Figure 1
From: Recycling of a selectable marker with a self-excisable plasmid in Pichia pastoris
Schemes of Cre/loxP zeocin-resistance selectable marker recycling vectors. Filled arrows and boxes, plasmid elements: MCS, multiple unique cloning sites; AOX1 TT, AOX1 transcription termination; ZeoR, zeocin-resistance marker; ori, bacterial replication origin. (a) The vector using the AOX1 promoter for gene expression; (b) vector using the GAP promoter for gene expression; (c) strategy for selectable marker recycling using the vectors (e.g., plasmid C-Phy). The linearized plasmid was introduced into P. pastoris cells by transformation and integrated into the genome through the his4 locus. After methanol induction, the Cre-ZeoR cassette was excised through recombination between lox71 and lox66, leaving the double-mutant lox72. Chromosomal integrations and Cre-ZeoR cassette excisions can be verified by PCR using primer pairs P1/P2 and P3/P4, respectively.
