The cleavage position in the reading frame for cloning sites is (where 3 = between triplets): HindIII-2; SphI-3; PstI-3; SalI-2; XbaI-2; BamHI-2; SmaI-3; KpnI-3; SacI-3; EcoRI-2. The SacI site is not unique. Cloning into the HindIII, SphI, PstI, SalI, 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-7.1; SalI-7.1; PstI-7.1; HindIII-7.1. One of 3 promoter-cloning, YI type shuttle vectors (ATCC 37755 - 37757) with URA3 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'AA GCT TGC ATG CCT GCA GGT CGA CTC TAG AGG ATC CCC GGG TAC CGA GCT CGA ATT CCC AGC TTC GAT CCC3', from nucleotide 1 of the MCS through CCC for amino acid 8 of beta-galactosidase. |
