Expression vector and Stop codon consideration - (Sep/27/2008 )
I have to clone the PCR product into pGMET easy vector for just stock, and then, have to subcloned into pCDNA5 Topo vector T/A (Invitrogen). I like to get some suggestions from you all:
* Should I consider stop codon when designing primer of my Gene of Interest (GOI) for pGMET easy vector before subcloning into pCDNA5. I like to detect V5 epitope incorporated in pCDNA5 Topo expression vector using anti-V5 antibody by Western blot. V5 epitope is located just after PCR product (GOI).
* If stop codon is added, is it possible that required results can be achieved?
Thanks in advance
of course you have to take into account that!!! the codon stop must be 3' after de epitope V5. The comercial vectors have it.
if you want to express the gene that you clone you must to revise carefully the sequence . I advice you to write the sequence interest plus the vector
and translate to verify that the recombinat protein will have all that you want.
sorry my english is ugly
Vector NTI has a useful too of translating genes into amino acid sequence. If you make a mistake and end your protein short, you will see that your protein ends before the gene ends at the point it is suppose too.
And yes, you have to watch for ATG (start codon) and location of stop codons in relation to the start codon.