adding kozak sequence to primer

[Azam Rahimpour]

Hi
I want to add kozak sequence to my primer for mamamlian expression but I know the consensus kozak sequence is gccgcc(a/g)ccAUGg but the second codon in my gene is started with a not g. should I convert it to g  to make it identical to kozak or using just gccgcc(a/g)ccAUG is OK?
best

[Cathal Garvey]

Using the Kozak you have is just fine, I'd say. It's a "Consensus" sequence,
which means the standard Kozak you find online is the average of hundreds or
thousands of sequenced Kozaks from natural sequences. Slight deviations from
the consensus are the norm, and shouldn't have a huge impact. In fact, it
may be an important part of the gene's natural regulation.

If you have the time and resources to replace the nucleotide and bring it to
full consensus, it might be worth it just to have the sequence on hand in
the future. And it might look better on a paper, though personally I
wouldn't nitpick such small details if I were reviewing it.

[Peter Ellis]

I think it would be a very bad idea to change it. It's a non-synonymous
substitution, which means you have a non-zero risk of altering your
protein's function. Meanwhile, one presumes that the native gene gets
translated fine despite not being a perfect match to the consensus. So
why change it?

Peter