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Modular Cloning (MoClo) Guide

Modular Cloning (MoClo) is a system for efficiently assembling multiple DNA parts into functional plasmids. It is a powerful method for creating many plasmids from different combinations of a set of components (Weber et al., 2011). Based on Golden Gate plasmid assembly, MoClo exploits the ability of Type IIS restriction enzymes BsaI and BpiI/BbsI to cut outside their recognition sites, creating DNA fragments with an overhang of 4 bp. By incorporating standard overhang sequences at the restriction cut sites flanking each module, multiple modules with complementary overhangs can be digested and ligated in a single step resulting in a 4-bp fusion site in between (Figure 1). MoClo can be used for a variety of applications, from building synthetic gene expression circuits to engineering metabolic pathways, assembling gene-editing tools, and more.

The MoClo system consists of three sets of cloning vectors (Level 0, 1, and 2) which can be used in successive assembly steps. Before beginning, scientists insert fragments of DNA containing basic parts (promoters, UTRs, coding sequences, terminators, etc.) flanked by unique, standardized sites into individual Level 0 plasmids, or choose from a growing number of kits containing standardized modules.

In the first assembly step, compatible Level 0 parts are digested and directionally assembled into a Level 1 vector creating a single transcriptional unit (for example, a promoter, a 5' UTR, a signal peptide or fusion tag, a protein-coding gene, and a terminator, as in Figure 1). Next, up to six Level 1 modules can be assembled into a Level 2 vector, forming a functional genetic circuit. Level 2 vectors are often designed with flexibility to allow for additional iterations of assembly. Combining multiple Level 2 vectors permits the creation of even more complex constructs constrained only by the ability of E. coli to maintain the final plasmid after transformation.

A series of schematics of genetic parts. A: “Level 0 library of basic modules”, with nine labeled blocks for each type of element: Promoters, 5’UTRs, Signal peptides, CDSs, and Terminators. Five elements (one each of P, U, SP, CDS, and T) are highlighted and then joined together into a linear “Level 1 transcription unit”. This transcription unit is then shown joined together with several transcription units as a “Level 2 multigene construct”. B: Detailed view showing the 4-base-pair fusion sites flanking each level 0 part. P has 5’ GGAG and 3’ TACT; U has 5’ TACT and 3’ AATG; SP has 5’AATG and 3’ AGGT; and so on. C: Parts joined together with a single fusion site between each element: GGAG between the backbone and P; TACT between P and U, AATG between U and SP, and so on. Additional details and sequences are available in Weber 2011 and the plasmids of the MoClo Toolkit.
Figure 1: Overview of the MoClo Toolkit components and assembly. Image reproduced from Weber et al. (2011) under CC BY license.

MoClo Plasmid Kits Available from Addgene

Browse or search the table below to find MoClo kits in our collection.

ID Kit Description Items PI

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Additional Resources

Guide to DNA Assembly Techniques by Nicola Patron at The Sainsbury Laboratory

References

Weber, E., Engler, C., Gruetzner, R., Werner, S., Marillonnet, S. (2011). A modular cloning system for standardized assembly of multigene constructs. PLoS One, 5(2), e16765.https://doi.org/10.1371/journal.pone.0016765 PMID: 21364738

Engler, C., Youles, M., Gruetzner, R., Ehnert, T. M., Werner, S., Jones, J. D., Patron, N. J., & Marillonnet, S. (2014). A golden gate modular cloning toolbox for plants. PLoS One, 5(2), e16765.https://doi.org/10.1021/sb4001504 PMID: 24933124

Credits

Contributing Authors
Written and reviewed by the Scientific Curation team at Addgene.
Last Updated
Content last reviewed on 16 June 2026. Catalog items are updated more frequently.