The first step in the GoldenBraid assembly strategy is to adapt your DNA building block to the GB standard. It implies the removal of the internal restriction sites for the enzymes used in GB (BsaI, BsmBI and BtgZI) and the addition of the appropriate 4-nt flanking overhangs .
This process is referred to as domestication and we offer three different domestication strategies:
1. The GB Domesticator uses PCR mutagenesis followed by typeIIS-based assembly of mutagenized fragments to domesticate the sequence.
2. The Phylogenetic Search, searches the databases for the closest orthologous genes containing minimum internal sites.
3. The Synthetic strategy provides the domesticated sequence in a single DNA fragment, ready to order it and clone it into the pUPD vector.