pTOC-KR: a positive selection cloning vector based on the ParE toxin

Several prokaryotic cloning vectors have been developed to clone foreign DNA in bacteria. The insertion inactivation of β-galactosidase activity, for instance, is a common screening method of identifying recombinant DNA molecules in many vectors (1,2). However, this system poses several problems. For one, many vectors can self-ligate and give false transformants. Although β-galactosidase is widely used as a screening marker, it is limited to use with specially mutated Escherichia coli hosts for α-complementation. The color of the colonies is also hard to distinguish when the blue/white selection system is used. This system also requires expensive reagents: 5-bromo4-chloro-3-indolyl-β-D-galactosidase (X-Gal) and isopropylthio-β-D-thiogalactopyranoside (IPTG). To address these problems, several positive selection vector systems have been developed based on the toxinantitoxin systems parD (Kis/Kid) of plasmid R1 (3) and on the CcdAB system of plasmid F (4). Similar positive selection cloning vectors using the transcriptional factor GATA-1 (5) or a cellulase gene (CelA) as screening marker (6) have also been developed. Nonetheless, the application of these systems is also limited; in most cases, they are host-limited and their cloning