Restriction digests of the clone give the following sizes (kb): BamHI--8.4, 2.1; BamHI/XhoI--4.9, 3.3, 2.2. To convert the host phenotype from LEU2 to TRP1, transform with the XhoI+HpaI digested vector and select for Trp+ transformants. Some combinations of marker swap plasmids and target locus may result in relatively high reversion rates. In most but not all cases the frequencies of successful convertants are greater than 30%. A marker swap vector designed to change the S. cerevisiae host phenotype by one-step gene disruption of the LEU2 gene with the TRP1 and ampR markers. When swapping markers on an episomal plasmid, appropriate phenotype may result from loss of the plasmid unless a second selectable or scorable marker is used to ensure plasmid maintenance. Recommendation for verification: XhoI+HpaI--6.1, 4.4; BamHI+XhoI--5.1, 3.4, 2.0; BamHI--8.5, 2.0; HindIII+XhoI--6.3, 4.2. Vector was constructed by replacing an internal EcoRI-EcoRV fragment of LEU2 with an XhoI-EcoRI fragment containing the TRP1 and ampR coding sequences. |
