Plasmids

WT CFTR and coCFTR1 (GenBank: MW222115) in the pcDNA3.1(+) vector were kind gifts from Drs. Jae Seok Yoon and William R. Skach. Only the first 356 amino acids of coCFTR1 were codon optimized. coCFTR1 contains a valine-for-methionine substitution at position 1475, which does not affect protein function.73 Codon optimization and synthesis of coCFTR2 (GenBank: MW116826) were commercially performed by GenScript in the Bluescript plasmid. coCFTR2 contains the leucine-for-phenylalanine substitution at position 833 present in the originally reported CFTR sequence,1 which increases protein solubility but does not affect protein structure.74 Codon optimization of coCFTR3 (GenBank: MW116827) was performed using JCat55 and was commercially synthesized by GenScript in the HIV-PGK-coCFTR3 vector. coCFTR2 and coCFTR3 were subcloned into pcDNA3.1(+). HIV-PGK-WTCFTR and HIV-EF1α-GFP were synthesized by GenScript and used to clone HIV-PGK-GFP, HIV-PGK-coCFTR3, and HIV-EF1α-WTCFTR. The HIV-based vectors used are second generation, self-inactivating (SIN), and contain a human ankyrin 1 element in the reverse orientation within the 3′ long terminal repeat (LTR) that improves long-term expression by avoiding silencing.75^,76