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. 2023 Apr 25;13:6745. doi: 10.1038/s41598-023-33448-w

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© The Author(s) 2023

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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Multiplexed fluorescence microscopy of nine validation sequences in coronal sections of the auditory cortex. (A) Positive control with all fluorophore variants (mTFP, EGFP, Venus, and tagRFP) driven by CMV promoter. Six neocortical layers indicated on DAPI image. (B) Zoomed in view of example region. (C) Cumulative distribution across cells of the relative fluorescence intensity of EGFP, Venus, and tagRFP compared to mTFP (relative expression strength) for the CMV-driven control constructs (mean across images ± SD). (D–L) Analogous to (A–C), but for the nine validation sequences examined, divided into three sets of three sequences (D–F, G–I, J–L). Each set also contains a CMV promoter-driven mTFP reporter, to which all the others are compared (F, I, L). VS33 drives high expression levels (G, H, I).