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Modular Cloning (MoClo) Guide


Synthetic biologists have developed a strategy of modular cloning, called MoClo, to efficiently assemble multiple DNA parts into functional plasmids. It is a powerful and very efficient method for creating many plasmids from different combinations of a common set of components (Weber et al., 2011). This method (based on Golden Gate plasmid assembly) exploits the ability of Type IIS restriction enzymes BsaI and BpiI/BbsI to cut outside their recognition site, leaving DNA fragments with an overhang of 4 bp. If each DNA module is flanked with a standard overhang sequence at the restriction cut site, multiple modules with complementary overhangs can be digested and ligated in a single step resulting in a 4-bp fusion site in between.

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 fusion sites into individual Level 0 plasmids, or choose from a growing number of libraries containing pre-constructed standardized modules.

In the first assembly step, compatible Level 0 parts are directionally assembled into a Level 1 vector creating a single transcriptional unit (for example, a promoter, a 5'UTR, a signal peptide, 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.

MoClo can be used across a variety of applications in various organisms—like building synthetic genetic circuits, metabolic engineering, assembling gene-editing tools, and more. Explore the following list of MoClo plasmid kits that have been deposited with Addgene.

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 from Weber et al., 2011.

MoClo Plasmid Kits Available from Addgene

Plant Expression

Yeast and Fungal Expression

Bacterial Expression

Mammalian Expression

Genome Engineering


  • Golden GATEway Cloning Kit (deposited by Joachim Wittbrodt) - A toolkit that combines Golden Gate and Multisite Gateway cloning. This system was specifically designed for generating zebrafish transgenesis constructs, but is also suitable for creating fusion proteins for use in many different model organisms.

  • Diatom uLoop Assembly Kit (deposited by Christopher Dupont) - 95 plasmids including backbones, fluorescent proteins, protein tags, and signal peptides, for use in a range of organisms including diatoms, yeast, plants and bacteria.

References and More Resources

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

  • Weber E, Engler C, Gruetzner R, Werner S, Marillonnet S. A modular cloning system for standardized assembly of multigene constructs. PLoS One. 2011 Feb 18;6(2):e16765. doi: 10.1371/journal.pone.0016765. PMID: 21364738 .

  • Engler C, Youles M, Gruetzner R, Ehnert TM, Werner S, Jones JD, Patron NJ, Marillonnet S. A golden gate modular cloning toolbox for plants. ACS Synth Biol. 2014 Nov 21;3(11):839-43. doi: 10.1021/sb4001504. PMID: 24933124 .