000160937 001__ 160937 000160937 005__ 20251017144632.0 000160937 0247_ $$2doi$$a10.1093/nar/gkaf386 000160937 0248_ $$2sideral$$a144066 000160937 037__ $$aART-2025-144066 000160937 041__ $$aeng 000160937 100__ $$aLeonte, Radu Cristian 000160937 245__ $$aIVA Prime: automated primer design for <i>in vivo</i> assembly cloning 000160937 260__ $$c2025 000160937 5060_ $$aAccess copy available to the general public$$fUnrestricted 000160937 5203_ $$aMolecular 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. 000160937 536__ $$9info:eu-repo/grantAgreement/ES/MCINN/AEI/RYC2018-025720-I$$9info:eu-repo/grantAgreement/ES/MICINN-AEI/PID2019-106284GA-I00$$9info:eu-repo/grantAgreement/ES/MICINN-FEDER/PID2020-113359GA-I00$$9info:eu-repo/grantAgreement/ES/MICINN/RYC2018-025731-I 000160937 540__ $$9info:eu-repo/semantics/openAccess$$aby-nc$$uhttps://creativecommons.org/licenses/by-nc/4.0/deed.es 000160937 655_4 $$ainfo:eu-repo/semantics/article$$vinfo:eu-repo/semantics/publishedVersion 000160937 700__ $$aFischer, Patrick David 000160937 700__ $$0(orcid)0000-0003-2044-4795$$aHerguedas, Beatriz$$uUniversidad de Zaragoza 000160937 700__ $$0(orcid)0000-0002-4254-3148$$aGarcía-Nafría, Javier$$uUniversidad de Zaragoza 000160937 700__ $$aHiller, Sebastian 000160937 7102_ $$11002$$2060$$aUniversidad de Zaragoza$$bDpto. Bioq.Biolog.Mol. Celular$$cÁrea Bioquímica y Biolog.Mole. 000160937 773__ $$g(2025), [8 pp.]$$pNucleic acids res.$$tNucleic Acids Research$$x0305-1048 000160937 8564_ $$s1516194$$uhttps://zaguan.unizar.es/record/160937/files/texto_completo.pdf$$yVersión publicada 000160937 8564_ $$s2314435$$uhttps://zaguan.unizar.es/record/160937/files/texto_completo.jpg?subformat=icon$$xicon$$yVersión publicada 000160937 909CO $$ooai:zaguan.unizar.es:160937$$particulos$$pdriver 000160937 951__ $$a2025-10-17-14:26:47 000160937 980__ $$aARTICLE