Restriction digests of the clone give the following sizes (kb): BglII--9.6; EcoRI--5.2, 4.4; BglII/EcoRI--4.6, 4.4, 0.6. The two step selection process requires a ura3 transformation host (this host can be created using pJL164 (ATCC 87471)). After transformation with the EcoRI/BglII digested plasmid, URA3 integrants are selected on ura- plates. The deletion strain is then recovered by selection on 5-FOA plates (loss of URA3 marker by a homologous recombination event between the two hisG repeats). 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 yeast strains with a trp1 auxotrophic marker deletion. The 4.6 kb EcoRI/BglII insert contains two direct repeats of the Salmonella hisG gene flanking URA3 plus TRP1 sequences flanking the hisG-URA3-hisG sequence. The plasmid was constructed by inserting the 3.8 kb BamHI-BglII hisG-URA3-hisG fragment into the modified EcoRV site within the TRP1 gene of YEp7. |
