Site-specific gene cassette insertion by combining artificial restriction DNA cutter and single-stranded DNA specific endonuclease

An artificial restriction DNA cutter (ARCUT), which was composed of Ce(IV)/EDTA complex and a pair of pseudo-complementary peptide nucleic acid, was used for construction of recombinant plasmid vector. The plasmid vector was cut by ARCUT only at target site, and resultant specific overhangs were blunted by treatment with Mung Bean Nuclease and Klenow fragment. This fragment having blunt ends at both termini was ligated with the gene of enhanced green fluorescent protein (EGFP) prepared by PCR. The resultant recombinant vector was translated and the fluorescent protein was successfully expressed in cells. Throughout the manipulation, neither natural restriction enzymes nor homologous recombination was employed. Thus, this strategy has promising applicability to construction of still larger vectors.