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Yeast. 2015 May;32(5):423-38. doi: 10.1002/yea.3069. Epub 2015 Mar 19.

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

Author information

1
Department of Molecular Physiology and Biophysics, University of Iowa, IA, USA.

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.

PMID:
25688547
PMCID:
PMC4454448
DOI:
10.1002/yea.3069
[Indexed for MEDLINE]
Free PMC Article

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