title: Self-excising integrative yeast plasmid vectors containing an intronated recombinase gene.
abstract: Site-specific recombinases are widely used for selectable marker recycling in molecular-genetic manipulations with eukaryotic cells. This usually involves the use of two genetic constructs, one of which possesses a selectable marker flanked by the recombinase recognition sequences, while the other one bears the recombinase gene. Combining the recombinase gene with its recognition sequences in one plasmid is usually avoided, as it may lead to undesirable recombination due to promoter leakage, while the plasmid is maintained in Escherichia coli cells. Here, we describe yeast vectors possessing Cre recombinase genes under control of regulatable yeast promoters and loxP sequences for the in vivo vector backbone excision. The plasmid stability in E. coli is ensured by the presence of an intron in the recombinase gene. Applicability of these vectors was validated by disruptions of the Hansenula polymorpha PMC1 and Saccharomyces cerevisiae HSP104 and PRB1 genes.
authors.affiliation: A.N. Bach Institute of Biochemistry, Russian Academy of Science, Moscow, Russia.
authors.forename: Michael
authors.initials: M
authors.lastname: Agaphonov
authors.forename: Alexander
authors.initials: A
authors.lastname: Alexandrov
mesh_terms: D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D055786:Gene Knockout Techniques; D005822:Genetic Vectors; D005827:Genetics, Microbial / Q000379:methods; D042822:Genomic Instability; D008967:Molecular Biology / Q000379:methods*; D008969:Molecular Sequence Data; D010957:Plasmids; D045522:Recombinases / Q000235:genetics* / Q000378:metabolism*; D011995:Recombination, Genetic; D004718:Saccharomycetales / Q000235:genetics*; D017422:Sequence Analysis, DNA
chemical_list: D004271:DNA, Fungal; D045522:Recombinases
keywords: Cre recombinase; gene disruption; plasmid integration; selectable marker recycling; yeast