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Virologica Sinica, 31 (2) : 160, 2016
Research Article
Assembly of long DNA sequences using a new synthetic Escherichia coli-yeast shuttle vector
State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China
 Correspondence: xiaogf@wh.iov.cn
(5683.38KB)  (137.26KB)  
Abstract
Synthetic biology is a newly developed field of research focused on designing and rebuilding novel biomolecular components, circuits, and networks. Synthetic biology can also help understand biological principles and engineer complex artificial metabolic systems. DNA manipulation on a large genome-wide scale is an inevitable challenge, but a necessary tool for synthetic biology. To improve the methods used for the synthesis of long DNA fragments, here we constructed a novel shuttle vector named pGF (plasmid Genome Fast) for DNA assembly in vivo. The BAC plasmid pCC1BAC, which can accommodate large DNA molecules, was chosen as the backbone. The sequence of the yeast artificial chromosome (YAC) regulatory element CEN6-ARS4 was synthesized and inserted into the plasmid to enable it to replicate in yeast. The selection sequence HIS3, obtained by polymerase chain reaction (PCR) from the plasmid pBS313, was inserted for screening. This new synthetic shuttle vector can mediate the transformation-associated recombination (TAR) assembly of large DNA fragments in yeast, and the assembled products can be transformed into Escherichia coli for further amplification. We also conducted in vivo DNA assembly using pGF and yeast homologous recombination and constructed a 31-kb long DNA sequence from the cyanophage PP genome. Our findings show that this novel shuttle vector would be a useful tool for efficient genome-scale DNA reconstruction.
Received: 21 Jan 2016  Accepted: 17 Mar 2016  Published online: 11 Apr 2016
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