title: 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.
authors: Department of Molecular Physiology and Biophysics, University of Iowa, IA, USA.
authors: Chris
authors: C
authors: MacDonald
authors: Robert C
authors: RC
authors: Piper
mesh_terms: D003062:Codon; D025141:Drug Resistance, Fungal; D005819:Genetic Markers; D019426:Integrases / Q000235:genetics* / Q000378:metabolism; D008727:Methotrexate / Q000494:pharmacology*; D010957:Plasmids / Q000235:genetics* / Q000378:metabolism; D015202:Protein Engineering; D011691:Puromycin / Q000494:pharmacology*; D012441:Saccharomyces cerevisiae / Q000187:drug effects* / Q000235:genetics* / Q000378:metabolism
chemical_list: D003062:Codon; D005819:Genetic Markers; D011691:Puromycin; C045073:Cre recombinase; D019426:Integrases; D008727:Methotrexate