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Directed Evolution Library Creation pp 213–224Cite as

USER Friendly DNA Recombination (USERec): Gene Library Construction Requiring Minimal Sequence Homology

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1179))

Abstract

USER friendly DNA recombination (USERec) is based on near homology-independent recombination of DNA fragments (~40–400 bp) that are efficiently reassembled into full-length genes, proven for up to ten fragments. USERec requires a minimal crossover sequence (a 5′-AN4–8 T-3′ motif) of the fragments that can be implemented wherever structural or functional comparisons suggest a fragment boundary. The greatly reduced sequence constraints of this method facilitate directional assembly of gene fragments for applications such as exon or domain shuffling, loop grafting, reassembly of natural modular biosynthetic assembly lines, and rearrangement of structurally (but not sequence) homologous proteins.

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Acknowledgments

This research was supported by the Medical Research Council. B. V. was supported by a fellowship from the EU Marie Curie Early-Stage Training Site ChemBioCam. F. H. is an ERC Starting Investigator. We thank David Ackerley for his thorough editing of this article.

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Authors and Affiliations

  1. Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge, CB2 1GA, UK

    Benoit Villiers & Florian Hollfelder

Authors
  1. Benoit Villiers
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  2. Florian Hollfelder
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Corresponding author

Correspondence to Florian Hollfelder .

Editor information

Editors and Affiliations

  1. Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia, Brisbane, Queensland, Australia

    Elizabeth M.J. Gillam

  2. Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada

    Janine N. Copp

  3. School of Biological Sciences, Victoria University, Wellington, New Zealand

    David Ackerley

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Villiers, B., Hollfelder, F. (2014). USER Friendly DNA Recombination (USERec): Gene Library Construction Requiring Minimal Sequence Homology. In: Gillam, E., Copp, J., Ackerley, D. (eds) Directed Evolution Library Creation. Methods in Molecular Biology, vol 1179. Springer, New York, NY. https://doi.org/10.1007/978-1-4939-1053-3_15

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  • DOI: https://doi.org/10.1007/978-1-4939-1053-3_15

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  • Publisher Name: Springer, New York, NY

  • Print ISBN: 978-1-4939-1052-6

  • Online ISBN: 978-1-4939-1053-3

  • eBook Packages: Springer Protocols

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