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Link to original content: http://www.addgene.org/cloning/moclo/
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Modular Cloning (MoClo) Guide

Overview

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. 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.

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.

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

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

Kit name Type PI Description
MoClo Toolkit Plant Expression Sylvestre Marillonnet 95 plasmids for cloning and assembling standardized parts to create eukaryotic multigene constructs.
MoClo Plant Parts Kit Plant Expression Nicola Patron 95 standardized parts specific for plant synthetic biology.
MoClo Plant Parts II and Infrastructure Kit Plant Expression Johannes Stuttmann 95 additional plasmids for hierarchical assembly of plant multigene constructs.
MoClo Plant Parts III: Transformation & Genome Engineering Kit Plant Expression, CRISPR Daniel Voytas 93 additional plasmids including regulatory elements, coding sequences, CRISPR parts
GreenGate Cloning System Plant Expression Jan Lohmann Plasmids to create plant expression vectors containing several cassettes and generate multi-construct transgenic plants.
GreenGate 2.0 Toolkit Plant Expression Alexis Maizel 68 plasmids to extend the GreenGate Cloning System (Lohmann) with additional Level 0 modules and components to create plasmids with multiple transcriptional units.
GoldenBraid 2.0 Kit Plant Expression, CRISPR Diego Orzaez 94 plasmids using the GoldenBraid framework for plant synthetic biology and multigene engineering, including CRISPR/Cas9 components.
MoChlo: Modular Cloning Chloroplast Toolbox Plant Expression Scott Lenaghan 122 plasmids with chloroplast-specific genetic modules, including destination vectors specific for tobacco (N. tabacum) or potato (S. tuberosum).
Joint Modular Cloning (JMC) Toolkit Plant Expression Daniel Voytas 76 plasmids for assembling plant transformation vectors for overexpression, gene editing, functional genomics, and more complex metabolic engineering and synthetic biology, compatible with PhytoBrick parts.
MoBiFC - Modular Bimolecular Fluorescence Complementation Toolkit Plant Expression Benjamin Field 50 plasmids for assembling bimolecular fluorescence complementation experiments to analyze protein-protein interactions in plants.
MoClo-YTK Yeast Expression John Dueber 96 standardized and characterized parts that can be used for bottom-up hierarchical assembly of single and multi-gene constructs for S. cerevisiae expression.
Multiplex Yeast Toolkit (MYT) Yeast Expression, CRISPR Tom Ellis 96 plasmids to extend the MoClo-YTK (Dueber) for genetic engineering of S. cerevisiae, enabling larger multi-gene constructs, multiplexed CRISPR editing, and more.
Fungal Toolkit for Modular Cloning (FTK) Fungal Expression, CRISPR Arnold Driessen 96 plasmids (including CRISPR/Cas9 components) for expression or gene editing in filamentous fungi such as Penicillium or Aspergillus species.
MoClo Yeast Secretion and Display (YSD) Toolkit Yeast Expression Paul Young 34 plasmids that can be used in conjunction with the MoClo Yeast Toolkit (YTK) to optimize secretion and cell surface display of proteins of interest.
Yeast Secrete and Detect Yeast Expression Sylvestre Marillonnet Plasmids to generate transcription units for protein secretion in yeasts S. cerevisiae and P. pastoris (a.k.a. K. phaffii).
MoClo Pichia Toolkit Yeast Expression Volker Sieber Plasmids with control elements and fluorescent reporters to be used in combination with the MoClo-YTK toolkit for protein expression and secretion in P. pastoris (a.k.a. K. phaffii).
GoldenPiCS Kit Yeast Expression Brigitte Gasser, Diethard Mattanovich, and Michael Sauer 62 plasmids for advanced strain engineering in P. pastoris (a.k.a. K. phaffii), including overexpression plasmids with up to eight transcription units.
Yarrowia lipolytica Golden Gate tool kit Yeast Expression Tristan Rossignol 64 plasmids to assemble and integrate up to three transcription units in the oleaginous yeast Y. lipolytica.
EXPRESSYALI Yeast Expression Irina Borodina Plasmids for assembly of single and multi-gene constructs for integration into Y. lipolytica chromosomal loci.
Yeast GPCR-sensor Toolkit Yeast Expression Tom Ellis Plasmids for constructing highly-tunable GPCR-based biosensors in yeast (requires MoClo-YTK parts for use).
Yeast GoldenBraid Cloning System and Toolkit Yeast Expression Elena Caro Bernat Plasmids for generating transcriptional units using the GoldenBraid system, for integration in S. cerevisiae.
Easy-MISE Toolkit Yeast Expression Paola Branduardi Easy-Modular Integrative fuSion-ready Expression toolkit for improved heterologous protein production in S. cerevisiae.
EcoFlex MoClo Toolkit Bacterial Expression Paul Freemont 78 plasmids including constitutive promoters, T7 expression, RBS strength variants, synthetic terminators, protein purification tags and fluorescence proteins for use in E. coli for a variety of applications.
CIDAR MoClo Parts Kit Bacterial Expression Douglas Densmore A collection of 93 modular plasmids and enhanced MoClo protocols for rapid one-pot, multipart assembly, combinatorial design and expression tuning in E. coli.
CIDAR MoClo Extension, Volume I Bacterial Expression Richard Murray 110 plasmids designed to quickly and efficiently build a wide variety of gene expression constructs using the CIDAR-standard MoClo system.
openCIDAR MoClo Toolkit Bacterial Expression Eric Young Broad host range vectors compatible with the CIDAR MoClo cloning standard, including a combinatorial library of constructs expressing GFP at 18 levels for rapid characterization in new organisms.
Mobius Assembly Vector Toolkit Bacterial Expression Naomi Nakayama A toolkit with both high cloning capacity and vector simplicity, compatible with other MoClo, GoldenBraid, and PhytoBricks standards.
CyanoGate Kit Bacterial Expression Alistair McCormick Kit containing 96 parts and acceptor vectors for generating multi-part assemblies in either integrative or self-replicating plasmid vectors for working in cyanobacteria.
Cultivarium POSSUM Toolkit Bacterial Expression, Fungal Expression Nili Ostrov, Charlie Gilbert, and Cultivarium Tools 189 plasmids, including origins of replication and selection markers for a variety of bacterial and fungal species for engineering non-model microbes.
AspFlex Kit Bacterial Expression, CRISPR Veronica Godoy-Carter Plasmids for protein expression and CRISPRi experiments in Acinetobacter spp. and Acinetobacter baumannii.
Komagataeibacter Tool Kit (KTK) Bacterial Expression Tom Ellis DNA parts to create multigene constructs expressed in the cellulose-producing Komagataeibacter species.
Ralstonia-GG-Kit Bacterial Expression Thomas Lahaye Plasmids to generate gene knockout and complementation constructs for use in Ralstonia solanacearum.
Ubigate Collection Bacterial Expression Marco Trujillo Plasmids for reconstituting the ubiquitination cascades of different organisms in E. coli.
MetClo DNA Assembly Kit Bacterial Expression Christopher A. O'Callaghan Toolkit for assembling large DNA fragments based on methylation-switching.
The Mammalian Toolkit Mammalian Expression Hana El-Samad 253 plasmids that allow for the assembly of cellular circuits using Golden Gate-based cloning methods for use in mammalian cells. These can be used with plasmids from the MoClo-YTK.
EMMA: Extensible Mammalian Modular Assembly Toolkit Mammalian Expression Yizhi Cai 95 plasmids to generate mammalian expression vectors for transient or stable transfections.
PUF Assembly Kit Mammalian Expression, Bacterial Expression Huimin Zhao Plasmids to assemble custom-made RNA-binding proteins based on the Pumilio and FBF (PUF) homology domain, for mammalian or E. coli expression.
T2A Split/Link TurboID Kit Mammalian Expression Sven Eyckerman Plasmids to assemble mammalian expression vectors for proximity labeling experiments.
Multiplex CRISPR/Cas9 Assembly Systems Genome Engineering, CRISPR, Mammalian Expression Takashi Yamamoto Plasmids for construction of all-in-one CRISPR/Cas9 vectors expressing multiple gRNAs (up to seven) with a Cas9 nuclease, nickase, dCas9, or dCas9-FokI fusion for use in human or mouse systems.
Fragmid Genome Engineering, CRISPR, Mammalian Expression, Drosophila John Doench 180-plasmid toolkit for rapid creation of CRISPR vectors compatible with various delivery approaches, enzymes, and mechanisms.
zCas9i cloning kit: MoClo-compatible CRISPR/Cas9 cloning kit with intronized Cas9 Genome Engineering, CRISPR Sylvestre Marillonnet and Johannes Stuttmann 73 plasmids for assembly of CRISPR/Cas9 constructs for use in plants. The constructs are made with zCas9i, a high efficiency intron-optimized SpCas9-coding gene.
MoClo CRISPR/Cas Toolkit for Plants Genome Engineering, CRISPR, Plant Expression Vladimir Nekrasov 95 plasmids including CRISPR/Cas nucleases, base editors, gRNA backbones, and promoters for genome editing in monocots and dicots.
pDGE Dicot Genome Editing Kit Genome Engineering, CRISPR, Plant Expression Johannes Stuttmann Plasmids for assembly of constructs for CRISPR genome editing in dicotyledonous plants.
CRISPi Kit Genome Engineering, CRISPR, Yeast Expression Brigitte Gasser Plasmids to perform CRISPR/Cas9 mediated genome editing in the yeast P. pastoris (a.k.a. K. phaffii).
NT-CRISPR Plasmid Collection Genome Engineering, CRISPR, Bacterial Expression Anke Becker Plasmids to perform genome engineering in Vibrio natriegens.
TALE Toolbox Genome Engineering, TALEN Feng Zhang Plasmids for building TALE nucleases (TALENs) and transcription factors (TALE-TFs) for genome editing and transcription modulation applications
Golden Gate TALEN and TAL Effector Kit 2.0 Genome Engineering, TALEN Daniel Voytas and Adam Bogdanove Plasmid toolkit to efficiently assemble TALEN constructs with custom repeat arrays, containing anywhere between 12 and 31 repeats.
Golden Gate TALEN Accessory Pack Genome Engineering, TALEN Takashi Yamamoto Nine pFUS array vectors and destination vectors designed for use with the Golden Gate TALEN and TAL Effector Kit (Voytas and Bogdanove).
Platinum Gate TALEN Kit Genome Engineering, TALEN Takashi Yamamoto Plasmids for construction of highly-active Platinum TALENs for use in a wide variety of target organisms.
TALEN Kit Genome Engineering, TALEN Stephen Ekker 256 plasmids encoding all possible combinations of pFUS_B4 clones for Golden Gate assembly of 15-RVD TALENs.
FusX TALEN Assembly System Kit Genome Engineering, TALEN Stephen Ekker 336 plasmids of pre-assembled pFusX clones allowing for Golden Gate assembly of 15-16 RVD TALEN inserts in one cloning step rather than two.
Golden GATEway Cloning Kit Other, Zebrafish Expression 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 Other, Yeast Expression, Plant Expression, Bacterial expression 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 .