Patent ID: 8911999

Claim:
A method of producing a selectable marker gene-free plasmid comprising the steps of: a) culturing a plasmid containing a selectable marker gene flanked by site specific recombinase target sites selected from Ecdif, cer, psi, pif and mwr in a first host cell environment which is incapable of effecting recombination between the site specific recombinase target sites, wherein the first host cell environment comprises an inactivating mutation in one or more of the genes encoding PepA, ArgR and ArcA; and b) subsequently culturing the plasmid in a second host cell environment which is capable of effecting recombination between the site specific recombinase target sites, such that the selectable marker gene is excised, wherein the second host cell environment contains active versions of PepA and ArgR or ArcA, and comprises a site specific recombinase selected from XerC and XerD.