I want clone two ORFs within a single vector using the 2A peptide instead of the IRES. I have a few questions.
1. Does the downstream ORF need to be in frame with the upstream ORF? This is not a requirement for the IRES. Using the 2A, are ORF-1 and ORF-2 basically a single ORF that undergoes post-translational cleavage?
2. Do I need to remove the stop codon from the upstream ORF? If O1 and O2 are a single ORF then I think the first stop codon needs to be removed.
3. Does the downstream ORF need a start codon/Kozak sequence? This is required for the ORF downstream of an IRES but not sure about the 2A. I do not think the start codon/Kozak would be necessary if the translation is continuous between ORF1 and ORF2.