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cloning membrane protein with 2 tags - (Apr/04/2006 )

Hi, everybody!

I want to clone the membrane protein with 2 tags (one in the middle of the protein and other on C terminus) for visualization.
How can I do this in simple way?


for c terminus, a PCR is ok.

but both tagging may be done in closely one step. First PCR of the first fragment. Fwd primer is classical and reverse primers contains a unique restiction site, the flag, and the annealing part.

Seond part of the gene is done by : fwd primer holds the unique restriction site inserted by the FLAG-rev primer and the reverse primer contains the second flag...
i've attached the picture


Thanks a lot. It looks pretty good.