The TM_dTomato probe was made by modifying the Triple mutant construct reported previously14 (link). A BamHI restriction site was added downstream of the VSD to facilitate cloning of different linker lengths. A single copy variant of tdTomato was synthesized with a 5′ BamHI site and a 3′ stop codon followed by a XhoI site (Integrated DNA Technology, USA)21 (link).
Single-step PCR was used to systematically decrease the length of the linker between the VSD and the FP. Final constructs were ligated into pcDNA3.1/Hygro (+) backbone vector (Invitrogen, USA).
Arginine scanning mutagenesis of the linker region was done by a two-step PCR process using LK7 and LK11 as template DNA. Primers were designed to introduce arginine mutations in each amino acid site of the linker. Constructs tested in the arginine scanning mutagenesis of dTomato were generated by two-step PCR using TM_dTomato as template DNA.
DNA of FlicR1 was obtained from Addgene (#74142; Addgene, USA) and cloned into pcDNA3.1/Hygro (+) backbone vector (Invitrogen, USA). All DNA constructs were verified by DNA sequencing (Cosmogenetech, Republic of Korea).
Single-step PCR was used to systematically decrease the length of the linker between the VSD and the FP. Final constructs were ligated into pcDNA3.1/Hygro (+) backbone vector (Invitrogen, USA).
Arginine scanning mutagenesis of the linker region was done by a two-step PCR process using LK7 and LK11 as template DNA. Primers were designed to introduce arginine mutations in each amino acid site of the linker. Constructs tested in the arginine scanning mutagenesis of dTomato were generated by two-step PCR using TM_dTomato as template DNA.
DNA of FlicR1 was obtained from Addgene (#74142; Addgene, USA) and cloned into pcDNA3.1/Hygro (+) backbone vector (Invitrogen, USA). All DNA constructs were verified by DNA sequencing (Cosmogenetech, Republic of Korea).