In the present study, a donor plasmid derived from pUC19 and two recipient plasmids, which had been modified from the donor plasmid and contained the red fluorescence protein gene mCherry as a reporter gene downstream of the hybrid tac promoter with the -35 region deletion mutation, were constructed. The complete genome sequence of coxsackievirus A10 downstream of the T7 promoter was divided into 7 fragments and synthesized by overlap extension PCR and the DNAworks program. Using the Golden Gate cloning strategy, the 7 fragments were then cloned into the donor plasmid and transferred to the recipient plasmid upstream of the deletion mutation tac promoter in a defined order and orientation without any deletions or insertions at the junction sites. Because the -35 region of the tac promoter was introduced into the 3' end of the last fragment during construction, the hybrid promoter was reconstructed to promote expression of mCherry, which facilitated the selection of colonies with the complete genome of coxsackievirus A10 to generate an infectious cDNA clone via reverse genetic engineering.

中文翻译：

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基于金门克隆结合启动子重建的柯萨奇病毒A10全基因组从头合成


在本研究中，来自pUC19的供体质粒和两个受体质粒，其已从供体质粒修改并包含红色荧光蛋白基因mCherry作为具有-35区域缺失突变的杂合tac启动子下游的报告基因，被建造。将柯萨奇病毒A10 T7启动子下游的完整基因组序列分为7个片段，并通过重叠延伸PCR和DNAworks程序合成。使用 Golden Gate 克隆策略，将 7 个片段克隆到供体质粒中，并以确定的顺序和方向转移到缺失突变 tac 启动子上游的受体质粒，在连接位点没有任何删除或插入。由于在构建时将tac启动子的-35区域引入到最后一个片段的3'端，因此重建了杂合启动子以促进mCherry的表达，这有利于选择具有柯萨奇病毒A10完整基因组的菌落，以产生通过反向基因工程克隆感染性 cDNA。