GEOapplication/xmlftp://ftp.ncbi.nlm.nih.gov/geo/series/GSE318nnn/GSE318900/primaryOK200TranscriptomicsEscherichia coli DH5[alpha] OtherExpression profiling by high throughput sequencinghttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE318900GEOGSEfalseDecoding and Rewiring Promoter Architecture Using Large Language Models and Diffusion Frameworks: High-Throughput Promoter Strength Sequencing Source DataHigh-performance promoters are essential tools for precisely regulating gene expres-sion, yet their rational design within the vast combinatorial sequence space remains a major challenge. Here, we present a hybrid framework that integrates a large lan-guage model (LLM) with a diffusion model to enable data-driven and interpretable promoter design. The fine-tuned LLM predicts promoter strength with high accuracy and, through pseudo-sequence mutations, identifies biologically essential core motifs. A diffusion model is then conditioned on these motifs to reconstruct non-core regions and generate complete promoter sequences. We experimentally validated this approach in E. coli by high-throughput barcoded promoter activity sequencing: over 90% of the generated promoters showed measurable activity, and the best variants achieved ap-proximately ∼20-fold higher expression than the benchmark promoter (BBa_J23119). By explicitly coupling interpretability with generative design, this strategy provides a generalizable path to accelerate synthetic biology efforts and advance large-scale regu-latory sequence engineering.2026/02/16GSE318900GSM9505671GSM9505672GSM9505675GSM9505676GSM9505673GSM950567436593318900Escherichia coli DH5[alpha]