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Construction of linear functional expression elements with DNA fragments created by site-specific DNA nickase, N.Bpu10 I, and exonuclease III.
A method to assemble linear expression elements for rapid gene expression is described. Primers containing target specific sequences and N.Bpu10 I nickase recognition sites were used to amplify promoter, open reading frame and terminator fragments. Amplified fragments were treated with N.Bpu10 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.Wen Xin, Yi-Ming Zhang, Jin-Hua Xiao, Da-Wei Huang
