Isolation and Cloning of SL-BBI cDNA

The biosynthetic precursor cDNA of SL-BBI was obtained as previously described [25 (link)]. Briefly, the Dynabeads^® mRNA DIRECT^TM Kit (Dynal Biotech, Merseyside, UK) was used to isolate the mRNA from the lyophilised skin secretion. The first-strand cDNA was synthesised by the BD SMART^TM RACE cDNA Amplification Kit (BD Bioscience Clontech, UK) and used to construct the cDNA library through 3′ RACE-PCR. For SL-BBI, the 3′-RACE reactions employed a NUP primer (supplied with the kit) and a degenerate sense primer (3′-RACE: 5′-GAWYYAYYHRAGCCYAAADATGTTCA-3′, R = A/G, V = A/C/G, N = A/C/T/G, Y = C/T, S = C/G, W = A/T) was designed based on the highly conserved nucleic acid sequences of the 5′-untranslated region of the protease inhibitory peptide published previously. The purified products were cloned by pGEM^®-T Easy Vector Kit (Promega Corporation, Madison WI, USA) and sequenced through an ABI3730 automated sequencer (Applied Biosystems, Foster City, CA, USA).