FIG. 1.
Schematic overview of markerless replacement using shuttle mutagenesis with the temperature-sensitive plasmid pST76K_SR (1, 44, 54). In the first RecA-mediated recombination via one of the homologous regions flanking the region of interest, the entire shuttle plasmid integrates into the target (BAC) DNA. Cointegrates were selected at 42°C on kanamycin-containing media (selection for the presence of the shuttle plasmid) and chloramphenicol (for BAC20 maintenance). A second RecA-mediated recombination resolved the cointegrates and resulted in the parental sequence or the desired mutated sequences. The sacB product confers sensitivity to sucrose of the clones harboring pST76K_SR, which was used to select for resolved cointegrates. In the given example, the asterisk marks the modified start codon (ACG) of UL44 sequence which was reinserted into 20ΔgC. The positions of the BamHI (B) and PstI (P) sites are shown.