Plasmids constructed to introduce an unmarked mutation into a large gene of non-competent bacteria. (A, B) Multiple cloning sites (MCS) of pJQ200sk and pK18mob were substituted with that of pLOI2224, generating pJQFRT and pKFRT, respectively. The pJQFRT plasmid contains a single FRT site adjacent to a multiple cloning site; p15A origin, a replication origin of E. coli; oriT, origin of transfer; SacB, a counter-selection marker; and GmR, a gentamicin resistance marker. The arrows indicate the primers used in PCR to amplify the substitute MCS. The nucleotide sequences of these primers are shown in Table 2. (C) A cassette containing tetR- Ptet promoter and flp recombinase amplified by PCR from pFT-A was ligated with the inverse-PCR product of pKFRT. The resultant pKFRT/FLP plasmid contains a single FRT site adjacent to a multiple cloning site; TetR-FLP, flp recombinase gene under the control of the tetR regulation system; KmR, a kanamycin resistance marker; oriT, origin of transfer; and ColE1 origin, a replication origin of E. coli.