Cloning TMEM16 Constructs with His6-N2B Tag

cDNAs were cloned into the expression vectors peGFP-N1, peGFP-C1, and pCMV-Sport6 (Supplementary Table 2) according to our research goals. The Mus musculus TMEM16F (Ano6, NP_780553.2) and TMEM16B (Ano2, NP_705817.1) consists of 911 a.a. and 913 a.a., respectively. The DNA plasmids were constructed as described previously^{47 (link)}. A His6-N2B tag (210 a.a.) that is composed of six histidines (His6) and an N2B module of giant muscle protein titin is added at the N- or C-terminus of the cDNA according to our research purpose. These TMEM16 constructs with His6-N2B tag were made and their sequences were confirmed by Genewiz Company in Suzhou, China. The His6-N2B tag doesn’t display any unfolding events under AFM stretching as described before^{47 (link),58 (link)}.

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Ye Z., Galvanetto N., Puppulin L., Pifferi S., Flechsig H., Arndt M., Triviño C.A., Di Palma M., Guo S., Vogel H., Menini A., Franz C.M., Torre V, & Marchesi A. (2024). Structural heterogeneity of the ion and lipid channel TMEM16F. Nature Communications, 15, 110.

Publication 2024

Corresponding Organization :

Other organizations : Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Scuola Internazionale Superiore di Studi Avanzati, University of Zurich, Life Science Institute, Kanazawa University, Ca' Foscari University of Venice, Marche Polytechnic University, École Polytechnique Fédérale de Lausanne, AREA Science Park

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Variable analysis

independent variables

Expression vectors used: peGFP-N1, peGFP-C1, and pCMV-Sport6
Addition of His6-N2B tag at the N- or C-terminus of the cDNA

dependent variables

Not explicitly mentioned

control variables

The His6-N2B tag doesn't display any unfolding events under AFM stretching

controls

Positive control: None mentioned
Negative control: None mentioned

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