Map of RK2 showing region replaced by the anti-F cassette and showing the targets of the anti-F functions.

The other functions marked are: oriV, the vegetative replication origin; oriT, the transfer origin; tra and trb regions encoding proteins for DNA processing and mating bridge formation during transfer; trfA, encoding the replication protein that activates oriV; ccr/par, the central control region that regulates transcription of RK2 backbone genes and also encodes active partitioning functions; psk/mrs, encoding addiction and multimer resolution functions. Mobile elements Tn1 and IS21 are associated with ampicillin and kanamycin resistance genes. Recombineering was used to replace aphA and IS21 in RK2 with the anti-F cassette that blocks the repFIA, repFIB and repFII replicons and neutralises the effect of the flmABC and letAB addiction loci [12].