Restriction sites available for inverse PCR rescue include MboI (flanking EcoRI by 107 and 77 bp), RsaI (flanking EcoRI by 297 and 352 bp), and TaqI (flanking EcoRI by 508 and 869 bp). Restriction digests of the clone give the following sizes (kb): BamHI--18.0, 1.8; EcoRI--20.0; HindIII--5.4, 4.5, 3.3, 2.7, 1.7, 1.4. Induced transcription from a GAL1 promoter inactivates the centromere and increases copy number. Additional amplification results from growth in media containing methotrexate, sulfanilamide and thymidine. This YAC cloning vector has bacterial replication origins and selectable markers on both arms to permit rescue of end subclones from each end in Escherichia coli. This YAC cloning vector has a conditional centromere for copy number amplification of yeast artificial chromosomes, T3 and T7 promoters, restriction sites for inverse PCR rescue, and a replication origin and markers functional in mammalian cells. |
