Pmgfp p2a k0 p2a rfp

To generate the Kcnj10 CDS containing a fluorescent reporter, a control cassette was first created by replacing the BspEI/KpnI segment of the pmGFP-P2A-K0-P2A-RFP (Addgene plasmids 105686) with a linker containing a P2A site, a Flag coding sequence, and the EcoRI and NotI restriction sites. A gene block (Integrated DNA Technologies) encoding mKir4.1 (AAI41089.1) without a stop codon was then inserted at the EcoRI/NotI sites of this control cassette in frame with both the GFP and mCherry coding sequences. The psiCHECK-2 vector (Promega, C8021) was used to build the dual luciferase reporters with Kcnj10 UTRs. The UTR sequences of mouse Kcnj10 mRNA (NM_001039484.1 and AB039879.1) were synthesized as gBlocks and inserted at the NheI site of the psiCHECK-2 vector. The 3′UTR of the Kcnj10 mRNA (AB039879.1) was inserted as a gBlock into the XhoI and NotI restriction sites in the psiCHECK-2 vector downstream of the Renilla luciferase reporter gene. The truncated versions of the Kcnj10 5′ UTR (1–146; 127–242; 95–242; 95–191; 1–191; 181–242) were inserted as NheI/NheI PCR fragments into the psiCHECK-2 vector at the 5′ end of the Renilla luciferase gene. The sequences of the primers and gBlocks used for subcloning are listed in Table S4.