The pYC plasmids, a series of cassette-based yeast plasmid vectors providing means of counter-selection.

Abstract

A series of 24 general-purpose yeast plasmid vectors has been constructed. The plasmid series is composed of inter-replaceable cassettes, allowing for easy interconversion of plasmid types. In addition to the usual replication origins, selectable markers and multiple cloning sites (MCS), cassettes dedicated to counter-selection have been constructed. A pair of unique 8 bp restriction enzyme recognition sites flank each type of cassette, FseI in the case of yeast replication origins, AscI in the case of selectable markers, PacI in the case of counter-selectable markers and NotI in the case of the MCS. Thus, any given cassette can be replaced by another cassette of the same type, facilitating interconversion of any given plasmid from one type to another, even after the insertion of DNA into the MCS. Hence, the plasmids have been named pYC for 'yeast cassettes'. The cassettes consist of either NONE, CEN4/ARS or 2micro as replication origin, either URA3, MET2-CA (Lg-MET2) or the G418 resistance gene (the apt1 gene from bacterial transposon Tn903, encoding aminoglycoside phosphotransferase) as selectable markers, either NONE, PMET25-PKA3 or PCHA1-PKA3 as counter-selectable marker, and the MCS, containing recognition sites for AflII, AvrII, BspEI, PmeI, SacII, SalI, SunI, BamHI, EcoRI, HindIII, KpnI, MluI, NarI and SacI (of which the seven first are unique in all plasmids). The counter-selectable markers consist of the PKA3 gene under control of the conditional MET25 or CHA1 promoters. At activating conditions these promoters express the PKA3 gene at toxic levels, facilitating easy selection for loss of plasmid or 'loop-out' of plasmid DNA sequence after genomic integration.