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.
Plasmid maps and sequences for constructs used in the study "Intein-assisted bisection mapping systematically splits proteins for Boolean logic and inducibility engineering"
pSEVA321-PBAD(SapI-)-B30-SrpR(1-54)-KLG-M86N-T SrpRN-M86N expression plasmid, to prove either lobe alone does not cause repression
Creator: Trevor Y. H. Ho Principal Investigator: Baojun Wang BioSafety Level: Level 1 Backbone: pSEVA321 Origin of replication: RK2 Selection Markers: Chloramphenicol
pSB3T5(BsaI-)-PBAD(SapI-)-B30-SrpR(1-22)-BsaI-BsaI-SrpR(192-213)-Ter Expression vector to generate ORF insertion library in IBM on SrpR
Creator: Trevor Y. H. Ho Principal Investigator: Baojun Wang BioSafety Level: Level 1 Backbone: pSB3T5 Origin of replication: p15A Selection Markers: Tetracycline
pSB3T5(BsaI-)-PBAD(SapI-)-B32-bla-Ter beta-lactamase expression plasmid. Activity reference in IBM experimenton beta-lactamase
pSB3T5(BsaI-)-PPhlF-RiboJ-BCD1-SYNZIP18-5aa-mCherry*(157-236)-H6-Ter Expression plasmid for SYNZIP18-mCherry C-lobe. Used in mCherry BiFC experiment
pSB3T5(BsaI-)-PBAD(SapI-)-B30-mCherry*(1-188)-KNG-M86N-cat-Plux2-B32-M86C-S-mCherry*(189-236)-H6-T Expression plasmid for split mCherry construct derived from IBM, to prove protein splicing by M86 intein
Creator: Trevor Y. H. Ho Principal Investigator: Baojun Wang BioSafety Level: Level 1 Backbone: pSB3T5 Origin of replication: p15A Selection Markers: Tetracycline/ Chloramphenicol
pSB3T5(BsaI-)-PBAD(SapI-)-B33-ECF20(1-101)-2aa-acVHH-T-cat-PBAD(SapI-)-RiboJ-BCD22-acVHH-2aa-ECF20(102-193)-T Expression plasmid for ECF20 spit at 101/102 fused with acVHH via 2 aa linker
pSB1C3(BsmBI-,BsaI+)-BsaI-TetR(20-188)-BsaI Staging plasmid for IBM on TetR
Creator: Trevor Y. H. Ho Principal Investigator: Baojun Wang BioSafety Level: Level 1 Backbone: pSB1C3 Origin of replication: pMB1 Selection Markers: Chloramphenicol
pSEVA321-PBAD(SapI-)-B30-SrpR(1-170)-LRG-M86N-T SrpRN-M86N expression plasmid, to prove either lobe alone does not cause repression
pSB3T5(BsaI-)-PBAD(SapI-)-B30-mCherry*(1-192)-G-M86(1-17)-PI-10aa-acVHH-T-cat-PBAD(SapI-)-RiboJ-BCD1-acVHH-10aa-M86(18-154)-S-mCherry*(193-236)-H6-T Expression plasmid for mCherry split at 192/193 split by M86 intein split at 17/18 by acVHH
pSB3T5(BsaI-)-PBAD(SapI-)-B33-ECF20(1-101)-T-cat-PBAD(SapI-)-RiboJ-BCD22-ECF20(102-193)-T Expression plasmid for ECF20 spit at 101/102
pSEVA321-PBAD(SapI-)-B30-SrpR(1-169)-GLG-M86N-T SrpRN-M86N expression plasmid, to prove either lobe alone does not cause repression
pSB3T5(BsaI-)-PPhlF-RiboJ-BCD1-SYNZIP18-5aa-mCherry*(177-236)-H6-Ter Expression plasmid for SYNZIP18-mCherry C-lobe. Used in mCherry BiFC experiment
pSB3T5(BsaI-)-PBAD(SapI-)-B30-mCherry*(1-171)-LNG-M86N-cat-Plux2-B32-M86C-S-mCherry*(172-236)-H6-T Expression plasmid for split mCherry construct derived from IBM, to prove protein splicing by M86 intein
pSEVA471(BsbI+) Empty plasmid for generating autofluorescence control strains
Creator: Trevor Y. H. Ho Principal Investigator: Baojun Wang BioSafety Level: Level 1 Backbone: pSEVA471 Origin of replication: pSC101 Selection Markers: Spectinomycin
pSB3T5(BsaI-)-PBAD(SapI-)-B30-mCherry*(1-192)-G-deltaI-SMTR(WT)-S-mCherry*(193-236)-H6-Ter Expression plasmid for mCherry split at 192/193 inserted with Mtu RecA ?I-SMTR
pSB3T5(BsaI-)-PBAD(SapI-)-B30-mCherry*(1-185)-YNG-M86N-cat-Plux2-B32-M86C-S-mCherry*(186-236)-H6-T Expression plasmid for split mCherry construct derived from IBM, to prove protein splicing by M86 intein
pSB3K3-TT-PBAD(SapI-)-B30-His-FKBP-14aa-T7RNAP(181+)-T Expression plasmid for split, evolved T7 RNA polymerase C-lobe fused to FKBP
Creator: Trevor Y. H. Ho Principal Investigator: Baojun Wang BioSafety Level: Level 1 Backbone: pSB3K3 Origin of replication: p15A Selection Markers: Kanamycin
pSB1A3-PBAD(SapI-)-B30-mCherry*(1-192)-5aa-SYNZIP17-Ter Expression plasmid for mCherry N-lobe-SYNZIP17. Used in mCherry BiFC experiment
Creator: Trevor Y. H. Ho Principal Investigator: Baojun Wang BioSafety Level: Level 1 Backbone: pSB1A3 Origin of replication: pMB1 Selection Markers: Ampicllin
pSB3A5*(BsaI-)-PBAD(SapI-)-tet1-TetR-Ter Expression plasmid for TetR
Creator: Trevor Y. H. Ho Principal Investigator: Baojun Wang BioSafety Level: Level 1 Backbone: pSB3A5* Origin of replication: p15A Selection Markers: Ampicillin
pSB3T5(BsaI-)-PPhlF-RiboJ-B32-M86C-S-SrpR(170-213)-Ter M86C-SrpRC expression plasmid, to prove either lobe alone does not cause repression
pTHSSd_1V-WTR1R2-BbsI(G)-kanR-lacZalpha-SapI-WTR1R2 Plasmid carrying engineered transposon for IBM
Creator: Trevor Y. H. Ho Principal Investigator: Baojun Wang BioSafety Level: Level 1 Backbone: pTHSSd_1 Origin of replication: pMB1 Selection Markers: Kanamycin / Chloramphenicol
pSB3T5(BsaI-)-PPhlF-RiboJ-B32-M86C-S-SrpR(177-213)-Ter M86C-SrpRC expression plasmid, to prove either lobe alone does not cause repression
pSB3T5(BsaI-)-PBAD(SapI-)-B30-mCherry*(1-192)-GAEY-e-NpuNcage-FKBP-T-cat-PBAD(SapI-)-RiboJ-BCD1-FRB-NpuCcage-CFNS-mCherry*(193-236)-H6-Ter Expression plasmid for mCherry split at 192/193 inserted with Npu DnaE, caged intein fused with FKBP/FRB
pSB4A3-T-PT7-B30-mScarlet-I-3T T7 RNAP polymerase reporter plasmid
Creator: Trevor Y. H. Ho Principal Investigator: Baojun Wang BioSafety Level: Level 1 Backbone: pSB4A3 Origin of replication: pSC101 Selection Markers: Ampicllin
pSB3T5(BsaI-)-PBAD(SapI-)-B30-mCherry*(1-192)-QLG-M86N-cat-Plux2-B32-M86C-S-mCherry*(193-236)-H6-T Expression plasmid for split mCherry construct derived from IBM, to prove protein splicing by M86 intein
pSB3T5(BsaI-)-PBAD(SapI-)-B30-mCherry*-T mCherry expression plasmid. Activity reference in IBM experimenton mCherry
pSEVA321-PBAD(SapI-)-B30-SrpR(1-77)-LGG-M86N-T SrpRN-M86N expression plasmid, to prove either lobe alone does not cause repression
pSB3T5(BsaI-)-PBAD(SapI-)-B30-mCherry*(1-191)-VHG-M86N-cat-Plux2-B32-M86C-S-mCherry*(192-236)-H6-T Expression plasmid for split mCherry construct derived from IBM, to prove protein splicing by M86 intein
pSB3A5*(BsaI-)-PBAD(SapI-)-B30-H6-TetR(1-166)-5aa-SYNZIP17-BCD1-SYNZIP18-5aa-TetR(167-207)-HA-Ter Expression plasmid for tagged TetR split at 166/167 with SYNZIP17 & SYNZIP18
pSB3T5(BsaI-)-PPhlF-RiboJ-BCD1-SYNZIP18-5aa-mCherry*(174-236)-H6-Ter Expression plasmid for SYNZIP18-mCherry C-lobe. Used in mCherry BiFC experiment
pSB1A3-PBAD(SapI-)-B30-mCherry*(1-159)-5aa-SYNZIP17-Ter Expression plasmid for mCherry N-lobe-SYNZIP17. Used in mCherry BiFC experiment
pSB3T5(BsaI-)-PBAD(SapI-)-B30-mCherry*(1-156)-PDG-M86N-cat-Plux2-B32-M86C-S-mCherry*(157-236)-H6-T Expression plasmid for split mCherry construct derived from IBM, to prove protein splicing by M86 intein
pSB1A3-BbsI-GY-gp-41-1N-cat-PPhlF-RiboJ-B32-gp41-1C-SS-SapI Plasmid carrying Golden Gate substitution insert for IBM, gp41-1 intien, using PPhlF as C-lobe promoter
Creator: Trevor Y. H. Ho Principal Investigator: Baojun Wang BioSafety Level: Level 1 Backbone: pSB1A3 Origin of replication: pMB1 Selection Markers: Ampicillin / Chloramphenicol
pSB3T5(BsaI-)-PBAD-B30-mCherry*(1-159)-GAG-M86N-cat-Plux2-B32-M86C-S-mCherry*(160-236)-T Outcome simulation control for IBM on mCherry
pSEVA321-PBAD(SapI-)-B30-SrpR(1-171)-RSG-M86N-T SrpRN-M86N expression plasmid, to prove either lobe alone does not cause repression
pSB3A5*(BsaI-)(empty) Empty plasmid for generating autofluorescence control strains
pSB3A5*(BsaI-)-PBAD(SapI-)-B30-H6-TetR-HA-Ter Expression plasmid for tagged TetR
pSB3A5*(BsaI-)-PBAD(SapI-)-tet1-TetR(1-20)-BsaI-BsaI-TetR(188-207)-Ter Expression vector to generate ORF insertion library in IBM on TetR
pSB3T5(BsaI-)-PBAD(SapI-)-B33-ECF20(1-101)-G-M86(1-85)-ELG-ER-LBD-S-M86(86-154)-S-ECF20(102-193)-T Expression plasmid for ECF20 split at 101/102 inserted with M86 intein split at 85/86 by ER-LBD
pSEVA171-PSrpR-B30-mScarlet-I-Ter SrpR reporter plasmid
Creator: Trevor Y. H. Ho Principal Investigator: Baojun Wang BioSafety Level: Level 1 Backbone: pSEVA171 Origin of replication: pSC101 Selection Markers: Ampicillin
pSB3T5(BsaI-)-PBAD(SapI-)-B30-mCherry*(1-173)-DGG-M86N-cat-Plux2-B32-M86C-S-mCherry*(174-236)-H6-T Expression plasmid for split mCherry construct derived from IBM, to prove protein splicing by M86 intein
pSB3T5(BsaI-)-PBAD(SapI-)-B30-mCherry*(1-192)-G-M86(1-39)-LD-10aa-acVHH-T-cat-PBAD(SapI-)-RiboJ-BCD1-acVHH-10aa-M86(40-154)-S-mCherry*(193-236)-H6-T Expression plasmid for mCherry split at 192/193 split by M86 intein split at 39/40 by acVHH
pSB1A3-PBAD(SapI-)-B30-mCherry*(1-185)-5aa-SYNZIP17-Ter Expression plasmid for mCherry N-lobe-SYNZIP17. Used in mCherry BiFC experiment
pSB1A3-PBAD(SapI-)-B30-mCherry*(1-185)-Ter Expression plasmid for mCherry N-lobe. To proof mCherry split parts do not self-dimerize in mCherry BiFC experiment
pSB3T5(BsaI-)-PPhlF-RiboJ-BCD1-SYNZIP18-5aa-mCherry*(122-236)-H6-Ter Expression plasmid for SYNZIP18-mCherry C-lobe. Used in mCherry BiFC experiment
pSB3T5(BsaI-)-PBAD(SapI-)-B30-mCherry*(1-190)-PVG-M86N-cat-Plux2-B32-M86C-S-mCherry*(191-236)-H6-T Expression plasmid for split mCherry construct derived from IBM, to prove protein splicing by M86 intein
pSB1C3(BsmBI-,BsaI+)-BsaI-SrpR(23-192)-BsaI Staging plasmid for IBM on SrpR
pSEVA321-PBAD(SapI-)-B30-SrpR(1-75)-PDG-M86N-T SrpRN-M86N expression plasmid, to prove either lobe alone does not cause repression
pSB3T5(BsaI-)-PBAD(SapI-)-B30-mCherry*(1-192)-GAEY-e-NpuNcage-acVHH-T-cat-PBAD(SapI-)-RiboJ-BCD1-acVHH-NpuCcage-CFNS-mCherry*(193-236)-H6-Ter Expression plasmid for mCherry split at 192/193 inserted with Npu DnaE, caged intein fused with acVHH
pSB1A3-PBAD(SapI-)-B30-mCherry*(1-191)-Ter Expression plasmid for mCherry N-lobe. To proof mCherry split parts do not self-dimerize in mCherry BiFC experiment
pSB3T5(BsaI-)-PBAD(SapI-)-B30-mCherry*(1-159)-GAG-M86N-cat-Plux2-B32-M86C-S-mCherry*(160-236)-H6-T Expression plasmid for split mCherry construct as positive control for splicing by M86 intein
pSB3T5(BsaI-)-PBAD(SapI-)-B33-ECF20(1-101)-G-M86-S-ECF20(102-193)-T Expression plasmid for ECF20 inserted with cis-M86 intein. Activity reference in acVHH-assisted bisection mapping experiment
pSB3T5(BsaI-)-PBAD(SapI-)-B30-mCherry*(1-192)-GCS-mCherry*(193-236)-H6-Ter Expression plasmid for mCherry split at 192/193 inserted with extein junction of Mtu RecA
pSB3T5(BsaI-)-PBAD(SapI-)-B33-ECF20(1-101)-G-M86(1-119)-WDG-ER-LBD-S-M86(120-154)-S-ECF20(102-193)-T Expression plasmid for ECF20 split at 101/102 inserted with M86 intein split at 119/120 by ER-LBD
pSB4A3(empty) Empty plasmid for generating autofluorescence control strains
Creator: Trevor Y. H. Ho Principal Investigator: Baojun Wang BioSafety Level: Level 1 Backbone: pSB4A3 Origin of replication: pSC101 Selection Markers: Ampicillin
pSB1A3-BbsI-G-ER-LBD-S-SapI Plasmid carrying Golden Gate substitution insert for ER-LBD domain-insertion mapping
Creator: Trevor Y. H. Ho Principal Investigator: Baojun Wang BioSafety Level: Level 1 Backbone: pSB1A3 Origin of replication: pMB1 Selection Markers: Ampicillin
pSB3T5(BsaI-)-PPhlF-RiboJ-BCD1-SYNZIP18-5aa-mCherry*(192-236)-H6-Ter Expression plasmid for SYNZIP18-mCherry C-lobe. Used in mCherry BiFC experiment
pSB1A3-PBAD(SapI-)-B30-mCherry*(1-173)-Ter Expression plasmid for mCherry N-lobe. To proof mCherry split parts do not self-dimerize in mCherry BiFC experiment
pSB3T5(BsaI-)-PBAD(SapI-)-B30-mCherry*(1-121)-EFG-M86N-cat-Plux2-B32-M86C-S-mCherry*(122-236)-H6-T Expression plasmid for split mCherry construct derived from IBM, to prove protein splicing by M86 intein
pSB3T5(BsaI-)-PPhlF-RiboJ-B32-M86C-S-SrpR(172-213)-Ter M86C-SrpRC expression plasmid, to prove either lobe alone does not cause repression
pSEVA321-PBAD(SapI-)-B30-SrpR(1-52)-NGG-M86N-T SrpRN-M86N expression plasmid, to prove either lobe alone does not cause repression
pSB1A3-PBAD(SapI-)-B30-mCherry*(1-156)-Ter Expression plasmid for mCherry N-lobe. To proof mCherry split parts do not self-dimerize in mCherry BiFC experiment
pSB3T5(BsaI-)-PPhlF-RiboJ-B32-M86C-S-SrpR(110-213)-Ter M86C-SrpRC expression plasmid, to prove either lobe alone does not cause repression
pSB3T5(BsaI-)-PBAD(SapI-)-B33-ECF20(1-101)-5aa-acVHH-T-cat-PBAD(SapI-)-RiboJ-BCD22-acVHH-5aa-ECF20(102-193)-T Expression plasmid for ECF20 spit at 101/102 fused with acVHH via 5 aa linker
pSB3T5(BsaI-)-PBAD(SapI-)-B30-mCherry*(1-158)-DGG-M86N-cat-Plux2-B32-M86C-S-mCherry*(159-236)-H6-T Expression plasmid for split mCherry construct derived from IBM, to prove protein splicing by M86 intein
pSEVA321-PBAD(SapI-)-B30-SrpR(1-114)-SGG-M86N-T SrpRN-M86N expression plasmid, to prove either lobe alone does not cause repression
pSB3A5*(BsaI-)-PBAD(SapI-)-B30-H6-TetR(1-112)-5aa-SYNZIP17-BCD1-SYNZIP18-5aa-TetR(113-207)-HA-Ter Expression plasmid for tagged TetR split at 112/113 with SYNZIP17 & SYNZIP18
pSB1C3(BsmBI-)-BsaI-bla(28-285)-BsaI Staging plasmid for IBM on beta-lactamase
pSB3T5(BsaI-)-PBAD(SapI-)-B33-ECF20(1-101)-G-M86(1-78)-DGG-ER-LBD-S-M86(79-154)-S-ECF20(102-193)-T Expression plasmid for ECF20 split at 101/102 inserted with M86 intein split at 78/79 by ER-LBD
pSB3T5(BsaI-)-PPhlF-RiboJ-BCD1-SYNZIP18-5aa-mCherry*(160-236)-H6-Ter Expression plasmid for SYNZIP18-mCherry C-lobe. Used in mCherry BiFC experiment
pSB3T5(BsaI-)-PBAD(SapI-)-B30-mCherry*-H6-Ter mCherry expression plasmid. Activity reference in mCherry BiFC experiment
pSB1A3-PBAD(SapI-)-B30-mCherry*(1-121)-Ter Expression plasmid for mCherry N-lobe. To proof mCherry split parts do not self-dimerize in mCherry BiFC experiment
pSB3T5(BsaI-)-PPhlF-RiboJ-B32-M86C-S-SrpR(78-213)-Ter M86C-SrpRC expression plasmid, to prove either lobe alone does not cause repression
pSB3T5(BsaI-)-PBAD(SapI-)-B33-ECF20(1-101)-15aa-acVHH-T-cat-PBAD(SapI-)-RiboJ-BCD22-acVHH-15aa-ECF20(102-193)-T Expression plasmid for ECF20 spit at 101/102 fused with acVHH via 15 aa linker
pSEVA321-PBAD(SapI-)-B30-SrpR(1-176)-QAG-M86N-T SrpRN-M86N expression plasmid, to prove either lobe alone does not cause repression
pSB3T5(BsaI-)-PPhlF-RiboJ-B32-M86C-S-SrpR(112-213)-Ter M86C-SrpRC expression plasmid, to prove either lobe alone does not cause repression
pSB3T5(BsaI-)-PPhlF-RiboJ-BCD1-SYNZIP18-5aa-mCherry*(193-236)-H6-Ter Expression plasmid for SYNZIP18-mCherry C-lobe. Used in mCherry BiFC experiment
pSB3T5(BsaI-)-PBAD(SapI-)-B30-mCherry(1-192)-G-M86(1-100)-10aa-acVHH-T-cat-PBAD(SapI-)-RiboJ-BCD1-acVHH-10aa-M86(101-154)-S-mCherry(193-236)-H6-T Expression plasmid for mCherry split at 192/193 split by M86 intein split at 100/101 by acVHH
pSB1C3(BsmBI-)-BsaI-mCherry*(27-218)-BsaI Staging plasmid for IBM on mCherry
pSB3T5(BsaI-)-PPhlF-RiboJ-B32-M86C-S-SrpR(55-213)-Ter M86C-SrpRC expression plasmid, to prove either lobe alone does not cause repression
pSEVA321 Empty plasmid for generating autofluorescence control strains
pSB1A3-PBAD(SapI-)-B30-mCherry*(1-156)-5aa-SYNZIP17-Ter Expression plasmid for mCherry N-lobe-SYNZIP17. Used in mCherry BiFC experiment
pSB3T5(BsaI-)-PBAD(SapI-)-B33-ECF20(1-101)-G-M86(1-77)-IDG-ER-LBD-S-M86(78-154)-S-ECF20(102-193)-T Expression plasmid for ECF20 split at 101/102 inserted with M86 intein split at 77/78 by ER-LBD
pSB1A3-PBAD(SapI-)-B30-mCherry*(1-176)-5aa-SYNZIP17-Ter Expression plasmid for mCherry N-lobe-SYNZIP17. Used in mCherry BiFC experiment
pSB3K3-TT-PBAD-T Empty plasmid for generating autofluorescence control strains
pSB3T5(BsaI-)-PBAD(SapI-)-B33-ECF20(1-101)-G-M86(1-76)-TIG-ER-LBD-S-M86(77-154)-S-ECF20(102-193)-T Expression plasmid for ECF20 split at 101/102 inserted with M86 intein split at 76/77 by ER-LBD
pSB1A3-BbsI-M86N-cat-Plux2-B32-M86C-SapI Plasmid carrying Golden Gate substitution insert for IBM, M86 intein, using Plux2 as C-lobe promoter
pSB1A3-PBAD(SapI-)-B30-mCherry*(1-173)-5aa-SYNZIP17-Ter Expression plasmid for mCherry N-lobe-SYNZIP17. Used in mCherry BiFC experiment
pSEVA471-PTet*-RiboJ10-B64-mScarlet-I-Ter TetR reporter plasmid
pSB4A3-PECF20-B30-mScarlet-I-B15 ECF20 reporter plasmid
pSB3T5(BsaI-)-PBAD(SapI-)-B30-mCherry*(1-192)-GAEYCFNS-mCherry*(193-236)-H6-Ter Expression plasmid for mCherry split at 192/193 inserted with extein junction of Npu DnaE
pSB3T5(BsaI-)-PBAD(SapI-)-B30-mCherry*(1-192)-G-M86(1-22)-LG-10aa-acVHH-T-cat-PBAD(SapI-)-RiboJ-BCD1-acVHH-10aa-M86(23-154)-S-mCherry*(193-236)-H6-T Expression plasmid for mCherry split at 192/193 split by M86 intein split at 22/23 by acVHH
pSB1A3-PBAD(SapI-)-B30-mCherry*(1-192)-Ter Expression plasmid for mCherry N-lobe. To proof mCherry split parts do not self-dimerize in mCherry BiFC experiment
pSB3T5(BsaI-)-PBAD(SapI-)-B30-mCherry*(1-27)-BsaI-BsaI-mCherry*(218-236)-T Expression vector to generate ORF insertion library in IBM on mCherry
pSB3T5(BsaI-)-PBAD(SapI-)-B33-ECF20(1-101)-G-M86(1-6)-BsaI-BsaI-M86(149-154)-S-ECF20(102-193)-T Expression vector to generate ORF insertion library in acVHH-assisted bisection mapping experiment
pSEVA171 Empty plasmid for generating autofluorescence control strains
pSB3T5(BsaI-)-PBAD(SapI-)-B30-mCherry*(1-160)-ALG-M86N-cat-Plux2-B32-M86C-S-mCherry*(161-236)-H6-T Expression plasmid for split mCherry construct derived from IBM, to prove protein splicing by M86 intein
pSEVA321-PBAD(SapI-)-B30-His-T7RNAP(29-1)-15aa-acVHH-T Expression plasmid for split, evolved T7 RNA polymerase N-lobe fused to acVHH
pSB3T5(BsaI-)-PBAD(SapI-)-B33-ECF20(1-101)-10aa-acVHH-T-cat-PBAD(SapI-)-RiboJ-BCD22-acVHH-10aa-ECF20(102-193)-T Expression plasmid for ECF20 spit at 101/102 fused with acVHH via 10 aa linker
pSB1A3-PBAD(SapI-)-B30-mCherry*(1-121)-5aa-SYNZIP17-Ter Expression plasmid for mCherry N-lobe-SYNZIP17. Used in mCherry BiFC experiment
pSEVA321-PBAD(SapI-)-B30-SrpR(1-111)-LDG-M86N-T SrpRN-M86N expression plasmid, to prove either lobe alone does not cause repression
pSB3A5*(BsaI-)-PBAD(SapI-)-B30-H6-TetR(1-70)-5aa-SYNZIP17-BCD1-SYNZIP18-5aa-TetR(71-207)-HA-Ter Expression plasmid for tagged TetR split at 70/71 with SYNZIP17 & SYNZIP18
pSEVA321-PBAD(SapI-)-B30-SrpR(1-109)-QGG-M86N-T SrpRN-M86N expression plasmid, to prove either lobe alone does not cause repression
pSB3T5(BsaI-)-PPhlF-RiboJ-B32-M86C-S-SrpR(53-213)-Ter M86C-SrpRC expression plasmid, to prove either lobe alone does not cause repression
pSEVA321-PBAD(SapI-)-B30-SrpR(1-116)-LIG-M86N-T SrpRN-M86N expression plasmid, to prove either lobe alone does not cause repression
pSB1A3-BbsI-10aa-acVHH-T-cat-PBAD(SapI)-RiboJ-BCD22-acVHH-10aa-SapI Plasmid carrying Golden Gate substitution insert for acVHH-assisted bisection mapping
pSB3K3-TT-PBAD(SapI-)-B30-His-acVHH-15aa-T7RNAP(181+)-T Expression plasmid for split, evolved T7 RNA polymerase C-lobe fused to acVHH
pSB3T5(BsaI-)-PPhlF-RiboJ-B32-M86C-S-SrpR(76-213)-Ter M86C-SrpRC expression plasmid, to prove either lobe alone does not cause repression
pSB3T5(BsaI-)-PBAD(SapI-)-B33-ECF20(1-101)-G-M86(1-118)-YCG-ER-LBD-S-M86(119-154)-S-ECF20(102-193)-T Expression plasmid for ECF20 split at 101/102 inserted with M86 intein split at 118/119 by ER-LBD
pSB1A3-BbsI-M86N-T-cat-PPhlF-RiboJ-B32-M86C-SapI Plasmid carrying Golden Gate substitution insert for IBM, M86 intien, using PPhlF as C-lobe promoter
pSB3T5(BsaI-)-PBAD(SapI-)-B32-bla(1-28)-BsaI-BsaI-bla(285-286)-Ter Expression vector to generate ORF insertion library in IBM on beta-lactamase
pSB3T5(BsaI-)-PBAD(SapI-)-B30-SrpR-Ter Expression plasmid for SrpR
pSB3T5(empty) Empty plasmid for generating autofluorescence control strains
pSB3T5(BsaI-)-PBAD(SapI-)-B33-ECF20-T ECF20 expression plasmid. Activity reference in IBM experiment on ECF20
pSB3T5(BsaI-)-PBAD(SapI-)-B30-mCherry*(1-192)-G-37R3-2-S-mCherry*(193-236)-H6-Ter Expression plasmid for mCherry split at 192/193 inserted with Mtu RecA 37R3-2
pSB3T5(BsaI-)-PBAD(SapI-)-B33-ECF20(1-101)-G-M86(1-91)-HIG-ER-LBD-S-M86(92-154)-S-ECF20(102-193)-T Expression plasmid for ECF20 split at 101/102 inserted with M86 intein split at 91/92 by ER-LBD
pSB3T5(BsaI-)-PPhlF-RiboJ-B32-M86C-S-SrpR(115-213)-Ter M86C-SrpRC expression plasmid, to prove either lobe alone does not cause repression
pSB1A3-PBAD(SapI-)-B30-mCherry*(1-159)-Ter Expression plasmid for mCherry N-lobe. To proof mCherry split parts do not self-dimerize in mCherry BiFC experiment
pSEVA321-PBAD(SapI-)-B30-SrpR(1-175)-QHG-M86N-T SrpRN-M86N expression plasmid, to prove either lobe alone does not cause repression
pSB3T5(BsaI-)-PBAD(SapI-)-B30-mCherry*(1-194)-PGG-M86N-cat-Plux2-B32-M86C-S-mCherry*(195-236)-H6-T Expression plasmid for split mCherry construct derived from IBM, to prove protein splicing by M86 intein
pSB3T5(BsaI-)-PBAD(SapI-)-B33-ECF20(1-5)-BsaI-BsaI-ECF20(187-193)-Ter Expression vector to generate ORF insertion library in IBM on ECF20
pSB3T5(BsaI-)-PPhlF-RiboJ-BCD1-SYNZIP18-5aa-mCherry*(186-236)-H6-Ter Expression plasmid for SYNZIP18-mCherry C-lobe. Used in mCherry BiFC experiment
pSB3T5(BsaI-)-PBAD(SapI-)-B30-mCherry*(1-192)-G-M86-S-mCherry*(193-236)-H6-T Expression plasmid for mCherry split at 192/193 inserted with cis-M86 intein
pSB3T5(BsaI-)-PPhlF-RiboJ-B32-M86C-S-SrpR(171-213)-Ter M86C-SrpRC expression plasmid, to prove either lobe alone does not cause repression
pSB3T5(BsaI-)-PBAD(SapI-)-B30-mCherry*(1-187)-ANG-M86N-cat-Plux2-B32-M86C-S-mCherry*(188-236)-H6-T Expression plasmid for split mCherry construct derived from IBM, to prove protein splicing by M86 intein
pSB3T5(BsaI-)-PBAD(SapI-)-B30-mCherry*(1-176)-HYG-M86N-cat-Plux2-B32-M86C-S-mCherry*(177-236)-H6-T Expression plasmid for split mCherry construct derived from IBM, to prove protein splicing by M86 intein
pSB3T5(BsaI-)-PBAD(SapI-)-B30-mCherry*(1-174)-GGG-M86N-cat-Plux2-B32-M86C-S-mCherry*(175-236)-H6-T Expression plasmid for split mCherry construct derived from IBM, to prove protein splicing by M86 intein
pSEVA321-PBAD(SapI-)-B30-His-T7RNAP(29-1)-14aa-FRB-T Expression plasmid for split, evolved T7 RNA polymerase N-lobe fused to FRB
pSB3T5(BsaI-)-PBAD(SapI-)-B30-mCherry*(1-192)-G-M86(1-6)-BsaI-BsaI-M86(149-154)-S-mCherry*(193-236)-H6-T Expression vector to generate ORF insertion library in ER-LBD domain-insertion mapping
pSB1C3(BsmBI-,BsaI+)-BsaI-ECF20(6-188)-BsaI_v3 Staging plasmid for IBM on ECF20
pSB3T5(BsaI-)-PPhlF-RiboJ-B32-M86C-S-SrpR(117-213)-Ter M86C-SrpRC expression plasmid, to prove either lobe alone does not cause repression
pSB3T5(BsaI-)-PPhlF-RiboJ-B32-M86C-S-SrpR(176-213)-Ter M86C-SrpRC expression plasmid, to prove either lobe alone does not cause repression
pSB1A3-PBAD(SapI-)-B30-mCherry*(1-191)-5aa-SYNZIP17-Ter Expression plasmid for mCherry N-lobe-SYNZIP17. Used in mCherry BiFC experiment
pSB1A3-PBAD(SapI-)-B30-mCherry*(1-176)-Ter Expression plasmid for mCherry N-lobe. To proof mCherry split parts do not self-dimerize in mCherry BiFC experiment
pSB1C3(BsmBI-,BsaI+)-BsaI-M86(6-149)-BsaI Staging plasmid for insertion on the M86 intein