Hello all,

Apologies if there is a simple answer to this that I'm just not seeing. I am designing a crispr/cas9 construct and want to use existing plasmids we have as the backbone. I am trying to use a single promoter to drive mcherry and cas9 expression as a single transcript with a t2a skip between them. If I have a second kozak sequence after the t2a skip (so, promoter- kozak-mcherry- t2a-kozak- cas9) will this somehow adversely effect expression? I know generally there is only one kozak sequence at the beginning of the transcript. I can't really get rid of the second kozak sequence in front of the cas9 sequence very easily (long story, restriction enzymes etc).

So would this cause issues in expression?