Patent ID: 8101355

Claim:
A method for cloning and expressing a target gene by homologous recombination, the method comprising the steps of: (a) performing homologous recombination with a recombinant vector and a linear insert for Target Gene and RBS (iTGR) DNA fragment, wherein the recombinant vector comprises: (i) a promoter, (ii) an upstream ribosome recognition sequence of the vector, (iii) a first selection marker gene having deletions of its ribosome recognition sequence (downstream Ribosome Binding Site) and its start codon, and thus having no expression ability, and (iv) a second selection marker gene that can be expressed, and wherein the linear iTGR DNA fragment comprises: (i) a base sequence of a target gene, (ii) a homologous sequence to a region downstream of the promoter and to the upstream ribosome recognition sequence in said vector, which are located before the base sequence of the target gene, and (iii) a downstream ribosome recognition sequence of the first selection marker gene, a translational start codon, and a homologous sequence to 5′-terminus sequence of the first selection marker gene, which are located behind the base sequence of target gene, and the linear iTGR DNA fragment can be inserted between the upstream ribosome recognition sequence in the vector and the first selection marker gene by the homologous recombination to express the target gene and the first selection marker gene, and (b) expressing the target gene by selecting a microorganism expressing the first selection marker gene and the second selection marker gene as a transformed microorganism introduced with the target gene.