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. 2018 Jan 15;8:758. doi: 10.1038/s41598-018-19238-9

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Construction of the auxotrophic strain. (A) Amplification of the three fragments to be fused: URA3 upstream region, SUC2 expression cassette and URA3 downstream region, (B) Overlap extension PCR fusing URAup, SUC2 expression cassette and URA down to form the URA disruption cassette, (C) Disruption of URA3 by double crossing over (homologous recombination) using SUC2 as a selective marker.