FAQ: What if there is an internal BsaI site(s) in my inserts?

Golden Gate Assembly with single inserts can tolerate a single internal site simply because the ligase can complete successfully with the BsaI restriction enzyme, although the efficiency of assembly will decrease. For multiple insert assemblies it is best to use site-directed mutagenesis to eliminate the internal site(s), or consider another assembly approach such as NEBuilder® HiFi DNA Assembly. This alternative aproach can be used for assemblies involving up to 5 inserts.