Codon Design Online (CoDOn): a comprehensive web application of synthetic gene design with multi-criteria and multi-parameters for heterologous expression

Codon optimization is a routine yet high-impact step in de novo gene design for heterologous expression and serves as an important tool in synthetic biology. Protein expression output depends on multiple sequence-level determinants, such as global codon-usage, codon-pair context, initial mRNA folding, and motif/repeat content. However, many existing tools still provide limited transparency or restricted design flexibility. We developed Codon Design Online (CoDOn), a publicly accessible web application that formulates codon optimization as an explicit multi-objective design task and uses the non-dominated sorting genetic algorithm II (NSGA-II) to generate diverse Pareto-optimal coding sequences. CoDOn supports various design criteria that capture both host-level codon usage and local translation context, including individual codon usage (ICU), codon context (CC), codon adaptation index (CAI) and hidden stop codons. Users can also impose sequence-level constraints, e.g., GC/GC3 composition targets, 5′-proximal mRNA folding energy, and motif/repeat exclusion. Importantly, the platform features a modular interface that enables intuitive, interactive parameter configuration and result exploration through Pareto plots, codon-usage radar charts and tables, codon-pair heatmaps, and exportable reports. We showcase the utility of the platform using representative design cases in E. coli and S. cerevisiae under realistic practical constraints. CoDOn turns codon optimization from a black-box, single-output procedure into a transparent, user-configurable decision process based on Pareto trade-offs, supporting customizable gene design across hosts and applications. The CoDOn web service is available at https://codondesign.com/ .