FAQ: What if there are internal BsaI and BsmBI sites in my insert sequences?

Either use site-directed mutagenesis to eliminate the internal sites beforehand, or utilize the internal sites as junctions between fragments using primers to introduce the silent mutations into the sequence simultaneously during part generation. Or screen your sequences for the absence of other Type IIS restriction sites that could allow an alternative Type IIS restriction enzyme to be used such as BbsI, SapI/BspQI or BtgZI (building your assembly reactions using individual restriction enzyme and T4 DNA Ligase stocks), or consider another assembly approach such as NEBuilder® HiFi DNA Assembly if the assembly will involve 5 or less inserts.

Please refer to our Golden Gate Domestication Tutorial.