Golden Gate Assembly

The efficient and seamless assembly of DNA fragments, commonly referred to as Golden Gate assembly (1,2), has its origins in 1996, when for the first time it was shown that multiple inserts could be assembled into a vector backbone using only the sequential (3) or simultaneous (4) activities of a single type IIS restriction enzyme and T4 DNA ligase. Golden Gate Assembly and its derivative methods exploit the ability of Type IIS restriction endonucleases (REases) to cleave DNA outside of the recognition sequence. The inserts and cloning vectors are designed to place the Type IIS recognition site distal to the cleavage site, such that the Type IIS REase can remove the recognition sequence from the assembly. The advantages of such an arrangement are three-fold:

  1. The overhang sequence created is not dictated by the REase, and therefore no scar sequence is introduced.
  2. The fragment-specific sequence of the overhangs allows orderly assembly of multiple fragments simultaneously.
  3. The restriction site is eliminated from the ligated product, so digestion and ligation can be carried out simultaneously.

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®v2, 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.

NEB has developed an optimized mix for performing Golden Gate Assembly.

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.

Golden Gate Assembly Workflow

In its simplest form, Golden Gate Assembly requires a Type IIS recognition site, in this case, BsaI (GGTCTC), added to both ends of a dsDNA fragment. After digestion, these sites are left behind, with each fragment bearing the designed 4-base overhangs that direct the assembly.


  1. Engler, C., Kandzia, R., and Marillonnet, S. (2008) PLoS ONE 3, e3647.
  2. Engler, C., et al. (2009) PLoS ONE 4, e5553.
  3. Lee, J.H. et al, (1996) Genetic Analysis: Biomolecular Engineering, 13: 139-145.
  4. Padgett, K.A. and Sorge, J.A. (1996) Gene, 168: 31-35.
  5. Werner, S., et al. (2012) Bioeng. Bugs 3, 38-43.
  6. Scior, A., et al. (2011) BMC Biotechnol. 11, 87.
  7. Yan, P., et al. (2012) Anal. Biochem. 430, 65-67.
  8. Sanjana, N. E., et al. (2012) Nat. Protoc. 7, 171-192.

Protocols for Golden Gate Assembly

    Publications related to Golden Gate Assembly

  1. Sakuma T, Nishikawa A, Kume S, Chayama K, Yamamoto T 2014. Multiplex genome engineering in human cells using all-in-one CRISPR/Cas9 vector system Sci Rep. 4, PubMedID: 24954249, DOI: 10.1038/srep05400
  2. Binder A, Lambert J, Morbitzer R, Popp C, Ott T, Lahaye T, Parniske M 2014. A Modular Plasmid Assembly Kit for Multigene Expression, Gene Silencing and Silencing Rescue in Plants PLoS One. 9(2), PubMedID: 24551083, DOI: 10.1371/journal.pone.0088218
  3. Feng Y, Zhang S, Huang X 2014. A robust TALENs system for highly efficient mammalian genome editing Sci Rep. 4, PubMedID: 24407151, DOI: 10.1038/srep03632
  4. Abil Z, Denard CA, Zhao H 2014. Modular assembly of designer PUF proteins for specific post-transcriptional regulation of endogenous RNA J Biol Eng. 8(1), PubMedID: 24581042, DOI: 10.1186/1754-1611-8-7
  5. Alejandro Sarrion-Perdigones, Marta Vazquez-Vilar, Jorge Palací, Bas Castelijns, Javier Forment, Peio Ziarsolo, José Blanca, Antonio Granell, Diego Orzaez 2013. GoldenBraid 2.0: A Comprehensive DNA Assembly Framework for Plant Synthetic Biology Plant Physiology. 162(3), PubMedID: 23669743, DOI:

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    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 reassembly. View a list of TypeIIS enzymes