Molecular cloning through in vivo assembly (IVA) is an efficient homology-based approach that can achieve complex cloning operations in a single step, bypassing the need for enzymes and reducing hands-on time and costs. However, primer design remains the most demanding task, requiring substantial expertise and a source of human error. Here, we introduce "IVA Prime," an intuitive online tool for automated primer design for IVA cloning, which reduces the workload and avoids errors. We describe how IVA Prime generates optimized primers for all cloning operations, i.e. mutagenesis, insertions, deletions, and subcloning. IVA Prime provides an intuitive interface and robust algorithms, thus mitigating the complexities associated with primer design into a single mouse click. A set of thoroughly tested default parameters allows both inexperienced users and experts to generate ready-to-order primer sequences for a standardized cloning protocol with high success rate. We describe the method and demonstrate its efficiency and reliability with example applications. IVA Prime is freely available at https://www.ivaprime.com.
© The Author(s) 2025. Published by Oxford University Press on behalf of Nucleic Acids Research.