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- Look for BsaI cut sites in your sequence using Geneious. Use Restriction Analysis to find the BsaI sites (under Advanced) and apply them to your sequence.
- Select the CDS annotation for your sequence, and check the BsaI sites to see in which codons they occur.
- Look for alternate codons that code for the same amino acid. Make sure that the codon that you end up with after mutating has a higher frequency than the existing codon. You can find a table of frequencies for humans here: http://www.genscript.com/cgi-bin/tools/codon_freq_table
- If you can't find a codon with a higher frequency, then use a lower frequency codon but you might have to mutate it back after doing the Golden Gate reaction.
Here are the changes that we made (4/29/2014):
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