GCGGTACGGA CCCGTCGTCC 15780
GGGAGGGCGT GCAGCGCCGG GGCGATCTCC CGCCCGGTGA CGCCGAGGGT GCGCGGCGAC 15840
GCCCAGAGCA GGACCGTGTC CGGCACGGTG GCCACGGCGA ACAGCGCGGC CAGCGCGGGG 15900
AGTCCGGCGG TGGGGTCGAT CGCGTACGCG CCGCCGGGAC GCACCTGCGC GGCCAGGTCG 15960
CGGCCGGCGG TGAGCAGGTC GTCCCAGGCG GCGAAGTCGC CGCCCCAGCG GACGCCGGTC 16020
ACGGCACCGG CCCGGCCCGC CGGGAGGTGA CGGGGGGTCA GGAAGCGCTG GAACGCTGGT 16080
CGGCCCATGT CACCCACCCC TCGATCGTGC GCGGCGCGTC GGGCAGTTCG GGCAGGGTCC 16140
GCCCGGGTGC CTGCCGGGCG AAGTACGACA GGACGACGAC CGTCTCCAGC GAGTCGAGCC 16200
GCCGCCGCGG GTCGTCCTCC TGGAGCAGCC CGGCCAGCTC CTCCTGCAGG TCCGGTACGC 16260
CGTCCGGCGT CCGGGGTGTG GTGCGCTGAG ATGTCACGTG TCGCCCTTCT GCGTCGTTCG 16320
GGGACCGGCG GGTGCGTCGT GCGCGGTGCG CCAGAAGTCC TCGACCGCCT CGTTGAACTC 16380
CCGCGGCCGG TCGTGGCCGA CCAGATGGCC GGCGCCGGGG ACGACCCGGC CGCGGACCCG 16440
GCCGCCCCAG CCGGGCGCCT GCTCGGCCGC CGCGCGTTCG ACGGACTTCA GGGAGCCGTT 16500
GAGGACGAGC GTCGGGACGG TGAGCGCGGC CACGTCCGCC GCGCGGACCA GCGCGGTCCG 16560
CTCGAAGTCC CGCTCGACAT GCCCGGTGAC CGCGTCGAGC GTCCGCTTCC ACCGGGTGCC 16620
GTGCAGCTGC TCGTACTCGG CGGCCAGGGC CGGCTGCTGC GCGGCGAGGC CCTCGAAACC 16680
GGCGATCAGC GAGAGGAACG CGTCGCGGGA GACGTCCGGC GCGAACCCGG TCAGCACCAG 16740
CGAGGAGACC AGGTCGGGCC GGGTGGCCGC GCAGCGGTGG GCGAGCGGCC CTCCCAGGTA 16800
GGAGGCGCCG ATCAGCCGGC CGGGCCCGAA GCGTTCCAGC ACGGCGACGA GGTAGCGCAG 16860
CGCGTCGTCG AAGTAGTCCT CGGCGGCGTC ACGGCGGCAG CGGCCGTGAC CGGGCAGATC 16920
CACGGGTACG GTCCGCAGCC GGCCGTGCCA GGCCCGGATC TGCGCGGCGA AGTGCCCGTG 16980
GCCGGTGCCC AGCAGCCCGT GCACCAGGTA GACGGTGGGC GTCTCCGGGC CCGTCGACAT 17040
CCCGGTGTGC ATCGGCGTCT CCGTGTCCCT CGGCGTCTCG GTGTCCATCG GCGTCTCCGC 17100
GTCCACCGGT GCCTCAGACC GCCCGGGACG CCTCGCCCGC GGGGTTCACG AAGTGGTCGA 17160
CGGGTGTGCC GGACGCCTCG ATACGGCCGA GGATGTGCCG CGGCCGGCGC AGGTCGGCGA 17220
CGAGGAACTG GGAGGACTTC TCGTCGTTGC GGGCCTCGCA GCCGGCCCGG TAGGGGGCGA 17280
CGTCGGGGCT GCCGGGCGAG ATCACGCCGG CCAGGCCCAG CGCGACGTCC TGGAACTCCT 17340
GCGGCTCCCG CGTGGTGAGG ACGAGCCGCT GCTCGGGACA GGTGAAGGCG ACCACCGACA 17400
TCAGCCGCAG GTAGTCGTCG TCGCCGACCC GCGTGGTGTC CCGGGACTTC ATGGCCGGCC 17460
GCATCCGCGG CACCGACAGG TCGGCGGTCG CGCCCCGGGA GCGCAGGTGG TCGCCGTGCG 17520
CGACCAGGCT CACCAGCTCG GCGGAGAGGT CGTCGTGCAG CCCGACCAGC ACCCCGGGGT 17580
TCACATACCG GTAGCCGGCG TCCAGCCAGC GGTCGAAGGA GACCACCCGG CGGTCGAAGT 17640
CGGCCTTGGG CACCCCGACG GAGGTCTTGC CCATGAAGCG GCGGTACGTC TCGCGGTCGT 17700
AGCTCTCCTG GAACACGCAC ATGGTGACCG GGTCCTCCCG GCCGATCCAC TCGCCGAGGA 17760
CGTCGATCTC GTCCTGCTCC ATGCTGCCGA TGTTGAAGTA GACCCGCTCG AAGCCCAGGT 17820
CCAGGGCGGT GCGGATGGCC CAGCCGATCC GGAAGGCGGA GGCGAGCCGG GTGTGCTTGT 17880
CCTCGTACTC GCCGGTGAGG AAGCCGACCC CGCGGACGCC CTCGTGGTGG TAGAGGATCT 17940
CCAGCTGCTC GGTGATCTCC TTGCGGCCGG AGAACTTGCG GTCGAGGCGG TGGTTGCCCT 18000
TCCGCATGGA GCACATCTTG CACTCGGAGT CGCAGTAGTT GGTGGTGTAG AGCGGCACGA 18060
AGGTGTGCAG CCGGGGGCGG CGGGCCCCGC ACCGGGCCTC CGCGGCCGCC TGCAGCTCGG 18120
CGGTGCCGAT CGAGCGGTCC TCCCACAGGG CGAGCGCGAC CGCCGCCCGC GACTCGGCCG 18180
GCTCCTCGCC CTCGGCGAGG GCGAGGACGG CACGGGTGTC CACCGTGGCC GCCTCGGCGC 18240
GTACGTCCTC CAGCTCCGGC AGGACGAAGT CACCGGTCAT TGCGTGCGAG GTCATTGCCT 18300
GGGAGTTCTG CGTCATGTGA ATTCCTCTGC GAACGGCCGG AAGGGACCCG GCCCATGTAA 18360
CGCCACCCGG GAATTCCGAA CAAGACACCG AGGAAACGGA GCACTCTTTC TCCGTTACCC 18420
TCCCCACCTG CGCATTCACC CAGCCCTGAA CCACCTCCAC GGCGGTTGTG CAGCAGACCC 18480
GAAGCGCATG GTTAGCAGCG CTTTAGCGGC CTTTTTGTGC CGTCACAGCA GTACAGCAGC 18540
GGGGAATCAG CAGAAATGCA AAAGGGCCGG AATCCTTGAC TCACGGAAGA CCCGCTGCAT 18600
AGCCTCCGAG GCACGCGCTG CAGGAAATGC GCGAAGTAAA TTACCGTCGA CATGGAAGGG 18660
GTTTCCGGTG GACGCTGCAC ACCTGTCCGA CCTGGACATC GACGCTCTCG AGATCTCCGA 18720
GTTCCTGGAC GAGAGCCGAC TGGAGGACAG CGAGGTCGTG GCCAAGGTCA TGTCGGCCTC 18780
GTGCACCACC TGCGAGTGCT GCTGTTCCTG CTCCTCCTGA TGGAGACGGC GCCCTGTACC 18840
GGAGCGATCC GGTACAGGGC GCCGCTTTTC TCGACCCGAA AGGAATCCGC ATGCGGCAGA 18900
ACCTGCTGAT GATCTATGTG CACATCCCTT TCTGCCATTC GAAGTGCACG TTCTGCGACT 18960
GGGTCCAGGC CATCCCGACG AAGGATCTCC TGCGCAAGCC CGGTGACTCG GTGCGCCAGA 19020
AGTACATCTC CGCACTCTGC GCGGAAATCG CAGAGCGGGG CGCGATGCAT CGCGCGGCAG 19080
GTGACATCCC CCATGTCCTC TACTGGGGCG GCGGCACGGC CAGCAGCCTC GACGAGCAGG 19140
AGACCGCGGC GGTCATGGAG GCGCTGCACT CCTCCTTCGA CATGAGCACG GTGGCCGAGG 19200
CGACCATCGA GTGCAGCCCG GACACCGTCG ACGAGCGGAA GCTGGCCTTC TACCGCGGTC 19260
TCGGATTCAA CCGGGTCTCC AGCGGCGTGC AGTCCTTCGA CGACGACCGG CTGCGCCGCC 19320
TGGGCCGCCG GCACACCGCG GAGCAGGCCG GGCGCATCGT GCACGCCGCG CGGGCTGCGG 19380
GTTTCGAGGA CGTCTCCATC GACATCATGT CCGGCTTCCC CGACCAGGAG GCCGACGAGC 19440
TGGACCGGAC CGTCGACCGT GCCCTTGAGC TGCCGGTCAA CCACCTCTCG CTGTACTCGT 19500
TCCGGCCCAC CCCGGGCACG TTCATGCGCC GCCGGATGGA CTCCTCGGAG CGGCGGACGT 19560
ACCTGCGCCG CCAGCAGGCG CTGTTCACGC GGGCCCGCCG GGCGATCGAG GGCTTCGGCC 19620
TCAGCGAGTA CGCCAACGGC TACTTCGGCA AGGTGTCGCC GTTCGCCTCC ATGTACTTCC 19680
AGCACCGCGC CGACACCGTC GGCCTGGGCT CGGGCGCGAT CTCCCTGGTG GACCAGCGGT 19740
TCAAGTCCCA CCAGAAGGGC CTGCTGCACT CCTACGTCGA CGATCCGCTC GCCTTCGACA 19800
TCGACGTGCC GGCGGGCCAG GACCGCGTCC TGGTGTCGCT GCTGCAGGCC GGGCTCGCCA 19860
TGTTCGACGG CATCCCCCGC GAGGACTGGC GGCAGCGCAC CGGCACGGAC CTCGCCGAGG 19920
TGCTGCTGCG GCCCACGGTC GCGCCCCTCG CGGACTTCCT GCGCGGCCGC GGCCTGGTCG 19980
AGGACGACCA CGGCATCCGC CTGCCGCGTG ACATCGCCGG TCTCACCCTG ATCGAGCTGG 20040
CCTTCGAGAT GGCCATGTCC CAGCCGGAGC TGGTGTGACC TCGGGACCGG GGCAGGCCCC 20100
GGCCGAGGCG GCACACGCAG CCGGGGCGGC CTGGCTGGAG ATCGGGCTCG ACGCCCCCGC 20160
CGACGCCGTA CCCGCCCTGG TCGCCGGTGT CGTGCGGCCG CTGCTGCGGG AGCCCGCGGA 20220
GCCGGGGGCC GAGCCCGTGC CCGGGTTCTT CCTGCGGGGC GTCGGCGCGG CCCAGCCCGC 20280
CCTGGTCGTC CAGCTGGAGG TGACCCCCGG CACGGACCTC GCCGAGCCGT ACGCCGCCCG 20340
GGCCCGCGCG CTCGCCGCCG GGCTGGGCCT CCCCGTGCAG GTCGCGGCCG GCCGGGCGAC 20400
GCTCGTCCCG CTCGCCGGCT CGGTCTTCGC GGGGGCGGCC CTCGGCCCGG TGACCCGCGC 20460
CGCGCTGGCC GCCGTATGCC CCGCGCTGCT CACCGCCACC GAGGCCGCGG AACAGGGCAG 20520
GCCCGCCCTG CTGGCCAGTG CCGCCGAACT CATGTCCGCG CATCTGCGCG CGGTCAGCGT 20580
CTCCGCGGCC CCCGGCCCCC GCCAGTGGGA GGAACTGCGC GAGGGCGTGC CGCTCGGCTT 20640
CCTCAGCTAC CGCTCGCACG CGGAGGCGTT CCTGGCCTCC AGCCGCGACC CGAAGGCCGC 20700
GCAGGCGATG ATGGACGCGA AGTACACCCG CGCCGCGGCC ACCCTGGAGC GCCTGGTCGA 20760
CGGCGTCCTC ACCCAGTGCG AGGAGCGCGG CCCGGTGGTC TCCCTGCCGG CGCGGCAGTG 20820
GTACGAGGCG ATGCGCGCCG CCAAGCCCGC GGTGACCGAG CTGTTCCGCG CCGGAACCGA 20880
CCTCGCCCTG GACACCGAGG AGCAGCCCCC GGACACCGGC CCGGACGGCA AGGGCCTGTC 20940
CGAAAGCGCG TTCCACCGCA TTGTCGAGGG CTCCGACGGG CTGCGTGACT TCCTCGACCG 21000
CGACCCGTCC TTTCTGGCCA CCCGACTGCT GACCAGCCTG CTCTACCTTT CCCTGAGCAG 21060
CGTCGGCATC GCCCTGGCGG AACGGTATTT CCTCTGCTAC GCGGTGAGCC GCGCCTGTGA 21120
GTCGATCTTC GACACCGACG CCCTCACCGT CCTGTCCGGT CTGGCCCGGA CCTCCCTGGC 21180
GTCCTGACGC TCCGACACCC TCCCTGACGT CGGTTTTCGG CCGAATCCAT GCCCGGAACC 21240
CGTGATCGAC GGGCCGGGCG GGACCGGCCT CTTCCCATGC ACCGGGCGCT CCCCGCAGAA 21300
TCACGTTCCG ATATAAGGCC CGACCGGATA CCGCCAAGCG GCGATCCGGC ACCGGGACAC 21360
ATGAAGTAGC ATCGCGCCCA GATAAGGACA TTCACCGCCA GAAACGTTCA AAAAGATCAC 21420
CGAGAGATCA CTGACCGAGC CTGCTCCACC ACGAGCAATG GGGGAACATG AAAGCTCAAG 21480
AGCTGGAACG TGAGGCGTGA TCCCGGCAGG GCGCCGCACG GCGCGGTCAT CCCGCGCCCA 21540
CGCGGCGCCC TGCCGCGTAT GCCGGTTTCC CCACGTCGCT TCCACACACC GGGAGATGCG 21600
ATGTCGTCAG CACGGTCGAT CCACTTGTGC ACCACCGCAA CGGGGGAGTT CGATGGAGAT 21660
CGATGTCCTG GGACCGCTTG AGGTCTCGCA GAAGTCCGCT TCGATTGTCC CGACGGCTTC 21720
CAAGCCGCGC ACCGTCCTGG CGATGCTCGC CGTCTACACC AACCGGCTGG TCCCCCTGCC 21780
CTCCCTCATG GGAGAACTCT GGGGACCCGA ACCGCCGGCC ACCGCGAAGA CGGCCGTACA 21840
GGGGTACATC CTGCATCTGC GCAAACGTAT CGCCGAGTCC GCCGCGCGCC GCCCACCGGG 21900
CCAGTTCCCC GAGGCCAAGG ACATCCTGGT CACCCTCCCC GGCGGCTATC TGCTGCGCGC 21960
CCCGGGAACC GCGGTGGTCC TGGAGCAGTT CGAGCAGCTG GCCTGCGCCG GACACCGCGC 22020
CCGGGAGAAG GGCGACTTCG AGGCCGCCTC GCGGAGCTTC ACCGAGGCCC TCGGCCTGTG 22080
GCGCGGCCGC GCCCTCGCCG ACGTTGAGGT GGGCCCCCAG CTGGGCATAG AGGTGCAGCG 22140
GATCGAGGAG GCCCGGCTCA ACGTCCTCGA CCGCAGGATC GAGGCCGACC TCCGGCTCGG 22200
CCGCCATCAC GAACTCCTCG GTGAGCTGCG GAGCGTCACC GCCCACCACC CCACCCATGA 22260
GGGCTTCTGC GCCCACCTCA TGCTGGCGCT CTACCGCTCC GGCCGCCGCT GCGAGGCACT 22320
CGACGCCTAC CAGCGCATGC GCACCACGCT GGTGAACGAA CTGGGACTGG AACCCTCCCC 22380
GTCCCTGCGC CGCCTCCAGC GCTCCCTGCT GGTCTCCGAC CGCGAACTCG ACGAACTCAA 22440
CGTCACCTGG GACGTCGCCA CGTTCTGACC CCGCCCGGCG AACGCCGAAG GGCGGCCACC 22500
CCGCACGGGG TGACCGCCCT TCGTCTCATG CGATCGCTCG CTTACTGGTT GTACGGACCG 22560
TAGTCGTAGT CCTCCAGCGG AACGGCCTGG CCGGAGCCCG TGCCGAACGG CGAGTAGTCG 22620
ATGTCGTCGT AGCCGACGGC CGAGTACATC GCGGCCTTGG CCTCCTCGGT CGGCTCGACC 22680
CGGATGTTGC GGTAGCGGGA CAGACCCGTA CCGGCCGGGA TGAGCTTACC GATGATGACG 22740
TTCTCCTTGA GGCCGATCAG GGAGTCGGAC TTGGCGTTGA TCGCCGCGTC CGTGAGGACC 22800
CTGGTCGTCT CCTGGAAGGA CGCCGCCGAC AGCCAGGACT CGGTCGCCAG CGAGGCCTTG 22860
GTGATACCCA TGAGCTGCGG ACGACCGGAG GCCGGGTGAC CGCCCTCCTG GACCACACGA 22920
CGGTTCTCGT GCTCGAACTT CGAGCGCTCG ACCAGCTCAC CGGGCAGCAG CTCGGCGTCG 22980
CCGGACTCGA TGATCGTCAC ACGGCGGAGC ATCTGCCGGA TGATGATCTC GATGTGCTTG 23040
TCGTGGATCG ACACACCCTG CGAGTTGTAC ACCTTCTGGA CCTCGCCGAC CAGGTGGACC 23100
TGGACGGCAC GCTGGCCCAG GATGCGCAGC ACGTCGTGCG GGTTGGTGGC ACCCACGGTG 23160
AGCTTCTGGC CCACCTCGAC GTGCTCGCCC TCGCTGACCA GGAGACGGGC GCGCTTCGAG 23220
ATCGGGTACG CCGTCTCGTC GCTGCCGTCG TCCGGCGTGA TGACGATCTT CTTGGTCTTC 23280
TCGGTCTCCT CGATCCGCAC GCGGCCGGAG GCCTCGGAGA TCGGGGCGAC ACCCTTCGGG 23340
GTACGGGCCT CGAAGAGCTC GACGACACGC GGCAGACCCT GGGTGATGTC GTCACCGGCC 23400
ACACCACCGG TGTGGAAGGT ACGCATCGTC AGCTGGGTTC CGGGCTCACC GATGGACTGG 23460
GCGGCGATGA TGCCGACCGC CTCACCGATG TCGACCAGCT TGCCGGTGGC GAGCGAACGG 23520
CCGTAGCACA TCGCGCAGGT ACCGACGGCG GACTCGCAGG TCAGGACCGA GCGGGTCTTG 23580
ACCTCCTCGA CGCCGCGCTG GACGAGCTCC TCGATGAGGA CGTCGCCCAG GTCGGTGCCG 23640
GCCGGGGCCA GCACCTGGCC GTCGACCACG ATGTCCTCGG CGAGGCAGCG CGCGTACACG 23700
GACGTCTCGA CGTCGTCCGC CTTGCGCAGC ACGCCGTCCG CGCCCCGCTC CGCGATCTTG 23760
AGCTTGAGGC CGCGGTCGGT GCCGCAGTCC TCCTCGCGGA TGATGACGTC CTGGGAGACG 23820
TCGACCAGAC GACGGGTGAG GTAACCCGAG TCGGCGGTAC GCAGAGCGGT GTCCGCCAGA 23880
CCCTTACGGG CACCGTGCGT GGAGATGAAG TACTCCAGCA CGGACAGACC CTCACGGAAC 23940
GAGGCCTTGA TGGGCCGCGG GATCGTCTCG TTCTTCGCGT TCGACACCAG ACCACGCATA 24000
CCGGCGATCT GACGCATCTG CATCATGTTG CCTCGTGCAC CCGAGTTCAC CATCATGAAG 24060
ATCGGGTTGG TCTTCGGGAA GTTGTCGTTC ATCGCCTCGG CGACCTCGTT GGTCGCCTTG 24120
GTCCAGATCG CGATGAGCTC CTGCGTGCGC TCGTCCTTGG TGATCAGACC GCGCTCGTAC 24180
TGCTTCTGGA CCTTCTCGTC CTGCGCCTCG TAGCCCTTGA CGATCTCCTT CTTCGCCTCG 24240
GGAACGACGA CGTCGGAGAT GGCCACGGTG ACACCGGAAC GGGTCGCCCA GTAGAAGCCG 24300
GACGCCTTCA GGTTGTCGAG CGTCGCCGCC ACGATGACCT TCGGGTAGCG CTCGGCGAGG 24360
TCGTTGACGA TCTCGGAGAG CTGCTTCTTG CCGACCTCGT AGTCGACGAA CGGGTAGTCC 24420
TCGGGCAGCA GCTCGTTGAA GAGCGCACGG CCCAGCGTGG TCTTCAGGGT GAAGCTGTCA 24480
CCCTGCTGCC ACTCCGGCTC GCCCTCCTCG CGCACCGGCG GGACCCAGCC GCGCGGCGGG 24540
ATGGTGCCCA CCGGGAAGCG GATGTGCACC GGCGCCTGCA GCGACAGCTC GCCCGCGTCG 24600
AACGCCATGA TCGCCTCGGC GACCGACGCG AACGCACGGT CCTCGCCCTT GACGCCCCGG 24660
CCCTCGGCGT CCGTGGTGAG GAAGAACAGA CCGAGGACCA TGTCCTGCGT CGGCATCGTC 24720
ACCGGACGGC CGTCGGCCGG CTTGAGGATG TTGTTCGAGG ACAGCATCAG GATGCGGGCC 24780
TCGGCCTGCG CCTCCGCGGA CAGCGGCAGG TGCACGGCCA TCTGGTCACC GTCGAAGTCC 24840
GCGTTGAACG CGGTGCAGAC GAGCGGGTGG ATCTGGATGG CCTTGCCCTC GACCAGCTGC 24900
GGCTCGAAGG CCTGGATGCC CAGGCGGTGC AGGGTGGGCG CACGGTTCAG CAGCACCGGG 24960
TGCTCGGCGA TGACCTCTTC GAGGACGTCG TACACCACGG TGCGGCCACG CTCGACCATC 25020
CGCTTGGCGC TCTTGATGTT CTGCGCGTGG TTCAGGTCGA CCAGGCGCTT CATCACGAAC 25080
GGCTTGAACA GCTCCAGCGC CATCGCCTTC GGCAGACCGC ACTGGTGCAG CTTCAGCTGC 25140
GGACCGACGA CGATCACGGA ACGCGCCGAG TAGTCGACAC GCTTGCCGAG CAGGTTCTGA 25200
CGGAAGCGGC CCTGCTTGCC CTTCAGCATG TCGCTGAGGG ACTTCAGCGG GCGGTTGCCG 25260
GGACCGGTCA CCGGCCGGCC GCGACGACCG TTGTCGAAGA GGGCGTCAAC AGCCTCCTGA 25320
AGCATGCGCT TCTCGTTGTT GACGATGATC TCCGGAGCAC CCAGGTCGAG CAGGCGCTTC 25380
AGACGGTTGT TGCGGTTGAT GACACGGCGG TACAGGTCGT TCAGGTCGGA GGTCGCGAAG 25440
CGGCCACCGT CCAGCTGCAC CATCGGACGC AGGTCCGGCG GGATGACCGG GACGCAGTCC 25500
AGGACCATGC CCTTGGGGCT GTTGGAGGTC TGCAGGAACG CCGAGACGAC CTTCAGGCGC 25560
TTGAGCGCAC GGGTCTTCTT CTGGCCCTTG CCGGTGCGGA TGATCTCCCG GAGCTTCTCC 25620
GCCTCCTCCT CCAGGTCGAA GGACTCCAGG CGCTTCTGCA GCGCCGCGGC ACCCATCGAA 25680
CCGTCGAAGT ACGTGCCGAA GCGGTCACGC AGCTCGCGGT AGAGCAGCTC GTCGCCCTCC 25740
AGGTCCTGGA CCTTGAGGTT CTTGAAGCGG GTCCACACCT CGTCGAGGCG GTCGATCTCG 25800
CGCTGCGCAC GGTCGCGCAG CTGCTTCATC TCACGCTCGG CACCCTCGCG CACCTTGCGG 25860
CGCACGTCGG CCTTGGCACC CTCGGCCTCC AGCTCGGCCA GGTCGGCCTC GAGCTTCTTG 25920
GCGCGGGCCT CCAGGTCGGC GTCCCGGCGG TTCTCGATCT GCTGGCGCTC GACCGAGACG 25980
TGCGCCTCCA GGGAGGGCAG GTCGCGGGTA CGGCGCTCCT CGTCGACGTA CGTGATCATG 26040
TACGCCGCGA AGTAGATGAC CTTCTCCAGG TCCTTCGGGG CGAGGTCGAG CAGGTAGCCC 26100
AGCCGCGACG GAACACCCTT GAAGTACCAG ATGTGCGTGA CGGGGGCGGC CAGCTCGATG 26160
TGGCCCATCC GCTCACGGCG CACCTTGGCG CGGGTGACCT CGACGCCACA GCGCTCGCAG 26220
ATGATGCCCT TGAAGCGGAC GCGCTTGTAC TTGCCGCAGT AGCACTCCCA GTCCCGGGTC 26280
GGACCGAAGA TCTTCTCGCA GAAGAGTCCG TCCTTTTCGG GCTTGAGCGT GCGGTAGTTG 26340
ATGGTCTCCG GCTTCTTGAC CTCGCCGTGG CTCCACTGAC GGATGTCGTC AGCGGTGGCC 26400
AGACCGATCC GGAGCTCGTC GAAGAAGTTG ACGTCGAGCA CTATGCGTCA ATCCCTCTCA 26460
GGGTTGTAAG TCTTGGGGTC TGAAACGGGG GTCCTGGGGC CGGCCGGGGA CTCAGCGGAA 26520
TGCACCGAGT CCCCGTACCG GACTCCCGTC AGACCTCTTC GACGCTGCTC GGCTCGCGCC 26580
GGGACAGGTC GATACCGAGC TCCTCCGCAG CGCGGAAGAC GTCCTCGTCG GTGTCGCGCA 26640
TCTCGATGGA CATGCCGTCC GAGGACAGCA CCTCCACGTT GAGGCACAGG GACTGCATTT 26700
CCTTGATGAG CACCTTGAAG GACTCGGGAA TGCCGGGCTC AGGGATGTTC TCGCCCTTGA 26760
CGATGGCCTC GTAGACCTTC ACGCGGCCGG TGACGTCGTC GGACTTGATG GTCAGCAGCT 26820
CCTGGAGGGC GTAGGCGGCG CCGTACGCCT CCAGCGCCCA CACCTCCATC TCACCGAAGC 26880
GCTGGCCACC GAACTGGGCC TTACCACCCA GCGGCTGCTG GGTGATCATC GAGTACGGAC 26940
CGGTCGAGCG GGCGTGCAGC TTGTCGTCGA CCAGGTGGTG CAGCTTCAGG ATGTACATGT 27000
AGCCGACCGA GATCGGGTCC GGGAACGGCT CACCGCTACG GCCGTCGAAC AGTCGCGCCT 27060
TGCCGGACGG GAGCACCATG CGCTCGCCGT CGCGGTTCGG GATGGTGTGC TGCAGCAGAC 27120
CCGCGAGCTC GTCCTCACGC GCACCGTCGA AGACCGGGGT GGCGACGTTG GTGCCGGGCT 27180
CGACCTTGTC GGCGCCGATC GCCTGGAGGC GCTGCGCCCA CTCGTCCGCC AGGCCGGAGA 27240
CGTCCCAGCC GCGGCTGGCG AGCCAGCCGA GGTGGATCTC CAGGACCTGT CCCGGGTTCA 27300
TTCGGGACGG CACACCCAGC GGGTTGAGGA TGATGTCGAC CGGGGTGCCG TCCTCCAGGA 27360
ACGGCATGTC CTCGATCGGC AGGATCTTGG AGATGACACC CTTGTTGCCG TGACGGCCGG 27420
CGAGCTTGTC ACCGTCGGTG ATCTTGCGCT TCTGCGCGAC GTAGACGCGG ACCAGCTGGT 27480
TCACACCCGG CGGCAGCTCG TCGCCCTCCT CGCGGTCGAA GACGCGCACA CCGATGACCT 27540
TGCCGGTCTC GCCGTGCGGC ACCTTCAGCG AGGTGTCACG GACCTCACGG GCCTTCTCAC 27600
CGAAGATGGC GCGCAGCAGG CGCTCCTCCG GCGTCAGCTC GGTCTCACCC TTGGGCGTGA 27660
CCTTGCCGAC GAGGATGTCA CCGGCGATGA CCTCGGCACC GATGCGGATG ATGCCGCGCT 27720
CGTCGAGGTC GGCGAGGACC TCCTCGGAGA CGTTCGGGAT GTCCCGGGTG ATCTCCTCGG 27780
GGCCGAGCTT GGTGTCACGG GCGTCGACCT CGTGCTCCTC GATGTGGATC GAGGAGAGGA 27840
CGTCGTCCTG CACGAGGCGC TGCGACAGGA TGATCGCGTC CTCGTAGTTG TGACCCTCCC 27900
ACGGCATGAA CGCCACGAGC AGGTTCTTGC CGAGCGCCAT CTCGCCGTTC TGGGTGGCCG 27960
GGCCGTCGGC GAGCACCTGG CCCGTGATGA CGCGGTCGCC CTCGTTGACG ATGACCTTCT 28020
GGTTGACCGA GGTGCCCTGG TTGGACCGGG AGAACTTGGC CAGGCGGTAC GTGATGTACG 28080
TGCCGTCGTC GTTGGCGGTG GTGATGTAGT CCGCGGAGAC CTCCTGGACC ACACCGTCCT 28140
TCTCGGCCTT GACCACGTCG CCGGCGTCGA CGGCGGAGCG GTACTCCATG CCGGTGCCGA 28200
CGAGCGGGGC CTCGGACTTA ATCAGCGGCA CGGCCTGGCG CATCATGTTC GCGCCCATGA 28260
GGGCACGGTT GGCGTCGTCG TGCTCGAGGA ACGGGATCAT GGCGGTCGCG ACCGACACCA 28320
TCTGGCGCGG CGAGACGTCC ATGTAGTCCA CGTCCTCGGG GGCGACGTAG TCGACCTCGC 28380
CGCCACGGCG GCGGACCAGG ACGCGGGCCT CCTCGAACCG CATGTCGGCG GTGAGCGGCG 28440
CGTTGGCCTG CGCGATGACG AAGCGGTCCT CCTCGTCGGC GGTCAGGTAG TTCACCTCGT 28500
CGGTGACCTG GCCGTCGACG ACCTTGCGGT ACGGGGTCTC GACGAAGCCG AACGCGTTGA 28560
CGCGGCCGTA GGTGGCGAGC GAGCCGATCA GACCGATGTT CGGGCCTTCG GGGGTCTCGA 28620
TCGGGCACAT GCGGCCGTAG TGCGACGGGT GGACGTCACG GACCTCGAAG CCGGCCCGCT 28680
CACGGGAGAG ACCACCCGGG CCGAGCGCGG ACAGACGACG CTTGTGCGTC AGCCCGGACA 28740
GCGGGTTGTT CTGGTCCATG AACTGGGACA GCTGGCTGGT GCCGAAGAAC TCCTTGATGG 28800
AGGCGACGAC CGGCCGGATG TTGATCAGGG TCTGCGGCGT GATCGCCTCG ACGTCCTGGG 28860
TCGTCATCCG CTCGCGGACG ACACGCTCCA TACGGGCCAG ACCCGTGCGG ACCTGGTTCT 28920
GGATGAGCTC GCCGACGCTG CGCAGGCGGC GGTTGCCGAA GTGGTCGATG TCGTCGGTCT 28980
CGACGACGAC CGGCTGGCCG TTGTCGCCGA CGGTCTCGGT CTCACCGGCG TGCAGCTTCA 29040
CCAGGTACTT GATCGTCGCG ATGATGTCCT CGACGGTCAG GATCCCGGCG TCCAGCGGGG 29100
TGTCCGTGCC CAGCTTCTTG TTGACCTTGT AGCGGCCGAC CTTGGCCAGG TCGTAGCGCT 29160
TGGGGTTGAA GTAGAGGTTC TCCAGCAGCG TCTGCGCGGC CTCACGCGTG GGGGGCTCGC 29220
CCGGGCGCAG CTTGCGGTAG ATGTCGAGCA GCGCGTCGTC CTGGCCCTGG GTGTGGTCCT 29280
TCTCCAGGGT GGCGCGCATG GACTCGTACT CGCCGAACTC CTCGAGGATC TGCTCGGTGG 29340
TCCAGCCGAG CGCCTTGAGC AGGACGGTCA CGGACTGCTT GCGCTTGCGG TCGATGCGGA 29400
CACCGACCAT GTCGCGCTTG TCGATCTCCA TCTCCAGCCA GGCACCCCGG GACGGGATGA 29460
TCTTGGCGGA GAAGATGTCC TTGTCGGACG TCTTGTCGAT GCTGGAGTCG AAGTAGACAC 29520
CGGGGGAACG GACCAGCTGC GACACCACGA CACGCTCGGT GCCGTTGATG ACGAACGTGC 29580
CCTTGTTCGT CATGAGCGGG AAGTCGCCCA TGAAGACGGT CTGGGACTTG ATCTCGCCGG 29640
TCTCGTTGTT GGTGAACTCG GCCGTGACGA AGAGCGGCGC GGCGTAGGTG AAGTCGCGCT 29700
CCTTGCACTC GTCGATCGAG TTCTTCGGCG GCTCGAAGCG GTGGTCGCGG AAGGTCAGCG 29760
ACATCGACCC CGAGAAGTCC TCGATCGGGG AGATCTCCTC GAAGATCTCC TCGAGGCCGG 29820
ACTTGGTGGG GACGTCCTGA CCGTTCTCGA GAGCCTCCTC GACCCGACTC TGCCAGGCGG 29880
TGTTGCCGAG CAGCCAGTCA AAGCTCTCGG TCTGCAGCGC GAGCAGGTTC GGAACCTCGA 29940
GAGGCTCCTT GATCTTTGCA AAGGAGATGC GCAGCGGGGC GGTGCTGGCG CCGTTGTTCG 30000
TATTCGCGGT CGAGGCAGTG CGCGAGGCGG CCAAGAGGGG GTCCTTCCGA GGGCTCGGAC 30060
TCACTACGCG CGTACCGGCC CCTCCCCCGT ACACAGAGAC AGAGATCCCA GGTCGGAGAT 30120
CATCTGTCGG TCAGTGCTCG AGTGAGGGCA GACCCCTGGT GACGGGCAGG GGACAGCTAA 30180
CAGGCAGCGC AAAGGGTCAG TGTAGCCACA AGGCACACTG ATGTCCAGTC CCGCATTTTC 30240
GGAGACTCCA CTGTTCTCAA CGCCTGCGGC ATGCCTGCCC TCAACGCACG TTGATACTGC 30300
CCTCTTCGTC GTCGATCCAT GCCTCGGATT CGGATCCTTG TGACGACGCG TCCTGAGAAT 30360
TGCGCGCTGC GTGCGGTTCG TCAAGGCCCC CCTTGCCCGA ACCGGGGCGG CCGAGACACG 30420
ACGAAGATCA CCTTACCCCT CACGAACACA GGTGCAAGGC AGCCCGGGCC GTCCCCCGGG 30480
GAACGCCGAA GGGCGACCAC CCGGATGGAT GATCGCCCTT CGGTGCGTTC GCGTTACAGC 30540
CCCTGAGGGC CGTTCATGCG GTCGCGAAGG TCTTACTTGA CCTCGACGGA GGCGCCGGCG 30600
CCCTTGAGGG CCTCGGCGGC CTTGTCGGCC TGCTCCTTGT TGACCTTCTC GAGGACCGGC 30660
TTCGGGGTGC CGTCGACGAG GTCCTTGGCC TCCTTCAGGC CCAGGGAGGT CAGCTCACGC 30720
ACGACCTTGA TGACCTGGAT CTTCTTGTCG CCGGCGCCGG TGAGGATGAC GTCGAACTCG 30780
TCCTTCTCCT CCTCGGCCTC GGCGGCGGCG CCACCGGCGG CACCACCGGC GACGACGACC 30840
GGCGCGGCAG CGGCGGCGGT GACGTCGAAC TTCTCCTCGA ACGCCTTCAC GAACTCGGAG 30900
AGCTCGATGA GGGTCATCTC CTCGAACTGC GCGAGCAGCT CGTCCTGGGT CAGCTTCGCC 30960
ATGATGGCGG TCCTTCCACT CAATTCGGCA GGTGCCGGAT GTACTGGATA AGGCGGGCGT 31020
ACGTCGGGCC CGCTAGGACC CTCGCCGGTC GGTCGGCGTG GGTCGGAAAG CGAGCCGAAT 31080
TACTCGGCAC CGCCCTGCTC GGCCTGCTTG GCGCGCAGCG CGTCCACGGT GCGGACGAGC 31140
TTCGACGGCA GCGCCTGGAA GACGGAGGCA GCCTGGGACT GCTTCGCCTT GAAGGCACCG 31200
GCCAGCTTGC TGAGCAGAAC CTCGCGGGAC TCGAGGTCCG CAAGCTTCTT GATCTCGTCG 31260
GCGGACAGCG CCTTGCCGTC AAGGACACCG CCCTTGATGA CGAGATTCGG GTTTTCCTTG 31320
GCGAAGTCAC GGAGACCCTT CGCCGACTCC ACCGGGTCAC CGGTGACGAA GGCGACAGCC 31380
GTCGGACCGT TGAACAGGTC GTCCAGCGTC GTGATCCCGG CCTCGTTGGC CGCAATCTTG 31440
GTCAGCGTGT TCTTCACCAC GGCGTACTGG GCGTTCTCAC CGAGTGCGAG ACGCAGCTTC 31500
CTGGTCTGCG CGACGGTGAG ACCGCGGTAC TCGGTCAGCA CGGCAGCAGA CGAGTTGCGG 31560
AACTTGTCCG TCAGCTCGGC AACCGCGGCA GCCTTGTCGG GCCTCGCCAT AGAGCCTCGG 31620
CCTCCTTCCG GGTGATTCGG ACCGCGCGGA CCCGAAGGAG GACTGGGAAA AACGAAACGC 31680
CCCGGCGCAG GCGCACGGGG CGGACTCAAC CGGTTCGCAC GTGCGTGAAC ACACGTATGC 31740
TCCGGGAGTT CTTCCACAGT CACCTGCGCG GGTCGTCCGC ATTGAACAGC GGATCCTTCG 31800
GCCACCGACC CCGAATGGGG CACGGCAACG ACCAGCGGTC TTTGGCTTCT GTAGGAGAGT 31860
ACGGGACGGG TACGCCGTCA AGCAAATCGC CCCTACGGGC GTCAGCCCTG GACGCCCTTC 31920
ATCATCTCGG CGAGGTCGGC GACGTCCTTC GCCGGCGGCG CCGTGACGGT GACGGGCTTG 31980
TTCACGTCGT AGAAGGTGAT CGTCATGTCG AGCGGGCCCT TGTCGGCGTC GCCGCGCATC 32040
CGGAACCGCT TGGTGTGGTC CTCGCCGTCG ACCCACAGGT CCATGGAGAG CTTGTCGATG 32100
CCCAGCTTCT CGAACTCGTC GAGGCTCTTC TGCCGCTTGT CGCGACTCGC CTTGTCGGTG 32160
CCCTTCAGCG ACTTCTCCAG GGCGTCGAGG GTGGCGGTGC CCTGGTAGTG GGTGGTCTTC 32220
ACCCCGTCGA CGGTCTCGGC GCCGACCTCG CGCACGTCGT CGGCGCCGGC CAGGAAGGTG 32280
GACTGGGCCG CCGGGTTCTG CTCGGCCGTG GCCCCGGCCC CGGCGAGCGG GTTGGCCTCC 32340
TTGCCCGCCT TGGACATGTC GAACTTGATC CAGCTCTTGC CGTCCATCTC GCCGGCCGCC 32400
TCGGCGCCAC CGTTGATGTA CATCGCCTTG TCCACGAACC GGATCTCGAC CTGTCCGGCG 32460
TCGGCCTGGT CCTCGGGCGT GATCTTCATG CTGAGGGCGG GCGGCTTCGT GCTCATCGCG 32520
GCCTCGCCGC GCACCCGGCC CTCGCCGGGC ACCCGGCCGT CGAGCCGGTA GCGGAACGAG 32580
GTGACGGCGT CGGCGTTCTT CGCGGCCCGG GCGACGGCCG CCGCGGGCTC CAGGTCCGGA 32640
GACTGCTCGC CCTTGCCGCA GCCGACGGCA CCCGCCAGGA GCAGCCCGGC GAGGGCAGCG 32700
GTGGACACGG CACGCTTCAT CGATTTCCCC CCCAAGGAAA CACATGGCCG CCACACAGCG 32760
GCCGGACGAG CCTAACCCGC GGGTTACCGA TCAGTCGCTC GATTTGTCAG GACCCCTGGC 32820
CGCCCATCAG TGCCTCGAAG TCCTCGGTGT CCCCGTCCGG CGGCCGCTGT ACGGCGACCC 32880
GCACGCCGTA GGTGTCGTAG TACGCCGTCT GGGTCATCGG GCCGGACGTC ATCTCGGCCT 32940
TCTCCACCTT CTTGACCAGC AGATTCTTCT CGTCGACCCA GATGTCGACC TGCTCCGCGG 33000
TGACCCCGGC GTCGGTGAGC TGCCGGCGCA GGGCCACGTC GCCGAGCGCG GAGACGTCGA 33060
CGGTGCCGGT GTAGTGCCGG GTGGCGCGGC CCCCTACCGT CTCCCGGCCC GTCTCGCGCA 33120
CCGTGCCGGA GGCCAGCAGG AGCCGCACGG GCAGCACGGG CGCGGTGTCG CGGAGCTGGT 33180
CGCGGAGGTA GGCGCCGGAG TCGCCGGGCA GCTCGGCGAG GTCGTCGAAG TCGTAGCGGA 33240
TCCAGTGCCG GCCGCCCATC CGCCGGGCGA AGTCCTCGCC GACCCGGGCG TAGTAGGCGT 33300
CGGGCAGCAG ACGGGCCTCC ATGGTGGTGG TGCCGAGCTT GCGCAGGGGC TCGGTGAGGC 33360
TGCCGCCTGT GTAGGTGATG CGCAGGGTGC CGGTGGAGTC GCCGGCCCAG TCCAGGGCGC 33420
CCTCGGTGCG GGTGGAGAGC AGGTCGCCCA TGGCCGTGGT GGAGCTGATC CGGGCCGAGC 33480
CCGCCCCGGC GACGGCCCGC TCGACGGCGC GCAGCGCGTC CGCCGTACGG GGATCGGGGC 33540
GCGGTGCGGC GCTCACGGTG CCGCCCGTGG CGTCCGGGCG GGCGGCGCCG TCGTCGGAGC 33600
CGCCGCACCC GGTCAGGGCC GTCAGCGCGG CGACGGCGGC CAGCAGGGCC GTCGTGGTCG 33660
GCGCGGCTCC CCGCGGGCGG GCGGTGCCCC TGTGCCTCAT GTCTCCCCCT CGGTCCCCGT 33720
GATCGCCACG CTAACCCGGC GAGGGGCCCC GGGGGTACGG GAACGGGCCC CGCACCTGGG 33780
AAGGTGCGGG GCCCGTGACC GACTCGGTGA GCCGTCAGGC TCAGACCGCG GCCGGGTCCT 33840
CCTCGACGAG GAGGTTGCGG GTGCGGTTCG GGTCGACGGG GATGCCGGGG CCCATCGTGG 33900
TGGTGATGGC GGCCTTCTTG ATGTAGCGAC CCTTGGCGGC GGACGGCTTC AGACGGAGGA 33960
TCTCCTCCAG CGCGGCGCCG TAGTTCTCCA CCAGCTTGGC GTCGTCGAAG GACGCCTTGC 34020
CGATGATGAA GTGCAGGTTC GAGTGCTTGT CGACGCGGAA CTCGATCTTG CCGCCCTTGA 34080
TGTCGGTGAC GGCCTTGGTC ACGTCGGGGG TGACGGTGCC GGTCTTCGGG TTCGGCATCA 34140
GACCACGGGG ACCGAGCACG CGGCCGAGGC GGCCGACCTT GCCCATGAGG TCCGGGGTGG 34200
CGACGACGGC GTCGAAGTCC AGGCGGCCCT TGGAGACCTC GTCGATCAGC TCGTCGGCGC 34260
CGACGATGTC GGCGCCCGCG GCACGCGCGG CCTCGGCACG GTCGCCGGTC GCGAAGACCA 34320
GGACCCGGGC GGTCTTACCG GTGCCGTGCG GAAGGTTCAC GGTGCCACGG ACCATCTGGT 34380
CGGCCTTGCG CGGGTCGACA CCCAGACGGA AGGCGACCTC GACGGTGGCG TCGAACTTGG 34440
TCGTGGAGGT CTCCTTGGCG AGACGGACGG CCTCGAGCGG GGCGTACAGC TTCTCCCGGT 34500
CGACCTTGGC GTCCGCAGCC TTGAGAGCCT TGCTGCGCTT GCTCACGTGA TGCTCCTGTG 34560
TTGGCTAGGA GTCGTGGTAC GGGCCGAGCA GGCCCTTCCA CGCTGTTCTG CAGGGGGGTT 34620
GAGGTCAGCC CTCGACGGTG ACGCCCATGG AACGGGCGGT ACCCTCGATG ATCTTCGCCG 34680
CGGCGTCCAG GTCGTTGGCG TTGAGGTCGG GCA 34713
<210>2
<211>151
<212>PRT
<213>Steptomyces actuosus ATCC25421
<400>1
Met Thr Glu His Pro Ala Gln Gln Leu Tyr Cys Thr Val Val Leu Trp
1 5 10 15
Asp Leu Ser Arg Ser Ala Ala Thr Val Ala Ser Leu Arg Ala Tyr Leu
20 25 30
Arg Asp His Ala Val Asp Ala Tyr Thr Thr Val Pro Gly Leu Arg Gln
35 40 45
Lys Thr Trp Ile Ser Ser Thr Gly Pro Glu Gly Glu Gln Trp Gly Ala
50 55 60
Val Tyr Leu Trp Asp Ser Pro Glu Ala Ala Tyr Gly Arg Pro Pro Gly
65 70 75 80
Val Ser Lys Val Val Glu Leu Ile Gly Tyr Arg Pro Thr Glu Arg Arg
85 90 95
Tyr Tyr Ser Val Glu Ala Ala Thr Glu Gly Pro Ala Ala Ala Ala Ala
100 105 110
Pro Phe Gly Lys Gly Leu Gly Leu Ala Phe Asp Pro Ala Ser Pro Glu
115 120 125
Pro Leu Thr Arg Pro Gln Glu Phe Val Pro Pro Gly Ala Asp Ala Phe
130 135 140
Ile Pro Ser Arg Pro Pro Ala
145 150
<210>3
<211>455
<212>PRT
<213>Streptomyces actuosus ATCC25421
<400>1
Met Asp Glu Ser Pro Thr Tyr Pro Phe Glu Gln Lys Cys Pro Phe Ala
1 5 10 15
Pro Pro Asp Glu Leu Val Arg Arg Arg Asp Glu Ala Pro Val Ser Gln
20 25 30
Ile Arg Leu Ala Asn Gly Ala Thr Ala Trp Leu Val Thr Arg His Asp
35 40 45
Asp Val Arg Thr Val Leu Thr Asp Pro Arg Phe Ser Arg Ala Ala Ile
50 55 60
Arg Ala Gly Ala Leu Arg Gly Gly Pro Pro Gly Pro Gly Gly Pro Gly
65 70 75 80
Gly Ser Ala Glu Gly Ala Pro Gln Gly Gly Trp Pro Gly Gly Pro Gly
85 90 95
Gly Ser Ala Gly Gly Ala Pro Gln Gly Gly Trp Pro Gly Gly Val Pro
100 105 110
Gly Pro Gly Gly Pro Gly Gly Ala Ser Val Gly Pro Gly Ala Pro Gly
115 120 125
Lys Gly Pro Gly Gln Arg Phe Asp Phe Gly Met Ala Leu Ala Asp Pro
130 135 140
Glu Arg His Ala Arg Trp Arg Arg Thr Leu Ala Thr Val Leu Thr Pro
145 150 155 160
Arg His Ala Glu Ser Leu Arg Lys Ala Val Gly Gln Ala Val Asp Glu
165 170 175
Val Leu Asp Gly Leu Ala Glu Ala Gly Pro Asp Pro Val Asp Leu Val
180 185 190
Ser Gly Phe Ala Tyr Gln Val Pro Val Arg Val Leu Cys Glu Leu Phe
195 200 205
Asp Leu Pro Glu Glu Leu Arg Pro Gly Leu Trp Gly Trp Ala Ala Gln
210 215 220
Val Arg Ala Ala Ala Pro Ser Thr Ala Ala Phe Gly Ala Ala Met Gly
225 230 235 240
Ala Leu Phe Gly Val Ala Arg Gln Leu Val Glu Lys Glu Leu Ala Ala
245 250 255
Pro Gly Asp Gly Leu Ile Gly Ser Leu Ile Ala Gly Leu Asp Glu Asp
260 265 270
Ala Arg Pro Ser Ala Asp Glu Leu Val Ser Thr Val Val Leu Leu Ala
275 280 285
Thr Ala Gly Tyr Glu Ser Thr Ala Val Gln Phe Ala Asn Gly Leu Leu
290 295 300
Ala Leu Phe Gln His Pro Asp Gln Leu Ala Arg Leu Arg Asp Gly Thr
305 310 315 320
Val Thr Glu Ala Ala Ala Val Glu Glu Ile Leu Arg Tyr Ala Gln Ala
325 330 335
Gly Thr Gly Phe Ala Gly Thr Thr Val Thr Thr Glu Glu Val Thr Leu
340 345 350
Gly Gly Ile Thr Leu Pro Glu Gly Ala Thr Val Phe Ile Ser Leu Asp
355 360 365
Ser Ala Ala Arg Asp Glu Arg His Val Asp Arg Pro Asp Thr Phe Asp
370 375 380
Leu Ala Arg Gly Ala Ala Arg Thr His Leu Thr Phe Gly Ala Gly Ala
385 390 395 400
His Tyr Cys Leu Gly Ser Ser Leu Ala Arg Val Glu Leu Gln Glu Gly
405 410 415
Phe Gly Arg Leu Leu Arg Arg Phe Pro Ala Leu Thr Pro Ala Ala Asp
420 425 430
Pro Ala Ala Val Glu Phe Thr Ala Asn Leu Phe His Arg Tyr Pro Arg
435 440 445
Glu Leu Lys Ala Val Leu Arg
450 455
<210>4
<211>408
<212>PRT
<213>Streptomyces actuosus ATCC25421
<400>1
Val Asp Ile Glu Ala Arg Thr Tyr Pro Phe Thr Arg His Thr Pro Phe
1 5 10 15
Glu Met Pro Glu Glu Phe Ala Trp Leu Arg Glu Asn Arg Pro Ile Ala
20 25 30
Gln Val Arg Leu Ala Thr Gly Asp Thr Ala Trp Leu Ile Thr Arg Tyr
35 40 45
Glu Asp Val Arg Thr Ala Leu Thr Asp Pro Arg Phe Ser Arg Thr Ile
50 55 60
Asn Arg Glu Gly Ala Ala Arg Val Asp Thr Gly Phe Gln Ala Asp Ala
65 70 75 80
Asp Ser Pro Val Phe Asn Phe Gly Gly Ser Ile Ser Glu Pro Pro Gly
85 90 95
His Thr Arg Trp Arg Arg Leu Val Asn Gln Ala Phe Thr Ala Arg His
100 105 110
Ala Glu Ser Met Arg Pro Ala Val Ala Ala His Thr Asp Ala Leu Leu
115 120 125
Asp Asp Leu Glu Ser Ala Gly Gly Arg Phe Asp Leu Met Ala Asp Tyr
130 135 140
Ala Tyr Gln Leu Pro Ile Arg Val Ile Cys Asp Leu Leu Gly Leu Glu
145 150 155 160
Gln Thr Ala Arg Pro Glu Phe Ser Ala Leu Ala Ala Gln Ile Thr Arg
165 170 175
Arg Asp Phe Thr Ser Ser Phe Val Glu Phe Gly Gln Ala Leu Gln Ala
180 185 190
Ile Gly Arg Tyr Ala Ile Gly Leu Ile Val Arg Lys Arg Gln Asn Leu
195 200 205
Gly Asp Asp Leu Leu Ser Thr Leu Ile Gly Leu Thr Asp Glu Glu Asp
210 215 220
Gly Arg Leu Ser Asn Glu Glu Leu Val Ser Thr Val Ile Leu Leu Leu
225 230 235 240
Met Ala Gly Tyr Glu Ser Thr Ala Val Gln Leu Gly Asn Ala Phe Tyr
245 250 255
Ala Leu Phe Arg Asp Pro Ser Gln Met Arg Arg Leu Arg Glu Glu Pro
260 265 270
Ala Leu Ile Gly Gln Ala Val Glu Glu Ile Leu Arg Tyr Ala Gln Met
275 280 285
Gly Thr Gly Tyr Ala Ile Ala Lys Phe Thr Thr Glu Asp Val Glu Leu
290 295 300
Ser Gly Gly Thr Val Pro Ala Gly Ser Thr Val Phe Val Ser Leu Ala
305 310 315 320
Ser Ala Asn Arg Asp Glu Lys Val Phe Gly Glu Asp Ala Asp Arg Phe
325 330 335
Asp Val Gly Arg Gly Cys Ala His Arg Gln Thr Ala Phe Gly Tyr Gly
340 345 350
Pro His Tyr Cys Leu Gly Ala Ala Leu Ala Arg Val Glu Met Gln Glu
355 360 365
Gly Ile Ala Arg Met Leu Asn Arg Phe Pro Asp Leu Arg Phe Asp Gly
370 375 380
Pro Asp Leu Asp Gly Val Pro Leu Ala Ser Asn Leu Phe Thr Phe Tyr
385 390 395 400
Pro Ala Glu Leu Pro Val Arg Ile
405
<210>5
<211>345
<212>PRT
<213>Streptomyces actuosus ATCC25421
<400>1
Met Pro Ala Pro Glu Thr Gly Pro Trp His Ser Leu His Leu His Arg
1 5 10 15
Tyr Ala Gly Gln Asp Ala Phe Leu Val Asp Gly Leu Ala Pro Val Leu
20 25 30
Ala Pro Leu His Ala Ser Gly Ala Leu Glu Ser Ser Phe Phe Leu Arg
35 40 45
Tyr Trp Gln Gly Gly His His Ile Arg Leu Arg Leu Arg Pro Ala Ala
50 55 60
His Asp Pro Glu Gln Ala Ala Arg Thr Val Arg Glu Val Ala Asp Arg
65 70 75 80
Leu Ala Gly His Leu Ala Ala His Pro Gly Gly Tyr Gly Asp Leu Asp
85 90 95
Pro Glu Glu Phe Arg Glu Ala Gln Leu Thr Met Ala Ala Leu Glu Ser
100 105 110
Glu Ala Thr Gly Glu Leu Leu Pro Pro Asp Thr Val His Pro Ala Arg
115 120 125
Tyr Glu Pro Glu Tyr Gly Lys Tyr Gly Gly Pro Glu Gly Val Ala Val
130 135 140
Ala Glu Arg Phe Phe Asp Arg Ser Ser Ala Val Ala Leu Ser Ala Leu
145 150 155 160
Arg Ala Ile Gly Asp Arg Pro Ala Arg Arg Leu Gly Ala Ala Phe Thr
165 170 175
Met Thr Leu Arg Gly Leu Cys Ala Ala Arg Leu Ser Pro Pro Ala Met
180 185 190
Ala Gly Phe Leu Ala His Tyr Cys Ala Val Trp Ser Pro Tyr Val Phe
195 200 205
Asp Ser Phe Leu Asp Thr Trp Pro Glu Leu Leu Asp Gln Arg Arg Gly
210 215 220
Pro Ala Ala Ala His Leu Arg Pro Leu Leu Ala Ala Ala Gln Pro Pro
225 230 235 240
Asp Asp Pro Tyr Gly Arg Ala Val Gly Glu Ala Trp Gln Ala Leu His
245 250 255
Thr Ala Ala Asp Thr Val Leu Pro Ala Val Thr Leu Gly Gly Pro Asp
260 265 270
Ala Pro Ala Glu Arg Arg Arg Gln Ile Val Leu Leu Ser Tyr Leu His
275 280 285
Thr His Asn Asn Arg Leu Gly Leu Ile Pro Glu Gln Glu Ala Phe Leu
290 295 300
Gly Tyr Leu Gly His His Val Val Ala Asp Cys Ala Gly Leu Ala Pro
305 310 315 320
Asp Ala Gly Leu Leu Asp Arg Val Arg Asp His Arg Arg Glu Arg Leu
325 330 335
Ala Ala His Leu Pro Ser Gly Gln His
340 345
<210>6
<211>920
<212>PRT
<213>Streptomyces actuosus ATCC25421
<400>1
Val Pro Gly Ala Pro Asp Ala Ala Gly Ala Pro Asp Gly Phe Ser Pro
1 5 10 15
Tyr Tyr Leu Tyr Arg Arg Gly Thr Leu Gly Pro Ser Glu Leu Ala Ala
20 25 30
Leu Thr Pro Ala Arg Thr Trp Ala Leu Leu Ala Glu Ala Glu Glu Thr
35 40 45
Arg Gln Arg Arg Glu Glu Leu Arg Gly Arg Leu Glu Asp Ala Leu His
50 55 60
Ala Ala Val Pro Glu Leu Pro Ala Asp Arg Arg His Glu Leu Leu Arg
65 70 75 80
Leu Arg Arg Asp Ile His Asn Asp Arg Val Pro Gly Val Pro Asp Ala
85 90 95
Ala Arg Leu Leu Asp Pro Ala Ser Arg Glu Leu Leu Glu Asp Trp Leu
100 105 110
Arg His Arg Ala Glu Gly Asn Arg Leu His Lys Glu Ala Glu Ala Ala
115 120 125
Leu Ala Ala Glu Leu Asp Ala Gly Arg Arg Ala Leu Ala Ala Ile Ala
130 135 140
Thr Gly Glu Phe Phe Gln Arg Gly Leu Gln Leu Ser Asp Glu Arg Thr
145 150 155 160
Trp Arg Thr Val Thr Glu Trp Ala Ala Asp Pro Phe Ser Ala Arg Arg
165 170 175
Lys Pro Ser Lys Arg Arg Arg Ala Glu Asn Thr Leu Thr Ser Phe Ala
180 185 190
Tyr Arg Val Ala Leu Lys Pro Ser Pro Phe Ala Ser Phe Thr Glu Ile
195 200 205
Gly Ala Ala Pro Trp Thr Pro Ala Gly Asp Gly Thr Ala Pro Ala Asp
210 215 220
Arg Pro Pro Ala Arg Pro Val Gln Ala Arg Leu Ser Ala Gly Leu Leu
225 230 235 240
Ser Trp Met Thr Tyr Glu Leu His Arg Leu Asp Arg Ala Asp Glu Leu
245 250 255
Met Arg Ile Arg Leu Asn His Ser Leu Leu Val Arg Glu Glu Gln Ala
260 265 270
Leu Cys Val Arg Arg Ala Pro Asp Gly Ala Pro Glu Ala Ala Tyr Gly
275 280 285
Thr Ala Gln Val Val Thr Ala Arg Asp Thr Gly Leu Leu Arg Leu Leu
290 295 300
Arg Ser Leu Leu Ala Asp Gly Gly Leu Pro Glu Arg Glu Leu Arg Glu
305 310 315 320
Arg Leu Ala Ala Ala Gly Leu Ser Pro Gln Ala Ala Ala Thr Ala Leu
325 330 335
Asp Lys Leu Val Arg Ala Gly Ile Cys Glu Arg Gly Leu Gly Ile Pro
340 345 350
Asp Gln His Pro Arg Pro Ala Leu Ala Val Ala Gln Arg Leu Arg Thr
355 360 365
Leu Asp Thr Asp Gln Ala Gly Arg Cys Ala Val Val Phe Glu Arg Leu
370 375 380
Gln Ala Ala Glu Asp Ala Phe Pro Ala Ala Pro Val Arg Arg Arg Ala
385 390 395 400
Ala Leu Leu Ala Glu Ile Arg Glu Gln Val Ala Val Phe Val Glu Ala
405 410 415
Val Gly Cys Arg Ala Pro Ala Pro Glu Ala Met Arg Ser Val Val Tyr
420 425 430
Glu Asp Val Gly Thr Arg Glu Pro Ala His Ser Trp His Pro Asp Leu
435 440 445
Leu His Ala Asn Arg Trp Ala Leu Glu Leu Phe Gln Arg Ile Val Pro
450 455 460
Val Leu Asp Asp Ala Ser Val Glu Lys Ala Gly Leu Tyr Ala Phe Phe
465 470 475 480
Ala Arg His Phe Gly Ala Ala Asp Gly Asp Gly Asp Val Pro Phe Ile
485 490 495
Glu Phe Tyr Arg Arg Phe Ala Ala Leu Pro Pro Ala Glu Ala Ser Ala
500 505 510
Val Ala Ser Gly Val Gly Asp Pro His Ser Asp Arg Ile Arg Arg Leu
515 520 525
Arg Ala Asp Phe Ala Asp Leu Leu Arg Thr Glu Leu Arg Ala His Asp
530 535 540
Ser Ala His Thr Gly Ala Leu Arg Leu Asp Ala Glu Arg Leu Arg Ala
545 550 555 560
Phe Ala Asp Arg Leu Pro Ala Glu Val Thr Pro Trp Arg Ser Thr Ala
565 570 575
Tyr Arg Met Gln Phe Thr Ala Glu Pro Glu Arg Pro Tyr Ala Val Val
580 585 590
Asn Gly Val Thr Thr Gly His Gly Val Phe Phe Ser Arg Phe Cys Asp
595 600 605
Leu Leu Glu Pro Asp Gly Pro His Glu Trp Ser Leu Thr Glu Ala Leu
610 615 620
Arg Gly His Ile Ala Arg Thr Thr Pro Arg Gln Cys Asp Ile Thr Ala
625 630 635 640
Val Leu Gly Leu Asn Phe Asn Leu His Pro Arg Leu Ser Pro Tyr Glu
645 650 655
Leu Val Tyr Pro Gly Ser Val Pro Arg Ala Ala Asp Glu His Thr Leu
660 665 670
Thr Leu Ala Asp Leu Ala Val Arg Ala Asp Pro Ala Arg Arg Thr Leu
675 680 685
Ala Leu Val Ser Thr Arg Asp Gly Gln Pro Leu Asp Leu Val Pro Leu
690 695 700
Asn Phe Leu Tyr Pro Ala Ala Ala Pro Gly Leu Tyr Arg Leu Leu Cys
705 710 715 720
Ala Phe Ala Pro Thr Arg Thr Tyr Arg Gly Gly Leu Trp Asp Gln Leu
725 730 735
Asp Arg Ala Asp Ala Glu Ala Gly Arg Ala Ala Gly Arg Thr Gly Val
740 745 750
Pro Ala Thr His Arg Ser Leu Pro Arg Val Leu Leu Gly Asp Leu Val
755 760 765
Leu Asp Arg Ala Ser Trp Arg Leu Pro Ala Ala Asp Val Pro Asp Thr
770 775 780
Asp Gly Leu Glu Arg Gln Glu Ala Ala Ala Leu Ala Ser Phe Asp Arg
785 790 795 800
Trp Leu Gly Gln Arg Gly Ile Pro Arg His Thr Phe Phe Arg Leu Thr
805 810 815
Thr Pro Pro Pro Val Pro Ala Gly Glu Arg Asp Leu Leu Ala Glu Thr
820 825 830
Arg Gln Trp Ala Leu Glu Ala Arg Thr Ala Arg Leu His Lys Pro His
835 840 845
Tyr Leu Asp Ala Arg Asn Pro Phe Leu Leu Gln Val Phe Ala Arg Arg
850 855 860
Leu Ala Glu Ala Gly Pro Asp Ala Thr Val Thr Phe Gln Glu Cys Leu
865 870 875 880
Pro His Ala Gly Asp Leu Asp Gly Arg Thr Ser Gly Ala Glu Glu Phe
885 890 895
Phe Val Glu Tyr Thr Leu Gly Thr Pro Ala Pro Ala Gly Thr Gln Ala
900 905 910
Thr Glu Asp Ala His Ala Arg Pro
915 920
<210>7
<211>549
<212>PRT
<213>Streptomyces actuosus ATCC25421
<400>1
Met Thr Thr Asp Glu Ala Tyr Thr Tyr Thr Thr Gly Leu Arg Leu Asp
1 5 10 15
Pro Arg Ser Ala Asn Pro Asp Gly Trp Arg Val Asp Trp Ala Asp Gly
20 25 30
Pro Trp Pro Val Lys Val Tyr Ser Gly Ala Arg Arg Leu Pro Leu Arg
35 40 45
Pro Asp Gly Pro Pro Pro Leu Ala Ala Leu His Arg Leu Leu His Gly
50 55 60
Gly Phe Ala Val Ser Arg Ile Arg Thr Asp Pro Ser Gly Gly Ile Ala
65 70 75 80
Ala Thr Pro Ala Asp Pro Arg Pro His His Gly Pro Glu Val Gln Leu
85 90 95
Arg Arg Pro Val Pro Ser Gly Gly Ala Met Tyr Pro Thr Glu Val Tyr
100 105 110
Ala Ala Leu Thr Ala Thr Gly Gln Val Cys His Tyr Asp Pro Tyr Arg
115 120 125
His Glu Leu Thr Val Leu Ala Gly Gly Asp Ala Ala Ala Arg Leu Arg
130 135 140
Ala Ala Leu His Leu Pro Thr Glu Ala Ala Pro Ala Ala Val Leu Val
145 150 155 160
Leu Thr Ser Arg Phe Trp Lys Asn Phe Tyr Lys Tyr Gly Asp Phe Ala
165 170 175
Thr Arg Leu Gly Leu Val Asp Ala Gly Val Ala Leu Gly Arg Ala Ala
180 185 190
Arg Leu Ala Arg Ala Glu Trp Glu His Ala Glu Val Arg Thr Val Phe
195 200 205
Asp Asp Glu Ala Val His Ala Cys Leu Gly Leu Asp Gly Glu Glu Glu
210 215 220
Asn Ala Trp Ala Ala Val Thr Leu Gly Pro Tyr Leu Pro Tyr Arg Ala
225 230 235 240
Asp Thr Gly Gly Pro Asp Ala Pro Pro Arg Pro Ala Leu Leu Glu Arg
245 250 255
Ser Arg Thr Val Arg Arg Ser Asp Arg Phe Thr Ala Phe Gln Arg Ala
260 265 270
Ala Arg Glu Asp Thr Ser Thr Ala Ala Pro Ala Arg Pro Pro Glu Asp
275 280 285
Ala Val Pro Ala Thr Ala Pro Gly Pro Val Pro Gly Asp Ala Gly Pro
290 295 300
Ala Ala Ala Pro Gly Pro Val His Glu Asp Ala Val Ser Ala Ala Ala
305 310 315 320
Pro Arg Pro Val Pro Gly Asp Ala Val Pro Val Gly Pro Val Pro Leu
325 330 335
Pro Ala Pro Arg Ala Leu Asp Leu Leu Ala Ala Glu Thr Ala Ala Arg
340 345 350
Arg Phe Ser Arg Gly Arg Leu Phe Thr Gly Ala Glu Ala Asp Gly Glu
355 360 365
Ala Leu Ala Gly Val Leu Gly Gln Ala Ala Glu Ala Leu Arg Ala Leu
370 375 380
Ala Gly Ala Gly Ala Asp Gly Pro Ala Gly Trp Ala Ala Arg Thr Arg
385 390 395 400
Leu Tyr Cys Ala Val His Arg Val Arg Gly Val Pro Pro Gly Trp Tyr
405 410 415
Arg Tyr Ala His Glu Leu Gly Ala Leu Leu Pro Val Gly Glu Gly Thr
420 425 430
Gly Pro Gly Ser Ala Arg Arg Val Gln Glu Ala Leu Phe Ala Ala Ser
435 440 445
Phe Asn Ala Glu Leu Ala Ala Phe Thr Val His Pro Val Thr Pro Ala
450 455 460
Asp Trp Arg Pro Ala Gly Gly Pro Arg Ala Tyr Arg Ala Gln Gln Leu
465 470 475 480
Ala Val Gly Ala Ala Ile Glu Ala Val Thr Leu Ala Ala Ala Ala Glu
485 490 495
Gly Leu Ser Gly His Ala Val Leu Gly Phe Asp Val Thr Arg Ile Asp
500 505 510
Thr Ala Tyr Gly Leu Asp Gly Gly Ala Asp Thr Asp Gly Gly Thr Gln
515 520 525
Ala Gln Ile Cys Val Gly Ala Val Arg Pro Asp Pro Asn Trp Glu Ile
530 535 540
Ala Val Met Pro Arg
545
<210>8
<211>656
<212>PRT
<213>Streptomyces actuosus ATCC25421
<400>1
Met Ser Thr Ala Thr Thr Pro Gln Pro Arg Thr Gly Pro Ala Gly Pro
1 5 10 15
Pro Val Val Val Gly Arg Gly Val Leu Ala Glu His Leu Val Arg Arg
20 25 30
Leu Gly Arg Asp Asp Thr Pro Asp Pro Asp Gly Gly Ala Arg Phe Gly
35 40 45
Arg Ser Ser Gly Ala Thr Val Leu Val Ala Gly Leu Asp Gly Leu Gly
50 55 60
Glu Phe Gln Asp Thr Val Val Asp Cys Leu Ala Thr Gly Arg Ser Leu
65 70 75 80
Leu Phe Val Gly Ser Trp Arg Ser Leu Val Tyr Ile Gly Pro Val Trp
85 90 95
Arg Pro Gly Thr Gln Gly Cys Pro Arg Cys Leu Val Thr Arg Thr Ala
100 105 110
Asn Ser Pro Phe Gly Pro Gly Leu Glu Gly Asp Ser Leu Ala Glu Ser
115 120 125
Trp Pro His Gly Ala Asp Val Arg Ile Trp Gly Pro Gly Val Leu Arg
130 135 140
Leu Val Glu Glu Tyr Val Arg Ala Arg Leu Ala Ser Cys Lys Gly Pro
145 150 155 160
Ala Gly Pro Ala Asp Gly Arg Val Leu Val Leu Asp Gly Ala Ala Gly
165 170 175
Thr Val Glu Gln Gln Thr Leu Leu Pro Asp Ser Gly Cys Pro Asn Cys
180 185 190
Gly Ser Leu Arg Glu Asp Thr Val Pro Ala Leu Thr Pro Ala Ala Val
195 200 205
Pro Leu Thr Lys Leu Thr Pro Ala Thr Leu Arg Thr Gly Arg Leu Pro
210 215 220
Ala Asp Ala Val Arg Gly Asp Tyr Leu Tyr Ala Gly Leu Gly Leu Phe
225 230 235 240
Lys Glu Leu Arg Gln Asp Leu Gln Ser Pro Phe Gly Ala Cys Ser Val
245 250 255
Glu Leu Pro Pro Arg Trp Gly Arg Arg Glu Pro Ala Ile Gly Arg Ala
260 265 270
Ala Asp Tyr Ala Thr Ser Arg Thr Val Ala Val Leu Glu Gly Leu Glu
275 280 285
Arg Tyr Ala Gly Leu His Arg Gly Gly Thr Leu Pro Pro Val Arg Ala
290 295 300
Arg Tyr Ala Asp Val Ala Asp Gln Ala Leu Tyr Pro Pro Asp Leu Gly
305 310 315 320
Thr His Pro Glu Glu Ser Tyr Ala Ser Glu Gly Phe Arg Tyr Arg Pro
325 330 335
Phe Asp Pro Gly Thr Glu Ile Asp Trp Val Gly Ala Tyr Ser Phe Arg
340 345 350
Arg Asp Gly Arg Val Leu Val Pro Glu Arg Ala Ala Phe Trp Gly Pro
355 360 365
Arg His Asp Asp Glu Ile Ser Phe Phe Tyr Asp Thr Ser Asn Gly Cys
370 375 380
Ala Leu Gly Asn Ser Val Glu Glu Ala Val Leu His Gly Leu Arg Glu
385 390 395 400
Leu Ala Glu Arg Asp Ala Phe Leu Leu Thr Trp Tyr Arg Glu Leu Asp
405 410 415
Val Pro Glu Val Ala Leu Asp GlyThr Ser Pro Ala Leu Asp His Leu
420 425 430
Leu Ala Lys Ser Arg Leu Phe Thr Gly Phe Asp Phe Arg Cys Phe Asp
435 440 445
Ala Thr Met Glu Tyr Gly Val Pro Ala Leu Leu Leu Thr Ala Glu Asn
450 455 460
Asp Ser Gly Asp Gly Pro Arg Leu Phe Ala Gly Cys Gly Ala His Pro
465 470 475 480
Asp Pro Val Gln Ala Val Thr Gly Ala Leu His Glu Leu Val Gly Thr
485 490 495
Val Leu Ala Thr Arg Asp Ala Tyr Glu Arg Arg Arg Pro Asp Ala Leu
500 505 510
Arg Met Leu Ala Asp Pro Phe Leu Ile Arg Arg Met Glu Asp His Ser
515 520 525
Thr Val Gly Ala Leu Pro Glu Ala Arg Asp Arg Phe Ser Phe Leu Leu
530 535 540
Asp Arg Pro Arg Thr Gly Ala Pro Val Pro Leu Gly Arg Val Arg Ser
545 550 555 560
Thr Leu Arg Thr Gln Asp Ala Asp Leu Arg Ala Asp Leu Tyr Ala Ala
565 570 575
Val Ser Gly Val Leu Asp Cys Gly Leu Asp Val Leu Val Val Asp Gln
580 585 590
Thr Met Pro Glu Leu Arg Arg Asn Gly Leu His Cys Val Arg Val Leu
595 600 605
Val Pro Gly Leu Ile Pro Met Thr Phe Gly His Arg Asn Arg Arg Thr
610 615 620
Cys Gly Leu Pro Arg Leu Thr Glu Gly Thr Thr Leu Pro Tyr Arg Ser
625 630 635 640
Leu Leu Ala Pro Gly Arg Glu Ile Gly Ala Val Pro His Pro Phe Pro
645 650 655
<210>9
<211>654
<212>PRT
<213>Streptomyces actuosus ATCC25421
<400>1
Met Thr Asp Val Ala Ala Asp Arg Ala Gln Pro Ala Gly Pro Ala Ala
1 5 10 15
Asp Pro Leu Thr Asp Thr Leu Thr Arg Thr Tyr Arg Leu Arg Ala Asp
20 25 30
Ala Phe Tyr Val Arg Gln Gln Asp Gly Val Trp Leu Gly Asn Asn Thr
35 40 45
Gly Ser Phe Ser Val Arg Gly Gln Gly Ala Tyr Gln Leu Val Ser Ser
50 55 60
Leu Phe Ala Gly Leu Asp Gly Glu Arg Thr Leu Gln Asp Leu Tyr Gly
65 70 75 80
Asp Leu Pro Asp Arg Ala Arg Arg Ser Val Leu Gly Leu Val Arg Ala
85 90 95
Met Leu Arg Asn Gly Phe Ile Lys Glu Val Ala His Pro Val Glu Pro
100 105 110
Val Pro Gly Trp Met Arg Glu Arg Tyr Ala Thr His Leu Ala Phe Leu
115 120 125
Glu His His Ala Asp Arg Pro Val Thr Arg Leu Gln Gln Val Arg Thr
130 135 140
Ala Arg Val Val Cys Ala Gly Arg Gly Thr Ala Leu His Ala Leu Leu
145 150 155 160
Asp Ala Leu Arg Glu Phe Gly Ile Ala Arg Leu Asp Val Val Pro Asp
165 170 175
Gly Asp Asp Asp Leu Thr Ala Val Gln Gln Val Leu Lys Glu Thr Ala
180 185 190
Ala Ala Asp Pro Gly Ala Arg Trp Arg Leu Arg Asp Pro Leu Ala Ala
195 200 205
Asp Gly Pro Ala Ala Leu Ala Gly His Pro Asp Val Glu Gly Ala Asp
210 215 220
Ala Val Leu Leu Ala Tyr Asp Ser Ala Asp Ala Ala Ala Leu Ala Arg
225 230 235 240
Ser Gln His Ala Leu Trp Arg Asp Gly Val Thr Val Gly Val Leu Ala
245 250 255
Arg Cys Gly Asp Phe Val Thr Ala Leu Glu Pro Gly Leu Gly Thr Pro
260 265 270
Tyr Cys Trp Glu Cys Val His Arg Ser Ile Ala Val Arg Ala Thr Gly
275 280 285
Asp Thr Ala Gly Leu Ala Pro Ala Val Ala Pro Ala Ala Val Gly Ala
290 295 300
Leu Arg Val Ala Gln His Thr Phe Ala Arg Leu Ala Gly Val Arg Pro
305 310 315 320
Pro Gly Asp Lys Pro Val Thr Thr Val Glu Pro Leu Val Pro Ala Val
325 330 335
Arg Gly His Ala Ala Arg Arg His Pro Arg Cys Pro His His Glu Pro
340 345 350
Ala Val Pro Arg Arg Leu Pro Pro Ala Gly Ala Ala Ala Pro Glu Asp
355 360 365
Ala Val Arg Pro Asp Ile Pro Arg Ser Glu Asp Pro Pro Glu Arg Val
370 375 380
Arg Ile Ser Asp Arg Ile Val Ala Ala Cys Ala Ala Leu Thr Asp Ala
385 390 395 400
Val Thr Gly Pro Leu Leu Ala Leu Gly Glu Glu Asp Leu Ala Gln Leu
405 410 415
Pro Leu Ser Ala Ser Ala Cys Gln Val Ala Asp Pro Asp Gly Gly Ala
420 425 430
Asp Ala Pro Ala Ala Leu Gly Val Val Cys Arg Ala Leu Ser Pro Arg
435 440 445
Glu Ala Arg Asn Gln Val Val Leu Cys Ala Val Glu Ser Leu Ala Gly
450 455 460
Arg Leu Thr Ala Gly Asp Ala Arg Tyr Gly Ala Val Gly Ala Gly Trp
465 470 475 480
Ser Leu Gly Glu Ala Arg Leu Arg Ala Arg Leu His Ala Ala Leu Gly
485 490 495
Arg Pro Thr Pro Asp Leu His Trp Ala Pro Ala Pro Glu Glu Pro Pro
500 505 510
Pro Gly Asp Thr Ala Ala Gly Tyr Leu Ala Gly Val Leu Ala Ala Glu
515 520 525
Gly Thr Pro Trp Thr Ala Thr Ala Ala Glu Glu Leu Pro Asp Gly Val
530 535 540
Val Arg Ala His Val Arg Thr Arg Asp Gly Ala Val Thr Ala Gly Val
545 550 555 560
Gly Thr Asp Arg Glu Arg Ala Val Gly His Ala Leu Leu Asn Ala Val
565 570 575
Ala Arg Val Leu Pro Leu Pro Gly Asn Ala Gln Glu Ala Thr Ala Phe
580 585 590
Leu Ala Pro Ala Thr Ala Thr Trp Thr Ala Ala Pro Thr Ala Glu Gly
595 600 605
Glu Arg Glu Ile Thr Asp Leu Leu Pro Phe Leu Thr Asp Thr Asp Thr
610 615 620
Gly Thr Gly Thr Gly Thr Gly Thr Gly Ser Gly Gly Glu Ala Gly Ala
625 630 635 640
Arg Val Arg Val Val Ala Leu Pro Val Ser Glu Glu Ala Ser
645 650
<210>10
<211>444
<212>PRT
<213>Streptomyces actuosus ATCC25421
<400>1
Val Gly Asp Met Gly Arg Pro Ala Phe Gln Arg Phe Leu Thr Pro Arg
1 5 10 15
His Leu Pro Ala Gly Arg Ala Gly Ala Val Thr Gly Val Arg Trp Gly
20 25 30
Gly Asp Phe Ala Ala Trp Asp Asp Leu Leu Thr Ala Gly Arg Asp Leu
35 40 45
Ala Ala Gln Val Arg Pro Gly Gly Ala Tyr Ala Ile Asp Pro Thr Ala
50 55 60
Gly Leu Pro Ala Leu Ala Ala Leu Phe Ala Val Ala Thr Val Pro Asp
65 70 75 80
Thr Val Leu Leu Trp Ala Ser Pro Arg Thr Leu Gly Val Thr Gly Arg
85 90 95
Glu Ile Ala Pro Ala Leu His Ala Leu Pro Asp Asp Gly Ser Val Pro
100 105 110
Leu Ala Ala Gln Glu Arg Pro Leu Trp Gly Val Cys Thr Ser Gly Ser
115 120 125
Ser Gly Ala Pro Lys Val Ala Val Gly Pro Ala Asp Glu Trp Glu Gln
130 135 140
Ile Ala Leu His Ala Glu Ala Ala Met Tyr Ala Asp Ala Phe Pro Ala
145 150 155 160
Gly Pro Pro Glu Ala Leu Ala Thr Cys Leu Pro Leu Gly Phe Ser Ala
165 170 175
Ala Phe Phe Met Cys Val Leu Pro Ala Leu Tyr Leu Lys Arg Asp Leu
180 185 190
Val Val His Pro Pro His Asp Trp Ser Pro Leu Tyr Asp Leu Ala Arg
195 200 205
Asp Arg Arg Val Leu Ala Leu Gly Val Pro Ala Leu Ala Ala Ala Ala
210 215 220
Cys Leu Ser Ala Pro Ala Ala Thr Asp Leu Gly Ser Val Ala Leu Phe
225 230 235 240
Leu Gly Gly Gly His Leu Ser Ala Pro Arg Val Glu Leu Ile Arg Arg
245 250 255
His Phe Thr Gly Ala Ala Val Ser Asn Leu Tyr Gly Thr Ala Glu Thr
260 265 270
Gly Ala Ile Ala Leu Asp His Asp Pro Gly His Asn Arg His Val Gly
275 280 285
Arg Pro Ile Pro Gly Lys Ser Val Trp Leu Thr Gly Thr Asp Glu Arg
290 295 300
Gly Ile Gly Thr Val Ala Val Ala Gly Pro Gly Cys Cys Arg Arg Thr
305 310 315 320
Trp Arg Pro Gly Ser Pro Pro Ser Ala Pro Ala Asp His Val Thr Gly
325 330 335
Thr Asp Tyr Gly Arg Phe Asp Ala Asp Gly Asn Leu Cys Leu Glu Gly
340 345 350
Arg Leu Asp Gly Ala Glu Lys Leu Ala Gly Val Leu Val Arg Pro Arg
355 360 365
Glu Ile Glu Arg His Val Leu Ala Leu Asp Gly Val Ser Asp Val Arg
370 375 380
Val Thr Val Glu Thr Ala Pro Thr Gly Leu Glu Phe Leu Ala Ala Thr
385 390 395 400
Val Val Gly Ser Val Asp Ala Asp Thr Val Arg Ala His Cys Ala Ala
405 410 415
Leu Pro Glu Gln His Arg Pro Ser Arg Ile Ser Cys Ala Ser Glu Gln
420 425 430
Glu Ala Ala Thr Val Tyr Ser Ala His Gly Lys Leu
435 440
<210>11
<211>79
<212>PRT
<213>Streptomyces actuosus ATCC25421
<400>1
Val Thr Ser Gln Arg Thr Thr Pro Arg Thr Pro Asp Gly Val Pro Asp
1 5 10 15
Leu Gln Glu Glu Leu Ala Gly Leu Leu Gln Glu Asp Asp Pro Arg Arg
20 25 30
Arg Leu Asp Ser Leu Glu Thr Val Val Val Leu Ser Tyr Phe Ala Arg
35 40 45
Gln Ala Pro Gly Arg Thr Leu Pro Glu Leu Pro Asp Ala Pro Arg Thr
50 55 60
Ile Glu Gly Trp Val Thr Trp Ala Asp Gln Arg Ser Ser Ala Ser
65 70 75
<210>12
<211>270
<212>PRT
<213>Streptomyces actuosus ATCC25421
<400>1
Val Asp Ala Glu Thr Pro Met Asp Thr Glu Thr Pro Arg Asp Thr Glu
1 5 10 15
Thr Pro Met His Thr Gly Met Ser Thr Gly Pro Glu Thr Pro Thr Val
20 25 30
Tyr Leu Val His Gly Leu Leu Gly Thr Gly His Gly His Phe Ala Ala
35 40 45
Gln Ile Arg Ala Trp His Gly Arg Leu Arg Thr Val Pro Val Asp Leu
50 55 60
Pro Gly His Gly Arg Cys Arg Arg Asp Ala Ala Glu Asp Tyr Phe Asp
65 70 75 80
Asp Ala Leu Arg Tyr Leu Val Ala Val Leu Glu Arg Phe Gly Pro Gly
85 90 95
Arg Leu Ile Gly Ala Ser Tyr Leu Gly Gly Pro Leu Ala His Arg Cys
100 105 110
Ala Ala Thr Arg Pro Asp Leu Val Ser Ser Leu Val Leu Thr Gly Phe
115 120 125
Ala Pro Asp Val Ser Arg Asp Ala Phe Leu Ser Leu Ile Ala Gly Phe
130 135 140
Glu Gly Leu Ala Ala Gln Gln Pro Ala Leu Ala Ala Glu Tyr Glu Gln
145 150 155 160
Leu His Gly Thr Arg Trp Lys Arg Thr Leu Asp Ala Val Thr Gly His
165 170 175
Val Glu Arg Asp Phe Glu Arg Thr Ala Leu Val Arg Ala Ala Asp Val
180 185 190
Ala Ala Leu Thr Val Pro Thr Leu Val Leu Asn Gly Ser Leu Lys Ser
195 200 205
Val Glu Arg Ala Ala Ala Glu Gln Ala Pro Gly Trp Gly Gly Arg Val
210 215 220
Arg Gly Arg Val Val Pro Gly Ala Gly His Leu Val Gly His Asp Arg
225 230 235 240
Pro Arg Glu Phe Asn Glu Ala Val Glu Asp Phe Trp Arg Thr Ala His
245 250 255
Asp Ala Pro Ala Gly Pro Arg Thr Thr Gln Lys Gly Asp Thr
260 265 270
<210>13
<211>400
<212>PRT
<213>Streptomyces actuosus ATCC25421
<400>1
Met Thr Gln Asn Ser Gln Ala Met Thr Ser His Ala Met Thr Gly Asp
1 5 10 15
Phe Val Leu Pro Glu Leu Glu Asp Val Arg Ala Glu Ala Ala Thr Val
20 25 30
Asp Thr Arg Ala Val Leu Ala Leu Ala Glu Gly Glu Glu Pro Ala Glu
35 40 45
Ser Arg Ala Ala Val Ala Leu Ala Leu Trp Glu Asp Arg Ser Ile Gly
50 55 60
Thr Ala Glu Leu Gln Ala Ala Ala Glu Ala Arg Cys Gly Ala Arg Arg
65 70 75 80
Pro Arg Leu His Thr Phe Val Pro Leu Tyr Thr Thr Asn Tyr Cys Asp
85 90 95
Ser Glu Cys Lys Met Cys Ser Met Arg Lys Gly Asn His Arg Leu Asp
100 105 110
Arg Lys Phe Ser Gly Arg Lys Glu Ile Thr Glu Gln Leu Glu Ile Leu
115 120 125
Tyr His His Glu Gly Val Arg Gly Val Gly Phe Leu Thr Gly Glu Tyr
130 135 140
Glu Asp Lys His Thr Arg Leu Ala Ser Ala Phe Arg Ile Gly Trp Ala
145 150 155 160
Ile Arg Thr Ala Leu Asp Leu Gly Phe Glu Arg Val Tyr Phe Asn Ile
165 170 175
Gly Ser Met Glu Gln Asp Glu Ile Asp Val Leu Gly Glu Trp Ile Gly
180 185 190
Arg Glu Asp Pro Val Thr Met Cys Val Phe Gln Glu Ser Tyr Asp Arg
195 200 205
Glu Thr Tyr Arg Arg Phe Met Gly Lys Thr Ser Val Gly Val Pro Lys
210 215 220
Ala Asp Phe Asp Arg Arg Val Val Ser Phe Asp Arg Trp Leu Asp Ala
225 230 235 240
Gly Tyr Arg Tyr Val Asn Pro Gly Val Leu Val Gly Leu His Asp Asp
245 250 255
Leu Ser Ala Glu Leu Val Ser Leu Val Ala His Gly Asp His Leu Arg
260 265 270
Ser Arg Gly Ala Thr Ala Asp Leu Ser Val Pro Arg Met Arg Pro Ala
275 280 285
Met Lys Ser Arg Asp Thr Thr Arg Val Gly Asp Asp Asp Tyr Leu Arg
290 295 300
Leu Met Ser Val Val Ala Phe Thr Cys Pro Glu Gln Arg Leu Val Leu
305 310 315 320
Thr Thr Arg Glu Pro Gln Glu Phe Gln Asp Val Ala Leu Gly Leu Ala
325 330 335
Gly Val Ile Ser Pro Gly Ser Pro Asp Val Ala Pro Tyr Arg Ala Gly
340 345 350
Cys Glu Ala Arg Asn Asp Glu Lys Ser Ser Gln Phe Leu Val Ala Asp
355 360 365
Leu Arg Arg Pro Arg His Ile Leu Gly Arg Ile Glu Ala Ser Gly Thr
370 375 380
Pro Val Asp His Phe Val Asn Pro Ala Gly Glu Ala Ser Arg Ala Val
385 390 395 400
<210>14
<211>50
<212>PRT
<213>Streptomyces actuosus ATCC25421
<400>1
Val Asp Ala Ala His Leu Ser Asp Leu Asp Ile Asp Ala Leu Glu Ile
1 5 10 15
Ser Glu Phe Leu Asp Glu Ser Arg Leu Glu Asp Ser Glu Val Val Ala
20 25 30
Lys Val Met Ser Ala Ser Cys Thr Thr Cys Glu Cys Cys Cys Ser Cys
35 40 45
Ser Ser
50
<210>15
<211>395
<212>PRT
<213>Streptomyces actuosus ATCC25421
<400>1
Met Arg Gln Asn Leu Leu Met Ile Tyr Val His Ile Pro Phe Cys His
1 5 10 15
Ser Lys Cys Thr Phe Cys Asp Trp Val Gln Ala Ile Pro Thr Lys Asp
20 25 30
Leu Leu Arg Lys Pro Gly Asp Ser Val Arg Gln Lys Tyr Ile Ser Ala
35 40 45
Leu Cys Ala Glu Ile Ala Glu Arg Gly Ala Met His Arg Ala Ala Gly
50 55 60
Asp Ile Pro His Val Leu Tyr Trp Gly Gly Gly Thr Ala Ser Ser Leu
65 70 75 80
Asp Glu Gln Glu Thr Ala Ala Val Met Glu Ala Leu His Ser Ser Phe
85 90 95
Asp Met Ser Thr Val Ala Glu Ala Thr Ile Glu Cys Ser Pro Asp Thr
100 105 110
Val Asp Glu Arg Lys Leu Ala Phe Tyr Arg Gly Leu Gly Phe Asn Arg
115 120 125
Val Ser Ser Gly Val Gln Ser Phe Asp Asp Asp Arg Leu Arg Arg Leu
130 135 140
Gly Arg Arg His Thr Ala Glu Gln Ala Gly Arg Ile Val His Ala Ala
145 150 155 160
Arg Ala Ala Gly Phe Glu Asp Val Ser Ile Asp Ile Met Ser Gly Phe
165 170 175
Pro Asp Gln Glu Ala Asp Glu Leu Asp Arg Thr Val Asp Arg Ala Leu
180 185 190
Glu Leu Pro Val Asn His Leu Ser Leu Tyr Ser Phe Arg Pro Thr Pro
195 200 205
Gly Thr Phe Met Arg Arg Arg Met Asp Ser Ser Glu Arg Arg Thr Tyr
210 215 220
Leu Arg Arg Gln Gln Ala Leu Phe Thr Arg Ala Arg Arg Ala Ile Glu
225 230 235 240
Gly Phe Gly Leu Ser Glu Tyr Ala Asn Gly Tyr Phe Gly Lys Val Ser
245 250 255
Pro Phe Ala Ser Met Tyr Phe Gln His Arg Ala Asp Thr Val Gly Leu
260 265 270
Gly Ser Gly Ala Ile Ser Leu Val Asp Gln Arg Phe Lys Ser His Gln
275 280 285
Lys Gly Leu Leu His Ser Tyr Val Asp Asp Pro Leu Ala Phe Asp Ile
290 295 300
Asp Val Pro Ala Gly Gln Asp Arg Val Leu Val Ser Leu Leu Gln Ala
305 310 315 320
Gly Leu Ala Met Phe Asp Gly Ile Pro Arg Glu Asp Trp Arg Gln Arg
325 330 335
Thr Gly Thr Asp Leu Ala Glu Val Leu Leu Arg Pro Thr Val Ala Pro
340 345 350
Leu Ala Asp Phe Leu Arg Gly Arg Gly Leu Val Glu Asp Asp His Gly
355 360 365
Ile Arg Leu Pro Arg Asp Ile Ala Gly Leu Thr Leu Ile Glu Leu Ala
370 375 380
Phe Glu Met Ala Met Ser Gln Pro Glu Leu Val
385 390 395
<210>16
<211>370
<212>PRT
<213>Steptomyces actuosus ATCC25421
<400>1
Val Thr Ser Gly Pro Gly Gln Ala Pro Ala Glu Ala Ala His Ala Ala
1 5 10 15
Gly Ala Ala Trp Leu Glu Ile Gly Leu Asp Ala Pro Ala Asp Ala Val
20 25 30
Pro Ala Leu Val Ala Gly Val Val Arg Pro Leu Leu Arg Glu Pro Ala
35 40 45
Glu Pro Gly Ala Glu Pro Val Pro Gly Phe Phe Leu Arg Gly Val Gly
50 55 60
Ala Ala Gln Pro Ala Leu Val Val Gln Leu Glu Val Thr Pro Gly Thr
65 70 75 80
Asp Leu Ala Glu Pro Tyr Ala Ala Arg Ala Arg Ala Leu Ala Ala Gly
85 90 95
Leu Gly Leu Pro Val Gln Val Ala Ala Gly Arg Ala Thr Leu Val Pro
100 105 110
Leu Ala Gly Ser Val Phe Ala Gly Ala Ala Leu Gly Pro Val Thr Arg
115 120 125
Ala Ala Leu Ala Ala Val Cys Pro Ala Leu Leu Thr Ala Thr Glu Ala
130 135 140
Ala Glu Gln Gly Arg Pro Ala Leu Leu Ala Ser Ala Ala Glu Leu Met
145 150 155 160
Ser Ala His Leu Arg Ala Val Ser Val Ser Ala Ala Pro Gly Pro Arg
165 170 175
Gln Trp Glu Glu Leu Arg Glu Gly Val Pro Leu Gly Phe Leu Ser Tyr
180 185 190
Arg Ser His Ala Glu Ala Phe Leu Ala Ser Ser Arg Asp Pro Lys Ala
195 200 205
Ala Gln Ala Met Met Asp Ala Lys Tyr Thr Arg Ala Ala Ala Thr Leu
210 215 220
Glu Arg Leu Val Asp Gly Val Leu Thr Gln Cys Glu Glu Arg Gly Pro
225 230 235 240
Val Val Ser Leu Pro Ala Arg Gln Trp Tyr Glu Ala Met Arg Ala Ala
245 250 255
Lys Pro Ala Val Thr Glu Leu Phe Arg Ala Gly Thr Asp Leu Ala Leu
260 265 270
Asp Thr Glu Glu Gln Pro Pro Asp Thr Gly Pro Asp Gly Lys Gly Leu
275 280 285
Ser Glu Ser Ala Phe His Arg Ile Val Glu Gly Ser Asp Gly Leu Arg
290 295 300
Asp Phe Leu Asp Arg Asp Pro Ser Phe Leu Ala Thr Arg Leu Leu Thr
305 310 315 320
Ser Leu Leu Tyr Leu Ser Leu Ser Ser Val Gly Ile Ala Leu Ala Glu
325 330 335
Arg Tyr Phe Leu Cys Tyr Ala Val Ser Arg Ala Cys Glu Ser Ile Phe
340 345 350
Asp Thr Asp Ala Leu Thr Val Leu Ser Gly Leu Ala Arg Thr Ser Leu
355 360 365
Ala Ser
370
<210>17
<211>323
<212>PRT
<213>Streptomyces actuosus ATCC25421
<400>1
Val Ile Pro Ala Gly Arg Arg Thr Ala Arg Ser Ser Arg Ala His Ala
1 5 10 15
Ala Pro Cys Arg Val Cys Arg Phe Pro His Val Ala Ser Thr His Arg
20 25 30
Glu Met Arg Cys Arg Gln His Gly Arg Ser Thr Cys Ala Pro Pro Gln
35 40 45
Arg Gly Ser Ser Met Glu Ile Asp Val Leu Gly Pro Leu Glu Val Ser
50 55 60
Gln Lys Ser Ala Ser Ile Val Pro Thr Ala Ser Lys Pro Arg Thr Val
65 70 75 80
Leu Ala Met Leu Ala Val Tyr Thr Asn Arg Leu Val Pro Leu Pro Ser
85 90 95
Leu Met Gly Glu Leu Trp Gly Pro Glu Pro Pro Ala Thr Ala Lys Thr
100 105 110
Ala Val Gln Gly Tyr Ile Leu His Leu Arg Lys Arg Ile Ala Glu Ser
115 120 125
Ala Ala Arg Arg Pro Pro Gly Gln Phe Pro Glu Ala Lys Asp Ile Leu
130 135 140
Val Thr Leu Pro Gly Gly Tyr Leu Leu Arg Ala Pro Gly Thr Ala Val
145 150 155 160
Val Leu Glu Gln Phe Glu Gln Leu Ala Cys Ala Gly His Arg Ala Arg
165 170 175
Glu Lys Gly Asp Phe Glu Ala Ala Ser Arg Ser Phe Thr Glu Ala Leu
180 185 190
Gly Leu Trp Arg Gly Arg Ala Leu Ala Asp Val Glu Val Gly Pro Gln
195 200 205
Leu Gly Ile Glu Val Gln Arg Ile Glu Glu Ala Arg Leu Asn Val Leu
210 215 220
Asp Arg Arg Ile Glu Ala Asp Leu Arg Leu Gly Arg His His Glu Leu
225 230 235 240
Leu Gly Glu Leu Arg Ser Val Thr Ala His His Pro Thr His Glu Gly
245 250 255
Phe Cys Ala His Leu Met Leu Ala Leu Tyr Arg Ser Gly Arg Arg Cys
260 265 270
Glu Ala Leu Asp Ala Tyr Gln Arg Met Arg Thr Thr Leu Val Asn Glu
275 280 285
Leu Gly Leu Glu Pro Ser Pro Ser Leu Arg Arg Leu Gln Arg Ser Leu
290 295 300
Leu Val Ser Asp Arg Glu Leu Asp Glu Leu Asn Val Thr Trp Asp Val
305 310 315 320
Ala Thr Phe
Claims (3)
1.一种抗革兰氏阳性菌活性的抗生素-诺丝七肽的生物合成基因,其特征在于,所述的基因选自下组:
1)nosA基因:
nosA的核苷酸序列为SEQ ID NO:1所示的核苷酸序列第2704-2249个碱基,长度为456个碱基对,编码151个氨基酸的蛋白,其氨基酸序列如SEQ ID NO:2所示;
2)nosB基因:
nosB的核苷酸序列为SEQ ID NO:1所示的核苷酸序列第4068-2701个碱基,长度为1368个碱基对,编码455个氨基酸蛋白,其氨基酸序列如SEQ ID NO:3所示;
3)nosC基因:
nosC的核苷酸序列为SEQ ID NO:1所示的核苷酸序列第5309-4083个碱基,长度为1227个碱基对,编码408个氨基酸的蛋白,其氨基酸序列如SEQ ID NO:4所示。
2.如权利要求1所述的抗革兰氏阳性菌活性的抗生素-诺丝七肽的生物合成基因所编码的蛋白,其特征在于,所述的蛋白选自下组:
1)nosA基因编码的蛋白,其氨基酸序列如SEQ ID NO:2所示;
2)nosB基因编码的蛋白,其氨基酸序列如SEQ ID NO:3所示;和
3)nosC基因编码的蛋白,其氨基酸序列如SEQ ID NO:4所示。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN2009100534277A CN101586112B (zh) | 2009-06-19 | 2009-06-19 | 诺丝七肽的生物合成基因簇 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN2009100534277A CN101586112B (zh) | 2009-06-19 | 2009-06-19 | 诺丝七肽的生物合成基因簇 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN101586112A CN101586112A (zh) | 2009-11-25 |
CN101586112B true CN101586112B (zh) | 2011-06-22 |
Family
ID=41370542
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN2009100534277A Active CN101586112B (zh) | 2009-06-19 | 2009-06-19 | 诺丝七肽的生物合成基因簇 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN101586112B (zh) |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104024272B (zh) * | 2011-10-12 | 2018-04-20 | 赛诺菲 | 用于浅灰霉素和甲基浅灰霉素的生物合成的基因簇 |
CN103304628B (zh) * | 2013-06-06 | 2016-01-06 | 中国药科大学 | 诺西肽衍生物及其用途 |
CN105861523B (zh) * | 2016-01-22 | 2019-07-16 | 中国农业科学院蔬菜花卉研究所 | 参与白灰制菌素a或b合成的基因簇 |
CN109580864B (zh) * | 2019-02-18 | 2019-09-06 | 华南农业大学 | 一种动物性食品中那西肽残留的检测方法 |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2009019289A1 (en) * | 2007-08-09 | 2009-02-12 | Novartis Ag | Thiopeptide precursor protein, gene encoding it and uses thereof |
-
2009
- 2009-06-19 CN CN2009100534277A patent/CN101586112B/zh active Active
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2009019289A1 (en) * | 2007-08-09 | 2009-02-12 | Novartis Ag | Thiopeptide precursor protein, gene encoding it and uses thereof |
Non-Patent Citations (4)
Title |
---|
JP昭62-195286A 1987.08.28 |
Mocek, U等.Biosynthesis of the modified peptide antibiotic nosiheptide in Streptomyces actuosus.《J. Am. Chem. Soc.》.1993,第115卷7557–7568. * |
周佩等.诺西肤突变生物合成的研究(I).《中国抗生素杂志》.1995,第20卷(第3期),159-162,168. * |
王辂等.噻唑肽类抗生素研究进展.《中国抗生素杂志》.2008,第33卷(第2期),69-74,105. * |
Also Published As
Publication number | Publication date |
---|---|
CN101586112A (zh) | 2009-11-25 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
DK2271666T3 (da) | Nrps-pks-gengruppe og dens manipulation og anvendelighed | |
KR20190099396A (ko) | 화합물의 생산을 위한 조성물 및 방법 | |
CN101275141B (zh) | 阿嗪霉素的生物合成基因簇 | |
CN107868789B (zh) | 可利霉素生物合成基因簇 | |
KR20100049580A (ko) | 티오펩티드 전구체 단백질, 그를 코딩하는 유전자 및 그의 용도 | |
CN101586112B (zh) | 诺丝七肽的生物合成基因簇 | |
CN101157929A (zh) | 番红霉素的生物合成基因簇 | |
CN101818158B (zh) | Fr901464的生物合成基因簇 | |
CN101445803A (zh) | 硫链丝菌素的生物合成基因簇 | |
CN101691575B (zh) | 一种萨菲菌素的生物合成基因簇 | |
CN101184838A (zh) | 基因重组微生物及使用这些微生物的大环内酯类化合物的制备方法 | |
CN107794286B (zh) | 一种环脂肽类化合物生物合成基因簇及其激活方法与应用 | |
CN107881205B (zh) | 双环霉素生物合成中氧化酶的功能及其应用 | |
CN107540682B (zh) | 曲张链丝菌素衍生物及其制备方法和应用 | |
CN111378008B (zh) | 脂肽类化合物Totopotensamides及其制备方法和应用 | |
CN106676115B (zh) | 2’-氯代喷司他丁和2’-氨基-2’-脱氧腺苷生物合成基因簇及其应用 | |
CN107164394B (zh) | 一种非典型角环素类化合物nenestatin A的生物合成基因簇及其应用 | |
CN112359048B (zh) | 一种吕宋肽菌素c的制备方法 | |
CN101962647B (zh) | 诺卡噻唑菌素的生物合成基因簇及其应用 | |
CN102517309A (zh) | 一种手霉素类抗生素生物合成基因簇 | |
CN110551739A (zh) | 吡唑霉素生物合成基因簇、重组菌及其应用 | |
CN106188093B (zh) | 一种雷帕霉素结构类似物及其制备方法 | |
CN101812472B (zh) | 米多霉素生物合成基因簇 | |
CN115247179B (zh) | 一种聚酮化合物骨架及其后修饰物的生物合成基因簇及其应用 | |
CN107541523B (zh) | 曲张链丝菌素生物合成基因簇及其应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
C14 | Grant of patent or utility model | ||
GR01 | Patent grant | ||
TR01 | Transfer of patent right |
Effective date of registration: 20230608 Address after: 202152 room 196-8, building 3, No. 1999, Beixing highway, Sanxing Town, Chongming District, Shanghai (Shanghai yuhaitang science and Technology Park) Patentee after: Guoke Xinyan international technology transfer Co.,Ltd. Address before: 200032 Shanghai city Xuhui District Fenglin Road No. 354 Patentee before: SHANGHAI INSTITUTE OF ORGANIC CHEMISTRY, CHINESE ACADEMY OF SCIENCES |
|
TR01 | Transfer of patent right |