Construction of linear functional expression elements with DNA fragments created by site-specific DNA nickase,N,pu 10 I,and exonuclease III

摘要

A method to assemble linear expression elements for rapid gene expression is described.Primers containing target specific sequences and N.Bpu 10 I nickase recognition sites were used to amplify promoter,open reading frame and terminator ragments.Amplified fragments were treated with N.Bpu 10 I nickase and exonuclease III to generate overhangs for directional ligation.These fragments were ligated and further amplified with element-specific primers.The amplified DNA was transfected into mammalian cells for gene expression.