I have a question about the plasmid qTAG-C-sTagRFP-Puro (Addgene #207723) from Professor Pelletier.
On the figure attached (Benchling software for sequence analysis), we can see that upon traduction of the cassette, there is a STOP (*) in the lox71 and a gap (one nucleotide A, blank) just before the P2A sequence. Will this be a problem for the correct production of the proteins ? Will this configuration lead to the production of RFP-V5tag and then no puromycinR ? If no puromycinR protein is produced, then the cells will not survive to puromycin treatment.
Thank you in advance or your time and valuable inputs.
Yannick Frey Thanks a lot Yannick for your kind answer. I wonder if this premature STOP + 1 bp insertion & frameshift is a normal feature for the functionning of the P2A sequence. Maybe the P2A sequence needs to have a STOP before P2A, to enable its normal function ?
Yannick Frey Thank you for your answer. This is very clear and I fear that this premature STOP will indeed STOP the proper traduction of proteins.
Hi Philip and Yannick,
Thank you to you both for your inputs on that question. I sent the plasmid to sequencing service (with M13 F and R primers) and I will let you know as soon as I get the sequences. In the meanwhile, Addgene has accepted to send me for free another plasmid of the suite, the RFP-blasticidine which does not display any problem.
I will write to you soon.
Carole
Hi both,
I got the sequencing results (see the file) and unfortunately, we have the mutation in the plasmid. Therefore I will ask a replacement from Addgene.
Thank you to you both for your help.
Hi Philip, I will indeed order the scarlet version for a replacement, and this will be ok. Thank you again for advising.