(A) Vector pYES2/GS for expression in yeast cells includes theGAL1 promoter for inducible expression in appropriate S. cerevisiae strains; the 2 μ origin of replication for maintenance of high copy number and improved expression; theURA3 gene for stable selection of transformants in ura3 S. cerevisiae strains. (B) Vector pcDNA3.1/GS for expression in mammalian cells includes the CMV promoter and enhancer sequences for high-level transient or stable expression; and the multipurpose Zeocin resistance antibiotic for selection of bacterial transformants and of stably transfected mammalian cells. Both vectors also include the V5 epitope tag after the insert to allow detection of proteins for which antibodies are not available, a carboxy-terminal polyhistidine tag (His)6 to allow metal-affinity protein purification, and the T7 promoter to enable in vitro transcription and translation of the target gene. (C) Schematic representation of the topoisomerase I-mediated cloning process. Phases are described in the text.
