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首页> 外文期刊>Plasmid: An International Journal Devoted to Extrachromosomal Gene Systems >Vectors for ligation-independent construction of lacZ gene fusions and cloning of PCR products using a nicking endonuclease.
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Vectors for ligation-independent construction of lacZ gene fusions and cloning of PCR products using a nicking endonuclease.

机译：用于连接非依赖性构建lacZ基因融合物和使用切口核酸内切酶克隆PCR产物的载体。

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摘要

Several ligation-independent cloning methods have been developed that offer advantages for construction of recombinant plasmids at high efficiency while minimizing cloning artifacts. Here we report new plasmid vectors that use the nicking endonuclease Nt.BspQI to generate extended single stranded tails for direct cloning of PCR products. The vectors include pLacCOs1, a ColE1-derivative plasmid imparting resistance to ampicillin, which allows facile construction of lacZ translational fusions and pKanCOs1, a pSC101-derivative cloning vector that imparts resistance to kanamycin, for cloning of PCR amplicons from genomic DNA as well as from ampicillin-based plasmids. We have successfully used these plasmids to directionally clone and characterize bacterial promoters that exhibit temperature regulated expression, as well as for cloning a variety of PCR products. In all cases, constructs with the correct configurations were generated at high efficiency and with a minimal number of manipulations. The cloning vectors can also be easily modified to incorporate additional reporter genes or to express epitope-tagged gene products.
机译：已经开发了几种不依赖连接的克隆方法，这些方法为高效构建重组质粒提供了优势，同时最大程度地减少了克隆假象。在这里，我们报告了新的质粒载体，该载体使用切口核酸内切酶Nt.BspQI生成扩展的单链尾巴，以直接克隆PCR产物。载体包括pLacCOs1（对氨苄青霉素具有抗性的ColE1衍生质粒，可轻松构建lacZ翻译融合体）和pKanCOs1（对卡那霉素具有抗性的pSC101衍生克隆载体），用于从基因组DNA以及从基因组DNA克隆PCR扩增子。基于氨苄青霉素的质粒。我们已经成功地使用这些质粒定向克隆并鉴定了表现出温度调节表达的细菌启动子，并克隆了多种PCR产物。在所有情况下，均以高效且最少的操作次数生成具有正确构型的构建体。克隆载体也可以容易地修饰以掺入其他报道基因或表达表位标记的基因产物。

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