With constant advances both in the development of new enzymes (e.g., PaqCI®BsaI-HF®v2 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.

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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

Fidelity decreases as the complexity of Golden Gate Assembly increases. Visit our protocols for multi-fragment assemblies to the right.

Fidelity vs. Complexity of Golden Gate Assembly

The percentage of colonies harboring correctly assembled constructs is shown for one-pot Golden Gate Assembly reactions containing: 12, 24, 35 and 52 fragments. Error bars depict the standard deviation of at least 3 experimental replicates; this data has been reported in several recent manuscripts:

Pryor, J.M., et al. (2022) ACS Synth. Biol. e0525
Pryor, J. M. et al. (2020). PLoS Onee8592
Potapov, V. et al. (2018). ACS Synth. Biol., e0333 

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, 2, 3, 4). 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 and learn how these tools and research are enabling advancements in synthetic biology.


Icon for Multiple Publications GG_LP_webinar_icon  GG_LP_LigaseFidelityTool_icon 

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 rCutSmartBuffer), PaqCI (an AarI isoschizomer with a 7 basepair recognition sequence) 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 PaqCIBbsI, BbsI-HF and SapI, view our Type IIS restriction enzyme table.




  1. Potapov, V. et. al. (2018) ACS Synth. Biol., e0333.
  2. Pryor, J. M. et al. (2020) PLoS Onee8592.
  3. Pryor, J.M., et al. (2022) ACS Synth. Biol. e0525.
  4. Sikkema A.P. et al. (2023) Current Protocols. e882.

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 The use of these products may require you to obtain additional third party intellectual property rights for certain applications.

Featured Products:

NEBNext_portal_product_icon NEBridge® 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 – 50+ inserts/modules using the Golden Gate approach.
NEBNext_portal_product_icon NEBridge® Ligase Master Mix
NEBridge Ligase Master Mix is a 3X master mix for Golden Gate Assembly. Designed for use with NEB Type IIS restriction enzymes, this master mix contains T4 DNA Ligase in an optimized reaction buffer with a proprietary ligation enhancer.
NEBNext_portal_product_icon Esp3I
An isoschizomer of BsmBI that is recommended for use at 37°C and is supplied with rCutSmart 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.
New Type IIS restriction enzyme with 7 basepair recognition sequence (AarI isoschizomer).


Golden Gate Assembly Brochure


Read our Golden Gate Assembly brochure for tips, tools and products to help you achieve your complex (50+) fragment assemblies.