T-vectors have been widely used in molecular cloning [1-3].Compared with common vectors,molecular cloning with T-vectors escapes the restriction endonuclease digestion process,decreases the time of DNA purification and thus is time-saving and cost-effective.There are two ways to produce T-vectors:(i) cutting the target vector with EcoRV and then adding a thymine residue to the 3’-blunt end with Taq DNA polymerase [4];and (ii) inserting a DNA cassette containing XcmI or AhdI recognition sites at its both ends and then splitting the recombinant vector with them later [5-7].However,only a small amount of the T-vector can be produced by these methods.What’s more,the residual undigested primary vector may cause false-positive clones.Therefore,T-vectors used in labs are usually bought from biotechnology companies providing with small amounts,thus limiting their application in conventional molecular cloning experiments.