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Puromycin‐ and methotrexate‐resistance cassettes and optimized Cre‐recombinase expression plasmids for use in yeast
Author(s) -
MacDonald Chris,
Piper Robert C.
Publication year - 2015
Publication title -
yeast
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.923
H-Index - 102
eISSN - 1097-0061
pISSN - 0749-503X
DOI - 10.1002/yea.3069
Subject(s) - biology , selectable marker , plasmid , cre recombinase , puromycin , recombinase , genetics , saccharomyces cerevisiae , gene , yeast , codon usage bias , flp frt recombination , dihydrofolate reductase , microbiology and biotechnology , genome , transgene , protein biosynthesis , recombination , genetic recombination , genetically modified mouse
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.