Construction of a restriction-endonuclease-Eam1105I-generated T-vector for high-throughput cloning and expression

A novel T-vector was constructed that could be used for direct cloning and expression of PCR-amplified cDNA. The technique was based on the insertion into the parent vector of two endonuclease-Eam1105I restriction sequences spaced by an expression cassette of the full-length beta-galactosidase, which helped to improve cloning efficiency and to minimize the non-recombinant background of the T-vector when used to clone PCR products. Moreover, this method took advantage of the reconstitution of the rarest restriction sequence of MssI to enable directional cloning. These advantages make the T-vector suitable for high-throughput expression and analysis.