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. 2020 Oct 15;10:17479. doi: 10.1038/s41598-020-74474-2

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© The Author(s) 2020

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Flp-TAL variants target FRT-like sequences FL-61, FL-63, and FL-71 in their native chromosomal environment. (A) Schematics of the integration assays. The reporter pTarget can be integrated either into FL-61, FL-63, or FL-71 depending on the specificity of the Flp-TAL recombinase and the version of pTarget: FL-61/FL-71 or FL-63/FL-71. Upon integration of pTarget into FL-61 or FL-63 the resultant cells become hygromycin resistant and red, while upon integration into FL-71, the cells become hygromycin resistant and green (images of the individual expanded hygro^R/red and hygro^R/green colonies are shown as examples). The analysis of the individual colonies was performed in two biological replicates. LJ and RJ mark the locations of the left and right junctions of the integrated reporter and genomic DNA, respectively; diagnostic PCR at these locations was used to determine the authenticity of the reporter integration. (B) PCR analysis of the pooled hygro^R colonies generated in the experiments with the Flp-TAL recombinase, the ‘plain’ recombinase variant, and the ‘empty’ expression vector. LJ, RJ, the PCR analysis of the left and right junctions of pTarget integrated into the respective genomic sequences; M, DNA ladder (NEB, 2-log). (C) PCR analysis of the pooled hygro^R colonies generated in the integration experiments with three combinations of the FV71-TAL recombinases: FV71-TAL(L24) + FV71-TAL(R15), FV71-TAL(L18) + FV71-TAL(R15) and FV71-TAL(L15) + FV71-TAL(R15). The diagnostic PCR analysis was performed at the right junction.