I am planning to insert a promoter into a pDsRed-Express2 vector (see attachment) upstream of the lac regulatory features. I will be transfecting a non-lactose fermenting bacteria with this recombinant plasmid.
I wanted to ask if anyone sees a potential problem that the lac regulatory features might pose when the transcriptional machinery meets this sequence. I know that when transcription starts from within the promoter that the downstream features (rest of the promoter, operator, etc.) will be transcribed into the mRNA transcript, but I wasn't totally sure what would happen if the transcription starts from further upstream.
I want the mRNA transcript to be made all the way from the upstream promoter, past the lac promoter, and all the way to the end of the RFP gene so that translation of the RFP gene can be successfully carried out as a reporter of upstream promoter activity.
Does anyone see any potential early termination of transcription that would occur with this configuration before the desired transcript is formed? Maybe a potential problem with the lac reg features or a transcription termination sequence somewhere...
Thank you all in advance,