I am inserting an shRNA loop into the pLKO.1 vector. The shRNA will be under the control of the U6 transcriptional promoter in this vector. From what I understand the transcriptional start site lies 23nt downstream of the TATA promotor and that the. However, the AgeI restriction site ends 25nt downstream of the U6 promoter leaving a 2nt (GG) gap. I have included an image showing the locations of these elements.
I am asking if a gap is in fact left between the end of the promotor and the beginning of the shRNA sequence in the pLKO.1 vector and if so why?
Hi, as you can search on the internet , the sequence of ageI r.e. recognizes 5’-accggt -3’ . Actually there is no gap formation in the plasmid because enzyme creates sticky ends in the plasmid. CCA site of the start is included in the enzyme cutting site.
Samuel,
I have had the same question as you before, and have come to the conclusion that it is for ease of cloning and to ensure that people using this vector don't forget to start their shRNA with a "G", as the U6 promoter prefers to start transcription with this base at the +23 position. All in all, the two G's will indeed be transcribed and will freely hang off of the 5' end as unpaired bases, just as the two U's from the poly-T terminator signal are left behind on the 3' end of the shRNA. It must not affect the shRNA quality too much, as most popular vectors employ this strategy, but I do not like it and often just make my own plasmids instead as I like keeping things clean.
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