Restriction digests of the clone give the following sizes (kb): HindIII--2.7, 1.4; KpnI--4.1; SmaI--3.1, 1.0. Expression of ble (bleomycin resistance) is regulated by the promoter and terminator regions of the am gene (amination, glutamate dehydrogenase) from N. crassa. Shuttle vector conferring bleomycin and phleomycin resistance. That expression was improved by modifying the 17 bp before the initiation codon in pAB366 to match the sequence of the am gene. The am::ble fusion cassette can be removed by digestion with either HindIII, SphI, XbaI or BamHI or any combination of them. The order of the major features in this plasmid is: pUC18 - MCS (HindIII to BamHI) - am promoter - ble - XbaI - am 3' region - EcoRI - MCS (HindIII to EcoRI) - pUC18. |
