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SORTASE-LABELLED CLOSTRIDIUM NEUROTOXINS

20220118113 · 2022-04-21

    Inventors

    Cpc classification

    International classification

    Abstract

    The present invention relates to a method for preparing a labelled polypeptide, the method comprising: a. providing a polypeptide comprising: i. a sortase acceptor site or a sortase donor site; ii. a non-cytotoxic protease or a proteolytically inactive mutant thereof; iii. a Targeting Moiety (TM) that is capable of binding to a Binding Site on a target cell; and iv. a translocation domain; b. incubating the polypeptide with: a sortase; and a labelled substrate comprising a sortase donor site or a sortase acceptor site, respectively, and a conjugated detectable label; wherein the sortase catalyses: conjugation between an amino acid of the sortase acceptor site of the polypeptide and an amino acid of the sortase donor site of the labelled substrate; or conjugation between an amino acid of the sortase acceptor site of the labelled substrate and an amino acid of the sortase donor site of the polypeptide; thereby labelling the polypeptide; and c. obtaining the labelled polypeptide. The invention also relates to polypeptides for labelling, labelled polypeptides, nucleic acids encoding said polypeptides, and methods of using and manufacturing said polypeptides.

    Claims

    1. A method for preparing a labelled polypeptide, the method comprising: a. providing a polypeptide comprising: i. a sortase acceptor site or a sortase donor site; ii. a non-cytotoxic protease or a proteolytically inactive mutant thereof; iii. a Targeting Moiety (TM) that is capable of binding to a Binding Site on a target cell; and iv. a translocation domain; b. incubating the polypeptide with: a sortase; and a labelled substrate comprising a sortase donor site or a sortase acceptor site, respectively, and a conjugated detectable label;  wherein the sortase catalyses: conjugation between an amino acid of the sortase acceptor site of the polypeptide and an amino acid of the sortase donor site of the labelled substrate; or conjugation between an amino acid of the sortase acceptor site of the labelled substrate and an amino acid of the sortase donor site of the polypeptide;  thereby labelling the polypeptide; and c. obtaining the labelled polypeptide.

    2. A polypeptide for labelling using a sortase, the polypeptide comprising: i. a sortase acceptor or donor site; ii. a non-cytotoxic protease that is capable of cleaving a protein of the exocytic fusion apparatus in a target cell or a proteolytically inactive mutant thereof; iii. a Targeting Moiety (TM) that is capable of binding to a Binding Site on a target cell; and iv. a translocation domain that is capable of translocating the non-cytotoxic protease from within an endosome, across the endosomal membrane and into the cytosol of the target cell; wherein when the polypeptide comprises a sortase donor site, the sortase donor site is located at an N-terminus of the polypeptide, and wherein when the sortase donor site comprises G.sub.n or A.sub.n, n is at least 2; and wherein the N-terminal residue of the donor site is the N-terminal residue of the polypeptide; or wherein the polypeptide comprises one or more amino acid residues N-terminal to the sortase donor site and a cleavable site, which when cleaved exposes the N-terminus of the sortase donor site.

    3. The method according to claim 1 or polypeptide according to claim 2, wherein the sortase acceptor or donor site is located C-terminal to the TM or wherein the sortase acceptor or donor site is located N-terminal to the non-cytotoxic protease or proteolytically inactive mutant thereof.

    4. The method or polypeptide according to any one of the preceding claims, wherein: the sortase acceptor site comprises (or consists of) L(A/P/S)X(T/S/A/C)(G/A), NPQTN, YPRTG, IPQTG, VPDTG, or LPXTGS, wherein X is any amino acid, and/or wherein the sortase donor site comprises (or consists of) G.sub.n or A.sub.n, wherein n is at least 1.

    5. The method or polypeptide according to any one of the preceding claims, wherein: the sortase acceptor site comprises (or consists of) L(A/P/S)X(T/S/A/C)G, wherein X is any amino acid, NPQTN, YPRTG, IPQTG, VPDTG, or LPXTGS, wherein X is any amino acid, and/or wherein the sortase donor site comprises (or consists of) G.sub.n, wherein n is at least 1.

    6. The method or polypeptide according to any one of the preceding claims, wherein the sortase is Sortase A (SrtA).

    7. The method or polypeptide according to any one of the preceding claims, wherein the polypeptide comprises: at least two sortase acceptor sites; at least two sortase donor sites; or at least one sortase acceptor site and at least one sortase donor site.

    8. The method or polypeptide according to claim 7, wherein the at least two sites are different, preferably wherein the at least two sites have different amino acid sequences.

    9. The method or polypeptide according to claim 7 or 8, wherein: a first sortase acceptor or donor site is located C-terminal to the TM and a second sortase acceptor or donor site is located N-terminal to the non-cytotoxic protease or proteolytically inactive mutant thereof; or a first sortase acceptor or donor site is located N-terminal to the non-cytotoxic protease or proteolytically inactive mutant thereof and a second sortase acceptor or donor site is located C-terminal to the TM.

    10. The method or polypeptide according to any one of the proceeding claims, wherein the polypeptide comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 2, 4 or 40.

    11. The method or polypeptide according to any one of the proceeding claims, wherein the polypeptide comprises a polypeptide sequence having at least 80% sequence identity to SEQ ID NO: 2, 4 or 40.

    12. The method or polypeptide according to any one of the proceeding claims, wherein the polypeptide comprises a polypeptide sequence having at least 90% sequence identity to SEQ ID NO: 2, 4 or 40.

    13. The method or polypeptide according to any one of the proceeding claims, wherein the polypeptide comprises (preferably consists of) a polypeptide sequence shown as SEQ ID NO: 2, 4 or 40.

    14. A labelled polypeptide, the polypeptide comprising: i. a detectable label conjugated to the polypeptide; ii. an amino acid sequence that comprises L(A/P/S)X(T/S/A/C)G.sub.n, wherein X is any amino acid and n is at least 1, L(A/P/S)X(T/S/A/C)A.sub.n, wherein X is any amino acid and n is at least 1, NPQTN, YPRTG, IPQTG, VPDTG, LPXTGS, wherein X is any amino acid, NPKTG, XPETG, LGATG, IPNTG, IPETG, NSKTA, NPQTG, NAKTN, NPQSS, LPXTX, wherein X is any amino acid, NPX.sub.1TX.sub.2, wherein X, is Lys or Gln and X.sub.2 is Asn, Asp or Gly, X.sub.1PX.sub.2X.sub.3G, wherein X.sub.1 is Leu, Ile, Val or Met, X.sub.2 is any amino acid and X.sub.3 is Ser, Thr or Ala, LPEX.sub.1G, wherein X, is Ala, Cys or Ser, LPXS, LAXT, MPXT, MPXTG, LAXS, NPXT, NPXTG, NAXT, NAXTG, NAXS, NAXSG, LPXP, LPXPG, wherein X is any amino acid, LRXTG.sub.n or LPAXG.sub.n, wherein X is any amino acid and n is at least 1; iii. a non-cytotoxic protease or a proteolytically inactive mutant thereof; iv. a Targeting Moiety (TM) that is capable of binding to a Binding Site on a target cell; and v. a translocation domain.

    15. The labelled polypeptide according to claim 14, wherein the amino acid sequence that comprises L(A/P/S)X(T/S/A/C)G.sub.n, L(A/P/S)X(T/S/A/C)A.sub.n, NPQTN, YPRTG, IPQTG, VPDTG, LPXTGS, NPKTG, XPETG, LGATG, IPNTG, IPETG, NSKTA, NPQTG, NAKTN, NPQSS, LPXTX, NPX.sub.1TX.sub.2, X.sub.1PX.sub.2X.sub.3G, LPEX.sub.1G, LPXS, LAXT, MPXT, MPXTG, LAXS, NPXT, NPXTG, NAXT, NAXTG, NAXS, NAXSG, LPXP, LPXPG, LRXTG.sub.n, or LPAXG.sub.n wherein X is any amino acid and n is at least 1 is located C-terminal to the TM or wherein the an amino acid sequence that comprises L(A/P/S)X(T/S/A/C)G.sub.n, L(A/P/S)X(T/S/A/C)A.sub.n, NPQTN, YPRTG, IPQTG, VPDTG, LPXTGS, NPKTG, XPETG, LGATG, IPNTG, IPETG, NSKTA, NPQTG, NAKTN, NPQSS, LPXTX, NPX.sub.1TX.sub.2, X.sub.1PX.sub.2X.sub.3G, LPEX.sub.1G, LPXS, LAXT, MPXT, MPXTG, LAXS, NPXT, NPXTG, NAXT, NAXTG, NAXS, NAXSG, LPXP, LPXPG, wherein X is any amino acid, LRXTG.sub.n, or LPAXG.sub.n wherein X is any amino acid and n is at least 1 is located N-terminal to the non-cytotoxic protease or proteolytically inactive mutant thereof.

    16. The labelled polypeptide according to claim 14 or 15 comprising a further detectable label conjugated to the polypeptide and a further amino acid sequence that comprises L(A/P/S)X(T/S/A/C)G.sub.n, wherein X is any amino acid and n is at least 1, L(A/P/S)X(T/S/A/C)A.sub.n, wherein X is any amino acid and n is at least 1, NPQTN, YPRTG, IPQTG, VPDTG, LPXTGS, wherein X is any amino acid, NPKTG, XPETG, LGATG, IPNTG, IPETG, NSKTA, NPQTG, NAKTN, NPQSS, LPXTX, NPX.sub.1TX.sub.2, X.sub.1PX.sub.2X.sub.3G, LPEX.sub.1G, LPXS, LAXT, MPXT, MPXTG, LAXS, NPXT, NPXTG, NAXT, NAXTG, NAXS, NAXSG, LPXP, LPXPG, LRXTG.sub.n or LPAXG.sub.n.

    17. The labelled polypeptide according to claim 16, wherein the (first) amino acid sequence is different to the further (second) amino acid sequence.

    18. The labelled polypeptide according to claim 16 or 17, wherein: the (first) amino acid sequence is located C-terminal to the TM and the further (second) amino acid sequence is located N-terminal to the non-cytotoxic protease or proteolytically inactive mutant thereof; or the (first) amino acid sequence is located N-terminal to the non-cytotoxic protease or proteolytically inactive mutant thereof and the further (second) amino acid sequence is located C-terminal to the TM.

    19. The labelled polypeptide according to any one of claims 14-18, wherein the polypeptide comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 2, 4, 26 or 40.

    20. The labelled polypeptide according to any one of claims 14-19, wherein the polypeptide comprises a polypeptide sequence having at least 80% sequence identity to SEQ ID NO: 2, 4, 26 or 40.

    21. The labelled polypeptide according to any one of claims 14-20, wherein the polypeptide comprises a polypeptide sequence having at least 90% sequence identity to SEQ ID NO: 2, 4, 26 or 40.

    22. The labelled polypeptide according to any one of claims 14-21, wherein the polypeptide comprises (preferably consists of) a polypeptide sequence shown as SEQ ID NO: 26.

    23. The method, polypeptide or labelled polypeptide according to any one of the preceding claims, wherein the non-cytotoxic protease comprises a clostridial neurotoxin L-chain.

    24. The method, polypeptide or labelled polypeptide according to any one of the preceding claims, wherein the translocation domain comprises a clostridial neurotoxin translocation domain.

    25. The method, polypeptide or labelled polypeptide according to any one of the preceding claims, wherein the polypeptide lacks a functional H.sub.C domain of a clostridial neurotoxin.

    26. The method, polypeptide or labelled polypeptide according to any one of claims 1-24, wherein the TM is a clostridial neurotoxin H.sub.C peptide.

    27. The method, polypeptide or labelled polypeptide according to any one of claims 1-24 or 26, wherein the polypeptide is a clostridial neurotoxin.

    28. The method, polypeptide or labelled polypeptide according to any one of claims 1-24 or 26-27, wherein the polypeptide is a botulinum neurotoxin (BoNT).

    29. The method, polypeptide or labelled polypeptide according to any one of the preceding claims, wherein the polypeptide comprises a botulinum neurotoxin L-chain or proteolytically inactive mutant thereof.

    30. The method, polypeptide or labelled polypeptide according to any one of claims 1-24 or 26-29, wherein the polypeptide comprises of a botulinum neurotoxin H-chain.

    31. The method, polypeptide or labelled polypeptide according to any one of claims 1-24 or 26-30, wherein the polypeptide is selected from: BoNT/A, BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/F, BoNT/G, BoNT/X or TeNT.

    32. A labelled polypeptide obtainable by the method according to any one of claim 1 or 3-13 or 23-31.

    33. The method or labelled polypeptide according to any one of claim 1 or 3-32, wherein the labelled polypeptide does not exhibit reduced potency when compared to an equivalent unlabelled polypeptide.

    34. The method or labelled polypeptide according to any one of claim 1 or 3-33, wherein the labelled polypeptide demonstrates similar cell binding, translocation, and SNARE protein cleavage when compared to an equivalent unlabelled polypeptide.

    35. The method or labelled polypeptide according to any one of claim 1 or 3-34, wherein the labelled polypeptide demonstrates improved cell binding, translocation, and/or SNARE protein cleavage when compared to an equivalent unlabelled polypeptide.

    36. The method or labelled polypeptide according to any one of claim 1 or 3-35, wherein the labelled polypeptide demonstrates improved cell binding, translocation, and SNARE protein cleavage when compared to an equivalent unlabelled polypeptide.

    37. A method for assaying a polypeptide, the method comprising: a. contacting a target cell with the labelled polypeptide according to any one of claims 14-36; and b. detecting the detectable label.

    38. A nucleic acid encoding the polypeptide according to any one of claims 2-13 or 23-31.

    39. The nucleic acid according to claim 38, wherein the nucleic acid comprises a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO: 1, 3 or 39.

    40. The nucleic acid according to claim 38 or 39, wherein the nucleic acid comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 1, 3 or 39.

    41. The nucleic acid according to any one of claims 38-40, wherein the nucleic acid comprises a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 1, 3 or 39.

    42. The nucleic acid according to any one of claims 38-41, wherein the nucleic acid comprises (preferably consists of) a nucleic acid sequence shown as SEQ ID NO: 1, 3 or 39.

    43. A method for manufacturing a polypeptide for labelling using a sortase, the method comprising: a. providing a nucleic acid sequence encoding a polypeptide, wherein the polypeptide comprises: i. a non-cytotoxic protease or a proteolytically inactive mutant thereof; ii. a Targeting Moiety (TM) that is capable of binding to a Binding Site on a target cell; and iii. a translocation domain; and b. introducing a sortase acceptor or donor site into said nucleic acid, thereby producing a modified nucleic acid that encodes a polypeptide comprising a sortase acceptor or donor site; and c. optionally expressing the modified nucleic acid in a host cell; and d. optionally obtaining the expressed polypeptide.

    44. The method according to claim 43, wherein the nucleic acid of step a. comprises a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO: 5 or 7.

    45. The method according to claim 43 or 44, wherein the nucleic acid of step a. comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 5 or 7.

    46. The method according to any one of claims 43-45, wherein the nucleic acid of step a. comprises a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 5 or 7.

    47. The method according to any one of claims 43-46, wherein the nucleic acid of step a. comprises (preferably consists of) a nucleic acid sequence shown as SEQ ID NO: 5 or 7.

    48. The method according to any one of claims 43-47, wherein the modified nucleic acid comprises a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO: 1, 3 or 39.

    49. The method according to any one of claims 43-48, wherein the modified nucleic acid comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 1, 3 or 39.

    50. The method according to any one of claims 43-49, wherein the modified nucleic acid comprises a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 1, 3 or 39.

    51. The method according to any one of claims 43-50, wherein the modified nucleic acid comprises (preferably consists of) a nucleic acid sequence shown as SEQ ID NO: 1, 3 or 39.

    52. The method according to any one of claims 43-51, wherein the modified nucleic acid expresses a polypeptide comprising a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 2, 4, 26 or 40.

    53. The method according to any one of claims 43-52, wherein the modified nucleic acid expresses a polypeptide comprising a polypeptide sequence having at least 80% sequence identity to SEQ ID NO: 2, 4, 26 or 40.

    54. The method according to any one of claims 43-53, wherein the modified nucleic acid expresses a polypeptide comprising a polypeptide sequence having at least 90% sequence identity to SEQ ID NO: 2, 4, 26 or 40.

    55. The method according to any one of claims 43-54, wherein the modified nucleic acid expresses a polypeptide comprising (preferably consisting of) a polypeptide sequence shown as SEQ ID NO: 2, 4, 26 or 40.

    56. A method for preparing a labelled polypeptide, the method comprising: a. providing a polypeptide comprising: i. a transpeptidase or ligase acceptor site or a transpeptidase or ligase donor site; ii. a non-cytotoxic protease or a proteolytically inactive mutant thereof; iii. a Targeting Moiety (TM) that is capable of binding to a Binding Site on a target cell; and iv. a translocation domain; b. incubating the polypeptide with: a transpeptidase or ligase; and a labelled substrate comprising a transpeptidase or ligase donor site or a transpeptidase or ligase acceptor site, respectively, and a conjugated detectable label;  wherein the transpeptidase or ligase catalyses: conjugation between an amino acid of the transpeptidase or ligase acceptor site of the polypeptide and an amino acid of the transpeptidase or ligase donor site of the labelled substrate; or conjugation between an amino acid of the transpeptidase or ligase acceptor site of the labelled substrate and an amino acid of the transpeptidase or ligase donor site of the polypeptide;  thereby labelling the polypeptide; and c. obtaining the labelled polypeptide.

    57. The method according to claim 56, wherein the ligase is butelase, PATG, PCY1 or POPB.

    58. The method according to claim 56 or 57, wherein the ligase is butelase, preferably Butelase 1.

    59. A polypeptide for labelling using a butelase, the polypeptide comprising: i. a butelase acceptor or donor site; ii. a non-cytotoxic protease that is capable of cleaving a protein of the exocytic fusion apparatus in a target cell or a proteolytically inactive mutant thereof; iii. a Targeting Moiety (TM) that is capable of binding to a Binding Site on a target cell; and iv. a translocation domain that is capable of translocating the non-cytotoxic protease from within an endosome, across the endosomal membrane and into the cytosol of the target cell; wherein when the polypeptide comprises a butelase donor site, the butelase donor site is located at an N-terminus of the polypeptide; and wherein the N-terminal residue of the donor site is the N-terminal residue of the polypeptide; or wherein the polypeptide comprises one or more amino acid residues N-terminal to the butelase donor site and a cleavable site, which when cleaved exposes the N-terminus of the butelase donor site.

    60. A labelled polypeptide, the polypeptide comprising: i. a detectable label conjugated to the polypeptide; ii. an amino acid sequence that comprises Asn/Asp-Xaa-(Ile/Leu/Val/Cys), wherein Xaa is any amino acid apart from proline; iii. a non-cytotoxic protease or a proteolytically inactive mutant thereof; iv. a Targeting Moiety (TM) that is capable of binding to a Binding Site on a target cell; and v. a translocation domain.

    61. The method, polypeptide or labelled polypeptide according to any one of claims 1-37 or 43-60, wherein the detectable label is a fluorophore.

    62. The method, polypeptide or labelled polypeptide according to claim 61, wherein the fluorophore is selected from: HiLyte, AlexaFluor, Atto, Quantum Dots, and Janelia Fluor.

    63. The method or labelled polypeptide according to any one of claims 1, 3-37, 43-58 or 60-62, wherein the labelled polypeptide comprises two or more detectable labels.

    64. The method or labelled polypeptide according to claim 63, wherein the two or more detectable labels are different fluorophores.

    65. The method or polypeptide according to any one of claims 1-13, 23-31, 33-36, 43-55, or 61-64, wherein the sortase acceptor site comprises (or consists of) NPKTG, XPETG, LGATG, IPNTG, IPETG, NSKTA, NPQTG, NAKTN, NPQSS, LPXTX, wherein X is any amino acid, NPX.sub.1TX.sub.2, wherein X.sub.1 is Lys or Gln and X.sub.2 is Asn, Asp or Gly, X.sub.1PX.sub.2X.sub.3G, wherein X, is Leu, Ile, Val or Met, X.sub.2 is any amino acid and X.sub.3 is Ser, Thr or Ala, LPEX.sub.1G, wherein X.sub.1 is Ala, Cys or Ser, LPXS, LAXT, MPXT, MPXTG, LAXS, NPXT, NPXTG, NAXT, NAXTG, NAXS, NAXSG, LPXP, LPXPG, LRXTG or LPAXG wherein X is any amino acid.

    Description

    BRIEF DESCRIPTION OF THE DRAWINGS

    [0601] Embodiments of the invention will now be described, by way of example only, with reference to the following Figures and Examples.

    [0602] FIG. 1 shows a schematic representation of the dual-labelling strategy of liganded polypeptides. The protein contains a SrtA recognition site at the C-terminal followed by a Strep-tag. At the N-terminal the protein contains a stretch of glycine protected by TEV cleavage site. A peptide containing a stretch of glycine attached to a fluorophore of choice and a second peptide containing the SrtA recognition site and 6 His tag (HT) were also generated. The two different SrtA enzymes allow site-specific labelling of fluorophores of different colours at the N- and C-termini.

    [0603] FIG. 2 shows a SNAP-25 cleavage assay of unlabelled, single and dual-labelled polypeptides. A. SNAP-25 cleavage in cortical neurons by 3, 10, 30, 100, 300 and 1000 nM unlabelled EGF-liganded polypeptide, TxRed labelled EGF-polypeptide, SNAP594-labelled EGF-liganded polypeptide, single SrtA-mediated labelled EGF-liganded polypeptide and dual SrtA-labelled EGF-liganded polypeptide. As a control a polypeptide without the ligand (unliganded) was used for all concentrations. Exposure to the polypeptides was performed for 24 h. B. SNAP-25 cleavage in cortical neurons by 3, 10, 30, 100, 300 and 1000 nM unlabelled nociceptin-liganded polypeptide and dual SrtA-mediated labelled nociceptin-polypeptide. As a control a polypeptide without the ligand (unliganded) was used for all concentrations. Exposure to the polypeptides was performed for 24 h.

    [0604] FIG. 3 shows live confocal imaging of dual-labelled EGF-liganded polypeptide. A. Snapshot of confocal live imaging recording of A549 cells treated with an EGF-liganded polypeptide labelled with HF555 at the N-terminal and HF488 at the C-terminal. The images (right) are snapshots of the boxed area shown on large image (left) taken at different intervals starting from 0.5 minutes after addition of the protein. Formation of the agglomerates characteristic of this polypeptide can be seen from 3 minutes onwards. B. Snapshot of confocal live imaging recording of A549 cells treated with an EGF-liganded polypeptide labelled with HF555 at the N-terminal and HF488 at the C-terminal. The images (right) are snapshots of the boxed area shown on large image (left) taken at different intervals starting from 30 minutes after addition of the protein. Disappearance of the agglomerates can be seen from 45 minutes onwards.

    [0605] FIG. 4 shows a schematic representation of a dual-labelled full length proteolytically inactivate mutant of BoNT/A1, referred to as BoNT/A(0). The sortase donor and acceptor sites and protocol are the same as those of FIG. 1.

    [0606] FIG. 5 shows SDS-PAGE analysis of a dual-labelled proteolytically inactivated BoNT/A (BoNT/A(0)) imaged using fluorescence (left) and Coomassie staining (right). Lanes 1 and 4 show the protein ladder, lanes 2 and 5 non-reduced dual-labelled BoNT/A(0) and lanes 3 and 6 show reduced dual-labelled (L-chain bottom and H-chain top) BoNT/A(0).

    [0607] FIG. 6 shows timelapse single molecule TIRF microscopy images of single labelled BoNT/A(0) recorded at 5 second intervals. The white arrow shows the moving single molecule throughout time in seconds.

    SEQUENCE LISTING

    [0608] Where an initial Met amino acid residue or a corresponding initial codon is indicated in any of the following SEQ ID NOs, said residue/codon is optional. In the event of any differences between the sequences described in the description and those of the ST.25 Sequence Listing, the sequences in the description shall prevail.

    [0609] SEQ ID NO: 1—Nucleotide sequence of EGF-liganded (EGF TM) polypeptide with dual-labelling SrtA sites

    [0610] SEQ ID NO: 2—Polypeptide sequence of EGF-liganded (EGF TM) polypeptide with dual-labelling SrtA sites

    [0611] SEQ ID NO: 3—Nucleotide sequence of nociceptin-liganded (nociceptin TM) polypeptide with dual-labelling SrtA sites

    [0612] SEQ ID NO: 4—Polypeptide sequence of nociceptin-liganded (nociceptin TM) polypeptide with dual-labelling SrtA sites

    [0613] SEQ ID NO: 5—Nucleotide sequence of EGF-liganded (EGF TM) polypeptide

    [0614] SEQ ID NO: 6—Polypeptide sequence of EGF-liganded (EGF TM) polypeptide

    [0615] SEQ ID NO: 7—Nucleotide sequence of nociceptin-liganded (nociceptin TM) polypeptide

    [0616] SEQ ID NO: 8—Polypeptide sequence of nociceptin-liganded (nociceptin TM) polypeptide

    [0617] SEQ ID NO: 9—Nucleotide sequence of EGF-liganded polypeptide GFP-tagged

    [0618] SEQ ID NO: 10—Polypeptide sequence of EGF-liganded polypeptide GFP-tagged

    [0619] SEQ ID NO: 11—Nucleotide sequence of EGF-liganded polypeptide SNAP tagged

    [0620] SEQ ID NO: 12—Polypeptide sequence of EGF-liganded polypeptide SNAP tagged

    [0621] SEQ ID NO: 13—Nucleotide sequence of Sortase A (LPESG-targeting)

    [0622] SEQ ID NO: 14—Polypeptide sequence of Sortase A (LPESG-targeting)

    [0623] SEQ ID NO: 15—Nucleotide sequence of Sortase A (LAETG-targeting)

    [0624] SEQ ID NO: 16—Polypeptide sequence of Sortase A (LAETG-targeting)

    [0625] SEQ ID NO: 17—BoNT/A—UniProt P10845

    [0626] SEQ ID NO: 18—BoNT/B—UniProt P10844

    [0627] SEQ ID NO: 19—BoNT/C—UniProt P18640

    [0628] SEQ ID NO: 20—BoNT/D—UniProt P19321

    [0629] SEQ ID NO: 21—BoNT/E—UniProt Q00496

    [0630] SEQ ID NO: 22—BoNT/F—UniProt A7GBG3

    [0631] SEQ ID NO: 23—BoNT/G—UniProt Q60393

    [0632] SEQ ID NO: 24—Polypeptide Sequence of BoNT/X

    [0633] SEQ ID NO: 25—TeNT—UniProt P04958

    [0634] SEQ ID NO: 26—Polypeptide sequence of labelled EGF TM polypeptide

    [0635] SEQ ID NO: 27—Polypeptide sequence of C. ternatea butelase 1 (plus signal peptide)

    [0636] SEQ ID NO: 28—Polypeptide sequence of C. ternatea butelase 1 (minus signal peptide)

    [0637] SEQ ID NO: 29—Peptide with conjugated detectable label and sortase donor site

    [0638] SEQ ID NO: 30—Peptide with conjugated detectable label and sortase acceptor site

    [0639] SEQ ID NO: 31—Polypeptide sequence of Staphylococcus aureus Sortase A

    [0640] SEQ ID NO: 32—Polypeptide sequence of Staphylococcus aureus Sortase B

    [0641] SEQ ID NO: 33—Polypeptide sequence of Streptococcus pneumoniae Sortase A

    [0642] SEQ ID NO: 34—Polypeptide sequence of Streptococcus pneumoniae Sortase B

    [0643] SEQ ID NO: 35—Polypeptide sequence of Streptococcus pneumoniae Sortase C

    [0644] SEQ ID NO: 36—Polypeptide sequence of Streptococcus pneumoniae Sortase D

    [0645] SEQ ID NO: 37—Polypeptide sequence of Streptococcus pyogenes Sortase A

    [0646] SEQ ID NO: 38—Polypeptide sequence of proteolytically inactive mutant BoNT/A(0)

    [0647] SEQ ID NO: 39—Nucleotide sequence of full length proteolytically inactive mutant BoNT/A(0) with dual-labelling SrtA sites

    [0648] SEQ ID NO: 40—Polypeptide sequence of full length proteolytically inactive mutant BoNT/A(O) with dual-labelling SrtA sites

    [0649] SEQ ID NO: 41—Polypeptide sequence of Prochloron didemni PATG

    [0650] SEQ ID NO: 42—Polypeptide sequence of Saponaria vaccaria PCY1

    [0651] SEQ ID NO: 43—Polypeptide sequence of Galerina marginata POPB

    [0652] SEQ ID NO: 44—Polypeptide sequence of Oldenlandia affinis Butelase homologue OaAEP1b (plus signal peptide)

    [0653] SEQ ID NO: 45—Polypeptide sequence of Oldenlandia affinis Butelase homologue OaAEP1b (minus signal peptide)

    TABLE-US-00005 Nucleotide sequence of EGF−liganded polypeptide with dual−labelling SrtA sites SEQ ID NO: 1 TGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGA CCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTC GCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCA CCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTT TTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTC AACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAA TGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTAGGTGGCAC TTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGC TCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATT ATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATA GGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTC CCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGG CAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCAC TCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTA AAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATT TTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTA ACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAG TTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTC TGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCC ATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGT TGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGACCA AAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCT TGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGT TTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATAC CAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACA TACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTT GGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGC CCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACG CTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAG GGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGC GTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTA CGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATCGGCGATAATGGCCTGCTTCTCGCCGAAACGTT TGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGG CCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTG TCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACC GGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGC TAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCA TTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTC ACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTC CACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGC TGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATG GCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATT CAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCT GAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAAT GGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACC GTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAA CATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCA CTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCAT CGACAGGACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCG CGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCC ACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAAC GTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGT ATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCG CGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTA GGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAG ATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAG CCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTG TGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAATTAA TACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCAAGAAATAATTTTGTTTAACTTT AAGAAGGAGATATACATATgggatccatgGAGAACCTGTATTTTCAGGGCGGCGGTGGCAGCGGCGGC AGCGGCGGCAGCcctttcgttaacaaacagttcaactataaagacccagttaacggtgttgacattgc ttacatcaaaatcccgaacgctggccagatgcagccggtaaaggcattcaaaatccacaacaaaatct gggttatcccggaacgtgatacctttactaacccggaagaaggtgacctgaacccgccaccggaagcg aaacaggtgccggtatcttactatgactccacctacctgtctaccgataacgaaaaggacaactacct gaaaggtgttactaaactgttcgagcgtatttactccaccgacctgggccgtatgctgctgactagca tcgttcgcggtatcccgttctggggcggttctaccatcgataccgaactgaaagtaatcgacactaac tgcatcaacgttattcagccggacggttcctatcgttccgaagaactgaacctggtgatcatcggccc gtctgctgatatcatccagttcgagtgtaagagctttggtcacgaagttctgaacctcacccgtaacg gctacggttccactcagtacatccgtttctctccggacttcaccttcggttttgaagaatccctggaa gtagacacgaacccactgctgggcgctggtaaattcgcaactgatcctgcggttaccctggctcacga actgattcatgcaggccaccgcctgtacggtatcgccatcaatccgaaccgtgtcttcaaagttaaca ccaacgcgtattacgagatgtccggtctggaagttagcttcgaagaactgcgtacttttggcggtcac gacgctaaattcatcgactctctgcaagaaaacgagttccgtctgtactactataacaagttcaaaga tatcgcatccaccctgaacaaagcgaaatccatcgtgggtaccactgcttctctccagtacatgaaga acgtttttaaagaaaaatacctgctcagcgaagacacctccggcaaattctctgtagacaagttgaaa ttcgataaactttacaaaatgctgactgaaatttacaccgaagacaacttcgttaagttctttaaagt tctgaaccgcaaaacctatctgaacttcgacaaggcagtattcaaaatcaacatcgtgccgaaagtta actacactatctacgatggtttcaacctgcgtaacaccaacctggctgctaattttaacggccagaac acggaaatcaacaacatgaacttcacaaaactgaaaaacttcactggtctgttcgagttttacaagct gctgtgcgtcgacggcatcattacctccaaaactaaatctctgatagaaggtagaaacaaagcgctga acctgcagtgtatcaaggttaacaactgggatttattcttcagcccgagtgaagacaacttcaccaac gacctgaacaaaggtgaagaaatcacctcagatactaacatcgaagcagccgaagaaaacatctcgct agacctgatccagcagtactacctgacctttaatttcgacaacgagccggaaaacatttctatcgaaa acctgagctctgatatcatcggccagctggaactgatgccgaacatcgaacgtttcccaaacggtaaa aagtacgagctggacaaatataccatgttccactacctgcgcgcgcaggaatttgaacacggcaaatc ccgtatcgcactgactaactccgttaacgaagctctgctcaacccgtcccgtgtatacaccttcttct ctagcgactacgtgaaaaaggtcaacaaagcgactgaagctgcaatgttcttgggttgggttgaacag cttgtttatgattttaccgacgagacgtccgaagtatctactaccgacaaaattgcggatatcactat catcatcccgtacatcggtccggctctgaacattggcaacatgctgtacaaagacgacttcgttggcg cactgatcttctccggtgcggtgatcctgctggagttcatcccggaaatcgccatcccggtactgggc acctttgctctggtttcttacattgcaaacaaggttctgactgtacaaaccatcgacaacgcgctgag caaacgtaacgaaaaatgggatgaagtttacaaatatatcgtgaccaactggctggctaaggttaata ctcagatcgacctcatccgcaaaaaaatgaaagaagcactggaaaaccaggcggaagctaccaaggca atcattaactaccagtacaaccagtacaccgaggaagaaaaaaacaacatcaacttcaacatcgacga tctgtcctctaaactgaacgaatccatcaacaaagctatgatcaacatcaacaagttcctgaaccagt gctctgtaagctatctgatgaactccatgatcccgtacggtgttaaacgtctggaggacttcgatgcg tctctgaaagacgccctgctgaaatacatttacgacaaccgtggcactctgatcggtcaggttgatcg tctgaaggacaaagtgaacaataccttatcgaccgacatcccttttcagctcagtaaatatgtcgata accaacgccttttgtccactctagaaggcggTGGCGGTAGCGGTGGCGGTGGCAGCGGCGGTGGCGGT AGCGCACTAGacAACAGCGACCCTAAATGCCCACTgAGTCATGAAGGATACTGCCTTAATGATGGTGT TTGTATGTACATAGGAACATTGGACCGTTATGCTTGCAATTGTGTAGTGGGCTATGTCGGGGAAAGGT GTCAATATCGAGATCTCAAGCTGGCAGAGTTAAGAgggctagaagcaGGCGGCAGCGGCGGCGGCAGC GGCCTGCCCGAAAGCGGTGGCGGATCTGCTTGGTCTCACCCGCAGTTCGAAAAAGGTGGTGGTTCTGG TGGTGGTTCTGGTGGTTCTGCTTGGTCTCACCCGCAGTTCGAAAAAtaatgaAAGCTTGCGGCCGCAC TCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCT GCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTT GCTGAAAGGAGGAACTATATCCGGAT Polypeptide sequence of EGF-liganded polypeptide with dual-labelling SrtA sites SEQ ID NO: 2 MENLYFQGGGGSGGSGGSPFVNKQFKYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHKKIWVIPERDTF TNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWG GSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIR FSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSG LEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLL SEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFN LRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVDGIITSKTKSLIEGRNKALNLQCIKVNN WDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQ LELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVN KATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVI LLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKK MKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNS MIPYGVKRLEDFDASLKDALLKYIYDMRGTLIGQvDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTLE GGGGSGGGGSGGGGSALDNSDPKCPLSHEGYCLNDGVCMYIGTLDRYACNCWGYVGERCQYRDLKLA ELRGLEAGGSGGGSGLPESGGGSAWSHPQFEKGGGSGGGSGGSAWSHPQFEK Nucleotide sequence of nociceptin-liganded polypeptide with dual- labelling SrtA sites SEQ ID NO: 3 TGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGA CCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTC GCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCA CCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTT TTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTC AACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAA TGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTAGGTGGCAC TTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGC TCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATT ATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATA GGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTC CCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGG CAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCAC TCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTA AAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATT TTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTA ACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAG TTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTC TGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCC ATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGT TGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGACCA AAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCT TGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGT TTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATAC CAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACA TACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTT GGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGC CCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACG CTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAG GGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGC GTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTA CGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATCGGCGATAATGGCCTGCTTCTCGCCGAAACGTT TGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGG CCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTG TCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACC GGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGC TAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCA TTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTC ACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTC CACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGC TGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATG GCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATT CAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCT GAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAAT GGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACC GTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAA CATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCA CTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCAT CGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCG CGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCC ACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAAC GTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGT ATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCG CGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTA GGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAG ATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAG CCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTG TGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAATTAA TACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCAAGAAATAATTTTGTTTAACTTT AAGAAGGAGATATACATatgGAGAACCTGTATTTTCAGGGCGGCGGTGGCAGCGGCGGCAGCGGCGGC AGCGGCAGCATGcctTTTGTGAACAAACAGTTCAACTATAAGGATCCGGTTAATGGTGTGGATATCGC CTATATCAAAATTCCGAATGCAGGTCAGATGCAGCCGGTTAAAGCCTTTAAAATCCATAACAAAATTT GGGTGATTCCGGAACGTGATACCTTTACCAATCCGGAAGAAGGTGATCTGAATCCGCCTCCGGAAGCA AAACAGGTTCCGGTTAGCTATTATGATAGCACCTATCTGAGCACCGATAACGAGAAAGATAACTATCT GAAAGGTGTGACCAAACTGTTTGAACGCATTTATAGTACCGATCTGGGTCGTATGCTGCTGACCAGCA TTGTTCGTGGTATTCCGTTTTGGGGTGGTAGCACCATTGATACCGAACTGAAAGTTATTGACACCAAC TGCATTAATGTGATTCAGCCGGATGGTAGCTATCGTAGCGAAGAACTGAATCTGGTTATTATTGGTCC GAGCGCAGATATCATTCAGTTTGAATGTAAATCCTTTGGCCACGAAGTTCTGAATCTGACCCGTAATG GTTATGGTAGTACCCAGTATATTCGTTTCAGTCCGGATTTTACCTTTGGCTTTGAAGAAAGCCTGGAA GTTGATACAAATCCGCTGTTAGGTGCAGGTAAATTTGCAACCGATCCGGCAGTTACCCTGGCACATGA ACTGATTCATGCCGGTCATCGTCTGTATGGTATTGCAATTAATCCGAACCGTGTGTTCAAAGTGAATA CCAACGCATATTATGAAATGAGCGGTCTGGAAGTGTCATTTGAAGAACTGCGTACCTTTGGTGGTCAT GATGCCAAATTTATCGATAGCCTGCAAGAAAATGAATTTCGCCTGTACTACTATAACAAATTCAAGGA TATTGCGAGCACCCTGAATAAAGCCAAAAGCATTGTTGGCACCACCGCAAGCCTGCAGTATATGAAAA ATGTGTTTAAAGAAAAATATCTGCTGAGCGAAGATACCAGCGGTAAATTTAGCGTTGACAAACTGAAA TTCGATAAACTGTACAAGATGCTGACCGAGATTTATACCGAAGATAACTTCGTGAAGTTTTTCAAAGT GCTGAACCGCAAAACCTACCTGAACTTTGATAAAGCCGTGTTCAAAATCAACATCGTGCCGAAAGTGA ACTATACCATCTATGATGGTTTTAACCTGCGCAATACCAATCTGGCAGCAAACTTTAATGGTCAGAAC ACCGAAATCAACAACATGAACTTTACCAAACTGAAGAACTTCACCGGTCTGTTCGAATTTTACAAACT GCTGTGTGTGGATGGCATTATTACCAGCAAAACCAAATCCGATGATGACGATAAATTCGGTGGTTTTA CCGGTGCACGTAAAAGCGCACGTAAACGTAAAAATCAGGCACTGGCAGGCGGTGGTGGTAGCGGTGGC GGTGGTTCAGGTGGTGGTGGCTCAGCACTGGTTCTGCAGTGTATTAAAGTTAATAACTGGGACCTGTT TTTTAGCCCGAGCGAGGATAATTTCACCAACGATCTGAACAAAGGCGAAGAAATTACCAGCGATACCA ATATTGAAGCAGCCGAAGAAAACATTAGCCTGGATCTGATTCAGCAGTATTATCTGACCTTCAACTTC GATAATGAGCCGGAAAATATCAGCATTGAAAACCTGAGCAGCGATATTATTGGCCAGCTGGAkCTGAT GCCGAATATTGAACGTTTTCCGAACGGCAAAAAATACGAGCTGGATAAATACACCATGTTCCATTATC TGCGTGCCCAAGAATTTGAACATGGTAAAAGCCGTATTGCACTGACCAATAGCGTTAATGAAGCACTG CTGAACCCGAGCCGTGTTTATACCTTTTTTAGCAGCGATTACGTGAAAAAGGTTAACAAAGCAACCGA AGCAGCCATGTTTTTAGGTTGGGTTGAACAGCTGGTTTATGATTTCACCGATGAAACCAGCGAAGTTA GCACCACCGATAAAATTGCAGATATTACCATCATCATCCCGTATATCGGTCCGGCACTGAATATTGGC AATATGCTGTATAAAGACGATTTTGTGGGTGCCCTGATCTTTAGCGGTGCAGTTATTCTGCTGGAATT TATTCCGGAAATTGCCATTCCGGTTCTGGGCACCTTTGCACTGGTGAGCTATATTGCAAATAAAGTTC TGACCGTGCAGACCATCGATAATGCACTGAGCAAACGTAACGAAAAATGGGATGAAGTGTACAAGTAT ATCGTGACCAATTGGCTGGCAAAAGTTAACACCCAGATTGACCTGATTCGCAAGAAGATGAAAGAAGC ACTGGAAAACCAGGCAGAAGCAACCAAAGCCATTATTAACTATCAGTACAACCAGTACACCGAAGAAG AGAAGAATAACATCAACTTCAACATCGATGATCTGAGCAGCAAGCTGAATGAAAGCATCAACAAAGCC ATGATCAACATTAACAAATTTCTGAATCAGTGCAGCGTGAGCTATCTGATGAATAGCATGATTCCGTA TGGTGTGAAACGTCTGGAAGATTTTGATGCAAGCCTGAAAGATGCCCTGCTGAAATATATCTATGATA ATCGTGGCACCCTGATTGGTCAGGTTGATCGTCTGAAAGATAAAGTGAACAACACCCTGAGTACCGAT ATTCCTTTTCAGCTGAGCAAATATGTGGATAATCAGCGTCTGCTGAGTACCCTGGATGGCGGCAGCGG CGGCGGCAGCGGCCTGCCCGAAAGCGGTGGCGGATCTGCTTGGTCTCACCCGCAGTTCGAAAAAGGTG GTGGTTCTGGTGGTGGTTCTGGTGGTTCTGCTTGGTCTCACCCGCAGTTCGAAAAAtaatgaAAGCTT GCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGC TGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGA GGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT Polypeptide sequence of nociceptin-liganded polypeptide with dual- labelling SrtA sites SEQ ID NO: 4 MENLYFQGGGGSGGSGGSGSMPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPER DTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIP FWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQ YIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYE MSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEK YLLSFDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYD GFKLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVDGIITSKTKSDDDDKFGGFTGARKS ARKRKNQALAGGGGSGGGGSGGGGSALVLQCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAE ENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEF EHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKI ADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIFEIAIPVLGTFALVSYIANKVLTVQTI DNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNIM FNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLI GQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTLDGGSGGGSGLPESGGGSAWSHPQFEKGGG Nucleotide sequence of EGF-liganded polypeptide SEQ ID NO: 5 TGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGA CCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTC GCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCA CCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTT TTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTC AACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAA TGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTAGGTGGCAC TTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGC TCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATT ATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATA GGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTC CCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGG CAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCAC TCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTA AAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATT TTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTA ACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAG TTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTC TGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCC ATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGT TGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGACCA AAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCT TGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGT TTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATAC CAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACA TACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTT GGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGC CCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACG CTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAG GGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGC GTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTA CGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATCGGCGATAATGGCCTGCTTCTCGCCGAAACGTT TGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGG CCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTG TCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACC GGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGC TAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCA TTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTC ACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTC CACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGC TGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATG GCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATT CAGGATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCT GAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAAT GGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACC GTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAA CATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCA CTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCAT CGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCG CGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCC ACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAAC GTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGT ATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCG CGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTA GGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAG ATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAG CCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTG TGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAATTAA TACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCAAGAAATAATTTTGTTTAACTTT AAGAAGGAGATATACATATgggatccatggagttcgttaacaaacagttcaactataaagacccagtt aacggtgttgacattgcttacatcaaaatcccgaacgctggccagatgcagccggtaaaggcattcaa aatccacaacaaaatctgggttatcccggaacgtgatacctttactaacccggaagaaggtgacctga acccgccaccggaagcgaaacaggtgccggtatcttactatgactccacctacctgtctaccgataac gaaaaggacaactacctgaaaggtgttactaaactgttcgagcgtatttactccaccgacctgggccg tatgctgctgactagcatcgttcgcggtatcccgttctggggcggttctaccatcgataccgaactga aagtaatcgacactaactgcatcaacgttattcagccggacggttcctatcgttccgaagaactgaac ctggtgatcatcggcccgtctgctgatatcatccagttcgagtgtaagagctttggtcacgaagttct gaacctcacccgtaacggctacggttccactcagtacatccgtttctctccggacttcaccttcggtt ttgaagaatccctggaagtagacacgaacccactgctgggcgctggtaaattcgcaactgatcctgcg gttaccctggctcacgaactgattcatgcaggccaccgcctgtacggtatcgccatcaatccgaaccg tgtcttcaaagttaacaccaacgcgtattacgagatgtccggtctggaagttagcttcgaagaactgc gtacttttggcggtcacgacgctaaattcatcgactctctgcaagaaaacgagttccgtctgtactac tataacaagttcaaagatatcgcatccaccctgaacaaagcgaaatccatcgtgggtaccactgcttc tctccagtacatgaagaacgtttttaaagaaaaatacctgctcagcgaagacacctccggcaaattct ctgtagacaagttgaaattcgataaactttacaaaatgctgactgaaatttacaccgaagacaacttc gttaagttctttaaagttctgaaccgcaaaacctatctgaacttcgacaaggcagtattcaaaatcaa catcgtgccgaaagttaactacactatctacgatggtttcaacctgcgtaacaccaacctggctgcta attttaacggccagaacacggaaatcaacaacatgaacttcacaaaactgaaaaacttcactggtctg ttcgagttttacaagctgctgtgcgtcgacggcatcattacctccaaaactaaatctctgatagaagg tagaaacaaagcgctgaacctgcagtgtatcaaggttaacaactgggatttattcttcagcccgagtg aagacaacttcaccaacgacctgaacaaaggtgaagaaatcacctcagatactaacatcgaagcagcc gaagaaaacatctcgctggacctgatccagcagtactacctgacctttaatttcgacaacgagccgga aaacatttctatcgaaaacctgagctctgatatcatcggccagctggaactgatgccgaacatcgaac gtttcccaaacggtaaaaagtacgagctggacaaatataccatgttccactacctgcgcgcgcaggaa tttgaacacggcaaatcccgtatcgcactgactaactccgttaacgaagctctgctcaacccgtcccg tgtatacaccttcttctctagcgactacgtgaaaaaggtcaacaaagcgactgaagctgcaatgttct tgggttgggttgaacagcttgtttatgattttaccgacgagacgtccgaagtatctactaccgacaaa attgcggatatcactatcatcatcccgtacatcggtccggctctgaacattggcaacatgctgtacaa agacgacttcgttggcgcactgatcttctccggtgcggtgatcctgctggagttcatcccggaaatcg ccatcccggtactaggcacctttgctctggtttcttacattgcaaacaaggttctgactgtacaaacc atcgacaacgcgctgagcaaacgtaacgaaaaatgggatgaagtttacaaatatatcgtgaccaactg gctggctaaggttaatactcagatcgacctcatccgcaaaaaaatgaaagaagcactggaaaaccagg cggaagctaccaaggcaatcattaactaccagtacaaccagtacaccgaggaagaaaaaaacaacatc aacttcaacatcgacgatctgtcctctaaactgaacgaatccatcaacaaagctatgatcaacatcaa caagttcctgaaccagtgctctgtaagctatctgatgaactccatgatcccgtacggtgttaaacgtc tggaggacttcgatgcgtctctgaaagacgccctgctgaaatacatttacgacaaccgtggcactctg atcggtcaggttgatcgtctgaaggacaaagtgaacaataccttatcgaccgacatcccttttcagct cagtaaatatgtcgataaccaacgccttttgtccactctagaaggcggTGGCGGTAGCGGTGGCGGTG GCAGCGGCGGTGGCGGTAGCGCACTAGacAACAGCGACCCTAAATGCCCACTgAGTCATGAAGGATAC TGCCTTAATGATGGTGTTTGTATGTACATAGGAACATTGGACCGTTATGCTTGCAATTGTGTAGTGGG CTATGTCGGGGAAAGGTGTCAATATCGAGATCTCAAGCTGGCAGAGTTAAGAgggctagaagcaCACC ATCATCACcaccatcaccatcaccattaatgaAAGCTTGCGGCCGCACTCGAGCACCACCACCACCAC CACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATA ACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATAT CCGGAT Polypeptide sequence of EGF-liganded polypeptide SEQ ID NO: 6 MEFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQV PVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTKCIN VIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDT NPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAK FIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDK LYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEI NNMNFTKLKNFTGLFEFYKLLCVDGIITSKTKSLIEGRNKALNLQCIKVNNWDLFFSPSEDNFTNDLN KGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYE LDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVY DFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFA LVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIIN YQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLK DALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTLEGGGGSGGGGSGGGGSAL DNSDPKCPLSHEGYCLNDGVCMYIGTLDRYACNCVVGYVGERCQYRDLKLAELRGLEAHHHHHHHHHH Nucleotide sequence of nociceptin-liganded polypeptide SEQ ID NO: 7 TGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGA CCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTC GCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCA CCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTT TTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTC AACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAA TGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTAGGTGGCAC TTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGC TCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATT ATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATA GGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTC CCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGG CAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCAC TCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTA AAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATT TTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTA ACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAG TTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTC TGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCC ATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGT TGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGACCA AAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCT TGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGT TTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATAC CAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACA TACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTT GGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGC CCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACG CTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAG GGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGC GTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTA CGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATCGGCGATAATGGCCTGCTTCTCGCCGAAACGTT TGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGG CCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTG TCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACC GGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGC TAACTTACATTAATTGCGTTGCGCTGACTGCCCGCTTTCCAGTCGGGAAAGCTGTCGTGCCAGCTGCA TTAATGAATGGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTC ACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTC CACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGC TGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATG GCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATT CAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCT GAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAAT GGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACC GTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAA CATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCA CTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCAT CGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCG CGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCC ACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAAC GTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGT ATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCG CGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTA GGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAG ATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAG CCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTG TGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAATTAA TACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCAAGAAATAATTTTGTTTAACTTT AAGAAGGAGATATACATATGGGCAGCATGGAATTTGTGAACAAACAGTTCAACTATAAGGATCCGGTT AATGGTGTGGATATCGCCTATATCAAAATTCCGAATGCAGGTCAGATGCAGCCGGTTAAAGCCTTTAA TATTGCAAATAAAGTTCTGACCGTGCAGACCATCGATAATGCACTGAGCAAACGTAACGAAAAATGGG ATGAAGTGTACAAGTATATCGTGACCAATTGGCTGGCAAAAGTTAACACCCAGATTGACCTGATTCGC AAGAAGATGAAAGAAGCACTGGAAAACCAGGCAGAAGCAACCAAAGCCATTATTAACTATCAGTACAA CCAGTACACCGAAGAAGAGAAGAATAACATCAACTTCAACATCGATGATCTGAGCAGCAAGCTGAATG AAAGCATCAACAAAGCCATGATCAACATTAACAAATTTCTGAATCAGTGCAGCGTGAGCTATCTGATG AATAGCATGATTCCGTATGGTGTGAAACGTCTGGAAGATTTTGATGCAAGCCTGAAAGATGCCCTGCT GAAATATATCTATGATAATCGTGGCACCCTGATTGGTCAGGTTGATCGTCTGAAAGATAAAGTGAACA ACACCCTGAGTACCGATATTCCTTTTCAGCTGAGCAAATATGTGGATAATCAGCGTCTGCTGAGTACC CTGGATCATCATCACCATCACCACTAAAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTG AGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAG CATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGA T Polypeptide sequence of nociceptin-liganded polypeptide SEQ ID NO: 8 MGSMEFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTETNPEEGDLNPPPEA KQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTN CINVIQPDGSYRSEELNLVTIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLE VDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGH DAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLK FDKLYKMLTEIYTEDNEVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQN TEINNMNFTKLKNFTGLFEFYKLLCVDGIITSKTKSDDDDKFGGFTGARKSARKRKNQALAGGGGSGG GGSGGGGSALVLQCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNF DNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEAL LNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIG NMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKY IVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKA MININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTD IPFQLSKYVDNQRLLSTLDHHHHHH Nucleotide sequence of EGF-liganded polypeptide GFP-tagged SEQ ID NO: 9 TGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGA CCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTC GCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCA CCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTT TTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTC AACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAA TGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTAGGTGGCAC TTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGC TCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATT ATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATA GGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTC CCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGG CAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCAC TCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTA AAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATT TTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTA ACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAG TGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCC ATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGT TGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGACCA AAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCT TGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGT TTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATAC CAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACA TACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTT GGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGC CCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACG CTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAG GGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGC GTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTA CGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATCGGCGATAATGGCCTGCTTCTCGCCGAAACGTT TGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGG CCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTG TCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACC GGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGC TAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCA TTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTC ACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTC CACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGC TGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATG GCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATT CAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCT GAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAAT GGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACC GTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAA CATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCA CTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCAT CGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCG CGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCC ACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAAC GTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGT ATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCG CGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTA GGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAG ATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAG CCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTG TGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAATTAA TACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCAAGAAATAATTTTGTTTAACTTT AAGAAGGAGATATACATATgATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTG GTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCAC CTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCG TGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTC TTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTA CAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCG ACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTAT ATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGG CAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCG ACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTC CTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCACGGCATGGACGAGCTGTACAAGGGCGGCAGCGG CGGCGGCAGCGGCGGCggatccatggagttcgttaacaaacagttcaactataaagacccagttaacg gtgttgacattgcttacatcaaaatcccgaacgctggccagatgcagccggtaaaggcattcaaaatc cacaacaaaatctgggttatcccggaacgtgatacctttactaacccggaagaaggtgacctgaaccc gccaccggaagcgaaacaggtgccggtatcttactatgactccacctacctgtctaccgataacgaaa aggacaactacctgaaaggtgttactaaactgttcgagcgtatttactccaccgacctgggccgtatg ctgctgactagcatcgttcgcggtatcccgttctggggcggttctaccatcgataccgaactgaaagt aatcgacactaactgcatcaacgttattcagccggacggttcctatcgttccgaagaactgaacctgg tgatcatcggcccgtctgctgatatcatccagttcgagtgtaagagctttggtcacgaagttctgaac ctcacccgtaacggctacggttccactcagtacatccgtttctctccggacttcaccttcggttttga agaatccctggaagtagacacgaacccactgctgggcgctggtaaattcgcaactgatcctgcggtta ccctggctcacgaactgattcatgcaggccaccgcctgtacggtatcgccatcaatccgaaccgtgtc ttcaaagttaacaccaacgcgtattacgagatgtccggtctggaagttagcttcgaagaactgcgtac ttttggcggtcacgacgctaaattcatcgactctctgcaagaaaacgagttccgtctgtactactata acaagttcaaagatatcgcatccaccctgaacaaagcgaaatccatcgtgggtaccactgcttctctc cagtacatgaagaacgtttttaaagaaaaatacctgctcagcgaagacacctccggcaaattctctgt agacaagttgaaattcgataaactttacaaaatgctgactgaaatttacaccgaagacaacttcgtta agttctttaaagttctgaaccgcaaaacctatctgaacttcgacaaggcagtattcaaaatcaacatc gtgccgaaagttaactacactatctacgatggtttcaacctgcgtaacaccaacctggctgctaattt taacggccagaacacggaaatcaacaacatgaacttcacaaaactgaaaaacttcactggtctgttcg agttttacaagctgctgtgcgtcgacggcatcattacctccaaaactaaatctctgatagaaggtaga aacaaagcgctgaacctgcagtgtatcaaggttaacaactgggatttattcttcagcccgagtgaaga caacttcaccaacgacctgaacaaaggtgaagaaatcacctcagatactaacatcgaagcagccgaag aaaacatctcgctggacctgatccagcagtactacctgacctttaatttcgacaacgagccggaaaac atttctatcgaaaacctgagctctgatatcatcggccagctggaactgatgccaaacatcgaacgttt cccaaacggtaaaaagtacgagctggacaaatataccatgttccactacctgcgcgcgcaggaatttg aacacggcaaatcccgtatcgcactgactaactccgttaacgaagctctgctcaacccgtcccgtgta tacaccttcttctctagcgactacgtgaaaaaggtcaacaaagcgactgaagctgcaatgttcttggg ttgggttgaacagcttgtttatgattttaccgacgagacgtccgaagtatctactaccgacaaaattg cggatatcactatcatcatcccgtacatcggtccggctctgaacattggcaacatgctgtacaaagac gacttcgttggcgcactgatcttctccggtgcggtgatcctgctggsgttcatcccggaaatcgccat cccggtactgggcacctttgctctggtttcttacattgcaaacaaggttctgactgtacaaaccatcg acaacgcgctgagcaaacgtaacgaaaaatgggatgaagtttacaaatatatcgtgaccaactggctg gctaaggttaatactcagatcgacctcatccgcaaaaaaatgaaagaagcactggaaaaccaggcgga agctaccaaggcaatcattaactaccagtacaaccagtacaccgaggaagaaaaaaacaacatcaact tcaacatcgacgatctgtcctctaaactgaacgaatccatcaacaaagctatgatcaacatcaacaag ttcctgaaccagtgctctgtaagctatctgatgaactccatgatcccgtacggtgttaaacgtctgga ggacttcgatgcgtctctgaaagacgccctgctgaaatacatttacgacaaccgtggcactctgatcg gtcaggttgatcgtctgaaggacaaagtgaacaataccttatcgaccgacatcccttttcagctcagt aaatatgtcgataaccaacgccttttgtccactctagaaggcggTGGCGGTAGCGGTGGCGGTGGCAG CGGCGGTGGCGGTAGCGCACTAGacAACAGCGACCCTAAATGCCCACTaAGTCATGAAGGATACTGCC TTAATGATGGTGTTTGTATGTACATAGGAACATTGGACCGTTATGCTTGCAATTGTGTAGTGGGCTAT GTCGGGGAAAGGTGTCAATATCGAGATCTCAAGCTGGCAGAGTTAAGAgggctagaagcaCACCATCA TCACcaccatcaccatcaccattaatgaAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACT GAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTA GCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGG AT Polypeptide sequence of EGF-liganded polypeptide GFP-tagged SEQ ID NO: 10 MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYG VQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGN ILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTFIGDGPVLLPDNHYLST QSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYKGGSGGGSGGGSMEFVNKQFNYKDPVNGVDIAYI KIPNAGQMQPVKAFKIHNKIWVTPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKG VTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSA DIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELI HAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIA STLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLN RKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLC VDGIITSKTKSLIEGRNKALNLQCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDL IQQYYLTFNFDNEPENISIENLSSDIIGQLELMPMIERFPNGKKYELDKYTMFKYLRAQEFEHGKSRI ALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIII PYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKR NEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLS SKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKBALLKYIYDNRGTLIGQVDRLK DKVNNTLSTDIPFQLSKYVDNQRLLSTLEGGGGSGGGGSGGGGSALDNSDPKCPLSHEGYCLNDGVCM YIGTLDRYACNCWGYVGERCQYRDLKLAELRGLEAHHHHHHHHHH Nucleotide sequence of EGF-liganded polypeptide SNAP tagged SEQ ID NO: 11 TGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGA CCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTC GCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCA CCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTT TTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTC AACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAA TGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTAGGTGGCAC TTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGC TCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATT ATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATA GGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTC CCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGG CAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCAC TCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTA aaaggacaattacaaacaggaatcgaatgcaaccggcgcaggaacactgccagcgcatcaacaatatt TTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTA ACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAG TTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTC TGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCC ATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGT TGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGACCA AAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCT TGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGT TTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATAC CAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACA TACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTT GGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGC CCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACG CTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAG GGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGC GTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTA CGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATCGGCGATAATGGCCTGCTTCTCGCCGAAACGTT TGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGG CCGATGATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTG TCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACC GGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGC TAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCA TTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTC ACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTC CACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGC TGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATG GCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATT CAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCT GAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAAT GGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACC GTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAA cattagtgcaggcagcttccacagcaatggcatcctggtcatccagcggatagttaatgatcagccca CTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCAT CGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCG CGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCC ACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAAC GTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGT ATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCG CGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTA GGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAG ATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAG CCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTG TGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAATTAA TACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCAAGAAATAATTTTGTTTAACTTT AAGAAGGAGATATACATATgATGGACAAAGACTGCGAAATGAAGCGCACCACCCTGGATAGCCCTCTG GGCAAGCTGGAACTGTCTGGGTGCGAACAGGGCCTGCACCGTATCATCTTCCTGGGCAAAGGAACATC TGCCGCCGACGCCGTGGAAGTGCCTGCCCCAGCCGCCGTGCTGGGCGGACCAGAGCCACTGATGCAGG CCACCGCCTGGCTCAACGCCTACTTTCACCAGCCTGAGGCCATCGAGGAGTTCCCTGTGCCAGCCCTG CACCACCCAGTGTTCCAGCAGGAGAGCTTTACCCGCCAGGTGCTGTGGAAACTGCTGAAAGTGGTGAA GTTCGGAGAGGTCATCAGCTACAGCCACCTGGCCGCCCTGGCCGGCAATCCCGCCGCCACCGCCGCCG TGAAAACCGCCCTGAGCGGAAATCCCGTGCCCATTCTGATCCCCTGCCACCGGGTGGTGCAGGGCGAC CTGGACGTGGGGGGCTACGAGGGCGGGCTCGCCGTGAAAGAGTGGCTGCTGGCCCACGAGGGCCACAG ACTGGGCAAGCCTGGGCTGGGTGGCGGCAGCGGCGGCGGCAGCGGCGGCggatccatggagttcgtta acaaacagttcaactataaagacccagttaacggtgttgacattgcttacatcaaaatcccgaacgct ggccagatgcagccggtaaaggcattcaaaatccacaacaaaatctgggttatcccggaacgtgatac ctttactaacccggaagaaggtgacctgaacccgccaccggaagcgaaacaggtgccggtatcttact atgactccacctacctgtctaccgataacgaaaaggacaactacctgaaaggtgttactaaactgttc gagcgtatttactccaccgacctgggccgtatgctgctgactagcatcgttcgcggtatcccgttctg gggcggttctaccatcgataccgaactgaaagtaatcgacactaactgcatcaacgttattcagccgg acggttcctatcgttccgaagaactgaacctggtgatcatcggcccgtctgctgatatcatccagttc gagtgtaagagctttggtcacgaagttctgaacctcacccgtaacggctacggttccactcagtacat ccgtttctctccggacttcaccttcggttttgaagaatccctggaagtagacacgaacccactgctgg gcgctggtaaattcgcaactgatcctgcggttaccctggctcacgaactgattcatgcaggccaccgc ctgtacggtatcgccatcaatccgaaccgtgtcttcaaagttaacaccaacgcgtattacgagatgtc cggtctggaagttagcttcgaagaactgcgtacttttggcggtcacgacgctaaattcatcgactctc tgcaagaaaacgagttccgtctgtactactataacaagttcaaagatatcgcatccaccctgaacaaa gcgaaatccatcgtgggtaccactgcttctctccagtacatgaagaacgtttttaaagaaaaatacct gctcagcgaagacacctccggcaaattctctgtagacaagttgaaattcgataaactttacaaaatgc tgactgaaatttacaccgaagacaacttcgttaagttctttaaagttctgaaccgcaaaacctatctg aacttcgacaaggcagtattcaaaatcaacatcgtgccgaaagttaactacactatctacgatggttt caacctgcgtaacaccaacctggctgctaattttaacggccagaacacggaaatcaacaacatgaact tcacaaaactgaaaaacttcactggtctgttcgagttttacaagctgctgtgcgtcgacggcatcatt acctccaaaactaaatctctgatagaaggtagaaacaaagcgctgaacctgcagtgtatcaaggttaa caactgggatttattcttcagcccgagtgaagacaacttcaccaacgacctgaacaaaggtgaagaaa tcacctcagatactaacatcgaagcagccgaagaaaacatctcgctggacctgatccagcagtactac ctgacctttaatttcgacaacgagccggaaaacatttctatcgaaaacctgagctctgatatcatcgg ccagctggaactgatgccgaacatcgaacgtttcccaaacggtaaaaagtacgagctggacaaatata ccatgttccactacctgcgcgcgcaggaatttgaacacggcaaatcccgtatcgcactgactaactcc gttaacgaagctctgctcaacccgtcccgtgtatacaccttcttctctagcgactacgtgaaaaaggt caacaaagcgactgaagctgcaatgttcttgggttgggttgaacagcttgtttatgattttaccgacg agacgtccgaagtatctactaccgacaaaattgcggatatcactatcatcatcccgtacatcggtccg gctctgaacattggcaacatgctgtacaaagacgacttcgttggcgcactgatcttctccggtgcggt gatcctgctggagttcatcccggaaatcgccatcccggtactgggcacctttgctctggtttcttaca ttgcaaacaaggttctgactgtacaaaccatcgacaacgcgctgagcaaacgtaacgaaaaatgggat gaagtttacaaatatatcgtgaccaactggctggctaaggttaatactcagatcgacctcatccgcaa aaaaatgaaagaagcactggaaaaccaggcggaagctaccaaggcaatcattaactaccagtacaacc agtacaccgaggaagaaaaaaacaacatcaacttcaacatcgacgatctgtcctctaaactgaacgaa tccatcaacaaagctatgatcaacatcaacaagttcctgaaccagtgctctgtaagctatctgatgaa ctccatgatcccgtacggtgttaaacgtctggaggacttcgatgcgtctctgaaagacgccctgctga aatacatttacgacaaccgtggcactctgatcggtcaggttgatcgtctgaaggacaaagtgaacaat accttatcgaccgacatcccttttcagctcagtaaatatgtcgataaccaacgccttttgtccactct agaaggcggTGGCGGTAGCGGTGGCGGTGGCAGCGGCGGTGGCGGTAGCGCACTAGacAACAGCGACC CTAAATGCCCACTaAGTCATGAAGGATACTGCCTTAATGATGGTGTTTGTATGTACATAGGAACATTG GACCGTTATGCTTGCAATTGTGTAGTGGGCTATGTCGGGGAAAGGTGTCAATATCGAGATCTCAAGCT GGCAGAGTTAAGAgggctagaagcaCACCATCATCACcaccatcaccatcaccattaatgaAAGCTTG CGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCT GAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAG GGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT Polypeptide sequence of EGF-liganded polypeptide SNAP tagged SEQ ID NO: 12 MDKDCEMKRTTLDSPLGKLELSGCEQGLHRIIFLGKGTSAADAVEVPAPAAVLGGPEPLMQATAWLNA YFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLKVVKFGEVISYSHLAALAGNPAATAAVKTALSG NPVPILIPCHRVVQGDLDVGGYEGGLAVKEWLLAHEGHRLGKPGLGGGSGGGSGGGSMEFVNKQFNYK DPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLS TDKSKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSE ELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFAT DPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFR LYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTE DNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNF TGLFEFYKLLCVDGIITSKTKSLIEGRNKALNLQCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNI EAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLR AQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVST TDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLT VQTIDNALSKRNEKWDEVYKYIVTKWLAKVNTQIDLIRKKMKEALEMQAEATKAIINYQYNQYTEEEK NNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNR GTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTLEGGGGSGGGGSGGGGSALDNSDPKCPLSH EGYCLNDGVCMYIGTLDRYACNCVVGYVGERCQYRDLKLAELRGLEAHHHHHHHHHH Nucleotide sequence of Sortase A (LPESG-targeting) SEQ ID NO: 13 TGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGA CCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTC GCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCA CCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTT TTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTC AACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAA TGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTAGGTGGCAC TTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGC TCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATT ATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATA GGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTC CCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGG CAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCAC TCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTA AAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGGAGGAACACTGCCAGCGCATCAAGAATATT TTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTA ACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAG TTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTC TGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCC ATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGT TGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGACCA AAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCT TGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGT TTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATAC CAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACA TACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTT GGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGC CCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACG CTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAG GGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGC GTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTA CGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATCGGCGATAATGGCCTGCTTCTCGCCGAAACGTT TGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGG CCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTG TCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACC GGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGC TAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCA TTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTC ACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTC CACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGC TGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATG GCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATT CAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCT GAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAAT GGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACC GTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAA CATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCA CTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCAT CGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCG CGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCC ACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAAC GTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGT ATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCG CGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTA GGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAG ATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAG CCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTG TGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAATTAA TACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCAAGAAATAATTTTGTTTAACTTT AAGAAGGAGATATCATATGCAGGCAAAACCGCAGATTCCGAAAGATAAAAGCAAAGTGGCAGGCTATA TTGAAATTCCGGATGCCGATATTAAAGAACCGGTTTATCCGGGTCCTGCAACACGTGAACAGCTGGAT CGTGGTGTTTGTTTTGTTGAAGAAAATGAGAGCCTGGATGATCAGAACATTAGCATTACCGGTCATAC CGCAATTGATCGTCCGAATTATCAGTTTACCAATCTGCGTGCAGCCAAACCGGGTAGCATGGTTTATC TGAAAGTTGGTAATGAAACCCGCATCTACAAAATGACCAGCATTCGTAATGTTAAACCGACCGCAGTT GGTGTTCTGGATGAACAAAAAGGTAAAGATAAACAGCTGACCCTGGTTACCTGTGATGATTATAACTT TGAAACCGGTGTTTGGGAAACGCGCAAAATCTTTGTTGCAACCGAAGTTAAACATCACCATCACCACC ATCATCATCACCATTAAAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCT GCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCT TGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT Polypeptide sequence of Sortase A (LPESG-targeting) SEQ ID NO: 14 MQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATREQLDRGVCFVEENESLDDQNISITGHTAIDRP NYQFTNLRAAKPGSMVYLKVGNETRIYKMTSIRNVKPTAVGVLDEQKGKDKQLTLVTCDDYNFETGVW ETRKIFVATEVKHHHKHHHHHH Nucleotide sequence of Sortase A (LAETG-targeting) SEQ ID NO: 15 TGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGA CCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTC GCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCA CCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTT TTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTC AACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAA TGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTAGGTGGCAC TTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGC TCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATT ATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATA GGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTC CCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGG CAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCAC TCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTA AAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATT TTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTA ACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAG TTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTC TGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCC ATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGT TGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGACCA AAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCT TGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGT TTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATAC CAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACA TACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTT GGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGC CCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACG CTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAG GGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGC GTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTA CGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATCGGCGATAATGGCCTGCTTCTCGCCGAAACGTT TGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGG CCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTG TCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACC GGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGC TAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCA TTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTC ACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTC CACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGC TGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATG GCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATT CAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCT GAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAAT GGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACC GTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAA CATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCA CTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCAT CGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCG CGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCC ACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAAC GTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGT ATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCG CGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTA GGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAG ATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAG CCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTG TGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAATTAA TACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCAAGAAATAATTTTGTTTAACTTT AAGAAGGAGATATACATATGCAGGCAAAACCGCAGATTCCGAAAGATAAAAGCAAAGTGGCAGGCTAT ATTGAAATTCCGGATGCCGATATTAAAGAACCGGTTTATCCGGGTCCTGCAACACGTGAACAGCTGAA TCGTGGTGTTTGTTTTCACGATGAAAATGAGAGCCTGGATGATCAGAATATTAGCATTGCAGGCCATA CCTTTATTGATCGTCCGAATTATCAGTTCACCAATCTGAAAGCAGCAAAACCGGGTAGCATGGTTTAT TTCAAAGTTGGTAATGAAACCCGCATCTACAAAATGACCAGCATTCGTAAAGTTCATCCGAATGCAGT TGGTGTTCTGGATGAACAAGAAGGCAAAGATAAACAGCTGACCCTGGTTACCTGTGATGATTATAACG AAGAAACCGGTGTTTGGGAAAGCCGTAAAATCTTTGTTGCAACCGAAGTGAAACATCATCACCACCAT CACCATCATCATCACTAAAAGCTTGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGC TGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCC TTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT Polypeptide sequence of Sortase A (LAETG-targeting) SEQ ID NO: 16 MQAKPQIPKDKSKVAGYIEIPDADIKEPVYPGPATREQLKRGVCFHDENESLDDQNISIAGHTFIDRP NYQFTNLKAAKPGSMVYFKVGNETRIYKMTSIRKVHPNAVGVLDEQEGKDKQLTLVTCDDYNEETGVW ESRKIFVATEVKHHHHHHHHHH BoNT/A-UniProt P10845 SEQ ID NO: 17 MPFVNKQFNYKDPVNGVDIAYIKIPNVGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQV PVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCIN VIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDT NPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAK FIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDK LYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEI NNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLN KGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYE LDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVY DFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFA LVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIIN YQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLK DALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYE SNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFKLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKY FNSISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTIT NNPANNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLY DNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYLNSSLYR GTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSALEIPDVGNLSQVVVMK SKNDQGITNKCKMNLQDNNGNDIGFIGFHQFNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWG ERPL BoNT/B-UniProt P10844 SEQ ID NO: 18 MPVTINNFNYNDPIDNNNIIMMEPPFARGTGRYYKAFKITDRIWIIPERYTFGYKPEDFNKSSGIFNR DVCEYYDPDYLNTNDKKKIFLQTMIKLFNRIKSKPLGEKLLEMIIMGIPYLGDRRVPLEEFNTNIASV TVNKLISNPGEVERKKGIFANLIIFGPGPVLNENETIDIGIQNHFASREGFGGIMQMKFCPEYVSVFN NVQENKGASIFNRRGYFSDPALILMHELIKVLHGLYGIKVDDLPIVPNEKKFFMQSTDAIQAEELYTF GGQDPSIITPSTDKSIYDKVLQNFRGIVDRLNKVLVCISDPNININIYKNKFKDKYKFVEDSEGKYSI DVESFDKLYKSLMFGFTETNIAENYKIKTRASYFSDSLPPVKIKKLLDNEIYTIEEGFNISDKDMEKE YRGQNKAINKQAYEEISKEHLAVYKIQMCKSVKAPGICIDVDNEDLFFIADKNSFSDDLSKNERIEYN TQSNYIENDFPINELILDTDLISKIELPSENTESLTDFNVDVPVYEKQFAIKKIFTDEMTIFQYLYSQ TFPLDIRDISLTSSFDDALLFSNKVYSFFSMDYIKTANKVVEAGLFAGWVKQIVNDFVIEANKSNTMD KIADISLIVPYIGLALNVGNETAKGNFENAFEIAGASILLEFIPELLIPVVGAFLLESYIDNKNKIIK TIDNALTKRNEKWSDMYGLIVAQWLSTVNTQFYTIKEGMYKALNYQAQALEEIIKYRYNIYSEKEKSN INIDFNDINSKLKEGINQAIDNINNFINGCSVSYLMKKMIPLAVEKLLDFDNTLKKNLLNYIDENKLY LIGSAEYEKSKVNKYLKTIMPFDLSIYTNDTILIEMFNKYNSEILNNIILNLRYKDNNLIDLSGYGAK VEVYDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPKYKNDGIQNYIHNEYTI INCMKNNSGWKISIRGNRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVTITNNLNNAKIYING KLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTELSQSNIEERYKIQSYSEYLKDFW GNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSKYNQNSKYINYRDLYIGEKFIIRRKSNSQSI NDDIVRKEDYIYLDFFNLNQEWRVYTYKYFKKEEEKLFLAPISDSDEFYNTIQIKEYDEQPTYSCQLL FKKDEESTDEIGLIGIHRFYESGIVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTE BoNT/C-UniProt P18640 SEQ ID NO: 19 MPITINNFNYSDPVDNKNILYLDTHLNTLANEPEKAFRITGNIWVIPDRFSRNSNPNLNKPPRVTSPK SGYYDPNYLSTDSDKDPFLKEIIKLFKRINSREIGEELIYRLSTDIPFPGNNNTPINTFDFDVDFNSV DVKTRQGNNWVKTGSINPSVIITGPRENIIDPETSTFKLTNNTFAAQEGFGALSIISISPRFMLTYSN ATNDVGEGRFSKSEFCMDPILILMHELNHAMHNLYGIAIPNDQTISSVTSNIFYSQYNVKLEYAEIYA FGGPTIDLIPKSARKYFEEKALDYYRSIAKRLNSITTANPSSFNKYIGEYKQKLIRKYRFVVESSGEV TVNRNKFVELYNELTQIFTEFNYAKIYNVQNRKIYLSNVYTPVTANILDDNVYDIQNGFNIPKSNLNV LFMGQNLSRNPALRKVNPENMLYLFTKFCHKAIDGRSLYNKTLDCRELLVKNTDLPFIGDISDVKTDI FLRKDINEETEVIYYPDNVSVDQVILSKNTSEHGQLDLLYFSIDSESEILPGENQVFYDNRTQNVDYL NSYYYLESQKLSDNVEDFTFTRSIEEALDNSAKVYTYFPTLANKVNAGVQGGLFLMWANDVVEDFTTN ILRKDTLDKISDVSAIIPYIGPALNISMSVRRGNFTEAFAVTGVTILLEAFPEFTIPALGAFVIYSKV QERNEIIKTIDNCLEQRIKRWKDSYEWMMGTWLSRIITQFNNISYQMYDSLNYQAGAIKAKIDLEYKK YSGSDKENIKSQVENLKNSLDVKISEAMNNINKFIRECSVTYLFKNMLPKVIDELNEFDRNTKAKLIN LIDSHNIILVGEVDKLKAKVNNSFQNTIPFNIFSYTNNSLLKDIINEYFNNINDSKILSLQNRKNTLV DTSGYNAEVSEEGDVQLNPIFPFDFKLGSSGEDRGKVIVTQNENIVYNSMYESFSISFWIRINKWVSN LPGYTIIDSVKNNSGWSIGIISNFLVFTLKQNEDSEQSINFSYDISNNAPGYNKWFFVTVTNNMMGNM KIYINGKLIDTIKVKELTGINFSKTITFEINKIPDTGLITSDSDNINMWIRDFYIFAKELDGKDINIL FNSLQYTNVVKDYWGNDLRYNKEYYMVNIDYLNRYMYANSRQIVFNTRRNNNDFNEGYKIIIKRIRGN TNDTRVRGGDILYFDMTINNKAYNLFMKNETMYADNHSTEDIYAIGLREQTKDINDNIIFQIQPMNNT YYYASQIFKSNFNGENISGICSIGTYRFRLGGDWYRHNYLVPTVKQGNYASLLESTSTHWGFVPVSE BoNT/D-UniProt P19321 SEQ ID NO: 20 MTWPVKDFNYSDPVNDNDILYLRIPQNKLITTPVKAFMITQNIWVIPERFSSDTNPSLSKPPRPTSKY QSYYDPSYLSTDEQKDTFLKGIIKLFKRINERDIGKKLINYLVVGSPFMGDSSTPEDTFDFTRHTTNI AVEKFENGSWKVTNIITPSVLIFGPLPNILDYTASLTLQGQQSNPSFEGFGTLSILKVAPEFLLTFSD VTSNQSSAVLGKSIFCMDPVIALMHELTHSLHQLYGINIPSDKRIRPQVSEGFFSQDGPNVQFEELYT FGGLDVEIIPQIERSQLREKALGHYKDIAKRLNNINKTIPSSWISNIDKYKKIFSEKYNFDKDNTGNF VVNIDKFNSLYSDLTNVMSEVVYSSQYNVKNRTHYFSRHYLPVFANILDDNIYTIRDGFNLTNKGFNI ENSGQNIERNPALQKLSSESVVDLFTKVCLRLTKNSRDDSTCIKVKNNRLPYVADKDSISQEIFENKI ITDETNVQNYSDKFSLDESILDGQVPINPEIVDPLLPNVNMEPLNLPGEEIVFYDDITKYVDYLNSYY YLESQKLSNNVENITLTTSVEEALGYSNKIYTFLPSLAEKVNKGVQAGLFLNWANEVVEDFTTNIMKK DTLDKISDVSVIIPYIGPALNIGNSALRGNFNQAFATAGVAFLLEGFPEFTIPALGVFTFYSSIQERE KIIKTIENCLEQRVKRWKDSYQWMVSKWLSRITTQFNHINYQMYDSLSYQADAIKAKIDLEYKKYSGS DKENIKSQVENLKNSLDVKISEAMNNINKFIRECSVTYLFKNMLPKVIDELNKFDLRTKTELINLIDS HNIILVGEVDRLKAKVNESFENTMPFNIFSYTNNSLLKDIINEYFNSINDSKILSLQNKKNALVDTSG YNAEVRVGDMVQLNTIYTNDFKLSSSGDKIIVNLNNNILYSAIYENSSVSFWIKISKDLTNSHNEYTI INSIEQNSGWKLCIRNGNIEWILQDVNRKYKSLIFDYSESLSHTGYTNKWFFVTITNNIMGYMKLYIN GELKQSQKIEDLDEVKLDKTIVFGIDENIDENQMLWIRDFNIFSKELSNEDINIVYEGQILRNVIKDY WGKPLKFDTEYYIINDNYIDRYIAPESNVLVLVQYPDRSKLYTGNPITIKSVSDKNPYSRILNGDNII LHMLYNSRKYMIIRDTDTIYATQGGECSQNCVYALKLQSNLGNYGIGIFSIKNIVSKNKYCSQIFSSF RENTMLLADIYKPWRFSFKNAYTPVAVTNYETKLLSTSSFWKFISRDPGWVE BoNT/E-UniProt Q00496 SEQ ID NO: 21 MPKINSFNYNDPVNDRTILYIKPGGCQEFYKSFNIMKNIWIIPERNVIGTTPQDFHPPTSLKNGDSSY YDPNYLQSDEEKDRFLKIVTKIFNRINNWLSGGILLEELSKANPYLGNDNTPDNQFHIGDASAVEIKF SNGSQDILLPNVIIMGAEPDLFETNSSNISLRNNYMPSNHRFGSIAIVTFSPEYSFRFNDNCMNEFIQ DPALTLMHELIHSLHGLYGAKGITTKYTITQKQNPLITNIRGTNIEEFLTFGGTDLNIITSAQSNDIY TNLLADYKKIASKLSKVQVSNPLLNPYKDVFEAKYGLDKDASGIYSVNINKFNDIFKKLYSFTEFDLR TKFQVKCRQTYIGQYKYFKLSNLLNDSIYNISEGYNINNLKVNFRGQNANLNPRIITPITGRGLVKKI IRFCKNIVSVKGIRKSICIEINNGELFFVASENSYNDDNINTPKEIDDTVTSNNNYENDLDQVILNFN SESAPGLSDEKLNLTIQNDAYIPKYDSNGTSDIEQHDVNELNVFFYLDAQKVPEGENNVNLTSSIDTA LLEQPKIYTFFSSEFINNVNKPVQAALFVSWIQQVLVDFTTEANQKSTVDKIADISIVVFYIGLALNI GNEAQKGNFKDALELLGAGILLEFEPELLIPTILVFTIKSFLGSSDNKNKVIKAINNALKERDEKWKE VYSFIVSNWMTKINTQFNKRKEQMYQALQNQVNAIKTIIESKYNSYTLEEKNELTNKYDIKQIENELN QKVSIAMNNIDRFLTESSISYLMKIINEVKINKLREYDEMVKTYLLMYIIQHGSILGESQQELNSMVT DTLNNSIPFKLSSYTDDKILISYFNKFFKRIKSSSVLNMRYKNDKYVDTSGYDSNININGDVYKYPTN KNQFGIYNDKLSEVNISQNDYIIYDNKYKNFSISFWVRIPNYDNKIVNVNNEYTIINCMRDNNSGWKV SLNHNEIIWTFEDNRGINQKLAFNYGNANGISDYINKWIFVTITNDRLGDSKLYINGNLIDQKSILNL GNIHVSDMILFKIVNCSYTRYIGIRYFNIFDKELDETEIQTLYSNEPNTNILKDFWGNYLLYDKEYYL LNVXKPNNFIDRRKDSTLSINNIRSTILLANRLYSGIKVKIQRVNNSSTNDNLVRKNDQVYIKFVASK THLFPLYADTATTNKEKTIKISSSGNRFNQVVVMNSVGNCTMNFKNNNGNNIGLLGFKADTVVASTWY YTHMRDHTNSNGCFWNFISEEHGWQEK BoNT/F-UniProt A7GBG3 SEQ ID NO: 22 MPVVINSFNYNDPVNDDTILYMQIPYSEKSKKYYKAFEIMRNVWIIPERNTIGTDPSDFDPPASLENG SSAYYDPNYLTTDAEKDRYLKTTIKLFKRINSNPAGEVLLQEISYAKPYLGNEHTPINEFHPVTRTTS VNIKSSTNVKSSIILNLLVLGAGPDIFENSSYPVRKLMDSGGVYDPSNDGFGSINIVTFSPEYEYTFN DISGGYNSSTESFIADPAISLAHELIHALHGLYGARGVTYKETIKVKQAPLMIAEKPIRLEEFLTFGG QDLNIITSAMKEKIYNNLLANYEKIATRLSRVNSAPPEYDINEYKDYFQWKYGLDKNADGSYTVNENK FNEIYKKLYSFTEIDLANKFKVKCRNTYFIKYGFLKVPNLLDDDIYTVSEGFNIGKLAVNNRGQNIKL NPKIIDSIPDKGLVEKIVKFCKSVIPRKGTKAPPRLCIRVNNRELFFVASESSYNENDINTPKEIDDT TNLNNNYRNNLDEVILDYNSETIPQISNQTLNTLVQDDSYVPRYDSNGTSEIEEHNVVDLNVFFYLHA QKVPEGETNISLTSSIDTALSEESQVYTFFSSEFINTINKPVHAALFISWINQVIRDFTTEATQKSTF DKIADISLVVPYVGLALNIGNEVQKENFKEAFELLGAGILLEEVPELLIPTILVFTIKSFIGSSENKN KIIKAINNSLMERETKWKEIYSWIVSNWLTRINTQFNKRKEQMYQALQNQVDAIKTVIEYKYNNYTSD ERNRLESEYNINNIREELNKKVSLAMENIERFITESSIFYLMKLINEAKVSKLREYDEGVKEYLLDYI SEHRSILGNSVQELNDLVTSTLNNSIPFELSSYTHDKILILYFNKLYKKIKDNSILDMRYENNKFIDI SGYGSNISINGDVYIYSTNRNQFGIYSSKPSEVNIAQNNDIIYNGRYQNFSISFWVRIPKYFNKVNLN NEYTIIDCIRNKNSGWKISLNYNKIIWTLQDTAGNKQKLVFNYTQMISISDYINKWIFVTITNNRLGN SRIYINGNLIDEKSISNLGDIHVSDNILFKIVGCNDTRYVGIRYFKVFDTELGKTEIETLYSDEPDPS ILKDFWGNYLLYNKRYYLLNLLRTDKSITQNSNFLNINQQRGVYQKPNIFSNTRLYTGVEVIIRKNGS TDISNTDNFVRKNDLAYINVVDRDVEYRLYADISIAKPEKIIKLIRTSNSNNSLGQIIVMDSIGNNCT MNFQNNNGGNIGLLGFHSNNLVASSWYYNNIRKNTSSNGCFWSFISKEHGWQEN BoNT/G-UniProt Q60393 SEQ ID NO: 23 MPVNIKXFNYNDPINNDDIIMMEPFNDPGPGTYYKAFRIIDRIWIVPERFTYGFQPDQFNASTGVFSK DVYEYYDPTYLKTDAEKDKFLKTMIKLFNRINSKPSGQRLLDMIVDAIPYLGNASTPPDKFAANVANV SINKKIIQPGAEDQIKGLMTNLIIFGPGPVLSDNFTDSMIMNGHSPISEGFGARMMIRFCPSCLNVFN NVQENKDTSIFSRRAYFADPALTLMHELIHVLHGLYGIKISNLPITPNTKEFFMQHSDPVQAEELYTF GGHDPSVISPSTDMNIYNKALQNFQDIANRLNIVSSAQGSGIDISLYKQIYKNKYDFVEDPNGKYSVD KDKFDKLYKALMFGFTETNLAGEYGIKTRYSYFSEYLPPIKTEKLLDNTIYTQNEGFNIASKNLKTEF NGQNKAVNKEAYEEISLEKLVIYRIAMCKPVMYKNTGKSEQCIIVNNEDLFFIANKDSFSKDLAKAET IAYNTQNNTIENNFSIDQLILDNDLSSGIDLPNENTEPFTNFDDIDIPVYIKQSALKKIFVDGDSLFE YLHAQTFPSNIENLQLTNSLNDALRNNNKVYTFFSTNLVEKANTVVGASLFVNWVKGVIDDFTSESTQ KSTIDKVSDVSIIIPYIGPALNVGNETAKENFKNAFEIGGAAILMEFIPELIVPIVGFFTLESYVGNK GHIIMTISNALKKRDQKWTDMYGLIVSQWLSTVNTQFYTIKERMYNALNNQSQAIEKIIEDQYNRYSE EDKMNINIDFNDIDFKLNQSINLAINNIDDFINQCSISYLMNRMIPLAVKKLKDFDDNLKRDLLEYID TNELYLLDEVNILKSKVNRHLKDSIPFDLSLYTKDTILIQVFNNYISNISSNAILSLSYRGGRLIDSS GYGATMNVGSDVIFNDIGNGQFKLNNSENSNITAHQSKFVVYDSMFDNFSINFWVRTPKYNNNDIQTY LQNEYTIISCIKNDSGWKVSIKGNRIIWTLIDVNAKSKSIFFEYSIKDKISDYIKKWFSITITNDRLG NANIYINGSLKKSEKILNLDRINSSNDIDFKLINCTDTTKFVWIKDFNIFGRELNATEVSSLYWIQSS TNTLKDFWGKPLRYDTQYYLFNQGMQNIYIKYFSKASMGETAPRTNFNNAAINYQNLYLGLRFIIKKA SNSRNINNDNIVREGDYIYLNIDNISDESYRVYVLVNSKEIQTQLFLAPINDDPTFYDVLQIKKYYEK TTYNCQILCEKDTKTFGLFGIGKFVKDYGYVWDTYDNYFCISQWYLRRISEMINKLRLGCNWQFIPVD EGWTE Polypeptide Sequence of BoNT/X SEQ ID NO: 24 MKLSINKFNYNDPIDGINVITMRPPRHSDKINKGKGPFKAFQVIKNIWIVPERYNFTNNTNDLNIPSE PIMEADAIYNPNYLNTPSEKDEFLQGVIKVLERIKSKPEGEKLLELISSSIPLPLVSNGALTLSDNET IAYQENNNIVSNLQANLVIYGPGPDIANNATYGLYSTPISNGEGTLSEVSFSPFYLKPFDESYGNYRS LVNIVNKFVKREFAPDPASTLMHELVHVTHNLYGISNRNFYYNFDTGKIETSRQQNSLIFEELLTFGG IDSKAISSLIIKKIIETAKNNYTTLISERLNTVTVENDLLKYIKNKIPVQGRLGNFKLDTAEFEKKLN TILFVLNESNLAQRFSILVRKHYLKERPIDPIYVNILDDNSYSTLEGFNISSQGSNDFQGQLLESSYF EKIESNALRAFIKICPRNGLLYNAIYRNSKNYLNNIDLEDKKTTSKTNVSYPCSLLNGCIEVENKDLF LISNKDSLNDINLSEEKIKPETTVFFKDKLPPQDITLSNYDFTEANSIPSISQQNILERNEELYEPIR NSLFEIKTIYVDKLTTFHFLEAQNIDESIDSSKIRVELTDSVDEALSNPNKVYSPFKNMSNTINSIET GITSTYIFYQWLRSIVKDFSDETGKIDVIDKSSDTLAIVPYIGPLLNIGNDIRHGDFVGAIELAGITA LLEYVPEFTIPILVGLEVIGGELAREQVEAIVNNALDKRDQKWAEVYNITKAQWWGTIHLQINTRLAH TYKALSRQANAIKMNMEFQLANYKGNIDDKAKIKNAISETEILLNKSVEQAMKNTEKFMIKLSNSYLT KEMIPKVQDNLKNFDLETKKTLDKFIKEKEDILGTNLSSSLRRKVSIRLKKNIAFDINDIPFSEFDDL INQYKKEIEDYEVLNLGAEDGKIKDLSGTTSDINIGSDIELADGRENKAIKIKGSENSTIKIAMNKYL RFSATDNFSISFWIKHPKPTNLLNKGIEYTLVENFNQRGWKISIQDSKLIWYLRDHNNSIKIVTPDYI AFNGWNLITITNNRSKGSIVYVNGSKIEEKDISSIWNTEVDDPIIFRLKNNRDTQAFTLLDQFSIYRK ELNQNEVVKLYNYYFNSNYIRDIWGNPLQYNKKYYLQTQDKPGKGLIREYWSSFGYDYVILSDSKTIT FPNNIRYGALYNGSKVLIKNSKKLDGLVRNKDFIQLEIDGYNMGISADRFNEDTNYIGTTYGTTHDLT TDFEIIQRQEKYRNYCQLKTPYNIFHKSGLMSTETSKPTFHDYRDWVYSSAWYFQNYENLNLRKHTKT NWYFIPKDEGWDED TeNT-UniProt P04958 SEQ ID NO: 25 MPITINNFRYSDPVNNDTIIMMEPPYCKGLDIYYKAFKITDRIWIVPERYEFGTKPEDFNPPSSLIEG ASEYYDPNYLRTDSDKDFFLQTMVKLFNRIKNNVAGEALLDKIINAIPYLGNSYSLLDKFDTNSNSVS FNLLEQDPSGATTKSAMLTNLIIFGPGPVLNKNEVRGIVLRVDNKNYFPCRDGFGSIMQMAFCPEYVP TFDNVIENITSLTIGKSKYFQDPALLLMHELIHVLHGLYGMQVSSHEIIPSKQEIYMQHTYPISAEEL FTFGGQDANLISIDIKNDLYEKTLNDYKAIANKLSQVTSCNDPNIDIDSYKQIYQQKYQFDKDSNGQY IVNEDKFQILYNSIMYGFTEIELGKKFNIKTRLSYFSMNHDPVKIPNLLDDTIYNDTEGFNIESKDLK SEYKGQNMRVNTNAFRNVDGSGLVSKLIGLCKKIIPPTNIRENLYNRTASLTDLGGELCIKIKNEDLT FIAEKNSFSEEPFQDEIVSYNTKNKPLNFNYSLDKIIVDYNLQSKITLPNDRTTPVTKGIPYAPEYKS NAASTIElHNIDDNTIYQYLYAQKSPTTLQRITMTNSVDDALINSTKIYSYFPSVISKVNQGAQGILF LQWVRDIIDDFTNESSQKTTIDKISDVSTIVPYIGPALNIVKQGYEGNFIGALETTGVVLLLEYIPEI TLPVIAALSIAESSTQKEKIIKTIDNFLEKRYEKWIEVYKLVKAKWLGTVNTQFQKRSYQMYRSLEYQ VDAIKKIIDYEYKIYSGPDKEQIADEINNLKNKLEEKAKKAMININIFMRESSRSFLVNQMINEAKKQ LLEFDTQSKNILMQYIKANSKFIGITELKKLESKINKVFSTPIPFSYSKNLDCWVDNEEDIDVILKKS TILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTV SFWLRVPKVSASHLEQYGTNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLP DKFMAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDK FRIFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPS YTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRI LRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFN HLKDKILGCDWYFVPTDEGWTND Polypeptide sequence of labelled EGF TM polypeptide SEQ ID NO: 26 *HHHHHHLAETGGSGGSGGSEFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERD TFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPF WGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQY IRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEM SGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYKKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKY LLSEDTSGKF3VDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDG FNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVDGIITSKTKSLIEGRNKALNLQCIKV NNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDII GQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKK VNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGA VILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIR KKMKEALENQAEATKAIINYQYNQYTEEEKNNIKFNIDDLSSKLNESINKAMININKFLNQCSVSYLM NSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLST LEGGGGSGGGGSGGGGSALDNSDPKCPLSHEGYCLNDGVCMYIGTLDRYACNCVVGYVGERCQYRDLK LAELRGLEAGGSGGGSGLPESGK† * = HiLyte555; † = HiLyte488 Polupeptide sequence of C. ternatea butelase 1 (plus signal peptide) SEQ ID NO: 27 MKNPLAILFLIATVVAVVSGIRDDFLRLPSQASKFFQADDNVEGTRWAVLVAGSKGYVNYRHQADVCH AYQILKKGGLKDENIIVFMYDDIAYNESNPHPGVIINHPYGSDVYKGVPKDYVGEDINPPNFYAVLLA NKSALTGTGSGKVLDSGPNDHVFIYYTDHGGAGVLGMPSKPYIAASDLNDVLKKKHASGTYKSIVFYV ESCESGSFMDGLLPEDHNIYVMGASDTGESSWVTYCPLQHPSPPPEYDVCVGDLFSVAWLEDCDVHNL QTETFQQQYEVVKNKTIVALIEDGTHVVQYGDVGLSKQTLFVYMGTDPANDNNTFTDKNSLGTPRKAV SQRDADLIHYWEKYRRAPEGSSRKAEAKKQLREVMAHRMHIDNSVKHIGKLLFGIEKGHKMLNNVRPA GLPVVDDWDCFKTLIRTFETHCGSLSEYGMKHMRSFANLCNAGIRKEQMAEASAQACVSIPDNPWSSL HAGFSV Polypeptide sequence of C. ternatea butelase 1 (minus signal peptide) SEQ ID NO: 28 IRDDFLRLPSQASKFFQADDNVEGTRWAVLVAGSKGYVNYRHQADVCKAYQILKKGGLKDENIIVFMY DDIAYNESNPHPGVIINHPYGSDVYKGVPKDYVGEDINPPNFYAVLIANKSALTGTGSGKVLDSGPND HVFIYYTDHGGAGVLGMPSKPYIAASDLNDVLKKKHASGTYKSIVFYVESCESGSMFDGLLPEDHNIY VMGASDTGESSWVTYCPLQKPSPPPEYDVCVGDLFSVAWLEDCDVHNLQTETFQQQYEVVKNKTIVAL IEDGTHVVQYGDVGLSKQTLFVYMGTDPANDNNTFTDKNSLGTPRKAVSQRDADLIHYWEKYRRAPEG SSRKAEAKKQLREVMAHRMHIDNSVKHIGKLLFGIEKGHKMLNNVRPAGLPVVDDWDCFKTLIRTFET HCGSLSEYGMKHMRSFANLCNAGIRKEQMAEASAQACVSIPDNPWSSLHAGFSV Peptide with conjugated detectable label and sortase donor site SEQ ID NO: 29 GGGGK† † = HiLyte488 Peptide with conjugated detectable label and sortase acceptor site SEQ ID NO: 30 *HHHHHHLAETGGG * = HiLyte555 Polypeptide sequence of Staphylococcus aureus Sortase A SEQ ID NO: 31 MKKWTNRLMTIAGVVLILVAAYLFAKPHIDNYLHDKDKDEKTEQYDKNVKEQASKDKKQQAKPQIPKD KSKVAGYIEIPDADIKEPVYPGPATPEQLNRGVSFAEENESLDDQNISIAGKTFIDRPNYQFTNLKAA KKGSMVYFKVGNETRKYKMTSIRDVKPTDVGVLDEQKGKDKQLTLITCDDYNEKTGVWEKRKIFVATE VK Polypeptide sequence of Staphylococcus aureus Sortase B SEQ ID NO: 32 MRMKRFLTIVQILLVVTIIIFGYKIVQTYIEDKQERANYEKLQQKFQMLMSKHQEHVRPQFESLEKIN KDIVGWIKLSGTSLNYPVLQGKTNHDYLNLDFEREHRRKGSIFMDFRNSLKNLNHNTILYGHHVGDNT MFDVLEDYLKQSFYEKHKIIEFDNKYGKYQLQVFSAYKTTTKDNYIRTDFENDQDYQQFLDETKRKSV INSDVNVTVKDRIMTLSTCEDAYSETTKRIVVVAKIIKVS Polypeptide sequence of Streptococcus pneumoniae Sortase A SEQ ID NO: 33 MEKLYIHLKNLRKVAVVMLLVFTTFYLLLMFLNQSDNQEIAKNIEKFNDSVIVAKTDNTKADIKEIEK NIEKVRKIEGGNVERVNQLTSENEKVKENIDLNIEEEIIENSYKSLETTDNFEKLGIIEIPKIDLNLS IFKGKPFVNTKNRQDTMLYGAVTNKKNQKMGRENYVLASHIISNSNLLFTSINQLEKGDVTTLKDSEY SYQYTVYNNFIVSKDETWILNDIKDYSILTLYTCYDDSTKLPENRWIRAVLTDIN Polypeptide sequence of Streptococcus pneumoniae Sortase B SEQ ID NO: 34 MAKTKKQKRNNLLLGVVFFIGXAVMAYPLVSRLYYRVESNQQIADFDKEKATLDEADIDEPWKLAQAF NDSLNNVVSGDPWSEEMKKKGRAEYARMLEIHERMGHVEIPAIDVDLPVYAGTAEEVLQQGAGHLEGT SLPIGGNSTHAVITAHTGLPTAKMFTDLTKLKVGDKFYVHNIKEVMAYQVDQVKVIEPTNFDDLLIVP GHDYVTLLTCTPYMINTHRLLVRGHRIPYVAEVEEEFIAANKLSHLYRYLFYVAVGLIVILLWIIRRL RKKKRQSERALKALKEATKEVKVEDE wherein X is Met or Ile. Polypeptide sequence of Streptococcus pneumoniae Sortase C SEQ ID NO: 35 MDNSRRSRKKGTKKKKHPLILLLIFLVGFAVAIYPLVSRYYYRIESNEVIKEFDETVSQMDKAELEER WRLAQAFNATLKPSEILDPFTEQEKKKGVSEYANMLKVHERIGYVEIPAIDQEIPMYVGTSEDILQKG AGLLEGASLPVGGKNTHTVITAHRGLPTAELFSQLDKMKKGDIFYLHVLDQVLAYQVDQIVTVEPNDF EPVLIQHGEDYATLLTCTPYMINSHRLLVRGKRIPYTAPIAERMRAVRERGQFWLWLLLGAMAVILLL LYRVYRNRRIVKGLEKQLEGRHVKD Polypeptide sequence of Streptococcus pneumoniae Sortase D SEQ ID NO: 36 MSRTKLRALLGYLLMLVACLIPIYCFGQMVLQSLGQVKGHATFVKSMTTEMYQEQQNHSLAYNQRIAS QNRIVDPFLAEGYEVNYQVSDDPDAVYGYLSIPSLEIMEPVYLGADYHHLGMGLAHVDGTPLPMDGTG IRSVIAGHRAEPSHVFFRHLDQLKVGDALYYDNGQEIVEYQMMDTEIILPSEWEKLESVSSKNIMTLI TCDPIPTFNKRLLVNFERVAVYQKSDPQTAAVARVAFTKEGQSVSRVATSQWLYPGLVVIAFLGILFV LWKLARLLRGK Polypeptide sequence of Streptococcus pyogenes Sortase A SEQ ID NO: 37 MVKKQKRRKIKSMSWARKLLIAVLLILGLALLFNKPIRNTLIARNSNKYQVTKVSKKQIKKNKEAKST FDFQAVEPVSTESVLQAQMAAQQLPVIGGIAIPELGINLPIFKGLGNTELIYGAGTMKEEQVMGGENN YSLASHHIFGITGSSQMLFSPLERAQNGMSIYLTDKEKIYEYIIKDVFTVAPERVDVIDDTAGLKEVT LVTCTDIEATERIIVKGELKTEYDFDKAPADVLKAFNHSYNQVST Polypeptide sequence of proteolytically inactive mutant BoNT/A(0) SEQ ID NO: 38 MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQV PVSYYDSTYLSTDNSKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCIN VIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDT NPLLGAGKFATDPAVTLAHQLIYAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAK FIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDK LYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEI NNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLN KGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYE LDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVY DFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFA LVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIIN YQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLK DALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYE SNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKY FNSISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTIT NNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLY DNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYLNSSLYR GTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSALEIPDVGNLSQVVVMK SKNDQGITNKCKMNLQDNNGNDIGFIGFHQFNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWG ERPL Nucleotide sequence of full length proteolytically inactive mutant BoNT/A(0) with dual-labelling SrtA sites SEQ ID NO: 39 ATGGAGAACCTGTATTTTCAGGGCGGCGGTGGCAGCGGCGGCAGCGGCGGCAGCCCGTTTGTGAACAA GCAGTTCAACTATAAAGATCCGGTTAATGGTGTGGATATCGCCTATATCAAAATTCCGAATGCAGGTC AGATGCAGCCGGTTAAAGCCTTTAAAATCCATAACAAAATTTGGGTGATTCCGGAACGTGATACCTTT ACCAATCCGGAAGAAGGTGATCTGAATCCGCCTCCGGAAGCAAAACAGGTTCCGGTTAGCTATTATGA TAGCACCTATCTGAGCACCGATAACGAGAAAGATAACTATCTGAAAGGTGTGACCAAACTGTTTGAAC GCATTTATAGTACCGATCTGGGTCGTATGCTGCTGACCAGCATTGTTCGTGGTATTCCGTTTTGGGGT GGTAGCACCATTGATACCGAACTGAAAGTTATTGACACCAACTGCATTAATGTGATTCAGCCGGATGG TAGCTATCGTAGCGAAGAACTGAATCTGGTTATTATTGGTCCGAGCGCAGATATCATTCAGTTTGAAT GTAAAAGCTTTGGCCACGAAGTTCTGAATCTGACCCGTAATGGTTATGGTAGTACCCAGTATATTCGT TTCAGTCCGGATTTTACCTTTGGCTTTGAAGAAAGCCTGGAAGTTGATACAAATCCGCTGTTAGGTGC AGGTAAATTTGCAACCGATCCGGCAGTTACCCTGGCACACCAGCTGATTTATGCCGGTCATCGTCTGT ATGGTATTGCCATTAATCCGAATCGTGTGTTCAAAGTGAATACCAACGCCTATTATGAAATGAGCGGT CTGGAAGTGAGTTTTGAAGAACTGCGTACCTTTGGTGGTCATGATGCCAAATTTATCGATAGCCTGCA AGAAAATGAATTTCGCCTGTACTACTATAACAAATTCAAGGATATTGCGAGCACCCTGAATAAAGCCA AAAGCATTGTTGGCACCACCGCAAGCCTGCAGTATATGAAAAATGTGTTTAAAGAAAAATATCTGCTG AGCGAAGATACCAGCGGTAAATTTAGCGTTGACAAACTGAAATTCGATAAACTGTACAAGATGCTGAC CGAGATTTATACCGAAGATAACTTCGTGAAGTTTTTCAAAGTGCTGAACCGCAAAACCTACCTGAACT TTGATAAAGCCGTGTTCAAAATCAACATCGTGCCGAAAGTGAACTATACCATCTATGATGGTTTTAAC CTGCGCAATACCAATCTGGCAGCAAACTTTAATGGTCAGAACACCGAAATCAACAACATGAACTTTAC CAAACTGAAGAACTTCACCGGTCTGTTCGAATTTTACAAACTGCTGTGTGTTCGTGGCATTATTACCA GCAAAACCAAAAGTCTGGATAAAGGCTACAATAAAGCCCTGAATGATCTGTGCATTAAGGTGAATAAT TGGGACCTGTTTTTTAGCCCGAGCGAGGATAATTTCACCAACGATCTGAACAAAGGCGAAGAAATTAC CAGCGATACCAATATTGAAGCAGCCGAAGAAAACATTAGCCTGGATCTGATTCAGCAGTATTATCTGA CCTTCAACTTCGATAATGAGCCGGAAAATATCAGCATTGAA&ACCTGAGCAGCGATATTATTGGCCAG CTGGAACTGATGCCGAATATTGAACGTTTTCCGAACGGCAAAAAATACGAGCTGGATAAATACACCAT GTTCCATTATCTGCGTGCCCAAGAATTTGAACATGGTAAAAGCCGTATTGCACTGACCAATAGCGTTA ATGAAGCACTGCTCAACCCGAGCCGTGTTTATACCTTTTTTAGCAGCGATTACGTGAAAAAGGTTAAC AAAGCAACCGAAGCAGCCATGTTTTTAGGTTGGGTTGAACAGCTGGTTTATGATTTCACCGATGAAAC CAGCGAAGTTAGCACCACCGATAAAATTGCAGATATTACCATCATCATCCCGTATATCGGTCCGGCAC TGAATATTGGCAATATGCTGTATAAAGACGATTTTGTGGGTGCCCTGATTTTTAGCGGTGCAGTTATT CTGCTGGAATTTATTCCGGAAATTGCCATTCCGGTTCTGGGCACCTTTGCACTGGTGAGCTATATTGC AAATAAAGTTCTGACCGTGCAGACCATCGATAATGCACTGAGCAAACGTAACGAAAAATGGGATGAAG TGTACAAGTATATCGTGACCAATTGGCTGGCAAAAGTTAACACCCAGATTGACCTGATTCGCAAGAAG ATGAAAGAAGCACTGGAAAATCAGGCAGAAGCAACCAAAGCCATTATCAACTATCAGTATAACCAGTA CACCGAAGAAGAGAAAAATAACATCAACTTCAACATCGAGGATCTGTCCAGCAAACTGAACGAAAGCA TCAACAAAGCCATGATTAACATTAACAAATTTCTGAACCAGTGCAGCGTGAGCTATCTGATGAATAGC ATGATTCCGTATGGTGTGAAACGTCTGGAAGATTTTGATGCAAGCCTGAAAGATGCCCTGCTGAAATA TATCTATGATAATCGTGGCACCCTGATTGGTCAGGTTGATCGTCTGAAAGATAAAGTGAACAACACCC TGAGTACCGATATTCCTTTTCAGCTGAGCAAATATGTGGATAATCAGCGTCTGCTGTCAACCTTTACC GAATACATTAAGAACATCATCAACACCAGCATTCTGAACCTGCGTTATGAAAGCAATCATCTGATTGA TCTGAGCCGTTATGCCAGCAAAATCAATATAGGCAGCAAGGTTAACTTCGACCCGATTGACAAAAATC AGATACAGCTGTTTAATCTGGAAAGCAGCAAAATTGAGGTGATCCTGAAAAACGCCATTGTGTATAAT AGCATGTACGAGAATTTCTCGACCAGCTTTTGGATTCGTATCCCGAAATACTTTAATAGCATCAGCCT GAACAACGAGTACACCATTATTAACTGCATGGAAAACAATAGCGGCTGGAAAGTTAGCCTGAATTATG GCGAAATTATCTGGACCCTGCAGGATACCCAAGAAATCAAACAGCGTGTGGTTTTCAAATACAGCCAG ATGATTAATATCAGCGACTATATCAACCGCTGGATTTTTGTGACCATTACCAATAATCGCCTGAATAA CAGCAAGATCTATATTAACGGTCGTCTGATTGACCAGAAACCGATTAGTAATCTGGGTAATATTCATG CGAGCAACAACATCATGTTTAAACTGGATGGTTGTCGTGATACCCATCGTTATATTTGGATCAAGTAC TTCAACCTGTTCGATAAAGAGTTGAACGAAAAAGAAATTAAAGACCTGTATGATAACCAGAGCAACAG CGGTATTCTGAAGGATTTTTGGGGAGATTATCTGCAGTATGACAAACCGTATTATATGCTGAATCTGT ACGACCCGAATAAATACGTGGATGTGAATAATGTTGGCATCCGTGGTTATATGTACCTGAAAGGTCCG CGTGGTAGCGTTATGACCACAAACATTTATCTGAATAGCAGCCTGTATCGCGGAACCAAATTCATCAT TAAAAAGTATGCCAGCGGCAACAAGGATAATATTGTGCGTAATAATGATCGCGTGTACATTAACGTTG TGGTGAAGAATAAAGAATATCGCCTGGCAACCAATGCAAGCCAGGCAGGCGTTGAAAAAATTCTGAGT GCCCTGGAAATTCCGGATGTTGGTAATCTGAGCCAGGTTGTTGTGATGAAAAGCAAAAATGATCAGGG CATCACCAACAAGTGCAAAATGAATCTGCAGGACAATAACGGCAACGATATTGGTTTTATTGGCTTCC ACCAGTTCAACAATATTGCGAAACTGGTTGCAAGCAATTGGTATAATCGTCAGATTGAACGTAGCAGT CGTACCCTGGGTTGTAGCTGGGAATTTATCCCTGTGGATGATGGTTGGGGTGAACGTCCGCTGGGCGG CAGCGGCGGCGGCAGCGGCCTGCCCGAAAGCGGTGGCGGATCTGCTTGGTCTCACCCGCAGTTCGAAA AAGGTGGTGGTTCTGGTGGTGGTTCTGGTGGTTCTGCTTGGTCTCACCCGCAGTTCGAAAAATAATGA Polypeptide sequence of full length proteolytically inactive mutant BoNT/A(0) with dual-labelling SrtA sites SEQ ID NO: 40 MENLYFQGGGGSGGSGGSPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTF TNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWG GSTIDTELKVIDTNCINVIQPDGSYRSESLNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIR FSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHQLIYAGHRLYGIAINPNRVFKVNTNAYYEMSG LEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLL SEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFN LRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNN WDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQ LELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVN KATEAAMFLGWVEQLVYDFTDETSEVSTTBKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVI LLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKK MKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNS MIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFT EYIKNIINTSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYN SMYENFSTSFWIRIPKYFNSISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTQEIKQRVVFKYSQ MINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKY FNLFDKELNEKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGP RGSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILS ALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQFNNIAKLVASNWYNRQIERSS RTLGCSWEFIPVDDGWGERPLGGSGGGSGLPESGGGSAWSHPQFEKGGGSGGGSGGSAWSHPQFEK Polypeptide sequence of Prochloron didemni PATG SEQ ID NO: 41 MFSIMITIDYPFTVSLNRDIQVTSTEDYYTLQVTESDPSAWLTFATTPAMDMAFDHLKAGTTTESLVQ TLAELGGPAAREQFALTLQQLDERGWLSYAVLPLAEAIPMVESAELNLPGNPHWMETGVTLSRFAYQH PYEGTMVLESPLSKFRVKLLDWRASALLAQLAQPQTLGTIAPPPYLGPETAYQFLNLLWATGFLASDH EPVSLQLWDFHNLLFHSRSRLGRHDYPGTDLNVDNWSDFPVVKPPMSDRIVPLPRPNLEALMSNDATL TEAIETRKSVREYDDDNPITIEQLGELLYRAARVTKLLSPEERFGKLWQQNKPVFEEAGVDEGEFSHR PYPGGGAMYELEIYPVVRLCQGLSQGVYHYDPLNHQLEQIVESKDDIFAVSGSPLASKLGPHVLLVIT ARFGRLFRLYRSVAYALVLKHVGVLQQNLYLVATNMGLAPCAGGAGDSDAEAQVTGIDYVEESAVGEF ILGSLASEVESDVVEGEDEIESAGVSASEVESSATKQKVALHPHDLDERIPGLADLHNQTLGDPQITI VIIDGDPDYTLSCFEGAEVSKVFPYWHEPAEPITPEDYAAFQSIRDQGLKGKEKEEALEAVIPDTKDR IVLNDHACHVTSTIVGQEHSPVFGIAPNCRVINMPQDAVIRGNYDDVMSPLNLARAIDLALELGANII HCAFCRPTQTSEGEEILVQAIKKCQDNNVLIVSPTGNNSNESWCLPAVLPGTLAVGAAKVDGTPCHFS MWGGNNTKEGILAPGEEILGAQPCTEEPVRLTGTSMAAPVMTGISALLMSLQVQQGKPVDAEAVRTAL LKTAIPCDPEVVEEPERGLRGFVNIPGAMKVLFGQPSVTVSFAGGQATRTEHPGYATVAPASIPSPMA ERATPAVQAATATEMVIAPSTEPANPATVEASTAFSGNVYALGTIGYDFGDEARRDTFKERMADPYDA RQMVDYLDRNPDEARSLIWTLNLEGDVIYALDPKGPFATNVYEIFLQMLAGQLEPETSABFIERLSVP ARRTTRTVELFSGEVMPVVNVPDPRGMYGWNVNALVDAALATVEYEEADEDSLRQGLTAFLNRVYHDL HNLGQTSRDRALNFTVTNTFQAASTFAQAIASGRQLDTIEVNKSPYCRLNSDCWDVLLTFYDPEKGRR SRRVFRFTLDWYVLPVTVGSIKSWSLPGKGTVSK Polypeptide sequence of Saponaria vaccaria PCY1 SEQ ID NO: 42 MATSGFSKPLHYPPVRRDETVVDDYFGVKVADPYRWLEDPNSEETKEFVDNQEKLANSVLEECELIDK FKQKIIDFVNFPRCGVPFRRANKYFKFYNSGLQAQNVFQMQDDLDGKPEVLYDPNLREGGRSGLSLYS VSEDAKYFAFGIHSGLTEWVTIKILKTEDRSYLPDTLEWVKFSPAIWTHDNKGFFYCPYPPLKEGEDH MTRSAVNQEARYHFLGTDQSEDILLWRDLENPAHHLKCQITDDGKYFLLYILDGCDDANKVYCLDLTK LPNGLESFRGREDSAPFMKLIDSFDASYTAIANDGSVFTFQTNKDAPRKKLVRVDLNNPSVWTDLVPE SKKDLLESAHAVNENQLILRYLSDVKHVLEIRDLESGALQHRLPIDIGSVDGITARRRDSVVFFKFTS ILTPGIVYQCDLKNDPTQLKIFRESVVPDFDRSEFEVKQVFVPSKDGTKIPIFIAARKGISLDGSHPC EMHGYGGFGINMMPTFSASRIVFLKHLGGVFCLANIRGGGEYGEEWHKAGFRDKKQNVFDDFISAAEY LISSGYTKARRVAIEGGSNGGLLVAACINQRPDLFGCAEANCGVMDMLRFHKFTLGYLWTGDYGCSDK EEEFKWLIKYSPIHNVRRPWEQPGNEETQYPATMILTADHDDRVVPLHSFKLLATMQHVLCTSLEDSP QKNPIIARIQRKAAHYGRATMTQIAEVADRYGFMAKALEAPWID Polypeptide sequence of Galerina marginata POPB SEQ ID NO: 43 MSSVTWAPGNYPSTRRSDHVDTYQSASKGEVPVPDPYQWLEESTDEVDKWTTAQADLAQSYLDQNADI QKLAEKFRASRNYAKFSAPTLLDDGHWYWFYNRGLQSQSVLYRSKEPALPDFSKGDDNVGDVFFDPNV LAADGSAGMVLCKFSPDGKFFAYAVSHLGGDYSTIYVTSTSSPLSQASVAQGVDGRLSDEVKWFKFST IIWTKDSKGFLYQRYPARERHEGTRSDRNAMMCYHKVGTTQEEDIIVYQDNEHPEWIYGADTSEDGKY LYLYQFKDTSKKNLLWVAELDEDGVKSGIHWRKVVNEYAADYNIITNHGSLVYIKTNLNAPQYKVITI DLSKDEPElRDFIPEEKDAKLAQVNCANEEYFVAIYKRNVKDEIYLYSKAGVQLTRLAPDFVGAASIA NRQKQTHFFLTLSGFNTPGTIARYDFTAPETQRFSILRTTKVNELDPDDFESTQVWYESKDGTKIPMF IVRHKSTKFDGTAAAIQYGYGGFATSADPFFSPIILTFLQTYGAIFAVPSIRGGGEFGEEWHKGGRRE TKVNTFDDFIAAAQFLVKNKYAAPGKVAINGASNGGLLVMGSIVRAPEGTFGAAVPEGGYADLLKFHK FTGGQAWISEYGNPSIPEEFDYIYPLSPVHNVRTDKVMPATLITVNIGDGRVVPMHSFKFIATLQHNV PQNPHPLLIKIDKSWLGHGMGKPTDKNVKDAADKWGFIARALGLELKTVE Polypeptide sequence of Oldenlandia affinis Butelase homologue OaAEP1b (plus signal peptide) SEQ ID NO: 44 MVRYLAGAVLLLVVLSVAAAVSGARDGDYLHLPSEVSRFFRPQETNDDHGEDSVGTRWAVLIAGSKGY ANYRHQAGVCHAYQILKRGrGLKDENIVVFMYDDIAYNESNPRPGVIINSPHGSDVYAGVPKDYTGEE VNAKNFLAAILGNKSAITGGSGKVVDSGPNDHIFIYYTDHGAAGVIGMPSKPYLYADELNDALKKKHA SGTYKSLVFYLEACESGSMFEGILPEDLNIYALTSTNTTESSWCYYCPAQENPPPPEYWVCLGDLFSV AWLEDSDVQNSWYETLNQQYHHVDKRISHASHATQYGNLKLGEEGLFVYMGSNPANDNYTSLDGNALT PSSIVVNQRDADLLHLWEKFRKAPEGSARKEVAQTQIFKAMSKRVHIDSSIKLIGKLLFGIEKCTEIL NAVRPAGQPLVDDWACLRSLVGTFETHCGSLSEYGMRHTRTIANICNAGISEEQMAEAASQACASIP Polypeptide sequence of Oldenlandia affinis Butelase homologue OaAEP1b (minus signal peptide) SEQ ID NO: 45 ARDGDYLHLPSEVSRFFRPQETNDDHGEDSVGTRWAVLIAGSKGYANYRHQAGVCHAYQILKRGGLKD ENIVVFMYDDIAYNESNPRPGVIINSPHGSDVYAGVPKDYTGEEVNAKNFLAAILGNKSAITGGSGKV VDSGPNDHIFIYYTDHGAAGVIGMPSKPYLYADELNDALKKKHASGTYKSLVFYLEACESGSMFEGIL PEDLNIYALTSTNTTESSWCYYCPAQENPPPPEYNVCLGDLFSVAWLEDSDVQNSWYETLNQQYHHVD KRISHASHATQYGNLKLGEEGLFVYMGSNPANDNYTSLDGNALTPSSIVVNQRDADLLHLWEKFRKAP EGSARKEVAQTQIFKAMSHRVHIDSSIKLIGKLLFGIEKCTEILNAVRPAGQPLVDDWACLRSLVGTF ETHCGSLSEYGMRHTRTIANICNAGISEEQMAEAASQACASIP

    EXAMPLES

    Example 1

    [0654] Design of Texas Red, eGFP, SNAP and SrtA-Mediated Single and Dual Labelled EGF-Liganded Polypeptide

    [0655] Several strategies for the labelling of polypeptides were attempted. The aim was to obtain a labelled version of the polypeptide which did not affect its structural characteristics and its ability to traffic into cells and cleave SNARE proteins effectively and in a similar manner to the unlabelled version.

    [0656] 4 different labelling strategies of an EGF-liganded polypeptide (Fonfria, E., S. Donald and V. A. Cadd (2016). “Botulinum neurotoxin A and an engineered derivate targeted secretion inhibitor (TSI) A enter cells via different vesicular compartments.” J Recept Signal Transduct Res 36(1): 79-88) were attempted. Following cloning, when necessary, the polypeptide was recombinantly expressed and purified using standard procedures, as previously published (Masuyer, G., M. Beard, V. A. Cadd, J. A. Chaddock and K. R. Acharya (2011). “Structure and activity of a functional derivative of Clostridium botulinum neurotoxin B.” J Struct Biol 174(1): 52-57, Somm, E., N. Bonnet, A. Martinez, P. M. Marks, V. A. Cadd, M. Elliott, A. Toulotte, S. L. Ferrari, R. Rizzoli, P. S. Huppi, E. Harper, S. Melmed, R. Jones and M. L. Aubert (2012). “A botulinum toxin-derived targeted secretion inhibitor downregulates the GH/IGF1 axis.” J Clin Invest 122(9): 3295-3306). Briefly, the polypeptide was expressed recombinantly in E. coli competent bacteria. The expressed polypeptide was purified using an affinity column followed by anion exchange chromatography, enzymatic activation to generate a di-chain complex and finally a polishing step using hydrophobic interaction. [0657] 1. Unmodified EGF-liganded polypeptide, purified as described above was labelled using the Texas Red-X Protein Labelling Kit (Thermo Fisher Scientific) according to the manufacturer's protocol. Successful labelling of the protein was confirmed by confocal microscopy and live imaging. The nucleotide and polypeptide sequences for the polypeptide used for labelling are shown as SEQ ID NOs: 5 and 6, respectively. [0658] 2. EGF-liganded polypeptide was tagged at the N-terminal with an enhanced green fluorescent protein (eGFP) by standard cloning procedures. The nucleotide and polypeptide sequences are shown as SEQ ID NOs: 9 and 10, respectively. Protein expression and purification was performed as indicated above. After expression, purification of the eGFP-tagged EGF-liganded polypeptide was attempted unsuccessfully. [0659] 3. EGF-liganded polypeptide was tagged at the N-terminal with a SNAP-tag substrate (New England Biolabs) by standard cloning procedures. The nucleotide and polypeptide sequences are shown as SEQ ID NOs: 11 and 12, respectively. Expression and purification of this protein was successful. Labelling of the SNAP-tagged EGF-liganded polypeptide was performed using SNAP-Surface 594 fluorescent substrate (New England Biolabs) according to the manufacturer's protocol. Successful labelling of the protein was confirmed by confocal microscopy and live imaging. [0660] 4. Attempts were also made to generate polypeptides containing non-natural amino acids for site-specific labelling. However, these attempts were unsuccessful due to expression and/or purification difficulties. [0661] 5. EGF-liganded polypeptide (i.e. a polypeptide having an EGF TM) was tagged with two different Sortase A (SrtA) recognition sites, one at the N-terminus and one at the C-terminus. The use of SrtA allowed conjugation of two fluorophores of different colours on the same protein. The polypeptide was constructed as illustrated in FIG. 1. Two mutated versions of SrtA (Dorr, B. M., H. O. Ham, C. An, E. L. Chaikof and D. R. Liu (2014). “Reprogramming the specificity of sortase enzymes.” Proc Natl Acad Sci USA 111(37): 13343-13348) were chosen (SEQ ID NOs: 14 and 16). These have been shown to be 100% specific for their respective recognition sites. The EGF-liganded polypeptide was cloned with the LPESG recognition site of the first SrtA at the C-terminal, followed by a double StrepTag recognition site (IBA-lifesciences) which allows the initial affinity-mediated purification of the protein. The nucleotide and polypeptide sequences are shown as SEQ ID NOs: 1 and 2, respectively. Separately, a peptide containing a stretch of glycine residues conjugated to a fluorophore of choice was obtained (Eurogentec). The sequence of this peptide was: GGGGK(HF488) (SEQ ID NO: 29). During the SrtA-mediated reaction, the glycine of the LPESG site was cleaved by SrtA (SEQ ID NO: 14) and the stretch of glycines present on the fluorescent peptide recognized by SrtA and used to mediate the conjugation between the polypeptide and the peptide. This generated a fluorescently single-labelled EGF-liganded polypeptide. To note is the fact that the labelled polypeptide no longer possessed the StrepTag and a reverse affinity-mediated purification step was used to select the labelled portion of the polypeptide. For dual-labelling the EGF-liganded polypeptide, a stretch of 3 glycine residues was cloned at the N-terminal site of the polypeptide following the starting codon and a Tobacco Etch Virus (TEV) cleavage recognition site. The TEV site was introduced to help protect the stretch of glycine residues from protein circularization during the initial C-terminal SrtA reaction detailed above. Separately, a peptide containing the LAETG recognition site conjugated to a fluorophore of choice was obtained (Eurogentec). The sequence of this peptide was: HiLyte Fluor™ 555-HHHHHHLAETGGG (SEQ ID NO: 30). In addition, a 6 His-Tag (6HT) was positioned before the LAETG site for ease of protein purification following SrtA reaction (SEQ ID NO: 16). The SrtA reaction was conducted similarly to the C-terminal site and the final dual-labelled EGF-liganded protein was purified using a His affinity purification step. Successful single- and dual-labelling of the protein was confirmed by SDS-PAGE gel electrophoresis, confocal microscopy and live imaging.

    [0662] Sortase A (SrtA) proteins possessing a C-terminal His Tag were expressed in competent E. coli bacteria and purified using an affinity capture column.

    [0663] Sortase conjugation of the polypeptide and the fluorescent peptides was performed overnight at 4° C. using a ratio of 1 to 2 to 20 equivalents of polypeptide to SrtA to fluorescent peptide, respectively.

    [0664] In the present Example, the EGF-liganded polypeptide was conjugated with a HiLyte 555 fluorophore at the C-terminal translocation-ligand portion and a HiLyte 488 fluorophore at the N-terminal light chain portion. The expression of the polypeptide containing the SrtA recognition sites and the two variants of SrtA was successful. Advantageously, by generating a polypeptide capable of being labelled with two different colour fluorophores, the trafficking mechanisms of both the light-chain (containing the non-cytotoxic protease) and the translocation-ligand portions of the protein could be visualised.

    Example 2

    Design of SrtA-Mediated Dual Labelled Nociceptin-Liganded Polypeptide

    [0665] A polypeptide possessing a nociceptin ligand TM (nociceptin-liganded polypeptide) was generated for dual fluorescent-labelling using the strategy used for the EGF-liganded polypeptide. The design, purification and fluorescent peptides used for the dual-labelling of this polypeptide were exactly the same as for the EGF-liganded polypeptide. Successful dual-labelling of the polypeptide was confirmed by SDS-PAGE gel electrophoresis, confocal microscopy and live imaging. The nucleotide and polypeptide sequences for the polypeptide containing the sortase sites are shown as SEQ ID NOs: 3 and 4, respectively.

    [0666] Validation of the Labelled Proteins Using SNAP25 Cleavage Assay

    [0667] In order to determine that labelling of the liganded polypeptides does not affect their ability to bind to their respective receptors, trafficking into cells and translocation, a SNAP25 cleavage assay was performed to determine the relative potency of the labelled polypeptides compared to the unlabelled versions. A similar potency profile would suggest that the labelled polypeptide is trafficked similarly to the unlabelled version. The SNAP25 cleavage assay was performed as described previously (Fonfria, E., S. Donald and V. A. Cadd (2016). “Botulinum neurotoxin A and an engineered derivate targeted secretion inhibitor (TSI) A enter cells via different vesicular compartments.” J Recept Signal Transduct Res 36(1): 79-88). Briefly, cortical neurons were treated with 3-1000 nM of each labelled and unlabelled protein for 24 hours. Following treatment, cells were harvested in NuPAGE lysis buffer (Thermo Fischer Scientific) supplemented with 0.1M dithiothreitol and 250 units/ml benzonase (Sigma). Lysates were separated by SDS-PAGE and subjected to Western blotting using primary antibodies against SNAP-25 (Sigma). These antibodies enable recognition of both the cleaved and uncleaved portion of SNAP25. Relative potency was determined by the proportion of cleaved SNAP25 versus uncleaved SNAP25 (FIG. 2). FIG. 2A shows the dose response potency of the EGF-liganded polypeptide. In comparison to the unlabelled polypeptide, the Texas Red and SNAP594 labelled versions showed a strong reduction in potency with values similar to the unliganded control polypeptide. In contrast, the SrtA-mediated single and dual-labelled polypeptides showed similar potencies to the unlabelled version demonstrating that this labelling strategy does not affect the protein architecture and its cellular trafficking mechanisms. Similarly, dual-labelling of the nociception-liganded polypeptide did not affect its potency in cortical neurons (FIG. 2B) compared to the unlabelled control polypeptide.

    [0668] In summary, simple and straightforward tagging techniques such as non-site specific labelling using a Texas Red dye and a SNAP Tag, site specific version were initially trialled. However, although these labelling strategies were successful they were shown to affect the potency of the polypeptides when compared to the unlabelled counterpart suggesting that the addition of several fluorescent molecules, in the case of Texas Red or a SNAP tag affected the trafficking properties of the labelled polypeptide. An attempt at generating an eGFP-tagged EGF-liganded polypeptide was unsuccessful due to the lack of expression of the tagged protein. In stark contrast SNAP25 cleavage assays confirm that the addition of the two fluorophores on the EGF-liganded and nociception-liganded polypeptides did not affect their potencies suggesting that the mechanisms of actions of the labelled polypeptides are similar to their unlabelled counterparts. This was surprising in view of the negative impact SNAP and Texas Red labelling had on potency.

    Example 3

    Visualization of a Dual-Labelled EGF-Liganded Polypeptide in Immortalized Cell Lines

    [0669] The dual-labelling SrtA-mediated technique was chosen as an optimal strategy for the labelling of polypeptides of the invention. In order to visualize the labelled polypeptide in mammalian cells, 3D live confocal microscopy was performed. Human adenocarcinoma lung cells (A549) were treated with 50 nM dual-labelled EGF-liganded polypeptide and imaged continuously over time using a Zeiss 880 confocal microscope equipped with AiryScan (Zeiss). For these experiments, the EGF-liganded polypeptide was labelled at the N-terminal with a HiLyte 555 fluorophore (AnaSpec) and at the C-terminal with a HiLyte 488 fluorophore (AnaSpec). FIG. 3 shows snapshot images of the dual-coloured agglomerates formed by the EGF-liganded polypeptide during internalization in A549 cells. From FIG. 3A it can be seen that the agglomerates appeared 3 minutes after addition of the polypeptide to the cells and their size and the amount increased over time. In FIG. 3B, the disappearance of the fluorescent agglomerate is shown over time with a total disappearance at 65 minutes after addition of the polypeptide.

    [0670] The live imaging performed using the dual-labelled EGF-liganded polypeptide clearly validated the labelling technique and the ability to monitor live internalisation and trafficking of the labelled polypeptides.

    [0671] Having demonstrated that sortase-labelling is advantageous and does not affect potency, this can now be applied to other clostridial neurotoxins, including BoNT serotypes (and derivatives).

    Example 4

    [0672] Design of SrtA-Mediated Dual-Labelled BoNT/A Polypeptide

    [0673] Full length proteolytically inactive mutant BoNT/A(0) (SEQ ID NO: 38) was modified to allow for dual fluorescent-labelling using sortase (see FIG. 4). The dual-labelled polypeptide sequence is shown as SEQ ID NO: 40, while the nucleotide sequence encoding said polypeptide is shown as SEQ ID NO: 39. The design, purification and fluorescent peptides used for the dual-labelling of SEQ ID NO: 40 were the same as for the EGF-liganded polypeptide in Example 1. Successful dual-labelling of the polypeptide was confirmed by SDS-PAGE (FIG. 5). In more detail, by using Coomassie staining, both bands representing the L-chain and H-chain domains of the polypeptide could be visualised, while exposure of the gel to UV light demonstrated (by way of fluorescence) the successful labelling of both the L-chain and H-chain.

    Example 5

    [0674] Visualization of a Single-Labelled BoNT/A(0) Polypeptide in Primary Cortical Neurons

    [0675] In order to visualize a labelled BoNT/A(0) polypeptide in primary neuronal cells, single molecule live TIRF microscopy was performed in neurons treated therewith. Primary cortical neurons were treated with 1 nM single-labelled BoNT/A(0) polypeptide and imaged continuously over time using a custom made single molecule TIRF microscope. For these experiments, the BoNT/A(0) polypeptide was labelled at the N-terminal with either a HiLyte 555 or HiLyte 488 fluorophore (AnaSpec). FIG. 6 shows timelapse images of the single-coloured molecule of BoNT/A(0) being trafficked into primary cortical neurons. From FIG. 6 it can be seen that the single BoNT/A(0) molecule (white arrow) moves rapidly within the chosen neuronal region. The single molecule live TIRF imaging of a single-labelled BoNT/A(0) polypeptide clearly demonstrates that single molecules of BoNT/A(0) trafficking into neurons can be visualized with specialized, high resolution microscopy techniques.

    [0676] Having demonstrated that single-labelling of BoNT/A(0) can be visualised at a single molecule level in primary neurons, this method can now be applied to other clostridial neurotoxin serotypes and derivatives, including those having non-cytotoxic protease activity.

    [0677] All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in biochemistry and biotechnology or related fields are intended to be within the scope of the following claims.