Primers B and F and primers C and G contained an overhang region homologous to the ultimate 5′ and 3′ regions of the lox66-P32-cat-lox71 cassette (amplified with primers I and J), respectively, to enable the joining of the three PCR products in a splicing-by-overlap-extension (SOEing) PCR (26) with primers E and H for ctsR and A and D for hrcA (see Table S2).