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. 2022 Dec 9;54(12):2188–2199. doi: 10.1038/s12276-022-00891-0

Fig. 1. SCON is a novel cKO approach that shows desired intronic functionality in vitro.

Fig. 1

a Sequence of SCON before and after recombination. The intact SCON is 189 bp long, and the sequence is annotated as follows: nucleotides highlighted in green and dark orange at the two ends represent the splice donor and splice acceptor, respectively; nucleotides highlighted in dark yellow represent the putative branch points; sequences that are underlined represent the polypyrimidine tract; nucleotides in blue and red represent loxP sites consisting of 13 bp recognition sites and 8 bp spacers, respectively. In the recombined SCON form, which is 55 bp long, the remaining components include the splice donor, one loxP, polypyrimidine tract, and the splice acceptor; the three coding frames contain stop codons that are indicated by a box and an asterisk over the nucleotides. b Schematic diagram of the SCON functionality test in an eGFP overexpression construct including intact eGFP, eG-SCON-FP, and recombinant eG-SC-FP. SD, splice donor; BP, branch point; SA, splice acceptor. c Images of transfected HEK293T cells on Day 1 with intact eGFP, eG-SCON-FP and recombinant eG-SC-FP. All constructs were cotransfected with an mCherry overexpression plasmid. Scale bar, 1 mm. d, e Histograms of the flow cytometry analysis of transfected HEK293T cells showing comparisons between eGFP (red) and eG-SCON-FP (blue) (d), between eGFP (red) and eG-DECAI-FP (blue) (e), and the respective recombined forms eG-SC-FP and eG-DE-FP (yellow) (d, e). f Flow cytometry analysis of mouse ES cells with integrated piggyBac-eG-SCON-FP transfected with Cre-expressing plasmid (yellow) or empty vector (blue).