How to clone a 2A peptide between two ORFs? - (Mar/29/2015 )

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. 

Thanks! 

1. The downstream ORF is not dependent on upstream ORF translation(5' Cap dependent translation), There are two different Ribosmome bind to 5' Cap(ORF1) and IRES(OFR2) element.

When you using 2A to link upstream ORF, you have to make sure 2A and upstream ORF in same frame(because 2A base on post-translational cleavage).

2. If you using 2A link O1 and O2=>Yes!! remove the O1 stop codon.

3. If you using 2A link O1 and O2=> the downstream ORF is not require start and additional Kozak sequence. 

Good luck~

Thank you for the clarification Robin Shen!