Plasmid Cloning

The scAAV2-CMV-GFP-(miR-1BS)₃ transgene plasmid was generated using the scAAV2-CMV-GFP plasmid and the rAAV9CBnLacZ-(miR-1BS)₃ plasmid that has three miR-1 binding sites (miR-1BS)₃.[24] Site-directed mutagenesis was used to insert a SpeI restriction site between the GFP stop codon and the SV40 polyadenylation signal of the scAAV2-CMV-GFP plasmid. PCR was used to amplify (miR-1BS)₃ from the rAAV9CBnLacZ-(miR-1BS)₃ plasmid while adding a NotI restriction site to the 5’ end of the amplicon and a SpeI restriction site to the 3’ end. The amplicon was ligated into the backbone followed by bacterial transformation into NEB10 competent cells. Plasmids were extracted and purified using the Zyppy Miniprep Kit (Zymo Research) then sequence verified. All Provector plasmids were generated as described previously.[20] Briefly, ‘peptide lock’ inserts were prepared for ligation insertion through the annealing and phosphorylation of synthesized oligos (see Table S1 for sequences, Integrated DNA Technologies). The Scrambled peptide sequence was generated by randomly scrambling the PLGLAR peptide cleavage sequence.