IVA Prime: Automatic Primer Design for In Vivo DNA Assembly

In vivo DNA assembly performs seamless DNA assembly in a simple and cost-effective manner, streamlining all molecular cloning protocols. It relies on the fusion of linear DNA fragments with flanking homologous sequences inside bacteria, following a recA-independent recombination pathway present in all laboratory strains. While protocols are simple, in vivo assembly (IVA) cloning requires precise primer design that can be a hurdle for inexperienced researchers. This hurdle is removed by IVA Prime, an intuitive online and desktop application for automatic design of IVA primers in a ready-to-order format, streamlining all stages of molecular cloning in plasmid DNA. In this chapter, we provide detailed instructions on how to use IVA Prime for automatic primer design, including all types of plasmid modifications such as insertions, deletions, site-directed mutagenesis, and subcloning. We also describe additional tools available in IVA Prime that can aid in construct design, such as automatic purification tag insertion or codon optimization, while highlighting potential pitfalls that must be considered in experimental design.