I'm trying to clone several genes into one plasmid, and was looking at using a 2A self cleaving sequence because its short. So:
start-Gene A -- 2A-- Gene B - stop
As far as the actual cloning - since the 2A sequence is around 60 bp, it seems economical to actually purchase 2 oligos corresponding to top and bottom and adapt a stitch-PCR technique to generate this construct in 2 steps with a high fidelity polymerase (like Q5) by designing overhangs (about 6-8 bp each) in the original oligos. i.e:
Gene A........ .....2AOligo2.......
.....2AOligo 1 ....... ....Gene B...
Does anyone have any particular experience doing this sort of thing or able to recommend a more effective design? I'm not sure if this will even work!