A novel vector for positive selection of inserts harboring an open reading frame by translational coupling

Abstract

We have developed a novel vector, pTCS, as a tool for efficient selection of open reading frame (ORF)-containing inserts. In pTCS clones containing an insert with an ORF, a downstream marker gene (immE3, conferring resistance to colicin) is activated via translational coupling with the insert, and transformed cells can then be selected by exposure to colicin E3. Our method differs from previous methods in that the marker gene is activated without protein fusion, and that selection occurs irrespective of the reading frame of the insert.