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- 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.
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