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

Publication date: Available online 27 March 2019Source: PlasmidAuthor(s): Meng Li, Xinguo Li, Xiaoqi Chen, Shuo ShenAbstractIn 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 a T7 promoter was divided into 7 fragments and synthesized with overlap extension PCR and the DNAworks program. Then, using the Golden Gate cloning strategy, the 7 fragments were cloned into the donor plasmid and transferred to the recipient plasmid upstream of the deletion mutation tac promoter in the 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 in the synthetic process, the hybrid promoter was reconstructed and promoted the expression of mCherry, which facilitated the selection of colonies with the complete genome of coxsackievirus A10 to construct the infectious cDNA clone via reverse genetic engineering.

Source: Plasmid - March 28, 2019 Category: Biotechnology Source Type: research