Background: Introduction of new antibiotic resistance genes in the plasmids of interest is a frequent task in molecular cloning practice. Classical approaches involving digestion with restriction endonucleases and ligation are time-consuming.
Findings: We have created a set of insertion vectors (pINS) carrying genes that provide resistance to various antibiotics (puromycin, blasticidin and G418) and containing a loxP site. Each vector (pINS-Puro, pINS-Blast or pINS-Neo) contains either a chloramphenicol or a kanamycin resistance gene and is unable to replicate in most E. coli strains as it contains a conditional R6Kgamma replication origin. Introduction of the antibiotic resistance genes into the vector of interest is achieved by Cre-mediated recombination between the replication-incompetent pINS and a replication-competent target vector. The recombination mix is then transformed into E. coli and selected by the resistance marker (kanamycin or chloramphenicol) present in pINS, which allows to recover the recombinant plasmids with 100% efficiency.
Conclusion: Here we propose a simple strategy that allows to introduce various antibiotic-resistance genes into any plasmid containing a replication origin, an ampicillin resistance gene and a loxP site.