String of A's (15-20nt) w/out polyA signal in middle of rlentiviral vector; - (Jun/26/2018 )
I know this might be a stupid question, but just want confirmation either way before going through the building and validation of a few new vectors for single cell sequencing.
I am want to insert a string of A's (no polyA signal) for polyA RNA capture on a gene of interest in the middle of my lentiviral vector that also has a different promoter driving selection downstream.
Basic example lentiviral vector (expression)
5'LTR--EF1a-GOI-P2A-BC(18nt)-N(280)-A(15-20)--PGK--Puro--3'LTR
I don't think this will affect the production of a full length viral transcript and packaging and might work for detecting the BC for single cell sequencing, but I am not 100% sure.
If anyone with more plasmid/lentiviral biology background can provide an answer that they believe this design would or would not work, I would greatly appreciate the input.
Thank you very much for your time and thoughts!
Hi! first let me get this straight- you want a string of A's in your RNA transcript (to mimic polyadenlyation, even though no polyadenylation signal is present). It seems to me you'd have to build a string of T's into your sequence if you want them to transcribe as A's. But I'm not sure either. But think about it!