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:
...
protein1 Q site protein2
PirB - goldengate the two pieces into a pENTR before PCRing the linkers on.