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DNA which the code does the cloning of the SnaBI restriction endonuclease and the SnaBI restriction endonuclease which includes the array of mannered

机译:编码序列的DNA可克隆SnaBI限制性核酸内切酶和SnaBI限制性核酸内切酶,包括一系列引物

摘要

The methylase selection method was used to clone the SnaBI methylase gene (snaBIM) from Sphaerotilus natans (ATCC 15291). An active SnaBI methylase was cloned in E. coli using pSnaBI-2, a pUC19 derivative containing two SnaBI sites. Because methylase and restriction genes are usually located alongside each other in restriction-modification systems, efforts were made to clone the downstream DNA by inverse PCR. The missing portion of the SnaBI endonuclease was cloned by inverse PCR. A control, or C, protein was identified using the same technique. The two open reading frames snaBIR (ORF1) and snaBIC (ORF2) were orientated towards the SnaBI methylase gene (ORF). Attempts to establish a snaBIR-recombinant plasmid expressing the SnaBI endonuclease in E.coli modified with SnaBI methylase failed. Overexpression of the SnaBI endonuclease in E. coli required the use of the heterospecific BsaAI methylase. IMAGE
机译:甲基化酶的选择方法被用于克隆来自Sphaerotilus natans(ATCC 15291)的SnaBI甲基化酶基因(snaBIM)。使用pSnaBI-2(一种含有两个SnaBI位点的pUC19衍生物)将活性SnaBI甲基化酶克隆到大肠杆菌中。由于甲基酶和限制性基因通常在限制性修饰系统中并排放置,因此人们努力通过反向PCR克隆下游DNA。通过反向PCR克隆了SnaBI核酸内切酶的缺失部分。使用相同的技术鉴定了对照蛋白或C蛋白。两个开放阅读框snaBIR(ORF1)和snaBIC(ORF2)面向SnaBI甲基化酶基因(ORF)。试图建立在SnaBI甲基化酶修饰的大肠杆菌中表达SnaBI核酸内切酶的snaBIR重组质粒的尝试失败了。 SnaBI核酸内切酶在大肠杆菌中的过表达需要使用异源特异性BsaAI甲基化酶。 <图像>

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