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Background Multiepitope mRNA vaccines present a scalable strategy for cancer immunotherapy by targeting multiple tumor antigens. However, key challenges persist: identifying immunodominant, broadly presented epitopes across diverse HLA alleles, strategically positioning these epitopes within synthetic mRNAs, and ensuring stable single-frame translation to enhance MHC-I presentation for CD8+ T cell activation. Here, we investigate epitope positioning within a synthetic mRNA affects antigen processing and immunogenicity.Methods To evaluate the impact of epitope positioning, we designed a model mRNA construct (FluM1-EGFP Multitag, FEM) encoding EGFP fused to the influenza M1 epitope (GILGFVFTL) in three configurations: N-terminal (FEM-N), C-terminal (FEM-C), and both termini (FEM-NC). The dual-terminal construct incorporated quadrivalent M1 epitopes to increase epitope density. Transfection efficiency was measured in HEK293T cells. Protein expression was confirmed by immunoblotting, and LC-MS/MS was used to validate peptide coverage. Proteasomal dependency was assessed using MG132. Antigen presentation and immunogenicity were assessed using HLA-A02:01+ dendritic cells derived from human PBMCs and THP-1 cells. CD8+ T cell responses were evaluated using HLA-A02:01 tetramer staining and IFN-γ ELISPOT.Results All mRNA constructs—including FEM-N, FEM-C, and FEM-NC—achieved >94% transfection efficiency in HEK293T cells comparable to EGFP-only mRNA controls, indicating that epitope insertion did not impair expression. Immunoblotting confirmed M1 protein expression only in the C-terminal and dual-terminal designs while LC-MS/MS detected M1 from all three constructs, confirming expression despite lack of immunoblot signal in FEM-N. CD8+ T cell activation varied significantly by epitope position: C-terminal M1 placement led to 2.8% tetramer+ CD8+ cells, versus 1.18% for N-terminal (2.4-fold increase); dual-terminal placement induced 4.32 (3.7-fold vs. N-terminal), comparable to the peptide controls which resulted in the highest activation at 4.91%. IFN-γ ELISPOT corroborated these findings, for dual-terminal FEM (4.54-fold) increases in spot-forming units relative to EGFP-only mRNA controls. Proteasome inhibition abolished CD8+ activation for all three positional constructs.Conclusions Epitope positioning within this multiepitope mRNA construct significantly alters antigen expression and immunogenicity. C-terminal or dual-terminal replicate M1 epitope expression improve CD8+ T cell responses. The impact of intervening sequences and intra-allelic epitope competition will be assessed. These findings provide mechanistic insights to guide rational design of mRNA-based immunotherapies.Ethics Approval This study obtained under institutional oversight at Arizona State University. All procedures complied with institutional guidelines, and informed consent was obtained from all donors through an IRB-approved protocol or exemption. The study was reviewed by the Arizona State University Institutional Review Board. Full protocol details are available upon request.