The net result is the ordered and seamless assembly of DNA fragments in one reaction.
The accuracy of the assembly is dependent on the length of the overhang sequences. Therefore, Type IIS REases that create 4-base overhangs (such as BsaI/BsaI-HF®, BbsI/BbsI-HF, BsmBI and Esp3I) are preferred. Insert assembly calls for careful design of overhangs to direct the assembly, as well as verification that the Type IIS REase sites used are not present in the fragments for the assembly of the expected product. The use of web tools such as the NEB Golden Gate Assembly Tool greatly simplifies both processes, making Golden Gate Assembly a robust technology that assembles multiple DNA fragments(5), even if repetitive elements are present(6) and can, if wished, introduce multiple site-directed mutations(7). Golden Gate Assembly has been widely used in the construction of custom-specific TALENs for in vivo gene editing(8), among other applications.
To help select the best DNA assembly method for your needs, please use our Synthetic Biology/DNA Assembly Selection Chart.
Visit Type IIS Restriction Enzymes for a comprehensive list of all Type IIS enzymes available from NEB and their characteristics.
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