Fusion proteins (Using GoldenGate):

Add Q sites using primers.

  • Make sure to add at least 1 extra nucleotide before the BsaI site (6 is standard). Make sure that the extra nucleotides at the end of the primers are NOT complementary.
  • Make sure to add/keep the Kozak sequence.
  • Make sure to get rid of the stop codon of the first protein (all proteins except the last protein).
  • Make sure the two proteins are in frame.
  • Check binding site melting temperature at the NEB website. Check hairpin structures in Geneious.

 

If the BsaI cleavage is scarless, then there should not be a frameshift. (Someone verify this.)

 

 

Fixing frameshifts:


If the fusion protein looks like this:

(NNN)(NNN)(NNN)(GGGG)(ATG)(NNN)(NNN)(NNN)

  protein1                    Q site             protein2

 

Add nucleotides:

(NNN)(NNN)(NNN)(TWO nucleotides)(GGGG)(ATG)(NNN)(NNN)(NNN)

  protein1                                                       Q site             protein2

OR

(NNN)(NNN)(NNN)(GGGG)(TWO nucleotides)(ATG)(NNN)(NNN)(NNN)

  protein1                    Q site                                                  protein2

 

 

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