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 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