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Standardized modular assembly of polycistronic operons with Modular Cloning (MoClo) using the In-Cloning toolkit

Modular Cloning (MoClo) toolkits enable the rapid assembly of multigene constructs. They are based on Golden Gate cloning, which uses Type IIS restriction enzymes that cut outside their recognition site. Since recognition and cut sequence are decoupled, overhangs created by Type IIS restriction enzy...

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Bibliographic Details
Main Authors: Vries, Jelmar de (Author) , Lindeboom, Timon A. (Author) , Vries, Stijn T. de (Author) , Schindler, Daniel (Author) , Hoffmann, Stefan A. (Author)
Format: Article (Journal)
Language:English
Published: September 2nd, 2025
In: JoVE journal. Neuroscience
Year: 2025, Issue: 223, Pages: ?
ISSN:1940-087X
DOI:10.3791/68103
Online Access:Verlag, lizenzpflichtig, Volltext: https://doi.org/10.3791/68103
Verlag, lizenzpflichtig, Volltext: https://app.jove.com/t/68103/standardized-modular-assembly-polycistronic-operons-with-modular
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Author Notes:Jelmar de Vries, Timon A. Lindeboom, Stijn T. de Vries, Daniel Schindler, Stefan A. Hoffmann
Description
Summary:Modular Cloning (MoClo) toolkits enable the rapid assembly of multigene constructs. They are based on Golden Gate cloning, which uses Type IIS restriction enzymes that cut outside their recognition site. Since recognition and cut sequence are decoupled, overhangs created by Type IIS restriction enzymes can be deliberately chosen, and cloning strategies typically prevent cutting of correctly joined DNA fragments. This allows highly efficient assemblies of multiple DNA fragments in a single reaction. In MoClo, individual functional DNA parts such as promoters, ribosomal binding sites, coding sequences, and terminators, as well as higher-order assemblies, are assigned standardized overhangs, such that reusable parts libraries can be created. The majority of bacterial MoClo toolkits are designed for cloning monocistronic transcriptional units and do not provide a structured path for the assembly of polycistronic operons. This protocol demonstrates the assembly of polycistronic transcription units with the In- & Out-Cloning toolkit. The same approach is transferable to other MoClo toolkits.
Item Description:Gesehen am 23.03.2026
Physical Description:Online Resource
ISSN:1940-087X
DOI:10.3791/68103

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