CN107075511B - 合成子的形成 - Google Patents

合成子的形成 Download PDF

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CN107075511B
CN107075511B CN201580058526.8A CN201580058526A CN107075511B CN 107075511 B CN107075511 B CN 107075511B CN 201580058526 A CN201580058526 A CN 201580058526A CN 107075511 B CN107075511 B CN 107075511B
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P-C·赫塞
L·孙
T·C·小埃文斯
T·B·戴维斯
A·加德纳
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Abstract

除了其它，本公开提供一种组合物，其包括：5’核酸外切酶；链置换聚合酶；和任选地，单链DNA结合蛋白质和/或连接酶。另外还描述形成合成子的多核苷酸组装方法以及用于进行该方法的试剂盒。

Description

合成子的形成
背景技术
合成生物学依赖于由组分部分构建新的DNA的能力。双链(ds)DNA分子已通过在第一DNA双链的两端产生交错末端得以组装。使用限制性核酸内切酶、或者通过使用核酸外切酶消化、或者通过T4DNA聚合酶，然后杂交，以及任选地将第二DNA双链与第一双链连接，已经实现这一点。在反应混合物中使用核酸外切酶和连接酶的技术还指定使用非链置换聚合酶。
发明内容
除了其它，本公开提供一种组合物，其包括：i.5’-3’核酸外切酶；ii.链置换聚合酶；iii.单链(ss)DNA结合蛋白质；和iv.非天然存在的缓冲剂，其中组合物不包括拥挤剂和/或非链置换聚合酶。该组合物可以用于将多核苷酸组装到合成子(synthon)中。取决于出于在包含其自身连接酶的细菌细胞中克隆的目的进行组装，还是出于不包括细菌中克隆步骤的目的进行组装，组合物的实施方式任选地包含连接酶。
在先描述的组装方法需要非链置换聚合酶，并进一步额外地需要拥挤剂(crowding agent)(例如，参见US 8,968,999)。与现有技术相反，本发明的实施方式表明，当与5’-3’核酸外切酶一起使用时，链置换聚合酶相对于非链置换聚合酶具有优势。相对于包括拥挤剂，这一组合倾向于包括ss结合蛋白质，这与声称拥挤剂的使用比包括使用单链结合蛋白质的替代方案要有效4倍的现有技术教导相反。本发明的实施方式提供组合物、方法和试剂盒，其在单步方法中和/或在单一反应容器中提供由包括ds和/或ss核酸分子的寡核苷酸和多核苷酸来组装和克隆功能性基因和合成子的效率得以增加。这些实施方式不依赖于拥挤剂，也不需要非链置换聚合酶用于填充两分子退火之后存在的间隙。例如，当作用于ds DNA的5’-3’核酸外切酶产生可以与来自另一分子的3’ss DNA突出端有效退火的3’ssDNA突出端时，链置换聚合酶可以填充分子退火之后留下的间隙。链置换DNA聚合酶和5’-3’核酸外切酶活性的组合产生在接合位点处或附近包含切口的双链合成子。这一切口可以通过连接酶在体外密封，或者通过内源性细胞连接酶在体内密封。此外，在反应混合物中包括ss DNA结合蛋白质使得能够有效地组装相对低浓度的核酸片段，从而在不损失效率或者不损失接合准确性的情况下节约成本。
在一些组合物实施方式中，链置换聚合酶是B族聚合酶。链置换聚合酶应当优选地在相同反应条件下(例如，使用比如图1A～1E中所述的试验)链置换活性大于以
Figure BDA0001280816920000021
聚合酶(Thermo Fisher，Waltham，MA)(通常描述为非链置换的)所观察者。在本发明的组合物、方法和试剂盒中，主要使用链置换聚合酶的链置换活性。在一些实施方式中，链置换聚合酶可以是非天然存在的，例如，链置换聚合酶可以是突变体。突变体的实例包括具有一个或更多个氨基酸替换的聚合酶，非天然存在的聚合酶可以或者另外是具有不相关氨基酸序列的部分的融合蛋白，其中融合聚合酶在自然界不存在。优选地，链置换聚合酶在50℃或更高下稳定，并且因此可以称作热稳定性链置换聚合酶。在一些情形中，链置换聚合酶是具有不相关或异源性DNA结合结构域的融合聚合酶。在一些实施方式中，聚合酶部分的氨基酸序列与SEQ ID NO:102的同一性可以为至少90％或95％或98％或99％。在另一个实施方式中，聚合酶的氨基酸序列与SEQ ID NO:1的同一性可以为至少90％或95％或98％或99％或100％，优选至少90％。在另一个实施方式中，聚合酶的氨基酸序列与SEQ ID NO:33至SEQID NO:55中任意者的同一性可以为至少90％或95％或98％或99％或100％，优选至少90％。在一些实施方式中，DNA结合结构域部分的氨基酸序列与SEQ ID NO:2的同一性可以为至少90％或95％或98％或99％。在另一个实施方式中，聚合酶的氨基酸序列与SEQ ID NO:1、SEQID NO:3、SEQ ID NO:56至SEQ ID NO:96或SEQ ID NO:102中任意者的同一性可以为至少90％或95％或98％或99％或100％，优选至少90％。在另一个实施方式中，本文所述的任意聚合酶结构域部分可以与本文所述的任意DNA结合结构域组合，其条件是聚合酶部分和DNA结合结构域是异源性的。例如，在其他实施方式中，融合蛋白的氨基酸序列与SEQ ID NO:1和SEQ ID NO:2的同一性可以为至少90％或95％或99％或100％，优选至少90％。在其他实施方式中，融合蛋白与SEQ ID NO:3的序列同一性可以为至少90％或95％或98％或99％或100％，优选至少90％。链置换聚合酶可以具有或不具有3’-5’核酸外切酶活性。链置换聚合酶具有3’-5’核酸外切酶活性时，多核苷酸接合可以通过使用包括本文例示者的条件来平衡3’-5’核酸外切酶活性、5’-3’聚合活性和链置换活性加以优化。组装的效率和准确性可以使用本文所述的试验来确认(例如，参见图3A和3B)。在一些实施方式中，聚合酶不是Phusion、9°N、Pfu或Vent或者氨基酸序列与Phusion或野生型9°N、Pfu或Vent的同一性为至少90％的聚合酶。在一些实施方式中，聚合酶是热稳定性的，即，在至少40℃或至少50℃的温度下有活性。与链置换聚合酶相反，一些聚合酶比如Taq DNA聚合酶经由5’→3’核酸外切酶活性降解遇到的下游链。该活性用于切口翻译方案。因此Taq DNA聚合酶不包括在链置换聚合酶的定义中。
确定合成子形成的效率和准确性的试验描述于实施例中，并示于图3A～B。设计的组装片段编码lacl和lacZ蛋白质，如果DNA片段组装正确，其产生蓝色菌落。因此，过夜板“蓝色”菌落的数目指示组装的效率和准确性。在不存在蓝色的情况下，可能发生有效的组装，但接合/延伸区的错误阻止了表达。当合成子得以组装、并且然后克隆到宿主细胞中，合成子形成的效率和准确性转化成每个克隆将包含正确组装的合成子的置信度。借助该置信度，仅需要对一个或多个复制的克隆进行测序，以确认合成子的存在。这样降低了对可能包含错误的克隆进行测序的成本和不便。在一个实施方式中，至少80％或者另外可选地至少90％的克隆将会包含正确组装的合成子。
在一些实施方式中，使用本文所述组合物的方法能够达到基本上超过最小需求的产率。例如，在单一转化事件中可以制备至多5,000或10,000个克隆。如果组装的目的不是制备合成子文库，而是制备单例合成子，则可以使用更低的核酸片段和试剂起始量，甚至其低于本文提供的范围。适合用于组装混合物的浓度范围的实例包括以下：0.02nM～100nM的DNA片段或者例如0.2nM～10nM DNA可以加入到反应容器中的试剂混合物中。在一个实施方式中，尽管可以使用更高或更低的比例，载体DNA与DNA片段以1:1的比例包括在内。与对于ds DNA所选择的浓度相比较，可以优选更高的ss DNA浓度。反应容器中的试剂混合物还可以包括0.0004U/μl～0.064U/μl的5’-3’核酸外切酶(例如0.0004U/μl～0.01U/μl)；0.5U/μl～32U/μl任选的连接酶(例如1U/μl～10U/μl)；0.0025U/μl～0.25U/μl链置换聚合酶(例如0.005U/μl～0.1U/μl)；和0.001μg/μl～0.1μg/μl的ss结合蛋白质(例如0.01μg/μl～0.5μg/μl)(单位对应于制造商(New England Biolabs,Ipswich,MA)所指定者)。
5’-3’核酸外切酶的量能够根据核酸片段重叠长度和每个片段的大小加以进一步优化。例如，5’-3’核酸外切酶的量可以在核酸片段长度大于80个核苷酸的范围内增加。指定范围内链置换聚合酶的绝对浓度并非关键。
尽管已知有许多其他的ss DNA结合蛋白质，并且其可以用于组合物中，组合物中使用的ss DNA结合蛋白质可以是大肠杆菌(E.coli)recA、T7基因2.5产物、RedB(来自噬菌体λ)或RecT(来自Rac原噬菌体)、ET SSB(极端热稳定的单链DNA结合蛋白质)或者与SEQ IDNO:100的序列同一性为90％的ss结合蛋白质。如通过菌落数目所测量，比起在否则不存在ss结合蛋白质的情况下所发生的，包括ss结合蛋白质提高了特别是对于具有更长重叠序列(例如，至少20个核苷酸)的核酸片段的组装效率。
任选的连接酶可以是依赖于NAD+的连接酶，比如Taq连接酶，或者依赖于ATP的连接酶，比如T4连接酶。然而，对于PCR，由于ATP能够抑制后续的合成子扩增中使用的Taq聚合酶，使用依赖于NAD+的连接酶是方便的。合适的连接酶的实例包括与SEQ ID NO:101的序列同一性为至少90％的蛋白质。
在此使用的5’-3’核酸外切酶可以是具有5’-3’核酸外切酶活性以及ss核酸内切酶活性的酶(参见，例如，Garforth等人,PNAS,96,38-43(1999))。具有核酸外切酶和ss核酸内切酶活性的5’-3’核酸外切酶的实例包括T5核酸外切酶、以及其同源物和变体。在一个实施例中，5’-3’核酸外切酶与SEQ ID NO:98的氨基酸序列同一性为至少90％。没有要求在用链置换聚合酶接合多核苷酸之前使5’-3’核酸外切酶变性。因此，在实施例中描述了使用热稳定性5’-3’核酸外切酶。
在一些实施方式中，组合物还可以包括dNTP(即，dGTP、dATP、dGTP和dTTP的混合物)，并且，在一些实施方式中，当使用T5 5’-3’核酸外切酶时，组合物还可以包括钾盐，比如KCl(例如，浓度范围7mM～150mM)。
概括地讲，提供一种制备合成子的方法。在一些实施方式中，该方法可以包括在合适的反应条件下将本文所述组合物的一种实施方式与一组多核苷酸和/或寡核苷酸一起孵育，上述组合物的实施方式包括本文所述的链置换聚合酶和5’-3’核酸外切酶和任选的连接酶(如果反应在体外或者在不含连接酶的细胞或有机体中的体内)，并且还可以包含ss结合蛋白质，在上述的一组多核苷酸和/或寡核苷酸中至少一种或一些组成员具有与一种或一些其他组成员重叠的序列。在一些实施方式中，多核苷酸或寡核苷酸可以是ds DNA，例如，重叠PCR产物或重叠限制片段。在其他实施方式中，多核苷酸可以是ss DNA或RNA。在一些实施方式中，该多核苷酸组可以包括ss DNA或RNA。在一些实施方式中，该多核苷酸组可以包括ds多核苷酸。在一些实施方式中，该多核苷酸组可以包括至少一种ds多核苷酸和至少一种ss多核苷酸。在一些实施方式中，该多核苷酸组可以包括除了在亚群成员之间变化的子序列以外具有相同序列的多核苷酸亚群。在其他实施方式中，该多核苷酸组可以包括ss或ds多核苷酸，或者在其用于接合目的的末端具有重叠区、但形成合成子的内部序列不同的多核苷酸。因此，在本发明的方法的一个实施方式中，该多核苷酸组中的多核苷酸为ds；比如，其中ds多核苷酸是重叠PCR产物或重叠限制片段或者由ss多核苷酸组装。在本发明的方法的一另外可选的实施方式中，合成子由该多核苷酸组中ss的多核苷酸组装。在本发明的方法的另一另外可选的实施方式中，合成子由包括至少一种ds多核苷酸和至少一种ss寡核苷酸的混合物的一组多核苷酸组装。在本发明的方法的实施方式中，合成子由包括在除亚群成员之间变化的子序列以外彼此相同的多核苷酸亚群的一组多核苷酸组装。
方法的实施方式可以用于制备各种合成子，包括编码序列、载体、用于基因工程和表达组件的引导分子(guide molecule)。
在组装之前，初始ds多核苷酸的长度可以在100碱基至30kb的范围内，尽管该范围以外的多核苷酸也可以在某些情形中使用。例如，在一些实施方式中，单个片段大小可以至多20kb～30kb或更长，或者短至30碱基～500碱基。而且，在一些实施方式中，不同大小的片段可以在组装反应中接合。在一个实施例中，将长多核苷酸(例如，长度5kb～20kb的片段)与短核苷酸(例如，长度100碱基～500碱基的片段)接合。新组装的合成子可以使用单分子测序方法直接进行测序，或者在克隆或扩增之后进行测序。
在一个实施方式中，该组成员可以包含长度小于2kb的重叠序列，例如，在15～200个核苷酸的范围内，例如，20～100个核苷酸。
在一个实施方式中，提供一种组合物，其中组合物具有5’-3’核酸外切酶；链置换聚合酶；和包含浓度范围7mM～150mM(例如20mM～50mM)的钾盐(比如KCl)的缓冲液。除钾盐以外，还可以使用范围10mM～100mM(比如20mM)的钠盐(例如氯化钠)。组合物中可以包括ss结合蛋白质。在一些实施方式中，组合物不含拥挤剂，比如聚乙二醇(PEG)、Ficoll或葡聚糖。在一些实施方式中，组合物不含非链置换聚合酶。在另一个实施方式中，用于形成合成子的组合物中包括多核苷酸和/或寡核苷酸片段。
在方法的另一个实施方式中，一组寡核苷酸可以在不存在非链置换聚合酶的情况下使用一种组合物来接合，上述组合物包括除ss结合蛋白质以外或者代替ss结合蛋白质的拥挤剂比如聚乙二醇(PEG)、Ficoll或葡聚糖，和至少7mM钾盐比如KCl，连同链置换聚合酶和5’-3’核酸外切酶。在一个实施方式中，钾盐浓度低于150mM，例如，20mM～50mM。
另外提供一种用于多核苷酸组装的试剂盒，其包括：i.5’-3’核酸外切酶；ii.任选的连接酶；iii.链置换聚合酶：和iv.ss DNA结合蛋白质。在某些实施方式中，例如，试剂盒还可以包括dNTP和/或缓冲剂。试剂盒的组分可以在单独的容器中(例如，一个或多个不同的反应管)，或者，试剂盒的组分可以在单一容器中。组分可以是冻干的，或者是溶液状态的，或者一部分冻干并且一部分为溶液状态。组分可以部分或全部固定化在固体表面比如球珠(bead)上或者反应室表面上，或者是溶液状态的。可以将组分加入到部分或全部固定化的或者溶液状态的靶标多核苷酸中。在一些实施方式中，试剂盒可以包含一种或多种试剂盒组分混合物。在一些实施方式中，试剂盒不包含非链置换聚合酶或拥挤剂。
在一个实施方式中，提供与SEQ ID:1的序列同一性为至少80％、85％、90％或95％的聚合酶用于组装混合物。在另一个实施方式中，提供结合结构域与SEQ ID:2的序列同一性为至少80％、85％、90％或95％的聚合酶用于组装组合物。在另一个实施方式中，提供与SEQ ID:3的序列同一性为至少80％、85％、90％或95％的聚合酶用于组装混合物。这些组合物可以在其中聚合酶为链置换性的反应条件下使用。组合物可以在其中与聚合酶活性有关的任何3’核酸外切酶活性呈活性的反应条件下使用。
组装反应可以使用ss或ds核酸发生。可以组装任意数目的片段，例如2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20或更多个片段。可以将化学合成的ss多核苷酸组装到ds重叠片段中，并且与线性化载体的末端杂交并连接，以形成适合于克隆的合成子。另外可选地，可以将双链片段组装到合成子中，并插入到用于克隆或PCR或恒温扩增的载体中(参见，例如，图2A～2C)。通过将核酸片段与线性化ds载体的3’ss末端杂交，还可以将ss核酸片段直接插入到载体中(参见，例如，图7)。ss核酸可以在ss核酸片段与线性化载体的3’ss末端杂交的同时、在其之前或之后通过重叠互补末端来组装。在插入到载体中用于克隆之前，组装的片段可以通过PCR或恒温方法进行扩增。核酸片段可以包含随机化核苷酸序列或简并密码使得能够形成包含在可变区的每个核苷酸位置处的代表性变体的文库。随机序列可以位于每个末端处的给定序列之间。在一个实施方式中，位于指定序列之间的随机序列可以用于与第二核酸片段(例如第二ss基因组多核苷酸)或者线性化载体的末端杂交。在一个实施例中，随机序列与用于将Cas9蛋白质引导至用于基因编辑的靶标核酸的靶标基因组序列杂交(参见，例如，图9A～9C)。因此，根据本发明这一方面，该方法可以用于将一组多核苷酸的至少一种成员与第二核酸片段(比如第二ss基因组多核苷酸)或者与线性化载体末端杂交。例如，该方法可以用于将该多核苷酸组的至少一种成员与靶标基因组序列杂交，以在基因编辑方法中将Cas核酸内切酶比如Cas9引导至靶标基因组核酸。
在一些实施方式中，提供一种将多种多核苷酸组装到合成子的方法，其包括：将多种多核苷酸与包括5’-3’核酸外切酶、链置换聚合酶、任选的连接酶、ss结合蛋白质和缓冲剂的组合物合并，其中每种多核苷酸均在一条链上具有3’ss末端多核苷酸序列：其可以与第二多核苷酸上的互补性ss互补序列杂交，并且可以任选地在单一容器中在恒温条件下接合以形成连续的双链多核苷酸。合成子可以进一步在其末端与用于扩增和/或克隆的线性化质粒的末端接合。
在一些实施方式中，整个组装方法可以作为“一步”反应(在单一管中，其不必在反应开始之后的期间打开)进行。在一个实施例中，组分在反应容器中混合在一起，并在40℃至60℃的温度下孵育一段时间，例如，5分钟至12小时，从而制备合成子。
在一方面，该方法包括在要通过聚合酶接合的多核苷酸中进行链置换的步骤，上述聚合酶与SEQ ID NO:1或SEQ ID NO:102和/或SEQ ID NO:2或SEQ ID NO:3的氨基酸序列同一性为至少90％。在方法实施方式中，不要求有额外的3’-5’核酸外切酶回切(chewback)步骤。另一方面还包括通过在反应混合物中包括最小浓度为7mM的钾盐提高组装反应效率，其中钾盐的实例有KCl。
在一方面，提供一种方法，其中多核苷酸包含给定的序列末端之间的随机序列。在另一方面，该方法还包括筛选具有与基因组DNA杂交的活性的随机序列以及鉴定具有杂交活性的随机序列。在另一方面，该方法还包括通过转录具有杂交活性的随机序列以形成RNA，以及在Cas核酸内切酶的存在下使用用于基因编辑的RNA，从而进行基因编辑。
在本发明的组合物、试剂盒或方法的一个实施方式中，本发明的组合物、试剂盒或方法中使用的链置换聚合酶可以是非天然存在的，比如，突变体或融合蛋白。在本发明的组合物、试剂盒或方法中，非天然链置换融合蛋白酶的特征可以在于聚合酶部分的氨基酸组成与SEQ ID NO:33至SEQ ID NO:55、或SEQ ID NO:1中任意者的同一性为至少90％或95％或99％或100％，或者DNA结合部分的氨基酸组成与SEQ ID NO:56至SEQ ID NO:98或SEQ IDNO:2中任意者的同一性为至少90％或95％或99％或100％。在一个实施方式中，氨基酸序列与SEQ ID NO:102的同一性可以为90％或95％或98％或99％的聚合酶部分，其与选自氨基酸序列与SEQ ID NO:56至SEQ ID NO:98中任意者的同一性为至少90％或95％或99％或100％的多肽中的异源性DNA结合部分融合。
概括来讲，在一方面，提供一种制剂，其包括氨基酸序列与SEQ ID NO:3的序列同一性为至少90％的组合物。该制剂还可以包括5’-3’核酸外切酶，例如，T5核酸外切酶。该制剂还可以包括ss DNA结合蛋白质，例如，ET SSB、大肠杆菌recA、T7基因2.5产物、噬菌体λRedB或Rac原噬菌体RecT，更具体地，包括热稳定性ss结合蛋白质，比如ET SSB。该制剂还可以包括连接酶。在一方面，包括组合物的制剂还可以包括ss结合结构域和5’-3’核酸外切酶，其中该制剂不包括拥挤剂和/或非链置换聚合酶。该制剂还可以包括钾盐。
在一方面，包括组合物的制剂还可以包括一组中的多种多核苷酸，其中所述组中的至少一种多核苷酸的序列与所述组中另一种多核苷酸重叠；并且其中多核苷酸选自：(i)ds多核苷酸；(ii)ss寡核苷酸；(iii)至少一种ds多核苷酸和至少一种ss寡核苷酸；和(iv)除了在亚群成员之间变化的序列以外，否则彼此相同的多核苷酸亚群。在一方面，多核苷酸组具有至少3种成员或至少4种成员或至少5种成员。
通常，用于制备合成子的方法包括：在合适的反应条件下将还包括5’-3’核酸外切酶以及任选的连接酶和ss DNA结合蛋白质的权利要求1的组合物与形成一组的多种多核苷酸一起孵育，其中所述组的成员具有重叠序列；以及将至少两种多核苷酸接合，以制备合成子。在一方面，该多核苷酸组包含至少3种成员或至少4种成员或至少5种成员。在一方面，该制剂还包括连接酶。一方面，该制剂还包括ss DNA结合蛋白质。在一方面，多核苷酸为ds，并且ds多核苷酸是重叠PCR产物、重叠限制片段或者由ss寡核苷酸组装。在一方面，多核苷酸是ss寡核苷酸。在一方面，多核苷酸组包括至少一种ds多核苷酸和至少一种ss寡核苷酸。
概括来讲，本发明提供一种试剂盒，其包括根据权利要求1的制剂和5’-3’核酸外切酶，如例如T5核酸外切酶。在一方面，试剂盒还可以包括ss结合蛋白质。在另一方面，试剂盒可以包括连接酶。在另一方面，试剂盒可以包括缓冲剂。在一方面，试剂盒不包括拥挤剂。在一方面，组合物和5’-3’核酸外切酶在相同容器中。在另一方面，组合物和5’-3’核酸外切酶在不同容器中，任选地是在适合于合并在单一容器中的缓冲剂中。
概括来讲，本发明提供一种用于组装合成子的组合物，其包括：5’-3’核酸外切酶，其在一方面具有ss核酸内切酶活性，例如，与SEQ ID NO:98的序列同一性为90％；链置换聚合酶，其包括B族链置换聚合酶，其优选为非天然存在的，比如源自天然存在的聚合酶的突变体或融合蛋白，其可以另外地为热稳定性的；任选的ss DNA结合蛋白质，例如，ss DNA结合蛋白质为ET SSB、大肠杆菌recA、T7基因2.5产物、噬菌体λRedB或Rac原噬菌体RecT；和非天然存在的缓冲剂，其中组合物不包括拥挤剂和/或非链置换聚合酶。在一方面，组合物还包括连接酶和/或ss结合结构域。在一方面，组合物包括一组至少两种多核苷酸(多种多核苷酸)。在一方面，组合物不包括非链置换聚合酶。在另一方面，组合物不包括9°N、Phusion、Vent或Pfu DNA聚合酶。
在一方面，组合物中的链置换聚合酶是融合蛋白，其中聚合酶部分的氨基酸序列与SEQ ID NO:1或SEQ ID NO:102或SEQ ID NO:33至SEQ ID NO:55中任意者的同一性为至少90％。例如，融合蛋白的氨基酸序列与SEQ ID NO:1或SEQ ID NO:102和SEQ ID NO:2的同一性可以为至少90％。例如，链置换聚合酶与SEQ ID NO:3的序列同一性可以为至少90％。在一方面，组合物可以包括浓度至少7mM的钾盐。在一方面，组合物可以包括一组多核苷酸，其中所述组中的至少一种多核苷酸具有与所述组中另一种多核苷酸重叠的序列，并且其中多核苷酸选自：(i)ds多核苷酸；(ii)ss寡核苷酸；(iii)至少一种ds多核苷酸和至少一种ss寡核苷酸；和(iv)除了在亚群成员之间变化的序列以外，否则彼此相同的多核苷酸亚群。在一方面，该多核苷酸组中至少一种成员包含位于每个末端处给定序列之间的用于与第二ss基因组多核苷酸杂交的随机序列，其中，例如，随机序列为ss，并且其能够与用于将Cas蛋白质引导至用于基因编辑的靶标基因组核酸的靶标基因组序列杂交。
概括来讲，本发明提供用于形成合成子的方法，其包括在合适的反应条件下孵育包含一组具有重叠序列的多核苷酸的任意上述组合物；以及将至少一些多核苷酸与其他多核苷酸接合，以制备合成子。在该方法的在一方面，所述组中全部或部分多核苷酸为ds。在另一方面，ds多核苷酸是重叠PCR产物；重叠限制片段或者由互补ss寡核苷酸组装的合成ds分子，其中这些寡核苷酸可以在合成器中制备。在一方面，所述组中的全部或部分多核苷酸是ss寡核苷酸。在一方面，该多核苷酸组包括至少一种ds多核苷酸和至少一种ss寡核苷酸。在一方面，该多核苷酸组包括除了在亚群成员之间变化的序列以外，否则彼此相同的多核苷酸亚群。在一方面，多核苷酸的重叠序列长度小于2千碱基。在该方法的一方面，链置换聚合酶包括与SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:33至SEQ ID NO:96或SEQID NO:102中任意者的同一性为至少90％的氨基酸序列。在该方法的一方面，该多核苷酸组中至少一种成员包含给定的序列末端之间的随机序列。该方法的另一方面包括筛选具有与基因组DNA杂交的活性的随机序列以及鉴定具有杂交活性的随机序列。该方法的另一方面包括通过转录具有杂交活性的随机序列以形成RNA，以及在Cas蛋白质的存在下使用用于基因编辑的RNA，从而进行基因编辑。
概括来讲，本发明提供一种用于多核苷酸组装的试剂盒，其包括：5’-3’核酸外切酶；链置换聚合酶；和任选的ss DNA结合蛋白质，其中该试剂盒任选地不包括拥挤剂和/或非链置换聚合酶。在一方面，该试剂盒包括连接酶。在另一方面，该试剂盒包括dNTP。在另一方面，该试剂盒包括缓冲剂。在另一方面，该试剂盒的单个组分可以在相同的容器或单独的容器中，比如一个或多个不同的储存或反应容器。
概括来讲，本发明提供一种组合物，其包括聚合酶融合蛋白，其中聚合酶融合蛋白包括与SEQ ID NO:2、SEQ ID NO:56至SEQ ID NO:96中任意者的同一性为至少90％的氨基酸序列和异源性聚合酶结构域。在一方面，聚合酶融合蛋白包括与SEQ ID NO:2中任意者的同一性为至少90％的氨基酸序列；和异源性聚合酶结构域。
概括来讲，本发明提供一种包括聚合酶融合蛋白的组合物，其中聚合酶融合蛋白包括氨基酸序列与SEQ ID NO:1、SEQ ID NO:33至SEQ ID NO:55或SEQ ID NO:102中任意者的同一性为至少90％的聚合酶结构域；和异源性DNA结合结构域。在一方面，聚合酶融合蛋白包括氨基酸序列与SEQ ID NO:1的同一性为至少90％的聚合酶结构域；和异源性DNA结合结构域。在一方面，聚合酶融合蛋白具有氨基酸序列与SEQ ID NO:102的同一性为至少90％的聚合酶结构域；和异源性DNA结合结构域。在一方面，聚合酶融合蛋白的氨基酸序列与SEQID NO:3的同一性为至少90％。
在一方面，上述组合物还包括5’-3’核酸外切酶，比如T5核酸外切酶。在一方面，组合物还包括单链DNA结合蛋白质，例如选自ET SSB、大肠杆菌recA、T7基因2.5产物、噬菌体λRedB或Rac原噬菌体RecT中的单链结合蛋白质。在一方面，组合物可以包括连接酶。在一方面，连接酶是热稳定性的。在一方面，组合物不包括拥挤剂和/或非链置换聚合酶。在另一方面，组合物还包括dNTP。在另一方面，组合物还包括浓度至少7mM的钾盐。组合物的一方面包括一组多核苷酸，其中所述组中的至少一种多核苷酸的序列与所述组中另一种多核苷酸重叠；并且其中多核苷酸选自：(i)ds多核苷酸；(ii)ss寡核苷酸；(iii)至少一种ds多核苷酸和至少一种ss寡核苷酸；和(iv)除了在亚群成员之间变化的序列以外，否则彼此相同的多核苷酸亚群。
概括来讲，本发明提供一种用于制备合成子的方法，其包括将一组多核苷酸与特征在于上文所述的包括聚合酶的组合物一起孵育，其中单个多核苷酸包含与其他多核苷酸中的序列重叠的序列，其中不同多核苷酸的重叠序列能够在合适的反应条件下交叉杂交，并且，例如其中重叠区小于2千碱基，其中组合物还包括5’-3’核酸外切酶和任选的连接酶和ss DNA结合蛋白质；以及将多核苷酸接合，以制备合成子。
在不同的方面，组合物包括连接酶；和/或ss DNA结合蛋白质。在另一方面，所述组中一种或多种多核苷酸为ds，其中ds多核苷酸是PCR产物、重叠限制片段或者由ss寡核苷酸组装；和/或一种或多种多核苷酸是ss寡核苷酸；和/或该多核苷酸组包括至少一种ds多核苷酸和至少一种ss寡核苷酸。
概括来讲，本发明提供一种用于多核苷酸组装的试剂盒，其包括上述聚合酶融合蛋白，和5’-3’核酸外切酶；和ss DNA结合蛋白质。在一方面，该试剂盒可以包括任意或全部的连接酶、dNTP和缓冲剂，其中试剂盒组分可以在相同容器中或不同容器中。
附图说明
技术人员将会理解到，下述附图仅仅出于说明目的。附图无意以任何方式对本发明教导的范围加以限定。
图1A～1E示出实施例1所述的试验如何区分链置换和非链置换聚合酶。该试验确认，T4DNA聚合酶是非链置换的，并在封闭寡核苷酸(blocking oligonucleotide)(2)处在44个核苷酸长度时终止模板DNA的合成(图1C)，而Bst聚合酶(图1D)和非天然聚合酶(图1E)是链置换的，并可以通过置换封闭寡核苷酸(长度27个核苷酸)继续FAM引物(1)的DNA合成。
图1A示出以下酶试验中使用的DNA模板上引物(1)和封闭寡核苷酸(2)的序列和位置。
图1B示出毛细管电泳之后得到的样品中观察到的荧光，其中不加入酶，并且起始原料在与24个核苷酸的FAM引物对应的位置处形成峰。
图1C示出加入T4 DNA聚合酶的结果。引物延长至最终长度44个核苷酸(24个核苷酸加上20个核苷酸)，但由封闭寡核苷酸终止。
图1D示出，Bst DNA聚合酶(大的片段)对封闭寡核苷酸进行链置换，并通过将引物延长至总长度72个核苷酸(24+20+27+dA)对模板进行拷贝。
图1E示出作为校对聚合酶的B族链置换DNA聚合酶对封闭寡核苷酸进行链置换，并通过将引物延长71个核苷酸(24+20+27)对模板进行拷贝。
图2A～2C示出DNA组装方法中的步骤。
图2A示出将5种片段中每一个的扩增子加入到具有氨苄青霉素抗性标志物的5种质粒中。用产生具有重叠区以及两侧为NotI限制性位点的扩增子的引物对5种片段进行初始扩增。NotI切割产生粘性末端。NotI限制性位点(3)允许由载体释放出每种扩增子。限制性酶切片段与相邻片段具有80个碱基对重叠区(4)。在图2C中，出于方便及降低成本，第一片段和相邻的试剂载体末端以及最后片段和相邻的试剂载体末端之间的重叠为15～25核苷酸，例如，20核苷酸，但这并不意在是限制性的。
图2B示出NotI切割的任选测序的片段(5)(由载体回收到的扩增子)，然后将其在单个反应容器中用包括T5/5’-3’核酸外切酶、具有3’-5’核酸外切酶活性的DNA聚合酶、ss结合蛋白质ET SSB(New England Biolabs,Ipswich,MA)和DNA连接酶的酶混合物处理(6)～(8)。尽管在此使用NotI，取决于方便性，也可以使用其他的限制性核酸内切酶以用于切割。可以用两种或更多种限制性核酸内切酶进行双消化。例如，已经发现，用两种限制性核酸内切酶对载体DNA进行双消化降低来自未切割载体的本底。将重叠ss DNA序列与相邻片段杂交。T5核酸外切酶在每个片段上将DNA链从5’到3’回切(chew back)，以暴露3’ss区(6)，这促使片段在ss结合蛋白质的存在下一起退火(7)。通过与链置换聚合酶相关的3’-5’核酸外切酶活性的手段，达到2个碱基翼(flap)的除去，然后通过链置换聚合酶进行延长，以充填组装产物中的间隙(8)。任何残余的切口或5’翼可以通过连接酶和/或T5核酸外切酶修复。
图2C示出现在按顺序接合并插入到用于转化到细菌宿主中的携带氯霉素抗性基因(Cam)的第二质粒中的5个片段(片段(Frag)1至片段5)。
图3A和3B示出，使用氯霉素板以选择在板上生长的菌落，并且在IPTG和X-Gal的存在下，包含laclZ基因的那些菌落产生蓝色菌落。试验提供了对其中基因以功能性形式有效组装的克隆的定量评价。
图3A示出仅有氯霉素。
图3B示出氯霉素+IPTG+Xgal。
图4示出质粒事实上确实包含整个基因。对图2B所示的组装产物进行PCR扩增，以确认所有片段在转化之前得以接合和连接。条带1和2是重复的PCR结果。条带M是来自NewEngland Biolabs,Ipswich,MA的2-log DNA梯状带(ladder)。
图5示出通过菌落数目测定的组装混合物中KCl的影响。缓冲液中使用的KCl浓度增加，表现出组装混合物中使用链置换聚合酶的组装的准确性/效率增加。左边的柱状图(T26)不含KCl，而右边的柱状图(T26K)包含25mM KCl，其示出效率提高1.5倍。无论组装条件如何，这一提高均发生。如果在不存在ss结合蛋白质的情况下使用PEG或其他拥挤剂，预计有类似的相对效率提高。
图6示出根据制造商所提供的方案，实施例2中所述的混合物(链置换聚合酶/ss结合蛋白质/5’-3’核酸外切酶/连接酶)(Mix 1)和市售Gibson
Figure BDA0001280816920000151
Mix(GAMM)(非链置换聚合酶和聚乙二醇)(Synthetic Genomics,La Jolla,CA/New England Biolabs,Ipswich,MA)之间的比较。Mix 1产生显著更高的DNA组装和转化效率。
图7示出ss DNA寡核苷酸和ds DNA片段之间的DNA组装的概略图。将ss靶标DNA寡核苷酸插入到DNA载体中。已合成出ss靶标DNA寡核苷酸，以在每个末端上具有20～30个核苷酸的与3’载体末端的重叠区。然而，该寡核苷酸的尺寸可以具有小于20个核苷酸的重叠区，例如小于15个核苷酸或小于10个核苷酸，或者另外可选地大于30个核苷酸，例如，至少40个或50个或60个核苷酸或更多。在重叠区之外，该寡核苷酸优选地具有位于末端之间的不重叠的1个或更多个核苷酸。将包含5’-3’核酸外切酶、链置换聚合酶、连接酶和ss结合蛋白质的组装主混合物加入到ss寡核苷酸和载体的混合物中，以使得ds DNA载体的5’末端得以回切(chewed back)，以产生ss突出端(9)。然后ss DNA的3’末端能够退火至载体的5’末端，然后DNA聚合酶对ss模板进行复制，以填充空隙，并产生平端ds DNA。将切口通过连接酶密封(10)。再次，核酸外切酶(在此为T5核酸外切酶)这次在靶标DNA的平端回切5’末端，产生3’ss区，以使得互补序列退火，并完成靶标DNA ds整合到DNA载体中(11)。片段可以借助填充空隙的DNA聚合酶和密封切口的连接酶退火(12)以制备合成子。
图8A～8C示出由短ss寡核苷酸桥接ds DNA的工作流程。(方案描述于图7中)。
图8A提供用于整合到在此示出为具有OFP报告子的CRISPR核酸酶载体的ds DNA载体中的短ss寡核苷酸序列的实例。
图8B示出的工作流程起始于ss寡核苷酸和ds CRISPR核酸酶载体(9424bp)，其用5’-3’核酸外切酶、链置换聚合酶、连接酶和ss结合蛋白质处理(13)，以产生完整的ds环状DNA。将该DNA转化到感受态细胞中(14)。过夜孵育之后，通过微制备对菌落进行分析，然后进行质粒测序(15)。图8C示出插入的序列和相邻序列、U6启动子序列(载体)、设计的ss寡核苷酸(71聚体(mer))的序列和骨架(scaffold)模板特异性序列(载体)。将在每个末端包括25个核苷酸的重叠区(黑体示出的靶标DNA的21核苷酸)的ss寡核苷酸(71聚体)适当地整合到宿主细胞中的载体中。
图9A～9C示出，与图8中类似的工作流程可以用于在重叠端之间具有简并碱基的ss寡核苷酸。同样，图9A中的起始序列示出于工作流程(图9B)之上，并且来自组装库的菌落的Sanger测序结果示出于下方(图9C)，实线是指sgRNA靶标序列库。sgRNA靶标序列包含21个可变核苷酸位置，提供421种变体。该库包含每种可能的变体，并且每种变体适于克隆反映重叠末端和载体之间序列的简并性。图9A示出包含简并碱基的ss寡核苷酸的序列。将图9A的sgRNA靶标序列插入在载体的U6启动子序列和骨架模板特异性序列之间(16)，将其转化到宿主细胞中(17)，并通过微制备和测序进行合成子分析(18)，如上文及在此所述。对来自组装库的克隆进行Sanger测序。图9C提供序列的实例。
图10示出将组装反应产物转化到大肠杆菌中之后选自一板的187个菌落的结果。对每个菌落进行PCR扩增和测序，以确认ss DNA的插入，并对简并碱基的分布进行分析。在此示出的结果确认，不同的菌落确实包含不同的简并序列。未检测到偏性。首先通过将序列转化成fastq文件，然后在github上使用来自fastx工具包的fastx_quality_stats工具，进行分析。使用来自Berkeley的weblogo产生序列标识。
术语说明
除非另外指出，本文使用的所有科技术语的含义与本发明所属领域普通技术人员所通常理解的相同。Singleton等人，DICTIONARY OF MICROBIOLOGY AND MOLECULARBIOLOGY，第二版，John Wiley and Sons,New York(1994)和Hale&Markham,THE HARPERCOLLINS DICTIONARY OF BIOLOGY,Harper Perennial,N.Y.(1991)为技术人员提供了许多本文所用术语的通常含义。为清楚和便于参考起见，以下仍对某些术语加以定义。
如本文所用，如基因合成领域所用的术语“合成子”是指多核苷酸组装体。多核苷酸组装可以包括组装一定大小的重叠片段，其可以在寡核苷酸合成器上制备，对于每个合成的多核苷酸，目前通常为2000～3000个碱基。另外可选地，重叠片段可以由连接有接头(adaptor)以提供重叠序列的天然存在的核酸通过PCR获得。出于组装目的，对每个片段的大小没有限定。依赖于末端处的重叠序列，可以将许多片段端到端地组装，使得能够准确、有效地产生具有任意所需长度的构建体。优选地，合成子是由较短的多核苷酸的组装体形成的不含间隙和切口的较长的连续多核苷酸。然而，由核酸片段组装体产生的合成子的长度不限定于任何具体的大小。
如本文所用，术语“5’-3’核酸外切酶”是指从5’末端，即以5’至3’方向降解DNA的核酸外切酶。所关注的5’-3’核酸外切酶可以在平端处以及在某些实施方式中在3’和/或5’突出端处从ds DNA链的5’末端除去核苷酸。T5核酸外切酶、λ核酸外切酶和T7核酸外切酶均为5’-3’核酸外切酶的实例。在某些实施方式中，优选T5核酸外切酶。T5核酸外切酶额外地具有ss核酸内切酶活性。
如本文所用，术语“连接酶”是指可以具体地在切口处将DNA分子的3’末端与另一DNA分子的5’末端共价接合的酶。尽管已知有许多其他的连接酶并可以在本文使用，连接酶的实例包括T7连接酶、T4DNA连接酶、大肠杆菌DNA连接酶和Taq连接酶。
如本文所用，术语“链置换聚合酶”是指能够置换酶下游的一个或多个核苷酸比如至少10个或100个或更多个核苷酸的聚合酶。链置换聚合酶可以与Phusion区分，其中Phusion在本领域公认的定义是非链置换聚合酶。在一些实施方式中，在至少50℃或至少55℃的温度下，链置换聚合酶是稳定、有活性的(包括链置换活性)。Taq聚合酶是切口翻译聚合酶，因此，其不是链置换聚合酶。
如本文所用，术语“单链(ss)DNA结合蛋白质”是指与ss DNA结合、防止早熟退火、保护ss DNA不被核酸酶和聚合酶消化、和/或去除DNA二级结构以使其他酶对其有效发挥功能的蛋白质。优选在本文所述的组合物中包括ss结合蛋白质，以优化合成子形成的效率。尽管已知有许多其他者，并可以在本文使用，例如λ噬菌体RedB、Rac原噬菌体的RecT和下文所列的序列，ss DNA结合蛋白质的实例有T4基因32蛋白质、大肠杆菌SSB、T7gp2.5SSB、和噬菌体phi29SSB、以及ET SSB。在一些情形中可以使用在50℃下稳定的热稳定性ss DNA结合蛋白质。因此，在本发明的组合物、试剂盒或方法的一个实施方式中，ss DNA结合蛋白质为T4基因32蛋白质、大肠杆菌SSB、T7gp2.5SSB、噬菌体phi29SSB、ET SSB、λ噬菌体RedB或Rac原噬菌体RecT。在一个实施方式中，ss DNA结合蛋白质是ET SSB。在本发明的组合物、试剂盒或方法的一个实施方式中，ss DNA结合蛋白质是热稳定性的(即，在40℃～60℃下稳定)。
如本文所用，术语“缓冲剂”是指当向溶液中加入酸或碱时使得溶液抵抗pH改变的试剂。本发明的组合物、试剂盒和方法中可以使用的合适的非天然存在的缓冲剂的实例包括，例如，Tris、HEPES、TAPS、MOPS、两性离子缓冲剂(tricine)或MES。
术语“非天然存在的”是指组合物在自然界中不存在。
本文所述的任何蛋白质均可以是非天然存在的，其中术语“非天然存在的”是指蛋白质具有与其天然状态下不同的氨基酸序列和/或翻译后修饰方式。例如，非天然存在的蛋白质可以在蛋白质的N-末端、C-末端和/或在N-末端和C-末端之间具有一个或多个氨基酸替换、缺失或插入。“非天然存在的”蛋白质的氨基酸序列可以与天然存在的氨基酸序列不同(即，与天然存在的蛋白质的氨基酸序列的同一性小于100％)，但与天然存在的氨基酸序列的同一性为至少80％、至少85％、至少90％、至少95％、至少97％、至少98％或至少99％。在某些情形中，如果由不同的(例如，细菌)细胞制备非天然存在的蛋白质，其可以包含N-末端甲硫氨酸或者可以缺少一个或多个翻译后修饰(例如，糖基化、磷酸化等)。“突变的”蛋白质相对于野生型蛋白质可以具有一个或多个氨基酸替换，并且可以包括“融合”蛋白。术语“融合蛋白”是指由多种在天然状态下未接合的多肽组分组成的蛋白质。融合蛋白可以是2个、3个或甚至4个或更多个不同蛋白质的组合。术语多肽包括融合蛋白，包括，但不限于，两个或更多个异源性氨基酸序列的融合，多肽与以下的融合：异源性靶标序列、连接序列(linker)、免疫学标签、可检测的融合配偶体比如荧光蛋白、β-半乳糖苷酶、荧光素酶等，等等。融合蛋白可以具有一个或多个加在蛋白质N-末端、C-末端和/或中间部分的异源性结构域。如果融合蛋白的两部分是“异源性”的，则它们在其天然状态下不是相同蛋白质的一部分。
在上下文述及核酸时，术语“非天然存在的”是指核酸包含：a)与天然状态下的核酸不同的核苷酸序列(即，与天然存在的核酸序列的序列同一性小于100％)，b)一个或多个非天然存在的核苷酸单体(其可以产生不是G、A、T或C的非天然骨架或糖)，和/或c)可以在核酸的5’-末端、3’-末端和/或在5’-和3’-末端之间包含一个或多个其他修饰(即，加入的标记或其他部分)。
在上下文述及制剂时，术语“非天然存在的”是指：a)例如由于组分在不同的位置、在不同的细胞中或在不同的细胞区室中而非通过自然组合，这样组分的组合物；b)其相对浓度在自然界未发现的组分的组合物；c)缺少某些通常在自然界中与组分之一关联的组分的组合物；d)自然界中未发现的形式例如干燥、冻干、结晶或含水形式的组合物；和/或e)包含自然界中未发现的组分的组合物。例如，制剂可以包含自然界中未发现的“非天然存在的”缓冲剂(例如，Tris、HEPES、TAPS、MOPS、两性离子缓冲剂或MES)、洗涤剂、染料、反应促进剂或抑制剂、氧化剂、还原剂、溶剂或防腐剂。
可能需要使用具有3’核酸外切酶活性的链置换聚合酶。尽管无意拘泥于理论，但需要3’核酸外切酶，以除去双链3’末端上的侧翼序列(flap sequence)，其中侧翼序列可以是酶切的产物，以便从其置于的质粒中提取靶标多核苷酸。这是如实施例中所述的使用NotI的情形。然而，如果使用在切除的片段上产生平端的限制性核酸内切酶，可以不要求3’核酸外切酶活性。
3’核酸外切酶活性可以通过使用标准DNA模板和引物加以常规测定，其中引物具有或不具有非杂交3’核苷酸。如果聚合酶具有3’核酸外切酶活性，则使用任一引物对将会检测到扩增子。如果聚合酶缺少3’核酸外切酶活性，则使用具有非杂交3’核苷酸的引物将检测不到扩增子。
如本文所用，术语“钾盐”是指包括但不限于KCl的钾盐。术语“钠盐”是指包括但不限于NaCl的钠盐。
如本文所用，术语“多核苷酸”包括寡核苷酸，并且其是指任意长度的核酸。多核苷酸可以是DNA或RNA。除非指定，多核苷酸可以为ss或ds。多核苷酸可以是合成的，例如，在DNA合成器中合成，或者是天然存在的，例如，提取自天然的来源，或者源自克隆或扩增的材料。本文所指的多核苷酸可以包含修饰的碱基。
如本文所用，术语“多核苷酸组”或“一组多核苷酸”是指至少两种多核苷酸的集合。在一些实施方式中，一组多核苷酸可以包括至少5种、至少10种、至少12种、或至少15种或更多种多核苷酸。
如本文所用，术语“重叠序列”是指在两个多核苷酸中互补的序列，并且其中重叠序列为ss，在一种多核苷酸上，其可以与另一种多核苷酸上另一重叠互补ss区杂交。例如，在一组多核苷酸中，重叠序列可以在至少5、10、15或更多种多核苷酸中互补。重叠序列可以在两个不同分子的3’末端处(例如，两个ss寡核苷酸的3’末端，或者第一ds多核苷酸顶部链的3’末端和第二ds分子底部链的3’末端)或者与其接近(例如，大约5、10、20个核苷酸以内)，其中，如果非重叠序列在3’末端处，则非重叠序列可以使用聚合酶的3’-5’核酸外切酶活性去除。重叠序列的长度可以变化，在一些情形中，长度可以为至少12个核苷酸(例如，长度至少15个、20个或更多个核苷酸)，和/或长度可以至多100个核苷酸(例如，长度至多50个、至多30个、至多20个或至多15个核苷酸)。另外可选地，多核苷酸组中的重叠序列可以为2kb或更小，或者1kb或更小，或者小于900碱基、800碱基、700碱基、600碱基、500碱基、400碱基、300碱基、200碱基或100碱基。优选地，重叠序列长度在15个核苷酸至80个核苷酸范围内，例如，至多20个、至多25个、至多30个、至多35个、至多40个、至多45个、至多50个、至多55个、至多60个、至多65个、至多70个、至多75个或至多80个核苷酸。重叠的最小长度可以通过Tm定义，其优选地等于或大于48℃。
如本文所用，术语“多核苷酸组装”是指2个或更多个、4个或更多个、6个或更多个、8个或更多个、10个或更多个、12或更多个、15或更多个多核苷酸，例如4个或更多个多核苷酸彼此接合产生更长的多核苷酸的反应。在许多实施方式中，多核苷酸组装反应的产物，即“组装的多核苷酸”或“合成子”应当包含一个拷贝的每种重叠序列。
如本文所用，术语“在合适的反应条件下孵育”是指保持反应在合适的温度和时间，以实现所需的结果，即多核苷酸组装。适合用于本发明方法的酶和试剂的反应条件是已知的(例如，如本文实施例中所述)，因此，本发明方法的合适的反应条件可以很容易确定。这些反应条件可以根据所用的酶而改变(例如，取决于其最佳温度等)。
如本文所用，术语“等温”是指对于组装进行不要求主动调节温度的温度条件。水浴或加热块温度无关紧要的变化在术语等温的含义范围之内。例如，术语“等温”可以指反应开始之后不要求热变性步骤的反应条件。更具体地，等温方法不涉及热循环，即在高于90℃的变性温度和退火/延长温度之间的循环。等温条件通常涉及在低于90℃的温度下孵育一段时间(例如，5分钟至12小时或更长时间)。在一个实施方式中，等温扩增反应在30℃～75℃，例如40℃～60℃范围内的温度下进行。
如本文所用，术语“接合”是指在两个序列之间产生共价连接。
如本文所用，术语“组合物”是指除了列出的那些之外，可以包含其它试剂例如甘油、盐、dNTP等的试剂的组合。组合物可以是任意形式的，例如，含水的或冻干的，并且可以是任意状态的(例如，冷冻的或液体形式)。
如本文所用，“载体”是片段或合成子可以整合到其中使得可以在宿主细胞中复制改造载体的合适的DNA。线性化载体可以通过环状载体的限制性核酸内切酶消化或者通过PCR产生。片段和/或线性化载体的浓度可以通过凝胶电泳或其他方式测定。
本文使用的任何一种或多种蛋白质(例如，连接酶、SSBP、5’-3’核酸外切酶或聚合酶等)可以是温度敏感的或热稳定的，其中，如本文所用，术语“温度敏感”是指酶在65℃的温度下10分钟之后丧失其活性的至少95％，并且术语“热稳定”是指酶在65℃的温度下10分钟之后保持其活性的至少95％。
具体实施方式
在对各种实施方式进行更详细的说明之前，应当理解到，本公开的教导不限于所述的具体实施方式，因此，其当然可以变化。还应当理解到，本文所用的术语仅出于说明具体实施方式的目的，而无意于进行限定，因为本发明教导的范围将仅由其权利要求书进行限定。
尽管本发明的教导结合各种实施方式进行说明，但无意于将本发明的教导限制于这些实施方式。相反，如本领域技术人员将会理解到，本发明的教导包括各种替代方式、修改方式和等同方式。
在提供数值范围时，应当理解到，位于该范围上限和下限之间的每个中间值(每个中间值精确到下限的单位的十分之一，除非上下文中有清楚地相反表示)，以及任何其他所述的或在其所述范围内的中间值均包括在本公开以内。
尽管在实施或测试本发明教导内容时也可以使用与本文所述的那些类似或等同的任何方法和材料，但是现对一些示例性方法和材料加以说明。
任何出版物的引用是出于其公开早于申请日，不应当理解成认可本发明的权利要求由于在先发明而无权早于这些出版物。而且，所提供出版物的日期可能与实际的公开日期不同，其需要独立地确认。
必须注意到，如本文和其权利要求中所用的，单数形式“一个”、“一种”和“该”包括复数的指示物，除非上下文中有清楚地相反表示。还注意到，权利要求可以撰写成排除任何任选的要素。这样，该声明意在用作结合权利要求要素的引用使用这些排除性术语如“仅仅”、“仅有”等、或者使用“否定”限定的前提基础。
如阅读本公开时对本领域技术人员将会显而易见的是，本文说明和例示的每个单独的实施方式均具有独立的组分和特征，其可以在不脱离本发明教导的范围或精神的前提下很容易地与任何其他若干实施方式的特征分开或组合。任何所引用的方法均可以以所引用事件的顺序或者以任何其他逻辑上可能的顺序实施。
在多核苷酸组中重叠的序列可以是任何合适的长度，比如，2kb或更小，或者1kb或更小，或者小于900碱基、800碱基、700碱基、600碱基、500碱基、400碱基、300碱基、200碱基或100碱基。重叠区可以少至8个核苷酸。优选地，重叠序列长度在15个核苷酸～80个核苷酸范围内，例如，至多20、至多25、至多30、至多35、至多40、至多45、至多50、至多55、至多60、至多65、至多70、至多75或者至多80个核苷酸。例如，最小重叠长度可以由Tm定义，其优选等于或大于48℃。
合成的寡核苷酸和多核苷酸在其用于合成子组装之前可以包含其合成过程中产生的错误。为了在组装之前校正这些错误，期望进行错配修复步骤。就此而言，已经说明了多种在组装之前实现合成核酸的错配修复的方法。合成核酸的种群可以具有随机错误，使得制剂的变性和复性可以显示错配。已经从自然界分离出的蛋白质，比如mutHLS、cel-1核酸酶、T7endo 1、uvrD、T4EndoVII、大肠杆菌EndoV(参见US 7,851,192和US 8,048,664)，可以选择性地与包含错配的DNA双链结合；在错配碱基处切割核酸；并任选地基于模板核苷酸序列用正确碱基替代。
尽管本领域中有教导非链置换聚合酶必须与ss结合蛋白质、5’-3’核酸外切酶和连接酶一起使用以组装DNA片段，本文出人意料地显示，链置换聚合酶可以在发生链置换的条件下使用，并且在起始多核苷酸片段出人意料的低浓度下有效，以有效地由多个片段产生单个核酸。
可以用于本发明的组装混合物、组合物、试剂盒或方法的实施方式中的链置换聚合酶的实例包括B族聚合酶成员，比如(但不限于)表1中所确认的任意者(SEQ ID NO:33至SEQ ID NO:55)。此外，可以使用这些聚合酶的融合，例如，在多种聚合酶和/或ss结合结构域(比如，如表2所示)(SEQ ID NO:56至SEQ ID NO:97)之间的融合。在实施方式中，表1中的任何聚合酶部分或者与表1中任意这些蛋白质部分的氨基酸序列同一性为至少80％、85％、90％、95％、98％、99％或100％的蛋白质均可以在N-末端处或C-末端处与表2中所述的任何DNA结合结构域或者与表2中任意DNA结合部分的氨基酸序列同一性为至少80％、85％、90％、95％、98％、99％或100％的蛋白质的部分来融合，以形成本文使用的链置换融合聚合酶。DNA结合结构域可以任选地融合在聚合酶的N-末端或C-末端。
也可以使用如通过本文提供的试验(参见，例如图1A～1E和实施例1)确定显示为链置换性的其他聚合酶变体或新的分离物。这些来源发现的聚合酶的序列通过GenBank很容易获得。由于链置换序列的高度保守性，任何与这些野生型聚合酶的氨基酸序列同一性为80％、85％、90％或95％的变体可以预计具有链置换特性，其可以在预选择的缓冲剂中在实施例1提供的试验中快速并容易地验证，而无需过度实验。
在一个实施方式中，本发明的反应混合物、组合物、试剂盒或方法包括或使用与SEQ ID NO:1或SEQ ID NO:102的序列同一性为至少80％、85％、90％、95％、98％、99％或100％(例如，与SEQ ID NO:1或SEQ ID NO:102的序列同一性为100％)的链置换聚合酶。在另一个实施方式中，本发明的反应混合物、组合物、试剂盒或方法包括或使用结合结构域与SEQ ID NO:2的序列同一性为至少80％、85％、90％、95％、98％、99％或100％(例如，与SEQID NO:2的序列同一性为100％)的聚合酶。在另一个实施方式中，本发明的反应混合物、组合物、试剂盒或方法包括或使用与SEQ ID NO:1或SEQ ID NO:102并且与SEQ ID NO:2或SEQID NO:3或SEQ ID NO:33至SEQ ID NO:97中任意者的序列同一性为至少80％、85％、90％、95％、98％、99％或100％(例如，与SEQ ID NO:1或SEQ ID NO:102并且与SEQ ID NO:2或SEQID NO:3或SEQ ID NO:33至SEQ ID NO:97中任意者的序列同一性为100％)的聚合酶。这些组合物可以在其中聚合酶为链置换性的反应条件下使用。组合物可以在其中任何与聚合酶活性有关的3’-5’核酸外切酶活性是有活性的反应条件下使用。当在反应中使用限制性酶比如NotI时这一点会有帮助。在该情形中，3’-5’核酸外切酶可以除去双链3’-末端上的侧翼序列。然而，如果使用在切除的片段上产生平端的限制性核酸内切酶，则可以不要求有3’-5’核酸外切酶活性。组装反应可以在等温条件下进行。在一个实施方式中，等温条件为50℃。
表1.聚合酶列表
Methanocaldococcus vulcanius M7 SP-13 gi|502573182 SEQ ID NO:33
Archaeoglobus fulgidus DSM 4304 SP-16 gi|499180464 SEQ ID NO:34
Archaeoglobus profundus DSM 5631 SP-17 gi|502704426 SEQ ID NO:35
Caldicellulosiruptor hydrothermalis 108 SP-19 gi|503168530 SEQ ID NO:36
Desulfurococcus mucosus DSM 2162 SP-27 gi|503328138 SEQ ID NO:37
Pyrolobus fumarii SP-29 gi|503791850 SEQ ID NO:38
Pyrobaculum oguniense CH SP-30 gi|379003208 SEQ ID NO:39
Staphylothermus marinus F1 SP-33 gi|500164563 SEQ ID NO:40
Pyrococcus yayaosii CH1 SP-42 gi|503672202 SEQ ID NO:41
Thermococcus sp.AM4-del SP-43 gi|503888003 SEQ ID NO:42
Thermococcus hydrothermalis SP-44 gi|17375628 SEQ ID NO:43
Thermococcus thioreducens SP-45 gi|117958105 SEQ ID NO:44
Thermococcus waiotapuensis SP-46 gi|378813034 SEQ ID NO:45
Thermococcus sibiricus MM 739 SP-47 gi|506329477 SEQ ID NO:46
Pyrococcus glycovorans SP-48 gi|7288074 SEQ ID NO:47
Pyrococcus sp.NA2 SP-49 gi|503513858 SEQ ID NO:48
Ferroglobus placidus DSM 10642 SP-61 gi|502730992 SEQ ID NO:49
Palaeococcus ferrophilus DSM 13482 SP-5 gi|851288004 SEQ ID NO:50
Thermococcus gammatolerans EJ3 SP-50 gi|506339349 SEQ ID NO:51
Thermococcus celericrescens SP-51 gi|332308985 SEQ ID NO:52
Vulcanisaeta distributa DSM 14429 SP-60 gi|503101260 SEQ ID NO:53
Methanopyrus kandleri AV19 SP-7 gi|20094475 SEQ ID NO:54
Thermoproteus neutrophilus V24Sta SP-9 gi|171185774 SEQ ID NO:55
表2.DNA结合蛋白质
Figure BDA0001280816920000251
Figure BDA0001280816920000261
在本发明的实施方式中，反应混合物、组合物、试剂盒或方法可以包括或使用5’-3’核酸外切酶，比如T5/5’-3’核酸外切酶，其对温度敏感，并可以通过将温度升高到50℃以上失活。在一个实施方式中，5’-3’核酸外切酶具有核酸外切酶活性以及ss核酸内切酶活性。在一些实施方式中，反应混合物还可以包括连接酶，例如要求NAD+的连接酶和/或热稳定性连接酶，例如Taq连接酶。在优选的实施方式中，反应混合物可以包括ss结合蛋白质。ss结合蛋白质可以是热稳定性的，例如，ET SSB。组装反应可以在等温条件下进行。
在某些实施方式中，任选使用连接酶。例如，当将组装的片段直接导入到载体中用于宿主细胞转化时，不需要有连接酶，因为宿主细胞比如大肠杆菌能够在体内修复切口。然而，如果出于在转化之前确认正确组装的目的对组装的片段进行扩增，则期望使用连接酶来封闭切口，并且使聚合酶能够扩增整个靶标DNA。
单个片段的克隆可以使用其序列得自任何数据库或出版物的化学合成的多核苷酸片段，其中多核苷酸片段具有重叠序列。通过将多核苷酸插入到质粒中邻近适用于切割所插入多核苷酸的限制性内切酶位点的位点中，可以在质粒中克隆这些片段。
可以使用任何质粒。本实施例使用包含氯霉素基因作为选择标志物的市售pACYC184。可以使用任何选择标志物代替氯霉素抗性基因。类似地，可以选择任何切割酶的特异性识别位点，只要该切割酶能够在寡核苷酸末端特异性切割以产生交错末端或平端，其中除了邻近所关注的片段的末端的工程化的位置以外，特异性切割位点不发生在所关注的片段中。在本实施例中，已经通过DNA合成的方法将产生交错末端的8碱基切割者NotI的识别位点(CGCCGGCG)引入邻近于所关注的多核苷酸。但是，该位点可以存在于所选择的质粒中，或者通过扩增引物加在所关注的合成寡核苷酸上。特异性切割酶的实例包括限制性核酸内切酶和归巢(homing)核酸内切酶。
一旦所关注的寡核苷酸或DNA片段已经化学合成或由现有DNA克隆或扩增并克隆到具有选择标志物的载体中，则优选通过酶切割将其切除。然后将已经合成或扩增以便包括与其要接合的相邻片段或寡核苷酸的重叠序列的片段或寡核苷酸在组装反应中组装。
在所选择的杂交条件下，反应混合物中的5’-3’核酸外切酶(例如，浓度范围0.004-0.016U/μl)回切(chew back)片段或寡核苷酸5’末端处的任意ss区，并继续回切经过重叠序列区，并可以进一步继续有限的距离(例如，至少100碱基)，以提供3’ss区(参见，例如，图2A-2C和图7)。同时，通过图1A-1E和实施例1的试验确定的链置换聚合酶(例如，浓度范围0.005U/μl-0.5U/μl)修复杂交的ds区和任何残余ss区之间残留的间隙。由于聚合酶是链置换性的，其可以置换额外的下游序列，以形成ss侧翼。但是，T5核酸外切酶的ss核酸内切酶活性将会除去该侧翼，并且任何相关的切口均可以通过连接酶(例如，浓度范围0.001U/μl-20U/μl)修复。
片段一经组装到更大一条DNA中，将其在选择性压力下在宿主细胞菌落中克隆，来自这些菌落的DNA可以从载体中释放，并再次与其他片段组装，并转化到宿主细胞中，从而多次延长DNA的大小。宿主细胞可以是感受态细菌细胞，或者可以是酵母细胞或其它真核细胞。
本文所述的组装方法经发现非常有效。例如，可以使用0.02nM-100nM寡核苷酸(ss)或DNA片段(ds)来组装更大的片段，其中反应中使用的ss寡核苷酸的浓度可以比类似组装反应中使用的ds DNA片段的量高至多大约50倍。类似地，可以使用等摩尔浓度的包含单一片段和选择标志物的质粒和类似量的包含组装片段的具有不同选择标志物的载体。这些量意在用于指导，但无论组装效率是否提高，这些量均可以降低。例如，如通过使用lac1Z作为指示物的组装体的菌落数目所测定，加入钾盐KCl能够将产生组装体的效率增加1.5倍(参见，例如，图5)。
用于两个ds DNA分子之间或者导入线性化载体的ss靶标寡核苷酸的组装过程也是非常高效的。本文提供了一个无意于限定的实例，其使用特定/随机序列来识别可引入到细胞中以确定表型改变的CRISPR-Cas基因编辑方案的向导RNA。最初，什么序列可以适合于实现该目的可能并不知晓。包含简并序列的文库的产生使得该类型的分析成为可能。基于CRISPR/Cas-9的基因编辑在基因组编辑领域迅速普及。由于最常用的包含Cas9的质粒的尺寸，将sgRNA或sgRNA文库构建至Cas9/sgRNA表达载体中会很繁琐。使用ss DNA寡核苷酸，该方法解决了这一问题。
在单独的实施方式中，可以将表2的任何DNA结合结构域与Bst聚合酶、Bst大片段或其突变体融合(参见，例如，US 8,993,298和US 2015/0152396，其包括本文描述并要求保护的所有Bst变体)。
试剂盒
本公开另外提供用于实施上述方法的试剂盒。在某些实施方式中，试剂盒可以包含：i.5’-3’核酸外切酶，ii.任选的连接酶，iii.链置换聚合酶：和iv.ss DNA结合蛋白质。试剂盒组分可以合并在一容器中，或者每个组分可以在其各自的容器中。例如，试剂盒组分可以合并在单个反应管中、或者一个或多个不同的反应管中。上文描述了该试剂盒组分进一步的细节。根据如何实施所述方法，试剂盒还可以包含上文和下文中所述的可以用于该方法的其他试剂，例如，错配修复酶，比如mutHLS、cel-1核酸酶、T7 endo 1、uvrD、T4EndoVII、大肠杆菌EndoV、缓冲剂、dNTP、向其中插入合成子的质粒和/或接收质粒的感受态细胞、对照物等。在一些实施方式中，试剂盒不含非链置换聚合酶和/或拥挤剂。
除上述组分以外，试剂盒还包括使用试剂盒组分以实施本方法的说明书。用于实施本方法的说明书通常记录在合适的记录介质上。例如，说明书可以打印在比如纸或塑料等的介质上。这样，说明书可以作为包装说明书存在于试剂盒中、在试剂盒或其组分的容器标签中(即，与包装或分包装关联)等。在其他实施方式中，说明书作为电子存储数据文件存在，存在于合适的计算机可读存储介质中，例如，CD-ROM、软盘等。在又一其他的实施方式中，实际的说明书不存在于试剂盒中，而是提供用于从远程资源例如经由互联网获得的方法。该实施方式的实例为包括可以浏览说明书和/或可以从其下载说明书的网络地址的试剂盒。就说明书而言，获取说明书的这一方法记录在合适的基质上。
本文所述的用于组装片段并形成合成子的组合物、试剂盒和方法产生ds完全密封DNA的产物，其可以用作PCR、RCA的模板、或许多种其他分子生物学应用，包括直接转化感受态细菌或转染真核宿主细胞。
为了进一步说明本发明，提供以下具体实施例，应理解到其提供是为了对本发明进行说明，而不应理解成以任何方式限定其范围。
本文引用的所有参考文献包括2014年8月27日提交的美国临时申请第62/042,527号、2015年7月7日提交的第62/189,599号加上2015年7月16日提交的第62/193,168号，将其并入以供参考。
实施例
实施例1：确定聚合酶链置换特性的试验
开发一种区分链置换和非链置换聚合酶的试验——制备包含10nM FAM-引物/模板/封闭寡核苷酸、
Figure BDA0001280816920000301
缓冲液(New England Biolabs,Ipswich,MA)(图1A)和0.1mM dNTP的10μl反应物。图1B是作为对照的不存在任何聚合酶的FAM标记引物。将链置换DNA聚合酶加入到反应物中，并在50℃下与稀释10倍的1μl样品一起孵育30分钟，并通过毛细管电泳进行分析，FAM引物通过被置换的封闭寡核苷酸得以延长。结果示出于图1D-1E中。图2D中确定的链置换聚合酶Bst聚合酶的峰位置与非天然聚合酶SPB49F观察到的峰对应。少量迁移的原因在于3’-5’核酸外切酶活性引起的SPB49F产生的平端，这在Bst聚合酶中并不存在，使得Bst聚合酶复制的产物具有3’dA。图1C示出了非链置换聚合酶-T4DNA聚合酶的产物，其中合成在封闭引物处终止。
实施例2：使用链置换聚合酶由6片段合成大的DNA分子并使用链置换聚合酶确认 组装有效
使用
Figure BDA0001280816920000302
PCR克隆试剂盒(New England Biolabs,Ipswich,MA)分别由PCR产物(片段(Frags)1、2、3、4、5，其一起覆盖Lacl-和LacZ基因区)构建质粒A、B、C、D和E。
在该实验中，使用以下浓度的整合到单独质粒中的5个不同片段：50ng的各PCR(“片段”来源)，和25ng的包含氨苄青霉素的质粒pMiniTTM载体(NEB#E1202)。首先使用PCR对5种用于组装的片段进行扩增。制备Lacl-lacZ DNA片段组装系统中使用的引物如下：
5PLacIZ-pACYC184VF1 ttggtctggtgtcaaaaatgaATCGTCACGGCGATTTATG(SEQ ID NO：4)
5PLacIZ-pACYC184VR1 gggtcattttcggcgaggacTGCATCAACGCATATAGCG(SEQ ID NO：5)
Not-IZ F1 GCGGCCGCgtcctcgccgaaaatgacccagag(SEQ ID NO：6)
Not-IZ R1 GCGGCCGCtggtgtcgatggtagaacgaagcg(SEQ ID NO：7)
Not-IZ F2 GCGGCCGCcccactgacgcgttgcgcgagaag(SEQ ID NO：8)
Not-IZ R2 GCGGCCGCggctgcgcaactgttgggaagggc(SEQ ID NO：9)
Not-IZ F3 GCGGCCGCtgcagcacatccccctttcgccag(SEQ ID NO：10)
Not-IZ R3 GCGGCCGCatgatgctcgtgacggttaacgcc(SEQ ID NO：11)
Not-IZ F4 GCGGCCGCaggtgcggattgaaaatggtctgc(SEQ ID NO：12)
Not-IZ R4 GCGGCCGCtcaccgcttgccagcggcttacca(SEQ ID NO：13)
Not-IZ F5 GCGGCCGCgaatacctgttccgtcatagcgat(SEQ ID NO：14)
Not-IZ R5 GCGGCCGCtcatttttgacaccagaccaactgg(SEQ ID NO：15)
将扩增的片段克隆并测序，以确认扩增过程中没有引入错误。
PCR扩增的片段1的序列(SEQ ID NO：16)：
GCGGCCGCgtcctcgccgaaaatgacccagagcgctgccggcacctgtcctacgagttgcatgataaagaagacagtcataagtgcggcgacgatagtcatgccccgcgcccaccggaaggagctgactgggttgaaggctctcaagggcatcggtcgagatcccggtgcctaatgagtgagctaacttacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgccagggtggtttttcttttcaccagtgagacgggcaacagctgattgcccttcaccgcctggccctgagagagttgcagcaagcggtccacgctggtttgccccagcaggcgaaaatcctgtttgatggtggttaacggcgggatataaccaacgcgcagcccggactcggtaatatcccactaccgagatatccgcaccaacgcgcagcccggactcggtaatggcgcgcattgcgcccagcgccatctgatcgttggcaaccagcatcgcagtgggaacgatgccctcattcagcatttgcatggtttgttgaaaaccggacatggcactccagtcgccttcccgttccgctatcggctgaatttgattgcgagtgagatatttatgccagccagccagacgcagacgcgccgagacagaacttaatgggcccgctaacagcgcgatttgctggtgacccaatgcgaccagatgctccacgcccagtcgcgtaccgtcttcatgggagaaaataatactgttgatgggtgtctggtcagagacatcaagaaataacgccggaacattagtgcaggcagcttccacagcaatggcatcctggtcatccagcggatagttaatgatcagcccactgacgcgttgcgcgagaagattgtgcaccgccgctttacaggcttcgacgccgcttcgttctaccatcgacaccaGCGGCCGC
PCR扩增的片段2的序列(SEQ ID NO:17)：
GCGGCCGCcccactgacgcgttgcgcgagaagattgtgcaccgccgctttacaggcttcgacgccgcttcgttctaccatcgacaccaccacgctggcacccagttgatcggcgcgagatttaatcgccgcgacaatttgcgacggcgcgtgcagggccagactggaggtggcaacgccaatcagcaacgactgtttgcccgccagttgttgtgccacgcggttgggaatgtaattcagctccgccatcgccgcttccactttttcccgcgttttcgcagaaacgtggctggcctggttcaccacgcgggaaacggtctgataagagacaccggcatactctgcgacatcgtataacgttactggtttcacattcaccaccctgaattgactctcttccgggcgctatcatgccataccgcgaaaggttttgcgccattcgatggtgtccgggatctcgacgctctcccttatgcgactcctgcattaggaagcagcccagtagtaggttgaggccgttgagcaccgccgccgcaaggaatggtgcatgcaaggagatggcgcccaacagtcccccggccacggggcctgccaccatacccacgccgaaacaagcgctcatgagcccgaagtggcgagcccgatcttccccatcggtgatgtcggcgatataggcgccagcaaccgcacctgtggcgccggtgatgccggccacgatgcgtccggcgtagaggatcgagatctcgatcccgcgaaattaatacgactcactataggggaattgtgagcggataacaattcccctctagaaataattttgtttaactttaagaaggagatatacatatgaccatgattacggattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagccGCGGCCGC
PCR片段3的序列(SEQ ID NO:18)：
GCGGCCGCtgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgctttgcctggtttccggcaccagaagcggtgccggaaagctggctggagtgcgatcttcctgaggccgatactgtcgtcgtcccctcaaactggcagatgcacggttacgatgcgcccatctacaccaacgtgacctatcccattacggtcaatccgccgtttgttcccacggagaatccgacgggttgttactcgctcacatttaatgttgatgaaagctggctacaggaaggccagacgcgaattatttttgatggcgttaactcggcgtttcatctgtggtgcaacgggcgctgggtcggttacggccaggacagtcgtttgccgtctgaatttgacctgagcgcatttttacgcgccggagaaaaccgcctcgcggtgatggtgctgcgctggagtgacggcagttatctggaagatcaggatatgtggcggatgagcggcattttccgtgacgtctcgttgctgcataaaccgactacacaaatcagcgatttccatgttgccactcgctttaatgatgatttcagccgcgctgtactggaggctgaagttcagatgtgcggcgagttgcgtgactacctacgggtaacagtttctttatggcagggtgaaacgcaggtcgccagcggcaccgcgcctttcggcggtgaaattatcgatgagcgtggtggttatgccgatcgcgtcacactacgtctgaacgtcgaaaacccgaaactgtggagcgccgaaatcccgaatctctatcgtgcggtggttgaactgcacaccgccgacggcacgctgattgaagcagaagcctgcgatgtcggtttccgcgaggtgcggattgaaaatggtctgctgctgctgaacggcaagccgttgctgattcgaggcgttaaccgtcacgagcatcatGCGGCCGC
PCR片段4的序列(SEQ ID NO:19)：
GCGGCCGCaggtgcggattgaaaatggtctgctgctgctgaacggcaagccgttgctgattcgaggcgttaaccgtcacgagcatcatcctctgcatggtcaggtcatggatgagcagacgatggtgcaggatatcctgctgatgaagcagaacaactttaacgccgtgcgctgttcgcattatccgaaccatccgctgtggtacacgctgtgcgaccgctacggcctgtatgtggtggatgaagccaatattgaaacccacggcatggtgccaatgaatcgtctgaccgatgatccgcgctggctaccggcgatgagcgaacgcgtaacgcgaatggtgcagcgcgatcgtaatcacccgagtgtgatcatctggtcgctggggaatgaatcaggccacggcgctaatcacgacgcgctgtatcgctggatcaaatctgtcgatccttcccgcccggtgcagtatgaaggcggcggagccgacaccacggccaccgatattatttgcccgatgtacgcgcgcgtggatgaagaccagcccttcccggctgtgccgaaatggtccatcaaaaaatggctttcgctacctggagagacgcgcccgctgatcctttgcgaatacgcccacgcgatgggtaacagtcttggcggtttcgctaaatactggcaggcgtttcgtcagtatccccgtttacagggcggcttcgtctgggactgggtggatcagtcgctgattaaatatgatgaaaacggcaacccgtggtcggcttacggcggtgattttggcgatacgccgaacgatcgccagttctgtatgaacggtctggtctttgccgaccgcacgccgcatccagcgctgacggaagcaaaacaccagcagcagtttttccagttccgtttatccgggcaaaccatcgaagtgaccagcgaatacctgttccgtcatagcgataacgagctcctgcactggatggtggcgctggatggtaagccgctggcaagcggtgaGCGGCCGC
PCR片段5的序列(SEQ ID NO:20)：
GCGGCCGCgaatacctgttccgtcatagcgataacgagctcctgcactggatggtggcgctggatggtaagccgctggcaagcggtgaagtgcctctggatgtcgctccacaaggtaaacagttgattgaactgcctgaactaccgcagccggagagcgccgggcaactctggctcacagtacgcgtagtgcaaccgaacgcgaccgcatggtcagaagccgggcacatcagcgcctggcagcagtggcgtctggcggaaaacctcagtgtgacgctccccgccgcgtcccacgccatcccgcatctgaccaccagcgaaatggatttttgcatcgagctgggtaataagcgttggcaatttaaccgccagtcaggctttctttcacagatgtggattggcgataaaaaacaactgctgacgccgctgcgcgatcagttcacccgtgcaccgctggataacgacattggcgtaagtgaagcgacccgcattgaccctaacgcctgggtcgaacgctggaaggcggcgggccattaccaggccgaagcagcgttgttgcagtgcacggcagatacacttgctgatgcggtgctgattacgaccgctcacgcgtggcagcatcaggggaaaaccttatttatcagccggaaaacctaccggattgatggtagtggtcaaatggcgattaccgttgatgttgaagtggcgagcgatacaccgcatccggcgcggattggcctgaactgccagctggcgcaggtagcagagcgggtaaactggctcggattagggccgcaagaaaactatcccgaccgccttactgccgcctgttttgaccgctgggatctgccattgtcagacatgtataccccgtacgtcttcccgagcgaaaacggtctgcgctgcgggacgcgcgaattgaattatggcccacaccagtggcgcggcgacttccagttcaacatcagccgctacagtcaacagcaactgatggaaaccagccatcgccatctgctgcacgcggaagaaggcacatggctgaatatcgacggtttccatatggggattggtggcgacgactcctggagcccgtcagtatcggcggaattccagctgagcgccggtcgctaccattaccagttggtctggtgtcaaaaatgaGCGGCCGC
按设计的最终组装的顺序(片段1和2之间、2和3之间、3和4之间、4和5之间)，5个片段各自与相邻片段具有80bp的重叠区。片段1和5还与载体末端共享20bp重叠。可以使用任何可用的载体，例如，pACYC184(New England Biolabs,Ipswich,MA)。通过反向PCR制备pACYC184载体，其使得可以在用
Figure BDA0001280816920000341
(New England Biolabs,Ipswich,MA)处理和热灭活之后在上述组装混合物的存在下进行片段1-5的组装(参见图2A-2C)。
在组装过程中，从阴影区域延伸的核苷酸通过T5核酸外切酶降解，同时灰色显示的核苷酸通过聚合酶除去。片段组装并转化到大肠杆菌中之后，在具有IPTG和X-Gal的平板上记录通过蓝色/白色选择确定的富有成效组装。
将T5核酸外切酶、Taq连接酶、链置换DNA聚合酶和ss结合蛋白质(ET SSB)在缓冲液中合并在反应混合物中，以形成Mix 1。这些酶均获自New England Biolabs,Ipswich,MA。将5种150ng NotI-HF-消化的质粒(质粒A、B、C、D和E)与105ng载体以及与Mix 1或GAMM混合成总体积20μl。将反应物在50℃下孵育60分钟。使用2μl组装的产物转化到NEB 5-α(New England Biolabs,Ipswich,MA)感受态细胞中。然后将细胞涂布在包含氯霉素的平板上。阳性组装体可以在具有氯霉素+IPTG+X-Gal的平板上作为蓝色菌落识别出，并在37℃下孵育过夜。
转化之前确认所有片段均接合并连接的组装产物的PCR包括以下步骤：在确保5片段和载体连接在一起的PCR中使用1μl组装产物。使用在载体上退火的成对PCR引物扩增整个组装的LaclZ基因(5.3kb)。条带1和2是复制PCR结果。条带M是来自New EnglandBiolabs,Ipswich,MA的2-log DNA梯状带(参见图4)。
测序结果获自选取的8个菌落，并且出于Sanger测序的目的将质粒DNA提纯。使用6个引物对4.8kb片段进行测序。片段之间的接合序列以及从重叠区的延长区均显示出小于2％的序列错误。
用于对组装的DNA进行测序的引物：
Figure BDA0001280816920000342
Figure BDA0001280816920000351
实施例3：将单链寡核苷酸组装到线性化载体或两个不同的ds DNA中
与用于靶向智人(H.sapiens)的基因的sgRNA对应的寡核苷酸设计如下：
1.筛选具有所需靶标序列的PAM序列。例如，以下中的NGG
5’GCGAAGAACCTCTTCCCAAGANGG3’(SEQ ID NO:27)
2.设计包含两侧为部分U6启动子序列和骨架RNA序列的21核苷酸靶标序列的71-碱基ss DNA寡核苷酸。
参见例如图8A-C，其中ss寡核苷酸确定为：
5’ATCTTGTGGAAAGGACGAAACACCGGCGAAGAACCTCTTCCCAAGAGTTTTAGAGCTAGAAATAGCAAGTT3’(SEQ ID NO:28)
或者图9A-C，其中ss寡核苷酸设计成产生以下随机文库：
5’ATCTTGTGGAAAGGACGAAACACCGN21GTTTTAGAGCTAGAAATAGCAAGTT3’(SEQ ID NO:29)
3.在1×NEBuffer 2(New England Biolabs,Ipswich,MA)中制备ss DNA寡核苷酸，最终浓度为0.2μM。
4.形成包含5μl ss DNA寡核苷酸(0.2μM)、30ng限制性内切酶-线性化载体和ddH2O的10μl反应混合物。
5.适合用于上述方法的载体是来自Life Technology的ds载体(
Figure BDA0001280816920000352
具有OFP报告子的CRISPR核酸酶载体试剂盒，目录号：A21174)。其他载体如质粒#42230(pX330-U6-Chimeric_BB-CBh-hSpCas9)由Addgene提供(具体参见https://www.addgene.org/42230/)。另外可选地，可以使用在U6启动子的控制下包含sgRNA骨架的任意质粒。
6.将包含ss结合蛋白质、连接酶、核酸外切酶和聚合酶的10μl主体混合物加入到反应混合物中，并将组装反应物在50℃下孵育1小时。
7.将NEB 10-β感受态大肠杆菌遵照制制造商(New England Biolabs)的方案用2μl组装产物转化。
8.将100μl转化的细胞涂布在具有氨苄青霉素抗生素的平板上，并在37℃下孵育过夜。
9.选取10个菌落使其生长，并对质粒DNA进行提纯用于测序。
与常规的必须合成两种寡核苷酸并将其再退火的克隆方法不同，该实施例提供一种简单的方式来设计寡核苷酸并将其用所需载体进行组装，其相对于常规方法表现出显著的改善，特别是在节约时间、便于使用和成本方面。
SEQ ID NO:1
MILDADYITEDGKPIIRLFKKENGRFKVEYDRNFRPYIYALLKDDSAIDDVRKITSERHGKVVRVIDVEKVKKKFLGRPIEVWKLYFEHPQDVPAMRDKIREHPAVIDIFEYDIPFAKRYLIDKGLIPMEGNEELTFLAVDIETLYHEGEEFGKGPIIMISYADEEGAKVITWKKIDLPYVEVVANEREMIKRLIKVIREKDPDVIITYNGDNFDFPYLLKRAEKLGMKLPLGRDNSEPKMQRLGDSLAVEIKGRIHFDLFPVIRRTINLPTYTLEAVYEAIFGKQKEKVYPHEIAEAWETGKGLERVAKYSMEDAKVTYELGKEFFPMEAQLARLVGQPLWDVSRSSTGNLVEWYLLRKAYERNELAPNKPDEREYERRLRESYEGGYVKEPERGLWEGIVSLDFRSLYPSIIITHNVSPDTLNKEGCGEYDEAPEVGHRFCKDFPGFIPSLLGSLLEERQKIKKRMKESKDPVERKLLDYRQRAIKILANSFYGYYGYAKARWYCKECAESVTAWGRQYIELVRRELEERGFKVLYIDTDGLYATIPGEKNWEEIKRRALEFVNYINSKLPGILELEYEGFYTRGFFVTKKKYALIDEEGKIVTRGLEIVRRDWSEIAKETQAKVLEAILKHGNVEEAVKIVKEVTEKLSNYEIPVEKLVIYEQITRPLNEYKAIGPHVAVAKRLAAKGIKIKPGMVIGYVVLRGDGPISKRAIAIEEFDGKKHKYDAEYYIENQVLPAVERILKAFGYKREDLRWQKTKQVGLGAWLKVKKS
SEQ ID NO:2
IINPQARLTPLELEILEIIKQKKSITITEIKEILSERRKSEYPLSLVSEYISRLERKGYVKKIAKGRKKFVEALI
SEQ ID NO:3
MILDADYITEDGKPIIRLFKKENGRFKVEYDRNFRPYIYALLKDDSAIDDVRKITSERHGKVVRVIDVEKVKKKFLGRPIEVWKLYFEHPQDVPAMRDKIREHPAVIDIFEYDIPFAKRYLIDKGLIPMEGNEELTFLAVDIETLYHEGEEFGKGPIIMISYADEEGAKVITWKKIDLPYVEVVANEREMIKRLIKVIREKDPDVIITYNGDNFDFPYLLKRAEKLGMKLPLGRDNSEPKMQRLGDSLAVEIKGRIHFDLFPVIRRTINLPTYTLEAVYEAIFGKQKEKVYPHEIAEAWETGKGLERVAKYSMEDAKVTYELGKEFFPMEAQLARLVGQPLWDVSRSSTGNLVEWYLLRKAYERNELAPNKPDEREYERRLRESYEGGYVKEPERGLWEGIVSLDFRSLYPSIIITHNVSPDTLNKEGCGEYDEAPEVGHRFCKDFPGFIPSLLGSLLEERQKIKKRMKESKDPVERKLLDYRQRAIKILANSFYGYYGYAKARWYCKECAESVTAWGRQYIELVRRELEERGFKVLYIDTDGLYATIPGEKNWEEIKRRALEFVNYINSKLPGILELEYEGFYTRGFFVTKKKYALIDEEGKIVTRGLEIVRRDWSEIAKETQAKVLEAILKHGNVEEAVKIVKEVTEKLSNYEIPVEKLVIYEQITRPLNEYKAIGPHVAVAKRLAAKGIKIKPGMVIGYVVLRGDGPISKRAIAIEEFDGKKHKYDAEYYIENQVLPAVERILKAFGYKREDLRWQKTKQVGLGAWLKVKKSGTGGGGIINPQARLTPLELEILEIIKQKKSITITEIKEILSERRKSEYPLSLVSEYISRLERKGYVKKIAKGRKKFVEALI
SEQ ID NO:33
MKEKAPKIDALIDCTYKTEDNRAVIYLYLLENILKDREFSPYFYVEMLKDRIEKEDIDKIKEFLLKEDLLKFVENLEVVNKTILKKEKEIVKIIATHPQRVPKLRKIKECDIVKEIYEHDIPFAKRYLIDSDIVPMTYWDFENRKQVSIEIPKLKTVSFDMEVYNRDTEPDPEKDPILMASFWDDNGGKVITYKHFDHSNIEVVNSEKDLIKKIVEMLRQYDVIFTYNGDNFDFPYLKARAKIYGIDIKLGRDGEELKIKRGGMEFRSYIPGRVHIDLYPISRRLLKLTKYTLEDVVYNLFGIEKLKIPHTKIVDYWANNDKILIEYSLQDAKYTHKIGKYFFPLEVMFSRIVNQTPFEITRMSSGQMVEYLLMKNAFKENMIVPNKPDEKEYRKRLLTSYEGGYVKEPEKGMFEDIISMDFRCHPRGTKVIVKNNGLTDIENVKVGDYVLGIDGWQKVKRVWKYPYNGFLVNVNGLKSTPNHKIPVIKKENGKDRVIDVSSIYLLNLKGCKILKIKNFESIGMFGKIFKKDTKIKKVKGLLEKIAYIDPREGLVIKVKNEKEDIFKTVIPILKELNILYKQVDEKTIIIDSIDGLLKYIVTIGFNDKNEEKIKEIIKEKSFLEFKELEDIKISIEEYEGYVYDLTLEGRPYYFANGILTHNSLYPSIIIAYNISPETLDCECCKDISEKILGHWFCKKREGLIPKTLRGLIERRINIKNKMKKMESEKEINEEYNLLDYEQRSLKILANSVYGYLAFPRARFYSRECAEVITYLGRKYILETIEEAEKFGFKVIYADSVVKDAKVIIKEDGKIKEIKIEDLFKKVDYTIGDKEYCILNNVETLTIEDTKLVWRKVPYIMRHRTNKKIYRVKVKDRYVDITEDHSIIGVKNNKLVELKPTEIKDDETKLIILNKDLKSYNFASVEEINCIKYSDYVYDIEVENTHRFFANGILVHNTDGFYAVWKEKISKDDLIKKALEFVKYINSKLPGTMELEFEGYFKRGIFITKKRYALIDENGRVIVKGLEFVRRDWSNLARITQRRVLEALLLEGDINKAKKAIQDVIKDLREKKIKKEDLIIYTQLTKNPNEYKTTAPHVEIAKKMMREGKKIKIGDVIGYIIVKGSKSISERAKLPEEVSIEEIDVNYYIDNQILPPVLRIMEAVGVSKNELKKEGTQLTLDRFLK
SEQ ID NO:34
MERVEGWLIDADYETIGGKAVVRLWCKDDQGIFVAYDYNFDPYFYVIGVDEDILKNAATSTRREVIKLKSFEKAQLKTLGREVEGYIVYAHHPQHVPKLRDYLSQFGDVREADIPFAYRYLIDKDLACMDGIAIEGEKQGGVIRSYKIEKVERIPRMEFPELKMLVFDCEMLSSFGMPEPEKDPIIVISVKTNDDDEIILTGDERKIISDFVKLIKSYDPDIIVGYNQDAFDWPYLRKRAERWNIPLDVGRDGSNVVFRGGRPKITGRLNVDLYDIAMRISDIKIKKLENVAEFLGTKIEIADIEAKDIYRYWSRGEKEKVLNYARQDAINTYLIAKELLPMHYELSKMIRLPVDDVTRMGRGKQVDWLLLSEAKKIGEIAPNPPEHAESYEGAFVLEPERGLHENVACLDFASMYPSIMIAFNISPDTYGCRDDCYEAPEVGHKFRKSPDGFFKRILRMLIEKRRELKVELKNLSPESSEYKLLDIKQQTLKVLTNSFYGYMGWNLARWYCHPCAEATTAWGRHFIRTSAKIAESMGFKVLYGDTDSIFVTKAGMTKEDVDRLIDKLHEELPIQIEVDEYYSAIFFVEKKRYAGLTEDGRLVVKGLEVRRGDWCELAKKVQREVIEVILKEKNPEKALSLVKDVILRIKEGKVSLEEVVIYKGLTKKPSKYESMQAHVKAALKAREMGIIYPVSSKIGYVIVKGSGNIGDRAYPIDLIEDFDGENLRIKTKSGIEIKKLDKDYYIDNQIIPSVLRILERFGYTEASLKGSSQMSLDSFFS
SEQ ID NO:35
MIKAWLLDVDYVTENDRAVIRLWCKDDKGVFVAYDRNFLPYFYVIGCKAEDVMKVKVRTNEGIITPLKVEEIEAKSLGKPIKALKVYTRHPQHVPKLREEIKKFAEVREADIPFAYRYLIDKDLACMDGIEIEPIAVKEGVLRAYEVRSVRRVEKKGFPDLKILAFDCEMLAQFMPDPEKDPIIAIAVKCGDFEEVLHGDERDILRRFVSIIKEQDPDIIVGYNQDNFDWPYVKKRAEKFGIRLDIGRDRSEISFRGGRPKIAGRLNVDLYDIALKIPDVKIKTLKKVAEFLGAKVEEEDIEGRDIYKCWMRGEKEKVFKHVLNDVLTTYRLALELLPMHYELSRMIRLPLDDVARLGRGKQVDYFLLSEAKKINEIAPNPPEIEESYEGAFVLEPARGLHENVACLDFASMYPSIMINFNISPDTLVKGECEDCYVAPEVGHKFRKSPDGFFKRILKMLIEKRREMKRQMKELDPDSEDYKLLDIKQQTLKVLTNSFYGYTGWNLARWYCRECAEATTAWGRYFIKRAVKIAESMGFEVLYGDTDSLFIKKNKLNLKDLEKECLKLIDVISKELPIQLEIDEFYKAIFFVEKKRYAGLTDDDRIVVKGLEVRRGDWCELAKRVQREVIEIILRERNPDKALKFVKNVIEEIKEGKFKLEDYVIYKGLTKKPDKYESKQAHVKAALRAMEMGIYYPIGTKVGFVIVKGGGSISDRAYPIELIEEFDGENLKIRTPSGIMVKKIDKDYYIDHQIIPAVMRILERFGYTEASLKTTIQKTLFDFT
SEQ ID NO:36
MKLVIFDGNSILYRAFFALPELTTSSNIPTNAIYGFINVILKYLEQEKPDYIAVAFDKRGREARKSEYQEYKANRKPMPDNLQVQIPYVREILYALNIPIVEFEGYEADDVIGSLVNKFKNTGLDIVIITGDRDTLQLLDKNVVVKIVSTKFDRTMEDLYTIENIKEKYGVWANQVPDYKALVGDQSDNIPGVKGIGEKSAQKLLEEYSSLEEIYQNLDKIKGSIREKLEAGKDMAFLSKRLATIVCDLPLNVNLEDLRTKEWNKERLYEILVQLEFKSIIKRLGLSENIQFEFVQQRTDIPDVEQRELESISRIRSKEIPLMFVQDEKCFYLYDQESNTVFVTRDRHLVEEILKSDTVKIVYDLKNIFHQLNLEDTDNIKNCEDVMIASYVLDSTRSSYELETLFVSYLNTDIEAVKKDKKMVSVVLLKRLWDDLLRLIDLNSCQFLYENIERPLIPVLYEMEKTGFKVDRDALLQYTKEIENKILKLETQIYQIAGEWFNINSPKQLSYILFEKLKLPVIKKTKTGYSTDAEVLEEFDKHEIVPLILDYRMYTKILTTYCQGLLQAINPSSGRVHTTFIQTGTATGRLASSDPNLQNIPVKYDEGKLIRKVFVPEEGHVLIDADYSQIELRILAHISEDERLINAFKNNIDIHSQTAAEVFGVDIADVTPEMRSQAKAVNFGIVYGISDYGLARDIKISRKEAAEFINKYFERYPKVKEYLDNIVRFARENGYVLTLFNRKRYVKDIKSANRNARSYAERIAMNSPIQGSAADIMKLAMIKVYQKLKENNLKSKIILQVHDELLIEAPYEEKDIVKRIVKREMENAVALKVPLVVEVKEGLNWYETK
SEQ ID NO:37
MEKRVYLVDITYGLVGNSPEIRMFGVDENGEKVVILDRGFRPYFYVIPEEGFEDQVARVIGKMQNVIKADVTERRLFGKPIKVVKVTVTVPDKVRELRDRVKSIQHVKEVLEADIRFYIRYMIDNDIRPGWLMFSNLKPVDNKIGGVSNVYLTETPPTSLDLGIMPRLNYMALDIEVYNPRGTPDPKRDPIIIIALANSNGDVKLLTLDNYKHEREMLNDMMSVIKEWDPDVLFGYNSNKFDMPYLVNRADALNVKLQLSKYGTPPEQSVYGHWSIIGRAHIDLYNFIEDMTDVKRKSLDYVAEYFGVMKRSERVNIPGHRIYQYWDDEGKRSQLIKYARDDVLSTLGLGKILLPYAMQLASVSGLPLDQVGPASVGSRVEMMIMHEAYKMGELAPNRVERPYETYKGAIVLEPKPGIHYNIAVLDFSSMYPNIMLKYNISPDTLVLDSSEGDYYTAPEVGYRFRKSPRGLYASLLQKLIEARREARDEMRNYPEGSFEWVLLNERQRALKIMANAMYGYCGWLGARWYIREVAESVTAWGRYLLKTAMSMAKERGLTVIYGDTDSLFVTYDKDKVADIISRINEMGFEVKIDKVYSKLIFTESKKRYIGLTADGEVDIVGFEAVRGDWSELARNVQERVAELVLRESVDEAVKYVKSVIDDLRNYRFTIDDVIIWKTLDKDINEYKAIQPHVVAARRLMEKGYVVNKGDTVGFVIVKDSGDKLTQRAYPYVFINDVKEIDVDYYVEKQVIPAALRILEVFGVNEAALLGKTGKSILDYFH
SEQ ID NO:38
MTEVVFTVLDSSYEVVGKEPQVIIWGIAENGERVVLIDRSFRPYFYALLAPGADPKQVAQRIRALSRPKSPIIGVEDDKRKYFGRPRRVLRIRTVLPEAVREYRELVKNVDGVEDVLEADIRFAMRYLIDHDLFPFTWYRVEAEPLENKMGFRVDKVYLVKSRPEPLYGEALAPTKLPDLRILAFDIEVYSKQGSPRPERDPVIVIAVKTDDGDEVLFIAEGKDDRKPIREFVEYVKRYDPDIIVGYNNNHFDWPYLLRRARILGIKLDVTRRVGAEPTTSVHGHVSVPGRLNVDLYDYAEEMPEIKIKSLEEVAEYLGVMKKSERVIINWWEIPDYWDDPKKRPLLLQYARDDVRATYGLAEKILPFAIQLSYVTGLPLDQVGAMSVGFRLEWYLIRAAFKMKELVPNRVERPEETYRGAIVLEPLRGVHENIAVLDFSSMYPNIMIKYNVGPDTLVRPGEECGECGCWEAPEVKHRFRRCPPGFFKTVLERLLELRKRVRAEMKKYPPDSPEYRLLDERQKALKVLANASYGYMGWSGARWYCRECAEAVTAWGRHLIRTAINIARKLGLKVIYGDTDSLFVTYDPEKVEKFIKIIEEELGFEIKLEKVYKRVFFTEAKKRYAGLLEDGRIDIVGFEAVRGDWCELAKEVQTKVVEIVLKTSDVNKAVEYVRKIVKELEEGKVPIEKLVIWKTLSKRLEEYTTEAPHVVAAKRMLSAGYRVSPGDKIGYVIVKGGGRISQRAWPYFMVKDPSQIDVTYYVDHQIIPAALRILGYFGITEKKLKASATGQKTLFDFLAKKSK
SEQ ID NO:39
MEIRVWPLDVTYIVVGGVPEVRVFGIAEGGERVVLADRSFRPYFYVDCAGCDPHAVKTHLGRTAPVEGVELVERRFLGRPRQFLKVVAKIPEDVRRLREAASTIPGVRGVYEADIRFYMRYVIDMGVVPCSWNVAEVEVADEKLGSLPVYRVVKWGGAVEGFPPPLRVLAFDIEVYNERGTPDPARDPIVMIAVQSSDGRLEVFEASGRDDRGVLRSFVEYVRSFDPDVVVGYNSNNFDWPYLAERAKAVGVPLRVDRLGGAPQQSVYGHWSVLGRANVDLYNIVDEFPEIKLKTLDRVAEYFGVMRRDERVLIPGHKIYEYWRDPSKRPLLRQYVIDDVRSTYGLAERLLPFLIQLSSVSGLPLDQVAAASVGNRVEWMLLRYAYGLGEVAPNREEREYEPYKGAIVLEPKPGLYSDVLVLDFSSMYPNVMMRYNLSPDTYLEPGEPDPPEGVYVAPEVGHRFRKEPPGFIPQVLRRLVALRRAVREEMKKYQPDTPEYRVLDERQKALKIMANAMYGYTGWVGARWYKKEVAESVTAFARAILKDVIDYARRLGIVVIYGDTDSLFVKKGGDLEKLARYVDEKYGIEIKVDKDYEKVLFTEAKKRYAGLLRDGRIDIVGFEVVRGDWSELAKEVQLKVIELILKARDLSEARQRVIKYVKDVIERLKSGKFDLDDLIIWKTLDKDLGEYKAYPPHVRAALILKKKGYKVGRGTTIGYVVVKGGEKVSERSLPYILVDDLAKIDVDYYIEKQVIPAALRIAEVIGVKEGDLRAGRSEKSLLDFFE
SEQ ID NO:40
MSEKINLEFYFLDNSYEVIGNEPHIIIWGITRDGRRVLLRDRRFRPYFYAILKDKVNIEDLARKIRTYSDPKSPIIGVEPVEKKYFGRKVSALKIITMIPEYVRKYREKIKSLPEVLEVVEADIRFSIRYIIDHDLRPCGWHVAEVVEVPKKPIYRVDAEYEIIGDIKPLEQTLQPDLRIIAFDIEVYNKSGTPRPQTDPIIIIGIMNNNGDIKQFLANKYDDKISVEEFVNYVKTFDPDIIVGYNTDGFDWPYLIERSKYIGVKLDVTRRVGATPRTSTYGHISVPGRLNTDLYHFAEEIPEVKVKSLENVAEYLGVMKKSERVIIEYIDIPKYWDDEKLRPKLLQYNIDDVKSTYGLAEKFLPFAMQLSNITGLPLDQVGAASVGFRLEWYLMREAFRYGELVPNRVERAAESYRGAVVLKPVKGVHENIAVLDFSSMYPNIMIKYNVGPDTIVRNEKCNPDKHNIAPEVGHCFRKEPPGFFKRVLETLLRLRKQIKSEMKKYPPTSYEYRLLDERQKAVKVLANATYGYMGWIHARWYCRECAEAVTAWGRQTIKSAIELARKLGLKVIYGDTDSLFVTYDKDKVEKLIELIQTKLGFEIKIDKIYKRVFFTEAKKRYAGLLEDGRIDIVGFEAVRGDWAEIAKEVQEKVTEILLKENSIDKAIEYVRQVIADLKAGKIPLDKLIIWKTLSKRIEEYSVDAPHVVAAKKLIKAGIKVSTNDKIGYVILKGGGKISSRAEPYIFVKDPKLIDTEYYVDHQIVPAALRILNYFGVTETQLKRAAASAGQKSLFDFFGGKK
SEQ ID NO:41
MILDADYITENGKPVVRIFKKENGEFKVEYDRSFRPYIYALLRDDSAIEDIKKITAERHGKVVRVVEAEKVRKKFLGRPIEVWKLYFEHPQDVPAIREKIREHPAVIDIFEYDIPFAKRYLIDKGLIPMEGNEELKLLAFDIETLYHEGDEFGSGPIIMISYADEKGAKVITWKGVDLPYVEVVSSEREMIKRFLRVIREKDPDVIITYNGDNFDFPYLLKRAEKLGMKLPIGRDGSEPKMQRMGDGFAVEVKGRIHFDIYPVIRRTINLPTYTLEAVYEAVFGRPKEKVYPNEIARAWENCKGLERVAKYSMEDAKVTYELGREFFPMEAQLARLVGQPVWDVSRSSTGNLVEWFLLRKAYERNELAPNRPDEREYERRLRESYEGGYVKEPEKGLWEGIIYLDFRSLYPSIIITHNISPDTLNKEGCNSYDVAPKVGHRFCKDFPGFIPSLLGQLLDERQKIKRKMKATIDPIERKLLDYRQRAIKILANSYYGYYGYAKARWYCKECAESVTAWGREYIELVSRELEKRGFKVLYIDTDGLYATIPGSREWDKIKERALEFVKYINARLPGLLELEYEGFYKRGFFVTKKKYALIDEEGKIITRGLEIVRRDWSEIAKETQARVLEAILKEGNLEKAVKIVKEVTEKLSKYEVPPEKLVIYEQITRDLKDYKAVGPHVAVAKRLAARGIKVRPGMVIGYLVLRGDGPISRRAIPAEEFDPSRHKYDAEYYIENQVLPAVLRILEAFGYRKEDLRYQKTRQAGLDAWLKRKASL
SEQ ID NO:42
MILDTDYITENGKPVIRVFKKENGEFKIEYDRTFEPYFYALLKDDSAIEDVKKVTAKRHGTVVRVKRAEKVQRKFLGRPIEVWKLYFTHPQDVPAIRDKIRAHPAVIDIYEYDIPFAKRYLIDKGLIPMEGDEELTMLAFDIETLYHEGEEFGTGPILMISYADGSEARVITWKKIDLPYVDVVSTEKEMIKRFLRIVKEKDPDVLITYNGDNFDFAYLKKRCEKLGIKFTLGRDGSEPKIQRMGDRFAVEVKGRIHFDLYPVIRRTINLPTYTLEAVYEAVFGKPKEKVYAEEIAQAWESGEGLERVARYSMEDAKVTYELGREFFPMEAQLSRLIGQSLWDVSRSSTGNLVEWFLLRKAYERNELAPNKPDEKELARRRGGYAGGYVKEPERGLWDNIVYLDFRSLYPSIIITHNVSPDTLNREGCKEYDVAPEVGHKFCKDFPGFIPSLLGDLLEERQKIKRKMKATVDPLEKKLLDYRQRAIKILANSYYGYYGYAKARWYCRECAESVTAWGREYIETTIREIEEKFGFKVLYADTDGFFATIPGADAETVKKKAKEFLKYINAKLPGLLELEYEGFYVRGFFVTKKKYAVIDEEGKITTRGLEIVRRDWSEIAKETQARVLEAILKHGDVEEAVRIVKEVTEKLSKYEVPPEKLVIHEQITRDLRDYKATGPHVAVAKRLAARGVKIRPGTVISYIVLKGSGRIGDRAIPFDEFDPTKHRYDAEYYIENQVLPAVERILKAFGYRKEDLRYQKTKQVGLGAWLKVKK
SEQ ID NO:43
MILDTDYITEDGKPVIRIFKKENGEFKIEYDREFEPYIYALLKDDSAIEEVKKITAGRHGRVVKVKRAEKVKKKFLGRPIEVWKLYFTHPQDVPAIRDEIRRHSAVVDIYEYDIPFAKRYLIDKGLIPMEGDEELKMMSFDIETLYHEGEEFGTGPILMISYADEGEARVITWKKIDLPYVEVVSTEKEMIKRFLKVVKEKDPDVLITYNGDNFDFAYLKKRCEKIGIKFTLRRDGSEPKIQRMGDRFAVEVKGRIHFDLYPVIRRTINLPTYTLEAVYEAVFGTPKEKVYPEEITTAWETGEGLERVARYSMEDAKVTYELGREFFPMEAQLSRLIGQSLWDVSRSSTGNLVEWFLLRKAYERNEIAPNKPDERELARRRGGYAGGYVKEPERGLWDNIVYLDFMSLYPSIIITHNVSPDTFNREGCKEYDTAPQVGHKFCKDVQGFIPSLLGALLDERQKIKKRMKASIDPLEKKLLDYRQKAIKILANSYYGYYGYARARWYCKECAESVTAWGRDYIETTIHEIEERFGFKVLYADTDGFFATIPGADAETVKKKAKEFLKYINAKLPGLLELEYEGFYVRGFFVTKKKYAVIDEEGKITTRGLEIVRRDWSEIAKETQARVLEAILRHGDVEEAVRIVKDVTEKLSKYEVPPEKLVIHEQITRELKDYKATGPHVAIAKRLAARGIKIRPGTVISYIVLKGSGRIGDRAIPFDEFDPTKHRYDAEYYIENQVLPAVERILKAFGYKKEELRYQKTRQVGLGAWLKLKGKK
SEQ ID NO:44
MILDADYITEDGKPVVRIFKKENGEFKIEYDREFEPYIYALLRDDSAIEEIKKITADRHGKVVKVKRAEKVQKKFLGRPIEVWKLYFTHPQDVPAIRDEIRKHPAVVDIYEYDIPFAKRYLIDKGLIPMEGDEELKMLAFDIETLYHEGEEFGTGPILMISYADEDGARVITWKKIDLPYVDVVSTEKEMIKRFLKVVKEKDPDVLITYNGDNFDFAYLKKRCEKLGIKFTLGRDGSEPKIQRMGDRFAVEVKGRIHFDLYPLIRRTINLPTYTLEAVYEAVFGKPKEKVYAEEIALAWESGEGLERVARYSMEDAKVTFELGREFFPMEAQLSRLIGQSLWDVSRSSTGNLVEWFLLRKAYERNELAPNKPNERELARRRGGYAGGYVKEPERGLWDNIVYLDFRSLYPSIIITHNVSPDTLNREGCKEYDRAPQVGHKFCKDVPGFIPSLLGSLLDERQKIKRKMKATIDPIEKKLLDYRQRAIKILANSYYGYYGYARARWYCRECAESVTAWGREYIEMAIRELEEKFGFKVLYADTDGLHATIPGADAETVKKKAMEFLKYINPKLPGLLELEYEGFYARGFFVTKKKYAVIDEEGKITTRGLEIVRRDWSEIAKETQARVLEAILRHGDVEEAVRIVKEVTEKLSKYEVPPEKLVIHEQITRELKDYRATGPHVAIAKRLAKRGIKIRPGTVISYIVLKGSGRIGDRAIPFDEFDPTKHKYDAEYYIENQVLPAVERVLKAFGYRKDDLRYQKTRQVGLGAWLKVKKR
SEQ ID NO:45
MILDADYITEDGKPVIRVFKKEKGEFKINYDRDFEPYIYALLKDDSAIEDIKKITAERHGTTVRVTRAERVKKKFLGRPVEVWKLYFTHPQDVPAIRDKIREHPAVVDIYEYDIPFAKRYLIDKGLIPMEGNEELRMLAFDIETLYHEGEEFGEGPILMISYADEEGARVITWKNIDLPYVESVSTEKEMIKRFLKVIQEKDPDVLITYNGDNFDFAYLKKRSETLGVKFILGRDGSEPKIQRMGDRFAVEVKGRIHFDLYPVIRRTINLPTYTLETVYEAIFGQPKEKVYAEEIAQAWESGEGLERVARYSMEDAKATYELGKEFFPMEAQLSRLVGQSLWDVSRSSTGNLVEWFLLRKAYERNELAPNKPDERELARRAESYAGGYVKEPEKGLWENIVYLDYKSLYPSIIITHNVSPDTLNREGCREYDVAPQVGHRFCKDFPGFIPSLLGDLLEERQKVKKKMKATVDPIERKLLDYRQRAIKILANSYYGYYGYANARWYCRECAESVTAWGRQYIETTMREIEEKFGFKVLYADTDGFFATIPGADAETVKKKTKEFLNYINPRLPGLLELEYEGFYRRGFFVTKKKYAVIDEEDKITTRGLEIVRRDWSEIAKETQARVLEAILKHGDVEEAVRIVKEVTEKLSRYEVPPEKLVIYEQITRNLRDYRATGPHVAVAKRLAARGIKIRPGTVISYIVLKGPGRVGDRAIPFDEFDPAKHRYDAEYYIENQVLPAVERILRAFGYRKEDLRYQKTKQAGLGAWLKPKT
SEQ ID NO:46
MILGADYITKDGKPIVRIFKKENGEFKIELDPHFQPYIYALLSEDSAIDEIKQIKGERHGKTVRIVDAVKVEKKFLKKPVKVWKLILEHPQDVPAIRNKIREHPAVQDIYEYDIPFAKRYLIDNGLIPMEGDEELKMLAFDIETFYHEGDEFGKGEIIMISYADEEGARVITWKNIDLPYVDVVSNEREMIKRFIQIIKEKDPDVIITYNGDNFDLPYLIKRAEKLGLRLILSRDNENPVPKIQRMGNSFAVEIKGRIHFDLFPVVKRAVNLPTYTLEAVYETVLGKHKSKLGAEEIAAIWETEEGLKKLAQYSMEDAKATYELGREFFPMEVELAKLIGQSVWDVSRSSTGNLVEWYMLRVAYERNELAPNRPSDEEYKRRLRTTYLGGYVKEPERGLWENIIYLDFRSLYPSIIVTHNVSPDTLERKGCQNYDVAPIVGYKFCKDFSGFIPSILEDLIETRQKIKKEMKSTIDPIKKKMLDYRQRAVKLLANSYYGYMGYPKARWYSKECAESVTAWGRHYIEMTIREIEEKFGFKVLYADTDGFYATIPGADPETIKKKAKEFLNYINSKLPGLLELEYEGFYLRGFFVTKKRYAVIDEEEKITTRGLEVVRRDWSEIAKETQARVLEAILREGSVEKAVEIVKEVVEAITKYKVPLEKLIIHEQITRELRDYKAVGPHVAIAKRLAAKGIKIKPGTIISYIVLRGSGKISDRVVLLTEYDPRKHKYDPDYYIENQVLPAVLRILEAFGYRKEDLKYQSSKQTGLESWLKK
SEQ ID NO:47
MILDADYITEDGKPIIRIFKKENGEFKVEYDRNFRPYIYALLKDDSQIDEVKKITAERHGKIVRIVDVEKVKKKFLGRPIEVWKLYFEHPQDVPAIRDKIREHPAVVDIFEYDIPFAKRYLIDKGLIPMEGDEELKLLAFDIETLYHEGEEFAKGPIIMISYADEEGAKVITWKKVDLPYVEVVSSEREMIKRFLKVIREKDPDVIITYNGDSFDLPYLVKRAEKLGIKLPLGRDGSEPKMQRLGDMTAVEIKGRIHFDLYHVIRRTINLPTYTLEAVYEAIFGKPKEKVYAHEIAEAWETGKGLERVAKYSMEDAKVTYELGREFFPMEAQLSRLVGQPLWDVSRSSTGNLVEWYLLRKAYERNELAPNKPDEREYERRLRESYAGGYVKEPEKGLWEGLVSLDFRSLYPSIIITHNVSPDTLNREGCMEYDVAPEVKHKFCKDFPGFIPSLLKRLLDERQEIKRRMKASKDPIEKKMLDYRQRAIKILANSYYGYYGYAKARWYCKECAESVTAWGREYIEFVRKELEEKFGFKVLYIDTDGLYATIPGAKPEEIKRKALEFVEYINAKLPGLLELEYEGFYVRGFFVTKKKYALIDEEGKIITRGLEIVRRDWSEIAKETQAKVLEAILKHGNVEEAVKIVKEVTEKLSKYEIPPEKLVIYEQITRPLHEYKAIGPHVAVAKRLAARGVKVRPGMVIGYIVLRGDGPISKRAILAEEFDPRKHKYDAEYYIENQVLPAVLRILEAFGYRKEDLRWQKTKQTGLTAWLNVKKK
SEQ ID NO:48
MILDADYITEDGKPIIRLFKKENGRFKVEYDRNFRPYIYALLKDDSAIDDVRKITSERHGKVVRVIDVEKVKKKFLGRPIEVWKLYFEHPQDVPAMRDKIREHPAVIDIFEYDIPFAKRYLIDKGLIPMEGNEELTFLAVDIETLYHEGEEFGKGPIIMISYADEEGAKVITWKKIDLPYVEVVANEREMIKRLIKVIREKDPDVIITYNGDNFDFPYLLKRAEKLGMKLPLGRDNSEPKMQRLGDSLAVEIKGRIHFDLFPVIRRTINLPTYTLEAVYEAIFGKQKEKVYPHEIAEAWETGKGLERVAKYSMEDAKVTYELGKEFFPMEAQLARLVGQPLWDVSRSSTGNLVEWYLLRKAYERNELAPNKPDEREYERRLRESYEGGYVKEPERGLWEGIVSLDFRSLYPSIIITHNVSPDTLNKEGCGEYDEAPEVGHRFCKDFPGFIPSLLGSLLEERQKIKKRMKESKDPVERKLLDYRQRAIKILANSFYGYYGYAKARWYCKECAESVTAWGRQYIELVRRELEERGFKVLYIDTDGLYATIPGEKNWEEIKRRALEFVNYINSKLPGILELEYEGFYTRGFFVTKKKYALIDEEGKIVTRGLEIVRRDWSEIAKETQAKVLEAILKHGNVEEAVKIVKEVTEKLSNYEIPVEKLVIYEQITRPLNEYKAIGPHVAVAKRLAAKGIKIKPGMVIGYVVLRGDGPISKRAIAIEEFDGKKHKYDAEYYIENQVLPAVERILKAFGYKREDLRWQKTKQVGLGAWLKVKKS
SEQ ID NO:49
MEGWLLDADYITAEDGRAVVRLWCKDFDGNTFVVYDRNFQPYFYAFKNGLSKEDIEKIVVKSREGVIKPFKVEEVRRKVFGKEVEVFKIYAYHPQHVPKLREELKKITEVREADIPFAYRYLIDKDLACMDGIRVEGKVREERGLKVIDAEHVERFEIPLPEPKVLAFDCEMLTELGMPDPEKDKIIIIGVKCGDFEEIITGNEREILLRFVEIIKEQDPDVIVGYNQDNFDWPYIRKRAEKLSVKLNIGRDGSEISFRGGRPKIAGRLNVDLYDIAMKLDVKVKTLENVAEFLGRKVELADIEAKDIYKRWTSGDKESVLKYSKQDVLNTYFIAEELLPMHYELSRMIRIPTDDVARIGRGKQVDWFLLSEAYKIGEIAPNPAEVEESYEGAFVLEPSRGLHKNVVCLDFASMYPSIMIAYNISPDTYVFGKCDDCYVAPEVGHKFRKHPDGFFKRILKMLIEKRREIKNQMKSLDRNSREYLLLNIKQQTLKILTNSFYGYTGWSGARWYCRQCAEATTAWGRHLIKSAVEIAKKLGFEVLYGDTDSIFVKKGNLSLEKIRGEVEKLIEEISEKFPVQIEVDEYYKTIFFVEKKRYAGLTEDGILVVKGLEVRRGDWCELAKEVQKKVIEIILKEENPEKAAEYVRKVINDIKSGKVKLEDVVIYKGLTKRPDKYESKQAHVKAALRAMELGIVYNVGSKVGFVVVEGAGNVGDRAYPIDLIEEFDGENLVIRTRSGSIVKKLDKDYYINHQIIPSVLRILERFGYNEASLKGATQKTLDAFW
SEQ ID NO:50
MILDADYITENGKPVVRIFKKENGEFKVEYDRNFEPYIYALLKDDSAIEEIKKITAERHGTVVRITKAEKVERKFLGRPVEVWKLYFTHPQDVPAIRDKIRSHPAVVDIYEYDIPFAKRYLIDKGLVPMEGDEELKMLAFDIETLYHEGEEFAEGPILMISYADESEARVITWKKVDLPYVDAVSTEKDMIKAFLRVVKEKDPDVLITYNGDNFDFAYLKKRCEKLGVKFILGRDGSEPKIQRMGDRFAVDVKGRIHFDLYPVIRRTINLPTYTLEAVYEAIFGRPKEKVYAEEIAQAWETNEGLERVARYSMEDAKVTYELGKEFFPMEAQLSRLIGQPLWDVSRSSTGNLVEWFLLRKAYERNELAPNKPSGREYDERRGGYAGGYVKEPEKGLWENIVYLDYKSLYPSIIITHNVSPDTLNREGCKEYDVAPQVGHRFCKDFPGFIPSLLGDLLEERQKIKRKMKATIDPIERRLLDYRQRAIKILANSYYGYYGYARARWYCKECAESVTAWGREYIEMSIREIEEKYGFKVLYADTDGFHATIPGEDAETIKKKAMEFLKYINSKLPGALELEYEGFYRRGFFVTKKKYAVIDEEGKITTRGLEIVRRDWSEIAKETQARVLEALLKDGNVEEAVSIVKEVTEKLSKYEVPPEKLVIHEQITRELKDYKATGPHVAIAKRLAARGVKIRPGTVISYIVLKGSGRIGDRAIPFDEFDPAKHRYDAEYYIENQVLPAVERILKAFGYRKEDLRYQKTRQVGLGAWLKPKGKK
SEQ ID NO:51
MILDTDYITENGKPVIRVFKKENGEFKIEYDRTFEPYFYALLKDDSAIEDVKKVTAKRHGAVVKVKRAEKVQRKFLGRPIEVWKLYFTHPQDVPAIRDKIRAHPAVVDIYEYDIPFAKRYLIDKGLIPMEGDEELRMLAFDIETLYHEGEEFGTGPILMISYADENEARVITWKKIDLPYVDVVSTEKEMIKRFLRVVKEKDPDVLITYNGDNFDFAYLKKRCEKLGIKFTLGRDGSEPKIQRMGDRFAVEVKGRIHFDLYPVIRRTINLPTYTLEAVYEAVFGKPKEKVYAEEITEAWESGEGLERVARYSMEDAKVTYELGREFFPMEAQLSRLIGQSLWDVSRSSTGNLVEWFLLRKAYERNELAPNKPDERELARRRESYAGGYVKEPERGLWDNIVYLDFRSLYPSIIITHNVSPDTLNREGCKEYDVAPEVGHKFCKDFPGFIPSLLGDLLEERQKIKRKMKATIDPLEKKLLDYRQRAIKILANSFYGYYGYAKARWYCKECAESVTAWGREYIETTIREIEEKFGFKVLYADTDGFFATIPGADAETVKKKAKEFLKYINAKLPGLLELEYEGFYVRGFFVTKKKYAVIDEEGKITTRGLEIVRRDWSEIAKETQARVLEAILKHGDVEEAVRIVKEVTEKLSKYEVPPEKLVIHEQITRDLRDYKATGPHVAVAKRLAAKGVKIRPGTVISYIVLKGSGRIGDRAIPADEFDPTKHRYDAEYYIENQVLPAVERILKAFGYRKEDLRYQKTKQVGLGAWLKVKGKK
SEQ ID NO:52
MILDTDYITEDGKPVIRIFKKDNGEFKIEYDRNFEPYIYALLRDDSAIEDVKKITAERHGRVVKVKRAEKVKKKFLGRPVEVWKLYFTRPQDVPAIRDRIRAHPAVVDIYEYDIPFAKRYLIDKGIIPMEGDEELKMLAFDIETLYHEGEEFAEGPILMISYADENEARVITWKKIDLPFVDVVSTEKEMIKRFLKVIKEKDPDVLITYNGDNFDFAYLKKRCEKFGIKFTLGRDGSDPKIQRMGDRFAVEVKGRIHFDLYPVILRTVNLPTYTLEAVYEAIFGTPKEKVYPEEITTAWETGEGLERVARYSMEDAKVTFELGREFFPMEAQLSRLVGQSFWDVSRSSTGNLVEWFLLRKAYERNELAPNKPDERELVRRRNSYTGGYVKEPERGLWDNIVYLDFRSLYPSIIITHNVSPDTLNREGCKEYDEAPQVGHKFCKDFPGFIPSLLGNLLDERQKIKKRMKATIDPLEKKLLDYRQRAIKILANSYYGYYAYARARWYCKECAESVTAWGREYIEMSIREIEEKYGFKVLYADTDGFHATIPGADAETVKKKAMEFLKYINAKLPGALELEYEGFYKRGFFVTKKKYAVIDEEGKITTRGLEIVRRDWSEIAKETQARVLEALLKDGNVEEAVSIVKEVTEKLGKYEVPPEKLVIHEQITRELKDYKATGPHVAIAKRLAARGVKIRPGTVISYIVLKGSGRIGDRAIPFDEFDPTKHRYDAEYYIENQVLPAVERILKAFGYRAEDLRYQKTRQVGLGVWLQPKGKK
SEQ ID NO:53
MELAFWLLDITYGVIGNTPELRLFGITDDGKRVLVLDRSFRPYFYVIPSGDVNAVFNNVKRKLEGKVLNVEVIKRKMFGNEVDAIRVTATIPEKVRELRELAAEVPGVEDVLEADIRFSQRYLLDMGVKPSNWIVVDQCEEVKGNYQVDLVCLAKSRPRMIEEHKLPSFRVLAFDIEVYNPRGMPNPDRDPVIIISTMTKEDGVKMFVVDDNKNDAKIIREFLDYFRKYDPDIVVGYNNNGFDWPYLVNRSSRVGVRLALSRMGNPPEPSVYGHWSIIGRANVDLYNFIEEISEIKVKSLDRAAEFFGIMKRSERVLIPGHRIHEYWDDKNKRDLLLKYARDDVVSTYGLAEKLLPFAIQLSSISGLPLDQVGAASVGARVEWMIFYEAVKRGELAPNREERPYETYKGAVVLEPRPGLHENIAVIDFSSMYPSIMMKYNVSPDTLVLGDCGDCYVAPEVNYKFRRSPEGLYPGLLRILVESRRRVRDLMKKYPENSPEWVLLNERQRALKVMANAMYGYCGWLGARWYRREVAEAVTAWGRNLLRTVIEKARSLGLPIIYGDTDSLFVRNISDKVDALINYVNNELGFEVKVDKVYRRVLFTEAKKRYVGLTVEGEVDIVGFEAVRGDWAEIAKDVQENVAEIVLTTGDVGKAISYVKSVIDKVKAYQFDIDDVIIWKTLDKSLNEYKVLTPHVAAAKQLVEAGYKVGKGDMIGYVVVKGGGAKLAYKVKPYILIKDIREVDVDYYVEKQIVPAAMRILEVLGVKESQLMEGKAGKSILDYFS
SEQ ID NO:54
MLRTVWVDYARKGEPDVILVGRREDGNPAALVVKGFRPYFYAEVEDGFDPSEVERLSGVVEVEEVLLEHPYGGDRVELLRIVATYPKVVPKLREQVKKLDGVKEVYEADIPFVRRAAVDLNLPPASEVDVSDLDTGSWSGLPAYFADVEDARELDHRPYPIEDLVVASFDLEVLAEPGTTIKGASGPIIAISFAYSTPDGERRNYVITWKGEDESFEVDGVETEVIVCRSEAAALRRFFDEFRRVDPDVVFTYNGDEFDLPYLQHRAGKLGIDVSPLARPAGKRGIILKHGGGRYASDIFGRAHVDLYHTARKNLKLERFTLEEAVKDVLGVEKEEMELADINEAWKRGNLDELMRYSAEDAHYTLELGLELAQVELELSYLTRLPLPDATRFSFGQLAEWRAIYKARQEDILVPNKPTRDEYKRRRRKAYKGAIVFEPEIGLHENVVCVDFASLYPNVMVAHNISPDTFDCDCCPRVTVEEVDDPTDATVAPDVGHKFCKRRKGFFPRLVEGLIERRRELKRRLRKLDTESHPHEAKILDVRQQAYKVLANSYYGYMGWANARWFCRECAESVTAWGRYYISEVRRIAEEKYGLKVVYGDTDSLFVKLPDADLEETIERVKEFLKEVNGRLPVELELEDAYKRILFVTKKKYAGYTEDGKIVTKGLEVVRRDWAPIARETQRRVLKRILADNDPEAALKEIHEVLERLKSGDVDIDELAVTSQLTKKPSEYVQKGPHVRAALRLARHLGVEPEPGTIVRYVIVRGPGSVSDKAYPVELVREEGKEPDVDYYIEHQILPAVERIMRAIGYSRGQIVGETASQKTLDQFFG
SEQ ID NO:55
MELKIWPLDVTYAVVGGYPEVRVFGLTEGGGRVVLVDRSFKPYFYVDCPTCEVGVVKSSLSRVAPVDEVSAAERRFLGRPRRFLMVVARVPEDVRRLREAAAQIPGVAGVYEADIRFYMRYMIDVGLLPCSWNRAEVEGGGKVGGLPQYTVVQWLGPAGGFPPPLRVLAFDIEVYNERGTPDPARDPVVMIAVKTDDGREEVFEAEGRDDRGVLRSFVEFVKSYDPDVVVGYNSNGFDWPYLAGRARAIGVPLRVDRLGGLPQQSVYGHWSIVGRANVDLYGIVEEFPEIKLKTLDRVAEYFGVMRREERVLIPGHKIYEYWRDPGKRPLLRQYVLDDVRSTLGLADKLLPFLIQLSSVSGLPLDQVAAASVGNRVEWMLLRYAYRLGEVAPNREEREYEPYKGAIVLEPKPGMYEDVLVLDFSSMYPNIMMKYNLSPDTYLEPGEPDPPEGVNAAPEVGHRFRRSPLGFVPQVLKSLVELRKAVREEAKRYPPDSPEFRILDERQRALKVMANAMYGYLGWVGARWYKREVAESVTAFARAILKDVIEQARRLGIVVVYGDTDSLFVKKHVNVDKLIQYVEEKYGIEIKVDKDYAKVLFTEAKKRYAGLLRDGRIDIVGFEVVRGDWSELAKEVQLKVVEIILNSRDVAEARRRVTQYVREIIERLREYKFNVDDLIIWKTLDKELGEYKAYPPHVHAALILKRHGYKVGKGNMVGYVVVKGGGKISEKALPYILLDDVKKIDVEYYIERQIIPAALRIAEVIGVKEADLKTGKSERSLLDFF
SEQ ID NO:56
MKTFLTEQQIKVLMLRAKGYKQSEIAKILGTSRANVSILEKRAMEKIEKARNTLLLWEQINSKVIVEIKAGEDIFSIPEKFFKKADKVGVKVPYSTAEIITFLVEHAPVEDRLAKRDFVLFLDSKNKLRIGDCLVIEEIKED
SEQ ID NO:57
MPITKVTRNYQITIPAEIRKALGIKEGELLEVRLENGKIIIERLKKERKTLKLGKKLTLEEIEKAIEEGMKQCMQ
SEQ ID NO:58
TKIEILRLLKEREMYAYEIWSLLGKPLKYQAVHQHIKELLELGLVEQAYRKGKRVYYKITEKGLRILQNFEDLENI
SEQ ID NO:59
MNTGAQGVSEMSRMKIISVQLPQSLIHGLDALVKRGIYPNRSEAIRVAIRELLKKELYKEEIQEEIPEYVVK
SEQ ID NO:60
VIIPRPIDPRDIRRIRKELGITQEELARKAGVTQAYIAKLEAGKVDPRLSTFNKILRALIECQKAKI
SEQ ID NO:61
NNCECMVVKEKLYTVKQASEILGVHPKTIQKWDREGKIKTVRTPGGRRRIPESEIKRLLGISEEK
SEQ ID NO:62
MLKDSAPKRKILEELRKGETVSGDYLASKLGVSRVAIWKHIRELKELGYGIIADKKGYKLVYEPKKPYPWE
SEQ ID NO:63
MIDERDKIILEILEKDARTPFTEIAKKLGISETAVRKRVKALEEKGIIEGYTIKINPKKLGYSLVTITGVDTKPEKLFEVAEKLKE
SEQ ID NO:64
MEIDDLDRKILSLLIEDSRLSYREIAKKLNVAVGTIYNRIKKLEDMGVIQGFTVKLNYEKLGYELTAIIGIKAQGKK
SEQ ID NO:65
EMLWMYILKLLKDRPMYAYEIRNELKKRFGFEPATVSSYVVLYRLEEGGYVSSEWHESEAGRPSRKYYRLTEKGEKLLEKGIETIEDVLNMLKS
SEQ ID NO:66
MKVSKATASKVLRSLENKGIVERERRGKTYLVRLTNKGLELLEEISKAGKELDEKIFAEMSVDERIVL
SEQ ID NO:67
SEDYMLQNRRKVLAKVLELLNYNPKALNISELARMFGVSRDTIYNDIQQIIKNVEV
SEQ ID NO:68
SKEISRFLKVISNPIRYGILKMLNDRWMCVCLISEALEIDQTLVSHHIRILKELDLLEERKEGKLRFYRTNKEKLREYLEKVLEDFNHGTSKGS
SEQ ID NO:69
MCRKDVMIISDPKQIKALSDPTRVKILELLRYHPMTVSEISRVIGKDKSTIYRHIKALEEAGLVEEVEKIGNETVYGR
SEQ ID NO:70
MEPVEFKLNQKGIKSILPTMEAEIMEYMWEIKEATAGEVYEYMKTKYPEIRRSTVSILMNRLCERGLLKRRMEKGKGGIRYVYSITTTREEFERKVVEKIIESLMMNFREATFAYLSKINKK
SEQ ID NO:71
MKKSNLDLLILLAKAGGIEKEILTTSRELSKMLNVSPQTIVRWLEDLEKDGLIKKSESRKGTLVTITEEGVKFLEKLHEELSDALYR
SEQ ID NO:72
MEIPPEISHALSEIGFTKYEILTYWTLLVYGPSTAKEISTKSGIPYNRVYDTISSLKLRGFVTEIEGTPKVYAAYSPRIAFFRFKKELEDIMKKLEIELNNVKK
SEQ ID NO:73
IINPQARLTPLELEILEIIKQKKSITITEIKEILSERRKSEYPLSLVSEYISRLERKGYVKKIAKGRKKFVEALI
SEQ ID NO:74
GIDVVIPEIKHDPIARDIVKILFDLRRANVSQIARELKGRRGKASRNTVRKKLKELEKLGVVKEVPGERGSVYTLSREVVKKWLDLIGIPINLL
SEQ ID NO:75
MTKRVKVITDPEVIKVMLEDTRRKILQLLRNREMTISQLSEILGKMPQTIYHHIEKLKEAGLVEVKR
SEQ ID NO:76
MEEIKEIMKSHTLGNPVRLGIMIYLFPRRRAPFSHIQKALDLTPGNLDSHIKVLEKHGFVRTYKVIADRPRTMVEITDYGMEETRKFLSHLKTVIDAIHF
SEQ ID NO:77
MGEELNRLLDVLGNETRRRILFLLTKRPYFVSELSRELGVGQKAVLEHLRILEEAGLIESRVEKIPRGRPRKYYMIKKGLRLEILLTPTLFGSEMYEAK
SEQ ID NO:78
MRRMDKVDLQLIKILSQNSRLTYRELAEMLGTTRQRVARKVDKLKKLGIIRKFTIIPNLEK
SEQ ID NO:79
GRKVRTQQNEILNLLNEKEKAVLRAILEHGGEIKQEDLPELVGYSRPTISKVIQELENKGLIKREKSGKTFVVKIERKIKLD
SEQ ID NO:80
KSLQRFLRRNTTSIKHLSEITGVARNRLSDILNGKTQKIRGETLRKIAKAFEKSNILSF
SEQ ID NO:81
DVIQRIKEKYDEFTNAEKKIADTILSDPKGIIESSISDLSEKAGVKSEASVVKFYKKLGLNSFQQFKVLLAQSISRAPLEIVYEDVSSEDDTKTITEKIFKATVRAI
SEQ ID NO:82
KIRDKILNVYTQFSPAERKVADYVLERPDDVIHYSITEFAKIVGVSETTIHRMIKKLDFEGYQAFKIALARELSGLEETIERRDFIDEEIDILRRLKDTLD
SEQ ID NO:83
KRRPTINDVAKLAGVSISTVSRYLKDPSQVSEKLGERIREAIKKLGYKPNKIAQGLRTGD
SEQ ID NO:84
MASIKDVAKLAGVSIATVSRVINGYNNVSEETRKKVIDAIRKLNYHPVYAVKGAVLKR
SEQ ID NO:85
MKKKYVTIRDIAEKAGVSINTVSRALNNKPDISEETRRKILKIAQELGYVKNATASSLRSK
SEQ ID NO:86
MPTIEDVAKLAGVSIATVSRVINGSGYVSEKTRYKVWKAIEELGYKPEISAKLLASKG
SEQ ID NO:87
MRIGEKLRKLRLSRGLTQEELAERTDLSRSFISQLESDKTSPSIDTLERILEALGTDLKHF
SEQ ID NO:88
MHMKTVRQERLKSIVRILERSKEPVSGAQLAEELSVSRQVIVQDIAYLRSLGYNIVATPRGYVLAGG
SEQ ID NO:89
MNTLKKAFEILDFIVKNPGDVSVSEIAEKFNMSVSNAYKYMVVLEEKGFVLRKKDKRYVPGYKLIEYGSFVLRRF
SEQ ID NO:90
MKISKKRRQELIRKIIHEKKISNQFQIVEELKKYGIKAVQPTVARDLKEIGAVKIMDESGNYVYKLLDETPVIDPWKELKR
SEQ ID NO:91
MHKKLNPKSMKRENKKMVLRYLIESGPHSRVEIARKTGLAQSAIWRIIEELVNEGLVEEKGTATGRRRKAVTYGPTRSFITS
SEQ ID NO:92
MPSPLLRRENKIKILRYILKNGKTTRNQLASNLNLAHSTLSYIIDELLDEGFLVFEEIKKKRGRPYQILSVNPEKFTAI
SEQ ID NO:93
MKEERLKEILDIVDRNGFISMKDLQEQLGVSMITVRRDVAELVKRNLVKKVHGGIRKVNYFEKETDFMKRLSINREAKE
SEQ ID NO:94
MFTMRSEYALRLMIVMAKEYGNYLSMTEILEKAKQSVPREFAEKILYTLKKAGLVKTRRGKSGGYMLSRPPKEIKVSEIVFLLDRKSKVFFDMPGCPDELDCVIRALWKRVENEIEKILSGVTLEDLVREQEEKMKQ
SEQ ID NO:95
MRDTKGHLKFLVLHIISQQPSHGYYIMKKISQIIGAEPPSPGALYPILSSLRKQKYIETYNEGKRKVYRLTDKGRKYLEEHKEEIKKALDFAERF
SEQ ID NO:96
MRHRGGRGFRGWWLASTILLLVAEKPSHGYELAERLAEFGIEIPGIGHMGNIYRVLADLEESGFLSTEWDTTVSPPRKIYRITPQGKLYLREILRSLEDMKRRIETLEERIKRVLQEE
SEQ ID NO:97
MLSKRDAILKAAVEVFGKKGYDRATTDEIAEKAGVAKGLIFHYFKNKEELYYQAYMSVTEKLQKEFENFL
SEQ ID NO:98
MSKSWGKFIEEEEAEMASRRNLMIVDGTNLGFRFKHNNSKKPFASSYVSTIQSLAKSYSARTTIVLGDKGKSVFRLEHLPEYKGNRDEKYAQRTEEEKALDEQFFEYLKDAFELCKTTFPTFTIRGVEADDMAAYIVKLIGHLYDHVWLISTDGDWDTLLTDKVSRFSFTTRREYHLRDMYEHHNVDDVEQFISLKAIMGDLGDNIRGVEGIGAKRGYNIIREFGNVLDIIDQLPLPGKQKYIQNLNASEELLFRNLILVDLPTYCVDAIAAVGQDVLDKFTKDILEIAEQ
SEQ ID NO:100
MEEKVGNLKPNMESVNVTVRVLEASEARQIQTKNGVRTISEAIVGDETGRVKLTLWGKHAGSIKEGQVVKIENAWTTAFKGQVQLNAGSKTKIAEASEDGFPESSQIPENTPTAPQQMRGGGRGFRGGGRRYGRRGGRRQENEEGEEE
SEQ ID NO:101
MTLEEARKRVNELRDLIRYHNYRYYVLADPISDAEYDRLLRELKELEERFPELKSPDSPTLQVGARPLEATFRPVRHPTRMYSLDNAFNLDELKAFEERIERALGRKGPFAYTVEHKVDGLSVNLYYEEGVLVYGATRGDGEVGEEVTQNLLTIPTIPRRLKGVPERLEVRGEMPIEAFLRLNEELEERGERIFKNPRNAAAGSLRQKDPRITAKRGLRATFYALGLGLEEVEREGVATQFALLHWLKEKGFPVEHGYARAVGAEGVEAVYQDWLKKRRALPFEADGVVVKLDELALWRELGYTARAPRFAIAYKFPAEEKETRLLDVVFQVGRTGRVTPVGILEPVFLEGSEVSRVTLHNESYIEELDIRIGDWVLVHKAGGVIPEVLRVLKERRTGEERPIRWPETCPECGHRLLKEGKVHRCPNPLCPAKRFEAIRHFASRKAMDIQGLGEKLIERLLEKGLVKDVADLYRLRKEDLVGLERMGEKSAQNLLRQIEESKKRGLERLLYALGLPGVGEVLARNLAARFGNMDRLLEASLEELLEVEEVGELTARAILETLKDPAFRDLVRRLKEAGVEMEAKEKGGEALKGLTFVITGELSRPREEVKALLRRLGAKVTDSVSRKTSYLVVGENPGSKLEKARALGVPTLTEEELYRLLEARTGKKAEELV
SEQ ID NO:102
MILDADYITEDGKPIIRIFKKENGEFKVEYDRNFRPYIYALLKDDSAIDDVKKITAERHGKVVRVVDVEKVKKKFLGRPIEVWKLYFEHPQDVPAIRDKIREHPAVIDIFEYDIPFAKRYLIDKGLIPMEGNEELKLLAFDIETLYHEGEEFGKGPIIMISYADEEGAKVITWKKVDLPYVEVVSSEREMIKRFLKVIREKDPDVIITYNGDNFDFPYLLKRAEKLGMKLPLGRDGSEPKMQRLGDSLAVEIKGRIHFDLYPVIRRTINLPTYTLEAVYEAIFGKPKEKVYPHEIAEAWETGKGLERVAKYSMEDAKVTYELGREFFPMEAQLARLVGQPLWDVSRSSTGNLVEWYLLRKAYERNELAPNKPDEREYERRLRESYEGGYVKEPEKGLWEGIVSLDFRSLYPSIIITHNVSPDTLNKEGCGEYDVAPEVGHRFCKDFPGFIPSLLGSLLDERQKIKRRMKASKDPIERKLLDYRQRAIKILANSYYGYYGYAKARWYCKECAESVTAWGREYIELVRRELEERGFKVLYIDTDGLYATIPGEKNWEEIKRRALEFVNYINAKLPGLLELEYEGFYTRGFFVTKKKYALIDEEGKIITRGLEIVRRDWSEIAKETQAKVLEAILKHGNVEEAVKIVKEVTEKLSKYEIPPEKLVIYEQITRPLNEYKAIGPHVAVAKRLAARGIKVRPGMVIGYVVLRGDGPISKRAIAAEEFDPKKHKYDAEYYIENQVLPAVLRILEAFGYRKEDLRWQKTKQVGLGAWLKVKKS
序列表
<110> 新英格兰生物实验室公司
<120> 合成子的形成
<130> NEB-380-PCT
<150> 62/042,527
<151> 2014-08-27
<150> 62/189,599
<151> 2015-07-07
<150> 62/193,168
<151> 2015-07-16
<160> 102
<170> PatentIn版本3.5
<210> 1
<211> 775
<212> PRT
<213> 人工序列
<220>
<223> 合成的构建体
<400> 1
Met Ile Leu Asp Ala Asp Tyr Ile Thr Glu Asp Gly Lys Pro Ile Ile
1 5 10 15
Arg Leu Phe Lys Lys Glu Asn Gly Arg Phe Lys Val Glu Tyr Asp Arg
20 25 30
Asn Phe Arg Pro Tyr Ile Tyr Ala Leu Leu Lys Asp Asp Ser Ala Ile
35 40 45
Asp Asp Val Arg Lys Ile Thr Ser Glu Arg His Gly Lys Val Val Arg
50 55 60
Val Ile Asp Val Glu Lys Val Lys Lys Lys Phe Leu Gly Arg Pro Ile
65 70 75 80
Glu Val Trp Lys Leu Tyr Phe Glu His Pro Gln Asp Val Pro Ala Met
85 90 95
Arg Asp Lys Ile Arg Glu His Pro Ala Val Ile Asp Ile Phe Glu Tyr
100 105 110
Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Lys Gly Leu Ile Pro
115 120 125
Met Glu Gly Asn Glu Glu Leu Thr Phe Leu Ala Val Asp Ile Glu Thr
130 135 140
Leu Tyr His Glu Gly Glu Glu Phe Gly Lys Gly Pro Ile Ile Met Ile
145 150 155 160
Ser Tyr Ala Asp Glu Glu Gly Ala Lys Val Ile Thr Trp Lys Lys Ile
165 170 175
Asp Leu Pro Tyr Val Glu Val Val Ala Asn Glu Arg Glu Met Ile Lys
180 185 190
Arg Leu Ile Lys Val Ile Arg Glu Lys Asp Pro Asp Val Ile Ile Thr
195 200 205
Tyr Asn Gly Asp Asn Phe Asp Phe Pro Tyr Leu Leu Lys Arg Ala Glu
210 215 220
Lys Leu Gly Met Lys Leu Pro Leu Gly Arg Asp Asn Ser Glu Pro Lys
225 230 235 240
Met Gln Arg Leu Gly Asp Ser Leu Ala Val Glu Ile Lys Gly Arg Ile
245 250 255
His Phe Asp Leu Phe Pro Val Ile Arg Arg Thr Ile Asn Leu Pro Thr
260 265 270
Tyr Thr Leu Glu Ala Val Tyr Glu Ala Ile Phe Gly Lys Gln Lys Glu
275 280 285
Lys Val Tyr Pro His Glu Ile Ala Glu Ala Trp Glu Thr Gly Lys Gly
290 295 300
Leu Glu Arg Val Ala Lys Tyr Ser Met Glu Asp Ala Lys Val Thr Tyr
305 310 315 320
Glu Leu Gly Lys Glu Phe Phe Pro Met Glu Ala Gln Leu Ala Arg Leu
325 330 335
Val Gly Gln Pro Leu Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu
340 345 350
Val Glu Trp Tyr Leu Leu Arg Lys Ala Tyr Glu Arg Asn Glu Leu Ala
355 360 365
Pro Asn Lys Pro Asp Glu Arg Glu Tyr Glu Arg Arg Leu Arg Glu Ser
370 375 380
Tyr Glu Gly Gly Tyr Val Lys Glu Pro Glu Arg Gly Leu Trp Glu Gly
385 390 395 400
Ile Val Ser Leu Asp Phe Arg Ser Leu Tyr Pro Ser Ile Ile Ile Thr
405 410 415
His Asn Val Ser Pro Asp Thr Leu Asn Lys Glu Gly Cys Gly Glu Tyr
420 425 430
Asp Glu Ala Pro Glu Val Gly His Arg Phe Cys Lys Asp Phe Pro Gly
435 440 445
Phe Ile Pro Ser Leu Leu Gly Ser Leu Leu Glu Glu Arg Gln Lys Ile
450 455 460
Lys Lys Arg Met Lys Glu Ser Lys Asp Pro Val Glu Arg Lys Leu Leu
465 470 475 480
Asp Tyr Arg Gln Arg Ala Ile Lys Ile Leu Ala Asn Ser Phe Tyr Gly
485 490 495
Tyr Tyr Gly Tyr Ala Lys Ala Arg Trp Tyr Cys Lys Glu Cys Ala Glu
500 505 510
Ser Val Thr Ala Trp Gly Arg Gln Tyr Ile Glu Leu Val Arg Arg Glu
515 520 525
Leu Glu Glu Arg Gly Phe Lys Val Leu Tyr Ile Asp Thr Asp Gly Leu
530 535 540
Tyr Ala Thr Ile Pro Gly Glu Lys Asn Trp Glu Glu Ile Lys Arg Arg
545 550 555 560
Ala Leu Glu Phe Val Asn Tyr Ile Asn Ser Lys Leu Pro Gly Ile Leu
565 570 575
Glu Leu Glu Tyr Glu Gly Phe Tyr Thr Arg Gly Phe Phe Val Thr Lys
580 585 590
Lys Lys Tyr Ala Leu Ile Asp Glu Glu Gly Lys Ile Val Thr Arg Gly
595 600 605
Leu Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu Thr Gln
610 615 620
Ala Lys Val Leu Glu Ala Ile Leu Lys His Gly Asn Val Glu Glu Ala
625 630 635 640
Val Lys Ile Val Lys Glu Val Thr Glu Lys Leu Ser Asn Tyr Glu Ile
645 650 655
Pro Val Glu Lys Leu Val Ile Tyr Glu Gln Ile Thr Arg Pro Leu Asn
660 665 670
Glu Tyr Lys Ala Ile Gly Pro His Val Ala Val Ala Lys Arg Leu Ala
675 680 685
Ala Lys Gly Ile Lys Ile Lys Pro Gly Met Val Ile Gly Tyr Val Val
690 695 700
Leu Arg Gly Asp Gly Pro Ile Ser Lys Arg Ala Ile Ala Ile Glu Glu
705 710 715 720
Phe Asp Gly Lys Lys His Lys Tyr Asp Ala Glu Tyr Tyr Ile Glu Asn
725 730 735
Gln Val Leu Pro Ala Val Glu Arg Ile Leu Lys Ala Phe Gly Tyr Lys
740 745 750
Arg Glu Asp Leu Arg Trp Gln Lys Thr Lys Gln Val Gly Leu Gly Ala
755 760 765
Trp Leu Lys Val Lys Lys Ser
770 775
<210> 2
<211> 75
<212> PRT
<213> 激烈火球菌（Pyrococcus furiosus）
<400> 2
Ile Ile Asn Pro Gln Ala Arg Leu Thr Pro Leu Glu Leu Glu Ile Leu
1 5 10 15
Glu Ile Ile Lys Gln Lys Lys Ser Ile Thr Ile Thr Glu Ile Lys Glu
20 25 30
Ile Leu Ser Glu Arg Arg Lys Ser Glu Tyr Pro Leu Ser Leu Val Ser
35 40 45
Glu Tyr Ile Ser Arg Leu Glu Arg Lys Gly Tyr Val Lys Lys Ile Ala
50 55 60
Lys Gly Arg Lys Lys Phe Val Glu Ala Leu Ile
65 70 75
<210> 3
<211> 856
<212> PRT
<213> 人工序列
<220>
<223> 合成的构建体
<400> 3
Met Ile Leu Asp Ala Asp Tyr Ile Thr Glu Asp Gly Lys Pro Ile Ile
1 5 10 15
Arg Leu Phe Lys Lys Glu Asn Gly Arg Phe Lys Val Glu Tyr Asp Arg
20 25 30
Asn Phe Arg Pro Tyr Ile Tyr Ala Leu Leu Lys Asp Asp Ser Ala Ile
35 40 45
Asp Asp Val Arg Lys Ile Thr Ser Glu Arg His Gly Lys Val Val Arg
50 55 60
Val Ile Asp Val Glu Lys Val Lys Lys Lys Phe Leu Gly Arg Pro Ile
65 70 75 80
Glu Val Trp Lys Leu Tyr Phe Glu His Pro Gln Asp Val Pro Ala Met
85 90 95
Arg Asp Lys Ile Arg Glu His Pro Ala Val Ile Asp Ile Phe Glu Tyr
100 105 110
Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Lys Gly Leu Ile Pro
115 120 125
Met Glu Gly Asn Glu Glu Leu Thr Phe Leu Ala Val Asp Ile Glu Thr
130 135 140
Leu Tyr His Glu Gly Glu Glu Phe Gly Lys Gly Pro Ile Ile Met Ile
145 150 155 160
Ser Tyr Ala Asp Glu Glu Gly Ala Lys Val Ile Thr Trp Lys Lys Ile
165 170 175
Asp Leu Pro Tyr Val Glu Val Val Ala Asn Glu Arg Glu Met Ile Lys
180 185 190
Arg Leu Ile Lys Val Ile Arg Glu Lys Asp Pro Asp Val Ile Ile Thr
195 200 205
Tyr Asn Gly Asp Asn Phe Asp Phe Pro Tyr Leu Leu Lys Arg Ala Glu
210 215 220
Lys Leu Gly Met Lys Leu Pro Leu Gly Arg Asp Asn Ser Glu Pro Lys
225 230 235 240
Met Gln Arg Leu Gly Asp Ser Leu Ala Val Glu Ile Lys Gly Arg Ile
245 250 255
His Phe Asp Leu Phe Pro Val Ile Arg Arg Thr Ile Asn Leu Pro Thr
260 265 270
Tyr Thr Leu Glu Ala Val Tyr Glu Ala Ile Phe Gly Lys Gln Lys Glu
275 280 285
Lys Val Tyr Pro His Glu Ile Ala Glu Ala Trp Glu Thr Gly Lys Gly
290 295 300
Leu Glu Arg Val Ala Lys Tyr Ser Met Glu Asp Ala Lys Val Thr Tyr
305 310 315 320
Glu Leu Gly Lys Glu Phe Phe Pro Met Glu Ala Gln Leu Ala Arg Leu
325 330 335
Val Gly Gln Pro Leu Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu
340 345 350
Val Glu Trp Tyr Leu Leu Arg Lys Ala Tyr Glu Arg Asn Glu Leu Ala
355 360 365
Pro Asn Lys Pro Asp Glu Arg Glu Tyr Glu Arg Arg Leu Arg Glu Ser
370 375 380
Tyr Glu Gly Gly Tyr Val Lys Glu Pro Glu Arg Gly Leu Trp Glu Gly
385 390 395 400
Ile Val Ser Leu Asp Phe Arg Ser Leu Tyr Pro Ser Ile Ile Ile Thr
405 410 415
His Asn Val Ser Pro Asp Thr Leu Asn Lys Glu Gly Cys Gly Glu Tyr
420 425 430
Asp Glu Ala Pro Glu Val Gly His Arg Phe Cys Lys Asp Phe Pro Gly
435 440 445
Phe Ile Pro Ser Leu Leu Gly Ser Leu Leu Glu Glu Arg Gln Lys Ile
450 455 460
Lys Lys Arg Met Lys Glu Ser Lys Asp Pro Val Glu Arg Lys Leu Leu
465 470 475 480
Asp Tyr Arg Gln Arg Ala Ile Lys Ile Leu Ala Asn Ser Phe Tyr Gly
485 490 495
Tyr Tyr Gly Tyr Ala Lys Ala Arg Trp Tyr Cys Lys Glu Cys Ala Glu
500 505 510
Ser Val Thr Ala Trp Gly Arg Gln Tyr Ile Glu Leu Val Arg Arg Glu
515 520 525
Leu Glu Glu Arg Gly Phe Lys Val Leu Tyr Ile Asp Thr Asp Gly Leu
530 535 540
Tyr Ala Thr Ile Pro Gly Glu Lys Asn Trp Glu Glu Ile Lys Arg Arg
545 550 555 560
Ala Leu Glu Phe Val Asn Tyr Ile Asn Ser Lys Leu Pro Gly Ile Leu
565 570 575
Glu Leu Glu Tyr Glu Gly Phe Tyr Thr Arg Gly Phe Phe Val Thr Lys
580 585 590
Lys Lys Tyr Ala Leu Ile Asp Glu Glu Gly Lys Ile Val Thr Arg Gly
595 600 605
Leu Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu Thr Gln
610 615 620
Ala Lys Val Leu Glu Ala Ile Leu Lys His Gly Asn Val Glu Glu Ala
625 630 635 640
Val Lys Ile Val Lys Glu Val Thr Glu Lys Leu Ser Asn Tyr Glu Ile
645 650 655
Pro Val Glu Lys Leu Val Ile Tyr Glu Gln Ile Thr Arg Pro Leu Asn
660 665 670
Glu Tyr Lys Ala Ile Gly Pro His Val Ala Val Ala Lys Arg Leu Ala
675 680 685
Ala Lys Gly Ile Lys Ile Lys Pro Gly Met Val Ile Gly Tyr Val Val
690 695 700
Leu Arg Gly Asp Gly Pro Ile Ser Lys Arg Ala Ile Ala Ile Glu Glu
705 710 715 720
Phe Asp Gly Lys Lys His Lys Tyr Asp Ala Glu Tyr Tyr Ile Glu Asn
725 730 735
Gln Val Leu Pro Ala Val Glu Arg Ile Leu Lys Ala Phe Gly Tyr Lys
740 745 750
Arg Glu Asp Leu Arg Trp Gln Lys Thr Lys Gln Val Gly Leu Gly Ala
755 760 765
Trp Leu Lys Val Lys Lys Ser Gly Thr Gly Gly Gly Gly Ile Ile Asn
770 775 780
Pro Gln Ala Arg Leu Thr Pro Leu Glu Leu Glu Ile Leu Glu Ile Ile
785 790 795 800
Lys Gln Lys Lys Ser Ile Thr Ile Thr Glu Ile Lys Glu Ile Leu Ser
805 810 815
Glu Arg Arg Lys Ser Glu Tyr Pro Leu Ser Leu Val Ser Glu Tyr Ile
820 825 830
Ser Arg Leu Glu Arg Lys Gly Tyr Val Lys Lys Ile Ala Lys Gly Arg
835 840 845
Lys Lys Phe Val Glu Ala Leu Ile
850 855
<210> 4
<211> 40
<212> DNA
<213> 人工序列
<220>
<223> 合成的构建体
<400> 4
ttggtctggt gtcaaaaatg aatcgtcacg gcgatttatg 40
<210> 5
<211> 39
<212> DNA
<213> 人工序列
<220>
<223> 合成的构建体
<400> 5
gggtcatttt cggcgaggac tgcatcaacg catatagcg 39
<210> 6
<211> 32
<212> DNA
<213> 人工序列
<220>
<223> 合成的构建体
<400> 6
gcggccgcgt cctcgccgaa aatgacccag ag 32
<210> 7
<211> 32
<212> DNA
<213> 人工序列
<220>
<223> 合成的构建体
<400> 7
gcggccgctg gtgtcgatgg tagaacgaag cg 32
<210> 8
<211> 32
<212> DNA
<213> 人工序列
<220>
<223> 合成的构建体
<400> 8
gcggccgccc cactgacgcg ttgcgcgaga ag 32
<210> 9
<211> 32
<212> DNA
<213> 人工序列
<220>
<223> 合成的构建体
<400> 9
gcggccgcgg ctgcgcaact gttgggaagg gc 32
<210> 10
<211> 32
<212> DNA
<213> 人工序列
<220>
<223> 合成的构建体
<400> 10
gcggccgctg cagcacatcc ccctttcgcc ag 32
<210> 11
<211> 32
<212> DNA
<213> 人工序列
<220>
<223> 合成的构建体
<400> 11
gcggccgcat gatgctcgtg acggttaacg cc 32
<210> 12
<211> 32
<212> DNA
<213> 人工序列
<220>
<223> 合成的构建体
<400> 12
gcggccgcag gtgcggattg aaaatggtct gc 32
<210> 13
<211> 32
<212> DNA
<213> 人工序列
<220>
<223> 合成的构建体
<400> 13
gcggccgctc accgcttgcc agcggcttac ca 32
<210> 14
<211> 32
<212> DNA
<213> 人工序列
<220>
<223> 合成的构建体
<400> 14
gcggccgcga atacctgttc cgtcatagcg at 32
<210> 15
<211> 33
<212> DNA
<213> 人工序列
<220>
<223> 合成的构建体
<400> 15
gcggccgctc atttttgaca ccagaccaac tgg 33
<210> 16
<211> 1016
<212> DNA
<213> 人工序列
<220>
<223> 合成的构建体
<400> 16
gcggccgcgt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc 60
atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag 120
gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg 180
agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg 240
tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc 300
cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg 360
gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg 420
tttgatggtg gttaacggcg ggatataacc aacgcgcagc ccggactcgg taatatccca 480
ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca 540
gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt 600
gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct 660
gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag 720
aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca 780
cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag 840
agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct 900
ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca 960
ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccagc ggccgc 1016
<210> 17
<211> 1016
<212> DNA
<213> 人工序列
<220>
<223> 合成的构建体
<400> 17
gcggccgccc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg 60
acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat 120
ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca 180
atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc 240
tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc 300
accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt 360
actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg 420
cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc 480
ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa 540
tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc 600
cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat 660
gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg 720
tccggcgtag aggatcgaga tctcgatccc gcgaaattaa tacgactcac tataggggaa 780
ttgtgagcgg ataacaattc ccctctagaa ataattttgt ttaactttaa gaaggagata 840
tacatatgac catgattacg gattcactgg ccgtcgtttt acaacgtcgt gactgggaaa 900
accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta 960
atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagccgc ggccgc 1016
<210> 18
<211> 1016
<212> DNA
<213> 人工序列
<220>
<223> 合成的构建体
<400> 18
gcggccgctg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc 60
gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcgctttgc ctggtttccg 120
gcaccagaag cggtgccgga aagctggctg gagtgcgatc ttcctgaggc cgatactgtc 180
gtcgtcccct caaactggca gatgcacggt tacgatgcgc ccatctacac caacgtgacc 240
tatcccatta cggtcaatcc gccgtttgtt cccacggaga atccgacggg ttgttactcg 300
ctcacattta atgttgatga aagctggcta caggaaggcc agacgcgaat tatttttgat 360
ggcgttaact cggcgtttca tctgtggtgc aacgggcgct gggtcggtta cggccaggac 420
agtcgtttgc cgtctgaatt tgacctgagc gcatttttac gcgccggaga aaaccgcctc 480
gcggtgatgg tgctgcgctg gagtgacggc agttatctgg aagatcagga tatgtggcgg 540
atgagcggca ttttccgtga cgtctcgttg ctgcataaac cgactacaca aatcagcgat 600
ttccatgttg ccactcgctt taatgatgat ttcagccgcg ctgtactgga ggctgaagtt 660
cagatgtgcg gcgagttgcg tgactaccta cgggtaacag tttctttatg gcagggtgaa 720
acgcaggtcg ccagcggcac cgcgcctttc ggcggtgaaa ttatcgatga gcgtggtggt 780
tatgccgatc gcgtcacact acgtctgaac gtcgaaaacc cgaaactgtg gagcgccgaa 840
atcccgaatc tctatcgtgc ggtggttgaa ctgcacaccg ccgacggcac gctgattgaa 900
gcagaagcct gcgatgtcgg tttccgcgag gtgcggattg aaaatggtct gctgctgctg 960
aacggcaagc cgttgctgat tcgaggcgtt aaccgtcacg agcatcatgc ggccgc 1016
<210> 19
<211> 1016
<212> DNA
<213> 人工序列
<220>
<223> 合成的构建体
<400> 19
gcggccgcag gtgcggattg aaaatggtct gctgctgctg aacggcaagc cgttgctgat 60
tcgaggcgtt aaccgtcacg agcatcatcc tctgcatggt caggtcatgg atgagcagac 120
gatggtgcag gatatcctgc tgatgaagca gaacaacttt aacgccgtgc gctgttcgca 180
ttatccgaac catccgctgt ggtacacgct gtgcgaccgc tacggcctgt atgtggtgga 240
tgaagccaat attgaaaccc acggcatggt gccaatgaat cgtctgaccg atgatccgcg 300
ctggctaccg gcgatgagcg aacgcgtaac gcgaatggtg cagcgcgatc gtaatcaccc 360
gagtgtgatc atctggtcgc tggggaatga atcaggccac ggcgctaatc acgacgcgct 420
gtatcgctgg atcaaatctg tcgatccttc ccgcccggtg cagtatgaag gcggcggagc 480
cgacaccacg gccaccgata ttatttgccc gatgtacgcg cgcgtggatg aagaccagcc 540
cttcccggct gtgccgaaat ggtccatcaa aaaatggctt tcgctacctg gagagacgcg 600
cccgctgatc ctttgcgaat acgcccacgc gatgggtaac agtcttggcg gtttcgctaa 660
atactggcag gcgtttcgtc agtatccccg tttacagggc ggcttcgtct gggactgggt 720
ggatcagtcg ctgattaaat atgatgaaaa cggcaacccg tggtcggctt acggcggtga 780
ttttggcgat acgccgaacg atcgccagtt ctgtatgaac ggtctggtct ttgccgaccg 840
cacgccgcat ccagcgctga cggaagcaaa acaccagcag cagtttttcc agttccgttt 900
atccgggcaa accatcgaag tgaccagcga atacctgttc cgtcatagcg ataacgagct 960
cctgcactgg atggtggcgc tggatggtaa gccgctggca agcggtgagc ggccgc 1016
<210> 20
<211> 1168
<212> DNA
<213> 人工序列
<220>
<223> 合成的构建体
<400> 20
gcggccgcga atacctgttc cgtcatagcg ataacgagct cctgcactgg atggtggcgc 60
tggatggtaa gccgctggca agcggtgaag tgcctctgga tgtcgctcca caaggtaaac 120
agttgattga actgcctgaa ctaccgcagc cggagagcgc cgggcaactc tggctcacag 180
tacgcgtagt gcaaccgaac gcgaccgcat ggtcagaagc cgggcacatc agcgcctggc 240
agcagtggcg tctggcggaa aacctcagtg tgacgctccc cgccgcgtcc cacgccatcc 300
cgcatctgac caccagcgaa atggattttt gcatcgagct gggtaataag cgttggcaat 360
ttaaccgcca gtcaggcttt ctttcacaga tgtggattgg cgataaaaaa caactgctga 420
cgccgctgcg cgatcagttc acccgtgcac cgctggataa cgacattggc gtaagtgaag 480
cgacccgcat tgaccctaac gcctgggtcg aacgctggaa ggcggcgggc cattaccagg 540
ccgaagcagc gttgttgcag tgcacggcag atacacttgc tgatgcggtg ctgattacga 600
ccgctcacgc gtggcagcat caggggaaaa ccttatttat cagccggaaa acctaccgga 660
ttgatggtag tggtcaaatg gcgattaccg ttgatgttga agtggcgagc gatacaccgc 720
atccggcgcg gattggcctg aactgccagc tggcgcaggt agcagagcgg gtaaactggc 780
tcggattagg gccgcaagaa aactatcccg accgccttac tgccgcctgt tttgaccgct 840
gggatctgcc attgtcagac atgtataccc cgtacgtctt cccgagcgaa aacggtctgc 900
gctgcgggac gcgcgaattg aattatggcc cacaccagtg gcgcggcgac ttccagttca 960
acatcagccg ctacagtcaa cagcaactga tggaaaccag ccatcgccat ctgctgcacg 1020
cggaagaagg cacatggctg aatatcgacg gtttccatat ggggattggt ggcgacgact 1080
cctggagccc gtcagtatcg gcggaattcc agctgagcgc cggtcgctac cattaccagt 1140
tggtctggtg tcaaaaatga gcggccgc 1168
<210> 21
<211> 24
<212> DNA
<213> 人工序列
<220>
<223> 合成的构建体
<400> 21
aaaaccaccc tggcgcccaa tacg 24
<210> 22
<211> 24
<212> DNA
<213> 人工序列
<220>
<223> 合成的构建体
<400> 22
cccggactcg gtaatggcgc gcat 24
<210> 23
<211> 24
<212> DNA
<213> 人工序列
<220>
<223> 合成的构建体
<400> 23
ggaagcagcc cagtagtagg ttga 24
<210> 24
<211> 24
<212> DNA
<213> 人工序列
<220>
<223> 合成的构建体
<400> 24
ggtgctgcgc tggagtgacg gcag 24
<210> 25
<211> 24
<212> DNA
<213> 人工序列
<220>
<223> 合成的构建体
<400> 25
cggccaccga tattatttgc ccga 24
<210> 26
<211> 24
<212> DNA
<213> 人工序列
<220>
<223> 合成的构建体
<400> 26
gattagggcc gcaagaaaac tatc 24
<210> 27
<211> 24
<212> DNA
<213> 人工序列
<220>
<223> 合成的构建体
<220>
<221> 尚未归类的特征
<222> (22)..(22)
<223> n为a, c, g或t
<400> 27
gcgaagaacc tcttcccaag angg 24
<210> 28
<211> 71
<212> DNA
<213> 人工序列
<220>
<223> 合成的构建体
<400> 28
atcttgtgga aaggacgaaa caccggcgaa gaacctcttc ccaagagttt tagagctaga 60
aatagcaagt t 71
<210> 29
<211> 71
<212> DNA
<213> 人工序列
<220>
<223> 合成的构建体
<220>
<221> 尚未归类的特征
<222> (26)..(46)
<223> n为a, c, g或t
<400> 29
atcttgtgga aaggacgaaa caccgnnnnn nnnnnnnnnn nnnnnngttt tagagctaga 60
aatagcaagt t 71
<210> 30
<211> 24
<212> DNA
<213> 人工序列
<220>
<223> 合成的构建体
<400> 30
cgccagggtt ttcccagtca cgac 24
<210> 31
<211> 27
<212> DNA
<213> 人工序列
<220>
<223> 合成的构建体
<400> 31
gccaagcttg catgcctgca ggtcgac 27
<210> 32
<211> 71
<212> DNA
<213> 人工序列
<220>
<223> 合成的构建体
<400> 32
gcggtcccaa aagggtcagt gctgcaacat tttgctgccg gtcacggttc gaacgtacgg 60
acgtccagct g 71
<210> 33
<211> 1184
<212> PRT
<213> Methanocaldococcus vulcanius
<400> 33
Met Lys Glu Lys Ala Pro Lys Ile Asp Ala Leu Ile Asp Cys Thr Tyr
1 5 10 15
Lys Thr Glu Asp Asn Arg Ala Val Ile Tyr Leu Tyr Leu Leu Glu Asn
20 25 30
Ile Leu Lys Asp Arg Glu Phe Ser Pro Tyr Phe Tyr Val Glu Met Leu
35 40 45
Lys Asp Arg Ile Glu Lys Glu Asp Ile Asp Lys Ile Lys Glu Phe Leu
50 55 60
Leu Lys Glu Asp Leu Leu Lys Phe Val Glu Asn Leu Glu Val Val Asn
65 70 75 80
Lys Thr Ile Leu Lys Lys Glu Lys Glu Ile Val Lys Ile Ile Ala Thr
85 90 95
His Pro Gln Arg Val Pro Lys Leu Arg Lys Ile Lys Glu Cys Asp Ile
100 105 110
Val Lys Glu Ile Tyr Glu His Asp Ile Pro Phe Ala Lys Arg Tyr Leu
115 120 125
Ile Asp Ser Asp Ile Val Pro Met Thr Tyr Trp Asp Phe Glu Asn Arg
130 135 140
Lys Gln Val Ser Ile Glu Ile Pro Lys Leu Lys Thr Val Ser Phe Asp
145 150 155 160
Met Glu Val Tyr Asn Arg Asp Thr Glu Pro Asp Pro Glu Lys Asp Pro
165 170 175
Ile Leu Met Ala Ser Phe Trp Asp Asp Asn Gly Gly Lys Val Ile Thr
180 185 190
Tyr Lys His Phe Asp His Ser Asn Ile Glu Val Val Asn Ser Glu Lys
195 200 205
Asp Leu Ile Lys Lys Ile Val Glu Met Leu Arg Gln Tyr Asp Val Ile
210 215 220
Phe Thr Tyr Asn Gly Asp Asn Phe Asp Phe Pro Tyr Leu Lys Ala Arg
225 230 235 240
Ala Lys Ile Tyr Gly Ile Asp Ile Lys Leu Gly Arg Asp Gly Glu Glu
245 250 255
Leu Lys Ile Lys Arg Gly Gly Met Glu Phe Arg Ser Tyr Ile Pro Gly
260 265 270
Arg Val His Ile Asp Leu Tyr Pro Ile Ser Arg Arg Leu Leu Lys Leu
275 280 285
Thr Lys Tyr Thr Leu Glu Asp Val Val Tyr Asn Leu Phe Gly Ile Glu
290 295 300
Lys Leu Lys Ile Pro His Thr Lys Ile Val Asp Tyr Trp Ala Asn Asn
305 310 315 320
Asp Lys Ile Leu Ile Glu Tyr Ser Leu Gln Asp Ala Lys Tyr Thr His
325 330 335
Lys Ile Gly Lys Tyr Phe Phe Pro Leu Glu Val Met Phe Ser Arg Ile
340 345 350
Val Asn Gln Thr Pro Phe Glu Ile Thr Arg Met Ser Ser Gly Gln Met
355 360 365
Val Glu Tyr Leu Leu Met Lys Asn Ala Phe Lys Glu Asn Met Ile Val
370 375 380
Pro Asn Lys Pro Asp Glu Lys Glu Tyr Arg Lys Arg Leu Leu Thr Ser
385 390 395 400
Tyr Glu Gly Gly Tyr Val Lys Glu Pro Glu Lys Gly Met Phe Glu Asp
405 410 415
Ile Ile Ser Met Asp Phe Arg Cys His Pro Arg Gly Thr Lys Val Ile
420 425 430
Val Lys Asn Asn Gly Leu Thr Asp Ile Glu Asn Val Lys Val Gly Asp
435 440 445
Tyr Val Leu Gly Ile Asp Gly Trp Gln Lys Val Lys Arg Val Trp Lys
450 455 460
Tyr Pro Tyr Asn Gly Phe Leu Val Asn Val Asn Gly Leu Lys Ser Thr
465 470 475 480
Pro Asn His Lys Ile Pro Val Ile Lys Lys Glu Asn Gly Lys Asp Arg
485 490 495
Val Ile Asp Val Ser Ser Ile Tyr Leu Leu Asn Leu Lys Gly Cys Lys
500 505 510
Ile Leu Lys Ile Lys Asn Phe Glu Ser Ile Gly Met Phe Gly Lys Ile
515 520 525
Phe Lys Lys Asp Thr Lys Ile Lys Lys Val Lys Gly Leu Leu Glu Lys
530 535 540
Ile Ala Tyr Ile Asp Pro Arg Glu Gly Leu Val Ile Lys Val Lys Asn
545 550 555 560
Glu Lys Glu Asp Ile Phe Lys Thr Val Ile Pro Ile Leu Lys Glu Leu
565 570 575
Asn Ile Leu Tyr Lys Gln Val Asp Glu Lys Thr Ile Ile Ile Asp Ser
580 585 590
Ile Asp Gly Leu Leu Lys Tyr Ile Val Thr Ile Gly Phe Asn Asp Lys
595 600 605
Asn Glu Glu Lys Ile Lys Glu Ile Ile Lys Glu Lys Ser Phe Leu Glu
610 615 620
Phe Lys Glu Leu Glu Asp Ile Lys Ile Ser Ile Glu Glu Tyr Glu Gly
625 630 635 640
Tyr Val Tyr Asp Leu Thr Leu Glu Gly Arg Pro Tyr Tyr Phe Ala Asn
645 650 655
Gly Ile Leu Thr His Asn Ser Leu Tyr Pro Ser Ile Ile Ile Ala Tyr
660 665 670
Asn Ile Ser Pro Glu Thr Leu Asp Cys Glu Cys Cys Lys Asp Ile Ser
675 680 685
Glu Lys Ile Leu Gly His Trp Phe Cys Lys Lys Arg Glu Gly Leu Ile
690 695 700
Pro Lys Thr Leu Arg Gly Leu Ile Glu Arg Arg Ile Asn Ile Lys Asn
705 710 715 720
Lys Met Lys Lys Met Glu Ser Glu Lys Glu Ile Asn Glu Glu Tyr Asn
725 730 735
Leu Leu Asp Tyr Glu Gln Arg Ser Leu Lys Ile Leu Ala Asn Ser Val
740 745 750
Tyr Gly Tyr Leu Ala Phe Pro Arg Ala Arg Phe Tyr Ser Arg Glu Cys
755 760 765
Ala Glu Val Ile Thr Tyr Leu Gly Arg Lys Tyr Ile Leu Glu Thr Ile
770 775 780
Glu Glu Ala Glu Lys Phe Gly Phe Lys Val Ile Tyr Ala Asp Ser Val
785 790 795 800
Val Lys Asp Ala Lys Val Ile Ile Lys Glu Asp Gly Lys Ile Lys Glu
805 810 815
Ile Lys Ile Glu Asp Leu Phe Lys Lys Val Asp Tyr Thr Ile Gly Asp
820 825 830
Lys Glu Tyr Cys Ile Leu Asn Asn Val Glu Thr Leu Thr Ile Glu Asp
835 840 845
Thr Lys Leu Val Trp Arg Lys Val Pro Tyr Ile Met Arg His Arg Thr
850 855 860
Asn Lys Lys Ile Tyr Arg Val Lys Val Lys Asp Arg Tyr Val Asp Ile
865 870 875 880
Thr Glu Asp His Ser Ile Ile Gly Val Lys Asn Asn Lys Leu Val Glu
885 890 895
Leu Lys Pro Thr Glu Ile Lys Asp Asp Glu Thr Lys Leu Ile Ile Leu
900 905 910
Asn Lys Asp Leu Lys Ser Tyr Asn Phe Ala Ser Val Glu Glu Ile Asn
915 920 925
Cys Ile Lys Tyr Ser Asp Tyr Val Tyr Asp Ile Glu Val Glu Asn Thr
930 935 940
His Arg Phe Phe Ala Asn Gly Ile Leu Val His Asn Thr Asp Gly Phe
945 950 955 960
Tyr Ala Val Trp Lys Glu Lys Ile Ser Lys Asp Asp Leu Ile Lys Lys
965 970 975
Ala Leu Glu Phe Val Lys Tyr Ile Asn Ser Lys Leu Pro Gly Thr Met
980 985 990
Glu Leu Glu Phe Glu Gly Tyr Phe Lys Arg Gly Ile Phe Ile Thr Lys
995 1000 1005
Lys Arg Tyr Ala Leu Ile Asp Glu Asn Gly Arg Val Ile Val Lys
1010 1015 1020
Gly Leu Glu Phe Val Arg Arg Asp Trp Ser Asn Leu Ala Arg Ile
1025 1030 1035
Thr Gln Arg Arg Val Leu Glu Ala Leu Leu Leu Glu Gly Asp Ile
1040 1045 1050
Asn Lys Ala Lys Lys Ala Ile Gln Asp Val Ile Lys Asp Leu Arg
1055 1060 1065
Glu Lys Lys Ile Lys Lys Glu Asp Leu Ile Ile Tyr Thr Gln Leu
1070 1075 1080
Thr Lys Asn Pro Asn Glu Tyr Lys Thr Thr Ala Pro His Val Glu
1085 1090 1095
Ile Ala Lys Lys Met Met Arg Glu Gly Lys Lys Ile Lys Ile Gly
1100 1105 1110
Asp Val Ile Gly Tyr Ile Ile Val Lys Gly Ser Lys Ser Ile Ser
1115 1120 1125
Glu Arg Ala Lys Leu Pro Glu Glu Val Ser Ile Glu Glu Ile Asp
1130 1135 1140
Val Asn Tyr Tyr Ile Asp Asn Gln Ile Leu Pro Pro Val Leu Arg
1145 1150 1155
Ile Met Glu Ala Val Gly Val Ser Lys Asn Glu Leu Lys Lys Glu
1160 1165 1170
Gly Thr Gln Leu Thr Leu Asp Arg Phe Leu Lys
1175 1180
<210> 34
<211> 781
<212> PRT
<213> Archaeoglobus fulgidus
<400> 34
Met Glu Arg Val Glu Gly Trp Leu Ile Asp Ala Asp Tyr Glu Thr Ile
1 5 10 15
Gly Gly Lys Ala Val Val Arg Leu Trp Cys Lys Asp Asp Gln Gly Ile
20 25 30
Phe Val Ala Tyr Asp Tyr Asn Phe Asp Pro Tyr Phe Tyr Val Ile Gly
35 40 45
Val Asp Glu Asp Ile Leu Lys Asn Ala Ala Thr Ser Thr Arg Arg Glu
50 55 60
Val Ile Lys Leu Lys Ser Phe Glu Lys Ala Gln Leu Lys Thr Leu Gly
65 70 75 80
Arg Glu Val Glu Gly Tyr Ile Val Tyr Ala His His Pro Gln His Val
85 90 95
Pro Lys Leu Arg Asp Tyr Leu Ser Gln Phe Gly Asp Val Arg Glu Ala
100 105 110
Asp Ile Pro Phe Ala Tyr Arg Tyr Leu Ile Asp Lys Asp Leu Ala Cys
115 120 125
Met Asp Gly Ile Ala Ile Glu Gly Glu Lys Gln Gly Gly Val Ile Arg
130 135 140
Ser Tyr Lys Ile Glu Lys Val Glu Arg Ile Pro Arg Met Glu Phe Pro
145 150 155 160
Glu Leu Lys Met Leu Val Phe Asp Cys Glu Met Leu Ser Ser Phe Gly
165 170 175
Met Pro Glu Pro Glu Lys Asp Pro Ile Ile Val Ile Ser Val Lys Thr
180 185 190
Asn Asp Asp Asp Glu Ile Ile Leu Thr Gly Asp Glu Arg Lys Ile Ile
195 200 205
Ser Asp Phe Val Lys Leu Ile Lys Ser Tyr Asp Pro Asp Ile Ile Val
210 215 220
Gly Tyr Asn Gln Asp Ala Phe Asp Trp Pro Tyr Leu Arg Lys Arg Ala
225 230 235 240
Glu Arg Trp Asn Ile Pro Leu Asp Val Gly Arg Asp Gly Ser Asn Val
245 250 255
Val Phe Arg Gly Gly Arg Pro Lys Ile Thr Gly Arg Leu Asn Val Asp
260 265 270
Leu Tyr Asp Ile Ala Met Arg Ile Ser Asp Ile Lys Ile Lys Lys Leu
275 280 285
Glu Asn Val Ala Glu Phe Leu Gly Thr Lys Ile Glu Ile Ala Asp Ile
290 295 300
Glu Ala Lys Asp Ile Tyr Arg Tyr Trp Ser Arg Gly Glu Lys Glu Lys
305 310 315 320
Val Leu Asn Tyr Ala Arg Gln Asp Ala Ile Asn Thr Tyr Leu Ile Ala
325 330 335
Lys Glu Leu Leu Pro Met His Tyr Glu Leu Ser Lys Met Ile Arg Leu
340 345 350
Pro Val Asp Asp Val Thr Arg Met Gly Arg Gly Lys Gln Val Asp Trp
355 360 365
Leu Leu Leu Ser Glu Ala Lys Lys Ile Gly Glu Ile Ala Pro Asn Pro
370 375 380
Pro Glu His Ala Glu Ser Tyr Glu Gly Ala Phe Val Leu Glu Pro Glu
385 390 395 400
Arg Gly Leu His Glu Asn Val Ala Cys Leu Asp Phe Ala Ser Met Tyr
405 410 415
Pro Ser Ile Met Ile Ala Phe Asn Ile Ser Pro Asp Thr Tyr Gly Cys
420 425 430
Arg Asp Asp Cys Tyr Glu Ala Pro Glu Val Gly His Lys Phe Arg Lys
435 440 445
Ser Pro Asp Gly Phe Phe Lys Arg Ile Leu Arg Met Leu Ile Glu Lys
450 455 460
Arg Arg Glu Leu Lys Val Glu Leu Lys Asn Leu Ser Pro Glu Ser Ser
465 470 475 480
Glu Tyr Lys Leu Leu Asp Ile Lys Gln Gln Thr Leu Lys Val Leu Thr
485 490 495
Asn Ser Phe Tyr Gly Tyr Met Gly Trp Asn Leu Ala Arg Trp Tyr Cys
500 505 510
His Pro Cys Ala Glu Ala Thr Thr Ala Trp Gly Arg His Phe Ile Arg
515 520 525
Thr Ser Ala Lys Ile Ala Glu Ser Met Gly Phe Lys Val Leu Tyr Gly
530 535 540
Asp Thr Asp Ser Ile Phe Val Thr Lys Ala Gly Met Thr Lys Glu Asp
545 550 555 560
Val Asp Arg Leu Ile Asp Lys Leu His Glu Glu Leu Pro Ile Gln Ile
565 570 575
Glu Val Asp Glu Tyr Tyr Ser Ala Ile Phe Phe Val Glu Lys Lys Arg
580 585 590
Tyr Ala Gly Leu Thr Glu Asp Gly Arg Leu Val Val Lys Gly Leu Glu
595 600 605
Val Arg Arg Gly Asp Trp Cys Glu Leu Ala Lys Lys Val Gln Arg Glu
610 615 620
Val Ile Glu Val Ile Leu Lys Glu Lys Asn Pro Glu Lys Ala Leu Ser
625 630 635 640
Leu Val Lys Asp Val Ile Leu Arg Ile Lys Glu Gly Lys Val Ser Leu
645 650 655
Glu Glu Val Val Ile Tyr Lys Gly Leu Thr Lys Lys Pro Ser Lys Tyr
660 665 670
Glu Ser Met Gln Ala His Val Lys Ala Ala Leu Lys Ala Arg Glu Met
675 680 685
Gly Ile Ile Tyr Pro Val Ser Ser Lys Ile Gly Tyr Val Ile Val Lys
690 695 700
Gly Ser Gly Asn Ile Gly Asp Arg Ala Tyr Pro Ile Asp Leu Ile Glu
705 710 715 720
Asp Phe Asp Gly Glu Asn Leu Arg Ile Lys Thr Lys Ser Gly Ile Glu
725 730 735
Ile Lys Lys Leu Asp Lys Asp Tyr Tyr Ile Asp Asn Gln Ile Ile Pro
740 745 750
Ser Val Leu Arg Ile Leu Glu Arg Phe Gly Tyr Thr Glu Ala Ser Leu
755 760 765
Lys Gly Ser Ser Gln Met Ser Leu Asp Ser Phe Phe Ser
770 775 780
<210> 35
<211> 783
<212> PRT
<213> Archaeoglobus profundus
<400> 35
Met Ile Lys Ala Trp Leu Leu Asp Val Asp Tyr Val Thr Glu Asn Asp
1 5 10 15
Arg Ala Val Ile Arg Leu Trp Cys Lys Asp Asp Lys Gly Val Phe Val
20 25 30
Ala Tyr Asp Arg Asn Phe Leu Pro Tyr Phe Tyr Val Ile Gly Cys Lys
35 40 45
Ala Glu Asp Val Met Lys Val Lys Val Arg Thr Asn Glu Gly Ile Ile
50 55 60
Thr Pro Leu Lys Val Glu Glu Ile Glu Ala Lys Ser Leu Gly Lys Pro
65 70 75 80
Ile Lys Ala Leu Lys Val Tyr Thr Arg His Pro Gln His Val Pro Lys
85 90 95
Leu Arg Glu Glu Ile Lys Lys Phe Ala Glu Val Arg Glu Ala Asp Ile
100 105 110
Pro Phe Ala Tyr Arg Tyr Leu Ile Asp Lys Asp Leu Ala Cys Met Asp
115 120 125
Gly Ile Glu Ile Glu Pro Ile Ala Val Lys Glu Gly Val Leu Arg Ala
130 135 140
Tyr Glu Val Arg Ser Val Arg Arg Val Glu Lys Lys Gly Phe Pro Asp
145 150 155 160
Leu Lys Ile Leu Ala Phe Asp Cys Glu Met Leu Ala Gln Phe Met Pro
165 170 175
Asp Pro Glu Lys Asp Pro Ile Ile Ala Ile Ala Val Lys Cys Gly Asp
180 185 190
Phe Glu Glu Val Leu His Gly Asp Glu Arg Asp Ile Leu Arg Arg Phe
195 200 205
Val Ser Ile Ile Lys Glu Gln Asp Pro Asp Ile Ile Val Gly Tyr Asn
210 215 220
Gln Asp Asn Phe Asp Trp Pro Tyr Val Lys Lys Arg Ala Glu Lys Phe
225 230 235 240
Gly Ile Arg Leu Asp Ile Gly Arg Asp Arg Ser Glu Ile Ser Phe Arg
245 250 255
Gly Gly Arg Pro Lys Ile Ala Gly Arg Leu Asn Val Asp Leu Tyr Asp
260 265 270
Ile Ala Leu Lys Ile Pro Asp Val Lys Ile Lys Thr Leu Lys Lys Val
275 280 285
Ala Glu Phe Leu Gly Ala Lys Val Glu Glu Glu Asp Ile Glu Gly Arg
290 295 300
Asp Ile Tyr Lys Cys Trp Met Arg Gly Glu Lys Glu Lys Val Phe Lys
305 310 315 320
His Val Leu Asn Asp Val Leu Thr Thr Tyr Arg Leu Ala Leu Glu Leu
325 330 335
Leu Pro Met His Tyr Glu Leu Ser Arg Met Ile Arg Leu Pro Leu Asp
340 345 350
Asp Val Ala Arg Leu Gly Arg Gly Lys Gln Val Asp Tyr Phe Leu Leu
355 360 365
Ser Glu Ala Lys Lys Ile Asn Glu Ile Ala Pro Asn Pro Pro Glu Ile
370 375 380
Glu Glu Ser Tyr Glu Gly Ala Phe Val Leu Glu Pro Ala Arg Gly Leu
385 390 395 400
His Glu Asn Val Ala Cys Leu Asp Phe Ala Ser Met Tyr Pro Ser Ile
405 410 415
Met Ile Asn Phe Asn Ile Ser Pro Asp Thr Leu Val Lys Gly Glu Cys
420 425 430
Glu Asp Cys Tyr Val Ala Pro Glu Val Gly His Lys Phe Arg Lys Ser
435 440 445
Pro Asp Gly Phe Phe Lys Arg Ile Leu Lys Met Leu Ile Glu Lys Arg
450 455 460
Arg Glu Met Lys Arg Gln Met Lys Glu Leu Asp Pro Asp Ser Glu Asp
465 470 475 480
Tyr Lys Leu Leu Asp Ile Lys Gln Gln Thr Leu Lys Val Leu Thr Asn
485 490 495
Ser Phe Tyr Gly Tyr Thr Gly Trp Asn Leu Ala Arg Trp Tyr Cys Arg
500 505 510
Glu Cys Ala Glu Ala Thr Thr Ala Trp Gly Arg Tyr Phe Ile Lys Arg
515 520 525
Ala Val Lys Ile Ala Glu Ser Met Gly Phe Glu Val Leu Tyr Gly Asp
530 535 540
Thr Asp Ser Leu Phe Ile Lys Lys Asn Lys Leu Asn Leu Lys Asp Leu
545 550 555 560
Glu Lys Glu Cys Leu Lys Leu Ile Asp Val Ile Ser Lys Glu Leu Pro
565 570 575
Ile Gln Leu Glu Ile Asp Glu Phe Tyr Lys Ala Ile Phe Phe Val Glu
580 585 590
Lys Lys Arg Tyr Ala Gly Leu Thr Asp Asp Asp Arg Ile Val Val Lys
595 600 605
Gly Leu Glu Val Arg Arg Gly Asp Trp Cys Glu Leu Ala Lys Arg Val
610 615 620
Gln Arg Glu Val Ile Glu Ile Ile Leu Arg Glu Arg Asn Pro Asp Lys
625 630 635 640
Ala Leu Lys Phe Val Lys Asn Val Ile Glu Glu Ile Lys Glu Gly Lys
645 650 655
Phe Lys Leu Glu Asp Tyr Val Ile Tyr Lys Gly Leu Thr Lys Lys Pro
660 665 670
Asp Lys Tyr Glu Ser Lys Gln Ala His Val Lys Ala Ala Leu Arg Ala
675 680 685
Met Glu Met Gly Ile Tyr Tyr Pro Ile Gly Thr Lys Val Gly Phe Val
690 695 700
Ile Val Lys Gly Gly Gly Ser Ile Ser Asp Arg Ala Tyr Pro Ile Glu
705 710 715 720
Leu Ile Glu Glu Phe Asp Gly Glu Asn Leu Lys Ile Arg Thr Pro Ser
725 730 735
Gly Ile Met Val Lys Lys Ile Asp Lys Asp Tyr Tyr Ile Asp His Gln
740 745 750
Ile Ile Pro Ala Val Met Arg Ile Leu Glu Arg Phe Gly Tyr Thr Glu
755 760 765
Ala Ser Leu Lys Thr Thr Ile Gln Lys Thr Leu Phe Asp Phe Thr
770 775 780
<210> 36
<211> 849
<212> PRT
<213> Caldicellulosiruptor hydrothermalis
<400> 36
Met Lys Leu Val Ile Phe Asp Gly Asn Ser Ile Leu Tyr Arg Ala Phe
1 5 10 15
Phe Ala Leu Pro Glu Leu Thr Thr Ser Ser Asn Ile Pro Thr Asn Ala
20 25 30
Ile Tyr Gly Phe Ile Asn Val Ile Leu Lys Tyr Leu Glu Gln Glu Lys
35 40 45
Pro Asp Tyr Ile Ala Val Ala Phe Asp Lys Arg Gly Arg Glu Ala Arg
50 55 60
Lys Ser Glu Tyr Gln Glu Tyr Lys Ala Asn Arg Lys Pro Met Pro Asp
65 70 75 80
Asn Leu Gln Val Gln Ile Pro Tyr Val Arg Glu Ile Leu Tyr Ala Leu
85 90 95
Asn Ile Pro Ile Val Glu Phe Glu Gly Tyr Glu Ala Asp Asp Val Ile
100 105 110
Gly Ser Leu Val Asn Lys Phe Lys Asn Thr Gly Leu Asp Ile Val Ile
115 120 125
Ile Thr Gly Asp Arg Asp Thr Leu Gln Leu Leu Asp Lys Asn Val Val
130 135 140
Val Lys Ile Val Ser Thr Lys Phe Asp Arg Thr Met Glu Asp Leu Tyr
145 150 155 160
Thr Ile Glu Asn Ile Lys Glu Lys Tyr Gly Val Trp Ala Asn Gln Val
165 170 175
Pro Asp Tyr Lys Ala Leu Val Gly Asp Gln Ser Asp Asn Ile Pro Gly
180 185 190
Val Lys Gly Ile Gly Glu Lys Ser Ala Gln Lys Leu Leu Glu Glu Tyr
195 200 205
Ser Ser Leu Glu Glu Ile Tyr Gln Asn Leu Asp Lys Ile Lys Gly Ser
210 215 220
Ile Arg Glu Lys Leu Glu Ala Gly Lys Asp Met Ala Phe Leu Ser Lys
225 230 235 240
Arg Leu Ala Thr Ile Val Cys Asp Leu Pro Leu Asn Val Asn Leu Glu
245 250 255
Asp Leu Arg Thr Lys Glu Trp Asn Lys Glu Arg Leu Tyr Glu Ile Leu
260 265 270
Val Gln Leu Glu Phe Lys Ser Ile Ile Lys Arg Leu Gly Leu Ser Glu
275 280 285
Asn Ile Gln Phe Glu Phe Val Gln Gln Arg Thr Asp Ile Pro Asp Val
290 295 300
Glu Gln Arg Glu Leu Glu Ser Ile Ser Arg Ile Arg Ser Lys Glu Ile
305 310 315 320
Pro Leu Met Phe Val Gln Asp Glu Lys Cys Phe Tyr Leu Tyr Asp Gln
325 330 335
Glu Ser Asn Thr Val Phe Val Thr Arg Asp Arg His Leu Val Glu Glu
340 345 350
Ile Leu Lys Ser Asp Thr Val Lys Ile Val Tyr Asp Leu Lys Asn Ile
355 360 365
Phe His Gln Leu Asn Leu Glu Asp Thr Asp Asn Ile Lys Asn Cys Glu
370 375 380
Asp Val Met Ile Ala Ser Tyr Val Leu Asp Ser Thr Arg Ser Ser Tyr
385 390 395 400
Glu Leu Glu Thr Leu Phe Val Ser Tyr Leu Asn Thr Asp Ile Glu Ala
405 410 415
Val Lys Lys Asp Lys Lys Met Val Ser Val Val Leu Leu Lys Arg Leu
420 425 430
Trp Asp Asp Leu Leu Arg Leu Ile Asp Leu Asn Ser Cys Gln Phe Leu
435 440 445
Tyr Glu Asn Ile Glu Arg Pro Leu Ile Pro Val Leu Tyr Glu Met Glu
450 455 460
Lys Thr Gly Phe Lys Val Asp Arg Asp Ala Leu Leu Gln Tyr Thr Lys
465 470 475 480
Glu Ile Glu Asn Lys Ile Leu Lys Leu Glu Thr Gln Ile Tyr Gln Ile
485 490 495
Ala Gly Glu Trp Phe Asn Ile Asn Ser Pro Lys Gln Leu Ser Tyr Ile
500 505 510
Leu Phe Glu Lys Leu Lys Leu Pro Val Ile Lys Lys Thr Lys Thr Gly
515 520 525
Tyr Ser Thr Asp Ala Glu Val Leu Glu Glu Phe Asp Lys His Glu Ile
530 535 540
Val Pro Leu Ile Leu Asp Tyr Arg Met Tyr Thr Lys Ile Leu Thr Thr
545 550 555 560
Tyr Cys Gln Gly Leu Leu Gln Ala Ile Asn Pro Ser Ser Gly Arg Val
565 570 575
His Thr Thr Phe Ile Gln Thr Gly Thr Ala Thr Gly Arg Leu Ala Ser
580 585 590
Ser Asp Pro Asn Leu Gln Asn Ile Pro Val Lys Tyr Asp Glu Gly Lys
595 600 605
Leu Ile Arg Lys Val Phe Val Pro Glu Glu Gly His Val Leu Ile Asp
610 615 620
Ala Asp Tyr Ser Gln Ile Glu Leu Arg Ile Leu Ala His Ile Ser Glu
625 630 635 640
Asp Glu Arg Leu Ile Asn Ala Phe Lys Asn Asn Ile Asp Ile His Ser
645 650 655
Gln Thr Ala Ala Glu Val Phe Gly Val Asp Ile Ala Asp Val Thr Pro
660 665 670
Glu Met Arg Ser Gln Ala Lys Ala Val Asn Phe Gly Ile Val Tyr Gly
675 680 685
Ile Ser Asp Tyr Gly Leu Ala Arg Asp Ile Lys Ile Ser Arg Lys Glu
690 695 700
Ala Ala Glu Phe Ile Asn Lys Tyr Phe Glu Arg Tyr Pro Lys Val Lys
705 710 715 720
Glu Tyr Leu Asp Asn Ile Val Arg Phe Ala Arg Glu Asn Gly Tyr Val
725 730 735
Leu Thr Leu Phe Asn Arg Lys Arg Tyr Val Lys Asp Ile Lys Ser Ala
740 745 750
Asn Arg Asn Ala Arg Ser Tyr Ala Glu Arg Ile Ala Met Asn Ser Pro
755 760 765
Ile Gln Gly Ser Ala Ala Asp Ile Met Lys Leu Ala Met Ile Lys Val
770 775 780
Tyr Gln Lys Leu Lys Glu Asn Asn Leu Lys Ser Lys Ile Ile Leu Gln
785 790 795 800
Val His Asp Glu Leu Leu Ile Glu Ala Pro Tyr Glu Glu Lys Asp Ile
805 810 815
Val Lys Arg Ile Val Lys Arg Glu Met Glu Asn Ala Val Ala Leu Lys
820 825 830
Val Pro Leu Val Val Glu Val Lys Glu Gly Leu Asn Trp Tyr Glu Thr
835 840 845
Lys
<210> 37
<211> 781
<212> PRT
<213> Desulfurococcus mucosus
<400> 37
Met Glu Lys Arg Val Tyr Leu Val Asp Ile Thr Tyr Gly Leu Val Gly
1 5 10 15
Asn Ser Pro Glu Ile Arg Met Phe Gly Val Asp Glu Asn Gly Glu Lys
20 25 30
Val Val Ile Leu Asp Arg Gly Phe Arg Pro Tyr Phe Tyr Val Ile Pro
35 40 45
Glu Glu Gly Phe Glu Asp Gln Val Ala Arg Val Ile Gly Lys Met Gln
50 55 60
Asn Val Ile Lys Ala Asp Val Thr Glu Arg Arg Leu Phe Gly Lys Pro
65 70 75 80
Ile Lys Val Val Lys Val Thr Val Thr Val Pro Asp Lys Val Arg Glu
85 90 95
Leu Arg Asp Arg Val Lys Ser Ile Gln His Val Lys Glu Val Leu Glu
100 105 110
Ala Asp Ile Arg Phe Tyr Ile Arg Tyr Met Ile Asp Asn Asp Ile Arg
115 120 125
Pro Gly Trp Leu Met Phe Ser Asn Leu Lys Pro Val Asp Asn Lys Ile
130 135 140
Gly Gly Val Ser Asn Val Tyr Leu Thr Glu Thr Pro Pro Thr Ser Leu
145 150 155 160
Asp Leu Gly Ile Met Pro Arg Leu Asn Tyr Met Ala Leu Asp Ile Glu
165 170 175
Val Tyr Asn Pro Arg Gly Thr Pro Asp Pro Lys Arg Asp Pro Ile Ile
180 185 190
Ile Ile Ala Leu Ala Asn Ser Asn Gly Asp Val Lys Leu Leu Thr Leu
195 200 205
Asp Asn Tyr Lys His Glu Arg Glu Met Leu Asn Asp Met Met Ser Val
210 215 220
Ile Lys Glu Trp Asp Pro Asp Val Leu Phe Gly Tyr Asn Ser Asn Lys
225 230 235 240
Phe Asp Met Pro Tyr Leu Val Asn Arg Ala Asp Ala Leu Asn Val Lys
245 250 255
Leu Gln Leu Ser Lys Tyr Gly Thr Pro Pro Glu Gln Ser Val Tyr Gly
260 265 270
His Trp Ser Ile Ile Gly Arg Ala His Ile Asp Leu Tyr Asn Phe Ile
275 280 285
Glu Asp Met Thr Asp Val Lys Arg Lys Ser Leu Asp Tyr Val Ala Glu
290 295 300
Tyr Phe Gly Val Met Lys Arg Ser Glu Arg Val Asn Ile Pro Gly His
305 310 315 320
Arg Ile Tyr Gln Tyr Trp Asp Asp Glu Gly Lys Arg Ser Gln Leu Ile
325 330 335
Lys Tyr Ala Arg Asp Asp Val Leu Ser Thr Leu Gly Leu Gly Lys Ile
340 345 350
Leu Leu Pro Tyr Ala Met Gln Leu Ala Ser Val Ser Gly Leu Pro Leu
355 360 365
Asp Gln Val Gly Pro Ala Ser Val Gly Ser Arg Val Glu Met Met Ile
370 375 380
Met His Glu Ala Tyr Lys Met Gly Glu Leu Ala Pro Asn Arg Val Glu
385 390 395 400
Arg Pro Tyr Glu Thr Tyr Lys Gly Ala Ile Val Leu Glu Pro Lys Pro
405 410 415
Gly Ile His Tyr Asn Ile Ala Val Leu Asp Phe Ser Ser Met Tyr Pro
420 425 430
Asn Ile Met Leu Lys Tyr Asn Ile Ser Pro Asp Thr Leu Val Leu Asp
435 440 445
Ser Ser Glu Gly Asp Tyr Tyr Thr Ala Pro Glu Val Gly Tyr Arg Phe
450 455 460
Arg Lys Ser Pro Arg Gly Leu Tyr Ala Ser Leu Leu Gln Lys Leu Ile
465 470 475 480
Glu Ala Arg Arg Glu Ala Arg Asp Glu Met Arg Asn Tyr Pro Glu Gly
485 490 495
Ser Phe Glu Trp Val Leu Leu Asn Glu Arg Gln Arg Ala Leu Lys Ile
500 505 510
Met Ala Asn Ala Met Tyr Gly Tyr Cys Gly Trp Leu Gly Ala Arg Trp
515 520 525
Tyr Ile Arg Glu Val Ala Glu Ser Val Thr Ala Trp Gly Arg Tyr Leu
530 535 540
Leu Lys Thr Ala Met Ser Met Ala Lys Glu Arg Gly Leu Thr Val Ile
545 550 555 560
Tyr Gly Asp Thr Asp Ser Leu Phe Val Thr Tyr Asp Lys Asp Lys Val
565 570 575
Ala Asp Ile Ile Ser Arg Ile Asn Glu Met Gly Phe Glu Val Lys Ile
580 585 590
Asp Lys Val Tyr Ser Lys Leu Ile Phe Thr Glu Ser Lys Lys Arg Tyr
595 600 605
Ile Gly Leu Thr Ala Asp Gly Glu Val Asp Ile Val Gly Phe Glu Ala
610 615 620
Val Arg Gly Asp Trp Ser Glu Leu Ala Arg Asn Val Gln Glu Arg Val
625 630 635 640
Ala Glu Leu Val Leu Arg Glu Ser Val Asp Glu Ala Val Lys Tyr Val
645 650 655
Lys Ser Val Ile Asp Asp Leu Arg Asn Tyr Arg Phe Thr Ile Asp Asp
660 665 670
Val Ile Ile Trp Lys Thr Leu Asp Lys Asp Ile Asn Glu Tyr Lys Ala
675 680 685
Ile Gln Pro His Val Val Ala Ala Arg Arg Leu Met Glu Lys Gly Tyr
690 695 700
Val Val Asn Lys Gly Asp Thr Val Gly Phe Val Ile Val Lys Asp Ser
705 710 715 720
Gly Asp Lys Leu Thr Gln Arg Ala Tyr Pro Tyr Val Phe Ile Asn Asp
725 730 735
Val Lys Glu Ile Asp Val Asp Tyr Tyr Val Glu Lys Gln Val Ile Pro
740 745 750
Ala Ala Leu Arg Ile Leu Glu Val Phe Gly Val Asn Glu Ala Ala Leu
755 760 765
Leu Gly Lys Thr Gly Lys Ser Ile Leu Asp Tyr Phe His
770 775 780
<210> 38
<211> 803
<212> PRT
<213> Pyrolobus fumarii
<400> 38
Met Thr Glu Val Val Phe Thr Val Leu Asp Ser Ser Tyr Glu Val Val
1 5 10 15
Gly Lys Glu Pro Gln Val Ile Ile Trp Gly Ile Ala Glu Asn Gly Glu
20 25 30
Arg Val Val Leu Ile Asp Arg Ser Phe Arg Pro Tyr Phe Tyr Ala Leu
35 40 45
Leu Ala Pro Gly Ala Asp Pro Lys Gln Val Ala Gln Arg Ile Arg Ala
50 55 60
Leu Ser Arg Pro Lys Ser Pro Ile Ile Gly Val Glu Asp Asp Lys Arg
65 70 75 80
Lys Tyr Phe Gly Arg Pro Arg Arg Val Leu Arg Ile Arg Thr Val Leu
85 90 95
Pro Glu Ala Val Arg Glu Tyr Arg Glu Leu Val Lys Asn Val Asp Gly
100 105 110
Val Glu Asp Val Leu Glu Ala Asp Ile Arg Phe Ala Met Arg Tyr Leu
115 120 125
Ile Asp His Asp Leu Phe Pro Phe Thr Trp Tyr Arg Val Glu Ala Glu
130 135 140
Pro Leu Glu Asn Lys Met Gly Phe Arg Val Asp Lys Val Tyr Leu Val
145 150 155 160
Lys Ser Arg Pro Glu Pro Leu Tyr Gly Glu Ala Leu Ala Pro Thr Lys
165 170 175
Leu Pro Asp Leu Arg Ile Leu Ala Phe Asp Ile Glu Val Tyr Ser Lys
180 185 190
Gln Gly Ser Pro Arg Pro Glu Arg Asp Pro Val Ile Val Ile Ala Val
195 200 205
Lys Thr Asp Asp Gly Asp Glu Val Leu Phe Ile Ala Glu Gly Lys Asp
210 215 220
Asp Arg Lys Pro Ile Arg Glu Phe Val Glu Tyr Val Lys Arg Tyr Asp
225 230 235 240
Pro Asp Ile Ile Val Gly Tyr Asn Asn Asn His Phe Asp Trp Pro Tyr
245 250 255
Leu Leu Arg Arg Ala Arg Ile Leu Gly Ile Lys Leu Asp Val Thr Arg
260 265 270
Arg Val Gly Ala Glu Pro Thr Thr Ser Val His Gly His Val Ser Val
275 280 285
Pro Gly Arg Leu Asn Val Asp Leu Tyr Asp Tyr Ala Glu Glu Met Pro
290 295 300
Glu Ile Lys Ile Lys Ser Leu Glu Glu Val Ala Glu Tyr Leu Gly Val
305 310 315 320
Met Lys Lys Ser Glu Arg Val Ile Ile Asn Trp Trp Glu Ile Pro Asp
325 330 335
Tyr Trp Asp Asp Pro Lys Lys Arg Pro Leu Leu Leu Gln Tyr Ala Arg
340 345 350
Asp Asp Val Arg Ala Thr Tyr Gly Leu Ala Glu Lys Ile Leu Pro Phe
355 360 365
Ala Ile Gln Leu Ser Tyr Val Thr Gly Leu Pro Leu Asp Gln Val Gly
370 375 380
Ala Met Ser Val Gly Phe Arg Leu Glu Trp Tyr Leu Ile Arg Ala Ala
385 390 395 400
Phe Lys Met Lys Glu Leu Val Pro Asn Arg Val Glu Arg Pro Glu Glu
405 410 415
Thr Tyr Arg Gly Ala Ile Val Leu Glu Pro Leu Arg Gly Val His Glu
420 425 430
Asn Ile Ala Val Leu Asp Phe Ser Ser Met Tyr Pro Asn Ile Met Ile
435 440 445
Lys Tyr Asn Val Gly Pro Asp Thr Leu Val Arg Pro Gly Glu Glu Cys
450 455 460
Gly Glu Cys Gly Cys Trp Glu Ala Pro Glu Val Lys His Arg Phe Arg
465 470 475 480
Arg Cys Pro Pro Gly Phe Phe Lys Thr Val Leu Glu Arg Leu Leu Glu
485 490 495
Leu Arg Lys Arg Val Arg Ala Glu Met Lys Lys Tyr Pro Pro Asp Ser
500 505 510
Pro Glu Tyr Arg Leu Leu Asp Glu Arg Gln Lys Ala Leu Lys Val Leu
515 520 525
Ala Asn Ala Ser Tyr Gly Tyr Met Gly Trp Ser Gly Ala Arg Trp Tyr
530 535 540
Cys Arg Glu Cys Ala Glu Ala Val Thr Ala Trp Gly Arg His Leu Ile
545 550 555 560
Arg Thr Ala Ile Asn Ile Ala Arg Lys Leu Gly Leu Lys Val Ile Tyr
565 570 575
Gly Asp Thr Asp Ser Leu Phe Val Thr Tyr Asp Pro Glu Lys Val Glu
580 585 590
Lys Phe Ile Lys Ile Ile Glu Glu Glu Leu Gly Phe Glu Ile Lys Leu
595 600 605
Glu Lys Val Tyr Lys Arg Val Phe Phe Thr Glu Ala Lys Lys Arg Tyr
610 615 620
Ala Gly Leu Leu Glu Asp Gly Arg Ile Asp Ile Val Gly Phe Glu Ala
625 630 635 640
Val Arg Gly Asp Trp Cys Glu Leu Ala Lys Glu Val Gln Thr Lys Val
645 650 655
Val Glu Ile Val Leu Lys Thr Ser Asp Val Asn Lys Ala Val Glu Tyr
660 665 670
Val Arg Lys Ile Val Lys Glu Leu Glu Glu Gly Lys Val Pro Ile Glu
675 680 685
Lys Leu Val Ile Trp Lys Thr Leu Ser Lys Arg Leu Glu Glu Tyr Thr
690 695 700
Thr Glu Ala Pro His Val Val Ala Ala Lys Arg Met Leu Ser Ala Gly
705 710 715 720
Tyr Arg Val Ser Pro Gly Asp Lys Ile Gly Tyr Val Ile Val Lys Gly
725 730 735
Gly Gly Arg Ile Ser Gln Arg Ala Trp Pro Tyr Phe Met Val Lys Asp
740 745 750
Pro Ser Gln Ile Asp Val Thr Tyr Tyr Val Asp His Gln Ile Ile Pro
755 760 765
Ala Ala Leu Arg Ile Leu Gly Tyr Phe Gly Ile Thr Glu Lys Lys Leu
770 775 780
Lys Ala Ser Ala Thr Gly Gln Lys Thr Leu Phe Asp Phe Leu Ala Lys
785 790 795 800
Lys Ser Lys
<210> 39
<211> 786
<212> PRT
<213> Pyrobaculum oguniense
<400> 39
Met Glu Ile Arg Val Trp Pro Leu Asp Val Thr Tyr Ile Val Val Gly
1 5 10 15
Gly Val Pro Glu Val Arg Val Phe Gly Ile Ala Glu Gly Gly Glu Arg
20 25 30
Val Val Leu Ala Asp Arg Ser Phe Arg Pro Tyr Phe Tyr Val Asp Cys
35 40 45
Ala Gly Cys Asp Pro His Ala Val Lys Thr His Leu Gly Arg Thr Ala
50 55 60
Pro Val Glu Gly Val Glu Leu Val Glu Arg Arg Phe Leu Gly Arg Pro
65 70 75 80
Arg Gln Phe Leu Lys Val Val Ala Lys Ile Pro Glu Asp Val Arg Arg
85 90 95
Leu Arg Glu Ala Ala Ser Thr Ile Pro Gly Val Arg Gly Val Tyr Glu
100 105 110
Ala Asp Ile Arg Phe Tyr Met Arg Tyr Val Ile Asp Met Gly Val Val
115 120 125
Pro Cys Ser Trp Asn Val Ala Glu Val Glu Val Ala Asp Glu Lys Leu
130 135 140
Gly Ser Leu Pro Val Tyr Arg Val Val Lys Trp Gly Gly Ala Val Glu
145 150 155 160
Gly Phe Pro Pro Pro Leu Arg Val Leu Ala Phe Asp Ile Glu Val Tyr
165 170 175
Asn Glu Arg Gly Thr Pro Asp Pro Ala Arg Asp Pro Ile Val Met Ile
180 185 190
Ala Val Gln Ser Ser Asp Gly Arg Leu Glu Val Phe Glu Ala Ser Gly
195 200 205
Arg Asp Asp Arg Gly Val Leu Arg Ser Phe Val Glu Tyr Val Arg Ser
210 215 220
Phe Asp Pro Asp Val Val Val Gly Tyr Asn Ser Asn Asn Phe Asp Trp
225 230 235 240
Pro Tyr Leu Ala Glu Arg Ala Lys Ala Val Gly Val Pro Leu Arg Val
245 250 255
Asp Arg Leu Gly Gly Ala Pro Gln Gln Ser Val Tyr Gly His Trp Ser
260 265 270
Val Leu Gly Arg Ala Asn Val Asp Leu Tyr Asn Ile Val Asp Glu Phe
275 280 285
Pro Glu Ile Lys Leu Lys Thr Leu Asp Arg Val Ala Glu Tyr Phe Gly
290 295 300
Val Met Arg Arg Asp Glu Arg Val Leu Ile Pro Gly His Lys Ile Tyr
305 310 315 320
Glu Tyr Trp Arg Asp Pro Ser Lys Arg Pro Leu Leu Arg Gln Tyr Val
325 330 335
Ile Asp Asp Val Arg Ser Thr Tyr Gly Leu Ala Glu Arg Leu Leu Pro
340 345 350
Phe Leu Ile Gln Leu Ser Ser Val Ser Gly Leu Pro Leu Asp Gln Val
355 360 365
Ala Ala Ala Ser Val Gly Asn Arg Val Glu Trp Met Leu Leu Arg Tyr
370 375 380
Ala Tyr Gly Leu Gly Glu Val Ala Pro Asn Arg Glu Glu Arg Glu Tyr
385 390 395 400
Glu Pro Tyr Lys Gly Ala Ile Val Leu Glu Pro Lys Pro Gly Leu Tyr
405 410 415
Ser Asp Val Leu Val Leu Asp Phe Ser Ser Met Tyr Pro Asn Val Met
420 425 430
Met Arg Tyr Asn Leu Ser Pro Asp Thr Tyr Leu Glu Pro Gly Glu Pro
435 440 445
Asp Pro Pro Glu Gly Val Tyr Val Ala Pro Glu Val Gly His Arg Phe
450 455 460
Arg Lys Glu Pro Pro Gly Phe Ile Pro Gln Val Leu Arg Arg Leu Val
465 470 475 480
Ala Leu Arg Arg Ala Val Arg Glu Glu Met Lys Lys Tyr Gln Pro Asp
485 490 495
Thr Pro Glu Tyr Arg Val Leu Asp Glu Arg Gln Lys Ala Leu Lys Ile
500 505 510
Met Ala Asn Ala Met Tyr Gly Tyr Thr Gly Trp Val Gly Ala Arg Trp
515 520 525
Tyr Lys Lys Glu Val Ala Glu Ser Val Thr Ala Phe Ala Arg Ala Ile
530 535 540
Leu Lys Asp Val Ile Asp Tyr Ala Arg Arg Leu Gly Ile Val Val Ile
545 550 555 560
Tyr Gly Asp Thr Asp Ser Leu Phe Val Lys Lys Gly Gly Asp Leu Glu
565 570 575
Lys Leu Ala Arg Tyr Val Asp Glu Lys Tyr Gly Ile Glu Ile Lys Val
580 585 590
Asp Lys Asp Tyr Glu Lys Val Leu Phe Thr Glu Ala Lys Lys Arg Tyr
595 600 605
Ala Gly Leu Leu Arg Asp Gly Arg Ile Asp Ile Val Gly Phe Glu Val
610 615 620
Val Arg Gly Asp Trp Ser Glu Leu Ala Lys Glu Val Gln Leu Lys Val
625 630 635 640
Ile Glu Leu Ile Leu Lys Ala Arg Asp Leu Ser Glu Ala Arg Gln Arg
645 650 655
Val Ile Lys Tyr Val Lys Asp Val Ile Glu Arg Leu Lys Ser Gly Lys
660 665 670
Phe Asp Leu Asp Asp Leu Ile Ile Trp Lys Thr Leu Asp Lys Asp Leu
675 680 685
Gly Glu Tyr Lys Ala Tyr Pro Pro His Val Arg Ala Ala Leu Ile Leu
690 695 700
Lys Lys Lys Gly Tyr Lys Val Gly Arg Gly Thr Thr Ile Gly Tyr Val
705 710 715 720
Val Val Lys Gly Gly Glu Lys Val Ser Glu Arg Ser Leu Pro Tyr Ile
725 730 735
Leu Val Asp Asp Leu Ala Lys Ile Asp Val Asp Tyr Tyr Ile Glu Lys
740 745 750
Gln Val Ile Pro Ala Ala Leu Arg Ile Ala Glu Val Ile Gly Val Lys
755 760 765
Glu Gly Asp Leu Arg Ala Gly Arg Ser Glu Lys Ser Leu Leu Asp Phe
770 775 780
Phe Glu
785
<210> 40
<211> 801
<212> PRT
<213> Staphylothermus marinus
<400> 40
Met Ser Glu Lys Ile Asn Leu Glu Phe Tyr Phe Leu Asp Asn Ser Tyr
1 5 10 15
Glu Val Ile Gly Asn Glu Pro His Ile Ile Ile Trp Gly Ile Thr Arg
20 25 30
Asp Gly Arg Arg Val Leu Leu Arg Asp Arg Arg Phe Arg Pro Tyr Phe
35 40 45
Tyr Ala Ile Leu Lys Asp Lys Val Asn Ile Glu Asp Leu Ala Arg Lys
50 55 60
Ile Arg Thr Tyr Ser Asp Pro Lys Ser Pro Ile Ile Gly Val Glu Pro
65 70 75 80
Val Glu Lys Lys Tyr Phe Gly Arg Lys Val Ser Ala Leu Lys Ile Ile
85 90 95
Thr Met Ile Pro Glu Tyr Val Arg Lys Tyr Arg Glu Lys Ile Lys Ser
100 105 110
Leu Pro Glu Val Leu Glu Val Val Glu Ala Asp Ile Arg Phe Ser Ile
115 120 125
Arg Tyr Ile Ile Asp His Asp Leu Arg Pro Cys Gly Trp His Val Ala
130 135 140
Glu Val Val Glu Val Pro Lys Lys Pro Ile Tyr Arg Val Asp Ala Glu
145 150 155 160
Tyr Glu Ile Ile Gly Asp Ile Lys Pro Leu Glu Gln Thr Leu Gln Pro
165 170 175
Asp Leu Arg Ile Ile Ala Phe Asp Ile Glu Val Tyr Asn Lys Ser Gly
180 185 190
Thr Pro Arg Pro Gln Thr Asp Pro Ile Ile Ile Ile Gly Ile Met Asn
195 200 205
Asn Asn Gly Asp Ile Lys Gln Phe Leu Ala Asn Lys Tyr Asp Asp Lys
210 215 220
Ile Ser Val Glu Glu Phe Val Asn Tyr Val Lys Thr Phe Asp Pro Asp
225 230 235 240
Ile Ile Val Gly Tyr Asn Thr Asp Gly Phe Asp Trp Pro Tyr Leu Ile
245 250 255
Glu Arg Ser Lys Tyr Ile Gly Val Lys Leu Asp Val Thr Arg Arg Val
260 265 270
Gly Ala Thr Pro Arg Thr Ser Thr Tyr Gly His Ile Ser Val Pro Gly
275 280 285
Arg Leu Asn Thr Asp Leu Tyr His Phe Ala Glu Glu Ile Pro Glu Val
290 295 300
Lys Val Lys Ser Leu Glu Asn Val Ala Glu Tyr Leu Gly Val Met Lys
305 310 315 320
Lys Ser Glu Arg Val Ile Ile Glu Tyr Ile Asp Ile Pro Lys Tyr Trp
325 330 335
Asp Asp Glu Lys Leu Arg Pro Lys Leu Leu Gln Tyr Asn Ile Asp Asp
340 345 350
Val Lys Ser Thr Tyr Gly Leu Ala Glu Lys Phe Leu Pro Phe Ala Met
355 360 365
Gln Leu Ser Asn Ile Thr Gly Leu Pro Leu Asp Gln Val Gly Ala Ala
370 375 380
Ser Val Gly Phe Arg Leu Glu Trp Tyr Leu Met Arg Glu Ala Phe Arg
385 390 395 400
Tyr Gly Glu Leu Val Pro Asn Arg Val Glu Arg Ala Ala Glu Ser Tyr
405 410 415
Arg Gly Ala Val Val Leu Lys Pro Val Lys Gly Val His Glu Asn Ile
420 425 430
Ala Val Leu Asp Phe Ser Ser Met Tyr Pro Asn Ile Met Ile Lys Tyr
435 440 445
Asn Val Gly Pro Asp Thr Ile Val Arg Asn Glu Lys Cys Asn Pro Asp
450 455 460
Lys His Asn Ile Ala Pro Glu Val Gly His Cys Phe Arg Lys Glu Pro
465 470 475 480
Pro Gly Phe Phe Lys Arg Val Leu Glu Thr Leu Leu Arg Leu Arg Lys
485 490 495
Gln Ile Lys Ser Glu Met Lys Lys Tyr Pro Pro Thr Ser Tyr Glu Tyr
500 505 510
Arg Leu Leu Asp Glu Arg Gln Lys Ala Val Lys Val Leu Ala Asn Ala
515 520 525
Thr Tyr Gly Tyr Met Gly Trp Ile His Ala Arg Trp Tyr Cys Arg Glu
530 535 540
Cys Ala Glu Ala Val Thr Ala Trp Gly Arg Gln Thr Ile Lys Ser Ala
545 550 555 560
Ile Glu Leu Ala Arg Lys Leu Gly Leu Lys Val Ile Tyr Gly Asp Thr
565 570 575
Asp Ser Leu Phe Val Thr Tyr Asp Lys Asp Lys Val Glu Lys Leu Ile
580 585 590
Glu Leu Ile Gln Thr Lys Leu Gly Phe Glu Ile Lys Ile Asp Lys Ile
595 600 605
Tyr Lys Arg Val Phe Phe Thr Glu Ala Lys Lys Arg Tyr Ala Gly Leu
610 615 620
Leu Glu Asp Gly Arg Ile Asp Ile Val Gly Phe Glu Ala Val Arg Gly
625 630 635 640
Asp Trp Ala Glu Ile Ala Lys Glu Val Gln Glu Lys Val Thr Glu Ile
645 650 655
Leu Leu Lys Glu Asn Ser Ile Asp Lys Ala Ile Glu Tyr Val Arg Gln
660 665 670
Val Ile Ala Asp Leu Lys Ala Gly Lys Ile Pro Leu Asp Lys Leu Ile
675 680 685
Ile Trp Lys Thr Leu Ser Lys Arg Ile Glu Glu Tyr Ser Val Asp Ala
690 695 700
Pro His Val Val Ala Ala Lys Lys Leu Ile Lys Ala Gly Ile Lys Val
705 710 715 720
Ser Thr Asn Asp Lys Ile Gly Tyr Val Ile Leu Lys Gly Gly Gly Lys
725 730 735
Ile Ser Ser Arg Ala Glu Pro Tyr Ile Phe Val Lys Asp Pro Lys Leu
740 745 750
Ile Asp Thr Glu Tyr Tyr Val Asp His Gln Ile Val Pro Ala Ala Leu
755 760 765
Arg Ile Leu Asn Tyr Phe Gly Val Thr Glu Thr Gln Leu Lys Arg Ala
770 775 780
Ala Ala Ser Ala Gly Gln Lys Ser Leu Phe Asp Phe Phe Gly Gly Lys
785 790 795 800
Lys
<210> 41
<211> 776
<212> PRT
<213> Pyrococcus yayaosii
<400> 41
Met Ile Leu Asp Ala Asp Tyr Ile Thr Glu Asn Gly Lys Pro Val Val
1 5 10 15
Arg Ile Phe Lys Lys Glu Asn Gly Glu Phe Lys Val Glu Tyr Asp Arg
20 25 30
Ser Phe Arg Pro Tyr Ile Tyr Ala Leu Leu Arg Asp Asp Ser Ala Ile
35 40 45
Glu Asp Ile Lys Lys Ile Thr Ala Glu Arg His Gly Lys Val Val Arg
50 55 60
Val Val Glu Ala Glu Lys Val Arg Lys Lys Phe Leu Gly Arg Pro Ile
65 70 75 80
Glu Val Trp Lys Leu Tyr Phe Glu His Pro Gln Asp Val Pro Ala Ile
85 90 95
Arg Glu Lys Ile Arg Glu His Pro Ala Val Ile Asp Ile Phe Glu Tyr
100 105 110
Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Lys Gly Leu Ile Pro
115 120 125
Met Glu Gly Asn Glu Glu Leu Lys Leu Leu Ala Phe Asp Ile Glu Thr
130 135 140
Leu Tyr His Glu Gly Asp Glu Phe Gly Ser Gly Pro Ile Ile Met Ile
145 150 155 160
Ser Tyr Ala Asp Glu Lys Gly Ala Lys Val Ile Thr Trp Lys Gly Val
165 170 175
Asp Leu Pro Tyr Val Glu Val Val Ser Ser Glu Arg Glu Met Ile Lys
180 185 190
Arg Phe Leu Arg Val Ile Arg Glu Lys Asp Pro Asp Val Ile Ile Thr
195 200 205
Tyr Asn Gly Asp Asn Phe Asp Phe Pro Tyr Leu Leu Lys Arg Ala Glu
210 215 220
Lys Leu Gly Met Lys Leu Pro Ile Gly Arg Asp Gly Ser Glu Pro Lys
225 230 235 240
Met Gln Arg Met Gly Asp Gly Phe Ala Val Glu Val Lys Gly Arg Ile
245 250 255
His Phe Asp Ile Tyr Pro Val Ile Arg Arg Thr Ile Asn Leu Pro Thr
260 265 270
Tyr Thr Leu Glu Ala Val Tyr Glu Ala Val Phe Gly Arg Pro Lys Glu
275 280 285
Lys Val Tyr Pro Asn Glu Ile Ala Arg Ala Trp Glu Asn Cys Lys Gly
290 295 300
Leu Glu Arg Val Ala Lys Tyr Ser Met Glu Asp Ala Lys Val Thr Tyr
305 310 315 320
Glu Leu Gly Arg Glu Phe Phe Pro Met Glu Ala Gln Leu Ala Arg Leu
325 330 335
Val Gly Gln Pro Val Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu
340 345 350
Val Glu Trp Phe Leu Leu Arg Lys Ala Tyr Glu Arg Asn Glu Leu Ala
355 360 365
Pro Asn Arg Pro Asp Glu Arg Glu Tyr Glu Arg Arg Leu Arg Glu Ser
370 375 380
Tyr Glu Gly Gly Tyr Val Lys Glu Pro Glu Lys Gly Leu Trp Glu Gly
385 390 395 400
Ile Ile Tyr Leu Asp Phe Arg Ser Leu Tyr Pro Ser Ile Ile Ile Thr
405 410 415
His Asn Ile Ser Pro Asp Thr Leu Asn Lys Glu Gly Cys Asn Ser Tyr
420 425 430
Asp Val Ala Pro Lys Val Gly His Arg Phe Cys Lys Asp Phe Pro Gly
435 440 445
Phe Ile Pro Ser Leu Leu Gly Gln Leu Leu Asp Glu Arg Gln Lys Ile
450 455 460
Lys Arg Lys Met Lys Ala Thr Ile Asp Pro Ile Glu Arg Lys Leu Leu
465 470 475 480
Asp Tyr Arg Gln Arg Ala Ile Lys Ile Leu Ala Asn Ser Tyr Tyr Gly
485 490 495
Tyr Tyr Gly Tyr Ala Lys Ala Arg Trp Tyr Cys Lys Glu Cys Ala Glu
500 505 510
Ser Val Thr Ala Trp Gly Arg Glu Tyr Ile Glu Leu Val Ser Arg Glu
515 520 525
Leu Glu Lys Arg Gly Phe Lys Val Leu Tyr Ile Asp Thr Asp Gly Leu
530 535 540
Tyr Ala Thr Ile Pro Gly Ser Arg Glu Trp Asp Lys Ile Lys Glu Arg
545 550 555 560
Ala Leu Glu Phe Val Lys Tyr Ile Asn Ala Arg Leu Pro Gly Leu Leu
565 570 575
Glu Leu Glu Tyr Glu Gly Phe Tyr Lys Arg Gly Phe Phe Val Thr Lys
580 585 590
Lys Lys Tyr Ala Leu Ile Asp Glu Glu Gly Lys Ile Ile Thr Arg Gly
595 600 605
Leu Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu Thr Gln
610 615 620
Ala Arg Val Leu Glu Ala Ile Leu Lys Glu Gly Asn Leu Glu Lys Ala
625 630 635 640
Val Lys Ile Val Lys Glu Val Thr Glu Lys Leu Ser Lys Tyr Glu Val
645 650 655
Pro Pro Glu Lys Leu Val Ile Tyr Glu Gln Ile Thr Arg Asp Leu Lys
660 665 670
Asp Tyr Lys Ala Val Gly Pro His Val Ala Val Ala Lys Arg Leu Ala
675 680 685
Ala Arg Gly Ile Lys Val Arg Pro Gly Met Val Ile Gly Tyr Leu Val
690 695 700
Leu Arg Gly Asp Gly Pro Ile Ser Arg Arg Ala Ile Pro Ala Glu Glu
705 710 715 720
Phe Asp Pro Ser Arg His Lys Tyr Asp Ala Glu Tyr Tyr Ile Glu Asn
725 730 735
Gln Val Leu Pro Ala Val Leu Arg Ile Leu Glu Ala Phe Gly Tyr Arg
740 745 750
Lys Glu Asp Leu Arg Tyr Gln Lys Thr Arg Gln Ala Gly Leu Asp Ala
755 760 765
Trp Leu Lys Arg Lys Ala Ser Leu
770 775
<210> 42
<211> 773
<212> PRT
<213> Thermococcus sp.AM4-del
<400> 42
Met Ile Leu Asp Thr Asp Tyr Ile Thr Glu Asn Gly Lys Pro Val Ile
1 5 10 15
Arg Val Phe Lys Lys Glu Asn Gly Glu Phe Lys Ile Glu Tyr Asp Arg
20 25 30
Thr Phe Glu Pro Tyr Phe Tyr Ala Leu Leu Lys Asp Asp Ser Ala Ile
35 40 45
Glu Asp Val Lys Lys Val Thr Ala Lys Arg His Gly Thr Val Val Arg
50 55 60
Val Lys Arg Ala Glu Lys Val Gln Arg Lys Phe Leu Gly Arg Pro Ile
65 70 75 80
Glu Val Trp Lys Leu Tyr Phe Thr His Pro Gln Asp Val Pro Ala Ile
85 90 95
Arg Asp Lys Ile Arg Ala His Pro Ala Val Ile Asp Ile Tyr Glu Tyr
100 105 110
Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Lys Gly Leu Ile Pro
115 120 125
Met Glu Gly Asp Glu Glu Leu Thr Met Leu Ala Phe Asp Ile Glu Thr
130 135 140
Leu Tyr His Glu Gly Glu Glu Phe Gly Thr Gly Pro Ile Leu Met Ile
145 150 155 160
Ser Tyr Ala Asp Gly Ser Glu Ala Arg Val Ile Thr Trp Lys Lys Ile
165 170 175
Asp Leu Pro Tyr Val Asp Val Val Ser Thr Glu Lys Glu Met Ile Lys
180 185 190
Arg Phe Leu Arg Ile Val Lys Glu Lys Asp Pro Asp Val Leu Ile Thr
195 200 205
Tyr Asn Gly Asp Asn Phe Asp Phe Ala Tyr Leu Lys Lys Arg Cys Glu
210 215 220
Lys Leu Gly Ile Lys Phe Thr Leu Gly Arg Asp Gly Ser Glu Pro Lys
225 230 235 240
Ile Gln Arg Met Gly Asp Arg Phe Ala Val Glu Val Lys Gly Arg Ile
245 250 255
His Phe Asp Leu Tyr Pro Val Ile Arg Arg Thr Ile Asn Leu Pro Thr
260 265 270
Tyr Thr Leu Glu Ala Val Tyr Glu Ala Val Phe Gly Lys Pro Lys Glu
275 280 285
Lys Val Tyr Ala Glu Glu Ile Ala Gln Ala Trp Glu Ser Gly Glu Gly
290 295 300
Leu Glu Arg Val Ala Arg Tyr Ser Met Glu Asp Ala Lys Val Thr Tyr
305 310 315 320
Glu Leu Gly Arg Glu Phe Phe Pro Met Glu Ala Gln Leu Ser Arg Leu
325 330 335
Ile Gly Gln Ser Leu Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu
340 345 350
Val Glu Trp Phe Leu Leu Arg Lys Ala Tyr Glu Arg Asn Glu Leu Ala
355 360 365
Pro Asn Lys Pro Asp Glu Lys Glu Leu Ala Arg Arg Arg Gly Gly Tyr
370 375 380
Ala Gly Gly Tyr Val Lys Glu Pro Glu Arg Gly Leu Trp Asp Asn Ile
385 390 395 400
Val Tyr Leu Asp Phe Arg Ser Leu Tyr Pro Ser Ile Ile Ile Thr His
405 410 415
Asn Val Ser Pro Asp Thr Leu Asn Arg Glu Gly Cys Lys Glu Tyr Asp
420 425 430
Val Ala Pro Glu Val Gly His Lys Phe Cys Lys Asp Phe Pro Gly Phe
435 440 445
Ile Pro Ser Leu Leu Gly Asp Leu Leu Glu Glu Arg Gln Lys Ile Lys
450 455 460
Arg Lys Met Lys Ala Thr Val Asp Pro Leu Glu Lys Lys Leu Leu Asp
465 470 475 480
Tyr Arg Gln Arg Ala Ile Lys Ile Leu Ala Asn Ser Tyr Tyr Gly Tyr
485 490 495
Tyr Gly Tyr Ala Lys Ala Arg Trp Tyr Cys Arg Glu Cys Ala Glu Ser
500 505 510
Val Thr Ala Trp Gly Arg Glu Tyr Ile Glu Thr Thr Ile Arg Glu Ile
515 520 525
Glu Glu Lys Phe Gly Phe Lys Val Leu Tyr Ala Asp Thr Asp Gly Phe
530 535 540
Phe Ala Thr Ile Pro Gly Ala Asp Ala Glu Thr Val Lys Lys Lys Ala
545 550 555 560
Lys Glu Phe Leu Lys Tyr Ile Asn Ala Lys Leu Pro Gly Leu Leu Glu
565 570 575
Leu Glu Tyr Glu Gly Phe Tyr Val Arg Gly Phe Phe Val Thr Lys Lys
580 585 590
Lys Tyr Ala Val Ile Asp Glu Glu Gly Lys Ile Thr Thr Arg Gly Leu
595 600 605
Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu Thr Gln Ala
610 615 620
Arg Val Leu Glu Ala Ile Leu Lys His Gly Asp Val Glu Glu Ala Val
625 630 635 640
Arg Ile Val Lys Glu Val Thr Glu Lys Leu Ser Lys Tyr Glu Val Pro
645 650 655
Pro Glu Lys Leu Val Ile His Glu Gln Ile Thr Arg Asp Leu Arg Asp
660 665 670
Tyr Lys Ala Thr Gly Pro His Val Ala Val Ala Lys Arg Leu Ala Ala
675 680 685
Arg Gly Val Lys Ile Arg Pro Gly Thr Val Ile Ser Tyr Ile Val Leu
690 695 700
Lys Gly Ser Gly Arg Ile Gly Asp Arg Ala Ile Pro Phe Asp Glu Phe
705 710 715 720
Asp Pro Thr Lys His Arg Tyr Asp Ala Glu Tyr Tyr Ile Glu Asn Gln
725 730 735
Val Leu Pro Ala Val Glu Arg Ile Leu Lys Ala Phe Gly Tyr Arg Lys
740 745 750
Glu Asp Leu Arg Tyr Gln Lys Thr Lys Gln Val Gly Leu Gly Ala Trp
755 760 765
Leu Lys Val Lys Lys
770
<210> 43
<211> 775
<212> PRT
<213> Thermococcus hydrothermalis
<400> 43
Met Ile Leu Asp Thr Asp Tyr Ile Thr Glu Asp Gly Lys Pro Val Ile
1 5 10 15
Arg Ile Phe Lys Lys Glu Asn Gly Glu Phe Lys Ile Glu Tyr Asp Arg
20 25 30
Glu Phe Glu Pro Tyr Ile Tyr Ala Leu Leu Lys Asp Asp Ser Ala Ile
35 40 45
Glu Glu Val Lys Lys Ile Thr Ala Gly Arg His Gly Arg Val Val Lys
50 55 60
Val Lys Arg Ala Glu Lys Val Lys Lys Lys Phe Leu Gly Arg Pro Ile
65 70 75 80
Glu Val Trp Lys Leu Tyr Phe Thr His Pro Gln Asp Val Pro Ala Ile
85 90 95
Arg Asp Glu Ile Arg Arg His Ser Ala Val Val Asp Ile Tyr Glu Tyr
100 105 110
Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Lys Gly Leu Ile Pro
115 120 125
Met Glu Gly Asp Glu Glu Leu Lys Met Met Ser Phe Asp Ile Glu Thr
130 135 140
Leu Tyr His Glu Gly Glu Glu Phe Gly Thr Gly Pro Ile Leu Met Ile
145 150 155 160
Ser Tyr Ala Asp Glu Gly Glu Ala Arg Val Ile Thr Trp Lys Lys Ile
165 170 175
Asp Leu Pro Tyr Val Glu Val Val Ser Thr Glu Lys Glu Met Ile Lys
180 185 190
Arg Phe Leu Lys Val Val Lys Glu Lys Asp Pro Asp Val Leu Ile Thr
195 200 205
Tyr Asn Gly Asp Asn Phe Asp Phe Ala Tyr Leu Lys Lys Arg Cys Glu
210 215 220
Lys Ile Gly Ile Lys Phe Thr Leu Arg Arg Asp Gly Ser Glu Pro Lys
225 230 235 240
Ile Gln Arg Met Gly Asp Arg Phe Ala Val Glu Val Lys Gly Arg Ile
245 250 255
His Phe Asp Leu Tyr Pro Val Ile Arg Arg Thr Ile Asn Leu Pro Thr
260 265 270
Tyr Thr Leu Glu Ala Val Tyr Glu Ala Val Phe Gly Thr Pro Lys Glu
275 280 285
Lys Val Tyr Pro Glu Glu Ile Thr Thr Ala Trp Glu Thr Gly Glu Gly
290 295 300
Leu Glu Arg Val Ala Arg Tyr Ser Met Glu Asp Ala Lys Val Thr Tyr
305 310 315 320
Glu Leu Gly Arg Glu Phe Phe Pro Met Glu Ala Gln Leu Ser Arg Leu
325 330 335
Ile Gly Gln Ser Leu Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu
340 345 350
Val Glu Trp Phe Leu Leu Arg Lys Ala Tyr Glu Arg Asn Glu Ile Ala
355 360 365
Pro Asn Lys Pro Asp Glu Arg Glu Leu Ala Arg Arg Arg Gly Gly Tyr
370 375 380
Ala Gly Gly Tyr Val Lys Glu Pro Glu Arg Gly Leu Trp Asp Asn Ile
385 390 395 400
Val Tyr Leu Asp Phe Met Ser Leu Tyr Pro Ser Ile Ile Ile Thr His
405 410 415
Asn Val Ser Pro Asp Thr Phe Asn Arg Glu Gly Cys Lys Glu Tyr Asp
420 425 430
Thr Ala Pro Gln Val Gly His Lys Phe Cys Lys Asp Val Gln Gly Phe
435 440 445
Ile Pro Ser Leu Leu Gly Ala Leu Leu Asp Glu Arg Gln Lys Ile Lys
450 455 460
Lys Arg Met Lys Ala Ser Ile Asp Pro Leu Glu Lys Lys Leu Leu Asp
465 470 475 480
Tyr Arg Gln Lys Ala Ile Lys Ile Leu Ala Asn Ser Tyr Tyr Gly Tyr
485 490 495
Tyr Gly Tyr Ala Arg Ala Arg Trp Tyr Cys Lys Glu Cys Ala Glu Ser
500 505 510
Val Thr Ala Trp Gly Arg Asp Tyr Ile Glu Thr Thr Ile His Glu Ile
515 520 525
Glu Glu Arg Phe Gly Phe Lys Val Leu Tyr Ala Asp Thr Asp Gly Phe
530 535 540
Phe Ala Thr Ile Pro Gly Ala Asp Ala Glu Thr Val Lys Lys Lys Ala
545 550 555 560
Lys Glu Phe Leu Lys Tyr Ile Asn Ala Lys Leu Pro Gly Leu Leu Glu
565 570 575
Leu Glu Tyr Glu Gly Phe Tyr Val Arg Gly Phe Phe Val Thr Lys Lys
580 585 590
Lys Tyr Ala Val Ile Asp Glu Glu Gly Lys Ile Thr Thr Arg Gly Leu
595 600 605
Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu Thr Gln Ala
610 615 620
Arg Val Leu Glu Ala Ile Leu Arg His Gly Asp Val Glu Glu Ala Val
625 630 635 640
Arg Ile Val Lys Asp Val Thr Glu Lys Leu Ser Lys Tyr Glu Val Pro
645 650 655
Pro Glu Lys Leu Val Ile His Glu Gln Ile Thr Arg Glu Leu Lys Asp
660 665 670
Tyr Lys Ala Thr Gly Pro His Val Ala Ile Ala Lys Arg Leu Ala Ala
675 680 685
Arg Gly Ile Lys Ile Arg Pro Gly Thr Val Ile Ser Tyr Ile Val Leu
690 695 700
Lys Gly Ser Gly Arg Ile Gly Asp Arg Ala Ile Pro Phe Asp Glu Phe
705 710 715 720
Asp Pro Thr Lys His Arg Tyr Asp Ala Glu Tyr Tyr Ile Glu Asn Gln
725 730 735
Val Leu Pro Ala Val Glu Arg Ile Leu Lys Ala Phe Gly Tyr Lys Lys
740 745 750
Glu Glu Leu Arg Tyr Gln Lys Thr Arg Gln Val Gly Leu Gly Ala Trp
755 760 765
Leu Lys Leu Lys Gly Lys Lys
770 775
<210> 44
<211> 774
<212> PRT
<213> Thermococcus thioreducens
<400> 44
Met Ile Leu Asp Ala Asp Tyr Ile Thr Glu Asp Gly Lys Pro Val Val
1 5 10 15
Arg Ile Phe Lys Lys Glu Asn Gly Glu Phe Lys Ile Glu Tyr Asp Arg
20 25 30
Glu Phe Glu Pro Tyr Ile Tyr Ala Leu Leu Arg Asp Asp Ser Ala Ile
35 40 45
Glu Glu Ile Lys Lys Ile Thr Ala Asp Arg His Gly Lys Val Val Lys
50 55 60
Val Lys Arg Ala Glu Lys Val Gln Lys Lys Phe Leu Gly Arg Pro Ile
65 70 75 80
Glu Val Trp Lys Leu Tyr Phe Thr His Pro Gln Asp Val Pro Ala Ile
85 90 95
Arg Asp Glu Ile Arg Lys His Pro Ala Val Val Asp Ile Tyr Glu Tyr
100 105 110
Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Lys Gly Leu Ile Pro
115 120 125
Met Glu Gly Asp Glu Glu Leu Lys Met Leu Ala Phe Asp Ile Glu Thr
130 135 140
Leu Tyr His Glu Gly Glu Glu Phe Gly Thr Gly Pro Ile Leu Met Ile
145 150 155 160
Ser Tyr Ala Asp Glu Asp Gly Ala Arg Val Ile Thr Trp Lys Lys Ile
165 170 175
Asp Leu Pro Tyr Val Asp Val Val Ser Thr Glu Lys Glu Met Ile Lys
180 185 190
Arg Phe Leu Lys Val Val Lys Glu Lys Asp Pro Asp Val Leu Ile Thr
195 200 205
Tyr Asn Gly Asp Asn Phe Asp Phe Ala Tyr Leu Lys Lys Arg Cys Glu
210 215 220
Lys Leu Gly Ile Lys Phe Thr Leu Gly Arg Asp Gly Ser Glu Pro Lys
225 230 235 240
Ile Gln Arg Met Gly Asp Arg Phe Ala Val Glu Val Lys Gly Arg Ile
245 250 255
His Phe Asp Leu Tyr Pro Leu Ile Arg Arg Thr Ile Asn Leu Pro Thr
260 265 270
Tyr Thr Leu Glu Ala Val Tyr Glu Ala Val Phe Gly Lys Pro Lys Glu
275 280 285
Lys Val Tyr Ala Glu Glu Ile Ala Leu Ala Trp Glu Ser Gly Glu Gly
290 295 300
Leu Glu Arg Val Ala Arg Tyr Ser Met Glu Asp Ala Lys Val Thr Phe
305 310 315 320
Glu Leu Gly Arg Glu Phe Phe Pro Met Glu Ala Gln Leu Ser Arg Leu
325 330 335
Ile Gly Gln Ser Leu Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu
340 345 350
Val Glu Trp Phe Leu Leu Arg Lys Ala Tyr Glu Arg Asn Glu Leu Ala
355 360 365
Pro Asn Lys Pro Asn Glu Arg Glu Leu Ala Arg Arg Arg Gly Gly Tyr
370 375 380
Ala Gly Gly Tyr Val Lys Glu Pro Glu Arg Gly Leu Trp Asp Asn Ile
385 390 395 400
Val Tyr Leu Asp Phe Arg Ser Leu Tyr Pro Ser Ile Ile Ile Thr His
405 410 415
Asn Val Ser Pro Asp Thr Leu Asn Arg Glu Gly Cys Lys Glu Tyr Asp
420 425 430
Arg Ala Pro Gln Val Gly His Lys Phe Cys Lys Asp Val Pro Gly Phe
435 440 445
Ile Pro Ser Leu Leu Gly Ser Leu Leu Asp Glu Arg Gln Lys Ile Lys
450 455 460
Arg Lys Met Lys Ala Thr Ile Asp Pro Ile Glu Lys Lys Leu Leu Asp
465 470 475 480
Tyr Arg Gln Arg Ala Ile Lys Ile Leu Ala Asn Ser Tyr Tyr Gly Tyr
485 490 495
Tyr Gly Tyr Ala Arg Ala Arg Trp Tyr Cys Arg Glu Cys Ala Glu Ser
500 505 510
Val Thr Ala Trp Gly Arg Glu Tyr Ile Glu Met Ala Ile Arg Glu Leu
515 520 525
Glu Glu Lys Phe Gly Phe Lys Val Leu Tyr Ala Asp Thr Asp Gly Leu
530 535 540
His Ala Thr Ile Pro Gly Ala Asp Ala Glu Thr Val Lys Lys Lys Ala
545 550 555 560
Met Glu Phe Leu Lys Tyr Ile Asn Pro Lys Leu Pro Gly Leu Leu Glu
565 570 575
Leu Glu Tyr Glu Gly Phe Tyr Ala Arg Gly Phe Phe Val Thr Lys Lys
580 585 590
Lys Tyr Ala Val Ile Asp Glu Glu Gly Lys Ile Thr Thr Arg Gly Leu
595 600 605
Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu Thr Gln Ala
610 615 620
Arg Val Leu Glu Ala Ile Leu Arg His Gly Asp Val Glu Glu Ala Val
625 630 635 640
Arg Ile Val Lys Glu Val Thr Glu Lys Leu Ser Lys Tyr Glu Val Pro
645 650 655
Pro Glu Lys Leu Val Ile His Glu Gln Ile Thr Arg Glu Leu Lys Asp
660 665 670
Tyr Arg Ala Thr Gly Pro His Val Ala Ile Ala Lys Arg Leu Ala Lys
675 680 685
Arg Gly Ile Lys Ile Arg Pro Gly Thr Val Ile Ser Tyr Ile Val Leu
690 695 700
Lys Gly Ser Gly Arg Ile Gly Asp Arg Ala Ile Pro Phe Asp Glu Phe
705 710 715 720
Asp Pro Thr Lys His Lys Tyr Asp Ala Glu Tyr Tyr Ile Glu Asn Gln
725 730 735
Val Leu Pro Ala Val Glu Arg Val Leu Lys Ala Phe Gly Tyr Arg Lys
740 745 750
Asp Asp Leu Arg Tyr Gln Lys Thr Arg Gln Val Gly Leu Gly Ala Trp
755 760 765
Leu Lys Val Lys Lys Arg
770
<210> 45
<211> 773
<212> PRT
<213> Thermococcus waiotapuensis
<400> 45
Met Ile Leu Asp Ala Asp Tyr Ile Thr Glu Asp Gly Lys Pro Val Ile
1 5 10 15
Arg Val Phe Lys Lys Glu Lys Gly Glu Phe Lys Ile Asn Tyr Asp Arg
20 25 30
Asp Phe Glu Pro Tyr Ile Tyr Ala Leu Leu Lys Asp Asp Ser Ala Ile
35 40 45
Glu Asp Ile Lys Lys Ile Thr Ala Glu Arg His Gly Thr Thr Val Arg
50 55 60
Val Thr Arg Ala Glu Arg Val Lys Lys Lys Phe Leu Gly Arg Pro Val
65 70 75 80
Glu Val Trp Lys Leu Tyr Phe Thr His Pro Gln Asp Val Pro Ala Ile
85 90 95
Arg Asp Lys Ile Arg Glu His Pro Ala Val Val Asp Ile Tyr Glu Tyr
100 105 110
Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Lys Gly Leu Ile Pro
115 120 125
Met Glu Gly Asn Glu Glu Leu Arg Met Leu Ala Phe Asp Ile Glu Thr
130 135 140
Leu Tyr His Glu Gly Glu Glu Phe Gly Glu Gly Pro Ile Leu Met Ile
145 150 155 160
Ser Tyr Ala Asp Glu Glu Gly Ala Arg Val Ile Thr Trp Lys Asn Ile
165 170 175
Asp Leu Pro Tyr Val Glu Ser Val Ser Thr Glu Lys Glu Met Ile Lys
180 185 190
Arg Phe Leu Lys Val Ile Gln Glu Lys Asp Pro Asp Val Leu Ile Thr
195 200 205
Tyr Asn Gly Asp Asn Phe Asp Phe Ala Tyr Leu Lys Lys Arg Ser Glu
210 215 220
Thr Leu Gly Val Lys Phe Ile Leu Gly Arg Asp Gly Ser Glu Pro Lys
225 230 235 240
Ile Gln Arg Met Gly Asp Arg Phe Ala Val Glu Val Lys Gly Arg Ile
245 250 255
His Phe Asp Leu Tyr Pro Val Ile Arg Arg Thr Ile Asn Leu Pro Thr
260 265 270
Tyr Thr Leu Glu Thr Val Tyr Glu Ala Ile Phe Gly Gln Pro Lys Glu
275 280 285
Lys Val Tyr Ala Glu Glu Ile Ala Gln Ala Trp Glu Ser Gly Glu Gly
290 295 300
Leu Glu Arg Val Ala Arg Tyr Ser Met Glu Asp Ala Lys Ala Thr Tyr
305 310 315 320
Glu Leu Gly Lys Glu Phe Phe Pro Met Glu Ala Gln Leu Ser Arg Leu
325 330 335
Val Gly Gln Ser Leu Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu
340 345 350
Val Glu Trp Phe Leu Leu Arg Lys Ala Tyr Glu Arg Asn Glu Leu Ala
355 360 365
Pro Asn Lys Pro Asp Glu Arg Glu Leu Ala Arg Arg Ala Glu Ser Tyr
370 375 380
Ala Gly Gly Tyr Val Lys Glu Pro Glu Lys Gly Leu Trp Glu Asn Ile
385 390 395 400
Val Tyr Leu Asp Tyr Lys Ser Leu Tyr Pro Ser Ile Ile Ile Thr His
405 410 415
Asn Val Ser Pro Asp Thr Leu Asn Arg Glu Gly Cys Arg Glu Tyr Asp
420 425 430
Val Ala Pro Gln Val Gly His Arg Phe Cys Lys Asp Phe Pro Gly Phe
435 440 445
Ile Pro Ser Leu Leu Gly Asp Leu Leu Glu Glu Arg Gln Lys Val Lys
450 455 460
Lys Lys Met Lys Ala Thr Val Asp Pro Ile Glu Arg Lys Leu Leu Asp
465 470 475 480
Tyr Arg Gln Arg Ala Ile Lys Ile Leu Ala Asn Ser Tyr Tyr Gly Tyr
485 490 495
Tyr Gly Tyr Ala Asn Ala Arg Trp Tyr Cys Arg Glu Cys Ala Glu Ser
500 505 510
Val Thr Ala Trp Gly Arg Gln Tyr Ile Glu Thr Thr Met Arg Glu Ile
515 520 525
Glu Glu Lys Phe Gly Phe Lys Val Leu Tyr Ala Asp Thr Asp Gly Phe
530 535 540
Phe Ala Thr Ile Pro Gly Ala Asp Ala Glu Thr Val Lys Lys Lys Thr
545 550 555 560
Lys Glu Phe Leu Asn Tyr Ile Asn Pro Arg Leu Pro Gly Leu Leu Glu
565 570 575
Leu Glu Tyr Glu Gly Phe Tyr Arg Arg Gly Phe Phe Val Thr Lys Lys
580 585 590
Lys Tyr Ala Val Ile Asp Glu Glu Asp Lys Ile Thr Thr Arg Gly Leu
595 600 605
Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu Thr Gln Ala
610 615 620
Arg Val Leu Glu Ala Ile Leu Lys His Gly Asp Val Glu Glu Ala Val
625 630 635 640
Arg Ile Val Lys Glu Val Thr Glu Lys Leu Ser Arg Tyr Glu Val Pro
645 650 655
Pro Glu Lys Leu Val Ile Tyr Glu Gln Ile Thr Arg Asn Leu Arg Asp
660 665 670
Tyr Arg Ala Thr Gly Pro His Val Ala Val Ala Lys Arg Leu Ala Ala
675 680 685
Arg Gly Ile Lys Ile Arg Pro Gly Thr Val Ile Ser Tyr Ile Val Leu
690 695 700
Lys Gly Pro Gly Arg Val Gly Asp Arg Ala Ile Pro Phe Asp Glu Phe
705 710 715 720
Asp Pro Ala Lys His Arg Tyr Asp Ala Glu Tyr Tyr Ile Glu Asn Gln
725 730 735
Val Leu Pro Ala Val Glu Arg Ile Leu Arg Ala Phe Gly Tyr Arg Lys
740 745 750
Glu Asp Leu Arg Tyr Gln Lys Thr Lys Gln Ala Gly Leu Gly Ala Trp
755 760 765
Leu Lys Pro Lys Thr
770
<210> 46
<211> 774
<212> PRT
<213> Thermococcus sibiricus
<400> 46
Met Ile Leu Gly Ala Asp Tyr Ile Thr Lys Asp Gly Lys Pro Ile Val
1 5 10 15
Arg Ile Phe Lys Lys Glu Asn Gly Glu Phe Lys Ile Glu Leu Asp Pro
20 25 30
His Phe Gln Pro Tyr Ile Tyr Ala Leu Leu Ser Glu Asp Ser Ala Ile
35 40 45
Asp Glu Ile Lys Gln Ile Lys Gly Glu Arg His Gly Lys Thr Val Arg
50 55 60
Ile Val Asp Ala Val Lys Val Glu Lys Lys Phe Leu Lys Lys Pro Val
65 70 75 80
Lys Val Trp Lys Leu Ile Leu Glu His Pro Gln Asp Val Pro Ala Ile
85 90 95
Arg Asn Lys Ile Arg Glu His Pro Ala Val Gln Asp Ile Tyr Glu Tyr
100 105 110
Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Asn Gly Leu Ile Pro
115 120 125
Met Glu Gly Asp Glu Glu Leu Lys Met Leu Ala Phe Asp Ile Glu Thr
130 135 140
Phe Tyr His Glu Gly Asp Glu Phe Gly Lys Gly Glu Ile Ile Met Ile
145 150 155 160
Ser Tyr Ala Asp Glu Glu Gly Ala Arg Val Ile Thr Trp Lys Asn Ile
165 170 175
Asp Leu Pro Tyr Val Asp Val Val Ser Asn Glu Arg Glu Met Ile Lys
180 185 190
Arg Phe Ile Gln Ile Ile Lys Glu Lys Asp Pro Asp Val Ile Ile Thr
195 200 205
Tyr Asn Gly Asp Asn Phe Asp Leu Pro Tyr Leu Ile Lys Arg Ala Glu
210 215 220
Lys Leu Gly Leu Arg Leu Ile Leu Ser Arg Asp Asn Glu Asn Pro Val
225 230 235 240
Pro Lys Ile Gln Arg Met Gly Asn Ser Phe Ala Val Glu Ile Lys Gly
245 250 255
Arg Ile His Phe Asp Leu Phe Pro Val Val Lys Arg Ala Val Asn Leu
260 265 270
Pro Thr Tyr Thr Leu Glu Ala Val Tyr Glu Thr Val Leu Gly Lys His
275 280 285
Lys Ser Lys Leu Gly Ala Glu Glu Ile Ala Ala Ile Trp Glu Thr Glu
290 295 300
Glu Gly Leu Lys Lys Leu Ala Gln Tyr Ser Met Glu Asp Ala Lys Ala
305 310 315 320
Thr Tyr Glu Leu Gly Arg Glu Phe Phe Pro Met Glu Val Glu Leu Ala
325 330 335
Lys Leu Ile Gly Gln Ser Val Trp Asp Val Ser Arg Ser Ser Thr Gly
340 345 350
Asn Leu Val Glu Trp Tyr Met Leu Arg Val Ala Tyr Glu Arg Asn Glu
355 360 365
Leu Ala Pro Asn Arg Pro Ser Asp Glu Glu Tyr Lys Arg Arg Leu Arg
370 375 380
Thr Thr Tyr Leu Gly Gly Tyr Val Lys Glu Pro Glu Arg Gly Leu Trp
385 390 395 400
Glu Asn Ile Ile Tyr Leu Asp Phe Arg Ser Leu Tyr Pro Ser Ile Ile
405 410 415
Val Thr His Asn Val Ser Pro Asp Thr Leu Glu Arg Lys Gly Cys Gln
420 425 430
Asn Tyr Asp Val Ala Pro Ile Val Gly Tyr Lys Phe Cys Lys Asp Phe
435 440 445
Ser Gly Phe Ile Pro Ser Ile Leu Glu Asp Leu Ile Glu Thr Arg Gln
450 455 460
Lys Ile Lys Lys Glu Met Lys Ser Thr Ile Asp Pro Ile Lys Lys Lys
465 470 475 480
Met Leu Asp Tyr Arg Gln Arg Ala Val Lys Leu Leu Ala Asn Ser Tyr
485 490 495
Tyr Gly Tyr Met Gly Tyr Pro Lys Ala Arg Trp Tyr Ser Lys Glu Cys
500 505 510
Ala Glu Ser Val Thr Ala Trp Gly Arg His Tyr Ile Glu Met Thr Ile
515 520 525
Arg Glu Ile Glu Glu Lys Phe Gly Phe Lys Val Leu Tyr Ala Asp Thr
530 535 540
Asp Gly Phe Tyr Ala Thr Ile Pro Gly Ala Asp Pro Glu Thr Ile Lys
545 550 555 560
Lys Lys Ala Lys Glu Phe Leu Asn Tyr Ile Asn Ser Lys Leu Pro Gly
565 570 575
Leu Leu Glu Leu Glu Tyr Glu Gly Phe Tyr Leu Arg Gly Phe Phe Val
580 585 590
Thr Lys Lys Arg Tyr Ala Val Ile Asp Glu Glu Glu Lys Ile Thr Thr
595 600 605
Arg Gly Leu Glu Val Val Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu
610 615 620
Thr Gln Ala Arg Val Leu Glu Ala Ile Leu Arg Glu Gly Ser Val Glu
625 630 635 640
Lys Ala Val Glu Ile Val Lys Glu Val Val Glu Ala Ile Thr Lys Tyr
645 650 655
Lys Val Pro Leu Glu Lys Leu Ile Ile His Glu Gln Ile Thr Arg Glu
660 665 670
Leu Arg Asp Tyr Lys Ala Val Gly Pro His Val Ala Ile Ala Lys Arg
675 680 685
Leu Ala Ala Lys Gly Ile Lys Ile Lys Pro Gly Thr Ile Ile Ser Tyr
690 695 700
Ile Val Leu Arg Gly Ser Gly Lys Ile Ser Asp Arg Val Val Leu Leu
705 710 715 720
Thr Glu Tyr Asp Pro Arg Lys His Lys Tyr Asp Pro Asp Tyr Tyr Ile
725 730 735
Glu Asn Gln Val Leu Pro Ala Val Leu Arg Ile Leu Glu Ala Phe Gly
740 745 750
Tyr Arg Lys Glu Asp Leu Lys Tyr Gln Ser Ser Lys Gln Thr Gly Leu
755 760 765
Glu Ser Trp Leu Lys Lys
770
<210> 47
<211> 775
<212> PRT
<213> Pyrococcus glycovorans
<400> 47
Met Ile Leu Asp Ala Asp Tyr Ile Thr Glu Asp Gly Lys Pro Ile Ile
1 5 10 15
Arg Ile Phe Lys Lys Glu Asn Gly Glu Phe Lys Val Glu Tyr Asp Arg
20 25 30
Asn Phe Arg Pro Tyr Ile Tyr Ala Leu Leu Lys Asp Asp Ser Gln Ile
35 40 45
Asp Glu Val Lys Lys Ile Thr Ala Glu Arg His Gly Lys Ile Val Arg
50 55 60
Ile Val Asp Val Glu Lys Val Lys Lys Lys Phe Leu Gly Arg Pro Ile
65 70 75 80
Glu Val Trp Lys Leu Tyr Phe Glu His Pro Gln Asp Val Pro Ala Ile
85 90 95
Arg Asp Lys Ile Arg Glu His Pro Ala Val Val Asp Ile Phe Glu Tyr
100 105 110
Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Lys Gly Leu Ile Pro
115 120 125
Met Glu Gly Asp Glu Glu Leu Lys Leu Leu Ala Phe Asp Ile Glu Thr
130 135 140
Leu Tyr His Glu Gly Glu Glu Phe Ala Lys Gly Pro Ile Ile Met Ile
145 150 155 160
Ser Tyr Ala Asp Glu Glu Gly Ala Lys Val Ile Thr Trp Lys Lys Val
165 170 175
Asp Leu Pro Tyr Val Glu Val Val Ser Ser Glu Arg Glu Met Ile Lys
180 185 190
Arg Phe Leu Lys Val Ile Arg Glu Lys Asp Pro Asp Val Ile Ile Thr
195 200 205
Tyr Asn Gly Asp Ser Phe Asp Leu Pro Tyr Leu Val Lys Arg Ala Glu
210 215 220
Lys Leu Gly Ile Lys Leu Pro Leu Gly Arg Asp Gly Ser Glu Pro Lys
225 230 235 240
Met Gln Arg Leu Gly Asp Met Thr Ala Val Glu Ile Lys Gly Arg Ile
245 250 255
His Phe Asp Leu Tyr His Val Ile Arg Arg Thr Ile Asn Leu Pro Thr
260 265 270
Tyr Thr Leu Glu Ala Val Tyr Glu Ala Ile Phe Gly Lys Pro Lys Glu
275 280 285
Lys Val Tyr Ala His Glu Ile Ala Glu Ala Trp Glu Thr Gly Lys Gly
290 295 300
Leu Glu Arg Val Ala Lys Tyr Ser Met Glu Asp Ala Lys Val Thr Tyr
305 310 315 320
Glu Leu Gly Arg Glu Phe Phe Pro Met Glu Ala Gln Leu Ser Arg Leu
325 330 335
Val Gly Gln Pro Leu Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu
340 345 350
Val Glu Trp Tyr Leu Leu Arg Lys Ala Tyr Glu Arg Asn Glu Leu Ala
355 360 365
Pro Asn Lys Pro Asp Glu Arg Glu Tyr Glu Arg Arg Leu Arg Glu Ser
370 375 380
Tyr Ala Gly Gly Tyr Val Lys Glu Pro Glu Lys Gly Leu Trp Glu Gly
385 390 395 400
Leu Val Ser Leu Asp Phe Arg Ser Leu Tyr Pro Ser Ile Ile Ile Thr
405 410 415
His Asn Val Ser Pro Asp Thr Leu Asn Arg Glu Gly Cys Met Glu Tyr
420 425 430
Asp Val Ala Pro Glu Val Lys His Lys Phe Cys Lys Asp Phe Pro Gly
435 440 445
Phe Ile Pro Ser Leu Leu Lys Arg Leu Leu Asp Glu Arg Gln Glu Ile
450 455 460
Lys Arg Arg Met Lys Ala Ser Lys Asp Pro Ile Glu Lys Lys Met Leu
465 470 475 480
Asp Tyr Arg Gln Arg Ala Ile Lys Ile Leu Ala Asn Ser Tyr Tyr Gly
485 490 495
Tyr Tyr Gly Tyr Ala Lys Ala Arg Trp Tyr Cys Lys Glu Cys Ala Glu
500 505 510
Ser Val Thr Ala Trp Gly Arg Glu Tyr Ile Glu Phe Val Arg Lys Glu
515 520 525
Leu Glu Glu Lys Phe Gly Phe Lys Val Leu Tyr Ile Asp Thr Asp Gly
530 535 540
Leu Tyr Ala Thr Ile Pro Gly Ala Lys Pro Glu Glu Ile Lys Arg Lys
545 550 555 560
Ala Leu Glu Phe Val Glu Tyr Ile Asn Ala Lys Leu Pro Gly Leu Leu
565 570 575
Glu Leu Glu Tyr Glu Gly Phe Tyr Val Arg Gly Phe Phe Val Thr Lys
580 585 590
Lys Lys Tyr Ala Leu Ile Asp Glu Glu Gly Lys Ile Ile Thr Arg Gly
595 600 605
Leu Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu Thr Gln
610 615 620
Ala Lys Val Leu Glu Ala Ile Leu Lys His Gly Asn Val Glu Glu Ala
625 630 635 640
Val Lys Ile Val Lys Glu Val Thr Glu Lys Leu Ser Lys Tyr Glu Ile
645 650 655
Pro Pro Glu Lys Leu Val Ile Tyr Glu Gln Ile Thr Arg Pro Leu His
660 665 670
Glu Tyr Lys Ala Ile Gly Pro His Val Ala Val Ala Lys Arg Leu Ala
675 680 685
Ala Arg Gly Val Lys Val Arg Pro Gly Met Val Ile Gly Tyr Ile Val
690 695 700
Leu Arg Gly Asp Gly Pro Ile Ser Lys Arg Ala Ile Leu Ala Glu Glu
705 710 715 720
Phe Asp Pro Arg Lys His Lys Tyr Asp Ala Glu Tyr Tyr Ile Glu Asn
725 730 735
Gln Val Leu Pro Ala Val Leu Arg Ile Leu Glu Ala Phe Gly Tyr Arg
740 745 750
Lys Glu Asp Leu Arg Trp Gln Lys Thr Lys Gln Thr Gly Leu Thr Ala
755 760 765
Trp Leu Asn Val Lys Lys Lys
770 775
<210> 48
<211> 775
<212> PRT
<213> Pyrococcus sp. NA2
<400> 48
Met Ile Leu Asp Ala Asp Tyr Ile Thr Glu Asp Gly Lys Pro Ile Ile
1 5 10 15
Arg Leu Phe Lys Lys Glu Asn Gly Arg Phe Lys Val Glu Tyr Asp Arg
20 25 30
Asn Phe Arg Pro Tyr Ile Tyr Ala Leu Leu Lys Asp Asp Ser Ala Ile
35 40 45
Asp Asp Val Arg Lys Ile Thr Ser Glu Arg His Gly Lys Val Val Arg
50 55 60
Val Ile Asp Val Glu Lys Val Lys Lys Lys Phe Leu Gly Arg Pro Ile
65 70 75 80
Glu Val Trp Lys Leu Tyr Phe Glu His Pro Gln Asp Val Pro Ala Met
85 90 95
Arg Asp Lys Ile Arg Glu His Pro Ala Val Ile Asp Ile Phe Glu Tyr
100 105 110
Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Lys Gly Leu Ile Pro
115 120 125
Met Glu Gly Asn Glu Glu Leu Thr Phe Leu Ala Val Asp Ile Glu Thr
130 135 140
Leu Tyr His Glu Gly Glu Glu Phe Gly Lys Gly Pro Ile Ile Met Ile
145 150 155 160
Ser Tyr Ala Asp Glu Glu Gly Ala Lys Val Ile Thr Trp Lys Lys Ile
165 170 175
Asp Leu Pro Tyr Val Glu Val Val Ala Asn Glu Arg Glu Met Ile Lys
180 185 190
Arg Leu Ile Lys Val Ile Arg Glu Lys Asp Pro Asp Val Ile Ile Thr
195 200 205
Tyr Asn Gly Asp Asn Phe Asp Phe Pro Tyr Leu Leu Lys Arg Ala Glu
210 215 220
Lys Leu Gly Met Lys Leu Pro Leu Gly Arg Asp Asn Ser Glu Pro Lys
225 230 235 240
Met Gln Arg Leu Gly Asp Ser Leu Ala Val Glu Ile Lys Gly Arg Ile
245 250 255
His Phe Asp Leu Phe Pro Val Ile Arg Arg Thr Ile Asn Leu Pro Thr
260 265 270
Tyr Thr Leu Glu Ala Val Tyr Glu Ala Ile Phe Gly Lys Gln Lys Glu
275 280 285
Lys Val Tyr Pro His Glu Ile Ala Glu Ala Trp Glu Thr Gly Lys Gly
290 295 300
Leu Glu Arg Val Ala Lys Tyr Ser Met Glu Asp Ala Lys Val Thr Tyr
305 310 315 320
Glu Leu Gly Lys Glu Phe Phe Pro Met Glu Ala Gln Leu Ala Arg Leu
325 330 335
Val Gly Gln Pro Leu Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu
340 345 350
Val Glu Trp Tyr Leu Leu Arg Lys Ala Tyr Glu Arg Asn Glu Leu Ala
355 360 365
Pro Asn Lys Pro Asp Glu Arg Glu Tyr Glu Arg Arg Leu Arg Glu Ser
370 375 380
Tyr Glu Gly Gly Tyr Val Lys Glu Pro Glu Arg Gly Leu Trp Glu Gly
385 390 395 400
Ile Val Ser Leu Asp Phe Arg Ser Leu Tyr Pro Ser Ile Ile Ile Thr
405 410 415
His Asn Val Ser Pro Asp Thr Leu Asn Lys Glu Gly Cys Gly Glu Tyr
420 425 430
Asp Glu Ala Pro Glu Val Gly His Arg Phe Cys Lys Asp Phe Pro Gly
435 440 445
Phe Ile Pro Ser Leu Leu Gly Ser Leu Leu Glu Glu Arg Gln Lys Ile
450 455 460
Lys Lys Arg Met Lys Glu Ser Lys Asp Pro Val Glu Arg Lys Leu Leu
465 470 475 480
Asp Tyr Arg Gln Arg Ala Ile Lys Ile Leu Ala Asn Ser Phe Tyr Gly
485 490 495
Tyr Tyr Gly Tyr Ala Lys Ala Arg Trp Tyr Cys Lys Glu Cys Ala Glu
500 505 510
Ser Val Thr Ala Trp Gly Arg Gln Tyr Ile Glu Leu Val Arg Arg Glu
515 520 525
Leu Glu Glu Arg Gly Phe Lys Val Leu Tyr Ile Asp Thr Asp Gly Leu
530 535 540
Tyr Ala Thr Ile Pro Gly Glu Lys Asn Trp Glu Glu Ile Lys Arg Arg
545 550 555 560
Ala Leu Glu Phe Val Asn Tyr Ile Asn Ser Lys Leu Pro Gly Ile Leu
565 570 575
Glu Leu Glu Tyr Glu Gly Phe Tyr Thr Arg Gly Phe Phe Val Thr Lys
580 585 590
Lys Lys Tyr Ala Leu Ile Asp Glu Glu Gly Lys Ile Val Thr Arg Gly
595 600 605
Leu Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu Thr Gln
610 615 620
Ala Lys Val Leu Glu Ala Ile Leu Lys His Gly Asn Val Glu Glu Ala
625 630 635 640
Val Lys Ile Val Lys Glu Val Thr Glu Lys Leu Ser Asn Tyr Glu Ile
645 650 655
Pro Val Glu Lys Leu Val Ile Tyr Glu Gln Ile Thr Arg Pro Leu Asn
660 665 670
Glu Tyr Lys Ala Ile Gly Pro His Val Ala Val Ala Lys Arg Leu Ala
675 680 685
Ala Lys Gly Ile Lys Ile Lys Pro Gly Met Val Ile Gly Tyr Val Val
690 695 700
Leu Arg Gly Asp Gly Pro Ile Ser Lys Arg Ala Ile Ala Ile Glu Glu
705 710 715 720
Phe Asp Gly Lys Lys His Lys Tyr Asp Ala Glu Tyr Tyr Ile Glu Asn
725 730 735
Gln Val Leu Pro Ala Val Glu Arg Ile Leu Lys Ala Phe Gly Tyr Lys
740 745 750
Arg Glu Asp Leu Arg Trp Gln Lys Thr Lys Gln Val Gly Leu Gly Ala
755 760 765
Trp Leu Lys Val Lys Lys Ser
770 775
<210> 49
<211> 785
<212> PRT
<213> Ferroglobus placidus
<400> 49
Met Glu Gly Trp Leu Leu Asp Ala Asp Tyr Ile Thr Ala Glu Asp Gly
1 5 10 15
Arg Ala Val Val Arg Leu Trp Cys Lys Asp Phe Asp Gly Asn Thr Phe
20 25 30
Val Val Tyr Asp Arg Asn Phe Gln Pro Tyr Phe Tyr Ala Phe Lys Asn
35 40 45
Gly Leu Ser Lys Glu Asp Ile Glu Lys Ile Val Val Lys Ser Arg Glu
50 55 60
Gly Val Ile Lys Pro Phe Lys Val Glu Glu Val Arg Arg Lys Val Phe
65 70 75 80
Gly Lys Glu Val Glu Val Phe Lys Ile Tyr Ala Tyr His Pro Gln His
85 90 95
Val Pro Lys Leu Arg Glu Glu Leu Lys Lys Ile Thr Glu Val Arg Glu
100 105 110
Ala Asp Ile Pro Phe Ala Tyr Arg Tyr Leu Ile Asp Lys Asp Leu Ala
115 120 125
Cys Met Asp Gly Ile Arg Val Glu Gly Lys Val Arg Glu Glu Arg Gly
130 135 140
Leu Lys Val Ile Asp Ala Glu His Val Glu Arg Phe Glu Ile Pro Leu
145 150 155 160
Pro Glu Pro Lys Val Leu Ala Phe Asp Cys Glu Met Leu Thr Glu Leu
165 170 175
Gly Met Pro Asp Pro Glu Lys Asp Lys Ile Ile Ile Ile Gly Val Lys
180 185 190
Cys Gly Asp Phe Glu Glu Ile Ile Thr Gly Asn Glu Arg Glu Ile Leu
195 200 205
Leu Arg Phe Val Glu Ile Ile Lys Glu Gln Asp Pro Asp Val Ile Val
210 215 220
Gly Tyr Asn Gln Asp Asn Phe Asp Trp Pro Tyr Ile Arg Lys Arg Ala
225 230 235 240
Glu Lys Leu Ser Val Lys Leu Asn Ile Gly Arg Asp Gly Ser Glu Ile
245 250 255
Ser Phe Arg Gly Gly Arg Pro Lys Ile Ala Gly Arg Leu Asn Val Asp
260 265 270
Leu Tyr Asp Ile Ala Met Lys Leu Asp Val Lys Val Lys Thr Leu Glu
275 280 285
Asn Val Ala Glu Phe Leu Gly Arg Lys Val Glu Leu Ala Asp Ile Glu
290 295 300
Ala Lys Asp Ile Tyr Lys Arg Trp Thr Ser Gly Asp Lys Glu Ser Val
305 310 315 320
Leu Lys Tyr Ser Lys Gln Asp Val Leu Asn Thr Tyr Phe Ile Ala Glu
325 330 335
Glu Leu Leu Pro Met His Tyr Glu Leu Ser Arg Met Ile Arg Ile Pro
340 345 350
Thr Asp Asp Val Ala Arg Ile Gly Arg Gly Lys Gln Val Asp Trp Phe
355 360 365
Leu Leu Ser Glu Ala Tyr Lys Ile Gly Glu Ile Ala Pro Asn Pro Ala
370 375 380
Glu Val Glu Glu Ser Tyr Glu Gly Ala Phe Val Leu Glu Pro Ser Arg
385 390 395 400
Gly Leu His Lys Asn Val Val Cys Leu Asp Phe Ala Ser Met Tyr Pro
405 410 415
Ser Ile Met Ile Ala Tyr Asn Ile Ser Pro Asp Thr Tyr Val Phe Gly
420 425 430
Lys Cys Asp Asp Cys Tyr Val Ala Pro Glu Val Gly His Lys Phe Arg
435 440 445
Lys His Pro Asp Gly Phe Phe Lys Arg Ile Leu Lys Met Leu Ile Glu
450 455 460
Lys Arg Arg Glu Ile Lys Asn Gln Met Lys Ser Leu Asp Arg Asn Ser
465 470 475 480
Arg Glu Tyr Leu Leu Leu Asn Ile Lys Gln Gln Thr Leu Lys Ile Leu
485 490 495
Thr Asn Ser Phe Tyr Gly Tyr Thr Gly Trp Ser Gly Ala Arg Trp Tyr
500 505 510
Cys Arg Gln Cys Ala Glu Ala Thr Thr Ala Trp Gly Arg His Leu Ile
515 520 525
Lys Ser Ala Val Glu Ile Ala Lys Lys Leu Gly Phe Glu Val Leu Tyr
530 535 540
Gly Asp Thr Asp Ser Ile Phe Val Lys Lys Gly Asn Leu Ser Leu Glu
545 550 555 560
Lys Ile Arg Gly Glu Val Glu Lys Leu Ile Glu Glu Ile Ser Glu Lys
565 570 575
Phe Pro Val Gln Ile Glu Val Asp Glu Tyr Tyr Lys Thr Ile Phe Phe
580 585 590
Val Glu Lys Lys Arg Tyr Ala Gly Leu Thr Glu Asp Gly Ile Leu Val
595 600 605
Val Lys Gly Leu Glu Val Arg Arg Gly Asp Trp Cys Glu Leu Ala Lys
610 615 620
Glu Val Gln Lys Lys Val Ile Glu Ile Ile Leu Lys Glu Glu Asn Pro
625 630 635 640
Glu Lys Ala Ala Glu Tyr Val Arg Lys Val Ile Asn Asp Ile Lys Ser
645 650 655
Gly Lys Val Lys Leu Glu Asp Val Val Ile Tyr Lys Gly Leu Thr Lys
660 665 670
Arg Pro Asp Lys Tyr Glu Ser Lys Gln Ala His Val Lys Ala Ala Leu
675 680 685
Arg Ala Met Glu Leu Gly Ile Val Tyr Asn Val Gly Ser Lys Val Gly
690 695 700
Phe Val Val Val Glu Gly Ala Gly Asn Val Gly Asp Arg Ala Tyr Pro
705 710 715 720
Ile Asp Leu Ile Glu Glu Phe Asp Gly Glu Asn Leu Val Ile Arg Thr
725 730 735
Arg Ser Gly Ser Ile Val Lys Lys Leu Asp Lys Asp Tyr Tyr Ile Asn
740 745 750
His Gln Ile Ile Pro Ser Val Leu Arg Ile Leu Glu Arg Phe Gly Tyr
755 760 765
Asn Glu Ala Ser Leu Lys Gly Ala Thr Gln Lys Thr Leu Asp Ala Phe
770 775 780
Trp
785
<210> 50
<211> 775
<212> PRT
<213> Palaeococcus ferrophilus
<400> 50
Met Ile Leu Asp Ala Asp Tyr Ile Thr Glu Asn Gly Lys Pro Val Val
1 5 10 15
Arg Ile Phe Lys Lys Glu Asn Gly Glu Phe Lys Val Glu Tyr Asp Arg
20 25 30
Asn Phe Glu Pro Tyr Ile Tyr Ala Leu Leu Lys Asp Asp Ser Ala Ile
35 40 45
Glu Glu Ile Lys Lys Ile Thr Ala Glu Arg His Gly Thr Val Val Arg
50 55 60
Ile Thr Lys Ala Glu Lys Val Glu Arg Lys Phe Leu Gly Arg Pro Val
65 70 75 80
Glu Val Trp Lys Leu Tyr Phe Thr His Pro Gln Asp Val Pro Ala Ile
85 90 95
Arg Asp Lys Ile Arg Ser His Pro Ala Val Val Asp Ile Tyr Glu Tyr
100 105 110
Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Lys Gly Leu Val Pro
115 120 125
Met Glu Gly Asp Glu Glu Leu Lys Met Leu Ala Phe Asp Ile Glu Thr
130 135 140
Leu Tyr His Glu Gly Glu Glu Phe Ala Glu Gly Pro Ile Leu Met Ile
145 150 155 160
Ser Tyr Ala Asp Glu Ser Glu Ala Arg Val Ile Thr Trp Lys Lys Val
165 170 175
Asp Leu Pro Tyr Val Asp Ala Val Ser Thr Glu Lys Asp Met Ile Lys
180 185 190
Ala Phe Leu Arg Val Val Lys Glu Lys Asp Pro Asp Val Leu Ile Thr
195 200 205
Tyr Asn Gly Asp Asn Phe Asp Phe Ala Tyr Leu Lys Lys Arg Cys Glu
210 215 220
Lys Leu Gly Val Lys Phe Ile Leu Gly Arg Asp Gly Ser Glu Pro Lys
225 230 235 240
Ile Gln Arg Met Gly Asp Arg Phe Ala Val Asp Val Lys Gly Arg Ile
245 250 255
His Phe Asp Leu Tyr Pro Val Ile Arg Arg Thr Ile Asn Leu Pro Thr
260 265 270
Tyr Thr Leu Glu Ala Val Tyr Glu Ala Ile Phe Gly Arg Pro Lys Glu
275 280 285
Lys Val Tyr Ala Glu Glu Ile Ala Gln Ala Trp Glu Thr Asn Glu Gly
290 295 300
Leu Glu Arg Val Ala Arg Tyr Ser Met Glu Asp Ala Lys Val Thr Tyr
305 310 315 320
Glu Leu Gly Lys Glu Phe Phe Pro Met Glu Ala Gln Leu Ser Arg Leu
325 330 335
Ile Gly Gln Pro Leu Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu
340 345 350
Val Glu Trp Phe Leu Leu Arg Lys Ala Tyr Glu Arg Asn Glu Leu Ala
355 360 365
Pro Asn Lys Pro Ser Gly Arg Glu Tyr Asp Glu Arg Arg Gly Gly Tyr
370 375 380
Ala Gly Gly Tyr Val Lys Glu Pro Glu Lys Gly Leu Trp Glu Asn Ile
385 390 395 400
Val Tyr Leu Asp Tyr Lys Ser Leu Tyr Pro Ser Ile Ile Ile Thr His
405 410 415
Asn Val Ser Pro Asp Thr Leu Asn Arg Glu Gly Cys Lys Glu Tyr Asp
420 425 430
Val Ala Pro Gln Val Gly His Arg Phe Cys Lys Asp Phe Pro Gly Phe
435 440 445
Ile Pro Ser Leu Leu Gly Asp Leu Leu Glu Glu Arg Gln Lys Ile Lys
450 455 460
Arg Lys Met Lys Ala Thr Ile Asp Pro Ile Glu Arg Arg Leu Leu Asp
465 470 475 480
Tyr Arg Gln Arg Ala Ile Lys Ile Leu Ala Asn Ser Tyr Tyr Gly Tyr
485 490 495
Tyr Gly Tyr Ala Arg Ala Arg Trp Tyr Cys Lys Glu Cys Ala Glu Ser
500 505 510
Val Thr Ala Trp Gly Arg Glu Tyr Ile Glu Met Ser Ile Arg Glu Ile
515 520 525
Glu Glu Lys Tyr Gly Phe Lys Val Leu Tyr Ala Asp Thr Asp Gly Phe
530 535 540
His Ala Thr Ile Pro Gly Glu Asp Ala Glu Thr Ile Lys Lys Lys Ala
545 550 555 560
Met Glu Phe Leu Lys Tyr Ile Asn Ser Lys Leu Pro Gly Ala Leu Glu
565 570 575
Leu Glu Tyr Glu Gly Phe Tyr Arg Arg Gly Phe Phe Val Thr Lys Lys
580 585 590
Lys Tyr Ala Val Ile Asp Glu Glu Gly Lys Ile Thr Thr Arg Gly Leu
595 600 605
Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu Thr Gln Ala
610 615 620
Arg Val Leu Glu Ala Leu Leu Lys Asp Gly Asn Val Glu Glu Ala Val
625 630 635 640
Ser Ile Val Lys Glu Val Thr Glu Lys Leu Ser Lys Tyr Glu Val Pro
645 650 655
Pro Glu Lys Leu Val Ile His Glu Gln Ile Thr Arg Glu Leu Lys Asp
660 665 670
Tyr Lys Ala Thr Gly Pro His Val Ala Ile Ala Lys Arg Leu Ala Ala
675 680 685
Arg Gly Val Lys Ile Arg Pro Gly Thr Val Ile Ser Tyr Ile Val Leu
690 695 700
Lys Gly Ser Gly Arg Ile Gly Asp Arg Ala Ile Pro Phe Asp Glu Phe
705 710 715 720
Asp Pro Ala Lys His Arg Tyr Asp Ala Glu Tyr Tyr Ile Glu Asn Gln
725 730 735
Val Leu Pro Ala Val Glu Arg Ile Leu Lys Ala Phe Gly Tyr Arg Lys
740 745 750
Glu Asp Leu Arg Tyr Gln Lys Thr Arg Gln Val Gly Leu Gly Ala Trp
755 760 765
Leu Lys Pro Lys Gly Lys Lys
770 775
<210> 51
<211> 775
<212> PRT
<213> Thermococcus gammatolerans
<400> 51
Met Ile Leu Asp Thr Asp Tyr Ile Thr Glu Asn Gly Lys Pro Val Ile
1 5 10 15
Arg Val Phe Lys Lys Glu Asn Gly Glu Phe Lys Ile Glu Tyr Asp Arg
20 25 30
Thr Phe Glu Pro Tyr Phe Tyr Ala Leu Leu Lys Asp Asp Ser Ala Ile
35 40 45
Glu Asp Val Lys Lys Val Thr Ala Lys Arg His Gly Ala Val Val Lys
50 55 60
Val Lys Arg Ala Glu Lys Val Gln Arg Lys Phe Leu Gly Arg Pro Ile
65 70 75 80
Glu Val Trp Lys Leu Tyr Phe Thr His Pro Gln Asp Val Pro Ala Ile
85 90 95
Arg Asp Lys Ile Arg Ala His Pro Ala Val Val Asp Ile Tyr Glu Tyr
100 105 110
Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Lys Gly Leu Ile Pro
115 120 125
Met Glu Gly Asp Glu Glu Leu Arg Met Leu Ala Phe Asp Ile Glu Thr
130 135 140
Leu Tyr His Glu Gly Glu Glu Phe Gly Thr Gly Pro Ile Leu Met Ile
145 150 155 160
Ser Tyr Ala Asp Glu Asn Glu Ala Arg Val Ile Thr Trp Lys Lys Ile
165 170 175
Asp Leu Pro Tyr Val Asp Val Val Ser Thr Glu Lys Glu Met Ile Lys
180 185 190
Arg Phe Leu Arg Val Val Lys Glu Lys Asp Pro Asp Val Leu Ile Thr
195 200 205
Tyr Asn Gly Asp Asn Phe Asp Phe Ala Tyr Leu Lys Lys Arg Cys Glu
210 215 220
Lys Leu Gly Ile Lys Phe Thr Leu Gly Arg Asp Gly Ser Glu Pro Lys
225 230 235 240
Ile Gln Arg Met Gly Asp Arg Phe Ala Val Glu Val Lys Gly Arg Ile
245 250 255
His Phe Asp Leu Tyr Pro Val Ile Arg Arg Thr Ile Asn Leu Pro Thr
260 265 270
Tyr Thr Leu Glu Ala Val Tyr Glu Ala Val Phe Gly Lys Pro Lys Glu
275 280 285
Lys Val Tyr Ala Glu Glu Ile Thr Glu Ala Trp Glu Ser Gly Glu Gly
290 295 300
Leu Glu Arg Val Ala Arg Tyr Ser Met Glu Asp Ala Lys Val Thr Tyr
305 310 315 320
Glu Leu Gly Arg Glu Phe Phe Pro Met Glu Ala Gln Leu Ser Arg Leu
325 330 335
Ile Gly Gln Ser Leu Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu
340 345 350
Val Glu Trp Phe Leu Leu Arg Lys Ala Tyr Glu Arg Asn Glu Leu Ala
355 360 365
Pro Asn Lys Pro Asp Glu Arg Glu Leu Ala Arg Arg Arg Glu Ser Tyr
370 375 380
Ala Gly Gly Tyr Val Lys Glu Pro Glu Arg Gly Leu Trp Asp Asn Ile
385 390 395 400
Val Tyr Leu Asp Phe Arg Ser Leu Tyr Pro Ser Ile Ile Ile Thr His
405 410 415
Asn Val Ser Pro Asp Thr Leu Asn Arg Glu Gly Cys Lys Glu Tyr Asp
420 425 430
Val Ala Pro Glu Val Gly His Lys Phe Cys Lys Asp Phe Pro Gly Phe
435 440 445
Ile Pro Ser Leu Leu Gly Asp Leu Leu Glu Glu Arg Gln Lys Ile Lys
450 455 460
Arg Lys Met Lys Ala Thr Ile Asp Pro Leu Glu Lys Lys Leu Leu Asp
465 470 475 480
Tyr Arg Gln Arg Ala Ile Lys Ile Leu Ala Asn Ser Phe Tyr Gly Tyr
485 490 495
Tyr Gly Tyr Ala Lys Ala Arg Trp Tyr Cys Lys Glu Cys Ala Glu Ser
500 505 510
Val Thr Ala Trp Gly Arg Glu Tyr Ile Glu Thr Thr Ile Arg Glu Ile
515 520 525
Glu Glu Lys Phe Gly Phe Lys Val Leu Tyr Ala Asp Thr Asp Gly Phe
530 535 540
Phe Ala Thr Ile Pro Gly Ala Asp Ala Glu Thr Val Lys Lys Lys Ala
545 550 555 560
Lys Glu Phe Leu Lys Tyr Ile Asn Ala Lys Leu Pro Gly Leu Leu Glu
565 570 575
Leu Glu Tyr Glu Gly Phe Tyr Val Arg Gly Phe Phe Val Thr Lys Lys
580 585 590
Lys Tyr Ala Val Ile Asp Glu Glu Gly Lys Ile Thr Thr Arg Gly Leu
595 600 605
Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu Thr Gln Ala
610 615 620
Arg Val Leu Glu Ala Ile Leu Lys His Gly Asp Val Glu Glu Ala Val
625 630 635 640
Arg Ile Val Lys Glu Val Thr Glu Lys Leu Ser Lys Tyr Glu Val Pro
645 650 655
Pro Glu Lys Leu Val Ile His Glu Gln Ile Thr Arg Asp Leu Arg Asp
660 665 670
Tyr Lys Ala Thr Gly Pro His Val Ala Val Ala Lys Arg Leu Ala Ala
675 680 685
Lys Gly Val Lys Ile Arg Pro Gly Thr Val Ile Ser Tyr Ile Val Leu
690 695 700
Lys Gly Ser Gly Arg Ile Gly Asp Arg Ala Ile Pro Ala Asp Glu Phe
705 710 715 720
Asp Pro Thr Lys His Arg Tyr Asp Ala Glu Tyr Tyr Ile Glu Asn Gln
725 730 735
Val Leu Pro Ala Val Glu Arg Ile Leu Lys Ala Phe Gly Tyr Arg Lys
740 745 750
Glu Asp Leu Arg Tyr Gln Lys Thr Lys Gln Val Gly Leu Gly Ala Trp
755 760 765
Leu Lys Val Lys Gly Lys Lys
770 775
<210> 52
<211> 775
<212> PRT
<213> Thermococcus celericrescens
<400> 52
Met Ile Leu Asp Thr Asp Tyr Ile Thr Glu Asp Gly Lys Pro Val Ile
1 5 10 15
Arg Ile Phe Lys Lys Asp Asn Gly Glu Phe Lys Ile Glu Tyr Asp Arg
20 25 30
Asn Phe Glu Pro Tyr Ile Tyr Ala Leu Leu Arg Asp Asp Ser Ala Ile
35 40 45
Glu Asp Val Lys Lys Ile Thr Ala Glu Arg His Gly Arg Val Val Lys
50 55 60
Val Lys Arg Ala Glu Lys Val Lys Lys Lys Phe Leu Gly Arg Pro Val
65 70 75 80
Glu Val Trp Lys Leu Tyr Phe Thr Arg Pro Gln Asp Val Pro Ala Ile
85 90 95
Arg Asp Arg Ile Arg Ala His Pro Ala Val Val Asp Ile Tyr Glu Tyr
100 105 110
Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Lys Gly Ile Ile Pro
115 120 125
Met Glu Gly Asp Glu Glu Leu Lys Met Leu Ala Phe Asp Ile Glu Thr
130 135 140
Leu Tyr His Glu Gly Glu Glu Phe Ala Glu Gly Pro Ile Leu Met Ile
145 150 155 160
Ser Tyr Ala Asp Glu Asn Glu Ala Arg Val Ile Thr Trp Lys Lys Ile
165 170 175
Asp Leu Pro Phe Val Asp Val Val Ser Thr Glu Lys Glu Met Ile Lys
180 185 190
Arg Phe Leu Lys Val Ile Lys Glu Lys Asp Pro Asp Val Leu Ile Thr
195 200 205
Tyr Asn Gly Asp Asn Phe Asp Phe Ala Tyr Leu Lys Lys Arg Cys Glu
210 215 220
Lys Phe Gly Ile Lys Phe Thr Leu Gly Arg Asp Gly Ser Asp Pro Lys
225 230 235 240
Ile Gln Arg Met Gly Asp Arg Phe Ala Val Glu Val Lys Gly Arg Ile
245 250 255
His Phe Asp Leu Tyr Pro Val Ile Leu Arg Thr Val Asn Leu Pro Thr
260 265 270
Tyr Thr Leu Glu Ala Val Tyr Glu Ala Ile Phe Gly Thr Pro Lys Glu
275 280 285
Lys Val Tyr Pro Glu Glu Ile Thr Thr Ala Trp Glu Thr Gly Glu Gly
290 295 300
Leu Glu Arg Val Ala Arg Tyr Ser Met Glu Asp Ala Lys Val Thr Phe
305 310 315 320
Glu Leu Gly Arg Glu Phe Phe Pro Met Glu Ala Gln Leu Ser Arg Leu
325 330 335
Val Gly Gln Ser Phe Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu
340 345 350
Val Glu Trp Phe Leu Leu Arg Lys Ala Tyr Glu Arg Asn Glu Leu Ala
355 360 365
Pro Asn Lys Pro Asp Glu Arg Glu Leu Val Arg Arg Arg Asn Ser Tyr
370 375 380
Thr Gly Gly Tyr Val Lys Glu Pro Glu Arg Gly Leu Trp Asp Asn Ile
385 390 395 400
Val Tyr Leu Asp Phe Arg Ser Leu Tyr Pro Ser Ile Ile Ile Thr His
405 410 415
Asn Val Ser Pro Asp Thr Leu Asn Arg Glu Gly Cys Lys Glu Tyr Asp
420 425 430
Glu Ala Pro Gln Val Gly His Lys Phe Cys Lys Asp Phe Pro Gly Phe
435 440 445
Ile Pro Ser Leu Leu Gly Asn Leu Leu Asp Glu Arg Gln Lys Ile Lys
450 455 460
Lys Arg Met Lys Ala Thr Ile Asp Pro Leu Glu Lys Lys Leu Leu Asp
465 470 475 480
Tyr Arg Gln Arg Ala Ile Lys Ile Leu Ala Asn Ser Tyr Tyr Gly Tyr
485 490 495
Tyr Ala Tyr Ala Arg Ala Arg Trp Tyr Cys Lys Glu Cys Ala Glu Ser
500 505 510
Val Thr Ala Trp Gly Arg Glu Tyr Ile Glu Met Ser Ile Arg Glu Ile
515 520 525
Glu Glu Lys Tyr Gly Phe Lys Val Leu Tyr Ala Asp Thr Asp Gly Phe
530 535 540
His Ala Thr Ile Pro Gly Ala Asp Ala Glu Thr Val Lys Lys Lys Ala
545 550 555 560
Met Glu Phe Leu Lys Tyr Ile Asn Ala Lys Leu Pro Gly Ala Leu Glu
565 570 575
Leu Glu Tyr Glu Gly Phe Tyr Lys Arg Gly Phe Phe Val Thr Lys Lys
580 585 590
Lys Tyr Ala Val Ile Asp Glu Glu Gly Lys Ile Thr Thr Arg Gly Leu
595 600 605
Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu Thr Gln Ala
610 615 620
Arg Val Leu Glu Ala Leu Leu Lys Asp Gly Asn Val Glu Glu Ala Val
625 630 635 640
Ser Ile Val Lys Glu Val Thr Glu Lys Leu Gly Lys Tyr Glu Val Pro
645 650 655
Pro Glu Lys Leu Val Ile His Glu Gln Ile Thr Arg Glu Leu Lys Asp
660 665 670
Tyr Lys Ala Thr Gly Pro His Val Ala Ile Ala Lys Arg Leu Ala Ala
675 680 685
Arg Gly Val Lys Ile Arg Pro Gly Thr Val Ile Ser Tyr Ile Val Leu
690 695 700
Lys Gly Ser Gly Arg Ile Gly Asp Arg Ala Ile Pro Phe Asp Glu Phe
705 710 715 720
Asp Pro Thr Lys His Arg Tyr Asp Ala Glu Tyr Tyr Ile Glu Asn Gln
725 730 735
Val Leu Pro Ala Val Glu Arg Ile Leu Lys Ala Phe Gly Tyr Arg Ala
740 745 750
Glu Asp Leu Arg Tyr Gln Lys Thr Arg Gln Val Gly Leu Gly Val Trp
755 760 765
Leu Gln Pro Lys Gly Lys Lys
770 775
<210> 53
<211> 784
<212> PRT
<213> Vulcanisaeta distributa
<400> 53
Met Glu Leu Ala Phe Trp Leu Leu Asp Ile Thr Tyr Gly Val Ile Gly
1 5 10 15
Asn Thr Pro Glu Leu Arg Leu Phe Gly Ile Thr Asp Asp Gly Lys Arg
20 25 30
Val Leu Val Leu Asp Arg Ser Phe Arg Pro Tyr Phe Tyr Val Ile Pro
35 40 45
Ser Gly Asp Val Asn Ala Val Phe Asn Asn Val Lys Arg Lys Leu Glu
50 55 60
Gly Lys Val Leu Asn Val Glu Val Ile Lys Arg Lys Met Phe Gly Asn
65 70 75 80
Glu Val Asp Ala Ile Arg Val Thr Ala Thr Ile Pro Glu Lys Val Arg
85 90 95
Glu Leu Arg Glu Leu Ala Ala Glu Val Pro Gly Val Glu Asp Val Leu
100 105 110
Glu Ala Asp Ile Arg Phe Ser Gln Arg Tyr Leu Leu Asp Met Gly Val
115 120 125
Lys Pro Ser Asn Trp Ile Val Val Asp Gln Cys Glu Glu Val Lys Gly
130 135 140
Asn Tyr Gln Val Asp Leu Val Cys Leu Ala Lys Ser Arg Pro Arg Met
145 150 155 160
Ile Glu Glu His Lys Leu Pro Ser Phe Arg Val Leu Ala Phe Asp Ile
165 170 175
Glu Val Tyr Asn Pro Arg Gly Met Pro Asn Pro Asp Arg Asp Pro Val
180 185 190
Ile Ile Ile Ser Thr Met Thr Lys Glu Asp Gly Val Lys Met Phe Val
195 200 205
Val Asp Asp Asn Lys Asn Asp Ala Lys Ile Ile Arg Glu Phe Leu Asp
210 215 220
Tyr Phe Arg Lys Tyr Asp Pro Asp Ile Val Val Gly Tyr Asn Asn Asn
225 230 235 240
Gly Phe Asp Trp Pro Tyr Leu Val Asn Arg Ser Ser Arg Val Gly Val
245 250 255
Arg Leu Ala Leu Ser Arg Met Gly Asn Pro Pro Glu Pro Ser Val Tyr
260 265 270
Gly His Trp Ser Ile Ile Gly Arg Ala Asn Val Asp Leu Tyr Asn Phe
275 280 285
Ile Glu Glu Ile Ser Glu Ile Lys Val Lys Ser Leu Asp Arg Ala Ala
290 295 300
Glu Phe Phe Gly Ile Met Lys Arg Ser Glu Arg Val Leu Ile Pro Gly
305 310 315 320
His Arg Ile His Glu Tyr Trp Asp Asp Lys Asn Lys Arg Asp Leu Leu
325 330 335
Leu Lys Tyr Ala Arg Asp Asp Val Val Ser Thr Tyr Gly Leu Ala Glu
340 345 350
Lys Leu Leu Pro Phe Ala Ile Gln Leu Ser Ser Ile Ser Gly Leu Pro
355 360 365
Leu Asp Gln Val Gly Ala Ala Ser Val Gly Ala Arg Val Glu Trp Met
370 375 380
Ile Phe Tyr Glu Ala Val Lys Arg Gly Glu Leu Ala Pro Asn Arg Glu
385 390 395 400
Glu Arg Pro Tyr Glu Thr Tyr Lys Gly Ala Val Val Leu Glu Pro Arg
405 410 415
Pro Gly Leu His Glu Asn Ile Ala Val Ile Asp Phe Ser Ser Met Tyr
420 425 430
Pro Ser Ile Met Met Lys Tyr Asn Val Ser Pro Asp Thr Leu Val Leu
435 440 445
Gly Asp Cys Gly Asp Cys Tyr Val Ala Pro Glu Val Asn Tyr Lys Phe
450 455 460
Arg Arg Ser Pro Glu Gly Leu Tyr Pro Gly Leu Leu Arg Ile Leu Val
465 470 475 480
Glu Ser Arg Arg Arg Val Arg Asp Leu Met Lys Lys Tyr Pro Glu Asn
485 490 495
Ser Pro Glu Trp Val Leu Leu Asn Glu Arg Gln Arg Ala Leu Lys Val
500 505 510
Met Ala Asn Ala Met Tyr Gly Tyr Cys Gly Trp Leu Gly Ala Arg Trp
515 520 525
Tyr Arg Arg Glu Val Ala Glu Ala Val Thr Ala Trp Gly Arg Asn Leu
530 535 540
Leu Arg Thr Val Ile Glu Lys Ala Arg Ser Leu Gly Leu Pro Ile Ile
545 550 555 560
Tyr Gly Asp Thr Asp Ser Leu Phe Val Arg Asn Ile Ser Asp Lys Val
565 570 575
Asp Ala Leu Ile Asn Tyr Val Asn Asn Glu Leu Gly Phe Glu Val Lys
580 585 590
Val Asp Lys Val Tyr Arg Arg Val Leu Phe Thr Glu Ala Lys Lys Arg
595 600 605
Tyr Val Gly Leu Thr Val Glu Gly Glu Val Asp Ile Val Gly Phe Glu
610 615 620
Ala Val Arg Gly Asp Trp Ala Glu Ile Ala Lys Asp Val Gln Glu Asn
625 630 635 640
Val Ala Glu Ile Val Leu Thr Thr Gly Asp Val Gly Lys Ala Ile Ser
645 650 655
Tyr Val Lys Ser Val Ile Asp Lys Val Lys Ala Tyr Gln Phe Asp Ile
660 665 670
Asp Asp Val Ile Ile Trp Lys Thr Leu Asp Lys Ser Leu Asn Glu Tyr
675 680 685
Lys Val Leu Thr Pro His Val Ala Ala Ala Lys Gln Leu Val Glu Ala
690 695 700
Gly Tyr Lys Val Gly Lys Gly Asp Met Ile Gly Tyr Val Val Val Lys
705 710 715 720
Gly Gly Gly Ala Lys Leu Ala Tyr Lys Val Lys Pro Tyr Ile Leu Ile
725 730 735
Lys Asp Ile Arg Glu Val Asp Val Asp Tyr Tyr Val Glu Lys Gln Ile
740 745 750
Val Pro Ala Ala Met Arg Ile Leu Glu Val Leu Gly Val Lys Glu Ser
755 760 765
Gln Leu Met Glu Gly Lys Ala Gly Lys Ser Ile Leu Asp Tyr Phe Ser
770 775 780
<210> 54
<211> 830
<212> PRT
<213> Methanopyrus kandleri
<400> 54
Met Leu Arg Thr Val Trp Val Asp Tyr Ala Arg Lys Gly Glu Pro Asp
1 5 10 15
Val Ile Leu Val Gly Arg Arg Glu Asp Gly Asn Pro Ala Ala Leu Val
20 25 30
Val Lys Gly Phe Arg Pro Tyr Phe Tyr Ala Glu Val Glu Asp Gly Phe
35 40 45
Asp Pro Ser Glu Val Glu Arg Leu Ser Gly Val Val Glu Val Glu Glu
50 55 60
Val Leu Leu Glu His Pro Tyr Gly Gly Asp Arg Val Glu Leu Leu Arg
65 70 75 80
Ile Val Ala Thr Tyr Pro Lys Val Val Pro Lys Leu Arg Glu Gln Val
85 90 95
Lys Lys Leu Asp Gly Val Lys Glu Val Tyr Glu Ala Asp Ile Pro Phe
100 105 110
Val Arg Arg Ala Ala Val Asp Leu Asn Leu Pro Pro Ala Ser Glu Val
115 120 125
Asp Val Ser Asp Leu Asp Thr Gly Ser Trp Ser Gly Leu Pro Ala Tyr
130 135 140
Phe Ala Asp Val Glu Asp Ala Arg Glu Leu Asp His Arg Pro Tyr Pro
145 150 155 160
Ile Glu Asp Leu Val Val Ala Ser Phe Asp Leu Glu Val Leu Ala Glu
165 170 175
Pro Gly Thr Thr Ile Lys Gly Ala Ser Gly Pro Ile Ile Ala Ile Ser
180 185 190
Phe Ala Tyr Ser Thr Pro Asp Gly Glu Arg Arg Asn Tyr Val Ile Thr
195 200 205
Trp Lys Gly Glu Asp Glu Ser Phe Glu Val Asp Gly Val Glu Thr Glu
210 215 220
Val Ile Val Cys Arg Ser Glu Ala Ala Ala Leu Arg Arg Phe Phe Asp
225 230 235 240
Glu Phe Arg Arg Val Asp Pro Asp Val Val Phe Thr Tyr Asn Gly Asp
245 250 255
Glu Phe Asp Leu Pro Tyr Leu Gln His Arg Ala Gly Lys Leu Gly Ile
260 265 270
Asp Val Ser Pro Leu Ala Arg Pro Ala Gly Lys Arg Gly Ile Ile Leu
275 280 285
Lys His Gly Gly Gly Arg Tyr Ala Ser Asp Ile Phe Gly Arg Ala His
290 295 300
Val Asp Leu Tyr His Thr Ala Arg Lys Asn Leu Lys Leu Glu Arg Phe
305 310 315 320
Thr Leu Glu Glu Ala Val Lys Asp Val Leu Gly Val Glu Lys Glu Glu
325 330 335
Met Glu Leu Ala Asp Ile Asn Glu Ala Trp Lys Arg Gly Asn Leu Asp
340 345 350
Glu Leu Met Arg Tyr Ser Ala Glu Asp Ala His Tyr Thr Leu Glu Leu
355 360 365
Gly Leu Glu Leu Ala Gln Val Glu Leu Glu Leu Ser Tyr Leu Thr Arg
370 375 380
Leu Pro Leu Pro Asp Ala Thr Arg Phe Ser Phe Gly Gln Leu Ala Glu
385 390 395 400
Trp Arg Ala Ile Tyr Lys Ala Arg Gln Glu Asp Ile Leu Val Pro Asn
405 410 415
Lys Pro Thr Arg Asp Glu Tyr Lys Arg Arg Arg Arg Lys Ala Tyr Lys
420 425 430
Gly Ala Ile Val Phe Glu Pro Glu Ile Gly Leu His Glu Asn Val Val
435 440 445
Cys Val Asp Phe Ala Ser Leu Tyr Pro Asn Val Met Val Ala His Asn
450 455 460
Ile Ser Pro Asp Thr Phe Asp Cys Asp Cys Cys Pro Arg Val Thr Val
465 470 475 480
Glu Glu Val Asp Asp Pro Thr Asp Ala Thr Val Ala Pro Asp Val Gly
485 490 495
His Lys Phe Cys Lys Arg Arg Lys Gly Phe Phe Pro Arg Leu Val Glu
500 505 510
Gly Leu Ile Glu Arg Arg Arg Glu Leu Lys Arg Arg Leu Arg Lys Leu
515 520 525
Asp Thr Glu Ser His Pro His Glu Ala Lys Ile Leu Asp Val Arg Gln
530 535 540
Gln Ala Tyr Lys Val Leu Ala Asn Ser Tyr Tyr Gly Tyr Met Gly Trp
545 550 555 560
Ala Asn Ala Arg Trp Phe Cys Arg Glu Cys Ala Glu Ser Val Thr Ala
565 570 575
Trp Gly Arg Tyr Tyr Ile Ser Glu Val Arg Arg Ile Ala Glu Glu Lys
580 585 590
Tyr Gly Leu Lys Val Val Tyr Gly Asp Thr Asp Ser Leu Phe Val Lys
595 600 605
Leu Pro Asp Ala Asp Leu Glu Glu Thr Ile Glu Arg Val Lys Glu Phe
610 615 620
Leu Lys Glu Val Asn Gly Arg Leu Pro Val Glu Leu Glu Leu Glu Asp
625 630 635 640
Ala Tyr Lys Arg Ile Leu Phe Val Thr Lys Lys Lys Tyr Ala Gly Tyr
645 650 655
Thr Glu Asp Gly Lys Ile Val Thr Lys Gly Leu Glu Val Val Arg Arg
660 665 670
Asp Trp Ala Pro Ile Ala Arg Glu Thr Gln Arg Arg Val Leu Lys Arg
675 680 685
Ile Leu Ala Asp Asn Asp Pro Glu Ala Ala Leu Lys Glu Ile His Glu
690 695 700
Val Leu Glu Arg Leu Lys Ser Gly Asp Val Asp Ile Asp Glu Leu Ala
705 710 715 720
Val Thr Ser Gln Leu Thr Lys Lys Pro Ser Glu Tyr Val Gln Lys Gly
725 730 735
Pro His Val Arg Ala Ala Leu Arg Leu Ala Arg His Leu Gly Val Glu
740 745 750
Pro Glu Pro Gly Thr Ile Val Arg Tyr Val Ile Val Arg Gly Pro Gly
755 760 765
Ser Val Ser Asp Lys Ala Tyr Pro Val Glu Leu Val Arg Glu Glu Gly
770 775 780
Lys Glu Pro Asp Val Asp Tyr Tyr Ile Glu His Gln Ile Leu Pro Ala
785 790 795 800
Val Glu Arg Ile Met Arg Ala Ile Gly Tyr Ser Arg Gly Gln Ile Val
805 810 815
Gly Glu Thr Ala Ser Gln Lys Thr Leu Asp Gln Phe Phe Gly
820 825 830
<210> 55
<211> 784
<212> PRT
<213> Thermoproteus neutrophilus
<400> 55
Met Glu Leu Lys Ile Trp Pro Leu Asp Val Thr Tyr Ala Val Val Gly
1 5 10 15
Gly Tyr Pro Glu Val Arg Val Phe Gly Leu Thr Glu Gly Gly Gly Arg
20 25 30
Val Val Leu Val Asp Arg Ser Phe Lys Pro Tyr Phe Tyr Val Asp Cys
35 40 45
Pro Thr Cys Glu Val Gly Val Val Lys Ser Ser Leu Ser Arg Val Ala
50 55 60
Pro Val Asp Glu Val Ser Ala Ala Glu Arg Arg Phe Leu Gly Arg Pro
65 70 75 80
Arg Arg Phe Leu Met Val Val Ala Arg Val Pro Glu Asp Val Arg Arg
85 90 95
Leu Arg Glu Ala Ala Ala Gln Ile Pro Gly Val Ala Gly Val Tyr Glu
100 105 110
Ala Asp Ile Arg Phe Tyr Met Arg Tyr Met Ile Asp Val Gly Leu Leu
115 120 125
Pro Cys Ser Trp Asn Arg Ala Glu Val Glu Gly Gly Gly Lys Val Gly
130 135 140
Gly Leu Pro Gln Tyr Thr Val Val Gln Trp Leu Gly Pro Ala Gly Gly
145 150 155 160
Phe Pro Pro Pro Leu Arg Val Leu Ala Phe Asp Ile Glu Val Tyr Asn
165 170 175
Glu Arg Gly Thr Pro Asp Pro Ala Arg Asp Pro Val Val Met Ile Ala
180 185 190
Val Lys Thr Asp Asp Gly Arg Glu Glu Val Phe Glu Ala Glu Gly Arg
195 200 205
Asp Asp Arg Gly Val Leu Arg Ser Phe Val Glu Phe Val Lys Ser Tyr
210 215 220
Asp Pro Asp Val Val Val Gly Tyr Asn Ser Asn Gly Phe Asp Trp Pro
225 230 235 240
Tyr Leu Ala Gly Arg Ala Arg Ala Ile Gly Val Pro Leu Arg Val Asp
245 250 255
Arg Leu Gly Gly Leu Pro Gln Gln Ser Val Tyr Gly His Trp Ser Ile
260 265 270
Val Gly Arg Ala Asn Val Asp Leu Tyr Gly Ile Val Glu Glu Phe Pro
275 280 285
Glu Ile Lys Leu Lys Thr Leu Asp Arg Val Ala Glu Tyr Phe Gly Val
290 295 300
Met Arg Arg Glu Glu Arg Val Leu Ile Pro Gly His Lys Ile Tyr Glu
305 310 315 320
Tyr Trp Arg Asp Pro Gly Lys Arg Pro Leu Leu Arg Gln Tyr Val Leu
325 330 335
Asp Asp Val Arg Ser Thr Leu Gly Leu Ala Asp Lys Leu Leu Pro Phe
340 345 350
Leu Ile Gln Leu Ser Ser Val Ser Gly Leu Pro Leu Asp Gln Val Ala
355 360 365
Ala Ala Ser Val Gly Asn Arg Val Glu Trp Met Leu Leu Arg Tyr Ala
370 375 380
Tyr Arg Leu Gly Glu Val Ala Pro Asn Arg Glu Glu Arg Glu Tyr Glu
385 390 395 400
Pro Tyr Lys Gly Ala Ile Val Leu Glu Pro Lys Pro Gly Met Tyr Glu
405 410 415
Asp Val Leu Val Leu Asp Phe Ser Ser Met Tyr Pro Asn Ile Met Met
420 425 430
Lys Tyr Asn Leu Ser Pro Asp Thr Tyr Leu Glu Pro Gly Glu Pro Asp
435 440 445
Pro Pro Glu Gly Val Asn Ala Ala Pro Glu Val Gly His Arg Phe Arg
450 455 460
Arg Ser Pro Leu Gly Phe Val Pro Gln Val Leu Lys Ser Leu Val Glu
465 470 475 480
Leu Arg Lys Ala Val Arg Glu Glu Ala Lys Arg Tyr Pro Pro Asp Ser
485 490 495
Pro Glu Phe Arg Ile Leu Asp Glu Arg Gln Arg Ala Leu Lys Val Met
500 505 510
Ala Asn Ala Met Tyr Gly Tyr Leu Gly Trp Val Gly Ala Arg Trp Tyr
515 520 525
Lys Arg Glu Val Ala Glu Ser Val Thr Ala Phe Ala Arg Ala Ile Leu
530 535 540
Lys Asp Val Ile Glu Gln Ala Arg Arg Leu Gly Ile Val Val Val Tyr
545 550 555 560
Gly Asp Thr Asp Ser Leu Phe Val Lys Lys His Val Asn Val Asp Lys
565 570 575
Leu Ile Gln Tyr Val Glu Glu Lys Tyr Gly Ile Glu Ile Lys Val Asp
580 585 590
Lys Asp Tyr Ala Lys Val Leu Phe Thr Glu Ala Lys Lys Arg Tyr Ala
595 600 605
Gly Leu Leu Arg Asp Gly Arg Ile Asp Ile Val Gly Phe Glu Val Val
610 615 620
Arg Gly Asp Trp Ser Glu Leu Ala Lys Glu Val Gln Leu Lys Val Val
625 630 635 640
Glu Ile Ile Leu Asn Ser Arg Asp Val Ala Glu Ala Arg Arg Arg Val
645 650 655
Thr Gln Tyr Val Arg Glu Ile Ile Glu Arg Leu Arg Glu Tyr Lys Phe
660 665 670
Asn Val Asp Asp Leu Ile Ile Trp Lys Thr Leu Asp Lys Glu Leu Gly
675 680 685
Glu Tyr Lys Ala Tyr Pro Pro His Val His Ala Ala Leu Ile Leu Lys
690 695 700
Arg His Gly Tyr Lys Val Gly Lys Gly Asn Met Val Gly Tyr Val Val
705 710 715 720
Val Lys Gly Gly Gly Lys Ile Ser Glu Lys Ala Leu Pro Tyr Ile Leu
725 730 735
Leu Asp Asp Val Lys Lys Ile Asp Val Glu Tyr Tyr Ile Glu Arg Gln
740 745 750
Ile Ile Pro Ala Ala Leu Arg Ile Ala Glu Val Ile Gly Val Lys Glu
755 760 765
Ala Asp Leu Lys Thr Gly Lys Ser Glu Arg Ser Leu Leu Asp Phe Phe
770 775 780
<210> 56
<211> 142
<212> PRT
<213> 激烈火球菌
<400> 56
Met Lys Thr Phe Leu Thr Glu Gln Gln Ile Lys Val Leu Met Leu Arg
1 5 10 15
Ala Lys Gly Tyr Lys Gln Ser Glu Ile Ala Lys Ile Leu Gly Thr Ser
20 25 30
Arg Ala Asn Val Ser Ile Leu Glu Lys Arg Ala Met Glu Lys Ile Glu
35 40 45
Lys Ala Arg Asn Thr Leu Leu Leu Trp Glu Gln Ile Asn Ser Lys Val
50 55 60
Ile Val Glu Ile Lys Ala Gly Glu Asp Ile Phe Ser Ile Pro Glu Lys
65 70 75 80
Phe Phe Lys Lys Ala Asp Lys Val Gly Val Lys Val Pro Tyr Ser Thr
85 90 95
Ala Glu Ile Ile Thr Phe Leu Val Glu His Ala Pro Val Glu Asp Arg
100 105 110
Leu Ala Lys Arg Asp Phe Val Leu Phe Leu Asp Ser Lys Asn Lys Leu
115 120 125
Arg Ile Gly Asp Cys Leu Val Ile Glu Glu Ile Lys Glu Asp
130 135 140
<210> 57
<211> 75
<212> PRT
<213> 激烈火球菌
<400> 57
Met Pro Ile Thr Lys Val Thr Arg Asn Tyr Gln Ile Thr Ile Pro Ala
1 5 10 15
Glu Ile Arg Lys Ala Leu Gly Ile Lys Glu Gly Glu Leu Leu Glu Val
20 25 30
Arg Leu Glu Asn Gly Lys Ile Ile Ile Glu Arg Leu Lys Lys Glu Arg
35 40 45
Lys Thr Leu Lys Leu Gly Lys Lys Leu Thr Leu Glu Glu Ile Glu Lys
50 55 60
Ala Ile Glu Glu Gly Met Lys Gln Cys Met Gln
65 70 75
<210> 58
<211> 76
<212> PRT
<213> 激烈火球菌
<400> 58
Thr Lys Ile Glu Ile Leu Arg Leu Leu Lys Glu Arg Glu Met Tyr Ala
1 5 10 15
Tyr Glu Ile Trp Ser Leu Leu Gly Lys Pro Leu Lys Tyr Gln Ala Val
20 25 30
His Gln His Ile Lys Glu Leu Leu Glu Leu Gly Leu Val Glu Gln Ala
35 40 45
Tyr Arg Lys Gly Lys Arg Val Tyr Tyr Lys Ile Thr Glu Lys Gly Leu
50 55 60
Arg Ile Leu Gln Asn Phe Glu Asp Leu Glu Asn Ile
65 70 75
<210> 59
<211> 72
<212> PRT
<213> 激烈火球菌
<400> 59
Met Asn Thr Gly Ala Gln Gly Val Ser Glu Met Ser Arg Met Lys Ile
1 5 10 15
Ile Ser Val Gln Leu Pro Gln Ser Leu Ile His Gly Leu Asp Ala Leu
20 25 30
Val Lys Arg Gly Ile Tyr Pro Asn Arg Ser Glu Ala Ile Arg Val Ala
35 40 45
Ile Arg Glu Leu Leu Lys Lys Glu Leu Tyr Lys Glu Glu Ile Gln Glu
50 55 60
Glu Ile Pro Glu Tyr Val Val Lys
65 70
<210> 60
<211> 67
<212> PRT
<213> 激烈火球菌
<400> 60
Val Ile Ile Pro Arg Pro Ile Asp Pro Arg Asp Ile Arg Arg Ile Arg
1 5 10 15
Lys Glu Leu Gly Ile Thr Gln Glu Glu Leu Ala Arg Lys Ala Gly Val
20 25 30
Thr Gln Ala Tyr Ile Ala Lys Leu Glu Ala Gly Lys Val Asp Pro Arg
35 40 45
Leu Ser Thr Phe Asn Lys Ile Leu Arg Ala Leu Ile Glu Cys Gln Lys
50 55 60
Ala Lys Ile
65
<210> 61
<211> 65
<212> PRT
<213> 激烈火球菌
<400> 61
Asn Asn Cys Glu Cys Met Val Val Lys Glu Lys Leu Tyr Thr Val Lys
1 5 10 15
Gln Ala Ser Glu Ile Leu Gly Val His Pro Lys Thr Ile Gln Lys Trp
20 25 30
Asp Arg Glu Gly Lys Ile Lys Thr Val Arg Thr Pro Gly Gly Arg Arg
35 40 45
Arg Ile Pro Glu Ser Glu Ile Lys Arg Leu Leu Gly Ile Ser Glu Glu
50 55 60
Lys
65
<210> 62
<211> 71
<212> PRT
<213> 激烈火球菌
<400> 62
Met Leu Lys Asp Ser Ala Pro Lys Arg Lys Ile Leu Glu Glu Leu Arg
1 5 10 15
Lys Gly Glu Thr Val Ser Gly Asp Tyr Leu Ala Ser Lys Leu Gly Val
20 25 30
Ser Arg Val Ala Ile Trp Lys His Ile Arg Glu Leu Lys Glu Leu Gly
35 40 45
Tyr Gly Ile Ile Ala Asp Lys Lys Gly Tyr Lys Leu Val Tyr Glu Pro
50 55 60
Lys Lys Pro Tyr Pro Trp Glu
65 70
<210> 63
<211> 86
<212> PRT
<213> 激烈火球菌
<400> 63
Met Ile Asp Glu Arg Asp Lys Ile Ile Leu Glu Ile Leu Glu Lys Asp
1 5 10 15
Ala Arg Thr Pro Phe Thr Glu Ile Ala Lys Lys Leu Gly Ile Ser Glu
20 25 30
Thr Ala Val Arg Lys Arg Val Lys Ala Leu Glu Glu Lys Gly Ile Ile
35 40 45
Glu Gly Tyr Thr Ile Lys Ile Asn Pro Lys Lys Leu Gly Tyr Ser Leu
50 55 60
Val Thr Ile Thr Gly Val Asp Thr Lys Pro Glu Lys Leu Phe Glu Val
65 70 75 80
Ala Glu Lys Leu Lys Glu
85
<210> 64
<211> 77
<212> PRT
<213> 激烈火球菌
<400> 64
Met Glu Ile Asp Asp Leu Asp Arg Lys Ile Leu Ser Leu Leu Ile Glu
1 5 10 15
Asp Ser Arg Leu Ser Tyr Arg Glu Ile Ala Lys Lys Leu Asn Val Ala
20 25 30
Val Gly Thr Ile Tyr Asn Arg Ile Lys Lys Leu Glu Asp Met Gly Val
35 40 45
Ile Gln Gly Phe Thr Val Lys Leu Asn Tyr Glu Lys Leu Gly Tyr Glu
50 55 60
Leu Thr Ala Ile Ile Gly Ile Lys Ala Gln Gly Lys Lys
65 70 75
<210> 65
<211> 94
<212> PRT
<213> 激烈火球菌
<400> 65
Glu Met Leu Trp Met Tyr Ile Leu Lys Leu Leu Lys Asp Arg Pro Met
1 5 10 15
Tyr Ala Tyr Glu Ile Arg Asn Glu Leu Lys Lys Arg Phe Gly Phe Glu
20 25 30
Pro Ala Thr Val Ser Ser Tyr Val Val Leu Tyr Arg Leu Glu Glu Gly
35 40 45
Gly Tyr Val Ser Ser Glu Trp His Glu Ser Glu Ala Gly Arg Pro Ser
50 55 60
Arg Lys Tyr Tyr Arg Leu Thr Glu Lys Gly Glu Lys Leu Leu Glu Lys
65 70 75 80
Gly Ile Glu Thr Ile Glu Asp Val Leu Asn Met Leu Lys Ser
85 90
<210> 66
<211> 68
<212> PRT
<213> 激烈火球菌
<400> 66
Met Lys Val Ser Lys Ala Thr Ala Ser Lys Val Leu Arg Ser Leu Glu
1 5 10 15
Asn Lys Gly Ile Val Glu Arg Glu Arg Arg Gly Lys Thr Tyr Leu Val
20 25 30
Arg Leu Thr Asn Lys Gly Leu Glu Leu Leu Glu Glu Ile Ser Lys Ala
35 40 45
Gly Lys Glu Leu Asp Glu Lys Ile Phe Ala Glu Met Ser Val Asp Glu
50 55 60
Arg Ile Val Leu
65
<210> 67
<211> 56
<212> PRT
<213> 激烈火球菌
<400> 67
Ser Glu Asp Tyr Met Leu Gln Asn Arg Arg Lys Val Leu Ala Lys Val
1 5 10 15
Leu Glu Leu Leu Asn Tyr Asn Pro Lys Ala Leu Asn Ile Ser Glu Leu
20 25 30
Ala Arg Met Phe Gly Val Ser Arg Asp Thr Ile Tyr Asn Asp Ile Gln
35 40 45
Gln Ile Ile Lys Asn Val Glu Val
50 55
<210> 68
<211> 94
<212> PRT
<213> 激烈火球菌
<400> 68
Ser Lys Glu Ile Ser Arg Phe Leu Lys Val Ile Ser Asn Pro Ile Arg
1 5 10 15
Tyr Gly Ile Leu Lys Met Leu Asn Asp Arg Trp Met Cys Val Cys Leu
20 25 30
Ile Ser Glu Ala Leu Glu Ile Asp Gln Thr Leu Val Ser His His Ile
35 40 45
Arg Ile Leu Lys Glu Leu Asp Leu Leu Glu Glu Arg Lys Glu Gly Lys
50 55 60
Leu Arg Phe Tyr Arg Thr Asn Lys Glu Lys Leu Arg Glu Tyr Leu Glu
65 70 75 80
Lys Val Leu Glu Asp Phe Asn His Gly Thr Ser Lys Gly Ser
85 90
<210> 69
<211> 78
<212> PRT
<213> 激烈火球菌
<400> 69
Met Cys Arg Lys Asp Val Met Ile Ile Ser Asp Pro Lys Gln Ile Lys
1 5 10 15
Ala Leu Ser Asp Pro Thr Arg Val Lys Ile Leu Glu Leu Leu Arg Tyr
20 25 30
His Pro Met Thr Val Ser Glu Ile Ser Arg Val Ile Gly Lys Asp Lys
35 40 45
Ser Thr Ile Tyr Arg His Ile Lys Ala Leu Glu Glu Ala Gly Leu Val
50 55 60
Glu Glu Val Glu Lys Ile Gly Asn Glu Thr Val Tyr Gly Arg
65 70 75
<210> 70
<211> 122
<212> PRT
<213> 激烈火球菌
<400> 70
Met Glu Pro Val Glu Phe Lys Leu Asn Gln Lys Gly Ile Lys Ser Ile
1 5 10 15
Leu Pro Thr Met Glu Ala Glu Ile Met Glu Tyr Met Trp Glu Ile Lys
20 25 30
Glu Ala Thr Ala Gly Glu Val Tyr Glu Tyr Met Lys Thr Lys Tyr Pro
35 40 45
Glu Ile Arg Arg Ser Thr Val Ser Ile Leu Met Asn Arg Leu Cys Glu
50 55 60
Arg Gly Leu Leu Lys Arg Arg Met Glu Lys Gly Lys Gly Gly Ile Arg
65 70 75 80
Tyr Val Tyr Ser Ile Thr Thr Thr Arg Glu Glu Phe Glu Arg Lys Val
85 90 95
Val Glu Lys Ile Ile Glu Ser Leu Met Met Asn Phe Arg Glu Ala Thr
100 105 110
Phe Ala Tyr Leu Ser Lys Ile Asn Lys Lys
115 120
<210> 71
<211> 87
<212> PRT
<213> 激烈火球菌
<400> 71
Met Lys Lys Ser Asn Leu Asp Leu Leu Ile Leu Leu Ala Lys Ala Gly
1 5 10 15
Gly Ile Glu Lys Glu Ile Leu Thr Thr Ser Arg Glu Leu Ser Lys Met
20 25 30
Leu Asn Val Ser Pro Gln Thr Ile Val Arg Trp Leu Glu Asp Leu Glu
35 40 45
Lys Asp Gly Leu Ile Lys Lys Ser Glu Ser Arg Lys Gly Thr Leu Val
50 55 60
Thr Ile Thr Glu Glu Gly Val Lys Phe Leu Glu Lys Leu His Glu Glu
65 70 75 80
Leu Ser Asp Ala Leu Tyr Arg
85
<210> 72
<211> 104
<212> PRT
<213> Thermococcaceae
<400> 72
Met Glu Ile Pro Pro Glu Ile Ser His Ala Leu Ser Glu Ile Gly Phe
1 5 10 15
Thr Lys Tyr Glu Ile Leu Thr Tyr Trp Thr Leu Leu Val Tyr Gly Pro
20 25 30
Ser Thr Ala Lys Glu Ile Ser Thr Lys Ser Gly Ile Pro Tyr Asn Arg
35 40 45
Val Tyr Asp Thr Ile Ser Ser Leu Lys Leu Arg Gly Phe Val Thr Glu
50 55 60
Ile Glu Gly Thr Pro Lys Val Tyr Ala Ala Tyr Ser Pro Arg Ile Ala
65 70 75 80
Phe Phe Arg Phe Lys Lys Glu Leu Glu Asp Ile Met Lys Lys Leu Glu
85 90 95
Ile Glu Leu Asn Asn Val Lys Lys
100
<210> 73
<211> 75
<212> PRT
<213> 激烈火球菌
<400> 73
Ile Ile Asn Pro Gln Ala Arg Leu Thr Pro Leu Glu Leu Glu Ile Leu
1 5 10 15
Glu Ile Ile Lys Gln Lys Lys Ser Ile Thr Ile Thr Glu Ile Lys Glu
20 25 30
Ile Leu Ser Glu Arg Arg Lys Ser Glu Tyr Pro Leu Ser Leu Val Ser
35 40 45
Glu Tyr Ile Ser Arg Leu Glu Arg Lys Gly Tyr Val Lys Lys Ile Ala
50 55 60
Lys Gly Arg Lys Lys Phe Val Glu Ala Leu Ile
65 70 75
<210> 74
<211> 94
<212> PRT
<213> 激烈火球菌
<400> 74
Gly Ile Asp Val Val Ile Pro Glu Ile Lys His Asp Pro Ile Ala Arg
1 5 10 15
Asp Ile Val Lys Ile Leu Phe Asp Leu Arg Arg Ala Asn Val Ser Gln
20 25 30
Ile Ala Arg Glu Leu Lys Gly Arg Arg Gly Lys Ala Ser Arg Asn Thr
35 40 45
Val Arg Lys Lys Leu Lys Glu Leu Glu Lys Leu Gly Val Val Lys Glu
50 55 60
Val Pro Gly Glu Arg Gly Ser Val Tyr Thr Leu Ser Arg Glu Val Val
65 70 75 80
Lys Lys Trp Leu Asp Leu Ile Gly Ile Pro Ile Asn Leu Leu
85 90
<210> 75
<211> 67
<212> PRT
<213> 激烈火球菌
<400> 75
Met Thr Lys Arg Val Lys Val Ile Thr Asp Pro Glu Val Ile Lys Val
1 5 10 15
Met Leu Glu Asp Thr Arg Arg Lys Ile Leu Gln Leu Leu Arg Asn Arg
20 25 30
Glu Met Thr Ile Ser Gln Leu Ser Glu Ile Leu Gly Lys Met Pro Gln
35 40 45
Thr Ile Tyr His His Ile Glu Lys Leu Lys Glu Ala Gly Leu Val Glu
50 55 60
Val Lys Arg
65
<210> 76
<211> 100
<212> PRT
<213> 激烈火球菌
<400> 76
Met Glu Glu Ile Lys Glu Ile Met Lys Ser His Thr Leu Gly Asn Pro
1 5 10 15
Val Arg Leu Gly Ile Met Ile Tyr Leu Phe Pro Arg Arg Arg Ala Pro
20 25 30
Phe Ser His Ile Gln Lys Ala Leu Asp Leu Thr Pro Gly Asn Leu Asp
35 40 45
Ser His Ile Lys Val Leu Glu Lys His Gly Phe Val Arg Thr Tyr Lys
50 55 60
Val Ile Ala Asp Arg Pro Arg Thr Met Val Glu Ile Thr Asp Tyr Gly
65 70 75 80
Met Glu Glu Thr Arg Lys Phe Leu Ser His Leu Lys Thr Val Ile Asp
85 90 95
Ala Ile His Phe
100
<210> 77
<211> 99
<212> PRT
<213> 激烈火球菌
<400> 77
Met Gly Glu Glu Leu Asn Arg Leu Leu Asp Val Leu Gly Asn Glu Thr
1 5 10 15
Arg Arg Arg Ile Leu Phe Leu Leu Thr Lys Arg Pro Tyr Phe Val Ser
20 25 30
Glu Leu Ser Arg Glu Leu Gly Val Gly Gln Lys Ala Val Leu Glu His
35 40 45
Leu Arg Ile Leu Glu Glu Ala Gly Leu Ile Glu Ser Arg Val Glu Lys
50 55 60
Ile Pro Arg Gly Arg Pro Arg Lys Tyr Tyr Met Ile Lys Lys Gly Leu
65 70 75 80
Arg Leu Glu Ile Leu Leu Thr Pro Thr Leu Phe Gly Ser Glu Met Tyr
85 90 95
Glu Ala Lys
<210> 78
<211> 61
<212> PRT
<213> 激烈火球菌
<400> 78
Met Arg Arg Met Asp Lys Val Asp Leu Gln Leu Ile Lys Ile Leu Ser
1 5 10 15
Gln Asn Ser Arg Leu Thr Tyr Arg Glu Leu Ala Glu Met Leu Gly Thr
20 25 30
Thr Arg Gln Arg Val Ala Arg Lys Val Asp Lys Leu Lys Lys Leu Gly
35 40 45
Ile Ile Arg Lys Phe Thr Ile Ile Pro Asn Leu Glu Lys
50 55 60
<210> 79
<211> 82
<212> PRT
<213> 激烈火球菌
<400> 79
Gly Arg Lys Val Arg Thr Gln Gln Asn Glu Ile Leu Asn Leu Leu Asn
1 5 10 15
Glu Lys Glu Lys Ala Val Leu Arg Ala Ile Leu Glu His Gly Gly Glu
20 25 30
Ile Lys Gln Glu Asp Leu Pro Glu Leu Val Gly Tyr Ser Arg Pro Thr
35 40 45
Ile Ser Lys Val Ile Gln Glu Leu Glu Asn Lys Gly Leu Ile Lys Arg
50 55 60
Glu Lys Ser Gly Lys Thr Phe Val Val Lys Ile Glu Arg Lys Ile Lys
65 70 75 80
Leu Asp
<210> 80
<211> 59
<212> PRT
<213> 海栖热袍菌（Thermotoga maritima）
<400> 80
Lys Ser Leu Gln Arg Phe Leu Arg Arg Asn Thr Thr Ser Ile Lys His
1 5 10 15
Leu Ser Glu Ile Thr Gly Val Ala Arg Asn Arg Leu Ser Asp Ile Leu
20 25 30
Asn Gly Lys Thr Gln Lys Ile Arg Gly Glu Thr Leu Arg Lys Ile Ala
35 40 45
Lys Ala Phe Glu Lys Ser Asn Ile Leu Ser Phe
50 55
<210> 81
<211> 107
<212> PRT
<213> 栖热袍菌
<400> 81
Asp Val Ile Gln Arg Ile Lys Glu Lys Tyr Asp Glu Phe Thr Asn Ala
1 5 10 15
Glu Lys Lys Ile Ala Asp Thr Ile Leu Ser Asp Pro Lys Gly Ile Ile
20 25 30
Glu Ser Ser Ile Ser Asp Leu Ser Glu Lys Ala Gly Val Lys Ser Glu
35 40 45
Ala Ser Val Val Lys Phe Tyr Lys Lys Leu Gly Leu Asn Ser Phe Gln
50 55 60
Gln Phe Lys Val Leu Leu Ala Gln Ser Ile Ser Arg Ala Pro Leu Glu
65 70 75 80
Ile Val Tyr Glu Asp Val Ser Ser Glu Asp Asp Thr Lys Thr Ile Thr
85 90 95
Glu Lys Ile Phe Lys Ala Thr Val Arg Ala Ile
100 105
<210> 82
<211> 101
<212> PRT
<213> 海栖热袍菌
<400> 82
Lys Ile Arg Asp Lys Ile Leu Asn Val Tyr Thr Gln Phe Ser Pro Ala
1 5 10 15
Glu Arg Lys Val Ala Asp Tyr Val Leu Glu Arg Pro Asp Asp Val Ile
20 25 30
His Tyr Ser Ile Thr Glu Phe Ala Lys Ile Val Gly Val Ser Glu Thr
35 40 45
Thr Ile His Arg Met Ile Lys Lys Leu Asp Phe Glu Gly Tyr Gln Ala
50 55 60
Phe Lys Ile Ala Leu Ala Arg Glu Leu Ser Gly Leu Glu Glu Thr Ile
65 70 75 80
Glu Arg Arg Asp Phe Ile Asp Glu Glu Ile Asp Ile Leu Arg Arg Leu
85 90 95
Lys Asp Thr Leu Asp
100
<210> 83
<211> 60
<212> PRT
<213> 海栖热袍菌
<400> 83
Lys Arg Arg Pro Thr Ile Asn Asp Val Ala Lys Leu Ala Gly Val Ser
1 5 10 15
Ile Ser Thr Val Ser Arg Tyr Leu Lys Asp Pro Ser Gln Val Ser Glu
20 25 30
Lys Leu Gly Glu Arg Ile Arg Glu Ala Ile Lys Lys Leu Gly Tyr Lys
35 40 45
Pro Asn Lys Ile Ala Gln Gly Leu Arg Thr Gly Asp
50 55 60
<210> 84
<211> 58
<212> PRT
<213> 海栖热袍菌
<400> 84
Met Ala Ser Ile Lys Asp Val Ala Lys Leu Ala Gly Val Ser Ile Ala
1 5 10 15
Thr Val Ser Arg Val Ile Asn Gly Tyr Asn Asn Val Ser Glu Glu Thr
20 25 30
Arg Lys Lys Val Ile Asp Ala Ile Arg Lys Leu Asn Tyr His Pro Val
35 40 45
Tyr Ala Val Lys Gly Ala Val Leu Lys Arg
50 55
<210> 85
<211> 61
<212> PRT
<213> 海栖热袍菌
<400> 85
Met Lys Lys Lys Tyr Val Thr Ile Arg Asp Ile Ala Glu Lys Ala Gly
1 5 10 15
Val Ser Ile Asn Thr Val Ser Arg Ala Leu Asn Asn Lys Pro Asp Ile
20 25 30
Ser Glu Glu Thr Arg Arg Lys Ile Leu Lys Ile Ala Gln Glu Leu Gly
35 40 45
Tyr Val Lys Asn Ala Thr Ala Ser Ser Leu Arg Ser Lys
50 55 60
<210> 86
<211> 58
<212> PRT
<213> 栖热袍菌
<400> 86
Met Pro Thr Ile Glu Asp Val Ala Lys Leu Ala Gly Val Ser Ile Ala
1 5 10 15
Thr Val Ser Arg Val Ile Asn Gly Ser Gly Tyr Val Ser Glu Lys Thr
20 25 30
Arg Tyr Lys Val Trp Lys Ala Ile Glu Glu Leu Gly Tyr Lys Pro Glu
35 40 45
Ile Ser Ala Lys Leu Leu Ala Ser Lys Gly
50 55
<210> 87
<211> 61
<212> PRT
<213> 海栖热袍菌
<400> 87
Met Arg Ile Gly Glu Lys Leu Arg Lys Leu Arg Leu Ser Arg Gly Leu
1 5 10 15
Thr Gln Glu Glu Leu Ala Glu Arg Thr Asp Leu Ser Arg Ser Phe Ile
20 25 30
Ser Gln Leu Glu Ser Asp Lys Thr Ser Pro Ser Ile Asp Thr Leu Glu
35 40 45
Arg Ile Leu Glu Ala Leu Gly Thr Asp Leu Lys His Phe
50 55 60
<210> 88
<211> 67
<212> PRT
<213> 海栖热袍菌
<400> 88
Met His Met Lys Thr Val Arg Gln Glu Arg Leu Lys Ser Ile Val Arg
1 5 10 15
Ile Leu Glu Arg Ser Lys Glu Pro Val Ser Gly Ala Gln Leu Ala Glu
20 25 30
Glu Leu Ser Val Ser Arg Gln Val Ile Val Gln Asp Ile Ala Tyr Leu
35 40 45
Arg Ser Leu Gly Tyr Asn Ile Val Ala Thr Pro Arg Gly Tyr Val Leu
50 55 60
Ala Gly Gly
65
<210> 89
<211> 75
<212> PRT
<213> 海栖热袍菌
<400> 89
Met Asn Thr Leu Lys Lys Ala Phe Glu Ile Leu Asp Phe Ile Val Lys
1 5 10 15
Asn Pro Gly Asp Val Ser Val Ser Glu Ile Ala Glu Lys Phe Asn Met
20 25 30
Ser Val Ser Asn Ala Tyr Lys Tyr Met Val Val Leu Glu Glu Lys Gly
35 40 45
Phe Val Leu Arg Lys Lys Asp Lys Arg Tyr Val Pro Gly Tyr Lys Leu
50 55 60
Ile Glu Tyr Gly Ser Phe Val Leu Arg Arg Phe
65 70 75
<210> 90
<211> 81
<212> PRT
<213> 海栖热袍菌
<400> 90
Met Lys Ile Ser Lys Lys Arg Arg Gln Glu Leu Ile Arg Lys Ile Ile
1 5 10 15
His Glu Lys Lys Ile Ser Asn Gln Phe Gln Ile Val Glu Glu Leu Lys
20 25 30
Lys Tyr Gly Ile Lys Ala Val Gln Pro Thr Val Ala Arg Asp Leu Lys
35 40 45
Glu Ile Gly Ala Val Lys Ile Met Asp Glu Ser Gly Asn Tyr Val Tyr
50 55 60
Lys Leu Leu Asp Glu Thr Pro Val Ile Asp Pro Trp Lys Glu Leu Lys
65 70 75 80
Arg
<210> 91
<211> 82
<212> PRT
<213> 海栖热袍菌
<400> 91
Met His Lys Lys Leu Asn Pro Lys Ser Met Lys Arg Glu Asn Lys Lys
1 5 10 15
Met Val Leu Arg Tyr Leu Ile Glu Ser Gly Pro His Ser Arg Val Glu
20 25 30
Ile Ala Arg Lys Thr Gly Leu Ala Gln Ser Ala Ile Trp Arg Ile Ile
35 40 45
Glu Glu Leu Val Asn Glu Gly Leu Val Glu Glu Lys Gly Thr Ala Thr
50 55 60
Gly Arg Arg Arg Lys Ala Val Thr Tyr Gly Pro Thr Arg Ser Phe Ile
65 70 75 80
Thr Ser
<210> 92
<211> 79
<212> PRT
<213> 海栖热袍菌
<400> 92
Met Pro Ser Pro Leu Leu Arg Arg Glu Asn Lys Ile Lys Ile Leu Arg
1 5 10 15
Tyr Ile Leu Lys Asn Gly Lys Thr Thr Arg Asn Gln Leu Ala Ser Asn
20 25 30
Leu Asn Leu Ala His Ser Thr Leu Ser Tyr Ile Ile Asp Glu Leu Leu
35 40 45
Asp Glu Gly Phe Leu Val Phe Glu Glu Ile Lys Lys Lys Arg Gly Arg
50 55 60
Pro Tyr Gln Ile Leu Ser Val Asn Pro Glu Lys Phe Thr Ala Ile
65 70 75
<210> 93
<211> 79
<212> PRT
<213> 海栖热袍菌
<400> 93
Met Lys Glu Glu Arg Leu Lys Glu Ile Leu Asp Ile Val Asp Arg Asn
1 5 10 15
Gly Phe Ile Ser Met Lys Asp Leu Gln Glu Gln Leu Gly Val Ser Met
20 25 30
Ile Thr Val Arg Arg Asp Val Ala Glu Leu Val Lys Arg Asn Leu Val
35 40 45
Lys Lys Val His Gly Gly Ile Arg Lys Val Asn Tyr Phe Glu Lys Glu
50 55 60
Thr Asp Phe Met Lys Arg Leu Ser Ile Asn Arg Glu Ala Lys Glu
65 70 75
<210> 94
<211> 137
<212> PRT
<213> 海栖热袍菌
<400> 94
Met Phe Thr Met Arg Ser Glu Tyr Ala Leu Arg Leu Met Ile Val Met
1 5 10 15
Ala Lys Glu Tyr Gly Asn Tyr Leu Ser Met Thr Glu Ile Leu Glu Lys
20 25 30
Ala Lys Gln Ser Val Pro Arg Glu Phe Ala Glu Lys Ile Leu Tyr Thr
35 40 45
Leu Lys Lys Ala Gly Leu Val Lys Thr Arg Arg Gly Lys Ser Gly Gly
50 55 60
Tyr Met Leu Ser Arg Pro Pro Lys Glu Ile Lys Val Ser Glu Ile Val
65 70 75 80
Phe Leu Leu Asp Arg Lys Ser Lys Val Phe Phe Asp Met Pro Gly Cys
85 90 95
Pro Asp Glu Leu Asp Cys Val Ile Arg Ala Leu Trp Lys Arg Val Glu
100 105 110
Asn Glu Ile Glu Lys Ile Leu Ser Gly Val Thr Leu Glu Asp Leu Val
115 120 125
Arg Glu Gln Glu Glu Lys Met Lys Gln
130 135
<210> 95
<211> 95
<212> PRT
<213> Thermotoga naphthophila
<400> 95
Met Arg Asp Thr Lys Gly His Leu Lys Phe Leu Val Leu His Ile Ile
1 5 10 15
Ser Gln Gln Pro Ser His Gly Tyr Tyr Ile Met Lys Lys Ile Ser Gln
20 25 30
Ile Ile Gly Ala Glu Pro Pro Ser Pro Gly Ala Leu Tyr Pro Ile Leu
35 40 45
Ser Ser Leu Arg Lys Gln Lys Tyr Ile Glu Thr Tyr Asn Glu Gly Lys
50 55 60
Arg Lys Val Tyr Arg Leu Thr Asp Lys Gly Arg Lys Tyr Leu Glu Glu
65 70 75 80
His Lys Glu Glu Ile Lys Lys Ala Leu Asp Phe Ala Glu Arg Phe
85 90 95
<210> 96
<211> 118
<212> PRT
<213> 海栖热袍菌
<400> 96
Met Arg His Arg Gly Gly Arg Gly Phe Arg Gly Trp Trp Leu Ala Ser
1 5 10 15
Thr Ile Leu Leu Leu Val Ala Glu Lys Pro Ser His Gly Tyr Glu Leu
20 25 30
Ala Glu Arg Leu Ala Glu Phe Gly Ile Glu Ile Pro Gly Ile Gly His
35 40 45
Met Gly Asn Ile Tyr Arg Val Leu Ala Asp Leu Glu Glu Ser Gly Phe
50 55 60
Leu Ser Thr Glu Trp Asp Thr Thr Val Ser Pro Pro Arg Lys Ile Tyr
65 70 75 80
Arg Ile Thr Pro Gln Gly Lys Leu Tyr Leu Arg Glu Ile Leu Arg Ser
85 90 95
Leu Glu Asp Met Lys Arg Arg Ile Glu Thr Leu Glu Glu Arg Ile Lys
100 105 110
Arg Val Leu Gln Glu Glu
115
<210> 97
<211> 70
<212> PRT
<213> 海栖热袍菌
<400> 97
Met Leu Ser Lys Arg Asp Ala Ile Leu Lys Ala Ala Val Glu Val Phe
1 5 10 15
Gly Lys Lys Gly Tyr Asp Arg Ala Thr Thr Asp Glu Ile Ala Glu Lys
20 25 30
Ala Gly Val Ala Lys Gly Leu Ile Phe His Tyr Phe Lys Asn Lys Glu
35 40 45
Glu Leu Tyr Tyr Gln Ala Tyr Met Ser Val Thr Glu Lys Leu Gln Lys
50 55 60
Glu Phe Glu Asn Phe Leu
65 70
<210> 98
<211> 291
<212> PRT
<213> 人工序列
<220>
<223> 合成的构建体
<400> 98
Met Ser Lys Ser Trp Gly Lys Phe Ile Glu Glu Glu Glu Ala Glu Met
1 5 10 15
Ala Ser Arg Arg Asn Leu Met Ile Val Asp Gly Thr Asn Leu Gly Phe
20 25 30
Arg Phe Lys His Asn Asn Ser Lys Lys Pro Phe Ala Ser Ser Tyr Val
35 40 45
Ser Thr Ile Gln Ser Leu Ala Lys Ser Tyr Ser Ala Arg Thr Thr Ile
50 55 60
Val Leu Gly Asp Lys Gly Lys Ser Val Phe Arg Leu Glu His Leu Pro
65 70 75 80
Glu Tyr Lys Gly Asn Arg Asp Glu Lys Tyr Ala Gln Arg Thr Glu Glu
85 90 95
Glu Lys Ala Leu Asp Glu Gln Phe Phe Glu Tyr Leu Lys Asp Ala Phe
100 105 110
Glu Leu Cys Lys Thr Thr Phe Pro Thr Phe Thr Ile Arg Gly Val Glu
115 120 125
Ala Asp Asp Met Ala Ala Tyr Ile Val Lys Leu Ile Gly His Leu Tyr
130 135 140
Asp His Val Trp Leu Ile Ser Thr Asp Gly Asp Trp Asp Thr Leu Leu
145 150 155 160
Thr Asp Lys Val Ser Arg Phe Ser Phe Thr Thr Arg Arg Glu Tyr His
165 170 175
Leu Arg Asp Met Tyr Glu His His Asn Val Asp Asp Val Glu Gln Phe
180 185 190
Ile Ser Leu Lys Ala Ile Met Gly Asp Leu Gly Asp Asn Ile Arg Gly
195 200 205
Val Glu Gly Ile Gly Ala Lys Arg Gly Tyr Asn Ile Ile Arg Glu Phe
210 215 220
Gly Asn Val Leu Asp Ile Ile Asp Gln Leu Pro Leu Pro Gly Lys Gln
225 230 235 240
Lys Tyr Ile Gln Asn Leu Asn Ala Ser Glu Glu Leu Leu Phe Arg Asn
245 250 255
Leu Ile Leu Val Asp Leu Pro Thr Tyr Cys Val Asp Ala Ile Ala Ala
260 265 270
Val Gly Gln Asp Val Leu Asp Lys Phe Thr Lys Asp Ile Leu Glu Ile
275 280 285
Ala Glu Gln
290
<210> 99
<211> 53
<212> DNA
<213> 人工序列
<220>
<223> 合成的构建体
<220>
<221> 尚未归类的特征
<222> (17)..(37)
<223> n为a, c, g或t
<400> 99
tttctagctc taaaacnnnn nnnnnnnnnn nnnnnnncgg tgtttcgtcc ttt 53
<210> 100
<211> 148
<212> PRT
<213> 硫矿硫化叶菌（Sulfolobus solfataricus）
<400> 100
Met Glu Glu Lys Val Gly Asn Leu Lys Pro Asn Met Glu Ser Val Asn
1 5 10 15
Val Thr Val Arg Val Leu Glu Ala Ser Glu Ala Arg Gln Ile Gln Thr
20 25 30
Lys Asn Gly Val Arg Thr Ile Ser Glu Ala Ile Val Gly Asp Glu Thr
35 40 45
Gly Arg Val Lys Leu Thr Leu Trp Gly Lys His Ala Gly Ser Ile Lys
50 55 60
Glu Gly Gln Val Val Lys Ile Glu Asn Ala Trp Thr Thr Ala Phe Lys
65 70 75 80
Gly Gln Val Gln Leu Asn Ala Gly Ser Lys Thr Lys Ile Ala Glu Ala
85 90 95
Ser Glu Asp Gly Phe Pro Glu Ser Ser Gln Ile Pro Glu Asn Thr Pro
100 105 110
Thr Ala Pro Gln Gln Met Arg Gly Gly Gly Arg Gly Phe Arg Gly Gly
115 120 125
Gly Arg Arg Tyr Gly Arg Arg Gly Gly Arg Arg Gln Glu Asn Glu Glu
130 135 140
Gly Glu Glu Glu
145
<210> 101
<211> 673
<212> PRT
<213> 嗜热菌（Thermus thermophilus
）
<400> 101
Met Thr Leu Glu Glu Ala Arg Lys Arg Val Asn Glu Leu Arg Asp Leu
1 5 10 15
Ile Arg Tyr His Asn Tyr Arg Tyr Tyr Val Leu Ala Asp Pro Ile Ser
20 25 30
Asp Ala Glu Tyr Asp Arg Leu Leu Arg Glu Leu Lys Glu Leu Glu Glu
35 40 45
Arg Phe Pro Glu Leu Lys Ser Pro Asp Ser Pro Thr Leu Gln Val Gly
50 55 60
Ala Arg Pro Leu Glu Ala Thr Phe Arg Pro Val Arg His Pro Thr Arg
65 70 75 80
Met Tyr Ser Leu Asp Asn Ala Phe Asn Leu Asp Glu Leu Lys Ala Phe
85 90 95
Glu Glu Arg Ile Glu Arg Ala Leu Gly Arg Lys Gly Pro Phe Ala Tyr
100 105 110
Thr Val Glu His Lys Val Asp Gly Leu Ser Val Asn Leu Tyr Tyr Glu
115 120 125
Glu Gly Val Leu Val Tyr Gly Ala Thr Arg Gly Asp Gly Glu Val Gly
130 135 140
Glu Glu Val Thr Gln Asn Leu Leu Thr Ile Pro Thr Ile Pro Arg Arg
145 150 155 160
Leu Lys Gly Val Pro Glu Arg Leu Glu Val Arg Gly Glu Met Pro Ile
165 170 175
Glu Ala Phe Leu Arg Leu Asn Glu Glu Leu Glu Glu Arg Gly Glu Arg
180 185 190
Ile Phe Lys Asn Pro Arg Asn Ala Ala Ala Gly Ser Leu Arg Gln Lys
195 200 205
Asp Pro Arg Ile Thr Ala Lys Arg Gly Leu Arg Ala Thr Phe Tyr Ala
210 215 220
Leu Gly Leu Gly Leu Glu Glu Val Glu Arg Glu Gly Val Ala Thr Gln
225 230 235 240
Phe Ala Leu Leu His Trp Leu Lys Glu Lys Gly Phe Pro Val Glu His
245 250 255
Gly Tyr Ala Arg Ala Val Gly Ala Glu Gly Val Glu Ala Val Tyr Gln
260 265 270
Asp Trp Leu Lys Lys Arg Arg Ala Leu Pro Phe Glu Ala Asp Gly Val
275 280 285
Val Val Lys Leu Asp Glu Leu Ala Leu Trp Arg Glu Leu Gly Tyr Thr
290 295 300
Ala Arg Ala Pro Arg Phe Ala Ile Ala Tyr Lys Phe Pro Ala Glu Glu
305 310 315 320
Lys Glu Thr Arg Leu Leu Asp Val Val Phe Gln Val Gly Arg Thr Gly
325 330 335
Arg Val Thr Pro Val Gly Ile Leu Glu Pro Val Phe Leu Glu Gly Ser
340 345 350
Glu Val Ser Arg Val Thr Leu His Asn Glu Ser Tyr Ile Glu Glu Leu
355 360 365
Asp Ile Arg Ile Gly Asp Trp Val Leu Val His Lys Ala Gly Gly Val
370 375 380
Ile Pro Glu Val Leu Arg Val Leu Lys Glu Arg Arg Thr Gly Glu Glu
385 390 395 400
Arg Pro Ile Arg Trp Pro Glu Thr Cys Pro Glu Cys Gly His Arg Leu
405 410 415
Leu Lys Glu Gly Lys Val His Arg Cys Pro Asn Pro Leu Cys Pro Ala
420 425 430
Lys Arg Phe Glu Ala Ile Arg His Phe Ala Ser Arg Lys Ala Met Asp
435 440 445
Ile Gln Gly Leu Gly Glu Lys Leu Ile Glu Arg Leu Leu Glu Lys Gly
450 455 460
Leu Val Lys Asp Val Ala Asp Leu Tyr Arg Leu Arg Lys Glu Asp Leu
465 470 475 480
Val Gly Leu Glu Arg Met Gly Glu Lys Ser Ala Gln Asn Leu Leu Arg
485 490 495
Gln Ile Glu Glu Ser Lys Lys Arg Gly Leu Glu Arg Leu Leu Tyr Ala
500 505 510
Leu Gly Leu Pro Gly Val Gly Glu Val Leu Ala Arg Asn Leu Ala Ala
515 520 525
Arg Phe Gly Asn Met Asp Arg Leu Leu Glu Ala Ser Leu Glu Glu Leu
530 535 540
Leu Glu Val Glu Glu Val Gly Glu Leu Thr Ala Arg Ala Ile Leu Glu
545 550 555 560
Thr Leu Lys Asp Pro Ala Phe Arg Asp Leu Val Arg Arg Leu Lys Glu
565 570 575
Ala Gly Val Glu Met Glu Ala Lys Glu Lys Gly Gly Glu Ala Leu Lys
580 585 590
Gly Leu Thr Phe Val Ile Thr Gly Glu Leu Ser Arg Pro Arg Glu Glu
595 600 605
Val Lys Ala Leu Leu Arg Arg Leu Gly Ala Lys Val Thr Asp Ser Val
610 615 620
Ser Arg Lys Thr Ser Tyr Leu Val Val Gly Glu Asn Pro Gly Ser Lys
625 630 635 640
Leu Glu Lys Ala Arg Ala Leu Gly Val Pro Thr Leu Thr Glu Glu Glu
645 650 655
Leu Tyr Arg Leu Leu Glu Ala Arg Thr Gly Lys Lys Ala Glu Glu Leu
660 665 670
Val
<210> 102
<211> 775
<212> PRT
<213> 人工序列
<220>
<223> 合成的构建体
<400> 102
Met Ile Leu Asp Ala Asp Tyr Ile Thr Glu Asp Gly Lys Pro Ile Ile
1 5 10 15
Arg Ile Phe Lys Lys Glu Asn Gly Glu Phe Lys Val Glu Tyr Asp Arg
20 25 30
Asn Phe Arg Pro Tyr Ile Tyr Ala Leu Leu Lys Asp Asp Ser Ala Ile
35 40 45
Asp Asp Val Lys Lys Ile Thr Ala Glu Arg His Gly Lys Val Val Arg
50 55 60
Val Val Asp Val Glu Lys Val Lys Lys Lys Phe Leu Gly Arg Pro Ile
65 70 75 80
Glu Val Trp Lys Leu Tyr Phe Glu His Pro Gln Asp Val Pro Ala Ile
85 90 95
Arg Asp Lys Ile Arg Glu His Pro Ala Val Ile Asp Ile Phe Glu Tyr
100 105 110
Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Lys Gly Leu Ile Pro
115 120 125
Met Glu Gly Asn Glu Glu Leu Lys Leu Leu Ala Phe Asp Ile Glu Thr
130 135 140
Leu Tyr His Glu Gly Glu Glu Phe Gly Lys Gly Pro Ile Ile Met Ile
145 150 155 160
Ser Tyr Ala Asp Glu Glu Gly Ala Lys Val Ile Thr Trp Lys Lys Val
165 170 175
Asp Leu Pro Tyr Val Glu Val Val Ser Ser Glu Arg Glu Met Ile Lys
180 185 190
Arg Phe Leu Lys Val Ile Arg Glu Lys Asp Pro Asp Val Ile Ile Thr
195 200 205
Tyr Asn Gly Asp Asn Phe Asp Phe Pro Tyr Leu Leu Lys Arg Ala Glu
210 215 220
Lys Leu Gly Met Lys Leu Pro Leu Gly Arg Asp Gly Ser Glu Pro Lys
225 230 235 240
Met Gln Arg Leu Gly Asp Ser Leu Ala Val Glu Ile Lys Gly Arg Ile
245 250 255
His Phe Asp Leu Tyr Pro Val Ile Arg Arg Thr Ile Asn Leu Pro Thr
260 265 270
Tyr Thr Leu Glu Ala Val Tyr Glu Ala Ile Phe Gly Lys Pro Lys Glu
275 280 285
Lys Val Tyr Pro His Glu Ile Ala Glu Ala Trp Glu Thr Gly Lys Gly
290 295 300
Leu Glu Arg Val Ala Lys Tyr Ser Met Glu Asp Ala Lys Val Thr Tyr
305 310 315 320
Glu Leu Gly Arg Glu Phe Phe Pro Met Glu Ala Gln Leu Ala Arg Leu
325 330 335
Val Gly Gln Pro Leu Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu
340 345 350
Val Glu Trp Tyr Leu Leu Arg Lys Ala Tyr Glu Arg Asn Glu Leu Ala
355 360 365
Pro Asn Lys Pro Asp Glu Arg Glu Tyr Glu Arg Arg Leu Arg Glu Ser
370 375 380
Tyr Glu Gly Gly Tyr Val Lys Glu Pro Glu Lys Gly Leu Trp Glu Gly
385 390 395 400
Ile Val Ser Leu Asp Phe Arg Ser Leu Tyr Pro Ser Ile Ile Ile Thr
405 410 415
His Asn Val Ser Pro Asp Thr Leu Asn Lys Glu Gly Cys Gly Glu Tyr
420 425 430
Asp Val Ala Pro Glu Val Gly His Arg Phe Cys Lys Asp Phe Pro Gly
435 440 445
Phe Ile Pro Ser Leu Leu Gly Ser Leu Leu Asp Glu Arg Gln Lys Ile
450 455 460
Lys Arg Arg Met Lys Ala Ser Lys Asp Pro Ile Glu Arg Lys Leu Leu
465 470 475 480
Asp Tyr Arg Gln Arg Ala Ile Lys Ile Leu Ala Asn Ser Tyr Tyr Gly
485 490 495
Tyr Tyr Gly Tyr Ala Lys Ala Arg Trp Tyr Cys Lys Glu Cys Ala Glu
500 505 510
Ser Val Thr Ala Trp Gly Arg Glu Tyr Ile Glu Leu Val Arg Arg Glu
515 520 525
Leu Glu Glu Arg Gly Phe Lys Val Leu Tyr Ile Asp Thr Asp Gly Leu
530 535 540
Tyr Ala Thr Ile Pro Gly Glu Lys Asn Trp Glu Glu Ile Lys Arg Arg
545 550 555 560
Ala Leu Glu Phe Val Asn Tyr Ile Asn Ala Lys Leu Pro Gly Leu Leu
565 570 575
Glu Leu Glu Tyr Glu Gly Phe Tyr Thr Arg Gly Phe Phe Val Thr Lys
580 585 590
Lys Lys Tyr Ala Leu Ile Asp Glu Glu Gly Lys Ile Ile Thr Arg Gly
595 600 605
Leu Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu Thr Gln
610 615 620
Ala Lys Val Leu Glu Ala Ile Leu Lys His Gly Asn Val Glu Glu Ala
625 630 635 640
Val Lys Ile Val Lys Glu Val Thr Glu Lys Leu Ser Lys Tyr Glu Ile
645 650 655
Pro Pro Glu Lys Leu Val Ile Tyr Glu Gln Ile Thr Arg Pro Leu Asn
660 665 670
Glu Tyr Lys Ala Ile Gly Pro His Val Ala Val Ala Lys Arg Leu Ala
675 680 685
Ala Arg Gly Ile Lys Val Arg Pro Gly Met Val Ile Gly Tyr Val Val
690 695 700
Leu Arg Gly Asp Gly Pro Ile Ser Lys Arg Ala Ile Ala Ala Glu Glu
705 710 715 720
Phe Asp Pro Lys Lys His Lys Tyr Asp Ala Glu Tyr Tyr Ile Glu Asn
725 730 735
Gln Val Leu Pro Ala Val Leu Arg Ile Leu Glu Ala Phe Gly Tyr Arg
740 745 750
Lys Glu Asp Leu Arg Trp Gln Lys Thr Lys Gln Val Gly Leu Gly Ala
755 760 765
Trp Leu Lys Val Lys Lys Ser
770 775

Claims (68)

1.一种用于组装合成子的组合物，其包括：
(a)核酸外切酶，其为T5核酸外切酶、与SEQ ID NO:98相同的5’-3’核酸外切酶、或具有单链核酸内切酶活性的5’-3’核酸外切酶；
(b)链置换聚合酶，其包含选自SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:33-98或SEQ ID NO:102中任意者的氨基酸序列；和
(c)任选地，单链DNA结合蛋白质。
2.根据权利要求1所述的组合物，其还包括连接酶和/或单链结合结构域。
3.根据权利要求1-2中任一项所述的组合物，其还包括一组至少两种多核苷酸。
4.根据权利要求1-2中任一项所述的组合物，其中所述链置换聚合酶为非天然存在。
5.根据权利要求4所述的组合物，其中所述链置换聚合酶为突变体或融合蛋白。
6.根据权利要求1-2和5中任一项所述的组合物，其中所述链置换聚合酶对热稳定。
7.根据权利要求1-2和5中任一项所述的组合物，其中所述链置换聚合酶为B族聚合酶，并且所述组合物不包括非链置换聚合酶。
8.根据权利要求1-2和5中任一项所述的组合物，其中不包括9°N、Phusion、Vent或PfuDNA聚合酶。
9.根据权利要求1-2和5中任一项所述的组合物，其中所述链置换聚合酶为融合蛋白，其中聚合酶部分具有选自SEQ ID NO:1或SEQ ID NO:102、或SEQ ID NO:33至SEQ ID NO:55中任意者的氨基酸序列。
10.根据权利要求9所述的组合物，其中所述融合蛋白具有选自SEQ ID NO:1和SEQ IDNO:2的氨基酸序列。
11.根据权利要求1-2和5中任一项所述的组合物，其中所述链置换聚合酶具有SEQ IDNO:3的氨基酸序列。
12.根据权利要求1-2和5中任一项所述的组合物，其中所述单链DNA结合蛋白质为极端热稳定的单链DNA结合蛋白质(ET SSB)、大肠杆菌recA、T7基因2.5产物、噬菌体λRedB或Rac原噬菌体RecT。
13.根据权利要求1-2和5中任一项所述的组合物，其中所述5’-3’核酸外切酶具有单链核酸内切酶活性。
14.根据权利要求1-2和5中任一项所述的组合物，还包含非天然存在的缓冲剂。
15.根据权利要求1-2和5中任一项所述的组合物，其还包括浓度为至少7mM的钾盐。
16.根据权利要求1-2和5中任一项所述的组合物，其中所述组合物不包括拥挤剂和/或非链置换聚合酶。
17.根据权利要求1-2和5中任一项所述的组合物，其还包括一组多核苷酸，其中所述组中的至少一种多核苷酸的序列与所述组中另一多核苷酸重叠；并且其中所述多核苷酸选自由以下组成的组：(i)双链多核苷酸；(ii)单链寡核苷酸；(iii)至少一种双链多核苷酸和至少一种单链寡核苷酸；和(iv)除了在亚群成员之间变化的序列以外，否则彼此相同的多核苷酸亚群。
18.根据权利要求17所述的组合物，其中所述多核苷酸组的至少一种成员包含位于各末端处给定序列之间的用于与第二单链基因组多核苷酸杂交的随机序列。
19.根据权利要求18所述的组合物，其中所述随机序列为单链，并且其能够与用于将Cas蛋白质引导至用于基因编辑的靶标基因组核酸的靶标基因组序列杂交。
20.一种用于形成合成子的方法，其包括：
将根据权利要求1～16中任一项所述的组合物与具有能够在合适的反应条件下杂交的重叠序列的根据权利要求17～19中任一项所述的一组多核苷酸一起孵育；以及将至少一些所述多核苷酸与其他多核苷酸接合，以制备合成子。
21.根据权利要求20所述的方法，其中所述组中所述多核苷酸的全部或部分为双链。
22.根据权利要求21所述的方法，其中所述双链多核苷酸为重叠PCR产物；重叠限制片段或者由互补单链寡核苷酸组装的合成双链分子。
23.根据权利要求20所述的方法，其中所述组中所述多核苷酸的全部或部分为单链寡核苷酸。
24.根据权利要求20所述的方法，其中所述多核苷酸组包括至少一种双链多核苷酸和至少一种单链寡核苷酸。
25.根据权利要求20所述的方法，其中所述多核苷酸组包括除了在亚群成员之间变化的序列以外，否则彼此相同的多核苷酸亚群。
26.根据权利要求20-25中任一项所述的方法，其中所述多核苷酸的重叠序列的长度小于2千碱基。
27.根据权利要求20-25中任一项所述的方法，其中所述链置换聚合酶包括与SEQ IDNO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:33至SEQ ID NO:96、或SEQ ID NO:102中任意者相同的氨基酸序列。
28.根据权利要求20-25中任一项所述的方法，其中所述多核苷酸组的至少一种成员包含在给定序列末端之间的随机序列。
29.根据权利要求28所述的方法，其还包括筛选具有与基因组DNA杂交的活性的随机序列以及鉴定具有杂交活性的随机序列。
30.根据权利要求28所述的方法，其还包括通过转录具有杂交活性的随机序列以形成RNA，以及在Cas蛋白质的存在下使用用于基因编辑的所述RNA，从而进行基因编辑。
31.根据权利要求29所述的方法，其还包括通过转录具有杂交活性的随机序列以形成RNA，以及在Cas蛋白质的存在下使用用于基因编辑的所述RNA，从而进行基因编辑。
32.一种用于多核苷酸组装的试剂盒，其包括：
(a)T5核酸外切酶、与SEQ ID NO:98相同的5’-3’核酸外切酶、或具有单链核酸内切酶活性的5’-3’核酸外切酶；
(b)链置换聚合酶，其包含选自SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:33-98或SEQ ID NO:102中任意者的氨基酸序列：和
(c)任选地，单链DNA结合蛋白质。
33.根据权利要求32所述的试剂盒，其还包括连接酶。
34.根据权利要求32或33所述的试剂盒，其还包括dNTP。
35.根据权利要求32或33所述的试剂盒，其还包括缓冲剂。
36.根据权利要求34所述的试剂盒，其还包括缓冲剂。
37.根据权利要求32或33所述的试剂盒，其中(a)至(c)在一个或多个不同的容器中，比如在一个或多个不同的储存或反应容器中。
38.一种组合物，其包括聚合酶融合蛋白，其中所述聚合酶融合蛋白包括：
i.选自SEQ ID NO:2或SEQ ID NO:56至SEQ ID NO:98中任意者的氨基酸序列；和
ii.异源性聚合酶结构域。
39.根据权利要求38所述的组合物，其中所述聚合酶融合蛋白包括：
i.SEQ ID NO:2的氨基酸序列；和
ii.异源性聚合酶结构域。
40.一种组合物，其包括聚合酶融合蛋白，其中所述聚合酶融合蛋白包括：
(a)聚合酶结构域，其具有选自SEQ ID NO:1、SEQ ID NO:102、或SEQ ID NO:33至SEQID NO:55中任意者的氨基酸序列；和
(b)异源性DNA结合结构域。
41.根据权利要求40所述的组合物，其包括聚合酶融合蛋白，其中所述聚合酶融合蛋白包括：
(a)聚合酶结构域，其具有SEQ ID NO:1的氨基酸序列；和
(b)异源性DNA结合结构域。
42.根据权利要求40所述的组合物，其包括聚合酶融合蛋白，其中所述聚合酶融合蛋白包括：
(a)聚合酶结构域，其具有SEQ ID NO:102的氨基酸序列；和
(b)异源性DNA结合结构域。
43.根据权利要求38-42中任一项所述的组合物，其中所述聚合酶融合蛋白具有SEQ IDNO:3的氨基酸序列。
44.根据权利要求38-42中任一项所述的组合物，其中所述组合物还包括5’-3’核酸外切酶。
45.根据权利要求44所述的组合物，其中所述5’-3’核酸外切酶为T5核酸外切酶。
46.根据权利要求38-42和45中任一项所述的组合物，其还包括单链DNA结合蛋白质。
47.根据权利要求46所述的组合物，其中所述单链DNA结合蛋白质为极端热稳定的单链DNA结合蛋白质(ET SSB)、大肠杆菌recA、T7基因2.5产物、噬菌体λRedB或Rac原噬菌体RecT。
48.根据权利要求38-42、45和47中任一项所述的组合物，其还包括连接酶。
49.根据权利要求48所述的组合物，其中所述连接酶对热稳定。
50.根据权利要求38-42、45、47和49中任一项所述的组合物，其中所述组合物不包括拥挤剂和/或非链置换聚合酶。
51.根据权利要求38-42、45、47和49中任一项所述的组合物，其还包括dNTP。
52.根据权利要求38-42、45、47和49中任一项所述的组合物，其还包括浓度为至少7mM的钾盐。
53.根据权利要求38-42、45、47和49中任一项所述的组合物，其还包括一组多核苷酸，其中所述组中的至少一种多核苷酸的序列与所述组中另一多核苷酸重叠；并且其中所述多核苷酸选自：(i)双链多核苷酸；(ii)单链寡核苷酸；(iii)至少一种双链多核苷酸和至少一种单链寡核苷酸；和(iv)除了在亚群成员之间变化的序列以外，否则彼此相同的多核苷酸亚群。
54.一种用于制备合成子的方法，其包括：
将一组多核苷酸与特征在于根据权利要求1-19、38-53中任一项所述的包括聚合酶的组合物一起孵育，以及将所述多核苷酸接合以制备合成子，其中单个多核苷酸包含与其他多核苷酸中的序列重叠的序列，其中不同多核苷酸的重叠序列能够在合适的反应条件下交叉杂交，其中所述组合物还包括核酸外切酶和任选的连接酶和单链DNA结合蛋白质，
其中所述核酸外切酶是T5核酸外切酶、与SEQ ID NO:98相同的5’-3’核酸外切酶、或具有单链核酸内切酶活性的5’-3’核酸外切酶。
55.根据权利要求54所述的方法，其中所述组合物还包括连接酶。
56.根据权利要求54或55所述的方法，其中所述组合物还包括单链DNA结合蛋白质。
57.根据权利要求54或55所述的方法，其中所述组中的一种或多种多核苷酸为双链。
58.根据权利要求57所述的方法，其中所述双链多核苷酸为重叠PCR产物、重叠限制片段或者由单链寡核苷酸组装。
59.根据权利要求54或55中任一项所述的方法，其中所述多核苷酸为单链寡核苷酸。
60.根据权利要求54或55所述的方法，其中所述多核苷酸组包括至少一种双链多核苷酸和至少一种单链寡核苷酸。
61.根据权利要求54或55中任一项所述的方法，其中所述的重叠序列的长度小于2千碱基。
62.一种用于多核苷酸组装的试剂盒，其包括：
(a)根据权利要求38-42中任一项的聚合酶融合蛋白；
(b)核酸外切酶，所述核酸外切酶是T5核酸外切酶、与SEQ ID NO:98相同的5’-3’核酸外切酶、或具有单链核酸内切酶活性的5’-3’核酸外切酶；和
(c)任选地，单链DNA结合蛋白质。
63.根据权利要求62所述的试剂盒，其还包括连接酶。
64.根据权利要求62或63所述的试剂盒，其还包括dNTP。
65.根据权利要求62或63所述的试剂盒，其还包括缓冲剂。
66.根据权利要求64所述的试剂盒，其还包括缓冲剂。
67.根据权利要求62或63所述的试剂盒，其中(a)至(c)在相同的容器中。
68.根据权利要求62或63所述的试剂盒，其中(a)至(c)在不同的容器中。
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