Restriction digests of the clone give the following sizes (kb): HindIII--7.0, 1.2; XbaI--8.2; PstI--8.2. The LEU2-d and 2 micron sequences are between the unique cloning sites, and thus the adjacent SacI, BamHI, and XbaI sites can be used for directional cloning, and similarly the SphI and SalI sites can be used. Selection for URA3 expression produces a plasmid copy number of 20-40; growth on YC minus leucine produces a copy number of 100-200. One of 9 YE-type shuttle vectors (ATCC 87541, 87554, 87560 an d 87640 - 87645) that have the leu2-d gene, which is correlated with high copy number in S. cerevisiae, when grown in medium lacking leucine. Vector was constructed by inserting 1) URA3 HindIII fragment into HindIII site; 2) a SpeI fragment containing 2 micron ori and 3) the leu2-d gene into the XbaI site of pUC119. Both inserted yeast genes have the opposite orientation of the ampR gene. |
