Features (with orientation and position when available):
replicon: f1, →
marker(s): cmlR, ←
replicon: pMB1
restriction site: XhoI, SalI
Applications
YI-type (integrating) shuttle vector
host modification
marker swap vector HIS3 --> URA3
Comments
Restriction digests of the clone give the following sizes (kb): HindIII--6.5; HindIII/PstI--5.0, 1.1, 0.5.
To convert the host phenotype from HIS3 to URA3, transform with the SmaI+XhoI 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 HIS3 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 HindIII fragment of HIS3 with a SmaI fragment containing the URA3 and kanR coding sequences. URA3 and HIS3 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
