Figure 1.

Generation of Minicircle plasmid DNA. (a) The expression cassette was excised from the first-generation plasmid vector, pCMV-GL3enh and ligated into the parental plasmid, pMC-Gl3-Enhancer. After the addition of arabinose, the parental vector is cleaved, and the progeny minicircle (C) and backbone, containing the bacterial origin of replication and antibiotic resistance, are religated. The backbone sequence contains several engineered I-SecI restriction sites that ultimately lead to the degradation of the parental DNA but not the Minicircle DNA. (b) DNA gel of the minicircle prep shows the intact parental vector (7.7 kb) in the absence (−) of arabinose, and the minicircle in the presence (+) of arabinose (3.29 kb, note the absence of the degraded backbone). Lane M indicates the reference ladder. Plasmids were cut by EcoRV to achieve linearization prior to electrophoresis.