Assistance with pIRES bicistronic vectors - Help? (Oct/19/2005 )
I'm wonding about putting a gene of interest into a Clontech pIRES2-acGFP vector. I know that I should not include the polyadenylation signal sequence in the insert (AATAAA). Currently, the sequence I have does not harbor the signal sequence, but it does include a 25-nt 'A' tail.
Do you think that this 25nt 'A' tail will terminate transcription so that the acGFP in the second reading frame will not be expressed?
We have had very good luck with this vector when using it for another gene of interest (albeit one without a short poly-A tail, nor a polyadenylation signal sequence).
Another another note, I am wondering why the clone we bought has a short poly-A tail without the signal sequence. I suspect it was cloned using a poly-T primer from some cDNA library. However, the sequence is missing the last 60nt from the consensus sequence for the cDNA. It is towards the end (20nt from the end) of the consensus sequence where we see the normal polyadenylation signal sequence.
I know the simple solution could be to PCR the gene with the reverse primer between the stop codon and the short poly-A tail, but I wondered if it would work when inserted with the short poly-A attached.
honnestly, try both... poly A repetition is not by itself a polyadenylation sgnal if i'm not wrong...