USER® Cloning

The USER-friendly DNA engineering method allows combining multiple PCR fragment assembly, nucleotide sequence alteration and directional cloning. In this approach, target DNA molecules and cloning vector are generated by PCR with 6-10 bases of homology between the neighboring fragments. PCR primers contain a single deoxyuracil residue (dU) flanking the 3’ end of the homology region, and can be designed to accommodate nucleotide substitutions, insertions and/or deletions. The primers are then used to amplify the vector and target DNA in discrete overlapping fragments that incorporate a dU at each end. The subsequent treatment of PCR fragments with USER Enzyme creates a single nucleotide gap at each location of dU resulting in PCR fragments flanked with single-stranded extensions that allow seamless and directional assembly of customized DNA molecules into a linearized vector. In this way, multi-fragment assemblies, as well as various mutagenic changes, can all be accomplished in a single-format experiment.

USER Workflow


Effect of PCR product concentration on the cloning efficiency



  1. Bitinaite J. et al. (2007) Nucl.Acids Res.35,1992-2002.
  2. Bitinaite, J. and Nichols, N.M. (2009) Curr Protoc Mol Biol., Chapter 3, Unit 3.21.1-3.21.16
  3. Vaisvila, R. and Bitinaite, J. (2013) Methods Mol.Biol. 978, 165-171.

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USER® Cloning includes these areas of focus:

Applications of USER® and Thermolabile USER II Enzymes

Protocols for USER® Cloning

    Publications related to USER® Cloning

  1. Wang S, Li W, Wang S, Hu B 2014. Rapid and Efficient Assembly of Transcription Activator-Like Effector Genes by USER Cloning J Genet Genomics. 41(6), PubMedID: 24976123, DOI: 10.1016/j.jgg.2014.05.002
  2. Lund AM, Kildegaard HF, Petersen MB, Rank J, Hansen BG, Andersen MR, Mortensen UH 2014. A versatile system for USER cloning-based assembly of expression vectors for mammalian cell engineering PLoS One. 9(5), PubMedID: 24879460, DOI: 10.1371/journal.pone.0096693
  3. Villiers BR, Stein V, Hollfelder F 2010. USER friendly DNA recombination (USERec): a simple and flexible near homology-independent method for gene library construction Protein Eng Des Sel. 23(1), PubMedID: 19897542, DOI: 10.1093/protein/gzp063