The cleavage position in the reading frame for cloning sites is (where 3 = between triplets): EcoRI-2; SacI-3; KpnI-3; SmaI-2; BamHI-2; XbaI-2; SalI-2; PstI-3; SphI-3; HindIII-2. The EcoRI, SacI, and KpnI sites are not unique. Cloning into the EcoRI, SacI, KpnI SmaI, BamHI, or XbaI sites leads to a TAG stop codon within the downstream XbaI site of the multiple cloning region. Restriction digests of the clone give the following sizes (kb): EcoRI--4.1, 2.9; SalI--7.0; PstI--7.0; HindIII--7.0. One of 3 promoter-cloning, YI type shuttle vectors (ATCC 37752- 37754) with LEU2 selection in Saccharomyces cerevisiae, a beta-galactosidase reporter gene and multiple cloning sites differing in reading frame. The sequence and reading frame of the multiple cloning sequence is: 5'GA ATT CGA GCT CGG TAC CCG GGG ATC CTC TAG AGT CGA CCT GCA GGC ATG CAA GCT TGC GAT CCC 3', from nucleotide 1 of the MCS through CCC for amino acid 8 of beta-galactosidase. |
