Pcdna5 frt plasmid

The sequence of turboGFP variant (Evrogen, Moscow, Russia) was fused to the C-terminal of human AQP3 (UniProtKB/Swiss-Prot #Q92482.2) in silico using Lasergene II software (DNASTAR, Madison, WI). The full cDNA containing AQP3-GFP was then synthesized by Genscript (Piscatawas, NJ). The cDNA was subcloned into a pcDNA5/FRT plasmid (Invitrogen, Carlsbad, CA) using restriction enzymes Hind3/EcoRV. Each murine TMC1 (Pubmed #NP_083229.1) construct was also based on custom synthesis by Genscript with its known ER retention motifs KK or RRR substituted for alanines, and codon optimized for mammalian expression in HEK293 cells. Each Tmc1 based sequence was then subcloned into the AQP3-GFP-pcDNA5/FRT plasmid using Hpa1/EcoRV restriction sites. The palmitoylated GFP was custom synthesized, codon optimized for mammalian HEK293 expression and subcloned into pcDNA5/FRT plasmid using Nhe1/EcoRV restriction sites by Genscript. All constructs were confirmed by sequencing.
To express TMC1-YFP construct, the C-terminus of coding region of mouse Tmc1 was fused to the coding sequence of mVenus reporter using Hind III and BamHI sites in an mVenus-N1 vector^{36 (link)}. Sequencing of this construct confirmed the identity as murine Tmc1 (Pumbed NM_028953).

The AQP3-tGFP-pcDNA5/FRT plasmid was synthesized as previously described^{17 (link)}. The full cDNA containing human AQP6-tGFP (NP_001643) was synthesized by Biomatik (Wilmington, DE) and tGFP-AQP6 (rat NP_071517.1) by Genscript (Piscatawas, NJ). In the tGFP-AQP6 construct, the tGFP was separated from AQP6 by the following peptide linker which included a Precision Protease site (underlined in blue): AASAVNGSLEVLFQGPAA, and containing Afe1 and Hpa1 restriction sites, as well as a 10× C-terminal Histamine Tag (Histag) as shown in Fig. 1A (in black). For the AQP6-tGFP construct, the tGFP was separated from AQP6 by the following peptide linker (Fig. 1B in blue) which included a Precision Protease site: GGSLEVLFQGPAA and a c-terminal 10× Histag (in black) as shown in Fig. 1B.
The AQP6-tGFP construct was subcloned into a pcDNA5/FRT plasmid (Invitrogen, Carlsbad, CA) using restriction enzymes BamH1/EcoRV while the AQP6 was subcloned using Hpa1/EcoRI into a pcDNA5/FRT plasmid containing tGFP. Each construct was custom synthesized by Genscript, codon optimized for mammalian expression in HEK293 cells. Each AQP6-based sequence was then subcloned into the AQP3-tGFP-pcDNA5/FRT plasmid using Hpa1/EcoRV restriction sites. All constructs were confirmed by sequencing.

For generation of the integration plasmid pDF_FRT, eGFP and mCherry expressed from a shared CMV enhancer/promoter element (pBI-CMV1; Clontech), were inserted into the NotI and NheI sites of the pcDNA5/FRT plasmid (Invitrogen). pDF_FRT_UCP3_wt and _MUTI/II were generated by hybridization of complementary oligonucleotides and insertion downstream of eGFP into the NotI and HindIII restriction sites of pDF_FRT.
The complete vector sequences are available upon request.

DNAs encoding full-length human α1 and α2 were amplified with primers designed to encode a 5′ KpnI site, and a 3′ FLAG-tag followed by an XhoI site. The resulting PCR products were cloned into the pcDNA5/FRT plasmid (Invitrogen). Stable cell lines were generated and cultured as described previously [19 (link)].