Kozak sequence - (Jul/07/2008 )
I have a gene to which I have to attach KOZAK sequence. After ATG my gene has A. Should I change A to G to get G after start codon ATG. This will change Ile (ATC) to Val (GTC). Is this valid?
Other thing I want to ask is adding HA tag after Kozak sequence. Can I add HA tag (TAC CCC TAC GAC GTG CCC GAC TAC GCC) after ATG or do I need G after ATG (see above). In that case I will have to add one extra amino acid like Ala (GCC).
GCC ACC ATG GCC TAC CCC TAC GAC GTG CCC GAC TAC GCC or can I just add
GCC ACC ATG TAC CCC TAC GAC GTG CCC GAC TAC GCC then my gene sequence
Generally the ACC before the ATG is the most important and I really don't see any difference in expression without the G after ATG. My rule of thumb is, if you are only trying to express the gene (No tag) then don't mess with the amino acids and leave out the G after ATG, but if I'm including a tag then one extra residue before the HA tag won't really hurt anything. I'd go ahead and add the extra residue on this one. By the way, depending on what you are going to do, there are some great primary-conjugated HA antibodies out there. I use a HA-alexa488 that is wonderful!