Restriction digests of the clone give the following sizes (kb): XbaI/PstI--4.5, 4.0, 2.6. The ClaI fragment (which includes pBR322 ClaI/EcoRI sequence) contains the following restriction sites (approximate kb from the vector-derived end): EcoRI--0.02, 1.8; XmnI--0.9, 3.9; HindIII--3.3, 3.5; BanII--4.1; KpnI--4.1. To optimize production, the plasmid should be transformed into E. coli MM294, grown at 30C to late log phase, and then shifted to 42C for an additional 3 hr. The vector is pUC19 modified by insertion of a 4.3 kb BamHI fragment containing bacteriophage lambda N, PR, cI857, and PR sequences. The PL promoter drives transcription of the insert. Distributed in aliquots of 100 ng dried. |
