Constraint-Aware Coding-Sequence Design for Nicotiana benthamiana: A 49,257-Record Benchmark and Codon-Reference Sensitivity Analysis

Synonymous coding DNA sequence (CDS) design for plant expression constructs must balance host codon adaptation against guanine–cytosine (GC) composition, restriction-site avoidance, and sequence-integrity constraints, yet benchmark outcomes can depend strongly on the chosen codon- usage reference. Using FactorForge v3.2.0, an open-source CDS design and sequence-review workflow, we evaluated four rule-based design presets (Balanced, High-CAI, GC-target, and Assembly-friendly) on 49,257 reference-derived Nicotiana benthamiana CDS/protein records from the Sol Genomics Network (SGN) QLD183 resource (seed 320), scoring sequence integrity, a historical 55–65% GC window, and absence of BsaI/BpiI/BsmBI sites. With the legacy packaged codon reference, Assembly-friendly passed all three criteria for 63.37% of records; with three genome-derived references its combined pass rate was 0.00% after rounding, whereas GC-target attained 22.68% in each derived condition. A separate v3.3.0 dynamic-programming (DP) execution maximizing the codon adaptation index (CAI) within the same GC window found a GC-compliant sequence for 99.83% of records (mean CAI 0.9989), but 28.21% passed all criteria because Type IIS sites are outside the DP objective. These in-silico results characterize candidate generation under a historical policy. Evaluation with the v3.3.0 default settings, against external tools, and in the laboratory remains to be done.