Objective: To construct tetracycline-dependent regulatory system in one vector

which contains two multiple cloning sites(MCS) for the convenient cloning of two target genes.Methods: The tetracycline(Tet)-dependent regulatory fragment(TO)(between NdeI and SacII sites) was amplified from pcDNA4TM/TO vector with PCR

which was inserted into pUC118 vector and sequenced.The counterpart fragment in pVITRO3 was replaced with correct Tet-dependent regulatory fragment

namely pVITRO3-TO vector.The Tet repressor gene(TR) encoding the Tet repressor from pcDNA6/TR vector was incorporating into the backbone of the pVITRO3-TO.Result: The tetracycline(Tet)-dependent regulatory fragment and the Tet repressor gene were amplified successfully with PCR from its template vectors and were subcloned.The clones with correct sequence were selected by sequencing the picked 2 clones.The target vector was constructed successfully by incorporating TO and TR into the backbone of pVITRO3 vector.Conclusion: Tetracycline-dependent regulatory double-expressing system in one vector was constructed initially.