With constant advances both in the development of new enzymes (e.g., BsaI-HFv2 and BsmBI-v2) and research on maximizing enzyme functionality (e.g., ligase fidelity), NEB has become the industry leader in pushing the limits of Golden Gate Assembly (and related methods such as MoClo, GoldenBraid, Mobius Assembly and Loop Assembly). NEB has all the products and information you need to perform complex assemblies, as demonstrated with 20+ fragment assemblies exhibiting high efficiencies and >90% accuracy, along with low backgrounds.


GG_LP_techNote_icon Golden Gate Assembly Workflow Diagram for complex assemblies GG_LP_GoldenGateTool_icon GG_LP_MoClo_icon


Golden Gate Assembly Workflow for complex assemblies

Golden Gate Assembly Workflow


Advances in ligase fidelity

Research at NEB has led to an increased understanding of ligase fidelity, including the development of a comprehensive method for profiling end-joining ligation fidelity in order to predict which overhangs have improved fidelity (1). Their research has enabled complex fragment assemblies with high efficiencies and >90% accuracy. Use our Ligase Fidelity Tools to design high fidelity Golden Gate Assemblies under various experimental conditions.


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Type IIS restriction enzymes for Golden Gate Assembly 

NEB offers more Type IIS (i.e., recognize asymmetric DNA sequences and cleave outside of their recognition sequence) restriction enzymes than any other supplier, many of which are used in Golden Gate Assembly. NEB is pleased to offer: Esp3I, an isoschizomer of BsmBI that is recommended for use at 37°C and is supplied with CutSmart® Buffer, and the improved BsaI-HFv2 and BsmBI-v2, which are optimized for Golden Gate Assembly.

For information about other Type IIS restriction enzymes used in Golden Gate Assembly, such as BbsI, BbsI-HF and SapI, view our Type IIS restriction enzyme table.

For a limited time, try a sample of BsaI-HFv2 and see how it works for you!

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1. Potapov, V. et. al. “Comprehensive Profiling of Four Base Overhang Ligation Fidelity by T4 DNA Ligase and Application to DNA Assembly.” ACS Synth. Biol. 2018. DOI: 10.1021/acssynbio.8b00333.

One or more of these products are covered by patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc. For more information, please email us at [email protected]. The use of these products may require you to obtain additional third party intellectual property rights for certain applications.

Golden Gate Assembly Brochure


Read our Golden Gate Assembly brochure for tips, tools and products to help you achieve your complex (20+) fragment assemblies.

Featured products:

NEBNext_portal_product_icon NEB® Golden Gate Assembly Kit (BsmBI-v2)
Contains an optimized mix of BsmBI-v2 (optimized for Golden Gate Assembly) and T4 DNA Ligase. Can direct the accurate assembly of 2 – 20+ inserts/modules using the Golden Gate approach.
NEBNext_portal_product_icon Esp3I
An isoschizomer of BsmBI that is recommended for use at 37°C and is supplied with CutSmart Buffer, adding flexibility to your reaction setup
NEBNext_portal_product_icon BsaI-HFv2
New version of BsaI-HF that is optimized for Golden Gate Assembly.
NEBNext_portal_product_icon BsmBI-v2
New version of BsmBI that is optimized for Golden Gate Assembly.