Golden Gate Assembly

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.

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

GoldenGate_52Fragment_CorrectColonies_0221 — 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 One*, e8592
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). 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.

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 rCutSmart^™Buffer), 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 PaqCI, BbsI, BbsI-HF and SapI, view our Type IIS restriction enzyme table.

Potapov, V. et. al. (2018) ACS Synth. Biol., e0333.
Pryor, J. M. et al. (2020) PLoS One, e8592.
Pryor, J.M., et al. (2022) ACS Synth. Biol. e0525.

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

Featured Products:

	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.
	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.
	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
	BsaI-HFv2 New version of BsaI-HF that is optimized for Golden Gate Assembly.
	BsmBI-v2 New version of BsmBI that is optimized for Golden Gate Assembly.
	PaqCI New Type IIS restriction enzyme with 7 basepair recognition sequence (AarI isoschizomer).

Featured Protocols:

Videos

Golden Gate Assembly Workflow

Find out how Golden Gate Assembly can be used to quickly join multiple DNA fragments.
Restriction Enzymes in Golden Gate Assembly

Type IIS restriction enzymes have both recognition and binding sites, but cut downstream of the recognition site, creating 4-base overhangs ideal for re-assembly. View a list of TypeIIS enzymes.
Listen to DAD Informatics tools and NEB enzymes to enable complex one-pot Golden Gate Assemblies

Advances in understanding ligase fidelity, bias, and efficiency in Golden Gate Assembly allows for assembly of more than 50 fragments in a single reaction.
Listen to DAD (Data-optimized Assembly Design) when constructing high-complexity Golden Gate Assembly targets

This webinar introduces insights and modified protocols for Golden Gate Assembly enabling 50+ DNA fragments to be faithfully assembled in a single reaction.
Golden Gate Assembly Domestication Tutorial

This video discusses Domestication, or the removal of Type IIS cut sites naturally occurring in vector or insert sequences, as it relates to Golden Gate Assembly.
Golden Gate Assembly Tool Tutorial

This video demonstrates how to use the Golden Gate Assembly Tool, we will walk through selecting insert and plasmids, primer design to make amplicon inserts.