CN113774032B - 重组t4连接酶突变体、编码dna及ngs建库方法 - Google Patents
重组t4连接酶突变体、编码dna及ngs建库方法 Download PDFInfo
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Abstract
本发明在野生型T4DL的基础上，进行了Q19K、L63T、E88R、P127K、K159S、K225A、F233A、A237R、D371W、E440K、T451K、D452P多个点突变；在突变体的两端包含两个双链DNA结合域；双链DNA结合域与T4DL之间的连接使用多肽桥。最终获得了一系列低偏好性和高效率的重组DNA连接酶突变体T4DLm。并公开了其编码DNA和NGS建库方法。T4DLm在平末端连接上具有显著的优势，连接效率高达95%，且偏好性极低。利用T4DLm开发了高效简便的新型NGS建库技术，具有耗时短、文库产量更高、均一性更好、文库自连更低、捕获测序数据覆盖深度更好等明显的优势，非常适用于临床样本的NGS检测，尤其是肿瘤样本的检测。
Description
技术领域
本发明涉及一种重组T4连接酶突变体、编码DNA及NGS建库方法，属于生物技术领域。
背景技术
T4 DNA连接酶（T4 DNA ligase, T4DL）由T4嗜菌体30基因编码的一类双链DNA连接酶。从1979年被Murray N E、Bruce S A和Murray K发现以来，T4DL广泛被应用在基因工程和诊断的各个领域，包括基因克隆、高通量测序和高通量筛选等。因此，T4DL的功能改造一直是酶改造领域的重点关注点，具有重要的商业和科研价值。
尽管现有的T4DL及突变体在基因工程和诊断领域具有广泛的应用，但目前的T4DL及突变体仍具有很大的缺陷。这些T4 DL的粘性末端连接效率只有60%，平末端连接效率低于30%。连接效率的缺陷极大地限制了T4DL的应用价值，因此，市面上一直急需一款可以进行高效率DNA连接的T4DL，尤其是平末端接头的连接。
NGS诊断技术是基因诊断的热门技术之一，是DNA和RNA诊断的关键。NGS建库指的是将DNA或RNA模板转换成具有测序仪识别的特定序列结构，是NGS诊断的关键环节。不论是DNA建库还是RNA建库，末端修复、接头连接都是必不可少的步骤。末端修复包括外切酶将3’端突出末端切平、5’端突出末端补平、5’端磷酸化、3’端去磷酸化，这个步骤都依赖于3’-5’DNA外切酶、T4 DNA聚合酶和T4磷酸激酶来介导，反应温度在30℃左右。此外，末端修复过程还需要利用Taq酶在72℃进行3’端末端加A，以保证下游T4DL介导的接头和底物的TA连接顺利进行。这种建库流程不仅受到T4DL连接效率的限制，也受到3’端末端加A效率的限制（Taq酶的加A效率只有70%），最终影响整个NGS建库过程的效率。使用平末端连接不仅可以省去加A的步骤和时长，也可以避免加A效率过低对建库整体效率的影响。因此，探寻高效率的平末端连接方式，是NGS建库领域的重要关注点。
近年来，为了提升T4DL的平末端连接，研究者们对T4DL的活性位点进行了一系列的改造，其中K159是T4DL的关键活性位点，介导双链DNA连接过程中中间产物的形成。K159L和K159C都会造成连接酶活性缺失。意外的是，K159L和K159C改变了T4DL的连接酶识别底物类型，对传统的5’磷酸底物没有连接活性，但对5’端预腺苷酰化的底物具有很强的亲和力和连接效率（平末端连接效率高达70%以上），这与连接酶的反应动力学机制是紧密相连的。因此，最近也有公司试图利用T4DL K159突变体的连接底物特异性这个特点来进行NGS平末端建库，但由于不是粘性末端能够通过碱基互补配对来稳定两端DNA底物，平末端连接效率存在显著的偏好性和差异性，不同末端的连接效率存在极大的差异，这导致了严重的建库偏好性，因此难以应用在NGS建库上。
发明内容
本发明的目的是提供一种重组T4连接酶突变体，系在野生型T4DL进行点突变而成。突变位点为：K159S、Q19K、L63T、E88R、P127K、K225A、F233A、A237R、D371W、E440K、T451K和D452P，重组T4连接酶突变体的氨基酸序列如SEQ ID No. 8所示，对平末端的DNA底物有良好的连接效率。在突变体一端或两端通过多肽桥重组双链DNA结合域，可以显著降低重组T4连接酶对底物DNA末端序列识别偏好性。其中的多肽桥从GGGGS多肽、PAPAPAPA多肽、GQGQGQGQ多肽、AAAAA多肽中选择，双链DNA结合域从p50、cTF、DSBA、H6、HNS或HTH中选择。
本发明还公开了上述重组T4连接酶突变体的编码DNA，其核酸序列如SEQ IDNo.26-42所示。
本发明还公开了一种NGS建库方法，其特征在于其步骤包括：
（1）采用平末端双链DNA片段化酶将DNA片段化；
（2）上述的重组T4连接酶突变体介导的腺苷酰化接头连接；
（3）磁珠回收DNA；
（4）缺口修复；
（5）文库扩增及回收。
优选的，步骤（1）中的平末端双链DNA片段化酶为脱氧核糖核酸I、创伤弧菌核酸酶、全能核酸酶、微球菌核酸酶、热敏感性碱性磷酸酶或T4 DNA聚合酶中的一种或数种的混合物。
优选的，步骤（1）的DNA片段化反应温度为37℃，反应时间为3-15 min。
优选的，步骤（2）中采用的接头为接头A和接头B的退火产物，所述接头A的序列为：/App/AGATCGGAAGAGCACACGTCTGAACTCCAGT/ddC/，接头B的序列为：ACACTCTTTCCCTACACGACGCTCTTCCGATCT-p。
优选的，步骤（2）中接头反应的反应温度为20℃，反应时间为15 min。
优选的，步骤（4）中采用T4磷酸激酶和E.coli连接酶进行缺口修复。
本发明在野生型T4DL的基础上，进行了Q19K、L63T、E88R、P127K、K159S、K225A、F233A、A237R、D371W、E440K、T451K、D452P等多个点突变；在突变体的两端包含两个双链DNA结合域；双链DNA结合域与T4DL之间的连接使用多肽桥。最终获得了一系列低偏好性和高效率的重组DNA连接酶突变体T4DLm。T4DLm在平末端连接上具有显著的优势，连接效率高达95%，且偏好性极低。利用T4DLm开发了高效简便的新型NGS建库技术，具有耗时短、文库产量更高、均一性更好、文库自连更低、捕获测序数据覆盖深度更好等明显的优势，非常适用于临床样本的NGS检测，尤其是肿瘤样本的检测。
附图说明
图1 T4连接酶重组突变体对粘性末端连接效率的测定。
图2 T4连接酶重组突变体对平末端连接效率的测定。
图3 T4连接酶重组突变体进行平末端连接建库的文库产量。
图4 T4连接酶重组突变体进行平末端连接建库的接头末端序列占比。
图5 T4连接酶重组突变体进行平末端连接建库的接头末端序列均一性。
图6 T4连接酶重组突变体进行平末端连接建库的DNA末端序列占比。
图7 T4连接酶重组突变体进行平末端连接建库的DNA末端序列均一性。
图8 基于T4DLm的简便高效的新型NGS建库流程及原理示意图。
图9 基于T4DLm的简便高效的新型NGS建库的凝胶电泳图。
图10 基于T4DLm的简便高效的新型NGS建库的文库产量。
图11 基于T4DLm的简便高效的新型NGS建库与传统NGS建库的文库产量比较。
图12 基于T4DLm的简便高效的新型NGS建库与传统NGS建库测序结果与理论值相关性比较。
图13 基于T4DLm的简便高效的新型NGS建库与传统NGS建库在捕获测序中的测序深度比较。
图14 基于T4DLm的简便高效的新型NGS建库与传统NGS建库在捕获测序中的捕获效率比较。
图15 基于T4DLm的简便高效的新型NGS建库与传统NGS建库在捕获测序中的捕获均一性比较。
具体实施方式
通过以下详细说明结合附图可以进一步理解本发明的特点和优点。所提供的实施例仅是对本发明方法的说明，而不以任何方式限制本发明揭示的其余内容。本实施例所使用的探针和引物序列及修饰如表1所示，N为随机碱基，即A、T、C、G中任意一种碱基。
表1
| 序号 | 序列名称 | 5’-3’ | 修饰类型 |
| 1 | P5-Conhensive | ACACTCTTTCCCTACACGACGCTCTTCCGATCTTCGA | 5’端NH2C6修饰 |
| 2 | P5-Blunt | ACACTCTTTCCCTACACGACGCTCTTCCGATCT | 5’端NH2C6修饰 |
| 3 | P-P7 | AGATCGGAAGAGCACACGTCTGAACTCCAGTC | 5’端磷酸化修饰，3’端NH2C6修饰 |
| 4 | App-P7 | AGATCGGAAGAGCACACGTCTGAACTCCAGTC | 5’端App修饰，3’端NH2C6修饰 |
| 5 | qPCR-F | ACACTCTTTCCCTACACGACGCTCTTCCGATCT | |
| 6 | qPCR-R | GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC | |
| 7 | UMI-Adapotor-F1 | ACACTCTTTCCCTACACGACGCTCTTCCGATCTAA | 3’端磷酸化修饰 |
| 8 | UMI-Adapotor-F2 | ACACTCTTTCCCTACACGACGCTCTTCCGATCTAG | 3’端磷酸化修饰 |
| 9 | UMI-Adapotor-F3 | ACACTCTTTCCCTACACGACGCTCTTCCGATCTAC | 3’端磷酸化修饰 |
| 10 | UMI-Adapotor-F4 | ACACTCTTTCCCTACACGACGCTCTTCCGATCTAT | 3’端磷酸化修饰 |
| 11 | UMI-Adapotor-F5 | ACACTCTTTCCCTACACGACGCTCTTCCGATCTGA | 3’端磷酸化修饰 |
| 12 | UMI-Adapotor-F6 | ACACTCTTTCCCTACACGACGCTCTTCCGATCTGG | 3’端磷酸化修饰 |
| 13 | UMI-Adapotor-F7 | ACACTCTTTCCCTACACGACGCTCTTCCGATCTGC | 3’端磷酸化修饰 |
| 14 | UMI-Adapotor-F8 | ACACTCTTTCCCTACACGACGCTCTTCCGATCTGT | 3’端磷酸化修饰 |
| 15 | UMI-Adapotor-F9 | ACACTCTTTCCCTACACGACGCTCTTCCGATCTCA | 3’端磷酸化修饰 |
| 16 | UMI-Adapotor-F10 | ACACTCTTTCCCTACACGACGCTCTTCCGATCTCG | 3’端磷酸化修饰 |
| 17 | UMI-Adapotor-F11 | ACACTCTTTCCCTACACGACGCTCTTCCGATCTCC | 3’端磷酸化修饰 |
| 18 | UMI-Adapotor-F12 | ACACTCTTTCCCTACACGACGCTCTTCCGATCTCT | 3’端磷酸化修饰 |
| 19 | UMI-Adapotor-F13 | ACACTCTTTCCCTACACGACGCTCTTCCGATCTTA | 3’端磷酸化修饰 |
| 20 | UMI-Adapotor-F14 | ACACTCTTTCCCTACACGACGCTCTTCCGATCTTG | 3’端磷酸化修饰 |
| 21 | UMI-Adapotor-F15 | ACACTCTTTCCCTACACGACGCTCTTCCGATCTTC | 3’端磷酸化修饰 |
| 22 | UMI-Adapotor-F16 | ACACTCTTTCCCTACACGACGCTCTTCCGATCTTT | 3’端磷酸化修饰 |
| 23 | UMI-Adapotor-R1 | TTAGATCGGAAGAGCACACGTCTGAACTCCAGTC | 5’端App修饰，3’端NH2C6修饰 |
| 24 | UMI-Adapotor-R2 | CTAGATCGGAAGAGCACACGTCTGAACTCCAGTC | 5’端App修饰，3’端NH2C6修饰 |
| 25 | UMI-Adapotor-R3 | GTAGATCGGAAGAGCACACGTCTGAACTCCAGTC | 5’端App修饰，3’端NH2C6修饰 |
| 26 | UMI-Adapotor-R4 | ATAGATCGGAAGAGCACACGTCTGAACTCCAGTC | 5’端App修饰，3’端NH2C6修饰 |
| 27 | UMI-Adapotor-R5 | TCAGATCGGAAGAGCACACGTCTGAACTCCAGTC | 5’端App修饰，3’端NH2C6修饰 |
| 28 | UMI-Adapotor-R6 | CCAGATCGGAAGAGCACACGTCTGAACTCCAGTC | 5’端App修饰，3’端NH2C6修饰 |
| 29 | UMI-Adapotor-R7 | GCAGATCGGAAGAGCACACGTCTGAACTCCAGTC | 5’端App修饰，3’端NH2C6修饰 |
| 30 | UMI-Adapotor-R8 | ACAGATCGGAAGAGCACACGTCTGAACTCCAGTC | 5’端App修饰，3’端NH2C6修饰 |
| 31 | UMI-Adapotor-R9 | TGAGATCGGAAGAGCACACGTCTGAACTCCAGTC | 5’端App修饰，3’端NH2C6修饰 |
| 32 | UMI-Adapotor-R10 | CGAGATCGGAAGAGCACACGTCTGAACTCCAGTC | 5’端App修饰，3’端NH2C6修饰 |
| 33 | UMI-Adapotor-R11 | GGAGATCGGAAGAGCACACGTCTGAACTCCAGTC | 5’端App修饰，3’端NH2C6修饰 |
| 34 | UMI-Adapotor-R12 | AGAGATCGGAAGAGCACACGTCTGAACTCCAGTC | 5’端App修饰，3’端NH2C6修饰 |
| 35 | UMI-Adapotor-R13 | TAAGATCGGAAGAGCACACGTCTGAACTCCAGTC | 5’端App修饰，3’端NH2C6修饰 |
| 36 | UMI-Adapotor-R14 | CAAGATCGGAAGAGCACACGTCTGAACTCCAGTC | 5’端App修饰，3’端NH2C6修饰 |
| 37 | UMI-Adapotor-R15 | GAAGATCGGAAGAGCACACGTCTGAACTCCAGTC | 5’端App修饰，3’端NH2C6修饰 |
| 38 | UMI-Adapotor-R16 | AAAGATCGGAAGAGCACACGTCTGAACTCCAGTC | 5’端App修饰，3’端NH2C6修饰 |
| 39 | PCR-F1 | AAAGACGAACTGTTTCAAGGCTTCT | |
| 40 | PCR-F2 | AAGGACGAACTGTTTCAAGGCTTCT | |
| 41 | PCR-F3 | AACGACGAACTGTTTCAAGGCTTCT | |
| 42 | PCR-F4 | AATGACGAACTGTTTCAAGGCTTCT | |
| 43 | PCR-F5 | AGAGACGAACTGTTTCAAGGCTTCT | |
| 44 | PCR-F6 | AGGGACGAACTGTTTCAAGGCTTCT | |
| 45 | PCR-F7 | AGCGACGAACTGTTTCAAGGCTTCT | |
| 46 | PCR-F8 | AGTGACGAACTGTTTCAAGGCTTCT | |
| 47 | PCR-F9 | ACAGACGAACTGTTTCAAGGCTTCT | |
| 48 | PCR-F10 | ACGGACGAACTGTTTCAAGGCTTCT | |
| 49 | PCR-F11 | ACCGACGAACTGTTTCAAGGCTTCT | |
| 50 | PCR-F12 | ACTGACGAACTGTTTCAAGGCTTCT | |
| 51 | PCR-F13 | ATAGACGAACTGTTTCAAGGCTTCT | |
| 52 | PCR-F14 | ATGGACGAACTGTTTCAAGGCTTCT | |
| 53 | PCR-F15 | ATCGACGAACTGTTTCAAGGCTTCT | |
| 54 | PCR-F16 | ATTGACGAACTGTTTCAAGGCTTCT | |
| 55 | PCR-F17 | GAAGACGAACTGTTTCAAGGCTTCT | |
| 56 | PCR-F18 | GAGGACGAACTGTTTCAAGGCTTCT | |
| 57 | PCR-F19 | GACGACGAACTGTTTCAAGGCTTCT | |
| 58 | PCR-F20 | GATGACGAACTGTTTCAAGGCTTCT | |
| 59 | PCR-F21 | GGAGACGAACTGTTTCAAGGCTTCT | |
| 60 | PCR-F22 | GGGGACGAACTGTTTCAAGGCTTCT | |
| 61 | PCR-F23 | GGCGACGAACTGTTTCAAGGCTTCT | |
| 62 | PCR-F24 | GGTGACGAACTGTTTCAAGGCTTCT | |
| 63 | PCR-F25 | GCAGACGAACTGTTTCAAGGCTTCT | |
| 64 | PCR-F26 | GCGGACGAACTGTTTCAAGGCTTCT | |
| 65 | PCR-F27 | GCCGACGAACTGTTTCAAGGCTTCT | |
| 66 | PCR-F28 | GCTGACGAACTGTTTCAAGGCTTCT | |
| 67 | PCR-F29 | GTAGACGAACTGTTTCAAGGCTTCT | |
| 68 | PCR-F30 | GTGGACGAACTGTTTCAAGGCTTCT | |
| 69 | PCR-F31 | GTCGACGAACTGTTTCAAGGCTTCT | |
| 70 | PCR-F32 | GTTGACGAACTGTTTCAAGGCTTCT | |
| 71 | PCR-F33 | CAAGACGAACTGTTTCAAGGCTTCT | |
| 72 | PCR-F34 | CAGGACGAACTGTTTCAAGGCTTCT | |
| 73 | PCR-F35 | CACGACGAACTGTTTCAAGGCTTCT | |
| 74 | PCR-F36 | CATGACGAACTGTTTCAAGGCTTCT | |
| 75 | PCR-F37 | CGAGACGAACTGTTTCAAGGCTTCT | |
| 76 | PCR-F38 | CGGGACGAACTGTTTCAAGGCTTCT | |
| 77 | PCR-F39 | CGCGACGAACTGTTTCAAGGCTTCT | |
| 78 | PCR-F40 | CGTGACGAACTGTTTCAAGGCTTCT | |
| 79 | PCR-F41 | CCAGACGAACTGTTTCAAGGCTTCT | |
| 80 | PCR-F42 | CCGGACGAACTGTTTCAAGGCTTCT | |
| 81 | PCR-F43 | CCCGACGAACTGTTTCAAGGCTTCT | |
| 82 | PCR-F44 | CCTGACGAACTGTTTCAAGGCTTCT | |
| 83 | PCR-F45 | CTAGACGAACTGTTTCAAGGCTTCT | |
| 84 | PCR-F46 | CTGGACGAACTGTTTCAAGGCTTCT | |
| 85 | PCR-F47 | CTCGACGAACTGTTTCAAGGCTTCT | |
| 86 | PCR-F48 | CTTGACGAACTGTTTCAAGGCTTCT | |
| 87 | PCR-F49 | TAAGACGAACTGTTTCAAGGCTTCT | |
| 88 | PCR-F50 | TAGGACGAACTGTTTCAAGGCTTCT | |
| 89 | PCR-F51 | TACGACGAACTGTTTCAAGGCTTCT | |
| 90 | PCR-F52 | TATGACGAACTGTTTCAAGGCTTCT | |
| 91 | PCR-F53 | TGAGACGAACTGTTTCAAGGCTTCT | |
| 92 | PCR-F54 | TGGGACGAACTGTTTCAAGGCTTCT | |
| 94 | PCR-F55 | TGCGACGAACTGTTTCAAGGCTTCT | |
| 95 | PCR-F56 | TGTGACGAACTGTTTCAAGGCTTCT | |
| 96 | PCR-F57 | TCAGACGAACTGTTTCAAGGCTTCT | |
| 97 | PCR-F58 | TCGGACGAACTGTTTCAAGGCTTCT | |
| 98 | PCR-F59 | TCCGACGAACTGTTTCAAGGCTTCT | |
| 99 | PCR-F60 | TCTGACGAACTGTTTCAAGGCTTCT | |
| 100 | PCR-F61 | TTAGACGAACTGTTTCAAGGCTTCT | |
| 101 | PCR-F62 | TTGGACGAACTGTTTCAAGGCTTCT | |
| 102 | PCR-F63 | TTCGACGAACTGTTTCAAGGCTTCT | |
| 103 | PCR-F64 | TTTGACGAACTGTTTCAAGGCTTCT | |
| 104 | PCR-R | TGGAATGGAGAGCCATTCAAC |
实施例1：重组连接酶突变体的设计。
在本实施例中，我们利用大肠杆菌表达系统纯化了野生型T4 DNA连接酶和重组突变体，包括：
（1）野生型T4 DNA连接酶T4DLWT，蛋白序列见SEQ NO: 1，cDNA序列见SEQ NO: 25；
（2）T4 DNA连接酶突变体T4DLm1（Q19K、L63T、E88R、P127K、K225A、F233A、A237R、D371W、E440K、T451K、D452P），蛋白序列见SEQ NO: 2，cDNA序列见SEQ NO: 26；
（3）T4 DNA连接酶突变体T4DLm2（K159S），蛋白序列见SEQ NO: 3，cDNA序列见SEQNO: 27；
（4）T4 DNA连接酶突变体T4DLm3（K159L），蛋白序列见SEQ NO: 4，cDNA序列见SEQNO: 28；
（5）T4 DNA连接酶突变体T4DLm4（K159C），蛋白序列见SEQ NO: 5，cDNA序列见SEQNO: 29；
（6）T4 DNA连接酶突变体T4DLm5（K159A），蛋白序列见SEQ NO: 6，cDNA序列见SEQNO: 30；
（7）T4 DNA连接酶突变体T4DLm6（K159W），蛋白序列见SEQ NO: 7，cDNA序列见SEQNO: 31；
（8）T4 DNA连接酶突变体T4DLm7（K159S、Q19K、L63T、E88R、P127K、K225A、F233A、A237R、D371W、E440K、T451K、D452P），蛋白序列见SEQ NO: 8，cDNA序列见SEQ NO: 32；
（9）T4 DNA连接酶重组突变体T4DLm7-GGGGS-p50，蛋白序列见SEQ NO: 15，cDNA序列见SEQ NO: 33；
（10）T4 DNA连接酶重组突变体T4DLm7-GGGGS-cTF，蛋白序列见SEQ NO: 16，cDNA序列见SEQ NO: 34；
（11）T4 DNA连接酶重组突变体T4DLm7-GGGGS-DSBA，蛋白序列见SEQ NO: 17，cDNA序列见SEQ NO: 35；
（12）T4 DNA连接酶重组突变体T4DLm7-GGGGS-H6，蛋白序列见SEQ NO: 18，cDNA序列见SEQ NO: 36；
（13）T4 DNA连接酶重组突变体T4DLm7-GGGGS-HNS，蛋白序列见SEQ NO: 19，cDNA序列见SEQ NO: 37；
（14）T4 DNA连接酶重组突变体T4DLm7-GGGGS-HTH，蛋白序列见SEQ NO: 20，cDNA序列见SEQ NO: 38；
（15）T4 DNA连接酶重组突变体H6-GGGGS-T4DLm7-GGGGS-H6，蛋白序列见SEQ NO:21，cDNA序列见SEQ NO: 39；
（16）T4 DNA连接酶重组突变体H6-PAPAPAPA-T4DLm7-PAPAPAPA-H6，蛋白序列见SEQ NO: 22，cDNA序列见SEQ NO: 40；
（17）T4 DNA连接酶重组突变体H6-GQGQGQGQ-T4DLm7-GQGQGQGQ-H6，蛋白序列见SEQ NO: 23，cDNA序列见SEQ NO: 41；
（18）T4 DNA连接酶重组突变体H6-AAAAA-T4DLm7-AAAAA-H6（即为T4DLm），蛋白序列见SEQ NO: 24，cDNA序列见SEQ NO: 42。
实施例2：粘性末端和平末端标准品的制备，
在本实施例中，我们制备了用于T4 DNA 连接酶连接效率测试的双链DNA标准品片段。具体实施方式如下：
用1×退火缓冲液（10 mM Tris-HCl（pH7.9），50 mM NaCl）溶解各引物（表1的序号1-4）。
磷酸化粘性末端：取50 ul 200 uM P5-Conhensive和50 ul 200 uM P-P7。 85℃2 min，85-15℃ 0.1℃/s，保存于15℃。
腺苷酰化粘性末端：取50 ul 200 uM P5-Conhensive和50 ul 200 uM App-P7。85℃ 2 min，85-15℃ 0.1℃/s，保存于15℃。
磷酸化平末端：取50 ul 200 uM P5-Blunt和50 ul 200 uM P-P7。 85℃ 2 min，85-15℃ 0.1℃/s，保存于15℃。
腺苷酰化平末端：取50 ul 200 uM P5-Blunt和50 ul 200 uM App-P7。 85℃ 2min，85-15℃ 0.1℃/s，保存于15℃。
其中磷酸化的粘性末端和平末端标准品主要应用于T4DLwt和T4DLm1的活性测试，其他腺苷酰化的粘性末端和平末端标准品主要用于其他重组T4 DNA连接酶突变体的活性测试。
实施例3：重组连接酶突变体对粘性末端的连接效率测试。
在本实施例中，我们利用qPCR验证了重组连接酶突变体在粘性末端上的连接效率。具体实施方式如下：
取10 ng退火好的磷酸化粘性末端DNA标准品，在1×磷酸化接头连接酶缓冲液（50mM Tris-HCl（pH 8.0），50 mM NaCl，10 mM MgCl2，10 mM DTT，1 mM ATP，6% PEG8000）和1ug T4DLwt或T4DLm1，20℃反应15 min。加水稀释100倍后，用qPCR-F和qPCR-R进行qPCR检测。
取10 ng退火好的腺苷酰化粘性末端DNA标准品，在1×腺苷酰化接头连接酶缓冲液（50 mM Tris-HCl（pH 8.0），50 mM NaCl，10 mM MgCl2，10 mM DTT，6% PEG8000）和1 ugT4DLm2—6以及实施例1中（9-18，SEQ ID No：15-24）的重组融合蛋白中的一种，20℃反应15min。加水稀释100倍后，用qPCR-F和qPCR-R进行qPCR检测。
结果如图1所述，T4 DNA连接酶突变体T4DLm1能够显著增强T4 DNA连接酶对5’端磷酸化DNA粘性末端底物的连接效率。当对K159位点进行突变后，T4 DNA连接酶突变体能够显著增强5’端App修饰DNA粘性末端底物的连接效率（T4DLm2-6），其中K159S突变体（T4DLm2）效果最明显。组合T4DLm1和T4DLm2突变位点的T4DLm7突变体能够进一步提高T4DL对5’端App修饰DNA粘性末端底物的连接效率。
在5’端App修饰DNA粘性末端底物的连接效率上，融合双端双链DNA结合域的重组T4DL突变体>融合单端双链DNA结合域的重组T4DL突变体>未融合的T4DL突变体。双链DNA结合域包括p50、cTF、DSBA、H6、HNS和HTH，其中H6的效果最好。H6与T4DL中间的多肽桥可以使用GGGGS多肽、PAPAPAPA多肽、GQGQGQGQ多肽和AAAAA多肽，其中AAAAA多肽的效果最佳。
实施例4：重组连接酶突变体对平末端的连接效率测试。
在本实施例中，我们利用qPCR验证了重组连接酶突变体在平末端上的连接效率。具体实施方式如下：
取10 ng退火好的磷酸化平末端DNA标准品，在1×磷酸化接头连接酶缓冲液（50mM Tris-HCl（pH 8.0），50 mM NaCl，10 mM MgCl2，10 mM DTT，1 mM ATP，6% PEG8000）和1ug T4DLwt或T4DLm1，20℃反应15 min。加水稀释100倍后，用qPCR-F和qPCR-R进行qPCR检测。
取10 ng退火好的腺苷酰化平末端DNA标准品，在1×腺苷酰化接头连接酶缓冲液（50 mM Tris-HCl（pH 8.0），50 mM NaCl，10 mM MgCl2，10 mM DTT，6% PEG8000）和1 ugT4DLm2—6以及实施例1中（9-18，SEQ ID No：15-24）的重组融合蛋白中的一种，20℃反应15min。加水稀释100倍后，用qPCR-F和qPCR-R进行qPCR检测。
结果如图2所述，T4 DNA连接酶突变体T4DLm1能够显著增强T4 DNA连接酶对5’端磷酸化DNA平末端底物的连接效率。当对K159位点进行突变后，T4 DNA连接酶突变体能够非常显著增强5’端App修饰DNA平末端底物的连接效率（T4DLm2-6），其中K159S突变体（T4DLm2）效果最明显。组合T4DLm1和T4DLm2突变位点的T4DLm7突变体能够进一步提高T4DL对5’端App修饰DNA平末端底物的连接效率。
在5’端App修饰DNA平末端底物的连接效率上，融合双端双链DNA结合域的重组T4DL突变体>融合单端双链DNA结合域的重组T4DL突变体>未融合的T4DL突变体。双链DNA结合域包括p50、cTF、DSBA、H6、HNS和HTH，其中H6的效果最好。H6与T4DL中间的多肽桥可以使用GGGGS多肽、PAPAPAPA多肽、GQGQGQGQ多肽和AAAAA多肽，其中AAAAA多肽的效果最佳。
如图1和图2所示，融合双端双链DNA结合域的重组T4DL突变体对平末端连接效率的增强效果要显著高于对粘性末端的连接效率。尽管野生型T4DL对粘性末端的连接效率（0.361）要明显高于平末端（0.115），改造后的T4DLm对平末端的连接效率（0.962）与粘性末端（0.971）差异不大。这说明经我们改造后的T4DLm对平末端接头的连接具有最佳的增强效果，能够有效解决平末端连接效率低的问题。
实施例5：平末端连接偏好性测试。
在本实施中，我们公布了基于T4DLm的简便高效的新型NGS建库技术，并用此方法测定了T4DLm2、T4DLm7和H6-AAAAA-T4DLm7-AAAAA-H6（T4DLm）的连接序列偏好性。具体实施方式如下：
（1）制备具有多样化等分子量末端序列的PCR混合物DNA标准品。我们设计了末端含有3个多样性碱基的PCR正向引物，共64种正向引物（见表1的PCR-F1-F64）。将64种PCR-F分别与PCR-R一一配对，扩增lambda DNA。使用翌圣生物的高保真DNA聚合酶预混液进行PCR扩增。将扩增产物使用0.6倍体积翌圣生物的DNA select beads进行PCR产物回收，回收后产物在Qubit上测定回收DNA产物浓度。并按照等质量混匀，获得具有相同分子数的64种不同3碱基末端的DNA标准品。
（2）制备具有多样化等分子量末端序列的腺苷酰化双链DNA接头。我们设计了末端含有2个多样性碱基的一一配对腺苷酰化接头，共16对引物（见表1的UMI-Adaptor-F1-F16和UMI-Adaptor-R1-R16）。将16种UMI-Adaptor-F分别与UMI-Adaptor-R一一配对，用1×退火缓冲液（10 mM Tris-HCl（pH7.9），50 mM NaCl）溶解各引物至100 uM，将对应的UMI-Adaptor-F分别与UMI-Adaptor-R等体积混匀，85℃ 2 min，85-15℃ 0.1℃/s，保存于15℃。将退火好的接头使用1×退火缓冲液稀释成10 uM备用。
（3）T4DL突变体介导的腺苷酰化接头连接。取步骤（1）中制备好的具有多样化末端的DNA标准品20 ng，加入步骤（2）中制备好的具有多样化末端的腺苷酰化接头 5 ul，在1×腺苷酰化接头连接酶缓冲液（50 mM Tris-HCl（pH 8.0），50 mM NaCl，10 mM MgCl2，10 mMDTT，6% PEG8000）和1 ug T4DLm2—6以及实施例1中（9-18，SEQ ID No：15-24）的重组融合蛋白中的一种，20℃反应15 min。
（4）磁珠回收DNA。在反应体系中加入0.6倍体积的翌圣生物DNA回收磁珠，混匀后置于室温孵育5 min。将PCR管置于磁力架上，待溶液澄清后，吸去上清，加入200 ul新鲜配制的70%乙醇清洗2次后，室温晾3 min。
（5）缺口修复。使用10 ul 缺口修复反应液（20 mM三羟甲基氨基甲烷、5 mM氯化镁、0.2 mM烟酰胺腺嘌呤二核苷酸（氧化型）、10 mM三磷酸腺苷、10 U T4 PNK和10 UE.coli连接酶）悬浮磁珠，37℃反应15 min后，98℃ 2 min，离心取上清。
（6）文库扩增及回收。使用翌圣生物的DNA建库试剂盒中的2×高保真酶预混液和illumina index引物进行文库扩增。扩增程序见说明书，7个扩增循环。扩增结束后，在反应体系中加入0.8倍体积的翌圣生物DNA回收磁珠，混匀后置于室温孵育5 min。将PCR管置于磁力架上，待溶液澄清后，吸去上清，加入200 ul新鲜配制的70%乙醇清洗2次后，室温晾3min。使用22 ul ddH2O悬浮磁珠，室温静置5 min后，将PCR管置于磁力架上，待溶液澄清后，吸取20 ul上清。Qubit测定文库浓度后，在NovaSeq 6000进行文库测序。
文库产量如图3所述，融合了双链DNA结合域的T4DL重组突变体能够显著提高DNA文库的产量。我们分析了T4DL对腺苷酰化接头的平末端序列偏好性，发现T4DL K159突变体均具有极严重的GC偏好性，连接GC末端接头的效率和占比是AT末端接头效率的10倍以上，这极大了限制了T4DL K159均突变体在DNA平末端连接建库上的使用（图4）。我们将T4DL突变体两端融合上双链DNA结合域后，偏好性得到了明显的改善，尤其是H6结构域最佳，且结构域与T4DL 突变体的连接多肽桥AAAAA使用时效果最好，连接GC末端接头的效率和占比与AT末端接头效率变化波动不超过2倍（图4）。我们对接头连接偏好性用均一性进行量化分析，结果见图5，我们改造后的T4DL重组突变体能够将连接均一性提高10倍以上。这证明我们的改造方向既可以提高连接效率和文库产量，又可以显著改善连接均一性，有效降低由连接造成的建库偏好性。
此外，我们也分析了T4DL重组突变体对DNA平末端序列的偏好性，结果显示与T4DL突变体对接头末端序列的偏好性类似，T4DL突变体对DNA平末端也表现出很明显的GC偏好性，改造后的T4DLm能够有效去除这种GC偏好性（图6）。我们同样对DNA末端偏好性用均一性进行量化分析，结果见图7，我们改造后的T4DL重组突变体能够将连接均一性提高10倍以上。这证明我们的改造方向能够显著降低连接酶对底物DNA末端序列识别偏好性。
实施例6：基于T4DLm的简便高效的新型NGS建库技术
在本实施中，我们公布了基于T4DLm的简便高效的新型NGS建库技术（示意图8），整个过程低于2 h，操作简便、连接效率和建库产量更高。具体实施方式如下：
（1）DNA片段化。使用表2的片段化酶反应体系:20 ng DNA在60 ul体系中进行片段化。
表2 片段化体系组成。
片段化体系1：50 mM Tris-HCl，50 mM NaCl，2 mM MgCl2，0.5 U脱氧核糖核酸I(NEB)，1 U热敏感性碱性磷酸酶（ThermoFisher）和1 U T4 DNA聚合酶(NEB)。
片段化体系2：50 mM Tris-HCl，50 mM NaCl，2 mM MgCl2，0.5 U创伤弧菌核酸酶(NEB)，1 U热敏感性碱性磷酸酶（ThermoFisher）和1 U T4 DNA聚合酶(NEB)。
片段化体系3：50 mM Tris-HCl，50 mM NaCl，2 mM MgCl2，0.5 U全能核酸酶(NEB)，1 U热敏感性碱性磷酸酶（ThermoFisher）和1 U T4 DNA聚合酶(NEB)。
片段化体系4：50 mM Tris-HCl，50 mM NaCl，2 mM MgCl2，0.5 U微球菌核酸酶(NEB)，1 U热敏感性碱性磷酸酶（ThermoFisher）和1 U T4 DNA聚合酶(NEB)。
（2）T4DL突变体介导的腺苷酰化接头连接。加入实施例5步骤（2）中制备好的具有多样化末端的腺苷酰化接头 5 ul，在30 ul腺苷酰化接头连接酶缓冲液（66.7 mM Tris-HCl（pH 8.0），66.7 mM NaCl，30 mM MgCl2，30 mM DTT，20% PEG8000）和1 ug T4DLm2—6以及实施例1中（9-18，SEQ ID No：15-24）的重组融合蛋白中的一种，20℃反应15 min。
（3）磁珠回收DNA。在反应体系中加入0.6倍体积的翌圣生物DNA回收磁珠，混匀后置于室温孵育5 min。将PCR管置于磁力架上，待溶液澄清后，吸去上清，加入200 ul新鲜配制的70%乙醇清洗2次后，室温晾3 min。
（4）缺口修复。使用10 ul 缺口修复反应液（20 mM三羟甲基氨基甲烷、5 mM氯化镁、0.2 mM烟酰胺腺嘌呤二核苷酸（氧化型）、10 mM三磷酸腺苷、10 U T4 PNK和10 UE.coli连接酶）悬浮磁珠，37℃反应15 min后，98℃ 2 min，离心取上清。
（5）文库扩增及回收。使用翌圣生物的DNA建库试剂盒中的2×高保真酶预混液和illumina index引物进行文库扩增。扩增程序见说明书，7个扩增循环。扩增结束后，在反应体系中加入0.8倍体积的翌圣生物DNA回收磁珠，混匀后置于室温孵育5 min。将PCR管置于磁力架上，待溶液澄清后，吸去上清，加入200 ul新鲜配制的70%乙醇清洗2次后，室温晾3min。使用22 ul ddH2O悬浮磁珠，室温静置5 min后，将PCR管置于磁力架上，待溶液澄清后，吸取20 ul上清。Qubit测定文库浓度后，在NovaSeq 6000进行文库测序。
结果如图9-图10所示，使用片段化酶体系4具有更好的片段化效果和文库产量。
实施例7：基于T4DLm的简便高效的新型NGS建库技术与传统DNA建库性能对比
在本实施例中，我们对比了不同DNA投入量的两种建库方法性能对比。我们将人类基因组DNA、酵母基因组DNA和大肠杆菌基因组DNA按照10:3:1的质量比例进行混合，获得具有复杂物种的DNA标准品。基于T4DLm的简便高效的新型NGS建库技术具体实施方式见实施例6，传统DNA建库具体实施方式按照NEB的酶切法DNA建库试剂盒进行建库。获得的文库经Qubit测定DNA浓度后，在Illumina NovaSeq 6000测序平台上进行测序。同时，将获得的文库利用IDT的杂交基因捕获试剂盒进行xGen Inherited Diseases Panel的捕获测序，分析不同建库方法获得文库的捕获特异性、深度、均一性和效率。
文库产量如图11所示，基于T4DLm的新型NGS建库技术比传统DNA建库具有更高的文库产量，这说明基于T4DLm的新型NGS建库技术对DNA模板的利用率更高。图12的相关性结果表明基于T4DLm的新型NGS建库技术获得的数据与DNA模板中的理论各DNA投入比具有更好的相关性。我们利用基因捕获测序验证了模板利用率效果，结果显示基于T4DLm的新型NGS建库技术在基因捕获测序结果上具有更好的测序深度、捕获效率和均一性（图13-图15）。这些结果表明基于T4DLm的新型NGS建库技术具有更低的建库偏好性和更高的建库效率。
综上，我们开发了一款新型的重组T4 DNA连接酶突变体，在野生型T4DL（SEQ NO:1）上，进行了Q19K、L63T、E88R、P127K、K159S、K225A、F233A、A237R、D371W、E440K、T451K、D452P等多个点突变；在突变体的两端包含两个双链DNA结合域；双链DNA结合域与T4DL之间的连接使用多肽桥。最终获得了一种低偏好性和高效率的重组DNA连接酶突变体T4DLm。T4DLm在平末端连接上具有显著的优势，连接效率高达95%，且偏好性极低。我们利用T4DLm开发了高效简便的新型NGS建库技术，具有耗时短、文库产量更高、均一性更好、文库自连更低、捕获测序数据覆盖深度更好等明显的优势，非常适用于临床样本的NGS检测，尤其是肿瘤样本的检测。
序列表
<110> 翌圣生物科技（上海）股份有限公司
<120> 重组T4连接酶突变体、编码DNA及NGS建库方法
<141> 2021-09-07
<160> 42
<170> SIPOSequenceListing 1.0
<210> 1
<211> 487
<212> PRT
<213> artifical sequence
<400> 1
Met Ile Leu Lys Ile Leu Asn Glu Ile Ala Ser Ile Gly Ser Thr Lys
1 5 10 15
Gln Lys Gln Ala Ile Leu Glu Lys Asn Lys Asp Asn Glu Leu Leu Lys
20 25 30
Arg Val Tyr Arg Leu Thr Tyr Ser Arg Gly Leu Gln Tyr Tyr Ile Lys
35 40 45
Lys Trp Pro Lys Pro Gly Ile Ala Thr Gln Ser Phe Gly Met Leu Thr
50 55 60
Leu Thr Asp Met Leu Asp Phe Ile Glu Phe Thr Leu Ala Thr Arg Lys
65 70 75 80
Leu Thr Gly Asn Ala Ala Ile Glu Glu Leu Thr Gly Tyr Ile Thr Asp
85 90 95
Gly Lys Lys Asp Asp Val Glu Val Leu Arg Arg Val Met Met Arg Asp
100 105 110
Leu Glu Cys Gly Ala Ser Val Ser Ile Ala Asn Lys Val Trp Pro Gly
115 120 125
Leu Ile Pro Glu Gln Pro Gln Met Leu Ala Ser Ser Tyr Asp Glu Lys
130 135 140
Gly Ile Asn Lys Asn Ile Lys Phe Pro Ala Phe Ala Gln Leu Lys Ala
145 150 155 160
Asp Gly Ala Arg Cys Phe Ala Glu Val Arg Gly Asp Glu Leu Asp Asp
165 170 175
Val Arg Leu Leu Ser Arg Ala Gly Asn Glu Tyr Leu Gly Leu Asp Leu
180 185 190
Leu Lys Glu Glu Leu Ile Lys Met Thr Ala Glu Ala Arg Gln Ile His
195 200 205
Pro Glu Gly Val Leu Ile Asp Gly Glu Leu Val Tyr His Glu Gln Val
210 215 220
Lys Lys Glu Pro Glu Gly Leu Asp Phe Leu Phe Asp Ala Tyr Pro Glu
225 230 235 240
Asn Ser Lys Ala Lys Glu Phe Ala Glu Val Ala Glu Ser Arg Thr Ala
245 250 255
Ser Asn Gly Ile Ala Asn Lys Ser Leu Lys Gly Thr Ile Ser Glu Lys
260 265 270
Glu Ala Gln Cys Met Lys Phe Gln Val Trp Asp Tyr Val Pro Leu Val
275 280 285
Glu Ile Tyr Ser Leu Pro Ala Phe Arg Leu Lys Tyr Asp Val Arg Phe
290 295 300
Ser Lys Leu Glu Gln Met Thr Ser Gly Tyr Asp Lys Val Ile Leu Ile
305 310 315 320
Glu Asn Gln Val Val Asn Asn Leu Asp Glu Ala Lys Val Ile Tyr Lys
325 330 335
Lys Tyr Ile Asp Gln Gly Leu Glu Gly Ile Ile Leu Lys Asn Ile Asp
340 345 350
Gly Leu Trp Glu Asn Ala Arg Ser Lys Asn Leu Tyr Lys Phe Lys Glu
355 360 365
Val Ile Asp Val Asp Leu Lys Ile Val Gly Ile Tyr Pro His Arg Lys
370 375 380
Asp Pro Thr Lys Ala Gly Gly Phe Ile Leu Glu Ser Glu Cys Gly Lys
385 390 395 400
Ile Lys Val Asn Ala Gly Ser Gly Leu Lys Asp Lys Ala Gly Val Lys
405 410 415
Ser His Glu Leu Asp Arg Thr Arg Ile Met Glu Asn Gln Asn Tyr Tyr
420 425 430
Ile Gly Lys Ile Leu Glu Cys Glu Cys Asn Gly Trp Leu Lys Ser Asp
435 440 445
Gly Arg Thr Asp Tyr Val Lys Leu Phe Leu Pro Ile Ala Ile Arg Leu
450 455 460
Arg Glu Asp Lys Thr Lys Ala Asn Thr Phe Glu Asp Val Phe Gly Asp
465 470 475 480
Phe His Glu Val Thr Gly Leu
485
<210> 2
<211> 487
<212> PRT
<213> artifical sequence
<400> 2
Met Ile Leu Lys Ile Leu Asn Glu Ile Ala Ser Ile Gly Ser Thr Lys
1 5 10 15
Gln Lys Lys Ala Ile Leu Glu Lys Asn Lys Asp Asn Glu Leu Leu Lys
20 25 30
Arg Val Tyr Arg Leu Thr Tyr Ser Arg Gly Leu Gln Tyr Tyr Ile Lys
35 40 45
Lys Trp Pro Lys Pro Gly Ile Ala Thr Gln Ser Phe Gly Met Thr Thr
50 55 60
Leu Thr Asp Met Leu Asp Phe Ile Glu Phe Thr Leu Ala Thr Arg Lys
65 70 75 80
Leu Thr Gly Asn Ala Ala Ile Arg Glu Leu Thr Gly Tyr Ile Thr Asp
85 90 95
Gly Lys Lys Asp Asp Val Glu Val Leu Arg Arg Val Met Met Arg Asp
100 105 110
Leu Glu Cys Gly Ala Ser Val Ser Ile Ala Asn Lys Val Trp Lys Gly
115 120 125
Leu Ile Pro Glu Gln Pro Gln Met Leu Ala Ser Ser Tyr Asp Glu Lys
130 135 140
Gly Ile Asn Lys Asn Ile Lys Phe Pro Ala Phe Ala Gln Leu Lys Ala
145 150 155 160
Asp Gly Ala Arg Cys Phe Ala Glu Val Arg Gly Asp Glu Leu Asp Asp
165 170 175
Val Arg Leu Leu Ser Arg Ala Gly Asn Glu Tyr Leu Gly Leu Asp Leu
180 185 190
Leu Lys Glu Glu Leu Ile Lys Met Thr Ala Glu Ala Arg Gln Ile His
195 200 205
Pro Glu Gly Val Leu Ile Asp Gly Glu Leu Val Tyr His Glu Gln Val
210 215 220
Ala Lys Glu Pro Glu Gly Leu Asp Ala Leu Phe Asp Arg Tyr Pro Glu
225 230 235 240
Asn Ser Lys Ala Lys Glu Phe Ala Glu Val Ala Glu Ser Arg Thr Ala
245 250 255
Ser Asn Gly Ile Ala Asn Lys Ser Leu Lys Gly Thr Ile Ser Glu Lys
260 265 270
Glu Ala Gln Cys Met Lys Phe Gln Val Trp Asp Tyr Val Pro Leu Val
275 280 285
Glu Ile Tyr Ser Leu Pro Ala Phe Arg Leu Lys Tyr Asp Val Arg Phe
290 295 300
Ser Lys Leu Glu Gln Met Thr Ser Gly Tyr Asp Lys Val Ile Leu Ile
305 310 315 320
Glu Asn Gln Val Val Asn Asn Leu Asp Glu Ala Lys Val Ile Tyr Lys
325 330 335
Lys Tyr Ile Asp Gln Gly Leu Glu Gly Ile Ile Leu Lys Asn Ile Asp
340 345 350
Gly Leu Trp Glu Asn Ala Arg Ser Lys Asn Leu Tyr Lys Phe Lys Glu
355 360 365
Val Ile Trp Val Asp Leu Lys Ile Val Gly Ile Tyr Pro His Arg Lys
370 375 380
Asp Pro Thr Lys Ala Gly Gly Phe Ile Leu Glu Ser Glu Cys Gly Lys
385 390 395 400
Ile Lys Val Asn Ala Gly Ser Gly Leu Lys Asp Lys Ala Gly Val Lys
405 410 415
Ser His Glu Leu Asp Arg Thr Arg Ile Met Glu Asn Gln Asn Tyr Tyr
420 425 430
Ile Gly Lys Ile Leu Glu Cys Lys Cys Asn Gly Trp Leu Lys Ser Asp
435 440 445
Gly Arg Lys Pro Tyr Val Lys Leu Phe Leu Pro Ile Ala Ile Arg Leu
450 455 460
Arg Glu Asp Lys Thr Lys Ala Asn Thr Phe Glu Asp Val Phe Gly Asp
465 470 475 480
Phe His Glu Val Thr Gly Leu
485
<210> 3
<211> 487
<212> PRT
<213> artifical sequence
<400> 3
Met Ile Leu Lys Ile Leu Asn Glu Ile Ala Ser Ile Gly Ser Thr Lys
1 5 10 15
Gln Lys Gln Ala Ile Leu Glu Lys Asn Lys Asp Asn Glu Leu Leu Lys
20 25 30
Arg Val Tyr Arg Leu Thr Tyr Ser Arg Gly Leu Gln Tyr Tyr Ile Lys
35 40 45
Lys Trp Pro Lys Pro Gly Ile Ala Thr Gln Ser Phe Gly Met Leu Thr
50 55 60
Leu Thr Asp Met Leu Asp Phe Ile Glu Phe Thr Leu Ala Thr Arg Lys
65 70 75 80
Leu Thr Gly Asn Ala Ala Ile Glu Glu Leu Thr Gly Tyr Ile Thr Asp
85 90 95
Gly Lys Lys Asp Asp Val Glu Val Leu Arg Arg Val Met Met Arg Asp
100 105 110
Leu Glu Cys Gly Ala Ser Val Ser Ile Ala Asn Lys Val Trp Pro Gly
115 120 125
Leu Ile Pro Glu Gln Pro Gln Met Leu Ala Ser Ser Tyr Asp Glu Lys
130 135 140
Gly Ile Asn Lys Asn Ile Lys Phe Pro Ala Phe Ala Gln Leu Ser Ala
145 150 155 160
Asp Gly Ala Arg Cys Phe Ala Glu Val Arg Gly Asp Glu Leu Asp Asp
165 170 175
Val Arg Leu Leu Ser Arg Ala Gly Asn Glu Tyr Leu Gly Leu Asp Leu
180 185 190
Leu Lys Glu Glu Leu Ile Lys Met Thr Ala Glu Ala Arg Gln Ile His
195 200 205
Pro Glu Gly Val Leu Ile Asp Gly Glu Leu Val Tyr His Glu Gln Val
210 215 220
Lys Lys Glu Pro Glu Gly Leu Asp Phe Leu Phe Asp Ala Tyr Pro Glu
225 230 235 240
Asn Ser Lys Ala Lys Glu Phe Ala Glu Val Ala Glu Ser Arg Thr Ala
245 250 255
Ser Asn Gly Ile Ala Asn Lys Ser Leu Lys Gly Thr Ile Ser Glu Lys
260 265 270
Glu Ala Gln Cys Met Lys Phe Gln Val Trp Asp Tyr Val Pro Leu Val
275 280 285
Glu Ile Tyr Ser Leu Pro Ala Phe Arg Leu Lys Tyr Asp Val Arg Phe
290 295 300
Ser Lys Leu Glu Gln Met Thr Ser Gly Tyr Asp Lys Val Ile Leu Ile
305 310 315 320
Glu Asn Gln Val Val Asn Asn Leu Asp Glu Ala Lys Val Ile Tyr Lys
325 330 335
Lys Tyr Ile Asp Gln Gly Leu Glu Gly Ile Ile Leu Lys Asn Ile Asp
340 345 350
Gly Leu Trp Glu Asn Ala Arg Ser Lys Asn Leu Tyr Lys Phe Lys Glu
355 360 365
Val Ile Asp Val Asp Leu Lys Ile Val Gly Ile Tyr Pro His Arg Lys
370 375 380
Asp Pro Thr Lys Ala Gly Gly Phe Ile Leu Glu Ser Glu Cys Gly Lys
385 390 395 400
Ile Lys Val Asn Ala Gly Ser Gly Leu Lys Asp Lys Ala Gly Val Lys
405 410 415
Ser His Glu Leu Asp Arg Thr Arg Ile Met Glu Asn Gln Asn Tyr Tyr
420 425 430
Ile Gly Lys Ile Leu Glu Cys Glu Cys Asn Gly Trp Leu Lys Ser Asp
435 440 445
Gly Arg Thr Asp Tyr Val Lys Leu Phe Leu Pro Ile Ala Ile Arg Leu
450 455 460
Arg Glu Asp Lys Thr Lys Ala Asn Thr Phe Glu Asp Val Phe Gly Asp
465 470 475 480
Phe His Glu Val Thr Gly Leu
485
<210> 4
<211> 487
<212> PRT
<213> artifical sequence
<400> 4
Met Ile Leu Lys Ile Leu Asn Glu Ile Ala Ser Ile Gly Ser Thr Lys
1 5 10 15
Gln Lys Gln Ala Ile Leu Glu Lys Asn Lys Asp Asn Glu Leu Leu Lys
20 25 30
Arg Val Tyr Arg Leu Thr Tyr Ser Arg Gly Leu Gln Tyr Tyr Ile Lys
35 40 45
Lys Trp Pro Lys Pro Gly Ile Ala Thr Gln Ser Phe Gly Met Leu Thr
50 55 60
Leu Thr Asp Met Leu Asp Phe Ile Glu Phe Thr Leu Ala Thr Arg Lys
65 70 75 80
Leu Thr Gly Asn Ala Ala Ile Glu Glu Leu Thr Gly Tyr Ile Thr Asp
85 90 95
Gly Lys Lys Asp Asp Val Glu Val Leu Arg Arg Val Met Met Arg Asp
100 105 110
Leu Glu Cys Gly Ala Ser Val Ser Ile Ala Asn Lys Val Trp Pro Gly
115 120 125
Leu Ile Pro Glu Gln Pro Gln Met Leu Ala Ser Ser Tyr Asp Glu Lys
130 135 140
Gly Ile Asn Lys Asn Ile Lys Phe Pro Ala Phe Ala Gln Leu Leu Ala
145 150 155 160
Asp Gly Ala Arg Cys Phe Ala Glu Val Arg Gly Asp Glu Leu Asp Asp
165 170 175
Val Arg Leu Leu Ser Arg Ala Gly Asn Glu Tyr Leu Gly Leu Asp Leu
180 185 190
Leu Lys Glu Glu Leu Ile Lys Met Thr Ala Glu Ala Arg Gln Ile His
195 200 205
Pro Glu Gly Val Leu Ile Asp Gly Glu Leu Val Tyr His Glu Gln Val
210 215 220
Lys Lys Glu Pro Glu Gly Leu Asp Phe Leu Phe Asp Ala Tyr Pro Glu
225 230 235 240
Asn Ser Lys Ala Lys Glu Phe Ala Glu Val Ala Glu Ser Arg Thr Ala
245 250 255
Ser Asn Gly Ile Ala Asn Lys Ser Leu Lys Gly Thr Ile Ser Glu Lys
260 265 270
Glu Ala Gln Cys Met Lys Phe Gln Val Trp Asp Tyr Val Pro Leu Val
275 280 285
Glu Ile Tyr Ser Leu Pro Ala Phe Arg Leu Lys Tyr Asp Val Arg Phe
290 295 300
Ser Lys Leu Glu Gln Met Thr Ser Gly Tyr Asp Lys Val Ile Leu Ile
305 310 315 320
Glu Asn Gln Val Val Asn Asn Leu Asp Glu Ala Lys Val Ile Tyr Lys
325 330 335
Lys Tyr Ile Asp Gln Gly Leu Glu Gly Ile Ile Leu Lys Asn Ile Asp
340 345 350
Gly Leu Trp Glu Asn Ala Arg Ser Lys Asn Leu Tyr Lys Phe Lys Glu
355 360 365
Val Ile Asp Val Asp Leu Lys Ile Val Gly Ile Tyr Pro His Arg Lys
370 375 380
Asp Pro Thr Lys Ala Gly Gly Phe Ile Leu Glu Ser Glu Cys Gly Lys
385 390 395 400
Ile Lys Val Asn Ala Gly Ser Gly Leu Lys Asp Lys Ala Gly Val Lys
405 410 415
Ser His Glu Leu Asp Arg Thr Arg Ile Met Glu Asn Gln Asn Tyr Tyr
420 425 430
Ile Gly Lys Ile Leu Glu Cys Glu Cys Asn Gly Trp Leu Lys Ser Asp
435 440 445
Gly Arg Thr Asp Tyr Val Lys Leu Phe Leu Pro Ile Ala Ile Arg Leu
450 455 460
Arg Glu Asp Lys Thr Lys Ala Asn Thr Phe Glu Asp Val Phe Gly Asp
465 470 475 480
Phe His Glu Val Thr Gly Leu
485
<210> 5
<211> 487
<212> PRT
<213> artifical sequence
<400> 5
Met Ile Leu Lys Ile Leu Asn Glu Ile Ala Ser Ile Gly Ser Thr Lys
1 5 10 15
Gln Lys Gln Ala Ile Leu Glu Lys Asn Lys Asp Asn Glu Leu Leu Lys
20 25 30
Arg Val Tyr Arg Leu Thr Tyr Ser Arg Gly Leu Gln Tyr Tyr Ile Lys
35 40 45
Lys Trp Pro Lys Pro Gly Ile Ala Thr Gln Ser Phe Gly Met Leu Thr
50 55 60
Leu Thr Asp Met Leu Asp Phe Ile Glu Phe Thr Leu Ala Thr Arg Lys
65 70 75 80
Leu Thr Gly Asn Ala Ala Ile Glu Glu Leu Thr Gly Tyr Ile Thr Asp
85 90 95
Gly Lys Lys Asp Asp Val Glu Val Leu Arg Arg Val Met Met Arg Asp
100 105 110
Leu Glu Cys Gly Ala Ser Val Ser Ile Ala Asn Lys Val Trp Pro Gly
115 120 125
Leu Ile Pro Glu Gln Pro Gln Met Leu Ala Ser Ser Tyr Asp Glu Lys
130 135 140
Gly Ile Asn Lys Asn Ile Lys Phe Pro Ala Phe Ala Gln Leu Cys Ala
145 150 155 160
Asp Gly Ala Arg Cys Phe Ala Glu Val Arg Gly Asp Glu Leu Asp Asp
165 170 175
Val Arg Leu Leu Ser Arg Ala Gly Asn Glu Tyr Leu Gly Leu Asp Leu
180 185 190
Leu Lys Glu Glu Leu Ile Lys Met Thr Ala Glu Ala Arg Gln Ile His
195 200 205
Pro Glu Gly Val Leu Ile Asp Gly Glu Leu Val Tyr His Glu Gln Val
210 215 220
Lys Lys Glu Pro Glu Gly Leu Asp Phe Leu Phe Asp Ala Tyr Pro Glu
225 230 235 240
Asn Ser Lys Ala Lys Glu Phe Ala Glu Val Ala Glu Ser Arg Thr Ala
245 250 255
Ser Asn Gly Ile Ala Asn Lys Ser Leu Lys Gly Thr Ile Ser Glu Lys
260 265 270
Glu Ala Gln Cys Met Lys Phe Gln Val Trp Asp Tyr Val Pro Leu Val
275 280 285
Glu Ile Tyr Ser Leu Pro Ala Phe Arg Leu Lys Tyr Asp Val Arg Phe
290 295 300
Ser Lys Leu Glu Gln Met Thr Ser Gly Tyr Asp Lys Val Ile Leu Ile
305 310 315 320
Glu Asn Gln Val Val Asn Asn Leu Asp Glu Ala Lys Val Ile Tyr Lys
325 330 335
Lys Tyr Ile Asp Gln Gly Leu Glu Gly Ile Ile Leu Lys Asn Ile Asp
340 345 350
Gly Leu Trp Glu Asn Ala Arg Ser Lys Asn Leu Tyr Lys Phe Lys Glu
355 360 365
Val Ile Asp Val Asp Leu Lys Ile Val Gly Ile Tyr Pro His Arg Lys
370 375 380
Asp Pro Thr Lys Ala Gly Gly Phe Ile Leu Glu Ser Glu Cys Gly Lys
385 390 395 400
Ile Lys Val Asn Ala Gly Ser Gly Leu Lys Asp Lys Ala Gly Val Lys
405 410 415
Ser His Glu Leu Asp Arg Thr Arg Ile Met Glu Asn Gln Asn Tyr Tyr
420 425 430
Ile Gly Lys Ile Leu Glu Cys Glu Cys Asn Gly Trp Leu Lys Ser Asp
435 440 445
Gly Arg Thr Asp Tyr Val Lys Leu Phe Leu Pro Ile Ala Ile Arg Leu
450 455 460
Arg Glu Asp Lys Thr Lys Ala Asn Thr Phe Glu Asp Val Phe Gly Asp
465 470 475 480
Phe His Glu Val Thr Gly Leu
485
<210> 6
<211> 487
<212> PRT
<213> artifical sequence
<400> 6
Met Ile Leu Lys Ile Leu Asn Glu Ile Ala Ser Ile Gly Ser Thr Lys
1 5 10 15
Gln Lys Gln Ala Ile Leu Glu Lys Asn Lys Asp Asn Glu Leu Leu Lys
20 25 30
Arg Val Tyr Arg Leu Thr Tyr Ser Arg Gly Leu Gln Tyr Tyr Ile Lys
35 40 45
Lys Trp Pro Lys Pro Gly Ile Ala Thr Gln Ser Phe Gly Met Leu Thr
50 55 60
Leu Thr Asp Met Leu Asp Phe Ile Glu Phe Thr Leu Ala Thr Arg Lys
65 70 75 80
Leu Thr Gly Asn Ala Ala Ile Glu Glu Leu Thr Gly Tyr Ile Thr Asp
85 90 95
Gly Lys Lys Asp Asp Val Glu Val Leu Arg Arg Val Met Met Arg Asp
100 105 110
Leu Glu Cys Gly Ala Ser Val Ser Ile Ala Asn Lys Val Trp Pro Gly
115 120 125
Leu Ile Pro Glu Gln Pro Gln Met Leu Ala Ser Ser Tyr Asp Glu Lys
130 135 140
Gly Ile Asn Lys Asn Ile Lys Phe Pro Ala Phe Ala Gln Leu Ala Ala
145 150 155 160
Asp Gly Ala Arg Cys Phe Ala Glu Val Arg Gly Asp Glu Leu Asp Asp
165 170 175
Val Arg Leu Leu Ser Arg Ala Gly Asn Glu Tyr Leu Gly Leu Asp Leu
180 185 190
Leu Lys Glu Glu Leu Ile Lys Met Thr Ala Glu Ala Arg Gln Ile His
195 200 205
Pro Glu Gly Val Leu Ile Asp Gly Glu Leu Val Tyr His Glu Gln Val
210 215 220
Lys Lys Glu Pro Glu Gly Leu Asp Phe Leu Phe Asp Ala Tyr Pro Glu
225 230 235 240
Asn Ser Lys Ala Lys Glu Phe Ala Glu Val Ala Glu Ser Arg Thr Ala
245 250 255
Ser Asn Gly Ile Ala Asn Lys Ser Leu Lys Gly Thr Ile Ser Glu Lys
260 265 270
Glu Ala Gln Cys Met Lys Phe Gln Val Trp Asp Tyr Val Pro Leu Val
275 280 285
Glu Ile Tyr Ser Leu Pro Ala Phe Arg Leu Lys Tyr Asp Val Arg Phe
290 295 300
Ser Lys Leu Glu Gln Met Thr Ser Gly Tyr Asp Lys Val Ile Leu Ile
305 310 315 320
Glu Asn Gln Val Val Asn Asn Leu Asp Glu Ala Lys Val Ile Tyr Lys
325 330 335
Lys Tyr Ile Asp Gln Gly Leu Glu Gly Ile Ile Leu Lys Asn Ile Asp
340 345 350
Gly Leu Trp Glu Asn Ala Arg Ser Lys Asn Leu Tyr Lys Phe Lys Glu
355 360 365
Val Ile Asp Val Asp Leu Lys Ile Val Gly Ile Tyr Pro His Arg Lys
370 375 380
Asp Pro Thr Lys Ala Gly Gly Phe Ile Leu Glu Ser Glu Cys Gly Lys
385 390 395 400
Ile Lys Val Asn Ala Gly Ser Gly Leu Lys Asp Lys Ala Gly Val Lys
405 410 415
Ser His Glu Leu Asp Arg Thr Arg Ile Met Glu Asn Gln Asn Tyr Tyr
420 425 430
Ile Gly Lys Ile Leu Glu Cys Glu Cys Asn Gly Trp Leu Lys Ser Asp
435 440 445
Gly Arg Thr Asp Tyr Val Lys Leu Phe Leu Pro Ile Ala Ile Arg Leu
450 455 460
Arg Glu Asp Lys Thr Lys Ala Asn Thr Phe Glu Asp Val Phe Gly Asp
465 470 475 480
Phe His Glu Val Thr Gly Leu
485
<210> 7
<211> 487
<212> PRT
<213> artifical sequence
<400> 7
Met Ile Leu Lys Ile Leu Asn Glu Ile Ala Ser Ile Gly Ser Thr Lys
1 5 10 15
Gln Lys Gln Ala Ile Leu Glu Lys Asn Lys Asp Asn Glu Leu Leu Lys
20 25 30
Arg Val Tyr Arg Leu Thr Tyr Ser Arg Gly Leu Gln Tyr Tyr Ile Lys
35 40 45
Lys Trp Pro Lys Pro Gly Ile Ala Thr Gln Ser Phe Gly Met Leu Thr
50 55 60
Leu Thr Asp Met Leu Asp Phe Ile Glu Phe Thr Leu Ala Thr Arg Lys
65 70 75 80
Leu Thr Gly Asn Ala Ala Ile Glu Glu Leu Thr Gly Tyr Ile Thr Asp
85 90 95
Gly Lys Lys Asp Asp Val Glu Val Leu Arg Arg Val Met Met Arg Asp
100 105 110
Leu Glu Cys Gly Ala Ser Val Ser Ile Ala Asn Lys Val Trp Pro Gly
115 120 125
Leu Ile Pro Glu Gln Pro Gln Met Leu Ala Ser Ser Tyr Asp Glu Lys
130 135 140
Gly Ile Asn Lys Asn Ile Lys Phe Pro Ala Phe Ala Gln Leu Trp Ala
145 150 155 160
Asp Gly Ala Arg Cys Phe Ala Glu Val Arg Gly Asp Glu Leu Asp Asp
165 170 175
Val Arg Leu Leu Ser Arg Ala Gly Asn Glu Tyr Leu Gly Leu Asp Leu
180 185 190
Leu Lys Glu Glu Leu Ile Lys Met Thr Ala Glu Ala Arg Gln Ile His
195 200 205
Pro Glu Gly Val Leu Ile Asp Gly Glu Leu Val Tyr His Glu Gln Val
210 215 220
Lys Lys Glu Pro Glu Gly Leu Asp Phe Leu Phe Asp Ala Tyr Pro Glu
225 230 235 240
Asn Ser Lys Ala Lys Glu Phe Ala Glu Val Ala Glu Ser Arg Thr Ala
245 250 255
Ser Asn Gly Ile Ala Asn Lys Ser Leu Lys Gly Thr Ile Ser Glu Lys
260 265 270
Glu Ala Gln Cys Met Lys Phe Gln Val Trp Asp Tyr Val Pro Leu Val
275 280 285
Glu Ile Tyr Ser Leu Pro Ala Phe Arg Leu Lys Tyr Asp Val Arg Phe
290 295 300
Ser Lys Leu Glu Gln Met Thr Ser Gly Tyr Asp Lys Val Ile Leu Ile
305 310 315 320
Glu Asn Gln Val Val Asn Asn Leu Asp Glu Ala Lys Val Ile Tyr Lys
325 330 335
Lys Tyr Ile Asp Gln Gly Leu Glu Gly Ile Ile Leu Lys Asn Ile Asp
340 345 350
Gly Leu Trp Glu Asn Ala Arg Ser Lys Asn Leu Tyr Lys Phe Lys Glu
355 360 365
Val Ile Asp Val Asp Leu Lys Ile Val Gly Ile Tyr Pro His Arg Lys
370 375 380
Asp Pro Thr Lys Ala Gly Gly Phe Ile Leu Glu Ser Glu Cys Gly Lys
385 390 395 400
Ile Lys Val Asn Ala Gly Ser Gly Leu Lys Asp Lys Ala Gly Val Lys
405 410 415
Ser His Glu Leu Asp Arg Thr Arg Ile Met Glu Asn Gln Asn Tyr Tyr
420 425 430
Ile Gly Lys Ile Leu Glu Cys Glu Cys Asn Gly Trp Leu Lys Ser Asp
435 440 445
Gly Arg Thr Asp Tyr Val Lys Leu Phe Leu Pro Ile Ala Ile Arg Leu
450 455 460
Arg Glu Asp Lys Thr Lys Ala Asn Thr Phe Glu Asp Val Phe Gly Asp
465 470 475 480
Phe His Glu Val Thr Gly Leu
485
<210> 8
<211> 487
<212> PRT
<213> artifical sequence
<400> 8
Met Ile Leu Lys Ile Leu Asn Glu Ile Ala Ser Ile Gly Ser Thr Lys
1 5 10 15
Gln Lys Lys Ala Ile Leu Glu Lys Asn Lys Asp Asn Glu Leu Leu Lys
20 25 30
Arg Val Tyr Arg Leu Thr Tyr Ser Arg Gly Leu Gln Tyr Tyr Ile Lys
35 40 45
Lys Trp Pro Lys Pro Gly Ile Ala Thr Gln Ser Phe Gly Met Thr Thr
50 55 60
Leu Thr Asp Met Leu Asp Phe Ile Glu Phe Thr Leu Ala Thr Arg Lys
65 70 75 80
Leu Thr Gly Asn Ala Ala Ile Arg Glu Leu Thr Gly Tyr Ile Thr Asp
85 90 95
Gly Lys Lys Asp Asp Val Glu Val Leu Arg Arg Val Met Met Arg Asp
100 105 110
Leu Glu Cys Gly Ala Ser Val Ser Ile Ala Asn Lys Val Trp Lys Gly
115 120 125
Leu Ile Pro Glu Gln Pro Gln Met Leu Ala Ser Ser Tyr Asp Glu Lys
130 135 140
Gly Ile Asn Lys Asn Ile Lys Phe Pro Ala Phe Ala Gln Leu Ser Ala
145 150 155 160
Asp Gly Ala Arg Cys Phe Ala Glu Val Arg Gly Asp Glu Leu Asp Asp
165 170 175
Val Arg Leu Leu Ser Arg Ala Gly Asn Glu Tyr Leu Gly Leu Asp Leu
180 185 190
Leu Lys Glu Glu Leu Ile Lys Met Thr Ala Glu Ala Arg Gln Ile His
195 200 205
Pro Glu Gly Val Leu Ile Asp Gly Glu Leu Val Tyr His Glu Gln Val
210 215 220
Ala Lys Glu Pro Glu Gly Leu Asp Ala Leu Phe Asp Arg Tyr Pro Glu
225 230 235 240
Asn Ser Lys Ala Lys Glu Phe Ala Glu Val Ala Glu Ser Arg Thr Ala
245 250 255
Ser Asn Gly Ile Ala Asn Lys Ser Leu Lys Gly Thr Ile Ser Glu Lys
260 265 270
Glu Ala Gln Cys Met Lys Phe Gln Val Trp Asp Tyr Val Pro Leu Val
275 280 285
Glu Ile Tyr Ser Leu Pro Ala Phe Arg Leu Lys Tyr Asp Val Arg Phe
290 295 300
Ser Lys Leu Glu Gln Met Thr Ser Gly Tyr Asp Lys Val Ile Leu Ile
305 310 315 320
Glu Asn Gln Val Val Asn Asn Leu Asp Glu Ala Lys Val Ile Tyr Lys
325 330 335
Lys Tyr Ile Asp Gln Gly Leu Glu Gly Ile Ile Leu Lys Asn Ile Asp
340 345 350
Gly Leu Trp Glu Asn Ala Arg Ser Lys Asn Leu Tyr Lys Phe Lys Glu
355 360 365
Val Ile Trp Val Asp Leu Lys Ile Val Gly Ile Tyr Pro His Arg Lys
370 375 380
Asp Pro Thr Lys Ala Gly Gly Phe Ile Leu Glu Ser Glu Cys Gly Lys
385 390 395 400
Ile Lys Val Asn Ala Gly Ser Gly Leu Lys Asp Lys Ala Gly Val Lys
405 410 415
Ser His Glu Leu Asp Arg Thr Arg Ile Met Glu Asn Gln Asn Tyr Tyr
420 425 430
Ile Gly Lys Ile Leu Glu Cys Lys Cys Asn Gly Trp Leu Lys Ser Asp
435 440 445
Gly Arg Lys Pro Tyr Val Lys Leu Phe Leu Pro Ile Ala Ile Arg Leu
450 455 460
Arg Glu Asp Lys Thr Lys Ala Asn Thr Phe Glu Asp Val Phe Gly Asp
465 470 475 480
Phe His Glu Val Thr Gly Leu
485
<210> 9
<211> 522
<212> PRT
<213> artifical sequence
<400> 9
Arg Glu Ile Leu Asn Pro Pro Glu Lys Glu Thr Gln Gly Glu Gly Pro
1 5 10 15
Ser Leu Phe Met Ala Ser Thr Lys Thr Glu Ala Ile Ala Pro Ala Ser
20 25 30
Thr Met Glu Asp Lys Glu Glu Asp Val Gly Phe Gln Asp Asn Leu Phe
35 40 45
Leu Glu Lys Ala Leu Gln Leu Ala Lys Arg His Ala Asn Ala Leu Phe
50 55 60
Asp Tyr Ala Val Thr Gly Asp Val Lys Met Leu Leu Ala Val Gln Arg
65 70 75 80
His Leu Thr Ala Val Gln Asp Glu Asn Gly Asp Ser Val Leu His Leu
85 90 95
Ala Ile Ile His Leu His Ala Gln Leu Val Arg Asp Leu Leu Glu Val
100 105 110
Thr Ser Gly Ser Ile Ser Asp Asp Ile Ile Asn Met Arg Asn Asp Leu
115 120 125
Tyr Gln Thr Pro Leu His Leu Ala Val Ile Thr Lys Gln Glu Asp Val
130 135 140
Val Glu Asp Leu Leu Arg Val Gly Ala Asp Leu Ser Leu Leu Asp Arg
145 150 155 160
Trp Gly Asn Ser Val Leu His Leu Ala Ala Lys Glu Gly His Asp Lys
165 170 175
Ile Leu Gly Val Leu Leu Lys Asn Ser Lys Ala Ala Leu Leu Ile Asn
180 185 190
His Pro Asn Gly Glu Gly Leu Asn Ala Ile His Ile Ala Val Met Ser
195 200 205
Asn Ser Leu Ser Cys Leu Gln Leu Leu Val Ala Ala Gly Ala Glu Val
210 215 220
Asn Ala Gln Glu Gln Lys Ser Gly Arg Thr Ala Leu His Leu Ala Val
225 230 235 240
Glu Tyr Asp Asn Ile Ser Leu Ala Gly Cys Leu Leu Leu Glu Gly Asp
245 250 255
Ala Leu Val Asp Ser Thr Thr Tyr Asp Gly Thr Thr Pro Leu His Ile
260 265 270
Ala Ala Gly Arg Gly Ser Thr Arg Leu Ala Ala Leu Leu Lys Ala Ala
275 280 285
Gly Ala Asp Pro Leu Val Glu Asn Phe Glu Pro Leu Tyr Asp Leu Asp
290 295 300
Asp Ser Trp Glu Lys Ala Gly Glu Asp Glu Gly Val Val Pro Gly Thr
305 310 315 320
Thr Pro Leu Asp Met Ala Ala Asn Trp Gln Val Phe Asp Ile Leu Asn
325 330 335
Gly Lys Pro Tyr Glu Pro Val Phe Thr Ser Asp Asp Ile Leu Pro Gln
340 345 350
Gly Asp Ile Lys Gln Leu Thr Glu Asp Thr Arg Leu Gln Leu Cys Lys
355 360 365
Leu Leu Glu Ile Pro Asp Pro Asp Lys Asn Trp Ala Thr Leu Ala Gln
370 375 380
Lys Leu Gly Leu Gly Ile Leu Asn Asn Ala Phe Arg Leu Ser Pro Ala
385 390 395 400
Pro Ser Lys Thr Leu Met Asp Asn Tyr Glu Val Ser Gly Gly Thr Ile
405 410 415
Lys Glu Leu Val Glu Ala Leu Arg Gln Met Gly Tyr Thr Glu Ala Ile
420 425 430
Glu Val Ile Gln Ala Ala Phe Arg Thr Pro Glu Thr Thr Ala Ser Ser
435 440 445
Pro Val Thr Thr Ala Gln Ala His Leu Leu Pro Leu Ser Ser Ser Ser
450 455 460
Thr Arg Gln His Ile Asp Glu Leu Arg Asp Asn Asp Ser Val Cys Asp
465 470 475 480
Ser Gly Val Glu Thr Ser Phe Arg Lys Leu Ser Phe Ser Glu Ser Leu
485 490 495
Thr Gly Asp Gly Pro Leu Leu Ser Leu Asn Lys Met Pro His Asn Tyr
500 505 510
Gly Gln Asp Gly Pro Ile Glu Gly Lys Ile
515 520
<210> 10
<211> 295
<212> PRT
<213> artifical sequence
<400> 10
Asp Trp Gln Leu Pro Ser His Ser Gly Pro Tyr Glu Leu Arg Ile Glu
1 5 10 15
Val Gln Pro Lys Ser His His Arg Ala His Tyr Glu Thr Glu Gly Ser
20 25 30
Arg Gly Ala Val Lys Ala Ser Ala Gly Gly His Pro Ile Val Gln Leu
35 40 45
His Gly Tyr Leu Glu Asn Glu Pro Leu Thr Leu Gln Leu Phe Ile Gly
50 55 60
Thr Ala Asp Asp Arg Leu Leu Arg Pro His Ala Phe Tyr Gln Val His
65 70 75 80
Arg Ile Thr Gly Lys Thr Val Ser Thr Thr Ser His Glu Ile Ile Leu
85 90 95
Ser Asn Thr Lys Val Leu Glu Ile Pro Leu Leu Pro Glu Asn Asn Met
100 105 110
Arg Ala Ile Ile Asp Cys Ala Gly Ile Leu Lys Leu Arg Asn Ser Asp
115 120 125
Ile Glu Leu Arg Lys Gly Glu Thr Asp Ile Gly Arg Lys Asn Thr Arg
130 135 140
Val Arg Leu Val Phe Arg Val His Ile Pro Gln Pro Asn Gly Arg Thr
145 150 155 160
Leu Ser Leu Gln Val Ala Ser Asn Pro Ile Glu Cys Ser Gln Arg Ser
165 170 175
Ala Ser Asn Leu Lys Ile Val Arg Met Asp Arg Thr Ala Gly Cys Val
180 185 190
Thr Gly Gly Glu Glu Ile Tyr Leu Leu Cys Asp Lys Val Gln Lys Asp
195 200 205
Asp Ile Gln Ile Arg Phe Tyr Glu Glu Glu Glu Asn Gly Gly Val Trp
210 215 220
Glu Gly Phe Gly Asp Phe Ser Pro Thr Asp Val His Arg Gln Phe Ala
225 230 235 240
Ile Val Phe Lys Thr Pro Lys Tyr Lys Asp Ile Asn Ile Thr Lys Pro
245 250 255
Ala Ser Val Phe Val Gln Leu Arg Arg Lys Ser Asp Leu Glu Thr Ser
260 265 270
Glu Pro Lys Pro Phe Leu Tyr Tyr Pro Glu Ile Lys Asp Lys Glu Glu
275 280 285
Val Gln Arg Lys Arg Gln Lys
290 295
<210> 11
<211> 88
<212> PRT
<213> artifical sequence
<400> 11
Ala Lys Lys Glu Met Val Glu Phe Asp Glu Ala Ile His Gly Glu Asp
1 5 10 15
Leu Ala Lys Phe Ile Lys Glu Ala Ser Asp His Lys Leu Lys Ile Ser
20 25 30
Gly Tyr Asn Glu Leu Ile Lys Asp Ile Arg Ile Arg Ala Lys Asp Glu
35 40 45
Leu Gly Val Asp Gly Lys Met Phe Asn Arg Leu Leu Ala Leu Tyr His
50 55 60
Lys Asp Asn Arg Asp Val Phe Glu Ala Glu Thr Glu Glu Val Val Glu
65 70 75 80
Leu Tyr Asp Thr Val Phe Ser Lys
85
<210> 12
<211> 103
<212> PRT
<213> artifical sequence
<400> 12
Ala Lys Met Met Gln Arg Glu Ile Thr Lys Thr Thr Val Asn Val Ala
1 5 10 15
Lys Met Val Met Val Asp Gly Glu Val Gln Val Glu Gln Leu Pro Ser
20 25 30
Glu Thr Phe Val Gly Asn Leu Thr Met Glu Gln Ala Gln Trp Arg Met
35 40 45
Lys Arg Lys Tyr Lys Gly Glu Pro Val Gln Val Val Ser Val Glu Pro
50 55 60
Asn Thr Glu Val Tyr Glu Leu Pro Val Glu Lys Phe Leu Glu Val Ala
65 70 75 80
Thr Val Arg Val Glu Lys Asp Glu Asp Gln Glu Glu Gln Thr Glu Ala
85 90 95
Pro Glu Glu Gln Val Ala Glu
100
<210> 13
<211> 136
<212> PRT
<213> artifical sequence
<400> 13
Ser Glu Ala Leu Lys Ile Leu Asn Asn Ile Arg Thr Leu Arg Ala Gln
1 5 10 15
Ala Arg Glu Cys Thr Leu Glu Thr Leu Glu Glu Met Leu Glu Lys Leu
20 25 30
Glu Val Val Val Asn Glu Arg Arg Glu Glu Glu Ser Ala Ala Ala Ala
35 40 45
Glu Val Glu Glu Arg Thr Arg Lys Leu Gln Gln Tyr Arg Glu Met Leu
50 55 60
Ile Ala Asp Gly Ile Asp Pro Asn Glu Leu Leu Asn Ser Leu Ala Ala
65 70 75 80
Val Lys Ser Gly Thr Lys Ala Lys Arg Ala Gln Arg Pro Ala Lys Tyr
85 90 95
Ser Tyr Val Asp Glu Asn Gly Glu Thr Lys Thr Trp Thr Gly Gln Gly
100 105 110
Arg Thr Pro Ala Val Ile Lys Lys Ala Met Asp Glu Gln Gly Lys Ser
115 120 125
Leu Asp Asp Phe Leu Ile Lys Gln
130 135
<210> 14
<211> 59
<212> PRT
<213> artifical sequence
<400> 14
Ala Thr Ile Lys Asp Val Ala Lys Arg Ala Asn Val Ser Thr Thr Thr
1 5 10 15
Val Ser His Val Ile Asn Lys Thr Arg Phe Val Ala Glu Glu Thr Arg
20 25 30
Asn Ala Val Trp Ala Ala Ile Lys Glu Leu His Tyr Ser Pro Ser Ala
35 40 45
Val Ala Arg Ser Leu Lys Val Asn His Thr Lys
50 55
<210> 15
<211> 1014
<212> PRT
<213> artifical sequence
<400> 15
Met Ile Leu Lys Ile Leu Asn Glu Ile Ala Ser Ile Gly Ser Thr Lys
1 5 10 15
Gln Lys Lys Ala Ile Leu Glu Lys Asn Lys Asp Asn Glu Leu Leu Lys
20 25 30
Arg Val Tyr Arg Leu Thr Tyr Ser Arg Gly Leu Gln Tyr Tyr Ile Lys
35 40 45
Lys Trp Pro Lys Pro Gly Ile Ala Thr Gln Ser Phe Gly Met Thr Thr
50 55 60
Leu Thr Asp Met Leu Asp Phe Ile Glu Phe Thr Leu Ala Thr Arg Lys
65 70 75 80
Leu Thr Gly Asn Ala Ala Ile Arg Glu Leu Thr Gly Tyr Ile Thr Asp
85 90 95
Gly Lys Lys Asp Asp Val Glu Val Leu Arg Arg Val Met Met Arg Asp
100 105 110
Leu Glu Cys Gly Ala Ser Val Ser Ile Ala Asn Lys Val Trp Lys Gly
115 120 125
Leu Ile Pro Glu Gln Pro Gln Met Leu Ala Ser Ser Tyr Asp Glu Lys
130 135 140
Gly Ile Asn Lys Asn Ile Lys Phe Pro Ala Phe Ala Gln Leu Ser Ala
145 150 155 160
Asp Gly Ala Arg Cys Phe Ala Glu Val Arg Gly Asp Glu Leu Asp Asp
165 170 175
Val Arg Leu Leu Ser Arg Ala Gly Asn Glu Tyr Leu Gly Leu Asp Leu
180 185 190
Leu Lys Glu Glu Leu Ile Lys Met Thr Ala Glu Ala Arg Gln Ile His
195 200 205
Pro Glu Gly Val Leu Ile Asp Gly Glu Leu Val Tyr His Glu Gln Val
210 215 220
Ala Lys Glu Pro Glu Gly Leu Asp Ala Leu Phe Asp Arg Tyr Pro Glu
225 230 235 240
Asn Ser Lys Ala Lys Glu Phe Ala Glu Val Ala Glu Ser Arg Thr Ala
245 250 255
Ser Asn Gly Ile Ala Asn Lys Ser Leu Lys Gly Thr Ile Ser Glu Lys
260 265 270
Glu Ala Gln Cys Met Lys Phe Gln Val Trp Asp Tyr Val Pro Leu Val
275 280 285
Glu Ile Tyr Ser Leu Pro Ala Phe Arg Leu Lys Tyr Asp Val Arg Phe
290 295 300
Ser Lys Leu Glu Gln Met Thr Ser Gly Tyr Asp Lys Val Ile Leu Ile
305 310 315 320
Glu Asn Gln Val Val Asn Asn Leu Asp Glu Ala Lys Val Ile Tyr Lys
325 330 335
Lys Tyr Ile Asp Gln Gly Leu Glu Gly Ile Ile Leu Lys Asn Ile Asp
340 345 350
Gly Leu Trp Glu Asn Ala Arg Ser Lys Asn Leu Tyr Lys Phe Lys Glu
355 360 365
Val Ile Trp Val Asp Leu Lys Ile Val Gly Ile Tyr Pro His Arg Lys
370 375 380
Asp Pro Thr Lys Ala Gly Gly Phe Ile Leu Glu Ser Glu Cys Gly Lys
385 390 395 400
Ile Lys Val Asn Ala Gly Ser Gly Leu Lys Asp Lys Ala Gly Val Lys
405 410 415
Ser His Glu Leu Asp Arg Thr Arg Ile Met Glu Asn Gln Asn Tyr Tyr
420 425 430
Ile Gly Lys Ile Leu Glu Cys Lys Cys Asn Gly Trp Leu Lys Ser Asp
435 440 445
Gly Arg Lys Pro Tyr Val Lys Leu Phe Leu Pro Ile Ala Ile Arg Leu
450 455 460
Arg Glu Asp Lys Thr Lys Ala Asn Thr Phe Glu Asp Val Phe Gly Asp
465 470 475 480
Phe His Glu Val Thr Gly Leu Gly Gly Gly Gly Ser Arg Glu Ile Leu
485 490 495
Asn Pro Pro Glu Lys Glu Thr Gln Gly Glu Gly Pro Ser Leu Phe Met
500 505 510
Ala Ser Thr Lys Thr Glu Ala Ile Ala Pro Ala Ser Thr Met Glu Asp
515 520 525
Lys Glu Glu Asp Val Gly Phe Gln Asp Asn Leu Phe Leu Glu Lys Ala
530 535 540
Leu Gln Leu Ala Lys Arg His Ala Asn Ala Leu Phe Asp Tyr Ala Val
545 550 555 560
Thr Gly Asp Val Lys Met Leu Leu Ala Val Gln Arg His Leu Thr Ala
565 570 575
Val Gln Asp Glu Asn Gly Asp Ser Val Leu His Leu Ala Ile Ile His
580 585 590
Leu His Ala Gln Leu Val Arg Asp Leu Leu Glu Val Thr Ser Gly Ser
595 600 605
Ile Ser Asp Asp Ile Ile Asn Met Arg Asn Asp Leu Tyr Gln Thr Pro
610 615 620
Leu His Leu Ala Val Ile Thr Lys Gln Glu Asp Val Val Glu Asp Leu
625 630 635 640
Leu Arg Val Gly Ala Asp Leu Ser Leu Leu Asp Arg Trp Gly Asn Ser
645 650 655
Val Leu His Leu Ala Ala Lys Glu Gly His Asp Lys Ile Leu Gly Val
660 665 670
Leu Leu Lys Asn Ser Lys Ala Ala Leu Leu Ile Asn His Pro Asn Gly
675 680 685
Glu Gly Leu Asn Ala Ile His Ile Ala Val Met Ser Asn Ser Leu Ser
690 695 700
Cys Leu Gln Leu Leu Val Ala Ala Gly Ala Glu Val Asn Ala Gln Glu
705 710 715 720
Gln Lys Ser Gly Arg Thr Ala Leu His Leu Ala Val Glu Tyr Asp Asn
725 730 735
Ile Ser Leu Ala Gly Cys Leu Leu Leu Glu Gly Asp Ala Leu Val Asp
740 745 750
Ser Thr Thr Tyr Asp Gly Thr Thr Pro Leu His Ile Ala Ala Gly Arg
755 760 765
Gly Ser Thr Arg Leu Ala Ala Leu Leu Lys Ala Ala Gly Ala Asp Pro
770 775 780
Leu Val Glu Asn Phe Glu Pro Leu Tyr Asp Leu Asp Asp Ser Trp Glu
785 790 795 800
Lys Ala Gly Glu Asp Glu Gly Val Val Pro Gly Thr Thr Pro Leu Asp
805 810 815
Met Ala Ala Asn Trp Gln Val Phe Asp Ile Leu Asn Gly Lys Pro Tyr
820 825 830
Glu Pro Val Phe Thr Ser Asp Asp Ile Leu Pro Gln Gly Asp Ile Lys
835 840 845
Gln Leu Thr Glu Asp Thr Arg Leu Gln Leu Cys Lys Leu Leu Glu Ile
850 855 860
Pro Asp Pro Asp Lys Asn Trp Ala Thr Leu Ala Gln Lys Leu Gly Leu
865 870 875 880
Gly Ile Leu Asn Asn Ala Phe Arg Leu Ser Pro Ala Pro Ser Lys Thr
885 890 895
Leu Met Asp Asn Tyr Glu Val Ser Gly Gly Thr Ile Lys Glu Leu Val
900 905 910
Glu Ala Leu Arg Gln Met Gly Tyr Thr Glu Ala Ile Glu Val Ile Gln
915 920 925
Ala Ala Phe Arg Thr Pro Glu Thr Thr Ala Ser Ser Pro Val Thr Thr
930 935 940
Ala Gln Ala His Leu Leu Pro Leu Ser Ser Ser Ser Thr Arg Gln His
945 950 955 960
Ile Asp Glu Leu Arg Asp Asn Asp Ser Val Cys Asp Ser Gly Val Glu
965 970 975
Thr Ser Phe Arg Lys Leu Ser Phe Ser Glu Ser Leu Thr Gly Asp Gly
980 985 990
Pro Leu Leu Ser Leu Asn Lys Met Pro His Asn Tyr Gly Gln Asp Gly
995 1000 1005
Pro Ile Glu Gly Lys Ile
1010
<210> 16
<211> 787
<212> PRT
<213> artifical sequence
<400> 16
Met Ile Leu Lys Ile Leu Asn Glu Ile Ala Ser Ile Gly Ser Thr Lys
1 5 10 15
Gln Lys Lys Ala Ile Leu Glu Lys Asn Lys Asp Asn Glu Leu Leu Lys
20 25 30
Arg Val Tyr Arg Leu Thr Tyr Ser Arg Gly Leu Gln Tyr Tyr Ile Lys
35 40 45
Lys Trp Pro Lys Pro Gly Ile Ala Thr Gln Ser Phe Gly Met Thr Thr
50 55 60
Leu Thr Asp Met Leu Asp Phe Ile Glu Phe Thr Leu Ala Thr Arg Lys
65 70 75 80
Leu Thr Gly Asn Ala Ala Ile Arg Glu Leu Thr Gly Tyr Ile Thr Asp
85 90 95
Gly Lys Lys Asp Asp Val Glu Val Leu Arg Arg Val Met Met Arg Asp
100 105 110
Leu Glu Cys Gly Ala Ser Val Ser Ile Ala Asn Lys Val Trp Lys Gly
115 120 125
Leu Ile Pro Glu Gln Pro Gln Met Leu Ala Ser Ser Tyr Asp Glu Lys
130 135 140
Gly Ile Asn Lys Asn Ile Lys Phe Pro Ala Phe Ala Gln Leu Ser Ala
145 150 155 160
Asp Gly Ala Arg Cys Phe Ala Glu Val Arg Gly Asp Glu Leu Asp Asp
165 170 175
Val Arg Leu Leu Ser Arg Ala Gly Asn Glu Tyr Leu Gly Leu Asp Leu
180 185 190
Leu Lys Glu Glu Leu Ile Lys Met Thr Ala Glu Ala Arg Gln Ile His
195 200 205
Pro Glu Gly Val Leu Ile Asp Gly Glu Leu Val Tyr His Glu Gln Val
210 215 220
Ala Lys Glu Pro Glu Gly Leu Asp Ala Leu Phe Asp Arg Tyr Pro Glu
225 230 235 240
Asn Ser Lys Ala Lys Glu Phe Ala Glu Val Ala Glu Ser Arg Thr Ala
245 250 255
Ser Asn Gly Ile Ala Asn Lys Ser Leu Lys Gly Thr Ile Ser Glu Lys
260 265 270
Glu Ala Gln Cys Met Lys Phe Gln Val Trp Asp Tyr Val Pro Leu Val
275 280 285
Glu Ile Tyr Ser Leu Pro Ala Phe Arg Leu Lys Tyr Asp Val Arg Phe
290 295 300
Ser Lys Leu Glu Gln Met Thr Ser Gly Tyr Asp Lys Val Ile Leu Ile
305 310 315 320
Glu Asn Gln Val Val Asn Asn Leu Asp Glu Ala Lys Val Ile Tyr Lys
325 330 335
Lys Tyr Ile Asp Gln Gly Leu Glu Gly Ile Ile Leu Lys Asn Ile Asp
340 345 350
Gly Leu Trp Glu Asn Ala Arg Ser Lys Asn Leu Tyr Lys Phe Lys Glu
355 360 365
Val Ile Trp Val Asp Leu Lys Ile Val Gly Ile Tyr Pro His Arg Lys
370 375 380
Asp Pro Thr Lys Ala Gly Gly Phe Ile Leu Glu Ser Glu Cys Gly Lys
385 390 395 400
Ile Lys Val Asn Ala Gly Ser Gly Leu Lys Asp Lys Ala Gly Val Lys
405 410 415
Ser His Glu Leu Asp Arg Thr Arg Ile Met Glu Asn Gln Asn Tyr Tyr
420 425 430
Ile Gly Lys Ile Leu Glu Cys Lys Cys Asn Gly Trp Leu Lys Ser Asp
435 440 445
Gly Arg Lys Pro Tyr Val Lys Leu Phe Leu Pro Ile Ala Ile Arg Leu
450 455 460
Arg Glu Asp Lys Thr Lys Ala Asn Thr Phe Glu Asp Val Phe Gly Asp
465 470 475 480
Phe His Glu Val Thr Gly Leu Gly Gly Gly Gly Ser Asp Trp Gln Leu
485 490 495
Pro Ser His Ser Gly Pro Tyr Glu Leu Arg Ile Glu Val Gln Pro Lys
500 505 510
Ser His His Arg Ala His Tyr Glu Thr Glu Gly Ser Arg Gly Ala Val
515 520 525
Lys Ala Ser Ala Gly Gly His Pro Ile Val Gln Leu His Gly Tyr Leu
530 535 540
Glu Asn Glu Pro Leu Thr Leu Gln Leu Phe Ile Gly Thr Ala Asp Asp
545 550 555 560
Arg Leu Leu Arg Pro His Ala Phe Tyr Gln Val His Arg Ile Thr Gly
565 570 575
Lys Thr Val Ser Thr Thr Ser His Glu Ile Ile Leu Ser Asn Thr Lys
580 585 590
Val Leu Glu Ile Pro Leu Leu Pro Glu Asn Asn Met Arg Ala Ile Ile
595 600 605
Asp Cys Ala Gly Ile Leu Lys Leu Arg Asn Ser Asp Ile Glu Leu Arg
610 615 620
Lys Gly Glu Thr Asp Ile Gly Arg Lys Asn Thr Arg Val Arg Leu Val
625 630 635 640
Phe Arg Val His Ile Pro Gln Pro Asn Gly Arg Thr Leu Ser Leu Gln
645 650 655
Val Ala Ser Asn Pro Ile Glu Cys Ser Gln Arg Ser Ala Ser Asn Leu
660 665 670
Lys Ile Val Arg Met Asp Arg Thr Ala Gly Cys Val Thr Gly Gly Glu
675 680 685
Glu Ile Tyr Leu Leu Cys Asp Lys Val Gln Lys Asp Asp Ile Gln Ile
690 695 700
Arg Phe Tyr Glu Glu Glu Glu Asn Gly Gly Val Trp Glu Gly Phe Gly
705 710 715 720
Asp Phe Ser Pro Thr Asp Val His Arg Gln Phe Ala Ile Val Phe Lys
725 730 735
Thr Pro Lys Tyr Lys Asp Ile Asn Ile Thr Lys Pro Ala Ser Val Phe
740 745 750
Val Gln Leu Arg Arg Lys Ser Asp Leu Glu Thr Ser Glu Pro Lys Pro
755 760 765
Phe Leu Tyr Tyr Pro Glu Ile Lys Asp Lys Glu Glu Val Gln Arg Lys
770 775 780
Arg Gln Lys
785
<210> 17
<211> 580
<212> PRT
<213> artifical sequence
<400> 17
Met Ile Leu Lys Ile Leu Asn Glu Ile Ala Ser Ile Gly Ser Thr Lys
1 5 10 15
Gln Lys Lys Ala Ile Leu Glu Lys Asn Lys Asp Asn Glu Leu Leu Lys
20 25 30
Arg Val Tyr Arg Leu Thr Tyr Ser Arg Gly Leu Gln Tyr Tyr Ile Lys
35 40 45
Lys Trp Pro Lys Pro Gly Ile Ala Thr Gln Ser Phe Gly Met Thr Thr
50 55 60
Leu Thr Asp Met Leu Asp Phe Ile Glu Phe Thr Leu Ala Thr Arg Lys
65 70 75 80
Leu Thr Gly Asn Ala Ala Ile Arg Glu Leu Thr Gly Tyr Ile Thr Asp
85 90 95
Gly Lys Lys Asp Asp Val Glu Val Leu Arg Arg Val Met Met Arg Asp
100 105 110
Leu Glu Cys Gly Ala Ser Val Ser Ile Ala Asn Lys Val Trp Lys Gly
115 120 125
Leu Ile Pro Glu Gln Pro Gln Met Leu Ala Ser Ser Tyr Asp Glu Lys
130 135 140
Gly Ile Asn Lys Asn Ile Lys Phe Pro Ala Phe Ala Gln Leu Ser Ala
145 150 155 160
Asp Gly Ala Arg Cys Phe Ala Glu Val Arg Gly Asp Glu Leu Asp Asp
165 170 175
Val Arg Leu Leu Ser Arg Ala Gly Asn Glu Tyr Leu Gly Leu Asp Leu
180 185 190
Leu Lys Glu Glu Leu Ile Lys Met Thr Ala Glu Ala Arg Gln Ile His
195 200 205
Pro Glu Gly Val Leu Ile Asp Gly Glu Leu Val Tyr His Glu Gln Val
210 215 220
Ala Lys Glu Pro Glu Gly Leu Asp Ala Leu Phe Asp Arg Tyr Pro Glu
225 230 235 240
Asn Ser Lys Ala Lys Glu Phe Ala Glu Val Ala Glu Ser Arg Thr Ala
245 250 255
Ser Asn Gly Ile Ala Asn Lys Ser Leu Lys Gly Thr Ile Ser Glu Lys
260 265 270
Glu Ala Gln Cys Met Lys Phe Gln Val Trp Asp Tyr Val Pro Leu Val
275 280 285
Glu Ile Tyr Ser Leu Pro Ala Phe Arg Leu Lys Tyr Asp Val Arg Phe
290 295 300
Ser Lys Leu Glu Gln Met Thr Ser Gly Tyr Asp Lys Val Ile Leu Ile
305 310 315 320
Glu Asn Gln Val Val Asn Asn Leu Asp Glu Ala Lys Val Ile Tyr Lys
325 330 335
Lys Tyr Ile Asp Gln Gly Leu Glu Gly Ile Ile Leu Lys Asn Ile Asp
340 345 350
Gly Leu Trp Glu Asn Ala Arg Ser Lys Asn Leu Tyr Lys Phe Lys Glu
355 360 365
Val Ile Trp Val Asp Leu Lys Ile Val Gly Ile Tyr Pro His Arg Lys
370 375 380
Asp Pro Thr Lys Ala Gly Gly Phe Ile Leu Glu Ser Glu Cys Gly Lys
385 390 395 400
Ile Lys Val Asn Ala Gly Ser Gly Leu Lys Asp Lys Ala Gly Val Lys
405 410 415
Ser His Glu Leu Asp Arg Thr Arg Ile Met Glu Asn Gln Asn Tyr Tyr
420 425 430
Ile Gly Lys Ile Leu Glu Cys Lys Cys Asn Gly Trp Leu Lys Ser Asp
435 440 445
Gly Arg Lys Pro Tyr Val Lys Leu Phe Leu Pro Ile Ala Ile Arg Leu
450 455 460
Arg Glu Asp Lys Thr Lys Ala Asn Thr Phe Glu Asp Val Phe Gly Asp
465 470 475 480
Phe His Glu Val Thr Gly Leu Gly Gly Gly Gly Ser Ala Lys Lys Glu
485 490 495
Met Val Glu Phe Asp Glu Ala Ile His Gly Glu Asp Leu Ala Lys Phe
500 505 510
Ile Lys Glu Ala Ser Asp His Lys Leu Lys Ile Ser Gly Tyr Asn Glu
515 520 525
Leu Ile Lys Asp Ile Arg Ile Arg Ala Lys Asp Glu Leu Gly Val Asp
530 535 540
Gly Lys Met Phe Asn Arg Leu Leu Ala Leu Tyr His Lys Asp Asn Arg
545 550 555 560
Asp Val Phe Glu Ala Glu Thr Glu Glu Val Val Glu Leu Tyr Asp Thr
565 570 575
Val Phe Ser Lys
580
<210> 18
<211> 595
<212> PRT
<213> artifical sequence
<400> 18
Met Ile Leu Lys Ile Leu Asn Glu Ile Ala Ser Ile Gly Ser Thr Lys
1 5 10 15
Gln Lys Lys Ala Ile Leu Glu Lys Asn Lys Asp Asn Glu Leu Leu Lys
20 25 30
Arg Val Tyr Arg Leu Thr Tyr Ser Arg Gly Leu Gln Tyr Tyr Ile Lys
35 40 45
Lys Trp Pro Lys Pro Gly Ile Ala Thr Gln Ser Phe Gly Met Thr Thr
50 55 60
Leu Thr Asp Met Leu Asp Phe Ile Glu Phe Thr Leu Ala Thr Arg Lys
65 70 75 80
Leu Thr Gly Asn Ala Ala Ile Arg Glu Leu Thr Gly Tyr Ile Thr Asp
85 90 95
Gly Lys Lys Asp Asp Val Glu Val Leu Arg Arg Val Met Met Arg Asp
100 105 110
Leu Glu Cys Gly Ala Ser Val Ser Ile Ala Asn Lys Val Trp Lys Gly
115 120 125
Leu Ile Pro Glu Gln Pro Gln Met Leu Ala Ser Ser Tyr Asp Glu Lys
130 135 140
Gly Ile Asn Lys Asn Ile Lys Phe Pro Ala Phe Ala Gln Leu Ser Ala
145 150 155 160
Asp Gly Ala Arg Cys Phe Ala Glu Val Arg Gly Asp Glu Leu Asp Asp
165 170 175
Val Arg Leu Leu Ser Arg Ala Gly Asn Glu Tyr Leu Gly Leu Asp Leu
180 185 190
Leu Lys Glu Glu Leu Ile Lys Met Thr Ala Glu Ala Arg Gln Ile His
195 200 205
Pro Glu Gly Val Leu Ile Asp Gly Glu Leu Val Tyr His Glu Gln Val
210 215 220
Ala Lys Glu Pro Glu Gly Leu Asp Ala Leu Phe Asp Arg Tyr Pro Glu
225 230 235 240
Asn Ser Lys Ala Lys Glu Phe Ala Glu Val Ala Glu Ser Arg Thr Ala
245 250 255
Ser Asn Gly Ile Ala Asn Lys Ser Leu Lys Gly Thr Ile Ser Glu Lys
260 265 270
Glu Ala Gln Cys Met Lys Phe Gln Val Trp Asp Tyr Val Pro Leu Val
275 280 285
Glu Ile Tyr Ser Leu Pro Ala Phe Arg Leu Lys Tyr Asp Val Arg Phe
290 295 300
Ser Lys Leu Glu Gln Met Thr Ser Gly Tyr Asp Lys Val Ile Leu Ile
305 310 315 320
Glu Asn Gln Val Val Asn Asn Leu Asp Glu Ala Lys Val Ile Tyr Lys
325 330 335
Lys Tyr Ile Asp Gln Gly Leu Glu Gly Ile Ile Leu Lys Asn Ile Asp
340 345 350
Gly Leu Trp Glu Asn Ala Arg Ser Lys Asn Leu Tyr Lys Phe Lys Glu
355 360 365
Val Ile Trp Val Asp Leu Lys Ile Val Gly Ile Tyr Pro His Arg Lys
370 375 380
Asp Pro Thr Lys Ala Gly Gly Phe Ile Leu Glu Ser Glu Cys Gly Lys
385 390 395 400
Ile Lys Val Asn Ala Gly Ser Gly Leu Lys Asp Lys Ala Gly Val Lys
405 410 415
Ser His Glu Leu Asp Arg Thr Arg Ile Met Glu Asn Gln Asn Tyr Tyr
420 425 430
Ile Gly Lys Ile Leu Glu Cys Lys Cys Asn Gly Trp Leu Lys Ser Asp
435 440 445
Gly Arg Lys Pro Tyr Val Lys Leu Phe Leu Pro Ile Ala Ile Arg Leu
450 455 460
Arg Glu Asp Lys Thr Lys Ala Asn Thr Phe Glu Asp Val Phe Gly Asp
465 470 475 480
Phe His Glu Val Thr Gly Leu Gly Gly Gly Gly Ser Ala Lys Met Met
485 490 495
Gln Arg Glu Ile Thr Lys Thr Thr Val Asn Val Ala Lys Met Val Met
500 505 510
Val Asp Gly Glu Val Gln Val Glu Gln Leu Pro Ser Glu Thr Phe Val
515 520 525
Gly Asn Leu Thr Met Glu Gln Ala Gln Trp Arg Met Lys Arg Lys Tyr
530 535 540
Lys Gly Glu Pro Val Gln Val Val Ser Val Glu Pro Asn Thr Glu Val
545 550 555 560
Tyr Glu Leu Pro Val Glu Lys Phe Leu Glu Val Ala Thr Val Arg Val
565 570 575
Glu Lys Asp Glu Asp Gln Glu Glu Gln Thr Glu Ala Pro Glu Glu Gln
580 585 590
Val Ala Glu
595
<210> 19
<211> 628
<212> PRT
<213> artifical sequence
<400> 19
Met Ile Leu Lys Ile Leu Asn Glu Ile Ala Ser Ile Gly Ser Thr Lys
1 5 10 15
Gln Lys Lys Ala Ile Leu Glu Lys Asn Lys Asp Asn Glu Leu Leu Lys
20 25 30
Arg Val Tyr Arg Leu Thr Tyr Ser Arg Gly Leu Gln Tyr Tyr Ile Lys
35 40 45
Lys Trp Pro Lys Pro Gly Ile Ala Thr Gln Ser Phe Gly Met Thr Thr
50 55 60
Leu Thr Asp Met Leu Asp Phe Ile Glu Phe Thr Leu Ala Thr Arg Lys
65 70 75 80
Leu Thr Gly Asn Ala Ala Ile Arg Glu Leu Thr Gly Tyr Ile Thr Asp
85 90 95
Gly Lys Lys Asp Asp Val Glu Val Leu Arg Arg Val Met Met Arg Asp
100 105 110
Leu Glu Cys Gly Ala Ser Val Ser Ile Ala Asn Lys Val Trp Lys Gly
115 120 125
Leu Ile Pro Glu Gln Pro Gln Met Leu Ala Ser Ser Tyr Asp Glu Lys
130 135 140
Gly Ile Asn Lys Asn Ile Lys Phe Pro Ala Phe Ala Gln Leu Ser Ala
145 150 155 160
Asp Gly Ala Arg Cys Phe Ala Glu Val Arg Gly Asp Glu Leu Asp Asp
165 170 175
Val Arg Leu Leu Ser Arg Ala Gly Asn Glu Tyr Leu Gly Leu Asp Leu
180 185 190
Leu Lys Glu Glu Leu Ile Lys Met Thr Ala Glu Ala Arg Gln Ile His
195 200 205
Pro Glu Gly Val Leu Ile Asp Gly Glu Leu Val Tyr His Glu Gln Val
210 215 220
Ala Lys Glu Pro Glu Gly Leu Asp Ala Leu Phe Asp Arg Tyr Pro Glu
225 230 235 240
Asn Ser Lys Ala Lys Glu Phe Ala Glu Val Ala Glu Ser Arg Thr Ala
245 250 255
Ser Asn Gly Ile Ala Asn Lys Ser Leu Lys Gly Thr Ile Ser Glu Lys
260 265 270
Glu Ala Gln Cys Met Lys Phe Gln Val Trp Asp Tyr Val Pro Leu Val
275 280 285
Glu Ile Tyr Ser Leu Pro Ala Phe Arg Leu Lys Tyr Asp Val Arg Phe
290 295 300
Ser Lys Leu Glu Gln Met Thr Ser Gly Tyr Asp Lys Val Ile Leu Ile
305 310 315 320
Glu Asn Gln Val Val Asn Asn Leu Asp Glu Ala Lys Val Ile Tyr Lys
325 330 335
Lys Tyr Ile Asp Gln Gly Leu Glu Gly Ile Ile Leu Lys Asn Ile Asp
340 345 350
Gly Leu Trp Glu Asn Ala Arg Ser Lys Asn Leu Tyr Lys Phe Lys Glu
355 360 365
Val Ile Trp Val Asp Leu Lys Ile Val Gly Ile Tyr Pro His Arg Lys
370 375 380
Asp Pro Thr Lys Ala Gly Gly Phe Ile Leu Glu Ser Glu Cys Gly Lys
385 390 395 400
Ile Lys Val Asn Ala Gly Ser Gly Leu Lys Asp Lys Ala Gly Val Lys
405 410 415
Ser His Glu Leu Asp Arg Thr Arg Ile Met Glu Asn Gln Asn Tyr Tyr
420 425 430
Ile Gly Lys Ile Leu Glu Cys Lys Cys Asn Gly Trp Leu Lys Ser Asp
435 440 445
Gly Arg Lys Pro Tyr Val Lys Leu Phe Leu Pro Ile Ala Ile Arg Leu
450 455 460
Arg Glu Asp Lys Thr Lys Ala Asn Thr Phe Glu Asp Val Phe Gly Asp
465 470 475 480
Phe His Glu Val Thr Gly Leu Gly Gly Gly Gly Ser Ser Glu Ala Leu
485 490 495
Lys Ile Leu Asn Asn Ile Arg Thr Leu Arg Ala Gln Ala Arg Glu Cys
500 505 510
Thr Leu Glu Thr Leu Glu Glu Met Leu Glu Lys Leu Glu Val Val Val
515 520 525
Asn Glu Arg Arg Glu Glu Glu Ser Ala Ala Ala Ala Glu Val Glu Glu
530 535 540
Arg Thr Arg Lys Leu Gln Gln Tyr Arg Glu Met Leu Ile Ala Asp Gly
545 550 555 560
Ile Asp Pro Asn Glu Leu Leu Asn Ser Leu Ala Ala Val Lys Ser Gly
565 570 575
Thr Lys Ala Lys Arg Ala Gln Arg Pro Ala Lys Tyr Ser Tyr Val Asp
580 585 590
Glu Asn Gly Glu Thr Lys Thr Trp Thr Gly Gln Gly Arg Thr Pro Ala
595 600 605
Val Ile Lys Lys Ala Met Asp Glu Gln Gly Lys Ser Leu Asp Asp Phe
610 615 620
Leu Ile Lys Gln
625
<210> 20
<211> 551
<212> PRT
<213> artifical sequence
<400> 20
Met Ile Leu Lys Ile Leu Asn Glu Ile Ala Ser Ile Gly Ser Thr Lys
1 5 10 15
Gln Lys Lys Ala Ile Leu Glu Lys Asn Lys Asp Asn Glu Leu Leu Lys
20 25 30
Arg Val Tyr Arg Leu Thr Tyr Ser Arg Gly Leu Gln Tyr Tyr Ile Lys
35 40 45
Lys Trp Pro Lys Pro Gly Ile Ala Thr Gln Ser Phe Gly Met Thr Thr
50 55 60
Leu Thr Asp Met Leu Asp Phe Ile Glu Phe Thr Leu Ala Thr Arg Lys
65 70 75 80
Leu Thr Gly Asn Ala Ala Ile Arg Glu Leu Thr Gly Tyr Ile Thr Asp
85 90 95
Gly Lys Lys Asp Asp Val Glu Val Leu Arg Arg Val Met Met Arg Asp
100 105 110
Leu Glu Cys Gly Ala Ser Val Ser Ile Ala Asn Lys Val Trp Lys Gly
115 120 125
Leu Ile Pro Glu Gln Pro Gln Met Leu Ala Ser Ser Tyr Asp Glu Lys
130 135 140
Gly Ile Asn Lys Asn Ile Lys Phe Pro Ala Phe Ala Gln Leu Ser Ala
145 150 155 160
Asp Gly Ala Arg Cys Phe Ala Glu Val Arg Gly Asp Glu Leu Asp Asp
165 170 175
Val Arg Leu Leu Ser Arg Ala Gly Asn Glu Tyr Leu Gly Leu Asp Leu
180 185 190
Leu Lys Glu Glu Leu Ile Lys Met Thr Ala Glu Ala Arg Gln Ile His
195 200 205
Pro Glu Gly Val Leu Ile Asp Gly Glu Leu Val Tyr His Glu Gln Val
210 215 220
Ala Lys Glu Pro Glu Gly Leu Asp Ala Leu Phe Asp Arg Tyr Pro Glu
225 230 235 240
Asn Ser Lys Ala Lys Glu Phe Ala Glu Val Ala Glu Ser Arg Thr Ala
245 250 255
Ser Asn Gly Ile Ala Asn Lys Ser Leu Lys Gly Thr Ile Ser Glu Lys
260 265 270
Glu Ala Gln Cys Met Lys Phe Gln Val Trp Asp Tyr Val Pro Leu Val
275 280 285
Glu Ile Tyr Ser Leu Pro Ala Phe Arg Leu Lys Tyr Asp Val Arg Phe
290 295 300
Ser Lys Leu Glu Gln Met Thr Ser Gly Tyr Asp Lys Val Ile Leu Ile
305 310 315 320
Glu Asn Gln Val Val Asn Asn Leu Asp Glu Ala Lys Val Ile Tyr Lys
325 330 335
Lys Tyr Ile Asp Gln Gly Leu Glu Gly Ile Ile Leu Lys Asn Ile Asp
340 345 350
Gly Leu Trp Glu Asn Ala Arg Ser Lys Asn Leu Tyr Lys Phe Lys Glu
355 360 365
Val Ile Trp Val Asp Leu Lys Ile Val Gly Ile Tyr Pro His Arg Lys
370 375 380
Asp Pro Thr Lys Ala Gly Gly Phe Ile Leu Glu Ser Glu Cys Gly Lys
385 390 395 400
Ile Lys Val Asn Ala Gly Ser Gly Leu Lys Asp Lys Ala Gly Val Lys
405 410 415
Ser His Glu Leu Asp Arg Thr Arg Ile Met Glu Asn Gln Asn Tyr Tyr
420 425 430
Ile Gly Lys Ile Leu Glu Cys Lys Cys Asn Gly Trp Leu Lys Ser Asp
435 440 445
Gly Arg Lys Pro Tyr Val Lys Leu Phe Leu Pro Ile Ala Ile Arg Leu
450 455 460
Arg Glu Asp Lys Thr Lys Ala Asn Thr Phe Glu Asp Val Phe Gly Asp
465 470 475 480
Phe His Glu Val Thr Gly Leu Gly Gly Gly Gly Ser Ala Thr Ile Lys
485 490 495
Asp Val Ala Lys Arg Ala Asn Val Ser Thr Thr Thr Val Ser His Val
500 505 510
Ile Asn Lys Thr Arg Phe Val Ala Glu Glu Thr Arg Asn Ala Val Trp
515 520 525
Ala Ala Ile Lys Glu Leu His Tyr Ser Pro Ser Ala Val Ala Arg Ser
530 535 540
Leu Lys Val Asn His Thr Lys
545 550
<210> 21
<211> 703
<212> PRT
<213> artifical sequence
<400> 21
Met Ala Lys Met Met Gln Arg Glu Ile Thr Lys Thr Thr Val Asn Val
1 5 10 15
Ala Lys Met Val Met Val Asp Gly Glu Val Gln Val Glu Gln Leu Pro
20 25 30
Ser Glu Thr Phe Val Gly Asn Leu Thr Met Glu Gln Ala Gln Trp Arg
35 40 45
Met Lys Arg Lys Tyr Lys Gly Glu Pro Val Gln Val Val Ser Val Glu
50 55 60
Pro Asn Thr Glu Val Tyr Glu Leu Pro Val Glu Lys Phe Leu Glu Val
65 70 75 80
Ala Thr Val Arg Val Glu Lys Asp Glu Asp Gln Glu Glu Gln Thr Glu
85 90 95
Ala Pro Glu Glu Gln Val Ala Glu Gly Gly Gly Gly Ser Ile Leu Lys
100 105 110
Ile Leu Asn Glu Ile Ala Ser Ile Gly Ser Thr Lys Gln Lys Lys Ala
115 120 125
Ile Leu Glu Lys Asn Lys Asp Asn Glu Leu Leu Lys Arg Val Tyr Arg
130 135 140
Leu Thr Tyr Ser Arg Gly Leu Gln Tyr Tyr Ile Lys Lys Trp Pro Lys
145 150 155 160
Pro Gly Ile Ala Thr Gln Ser Phe Gly Met Thr Thr Leu Thr Asp Met
165 170 175
Leu Asp Phe Ile Glu Phe Thr Leu Ala Thr Arg Lys Leu Thr Gly Asn
180 185 190
Ala Ala Ile Arg Glu Leu Thr Gly Tyr Ile Thr Asp Gly Lys Lys Asp
195 200 205
Asp Val Glu Val Leu Arg Arg Val Met Met Arg Asp Leu Glu Cys Gly
210 215 220
Ala Ser Val Ser Ile Ala Asn Lys Val Trp Lys Gly Leu Ile Pro Glu
225 230 235 240
Gln Pro Gln Met Leu Ala Ser Ser Tyr Asp Glu Lys Gly Ile Asn Lys
245 250 255
Asn Ile Lys Phe Pro Ala Phe Ala Gln Leu Ser Ala Asp Gly Ala Arg
260 265 270
Cys Phe Ala Glu Val Arg Gly Asp Glu Leu Asp Asp Val Arg Leu Leu
275 280 285
Ser Arg Ala Gly Asn Glu Tyr Leu Gly Leu Asp Leu Leu Lys Glu Glu
290 295 300
Leu Ile Lys Met Thr Ala Glu Ala Arg Gln Ile His Pro Glu Gly Val
305 310 315 320
Leu Ile Asp Gly Glu Leu Val Tyr His Glu Gln Val Ala Lys Glu Pro
325 330 335
Glu Gly Leu Asp Ala Leu Phe Asp Arg Tyr Pro Glu Asn Ser Lys Ala
340 345 350
Lys Glu Phe Ala Glu Val Ala Glu Ser Arg Thr Ala Ser Asn Gly Ile
355 360 365
Ala Asn Lys Ser Leu Lys Gly Thr Ile Ser Glu Lys Glu Ala Gln Cys
370 375 380
Met Lys Phe Gln Val Trp Asp Tyr Val Pro Leu Val Glu Ile Tyr Ser
385 390 395 400
Leu Pro Ala Phe Arg Leu Lys Tyr Asp Val Arg Phe Ser Lys Leu Glu
405 410 415
Gln Met Thr Ser Gly Tyr Asp Lys Val Ile Leu Ile Glu Asn Gln Val
420 425 430
Val Asn Asn Leu Asp Glu Ala Lys Val Ile Tyr Lys Lys Tyr Ile Asp
435 440 445
Gln Gly Leu Glu Gly Ile Ile Leu Lys Asn Ile Asp Gly Leu Trp Glu
450 455 460
Asn Ala Arg Ser Lys Asn Leu Tyr Lys Phe Lys Glu Val Ile Trp Val
465 470 475 480
Asp Leu Lys Ile Val Gly Ile Tyr Pro His Arg Lys Asp Pro Thr Lys
485 490 495
Ala Gly Gly Phe Ile Leu Glu Ser Glu Cys Gly Lys Ile Lys Val Asn
500 505 510
Ala Gly Ser Gly Leu Lys Asp Lys Ala Gly Val Lys Ser His Glu Leu
515 520 525
Asp Arg Thr Arg Ile Met Glu Asn Gln Asn Tyr Tyr Ile Gly Lys Ile
530 535 540
Leu Glu Cys Lys Cys Asn Gly Trp Leu Lys Ser Asp Gly Arg Lys Pro
545 550 555 560
Tyr Val Lys Leu Phe Leu Pro Ile Ala Ile Arg Leu Arg Glu Asp Lys
565 570 575
Thr Lys Ala Asn Thr Phe Glu Asp Val Phe Gly Asp Phe His Glu Val
580 585 590
Thr Gly Leu Gly Gly Gly Gly Ser Ala Lys Met Met Gln Arg Glu Ile
595 600 605
Thr Lys Thr Thr Val Asn Val Ala Lys Met Val Met Val Asp Gly Glu
610 615 620
Val Gln Val Glu Gln Leu Pro Ser Glu Thr Phe Val Gly Asn Leu Thr
625 630 635 640
Met Glu Gln Ala Gln Trp Arg Met Lys Arg Lys Tyr Lys Gly Glu Pro
645 650 655
Val Gln Val Val Ser Val Glu Pro Asn Thr Glu Val Tyr Glu Leu Pro
660 665 670
Val Glu Lys Phe Leu Glu Val Ala Thr Val Arg Val Glu Lys Asp Glu
675 680 685
Asp Gln Glu Glu Gln Thr Glu Ala Pro Glu Glu Gln Val Ala Glu
690 695 700
<210> 22
<211> 709
<212> PRT
<213> artifical sequence
<400> 22
Met Ala Lys Met Met Gln Arg Glu Ile Thr Lys Thr Thr Val Asn Val
1 5 10 15
Ala Lys Met Val Met Val Asp Gly Glu Val Gln Val Glu Gln Leu Pro
20 25 30
Ser Glu Thr Phe Val Gly Asn Leu Thr Met Glu Gln Ala Gln Trp Arg
35 40 45
Met Lys Arg Lys Tyr Lys Gly Glu Pro Val Gln Val Val Ser Val Glu
50 55 60
Pro Asn Thr Glu Val Tyr Glu Leu Pro Val Glu Lys Phe Leu Glu Val
65 70 75 80
Ala Thr Val Arg Val Glu Lys Asp Glu Asp Gln Glu Glu Gln Thr Glu
85 90 95
Ala Pro Glu Glu Gln Val Ala Glu Pro Ala Pro Ala Pro Ala Pro Ala
100 105 110
Ile Leu Lys Ile Leu Asn Glu Ile Ala Ser Ile Gly Ser Thr Lys Gln
115 120 125
Lys Lys Ala Ile Leu Glu Lys Asn Lys Asp Asn Glu Leu Leu Lys Arg
130 135 140
Val Tyr Arg Leu Thr Tyr Ser Arg Gly Leu Gln Tyr Tyr Ile Lys Lys
145 150 155 160
Trp Pro Lys Pro Gly Ile Ala Thr Gln Ser Phe Gly Met Thr Thr Leu
165 170 175
Thr Asp Met Leu Asp Phe Ile Glu Phe Thr Leu Ala Thr Arg Lys Leu
180 185 190
Thr Gly Asn Ala Ala Ile Arg Glu Leu Thr Gly Tyr Ile Thr Asp Gly
195 200 205
Lys Lys Asp Asp Val Glu Val Leu Arg Arg Val Met Met Arg Asp Leu
210 215 220
Glu Cys Gly Ala Ser Val Ser Ile Ala Asn Lys Val Trp Lys Gly Leu
225 230 235 240
Ile Pro Glu Gln Pro Gln Met Leu Ala Ser Ser Tyr Asp Glu Lys Gly
245 250 255
Ile Asn Lys Asn Ile Lys Phe Pro Ala Phe Ala Gln Leu Ser Ala Asp
260 265 270
Gly Ala Arg Cys Phe Ala Glu Val Arg Gly Asp Glu Leu Asp Asp Val
275 280 285
Arg Leu Leu Ser Arg Ala Gly Asn Glu Tyr Leu Gly Leu Asp Leu Leu
290 295 300
Lys Glu Glu Leu Ile Lys Met Thr Ala Glu Ala Arg Gln Ile His Pro
305 310 315 320
Glu Gly Val Leu Ile Asp Gly Glu Leu Val Tyr His Glu Gln Val Ala
325 330 335
Lys Glu Pro Glu Gly Leu Asp Ala Leu Phe Asp Arg Tyr Pro Glu Asn
340 345 350
Ser Lys Ala Lys Glu Phe Ala Glu Val Ala Glu Ser Arg Thr Ala Ser
355 360 365
Asn Gly Ile Ala Asn Lys Ser Leu Lys Gly Thr Ile Ser Glu Lys Glu
370 375 380
Ala Gln Cys Met Lys Phe Gln Val Trp Asp Tyr Val Pro Leu Val Glu
385 390 395 400
Ile Tyr Ser Leu Pro Ala Phe Arg Leu Lys Tyr Asp Val Arg Phe Ser
405 410 415
Lys Leu Glu Gln Met Thr Ser Gly Tyr Asp Lys Val Ile Leu Ile Glu
420 425 430
Asn Gln Val Val Asn Asn Leu Asp Glu Ala Lys Val Ile Tyr Lys Lys
435 440 445
Tyr Ile Asp Gln Gly Leu Glu Gly Ile Ile Leu Lys Asn Ile Asp Gly
450 455 460
Leu Trp Glu Asn Ala Arg Ser Lys Asn Leu Tyr Lys Phe Lys Glu Val
465 470 475 480
Ile Trp Val Asp Leu Lys Ile Val Gly Ile Tyr Pro His Arg Lys Asp
485 490 495
Pro Thr Lys Ala Gly Gly Phe Ile Leu Glu Ser Glu Cys Gly Lys Ile
500 505 510
Lys Val Asn Ala Gly Ser Gly Leu Lys Asp Lys Ala Gly Val Lys Ser
515 520 525
His Glu Leu Asp Arg Thr Arg Ile Met Glu Asn Gln Asn Tyr Tyr Ile
530 535 540
Gly Lys Ile Leu Glu Cys Lys Cys Asn Gly Trp Leu Lys Ser Asp Gly
545 550 555 560
Arg Lys Pro Tyr Val Lys Leu Phe Leu Pro Ile Ala Ile Arg Leu Arg
565 570 575
Glu Asp Lys Thr Lys Ala Asn Thr Phe Glu Asp Val Phe Gly Asp Phe
580 585 590
His Glu Val Thr Gly Leu Pro Ala Pro Ala Pro Ala Pro Ala Ala Lys
595 600 605
Met Met Gln Arg Glu Ile Thr Lys Thr Thr Val Asn Val Ala Lys Met
610 615 620
Val Met Val Asp Gly Glu Val Gln Val Glu Gln Leu Pro Ser Glu Thr
625 630 635 640
Phe Val Gly Asn Leu Thr Met Glu Gln Ala Gln Trp Arg Met Lys Arg
645 650 655
Lys Tyr Lys Gly Glu Pro Val Gln Val Val Ser Val Glu Pro Asn Thr
660 665 670
Glu Val Tyr Glu Leu Pro Val Glu Lys Phe Leu Glu Val Ala Thr Val
675 680 685
Arg Val Glu Lys Asp Glu Asp Gln Glu Glu Gln Thr Glu Ala Pro Glu
690 695 700
Glu Gln Val Ala Glu
705
<210> 23
<211> 709
<212> PRT
<213> artifical sequence
<400> 23
Met Ala Lys Met Met Gln Arg Glu Ile Thr Lys Thr Thr Val Asn Val
1 5 10 15
Ala Lys Met Val Met Val Asp Gly Glu Val Gln Val Glu Gln Leu Pro
20 25 30
Ser Glu Thr Phe Val Gly Asn Leu Thr Met Glu Gln Ala Gln Trp Arg
35 40 45
Met Lys Arg Lys Tyr Lys Gly Glu Pro Val Gln Val Val Ser Val Glu
50 55 60
Pro Asn Thr Glu Val Tyr Glu Leu Pro Val Glu Lys Phe Leu Glu Val
65 70 75 80
Ala Thr Val Arg Val Glu Lys Asp Glu Asp Gln Glu Glu Gln Thr Glu
85 90 95
Ala Pro Glu Glu Gln Val Ala Glu Gly Gln Gly Gln Gly Gln Gly Gln
100 105 110
Ile Leu Lys Ile Leu Asn Glu Ile Ala Ser Ile Gly Ser Thr Lys Gln
115 120 125
Lys Lys Ala Ile Leu Glu Lys Asn Lys Asp Asn Glu Leu Leu Lys Arg
130 135 140
Val Tyr Arg Leu Thr Tyr Ser Arg Gly Leu Gln Tyr Tyr Ile Lys Lys
145 150 155 160
Trp Pro Lys Pro Gly Ile Ala Thr Gln Ser Phe Gly Met Thr Thr Leu
165 170 175
Thr Asp Met Leu Asp Phe Ile Glu Phe Thr Leu Ala Thr Arg Lys Leu
180 185 190
Thr Gly Asn Ala Ala Ile Arg Glu Leu Thr Gly Tyr Ile Thr Asp Gly
195 200 205
Lys Lys Asp Asp Val Glu Val Leu Arg Arg Val Met Met Arg Asp Leu
210 215 220
Glu Cys Gly Ala Ser Val Ser Ile Ala Asn Lys Val Trp Lys Gly Leu
225 230 235 240
Ile Pro Glu Gln Pro Gln Met Leu Ala Ser Ser Tyr Asp Glu Lys Gly
245 250 255
Ile Asn Lys Asn Ile Lys Phe Pro Ala Phe Ala Gln Leu Ser Ala Asp
260 265 270
Gly Ala Arg Cys Phe Ala Glu Val Arg Gly Asp Glu Leu Asp Asp Val
275 280 285
Arg Leu Leu Ser Arg Ala Gly Asn Glu Tyr Leu Gly Leu Asp Leu Leu
290 295 300
Lys Glu Glu Leu Ile Lys Met Thr Ala Glu Ala Arg Gln Ile His Pro
305 310 315 320
Glu Gly Val Leu Ile Asp Gly Glu Leu Val Tyr His Glu Gln Val Ala
325 330 335
Lys Glu Pro Glu Gly Leu Asp Ala Leu Phe Asp Arg Tyr Pro Glu Asn
340 345 350
Ser Lys Ala Lys Glu Phe Ala Glu Val Ala Glu Ser Arg Thr Ala Ser
355 360 365
Asn Gly Ile Ala Asn Lys Ser Leu Lys Gly Thr Ile Ser Glu Lys Glu
370 375 380
Ala Gln Cys Met Lys Phe Gln Val Trp Asp Tyr Val Pro Leu Val Glu
385 390 395 400
Ile Tyr Ser Leu Pro Ala Phe Arg Leu Lys Tyr Asp Val Arg Phe Ser
405 410 415
Lys Leu Glu Gln Met Thr Ser Gly Tyr Asp Lys Val Ile Leu Ile Glu
420 425 430
Asn Gln Val Val Asn Asn Leu Asp Glu Ala Lys Val Ile Tyr Lys Lys
435 440 445
Tyr Ile Asp Gln Gly Leu Glu Gly Ile Ile Leu Lys Asn Ile Asp Gly
450 455 460
Leu Trp Glu Asn Ala Arg Ser Lys Asn Leu Tyr Lys Phe Lys Glu Val
465 470 475 480
Ile Trp Val Asp Leu Lys Ile Val Gly Ile Tyr Pro His Arg Lys Asp
485 490 495
Pro Thr Lys Ala Gly Gly Phe Ile Leu Glu Ser Glu Cys Gly Lys Ile
500 505 510
Lys Val Asn Ala Gly Ser Gly Leu Lys Asp Lys Ala Gly Val Lys Ser
515 520 525
His Glu Leu Asp Arg Thr Arg Ile Met Glu Asn Gln Asn Tyr Tyr Ile
530 535 540
Gly Lys Ile Leu Glu Cys Lys Cys Asn Gly Trp Leu Lys Ser Asp Gly
545 550 555 560
Arg Lys Pro Tyr Val Lys Leu Phe Leu Pro Ile Ala Ile Arg Leu Arg
565 570 575
Glu Asp Lys Thr Lys Ala Asn Thr Phe Glu Asp Val Phe Gly Asp Phe
580 585 590
His Glu Val Thr Gly Leu Gly Gln Gly Gln Gly Gln Gly Gln Ala Lys
595 600 605
Met Met Gln Arg Glu Ile Thr Lys Thr Thr Val Asn Val Ala Lys Met
610 615 620
Val Met Val Asp Gly Glu Val Gln Val Glu Gln Leu Pro Ser Glu Thr
625 630 635 640
Phe Val Gly Asn Leu Thr Met Glu Gln Ala Gln Trp Arg Met Lys Arg
645 650 655
Lys Tyr Lys Gly Glu Pro Val Gln Val Val Ser Val Glu Pro Asn Thr
660 665 670
Glu Val Tyr Glu Leu Pro Val Glu Lys Phe Leu Glu Val Ala Thr Val
675 680 685
Arg Val Glu Lys Asp Glu Asp Gln Glu Glu Gln Thr Glu Ala Pro Glu
690 695 700
Glu Gln Val Ala Glu
705
<210> 24
<211> 703
<212> PRT
<213> artifical sequence
<400> 24
Met Ala Lys Met Met Gln Arg Glu Ile Thr Lys Thr Thr Val Asn Val
1 5 10 15
Ala Lys Met Val Met Val Asp Gly Glu Val Gln Val Glu Gln Leu Pro
20 25 30
Ser Glu Thr Phe Val Gly Asn Leu Thr Met Glu Gln Ala Gln Trp Arg
35 40 45
Met Lys Arg Lys Tyr Lys Gly Glu Pro Val Gln Val Val Ser Val Glu
50 55 60
Pro Asn Thr Glu Val Tyr Glu Leu Pro Val Glu Lys Phe Leu Glu Val
65 70 75 80
Ala Thr Val Arg Val Glu Lys Asp Glu Asp Gln Glu Glu Gln Thr Glu
85 90 95
Ala Pro Glu Glu Gln Val Ala Glu Ala Ala Ala Ala Ala Ile Leu Lys
100 105 110
Ile Leu Asn Glu Ile Ala Ser Ile Gly Ser Thr Lys Gln Lys Lys Ala
115 120 125
Ile Leu Glu Lys Asn Lys Asp Asn Glu Leu Leu Lys Arg Val Tyr Arg
130 135 140
Leu Thr Tyr Ser Arg Gly Leu Gln Tyr Tyr Ile Lys Lys Trp Pro Lys
145 150 155 160
Pro Gly Ile Ala Thr Gln Ser Phe Gly Met Thr Thr Leu Thr Asp Met
165 170 175
Leu Asp Phe Ile Glu Phe Thr Leu Ala Thr Arg Lys Leu Thr Gly Asn
180 185 190
Ala Ala Ile Arg Glu Leu Thr Gly Tyr Ile Thr Asp Gly Lys Lys Asp
195 200 205
Asp Val Glu Val Leu Arg Arg Val Met Met Arg Asp Leu Glu Cys Gly
210 215 220
Ala Ser Val Ser Ile Ala Asn Lys Val Trp Lys Gly Leu Ile Pro Glu
225 230 235 240
Gln Pro Gln Met Leu Ala Ser Ser Tyr Asp Glu Lys Gly Ile Asn Lys
245 250 255
Asn Ile Lys Phe Pro Ala Phe Ala Gln Leu Ser Ala Asp Gly Ala Arg
260 265 270
Cys Phe Ala Glu Val Arg Gly Asp Glu Leu Asp Asp Val Arg Leu Leu
275 280 285
Ser Arg Ala Gly Asn Glu Tyr Leu Gly Leu Asp Leu Leu Lys Glu Glu
290 295 300
Leu Ile Lys Met Thr Ala Glu Ala Arg Gln Ile His Pro Glu Gly Val
305 310 315 320
Leu Ile Asp Gly Glu Leu Val Tyr His Glu Gln Val Ala Lys Glu Pro
325 330 335
Glu Gly Leu Asp Ala Leu Phe Asp Arg Tyr Pro Glu Asn Ser Lys Ala
340 345 350
Lys Glu Phe Ala Glu Val Ala Glu Ser Arg Thr Ala Ser Asn Gly Ile
355 360 365
Ala Asn Lys Ser Leu Lys Gly Thr Ile Ser Glu Lys Glu Ala Gln Cys
370 375 380
Met Lys Phe Gln Val Trp Asp Tyr Val Pro Leu Val Glu Ile Tyr Ser
385 390 395 400
Leu Pro Ala Phe Arg Leu Lys Tyr Asp Val Arg Phe Ser Lys Leu Glu
405 410 415
Gln Met Thr Ser Gly Tyr Asp Lys Val Ile Leu Ile Glu Asn Gln Val
420 425 430
Val Asn Asn Leu Asp Glu Ala Lys Val Ile Tyr Lys Lys Tyr Ile Asp
435 440 445
Gln Gly Leu Glu Gly Ile Ile Leu Lys Asn Ile Asp Gly Leu Trp Glu
450 455 460
Asn Ala Arg Ser Lys Asn Leu Tyr Lys Phe Lys Glu Val Ile Trp Val
465 470 475 480
Asp Leu Lys Ile Val Gly Ile Tyr Pro His Arg Lys Asp Pro Thr Lys
485 490 495
Ala Gly Gly Phe Ile Leu Glu Ser Glu Cys Gly Lys Ile Lys Val Asn
500 505 510
Ala Gly Ser Gly Leu Lys Asp Lys Ala Gly Val Lys Ser His Glu Leu
515 520 525
Asp Arg Thr Arg Ile Met Glu Asn Gln Asn Tyr Tyr Ile Gly Lys Ile
530 535 540
Leu Glu Cys Lys Cys Asn Gly Trp Leu Lys Ser Asp Gly Arg Lys Pro
545 550 555 560
Tyr Val Lys Leu Phe Leu Pro Ile Ala Ile Arg Leu Arg Glu Asp Lys
565 570 575
Thr Lys Ala Asn Thr Phe Glu Asp Val Phe Gly Asp Phe His Glu Val
580 585 590
Thr Gly Leu Ala Ala Ala Ala Ala Ala Lys Met Met Gln Arg Glu Ile
595 600 605
Thr Lys Thr Thr Val Asn Val Ala Lys Met Val Met Val Asp Gly Glu
610 615 620
Val Gln Val Glu Gln Leu Pro Ser Glu Thr Phe Val Gly Asn Leu Thr
625 630 635 640
Met Glu Gln Ala Gln Trp Arg Met Lys Arg Lys Tyr Lys Gly Glu Pro
645 650 655
Val Gln Val Val Ser Val Glu Pro Asn Thr Glu Val Tyr Glu Leu Pro
660 665 670
Val Glu Lys Phe Leu Glu Val Ala Thr Val Arg Val Glu Lys Asp Glu
675 680 685
Asp Gln Glu Glu Gln Thr Glu Ala Pro Glu Glu Gln Val Ala Glu
690 695 700
<210> 25
<211> 1461
<212> DNA
<213> artifical sequence
<400> 25
atgattctga agatcctgaa cgaaattgcc agcatcggta gcaccaaaca aaaacaggcg 60
attctggaaa aaaacaaaga caacgaactg ctgaaacgcg tgtatcgcct gacttactct 120
cgtggtctgc aatactatat taagaagtgg ccgaaaccag gtattgcgac tcagtctttc 180
ggcatgctga ccctgaccga tatgctggac ttcatcgaat tcaccctggc aactcgcaaa 240
ctgaccggca acgcagcgat tgaagaactg accggctata tcaccgatgg caaaaaagac 300
gatgttgaag tcctgcgccg cgtcatgatg cgcgatctgg aatgcggcgc tagcgtttct 360
atcgctaaca aagtgtggcc aggtctgatt ccggaacagc cgcagatgct ggctagctcc 420
tacgacgaaa agggcattaa caaaaacatt aaattccctg cgttcgcaca gctgaaagcc 480
gacggtgctc gctgtttcgc ggaagttcgt ggcgacgaac tggacgatgt acgtctgctg 540
tcccgtgcgg gtaacgaata cctgggtctg gacctgctga aggaagaact gatcaaaatg 600
accgctgaag cacgccagat ccatccggaa ggtgtgctga ttgacggtga gctggtctac 660
cacgaacagg ttaaaaaaga acctgaaggc ctggatttcc tgttcgacgc ttacccggaa 720
aattccaaag cgaaggaatt cgcagaagtg gcggaatccc gtaccgcgtc taacggtatc 780
gccaacaaat ccctgaaggg caccatttct gaaaaagaag ctcaatgtat gaaattccag 840
gtctgggatt atgtcccgct ggttgaaatt tactctctgc cggcattccg tctgaaatac 900
gatgttcgct ttagcaaact ggaacagatg acttctggct acgacaaggt gatcctgatc 960
gaaaaccagg tggtaaacaa cctggacgag gctaaagtaa tttataaaaa atacatcgac 1020
cagggcctgg aaggtatcat cctgaaaaac atcgatggcc tgtgggaaaa cgcccgttcc 1080
aaaaacctgt ataaattcaa agaagtgatc gatgtggacc tgaaaatcgt gggtatttat 1140
ccgcaccgta aagatccgac caaagctggc ggcttcatcc tggaatccga atgcggcaaa 1200
atcaaagtca atgcaggtag cggcctgaaa gataaagctg gtgtcaaatc tcatgaactg 1260
gaccgtaccc gtatcatgga aaaccagaac tactatatcg gcaaaatcct ggaatgcgaa 1320
tgcaacggct ggctgaaaag cgatggccgt actgactatg tgaaactgtt cctgccgatc 1380
gcgattcgtc tgcgcgagga caagactaaa gctaacacgt ttgaagacgt gttcggcgac 1440
tttcatgaag taaccggtct g 1461
<210> 26
<211> 1461
<212> DNA
<213> artifical sequence
<400> 26
atgatcctga aaattctgaa cgaaatcgcc agcatcggta gcaccaaaca gaaaaaagcg 60
atcctggaga aaaacaaaga caacgaactg ctgaaacgtg tttaccgtct gacttattcc 120
cgtggcctgc agtactacat taaaaaatgg ccgaaaccgg gcattgccac ccagtccttt 180
ggtatgacta ccctgactga catgctggat ttcattgaat tcaccctggc tacccgtaag 240
ctgaccggca acgcagctat ccgtgaactg accggctaca ttaccgacgg taaaaaagat 300
gacgttgaag ttctgcgccg tgtaatgatg cgtgatctgg aatgcggtgc atccgtatct 360
atcgcgaaca aagtttggaa aggtctgatc cctgagcaac cgcagatgct ggcatcttcc 420
tacgatgaga aaggtatcaa caaaaacatc aaatttccgg cgtttgcaca gctgaaagct 480
gatggcgcgc gttgtttcgc tgaagttcgt ggcgatgaac tggatgatgt acgtctgctg 540
agccgtgcgg gcaacgaata cctgggcctg gatctgctga aggaggaact gatcaaaatg 600
actgcagaag cccgccagat ccatccagaa ggcgtactga tcgacggcga gctggtgtat 660
catgaacagg ttgctaaaga accggaaggt ctggatgctc tgttcgaccg ttacccggag 720
aatagcaaag caaaagaatt cgccgaagtg gcggaatctc gtaccgcgtc caacggcatc 780
gctaacaaat ccctgaaagg taccatctcc gaaaaagaag ctcaatgcat gaaattccag 840
gtgtgggatt atgtcccgct ggtcgaaatt tactccctgc cggctttccg tctgaaatac 900
gacgtacgtt tcagcaagct ggaacagatg accagcggtt acgataaggt aatcctgatc 960
gaaaaccagg ttgttaacaa cctggatgaa gcgaaagtca tttacaaaaa atacatcgat 1020
cagggtctgg aaggcattat tctgaaaaac atcgacggtc tgtgggaaaa cgctcgttct 1080
aaaaatctgt acaagtttaa agaagttatt tgggtcgatc tgaaaatcgt gggcatctat 1140
ccgcaccgca aggatccaac caaagcgggt ggcttcattc tggagtccga atgtggcaaa 1200
atcaaagtta acgcaggttc tggtctgaaa gataaagctg gtgttaaatc tcacgaactg 1260
gatcgtaccc gtatcatgga aaaccagaac tattatatcg gtaaaatcct ggaatgcaaa 1320
tgcaacggct ggctgaaatc cgacggccgt aagccatacg tgaaactgtt cctgccgatc 1380
gctattcgtc tgcgtgagga caaaaccaaa gcaaacactt tcgaagatgt gttcggtgac 1440
ttccacgaag taactggtct g 1461
<210> 27
<211> 1461
<212> DNA
<213> artifical sequence
<400> 27
atgattctga agatcctgaa cgaaattgcc agcatcggta gcaccaaaca aaaacaggcg 60
attctggaaa aaaacaaaga caacgaactg ctgaaacgcg tgtatcgcct gacttactct 120
cgtggtctgc aatactatat taagaagtgg ccgaaaccag gtattgcgac tcagtctttc 180
ggcatgctga ccctgaccga tatgctggac ttcatcgaat tcaccctggc aactcgcaaa 240
ctgaccggca acgcagcgat tgaagaactg accggctata tcaccgatgg caaaaaagac 300
gatgttgaag tcctgcgccg cgtcatgatg cgcgatctgg aatgcggcgc tagcgtttct 360
atcgctaaca aagtgtggcc aggtctgatt ccggaacagc cgcagatgct ggctagctcc 420
tacgacgaaa agggcattaa caaaaacatt aaattccctg cgttcgcaca gctgagcgcc 480
gacggtgctc gctgtttcgc ggaagttcgt ggcgacgaac tggacgatgt acgtctgctg 540
tcccgtgcgg gtaacgaata cctgggtctg gacctgctga aggaagaact gatcaaaatg 600
accgctgaag cacgccagat ccatccggaa ggtgtgctga ttgacggtga gctggtctac 660
cacgaacagg ttaaaaaaga acctgaaggc ctggatttcc tgttcgacgc ttacccggaa 720
aattccaaag cgaaggaatt cgcagaagtg gcggaatccc gtaccgcgtc taacggtatc 780
gccaacaaat ccctgaaggg caccatttct gaaaaagaag ctcaatgtat gaaattccag 840
gtctgggatt atgtcccgct ggttgaaatt tactctctgc cggcattccg tctgaaatac 900
gatgttcgct ttagcaaact ggaacagatg acttctggct acgacaaggt gatcctgatc 960
gaaaaccagg tggtaaacaa cctggacgag gctaaagtaa tttataaaaa atacatcgac 1020
cagggcctgg aaggtatcat cctgaaaaac atcgatggcc tgtgggaaaa cgcccgttcc 1080
aaaaacctgt ataaattcaa agaagtgatc gatgtggacc tgaaaatcgt gggtatttat 1140
ccgcaccgta aagatccgac caaagctggc ggcttcatcc tggaatccga atgcggcaaa 1200
atcaaagtca atgcaggtag cggcctgaaa gataaagctg gtgtcaaatc tcatgaactg 1260
gaccgtaccc gtatcatgga aaaccagaac tactatatcg gcaaaatcct ggaatgcgaa 1320
tgcaacggct ggctgaaaag cgatggccgt actgactatg tgaaactgtt cctgccgatc 1380
gcgattcgtc tgcgcgagga caagactaaa gctaacacgt ttgaagacgt gttcggcgac 1440
tttcatgaag taaccggtct g 1461
<210> 28
<211> 1461
<212> DNA
<213> artifical sequence
<400> 28
atgattctga agatcctgaa cgaaattgcc agcatcggta gcaccaaaca aaaacaggcg 60
attctggaaa aaaacaaaga caacgaactg ctgaaacgcg tgtatcgcct gacttactct 120
cgtggtctgc aatactatat taagaagtgg ccgaaaccag gtattgcgac tcagtctttc 180
ggcatgctga ccctgaccga tatgctggac ttcatcgaat tcaccctggc aactcgcaaa 240
ctgaccggca acgcagcgat tgaagaactg accggctata tcaccgatgg caaaaaagac 300
gatgttgaag tcctgcgccg cgtcatgatg cgcgatctgg aatgcggcgc tagcgtttct 360
atcgctaaca aagtgtggcc aggtctgatt ccggaacagc cgcagatgct ggctagctcc 420
tacgacgaaa agggcattaa caaaaacatt aaattccctg cgttcgcaca gctgctggcc 480
gacggtgctc gctgtttcgc ggaagttcgt ggcgacgaac tggacgatgt acgtctgctg 540
tcccgtgcgg gtaacgaata cctgggtctg gacctgctga aggaagaact gatcaaaatg 600
accgctgaag cacgccagat ccatccggaa ggtgtgctga ttgacggtga gctggtctac 660
cacgaacagg ttaaaaaaga acctgaaggc ctggatttcc tgttcgacgc ttacccggaa 720
aattccaaag cgaaggaatt cgcagaagtg gcggaatccc gtaccgcgtc taacggtatc 780
gccaacaaat ccctgaaggg caccatttct gaaaaagaag ctcaatgtat gaaattccag 840
gtctgggatt atgtcccgct ggttgaaatt tactctctgc cggcattccg tctgaaatac 900
gatgttcgct ttagcaaact ggaacagatg acttctggct acgacaaggt gatcctgatc 960
gaaaaccagg tggtaaacaa cctggacgag gctaaagtaa tttataaaaa atacatcgac 1020
cagggcctgg aaggtatcat cctgaaaaac atcgatggcc tgtgggaaaa cgcccgttcc 1080
aaaaacctgt ataaattcaa agaagtgatc gatgtggacc tgaaaatcgt gggtatttat 1140
ccgcaccgta aagatccgac caaagctggc ggcttcatcc tggaatccga atgcggcaaa 1200
atcaaagtca atgcaggtag cggcctgaaa gataaagctg gtgtcaaatc tcatgaactg 1260
gaccgtaccc gtatcatgga aaaccagaac tactatatcg gcaaaatcct ggaatgcgaa 1320
tgcaacggct ggctgaaaag cgatggccgt actgactatg tgaaactgtt cctgccgatc 1380
gcgattcgtc tgcgcgagga caagactaaa gctaacacgt ttgaagacgt gttcggcgac 1440
tttcatgaag taaccggtct g 1461
<210> 29
<211> 1461
<212> DNA
<213> artifical sequence
<400> 29
atgattctga agatcctgaa cgaaattgcc agcatcggta gcaccaaaca aaaacaggcg 60
attctggaaa aaaacaaaga caacgaactg ctgaaacgcg tgtatcgcct gacttactct 120
cgtggtctgc aatactatat taagaagtgg ccgaaaccag gtattgcgac tcagtctttc 180
ggcatgctga ccctgaccga tatgctggac ttcatcgaat tcaccctggc aactcgcaaa 240
ctgaccggca acgcagcgat tgaagaactg accggctata tcaccgatgg caaaaaagac 300
gatgttgaag tcctgcgccg cgtcatgatg cgcgatctgg aatgcggcgc tagcgtttct 360
atcgctaaca aagtgtggcc aggtctgatt ccggaacagc cgcagatgct ggctagctcc 420
tacgacgaaa agggcattaa caaaaacatt aaattccctg cgttcgcaca gctgtgtgcc 480
gacggtgctc gctgtttcgc ggaagttcgt ggcgacgaac tggacgatgt acgtctgctg 540
tcccgtgcgg gtaacgaata cctgggtctg gacctgctga aggaagaact gatcaaaatg 600
accgctgaag cacgccagat ccatccggaa ggtgtgctga ttgacggtga gctggtctac 660
cacgaacagg ttaaaaaaga acctgaaggc ctggatttcc tgttcgacgc ttacccggaa 720
aattccaaag cgaaggaatt cgcagaagtg gcggaatccc gtaccgcgtc taacggtatc 780
gccaacaaat ccctgaaggg caccatttct gaaaaagaag ctcaatgtat gaaattccag 840
gtctgggatt atgtcccgct ggttgaaatt tactctctgc cggcattccg tctgaaatac 900
gatgttcgct ttagcaaact ggaacagatg acttctggct acgacaaggt gatcctgatc 960
gaaaaccagg tggtaaacaa cctggacgag gctaaagtaa tttataaaaa atacatcgac 1020
cagggcctgg aaggtatcat cctgaaaaac atcgatggcc tgtgggaaaa cgcccgttcc 1080
aaaaacctgt ataaattcaa agaagtgatc gatgtggacc tgaaaatcgt gggtatttat 1140
ccgcaccgta aagatccgac caaagctggc ggcttcatcc tggaatccga atgcggcaaa 1200
atcaaagtca atgcaggtag cggcctgaaa gataaagctg gtgtcaaatc tcatgaactg 1260
gaccgtaccc gtatcatgga aaaccagaac tactatatcg gcaaaatcct ggaatgcgaa 1320
tgcaacggct ggctgaaaag cgatggccgt actgactatg tgaaactgtt cctgccgatc 1380
gcgattcgtc tgcgcgagga caagactaaa gctaacacgt ttgaagacgt gttcggcgac 1440
tttcatgaag taaccggtct g 1461
<210> 30
<211> 1461
<212> DNA
<213> artifical sequence
<400> 30
atgattctga agatcctgaa cgaaattgcc agcatcggta gcaccaaaca aaaacaggcg 60
attctggaaa aaaacaaaga caacgaactg ctgaaacgcg tgtatcgcct gacttactct 120
cgtggtctgc aatactatat taagaagtgg ccgaaaccag gtattgcgac tcagtctttc 180
ggcatgctga ccctgaccga tatgctggac ttcatcgaat tcaccctggc aactcgcaaa 240
ctgaccggca acgcagcgat tgaagaactg accggctata tcaccgatgg caaaaaagac 300
gatgttgaag tcctgcgccg cgtcatgatg cgcgatctgg aatgcggcgc tagcgtttct 360
atcgctaaca aagtgtggcc aggtctgatt ccggaacagc cgcagatgct ggctagctcc 420
tacgacgaaa agggcattaa caaaaacatt aaattccctg cgttcgcaca gctggcagcc 480
gacggtgctc gctgtttcgc ggaagttcgt ggcgacgaac tggacgatgt acgtctgctg 540
tcccgtgcgg gtaacgaata cctgggtctg gacctgctga aggaagaact gatcaaaatg 600
accgctgaag cacgccagat ccatccggaa ggtgtgctga ttgacggtga gctggtctac 660
cacgaacagg ttaaaaaaga acctgaaggc ctggatttcc tgttcgacgc ttacccggaa 720
aattccaaag cgaaggaatt cgcagaagtg gcggaatccc gtaccgcgtc taacggtatc 780
gccaacaaat ccctgaaggg caccatttct gaaaaagaag ctcaatgtat gaaattccag 840
gtctgggatt atgtcccgct ggttgaaatt tactctctgc cggcattccg tctgaaatac 900
gatgttcgct ttagcaaact ggaacagatg acttctggct acgacaaggt gatcctgatc 960
gaaaaccagg tggtaaacaa cctggacgag gctaaagtaa tttataaaaa atacatcgac 1020
cagggcctgg aaggtatcat cctgaaaaac atcgatggcc tgtgggaaaa cgcccgttcc 1080
aaaaacctgt ataaattcaa agaagtgatc gatgtggacc tgaaaatcgt gggtatttat 1140
ccgcaccgta aagatccgac caaagctggc ggcttcatcc tggaatccga atgcggcaaa 1200
atcaaagtca atgcaggtag cggcctgaaa gataaagctg gtgtcaaatc tcatgaactg 1260
gaccgtaccc gtatcatgga aaaccagaac tactatatcg gcaaaatcct ggaatgcgaa 1320
tgcaacggct ggctgaaaag cgatggccgt actgactatg tgaaactgtt cctgccgatc 1380
gcgattcgtc tgcgcgagga caagactaaa gctaacacgt ttgaagacgt gttcggcgac 1440
tttcatgaag taaccggtct g 1461
<210> 31
<211> 1461
<212> DNA
<213> artifical sequence
<400> 31
atgattctga agatcctgaa cgaaattgcc agcatcggta gcaccaaaca aaaacaggcg 60
attctggaaa aaaacaaaga caacgaactg ctgaaacgcg tgtatcgcct gacttactct 120
cgtggtctgc aatactatat taagaagtgg ccgaaaccag gtattgcgac tcagtctttc 180
ggcatgctga ccctgaccga tatgctggac ttcatcgaat tcaccctggc aactcgcaaa 240
ctgaccggca acgcagcgat tgaagaactg accggctata tcaccgatgg caaaaaagac 300
gatgttgaag tcctgcgccg cgtcatgatg cgcgatctgg aatgcggcgc tagcgtttct 360
atcgctaaca aagtgtggcc aggtctgatt ccggaacagc cgcagatgct ggctagctcc 420
tacgacgaaa agggcattaa caaaaacatt aaattccctg cgttcgcaca gctgtgggcc 480
gacggtgctc gctgtttcgc ggaagttcgt ggcgacgaac tggacgatgt acgtctgctg 540
tcccgtgcgg gtaacgaata cctgggtctg gacctgctga aggaagaact gatcaaaatg 600
accgctgaag cacgccagat ccatccggaa ggtgtgctga ttgacggtga gctggtctac 660
cacgaacagg ttaaaaaaga acctgaaggc ctggatttcc tgttcgacgc ttacccggaa 720
aattccaaag cgaaggaatt cgcagaagtg gcggaatccc gtaccgcgtc taacggtatc 780
gccaacaaat ccctgaaggg caccatttct gaaaaagaag ctcaatgtat gaaattccag 840
gtctgggatt atgtcccgct ggttgaaatt tactctctgc cggcattccg tctgaaatac 900
gatgttcgct ttagcaaact ggaacagatg acttctggct acgacaaggt gatcctgatc 960
gaaaaccagg tggtaaacaa cctggacgag gctaaagtaa tttataaaaa atacatcgac 1020
cagggcctgg aaggtatcat cctgaaaaac atcgatggcc tgtgggaaaa cgcccgttcc 1080
aaaaacctgt ataaattcaa agaagtgatc gatgtggacc tgaaaatcgt gggtatttat 1140
ccgcaccgta aagatccgac caaagctggc ggcttcatcc tggaatccga atgcggcaaa 1200
atcaaagtca atgcaggtag cggcctgaaa gataaagctg gtgtcaaatc tcatgaactg 1260
gaccgtaccc gtatcatgga aaaccagaac tactatatcg gcaaaatcct ggaatgcgaa 1320
tgcaacggct ggctgaaaag cgatggccgt actgactatg tgaaactgtt cctgccgatc 1380
gcgattcgtc tgcgcgagga caagactaaa gctaacacgt ttgaagacgt gttcggcgac 1440
tttcatgaag taaccggtct g 1461
<210> 32
<211> 1461
<212> DNA
<213> artifical sequence
<400> 32
atgatcctga aaattctgaa cgaaatcgcc agcatcggta gcaccaaaca gaaaaaagcg 60
atcctggaga aaaacaaaga caacgaactg ctgaaacgtg tttaccgtct gacttattcc 120
cgtggcctgc agtactacat taaaaaatgg ccgaaaccgg gcattgccac ccagtccttt 180
ggtatgacta ccctgactga catgctggat ttcattgaat tcaccctggc tacccgtaag 240
ctgaccggca acgcagctat ccgtgaactg accggctaca ttaccgacgg taaaaaagat 300
gacgttgaag ttctgcgccg tgtaatgatg cgtgatctgg aatgcggtgc atccgtatct 360
atcgcgaaca aagtttggaa aggtctgatc cctgagcaac cgcagatgct ggcatcttcc 420
tacgatgaga aaggtatcaa caaaaacatc aaatttccgg cgtttgcaca gctgagcgct 480
gatggcgcgc gttgtttcgc tgaagttcgt ggcgatgaac tggatgatgt acgtctgctg 540
agccgtgcgg gcaacgaata cctgggcctg gatctgctga aggaggaact gatcaaaatg 600
actgcagaag cccgccagat ccatccagaa ggcgtactga tcgacggcga gctggtgtat 660
catgaacagg ttgctaaaga accggaaggt ctggatgctc tgttcgaccg ttacccggag 720
aatagcaaag caaaagaatt cgccgaagtg gcggaatctc gtaccgcgtc caacggcatc 780
gctaacaaat ccctgaaagg taccatctcc gaaaaagaag ctcaatgcat gaaattccag 840
gtgtgggatt atgtcccgct ggtcgaaatt tactccctgc cggctttccg tctgaaatac 900
gacgtacgtt tcagcaagct ggaacagatg accagcggtt acgataaggt aatcctgatc 960
gaaaaccagg ttgttaacaa cctggatgaa gcgaaagtca tttacaaaaa atacatcgat 1020
cagggtctgg aaggcattat tctgaaaaac atcgacggtc tgtgggaaaa cgctcgttct 1080
aaaaatctgt acaagtttaa agaagttatt tgggtcgatc tgaaaatcgt gggcatctat 1140
ccgcaccgca aggatccaac caaagcgggt ggcttcattc tggagtccga atgtggcaaa 1200
atcaaagtta acgcaggttc tggtctgaaa gataaagctg gtgttaaatc tcacgaactg 1260
gatcgtaccc gtatcatgga aaaccagaac tattatatcg gtaaaatcct ggaatgcaaa 1320
tgcaacggct ggctgaaatc cgacggccgt aagccatacg tgaaactgtt cctgccgatc 1380
gctattcgtc tgcgtgagga caaaaccaaa gcaaacactt tcgaagatgt gttcggtgac 1440
ttccacgaag taactggtct g 1461
<210> 33
<211> 3042
<212> DNA
<213> artifical sequence
<400> 33
atgatcctga aaattctgaa cgaaatcgcc agcatcggta gcaccaaaca gaaaaaagcg 60
atcctggaga aaaacaaaga caacgaactg ctgaaacgtg tttaccgtct gacttattcc 120
cgtggcctgc agtactacat taaaaaatgg ccgaaaccgg gcattgccac ccagtccttt 180
ggtatgacta ccctgactga catgctggat ttcattgaat tcaccctggc tacccgtaag 240
ctgaccggca acgcagctat ccgtgaactg accggctaca ttaccgacgg taaaaaagat 300
gacgttgaag ttctgcgccg tgtaatgatg cgtgatctgg aatgcggtgc atccgtatct 360
atcgcgaaca aagtttggaa aggtctgatc cctgagcaac cgcagatgct ggcatcttcc 420
tacgatgaga aaggtatcaa caaaaacatc aaatttccgg cgtttgcaca gctgagcgct 480
gatggcgcgc gttgtttcgc tgaagttcgt ggcgatgaac tggatgatgt acgtctgctg 540
agccgtgcgg gcaacgaata cctgggcctg gatctgctga aggaggaact gatcaaaatg 600
actgcagaag cccgccagat ccatccagaa ggcgtactga tcgacggcga gctggtgtat 660
catgaacagg ttgctaaaga accggaaggt ctggatgctc tgttcgaccg ttacccggag 720
aatagcaaag caaaagaatt cgccgaagtg gcggaatctc gtaccgcgtc caacggcatc 780
gctaacaaat ccctgaaagg taccatctcc gaaaaagaag ctcaatgcat gaaattccag 840
gtgtgggatt atgtcccgct ggtcgaaatt tactccctgc cggctttccg tctgaaatac 900
gacgtacgtt tcagcaagct ggaacagatg accagcggtt acgataaggt aatcctgatc 960
gaaaaccagg ttgttaacaa cctggatgaa gcgaaagtca tttacaaaaa atacatcgat 1020
cagggtctgg aaggcattat tctgaaaaac atcgacggtc tgtgggaaaa cgctcgttct 1080
aaaaatctgt acaagtttaa agaagttatt tgggtcgatc tgaaaatcgt gggcatctat 1140
ccgcaccgca aggatccaac caaagcgggt ggcttcattc tggagtccga atgtggcaaa 1200
atcaaagtta acgcaggttc tggtctgaaa gataaagctg gtgttaaatc tcacgaactg 1260
gatcgtaccc gtatcatgga aaaccagaac tattatatcg gtaaaatcct ggaatgcaaa 1320
tgcaacggct ggctgaaatc cgacggccgt aagccatacg tgaaactgtt cctgccgatc 1380
gctattcgtc tgcgtgagga caaaaccaaa gcaaacactt tcgaagatgt gttcggtgac 1440
ttccacgaag taactggtct gggtggtggt ggtagccgtg aaatcctgaa tccaccagaa 1500
aaagaaaccc agggcgaagg tccgtccctg tttatggctt ctaccaaaac cgaagcaatc 1560
gcgccggcgt ccactatgga agataaagaa gaagatgtgg gcttccagga taacctgttt 1620
ctggaaaaag cactgcaact ggctaaacgt cacgcgaacg ctctgttcga ttatgcggtt 1680
actggtgacg taaaaatgct gctggcggtt cagcgtcacc tgactgcggt ccaagatgaa 1740
aatggtgact ccgtcctgca tctggcgatc atccacctgc acgctcagct ggtgcgtgat 1800
ctgctggagg ttacctctgg ttctatctcc gatgacatca tcaatatgcg taacgacctg 1860
tatcagaccc cgctgcatct ggcggtgatt actaaacagg aagacgtagt agaagacctg 1920
ctgcgtgtgg gcgcagatct gtctctgctg gatcgttggg gtaacagcgt actgcacctg 1980
gcggcgaaag aaggtcatga taaaatcctg ggtgtcctgc tgaagaactc taaggcggct 2040
ctgctgatca accacccgaa cggtgaaggt ctgaacgcta tccacattgc cgttatgagc 2100
aactccctgt cttgtctgca gctgctggtg gctgctggcg ctgaagttaa cgctcaggag 2160
cagaagagcg gtcgtaccgc cctgcacctg gcagttgaat acgataacat ttccctggcg 2220
ggttgtctgc tgctggaagg cgacgcactg gttgacagca ctacctacga tggtactact 2280
ccgctgcaca ttgctgccgg tcgtggtagc actcgtctgg ctgctctgct gaaagctgct 2340
ggcgcggatc cactggtcga aaacttcgaa ccgctgtacg acctggacga ctcttgggaa 2400
aaagccggtg aggacgaagg tgttgtaccg ggtaccactc cgctggatat ggctgcaaat 2460
tggcaggtgt tcgatatcct gaacggtaaa ccatatgaac cggttttcac gtccgacgac 2520
atcctgccac aaggtgacat caaacagctg accgaagata ctcgtctgca gctgtgcaaa 2580
ctgctggaga tcccggaccc ggataaaaac tgggcaacgc tggctcagaa actgggtctg 2640
ggtatcctga acaacgcatt ccgtctgagc ccggcaccga gcaaaactct gatggacaac 2700
tacgaagtct ctggtggtac catcaaggaa ctggttgagg cactgcgtca gatgggctac 2760
acggaagcga tcgaagtgat ccaggcggcg ttccgtactc cggaaaccac cgctagctct 2820
ccagtaacta ctgctcaagc gcacctgctg ccgctgtctt cttcctctac tcgccagcac 2880
atcgacgaac tgcgtgacaa tgactctgtt tgcgacagcg gtgtggaaac cagctttcgc 2940
aaactgtctt tctctgaatc cctgactggc gatggtccgc tgctgagcct gaacaaaatg 3000
ccacataatt acggccagga tggcccgatt gaaggtaaga tc 3042
<210> 34
<211> 2361
<212> DNA
<213> artifical sequence
<400> 34
atgatcctga aaattctgaa cgaaatcgcc agcatcggta gcaccaaaca gaaaaaagcg 60
atcctggaga aaaacaaaga caacgaactg ctgaaacgtg tttaccgtct gacttattcc 120
cgtggcctgc agtactacat taaaaaatgg ccgaaaccgg gcattgccac ccagtccttt 180
ggtatgacta ccctgactga catgctggat ttcattgaat tcaccctggc tacccgtaag 240
ctgaccggca acgcagctat ccgtgaactg accggctaca ttaccgacgg taaaaaagat 300
gacgttgaag ttctgcgccg tgtaatgatg cgtgatctgg aatgcggtgc atccgtatct 360
atcgcgaaca aagtttggaa aggtctgatc cctgagcaac cgcagatgct ggcatcttcc 420
tacgatgaga aaggtatcaa caaaaacatc aaatttccgg cgtttgcaca gctgagcgct 480
gatggcgcgc gttgtttcgc tgaagttcgt ggcgatgaac tggatgatgt acgtctgctg 540
agccgtgcgg gcaacgaata cctgggcctg gatctgctga aggaggaact gatcaaaatg 600
actgcagaag cccgccagat ccatccagaa ggcgtactga tcgacggcga gctggtgtat 660
catgaacagg ttgctaaaga accggaaggt ctggatgctc tgttcgaccg ttacccggag 720
aatagcaaag caaaagaatt cgccgaagtg gcggaatctc gtaccgcgtc caacggcatc 780
gctaacaaat ccctgaaagg taccatctcc gaaaaagaag ctcaatgcat gaaattccag 840
gtgtgggatt atgtcccgct ggtcgaaatt tactccctgc cggctttccg tctgaaatac 900
gacgtacgtt tcagcaagct ggaacagatg accagcggtt acgataaggt aatcctgatc 960
gaaaaccagg ttgttaacaa cctggatgaa gcgaaagtca tttacaaaaa atacatcgat 1020
cagggtctgg aaggcattat tctgaaaaac atcgacggtc tgtgggaaaa cgctcgttct 1080
aaaaatctgt acaagtttaa agaagttatt tgggtcgatc tgaaaatcgt gggcatctat 1140
ccgcaccgca aggatccaac caaagcgggt ggcttcattc tggagtccga atgtggcaaa 1200
atcaaagtta acgcaggttc tggtctgaaa gataaagctg gtgttaaatc tcacgaactg 1260
gatcgtaccc gtatcatgga aaaccagaac tattatatcg gtaaaatcct ggaatgcaaa 1320
tgcaacggct ggctgaaatc cgacggccgt aagccatacg tgaaactgtt cctgccgatc 1380
gctattcgtc tgcgtgagga caaaaccaaa gcaaacactt tcgaagatgt gttcggtgac 1440
ttccacgaag taactggtct gggtggtggt ggtagcgatt ggcagctgcc gtctcattct 1500
ggtccgtatg agctgcgtat tgaagtgcag ccgaaatctc accaccgtgc tcactatgaa 1560
acggaaggtt cccgtggtgc tgtcaaggct tctgcaggtg gtcacccaat tgttcagctg 1620
cacggttatc tggaaaacga gccgctgacc ctgcaactgt tcatcggtac tgctgatgac 1680
cgtctgctgc gtccacatgc tttttaccag gttcaccgta tcaccggtaa aaccgttagc 1740
actacttctc acgagattat cctgagcaac accaaagttc tggaaattcc gctgctgccg 1800
gaaaacaaca tgcgtgcaat tatcgactgt gctggcatcc tgaaactgcg taactctgac 1860
atcgaactgc gtaaaggtga aactgacatc ggtcgcaaaa acacccgtgt tcgcctggta 1920
tttcgcgttc acattccgca accgaatggt cgtactctgt ctctgcaggt cgcttctaac 1980
ccgattgagt gctctcaacg tagcgcttct aacctgaaga ttgttcgtat ggaccgcact 2040
gcgggttgtg ttaccggtgg cgaagaaatt tacctgctgt gcgacaaagt ccaaaaagac 2100
gatattcaga ttcgttttta cgaagaggaa gaaaatggcg gtgtttggga aggttttggc 2160
gatttctctc cgaccgacgt tcaccgtcag ttcgcgatcg tgttcaaaac cccgaaatac 2220
aaggacatca acatcaccaa gccggcaagc gtatttgtgc agctgcgtcg taaaagcgac 2280
ctggaaacta gcgaaccgaa gccgttcctg tattacccgg aaatcaaaga taaggaggaa 2340
gttcagcgca aacgtcagaa a 2361
<210> 35
<211> 1740
<212> DNA
<213> artifical sequence
<400> 35
atgatcctga aaattctgaa cgaaatcgcc agcatcggta gcaccaaaca gaaaaaagcg 60
atcctggaga aaaacaaaga caacgaactg ctgaaacgtg tttaccgtct gacttattcc 120
cgtggcctgc agtactacat taaaaaatgg ccgaaaccgg gcattgccac ccagtccttt 180
ggtatgacta ccctgactga catgctggat ttcattgaat tcaccctggc tacccgtaag 240
ctgaccggca acgcagctat ccgtgaactg accggctaca ttaccgacgg taaaaaagat 300
gacgttgaag ttctgcgccg tgtaatgatg cgtgatctgg aatgcggtgc atccgtatct 360
atcgcgaaca aagtttggaa aggtctgatc cctgagcaac cgcagatgct ggcatcttcc 420
tacgatgaga aaggtatcaa caaaaacatc aaatttccgg cgtttgcaca gctgagcgct 480
gatggcgcgc gttgtttcgc tgaagttcgt ggcgatgaac tggatgatgt acgtctgctg 540
agccgtgcgg gcaacgaata cctgggcctg gatctgctga aggaggaact gatcaaaatg 600
actgcagaag cccgccagat ccatccagaa ggcgtactga tcgacggcga gctggtgtat 660
catgaacagg ttgctaaaga accggaaggt ctggatgctc tgttcgaccg ttacccggag 720
aatagcaaag caaaagaatt cgccgaagtg gcggaatctc gtaccgcgtc caacggcatc 780
gctaacaaat ccctgaaagg taccatctcc gaaaaagaag ctcaatgcat gaaattccag 840
gtgtgggatt atgtcccgct ggtcgaaatt tactccctgc cggctttccg tctgaaatac 900
gacgtacgtt tcagcaagct ggaacagatg accagcggtt acgataaggt aatcctgatc 960
gaaaaccagg ttgttaacaa cctggatgaa gcgaaagtca tttacaaaaa atacatcgat 1020
cagggtctgg aaggcattat tctgaaaaac atcgacggtc tgtgggaaaa cgctcgttct 1080
aaaaatctgt acaagtttaa agaagttatt tgggtcgatc tgaaaatcgt gggcatctat 1140
ccgcaccgca aggatccaac caaagcgggt ggcttcattc tggagtccga atgtggcaaa 1200
atcaaagtta acgcaggttc tggtctgaaa gataaagctg gtgttaaatc tcacgaactg 1260
gatcgtaccc gtatcatgga aaaccagaac tattatatcg gtaaaatcct ggaatgcaaa 1320
tgcaacggct ggctgaaatc cgacggccgt aagccatacg tgaaactgtt cctgccgatc 1380
gctattcgtc tgcgtgagga caaaaccaaa gcaaacactt tcgaagatgt gttcggtgac 1440
ttccacgaag taactggtct gggtggtggt ggtagcgcga aaaaagaaat ggtggaattc 1500
gatgaagcta ttcatggtga agacctggcc aagttcatca aagaagcttc tgaccacaaa 1560
ctgaaaatta gcggctacaa cgaactgatc aaagatatcc gtatccgtgc gaaagatgag 1620
ctgggtgttg atggcaaaat gttcaatcgt ctgctggcgc tgtaccacaa agataaccgt 1680
gacgttttcg aagcggaaac cgaagaagtt gtggagctgt atgacaccgt gttctccaag 1740
<210> 36
<211> 1785
<212> DNA
<213> artifical sequence
<400> 36
atgatcctga aaattctgaa cgaaatcgcc agcatcggta gcaccaaaca gaaaaaagcg 60
atcctggaga aaaacaaaga caacgaactg ctgaaacgtg tttaccgtct gacttattcc 120
cgtggcctgc agtactacat taaaaaatgg ccgaaaccgg gcattgccac ccagtccttt 180
ggtatgacta ccctgactga catgctggat ttcattgaat tcaccctggc tacccgtaag 240
ctgaccggca acgcagctat ccgtgaactg accggctaca ttaccgacgg taaaaaagat 300
gacgttgaag ttctgcgccg tgtaatgatg cgtgatctgg aatgcggtgc atccgtatct 360
atcgcgaaca aagtttggaa aggtctgatc cctgagcaac cgcagatgct ggcatcttcc 420
tacgatgaga aaggtatcaa caaaaacatc aaatttccgg cgtttgcaca gctgagcgct 480
gatggcgcgc gttgtttcgc tgaagttcgt ggcgatgaac tggatgatgt acgtctgctg 540
agccgtgcgg gcaacgaata cctgggcctg gatctgctga aggaggaact gatcaaaatg 600
actgcagaag cccgccagat ccatccagaa ggcgtactga tcgacggcga gctggtgtat 660
catgaacagg ttgctaaaga accggaaggt ctggatgctc tgttcgaccg ttacccggag 720
aatagcaaag caaaagaatt cgccgaagtg gcggaatctc gtaccgcgtc caacggcatc 780
gctaacaaat ccctgaaagg taccatctcc gaaaaagaag ctcaatgcat gaaattccag 840
gtgtgggatt atgtcccgct ggtcgaaatt tactccctgc cggctttccg tctgaaatac 900
gacgtacgtt tcagcaagct ggaacagatg accagcggtt acgataaggt aatcctgatc 960
gaaaaccagg ttgttaacaa cctggatgaa gcgaaagtca tttacaaaaa atacatcgat 1020
cagggtctgg aaggcattat tctgaaaaac atcgacggtc tgtgggaaaa cgctcgttct 1080
aaaaatctgt acaagtttaa agaagttatt tgggtcgatc tgaaaatcgt gggcatctat 1140
ccgcaccgca aggatccaac caaagcgggt ggcttcattc tggagtccga atgtggcaaa 1200
atcaaagtta acgcaggttc tggtctgaaa gataaagctg gtgttaaatc tcacgaactg 1260
gatcgtaccc gtatcatgga aaaccagaac tattatatcg gtaaaatcct ggaatgcaaa 1320
tgcaacggct ggctgaaatc cgacggccgt aagccatacg tgaaactgtt cctgccgatc 1380
gctattcgtc tgcgtgagga caaaaccaaa gcaaacactt tcgaagatgt gttcggtgac 1440
ttccacgaag taactggtct gggtggtggt ggtagcgcca aaatgatgca gcgtgaaatc 1500
accaagacca ccgttaacgt agctaagatg gtgatggttg atggtgaagt tcaggttgag 1560
cagctgccgt ctgaaacctt cgtgggtaac ctgactatgg agcaggcgca gtggcgtatg 1620
aaacgcaaat acaaaggcga accagtgcag gttgtaagcg tggaaccgaa taccgaagtg 1680
tacgaactgc cggtggaaaa attcctggaa gttgccaccg tacgtgttga aaaagatgaa 1740
gaccaggaag aacagaccga agcaccggaa gagcaggttg ccgaa 1785
<210> 37
<211> 1884
<212> DNA
<213> artifical sequence
<400> 37
atgatcctga aaattctgaa cgaaatcgcc agcatcggta gcaccaaaca gaaaaaagcg 60
atcctggaga aaaacaaaga caacgaactg ctgaaacgtg tttaccgtct gacttattcc 120
cgtggcctgc agtactacat taaaaaatgg ccgaaaccgg gcattgccac ccagtccttt 180
ggtatgacta ccctgactga catgctggat ttcattgaat tcaccctggc tacccgtaag 240
ctgaccggca acgcagctat ccgtgaactg accggctaca ttaccgacgg taaaaaagat 300
gacgttgaag ttctgcgccg tgtaatgatg cgtgatctgg aatgcggtgc atccgtatct 360
atcgcgaaca aagtttggaa aggtctgatc cctgagcaac cgcagatgct ggcatcttcc 420
tacgatgaga aaggtatcaa caaaaacatc aaatttccgg cgtttgcaca gctgagcgct 480
gatggcgcgc gttgtttcgc tgaagttcgt ggcgatgaac tggatgatgt acgtctgctg 540
agccgtgcgg gcaacgaata cctgggcctg gatctgctga aggaggaact gatcaaaatg 600
actgcagaag cccgccagat ccatccagaa ggcgtactga tcgacggcga gctggtgtat 660
catgaacagg ttgctaaaga accggaaggt ctggatgctc tgttcgaccg ttacccggag 720
aatagcaaag caaaagaatt cgccgaagtg gcggaatctc gtaccgcgtc caacggcatc 780
gctaacaaat ccctgaaagg taccatctcc gaaaaagaag ctcaatgcat gaaattccag 840
gtgtgggatt atgtcccgct ggtcgaaatt tactccctgc cggctttccg tctgaaatac 900
gacgtacgtt tcagcaagct ggaacagatg accagcggtt acgataaggt aatcctgatc 960
gaaaaccagg ttgttaacaa cctggatgaa gcgaaagtca tttacaaaaa atacatcgat 1020
cagggtctgg aaggcattat tctgaaaaac atcgacggtc tgtgggaaaa cgctcgttct 1080
aaaaatctgt acaagtttaa agaagttatt tgggtcgatc tgaaaatcgt gggcatctat 1140
ccgcaccgca aggatccaac caaagcgggt ggcttcattc tggagtccga atgtggcaaa 1200
atcaaagtta acgcaggttc tggtctgaaa gataaagctg gtgttaaatc tcacgaactg 1260
gatcgtaccc gtatcatgga aaaccagaac tattatatcg gtaaaatcct ggaatgcaaa 1320
tgcaacggct ggctgaaatc cgacggccgt aagccatacg tgaaactgtt cctgccgatc 1380
gctattcgtc tgcgtgagga caaaaccaaa gcaaacactt tcgaagatgt gttcggtgac 1440
ttccacgaag taactggtct gggtggtggt ggtagctctg aagcactgaa gatcctgaac 1500
aacatccgta ccctgcgtgc acaggctcgt gaatgcaccc tggaaactct ggaagaaatg 1560
ctggaaaaac tggaagttgt agttaacgaa cgtcgtgagg aagaatccgc agcagcggcc 1620
gaagtggaag agcgtactcg taaactgcag cagtaccgcg aaatgctgat cgctgacggc 1680
atcgatccga atgagctgct gaacagcctg gcagctgtga aatctggcac caaagcgaaa 1740
cgcgcgcagc gtcctgcaaa atacagctac gtcgacgaga acggcgaaac taaaacctgg 1800
accggccagg gtcgtactcc agctgtcatc aaaaaagcga tggacgaaca gggtaaaagc 1860
ctggacgatt ttctgatcaa gcaa 1884
<210> 38
<211> 1492
<212> DNA
<213> artifical sequence
<400> 38
atgatcctga aaattctgaa cgaaatcgcc agcatcggta gcaccaaaca gaaaaaagcg 60
atcctggaga aaaacaaaga caacgaactg ctgaaacgtg tttaccgtct gacttattcc 120
cgtggcctgc agtactacat taaaaaatgg ccgaaaccgg gcattgccac ccagtccttt 180
ggtatgacta ccctgactga catgctggat ttcattgaat tcaccctggc tacccgtaag 240
ctgaccggca acgcagctat ccgtgaactg accggctaca ttaccgacgg taaaaaagat 300
gacgttgaag ttctgcgccg tgtaatgatg cgtgatctgg aatgcggtgc atccgtatct 360
atcgcgaaca aagtttggaa aggtctgatc cctgagcaac cgcagatgct ggcatcttcc 420
tacgatgaga aaggtatcaa caaaaacatc aaatttccgg cgtttgcaca gctgagcgct 480
gatggcgcgc gttgtttcgc tgaagttcgt ggcgatgaac tggatgatgt acgtctgctg 540
agccgtgcgg gcaacgaata cctgggcctg gatctgctga aggaggaact gatcaaaatg 600
actgcagaag cccgccagat ccatccagaa ggcgtactga tcgacggcga gctggtgtat 660
catgaacagg ttgctaaaga accggaaggt ctggatgctc tgttcgaccg ttacccggag 720
aatagcaaag caaaagaatt cgccgaagtg gcggaatctc gtaccgcgtc caacggcatc 780
gctaacaaat ccctgaaagg taccatctcc gaaaaagaag ctcaatgcat gaaattccag 840
gtgtgggatt atgtcccgct ggtcgaaatt tactccctgc cggctttccg tctgaaatac 900
gacgtacgtt tcagcaagct ggaacagatg accagcggtt acgataaggt aatcctgatc 960
gaaaaccagg ttgttaacaa cctggatgaa gcgaaagtca tttacaaaaa atacatcgat 1020
cagggtctgg aaggcattat tctgaaaaac atcgacggtc tgtgggaaaa cgctcgttct 1080
aaaaatctgt acaagtttaa agaagttatt tgggtcgatc tgaaaatcgt gggcatctat 1140
ccgcaccgca aggatccaac caaagcgggt ggcttcattc tggagtccga atgtggcaaa 1200
atcaaagtta acgcaggttc tggtctgaaa gataaagctg gtgttaaatc tcacgaactg 1260
gatcgtaccc gtatcatgga aaaccagaac tattatatcg gtaaaatcct ggaatgcaaa 1320
tgcaacggct ggctgaaatc cgacggccgt aagccatacg tgaaactgtt cctgccgatc 1380
gctattcgtc tgcgtgagga caaaaccaaa gcaaacactt tcgaagatgt gttcggtgac 1440
ttccacgaag taactggtct gggtggtggt ggtagcataa ttttataaaa at 1492
<210> 39
<211> 2109
<212> DNA
<213> artifical sequence
<400> 39
atggcgaaaa tgatgcagcg tgaaatcacg aagactaccg ttaacgttgc taaaatggtg 60
atggtggacg gtgaagttca ggtcgaacag ctgccgagcg aaaccttcgt aggcaacctg 120
accatggagc aagcacagtg gcgtatgaaa cgtaaatata agggtgaacc ggttcaggtc 180
gtatctgttg aaccgaacac ggaagtatac gagctgcctg tagaaaaatt cctggaagtt 240
gctaccgttc gtgtcgaaaa agacgaagat caggaggaac aaaccgaagc gccggaagaa 300
caggttgcgg aaggcggtgg cggttctatc ctgaaaattc tgaacgaaat cgccagcatc 360
ggtagcacca aacagaaaaa agcgatcctg gagaaaaaca aagacaacga actgctgaaa 420
cgtgtttacc gtctgactta ttcccgtggc ctgcagtact acattaaaaa atggccgaaa 480
ccgggcattg ccacccagtc ctttggtatg actaccctga ctgacatgct ggatttcatt 540
gaattcaccc tggctacccg taagctgacc ggcaacgcag ctatccgtga actgaccggc 600
tacattaccg acggtaaaaa agatgacgtt gaagttctgc gccgtgtaat gatgcgtgat 660
ctggaatgcg gtgcatccgt atctatcgcg aacaaagttt ggaaaggtct gatccctgag 720
caaccgcaga tgctggcatc ttcctacgat gagaaaggta tcaacaaaaa catcaaattt 780
ccggcgtttg cacagctgag cgctgatggc gcgcgttgtt tcgctgaagt tcgtggcgat 840
gaactggatg atgtacgtct gctgagccgt gcgggcaacg aatacctggg cctggatctg 900
ctgaaggagg aactgatcaa aatgactgca gaagcccgcc agatccatcc agaaggcgta 960
ctgatcgacg gcgagctggt gtatcatgaa caggttgcta aagaaccgga aggtctggat 1020
gctctgttcg accgttaccc ggagaatagc aaagcaaaag aattcgccga agtggcggaa 1080
tctcgtaccg cgtccaacgg catcgctaac aaatccctga aaggtaccat ctccgaaaaa 1140
gaagctcaat gcatgaaatt ccaggtgtgg gattatgtcc cgctggtcga aatttactcc 1200
ctgccggctt tccgtctgaa atacgacgta cgtttcagca agctggaaca gatgaccagc 1260
ggttacgata aggtaatcct gatcgaaaac caggttgtta acaacctgga tgaagcgaaa 1320
gtcatttaca aaaaatacat cgatcagggt ctggaaggca ttattctgaa aaacatcgac 1380
ggtctgtggg aaaacgctcg ttctaaaaat ctgtacaagt ttaaagaagt tatttgggtc 1440
gatctgaaaa tcgtgggcat ctatccgcac cgcaaggatc caaccaaagc gggtggcttc 1500
attctggagt ccgaatgtgg caaaatcaaa gttaacgcag gttctggtct gaaagataaa 1560
gctggtgtta aatctcacga actggatcgt acccgtatca tggaaaacca gaactattat 1620
atcggtaaaa tcctggaatg caaatgcaac ggctggctga aatccgacgg ccgtaagcca 1680
tacgtgaaac tgttcctgcc gatcgctatt cgtctgcgtg aggacaaaac caaagcaaac 1740
actttcgaag atgtgttcgg tgacttccac gaagtaactg gtctgggtgg tggtggtagc 1800
gccaaaatga tgcagcgtga aatcaccaag accaccgtta acgtagctaa gatggtgatg 1860
gttgatggtg aagttcaggt tgagcagctg ccgtctgaaa ccttcgtggg taacctgact 1920
atggagcagg cgcagtggcg tatgaaacgc aaatacaaag gcgaaccagt gcaggttgta 1980
agcgtggaac cgaataccga agtgtacgaa ctgccggtgg aaaaattcct ggaagttgcc 2040
accgtacgtg ttgaaaaaga tgaagaccag gaagaacaga ccgaagcacc ggaagagcag 2100
gttgccgaa 2109
<210> 40
<211> 2127
<212> DNA
<213> artifical sequence
<400> 40
atggcgaaaa tgatgcagcg tgaaatcacg aagactaccg ttaacgttgc taaaatggtg 60
atggtggacg gtgaagttca ggtcgaacag ctgccgagcg aaaccttcgt aggcaacctg 120
accatggagc aagcacagtg gcgtatgaaa cgtaaatata agggtgaacc ggttcaggtc 180
gtatctgttg aaccgaacac ggaagtatac gagctgcctg tagaaaaatt cctggaagtt 240
gctaccgttc gtgtcgaaaa agacgaagat caggaggaac aaaccgaagc gccggaagaa 300
caggttgcgg aaccggcgcc ggcgccggca ccggcaatcc tgaaaattct gaacgaaatc 360
gccagcatcg gtagcaccaa acagaaaaaa gcgatcctgg agaaaaacaa agacaacgaa 420
ctgctgaaac gtgtttaccg tctgacttat tcccgtggcc tgcagtacta cattaaaaaa 480
tggccgaaac cgggcattgc cacccagtcc tttggtatga ctaccctgac tgacatgctg 540
gatttcattg aattcaccct ggctacccgt aagctgaccg gcaacgcagc tatccgtgaa 600
ctgaccggct acattaccga cggtaaaaaa gatgacgttg aagttctgcg ccgtgtaatg 660
atgcgtgatc tggaatgcgg tgcatccgta tctatcgcga acaaagtttg gaaaggtctg 720
atccctgagc aaccgcagat gctggcatct tcctacgatg agaaaggtat caacaaaaac 780
atcaaatttc cggcgtttgc acagctgagc gctgatggcg cgcgttgttt cgctgaagtt 840
cgtggcgatg aactggatga tgtacgtctg ctgagccgtg cgggcaacga atacctgggc 900
ctggatctgc tgaaggagga actgatcaaa atgactgcag aagcccgcca gatccatcca 960
gaaggcgtac tgatcgacgg cgagctggtg tatcatgaac aggttgctaa agaaccggaa 1020
ggtctggatg ctctgttcga ccgttacccg gagaatagca aagcaaaaga attcgccgaa 1080
gtggcggaat ctcgtaccgc gtccaacggc atcgctaaca aatccctgaa aggtaccatc 1140
tccgaaaaag aagctcaatg catgaaattc caggtgtggg attatgtccc gctggtcgaa 1200
atttactccc tgccggcttt ccgtctgaaa tacgacgtac gtttcagcaa gctggaacag 1260
atgaccagcg gttacgataa ggtaatcctg atcgaaaacc aggttgttaa caacctggat 1320
gaagcgaaag tcatttacaa aaaatacatc gatcagggtc tggaaggcat tattctgaaa 1380
aacatcgacg gtctgtggga aaacgctcgt tctaaaaatc tgtacaagtt taaagaagtt 1440
atttgggtcg atctgaaaat cgtgggcatc tatccgcacc gcaaggatcc aaccaaagcg 1500
ggtggcttca ttctggagtc cgaatgtggc aaaatcaaag ttaacgcagg ttctggtctg 1560
aaagataaag ctggtgttaa atctcacgaa ctggatcgta cccgtatcat ggaaaaccag 1620
aactattata tcggtaaaat cctggaatgc aaatgcaacg gctggctgaa atccgacggc 1680
cgtaagccat acgtgaaact gttcctgccg atcgctattc gtctgcgtga ggacaaaacc 1740
aaagcaaaca ctttcgaaga tgtgttcggt gacttccacg aagtaactgg tctgccggcg 1800
ccggcgccgg caccggcagc caaaatgatg cagcgtgaaa tcaccaagac caccgttaac 1860
gtagctaaga tggtgatggt tgatggtgaa gttcaggttg agcagctgcc gtctgaaacc 1920
ttcgtgggta acctgactat ggagcaggcg cagtggcgta tgaaacgcaa atacaaaggc 1980
gaaccagtgc aggttgtaag cgtggaaccg aataccgaag tgtacgaact gccggtggaa 2040
aaattcctgg aagttgccac cgtacgtgtt gaaaaagatg aagaccagga agaacagacc 2100
gaagcaccgg aagagcaggt tgccgaa 2127
<210> 41
<211> 2127
<212> DNA
<213> artifical sequence
<400> 41
atggcgaaaa tgatgcagcg tgaaatcacg aagactaccg ttaacgttgc taaaatggtg 60
atggtggacg gtgaagttca ggtcgaacag ctgccgagcg aaaccttcgt aggcaacctg 120
accatggagc aagcacagtg gcgtatgaaa cgtaaatata agggtgaacc ggttcaggtc 180
gtatctgttg aaccgaacac ggaagtatac gagctgcctg tagaaaaatt cctggaagtt 240
gctaccgttc gtgtcgaaaa agacgaagat caggaggaac aaaccgaagc gccggaagaa 300
caggttgcgg aaggccaggg ccagggtcag ggccagatcc tgaaaattct gaacgaaatc 360
gccagcatcg gtagcaccaa acagaaaaaa gcgatcctgg agaaaaacaa agacaacgaa 420
ctgctgaaac gtgtttaccg tctgacttat tcccgtggcc tgcagtacta cattaaaaaa 480
tggccgaaac cgggcattgc cacccagtcc tttggtatga ctaccctgac tgacatgctg 540
gatttcattg aattcaccct ggctacccgt aagctgaccg gcaacgcagc tatccgtgaa 600
ctgaccggct acattaccga cggtaaaaaa gatgacgttg aagttctgcg ccgtgtaatg 660
atgcgtgatc tggaatgcgg tgcatccgta tctatcgcga acaaagtttg gaaaggtctg 720
atccctgagc aaccgcagat gctggcatct tcctacgatg agaaaggtat caacaaaaac 780
atcaaatttc cggcgtttgc acagctgagc gctgatggcg cgcgttgttt cgctgaagtt 840
cgtggcgatg aactggatga tgtacgtctg ctgagccgtg cgggcaacga atacctgggc 900
ctggatctgc tgaaggagga actgatcaaa atgactgcag aagcccgcca gatccatcca 960
gaaggcgtac tgatcgacgg cgagctggtg tatcatgaac aggttgctaa agaaccggaa 1020
ggtctggatg ctctgttcga ccgttacccg gagaatagca aagcaaaaga attcgccgaa 1080
gtggcggaat ctcgtaccgc gtccaacggc atcgctaaca aatccctgaa aggtaccatc 1140
tccgaaaaag aagctcaatg catgaaattc caggtgtggg attatgtccc gctggtcgaa 1200
atttactccc tgccggcttt ccgtctgaaa tacgacgtac gtttcagcaa gctggaacag 1260
atgaccagcg gttacgataa ggtaatcctg atcgaaaacc aggttgttaa caacctggat 1320
gaagcgaaag tcatttacaa aaaatacatc gatcagggtc tggaaggcat tattctgaaa 1380
aacatcgacg gtctgtggga aaacgctcgt tctaaaaatc tgtacaagtt taaagaagtt 1440
atttgggtcg atctgaaaat cgtgggcatc tatccgcacc gcaaggatcc aaccaaagcg 1500
ggtggcttca ttctggagtc cgaatgtggc aaaatcaaag ttaacgcagg ttctggtctg 1560
aaagataaag ctggtgttaa atctcacgaa ctggatcgta cccgtatcat ggaaaaccag 1620
aactattata tcggtaaaat cctggaatgc aaatgcaacg gctggctgaa atccgacggc 1680
cgtaagccat acgtgaaact gttcctgccg atcgctattc gtctgcgtga ggacaaaacc 1740
aaagcaaaca ctttcgaaga tgtgttcggt gacttccacg aagtaactgg tctgggccag 1800
ggccagggtc agggccaggc caaaatgatg cagcgtgaaa tcaccaagac caccgttaac 1860
gtagctaaga tggtgatggt tgatggtgaa gttcaggttg agcagctgcc gtctgaaacc 1920
ttcgtgggta acctgactat ggagcaggcg cagtggcgta tgaaacgcaa atacaaaggc 1980
gaaccagtgc aggttgtaag cgtggaaccg aataccgaag tgtacgaact gccggtggaa 2040
aaattcctgg aagttgccac cgtacgtgtt gaaaaagatg aagaccagga agaacagacc 2100
gaagcaccgg aagagcaggt tgccgaa 2127
<210> 42
<211> 2109
<212> DNA
<213> artifical sequence
<400> 42
atggcgaaaa tgatgcagcg tgaaatcacg aagactaccg ttaacgttgc taaaatggtg 60
atggtggacg gtgaagttca ggtcgaacag ctgccgagcg aaaccttcgt aggcaacctg 120
accatggagc aagcacagtg gcgtatgaaa cgtaaatata agggtgaacc ggttcaggtc 180
gtatctgttg aaccgaacac ggaagtatac gagctgcctg tagaaaaatt cctggaagtt 240
gctaccgttc gtgtcgaaaa agacgaagat caggaggaac aaaccgaagc gccggaagaa 300
caggttgcgg aagcggctgc agcggcaatc ctgaaaattc tgaacgaaat cgccagcatc 360
ggtagcacca aacagaaaaa agcgatcctg gagaaaaaca aagacaacga actgctgaaa 420
cgtgtttacc gtctgactta ttcccgtggc ctgcagtact acattaaaaa atggccgaaa 480
ccgggcattg ccacccagtc ctttggtatg actaccctga ctgacatgct ggatttcatt 540
gaattcaccc tggctacccg taagctgacc ggcaacgcag ctatccgtga actgaccggc 600
tacattaccg acggtaaaaa agatgacgtt gaagttctgc gccgtgtaat gatgcgtgat 660
ctggaatgcg gtgcatccgt atctatcgcg aacaaagttt ggaaaggtct gatccctgag 720
caaccgcaga tgctggcatc ttcctacgat gagaaaggta tcaacaaaaa catcaaattt 780
ccggcgtttg cacagctgag cgctgatggc gcgcgttgtt tcgctgaagt tcgtggcgat 840
gaactggatg atgtacgtct gctgagccgt gcgggcaacg aatacctggg cctggatctg 900
ctgaaggagg aactgatcaa aatgactgca gaagcccgcc agatccatcc agaaggcgta 960
ctgatcgacg gcgagctggt gtatcatgaa caggttgcta aagaaccgga aggtctggat 1020
gctctgttcg accgttaccc ggagaatagc aaagcaaaag aattcgccga agtggcggaa 1080
tctcgtaccg cgtccaacgg catcgctaac aaatccctga aaggtaccat ctccgaaaaa 1140
gaagctcaat gcatgaaatt ccaggtgtgg gattatgtcc cgctggtcga aatttactcc 1200
ctgccggctt tccgtctgaa atacgacgta cgtttcagca agctggaaca gatgaccagc 1260
ggttacgata aggtaatcct gatcgaaaac caggttgtta acaacctgga tgaagcgaaa 1320
gtcatttaca aaaaatacat cgatcagggt ctggaaggca ttattctgaa aaacatcgac 1380
ggtctgtggg aaaacgctcg ttctaaaaat ctgtacaagt ttaaagaagt tatttgggtc 1440
gatctgaaaa tcgtgggcat ctatccgcac cgcaaggatc caaccaaagc gggtggcttc 1500
attctggagt ccgaatgtgg caaaatcaaa gttaacgcag gttctggtct gaaagataaa 1560
gctggtgtta aatctcacga actggatcgt acccgtatca tggaaaacca gaactattat 1620
atcggtaaaa tcctggaatg caaatgcaac ggctggctga aatccgacgg ccgtaagcca 1680
tacgtgaaac tgttcctgcc gatcgctatt cgtctgcgtg aggacaaaac caaagcaaac 1740
actttcgaag atgtgttcgg tgacttccac gaagtaactg gtctggcggc tgcagcggca 1800
gccaaaatga tgcagcgtga aatcaccaag accaccgtta acgtagctaa gatggtgatg 1860
gttgatggtg aagttcaggt tgagcagctg ccgtctgaaa ccttcgtggg taacctgact 1920
atggagcagg cgcagtggcg tatgaaacgc aaatacaaag gcgaaccagt gcaggttgta 1980
agcgtggaac cgaataccga agtgtacgaa ctgccggtgg aaaaattcct ggaagttgcc 2040
accgtacgtg ttgaaaaaga tgaagaccag gaagaacaga ccgaagcacc ggaagagcag 2100
gttgccgaa 2109
Claims (14)
1.一种重组T4连接酶突变体，其特征在于其氨基酸序列如SEQ ID No . 8所示。
2.根据权利要求1所述的重组T4连接酶突变体，其特征在于在重组T4连接酶突变体的一端或者两端通过多肽桥重组双链DNA结合域。
3.根据权利要求2所述的重组T4连接酶突变体，其特征在于所述双链DNA结合域为p50、cTF、DSBA、H6、HNS或HTH。
4.根据权利要求3所述的重组T4连接酶突变体，其特征在于所述多肽桥为GGGGS多肽、PAPAPAPA多肽、GQGQGQGQ多肽或AAAAA多肽。
5.根据权利要求2所述的重组T4连接酶突变体，其特征在于其氨基酸序列如SEQ ID No.15-19、21-24中任一项所示。
6.根据权利要求2所述的重组T4连接酶突变体，其特征在于其氨基酸序列如SEQ ID No.20所示。
7.权利要求1所述的重组T4连接酶突变体的编码DNA，其特征在于其核酸序列如SEQ IDNo . 32所示。
8.权利要求5所述的重组T4连接酶突变体的编码DNA，其特征在于其核酸序列如SEQ IDNo .33-37、39-42中任一项所示。
9.一种NGS建库方法，其特征在于其步骤包括：
（1）采用平末端双链DNA片段化酶将DNA片段化；
（2）权利要求1-6中任一项所述的重组T4连接酶突变体介导的腺苷酰化接头连接；
（3）磁珠回收DNA；
（4）缺口修复；
（5）文库扩增及回收。
10.根据权利要求9所述的NGS建库方法，其特征在于：步骤（1）中的平末端双链DNA片段化酶为多种酶的混合物，包括热敏感性碱性磷酸酶和T4 DNA聚合酶，还包括脱氧核糖核酸I、创伤弧菌核酸酶、全能核酸酶、微球菌核酸酶中的一种或数种。
11.根据权利要求10所述的NGS建库方法，其特征在于：步骤（1）的DNA片段化反应温度为37℃，反应时间为3-15 min。
12.根据权利要求9所述的NGS建库方法，其特征在于：步骤（2）中采用的接头为接头A和接头B的退火产物，所述接头A的序列为：/App/AGATCGGAAGAGCACACGTCTGAACTCCAGT/ddC/，接头B的序列为：ACACTCTTTCCCTACACGACGCTCTTCCGATCT-p。
13.根据权利要求12所述的NGS建库方法，其特征在于：步骤（2）中接头反应的反应温度为20℃，反应时间为15 min。
14.根据权利要求9所述的NGS建库方法，其特征在于：步骤（4）中采用T4磷酸激酶和E.coli连接酶进行缺口修复。
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