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Universal Endonuclease – Cutting Edge Technology Gene technology is driven by the use of restriction endonucleases. Yet, constraints of limited sequence length and variation recognized by available restriction enzymes pose a major roadblock for synthetic biology. We developed the basis for universal restriction enzymes, primarily for routine cloning but also with potential for in vivo applications. We use a nucleotide cleavage domain fused to a binding domain, which recognizes a programmable adapter that mediates binding to DNA and thus cleavage. As adapter we use readily available modified oligonucleotides, as binding domain anticalins and as cleavage domain FokI moieties engineered for heterodimerization and activity. For cloning, this universal enzyme has merely to be mixed with the sequence specific oligonucleotide and the target DNA. Binding and release are addressed with thermocycling.