spaKSpaK Version (Component) spaRSpaR Version (Component) BBa_K1377001BBa_K1377001 Version 1 (Component)spaA backward + sortase A + spaB + spaC
RFC 37BBa_K245080 Version 1 (Component)scar Xba/Spe NgoMIV
BBa_K1351013BBa_K1351013 Version 1 (Component)spaS Subtilin (antimicrobial peptide)
spsEBO_31979 Version 1 (Component) SpsEBO_11138 Version 1 (Component) SprEBO_11502 Version 1 (Component) BBa_K1478000BBa_K1478000 Version 1 (Component)Expa4 plant secretion signal, localizes to extracellular space
BBa_K1351012BBa_K1351012 Version 1 (Component)spaS Subtilin (antimicrobial peptide, Freiburg standard)
speEBO_3478 Version 1 (Component) BBa_K104004BBa_K104004 Version 1 (Component)spaR CDS (Coding sequence)
BBa_K104006BBa_K104006 Version 1 (Component)spaK CDS (Coding Sequence)
BBa_K1931017BBa_K1931017 Version 1 (Component)T7-RBS-SpaC-Terminator
spac+efeBBa_K1065203 Version 1 (Component)Efe+Bba_B0015 in pSpac (BBa_K823026)
SPA Z domaBBa_K1947003 Version 1 (Component)SPA Z domain
BBa_J15506BBa_J15506 Version 1 (Component)spac promoter with lacI
BBa_K103004BBa_K103004 Version 1 (Component)protein Z<sub>SPA-1</sub>
BBa_K1377000BBa_K1377000 Version 1 (Component)promoter of pili A gene cluster + spaA front
pSBBs0KBBa_K823026 Version 1 (Component)pSB<sub>Bs</sub>0K-P<sub>spac</sub> (replicative Bacillus subtilis expression vector; IPTG inducible
Intein_assisted_Bisection_MappingIntein_assisted_Bisection_Mapping_collection Version 1 (Collection)Split inteins are powerful tools for seamless ligation of synthetic split proteins. Yet, their use remains limited because the already intricate split site identification problem is often complicated by the requirement of extein junction sequences. To address this, we augmented a mini-Mu transposon-based screening approach and devised the intein-assisted bisection mapping (IBM) method. IBM robustly revealed clusters of split sites on five proteins, converting them into AND or NAND logic gates. We further showed that the use of inteins expands functional sequence space for splitting a protein. We also demonstrated the utility of our approach over rational inference of split sites from secondary structure alignment of homologous proteins. Furthermore, the intein inserted at an identified site could be engineered by the transposon again to become partially chemically inducible, and to some extent enabled post-translational tuning on host protein function. Our work offers a generalizable and systematic route towards creating split protein-intein fusions and conditional inteins for protein activity control.