You are ordering DNA to use with a Golden Gate reaction (using BsaI) to make entry vectors.  But... your sequence has BsaI cut sites - oh no!  Little fear, you might be able to mutate the codons that make up the BsaI site to remove those sites without changing the amino acid sequence.

  1. 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.
  2. Select the CDS annotation for your sequence, and check the BsaI sites to see in which codons they occur.
  3. 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
  4. 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 (i.e., site-directed mutagenesis*) after doing the Golden Gate reaction.

 

Here are the changes that we made (4/29/2014):

PirB

  1. VVS: GTG GTC TCC -> GTG GTG TCC
    1. GTC frequency: 0.24
    2. GTG frequency: 0.47
  2. LVS:  CTG GTC TCA -> CTG GTG TCA
    1. GTC frequency: 0.24
    2. GTG frequency: 0.47
  3. ET:  GAG ACC -> GAA ACC
    1. GAG frequency: 0.58
    2. GAA frequency: 0.42
    3. WARNING: We replaced with a lower frequency codon

LilrB2

  1. VVS: GTG GTC TCC -> GTG GTG TCC
    1. GTC frequency: 0.24
    2. GTG frequency: 0.47

 

*If you really care, you can mutate these sites back using site-directed mutagenesis (e.g. this kit: http://www.genomics.agilent.com/en/Site-Directed-Mutagenesis/QuikChange-Lightning/?cid=AG-PT-175&tabId=AG-PR-1162)

  • No labels