N-terminal fusion length: The key to reliable and context-preserving regulatory sequence characterization

Regulatory sequences are commonly characterized using fluorescent reporters, yet how N-terminal coding context shapes these measurements has not been systematically quantified. Here, we evaluated the impact of N-terminal fusion length (45-180 bp) from four genes (lacZ, icd, zwf, bfp) on GFP reporter expression driven by 15 different promoter-RBS combinations in E. coli with normalized fluorescence, enzymatic activity assays and transcription analysis for a representative subset of constructs. Our results demonstrate that N-terminal fusion critically determines the reliability of regulatory-sequence characterization in target-gene-specific coding contexts, with strong gene- and length-dependent effects. Fusions as short as 45 bp failed to rescue context-sensitive cases. However, among the tested fusion lengths, fusions of 90 bp or longer achieved strong correlations (mean R² > 0.75) between reporter fluorescence and target protein activity. Among the factors examined, N-terminal mRNA secondary structure showed a closer association with these fusion-length-dependent effects than transcription or translation initiation changes. This practical, context-preserving fusion strategy provides cost-effective guidance for scalable and accurate regulatory sequence profiling.