RF-Cloning.org Forum

Hello everyone!

I'm new here. I've created a new thread for this topic, to make it cleaner and maybe get more attention (and help )

So the problem is the following:

I have a plasmid pIRES2-GFP - it can express two genes; one construct inserted in MCS and also GFP positioned after IRES. http://www.addgene.org/vector-database/3178/

I'd like to insert one gene (which contains also CFP) into MCS (with unique restriction ligation sites). In place of GFP I would like to insert RFP+1700 long gene (5ptase) with restriction free cloning. See image: http://oi58.tinypic.com/ayvh1k.jpg

The RF would be performed first, so GFP, CFP and RFP wouldn't interfere.

Now the question is; GFP has some identical sequence to RFP on 5' and 3' (about 22 bp). I could use the one on the 5' end in my advantage by primer construction.
The GFP-RFP identical sequence on their 3' end however may cause me some problems, since I would like to insert also 1700 bp of FKBP-5ptase after RFP as well not just RFP. Please see the picture: http://oi57.tinypic.com/14tc8zq.jpg

Does anyone have any suggestions? Should I be worried about this interference and maybe solve it by first RF cloning RFP in place of GFP and then RF clone FKBP-5ptase part?

Any help would be greatly appreciated!