Psbbi bla vector

A cell line that constitutively expresses mVenus (Kremers et al., 2006 (link)) harboring p.His148Gln and p.Ile152Leu (Galietta et al., 2001 (link)) was established in HEK293T cells using the Sleeping Beauty transposon system (Kowarz et al., 2015 (link)). Briefly, a cDNA encoding mVenus^{p.H148Q/p.I152L} was cloned in a pSBbi-Bla vector (60526, addgene), and introduced to HEK293T cells together with pCMV(CAT)T7-SB100 (34879, addgene) using Effectene transfection reagent (301425, Qiagen). Transfected cells were selected in a DMEM medium (11965, Thermo Fisher Scientific) supplemented with 10% FBS and 1 µg/mL blasticidin (A11139, Thermo Fisher Scientific). Cell lines that express recombinant human pendrin constructs (both wild-type and disease-associated missense pendrin variants) in a doxycycline dosage-dependent manner were established in a similar way using a pSBtet-Pur vector (60507, addgene). Transfected cells were selected in a DMEM medium supplemented with 10% FBS and 1 µg/mL puromycin (A11138, Thermo Fisher Scientific). For I⁻/Cl⁻ antiport assay, the pendrin-expressing pSBtet-Pur vectors were transfected in the HEK293T cell line that constitutively express mVenus^{p.H148Q/p.I152L} (see above).