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Review
. 2025:2850:481-500.
doi: 10.1007/978-1-0716-4220-7_27.

Advancements in Golden Gate Cloning: A Comprehensive Review

Jesús Laborda-Mansilla  1 , Eva García-Ruiz  2
Affiliations
Review

Advancements in Golden Gate Cloning: A Comprehensive Review

Jesús Laborda-Mansilla et al. Methods Mol Biol. 2025.

Abstract

Researchers have dedicated efforts to refining genetic part assembly techniques, responding to the demand for complex DNA constructs. The optimization efforts, targeting enhanced efficiency, fidelity, and modularity, have yielded streamlined protocols. Among these, Golden Gate cloning has gained prominence, offering a modular and hierarchical approach for constructing complex DNA fragments. This method is instrumental in establishing a repository of reusable parts, effectively reducing the costs and proving highly valuable for high-throughput DNA assembly projects. In this review, we delve into the main protocol of Golden Gate cloning, providing refined insights to enhance protocols and address potential challenges. Additionally, we perform a thorough evaluation of the primary modular cloning toolkits adopted by the scientific community. The discussion includes an exploration of recent advances and challenges in the field, providing a comprehensive overview of the current state of Golden Gate cloning.

Keywords: DNA assembly; Golden Gate cloning; Modular cloning; Synthetic biology; Type IIS restriction endonucleases (TIIS-RE).

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References

    1. Pryor JM, Potapov V, Bilotti K et al (2022) Rapid 40 kb Genome Construction from 52 Parts through Data-optimized Assembly Design. ACS Synth Biol 11:2036–2042. https://doi.org/10.1021/acssynbio.1c00525 - DOI - PubMed - PMC
    1. Gibson DG, Young L, Chuang RY et al (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6:343–345. https://doi.org/10.1038/nmeth.1318 - DOI - PubMed
    1. Schindler D, Dai J, Cai Y (2018) Synthetic genomics: a new venture to dissect genome fundamentals and engineer new functions. Curr Opin Chem Biol 46:56–62. https://doi.org/10.1016/j.cbpa.2018.04.002 - DOI - PubMed - PMC
    1. Cost GJ (2007) Enzymatic ligation assisted by nucleases: Simultaneous ligation and digestion promote the ordered assembly of DNA. Nat Protoc 2:2198–2202. https://doi.org/10.1038/nprot.2007.325 - DOI - PubMed
    1. Shetty RP, Endy D, Knight TF (2008) Engineering BioBrick vectors from BioBrick parts. J Biol Eng 2:1–12. https://doi.org/10.1186/1754-1611-2-5 - DOI

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