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Target Molecule

A. Target Molecule. Intron 51. Intron 52. 5‘SS. 3‘SS. 5‘SS. 3‘SS. 5‘ acGFP. Exon 52. 3‘ acGFP. 1nt. 183nt. 490nt. 720nt. 3‘ BD. 5‘ BD. RTM. Spacer. Spacer. 5‘SS. 3‘SS. int. acGFP. BP. pPT. 3‘ trans -splicing. 5‘ trans -splicing. 489nt. 184nt. int. acGFP. 5‘ acGFP.

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Target Molecule

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  1. A Target Molecule Intron 51 Intron 52 5‘SS 3‘SS 5‘SS 3‘SS 5‘ acGFP Exon 52 3‘ acGFP 1nt 183nt 490nt 720nt 3‘ BD 5‘ BD RTM Spacer Spacer 5‘SS 3‘SS int. acGFP BP pPT 3‘ trans-splicing 5‘ trans-splicing 489nt 184nt int. acGFP 5‘ acGFP 3‘ acGFP 1nt 720nt acGFP fusion mRNA B

  2. A target molecule & RTM version 1 A target molecule & RTM version 1 Intron 51 Intron 51 Intron 52 Intron 52 5‘SS 5‘SS 3‘SS 3‘SS 5‘SS 5‘SS 3‘SS 3‘SS 5‘ acGFP 5‘ acGFP Exon 52 Exon 52 3‘ acGFP 3‘ acGFP 1nt 1nt 183nt 183nt 490nt 490nt 720nt 720nt variable 3‘ BD variable 3‘ BD 5‘SS 5‘SS 3‘SS 3‘SS int. acGFP int. acGFP constant 5‘ BD constant 5‘ BD BP BP pPT pPT 489nt 489nt 184nt 184nt int. acGFP int. acGFP 5‘ acGFP 5‘ acGFP 3‘ acGFP 3‘ acGFP acGFP fusion mRNA acGFP fusion mRNA 1nt 720nt 1nt 720nt B target molecule & RTM version 2 B target molecule & RTM version 2 Intron 51 Intron 51 Intron 52 Intron 52 5‘SS 5‘SS 3‘SS 3‘SS 5‘SS 5‘SS 3‘SS 3‘SS 5‘ acGFP 5‘ acGFP Exon 52 Exon 52 3‘ acGFP 3‘ acGFP 1nt 1nt 183nt 183nt 490nt 490nt 720nt 720nt variable 5‘ BD variable 5‘ BD 5‘SS 5‘SS 3‘SS 3‘SS int. acGFP int. acGFP constant 3‘ BD constant 3‘ BD BP BP pPT pPT 489nt 489nt 184nt 184nt int. acGFP int. acGFP 5‘ acGFP 5‘ acGFP 3‘ acGFP 3‘ acGFP 1nt 720nt 1nt 720nt acGFP fusion mRNA acGFP fusion mRNA

  3. Exon 52 Intron 51 Intron 52 a RTM 0 a b RTM 1 c a RTM 2 d a e RTM 3 a f g RTM 4

  4. target RTM2 RTM4 RTM2 RTM4 H2O 1 target target 2 3 4 5 6 7 700bp 5‘ acGFP – Exon 52 – 3‘ acGFP (669bp)  acGFP fusion (585bp)  5‘ acGFP – 3‘ acGFP (279bp)  600bp 500bp 400bp 300bp 300bp 300bp 300bp acGFP 5 5 ‘ ‘ acGFP acGFP int. int. int. int. acGFP 3 3 ‘ ‘ acGFP acGFP // // // //

  5. A COL17A1 mini-gene Intron 51 Intron 52 3‘SS 5‘SS 3‘SS 5‘SS Exon 53 Exon 51 Exon 52 5‘BD 3‘BD RTM Spacer Spacer 3‘SS 5‘SS int. acGFP BP pPT 3‘ trans-splicing 5‘ trans-splicing int. acGFP Exon 51 Exon 53 COL17A1- acGFP fusion mRNA B mini-gene RTM4 RTM2 RTM4 RTM1 RTM3 mini-gene mini-genemini-genemini-gene • Exon 51-Exon 52-Exon 53 • Exon 51-int. acGFP-Exon 53 400bp 300bp 1 2 3 4 5 6 7

  6. A B C D E F G H

  7. sorted acGFP RTM2 RTM3 RTM1RTM2 RTM4 RTM0 RTM2 RTM4 target target target target target target target control 37kDa Annexin I (37kDa) acGFP ( 27kDa) 25kDa 1 2 3 4 5 6 7 8 9 10 11

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