Restriction digests of the clone give the following sizes (kb): BamHI--4.4 (doublet); BglII-uncut; EcoRI--8.8; PstI--8.8; HindIII--8.8. The T7 RNA polymerase expression construct, encoding gene 1 and lac regulatory sequences, can easily be excised as a 4.4 kb BamHI fragment for insertion into another construct. The insert size given is for that of the sum of the lac and gene 1 sequences. Gene1 was sequenced from T7 wild-type DNA. The junctions between gene 1 and lac were sequenced. Lac sequence is taken from GenBank J01637. pAR1219 produces large amounts of T7 RNA polymerase in a suitable host upon induction with IPTG. Large amounts of active T7 RNA polymerase can be purified from the strain BL21/pAR1219. The lac fragment contains the lacI promoter, lacI gene, lacUV5 promoter, lac operator, and the translation start site and first 147 amino acids of B-galactosidase. pAR1219 was made by inserting a 1724 bp HincII fragment from pMC1, containing lac control elements, into the BglII site of pAR1173. The construction consists of a fragment containing the T7 RNA polymerase gene (gene 1, including nucleotides 3145 to 5841 of T7 DNA) under the transcriptional regulation of lacI and the lacUV5 promoter. |
