Generation of AQP6-tGFP Fusion Constructs

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.