Schematic representation of cloning steps leading to the construction of the integration vector pLox2.

An amplicon resulting from the amplification of a genomic fragment was first subcloned into pLME vector in order to allow the integration of the loxP-ermB-loxP-tetM cassette in the intergenic region between loci ef_1597 and ef_1598. The resulting plasmid, pLox0, is then used to add a loxP-tetM cassette between these both genes. The obtained vector was named pLox1. A cassette ermB-loxP was finally cloned into pLox1 to obtain the integration plasmid pLox2. Only relevant restriction sites are indicated. ColE1 = colE1 origin; cat = CmR encoding gene from pNZ7125 [17]; ermB = erythromycin rRNA methylase gene from pUCB30 [51]; tetM = TetR encoding gene from p3Tet [27]. Thin arrows represent primers used in these constructions.