Plasmid maps for pIMC and IPTG inducible marker expression in pIMC3. The plasmid (A) pIMC was created by SOE PCR (see Material and Methods) from oligonucleotides described in Table 1. The backbone of pIMC, derived from pPL2  encodes the p15A low copy E. coli origin of replication and RP4 conjugative origin of transfer. No gram-positive origin of replication is present on the plasmid, therefore upon transformation, chromosomal integration into L. monocytogenes tRNAARG is directed by the Listeriophage PSA integrase. Antibiotic selection is supplied by the chloramphenicol acetytransferase (cat) fused to the highly expressed listerial promoter (Phelp) . Restriction sites labelled on pIMC are unique and for sequencing purposes, T3 and T7 primer binding sites are present before the Kpn I and after the Sac I restriction sites, respectively. The plasmid sequence is accessible under the EMBL nucleotide accession number AM940001. A comparison of chloramphenicol selection (7.5 μg/ml) is shown in (B), with pIMC exhibiting uniform colony size compared to pPL2 transformed EGDe. (C) Antibiotic markers (kanamycin (aphA3), erythromycin (ermAM) and tetracycline (tetM)) and the beta-glucuronidase marker (gusA) were subcloned from pIMK3 into pIMCa as a Sac I/Pst I fragment (see Materials and Methods).