Accurate DNA assembly and genome engineering with optimized uracil excision cloning

Mafalda Cavaleiro, Se Hyeuk Kim, Susanna Seppala, Morten Thrane Nielsen, Morten H. H. Nørholm

Research output: Contribution to journalJournal articleResearchpeer-review


Simple and reliable DNA editing by uracil excision (a.k.a. USER
cloning) has been described by several research groups, but the optimal design of
cohesive DNA ends for multigene assembly remains elusive. Here, we use two
model constructs based on expression of gfp and a four-gene pathway that
produces β-carotene to optimize assembly junctions and the uracil excision
protocol. By combining uracil excision cloning with a genomic integration
technology, we demonstrate that up to six DNA fragments can be assembled in a
one-tube reaction for direct genome integration with high accuracy, greatly
facilitating the advanced engineering of robust cell factories.
Original languageEnglish
JournalA C S Synthetic Biology
Issue number9
Pages (from-to)1042-1046
Number of pages5
Publication statusPublished - 2015


  • Molecular cloning
  • DNA assembly
  • Uracil excision cloning
  • Genome engineering


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