文献类型： 外文期刊
作者： Gao, XinZheng 1 ; Yan, Pu 2 ; Shen, Wentao 2 ; Li, Xiaoying 2 ; Zhou, Peng 2 ; Li, Yuenan 1 ;
作者机构： 1.Hainan Med Univ, Coll Sci, Haikou 571101, Peoples R China
2.Chinese Acad Trop Agr Sci, Inst Trop Biosci & Biotechnol, Anal & Testing Ctr, Haikou 571101, Peoples R China
关键词： Genetic module;Golden Gate cloning;Linear DNA;Parallel assembly;Plasmid construction
期刊名称：ANALYTICAL BIOCHEMISTRY （ 影响因子：3.365； 五年影响因子：3.049 ）
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收录情况： SCI
摘要： Construction of plasmids is the basic and pivotal technology in molecular biology. The common method for constructing plasmids is to cut DNA fragments by restriction enzymes and then join the resulting fragments using ligase. We present here a modified Golden Gate cloning method for modular construction of plasmids. Unlike the original Golden Gate cloning system for cloning from entry vector to expression vector, this method can be used to construct plasmids immediately from linear DNA fragments. After polymerase chain reaction (PCR) amplification for flanking with BsaI sites, multiple linear DNA components (modules) can be parallel assembled into a circle plasmid by a single restriction-ligation reaction using the method. This method is flexible to construct different types of plasmids because the modules can be freely selected and assembled in any combination. This method was applied successfully to construct a prokaryotic expression plasmid from four modules and a plant expression plasmid from five modules (fragments). The results suggest that this method provides a simple and flexible platform for modular construction of plasmids.
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