I want to express 4 genes in a single cell and so will have to clone all the 4 genes in a vector. During the expression, I want all the proteins to be expressed in the same amount, so I do not want the interspersing sequence like IRES/2A to affect the protein production. Usually for IRES, the sequence downstream to IRES is expressed lesser than the one upstream to it and in case of 2A, a portion of the 2A peptide still stays with the upstream and downstream peptides after the cleavage. Is there any better way to do this so that the proteins produced remain seamless?
I have seen the air-dry step only for the li-cor system, but I do not do the air-dry step, and the result is ok.
You could use a separate promoter for each gene. For bacteria, something like T7 promoter would be a reasonable choice.
For eukaryotes, promoters tend to be on a long side, so a binding site for a transactivator might be a better way to go. Also, recombination among long tandem sequences may be significant. As a transactivator, you could use dead Cas9 fused to a transcription activation domain (VP16, for example). Another possibility would be Gal4-VP16 fusion; you would need UAS upstream of the genes you want to express.
You will have to express the transactivator (and sgRNAs if you use Cas9) in addition to your genes, of course.
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