Restriction digests of the clone give the following sizes (kb): BamHI--8.0; EcoRI--2.1, 5.9; SacI--8.0. The factor Xa recognition site of pGEX-3X is maintained and precedes the BamHI cloning site. The BamHI site is in the 1st reading frame (The A in ggatcc is position 1.) The GAL10 RNA start site is disrupted, thus providing only one direction of transcription. Unless a start site for transcription is provided, little or no message should be made from inserts cloned into the GAL10 side of the promoter cassette. Do not use this side of the promoter unless you can insure that mRNA will be properly initiated by providing an RNA start site. Proteins that associate with the expressed GST fusion protein may co-purify after glutathione affinity chromatography. The polylinker sites listed may not be unique in this vector. YE-type, high copy number, galactose-inducible, GST fusion protein expression shuttle vector containing a divergent GAL1/GAL10 promoter cassette. Fusion proteins can be recovered by glutathione affinity chromatography. Can be used to produce ssDNA. Contains promoters for in vitro RNA synthesis, priming sites useful for sequencing, and yeast REP3 and FRT sequences. An in-frame stop codon occurs in the SpeI site, so 3' sites are not useful. |
