A Modified pBluescript-Based Vector for Facile Cloning and Transcription of RNAs

Abstract

We report here the generation of a vector that would permit efficient in vitro transcription of RNAs and ensure that unwanted additional sequences at the 59 termini are minimized in the final transcripts. Several vectors (such as the Bluescript and GEM series) are currently available for cloning genes under the control of a bacteriophage RNA polymerase promoter and subsequent in vitro transcription. During the cloning process, if recognition sequences for the type IIS restriction enzymes (e.g., FokI, BsmAI) are adroitly placed at the end of the coding region, templates with the desired 39 end for runoff transcription can be easily generated (1, 2). In contrast to the precision with which the 39 end can be engineered, the use of restriction enzymes for directional cloning downstream to the promoter sequence frequently results in additional nucleotides being introduced 59 to the sequence encoding gene of interest. Consequently, the 59 end of the resulting transcript has unwanted sequences. The commercially available LITMUS vectors (New England Biolabs) sought to address this limitation by engineering restriction sites into the 26 to 21 region of the T7 RNA polymerase promoter (PT7) sequence (3). However, this approach resulted in reductions in transcriptional efficiency owing to modifications in the consensus sequence of PT7 that is required for initiation (3, 4). Yet another solution to circumvent addition of unwanted 59 sequences in the transcript is by including the PT7 sequence in the sense primer used in PCR-based cloning of the gene of interest (1). Al-