Primer Design for Gene Inserted Counterclockwise into Plasmid - Please help...very basic question for a mol. biologist (Feb/04/2007 )

Hi,

I'm a person who works mostly with with biochemistry, so I'm a bit unsure when it comes to my theory in molecular biology. I am really in need of some rather basic technical advice.

I need to PCR out a gene from a plasmid that we have with an N-terminal HA-tag and I actually understand exactly how to do it except for one problem...I know how to do it for a gene inserted clockwise on a vector map. So, as I understand it, my 5' primer would have been "cc-RE site-HA-ATG....same as the coding sequence (sense strand)" and my 3' C-term primer would have been "cc-RE site-stop codon.... but the antisense of the sequence I want for the gene".

However, it's not the same when the gene is facing the other way right?

So for example....on the printout of the plasmid map, the sequence is listed as:

TCAGCAGCTT...........................................................TCACCAT
AGTCGTCGAA...........................................................AGTGGTA (this is my start codon) it's in a pCMV-SPORT vector so there is a CMV promoter 3' of the gene making it possible to express
in mammalian cells despite being inserted "backwards"
<------------- gene ----------------


Does that mean that my primers need to be the same as I listed, except the one that will bind to the C-term of the coding sequence is the same as the sequence encoding the correct amino acids and the one for the N-terminus of my protein should be backwards and the anti-sense of what the correct nucleotides are to encode the correct amino acids and HA-tag. Maybe that's not a very clear way to put it.

Any help someone could provide so that I don't mess up my primers and waste tons of time would be greatly appreciated!

My sincere thanks

Dan