Restriction digests of the clone give the following sizes (kb): EcoRI--4.4; BamHI--4.3, 0.2; AvaI--1.8, 1.65, 1.2 Cultures should be grown in minimal media for more reproducible induction of expression. Expression is induced in glycerol-containing media by addition of arabinose. Expression is repressed by addition of glucose or other catabolites. One of several tightly controlled expression vectors (ATCC 87393-87402) regulated by the arabinose operon. The vectors differ in replicon, antibiotic resistance marker, multiple cloning site and mechanism of initiation of translation. The following primers can be used for sequencing of cloned inserts: 5' primer (27 - 8 bp upstream of the NheI site) 5'-CTGTTTCTCCATACCCGTT-3'; and one of two 3' primers: 3' primer 1 (2 - 19 bp downstream of the HindIII site) 5'-CTCATCCGCCAAAACAG-3'; 3' primer 2 (17 - 33 bp downstream of the HindIII site) 5'-GGCTGAAAATCTTCTCT-3'. Vector contains an optimized Shine-Dalgarno ribosome binding site, translation initiation codon and Kozak sequence (relevant for expression in eukaryotic cells) which promote a high level of expression of cloned inserts. |
