10.1021/acssynbio.7b00017.s002 Bob Van Hove Bob Van Hove Chiara Guidi Chiara Guidi Lien De Wannemaeker Lien De Wannemaeker Jo Maertens Jo Maertens Marjan De Mey Marjan De Mey Recursive DNA Assembly Using Protected Oligonucleotide Duplex Assisted Cloning (PODAC) American Chemical Society 2017 sequence novel technique biology toolkit Protected Oligonucleotide Duplex Assisted Cloning PODAC DNA assembly methods CRISPR arrays iterative Golden Gate biosynthetic pathway restriction enzyme Methylated bases Costly PCR workflows design constraints oligonucleotide duplex Recursive DNA Assembly assembly reaction 2017-03-20 00:00:00 Dataset https://acs.figshare.com/articles/dataset/Recursive_DNA_Assembly_Using_Protected_Oligonucleotide_Duplex_Assisted_Cloning_PODAC_/4786096 A problem rarely tackled by current DNA assembly methods is the issue of cloning additional parts into an already assembled construct. Costly PCR workflows are often hindered by repeated sequences, and restriction based strategies impose design constraints for each enzyme used. Here we present Protected Oligonucleotide Duplex Assisted Cloning (PODAC), a novel technique that makes use of an oligonucleotide duplex for iterative Golden Gate cloning using only one restriction enzyme. Methylated bases confer protection from digestion during the assembly reaction and are removed during replication <i>in vivo</i>, unveiling a new cloning site in the process. We used this method to efficiently and accurately assemble a biosynthetic pathway and demonstrated its robustness toward sequence repeats by constructing artificial CRISPR arrays. As PODAC is readily amenable to standardization, it would make a useful addition to the synthetic biology toolkit.