Yes, and it's pretty common! Did this as a postdoc

Yes, it is possible and sometimes advantageous to create qPCR standard curve samples by inserting multiple target sequences into a single plasmid or DNA template. This approach is often used to simplify standard preparation in multiplex qPCR assays, where multiple genes are quantified simultaneously. By placing all target sequences in a single construct, you ensure uniform copy number across all targets, improving consistency and reducing variability in standard curves. This method also minimizes the need to prepare and quantify separate standards for each gene. However, careful design is crucial to avoid primer-dimer formation, non-specific amplification, or interference between targets. Each target region should be well separated within the construct, and the plasmid is typically linearized before use to ensure consistent amplification efficiency. This strategy is especially useful in diagnostic assays or gene expression studies where multiple genes must be accurately quantified using the same standard template.