CN116547379A - 具有增加的活性和热稳定性的逆转录酶突变体 - Google Patents
具有增加的活性和热稳定性的逆转录酶突变体 Download PDFInfo
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Abstract
本公开文本提供了莫洛尼氏鼠白血病病毒(MMLV)逆转录酶(RT酶)突变体。本公开文本提供了MMLV RT酶中用于诱变的合适的氨基酸位置以及用于使用MMLV RT酶突变体从RNA模板合成cDNA的方法和试剂盒。
Description
相关申请的交叉引用
本申请根据35 U.S.C.§119(e)要求2020年7月20日提交的美国临时申请号63/054,228的权益。将上文所列申请出于所有目的通过引用以其整体并入本文。
对电子序列表的引用
本申请含有已经以ASCII格式的文本文件电子提交并且通过引用以其整体特此并入的序列表。ASCII文本文件的名称为“20-1076-WO_Sequence-Listing_ST25_FINAL.txt”，是2021年7月19日创建的，大小为492千字节。
技术领域
本公开文本涉及莫洛尼氏鼠白血病病毒(MMLV)逆转录酶(RT酶)突变体。本公开文本还涉及MMLV RT酶中用于诱变的合适的氨基酸位置以及用于使用MMLV RT酶突变体以从RNA模板合成cDNA的方法。
背景技术
逆转录酶(RT酶)酶彻底改变了分子生物学。RT酶是逆转录聚合酶链式反应(RT-PCR)的关键组分，允许从RNA产生互补DNA(cDNA)。在逆转录反应中产生的cDNA可用于广泛的下游应用，包括定量PCR、基因表达分析、分离的RNA测序、基因克隆和cDNA文库创建。
RT酶(最先源自逆转录病毒)通过利用RNA依赖性聚合酶和RNA酶H(一种催化RNA/DNA双链体中RNA的裂解的非序列特异性核酸内切酶)来促进RNA逆转录为cDNA。这导致病毒复制并将病毒序列整合到宿主DNA中，从而允许病毒连同宿主DNA一起增殖。在实验室环境中，来自莫洛尼氏鼠白血病病毒(MMLV)、禽成髓细胞瘤病毒(AMV)和1型人类免疫缺陷病毒(HIV-1)的RT酶是用于cDNA合成的最常用的RT酶。
用于研究应用的RT酶通常是已经针对实验室程序进行优化的突变的多代MMLV和AMV RT酶。RT酶中的突变会改变酶的特性，包括热稳定性、RT酶活性、5'mRNA覆盖率和RNA酶H活性。
AMV RT酶是热稳定的，并且对热降解的敏感性较低(与MMLV RT酶相比)，并且对于具有强二级结构的RNA是优选的。此外，由于AMV RT酶的热稳定性，AMV RT酶通常适合与五千个碱基或更长的RNA分子一起使用。然而，解离强二级结构或长RNA链所需的高温会对RNA的完整性和转录保真度产生负面影响。AMV还具有降解RNA/DNA杂交体中的RNA的内在RNA酶活性，从而可以导致总cDNA减少和全长cDNA产量降低。
MMLV RT酶的特征在于RNA酶H活性低并且保真度更高(与AMV RT酶相比)。降低的RNA酶H活性允许MMLV RT酶可用于长RNA(>5kb)的逆转录。然而，MMLV RT酶的RNA酶H活性限制了体外合成长cDNA的效率。已经在MMLV RT酶中引入突变以降低RNA酶H活性。此外，由于MMLV RT酶活性的最佳温度为约37℃，因此所述酶缺乏有效逆转录具有强二级结构的RNA的能力。由于较低的酶活性，因此在升高的温度下使用MMLV RT酶会损害cDNA长度和产量。在反应混合物中用Mn2+替代Mg2+的MMLV RT酶突变体试图克服这些限制，但是其特点是效率低下和错误。
因此，尽管AMV和MMLV RT酶具有独特的特性，但是仍需要结合AMV和MMLV RT酶的有益属性的RT酶。与此一致，本申请公开了通过MMLV RT酶的合理诱变分离的MMLV RT酶突变体，与本领域中当前可用的RT酶(包括RNA酶H-构建体)相比，所述突变体展现出增加的RT酶活性和热稳定性。
发明内容
本公开文本提供了莫洛尼氏鼠白血病病毒(MMLV)逆转录酶(RT酶)突变体。本公开文本还提供了用于诱变的MMLV RT酶中的合适的氨基酸位置以及用于使用MMLV RT酶突变体从RNA模板合成cDNA的方法和试剂盒。
本公开文本的一方面提供了一种包含SEQ ID NO:637的氨基酸序列的分离的莫洛尼氏鼠白血病病毒(MMLV)逆转录酶(RT酶)突变体，其中所述MMLV RT酶突变体的氨基酸序列进一步包含至少一个氨基酸取代，所述至少一个氨基酸取代是：(a)位置61处的异亮氨酸至精氨酸、赖氨酸或甲硫氨酸取代(I61R、I61K或I61M)；(b)位置68处的谷氨酰胺至精氨酸、赖氨酸或异亮氨酸取代(Q68R、Q68K或Q68I)；(c)位置79处的谷氨酰胺至精氨酸、组氨酸或异亮氨酸取代(Q79R、Q79H或Q79I)；(d)位置99处的亮氨酸至精氨酸、赖氨酸或天冬酰胺取代(L99R、L99K或L99N)；(e)位置282处的谷氨酸至天冬氨酸、甲硫氨酸或色氨酸取代(E282D、E282M或E282W)；和/或(f)位置298处的精氨酸至丙氨酸取代(R298A)。
本公开文本的另一方面提供了一种包含SEQ ID NO:637的氨基酸序列的分离的莫洛尼氏鼠白血病病毒(MMLV)逆转录酶(RT酶)突变体，其中所述MMLV RT酶突变体的氨基酸序列进一步包含至少两个氨基酸取代，所述至少两个氨基酸取代是：(a)位置61处的异亮氨酸至精氨酸取代(I61R)；(b)位置68处的谷氨酰胺至精氨酸取代(Q68R)；(c)位置79处的谷氨酰胺至精氨酸取代(Q79R)；(d)位置99处的亮氨酸至精氨酸取代(L99R)；(e)位置282处的谷氨酸至天冬氨酸取代(E282D)；和/或(f)位置298处的精氨酸至丙氨酸取代(R298A)：(a)位置61处的异亮氨酸至精氨酸取代和位置282处的谷氨酸至天冬氨酸取代(I61R/E282D)；(b)取代位置99处的亮氨酸至精氨酸和位置282处的谷氨酸至天冬氨酸取代(L99R/E282D)；(c)位置68处的谷氨酰胺至精氨酸取代和位置282处的谷氨酸至天冬氨酸取代(Q68R/E282D)；(d)位置79处的谷氨酰胺至精氨酸取代和位置282处的谷氨酸至天冬氨酸取代(Q79R/E282D)；(e)位置282处的谷氨酸至天冬氨酸取代和位置298处的精氨酸至丙氨酸取代(E282D/R298A)；(f)位置61处的异亮氨酸至精氨酸取代和位置99处的亮氨酸至精氨酸取代(I61R/L99R)；(g)位置61处的异亮氨酸至精氨酸取代和位置68处的谷氨酰胺至精氨酸取代(I61R/Q68R)；(h)位置61处的异亮氨酸至精氨酸取代和位置79处的谷氨酰胺至精氨酸取代(I61R/Q79R)；(i)位置61处的异亮氨酸至精氨酸取代和位置298处的精氨酸至丙氨酸取代(I61R/R298A)；(j)位置68处的谷氨酰胺至精氨酸取代和位置99处的亮氨酸至精氨酸取代(Q68R/L99R)；(k)位置79处的谷氨酰胺至精氨酸取代和位置99处的亮氨酸至精氨酸取代(Q79R/L99R)；(l)取代位置99处的亮氨酸至精氨酸和位置298处的精氨酸至丙氨酸取代(L99R/R298A)；(m)位置68处的谷氨酰胺至精氨酸取代和位置79处的谷氨酰胺至精氨酸取代(Q68R/Q79R)；(n)位置68处的谷氨酰胺至精氨酸取代和位置298处的精氨酸至丙氨酸取代(Q68R/R298A)；或(o)位置79处的谷氨酰胺至精氨酸取代和位置298处的精氨酸至丙氨酸取代(Q79R/R298A)。
本公开文本的另一方面提供了一种包含SEQ ID NO:637的氨基酸序列的分离的莫洛尼氏鼠白血病病毒(MMLV)逆转录酶(RT酶)突变体，其中所述MMLV RT酶突变体的氨基酸序列进一步包含至少三个氨基酸取代，所述至少三个氨基酸取代是：(a)位置68处的谷氨酰胺至精氨酸取代(Q68R)；(b)位置79处的谷氨酰胺至精氨酸取代(Q79R)；(c)位置99处的亮氨酸至精氨酸取代(L99R)；和/或(d)位置282处的谷氨酸至天冬氨酸取代(E282D)：(a)位置68处的谷氨酰胺至精氨酸取代、位置99处的亮氨酸至精氨酸取代和位置282处的谷氨酸至天冬氨酸取代(Q68R/L99R/E282D)；(b)位置79处的谷氨酰胺至精氨酸取代、位置99处的亮氨酸至精氨酸取代和位置282处的谷氨酸至天冬氨酸取代(Q79R/L99R/E282D)；(c)位置68处的谷氨酰胺至精氨酸取代、位置68处的谷氨酰胺至精氨酸取代和位置282处的谷氨酸至天冬氨酸取代(Q68R/Q79R/E282D)；或(d)位置68处的谷氨酰胺至精氨酸取代、位置68处的谷氨酰胺至精氨酸取代和位置99处的亮氨酸至精氨酸取代(Q68R/Q79R/L99R)。
本公开文本的另一方面提供了一种包含SEQ ID NO:637的氨基酸序列的分离的莫洛尼氏鼠白血病病毒(MMLV)逆转录酶(RT酶)突变体，其中所述MMLV RT酶突变体的氨基酸序列进一步包含至少四个氨基酸取代，所述至少四个氨基酸取代是：(a)位置68处的谷氨酰胺至精氨酸、赖氨酸或异亮氨酸取代(Q68R、Q68K或Q68I)；(b)位置79处的谷氨酰胺至精氨酸、组氨酸或异亮氨酸取代(Q79R、Q79H或Q79I)；(c)位置99处的亮氨酸至精氨酸、赖氨酸或天冬酰胺取代(L99R、L99K或L99N)；(d)位置282处的谷氨酸至天冬氨酸、甲硫氨酸或色氨酸取代(E282D、E282M或E282W)；(a)位置68处的谷氨酰胺至精氨酸取代、位置79处的谷氨酰胺至精氨酸取代、位置99处的亮氨酸至精氨酸取代和位置282处的谷氨酸至天冬氨酸取代(Q68R/Q79R/L99R/E282D)；(b)位置68处的谷氨酰胺至精氨酸取代、位置79处的谷氨酰胺至精氨酸取代、位置99处的亮氨酸至赖氨酸取代和位置282处的谷氨酸至天冬氨酸取代(Q68R/Q79R/L99K/E282D)；(c)位置68处的谷氨酰胺至精氨酸取代、位置79处的谷氨酰胺至精氨酸取代、位置99处的亮氨酸至天冬酰胺取代和位置282处的谷氨酸至天冬氨酸取代(Q68R/Q79R/L99N/E282D)；(d)位置68处的谷氨酰胺至异亮氨酸取代、位置79处的谷氨酰胺至精氨酸取代、位置99处的亮氨酸至精氨酸取代和位置282处的谷氨酸至天冬氨酸取代(Q68I/Q79R/L99R/E282D)；(e)位置68处的谷氨酰胺至赖氨酸取代、位置79处的谷氨酰胺至精氨酸取代、位置99处的亮氨酸至精氨酸取代和位置282处的谷氨酸至天冬氨酸取代(Q68K/Q79R/L99R/E282D)；(f)位置68处的谷氨酰胺至精氨酸取代、位置79处的谷氨酰胺至组氨酸取代、位置99处的亮氨酸至精氨酸取代和位置282处的谷氨酸至天冬氨酸取代(Q68R/Q79H/L99R/E282D)；(g)位置68处的谷氨酰胺至精氨酸取代、位置79处的谷氨酰胺至异亮氨酸取代、位置99处的亮氨酸至精氨酸取代和位置282处的谷氨酸至天冬氨酸取代(Q68R/Q79I/L99R/E282D)；(h)位置68处的谷氨酰胺至精氨酸取代、位置79处的谷氨酰胺至精氨酸取代、位置99处的亮氨酸至精氨酸取代和位置282处的谷氨酸至甲硫氨酸取代(Q68R/Q79R/L99R/E282M)；(i)位置68处的谷氨酰胺至精氨酸取代、位置79处的谷氨酰胺至精氨酸取代、位置99处的亮氨酸至精氨酸取代和位置282处的谷氨酸至色氨酸取代(Q68R/Q79R/L99R/E282W)；(j)位置68处的谷氨酰胺至异亮氨酸取代、位置79处的谷氨酰胺至组氨酸取代、位置99处的亮氨酸至赖氨酸取代和位置282处的谷氨酸至甲硫氨酸取代(Q68I/Q79H/L99K/E282M)。
本公开文本的另一方面提供了一种包含SEQ ID NO:637的氨基酸序列的分离的莫洛尼氏鼠白血病病毒(MMLV)逆转录酶(RT酶)突变体，其中所述MMLV RT酶突变体的氨基酸序列进一步包含至少五个氨基酸取代，所述至少五个氨基酸取代是：(a)位置61处的异亮氨酸至赖氨酸或甲硫氨酸取代(I61K或I61M)；(b)位置68处的谷氨酰胺至精氨酸或异亮氨酸取代(Q68R或Q68I)；(c)位置79处的谷氨酰胺至精氨酸或组氨酸取代(Q79R或Q79H)；(d)位置99处的亮氨酸至精氨酸或赖氨酸取代(L99R或L99K)；(e)位置282处谷氨酸至天冬氨酸或甲硫氨酸取代(E282D或E282M)：(a)位置61处的异亮氨酸至赖氨酸取代、位置68处的谷氨酰胺至精氨酸取代、位置79处的谷氨酰胺至精氨酸取代、位置99处的亮氨酸至精氨酸取代和位置282处的谷氨酸至天冬氨酸取代(I61K/Q68R/Q79R/L99R/E282D)；(b)位置61处的异亮氨酸至甲硫氨酸取代、位置68处的谷氨酰胺至精氨酸取代、位置79处的谷氨酰胺至精氨酸取代、位置99处的亮氨酸至精氨酸取代和位置282处的谷氨酸至天冬氨酸取代(I61M/Q68R/Q79R/L99R/E282D)；或(c)位置61处的异亮氨酸至甲硫氨酸取代、位置68处的谷氨酰胺至异亮氨酸取代、位置79处的谷氨酰胺至组氨酸取代、位置99处的亮氨酸至赖氨酸取代和位置282处的谷氨酸至甲硫氨酸取代(I61M/Q68IR/Q79H/L99K/E282M)。
本公开文本的另一方面提供了一种包含SEQ ID NO:637的氨基酸序列的分离的莫洛尼氏鼠白血病病毒(MMLV)逆转录酶(RT酶)突变体，其中所述MMLV RT酶突变体的氨基酸序列进一步包含至少五个或更多个氨基酸取代，所述至少五个或更多个氨基酸取代是：(a)位置68处的谷氨酰胺至精氨酸、赖氨酸或异亮氨酸取代(Q68R、Q68K或Q68I)；(b)位置79处的谷氨酰胺至精氨酸、组氨酸或异亮氨酸取代(Q79R、Q79H或Q79I)；(c)位置99处的亮氨酸至精氨酸、赖氨酸或天冬酰胺取代(L99R、L99K或L99N)；(d)位置282处的谷氨酸至天冬氨酸、甲硫氨酸或色氨酸取代(E282D、E282M或E282W)；(e)位置299处的谷氨酰胺至谷氨酸取代；(f)位置332处的苏氨酸至谷氨酸取代；(g)位置433处的缬氨酸至精氨酸取代；(h)位置593处的异亮氨酸至谷氨酸取代；(a)位置68处的谷氨酰胺至精氨酸取代、位置79处的谷氨酰胺至精氨酸取代、位置99处的亮氨酸至精氨酸取代和位置282处的谷氨酸至天冬氨酸取代、位置299处的谷氨酰胺至谷氨酸取代、位置433处的缬氨酸至精氨酸取代和位置593处的异亮氨酸至谷氨酸取代(Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E)：(b)位置68处的谷氨酰胺至精氨酸取代、位置79处的谷氨酰胺至精氨酸取代、位置82处的亮氨酸至精氨酸取代、位置99处的亮氨酸至精氨酸取代和位置282处的谷氨酸至天冬氨酸取代、位置299处的谷氨酰胺至谷氨酸取代、位置433处的缬氨酸至精氨酸取代和位置593处的异亮氨酸至谷氨酸取代(Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E)；(c)位置68处的谷氨酰胺至精氨酸取代、位置79处的谷氨酰胺至精氨酸取代、位置82处的亮氨酸至精氨酸取代、位置99处的亮氨酸至精氨酸取代和位置282处的谷氨酸至天冬氨酸取代、位置299处的谷氨酰胺至谷氨酸取代、位置332处的苏氨酸至谷氨酸取代,和位置593处的异亮氨酸至谷氨酸取代(Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E)；(d)位置68处的谷氨酰胺至精氨酸取代、位置79处的谷氨酰胺至精氨酸取代、位置82处的亮氨酸至精氨酸取代、位置99处的亮氨酸至精氨酸取代和位置282处的谷氨酸至天冬氨酸取代、位置299处的谷氨酰胺至谷氨酸取代、位置332处的苏氨酸至谷氨酸取代、位置433处的缬氨酸至精氨酸取代和位置593处的异亮氨酸至谷氨酸取代(Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/I593E)。
本公开文本的另一方面提供了一种分离的核酸分子，所述分离的核酸分子包含编码本公开文本的MMLV RT酶突变体的核苷酸序列。
本公开文本的另一方面提供了一种组合物或试剂盒，所述组合物或试剂盒包含本公开文本的MMLV RT酶突变体。
本公开文本的其他方面提供了使用本公开文本的MMLV RT酶突变体合成互补脱氧核糖核酸(cDNA)的方法或进行逆转录聚合酶链式反应(RT-PCR)的方法。
本公开文本的具体实施方案将从以下更详细的描述和权利要求中变得清楚。
附图说明
图1A-图1C是示出了通过合理设计进行的逆转录酶诱变选择的示意图。在底物结合位点(图1A和图1B)或底物结合位点附近(图1C)选择用于诱变的氨基酸位置。
图2示出了对于MMLV RT在TB培养基中的BL21(DE3)细胞中的测试诱导结果的蛋白质印迹分析。泳道1-Precision Plus Protein未染色标准品(Bio Rad，目录号161-0363)，泳道2-时间＝0小时，泳道3-时间＝37℃下诱导后3小时，泳道4-时间＝0小时，泳道5-时间＝18℃下诱导后21小时。
具体实施方式
本公开文本涉及莫洛尼氏鼠白血病病毒(MMLV)逆转录酶(RT酶)突变体。本公开文本涉及用于诱变的MMLV RT酶中的合适的氨基酸位置以及用于使用MMLV RT酶突变体从RNA模板合成cDNA的方法和试剂盒。
如与野生型MMLV RT酶和某些MMLV RT酶突变体(包括本领域当前可用的RNA酶H-RT酶)相比，发现本公开文本的MMLV RT酶突变体(至少部分地通过MMLV RT酶的碱基构建体的合理诱变而被鉴定和分离)具有增加的RT酶活性和热稳定性。
现在将详细地引用要求保护的发明的示例性实施方案。虽然将结合示例性实施方案描述要求保护的发明，但是应当理解，它们并不旨在将要求保护的发明限制于那些实施方案。相反，它旨在覆盖可以包括在由所附权利要求书限定的要求保护的发明的精神和范围内的替代物、修改和等同物。
在不脱离要求保护的发明的精神或范围的情况下，本领域普通技术人员可以对本文所述的实施方案进行修改和变化。另外，尽管与本文所述的那些指代什么要说明类似或等同的方法和材料也可以用于本发明的实践或测试，但是本文描述了某些方法和材料，但是与本文所述的那些类似或等同的其他方法和材料也可用于要求保护的发明的实践。
此外，本文提供、公开或描述的任何组合物或方法可以与本文提供、公开或描述的任何其他组合物和方法中的一种或多种组合。
1.定义
除非另外定义，否则本文所用的所有技术和科学术语都具有要求保护的发明所属领域中的技术人员通常所理解的含义。本文使用的术语仅用于描述特定实施方案，并不旨在限制要求保护的发明。本文使用的所有技术和科学术语具有相同的含义。
以下参考文献向本领域技术人员提供了本文所用的许多术语的一般理解(除非本文其他地方定义)：Singleton等人,Dictionary of Microbiology and MolecularBiology,第3版(Wiley,2006)；Walker,The Cambridge Dictionary of Science andTechnology(Cambridge University Press,1990)；Rieger等人,Glossary of Genetics:Classical and Molecular,第5版(Springer Verlag,1991)；和Hale等人,Harper CollinsDictionary of Biology(HarperCollins Publishers,1991)。总体上，本文所述的程序或方法等是本领域中常用的方法。此类标准技术可在例如像以下的参考手册中找到：Green等人,Molecular Cloning:A Laboratory Manual,第4版(Cold Spring Harbor LaboratoryPress,2012)和Ausubel,Current Protocols in Molecular Biology(John Wiley&SonsInc.,2004)。
除非另有规定，否则以下术语可能具有下文赋予它们的含义。然而，应当理解，本领域普通技术人员已知或理解的其他含义也是可能的，并且在要求保护的发明的范围内。本文提及或讨论的所有出版物、专利申请、专利和其他参考文献都通过引用以其整体明确地并入。在冲突的情况下，以包括定义的本说明书为准。另外，材料、方法以及实施例仅仅是说明性的并不意在是限制性的。
除非上下文明确另外指示其他含义，否则如本文所用的单数形式“一个/一种(a)”、“一个/一种(an)”以及“所述(the)”包括复数指示物。
除非上下文明确另外指示其他含义，否则如本文所用的术语“或”意指术语“和/或”，并且可与其互换使用。
如本文所用，术语“包括”意指短语“包括但不限于”，并且可与其互换使用。
如本文所用，术语“如”意指短语“例如像”或“诸如但不限于”，并且可与其互换使用。
除非根据上下文明确说明或显而易见，否则如本文所用的术语“约”被理解为在本领域的正常公差范围内，例如，在平均值的两个标准差内。约可以理解为在所述值的10％、9％、8％、7％、6％、5％、4％、3％、2％、1％、0.5％、0.1％、0.05％或0.01％内。除非根据上下文另外清楚，否则本文提供的所有数值均可以被术语约修饰。
本文提供的范围应理解为是所述范围内所有值的简写。例如，范围1至50应理解为包括来自由1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49或50组成的组的任何数字、数字组合或子范围。
如本文所用，术语“核酸分子”和“多核苷酸”是指由核苷酸组成的聚合物或大生物分子。术语“核酸”包括脱氧核糖核酸(DNA)、核糖核酸(RNA)及其类似物。核酸分子的非限制性例子包括DNA(例如，基因组DNA、cDNA)、RNA分子(例如mRNA、rRNA、cRNA、tRNA)及其嵌合体。核酸分子可以通过克隆技术获得或使用本领域技术人员已知的技术合成。DNA可以是双链或单链的(编码链或非编码链，即反义的)。核酸骨架可以包含本领域已知的多种连接，包括糖-磷酸二酯连接、肽-核酸键(称为“肽核酸”(PNA))、硫代磷酸酯连接、甲基膦酸酯连接或其组合中的一种或多种。核酸的糖部分可以是核糖或脱氧核糖，或具有已知取代基(例如2'甲氧基取代基(含有2'-O-甲基呋喃核糖基部分)和/或2'卤化物取代基)的类似化合物。含氮碱基可以是常规碱基(腺嘌呤(A)、鸟嘌呤(G)、胸腺嘧啶(T)、胞嘧啶(C)和尿嘧啶(U))、其已知类似物(例如肌苷)、嘌呤或嘧啶碱基的已知衍生物、或其中骨架不包括一个或多个残基的含氮碱基的“无碱基”残基。核酸可以仅包含如在RNA和DNA中发现的常规糖、碱基和连接，或者可以包含常规组分和取代基两者(例如经由甲氧基骨架连接的常规碱基，或包含常规碱基和一个或多个碱基类似物的核酸)。如本领域技术人员通常理解的并且如本文所用，“分离的核酸分子”是指核苷酸的聚合物，并且包括但不限于DNA和RNA。
如本文所用，术语“探针”是指这样的核酸寡核苷酸，其在促进杂交的条件下与核酸或其互补体中的靶序列特异性杂交从而允许检测靶序列或其扩增的核酸。检测可以是直接的(即，通过探针与靶序列或扩增序列直接杂交进行)或间接的(即，通过探针与将探针与靶序列或扩增序列连接的中间分子结构杂交进行)。探针的“靶标”通常是指扩增的核酸序列内的序列(即，扩增的序列的子集)，其通过标准氢键合或“碱基配对”与探针序列的至少一部分特异性杂交。“充分互补”的序列允许探针序列与靶序列的稳定杂交，即使这两个序列不是完全互补的。探针可以被标记或不被标记。探针可以通过特定DNA序列的分子克隆产生，或者也可以合成。本领域技术人员可以容易地设计和使用在本文公开的方法中使用的探针。
如本文所用，术语“引物”是指这样的核酸寡核苷酸，其与核酸或其补体中的靶序列特异性杂交，并且其能够在模板依赖性过程中引发新生核酸的合成。引物可以以双链形式或单链形式提供。本领域技术人员可以容易地设计和使用在本文公开的方法中使用的引物。
用于本文公开的方法的探针或引物可以具有任何合适的长度，这取决于特定的测定形式和特定的需要以及所采用的靶向序列。例如，用于本文公开的方法的探针或引物的长度为至少10个核苷酸，或长度为至少15、20、25、30或多于30个核苷酸，并且它们可以调整为特别适合于所选择的核酸扩增系统和/或所使用的杂交系统。较长的探针和引物也在本公开文本的范围内。
“转录的多核苷酸”或“核苷酸转录物”是这样的多核苷酸(例如，mRNA、hnRNA、cDNA或此类RNA或cDNA的类似物)，其与成熟mRNA的全部或一部分互补或者具有高同一性百分比(例如，至少80％同一性)，所述成熟mRNA是通过本公开文本的标记物的转录和RNA转录物的正常转录后加工(例如，剪接)(如果有的话)和RNA转录物的逆转录制备的。
如本文所用，术语“逆转录酶”、“RT酶”或“RT”是指用于在称为“逆转录”的过程中从RNA模板产生互补(cDNA)的酶。如本文所用，术语逆转录酶也指展现出逆转录活性的任何酶。逆转录酶可以来源于多种来源，包括但不限于病毒(包括逆转录病毒)和展现出转录酶活性的DNA聚合酶。此类逆转录病毒包括但不限于莫洛尼氏鼠白血病病毒(MMLV)、禽成髓细胞瘤病毒(AMV)和人类免疫缺陷病毒(HIV)。
可以通过将RT酶在含有RNA模板和脱氧核苷酸的缓冲液中孵育来测量逆转录酶活性。本领域技术人员将认识到，可以使用多种条件来进行逆转录反应，并且存在多种方法来测量在逆转录期间产生的cDNA的量。
本公开文本的逆转录酶包括具有本文所述特性中的一种或组合的逆转录酶。此类特性包括但不限于例如当与碱基构建体相比时，增加的活性、增强的DNA合成、增强的稳定性或增强的热稳定性、降低的或消除的RNA酶H活性、降低的末端脱氧核苷酸转移酶活性、增加的准确性或增加的保真度、增加的特异性或改变的半衰期。如本文所用，术语“碱基构建体”是指从其制备本公开文本的RT酶突变体的初始RT酶(例如野生型RT酶或经修饰的野生型RT酶)。
如本文所用，术语“准确性”和“保真度”可互换使用，并且是指RT酶准确复制所需模板的能力；即RT酶在逆转录反应中准确执行cDNA合成的能力。可以通过确定不正确的核苷酸掺入合成的cDNA分子的频率(这可以称为酶的错误率)来评估逆转录酶的“保真度”或“准确性”。如本文所用，术语“增加的保真度”是指本公开文本的RT酶突变体，其展现出比碱基构建体的错误率低的错误率。例如，如本文公开的RT酶突变体可以展现出比RT酶碱基构建体的错误率低10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、100％或200％的错误率，或者其错误率是RT酶碱基构建体的错误率的至少1/2倍、1/3倍、1/4倍、1/5倍或1/10倍或少于1/10倍。
如本文所用，术语“特异性”是指在cDNA合成期间RT酶错误引发的减少。RT酶突变体的特异性可以通过在特定温度(包括较高温度)下进行逆转录反应，并且将该反应中的错误引发量与在相同条件下用野生型RT酶(或RT酶碱基构建体)进行的反应中的错误引发量进行比较来评估。
如本文关于本公开文本的RT酶分子所用的，术语“稳定的”和“热稳定的”可互换使用，并且是指耐热灭活并且在超过37℃的温度(例如，38℃、39℃、40℃、41℃、42℃、43℃、44℃、45℃、46℃、47℃、48℃、49℃、50℃、51℃、52℃、53℃、54℃、55℃、56℃、57℃、58℃、59℃、60℃、61℃、62℃、63℃、64℃、65℃、70℃或更高的温度)下保持有活性的酶。例如，在一个实施方案中，本公开文本提供了一种RT酶突变体，其在升高的温度下具有比碱基构建体RT酶的半衰期更长的半衰期的活性。因此，具有“增强的热稳定性”的RT酶突变体可以是指与碱基构建体RT酶相比，在约50℃至约90℃的温度下展现出热稳定性增加的本公开文本的RT酶突变体。在一些实施方案中，RT酶突变体的热稳定性是与碱基构建体RT酶的热稳定性相比的至少1.5倍或更大。使用碱基构建体和RT酶突变体反应的相同反应条件，比较了由碱基构建体和RT酶突变体产生的cDNA的比较。反应条件可以包括但不限于盐浓度、缓冲液浓度、pH、二价金属离子浓度、温度、三磷酸核苷浓度、模板浓度、RT酶浓度、引物浓度、时间以及在一步PCR中定量的PCR引物和探针浓度。
如本文所用，术语“增强的DNA合成”是指产生比碱基RT酶构建体更多的DNA(例如cDNA)的RT酶酶。在一些实施方案中，与碱基构建体RT酶相比，可以在标准反应条件下通过定量PCR测量DNA合成。与热稳定性的评估一致，在相似或相同的反应条件下进行定量比较，并且将使用碱基构建体RT酶合成的cDNA的量与使用RT酶突变体产生的cDNA的量进行比较(参见表4、5、6和7)。在一些实施方案中，具有增强的DNA合成的本公开文本的RT酶突变体可以产生比碱基构建体RT酶多约5％至约200％的cDNA。在一些实施方案中，具有增强的DNA合成的本公开文本的RT酶突变体的DNA合成比RT酶碱基构建体DNA合成多至少10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％、100％或200％，或者是RT酶碱基构建体DNA合成的至少2倍、3倍、4倍、5倍或10倍或多于10倍。
如本文所述，在一步程序或两步程序中评价逆转录酶活性。一步程序将逆转录和定量PCR组合在单个反应中。所述方法通过包括如实施例3中所述的基因表达主要混合物(Gene Expression Master Mix)、RT酶、RNA、荧光探针以及引物和探针来进行。两步程序包括逆转录，然后进行定量PCR。在逆转录步骤中，将RT酶添加到含有RNA、基因特异性引物、第一链合成缓冲液和RNase的混合物中。然后在第二步骤中定量所得cDNA，其中将所述cDNA与基因表达主要混合物、引物和探针以及荧光标记物组合。对在一步程序和两步程序中产生的cDNA进行定量，并且如本文表4、5、6和7所示报告平均值和标准差。
如本文所用，“RNA酶H活性”是指经由水解机制切割DNA-RNA双链体中的RNA，以产生5'磷酸末端寡核苷酸。RNA酶H活性不包括单链核酸、双链DNA或双链RNA的降解。如本文所用，短语“基本上缺乏RNA酶H活性”意指具有小于10％、5％、1％、0.5％或0.1％的野生型酶的活性。如本文所用，短语“缺乏RNA酶H活性”意指具有不可检测的RNA酶H活性或具有小于约1％、0.5％或0.1％的野生型酶的RNA酶H活性。
如本文所用，术语“突变”是指引入编码蛋白质的核酸序列中的改变，其改变所述蛋白质的氨基酸序列，包括但不限于取代、插入、缺失、点突变、转位、倒位、读框移位、无义突变、截短或者其他形式的变型。突变可能不会产生可辨别的变化或导致突变的蛋白质的新特性、功能或性状。本公开文本的RT酶突变体可以在编码所述RT酶突变体的核酸序列中具有一个或多个突变，导致所述RT酶突变体的氨基酸序列中的一个或多个突变。突变可以导致一个或多个氨基酸被取代为可替代的氨基酸残基，包括Ala、Arg、Asn、Asp、Cys、Gln、Glu、Gly、His、Ile、Leu、Lys、Met、Phe、Pro、Ser、Thr、Trp、Tyr和/或Val。所得氨基酸突变可以赋予RT酶突变体改变的功能和生物学特性，包括但不限于增加的活性、增强的DNA合成、增强的稳定性或增强的热稳定性、降低或消除的RNA酶H活性、降低的末端脱氧核苷酸转移酶活性、增加的准确性或增加的保真度、增加的特异性或改变的半衰期。
如本文所用，术语“检测”、“测定”等是指为鉴定作为RT酶活性的标记物的cDNA合成量而进行的测定。与使用RT酶碱基构建体检测到的标记物表达量或活性相比，在样品中检测到的标记物表达量或活性可以相同、减少或增加。本领域技术人员将理解，可以使用多种技术来定量cDNA的量。
如本文关于RT酶活性所用的术语“增加”是指与RT酶碱基构建体相比，RT酶突变体的RT酶活性水平。如果通过合成的cDNA的量测量或通过本领域已知的其他方法测量的RT酶突变体的RT酶活性的水平高于RT酶碱基构建体的活性，则所述RT酶突变体具有“增加的”RT酶活性。例如，如果RT酶突变体的RT酶活性比RT酶碱基构建体活性高至少10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或100％，或者是RT酶碱基构建体活性的至少2倍、3倍、4倍、5倍、或10倍或大于10倍，则所述RT酶突变体的RT酶活性增加。
如本文关于RT酶活性所用的术语“降低”是指与RT酶碱基构建体相比，RT酶突变体的RT酶活性水平。如果通过合成的cDNA的量测量或通过本领域已知的其他方法测量的RT酶突变体的RT酶活性的水平低于RT酶碱基构建体的活性，则所述RT酶突变体具有“降低的”RT酶活性。例如，如果RT酶突变体的RT酶活性比RT酶碱基构建体活性低至少95％、90％、85％、80％、75％、70％、65％、60％、55％、50％、45％、40％、35％、30％、25％、20％、15％、10％或5％，或者是RT酶碱基构建体活性的至少1/2倍、1/3倍、1/4倍、1/5倍、1/10倍或少于1/10倍，则所述RT酶突变体的RT酶活性降低。
如本文所用，术语“扩增”是指用于获得靶核酸序列或其补体或其片段的多个拷贝的任何已知的体外程序。体外扩增是指可以含有少于完整靶区序列或其补体的扩增的核酸的产生。已知的体外扩增方法包括例如转录介导的扩增、复制酶介导的扩增、聚合酶链式反应(PCR)扩增、连接酶链式反应(LCR)扩增和链置换扩增(SDA，包括多链置换扩增方法(MSDA))。复制酶介导的扩增使用自我复制RNA分子和复制酶如Q-β-复制酶。PCR扩增使用DNA聚合酶、引物和热循环来合成DNA或cDNA的两条互补链的多个拷贝。PCR涉及双链DNA分子的变性，然后是针对目的序列的DNA引物的退火，以及新形成的DNA链的扩增/延伸。LCR扩增使用至少四个单独的寡核苷酸，通过使用杂交、连接和变性的多个循环来扩增靶标及其互补链。SDA是这样一种方法，其中引物含有限制性核酸内切酶的识别位点，所述识别位点允许核酸内切酶将包含靶序列的半修饰DNA双链体的一条链产生切口，之后在一系列引物延伸和链置换步骤中扩增。本领域已知的其他链置换扩增方法(例如MSDA)不需要核酸内切酶切口。本领域技术人员将理解，本公开文本的寡核苷酸引物序列可以容易地用于任何基于通过聚合酶的引物延伸的体外扩增方法中。如本领域公知的，寡核苷酸被设计成在选定的条件下与互补序列结合。
如本文所用，“实时PCR”或“定量PCR”是指PCR方法，其中可使用荧光探针监测正在形成的产物的量，并通过追踪所产生的高于阈值水平的荧光信号来定量。实时PCR可以在一步反应(其包括在同时反应(即实时PCR或RT-PCR)中的逆转录步骤)或两步反应(其中连续进行逆转录步骤和PCR步骤)中进行。
如本文所用，术语“互补”是指两条核酸链的区域之间或同一核酸链的两个区域之间的序列互补性的广义概念。如果当所述两个区域以反向平行方式排列时，第一区域的至少一个核苷酸能够与第二区域的核苷酸进行碱基配对，则核酸的第一区域与相同或不同核酸的第二区域互补。在一些实施方案中，第一区域包括第一部分并且第二区域包括第二部分，由此当第一部分和第二部分以反向平行方式排列时，至少约50％、75％、80％、85％、90％、95％、96％、97％、98％或99％的第一部分的核苷酸能够与第二部分中的核苷酸进行碱基配对。在另一个实施方案中，第一部分的所有核苷酸能够与第二部分中的核苷酸进行碱基配对。
可以比对多肽序列和多核苷酸序列，并且可以使用公开可用的计算机算法相对于另一多肽或多核苷酸序列确定指定区域中相同氨基酸或核苷酸的百分比。多核苷酸序列或多肽序列的同一性百分比是通过以下方法来确定的：使用适当的算法(例如分别设置为默认参数的BLASTN或BLASTP)来比对多核苷酸和多肽序列；鉴定相对于比对部分的相同的核酸或氨基酸的数量；将相同核酸或氨基酸的数量除以本公开文本的多核苷酸的核酸或多肽的氨基酸的总数量；然后乘以100以确定同一性百分比。
如本文所用，术语“样品”和“生物样品”包括获自任何来源的样本或培养物。生物样品可以获自脑脊液、泪液、血液(包括任何血液产品，如全血、血浆、血清或血液的特定类型的细胞)、尿液、唾液等。生物样品还包括组织样品，如活检组织或先前已经固定(例如福尔马林快速冷冻、细胞学处理)的病理组织。
2.逆转录酶
本公开文本涉及莫洛尼氏鼠白血病病毒(MMLV)逆转录酶(RT酶)突变体。本公开文本的MMLV RT酶突变体是通过修饰MMLV RT酶碱基构建体的序列来制备(SEQ ID NO:637)。在一个实施方案中，本公开文本的MMLV RT酶突变体包含SEQ ID NO:637的氨基酸序列，其中所述MMLV RT酶突变体的氨基酸序列进一步包含至少一个氨基酸突变，所述至少一个氨基酸突变是：(a)位置61处的异亮氨酸至精氨酸、赖氨酸或甲硫氨酸取代(I61R、I61K或I61M)；(b)位置68处的谷氨酰胺至精氨酸、赖氨酸或异亮氨酸取代(Q68R、Q68K或Q68I)；(c)位置79处的谷氨酰胺至精氨酸、组氨酸或异亮氨酸取代(Q79R、Q79H或Q79I)；(d)位置99处的亮氨酸至精氨酸、赖氨酸或天冬酰胺取代(L99R、L99K或L99N)；(e)位置282处的谷氨酸至天冬氨酸、甲硫氨酸或色氨酸取代(E282D、E282M或E282W)；和/或(f)位置298处的精氨酸至丙氨酸取代(R298A)。
在另一个实施方案中，本公开文本的MMLV RT酶突变体包含SEQ ID NO:637的氨基酸序列，其中所述MMLV RT酶突变体的氨基酸序列进一步包含至少两个氨基酸取代，所述至少两个氨基酸取代是：(a)位置61处的异亮氨酸至精氨酸取代(I61R)；(b)位置68处的谷氨酰胺至精氨酸取代(Q68R)；(c)位置79处的谷氨酰胺至精氨酸取代(Q79R)；(d)位置99处的亮氨酸至精氨酸取代(L99R)；(e)位置282处的谷氨酸至天冬氨酸取代(E282D)；和/或(f)位置298处的精氨酸至丙氨酸取代(R298A)：(a)位置61处的异亮氨酸至精氨酸取代和位置282处的谷氨酸至天冬氨酸取代(I61R/E282D)；(b)取代位置99处的亮氨酸至精氨酸和位置282处的谷氨酸至天冬氨酸取代(L99R/E282D)；(c)位置68处的谷氨酰胺至精氨酸取代和位置282处的谷氨酸至天冬氨酸取代(Q68R/E282D)；(d)位置79处的谷氨酰胺至精氨酸取代和位置282处的谷氨酸至天冬氨酸取代(Q79R/E282D)；(e)位置282处的谷氨酸至天冬氨酸取代和位置298处的精氨酸至丙氨酸取代(E282D/R298A)；(f)位置61处的异亮氨酸至精氨酸取代和位置99处的亮氨酸至精氨酸取代(I61R/L99R)；(g)位置61处的异亮氨酸至精氨酸取代和位置68处的谷氨酰胺至精氨酸取代(I61R/Q68R)；(h)位置61处的异亮氨酸至精氨酸取代和位置79处的谷氨酰胺至精氨酸取代(I61R/Q79R)；(i)位置61处的异亮氨酸至精氨酸取代和位置298处的精氨酸至丙氨酸取代(I61R/R298A)；(j)位置68处的谷氨酰胺至精氨酸取代和位置99处的亮氨酸至精氨酸取代(Q68R/L99R)；(k)位置79处的谷氨酰胺至精氨酸取代和位置99处的亮氨酸至精氨酸取代(Q79R/L99R)；(l)取代位置99处的亮氨酸至精氨酸和位置298处的精氨酸至丙氨酸取代(L99R/R298A)；(m)位置68处的谷氨酰胺至精氨酸取代和位置79处的谷氨酰胺至精氨酸取代(Q68R/Q79R)；(n)位置68处的谷氨酰胺至精氨酸取代和位置298处的精氨酸至丙氨酸取代(Q68R/R298A)；或(o)位置79处的谷氨酰胺至精氨酸取代和位置298处的精氨酸至丙氨酸取代(Q79R/R298A)。
在另一个实施方案中，本公开文本的MMLV RT酶突变体包含SEQ ID NO:637的氨基酸序列，其中所述MMLV RT酶突变体的氨基酸序列进一步包含至少三个氨基酸取代，所述至少三个氨基酸取代是：(a)位置68处的谷氨酰胺至精氨酸取代(Q68R)；(b)位置79处的谷氨酰胺至精氨酸取代(Q79R)；(c)位置99处的亮氨酸至精氨酸取代(L99R)；和/或(d)位置282处的谷氨酸至天冬氨酸取代(E282D)：(a)位置68处的谷氨酰胺至精氨酸取代、位置99处的亮氨酸至精氨酸取代和位置282处的谷氨酸至天冬氨酸取代(Q68R/L99R/E282D)；(b)位置79处的谷氨酰胺至精氨酸取代、位置99处的亮氨酸至精氨酸取代和位置282处的谷氨酸至天冬氨酸取代(Q79R/L99R/E282D)；(c)位置68处的谷氨酰胺至精氨酸取代、位置68处的谷氨酰胺至精氨酸取代和位置282处的谷氨酸至天冬氨酸取代(Q68R/Q79R/E282D)；或(d)位置68处的谷氨酰胺至精氨酸取代、位置68处的谷氨酰胺至精氨酸取代和位置99处的亮氨酸至精氨酸取代(Q68R/Q79R/L99R)。
在另一个实施方案中，本公开文本的MMLV RT酶突变体包含SEQ ID NO:637的氨基酸序列，其中所述MMLV RT酶突变体的氨基酸序列进一步包含至少四个氨基酸取代，所述至少四个氨基酸取代是：(a)位置68处的谷氨酰胺至精氨酸、赖氨酸或异亮氨酸取代(Q68R、Q68K或Q68I)；(b)位置79处的谷氨酰胺至精氨酸、组氨酸或异亮氨酸取代(Q79R、Q79H或Q79I)；(c)位置99处的亮氨酸至精氨酸、赖氨酸或天冬酰胺取代(L99R、L99K或L99N)；(d)位置282处的谷氨酸至天冬氨酸、甲硫氨酸或色氨酸取代(E282D、E282M或E282W)：(a)位置68处的谷氨酰胺至精氨酸取代、位置79处的谷氨酰胺至精氨酸取代、位置99处的亮氨酸至精氨酸取代和位置282处的谷氨酸至天冬氨酸取代(Q68R/Q79R/L99R/E282D)；(b)位置68处的谷氨酰胺至精氨酸取代、位置79处的谷氨酰胺至精氨酸取代、位置99处的亮氨酸至赖氨酸取代和位置282处的谷氨酸至天冬氨酸取代(Q68R/Q79R/L99K/E282D)；(c)位置68处的谷氨酰胺至精氨酸取代、位置79处的谷氨酰胺至精氨酸取代、位置99处的亮氨酸至天冬酰胺取代和位置282处的谷氨酸至天冬氨酸取代(Q68R/Q79R/L99N/E282D)；(d)位置68处的谷氨酰胺至异亮氨酸取代、位置79处的谷氨酰胺至精氨酸取代、位置99处的亮氨酸至精氨酸取代和位置282处的谷氨酸至天冬氨酸取代(Q68I/Q79R/L99R/E282D)；(e)位置68处的谷氨酰胺至赖氨酸取代、位置79处的谷氨酰胺至精氨酸取代、位置99处的亮氨酸至精氨酸取代和位置282处的谷氨酸至天冬氨酸取代(Q68K/Q79R/L99R/E282D)；(f)位置68处的谷氨酰胺至精氨酸取代、位置79处的谷氨酰胺至组氨酸取代、位置99处的亮氨酸至精氨酸取代和位置282处的谷氨酸至天冬氨酸取代(Q68R/Q79H/L99R/E282D)；(g)位置68处的谷氨酰胺至精氨酸取代、位置79处的谷氨酰胺至异亮氨酸取代、位置99处的亮氨酸至精氨酸取代和位置282处的谷氨酸至天冬氨酸取代(Q68R/Q79I/L99R/E282D)；(h)位置68处的谷氨酰胺至精氨酸取代、位置79处的谷氨酰胺至精氨酸取代、位置99处的亮氨酸至精氨酸取代和位置282处的谷氨酸至甲硫氨酸取代(Q68R/Q79R/L99R/E282M)；(i)位置68处的谷氨酰胺至精氨酸取代、位置79处的谷氨酰胺至精氨酸取代、位置99处的亮氨酸至精氨酸取代和位置282处的谷氨酸至色氨酸取代(Q68R/Q79R/L99R/E282W)；或(j)位置68处的谷氨酰胺至异亮氨酸取代、位置79处的谷氨酰胺至组氨酸取代、位置99处的亮氨酸至赖氨酸取代和位置282处的谷氨酸至甲硫氨酸取代(Q68I/Q79H/L99K/E282M)。
在另一个实施方案中，本公开文本的MMLV RT酶突变体包含SEQ ID NO:637的氨基酸序列，其中所述MMLV RT酶突变体的氨基酸序列进一步包含至少五个氨基酸取代，所述至少五个氨基酸取代是：(a)位置61处的异亮氨酸至赖氨酸或甲硫氨酸取代(I61K或I61M)；(b)位置68处的谷氨酰胺至精氨酸或异亮氨酸取代(Q68R或Q68I)；(c)位置79处的谷氨酰胺至精氨酸或组氨酸取代(Q79R或Q79H)；(d)位置99处的亮氨酸至精氨酸或赖氨酸取代(L99R或L99K)；(e)位置282处谷氨酸至天冬氨酸或甲硫氨酸取代(E282D或E282M)：(a)位置61处的异亮氨酸至赖氨酸取代、位置68处的谷氨酰胺至精氨酸取代、位置79处的谷氨酰胺至精氨酸取代、位置99处的亮氨酸至精氨酸取代和位置282处的谷氨酸至天冬氨酸取代(I61K/Q68R/Q79R/L99R/E282D)；(b)位置61处的异亮氨酸至甲硫氨酸取代、位置68处的谷氨酰胺至精氨酸取代、位置79处的谷氨酰胺至精氨酸取代、位置99处的亮氨酸至精氨酸取代和位置282处的谷氨酸至天冬氨酸取代(I61M/Q68R/Q79R/L99R/E282D)；或(c)位置61处的异亮氨酸至甲硫氨酸取代、位置68处的谷氨酰胺至异亮氨酸取代、位置79处的谷氨酰胺至组氨酸取代、位置99处的亮氨酸至赖氨酸取代和位置282处的谷氨酸至甲硫氨酸取代(I61M/Q68IR/Q79H/L99K/E282M)。
在另一个实施方案中，本公开文本的MMLV RT酶突变体包含SEQ ID NO:637的氨基酸序列，其中所述MMLV RT酶突变体的氨基酸序列进一步包含至少五个或更多个氨基酸取代，所述至少五个或更多个氨基酸取代是：(a)位置68处的谷氨酰胺至精氨酸、赖氨酸或异亮氨酸取代(Q68R、Q68K或Q68I)；(b)位置79处的谷氨酰胺至精氨酸、组氨酸或异亮氨酸取代(Q79R、Q79H或Q79I)；(c)位置99处的亮氨酸至精氨酸、赖氨酸或天冬酰胺取代(L99R、L99K或L99N)；(d)位置282处的谷氨酸至天冬氨酸、甲硫氨酸或色氨酸取代(E282D、E282M或E282W)；(e)位置299处的谷氨酰胺至谷氨酸取代；(f)位置332处的苏氨酸至谷氨酸取代；(g)位置433处的缬氨酸至精氨酸取代；(h)位置593处的异亮氨酸至谷氨酸取代；(a)位置68处的谷氨酰胺至精氨酸取代、位置79处的谷氨酰胺至精氨酸取代、位置99处的亮氨酸至精氨酸取代和位置282处的谷氨酸至天冬氨酸取代、位置299处的谷氨酰胺至谷氨酸取代、位置433处的缬氨酸至精氨酸取代和位置593处的异亮氨酸至谷氨酸取代(Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E)：(b)位置68处的谷氨酰胺至精氨酸取代、位置79处的谷氨酰胺至精氨酸取代、位置82处的亮氨酸至精氨酸取代、位置99处的亮氨酸至精氨酸取代和位置282处的谷氨酸至天冬氨酸取代、位置299处的谷氨酰胺至谷氨酸取代、位置433处的缬氨酸至精氨酸取代和位置593处的异亮氨酸至谷氨酸取代(Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E)；(c)位置68处的谷氨酰胺至精氨酸取代、位置79处的谷氨酰胺至精氨酸取代、位置82处的亮氨酸至精氨酸取代、位置99处的亮氨酸至精氨酸取代和位置282处的谷氨酸至天冬氨酸取代、位置299处的谷氨酰胺至谷氨酸取代、位置332处的苏氨酸至谷氨酸取代,和位置593处的异亮氨酸至谷氨酸取代(Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E)；(d)位置68处的谷氨酰胺至精氨酸取代、位置79处的谷氨酰胺至精氨酸取代、位置82处的亮氨酸至精氨酸取代、位置99处的亮氨酸至精氨酸取代和位置282处的谷氨酸至天冬氨酸取代、位置299处的谷氨酰胺至谷氨酸取代、位置332处的苏氨酸至谷氨酸取代、位置433处的缬氨酸至精氨酸取代和位置593处的异亮氨酸至谷氨酸取代(Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/I593E)。
在一个实施方案中，所述RT酶突变体氨基酸序列包含选自表3、表8、表9、表12或表33的突变体。在一方面，所述RT酶突变体氨基酸序列包含选自以下的突变体：SEQ ID NO:638、SEQ ID NO:639、SEQ ID NO:640、SEQ ID NO:641、SEQ ID NO:642、SEQ ID NO:643、SEQID NO:644、SEQ ID NO:645、SEQ ID NO:646、SEQ ID NO:647、SEQ ID NO:648、SEQ ID NO:649、SEQ ID NO:650、SEQ ID NO:651、SEQ ID NO:652、SEQ ID NO:653、SEQ ID NO:654、SEQID NO:655、SEQ ID NO:656、SEQ ID NO:657、SEQ ID NO:658、SEQ ID NO:659、SEQ ID NO:660、SEQ ID NO:661、SEQ ID NO:662、SEQ ID NO:663、SEQ ID NO:664、SEQ ID NO:665、SEQID NO:666、SEQ ID NO:667、SEQ ID NO:668、SEQ ID NO:669、SEQ ID NO:679、SEQ ID NO:671、SEQ ID NO:672、SEQ ID NO:673、SEQ ID NO:674、SEQ ID NO:675、SEQ ID NO:676、SEQID NO:677、SEQ ID NO:678、SEQ ID NO:679、SEQ ID NO:670、SEQ ID NO:671、SEQ ID NO:672、SEQ ID NO:673、SEQ ID NO:674、SEQ ID NO:675、SEQ ID NO:676、SEQ ID NO:677、SEQID NO:678、SEQ ID NO:679、SEQ ID NO:680、SEQ ID NO:681、SEQ ID NO:682、SEQ ID NO:683、SEQ ID NO:684、SEQ ID NO:685、SEQ ID NO:686、SEQ ID NO:687、SEQ ID NO:688、SEQID NO:689、SEQ ID NO:690、SEQ ID NO:691、SEQ ID NO:692、SEQ ID NO:693、SEQ ID NO:694、SEQ ID NO:695、SEQ ID NO:696、SEQ ID NO:697、SEQ ID NO:698或SEQ ID NO:699。
在一个实施方案中，所述RT酶突变体氨基酸序列包含C末端延伸。在一方面，所述C末端延伸包含肽序列。在另一个实施方案中，分离的多肽编码具有C末端延伸的RT酶突变体。
要求保护的发明至少部分地基于这样的发现：如本文所公开的引入MMLV RT酶序列中的某些单和双氨基酸突变导致具有增加或增强的热稳定性和/或RT酶活性的MMLV RT酶。因此，本文还提供了用于合成MMLV RT酶突变体的方法和用于进行逆转录-聚合酶链式反应(RT-PCR)的方法。进一步提供了试剂盒，所述试剂盒包含分离的MMLV RT酶单、双、三或多个突变。
在某些实施方案中，所述突变的RT酶衍生自逆转录病毒莫洛尼氏鼠白血病病毒(MMLV)。在其他实施方案中，本公开文本的突变的RT酶可以通过以下方法衍生自除MMLV之外的逆转录病毒(如禽成髓细胞瘤病毒(AMV)或1型人类免疫缺陷病毒(HIV-1))的RT酶：将相同的突变引入获自其他逆转录病毒的RT酶碱基构建体中。
在某些实施方案中，本公开文本的RT酶突变体是通过本领域熟知的基因工程技术获得的。例如，可以使用定点诱变和随机诱变产生本公开文本的RT酶突变体。
在本公开文本的一个实施方案中，本公开文本的RT酶突变体是组合物的一部分。
3.诱变
本公开文本的RT酶突变体可以通过本文公开的或本领域已知的标准方法制备。在一个实施方案中，将RT酶碱基构建体的核酸序列(SEQ ID NO:637)进行修饰以产生编码RT酶突变体的核酸序列。本领域技术人员将认识到，具有适当菌株的菌落可用于产生和表达目的RT酶突变体，并且在细胞收获和蛋白质分离之后，RT酶突变体可用于cDNA合成技术。诱变和cDNA合成的非限制性例子描述于本文的实施例1-实施例3中。
如本文所用，术语“诱变”是指在细胞的核酸序列中引入遗传变化，然后其中所述改变由每个细胞遗传。本领域技术人员将理解，可以使用多种方法引入给定核酸序列中的突变。本领域技术人员将进一步认识到，诱变方法寻求使靶基因或靶多核苷酸突变。靶基因可以编码任一种或多种所需蛋白质。诱变方法通常使用带有所需序列修饰的合成寡核苷酸。将诱变的寡核苷酸使用体外酶促DNA合成掺入到DNA序列中，并且在突变体或野生型细菌中繁殖。
可以使用引物延伸诱变来实现定点诱变，其中将靶向突变引入模板多核苷酸的一个或多个所需位置。此技术需要使用含有相对于模板多核苷酸的一个或多个所需突变的特异性引物。诱变引物可以是合成的寡核苷酸或PCR产物。突变的引物可以包含一种或多种取代、缺失、添加或其组合。
也可以使用随机诱变产生突变的逆转录酶，其中在合成期间将突变引入诱变引物中。随机诱变的寡核苷酸也可以用作诱变引物。
在另一个实施方案中，本公开文本的突变的逆转录酶可以使用容易出错的滚环扩增(RCA)来开发。在此技术中，通过在MnCl2存在下进行RCA或者通过减少输入DNA的量来降低DNA聚合酶的保真度。
4.cDNA合成
本公开文本还涉及MMLV RT酶的活性，如通过本文公开的MMLV RT酶产生的cDNA的量测量。cDNA可以使用一步或两步程序制备，并且可以获自多种模板分子。如本文所用，术语“模板分子”是指携带用于制造新核酸链的遗传密码的生物分子。例如，在DNA复制中，将解开的双螺旋和每个单链DNA分子用作模板以合成互补链。从RNA逆转录生成cDNA。本领域技术人员将理解，cDNA分子可以由多种核酸模板分子制备。在一个实施方案中，所述核酸模板可以是单链或双链DNA。在一个实施方案中，RNA可用于cDNA合成。在某些实施方案中，与RT酶碱基构建体相比，本公开文本的MMLV RT酶突变体展现出增加的或增强的热稳定性和/或RT酶活性。在其他实施方案中，本公开文本的MMLV RT酶突变体展现出改变的半衰期、降低的或消除的RNA酶H活性、降低的末端脱氧核苷酸转移酶活性、增加的准确性或保真度、或增加的特异性。
本公开文本还提供了使用本公开文本的具有单或双氨基酸突变的MMLV RT酶突变体合成cDNA的方法。本公开文本的MMLV RT酶突变体可用于产生第一链cDNA或第一和第二链cDNA的方法中。本领域技术人员将理解，第一和第二链cDNA可以形成双链DNA分子，所述双链DNA分子可以包括全长cDNA序列和cDNA文库。
可以分离已经通过本公开文本的MMLV RT酶突变体逆转录的cDNA分子，或者含有所述cDNA分子的反应混合物可以直接用于下游应用或用于进一步分析或操纵。可用于实施本公开文本的方法的扩增方法描述于本文中并且是本领域熟知的。可以使用非特异性引物(如oligo-dT锚定引物或随机序列引物)、或者使用与所监测的每个遗传探针的RNA互补的靶特异性引物、或者使用热稳定的DNA聚合酶(如AMV RT酶或MMLV RT酶)进行逆转录反应。
扩增方法利用与对应于特定目的核苷酸序列的核酸选择性杂交的引物对，使所述引物对在允许选择性杂交的条件下与分离的核酸接触。杂交后，使核酸:引物复合物与一种或多种促进模板依赖性核酸合成的酶接触。进行多轮扩增，也称为“循环”，直到产生足够量的扩增产物。接下来，检测扩增产物。在某些方法中，可以通过视觉手段进行检测。可替代地，检测可以涉及经由化学发光、掺入的放射性标记或荧光标记的放射性闪烁扫描法或甚至经由使用电或热脉冲信号的系统来间接鉴定产物。
基于在具有所得“二寡核苷酸”序列的核酸的存在下连接两个(或更多个)寡核苷酸从而扩增二寡核苷酸的方法也可用于本公开文本的扩增步骤。
在本公开文本的一些实施方案中，所述检测过程可以利用杂交技术，例如，其中选择特异性引物或探针退火至目的靶生物标记物，并且此后进行选择性杂交的检测。如本领域公知的，寡核苷酸探针和引物可以通过考虑与其靶向序列杂交的熔点来设计。
本领域技术人员将认识到，使用本公开文本的MMLV RT酶突变体制备的cDNA分子可以用于多种另外的下游应用中。例如，扩增方法可以包括一步PCR、两步PCR、实时或定量PCR、热启动PCR、巢式PCR、降落PCR、差异显示PCR(DDRT-PCR)、微阵列技术、反向PCR、PCR末端的快速扩增(RACE或锚定PCR)、多重PCR和定点PCR诱变。用本公开文本的MMLV RT酶突变体创建的合成cDNA和cDNA文库可用于克隆和/或测序以进一步表征。本领域技术人员将认识到，使用用本公开文本的MMLV RT酶突变体制备的cDNA的核酸扩增可以包括本文未列出的另外的技术。
为了使杂交能够在上文呈现的方法下发生，寡核苷酸引物和探针应包含与目的序列的一部分具有至少70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或100％同一性的寡核苷酸序列。
5.生物样品
本公开文本的MMLV RT酶突变体和相关方法可以用可从中分离RNA或DNA的任何合适的生物样品来实施。在本公开文本的一个实施方案中，所述生物样品可以是获自患病或健康受试者的体液或组织。在本公开文本的一些实施方案中，生物样品可以是体液，包括但不限于全血、血浆、血清、粪便或尿液。在另一个实施方案中，本公开文本的方法可以用任何合适的样品来实施，所述样品是新鲜分离的、或者在从例如具有已知的诊断、治疗和/或结局史的受试者收集之后已经冷冻或储存的。样品可以通过任何非侵入性手段(例如像，细针穿刺或针活检)或者可替代地通过侵入性方法(包括例如，手术活检)收集。在此类实施方案中，可以在分析之前从生物样品(例如，血清)中提取RNA或DNA。RNA和DNA提取的方法是本领域熟知的。
许多用于从体液或组织(例如血清)中提取RNA(即总RNA或mRNA)的试剂盒是本领域已知的并且是可商购获得的。本领域普通技术人员可以容易地选择用于特定情况的合适的试剂盒。
在本公开文本的某些实施方案中，在提取之后，将mRNA扩增并且转录成cDNA，然后所述cDNA可以用作通过适当的RNA聚合酶进行多轮转录的模板。可用于实施本公开文本的方法的扩增方法描述于本文中并且是本领域熟知的。可以使用非特异性引物(如oligo-dT锚定引物或随机序列引物)、或者使用与所监测的每个遗传探针的RNA互补的靶特异性引物、或者使用热稳定的DNA聚合酶(如本公开文本的MMLV RT酶或MMLV RT酶突变体)进行逆转录反应。
在某些实施方案中，将从生物样品分离的RNA(例如，在扩增和/或转化为cDNA或cRNA之后)用可检测试剂标记，然后进行分析。可检测试剂的作用是促进RNA的检测或允许杂交的核酸片段(例如，在基于阵列的测定中与遗传探针杂交的核酸片段)的可视化。在一些实施方案中，选择可检测试剂，使得其产生可以测量的信号，并且其强度与存在于被分析的样品中的标记核酸的量相关。
用于标记核酸分子的方法是本领域熟知的。标记方案和标记检测技术的综述可以在Kricka,Ann.Clin.Biochem.39:114-29(2002)；van Gijlswijk等人,ExpertRev.Mol.Diagn.1:81-91(2001)；和Joos等人,J.Biotechnol.35:135-53(1994)中找到。标准的核酸标记方法包括掺入放射性试剂；荧光染料或酶的直接附接；核酸片段的化学修饰，使其可以通过免疫化学或其他亲和反应进行检测；和酶介导的标记方法，如随机引发、切口平移、PCR和末端转移酶的拖尾。
各种可检测试剂中的任一种可用于实施本公开文本的方法。合适的可检测试剂包括但不限于各种配体、放射性核素、荧光染料、化学发光剂、微粒(例如像，量子点、纳米晶体和磷光体)、酶(例如像，ELISA中使用的那些，即辣根过氧化物酶、β-半乳糖苷酶、萤光素酶和碱性磷酸酶)、比色标记、磁性标记、生物素、地高辛(dioxigenin)或可用抗血清或单克隆抗体的其他半抗原和蛋白质。
6.试剂盒
本公开文本还提供了用于逆转录或相关技术的试剂盒。这些试剂盒包括以下的一种或多种：MMLV RT酶突变体酶、用于进行逆转录酶反应的试剂和缓冲液、盒、小瓶管、安瓿等。试剂盒还可以包括使用试剂盒来实施本文公开的任何方法或本领域技术人员已知的其他方法的说明书。
实施例
通过以下实施例进一步说明要求保护的发明，所述实施例不应被解释为限制。本领域技术人员将认识到，要求保护的发明可以通过所公开的结构、材料、组合物和方法的变化来实施，并且这种变化被认为在要求保护的发明的范围内。
本文所述的RT酶在大肠杆菌(E.coli)中过表达，纯化至均一，并且在逆转录酶定量PCR(RT-qPCR)的情况下测试其增强RNA检测的能力。
实施例1.通过定点诱变制备逆转录酶突变体
a.从碱基构建体(RNA酶H-构建体)创建的MMLV R T酶突变体的克隆
通过以下制备MMLV RT酶突变体，首先将三个突变(D524G、E562Q和D583N)引入野生型或天然存在的MMLV RT酶的氨基酸序列中，以制备MMLV RT酶碱基构建体(SEQ ID NO:637)。已经证明SuperScript II RT酶(Invitrogen)中包含的三个突变降低RNA酶H活性(参见美国专利号5,405,776)。将MMLV RT酶碱基构建体针对大肠杆菌表达进行优化，并且以基因片段(Integrated DNA Technologies)或通过具有适当纯化标签的定制基因合成获得。使用标准PCR条件和引物扩增后续基因(参见表1)。使用QIAquick PCR纯化试剂盒(Qiagen，目录号28104)使扩增的DNA经受纯化，然后将基因片段组装到pET28b表达质粒中。分离质粒DNA并且对其测序以验证转化到大肠杆菌细胞后的所需序列。通过合理设计(图1A-图1C)选择MMLV RT酶突变，并且使用标准PCR条件和引物(参见表1)通过定点诱变将其引入。将所得质粒转化到大肠杆菌BL21(DE3)细胞中以进行表达。
表1.用于将MMLV RT酶碱基构建体和突变体克隆到pET28b中的引物序列。
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实施例2：用于筛选增加的活性和热稳定性的逆转录酶突变体的制备
a.MMLV RT酶和突变变体的过表达
使用测试诱导来确定最佳生长条件。使用具有适当菌株的菌落接种具有卡那霉素(0.05mg/mL)的Terrific Broth(TB)培养基(50mL)，并且在37℃下生长直到达到约0.9的OD。将50mL培养物分成两半以适应两个诱导温度。将IPTG(1M；12.5μL)用于诱导蛋白质表达，然后在两个诱导温度下生长21小时。在3和21小时取等分试样(针对1.25的OD归一化)，在13,000x g下持续一分钟后收获细胞，并且将收获的细胞在-20℃下储存。将细胞重新悬浮于1x SDS-PAGE运行缓冲液(270μL)和5x SDS-PAGE加载染料(70μL)中。将样品煮沸5分钟，超声，并且加载(15μL)到4％-20％的TGX Stain-FreeTM蛋白质凝胶(Bio Rad，目录号4568094)上。SDS-PAGE图像示于图2中。
b.MMLV RT酶和突变变体的表达和纯化
使用具有适当菌株的菌落接种具有卡那霉素(0.05mg/mL)的TB培养基(1mL，在96孔深孔板中)，并且在37℃下生长直至达到约0.9的OD，然后将板在冰上冷却5分钟。通过添加100mM IPTG(5μL)然后在18℃下生长21小时来诱导蛋白质表达。通过在4,700x g下旋转样品10分钟来收获细胞。
将细胞沉淀物重新悬浮于裂解缓冲液(50mM NaPO4(pH 7.8)、5％甘油、300mMNaCl和10mM咪唑)中，并且通过添加1x(Millipore Sigma，目录号70921)裂解，并且在室温下在直立圆筒混合机上孵育15分钟。通过将裂解物在4℃下在16,000x g下离心20分钟来去除细胞碎片。
将澄清的裂解物施加到HisPurTMNi-NTA旋转板(ThermoFisher，目录号88230)上。将树脂用筛选His结合缓冲液(50mM NaPO4(pH 7.8)、5％甘油、300mM NaCl和10mM咪唑)平衡并且加载样品。将样品用筛选His洗涤缓冲液(50mM NaPO4(pH 7.8)、5％甘油、300mMNaCl和25mM咪唑)洗涤三次，并且使用筛选His洗脱缓冲液(50mM NaPO4(pH 7.8)、5％甘油、300mM NaCl和250mM咪唑)洗脱。将纯化的蛋白质针对设定浓度(100nM)归一化以用于测试目的。
实施例3：逆转录酶突变体的评价
a.RT酶突变体合成DNA的能力的评价
将突变体RT酶从纯化的总RNA(DNA酶化的，从HeLa细胞分离)合成cDNA的能力与MMLV RT酶碱基构建体(RNA酶H-构建体)进行比较。以两种形式测试了突变体MMLV RT酶：(1)用基因特异性引物进行标准两步cDNA合成，然后进行qPCR，和(2)在Integrated DNATechnologies基因表达主要混合物(GEM)中一步添加RT酶。
b.标准两步程序
将RT酶(2μL，100nM)添加到含有RNA(50ng)、dNTP(100μM)、基因特异性引物集(500nM；参见表2)、第一链合成缓冲液(1x，50mM Tris-HCl(pH 8.3)、75mM KCl、3mM MgCl2、10mM DTT)和SuperaseIN(0.17U/μL)的体积为50μL的反应混合物中。使反应在50℃下进行15分钟，然后在80℃下孵育10分钟。
使用鉴定人细胞中SFRS9基因的测定，通过qPCR扩增对通过RT酶突变体合成的cDNA进行定量。测定主要混合物组成包括GEM(1x)、ROX(50nM)、SFRS9引物集(500nM；参见表2)和SFRS9探针(250nM；参见表2)。将测定主要混合物和合成的cDNA以4:1比率混合以获得20μL的最终体积。将反应在以下循环条件下在qPCR(QuantStudio)上运行40个循环：95℃保持3分钟，95℃保持15秒以及60℃保持一分钟。
表2.用于qPCR测定的引物和探针序列。
c.GEM中的一步程序
将RT酶(1μL，100nM)添加到含有RNA(10ng)、GEM(1x)、ROX(50nM)、SFRS9引物集(500nM；参见表2)和SFRS9探针(250nM；参见表2)的最终体积为20μL的反应混合物中。将反应使用以下循环条件在qPCR机器(QuantStudio)上运行40个循环：60℃保持15分钟，95℃保持3分钟，95℃保持15秒以及60℃持续一分钟。
d.MMLV RT酶碱基构建体和单突变变体
如实施例1所述，MMLV RT酶单突变变体是使用标准PCR条件和引物通过定点诱变将选定的突变引入MMLV RT酶碱基构建体中制备的。MMLV RT酶碱基构建体和单突变变体的序列示于表3。本领域技术人员将理解SEQ ID NO:637中所示的MMLV RT酶氨基酸序列是野生型或天然存在的MMLV RT酶的全长氨基酸序列的截短形式。此外，本领域普通技术人员将理解，需要甲硫氨酸残基来重组产生本公开文本的MMLV RT酶碱基构建体和突变体，并且因此，本文公开的MMLV RT酶序列(参见例如表3、表8和表9)包含氨基酸序列的N末端处的甲硫氨酸残基。然而，关于本公开文本，并且出于鉴定和编号已经引入突变的MMLV RT酶氨基酸序列中的残基的目的，此甲硫氨酸残基被认为是氨基酸残基0(即，不计算)并且第二氨基酸残基(例如SEQ ID NO:637中所示的MMLV RT酶碱基构建体中的苏氨酸)被认为是氨基酸残基1。
表3.MMLV RT酶碱基构建体和单突变MMLV RT酶构建体的序列。
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e.实验结果
根据标准曲线通过拷贝数输出分析并报告MMLV RT酶碱基构建体和MMLV RT酶单突变变体的两步反应和一步反应(参见表4和表5)。与MMLV RT酶碱基构建体相比，发现六个单突变MMLV RT酶变体展现出增加的总体活性和热稳定性。六个单突变MMLV RT酶变体如下：I61R、Q68R、Q79R、L99R、E282D和R298A。
表4.通过MMLV RT单突变体的两步cDNA合成。经由qPCR人归一化测定产生数据并且将其通过拷贝数翻译。
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表5.通过MMLV RT单突变体的一步cDNA合成。经由qPCR人归一化测定产生数据，并且将数据通过拷贝数翻译。
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实施例4：逆转录酶单突变体的延伸
进一步评价包围实施例3中鉴定的MMLV RT酶单突变体的氨基酸位置，以包括在该位置的所有可能的氨基酸取代。将单突变体如实施例1和2中所述进行克隆、过表达和纯化，并且如实施例3中所述进行评价。根据标准曲线通过拷贝数输出分析并报告MMLV RT酶碱基构建体和MMLV RT酶双突变变体的两步反应和一步反应(参见表6和表7)。与MMLV RT酶碱基构建体相比，发现十个单突变MMLV RT酶变体(参见表8)展现出增加的总体活性和热稳定性。十个单突变MMLV RT酶变体如下：I61K、I61M、Q68I、Q68K、Q79H、Q79I、L99K、L99N、E282M和E282W。
表6.通过MMLV RT单突变体的两步cDNA合成。经由qPCR人归一化测定产生数据并且将其通过拷贝数翻译。
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表7.通过MMLV RT单突变体的一步cDNA合成。经由qPCR人归一化测定产生数据，并且将数据通过拷贝数翻译。
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表8.单突变MMLV RT酶变体的序列。
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实施例5：具有增强的活性的逆转录酶突变体的堆叠
a.MMLV RT酶双突变体
与MMLV RT酶碱基构建体(RNA酶H-构建体)相比，将实施例3中鉴定的MMLV RT酶单突变体堆叠以进一步改善MMLV RT酶从纯化的总RNA(DNA酶化的，从HeLa细胞分离)合成cDNA的能力。将十五个MMLV RT酶双突变变体(参见表9)如实施例1和2中所述进行克隆、过表达和纯化，并且如实施例3中所述进行评价。根据标准曲线通过拷贝数输出分析并报告MMLV RT酶碱基构建体和MMLV RT酶双突变变体的两步反应和一步反应(参见表10和表11)。
与其他MMLV RT酶双突变变体相比，发现十五个MMLV RT酶双突变变体中有四个展现出增加的总体活性和热稳定性，并且与MMLV RT酶碱基构建体相比，几乎所有的MMLV RT酶双突变变体均展现出增加的总体活性和热稳定性。发现展现出最高总体活性的四个MMLVRT酶双突变变体是E282D/L99R、L99R/Q68R、L99R/Q79R和Q68R/Q79R。
表9.双突变MMLV RT酶变体的序列。
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表10.通过MMLV RT双突变体的两步cDNA合成。经由qPCR人归一化测定产生数据，并且将数据通过拷贝数翻译。
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表11.通过MMLV RT双突变体的一步cDNA合成。经由qPCR人归一化测定产生数据，并且将数据通过拷贝数翻译。
| MMLV-RT变体 | 数量平均值 | 数量标准差 |
| MMLV-II | 859.127 | 24.795 |
| MMLV-II E282D/I61R | 2,948.906 | 49.177 |
| MMLV-II E282D/L99R | 4,814.957 | 239.110 |
| MMLV-II E282D/Q68R | 3,709.046 | 131.434 |
| MMLV-II E282D/Q79R | 3,694.187 | 98.772 |
| MMLV-II E282D/R298A | 794.643 | 39.913 |
| MMLV-II I61R/L99R | 3,443.713 | 180.210 |
| MMLV-II I61R/Q68R | 3,525.138 | 112.288 |
| MMLV-II I61R/Q79R | 3,125.990 | 120.996 |
| MMLV-II I61R/R298A | 2,006.208 | 83.559 |
| MMLV-II L99R/Q68R | 6,755.852 | 102.788 |
| MMLV-II L99R/Q79R | 6,709.502 | 35.997 |
| MMLV-II L99R/R298A | 2,128.451 | 55.565 |
| MMLV-II Q68R/Q79R | 6,343.821 | 140.779 |
| MMLV-II Q68R/R298A | 2,406.470 | 74.117 |
| MMLV-II Q79R/R298A | 2,301.759 | 22.849 |
| MMLV-IV | 15,411.857 | 333.388 |
b.MMLV RT酶三突变体和更多突变体的克隆
在双突变变体之后，将MMLV RT酶单突变体进一步堆叠以改善MMLV RT酶与MMLVRT酶碱基构建体(RNA酶H-构建体)相比从纯化的总RNA(DNA酶化的，从HeLa细胞分离)合成cDNA的能力。如实施例1所述，克隆十七个MMLV RT酶三突变或更多突变变体(参见表12)。
表12.三突变或更多突变MMLV RT酶变体的序列。
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c.MMLV RT酶和突变变体的表达和纯化
将具有适当菌株的菌落用于接种具有卡那霉素(0.05mg/mL)的TB培养基(200mL)，并且在37℃下生长直至达到约0.9的OD，然后将烧瓶在4℃下冷却30分钟。通过添加1M IPTG(100μL)然后在18℃下生长21小时诱导蛋白质表达。通过在4,700x g下旋转样品10分钟来收获细胞。
将细胞沉淀物重悬于裂解缓冲液(50mM NaPO4(pH 7.8)、5％甘油、300mM NaCl、10mM咪唑、5mM DTT、0.01％n-辛基-β-D-吡喃葡萄糖苷、DNA酶I、10mM CaCl2、溶菌酶(1mg/mL)和蛋白酶抑制剂)中。将样品在15-20kpsi下预冷却至4℃的Avestin Emulsiflex C3上进行裂解，其中通过三次。通过将裂解物在4℃下在16,000x g下离心30分钟来去除细胞碎片。
将澄清的裂解物施加到HisTrap HP柱(Cytiva Life Sciences，目录号17524701)上。将树脂用MMLV His结合缓冲液(50mM NaPO4(pH 7.8)、5％甘油、0.3M NaCl、10mM咪唑、1mM DTT和0.01％IGEPAL-CA)平衡，然后加载样品。将样品用MMLV His结合缓冲液洗涤，然后用25％B洗涤(B＝MMLV His洗脱缓冲液＝50mM NaPO4，pH 7.8，5％甘油，0.3M NaCl，250mM咪唑，1mM DTT和0.01％IGEPAL-CA)洗涤。将样品用100％B洗脱10个CV于45mL级分中。
将纯化的蛋白质施加到HiTrap肝素HP柱(Cytiva Life Sciences，目录号17040601)上。将树脂用MMLV肝素结合缓冲液(50mM Tris HCl pH 8.5，75mM NaCl，1mMDTT，5％甘油和0.01％IGEPAL-CA)平衡，然后加载样品。将样品用MLV肝素结合缓冲液洗涤，然后用25％B洗涤(B＝MLV肝素洗脱缓冲液)洗涤。将样品用60％B洗脱10个CV于45mL级分中。
将纯化的蛋白质施加到Bio-ScaleTMMini CHTTM盒(Bio-Rad Laboratories，目录号7324322)上。将树脂用1M NaOH洗涤，然后用MMLV肝素结合缓冲液平衡并且加载样品。将样品用MLV肝素洗脱缓冲液洗涤，然后用MMLV肝素结合缓冲液洗涤。将样品线性洗脱15个CV至100％B2(B2＝MMLV HA洗脱缓冲液＝250mM KPO4(pH 7.5)、1mM DTT、5％甘油和0.01％IGEPAL-CA)于5mL级分中。
将含有纯化的蛋白质的级分合并并且在MMLV储存缓冲液(50mM Tris-HCl(pH7.5)、100mM NaCl、1mM DTT、50％(v/v)甘油)中透析。
d.纯化的MMLV RT酶突变变体通过基因特异性引发合成DNA的能力的评价
如实施例3中所述评价MMLV RT酶碱基构建体和MMLV RT酶突变变体。将两步反应和一步反应的温度分别调整为55℃和60℃。通过qPCR的Ct输出分析并报告MMLV RT酶碱基构建体和MMLV RT酶突变变体的两步反应和一步反应(参见表13和表14)。
与其他MMLV RT酶堆叠突变变体相比，发现十七个MMLV RT酶三突变或更多突变变体中有六个展现出增加的总体活性和热稳定性，并且与MMLV RT酶碱基构建体相比，几乎所有的MMLV RT酶堆叠突变变体均展现出增加的总体活性和热稳定性。发现展现出最高的总体活性的MMLV RT酶突变变体为Q68R/L99R、Q68R/Q79R/L99R、Q68R/Q79R/L99R/E282D、Q68R/Q79R/L99K/E282D、Q68R/Q79R/L99R/E282W、I61M/Q68R/Q79R/L99R/E282D和Q68I/Q79H/L99K/E282M。
表13.由MMLV RT三突变体和更多突变体的两步cDNA合成。经由qPCR人归一化测定产生数据，并且将数据通过Ct值报告。
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表14.通过MMLV RT三突变体和更多突变体的一步cDNA合成。经由qPCR人归一化测定产生数据，并且将数据通过Ct值报告。
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e.纯化的MMLV RT酶突变变体通过oligo-dT或随机引发合成DNA的能力的评价
如实施例3中所述评价MMLV RT酶碱基构建体和MMLV RT酶突变变体。调整两步反应的oligo-dT或随机六聚体引发条件，并且将RT酶浓度针对31nM归一化。通过qPCR的Ct输出分析并报告MMLV RT酶碱基构建体和MMLV RT酶突变变体的两步反应(参见表15和表16)。
与其他MMLV RT酶堆叠突变变体相比，发现十七个MMLV RT酶三突变或更多突变变体中有九个展现出增加的总体活性和热稳定性，并且与MMLV RT酶碱基构建体相比，几乎所有的MMLV RT酶堆叠突变变体均展现出增加的总体活性和热稳定性。发现展现出最高总体活性的MMLV RT酶突变变体为Q79R/L99R/E282D、Q68R/Q79R/L99R、Q68R/Q79R/L99R/E282D、Q68R/Q79R/L99K/E282D、Q68R/Q79R/L99N/E282D、Q68K/Q79R/L99R/E282D、Q68R/Q79R/L99R/E282M、I61K/Q68R/Q79R/L99R/E282D和I61M/Q68R/Q79R/L99R/E282D。
表15.通过Oligo-dT引发经由MMLV RT三突变体和更多突变体的两步cDNA合成。经由qPCR人归一化测定产生数据，并且将数据通过Ct值报告。
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表16.通过随机六聚体引发经由MMLV RT三突变体和更多突变体的两步cDNA合成。
经由qPCR人归一化测定产生数据，并且将数据通过Ct值报告。
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f.纯化的MMLV RT酶突变变体在宽温度范围内合成DNA的能力的评价如实施例3中所述评价MMLV RT酶碱基构建体和MMLV RT酶突变变体。针对两步反应调整Oligo-dT或随机六聚体引发条件和反应温度，并且将RT酶浓度相对于31nM归一化。通过qPCR的Ct输出分析并报告MMLV RT酶碱基构建体和MMLV RT酶突变变体的两步反应(参见表17和表18)。
发现无论使用哪种引发方法，与其他MMLV RT酶堆叠突变变体相比，九个MMLV RT酶三突变或更多突变变体中有六个在宽范围的温度(跨越从37.0℃至65℃)下展现出高的总体活性。与MMLV RT酶碱基构建体相比，所有MMLV RT酶堆叠的突变变体均展现出增加的总体活性和热稳定性。发现在宽范围的温度下展现出最高的总体活性的六个MMLV RT酶突变变体为Q68R/Q79R/L99R、Q68R/Q79R/L99R/E282D、Q68R/Q79R/L99K/E282D、Q68R/Q79R/L99N/E282D、I61K/Q68R/Q79R/L99R/E282D和I61M/Q68R/Q79R/L99R/E282D。
表17.通过Oligo-dT引发经由MMLV RT三突变体和更多突变体的两步cDNA合成。经由qPCR人归一化测定产生数据，并且将数据通过Ct值报告。
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表18.通过随机六聚体引发经由MMLV RT三突变体和更多突变体的两步cDNA合成。
经由qPCR人归一化测定产生数据，并且将数据通过Ct值报告。
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实施例6：通过oligo dT或随机引发进行逆转录酶突变体评价
此实施例展示了用于评价每个突变RT酶与MMLV RT酶的碱基构建体相比从纯化的总RNA(DNA酶化的，从HeLa细胞分离)合成cDNA的能力的程序。使用如实施例5所述的标准两步cDNA合成通过两种引发条件测试突变体MMLV RT酶：仅oligo dT和随机六聚体引发。通过Ct值分析并报告反应(表19和表20)。与碱基构建体相比，MMLV RT酶的四个突变变体使用oligo dT引发显示出总体活性的增加：Q299E、T332E和V433R。与碱基构建体相比，MMLV RT酶的八个突变变体使用随机引发显示出总体活性的增加：P76R、L82R、I125R、Y271A、L280A、L280R、T328R和V433R。
表19使用oligo dT引发通过MMLV-RT单突变体的两步cDNA合成。经由qPCR人归一化测定产生数据，并且将数据通过Ct值报告。
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表20.使用随机引发通过MMLV-RT单突变体的两步cDNA合成。经由qPCR人归一化测定产生数据，并且将数据通过Ct值报告。
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实施例7.通过基因特异性引发进行逆转录酶突变体评价
此实施例展示了用于评价每个突变体RT酶与MMLV RT酶的碱基构建体相比从纯化的RNA超聚体(ultramer)(Integrated DNA Technologies)合成cDNA的能力的程序。如实施例5所述，通过在GEM中一步添加RT酶来测试突变体MMLV RT酶。通过Ct值分析并报告反应(表21)。与碱基构建体相比，MMLV RT酶的十二个突变变体显示总体活性的增加：H77A、D83E、D83R、Y271E、Q299E、G308E、F396A、V433R、I593E、I597A和I597R。
表21通过基因特异性引发经由MMLV-RT单突变体的一步cDNA合成。经由qPCR人归一化测定产生数据，并且将数据通过Ct值报告。
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实施例8.具有增强的活性的逆转录酶突变体的进一步堆叠。
此实施例展示了用于堆叠实施例6-7中发现的增强的突变体的程序，以进一步改善MM LV RT酶与碱基构建体和先前发现的含有突变Q68R/Q79R/L99R/E282D的突变体MMLVRT酶相比从纯化的总RNA(DNA酶化的，从HeLa细胞分离)合成cDNA的能力。将堆叠的突变体MMLV RT酶如实施例1-2所述进行克隆、过表达和纯化，并且如实施例6-7所述进行测试。通过Ct值分析并报告两步和一步反应两者(表22-表24)。与碱基构建体相比，MMLV RT酶的八个堆叠突变变体中有六个增加了总体活性和热稳定性：Q68R/Q79R/L99R/E282D/V433R、Q68R/Q79R/L99R/E282D/I593E、Q68R/Q79R/L99R/E282D/Q299E、Q68R/Q79R/L99R/E282D/T332E、Q68R/L82R/L99R/E282D和Q68R/Q79R/L82R/L99R/E282D。随后，与先前鉴定的突变体RT酶(Q68R/Q79R/L99R/E282D)相比，MMLV RT酶的这六个堆叠突变变体中有四个增加了总体活性和热稳定性：Q68R/Q79R/L99R/E282D/I593E、Q68R/Q79R/L99R/E282D/Q299E、Q68R/L82R/L99R/E282D和Q68R/Q79R/L82R/L99R/E282D。
在这些堆叠突变变体之后，将MMLV RT酶突变体进一步堆叠以改善MMLV RT酶与MMLV RT酶碱基构建体(RNA酶H-构建体)相比从纯化的总RNA(DNA酶化的，从HeLa细胞分离)合成cDNA的能力。将八个MMLV RT酶六突变或更多突变变体如实施例1所述进行克隆，如并且实施例5所述进行过表达和纯化。
如实施例3中所述评价MMLV RT酶碱基构建体和MMLV RT酶突变变体。将两步反应和一步反应的温度分别调节至42℃/55℃和50℃/60℃。将两步第一链合成缓冲液从50mMTris-盐酸盐(pH 8.3)、75mM氯化钾、3mM氯化镁和10mM DTT改良为50mM乙酸钾、20mM Tris-乙酸盐(pH 7.0)、10mM乙酸镁、100μg/ml牛血清白蛋白和10mM DTT。通过qPCR的Ct输出分析并报告MMLV RT酶碱基构建体和MMLV RT酶突变变体的两步反应和一步反应(表22-表24)。
与其他MMLV RT酶堆叠突变变体相比，发现十一个MMLV RT酶六突变或更多突变变体中有四个展现出增加的总体活性和热稳定性，并且与MMLV RT酶碱基构建体相比，几乎所有的MMLV RT酶堆叠突变变体均展现出增加的总体活性和热稳定性。发现展现出最高总体活性的MMLV RT酶突变变体为Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E、Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E、Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E和Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/I593E。
表22.使用oligo dT引发通过MMLV-RT堆叠突变体的两步cDNA合成。经由qPCR人归一化测定产生数据，并且将数据通过Ct值报告。
| MMLV-RT变体 | Ct平均值 | Ct标准差 |
| MMLV-II | 37.388 | 0.396 |
| MMLV-II Q68R/Q79R/L99R/E282D/V433R | 29.215 | 0.113 |
| MMLV-II Q68R/Q79R/L99R/E282D/I593E | 33.563 | 0.118 |
| MMLV-II Q68R/Q79R/L99R/E282D/Q299E | 31.902 | 0.169 |
| MMLV-II Q68R/Q79R/L99R/E282D/T332E | 33.988 | 0.108 |
| MMLV-II Q68R/Q79R/L99R/L280R | 40.000 | 0.000 |
| MMLV-II Q68R/Q79R/L99R/L280R/E282D | 40.000 | 0.000 |
| MMLV-II Q68R/L82R/L99R/E282D | 39.259 | 1.047 |
| MMLV-II Q68R/Q79R/L82R/L99R/E282D | 30.623 | 0.076 |
| MMLV-IV | 25.880 | 0.023 |
表23.使用随机引发通过MMLV-RT堆叠突变体的两步cDNA合成。经由qPCR人归一化测定产生数据，并且将数据通过Ct值报告。
表24.通过基因特异性引发经由MMLV-RT堆叠突变体的一步cDNA合成。经由qPCR人归一化测定产生数据，并且将数据通过Ct值报告。
| MMLV-RT变体 | Ct平均值 | Ct标准差 |
| MMLV-II | 33.027 | 0.048 |
| MMLV-II Q68R/Q79R/L99R/E282D/V433R | 29.937 | 0.040 |
| MMLV-II Q68R/Q79R/L99R/E282D/I593E | 28.724 | 0.081 |
| MMLV-II Q68R/Q79R/L99R/E282D/Q299E | 29.341 | 0.022 |
| MMLV-II Q68R/Q79R/L99R/E282D/T332E | 30.330 | 0.036 |
| MMLV-II Q68R/Q79R/L99R/L280R | 40.000 | 0.000 |
| MMLV-II Q68R/Q79R/L99R/L280R/E282D | 40.000 | 0.000 |
| MMLV-II Q68R/L82R/L99R/E282D | 30.559 | 0.045 |
| MMLV-II Q68R/Q79R/L82R/L99R/E282D | 30.097 | 0.033 |
| MMLV-IV | 28.975 | 0.012 |
a.纯化的MMLV RT酶突变变体在宽温度范围内合成DNA的能力的评价
如实施例5中所述评价MMLV RT酶碱基构建体、MMLV RT酶突变变体。针对两步反应调整Oligo-dT或随机六聚体引发条件和反应温度，并且将RT酶浓度相对于31nM归一化。通过qPCR的Ct输出分析并报告MMLV RT酶碱基构建体和MMLV RT酶突变变体的两步反应(参见表25和表26)。
发现无论使用哪种引发方法，与MMLV RT酶碱基构建体相比，五个MMLV RT酶突变体在宽温度范围(跨越从37.0℃至51℃)内展现出高的总体活性。与MMLV RT酶碱基构建体相比，所有MMLV RT酶堆叠的突变变体均展现出增加的总体活性和热稳定性。发现在宽温度范围内展现出最高总体活性的五个MMLV RT酶突变变体为Q68R/Q79R/L99R/E282D、Q68R/Q79R/L99R/E282D/Q299E/V433R/I593E、Q68R/Q79R/L82R/L99R/E282D/Q299E/V433R/I593E、Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/I593E和Q68R/Q79R/L82R/L99R/E282D/Q299E/T332E/V433R/I593E。
表25.通过Oligo-dT引发经由MMLV RT四突变体或更多突变体的两步cDNA合成。经由qPCR人归一化测定产生数据，并且将数据通过Ct值报告。
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/>
/>
表26.通过随机引发经由MMLV RT四突变体和更多突变体的两步cDNA合成。经由qPCR人归一化测定产生数据，并且将数据通过Ct值报告。
/>
/>
/>
/>
实施例9：逆转录酶单突变体的延伸
进一步评价包围实施例6和7中鉴定的MMLV RT酶单突变体的氨基酸位置，以包括该位置处的所有可能的氨基酸取代。将单突变体如实施例1和2中所述进行克隆、过表达和纯化，并且如实施例6和7中所述进行评价。通过qPCR的Ct输出分析并报告MMLV RT酶碱基构建体和MMLV RT酶双突变变体的两步反应和一步反应(表27-29)。与MMLV RT酶碱基构建体相比，发现多个单突变MMLV RT酶变体展现出增加的总体活性和热稳定性。其中最普遍的是：L82F、L82K、L82T、L82Y、L280I、T332V、V433K、V433N和I593W。
表27.使用Oligo-dT引发通过MMLV-RT单突变体的两步cDNA合成。经由qPCR人归一化测定产生数据，并且将数据通过Ct值报告。
/>
/>
表28.使用随机引发通过MMLV-RT单突变体的两步cDNA合成。经由qPCR人归一化测定产生数据，并且将数据通过Ct值报告。
/>
/>
/>
表29.通过基因特异性引发经由MMLV-RT单突变体的一步cDNA合成。经由qPCR人归一化测定产生数据，并且将数据通过Ct值报告。
/>
/>
表30.使用oligo dT引发通过MMLV-RT堆叠突变体的两步cDNA合成。经由qPCR人归一化测定产生数据，并且将数据通过Ct值报告。
/>
表31.使用随机引发通过MMLV-RT堆叠突变体的两步cDNA合成。经由qPCR人归一化测定产生数据，并且将数据通过Ct值报告。
/>
表32.通过基因特异性引发经由MMLV-RT堆叠突变体的一步cDNA合成。经由qPCR人归一化测定产生数据，并且将数据通过Ct值报告。
/>
/>
表33.四突变或更多突变MMLV RT酶变体的序列。
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/>
/>
/>
/>
文献目录：
1.Coffin et al.,"The discovery of reverse transcriptase,"Ann.Rev.Virol.3(1):29-51(2016).
2.Hogrefe et al.,"Mutant reverse transcriptase and methods of use,"U.S.Patent No.9,783,791.
3.Kotewicz et al.,"Cloned genes encoding reverse transcriptaselacking RNase H activity,"U.S.Patent No.5,405,776.
4.Kotewicz et al.,"Isolation of cloned Moloney murine leukemia virusreverse transcriptase lacking ribonuclease H activity,"Nucleic Acids Res.16(1):265-77(1988).
SEQUENCE LISTING
<110> 综合基因技术公司
<120> 具有增加的活性和热稳定性的逆转录酶突变体
<130> 20-1076-WO
<150> US 63/054,228
<151> 2020-07-20
<160> 699
<170> PatentIn version 3.5
<210> 1
<211> 69
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 1
ggtatatctc cttcttaaag ttaaacaaaa ttatttctag aggggaatga tccggctgct 60
aacaaagcc 69
<210> 2
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 2
gatccggctg ctaacaaagc c 21
<210> 3
<211> 52
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 3
ttttgtttaa ctttaagaag gagatatacc atgggcagca gccatcatca tc 52
<210> 4
<211> 59
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 4
gcagccaact cagcttcctt tcgggctttg ttaaaaatgc tcgctagtgt agggagagc 59
<210> 5
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 5
aagcaccgtt gatcatcccg ttagcggcaa cgtctacacc tgtctctatc aaac 54
<210> 6
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 6
aagcaccgtt gatcatcccg ttacgtgcaa cgtctacacc tgtctctatc aaac 54
<210> 7
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 7
aagcaccgtt gatcatcccg ttagaagcaa cgtctacacc tgtctctatc aaac 54
<210> 8
<211> 57
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 8
ccgttgatca tcccgttaaa ggcagcgtct acacctgtct ctatcaaaca gtacccc 57
<210> 9
<211> 57
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 9
ccgttgatca tcccgttaaa ggcacgttct acacctgtct ctatcaaaca gtacccc 57
<210> 10
<211> 57
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 10
ccgttgatca tcccgttaaa ggcagaatct acacctgtct ctatcaaaca gtacccc 57
<210> 11
<211> 56
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 11
atcatcccgt taaaggcaac gtctgcgcct gtctctatca aacagtaccc catgag 56
<210> 12
<211> 56
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 12
atcatcccgt taaaggcaac gtctcgtcct gtctctatca aacagtaccc catgag 56
<210> 13
<211> 56
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 13
atcatcccgt taaaggcaac gtctgaacct gtctctatca aacagtaccc catgag 56
<210> 14
<211> 58
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 14
ccgttaaagg caacgtctac acctgcgtct atcaaacagt accccatgag tcaagagg 58
<210> 15
<211> 58
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 15
ccgttaaagg caacgtctac acctcgttct atcaaacagt accccatgag tcaagagg 58
<210> 16
<211> 58
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 16
ccgttaaagg caacgtctac acctgaatct atcaaacagt accccatgag tcaagagg 58
<210> 17
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 17
taaaggcaac gtctacacct gtctctgcga aacagtaccc catgagtcaa gagg 54
<210> 18
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 18
taaaggcaac gtctacacct gtctctcgta aacagtaccc catgagtcaa gagg 54
<210> 19
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 19
taaaggcaac gtctacacct gtctctgaaa aacagtaccc catgagtcaa gagg 54
<210> 20
<211> 51
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 20
ggcaacgtct acacctgtct ctatcgcgca gtaccccatg agtcaagagg c 51
<210> 21
<211> 51
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 21
ggcaacgtct acacctgtct ctatccgtca gtaccccatg agtcaagagg c 51
<210> 22
<211> 51
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 22
ggcaacgtct acacctgtct ctatcgaaca gtaccccatg agtcaagagg c 51
<210> 23
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 23
ctgtctctat caaacagtac cccatgagtg cggaggcccg cctggg 46
<210> 24
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 24
ctgtctctat caaacagtac cccatgagtc gtgaggcccg cctggg 46
<210> 25
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 25
ctgtctctat caaacagtac cccatgagtg aagaggcccg cctggg 46
<210> 26
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 26
ggcccgcctg gggattgcgc cacatattca gcgcttgctg gacca 45
<210> 27
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 27
ggcccgcctg gggattcgtc cacatattca gcgcttgctg gacca 45
<210> 28
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 28
ggcccgcctg gggattgaac cacatattca gcgcttgctg gacca 45
<210> 29
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 29
cgcctgggga ttaagccaca tattgcgcgc ttgctggacc agggg 45
<210> 30
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 30
cgcctgggga ttaagccaca tattcgtcgc ttgctggacc agggg 45
<210> 31
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 31
cgcctgggga ttaagccaca tattgaacgc ttgctggacc agggg 45
<210> 32
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 32
ccgtggaaca ccccccttgc gcccgtgaaa aagccaggta caaac 45
<210> 33
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 33
ccgtggaaca ccccccttcg tcccgtgaaa aagccaggta caaac 45
<210> 34
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 34
ccgtggaaca ccccccttga acccgtgaaa aagccaggta caaac 45
<210> 35
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 35
cacccccctt ctgcccgcga aaaagccagg tacaaacgat tatcgtcc 48
<210> 36
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 36
cacccccctt ctgccccgta aaaagccagg tacaaacgat tatcgtcc 48
<210> 37
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 37
cacccccctt ctgcccgaaa aaaagccagg tacaaacgat tatcgtcc 48
<210> 38
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 38
cccccttctg cccgtggcga agccaggtac aaacgattat cgtcc 45
<210> 39
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 39
cccccttctg cccgtgcgta agccaggtac aaacgattat cgtcc 45
<210> 40
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 40
cccccttctg cccgtggaaa agccaggtac aaacgattat cgtcc 45
<210> 41
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 41
cccccttctg cccgtgaaag cgccaggtac aaacgattat cgtccagtt 49
<210> 42
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 42
cccccttctg cccgtgaaac gtccaggtac aaacgattat cgtccagtt 49
<210> 43
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 43
cccccttctg cccgtgaaag aaccaggtac aaacgattat cgtccagtt 49
<210> 44
<211> 51
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 44
gcccgtgaaa aagccaggtg cgaacgatta tcgtccagtt caagatcttc g 51
<210> 45
<211> 51
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 45
gcccgtgaaa aagccaggtc gtaacgatta tcgtccagtt caagatcttc g 51
<210> 46
<211> 51
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 46
gcccgtgaaa aagccaggtg aaaacgatta tcgtccagtt caagatcttc g 51
<210> 47
<211> 52
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 47
cccgtgaaaa agccaggtac agcggattat cgtccagttc aagatcttcg cg 52
<210> 48
<211> 52
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 48
cccgtgaaaa agccaggtac acgtgattat cgtccagttc aagatcttcg cg 52
<210> 49
<211> 52
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 49
cccgtgaaaa agccaggtac agaagattat cgtccagttc aagatcttcg cg 52
<210> 50
<211> 50
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 50
cgtgaaaaag ccaggtacaa acgatgcgcg tccagttcaa gatcttcgcg 50
<210> 51
<211> 50
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 51
cgtgaaaaag ccaggtacaa acgatcgtcg tccagttcaa gatcttcgcg 50
<210> 52
<211> 50
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 52
cgtgaaaaag ccaggtacaa acgatgaacg tccagttcaa gatcttcgcg 50
<210> 53
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 53
cgtgaaaaag ccaggtacaa acgattatgc gccagttcaa gatcttcgcg agg 53
<210> 54
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 54
cgtgaaaaag ccaggtacaa acgattataa accagttcaa gatcttcgcg agg 53
<210> 55
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 55
cgtgaaaaag ccaggtacaa acgattatga accagttcaa gatcttcgcg agg 53
<210> 56
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 56
gccaggtaca aacgattatc gtccagcgca agatcttcgc gaggtcaaca aac 53
<210> 57
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 57
gccaggtaca aacgattatc gtccacgtca agatcttcgc gaggtcaaca aac 53
<210> 58
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 58
gccaggtaca aacgattatc gtccagaaca agatcttcgc gaggtcaaca aac 53
<210> 59
<211> 51
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 59
agttcaagat cttcgcgagg tcaacgcgcg cgtagaagac atccatccga c 51
<210> 60
<211> 51
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 60
agttcaagat cttcgcgagg tcaaccgtcg cgtagaagac atccatccga c 51
<210> 61
<211> 51
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 61
agttcaagat cttcgcgagg tcaacgaacg cgtagaagac atccatccga c 51
<210> 62
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 62
gcgaggtcaa caaacgcgta gcggacatcc atccgactgt acctaatcc 49
<210> 63
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 63
gcgaggtcaa caaacgcgta cgtgacatcc atccgactgt acctaatcc 49
<210> 64
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 64
gcgaggtcaa caaacgcgta gatgacatcc atccgactgt acctaatcc 49
<210> 65
<211> 59
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 65
acgcgtagaa gacatccatc cggtggtacc taatccttat aatctgttat caggcctgc 59
<210> 66
<211> 59
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 66
acgcgtagaa gacatccatc cgcgtgtacc taatccttat aatctgttat caggcctgc 59
<210> 67
<211> 59
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 67
acgcgtagaa gacatccatc cggaagtacc taatccttat aatctgttat caggcctgc 59
<210> 68
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 68
cgtctgcccc agggctttgc gaacagcccc acattgttcg atgaa 45
<210> 69
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 69
cgtctgcccc agggctttcg taacagcccc acattgttcg atgaa 45
<210> 70
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 70
cgtctgcccc agggctttga aaacagcccc acattgttcg atgaa 45
<210> 71
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 71
agaaggtcaa cgttggctga ctgcggcgcg taaggagacc gtaatg 46
<210> 72
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 72
agaaggtcaa cgttggctga ctcgtgcgcg taaggagacc gtaatg 46
<210> 73
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 73
agaaggtcaa cgttggctga ctgatgcgcg taaggagacc gtaatg 46
<210> 74
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 74
gaaggtcaac gttggctgac tgaagtgcgt aaggagaccg taatggggc 49
<210> 75
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 75
gaaggtcaac gttggctgac tgaacgtcgt aaggagaccg taatggggc 49
<210> 76
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 76
gaaggtcaac gttggctgac tgaagaacgt aaggagaccg taatggggc 49
<210> 77
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 77
gcgtaaggag accgtaatgg gggcgcctac gcctaagacg ccacg 45
<210> 78
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 78
gcgtaaggag accgtaatgg ggcgtcctac gcctaagacg ccacg 45
<210> 79
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 79
gcgtaaggag accgtaatgg gggaacctac gcctaagacg ccacg 45
<210> 80
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 80
gagaccgtaa tggggcagcc tgcgcctaag acgccacgcc agttg 45
<210> 81
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 81
gagaccgtaa tggggcagcc tcgtcctaag acgccacgcc agttg 45
<210> 82
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 82
gagaccgtaa tggggcagcc tgaacctaag acgccacgcc agttg 45
<210> 83
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 83
gtaatggggc agcctacgcc tgcgacgcca cgccagttgc gtgaa 45
<210> 84
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 84
gtaatggggc agcctacgcc tcgtacgcca cgccagttgc gtgaa 45
<210> 85
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 85
gtaatggggc agcctacgcc tgaaacgcca cgccagttgc gtgaa 45
<210> 86
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 86
tggggcagcc tacgcctaag gcgccacgcc agttgcgtga atttt 45
<210> 87
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 87
tggggcagcc tacgcctaag cgtccacgcc agttgcgtga atttt 45
<210> 88
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 88
tggggcagcc tacgcctaag gaaccacgcc agttgcgtga atttt 45
<210> 89
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 89
gcctacgcct aagacgccag cgcagttgcg tgaatttttg ggcacag 47
<210> 90
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 90
gcctacgcct aagacgccaa aacagttgcg tgaatttttg ggcacag 47
<210> 91
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 91
gcctacgcct aagacgccag aacagttgcg tgaatttttg ggcacag 47
<210> 92
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 92
cctaagacgc cacgccagtt ggcggaattt ttgggcacag cggga 45
<210> 93
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 93
cctaagacgc cacgccagtt gaaagaattt ttgggcacag cggga 45
<210> 94
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 94
cctaagacgc cacgccagtt ggaagaattt ttgggcacag cggga 45
<210> 95
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 95
gcacccctgt accccttaac agcgacaggg acgcttttca actgg 45
<210> 96
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 96
gcacccctgt accccttaac acgtacaggg acgcttttca actgg 45
<210> 97
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 97
gcacccctgt accccttaac agaaacaggg acgcttttca actgg 45
<210> 98
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 98
gtttgataga gacaggtgta gacgttgccg ctaacgggat gatcaacggt gctt 54
<210> 99
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 99
gtttgataga gacaggtgta gacgttgcac gtaacgggat gatcaacggt gctt 54
<210> 100
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 100
gtttgataga gacaggtgta gacgttgctt ctaacgggat gatcaacggt gctt 54
<210> 101
<211> 57
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 101
ggggtactgt ttgatagaga caggtgtaga cgctgccttt aacgggatga tcaacgg 57
<210> 102
<211> 57
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 102
ggggtactgt ttgatagaga caggtgtaga acgtgccttt aacgggatga tcaacgg 57
<210> 103
<211> 57
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 103
ggggtactgt ttgatagaga caggtgtaga ttctgccttt aacgggatga tcaacgg 57
<210> 104
<211> 56
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 104
ctcatggggt actgtttgat agagacaggc gcagacgttg cctttaacgg gatgat 56
<210> 105
<211> 56
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 105
ctcatggggt actgtttgat agagacagga cgagacgttg cctttaacgg gatgat 56
<210> 106
<211> 56
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 106
ctcatggggt actgtttgat agagacaggt tcagacgttg cctttaacgg gatgat 56
<210> 107
<211> 58
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 107
cctcttgact catggggtac tgtttgatag acgcaggtgt agacgttgcc tttaacgg 58
<210> 108
<211> 58
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 108
cctcttgact catggggtac tgtttgatag aacgaggtgt agacgttgcc tttaacgg 58
<210> 109
<211> 58
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 109
cctcttgact catggggtac tgtttgatag attcaggtgt agacgttgcc tttaacgg 58
<210> 110
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 110
cctcttgact catggggtac tgtttcgcag agacaggtgt agacgttgcc ttta 54
<210> 111
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 111
cctcttgact catggggtac tgtttacgag agacaggtgt agacgttgcc ttta 54
<210> 112
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 112
cctcttgact catggggtac tgtttttcag agacaggtgt agacgttgcc ttta 54
<210> 113
<211> 51
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 113
gcctcttgac tcatggggta ctgcgcgata gagacaggtg tagacgttgc c 51
<210> 114
<211> 51
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 114
gcctcttgac tcatggggta ctgacggata gagacaggtg tagacgttgc c 51
<210> 115
<211> 51
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 115
gcctcttgac tcatggggta ctgttcgata gagacaggtg tagacgttgc c 51
<210> 116
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 116
ctgtctctat caaacagtac cccatgagtg cggaggcccg cctggg 46
<210> 117
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 117
ctgtctctat caaacagtac cccatgagtc gtgaggcccg cctggg 46
<210> 118
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 118
ctgtctctat caaacagtac cccatgagtg aagaggcccg cctggg 46
<210> 119
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 119
tggtccagca agcgctgaat atgtggcgca atccccaggc gggcc 45
<210> 120
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 120
tggtccagca agcgctgaat atgtggacga atccccaggc gggcc 45
<210> 121
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 121
tggtccagca agcgctgaat atgtggttca atccccaggc gggcc 45
<210> 122
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 122
cccctggtcc agcaagcgcg caatatgtgg cttaatcccc aggcg 45
<210> 123
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 123
cccctggtcc agcaagcgac gaatatgtgg cttaatcccc aggcg 45
<210> 124
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 124
cccctggtcc agcaagcgtt caatatgtgg cttaatcccc aggcg 45
<210> 125
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 125
gtttgtacct ggctttttca cgggcgcaag gggggtgttc cacgg 45
<210> 126
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 126
gtttgtacct ggctttttca cgggacgaag gggggtgttc cacgg 45
<210> 127
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 127
gtttgtacct ggctttttca cgggttcaag gggggtgttc cacgg 45
<210> 128
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 128
ggacgataat cgtttgtacc tggctttttc gcgggcagaa ggggggtg 48
<210> 129
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 129
ggacgataat cgtttgtacc tggcttttta cggggcagaa ggggggtg 48
<210> 130
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 130
ggacgataat cgtttgtacc tggctttttt tcgggcagaa ggggggtg 48
<210> 131
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 131
ggacgataat cgtttgtacc tggcttcgcc acgggcagaa ggggg 45
<210> 132
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 132
ggacgataat cgtttgtacc tggcttacgc acgggcagaa ggggg 45
<210> 133
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 133
ggacgataat cgtttgtacc tggcttttcc acgggcagaa ggggg 45
<210> 134
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 134
aactggacga taatcgtttg tacctggcgc tttcacgggc agaaggggg 49
<210> 135
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 135
aactggacga taatcgtttg tacctggacg tttcacgggc agaaggggg 49
<210> 136
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 136
aactggacga taatcgtttg tacctggttc tttcacgggc agaaggggg 49
<210> 137
<211> 51
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 137
cgaagatctt gaactggacg ataatcgttc gcacctggct ttttcacggg c 51
<210> 138
<211> 51
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 138
cgaagatctt gaactggacg ataatcgtta cgacctggct ttttcacggg c 51
<210> 139
<211> 51
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 139
cgaagatctt gaactggacg ataatcgttt tcacctggct ttttcacggg c 51
<210> 140
<211> 52
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 140
cgcgaagatc ttgaactgga cgataatccg ctgtacctgg ctttttcacg gg 52
<210> 141
<211> 52
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 141
cgcgaagatc ttgaactgga cgataatcac gtgtacctgg ctttttcacg gg 52
<210> 142
<211> 52
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 142
cgcgaagatc ttgaactgga cgataatctt ctgtacctgg ctttttcacg gg 52
<210> 143
<211> 50
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 143
cgcgaagatc ttgaactgga cgcgcatcgt ttgtacctgg ctttttcacg 50
<210> 144
<211> 50
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 144
cgcgaagatc ttgaactgga cgacgatcgt ttgtacctgg ctttttcacg 50
<210> 145
<211> 50
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 145
cgcgaagatc ttgaactgga cgttcatcgt ttgtacctgg ctttttcacg 50
<210> 146
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 146
cctcgcgaag atcttgaact ggcgcataat cgtttgtacc tggctttttc acg 53
<210> 147
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 147
cctcgcgaag atcttgaact ggtttataat cgtttgtacc tggctttttc acg 53
<210> 148
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 148
cctcgcgaag atcttgaact ggttcataat cgtttgtacc tggctttttc acg 53
<210> 149
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 149
gtttgttgac ctcgcgaaga tcttgcgctg gacgataatc gtttgtacct ggc 53
<210> 150
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 150
gtttgttgac ctcgcgaaga tcttgacgtg gacgataatc gtttgtacct ggc 53
<210> 151
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 151
gtttgttgac ctcgcgaaga tcttgttctg gacgataatc gtttgtacct ggc 53
<210> 152
<211> 51
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 152
gtcggatgga tgtcttctac gcgcgcgttg acctcgcgaa gatcttgaac t 51
<210> 153
<211> 51
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 153
gtcggatgga tgtcttctac gcgacggttg acctcgcgaa gatcttgaac t 51
<210> 154
<211> 51
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 154
gtcggatgga tgtcttctac gcgttcgttg acctcgcgaa gatcttgaac t 51
<210> 155
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 155
ggattaggta cagtcggatg gatgtccgct acgcgtttgt tgacctcgc 49
<210> 156
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 156
ggattaggta cagtcggatg gatgtcacgt acgcgtttgt tgacctcgc 49
<210> 157
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 157
ggattaggta cagtcggatg gatgtcatct acgcgtttgt tgacctcgc 49
<210> 158
<211> 59
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 158
gcaggcctga taacagatta taaggattag gtaccaccgg atggatgtct tctacgcgt 59
<210> 159
<211> 59
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 159
gcaggcctga taacagatta taaggattag gtacacgcgg atggatgtct tctacgcgt 59
<210> 160
<211> 59
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 160
gcaggcctga taacagatta taaggattag gtacttccgg atggatgtct tctacgcgt 59
<210> 161
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 161
ttcatcgaac aatgtggggc tgttcgcaaa gccctggggc agacg 45
<210> 162
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 162
ttcatcgaac aatgtggggc tgttacgaaa gccctggggc agacg 45
<210> 163
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 163
ttcatcgaac aatgtggggc tgttttcaaa gccctggggc agacg 45
<210> 164
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 164
cattacggtc tccttacgcg ccgcagtcag ccaacgttga ccttct 46
<210> 165
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 165
cattacggtc tccttacgcg cacgagtcag ccaacgttga ccttct 46
<210> 166
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 166
cattacggtc tccttacgcg catcagtcag ccaacgttga ccttct 46
<210> 167
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 167
gccccattac ggtctcctta cgcacttcag tcagccaacg ttgaccttc 49
<210> 168
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 168
gccccattac ggtctcctta cgacgttcag tcagccaacg ttgaccttc 49
<210> 169
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 169
gccccattac ggtctcctta cgttcttcag tcagccaacg ttgaccttc 49
<210> 170
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 170
cgtggcgtct taggcgtagg cgcccccatt acggtctcct tacgc 45
<210> 171
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 171
cgtggcgtct taggcgtagg acgccccatt acggtctcct tacgc 45
<210> 172
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 172
cgtggcgtct taggcgtagg ttcccccatt acggtctcct tacgc 45
<210> 173
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 173
caactggcgt ggcgtcttag gcgcaggctg ccccattacg gtctc 45
<210> 174
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 174
caactggcgt ggcgtcttag gacgaggctg ccccattacg gtctc 45
<210> 175
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 175
caactggcgt ggcgtcttag gttcaggctg ccccattacg gtctc 45
<210> 176
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 176
ttcacgcaac tggcgtggcg tcgcaggcgt aggctgcccc attac 45
<210> 177
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 177
ttcacgcaac tggcgtggcg tacgaggcgt aggctgcccc attac 45
<210> 178
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 178
ttcacgcaac tggcgtggcg tttcaggcgt aggctgcccc attac 45
<210> 179
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 179
aaaattcacg caactggcgt ggcgccttag gcgtaggctg cccca 45
<210> 180
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 180
aaaattcacg caactggcgt ggacgcttag gcgtaggctg cccca 45
<210> 181
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 181
aaaattcacg caactggcgt ggttccttag gcgtaggctg cccca 45
<210> 182
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 182
ctgtgcccaa aaattcacgc aactgcgctg gcgtcttagg cgtaggc 47
<210> 183
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 183
ctgtgcccaa aaattcacgc aactgttttg gcgtcttagg cgtaggc 47
<210> 184
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 184
ctgtgcccaa aaattcacgc aactgttctg gcgtcttagg cgtaggc 47
<210> 185
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 185
tcccgctgtg cccaaaaatt ccgccaactg gcgtggcgtc ttagg 45
<210> 186
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 186
tcccgctgtg cccaaaaatt ctttcaactg gcgtggcgtc ttagg 45
<210> 187
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 187
tcccgctgtg cccaaaaatt cttccaactg gcgtggcgtc ttagg 45
<210> 188
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 188
ccagttgaaa agcgtccctg tcgctgttaa ggggtacagg ggtgc 45
<210> 189
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 189
ccagttgaaa agcgtccctg tacgtgttaa ggggtacagg ggtgc 45
<210> 190
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 190
ccagttgaaa agcgtccctg tttctgttaa ggggtacagg ggtgc 45
<210> 191
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 191
taaaggcaac gtctacacct gtctctggca aacagtaccc catgagtcaa gagg 54
<210> 192
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 192
cctcttgact catggggtac tgtttgccag agacaggtgt agacgttgcc ttta 54
<210> 193
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 193
taaaggcaac gtctacacct gtctctctga aacagtaccc catgagtcaa gagg 54
<210> 194
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 194
cctcttgact catggggtac tgtttcagag agacaggtgt agacgttgcc ttta 54
<210> 195
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 195
taaaggcaac gtctacacct gtctctgtga aacagtaccc catgagtcaa gagg 54
<210> 196
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 196
cctcttgact catggggtac tgtttcacag agacaggtgt agacgttgcc ttta 54
<210> 197
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 197
taaaggcaac gtctacacct gtctctccga aacagtaccc catgagtcaa gagg 54
<210> 198
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 198
cctcttgact catggggtac tgtttcggag agacaggtgt agacgttgcc ttta 54
<210> 199
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 199
taaaggcaac gtctacacct gtctctatga aacagtaccc catgagtcaa gagg 54
<210> 200
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 200
cctcttgact catggggtac tgtttcatag agacaggtgt agacgttgcc ttta 54
<210> 201
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 201
taaaggcaac gtctacacct gtctctagca aacagtaccc catgagtcaa gagg 54
<210> 202
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 202
cctcttgact catggggtac tgtttgctag agacaggtgt agacgttgcc ttta 54
<210> 203
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 203
taaaggcaac gtctacacct gtctctacca aacagtaccc catgagtcaa gagg 54
<210> 204
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 204
cctcttgact catggggtac tgtttggtag agacaggtgt agacgttgcc ttta 54
<210> 205
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 205
taaaggcaac gtctacacct gtctcttgca aacagtaccc catgagtcaa gagg 54
<210> 206
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 206
cctcttgact catggggtac tgtttgcaag agacaggtgt agacgttgcc ttta 54
<210> 207
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 207
taaaggcaac gtctacacct gtctctttta aacagtaccc catgagtcaa gagg 54
<210> 208
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 208
cctcttgact catggggtac tgtttaaaag agacaggtgt agacgttgcc ttta 54
<210> 209
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 209
taaaggcaac gtctacacct gtctcttata aacagtaccc catgagtcaa gagg 54
<210> 210
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 210
cctcttgact catggggtac tgtttataag agacaggtgt agacgttgcc ttta 54
<210> 211
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 211
taaaggcaac gtctacacct gtctctcata aacagtaccc catgagtcaa gagg 54
<210> 212
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 212
cctcttgact catggggtac tgtttatgag agacaggtgt agacgttgcc ttta 54
<210> 213
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 213
taaaggcaac gtctacacct gtctcttgga aacagtaccc catgagtcaa gagg 54
<210> 214
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 214
cctcttgact catggggtac tgtttccaag agacaggtgt agacgttgcc ttta 54
<210> 215
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 215
taaaggcaac gtctacacct gtctctgata aacagtaccc catgagtcaa gagg 54
<210> 216
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 216
cctcttgact catggggtac tgtttatcag agacaggtgt agacgttgcc ttta 54
<210> 217
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 217
taaaggcaac gtctacacct gtctctaaca aacagtaccc catgagtcaa gagg 54
<210> 218
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 218
cctcttgact catggggtac tgtttgttag agacaggtgt agacgttgcc ttta 54
<210> 219
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 219
taaaggcaac gtctacacct gtctctcaga aacagtaccc catgagtcaa gagg 54
<210> 220
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 220
cctcttgact catggggtac tgtttctgag agacaggtgt agacgttgcc ttta 54
<210> 221
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 221
taaaggcaac gtctacacct gtctctaaaa aacagtaccc catgagtcaa gagg 54
<210> 222
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 222
cctcttgact catggggtac tgttttttag agacaggtgt agacgttgcc ttta 54
<210> 223
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 223
ctgtctctat caaacagtac cccatgagtg gcgaggcccg cctggg 46
<210> 224
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 224
cccaggcggg cctcgccact catggggtac tgtttgatag agacag 46
<210> 225
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 225
ctgtctctat caaacagtac cccatgagtc tggaggcccg cctggg 46
<210> 226
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 226
cccaggcggg cctccagact catggggtac tgtttgatag agacag 46
<210> 227
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 227
ctgtctctat caaacagtac cccatgagta ttgaggcccg cctggg 46
<210> 228
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 228
cccaggcggg cctcaatact catggggtac tgtttgatag agacag 46
<210> 229
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 229
ctgtctctat caaacagtac cccatgagtg tggaggcccg cctggg 46
<210> 230
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 230
cccaggcggg cctccacact catggggtac tgtttgatag agacag 46
<210> 231
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 231
ctgtctctat caaacagtac cccatgagtc cggaggcccg cctggg 46
<210> 232
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 232
cccaggcggg cctccggact catggggtac tgtttgatag agacag 46
<210> 233
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 233
ctgtctctat caaacagtac cccatgagta tggaggcccg cctggg 46
<210> 234
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 234
cccaggcggg cctccatact catggggtac tgtttgatag agacag 46
<210> 235
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 235
ctgtctctat caaacagtac cccatgagta gcgaggcccg cctggg 46
<210> 236
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 236
cccaggcggg cctcgctact catggggtac tgtttgatag agacag 46
<210> 237
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 237
ctgtctctat caaacagtac cccatgagta ccgaggcccg cctggg 46
<210> 238
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 238
cccaggcggg cctcggtact catggggtac tgtttgatag agacag 46
<210> 239
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 239
ctgtctctat caaacagtac cccatgagtt gcgaggcccg cctggg 46
<210> 240
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 240
cccaggcggg cctcgcaact catggggtac tgtttgatag agacag 46
<210> 241
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 241
ctgtctctat caaacagtac cccatgagtt ttgaggcccg cctggg 46
<210> 242
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 242
cccaggcggg cctcaaaact catggggtac tgtttgatag agacag 46
<210> 243
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 243
ctgtctctat caaacagtac cccatgagtt atgaggcccg cctggg 46
<210> 244
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 244
cccaggcggg cctcataact catggggtac tgtttgatag agacag 46
<210> 245
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 245
ctgtctctat caaacagtac cccatgagtc atgaggcccg cctggg 46
<210> 246
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 246
cccaggcggg cctcatgact catggggtac tgtttgatag agacag 46
<210> 247
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 247
ctgtctctat caaacagtac cccatgagtt gggaggcccg cctggg 46
<210> 248
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 248
cccaggcggg cctcccaact catggggtac tgtttgatag agacag 46
<210> 249
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 249
ctgtctctat caaacagtac cccatgagtg atgaggcccg cctggg 46
<210> 250
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 250
cccaggcggg cctcatcact catggggtac tgtttgatag agacag 46
<210> 251
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 251
ctgtctctat caaacagtac cccatgagta acgaggcccg cctggg 46
<210> 252
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 252
cccaggcggg cctcgttact catggggtac tgtttgatag agacag 46
<210> 253
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 253
ctgtctctat caaacagtac cccatgagta aagaggcccg cctggg 46
<210> 254
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 254
cccaggcggg cctctttact catggggtac tgtttgatag agacag 46
<210> 255
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 255
cgcctgggga ttaagccaca tattggccgc ttgctggacc agggg 45
<210> 256
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 256
cccctggtcc agcaagcggc caatatgtgg cttaatcccc aggcg 45
<210> 257
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 257
cgcctgggga ttaagccaca tattctgcgc ttgctggacc agggg 45
<210> 258
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 258
cccctggtcc agcaagcgca gaatatgtgg cttaatcccc aggcg 45
<210> 259
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 259
cgcctgggga ttaagccaca tattattcgc ttgctggacc agggg 45
<210> 260
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 260
cccctggtcc agcaagcgaa taatatgtgg cttaatcccc aggcg 45
<210> 261
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 261
cgcctgggga ttaagccaca tattgtgcgc ttgctggacc agggg 45
<210> 262
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 262
cccctggtcc agcaagcgca caatatgtgg cttaatcccc aggcg 45
<210> 263
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 263
cgcctgggga ttaagccaca tattccgcgc ttgctggacc agggg 45
<210> 264
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 264
cccctggtcc agcaagcgcg gaatatgtgg cttaatcccc aggcg 45
<210> 265
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 265
cgcctgggga ttaagccaca tattatgcgc ttgctggacc agggg 45
<210> 266
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 266
cccctggtcc agcaagcgca taatatgtgg cttaatcccc aggcg 45
<210> 267
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 267
cgcctgggga ttaagccaca tattagccgc ttgctggacc agggg 45
<210> 268
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 268
cccctggtcc agcaagcggc taatatgtgg cttaatcccc aggcg 45
<210> 269
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 269
cgcctgggga ttaagccaca tattacccgc ttgctggacc agggg 45
<210> 270
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 270
cccctggtcc agcaagcggg taatatgtgg cttaatcccc aggcg 45
<210> 271
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 271
cgcctgggga ttaagccaca tatttgccgc ttgctggacc agggg 45
<210> 272
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 272
cccctggtcc agcaagcggc aaatatgtgg cttaatcccc aggcg 45
<210> 273
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 273
cgcctgggga ttaagccaca tatttttcgc ttgctggacc agggg 45
<210> 274
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 274
cccctggtcc agcaagcgaa aaatatgtgg cttaatcccc aggcg 45
<210> 275
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 275
cgcctgggga ttaagccaca tatttatcgc ttgctggacc agggg 45
<210> 276
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 276
cccctggtcc agcaagcgat aaatatgtgg cttaatcccc aggcg 45
<210> 277
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 277
cgcctgggga ttaagccaca tattcatcgc ttgctggacc agggg 45
<210> 278
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 278
cccctggtcc agcaagcgat gaatatgtgg cttaatcccc aggcg 45
<210> 279
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 279
cgcctgggga ttaagccaca tatttggcgc ttgctggacc agggg 45
<210> 280
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 280
cccctggtcc agcaagcgcc aaatatgtgg cttaatcccc aggcg 45
<210> 281
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 281
cgcctgggga ttaagccaca tattgatcgc ttgctggacc agggg 45
<210> 282
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 282
cccctggtcc agcaagcgat caatatgtgg cttaatcccc aggcg 45
<210> 283
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 283
cgcctgggga ttaagccaca tattaaccgc ttgctggacc agggg 45
<210> 284
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 284
cccctggtcc agcaagcggt taatatgtgg cttaatcccc aggcg 45
<210> 285
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 285
cgcctgggga ttaagccaca tattaaacgc ttgctggacc agggg 45
<210> 286
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 286
cccctggtcc agcaagcgtt taatatgtgg cttaatcccc aggcg 45
<210> 287
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 287
ccgtggaaca ccccccttgg ccccgtgaaa aagccaggta caaac 45
<210> 288
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 288
gtttgtacct ggctttttca cggggccaag gggggtgttc cacgg 45
<210> 289
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 289
ccgtggaaca ccccccttat tcccgtgaaa aagccaggta caaac 45
<210> 290
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 290
gtttgtacct ggctttttca cgggaataag gggggtgttc cacgg 45
<210> 291
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 291
ccgtggaaca ccccccttgt gcccgtgaaa aagccaggta caaac 45
<210> 292
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 292
gtttgtacct ggctttttca cgggcacaag gggggtgttc cacgg 45
<210> 293
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 293
ccgtggaaca ccccccttcc gcccgtgaaa aagccaggta caaac 45
<210> 294
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 294
gtttgtacct ggctttttca cgggcggaag gggggtgttc cacgg 45
<210> 295
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 295
ccgtggaaca ccccccttat gcccgtgaaa aagccaggta caaac 45
<210> 296
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 296
gtttgtacct ggctttttca cgggcataag gggggtgttc cacgg 45
<210> 297
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 297
ccgtggaaca ccccccttag ccccgtgaaa aagccaggta caaac 45
<210> 298
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 298
gtttgtacct ggctttttca cggggctaag gggggtgttc cacgg 45
<210> 299
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 299
ccgtggaaca ccccccttac ccccgtgaaa aagccaggta caaac 45
<210> 300
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 300
gtttgtacct ggctttttca cgggggtaag gggggtgttc cacgg 45
<210> 301
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 301
ccgtggaaca cccccctttg ccccgtgaaa aagccaggta caaac 45
<210> 302
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 302
gtttgtacct ggctttttca cggggcaaag gggggtgttc cacgg 45
<210> 303
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 303
ccgtggaaca cccccctttt tcccgtgaaa aagccaggta caaac 45
<210> 304
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 304
gtttgtacct ggctttttca cgggaaaaag gggggtgttc cacgg 45
<210> 305
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 305
ccgtggaaca ccccccttta tcccgtgaaa aagccaggta caaac 45
<210> 306
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 306
gtttgtacct ggctttttca cgggataaag gggggtgttc cacgg 45
<210> 307
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 307
ccgtggaaca ccccccttca tcccgtgaaa aagccaggta caaac 45
<210> 308
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 308
gtttgtacct ggctttttca cgggatgaag gggggtgttc cacgg 45
<210> 309
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 309
ccgtggaaca cccccctttg gcccgtgaaa aagccaggta caaac 45
<210> 310
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 310
gtttgtacct ggctttttca cgggccaaag gggggtgttc cacgg 45
<210> 311
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 311
ccgtggaaca ccccccttga tcccgtgaaa aagccaggta caaac 45
<210> 312
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 312
gtttgtacct ggctttttca cgggatcaag gggggtgttc cacgg 45
<210> 313
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 313
ccgtggaaca ccccccttaa ccccgtgaaa aagccaggta caaac 45
<210> 314
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 314
gtttgtacct ggctttttca cggggttaag gggggtgttc cacgg 45
<210> 315
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 315
ccgtggaaca ccccccttca gcccgtgaaa aagccaggta caaac 45
<210> 316
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 316
gtttgtacct ggctttttca cgggctgaag gggggtgttc cacgg 45
<210> 317
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 317
ccgtggaaca ccccccttaa acccgtgaaa aagccaggta caaac 45
<210> 318
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 318
gtttgtacct ggctttttca cgggtttaag gggggtgttc cacgg 45
<210> 319
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 319
agaaggtcaa cgttggctga ctggcgcgcg taaggagacc gtaatg 46
<210> 320
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 320
cattacggtc tccttacgcg cgccagtcag ccaacgttga ccttct 46
<210> 321
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 321
agaaggtcaa cgttggctga ctctggcgcg taaggagacc gtaatg 46
<210> 322
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 322
cattacggtc tccttacgcg ccagagtcag ccaacgttga ccttct 46
<210> 323
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 323
agaaggtcaa cgttggctga ctattgcgcg taaggagacc gtaatg 46
<210> 324
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 324
cattacggtc tccttacgcg caatagtcag ccaacgttga ccttct 46
<210> 325
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 325
agaaggtcaa cgttggctga ctgtggcgcg taaggagacc gtaatg 46
<210> 326
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 326
cattacggtc tccttacgcg ccacagtcag ccaacgttga ccttct 46
<210> 327
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 327
agaaggtcaa cgttggctga ctccggcgcg taaggagacc gtaatg 46
<210> 328
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 328
cattacggtc tccttacgcg ccggagtcag ccaacgttga ccttct 46
<210> 329
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 329
agaaggtcaa cgttggctga ctatggcgcg taaggagacc gtaatg 46
<210> 330
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 330
cattacggtc tccttacgcg ccatagtcag ccaacgttga ccttct 46
<210> 331
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 331
agaaggtcaa cgttggctga ctagcgcgcg taaggagacc gtaatg 46
<210> 332
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 332
cattacggtc tccttacgcg cgctagtcag ccaacgttga ccttct 46
<210> 333
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 333
agaaggtcaa cgttggctga ctaccgcgcg taaggagacc gtaatg 46
<210> 334
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 334
cattacggtc tccttacgcg cggtagtcag ccaacgttga ccttct 46
<210> 335
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 335
agaaggtcaa cgttggctga cttgcgcgcg taaggagacc gtaatg 46
<210> 336
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 336
cattacggtc tccttacgcg cgcaagtcag ccaacgttga ccttct 46
<210> 337
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 337
agaaggtcaa cgttggctga cttttgcgcg taaggagacc gtaatg 46
<210> 338
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 338
cattacggtc tccttacgcg caaaagtcag ccaacgttga ccttct 46
<210> 339
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 339
agaaggtcaa cgttggctga cttatgcgcg taaggagacc gtaatg 46
<210> 340
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 340
cattacggtc tccttacgcg cataagtcag ccaacgttga ccttct 46
<210> 341
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 341
agaaggtcaa cgttggctga ctcatgcgcg taaggagacc gtaatg 46
<210> 342
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 342
cattacggtc tccttacgcg catgagtcag ccaacgttga ccttct 46
<210> 343
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 343
agaaggtcaa cgttggctga cttgggcgcg taaggagacc gtaatg 46
<210> 344
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 344
cattacggtc tccttacgcg cccaagtcag ccaacgttga ccttct 46
<210> 345
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 345
agaaggtcaa cgttggctga ctaacgcgcg taaggagacc gtaatg 46
<210> 346
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 346
cattacggtc tccttacgcg cgttagtcag ccaacgttga ccttct 46
<210> 347
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 347
agaaggtcaa cgttggctga ctcaggcgcg taaggagacc gtaatg 46
<210> 348
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 348
cattacggtc tccttacgcg cctgagtcag ccaacgttga ccttct 46
<210> 349
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 349
agaaggtcaa cgttggctga ctaaagcgcg taaggagacc gtaatg 46
<210> 350
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 350
cattacggtc tccttacgcg ctttagtcag ccaacgttga ccttct 46
<210> 351
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 351
gcctacgcct aagacgccag gccagttgcg tgaatttttg ggcacag 47
<210> 352
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 352
ctgtgcccaa aaattcacgc aactggcctg gcgtcttagg cgtaggc 47
<210> 353
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 353
gcctacgcct aagacgccac tgcagttgcg tgaatttttg ggcacag 47
<210> 354
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 354
ctgtgcccaa aaattcacgc aactgcagtg gcgtcttagg cgtaggc 47
<210> 355
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 355
gcctacgcct aagacgccaa ttcagttgcg tgaatttttg ggcacag 47
<210> 356
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 356
ctgtgcccaa aaattcacgc aactgaattg gcgtcttagg cgtaggc 47
<210> 357
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 357
gcctacgcct aagacgccag tgcagttgcg tgaatttttg ggcacag 47
<210> 358
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 358
ctgtgcccaa aaattcacgc aactgcactg gcgtcttagg cgtaggc 47
<210> 359
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 359
gcctacgcct aagacgccac cgcagttgcg tgaatttttg ggcacag 47
<210> 360
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 360
ctgtgcccaa aaattcacgc aactgcggtg gcgtcttagg cgtaggc 47
<210> 361
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 361
gcctacgcct aagacgccaa tgcagttgcg tgaatttttg ggcacag 47
<210> 362
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 362
ctgtgcccaa aaattcacgc aactgcattg gcgtcttagg cgtaggc 47
<210> 363
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 363
gcctacgcct aagacgccaa gccagttgcg tgaatttttg ggcacag 47
<210> 364
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 364
ctgtgcccaa aaattcacgc aactggcttg gcgtcttagg cgtaggc 47
<210> 365
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 365
gcctacgcct aagacgccaa cccagttgcg tgaatttttg ggcacag 47
<210> 366
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 366
ctgtgcccaa aaattcacgc aactgggttg gcgtcttagg cgtaggc 47
<210> 367
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 367
gcctacgcct aagacgccat gccagttgcg tgaatttttg ggcacag 47
<210> 368
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 368
ctgtgcccaa aaattcacgc aactggcatg gcgtcttagg cgtaggc 47
<210> 369
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 369
gcctacgcct aagacgccat ttcagttgcg tgaatttttg ggcacag 47
<210> 370
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 370
ctgtgcccaa aaattcacgc aactgaaatg gcgtcttagg cgtaggc 47
<210> 371
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 371
gcctacgcct aagacgccat atcagttgcg tgaatttttg ggcacag 47
<210> 372
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 372
ctgtgcccaa aaattcacgc aactgatatg gcgtcttagg cgtaggc 47
<210> 373
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 373
gcctacgcct aagacgccac atcagttgcg tgaatttttg ggcacag 47
<210> 374
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 374
ctgtgcccaa aaattcacgc aactgatgtg gcgtcttagg cgtaggc 47
<210> 375
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 375
gcctacgcct aagacgccat ggcagttgcg tgaatttttg ggcacag 47
<210> 376
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 376
ctgtgcccaa aaattcacgc aactgccatg gcgtcttagg cgtaggc 47
<210> 377
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 377
gcctacgcct aagacgccag atcagttgcg tgaatttttg ggcacag 47
<210> 378
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 378
ctgtgcccaa aaattcacgc aactgatctg gcgtcttagg cgtaggc 47
<210> 379
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 379
gcctacgcct aagacgccaa accagttgcg tgaatttttg ggcacag 47
<210> 380
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 380
ctgtgcccaa aaattcacgc aactggtttg gcgtcttagg cgtaggc 47
<210> 381
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 381
gcctacgcct aagacgccac agcagttgcg tgaatttttg ggcacag 47
<210> 382
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 382
ctgtgcccaa aaattcacgc aactgctgtg gcgtcttagg cgtaggc 47
<210> 383
<211> 62
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 383
aggcaacgtc tacacctgtc tctcgtaaac agtaccccat gagtcgtgag gcccgcctgg 60
gg 62
<210> 384
<211> 62
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 384
ccccaggcgg gcctcacgac tcatggggta ctgtttacga gagacaggtg tagacgttgc 60
ct 62
<210> 385
<211> 51
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 385
aggcaacgtc tacacctgtc tctaaaaaac agtaccccat gagtcgtgag g 51
<210> 386
<211> 51
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 386
cctcacgact catggggtac tgttttttag agacaggtgt agacgttgcc t 51
<210> 387
<211> 51
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 387
aggcaacgtc tacacctgtc tctatgaaac agtaccccat gagtcgtgag g 51
<210> 388
<211> 51
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 388
cctcacgact catggggtac tgtttcatag agacaggtgt agacgttgcc t 51
<210> 389
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 389
aggcaacgtc tacacctgtc tctatgaaac agtaccccat gagtattgag gcc 53
<210> 390
<400> 390
000
<210> 391
<400> 391
000
<210> 392
<400> 392
000
<210> 393
<211> 44
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 393
gtctctatca aacagtaccc catggcgcaa gaggcccgcc tggg 44
<210> 394
<211> 44
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 394
gtctctatca aacagtaccc catgcgtcaa gaggcccgcc tggg 44
<210> 395
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 395
catgagtcaa gaggcccgcg aggggattaa gccacatatt cagcg 45
<210> 396
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 396
gagtcaagag gcccgcctgg cgattaagcc acatattcag cgcttgc 47
<210> 397
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 397
gagtcaagag gcccgcctgc gtattaagcc acatattcag cgcttgc 47
<210> 398
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 398
gagtcaagag gcccgcctgg agattaagcc acatattcag cgcttgc 47
<210> 399
<211> 44
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 399
ggcccgcctg gggattaagg cgcatattca gcgcttgctg gacc 44
<210> 400
<211> 44
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 400
ggcccgcctg gggattaagc gtcatattca gcgcttgctg gacc 44
<210> 401
<211> 44
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 401
ggcccgcctg gggattaagg agcatattca gcgcttgctg gacc 44
<210> 402
<211> 43
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 402
ccgcctgggg attaagccag cgattcagcg cttgctggac cag 43
<210> 403
<211> 43
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 403
ccgcctgggg attaagccac gtattcagcg cttgctggac cag 43
<210> 404
<211> 43
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 404
ccgcctgggg attaagccag agattcagcg cttgctggac cag 43
<210> 405
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 405
gattaagcca catattcagc gcttggcgga ccaggggatc ttggtcc 47
<210> 406
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 406
gattaagcca catattcagc gcttgcgtga ccaggggatc ttggtcc 47
<210> 407
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 407
gattaagcca catattcagc gcttggagga ccaggggatc ttggtcc 47
<210> 408
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 408
gccacatatt cagcgcttgc tggcgcaggg gatcttggtc ccatg 45
<210> 409
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 409
gccacatatt cagcgcttgc tgcgtcaggg gatcttggtc ccatg 45
<210> 410
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 410
gccacatatt cagcgcttgc tggagcaggg gatcttggtc ccatg 45
<210> 411
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 411
aggtcaacaa acgcgtagaa gacgcgcatc cgactgtacc taatccttat aat 53
<210> 412
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 412
aggtcaacaa acgcgtagaa gaccgtcatc cgactgtacc taatccttat aat 53
<210> 413
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 413
aggtcaacaa acgcgtagaa gacgagcatc cgactgtacc taatccttat aat 53
<210> 414
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 414
gcgtagaaga catccatccg actgcgccta atccttataa tctgttatca ggc 53
<210> 415
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 415
gcgtagaaga catccatccg actcgtccta atccttataa tctgttatca ggc 53
<210> 416
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 416
gcgtagaaga catccatccg actgagccta atccttataa tctgttatca ggc 53
<210> 417
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 417
agggctttaa aaacagcccc acagcgttcg atgaagcact tcaccgtga 49
<210> 418
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 418
agggctttaa aaacagcccc acacgtttcg atgaagcact tcaccgtga 49
<210> 419
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 419
agggctttaa aaacagcccc acagagttcg atgaagcact tcaccgtga 49
<210> 420
<211> 52
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 420
tttaaaaaca gccccacatt gttcgatgcg gcacttcacc gtgacttagc ag 52
<210> 421
<211> 52
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 421
tttaaaaaca gccccacatt gttcgatcgt gcacttcacc gtgacttagc ag 52
<210> 422
<211> 52
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 422
tttaaaaaca gccccacatt gttcgatgat gcacttcacc gtgacttagc ag 52
<210> 423
<211> 51
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 423
cacattgttc gatgaagcac ttcacgcgga cttagcagac ttccgtatcc a 51
<210> 424
<211> 51
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 424
cacattgttc gatgaagcac ttcacaaaga cttagcagac ttccgtatcc a 51
<210> 425
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 425
gatgaagcac ttcaccgtga cttagaggac ttccgtatcc aacacccag 49
<210> 426
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 426
aagcacttca ccgtgactta gcagcgttcc gtatccaaca cccagactt 49
<210> 427
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 427
aagcacttca ccgtgactta gcacgtttcc gtatccaaca cccagactt 49
<210> 428
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 428
aagcacttca ccgtgactta gcagagttcc gtatccaaca cccagactt 49
<210> 429
<211> 51
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 429
cacttcaccg tgacttagca gacgcgcgta tccaacaccc agacttaatt c 51
<210> 430
<211> 51
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 430
cacttcaccg tgacttagca gaccgtcgta tccaacaccc agacttaatt c 51
<210> 431
<211> 51
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 431
cacttcaccg tgacttagca gacgagcgta tccaacaccc agacttaatt c 51
<210> 432
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 432
ttcaccgtga cttagcagac ttcgcgatcc aacacccaga cttaattctg tta 53
<210> 433
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 433
ttcaccgtga cttagcagac ttcaaaatcc aacacccaga cttaattctg tta 53
<210> 434
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 434
ttcaccgtga cttagcagac ttcgagatcc aacacccaga cttaattctg tta 53
<210> 435
<211> 52
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 435
ccgtgactta gcagacttcc gtgcgcaaca cccagactta attctgttac ag 52
<210> 436
<211> 52
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 436
ccgtgactta gcagacttcc gtcgtcaaca cccagactta attctgttac ag 52
<210> 437
<211> 52
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 437
ccgtgactta gcagacttcc gtgagcaaca cccagactta attctgttac ag 52
<210> 438
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 438
gtgacttagc agacttccgt atcgcgcacc cagacttaat tctgttacag tat 53
<210> 439
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 439
gtgacttagc agacttccgt atccgtcacc cagacttaat tctgttacag tat 53
<210> 440
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 440
gtgacttagc agacttccgt atcgagcacc cagacttaat tctgttacag tat 53
<210> 441
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 441
agcaaaaggc gtatcaggag atcgcgcaag ctttgttgac cgcaccc 47
<210> 442
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 442
agcaaaaggc gtatcaggag atccgtcaag ctttgttgac cgcaccc 47
<210> 443
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 443
agcaaaaggc gtatcaggag atcgagcaag ctttgttgac cgcaccc 47
<210> 444
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 444
cgtatcagga gatcaaacaa gctttggcga ccgcacccgc gttggg 46
<210> 445
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 445
cgtatcagga gatcaaacaa gctttgcgta ccgcacccgc gttggg 46
<210> 446
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 446
cgtatcagga gatcaaacaa gctttggaga ccgcacccgc gttggg 46
<210> 447
<211> 42
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 447
gttggctgac tgaagcgcgt gcggagaccg taatggggca gc 42
<210> 448
<211> 42
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 448
gttggctgac tgaagcgcgt cgtgagaccg taatggggca gc 42
<210> 449
<211> 42
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 449
gttggctgac tgaagcgcgt gaggagaccg taatggggca gc 42
<210> 450
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 450
tacgcctaag acgccacgcg cgttgcgtga atttttgggc acagc 45
<210> 451
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 451
tacgcctaag acgccacgcc gtttgcgtga atttttgggc acagc 45
<210> 452
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 452
tacgcctaag acgccacgcg agttgcgtga atttttgggc acagc 45
<210> 453
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 453
gcgtgaattt ttgggcacag cggcgttctg tcgtttatgg attcctggg 49
<210> 454
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 454
gcgtgaattt ttgggcacag cgcgtttctg tcgtttatgg attcctggg 49
<210> 455
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 455
gcgtgaattt ttgggcacag cggagttctg tcgtttatgg attcctggg 49
<210> 456
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 456
gggcacagcg ggattctgtg cgttatggat tcctgggttc gctga 45
<210> 457
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 457
gggcacagcg ggattctgta aattatggat tcctgggttc gctga 45
<210> 458
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 458
gggcacagcg ggattctgtg agttatggat tcctgggttc gctga 45
<210> 459
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 459
gtcaaaaaca ggtaaagtac cttggggcgt tgctgaaaga aggtcaacgt tgg 53
<210> 460
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 460
gtcaaaaaca ggtaaagtac cttgggcgtt tgctgaaaga aggtcaacgt tgg 53
<210> 461
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 461
gtcaaaaaca ggtaaagtac cttggggagt tgctgaaaga aggtcaacgt tgg 53
<210> 462
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 462
tgctgaaaga aggtcaacgt tgggcgactg aagcgcgtaa ggagacc 47
<210> 463
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 463
tgctgaaaga aggtcaacgt tggcgtactg aagcgcgtaa ggagacc 47
<210> 464
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 464
tgctgaaaga aggtcaacgt tgggagactg aagcgcgtaa ggagacc 47
<210> 465
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 465
tttgttgacc gcacccgcgg cgggtcttcc ggatttaacc aagcc 45
<210> 466
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 466
tttgttgacc gcacccgcgc gtggtcttcc ggatttaacc aagcc 45
<210> 467
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 467
tttgttgacc gcacccgcgg agggtcttcc ggatttaacc aagcc 45
<210> 468
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 468
ctgcacccct gtacccctta gcgaaaacag ggacgctttt caactgg 47
<210> 469
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 469
ctgcacccct gtacccctta cgtaaaacag ggacgctttt caactgg 47
<210> 470
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 470
ctgcacccct gtacccctta gagaaaacag ggacgctttt caactgg 47
<210> 471
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 471
cccctgtacc ccttaacaaa aacagcgacg cttttcaact gggggcc 47
<210> 472
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 472
cccctgtacc ccttaacaaa aacacgtacg cttttcaact gggggcc 47
<210> 473
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 473
cccctgtacc ccttaacaaa aacagagacg cttttcaact gggggcc 47
<210> 474
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 474
ctgtacccct taacaaaaac aggggcgctt ttcaactggg ggccagac 48
<210> 475
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 475
ctgtacccct taacaaaaac agggcgtctt ttcaactggg ggccagac 48
<210> 476
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 476
ctgtacccct taacaaaaac aggggagctt ttcaactggg ggccagac 48
<210> 477
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 477
ccttaacaaa aacagggacg cttttcgcgt gggggccaga ccagcaaa 48
<210> 478
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 478
ccttaacaaa aacagggacg cttttccgtt gggggccaga ccagcaaa 48
<210> 479
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 479
cttccggatt taaccaagcc ctttgcgctg ttcgttgatg aaaaacaggg atat 54
<210> 480
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 480
cttccggatt taaccaagcc ctttcgtctg ttcgttgatg aaaaacaggg atat 54
<210> 481
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 481
cttccggatt taaccaagcc ctttgatctg ttcgttgatg aaaaacaggg atat 54
<210> 482
<211> 55
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 482
gatttaacca agccctttga gctggcggtt gatgaaaaac agggatatgc aaaag 55
<210> 483
<211> 55
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 483
gatttaacca agccctttga gctgcgtgtt gatgaaaaac agggatatgc aaaag 55
<210> 484
<211> 55
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 484
gatttaacca agccctttga gctggaggtt gatgaaaaac agggatatgc aaaag 55
<210> 485
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 485
cccaaaagtt aggcccgtgg gcgcgccctg ttgcttactt gagtaa 46
<210> 486
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 486
cccaaaagtt aggcccgtgg aaacgccctg ttgcttactt gagtaa 46
<210> 487
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 487
cccaaaagtt aggcccgtgg gagcgccctg ttgcttactt gagtaa 46
<210> 488
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 488
agttgacgat gggtcaaccc ttagcgatct tggctccaca tgctgtaga 49
<210> 489
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 489
agttgacgat gggtcaaccc ttacgtatct tggctccaca tgctgtaga 49
<210> 490
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 490
agttgacgat gggtcaaccc ttagagatct tggctccaca tgctgtaga 49
<210> 491
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 491
ggatcgtgta caatttggac cagttgcggc tttgaatcca gctactttgc ttc 53
<210> 492
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 492
ggatcgtgta caatttggac cagttcgtgc tttgaatcca gctactttgc ttc 53
<210> 493
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 493
ggatcgtgta caatttggac cagttgaggc tttgaatcca gctactttgc ttc 53
<210> 494
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 494
cgttatgctt ttgcaacagc gcatgcgcat ggcgaaattt accgccgc 48
<210> 495
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 495
cgttatgctt ttgcaacagc gcatcgtcat ggcgaaattt accgccgc 48
<210> 496
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 496
cgttatgctt ttgcaacagc gcatgagcat ggcgaaattt accgccgc 48
<210> 497
<211> 43
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 497
gcaacagcgc atatccatgg cgcgatttac cgccgccgtg gtc 43
<210> 498
<211> 43
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 498
gcaacagcgc atatccatgg ccgtatttac cgccgccgtg gtc 43
<210> 499
<211> 43
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 499
gcaacagcgc atatccatgg cgatatttac cgccgccgtg gtc 43
<210> 500
<211> 44
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 500
caacagcgca tatccatggc gaagcgtacc gccgccgtgg tctg 44
<210> 501
<211> 44
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 501
caacagcgca tatccatggc gaacgttacc gccgccgtgg tctg 44
<210> 502
<211> 44
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 502
caacagcgca tatccatggc gaagagtacc gccgccgtgg tctg 44
<210> 503
<211> 41
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 503
agcggaggct cgtggaaacg cgatggcgga ccaagctgcc c 41
<210> 504
<211> 41
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 504
agcggaggct cgtggaaaca aaatggcgga ccaagctgcc c 41
<210> 505
<211> 41
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 505
agcggaggct cgtggaaacg agatggcgga ccaagctgcc c 41
<210> 506
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 506
gtggaaaccg tatggcggac gcggctgccc gtaaggcggc 40
<210> 507
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 507
gtggaaaccg tatggcggac cgtgctgccc gtaaggcggc 40
<210> 508
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 508
gtggaaaccg tatggcggac gaggctgccc gtaaggcggc 40
<210> 509
<211> 42
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 509
tatggcggac caagctgccg cgaaggcggc gatcacagag ac 42
<210> 510
<211> 42
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 510
tatggcggac caagctgcca aaaaggcggc gatcacagag ac 42
<210> 511
<211> 42
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 511
tatggcggac caagctgccg agaaggcggc gatcacagag ac 42
<210> 512
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 512
gcaagcgctg aatatgtggc ttaatcgcca ggcgggcctc ttgactc 47
<210> 513
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 513
gcaagcgctg aatatgtggc ttaatacgca ggcgggcctc ttgactc 47
<210> 514
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 514
gcaagcgctg aatatgtggc ttaatctcca ggcgggcctc ttgactc 47
<210> 515
<211> 44
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 515
ggtccagcaa gcgctgaata tgcgccttaa tccccaggcg ggcc 44
<210> 516
<211> 44
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 516
ggtccagcaa gcgctgaata tgacgcttaa tccccaggcg ggcc 44
<210> 517
<211> 44
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 517
ggtccagcaa gcgctgaata tgctccttaa tccccaggcg ggcc 44
<210> 518
<211> 43
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 518
ctggtccagc aagcgctgaa tcgctggctt aatccccagg cgg 43
<210> 519
<211> 43
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 519
ctggtccagc aagcgctgaa tacgtggctt aatccccagg cgg 43
<210> 520
<211> 43
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 520
ctggtccagc aagcgctgaa tctctggctt aatccccagg cgg 43
<210> 521
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 521
ggaccaagat cccctggtcc gccaagcgct gaatatgtgg cttaatc 47
<210> 522
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 522
ggaccaagat cccctggtca cgcaagcgct gaatatgtgg cttaatc 47
<210> 523
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 523
ggaccaagat cccctggtcc tccaagcgct gaatatgtgg cttaatc 47
<210> 524
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 524
catgggacca agatcccctg cgccagcaag cgctgaatat gtggc 45
<210> 525
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 525
catgggacca agatcccctg acgcagcaag cgctgaatat gtggc 45
<210> 526
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 526
catgggacca agatcccctg ctccagcaag cgctgaatat gtggc 45
<210> 527
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 527
attataagga ttaggtacag tcggatgcgc gtcttctacg cgtttgttga cct 53
<210> 528
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 528
attataagga ttaggtacag tcggatgacg gtcttctacg cgtttgttga cct 53
<210> 529
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 529
attataagga ttaggtacag tcggatgctc gtcttctacg cgtttgttga cct 53
<210> 530
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 530
gcctgataac agattataag gattaggcgc agtcggatgg atgtcttcta cgc 53
<210> 531
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 531
gcctgataac agattataag gattaggacg agtcggatgg atgtcttcta cgc 53
<210> 532
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 532
gcctgataac agattataag gattaggctc agtcggatgg atgtcttcta cgc 53
<210> 533
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 533
tcacggtgaa gtgcttcatc gaacgctgtg gggctgtttt taaagccct 49
<210> 534
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 534
tcacggtgaa gtgcttcatc gaaacgtgtg gggctgtttt taaagccct 49
<210> 535
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 535
tcacggtgaa gtgcttcatc gaactctgtg gggctgtttt taaagccct 49
<210> 536
<211> 52
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 536
ctgctaagtc acggtgaagt gccgcatcga acaatgtggg gctgttttta aa 52
<210> 537
<211> 52
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 537
ctgctaagtc acggtgaagt gcacgatcga acaatgtggg gctgttttta aa 52
<210> 538
<211> 52
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 538
ctgctaagtc acggtgaagt gcatcatcga acaatgtggg gctgttttta aa 52
<210> 539
<211> 51
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 539
tggatacgga agtctgctaa gtccgcgtga agtgcttcat cgaacaatgt g 51
<210> 540
<211> 51
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 540
tggatacgga agtctgctaa gtctttgtga agtgcttcat cgaacaatgt g 51
<210> 541
<211> 51
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 541
tggatacgga agtctgctaa gtcctcgtga agtgcttcat cgaacaatgt g 51
<210> 542
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 542
aagtctgggt gttggatacg gaacgctgct aagtcacggt gaagtgctt 49
<210> 543
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 543
aagtctgggt gttggatacg gaaacgtgct aagtcacggt gaagtgctt 49
<210> 544
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 544
aagtctgggt gttggatacg gaactctgct aagtcacggt gaagtgctt 49
<210> 545
<211> 51
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 545
gaattaagtc tgggtgttgg atacgcgcgt ctgctaagtc acggtgaagt g 51
<210> 546
<211> 51
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 546
gaattaagtc tgggtgttgg atacgacggt ctgctaagtc acggtgaagt g 51
<210> 547
<211> 51
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 547
gaattaagtc tgggtgttgg atacgctcgt ctgctaagtc acggtgaagt g 51
<210> 548
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 548
taacagaatt aagtctgggt gttggatcgc gaagtctgct aagtcacggt gaa 53
<210> 549
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 549
taacagaatt aagtctgggt gttggatttt gaagtctgct aagtcacggt gaa 53
<210> 550
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 550
taacagaatt aagtctgggt gttggatctc gaagtctgct aagtcacggt gaa 53
<210> 551
<211> 52
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 551
ctgtaacaga attaagtctg ggtgttgcgc acggaagtct gctaagtcac gg 52
<210> 552
<211> 52
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 552
ctgtaacaga attaagtctg ggtgttgacg acggaagtct gctaagtcac gg 52
<210> 553
<211> 52
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 553
ctgtaacaga attaagtctg ggtgttgctc acggaagtct gctaagtcac gg 52
<210> 554
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 554
atactgtaac agaattaagt ctgggtgcgc gatacggaag tctgctaagt cac 53
<210> 555
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 555
atactgtaac agaattaagt ctgggtgacg gatacggaag tctgctaagt cac 53
<210> 556
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 556
atactgtaac agaattaagt ctgggtgctc gatacggaag tctgctaagt cac 53
<210> 557
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 557
gggtgcggtc aacaaagctt gcgcgatctc ctgatacgcc ttttgct 47
<210> 558
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 558
gggtgcggtc aacaaagctt gacggatctc ctgatacgcc ttttgct 47
<210> 559
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 559
gggtgcggtc aacaaagctt gctcgatctc ctgatacgcc ttttgct 47
<210> 560
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 560
cccaacgcgg gtgcggtcgc caaagcttgt ttgatctcct gatacg 46
<210> 561
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 561
cccaacgcgg gtgcggtacg caaagcttgt ttgatctcct gatacg 46
<210> 562
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 562
cccaacgcgg gtgcggtctc caaagcttgt ttgatctcct gatacg 46
<210> 563
<211> 42
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 563
gctgccccat tacggtctcc gcacgcgctt cagtcagcca ac 42
<210> 564
<211> 42
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 564
gctgccccat tacggtctca cgacgcgctt cagtcagcca ac 42
<210> 565
<211> 42
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 565
gctgccccat tacggtctcc tcacgcgctt cagtcagcca ac 42
<210> 566
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 566
gctgtgccca aaaattcacg caacgcgcgt ggcgtcttag gcgta 45
<210> 567
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 567
gctgtgccca aaaattcacg caaacggcgt ggcgtcttag gcgta 45
<210> 568
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 568
gctgtgccca aaaattcacg caactcgcgt ggcgtcttag gcgta 45
<210> 569
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 569
cccaggaatc cataaacgac agaacgccgc tgtgcccaaa aattcacgc 49
<210> 570
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 570
cccaggaatc cataaacgac agaaacgcgc tgtgcccaaa aattcacgc 49
<210> 571
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 571
cccaggaatc cataaacgac agaactccgc tgtgcccaaa aattcacgc 49
<210> 572
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 572
tcagcgaacc caggaatcca taacgcacag aatcccgctg tgccc 45
<210> 573
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 573
tcagcgaacc caggaatcca taatttacag aatcccgctg tgccc 45
<210> 574
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 574
tcagcgaacc caggaatcca taactcacag aatcccgctg tgccc 45
<210> 575
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 575
ccaacgttga ccttctttca gcaacgcccc aaggtacttt acctgttttt gac 53
<210> 576
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 576
ccaacgttga ccttctttca gcaaacgccc aaggtacttt acctgttttt gac 53
<210> 577
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 577
ccaacgttga ccttctttca gcaactcccc aaggtacttt acctgttttt gac 53
<210> 578
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 578
ggtctcctta cgcgcttcag tcgcccaacg ttgaccttct ttcagca 47
<210> 579
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 579
ggtctcctta cgcgcttcag tacgccaacg ttgaccttct ttcagca 47
<210> 580
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 580
ggtctcctta cgcgcttcag tctcccaacg ttgaccttct ttcagca 47
<210> 581
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 581
ggcttggtta aatccggaag acccgccgcg ggtgcggtca acaaa 45
<210> 582
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 582
ggcttggtta aatccggaag accacgcgcg ggtgcggtca acaaa 45
<210> 583
<211> 45
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 583
ggcttggtta aatccggaag accctccgcg ggtgcggtca acaaa 45
<210> 584
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 584
ccagttgaaa agcgtccctg ttttcgctaa ggggtacagg ggtgcag 47
<210> 585
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 585
ccagttgaaa agcgtccctg ttttacgtaa ggggtacagg ggtgcag 47
<210> 586
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 586
ccagttgaaa agcgtccctg ttttctctaa ggggtacagg ggtgcag 47
<210> 587
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 587
ggcccccagt tgaaaagcgt cgctgttttt gttaaggggt acagggg 47
<210> 588
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 588
ggcccccagt tgaaaagcgt acgtgttttt gttaaggggt acagggg 47
<210> 589
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 589
ggcccccagt tgaaaagcgt ctctgttttt gttaaggggt acagggg 47
<210> 590
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 590
gtctggcccc cagttgaaaa gcgcccctgt ttttgttaag gggtacag 48
<210> 591
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 591
gtctggcccc cagttgaaaa gacgccctgt ttttgttaag gggtacag 48
<210> 592
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 592
gtctggcccc cagttgaaaa gctcccctgt ttttgttaag gggtacag 48
<210> 593
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 593
tttgctggtc tggcccccac gcgaaaagcg tccctgtttt tgttaagg 48
<210> 594
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 594
tttgctggtc tggcccccaa cggaaaagcg tccctgtttt tgttaagg 48
<210> 595
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 595
tttgctggtc tggcccccac tcgaaaagcg tccctgtttt tgttaagg 48
<210> 596
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 596
atatccctgt ttttcatcaa cgaacagcgc aaagggcttg gttaaatccg gaag 54
<210> 597
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 597
atatccctgt ttttcatcaa cgaacagacg aaagggcttg gttaaatccg gaag 54
<210> 598
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 598
atatccctgt ttttcatcaa cgaacagatc aaagggcttg gttaaatccg gaag 54
<210> 599
<211> 55
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 599
cttttgcata tccctgtttt tcatcaaccg ccagctcaaa gggcttggtt aaatc 55
<210> 600
<211> 55
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 600
cttttgcata tccctgtttt tcatcaacac gcagctcaaa gggcttggtt aaatc 55
<210> 601
<211> 55
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 601
cttttgcata tccctgtttt tcatcaacct ccagctcaaa gggcttggtt aaatc 55
<210> 602
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 602
ttactcaagt aagcaacagg gcgcgcccac gggcctaact tttggg 46
<210> 603
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 603
ttactcaagt aagcaacagg gcgtttccac gggcctaact tttggg 46
<210> 604
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 604
ttactcaagt aagcaacagg gcgctcccac gggcctaact tttggg 46
<210> 605
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 605
tctacagcat gtggagccaa gatcgctaag ggttgaccca tcgtcaact 49
<210> 606
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 606
tctacagcat gtggagccaa gatacgtaag ggttgaccca tcgtcaact 49
<210> 607
<211> 49
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 607
tctacagcat gtggagccaa gatctctaag ggttgaccca tcgtcaact 49
<210> 608
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 608
gaagcaaagt agctggattc aaagccgcaa ctggtccaaa ttgtacacga tcc 53
<210> 609
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 609
gaagcaaagt agctggattc aaagcacgaa ctggtccaaa ttgtacacga tcc 53
<210> 610
<211> 53
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 610
gaagcaaagt agctggattc aaagcctcaa ctggtccaaa ttgtacacga tcc 53
<210> 611
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 611
gcggcggtaa atttcgccat gcgcatgcgc tgttgcaaaa gcataacg 48
<210> 612
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 612
gcggcggtaa atttcgccat gacgatgcgc tgttgcaaaa gcataacg 48
<210> 613
<211> 48
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 613
gcggcggtaa atttcgccat gctcatgcgc tgttgcaaaa gcataacg 48
<210> 614
<211> 43
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 614
gaccacggcg gcggtaaatc gcgccatgga tatgcgctgt tgc 43
<210> 615
<211> 43
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 615
gaccacggcg gcggtaaata cggccatgga tatgcgctgt tgc 43
<210> 616
<211> 43
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 616
gaccacggcg gcggtaaata tcgccatgga tatgcgctgt tgc 43
<210> 617
<211> 44
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 617
cagaccacgg cggcggtacg cttcgccatg gatatgcgct gttg 44
<210> 618
<211> 44
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 618
cagaccacgg cggcggtaac gttcgccatg gatatgcgct gttg 44
<210> 619
<211> 44
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 619
cagaccacgg cggcggtact cttcgccatg gatatgcgct gttg 44
<210> 620
<211> 41
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 620
gggcagcttg gtccgccatc gcgtttccac gagcctccgc t 41
<210> 621
<211> 41
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 621
gggcagcttg gtccgccatt ttgtttccac gagcctccgc t 41
<210> 622
<211> 41
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 622
gggcagcttg gtccgccatc tcgtttccac gagcctccgc t 41
<210> 623
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 623
gccgccttac gggcagccgc gtccgccata cggtttccac 40
<210> 624
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 624
gccgccttac gggcagcacg gtccgccata cggtttccac 40
<210> 625
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 625
gccgccttac gggcagcctc gtccgccata cggtttccac 40
<210> 626
<211> 42
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 626
gtctctgtga tcgccgcctt cgcggcagct tggtccgcca ta 42
<210> 627
<211> 42
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 627
gtctctgtga tcgccgcctt tttggcagct tggtccgcca ta 42
<210> 628
<211> 42
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 628
gtctctgtga tcgccgcctt ctcggcagct tggtccgcca ta 42
<210> 629
<211> 50
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 629
atttgctgaa agaaggtcaa cgttggcgta ctgatgcgcg taaggagacc 50
<210> 630
<211> 50
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 630
ggtctcctta cgcgcatcag tacgccaacg ttgaccttct ttcagcaaat 50
<210> 631
<211> 50
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 631
gggattaagc cacatattcg tcgcttgcgt gaccagggga tcttggtccc 50
<210> 632
<211> 50
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 632
gggaccaaga tcccctggtc acgcaagcga cgaatatgtg gcttaatccc 50
<210> 633
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 633
gtcgagtatc tcagaaaaga agaca 25
<210> 634
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 634
ctcggatgta ggaagtttca cc 22
<210> 635
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<220>
<221> misc_feature
<222> (1)..(1)
<223> 5' SUN
<220>
<221> misc_feature
<222> (10)..(11)
<223> ZEN molecule
<220>
<221> misc_feature
<222> (24)..(24)
<223> 3' IABkFQ
<400> 635
atgccctgcg taaactggat gaca 24
<210> 636
<211> 2055
<212> DNA
<213> Artificial Sequence
<220>
<223> Synthetic oligonucleotide
<400> 636
atgactttaa atattgagga tgagcatcgt ttacatgaga catcaaaaga acccgacgtg 60
agcttagggt caacgtggct ttctgacttc ccccaggcgt gggcggagac tggcggaatg 120
gggttagctg tccgccaagc accgttgatc atcccgttaa aggcaacgtc tacacctgtc 180
tctatcaaac agtaccccat gagtcaagag gcccgcctgg ggattaagcc acatattcag 240
cgcttgctgg accaggggat cttggtccca tgtcaatctc cgtggaacac cccccttctg 300
cccgtgaaaa agccaggtac aaacgattat cgtccagttc aagatcttcg cgaggtcaac 360
aaacgcgtag aagacatcca tccgactgta cctaatcctt ataatctgtt atcaggcctg 420
cccccatcgc accaatggta tacagtatta gacttgaaag acgcgttctt ttgcctgcgt 480
ctgcacccaa cgtctcagcc gctgtttgcg ttcgaatggc gtgatcctga aatgggaatt 540
tcgggtcagt taacctggac tcgtctgccc cagggcttta aaaacagccc cacattgttc 600
gatgaagcac ttcaccgtga cttagcagac ttccgtatcc aacacccaga cttaattctg 660
ttacagtatg ttgacgacct tttgttggcg gcaacgtctg aacttgactg tcagcaaggc 720
acacgcgcgt tattacaaac gttaggtaac ttaggatatc gtgcgtccgc gaaaaaggcg 780
caaatttgtc aaaaacaggt aaagtacctt gggtatttgc tgaaagaagg tcaacgttgg 840
ctgactgaag cgcgtaagga gaccgtaatg gggcagccta cgcctaagac gccacgccag 900
ttgcgtgaat ttttgggcac agcgggattc tgtcgtttat ggattcctgg gttcgctgaa 960
atggctgcac ccctgtaccc cttaacaaaa acagggacgc ttttcaactg ggggccagac 1020
cagcaaaagg cgtatcagga gatcaaacaa gctttgttga ccgcacccgc gttgggtctt 1080
ccggatttaa ccaagccctt tgagctgttc gttgatgaaa aacagggata tgcaaaagga 1140
gtattaaccc aaaagttagg cccgtggcgt cgccctgttg cttacttgag taaaaaattg 1200
gatcctgtcg cagcaggatg gccaccgtgc ttgcgtatgg tcgcggcaat tgccgttttg 1260
acaaaggatg caggtaagtt gacgatgggt caacccttag taatcttggc tccacatgct 1320
gtagaagcgt tagtaaagca gcccccagac cgctggcttt ctaatgcgcg catgacccac 1380
tatcaggcgc ttctgcttga tacggatcgt gtacaatttg gaccagttgt agctttgaat 1440
ccagctactt tgcttcccct tccagaagaa ggacttcagc acaattgttt agatattctg 1500
gccgaggcac atgggacgcg ccctgatttg acggatcagc cactgcctga tgccgaccat 1560
acatggtata ctggcggcag tagtcttctt caagaggggc aacgcaaggc gggagcagcc 1620
gtcactacgg agaccgaagt tatctgggcc aaagcgttac ccgcgggaac atccgcgcaa 1680
cgtgcacagt taatcgctct gacacaggcc ctgaagatgg cagagggcaa aaagttgaat 1740
gtctacacca actcacgtta tgcttttgca acagcgcata tccatggcga aatttaccgc 1800
cgccgtggtc tgctgactag tgagggtaag gaaattaaaa ataaagatga gattcttgcg 1860
ttgttaaaag ctttattctt accaaaacgc ctttcgatca ttcattgccc ggggcatcaa 1920
aagggtcact cagcggaggc tcgtggaaac cgtatggcgg accaagctgc ccgtaaggcg 1980
gcgatcacag agaccccgga tacatcaacg ctgttgatcg aaaacagctc tccctacact 2040
agcgagcatt tttaa 2055
<210> 637
<211> 684
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 637
Met Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys
1 5 10 15
Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln
20 25 30
Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro
35 40 45
Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln
50 55 60
Tyr Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln
65 70 75 80
Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn
85 90 95
Thr Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro
100 105 110
Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
115 120 125
Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His
130 135 140
Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg
145 150 155 160
Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro
165 170 175
Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
180 185 190
Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu
195 200 205
Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val
210 215 220
Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly
225 230 235 240
Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser
245 250 255
Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr
260 265 270
Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr
275 280 285
Val Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe
290 295 300
Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu
305 310 315 320
Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn
325 330 335
Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu
340 345 350
Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu
355 360 365
Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln
370 375 380
Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu
385 390 395 400
Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
405 410 415
Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro
420 425 430
Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro
435 440 445
Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu
450 455 460
Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn
465 470 475 480
Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys
485 490 495
Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp
500 505 510
Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser
515 520 525
Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu
530 535 540
Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln
545 550 555 560
Arg Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly
565 570 575
Lys Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala
580 585 590
His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu
595 600 605
Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala
610 615 620
Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln
625 630 635 640
Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala
645 650 655
Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu
660 665 670
Ile Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 638
<211> 684
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 638
Met Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys
1 5 10 15
Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln
20 25 30
Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro
35 40 45
Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Arg Lys Gln
50 55 60
Tyr Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln
65 70 75 80
Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn
85 90 95
Thr Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro
100 105 110
Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
115 120 125
Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His
130 135 140
Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg
145 150 155 160
Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro
165 170 175
Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
180 185 190
Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu
195 200 205
Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val
210 215 220
Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly
225 230 235 240
Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser
245 250 255
Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr
260 265 270
Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr
275 280 285
Val Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe
290 295 300
Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu
305 310 315 320
Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn
325 330 335
Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu
340 345 350
Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu
355 360 365
Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln
370 375 380
Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu
385 390 395 400
Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
405 410 415
Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro
420 425 430
Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro
435 440 445
Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu
450 455 460
Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn
465 470 475 480
Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys
485 490 495
Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp
500 505 510
Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser
515 520 525
Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu
530 535 540
Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln
545 550 555 560
Arg Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly
565 570 575
Lys Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala
580 585 590
His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu
595 600 605
Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala
610 615 620
Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln
625 630 635 640
Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala
645 650 655
Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu
660 665 670
Ile Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 639
<211> 684
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 639
Met Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys
1 5 10 15
Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln
20 25 30
Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro
35 40 45
Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln
50 55 60
Tyr Pro Met Ser Arg Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln
65 70 75 80
Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn
85 90 95
Thr Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro
100 105 110
Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
115 120 125
Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His
130 135 140
Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg
145 150 155 160
Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro
165 170 175
Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
180 185 190
Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu
195 200 205
Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val
210 215 220
Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly
225 230 235 240
Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser
245 250 255
Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr
260 265 270
Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr
275 280 285
Val Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe
290 295 300
Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu
305 310 315 320
Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn
325 330 335
Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu
340 345 350
Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu
355 360 365
Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln
370 375 380
Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu
385 390 395 400
Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
405 410 415
Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro
420 425 430
Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro
435 440 445
Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu
450 455 460
Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn
465 470 475 480
Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys
485 490 495
Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp
500 505 510
Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser
515 520 525
Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu
530 535 540
Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln
545 550 555 560
Arg Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly
565 570 575
Lys Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala
580 585 590
His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu
595 600 605
Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala
610 615 620
Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln
625 630 635 640
Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala
645 650 655
Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu
660 665 670
Ile Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 640
<211> 684
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 640
Met Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys
1 5 10 15
Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln
20 25 30
Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro
35 40 45
Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln
50 55 60
Tyr Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Arg
65 70 75 80
Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn
85 90 95
Thr Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro
100 105 110
Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
115 120 125
Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His
130 135 140
Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg
145 150 155 160
Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro
165 170 175
Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
180 185 190
Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu
195 200 205
Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val
210 215 220
Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly
225 230 235 240
Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser
245 250 255
Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr
260 265 270
Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr
275 280 285
Val Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe
290 295 300
Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu
305 310 315 320
Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn
325 330 335
Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu
340 345 350
Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu
355 360 365
Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln
370 375 380
Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu
385 390 395 400
Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
405 410 415
Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro
420 425 430
Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro
435 440 445
Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu
450 455 460
Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn
465 470 475 480
Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys
485 490 495
Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp
500 505 510
Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser
515 520 525
Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu
530 535 540
Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln
545 550 555 560
Arg Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly
565 570 575
Lys Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala
580 585 590
His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu
595 600 605
Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala
610 615 620
Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln
625 630 635 640
Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala
645 650 655
Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu
660 665 670
Ile Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 641
<211> 684
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 641
Met Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys
1 5 10 15
Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln
20 25 30
Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro
35 40 45
Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln
50 55 60
Tyr Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln
65 70 75 80
Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn
85 90 95
Thr Pro Arg Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro
100 105 110
Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
115 120 125
Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His
130 135 140
Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg
145 150 155 160
Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro
165 170 175
Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
180 185 190
Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu
195 200 205
Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val
210 215 220
Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly
225 230 235 240
Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser
245 250 255
Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr
260 265 270
Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr
275 280 285
Val Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe
290 295 300
Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu
305 310 315 320
Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn
325 330 335
Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu
340 345 350
Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu
355 360 365
Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln
370 375 380
Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu
385 390 395 400
Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
405 410 415
Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro
420 425 430
Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro
435 440 445
Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu
450 455 460
Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn
465 470 475 480
Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys
485 490 495
Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp
500 505 510
Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser
515 520 525
Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu
530 535 540
Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln
545 550 555 560
Arg Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly
565 570 575
Lys Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala
580 585 590
His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu
595 600 605
Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala
610 615 620
Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln
625 630 635 640
Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala
645 650 655
Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu
660 665 670
Ile Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 642
<211> 684
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 642
Met Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys
1 5 10 15
Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln
20 25 30
Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro
35 40 45
Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln
50 55 60
Tyr Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln
65 70 75 80
Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn
85 90 95
Thr Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro
100 105 110
Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
115 120 125
Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His
130 135 140
Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg
145 150 155 160
Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro
165 170 175
Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
180 185 190
Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu
195 200 205
Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val
210 215 220
Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly
225 230 235 240
Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser
245 250 255
Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr
260 265 270
Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Asp Ala Arg Lys Glu Thr
275 280 285
Val Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe
290 295 300
Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu
305 310 315 320
Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn
325 330 335
Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu
340 345 350
Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu
355 360 365
Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln
370 375 380
Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu
385 390 395 400
Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
405 410 415
Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro
420 425 430
Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro
435 440 445
Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu
450 455 460
Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn
465 470 475 480
Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys
485 490 495
Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp
500 505 510
Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser
515 520 525
Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu
530 535 540
Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln
545 550 555 560
Arg Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly
565 570 575
Lys Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala
580 585 590
His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu
595 600 605
Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala
610 615 620
Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln
625 630 635 640
Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala
645 650 655
Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu
660 665 670
Ile Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 643
<211> 684
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 643
Met Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys
1 5 10 15
Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln
20 25 30
Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro
35 40 45
Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln
50 55 60
Tyr Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln
65 70 75 80
Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn
85 90 95
Thr Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro
100 105 110
Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
115 120 125
Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His
130 135 140
Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg
145 150 155 160
Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro
165 170 175
Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
180 185 190
Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu
195 200 205
Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val
210 215 220
Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly
225 230 235 240
Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser
245 250 255
Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr
260 265 270
Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr
275 280 285
Val Met Gly Gln Pro Thr Pro Lys Thr Pro Ala Gln Leu Arg Glu Phe
290 295 300
Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu
305 310 315 320
Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn
325 330 335
Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu
340 345 350
Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu
355 360 365
Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln
370 375 380
Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu
385 390 395 400
Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
405 410 415
Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro
420 425 430
Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro
435 440 445
Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu
450 455 460
Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn
465 470 475 480
Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys
485 490 495
Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp
500 505 510
Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser
515 520 525
Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu
530 535 540
Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln
545 550 555 560
Arg Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly
565 570 575
Lys Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala
580 585 590
His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu
595 600 605
Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala
610 615 620
Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln
625 630 635 640
Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala
645 650 655
Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu
660 665 670
Ile Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 644
<211> 684
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 644
Met Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys
1 5 10 15
Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln
20 25 30
Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro
35 40 45
Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Lys Lys Gln
50 55 60
Tyr Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln
65 70 75 80
Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn
85 90 95
Thr Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro
100 105 110
Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
115 120 125
Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His
130 135 140
Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg
145 150 155 160
Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro
165 170 175
Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
180 185 190
Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu
195 200 205
Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val
210 215 220
Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly
225 230 235 240
Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser
245 250 255
Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr
260 265 270
Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr
275 280 285
Val Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe
290 295 300
Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu
305 310 315 320
Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn
325 330 335
Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu
340 345 350
Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu
355 360 365
Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln
370 375 380
Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu
385 390 395 400
Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
405 410 415
Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro
420 425 430
Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro
435 440 445
Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu
450 455 460
Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn
465 470 475 480
Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys
485 490 495
Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp
500 505 510
Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser
515 520 525
Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu
530 535 540
Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln
545 550 555 560
Arg Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly
565 570 575
Lys Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala
580 585 590
His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu
595 600 605
Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala
610 615 620
Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln
625 630 635 640
Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala
645 650 655
Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu
660 665 670
Ile Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 645
<211> 684
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 645
Met Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys
1 5 10 15
Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln
20 25 30
Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro
35 40 45
Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Met Lys Gln
50 55 60
Tyr Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln
65 70 75 80
Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn
85 90 95
Thr Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro
100 105 110
Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
115 120 125
Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His
130 135 140
Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg
145 150 155 160
Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro
165 170 175
Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
180 185 190
Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu
195 200 205
Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val
210 215 220
Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly
225 230 235 240
Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser
245 250 255
Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr
260 265 270
Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr
275 280 285
Val Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe
290 295 300
Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu
305 310 315 320
Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn
325 330 335
Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu
340 345 350
Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu
355 360 365
Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln
370 375 380
Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu
385 390 395 400
Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
405 410 415
Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro
420 425 430
Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro
435 440 445
Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu
450 455 460
Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn
465 470 475 480
Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys
485 490 495
Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp
500 505 510
Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser
515 520 525
Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu
530 535 540
Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln
545 550 555 560
Arg Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly
565 570 575
Lys Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala
580 585 590
His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu
595 600 605
Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala
610 615 620
Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln
625 630 635 640
Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala
645 650 655
Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu
660 665 670
Ile Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 646
<211> 684
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 646
Met Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys
1 5 10 15
Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln
20 25 30
Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro
35 40 45
Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln
50 55 60
Tyr Pro Met Ser Ile Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln
65 70 75 80
Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn
85 90 95
Thr Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro
100 105 110
Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
115 120 125
Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His
130 135 140
Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg
145 150 155 160
Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro
165 170 175
Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
180 185 190
Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu
195 200 205
Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val
210 215 220
Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly
225 230 235 240
Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser
245 250 255
Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr
260 265 270
Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr
275 280 285
Val Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe
290 295 300
Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu
305 310 315 320
Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn
325 330 335
Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu
340 345 350
Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu
355 360 365
Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln
370 375 380
Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu
385 390 395 400
Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
405 410 415
Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro
420 425 430
Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro
435 440 445
Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu
450 455 460
Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn
465 470 475 480
Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys
485 490 495
Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp
500 505 510
Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser
515 520 525
Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu
530 535 540
Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln
545 550 555 560
Arg Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly
565 570 575
Lys Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala
580 585 590
His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu
595 600 605
Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala
610 615 620
Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln
625 630 635 640
Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala
645 650 655
Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu
660 665 670
Ile Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 647
<211> 684
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 647
Met Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys
1 5 10 15
Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln
20 25 30
Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro
35 40 45
Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln
50 55 60
Tyr Pro Met Ser Lys Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln
65 70 75 80
Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn
85 90 95
Thr Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro
100 105 110
Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
115 120 125
Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His
130 135 140
Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg
145 150 155 160
Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro
165 170 175
Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
180 185 190
Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu
195 200 205
Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val
210 215 220
Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly
225 230 235 240
Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser
245 250 255
Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr
260 265 270
Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr
275 280 285
Val Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe
290 295 300
Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu
305 310 315 320
Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn
325 330 335
Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu
340 345 350
Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu
355 360 365
Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln
370 375 380
Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu
385 390 395 400
Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
405 410 415
Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro
420 425 430
Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro
435 440 445
Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu
450 455 460
Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn
465 470 475 480
Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys
485 490 495
Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp
500 505 510
Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser
515 520 525
Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu
530 535 540
Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln
545 550 555 560
Arg Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly
565 570 575
Lys Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala
580 585 590
His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu
595 600 605
Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala
610 615 620
Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln
625 630 635 640
Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala
645 650 655
Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu
660 665 670
Ile Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 648
<211> 684
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 648
Met Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys
1 5 10 15
Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln
20 25 30
Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro
35 40 45
Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln
50 55 60
Tyr Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile His
65 70 75 80
Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn
85 90 95
Thr Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro
100 105 110
Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
115 120 125
Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His
130 135 140
Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg
145 150 155 160
Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro
165 170 175
Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
180 185 190
Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu
195 200 205
Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val
210 215 220
Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly
225 230 235 240
Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser
245 250 255
Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr
260 265 270
Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr
275 280 285
Val Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe
290 295 300
Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu
305 310 315 320
Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn
325 330 335
Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu
340 345 350
Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu
355 360 365
Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln
370 375 380
Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu
385 390 395 400
Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
405 410 415
Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro
420 425 430
Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro
435 440 445
Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu
450 455 460
Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn
465 470 475 480
Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys
485 490 495
Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp
500 505 510
Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser
515 520 525
Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu
530 535 540
Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln
545 550 555 560
Arg Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly
565 570 575
Lys Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala
580 585 590
His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu
595 600 605
Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala
610 615 620
Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln
625 630 635 640
Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala
645 650 655
Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu
660 665 670
Ile Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 649
<211> 684
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 649
Met Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys
1 5 10 15
Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln
20 25 30
Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro
35 40 45
Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln
50 55 60
Tyr Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Ile
65 70 75 80
Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn
85 90 95
Thr Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro
100 105 110
Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
115 120 125
Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His
130 135 140
Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg
145 150 155 160
Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro
165 170 175
Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
180 185 190
Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu
195 200 205
Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val
210 215 220
Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly
225 230 235 240
Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser
245 250 255
Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr
260 265 270
Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr
275 280 285
Val Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe
290 295 300
Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu
305 310 315 320
Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn
325 330 335
Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu
340 345 350
Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu
355 360 365
Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln
370 375 380
Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu
385 390 395 400
Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
405 410 415
Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro
420 425 430
Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro
435 440 445
Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu
450 455 460
Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn
465 470 475 480
Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys
485 490 495
Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp
500 505 510
Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser
515 520 525
Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu
530 535 540
Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln
545 550 555 560
Arg Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly
565 570 575
Lys Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala
580 585 590
His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu
595 600 605
Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala
610 615 620
Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln
625 630 635 640
Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala
645 650 655
Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu
660 665 670
Ile Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 650
<211> 684
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 650
Met Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys
1 5 10 15
Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln
20 25 30
Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro
35 40 45
Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln
50 55 60
Tyr Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln
65 70 75 80
Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn
85 90 95
Thr Pro Leu Lys Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro
100 105 110
Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
115 120 125
Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His
130 135 140
Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg
145 150 155 160
Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro
165 170 175
Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
180 185 190
Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu
195 200 205
Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val
210 215 220
Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly
225 230 235 240
Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser
245 250 255
Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr
260 265 270
Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr
275 280 285
Val Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe
290 295 300
Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu
305 310 315 320
Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn
325 330 335
Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu
340 345 350
Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu
355 360 365
Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln
370 375 380
Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu
385 390 395 400
Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
405 410 415
Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro
420 425 430
Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro
435 440 445
Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu
450 455 460
Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn
465 470 475 480
Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys
485 490 495
Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp
500 505 510
Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser
515 520 525
Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu
530 535 540
Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln
545 550 555 560
Arg Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly
565 570 575
Lys Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala
580 585 590
His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu
595 600 605
Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala
610 615 620
Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln
625 630 635 640
Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala
645 650 655
Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu
660 665 670
Ile Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 651
<211> 684
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 651
Met Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys
1 5 10 15
Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln
20 25 30
Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro
35 40 45
Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln
50 55 60
Tyr Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln
65 70 75 80
Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn
85 90 95
Thr Pro Leu Asn Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro
100 105 110
Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
115 120 125
Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His
130 135 140
Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg
145 150 155 160
Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro
165 170 175
Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
180 185 190
Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu
195 200 205
Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val
210 215 220
Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly
225 230 235 240
Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser
245 250 255
Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr
260 265 270
Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr
275 280 285
Val Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe
290 295 300
Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu
305 310 315 320
Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn
325 330 335
Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu
340 345 350
Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu
355 360 365
Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln
370 375 380
Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu
385 390 395 400
Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
405 410 415
Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro
420 425 430
Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro
435 440 445
Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu
450 455 460
Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn
465 470 475 480
Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys
485 490 495
Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp
500 505 510
Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser
515 520 525
Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu
530 535 540
Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln
545 550 555 560
Arg Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly
565 570 575
Lys Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala
580 585 590
His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu
595 600 605
Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala
610 615 620
Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln
625 630 635 640
Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala
645 650 655
Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu
660 665 670
Ile Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 652
<211> 684
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 652
Met Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys
1 5 10 15
Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln
20 25 30
Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro
35 40 45
Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln
50 55 60
Tyr Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln
65 70 75 80
Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn
85 90 95
Thr Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro
100 105 110
Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
115 120 125
Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His
130 135 140
Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg
145 150 155 160
Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro
165 170 175
Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
180 185 190
Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu
195 200 205
Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val
210 215 220
Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly
225 230 235 240
Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser
245 250 255
Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr
260 265 270
Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Met Ala Arg Lys Glu Thr
275 280 285
Val Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe
290 295 300
Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu
305 310 315 320
Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn
325 330 335
Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu
340 345 350
Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu
355 360 365
Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln
370 375 380
Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu
385 390 395 400
Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
405 410 415
Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro
420 425 430
Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro
435 440 445
Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu
450 455 460
Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn
465 470 475 480
Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys
485 490 495
Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp
500 505 510
Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser
515 520 525
Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu
530 535 540
Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln
545 550 555 560
Arg Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly
565 570 575
Lys Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala
580 585 590
His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu
595 600 605
Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala
610 615 620
Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln
625 630 635 640
Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala
645 650 655
Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu
660 665 670
Ile Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 653
<211> 684
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 653
Met Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys
1 5 10 15
Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln
20 25 30
Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro
35 40 45
Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln
50 55 60
Tyr Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln
65 70 75 80
Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn
85 90 95
Thr Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro
100 105 110
Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
115 120 125
Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His
130 135 140
Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg
145 150 155 160
Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro
165 170 175
Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
180 185 190
Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu
195 200 205
Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val
210 215 220
Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly
225 230 235 240
Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser
245 250 255
Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr
260 265 270
Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Trp Ala Arg Lys Glu Thr
275 280 285
Val Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe
290 295 300
Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu
305 310 315 320
Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn
325 330 335
Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu
340 345 350
Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu
355 360 365
Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln
370 375 380
Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu
385 390 395 400
Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
405 410 415
Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro
420 425 430
Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro
435 440 445
Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu
450 455 460
Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn
465 470 475 480
Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys
485 490 495
Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp
500 505 510
Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser
515 520 525
Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu
530 535 540
Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln
545 550 555 560
Arg Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly
565 570 575
Lys Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala
580 585 590
His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu
595 600 605
Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala
610 615 620
Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln
625 630 635 640
Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala
645 650 655
Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu
660 665 670
Ile Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 654
<211> 684
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 654
Met Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys
1 5 10 15
Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln
20 25 30
Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro
35 40 45
Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Arg Lys Gln
50 55 60
Tyr Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln
65 70 75 80
Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn
85 90 95
Thr Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro
100 105 110
Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
115 120 125
Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His
130 135 140
Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg
145 150 155 160
Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro
165 170 175
Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
180 185 190
Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu
195 200 205
Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val
210 215 220
Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly
225 230 235 240
Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser
245 250 255
Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr
260 265 270
Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Asp Ala Arg Lys Glu Thr
275 280 285
Val Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe
290 295 300
Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu
305 310 315 320
Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn
325 330 335
Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu
340 345 350
Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu
355 360 365
Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln
370 375 380
Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu
385 390 395 400
Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
405 410 415
Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro
420 425 430
Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro
435 440 445
Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu
450 455 460
Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn
465 470 475 480
Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys
485 490 495
Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp
500 505 510
Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser
515 520 525
Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu
530 535 540
Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln
545 550 555 560
Arg Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly
565 570 575
Lys Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala
580 585 590
His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu
595 600 605
Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala
610 615 620
Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln
625 630 635 640
Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala
645 650 655
Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu
660 665 670
Ile Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 655
<211> 684
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 655
Met Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys
1 5 10 15
Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln
20 25 30
Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro
35 40 45
Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln
50 55 60
Tyr Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln
65 70 75 80
Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn
85 90 95
Thr Pro Arg Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro
100 105 110
Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
115 120 125
Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His
130 135 140
Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg
145 150 155 160
Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro
165 170 175
Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
180 185 190
Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu
195 200 205
Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val
210 215 220
Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly
225 230 235 240
Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser
245 250 255
Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr
260 265 270
Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Asp Ala Arg Lys Glu Thr
275 280 285
Val Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe
290 295 300
Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu
305 310 315 320
Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn
325 330 335
Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu
340 345 350
Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu
355 360 365
Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln
370 375 380
Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu
385 390 395 400
Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
405 410 415
Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro
420 425 430
Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro
435 440 445
Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu
450 455 460
Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn
465 470 475 480
Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys
485 490 495
Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp
500 505 510
Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser
515 520 525
Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu
530 535 540
Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln
545 550 555 560
Arg Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly
565 570 575
Lys Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala
580 585 590
His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu
595 600 605
Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala
610 615 620
Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln
625 630 635 640
Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala
645 650 655
Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu
660 665 670
Ile Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 656
<211> 684
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 656
Met Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys
1 5 10 15
Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln
20 25 30
Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro
35 40 45
Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln
50 55 60
Tyr Pro Met Ser Arg Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln
65 70 75 80
Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn
85 90 95
Thr Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro
100 105 110
Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
115 120 125
Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His
130 135 140
Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg
145 150 155 160
Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro
165 170 175
Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
180 185 190
Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu
195 200 205
Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val
210 215 220
Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly
225 230 235 240
Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser
245 250 255
Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr
260 265 270
Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Asp Ala Arg Lys Glu Thr
275 280 285
Val Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe
290 295 300
Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu
305 310 315 320
Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn
325 330 335
Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu
340 345 350
Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu
355 360 365
Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln
370 375 380
Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu
385 390 395 400
Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
405 410 415
Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro
420 425 430
Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro
435 440 445
Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu
450 455 460
Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn
465 470 475 480
Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys
485 490 495
Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp
500 505 510
Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser
515 520 525
Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu
530 535 540
Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln
545 550 555 560
Arg Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly
565 570 575
Lys Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala
580 585 590
His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu
595 600 605
Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala
610 615 620
Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln
625 630 635 640
Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala
645 650 655
Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu
660 665 670
Ile Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 657
<211> 684
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 657
Met Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys
1 5 10 15
Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln
20 25 30
Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro
35 40 45
Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln
50 55 60
Tyr Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Arg
65 70 75 80
Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn
85 90 95
Thr Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro
100 105 110
Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
115 120 125
Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His
130 135 140
Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg
145 150 155 160
Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro
165 170 175
Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
180 185 190
Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu
195 200 205
Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val
210 215 220
Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly
225 230 235 240
Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser
245 250 255
Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr
260 265 270
Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Asp Ala Arg Lys Glu Thr
275 280 285
Val Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe
290 295 300
Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu
305 310 315 320
Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn
325 330 335
Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu
340 345 350
Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu
355 360 365
Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln
370 375 380
Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu
385 390 395 400
Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
405 410 415
Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro
420 425 430
Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro
435 440 445
Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu
450 455 460
Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn
465 470 475 480
Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys
485 490 495
Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp
500 505 510
Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser
515 520 525
Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu
530 535 540
Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln
545 550 555 560
Arg Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly
565 570 575
Lys Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala
580 585 590
His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu
595 600 605
Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala
610 615 620
Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln
625 630 635 640
Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala
645 650 655
Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu
660 665 670
Ile Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 658
<211> 684
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 658
Met Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys
1 5 10 15
Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln
20 25 30
Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro
35 40 45
Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln
50 55 60
Tyr Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln
65 70 75 80
Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn
85 90 95
Thr Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro
100 105 110
Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
115 120 125
Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His
130 135 140
Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg
145 150 155 160
Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro
165 170 175
Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
180 185 190
Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu
195 200 205
Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val
210 215 220
Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly
225 230 235 240
Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser
245 250 255
Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr
260 265 270
Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Asp Ala Arg Lys Glu Thr
275 280 285
Val Met Gly Gln Pro Thr Pro Lys Thr Pro Ala Gln Leu Arg Glu Phe
290 295 300
Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu
305 310 315 320
Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn
325 330 335
Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu
340 345 350
Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu
355 360 365
Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln
370 375 380
Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu
385 390 395 400
Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
405 410 415
Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro
420 425 430
Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro
435 440 445
Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu
450 455 460
Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn
465 470 475 480
Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys
485 490 495
Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp
500 505 510
Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser
515 520 525
Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu
530 535 540
Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln
545 550 555 560
Arg Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly
565 570 575
Lys Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala
580 585 590
His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu
595 600 605
Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala
610 615 620
Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln
625 630 635 640
Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala
645 650 655
Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu
660 665 670
Ile Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 659
<211> 684
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 659
Met Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys
1 5 10 15
Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln
20 25 30
Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro
35 40 45
Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Arg Lys Gln
50 55 60
Tyr Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln
65 70 75 80
Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn
85 90 95
Thr Pro Leu Arg Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro
100 105 110
Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
115 120 125
Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His
130 135 140
Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg
145 150 155 160
Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro
165 170 175
Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
180 185 190
Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu
195 200 205
Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val
210 215 220
Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly
225 230 235 240
Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser
245 250 255
Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr
260 265 270
Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr
275 280 285
Val Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe
290 295 300
Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu
305 310 315 320
Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn
325 330 335
Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu
340 345 350
Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu
355 360 365
Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln
370 375 380
Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu
385 390 395 400
Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
405 410 415
Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro
420 425 430
Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro
435 440 445
Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu
450 455 460
Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn
465 470 475 480
Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys
485 490 495
Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp
500 505 510
Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser
515 520 525
Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu
530 535 540
Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln
545 550 555 560
Arg Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly
565 570 575
Lys Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala
580 585 590
His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu
595 600 605
Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala
610 615 620
Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln
625 630 635 640
Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala
645 650 655
Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu
660 665 670
Ile Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 660
<211> 683
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 660
Met Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys
1 5 10 15
Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln
20 25 30
Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro
35 40 45
Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Arg Lys Gln
50 55 60
Tyr Pro Met Ser Arg Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln
65 70 75 80
Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn
85 90 95
Thr Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro
100 105 110
Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
115 120 125
Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His
130 135 140
Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg
145 150 155 160
Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro
165 170 175
Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
180 185 190
Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu
195 200 205
Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val
210 215 220
Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly
225 230 235 240
Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser
245 250 255
Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr
260 265 270
Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr
275 280 285
Val Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe
290 295 300
Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu
305 310 315 320
Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn
325 330 335
Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu
340 345 350
Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu
355 360 365
Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln
370 375 380
Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu
385 390 395 400
Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
405 410 415
Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro
420 425 430
Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro
435 440 445
Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu
450 455 460
Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn
465 470 475 480
Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys
485 490 495
Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp
500 505 510
Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser
515 520 525
Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu
530 535 540
Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln
545 550 555 560
Arg Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly
565 570 575
Lys Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala
580 585 590
His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu
595 600 605
Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala
610 615 620
Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln
625 630 635 640
Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala
645 650 655
Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu
660 665 670
Ile Glu Asn Ser Ser Pro Tyr Thr Ser Glu His
675 680
<210> 661
<211> 684
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 661
Met Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys
1 5 10 15
Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln
20 25 30
Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro
35 40 45
Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Arg Lys Gln
50 55 60
Tyr Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Arg
65 70 75 80
Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn
85 90 95
Thr Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro
100 105 110
Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
115 120 125
Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His
130 135 140
Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg
145 150 155 160
Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro
165 170 175
Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
180 185 190
Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu
195 200 205
Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val
210 215 220
Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly
225 230 235 240
Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser
245 250 255
Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr
260 265 270
Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr
275 280 285
Val Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe
290 295 300
Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu
305 310 315 320
Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn
325 330 335
Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu
340 345 350
Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu
355 360 365
Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln
370 375 380
Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu
385 390 395 400
Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
405 410 415
Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro
420 425 430
Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro
435 440 445
Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu
450 455 460
Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn
465 470 475 480
Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys
485 490 495
Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp
500 505 510
Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser
515 520 525
Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu
530 535 540
Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln
545 550 555 560
Arg Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly
565 570 575
Lys Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala
580 585 590
His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu
595 600 605
Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala
610 615 620
Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln
625 630 635 640
Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala
645 650 655
Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu
660 665 670
Ile Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 662
<211> 684
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 662
Met Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys
1 5 10 15
Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln
20 25 30
Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro
35 40 45
Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Arg Lys Gln
50 55 60
Tyr Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln
65 70 75 80
Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn
85 90 95
Thr Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro
100 105 110
Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
115 120 125
Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His
130 135 140
Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg
145 150 155 160
Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro
165 170 175
Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
180 185 190
Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu
195 200 205
Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val
210 215 220
Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly
225 230 235 240
Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser
245 250 255
Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr
260 265 270
Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr
275 280 285
Val Met Gly Gln Pro Thr Pro Lys Thr Pro Ala Gln Leu Arg Glu Phe
290 295 300
Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu
305 310 315 320
Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn
325 330 335
Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu
340 345 350
Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu
355 360 365
Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln
370 375 380
Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu
385 390 395 400
Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
405 410 415
Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro
420 425 430
Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro
435 440 445
Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu
450 455 460
Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn
465 470 475 480
Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys
485 490 495
Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp
500 505 510
Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser
515 520 525
Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu
530 535 540
Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln
545 550 555 560
Arg Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly
565 570 575
Lys Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala
580 585 590
His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu
595 600 605
Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala
610 615 620
Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln
625 630 635 640
Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala
645 650 655
Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu
660 665 670
Ile Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 663
<211> 684
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 663
Met Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys
1 5 10 15
Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln
20 25 30
Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro
35 40 45
Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln
50 55 60
Tyr Pro Met Ser Arg Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln
65 70 75 80
Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn
85 90 95
Thr Pro Leu Arg Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro
100 105 110
Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
115 120 125
Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His
130 135 140
Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg
145 150 155 160
Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro
165 170 175
Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
180 185 190
Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu
195 200 205
Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val
210 215 220
Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly
225 230 235 240
Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser
245 250 255
Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr
260 265 270
Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr
275 280 285
Val Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe
290 295 300
Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu
305 310 315 320
Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn
325 330 335
Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu
340 345 350
Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu
355 360 365
Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln
370 375 380
Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu
385 390 395 400
Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
405 410 415
Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro
420 425 430
Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro
435 440 445
Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu
450 455 460
Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn
465 470 475 480
Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys
485 490 495
Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp
500 505 510
Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser
515 520 525
Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu
530 535 540
Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln
545 550 555 560
Arg Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly
565 570 575
Lys Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala
580 585 590
His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu
595 600 605
Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala
610 615 620
Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln
625 630 635 640
Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala
645 650 655
Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu
660 665 670
Ile Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 664
<211> 684
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 664
Met Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys
1 5 10 15
Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln
20 25 30
Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro
35 40 45
Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln
50 55 60
Tyr Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Arg
65 70 75 80
Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn
85 90 95
Thr Pro Leu Arg Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro
100 105 110
Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
115 120 125
Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His
130 135 140
Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg
145 150 155 160
Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro
165 170 175
Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
180 185 190
Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu
195 200 205
Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val
210 215 220
Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly
225 230 235 240
Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser
245 250 255
Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr
260 265 270
Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr
275 280 285
Val Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe
290 295 300
Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu
305 310 315 320
Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn
325 330 335
Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu
340 345 350
Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu
355 360 365
Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln
370 375 380
Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu
385 390 395 400
Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
405 410 415
Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro
420 425 430
Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro
435 440 445
Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu
450 455 460
Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn
465 470 475 480
Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys
485 490 495
Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp
500 505 510
Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser
515 520 525
Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu
530 535 540
Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln
545 550 555 560
Arg Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly
565 570 575
Lys Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala
580 585 590
His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu
595 600 605
Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala
610 615 620
Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln
625 630 635 640
Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala
645 650 655
Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu
660 665 670
Ile Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 665
<211> 684
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 665
Met Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys
1 5 10 15
Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln
20 25 30
Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro
35 40 45
Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln
50 55 60
Tyr Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln
65 70 75 80
Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn
85 90 95
Thr Pro Leu Arg Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro
100 105 110
Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
115 120 125
Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His
130 135 140
Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg
145 150 155 160
Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro
165 170 175
Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
180 185 190
Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu
195 200 205
Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val
210 215 220
Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly
225 230 235 240
Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser
245 250 255
Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr
260 265 270
Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr
275 280 285
Val Met Gly Gln Pro Thr Pro Lys Thr Pro Ala Gln Leu Arg Glu Phe
290 295 300
Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu
305 310 315 320
Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn
325 330 335
Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu
340 345 350
Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu
355 360 365
Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln
370 375 380
Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu
385 390 395 400
Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
405 410 415
Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro
420 425 430
Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro
435 440 445
Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu
450 455 460
Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn
465 470 475 480
Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys
485 490 495
Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp
500 505 510
Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser
515 520 525
Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu
530 535 540
Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln
545 550 555 560
Arg Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly
565 570 575
Lys Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala
580 585 590
His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu
595 600 605
Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala
610 615 620
Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln
625 630 635 640
Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala
645 650 655
Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu
660 665 670
Ile Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 666
<211> 684
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 666
Met Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys
1 5 10 15
Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln
20 25 30
Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro
35 40 45
Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln
50 55 60
Tyr Pro Met Ser Arg Glu Ala Arg Leu Gly Ile Lys Pro His Ile Arg
65 70 75 80
Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn
85 90 95
Thr Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro
100 105 110
Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
115 120 125
Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His
130 135 140
Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg
145 150 155 160
Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro
165 170 175
Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
180 185 190
Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu
195 200 205
Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val
210 215 220
Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly
225 230 235 240
Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser
245 250 255
Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr
260 265 270
Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr
275 280 285
Val Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe
290 295 300
Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu
305 310 315 320
Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn
325 330 335
Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu
340 345 350
Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu
355 360 365
Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln
370 375 380
Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu
385 390 395 400
Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
405 410 415
Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro
420 425 430
Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro
435 440 445
Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu
450 455 460
Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn
465 470 475 480
Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys
485 490 495
Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp
500 505 510
Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser
515 520 525
Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu
530 535 540
Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln
545 550 555 560
Arg Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly
565 570 575
Lys Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala
580 585 590
His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu
595 600 605
Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala
610 615 620
Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln
625 630 635 640
Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala
645 650 655
Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu
660 665 670
Ile Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 667
<211> 684
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 667
Met Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys
1 5 10 15
Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln
20 25 30
Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro
35 40 45
Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln
50 55 60
Tyr Pro Met Ser Arg Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln
65 70 75 80
Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn
85 90 95
Thr Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro
100 105 110
Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
115 120 125
Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His
130 135 140
Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg
145 150 155 160
Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro
165 170 175
Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
180 185 190
Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu
195 200 205
Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val
210 215 220
Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly
225 230 235 240
Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser
245 250 255
Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr
260 265 270
Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr
275 280 285
Val Met Gly Gln Pro Thr Pro Lys Thr Pro Ala Gln Leu Arg Glu Phe
290 295 300
Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu
305 310 315 320
Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn
325 330 335
Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu
340 345 350
Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu
355 360 365
Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln
370 375 380
Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu
385 390 395 400
Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
405 410 415
Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro
420 425 430
Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro
435 440 445
Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu
450 455 460
Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn
465 470 475 480
Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys
485 490 495
Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp
500 505 510
Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser
515 520 525
Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu
530 535 540
Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln
545 550 555 560
Arg Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly
565 570 575
Lys Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala
580 585 590
His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu
595 600 605
Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala
610 615 620
Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln
625 630 635 640
Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala
645 650 655
Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu
660 665 670
Ile Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 668
<211> 684
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 668
Met Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys
1 5 10 15
Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln
20 25 30
Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro
35 40 45
Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln
50 55 60
Tyr Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Arg
65 70 75 80
Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn
85 90 95
Thr Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro
100 105 110
Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
115 120 125
Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His
130 135 140
Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg
145 150 155 160
Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro
165 170 175
Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
180 185 190
Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu
195 200 205
Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val
210 215 220
Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly
225 230 235 240
Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser
245 250 255
Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr
260 265 270
Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr
275 280 285
Val Met Gly Gln Pro Thr Pro Lys Thr Pro Ala Gln Leu Arg Glu Phe
290 295 300
Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu
305 310 315 320
Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn
325 330 335
Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu
340 345 350
Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu
355 360 365
Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln
370 375 380
Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu
385 390 395 400
Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
405 410 415
Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro
420 425 430
Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro
435 440 445
Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu
450 455 460
Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn
465 470 475 480
Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys
485 490 495
Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp
500 505 510
Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser
515 520 525
Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu
530 535 540
Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln
545 550 555 560
Arg Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly
565 570 575
Lys Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala
580 585 590
His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu
595 600 605
Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala
610 615 620
Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln
625 630 635 640
Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala
645 650 655
Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu
660 665 670
Ile Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 669
<211> 684
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 669
Met Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys
1 5 10 15
Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln
20 25 30
Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro
35 40 45
Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln
50 55 60
Tyr Pro Met Ser Arg Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln
65 70 75 80
Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn
85 90 95
Thr Pro Leu Arg Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro
100 105 110
Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
115 120 125
Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His
130 135 140
Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg
145 150 155 160
Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro
165 170 175
Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
180 185 190
Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu
195 200 205
Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val
210 215 220
Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly
225 230 235 240
Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser
245 250 255
Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr
260 265 270
Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Asp Ala Arg Lys Glu Thr
275 280 285
Val Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe
290 295 300
Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu
305 310 315 320
Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn
325 330 335
Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu
340 345 350
Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu
355 360 365
Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln
370 375 380
Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu
385 390 395 400
Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
405 410 415
Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro
420 425 430
Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro
435 440 445
Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu
450 455 460
Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn
465 470 475 480
Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys
485 490 495
Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp
500 505 510
Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser
515 520 525
Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu
530 535 540
Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln
545 550 555 560
Arg Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly
565 570 575
Lys Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala
580 585 590
His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu
595 600 605
Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala
610 615 620
Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln
625 630 635 640
Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala
645 650 655
Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu
660 665 670
Ile Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 670
<211> 684
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 670
Met Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys
1 5 10 15
Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln
20 25 30
Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro
35 40 45
Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln
50 55 60
Tyr Pro Met Ser Gln Glu Ala Arg Leu Gly Ile Lys Pro His Ile Arg
65 70 75 80
Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn
85 90 95
Thr Pro Leu Arg Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro
100 105 110
Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
115 120 125
Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His
130 135 140
Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg
145 150 155 160
Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro
165 170 175
Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
180 185 190
Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu
195 200 205
Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val
210 215 220
Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly
225 230 235 240
Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser
245 250 255
Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr
260 265 270
Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Asp Ala Arg Lys Glu Thr
275 280 285
Val Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe
290 295 300
Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu
305 310 315 320
Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn
325 330 335
Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu
340 345 350
Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu
355 360 365
Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln
370 375 380
Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu
385 390 395 400
Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
405 410 415
Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro
420 425 430
Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro
435 440 445
Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu
450 455 460
Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn
465 470 475 480
Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys
485 490 495
Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp
500 505 510
Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser
515 520 525
Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu
530 535 540
Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln
545 550 555 560
Arg Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly
565 570 575
Lys Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala
580 585 590
His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu
595 600 605
Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala
610 615 620
Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln
625 630 635 640
Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala
645 650 655
Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu
660 665 670
Ile Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 671
<211> 684
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 671
Met Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys
1 5 10 15
Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln
20 25 30
Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro
35 40 45
Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln
50 55 60
Tyr Pro Met Ser Arg Glu Ala Arg Leu Gly Ile Lys Pro His Ile Arg
65 70 75 80
Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn
85 90 95
Thr Pro Leu Leu Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro
100 105 110
Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
115 120 125
Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His
130 135 140
Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg
145 150 155 160
Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro
165 170 175
Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
180 185 190
Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu
195 200 205
Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val
210 215 220
Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly
225 230 235 240
Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser
245 250 255
Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr
260 265 270
Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Asp Ala Arg Lys Glu Thr
275 280 285
Val Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe
290 295 300
Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu
305 310 315 320
Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn
325 330 335
Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu
340 345 350
Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu
355 360 365
Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln
370 375 380
Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu
385 390 395 400
Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
405 410 415
Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro
420 425 430
Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro
435 440 445
Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu
450 455 460
Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn
465 470 475 480
Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys
485 490 495
Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp
500 505 510
Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser
515 520 525
Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu
530 535 540
Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln
545 550 555 560
Arg Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly
565 570 575
Lys Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala
580 585 590
His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu
595 600 605
Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala
610 615 620
Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln
625 630 635 640
Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala
645 650 655
Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu
660 665 670
Ile Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 672
<211> 684
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 672
Met Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys
1 5 10 15
Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln
20 25 30
Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro
35 40 45
Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln
50 55 60
Tyr Pro Met Ser Arg Glu Ala Arg Leu Gly Ile Lys Pro His Ile Arg
65 70 75 80
Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn
85 90 95
Thr Pro Leu Arg Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro
100 105 110
Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
115 120 125
Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His
130 135 140
Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg
145 150 155 160
Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro
165 170 175
Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
180 185 190
Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu
195 200 205
Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val
210 215 220
Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly
225 230 235 240
Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser
245 250 255
Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr
260 265 270
Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Glu Ala Arg Lys Glu Thr
275 280 285
Val Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe
290 295 300
Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu
305 310 315 320
Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn
325 330 335
Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu
340 345 350
Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu
355 360 365
Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln
370 375 380
Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu
385 390 395 400
Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
405 410 415
Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro
420 425 430
Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro
435 440 445
Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu
450 455 460
Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn
465 470 475 480
Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys
485 490 495
Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp
500 505 510
Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser
515 520 525
Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu
530 535 540
Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln
545 550 555 560
Arg Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly
565 570 575
Lys Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala
580 585 590
His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu
595 600 605
Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala
610 615 620
Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln
625 630 635 640
Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala
645 650 655
Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu
660 665 670
Ile Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 673
<211> 684
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 673
Met Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys
1 5 10 15
Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln
20 25 30
Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro
35 40 45
Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln
50 55 60
Tyr Pro Met Ser Arg Glu Ala Arg Leu Gly Ile Lys Pro His Ile Arg
65 70 75 80
Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn
85 90 95
Thr Pro Leu Arg Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro
100 105 110
Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
115 120 125
Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His
130 135 140
Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg
145 150 155 160
Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro
165 170 175
Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
180 185 190
Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu
195 200 205
Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val
210 215 220
Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly
225 230 235 240
Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser
245 250 255
Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr
260 265 270
Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Asp Ala Arg Lys Glu Thr
275 280 285
Val Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe
290 295 300
Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu
305 310 315 320
Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn
325 330 335
Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu
340 345 350
Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu
355 360 365
Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln
370 375 380
Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu
385 390 395 400
Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
405 410 415
Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro
420 425 430
Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro
435 440 445
Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu
450 455 460
Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn
465 470 475 480
Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys
485 490 495
Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp
500 505 510
Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser
515 520 525
Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu
530 535 540
Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln
545 550 555 560
Arg Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly
565 570 575
Lys Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala
580 585 590
His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu
595 600 605
Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala
610 615 620
Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln
625 630 635 640
Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala
645 650 655
Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu
660 665 670
Ile Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 674
<211> 684
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 674
Met Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys
1 5 10 15
Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln
20 25 30
Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro
35 40 45
Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln
50 55 60
Tyr Pro Met Ser Arg Glu Ala Arg Leu Gly Ile Lys Pro His Ile Arg
65 70 75 80
Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn
85 90 95
Thr Pro Leu Lys Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro
100 105 110
Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
115 120 125
Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His
130 135 140
Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg
145 150 155 160
Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro
165 170 175
Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
180 185 190
Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu
195 200 205
Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val
210 215 220
Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly
225 230 235 240
Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser
245 250 255
Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr
260 265 270
Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Asp Ala Arg Lys Glu Thr
275 280 285
Val Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe
290 295 300
Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu
305 310 315 320
Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn
325 330 335
Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu
340 345 350
Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu
355 360 365
Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln
370 375 380
Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu
385 390 395 400
Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
405 410 415
Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro
420 425 430
Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro
435 440 445
Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu
450 455 460
Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn
465 470 475 480
Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys
485 490 495
Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp
500 505 510
Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser
515 520 525
Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu
530 535 540
Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln
545 550 555 560
Arg Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly
565 570 575
Lys Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala
580 585 590
His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu
595 600 605
Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala
610 615 620
Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln
625 630 635 640
Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala
645 650 655
Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu
660 665 670
Ile Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 675
<211> 684
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 675
Met Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys
1 5 10 15
Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln
20 25 30
Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro
35 40 45
Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln
50 55 60
Tyr Pro Met Ser Arg Glu Ala Arg Leu Gly Ile Lys Pro His Ile Arg
65 70 75 80
Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn
85 90 95
Thr Pro Leu Asn Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro
100 105 110
Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
115 120 125
Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His
130 135 140
Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg
145 150 155 160
Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro
165 170 175
Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
180 185 190
Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu
195 200 205
Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val
210 215 220
Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly
225 230 235 240
Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser
245 250 255
Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr
260 265 270
Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Asp Ala Arg Lys Glu Thr
275 280 285
Val Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe
290 295 300
Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu
305 310 315 320
Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn
325 330 335
Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu
340 345 350
Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu
355 360 365
Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln
370 375 380
Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu
385 390 395 400
Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
405 410 415
Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro
420 425 430
Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro
435 440 445
Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu
450 455 460
Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn
465 470 475 480
Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys
485 490 495
Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp
500 505 510
Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser
515 520 525
Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu
530 535 540
Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln
545 550 555 560
Arg Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly
565 570 575
Lys Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala
580 585 590
His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu
595 600 605
Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala
610 615 620
Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln
625 630 635 640
Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala
645 650 655
Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu
660 665 670
Ile Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 676
<211> 684
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 676
Met Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys
1 5 10 15
Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln
20 25 30
Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro
35 40 45
Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln
50 55 60
Tyr Pro Met Ser Ile Glu Ala Arg Leu Gly Ile Lys Pro His Ile Arg
65 70 75 80
Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn
85 90 95
Thr Pro Leu Arg Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro
100 105 110
Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
115 120 125
Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His
130 135 140
Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg
145 150 155 160
Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro
165 170 175
Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
180 185 190
Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu
195 200 205
Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val
210 215 220
Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly
225 230 235 240
Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser
245 250 255
Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr
260 265 270
Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Asp Ala Arg Lys Glu Thr
275 280 285
Val Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe
290 295 300
Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu
305 310 315 320
Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn
325 330 335
Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu
340 345 350
Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu
355 360 365
Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln
370 375 380
Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu
385 390 395 400
Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
405 410 415
Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro
420 425 430
Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro
435 440 445
Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu
450 455 460
Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn
465 470 475 480
Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys
485 490 495
Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp
500 505 510
Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser
515 520 525
Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu
530 535 540
Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln
545 550 555 560
Arg Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly
565 570 575
Lys Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala
580 585 590
His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu
595 600 605
Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala
610 615 620
Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln
625 630 635 640
Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala
645 650 655
Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu
660 665 670
Ile Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 677
<211> 684
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 677
Met Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys
1 5 10 15
Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln
20 25 30
Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro
35 40 45
Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln
50 55 60
Tyr Pro Met Ser Lys Glu Ala Arg Leu Gly Ile Lys Pro His Ile Arg
65 70 75 80
Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn
85 90 95
Thr Pro Leu Arg Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro
100 105 110
Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
115 120 125
Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His
130 135 140
Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg
145 150 155 160
Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro
165 170 175
Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
180 185 190
Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu
195 200 205
Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val
210 215 220
Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly
225 230 235 240
Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser
245 250 255
Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr
260 265 270
Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Asp Ala Arg Lys Glu Thr
275 280 285
Val Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe
290 295 300
Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu
305 310 315 320
Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn
325 330 335
Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu
340 345 350
Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu
355 360 365
Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln
370 375 380
Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu
385 390 395 400
Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
405 410 415
Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro
420 425 430
Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro
435 440 445
Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu
450 455 460
Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn
465 470 475 480
Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys
485 490 495
Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp
500 505 510
Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser
515 520 525
Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu
530 535 540
Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln
545 550 555 560
Arg Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly
565 570 575
Lys Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala
580 585 590
His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu
595 600 605
Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala
610 615 620
Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln
625 630 635 640
Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala
645 650 655
Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu
660 665 670
Ile Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 678
<211> 684
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 678
Met Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys
1 5 10 15
Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln
20 25 30
Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro
35 40 45
Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln
50 55 60
Tyr Pro Met Ser Arg Glu Ala Arg Leu Gly Ile Lys Pro His Ile His
65 70 75 80
Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn
85 90 95
Thr Pro Leu Arg Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro
100 105 110
Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
115 120 125
Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His
130 135 140
Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg
145 150 155 160
Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro
165 170 175
Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
180 185 190
Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu
195 200 205
Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val
210 215 220
Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly
225 230 235 240
Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser
245 250 255
Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr
260 265 270
Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Asp Ala Arg Lys Glu Thr
275 280 285
Val Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe
290 295 300
Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu
305 310 315 320
Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn
325 330 335
Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu
340 345 350
Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu
355 360 365
Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln
370 375 380
Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu
385 390 395 400
Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
405 410 415
Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro
420 425 430
Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro
435 440 445
Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu
450 455 460
Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn
465 470 475 480
Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys
485 490 495
Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp
500 505 510
Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser
515 520 525
Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu
530 535 540
Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln
545 550 555 560
Arg Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly
565 570 575
Lys Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala
580 585 590
His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu
595 600 605
Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala
610 615 620
Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln
625 630 635 640
Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala
645 650 655
Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu
660 665 670
Ile Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 679
<211> 684
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 679
Met Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys
1 5 10 15
Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln
20 25 30
Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro
35 40 45
Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln
50 55 60
Tyr Pro Met Ser Arg Glu Ala Arg Leu Gly Ile Lys Pro His Ile Ile
65 70 75 80
Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn
85 90 95
Thr Pro Leu Arg Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro
100 105 110
Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
115 120 125
Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His
130 135 140
Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg
145 150 155 160
Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro
165 170 175
Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
180 185 190
Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu
195 200 205
Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val
210 215 220
Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly
225 230 235 240
Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser
245 250 255
Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr
260 265 270
Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Asp Ala Arg Lys Glu Thr
275 280 285
Val Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe
290 295 300
Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu
305 310 315 320
Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn
325 330 335
Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu
340 345 350
Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu
355 360 365
Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln
370 375 380
Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu
385 390 395 400
Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
405 410 415
Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro
420 425 430
Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro
435 440 445
Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu
450 455 460
Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn
465 470 475 480
Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys
485 490 495
Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp
500 505 510
Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser
515 520 525
Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu
530 535 540
Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln
545 550 555 560
Arg Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly
565 570 575
Lys Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala
580 585 590
His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu
595 600 605
Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala
610 615 620
Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln
625 630 635 640
Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala
645 650 655
Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu
660 665 670
Ile Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 680
<211> 684
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 680
Met Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys
1 5 10 15
Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln
20 25 30
Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro
35 40 45
Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln
50 55 60
Tyr Pro Met Ser Arg Glu Ala Arg Leu Gly Ile Lys Pro His Ile Arg
65 70 75 80
Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn
85 90 95
Thr Pro Leu Arg Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro
100 105 110
Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
115 120 125
Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His
130 135 140
Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg
145 150 155 160
Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro
165 170 175
Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
180 185 190
Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu
195 200 205
Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val
210 215 220
Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly
225 230 235 240
Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser
245 250 255
Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr
260 265 270
Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Met Ala Arg Lys Glu Thr
275 280 285
Val Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe
290 295 300
Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu
305 310 315 320
Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn
325 330 335
Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu
340 345 350
Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu
355 360 365
Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln
370 375 380
Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu
385 390 395 400
Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
405 410 415
Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro
420 425 430
Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro
435 440 445
Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu
450 455 460
Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn
465 470 475 480
Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys
485 490 495
Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp
500 505 510
Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser
515 520 525
Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu
530 535 540
Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln
545 550 555 560
Arg Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly
565 570 575
Lys Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala
580 585 590
His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu
595 600 605
Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala
610 615 620
Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln
625 630 635 640
Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala
645 650 655
Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu
660 665 670
Ile Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 681
<211> 684
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 681
Met Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys
1 5 10 15
Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln
20 25 30
Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro
35 40 45
Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln
50 55 60
Tyr Pro Met Ser Arg Glu Ala Arg Leu Gly Ile Lys Pro His Ile Arg
65 70 75 80
Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn
85 90 95
Thr Pro Leu Arg Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro
100 105 110
Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
115 120 125
Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His
130 135 140
Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg
145 150 155 160
Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro
165 170 175
Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
180 185 190
Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu
195 200 205
Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val
210 215 220
Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly
225 230 235 240
Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser
245 250 255
Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr
260 265 270
Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Trp Ala Arg Lys Glu Thr
275 280 285
Val Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe
290 295 300
Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu
305 310 315 320
Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn
325 330 335
Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu
340 345 350
Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu
355 360 365
Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln
370 375 380
Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu
385 390 395 400
Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
405 410 415
Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro
420 425 430
Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro
435 440 445
Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu
450 455 460
Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn
465 470 475 480
Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys
485 490 495
Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp
500 505 510
Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser
515 520 525
Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu
530 535 540
Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln
545 550 555 560
Arg Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly
565 570 575
Lys Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala
580 585 590
His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu
595 600 605
Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala
610 615 620
Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln
625 630 635 640
Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala
645 650 655
Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu
660 665 670
Ile Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 682
<211> 684
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 682
Met Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys
1 5 10 15
Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln
20 25 30
Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro
35 40 45
Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Lys Lys Gln
50 55 60
Tyr Pro Met Ser Arg Glu Ala Arg Leu Gly Ile Lys Pro His Ile Arg
65 70 75 80
Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn
85 90 95
Thr Pro Leu Arg Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro
100 105 110
Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
115 120 125
Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His
130 135 140
Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg
145 150 155 160
Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro
165 170 175
Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
180 185 190
Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu
195 200 205
Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val
210 215 220
Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly
225 230 235 240
Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser
245 250 255
Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr
260 265 270
Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Asp Ala Arg Lys Glu Thr
275 280 285
Val Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe
290 295 300
Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu
305 310 315 320
Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn
325 330 335
Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu
340 345 350
Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu
355 360 365
Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln
370 375 380
Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu
385 390 395 400
Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
405 410 415
Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro
420 425 430
Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro
435 440 445
Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu
450 455 460
Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn
465 470 475 480
Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys
485 490 495
Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp
500 505 510
Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser
515 520 525
Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu
530 535 540
Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln
545 550 555 560
Arg Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly
565 570 575
Lys Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala
580 585 590
His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu
595 600 605
Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala
610 615 620
Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln
625 630 635 640
Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala
645 650 655
Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu
660 665 670
Ile Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 683
<211> 684
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 683
Met Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys
1 5 10 15
Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln
20 25 30
Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro
35 40 45
Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Met Lys Gln
50 55 60
Tyr Pro Met Ser Arg Glu Ala Arg Leu Gly Ile Lys Pro His Ile Arg
65 70 75 80
Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn
85 90 95
Thr Pro Leu Arg Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro
100 105 110
Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
115 120 125
Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His
130 135 140
Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg
145 150 155 160
Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro
165 170 175
Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
180 185 190
Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu
195 200 205
Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val
210 215 220
Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly
225 230 235 240
Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser
245 250 255
Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr
260 265 270
Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Asp Ala Arg Lys Glu Thr
275 280 285
Val Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe
290 295 300
Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu
305 310 315 320
Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn
325 330 335
Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu
340 345 350
Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu
355 360 365
Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln
370 375 380
Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu
385 390 395 400
Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
405 410 415
Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro
420 425 430
Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro
435 440 445
Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu
450 455 460
Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn
465 470 475 480
Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys
485 490 495
Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp
500 505 510
Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser
515 520 525
Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu
530 535 540
Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln
545 550 555 560
Arg Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly
565 570 575
Lys Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala
580 585 590
His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu
595 600 605
Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala
610 615 620
Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln
625 630 635 640
Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala
645 650 655
Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu
660 665 670
Ile Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 684
<211> 684
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 684
Met Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys
1 5 10 15
Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln
20 25 30
Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro
35 40 45
Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln
50 55 60
Tyr Pro Met Ser Ile Glu Ala Arg Leu Gly Ile Lys Pro His Ile His
65 70 75 80
Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn
85 90 95
Thr Pro Leu Lys Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro
100 105 110
Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
115 120 125
Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His
130 135 140
Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg
145 150 155 160
Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro
165 170 175
Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
180 185 190
Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu
195 200 205
Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val
210 215 220
Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly
225 230 235 240
Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser
245 250 255
Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr
260 265 270
Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Met Ala Arg Lys Glu Thr
275 280 285
Val Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe
290 295 300
Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu
305 310 315 320
Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn
325 330 335
Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu
340 345 350
Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu
355 360 365
Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln
370 375 380
Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu
385 390 395 400
Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
405 410 415
Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro
420 425 430
Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro
435 440 445
Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu
450 455 460
Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn
465 470 475 480
Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys
485 490 495
Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp
500 505 510
Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser
515 520 525
Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu
530 535 540
Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln
545 550 555 560
Arg Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly
565 570 575
Lys Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala
580 585 590
His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu
595 600 605
Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala
610 615 620
Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln
625 630 635 640
Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala
645 650 655
Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu
660 665 670
Ile Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 685
<211> 684
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 685
Met Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys
1 5 10 15
Glu Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln
20 25 30
Ala Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro
35 40 45
Leu Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Met Lys Gln
50 55 60
Tyr Pro Met Ser Ile Glu Ala Arg Leu Gly Ile Lys Pro His Ile His
65 70 75 80
Arg Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn
85 90 95
Thr Pro Leu Lys Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro
100 105 110
Val Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro
115 120 125
Thr Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His
130 135 140
Gln Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg
145 150 155 160
Leu His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro
165 170 175
Glu Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly
180 185 190
Phe Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu
195 200 205
Ala Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val
210 215 220
Asp Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly
225 230 235 240
Thr Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser
245 250 255
Ala Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr
260 265 270
Leu Leu Lys Glu Gly Gln Arg Trp Leu Thr Met Ala Arg Lys Glu Thr
275 280 285
Val Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe
290 295 300
Leu Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu
305 310 315 320
Met Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn
325 330 335
Trp Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu
340 345 350
Leu Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu
355 360 365
Leu Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln
370 375 380
Lys Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu
385 390 395 400
Asp Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala
405 410 415
Ile Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro
420 425 430
Leu Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro
435 440 445
Pro Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu
450 455 460
Leu Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn
465 470 475 480
Pro Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys
485 490 495
Leu Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp
500 505 510
Gln Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser
515 520 525
Leu Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu
530 535 540
Thr Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln
545 550 555 560
Arg Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly
565 570 575
Lys Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala
580 585 590
His Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu
595 600 605
Gly Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala
610 615 620
Leu Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln
625 630 635 640
Lys Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala
645 650 655
Ala Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu
660 665 670
Ile Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 686
<211> 683
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 686
Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys Glu
1 5 10 15
Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala
20 25 30
Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu
35 40 45
Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr
50 55 60
Pro Met Ser Arg Glu Ala Arg Leu Gly Ile Lys Pro His Ile Arg Arg
65 70 75 80
Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn Thr
85 90 95
Pro Leu Arg Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val
100 105 110
Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro Thr
115 120 125
Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His Gln
130 135 140
Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu
145 150 155 160
His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu
165 170 175
Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe
180 185 190
Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu Ala
195 200 205
Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp
210 215 220
Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr
225 230 235 240
Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala
245 250 255
Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu
260 265 270
Leu Lys Glu Gly Gln Arg Trp Leu Thr Asp Ala Arg Lys Glu Thr Val
275 280 285
Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe Leu
290 295 300
Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu Met
305 310 315 320
Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn Trp
325 330 335
Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu
340 345 350
Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu
355 360 365
Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys
370 375 380
Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp
385 390 395 400
Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile
405 410 415
Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu
420 425 430
Arg Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro Pro
435 440 445
Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu Leu
450 455 460
Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn Pro
465 470 475 480
Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys Leu
485 490 495
Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp Gln
500 505 510
Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser Leu
515 520 525
Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Thr
530 535 540
Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln Arg
545 550 555 560
Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly Lys
565 570 575
Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala His
580 585 590
Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly
595 600 605
Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala Leu
610 615 620
Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys
625 630 635 640
Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala
645 650 655
Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu Ile
660 665 670
Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 687
<211> 683
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 687
Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys Glu
1 5 10 15
Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala
20 25 30
Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu
35 40 45
Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr
50 55 60
Pro Met Ser Arg Glu Ala Arg Leu Gly Ile Lys Pro His Ile Arg Arg
65 70 75 80
Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn Thr
85 90 95
Pro Leu Arg Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val
100 105 110
Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro Thr
115 120 125
Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His Gln
130 135 140
Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu
145 150 155 160
His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu
165 170 175
Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe
180 185 190
Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu Ala
195 200 205
Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp
210 215 220
Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr
225 230 235 240
Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala
245 250 255
Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu
260 265 270
Leu Lys Glu Gly Gln Arg Trp Leu Thr Asp Ala Arg Lys Glu Thr Val
275 280 285
Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe Leu
290 295 300
Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu Met
305 310 315 320
Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn Trp
325 330 335
Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu
340 345 350
Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu
355 360 365
Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys
370 375 380
Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp
385 390 395 400
Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile
405 410 415
Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu
420 425 430
Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro Pro
435 440 445
Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu Leu
450 455 460
Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn Pro
465 470 475 480
Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys Leu
485 490 495
Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp Gln
500 505 510
Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser Leu
515 520 525
Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Thr
530 535 540
Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln Arg
545 550 555 560
Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly Lys
565 570 575
Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala His
580 585 590
Glu His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly
595 600 605
Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala Leu
610 615 620
Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys
625 630 635 640
Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala
645 650 655
Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu Ile
660 665 670
Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 688
<211> 683
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 688
Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys Glu
1 5 10 15
Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala
20 25 30
Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu
35 40 45
Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr
50 55 60
Pro Met Ser Arg Glu Ala Arg Leu Gly Ile Lys Pro His Ile Arg Arg
65 70 75 80
Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn Thr
85 90 95
Pro Leu Arg Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val
100 105 110
Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro Thr
115 120 125
Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His Gln
130 135 140
Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu
145 150 155 160
His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu
165 170 175
Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe
180 185 190
Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu Ala
195 200 205
Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp
210 215 220
Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr
225 230 235 240
Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala
245 250 255
Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu
260 265 270
Leu Lys Glu Gly Gln Arg Trp Leu Thr Asp Ala Arg Lys Glu Thr Val
275 280 285
Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Glu Leu Arg Glu Phe Leu
290 295 300
Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu Met
305 310 315 320
Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn Trp
325 330 335
Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu
340 345 350
Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu
355 360 365
Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys
370 375 380
Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp
385 390 395 400
Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile
405 410 415
Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu
420 425 430
Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro Pro
435 440 445
Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu Leu
450 455 460
Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn Pro
465 470 475 480
Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys Leu
485 490 495
Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp Gln
500 505 510
Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser Leu
515 520 525
Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Thr
530 535 540
Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln Arg
545 550 555 560
Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly Lys
565 570 575
Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala His
580 585 590
Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly
595 600 605
Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala Leu
610 615 620
Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys
625 630 635 640
Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala
645 650 655
Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu Ile
660 665 670
Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 689
<211> 683
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 689
Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys Glu
1 5 10 15
Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala
20 25 30
Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu
35 40 45
Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr
50 55 60
Pro Met Ser Arg Glu Ala Arg Leu Gly Ile Lys Pro His Ile Arg Arg
65 70 75 80
Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn Thr
85 90 95
Pro Leu Arg Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val
100 105 110
Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro Thr
115 120 125
Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His Gln
130 135 140
Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu
145 150 155 160
His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu
165 170 175
Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe
180 185 190
Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu Ala
195 200 205
Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp
210 215 220
Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr
225 230 235 240
Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala
245 250 255
Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu
260 265 270
Leu Lys Glu Gly Gln Arg Trp Leu Thr Asp Ala Arg Lys Glu Thr Val
275 280 285
Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe Leu
290 295 300
Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu Met
305 310 315 320
Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Glu Leu Phe Asn Trp
325 330 335
Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu
340 345 350
Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu
355 360 365
Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys
370 375 380
Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp
385 390 395 400
Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile
405 410 415
Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu
420 425 430
Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro Pro
435 440 445
Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu Leu
450 455 460
Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn Pro
465 470 475 480
Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys Leu
485 490 495
Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp Gln
500 505 510
Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser Leu
515 520 525
Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Thr
530 535 540
Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln Arg
545 550 555 560
Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly Lys
565 570 575
Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala His
580 585 590
Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly
595 600 605
Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala Leu
610 615 620
Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys
625 630 635 640
Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala
645 650 655
Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu Ile
660 665 670
Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 690
<211> 683
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 690
Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys Glu
1 5 10 15
Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala
20 25 30
Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu
35 40 45
Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr
50 55 60
Pro Met Ser Arg Glu Ala Arg Leu Gly Ile Lys Pro His Ile Arg Arg
65 70 75 80
Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn Thr
85 90 95
Pro Leu Arg Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val
100 105 110
Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro Thr
115 120 125
Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His Gln
130 135 140
Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu
145 150 155 160
His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu
165 170 175
Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe
180 185 190
Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu Ala
195 200 205
Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp
210 215 220
Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr
225 230 235 240
Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala
245 250 255
Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu
260 265 270
Leu Lys Glu Gly Gln Arg Trp Arg Thr Glu Ala Arg Lys Glu Thr Val
275 280 285
Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe Leu
290 295 300
Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu Met
305 310 315 320
Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn Trp
325 330 335
Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu
340 345 350
Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu
355 360 365
Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys
370 375 380
Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp
385 390 395 400
Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile
405 410 415
Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu
420 425 430
Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro Pro
435 440 445
Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu Leu
450 455 460
Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn Pro
465 470 475 480
Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys Leu
485 490 495
Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp Gln
500 505 510
Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser Leu
515 520 525
Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Thr
530 535 540
Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln Arg
545 550 555 560
Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly Lys
565 570 575
Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala His
580 585 590
Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly
595 600 605
Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala Leu
610 615 620
Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys
625 630 635 640
Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala
645 650 655
Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu Ile
660 665 670
Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 691
<211> 683
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 691
Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys Glu
1 5 10 15
Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala
20 25 30
Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu
35 40 45
Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr
50 55 60
Pro Met Ser Arg Glu Ala Arg Leu Gly Ile Lys Pro His Ile Arg Arg
65 70 75 80
Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn Thr
85 90 95
Pro Leu Arg Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val
100 105 110
Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro Thr
115 120 125
Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His Gln
130 135 140
Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu
145 150 155 160
His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu
165 170 175
Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe
180 185 190
Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu Ala
195 200 205
Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp
210 215 220
Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr
225 230 235 240
Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala
245 250 255
Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu
260 265 270
Leu Lys Glu Gly Gln Arg Trp Arg Thr Asp Ala Arg Lys Glu Thr Val
275 280 285
Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe Leu
290 295 300
Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu Met
305 310 315 320
Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn Trp
325 330 335
Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu
340 345 350
Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu
355 360 365
Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys
370 375 380
Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp
385 390 395 400
Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile
405 410 415
Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu
420 425 430
Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro Pro
435 440 445
Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu Leu
450 455 460
Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn Pro
465 470 475 480
Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys Leu
485 490 495
Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp Gln
500 505 510
Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser Leu
515 520 525
Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Thr
530 535 540
Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln Arg
545 550 555 560
Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly Lys
565 570 575
Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala His
580 585 590
Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly
595 600 605
Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala Leu
610 615 620
Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys
625 630 635 640
Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala
645 650 655
Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu Ile
660 665 670
Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 692
<211> 683
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 692
Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys Glu
1 5 10 15
Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala
20 25 30
Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu
35 40 45
Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr
50 55 60
Pro Met Ser Arg Glu Ala Arg Leu Gly Ile Lys Pro His Ile Gln Arg
65 70 75 80
Leu Arg Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn Thr
85 90 95
Pro Leu Arg Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val
100 105 110
Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro Thr
115 120 125
Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His Gln
130 135 140
Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu
145 150 155 160
His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu
165 170 175
Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe
180 185 190
Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu Ala
195 200 205
Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp
210 215 220
Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr
225 230 235 240
Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala
245 250 255
Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu
260 265 270
Leu Lys Glu Gly Gln Arg Trp Leu Thr Asp Ala Arg Lys Glu Thr Val
275 280 285
Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe Leu
290 295 300
Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu Met
305 310 315 320
Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn Trp
325 330 335
Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu
340 345 350
Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu
355 360 365
Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys
370 375 380
Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp
385 390 395 400
Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile
405 410 415
Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu
420 425 430
Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro Pro
435 440 445
Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu Leu
450 455 460
Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn Pro
465 470 475 480
Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys Leu
485 490 495
Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp Gln
500 505 510
Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser Leu
515 520 525
Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Thr
530 535 540
Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln Arg
545 550 555 560
Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly Lys
565 570 575
Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala His
580 585 590
Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly
595 600 605
Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala Leu
610 615 620
Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys
625 630 635 640
Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala
645 650 655
Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu Ile
660 665 670
Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 693
<211> 683
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 693
Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys Glu
1 5 10 15
Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala
20 25 30
Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu
35 40 45
Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr
50 55 60
Pro Met Ser Arg Glu Ala Arg Leu Gly Ile Lys Pro His Ile Arg Arg
65 70 75 80
Leu Arg Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn Thr
85 90 95
Pro Leu Arg Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val
100 105 110
Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro Thr
115 120 125
Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His Gln
130 135 140
Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu
145 150 155 160
His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu
165 170 175
Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe
180 185 190
Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu Ala
195 200 205
Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp
210 215 220
Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr
225 230 235 240
Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala
245 250 255
Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu
260 265 270
Leu Lys Glu Gly Gln Arg Trp Leu Thr Asp Ala Arg Lys Glu Thr Val
275 280 285
Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Gln Leu Arg Glu Phe Leu
290 295 300
Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu Met
305 310 315 320
Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn Trp
325 330 335
Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu
340 345 350
Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu
355 360 365
Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys
370 375 380
Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp
385 390 395 400
Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile
405 410 415
Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu
420 425 430
Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro Pro
435 440 445
Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu Leu
450 455 460
Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn Pro
465 470 475 480
Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys Leu
485 490 495
Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp Gln
500 505 510
Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser Leu
515 520 525
Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Thr
530 535 540
Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln Arg
545 550 555 560
Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly Lys
565 570 575
Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala His
580 585 590
Ile His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly
595 600 605
Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala Leu
610 615 620
Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys
625 630 635 640
Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala
645 650 655
Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu Ile
660 665 670
Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 694
<211> 683
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 694
Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys Glu
1 5 10 15
Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala
20 25 30
Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu
35 40 45
Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr
50 55 60
Pro Met Ser Arg Glu Ala Arg Leu Gly Ile Lys Pro His Ile Arg Arg
65 70 75 80
Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn Thr
85 90 95
Pro Leu Arg Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val
100 105 110
Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro Thr
115 120 125
Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His Gln
130 135 140
Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu
145 150 155 160
His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu
165 170 175
Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe
180 185 190
Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu Ala
195 200 205
Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp
210 215 220
Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr
225 230 235 240
Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala
245 250 255
Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu
260 265 270
Leu Lys Glu Gly Gln Arg Trp Leu Thr Asp Ala Arg Lys Glu Thr Val
275 280 285
Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Glu Leu Arg Glu Phe Leu
290 295 300
Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu Met
305 310 315 320
Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn Trp
325 330 335
Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu
340 345 350
Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu
355 360 365
Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys
370 375 380
Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp
385 390 395 400
Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile
405 410 415
Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu
420 425 430
Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro Pro
435 440 445
Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu Leu
450 455 460
Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn Pro
465 470 475 480
Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys Leu
485 490 495
Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp Gln
500 505 510
Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser Leu
515 520 525
Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Thr
530 535 540
Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln Arg
545 550 555 560
Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly Lys
565 570 575
Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala His
580 585 590
Glu His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly
595 600 605
Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala Leu
610 615 620
Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys
625 630 635 640
Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala
645 650 655
Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu Ile
660 665 670
Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 695
<211> 683
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 695
Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys Glu
1 5 10 15
Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala
20 25 30
Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu
35 40 45
Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr
50 55 60
Pro Met Ser Arg Glu Ala Arg Leu Gly Ile Lys Pro His Ile Arg Arg
65 70 75 80
Leu Arg Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn Thr
85 90 95
Pro Leu Arg Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val
100 105 110
Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro Thr
115 120 125
Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His Gln
130 135 140
Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu
145 150 155 160
His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu
165 170 175
Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe
180 185 190
Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu Ala
195 200 205
Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp
210 215 220
Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr
225 230 235 240
Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala
245 250 255
Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu
260 265 270
Leu Lys Glu Gly Gln Arg Trp Leu Thr Asp Ala Arg Lys Glu Thr Val
275 280 285
Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Glu Leu Arg Glu Phe Leu
290 295 300
Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu Met
305 310 315 320
Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn Trp
325 330 335
Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu
340 345 350
Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu
355 360 365
Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys
370 375 380
Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp
385 390 395 400
Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile
405 410 415
Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu
420 425 430
Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro Pro
435 440 445
Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu Leu
450 455 460
Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn Pro
465 470 475 480
Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys Leu
485 490 495
Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp Gln
500 505 510
Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser Leu
515 520 525
Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Thr
530 535 540
Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln Arg
545 550 555 560
Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly Lys
565 570 575
Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala His
580 585 590
Glu His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly
595 600 605
Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala Leu
610 615 620
Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys
625 630 635 640
Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala
645 650 655
Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu Ile
660 665 670
Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 696
<211> 683
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 696
Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys Glu
1 5 10 15
Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala
20 25 30
Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu
35 40 45
Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr
50 55 60
Pro Met Ser Arg Glu Ala Arg Leu Gly Ile Lys Pro His Ile Arg Arg
65 70 75 80
Leu Leu Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn Thr
85 90 95
Pro Leu Arg Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val
100 105 110
Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro Thr
115 120 125
Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His Gln
130 135 140
Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu
145 150 155 160
His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu
165 170 175
Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe
180 185 190
Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu Ala
195 200 205
Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp
210 215 220
Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr
225 230 235 240
Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala
245 250 255
Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu
260 265 270
Leu Lys Glu Gly Gln Arg Trp Leu Thr Asp Ala Arg Lys Glu Thr Val
275 280 285
Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Glu Leu Arg Glu Phe Leu
290 295 300
Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu Met
305 310 315 320
Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn Trp
325 330 335
Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu
340 345 350
Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu
355 360 365
Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys
370 375 380
Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp
385 390 395 400
Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile
405 410 415
Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu
420 425 430
Arg Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro Pro
435 440 445
Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu Leu
450 455 460
Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn Pro
465 470 475 480
Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys Leu
485 490 495
Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp Gln
500 505 510
Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser Leu
515 520 525
Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Thr
530 535 540
Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln Arg
545 550 555 560
Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly Lys
565 570 575
Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala His
580 585 590
Glu His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly
595 600 605
Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala Leu
610 615 620
Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys
625 630 635 640
Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala
645 650 655
Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu Ile
660 665 670
Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 697
<211> 683
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 697
Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys Glu
1 5 10 15
Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala
20 25 30
Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu
35 40 45
Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr
50 55 60
Pro Met Ser Arg Glu Ala Arg Leu Gly Ile Lys Pro His Ile Arg Arg
65 70 75 80
Leu Arg Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn Thr
85 90 95
Pro Leu Arg Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val
100 105 110
Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro Thr
115 120 125
Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His Gln
130 135 140
Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu
145 150 155 160
His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu
165 170 175
Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe
180 185 190
Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu Ala
195 200 205
Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp
210 215 220
Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr
225 230 235 240
Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala
245 250 255
Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu
260 265 270
Leu Lys Glu Gly Gln Arg Trp Leu Thr Asp Ala Arg Lys Glu Thr Val
275 280 285
Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Glu Leu Arg Glu Phe Leu
290 295 300
Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu Met
305 310 315 320
Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Thr Leu Phe Asn Trp
325 330 335
Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu
340 345 350
Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu
355 360 365
Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys
370 375 380
Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp
385 390 395 400
Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile
405 410 415
Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu
420 425 430
Arg Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro Pro
435 440 445
Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu Leu
450 455 460
Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn Pro
465 470 475 480
Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys Leu
485 490 495
Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp Gln
500 505 510
Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser Leu
515 520 525
Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Thr
530 535 540
Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln Arg
545 550 555 560
Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly Lys
565 570 575
Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala His
580 585 590
Glu His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly
595 600 605
Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala Leu
610 615 620
Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys
625 630 635 640
Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala
645 650 655
Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu Ile
660 665 670
Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 698
<211> 683
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 698
Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys Glu
1 5 10 15
Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala
20 25 30
Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu
35 40 45
Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr
50 55 60
Pro Met Ser Arg Glu Ala Arg Leu Gly Ile Lys Pro His Ile Arg Arg
65 70 75 80
Leu Arg Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn Thr
85 90 95
Pro Leu Arg Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val
100 105 110
Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro Thr
115 120 125
Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His Gln
130 135 140
Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu
145 150 155 160
His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu
165 170 175
Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe
180 185 190
Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu Ala
195 200 205
Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp
210 215 220
Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr
225 230 235 240
Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala
245 250 255
Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu
260 265 270
Leu Lys Glu Gly Gln Arg Trp Leu Thr Asp Ala Arg Lys Glu Thr Val
275 280 285
Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Glu Leu Arg Glu Phe Leu
290 295 300
Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu Met
305 310 315 320
Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Glu Leu Phe Asn Trp
325 330 335
Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu
340 345 350
Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu
355 360 365
Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys
370 375 380
Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp
385 390 395 400
Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile
405 410 415
Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu
420 425 430
Val Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro Pro
435 440 445
Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu Leu
450 455 460
Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn Pro
465 470 475 480
Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys Leu
485 490 495
Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp Gln
500 505 510
Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser Leu
515 520 525
Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Thr
530 535 540
Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln Arg
545 550 555 560
Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly Lys
565 570 575
Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala His
580 585 590
Glu His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly
595 600 605
Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala Leu
610 615 620
Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys
625 630 635 640
Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala
645 650 655
Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu Ile
660 665 670
Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
<210> 699
<211> 683
<212> PRT
<213> Artificial Sequence
<220>
<223> RTase mutant
<400> 699
Thr Leu Asn Ile Glu Asp Glu His Arg Leu His Glu Thr Ser Lys Glu
1 5 10 15
Pro Asp Val Ser Leu Gly Ser Thr Trp Leu Ser Asp Phe Pro Gln Ala
20 25 30
Trp Ala Glu Thr Gly Gly Met Gly Leu Ala Val Arg Gln Ala Pro Leu
35 40 45
Ile Ile Pro Leu Lys Ala Thr Ser Thr Pro Val Ser Ile Lys Gln Tyr
50 55 60
Pro Met Ser Arg Glu Ala Arg Leu Gly Ile Lys Pro His Ile Arg Arg
65 70 75 80
Leu Arg Asp Gln Gly Ile Leu Val Pro Cys Gln Ser Pro Trp Asn Thr
85 90 95
Pro Leu Arg Pro Val Lys Lys Pro Gly Thr Asn Asp Tyr Arg Pro Val
100 105 110
Gln Asp Leu Arg Glu Val Asn Lys Arg Val Glu Asp Ile His Pro Thr
115 120 125
Val Pro Asn Pro Tyr Asn Leu Leu Ser Gly Leu Pro Pro Ser His Gln
130 135 140
Trp Tyr Thr Val Leu Asp Leu Lys Asp Ala Phe Phe Cys Leu Arg Leu
145 150 155 160
His Pro Thr Ser Gln Pro Leu Phe Ala Phe Glu Trp Arg Asp Pro Glu
165 170 175
Met Gly Ile Ser Gly Gln Leu Thr Trp Thr Arg Leu Pro Gln Gly Phe
180 185 190
Lys Asn Ser Pro Thr Leu Phe Asp Glu Ala Leu His Arg Asp Leu Ala
195 200 205
Asp Phe Arg Ile Gln His Pro Asp Leu Ile Leu Leu Gln Tyr Val Asp
210 215 220
Asp Leu Leu Leu Ala Ala Thr Ser Glu Leu Asp Cys Gln Gln Gly Thr
225 230 235 240
Arg Ala Leu Leu Gln Thr Leu Gly Asn Leu Gly Tyr Arg Ala Ser Ala
245 250 255
Lys Lys Ala Gln Ile Cys Gln Lys Gln Val Lys Tyr Leu Gly Tyr Leu
260 265 270
Leu Lys Glu Gly Gln Arg Trp Leu Thr Asp Ala Arg Lys Glu Thr Val
275 280 285
Met Gly Gln Pro Thr Pro Lys Thr Pro Arg Glu Leu Arg Glu Phe Leu
290 295 300
Gly Thr Ala Gly Phe Cys Arg Leu Trp Ile Pro Gly Phe Ala Glu Met
305 310 315 320
Ala Ala Pro Leu Tyr Pro Leu Thr Lys Thr Gly Glu Leu Phe Asn Trp
325 330 335
Gly Pro Asp Gln Gln Lys Ala Tyr Gln Glu Ile Lys Gln Ala Leu Leu
340 345 350
Thr Ala Pro Ala Leu Gly Leu Pro Asp Leu Thr Lys Pro Phe Glu Leu
355 360 365
Phe Val Asp Glu Lys Gln Gly Tyr Ala Lys Gly Val Leu Thr Gln Lys
370 375 380
Leu Gly Pro Trp Arg Arg Pro Val Ala Tyr Leu Ser Lys Lys Leu Asp
385 390 395 400
Pro Val Ala Ala Gly Trp Pro Pro Cys Leu Arg Met Val Ala Ala Ile
405 410 415
Ala Val Leu Thr Lys Asp Ala Gly Lys Leu Thr Met Gly Gln Pro Leu
420 425 430
Arg Ile Leu Ala Pro His Ala Val Glu Ala Leu Val Lys Gln Pro Pro
435 440 445
Asp Arg Trp Leu Ser Asn Ala Arg Met Thr His Tyr Gln Ala Leu Leu
450 455 460
Leu Asp Thr Asp Arg Val Gln Phe Gly Pro Val Val Ala Leu Asn Pro
465 470 475 480
Ala Thr Leu Leu Pro Leu Pro Glu Glu Gly Leu Gln His Asn Cys Leu
485 490 495
Asp Ile Leu Ala Glu Ala His Gly Thr Arg Pro Asp Leu Thr Asp Gln
500 505 510
Pro Leu Pro Asp Ala Asp His Thr Trp Tyr Thr Gly Gly Ser Ser Leu
515 520 525
Leu Gln Glu Gly Gln Arg Lys Ala Gly Ala Ala Val Thr Thr Glu Thr
530 535 540
Glu Val Ile Trp Ala Lys Ala Leu Pro Ala Gly Thr Ser Ala Gln Arg
545 550 555 560
Ala Gln Leu Ile Ala Leu Thr Gln Ala Leu Lys Met Ala Glu Gly Lys
565 570 575
Lys Leu Asn Val Tyr Thr Asn Ser Arg Tyr Ala Phe Ala Thr Ala His
580 585 590
Glu His Gly Glu Ile Tyr Arg Arg Arg Gly Leu Leu Thr Ser Glu Gly
595 600 605
Lys Glu Ile Lys Asn Lys Asp Glu Ile Leu Ala Leu Leu Lys Ala Leu
610 615 620
Phe Leu Pro Lys Arg Leu Ser Ile Ile His Cys Pro Gly His Gln Lys
625 630 635 640
Gly His Ser Ala Glu Ala Arg Gly Asn Arg Met Ala Asp Gln Ala Ala
645 650 655
Arg Lys Ala Ala Ile Thr Glu Thr Pro Asp Thr Ser Thr Leu Leu Ile
660 665 670
Glu Asn Ser Ser Pro Tyr Thr Ser Glu His Phe
675 680
Claims (16)
1.一种包含SEQ ID NO:637的氨基酸序列的分离的莫洛尼氏鼠白血病病毒(MMLV)逆转录酶(RT酶)突变体，其中所述MMLV RT酶突变体的氨基酸序列进一步包含至少一个氨基酸取代，所述至少一个氨基酸取代是：
(a)位置61处的异亮氨酸至精氨酸取代(I61R)；
(b)位置68处的谷氨酰胺至精氨酸取代(Q68R)；
(c)位置79处的谷氨酰胺至精氨酸取代(Q79R)；
(d)位置99处的亮氨酸至精氨酸取代(L99R)；
(e)位置282处的谷氨酸至天冬氨酸取代(E282D)；
(f)位置298处的精氨酸至丙氨酸取代(R298A)；
(g)位置299处的谷氨酰胺至谷氨酸取代(Q299E)；
(h)位置332处的苏氨酸至谷氨酸取代(T332E)；
(i)位置433处的缬氨酸至精氨酸取代(V433R)；和/或
(j).位置593处的异亮氨酸至谷氨酸取代(I593E)。
2.根据权利要求1所述的分离的MMLV RT酶突变体，其中所述MMLV RT酶突变体包含SEQID NO:637-699中任一个所示的氨基酸序列。
3.根据权利要求2所述的分离的MMLV RT酶突变体，其中所述MMLV RT酶突变体包含SEQID NO:674所示的氨基酸序列。
4.一种包含SEQ ID NO:637的氨基酸序列的分离的莫洛尼氏鼠白血病病毒(MMLV)逆转录酶(RT酶)突变体，其中所述MMLV RT酶突变体的氨基酸序列进一步包含至少两个氨基酸取代，所述至少两个氨基酸取代是：
(a)位置61处的异亮氨酸至精氨酸取代和位置282处的谷氨酸至天冬氨酸取代(I61R/E282D)；
(b)取代位置99处的亮氨酸至精氨酸和位置282处的谷氨酸至天冬氨酸取代(L99R/E282D)；
(c)位置68处的谷氨酰胺至精氨酸取代和位置282处的谷氨酸至天冬氨酸取代(Q68R/E282D)；
(d)位置79处的谷氨酰胺至精氨酸取代和位置282处的谷氨酸至天冬氨酸取代(Q79R/E282D)；
(e)位置282处的谷氨酸至天冬氨酸取代和位置298处的精氨酸至丙氨酸取代(E282D/R298A)；
(f)位置61处的异亮氨酸至精氨酸取代和位置99处的亮氨酸至精氨酸取代(I61R/L99R)；
(g)位置61处的异亮氨酸至精氨酸取代和位置68处的谷氨酰胺至精氨酸取代(I61R/Q68R)；
(h)位置61处的异亮氨酸至精氨酸取代和位置79处的谷氨酰胺至精氨酸取代(I61R/Q79R)；
(i)位置61处的异亮氨酸至精氨酸取代和位置298处的精氨酸至丙氨酸取代(I61R/R298A)；
(j)位置68处的谷氨酰胺至精氨酸取代和位置99处的亮氨酸至精氨酸取代(Q68R/L99R)；
(k)位置79处的谷氨酰胺至精氨酸取代和位置99处的亮氨酸至精氨酸取代(Q79R/L99R)；
(l)取代位置99处的亮氨酸至精氨酸和位置298处的精氨酸至丙氨酸取代(L99R/R298A)；
(m)位置68处的谷氨酰胺至精氨酸取代和位置79处的谷氨酰胺至精氨酸取代(Q68R/Q79R)；
(n)位置68处的谷氨酰胺至精氨酸取代和位置298处的精氨酸至丙氨酸取代(Q68R/R298A)；或
(o)位置79处的谷氨酰胺至精氨酸取代和位置298处的精氨酸至丙氨酸取代(Q79R/R298A)。
5.根据权利要求4所述的分离的MMLV RT酶突变体，其中所述MMLV RT酶突变体包含SEQID NO:637-699中一个或多个的氨基酸序列。
6.根据权利要求1或4所述的MMLV RT酶突变体，其中所述MMLV RT酶突变体缺乏RNA酶H活性。
7.根据权利要求1或4所述的MMLV RT酶突变体，其中所述MMLV RT酶突变体具有以下特征中的至少一种：增强的DNA合成、增加的保真度或增强的热稳定性。
8.一种分离的核酸分子，所述分离的核酸分子包含编码根据权利要求1或4所述的MMLVRT酶突变体的核苷酸序列。
9.一种组合物，所述组合物包含根据权利要求1或4所述的分离的MMLV RT酶突变体。
10.根据权利要求9所述的组合物，其中所述分离的MMLV RT酶突变体缺乏RNA酶H活性。
11.根据权利要求9所述的组合物，其中所述分离的MMLV RT酶突变体具有以下特征中的至少一种：增强的DNA合成、增加的保真度或增强的热稳定性。
12.一种试剂盒，所述试剂盒包含根据权利要求1或4所述的分离的MMLV RT酶突变体。
13.根据权利要求12所述的试剂盒，其中所述分离的MMLV RT酶突变体缺乏RNA酶H活性。
14.根据权利要求12所述的试剂盒，其中所述分离的MMLV RT酶突变体具有以下特征中的至少一种：增强的DNA合成、增加的保真度或增强的热稳定性。
15.一种用于合成互补脱氧核糖核酸(cDNA)的方法，所述方法包括：
(a)提供根据权利要求1或4所述的分离的MMLV RT酶突变体；以及
(b)使所述分离的MMLV RT酶突变体与核酸模板接触以允许合成cDNA。
16.一种用于进行逆转录-聚合酶链式反应(RT-PCR)的方法，所述方法包括：
(a)提供根据权利要求1或4所述的分离的MMLV RT酶突变体；以及
(b)使所述分离的MMLV RT酶突变体与核酸模板接触以复制和扩增所述核酸模板。
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