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.

• 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 .