FIG. 3.
Schematic diagram of the Red/ET recombination approach for construction of pKOS272-166 (gdmA2A3-KR6° expression plasmid). In step 1, a linear DNA fragment containing the aphII gene and approximately 0.4 kb of the KR6° cassette flanking the gene marker was cotransformed with plasmid pKOS279-69 into competent Red/ET recombination cells. Kmr clones were selected. In step 2, a linear DNA fragment containing the KR6° cassette was coelectroporated with linearized pKOS272-153 cut by AvrII and NsiI. Aprr transformants were selected. ermE*P, mutated ermE gene promoter (1); int, integrase gene from streptomycete phage ΦC31.