Vector Construction- Tagging N-term - necessity of ATG in Gene of Interest (Sep/06/2005 )
I'm designing a new vector that has an ORF that will code for a gene of interest. I want to tag my Gene on the N-term. with HIS/MYC/HA etc. tag. I realize I need a ATG start site in frame with my tag. My question is: Do I need the second pre-existing ATG at the beginning of the gene? 1. Is it absolutly necessary? Does its presesence somehow inhibit the fusion of the tag?
Any input would be great.
i wouldn't think that it would be absolutely necessary to add the second ATG, as long as you have the first one before the tag and after the RBS. but sometimes the methionine at the begining of a protein is responsible for interactions that stabilize the secondary stucture of the protein.
it's presensce shouldn't effect the fusion tag. are you making this a cleavable affinity tag?
It is common practice to mutate the ATG in your gene of interest to ATC (Isoleucine) so as to discourage false starting at this internal MET (which would cause you to loose your tag). Also, most people put 2 or 3 "buffer" amino acids between the tag and the start site of your gene.
Good luck with it.