Puromycin‐ and methotrexate‐resistance cassettes and optimized Cre‐recombinase expression plasmids for use in yeast

Abstract Here we expand the set of tools for genetically manipulating Saccharomyces cerevisiae . We show that puromycin‐resistance can be achieved in yeast through expression of a bacterial puromycin‐resistance gene optimized to the yeast codon bias, which in turn serves as an easy‐to‐use dominant genetic marker suitable for gene disruption. We have constructed a similar DNA cassette expressing yeast codon‐optimized mutant human dihydrofolate reductase ( DHFR ), which confers resistance to methotrexate and can also be used as a dominant selectable marker. Both of these drug‐resistant marker cassettes are flanked by loxP sites, allowing for their excision from the genome following expression of Cre‐recombinase. Finally, we have created a series of plasmids for low‐level constitutive expression of Cre‐recombinase in yeast that allows for efficient excision of loxP ‐flanked markers. Copyright © 2015 John Wiley & Sons, Ltd.