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. 2004 Sep;48(9):3468–3476. doi: 10.1128/AAC.48.9.3468-3476.2004

FIG. 4.

FIG. 4.

Disruption of ORF3 and ORF5 by double crossing over. (A) ORF maps of “S. carzinostaticus” var. F-41 and disruptants showing the predicted insertion position of the kanamycin resistance marker and fragment sizes amplified with the illustrated primer set. (B) Colony PCR results with the PKS-specific primer set and pMO5 transformants as templates; transformants 1, 3, and 5 (lanes 1, 3, and 5, respectively) are disruptants obtained by single crossing over, whereas transformants 2 and 4 (lanes 2 and 4, respectively) are disruptants obtained by double crossing over. Lane 6, control for the wild type. (C) Colony PCR results with the Halo primer set and pMO7 transformants as templates; transformants 1, 5, and 6 (lanes 1, 5, and 6, respectively) are disruptants obtained by single crossing over, whereas transformants 2, 3, and 4 (lanes 2, 3, and 4, respectively) are disruptants obtained by double crossing over. (D) HPLC analysis with UV detection at 380 nm of mycelial extracts from “S. carzinostaticus” var. F-41, ORF5 disruptants 2 and 4.