Spontaneous C-cleavage of a mini-intein without its conserved N-terminal motif A

► We converted Ssp DnaX intein into a mini- and split intein. ► Its C-terminal 136-aa fragment underwent spontaneous C-cleavage. ► The N-terminal 15 aa of intein motif A are dispensable for C-cleavage. ► C-cleavage decreased by mutating conserved Thr in intein motif B. ► Findings indicated a robust intein structure in the absence of motif A and suggested a larger role of motif B.