Highly Efficient Single-Pot Scarless Golden Gate Assembly

Mohammad Hamedirad, Scott Weisberg, Ran Chao, Jiazhang Lian, Huimin Zhao

Research output: Contribution to journalArticlepeer-review


Golden Gate assembly is one of the most widely used DNA assembly methods due to its robustness and modularity. However, despite its popularity, the need for BsaI-free parts, the introduction of scars between junctions, as well as the lack of a comprehensive study on the linkers hinders its more widespread use. Here, we first developed a novel sequencing scheme to test the efficiency and specificity of 96 linkers of 4-bp length and experimentally verified these linkers and their effects on Golden Gate assembly efficiency and specificity. We then used this sequencing data to generate 200 distinct linker sets that can be used by the community to perform efficient Golden Gate assemblies of different sizes and complexity. We also present a single-pot scarless Golden Gate assembly and BsaI removal scheme and its accompanying assembly design software to perform point mutations and Golden Gate assembly. This assembly scheme enables scarless assembly without compromising efficiency by choosing optimized linkers near assembly junctions.

Original languageEnglish (US)
Pages (from-to)1047-1054
Number of pages8
JournalACS synthetic biology
Issue number5
StatePublished - May 17 2019


  • DNA assembly
  • Golden Gate assembly
  • synthetic biology

ASJC Scopus subject areas

  • Biomedical Engineering
  • Biochemistry, Genetics and Molecular Biology (miscellaneous)


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