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. 2024 Apr 30;43(12):2506–2525. doi: 10.1038/s44318-024-00102-8

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© The Author(s) 2024

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/. Creative Commons Public Domain Dedication waiver http://creativecommons.org/publicdomain/zero/1.0/ applies to the data associated with this article, unless otherwise stated in a credit line to the data, but does not extend to the graphical or creative elements of illustrations, charts, or figures. This waiver removes legal barriers to the re-use and mining of research data. According to standard scholarly practice, it is recommended to provide appropriate citation and attribution whenever technically possible.

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Figure EV2 — The HENMT1^∆C-T6B cDNA was cloned in the CAG-STOP-eGFP-Rosa26 TV plasmid by replacing the IRES sequence with the HENMT1^∆C-T6B cDNA fused in frame via a P2A peptide to the eGFP coding sequence to generate the CAG-STOP-HENMT1^∆C-T6B-eGFP-Rosa26 plasmid. A 3,709-bp PCR fragment containing a 1120-bp 5´ homology arm (HA) and a 1356-bp 3´ HA was used as double-stranded DNA repair template together with two sgRNAs to replace the miR17-92-IRES DNA sequences of the R26^LSL-miR17-92 allele²⁶ by CRISPR/Cas9-mediated genome editing in mouse 2-cell embryos, resulting in the R26^LSL-HenT6B allele. Cre-mediated deletion of the loxP-Stop-loxP (LSL) cassette leads to the expression of the HENMT1^∆C-T6B-P2A-eGFP gene from the ubiquitously transcribed CAG promoter of the R26^HenT6B allele. LoxP and frt sites are indicated by red and yellow arrowheads, respectively. The herpes simplex virus thymidine kinase (TK) promoter drives expression of the neomycin (Neo) resistance gene. Arrows indicate primers 1 and 2 used for genotyping of the R26^LSL-HenT6B allele and primers 3 and 4 used for genotyping of the wild-type R26 allele (see Methods). pA, polyadenylation sequence.