FIG 2.

Schematic illustration of the generation of CPXV knockout mutants using Red recombination in E. coli. PCR fragments containing a kanamycin resistance gene aphAI and a sequence coding for two alanines (GCCGCG), followed by a stop codon (TGA) were amplified from plasmid pACAA. Homologous flanking sequences (40 bp) were added through 5′ overhangs of each PCR primer. PCR products were inserted into the target genes of the CPXV sequence by Red recombination. Restriction sites in PCR products for DraIII, HindIII, NruI, PvuI, and XhoI are indicated.