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. 2018 Dec 5;20(3):447–459. doi: 10.1111/mpp.12766

Figure 6.

Figure 6

DMAT3 knock‐out in the pks1/ura3a/b mutant. (a) Schematic diagrams of the DMAT3 coding sequence (CDS) knock‐out experiments in the pks1/ura3a/b background. The pNK098 plasmid includes about 2 kb of upstream (Up) and downstream (Down) sequences around the DMAT3 locus. The Down sequence contains the complete DMAT3 CDS shown by a black box. Green arrows show homologous 500‐bp sequences for recombination originally from the downstream region of DMAT3 CDS. (b) Genomic DNA polymerase chain reaction (PCR) showed successful homologous recombination by pNK098, resulting in pNK098HR pks1/ura3a/b strains. The primer sets Po17/Po19 and Po18/20 generate 2826‐ and 2674‐bp bands, respectively, from pNK098HR pks1/ura3a/b, but not from the genome of pks1/ura3a/b. The 266‐bp bands corresponding to CHITIN SYNTHASE (CHS) were amplified to show the presence of genomic DNA. (c) Genomic DNA PCR showed the successful removal of the Tef::URA3B expression cassette and the DMAT3 CDS. The primer sets Po13/Po14 and Po21/Po22 generate 1200‐ and 540‐bp bands, respectively, from the genome of pNK098HR pks1/ura3a/b, but not from that of dmat3/pks1/ura3a/b. The primers used are listed in Table S3 (see Supporting Information). [Colour figure can be viewed at wileyonlinelibrary.com]