FIG 5.

Schematic of generation of repaired BAC clones. Sequence flanking the deleted sequence of the target ORF was amplified from wild-type BAC DNA and cloned into the pUC19 plasmid. The cloned fragment together with the ampicillin resistance gene was PCR amplified and inserted into the knockout BAC by Red recombination, with primers containing additional sequences homologous to the target locus. The ampicillin resistance cassette was removed by two-step Red recombination. CDS, coding sequence.