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. 2019 Oct 24;9:15275. doi: 10.1038/s41598-019-51730-8

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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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Candidate Anopheles coluzzii enhancers augment expression of a luciferase reporter. The six candidate enhancers from Fig. 1 were amplified from Anopheles coluzzii mosquitoes and cloned into the pGL-Gateway-DSCP plasmid carrying a basal core promoter and firefly luciferase reporter gene. The cloned candidates were tested for influence upon luciferase expression using a dual luciferase assay system to quantify luciferase activity above background, defined by the DLX negative control (horizontal dotted line). Each of the six tested candidates displayed normalized luciferase activity significantly above background (p < 0.005), thus validating the candidates as functional A. coluzzii enhancers. Each point represents an individual replicate measure of luciferase activity for the tested candidate. Bars indicate the median and 95% confidence intervals. X-axis indicates the name of the candidate enhancer according to the nearest gene (Table 1), y-axis indicates the relative luciferase activity for each measurement, expressed as firefly luciferase corrected to the renilla luciferase internal control value, and normalized for the value of the DLX negative control (see Methods).