PrimerWeaver: an integrated web server for primer design in molecular biology workflows

Primer design remains a fundamental yet non-trivial step in modern molecular biology workflows. Although numerous tools are available, they are often fragmented across disparate applications, constrained by commercial licensing, or dependent on external servers, limiting workflow integration and compromising sequence confidentiality. To address this challenge, we developed PrimerWeaver (https://ignea.lab.mcgill.ca/primerweaver), a free browser-based tool that integrates primer design, quality control, and support for diverse cloning workflows within a single platform. PrimerWeaver enables overlap PCR, site-directed mutagenesis, multiplex PCR, restriction enzyme-based cloning (including Type II and Golden Gate assembly), and homology-directed methods such as Gibson assembly and Uracil-Specific Excision Reagent (USER) cloning. Primers were generated by optimizing the 3' annealing region for efficient amplification while appending workflow-specific 5' sequences according to the selected application. In silico and wet lab validation across diverse cloning workflows demonstrated consistent results relative to established molecular cloning software. All calculations are performed locally in the user's browser without sequence upload, preserving data confidentiality and supporting reproducible performance across systems. Overall, PrimerWeaver streamlines primer workflows for users ranging from undergraduate students to experienced researchers by integrating multiple PCR and cloning strategies into a single browser-based platform.