Restriction digests of the clone give the following sizes (kb): BamHI--5.2, 3.2; EcoRI--5.0, 3.4; HindIII--7.0, 1.3. The two step selection process requires a ura3 transformation host (this host can be created using pJL164 (ATCC 87471)). After transformation with the BamHI digested plasmid, URA3 integrants are selected on ura- plates. The designer deletion strain is then recovered by selection on 5-FOA plates (loss of URA3 and ADE2 markers by a homologous recombination event between the two hisG repeats). The deleted host retains the coding sequence for six C-terminal amino acids of ADE2. E. coli containing this plasmid should be grown on media lacking pyrimidines to select for URA3-containing cells. This deleter vector is used to create designer yeast strains with a non-revertable ade2 auxotrophic marker deletion. The 5.2 kb BamHI insert contains two direct repeats of the Salmonella hisG gene flanking URA3 and about 700 bp of homology to sequences upstream and downstream of the ADE2 gene flanking the hisG-URA3-hisG sequence. |
