We amplified 5 ng of the HSS library with the following primers: STARR-Seq-AG-f and STARR-Seq-AG-r (Supplemental Table 8) using KAPA HiFi 2x Readymix (Kapa Biosystems) with a 65 °C annealing temperature and 30 s extension. These primers amplify both candidate enhancers and previously assigned degenerate barcodes and add homology arms to the ORI vector (Addgene 99296)22 (link). We followed the reaction in real time with Sybr Green (Thermo fisher scientific) and stopped the reaction before plateauing, at 13 cycles. We gel extracted the amplicon on a 1.5% agarose gel as described above. We then cloned the library in a 2:1 molar excess into 100 ng of the hSTARR-seq_ORI vector (addgene ID:99296), linearized with AgeI and SalI, using the NEBuilder HiFi DNA Assembly Cloning Kit (NEB), following the manufacturer’s protocol. We then transformed 10-beta electrocompetent cells (NEB C3020) with the plasmids in duplicate following the manufacturer’s protocol, along with a no insert negative control. We pooled the two positive transformations during recovery, plated 15 μL to estimate complexity and grew the remainder of the culture in 100 mL LB+Amp. The following day, we estimated the complexity as >500K and extracted the plasmid using the ZymoPURE II Plasmid Midiprep Kit (Zymo Research).