具体实施方式

下面结合附图所描述的实施方式对本发明做以详细说明，其中所有附图中相同的数字表示相同的特征。虽然附图中显示了本发明的具体实施例，然而应当理解，可以以各种形式实现本发明而不应被这里阐述的实施例所限制。相反，提供这些实施例是为了能够更透彻地理解本发明，并且能够将本发明的范围完整的传达给本领域的技术人员。

需要说明的是，在说明书及权利要求当中使用了某些词汇来指称特定组件。本领域技术人员应可以理解，技术人员可能会用不同名词来称呼同一个组件。本说明书及权利要求并不以名词的差异作为区分组件的方式，而是以组件在功能上的差异作为区分的准则。如在通篇说明书及权利要求当中所提及的“包含”或“包括”为开放式用语，故应解释成“包含但不限定于”。说明书后续描述为实施本发明的较佳实施方式，然而所述描述乃以说明书的一般原则为目的，并非用以限定本发明的范围。本发明的保护范围当视所附权利要求所界定者为准。

本发明提供了一种连接肽，其为R0、R1、R2或R3，其中，R0的氨基酸序列为AAA(SEQID NO:1)，R1的氨基酸序列为EAAAK(SEQ ID NO:2)，R2的氨基酸序列为(EAAAK)₂(SEQ IDNO:3)，R3的氨基酸序列为(EAAAK)₃(SEQ ID NO:4)。

所述连接肽是用于连接两个或以上目标蛋白质或蛋白质结构域的一段氨基酸序列，通常连接肽的长度为3-50个氨基酸。连接肽可以用于连接不同体系的两个或多个蛋白质、多肽、抗体等等，本领域技术人员可以根据所需要连接的蛋白质、多肽、抗体的性质等来选择适当的连接肽。

本发明提供了一种DNA分子，其编码本文所述的连接肽。

对于编码所述的连接肽的基因没有特别的限制，只要通过翻译能表达出相应的连接肽序列即可。例如，当连接肽为R0时，其核苷酸序列可以为如SEQ ID NO:5所示的核苷酸序列，其核苷酸序列如下：

GCGGCCGCG(SEQ ID NO:5)

当连接肽为R1时，其核苷酸序列可以为如SEQ ID NO:6所示的核苷酸序列，其核苷酸序列如下：

GAAGCGGCCGCGAAA(SEQ ID NO:6)

当连接肽为R2时，其核苷酸序列可以如SEQ ID NO:7所示的核苷酸序列，其核苷酸序列如下：GAAGCCGCGGCGAAAGAAGCGGCCGCGAAA(SEQ ID NO:7)。

当连接肽为R3时，其核苷酸序列可以如SEQ ID NO:8所示的核苷酸序列，其核苷酸序列如下：GAAGCCGCGGCGAAAGAAGCGGCCGCGAAAGAAGCCGCGGCGAAA(SEQ ID NO:8)。

本发明提供了上述所述的连接肽在辅酶再生中的应用，例如可以将所述连接肽用于连接醇脱氢酶(ADH)和NAD(P)H氧化酶(NOX)，并将该获得的融合蛋白用于辅酶再生。

本发明提供了一种融合蛋白，其选自下述中的一种：醇脱氢酶(以下用ADH表示)-R0-NAD(P)H氧化酶(以下用NOX表示)、醇脱氢酶(ADH)-R1-NAD(P)H氧化酶(NOX)、醇脱氢酶(ADH)-R2-NAD(P)H氧化酶(NOX)、醇脱氢酶(ADH)-R3-NAD(P)H氧化酶(NOX)、NAD(P)H氧化酶(NOX)-R0-醇脱氢酶(ADH)、NAD(P)H氧化酶(NOX)-R1-醇脱氢酶(ADH)、NAD(P)H氧化酶(NOX)-R2-醇脱氢酶(ADH)和NAD(P)H氧化酶(NOX)-R3-醇脱氢酶(ADH)。

当融合蛋白为ADH-R0-NOX时，其氨基酸序列如SEQ ID NO:9所示；当融合蛋白为ADH-R1-NOX时，其氨基酸序列如SEQ ID NO:10所示；当融合蛋白为ADH-R2-NOX时，其氨基酸序列如SEQ ID NO:11所示；当融合蛋白为ADH-R3-NOX时，其氨基酸序列如SEQ ID NO:12所示；当融合蛋白为NOX-R0-ADH时，其氨基酸序列如SEQ ID NO:13所示；当融合蛋白为NOX-R1-ADH时，其氨基酸序列如SEQ ID NO:14所示；当融合蛋白为NOX-R2-ADH时，其氨基酸序列如SEQ ID NO:15所示；当融合蛋白为NOX-R3-ADH时，其氨基酸序列如SEQ ID NO:16所示。

其中，所述醇脱氢酶(ADH)是一类重要的氧化还原酶，催化醇和醛/酮的相互转化，它们广泛分布于各种生物当中，对维持生物的正常生理功能有着重要作用。

NAD(P)H氧化酶(NOX)可催化NAD(P)H的氧化，通过两电子传递，将分子氧还原至过氧化氢，或通过四电子传递，将分子氧还原至水。NOX广泛分布于各种具有不同亲缘关系的物种当中，如人类、脊椎动物、植物、细菌以及古细菌，它们属于嘧啶核苷酸二硫氧化还原酶类，通常需要FAD(黄素腺嘌呤二核苷酸)作为第二个辅酶，共价地与N端结构域中高度保守的GXT(H/S)AG基元相结合。

本发明提供了一种核酸分子，其编码本文的融合蛋白。

对于编码所述的融合蛋白的核酸分子，本发明没有特别的限制，只要通过翻译能表达出相应的融合蛋白的序列即可。

例如，当融合蛋白为ADH-R0-NOX时，其核苷酸序列如SEQ ID NO:17所示；当融合蛋白为ADH-R1-NOX时，其核苷酸序列如SEQ ID NO:18所示；当融合蛋白为ADH-R2-NOX时，其核苷酸序列如SEQ ID NO:19所示；当融合蛋白为ADH-R3-NOX时，其核苷酸序列如SEQ ID NO:20所示；当融合蛋白为NOX-R0-ADH时，其核苷酸序列如SEQ ID NO:21所示；当融合蛋白为NOX-R1-ADH时，其核苷酸序列如SEQ ID NO:22所示；当融合蛋白为NOX-R2-ADH时，其核苷酸序列如SEQ ID NO:23所示；当融合蛋白为NOX-R3-ADH时，其核苷酸序列如SEQ ID NO:24所示。

本发明提供了一种载体，其包含编码上述融合蛋白的核酸分子。

本发明对于载体不作任何限制，其可以根据需要选择的合适的载体，例如，所述载体可以为质粒。

本发明提供了一种基因工程菌，其包括上述所述的载体。

对于基因工程菌，本发明不作任何限制，本领域技术人员可以根据需要进行确定，例如，所述基因工程菌可以为大肠杆菌。

本发明提供了一种融合蛋白的构建和表达的方法，其是将连接肽与目的蛋白质进行融合，所构建的融合蛋白是将两个目的蛋白(如ADH和NOX)通过连接肽按照一定顺序连接而得到的融合蛋白。

融合蛋白的构建和表达方法是本领域技术人员所熟知的，例如可以采用如下的步骤：

(1)将编码目的蛋白(如ADH和NOX)的基因与编码连接肽的基因实现串联连接，构建融合基因，所述的连接的方法包括：设计适当的引物，通过聚合酶链式反应(PCR)方法得到融合基因；也可以采用人工合成的方法直接合成所需的融合基因。

(2)将(1)中所述的融合基因插入到表达载体的多克隆位点区域，从而得到含有融合基因的表达载体；

(3)将(2)中所得到的含有融合基因的表达载体转化合适的宿主细胞，获得转化的宿主细胞；

(4)培养转化的宿主细胞，通过合适的方式诱导融合蛋白的表达；

(5)从(4)所述的细胞中提取、分离融合蛋白。

本发明提供了上述所述的融合蛋白在辅酶再生中的应用。

所述辅酶例如可以为NAD⁺以及NADP⁺，优选的，本发明通过连接肽将NOX融合在ADH的C端或N端，能够提高醇脱氢酶与NAD(P)H氧化酶的活性以及催化特异性常数，通过连接肽的长度调节合理控制两个酶之间的距离从而提高了辅酶的循环效率。

本发明使用NAD⁺是启动ADH催化的手性二级醇的氧化，而ADH在消耗NAD⁺的同时，会生成NADH，利用NOX氧化NADH将NAD⁺再生出来，从而实现了辅酶再生，其反应机理示意图如图3所示。由于ADH也可利用NADP⁺，NOX也可氧化NADPH，因此将上述表述中的NAD⁺替换成NADP⁺，相应地将NADH替换成NADPH同样成立。

本发明提供了上述所述的融合蛋白在手性二级醇生产中的应用。

优选的，所述二级醇为脂肪二级醇或者芳基二级醇，优选的,所述芳基二级醇可以为1-苯乙醇。

本发明通过使用ADH来实现手性二级醇的生产，而ADH在消耗NAD⁺的同时，会生成NADH，此时利用NOX氧化NADH就可以将NAD⁺再生出来，从而在实现手性二级醇生产的同时，实现了辅酶再生，其反应机理示意图如图3所示。由于ADH也可利用NADP⁺，NOX也可氧化NADPH，因此将上述表述中的NAD⁺替换成NADP⁺，相应地将NADH替换成NADPH同样成立。

本发明通过使用上述所述的融合蛋白，能够实现手性二级醇例如芳基二级醇或者脂肪二级醇完全的拆分，即实现了单一酶对具有光学活性的二级醇例如芳基二级醇或脂肪二级醇的高效制备。

本发明提供了一种辅酶再生的方法，其包括使用上述所述的融合蛋白将NAD(P)H再生为NAD(P)⁺。

优选的，所述融合蛋白为ADH-R2-NOX。

本发明提供了一种手性二级醇的生产方法，其包括使用上述所述的融合蛋白对包含(R)-二级醇和(S)-二级醇的混合物进行手性拆分以获得(R)-二级醇。

所述包含(R)-二级醇和(S)-二级醇的混合物既可以是外消旋体(即n_{(R)-二级醇}:n_{(S)-二级醇}＝1:1，也可以不是外消旋体(即二者的摩尔比例不同)，均可实现手性拆分得到(R)-二级醇。

优选的，所述二级醇为脂肪二级醇或芳基二级醇，优选的，所述芳基二级醇为1-苯乙醇，所述融合蛋白为ADH-R2-NOX。

实施例

下面列举实施例对本发明进行进一步的说明。

下列实施例中若无特殊说明，均为常规方法。其中，PCR相关试剂来自北京全式金公司；限制性内切酶均购自NEB公司(New England BioLabs)；T4 DNA连接酶购自宝生物工程有限公司(Takara)；质粒提取试剂盒购自Omega公司；PCR产物纯化，酶切产物纯化试剂盒购自上海华舜公司；辅酶NAD⁺及NADH购自Roche公司；其他分析纯化学试剂购自Sigma公司。

实施例1醇脱氢酶与NAD(P)H氧化酶的融合蛋白构建

本实施例中涉及的醇脱氢酶与NAD(P)H氧化酶均来源于超嗜热菌Thermococcuskodakarensis KOD1，分别命名为TkADH和TkNOX，在Genebank中的登录号分别为BAD85034.1及BAD84493.1。

TkADH和TkNOX的异源表达及纯化分别参见文献(Wu Xi,et al.Thermostablealcohol dehydrogenase from Thermococcus kodakarensis KOD1for enantioselectivebioconversion of aromatic secondary alcohols.Applied and EnvironmentalMicrobiology,2013,79:2209-2217.)与(Wu Xi,et al.Application of a novelthermostable NAD(P)H oxidase from hyperthermophilic archaeon for regenerationof both NAD⁺and NADP⁺.Biotechnology and Bioengineering,2012,109:53-62.)。

本发明涉及到的融合蛋白的构建，是通过引物和酶切位点的巧妙设计，通过PCR扩增的方法，将编码部分连接肽的基因分别连接至两个目的蛋白基因的一端，再通过酶切与连接，使两个目的蛋白基因之间插入完整的编码目标连接肽的基因，引物组合1中的上游引物如SEQ ID NO:25所示，下游引物如SEQ ID NO:26所示；

引物组合2中的上游引物如SEQ ID NO:27所示，下游引物如SEQ ID NO:28所示；

引物组合3中的上游引物如SEQ ID NO:25所示，下游引物如SEQ ID NO:29所示；

引物组合4中的上游引物如SEQ ID NO:30所示，下游引物如SEQ ID NO:28所示；

引物组合5中的上游引物如SEQ ID NO:25所示，下游引物如SEQ ID NO:31所示；

引物组合6中的上游引物如SEQ ID NO:32所示，下游引物如SEQ ID NO:28所示；

引物组合7中的上游引物如SEQ ID NO:25所示，下游引物如SEQ ID NO:33所示；

引物组合8中的上游引物如SEQ ID NO:34所示，下游引物如SEQ ID NO:28所示；

引物组合9中的上游引物如SEQ ID NO:35所示，下游引物如SEQ ID NO:36所示；

引物组合10中的上游引物如SEQ ID NO:37所示，下游引物如SEQ ID NO:38所示；

引物组合11中的上游引物如SEQ ID NO:35所示，下游引物如SEQ ID NO:39所示；

引物组合12中的上游引物如SEQ ID NO:40所示，下游引物如SEQ ID NO:38所示；

引物组合13中的上游引物如SEQ ID NO:35所示，下游引物如SEQ ID NO:41所示；

引物组合14中的上游引物如SEQ ID NO:42所示，下游引物如SEQ ID NO:38所示；

引物组合15中的上游引物如SEQ ID NO:35所示，下游引物如SEQ ID NO:43所示；

引物组合16中的上游引物如SEQ ID NO:44所示，下游引物如SEQ ID NO:38所示；

并且，核苷酸序列(下划线表示酶切位点)如下表所示：

表1用于融合蛋白表达的质粒构建引物列表

具体构建步骤如下：

以含有TkADH编码基因的序列为模板，利用引物组合1进行PCR反应扩增出连接部分连接肽序列的tkadh基因片段，其中，PCR扩增程序如下：98℃预变性30s，然后进入以下30个循环：98℃变性10s，63℃退火30s，72℃延伸45s；循环完毕，72℃延伸10min。

以含有TkNOX编码基因的序列为模版，利用引物组合2进行PCR反应扩增出连接剩余部分连接肽的tknox基因片段，其中，PCR扩增程序如下：98℃预变性30s，然后进入以下30个循环：98℃变性10s，63℃退火45s，72℃延伸45s；循环完毕，72℃延伸10min。

将扩增出的tkadh片段用Nde I及Not I，tknox片段用Not I及EcoR I，质粒pET-21b(+)(购自Novagen)用Nde I及EcoR I进行双酶切后回收，用T4连接酶连接三个片段，构建出含有目的融合蛋白编码基因的质粒pET-21b/ADH-R0-NOX，并进行酶切及测序验证质粒的正确性。

通过类似的方法，分别使用上述引物组合3-16，成功构建出质粒pET-21b/ADH-R1-NOX、pET-21b/ADH-R2-NOX、pET-21b/ADH-R3-NOX、pET-21b/NOX-R0-ADH、pET-21b/NOX-R1-ADH、pET-21b/pET-NOX-R2-ADH和pET-21b/NOX-R3-ADH；

将所述质粒转化大肠杆菌Rosetta(DE3)菌株(购自Novagen)，构建出工程菌Rosetta(DE3)/pET21b/ADH-R0-NOX、Rosetta(DE3)/pET21b/ADH-R1-NOX、Rosetta(DE3)/pET21b/ADH-R2-NOX、Rosetta(DE3)/pET21b/ADH-R3-NOX、Rosetta(DE3)/pET21b/NOX-R0-ADH、Rosetta(DE3)/pET21b/NOX-R1-ADH、Rosetta(DE3)/pET21b/NOX-R2-ADH和Rosetta(DE3)/pET21b/NOX-R3-ADH。

利用下述方法培养基因工程菌：基因工程菌的液体培养基使用LB培养基(含100μg·ml^-1氨苄青霉素及34μg·ml^-1氯霉素)，摇床转速为170rpm。并利用下述方法诱导培养基因工程菌：从固体平板上接种单菌落至LB培养基中，37℃预培养12-16h，将培养物以1％(v/v)的接种量转接至新鲜的LB培养基中，37℃培养3h左右，待菌体浓度OD₆₀₀达到0.6时，加入终浓度为0.05mM的IPTG进行诱导，并转入20℃空气浴摇床继续培养20h。培养结束后，8,000×g，4℃离心10min，收集菌体并用50mM Tris-HCl缓冲液(pH 8.5)洗涤三次，最后利用含0.1mM FAD，100mM NaCl的50mM Tris-HCl缓冲液(pH 8.5)重悬菌体。低温超声破碎所得重悬液，8,000×g，4℃离心10min收集上清获取粗酶液。将融合蛋白的粗酶液经过85℃热处理10min后，15,000×g，4℃离心30min，此步骤可以去除变性沉淀的蛋白，获得上清并浓缩纯化的融合蛋白。经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定(图1和图2)，粗酶液中含有目的融合蛋白，并且经过热处理可以达到较好的纯化效果，进一步的鉴定表明，获得的融合蛋白ADH-R0-NOX、ADH-R1-NOX、ADH-R2-NOX、ADH-R3-NOX、NOX-R0-ADH、NOX-R1-ADH、NOX-R2-ADH和NOX-R3-ADH同时具有TkADH和TkNOX本身具有的活性。

实施例2融合蛋白的活性分析

通过实施例1中的方法，构建并表达融合蛋白ADH-R0-NOX、ADH-R1-NOX、ADH-R2-NOX、ADH-R3-NOX、NOX-R0-ADH、NOX-R1-ADH、NOX-R2-ADH和NOX-R3-ADH，通过热处理的方式对融合蛋白进行纯化。为了考察这八种具有不同连接肽长度及融合方向的融合蛋白的活性的本征变化，分别在70℃对其进行醇脱氢酶活性以及NAD(P)H氧化酶活性的测定。

(1)醇脱氢酶活性的测定

融合蛋白中醇脱氢酶(ADH)的活性检测方法如下：反应体系为750μL，包括50mM甘氨酸-氢氧化钠缓冲液(pH 9.0)，100mM(RS)-1-苯乙醇，1mM NAD⁺，加入适量酶液(当测定融合蛋白活性时，酶液为融合蛋白；当测定单酶TkADH活性时，酶液为TkADH)后迅速混合均匀以起始反应，温度为70℃，利用分光光度计(Ultrospec 3100Pro,Amersham Biosciences,USA)测定1min内340nm下吸光度的变化，NAD⁺在340nm下无吸收，而NADH在340nm下的吸光系数为ε₃₄₀＝6.22mM^-1cm^-1。1U ADH的活性定义为每分钟还原1μmol NAD⁺所需要的酶量。

(2)NAD(P)H氧化酶活性的测定

融合蛋白中NAD(P)H氧化酶(NOX)的活性检测方法如下：反应体系为750μL，包括100mM Tris-HCl缓冲液(pH 7.0)，0.1mM FAD(黄素腺嘌呤二核苷酸)，0.2mM NADH，加入适量的酶液(当测定融合蛋白活性时，酶液为融合蛋白；当测定单酶TkNOX活性时，酶液为TkNOX)后迅速混合均匀以起始反应，温度为70℃，利用分光光度计(Ultrospec 3100Pro,Amersham Biosciences,USA)测定1min内340nm下吸光度的变化，NAD⁺在340nm下无吸收，而NADH在340nm下的吸光系数为ε₃₄₀＝6.22mM^-1cm^-1。1U NOX的活性定义为每分钟氧化1μmolNADH所需要的酶量。

其中，蛋白浓度是以牛血清蛋白(BSA)为标准蛋白，根据Bradford的标准方法测定。为了计算融合蛋白的比活性，需分别对融合蛋白进行胶密度扫描，估算出目标蛋白在总蛋白中所占的比例。

分别将经纯化的单酶TkADH(来源于Thermococcus kodakarensis KOD1的醇脱氢酶，在E.coli中异源表达)与TkNOX(来源于Thermococcus kodakarensis KOD1的NAD(P)H氧化酶，在E.coli中异源表达)的比活性设为100％，八种融合蛋白的比活性的绝对值如表2所示。

表2单酶及融合蛋白的ADH及NOX比活性

注:括号内的数值为融合蛋白的比活性相对于对应单酶的比活性的比值。

由表2可以看出，对于NOX融合在ADH的C端的情况下，当连接肽为R2时，融合蛋白ADH-R2-NOX的ADH部分与NOX部分在70℃下的比活性相比单酶TkADH与TkNOX分别提高了38％与27％；与连接肽为R3的融合蛋白相比，ADH部分比活性提高了20％，而NOX部分比活性持平。

而对于NOX融合在ADH的N端的情况下，当连接肽为R2时，融合蛋白NOX-R2-ADH的ADH部分与NOX部分在70℃下的比活性相比单酶TkADH与TkNOX分别提高了1％与116％；与连接肽为R3的融合蛋白相比，ADH部分与NOX部分比活性分别提高了87％与2％。

综上可见，无论NOX融合在ADH的C端或N端，连接肽R2的应用，使得融合蛋白中的ADH部分与NOX部分在70℃下的比活性相较单酶都有不同程度的提高，且都高于相对应的连接肽为R3的融合蛋白，而连接肽为R3的融合蛋白相对略差。

实施例3融合蛋白的动力学参数测定

为了尽可能减小底物在高温下挥发对实验造成的误差，以更好地比较融合蛋白与其等活性单酶混合体系的辅酶再生效率的差异，将反应温度设为40℃。首先测定了融合蛋白在40℃下分别对于辅酶NAD⁺及NADH的动力学参数。醇脱氢酶及NAD(P)H氧化酶的活性测定采用的缓冲液为辅酶再生反应将要采用的缓冲液——50mM Gly-NaOH缓冲液(pH 9.0)。

具体方法如下：对于ADH部分，缓冲液中加入100mM的(RS)-1-苯乙醇，在0-1.0mM的范围内改变NAD⁺的浓度，测定NAD⁺还原的初始速率；对于NOX部分，在0-0.25mM范围内改变NADH的浓度，测定NADH氧化的初始速率。将八种融合蛋白与TkADH在不同NAD⁺浓度下的初始反应速率以及八种融合蛋白与TkNOX在不同NADH浓度下的初始反应速率分别拟合米氏方程曲线，计算蛋白的动力学参数，结果如表3所示。

表3单酶及融合蛋白对辅酶的动力学参数

注:括号内的数值为融合蛋白的参数值相对于单酶的对应参数值的比值。

从表3可以看出，当连接肽为R3(氨基酸序列为(EAAAK)₃)时，对融合蛋白ADH-R3-NOX及NOX-R3-ADH的动力学分析表明，蛋白融合使ADH及NOX对辅酶的亲和性降低，且催化特异性常数k_cat/K_m均有所下降。

将连接肽替换为R2(氨基酸序列为(EAAAK)₂)后，对于NOX融合在ADH的C端的情况下，即ADH-R2-NOX，ADH部分对NAD⁺的亲和性以及NOX部分对NADH的亲和性相较连接肽为R3均有所提高。ADH-R2-NOX中的ADH部分与NOX部分的催化特异性常数k_cat/K_m值均较连接肽为R3有不同程度的提高(比例分别为46％与28％)，特别地，该值较单酶也有所提高(比例分别为23％与8％)。

将连接肽替换为R2(氨基酸序列为(EAAAK)₂)后，对于NOX融合在ADH的N端的情况下，NOX-R2-ADH中的ADH部分与NOX部分的催化特异性常数k_cat/K_m值均较连接肽为R3也有不同程度的提高(比例分别为60％与35％)。

综上，连接肽R2的应用对于融合蛋白催化特性的改善具有明显的成效。

实施例4融合蛋白与其等活性单酶混合体系的辅酶再生

辅酶再生体系总体积为0.18ml，缓冲液为50mM Gly-NaOH缓冲液(pH9.0)，其中包含20mM(RS)-1-苯乙醇，100mM NaCl，0.05mM NAD⁺，0.17mM FAD，以及适量的酶液(酶液为融合蛋白或者为融合蛋白的等活性单酶混合体系)。辅酶再生反应的示意图如图3所示，反应温度为40℃，1小时后，终止反应，利用乙酸乙酯萃取反应液并进行气相色谱(GC-2010plus,Shimazu,Kyoto,Japan)的分析，气相色谱柱为CP-Chirasil-DEX CB(Varian,USA)，载气为氦气，检测器为氢焰离子检测器(FID)，检测器温度为250℃，进样口SPL温度为220℃，柱温箱温度为100℃，(R)-1-苯乙醇与(S)-1-苯乙醇的保留时间分别为4.94min与5.60min。

根据实施例3的结果，当NOX融合在ADH的C端时，连接肽R2的应用对融合蛋白的动力学性质改善较为显著，因此辅酶再生反应采用融合蛋白ADH-R2-NOX体系，并使用ADH-R3-NOX体系，利用上述方法，将融合蛋白ADH-R2-NOX、ADH-R3-NOX与其各自等活性的TkADH及TkNOX的单酶混合体系进行辅酶再生效率的比较，并计算反应体系的总转化数(TotalTurnover Number,TTN)，即每摩尔起始加入的辅酶转化的1-苯乙醇的摩尔数，所得结果见表4。其中，表4中的所谓等活性单酶混合体系是指在固定每种融合蛋白量(ADH-R2-NOX或ADH-R3-NOX)的条件下，调节单酶TkADH及TkNOX的蛋白量，使之活性分别与各自ADH-R2-NOX或ADH-R3-NOX融合蛋白中ADH及NOX的活性相等，以ADH-R2-NOX为例，在确定了该融合蛋白中ADH和NOX各自的活性后，按照该活性添加与融合蛋白ADH-R2-NOX中ADH部分等活性的单酶，以及添加与融合蛋白ADH-R2-NOX中NOX部分等活性的单酶，以组成表4中的等活性单酶混合体系，同理ADH-R3-NOX也是同样的情况。

表4融合蛋白与其等活性单酶混合体系的辅酶再生效率比较

a:比例＝融合蛋白体系的TTN/各自对应的等活性单酶混合体系的TTN

由表4的结果可以看出，ADH-R2-NOX相比等活性单酶混合体系，辅酶再生效率提高了53％，这可能得益于合适的连接肽使得蛋白的活性位点接近，局部辅酶浓度提高，从而使两种蛋白具有更高的表观活性所致。而连接肽为R3时，ADH-R3-NOX相比等活性单酶混合体系辅酶再生效率略有下降，表明连接肽的长度控制对两酶之间辅酶循环效率起到了重要作用。

实施例5融合蛋白应用于手性芳基二级醇的生产

由于ADH-R2-NOX相较等活性单酶混合体系具有更高的氧化型辅酶循环效率，被进一步应用于手性醇的生产。反应体系总体积为1.2ml，缓冲液为50mM Gly-NaOH缓冲液(pH9.0)，其中包含20mM(RS)-1-苯乙醇，100mM NaCl，0.2mM NAD⁺，0.17mM FAD，以及适量的融合蛋白酶液，反应示意图如图3所示。反应温度为40℃，总反应时间为8h。反应在15ml的带盖试管中进行，样品采取三个平行，置于空气浴摇床中，转速为150rpm。经过合适的反应时间后，从试管中迅速取出100μl反应液置于冰上，利用乙酸乙酯萃取反应液并进行气相色谱的分析，分析条件与实施例4中的分析条件相同。反应过程中(S)-1-苯乙醇与(R)-1-苯乙醇的转化率随时间变化的曲线如图4所示。

从图4可以看出，利用ADH-R2-NOX融合蛋白体系，经过8小时的反应，(S)-1-苯乙醇的转化率为100％，而(R)-1-苯乙醇没有消耗，即20mM的(RS)-1-苯乙醇实现了完全的拆分，生成了对映体过量值(enantiomeric excess,ee)为>99.8％的(R)-1-苯乙醇，即实现了单一酶对具有光学活性的芳基二级醇的高效制备。

综上所述，本发明所提供的连接肽，可用于融合蛋白的分子改造。将醇脱氢酶与NAD(P)H氧化酶通过该连接肽融合，实现了醇脱氢酶与NAD(P)H氧化酶的活性提高，改善两酶的催化特性，通过连接肽的长度调节合理控制两酶之间的距离，实现辅酶循环效率的提高。进一步利用该融合蛋白催化包含(R)-二级醇和(S)-二级醇的混合物的动力学拆分，制备出光学纯的手性醇。

以上所述，仅是本发明的较佳实施例而已，并非是对本发明作其它形式的限制，任何熟悉本专业的技术人员可能利用上述揭示的技术内容加以变更或改型为等同变化的等效实施例。但是凡是未脱离本发明技术方案内容，依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与改型，仍属于本发明技术方案的保护范围。

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65 70 75 80

Arg Asp Asp Ile Phe Ile Ile Ser Lys Val Trp Pro Thr His Phe Gly

85 90 95

Tyr Glu Glu Ala Lys Arg Ala Ala Arg Ala Ser Ala Lys Arg Leu Gly

100 105 110

Thr Tyr Ile Asp Leu Tyr Leu Leu His Trp Pro Gly Asp Ser Trp Glu

115 120 125

Lys Ile Lys Glu Thr Leu His Ala Leu Glu Glu Leu Val Asp Glu Gly

130 135 140

Leu Ile Arg Tyr Ile Gly Val Ser Asn Phe Asp Leu Glu Leu Leu Lys

145 150 155 160

Arg Ser Gln Glu Ala Met Lys Lys Tyr Glu Ile Val Ala Asn Glu Val

165 170 175

Lys Tyr Ser Leu Lys Asp Arg Trp Pro Glu Thr Thr Gly Leu Leu Asp

180 185 190

Tyr Met Lys Arg Glu Gly Ile Ala Leu Ile Ala Tyr Thr Pro Leu Glu

195 200 205

Lys Gly Thr Leu Ala Arg Asn Glu Cys Leu Ala Glu Ile Gly Lys Lys

210 215 220

Tyr Gly Lys Thr Ala Ala Gln Val Ala Leu Asn Tyr Leu Ile Trp Glu

225 230 235 240

Glu Asn Val Ile Ala Ile Pro Lys Ala Gly Asn Lys Ala His Leu Glu

245 250 255

Glu Asn Phe Gly Ala Met Gly Trp Arg Leu Ser Lys Glu Asp Arg Glu

260 265 270

Asn Ala Arg Gly Cys Val Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys

275 280 285

Met Lys Ile Val Val Val Gly Ser Gly Thr Ala Gly Ser Asn Phe Ala

290 295 300

Leu Phe Met Arg Lys Leu Asp Arg Lys Ala Glu Ile Thr Val Ile Gly

305 310 315 320

Lys Glu Pro Thr Met Gln Tyr Ser Pro Cys Ala Leu Pro His Val Val

325 330 335

Ser Gly Thr Ile Glu Lys Pro Glu Asp Ile Ile Val Phe Pro Asn Glu

340 345 350

Phe Tyr Glu Lys Gln Lys Ile Asn Leu Met Leu Asn Thr Glu Ala Lys

355 360 365

Ala Ile Asp Arg Glu Arg Lys Val Val Val Thr Asp Lys Gly Glu Val

370 375 380

Pro Tyr Asp Lys Leu Val Leu Ala Val Gly Ser Lys Ala Phe Ile Pro

385 390 395 400

Pro Ile Lys Gly Val Glu Asn Glu Gly Val Phe Thr Leu Lys Ser Leu

405 410 415

Asp Asp Val Arg Arg Ile Lys Ala Tyr Ile Ala Glu Arg Lys Pro Lys

420 425 430

Lys Ala Val Val Ile Gly Ala Gly Leu Ile Gly Leu Glu Gly Ala Glu

435 440 445

Ala Phe Ala Lys Leu Gly Met Glu Val Leu Ile Val Glu Leu Met Asp

450 455 460

Arg Leu Met Pro Thr Met Leu Asp Lys Asp Thr Ala Lys Leu Val Gln

465 470 475 480

Ala Glu Met Glu Lys Tyr Gly Val Ser Phe Arg Phe Gly Val Gly Val

485 490 495

Ser Glu Ile Ile Gly Ser Pro Val Arg Ala Val Lys Ile Gly Asp Glu

500 505 510

Glu Val Pro Ala Asp Leu Val Leu Val Ala Thr Gly Val Arg Ala Asn

515 520 525

Thr Asp Leu Ala Lys Gln Ala Gly Leu Glu Val Asn Arg Gly Ile Val

530 535 540

Val Asn Glu His Leu Gln Thr Ser Asp Pro Glu Val Tyr Ala Ile Gly

545 550 555 560

Asp Cys Ala Glu Val Ile Asp Ala Val Thr Gly Lys Arg Thr Leu Ser

565 570 575

Gln Leu Gly Thr Ser Ala Val Arg Met Ala Lys Val Ala Ala Glu His

580 585 590

Ile Ala Gly Lys Asp Val Ser Phe Arg Pro Val Phe Asn Thr Ala Ile

595 600 605

Thr Glu Leu Phe Gly Leu Glu Ile Gly Thr Phe Gly Ile Thr Glu Glu

610 615 620

Arg Ala Lys Lys Glu Asp Ile Glu Val Ala Val Gly Lys Phe Lys Gly

625 630 635 640

Ser Thr Lys Pro Glu Tyr Tyr Pro Gly Gly Lys Pro Ile Thr Val Lys

645 650 655

Leu Ile Phe Arg Lys Ser Asp Arg Lys Leu Ile Gly Gly Gln Ile Val

660 665 670

Gly Gly Glu Arg Val Trp Gly Arg Ile Met Thr Leu Ser Ala Leu Ala

675 680 685

Gln Lys Gly Ala Thr Val Glu Asp Val Ala Tyr Leu Glu Thr Ala Tyr

690 695 700

Ala Pro Pro Ile Ser Pro Thr Ile Asp Pro Ile Thr Val Ala Ala Glu

705 710 715 720

Met Ala Gln Arg Lys Leu Arg

725

<210> 12

<211> 732

<212> PRT

<213> Artificial Sequence

<220>

<223> Artificial

<400> 12

Met Lys Lys Val Arg Ile Phe Asn Asp Leu Lys Trp Ile Gly Asp Asp

1 5 10 15

Lys Val Thr Ala Ile Gly Met Gly Thr Trp Gly Ile Gly Gly Tyr Glu

20 25 30

Ser Pro Asp Tyr Ser Lys Asp Asn Glu Ser Val Glu Val Leu Arg His

35 40 45

Gly Leu Glu Leu Gly Ile Asn Leu Ile Asp Thr Ala Glu Phe Tyr Gly

50 55 60

Ala Gly His Ser Glu Glu Leu Val Gly Glu Ala Ile Lys Glu Phe Glu

65 70 75 80

Arg Asp Asp Ile Phe Ile Ile Ser Lys Val Trp Pro Thr His Phe Gly

85 90 95

Tyr Glu Glu Ala Lys Arg Ala Ala Arg Ala Ser Ala Lys Arg Leu Gly

100 105 110

Thr Tyr Ile Asp Leu Tyr Leu Leu His Trp Pro Gly Asp Ser Trp Glu

115 120 125

Lys Ile Lys Glu Thr Leu His Ala Leu Glu Glu Leu Val Asp Glu Gly

130 135 140

Leu Ile Arg Tyr Ile Gly Val Ser Asn Phe Asp Leu Glu Leu Leu Lys

145 150 155 160

Arg Ser Gln Glu Ala Met Lys Lys Tyr Glu Ile Val Ala Asn Glu Val

165 170 175

Lys Tyr Ser Leu Lys Asp Arg Trp Pro Glu Thr Thr Gly Leu Leu Asp

180 185 190

Tyr Met Lys Arg Glu Gly Ile Ala Leu Ile Ala Tyr Thr Pro Leu Glu

195 200 205

Lys Gly Thr Leu Ala Arg Asn Glu Cys Leu Ala Glu Ile Gly Lys Lys

210 215 220

Tyr Gly Lys Thr Ala Ala Gln Val Ala Leu Asn Tyr Leu Ile Trp Glu

225 230 235 240

Glu Asn Val Ile Ala Ile Pro Lys Ala Gly Asn Lys Ala His Leu Glu

245 250 255

Glu Asn Phe Gly Ala Met Gly Trp Arg Leu Ser Lys Glu Asp Arg Glu

260 265 270

Asn Ala Arg Gly Cys Val Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys

275 280 285

Glu Ala Ala Ala Lys Met Lys Ile Val Val Val Gly Ser Gly Thr Ala

290 295 300

Gly Ser Asn Phe Ala Leu Phe Met Arg Lys Leu Asp Arg Lys Ala Glu

305 310 315 320

Ile Thr Val Ile Gly Lys Glu Pro Thr Met Gln Tyr Ser Pro Cys Ala

325 330 335

Leu Pro His Val Val Ser Gly Thr Ile Glu Lys Pro Glu Asp Ile Ile

340 345 350

Val Phe Pro Asn Glu Phe Tyr Glu Lys Gln Lys Ile Asn Leu Met Leu

355 360 365

Asn Thr Glu Ala Lys Ala Ile Asp Arg Glu Arg Lys Val Val Val Thr

370 375 380

Asp Lys Gly Glu Val Pro Tyr Asp Lys Leu Val Leu Ala Val Gly Ser

385 390 395 400

Lys Ala Phe Ile Pro Pro Ile Lys Gly Val Glu Asn Glu Gly Val Phe

405 410 415

Thr Leu Lys Ser Leu Asp Asp Val Arg Arg Ile Lys Ala Tyr Ile Ala

420 425 430

Glu Arg Lys Pro Lys Lys Ala Val Val Ile Gly Ala Gly Leu Ile Gly

435 440 445

Leu Glu Gly Ala Glu Ala Phe Ala Lys Leu Gly Met Glu Val Leu Ile

450 455 460

Val Glu Leu Met Asp Arg Leu Met Pro Thr Met Leu Asp Lys Asp Thr

465 470 475 480

Ala Lys Leu Val Gln Ala Glu Met Glu Lys Tyr Gly Val Ser Phe Arg

485 490 495

Phe Gly Val Gly Val Ser Glu Ile Ile Gly Ser Pro Val Arg Ala Val

500 505 510

Lys Ile Gly Asp Glu Glu Val Pro Ala Asp Leu Val Leu Val Ala Thr

515 520 525

Gly Val Arg Ala Asn Thr Asp Leu Ala Lys Gln Ala Gly Leu Glu Val

530 535 540

Asn Arg Gly Ile Val Val Asn Glu His Leu Gln Thr Ser Asp Pro Glu

545 550 555 560

Val Tyr Ala Ile Gly Asp Cys Ala Glu Val Ile Asp Ala Val Thr Gly

565 570 575

Lys Arg Thr Leu Ser Gln Leu Gly Thr Ser Ala Val Arg Met Ala Lys

580 585 590

Val Ala Ala Glu His Ile Ala Gly Lys Asp Val Ser Phe Arg Pro Val

595 600 605

Phe Asn Thr Ala Ile Thr Glu Leu Phe Gly Leu Glu Ile Gly Thr Phe

610 615 620

Gly Ile Thr Glu Glu Arg Ala Lys Lys Glu Asp Ile Glu Val Ala Val

625 630 635 640

Gly Lys Phe Lys Gly Ser Thr Lys Pro Glu Tyr Tyr Pro Gly Gly Lys

645 650 655

Pro Ile Thr Val Lys Leu Ile Phe Arg Lys Ser Asp Arg Lys Leu Ile

660 665 670

Gly Gly Gln Ile Val Gly Gly Glu Arg Val Trp Gly Arg Ile Met Thr

675 680 685

Leu Ser Ala Leu Ala Gln Lys Gly Ala Thr Val Glu Asp Val Ala Tyr

690 695 700

Leu Glu Thr Ala Tyr Ala Pro Pro Ile Ser Pro Thr Ile Asp Pro Ile

705 710 715 720

Thr Val Ala Ala Glu Met Ala Gln Arg Lys Leu Arg

725 730

<210> 13

<211> 720

<212> PRT

<213> Artificial Sequence

<220>

<223> Artificial

<400> 13

Met Lys Ile Val Val Val Gly Ser Gly Thr Ala Gly Ser Asn Phe Ala

1 5 10 15

Leu Phe Met Arg Lys Leu Asp Arg Lys Ala Glu Ile Thr Val Ile Gly

20 25 30

Lys Glu Pro Thr Met Gln Tyr Ser Pro Cys Ala Leu Pro His Val Val

35 40 45

Ser Gly Thr Ile Glu Lys Pro Glu Asp Ile Ile Val Phe Pro Asn Glu

50 55 60

Phe Tyr Glu Lys Gln Lys Ile Asn Leu Met Leu Asn Thr Glu Ala Lys

65 70 75 80

Ala Ile Asp Arg Glu Arg Lys Val Val Val Thr Asp Lys Gly Glu Val

85 90 95

Pro Tyr Asp Lys Leu Val Leu Ala Val Gly Ser Lys Ala Phe Ile Pro

100 105 110

Pro Ile Lys Gly Val Glu Asn Glu Gly Val Phe Thr Leu Lys Ser Leu

115 120 125

Asp Asp Val Arg Arg Ile Lys Ala Tyr Ile Ala Glu Arg Lys Pro Lys

130 135 140

Lys Ala Val Val Ile Gly Ala Gly Leu Ile Gly Leu Glu Gly Ala Glu

145 150 155 160

Ala Phe Ala Lys Leu Gly Met Glu Val Leu Ile Val Glu Leu Met Asp

165 170 175

Arg Leu Met Pro Thr Met Leu Asp Lys Asp Thr Ala Lys Leu Val Gln

180 185 190

Ala Glu Met Glu Lys Tyr Gly Val Ser Phe Arg Phe Gly Val Gly Val

195 200 205

Ser Glu Ile Ile Gly Ser Pro Val Arg Ala Val Lys Ile Gly Asp Glu

210 215 220

Glu Val Pro Ala Asp Leu Val Leu Val Ala Thr Gly Val Arg Ala Asn

225 230 235 240

Thr Asp Leu Ala Lys Gln Ala Gly Leu Glu Val Asn Arg Gly Ile Val

245 250 255

Val Asn Glu His Leu Gln Thr Ser Asp Pro Glu Val Tyr Ala Ile Gly

260 265 270

Asp Cys Ala Glu Val Ile Asp Ala Val Thr Gly Lys Arg Thr Leu Ser

275 280 285

Gln Leu Gly Thr Ser Ala Val Arg Met Ala Lys Val Ala Ala Glu His

290 295 300

Ile Ala Gly Lys Asp Val Ser Phe Arg Pro Val Phe Asn Thr Ala Ile

305 310 315 320

Thr Glu Leu Phe Gly Leu Glu Ile Gly Thr Phe Gly Ile Thr Glu Glu

325 330 335

Arg Ala Lys Lys Glu Asp Ile Glu Val Ala Val Gly Lys Phe Lys Gly

340 345 350

Ser Thr Lys Pro Glu Tyr Tyr Pro Gly Gly Lys Pro Ile Thr Val Lys

355 360 365

Leu Ile Phe Arg Lys Ser Asp Arg Lys Leu Ile Gly Gly Gln Ile Val

370 375 380

Gly Gly Glu Arg Val Trp Gly Arg Ile Met Thr Leu Ser Ala Leu Ala

385 390 395 400

Gln Lys Gly Ala Thr Val Glu Asp Val Ala Tyr Leu Glu Thr Ala Tyr

405 410 415

Ala Pro Pro Ile Ser Pro Thr Ile Asp Pro Ile Thr Val Ala Ala Glu

420 425 430

Met Ala Gln Arg Lys Leu Arg Ala Ala Ala Met Lys Lys Val Arg Ile

435 440 445

Phe Asn Asp Leu Lys Trp Ile Gly Asp Asp Lys Val Thr Ala Ile Gly

450 455 460

Met Gly Thr Trp Gly Ile Gly Gly Tyr Glu Ser Pro Asp Tyr Ser Lys

465 470 475 480

Asp Asn Glu Ser Val Glu Val Leu Arg His Gly Leu Glu Leu Gly Ile

485 490 495

Asn Leu Ile Asp Thr Ala Glu Phe Tyr Gly Ala Gly His Ser Glu Glu

500 505 510

Leu Val Gly Glu Ala Ile Lys Glu Phe Glu Arg Asp Asp Ile Phe Ile

515 520 525

Ile Ser Lys Val Trp Pro Thr His Phe Gly Tyr Glu Glu Ala Lys Arg

530 535 540

Ala Ala Arg Ala Ser Ala Lys Arg Leu Gly Thr Tyr Ile Asp Leu Tyr

545 550 555 560

Leu Leu His Trp Pro Gly Asp Ser Trp Glu Lys Ile Lys Glu Thr Leu

565 570 575

His Ala Leu Glu Glu Leu Val Asp Glu Gly Leu Ile Arg Tyr Ile Gly

580 585 590

Val Ser Asn Phe Asp Leu Glu Leu Leu Lys Arg Ser Gln Glu Ala Met

595 600 605

Lys Lys Tyr Glu Ile Val Ala Asn Glu Val Lys Tyr Ser Leu Lys Asp

610 615 620

Arg Trp Pro Glu Thr Thr Gly Leu Leu Asp Tyr Met Lys Arg Glu Gly

625 630 635 640

Ile Ala Leu Ile Ala Tyr Thr Pro Leu Glu Lys Gly Thr Leu Ala Arg

645 650 655

Asn Glu Cys Leu Ala Glu Ile Gly Lys Lys Tyr Gly Lys Thr Ala Ala

660 665 670

Gln Val Ala Leu Asn Tyr Leu Ile Trp Glu Glu Asn Val Ile Ala Ile

675 680 685

Pro Lys Ala Gly Asn Lys Ala His Leu Glu Glu Asn Phe Gly Ala Met

690 695 700

Gly Trp Arg Leu Ser Lys Glu Asp Arg Glu Asn Ala Arg Gly Cys Val

705 710 715 720

<210> 14

<211> 722

<212> PRT

<213> Artificial Sequence

<220>

<223> Artificial

<400> 14

Met Lys Ile Val Val Val Gly Ser Gly Thr Ala Gly Ser Asn Phe Ala

1 5 10 15

Leu Phe Met Arg Lys Leu Asp Arg Lys Ala Glu Ile Thr Val Ile Gly

20 25 30

Lys Glu Pro Thr Met Gln Tyr Ser Pro Cys Ala Leu Pro His Val Val

35 40 45

Ser Gly Thr Ile Glu Lys Pro Glu Asp Ile Ile Val Phe Pro Asn Glu

50 55 60

Phe Tyr Glu Lys Gln Lys Ile Asn Leu Met Leu Asn Thr Glu Ala Lys

65 70 75 80

Ala Ile Asp Arg Glu Arg Lys Val Val Val Thr Asp Lys Gly Glu Val

85 90 95

Pro Tyr Asp Lys Leu Val Leu Ala Val Gly Ser Lys Ala Phe Ile Pro

100 105 110

Pro Ile Lys Gly Val Glu Asn Glu Gly Val Phe Thr Leu Lys Ser Leu

115 120 125

Asp Asp Val Arg Arg Ile Lys Ala Tyr Ile Ala Glu Arg Lys Pro Lys

130 135 140

Lys Ala Val Val Ile Gly Ala Gly Leu Ile Gly Leu Glu Gly Ala Glu

145 150 155 160

Ala Phe Ala Lys Leu Gly Met Glu Val Leu Ile Val Glu Leu Met Asp

165 170 175

Arg Leu Met Pro Thr Met Leu Asp Lys Asp Thr Ala Lys Leu Val Gln

180 185 190

Ala Glu Met Glu Lys Tyr Gly Val Ser Phe Arg Phe Gly Val Gly Val

195 200 205

Ser Glu Ile Ile Gly Ser Pro Val Arg Ala Val Lys Ile Gly Asp Glu

210 215 220

Glu Val Pro Ala Asp Leu Val Leu Val Ala Thr Gly Val Arg Ala Asn

225 230 235 240

Thr Asp Leu Ala Lys Gln Ala Gly Leu Glu Val Asn Arg Gly Ile Val

245 250 255

Val Asn Glu His Leu Gln Thr Ser Asp Pro Glu Val Tyr Ala Ile Gly

260 265 270

Asp Cys Ala Glu Val Ile Asp Ala Val Thr Gly Lys Arg Thr Leu Ser

275 280 285

Gln Leu Gly Thr Ser Ala Val Arg Met Ala Lys Val Ala Ala Glu His

290 295 300

Ile Ala Gly Lys Asp Val Ser Phe Arg Pro Val Phe Asn Thr Ala Ile

305 310 315 320

Thr Glu Leu Phe Gly Leu Glu Ile Gly Thr Phe Gly Ile Thr Glu Glu

325 330 335

Arg Ala Lys Lys Glu Asp Ile Glu Val Ala Val Gly Lys Phe Lys Gly

340 345 350

Ser Thr Lys Pro Glu Tyr Tyr Pro Gly Gly Lys Pro Ile Thr Val Lys

355 360 365

Leu Ile Phe Arg Lys Ser Asp Arg Lys Leu Ile Gly Gly Gln Ile Val

370 375 380

Gly Gly Glu Arg Val Trp Gly Arg Ile Met Thr Leu Ser Ala Leu Ala

385 390 395 400

Gln Lys Gly Ala Thr Val Glu Asp Val Ala Tyr Leu Glu Thr Ala Tyr

405 410 415

Ala Pro Pro Ile Ser Pro Thr Ile Asp Pro Ile Thr Val Ala Ala Glu

420 425 430

Met Ala Gln Arg Lys Leu Arg Glu Ala Ala Ala Lys Met Lys Lys Val

435 440 445

Arg Ile Phe Asn Asp Leu Lys Trp Ile Gly Asp Asp Lys Val Thr Ala

450 455 460

Ile Gly Met Gly Thr Trp Gly Ile Gly Gly Tyr Glu Ser Pro Asp Tyr

465 470 475 480

Ser Lys Asp Asn Glu Ser Val Glu Val Leu Arg His Gly Leu Glu Leu

485 490 495

Gly Ile Asn Leu Ile Asp Thr Ala Glu Phe Tyr Gly Ala Gly His Ser

500 505 510

Glu Glu Leu Val Gly Glu Ala Ile Lys Glu Phe Glu Arg Asp Asp Ile

515 520 525

Phe Ile Ile Ser Lys Val Trp Pro Thr His Phe Gly Tyr Glu Glu Ala

530 535 540

Lys Arg Ala Ala Arg Ala Ser Ala Lys Arg Leu Gly Thr Tyr Ile Asp

545 550 555 560

Leu Tyr Leu Leu His Trp Pro Gly Asp Ser Trp Glu Lys Ile Lys Glu

565 570 575

Thr Leu His Ala Leu Glu Glu Leu Val Asp Glu Gly Leu Ile Arg Tyr

580 585 590

Ile Gly Val Ser Asn Phe Asp Leu Glu Leu Leu Lys Arg Ser Gln Glu

595 600 605

Ala Met Lys Lys Tyr Glu Ile Val Ala Asn Glu Val Lys Tyr Ser Leu

610 615 620

Lys Asp Arg Trp Pro Glu Thr Thr Gly Leu Leu Asp Tyr Met Lys Arg

625 630 635 640

Glu Gly Ile Ala Leu Ile Ala Tyr Thr Pro Leu Glu Lys Gly Thr Leu

645 650 655

Ala Arg Asn Glu Cys Leu Ala Glu Ile Gly Lys Lys Tyr Gly Lys Thr

660 665 670

Ala Ala Gln Val Ala Leu Asn Tyr Leu Ile Trp Glu Glu Asn Val Ile

675 680 685

Ala Ile Pro Lys Ala Gly Asn Lys Ala His Leu Glu Glu Asn Phe Gly

690 695 700

Ala Met Gly Trp Arg Leu Ser Lys Glu Asp Arg Glu Asn Ala Arg Gly

705 710 715 720

Cys Val

<210> 15

<211> 727

<212> PRT

<213> Artificial Sequence

<220>

<223> Artificial

<400> 15

Met Lys Ile Val Val Val Gly Ser Gly Thr Ala Gly Ser Asn Phe Ala

1 5 10 15

Leu Phe Met Arg Lys Leu Asp Arg Lys Ala Glu Ile Thr Val Ile Gly

20 25 30

Lys Glu Pro Thr Met Gln Tyr Ser Pro Cys Ala Leu Pro His Val Val

35 40 45

Ser Gly Thr Ile Glu Lys Pro Glu Asp Ile Ile Val Phe Pro Asn Glu

50 55 60

Phe Tyr Glu Lys Gln Lys Ile Asn Leu Met Leu Asn Thr Glu Ala Lys

65 70 75 80

Ala Ile Asp Arg Glu Arg Lys Val Val Val Thr Asp Lys Gly Glu Val

85 90 95

Pro Tyr Asp Lys Leu Val Leu Ala Val Gly Ser Lys Ala Phe Ile Pro

100 105 110

Pro Ile Lys Gly Val Glu Asn Glu Gly Val Phe Thr Leu Lys Ser Leu

115 120 125

Asp Asp Val Arg Arg Ile Lys Ala Tyr Ile Ala Glu Arg Lys Pro Lys

130 135 140

Lys Ala Val Val Ile Gly Ala Gly Leu Ile Gly Leu Glu Gly Ala Glu

145 150 155 160

Ala Phe Ala Lys Leu Gly Met Glu Val Leu Ile Val Glu Leu Met Asp

165 170 175

Arg Leu Met Pro Thr Met Leu Asp Lys Asp Thr Ala Lys Leu Val Gln

180 185 190

Ala Glu Met Glu Lys Tyr Gly Val Ser Phe Arg Phe Gly Val Gly Val

195 200 205

Ser Glu Ile Ile Gly Ser Pro Val Arg Ala Val Lys Ile Gly Asp Glu

210 215 220

Glu Val Pro Ala Asp Leu Val Leu Val Ala Thr Gly Val Arg Ala Asn

225 230 235 240

Thr Asp Leu Ala Lys Gln Ala Gly Leu Glu Val Asn Arg Gly Ile Val

245 250 255

Val Asn Glu His Leu Gln Thr Ser Asp Pro Glu Val Tyr Ala Ile Gly

260 265 270

Asp Cys Ala Glu Val Ile Asp Ala Val Thr Gly Lys Arg Thr Leu Ser

275 280 285

Gln Leu Gly Thr Ser Ala Val Arg Met Ala Lys Val Ala Ala Glu His

290 295 300

Ile Ala Gly Lys Asp Val Ser Phe Arg Pro Val Phe Asn Thr Ala Ile

305 310 315 320

Thr Glu Leu Phe Gly Leu Glu Ile Gly Thr Phe Gly Ile Thr Glu Glu

325 330 335

Arg Ala Lys Lys Glu Asp Ile Glu Val Ala Val Gly Lys Phe Lys Gly

340 345 350

Ser Thr Lys Pro Glu Tyr Tyr Pro Gly Gly Lys Pro Ile Thr Val Lys

355 360 365

Leu Ile Phe Arg Lys Ser Asp Arg Lys Leu Ile Gly Gly Gln Ile Val

370 375 380

Gly Gly Glu Arg Val Trp Gly Arg Ile Met Thr Leu Ser Ala Leu Ala

385 390 395 400

Gln Lys Gly Ala Thr Val Glu Asp Val Ala Tyr Leu Glu Thr Ala Tyr

405 410 415

Ala Pro Pro Ile Ser Pro Thr Ile Asp Pro Ile Thr Val Ala Ala Glu

420 425 430

Met Ala Gln Arg Lys Leu Arg Glu Ala Ala Ala Lys Glu Ala Ala Ala

435 440 445

Lys Met Lys Lys Val Arg Ile Phe Asn Asp Leu Lys Trp Ile Gly Asp

450 455 460

Asp Lys Val Thr Ala Ile Gly Met Gly Thr Trp Gly Ile Gly Gly Tyr

465 470 475 480

Glu Ser Pro Asp Tyr Ser Lys Asp Asn Glu Ser Val Glu Val Leu Arg

485 490 495

His Gly Leu Glu Leu Gly Ile Asn Leu Ile Asp Thr Ala Glu Phe Tyr

500 505 510

Gly Ala Gly His Ser Glu Glu Leu Val Gly Glu Ala Ile Lys Glu Phe

515 520 525

Glu Arg Asp Asp Ile Phe Ile Ile Ser Lys Val Trp Pro Thr His Phe

530 535 540

Gly Tyr Glu Glu Ala Lys Arg Ala Ala Arg Ala Ser Ala Lys Arg Leu

545 550 555 560

Gly Thr Tyr Ile Asp Leu Tyr Leu Leu His Trp Pro Gly Asp Ser Trp

565 570 575

Glu Lys Ile Lys Glu Thr Leu His Ala Leu Glu Glu Leu Val Asp Glu

580 585 590

Gly Leu Ile Arg Tyr Ile Gly Val Ser Asn Phe Asp Leu Glu Leu Leu

595 600 605

Lys Arg Ser Gln Glu Ala Met Lys Lys Tyr Glu Ile Val Ala Asn Glu

610 615 620

Val Lys Tyr Ser Leu Lys Asp Arg Trp Pro Glu Thr Thr Gly Leu Leu

625 630 635 640

Asp Tyr Met Lys Arg Glu Gly Ile Ala Leu Ile Ala Tyr Thr Pro Leu

645 650 655

Glu Lys Gly Thr Leu Ala Arg Asn Glu Cys Leu Ala Glu Ile Gly Lys

660 665 670

Lys Tyr Gly Lys Thr Ala Ala Gln Val Ala Leu Asn Tyr Leu Ile Trp

675 680 685

Glu Glu Asn Val Ile Ala Ile Pro Lys Ala Gly Asn Lys Ala His Leu

690 695 700

Glu Glu Asn Phe Gly Ala Met Gly Trp Arg Leu Ser Lys Glu Asp Arg

705 710 715 720

Glu Asn Ala Arg Gly Cys Val

725

<210> 16

<211> 732

<212> PRT

<213> Artificial Sequence

<220>

<223> Artificial

<400> 16

Met Lys Ile Val Val Val Gly Ser Gly Thr Ala Gly Ser Asn Phe Ala

1 5 10 15

Leu Phe Met Arg Lys Leu Asp Arg Lys Ala Glu Ile Thr Val Ile Gly

20 25 30

Lys Glu Pro Thr Met Gln Tyr Ser Pro Cys Ala Leu Pro His Val Val

35 40 45

Ser Gly Thr Ile Glu Lys Pro Glu Asp Ile Ile Val Phe Pro Asn Glu

50 55 60

Phe Tyr Glu Lys Gln Lys Ile Asn Leu Met Leu Asn Thr Glu Ala Lys

65 70 75 80

Ala Ile Asp Arg Glu Arg Lys Val Val Val Thr Asp Lys Gly Glu Val

85 90 95

Pro Tyr Asp Lys Leu Val Leu Ala Val Gly Ser Lys Ala Phe Ile Pro

100 105 110

Pro Ile Lys Gly Val Glu Asn Glu Gly Val Phe Thr Leu Lys Ser Leu

115 120 125

Asp Asp Val Arg Arg Ile Lys Ala Tyr Ile Ala Glu Arg Lys Pro Lys

130 135 140

Lys Ala Val Val Ile Gly Ala Gly Leu Ile Gly Leu Glu Gly Ala Glu

145 150 155 160

Ala Phe Ala Lys Leu Gly Met Glu Val Leu Ile Val Glu Leu Met Asp

165 170 175

Arg Leu Met Pro Thr Met Leu Asp Lys Asp Thr Ala Lys Leu Val Gln

180 185 190

Ala Glu Met Glu Lys Tyr Gly Val Ser Phe Arg Phe Gly Val Gly Val

195 200 205

Ser Glu Ile Ile Gly Ser Pro Val Arg Ala Val Lys Ile Gly Asp Glu

210 215 220

Glu Val Pro Ala Asp Leu Val Leu Val Ala Thr Gly Val Arg Ala Asn

225 230 235 240

Thr Asp Leu Ala Lys Gln Ala Gly Leu Glu Val Asn Arg Gly Ile Val

245 250 255

Val Asn Glu His Leu Gln Thr Ser Asp Pro Glu Val Tyr Ala Ile Gly

260 265 270

Asp Cys Ala Glu Val Ile Asp Ala Val Thr Gly Lys Arg Thr Leu Ser

275 280 285

Gln Leu Gly Thr Ser Ala Val Arg Met Ala Lys Val Ala Ala Glu His

290 295 300

Ile Ala Gly Lys Asp Val Ser Phe Arg Pro Val Phe Asn Thr Ala Ile

305 310 315 320

Thr Glu Leu Phe Gly Leu Glu Ile Gly Thr Phe Gly Ile Thr Glu Glu

325 330 335

Arg Ala Lys Lys Glu Asp Ile Glu Val Ala Val Gly Lys Phe Lys Gly

340 345 350

Ser Thr Lys Pro Glu Tyr Tyr Pro Gly Gly Lys Pro Ile Thr Val Lys

355 360 365

Leu Ile Phe Arg Lys Ser Asp Arg Lys Leu Ile Gly Gly Gln Ile Val

370 375 380

Gly Gly Glu Arg Val Trp Gly Arg Ile Met Thr Leu Ser Ala Leu Ala

385 390 395 400

Gln Lys Gly Ala Thr Val Glu Asp Val Ala Tyr Leu Glu Thr Ala Tyr

405 410 415

Ala Pro Pro Ile Ser Pro Thr Ile Asp Pro Ile Thr Val Ala Ala Glu

420 425 430

Met Ala Gln Arg Lys Leu Arg Glu Ala Ala Ala Lys Glu Ala Ala Ala

435 440 445

Lys Glu Ala Ala Ala Lys Met Lys Lys Val Arg Ile Phe Asn Asp Leu

450 455 460

Lys Trp Ile Gly Asp Asp Lys Val Thr Ala Ile Gly Met Gly Thr Trp

465 470 475 480

Gly Ile Gly Gly Tyr Glu Ser Pro Asp Tyr Ser Lys Asp Asn Glu Ser

485 490 495

Val Glu Val Leu Arg His Gly Leu Glu Leu Gly Ile Asn Leu Ile Asp

500 505 510

Thr Ala Glu Phe Tyr Gly Ala Gly His Ser Glu Glu Leu Val Gly Glu

515 520 525

Ala Ile Lys Glu Phe Glu Arg Asp Asp Ile Phe Ile Ile Ser Lys Val

530 535 540

Trp Pro Thr His Phe Gly Tyr Glu Glu Ala Lys Arg Ala Ala Arg Ala

545 550 555 560

Ser Ala Lys Arg Leu Gly Thr Tyr Ile Asp Leu Tyr Leu Leu His Trp

565 570 575

Pro Gly Asp Ser Trp Glu Lys Ile Lys Glu Thr Leu His Ala Leu Glu

580 585 590

Glu Leu Val Asp Glu Gly Leu Ile Arg Tyr Ile Gly Val Ser Asn Phe

595 600 605

Asp Leu Glu Leu Leu Lys Arg Ser Gln Glu Ala Met Lys Lys Tyr Glu

610 615 620

Ile Val Ala Asn Glu Val Lys Tyr Ser Leu Lys Asp Arg Trp Pro Glu

625 630 635 640

Thr Thr Gly Leu Leu Asp Tyr Met Lys Arg Glu Gly Ile Ala Leu Ile

645 650 655

Ala Tyr Thr Pro Leu Glu Lys Gly Thr Leu Ala Arg Asn Glu Cys Leu

660 665 670

Ala Glu Ile Gly Lys Lys Tyr Gly Lys Thr Ala Ala Gln Val Ala Leu

675 680 685

Asn Tyr Leu Ile Trp Glu Glu Asn Val Ile Ala Ile Pro Lys Ala Gly

690 695 700

Asn Lys Ala His Leu Glu Glu Asn Phe Gly Ala Met Gly Trp Arg Leu

705 710 715 720

Ser Lys Glu Asp Arg Glu Asn Ala Arg Gly Cys Val

725 730

<210> 17

<211> 2163

<212> DNA

<213> Artificial Sequence

<220>

<223> Artificial

<400> 17

atgaagaagg ttaggatttt taacgacctt aagtggatag gtgacgacaa agttacggcc 60

ataggcatgg gcacgtgggg aataggcggc tacgagagtc cagactattc aaaggataat 120

gagagcgttg aggttctaag gcatggcctc gagctcggaa taaacctcat agacaccgct 180

gagttctacg gggcaggaca ctcggaggag ctcgtaggag aggccataaa ggagttcgag 240

cgcgatgata ttttcatcat cagcaaagtg tggccgacac acttcggcta cgaggaagca 300

aagagggccg caagagcgag cgcaaagaga ctaggcactt atattgacct ttatctcctc 360

cactggcctg gcgacagttg ggagaagatc aaggagacgc tccacgcgct agaggagctc 420

gtcgacgagg ggctgatcag gtacatcggc gtcagcaact tcgacctcga gcttctcaag 480

agaagccagg aggcgatgaa gaagtacgag atagtcgcca acgaggtcaa gtactccctc 540

aaagaccgct ggccagaaac tacaggtctg ctcgactaca tgaagcgtga ggggattgcg 600

ctgatagcct acacgccgct tgaaaaggga accctcgcga gaaacgaatg tttggccgag 660

atcgggaaga aatacggtaa gacagccgct caggttgcgc tcaactacct catctgggag 720

gagaacgtca tagccatccc aaaggctgga aacaaggctc acctagagga gaacttcggt 780

gctatgggat ggagactctc aaaggaggat agagagaacg caagggggtg tgtcgcggcc 840

gcgatgaaaa tcgtcgtggt cggttctgga acagccggaa gcaacttcgc cctcttcatg 900

cgcaagcttg acaggaaggc cgagataacc gtcataggaa aggaaccaac gatgcagtac 960

tccccctgcg ccctgccgca cgtggtaagc ggcactatcg agaagcctga ggacattata 1020

gtctttccca acgagttcta cgagaagcag aagataaacc tcatgctgaa cacggaagca 1080

aaggcgatag acagagaaag gaaggttgta gtcacggata agggcgaagt cccgtacgac 1140

aagcttgttt tggccgttgg ttcaaaggca ttcattccgc cgattaaggg agttgagaac 1200

gagggggtct tcacactcaa gagcctcgac gacgttagga ggataaaagc ctacatagcc 1260

gagagaaagc cgaagaaggc cgtcgttatc ggagctggtc tcatcggcct tgagggcgcc 1320

gaggcctttg caaaacttgg aatggaagtt ctgattgtag agctgatgga caggcttatg 1380

cccacaatgc tcgacaagga cacggcaaag ctcgtccagg ctgagatgga gaagtacggc 1440

gtttccttcc gcttcggcgt cggcgtgagt gagatcatcg gaagccccgt cagggctgtc 1500

aaaataggcg acgaagaagt tcccgcagac ctcgtcttgg ttgcaaccgg ggtgagagcc 1560

aacaccgacc tcgccaaaca ggcagggctt gaagtgaaca ggggcatagt ggttaatgag 1620

cacctccaga cgagcgaccc ggaggtctac gcaataggcg actgtgctga agttatagac 1680

gccgtaactg gaaaaagaac tctcagtcag ctcggaactt ccgccgttag gatggccaag 1740

gtggccgcgg agcacatagc gggtaaggac gtctccttca gaccagtctt caacaccgct 1800

ataaccgagc tgtttggcct tgaaatcggc accttcggaa tcaccgagga gagggcaaag 1860

aaggaggaca tcgaggtagc ggtcggaaag ttcaaaggct cgaccaagcc agagtactat 1920

cccggaggca agcccataac cgtaaagctc atcttcagaa agtcagacag aaagcttatc 1980

ggcggccaga tagtcggcgg cgagagggtc tggggcagga taatgacgct ttctgccctc 2040

gctcaaaaag gcgcaacggt tgaggacgtt gcctacctgg aaacggccta cgccccgccg 2100

ataagtccga ccatagaccc gataacggtc gcggcagaga tggcccagag aaagctccgc 2160

tga 2163

<210> 18

<211> 2169

<212> DNA

<213> Artificial Sequence

<220>

<223> Artificial

<400> 18

atgaagaagg ttaggatttt taacgacctt aagtggatag gtgacgacaa agttacggcc 60

ataggcatgg gcacgtgggg aataggcggc tacgagagtc cagactattc aaaggataat 120

gagagcgttg aggttctaag gcatggcctc gagctcggaa taaacctcat agacaccgct 180

gagttctacg gggcaggaca ctcggaggag ctcgtaggag aggccataaa ggagttcgag 240

cgcgatgata ttttcatcat cagcaaagtg tggccgacac acttcggcta cgaggaagca 300

aagagggccg caagagcgag cgcaaagaga ctaggcactt atattgacct ttatctcctc 360

cactggcctg gcgacagttg ggagaagatc aaggagacgc tccacgcgct agaggagctc 420

gtcgacgagg ggctgatcag gtacatcggc gtcagcaact tcgacctcga gcttctcaag 480

agaagccagg aggcgatgaa gaagtacgag atagtcgcca acgaggtcaa gtactccctc 540

aaagaccgct ggccagaaac tacaggtctg ctcgactaca tgaagcgtga ggggattgcg 600

ctgatagcct acacgccgct tgaaaaggga accctcgcga gaaacgaatg tttggccgag 660

atcgggaaga aatacggtaa gacagccgct caggttgcgc tcaactacct catctgggag 720

gagaacgtca tagccatccc aaaggctgga aacaaggctc acctagagga gaacttcggt 780

gctatgggat ggagactctc aaaggaggat agagagaacg caagggggtg tgtcgaagcg 840

gccgcgaaaa tgaaaatcgt cgtggtcggt tctggaacag ccggaagcaa cttcgccctc 900

ttcatgcgca agcttgacag gaaggccgag ataaccgtca taggaaagga accaacgatg 960

cagtactccc cctgcgccct gccgcacgtg gtaagcggca ctatcgagaa gcctgaggac 1020

attatagtct ttcccaacga gttctacgag aagcagaaga taaacctcat gctgaacacg 1080

gaagcaaagg cgatagacag agaaaggaag gttgtagtca cggataaggg cgaagtcccg 1140

tacgacaagc ttgttttggc cgttggttca aaggcattca ttccgccgat taagggagtt 1200

gagaacgagg gggtcttcac actcaagagc ctcgacgacg ttaggaggat aaaagcctac 1260

atagccgaga gaaagccgaa gaaggccgtc gttatcggag ctggtctcat cggccttgag 1320

ggcgccgagg cctttgcaaa acttggaatg gaagttctga ttgtagagct gatggacagg 1380

cttatgccca caatgctcga caaggacacg gcaaagctcg tccaggctga gatggagaag 1440

tacggcgttt ccttccgctt cggcgtcggc gtgagtgaga tcatcggaag ccccgtcagg 1500

gctgtcaaaa taggcgacga agaagttccc gcagacctcg tcttggttgc aaccggggtg 1560

agagccaaca ccgacctcgc caaacaggca gggcttgaag tgaacagggg catagtggtt 1620

aatgagcacc tccagacgag cgacccggag gtctacgcaa taggcgactg tgctgaagtt 1680

atagacgccg taactggaaa aagaactctc agtcagctcg gaacttccgc cgttaggatg 1740

gccaaggtgg ccgcggagca catagcgggt aaggacgtct ccttcagacc agtcttcaac 1800

accgctataa ccgagctgtt tggccttgaa atcggcacct tcggaatcac cgaggagagg 1860

gcaaagaagg aggacatcga ggtagcggtc ggaaagttca aaggctcgac caagccagag 1920

tactatcccg gaggcaagcc cataaccgta aagctcatct tcagaaagtc agacagaaag 1980

cttatcggcg gccagatagt cggcggcgag agggtctggg gcaggataat gacgctttct 2040

gccctcgctc aaaaaggcgc aacggttgag gacgttgcct acctggaaac ggcctacgcc 2100

ccgccgataa gtccgaccat agacccgata acggtcgcgg cagagatggc ccagagaaag 2160

ctccgctga 2169

<210> 19

<211> 2184

<212> DNA

<213> Artificial Sequence

<220>

<223> Artificial

<400> 19

atgaagaagg ttaggatttt taacgacctt aagtggatag gtgacgacaa agttacggcc 60

ataggcatgg gcacgtgggg aataggcggc tacgagagtc cagactattc aaaggataat 120

gagagcgttg aggttctaag gcatggcctc gagctcggaa taaacctcat agacaccgct 180

gagttctacg gggcaggaca ctcggaggag ctcgtaggag aggccataaa ggagttcgag 240

cgcgatgata ttttcatcat cagcaaagtg tggccgacac acttcggcta cgaggaagca 300

aagagggccg caagagcgag cgcaaagaga ctaggcactt atattgacct ttatctcctc 360

cactggcctg gcgacagttg ggagaagatc aaggagacgc tccacgcgct agaggagctc 420

gtcgacgagg ggctgatcag gtacatcggc gtcagcaact tcgacctcga gcttctcaag 480

agaagccagg aggcgatgaa gaagtacgag atagtcgcca acgaggtcaa gtactccctc 540

aaagaccgct ggccagaaac tacaggtctg ctcgactaca tgaagcgtga ggggattgcg 600

ctgatagcct acacgccgct tgaaaaggga accctcgcga gaaacgaatg tttggccgag 660

atcgggaaga aatacggtaa gacagccgct caggttgcgc tcaactacct catctgggag 720

gagaacgtca tagccatccc aaaggctgga aacaaggctc acctagagga gaacttcggt 780

gctatgggat ggagactctc aaaggaggat agagagaacg caagggggtg tgtcgaagcc 840

gcggcgaaag aagcggccgc gaaaatgaaa atcgtcgtgg tcggttctgg aacagccgga 900

agcaacttcg ccctcttcat gcgcaagctt gacaggaagg ccgagataac cgtcatagga 960

aaggaaccaa cgatgcagta ctccccctgc gccctgccgc acgtggtaag cggcactatc 1020

gagaagcctg aggacattat agtctttccc aacgagttct acgagaagca gaagataaac 1080

ctcatgctga acacggaagc aaaggcgata gacagagaaa ggaaggttgt agtcacggat 1140

aagggcgaag tcccgtacga caagcttgtt ttggccgttg gttcaaaggc attcattccg 1200

ccgattaagg gagttgagaa cgagggggtc ttcacactca agagcctcga cgacgttagg 1260

aggataaaag cctacatagc cgagagaaag ccgaagaagg ccgtcgttat cggagctggt 1320

ctcatcggcc ttgagggcgc cgaggccttt gcaaaacttg gaatggaagt tctgattgta 1380

gagctgatgg acaggcttat gcccacaatg ctcgacaagg acacggcaaa gctcgtccag 1440

gctgagatgg agaagtacgg cgtttccttc cgcttcggcg tcggcgtgag tgagatcatc 1500

ggaagccccg tcagggctgt caaaataggc gacgaagaag ttcccgcaga cctcgtcttg 1560

gttgcaaccg gggtgagagc caacaccgac ctcgccaaac aggcagggct tgaagtgaac 1620

aggggcatag tggttaatga gcacctccag acgagcgacc cggaggtcta cgcaataggc 1680

gactgtgctg aagttataga cgccgtaact ggaaaaagaa ctctcagtca gctcggaact 1740

tccgccgtta ggatggccaa ggtggccgcg gagcacatag cgggtaagga cgtctccttc 1800

agaccagtct tcaacaccgc tataaccgag ctgtttggcc ttgaaatcgg caccttcgga 1860

atcaccgagg agagggcaaa gaaggaggac atcgaggtag cggtcggaaa gttcaaaggc 1920

tcgaccaagc cagagtacta tcccggaggc aagcccataa ccgtaaagct catcttcaga 1980

aagtcagaca gaaagcttat cggcggccag atagtcggcg gcgagagggt ctggggcagg 2040

ataatgacgc tttctgccct cgctcaaaaa ggcgcaacgg ttgaggacgt tgcctacctg 2100

gaaacggcct acgccccgcc gataagtccg accatagacc cgataacggt cgcggcagag 2160

atggcccaga gaaagctccg ctga 2184

<210> 20

<211> 2199

<212> DNA

<213> Artificial Sequence

<220>

<223> Artificial

<400> 20

atgaagaagg ttaggatttt taacgacctt aagtggatag gtgacgacaa agttacggcc 60

ataggcatgg gcacgtgggg aataggcggc tacgagagtc cagactattc aaaggataat 120

gagagcgttg aggttctaag gcatggcctc gagctcggaa taaacctcat agacaccgct 180

gagttctacg gggcaggaca ctcggaggag ctcgtaggag aggccataaa ggagttcgag 240

cgcgatgata ttttcatcat cagcaaagtg tggccgacac acttcggcta cgaggaagca 300

aagagggccg caagagcgag cgcaaagaga ctaggcactt atattgacct ttatctcctc 360

cactggcctg gcgacagttg ggagaagatc aaggagacgc tccacgcgct agaggagctc 420

gtcgacgagg ggctgatcag gtacatcggc gtcagcaact tcgacctcga gcttctcaag 480

agaagccagg aggcgatgaa gaagtacgag atagtcgcca acgaggtcaa gtactccctc 540

aaagaccgct ggccagaaac tacaggtctg ctcgactaca tgaagcgtga ggggattgcg 600

ctgatagcct acacgccgct tgaaaaggga accctcgcga gaaacgaatg tttggccgag 660

atcgggaaga aatacggtaa gacagccgct caggttgcgc tcaactacct catctgggag 720

gagaacgtca tagccatccc aaaggctgga aacaaggctc acctagagga gaacttcggt 780

gctatgggat ggagactctc aaaggaggat agagagaacg caagggggtg tgtcgaagcc 840

gcggcgaaag aagcggccgc gaaagaagcc gcggcgaaaa tgaaaatcgt cgtggtcggt 900

tctggaacag ccggaagcaa cttcgccctc ttcatgcgca agcttgacag gaaggccgag 960

ataaccgtca taggaaagga accaacgatg cagtactccc cctgcgccct gccgcacgtg 1020

gtaagcggca ctatcgagaa gcctgaggac attatagtct ttcccaacga gttctacgag 1080

aagcagaaga taaacctcat gctgaacacg gaagcaaagg cgatagacag agaaaggaag 1140

gttgtagtca cggataaggg cgaagtcccg tacgacaagc ttgttttggc cgttggttca 1200

aaggcattca ttccgccgat taagggagtt gagaacgagg gggtcttcac actcaagagc 1260

ctcgacgacg ttaggaggat aaaagcctac atagccgaga gaaagccgaa gaaggccgtc 1320

gttatcggag ctggtctcat cggccttgag ggcgccgagg cctttgcaaa acttggaatg 1380

gaagttctga ttgtagagct gatggacagg cttatgccca caatgctcga caaggacacg 1440

gcaaagctcg tccaggctga gatggagaag tacggcgttt ccttccgctt cggcgtcggc 1500

gtgagtgaga tcatcggaag ccccgtcagg gctgtcaaaa taggcgacga agaagttccc 1560

gcagacctcg tcttggttgc aaccggggtg agagccaaca ccgacctcgc caaacaggca 1620

gggcttgaag tgaacagggg catagtggtt aatgagcacc tccagacgag cgacccggag 1680

gtctacgcaa taggcgactg tgctgaagtt atagacgccg taactggaaa aagaactctc 1740

agtcagctcg gaacttccgc cgttaggatg gccaaggtgg ccgcggagca catagcgggt 1800

aaggacgtct ccttcagacc agtcttcaac accgctataa ccgagctgtt tggccttgaa 1860

atcggcacct tcggaatcac cgaggagagg gcaaagaagg aggacatcga ggtagcggtc 1920

ggaaagttca aaggctcgac caagccagag tactatcccg gaggcaagcc cataaccgta 1980

aagctcatct tcagaaagtc agacagaaag cttatcggcg gccagatagt cggcggcgag 2040

agggtctggg gcaggataat gacgctttct gccctcgctc aaaaaggcgc aacggttgag 2100

gacgttgcct acctggaaac ggcctacgcc ccgccgataa gtccgaccat agacccgata 2160

acggtcgcgg cagagatggc ccagagaaag ctccgctga 2199

<210> 21

<211> 2163

<212> DNA

<213> Artificial Sequence

<220>

<223> Artificial

<400> 21

atgaaaatcg tcgtggtcgg ttctggaaca gccggaagca acttcgccct cttcatgcgc 60

aagcttgaca ggaaggccga gataaccgtc ataggaaagg aaccaacgat gcagtactcc 120

ccctgcgccc tgccgcacgt ggtaagcggc actatcgaga agcctgagga cattatagtc 180

tttcccaacg agttctacga gaagcagaag ataaacctca tgctgaacac ggaagcaaag 240

gcgatagaca gagaaaggaa ggttgtagtc acggataagg gcgaagtccc gtacgacaag 300

cttgttttgg ccgttggttc aaaggcattc attccgccga ttaagggagt tgagaacgag 360

ggggtcttca cactcaagag cctcgacgac gttaggagga taaaagccta catagccgag 420

agaaagccga agaaggccgt cgttatcgga gctggtctca tcggccttga gggcgccgag 480

gcctttgcaa aacttggaat ggaagttctg attgtagagc tgatggacag gcttatgccc 540

acaatgctcg acaaggacac ggcaaagctc gtccaggctg agatggagaa gtacggcgtt 600

tccttccgct tcggcgtcgg cgtgagtgag atcatcggaa gccccgtcag ggctgtcaaa 660

ataggcgacg aagaagttcc cgcagacctc gtcttggttg caaccggggt gagagccaac 720

accgacctcg ccaaacaggc agggcttgaa gtgaacaggg gcatagtggt taatgagcac 780

ctccagacga gcgacccgga ggtctacgca ataggcgact gtgctgaagt tatagacgcc 840

gtaactggaa aaagaactct cagtcagctc ggaacttccg ccgttaggat ggccaaggtg 900

gccgcggagc acatagcggg taaggacgtc tccttcagac cagtcttcaa caccgctata 960

accgagctgt ttggccttga aatcggcacc ttcggaatca ccgaggagag ggcaaagaag 1020

gaggacatcg aggtagcggt cggaaagttc aaaggctcga ccaagccaga gtactatccc 1080

ggaggcaagc ccataaccgt aaagctcatc ttcagaaagt cagacagaaa gcttatcggc 1140

ggccagatag tcggcggcga gagggtctgg ggcaggataa tgacgctttc tgccctcgct 1200

caaaaaggcg caacggttga ggacgttgcc tacctggaaa cggcctacgc cccgccgata 1260

agtccgacca tagacccgat aacggtcgcg gcagagatgg cccagagaaa gctccgcgcg 1320

gccgcgatga agaaggttag gatttttaac gaccttaagt ggataggtga cgacaaagtt 1380

acggccatag gcatgggcac gtggggaata ggcggctacg agagtccaga ctattcaaag 1440

gataatgaga gcgttgaggt tctaaggcat ggcctcgagc tcggaataaa cctcatagac 1500

accgctgagt tctacggggc aggacactcg gaggagctcg taggagaggc cataaaggag 1560

ttcgagcgcg atgatatttt catcatcagc aaagtgtggc cgacacactt cggctacgag 1620

gaagcaaaga gggccgcaag agcgagcgca aagagactag gcacttatat tgacctttat 1680

ctcctccact ggcctggcga cagttgggag aagatcaagg agacgctcca cgcgctagag 1740

gagctcgtcg acgaggggct gatcaggtac atcggcgtca gcaacttcga cctcgagctt 1800

ctcaagagaa gccaggaggc gatgaagaag tacgagatag tcgccaacga ggtcaagtac 1860

tccctcaaag accgctggcc agaaactaca ggtctgctcg actacatgaa gcgtgagggg 1920

attgcgctga tagcctacac gccgcttgaa aagggaaccc tcgcgagaaa cgaatgtttg 1980

gccgagatcg ggaagaaata cggtaagaca gccgctcagg ttgcgctcaa ctacctcatc 2040

tgggaggaga acgtcatagc catcccaaag gctggaaaca aggctcacct agaggagaac 2100

ttcggtgcta tgggatggag actctcaaag gaggatagag agaacgcaag ggggtgtgtc 2160

tga 2163

<210> 22

<211> 2169

<212> DNA

<213> Artificial Sequence

<220>

<223> Artificial

<400> 22

atgaaaatcg tcgtggtcgg ttctggaaca gccggaagca acttcgccct cttcatgcgc 60

aagcttgaca ggaaggccga gataaccgtc ataggaaagg aaccaacgat gcagtactcc 120

ccctgcgccc tgccgcacgt ggtaagcggc actatcgaga agcctgagga cattatagtc 180

tttcccaacg agttctacga gaagcagaag ataaacctca tgctgaacac ggaagcaaag 240

gcgatagaca gagaaaggaa ggttgtagtc acggataagg gcgaagtccc gtacgacaag 300

cttgttttgg ccgttggttc aaaggcattc attccgccga ttaagggagt tgagaacgag 360

ggggtcttca cactcaagag cctcgacgac gttaggagga taaaagccta catagccgag 420

agaaagccga agaaggccgt cgttatcgga gctggtctca tcggccttga gggcgccgag 480

gcctttgcaa aacttggaat ggaagttctg attgtagagc tgatggacag gcttatgccc 540

acaatgctcg acaaggacac ggcaaagctc gtccaggctg agatggagaa gtacggcgtt 600

tccttccgct tcggcgtcgg cgtgagtgag atcatcggaa gccccgtcag ggctgtcaaa 660

ataggcgacg aagaagttcc cgcagacctc gtcttggttg caaccggggt gagagccaac 720

accgacctcg ccaaacaggc agggcttgaa gtgaacaggg gcatagtggt taatgagcac 780

ctccagacga gcgacccgga ggtctacgca ataggcgact gtgctgaagt tatagacgcc 840

gtaactggaa aaagaactct cagtcagctc ggaacttccg ccgttaggat ggccaaggtg 900

gccgcggagc acatagcggg taaggacgtc tccttcagac cagtcttcaa caccgctata 960

accgagctgt ttggccttga aatcggcacc ttcggaatca ccgaggagag ggcaaagaag 1020

gaggacatcg aggtagcggt cggaaagttc aaaggctcga ccaagccaga gtactatccc 1080

ggaggcaagc ccataaccgt aaagctcatc ttcagaaagt cagacagaaa gcttatcggc 1140

ggccagatag tcggcggcga gagggtctgg ggcaggataa tgacgctttc tgccctcgct 1200

caaaaaggcg caacggttga ggacgttgcc tacctggaaa cggcctacgc cccgccgata 1260

agtccgacca tagacccgat aacggtcgcg gcagagatgg cccagagaaa gctccgcgaa 1320

gcggccgcga aaatgaagaa ggttaggatt tttaacgacc ttaagtggat aggtgacgac 1380

aaagttacgg ccataggcat gggcacgtgg ggaataggcg gctacgagag tccagactat 1440

tcaaaggata atgagagcgt tgaggttcta aggcatggcc tcgagctcgg aataaacctc 1500

atagacaccg ctgagttcta cggggcagga cactcggagg agctcgtagg agaggccata 1560

aaggagttcg agcgcgatga tattttcatc atcagcaaag tgtggccgac acacttcggc 1620

tacgaggaag caaagagggc cgcaagagcg agcgcaaaga gactaggcac ttatattgac 1680

ctttatctcc tccactggcc tggcgacagt tgggagaaga tcaaggagac gctccacgcg 1740

ctagaggagc tcgtcgacga ggggctgatc aggtacatcg gcgtcagcaa cttcgacctc 1800

gagcttctca agagaagcca ggaggcgatg aagaagtacg agatagtcgc caacgaggtc 1860

aagtactccc tcaaagaccg ctggccagaa actacaggtc tgctcgacta catgaagcgt 1920

gaggggattg cgctgatagc ctacacgccg cttgaaaagg gaaccctcgc gagaaacgaa 1980

tgtttggccg agatcgggaa gaaatacggt aagacagccg ctcaggttgc gctcaactac 2040

ctcatctggg aggagaacgt catagccatc ccaaaggctg gaaacaaggc tcacctagag 2100

gagaacttcg gtgctatggg atggagactc tcaaaggagg atagagagaa cgcaaggggg 2160

tgtgtctga 2169

<210> 23

<211> 2184

<212> DNA

<213> Artificial Sequence

<220>

<223> Artificial

<400> 23

atgaaaatcg tcgtggtcgg ttctggaaca gccggaagca acttcgccct cttcatgcgc 60

aagcttgaca ggaaggccga gataaccgtc ataggaaagg aaccaacgat gcagtactcc 120

ccctgcgccc tgccgcacgt ggtaagcggc actatcgaga agcctgagga cattatagtc 180

tttcccaacg agttctacga gaagcagaag ataaacctca tgctgaacac ggaagcaaag 240

gcgatagaca gagaaaggaa ggttgtagtc acggataagg gcgaagtccc gtacgacaag 300

cttgttttgg ccgttggttc aaaggcattc attccgccga ttaagggagt tgagaacgag 360

ggggtcttca cactcaagag cctcgacgac gttaggagga taaaagccta catagccgag 420

agaaagccga agaaggccgt cgttatcgga gctggtctca tcggccttga gggcgccgag 480

gcctttgcaa aacttggaat ggaagttctg attgtagagc tgatggacag gcttatgccc 540

acaatgctcg acaaggacac ggcaaagctc gtccaggctg agatggagaa gtacggcgtt 600

tccttccgct tcggcgtcgg cgtgagtgag atcatcggaa gccccgtcag ggctgtcaaa 660

ataggcgacg aagaagttcc cgcagacctc gtcttggttg caaccggggt gagagccaac 720

accgacctcg ccaaacaggc agggcttgaa gtgaacaggg gcatagtggt taatgagcac 780

ctccagacga gcgacccgga ggtctacgca ataggcgact gtgctgaagt tatagacgcc 840

gtaactggaa aaagaactct cagtcagctc ggaacttccg ccgttaggat ggccaaggtg 900

gccgcggagc acatagcggg taaggacgtc tccttcagac cagtcttcaa caccgctata 960

accgagctgt ttggccttga aatcggcacc ttcggaatca ccgaggagag ggcaaagaag 1020

gaggacatcg aggtagcggt cggaaagttc aaaggctcga ccaagccaga gtactatccc 1080

ggaggcaagc ccataaccgt aaagctcatc ttcagaaagt cagacagaaa gcttatcggc 1140

ggccagatag tcggcggcga gagggtctgg ggcaggataa tgacgctttc tgccctcgct 1200

caaaaaggcg caacggttga ggacgttgcc tacctggaaa cggcctacgc cccgccgata 1260

agtccgacca tagacccgat aacggtcgcg gcagagatgg cccagagaaa gctccgcgaa 1320

gccgcggcga aagaagcggc cgcgaaaatg aagaaggtta ggatttttaa cgaccttaag 1380

tggataggtg acgacaaagt tacggccata ggcatgggca cgtggggaat aggcggctac 1440

gagagtccag actattcaaa ggataatgag agcgttgagg ttctaaggca tggcctcgag 1500

ctcggaataa acctcataga caccgctgag ttctacgggg caggacactc ggaggagctc 1560

gtaggagagg ccataaagga gttcgagcgc gatgatattt tcatcatcag caaagtgtgg 1620

ccgacacact tcggctacga ggaagcaaag agggccgcaa gagcgagcgc aaagagacta 1680

ggcacttata ttgaccttta tctcctccac tggcctggcg acagttggga gaagatcaag 1740

gagacgctcc acgcgctaga ggagctcgtc gacgaggggc tgatcaggta catcggcgtc 1800

agcaacttcg acctcgagct tctcaagaga agccaggagg cgatgaagaa gtacgagata 1860

gtcgccaacg aggtcaagta ctccctcaaa gaccgctggc cagaaactac aggtctgctc 1920

gactacatga agcgtgaggg gattgcgctg atagcctaca cgccgcttga aaagggaacc 1980

ctcgcgagaa acgaatgttt ggccgagatc gggaagaaat acggtaagac agccgctcag 2040

gttgcgctca actacctcat ctgggaggag aacgtcatag ccatcccaaa ggctggaaac 2100

aaggctcacc tagaggagaa cttcggtgct atgggatgga gactctcaaa ggaggataga 2160

gagaacgcaa gggggtgtgt ctga 2184

<210> 24

<211> 2199

<212> DNA

<213> Artificial Sequence

<220>

<223> Artificial

<400> 24

atgaaaatcg tcgtggtcgg ttctggaaca gccggaagca acttcgccct cttcatgcgc 60

aagcttgaca ggaaggccga gataaccgtc ataggaaagg aaccaacgat gcagtactcc 120

ccctgcgccc tgccgcacgt ggtaagcggc actatcgaga agcctgagga cattatagtc 180

tttcccaacg agttctacga gaagcagaag ataaacctca tgctgaacac ggaagcaaag 240

gcgatagaca gagaaaggaa ggttgtagtc acggataagg gcgaagtccc gtacgacaag 300

cttgttttgg ccgttggttc aaaggcattc attccgccga ttaagggagt tgagaacgag 360

ggggtcttca cactcaagag cctcgacgac gttaggagga taaaagccta catagccgag 420

agaaagccga agaaggccgt cgttatcgga gctggtctca tcggccttga gggcgccgag 480

gcctttgcaa aacttggaat ggaagttctg attgtagagc tgatggacag gcttatgccc 540

acaatgctcg acaaggacac ggcaaagctc gtccaggctg agatggagaa gtacggcgtt 600

tccttccgct tcggcgtcgg cgtgagtgag atcatcggaa gccccgtcag ggctgtcaaa 660

ataggcgacg aagaagttcc cgcagacctc gtcttggttg caaccggggt gagagccaac 720

accgacctcg ccaaacaggc agggcttgaa gtgaacaggg gcatagtggt taatgagcac 780

ctccagacga gcgacccgga ggtctacgca ataggcgact gtgctgaagt tatagacgcc 840

gtaactggaa aaagaactct cagtcagctc ggaacttccg ccgttaggat ggccaaggtg 900

gccgcggagc acatagcggg taaggacgtc tccttcagac cagtcttcaa caccgctata 960

accgagctgt ttggccttga aatcggcacc ttcggaatca ccgaggagag ggcaaagaag 1020

gaggacatcg aggtagcggt cggaaagttc aaaggctcga ccaagccaga gtactatccc 1080

ggaggcaagc ccataaccgt aaagctcatc ttcagaaagt cagacagaaa gcttatcggc 1140

ggccagatag tcggcggcga gagggtctgg ggcaggataa tgacgctttc tgccctcgct 1200

caaaaaggcg caacggttga ggacgttgcc tacctggaaa cggcctacgc cccgccgata 1260

agtccgacca tagacccgat aacggtcgcg gcagagatgg cccagagaaa gctccgcgaa 1320

gccgcggcga aagaagcggc cgcgaaagaa gccgcggcga aaatgaagaa ggttaggatt 1380

tttaacgacc ttaagtggat aggtgacgac aaagttacgg ccataggcat gggcacgtgg 1440

ggaataggcg gctacgagag tccagactat tcaaaggata atgagagcgt tgaggttcta 1500

aggcatggcc tcgagctcgg aataaacctc atagacaccg ctgagttcta cggggcagga 1560

cactcggagg agctcgtagg agaggccata aaggagttcg agcgcgatga tattttcatc 1620

atcagcaaag tgtggccgac acacttcggc tacgaggaag caaagagggc cgcaagagcg 1680

agcgcaaaga gactaggcac ttatattgac ctttatctcc tccactggcc tggcgacagt 1740

tgggagaaga tcaaggagac gctccacgcg ctagaggagc tcgtcgacga ggggctgatc 1800

aggtacatcg gcgtcagcaa cttcgacctc gagcttctca agagaagcca ggaggcgatg 1860

aagaagtacg agatagtcgc caacgaggtc aagtactccc tcaaagaccg ctggccagaa 1920

actacaggtc tgctcgacta catgaagcgt gaggggattg cgctgatagc ctacacgccg 1980

cttgaaaagg gaaccctcgc gagaaacgaa tgtttggccg agatcgggaa gaaatacggt 2040

aagacagccg ctcaggttgc gctcaactac ctcatctggg aggagaacgt catagccatc 2100

ccaaaggctg gaaacaaggc tcacctagag gagaacttcg gtgctatggg atggagactc 2160

tcaaaggagg atagagagaa cgcaaggggg tgtgtctga 2199

<210> 25

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> Artificial

<400> 25

gccgtcgcat atgaagaagg ttaggatttt taacg 35

<210> 26

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<223> Artificial

<400> 26

ttatttcagc ggccgcgaca cacccccttg cgttct 36

<210> 27

<211> 39

<212> DNA

<213> Artificial Sequence

<220>

<223> Artificial

<400> 27

aaatatatgc ggccgcgatg aaaatcgtcg tggtcggtt 39

<210> 28

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<223> Artificial

<400> 28

ctggaattct cagcggagct ttctctgggc cat 33

<210> 29

<211> 39

<212> DNA

<213> Artificial Sequence

<220>

<223> Artificial

<400> 29

ttatttcagc ggccgcttcg acacaccccc ttgcgttct 39

<210> 30

<211> 42

<212> DNA

<213> Artificial Sequence

<220>

<223> Artificial

<400> 30

aaatatatgc ggccgcgaaa atgaaaatcg tcgtggtcgg tt 42

<210> 31

<211> 54

<212> DNA

<213> Artificial Sequence

<220>

<223> Artificial

<400> 31

ttatttcagc ggccgcttct ttcgccgcgg cttcgacaca cccccttgcg ttct 54

<210> 32

<211> 42

<212> DNA

<213> Artificial Sequence

<220>

<223> Artificial

<400> 32

aaatatatgc ggccgcgaaa atgaaaatcg tcgtggtcgg tt 42

<210> 33

<211> 54

<212> DNA

<213> Artificial Sequence

<220>

<223> Artificial

<400> 33

ttatttcagc ggccgcttct ttcgccgcgg cttcgacaca cccccttgcg ttct 54

<210> 34

<211> 57

<212> DNA

<213> Artificial Sequence

<220>

<223> Artificial

<400> 34

aaatatatgc ggccgcgaaa gaagccgcgg cgaaaatgaa aatcgtcgtg gtcggtt 57

<210> 35

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<223> Artificial

<400> 35

gccgtcgcat atgaaaatcg tcgtggtcgg ttctg 35

<210> 36

<211> 39

<212> DNA

<213> Artificial Sequence

<220>

<223> Artificial

<400> 36

ttatttcagc ggccgcgcgg agctttctct gggccatct 39

<210> 37

<211> 42

<212> DNA

<213> Artificial Sequence

<220>

<223> Artificial

<400> 37

aaatatatgc ggccgcgatg aagaaggtta ggatttttaa cg 42

<210> 38

<211> 25

<212> DNA

<213> Artificial Sequence

<220>

<223> Artificial

<400> 38

ctggaattct cagacacacc ccctt 25

<210> 39

<211> 42

<212> DNA

<213> Artificial Sequence

<220>

<223> Artificial

<400> 39

ttatttcagc ggccgcttcg cggagctttc tctgggccat ct 42

<210> 40

<211> 45

<212> DNA

<213> Artificial Sequence

<220>

<223> Artificial

<400> 40

aaatatatgc ggccgcgaaa atgaagaagg ttaggatttt taacg 45

<210> 41

<211> 57

<212> DNA

<213> Artificial Sequence

<220>

<223> Artificial

<400> 41

ttatttcagc ggccgcttct ttcgccgcgg cttcgcggag ctttctctgg gccatct 57

<210> 42

<211> 45

<212> DNA

<213> Artificial Sequence

<220>

<223> Artificial

<400> 42

aaatatatgc ggccgcgaaa atgaagaagg ttaggatttt taacg 45

<210> 43

<211> 57

<212> DNA

<213> Artificial Sequence

<220>

<223> Artificial

<400> 43

ttatttcagc ggccgcttct ttcgccgcgg cttcgcggag ctttctctgg gccatct 57

<210> 44

<211> 60

<212> DNA

<213> Artificial Sequence

<220>

<223> Artificial

<400> 44

aaatatatgc ggccgcgaaa gaagccgcgg cgaaaatgaa gaaggttagg atttttaacg 60