FAQ: Which kit from NEB should I use for Golden Gate Assembly—the BsmBI-v2 kit (NEB #E1602) or the original BsaI-HFv2 kit (NEB #E1601)?

It depends on whether there are any internal sites for these enzymes in your insert sequences. Since internal sites need to be eliminated by site-directed mutagenesis, choose the kit based on the Type IIS restriction enzyme that has no, or the fewest, sites in your insert sequences. If your sequences have neither BsmBI nor BsaI sites, the BsmBI-v2 kit (NEB #E1602) would be the best choice as it supports the highest complex assembly performance yet developed at NEB both in terms of efficiency (number of transformants) and fidelity (% correct assemblies). However, both kits support complex 24 fragment assemblies.