Figure 4

Promoter replacement strategy. (A) The pPuzzle vector was used as backbone for cloning the promoter exchange cassette of the two genes ERO1 and PDI1. P _ERO part is a fragment of ~500 bp of the native ERO1 promoter, about −700 to −200 bp upstream of the ATG. Zeocin was used as selection marker, where loxP sites could be used for marker recycling. (B) After digestion with Asc I and BspH I the promoter exchange cassette was transformed into P. pastoris. It interacted there with the respective genomic DNA by homologous recombination and replaced the native promoter of the respective gene. The promoter exchange cassette integrated into the genomic DNA, whereby 200 bp of the native promoter were excised.