I am planning to clone a truncated version of a protein that includes only the extracellular domain, tagged with mCherry. Since the extracellular part lacks an ATG start codon, I am considering two options: 1. Adding an ATG in the forward primer. 2. Using the ATG already present in the vector, which originally drives the expression of GFP (I intend to remove GFP entirely and replace it with my gene of interest). Does it really matter which approach I choose, or is one method more efficient or reliable?
Do you want your protein to get exported out of the cell? If so you should include the signal peptide which is likely the beginning of the ORF, includinng the ATG.
I would add an ATG because with the ATG of the vector you will have some extra aminoacid from GFP (if I understood well ...)
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