The following unique restriction sites are found on this vector separated by (bp)(approx): BglI- 100- BamHI- 900- EcoRI- 600- XhoI- 300- SmaI- 250- HindIII- 1650. Restriction digests of the clone give the following sizes (kb): EcoRI--3.1; PvuI/BamHI--1.7, 1.4; BglI--3.1; PstI--3.1; HindIII--3.1. Plates equally well at 28C and 42C in E. coli K-12 deltaH1 hosts. Escherichia coli M5219 is Escherichia coli K-12 M72 lac(am) trp(am) rpsL lambda cI857 deltaH1 bio252. deltaH1 removes part of cro and all genes to the right of cro. bio252 removes all genes to the left of cIII. At 42C, N is expressed from the chromosome. Translation from the MS2 replicase is colinear with transcription from the PL promoter and thus is under PL control. Shows reduced plating efficiency in E. coli M5219 at 42C under antibiotic selection. This vector is used for expression of fused proteins with MS2 polymerase. ATG is in phase with GAT from the BamHI site, AAG from the HindIII site, TTA from the MstII site, CGC from the NruI site and GCT from the EspI site. The orientation of the PL promoter is clockwise with respect to the plasmid ori. This vector was constructed from pPLc28 (ATCC 31696) by inserting a 431 bp EcoRI/BamHI fragment coding for the ribosome binding site and the first 98 amino acids of the MS2 replicase (from pMS2-7) into pPLc28. |
