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. 2021 Sep 9;11:17928. doi: 10.1038/s41598-021-96986-1

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© The Author(s) 2021

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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Strategy to generate a chimeric P. berghei parasite line expressing a PvCSP-P. vivax-like protein as additional CSP. An additional copy line in which the PvCSP-P. vivax-like CDS (PVU09738) gene was introduced into the genome as an additional copy of the gene in the neutral 230p locus. The construct that contains the ‘PvCSP-P. vivax-like gene expression cassette’ was integrated into the 230p locus on chromosome 3 of the P. berghei ANKA GIMO mother line by GIMO transfection using negative selection (5-FC), resulting in the expression of the PvCSP-P. vivax-like gene under the control of the Pbuis4 gene promoter and transcriptional terminator sequences. This construct also expresses a GFP and firefly luciferase (LUC-IAV) fusion protein under control of the constitutive Pbeef1a promoter and is selectable marker (SM)-free. The construct is integrated into the neutral p230p locus by double crossover integration. Black arrows: location of PCR primers used for the diagnostic PCR analysis.