Transformation-associated recombination(TAR)has been widely used to assemble large DNA constructs.One of the significant obstacles hindering assembly efficiency is the presence of error-prone DNA repair pathways in yeast,which results in vector backbone recircularization or illegitimate recombination products.To increase TAR assembly efficiency,we prepared a dual-selective TAR vector,pGFCS,by adding a PADH1-URA3 cassette to a previously described yeast-bacteria shuttle vector,pGF,harboring a PHIS3-HIS3 cassette as a positive selection marker.This new cassette works as a negative selection marker to ensure that yeast harboring a recircularized vector cannot propagate in the presence of 5-fluoroorotic acid.To prevent pGFCS bearing ura3 from recombining with endogenous ura3-52 in the yeast genome,a highly transformable Saccharomyces cerevisiae strain,VL6-48B,was prepared by chromosomal substitution of ura3-52 with a transgene conferring resistance to blasticidin.A 55-kb genomic fragment of monkeypox virus encompassing primary detection targets for quantitative PCR was assembled by TAR using pGFCS in VL6-48B.The pGFCS-mediated TAR assembly showed a zero rate of vector recircularization and an average correct assembly yield of 79％indicating that the dual-selection strategy provides an efficient approach to optimizing TAR assembly.

State Key Laboratory of Virology,Wuhan Institute of Virology,Center for Biosafety Mega-Science,Chinese Academy of Sciences,Wuhan,430071,China;University of Chinese Academy of Sciences,Beijing 100049,China

[1]Lei Yang;Lingqian Tian;Leshan Li;Qiuhong Liu;Xiang Guo;Yuan Zhou;Rongjuan Pei;Xinwen Chen;Yun Wang-.Efficient assembly of a large fragment of monkeypox virus genome as a qPCR template using dual-selection based transformation-associated recombination)[J].中国病毒学,2022(03):341-347

recircularization,illegitimate,pGFCS,PADH1,URA3,pGF,PHIS3,HIS3,recircularized,fluoroorotic,ura3,VL6,48B,blasticidin

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