The unique EcoRI site in the vector occurs downstream of an insert into the SmaI site. Plasmid with insert can be linearized with EcoRI to generate a transcript. This is a vector for in vitro transcription from a modified lambda PR promoter. Distributed in aliquots of 187.5 ng (3.75 ul, 50 ng/ul). This vector contains the cI857 temperature sensitive mutant gene of the lambda cI repressor. Temperature control can be used to repress, in vivo, transcription of potentially deleterious genes during culture in Escherichia coli. The PR promoter in this vector has been modified to position the cleavage site of a unique SmaI site exactly at the initiation site of the PR promoter. Transcription will be initiated at the first nucleotide of any 5'-purine-terminated DNA fragment fused to the SmaI site. In some cases, significant yields of transcript initiated at the first nucleotide of an inserted 5'-pyrimidine-terminated DNA fragment can also be obtained using this vector. This was constructed by inserting a 0.9 kb ClaI-BamHI fragment from pCQV2 (containing lambdaPR and cI857) into M13mp9 (AccI/BamHI). This was used to make PstI/SmaI fragment with the SmaI site adjacent to the PR initiation site and inserted into pUC9. |
