Restriction digests of the clone give the following sizes (kb): BamHI--8.8; BamHI/XhoI--5.5, 3.2; EcoRI/HindIII--7.5, 1.2; HpaI/XhoI--4.6, 4.2.
To convert the host phenotype from LEU2 to URA3, transform with the XhoI+HpaI digested vector and select for Ura+ 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 URA3 and kanR 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.
Vector was constructed by replacing an internal EcoRV fragment of LEU2 with a SmaI fragment containing the URA3 and kanR coding sequences. URA3 and LEU2 are in the opposite orientation.
Media
ATCC Medium 1065 (see below) plus kanamycin (50.0 mcg/ml) plus chloramphenicol (25.0 mcg/ml) ATCC Medium 1065: Tryptone (Difco 0123), 10.0 g Yeast Extract (Difco 0127), 5.0 g NaCl, 10.0 g Distilled water, 1.0 L
