I am trying to detect expression of gene of interest by constructing a multicistronic vector that incorporates 'P2A' and 'tdTomato', so basically, modified gene would look something like: [gene of interest - P2A - tdTomato (or mCherry)]. While many literature points to the advantages of using P2A in enabling expression of genes on multicistronic vector, I realize they don't quite go in depth about the sequences in between P2A and the downstream reporter gene (tdTomato, in my case).

I have referred to some available P2A-tdTomato containing vectors donated on Addgene.com, and I've found varying 'linker residues' connecting P2A-tdTomato. For instance, one would have 'E F' residues between P2A and tdTomato, another would have 'P G P', and another would have none (P2A sequences continues directly into tdTomato).

Q. What are the factors that dictate the choice of 'linker residues' between P2A and tdTomato genes (or any other reporter genes in that matter)?

In other words, are 'linker residues' necessary to be placed between P2A and tdTomato sequences and if so, what are they?

I'd greatly appreciate if anybody could share their knowledge on this and provide some explanations! (accompanying resources would be plus appreciative :D)