a vector with two opposite XcmI restriction sites was developed for cloning and expression. This vector was verified by cloning and expressing the 14-3-3ZsGreen and hRBP protein.The DNA sequence of the T vector and the amino acid sequences of expressed proteins were confirmed by Western blot and MALDI-TOF peptide mass fingerprint. By cloning interested gene by using this T vector, we can identify the direction of the insert gene using one particular restriction enzyme digestion process.This methodology is easy to be manipulated to develop suitable constructs for use in various hosts and is appropriate for many kinds of applications because it requires no cofactors and is highly time- and cost-effective.