Expanded “assembly standards” for MoClo, GoldenBraid2.0 and other modular Golden Gate Assembly methods

What is Modular Cloning?

Modular Cloning, or better known as MoClo, is a Type IIS assembly method commonly used by synthetic biologists, including those from the plant and AgBio community, for the assembly of multiple biological parts to engineer new biological systems.  MoClo methods provide efficient assembly, and allow users to generate and reuse “parts for assemblies” so that laboratories can share assembly-ready sequences.

MoClo methods depend on Type IIS restriction enzymes (such as BsaI-HFv2, BbsI / BbsI-HF, and BsmBI-v2 / Esp3I) that leave 4-base overhangs.  New England Biolabs (NEB) has extensively studied the fidelity of ligation for all possible 4-base overhangs.  With this information, we are able to estimate the fidelity of any set of junctions (fusion sites). 

Based on Golden Gate Assembly, MoClo (and GoldenBraid 2.0) uses 3 levels of successive assembly.  The community has agreed upon a set of common standard overhangs for each level as indicated below, along with predicted fidelity: 


MoClo Standardized Assembly Overhangs*

  • Level 0 (Basic parts):  ACAT, TTGT   (94% fidelity)
  • Level 1 (Transcriptional units):  GGAG, TACT, CCAT, AATG, AGGT, TTCG, GCTT, GGTA, CGCT   (93% fidelity)
  • Level 2 (Multigene constructs): TGCC, GCAA, ACTA, TTAC, CAGA, TGTG, GAGC, GGGA   (95% fidelity)

Utilizing the gathered ligase fidelity information, NEB has expanded each level of assembly overhangs without sacrificing fidelity.  The expanded sets are listed below:


Expanded MoClo Standardized Assembly Overhangs

  •  Level 0 (Basic parts):  ACAT, TTGT, ACTG, GCTA, CCCA, AATA, ATTC, GTGA, CGCC, AAGA, AAAC, AACG, CTGC, GACC, AAGA, AAAC, AACG, CTGC, GACC, CTAA, ACCC, TACA, GGAA, CAAG, AGAG   (93% fidelity)
  • Level 1 (Transcriptional units):  GGAG, TACT, CCAT, AATG, AGGT, TTCG, GCTT, GGTA, CGCT, GAAA, TCAA, ATAA, GCGA, CGGC, GTCA, AACA, AAAT, GCAC, CTTA, TCCA  (92% fidelity)
  • Level 2 (Multigene constructs):  TGCC, GCAA, ACTA, TTAC, CAGA, TGTG, GAGC, GGGA, CGTA, CTTC, ATCC, ATAG, CCAG, AATC, ACCG, AAAA, AGAC, AGGG, TGAA, ATGA   (95% fidelity)

NEB research on ligase fidelity has led to the development of tools that aid in the design of high fidelity assemblies.  These tools can suggest additional high fidelity overhangs and are available at NEBeta Tools:

  • Ligase Fidelity Viewer™ (v2)
    Visualize overhang ligation preferences
  • GetSet
    Predict high-fidelity junction sets
  • SplitSet
    Split DNA sequence for scarless high-fidelity assembly

Versions of these tools are also referenced in the NEB Golden Gate Assembly Tool.

*These standardized assembly overhangs are part of the MoClo Toolkit referenced in this paper: A modular cloning system for standardized assembly of multigene constructs. Weber E, Engler C, Gruetzner R, Werner S, Marillonnet S. PLOS ONE. 2011 Feb 18;6(2):e16765. doi: 10.1371/journal.pone.0016765. PubMed PMID 21364738.