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Plasmid. 2019 May;103:17-24. doi: 10.1016/j.plasmid.2019.03.001. Epub 2019 Mar 27.

De novo synthesis of the complete genome of coxsackievirus A10 based on Golden Gate cloning combined with promoter reconstruction.

Author information

1
China National Biotech Group, Wuhan Institute of Biological Products Co., Ltd., Wuhan, People's Republic of China. Electronic address: 2008limeng.cool@163.com.
2
China National Biotech Group, Wuhan Institute of Biological Products Co., Ltd., Wuhan, People's Republic of China.

Abstract

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.

KEYWORDS:

Blue-white screening; Coxsackievirus cDNA genome synthesis; Golden Gate cloning; Promoter reconstruction; Red-white screening

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