Figure 1. Comparison of the new and old minicircle systems.

(a) An earlier version of the minicircle production system. (aI) Structure of the previous minicircle producer plasmid. BAD and araC, the promoter and the repressor gene of the inducible L–arabinose-araC.BAD system; øC31, bacteriophage øC31 integrase gene; attB and attP, the bacterial and phage attachment sites of the øC31 integrase; I-SceI, I-SceI homing endonuclease gene; I-SceIs, the I-SceI recognition site; AmpR, ampicillin resistance gene; ColE1, DNA replication origin. (aII) E. coli strain Top10 Invitrogen (Carlsbad, California) original strain used to produce minicircle. (aIII) Flowchart showing the minicircle production protocol. Each box represents a major step and the darkened boxes represent the steps required in addition to a routine plasmid production protocol. (b) The new minicircle system. (bI) Diagram of new minicircle parental plasmids and its conversion to minicircle DNA. pMC.hFIX, minicrcle producer plasmid; hFIX, human factor IX; sApoE, promoter/enhancer as described previously²; KanR, kanamycin resistance gene. Upon L-arabinose induction, øc31 is expressed to mediate the formation of minicircle and plasmid backbone circle and I-SceI to induce the destruction of plasmid backbone circle. (bII) The genetic modifications of the minicircle producing bacterial strain ZYCY10P3S2T. 10P3S2T = (1) 10 copies of BAD.øC31 cassette, which were integrated in 3 loci of the bacterial genome: 2 tandem copies at the ΔendA locus (Supplementary Figure 4c), and 4 copies at the araD (Supplementary Figure 5a) and galK (Supplementary Figure 6a) each; (2) 3 tandem copies of BAD.I-SceI cassette, which were integrated at UMU locus (Supplementary Figure 3a) and (3) 2 constitutively expressing L-arabinose transporter genes: one was araE gene driven by an artificial promoter cp8, which presented in strain BW27783¹⁰; the other was the bla-lacY A177C cassette, which was integrated at the lacY locus (Supplementary Figure 2); bla, beta-galactosidase gene promoter; lacY A177C, the missense mutant of lacY gene. (bIII) Flowchart showing the new minicircle production protocol. (c) Stepwise genetic modification of the bacterial genome to make the current ZYCY10P3S2T strain.