Fig. 4

Sequential splitting left and right edge of Chr2-6 replaced transformants. a Two splitting modules (One module synthesized from the plasmid pSJ70 contained CgHIS3 and the other module synthesized from the plasmid p3121 contained CEN4 as a centromere) were introduced into the host strain SJY 576 (Chr2-6 region replaced transformants) to split left edge of CgLEU2. Bottom part of a represents gel electrophoresis of colony PCR and Lane 1 to 10 represents 10 independent transformants were checked to amplify 1 kb band denoting the left edge of CgHIS3 and right edge of CgLEU2. Primers used for colony PCR are illustrated in a. b After splitting left edge of CgLEU2 in Chr2-6 region replaced transformants (SJY 577), we tried to split sequentially the right edge of CgLEU2. 2 splitting modules (One module synthesized from the plasmid pSJ23 contained URA3 and another module synthesized from the plasmid p3121 contain CEN4 as a centromere) were introduced into SJY 577 to split right edge of CgLEU2. Bottom part of b represents gel electrophoresis of colony PCR and 5 independent transformants were checked for amplifying 1.5 kb band denoting the right edge of CgLEU2 and left edge of URA3. Primers used for colony PCR are illustrated in b