Highly parallelized construction of DNA from low-cost oligonucleotide mixtures using Data-optimized Assembly Design and Golden Gate

Commercially synthesized genes are typically made using variations of homology-based cloning techniques, including polymerase cycling assembly from chemically synthesized microarray-derived oligonucleotides. Here we apply Data-optimized Assembly Design to the synthesis of hundreds of codon-optimized genes in both constitutive and inducible vectors using Golden Gate Assembly. Starting from oligonucleotide pools, we synthesize genes in three simple steps: 1) Amplification of parts belonging to individual assemblies in parallel from a single pool; 2) Golden Gate Assembly of parts for each construct; and 3) Transformation. We construct genes from receiving DNA to sequence confirmed isolates in as little as 4 days. By leveraging the ligation fidelity afforded by T4 DNA ligase, we expect to be able to construct a larger breadth of sequences not currently supported by homology-based methods which require stability of extensive single-stranded DNA overhangs. ### Competing Interest Statement The authors declare the following competing financial interest(s): When performing this research and drafting this manuscript, all authors were employees of New England Biolabs, a manufacturer and vendor of molecular biology reagents including DNA ligases and Type IIS restriction enzymes. New England Biolabs funded the work and paid the salaries of all authors.