Fig. 2.

Plasmid design and integration of plasmid constructs at the K. pastoris MET3 and MET5 loci. a Design of the MET3 and MET5 integration plasmids. The Kp_Δmet3 and Kp_Δmet5 targeting cassettes were assembled in the pFA6a-kanMX4 plasmid to produce plasmids pFA6a-Kp_Δmet3-kanMX4 and pFA6a-Kp_Δmet5-kanMX4, respectively. Plasmid components are not drawn to scale. b The plasmids pFA6a-Kp_Δmet3-kanMX4 and pFA6a-Kp_Δmet5-kanMX4 were linearized by digestion with SwaI to enable homologous recombination with the 5′ and 3′ IGRs of MET3 and MET5. The locations of control primers to confirm correct integration of the construct are indicated. DNA elements are not drawn to scale. c Confirmation of the correct integration of the linearized pFA6a-Kp_Δmet3-kanMX4 construct as demonstrated by PCR of genomic DNA using primers pFA6a fwd and KpMET3 3′ ctrl rev. d Confirmation of the removal of the endogenous MET3 locus as demonstrated by PCR of genomic DNA using primers KpMET3 ctrl fwd and KpMET3 3′ ctrl rev. e Confirmation of the correct integration of the linearized pFA6a-Kp_Δmet5-kanMX4 construct as demonstrated by PCR of genomic DNA using primers pFA6a fwd and KpMET5 3′ ctrl rev. f Confirmation of the removal of the endogenous MET5 locus as demonstrated by PCR of genomic DNA using primers KpMET5 ctrl fwd and KpMET5 3′ ctrl rev