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. 2015 Jun 19;11(8):1972–1982. doi: 10.1080/21645515.2015.1022008

Figure 1.

Figure 1.

Construction and representative expression of dbDNA™ PR8 and pDNA PR8 constructs. (A) Process of enzymatic production of dbDNA™. Rolling circle amplification of the double-stranded DNA template results in concatamers that are cleaved and joined by the protelomerase TelN to yield the covalently closed, double-stranded cassette. (B) Schematic of the linear double-stranded dbDNA™ PR8 construct with end terminal single-stranded DNA hairpins. The end product was treated with restriction enzymes and exonuclease to remove plasmid backbone sequences. (C) Schematic of pDNA PR8 construct, PR8.HA.ECRO. ECRO refers to the gene sequence containing an IgE leader sequence [e] and the target sequence is codon [c] and RNA [r] optimized [o]. The sequence of PR8 was cloned into the pVax1 mammalian expression vector. The CMV promoter, HA gene, BGH poly A signal, kanamycin resistance gene, and pUC origin are shown. (D) Representative in vitro expression of the dbDNA™ PR8 and pDNA PR8 constructs. Expression was confirmed using transfected RD cells and a HA-tagged antibody. An empty vector (pVax) was used as a negative control. Results were analyzed with confocal imaging. Expression is indicated by fluorescein isothiocyanate (FITC) staining (green).