Pureyield plasmid mini kit

The pGL3-enhancer vector was purchased from Promega (Madison, WI) and a canonical CMV promoter was inserted in the multiple cloning site. The entire expression cassette, including promoter, luciferase open reading frame, polyA, and enhancer were subcloned into the pAAV-MCS (Stratagene, La Jolla, CA) vector, resulting in pCMV-GL3enh. The expression cassette was then excised from pCMV-GL3enh and ligated into the pMC.BESPX-MCS1 vector (System Biosciences, Mountain View, CA; #MN100A-1), resulting in pMC-CMV-GL3enh (both vectors shown in Figure 1a). Full sequence information for both of these vectors is provided in the Supplementary Material. Minicircle construction was performed in the ZYCY10P3S2T bacterial strain per manufacturer’s instructions. Both vectors were prepared for experiments by purification using CsCl gradient centrifugation. Absorbance ratios were 1.89 (260/280) and 1.86 (260/230) for the plasmid and 1.98 (260/280) and 1.47 (260/230) for the minicircle. Endotoxin levels were measured in the final preps using a ToxinSensor kit (GenScript, Piscataway, NJ) according to manufacturer’s instructions. Endotoxin levels in the plasmid and minicircle were 0.082 and 0.041 EU/ml, respectively and did not differ substantially from a plasmid purified using a PureYield Plasmid Mini kit (0.097 EU/ml) including the endotoxin removal step (Promega, Madison, WI).