So I recently started using CLC Main Workbench to do in silico cloning, instead of doing it by hand, and I came to realize the usefulness of being able to see all three potential reading frames at the same time.
This made me realize that sometimes you end up with a second methionine close to the start-codon-methionine. i.e., you have the sequence "ATG CCG TAT GCG" for instance, but if we shift that a single nucleotide, we get "A TGC CGT ATG CGx", so just a second potential reading frame a few bases downstream. I asked out residential cloning expert, and she said she would never care about that, but the literature states that 'leaky translation' is a common thing.
So my question to you guys: Would you ever do a silent mutations to remove any secondary methionines that are located close to the original one? Especially if you want to optimize expression levels of a protein for maximum output.