For efficient golden gate assembly, our team prepared a gene fragment that only contains BsaI sequences and its cleavage site so that the prefix and suffix could be flexibly changed and attached to both ends of the transcription unit, assembled using SapI. The advantage of this method is that it is possible to greatly increase the number of gene fragments that can be synthesized using the golden gate assembly at once without the need to use various plasmids by ordering a prefix/suffix fragment at a low price. According to NEB, it was confirmed that their high-fidelity restriction enzyme could synthesize up to 52 gene fragments at a time. The first BsaI prefix and the last BsaI suffix have EcoRI and BamHI cleavage sites for double digestion and ligation to pCambia 2300 vector for agrobacterium-mediated microalgae transformation.

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