Restriction digests of the clone give the following sizes (kb): EcoRV--5.1, 4.2, 3.0; BamHI--8.1, 4.2; HindIII--8.5, 3.8. The plasmid is maintained at approximately 200 copies per cell in haploid S. cerevisiae cells. LEU2-d is a promoter-defective allele of the yeast LEU2 gene, which increases plasmid copy number to 200-300 copies in the presence of LEU2 selection (host grown in medium lacking leucine). High copy number YE-type shuttle expression vector. Note that the XhoI, EcoRV and BamHI sites are not unique in this plasmid. The promoter consists of nt -610 to -157 of the GAL 1-10 upstream activation site, fused to the transcription initiation site (nt -158 to -1) of the alpha mating factor gene MFalpha1. The 3.65 kb HindIII fragment encodes the positive regulator protein GAL4, necessary for transcription from the GAL1-10 promoter. The PHO5 signal sequence (51 nt) was modified to use preferred Saccharomyces codons. The order of the major features in the plasmid is: HindIII - GAL4 - HindIII - Gal1-10 UAS - MFalpha1 transcription initiation sequence - PHO5 signal sequence - EcoRV - BamHI - GAPDH terminator - ampR - LEU2-d - 2 micron ori. |
