pcDNA3-SpmCherry_n and pcDNA3-SpmCherry_full

The pcDNA3-SpmCherry_n plasmid was constructed as follows: the 5′-split mCherry (1–396) sequence was amplified using pcDNA3-EF1 α-mCherry as a template. The mouse β-actin intron III sequence was amplified by PCR using genomic DNA from C57/B6J mouse. The mCherry (367–426)-P2A peptide-coding sequence (5′-GGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCT-3′) was fused to the 5′-end of the puromycin resistance gene by PCR amplification. The C402T silent mutation for introducing the CRISPR-Cas12a PAM was added in the PCR primer. All fragments were fused and inserted into HindIII–EcoRI sites of pcDNA3 using NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs), in accordance with the manufacturer’s protocol. For construction of the pcDNA3-SpmCherry_full plasmid, the 3′-split mCherry (427–708) sequence was amplified using pcDNA3-EF1α-mCherry as a template, and inserted into the HindIII site of pcDNA3-SpmCherry_n using NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs), in accordance with the manufacturer’s protocol.