Robinson, N. E.; Paul, J.-S.; Zhang, W.; Zhang, H.; Wang, S.; Zhao, T.; Abraham, E.; Simpson, B.; Wang, K.

Reliable and cost effective de novo DNA production has become central to studying and engineering biology. Short synthetic single-stranded DNA oligo pools offer substantially reduced costs at the sacrifice of yield and individual oligo isolation. Efficiently constructing longer synthetic double-stranded DNA molecules from oligo pools as the input has remained an engineering challenge with the potential to drastically reduce costs, labor, and experimental turn-around time. Here we show one-pot, parallel assembly of hundreds of DNA fragments simultaneously into dozens of defined constructs with high fidelity using Sidewinder. We designed a novel string-based bespoke barcode design algorithm which rapidly generates Sidewinder barcodes at unprecedented scale. We apply the new algorithm to Sidewinder using oligo pools, demonstrating construct-specific amplification from pooled assemblies with misconnection rates as low as 1 in 10,000,000. Further, we demonstrate universal amplification of pooled assemblies to generate a library of specific target sequences that we combine with in vitro hierarchical assembly to 12.5 kilobases.