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Epitope-Tagging Vectors for Functional Analysis in Yeast

nces: a ColE1ori-ampR fragment, which allows antibiotic selection and replication of the vector in E. coli for cloning; the 2-m sequence, which provides the origin of replication so that the vector can replicate autonomously in S. cerevisiae; an auxotrophic selectable marker gene (HIS, LEU2, TRP1, or URA3) to select and maintain the expression vector in yeast cells; GAL1 and GAL10 promoters in opposite orientation; a multiple cloning site (MCS); and a transcription termination sequence downstream of each promoter.

LEU2, TRP1, and HIS3 genes encode b-isopropylmalate dehydrogenase, N-(5-phosphoribosyl) anthranilate isomerase, and imidizoleglycerolphosphate dehydratase, respectively. These gene products are required for the biosynthesis of the amino acids leucine, tryptophan, and histidine, respectively. The URA3 gene encodes orotidine-5-phosphate decarboxylase, which is required for uracil biosynthesis. Plasmids bearing URA3 can be counter-selected by plating the cells on media containing 5-FOA (5-fluoroorotic acid). These features facilitate the functional analysis of genes by plasmid shuffling.3

Both the GAL1 and GAL10 promoters from S. cerevisiae are strictly regulated at the transcription level by the carbon source in the media. These promoters are tightly repressed when glucose is present in the media and are highly induced when galactose is the sole carbon source.4 In S. cerevisiae, the induction ratio of these promoters has been estimated to be greater than 1000 fold.5,6 The presence of both the GAL1 and GAL10 promoters in opposite orientation allows two genes to coexpress in the same host cell.

The pESC vectors also contain DNA sequences coding for epitope peptid
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