Ligation kit

Six different plasmids encoding near infrared (NIR)-BRET probes were constructed for the mammalian expression (Figure 3A).
The cDNA templates encoding fluorescent proteins (FPs), i.e., LSSmCherry, LSSmKate2 (S159T), LSSmNep (i.e., mNeptune with C162D mutation), and mCherry, were custom-synthesized by Eurofins Genetics (Tokyo, Japan) according to the precedent sequence information [30 (link),31 (link)].
First, a series of cDNA segments encoding three different fluorescent proteins (FP) were generated by PCR using corresponding primers to introduce unique restriction sites, HindIII/BamHI. Similarly, cDNA segments encoding ALuc47 (19–193 AA) and NanoLuc were synthesized by PCR using corresponding primers to introduce BamHI (KpnI)/XhoI at the 5′ and 3′ ends, respectively. The cDNA segments were double digested by the corresponding restriction enzymes (NEB, Ipswich, MA, USA), ligated using a ligation kit (Takara Bio), and subcloned into a respective enzyme-digested pcDNA3.1(+) vector (Invitrogen, Carlsbad, CA, USA) as illustrated in Figure 3A. After expression, the probes were named LSSmChe_A47, LSSmKate2_A47, LSSmNep_A47, mChe_NLuc, LSSmKate2_NLuc, and LSSmNep_NLuc for highlighting the components.
We used N-terminal secretion peptide (SP: 1–18 AA)–eliminated ALuc47 (i.e., 19–193 AA) in this study for excluding spontaneous secretion of the expressed fusion proteins from cells.

Wild-type or mutated Ins1/2 promoter cDNAs containing approximately 1000 bp of the 5′-flanking sequences of the Ins1/2 promoter regions were amplified using PCR with appropriate linker-containing primers, which were then used to replace the promoter region of the Rat Ins2 promoter–reporter (luciferase) plasmid^{37 (link)} using a ligation kit (TaKaRa, Tokyo, Japan) (Supplementary Fig. 3).