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. 2019 Jul 12;9:10131. doi: 10.1038/s41598-019-46171-2

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© The Author(s) 2019

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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Creating LII and LIII plasmids with the MK cloning system. (A) LI plasmids containing DNA parts (A-B, B-C, C-D, D-E, E-F, F-G positions) are mixed with a LII backbone in a BsaI cut-ligation to generate a LII plasmid. LII backbones contain a spectinomycin resistance cassette and a ccdB negative selection cassette. (B) An assembled LIII plasmid is created by mixing LII plasmids (1-2, 2-3, 3-4, 4-5, 5-6 positions) with a LIII backbone in a BpiI cut-ligation. LIII backbones contain kanamycin resistance and ccdB negative selection cassettes.