CN110551713A - 用于修饰梭状芽孢杆菌属细菌的优化的遗传工具 - Google Patents

用于修饰梭状芽孢杆菌属细菌的优化的遗传工具 Download PDF

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CN110551713A
CN110551713A CN201910439553.XA CN201910439553A CN110551713A CN 110551713 A CN110551713 A CN 110551713A CN 201910439553 A CN201910439553 A CN 201910439553A CN 110551713 A CN110551713 A CN 110551713A
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clostridium
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尼古拉斯·洛普斯费雷拉
弗朗索瓦·瓦塞尔斯
格拉迪斯·沙尔捷
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Abstract

本发明涉及用于修饰梭状芽孢杆菌属细菌的优化的遗传工具。具体地，本发明涉及一种包含至少两种不同核酸的遗传工具，其被优化以促进梭状芽孢杆菌属(Clostridium)的细菌、通常为产溶剂细菌的转化和通过同源重组进行的修饰。

Description

用于修饰梭状芽孢杆菌属细菌的优化的遗传工具
技术领域
本发明涉及一种包含至少两种不同核酸的遗传工具，其被优化以促进梭状芽孢杆菌属(Clostridium)的细菌，通常为梭状芽孢杆菌属(Clostridium)的产溶剂细菌的转化和通过同源重组进行的修饰。
背景技术
属于厚壁菌门梭状芽孢杆菌属(Clostridium)的细菌是革兰氏阳性、专性厌氧、形成内生孢子的杆菌。这个属含有许多由于它们的致病性或它们的工业和医学兴趣而被研究的物种。例如，破伤风梭状芽孢杆菌(Clostridium tetani)、肉毒梭状芽孢杆菌(Clostridium botulinum)、产气荚膜梭状芽孢杆菌(Clostridium perfringens)和难辨梭状芽孢杆菌(Clostridium difficile)分别是造成破伤风、肉毒中毒、气性坏疽和假膜性结肠炎的致病因子。诺维氏梭状芽孢杆菌(Clostridium novyi)和产芽孢梭状芽孢杆菌(Clostridium sporogenes)已被用于开发癌症疗法的研究中。同时，对人类不致病的其他物种例如丙酮丁醇梭状芽孢杆菌(Clostridium acetobutylicum)、丁酸梭状芽孢杆菌(Clostridium butyricum)和拜氏梭状芽孢杆菌(Clostridium beijerinckii)被用于发酵。
具有所谓的工业兴趣的梭状芽孢杆菌属(Clostridium)物种能够从葡萄糖到纤维素的广泛的各种不同糖和底物生产感兴趣的化合物例如酸和溶剂。生产溶剂的梭状芽孢杆菌属(Clostridium)细菌(“产溶剂细菌”)的生长被称为是双相的。酸(乙酸和丁酸)在指数生长期中生产。然后，当细胞生长停止并且细菌进入静止期时，它们生产溶剂。
梭状芽孢杆菌属(Clostridium)的大多数产溶剂菌株生产丙酮、丁醇和乙醇作为终产物。这些菌株被称为“ABE菌株”。例如菌株丙酮丁醇梭状芽孢杆菌(C.acetobutylicum)DSM 792(也被称为ATCC 824或LMG 5710)和拜氏梭状芽孢杆菌(C.beijerinckii)NCIMB8052就是这种情况。其他菌株也能将全部或一部分丙酮还原为异丙醇，并且被称为“IBE菌株”。例如菌株拜氏梭状芽孢杆菌(C.beijerinckii)DSM6423(也被称为NRRL B–593、LMG7814、LMG 7815)就是这种情况，其在基因组中具有编码将丙酮还原成异丙醇的伯醇/仲醇脱氢酶的adh基因。
尽管在工业中已使用超过一个世纪，但对属于梭状芽孢杆菌属(Clostridium)的细菌的了解长期以来受到在它们的遗传修饰中遇到的困难的限制。近年中已开发了各种不同的遗传工具来优化这个属的菌株，最新的一代是基于CRISPR(簇集规则间隔短回文重复序列)/Cas(CRISPR相关蛋白)技术的使用。这种方法是基于被称为核酸酶的酶(通常为Cas核酸酶例如化脓链球菌(Streptococcus pyogenes)的Cas9蛋白)的使用，所述酶由RNA分子指导，在DNA分子(感兴趣的靶序列)内制造双链切口。所述指导RNA(gRNA)序列将决定核酸酶切割位点，为它提供了非常高的特异性(图1)。
由于必需DNA分子中的双链切口对于生物体来说是致死的，因此它的存活将依赖于它修复所述切口的能力(参见例如Cui&Bikard，2016)。在梭状芽孢杆菌属(Clostridium)的细菌中，双链切口的修复依赖于同源重组机制，其需要被切割序列的完整拷贝。通过向细菌提供允许它进行这种修复同时修饰其原始序列的DNA片段，可以迫使所述微生物将所需的变化整合在它的基因组内。通过靶序列或PAM位点的修饰(图2)，所进行的修饰必须不再允许基因组DNA被Cas9–gRNA核糖核蛋白复合体靶向。
已经描述了不同方法尝试使这种遗传工具在梭状芽孢杆菌属(Clostridium)细菌中发挥作用。已知这些微生物由于低的转化和同源重组频率而难以遗传修饰。几种方法是基于在拜氏梭状芽孢杆菌(C.beijerinckii)和杨氏梭状芽孢杆菌(C.ljungdahlii)中组成性表达(Wang等，2015；Huang等，2016)或在拜氏梭状芽孢杆菌(C.beijerinckii)、C.saccharoperbutylacetonicum和自产乙醇梭状芽孢杆菌(C.authoethanogenum)中在诱导型启动子的控制之下的Cas9(Wang等，2016；Nagaraju等，2016；Wang等，2017)的使用。其他作者已描述了使用修饰版本的核酸酶Cas9n，其在基因组内制造单链而不是双链切口(Xu等，2015；Li等，2016)。这种选择是由于观察到在所试验的实验条件下Cas9的毒性对于它在梭状芽孢杆菌属(Clostridium)细菌中的使用来说过高。上述所有工具都是基于单一质粒的使用。最后，当在微生物的基因组中已鉴定到内源CRISPR/Cas系统时，也可以使用所述内源CRISPR/Cas系统，例如在巴氏梭状芽孢杆菌(C.pasteurianum)中(Pyne等，2016)。
除非它们使用(如在上述最后一种情况中那样)待修饰菌株的内源机制，否则基于CRISPR技术的工具具有显著限制可以插入到细菌基因组中的感兴趣的核酸的大小(根据Xu等，2015，约1.8kb为最佳)(并因此限制编码序列或基因的数目)的主要缺点。
本发明人最近开发并描述了用于修饰细菌、适合于梭状芽孢杆菌属(Clostridium)细菌的更高效的遗传工具，其基于两种不同的核酸、通常为两种质粒的使用(WO2017064439，Wasels等，2017和图3)，从而解决了这个问题。在特定实施方式中，该工具的第一核酸允许cas9的表达，并且特异性针对待制造的修饰的第二核酸含有一个或多个gRNA表达盒以及修复模板，允许将被Cas9靶向的细菌DNA的一部分用感兴趣的序列代替。通过将cas9和/或gRNA表达盒置于诱导型启动子的控制之下，这种系统的毒性受到限制。
最近，本发明人还成功地对在野生型状态下具有为细菌提供针对一种或多种抗生素的抗性的基因的细菌进行遗传修饰，以便使它们对所述抗生素敏感，这使得可以便于使用它们的基于至少两种核酸的使用的遗传工具。因此，他们能够遗传修饰拜氏梭状芽孢杆菌(C.beijerinckii)菌株DSM 6423，该菌株是异丙醇的天然生产菌。具体来说，他们成功地从这个菌株移除了在本说明书中被鉴定为“pNF2”的对所述菌株非必需的天然质粒(参见FR18/73492)。本发明人在本文中还发现并揭示了该质粒pNF2的移除使得在本发明的情形中可以获得遗传物质引入效率(即转化效率)提高约101至5x 103之间的倍数的拜氏梭状芽孢杆菌(C.beijerinckii)DSM 6423细菌。
本发明人在本文中描述了用于修饰梭状芽孢杆菌属(Clostridium)细菌的改进的遗传工具，所述遗传工具使得可以非常显著地提高所述细菌的转化效率，并因此以大且有用的量(特别是在选择用于工业规模生产的稳健菌株的情形中)获得感兴趣的突变(遗传修饰的)细菌。正如下文解释的，本发明人成功地改进了本发明的遗传工具，特别是通过使用质粒pNF2的一部分来设计特异性核酸，所述核酸带有用于修饰细菌的遗传物质和/或用于在细菌内表达在所述细菌的野生型版本中存在的遗传物质所缺乏的DNA序列的序列。这些核酸和新工具急剧提高了细菌的转化效率，特别是首先被清除了它们在野生型状态下含有的天然质粒的细菌的转化效率。
因此，本发明非常有利地促进了这些细菌的转化效率以及它们的使用，特别是在工业规模上的使用。
发明内容
本发明人在本发明的情形中并且首次描述了一种遗传工具，其允许优化梭状芽孢杆菌属(Clostridium)的细菌的转化和通过同源重组进行的遗传修饰，和/或在野生型状态下在所述细菌的遗传物质中部分或完全不存在的DNA序列在所述细菌内的表达。这种工具的特征通常在于：
i)它包含：
-编码至少一种DNA核酸内切酶例如Cas9酶的“第一”核酸，其中编码所述DNA核酸内切酶的序列被置于启动子的控制之下，和
-至少一种含有修复模板的“第二”核酸，其允许通过同源重组机制将被所述核酸内切酶靶向的细菌DNA的一部分用感兴趣的序列代替，
ii)所述核酸中的至少一者还编码一个或多个指导RNA(gRNA)或者所述遗传工具还包含一个或多个指导RNA，每个指导RNA包含结合DNA核酸内切酶的RNA结构和与所述细菌DNA的被靶向部分互补的序列，和
iii)所述核酸中的至少一者还包含置于诱导型启动子的控制之下的编码抗CRISPR蛋白的序列，或者所述遗传工具还包含置于诱导型启动子的控制之下的编码抗CRISPR蛋白的“第三”核酸。
在这种改进的工具中，至少一种核酸包含置于诱导型启动子的控制之下的编码抗CRISPR蛋白的序列(“acr”)。这种抗CRISPR蛋白抑制所述DNA核酸内切酶/指导RNA复合体的活性。所述蛋白的表达受到调控，以允许它只在所述细菌的转化步骤期间表达。
与现有技术的工具相比，这种工具具有大幅促进梭状芽孢杆菌属(Clostridium)细菌的转化以及因此以大且有用的量在工业规模上生产感兴趣的遗传修饰细菌的优点。
本发明人还在本发明的情形中并首次描述了促进细菌的转化(通过改进引入的所有遗传物质在所述细菌内的维持)的核酸(在本文中也被鉴定为“OPT”核酸)。所述OPT核酸包含：i)序列SEQ ID NO：126的全部或一部分，和ii)允许修饰细菌的遗传物质和/或在所述细菌内表达在所述细菌的野生型版本内存在的遗传物质中部分或完全不存在的DNA序列的序列。序列SEQ ID NO：126在本文中也被鉴定为“OREP”核酸。
本发明人已经成功地提高了核酸在细菌拜氏梭状芽孢杆菌(C.beijerinckii)DSM6423内的转化频率，特别是通过缺失所述细菌内的OREP序列并通过有利地使用该OREP序列的全部或一部分来构建允许修饰细菌的遗传物质和/或在所述细菌内表达在所述细菌的野生型版本内存在的遗传物质中部分或完全不存在的DNA序列的核酸和/或遗传工具。
所述OREP序列包含编码参与感兴趣的OPT核酸的复制的蛋白质的核苷酸序列(SEQID NO：127)。这种参与复制的蛋白质在本文中也被鉴定为“REP”蛋白((SEQ ID NO：128–“MNNNNTESEELKEQSQLLLDKCTKKKKKNPKFSSYIEPLVSKKLSERIKECGDFLQMLSDLNLENSKLHRASFCGNRFCPMCSWRIACKDSLEISILMEHLRKEESKEFIFLTLTTPNVKGADLDNSIKAYNKAFKKLMERKEVKSIVKGYIRKLEVTYNLDKSSKSYNTYHPHFHVVLAVNRSYFKKQNLYINHHRWLSLWQESTGDYSITQVDVRKAKINDYKEVYELAKYSAKDSDYLINREVFTVFYKSLKGKQVLVFSGLFKDAHKMYKNGELDLYKKLDTIEYAYMVSYNWLKKKYDTSNIRELTEEEKQKFNKNLIEDVDIE”)。在厚壁菌门中所述REP蛋白具有序列SEQ ID NO：129的被称为“COG 5655”的保守结构域(质粒滚环复制起始蛋白REP)。
因此，具体来说，本发明描述了一种遗传工具，其至少包含：
-编码至少一种DNA核酸内切酶的“第一”核酸，其中编码所述DNA核酸内切酶的序列被置于启动子的控制之下，和
-包含“OREP核酸序列”或由“OREP核酸序列”构成的“其他”核酸，即所述其他核酸包含下述序列或由下述序列构成：i)序列SEQ ID NO：126的全部或一部分，和ii)允许修饰细菌的遗传物质和/或在所述细菌内表达在所述细菌的野生型版本内存在的遗传物质中部分或完全不存在的DNA序列的序列。
在特定实施方式中，如上所述的“含有修复模板的第二核酸”包含这种“其他”核酸。
还描述了一种用于转化并通常用于例如通过同源重组遗传修饰梭状芽孢杆菌属(Clostridium)的细菌、通常为梭状芽孢杆菌属(Clostridium)的产溶剂细菌的方法，以及通过这种方法获得的(被转化和通常被遗传修饰的)细菌。这种方法包括下述步骤：
a)在所述抗CRISPR蛋白的表达诱导物存在下，在所述细菌内引入本发明的遗传工具，以及
b)将在步骤a)结束时获得的被转化的细菌在不含所述抗CRISPR蛋白的表达诱导物并通常允许所述DNA核酸内切酶/gRNA核糖核蛋白复合体表达的培养基上培养。
有利情况下，这种方法的实例包括通过在所述细菌中引入本文中所描述的遗传工具的全部或一部分、特别是核酸(“OPT核酸”)来转化所述细菌的步骤，所述OPT核酸包含下述序列或由下述序列构成：i)序列SEQ ID NO：126的全部或一部分，和ii)允许修饰细菌的遗传物质和/或在所述细菌内表达在所述细菌的野生型版本内存在的遗传物质中部分或完全不存在的DNA序列的序列。
本发明人还描述了一种试剂盒，其用于转化并优选地遗传修饰梭状芽孢杆菌属(Clostridium)的细菌或使用梭状芽孢杆菌属(Clostridium)的细菌生产至少一种溶剂例如溶剂的混合物。这种试剂盒包含本文中所描述的核酸或本文中所描述的遗传工具的元件，和适用于所述工具中使用的所选抗CRISPR蛋白的表达的诱导型启动子的至少一种诱导物。在特定实施方式中，所述试剂盒包含本文中所描述的遗传工具的全部或一些元件。
还描述了在本文中首次公开的核酸或遗传工具的用途，其用于转化并任选地遗传修饰梭状芽孢杆菌属(Clostridium)的细菌，例如在野生型状态下具有细菌染色体和不同于染色体DNA的至少一种DNA分子(通常为天然质粒)两者的梭状芽孢杆菌属(Clostridium)的细菌。
还描述了在本文中首次公开的核酸或遗传工具、用于转化并优选地通常通过同源重组遗传修饰梭状芽孢杆菌属(Clostridium)的细菌的方法、通过所述方法获得的细菌和/或试剂盒的用途，其能够用于优选地在工业规模上生产溶剂或溶剂的混合物，优选为丙酮、丁醇、乙醇、异丙醇或其混合物，通常为异丙醇/丁醇、丁醇/乙醇或异丙醇/乙醇混合物。
附图说明
下面的附图旨在更充分地说明本发明而不是限制它的范围。
图1：作为遗传工具用于基因组编辑的CRISPR/Cas9系统，其使用Cas9核酸酶在被gRNA指导的基因组DNA中产生一个或多个双链切口。
gRNA，指导RNA；PAM，前间区序列邻近基序。图从Jinek等，2012修改。
图2：通过同源重组修复Cas9诱导的双链切口。PAM，前间区序列邻近基序。
图3：CRISPR/Cas9在梭状芽孢杆菌属(Clostridium)中的使用。
ermB，红霉素抗性基因；catP(SEQ ID NO：70)，甲砜霉素/氯霉素抗性基因；tetR，其表达产物阻遏从Pcm–tetO2/1的转录的基因；Pcm–2tetO1和Pcm–tetO2/1，脱水四环素(aTc)诱导型启动子(Dong等，2012)；miniPthl，组成型启动子(Dong等，2012)。
图4：pCas9acr质粒图谱(SEQ ID NO：23)。
ermB，红霉素抗性基因；rep，大肠杆菌(E.coli)中的复制起点；repH，丙酮丁醇梭状芽孢杆菌(C.acetobutylicum)中的复制起点；Tthl，硫解酶终止子；miniPthl，组成型启动子(Dong等，2012)；Pcm–tetO2/1，被tetR的产物阻遏并且可以被脱水四环素(aTc)诱导的启动子(Dong等，2012)；Pbgal，被lacR的产物阻遏并且可以被乳糖诱导的启动子(Hartman等，2011)；acrIIA4，编码抗CRISPR蛋白AcrII14的基因；bgaR，其表达产物阻遏从Pbgal的转录的基因。
图5：含有pCas9ind(SEQ ID NO：22)或pCas9acr(SEQ ID NO：23)的丙酮丁醇梭状芽孢杆菌(C.acetobutylicum)DSM 792的相对转化率。频率被表示为在转化中使用的每μgDNA获得的转化体的数目，相对于pEC750C(SEQ ID NO：106)的转化频率，并代表了至少两个独立实验的平均值。
图6：在含有pCas9acr和用于靶向bdhB的gRNA的表达质粒的具有(SEQ ID NO：79和SEQ ID NO：80)或没有(SEQ ID NO：105)修复模板的菌株DSM 792转化体中CRISPR/Cas9系统的诱导。Em，红霉素；Tm，甲砜霉素；aTc，脱水四环素；ND，未稀释。
图7：通过CRISPR/DNA核酸内切酶系统修饰丙酮丁醇梭状芽孢杆菌(C.acetobutylicum)DSM792的bdh基因座。
A，bdh基因座的遗传组织。修复模板与基因组DNA之间的同源性用浅灰色平行四边形标出。还示出了引物V1和V2的杂交位点。
B，使用引物V1和V2扩增bdh基因座。M，2–log大小标志物(NEB)；P，pGRNA–ΔbdhAΔbdhB质粒；WT，野生型菌株。
图8：30种产溶剂梭状芽孢杆菌属(Clostridium)菌株根据Poehlein等，2017的分类。注意子进化枝拜氏梭状芽孢杆菌(C.beijerinckii)NRRL B–593在文献中也被鉴定为拜氏梭状芽孢杆菌(C.beijerinckii)DSM 6423。
图9：pCas9ind–ΔcatB质粒图谱。
图10：pCas9acr质粒图谱。
图11：pEC750S–uppHR质粒图谱。
图12：pEX–A2–gRNA–upp质粒图谱。
图13：pEC750S–Δupp质粒图谱。
图14：pEC750C–Δupp质粒图谱。
图15：pGRNA–pNF2图谱。
图16：在源自于拜氏梭状芽孢杆菌(C.beijerinckii)菌株DSM 6423的细菌转化的克隆中catB基因的PCR扩增。
如果所述菌株仍具有catB基因则扩增出约1.5kb，或者如果该基因缺失则扩增出约900bp。
图17：拜氏梭状芽孢杆菌(C.beijerinckii)菌株DSM 6423 WT和ΔcatB在2YTG培养基和2YTG甲砜霉素选择性培养基上的生长。
图18：在含有pCas9acr和用于靶向upp的gRNA的表达质粒的具有或没有修复基质的拜氏梭状芽孢杆菌(C.beijerinckii)菌株DSM 6423的转化体中CRISPR/Cas9acr系统的诱导。图例说明：Em，红霉素；Tm，甲砜霉素；aTc，脱水四环素；ND，未稀释。
图19：图19A示出了通过CRISPR/Cas9系统修饰拜氏梭状芽孢杆菌(C.beijerinckii)DSM 6423的upp基因座。图19A示出了upp基因座的遗传组织：基因，gRNA靶位点和修复基质，伴有在基因组DNA上的对应同源区域。用于PCR验证的引物(RH010和RH011)的杂交位点也被标出。
图19B示出了通过CRISPR/Cas9系统修饰拜氏梭状芽孢杆菌(C.beijerinckii)DSM6423的upp基因座。图19B示出了使用引物RH010和RH011扩增upp基因座。在野生型基因的情况下预期扩增出1680bp，与此相比修饰的upp基因为1090bp。M，100bp–3kb大小标志物(Lonza)；WT，野生型菌株。
图20：验证拜氏梭状芽孢杆菌(C.beijerinckii)菌株6423ΔcatB中存在质粒pCas9ind的PCR扩增。
图21：在诱导之前(阳性对照1和2)，然后在含有aTc的培养基上从CRISPR–Cas9系统诱导后，验证天然pNF2质粒的存在或不存在的PCR扩增(≈900bp)。
图22：基于两种质粒的使用、适用于梭状芽孢杆菌属(Clostridium)细菌的用于修饰细菌的遗传工具(参考WO2017/064439，Wasels等，2017)。
图23：pCas9ind–gRNA_catB质粒图谱。
图24：在拜氏梭状芽孢杆菌(C.beijerinckii)菌株DSM6423中，20μg pCas9ind质粒的转化效率(以每μg转化的DNA观察到的菌落数为单位)。误差条表示三份生物学平行样的平均值的标准误差。
图25：pNF3质粒图谱。
图26：pEC751S质粒图谱。
图27：pNF3S质粒图谱。
图28：pNF3E质粒图谱。
图29：pNF3C质粒图谱。
图30：在拜氏梭状芽孢杆菌(C.beijerinckii)DSM 6423的三株菌株中，质粒pCas9ind的转化效率(以每μg转化的DNA观察到的菌落数为单位)。误差条对应于生物学平行样的平均值的标准偏差。
图31：在源自于拜氏梭状芽孢杆菌(C.beijerinckii)DSM 6423的两株菌株中，质粒pEC750C的转化效率(以每μg转化的DNA观察到的菌落数为单位)。误差条对应于生物学平行样的平均值的标准偏差。
图32：在拜氏梭状芽孢杆菌(C.beijerinckii)菌株DSM 6423ΔcatBΔpNF2中，质粒pEC750C、pNF3C、pFW01和pNF3E的转化效率(以每μg转化的DNA观察到的菌落数为单位)。误差条对应于三份生物学平行样的平均值的标准偏差。
图33：在拜氏梭状芽孢杆菌(C.beijerinckii)菌株NCIMB 8052中，质粒pFW01、pNF3E和pNF3S的转化效率(以每μg转化的DNA观察到的菌落数为单位)。
具体实施方式
尽管在工业中使用已超过一个世纪，但对产溶剂细菌、特别是属于梭状芽孢杆菌属(Clostridium)的产溶剂细菌的了解仍受到在它们的遗传修饰中遇到的困难的限制。例如，通常在基因组中具有编码将丙酮还原成异丙醇的伯醇/叔醇脱氢酶的adh基因的天然生产异丙醇的梭状芽孢杆菌属(Clostridium)的细菌，在遗传和功能两方面不同于能够在天然状态下进行ABE发酵的细菌。
本文中描述的遗传工具具有大幅促进用感兴趣的序列转化梭状芽孢杆菌属(Clostridium)的细菌以便改进其性质的优点。
这种工具的特征通常在于：
i)它包含：
-编码至少一种DNA核酸内切酶例如Cas9酶的“第一”核酸，其中编码所述DNA核酸内切酶的序列被置于启动子的控制之下，和
-至少一种含有修复模板的“第二”核酸，其允许通过同源重组机制将被所述核酸内切酶靶向的细菌DNA的一部分用感兴趣的序列代替，
ii)所述核酸中的至少一者还编码一个或多个指导RNA(gRNA)或者所述遗传工具还包含一个或多个指导RNA，每个指导RNA包含结合DNA核酸内切酶的RNA结构和与所述细菌DNA的被靶向部分互补的序列，和
iii)所述核酸中的至少一者还包含置于诱导型启动子的控制之下的编码抗CRISPR蛋白的序列，或者所述遗传工具还包含置于诱导型启动子的控制之下的编码抗CRISPR蛋白的“第三”核酸。
这种工具允许插入核酸序列的大片段。
本发明人描述的工具可用于转化和/或遗传修饰感兴趣的细菌，通常为本文中描述的属于梭状芽孢杆菌属(Clostridium)的细菌，优选为天然地能够(即在野生型状态下能够)生产异丙醇、特别是天然地能够进行IBE发酵的梭状芽孢杆菌属(Clostridium)的细菌，优选为天然地对一种或多种抗生素具有抗性的细菌，例如拜氏梭状芽孢杆菌(C.beijerinckii)。优选的细菌在野生型状态下具有细菌染色体和不同于染色体DNA的至少一种DNA分子两者。
表述“梭状芽孢杆菌属(Clostridium)的细菌”具体来说是指具有所谓的工业兴趣的梭状芽孢杆菌属(Clostridium)物种，通常为梭状芽孢杆菌属(Clostridium)的产溶剂或产乙酸细菌。表述“梭状芽孢杆菌属(Clostridium)的细菌”包含野生型细菌以及从其衍生的通过遗传修饰以改进其性能(例如过表达ctfA、ctfB和adc基因)的尚未暴露于CRISPR系统的菌株。
表述“具有工业兴趣的梭状芽孢杆菌属(Clostridium)物种”是指能够通过发酵从糖或单糖，通常为五碳糖例如木糖、阿拉伯糖或果糖，六碳糖例如葡萄糖和甘露糖，多糖例如纤维素或半纤维素，和/或可以被梭状芽孢杆菌属(Clostridium)的细菌同化并利用的任何其他碳源(例如CO、CO2和甲醇)生产溶剂和酸例如丁酸或乙酸的梭状芽孢杆菌属物种。感兴趣的产溶剂细菌的实例是生产丙酮、丁醇、乙醇和/或异丙醇的梭状芽孢杆菌属(Clostridium)细菌，例如在文献中被鉴定为“ABE菌株”[通过发酵生产丙酮、丁醇和乙醇的菌株]和“IBE”菌株[通过发酵生产异丙醇(通过还原丙酮)、丁醇和乙醇的菌株]的菌株。梭状芽孢杆菌属(Clostridium)的产溶剂细菌可以选自丙酮丁醇梭状芽孢杆菌(C.acetobutylicum)、解纤维梭状芽孢杆菌(C.cellulolyticum)、植物发酵梭状芽孢杆菌(C.phytofermentans)、拜氏梭状芽孢杆菌(C.beijerinckii)、糖丁酸梭状芽孢杆菌(C.saccharobutylicum)、C.saccharoperbutylacetonicum、产芽孢梭状芽孢杆菌(C.sporogenes)、丁酸梭状芽孢杆菌(C.butyricum)、金黄丁酸梭状芽孢杆菌(C.aurantibutyricum)和酪丁酸梭状芽孢杆菌(C.tyrobutyricum)，优选地选自丙酮丁醇梭状芽孢杆菌(C.acetobutylicum)、拜氏梭状芽孢杆菌(C.beijerinckii)、丁酸梭状芽孢杆菌(C.butyricum)、酪丁酸梭状芽孢杆菌(C.tyrobutyricum)和解纤维梭状芽孢杆菌(C.cellulolyticum)，甚至更优选地选自丙酮丁醇梭状芽孢杆菌(C.acetobutylicum)和拜氏梭状芽孢杆菌(C.beijerinckii)。
能够在野生型状态下生产异丙醇、特别是能够在野生型状态下IBE发酵的细菌可以是例如选自拜氏梭状芽孢杆菌(C.beijerinckii)、二醇梭状芽孢杆菌(C.diolis)、C.puniceum、丁酸梭状芽孢杆菌(C.butyricum)、C.saccharoperbutylacetonicum、肉毒梭状芽孢杆菌(C.botulinum)、德雷克氏梭状芽孢杆菌(C.drakei)、粪味梭状芽孢杆菌(C.scatologenes)、产气荚膜梭状芽孢杆菌(C.perfringens)和突尼斯梭状芽孢杆菌(C.tunisiense)的细菌，优选为选自拜氏梭状芽孢杆菌(C.beijerinckii)、二醇梭状芽孢杆菌(C.diolis)、C.puniceum和C.saccharoperbutylacetonicum的细菌。天然地能够生产异丙醇、特别是能够在野生型状态下IBE发酵的细菌，特别优选的是拜氏梭状芽孢杆菌(C.beijerinckii)。
感兴趣的产乙酸细菌是从CO2和H2生产酸和/或溶剂的细菌。梭状芽孢杆菌属(Clostridium)的产乙酸细菌可以例如选自乙酸梭状芽孢杆菌(C.aceticum)、嗜热乙酸梭状芽孢杆菌(C.thermoaceticum)、杨氏梭状芽孢杆菌(C.ljungdahlii)、自产乙醇梭状芽孢杆菌(C.autoethanogenum)、难辨梭状芽孢杆菌(C.difficile)、粪味梭状芽孢杆菌(C.scatologenes)和C.carboxydivorans。
在特定实施方式中，所考虑的梭状芽孢杆菌属(Clostridium)的细菌是“ABE菌株”，优选为丙酮丁醇梭状芽孢杆菌(C.acetobutylicum)菌株DSM 792(也被称为ATCC 824或LMG 5710)或拜氏梭状芽孢杆菌(C.beijerinckii)菌株NCIMB 8052。
在另一个特定实施方式中，所考虑的梭状芽孢杆菌属(Clostridium)的细菌是“IBE菌株”，优选为选自DSM 6423、LMG7814、LMG 7815、NRRL B–593、NCCB 27006的拜氏梭状芽孢杆菌(C.beijerinckii)的子进化枝或金黄丁酸梭状芽孢杆菌(C.aurantibutyricum)DSZM 793(Georges等，1983)，以及这些拜氏梭状芽孢杆菌(C.beijerinckii)或金黄丁酸梭状芽孢杆菌(C.aurantibutyricum)细菌的与菌株DSM 6423具有至少90％、95％、96％、97％、98％或99％同一性的子进化枝。特别优选的拜氏梭状芽孢杆菌(C.beijerinckii)细菌或拜氏梭状芽孢杆菌(C.beijerinckii)细菌的子进化枝不含质粒pNF2。
一方面子进化枝LMG 7814、LMG 7815、NRRL B–593和NCCB27006和另一方面DSZM793的相应的基因组，与子进化枝DSM 6423的基因组具有至少97％的序列同一性百分数。
本发明人进行了发酵试验，证实了子进化枝DSM 6423、LMG 7815和NCCB 27006的拜氏梭状芽孢杆菌(C.beijerinckii)细菌能够在野生型状态下生产异丙醇(参见表1)。
[表1]
使用天然生产异丙醇的拜氏梭状芽孢杆菌(C.beijerinckii)菌株DSM 6423、LMG7815和NCCB 27006的葡萄糖发酵试验的概述。在本发明的特别优选实施方式中，所述拜氏梭状芽孢杆菌(C.beijerinckii)细菌是子进化枝DSM 6423的细菌。
在本发明的另一个优选实施方式中，所述拜氏梭状芽孢杆菌(C.beijerinckii)细菌是拜氏梭状芽孢杆菌(C.beijerinckii)菌株IFP963ΔcatBΔpNF2(在2019年2月20日登记在BCCM–LMG保藏中心的保藏号LMG P–31277下)。
所述CRISPR/DNA核酸内切酶系统含有两个不同的必需组分，即i)核酸内切酶，在本发明的情况下是与CRISPR系统相关的核酸酶(Cas或“CRISPR相关蛋白”)，通常为Cas9，和ii)指导RNA。指导RNA是由细菌的CRISPR RNA(crRNA)和tracrRNA(反式激活CRISPR RNA)的组合构成的嵌合RNA(Jinek等，Science 2012)。所述gRNA在单一转录本中组合了crRNA的靶向特异性和所述tracrRNA的构象性质，所述crRNA对应于指导Cas蛋白的“间隔物序列”。当所述gRNA和Cas蛋白在细胞中同时表达时，可以使用提供的修复模板将所述靶基因组序列永久修饰。
在最近的实验中，本发明人成功地转化并遗传修饰了天然生产异丙醇的梭状芽孢杆菌属(Clostridium)的细菌即细菌拜氏梭状芽孢杆菌(C.beijerinckii)DSM 6423以及参比菌株丙酮丁醇梭状芽孢杆菌(C.acetobutylicum)DSM 792。
在实验部分中描述的某些工作在能够IBE发酵的菌株即拜氏梭状芽孢杆菌(C.beijerinckii)菌株DSM 6423内进行，所述菌株的基因组和转录组分析最近已被本发明人描述(Mátéde Gerando等，2018)。
在这个菌株的基因组的组装期间，本发明人发现除了染色体之外还存在可移动遗传元件(登记号PRJEB11626–https://www.ebi.ac.uk/ena/data/view/PRJEB11626)：两个天然质粒(pNF1和pNF2)和一个线性噬菌体(Φ6423)。
所述拜氏梭状芽孢杆菌(C.beijerinckii)菌株DSM 6423是天然对红霉素敏感但对甲砜霉素有抗性的。专利申请号FR18/73492描述了一种特定菌株拜氏梭状芽孢杆菌(C.beijerinckii)菌株DSM 6423ΔcatB，其被制造成甲砜霉素敏感的。在本发明的特定实施方式中，本发明人成功地从拜氏梭状芽孢杆菌(C.beijerinckii)菌株DSM 6423移除了它的天然pNF2质粒并获得了拜氏梭状芽孢杆菌(C.beijerinckii)菌株DSM6423ΔcatBΔpNF2。这个菌株在2019年2月20日在BCCM–LMG保藏中心登记在保藏号LMG P–31277下。本说明书还涉及其任何衍生的细菌、克隆、突变体或遗传修饰的版本。它也更广义地涉及在野生型状态下具有细菌染色体和不同于染色体DNA的至少一种DNA分子(在本文中被鉴定为“非染色体(细菌)DNA”或“天然(细菌)质粒”)的任何细菌，其使用本文中描述的核酸和/或遗传工具进行遗传修饰，使得不再包括它的非染色体DNA分子中的至少一种，通常为它的非染色体DNA分子中的几种(例如2、3、或4种非染色体DNA分子)，优选地所有非染色体DNA分子。
本发明人观察到所述天然pNF2质粒的移除对于另外的天然或合成的遗传元件(例如表达盒或表达质粒载体)的引入和维持具有显著优点。因此，菌株DSM 6423ΔcatBΔpNF2可以以比其野生型对应物或菌株DSM 6423ΔcatB高10至5x 103倍的效率转化。
因此，本发明人在本申请中描述了一种天然地能够(即在野生型状态下能够)生产异丙醇、特别是天然地能够IBE发酵的梭状芽孢杆菌属(Clostridium)的细菌，其已被遗传修饰，并且作为这种遗传修饰的结果，已经具体来说失去了至少一种天然质粒(即天然存在于所述细菌的野生型版本中的质粒)，优选地所有天然质粒，还描述了用于获得所述细菌的工具，特别是遗传工具。
这些工具具有大幅促进细菌的转化和遗传修饰的优点。由本发明人进行的实验证实了所述工具和更通常地本文中描述的用于遗传修饰梭状芽孢杆菌属(Clostridium)的细菌的技术的可能用途，所述梭状芽孢杆菌属(Clostridium)的细菌特别是能够在野生型状态下生产异丙醇、特别是进行IBE发酵的梭状芽孢杆菌属(Clostridium)的细菌，特别是带有编码负责对抗生素的抗性的酶的基因、特别是编码酰胺醇–O–乙酰基转移酶(amphenicol–O–acetyltransferase)例如氯霉素–O–乙酰基转移酶或甲砜霉素–O–乙酰基转移酶的基因的梭状芽孢杆菌属(Clostridium)的细菌。
因此，在特定实施方式中，本发明人成功地使天然携带(在野生型状态下携带)编码负责对酰胺醇(amphenicol)类抗生素的抗性的酶的基因的细菌对这些抗生素敏感。
其他优选的细菌在野生型状态下含有细菌染色体和不同于染色体DNA的至少一种DNA分子两者。
此外，优选的细菌在野生型状态下含有细菌染色体和不同于染色体DNA的至少一种DNA分子两者，以及赋予对抗生素的抗性的基因。在特定实施方式中，这个基因编码酰胺醇–O–乙酰基转移酶，例如氯霉素–O–乙酰基转移酶或甲砜霉素–O–乙酰基转移酶。
打算被转化并优选地遗传修饰的具体细菌优选为已暴露于使用本发明的核酸或遗传工具的第一转化步骤和第一遗传修饰步骤的细菌，所述暴露使得可以移除天然存在于野生型状态下的所述细菌内的至少一种染色体外DNA分子(通常为至少一种质粒)。
本发明人所描述的目的涉及一种核酸(在本文中被鉴定为“OPT”核酸)，其可以有利地用于通过提高所有引入的遗传物质在所述细菌内的维持来促进细菌的转化。这种OPT核酸包含i)序列SEQ ID NO：126(“OREP”序列)或其功能性变体的全部或一部分，和ii)允许修饰细菌的遗传物质和/或在所述细菌内表达在所述细菌的野生型版本内存在的遗传物质中部分或完全不存在的DNA序列的序列(在本文中也被鉴定为“感兴趣的序列”)。
所述OREP序列(SEQ ID NO：126)包含序列SEQ ID NO：127的核苷酸序列。序列SEQID NO：127优选地包含编码参与所述OPT核酸的复制的蛋白质的序列。被认为参与复制的蛋白质在本文中也被鉴定为“REP”蛋白(SEQ ID NO：128)。在厚壁菌门中所述REP蛋白具有序列SEQ ID NO：129的被称为“COG 5655”的保守结构域。
在特定实施方式中，所述OPT核酸包含所述OREP序列(SEQ ID NO：126)的一部分，通常为所述OREP序列的一个或多个片段，优选地至少包含编码所述REP蛋白(SEQ ID NO：128)或其功能性变体或片段(即参与复制的片段)的序列，通常为序列SEQ ID NO：127或其变体或编码在所述REP蛋白内参与OPT核酸的复制的片段的片段。所述OREP序列的编码所述REP蛋白内存在的参与OPT核酸的复制的片段的功能性片段，包含序列SEQ ID NO：129的结构域。这些编码所述REP蛋白的功能性片段及其变体的核酸片段的实例，可以由专业技术人员容易地制备。变体的典型实例与序列SEQ ID NO：127具有70％至100％之间、优选地85至99％之间、更优选地95至99％之间例如70、71、72、73、74、75、76,77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99或100％的序列同源性。
在优选实施方式中，所述OREP序列的功能性变体或片段编码参与所述OPT核酸的复制的蛋白质。
在本发明的优选实施方式中，所述OREP序列的功能性变体或片段除了包含编码参与所述OPT核酸(例如质粒样遗传构建物)的复制的蛋白质(例如REP蛋白)或其功能性变体或片段的序列之外，还包含1至150个碱基、优选地1至15个碱基的位点，例如富含A和T碱基的序列(Rajewska等)，优选为序列SEQ ID NO：118的pNF2质粒内存在的位点，所述位点允许容许所述OPT核酸复制的蛋白质的结合。
所述允许修饰细菌的遗传物质的感兴趣的序列通常为修饰模板，其允许例如通过同源重组机制将所述细菌的遗传物质的一部分用感兴趣的序列代替。所述允许修饰细菌的遗传物质的感兴趣的序列也可以是识别序列(至少部分结合)，并优选地在感兴趣的细菌的基因组中靶向、即识别并允许切割下述序列的至少一条链：i)靶序列，ii)控制靶序列的转录的序列，或iii)靶序列侧翼的序列。
所述允许在所述细菌内表达在所述细菌的野生型版本内存在的遗传物质中部分或完全不存在的DNA序列的感兴趣的序列，通常允许所述细菌表达在野生型状态下不能表达或不能以足够的量表达的一种或多种蛋白质。
根据特定情况，所述“OPT核酸”还包含iii)编码DNA核酸内切酶例如Cas9的序列，和/或iv)一个或多个指导RNA(gRNA)，每个gRNA包含结合DNA核酸内切酶的RNA结构和与所述细菌遗传物质的被靶向部分互补的序列。
根据另一种特定情况，所述“OPT核酸”在被Dam和Dcm甲基转移酶识别的基序处不具有甲基化。
优选地，所述“OPT核酸”选自表达盒和载体，优选为质粒，例如具有选自SEQ IDNO：119、SEQ ID NO：123、SEQ ID NO：124和SEQ ID NO：125的序列的质粒。
因此，具体描述了一种遗传工具，其至少包含：
-编码至少一种DNA核酸内切酶的第一核酸，其中编码所述DNA核酸内切酶的序列被置于启动子的控制之下，和
-另一个核酸(或“第n核酸”)，其包含“OPT”核酸序列或由“OPT”核酸序列构成，所述“OPT”核酸序列即包含下述序列的序列：i)序列SEQ ID NO：126(“OREP”)的全部或一部分；和ii)允许修饰细菌的遗传物质和/或在所述细菌内表达在所述细菌的野生型版本内存在的遗传物质中部分或完全不存在的DNA序列的序列，这种特定遗传工具的所述核酸中的至少一者还包含置于诱导型启动子的控制之下的编码抗CRISPR蛋白的序列，或者所述特定遗传工具还优选地包含置于诱导型启动子的控制之下的编码抗CRISPR蛋白的第三核酸。
在特定实施方式中，如上所述的含有修复模板的“第二”或“第n”核酸包含该“其他核酸”或由该“其他核酸”构成。
在另一个特定实施方式中，所述“第一核酸”还编码一个或多个指导RNA(gRNA)。
在本发明的意义上，术语“核酸”是指任何天然、合成、半合成或重组的DNA或RNA分子，其任选地被化学修饰(即包含非天然碱基、具有例如修饰的键、修饰的碱基和/或修饰的糖的修饰的核苷酸)或优化，以使从所述编码序列合成的转录本的密码子是在梭状芽孢杆菌属(Clostridium)的细菌中最常发现的密码子，以在其中使用它。在梭状芽孢杆菌属(Clostridium)的情况下，所述优化的密码子通常是富含腺嘌呤(“A”)和胸腺嘧啶(“T”)碱基的密码子。
本发明的遗传工具包含编码至少一种核酸内切酶、通常为Cas核酸酶例如Cas9或MAD7的第一核酸。“Cas9”是指Cas9(也被称为CRISPR相关蛋白9、Csn1或Csx12)蛋白或其功能性蛋白、肽或多肽片段，即能够与指导RNA相互作用并执行允许它在靶基因组的DNA中制造双链切口的酶(核酸酶)活性。因此，“Cas9”可以是指修饰的蛋白质，例如被截短以除去对于所述蛋白质的预定功能来说非必需的蛋白质结构域，特别是对于与gRNA的相互作用来说非必需的结构域。
另外，MAD7核酸酶(其氨基酸序列对应于序列SEQ ID NO：72)，也被鉴定为“Cas12”或“Cpf1”，可以有利地通过将它与专业技术人员已知的能够与这种核酸酶结合的一个或多个gRNA相组合，用于本发明的情形中(参见Garcia–Doval等，2017和Stella S.等，2017)。
根据一种特定情况，所述编码MAD7核酸酶的序列是被优化以容易地在梭状芽孢杆菌属(Clostridium)菌株中表达的序列，优选为序列SEQ ID NO：71。
在本发明的情形中使用的编码Cas9(完整蛋白或其片段)的序列可以从任何已知的Cas9蛋白获得(Makarova等，2011)。可用于本发明的Cas9蛋白的实例包括但不限于来自于化脓链球菌(S.pyogenes)、嗜热链球菌(Streptococcus thermophilus)、变异链球菌(Streptococcus mutans)、空肠弯曲杆菌(Campylobacter jejuni)、多杀巴氏杆菌(Pasteurella multocida)、新凶手弗朗西斯菌(Francisella novicida)、脑膜炎奈瑟氏菌(Neisseria meningitidis)、乳糖奈瑟氏菌(Neisseria lactamica)和嗜肺军团菌(Legionella pneumophila)的Cas9蛋白(参见Fonfara等，2013；Makarova等，2015)。
在特定实施方式中，由本发明的遗传工具的核酸之一编码的Cas9蛋白或其功能性蛋白、肽或多肽片段包含下述序列或由下述序列构成：氨基酸序列SEQ ID NO：75，或与其具有至少50％、优选地至少60％的同一性并至少含有占据氨基酸序列SEQ ID NO：75的10(“D10”)和840(“D840”)位置的两个天冬氨酸(“D”)的任何其他氨基酸序列。
在优选实施方式中，Cas9包含下述序列或由下述序列构成：由来自于化脓链球菌(S.pyogenes)菌株M1 GAS的cas9基因(NCBI登记号：NC_002737.2 SPy_1046，SEQ ID NO：76)或其已经历优化的版本(“优化的版本”)编码的Cas9蛋白(NCBI登记号：WP_010922251.1，SEQ ID NO：75)，所述优化产生含有被梭状芽孢杆菌属(Clostridium)的细菌优先使用的密码子、通常为富含腺嘌呤(“A”)和胸腺嘧啶(“T”)碱基的密码子的转录本，允许促进所述Cas9蛋白在这种细菌属中表达。这些优化的密码子遵守专业技术人员公知的特异性针对每种细菌菌株的密码子使用偏好性。
在本文件中描述的肽序列中，氨基酸根据下述命名法用它们的单字母密码表示：C：半胱氨酸；D：天冬氨酸；E：谷氨酸；F：苯丙氨酸；G：甘氨酸；H：组氨酸；I：异亮氨酸；K：赖氨酸；L：亮氨酸；M：甲硫氨酸；N：天冬酰胺；P：脯氨酸；Q：谷氨酰胺；R：精氨酸；S：丝氨酸；T：苏氨酸；V：缬氨酸；W：色氨酸和Y：酪氨酸。
根据特定实施方式，所述Cas9结构域由完整的Cas9蛋白、优选为化脓链球菌(S.pyogenes)的Cas9蛋白或其优化的版本构成。
存在于本发明的遗传工具的核酸之一内的编码DNA核酸内切酶例如Cas9的序列被置于启动子的控制之下。这种启动子可以是组成型启动子或诱导型启动子。在优选实施方式中，所述控制Cas9表达的启动子是诱导型启动子。
可用于本发明的组成型启动子的实例可以选自thl基因、ptb基因、adc基因、BCS操纵子的启动子或其衍生物，优选为有功能但更短(截短的)衍生物例如来自于丙酮丁醇梭状芽孢杆菌(C.acetobutylicum)的thl基因启动子的“miniPthl”衍生物(Dong等，2012)，或专业技术人员公知的允许蛋白质在梭状芽孢杆菌属(Clostridium)的细菌内表达的任何其他启动子。
可以在本发明的情形中使用的诱导型启动子的实例可以例如选自表达受到转录阻遏物TetR控制的启动子例如tetA基因(最初存在于大肠杆菌转座子Tn10上的四环素抗性基因)的启动子；表达受到L-阿拉伯糖控制的启动子例如ptk基因启动子(Zhang等，2015)，其优选地与丙酮丁醇梭状芽孢杆菌(C.acetobutylicum)的araR调控物表达盒相组合以便构建ARAi系统(Zhang等，2015)；表达受到昆布二糖(β–1,3葡萄糖二聚体)控制的启动子，例如celC基因启动子，优选地立即跟有阻遏基因glyR3和感兴趣的基因(Mearls等，2015)或celC基因启动子(Newcomb等，2011)；表达受到乳糖控制的启动子，例如bgaL基因启动子(Banerjee等，2014)；表达受到木糖控制的启动子，例如xylB基因启动子(Nariya等，2011)；和表达受到UV暴露控制的启动子，例如bcn基因启动子(Dupuy等，2005)。
衍生自上述启动子之一的启动子，优选为有功能但较短(截短)的衍生物，也可用于本发明的情形中。
可用于本发明的情形中的其他诱导型启动子也被描述在例如Ransom等(2015)、Currie等(2013)和Hartman等(2011)的文章中。
优选的诱导型启动子是源自于tetA的脱水四环素(aTc)诱导型启动子(aTc毒性比四环素更低并且能够在较低浓度下释放转录阻遏物TetR的抑制)，其选自Pcm–2tetO1和Pcm–2tetO2/1(Dong等，2012)。
另一种优选的诱导型启动子是木糖诱导型启动子，例如来自于难辨梭状芽孢杆菌(Clostridium difficile)630的xylB启动子(Nariya等，2011)。
另一种优选的诱导型启动子是乳糖诱导型启动子，例如bgaL基因的启动子(Banerjee等，2014)。
特定的感兴趣的核酸、通常为表达盒或载体，包含一个或多个表达盒，每个表达盒编码gRNA(指导RNA)。
在本发明的意义内，术语“指导RNA”或“gRNA”是指能够与DNA核酸内切酶例如Cas9相互作用以便将它导向细菌染色体的靶区域的RNA分子。切割的特异性由所述gRNA决定。正如上文解释的，每个gRNA包含两个区域：
–在gRNA的5'末端的第一区域(通常被称为“SDS”区域)，其与靶染色体区域互补并模拟内源CRISPR系统的crRNA，和
–在gRNA的3'末端的第二区域(通常被称为“手柄”区域)，其模拟tracrRNA(反式作用crRNA)与内源CRISPR系统的crRNA之间的碱基配对相互作用，并具有在3'方向上以基本上单链的序列结束的双链茎环结构。该第二区域对于所述gRNA与DNA核酸内切酶的结合来说是必不可少的。
所述gRNA的第一区域(“SDS”区域)随着被靶向的染色体序列而变。
所述gRNA的与靶染色体区域互补的“SDS”区域包含至少1个核苷酸，优选地至少1、2、3、4、5、10、15、20、25、30、35或40个核苷酸，通常为1至40个核苷酸。优选地，这个区域具有20、21、22、23、24、25、26、27、28、29或30个核苷酸的长度。
所述gRNA的第二区域(“手柄”区域)具有茎环(或发夹)结构。不同gRNA的手柄区域不依赖于所选的染色体靶。
根据特定实施方式，所述“手柄”区域包含至少1个核苷酸、优选地至少1、50、100、200、500和1000个核苷酸、通常1至1000个核苷酸的序列，或由所述序列构成。优选地，这个区域具有40至120个核苷酸的长度。
gRNA的总长度通常为50至1000个核苷酸，优选为80至200个核苷酸，更特别优选为90至120个核苷酸。根据特定实施方式，在本发明中使用的gRNA具有95至110个核苷酸的长度，例如约100或约110个核苷酸的长度。
专业技术人员可以使用公知的技术，根据待靶向的染色体区域容易地确定gRNA的序列和结构(参见例如DiCarlo等，2013的文章)。
细菌染色体内的被靶向DNA区域/部分/序列可以对应于非编码DNA的一部分或编码DNA的一部分。
在修饰给定序列的特定实施方式中，所述细菌DNA的被靶向部分对于细菌存活来说是必需的。例如，它对应于所述细菌染色体的任何区域或位于非染色体DNA上、例如在特定生长条件下微生物的存活所必需的可移动遗传元件上、例如在预期生长条件需要细菌在抗生素存在下生长时在含有所述抗生素抗性标志物的质粒上的任何区域。
在旨在移除在与所述微生物的培养相关的特定生长条件下非必需的遗传元件的另一个特定实施方式中，所述细菌DNA的被靶向部分可以对应于所述非染色体细菌DNA例如所述可移动遗传元件的任何区域。
梭状芽孢杆菌属(Clostridium)的细菌内被靶向部分的特定实例是在实验部分的实施例1中使用的序列。它们是例如编码bdhA(SEQ ID NO：77)和bdhB(SEQ ID NO：78)基因的序列。所述被靶向DNA区域/部分/序列后面跟有参与Cas9结合的前间区序列邻近基序(“PAM”)序列。
给定gRNA的“SDS”区域至少与所述细菌染色体内的被靶向DNA区域/部分/序列一致(100％同一性)或具有至少80％同一性，优选地85％、90％、95％、96％、97％、98％或99％同一性，并且能够与所述区域/部分/序列的互补序列的全部或一部分，通常与包含至少1个核苷酸、优选地至少1、2、3、4、5、10、15、20、25、30、35或40个核苷酸、通常在1至40个核苷酸之间的序列，优选为包含14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30个核苷酸的序列杂交。
在本发明的方法中，可以同时使用靶向序列(“靶序列”、“被靶向序列”或“被识别序列”)的一个或多个gRNA。这些不同的gRNA可以靶向染色体区域或可能存在于所述微生物内的可以相同或不同的属于非染色体细菌DNA的区域(例如可移动遗传元件)。
所述gRNA可以作为gRNA分子(成熟的或前体)、作为前体或作为编码所述gRNA的一个或多个核酸，引入到所述细菌细胞中。所述gRNA优选地作为编码所述gRNA的一个或多个核酸引入到所述细菌细胞中。
当所述一个或多个gRNA作为RNA分子直接引入到所述细胞中时，这些gRNA(成熟的或前体)可以含有修饰的核苷酸或化学修饰，其允许它们例如提高它们对核酸酶的抗性并因此提高它们在细胞中的寿命。具体来说，它们可以包含至少一个修饰的或非天然的核苷酸，例如具有修饰的碱基例如次黄嘌呤、甲基–5–脱氧胞苷、二甲基氨基–5–脱氧尿苷、脱氧尿苷、二氨基–2,6–嘌呤、溴–5–脱氧尿苷或允许杂交的任何其他修饰的碱基的核苷酸。按照本发明使用的gRNA也可以在核苷酸间连键的水平上修饰，例如硫代磷酸酯、H–膦酸酯或烷基–膦酸酯，或在骨架的水平上修饰，例如α–寡核苷酸、2′–O–烷基核糖或肽核酸(PNA)(Egholm等，1992)。
所述gRNA可以是天然RNA、合成的RNA或通过重组技术生产的RNA。这些gRNA可以通过专业技术人员已知的任何方法例如化学合成、体内转录或扩增技术来制备。
当所述gRNA作为一个或多个核酸引入到所述细菌细胞中时，编码所述gRNA的序列被置于表达启动子的控制之下。这个启动子可以是组成型或诱导型的。
当使用几个gRNA时，每个gRNA的表达可以由不同启动子控制。优选地，使用的启动子对于所有gRNA来说是相同的。在特定实施方式中，相同的启动子可用于允许打算表达的gRNA中的几个、例如仅仅几个或者换句话说全部或一部分的表达。
在优选实施方式中，控制所述gRNA表达的启动子是诱导型启动子。
可以在本发明的情形中使用的组成型启动子的实例可以选自thl基因、ptb基因或BCS操纵子的启动子或其衍生物，优选为miniPthl，或专业技术人员公知的允许在梭状芽孢杆菌属(Clostridium)内合成RNA(编码或非编码的)的任何其他启动子。
可以在本发明的情形中使用的诱导型启动子的实例可以选自tetA基因、xylA基因、lacI基因或bgaL基因的启动子或其衍生物，优选为2tetO1或tetO2/1。优选的诱导型启动子是2tetO1。
控制所述DNA核酸内切酶例如Cas9和所述gRNA的表达的启动子可以是相同或不同的，并且是组成型或诱导型的。在本发明的特定且优选的实施方式中，分别控制所述DNA核酸内切酶或gRNA的表达的启动子是不同的的启动子但可以被相同诱导物诱导。
如上所述的诱导型启动子使得可以有利地控制所述DNA核酸内切酶/gRNA核糖核蛋白复合体的作用并促进已经历所需遗传修饰的转化体的选择。
有利情况下，本发明的遗传工具包含编码至少一种抗CRISPR蛋白(在本文中也被鉴定为“抗CRISPR/DNA核酸内切酶蛋白”或“抗CRISPR/Cas9蛋白”)、即能够抑制或阻止/中和Cas的作用的蛋白质和/或能够抑制或阻止/中和CRISPR/Cas系统例如当所述核酸酶是Cas9核酸酶时的CRISPR/Cas II型系统的作用的蛋白质的序列。这个序列通常被置于与控制所述DNA核酸内切酶和/或gRNA的表达的启动子不同的诱导型启动子的控制之下，并且可以被另一种诱导物诱导。在优选实施方式中，所述编码抗CRISPR蛋白的序列通常也位于所述遗传工具中存在的至少两种核酸中的一者上。在特定实施方式中，所述编码抗CRISPR蛋白的序列位于不同于前两个核酸的核酸(通常为“第三核酸”)上。在另一个特定实施方式中，所述编码抗CRISPR蛋白的序列和编码所述抗CRISPR蛋白的转录阻遏物的序列两者被整合在细菌染色体中。
在优选实施方式中，所述编码抗CRISPR蛋白的序列被放置在所述遗传工具内编码所述DNA核酸内切酶的核酸(在本文中也被鉴定为“第一核酸”)上。在另一个实施方式中，所述编码抗CRISPR蛋白的序列被放置在所述遗传工具内与编码DNA核酸内切酶的核酸不同的核酸上，例如在本文中被鉴定为“第二核酸”的核酸上或在所述遗传工具中任选地包含的“第n”(通常为“第三”)核酸上。
所述抗CRISPR蛋白通常是“抗Cas9”蛋白，即能够抑制或阻止/中和Cas9的作用的蛋白质，和/或能够抑制或阻止/中和CRISPR/Cas9 II型系统的作用的蛋白质。
有利情况下，所述抗CRISPR蛋白是“抗Cas9”蛋白或“抗MAD7”蛋白，即能够抑制或阻止/中和Cas9或CAS7的作用的蛋白质。
有利情况下，所述抗CRISPR蛋白是“抗Cas9”蛋白，例如选自AcrIIA1、AcrIIA2、AcrIIA3、AcrIIA4、AcrIIA5、AcrIIC1、AcrIIC2和AcrIIC3(Pawluk等，2018)。优选地，所述“抗Cas9”蛋白是AcrIIA2或AcrIIA4。更优选地，所述“抗Cas9”蛋白是AcrIIA4。这种蛋白通常能够显著限制、理想情况下阻止Cas9的作用，例如通过结合到所述Cas9酶(Dong等，2017；Rauch等，2017)。
有利情况下，另一种有用的抗CRISPR蛋白是“抗MAD7”蛋白，例如AcrVA1(Marino等，2018)。
在优选实施方式中，所述抗CRISPR蛋白能够抑制、优选地中和所述DNA核酸内切酶的作用，优选地在将核酸序列从所述遗传工具引入到感兴趣的细菌菌株的阶段中。
控制编码所述抗CRISPR蛋白的序列的表达的启动子优选为诱导型启动子。所述诱导型启动子伴有组成性表达的基因，通常为负责允许从所述诱导型启动子转录阻遏的蛋白质的表达的基因。该启动子可以例如选自tetA基因、xylA基因、lacI基因或bgaL基因的启动子或其衍生物。
可以在本发明的情形中使用的诱导型启动子的实例是Pbgal启动子(乳糖诱导型)，其存在于所述遗传工具内，并与组成性表达并且其表达产物允许从Pbgal转录阻遏的bgaR基因一起存在于同一核酸上。在存在诱导物乳糖的情况下，所述Pbgal启动子的转录阻遏被释放，允许置于其下游的基因的转录。优选地，放置在下游的基因在本发明的情形中对应于编码抗CRISPR蛋白的基因例如acrIIA4。
所述控制抗CRISPR蛋白的表达的启动子使得可以有利地控制DNA核酸内切酶例如Cas9酶的作用，并因此促进梭状芽孢杆菌属(Clostridium)细菌的转化和已经历所需遗传修饰的转化体的生产。
在本发明的意义上，术语“核酸”是指任何天然、合成、半合成或重组的DNA或RNA分子，其任选地被化学修饰(即包含非天然碱基、具有例如修饰的键、修饰的碱基和/或修饰的糖的修饰的核苷酸)或优化，以使从所述编码序列合成的转录本的密码子是在梭状芽孢杆菌属(Clostridium)的细菌中最常发现的密码子，以便在其中使用它。正如在上面解释的，在梭状芽孢杆菌属(Clostridium)的情况下，所述优化的密码子通常是富含腺嘌呤(“A”)和胸腺嘧啶(“T”)碱基的密码子。
存在于本发明的遗传工具内的每个核酸、通常为“第一”核酸和“第二”或“第n”核酸，由不同的实体构成，并且例如对应于i)表达盒(或“构造”)，例如包含至少一个转录启动子的核酸，所述启动子被可操作连接(正如专业技术人员所理解的)到一个或多个感兴趣的序列(编码序列)，通常可操作连接到包含其表达产物对所述细菌内感兴趣的功能的性能有贡献的几个感兴趣的编码序列的操纵子，或者例如还包含激活序列和/或转录终止子的核酸；或ii)环状或线性的单链或双链载体例如质粒、噬菌体、粘粒、人工或合成的染色体，其包含如上所定义的一个或多个表达盒。优选地，所述载体是质粒。
所述感兴趣的核酸、通常为表达盒和载体，可以通过专业技术人员公知的常规技术来构建，并且可以包含一个或多个启动子、细菌复制起点(ORI序列)、终止序列、选择基因例如抗生素抗性基因和允许表达盒或载体靶向插入的序列(“侧翼区”)。此外，这些表达盒和表达载体可以使用专业技术人员公知的技术整合到基因组中。
感兴趣的ORI序列可以选自pIP404、pAMβ1、repH(丙酮丁醇梭状芽孢杆菌(C.acetobutylicum)中的复制起点)、ColE1或rep(大肠杆菌中的复制起点)或允许所述载体、通常为质粒维持在梭状芽孢杆菌属(Clostridium)细胞内的任何其他复制起点。
在本发明的情形中，优选的ORI序列是存在于质粒pNF2(SEQ ID NO：118)的OREP序列(SEQ ID NO：126)内的ORI序列。
感兴趣的终止序列可以选自adc或thl基因、bcs操纵子或专业技术人员公知的允许在梭状芽孢杆菌属(Clostridium)内停止转录的任何其他终止子的终止序列。
感兴趣的选择基因(抗性基因)可以选自ermB、catP、bla、tetA、tetM和/或用于针对氨苄青霉素、红霉素、氯霉素、甲砜霉素、壮观霉素、四环素或本领域技术人员公知的可用于选择梭状芽孢杆菌属(Clostridium)细菌的任何其他抗生素的抗性的任何其他基因。
特定载体包含一个或多个表达盒，每个表达盒编码gRNA。
在特定实施方式中，本发明涉及一种遗传工具，其包含序列为SEQ ID NO：23的质粒载体作为权利要求书中所鉴定的“第一”核酸。
在特定实施方式中，本发明涉及一种遗传工具，其包含序列选自序列SEQ ID NO：79、SEQ ID NO：80、SEQ ID NO：119、SEQ ID NO：123、SEQ ID NO：124和SEQ ID NO：125之一的质粒载体作为“第二”或“第n”核酸。
在另一个特定实施方式中，本发明涉及一种遗传工具，其包含序列选自序列SEQID NO：119、SEQ ID NO：123、SEQ ID NO：124和SEQ ID NO：125之一的质粒载体作为“OPT核酸”。在另一个特定实施方式中，所述遗传工具包含SEQ ID NO：23、79、80、119、123、124和125中的几个(例如至少2或3个)序列，所述序列彼此不同。
所述感兴趣的序列通过由所选修复模板指导的同源重组机制(根据CRISPR技术)引入到细菌基因组中。所述感兴趣的序列代替细菌基因组内的被靶向部分。因此，所述重组过程允许所述细菌基因组内被靶向部分的完全或部分修饰或缺失，或允许将核酸片段(在特定实施方式中大片段)插入到细菌基因组中。根据所需转化的本质，所选修复模板可以包括细菌基因组的被靶向序列的全部或一部分或其或多或少修饰的版本。与DNA的被靶向部分相似，所述模板本身因此可以包括一个或多个核酸序列或核酸序列部分，其对应于天然和/或合成的编码和/或非编码序列。所述模板也可以包括一个或多个“外来”序列，即在属于梭状芽孢杆菌属(Clostridium)的细菌的基因组或所述属的特定物种的基因组中天然不存在的序列。所述模板也可以包括如上所述的序列的组合。
本发明的遗传工具允许所述修复模板指导感兴趣的核酸并入到梭状芽孢杆菌属(Clostridium)细菌的细菌基因组中，所述感兴趣的核酸通常为DNA序列或序列部分，其包含至少1个碱基对(bp)，优选地至少1、2、3、4、5、10、15、20、50、100、1,000、10,000、100,000或1,000,000bp，通常在1bp至20kb之间或1bp至10kb之间，优选地10bp至10kb之间或1kb至10kb之间，例如1bp至5kb之间、2kb至5kb之间、或者2.5或3kb至5kb之间。
本发明人描述了感兴趣的核酸、通常为感兴趣的DNA序列的实例，其允许在细菌内表达在所述细菌的野生型版本中存在的遗传物质中部分或完全不存在的DNA序列。
在特定实施方式中，所述感兴趣的DNA序列的表达允许所述梭状芽孢杆菌属(Clostridium)的细菌发酵(通常同时地)几种不同糖，例如至少两种不同糖，通常为五碳糖(例如葡萄糖或甘露糖)和/或六碳糖(例如木糖、阿拉伯糖或果糖)中的至少两种不同糖，优选为至少三种不同糖，选自例如葡萄糖、木糖和甘露糖；葡萄糖、阿拉伯糖和甘露糖；以及葡萄糖、木糖和阿拉伯糖。
在另一个特定实施方式中，所述感兴趣的DNA序列编码至少一种感兴趣的产物，优选为促进梭状芽孢杆菌属(Clostridium)的细菌的溶剂生产的产物，通常为至少一种感兴趣的蛋白质，例如酶、膜蛋白如转运蛋白、其他蛋白(伴侣蛋白)的成熟蛋白质、转录因子或其组合。
在优选实施方式中，所述感兴趣的DNA序列促进溶剂生产，并通常选自i)编码酶例如参与醛向醇的转化的酶的序列，例如选自编码醇脱氢酶的序列(例如选自adh、adhE、adhE1、adhE2、bdhA、bdhB和bdhC的序列)、编码转移酶的序列(例如选自ctfA、ctfB、atoA和atoB的序列)、编码脱羧酶的序列(例如adc)、编码氢化酶的序列(例如选自etfA、etfB和hydA的序列)及其组合，ii)编码膜蛋白的序列，例如编码磷酸转移酶的序列(例如选自glcG、bglC、cbe4532、cbe4533、cbe4982、cbe4983、cbe0751的序列)，iii)编码转录因子的序列(例如选自sigE、sigF、sigG、sigH、sigK的序列)，和iv)它们的组合。
本发明人还描述了感兴趣的核酸的实例，其识别(至少部分结合)并优选地靶向、即识别并允许切割感兴趣的细菌基因组中下述序列的至少一条链：i)靶序列，ii)控制靶序列的转录的序列，或iii)靶序列侧翼的序列。
所述被识别的序列在本文中也被鉴定为“靶序列”或“被靶向序列”。
还描述了包含这种感兴趣的核酸或由所述核酸构成的遗传工具。在这种情况下，所述感兴趣的核酸通常存在于本文中描述的遗传工具的“第二”或“第n”核酸中。
在本说明书的情形中，所述感兴趣的核酸通常被用于从细菌的基因组中移除被识别序列或修饰它的表达，例如调节/调控它的表达，特别是抑制它，优选地修饰它以使所述细菌不能从所述序列表达蛋白质、特别是有功能的蛋白质。
当所述靶序列是编码允许感兴趣的细菌在含有其所赋予的抗性的抗生素的培养基中生长的酶的序列、控制这种序列的转录的序列或这种序列侧翼的序列时，所述抗生素通常是属于酰胺醇类的抗生素。在本说明书的情形中，感兴趣的酰胺醇的实例是氯霉素、甲砜霉素、叠氮氯霉素和氟苯尼考(Schwarz S.等，2004)，特别是氯霉素和甲砜霉素。
在特定实施方式中，所述感兴趣的核酸包含至少一个与靶序列互补的区域，其至少与细菌基因组内的被靶向DNA区域/部分/序列具有100％同一性或至少80％同一性，优选地85％、90％、95％、96％、97％、98％或99％同一性，并且能够杂交到所述区域/部分/序列的互补序列的全部或一部分，通常杂交到包含至少1个核苷酸，优选地至少1、2、3、4、5、10、14、15、20、25、30、35或40个核苷酸，通常为1、10或20至1000个核苷酸之间，例如1、10或20至900、800、700、600、500、400、300或200个核苷酸之间，1、10或20至100个核苷酸之间，1、10或20至50个核苷酸之间或1、10或20至40个核苷酸之间，例如10至40个核苷酸之间、10至30个核苷酸之间、10至20个核苷酸之间、20至30个核苷酸之间、15至40个核苷酸之间、15至30个核苷酸之间或15至20个核苷酸之间的序列，优选为包含14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30个核苷酸的序列。所述感兴趣的核酸内存在的靶序列的互补区可以对应于在本文中描述的CRISPR工具中使用的指导RNA(gRNA)的“SDS”区。
在描述的另一个特定实施方式中，所述感兴趣的核酸包含每个靶序列的至少两个互补区，其至少与细菌基因组内的所述被靶向DNA区域/部分/序列具有100％同一性或至少80％同一性，优选地85％、90％、95％、96％、97％、98％或99％同一性。这些区域能够杂交到所述区域/部分/序列的互补序列的全部或一部分，通常杂交到如上所述的包含至少1个核苷酸、优选地至少100个核苷酸、通常在100至1000个核苷酸之间的序列。与感兴趣的核酸内存在的靶序列互补的区域可以识别、优选地靶向本文中所描述的遗传修饰工具中的靶序列的5′和3′侧翼区域。
根据一种特定情况，所述靶序列是在感兴趣的细菌例如能够在含有一种或多种属于酰胺醇类的抗生素例如氯霉素和/或甲砜霉素的培养基中生长的梭状芽孢杆菌属(Clostridium)的细菌的基因组中编码酰胺醇–O–乙酰基转移酶例如氯霉素–O–乙酰基转移酶或甲砜霉素–O–乙酰基转移酶的序列、控制这种序列的转录的序列或在这种序列侧翼的序列。
所述被识别序列是例如对应于编码来自于拜氏梭状芽孢杆菌(C.beijerinckii)DSM 6423的氯霉素–O–乙酰基转移酶的catB基因(CIBE_3859)的序列SEQ ID NO：18，或与所述氯霉素–O–乙酰基转移酶具有至少70％、75％、80％、85％、90％或95％同一性的氨基酸序列，或包含序列SEQ ID NO：18的全部或至少70％、75％、80％、85％、90％、95％、96％、97％、98％或99％的序列。换句话说，所述被识别序列可以是包含序列SEQ ID NO：18的至少1个核苷酸、优选地至少1、2、3、4、5、10、15、20、25、30、35或40个核苷酸、通常1至40个核苷酸之间的序列，优选为包含序列SEQ ID NO：18的14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30个核苷酸的序列。
与序列SEQ ID NO：18编码的氯霉素–O–乙酰基转移酶具有至少70％同一性的氨基酸序列的实例对应于在NCBI数据库中在下述引用下鉴定的序列：WP_077843937.1，SEQ IDNO：44(WP_063843219.1)，SEQ ID NO：45(WP_078116092.1)，SEQ ID NO：46(WP_077840383.1)，SEQ ID NO：47(WP_077307770.1)，SEQ ID NO：48(WP_103699368.1)，SEQ IDNO：49(WP_087701812.1)，SEQ ID NO：50(WP_017210112.1)，SEQ ID NO：51(WP_077831818.1)，SEQ ID NO：52(WP_012059398.1)，SEQ ID NO：53(WP_077363893.1)，SEQ IDNO：54(WP_015393553.1)，SEQ ID NO：55(WP_023973814.1)，SEQ ID NO：56(WP_026887895.1)，SEQ ID NO：57(AWK51568.1)，SEQ ID NO：58(WP_003359882.1)，SEQ ID NO：59(WP_091687918.1)，SEQ ID NO：60(WP_055668544.1)，SEQ ID NO：61(KGK90159.1)，SEQID NO：62(WP_032079033.1)，SEQ ID NO：63(WP_029163167.1)，SEQ ID NO：64(WP_017414356.1)，SEQ ID NO：65(WP_073285202.1)，SEQ ID NO：66(WP_063843220.1)和SEQID NO：67(WP_021281995.1)。
与序列SEQ ID NO：18编码的氯霉素–O–乙酰基转移酶具有至少75％同一性的氨基酸序列的实例对应于序列WP_077843937.1、WP_063843219.1、WP_078116092.1、WP_077840383.1、WP_077307770.1、WP_103699368.1、WP_087701812.1、WP_017210112.1、WP_077831818.1、WP_012059398.1、WP_077363893.1、WP_015393553.1、WP_023973814.1、WP_026887895.1、AWK51568.1、WP_003359882.1、WP_091687918.1、WP_055668544.1和KGK90159.1。
与序列SEQ ID NO：18编码的氯霉素–O–乙酰基转移酶具有至少90％同一性的氨基酸序列的实例是WP_077843937.1、WP_063843219.1、WP_078116092.1、WP_077840383.1、WP_077307770.1、WP_103699368.1、WP_087701812.1、WP_017210112.1、WP_077831818.1、WP_012059398.1、WP_077363893.1、WP_015393553.1、WP_023973814.1、WP_026887895.1和AWK51568.1。
与序列SEQ ID NO：18编码的氯霉素–O–乙酰基转移酶具有至少95％同一性的氨基酸序列的实例对应于序列WP_077843937.1、WP_063843219.1、WP_078116092.1、WP_077840383.1、WP_077307770.1、WP_103699368.1、WP_087701812.1、WP_017210112.1、WP_077831818.1、WP_012059398.1、WP_077363893.1、WP_015393553.1、WP_023973814.1和WP_02688787895.1。
与序列SEQ ID NO：18编码的氯霉素–O–乙酰基转移酶具有至少99％同一性的优选氨基酸序列是WP_077843937.1、SEQ ID NO：44(WP_063843219.1)和SEQ ID NO：45(WP_078116092.1)。
与SEQ ID NO：18一致的具体序列是在NCBI数据库中被鉴定为WP_077843937.1的序列。
根据特定实例，所述靶序列是序列SEQ ID NO：68，其对应于编码来自于产气荚膜梭状芽孢杆菌(C.perfringens)的氯霉素–O–乙酰基转移酶的catQ基因，所述氯霉素–O–乙酰基转移酶的氨基酸序列对应于SEQ ID NO：66(WP_063843220.1)，或者是与所述氯霉素–O–乙酰基转移酶具有至少70％、75％、80％、80％、85％、90％或95％同一性的序列，或包含序列SEQ ID NO：68的全部或至少70％、75％、80％、85％、90％、95％、96％、97％、98％或99％的序列。
换句话说，所述被识别序列可以是包含序列SEQ ID NO：68的至少1个核苷酸、优选地至少1、2、3、4、5、10、15、20、25、30、35或40个核苷酸、通常在1至40个核苷酸之间的序列，优选为包含序列SEQ ID NO：68的14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30个核苷酸的序列。
根据另一个特定实例，所述被识别序列选自专业技术人员已知的天然存在于细菌内或人工引入到这种细菌中的核酸序列catB(SEQ ID NO：18)、catQ(SEQ ID NO：68)、catD(SEQ ID NO：69，Schwarz S.等，2004)或catP(SEQ ID NO：70，Schwarz S.等，2004)。
正如上文指示的，根据另一个特定实例，所述靶序列也可以是控制如上所述的编码序列(编码允许感兴趣的细菌在含有其赋予抗性的抗生素的培养基中生长的酶)的转录的序列，通常为启动子序列，例如catB基因的启动子序列(SEQ ID NO：73)或catQ基因的启动子序列(SEQ ID NO：74)。
然后所述感兴趣的核酸识别并因此通常能够结合到控制如上所述的编码序列的转录的序列。
根据另一个特定实例，所述靶序列可以是如上所述的编码序列侧翼的序列，例如序列SEQ ID NO：18的catB基因侧翼的序列或与其具有至少70％同一性的序列。这种侧翼序列通常包含1、10或20至1000个核苷酸，例如1、10或20至900、800、700、600、500、400、300或200个之间的核苷酸，1、10或20至100个之间的核苷酸，1、10或20至50个之间的核苷酸，或1、10或20至40个之间的核苷酸，例如10至40个之间的核苷酸、10至30个之间的核苷酸、10至20个之间的核苷酸、20至30个之间的核苷酸、15至40个之间的核苷酸、15至30个之间的核苷酸或15至20个之间的核苷酸。
根据特定情况，所述靶序列对应于在这种编码序列侧翼的成对序列，每个侧翼序列通常包含至少20个核苷酸，通常100至1000个之间的核苷酸，优选为200至800个之间的核苷酸。
在本说明书的情形中，用于转化和/或遗传修饰感兴趣的细菌的感兴趣的核酸的具体实例是i)识别编码序列，ii)控制编码序列的转录，或iii)在编码序列侧翼的DNA片段，所述编码序列编码细菌例如上述梭状芽孢杆菌属(Clostridium)细菌的基因组内感兴趣的酶，优选为酰胺醇–O–乙酰基转移酶例如氯霉素–O–乙酰基转移酶或甲砜霉素–O–乙酰基转移酶。
本发明的感兴趣的核酸的实例能够从细菌的基因组移除被识别序列(“靶序列”)或修饰它的表达例如调节它、特别是抑制它，优选地修饰它以使所述细菌不能从所述序列表达蛋白质例如酰胺醇–O–乙酰基转移酶，特别是有功能的蛋白质。
在所述编码酶的被识别序列是为所述细菌提供针对氯霉素和/或甲砜霉素抗性的序列的特定实施方式中，使用的选择基因不是氯霉素和/或甲砜霉素抗性基因，并且优选地不是catB、catQ、catD或catP基因。
在特定实施方式中，所述感兴趣的核酸包含一个或多个指导RNA(gRNA)，其靶向编码序列、控制编码序列的转录或在编码序列侧翼，所述编码序列编码感兴趣的酶、特别是酰胺醇–O–乙酰基转移酶，和/或包含修饰模板(在本文中也被鉴定为“编辑模板”)，例如用于移除或修饰所述靶序列的全部或一部分的模板，优选地用于抑制或阻遏靶序列的表达的目的，通常为包含与位于上述靶序列的上游和下游的序列同源(对应)的序列的模板，通常所述序列(与位于靶序列的上游和下游的所述序列同源的)各自包含10或20个碱基对至1000、1500或2000个碱基对，例如100、200、300、400或500个碱基对至1000、1200、1300、1400或1500个碱基对，优选为100至1500或100至1000个碱基对，甚至更优选为500至1000个碱基对或200至800个碱基对。
在特定实施方式中，用于转化和/或遗传修饰感兴趣的细菌的感兴趣的核酸是在被Dam和Dcm甲基转移酶识别的基序处不具有甲基化的核酸(从具有dam–dcm–基因型的大肠杆菌细菌制备)。
当所述待转化和/或遗传修饰的感兴趣的细菌是拜氏梭状芽孢杆菌(C.beijerinckii)细菌、特别是属于子进化枝DSM 6423、LMG 7814、LMG 7815、NRRL B–593和NCCB 27006之一的拜氏梭状芽孢杆菌(C.beijerinckii)细菌时，所述用作遗传工具的感兴趣的核酸例如质粒是在被Dam和Dcm甲基转移酶识别的基序处不具有甲基化的核酸，通常为其中GATC基序的腺苷(“A”)和/或CCWGG基序(W可以对应于腺苷(“A”)或胸腺嘧啶(“T”))的第二个胞嘧啶(“C”)被脱甲基化的核酸。
在被Dam和Dcm甲基转移酶识别的基序处不具有甲基化的核酸通常可以从具有dam–dcm–基因型的大肠杆菌细菌(例如大肠杆菌INV110，Invitrogen)制备。同一核酸可能具有其他甲基化，例如被EcoKI甲基转移酶甲基化，后者靶向基序AAC(N6)GTGC和GCAC(N6)GTT(N可以对应于任何碱基)的腺嘌呤(“A”)。
在特定实施方式中，所述被靶向序列对应于编码酰胺醇–O–乙酰基转移酶例如氯霉素–O–乙酰基转移酶的基因例如catB基因、控制该基因的转录的序列或在该基因侧翼的序列。
本发明人描述的感兴趣的特定核酸是例如载体，优选为质粒，例如在本说明书的实验部分(参见实施例2)中描述的序列SEQ ID NO：21的质粒pCas9ind–ΔcatB或序列SEQID NO：38的质粒pCas9ind–gRNA_catB，特别是所述序列的在被Dam和Dcm甲基转移酶识别的基序处不具有甲基化的版本。
本说明书还包括使用感兴趣的核酸转化和/或遗传修饰本文中所描述的感兴趣的细菌。
本发明还涉及一种用于转化并通常通过同源重组遗传修饰梭状芽孢杆菌属(Clostridium)的细菌、优选为梭状芽孢杆菌属(Clostridium)的产溶剂细菌的方法。有利情况下，这种方法包括通过将本申请中描述的本发明的遗传工具引入到所述细菌中来转化所述细菌的步骤，所述遗传工具优选为“OPT核酸”，其包含下述序列或由下述序列构成：i)序列SEQ ID NO：126(OREP)的全部或一部分，和ii)允许修饰细菌的遗传物质和/或在所述细菌内表达在所述细菌的野生型版本中存在的遗传物质中部分或完全不存在的DNA序列的序列。所述方法还可以包括获得、回收、选择或分离被转化的细菌、即具有所需的重组/修饰/优化的细菌的步骤。
有利情况下，本发明通常在所选的用于转化并优选地遗传修饰梭状芽孢杆菌属(Clostridium)的细菌的遗传修饰工具打算用于细菌例如拜氏梭状芽孢杆菌(C.beijerinckii)时实施，所述细菌在野生型状态下携带编码负责对一种或多种抗生素的抗性的酶的基因和/或在野生型状态下携带至少一种染色体外DNA序列，并且所述遗传工具的实施包括使用允许针对所述细菌在野生型状态下有抗性的抗生素的抗性标志物表达的核酸转化所述细菌的步骤，和/或使用所述抗生素(所述细菌在野生型状态下对其有抗性)来选择被转化和/或遗传修饰的细菌，优选地在所述细菌中选择已失去所述染色体外DNA序列的细菌的步骤。
可以使用本发明、通常使用CRISPR遗传修饰工具有利地获得的修饰在于移除不想要的序列例如编码为所述细菌提供针对一种或多种抗生素的抗性的酶的序列，或使该不想要的序列无功能。可以通过本发明有利地获得的另一种修饰在于对细菌进行遗传修饰以便改进它的性能，例如它在生产感兴趣的溶剂或溶剂混合物中的性能，所述细菌预先已通过本发明进行修饰，使所述细菌对它在野生型状态下有抗性的抗生素敏感，和/或从所述细菌中清除在所述细菌的野生型形式中存在的染色体外DNA序列。
本发明的方法是基于CRISPR(簇集规则间隔短回文重复序列)遗传工具/技术，特别是CRISPR/Cas(CRISPR相关蛋白)遗传工具的使用(实施)。
本发明可以使用常规CRISPR/Cas遗传工具，使用Wang等(2015)所描述的包含核酸酶、gRNA和修复模板的单一质粒来实施。
gRNA的序列和结构可以由专业技术人员根据待靶向的染色体区域或可移动遗传元件，使用公知的技术容易地确定(参见例如DiCarlo等，2013的文章)。
本发明人开发并描述了一种基于两种质粒的使用(参见WO2017/064439，Wasels等，2017和与本说明书相伴的图15)来修饰细菌的遗传工具，其适用于梭状芽孢杆菌属(Clostridium)的细菌，并且也可用于本发明的情形。
在特定实施方式中，该工具的“第一”质粒允许Cas核酸酶的表达，并且特异性针对待进行的修饰的“第二”核酸含有一个或多个gRNA表达盒(通常靶向细菌DNA的不同区域)，以及允许通过同源重组机制将被Cas靶向的细菌DNA的部分用感兴趣的序列代替的修复模板。所述cas基因和/或gRNA表达盒被置于专业技术人员已知的组成型或诱导型表达启动子、优选为诱导型启动子(例如在申请WO2017/064439中描述并通过参考并入本说明书的)、优选为不同但可通过相同诱导物诱导的启动子的控制之下。
可以使用的gRNA对应于本文中如上所述的gRNA。
本发明的用于转化和通常通过同源重组遗传修饰梭状芽孢杆菌属(Clostridium)的产溶剂细菌的特定方法，依次包括下述步骤：
a)在抗CRISPR蛋白的表达诱导物存在下，在所述细菌中引入本申请中所描述的核酸或遗传工具，和
b)将在步骤a)结束时获得的被转化的细菌，在不含抗CRISPR蛋白的表达诱导物的培养基上(或在不涉及所述诱导物的条件下)培养，通常允许所述DNA核酸内切酶/gRNA核糖核蛋白复合体、通常为Cas/gRNA的表达(以便停止所述抗CRISPR蛋白的生产并允许所述核酸内切酶的作用)。
将本发明的遗传工具的元件(核酸或gRNA)通过专业技术人员已知的任何直接或间接方法，例如通过转化、接合、微注射、转染、电穿孔等，优选地通过电穿孔(Mermelstein等，1993)，引入到所述细菌中。
所述抗CRISPR蛋白的表达诱导物以足以诱导所述表达的量存在。在Pbgal启动子的情况下，诱导物乳糖释放与BgaR蛋白的表达相关联的抗CRISPR蛋白的表达的抑制(转录阻遏)。
所述抗CRISPR蛋白的表达诱导物优选地以约1mM至约1 M之间、优选地约10mM至约100mM之间、例如约40mM的浓度使用。
在优选实施方式中，所述抗CRISPR蛋白能够抑制、优选地中和所述核酸酶的作用，优选地在将所述遗传工具的核酸序列引入到感兴趣的细菌菌株的阶段中。
正如在实验部分中证实的，本发明有利地允许用含有gRNA表达盒的任何核酸转化含有Cas9表达盒和用于抗CRISPR蛋白例如AcrIIA4的表达盒的梭状芽孢杆菌属(Clostridium)的细菌。
然后将在上述方法的步骤a)结束时获得的被转化的细菌在不含所述抗CRISPR蛋白的表达诱导物(以便停止所述抗CRISPR蛋白的生产并允许所述核酸酶的作用)的培养基上生长。
在特定实施方式中，所述方法还在步骤b)期间或之后包含以下步骤：当在所述遗传工具中存在控制核酸酶和/或指导RNA的表达的诱导型启动子时诱导所述启动子的表达，以便允许在所述遗传工具被引入到所述细菌中之后对所述细菌进行感兴趣的遗传修饰。诱导通常使用释放与所选诱导型启动子相关的表达抑制的物质来进行。
在存在时，所述诱导步骤可以在将本发明的遗传工具引入到靶细菌中之后，在专业技术人员已知的允许所述DNA核酸内切酶/gRNA核糖核蛋白复合体表达的培养基上通过任何培养方法来进行。它通过例如将所述细菌与以足够量存在的适合物质进行接触或通过暴露于UV光来进行。这种物质释放与所选诱导型启动子相关的表达抑制。当所选的启动子是选自Pcm–2tetO1和Pcm–tetO2/1的脱水四环素(aTc)诱导型启动子时，aTc优选地以约1ng/mL至约5000ng/mL之间、优选地约10ng/mL至1000ng/mL、10ng/mL至800ng/mL、10ng/mL至500ng/mL、100ng/mL或200ng/mL至约800ng/mL或1000ng/mL之间或约100ng/mL或200ng/mL至约500ng/mL、600ng/mL或700ng/mL之间，例如约50ng/mL、100ng/mL、150ng/mL、200ng/mL、250ng/mL、300ng/mL、350ng/mL、400ng/mL、450ng/mL、500ng/mL、550ng/mL、600ng/mL、650ng/mL、700ng/mL、750ng/mL或800ng/mL的浓度使用。在特定实施方式中，aTc优选地以约200ng/mL至约1000ng/mL之间或约200ng/mL至约800ng/mL之间，例如约500ng/mL的浓度使用。
在特定实施方式中，所述方法包含另外的步骤c)：移除含有所述修复模板的核酸(所述细菌细胞然后被认为是“清除了”所述核酸)和/或移除在步骤a)期间随着所述遗传工具引入的所述指导RNA或编码所述指导RNA的序列。
在另一个特定实施方式中，所述方法在步骤b)或步骤c)后包括一个或多个其他步骤：在所述抗CRISPR蛋白的表达诱导物存在下引入第n核酸例如第三、第四、第五等核酸，其含有与已经引入的修复模板不同的修复模板和一个或多个指导RNA表达盒，以允许将所述不同的修复模板中包含的感兴趣的序列整合到细菌基因组的被靶向区域中，在每个其他步骤之后跟随有将由此转化的细菌在不含所述抗CRISPR蛋白的表达诱导物的培养基上培养并通常允许所述Cas/gRNA核糖核蛋白复合体例如Cas9/gRNA的表达的步骤。
在本发明的方法的特定实施方式中，使用例如上文描述的核酸或遗传工具来转化所述细菌，所述核酸或遗传工具使用(例如编码)负责切开感兴趣的靶序列的至少一条链的酶，其中所述酶在特定实施方式中是核酸酶，优选为Cas核酸酶，优先选自Cas9酶和MAD7酶。在示例性实施方式中，所述感兴趣的靶序列是编码赋予所述细菌对一种或多种抗生素、优选为属于酰胺醇类的一种或多种抗生素的抗性的酶、通常为酰胺醇–O–乙酰基转移酶例如氯霉素–O–乙酰基转移酶的序列例如catB基因，控制所述编码序列的转录的序列或在所述编码序列侧翼的序列。
当使用时，所述抗CRISPR蛋白通常是如上所述的“抗Cas”蛋白。有利情况下，所述抗CRISPR蛋白是“抗Cas9”蛋白或“抗MAD7”蛋白。
与所述被靶向DNA序列(“被识别序列”)类似，所述编辑/修复模板自身可以包括对应于天然和/或合成的编码和/或非编码序列的一个或多个核酸序列或核酸序列的部分。所述模板也可以包括一个或多个“外来”序列，即在属于梭状芽孢杆菌属(Clostridium)的细菌的基因组中或所述属的特定物种的基因组中天然不存在的。所述模板也可以包括序列的组合。
在本发明中使用的遗传工具允许所述修复模板指导感兴趣的核酸并入到细菌基因组中，所述感兴趣的核酸通常为DNA序列或序列部分，其包含至少1个碱基对(bp)、优选地至少1、2、3、4、5、10、15、20、50、100、1000、10,000、100,000或1,000,000bp，通常在1bp至20kb之间例如2、3、4、5、6、7、8、9、10、11、12或13kb，或在1bp至10kb之间、优选地10bp至10kb之间或1kb至10kb之间例如1bp至5kb之间、2kb至5kb之间、或者2.5或3kb至5kb之间。
在特定实施方式中，所述感兴趣的DNA序列的表达允许属于厚壁菌门、特别是梭状芽孢杆菌属(Clostridium)、芽孢杆菌属(Bacillus)或乳杆菌属(Lactobacillus)的细菌发酵(通常同时地)几种不同糖，例如至少两种不同糖，通常为五碳糖(例如葡萄糖或甘露糖)和/或六碳糖(例如木糖、阿拉伯糖或果糖)中的至少两种不同糖，优选为至少三种不同糖，例如选自葡萄糖、木糖和甘露糖；葡萄糖、阿拉伯糖和甘露糖；以及葡萄糖、木糖和阿拉伯糖。
在另一个特定实施方式中，所述感兴趣的DNA序列编码至少一种感兴趣的产物，优选为促进所述被修饰细菌的溶剂生产的产物，通常为至少一种感兴趣的蛋白质，例如酶、膜蛋白例如转运蛋白、其他蛋白(伴侣蛋白)的成熟蛋白质、转录因子或其组合。
特别有利的是，本发明的遗传工具允许在一个步骤中即使用单一核酸(通常为本文中描述的“OPT核酸”、“第二”或“第n”核酸)或在几个步骤中即使用几个核酸(通常为本文中描述的“第二”和“第n”核酸)，优选地在一个步骤中引入小的和大的感兴趣的序列两者。
在本发明的特定实施方式中，所述核酸例如这种“第n”核酸和本文中描述的遗传工具，允许将所述细菌DNA的被靶向部分移除或用更短的(例如其中已缺失至少一个碱基对的序列)和/或无功能的序列代替。在本发明的特别优选的实施方式中，所述“第二”或“第n”核酸有利地将包含至少一个碱基对且多达2、3、4、5、6、7、8、9、10、11、12、13、14或15kb的感兴趣的核酸引入到所述细菌中，例如细菌基因组中。
取决于所使用的gRNA，可以将所述感兴趣的核酸在相同或不同的区域中插入到所述细菌染色体内。
借助于本发明，通常借助于本发明的遗传工具和方法，现在可以系统性地转化并有效(同源重组的高频率)、实质性(可以在细菌的基因组中并入大的感兴趣的核酸)和稳定地(不需要维持被转化的细菌与抗生素相接触)修饰梭状芽孢杆菌属(Clostridium)的细菌，以便获得感兴趣的被转化的细菌，例如与它们所源自的细菌相比具有基因型或表型差异的改进的突变体，通常为工业上有用的细菌，例如在溶剂或生物燃料的生产中有用的细菌。
本发明的另一个目的涉及使用所述方法获得和/或使用本发明的遗传工具转化并理想地遗传修饰的梭状芽孢杆菌属(Clostridium)的细菌，通常为梭状芽孢杆菌属(Clostridium)的产溶剂细菌，及其任何衍生细菌、克隆、突变体或遗传修饰的版本，及其用途。这种细菌表达使用修复模板通过同源重组引入到它的基因组中的感兴趣的核酸。这种细菌可以包含本发明的遗传工具的全部或一部分，通常为核酸酶例如Cas9或编码核酸酶例如Cas9的核酸。
由此通过本发明转化和/或遗传修饰的示例性细菌是不再表达为它提供对一种或多种抗生素的抗性的酶的细菌，特别是不再表达酰胺醇–O–乙酰基转移酶的细菌，例如在野生型状态下表达catB基因并且在通过本发明转化和/或遗传修饰后不含所述catB基因或不能表达所述catB基因的细菌。由此使得通过本发明转化和/或遗传修饰的细菌对酰胺醇例如本文中描述的酰胺醇、特别是对氯霉素或甲砜霉素敏感。
本发明的优选的遗传修饰的细菌的具体实例是在本说明书中被鉴定为拜氏梭状芽孢杆菌(C.beijerinckii)IFP962ΔcatB(在本文中也被鉴定为拜氏梭状芽孢杆菌(C.beijerinckii)DSM 6423ΔcatB)的细菌，其在2018年12月6日在比利时微生物合作保藏中心(Belgian Co–ordinated Collections of Micro–organisms)(“BCCM”，K.L.Ledeganckstraat35,B–9000 Gent–Belgium)登记在保藏号LMG P–31151之下。
本发明的优选的遗传修饰的细菌的另一个具体实例是在本说明书中被鉴定为拜氏梭状芽孢杆菌(C.beijerinckii)IFP963ΔcatBΔpNF2(在本文中也被鉴定为拜氏梭状芽孢杆菌(C.beijerinckii)DSM6423ΔcatBΔpNF2)的细菌，其在2019年2月20日在BCCM–LMG保藏中心登记在保藏号LMG P–31277之下。
本说明书还涉及所述细菌之一的任何衍生细菌、克隆、突变体或遗传修饰的版本，例如仍然对酰胺醇例如甲砜霉素和/或氯霉素敏感的任何衍生细菌、克隆、突变体或遗传修饰的版本。
根据特定实施方式，按照本发明转化和/或遗传修饰的细菌例如拜氏梭状芽孢杆菌(C.beijerinckii)DSM 6423ΔcatB或拜氏梭状芽孢杆菌(C.beijerinckii)DSM6423ΔcatBΔpNF2，仍然能够被转化并优选地遗传修饰。它可以使用核酸例如在本说明书中例如在实验部分中所描述的质粒来进行。可以有利地使用的示例性核酸是序列SEQ ID NO：23的质粒pCas9acr(描述在本说明书的实验部分中)或选自pCas9ind(SEQ ID NO：22)、pCas9cond(SEQ ID NO：133)和pMAD7(SEQ ID NO：134)的质粒。
本发明的特定情况涉及本文中描述的遗传修饰的细菌、优选为在编号LMG P–31151下保藏的细菌拜氏梭状芽孢杆菌(C.beijerinckii)IFP962ΔcatB、更优选为在编号LMG P–31277下登记的拜氏梭状芽孢杆菌(C.beijerinckii)IFP963ΔcatBΔpNF2或这些细菌之一的遗传修饰的版本的用途，其例如使用本文中描述的核酸、遗传工具或方法之一，由于被故意引入到它的基因组中的感兴趣的核酸的表达，用于优选地在工业规模上生产一种或多种溶剂、优选为至少异丙醇。
在特定实施方式中，使用本发明的方法和遗传工具获得的本发明的梭状芽孢杆菌属(Clostridium)的细菌能够生产一种或多种溶剂，这仅仅是由于故意引入到它的基因组中的感兴趣的核酸的表达。
本发明还涉及一种用于转化并通常遗传修饰梭状芽孢杆菌属(Clostridium)的细菌的试剂盒，其包含本文中描述的遗传工具的全部或部分元件，通常为i)编码一种DNA核酸内切酶例如Cas9的第一核酸，其中编码所述核酸内切酶的序列被置于启动子的控制之下，和ii)至少一种编码修复模板的第二核酸，其允许通过同源重组机制将所述细菌DNA的被核酸内切酶靶向的部分用感兴趣的序列代替，以及适合于所述工具中使用的所选抗CRISPR蛋白的表达的诱导型启动子的至少一种诱导物。所述试剂盒也可以包含适合于所述工具中任选地使用的所选诱导型启动子的一种或多种诱导物，以控制所述核酸内切酶和/或一个或多个指导RNA的表达。
还描述了一种试剂盒，其包含(i)本文中所描述的核酸，例如本文中描述的“OPT核酸”或识别细菌中的靶序列的DNA片段，和(ii)至少一种工具，优选为几种工具，其选自本文中描述的用于转化和通常遗传修饰这种细菌以便产生所述细菌的改进的变体的遗传修饰工具的元件，作为gRNA的核酸，作为修复基质的核酸，“OPT核酸”，至少一对引物例如在本发明的情形中描述的引物对，和用于表达由所述工具编码的蛋白质例如Cas9或MAD7核酸酶的诱导物。
本文中描述的用于转化和通常遗传修饰细菌的遗传修饰工具可以例如选自“OPT核酸”、CRISPR工具、基于II型内含子的使用的工具和等位基因交换工具，正如上文解释的。
在特定实施方式中，所述试剂盒包含本文中描述的遗传工具的全部或一些元件。
用于转化和优选地遗传修饰本文中描述的属于厚壁菌门的细菌或使用这种细菌生产至少一种溶剂例如溶剂的混合物的特定试剂盒包含含有下述序列或由下述序列构成的核酸：i)序列SEQ ID NO：126的全部或一部分，和ii)允许修饰细菌的遗传物质和/或在所述细菌内表达在所述细菌的野生型版本中存在的遗传物质中部分或完全不存在的DNA序列的序列；以及适合于在本文中描述的遗传工具中使用的所选抗CRISPR蛋白的表达的诱导型启动子的至少一种诱导物。
所述试剂盒也可以包含适合于在所述遗传工具中任选地使用的所选诱导型启动子的一种或多种诱导物，以控制所使用的核酸酶和/或一个或多个指导RNA的表达。
本发明的特定试剂盒允许表达含有标签的核酸内切酶例如Cas9或MAD7蛋白。
本发明的试剂盒还可以包含一种或多种消耗品例如培养基、至少一种梭状芽孢杆菌属(Clostridium)的感受态细菌(即被包装用于转化)、至少一种gRNA、核酸酶例如Cas9或MAD7蛋白、一种或多种选择分子或一套说明书。
本发明通常涉及一种用于执行本文中描述的转化和理想情况下遗传修饰的方法和/或使用梭状芽孢杆菌属(Clostridium)的细菌生产溶剂(至少一种溶剂)的试剂盒。
本发明还涉及本发明的核酸、遗传工具、方法或试剂盒的潜在用途，其用于转化并通常遗传修饰梭状芽孢杆菌属(Clostridium)的细菌，通常为梭状芽孢杆菌属(Clostridium)的产溶剂细菌，以例如产生梭状芽孢杆菌属(Clostridium)细菌的改进的变体。
本说明书特别涉及了本发明的试剂盒或该试剂盒的一种或多种元件的用途，其用于执行本文中描述的转化和理想情况下遗传修饰本文中描述的细菌的方法，所述细菌通常为梭状芽孢杆菌属(Clostridium)的细菌(例如在编号LMG P–31151下保藏的拜氏梭状芽孢杆菌(C.beijerinckii)IFP962ΔcatB)，优选为在野生型状态下具有细菌染色体和不同于染色体DNA的至少一种DNA分子(通常为天然质粒)两者的细菌，最优选为在编号LMG P–31277下保藏的细菌拜氏梭状芽孢杆菌(C.beijerinckii)IFP963ΔcatBΔpNF2细菌。
最后，本发明涉及本发明的核酸、遗传工具、方法、试剂盒或被转化的梭状芽孢杆菌属(Clostridium)的细菌的潜在用途，其特别是用于能够生产溶剂或生物燃料或其混合物，通常是在工业规模上。可以生产的溶剂通常是丙酮、丁醇、乙醇、异丙醇或其混合物，通常为乙醇/异丙醇、丁醇/异丙醇或乙醇/丁醇混合物，优选为异丙醇/丁醇混合物。
在特定实施方式中，所述乙醇/异丙醇混合物的比率至少等于1/4。该比率优选地在1/3至1之间，更优选地等于1。
在特定实施方式中，所述乙醇/丁醇混合物的比率至少等于1/4。该比率优选地在1/3至1之间，更优选地等于1。
在特定实施方式中，所述异丙醇/丁醇混合物的比率至少等于1/4。该比率优选地在1/3至1之间，更优选地等于1。
本发明的被转化的细菌的使用通常允许在工业规模上每年工业生产至少100吨丙酮、至少100吨乙醇、至少1000吨异丙醇、至少1800吨丁醇或至少40,000吨它们的混合物。
下面的实施例旨在更充分地说明本发明而不是限制它的范围。
实施例
实施例1
材料和方法
生长条件
丙酮丁醇梭状芽孢杆菌(C.acetobutylicum)DSM 792生长在2YTG培养基(16g/l胰蛋白胨，10g/l酵母提取物，5g/l葡萄糖，4g/l NaCl)中。大肠杆菌NEB10B生长在LB培养基(10g/l胰蛋白胨，5g/l酵母提取物，5g/l NaCl)中。固体培养基通过向液体培养基添加15g/l琼脂糖来制造。在必要时使用红霉素(在2YTG或LB培养基中浓度分别为40或500mg/l)、氯霉素(在固体或液体LB中分别为25或12.5mg/l)和甲砜霉素(在2YTG培养基中15mg/l)。
核酸的使用
使用的所有酶和试剂盒按照供应商的推荐来进行。
质粒构建
图4中示出的pCas9acr质粒(SEQ ID NO：23)通过将由Eurofins Genomes合成的含有在Pbgal启动子控制之下的bgaR和acrIIA4的片段(SEQ ID NO：1)克隆在pCas9ind载体(Wasels等，2017)的SacI位点处来构建。
pGRNAind质粒(SEQ ID NO：82)通过将由Eurofins Genomes合成的在启动子Pcm–2tetO1(Dong等，2012)的控制之下用于gRNA的表达盒(SEQ ID NO：83)克隆在pEC750C载体(SEQ ID NO：106)(Wasels等，2017)的SacI位点处来构建。
pGRNA–xylB(SEQ ID NO：102)、pGRNA–xylR(SEQ ID NO：103)、pGRNA–glcG(SEQ IDNO：104)和pGRNA–bdhB(SEQ ID NO：105)质粒通过将相应的引物对5′–TCATGATTTCTCCATATTAGCTAG–3′和5′–AAACCTAGCTAATATGGAGAAATC–3′、5′–TCATGTTACACTTGGAACAGGCGT–3′和5′–AAACACGCCTGTTCCAAGTGTAAC–3′、5′–TCATTTCCGGCAGTAGGATCCCCA–3′和5′–AAACTGGGGATCCTACTGCCGGAA–3′、5′–TCATGCTTATTACGACATAACACA–3′和5′–AAACTGTGTTATGTCGTAATAAGC–3′克隆在用BsaI消化的pGRNAind质粒(SEQ ID NO：82)内来构建。
pGRNA–ΔbdhB质粒(SEQ ID NO：79)通过将在一方面使用引物5′–ATGCATGGATCCAAACGAACCCAAAAAGAAAGTTTC–3′和5′–GGTTGATTTCAAATCTGTGTAAACCTACCG–3′，在另一方面使用5′–ACACAGATTTGAAATCAACCACTTTAACCC–3′和5′–ATGCATGTCGACTCTTAAGAACATGTATAAAGTATGG–3′获得的PCR产物的重叠PCR组装所获得的DNA片段，克隆在用BamHI和SacI消化的pGRNA–bdhB载体中来构建。
pGRNA–ΔbdhAΔbdhB质粒(SEQ ID NO：80)通过将在一方面使用引物引物5′–ATGCATGGATCCAAACGAACCCAAAAAGAAAGTTTC–3′和5′–GCTAAGTTTTAAATCTGTGTAAACCTACCG–3′，在另一方面使用5′–ACACAGATTTAAAACTTAGCATACTTCTTACC–3′和5′–ATGCATGTCGACCTTCTAATCTCCTCTACTATTTTAG–3′获得的PCR产物的重叠PCR组装所获得的DNA片段，克隆在用BamHI和SacI消化的pGRNA–bdhB载体中来构建。
转化
丙酮丁醇梭状芽孢杆菌(C.acetobutylicum)DSM 792按照Mermelstein等，1993描述的方案进行转化。已经含有Cas9表达质粒(pCas9ind或pCas9acr)的用含有gRNA表达盒的质粒转化的丙酮丁醇梭状芽孢杆菌(C.acetobutylicum)DSM 792转化体的选择，在含有红霉素(40mg/l)、甲砜霉素(15mg/l)和乳糖(40nM)的固体2YTG培养基上进行。
cas9表达的诱导
cas9表达的诱导通过将获得的转化体在含有红霉素(40mg/l)、甲砜霉素(15mg/l)和cas9和gRNA的表达诱导物aTc(1mg/l)的固体2YTG培养基上生长来实现。
bdh基因座的扩增
丙酮丁醇梭状芽孢杆菌(C.acetobutylicum)DSM 792基因组在bdhA和bdhB基因基因座处的编辑的验证，使用高保真DNA聚合酶(NEB)以及引物V1(5′–ACACATTGAAGGGAGCTTTT–3′)和V2(5′–GGCAAC AACATCAGGCCTTT–3′)，通过PCR来进行。
结果
转化效率
为了评估acrIIA4基因的插入对cas9表达质粒的转化频率的影响，将不同的gRNA表达质粒转化到含有pCas9ind(SEQ ID NO：22)或pCas9acr(SEQ ID NO：23)的菌株DSM 792中，并将转化体在增补有乳糖的培养基上选择。获得的转化频率呈现在图5中。
ΔbdhB和ΔbdhAΔbdhB突变体的产生
将含有用于靶向bdhB的gRNA的表达盒的靶向质粒(pGRNA–bdhB–SEQ ID NO：105)以及含有修复基质以允许缺失单独的bdhB基因(pGRNA–ΔbdhB–SEQ ID NO：79)或bdhA和bdhB基因(pGRNA–ΔbdhAΔbdhB–SEQ ID NO：80)的两种衍生质粒转化到含有pCas9ind(SEQID NO：22)或pCas9acr(SEQ ID NO：23)的菌株DSM 792中。得到的转化频率呈现在表2中：
表2：靶向bdhB的质粒对含有pCas9ind或pCas9acr的菌株DSM 792的转化频率。频率被表示为在转化中使用的每μg DNA获得的转化体的数目，并表示至少两个独立实验的平均值。
获得的转化体通过在增补有脱水四环素(aTc)的培养基上传代，经历诱导CRISPR/Cas9系统的表达的阶段(图6)。
在两个aTc抗性菌落的基因组DNA上，通过PCR确认了所需的修饰(图7)。
结论
在Wasels等，(2017)中描述的基于CRISPR/Cas9的遗传工具使用两种质粒：
–第一种质粒pCas9ind含有在aTc诱导型启动子的控制之下的cas9，并且
–源自于pEC750C的第二种质粒含有用于gRNA(置于第二个aTc诱导型启动子的控制之下)以及用于修复由所述系统引起的双链断裂的编辑模板的表达盒。
然而，本发明人观察到某些gRNA仍显得毒性过高，尽管使用了aTc诱导型启动子来控制它们的表达以及Cas9的表达，从而限制了所述遗传工具的细菌转化效率以及因此限制了染色体修饰的效率。
为了改进这种遗传工具，通过插入在乳糖诱导型启动子的控制之下的抗CRISPR基因acrIIA4，对所述cas9表达质粒进行改良。因此，不同的gRNA表达质粒的转化效率被显著提高，允许对所有试验的质粒获得转化体。
也可以使用不能被引入到含有pCas9ind的丙酮丁醇梭状芽孢杆菌(C.acetobutylicum)DSM 792中的质粒来编辑菌株DSM 792的基因组内的bdhB基因座。观察到的修饰频率与以前观察到的相同(Wasels等，2017)，其中100％的试验菌落被修饰。
总而言之，所述cas9表达质粒的改良允许更好地控制Cas9–gRNA核糖核蛋白复合体，有利地促进生产其中Cas9的作用可以被触发以便获得感兴趣的突变体的转化体。
实施例2
材料和方法
生长条件
拜氏梭状芽孢杆菌(C.beijerinckii)DSM 6423生长在2YTG培养基(16g/l胰蛋白胨，10g/l酵母提取物，5g/l葡萄糖，4g/l NaCl)中。大肠杆菌NEB10–beta和INV110生长在LB培养基(10g/l胰蛋白胨，5g/l酵母提取物，5g/l NaCl)中。固体培养基通过向液体培养基添加15g/l琼脂糖来制备。在必要时，使用红霉素(在2YTG或LB培养基中浓度分别为20或500mg/l)、氯霉素(在固体或液体LB中分别为25或12.5mg/l)、甲砜霉素(在2YTG培养基中15mg/l)或壮观霉素(在LB或2YTG培养基中浓度分别为100或650mg/l)。
核酸和质粒载体
使用的所有酶和试剂盒按照供应商的推荐来使用。
菌落PCR试验遵照下述流程：
将分离的拜氏梭状芽孢杆菌(C.beijerinckii)DSM 6423菌落重悬浮在100μL的10mM Tris，pH7.5，5mM EDTA中。将该溶液加热至98℃不搅拌10min。然后可以将0.5μL该细菌裂解物作为PCR基质，使用在含有Phire(Thermo Scientific)、Phusion(ThermoScientific)、Q5(NEB)或KAPA2G Robust(Sigma–Aldrich)聚合酶的10μL反应中。
用于所有构建的引物的名单(名称/DNA序列)详述如下：
ΔcatB_fwd：TGTTATGGATTATAAGCGGCTCGAGGACGTCAAACCATGTTAATCATTGC
ΔcatB_rev：AATCTATCACTGATAGGGACTCGAGCAATTTCACCAAAGAATTCGCTAGC
ΔcatB_gRNA_rev：AATCTATCACTGATAGGGACTCGAGGGGCAAAAGTGTAAAGACAAGCTTC
RH076：CATATAATAAAAGGAAACCTCTTGATCG
RH077：ATTGCCAGCCTAACACTTGG
RH001：ATCTCCATGGACGCGTGACGTCGACATAAGGTACCAGGAATTAGAGCAGC
RH002：TCTATCTCCAGCTCTAGACCATTATTATTCCTCCAAGTTTGCT
RH003：ATAATGGTCTAGAGCTGGAGATAGATTATTTGGTACTAAG
RH004：TATGACCATGATTACGAATTCGAGCTCGAAGCGCTTATTATTGCATTAGC
pEX–fwd：CAGATTGTACTGAGAGTGCACC
pEX–rev：GTGAGCGGATAACAATTTCACAC
pEC750C–fwd：CAATATTCCACAATATTATATTATAAGCTAGC
M13–rev：CAGGAAACAGCTATGAC
RH010：CGGATATTGCATTACCAGTAGC
RH011：TTATCAATCTCTTACACATGGAGC
RH025：TAGTATGCCGCCATTATTACGACA
RH134：GTCGACGTGGAATTGTGAGC
pNF2_fwd：GGGCGCACTTATACACCACC
pNF2_rev：TGCTACGCACCCCCTAAAGG
RH021：ACTTGGGTCGACCACGATAAAACAAGGTTTTAAGG
RH022：TACCAGGGATCCGTATTAATGTAACTATGATATCAATTCTTG
aad9–fwd2：ATGCATGGTCCCAATGAATAGGTTTACACTTACTTTAGTTTTATGG
aad9–rev：ATGCGAGTTAACAACTTCTAAAATCTGATTACCAATTAG
RH031：ATGCATGGATCCCAATGAATAGGTTTACACTTACTTTAGTTTTATGG
RH032：ATGCGAGAGCTCAACTTCTAAAATCTGATTACCAATTAG
RH138：ATGCATGGATCCGTCTGACAGTTACCAGGTCC
RH139：ATGCGAGAGCTCCAATTGTTCAAAAAAATAATGGCGGAG
RH140：ATGCATGGATCCCGGCAGTTTTTCTTTTTCGG
RH141：ATGCGAGAGCTCGGTTAAATACTAGTTTTTAGTTACAGAC
制备了下述质粒载体：
–1号质粒：pEX–A258–ΔcatB(SEQ ID NO：17)
它含有克隆到质粒pEX–A258中的ΔcatB合成DNA片段。该ΔcatB片段包含：i)在脱水四环素诱导型启动子的控制之下用于靶向来自于拜氏梭状芽孢杆菌(C.beijerinckii)DSM6423的catB基因(编码氯霉素–O–乙酰基转移酶的氯霉素抗性基因–SEQ ID NO：18)的指导RNA的表达盒(表达盒：SEQ ID NO：19)，和ii)包含位于所述catB基因的上游和下游的400个同源bp的编辑基质(SEQ ID NO：20)。
–2号质粒：pCas9ind–ΔcatB(参见图9和SEQ ID NO：21)
它含有通过PCR(引物ΔcatB_fwd和ΔcatB_rev)扩增并在用限制性酶XhoI消化不同DNA后克隆到pCas9ind(描述在专利申请WO2017/064439中–SEQ ID NO：22)中的ΔcatB片段。
–3号质粒：pCas9acr(参见图10和SEQ ID NO：23)
–4号质粒：pEC750S–uppHR(参见图11和SEQ ID NO：24)
它含有用于缺失upp基因并由upp基因上游和下游的两个同源DNA片段(尺寸分别为：500(SEQ ID NO：26)和377(SEQ ID NO：27)个碱基对)构成的修复基质(SEQ ID NO：25)。使用Gibson克隆系统(New England Biolabs,Gibson assembly Master Mix2X)获得组装物。为此，从菌株DSM 6423的基因组DNA(参见Matéde Gerando等，2018和登记号PRJEB11626(https://www.ebi.ac.uk/ena/data/view/PRJEB11626))，使用相应的引物RH001/RH002和RH003/RH004，通过PCR扩增所述上游和下游部分。然后将这两个片段组装在预先用限制性酶(SalI和SacI限制性酶)线性化的pEC750S中。
–5号质粒：pEX–A2–gRNA–upp(参见图12和SEQ ID NO：28)这个质粒包含插入到名为pEX–A2的复制质粒中的在组成型启动子(序列SEQ ID NO：30的非编码RNA)的控制之下的对应于用于靶向upp基因的指导RNA(靶向upp的前间区序列(SEQ ID NO：31))的表达盒(SEQID NO：29)的gRNA–upp DNA片段。
–6号质粒：pEC750S–Δupp(参见图13和SEQ ID NO：32)
它基于质粒pEC750S–uppHR(SEQ ID NO：24)，并另外含有包含在组成型启动子的控制之下用于靶向upp基因的指导RNA的表达盒的DNA片段。
将这个片段插入到pEX–A2中，被称为pEX–A2–gRNA–upp。然后使用引物pEX–fwd和pEX–rev通过PCR扩增所述插入片段，然后用限制性酶XhoI和NcoI消化。最后，通过连接到首先用相同的限制性酶消化的pEC750S–uppHR中来克隆这个片段，以获得pEC750S–Δupp。
–7号质粒：pEC750C–Δupp(参见图14和SEQ ID NO：33)
然后使用引物pEC750C–fwd和M13–rev扩增带有所述指导RNA和修复基质的表达盒。将扩增子用酶XhoI和SacI通过酶限制进行消化，然后通过酶连接克隆到pEC750C中，以获得pEC750C–Δupp。
–8号质粒：pGRNA–pNF2(参见图15和SEQ ID NO：34)
这个质粒是基于pEC750C，并含有用于靶向质粒pNF2(SEQ ID NO：118)的指导RNA的表达盒。
–9号质粒：pCas9ind–gRNA_catB(参见图23和SEQ ID NO：38)它含有通过PCR(引物ΔcatB_fwd和ΔcatB_gRNA_rev)扩增的编码靶向catB基因座的指导RNA在用限制性酶XhoI消化不同DNA并连接后克隆到pCas9ind(描述在专利申请WO2017/064439中)中的序列。
–10号质粒：pNF3(参见图25和SEQ ID NO：119)
它含有使用引物RH021和RH022扩增的pNF2的一部分，包括复制起点和编码质粒复制蛋白的基因(CIBE_p20001)。然后将该PCR产物克隆在质粒pUC19(SEQ ID NO：117)的SalI和BamHI限制性位点处。
–11号质粒：pEC751S(参见图26和SEQ ID NO：121)
它含有除了氯霉素抗性基因catP(SEQ ID NO：70)之外pEC750C(SEQ ID NO：106)的全部元件。所述抗性基因被提供壮观霉素抗性的粪肠球菌(Enterococcus faecalis)的aad9基因(SEQ ID NO：130)代替。该元件使用引物aad9–fwd2和aad9–rev从质粒pMTL007S–E1(SEQ ID NO：120)扩增，并被克隆到pEC750C的AvaII和HpaI位点中，代替catP基因(SEQID NO：70)。
–12号质粒：pNF3S(参见图27和SEQ ID NO：123)
它含有pNF3的全部元件，并在BamHI与SacI位点之间插入aad9基因(使用引物RH031和RH032从pEC751S扩增)。
–13号质粒：pNF3E(参见图28和SEQ ID NO：124)
它含有pNF3的全部元件，并插入在miniPthl启动子的控制之下的难辨梭状芽孢杆菌(Clostridium difficile)的ermB基因(SEQ ID NO：131)。这个元件使用引物RH138和RH139从pFW01扩增，并被克隆在pNF3E的BamHI与SacI位点之间。
–14号质粒：pNF3C(参见图29和SEQ ID NO：125)
它含有pNF3的全部元件，并插入产气荚膜梭状芽孢杆菌(Clostridiumperfringens)的catP基因(SEQ ID NO：70)。这个元件使用引物RH140和RH141从pEC750C扩增，并被克隆在pNF3E的BamHI与SacI位点之间。
结果1
拜氏梭状芽孢杆菌(C.beijerinckii)菌株DSM 6423的加工
所述质粒被引入到大肠杆菌dam–dcm–菌株(INV110，Invitrogen)中并在其中复制。这允许移除pCas9ind–ΔcatB质粒上的Dam和Dcm甲基化，然后按照Mermelstein等，(1993)描述的流程通过转化将它引入到菌株DSM 6423中，并进行了下述修改：将菌株在OD600为0.8时用较大量的质粒(20μg)转化，并使用下述电穿孔参数：100Ω，25μF，1400 V。在含有红霉素(20μg/mL)的皮氏培养皿上划线，产生含有pCas9ind–ΔcatB质粒的拜氏梭状芽孢杆菌(C.beijerinckii)DSM6423转化体。
cas9表达的诱导和拜氏梭状芽孢杆菌(C.beijerinckii)菌株DSM6423ΔcatB的产生
然后将几个红霉素抗性菌落挑取到100μL培养基(2YTG)中，并在培养基中连续稀释直至稀释倍数为104。对于每个菌落，将8μL每种稀释液沉积在含有红霉素和脱水四环素(200ng/mL)的皮氏培养皿上，以诱导编码Cas9核酸酶的基因的表达。
在提取基因组DNA后，使用引物RH076和RH077通过PCR验证在该培养皿上生长的克隆中catB基因的缺失(参见图16)。
拜氏梭状芽孢杆菌(C.beijerinckii)菌株DSM 6423ΔcatB对甲砜霉素的敏感性的验证
为了确保catB基因的缺失确实赋予了对甲砜霉素的新的敏感性，进行了在琼脂培养基上的比较性分析。在2YTG培养基上制备拜氏梭状芽孢杆菌(C.beijerinckii)DSM 6423和拜氏梭状芽孢杆菌(C.beijerinckii)DSM 6423ΔcatB的预培养物，然后将100μL这些预培养物铺在任选地以15mg/L的浓度增补有甲砜霉素的2YTG琼脂培养基上。图17显示只有初始的拜氏梭状芽孢杆菌(C.beijerinckii)DSM 6423菌株能够生长在增补有甲砜霉素的培养基上。
通过CRISPR–Cas9工具在拜氏梭状芽孢杆菌(C.beijerinckii)菌株DSM 6423ΔcatB中缺失upp基因
将拜氏梭状芽孢杆菌(C.beijerinckii)菌株DSM 6423ΔcatB的克隆首先用在被dam和dcm甲基转移酶识别的基序处不具有甲基化的pCas9acr载体(从具有dam–dcm–基因型的大肠杆菌细菌制备)转化。使用引物RH025和RH134，通过菌落PCR验证维持在拜氏梭状芽孢杆菌(C.beijerinckii)菌株DSM 6423中的质粒pCas9acr的存在。
然后将红霉素抗性克隆用事先脱甲基化的pEC750C–Δupp转化。将由此获得的菌落在含有红霉素(20μg/mL)、甲砜霉素(15μg/mL)和乳糖(40mM)的培养基上选择。
然后将这些克隆中的几个重悬浮在100μL培养基(2YTG)中，并在培养基中连续稀释(至104的稀释倍数)。将5微升每种稀释液置于含有红霉素、甲砜霉素和脱水四环素(200ng/mL)的皮氏培养皿上(参见图18)。
对于每个克隆，使用引物通过菌落PCR测试两个aTc抗性菌落，以扩增upp基因座(参见图19)。
通过CRISPR–Cas9工具在拜氏梭状芽孢杆菌(C.beijerinckii)菌株DSM 6423ΔcatB中缺失天然pNF2质粒
将拜氏梭状芽孢杆菌(C.beijerinckii)菌株DSM 6423ΔcatB的克隆首先用在被Dam和Dcm甲基转移酶识别的基序处不具有甲基化的载体pCas9ind(从具有dam–dcm–基因型的大肠杆菌细菌制备)转化。使用引物pCas9ind_fwd(SEQ ID NO：42)和pCas9ind_rev(SEQ IDNO：43)，通过PCR验证拜氏梭状芽孢杆菌(C.beijerinckii)菌株DSM 6423中质粒pCas9ind的存在(参见图20)。
然后将红霉素抗性克隆用于转化从具有dam–dcm–基因型的大肠杆菌细菌制备的pGRNA–pNF2。
将在含有红霉素(20μg/mL)和甲砜霉素(15μg/mL)的培养基上获得的几个菌落重悬浮在培养基中，并连续稀释至104的稀释倍数。将8微升每种稀释液置于含有红霉素、甲砜霉素和脱水四环素(200ng/mL)的皮氏培养皿上，以便诱导CRISPR/Cas9系统的表达。
使用引物pNF2_fwd(SEQ ID NO：39)和pNF2_rev(SEQ ID NO：40)，通过PCR验证天然pNF2质粒的不存在(参见图21)。
结论
在本工作中，本发明人成功地在拜氏梭状芽孢杆菌(Clostridium beijerinckii)菌株DSM 6423中引入并维持不同的质粒。它们能够在使用单一质粒的基础上，使用CRISPR–Cas9工具移除catB基因。获得的重组菌株对甲砜霉素的敏感性通过在琼脂培养基上的试验得以证实。
这个缺失允许他们更有效地使用在专利申请FR1854835中描述的需要两种质粒的CRISPR–Cas9工具。进行了两个实施例以证实本申请的重要性：upp基因的缺失和对拜氏梭状芽孢杆菌(Clostridium beijerinckii)菌株DSM 6423非必需的天然质粒的移除。
结果2
拜氏梭状芽孢杆菌(C.beijerinckii)菌株的转化
在大肠杆菌菌株NEB10–beta中制备的质粒也被用于转化拜氏梭状芽孢杆菌(C.beijerinckii)菌株NCIMB 8052。相反，对于拜氏梭状芽孢杆菌(C.beijerinckii)DSM6423来说，质粒被首先引入到大肠杆菌dam–dcm–菌株(INV110，Invitrogen)中并在其中复制。这允许在通过转化引入到菌株DSM 6423中之前移除感兴趣的质粒上的Dam和Dcm甲基化。
对于每种菌株来说，转化在其他方面相似地进行，即按照Mermelstein等，1992描述的流程并进行了下述修改：将菌株在OD600为0.6–0.8时用较大量的质粒(5–20μg)转化，并且电穿孔参数为100Ω、25μF、1400 V。在2YTG中再生3小时后，将所述细菌在含有所需抗生素(红霉素：20–40μg/mL；甲砜霉素：15μg/mL；壮观霉素：650μg/mL)的皮氏培养皿(2YTG琼脂)上划线。
拜氏梭状芽孢杆菌(C.beijerinckii)DSM 6423菌株的转化效率的比较转化在下述拜氏梭状芽孢杆菌(C.beijerinckii)菌株中，在生物学平行样中进行：DSM 6423野生型，DSM 6423ΔcatB和DSM 6423ΔcatBΔpNF2(图30)。为此目的，使用pCas9ind载体，其由于不允许良好的转化效率而尤其难以用于修饰细菌。它还含有为菌株提供对红霉素的抗性的基因，所有三种菌株都对这种抗生素敏感。
结果表明，由于失去天然pNF2质粒，转化效率提高约15–20倍。
也测试了提供甲砜霉素抗性的质粒pEC750C的转化效率，但仅仅在DSM 6423ΔcatB和DSM 6423ΔcatBΔpNF2菌株中，因为野生型菌株对这种抗生素具有抗性(图31)。对于这种质粒来说，转化效率的增加甚至更加明显(提高约2000倍)。
pNF3质粒与其他质粒的转化效率的比较
为了确定含有天然pNF2质粒的复制起点的质粒的转化效率，将质粒pNF3E和pNF3C引入到拜氏梭状芽孢杆菌(C.beijerinckii)菌株DSM 6423ΔcatBΔpNF2中。含有红霉素或氯霉素抗性基因的载体的使用允许根据抗性基因的本性比较载体的转化效率。也转化了质粒pFW01和pEC750C。这两种质粒含有针对不同抗生素(分别为红霉素和甲砜霉素)的抗性基因，并且通常被用于转化拜氏梭状芽孢杆菌(C.beijerinckii)和丙酮丁醇梭状芽孢杆菌(C.acetobutylicum)。
如图32中所示，基于pNF3的载体具有出色的转化效率，并且特别适合用于拜氏梭状芽孢杆菌(C.beijerinckii)DSM 6423ΔcatBΔpNF2中。具体来说，pNF3E(其含有红霉素抗性基因)与具有相同抗性基因的pFW01相比显示出显著更高的转化效率。该同一个质粒不能引入到野生型拜氏梭状芽孢杆菌(C.beijerinckii)DSM 6423菌株中(使用转化到生物学平行样中的5μg质粒获得0个菌落)，这证实了存在天然pNF2质粒的影响。
pNF3质粒在其他菌株/物种中的转化能力的验证
为了说明在其他产溶剂梭状芽孢杆菌属(Clostridium)菌株中使用这种新质粒的可能性，本发明人进行了质粒pFW01、pNF3E和pNF3S在ABE菌株拜氏梭状芽孢杆菌(C.beijerinckii)NCIMB 8052(图33)中的转化效率的比较性分析。由于菌株NCIMB 8052天然对甲砜霉素有抗性，因此使用提供对壮观霉素的抗性的pNF3S代替pNF3C。
结果显示，菌株NCIMB 8052可以用基于pNF3的质粒转化，这证明了这些载体在广义上适用于拜氏梭状芽孢杆菌(C.beijerinckii)物种。
也在参比菌株丙酮丁醇梭状芽孢杆菌(C.acetobutylicum)DSM792中测试了基于pNF3的一套合成载体的适用性。因此，转化试验显示了用质粒pNF3C转化这种菌株的可能性(转化效率为每μg转化的DNA观察到3个菌落，相比较而言，对质粒pEC750C来说为120个菌落/μg)。
pNF3质粒与申请FR18/73492中描述的遗传工具的相容性的验证
专利申请FR18/73492描述了ΔcatB菌株和需要使用红霉素抗性基因和甲砜霉素抗性基因的双质粒CRISPR/Cas9系统的使用。为了证实新的一套pNF3质粒的重要性，将载体pNF3C转化到已经含有pCas9acr质粒的菌株ΔcatB中。一式两份进行的所述转化显示出0.625±0.125个菌落/μg DNA(平均值±标准误差)的转化效率，这证明了基于pNF3C的载体可以与pCas9acr相组合用于ΔcatB菌株中。
与这些结果相平行，可以将质粒pNF2的包含其复制起点的部分(SEQ ID NO：118)成功地重新用于产生新的一套可以在需要时修饰的穿梭载体(SEQ ID NO：119、123、124和125)，特别是允许它们在大肠杆菌中复制以及将它们重新引入到拜氏梭状芽孢杆菌(C.beijerinckii)DSM 6423中。这些新的载体具有对于在例如拜氏梭状芽孢杆菌(C.beijerinckii)DSM 6423及其衍生株中进行遗传编辑来说有利的转化效率，特别是使用包含两种不同核酸的CRISPR/Cas9工具的遗传编辑。
也已在另一种拜氏梭状芽孢杆菌(C.beijerinckii)菌株(NCIMB8052)和梭状芽孢杆菌属(Clostridium)物种(特别是丙酮丁醇梭状芽孢杆菌(C.acetobutylicum))中成功地测试了这些新的载体，证实了它们在厚壁菌门的其他生物体中的适用性。也在芽孢杆菌属(Bacillus)上进行了试验。
结论
这些结果显示，天然pNF2质粒的抑制显著提高了含有它的细菌的转化频率(对于pFW01来说提高约15倍，对于pEC750C来说提高约2000倍)。在已知难以转化的梭状芽孢杆菌属(Clostridium)细菌的情况下，特别是对于天然具有低转化效率(低于5个菌落/μg质粒)的拜氏梭状芽孢杆菌(C.beijerinckii)菌株DSM 6423来说，这个结果是特别有趣的。
参考文献
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<213> 人工序列
<220>
<223> 引物M13-rev
<400> 12
caggaaacag ctatgac 17
<210> 13
<211> 22
<212> DNA
<213> 人工序列
<220>
<223> 引物RH010
<400> 13
cggatattgc attaccagta gc 22
<210> 14
<211> 24
<212> DNA
<213> 人工序列
<220>
<223> 引物RH011
<400> 14
ttatcaatct cttacacatg gagc 24
<210> 15
<211> 24
<212> DNA
<213> 人工序列
<220>
<223> 引物RH025
<400> 15
tagtatgccg ccattattac gaca 24
<210> 16
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 引物RH134
<400> 16
gtcgacgtgg aattgtgagc 20
<210> 17
<211> 3658
<212> DNA
<213> 人工序列
<220>
<223> pEX-A258-ΔcatB
<400> 17
ctcgagctgc agcaaaaaaa gcaccgactc ggtgccactt tttcaagttg ataacggact 60
agccttattt taacttgcta tttctagctc taaaactgtg gtctctcttt tcgttgatgg 120
tggaatgata agggtttgca ccttaatttc tcctattgag aaaatcgtct cttctcagac 180
gtcaaaccat gttaatcatt gcttttatca aaaataggat ccactctatc attgatagag 240
tttgaaactc tatcattgat agagtataat atctttgttc atgtacatca tgctatctgt 300
gagttttaga gctagaaata gcaagttaaa ataaggctag tccgttatca acttgaaaaa 360
gtggcaccga gtcggtgctt tttttgaagc ttgtctttac acttttgccc attaattttt 420
gagttcctta tttttaggga gcttttatta tttttatcat gaaaatttca taaaatactc 480
ataaactaag gatgtcttca taatcagatt agtactccat tttcaatcca tttaatctgg 540
gaatatgata ttttaattac gtattattta agatatatta acgtgtaata taataccccg 600
caaatattaa ttatcacata catatccccc ctttattggg gcattttttg tacccattat 660
tttagtattg tgcagtactt aaataaaaaa atgccgcaaa ttcattttta ttgaataatg 720
cggtatttct tctattcttt atttttatta ctctataaat aatgtaatca agacatgact 780
atctaaatat atgatatctt aattcataat tcgggcctcc taaaaatttt cgtaattcta 840
ttttagaagg cttttttccg tgacctagcc atttcaatct cctttttaca atgatattta 900
cgctttagtt tattatagca cattctgtaa taccgaacta ttcaattttc agagaccatt 960
ttttattgat tcataactta agaatactac gaattactct aatattttac tttttcttat 1020
ctcttgttat tttaacatcg gaattactac taatattaat ttttattttt ccatccgcat 1080
ttgctccaac atttttttaa ctatactttc cttttgttaa taaattatgt tattgttgaa 1140
caatataaga aaagtgcgta acatttttta ttaaaaataa ttaggtattt ctatctgtgg 1200
ggtaccctcg aggtggcagc tctagagcta gcgaattctt tggtgaaatt gttatccgct 1260
cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg 1320
agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct 1380
gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg 1440
gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc 1500
ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg 1560
aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct 1620
ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca 1680
gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct 1740
cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc 1800
gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt 1860
tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc 1920
cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc 1980
cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg 2040
gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc 2100
agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag 2160
cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga 2220
tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat 2280
tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag 2340
ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat 2400
cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc 2460
cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat 2520
accgcgcgaa ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag 2580
ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg 2640
ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc 2700
tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca 2760
acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg 2820
tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc 2880
actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta 2940
ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc 3000
aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg 3060
ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc 3120
cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc 3180
aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat 3240
actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag 3300
cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc 3360
ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa 3420
taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg 3480
acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca 3540
agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc 3600
atcagagcag attgtactga gagtttggca attggtcgac ctcgagggcg cgcccgta 3658
<210> 18
<211> 660
<212> DNA
<213> 拜氏梭状芽孢杆菌（Clostridium beijerinckii）
<400> 18
atgaatttta atttgataga tattaatcat tggagtagaa agccatactt tgaacattat 60
ttaaacaatg tgaaatgtac ttatagtatg actgccaata tagaaataac tgatttattg 120
tatgaaatta aacttaaaaa tattaaattt tatcctaccc ttatttatat gattgcaact 180
gtggttaata agcataaaga attccgtatt tgttttgatc atgaaggtag tttaggatat 240
tgggatagca tgaatccaag ctatactatt tttcataaag aaaacgaaac attttcaagt 300
atttggacgg aatataacaa aagtttttta cgtttttata gtgattatct tgacgatata 360
aaaaactatg gaaatatcat gaagtttact ccgaaatcaa atgaacctga caatacattt 420
tctgtatcaa gcattccttg ggtgagtttt acaggattta acttgaatgt gtataatgaa 480
ggaacatatt taattcctat ttttactgca ggaaagtatt tcaaacaaga aaataaaata 540
tttattccta tatcaataca agtacatcat gctatctgtg acggttatca tgctagtaga 600
tttattaatg aaatgcaaga attagcattt agttttcaag aatggttaga aaataaataa 660
<210> 19
<211> 160
<212> DNA
<213> 人工序列
<220>
<223> 表达盒d的表达ARNg
<400> 19
actctatcat tgatagagtt tgaaactcta tcattgatag agtataatat ctttgttcat 60
gtacatcatg ctatctgtga gttttagagc tagaaatagc aagttaaaat aaggctagtc 120
cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt 160
<210> 20
<211> 808
<212> DNA
<213> 人工序列
<220>
<223> 编辑模板
<400> 20
gtctttacac ttttgcccat taatttttga gttccttatt tttagggagc ttttattatt 60
tttatcatga aaatttcata aaatactcat aaactaagga tgtcttcata atcagattag 120
tactccattt tcaatccatt taatctggga atatgatatt ttaattacgt attatttaag 180
atatattaac gtgtaatata ataccccgca aatattaatt atcacataca tatcccccct 240
ttattggggc attttttgta cccattattt tagtattgtg cagtacttaa ataaaaaaat 300
gccgcaaatt catttttatt gaataatgcg gtatttcttc tattctttat ttttattact 360
ctataaataa tgtaatcaag acatgactat ctaaatatat gatatcttaa ttcataattc 420
gggcctccta aaaattttcg taattctatt ttagaaggct tttttccgtg acctagccat 480
ttcaatctcc tttttacaat gatatttacg ctttagttta ttatagcaca ttctgtaata 540
ccgaactatt caattttcag agaccatttt ttattgattc ataacttaag aatactacga 600
attactctaa tattttactt tttcttatct cttgttattt taacatcgga attactacta 660
atattaattt ttatttttcc atccgcattt gctccaacat ttttttaact atactttcct 720
tttgttaata aattatgtta ttgttgaaca atataagaaa agtgcgtaac attttttatt 780
aaaaataatt aggtatttct atctgtgg 808
<210> 21
<211> 9954
<212> DNA
<213> 人工序列
<220>
<223> pCas9ind-ΔcatB
<400> 21
catggataaa aagtacagta ttggtctaga cataggaact aactctgttg ggtgggctgt 60
tataacagat gaatataaag ttccatcaaa aaaatttaaa gtattaggaa acactgatag 120
acattcaata aaaaaaaact tgataggtgc tttattattc gattcaggag agactgctga 180
agctacacgt ttaaaaagaa cagctagacg tagatataca agaagaaaaa ataggatatg 240
ttatcttcaa gaaattttta gtaatgaaat ggcaaaagtt gatgattcat tctttcacag 300
actagaagaa agtttcttag ttgaagaaga taagaagcat gaaagacacc ctatttttgg 360
taatatcgta gatgaagtag catatcatga gaagtatcca actatctatc atttaagaaa 420
gaaattagtt gattctacag ataaagctga tctgagatta atatatttag ctttagctca 480
tatgattaaa tttagaggac attttttaat agaaggtgat ttaaacccag acaacagcga 540
tgtagataaa ttatttatcc aattagttca aacttataat caattattcg aagagaatcc 600
aattaatgca agtggtgtag acgctaaggc tatattatca gctagattat caaaatctag 660
aagattagaa aatctaatag ctcaacttcc tggagaaaag aaaaatggac tttttgggaa 720
cctaatagct ctctcactcg gactaacacc aaattttaaa agcaattttg atcttgctga 780
agacgcaaag ttacaactat caaaggatac atacgatgat gatttagata atttgttagc 840
tcaaataggt gatcaatatg ctgatttgtt tcttgcagca aaaaacttaa gtgatgcaat 900
tttactatca gatatactta gagtaaatac agaaataaca aaggctcctt tatcagcaag 960
tatgattaaa cgatatgatg agcatcatca agatttaaca ttattaaagg cacttgtaag 1020
acaacaatta ccagaaaaat ataaagaaat tttctttgat caatctaaaa atggatatgc 1080
tggatatata gacggtggag caagtcaaga agagttttat aaatttataa agcctatttt 1140
agaaaaaatg gatggaactg aagaattact tgttaaactt aacagagaag atttacttag 1200
aaaacaaaga acttttgata atggttcaat tcctcaccaa attcatttag gagaattaca 1260
tgctatacta agaagacaag aagattttta tccatttctt aaagataata gagaaaaaat 1320
tgaaaaaatt ttaactttta gaataccata ttatgtagga ccacttgcaa ggggaaattc 1380
aagatttgca tggatgacta gaaaatcaga agaaactata accccgtgga attttgaaga 1440
agtagtagat aaaggagcta gtgctcaatc atttatagaa agaatgacaa attttgataa 1500
gaatcttcct aacgaaaagg ttttgccaaa gcatagcctt ctttatgagt attttacagt 1560
ttataatgag cttactaaag taaaatacgt tacagaagga atgagaaaac cagcattttt 1620
gtctggtgaa caaaagaaag caatagtaga cctattattt aaaacaaata ggaaggttac 1680
cgtaaagcaa cttaaagaag attacttcaa aaaaattgaa tgctttgata gtgttgaaat 1740
atcaggagtt gaagatagat ttaatgcttc acttggtaca tatcacgatc tcttaaaaat 1800
tataaaagat aaggattttt tagataatga agaaaatgaa gatattcttg aagatatagt 1860
attaacattg acactttttg aagatagaga aatgatagaa gaaagattaa aaacatatgc 1920
acatcttttt gatgataagg ttatgaagca acttaaaaga agaagatata caggttgggg 1980
acgtttgtca agaaagctaa ttaatggtat tagagataaa caatcaggaa agactattct 2040
cgattttctt aaatcagatg gatttgctaa tagaaacttt atgcaattaa ttcatgatga 2100
ttctcttact ttcaaagagg atattcaaaa ggctcaagtt tctggacaag gcgatagctt 2160
acacgaacac attgctaacc ttgcagggag ccccgctatc aaaaaaggaa ttttacaaac 2220
agttaaagtt gtagatgaac ttgttaaagt tatgggaaga cacaaacctg agaatatagt 2280
tatagaaatg gccagagaaa atcaaacaac acaaaaagga caaaaaaatt ctagagagag 2340
aatgaagaga attgaagaag gaataaaaga gctaggatca caaatattaa aagaacatcc 2400
agttgaaaat actcaattgc aaaatgaaaa gttatatttg tattacttac aaaatggaag 2460
agatatgtat gttgatcaag aactcgatat taatagatta agtgactatg atgttgatca 2520
tattgttcct caatcatttt taaaagatga ttcaatcgat aacaaagtat taactagatc 2580
agataaaaat agaggaaagt cagataatgt accatctgaa gaagttgtta aaaaaatgaa 2640
gaactattgg agacaacttt taaatgcaaa gctaattaca caaagaaaat ttgacaattt 2700
aacaaaagca gaaagaggag gattaagcga attagacaaa gctggattta taaaaagaca 2760
acttgttgag acaagacaaa taactaagca tgttgctcaa atacttgatt caagaatgaa 2820
tacaaaatat gatgaaaatg ataaattaat cagagaagta aaagtaataa cattaaagtc 2880
aaaattagta tcagatttca gaaaggattt tcaattttac aaagttcgtg aaataaataa 2940
ctatcatcat gctcatgatg catacttaaa tgctgttgta ggaactgctc ttattaagaa 3000
atatcctaaa ctagaaagcg aatttgttta tggagattat aaagtttatg atgtgcgcaa 3060
aatgatcgcg aaatccgaac aagaaatcgg taaggctaca gcaaaatatt tcttttatag 3120
taatataatg aattttttta agacagaaat aactttggct aatggtgaaa tcagaaaaag 3180
accacttatc gaaacaaatg gagagacagg agaaatagta tgggataaag gaagagattt 3240
tgctactgtt agaaaagtac taagtatgcc acaagtaaat atcgtaaaga aaactgaagt 3300
tcaaactgga ggtttctcta aggaatcaat tttacctaag agaaattcag ataagttaat 3360
tgcaaggaaa aaagattggg acccaaaaaa atacggtggt tttgatagtc caacagttgc 3420
ctatagtgtt cttgtagtag cgaaagttga gaaaggtaag tcaaaaaagt tgaaaagcgt 3480
aaaagaactt cttggtatca caattatgga aagatcttca tttgaaaaaa atccaattga 3540
ctttttagaa gctaagggtt ataaagaagt taaaaaggat ttaatcataa aactaccaaa 3600
gtatagtcta tttgaactcg aaaacggaag aaaacgaatg ctcgctagcg caggagaact 3660
tcaaaaagga aatgaacttg cgctgccatc aaagtatgta aatttcttat atttagcttc 3720
tcattatgag aaattaaaag gatcaccaga ggataatgaa caaaagcaac tatttgtaga 3780
acaacacaaa cattatttag atgaaataat agaacaaata tctgaatttt ctaaaagagt 3840
tatacttgcc gacgcaaatc tagataaggt gctttcagcg tataataaac acagagataa 3900
accaataaga gaacaagcag aaaacattat ccatcttttt acattaacta atcttggtgc 3960
accagctgca tttaagtact ttgatacaac aatagataga aaaagataca catctactaa 4020
agaagtatta gacgcaactt taatacatca atctattaca gggctttatg aaacaagaat 4080
tgatttaagt caactaggcg gagattaagt cgacaaagta ttgttaaaaa taactctgta 4140
gaattataaa ttagttctac agagttattt tttgacccgg gtatattgat aaaaataata 4200
atagtgggta taattaagtt gttaggaggt tagttagaat gatgtcaaga ttagataaaa 4260
gtaaagtgat taacagcgca ttagagctgc ttaatgaggt cggaatcgaa ggtttaacaa 4320
cccgtaaact cgcccagaag ctaggtgtag agcagcctac attgtattgg catgtaaaaa 4380
ataagcgggc tttgctcgac gccttagcca ttgagatgtt agataggcac catactcact 4440
tttgcccttt agaaggggaa agctggcaag attttttacg taataacgct aaaagtttta 4500
gatgtgcttt actaagtcat cgcgatggag caaaagtaca tttaggtaca cggcctacag 4560
aaaaacagta tgaaactctc gaaaatcaat tagccttttt atgccaacaa ggtttttcac 4620
tagagaatgc attatatgca ctcagcgctg tggggcattt tactttaggt tgcgtattgg 4680
aagatcaaga gcatcaagtc gctaaagaag aaagggaaac acctactact gatagtatgc 4740
cgccattatt acgacaagct atcgaattat ttgatcacca aggtgcagag ccagccttct 4800
tattcggcct tgaattgatc atatgcggat tagaaaaaca acttaaatgt gaaagtgggt 4860
cttaaaagca gcataacctt tttccgtgat ggtaacttca cggtaaccaa gatgtcgagt 4920
tgagctcgaa ttcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca 4980
caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag 5040
tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt 5100
cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc 5160
gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg 5220
tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa 5280
agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg 5340
cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga 5400
ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg 5460
tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg 5520
gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc 5580
gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg 5640
gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca 5700
ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt 5760
ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag 5820
ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg 5880
gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc 5940
ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt 6000
tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt 6060
ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccag gtccactgcc 6120
gggcctcttg cgggatcaaa agaaaaacga aatgatacac caatcagtgc aaaaaaagat 6180
ataatgggag ataagacggt tcgtgttcgt gctgacttgc accatatcat aaaaatcgaa 6240
acagcaaaga atggcggaaa cgtaaaagaa gttatggaaa taagacttag aagcaaactt 6300
aagagtgtgt tgatagtgca gtatcttaaa attttgtata ataggaattg aagttaaatt 6360
agatgctaaa aatttgtaat taagaaggag tgattacatg aacaaaaata taaaatattc 6420
tcaaaacttt ttaacgagtg aaaaagtact caaccaaata ataaaacaat tgaatttaaa 6480
agaaaccgat accgtttacg aaattggaac aggtaaaggg catttaacga cgaaactggc 6540
taaaataagt aaacaggtaa cgtctattga attagacagt catctattca acttatcgtc 6600
agaaaaatta aaactgaata ctcgtgtcac tttaattcac caagatattc tacagtttca 6660
attccctaac aaacagaggt ataaaattgt tgggagtatt ccttaccatt taagcacaca 6720
aattattaaa aaagtggttt ttgaaagcca tgcgtctgac atctatctga ttgttgaaga 6780
aggattctac aagcgtacct tggatattca ccgaacacta gggttgctct tgcacactca 6840
agtctcgatt cagcaattgc ttaagctgcc agcggaatgc tttcatccta aaccaaaagt 6900
aaacagtgtc ttaataaaac ttacccgcca taccacagat gttccagata aatattggaa 6960
gctatatacg tactttgttt caaaatgggt caatcgagaa tatcgtcaac tgtttactaa 7020
aaatcagttt catcaagcaa tgaaacacgc caaagtaaac aatttaagta ccgttactta 7080
tgagcaagta ttgtctattt ttaatagtta tctattattt aacgggagga aataattcta 7140
tgagtcccta ggcaggcctc cgccattatt tttttgaaca attgacaatt catttcttat 7200
tttttattaa gtgatagtca aaaggcataa cagtgctgaa tagaaagaaa tttacagaaa 7260
agaaaattat agaatttagt atgattaatt atactcattt atgaatgttt aattgaatac 7320
aaaaaaaaat acttgttatg tattcaatta cgggttaaaa tatagacaag ttgaaaaatt 7380
taataaaaaa ataagtcctc agctcttata tattaagcta ccaacttagt atataagcca 7440
aaacttaaat gtgctaccaa cacatcaagc cgttagagaa ctctatctat agcaatattt 7500
caaatgtacc gacatacaag agaaacatta actatatata ttcaatttat gagattatct 7560
taacagatat aaatgtaaat tgcaataagt aagatttaga agtttatagc ctttgtgtat 7620
tggaagcagt acgcaaaggc ttttttattt gataaaaatt agaagtatat ttattttttc 7680
ataattaatt tatgaaaatg aaagggggtg agcaaagtga cagaggaaag cagtatctta 7740
tcaaataaca aggtattagc aatatcatta ttgactttag cagtaaacat tatgactttt 7800
atagtgcttg tagctaagta gtacgaaagg gggagcttta aaaagctcct tggaatacat 7860
agaattcata aattaattta tgaaaagaag ggcgtatatg aaaacttgta aaaattgcaa 7920
agagtttatt aaagatactg aaatatgcaa aatacattcg ttgatgattc atgataaaac 7980
agtagcaacc tattgcagta aatacaatga gtcaagatgt ttacataaag ggaaagtcca 8040
atgtattaat tgttcaaaga tgaaccgata tggatggtgt gccataaaaa tgagatgttt 8100
tacagaggaa gaacagaaaa aagaacgtac atgcattaaa tattatgcaa ggagctttaa 8160
aaaagctcat gtaaagaaga gtaaaaagaa aaaataattt atttattaat ttaatattga 8220
gagtgccgac acagtatgca ctaaaaaata tatctgtggt gtagtgagcc gatacaaaag 8280
gatagtcact cgcattttca taatacatct tatgttatga ttatgtgtcg gtgggacttc 8340
acgacgaaaa cccacaataa aaaaagagtt cggggtaggg ttaagcatag ttgaggcaac 8400
taaacaatca agctaggata tgcagtagca gaccgtaagg tcgttgttta ggtgtgttgt 8460
aatacatacg ctattaagat gtaaaaatac ggataccaat gaagggaaaa gtataatttt 8520
tggatgtagt ttgtttgttc atctatgggc aaactacgtc caaagccgtt tccaaatctg 8580
ctaaaaagta tatcctttct aaaatcaaag tcaagtatga aatcataaat aaagtttaat 8640
tttgaagtta ttatgatatt atgtttttct attaaaataa attaagtata tagaatagtt 8700
taataatagt atatacttaa tgtgataagt gtctgacagt gtcacagaaa ggatgattgt 8760
tatggattat aagcggctcg aggacgtcaa accatgttaa tcattgcttt tatcaaaaat 8820
aggatccact ctatcattga tagagtttga aactctatca ttgatagagt ataatatctt 8880
tgttcatgta catcatgcta tctgtgagtt ttagagctag aaatagcaag ttaaaataag 8940
gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt gaagcttgtc 9000
tttacacttt tgcccattaa tttttgagtt ccttattttt agggagcttt tattattttt 9060
atcatgaaaa tttcataaaa tactcataaa ctaaggatgt cttcataatc agattagtac 9120
tccattttca atccatttaa tctgggaata tgatatttta attacgtatt atttaagata 9180
tattaacgtg taatataata ccccgcaaat attaattatc acatacatat ccccccttta 9240
ttggggcatt ttttgtaccc attattttag tattgtgcag tacttaaata aaaaaatgcc 9300
gcaaattcat ttttattgaa taatgcggta tttcttctat tctttatttt tattactcta 9360
taaataatgt aatcaagaca tgactatcta aatatatgat atcttaattc ataattcggg 9420
cctcctaaaa attttcgtaa ttctatttta gaaggctttt ttccgtgacc tagccatttc 9480
aatctccttt ttacaatgat atttacgctt tagtttatta tagcacattc tgtaataccg 9540
aactattcaa ttttcagaga ccatttttta ttgattcata acttaagaat actacgaatt 9600
actctaatat tttacttttt cttatctctt gttattttaa catcggaatt actactaata 9660
ttaattttta tttttccatc cgcatttgct ccaacatttt tttaactata ctttcctttt 9720
gttaataaat tatgttattg ttgaacaata taagaaaagt gcgtaacatt ttttattaaa 9780
aataattagg tatttctatc tgtggggtac cctcgaggtg gcagctctag agctagcgaa 9840
ttctttggtg aaattgctcg agtccctatc agtgatagat tgaaactcta tcattgatag 9900
agtataatat ctttgttcat tagagcgata aacttgaatt tgagagggaa cttc 9954
<210> 22
<211> 8874
<212> DNA
<213> 人工序列
<220>
<223> pCas9ind
<400> 22
catggataaa aagtacagta ttggtctaga cataggaact aactctgttg ggtgggctgt 60
tataacagat gaatataaag ttccatcaaa aaaatttaaa gtattaggaa acactgatag 120
acattcaata aaaaaaaact tgataggtgc tttattattc gattcaggag agactgctga 180
agctacacgt ttaaaaagaa cagctagacg tagatataca agaagaaaaa ataggatatg 240
ttatcttcaa gaaattttta gtaatgaaat ggcaaaagtt gatgattcat tctttcacag 300
actagaagaa agtttcttag ttgaagaaga taagaagcat gaaagacacc ctatttttgg 360
taatatcgta gatgaagtag catatcatga gaagtatcca actatctatc atttaagaaa 420
gaaattagtt gattctacag ataaagctga tctgagatta atatatttag ctttagctca 480
tatgattaaa tttagaggac attttttaat agaaggtgat ttaaacccag acaacagcga 540
tgtagataaa ttatttatcc aattagttca aacttataat caattattcg aagagaatcc 600
aattaatgca agtggtgtag acgctaaggc tatattatca gctagattat caaaatctag 660
aagattagaa aatctaatag ctcaacttcc tggagaaaag aaaaatggac tttttgggaa 720
cctaatagct ctctcactcg gactaacacc aaattttaaa agcaattttg atcttgctga 780
agacgcaaag ttacaactat caaaggatac atacgatgat gatttagata atttgttagc 840
tcaaataggt gatcaatatg ctgatttgtt tcttgcagca aaaaacttaa gtgatgcaat 900
tttactatca gatatactta gagtaaatac agaaataaca aaggctcctt tatcagcaag 960
tatgattaaa cgatatgatg agcatcatca agatttaaca ttattaaagg cacttgtaag 1020
acaacaatta ccagaaaaat ataaagaaat tttctttgat caatctaaaa atggatatgc 1080
tggatatata gacggtggag caagtcaaga agagttttat aaatttataa agcctatttt 1140
agaaaaaatg gatggaactg aagaattact tgttaaactt aacagagaag atttacttag 1200
aaaacaaaga acttttgata atggttcaat tcctcaccaa attcatttag gagaattaca 1260
tgctatacta agaagacaag aagattttta tccatttctt aaagataata gagaaaaaat 1320
tgaaaaaatt ttaactttta gaataccata ttatgtagga ccacttgcaa ggggaaattc 1380
aagatttgca tggatgacta gaaaatcaga agaaactata accccgtgga attttgaaga 1440
agtagtagat aaaggagcta gtgctcaatc atttatagaa agaatgacaa attttgataa 1500
gaatcttcct aacgaaaagg ttttgccaaa gcatagcctt ctttatgagt attttacagt 1560
ttataatgag cttactaaag taaaatacgt tacagaagga atgagaaaac cagcattttt 1620
gtctggtgaa caaaagaaag caatagtaga cctattattt aaaacaaata ggaaggttac 1680
cgtaaagcaa cttaaagaag attacttcaa aaaaattgaa tgctttgata gtgttgaaat 1740
atcaggagtt gaagatagat ttaatgcttc acttggtaca tatcacgatc tcttaaaaat 1800
tataaaagat aaggattttt tagataatga agaaaatgaa gatattcttg aagatatagt 1860
attaacattg acactttttg aagatagaga aatgatagaa gaaagattaa aaacatatgc 1920
acatcttttt gatgataagg ttatgaagca acttaaaaga agaagatata caggttgggg 1980
acgtttgtca agaaagctaa ttaatggtat tagagataaa caatcaggaa agactattct 2040
cgattttctt aaatcagatg gatttgctaa tagaaacttt atgcaattaa ttcatgatga 2100
ttctcttact ttcaaagagg atattcaaaa ggctcaagtt tctggacaag gcgatagctt 2160
acacgaacac attgctaacc ttgcagggag ccccgctatc aaaaaaggaa ttttacaaac 2220
agttaaagtt gtagatgaac ttgttaaagt tatgggaaga cacaaacctg agaatatagt 2280
tatagaaatg gccagagaaa atcaaacaac acaaaaagga caaaaaaatt ctagagagag 2340
aatgaagaga attgaagaag gaataaaaga gctaggatca caaatattaa aagaacatcc 2400
agttgaaaat actcaattgc aaaatgaaaa gttatatttg tattacttac aaaatggaag 2460
agatatgtat gttgatcaag aactcgatat taatagatta agtgactatg atgttgatca 2520
tattgttcct caatcatttt taaaagatga ttcaatcgat aacaaagtat taactagatc 2580
agataaaaat agaggaaagt cagataatgt accatctgaa gaagttgtta aaaaaatgaa 2640
gaactattgg agacaacttt taaatgcaaa gctaattaca caaagaaaat ttgacaattt 2700
aacaaaagca gaaagaggag gattaagcga attagacaaa gctggattta taaaaagaca 2760
acttgttgag acaagacaaa taactaagca tgttgctcaa atacttgatt caagaatgaa 2820
tacaaaatat gatgaaaatg ataaattaat cagagaagta aaagtaataa cattaaagtc 2880
aaaattagta tcagatttca gaaaggattt tcaattttac aaagttcgtg aaataaataa 2940
ctatcatcat gctcatgatg catacttaaa tgctgttgta ggaactgctc ttattaagaa 3000
atatcctaaa ctagaaagcg aatttgttta tggagattat aaagtttatg atgtgcgcaa 3060
aatgatcgcg aaatccgaac aagaaatcgg taaggctaca gcaaaatatt tcttttatag 3120
taatataatg aattttttta agacagaaat aactttggct aatggtgaaa tcagaaaaag 3180
accacttatc gaaacaaatg gagagacagg agaaatagta tgggataaag gaagagattt 3240
tgctactgtt agaaaagtac taagtatgcc acaagtaaat atcgtaaaga aaactgaagt 3300
tcaaactgga ggtttctcta aggaatcaat tttacctaag agaaattcag ataagttaat 3360
tgcaaggaaa aaagattggg acccaaaaaa atacggtggt tttgatagtc caacagttgc 3420
ctatagtgtt cttgtagtag cgaaagttga gaaaggtaag tcaaaaaagt tgaaaagcgt 3480
aaaagaactt cttggtatca caattatgga aagatcttca tttgaaaaaa atccaattga 3540
ctttttagaa gctaagggtt ataaagaagt taaaaaggat ttaatcataa aactaccaaa 3600
gtatagtcta tttgaactcg aaaacggaag aaaacgaatg ctcgctagcg caggagaact 3660
tcaaaaagga aatgaacttg cgctgccatc aaagtatgta aatttcttat atttagcttc 3720
tcattatgag aaattaaaag gatcaccaga ggataatgaa caaaagcaac tatttgtaga 3780
acaacacaaa cattatttag atgaaataat agaacaaata tctgaatttt ctaaaagagt 3840
tatacttgcc gacgcaaatc tagataaggt gctttcagcg tataataaac acagagataa 3900
accaataaga gaacaagcag aaaacattat ccatcttttt acattaacta atcttggtgc 3960
accagctgca tttaagtact ttgatacaac aatagataga aaaagataca catctactaa 4020
agaagtatta gacgcaactt taatacatca atctattaca gggctttatg aaacaagaat 4080
tgatttaagt caactaggcg gagattaagt cgacaaagta ttgttaaaaa taactctgta 4140
gaattataaa ttagttctac agagttattt tttgacccgg gtatattgat aaaaataata 4200
atagtgggta taattaagtt gttaggaggt tagttagaat gatgtcaaga ttagataaaa 4260
gtaaagtgat taacagcgca ttagagctgc ttaatgaggt cggaatcgaa ggtttaacaa 4320
cccgtaaact cgcccagaag ctaggtgtag agcagcctac attgtattgg catgtaaaaa 4380
ataagcgggc tttgctcgac gccttagcca ttgagatgtt agataggcac catactcact 4440
tttgcccttt agaaggggaa agctggcaag attttttacg taataacgct aaaagtttta 4500
gatgtgcttt actaagtcat cgcgatggag caaaagtaca tttaggtaca cggcctacag 4560
aaaaacagta tgaaactctc gaaaatcaat tagccttttt atgccaacaa ggtttttcac 4620
tagagaatgc attatatgca ctcagcgctg tggggcattt tactttaggt tgcgtattgg 4680
aagatcaaga gcatcaagtc gctaaagaag aaagggaaac acctactact gatagtatgc 4740
cgccattatt acgacaagct atcgaattat ttgatcacca aggtgcagag ccagccttct 4800
tattcggcct tgaattgatc atatgcggat tagaaaaaca acttaaatgt gaaagtgggt 4860
cttaaaagca gcataacctt tttccgtgat ggtaacttca cggtaaccaa gatgtcgagt 4920
tgagctcgaa ttcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca 4980
caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag 5040
tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt 5100
cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc 5160
gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg 5220
tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa 5280
agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg 5340
cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga 5400
ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg 5460
tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg 5520
gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc 5580
gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg 5640
gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca 5700
ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt 5760
ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag 5820
ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg 5880
gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc 5940
ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt 6000
tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt 6060
ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccag gtccactgcc 6120
gggcctcttg cgggatcaaa agaaaaacga aatgatacac caatcagtgc aaaaaaagat 6180
ataatgggag ataagacggt tcgtgttcgt gctgacttgc accatatcat aaaaatcgaa 6240
acagcaaaga atggcggaaa cgtaaaagaa gttatggaaa taagacttag aagcaaactt 6300
aagagtgtgt tgatagtgca gtatcttaaa attttgtata ataggaattg aagttaaatt 6360
agatgctaaa aatttgtaat taagaaggag tgattacatg aacaaaaata taaaatattc 6420
tcaaaacttt ttaacgagtg aaaaagtact caaccaaata ataaaacaat tgaatttaaa 6480
agaaaccgat accgtttacg aaattggaac aggtaaaggg catttaacga cgaaactggc 6540
taaaataagt aaacaggtaa cgtctattga attagacagt catctattca acttatcgtc 6600
agaaaaatta aaactgaata ctcgtgtcac tttaattcac caagatattc tacagtttca 6660
attccctaac aaacagaggt ataaaattgt tgggagtatt ccttaccatt taagcacaca 6720
aattattaaa aaagtggttt ttgaaagcca tgcgtctgac atctatctga ttgttgaaga 6780
aggattctac aagcgtacct tggatattca ccgaacacta gggttgctct tgcacactca 6840
agtctcgatt cagcaattgc ttaagctgcc agcggaatgc tttcatccta aaccaaaagt 6900
aaacagtgtc ttaataaaac ttacccgcca taccacagat gttccagata aatattggaa 6960
gctatatacg tactttgttt caaaatgggt caatcgagaa tatcgtcaac tgtttactaa 7020
aaatcagttt catcaagcaa tgaaacacgc caaagtaaac aatttaagta ccgttactta 7080
tgagcaagta ttgtctattt ttaatagtta tctattattt aacgggagga aataattcta 7140
tgagtcccta ggcaggcctc cgccattatt tttttgaaca attgacaatt catttcttat 7200
tttttattaa gtgatagtca aaaggcataa cagtgctgaa tagaaagaaa tttacagaaa 7260
agaaaattat agaatttagt atgattaatt atactcattt atgaatgttt aattgaatac 7320
aaaaaaaaat acttgttatg tattcaatta cgggttaaaa tatagacaag ttgaaaaatt 7380
taataaaaaa ataagtcctc agctcttata tattaagcta ccaacttagt atataagcca 7440
aaacttaaat gtgctaccaa cacatcaagc cgttagagaa ctctatctat agcaatattt 7500
caaatgtacc gacatacaag agaaacatta actatatata ttcaatttat gagattatct 7560
taacagatat aaatgtaaat tgcaataagt aagatttaga agtttatagc ctttgtgtat 7620
tggaagcagt acgcaaaggc ttttttattt gataaaaatt agaagtatat ttattttttc 7680
ataattaatt tatgaaaatg aaagggggtg agcaaagtga cagaggaaag cagtatctta 7740
tcaaataaca aggtattagc aatatcatta ttgactttag cagtaaacat tatgactttt 7800
atagtgcttg tagctaagta gtacgaaagg gggagcttta aaaagctcct tggaatacat 7860
agaattcata aattaattta tgaaaagaag ggcgtatatg aaaacttgta aaaattgcaa 7920
agagtttatt aaagatactg aaatatgcaa aatacattcg ttgatgattc atgataaaac 7980
agtagcaacc tattgcagta aatacaatga gtcaagatgt ttacataaag ggaaagtcca 8040
atgtattaat tgttcaaaga tgaaccgata tggatggtgt gccataaaaa tgagatgttt 8100
tacagaggaa gaacagaaaa aagaacgtac atgcattaaa tattatgcaa ggagctttaa 8160
aaaagctcat gtaaagaaga gtaaaaagaa aaaataattt atttattaat ttaatattga 8220
gagtgccgac acagtatgca ctaaaaaata tatctgtggt gtagtgagcc gatacaaaag 8280
gatagtcact cgcattttca taatacatct tatgttatga ttatgtgtcg gtgggacttc 8340
acgacgaaaa cccacaataa aaaaagagtt cggggtaggg ttaagcatag ttgaggcaac 8400
taaacaatca agctaggata tgcagtagca gaccgtaagg tcgttgttta ggtgtgttgt 8460
aatacatacg ctattaagat gtaaaaatac ggataccaat gaagggaaaa gtataatttt 8520
tggatgtagt ttgtttgttc atctatgggc aaactacgtc caaagccgtt tccaaatctg 8580
ctaaaaagta tatcctttct aaaatcaaag tcaagtatga aatcataaat aaagtttaat 8640
tttgaagtta ttatgatatt atgtttttct attaaaataa attaagtata tagaatagtt 8700
taataatagt atatacttaa tgtgataagt gtctgacagt gtcacagaaa ggatgattgt 8760
tatggattat aagcggctcg agtccctatc agtgatagat tgaaactcta tcattgatag 8820
agtataatat ctttgttcat tagagcgata aacttgaatt tgagagggaa cttc 8874
<210> 23
<211> 10534
<212> DNA
<213> 人工序列
<220>
<223> pCas9acr
<400> 23
cgaattcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc 60
cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct 120
aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc 180
agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt 240
ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag 300
ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca 360
tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt 420
tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc 480
gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct 540
ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg 600
tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca 660
agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact 720
atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta 780
acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta 840
actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct 900
tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt 960
tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga 1020
tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca 1080
tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat 1140
caatctaaag tatatatgag taaacttggt ctgacagtta ccaggtccac tgccgggcct 1200
cttgcgggat caaaagaaaa acgaaatgat acaccaatca gtgcaaaaaa agatataatg 1260
ggagataaga cggttcgtgt tcgtgctgac ttgcaccata tcataaaaat cgaaacagca 1320
aagaatggcg gaaacgtaaa agaagttatg gaaataagac ttagaagcaa acttaagagt 1380
gtgttgatag tgcagtatct taaaattttg tataatagga attgaagtta aattagatgc 1440
taaaaatttg taattaagaa ggagtgatta catgaacaaa aatataaaat attctcaaaa 1500
ctttttaacg agtgaaaaag tactcaacca aataataaaa caattgaatt taaaagaaac 1560
cgataccgtt tacgaaattg gaacaggtaa agggcattta acgacgaaac tggctaaaat 1620
aagtaaacag gtaacgtcta ttgaattaga cagtcatcta ttcaacttat cgtcagaaaa 1680
attaaaactg aatactcgtg tcactttaat tcaccaagat attctacagt ttcaattccc 1740
taacaaacag aggtataaaa ttgttgggag tattccttac catttaagca cacaaattat 1800
taaaaaagtg gtttttgaaa gccatgcgtc tgacatctat ctgattgttg aagaaggatt 1860
ctacaagcgt accttggata ttcaccgaac actagggttg ctcttgcaca ctcaagtctc 1920
gattcagcaa ttgcttaagc tgccagcgga atgctttcat cctaaaccaa aagtaaacag 1980
tgtcttaata aaacttaccc gccataccac agatgttcca gataaatatt ggaagctata 2040
tacgtacttt gtttcaaaat gggtcaatcg agaatatcgt caactgttta ctaaaaatca 2100
gtttcatcaa gcaatgaaac acgccaaagt aaacaattta agtaccgtta cttatgagca 2160
agtattgtct atttttaata gttatctatt atttaacggg aggaaataat tctatgagtc 2220
cctaggcagg cctccgccat tatttttttg aacaattgac aattcatttc ttatttttta 2280
ttaagtgata gtcaaaaggc ataacagtgc tgaatagaaa gaaatttaca gaaaagaaaa 2340
ttatagaatt tagtatgatt aattatactc atttatgaat gtttaattga atacaaaaaa 2400
aaatacttgt tatgtattca attacgggtt aaaatataga caagttgaaa aatttaataa 2460
aaaaataagt cctcagctct tatatattaa gctaccaact tagtatataa gccaaaactt 2520
aaatgtgcta ccaacacatc aagccgttag agaactctat ctatagcaat atttcaaatg 2580
taccgacata caagagaaac attaactata tatattcaat ttatgagatt atcttaacag 2640
atataaatgt aaattgcaat aagtaagatt tagaagttta tagcctttgt gtattggaag 2700
cagtacgcaa aggctttttt atttgataaa aattagaagt atatttattt tttcataatt 2760
aatttatgaa aatgaaaggg ggtgagcaaa gtgacagagg aaagcagtat cttatcaaat 2820
aacaaggtat tagcaatatc attattgact ttagcagtaa acattatgac ttttatagtg 2880
cttgtagcta agtagtacga aagggggagc tttaaaaagc tccttggaat acatagaatt 2940
cataaattaa tttatgaaaa gaagggcgta tatgaaaact tgtaaaaatt gcaaagagtt 3000
tattaaagat actgaaatat gcaaaataca ttcgttgatg attcatgata aaacagtagc 3060
aacctattgc agtaaataca atgagtcaag atgtttacat aaagggaaag tccaatgtat 3120
taattgttca aagatgaacc gatatggatg gtgtgccata aaaatgagat gttttacaga 3180
ggaagaacag aaaaaagaac gtacatgcat taaatattat gcaaggagct ttaaaaaagc 3240
tcatgtaaag aagagtaaaa agaaaaaata atttatttat taatttaata ttgagagtgc 3300
cgacacagta tgcactaaaa aatatatctg tggtgtagtg agccgataca aaaggatagt 3360
cactcgcatt ttcataatac atcttatgtt atgattatgt gtcggtggga cttcacgacg 3420
aaaacccaca ataaaaaaag agttcggggt agggttaagc atagttgagg caactaaaca 3480
atcaagctag gatatgcagt agcagaccgt aaggtcgttg tttaggtgtg ttgtaataca 3540
tacgctatta agatgtaaaa atacggatac caatgaaggg aaaagtataa tttttggatg 3600
tagtttgttt gttcatctat gggcaaacta cgtccaaagc cgtttccaaa tctgctaaaa 3660
agtatatcct ttctaaaatc aaagtcaagt atgaaatcat aaataaagtt taattttgaa 3720
gttattatga tattatgttt ttctattaaa ataaattaag tatatagaat agtttaataa 3780
tagtatatac ttaatgtgat aagtgtctga cagtgtcaca gaaaggatga ttgttatgga 3840
ttataagcgg ctcgagtccc tatcagtgat agattgaaac tctatcattg atagagtata 3900
atatctttgt tcattagagc gataaacttg aatttgagag ggaacttcca tggataaaaa 3960
gtacagtatt ggtctagaca taggaactaa ctctgttggg tgggctgtta taacagatga 4020
atataaagtt ccatcaaaaa aatttaaagt attaggaaac actgatagac attcaataaa 4080
aaaaaacttg ataggtgctt tattattcga ttcaggagag actgctgaag ctacacgttt 4140
aaaaagaaca gctagacgta gatatacaag aagaaaaaat aggatatgtt atcttcaaga 4200
aatttttagt aatgaaatgg caaaagttga tgattcattc tttcacagac tagaagaaag 4260
tttcttagtt gaagaagata agaagcatga aagacaccct atttttggta atatcgtaga 4320
tgaagtagca tatcatgaga agtatccaac tatctatcat ttaagaaaga aattagttga 4380
ttctacagat aaagctgatc tgagattaat atatttagct ttagctcata tgattaaatt 4440
tagaggacat tttttaatag aaggtgattt aaacccagac aacagcgatg tagataaatt 4500
atttatccaa ttagttcaaa cttataatca attattcgaa gagaatccaa ttaatgcaag 4560
tggtgtagac gctaaggcta tattatcagc tagattatca aaatctagaa gattagaaaa 4620
tctaatagct caacttcctg gagaaaagaa aaatggactt tttgggaacc taatagctct 4680
ctcactcgga ctaacaccaa attttaaaag caattttgat cttgctgaag acgcaaagtt 4740
acaactatca aaggatacat acgatgatga tttagataat ttgttagctc aaataggtga 4800
tcaatatgct gatttgtttc ttgcagcaaa aaacttaagt gatgcaattt tactatcaga 4860
tatacttaga gtaaatacag aaataacaaa ggctccttta tcagcaagta tgattaaacg 4920
atatgatgag catcatcaag atttaacatt attaaaggca cttgtaagac aacaattacc 4980
agaaaaatat aaagaaattt tctttgatca atctaaaaat ggatatgctg gatatataga 5040
cggtggagca agtcaagaag agttttataa atttataaag cctattttag aaaaaatgga 5100
tggaactgaa gaattacttg ttaaacttaa cagagaagat ttacttagaa aacaaagaac 5160
ttttgataat ggttcaattc ctcaccaaat tcatttagga gaattacatg ctatactaag 5220
aagacaagaa gatttttatc catttcttaa agataataga gaaaaaattg aaaaaatttt 5280
aacttttaga ataccatatt atgtaggacc acttgcaagg ggaaattcaa gatttgcatg 5340
gatgactaga aaatcagaag aaactataac cccgtggaat tttgaagaag tagtagataa 5400
aggagctagt gctcaatcat ttatagaaag aatgacaaat tttgataaga atcttcctaa 5460
cgaaaaggtt ttgccaaagc atagccttct ttatgagtat tttacagttt ataatgagct 5520
tactaaagta aaatacgtta cagaaggaat gagaaaacca gcatttttgt ctggtgaaca 5580
aaagaaagca atagtagacc tattatttaa aacaaatagg aaggttaccg taaagcaact 5640
taaagaagat tacttcaaaa aaattgaatg ctttgatagt gttgaaatat caggagttga 5700
agatagattt aatgcttcac ttggtacata tcacgatctc ttaaaaatta taaaagataa 5760
ggatttttta gataatgaag aaaatgaaga tattcttgaa gatatagtat taacattgac 5820
actttttgaa gatagagaaa tgatagaaga aagattaaaa acatatgcac atctttttga 5880
tgataaggtt atgaagcaac ttaaaagaag aagatataca ggttggggac gtttgtcaag 5940
aaagctaatt aatggtatta gagataaaca atcaggaaag actattctcg attttcttaa 6000
atcagatgga tttgctaata gaaactttat gcaattaatt catgatgatt ctcttacttt 6060
caaagaggat attcaaaagg ctcaagtttc tggacaaggc gatagcttac acgaacacat 6120
tgctaacctt gcagggagcc ccgctatcaa aaaaggaatt ttacaaacag ttaaagttgt 6180
agatgaactt gttaaagtta tgggaagaca caaacctgag aatatagtta tagaaatggc 6240
cagagaaaat caaacaacac aaaaaggaca aaaaaattct agagagagaa tgaagagaat 6300
tgaagaagga ataaaagagc taggatcaca aatattaaaa gaacatccag ttgaaaatac 6360
tcaattgcaa aatgaaaagt tatatttgta ttacttacaa aatggaagag atatgtatgt 6420
tgatcaagaa ctcgatatta atagattaag tgactatgat gttgatcata ttgttcctca 6480
atcattttta aaagatgatt caatcgataa caaagtatta actagatcag ataaaaatag 6540
aggaaagtca gataatgtac catctgaaga agttgttaaa aaaatgaaga actattggag 6600
acaactttta aatgcaaagc taattacaca aagaaaattt gacaatttaa caaaagcaga 6660
aagaggagga ttaagcgaat tagacaaagc tggatttata aaaagacaac ttgttgagac 6720
aagacaaata actaagcatg ttgctcaaat acttgattca agaatgaata caaaatatga 6780
tgaaaatgat aaattaatca gagaagtaaa agtaataaca ttaaagtcaa aattagtatc 6840
agatttcaga aaggattttc aattttacaa agttcgtgaa ataaataact atcatcatgc 6900
tcatgatgca tacttaaatg ctgttgtagg aactgctctt attaagaaat atcctaaact 6960
agaaagcgaa tttgtttatg gagattataa agtttatgat gtgcgcaaaa tgatcgcgaa 7020
atccgaacaa gaaatcggta aggctacagc aaaatatttc ttttatagta atataatgaa 7080
tttttttaag acagaaataa ctttggctaa tggtgaaatc agaaaaagac cacttatcga 7140
aacaaatgga gagacaggag aaatagtatg ggataaagga agagattttg ctactgttag 7200
aaaagtacta agtatgccac aagtaaatat cgtaaagaaa actgaagttc aaactggagg 7260
tttctctaag gaatcaattt tacctaagag aaattcagat aagttaattg caaggaaaaa 7320
agattgggac ccaaaaaaat acggtggttt tgatagtcca acagttgcct atagtgttct 7380
tgtagtagcg aaagttgaga aaggtaagtc aaaaaagttg aaaagcgtaa aagaacttct 7440
tggtatcaca attatggaaa gatcttcatt tgaaaaaaat ccaattgact ttttagaagc 7500
taagggttat aaagaagtta aaaaggattt aatcataaaa ctaccaaagt atagtctatt 7560
tgaactcgaa aacggaagaa aacgaatgct cgctagcgca ggagaacttc aaaaaggaaa 7620
tgaacttgcg ctgccatcaa agtatgtaaa tttcttatat ttagcttctc attatgagaa 7680
attaaaagga tcaccagagg ataatgaaca aaagcaacta tttgtagaac aacacaaaca 7740
ttatttagat gaaataatag aacaaatatc tgaattttct aaaagagtta tacttgccga 7800
cgcaaatcta gataaggtgc tttcagcgta taataaacac agagataaac caataagaga 7860
acaagcagaa aacattatcc atctttttac attaactaat cttggtgcac cagctgcatt 7920
taagtacttt gatacaacaa tagatagaaa aagatacaca tctactaaag aagtattaga 7980
cgcaacttta atacatcaat ctattacagg gctttatgaa acaagaattg atttaagtca 8040
actaggcgga gattaagtcg acaaagtatt gttaaaaata actctgtaga attataaatt 8100
agttctacag agttattttt tgacccgggt atattgataa aaataataat agtgggtata 8160
attaagttgt taggaggtta gttagaatga tgtcaagatt agataaaagt aaagtgatta 8220
acagcgcatt agagctgctt aatgaggtcg gaatcgaagg tttaacaacc cgtaaactcg 8280
cccagaagct aggtgtagag cagcctacat tgtattggca tgtaaaaaat aagcgggctt 8340
tgctcgacgc cttagccatt gagatgttag ataggcacca tactcacttt tgccctttag 8400
aaggggaaag ctggcaagat tttttacgta ataacgctaa aagttttaga tgtgctttac 8460
taagtcatcg cgatggagca aaagtacatt taggtacacg gcctacagaa aaacagtatg 8520
aaactctcga aaatcaatta gcctttttat gccaacaagg tttttcacta gagaatgcat 8580
tatatgcact cagcgctgtg gggcatttta ctttaggttg cgtattggaa gatcaagagc 8640
atcaagtcgc taaagaagaa agggaaacac ctactactga tagtatgccg ccattattac 8700
gacaagctat cgaattattt gatcaccaag gtgcagagcc agccttctta ttcggccttg 8760
aattgatcat atgcggatta gaaaaacaac ttaaatgtga aagtgggtct taaaagcagc 8820
ataacctttt tccgtgatgg taacttcacg gtaaccaaga tgtcgagttg agctcttagt 8880
tcaactcact ttttaaggtg attgtttgca tgtcattata aaattcttct tcatcctcgt 8940
attcttgatt ccaaccgttt ttaaatgcag atatgaattt ttcaactatt gattcatttt 9000
cactttcaga aattacatac tcgtttccat cattattaac tctaataatt agctgtgtta 9060
tactattgct atccgtacca ctcaatttca ctgtgtaatc tttgtttttt atttctctaa 9120
ttaagtcatt aatattcatt tcagccctcc tgtgaaattg ttatccgctc acaattccac 9180
gtcgactacc gcggattcta gattctgcag tatcttcatg gtattcattt tttaatatca 9240
ttttaccctc ccaatacatt taaaataatt atgtattcat gaaacatgat tgtatattta 9300
agaaacataa ttccatataa atcatttttc aaaatagttt ttacccataa ttaaatgtta 9360
atatgtaaat taatctttta gaatagttaa aaagttctaa aatatgttat aatgtttctt 9420
ataatcttat aaattttaat aactaatata taaagatatt tctttaaaat attcttatat 9480
ttagaagaat ttattttaaa ataaaaagct tttatgttga taaactgctt tgcaaagctc 9540
tcatgtaaat gtttaatata agactactat aaaattggct aattttatag gttaggaggt 9600
agaaatgcaa atattgtgga aaaagtatgt taaagaaaac tttgaaatga atgtagatga 9660
atgtggtata gaacaaggta taccaggatt aggatataac tatgaagtat tgaaaaatgc 9720
tgttattcat tacgtaacta agggatatgg aacttttaaa tttaatggta aggtatataa 9780
cttaaaacaa ggtgatattt ttatactact aaaaggtatg caagttgagt atgtggcttc 9840
tattgatgat ccttgggaat actactggat aggatttagt ggttcaaatg ctaatgagta 9900
tttaaataga acttctatta ctaactcctg tgttgctaat tgtgaagaaa actcaaaaat 9960
tccacagata atattaaata tgtgcgaaat atcaaaaact tataatcctt caagatctga 10020
tgacatacta ttactaaaag aactttactc attattgtac gcacttatag aagaattccc 10080
aaaacctttt gaatacaaag ataaggaatt acacacatat attcaagatg ctcttaattt 10140
cattaattct aattacatgc atagcataac tgttcaagaa attgctgatt atgtgaactt 10200
aagtagaagt tatttatata aaatgttcat aaaaaacctt ggaatttctc ctcaaagata 10260
tttaataaac cttagaatgt acaaagccac ccttttatta aaaagcacta aacttcctat 10320
aggagaagtc gcaagtagtg taggttatag tgactccctg ttattttcaa aaactttttc 10380
aaaacatttt tcaatgtctc cactaaatta cagaaataat caagtaaata aaccaagtat 10440
ataaatttaa aatacagctt taaaacaaaa aaatttcaaa aataaaaagt ataacagagg 10500
cgtaaattaa aacctctgtt atactttttg agct 10534
<210> 24
<211> 5754
<212> DNA
<213> 人工序列
<220>
<223> pEC750S-uppHR
<400> 24
ataaggtacc aggaattaga gcagcgctat gttcagatac atttagtgct catgcaacaa 60
gagaacataa taatgctaat atattaacta tgggtcaaag ggttgttgga gcaggtcttg 120
ctttagatat agtaaaaaca tttatatcag ctaaatttga aggagatagg caccaaaaaa 180
gaatagataa gatttcagat attgaaaaaa agtatacaca ttagaaaaaa gcagctatgc 240
tgcaaataag atcaatttat attagaaaaa agcagctatg ctgcaaataa gatcaattta 300
tattagaaaa aagcagctat gctgcaaata agatcaattt atattagaaa aaagcagcta 360
tgctacaaat aagatcaatt tatattagaa aaaagtagct atgctgcaac aatattaatt 420
tatattacta gaaagctaaa tggggtatat aaatataaag ggctataaat actaaaagca 480
aacttggagg aataataatg gtctagagct ggagatagat tatttggtac taagtaatta 540
gtaatctatt agaattaaaa gctatctaca taagtttctg aatgacccaa gataatttta 600
ctggggggaa tatagaaaat ggagagacga gataagaaaa attattactt ggatattgct 660
gaaacagttt tagagagagg aacctgtcta aggagaaact atggttctat aattgttaaa 720
aatgatgaaa taatttctac tggatacaca ggagcaccta gaggtagaaa aaattgcatg 780
gatttgaata gttgcataag agaaaagttg aaagttccaa gaggtactca ttatgagttg 840
tgtaggagtg tacatagtga agctaatgca ataataagcg cttcgagctc gaattcgtaa 900
tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata 960
cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta 1020
attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa 1080
tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg 1140
ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag 1200
gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa 1260
ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 1320
cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 1380
ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 1440
accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 1500
catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 1560
gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 1620
tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 1680
agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 1740
actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 1800
gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc 1860
aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg 1920
gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca 1980
aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt 2040
atatatgagt aaacttggtc tgacagttac caaagctagc ttaatactag tatatactta 2100
atgtgataag tgtctgacag ctgaccggtc taaagaggtc cgccaatgaa atctataaat 2160
aaactaaatt aagtttattt aattaacaac tatggatata aaataggtac taatcaaaat 2220
agtgaggagg atatatttga atacatacga acaaattaat aaagtgaaaa aaatacttcg 2280
gaaacattta aaaaataacc ttattggtac ttacatgttt ggatcaggag ttgagagtgg 2340
actaaaacca aatagtgatc ttgacttttt agtcgtcgta tctgaaccat tgacagatca 2400
aagtaaagaa atacttatac aaaaaattag acctatttca aagaaaatag gagataaaag 2460
caacttacga tatattgaat taacaattat tattcagcaa gaaatggtac cgtggaatca 2520
tcctcccaaa caagaattta tttatggaga atggttacaa gagctttatg aacaaggata 2580
cattcctcag aaggaattaa attcagattt aaccataatg ctttaccaag caaaacgaaa 2640
aaataaaaga atatacggaa attatgactt agaggaatta ctacctgata ttccattttc 2700
tgatgtgaga agagccatta tggattcgtc agaggaatta atagataatt atcaggatga 2760
tgaaaccaac tctatattaa ctttatgccg tatgatttta actatggaca cgggtaaaat 2820
cataccaaaa gatattgcgg gaaatgcagt ggctgaatct tctccattag aacataggga 2880
gagaattttg ttagcagttc gtagttatct tggagagaat attgaatgga ctaatgaaaa 2940
tgtaaattta actataaact atttaaataa cagattaaaa aaattataaa aaaattgaaa 3000
aaatggtgga aacacttttt tcaatttttt tgttttatta tttaatattt gggaaatatt 3060
cattctaatt ggtaatcaga ttttagaagt tgttaacttc aggtttgtct gtaactaaaa 3120
actagtattt aacctaggat caaaaaaatt tccaataatc ccactctaag ccacaaacac 3180
gccctataaa atcccgcttt aatcccactt tgagacacat gtaatattac tttacgccct 3240
agtatagtga taatttttta cattcaatgc cacgcaaaaa aataaagggg cactataata 3300
aaagttcctt cggaactaac taaagtaaaa aattatcttt acaacctccc caaaaaaaag 3360
aacaggtaca aagtacccta taatacaagc gtaaaaaaaa tgagggtaaa aataaaaaaa 3420
taaaaaaata aaaaaataaa aaaataaaaa aataaaaaaa taaaaaaata taaaaataaa 3480
aaaatataaa aataaaaaaa tataaaaata aaaaaataaa aaaatataaa aataaaaaaa 3540
taaaaaaata taaaaatatt ttttatttaa agtttgaaaa aaattttttt atattatata 3600
atctttgaag aaaagaatat aaaaaatgag cctttataaa agcccatttt ttttcatata 3660
cgtaatatga cgttctaatg tttttattgg tacttctaac attagagtaa tttctttatt 3720
tttaaagcct ttttctttaa gggcttttat tttttttctt aatacattta attcctcttt 3780
ttttgttgct tttcctttag cttttaattg ctcttgataa ttttttttac ctctaatatt 3840
ttctcttctc ttatattcct ttttagaaat tattattgtc atatattttt gttcttcttc 3900
tgtaatttct aataactcta taagagtttc attcttatac ttatattgct tatttttatc 3960
taaataacat ctttcagcac ttctagttgc tcttataact tctctttcac ttaaatgttg 4020
tctaaacata ctattaagtt ctaaaacatc atttaatgcc ttctcaatgt cttctgtaaa 4080
gctacaaaga taatatctat ataaaaataa tataagctct ctgtgtcctt ttaaatcata 4140
ttctcttagt tcacaaagtt ttattatgtc ttgtattctt ccataatata aacttctttc 4200
tctataaata taatttattt tgcttggtct accctttttc ctttcatatg gttttaattc 4260
aggtaaaaat ccattttgta tttctcttaa gtcataaata tattcgtact catctaatat 4320
attgactact gtttttgatt tagagtttat acttcctgga actcttaata ttctcgttgc 4380
atctaaggct tgtctatctg ctccaaagta ttttaattga ttatataaat attcttgaac 4440
cgctttccat aatggtaatg ctttactagg tactgcattt attatccata ttaaatacat 4500
tcctcttcca ctatctatta catagtttgg tataggaata ctttgattaa aataattctt 4560
ttctaagtcc attaatacct ggtctttagt tttgccagtt ttataataat ccaagtctat 4620
aaacagtgta tttaactctt ttatattttc taatcgccta cacggcttat aaaaggtatt 4680
tagagttata tagatatttt catcactcat atctaaatct tttaattcag cgtatttata 4740
gtgccattgg ctatatcctt ttttatctat aacgctcctg gttatccacc ctttacttct 4800
actatgaata ttatctatat agttcttttt attcagcttt aatgcgtttc tcacttattc 4860
acctcccctt ctgtaaaact aagaaaatta tatcatattt tcaataatta ttaactattc 4920
ttaaactctt aataaaaaat agagtaagtc cccaattgaa acttaatcta ttttttatgt 4980
tttaatttat tatttttatt aaaatatttt aaactaaatt aaatgattct ttttaatttt 5040
ttactatttc attccataat atattactat aattatttac aaataatatt tcttcatttg 5100
taatatttag atgatttact aattttagtt tttatatatt aaataattaa tgtataattt 5160
atataaaaaa tcaaaggagc ttataaatta tgattatttc caaagatact aaagatttaa 5220
tttttttcaa ttttaacaat actttttgta atattatgtt taaatttaat tgtatttttt 5280
tcatataata aagccgttga agtaaaccaa tccattttcc ttatgatgtt attattaaat 5340
ttaagtttta taataatatc tttattatat ttattgtttt taaaaaaact agtgaaattt 5400
ctagtgaaat ttccggcttt attaaactta tttttaggaa ttttattttc attttcatct 5460
ttacaggatt tgattatatc tttaaatatg ttttatcaaa tattatcttt ttctaaattt 5520
atatatattt ttattatatt tattattata tatattttat ttttaagttt ctttctaaca 5580
gctattaaaa agaaacttaa aaataaaaac acgtactcta aaccaataaa taaaactatt 5640
tttattattg ctgccttgat tggaatagtt tttagtaaaa ttaatttcaa tattccacaa 5700
tattatatta taagctagca ggcctcgaga tctccatgga cgcgtgacgt cgac 5754
<210> 25
<211> 884
<212> DNA
<213> 人工序列
<220>
<223> 修复模板
<400> 25
ataaggtacc aggaattaga gcagcgctat gttcagatac atttagtgct catgcaacaa 60
gagaacataa taatgctaat atattaacta tgggtcaaag ggttgttgga gcaggtcttg 120
ctttagatat agtaaaaaca tttatatcag ctaaatttga aggagatagg caccaaaaaa 180
gaatagataa gatttcagat attgaaaaaa agtatacaca ttagaaaaaa gcagctatgc 240
tgcaaataag atcaatttat attagaaaaa agcagctatg ctgcaaataa gatcaattta 300
tattagaaaa aagcagctat gctgcaaata agatcaattt atattagaaa aaagcagcta 360
tgctacaaat aagatcaatt tatattagaa aaaagtagct atgctgcaac aatattaatt 420
tatattacta gaaagctaaa tggggtatat aaatataaag ggctataaat actaaaagca 480
aacttggagg aataataatg gtctagagct ggagatagat tatttggtac taagtaatta 540
gtaatctatt agaattaaaa gctatctaca taagtttctg aatgacccaa gataatttta 600
ctggggggaa tatagaaaat ggagagacga gataagaaaa attattactt ggatattgct 660
gaaacagttt tagagagagg aacctgtcta aggagaaact atggttctat aattgttaaa 720
aatgatgaaa taatttctac tggatacaca ggagcaccta gaggtagaaa aaattgcatg 780
gatttgaata gttgcataag agaaaagttg aaagttccaa gaggtactca ttatgagttg 840
tgtaggagtg tacatagtga agctaatgca ataataagcg cttc 884
<210> 26
<211> 500
<212> DNA
<213> 人工序列
<220>
<223> upp基因上游片段
<400> 26
ataaggtacc aggaattaga gcagcgctat gttcagatac atttagtgct catgcaacaa 60
gagaacataa taatgctaat atattaacta tgggtcaaag ggttgttgga gcaggtcttg 120
ctttagatat agtaaaaaca tttatatcag ctaaatttga aggagatagg caccaaaaaa 180
gaatagataa gatttcagat attgaaaaaa agtatacaca ttagaaaaaa gcagctatgc 240
tgcaaataag atcaatttat attagaaaaa agcagctatg ctgcaaataa gatcaattta 300
tattagaaaa aagcagctat gctgcaaata agatcaattt atattagaaa aaagcagcta 360
tgctacaaat aagatcaatt tatattagaa aaaagtagct atgctgcaac aatattaatt 420
tatattacta gaaagctaaa tggggtatat aaatataaag ggctataaat actaaaagca 480
aacttggagg aataataatg 500
<210> 27
<211> 377
<212> DNA
<213> 人工序列
<220>
<223> upp基因下游片段
<400> 27
gctggagata gattatttgg tactaagtaa ttagtaatct attagaatta aaagctatct 60
acataagttt ctgaatgacc caagataatt ttactggggg gaatatagaa aatggagaga 120
cgagataaga aaaattatta cttggatatt gctgaaacag ttttagagag aggaacctgt 180
ctaaggagaa actatggttc tataattgtt aaaaatgatg aaataatttc tactggatac 240
acaggagcac ctagaggtag aaaaaattgc atggatttga atagttgcat aagagaaaag 300
ttgaaagttc caagaggtac tcattatgag ttgtgtagga gtgtacatag tgaagctaat 360
gcaataataa gcgcttc 377
<210> 28
<211> 2666
<212> DNA
<213> 人工序列
<220>
<223> pEX-A2-gRNA-upp
<400> 28
ctcgagtatt tttgataaaa gcaatgatta acatggtttg acgtctgaga agagacgatt 60
ttctcaatag gagaaattaa ggtgcaaacc cttatcattc caccatgatc cacctgtagc 120
aagcatgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga 180
aaaagtggca ccgagtcggt gctttttttg ccatggacct gcttttgctc gcttggatcc 240
gaattcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca 300
taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct 360
cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac 420
gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc 480
tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt 540
tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg 600
ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg 660
agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat 720
accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta 780
ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct 840
gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc 900
ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa 960
gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg 1020
taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag 1080
tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt 1140
gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta 1200
cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc 1260
agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca 1320
cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa 1380
cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat 1440
ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct 1500
taccatctgg ccccagtgct gcaatgatac cgcgactccc acgctcaccg gctccagatt 1560
tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat 1620
ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta 1680
atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg 1740
gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt 1800
tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg 1860
cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg 1920
taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc 1980
ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa 2040
ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac 2100
cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt 2160
ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg 2220
gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa 2280
gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata 2340
aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca 2400
ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc 2460
gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt 2520
gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg 2580
ggtgtcgggg ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccaat 2640
tgggtaccga gctcgcggcc gcaagc 2666
<210> 29
<211> 203
<212> DNA
<213> 人工序列
<220>
<223> gRNA表达盒
<400> 29
tatttttgat aaaagcaatg attaacatgg tttgacgtct gagaagagac gattttctca 60
ataggagaaa ttaaggtgca aacccttatc attccaccat gatccacctg tagcaagcat 120
gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt 180
ggcaccgagt cggtgctttt ttt 203
<210> 30
<211> 100
<212> DNA
<213> 人工序列
<220>
<223> 组成型启动子
<400> 30
tatttttgat aaaagcaatg attaacatgg tttgacgtct gagaagagac gattttctca 60
ataggagaaa ttaaggtgca aacccttatc attccaccat 100
<210> 31
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 靶向upp的前间区序列
<400> 31
gatccacctg tagcaagcat 20
<210> 32
<211> 5954
<212> DNA
<213> 人工序列
<220>
<223> pEC750S-Δupp
<400> 32
ataaggtacc aggaattaga gcagcgctat gttcagatac atttagtgct catgcaacaa 60
gagaacataa taatgctaat atattaacta tgggtcaaag ggttgttgga gcaggtcttg 120
ctttagatat agtaaaaaca tttatatcag ctaaatttga aggagatagg caccaaaaaa 180
gaatagataa gatttcagat attgaaaaaa agtatacaca ttagaaaaaa gcagctatgc 240
tgcaaataag atcaatttat attagaaaaa agcagctatg ctgcaaataa gatcaattta 300
tattagaaaa aagcagctat gctgcaaata agatcaattt atattagaaa aaagcagcta 360
tgctacaaat aagatcaatt tatattagaa aaaagtagct atgctgcaac aatattaatt 420
tatattacta gaaagctaaa tggggtatat aaatataaag ggctataaat actaaaagca 480
aacttggagg aataataatg gtctagagct ggagatagat tatttggtac taagtaatta 540
gtaatctatt agaattaaaa gctatctaca taagtttctg aatgacccaa gataatttta 600
ctggggggaa tatagaaaat ggagagacga gataagaaaa attattactt ggatattgct 660
gaaacagttt tagagagagg aacctgtcta aggagaaact atggttctat aattgttaaa 720
aatgatgaaa taatttctac tggatacaca ggagcaccta gaggtagaaa aaattgcatg 780
gatttgaata gttgcataag agaaaagttg aaagttccaa gaggtactca ttatgagttg 840
tgtaggagtg tacatagtga agctaatgca ataataagcg cttcgagctc gaattcgtaa 900
tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata 960
cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta 1020
attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa 1080
tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg 1140
ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag 1200
gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa 1260
ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 1320
cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 1380
ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 1440
accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 1500
catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 1560
gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 1620
tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 1680
agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 1740
actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 1800
gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc 1860
aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg 1920
gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca 1980
aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt 2040
atatatgagt aaacttggtc tgacagttac caaagctagc ttaatactag tatatactta 2100
atgtgataag tgtctgacag ctgaccggtc taaagaggtc cgccaatgaa atctataaat 2160
aaactaaatt aagtttattt aattaacaac tatggatata aaataggtac taatcaaaat 2220
agtgaggagg atatatttga atacatacga acaaattaat aaagtgaaaa aaatacttcg 2280
gaaacattta aaaaataacc ttattggtac ttacatgttt ggatcaggag ttgagagtgg 2340
actaaaacca aatagtgatc ttgacttttt agtcgtcgta tctgaaccat tgacagatca 2400
aagtaaagaa atacttatac aaaaaattag acctatttca aagaaaatag gagataaaag 2460
caacttacga tatattgaat taacaattat tattcagcaa gaaatggtac cgtggaatca 2520
tcctcccaaa caagaattta tttatggaga atggttacaa gagctttatg aacaaggata 2580
cattcctcag aaggaattaa attcagattt aaccataatg ctttaccaag caaaacgaaa 2640
aaataaaaga atatacggaa attatgactt agaggaatta ctacctgata ttccattttc 2700
tgatgtgaga agagccatta tggattcgtc agaggaatta atagataatt atcaggatga 2760
tgaaaccaac tctatattaa ctttatgccg tatgatttta actatggaca cgggtaaaat 2820
cataccaaaa gatattgcgg gaaatgcagt ggctgaatct tctccattag aacataggga 2880
gagaattttg ttagcagttc gtagttatct tggagagaat attgaatgga ctaatgaaaa 2940
tgtaaattta actataaact atttaaataa cagattaaaa aaattataaa aaaattgaaa 3000
aaatggtgga aacacttttt tcaatttttt tgttttatta tttaatattt gggaaatatt 3060
cattctaatt ggtaatcaga ttttagaagt tgttaacttc aggtttgtct gtaactaaaa 3120
actagtattt aacctaggat caaaaaaatt tccaataatc ccactctaag ccacaaacac 3180
gccctataaa atcccgcttt aatcccactt tgagacacat gtaatattac tttacgccct 3240
agtatagtga taatttttta cattcaatgc cacgcaaaaa aataaagggg cactataata 3300
aaagttcctt cggaactaac taaagtaaaa aattatcttt acaacctccc caaaaaaaag 3360
aacaggtaca aagtacccta taatacaagc gtaaaaaaaa tgagggtaaa aataaaaaaa 3420
taaaaaaata aaaaaataaa aaaataaaaa aataaaaaaa taaaaaaata taaaaataaa 3480
aaaatataaa aataaaaaaa tataaaaata aaaaaataaa aaaatataaa aataaaaaaa 3540
taaaaaaata taaaaatatt ttttatttaa agtttgaaaa aaattttttt atattatata 3600
atctttgaag aaaagaatat aaaaaatgag cctttataaa agcccatttt ttttcatata 3660
cgtaatatga cgttctaatg tttttattgg tacttctaac attagagtaa tttctttatt 3720
tttaaagcct ttttctttaa gggcttttat tttttttctt aatacattta attcctcttt 3780
ttttgttgct tttcctttag cttttaattg ctcttgataa ttttttttac ctctaatatt 3840
ttctcttctc ttatattcct ttttagaaat tattattgtc atatattttt gttcttcttc 3900
tgtaatttct aataactcta taagagtttc attcttatac ttatattgct tatttttatc 3960
taaataacat ctttcagcac ttctagttgc tcttataact tctctttcac ttaaatgttg 4020
tctaaacata ctattaagtt ctaaaacatc atttaatgcc ttctcaatgt cttctgtaaa 4080
gctacaaaga taatatctat ataaaaataa tataagctct ctgtgtcctt ttaaatcata 4140
ttctcttagt tcacaaagtt ttattatgtc ttgtattctt ccataatata aacttctttc 4200
tctataaata taatttattt tgcttggtct accctttttc ctttcatatg gttttaattc 4260
aggtaaaaat ccattttgta tttctcttaa gtcataaata tattcgtact catctaatat 4320
attgactact gtttttgatt tagagtttat acttcctgga actcttaata ttctcgttgc 4380
atctaaggct tgtctatctg ctccaaagta ttttaattga ttatataaat attcttgaac 4440
cgctttccat aatggtaatg ctttactagg tactgcattt attatccata ttaaatacat 4500
tcctcttcca ctatctatta catagtttgg tataggaata ctttgattaa aataattctt 4560
ttctaagtcc attaatacct ggtctttagt tttgccagtt ttataataat ccaagtctat 4620
aaacagtgta tttaactctt ttatattttc taatcgccta cacggcttat aaaaggtatt 4680
tagagttata tagatatttt catcactcat atctaaatct tttaattcag cgtatttata 4740
gtgccattgg ctatatcctt ttttatctat aacgctcctg gttatccacc ctttacttct 4800
actatgaata ttatctatat agttcttttt attcagcttt aatgcgtttc tcacttattc 4860
acctcccctt ctgtaaaact aagaaaatta tatcatattt tcaataatta ttaactattc 4920
ttaaactctt aataaaaaat agagtaagtc cccaattgaa acttaatcta ttttttatgt 4980
tttaatttat tatttttatt aaaatatttt aaactaaatt aaatgattct ttttaatttt 5040
ttactatttc attccataat atattactat aattatttac aaataatatt tcttcatttg 5100
taatatttag atgatttact aattttagtt tttatatatt aaataattaa tgtataattt 5160
atataaaaaa tcaaaggagc ttataaatta tgattatttc caaagatact aaagatttaa 5220
tttttttcaa ttttaacaat actttttgta atattatgtt taaatttaat tgtatttttt 5280
tcatataata aagccgttga agtaaaccaa tccattttcc ttatgatgtt attattaaat 5340
ttaagtttta taataatatc tttattatat ttattgtttt taaaaaaact agtgaaattt 5400
ctagtgaaat ttccggcttt attaaactta tttttaggaa ttttattttc attttcatct 5460
ttacaggatt tgattatatc tttaaatatg ttttatcaaa tattatcttt ttctaaattt 5520
atatatattt ttattatatt tattattata tatattttat ttttaagttt ctttctaaca 5580
gctattaaaa agaaacttaa aaataaaaac acgtactcta aaccaataaa taaaactatt 5640
tttattattg ctgccttgat tggaatagtt tttagtaaaa ttaatttcaa tattccacaa 5700
tattatatta taagctagca cgcctcgagt atttttgata aaagcaatga ttaacatggt 5760
ttgacgtctg agaagagacg attttctcaa taggagaaat taaggtgcaa acccttatca 5820
ttccaccatg atccacctgt agcaagcatg ttttagagct agaaatagca agttaaaata 5880
aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttgccatgga 5940
cgcgtgacgt cgac 5954
<210> 33
<211> 5853
<212> DNA
<213> 人工序列
<220>
<223> pEC750C-Δupp
<400> 33
atcaaaaaaa tttccaataa tcccactcta agccacaaac acgccctata aaatcccgct 60
ttaatcccac tttgagacac atgtaatatt actttacgcc ctagtatagt gataattttt 120
tacattcaat gccacgcaaa aaaataaagg ggcactataa taaaagttcc ttcggaacta 180
actaaagtaa aaaattatct ttacaacctc cccaaaaaaa agaacaggta caaagtaccc 240
tataatacaa gcgtaaaaaa aatgagggta aaaataaaaa aataaaaaaa taaaaaaata 300
aaaaaataaa aaaataaaaa aataaaaaaa tataaaaata aaaaaatata aaaataaaaa 360
aatataaaaa taaaaaaata aaaaaatata aaaataaaaa aataaaaaaa tataaaaata 420
ttttttattt aaagtttgaa aaaaattttt ttatattata taatctttga agaaaagaat 480
ataaaaaatg agcctttata aaagcccatt ttttttcata tacgtaatat gacgttctaa 540
tgtttttatt ggtacttcta acattagagt aatttcttta tttttaaagc ctttttcttt 600
aagggctttt attttttttc ttaatacatt taattcctct ttttttgttg cttttccttt 660
agcttttaat tgctcttgat aatttttttt acctctaata ttttctcttc tcttatattc 720
ctttttagaa attattattg tcatatattt ttgttcttct tctgtaattt ctaataactc 780
tataagagtt tcattcttat acttatattg cttattttta tctaaataac atctttcagc 840
acttctagtt gctcttataa cttctctttc acttaaatgt tgtctaaaca tactattaag 900
ttctaaaaca tcatttaatg ccttctcaat gtcttctgta aagctacaaa gataatatct 960
atataaaaat aatataagct ctctgtgtcc ttttaaatca tattctctta gttcacaaag 1020
ttttattatg tcttgtattc ttccataata taaacttctt tctctataaa tataatttat 1080
tttgcttggt ctaccctttt tcctttcata tggttttaat tcaggtaaaa atccattttg 1140
tatttctctt aagtcataaa tatattcgta ctcatctaat atattgacta ctgtttttga 1200
tttagagttt atacttcctg gaactcttaa tattctcgtt gcatctaagg cttgtctatc 1260
tgctccaaag tattttaatt gattatataa atattcttga accgctttcc ataatggtaa 1320
tgctttacta ggtactgcat ttattatcca tattaaatac attcctcttc cactatctat 1380
tacatagttt ggtataggaa tactttgatt aaaataattc ttttctaagt ccattaatac 1440
ctggtcttta gttttgccag ttttataata atccaagtct ataaacagtg tatttaactc 1500
ttttatattt tctaatcgcc tacacggctt ataaaaggta tttagagtta tatagatatt 1560
ttcatcactc atatctaaat cttttaattc agcgtattta tagtgccatt ggctatatcc 1620
ttttttatct ataacgctcc tggttatcca ccctttactt ctactatgaa tattatctat 1680
atagttcttt ttattcagct ttaatgcgtt tctcacttat tcacctcccc ttctgtaaaa 1740
ctaagaaaat tatatcatat tttcaataat tattaactat tcttaaactc ttaataaaaa 1800
atagagtaag tccccaattg aaacttaatc tattttttat gttttaattt attattttta 1860
ttaaaatatt ttaaactaaa ttaaatgatt ctttttaatt ttttactatt tcattccata 1920
atatattact ataattattt acaaataata tttcttcatt tgtaatattt agatgattta 1980
ctaattttag tttttatata ttaaataatt aatgtataat ttatataaaa aatcaaagga 2040
gcttataaat tatgattatt tccaaagata ctaaagattt aatttttttc aattttaaca 2100
atactttttg taatattatg tttaaattta attgtatttt tttcatataa taaagccgtt 2160
gaagtaaacc aatccatttt ccttatgatg ttattattaa atttaagttt tataataata 2220
tctttattat atttattgtt tttaaaaaaa ctagtgaaat ttctagtgaa atttccggct 2280
ttattaaact tatttttagg aattttattt tcattttcat ctttacagga tttgattata 2340
tctttaaata tgttttatca aatattatct ttttctaaat ttatatatat ttttattata 2400
tttattatta tatatatttt atttttaagt ttctttctaa cagctattaa aaagaaactt 2460
aaaaataaaa acacgtactc taaaccaata aataaaacta tttttattat tgctgccttg 2520
attggaatag tttttagtaa aattaatttc aatattccac aatattatat tataagctag 2580
cacgcctcga gtatttttga taaaagcaat gattaacatg gtttgacgtc tgagaagaga 2640
cgattttctc aataggagaa attaaggtgc aaacccttat cattccacca tgatccacct 2700
gtagcaagca tgttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa 2760
cttgaaaaag tggcaccgag tcggtgcttt ttttgccatg gacgcgtgac gtcgacataa 2820
ggtaccagga attagagcag cgctatgttc agatacattt agtgctcatg caacaagaga 2880
acataataat gctaatatat taactatggg tcaaagggtt gttggagcag gtcttgcttt 2940
agatatagta aaaacattta tatcagctaa atttgaagga gataggcacc aaaaaagaat 3000
agataagatt tcagatattg aaaaaaagta tacacattag aaaaaagcag ctatgctgca 3060
aataagatca atttatatta gaaaaaagca gctatgctgc aaataagatc aatttatatt 3120
agaaaaaagc agctatgctg caaataagat caatttatat tagaaaaaag cagctatgct 3180
acaaataaga tcaatttata ttagaaaaaa gtagctatgc tgcaacaata ttaatttata 3240
ttactagaaa gctaaatggg gtatataaat ataaagggct ataaatacta aaagcaaact 3300
tggaggaata ataatggtct agagctggag atagattatt tggtactaag taattagtaa 3360
tctattagaa ttaaaagcta tctacataag tttctgaatg acccaagata attttactgg 3420
ggggaatata gaaaatggag agacgagata agaaaaatta ttacttggat attgctgaaa 3480
cagttttaga gagaggaacc tgtctaagga gaaactatgg ttctataatt gttaaaaatg 3540
atgaaataat ttctactgga tacacaggag cacctagagg tagaaaaaat tgcatggatt 3600
tgaatagttg cataagagaa aagttgaaag ttccaagagg tactcattat gagttgtgta 3660
ggagtgtaca tagtgaagct aatgcaataa taagcgcttc gagctcgaat tcgtaatcat 3720
ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag 3780
ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg 3840
cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa 3900
tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca 3960
ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg 4020
taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc 4080
agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc 4140
cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac 4200
tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc 4260
tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata 4320
gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc 4380
acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca 4440
acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag 4500
cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta 4560
gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg 4620
gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc 4680
agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt 4740
ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa 4800
ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat 4860
atgagtaaac ttggtctgac agttaccaaa gctagcttaa tactagtata tacttaatgt 4920
gataagtgtc tgacagctga ccggtctaaa gaggtcccta gcgcctacgg ggaatttgta 4980
tcgataaggg gtacaaattc ccactaagcg ctcggccggg gatcgatccc cgggtacgta 5040
cccggcagtt tttctttttc ggcaagtgtt caagaagtta ttaagtcggg agtgcagtcg 5100
aagtgggcaa gttgaaaaat tcacaaaaat gtggtataat atctttgttc attagagcga 5160
taaacttgaa tttgagaggg aacttagatg gtatttgaaa aaattgataa aaatagttgg 5220
aacagaaaag agtattttga ccactacttt gcaagtgtac cttgtaccta cagcatgacc 5280
gttaaagtgg atatcacaca aataaaggaa aagggaatga aactatatcc tgcaatgctt 5340
tattatattg caatgattgt aaaccgccat tcagagttta ggacggcaat caatcaagat 5400
ggtgaattgg ggatatatga tgagatgata ccaagctata caatatttca caatgatact 5460
gaaacatttt ccagcctttg gactgagtgt aagtctgact ttaaatcatt tttagcagat 5520
tatgaaagtg atacgcaacg gtatggaaac aatcatagaa tggaaggaaa gccaaatgct 5580
ccggaaaaca tttttaatgt atctatgata ccgtggtcaa ccttcgatgg ctttaatctg 5640
aatttgcaga aaggatatga ttatttgatt cctattttta ctatggggaa atattataaa 5700
gaagataaca aaattatact tcctttggca attcaagttc atcacgcagt atgtgacgga 5760
tttcacattt gccgttttgt aaacgaattg caggaattga taaatagtta acttcaggtt 5820
tgtctgtaac taaaaactag tatttaacct agg 5853
<210> 34
<211> 4966
<212> DNA
<213> 人工序列
<220>
<223> pGRNA-pNF2
<400> 34
agctcggtac ccggggatcc tctagagtcg acgtcacgcg tccatggaga tctcgaggcg 60
tgctagctta taatataata ttgtggaata ttgaaattaa ttttactaaa aactattcca 120
atcaaggcag caataataaa aatagtttta tttattggtt tagagtacgt gtttttattt 180
ttaagtttct ttttaatagc tgttagaaag aaacttaaaa ataaaatata tataataata 240
aatataataa aaatatatat aaatttagaa aaagataata tttgataaaa catatttaaa 300
gatataatca aatcctgtaa agatgaaaat gaaaataaaa ttcctaaaaa taagtttaat 360
aaagccggaa atttcactag aaatttcact agttttttta aaaacaataa atataataaa 420
gatattatta taaaacttaa atttaataat aacatcataa ggaaaatgga ttggtttact 480
tcaacggctt tattatatga aaaaaataca attaaattta aacataatat tacaaaaagt 540
attgttaaaa ttgaaaaaaa ttaaatcttt agtatctttg gaaataatca taatttataa 600
gctcctttga ttttttatat aaattataca ttaattattt aatatataaa aactaaaatt 660
agtaaatcat ctaaatatta caaatgaaga aatattattt gtaaataatt atagtaatat 720
attatggaat gaaatagtaa aaaattaaaa agaatcattt aatttagttt aaaatatttt 780
aataaaaata ataaattaaa acataaaaaa tagattaagt ttcaattggg gacttactct 840
attttttatt aagagtttaa gaatagttaa taattattga aaatatgata taattttctt 900
agttttacag aaggggaggt gaataagtga gaaacgcatt aaagctgaat aaaaagaact 960
atatagataa tattcatagt agaagtaaag ggtggataac caggagcgtt atagataaaa 1020
aaggatatag ccaatggcac tataaatacg ctgaattaaa agatttagat atgagtgatg 1080
aaaatatcta tataactcta aatacctttt ataagccgtg taggcgatta gaaaatataa 1140
aagagttaaa tacactgttt atagacttgg attattataa aactggcaaa actaaagacc 1200
aggtattaat ggacttagaa aagaattatt ttaatcaaag tattcctata ccaaactatg 1260
taatagatag tggaagagga atgtatttaa tatggataat aaatgcagta cctagtaaag 1320
cattaccatt atggaaagcg gttcaagaat atttatataa tcaattaaaa tactttggag 1380
cagatagaca agccttagat gcaacgagaa tattaagagt tccaggaagt ataaactcta 1440
aatcaaaaac agtagtcaat atattagatg agtacgaata tatttatgac ttaagagaaa 1500
tacaaaatgg atttttacct gaattaaaac catatgaaag gaaaaagggt agaccaagca 1560
aaataaatta tatttataga gaaagaagtt tatattatgg aagaatacaa gacataataa 1620
aactttgtga actaagagaa tatgatttaa aaggacacag agagcttata ttatttttat 1680
atagatatta tctttgtagc tttacagaag acattgagaa ggcattaaat gatgttttag 1740
aacttaatag tatgtttaga caacatttaa gtgaaagaga agttataaga gcaactagaa 1800
gtgctgaaag atgttattta gataaaaata agcaatataa gtataagaat gaaactctta 1860
tagagttatt agaaattaca gaagaagaac aaaaatatat gacaataata atttctaaaa 1920
aggaatataa gagaagagaa aatattagag gtaaaaaaaa ttatcaagag caattaaaag 1980
ctaaaggaaa agcaacaaaa aaagaggaat taaatgtatt aagaaaaaaa ataaaagccc 2040
ttaaagaaaa aggctttaaa aataaagaaa ttactctaat gttagaagta ccaataaaaa 2100
cattagaacg tcatattacg tatatgaaaa aaaatgggct tttataaagg ctcatttttt 2160
atattctttt cttcaaagat tatataatat aaaaaaattt ttttcaaact ttaaataaaa 2220
aatattttta tattttttta tttttttatt tttatatttt tttatttttt tatttttata 2280
tttttttatt tttatatttt tttattttta tattttttta tttttttatt tttttatttt 2340
tttatttttt tattttttta tttttttatt tttaccctca ttttttttac gcttgtatta 2400
tagggtactt tgtacctgtt cttttttttg gggaggttgt aaagataatt ttttacttta 2460
gttagttccg aaggaacttt tattatagtg cccctttatt tttttgcgtg gcattgaatg 2520
taaaaaatta tcactatact agggcgtaaa gtaatattac atgtgtctca aagtgggatt 2580
aaagcgggat tttatagggc gtgtttgtgg cttagagtgg gattattgga aatttttttg 2640
atcctaggtt aaatactagt ttttagttac agacaaacct gaagttaact atttatcaat 2700
tcctgcaatt cgtttacaaa acggcaaatg tgaaatccgt cacatactgc gtgatgaact 2760
tgaattgcca aaggaagtat aattttgtta tcttctttat aatatttccc catagtaaaa 2820
ataggaatca aataatcata tcctttctgc aaattcagat taaagccatc gaaggttgac 2880
cacggtatca tagatacatt aaaaatgttt tccggagcat ttggctttcc ttccattcta 2940
tgattgtttc cataccgttg cgtatcactt tcataatctg ctaaaaatga tttaaagtca 3000
gacttacact cagtccaaag gctggaaaat gtttcagtat cattgtgaaa tattgtatag 3060
cttggtatca tctcatcata tatccccaat tcaccatctt gattgattgc cgtcctaaac 3120
tctgaatggc ggtttacaat cattgcaata taataaagca ttgcaggata tagtttcatt 3180
cccttttcct ttatttgtgt gatatccact ttaacggtca tgctgtaggt acaaggtaca 3240
cttgcaaagt agtggtcaaa atactctttt ctgttccaac tatttttatc aattttttca 3300
aataccatct aagttccctc tcaaattcaa gtttatcgct ctaatgaaca aagatattat 3360
accacatttt tgtgaatttt tcaacttgcc cacttcgact gcactcccga cttaataact 3420
tcttgaacac ttgccgaaaa agaaaaactg ccgggtacgt acccggggat cgatccccgg 3480
ccgagcgctt agtgggaatt tgtacccctt atcgatacaa attccccgta ggcgctaggg 3540
acctctttag accggtcagc tgtcagacac ttatcacatt aagtatatac tagtattaag 3600
ctagctttgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact 3660
tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat 3720
cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc 3780
ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct 3840
accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg 3900
cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca 3960
cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc 4020
tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga 4080
taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac 4140
gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga 4200
agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag 4260
ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg 4320
acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag 4380
caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc 4440
tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc 4500
tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc 4560
aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag 4620
gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca 4680
ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag 4740
cggataacaa tttcacacag gaaacagcta tgaccatgat tacgaattcg agctcactct 4800
atcattgata gagtttgaaa ctctatcatt gatagagtat aatatctttg ttcatttaag 4860
ccatctacta aacaagtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta 4920
tcaacttgaa aaagtggcac cgagtcggtg ctttttttga agcttg 4966
<210> 35
<211> 400
<212> DNA
<213> 人工序列
<220>
<223> Fragment upstream of the catB gene
<400> 35
gtctttacac ttttgcccat taatttttga gttccttatt tttagggagc ttttattatt 60
tttatcatga aaatttcata aaatactcat aaactaagga tgtcttcata atcagattag 120
tactccattt tcaatccatt taatctggga atatgatatt ttaattacgt attatttaag 180
atatattaac gtgtaatata ataccccgca aatattaatt atcacataca tatcccccct 240
ttattggggc attttttgta cccattattt tagtattgtg cagtacttaa ataaaaaaat 300
gccgcaaatt catttttatt gaataatgcg gtatttcttc tattctttat ttttattact 360
ctataaataa tgtaatcaag acatgactat ctaaatatat 400
<210> 36
<211> 400
<212> DNA
<213> 人工序列
<220>
<223> catB基因下游的片段
<400> 36
aattcataat tcgggcctcc taaaaatttt cgtaattcta ttttagaagg cttttttccg 60
tgacctagcc atttcaatct cctttttaca atgatattta cgctttagtt tattatagca 120
cattctgtaa taccgaacta ttcaattttc agagaccatt ttttattgat tcataactta 180
agaatactac gaattactct aatattttac tttttcttat ctcttgttat tttaacatcg 240
gaattactac taatattaat ttttattttt ccatccgcat ttgctccaac atttttttaa 300
ctatactttc cttttgttaa taaattatgt tattgttgaa caatataaga aaagtgcgta 360
acatttttta ttaaaaataa ttaggtattt ctatctgtgg 400
<210> 37
<211> 218
<212> PRT
<213> 拜氏梭状芽孢杆菌（Clostridium beijerinckii）
<400> 37
Met Asn Phe Asn Leu Ile Asp Ile Asn His Trp Ser Arg Lys Pro Tyr
1 5 10 15
Phe Glu His Tyr Leu Asn Asn Val Lys Cys Thr Tyr Ser Met Thr Ala
20 25 30
Asn Ile Glu Ile Thr Asp Leu Leu Tyr Glu Ile Lys Leu Lys Asn Ile
35 40 45
Lys Phe Tyr Pro Thr Leu Ile Tyr Met Ile Ala Thr Val Val Asn Lys
50 55 60
His Lys Glu Phe Arg Ile Cys Asp His Glu Gly Ser Leu Gly Tyr Trp
65 70 75 80
Asp Ser Met Asn Pro Ser Tyr Thr Ile Phe His Lys Glu Asn Glu Thr
85 90 95
Phe Ser Ser Ile Trp Thr Glu Tyr Asn Lys Ser Phe Leu Arg Phe Tyr
100 105 110
Ser Asp Tyr Leu Asp Asp Ile Lys Asn Tyr Gly Asn Ile Met Lys Phe
115 120 125
Thr Pro Lys Ser Asn Glu Pro Asp Asn Thr Phe Ser Val Ser Ser Ile
130 135 140
Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Val Tyr Asn Glu Gly
145 150 155 160
Thr Tyr Leu Ile Pro Ile Phe Thr Ala Gly Lys Tyr Phe Lys Gln Glu
165 170 175
Asn Lys Ile Phe Ile Pro Ile Ser Ile Gln Val His His Ala Ile Cys
180 185 190
Asp Gly Tyr His Ala Ser Arg Phe Ile Asn Glu Met Gln Glu Leu Ala
195 200 205
Phe Ser Phe Gln Glu Trp Leu Glu Asn Lys
210 215
<210> 38
<211> 9113
<212> DNA
<213> 人工序列
<220>
<223> pCas9ind-gRNA_catB
<400> 38
catggataaa aagtacagta ttggtctaga cataggaact aactctgttg ggtgggctgt 60
tataacagat gaatataaag ttccatcaaa aaaatttaaa gtattaggaa acactgatag 120
acattcaata aaaaaaaact tgataggtgc tttattattc gattcaggag agactgctga 180
agctacacgt ttaaaaagaa cagctagacg tagatataca agaagaaaaa ataggatatg 240
ttatcttcaa gaaattttta gtaatgaaat ggcaaaagtt gatgattcat tctttcacag 300
actagaagaa agtttcttag ttgaagaaga taagaagcat gaaagacacc ctatttttgg 360
taatatcgta gatgaagtag catatcatga gaagtatcca actatctatc atttaagaaa 420
gaaattagtt gattctacag ataaagctga tctgagatta atatatttag ctttagctca 480
tatgattaaa tttagaggac attttttaat agaaggtgat ttaaacccag acaacagcga 540
tgtagataaa ttatttatcc aattagttca aacttataat caattattcg aagagaatcc 600
aattaatgca agtggtgtag acgctaaggc tatattatca gctagattat caaaatctag 660
aagattagaa aatctaatag ctcaacttcc tggagaaaag aaaaatggac tttttgggaa 720
cctaatagct ctctcactcg gactaacacc aaattttaaa agcaattttg atcttgctga 780
agacgcaaag ttacaactat caaaggatac atacgatgat gatttagata atttgttagc 840
tcaaataggt gatcaatatg ctgatttgtt tcttgcagca aaaaacttaa gtgatgcaat 900
tttactatca gatatactta gagtaaatac agaaataaca aaggctcctt tatcagcaag 960
tatgattaaa cgatatgatg agcatcatca agatttaaca ttattaaagg cacttgtaag 1020
acaacaatta ccagaaaaat ataaagaaat tttctttgat caatctaaaa atggatatgc 1080
tggatatata gacggtggag caagtcaaga agagttttat aaatttataa agcctatttt 1140
agaaaaaatg gatggaactg aagaattact tgttaaactt aacagagaag atttacttag 1200
aaaacaaaga acttttgata atggttcaat tcctcaccaa attcatttag gagaattaca 1260
tgctatacta agaagacaag aagattttta tccatttctt aaagataata gagaaaaaat 1320
tgaaaaaatt ttaactttta gaataccata ttatgtagga ccacttgcaa ggggaaattc 1380
aagatttgca tggatgacta gaaaatcaga agaaactata accccgtgga attttgaaga 1440
agtagtagat aaaggagcta gtgctcaatc atttatagaa agaatgacaa attttgataa 1500
gaatcttcct aacgaaaagg ttttgccaaa gcatagcctt ctttatgagt attttacagt 1560
ttataatgag cttactaaag taaaatacgt tacagaagga atgagaaaac cagcattttt 1620
gtctggtgaa caaaagaaag caatagtaga cctattattt aaaacaaata ggaaggttac 1680
cgtaaagcaa cttaaagaag attacttcaa aaaaattgaa tgctttgata gtgttgaaat 1740
atcaggagtt gaagatagat ttaatgcttc acttggtaca tatcacgatc tcttaaaaat 1800
tataaaagat aaggattttt tagataatga agaaaatgaa gatattcttg aagatatagt 1860
attaacattg acactttttg aagatagaga aatgatagaa gaaagattaa aaacatatgc 1920
acatcttttt gatgataagg ttatgaagca acttaaaaga agaagatata caggttgggg 1980
acgtttgtca agaaagctaa ttaatggtat tagagataaa caatcaggaa agactattct 2040
cgattttctt aaatcagatg gatttgctaa tagaaacttt atgcaattaa ttcatgatga 2100
ttctcttact ttcaaagagg atattcaaaa ggctcaagtt tctggacaag gcgatagctt 2160
acacgaacac attgctaacc ttgcagggag ccccgctatc aaaaaaggaa ttttacaaac 2220
agttaaagtt gtagatgaac ttgttaaagt tatgggaaga cacaaacctg agaatatagt 2280
tatagaaatg gccagagaaa atcaaacaac acaaaaagga caaaaaaatt ctagagagag 2340
aatgaagaga attgaagaag gaataaaaga gctaggatca caaatattaa aagaacatcc 2400
agttgaaaat actcaattgc aaaatgaaaa gttatatttg tattacttac aaaatggaag 2460
agatatgtat gttgatcaag aactcgatat taatagatta agtgactatg atgttgatca 2520
tattgttcct caatcatttt taaaagatga ttcaatcgat aacaaagtat taactagatc 2580
agataaaaat agaggaaagt cagataatgt accatctgaa gaagttgtta aaaaaatgaa 2640
gaactattgg agacaacttt taaatgcaaa gctaattaca caaagaaaat ttgacaattt 2700
aacaaaagca gaaagaggag gattaagcga attagacaaa gctggattta taaaaagaca 2760
acttgttgag acaagacaaa taactaagca tgttgctcaa atacttgatt caagaatgaa 2820
tacaaaatat gatgaaaatg ataaattaat cagagaagta aaagtaataa cattaaagtc 2880
aaaattagta tcagatttca gaaaggattt tcaattttac aaagttcgtg aaataaataa 2940
ctatcatcat gctcatgatg catacttaaa tgctgttgta ggaactgctc ttattaagaa 3000
atatcctaaa ctagaaagcg aatttgttta tggagattat aaagtttatg atgtgcgcaa 3060
aatgatcgcg aaatccgaac aagaaatcgg taaggctaca gcaaaatatt tcttttatag 3120
taatataatg aattttttta agacagaaat aactttggct aatggtgaaa tcagaaaaag 3180
accacttatc gaaacaaatg gagagacagg agaaatagta tgggataaag gaagagattt 3240
tgctactgtt agaaaagtac taagtatgcc acaagtaaat atcgtaaaga aaactgaagt 3300
tcaaactgga ggtttctcta aggaatcaat tttacctaag agaaattcag ataagttaat 3360
tgcaaggaaa aaagattggg acccaaaaaa atacggtggt tttgatagtc caacagttgc 3420
ctatagtgtt cttgtagtag cgaaagttga gaaaggtaag tcaaaaaagt tgaaaagcgt 3480
aaaagaactt cttggtatca caattatgga aagatcttca tttgaaaaaa atccaattga 3540
ctttttagaa gctaagggtt ataaagaagt taaaaaggat ttaatcataa aactaccaaa 3600
gtatagtcta tttgaactcg aaaacggaag aaaacgaatg ctcgctagcg caggagaact 3660
tcaaaaagga aatgaacttg cgctgccatc aaagtatgta aatttcttat atttagcttc 3720
tcattatgag aaattaaaag gatcaccaga ggataatgaa caaaagcaac tatttgtaga 3780
acaacacaaa cattatttag atgaaataat agaacaaata tctgaatttt ctaaaagagt 3840
tatacttgcc gacgcaaatc tagataaggt gctttcagcg tataataaac acagagataa 3900
accaataaga gaacaagcag aaaacattat ccatcttttt acattaacta atcttggtgc 3960
accagctgca tttaagtact ttgatacaac aatagataga aaaagataca catctactaa 4020
agaagtatta gacgcaactt taatacatca atctattaca gggctttatg aaacaagaat 4080
tgatttaagt caactaggcg gagattaagt cgacaaagta ttgttaaaaa taactctgta 4140
gaattataaa ttagttctac agagttattt tttgacccgg gtatattgat aaaaataata 4200
atagtgggta taattaagtt gttaggaggt tagttagaat gatgtcaaga ttagataaaa 4260
gtaaagtgat taacagcgca ttagagctgc ttaatgaggt cggaatcgaa ggtttaacaa 4320
cccgtaaact cgcccagaag ctaggtgtag agcagcctac attgtattgg catgtaaaaa 4380
ataagcgggc tttgctcgac gccttagcca ttgagatgtt agataggcac catactcact 4440
tttgcccttt agaaggggaa agctggcaag attttttacg taataacgct aaaagtttta 4500
gatgtgcttt actaagtcat cgcgatggag caaaagtaca tttaggtaca cggcctacag 4560
aaaaacagta tgaaactctc gaaaatcaat tagccttttt atgccaacaa ggtttttcac 4620
tagagaatgc attatatgca ctcagcgctg tggggcattt tactttaggt tgcgtattgg 4680
aagatcaaga gcatcaagtc gctaaagaag aaagggaaac acctactact gatagtatgc 4740
cgccattatt acgacaagct atcgaattat ttgatcacca aggtgcagag ccagccttct 4800
tattcggcct tgaattgatc atatgcggat tagaaaaaca acttaaatgt gaaagtgggt 4860
cttaaaagca gcataacctt tttccgtgat ggtaacttca cggtaaccaa gatgtcgagt 4920
tgagctcgaa ttcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca 4980
caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag 5040
tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt 5100
cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc 5160
gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg 5220
tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa 5280
agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg 5340
cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga 5400
ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg 5460
tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg 5520
gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc 5580
gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg 5640
gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca 5700
ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt 5760
ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag 5820
ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg 5880
gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc 5940
ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt 6000
tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt 6060
ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccag gtccactgcc 6120
gggcctcttg cgggatcaaa agaaaaacga aatgatacac caatcagtgc aaaaaaagat 6180
ataatgggag ataagacggt tcgtgttcgt gctgacttgc accatatcat aaaaatcgaa 6240
acagcaaaga atggcggaaa cgtaaaagaa gttatggaaa taagacttag aagcaaactt 6300
aagagtgtgt tgatagtgca gtatcttaaa attttgtata ataggaattg aagttaaatt 6360
agatgctaaa aatttgtaat taagaaggag tgattacatg aacaaaaata taaaatattc 6420
tcaaaacttt ttaacgagtg aaaaagtact caaccaaata ataaaacaat tgaatttaaa 6480
agaaaccgat accgtttacg aaattggaac aggtaaaggg catttaacga cgaaactggc 6540
taaaataagt aaacaggtaa cgtctattga attagacagt catctattca acttatcgtc 6600
agaaaaatta aaactgaata ctcgtgtcac tttaattcac caagatattc tacagtttca 6660
attccctaac aaacagaggt ataaaattgt tgggagtatt ccttaccatt taagcacaca 6720
aattattaaa aaagtggttt ttgaaagcca tgcgtctgac atctatctga ttgttgaaga 6780
aggattctac aagcgtacct tggatattca ccgaacacta gggttgctct tgcacactca 6840
agtctcgatt cagcaattgc ttaagctgcc agcggaatgc tttcatccta aaccaaaagt 6900
aaacagtgtc ttaataaaac ttacccgcca taccacagat gttccagata aatattggaa 6960
gctatatacg tactttgttt caaaatgggt caatcgagaa tatcgtcaac tgtttactaa 7020
aaatcagttt catcaagcaa tgaaacacgc caaagtaaac aatttaagta ccgttactta 7080
tgagcaagta ttgtctattt ttaatagtta tctattattt aacgggagga aataattcta 7140
tgagtcccta ggcaggcctc cgccattatt tttttgaaca attgacaatt catttcttat 7200
tttttattaa gtgatagtca aaaggcataa cagtgctgaa tagaaagaaa tttacagaaa 7260
agaaaattat agaatttagt atgattaatt atactcattt atgaatgttt aattgaatac 7320
aaaaaaaaat acttgttatg tattcaatta cgggttaaaa tatagacaag ttgaaaaatt 7380
taataaaaaa ataagtcctc agctcttata tattaagcta ccaacttagt atataagcca 7440
aaacttaaat gtgctaccaa cacatcaagc cgttagagaa ctctatctat agcaatattt 7500
caaatgtacc gacatacaag agaaacatta actatatata ttcaatttat gagattatct 7560
taacagatat aaatgtaaat tgcaataagt aagatttaga agtttatagc ctttgtgtat 7620
tggaagcagt acgcaaaggc ttttttattt gataaaaatt agaagtatat ttattttttc 7680
ataattaatt tatgaaaatg aaagggggtg agcaaagtga cagaggaaag cagtatctta 7740
tcaaataaca aggtattagc aatatcatta ttgactttag cagtaaacat tatgactttt 7800
atagtgcttg tagctaagta gtacgaaagg gggagcttta aaaagctcct tggaatacat 7860
agaattcata aattaattta tgaaaagaag ggcgtatatg aaaacttgta aaaattgcaa 7920
agagtttatt aaagatactg aaatatgcaa aatacattcg ttgatgattc atgataaaac 7980
agtagcaacc tattgcagta aatacaatga gtcaagatgt ttacataaag ggaaagtcca 8040
atgtattaat tgttcaaaga tgaaccgata tggatggtgt gccataaaaa tgagatgttt 8100
tacagaggaa gaacagaaaa aagaacgtac atgcattaaa tattatgcaa ggagctttaa 8160
aaaagctcat gtaaagaaga gtaaaaagaa aaaataattt atttattaat ttaatattga 8220
gagtgccgac acagtatgca ctaaaaaata tatctgtggt gtagtgagcc gatacaaaag 8280
gatagtcact cgcattttca taatacatct tatgttatga ttatgtgtcg gtgggacttc 8340
acgacgaaaa cccacaataa aaaaagagtt cggggtaggg ttaagcatag ttgaggcaac 8400
taaacaatca agctaggata tgcagtagca gaccgtaagg tcgttgttta ggtgtgttgt 8460
aatacatacg ctattaagat gtaaaaatac ggataccaat gaagggaaaa gtataatttt 8520
tggatgtagt ttgtttgttc atctatgggc aaactacgtc caaagccgtt tccaaatctg 8580
ctaaaaagta tatcctttct aaaatcaaag tcaagtatga aatcataaat aaagtttaat 8640
tttgaagtta ttatgatatt atgtttttct attaaaataa attaagtata tagaatagtt 8700
taataatagt atatacttaa tgtgataagt gtctgacagt gtcacagaaa ggatgattgt 8760
tatggattat aagcggctcg aggacgtcaa accatgttaa tcattgcttt tatcaaaaat 8820
aggatccact ctatcattga tagagtttga aactctatca ttgatagagt ataatatctt 8880
tgttcatgta catcatgcta tctgtgagtt ttagagctag aaatagcaag ttaaaataag 8940
gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt gaagcttgtc 9000
tttacacttt tgcccctcga gtccctatca gtgatagatt gaaactctat cattgataga 9060
gtataatatc tttgttcatt agagcgataa acttgaattt gagagggaac ttc 9113
<210> 39
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 引物pNF2
<400> 39
gggcgcactt atacaccacc 20
<210> 40
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 引物pNF2
<400> 40
tgctacgcac cccctaaagg 20
<210> 41
<211> 50
<212> DNA
<213> 人工序列
<220>
<223> ΔcatB_gRNA_rev
<400> 41
aatctatcac tgatagggac tcgaggggca aaagtgtaaa gacaagcttc 50
<210> 42
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 引物pCas9ind_fwd
<400> 42
agctcttgat ccggcaaaca 20
<210> 43
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 引物pCas9ind _rev
<400> 43
gcaaccctag tgttcggtga 20
<210> 44
<211> 219
<212> PRT
<213> 丁酸梭状芽孢杆菌（Clostridium butyricum）
<400> 44
Met Asn Phe Asn Leu Ile Asp Ile Asn His Trp Ser Arg Lys Pro Tyr
1 5 10 15
Phe Glu His Tyr Leu Asn Asn Val Lys Cys Thr Tyr Ser Met Thr Ala
20 25 30
Asn Ile Glu Ile Thr Asp Leu Leu Tyr Glu Ile Lys Leu Lys Asn Ile
35 40 45
Lys Phe Tyr Pro Thr Leu Ile Tyr Met Ile Ala Thr Val Val Asn Asn
50 55 60
His Lys Glu Phe Arg Ile Cys Phe Asp His Lys Gly Ser Leu Gly Tyr
65 70 75 80
Trp Asp Ser Met Asn Pro Ser Tyr Thr Ile Phe His Lys Glu Asn Glu
85 90 95
Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asn Lys Ser Phe Leu Arg Phe
100 105 110
Tyr Ser Asp Tyr Leu Asp Asp Ile Lys Asn Tyr Gly Asn Ile Met Lys
115 120 125
Phe Thr Pro Lys Ser Asn Glu Pro Asp Asn Thr Phe Ser Val Ser Ser
130 135 140
Ile Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Val Tyr Asn Glu
145 150 155 160
Gly Thr Tyr Leu Ile Pro Ile Phe Thr Ala Gly Lys Tyr Phe Lys Gln
165 170 175
Glu Asn Lys Ile Phe Ile Pro Ile Ser Ile Gln Val His His Ala Ile
180 185 190
Cys Asp Gly Tyr His Ala Ser Arg Phe Ile Asn Glu Met Gln Glu Leu
195 200 205
Ala Phe Ser Phe Gln Glu Trp Leu Glu Asn Lys
210 215
<210> 45
<211> 219
<212> PRT
<213> 拜氏梭状芽孢杆菌（Clostridium beijerinckii）
<400> 45
Met Asn Phe Asn Leu Ile Asp Ile Asn His Trp Ile Arg Lys Pro Tyr
1 5 10 15
Phe Glu His Tyr Leu Asn Asn Val Lys Cys Thr Tyr Ser Met Thr Ala
20 25 30
Asn Ile Glu Ile Thr Asp Leu Leu Tyr Glu Ile Lys Leu Lys Asn Ile
35 40 45
Lys Phe Tyr Pro Thr Leu Ile Tyr Met Ile Ala Thr Val Val Asn Asn
50 55 60
His Lys Glu Phe Arg Ile Cys Phe Asp His Lys Gly Ser Leu Gly Tyr
65 70 75 80
Trp Asp Ser Met Asn Pro Ser Tyr Thr Ile Phe His Lys Glu Asn Glu
85 90 95
Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asn Lys Ser Phe Leu Arg Phe
100 105 110
Tyr Ser Asp Tyr Leu Asp Asp Ile Lys Asn Tyr Gly Asn Ile Met Lys
115 120 125
Phe Thr Pro Lys Ser Asn Glu Pro Asp Asn Thr Phe Ser Val Ser Ser
130 135 140
Ile Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Val Tyr Asn Glu
145 150 155 160
Gly Thr Tyr Leu Ile Pro Ile Phe Thr Ala Gly Lys Tyr Phe Lys Gln
165 170 175
Glu Asn Lys Ile Phe Ile Pro Ile Ser Ile Gln Val His His Ala Ile
180 185 190
Cys Asp Gly Tyr His Ala Ser Arg Phe Ile Asn Glu Met Gln Glu Leu
195 200 205
Ala Phe Ser Phe Gln Glu Trp Leu Glu Asn Lys
210 215
<210> 46
<211> 219
<212> PRT
<213> 拜氏梭状芽孢杆菌（Clostridium beijerinckii）
<400> 46
Met Asn Phe Asn Leu Ile Asp Ile Asn His Trp Ser Arg Lys Pro Tyr
1 5 10 15
Phe Glu His Tyr Leu Asn Asn Val Lys Cys Thr Tyr Ser Met Thr Ala
20 25 30
Asn Ile Glu Ile Thr Asp Leu Leu Tyr Glu Ile Lys Leu Lys Asn Ile
35 40 45
Lys Phe Tyr Pro Thr Leu Ile Tyr Met Ile Ala Thr Val Val Asn Asn
50 55 60
His Glu Glu Phe Arg Ile Cys Phe Asp His Glu Gly Ser Leu Gly Tyr
65 70 75 80
Trp Asp Ser Met Asn Pro Ser Tyr Thr Ile Phe His Lys Glu Asn Glu
85 90 95
Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asn Lys Ser Phe Leu Arg Phe
100 105 110
Tyr Ser Asp Tyr Leu Asp Asp Ile Lys Asn Tyr Gly Asn Ile Met Lys
115 120 125
Phe Thr Pro Lys Ser Asn Glu Pro Asp Asn Thr Phe Ser Val Ser Ser
130 135 140
Ile Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Val Tyr Asn Glu
145 150 155 160
Gly Thr Tyr Leu Ile Pro Ile Phe Thr Ala Gly Lys Tyr Phe Lys Gln
165 170 175
Gly Asn Lys Val Phe Ile Pro Ile Ser Ile Gln Val His His Ala Ile
180 185 190
Cys Asp Gly Tyr His Ala Ser Arg Phe Ile Asn Glu Met Gln Glu Leu
195 200 205
Ala Phe Ser Phe Gln Glu Trp Leu Glu Asn Lys
210 215
<210> 47
<211> 219
<212> PRT
<213> 拜氏梭状芽孢杆菌（Clostridium beijerinckii）
<400> 47
Met Asn Phe Asn Leu Ile Asp Ile Asn His Trp Ser Arg Lys Pro Tyr
1 5 10 15
Phe Glu His Tyr Leu Asn Asn Val Lys Cys Thr Tyr Ser Met Thr Ala
20 25 30
Asn Ile Glu Ile Thr Asp Leu Leu Tyr Glu Ile Lys Leu Lys Asn Ile
35 40 45
Lys Phe Tyr Pro Thr Leu Ile Tyr Met Ile Ala Thr Val Val Asn Asn
50 55 60
His Lys Glu Phe Ser Ile Cys Phe Asp His Glu Gly Ser Leu Gly Tyr
65 70 75 80
Trp Asp Ser Met Asn Pro Ser Tyr Thr Ile Phe His Lys Glu Asn Glu
85 90 95
Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asn Glu Ser Phe Leu Arg Phe
100 105 110
Tyr Ser Asp Tyr Leu Asp Asp Ile Lys Asn Tyr Gly Asn Ile Met Lys
115 120 125
Phe Thr Pro Lys Ser Asn Glu Pro Asp Asn Thr Phe Ser Val Ser Ser
130 135 140
Ile Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Val Tyr Asn Glu
145 150 155 160
Gly Thr Tyr Leu Ile Pro Ile Phe Thr Ala Gly Lys Tyr Phe Lys Gln
165 170 175
Gly Asn Lys Ile Phe Ile Pro Ile Ser Ile Gln Val His His Ala Ile
180 185 190
Cys Asp Gly Tyr His Ala Ser Arg Phe Ile Asn Glu Met Gln Glu Leu
195 200 205
Ala Phe Ser Phe Gln Glu Trp Leu Glu Asn Lys
210 215
<210> 48
<211> 219
<212> PRT
<213> 人工序列
<220>
<223> 梭状芽孢杆菌属物种2-1
<400> 48
Met Asn Phe Asn Leu Ile Asp Ile Asn His Trp Ser Arg Lys Pro Tyr
1 5 10 15
Phe Glu His Tyr Leu Asn Asn Val Lys Cys Thr Tyr Ser Met Thr Ala
20 25 30
Asn Ile Glu Ile Thr Asp Leu Leu Tyr Glu Ile Lys Leu Lys Asn Ile
35 40 45
Lys Phe Tyr Pro Thr Leu Ile Tyr Met Ile Ala Asn Val Val Asn Asn
50 55 60
His Lys Glu Phe Arg Ile Cys Phe Asp His Asn Gly Ser Leu Gly Tyr
65 70 75 80
Trp Asp Ser Met Asn Pro Ser Tyr Thr Ile Phe His Lys Glu Asn Glu
85 90 95
Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asn Glu Ser Phe Leu Arg Phe
100 105 110
Tyr Ser Asp Tyr Leu Asp Asp Ile Lys Asn Tyr Gly Asn Ile Met Lys
115 120 125
Phe Thr Pro Lys Ser Asn Gln Pro Asp Asn Thr Phe Ser Val Ser Ser
130 135 140
Ile Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Val Tyr Asn Glu
145 150 155 160
Gly Thr Tyr Leu Ile Pro Ile Phe Thr Ala Gly Lys Tyr Phe Lys Gln
165 170 175
Glu Asn Lys Ile Phe Ile Pro Ile Ser Ile Gln Val His His Ala Ile
180 185 190
Cys Asp Gly Tyr His Ala Ser Arg Phe Ile Asn Glu Met Gln Glu Leu
195 200 205
Ala Phe Ser Phe Gln Glu Trp Leu Glu Asn Lys
210 215
<210> 49
<211> 219
<212> PRT
<213> 人工序列
<220>
<223> 二醇梭状芽孢杆菌（Clostridium diolis）
<400> 49
Met Asn Phe Asn Leu Ile Asp Ile Asn His Trp Ser Arg Lys Pro Tyr
1 5 10 15
Phe Glu His Tyr Leu Asn Asn Val Lys Cys Thr Tyr Ser Met Thr Ala
20 25 30
Asn Ile Glu Ile Thr Asp Leu Leu Tyr Glu Ile Lys Leu Lys Asn Ile
35 40 45
Lys Phe Tyr Pro Thr Leu Ile Tyr Met Ile Ala Asn Val Val Asn Asn
50 55 60
His Lys Glu Phe Arg Ile Cys Phe Asp His Asn Gly Ser Leu Gly Tyr
65 70 75 80
Trp Asp Ser Met Asn Pro Ser Tyr Thr Val Phe His Lys Glu Asn Glu
85 90 95
Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asn Glu Ser Phe Leu Arg Phe
100 105 110
Tyr Ser Asp Tyr Leu Asp Asp Ile Lys Asn Tyr Gly Asn Ile Met Lys
115 120 125
Phe Thr Pro Lys Ser Asn Glu Pro Asp Asn Thr Phe Ser Val Ser Ser
130 135 140
Ile Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Val Tyr Asn Glu
145 150 155 160
Gly Thr Tyr Leu Ile Pro Ile Phe Thr Ala Gly Lys Tyr Phe Lys Gln
165 170 175
Gly Asn Lys Ile Phe Ile Pro Ile Ser Ile Gln Val His His Ala Ile
180 185 190
Cys Asp Gly Tyr His Ala Ser Arg Phe Ile Asn Glu Met Gln Glu Leu
195 200 205
Ala Phe Ser Phe Gln Glu Trp Leu Glu Asn Lys
210 215
<210> 50
<211> 219
<212> PRT
<213> 拜氏梭状芽孢杆菌（Clostridium beijerinckii）
<400> 50
Met Asn Phe Asn Leu Ile Asp Ile Asn His Trp Ile Arg Lys Pro Tyr
1 5 10 15
Phe Glu His Tyr Leu Asn Asn Val Lys Cys Thr Tyr Ser Met Thr Ala
20 25 30
Asn Ile Glu Ile Thr Asp Leu Leu Tyr Glu Ile Lys Leu Lys Asn Ile
35 40 45
Lys Phe Tyr Pro Thr Leu Ile Tyr Met Ile Ala Thr Val Val Asn Asn
50 55 60
His Lys Glu Phe Arg Ile Cys Phe Asp His Lys Gly Ser Leu Gly Tyr
65 70 75 80
Trp Asp Ser Met Asn Pro Ser Tyr Thr Ile Phe His Lys Glu Asn Glu
85 90 95
Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asn Lys Ser Phe Leu Arg Phe
100 105 110
Tyr Ser Asp Tyr Leu Asp Asp Ile Lys Asn Tyr Gly Asn Ile Met Lys
115 120 125
Phe Thr Pro Lys Ser Asn Gln Pro Asp Asn Thr Phe Ser Val Ser Ser
130 135 140
Ile Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Val Asn Asn Glu
145 150 155 160
Gly Thr Tyr Leu Ile Pro Ile Phe Thr Ala Gly Lys Tyr Phe Lys Gln
165 170 175
Glu Asn Lys Ile Phe Ile Pro Ile Ser Ile Gln Val His His Ala Ile
180 185 190
Cys Asp Gly Tyr His Ala Ser Arg Phe Ile Asn Glu Met Gln Glu Leu
195 200 205
Ala Phe Ser Phe Lys Glu Trp Leu Glu Asn Lys
210 215
<210> 51
<211> 221
<212> PRT
<213> 拜氏梭状芽孢杆菌（Clostridium beijerinckii）
<400> 51
Met Asn Phe Asn Leu Ile Asp Ile Asn Asn Trp Ser Arg Lys Pro Tyr
1 5 10 15
Phe Glu His Tyr Leu Asn Asn Val Lys Cys Thr Tyr Ser Met Thr Ala
20 25 30
Asn Ile Glu Ile Thr Asp Leu Leu Tyr Glu Ile Lys Leu Lys Asn Ile
35 40 45
Lys Phe Tyr Pro Thr Leu Ile Tyr Met Ile Ala Asn Val Val Asn Asn
50 55 60
His Lys Glu Phe Arg Ile Cys Phe Asp His Glu Gly Ser Leu Gly Tyr
65 70 75 80
Trp Asp Ser Met Asn Pro Ser Tyr Thr Ile Phe His Lys Glu Asn Glu
85 90 95
Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asn Glu Ser Phe Leu Arg Phe
100 105 110
Tyr Ser Asp Tyr Leu Asp Asp Ile Lys Asn Tyr Gly Asn Ile Met Lys
115 120 125
Phe Thr Pro Lys Ser Asn Glu Pro Asp Asn Thr Phe Pro Val Ser Ser
130 135 140
Ile Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Val Tyr Asn Glu
145 150 155 160
Gly Thr Tyr Leu Ile Pro Ile Phe Thr Ala Gly Lys Tyr Phe Lys Gln
165 170 175
Gly Asn Lys Ile Phe Ile Pro Ile Ser Ile Gln Val His His Ala Ile
180 185 190
Cys Asp Gly Tyr His Ala Ser Arg Phe Ile Asn Glu Met Gln Glu Leu
195 200 205
Ala Phe Ser Phe Gln Glu Trp Leu Glu Asn Lys Tyr Ile
210 215 220
<210> 52
<211> 219
<212> PRT
<213> 拜氏梭状芽孢杆菌（Clostridium beijerinckii）
<400> 52
Met Asn Phe Asn Leu Ile Asp Ile Asn Asn Trp Ser Arg Lys Pro Tyr
1 5 10 15
Phe Glu His Tyr Leu Asn Asn Val Lys Cys Thr Tyr Ser Met Thr Ala
20 25 30
Asn Ile Glu Ile Thr Asp Leu Leu Tyr Glu Ile Lys Leu Lys Asn Ile
35 40 45
Lys Phe Tyr Pro Thr Leu Ile Tyr Met Ile Ala Thr Val Val Asn Asn
50 55 60
His Lys Glu Phe Arg Ile Cys Phe Asp His Asn Gly Ser Leu Gly Tyr
65 70 75 80
Trp Asp Ser Met Asn Pro Ser Tyr Thr Ile Phe His Lys Glu Asn Glu
85 90 95
Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asn Glu Ser Phe Leu Arg Phe
100 105 110
Tyr Ser Asp Tyr Leu Asp Asp Ile Lys Asn Tyr Gly Asn Ile Met Lys
115 120 125
Phe Thr Pro Lys Ser Asn Glu Pro Asp Asn Thr Phe Pro Val Ser Ser
130 135 140
Ile Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Val Tyr Asn Glu
145 150 155 160
Gly Thr Tyr Leu Ile Pro Ile Phe Thr Ala Gly Lys Tyr Phe Lys Gln
165 170 175
Gly Asn Lys Ile Phe Ile Pro Ile Ser Ile Gln Val His His Ala Ile
180 185 190
Cys Asp Gly Tyr His Ala Ser Arg Phe Ile Asn Glu Met Gln Glu Leu
195 200 205
Ala Phe Ser Phe Arg Glu Trp Leu Glu Asn Lys
210 215
<210> 53
<211> 219
<212> PRT
<213> Clostridium saccharoperbutylacetonicum
<400> 53
Met Asn Phe Asn Leu Ile Asp Ile Asn His Trp Ser Arg Lys Pro Tyr
1 5 10 15
Phe Glu His Tyr Leu Asn Thr Val Lys Cys Thr Tyr Ser Met Thr Ala
20 25 30
Asn Ile Glu Ile Thr Asp Leu Leu Tyr Glu Ile Lys Leu Lys Asn Ile
35 40 45
Lys Phe Tyr Pro Thr Leu Ile Tyr Met Ile Ala Thr Val Val Asn Asn
50 55 60
His Lys Glu Phe Arg Ile Cys Phe Asp His Lys Gly Ser Leu Gly Tyr
65 70 75 80
Trp Asp Ser Met Asn Pro Ser Tyr Thr Ile Phe His Lys Glu Asn Glu
85 90 95
Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asn Glu Ser Phe Leu Arg Phe
100 105 110
Tyr Ser Asp Tyr Ile Asp Asp Ile Lys Asn Tyr Gly Asn Ile Met Lys
115 120 125
Phe Thr Pro Lys Ser Asn Glu Pro Asp Asn Ile Phe Pro Val Ser Ser
130 135 140
Ile Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Val Tyr Asn Glu
145 150 155 160
Gly Thr Tyr Leu Ile Pro Ile Phe Thr Ala Gly Lys Tyr Phe Lys Gln
165 170 175
Glu Asn Lys Ile Phe Ile Pro Ile Ser Ile Gln Val His His Ala Val
180 185 190
Cys Asp Gly Tyr His Ala Ser Arg Phe Ile Asn Glu Met Gln Glu Leu
195 200 205
Ala Phe Ser Phe Gln Glu Trp Leu Glu Asn Lys
210 215
<210> 54
<211> 219
<212> PRT
<213> Clostridium saccharoperbutylacetonicum
<400> 54
Met Asn Phe Asn Leu Ile Asp Ile Asn His Trp Ser Arg Lys Pro Tyr
1 5 10 15
Phe Glu His Tyr Leu Asn Thr Val Lys Cys Thr Tyr Ser Met Thr Ala
20 25 30
Asn Ile Glu Ile Thr Asp Leu Leu Tyr Glu Ile Lys Leu Lys Asn Ile
35 40 45
Lys Phe Tyr Pro Thr Leu Ile Tyr Met Ile Ala Thr Val Val Asn Asn
50 55 60
His Lys Glu Phe Arg Ile Cys Phe Asp His Lys Gly Ser Leu Gly Tyr
65 70 75 80
Trp Asp Ser Met Asn Pro Ser Tyr Thr Ile Phe Tyr Lys Glu Asn Glu
85 90 95
Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asn Glu Ser Phe Leu Arg Phe
100 105 110
Tyr Ser Asp Tyr Ile Asp Asp Ile Lys Asn Tyr Gly Asn Ile Met Lys
115 120 125
Phe Thr Pro Lys Ser Asn Glu Pro Asp Asn Ile Phe Pro Val Ser Ser
130 135 140
Ile Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Val Tyr Asn Glu
145 150 155 160
Gly Thr Tyr Leu Ile Pro Ile Phe Thr Ala Gly Lys Tyr Phe Lys Gln
165 170 175
Glu Asn Lys Ile Phe Ile Pro Ile Ser Ile Gln Val His His Ala Val
180 185 190
Cys Asp Gly Tyr His Ala Ser Arg Phe Ile Asn Glu Met Gln Glu Leu
195 200 205
Ala Phe Ser Phe Gln Glu Trp Leu Glu Asn Lys
210 215
<210> 55
<211> 219
<212> PRT
<213> 拜氏梭状芽孢杆菌（Clostridium beijerinckii）
<400> 55
Met Asn Phe Asn Leu Ile Asp Ile Asn Asn Trp Ser Arg Lys Pro Tyr
1 5 10 15
Phe Glu His Tyr Leu Asn Asn Val Lys Cys Thr Tyr Ser Met Thr Ala
20 25 30
Asn Ile Glu Ile Thr Asp Leu Leu Tyr Glu Ile Lys Leu Lys Asn Ile
35 40 45
Lys Phe Tyr Pro Thr Leu Ile Tyr Met Ile Ala Thr Val Val Asn Asn
50 55 60
His Lys Glu Phe Arg Ile Cys Phe Asp His Asn Gly Ser Leu Gly Tyr
65 70 75 80
Trp Asp Ser Met Asn Pro Ser Tyr Thr Ile Phe His Lys Glu Asn Glu
85 90 95
Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asn Glu Ser Phe Leu Arg Phe
100 105 110
Tyr Ser Asp Tyr Leu Asp Asp Ile Lys Asn Tyr Gly Asn Ile Met Lys
115 120 125
Phe Thr Pro Lys Ser Asn Arg Ser Asp Asn Thr Phe Pro Val Ser Ser
130 135 140
Ile Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Val Tyr Asn Glu
145 150 155 160
Gly Thr Tyr Leu Ile Pro Ile Phe Thr Ala Gly Lys Tyr Phe Lys Gln
165 170 175
Gly Asn Lys Ile Phe Ile Pro Ile Ser Ile Gln Val His His Ala Ile
180 185 190
Cys Asp Gly Tyr His Ala Ser Arg Phe Ile Asn Glu Met Gln Glu Leu
195 200 205
Ala Phe Ser Phe Arg Glu Trp Leu Glu Asn Lys
210 215
<210> 56
<211> 221
<212> PRT
<213> 拜氏梭状芽孢杆菌（Clostridium beijerinckii）
<400> 56
Met Asn Phe Asn Leu Ile Asp Ile Asn His Trp Asn Arg Lys Pro Phe
1 5 10 15
Phe Glu His Tyr Leu Asn Asn Val Lys Cys Thr Tyr Ser Met Thr Ala
20 25 30
Asn Ile Glu Ile Thr Asp Leu Leu Tyr Glu Ile Lys Leu Lys Asn Ile
35 40 45
Lys Phe Tyr Pro Thr Leu Ile Tyr Met Ile Ala Thr Val Val Asn Asn
50 55 60
His Lys Glu Phe Arg Ile Cys Phe Asp His Lys Gly Ser Leu Gly Tyr
65 70 75 80
Trp Asp Ser Met Asn Pro Ser Tyr Thr Ile Phe His Glu Glu Asn Glu
85 90 95
Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asn Glu Ser Phe Leu Arg Phe
100 105 110
Tyr Ser Asp Tyr Leu Asp Asp Ile Lys Asn Tyr Gly Asn Ile Met Lys
115 120 125
Phe Thr Pro Lys Ser Asn Glu Pro Asp Asn Thr Phe Pro Val Ser Ser
130 135 140
Ile Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Val Cys Asn Glu
145 150 155 160
Gly Thr Tyr Leu Thr Pro Ile Phe Thr Ala Gly Lys Tyr Phe Lys Gln
165 170 175
Glu Asn Lys Ile Phe Ile Pro Ile Ser Ile Gln Val His His Ser Ile
180 185 190
Cys Asp Gly Tyr His Ala Ser Arg Phe Ile Asn Glu Met Gln Glu Leu
195 200 205
Ala Phe Ser Phe Gln Glu Trp Leu Glu Asn Lys Tyr Ile
210 215 220
<210> 57
<211> 219
<212> PRT
<213> 拜氏梭状芽孢杆菌（Clostridium beijerinckii）
<400> 57
Met Asn Phe Asn Leu Ile Asp Ile Lys His Trp Ser Arg Lys Pro Tyr
1 5 10 15
Phe Glu His Tyr Leu Asn Asn Val Lys Cys Thr Tyr Ser Met Thr Ala
20 25 30
Asn Ile Glu Ile Thr Asp Leu Leu Tyr Glu Ile Arg Leu Lys Asn Ile
35 40 45
Lys Leu Tyr Pro Thr Leu Ile Tyr Met Ile Ala Thr Val Val Asn Asn
50 55 60
His Lys Glu Phe Arg Thr Cys Phe Asp His Ser Gly Ser Leu Gly Tyr
65 70 75 80
Trp Asp Ser Met Ser Pro Ser Tyr Thr Ile Phe His Lys Glu Asn Glu
85 90 95
Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asn Glu Ser Phe Pro Arg Phe
100 105 110
Tyr Ser Asp Tyr Phe Asp Asp Ile Lys Asn Tyr Gly Asn Ile Met Lys
115 120 125
Phe Thr Pro Lys Leu Asn Glu Pro Asp Asn Thr Phe Pro Val Ser Ser
130 135 140
Ile Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Val Tyr Asn Glu
145 150 155 160
Gly Thr Tyr Leu Ile Pro Ile Phe Thr Thr Gly Lys Tyr Phe Lys Gln
165 170 175
Glu Asn Lys Met Phe Ile Pro Ile Ser Ile Gln Val His His Ala Ile
180 185 190
Cys Asp Gly Tyr His Ala Ser Arg Phe Ile Asn Glu Met Gln Glu Leu
195 200 205
Ala Phe Ser Phe Gln Asp Trp Leu Glu Asn Lys
210 215
<210> 58
<211> 219
<212> PRT
<213> 肉毒梭状芽孢杆菌（Clostridium botulinum）
<400> 58
Met Lys Phe Asn Leu Ile Asp Ile Glu His Trp Asn Arg Lys Pro Tyr
1 5 10 15
Phe Glu Tyr Tyr Leu His Ser Val Arg Cys Thr Tyr Ser Met Thr Ala
20 25 30
Asn Ile Glu Ile Thr Asn Leu Leu His Glu Ile Lys Leu Lys Lys Leu
35 40 45
Lys Leu Tyr Pro Thr Leu Ile Tyr Ile Ile Ala Thr Val Val Asn Asn
50 55 60
His Lys Glu Phe Arg Thr Cys Phe Asp Glu Asn Gly Asn Leu Gly Tyr
65 70 75 80
Trp Asp Ser Met Ser Pro Ser Tyr Thr Ile Phe His Lys Asp Asn Glu
85 90 95
Thr Phe Ser Ser Ile Trp Thr Asp Tyr Asp Glu Ser Phe Ser Cys Phe
100 105 110
Tyr Asn Asp Tyr Leu Asp Asp Ile Lys Asn Tyr Gly Ala Ile Met Lys
115 120 125
Phe Thr Pro Lys Leu Asn Glu Pro Ala Asn Thr Phe Pro Val Ser Ser
130 135 140
Ile Pro Trp Val Asn Phe Thr Gly Phe Asn Leu Asn Val Tyr Asn Asn
145 150 155 160
Gly Thr Tyr Leu Val Pro Ile Phe Thr Met Gly Lys Tyr Phe Glu Gln
165 170 175
Asn Asn Lys Ile Phe Ile Pro Met Ser Ile Gln Val His His Ala Val
180 185 190
Cys Asp Gly Tyr His Ile Ser Arg Phe Ile Asn Glu Val Gln Glu Leu
195 200 205
Ala Leu Asn Ser Gln Thr Trp Leu Lys His Lys
210 215
<210> 59
<211> 219
<212> PRT
<213> 人工序列
<220>
<223> Anaerocolumna aminovalerica
<400> 59
Met Lys Phe Asn Leu Ile Asp Ile Glu Asn Trp Asn Arg Lys Pro Tyr
1 5 10 15
Phe Glu His Tyr Leu Asn Ser Val Arg Cys Thr Tyr Ser Met Thr Ala
20 25 30
Asn Ile Glu Ile Thr Asn Leu Leu His Glu Ile Lys Leu Lys Asp Leu
35 40 45
Lys Leu Tyr Pro Thr Leu Ile Tyr Ile Leu Ala Thr Val Val Asn Asn
50 55 60
His Lys Glu Phe Arg Thr Cys Phe Asp Glu Asn Gly Asn Leu Gly Tyr
65 70 75 80
Trp Asp Ser Met Ser Pro Ser Tyr Thr Ile Phe His Lys Glu Asn Glu
85 90 95
Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asp Glu Ser Phe Ser Arg Phe
100 105 110
Tyr Thr Ala Tyr Leu Asp Asp Ile Lys Asn His Gly Asn Ile Met Lys
115 120 125
Phe Thr Pro Lys Leu Asn Glu Pro Ala Asn Thr Phe Pro Ile Ser Ser
130 135 140
Ile Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Val Tyr Asn Asp
145 150 155 160
Gly Lys Tyr Leu Leu Pro Ile Phe Thr Thr Gly Lys Tyr Phe Glu Gln
165 170 175
Asn Ser Lys Ile Phe Ile Pro Met Ser Val Gln Val His His Ala Val
180 185 190
Cys Asp Gly Tyr His Ile Ser Arg Phe Ile Asn Glu Val Gln Glu Val
195 200 205
Ile Leu Asn Tyr Gln Thr Trp Leu Gly Asp Lys
210 215
<210> 60
<211> 219
<212> PRT
<213> 人工序列
<220>
<223> Desnuesiella massiliensis
<400> 60
Met Lys Phe Asn Leu Ile Asp Ile Glu His Trp Asn Arg Lys Pro Tyr
1 5 10 15
Phe Glu His Tyr Leu Asn Ser Val Arg Cys Thr Tyr Ser Met Thr Ala
20 25 30
Asn Ile Glu Ile Thr Asn Leu Leu His Asp Ile Lys Leu Lys Lys Leu
35 40 45
Lys Leu Tyr Pro Thr Leu Ile Tyr Ile Ile Ala Thr Val Val Asn Asn
50 55 60
His Glu Glu Phe Arg Thr Cys Phe Tyr Glu Asn Gly Asn Leu Gly Tyr
65 70 75 80
Trp Asp Ser Met Ser Pro Ser Tyr Thr Ile Phe His Lys Asp Asn Glu
85 90 95
Thr Phe Ser Glu Ile Trp Ser Glu Tyr Asp Glu Ser Phe Ser Cys Phe
100 105 110
Tyr Ser Lys Tyr Leu Asp Asp Ile Lys Asn Tyr Gly Asp Ile Met Arg
115 120 125
Phe Thr Pro Lys Leu Asn Glu Pro Ala Asn Thr Phe Pro Ile Ser Cys
130 135 140
Ile Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Val Tyr Asn Asp
145 150 155 160
Gly Arg Tyr Leu Val Pro Ile Phe Thr Ile Gly Lys Tyr Phe Glu Gln
165 170 175
Asn Asn Lys Ile Phe Ile Pro Met Ser Ile Gln Val His His Ala Val
180 185 190
Cys Asp Gly Tyr His Thr Ser Arg Phe Ile Asn Glu Val Gln Glu Leu
195 200 205
Ala Leu Asn Ser Gln Thr Trp Leu Arg His Lys
210 215
<210> 61
<211> 219
<212> PRT
<213> 人工序列
<220>
<223> 梭状芽孢杆菌属物种HMP27
<400> 61
Met Lys Phe Asn Leu Ile Asp Thr Glu His Trp Asn Arg Lys Pro Tyr
1 5 10 15
Phe Glu His Tyr Leu Asn Ser Val Arg Cys Thr Tyr Ser Ile Thr Ala
20 25 30
Asn Ile Glu Ile Thr Asn Leu Leu His Asp Ile Lys Gln Lys Lys Leu
35 40 45
Lys Leu Tyr Pro Thr Phe Ile Tyr Ile Ile Ala Thr Val Val Asn Thr
50 55 60
His Lys Glu Phe Arg Thr Cys Phe Asp Glu Ser Gly Asn Leu Gly Tyr
65 70 75 80
Trp Asp Ser Met Ser Pro Ser Tyr Thr Ile Phe His Lys Asp Asn Glu
85 90 95
Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asp Lys Ser Phe Ser Cys Phe
100 105 110
Tyr Ser Lys Tyr Leu His Asp Ile Lys Asn Tyr Gly Asp Ile Met Ser
115 120 125
Phe Thr Pro Lys Leu Asn Glu Pro Ala Asn Thr Phe Pro Ile Ser Cys
130 135 140
Ile Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Ile Tyr Asn Asp
145 150 155 160
Gly Thr Tyr Leu Val Pro Ile Phe Thr Ile Gly Lys Tyr Phe Lys Gln
165 170 175
Ala Asp Lys Ile Leu Ile Pro Ile Ser Ile Gln Val His His Ala Val
180 185 190
Cys Asp Gly Tyr His Thr Ser Arg Phe Ile Asn Glu Val Gln Glu Leu
195 200 205
Ile Leu Asn Tyr Gln Thr Trp Leu Lys His Lys
210 215
<210> 62
<211> 219
<212> PRT
<213> 人工序列
<220>
<223> 德雷克氏梭状芽孢杆菌（Clostridium drakei）
<400> 62
Met Lys Phe Asn Leu Ile Asp Ile Glu Asn Trp Asn Arg Lys Pro Tyr
1 5 10 15
Phe Glu His Tyr Leu Asn Ala Val Arg Cys Thr Tyr Ser Met Thr Ala
20 25 30
Asn Ile Glu Ile Thr Gly Leu Leu Arg Glu Ile Lys Leu Lys Gly Leu
35 40 45
Lys Leu Tyr Pro Thr Leu Ile Tyr Ile Ile Thr Ala Val Ile Asn Arg
50 55 60
His Lys Glu Phe Arg Thr Cys Phe Asp Glu Asn Arg Lys Leu Gly Tyr
65 70 75 80
Trp Asp Ser Met Ser Pro Ser Tyr Thr Val Phe His Lys Glu Asp Glu
85 90 95
Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asp Glu Ser Phe Pro Arg Phe
100 105 110
Tyr Asp Asn Tyr Leu Asp Asp Ile Lys Ser Tyr Gly Asp Val Leu Lys
115 120 125
Phe Met Pro Lys Pro Asp Glu Pro Gly Asn Thr Phe Asn Val Ser Ser
130 135 140
Ile Pro Trp Val Asn Phe Thr Gly Phe Asn Leu Asn Ile Tyr Asn Asp
145 150 155 160
Ala Thr Tyr Leu Ile Pro Ile Phe Thr Met Gly Lys Phe Phe His Gln
165 170 175
Asp Asn Lys Ile Phe Ile Pro Met Ser Ile Gln Val His His Ala Val
180 185 190
Cys Asp Gly Tyr His Thr Ser Arg Phe Phe Asn Glu Val Gln Glu Leu
195 200 205
Ser Ser Asn Phe Glu Thr Trp Leu Asp Glu Lys
210 215
<210> 63
<211> 219
<212> PRT
<213> 粪味梭状芽孢杆菌（Clostridium scatologenes）
<400> 63
Met Lys Phe Asn Leu Ile Asp Ile Glu Asp Trp Asn Arg Lys Pro Tyr
1 5 10 15
Phe Glu His Tyr Leu Asn Ala Val Arg Cys Thr Tyr Ser Met Thr Ala
20 25 30
Asn Ile Glu Ile Thr Gly Leu Leu Arg Glu Ile Lys Leu Lys Gly Leu
35 40 45
Lys Leu Tyr Pro Thr Leu Ile Tyr Ile Ile Thr Ala Val Ile Asn Arg
50 55 60
His Lys Glu Phe Arg Thr Cys Phe Asp Glu Asn Arg Lys Leu Gly Tyr
65 70 75 80
Trp Asp Ser Met Ser Pro Ser Tyr Thr Val Phe His Lys Glu Asp Glu
85 90 95
Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asp Glu Ser Phe Pro Arg Phe
100 105 110
Tyr Asp Asn Tyr Leu Asp Asp Ile Lys Ser Tyr Gly Asp Val Leu Lys
115 120 125
Phe Met Pro Lys Pro Asp Glu Pro Gly Asn Thr Phe Asn Val Ser Ser
130 135 140
Ile Pro Trp Val Asn Phe Thr Gly Phe Asn Leu Asn Ile Tyr Asn Asp
145 150 155 160
Ala Thr Tyr Leu Ile Pro Ile Phe Thr Met Gly Lys Phe Phe His Gln
165 170 175
Asp Asn Lys Ile Phe Ile Pro Met Ser Ile Gln Val His His Ala Val
180 185 190
Cys Asp Gly Tyr His Thr Ser Arg Phe Phe Asn Glu Val Gln Glu Leu
195 200 205
Ser Ser Asn Phe Glu Thr Trp Leu Gly Glu Lys
210 215
<210> 64
<211> 219
<212> PRT
<213> 人工序列
<220>
<223> 突尼斯梭状芽孢杆菌（Clostridium tunisiense）
<400> 64
Met Lys Phe Asn Leu Ile Asp Thr Glu His Trp Asp Arg Lys Pro Tyr
1 5 10 15
Phe Glu His Tyr Phe Asn Ser Val Lys Cys Thr Tyr Ser Met Thr Ala
20 25 30
Asn Ile Glu Ile Thr Asn Leu Leu Asn His Ile Arg Leu Lys Lys Leu
35 40 45
Lys Leu Tyr Pro Thr Leu Ile Tyr Ile Ile Ala Thr Val Val Asn Asn
50 55 60
His Glu Glu Phe Arg Ile Cys Phe Asp Glu Asn Asn Asn Leu Gly Tyr
65 70 75 80
Trp Asp Ser Met Ser Pro Asn Tyr Thr Ile Phe His Glu Asp Asn Lys
85 90 95
Thr Phe Ser Ser Ile Trp Thr Glu Tyr Glu Glu Ser Phe Ser Gly Phe
100 105 110
Tyr Asn Lys Tyr Leu Glu Asp Ile Lys Thr Tyr Gly His Ile Met Ser
115 120 125
Phe Glu Pro Lys Leu Asn Glu Ser Thr Asn Thr Phe Pro Ile Ser Cys
130 135 140
Ile Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Ile Gln Asp Asp
145 150 155 160
Gly Thr Tyr Leu Thr Pro Ile Phe Thr Leu Gly Lys Tyr Phe Glu Gln
165 170 175
Asn Asn Lys Thr Phe Ile Pro Ile Ser Ile Gln Val His His Ala Val
180 185 190
Cys Asp Gly Tyr His Thr Ser Arg Phe Ile Asn Glu Val Gln Glu Leu
195 200 205
Ala Ser Asp Phe Gln Ile Trp Leu Thr Tyr Lys
210 215
<210> 65
<211> 219
<212> PRT
<213> 人工序列
<220>
<223> 毛螺菌科（Lachnospiraceae）
<400> 65
Met Lys Phe Asn Leu Ile Asp Ile Glu Asp Trp Asn Arg Lys Pro Tyr
1 5 10 15
Phe Glu His Tyr Leu Asn Ala Val Arg Cys Thr Tyr Ser Met Thr Ala
20 25 30
Asn Ile Glu Ile Thr Gly Leu Leu Arg Glu Ile Lys Leu Lys Gly Leu
35 40 45
Lys Leu Tyr Pro Thr Leu Ile Tyr Ile Ile Thr Thr Val Val Asn Arg
50 55 60
His Lys Glu Phe Arg Thr Cys Phe Asp Gln Lys Gly Lys Leu Gly Tyr
65 70 75 80
Trp Asp Ser Met Asn Pro Ser Tyr Thr Val Phe His Lys Asp Asn Glu
85 90 95
Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asp Glu Asn Phe Pro Arg Phe
100 105 110
Tyr Tyr Asn Tyr Leu Glu Asp Ile Arg Asn Tyr Ser Asp Val Leu Asn
115 120 125
Phe Met Pro Lys Thr Gly Glu Pro Ala Asn Thr Ile Asn Val Ser Ser
130 135 140
Ile Pro Trp Val Asn Phe Thr Gly Phe Asn Leu Asn Ile Tyr Asn Asp
145 150 155 160
Ala Thr Tyr Leu Ile Pro Ile Phe Thr Leu Gly Lys Tyr Phe Gln Gln
165 170 175
Asp Asn Lys Ile Leu Leu Pro Met Ser Val Gln Val His His Ala Val
180 185 190
Cys Asp Gly Tyr His Thr Ser Arg Phe Phe Asn Glu Ala Gln Glu Leu
195 200 205
Ala Ser Asn Tyr Glu Thr Trp Leu Gly Glu Lys
210 215
<210> 66
<211> 219
<212> PRT
<213> 产气荚膜梭状芽孢杆菌（Clostridium perfringens）
<400> 66
Met Lys Phe Asn Leu Ile Asp Ile Glu Asp Trp Asn Arg Lys Pro Tyr
1 5 10 15
Phe Glu His Tyr Leu Asn Ala Val Arg Cys Thr Tyr Ser Met Thr Ala
20 25 30
Asn Ile Glu Ile Thr Gly Leu Leu Arg Glu Ile Lys Leu Lys Gly Leu
35 40 45
Lys Leu Tyr Pro Thr Leu Ile Tyr Ile Ile Thr Thr Val Val Asn Arg
50 55 60
His Lys Glu Phe Arg Thr Cys Phe Asp Gln Lys Gly Lys Leu Gly Tyr
65 70 75 80
Trp Asp Ser Met Asn Pro Ser Tyr Thr Val Phe His Lys Asp Asn Glu
85 90 95
Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asp Glu Asn Phe Pro Arg Phe
100 105 110
Tyr Tyr Asn Tyr Leu Glu Asp Ile Arg Asn Tyr Ser Asp Val Leu Asn
115 120 125
Phe Met Pro Lys Thr Gly Glu Pro Ala Asn Thr Ile Asn Val Ser Ser
130 135 140
Ile Pro Trp Val Asn Phe Thr Gly Phe Asn Leu Asn Ile Tyr Asn Asp
145 150 155 160
Ala Thr Tyr Leu Ile Pro Ile Phe Thr Leu Gly Lys Tyr Phe Gln Gln
165 170 175
Asp Asn Lys Ile Leu Leu Pro Met Ser Val Gln Val His His Ala Val
180 185 190
Cys Asp Gly Tyr His Ile Ser Arg Phe Phe Asn Glu Ala Gln Glu Leu
195 200 205
Ala Ser Asn Tyr Glu Thr Trp Leu Gly Glu Lys
210 215
<210> 67
<211> 218
<212> PRT
<213> 人工序列
<220>
<223> 梭状芽孢杆菌属物种BL8
<400> 67
Met Lys Phe Asn Leu Ile Asp Ile Asp Gln Trp Asp Arg Lys Pro Tyr
1 5 10 15
Phe Glu His Tyr Phe Asn Ser Val Lys Cys Thr Tyr Ser Ile Thr Ala
20 25 30
Asn Ile Glu Ile Thr Asn Leu Leu Lys Asp Ile Lys Ile Thr Lys Leu
35 40 45
Lys Leu Tyr Pro Thr Leu Ile Tyr Ile Ile Ala Thr Val Ile Asn Asn
50 55 60
His Glu Glu Phe Arg Thr Cys Phe Asp Glu Asn Asn Asn Leu Gly Tyr
65 70 75 80
Trp Asp Ser Met Ser Pro Asn Tyr Thr Ile Phe His Glu Glu Thr Lys
85 90 95
Thr Phe Ser Asn Ile Trp Thr Glu Tyr Asp Lys Ser Phe Ser Gly Phe
100 105 110
Tyr Asn Lys Tyr Val Glu Asp Asn Lys Asn Tyr Gly Asn Ile Met Asn
115 120 125
Phe Asp Pro Lys Leu Asn Glu Pro Ala Asn Thr Phe Pro Ile Ser Cys
130 135 140
Ile Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Ile Gln Asp His
145 150 155 160
Gly Thr Tyr Leu Thr Pro Ile Phe Thr Leu Gly Lys Tyr Phe Glu Glu
165 170 175
Asn Asn Lys Val Phe Ile Pro Met Ser Ile Gln Val His His Ala Val
180 185 190
Cys Asp Gly Tyr His Thr Ser Arg Phe Ile Asn Glu Val Gln Glu Leu
195 200 205
Ala Ser Asn Ser Gln Ser Trp Leu Lys His
210 215
<210> 68
<211> 660
<212> DNA
<213> 产气荚膜梭状芽孢杆菌（Clostridium perfringens）
<400> 68
atgaaattta atttgataga tattgaggat tggaatagaa agccatactt tgagcattat 60
ttaaatgcgg ttaggtgcac ttacagtatg actgcaaata tagagataac tggtttactg 120
cgtgaaatta aacttaaggg cctgaaactg taccctacgc ttatttatat catcacaact 180
gtggttaacc gtcacaagga gttccgcacc tgttttgatc aaaaaggtaa gttaggatac 240
tgggatagta tgaacccaag ttatactgtc tttcataagg ataacgaaac tttttcaagt 300
atttggacag agtatgacga gaacttccca cgtttttact ataattacct tgaggatatt 360
agaaactata gcgacgtttt gaatttcatg cctaagacag gtgaacctgc taatacaatt 420
aatgtgtcca gcattccttg ggtgaatttt accggattca acctgaatat atacaatgat 480
gcaacatatc taatccctat ttttactttg ggtaagtatt ttcagcagga taataaaatt 540
ttattaccta tgtctgtaca ggtgcatcat gcggtttgcg acggttatca tataagcaga 600
ttttttaatg aggcacagga attagcgtca aattatgaga catggttagg agaaaaataa 660
<210> 69
<211> 624
<212> DNA
<213> 难辨梭状芽孢杆菌（Clostridium difficile）
<400> 69
atggtatttg aaaaaattga taaaaatagt tggaacagaa aagagtattt tgaccactac 60
tttgcaagtg taccttgtac atacagcatg accgttaaag tggatatcac acaaataaag 120
gaaaagggaa tgaaactata tcctgcaatg ctttattata ttgcaatgat tgtaaaccgc 180
cattcagagt ttaggacggc aatcaatcaa gatggtgaat tggggatata tgatgagatg 240
ataccaagct atacaatatt tcacaatgat actgaaacat tttccagcct ttggactgag 300
tgtaagtctg actttaaatc atttttagca gattatgaaa gtgatacgca acggtatgga 360
aacaatcata gaatggaagg aaagccaaat gctccggaaa acatttttaa tgtatctatg 420
ataccgtggt caaccttcga tggctttaat ctgaatttgc agaaaggata tgattatttg 480
attcctattt ttactatggg gaaatattat aaagaagata acaaaattat acttcctttg 540
gcaattcaag ttcatcacgc agtatgtgac ggatttcaca tttgccgttt tgtaaacgaa 600
ttgcaggaat tgataaatag ttaa 624
<210> 70
<211> 624
<212> DNA
<213> 产气荚膜梭状芽孢杆菌（Clostridium perfringens）
<400> 70
atggtatttg aaaaaattga taaaaatagt tggaacagaa aagagtattt tgaccactac 60
tttgcaagtg taccttgtac atacagcatg accgttaaag tggatatcac acaaataaag 120
gaaaagggaa tgaaactata tcctgcaatg ctttattata ttgcaatgat tgtaaaccgc 180
cattcagagt ttaggacggc aatcaatcaa gatggtgaat tggggatata tgatgagatg 240
ataccaagct atacaatatt tcacaatgat actgaaacat tttccagcct ttggactgag 300
tgtaagtctg actttaaatc atttttagca gattatgaaa gtgatacgca acggtatgga 360
aacaatcata gaatggaagg aaagccaaat gctccggaaa acatttttaa tgtatctatg 420
ataccgtggt caaccttcga tggctttaat ctgaatttgc agaaaggata tgattatttg 480
attcctattt ttactatggg gaaatattat aaagaagata acaaaattat acttcctttg 540
gcaattcaag ttcatcacgc agtatgtgac ggatttcaca tttgccgttt tgtaaacgaa 600
ttgcaggaat tgataaatag ttaa 624
<210> 71
<211> 3897
<212> DNA
<213> 人工序列
<220>
<223> 优化的MAD7
<400> 71
ctcgagtccc tatcagtgat agattgaaac tctatcattg atagagtata atatctttgt 60
tcattagagc gataaacttg aatttgagag ggaacttaga tgaacaacgg cacaaataat 120
tttcagaact tcatagggat atcaagtttg cagaaaacgt taagaaatgc tttaataccc 180
acggaaacca cgcaacagtt catagttaag aacggaataa ttaaagaaga tgagttaaga 240
ggcgagaaca gacagatttt aaaagatata atggatgact actacagagg attcatatct 300
gagactttaa gttctattga tgacatagat tggactagct tattcgaaaa aatggaaatt 360
cagttaaaaa atggtgataa taaagatacc ttaattaagg aacagacaga gtatagaaaa 420
gcaatacata aaaaatttgc gaacgacgat agatttaaga acatgtttag cgccaaatta 480
attagtgaca tattacctga atttgttata cacaacaata attattcggc atcagagaaa 540
gaggaaaaaa cccaggtgat aaaattgttt tcgagatttg cgactagctt taaagattac 600
ttcaagaaca gagcaaattg cttttcagcg gacgatattt catcaagcag ctgccataga 660
atagttaacg acaatgcaga gatattcttt tcaaatgcgt tagtttacag aagaatagta 720
aaatcgttaa gcaatgacga tataaacaaa atttcgggcg atatgaaaga ttcattaaaa 780
gaaatgagtt tagaagaaat atattcttac gagaagtatg gggaatttat tacccaggaa 840
ggcattagct tctataatga tatatgtggg aaagtgaatt cttttatgaa cttatattgt 900
cagaaaaata aagaaaacaa aaatttatac aaacttcaga aacttcacaa acagattcta 960
tgcattgcgg acactagcta tgaggttccg tataaatttg aaagtgacga ggaagtgtac 1020
caatcagtta acggcttcct tgataacatt agcagcaaac atatagttga aagattaaga 1080
aaaataggcg ataactataa cggctacaac ttagataaaa tttatatagt gtccaaattt 1140
tacgagagcg ttagccaaaa aacctacaga gactgggaaa caattaatac cgccttagaa 1200
attcattaca ataatatatt gccgggtaac ggtaaaagta aagccgacaa agtaaaaaaa 1260
gcggttaaga atgatttaca gaaatccata accgaaataa atgaactagt gtcaaactat 1320
aagttatgca gtgacgacaa cataaaagcg gagacttata tacatgagat tagccatata 1380
ttgaataact ttgaagcaca ggaattgaaa tacaatccgg aaattcacct agttgaatcc 1440
gagttaaaag cgagtgagct taaaaacgtg ttagacgtga taatgaatgc gtttcattgg 1500
tgttcggttt ttatgactga ggaacttgtt gataaagaca acaattttta tgcggaatta 1560
gaggagattt acgatgaaat ttatccagta attagtttat acaacttagt tagaaactac 1620
gttacccaga aaccgtacag cacgaaaaag attaaattga actttggaat accgacgtta 1680
gcagacggtt ggtcaaagtc caaagagtat tctaataacg ctataatatt aatgagagac 1740
aatttatatt atttaggcat atttaatgcg aagaataaac cggacaagaa gattatagag 1800
ggtaatacgt cagaaaataa gggtgactac aaaaagatga tttataattt gttaccgggt 1860
cccaacaaaa tgataccgaa agttttcttg agcagcaaga cgggggtgga aacgtataaa 1920
ccgagcgcct atatactaga ggggtataaa cagaataaac atataaagtc ttcaaaagac 1980
tttgatataa ctttctgtca tgatttaata gactacttca aaaactgtat tgcaattcat 2040
cccgagtgga aaaacttcgg ttttgatttt agcgacacca gtacttatga agacatttcc 2100
gggttttata gagaggtaga gttacaaggt tacaagattg attggacata cattagcgaa 2160
aaagacattg atttattaca ggaaaaaggt caattatatt tattccagat atataacaaa 2220
gatttttcga aaaaatcaac cgggaatgac aaccttcaca ccatgtactt aaaaaatctt 2280
ttctcagaag aaaatcttaa ggatatagtt ttaaaactta acggcgaagc ggaaatattc 2340
ttcaggaaga gcagcataaa gaacccaata attcataaaa aaggctcgat tttagttaac 2400
agaacctacg aagcagaaga aaaagaccag tttggcaaca ttcaaattgt gagaaaaaat 2460
attccggaaa acatttatca ggagttatac aaatacttca acgataaaag cgacaaagag 2520
ttatctgatg aagcagccaa attaaagaat gtagtgggac accacgaggc agcgacgaat 2580
atagttaagg actatagata cacgtatgat aaatacttcc ttcatatgcc tattacgata 2640
aatttcaaag ccaataaaac gggttttatt aatgatagga tattacagta tatagctaaa 2700
gaaaaagact tacatgtgat aggcattgat agaggcgaga gaaacttaat atacgtgtcc 2760
gtgattgata cttgtggtaa tatagttgaa cagaaaagct ttaacattgt aaacggctac 2820
gactatcaga taaaattaaa acaacaggag ggcgctagac agattgcgag aaaagaatgg 2880
aaagaaattg gtaaaattaa agagataaaa gagggctact taagcttagt aatacacgag 2940
atatctaaaa tggtaataaa atacaatgca attatagcga tggaggattt gtcttatggt 3000
tttaaaaaag ggagatttaa ggttgaaaga caagtttacc agaaatttga aaccatgtta 3060
ataaataaat taaactattt agtatttaaa gatatttcga ttaccgagaa tggcggttta 3120
ttaaaaggtt atcagttaac atacattcct gataaactta aaaacgtggg tcatcagtgc 3180
ggctgcattt tttatgtgcc tgctgcatac acgagcaaaa ttgatccgac caccggcttt 3240
gtgaatatat ttaaatttaa agacttaaca gtggacgcaa aaagagaatt cattaaaaaa 3300
tttgactcaa ttagatatga cagtgaaaaa aatttattct gctttacatt tgactacaat 3360
aactttatta cgcaaaacac ggttatgagc aaatcatcgt ggagtgtgta tacatacggc 3420
gtgagaataa aaagaagatt tgtgaacggc agattctcaa acgaaagtga taccattgac 3480
ataaccaaag atatggagaa aacgttggaa atgacggaca ttaactggag agatggccac 3540
gatcttagac aagacattat agattatgaa attgttcagc acatattcga aattttcaga 3600
ttaacagtgc aaatgagaaa ctccttgtct gaattagagg acagagatta cgatagatta 3660
atttcacctg tattaaacga aaataacatt ttttatgaca gcgcgaaagc gggggatgca 3720
cttcctaagg atgccgatgc aaatggtgcg tattgtattg cattaaaagg gttatatgaa 3780
attaaacaaa ttaccgaaaa ttggaaagaa gatggtaaat tttcgagaga taaattaaaa 3840
ataagcaata aagattggtt cgactttata cagaataaga gatatttata agtcgac 3897
<210> 72
<211> 1263
<212> PRT
<213> 人工序列
<220>
<223> MAD7
<400> 72
Met Asn Asn Gly Thr Asn Asn Phe Gln Asn Phe Ile Gly Ile Ser Ser
1 5 10 15
Leu Gln Lys Thr Leu Arg Asn Ala Leu Ile Pro Thr Glu Thr Thr Gln
20 25 30
Gln Phe Ile Val Lys Asn Gly Ile Ile Lys Glu Asp Glu Leu Arg Gly
35 40 45
Glu Asn Arg Gln Ile Leu Lys Asp Ile Met Asp Asp Tyr Tyr Arg Gly
50 55 60
Phe Ile Ser Glu Thr Leu Ser Ser Ile Asp Asp Ile Asp Trp Thr Ser
65 70 75 80
Leu Phe Glu Lys Met Glu Ile Gln Leu Lys Asn Gly Asp Asn Lys Asp
85 90 95
Thr Leu Ile Lys Glu Gln Thr Glu Tyr Arg Lys Ala Ile His Lys Lys
100 105 110
Phe Ala Asn Asp Asp Arg Phe Lys Asn Met Phe Ser Ala Lys Leu Ile
115 120 125
Ser Asp Ile Leu Pro Glu Phe Val Ile His Asn Asn Asn Tyr Ser Ala
130 135 140
Ser Glu Lys Glu Glu Lys Thr Gln Val Ile Lys Leu Phe Ser Arg Phe
145 150 155 160
Ala Thr Ser Phe Lys Asp Tyr Phe Lys Asn Arg Ala Asn Cys Phe Ser
165 170 175
Ala Asp Asp Ile Ser Ser Ser Ser Cys His Arg Ile Val Asn Asp Asn
180 185 190
Ala Glu Ile Phe Phe Ser Asn Ala Leu Val Tyr Arg Arg Ile Val Lys
195 200 205
Ser Leu Ser Asn Asp Asp Ile Asn Lys Ile Ser Gly Asp Met Lys Asp
210 215 220
Ser Leu Lys Glu Met Ser Leu Glu Glu Ile Tyr Ser Tyr Glu Lys Tyr
225 230 235 240
Gly Glu Phe Ile Thr Gln Glu Gly Ile Ser Phe Tyr Asn Asp Ile Cys
245 250 255
Gly Lys Val Asn Ser Phe Met Asn Leu Tyr Cys Gln Lys Asn Lys Glu
260 265 270
Asn Lys Asn Leu Tyr Lys Leu Gln Lys Leu His Lys Gln Ile Leu Cys
275 280 285
Ile Ala Asp Thr Ser Tyr Glu Val Pro Tyr Lys Phe Glu Ser Asp Glu
290 295 300
Glu Val Tyr Gln Ser Val Asn Gly Phe Leu Asp Asn Ile Ser Ser Lys
305 310 315 320
His Ile Val Glu Arg Leu Arg Lys Ile Gly Asp Asn Tyr Asn Gly Tyr
325 330 335
Asn Leu Asp Lys Ile Tyr Ile Val Ser Lys Phe Tyr Glu Ser Val Ser
340 345 350
Gln Lys Thr Tyr Arg Asp Trp Glu Thr Ile Asn Thr Ala Leu Glu Ile
355 360 365
His Tyr Asn Asn Ile Leu Pro Gly Asn Gly Lys Ser Lys Ala Asp Lys
370 375 380
Val Lys Lys Ala Val Lys Asn Asp Leu Gln Lys Ser Ile Thr Glu Ile
385 390 395 400
Asn Glu Leu Val Ser Asn Tyr Lys Leu Cys Ser Asp Asp Asn Ile Lys
405 410 415
Ala Glu Thr Tyr Ile His Glu Ile Ser His Ile Leu Asn Asn Phe Glu
420 425 430
Ala Gln Glu Leu Lys Tyr Asn Pro Glu Ile His Leu Val Glu Ser Glu
435 440 445
Leu Lys Ala Ser Glu Leu Lys Asn Val Leu Asp Val Ile Met Asn Ala
450 455 460
Phe His Trp Cys Ser Val Phe Met Thr Glu Glu Leu Val Asp Lys Asp
465 470 475 480
Asn Asn Phe Tyr Ala Glu Leu Glu Glu Ile Tyr Asp Glu Ile Tyr Pro
485 490 495
Val Ile Ser Leu Tyr Asn Leu Val Arg Asn Tyr Val Thr Gln Lys Pro
500 505 510
Tyr Ser Thr Lys Lys Ile Lys Leu Asn Phe Gly Ile Pro Thr Leu Ala
515 520 525
Asp Gly Trp Ser Lys Ser Lys Glu Tyr Ser Asn Asn Ala Ile Ile Leu
530 535 540
Met Arg Asp Asn Leu Tyr Tyr Leu Gly Ile Phe Asn Ala Lys Asn Lys
545 550 555 560
Pro Asp Lys Lys Ile Ile Glu Gly Asn Thr Ser Glu Asn Lys Gly Asp
565 570 575
Tyr Lys Lys Met Ile Tyr Asn Leu Leu Pro Gly Pro Asn Lys Met Ile
580 585 590
Pro Lys Val Phe Leu Ser Ser Lys Thr Gly Val Glu Thr Tyr Lys Pro
595 600 605
Ser Ala Tyr Ile Leu Glu Gly Tyr Lys Gln Asn Lys His Ile Lys Ser
610 615 620
Ser Lys Asp Phe Asp Ile Thr Phe Cys His Asp Leu Ile Asp Tyr Phe
625 630 635 640
Lys Asn Cys Ile Ala Ile His Pro Glu Trp Lys Asn Phe Gly Phe Asp
645 650 655
Phe Ser Asp Thr Ser Thr Tyr Glu Asp Ile Ser Gly Phe Tyr Arg Glu
660 665 670
Val Glu Leu Gln Gly Tyr Lys Ile Asp Trp Thr Tyr Ile Ser Glu Lys
675 680 685
Asp Ile Asp Leu Leu Gln Glu Lys Gly Gln Leu Tyr Leu Phe Gln Ile
690 695 700
Tyr Asn Lys Asp Phe Ser Lys Lys Ser Thr Gly Asn Asp Asn Leu His
705 710 715 720
Thr Met Tyr Leu Lys Asn Leu Phe Ser Glu Glu Asn Leu Lys Asp Ile
725 730 735
Val Leu Lys Leu Asn Gly Glu Ala Glu Ile Phe Phe Arg Lys Ser Ser
740 745 750
Ile Lys Asn Pro Ile Ile His Lys Lys Gly Ser Ile Leu Val Asn Arg
755 760 765
Thr Tyr Glu Ala Glu Glu Lys Asp Gln Phe Gly Asn Ile Gln Ile Val
770 775 780
Arg Lys Asn Ile Pro Glu Asn Ile Tyr Gln Glu Leu Tyr Lys Tyr Phe
785 790 795 800
Asn Asp Lys Ser Asp Lys Glu Leu Ser Asp Glu Ala Ala Lys Leu Lys
805 810 815
Asn Val Val Gly His His Glu Ala Ala Thr Asn Ile Val Lys Asp Tyr
820 825 830
Arg Tyr Thr Tyr Asp Lys Tyr Phe Leu His Met Pro Ile Thr Ile Asn
835 840 845
Phe Lys Ala Asn Lys Thr Gly Phe Ile Asn Asp Arg Ile Leu Gln Tyr
850 855 860
Ile Ala Lys Glu Lys Asp Leu His Val Ile Gly Ile Asp Arg Gly Glu
865 870 875 880
Arg Asn Leu Ile Tyr Val Ser Val Ile Asp Thr Cys Gly Asn Ile Val
885 890 895
Glu Gln Lys Ser Phe Asn Ile Val Asn Gly Tyr Asp Tyr Gln Ile Lys
900 905 910
Leu Lys Gln Gln Glu Gly Ala Arg Gln Ile Ala Arg Lys Glu Trp Lys
915 920 925
Glu Ile Gly Lys Ile Lys Glu Ile Lys Glu Gly Tyr Leu Ser Leu Val
930 935 940
Ile His Glu Ile Ser Lys Met Val Ile Lys Tyr Asn Ala Ile Ile Ala
945 950 955 960
Met Glu Asp Leu Ser Tyr Gly Phe Lys Lys Gly Arg Phe Lys Val Glu
965 970 975
Arg Gln Val Tyr Gln Lys Phe Glu Thr Met Leu Ile Asn Lys Leu Asn
980 985 990
Tyr Leu Val Phe Lys Asp Ile Ser Ile Thr Glu Asn Gly Gly Leu Leu
995 1000 1005
Lys Gly Tyr Gln Leu Thr Tyr Ile Pro Asp Lys Leu Lys Asn Val
1010 1015 1020
Gly His Gln Cys Gly Cys Ile Phe Tyr Val Pro Ala Ala Tyr Thr
1025 1030 1035
Ser Lys Ile Asp Pro Thr Thr Gly Phe Val Asn Ile Phe Lys Phe
1040 1045 1050
Lys Asp Leu Thr Val Asp Ala Lys Arg Glu Phe Ile Lys Lys Phe
1055 1060 1065
Asp Ser Ile Arg Tyr Asp Ser Glu Lys Asn Leu Phe Cys Phe Thr
1070 1075 1080
Phe Asp Tyr Asn Asn Phe Ile Thr Gln Asn Thr Val Met Ser Lys
1085 1090 1095
Ser Ser Trp Ser Val Tyr Thr Tyr Gly Val Arg Ile Lys Arg Arg
1100 1105 1110
Phe Val Asn Gly Arg Phe Ser Asn Glu Ser Asp Thr Ile Asp Ile
1115 1120 1125
Thr Lys Asp Met Glu Lys Thr Leu Glu Met Thr Asp Ile Asn Trp
1130 1135 1140
Arg Asp Gly His Asp Leu Arg Gln Asp Ile Ile Asp Tyr Glu Ile
1145 1150 1155
Val Gln His Ile Phe Glu Ile Phe Arg Leu Thr Val Gln Met Arg
1160 1165 1170
Asn Ser Leu Ser Glu Leu Glu Asp Arg Asp Tyr Asp Arg Leu Ile
1175 1180 1185
Ser Pro Val Leu Asn Glu Asn Asn Ile Phe Tyr Asp Ser Ala Lys
1190 1195 1200
Ala Gly Asp Ala Leu Pro Lys Asp Ala Asp Ala Asn Gly Ala Tyr
1205 1210 1215
Cys Ile Ala Leu Lys Gly Leu Tyr Glu Ile Lys Gln Ile Thr Glu
1220 1225 1230
Asn Trp Lys Glu Asp Gly Lys Phe Ser Arg Asp Lys Leu Lys Ile
1235 1240 1245
Ser Asn Lys Asp Trp Phe Asp Phe Ile Gln Asn Lys Arg Tyr Leu
1250 1255 1260
<210> 73
<211> 363
<212> DNA
<213> 人工序列
<220>
<223> 启动子CatB
<400> 73
taaaaaatgt tacgcacttt tcttatattg ttcaacaata acataattta ttaacaaaag 60
gaaagtatag ttaaaaaaat gttggagcaa atgcggatgg aaaaataaaa attaatatta 120
gtagtaattc cgatgttaaa ataacaagag ataagaaaaa gtaaaatatt agagtaattc 180
gtagtattct taagttatga atcaataaaa aatggtctct gaaaattgaa tagttcggta 240
ttacagaatg tgctataata aactaaagcg taaatatcat tgtaaaaagg agattgaaat 300
ggctaggtca cggaaaaaag ccttctaaaa tagaattacg aaaattttta ggaggcccga 360
att 363
<210> 74
<211> 322
<212> DNA
<213> 人工序列
<220>
<223> 启动子CATQ
<400> 74
ctgcgtacac atccagacat cgctttagag tatggtgaat taaagatgga gcgggcttat 60
cgattctcag aggatattga aggctactgc actggtaagg atgcatttgt aaagcaacta 120
gaaaaggatg ctttgcgatg gtggcaaact gtctgttagg aggttattct caaaggattg 180
caagaagcag ttgaggataa tccgtataac taactattac acattcttaa cattgctggt 240
ttgtatcggt agaataacac gaattaacaa aggatatatt ttgtagtagc aagtgtattt 300
gttttatatt ctatgaacct at 322
<210> 75
<211> 1368
<212> PRT
<213> 化脓链球菌（Streptococcus pyogenes）
<400> 75
Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val
1 5 10 15
Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe
20 25 30
Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile
35 40 45
Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu
50 55 60
Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys
65 70 75 80
Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser
85 90 95
Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys
100 105 110
His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr
115 120 125
His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp
130 135 140
Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His
145 150 155 160
Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro
165 170 175
Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr
180 185 190
Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala
195 200 205
Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn
210 215 220
Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn
225 230 235 240
Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe
245 250 255
Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp
260 265 270
Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp
275 280 285
Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp
290 295 300
Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser
305 310 315 320
Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys
325 330 335
Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe
340 345 350
Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser
355 360 365
Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp
370 375 380
Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg
385 390 395 400
Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu
405 410 415
Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe
420 425 430
Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile
435 440 445
Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp
450 455 460
Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu
465 470 475 480
Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr
485 490 495
Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser
500 505 510
Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys
515 520 525
Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln
530 535 540
Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr
545 550 555 560
Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp
565 570 575
Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly
580 585 590
Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp
595 600 605
Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr
610 615 620
Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala
625 630 635 640
His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr
645 650 655
Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp
660 665 670
Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe
675 680 685
Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe
690 695 700
Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu
705 710 715 720
His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly
725 730 735
Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly
740 745 750
Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln
755 760 765
Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile
770 775 780
Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro
785 790 795 800
Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu
805 810 815
Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg
820 825 830
Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys
835 840 845
Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg
850 855 860
Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys
865 870 875 880
Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys
885 890 895
Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp
900 905 910
Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr
915 920 925
Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp
930 935 940
Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser
945 950 955 960
Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg
965 970 975
Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val
980 985 990
Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe
995 1000 1005
Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala
1010 1015 1020
Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe
1025 1030 1035
Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala
1040 1045 1050
Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu
1055 1060 1065
Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val
1070 1075 1080
Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr
1085 1090 1095
Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys
1100 1105 1110
Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro
1115 1120 1125
Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val
1130 1135 1140
Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys
1145 1150 1155
Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser
1160 1165 1170
Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys
1175 1180 1185
Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu
1190 1195 1200
Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly
1205 1210 1215
Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val
1220 1225 1230
Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser
1235 1240 1245
Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys
1250 1255 1260
His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys
1265 1270 1275
Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala
1280 1285 1290
Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn
1295 1300 1305
Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala
1310 1315 1320
Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser
1325 1330 1335
Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr
1340 1345 1350
Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp
1355 1360 1365
<210> 76
<211> 4107
<212> DNA
<213> 化脓链球菌（Streptococcus pyogenes）
<400> 76
atggataaga aatactcaat aggcttagat atcggcacaa atagcgtcgg atgggcggtg 60
atcactgatg aatataaggt tccgtctaaa aagttcaagg ttctgggaaa tacagaccgc 120
cacagtatca aaaaaaatct tataggggct cttttatttg acagtggaga gacagcggaa 180
gcgactcgtc tcaaacggac agctcgtaga aggtatacac gtcggaagaa tcgtatttgt 240
tatctacagg agattttttc aaatgagatg gcgaaagtag atgatagttt ctttcatcga 300
cttgaagagt cttttttggt ggaagaagac aagaagcatg aacgtcatcc tatttttgga 360
aatatagtag atgaagttgc ttatcatgag aaatatccaa ctatctatca tctgcgaaaa 420
aaattggtag attctactga taaagcggat ttgcgcttaa tctatttggc cttagcgcat 480
atgattaagt ttcgtggtca ttttttgatt gagggagatt taaatcctga taatagtgat 540
gtggacaaac tatttatcca gttggtacaa acctacaatc aattatttga agaaaaccct 600
attaacgcaa gtggagtaga tgctaaagcg attctttctg cacgattgag taaatcaaga 660
cgattagaaa atctcattgc tcagctcccc ggtgagaaga aaaatggctt atttgggaat 720
ctcattgctt tgtcattggg tttgacccct aattttaaat caaattttga tttggcagaa 780
gatgctaaat tacagctttc aaaagatact tacgatgatg atttagataa tttattggcg 840
caaattggag atcaatatgc tgatttgttt ttggcagcta agaatttatc agatgctatt 900
ttactttcag atatcctaag agtaaatact gaaataacta aggctcccct atcagcttca 960
atgattaaac gctacgatga acatcatcaa gacttgactc ttttaaaagc tttagttcga 1020
caacaacttc cagaaaagta taaagaaatc ttttttgatc aatcaaaaaa cggatatgca 1080
ggttatattg atgggggagc tagccaagaa gaattttata aatttatcaa accaatttta 1140
gaaaaaatgg atggtactga ggaattattg gtgaaactaa atcgtgaaga tttgctgcgc 1200
aagcaacgga cctttgacaa cggctctatt ccccatcaaa ttcacttggg tgagctgcat 1260
gctattttga gaagacaaga agacttttat ccatttttaa aagacaatcg tgagaagatt 1320
gaaaaaatct tgacttttcg aattccttat tatgttggtc cattggcgcg tggcaatagt 1380
cgttttgcat ggatgactcg gaagtctgaa gaaacaatta ccccatggaa ttttgaagaa 1440
gttgtcgata aaggtgcttc agctcaatca tttattgaac gcatgacaaa ctttgataaa 1500
aatcttccaa atgaaaaagt actaccaaaa catagtttgc tttatgagta ttttacggtt 1560
tataacgaat tgacaaaggt caaatatgtt actgaaggaa tgcgaaaacc agcatttctt 1620
tcaggtgaac agaagaaagc cattgttgat ttactcttca aaacaaatcg aaaagtaacc 1680
gttaagcaat taaaagaaga ttatttcaaa aaaatagaat gttttgatag tgttgaaatt 1740
tcaggagttg aagatagatt taatgcttca ttaggtacct accatgattt gctaaaaatt 1800
attaaagata aagatttttt ggataatgaa gaaaatgaag atatcttaga ggatattgtt 1860
ttaacattga ccttatttga agatagggag atgattgagg aaagacttaa aacatatgct 1920
cacctctttg atgataaggt gatgaaacag cttaaacgtc gccgttatac tggttgggga 1980
cgtttgtctc gaaaattgat taatggtatt agggataagc aatctggcaa aacaatatta 2040
gattttttga aatcagatgg ttttgccaat cgcaatttta tgcagctgat ccatgatgat 2100
agtttgacat ttaaagaaga cattcaaaaa gcacaagtgt ctggacaagg cgatagttta 2160
catgaacata ttgcaaattt agctggtagc cctgctatta aaaaaggtat tttacagact 2220
gtaaaagttg ttgatgaatt ggtcaaagta atggggcggc ataagccaga aaatatcgtt 2280
attgaaatgg cacgtgaaaa tcagacaact caaaagggcc agaaaaattc gcgagagcgt 2340
atgaaacgaa tcgaagaagg tatcaaagaa ttaggaagtc agattcttaa agagcatcct 2400
gttgaaaata ctcaattgca aaatgaaaag ctctatctct attatctcca aaatggaaga 2460
gacatgtatg tggaccaaga attagatatt aatcgtttaa gtgattatga tgtcgatcac 2520
attgttccac aaagtttcct taaagacgat tcaatagaca ataaggtctt aacgcgttct 2580
gataaaaatc gtggtaaatc ggataacgtt ccaagtgaag aagtagtcaa aaagatgaaa 2640
aactattgga gacaacttct aaacgccaag ttaatcactc aacgtaagtt tgataattta 2700
acgaaagctg aacgtggagg tttgagtgaa cttgataaag ctggttttat caaacgccaa 2760
ttggttgaaa ctcgccaaat cactaagcat gtggcacaaa ttttggatag tcgcatgaat 2820
actaaatacg atgaaaatga taaacttatt cgagaggtta aagtgattac cttaaaatct 2880
aaattagttt ctgacttccg aaaagatttc caattctata aagtacgtga gattaacaat 2940
taccatcatg cccatgatgc gtatctaaat gccgtcgttg gaactgcttt gattaagaaa 3000
tatccaaaac ttgaatcgga gtttgtctat ggtgattata aagtttatga tgttcgtaaa 3060
atgattgcta agtctgagca agaaataggc aaagcaaccg caaaatattt cttttactct 3120
aatatcatga acttcttcaa aacagaaatt acacttgcaa atggagagat tcgcaaacgc 3180
cctctaatcg aaactaatgg ggaaactgga gaaattgtct gggataaagg gcgagatttt 3240
gccacagtgc gcaaagtatt gtccatgccc caagtcaata ttgtcaagaa aacagaagta 3300
cagacaggcg gattctccaa ggagtcaatt ttaccaaaaa gaaattcgga caagcttatt 3360
gctcgtaaaa aagactggga tccaaaaaaa tatggtggtt ttgatagtcc aacggtagct 3420
tattcagtcc tagtggttgc taaggtggaa aaagggaaat cgaagaagtt aaaatccgtt 3480
aaagagttac tagggatcac aattatggaa agaagttcct ttgaaaaaaa tccgattgac 3540
tttttagaag ctaaaggata taaggaagtt aaaaaagact taatcattaa actacctaaa 3600
tatagtcttt ttgagttaga aaacggtcgt aaacggatgc tggctagtgc cggagaatta 3660
caaaaaggaa atgagctggc tctgccaagc aaatatgtga attttttata tttagctagt 3720
cattatgaaa agttgaaggg tagtccagaa gataacgaac aaaaacaatt gtttgtggag 3780
cagcataagc attatttaga tgagattatt gagcaaatca gtgaattttc taagcgtgtt 3840
attttagcag atgccaattt agataaagtt cttagtgcat ataacaaaca tagagacaaa 3900
ccaatacgtg aacaagcaga aaatattatt catttattta cgttgacgaa tcttggagct 3960
cccgctgctt ttaaatattt tgatacaaca attgatcgta aacgatatac gtctacaaaa 4020
gaagttttag atgccactct tatccatcaa tccatcactg gtctttatga aacacgcatt 4080
gatttgagtc agctaggagg tgactga 4107
<210> 77
<211> 1170
<212> DNA
<213> 人工序列
<220>
<223> bdhA
<400> 77
atgctaagtt ttgattattc aataccaact aaagtttttt ttggaaaagg aaaaatagac 60
gtaattggag aagaaattaa gaaatatggc tcaagagtgc ttatagttta tggcggagga 120
agtataaaaa ggaacggtat atatgataga gcaacagcta tattaaaaga aaacaatata 180
gctttctatg aactttcagg agtagagcca aatcctagga taacaacagt aaaaaaaggc 240
atagaaatat gtagagaaaa taatgtggat ttagtattag caataggggg aggaagtgca 300
atagactgtt ctaaggtaat tgcagctgga gtttattatg atggcgatac atgggacatg 360
gttaaagatc catctaaaat aactaaagtt cttccaattg caagtatact tactctttca 420
gcaacagggt ctgaaatgga tcaaattgca gtaatttcaa atatggagac taatgaaaag 480
cttggagtag gacatgatga tatgagacct aaattttcag tgttagatcc tacatatact 540
tttacagtac ctaaaaatca aacagcagcg ggaacagctg acattatgag tcacaccttt 600
gaatcttact ttagtggtgt tgaaggtgct tatgtgcagg acggtatagc agaagcaatc 660
ttaagaacat gtataaagta tggaaaaata gcaatggaga agactgatga ttacgaggct 720
agagctaatt tgatgtgggc ttcaagttta gctataaatg gtctattatc acttggtaag 780
gatagaaaat ggagttgtca tcctatggaa cacgagttaa gtgcatatta tgatataaca 840
catggtgtag gacttgcaat tttaacacct aattggatgg aatatattct aaatgacgat 900
acacttcata aatttgtttc ttatggaata aatgtttggg gaatagacaa gaacaaagat 960
aactatgaaa tagcacgaga ggctattaaa aatacgagag aatactttaa ttcattgggt 1020
attccttcaa agcttagaga agttggaata ggaaaagata aactagaact aatggcaaag 1080
caagctgtta gaaattctgg aggaacaata ggaagtttaa gaccaataaa tgcagaggat 1140
gttcttgaga tatttaaaaa atcttattaa 1170
<210> 78
<211> 1173
<212> DNA
<213> 人工序列
<220>
<223> bdhB
<400> 78
gtggttgatt tcgaatattc aataccaact agaatttttt tcggtaaaga taagataaat 60
gtacttggaa gagagcttaa aaaatatggt tctaaagtgc ttatagttta tggtggagga 120
agtataaaga gaaatggaat atatgataaa gctgtaagta tacttgaaaa aaacagtatt 180
aaattttatg aacttgcagg agtagagcca aatccaagag taactacagt tgaaaaagga 240
gttaaaatat gtagagaaaa tggagttgaa gtagtactag ctataggtgg aggaagtgca 300
atagattgcg caaaggttat agcagcagca tgtgaatatg atggaaatcc atgggatatt 360
gtgttagatg gctcaaaaat aaaaagggtg cttcctatag ctagtatatt aaccattgct 420
gcaacaggat cagaaatgga tacgtgggca gtaataaata atatggatac aaacgaaaaa 480
ctaattgcgg cacatccaga tatggctcct aagttttcta tattagatcc aacgtatacg 540
tataccgtac ctaccaatca aacagcagca ggaacagctg atattatgag tcatatattt 600
gaggtgtatt ttagtaatac aaaaacagca tatttgcagg atagaatggc agaagcgtta 660
ttaagaactt gtattaaata tggaggaata gctcttgaga agccggatga ttatgaggca 720
agagccaatc taatgtgggc ttcaagtctt gcgataaatg gacttttaac atatggtaaa 780
gacactaatt ggagtgtaca cttaatggaa catgaattaa gtgcttatta cgacataaca 840
cacggcgtag ggcttgcaat tttaacacct aattggatgg agtatatttt aaataatgat 900
acagtgtaca agtttgttga atatggtgta aatgtttggg gaatagacaa agaaaaaaat 960
cactatgaca tagcacatca agcaatacaa aaaacaagag attactttgt aaatgtacta 1020
ggtttaccat ctagactgag agatgttgga attgaagaag aaaaattgga cataatggca 1080
aaggaatcag taaagcttac aggaggaacc ataggaaacc taagaccagt aaacgcctcc 1140
gaagtcctac aaatattcaa aaaatctgtg taa 1173
<210> 79
<211> 6560
<212> DNA
<213> 人工序列
<220>
<223> pGRNA-ΔbdhB
<400> 79
gatccccggg taccgagctc gaattcgtaa tcatggtcat agctgtttcc tgtgtgaaat 60
tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg 120
ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag 180
tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt 240
ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg 300
ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg 360
gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 420
gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga 480
cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 540
ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc 600
tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg 660
gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc 720
tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca 780
ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag 840
ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct 900
ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc 960
accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga 1020
tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca 1080
cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat 1140
taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac 1200
caaagctagc ttaatactag tatatactta atgtgataag tgtctgacag ctgaccggtc 1260
taaagaggtc cctagcgcct acggggaatt tgtatcgata aggggtacaa attcccacta 1320
agcgctcggc cggggatcga tccccgggta cgtacccggc agtttttctt tttcggcaag 1380
tgttcaagaa gttattaagt cgggagtgca gtcgaagtgg gcaagttgaa aaattcacaa 1440
aaatgtggta taatatcttt gttcattaga gcgataaact tgaatttgag agggaactta 1500
gatggtattt gaaaaaattg ataaaaatag ttggaacaga aaagagtatt ttgaccacta 1560
ctttgcaagt gtaccttgta cctacagcat gaccgttaaa gtggatatca cacaaataaa 1620
ggaaaaggga atgaaactat atcctgcaat gctttattat attgcaatga ttgtaaaccg 1680
ccattcagag tttaggacgg caatcaatca agatggtgaa ttggggatat atgatgagat 1740
gataccaagc tatacaatat ttcacaatga tactgaaaca ttttccagcc tttggactga 1800
gtgtaagtct gactttaaat catttttagc agattatgaa agtgatacgc aacggtatgg 1860
aaacaatcat agaatggaag gaaagccaaa tgctccggaa aacattttta atgtatctat 1920
gataccgtgg tcaaccttcg atggctttaa tctgaatttg cagaaaggat atgattattt 1980
gattcctatt tttactatgg ggaaatatta taaagaagat aacaaaatta tacttccttt 2040
ggcaattcaa gttcatcacg cagtatgtga cggatttcac atttgccgtt ttgtaaacga 2100
attgcaggaa ttgataaata gttaacttca ggtttgtctg taactaaaaa ctagtattta 2160
acctaggatc aaaaaaattt ccaataatcc cactctaagc cacaaacacg ccctataaaa 2220
tcccgcttta atcccacttt gagacacatg taatattact ttacgcccta gtatagtgat 2280
aattttttac attcaatgcc acgcaaaaaa ataaaggggc actataataa aagttccttc 2340
ggaactaact aaagtaaaaa attatcttta caacctcccc aaaaaaaaga acaggtacaa 2400
agtaccctat aatacaagcg taaaaaaaat gagggtaaaa ataaaaaaat aaaaaaataa 2460
aaaaataaaa aaataaaaaa ataaaaaaat aaaaaaatat aaaaataaaa aaatataaaa 2520
ataaaaaaat ataaaaataa aaaaataaaa aaatataaaa ataaaaaaat aaaaaaatat 2580
aaaaatattt tttatttaaa gtttgaaaaa aattttttta tattatataa tctttgaaga 2640
aaagaatata aaaaatgagc ctttataaaa gcccattttt tttcatatac gtaatatgac 2700
gttctaatgt ttttattggt acttctaaca ttagagtaat ttctttattt ttaaagcctt 2760
tttctttaag ggcttttatt ttttttctta atacatttaa ttcctctttt tttgttgctt 2820
ttcctttagc ttttaattgc tcttgataat tttttttacc tctaatattt tctcttctct 2880
tatattcctt tttagaaatt attattgtca tatatttttg ttcttcttct gtaatttcta 2940
ataactctat aagagtttca ttcttatact tatattgctt atttttatct aaataacatc 3000
tttcagcact tctagttgct cttataactt ctctttcact taaatgttgt ctaaacatac 3060
tattaagttc taaaacatca tttaatgcct tctcaatgtc ttctgtaaag ctacaaagat 3120
aatatctata taaaaataat ataagctctc tgtgtccttt taaatcatat tctcttagtt 3180
cacaaagttt tattatgtct tgtattcttc cataatataa acttctttct ctataaatat 3240
aatttatttt gcttggtcta ccctttttcc tttcatatgg ttttaattca ggtaaaaatc 3300
cattttgtat ttctcttaag tcataaatat attcgtactc atctaatata ttgactactg 3360
tttttgattt agagtttata cttcctggaa ctcttaatat tctcgttgca tctaaggctt 3420
gtctatctgc tccaaagtat tttaattgat tatataaata ttcttgaacc gctttccata 3480
atggtaatgc tttactaggt actgcattta ttatccatat taaatacatt cctcttccac 3540
tatctattac atagtttggt ataggaatac tttgattaaa ataattcttt tctaagtcca 3600
ttaatacctg gtctttagtt ttgccagttt tataataatc caagtctata aacagtgtat 3660
ttaactcttt tatattttct aatcgcctac acggcttata aaaggtattt agagttatat 3720
agatattttc atcactcata tctaaatctt ttaattcagc gtatttatag tgccattggc 3780
tatatccttt tttatctata acgctcctgg ttatccaccc tttacttcta ctatgaatat 3840
tatctatata gttcttttta ttcagcttta atgcgtttct cacttattca cctccccttc 3900
tgtaaaacta agaaaattat atcatatttt caataattat taactattct taaactctta 3960
ataaaaaata gagtaagtcc ccaattgaaa cttaatctat tttttatgtt ttaatttatt 4020
atttttatta aaatatttta aactaaatta aatgattctt tttaattttt tactatttca 4080
ttccataata tattactata attatttaca aataatattt cttcatttgt aatatttaga 4140
tgatttacta attttagttt ttatatatta aataattaat gtataattta tataaaaaat 4200
caaaggagct tataaattat gattatttcc aaagatacta aagatttaat ttttttcaat 4260
tttaacaata ctttttgtaa tattatgttt aaatttaatt gtattttttt catataataa 4320
agccgttgaa gtaaaccaat ccattttcct tatgatgtta ttattaaatt taagttttat 4380
aataatatct ttattatatt tattgttttt aaaaaaacta gtgaaatttc tagtgaaatt 4440
tccggcttta ttaaacttat ttttaggaat tttattttca ttttcatctt tacaggattt 4500
gattatatct ttaaatatgt tttatcaaat attatctttt tctaaattta tatatatttt 4560
tattatattt attattatat atattttatt tttaagtttc tttctaacag ctattaaaaa 4620
gaaacttaaa aataaaaaca cgtactctaa accaataaat aaaactattt ttattattgc 4680
tgccttgatt ggaatagttt ttagtaaaat taatttcaat attccacaat attatattat 4740
aagctagcac gcctcgagac tctatcattg atagagtttg aaactctatc attgatagag 4800
tataatatct ttgttcatgc ttattacgac ataacacagt tttagagcta gaaatagcaa 4860
gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt 4920
tgaagcttct cgagatctcc atggacgcgt gacgtcgact cttaagaaca tgtataaagt 4980
atggaaaaat agcaatggag aagactgatg attacgaggc tagagctaat ttgatgtggg 5040
cttcaagttt agctataaat ggtctattat cacttggtaa ggatagaaaa tggagttgtc 5100
atcctatgga acacgagtta agtgcatatt atgatataac acatggtgta ggacttgcaa 5160
ttttaacacc taattggatg gaatatattc taaatgacga tacacttcat aaatttgttt 5220
cttatggaat aaatgtttgg ggaatagaca agaacaaaga taactatgaa atagcacgag 5280
aggctattaa aaatacgaga gaatacttta attcattggg tattccttca aagcttagag 5340
aagttggaat aggaaaagat aaactagaac taatggcaaa gcaagctgtt agaaattctg 5400
gaggaacaat aggaagttta agaccaataa atgcagagga tgttcttgag atatttaaaa 5460
aatcttatta atagaaactg tagaggtatt tttataattt aaaagatgtt aaagagtgag 5520
gagtaatttt gttctaacgc ctcactcttt tcattttatg attaaatgta tgctgattta 5580
cgctaactta aatcctaaat aataacctaa tgttaatatt ttgtaacaaa tggataaaag 5640
cgtaaaaata ttattgtaat aattttaagt aggtttaaaa tatatataat gtagaagcat 5700
tcctacatta tattatttaa ataataatct aaacaggagg ggttaaagtg gttgatttca 5760
aatctgtgta aacctaccgg ggtttgggcg tagccattat attcatgaac tccaagaaag 5820
cagtatgcta gcaaagaaat aaaactcaaa gcagagagaa aatttagaca ttcaactata 5880
aataaaaaat accccccaaa gcattaatat cttggggagt attttttatt ttgaagtatt 5940
ctgttcagct aaatattctt ctaaggtaat acctctgttc ataatttctt gtgaggcagg 6000
aagaccgata tatcttacat gccatggctc aaaattatac tttgttatgt tttctttatc 6060
cttaggatat cttattatga aaccatattt accacaattt tgttgaagcc atttataaga 6120
atttgtattc ataaatccat catctaaaga agagtattcg gttgatagta agtccattgc 6180
caatccagtt tgatgctcac ttgtaccagg ttcagctaca tatttatcag cttcggcttt 6240
tccgtctcgt gctacttttt cattatataa tttttgctga tacgaataag gtctataacc 6300
tgaaacagct agaagtgtaa gaccatcctt tgatgctgca ttaaacatat tttcaagtcc 6360
tgttgcagct tcgctctcca tttgatttac attaggatca gaactactaa taaatttaac 6420
gttaggagtt ctcaaatttt gaggtatata gtttcctgat aatttacttt gcttgtttac 6480
aagtaggatg ttctgtttct ttacctcggg tttcttggct tgttttttag gtgtagaaac 6540
tttctttttg ggttcgtttg 6560
<210> 80
<211> 6560
<212> DNA
<213> 人工序列
<220>
<223> pGRNA_ΔbdhA_ΔbdhB
<400> 80
gatccccggg taccgagctc gaattcgtaa tcatggtcat agctgtttcc tgtgtgaaat 60
tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg 120
ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag 180
tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt 240
ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg 300
ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg 360
gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 420
gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga 480
cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 540
ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc 600
tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg 660
gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc 720
tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca 780
ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag 840
ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct 900
ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc 960
accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga 1020
tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca 1080
cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat 1140
taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac 1200
caaagctagc ttaatactag tatatactta atgtgataag tgtctgacag ctgaccggtc 1260
taaagaggtc cctagcgcct acggggaatt tgtatcgata aggggtacaa attcccacta 1320
agcgctcggc cggggatcga tccccgggta cgtacccggc agtttttctt tttcggcaag 1380
tgttcaagaa gttattaagt cgggagtgca gtcgaagtgg gcaagttgaa aaattcacaa 1440
aaatgtggta taatatcttt gttcattaga gcgataaact tgaatttgag agggaactta 1500
gatggtattt gaaaaaattg ataaaaatag ttggaacaga aaagagtatt ttgaccacta 1560
ctttgcaagt gtaccttgta cctacagcat gaccgttaaa gtggatatca cacaaataaa 1620
ggaaaaggga atgaaactat atcctgcaat gctttattat attgcaatga ttgtaaaccg 1680
ccattcagag tttaggacgg caatcaatca agatggtgaa ttggggatat atgatgagat 1740
gataccaagc tatacaatat ttcacaatga tactgaaaca ttttccagcc tttggactga 1800
gtgtaagtct gactttaaat catttttagc agattatgaa agtgatacgc aacggtatgg 1860
aaacaatcat agaatggaag gaaagccaaa tgctccggaa aacattttta atgtatctat 1920
gataccgtgg tcaaccttcg atggctttaa tctgaatttg cagaaaggat atgattattt 1980
gattcctatt tttactatgg ggaaatatta taaagaagat aacaaaatta tacttccttt 2040
ggcaattcaa gttcatcacg cagtatgtga cggatttcac atttgccgtt ttgtaaacga 2100
attgcaggaa ttgataaata gttaacttca ggtttgtctg taactaaaaa ctagtattta 2160
acctaggatc aaaaaaattt ccaataatcc cactctaagc cacaaacacg ccctataaaa 2220
tcccgcttta atcccacttt gagacacatg taatattact ttacgcccta gtatagtgat 2280
aattttttac attcaatgcc acgcaaaaaa ataaaggggc actataataa aagttccttc 2340
ggaactaact aaagtaaaaa attatcttta caacctcccc aaaaaaaaga acaggtacaa 2400
agtaccctat aatacaagcg taaaaaaaat gagggtaaaa ataaaaaaat aaaaaaataa 2460
aaaaataaaa aaataaaaaa ataaaaaaat aaaaaaatat aaaaataaaa aaatataaaa 2520
ataaaaaaat ataaaaataa aaaaataaaa aaatataaaa ataaaaaaat aaaaaaatat 2580
aaaaatattt tttatttaaa gtttgaaaaa aattttttta tattatataa tctttgaaga 2640
aaagaatata aaaaatgagc ctttataaaa gcccattttt tttcatatac gtaatatgac 2700
gttctaatgt ttttattggt acttctaaca ttagagtaat ttctttattt ttaaagcctt 2760
tttctttaag ggcttttatt ttttttctta atacatttaa ttcctctttt tttgttgctt 2820
ttcctttagc ttttaattgc tcttgataat tttttttacc tctaatattt tctcttctct 2880
tatattcctt tttagaaatt attattgtca tatatttttg ttcttcttct gtaatttcta 2940
ataactctat aagagtttca ttcttatact tatattgctt atttttatct aaataacatc 3000
tttcagcact tctagttgct cttataactt ctctttcact taaatgttgt ctaaacatac 3060
tattaagttc taaaacatca tttaatgcct tctcaatgtc ttctgtaaag ctacaaagat 3120
aatatctata taaaaataat ataagctctc tgtgtccttt taaatcatat tctcttagtt 3180
cacaaagttt tattatgtct tgtattcttc cataatataa acttctttct ctataaatat 3240
aatttatttt gcttggtcta ccctttttcc tttcatatgg ttttaattca ggtaaaaatc 3300
cattttgtat ttctcttaag tcataaatat attcgtactc atctaatata ttgactactg 3360
tttttgattt agagtttata cttcctggaa ctcttaatat tctcgttgca tctaaggctt 3420
gtctatctgc tccaaagtat tttaattgat tatataaata ttcttgaacc gctttccata 3480
atggtaatgc tttactaggt actgcattta ttatccatat taaatacatt cctcttccac 3540
tatctattac atagtttggt ataggaatac tttgattaaa ataattcttt tctaagtcca 3600
ttaatacctg gtctttagtt ttgccagttt tataataatc caagtctata aacagtgtat 3660
ttaactcttt tatattttct aatcgcctac acggcttata aaaggtattt agagttatat 3720
agatattttc atcactcata tctaaatctt ttaattcagc gtatttatag tgccattggc 3780
tatatccttt tttatctata acgctcctgg ttatccaccc tttacttcta ctatgaatat 3840
tatctatata gttcttttta ttcagcttta atgcgtttct cacttattca cctccccttc 3900
tgtaaaacta agaaaattat atcatatttt caataattat taactattct taaactctta 3960
ataaaaaata gagtaagtcc ccaattgaaa cttaatctat tttttatgtt ttaatttatt 4020
atttttatta aaatatttta aactaaatta aatgattctt tttaattttt tactatttca 4080
ttccataata tattactata attatttaca aataatattt cttcatttgt aatatttaga 4140
tgatttacta attttagttt ttatatatta aataattaat gtataattta tataaaaaat 4200
caaaggagct tataaattat gattatttcc aaagatacta aagatttaat ttttttcaat 4260
tttaacaata ctttttgtaa tattatgttt aaatttaatt gtattttttt catataataa 4320
agccgttgaa gtaaaccaat ccattttcct tatgatgtta ttattaaatt taagttttat 4380
aataatatct ttattatatt tattgttttt aaaaaaacta gtgaaatttc tagtgaaatt 4440
tccggcttta ttaaacttat ttttaggaat tttattttca ttttcatctt tacaggattt 4500
gattatatct ttaaatatgt tttatcaaat attatctttt tctaaattta tatatatttt 4560
tattatattt attattatat atattttatt tttaagtttc tttctaacag ctattaaaaa 4620
gaaacttaaa aataaaaaca cgtactctaa accaataaat aaaactattt ttattattgc 4680
tgccttgatt ggaatagttt ttagtaaaat taatttcaat attccacaat attatattat 4740
aagctagcac gcctcgagac tctatcattg atagagtttg aaactctatc attgatagag 4800
tataatatct ttgttcatgc ttattacgac ataacacagt tttagagcta gaaatagcaa 4860
gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt 4920
tgaagcttct cgagatctcc atggacgcgt gacgtcgacc ttctaatctc ctctactatt 4980
ttagggttag ctacattagc taaataggta atagctacag ttgtctttga attctcacct 5040
aaagtaagtt cttccacttt aaaatcagtg cttctaattt tttttcttaa aagggctaca 5100
tttgtggtta aagattcagt gaagccctct ctaggacctc ttattacagt ttcaacagtt 5160
ggttctgtta tagctctttc agggggtttt ccaatactta taataattgc tttactttca 5220
ccatctagga ataatgctat acttcctttt aaaatggaca atataacatc atccatgctt 5280
ttatatacat ttttatcatt aacagcaaaa attgattttg tatattcaaa tatgtttaaa 5340
tggggatggt tattgtaatc ttcttctata agttttttta taacagagga ttctattaca 5400
tcagattgga taagattatt tatgtagaca atcattgcag aaaaatttct attattagct 5460
attttaaatt ctctaatcgt taaatctgag caatttgtaa ataaggtttc tatagtatgt 5520
ttatttgttt taaggctagt tgaaaccgtc ttcgcgttat ttttagatgc ttcttcttta 5580
ttaaaaattt tattaaacaa cgaaaaattc accccctcaa tttatttata taatagtagt 5640
ttgcatgaaa tttcgttgtt tattcatatt agatgcttgt attaaaataa taaaatagta 5700
aaatataagt agacaaacta taaatctatt actaggaggt aagaagtatg ctaagtttta 5760
aatctgtgta aacctaccgg ggtttgggcg tagccattat attcatgaac tccaagaaag 5820
cagtatgcta gcaaagaaat aaaactcaaa gcagagagaa aatttagaca ttcaactata 5880
aataaaaaat accccccaaa gcattaatat cttggggagt attttttatt ttgaagtatt 5940
ctgttcagct aaatattctt ctaaggtaat acctctgttc ataatttctt gtgaggcagg 6000
aagaccgata tatcttacat gccatggctc aaaattatac tttgttatgt tttctttatc 6060
cttaggatat cttattatga aaccatattt accacaattt tgttgaagcc atttataaga 6120
atttgtattc ataaatccat catctaaaga agagtattcg gttgatagta agtccattgc 6180
caatccagtt tgatgctcac ttgtaccagg ttcagctaca tatttatcag cttcggcttt 6240
tccgtctcgt gctacttttt cattatataa tttttgctga tacgaataag gtctataacc 6300
tgaaacagct agaagtgtaa gaccatcctt tgatgctgca ttaaacatat tttcaagtcc 6360
tgttgcagct tcgctctcca tttgatttac attaggatca gaactactaa taaatttaac 6420
gttaggagtt ctcaaatttt gaggtatata gtttcctgat aatttacttt gcttgtttac 6480
aagtaggatg ttctgtttct ttacctcggg tttcttggct tgttttttag gtgtagaaac 6540
tttctttttg ggttcgtttg 6560
<210> 81
<211> 1654
<212> DNA
<213> 人工序列
<220>
<223> bgaR acrIIA4表达盒
<400> 81
aaaaagtata acagaggttt taatttacgc ctctgttata ctttttattt ttgaaatttt 60
tttgttttaa agctgtattt taaatttata tacttggttt atttacttga ttatttctgt 120
aatttagtgg agacattgaa aaatgttttg aaaaagtttt tgaaaataac agggagtcac 180
tataacctac actacttgcg acttctccta taggaagttt agtgcttttt aataaaaggg 240
tggctttgta cattctaagg tttattaaat atctttgagg agaaattcca aggtttttta 300
tgaacatttt atataaataa cttctactta agttcacata atcagcaatt tcttgaacag 360
ttatgctatg catgtaatta gaattaatga aattaagagc atcttgaata tatgtgtgta 420
attccttatc tttgtattca aaaggttttg ggaattcttc tataagtgcg tacaataatg 480
agtaaagttc ttttagtaat agtatgtcat cagatcttga aggattataa gtttttgata 540
tttcgcacat atttaatatt atctgtggaa tttttgagtt ttcttcacaa ttagcaacac 600
aggagttagt aatagaagtt ctatttaaat actcattagc atttgaacca ctaaatccta 660
tccagtagta ttcccaagga tcatcaatag aagccacata ctcaacttgc atacctttta 720
gtagtataaa aatatcacct tgttttaagt tatatacctt accattaaat ttaaaagttc 780
catatccctt agttacgtaa tgaataacag catttttcaa tacttcatag ttatatccta 840
atcctggtat accttgttct ataccacatt catctacatt catttcaaag ttttctttaa 900
catacttttt ccacaatatt tgcatttcta cctcctaacc tataaaatta gccaatttta 960
tagtagtctt atattaaaca tttacatgag agctttgcaa agcagtttat caacataaaa 1020
gctttttatt ttaaaataaa ttcttctaaa tataagaata ttttaaagaa atatctttat 1080
atattagtta ttaaaattta taagattata agaaacatta taacatattt tagaactttt 1140
taactattct aaaagattaa tttacatatt aacatttaat tatgggtaaa aactattttg 1200
aaaaatgatt tatatggaat tatgtttctt aaatatacaa tcatgtttca tgaatacata 1260
attattttaa atgtattggg agggtaaaat gatattaaaa aatgaatacc atgaagatac 1320
tgcagaatct agaatccgcg gtagtcgacg tggaattgtg agcggataac aatttcacag 1380
gagggctgaa atgaatatta atgacttaat tagagaaata aaaaacaaag attacacagt 1440
gaaattgagt ggtacggata gcaatagtat aacacagcta attattagag ttaataatga 1500
tggaaacgag tatgtaattt ctgaaagtga aaatgaatca atagttgaaa aattcatatc 1560
tgcatttaaa aacggttgga atcaagaata cgaggatgaa gaagaatttt ataatgacat 1620
gcaaacaatc accttaaaaa gtgagttgaa ctaa 1654
<210> 82
<211> 4984
<212> DNA
<213> 人工序列
<220>
<223> pGRNAind
<400> 82
caagcttcaa aaaaagcacc gactcggtgc cactttttca agttgataac ggactagcct 60
tattttaact tgctatttct agctctaaaa cagagaccgc tagcgatatc cccgggagat 120
ctggtctcaa tgaacaaaga tattatactc tatcaatgat agagtttcaa actctatcaa 180
tgatagagtg agctcgaatt cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta 240
tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc 300
ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg 360
aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg 420
tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg 480
gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa 540
cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc 600
gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc 660
aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag 720
ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct 780
cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta 840
ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc 900
cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc 960
agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt 1020
gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct 1080
gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc 1140
tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca 1200
agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta 1260
agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa 1320
atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaaag 1380
ctagcttaat actagtatat acttaatgtg ataagtgtct gacagctgac cggtctaaag 1440
aggtccctag cgcctacggg gaatttgtat cgataagggg tacaaattcc cactaagcgc 1500
tcggccgggg atcgatcccc gggtacgtac ccggcagttt ttctttttcg gcaagtgttc 1560
aagaagttat taagtcggga gtgcagtcga agtgggcaag ttgaaaaatt cacaaaaatg 1620
tggtataata tctttgttca ttagagcgat aaacttgaat ttgagaggga acttagatgg 1680
tatttgaaaa aattgataaa aatagttgga acagaaaaga gtattttgac cactactttg 1740
caagtgtacc ttgtacctac agcatgaccg ttaaagtgga tatcacacaa ataaaggaaa 1800
agggaatgaa actatatcct gcaatgcttt attatattgc aatgattgta aaccgccatt 1860
cagagtttag gacggcaatc aatcaagatg gtgaattggg gatatatgat gagatgatac 1920
caagctatac aatatttcac aatgatactg aaacattttc cagcctttgg actgagtgta 1980
agtctgactt taaatcattt ttagcagatt atgaaagtga tacgcaacgg tatggaaaca 2040
atcatagaat ggaaggaaag ccaaatgctc cggaaaacat ttttaatgta tctatgatac 2100
cgtggtcaac cttcgatggc tttaatctga atttgcagaa aggatatgat tatttgattc 2160
ctatttttac tatggggaaa tattataaag aagataacaa aattatactt cctttggcaa 2220
ttcaagttca tcacgcagta tgtgacggat ttcacatttg ccgttttgta aacgaattgc 2280
aggaattgat aaatagttaa cttcaggttt gtctgtaact aaaaactagt atttaaccta 2340
ggatcaaaaa aatttccaat aatcccactc taagccacaa acacgcccta taaaatcccg 2400
ctttaatccc actttgagac acatgtaata ttactttacg ccctagtata gtgataattt 2460
tttacattca atgccacgca aaaaaataaa ggggcactat aataaaagtt ccttcggaac 2520
taactaaagt aaaaaattat ctttacaacc tccccaaaaa aaagaacagg tacaaagtac 2580
cctataatac aagcgtaaaa aaaatgaggg taaaaataaa aaaataaaaa aataaaaaaa 2640
taaaaaaata aaaaaataaa aaaataaaaa aatataaaaa taaaaaaata taaaaataaa 2700
aaaatataaa aataaaaaaa taaaaaaata taaaaataaa aaaataaaaa aatataaaaa 2760
tattttttat ttaaagtttg aaaaaaattt ttttatatta tataatcttt gaagaaaaga 2820
atataaaaaa tgagccttta taaaagccca ttttttttca tatacgtaat atgacgttct 2880
aatgttttta ttggtacttc taacattaga gtaatttctt tatttttaaa gcctttttct 2940
ttaagggctt ttattttttt tcttaataca tttaattcct ctttttttgt tgcttttcct 3000
ttagctttta attgctcttg ataatttttt ttacctctaa tattttctct tctcttatat 3060
tcctttttag aaattattat tgtcatatat ttttgttctt cttctgtaat ttctaataac 3120
tctataagag tttcattctt atacttatat tgcttatttt tatctaaata acatctttca 3180
gcacttctag ttgctcttat aacttctctt tcacttaaat gttgtctaaa catactatta 3240
agttctaaaa catcatttaa tgccttctca atgtcttctg taaagctaca aagataatat 3300
ctatataaaa ataatataag ctctctgtgt ccttttaaat catattctct tagttcacaa 3360
agttttatta tgtcttgtat tcttccataa tataaacttc tttctctata aatataattt 3420
attttgcttg gtctaccctt tttcctttca tatggtttta attcaggtaa aaatccattt 3480
tgtatttctc ttaagtcata aatatattcg tactcatcta atatattgac tactgttttt 3540
gatttagagt ttatacttcc tggaactctt aatattctcg ttgcatctaa ggcttgtcta 3600
tctgctccaa agtattttaa ttgattatat aaatattctt gaaccgcttt ccataatggt 3660
aatgctttac taggtactgc atttattatc catattaaat acattcctct tccactatct 3720
attacatagt ttggtatagg aatactttga ttaaaataat tcttttctaa gtccattaat 3780
acctggtctt tagttttgcc agttttataa taatccaagt ctataaacag tgtatttaac 3840
tcttttatat tttctaatcg cctacacggc ttataaaagg tatttagagt tatatagata 3900
ttttcatcac tcatatctaa atcttttaat tcagcgtatt tatagtgcca ttggctatat 3960
ccttttttat ctataacgct cctggttatc caccctttac ttctactatg aatattatct 4020
atatagttct ttttattcag ctttaatgcg tttctcactt attcacctcc ccttctgtaa 4080
aactaagaaa attatatcat attttcaata attattaact attcttaaac tcttaataaa 4140
aaatagagta agtccccaat tgaaacttaa tctatttttt atgttttaat ttattatttt 4200
tattaaaata ttttaaacta aattaaatga ttctttttaa ttttttacta tttcattcca 4260
taatatatta ctataattat ttacaaataa tatttcttca tttgtaatat ttagatgatt 4320
tactaatttt agtttttata tattaaataa ttaatgtata atttatataa aaaatcaaag 4380
gagcttataa attatgatta tttccaaaga tactaaagat ttaatttttt tcaattttaa 4440
caatactttt tgtaatatta tgtttaaatt taattgtatt tttttcatat aataaagccg 4500
ttgaagtaaa ccaatccatt ttccttatga tgttattatt aaatttaagt tttataataa 4560
tatctttatt atatttattg tttttaaaaa aactagtgaa atttctagtg aaatttccgg 4620
ctttattaaa cttattttta ggaattttat tttcattttc atctttacag gatttgatta 4680
tatctttaaa tatgttttat caaatattat ctttttctaa atttatatat atttttatta 4740
tatttattat tatatatatt ttatttttaa gtttctttct aacagctatt aaaaagaaac 4800
ttaaaaataa aaacacgtac tctaaaccaa taaataaaac tatttttatt attgctgcct 4860
tgattggaat agtttttagt aaaattaatt tcaatattcc acaatattat attataagct 4920
agcacgcctc gagatctcca tggacgcgtg acgtcgactc tagaggatcc ccgggtaccg 4980
agct 4984
<210> 83
<211> 200
<212> DNA
<213> 人工序列
<220>
<223> gRNA表达盒
<400> 83
gagctcactc tatcattgat agagtttgaa actctatcat tgatagagta taatatcttt 60
gttcattgag accagatctc ccggggatat cgctagcggt ctctgtttta gagctagaaa 120
tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc 180
tttttttgaa gcttgagctc 200
<210> 84
<211> 24
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 84
tcatgatttc tccatattag ctag 24
<210> 85
<211> 24
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 85
aaacctagct aatatggaga aatc 24
<210> 86
<211> 24
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 86
tcatgttaca cttggaacag gcgt 24
<210> 87
<211> 24
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 87
aaacacgcct gttccaagtg taac 24
<210> 88
<211> 24
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 88
tcatttccgg cagtaggatc ccca 24
<210> 89
<211> 24
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 89
aaactgggga tcctactgcc ggaa 24
<210> 90
<211> 24
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 90
tcatgcttat tacgacataa caca 24
<210> 91
<211> 24
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 91
aaactgtgtt atgtcgtaat aagc 24
<210> 92
<211> 36
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 92
atgcatggat ccaaacgaac ccaaaaagaa agtttc 36
<210> 93
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 93
ggttgatttc aaatctgtgt aaacctaccg 30
<210> 94
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 94
acacagattt gaaatcaacc actttaaccc 30
<210> 95
<211> 37
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 95
atgcatgtcg actcttaaga acatgtataa agtatgg 37
<210> 96
<211> 36
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 96
atgcatggat ccaaacgaac ccaaaaagaa agtttc 36
<210> 97
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 97
gctaagtttt aaatctgtgt aaacctaccg 30
<210> 98
<211> 32
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 98
acacagattt aaaacttagc atacttctta cc 32
<210> 99
<211> 37
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 99
atgcatgtcg accttctaat ctcctctact attttag 37
<210> 100
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 100
acacattgaa gggagctttt 20
<210> 101
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 101
ggcaacaaca tcaggccttt 20
<210> 102
<211> 4966
<212> DNA
<213> 人工序列
<220>
<223> pGRNA-xylB
<400> 102
atcaaaaaaa tttccaataa tcccactcta agccacaaac acgccctata aaatcccgct 60
ttaatcccac tttgagacac atgtaatatt actttacgcc ctagtatagt gataattttt 120
tacattcaat gccacgcaaa aaaataaagg ggcactataa taaaagttcc ttcggaacta 180
actaaagtaa aaaattatct ttacaacctc cccaaaaaaa agaacaggta caaagtaccc 240
tataatacaa gcgtaaaaaa aatgagggta aaaataaaaa aataaaaaaa taaaaaaata 300
aaaaaataaa aaaataaaaa aataaaaaaa tataaaaata aaaaaatata aaaataaaaa 360
aatataaaaa taaaaaaata aaaaaatata aaaataaaaa aataaaaaaa tataaaaata 420
ttttttattt aaagtttgaa aaaaattttt ttatattata taatctttga agaaaagaat 480
ataaaaaatg agcctttata aaagcccatt ttttttcata tacgtaatat gacgttctaa 540
tgtttttatt ggtacttcta acattagagt aatttcttta tttttaaagc ctttttcttt 600
aagggctttt attttttttc ttaatacatt taattcctct ttttttgttg cttttccttt 660
agcttttaat tgctcttgat aatttttttt acctctaata ttttctcttc tcttatattc 720
ctttttagaa attattattg tcatatattt ttgttcttct tctgtaattt ctaataactc 780
tataagagtt tcattcttat acttatattg cttattttta tctaaataac atctttcagc 840
acttctagtt gctcttataa cttctctttc acttaaatgt tgtctaaaca tactattaag 900
ttctaaaaca tcatttaatg ccttctcaat gtcttctgta aagctacaaa gataatatct 960
atataaaaat aatataagct ctctgtgtcc ttttaaatca tattctctta gttcacaaag 1020
ttttattatg tcttgtattc ttccataata taaacttctt tctctataaa tataatttat 1080
tttgcttggt ctaccctttt tcctttcata tggttttaat tcaggtaaaa atccattttg 1140
tatttctctt aagtcataaa tatattcgta ctcatctaat atattgacta ctgtttttga 1200
tttagagttt atacttcctg gaactcttaa tattctcgtt gcatctaagg cttgtctatc 1260
tgctccaaag tattttaatt gattatataa atattcttga accgctttcc ataatggtaa 1320
tgctttacta ggtactgcat ttattatcca tattaaatac attcctcttc cactatctat 1380
tacatagttt ggtataggaa tactttgatt aaaataattc ttttctaagt ccattaatac 1440
ctggtcttta gttttgccag ttttataata atccaagtct ataaacagtg tatttaactc 1500
ttttatattt tctaatcgcc tacacggctt ataaaaggta tttagagtta tatagatatt 1560
ttcatcactc atatctaaat cttttaattc agcgtattta tagtgccatt ggctatatcc 1620
ttttttatct ataacgctcc tggttatcca ccctttactt ctactatgaa tattatctat 1680
atagttcttt ttattcagct ttaatgcgtt tctcacttat tcacctcccc ttctgtaaaa 1740
ctaagaaaat tatatcatat tttcaataat tattaactat tcttaaactc ttaataaaaa 1800
atagagtaag tccccaattg aaacttaatc tattttttat gttttaattt attattttta 1860
ttaaaatatt ttaaactaaa ttaaatgatt ctttttaatt ttttactatt tcattccata 1920
atatattact ataattattt acaaataata tttcttcatt tgtaatattt agatgattta 1980
ctaattttag tttttatata ttaaataatt aatgtataat ttatataaaa aatcaaagga 2040
gcttataaat tatgattatt tccaaagata ctaaagattt aatttttttc aattttaaca 2100
atactttttg taatattatg tttaaattta attgtatttt tttcatataa taaagccgtt 2160
gaagtaaacc aatccatttt ccttatgatg ttattattaa atttaagttt tataataata 2220
tctttattat atttattgtt tttaaaaaaa ctagtgaaat ttctagtgaa atttccggct 2280
ttattaaact tatttttagg aattttattt tcattttcat ctttacagga tttgattata 2340
tctttaaata tgttttatca aatattatct ttttctaaat ttatatatat ttttattata 2400
tttattatta tatatatttt atttttaagt ttctttctaa cagctattaa aaagaaactt 2460
aaaaataaaa acacgtactc taaaccaata aataaaacta tttttattat tgctgccttg 2520
attggaatag tttttagtaa aattaatttc aatattccac aatattatat tataagctag 2580
cacgcctcga gaagcttcaa aaaaagcacc gactcggtgc cactttttca agttgataac 2640
ggactagcct tattttaact tgctatttct agctctaaaa cctagctaat atggagaaat 2700
catgaacaaa gatattatac tctatcaatg atagagtttc aaactctatc aatgatagag 2760
tctcgagatc tccatggacg cgtgacgtcg actctagagg atccccgggt accgagctcg 2820
aattcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca 2880
cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa 2940
ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag 3000
ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc 3060
gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 3120
cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg 3180
tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc 3240
cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga 3300
aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct 3360
cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg 3420
gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 3480
ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat 3540
cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac 3600
aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac 3660
tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc 3720
ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt 3780
tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc 3840
ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg 3900
agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca 3960
atctaaagta tatatgagta aacttggtct gacagttacc aaagctagct taatactagt 4020
atatacttaa tgtgataagt gtctgacagc tgaccggtct aaagaggtcc ctagcgccta 4080
cggggaattt gtatcgataa ggggtacaaa ttcccactaa gcgctcggcc ggggatcgat 4140
ccccgggtac gtacccggca gtttttcttt ttcggcaagt gttcaagaag ttattaagtc 4200
gggagtgcag tcgaagtggg caagttgaaa aattcacaaa aatgtggtat aatatctttg 4260
ttcattagag cgataaactt gaatttgaga gggaacttag atggtatttg aaaaaattga 4320
taaaaatagt tggaacagaa aagagtattt tgaccactac tttgcaagtg taccttgtac 4380
ctacagcatg accgttaaag tggatatcac acaaataaag gaaaagggaa tgaaactata 4440
tcctgcaatg ctttattata ttgcaatgat tgtaaaccgc cattcagagt ttaggacggc 4500
aatcaatcaa gatggtgaat tggggatata tgatgagatg ataccaagct atacaatatt 4560
tcacaatgat actgaaacat tttccagcct ttggactgag tgtaagtctg actttaaatc 4620
atttttagca gattatgaaa gtgatacgca acggtatgga aacaatcata gaatggaagg 4680
aaagccaaat gctccggaaa acatttttaa tgtatctatg ataccgtggt caaccttcga 4740
tggctttaat ctgaatttgc agaaaggata tgattatttg attcctattt ttactatggg 4800
gaaatattat aaagaagata acaaaattat acttcctttg gcaattcaag ttcatcacgc 4860
agtatgtgac ggatttcaca tttgccgttt tgtaaacgaa ttgcaggaat tgataaatag 4920
ttaacttcag gtttgtctgt aactaaaaac tagtatttaa cctagg 4966
<210> 103
<211> 4966
<212> DNA
<213> 人工序列
<220>
<223> pGRNA-xylR
<400> 103
atcaaaaaaa tttccaataa tcccactcta agccacaaac acgccctata aaatcccgct 60
ttaatcccac tttgagacac atgtaatatt actttacgcc ctagtatagt gataattttt 120
tacattcaat gccacgcaaa aaaataaagg ggcactataa taaaagttcc ttcggaacta 180
actaaagtaa aaaattatct ttacaacctc cccaaaaaaa agaacaggta caaagtaccc 240
tataatacaa gcgtaaaaaa aatgagggta aaaataaaaa aataaaaaaa taaaaaaata 300
aaaaaataaa aaaataaaaa aataaaaaaa tataaaaata aaaaaatata aaaataaaaa 360
aatataaaaa taaaaaaata aaaaaatata aaaataaaaa aataaaaaaa tataaaaata 420
ttttttattt aaagtttgaa aaaaattttt ttatattata taatctttga agaaaagaat 480
ataaaaaatg agcctttata aaagcccatt ttttttcata tacgtaatat gacgttctaa 540
tgtttttatt ggtacttcta acattagagt aatttcttta tttttaaagc ctttttcttt 600
aagggctttt attttttttc ttaatacatt taattcctct ttttttgttg cttttccttt 660
agcttttaat tgctcttgat aatttttttt acctctaata ttttctcttc tcttatattc 720
ctttttagaa attattattg tcatatattt ttgttcttct tctgtaattt ctaataactc 780
tataagagtt tcattcttat acttatattg cttattttta tctaaataac atctttcagc 840
acttctagtt gctcttataa cttctctttc acttaaatgt tgtctaaaca tactattaag 900
ttctaaaaca tcatttaatg ccttctcaat gtcttctgta aagctacaaa gataatatct 960
atataaaaat aatataagct ctctgtgtcc ttttaaatca tattctctta gttcacaaag 1020
ttttattatg tcttgtattc ttccataata taaacttctt tctctataaa tataatttat 1080
tttgcttggt ctaccctttt tcctttcata tggttttaat tcaggtaaaa atccattttg 1140
tatttctctt aagtcataaa tatattcgta ctcatctaat atattgacta ctgtttttga 1200
tttagagttt atacttcctg gaactcttaa tattctcgtt gcatctaagg cttgtctatc 1260
tgctccaaag tattttaatt gattatataa atattcttga accgctttcc ataatggtaa 1320
tgctttacta ggtactgcat ttattatcca tattaaatac attcctcttc cactatctat 1380
tacatagttt ggtataggaa tactttgatt aaaataattc ttttctaagt ccattaatac 1440
ctggtcttta gttttgccag ttttataata atccaagtct ataaacagtg tatttaactc 1500
ttttatattt tctaatcgcc tacacggctt ataaaaggta tttagagtta tatagatatt 1560
ttcatcactc atatctaaat cttttaattc agcgtattta tagtgccatt ggctatatcc 1620
ttttttatct ataacgctcc tggttatcca ccctttactt ctactatgaa tattatctat 1680
atagttcttt ttattcagct ttaatgcgtt tctcacttat tcacctcccc ttctgtaaaa 1740
ctaagaaaat tatatcatat tttcaataat tattaactat tcttaaactc ttaataaaaa 1800
atagagtaag tccccaattg aaacttaatc tattttttat gttttaattt attattttta 1860
ttaaaatatt ttaaactaaa ttaaatgatt ctttttaatt ttttactatt tcattccata 1920
atatattact ataattattt acaaataata tttcttcatt tgtaatattt agatgattta 1980
ctaattttag tttttatata ttaaataatt aatgtataat ttatataaaa aatcaaagga 2040
gcttataaat tatgattatt tccaaagata ctaaagattt aatttttttc aattttaaca 2100
atactttttg taatattatg tttaaattta attgtatttt tttcatataa taaagccgtt 2160
gaagtaaacc aatccatttt ccttatgatg ttattattaa atttaagttt tataataata 2220
tctttattat atttattgtt tttaaaaaaa ctagtgaaat ttctagtgaa atttccggct 2280
ttattaaact tatttttagg aattttattt tcattttcat ctttacagga tttgattata 2340
tctttaaata tgttttatca aatattatct ttttctaaat ttatatatat ttttattata 2400
tttattatta tatatatttt atttttaagt ttctttctaa cagctattaa aaagaaactt 2460
aaaaataaaa acacgtactc taaaccaata aataaaacta tttttattat tgctgccttg 2520
attggaatag tttttagtaa aattaatttc aatattccac aatattatat tataagctag 2580
cacgcctcga gactctatca ttgatagagt ttgaaactct atcattgata gagtataata 2640
tctttgttca tgttacactt ggaacaggcg tgttttagag ctagaaatag caagttaaaa 2700
taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt ttttgaagct 2760
tctcgagatc tccatggacg cgtgacgtcg actctagagg atccccgggt accgagctcg 2820
aattcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca 2880
cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa 2940
ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag 3000
ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc 3060
gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 3120
cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg 3180
tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc 3240
cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga 3300
aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct 3360
cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg 3420
gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 3480
ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat 3540
cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac 3600
aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac 3660
tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc 3720
ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt 3780
tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc 3840
ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg 3900
agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca 3960
atctaaagta tatatgagta aacttggtct gacagttacc aaagctagct taatactagt 4020
atatacttaa tgtgataagt gtctgacagc tgaccggtct aaagaggtcc ctagcgccta 4080
cggggaattt gtatcgataa ggggtacaaa ttcccactaa gcgctcggcc ggggatcgat 4140
ccccgggtac gtacccggca gtttttcttt ttcggcaagt gttcaagaag ttattaagtc 4200
gggagtgcag tcgaagtggg caagttgaaa aattcacaaa aatgtggtat aatatctttg 4260
ttcattagag cgataaactt gaatttgaga gggaacttag atggtatttg aaaaaattga 4320
taaaaatagt tggaacagaa aagagtattt tgaccactac tttgcaagtg taccttgtac 4380
ctacagcatg accgttaaag tggatatcac acaaataaag gaaaagggaa tgaaactata 4440
tcctgcaatg ctttattata ttgcaatgat tgtaaaccgc cattcagagt ttaggacggc 4500
aatcaatcaa gatggtgaat tggggatata tgatgagatg ataccaagct atacaatatt 4560
tcacaatgat actgaaacat tttccagcct ttggactgag tgtaagtctg actttaaatc 4620
atttttagca gattatgaaa gtgatacgca acggtatgga aacaatcata gaatggaagg 4680
aaagccaaat gctccggaaa acatttttaa tgtatctatg ataccgtggt caaccttcga 4740
tggctttaat ctgaatttgc agaaaggata tgattatttg attcctattt ttactatggg 4800
gaaatattat aaagaagata acaaaattat acttcctttg gcaattcaag ttcatcacgc 4860
agtatgtgac ggatttcaca tttgccgttt tgtaaacgaa ttgcaggaat tgataaatag 4920
ttaacttcag gtttgtctgt aactaaaaac tagtatttaa cctagg 4966
<210> 104
<211> 4966
<212> DNA
<213> 人工序列
<220>
<223> pGRNA-glcG
<400> 104
agctcggtac ccggggatcc tctagagtcg acgtcacgcg tccatggaga tctcgaggcg 60
tgctagctta taatataata ttgtggaata ttgaaattaa ttttactaaa aactattcca 120
atcaaggcag caataataaa aatagtttta tttattggtt tagagtacgt gtttttattt 180
ttaagtttct ttttaatagc tgttagaaag aaacttaaaa ataaaatata tataataata 240
aatataataa aaatatatat aaatttagaa aaagataata tttgataaaa catatttaaa 300
gatataatca aatcctgtaa agatgaaaat gaaaataaaa ttcctaaaaa taagtttaat 360
aaagccggaa atttcactag aaatttcact agttttttta aaaacaataa atataataaa 420
gatattatta taaaacttaa atttaataat aacatcataa ggaaaatgga ttggtttact 480
tcaacggctt tattatatga aaaaaataca attaaattta aacataatat tacaaaaagt 540
attgttaaaa ttgaaaaaaa ttaaatcttt agtatctttg gaaataatca taatttataa 600
gctcctttga ttttttatat aaattataca ttaattattt aatatataaa aactaaaatt 660
agtaaatcat ctaaatatta caaatgaaga aatattattt gtaaataatt atagtaatat 720
attatggaat gaaatagtaa aaaattaaaa agaatcattt aatttagttt aaaatatttt 780
aataaaaata ataaattaaa acataaaaaa tagattaagt ttcaattggg gacttactct 840
attttttatt aagagtttaa gaatagttaa taattattga aaatatgata taattttctt 900
agttttacag aaggggaggt gaataagtga gaaacgcatt aaagctgaat aaaaagaact 960
atatagataa tattcatagt agaagtaaag ggtggataac caggagcgtt atagataaaa 1020
aaggatatag ccaatggcac tataaatacg ctgaattaaa agatttagat atgagtgatg 1080
aaaatatcta tataactcta aatacctttt ataagccgtg taggcgatta gaaaatataa 1140
aagagttaaa tacactgttt atagacttgg attattataa aactggcaaa actaaagacc 1200
aggtattaat ggacttagaa aagaattatt ttaatcaaag tattcctata ccaaactatg 1260
taatagatag tggaagagga atgtatttaa tatggataat aaatgcagta cctagtaaag 1320
cattaccatt atggaaagcg gttcaagaat atttatataa tcaattaaaa tactttggag 1380
cagatagaca agccttagat gcaacgagaa tattaagagt tccaggaagt ataaactcta 1440
aatcaaaaac agtagtcaat atattagatg agtacgaata tatttatgac ttaagagaaa 1500
tacaaaatgg atttttacct gaattaaaac catatgaaag gaaaaagggt agaccaagca 1560
aaataaatta tatttataga gaaagaagtt tatattatgg aagaatacaa gacataataa 1620
aactttgtga actaagagaa tatgatttaa aaggacacag agagcttata ttatttttat 1680
atagatatta tctttgtagc tttacagaag acattgagaa ggcattaaat gatgttttag 1740
aacttaatag tatgtttaga caacatttaa gtgaaagaga agttataaga gcaactagaa 1800
gtgctgaaag atgttattta gataaaaata agcaatataa gtataagaat gaaactctta 1860
tagagttatt agaaattaca gaagaagaac aaaaatatat gacaataata atttctaaaa 1920
aggaatataa gagaagagaa aatattagag gtaaaaaaaa ttatcaagag caattaaaag 1980
ctaaaggaaa agcaacaaaa aaagaggaat taaatgtatt aagaaaaaaa ataaaagccc 2040
ttaaagaaaa aggctttaaa aataaagaaa ttactctaat gttagaagta ccaataaaaa 2100
cattagaacg tcatattacg tatatgaaaa aaaatgggct tttataaagg ctcatttttt 2160
atattctttt cttcaaagat tatataatat aaaaaaattt ttttcaaact ttaaataaaa 2220
aatattttta tattttttta tttttttatt tttatatttt tttatttttt tatttttata 2280
tttttttatt tttatatttt tttattttta tattttttta tttttttatt tttttatttt 2340
tttatttttt tattttttta tttttttatt tttaccctca ttttttttac gcttgtatta 2400
tagggtactt tgtacctgtt cttttttttg gggaggttgt aaagataatt ttttacttta 2460
gttagttccg aaggaacttt tattatagtg cccctttatt tttttgcgtg gcattgaatg 2520
taaaaaatta tcactatact agggcgtaaa gtaatattac atgtgtctca aagtgggatt 2580
aaagcgggat tttatagggc gtgtttgtgg cttagagtgg gattattgga aatttttttg 2640
atcctaggtt aaatactagt ttttagttac agacaaacct gaagttaact atttatcaat 2700
tcctgcaatt cgtttacaaa acggcaaatg tgaaatccgt cacatactgc gtgatgaact 2760
tgaattgcca aaggaagtat aattttgtta tcttctttat aatatttccc catagtaaaa 2820
ataggaatca aataatcata tcctttctgc aaattcagat taaagccatc gaaggttgac 2880
cacggtatca tagatacatt aaaaatgttt tccggagcat ttggctttcc ttccattcta 2940
tgattgtttc cataccgttg cgtatcactt tcataatctg ctaaaaatga tttaaagtca 3000
gacttacact cagtccaaag gctggaaaat gtttcagtat cattgtgaaa tattgtatag 3060
cttggtatca tctcatcata tatccccaat tcaccatctt gattgattgc cgtcctaaac 3120
tctgaatggc ggtttacaat cattgcaata taataaagca ttgcaggata tagtttcatt 3180
cccttttcct ttatttgtgt gatatccact ttaacggtca tgctgtaggt acaaggtaca 3240
cttgcaaagt agtggtcaaa atactctttt ctgttccaac tatttttatc aattttttca 3300
aataccatct aagttccctc tcaaattcaa gtttatcgct ctaatgaaca aagatattat 3360
accacatttt tgtgaatttt tcaacttgcc cacttcgact gcactcccga cttaataact 3420
tcttgaacac ttgccgaaaa agaaaaactg ccgggtacgt acccggggat cgatccccgg 3480
ccgagcgctt agtgggaatt tgtacccctt atcgatacaa attccccgta ggcgctaggg 3540
acctctttag accggtcagc tgtcagacac ttatcacatt aagtatatac tagtattaag 3600
ctagctttgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact 3660
tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat 3720
cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc 3780
ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct 3840
accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg 3900
cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca 3960
cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc 4020
tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga 4080
taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac 4140
gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga 4200
agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag 4260
ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg 4320
acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag 4380
caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc 4440
tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc 4500
tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc 4560
aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag 4620
gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca 4680
ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag 4740
cggataacaa tttcacacag gaaacagcta tgaccatgat tacgaattcg agctcactct 4800
atcattgata gagtttgaaa ctctatcatt gatagagtat aatatctttg ttcatttccg 4860
gcagtaggat ccccagtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta 4920
tcaacttgaa aaagtggcac cgagtcggtg ctttttttga agcttg 4966
<210> 105
<211> 4938
<212> DNA
<213> 人工序列
<220>
<223> pGRNA-bdhB
<400> 105
atcaaaaaaa tttccaataa tcccactcta agccacaaac acgccctata aaatcccgct 60
ttaatcccac tttgagacac atgtaatatt actttacgcc ctagtatagt gataattttt 120
tacattcaat gccacgcaaa aaaataaagg ggcactataa taaaagttcc ttcggaacta 180
actaaagtaa aaaattatct ttacaacctc cccaaaaaaa agaacaggta caaagtaccc 240
tataatacaa gcgtaaaaaa aatgagggta aaaataaaaa aataaaaaaa taaaaaaata 300
aaaaaataaa aaaataaaaa aataaaaaaa tataaaaata aaaaaatata aaaataaaaa 360
aatataaaaa taaaaaaata aaaaaatata aaaataaaaa aataaaaaaa tataaaaata 420
ttttttattt aaagtttgaa aaaaattttt ttatattata taatctttga agaaaagaat 480
ataaaaaatg agcctttata aaagcccatt ttttttcata tacgtaatat gacgttctaa 540
tgtttttatt ggtacttcta acattagagt aatttcttta tttttaaagc ctttttcttt 600
aagggctttt attttttttc ttaatacatt taattcctct ttttttgttg cttttccttt 660
agcttttaat tgctcttgat aatttttttt acctctaata ttttctcttc tcttatattc 720
ctttttagaa attattattg tcatatattt ttgttcttct tctgtaattt ctaataactc 780
tataagagtt tcattcttat acttatattg cttattttta tctaaataac atctttcagc 840
acttctagtt gctcttataa cttctctttc acttaaatgt tgtctaaaca tactattaag 900
ttctaaaaca tcatttaatg ccttctcaat gtcttctgta aagctacaaa gataatatct 960
atataaaaat aatataagct ctctgtgtcc ttttaaatca tattctctta gttcacaaag 1020
ttttattatg tcttgtattc ttccataata taaacttctt tctctataaa tataatttat 1080
tttgcttggt ctaccctttt tcctttcata tggttttaat tcaggtaaaa atccattttg 1140
tatttctctt aagtcataaa tatattcgta ctcatctaat atattgacta ctgtttttga 1200
tttagagttt atacttcctg gaactcttaa tattctcgtt gcatctaagg cttgtctatc 1260
tgctccaaag tattttaatt gattatataa atattcttga accgctttcc ataatggtaa 1320
tgctttacta ggtactgcat ttattatcca tattaaatac attcctcttc cactatctat 1380
tacatagttt ggtataggaa tactttgatt aaaataattc ttttctaagt ccattaatac 1440
ctggtcttta gttttgccag ttttataata atccaagtct ataaacagtg tatttaactc 1500
ttttatattt tctaatcgcc tacacggctt ataaaaggta tttagagtta tatagatatt 1560
ttcatcactc atatctaaat cttttaattc agcgtattta tagtgccatt ggctatatcc 1620
ttttttatct ataacgctcc tggttatcca ccctttactt ctactatgaa tattatctat 1680
atagttcttt ttattcagct ttaatgcgtt tctcacttat tcacctcccc ttctgtaaaa 1740
ctaagaaaat tatatcatat tttcaataat tattaactat tcttaaactc ttaataaaaa 1800
atagagtaag tccccaattg aaacttaatc tattttttat gttttaattt attattttta 1860
ttaaaatatt ttaaactaaa ttaaatgatt ctttttaatt ttttactatt tcattccata 1920
atatattact ataattattt acaaataata tttcttcatt tgtaatattt agatgattta 1980
ctaattttag tttttatata ttaaataatt aatgtataat ttatataaaa aatcaaagga 2040
gcttataaat tatgattatt tccaaagata ctaaagattt aatttttttc aattttaaca 2100
atactttttg taatattatg tttaaattta attgtatttt tttcatataa taaagccgtt 2160
gaagtaaacc aatccatttt ccttatgatg ttattattaa atttaagttt tataataata 2220
tctttattat atttattgtt tttaaaaaaa ctagtgaaat ttctagtgaa atttccggct 2280
ttattaaact tatttttagg aattttattt tcattttcat ctttacagga tttgattata 2340
tctttaaata tgttttatca aatattatct ttttctaaat ttatatatat ttttattata 2400
tttattatta tatatatttt atttttaagt ttctttctaa cagctattaa aaagaaactt 2460
aaaaataaaa acacgtactc taaaccaata aataaaacta tttttattat tgctgccttg 2520
attggaatag tttttagtaa aattaatttc aatattccac aatattatat tataagctag 2580
cacgcctcga gtatattgat aaaaataata atagtgggta taattaagtt gttaggaggt 2640
tagttagagc ttattacgac ataacacagt tttagagcta gaaatagcaa gttaaaataa 2700
ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt tgaagcttgt 2760
cgactctaga ggatccccgg gtaccgagct cgaattcgta atcatggtca tagctgtttc 2820
ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt 2880
gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc 2940
ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg 3000
ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct 3060
cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca 3120
cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga 3180
accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc 3240
acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg 3300
cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat 3360
acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt 3420
atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc 3480
agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg 3540
acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg 3600
gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg 3660
gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg 3720
gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca 3780
gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga 3840
acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga 3900
tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt 3960
ctgacagtta ccaaagctag cttaatacta gtatatactt aatgtgataa gtgtctgaca 4020
gctgaccggt ctaaagaggt ccctagcgcc tacggggaat ttgtatcgat aaggggtaca 4080
aattcccact aagcgctcgg ccggggatcg atccccgggt acgtacccgg cagtttttct 4140
ttttcggcaa gtgttcaaga agttattaag tcgggagtgc agtcgaagtg ggcaagttga 4200
aaaattcaca aaaatgtggt ataatatctt tgttcattag agcgataaac ttgaatttga 4260
gagggaactt agatggtatt tgaaaaaatt gataaaaata gttggaacag aaaagagtat 4320
tttgaccact actttgcaag tgtaccttgt acctacagca tgaccgttaa agtggatatc 4380
acacaaataa aggaaaaggg aatgaaacta tatcctgcaa tgctttatta tattgcaatg 4440
attgtaaacc gccattcaga gtttaggacg gcaatcaatc aagatggtga attggggata 4500
tatgatgaga tgataccaag ctatacaata tttcacaatg atactgaaac attttccagc 4560
ctttggactg agtgtaagtc tgactttaaa tcatttttag cagattatga aagtgatacg 4620
caacggtatg gaaacaatca tagaatggaa ggaaagccaa atgctccgga aaacattttt 4680
aatgtatcta tgataccgtg gtcaaccttc gatggcttta atctgaattt gcagaaagga 4740
tatgattatt tgattcctat ttttactatg gggaaatatt ataaagaaga taacaaaatt 4800
atacttcctt tggcaattca agttcatcac gcagtatgtg acggatttca catttgccgt 4860
tttgtaaacg aattgcagga attgataaat agttaacttc aggtttgtct gtaactaaaa 4920
actagtattt aacctagg 4938
<210> 106
<211> 4790
<212> DNA
<213> 人工序列
<220>
<223> pEC750C
<400> 106
atcaaaaaaa tttccaataa tcccactcta agccacaaac acgccctata aaatcccgct 60
ttaatcccac tttgagacac atgtaatatt actttacgcc ctagtatagt gataattttt 120
tacattcaat gccacgcaaa aaaataaagg ggcactataa taaaagttcc ttcggaacta 180
actaaagtaa aaaattatct ttacaacctc cccaaaaaaa agaacaggta caaagtaccc 240
tataatacaa gcgtaaaaaa aatgagggta aaaataaaaa aataaaaaaa taaaaaaata 300
aaaaaataaa aaaataaaaa aataaaaaaa tataaaaata aaaaaatata aaaataaaaa 360
aatataaaaa taaaaaaata aaaaaatata aaaataaaaa aataaaaaaa tataaaaata 420
ttttttattt aaagtttgaa aaaaattttt ttatattata taatctttga agaaaagaat 480
ataaaaaatg agcctttata aaagcccatt ttttttcata tacgtaatat gacgttctaa 540
tgtttttatt ggtacttcta acattagagt aatttcttta tttttaaagc ctttttcttt 600
aagggctttt attttttttc ttaatacatt taattcctct ttttttgttg cttttccttt 660
agcttttaat tgctcttgat aatttttttt acctctaata ttttctcttc tcttatattc 720
ctttttagaa attattattg tcatatattt ttgttcttct tctgtaattt ctaataactc 780
tataagagtt tcattcttat acttatattg cttattttta tctaaataac atctttcagc 840
acttctagtt gctcttataa cttctctttc acttaaatgt tgtctaaaca tactattaag 900
ttctaaaaca tcatttaatg ccttctcaat gtcttctgta aagctacaaa gataatatct 960
atataaaaat aatataagct ctctgtgtcc ttttaaatca tattctctta gttcacaaag 1020
ttttattatg tcttgtattc ttccataata taaacttctt tctctataaa tataatttat 1080
tttgcttggt ctaccctttt tcctttcata tggttttaat tcaggtaaaa atccattttg 1140
tatttctctt aagtcataaa tatattcgta ctcatctaat atattgacta ctgtttttga 1200
tttagagttt atacttcctg gaactcttaa tattctcgtt gcatctaagg cttgtctatc 1260
tgctccaaag tattttaatt gattatataa atattcttga accgctttcc ataatggtaa 1320
tgctttacta ggtactgcat ttattatcca tattaaatac attcctcttc cactatctat 1380
tacatagttt ggtataggaa tactttgatt aaaataattc ttttctaagt ccattaatac 1440
ctggtcttta gttttgccag ttttataata atccaagtct ataaacagtg tatttaactc 1500
ttttatattt tctaatcgcc tacacggctt ataaaaggta tttagagtta tatagatatt 1560
ttcatcactc atatctaaat cttttaattc agcgtattta tagtgccatt ggctatatcc 1620
ttttttatct ataacgctcc tggttatcca ccctttactt ctactatgaa tattatctat 1680
atagttcttt ttattcagct ttaatgcgtt tctcacttat tcacctcccc ttctgtaaaa 1740
ctaagaaaat tatatcatat tttcaataat tattaactat tcttaaactc ttaataaaaa 1800
atagagtaag tccccaattg aaacttaatc tattttttat gttttaattt attattttta 1860
ttaaaatatt ttaaactaaa ttaaatgatt ctttttaatt ttttactatt tcattccata 1920
atatattact ataattattt acaaataata tttcttcatt tgtaatattt agatgattta 1980
ctaattttag tttttatata ttaaataatt aatgtataat ttatataaaa aatcaaagga 2040
gcttataaat tatgattatt tccaaagata ctaaagattt aatttttttc aattttaaca 2100
atactttttg taatattatg tttaaattta attgtatttt tttcatataa taaagccgtt 2160
gaagtaaacc aatccatttt ccttatgatg ttattattaa atttaagttt tataataata 2220
tctttattat atttattgtt tttaaaaaaa ctagtgaaat ttctagtgaa atttccggct 2280
ttattaaact tatttttagg aattttattt tcattttcat ctttacagga tttgattata 2340
tctttaaata tgttttatca aatattatct ttttctaaat ttatatatat ttttattata 2400
tttattatta tatatatttt atttttaagt ttctttctaa cagctattaa aaagaaactt 2460
aaaaataaaa acacgtactc taaaccaata aataaaacta tttttattat tgctgccttg 2520
attggaatag tttttagtaa aattaatttc aatattccac aatattatat tataagctag 2580
cacgcctcga gatctccatg gacgcgtgac gtcgactcta gaggatcccc gggtaccgag 2640
ctcgaattcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat 2700
tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag 2760
ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg 2820
ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc 2880
ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc 2940
agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa 3000
catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt 3060
tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg 3120
gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg 3180
ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag 3240
cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc 3300
caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa 3360
ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg 3420
taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc 3480
taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac 3540
cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg 3600
tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt 3660
gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt 3720
catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa 3780
atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaaagct agcttaatac 3840
tagtatatac ttaatgtgat aagtgtctga cagctgaccg gtctaaagag gtccctagcg 3900
cctacgggga atttgtatcg ataaggggta caaattccca ctaagcgctc ggccggggat 3960
cgatccccgg gtacgtaccc ggcagttttt ctttttcggc aagtgttcaa gaagttatta 4020
agtcgggagt gcagtcgaag tgggcaagtt gaaaaattca caaaaatgtg gtataatatc 4080
tttgttcatt agagcgataa acttgaattt gagagggaac ttagatggta tttgaaaaaa 4140
ttgataaaaa tagttggaac agaaaagagt attttgacca ctactttgca agtgtacctt 4200
gtacctacag catgaccgtt aaagtggata tcacacaaat aaaggaaaag ggaatgaaac 4260
tatatcctgc aatgctttat tatattgcaa tgattgtaaa ccgccattca gagtttagga 4320
cggcaatcaa tcaagatggt gaattgggga tatatgatga gatgatacca agctatacaa 4380
tatttcacaa tgatactgaa acattttcca gcctttggac tgagtgtaag tctgacttta 4440
aatcattttt agcagattat gaaagtgata cgcaacggta tggaaacaat catagaatgg 4500
aaggaaagcc aaatgctccg gaaaacattt ttaatgtatc tatgataccg tggtcaacct 4560
tcgatggctt taatctgaat ttgcagaaag gatatgatta tttgattcct atttttacta 4620
tggggaaata ttataaagaa gataacaaaa ttatacttcc tttggcaatt caagttcatc 4680
acgcagtatg tgacggattt cacatttgcc gttttgtaaa cgaattgcag gaattgataa 4740
atagttaact tcaggtttgt ctgtaactaa aaactagtat ttaacctagg 4790
<210> 107
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 107
acttgggtcg accacgataa aacaaggttt taagg 35
<210> 108
<211> 42
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 108
taccagggat ccgtattaat gtaactatga tatcaattct tg 42
<210> 109
<211> 46
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 109
atgcatggtc ccaatgaata ggtttacact tactttagtt ttatgg 46
<210> 110
<211> 39
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 110
atgcgagtta acaacttcta aaatctgatt accaattag 39
<210> 111
<211> 47
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 111
atgcatggat cccaatgaat aggtttacac ttactttagt tttatgg 47
<210> 112
<211> 39
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 112
atgcgagagc tcaacttcta aaatctgatt accaattag 39
<210> 113
<211> 32
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 113
atgcatggat ccgtctgaca gttaccaggt cc 32
<210> 114
<211> 39
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 114
atgcgagagc tccaattgtt caaaaaaata atggcggag 39
<210> 115
<211> 32
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 115
atgcatggat cccggcagtt tttctttttc gg 32
<210> 116
<211> 40
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 116
atgcgagagc tcggttaaat actagttttt agttacagac 40
<210> 117
<211> 2686
<212> DNA
<213> 人工序列
<220>
<223> pUC19
<400> 117
gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 60
cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct 120
cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat 180
tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagcttgc 240
atgcctgcag gtcgactcta gaggatcccc gggtaccgag ctcgaattca ctggccgtcg 300
ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac 360
atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac 420
agttgcgcag cctgaatggc gaatggcgcc tgatgcggta ttttctcctt acgcatctgt 480
gcggtatttc acaccgcata tggtgcactc tcagtacaat ctgctctgat gccgcatagt 540
taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc 600
cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt 660
caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg 720
ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc 780
gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac 840
aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt 900
tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag 960
aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg 1020
aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa 1080
tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc 1140
aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag 1200
tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa 1260
ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc 1320
taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg 1380
agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa 1440
caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa 1500
tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg 1560
gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag 1620
cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg 1680
caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt 1740
ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt 1800
aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac 1860
gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag 1920
atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg 1980
tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca 2040
gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga 2100
actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca 2160
gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc 2220
agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca 2280
ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa 2340
aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc 2400
cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc 2460
gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg 2520
cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat 2580
cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca 2640
gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaaga 2686
<210> 118
<211> 4282
<212> DNA
<213> 人工序列
<220>
<223> pNF2
<400> 118
ctggagagga ttgtccttat acttatcata agcatgaagg acttgttatt cctagataga 60
gaattaatta tgttaaagag atataataaa ctcattataa ttataatttt tagtataatt 120
attattgcaa ttttttcgta taaatatcta ataatgccaa aagagcatag aatagaaatt 180
tcaacattat caaacataga agtttttaaa tttaatagtt tttcaaagtt tagtaacgaa 240
aaaatgtata ctattaatga tagtgataag ttaataaaat tcaaaacact atttaataat 300
ttagataaat caaaagatat aaaaaagatt agtattccgg aaagtgaaaa tttaaatgca 360
tttaaatttt ctgcacatat aaaacttaac tttaactatg ttaataaaga tagccaaata 420
actgaaggtg cttttcttat gtatattttg gtagacaatt tagaagggaa gtcatatatg 480
acttttttag gacaagattc aagctatata ttagatagta atgaaactaa cattttaaga 540
gaaatattta tgaattcaga gattaattaa tttatgaatt cataaatatt atctaagcac 600
gataaaacaa ggttttaagg ataagaaaag tcatgagatt tatagtaaat cttgtgactt 660
tttttattga atagtagaga gagttcggaa gtataacacg ctatattctt gatattttta 720
gaatagcaag cattggattt gtcctgacac tttcccaaaa attaaggagt tattccttaa 780
accaaaaaga ttaatgtggg aacaaattta gtgtatccat ttttgaaggg cgcacttata 840
caccaccaaa atggtgtgtg cgaaatcttt aaaaaagatt tatcaaaaag cttttttaaa 900
gctgggacat ttagaaaatc aataatgttt tttgcccaat acgctagtct taaaatctgc 960
aaggttgata actatttagt cccaggtatt agaatggggc atatatatac aaagtatata 1020
tatgcgtaaa tatatgtggg actgtgggaa caaaattgcg tgctaaaatt gtattgaaaa 1080
ggtaatgaaa aggtcatgct ttggtattgc taacgtatag aaaaggtaat gaaaagctca 1140
tggttctata aaaaagatgt acccacgaaa ataataggct ttgcctattt ccccatgtaa 1200
tatgggggca gttttctctt atgctctttc ttaacatatt gaataaatac aaaatgcagc 1260
tttgtgggaa taaaaatatt tttgttttta ttcttatagt tagacaaaat tttaatcttt 1320
tttgtgctat aacaagatta aaatttgtgg gaacattaag aaatattgtt gtcacaaata 1380
aaaaggagag tgggaacaat tgctataaaa aacgcagaaa ttaagattag agttacaaaa 1440
gagcaaaaag aattatttaa gaaaattgca aaagctgaaa atatgagtat gagtgaattt 1500
attattgtga ccacagaata tttagccaga aaaaaagatg aaaatatgaa atcaaaagac 1560
atgatcgaga gaagagctgc gaagactgaa gaaaaaatta tgaagctaaa aaagaaacta 1620
aataaaaaca ggtaatatag attacagttt taagcttgtt ttccctatag actagagtaa 1680
atatataaat atacctgtca agggcttata agccccttta gggggtgcgt agcacccttg 1740
acaggtatat ttatatattt tagggtgcca ttaagggaaa caagctttaa aatgccttta 1800
aaggcatttt aaaataaata aaaaaaagat ggtttttacc atctttttta actcccgaaa 1860
gggagttctt tcttttcttg atactatacg taactatttc gatttgccct gaacctaatc 1920
aaagctagat aaattcagta ttagggcata aaaaaacttg ctttttcggg tggaaatctg 1980
tataatttaa attgcttaga taaaaattac caattccata cgaaaggagc aagttttaca 2040
taaggttaaa gccttatgtg aattctcatt taattacatg aataataata acacagaaag 2100
tgaagaatta aaagagcaaa gtcaactatt gcttgacaaa tgcacaaaaa agaaaaagaa 2160
aaatcctaaa tttagtagtt atatagaacc attagtaagc aagaaattat ctgaaagaat 2220
aaaggaatgt ggtgactttt tgcagatgtt atctgattta aaccttgaaa attcgaaact 2280
gcatagagca agtttttgtg gtaacagatt ttgtcctatg tgtagctggc gtattgcttg 2340
taaggatagt ttggaaatat ctattctcat ggagcattta cgcaaagagg aaagcaaaga 2400
atttatcttt ttgaccttaa caactccaaa tgtgaaaggt gcggaccttg ataattccat 2460
aaaagcatac aataaagcat ttaaaaagtt aatggaacgc aaagaggtca agagcatagt 2520
aaaaggctac ataagaaagc tagaagtaac ctataatttg gacaagagtt ccaaatcata 2580
taatacttat cacccacatt tccatgtggt actagcagtc aatagaagtt actttaaaaa 2640
gcaaaatcta tatataaacc atcatagatg gcttagtttg tggcaagagt caactggtga 2700
ttattcgata actcaagttg atgtaagaaa ggctaaaatt aacgattata aagaggttta 2760
tgagcttgct aagtattcgg ctaaggattc cgactattta atcaatagag aagtgtttac 2820
ggtattctac aaatctttaa agggtaaaca ggtacttgta tttagtggat tatttaaaga 2880
cgctcataaa atgtataaga atggagagct agatctgtat aagaagttgg atactatcga 2940
atatgcttat atggtaagtt ataactggct taaaaagaag tatgatactt caaatattag 3000
agaattaact gaggaagaaa agcagaaatt caataaaaat ttaatcgaag atgtggatat 3060
tgagtaggtg ggattatatc tcaccttttt tattgtcttt tcatgttgaa attttgacgc 3120
ttaatgcatg aagtattgac aagtttaaaa attacggttt ttaatcctta gttgattagc 3180
aggattatgg ccggaatgct ccgtccagtc ctgttaagga attaaaattc cctaaaaccc 3240
ttggctatga tttatagcga gaatcgtcaa ttaaaaattt aataggtgct atgaaagtcg 3300
attaataatt aattttaaaa tgcaatatga aacataatta caagaatttg acttttaata 3360
caagaattga tatcatagtt acattaatac atttattttg aagggggaaa atgttttatg 3420
aaaagactac ttaaactacc tattttatca ttattaggat tatttttaat tggatcaact 3480
ccaacattag ctttaactaa agataataat caaaatttag atactatgaa agtaaactta 3540
tatactgaaa cagtagatgt gtttgataaa gatgcattta aacaaacatt tactaataaa 3600
gatataaaat ttctagagga ttctttgaat gcaaaaataa attattcagg taaatctgtt 3660
acagtaacaa tgaaaaacaa aattaagcca tctactaaac aagggcttgt tttatatgta 3720
aatggaaaat cagttaatgt tgattcagat ggcagtataa aagtacctaa agatactaag 3780
aaaatttcta aattaaataa agataaatca atgatggatg gatcaatgat ggataaatca 3840
ttacatgatg agaattgtgt agtatcagat agtttttata atgctgatgt taataatata 3900
aattcaaaag aagcagaagc tgtatttaaa gtaagttctg gtgaattatt agctaaaatg 3960
gatgaaaaag aagatgatta catacaaaag aactcatcta aaattctagc agctgcttat 4020
cataagggat atggggacaa gtactatgaa ggagattggg ttcattgcaa taggtttaat 4080
ggtcaactta cagatgatgt tcactataat tggagaactg gaagtgtttc agaaaaagca 4140
gctgcaatga gaaattttta tggcagtgat tgtcatatag cattagttca agcaggtagt 4200
ggatgtacaa gtataggttc atgcgaatgc aatacagatc aaatagctgc gtattgttca 4260
ggtttcgtaa aagataaaaa ta 4282
<210> 119
<211> 5473
<212> DNA
<213> 人工序列
<220>
<223> pNF3
<400> 119
gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 60
cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct 120
cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat 180
tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagcttgc 240
atgcctgcag gtcgaccacg ataaaacaag gttttaagga taagaaaagt catgagattt 300
atagtaaatc ttgtgacttt ttttattgaa tagtagagag agttcggaag tataacacgc 360
tatattcttg atatttttag aatagcaagc attggatttg tcctgacact ttcccaaaaa 420
ttaaggagtt attccttaaa ccaaaaagat taatgtggga acaaatttag tgtatccatt 480
tttgaagggc gcacttatac accaccaaaa tggtgtgtgc gaaatcttta aaaaagattt 540
atcaaaaagc ttttttaaag ctgggacatt tagaaaatca ataatgtttt ttgcccaata 600
cgctagtctt aaaatctgca aggttgataa ctatttagtc ccaggtatta gaatggggca 660
tatatataca aagtatatat atgcgtaaat atatgtggga ctgtgggaac aaaattgcgt 720
gctaaaattg tattgaaaag gtaatgaaaa ggtcatgctt tggtattgct aacgtataga 780
aaaggtaatg aaaagctcat ggttctataa aaaagatgta cccacgaaaa taataggctt 840
tgcctatttc cccatgtaat atgggggcag ttttctctta tgctctttct taacatattg 900
aataaataca aaatgcagct ttgtgggaat aaaaatattt ttgtttttat tcttatagtt 960
agacaaaatt ttaatctttt ttgtgctata acaagattaa aatttgtggg aacattaaga 1020
aatattgttg tcacaaataa aaaggagagt gggaacaatt gctataaaaa acgcagaaat 1080
taagattaga gttacaaaag agcaaaaaga attatttaag aaaattgcaa aagctgaaaa 1140
tatgagtatg agtgaattta ttattgtgac cacagaatat ttagccagaa aaaaagatga 1200
aaatatgaaa tcaaaagaca tgatcgagag aagagctgcg aagactgaag aaaaaattat 1260
gaagctaaaa aagaaactaa ataaaaacag gtaatataga ttacagtttt aagcttgttt 1320
tccctataga ctagagtaaa tatataaata tacctgtcaa gggcttataa gcccctttag 1380
ggggtgcgta gcacccttga caggtatatt tatatatttt agggtgccat taagggaaac 1440
aagctttaaa atgcctttaa aggcatttta aaataaataa aaaaaagatg gtttttacca 1500
tcttttttaa ctcccgaaag ggagttcttt cttttcttga tactatacgt aactatttcg 1560
atttgccctg aacctaatca aagctagata aattcagtat tagggcataa aaaaacttgc 1620
tttttcgggt ggaaatctgt ataatttaaa ttgcttagat aaaaattacc aattccatac 1680
gaaaggagca agttttacat aaggttaaag ccttatgtga attctcattt aattacatga 1740
ataataataa cacagaaagt gaagaattaa aagagcaaag tcaactattg cttgacaaat 1800
gcacaaaaaa gaaaaagaaa aatcctaaat ttagtagtta tatagaacca ttagtaagca 1860
agaaattatc tgaaagaata aaggaatgtg gtgacttttt gcagatgtta tctgatttaa 1920
accttgaaaa ttcgaaactg catagagcaa gtttttgtgg taacagattt tgtcctatgt 1980
gtagctggcg tattgcttgt aaggatagtt tggaaatatc tattctcatg gagcatttac 2040
gcaaagagga aagcaaagaa tttatctttt tgaccttaac aactccaaat gtgaaaggtg 2100
cggaccttga taattccata aaagcataca ataaagcatt taaaaagtta atggaacgca 2160
aagaggtcaa gagcatagta aaaggctaca taagaaagct agaagtaacc tataatttgg 2220
acaagagttc caaatcatat aatacttatc acccacattt ccatgtggta ctagcagtca 2280
atagaagtta ctttaaaaag caaaatctat atataaacca tcatagatgg cttagtttgt 2340
ggcaagagtc aactggtgat tattcgataa ctcaagttga tgtaagaaag gctaaaatta 2400
acgattataa agaggtttat gagcttgcta agtattcggc taaggattcc gactatttaa 2460
tcaatagaga agtgtttacg gtattctaca aatctttaaa gggtaaacag gtacttgtat 2520
ttagtggatt atttaaagac gctcataaaa tgtataagaa tggagagcta gatctgtata 2580
agaagttgga tactatcgaa tatgcttata tggtaagtta taactggctt aaaaagaagt 2640
atgatacttc aaatattaga gaattaactg aggaagaaaa gcagaaattc aataaaaatt 2700
taatcgaaga tgtggatatt gagtaggtgg gattatatct cacctttttt attgtctttt 2760
catgttgaaa ttttgacgct taatgcatga agtattgaca agtttaaaaa ttacggtttt 2820
taatccttag ttgattagca ggattatggc cggaatgctc cgtccagtcc tgttaaggaa 2880
ttaaaattcc ctaaaaccct tggctatgat ttatagcgag aatcgtcaat taaaaattta 2940
ataggtgcta tgaaagtcga ttaataatta attttaaaat gcaatatgaa acataattac 3000
aagaatttga cttttaatac aagaattgat atcatagtta cattaatacg gatccccggg 3060
taccgagctc gaattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg 3120
ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag 3180
aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggcgcctga 3240
tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatatgg tgcactctca 3300
gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg 3360
acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct 3420
ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg 3480
gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt 3540
caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac 3600
attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa 3660
aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat 3720
tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc 3780
agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga 3840
gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg 3900
cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc 3960
agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag 4020
taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc 4080
tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg 4140
taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg 4200
acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac 4260
ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac 4320
cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg 4380
agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg 4440
tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg 4500
agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac 4560
tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg 4620
ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg 4680
tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc 4740
aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc 4800
tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt 4860
agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc 4920
taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact 4980
caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac 5040
agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag 5100
aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg 5160
gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg 5220
tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga 5280
gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt 5340
ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct 5400
ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg 5460
aggaagcgga aga 5473
<210> 120
<211> 9128
<212> DNA
<213> 人工序列
<220>
<223> pMTL007S-E1
<400> 120
gatcgggccc cctgcagggt gtagtagcct gtgaaataag taaggaaaaa aaagaagtaa 60
gtgttatata tgatgattat tttgtagatg tagataggat aatagaatcc atagaaaata 120
taggttatac agttatataa aaattacttt aaaaattaat aaaaacatgg taaaatataa 180
atcgtataaa gttgtgtaat ttttaagctt gagctcataa caatttcaca caggaaacag 240
ctatgaccat gattacggat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc 300
ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata 360
gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc 420
gctaataaag atcttgtaca atctgtagga gaacctatgg gaacgaaacg aaagcgatgc 480
cgagaatctg aatttaccaa gacttaacac taactgggga taccctaaac aagaatgcct 540
aatagaaagg aggaaaaagg ctatagcact agagcttgaa aatcttgcaa gggtacggag 600
tactcgtagt agtctgagaa gggtaacgcc ctttacatgg caaaggggta cagttattgt 660
gtactaaaat taaaaattga ttagggagga aaacctcaaa atgaaaccaa caatggcaat 720
tttagaaaga atcagtaaaa attcacaaga aaatatagac gaagttttta caagacttta 780
tcgttatctt ttacgtccag atatttatta cgtggcgacg cgtgcgactc atagaattat 840
ttcctcccgt taaataatag ataactatta aaaatagaca atacttgctc ataagtaacg 900
gtacttaaat tgtttacttt ggcgtgtttc attgcttgat gaaactgatt tttagtaaac 960
agttgacgat attctcgatt gacccatttt gaaacaaagt acgtatatag cttccaatat 1020
ttatctggaa catctgtggt atggcgggta agttttatta agacactgtt tacttttggt 1080
ttaggatgaa agcattccgc tggcagctta agcaattgct gaatcgagac ttgagtgtgc 1140
aagagcaacc ctagtgttcg gtgaatatcc aaggtacgct tgtagaatcc ttcttcaaca 1200
atcagataga tgtcagacgc atggctttca aaaaccactt ttttaataat ttgtgtgctt 1260
aaatggtaag gaatactccc aacaatttta tacctctgtt tgttagggaa ttgaaactgt 1320
agaatatctt ggtgaattaa agtgacacga gtattcagtt ttaatttttc tgacgataag 1380
ttgaatagat gactgtctaa ttcaatagac gttacctgtt tacttatttt agccagtttc 1440
gtcgttaaat gccctttacc tgttccaatt tcgtaaacgg tatcggtttc ttttaaattc 1500
aattgtttta ttatttggtt gagtactttt tcactcgtta aaaagttttg agaatatttt 1560
atatttttgt tcataccagc accagaagca ccagcatctc ttgggttaat tgaggcctga 1620
gtataaggtg acttatactt gtaatctatc taaacgggga acctctctag tagacaatcc 1680
cgtgctaaat tgtaggactg ccctttaata aatacttcta tatttaaaga ggtatttatg 1740
aaaagcggaa tttatcagat taaaaatact ttctctagag aaaatttcgt ctggattagt 1800
tacttatcgt gtaaaatctg ataaatggaa ttggttctac ataaatgcct aacgactatc 1860
cctttgggga gtagggtcaa gtgactcgaa acgatagaca acttgcttta acaagttgga 1920
gatatagtct gctctgcatg gtgacatgca gctggatata attccggggt aagattaacg 1980
accttatctg aacataatgc catatgaatc cctcctaatt tatacgtttt ctctaacaac 2040
ttaattatac ccactattat tatttttatc aatataacgc gttgggaaat ggcaatgata 2100
gcgaaacaac gtaaaactct tgttgtatgc tttcattgtc atcgtcacgt gattcataaa 2160
cacaagtgaa tgtcgacagt gaatttttac gaacgaacaa taacagagcc gtatactccg 2220
agaggggtac gtacggttcc cgaagagggt ggtgcaaacc agtcacagta atgtgaacaa 2280
ggcggtacct ccctacttca ccatatcatt ttctgcagcc ccctagaaat aattttgttt 2340
aactttaaga aggagatata catatatggc tagatcgtcc attccgacag catcgccagt 2400
cactatggcg tgctgctagc gctatatgcg ttgatgcaat ttctatgcac tcgtagtagt 2460
ctgagaaggg taacgccctt tacatggcaa aggggtacag ttattgtgta ctaaaattaa 2520
aaattgatta gggaggaaaa cctcaaaatg aaaccaacaa tggcaatttt agaaagaatc 2580
agtaaaaatt cacaagaaaa tatagacgaa gtttttacaa gactttatcg ttatctttta 2640
cgtccagata tttattacgt ggcgtatcaa aatttatatt ccaataaagg agcttccaca 2700
aaaggaatat tagatgatac agcggatggc tttagtgaag aaaaaataaa aaagattatt 2760
caatctttaa aagacggaac ttactatcct caacctgtac gaagaatgta tattgcaaaa 2820
aagaattcta aaaagatgag acctttagga attccaactt tcacagataa attgatccaa 2880
gaagctgtga gaataattct tgaatctatc tatgaaccgg tattcgaaga tgtgtctcac 2940
ggttttagac ctcaacgaag ctgtcacaca gctttgaaaa caatcaaaag agagtttggc 3000
ggcgcaagat ggtttgtgga gggagatata aaaggctgct tcgataatat agaccacgtt 3060
acactcattg gactcatcaa tcttaaaatc aaagatatga aaatgagcca attgatttat 3120
aaatttctaa aagcaggtta tctggaaaac tggcagtatc acaaaactta cagcggaaca 3180
cctcaaggtg gaattctatc tcctcttttg gccaacatct atcttcatga attggataag 3240
tttgttttac aactcaaaat gaagtttgac cgagaaagtc cagaaagaat aacacctgaa 3300
tatcgggagc tccacaatga gataaaaaga atttctcacc gtctcaagaa gttggagggt 3360
gaagaaaaag ctaaagttct tttagaatat caagaaaaac gtaaaagatt acccacactc 3420
ccctgtacct cacagacaaa taaagtattg aaatacgtcc ggtatgcgga cgacttcatt 3480
atctctgtta aaggaagcaa agaggactgt caatggataa aagaacaatt aaaacttttt 3540
attcataaca agctaaaaat ggaattgagt gaagaaaaaa cactcatcac acatagcagt 3600
caacccgctc gttttctggg atatgatata cgagtaagga gatctggaac gataaaacga 3660
tctggtaaag tcaaaaagag aacactcaat gggagtgtag aactccttat tcctcttcaa 3720
gacaaaattc gtcaatttat ttttgacaag aaaatagcta tccaaaagaa agatagctca 3780
tggtttccag ttcacaggaa atatcttatt cgttcaacag acttagaaat catcacaatt 3840
tataattctg aactccgcgg gatttgtaat tactacggtc tagcaagtaa ttttaaccag 3900
ctcaattatt ttgcttatct tatggaatac agctgtctaa aaacgatagc ctccaaacat 3960
aagggaacac tttcaaaaac catttccatg tttaaagatg gaagtggttc gtgggggatc 4020
ccgtatgaga taaagcaagg taagcagcgc cgttattttg caaattttag tgaatgtaaa 4080
tccccttatc aatttacgga tgagataagt caagctcctg tattgtatgg ctatgcccgg 4140
aatactcttg aaaacaggtt aaaagctaaa tgttgtgaat tatgtgggac gtctgatgaa 4200
aatacttcct atgaaattca ccatgtcaat aaggtcaaaa atcttaaagg caaagaaaaa 4260
tgggaaatgg caatgatagc gaaacaacgt aaaactcttg ttgtatgctt tcattgtcat 4320
cgtcacgtga ttcataaaca caagtgaatg tcgagcaccc gttctcggag cactgtccga 4380
ccgctttggc cgccgcccag tcctgctcgc ttcgctactt ggagccacta tcgactacgc 4440
gatcatggcg accacacccg tcctgtggat cgccaagccg ccgatggtag tgtggggtct 4500
ccccatgcga gagtagggaa ctgccaggca tcaaataaaa cgaaaggctc agtcgaaaga 4560
ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctcctgagta ggacaaatcc 4620
gccgggagcg gatttgaacg ttgcgaagca acggcccgga gggtggcggg caggacgccc 4680
gccataaact gccaggcatc aaattaagca gaaggccatc ctgacggatg gcctttttgc 4740
gtttctacaa actcttcctg tcgtcatatc tacaagccat ccccccacag atacgggcgc 4800
gccgccatta tttttttgaa caattgacaa ttcatttctt attttttatt aagtgatagt 4860
caaaaggcat aacagtgctg aatagaaaga aatttacaga aaagaaaatt atagaattta 4920
gtatgattaa ttatactcat ttatgaatgt ttaattgaat acaaaaaaaa atacttgtta 4980
tgtattcaat tacgggttaa aatatagaca agttgaaaaa tttaataaaa aaataagtcc 5040
tcagctctta tatattaagc taccaactta gtatataagc caaaacttaa atgtgctacc 5100
aacacatcaa gccgttagag aactctatct atagcaatat ttcaaatgta ccgacataca 5160
agagaaacat taactatata tattcaattt atgagattat cttaacagat ataaatgtaa 5220
attgcaataa gtaagattta gaagtttata gcctttgtgt attggaagca gtacgcaaag 5280
gcttttttat ttgataaaaa ttagaagtat atttattttt tcataattaa tttatgaaaa 5340
tgaaaggggg tgagcaaagt gacagaggaa agcagtatct tatcaaataa caaggtatta 5400
gcaatatcat tattgacttt agcagtaaac attatgactt ttatagtgct tgtagctaag 5460
tagtacgaaa gggggagctt taaaaagctc cttggaatac atagaattca taaattaatt 5520
tatgaaaaga agggcgtata tgaaaacttg taaaaattgc aaagagttta ttaaagatac 5580
tgaaatatgc aaaatacatt cgttgatgat tcatgataaa acagtagcaa cctattgcag 5640
taaatacaat gagtcaagat gtttacataa agggaaagtc caatgtatta attgttcaaa 5700
gatgaaccga tatggatggt gtgccataaa aatgagatgt tttacagagg aagaacagaa 5760
aaaagaacgt acatgcatta aatattatgc aaggagcttt aaaaaagctc atgtaaagaa 5820
gagtaaaaag aaaaaataat ttatttatta atttaatatt gagagtgccg acacagtatg 5880
cactaaaaaa tatatctgtg gtgtagtgag ccgatacaaa aggatagtca ctcgcatttt 5940
cataatacat cttatgttat gattatgtgt cggtgggact tcacgacgaa aacccacaat 6000
aaaaaaagag ttcggggtag ggttaagcat agttgaggca actaaacaat caagctagga 6060
tatgcagtag cagaccgtaa ggtcgttgtt taggtgtgtt gtaatacata cgctattaag 6120
atgtaaaaat acggatacca atgaagggaa aagtataatt tttggatgta gtttgtttgt 6180
tcatctatgg gcaaactacg tccaaagccg tttccaaatc tgctaaaaag tatatccttt 6240
ctaaaatcaa agtcaagtat gaaatcataa ataaagttta attttgaagt tattatgata 6300
ttatgttttt ctattaaaat aaattaagta tatagaatag tttaataata gtatatactt 6360
aatgtgataa gtgtctgaca gtgtcacaga aaggatgatt gttatggatt ataagcggcc 6420
ggcccaatga ataggtttac acttacttta gttttatgga aatgaaagat catatcatat 6480
ataatctaga ataaaattaa ctaaaataat tattatctag ataaaaaatt tagaagccaa 6540
tgaaatctat aaataaacta aattaagttt atttaattaa caactatgga tataaaatag 6600
gtactaatca aaatagtgag gaggatatat ttgaatacat acgaacaaat taataaagtg 6660
aaaaaaatac ttcggaaaca tttaaaaaat aaccttattg gtacttacat gtttggatca 6720
ggagttgaga gtggactaaa accaaatagt gatcttgact ttttagtcgt cgtatctgaa 6780
ccattgacag atcaaagtaa agaaatactt atacaaaaaa ttagacctat ttcaaagaaa 6840
ataggagata aaagcaactt acgatatatt gaattaacaa ttattattca gcaagaaatg 6900
gtaccgtgga atcatcctcc caaacaagaa tttatttatg gagaatggtt acaagagctt 6960
tatgaacaag gatacattcc tcagaaggaa ttaaattcag atttaaccat aatgctttac 7020
caagcaaaac gaaaaaataa aagaatatac ggaaattatg acttagagga attactacct 7080
gatattccat tttctgatgt gagaagagcc attatggatt cgtcagagga attaatagat 7140
aattatcagg atgatgaaac caactctata ttaactttat gccgtatgat tttaactatg 7200
gacacgggta aaatcatacc aaaagatatt gcgggaaatg cagtggctga atcttctcca 7260
ttagaacata gggagagaat tttgttagca gttcgtagtt atcttggaga gaatattgaa 7320
tggactaatg aaaatgtaaa tttaactata aactatttaa ataacagatt aaaaaaatta 7380
taaaaaaatt gaaaaaatgg tggaaacact tttttcaatt tttttgtttt attatttaat 7440
atttgggaaa tattcattct aattggtaat cagattttag aagtttaaac tcctttttga 7500
taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt 7560
agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca 7620
aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct 7680
ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta 7740
gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct 7800
aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc 7860
aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca 7920
gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga 7980
aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg 8040
aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt 8100
cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag 8160
cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt 8220
tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt 8280
tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga 8340
ggaagcggaa gagcgcccaa tacgcagggc cccctgcttc ggggtcatta tagcgatttt 8400
ttcggtatat ccatcctttt tcgcacgata tacaggattt tgccaaaggg ttcgtgtaga 8460
ctttccttgg tgtatccaac ggcgtcagcc gggcaggata ggtgaagtag gcccacccgc 8520
gagcgggtgt tccttcttca ctgtccctta ttcgcacctg gcggtgctca acgggaatcc 8580
tgctctgcga ggctggccgg ctaccgccgg cgtaacagat gagggcaagc ggatggctga 8640
tgaaaccaag ccaaccagga agggcagccc acctatcaag gtgtactgcc ttccagacga 8700
acgaagagcg attgaggaaa aggcggcggc ggccggcatg agcctgtcgg cctacctgct 8760
ggccgtcggc cagggctaca aaatcacggg cgtcgtggac tatgagcacg tccgcgagct 8820
ggcccgcatc aatggcgacc tgggccgcct gggcggcctg ctgaaactct ggctcaccga 8880
cgacccgcgc acggcgcggt tcggtgatgc cacgatcctc gccctgctgg cgaagatcga 8940
agagaagcag gacgagcttg gcaaggtcat gatgggcgtg gtccgcccga gggcagagcc 9000
atgacttttt tagccgctaa aacggccggg gggtgcgcgt gattgccaag cacgtcccca 9060
tgcgctccat caagaagagc gacttcgcgg agctggtgaa gtacatcacc gacgagcaag 9120
gcaagacc 9128
<210> 121
<211> 5002
<212> DNA
<213> 人工序列
<220>
<223> pEC751S
<400> 121
atcaaaaaaa tttccaataa tcccactcta agccacaaac acgccctata aaatcccgct 60
ttaatcccac tttgagacac atgtaatatt actttacgcc ctagtatagt gataattttt 120
tacattcaat gccacgcaaa aaaataaagg ggcactataa taaaagttcc ttcggaacta 180
actaaagtaa aaaattatct ttacaacctc cccaaaaaaa agaacaggta caaagtaccc 240
tataatacaa gcgtaaaaaa aatgagggta aaaataaaaa aataaaaaaa taaaaaaata 300
aaaaaataaa aaaataaaaa aataaaaaaa tataaaaata aaaaaatata aaaataaaaa 360
aatataaaaa taaaaaaata aaaaaatata aaaataaaaa aataaaaaaa tataaaaata 420
ttttttattt aaagtttgaa aaaaattttt ttatattata taatctttga agaaaagaat 480
ataaaaaatg agcctttata aaagcccatt ttttttcata tacgtaatat gacgttctaa 540
tgtttttatt ggtacttcta acattagagt aatttcttta tttttaaagc ctttttcttt 600
aagggctttt attttttttc ttaatacatt taattcctct ttttttgttg cttttccttt 660
agcttttaat tgctcttgat aatttttttt acctctaata ttttctcttc tcttatattc 720
ctttttagaa attattattg tcatatattt ttgttcttct tctgtaattt ctaataactc 780
tataagagtt tcattcttat acttatattg cttattttta tctaaataac atctttcagc 840
acttctagtt gctcttataa cttctctttc acttaaatgt tgtctaaaca tactattaag 900
ttctaaaaca tcatttaatg ccttctcaat gtcttctgta aagctacaaa gataatatct 960
atataaaaat aatataagct ctctgtgtcc ttttaaatca tattctctta gttcacaaag 1020
ttttattatg tcttgtattc ttccataata taaacttctt tctctataaa tataatttat 1080
tttgcttggt ctaccctttt tcctttcata tggttttaat tcaggtaaaa atccattttg 1140
tatttctctt aagtcataaa tatattcgta ctcatctaat atattgacta ctgtttttga 1200
tttagagttt atacttcctg gaactcttaa tattctcgtt gcatctaagg cttgtctatc 1260
tgctccaaag tattttaatt gattatataa atattcttga accgctttcc ataatggtaa 1320
tgctttacta ggtactgcat ttattatcca tattaaatac attcctcttc cactatctat 1380
tacatagttt ggtataggaa tactttgatt aaaataattc ttttctaagt ccattaatac 1440
ctggtcttta gttttgccag ttttataata atccaagtct ataaacagtg tatttaactc 1500
ttttatattt tctaatcgcc tacacggctt ataaaaggta tttagagtta tatagatatt 1560
ttcatcactc atatctaaat cttttaattc agcgtattta tagtgccatt ggctatatcc 1620
ttttttatct ataacgctcc tggttatcca ccctttactt ctactatgaa tattatctat 1680
atagttcttt ttattcagct ttaatgcgtt tctcacttat tcacctcccc ttctgtaaaa 1740
ctaagaaaat tatatcatat tttcaataat tattaactat tcttaaactc ttaataaaaa 1800
atagagtaag tccccaattg aaacttaatc tattttttat gttttaattt attattttta 1860
ttaaaatatt ttaaactaaa ttaaatgatt ctttttaatt ttttactatt tcattccata 1920
atatattact ataattattt acaaataata tttcttcatt tgtaatattt agatgattta 1980
ctaattttag tttttatata ttaaataatt aatgtataat ttatataaaa aatcaaagga 2040
gcttataaat tatgattatt tccaaagata ctaaagattt aatttttttc aattttaaca 2100
atactttttg taatattatg tttaaattta attgtatttt tttcatataa taaagccgtt 2160
gaagtaaacc aatccatttt ccttatgatg ttattattaa atttaagttt tataataata 2220
tctttattat atttattgtt tttaaaaaaa ctagtgaaat ttctagtgaa atttccggct 2280
ttattaaact tatttttagg aattttattt tcattttcat ctttacagga tttgattata 2340
tctttaaata tgttttatca aatattatct ttttctaaat ttatatatat ttttattata 2400
tttattatta tatatatttt atttttaagt ttctttctaa cagctattaa aaagaaactt 2460
aaaaataaaa acacgtactc taaaccaata aataaaacta tttttattat tgctgccttg 2520
attggaatag tttttagtaa aattaatttc aatattccac aatattatat tataagctag 2580
cacgcctcga gatctccatg gacgcgtgac gtcgactcta gaggatcccc gggtaccgag 2640
ctcgaattcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat 2700
tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag 2760
ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg 2820
ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc 2880
ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc 2940
agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa 3000
catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt 3060
tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg 3120
gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg 3180
ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag 3240
cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc 3300
caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa 3360
ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg 3420
taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc 3480
taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac 3540
cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg 3600
tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt 3660
gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt 3720
catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa 3780
atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaaagct agcttaatac 3840
tagtatatac ttaatgtgat aagtgtctga cagctgaccg gtctaaagag gtcccaatga 3900
ataggtttac acttacttta gttttatgga aatgaaagat catatcatat ataatctaga 3960
ataaaattaa ctaaaataat tattatctag ataaaaaatt tagaagccaa tgaaatctat 4020
aaataaacta aattaagttt atttaattaa caactatgga tataaaatag gtactaatca 4080
aaatagtgag gaggatatat ttgaatacat acgaacaaat taataaagtg aaaaaaatac 4140
ttcggaaaca tttaaaaaat aaccttattg gtacttacat gtttggatca ggagttgaga 4200
gtggactaaa accaaatagt gatcttgact ttttagtcgt cgtatctgaa ccattgacag 4260
atcaaagtaa agaaatactt atacaaaaaa ttagacctat ttcaaagaaa ataggagata 4320
aaagcaactt acgatatatt gaattaacaa ttattattca gcaagaaatg gtaccgtgga 4380
atcatcctcc caaacaagaa tttatttatg gagaatggtt acaagagctt tatgaacaag 4440
gatacattcc tcagaaggaa ttaaattcag atttaaccat aatgctttac caagcaaaac 4500
gaaaaaataa aagaatatac ggaaattatg acttagagga attactacct gatattccat 4560
tttctgatgt gagaagagcc attatggatt cgtcagagga attaatagat aattatcagg 4620
atgatgaaac caactctata ttaactttat gccgtatgat tttaactatg gacacgggta 4680
aaatcatacc aaaagatatt gcgggaaatg cagtggctga atcttctcca ttagaacata 4740
gggagagaat tttgttagca gttcgtagtt atcttggaga gaatattgaa tggactaatg 4800
aaaatgtaaa tttaactata aactatttaa ataacagatt aaaaaaatta taaaaaaatt 4860
gaaaaaatgg tggaaacact tttttcaatt tttttgtttt attatttaat atttgggaaa 4920
tattcattct aattggtaat cagattttag aagttgttaa cttcaggttt gtctgtaact 4980
aaaaactagt atttaaccta gg 5002
<210> 122
<211> 3907
<212> DNA
<213> 人工序列
<220>
<223> pFW01
<400> 122
tcgagatctc catggacgcg tgacgtcgac tctagaggat ccccgggtac cgagctcgaa 60
ttcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca 120
caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact 180
cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct 240
gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc 300
ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca 360
ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg 420
agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca 480
taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa 540
cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc 600
tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc 660
gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct 720
gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg 780
tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag 840
gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta 900
cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg 960
aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt 1020
tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt 1080
ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag 1140
attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat 1200
ctaaagtata tatgagtaaa cttggtctga cagttaccag gtccactgcc gggcctcttg 1260
cgggatcaaa agaaaaacga aatgatacac caatcagtgc aaaaaaagat ataatgggag 1320
ataagacggt tcgtgttcgt gctgacttgc accatatcat aaaaatcgaa acagcaaaga 1380
atggcggaaa cgtaaaagaa gttatggaaa taagacttag aagcaaactt aagagtgtgt 1440
tgatagtgca gtatcttaaa attttgtata ataggaattg aagttaaatt agatgctaaa 1500
aatttgtaat taagaaggag tgattacatg aacaaaaata taaaatattc tcaaaacttt 1560
ttaacgagtg aaaaagtact caaccaaata ataaaacaat tgaatttaaa agaaaccgat 1620
accgtttacg aaattggaac aggtaaaggg catttaacga cgaaactggc taaaataagt 1680
aaacaggtaa cgtctattga attagacagt catctattca acttatcgtc agaaaaatta 1740
aaactgaata ctcgtgtcac tttaattcac caagatattc tacagtttca attccctaac 1800
aaacagaggt ataaaattgt tgggagtatt ccttaccatt taagcacaca aattattaaa 1860
aaagtggttt ttgaaagcca tgcgtctgac atctatctga ttgttgaaga aggattctac 1920
aagcgtacct tggatattca ccgaacacta gggttgctct tgcacactca agtctcgatt 1980
cagcaattgc ttaagctgcc agcggaatgc tttcatccta aaccaaaagt aaacagtgtc 2040
ttaataaaac ttacccgcca taccacagat gttccagata aatattggaa gctatatacg 2100
tactttgttt caaaatgggt caatcgagaa tatcgtcaac tgtttactaa aaatcagttt 2160
catcaagcaa tgaaacacgc caaagtaaac aatttaagta ccgttactta tgagcaagta 2220
ttgtctattt ttaatagtta tctattattt aacgggagga aataattcta tgagtcccta 2280
ggcaggcctc cgccattatt tttttgaaca attgacaatt catttcttat tttttattaa 2340
gtgatagtca aaaggcataa cagtgctgaa tagaaagaaa tttacagaaa agaaaattat 2400
agaatttagt atgattaatt atactcattt atgaatgttt aattgaatac aaaaaaaaat 2460
acttgttatg tattcaatta cgggttaaaa tatagacaag ttgaaaaatt taataaaaaa 2520
ataagtcctc agctcttata tattaagcta ccaacttagt atataagcca aaacttaaat 2580
gtgctaccaa cacatcaagc cgttagagaa ctctatctat agcaatattt caaatgtacc 2640
gacatacaag agaaacatta actatatata ttcaatttat gagattatct taacagatat 2700
aaatgtaaat tgcaataagt aagatttaga agtttatagc ctttgtgtat tggaagcagt 2760
acgcaaaggc ttttttattt gataaaaatt agaagtatat ttattttttc ataattaatt 2820
tatgaaaatg aaagggggtg agcaaagtga cagaggaaag cagtatctta tcaaataaca 2880
aggtattagc aatatcatta ttgactttag cagtaaacat tatgactttt atagtgcttg 2940
tagctaagta gtacgaaagg gggagcttta aaaagctcct tggaatacat agaattcata 3000
aattaattta tgaaaagaag ggcgtatatg aaaacttgta aaaattgcaa agagtttatt 3060
aaagatactg aaatatgcaa aatacattcg ttgatgattc atgataaaac agtagcaacc 3120
tattgcagta aatacaatga gtcaagatgt ttacataaag ggaaagtcca atgtattaat 3180
tgttcaaaga tgaaccgata tggatggtgt gccataaaaa tgagatgttt tacagaggaa 3240
gaacagaaaa aagaacgtac atgcattaaa tattatgcaa ggagctttaa aaaagctcat 3300
gtaaagaaga gtaaaaagaa aaaataattt atttattaat ttaatattga gagtgccgac 3360
acagtatgca ctaaaaaata tatctgtggt gtagtgagcc gatacaaaag gatagtcact 3420
cgcattttca taatacatct tatgttatga ttatgtgtcg gtgggacttc acgacgaaaa 3480
cccacaataa aaaaagagtt cggggtaggg ttaagcatag ttgaggcaac taaacaatca 3540
agctaggata tgcagtagca gaccgtaagg tcgttgttta ggtgtgttgt aatacatacg 3600
ctattaagat gtaaaaatac ggataccaat gaagggaaaa gtataatttt tggatgtagt 3660
ttgtttgttc atctatgggc aaactacgtc caaagccgtt tccaaatctg ctaaaaagta 3720
tatcctttct aaaatcaaag tcaagtatga aatcataaat aaagtttaat tttgaagtta 3780
ttatgatatt atgtttttct attaaaataa attaagtata tagaatagtt taataatagt 3840
atatacttaa tgtgataagt gtctgacagt gtcacagaaa ggatgattgt tatggattat 3900
aagcggc 3907
<210> 123
<211> 6525
<212> DNA
<213> 人工序列
<220>
<223> pNF3S
<400> 123
gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 60
cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct 120
cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat 180
tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagcttgc 240
atgcctgcag gtcgaccacg ataaaacaag gttttaagga taagaaaagt catgagattt 300
atagtaaatc ttgtgacttt ttttattgaa tagtagagag agttcggaag tataacacgc 360
tatattcttg atatttttag aatagcaagc attggatttg tcctgacact ttcccaaaaa 420
ttaaggagtt attccttaaa ccaaaaagat taatgtggga acaaatttag tgtatccatt 480
tttgaagggc gcacttatac accaccaaaa tggtgtgtgc gaaatcttta aaaaagattt 540
atcaaaaagc ttttttaaag ctgggacatt tagaaaatca ataatgtttt ttgcccaata 600
cgctagtctt aaaatctgca aggttgataa ctatttagtc ccaggtatta gaatggggca 660
tatatataca aagtatatat atgcgtaaat atatgtggga ctgtgggaac aaaattgcgt 720
gctaaaattg tattgaaaag gtaatgaaaa ggtcatgctt tggtattgct aacgtataga 780
aaaggtaatg aaaagctcat ggttctataa aaaagatgta cccacgaaaa taataggctt 840
tgcctatttc cccatgtaat atgggggcag ttttctctta tgctctttct taacatattg 900
aataaataca aaatgcagct ttgtgggaat aaaaatattt ttgtttttat tcttatagtt 960
agacaaaatt ttaatctttt ttgtgctata acaagattaa aatttgtggg aacattaaga 1020
aatattgttg tcacaaataa aaaggagagt gggaacaatt gctataaaaa acgcagaaat 1080
taagattaga gttacaaaag agcaaaaaga attatttaag aaaattgcaa aagctgaaaa 1140
tatgagtatg agtgaattta ttattgtgac cacagaatat ttagccagaa aaaaagatga 1200
aaatatgaaa tcaaaagaca tgatcgagag aagagctgcg aagactgaag aaaaaattat 1260
gaagctaaaa aagaaactaa ataaaaacag gtaatataga ttacagtttt aagcttgttt 1320
tccctataga ctagagtaaa tatataaata tacctgtcaa gggcttataa gcccctttag 1380
ggggtgcgta gcacccttga caggtatatt tatatatttt agggtgccat taagggaaac 1440
aagctttaaa atgcctttaa aggcatttta aaataaataa aaaaaagatg gtttttacca 1500
tcttttttaa ctcccgaaag ggagttcttt cttttcttga tactatacgt aactatttcg 1560
atttgccctg aacctaatca aagctagata aattcagtat tagggcataa aaaaacttgc 1620
tttttcgggt ggaaatctgt ataatttaaa ttgcttagat aaaaattacc aattccatac 1680
gaaaggagca agttttacat aaggttaaag ccttatgtga attctcattt aattacatga 1740
ataataataa cacagaaagt gaagaattaa aagagcaaag tcaactattg cttgacaaat 1800
gcacaaaaaa gaaaaagaaa aatcctaaat ttagtagtta tatagaacca ttagtaagca 1860
agaaattatc tgaaagaata aaggaatgtg gtgacttttt gcagatgtta tctgatttaa 1920
accttgaaaa ttcgaaactg catagagcaa gtttttgtgg taacagattt tgtcctatgt 1980
gtagctggcg tattgcttgt aaggatagtt tggaaatatc tattctcatg gagcatttac 2040
gcaaagagga aagcaaagaa tttatctttt tgaccttaac aactccaaat gtgaaaggtg 2100
cggaccttga taattccata aaagcataca ataaagcatt taaaaagtta atggaacgca 2160
aagaggtcaa gagcatagta aaaggctaca taagaaagct agaagtaacc tataatttgg 2220
acaagagttc caaatcatat aatacttatc acccacattt ccatgtggta ctagcagtca 2280
atagaagtta ctttaaaaag caaaatctat atataaacca tcatagatgg cttagtttgt 2340
ggcaagagtc aactggtgat tattcgataa ctcaagttga tgtaagaaag gctaaaatta 2400
acgattataa agaggtttat gagcttgcta agtattcggc taaggattcc gactatttaa 2460
tcaatagaga agtgtttacg gtattctaca aatctttaaa gggtaaacag gtacttgtat 2520
ttagtggatt atttaaagac gctcataaaa tgtataagaa tggagagcta gatctgtata 2580
agaagttgga tactatcgaa tatgcttata tggtaagtta taactggctt aaaaagaagt 2640
atgatacttc aaatattaga gaattaactg aggaagaaaa gcagaaattc aataaaaatt 2700
taatcgaaga tgtggatatt gagtaggtgg gattatatct cacctttttt attgtctttt 2760
catgttgaaa ttttgacgct taatgcatga agtattgaca agtttaaaaa ttacggtttt 2820
taatccttag ttgattagca ggattatggc cggaatgctc cgtccagtcc tgttaaggaa 2880
ttaaaattcc ctaaaaccct tggctatgat ttatagcgag aatcgtcaat taaaaattta 2940
ataggtgcta tgaaagtcga ttaataatta attttaaaat gcaatatgaa acataattac 3000
aagaatttga cttttaatac aagaattgat atcatagtta cattaatacg gatcccaatg 3060
aataggttta cacttacttt agttttatgg aaatgaaaga tcatatcata tataatctag 3120
aataaaatta actaaaataa ttattatcta gataaaaaat ttagaagcca atgaaatcta 3180
taaataaact aaattaagtt tatttaatta acaactatgg atataaaata ggtactaatc 3240
aaaatagtga ggaggatata tttgaataca tacgaacaaa ttaataaagt gaaaaaaata 3300
cttcggaaac atttaaaaaa taaccttatt ggtacttaca tgtttggatc aggagttgag 3360
agtggactaa aaccaaatag tgatcttgac tttttagtcg tcgtatctga accattgaca 3420
gatcaaagta aagaaatact tatacaaaaa attagaccta tttcaaagaa aataggagat 3480
aaaagcaact tacgatatat tgaattaaca attattattc agcaagaaat ggtaccgtgg 3540
aatcatcctc ccaaacaaga atttatttat ggagaatggt tacaagagct ttatgaacaa 3600
ggatacattc ctcagaagga attaaattca gatttaacca taatgcttta ccaagcaaaa 3660
cgaaaaaata aaagaatata cggaaattat gacttagagg aattactacc tgatattcca 3720
ttttctgatg tgagaagagc cattatggat tcgtcagagg aattaataga taattatcag 3780
gatgatgaaa ccaactctat attaacttta tgccgtatga ttttaactat ggacacgggt 3840
aaaatcatac caaaagatat tgcgggaaat gcagtggctg aatcttctcc attagaacat 3900
agggagagaa ttttgttagc agttcgtagt tatcttggag agaatattga atggactaat 3960
gaaaatgtaa atttaactat aaactattta aataacagat taaaaaaatt ataaaaaaat 4020
tgaaaaaatg gtggaaacac ttttttcaat ttttttgttt tattatttaa tatttgggaa 4080
atattcattc taattggtaa tcagatttta gaagttgagc tcgaattcac tggccgtcgt 4140
tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca 4200
tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca 4260
gttgcgcagc ctgaatggcg aatggcgcct gatgcggtat tttctcctta cgcatctgtg 4320
cggtatttca caccgcatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt 4380
aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc 4440
ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc 4500
accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt 4560
taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg 4620
cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca 4680
ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt 4740
ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga 4800
aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga 4860
actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat 4920
gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca 4980
agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt 5040
cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac 5100
catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct 5160
aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga 5220
gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac 5280
aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat 5340
agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg 5400
ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc 5460
actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc 5520
aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg 5580
gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta 5640
atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg 5700
tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga 5760
tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt 5820
ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag 5880
agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa 5940
ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag 6000
tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca 6060
gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac 6120
cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa 6180
ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc 6240
agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg 6300
tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc 6360
ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc 6420
ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag 6480
ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaaga 6525
<210> 124
<211> 6554
<212> DNA
<213> 人工序列
<220>
<223> pNF3E
<400> 124
gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 60
cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct 120
cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat 180
tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagcttgc 240
atgcctgcag gtcgaccacg ataaaacaag gttttaagga taagaaaagt catgagattt 300
atagtaaatc ttgtgacttt ttttattgaa tagtagagag agttcggaag tataacacgc 360
tatattcttg atatttttag aatagcaagc attggatttg tcctgacact ttcccaaaaa 420
ttaaggagtt attccttaaa ccaaaaagat taatgtggga acaaatttag tgtatccatt 480
tttgaagggc gcacttatac accaccaaaa tggtgtgtgc gaaatcttta aaaaagattt 540
atcaaaaagc ttttttaaag ctgggacatt tagaaaatca ataatgtttt ttgcccaata 600
cgctagtctt aaaatctgca aggttgataa ctatttagtc ccaggtatta gaatggggca 660
tatatataca aagtatatat atgcgtaaat atatgtggga ctgtgggaac aaaattgcgt 720
gctaaaattg tattgaaaag gtaatgaaaa ggtcatgctt tggtattgct aacgtataga 780
aaaggtaatg aaaagctcat ggttctataa aaaagatgta cccacgaaaa taataggctt 840
tgcctatttc cccatgtaat atgggggcag ttttctctta tgctctttct taacatattg 900
aataaataca aaatgcagct ttgtgggaat aaaaatattt ttgtttttat tcttatagtt 960
agacaaaatt ttaatctttt ttgtgctata acaagattaa aatttgtggg aacattaaga 1020
aatattgttg tcacaaataa aaaggagagt gggaacaatt gctataaaaa acgcagaaat 1080
taagattaga gttacaaaag agcaaaaaga attatttaag aaaattgcaa aagctgaaaa 1140
tatgagtatg agtgaattta ttattgtgac cacagaatat ttagccagaa aaaaagatga 1200
aaatatgaaa tcaaaagaca tgatcgagag aagagctgcg aagactgaag aaaaaattat 1260
gaagctaaaa aagaaactaa ataaaaacag gtaatataga ttacagtttt aagcttgttt 1320
tccctataga ctagagtaaa tatataaata tacctgtcaa gggcttataa gcccctttag 1380
ggggtgcgta gcacccttga caggtatatt tatatatttt agggtgccat taagggaaac 1440
aagctttaaa atgcctttaa aggcatttta aaataaataa aaaaaagatg gtttttacca 1500
tcttttttaa ctcccgaaag ggagttcttt cttttcttga tactatacgt aactatttcg 1560
atttgccctg aacctaatca aagctagata aattcagtat tagggcataa aaaaacttgc 1620
tttttcgggt ggaaatctgt ataatttaaa ttgcttagat aaaaattacc aattccatac 1680
gaaaggagca agttttacat aaggttaaag ccttatgtga attctcattt aattacatga 1740
ataataataa cacagaaagt gaagaattaa aagagcaaag tcaactattg cttgacaaat 1800
gcacaaaaaa gaaaaagaaa aatcctaaat ttagtagtta tatagaacca ttagtaagca 1860
agaaattatc tgaaagaata aaggaatgtg gtgacttttt gcagatgtta tctgatttaa 1920
accttgaaaa ttcgaaactg catagagcaa gtttttgtgg taacagattt tgtcctatgt 1980
gtagctggcg tattgcttgt aaggatagtt tggaaatatc tattctcatg gagcatttac 2040
gcaaagagga aagcaaagaa tttatctttt tgaccttaac aactccaaat gtgaaaggtg 2100
cggaccttga taattccata aaagcataca ataaagcatt taaaaagtta atggaacgca 2160
aagaggtcaa gagcatagta aaaggctaca taagaaagct agaagtaacc tataatttgg 2220
acaagagttc caaatcatat aatacttatc acccacattt ccatgtggta ctagcagtca 2280
atagaagtta ctttaaaaag caaaatctat atataaacca tcatagatgg cttagtttgt 2340
ggcaagagtc aactggtgat tattcgataa ctcaagttga tgtaagaaag gctaaaatta 2400
acgattataa agaggtttat gagcttgcta agtattcggc taaggattcc gactatttaa 2460
tcaatagaga agtgtttacg gtattctaca aatctttaaa gggtaaacag gtacttgtat 2520
ttagtggatt atttaaagac gctcataaaa tgtataagaa tggagagcta gatctgtata 2580
agaagttgga tactatcgaa tatgcttata tggtaagtta taactggctt aaaaagaagt 2640
atgatacttc aaatattaga gaattaactg aggaagaaaa gcagaaattc aataaaaatt 2700
taatcgaaga tgtggatatt gagtaggtgg gattatatct cacctttttt attgtctttt 2760
catgttgaaa ttttgacgct taatgcatga agtattgaca agtttaaaaa ttacggtttt 2820
taatccttag ttgattagca ggattatggc cggaatgctc cgtccagtcc tgttaaggaa 2880
ttaaaattcc ctaaaaccct tggctatgat ttatagcgag aatcgtcaat taaaaattta 2940
ataggtgcta tgaaagtcga ttaataatta attttaaaat gcaatatgaa acataattac 3000
aagaatttga cttttaatac aagaattgat atcatagtta cattaatacg gatccgtctg 3060
acagttacca ggtccactgc cgggcctctt gcgggatcaa aagaaaaacg aaatgataca 3120
ccaatcagtg caaaaaaaga tataatggga gataagacgg ttcgtgttcg tgctgacttg 3180
caccatatca taaaaatcga aacagcaaag aatggcggaa acgtaaaaga agttatggaa 3240
ataagactta gaagcaaact taagagtgtg ttgatagtgc agtatcttaa aattttgtat 3300
aataggaatt gaagttaaat tagatgctaa aaatttgtaa ttaagaagga gtgattacat 3360
gaacaaaaat ataaaatatt ctcaaaactt tttaacgagt gaaaaagtac tcaaccaaat 3420
aataaaacaa ttgaatttaa aagaaaccga taccgtttac gaaattggaa caggtaaagg 3480
gcatttaacg acgaaactgg ctaaaataag taaacaggta acgtctattg aattagacag 3540
tcatctattc aacttatcgt cagaaaaatt aaaactgaat actcgtgtca ctttaattca 3600
ccaagatatt ctacagtttc aattccctaa caaacagagg tataaaattg ttgggagtat 3660
tccttaccat ttaagcacac aaattattaa aaaagtggtt tttgaaagcc atgcgtctga 3720
catctatctg attgttgaag aaggattcta caagcgtacc ttggatattc accgaacact 3780
agggttgctc ttgcacactc aagtctcgat tcagcaattg cttaagctgc cagcggaatg 3840
ctttcatcct aaaccaaaag taaacagtgt cttaataaaa cttacccgcc ataccacaga 3900
tgttccagat aaatattgga agctatatac gtactttgtt tcaaaatggg tcaatcgaga 3960
atatcgtcaa ctgtttacta aaaatcagtt tcatcaagca atgaaacacg ccaaagtaaa 4020
caatttaagt accgttactt atgagcaagt attgtctatt tttaatagtt atctattatt 4080
taacgggagg aaataattct atgagtccct aggcaggcct ccgccattat ttttttgaac 4140
aattggagct cgaattcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc 4200
gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa 4260
gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggcgcctg 4320
atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatatg gtgcactctc 4380
agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct 4440
gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc 4500
tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag 4560
ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg 4620
tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata 4680
cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga 4740
aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca 4800
ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat 4860
cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag 4920
agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc 4980
gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct 5040
cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca 5100
gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt 5160
ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat 5220
gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt 5280
gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta 5340
cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga 5400
ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt 5460
gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc 5520
gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct 5580
gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata 5640
ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt 5700
gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc 5760
gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg 5820
caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact 5880
ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg 5940
tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg 6000
ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac 6060
tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca 6120
cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga 6180
gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc 6240
ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct 6300
gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg 6360
agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct 6420
tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc 6480
tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc 6540
gaggaagcgg aaga 6554
<210> 125
<211> 6271
<212> DNA
<213> 人工序列
<220>
<223> pNF3C
<400> 125
gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 60
cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct 120
cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat 180
tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagcttgc 240
atgcctgcag gtcgaccacg ataaaacaag gttttaagga taagaaaagt catgagattt 300
atagtaaatc ttgtgacttt ttttattgaa tagtagagag agttcggaag tataacacgc 360
tatattcttg atatttttag aatagcaagc attggatttg tcctgacact ttcccaaaaa 420
ttaaggagtt attccttaaa ccaaaaagat taatgtggga acaaatttag tgtatccatt 480
tttgaagggc gcacttatac accaccaaaa tggtgtgtgc gaaatcttta aaaaagattt 540
atcaaaaagc ttttttaaag ctgggacatt tagaaaatca ataatgtttt ttgcccaata 600
cgctagtctt aaaatctgca aggttgataa ctatttagtc ccaggtatta gaatggggca 660
tatatataca aagtatatat atgcgtaaat atatgtggga ctgtgggaac aaaattgcgt 720
gctaaaattg tattgaaaag gtaatgaaaa ggtcatgctt tggtattgct aacgtataga 780
aaaggtaatg aaaagctcat ggttctataa aaaagatgta cccacgaaaa taataggctt 840
tgcctatttc cccatgtaat atgggggcag ttttctctta tgctctttct taacatattg 900
aataaataca aaatgcagct ttgtgggaat aaaaatattt ttgtttttat tcttatagtt 960
agacaaaatt ttaatctttt ttgtgctata acaagattaa aatttgtggg aacattaaga 1020
aatattgttg tcacaaataa aaaggagagt gggaacaatt gctataaaaa acgcagaaat 1080
taagattaga gttacaaaag agcaaaaaga attatttaag aaaattgcaa aagctgaaaa 1140
tatgagtatg agtgaattta ttattgtgac cacagaatat ttagccagaa aaaaagatga 1200
aaatatgaaa tcaaaagaca tgatcgagag aagagctgcg aagactgaag aaaaaattat 1260
gaagctaaaa aagaaactaa ataaaaacag gtaatataga ttacagtttt aagcttgttt 1320
tccctataga ctagagtaaa tatataaata tacctgtcaa gggcttataa gcccctttag 1380
ggggtgcgta gcacccttga caggtatatt tatatatttt agggtgccat taagggaaac 1440
aagctttaaa atgcctttaa aggcatttta aaataaataa aaaaaagatg gtttttacca 1500
tcttttttaa ctcccgaaag ggagttcttt cttttcttga tactatacgt aactatttcg 1560
atttgccctg aacctaatca aagctagata aattcagtat tagggcataa aaaaacttgc 1620
tttttcgggt ggaaatctgt ataatttaaa ttgcttagat aaaaattacc aattccatac 1680
gaaaggagca agttttacat aaggttaaag ccttatgtga attctcattt aattacatga 1740
ataataataa cacagaaagt gaagaattaa aagagcaaag tcaactattg cttgacaaat 1800
gcacaaaaaa gaaaaagaaa aatcctaaat ttagtagtta tatagaacca ttagtaagca 1860
agaaattatc tgaaagaata aaggaatgtg gtgacttttt gcagatgtta tctgatttaa 1920
accttgaaaa ttcgaaactg catagagcaa gtttttgtgg taacagattt tgtcctatgt 1980
gtagctggcg tattgcttgt aaggatagtt tggaaatatc tattctcatg gagcatttac 2040
gcaaagagga aagcaaagaa tttatctttt tgaccttaac aactccaaat gtgaaaggtg 2100
cggaccttga taattccata aaagcataca ataaagcatt taaaaagtta atggaacgca 2160
aagaggtcaa gagcatagta aaaggctaca taagaaagct agaagtaacc tataatttgg 2220
acaagagttc caaatcatat aatacttatc acccacattt ccatgtggta ctagcagtca 2280
atagaagtta ctttaaaaag caaaatctat atataaacca tcatagatgg cttagtttgt 2340
ggcaagagtc aactggtgat tattcgataa ctcaagttga tgtaagaaag gctaaaatta 2400
acgattataa agaggtttat gagcttgcta agtattcggc taaggattcc gactatttaa 2460
tcaatagaga agtgtttacg gtattctaca aatctttaaa gggtaaacag gtacttgtat 2520
ttagtggatt atttaaagac gctcataaaa tgtataagaa tggagagcta gatctgtata 2580
agaagttgga tactatcgaa tatgcttata tggtaagtta taactggctt aaaaagaagt 2640
atgatacttc aaatattaga gaattaactg aggaagaaaa gcagaaattc aataaaaatt 2700
taatcgaaga tgtggatatt gagtaggtgg gattatatct cacctttttt attgtctttt 2760
catgttgaaa ttttgacgct taatgcatga agtattgaca agtttaaaaa ttacggtttt 2820
taatccttag ttgattagca ggattatggc cggaatgctc cgtccagtcc tgttaaggaa 2880
ttaaaattcc ctaaaaccct tggctatgat ttatagcgag aatcgtcaat taaaaattta 2940
ataggtgcta tgaaagtcga ttaataatta attttaaaat gcaatatgaa acataattac 3000
aagaatttga cttttaatac aagaattgat atcatagtta cattaatacg gatcccggca 3060
gtttttcttt ttcggcaagt gttcaagaag ttattaagtc gggagtgcag tcgaagtggg 3120
caagttgaaa aattcacaaa aatgtggtat aatatctttg ttcattagag cgataaactt 3180
gaatttgaga gggaacttag atggtatttg aaaaaattga taaaaatagt tggaacagaa 3240
aagagtattt tgaccactac tttgcaagtg taccttgtac ctacagcatg accgttaaag 3300
tggatatcac acaaataaag gaaaagggaa tgaaactata tcctgcaatg ctttattata 3360
ttgcaatgat tgtaaaccgc cattcagagt ttaggacggc aatcaatcaa gatggtgaat 3420
tggggatata tgatgagatg ataccaagct atacaatatt tcacaatgat actgaaacat 3480
tttccagcct ttggactgag tgtaagtctg actttaaatc atttttagca gattatgaaa 3540
gtgatacgca acggtatgga aacaatcata gaatggaagg aaagccaaat gctccggaaa 3600
acatttttaa tgtatctatg ataccgtggt caaccttcga tggctttaat ctgaatttgc 3660
agaaaggata tgattatttg attcctattt ttactatggg gaaatattat aaagaagata 3720
acaaaattat acttcctttg gcaattcaag ttcatcacgc agtatgtgac ggatttcaca 3780
tttgccgttt tgtaaacgaa ttgcaggaat tgataaatag ttaacttcag gtttgtctgt 3840
aactaaaaac tagtatttaa ccgagctcga attcactggc cgtcgtttta caacgtcgtg 3900
actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca 3960
gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga 4020
atggcgaatg gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc 4080
gcatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac 4140
acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca 4200
gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga 4260
aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa 4320
taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt 4380
gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa 4440
tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta 4500
ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag 4560
taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca 4620
gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta 4680
aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc 4740
gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc 4800
ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca 4860
ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc 4920
acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca 4980
taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac 5040
tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg 5100
cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg 5160
ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg 5220
gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac 5280
gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc 5340
aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct 5400
aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc 5460
actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc 5520
gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg 5580
atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa 5640
atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc 5700
ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt 5760
gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa 5820
cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc 5880
tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc 5940
cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct 6000
ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat 6060
gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc 6120
tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg 6180
ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc 6240
gcagcgagtc agtgagcgag gaagcggaag a 6271
<210> 126
<211> 2793
<212> DNA
<213> 人工序列
<220>
<223> OREP
<400> 126
cacgataaaa caaggtttta aggataagaa aagtcatgag atttatagta aatcttgtga 60
ctttttttat tgaatagtag agagagttcg gaagtataac acgctatatt cttgatattt 120
ttagaatagc aagcattgga tttgtcctga cactttccca aaaattaagg agttattcct 180
taaaccaaaa agattaatgt gggaacaaat ttagtgtatc catttttgaa gggcgcactt 240
atacaccacc aaaatggtgt gtgcgaaatc tttaaaaaag atttatcaaa aagctttttt 300
aaagctggga catttagaaa atcaataatg ttttttgccc aatacgctag tcttaaaatc 360
tgcaaggttg ataactattt agtcccaggt attagaatgg ggcatatata tacaaagtat 420
atatatgcgt aaatatatgt gggactgtgg gaacaaaatt gcgtgctaaa attgtattga 480
aaaggtaatg aaaaggtcat gctttggtat tgctaacgta tagaaaaggt aatgaaaagc 540
tcatggttct ataaaaaaga tgtacccacg aaaataatag gctttgccta tttccccatg 600
taatatgggg gcagttttct cttatgctct ttcttaacat attgaataaa tacaaaatgc 660
agctttgtgg gaataaaaat atttttgttt ttattcttat agttagacaa aattttaatc 720
ttttttgtgc tataacaaga ttaaaatttg tgggaacatt aagaaatatt gttgtcacaa 780
ataaaaagga gagtgggaac aattgctata aaaaacgcag aaattaagat tagagttaca 840
aaagagcaaa aagaattatt taagaaaatt gcaaaagctg aaaatatgag tatgagtgaa 900
tttattattg tgaccacaga atatttagcc agaaaaaaag atgaaaatat gaaatcaaaa 960
gacatgatcg agagaagagc tgcgaagact gaagaaaaaa ttatgaagct aaaaaagaaa 1020
ctaaataaaa acaggtaata tagattacag ttttaagctt gttttcccta tagactagag 1080
taaatatata aatatacctg tcaagggctt ataagcccct ttagggggtg cgtagcaccc 1140
ttgacaggta tatttatata ttttagggtg ccattaaggg aaacaagctt taaaatgcct 1200
ttaaaggcat tttaaaataa ataaaaaaaa gatggttttt accatctttt ttaactcccg 1260
aaagggagtt ctttcttttc ttgatactat acgtaactat ttcgatttgc cctgaaccta 1320
atcaaagcta gataaattca gtattagggc ataaaaaaac ttgctttttc gggtggaaat 1380
ctgtataatt taaattgctt agataaaaat taccaattcc atacgaaagg agcaagtttt 1440
acataaggtt aaagccttat gtgaattctc atttaattac atgaataata ataacacaga 1500
aagtgaagaa ttaaaagagc aaagtcaact attgcttgac aaatgcacaa aaaagaaaaa 1560
gaaaaatcct aaatttagta gttatataga accattagta agcaagaaat tatctgaaag 1620
aataaaggaa tgtggtgact ttttgcagat gttatctgat ttaaaccttg aaaattcgaa 1680
actgcataga gcaagttttt gtggtaacag attttgtcct atgtgtagct ggcgtattgc 1740
ttgtaaggat agtttggaaa tatctattct catggagcat ttacgcaaag aggaaagcaa 1800
agaatttatc tttttgacct taacaactcc aaatgtgaaa ggtgcggacc ttgataattc 1860
cataaaagca tacaataaag catttaaaaa gttaatggaa cgcaaagagg tcaagagcat 1920
agtaaaaggc tacataagaa agctagaagt aacctataat ttggacaaga gttccaaatc 1980
atataatact tatcacccac atttccatgt ggtactagca gtcaatagaa gttactttaa 2040
aaagcaaaat ctatatataa accatcatag atggcttagt ttgtggcaag agtcaactgg 2100
tgattattcg ataactcaag ttgatgtaag aaaggctaaa attaacgatt ataaagaggt 2160
ttatgagctt gctaagtatt cggctaagga ttccgactat ttaatcaata gagaagtgtt 2220
tacggtattc tacaaatctt taaagggtaa acaggtactt gtatttagtg gattatttaa 2280
agacgctcat aaaatgtata agaatggaga gctagatctg tataagaagt tggatactat 2340
cgaatatgct tatatggtaa gttataactg gcttaaaaag aagtatgata cttcaaatat 2400
tagagaatta actgaggaag aaaagcagaa attcaataaa aatttaatcg aagatgtgga 2460
tattgagtag gtgggattat atctcacctt ttttattgtc ttttcatgtt gaaattttga 2520
cgcttaatgc atgaagtatt gacaagttta aaaattacgg tttttaatcc ttagttgatt 2580
agcaggatta tggccggaat gctccgtcca gtcctgttaa ggaattaaaa ttccctaaaa 2640
cccttggcta tgatttatag cgagaatcgt caattaaaaa tttaataggt gctatgaaag 2700
tcgattaata attaatttta aaatgcaata tgaaacataa ttacaagaat ttgactttta 2760
atacaagaat tgatatcata gttacattaa tac 2793
<210> 127
<211> 2793
<212> DNA
<213> 拜氏梭状芽孢杆菌（Clostridium beijerinckii）
<400> 127
cacgataaaa caaggtttta aggataagaa aagtcatgag atttatagta aatcttgtga 60
ctttttttat tgaatagtag agagagttcg gaagtataac acgctatatt cttgatattt 120
ttagaatagc aagcattgga tttgtcctga cactttccca aaaattaagg agttattcct 180
taaaccaaaa agattaatgt gggaacaaat ttagtgtatc catttttgaa gggcgcactt 240
atacaccacc aaaatggtgt gtgcgaaatc tttaaaaaag atttatcaaa aagctttttt 300
aaagctggga catttagaaa atcaataatg ttttttgccc aatacgctag tcttaaaatc 360
tgcaaggttg ataactattt agtcccaggt attagaatgg ggcatatata tacaaagtat 420
atatatgcgt aaatatatgt gggactgtgg gaacaaaatt gcgtgctaaa attgtattga 480
aaaggtaatg aaaaggtcat gctttggtat tgctaacgta tagaaaaggt aatgaaaagc 540
tcatggttct ataaaaaaga tgtacccacg aaaataatag gctttgccta tttccccatg 600
taatatgggg gcagttttct cttatgctct ttcttaacat attgaataaa tacaaaatgc 660
agctttgtgg gaataaaaat atttttgttt ttattcttat agttagacaa aattttaatc 720
ttttttgtgc tataacaaga ttaaaatttg tgggaacatt aagaaatatt gttgtcacaa 780
ataaaaagga gagtgggaac aattgctata aaaaacgcag aaattaagat tagagttaca 840
aaagagcaaa aagaattatt taagaaaatt gcaaaagctg aaaatatgag tatgagtgaa 900
tttattattg tgaccacaga atatttagcc agaaaaaaag atgaaaatat gaaatcaaaa 960
gacatgatcg agagaagagc tgcgaagact gaagaaaaaa ttatgaagct aaaaaagaaa 1020
ctaaataaaa acaggtaata tagattacag ttttaagctt gttttcccta tagactagag 1080
taaatatata aatatacctg tcaagggctt ataagcccct ttagggggtg cgtagcaccc 1140
ttgacaggta tatttatata ttttagggtg ccattaaggg aaacaagctt taaaatgcct 1200
ttaaaggcat tttaaaataa ataaaaaaaa gatggttttt accatctttt ttaactcccg 1260
aaagggagtt ctttcttttc ttgatactat acgtaactat ttcgatttgc cctgaaccta 1320
atcaaagcta gataaattca gtattagggc ataaaaaaac ttgctttttc gggtggaaat 1380
ctgtataatt taaattgctt agataaaaat taccaattcc atacgaaagg agcaagtttt 1440
acataaggtt aaagccttat gtgaattctc atttaattac atgaataata ataacacaga 1500
aagtgaagaa ttaaaagagc aaagtcaact attgcttgac aaatgcacaa aaaagaaaaa 1560
gaaaaatcct aaatttagta gttatataga accattagta agcaagaaat tatctgaaag 1620
aataaaggaa tgtggtgact ttttgcagat gttatctgat ttaaaccttg aaaattcgaa 1680
actgcataga gcaagttttt gtggtaacag attttgtcct atgtgtagct ggcgtattgc 1740
ttgtaaggat agtttggaaa tatctattct catggagcat ttacgcaaag aggaaagcaa 1800
agaatttatc tttttgacct taacaactcc aaatgtgaaa ggtgcggacc ttgataattc 1860
cataaaagca tacaataaag catttaaaaa gttaatggaa cgcaaagagg tcaagagcat 1920
agtaaaaggc tacataagaa agctagaagt aacctataat ttggacaaga gttccaaatc 1980
atataatact tatcacccac atttccatgt ggtactagca gtcaatagaa gttactttaa 2040
aaagcaaaat ctatatataa accatcatag atggcttagt ttgtggcaag agtcaactgg 2100
tgattattcg ataactcaag ttgatgtaag aaaggctaaa attaacgatt ataaagaggt 2160
ttatgagctt gctaagtatt cggctaagga ttccgactat ttaatcaata gagaagtgtt 2220
tacggtattc tacaaatctt taaagggtaa acaggtactt gtatttagtg gattatttaa 2280
agacgctcat aaaatgtata agaatggaga gctagatctg tataagaagt tggatactat 2340
cgaatatgct tatatggtaa gttataactg gcttaaaaag aagtatgata cttcaaatat 2400
tagagaatta actgaggaag aaaagcagaa attcaataaa aatttaatcg aagatgtgga 2460
tattgagtag gtgggattat atctcacctt ttttattgtc ttttcatgtt gaaattttga 2520
cgcttaatgc atgaagtatt gacaagttta aaaattacgg tttttaatcc ttagttgatt 2580
agcaggatta tggccggaat gctccgtcca gtcctgttaa ggaattaaaa ttccctaaaa 2640
cccttggcta tgatttatag cgagaatcgt caattaaaaa tttaataggt gctatgaaag 2700
tcgattaata attaatttta aaatgcaata tgaaacataa ttacaagaat ttgactttta 2760
atacaagaat tgatatcata gttacattaa tac 2793
<210> 128
<211> 329
<212> PRT
<213> 拜氏梭状芽孢杆菌（Clostridium beijerinckii）
<400> 128
Met Asn Asn Asn Asn Thr Glu Ser Glu Glu Leu Lys Glu Gln Ser Gln
1 5 10 15
Leu Leu Leu Asp Lys Cys Thr Lys Lys Lys Lys Lys Asn Pro Lys Phe
20 25 30
Ser Ser Tyr Ile Glu Pro Leu Val Ser Lys Lys Leu Ser Glu Arg Ile
35 40 45
Lys Glu Cys Gly Asp Phe Leu Gln Met Leu Ser Asp Leu Asn Leu Glu
50 55 60
Asn Ser Lys Leu His Arg Ala Ser Phe Cys Gly Asn Arg Phe Cys Pro
65 70 75 80
Met Cys Ser Trp Arg Ile Ala Cys Lys Asp Ser Leu Glu Ile Ser Ile
85 90 95
Leu Met Glu His Leu Arg Lys Glu Glu Ser Lys Glu Phe Ile Phe Leu
100 105 110
Thr Leu Thr Thr Pro Asn Val Lys Gly Ala Asp Leu Asp Asn Ser Ile
115 120 125
Lys Ala Tyr Asn Lys Ala Phe Lys Lys Leu Met Glu Arg Lys Glu Val
130 135 140
Lys Ser Ile Val Lys Gly Tyr Ile Arg Lys Leu Glu Val Thr Tyr Asn
145 150 155 160
Leu Asp Lys Ser Ser Lys Ser Tyr Asn Thr Tyr His Pro His Phe His
165 170 175
Val Val Leu Ala Val Asn Arg Ser Tyr Phe Lys Lys Gln Asn Leu Tyr
180 185 190
Ile Asn His His Arg Trp Leu Ser Leu Trp Gln Glu Ser Thr Gly Asp
195 200 205
Tyr Ser Ile Thr Gln Val Asp Val Arg Lys Ala Lys Ile Asn Asp Tyr
210 215 220
Lys Glu Val Tyr Glu Leu Ala Lys Tyr Ser Ala Lys Asp Ser Asp Tyr
225 230 235 240
Leu Ile Asn Arg Glu Val Phe Thr Val Phe Tyr Lys Ser Leu Lys Gly
245 250 255
Lys Gln Val Leu Val Phe Ser Gly Leu Phe Lys Asp Ala His Lys Met
260 265 270
Tyr Lys Asn Gly Glu Leu Asp Leu Tyr Lys Lys Leu Asp Thr Ile Glu
275 280 285
Tyr Ala Tyr Met Val Ser Tyr Asn Trp Leu Lys Lys Lys Tyr Asp Thr
290 295 300
Ser Asn Ile Arg Glu Leu Thr Glu Glu Glu Lys Gln Lys Phe Asn Lys
305 310 315 320
Asn Leu Ile Glu Asp Val Asp Ile Glu
325
<210> 129
<211> 256
<212> PRT
<213> 人工序列
<220>
<223> 共有COG5655
<400> 129
Met Cys Gln Lys Arg Ser Asp Tyr Ser Asp Glu Lys Ala Trp Leu Lys
1 5 10 15
Asp Lys Ser Lys Asp Gly Lys Val Glu Pro Trp Arg Glu Lys Lys Glu
20 25 30
Ala Asn Val Lys Tyr Phe Glu Leu Leu Lys Ile Leu Met Phe Lys Lys
35 40 45
Ala Glu Arg Val Tyr Arg Cys Asn Glu Leu Leu Glu Leu Gln Lys Val
50 55 60
Asn Glu Thr Gly Glu Asn Lys Leu Cys Pro Asn Trp Phe Cys Lys Ser
65 70 75 80
Leu Leu Cys Pro Met Cys Asn Trp Arg Lys Pro Met Lys Ser Asp Leu
85 90 95
Gln Asp Gly Leu Tyr Val Lys Arg Val Ile Ser Tyr Gly Pro Leu Leu
100 105 110
Lys Trp Lys His Leu Lys Leu Asn Leu Lys Asn Val Glu Asp Gly Asp
115 120 125
Leu Leu Asn Lys Ser Leu Asp Glu Met Ala Leu Gly Phe Lys Arg Thr
130 135 140
Met Gly Phe Lys Lys Ile Ala Lys Asn Phe Val Gly Phe Met Lys Ser
145 150 155 160
Thr Glu Ile Thr Tyr Asn Glu Lys Asp Asn Ser Tyr Asn Gln His Met
165 170 175
His Val Leu Phe Cys Ser Glu Gln Thr Tyr Phe Lys Asn Phe Ile Asn
180 185 190
Asn Thr Pro Gln Glu Phe Trp Asn Lys Arg Trp Ser Lys Ala Met Lys
195 200 205
Leu Asp Tyr Asp Pro Gln Val Met Lys Leu Trp Thr Met Tyr Lys Lys
210 215 220
Glu Ile Lys Asn Tyr Ile Gln Thr Ala Leu Gln Glu Thr Ala Lys Tyr
225 230 235 240
Asp Val Lys Asp Met Asp Ser Ala Thr Ile Asp Asp Glu Lys Ser Leu
245 250 255
<210> 130
<211> 768
<212> DNA
<213> 粪肠球菌（Enterococcus faecalis）
<400> 130
gtgaggagga tatatttgaa tacatacgaa caaattaata aagtgaaaaa aatacttcgg 60
aaacatttaa aaaataacct tattggtact tacatgtttg gatcaggagt tgagagtgga 120
ctaaaaccaa atagtgatct tgacttttta gtcgtcgtat ctgaaccatt gacagatcaa 180
agtaaagaaa tacttataca aaaaattaga cctatttcaa agaaaatagg agataaaagc 240
aacttacgat atattgaatt aacaattatt attcagcaag aaatggtacc gtggaatcat 300
cctcccaaac aagaatttat ttatggagaa tggttacaag agctttatga acaaggatac 360
attcctcaga aggaattaaa ttcagattta accataatgc tttaccaagc aaaacgaaaa 420
aataaaagaa tatacggaaa ttatgactta gaggaattac tacctgatat tccattttct 480
gatgtgagaa gagccattat ggattcgtca gaggaattaa tagataatta tcaggatgat 540
gaaaccaact ctatattaac tttatgccgt atgattttaa ctatggacac gggtaaaatc 600
ataccaaaag atattgcggg aaatgcagtg gctgaatctt ctccattaga acatagggag 660
agaattttgt tagcagttcg tagttatctt ggagagaata ttgaatggac taatgaaaat 720
gtaaatttaa ctataaacta tttaaataac agattaaaaa aattataa 768
<210> 131
<211> 738
<212> DNA
<213> 难辨梭状芽孢杆菌（Clostridium difficile）
<400> 131
atgaacaaaa atataaaata ttctcaaaac tttttaacga gtgaaaaagt actcaaccaa 60
ataataaaac aattgaattt aaaagaaacc gataccgttt acgaaattgg aacaggtaaa 120
gggcatttaa cgacgaaact ggctaaaata agtaaacagg taacgtctat tgaattagac 180
agtcatctat tcaacttatc gtcagaaaaa ttaaaactga atactcgtgt cactttaatt 240
caccaagata ttctacagtt tcaattccct aacaaacaga ggtataaaat tgttgggagt 300
attccttacc atttaagcac acaaattatt aaaaaagtgg tttttgaaag ccatgcgtct 360
gacatctatc tgattgttga agaaggattc tacaagcgta ccttggatat tcaccgaaca 420
ctagggttgc tcttgcacac tcaagtctcg attcagcaat tgcttaagct gccagcggaa 480
tgctttcatc ctaaaccaaa agtaaacagt gtcttaataa aacttacccg ccataccaca 540
gatgttccag ataaatattg gaagctatat acgtactttg tttcaaaatg ggtcaatcga 600
gaatatcgtc aactgtttac taaaaatcag tttcatcaag caatgaaaca cgccaaagta 660
aacaatttaa gtaccgttac ttatgagcaa gtattgtcta tttttaatag ttatctatta 720
tttaacggga ggaaataa 738
<210> 132
<211> 3792
<212> DNA
<213> 人工序列
<220>
<223> 用于枯草芽孢杆菌（B. subtilis）的优化的Mad7 CDS
<400> 132
atgaacaacg gcacaaataa ttttcagaac tttattggca tttcatcatt gcagaaaacg 60
ttaagaaatg ctttaattcc gacggaaaca acgcaacagt ttattgttaa aaacggaatt 120
attaaagaag atgaattaag aggcgaaaac agacagattt taaaagatat tatggatgac 180
tactacagag gatttatttc tgaaacatta tcatctattg atgacattga ttggacaagc 240
ttatttgaaa aaatggaaat tcagttaaaa aatggtgata ataaagatac attaattaaa 300
gaacagacag aatatagaaa agcaattcat aaaaaatttg cgaacgacga tagatttaaa 360
aacatgttta gcgccaaatt aatttcagac attttacctg aatttgttat tcataacaat 420
aattattcag catcagaaaa agaagaaaaa acacaggtga ttaaattgtt ttcaagattt 480
gcgacaagct ttaaagatta ctttaaaaac agagcaaatt gcttttcagc ggacgatatt 540
tcatcaagca gctgccatag aattgttaac gacaatgcag aaattttttt ttcaaatgcg 600
ttagtttaca gaagaattgt aaaatcatta agcaatgacg atattaacaa aatttcaggc 660
gatatgaaag attcattaaa agaaatgtca ttagaagaaa tttattctta cgaaaaatat 720
ggcgaattta ttacacagga aggcattagc ttttataatg atatttgtgg caaagtgaat 780
tcttttatga acttatattg tcagaaaaat aaagaaaaca aaaatttata caaacttcag 840
aaacttcata aacagattct gtgcattgcg gacacaagct atgaagttcc gtataaattt 900
gaatcagacg aagaagtgta ccaatcagtt aacggctttc ttgataacat tagcagcaaa 960
catattgttg aaagattaag aaaaattggc gataactata acggctacaa cttagataaa 1020
atttatattg tgtccaaatt ttacgaaagc gttagccaaa aaacatacag agactgggaa 1080
acaattaata cagccttaga aattcattac aataatattt tgccgggtaa cggtaaatca 1140
aaagccgaca aagtaaaaaa agcggttaaa aatgatttac agaaatccat tacagaaatt 1200
aatgaactgg tgtcaaacta taaattatgc tcagacgaca acattaaagc ggaaacatat 1260
attcatgaaa ttagccatat tttgaataac tttgaagcac aggaattgaa atacaatccg 1320
gaaattcatc tggttgaatc cgaattaaaa gcgtcagaac ttaaaaacgt gttagacgtg 1380
attatgaatg cgtttcattg gtgttcagtt tttatgacag aagaacttgt tgataaagac 1440
aacaattttt atgcggaatt agaagaaatt tacgatgaaa tttatccggt aatttcatta 1500
tacaacttag ttagaaacta cgttacacag aaaccgtaca gcacgaaaaa aattaaattg 1560
aactttggaa ttccgacgtt agcagacggt tggtcaaaat ccaaagaata ttctaataac 1620
gctattattt taatgagaga caatttatat tatttaggca tttttaatgc gaaaaataaa 1680
ccggacaaaa aaattattga aggtaatacg tcagaaaata aaggtgacta caaaaaaatg 1740
atttataatt tgttaccggg tccgaacaaa atgattccga aagttttttt gagcagcaaa 1800
acgggcgtgg aaacgtataa accgagcgcc tatattctgg aaggctataa acagaataaa 1860
catattaaat cttcaaaaga ctttgatatt acattttgtc atgatttaat tgactacttt 1920
aaaaactgta ttgcaattca tccggaatgg aaaaactttg gttttgattt tagcgacaca 1980
tcaacatatg aagacatttc cggcttttat agagaagtag aattacaagg ttacaaaatt 2040
gattggacat acattagcga aaaagacatt gatttattac aggaaaaagg tcaattatat 2100
ttatttcaga tttataacaa agatttttca aaaaaatcaa caggcaatga caaccttcat 2160
acaatgtact taaaaaatct tttttcagaa gaaaatctta aagatattgt tttaaaactt 2220
aacggcgaag cggaaatttt ttttagaaaa agcagcatta aaaacccgat tattcataaa 2280
aaaggctcaa ttttagttaa cagaacatac gaagcagaag aaaaagacca gtttggcaac 2340
attcaaattg tgagaaaaaa tattccggaa aacatttatc aggaattata caaatacttt 2400
aacgataaaa gcgacaaaga attatctgat gaagcagcca aattaaaaaa tgtagtggga 2460
catcatgaag cagcgacgaa tattgttaaa gactatagat acacgtatga taaatacttt 2520
cttcatatgc ctattacgat taattttaaa gccaataaaa cgggttttat taatgataga 2580
attttacagt atattgctaa agaaaaagac ttacatgtga ttggcattga tagaggcgaa 2640
agaaacttaa tttacgtgtc cgtgattgat acatgtggta atattgttga acagaaaagc 2700
tttaacattg taaacggcta cgactatcag attaaattaa aacaacagga aggcgctaga 2760
cagattgcga gaaaagaatg gaaagaaatt ggtaaaatta aagaaattaa agaaggctac 2820
ttaagcttag taattcatga aatttctaaa atggtaatta aatacaatgc aattattgcg 2880
atggaagatt tgtcttatgg ttttaaaaaa ggcagattta aagttgaaag acaagtttac 2940
cagaaatttg aaacaatgtt aattaataaa ttaaactatt tagtatttaa agatatttca 3000
attacagaaa atggcggttt attaaaaggt tatcagttaa catacattcc tgataaactt 3060
aaaaacgtgg gtcatcagtg cggctgcatt ttttatgtgc ctgctgcata cacgagcaaa 3120
attgatccga caacaggctt tgtgaatatt tttaaattta aagacttaac agtggacgca 3180
aaaagagaat ttattaaaaa atttgactca attagatatg actcagaaaa aaatttattt 3240
tgctttacat ttgactacaa taactttatt acgcaaaaca cggttatgag caaatcatca 3300
tggtcagtgt atacatacgg cgtgagaatt aaaagaagat ttgtgaacgg cagattttca 3360
aacgaatcag atacaattga cattacaaaa gatatggaaa aaacgttgga aatgacggac 3420
attaactgga gagatggcca tgatcttaga caagacatta ttgattatga aattgttcag 3480
catatttttg aaatttttag attaacagtg caaatgagaa actccttgtc tgaattagaa 3540
gacagagatt acgatagatt aatttcacct gtattaaacg aaaataacat tttttatgac 3600
agcgcgaaag cgggcgatgc acttcctaaa gatgccgatg caaatggtgc gtattgtatt 3660
gcattaaaag gcttatatga aattaaacaa attacagaaa attggaaaga agatggtaaa 3720
ttttcaagag ataaattaaa aattagcaat aaagattggt ttgactttat tcagaataaa 3780
agatatttat aa 3792
<210> 133
<211> 10469
<212> DNA
<213> 人工序列
<220>
<223> pCas9cond
<400> 133
catggataaa aagtacagta ttggtctaga cataggaact aactctgttg ggtgggctgt 60
tataacagat gaatataaag ttccatcaaa aaaatttaaa gtattaggaa acactgatag 120
acattcaata aaaaaaaact tgataggtgc tttattattc gattcaggag agactgctga 180
agctacacgt ttaaaaagaa cagctagacg tagatataca agaagaaaaa ataggatatg 240
ttatcttcaa gaaattttta gtaatgaaat ggcaaaagtt gatgattcat tctttcacag 300
actagaagaa agtttcttag ttgaagaaga taagaagcat gaaagacacc ctatttttgg 360
taatatcgta gatgaagtag catatcatga gaagtatcca actatctatc atttaagaaa 420
gaaattagtt gattctacag ataaagctga tctgagatta atatatttag ctttagctca 480
tatgattaaa tttagaggac attttttaat agaaggtgat ttaaacccag acaacagcga 540
tgtagataaa ttatttatcc aattagttca aacttataat caattattcg aagagaatcc 600
aattaatgca agtggtgtag acgctaaggc tatattatca gctagattat caaaatctag 660
aagattagaa aatctaatag ctcaacttcc tggagaaaag aaaaatggac tttttgggaa 720
cctaatagct ctctcactcg gactaacacc aaattttaaa agcaattttg atcttgctga 780
agacgcaaag ttacaactat caaaggatac atacgatgat gatttagata atttgttagc 840
tcaaataggt gatcaatatg ctgatttgtt tcttgcagca aaaaacttaa gtgatgcaat 900
tttactatca gatatactta gagtaaatac agaaataaca aaggctcctt tatcagcaag 960
tatgattaaa cgatatgatg agcatcatca agatttaaca ttattaaagg cacttgtaag 1020
acaacaatta ccagaaaaat ataaagaaat tttctttgat caatctaaaa atggatatgc 1080
tggatatata gacggtggag caagtcaaga agagttttat aaatttataa agcctatttt 1140
agaaaaaatg gatggaactg aagaattact tgttaaactt aacagagaag atttacttag 1200
aaaacaaaga acttttgata atggttcaat tcctcaccaa attcatttag gagaattaca 1260
tgctatacta agaagacaag aagattttta tccatttctt aaagataata gagaaaaaat 1320
tgaaaaaatt ttaactttta gaataccata ttatgtagga ccacttgcaa ggggaaattc 1380
aagatttgca tggatgacta gaaaatcaga agaaactata accccgtgga attttgaaga 1440
agtagtagat aaaggagcta gtgctcaatc atttatagaa agaatgacaa attttgataa 1500
gaatcttcct aacgaaaagg ttttgccaaa gcatagcctt ctttatgagt attttacagt 1560
ttataatgag cttactaaag taaaatacgt tacagaagga atgagaaaac cagcattttt 1620
gtctggtgaa caaaagaaag caatagtaga cctattattt aaaacaaata ggaaggttac 1680
cgtaaagcaa cttaaagaag attacttcaa aaaaattgaa tgctttgata gtgttgaaat 1740
atcaggagtt gaagatagat ttaatgcttc acttggtaca tatcacgatc tcttaaaaat 1800
tataaaagat aaggattttt tagataatga agaaaatgaa gatattcttg aagatatagt 1860
attaacattg acactttttg aagatagaga aatgatagaa gaaagattaa aaacatatgc 1920
acatcttttt gatgataagg ttatgaagca acttaaaaga agaagatata caggttgggg 1980
acgtttgtca agaaagctaa ttaatggtat tagagataaa caatcaggaa agactattct 2040
cgattttctt aaatcagatg gatttgctaa tagaaacttt atgcaattaa ttcatgatga 2100
ttctcttact ttcaaagagg atattcaaaa ggctcaagtt tctggacaag gcgatagctt 2160
acacgaacac attgctaacc ttgcagggag ccccgctatc aaaaaaggaa ttttacaaac 2220
agttaaagtt gtagatgaac ttgttaaagt tatgggaaga cacaaacctg agaatatagt 2280
tatagaaatg gccagagaaa atcaaacaac acaaaaagga caaaaaaatt ctagagagag 2340
aatgaagaga attgaagaag gaataaaaga gctaggatca caaatattaa aagaacatcc 2400
agttgaaaat actcaattgc aaaatgaaaa gttatatttg tattacttac aaaatggaag 2460
agatatgtat gttgatcaag aactcgatat taatagatta agtgactatg atgttgatca 2520
tattgttcct caatcatttt taaaagatga ttcaatcgat aacaaagtat taactagatc 2580
agataaaaat agaggaaagt cagataatgt accatctgaa gaagttgtta aaaaaatgaa 2640
gaactattgg agacaacttt taaatgcaaa gctaattaca caaagaaaat ttgacaattt 2700
aacaaaagca gaaagaggag gattaagcga attagacaaa gctggattta taaaaagaca 2760
acttgttgag acaagacaaa taactaagca tgttgctcaa atacttgatt caagaatgaa 2820
tacaaaatat gatgaaaatg ataaattaat cagagaagta aaagtaataa cattaaagtc 2880
aaaattagta tcagatttca gaaaggattt tcaattttac aaagttcgtg aaataaataa 2940
ctatcatcat gctcatgatg catacttaaa tgctgttgta ggaactgctc ttattaagaa 3000
atatcctaaa ctagaaagcg aatttgttta tggagattat aaagtttatg atgtgcgcaa 3060
aatgatcgcg aaatccgaac aagaaatcgg taaggctaca gcaaaatatt tcttttatag 3120
taatataatg aattttttta agacagaaat aactttggct aatggtgaaa tcagaaaaag 3180
accacttatc gaaacaaatg gagagacagg agaaatagta tgggataaag gaagagattt 3240
tgctactgtt agaaaagtac taagtatgcc acaagtaaat atcgtaaaga aaactgaagt 3300
tcaaactgga ggtttctcta aggaatcaat tttacctaag agaaattcag ataagttaat 3360
tgcaaggaaa aaagattggg acccaaaaaa atacggtggt tttgatagtc caacagttgc 3420
ctatagtgtt cttgtagtag cgaaagttga gaaaggtaag tcaaaaaagt tgaaaagcgt 3480
aaaagaactt cttggtatca caattatgga aagatcttca tttgaaaaaa atccaattga 3540
ctttttagaa gctaagggtt ataaagaagt taaaaaggat ttaatcataa aactaccaaa 3600
gtatagtcta tttgaactcg aaaacggaag aaaacgaatg ctcgctagcg caggagaact 3660
tcaaaaagga aatgaacttg cgctgccatc aaagtatgta aatttcttat atttagcttc 3720
tcattatgag aaattaaaag gatcaccaga ggataatgaa caaaagcaac tatttgtaga 3780
acaacacaaa cattatttag atgaaataat agaacaaata tctgaatttt ctaaaagagt 3840
tatacttgcc gacgcaaatc tagataaggt gctttcagcg tataataaac acagagataa 3900
accaataaga gaacaagcag aaaacattat ccatcttttt acattaacta atcttggtgc 3960
accagctgca tttaagtact ttgatacaac aatagataga aaaagataca catctactaa 4020
agaagtatta gacgcaactt taatacatca atctattaca gggctttatg aaacaagaat 4080
tgatttaagt caactaggcg gagattaagt cgacaaagta ttgttaaaaa taactctgta 4140
gaattataaa ttagttctac agagttattt tttgacccgg gtaccgagct cgaattcgta 4200
atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat 4260
acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt 4320
aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta 4380
atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc 4440
gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa 4500
ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa 4560
aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct 4620
ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac 4680
aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc 4740
gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc 4800
tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg 4860
tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga 4920
gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag 4980
cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta 5040
cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag 5100
agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg 5160
caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac 5220
ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc 5280
aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag 5340
tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc 5400
agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac 5460
gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc 5520
accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg 5580
tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag 5640
tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc 5700
acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac 5760
atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag 5820
aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac 5880
tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg 5940
agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc 6000
gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact 6060
ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg 6120
atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa 6180
tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt 6240
tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg 6300
tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga 6360
ctgccgggcc tcttgcggga tcaaaagaaa aacgaaatga tacaccaatc agtgcaaaaa 6420
aagatataat gggagataag acggttcgtg ttcgtgctga cttgcaccat atcataaaaa 6480
tcgaaacagc aaagaatggc ggaaacgtaa aagaagttat ggaaataaga cttagaagca 6540
aacttaagag tgtgttgata gtgcagtatc ttaaaatttt gtataatagg aattgaagtt 6600
aaattagatg ctaaaaattt gtaattaaga aggagtgatt acatgaacaa aaatataaaa 6660
tattctcaaa actttttaac gagtgaaaaa gtactcaacc aaataataaa acaattgaat 6720
ttaaaagaaa ccgataccgt ttacgaaatt ggaacaggta aagggcattt aacgacgaaa 6780
ctggctaaaa taagtaaaca ggtaacgtct attgaattag acagtcatct attcaactta 6840
tcgtcagaaa aattaaaact gaatactcgt gtcactttaa ttcaccaaga tattctacag 6900
tttcaattcc ctaacaaaca gaggtataaa attgttggga gtattcctta ccatttaagc 6960
acacaaatta ttaaaaaagt ggtttttgaa agccatgcgt ctgacatcta tctgattgtt 7020
gaagaaggat tctacaagcg taccttggat attcaccgaa cactagggtt gctcttgcac 7080
actcaagtct cgattcagca attgcttaag ctgccagcgg aatgctttca tcctaaacca 7140
aaagtaaaca gtgtcttaat aaaacttacc cgccatacca cagatgttcc agataaatat 7200
tggaagctat atacgtactt tgtttcaaaa tgggtcaatc gagaatatcg tcaactgttt 7260
actaaaaatc agtttcatca agcaatgaaa cacgccaaag taaacaattt aagtaccgtt 7320
acttatgagc aagtattgtc tatttttaat agttatctat tatttaacgg gaggaaataa 7380
ttctatgagt ccctaggccc aactaactca acgctagtag tggatttaat cccaaatgag 7440
ccaacagaac cagaaccaga aacagaatca gaacaagtaa cattggattt agaaatggaa 7500
gaagaaaaaa gcaatgactt cgtgtgaata atgcacgaaa tcgttgctta ttttttttta 7560
aaagcggtat actagatata acgaaacaac gaactgaata gaaacgaaaa aagagccatg 7620
acacatttat aaaatgtttg acgacatttt ataaatgcat agcccgataa gattgccaaa 7680
ccaacgctta tcagttagtc agatgaactc ttccctcgta agaagttatt taattaactt 7740
tgtttgaaga cggtatataa ccgtactatc attatatagg gaaatcagag agttttcaag 7800
tatctaagct actgaattta agaattgtta agcaatcaat cggaaatcgt ttgattgctt 7860
tttttgtatt catttataga aggtggagtt tgtatgaatc atgatgaatg taaaacttat 7920
ataaaaaata gtttattgga gataagaaaa ttagcaaata tctatacact agaaacgttt 7980
aagaaagagt tagaaaagag aaatatctac ttagaaacaa aatcagataa gtatttttct 8040
tcggaggggg aagattatat atataagtta atagaaaata acaaaataat ttattcgatt 8100
agtggaaaaa aattgactta taaaggaaaa aaatcttttt caaaacatgc aatattgaaa 8160
cagttgaatg aaaaagcaaa ccaagttaat taaacaacct attttatagg atttatagga 8220
aaggagaaca gctgaatgaa tatccctttt gttgtagaaa ctgtgcttca tgacggcttg 8280
ttaaagtaca aatttaaaaa tagtaaaatt cgctcaatca ctaccaagcc aggtaaaagc 8340
aaaggggcta tttttgcgta tcgctcaaaa tcaagcatga ttggcggtcg tggtgttgtt 8400
ctgacttccg aggaagcgat tcaagaaaat caagatacat ttacacattg gacacccaac 8460
gtttatcgtt atggaacgta tgcagacgaa aaccgttcat acacgaaagg acattctgaa 8520
aacaatttaa gacaaatcaa taccttcttt attgattttg atattcacac ggcaaaagaa 8580
actatttcag caagcgatat tttaacaacc gctattgatt taggttttat gcctactatg 8640
attatcaaat ctgataaagg ttatcaagca tattttgttt tagaaacgcc agtctatgtg 8700
acttcaaaat cagaatttaa atctgtcaaa gcagccaaaa taatttcgca aaatatccga 8760
gaatattttg gaaagtcttt gccagttgat ctaacgtgta atcattttgg tattgctcgc 8820
ataccaagaa cggacaatgt agaatttttt gatcctaatt accgttattc tttcaaagaa 8880
tggcaagatt ggtctttcaa acaaacagat aataagggct ttactcgttc aagtctaacg 8940
gttttaagcg gtacagaagg caaaaaacaa gtagatgaac cctggtttaa tctcttattg 9000
cacgaaacga aattttcagg agaaaagggt ttaatagggc gtaataacgt catgtttacc 9060
ctctctttag cctactttag ttcaggctat tcaatcgaaa cgtgcgaata taatatgttt 9120
gagtttaata atcgattaga tcaaccctta gaagaaaaag aagtaatcaa aattgttaga 9180
agtgcctatt cagaaaacta tcaaggggct aatagggaat acattaccat tctttgcaaa 9240
gcttgggtat caagtgattt aaccagtaaa gatttatttg tccgtcaagg gtggtttaaa 9300
ttcaagaaaa aaagaagcga acgtcaacgt gttcatttgt cagaatggaa agaagattta 9360
atggcttata ttagcgaaaa aagcgatgta tacaagcctt atttagtgac gaccaaaaaa 9420
gagattagag aagtgctagg cattcctgaa cggacattag ataaattgct gaaggtactg 9480
aaggcgaatc aggaaatttt ctttaagatt aaaccaggaa gaaatggtgg cattcaactt 9540
gctagtgtta aatcattgtt gctatcgatc attaaagtaa aaaaagaaga aaaagaaagc 9600
tatataaagg cgctgacaaa ttcttttgac ttagagcata cattcattca agagacttta 9660
aacaagctag cagaacgccc taaaacggac acacaactcg atttgtttag ctatgataca 9720
ggctgaaaat aaaacccgca ctatgccatt acatttatat ctatgatacg tgtttgtttt 9780
ttctttgctg tttagcgaat gattagcaga aatatacaga gtaagatttt aattaattat 9840
tagggggaga aggagagagt agcccgaaaa cttttagttg gcttggactg aacgaagtga 9900
gggaaaggct actaaaacgt cgaggggcag tgagagcgaa gcgaacactt gattttttaa 9960
ttttctatct tttataggtc attagagtat acttatttgt cctataaact atttagcagc 10020
ataatagatt tattgaatag gtcatttaag ttgagcatat tagaggagga aaatcttgga 10080
gaaatatttg aagaacccga ttacatggat tggattagtt cttgtggtta cgtggttttt 10140
aactaaaagt agtgaatttt tgatttttgg tgtgtgtgtc ttgttgttag tatttgctag 10200
tcaaagtgat taaatagaat tctagcgcca ttcgccattc aggctgcgca actgttggga 10260
agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc 10320
aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta aaacgacggc 10380
cagtgccaag cttgcatgcc tgcaggcctc gagtatattg ataaaaataa taatagtggg 10440
tataattaag ttgttaggag gttagttac 10469
<210> 134
<211> 8559
<212> DNA
<213> 人工序列
<220>
<223> pMAD7
<400> 134
tcgagtccct atcagtgata gattgaaact ctatcattga tagagtataa tatctttgtt 60
cattagagcg ataaacttga atttgagagg gaacttagat gaacaacggc acaaataatt 120
ttcagaactt catagggata tcaagtttgc agaaaacgtt aagaaatgct ttaataccca 180
cggaaaccac gcaacagttc atagttaaga acggaataat taaagaagat gagttaagag 240
gcgagaacag acagatttta aaagatataa tggatgacta ctacagagga ttcatatctg 300
agactttaag ttctattgat gacatagatt ggactagctt attcgaaaaa atggaaattc 360
agttaaaaaa tggtgataat aaagatacct taattaagga acagacagag tatagaaaag 420
caatacataa aaaatttgcg aacgacgata gatttaagaa catgtttagc gccaaattaa 480
ttagtgacat attacctgaa tttgttatac acaacaataa ttattcggca tcagagaaag 540
aggaaaaaac ccaggtgata aaattgtttt cgagatttgc gactagcttt aaagattact 600
tcaagaacag agcaaattgc ttttcagcgg acgatatttc atcaagcagc tgccatagaa 660
tagttaacga caatgcagag atattctttt caaatgcgtt agtttacaga agaatagtaa 720
aatcgttaag caatgacgat ataaacaaaa tttcgggcga tatgaaagat tcattaaaag 780
aaatgagttt agaagaaata tattcttacg agaagtatgg ggaatttatt acccaggaag 840
gcattagctt ctataatgat atatgtggga aagtgaattc ttttatgaac ttatattgtc 900
agaaaaataa agaaaacaaa aatttataca aacttcagaa acttcacaaa cagattctat 960
gcattgcgga cactagctat gaggttccgt ataaatttga aagtgacgag gaagtgtacc 1020
aatcagttaa cggcttcctt gataacatta gcagcaaaca tatagttgaa agattaagaa 1080
aaataggcga taactataac ggctacaact tagataaaat ttatatagtg tccaaatttt 1140
acgagagcgt tagccaaaaa acctacagag actgggaaac aattaatacc gccttagaaa 1200
ttcattacaa taatatattg ccgggtaacg gtaaaagtaa agccgacaaa gtaaaaaaag 1260
cggttaagaa tgatttacag aaatccataa ccgaaataaa tgaactagtg tcaaactata 1320
agttatgcag tgacgacaac ataaaagcgg agacttatat acatgagatt agccatatat 1380
tgaataactt tgaagcacag gaattgaaat acaatccgga aattcaccta gttgaatccg 1440
agttaaaagc gagtgagctt aaaaacgtgt tagacgtgat aatgaatgcg tttcattggt 1500
gttcggtttt tatgactgag gaacttgttg ataaagacaa caatttttat gcggaattag 1560
aggagattta cgatgaaatt tatccagtaa ttagtttata caacttagtt agaaactacg 1620
ttacccagaa accgtacagc acgaaaaaga ttaaattgaa ctttggaata ccgacgttag 1680
cagacggttg gtcaaagtcc aaagagtatt ctaataacgc tataatatta atgagagaca 1740
atttatatta tttaggcata tttaatgcga agaataaacc ggacaagaag attatagagg 1800
gtaatacgtc agaaaataag ggtgactaca aaaagatgat ttataatttg ttaccgggtc 1860
ccaacaaaat gataccgaaa gttttcttga gcagcaagac gggggtggaa acgtataaac 1920
cgagcgccta tatactagag gggtataaac agaataaaca tataaagtct tcaaaagact 1980
ttgatataac tttctgtcat gatttaatag actacttcaa aaactgtatt gcaattcatc 2040
ccgagtggaa aaacttcggt tttgatttta gcgacaccag tacttatgaa gacatttccg 2100
ggttttatag agaggtagag ttacaaggtt acaagattga ttggacatac attagcgaaa 2160
aagacattga tttattacag gaaaaaggtc aattatattt attccagata tataacaaag 2220
atttttcgaa aaaatcaacc gggaatgaca accttcacac catgtactta aaaaatcttt 2280
tctcagaaga aaatcttaag gatatagttt taaaacttaa cggcgaagcg gaaatattct 2340
tcaggaagag cagcataaag aacccaataa ttcataaaaa aggctcgatt ttagttaaca 2400
gaacctacga agcagaagaa aaagaccagt ttggcaacat tcaaattgtg agaaaaaata 2460
ttccggaaaa catttatcag gagttataca aatacttcaa cgataaaagc gacaaagagt 2520
tatctgatga agcagccaaa ttaaagaatg tagtgggaca ccacgaggca gcgacgaata 2580
tagttaagga ctatagatac acgtatgata aatacttcct tcatatgcct attacgataa 2640
atttcaaagc caataaaacg ggttttatta atgataggat attacagtat atagctaaag 2700
aaaaagactt acatgtgata ggcattgata gaggcgagag aaacttaata tacgtgtccg 2760
tgattgatac ttgtggtaat atagttgaac agaaaagctt taacattgta aacggctacg 2820
actatcagat aaaattaaaa caacaggagg gcgctagaca gattgcgaga aaagaatgga 2880
aagaaattgg taaaattaaa gagataaaag agggctactt aagcttagta atacacgaga 2940
tatctaaaat ggtaataaaa tacaatgcaa ttatagcgat ggaggatttg tcttatggtt 3000
ttaaaaaagg gagatttaag gttgaaagac aagtttacca gaaatttgaa accatgttaa 3060
taaataaatt aaactattta gtatttaaag atatttcgat taccgagaat ggcggtttat 3120
taaaaggtta tcagttaaca tacattcctg ataaacttaa aaacgtgggt catcagtgcg 3180
gctgcatttt ttatgtgcct gctgcataca cgagcaaaat tgatccgacc accggctttg 3240
tgaatatatt taaatttaaa gacttaacag tggacgcaaa aagagaattc attaaaaaat 3300
ttgactcaat tagatatgac agtgaaaaaa atttattctg ctttacattt gactacaata 3360
actttattac gcaaaacacg gttatgagca aatcatcgtg gagtgtgtat acatacggcg 3420
tgagaataaa aagaagattt gtgaacggca gattctcaaa cgaaagtgat accattgaca 3480
taaccaaaga tatggagaaa acgttggaaa tgacggacat taactggaga gatggccacg 3540
atcttagaca agacattata gattatgaaa ttgttcagca catattcgaa attttcagat 3600
taacagtgca aatgagaaac tccttgtctg aattagagga cagagattac gatagattaa 3660
tttcacctgt attaaacgaa aataacattt tttatgacag cgcgaaagcg ggggatgcac 3720
ttcctaagga tgccgatgca aatggtgcgt attgtattgc attaaaaggg ttatatgaaa 3780
ttaaacaaat taccgaaaat tggaaagaag atggtaaatt ttcgagagat aaattaaaaa 3840
taagcaataa agattggttc gactttatac agaataagag atatttataa gtcgacaaag 3900
tattgttaaa aataactctg tagaattata aattagttct acagagttat tttttgaccc 3960
gggtatattg ataaaaataa taatagtggg tataattaag ttgttaggag gttagttaga 4020
atgatgtcaa gattagataa aagtaaagtg attaacagcg cattagagct gcttaatgag 4080
gtcggaatcg aaggtttaac aacccgtaaa ctcgcccaga agctaggtgt agagcagcct 4140
acattgtatt ggcatgtaaa aaataagcgg gctttgctcg acgccttagc cattgagatg 4200
ttagataggc accatactca cttttgccct ttagaagggg aaagctggca agatttttta 4260
cgtaataacg ctaaaagttt tagatgtgct ttactaagtc atcgcgatgg agcaaaagta 4320
catttaggta cacggcctac agaaaaacag tatgaaactc tcgaaaatca attagccttt 4380
ttatgccaac aaggtttttc actagagaat gcattatatg cactcagcgc tgtggggcat 4440
tttactttag gttgcgtatt ggaagatcaa gagcatcaag tcgctaaaga agaaagggaa 4500
acacctacta ctgatagtat gccgccatta ttacgacaag ctatcgaatt atttgatcac 4560
caaggtgcag agccagcctt cttattcggc cttgaattga tcatatgcgg attagaaaaa 4620
caacttaaat gtgaaagtgg gtcttaaaag cagcataacc tttttccgtg atggtaactt 4680
cacggtaacc aagatgtcga gttgagctcg aattcgtaat catggtcata gctgtttcct 4740
gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt 4800
aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc 4860
gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg 4920
agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg 4980
gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca 5040
gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac 5100
cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac 5160
aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg 5220
tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac 5280
ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat 5340
ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag 5400
cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac 5460
ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt 5520
gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt 5580
atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc 5640
aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga 5700
aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac 5760
gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc 5820
cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct 5880
gacagttacc aggtccactg ccgggcctct tgcgggatca aaagaaaaac gaaatgatac 5940
accaatcagt gcaaaaaaag atataatggg agataagacg gttcgtgttc gtgctgactt 6000
gcaccatatc ataaaaatcg aaacagcaaa gaatggcgga aacgtaaaag aagttatgga 6060
aataagactt agaagcaaac ttaagagtgt gttgatagtg cagtatctta aaattttgta 6120
taataggaat tgaagttaaa ttagatgcta aaaatttgta attaagaagg agtgattaca 6180
tgaacaaaaa tataaaatat tctcaaaact ttttaacgag tgaaaaagta ctcaaccaaa 6240
taataaaaca attgaattta aaagaaaccg ataccgttta cgaaattgga acaggtaaag 6300
ggcatttaac gacgaaactg gctaaaataa gtaaacaggt aacgtctatt gaattagaca 6360
gtcatctatt caacttatcg tcagaaaaat taaaactgaa tactcgtgtc actttaattc 6420
accaagatat tctacagttt caattcccta acaaacagag gtataaaatt gttgggagta 6480
ttccttacca tttaagcaca caaattatta aaaaagtggt ttttgaaagc catgcgtctg 6540
acatctatct gattgttgaa gaaggattct acaagcgtac cttggatatt caccgaacac 6600
tagggttgct cttgcacact caagtctcga ttcagcaatt gcttaagctg ccagcggaat 6660
gctttcatcc taaaccaaaa gtaaacagtg tcttaataaa acttacccgc cataccacag 6720
atgttccaga taaatattgg aagctatata cgtactttgt ttcaaaatgg gtcaatcgag 6780
aatatcgtca actgtttact aaaaatcagt ttcatcaagc aatgaaacac gccaaagtaa 6840
acaatttaag taccgttact tatgagcaag tattgtctat ttttaatagt tatctattat 6900
ttaacgggag gaaataattc tatgagtccc taggcaggcc tccgccatta tttttttgaa 6960
caattgacaa ttcatttctt attttttatt aagtgatagt caaaaggcat aacagtgctg 7020
aatagaaaga aatttacaga aaagaaaatt atagaattta gtatgattaa ttatactcat 7080
ttatgaatgt ttaattgaat acaaaaaaaa atacttgtta tgtattcaat tacgggttaa 7140
aatatagaca agttgaaaaa tttaataaaa aaataagtcc tcagctctta tatattaagc 7200
taccaactta gtatataagc caaaacttaa atgtgctacc aacacatcaa gccgttagag 7260
aactctatct atagcaatat ttcaaatgta ccgacataca agagaaacat taactatata 7320
tattcaattt atgagattat cttaacagat ataaatgtaa attgcaataa gtaagattta 7380
gaagtttata gcctttgtgt attggaagca gtacgcaaag gcttttttat ttgataaaaa 7440
ttagaagtat atttattttt tcataattaa tttatgaaaa tgaaaggggg tgagcaaagt 7500
gacagaggaa agcagtatct tatcaaataa caaggtatta gcaatatcat tattgacttt 7560
agcagtaaac attatgactt ttatagtgct tgtagctaag tagtacgaaa gggggagctt 7620
taaaaagctc cttggaatac atagaattca taaattaatt tatgaaaaga agggcgtata 7680
tgaaaacttg taaaaattgc aaagagttta ttaaagatac tgaaatatgc aaaatacatt 7740
cgttgatgat tcatgataaa acagtagcaa cctattgcag taaatacaat gagtcaagat 7800
gtttacataa agggaaagtc caatgtatta attgttcaaa gatgaaccga tatggatggt 7860
gtgccataaa aatgagatgt tttacagagg aagaacagaa aaaagaacgt acatgcatta 7920
aatattatgc aaggagcttt aaaaaagctc atgtaaagaa gagtaaaaag aaaaaataat 7980
ttatttatta atttaatatt gagagtgccg acacagtatg cactaaaaaa tatatctgtg 8040
gtgtagtgag ccgatacaaa aggatagtca ctcgcatttt cataatacat cttatgttat 8100
gattatgtgt cggtgggact tcacgacgaa aacccacaat aaaaaaagag ttcggggtag 8160
ggttaagcat agttgaggca actaaacaat caagctagga tatgcagtag cagaccgtaa 8220
ggtcgttgtt taggtgtgtt gtaatacata cgctattaag atgtaaaaat acggatacca 8280
atgaagggaa aagtataatt tttggatgta gtttgtttgt tcatctatgg gcaaactacg 8340
tccaaagccg tttccaaatc tgctaaaaag tatatccttt ctaaaatcaa agtcaagtat 8400
gaaatcataa ataaagttta attttgaagt tattatgata ttatgttttt ctattaaaat 8460
aaattaagta tatagaatag tttaataata gtatatactt aatgtgataa gtgtctgaca 8520
gtgtcacaga aaggatgatt gttatggatt ataagcggc 8559

Claims (14)

1.一种遗传工具，其允许对梭状芽孢杆菌属(Clostridium)的细菌进行转化和通过同源重组进行遗传修饰，其特征在于
i)它包含：
-至少编码Cas9的第一核酸，其中编码Cas9的序列被置于启动子的控制之下，和
-至少一种含有修复模板的第二核酸，其允许通过同源重组机制将被Cas9靶向的细菌DNA的一部分用感兴趣的序列代替，
ii)所述核酸中的至少一者还编码一个或多个指导RNA(gRNA)，或者所述遗传工具还包含一个或多个指导RNA，每个指导RNA包含结合Cas9酶的RNA结构和与所述细菌DNA的被靶向部分互补的序列，和
iii)所述核酸中的至少一者还包含置于诱导型启动子的控制之下的编码抗CRISPR蛋白的序列，或者所述遗传工具还包含置于诱导型启动子的控制之下的编码抗CRISPR蛋白的第三核酸。
2.权利要求1的工具，其特征在于所述梭状芽孢杆菌属(Clostridium)的细菌是选自以下的产溶剂细菌：丙酮丁醇梭状芽孢杆菌(C.acetobutylicum)、解纤维梭状芽孢杆菌(C.cellulolyticum)、植物发酵梭状芽孢杆菌(C.phytofermentans)、拜氏梭状芽孢杆菌(C.beijerinckii)、糖丁酸梭状芽孢杆菌(C.saccharobutylicum)、C.saccharoperbutylacetonicum、产芽孢梭状芽孢杆菌(C.sporogenes)、丁酸梭状芽孢杆菌(C.butyricum)、金黄丁酸梭状芽孢杆菌(C.aurantibutyricum)、酪丁酸梭状芽孢杆菌(C.tyrobutyricum)。
3.权利要求2的工具，其特征在于当所述细菌是丙酮丁醇梭状芽孢杆菌(C.acetobutylicum)时，所述丙酮丁醇梭状芽孢杆菌(C.acetobutylicum)细菌是菌株DSM792(也被称为ATCC 824或LMG 5710)，并且当所述产溶剂细菌是拜氏梭状芽孢杆菌(C.beijerinckii)时，所述拜氏梭状芽孢杆菌(C.beijerinckii)细菌是菌株NCIMB 8052或菌株DSM 6423(也被称为NRRL B–593、LMG 7814或LMG 7815)。
4.权利要求1至3任一项的工具，其特征在于所述编码抗CRISPR蛋白的序列是由所述第一核酸携带的。
5.权利要求1至4任一项的工具，其特征在于所述抗CRISPR蛋白是AcrIIA2蛋白或AcrIIA4蛋白。
6.权利要求1至5任一项的工具，其特征在于所述感兴趣的DNA序列的表达允许所述梭状芽孢杆菌属(Clostridium)的细菌发酵六碳糖和/或五碳糖中的至少两种不同的糖。
7.权利要求1至6任一项的工具，其特征在于所述感兴趣的序列编码至少一种促进梭状芽孢杆菌属(Clostridium)的细菌的溶剂生产的产物，例如至少一种参与醛向醇的转化的酶、膜蛋白、转录因子或其组合。
8.权利要求1至7任一项的工具，其特征在于所述工具内存在的每种核酸属于不同的表达盒或不同的载体例如质粒。
9.一种用于转化并通过同源重组遗传修饰梭状芽孢杆菌属(Clostridium)的细菌的方法，其特征在于它包含通过在所述细菌中引入权利要求1至8任一项的遗传工具来转化所述细菌的步骤。
10.权利要求9的方法，其特征在于它包含下述步骤：
a)在抗CRISPR蛋白的表达诱导物存在下，在所述细菌中引入权利要求1至8任一项的遗传工具，以及
b)将在步骤a)结束时获得的被转化的细菌在不含所述抗CRISPR蛋白的表达诱导物的培养基上培养。
11.权利要求9或10的方法，其特征在于它包含另外的步骤c)：除去在步骤a)期间随着所述遗传工具引入的含有修复模板的核酸和/或指导RNA或编码指导RNA的序列。
12.权利要求9至11任一项的方法，其特征在于它在步骤b)或步骤c)之后包含一个或多个其他步骤：在所述抗CRISPR蛋白的表达诱导物存在下引入第n核酸，所述第n核酸含有与已经引入的修复模板不同的修复模板和一个或多个用于指导RNA的表达盒，以允许将所述不同的修复模板中包含的感兴趣的序列整合到细菌基因组的被靶向区域中，在每个其他步骤之后跟随有将由此转化的细菌在不含所述抗CRISPR蛋白的表达诱导物的培养基上培养并允许Cas9/gRNA核糖核蛋白复合体表达的步骤。
13.一种试剂盒，其用于转化并优选地遗传修饰梭状芽孢杆菌属(Clostridium)的细菌或使用梭状芽孢杆菌属(Clostridium)的细菌生产至少一种溶剂，所述试剂盒包含如权利要求1至8任一项中所描述的遗传工具的元件，和适用于所述工具中使用的所选抗CRISPR蛋白的表达的诱导型启动子的至少一种诱导物。
14.权利要求1至8任一项的遗传工具、权利要求9至12任一项的方法或权利要求13的试剂盒的用途，其能够用于在工业规模上生产溶剂或溶剂的混合物，优选为丙酮、丁醇、乙醇、异丙醇或其混合物，通常为异丙醇/丁醇混合物。
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