Cloning a fusion protein, do I remove the ATG codon. - (Aug/15/2014 )
Hi all,
I have a dilemma if anyone was kind and knowledgeable enough.
I am cloning a N-terminal fusion protein, and I am designing my primers so that I have to remove the ATG codon of my gene of interest.
My question --> is it appropriate to remove the ATG from your protein and rely on the transcription of you fused protein within the vector?
I am little stumped on why you have to remove the ATG in the first place?
Would appreciate some ideas.
Cheers
Yours Sincerely Liam Tegg
-Teapot_11-
You don't need to, but if you are using a short tag you run the risk that the RNA pol will skip over the tag and start transcription at the internal ATG. To get around this, you can add a Kozak (or Shine-Delgarno, depending on application) sequence at the ATG for the tag.
-bob1-