Pore monomers and their uses

Abstract

The present invention relates to novel pore monomers which have been elongated to increase the distance between two constrictions, pores formed from the pore monomers, and their uses in analyte detection and characterisation.

Description

[0001] PORE MONOMERS AND THEIR USES

[0002] TECHNICAL FIELD

[0003] The present invention relates to novel pore monomers which have been elongated to increase the distance between two constrictions, pores formed from the pore monomers, and their uses in analyte detection and characterisation.

[0004] BACKGROUND

[0005] Nanopore sensing is an approach to analyte detection and characterization that relies on the observation of individual binding or interaction events between the analyte molecules and an ion conducting channel. Two of the essential components of analyte characterization using nanopore sensing are (1) the control of analyte movement through the pore and (2) the discrimination of the composing building blocks as the analyte is moved through the pore. During nanopore sensing, the narrowest part of the pore (also known as a constriction) forms the most discriminating part of the nanopore with respect to the current signatures as a function of the passing analyte.

[0006] For polynucleotide analytes, nucleotide discrimination is achieved by measuring the current as the polynucleotide passes through the pore. Multiple nucleotides contribute to the observed current, so the height of the channel constriction and extent of the interaction with the polynucleotide affect the relationship between observed current and polynucleotide sequence. While the current range and signal-to-noise ratio for nucleotide discrimination have been improved through mutation of the transmembrane pores, a sequencing system would have higher performance if the current differences between nucleotides could be improved further. Accordingly, there is a need to identify novel ways to improve nanopore sensing features.

[0007] SUMMARY OF THE INVENTION

[0008] The inventors have surprisingly shown that it is possible to modulate the distance, such as increase or decrease the distance, between constrictions in a pore. The inventors have also surprisingly demonstrated that increasing the distance between at least two constrictions in a pore improves the ability of the pore to characterise, such as sequence, target polynucleotides. In particular, the increased distance between constrictions improves the ability of the pore to characterise or sequence polynucleotides that contain at least one homopolymeric stretch. The increased distance between constrictions may also improve the signal metrics. The increased distance between constrictions may increase the range and / or decrease the noise when characterising an analyte. A decreased noise can result in an increased signal-to-noise ratio (SNR). The at least two constrictions may be naturally present in the pore or one or more constrictions may be added to the pore using an accessory polypeptide. The invention provides a pore monomer capable of forming a pore comprising a channel, wherein the pore monomer comprises a first constriction region which forms part of a first constriction in the channel and a second constriction region which forms part of a second constriction in the channel, and wherein the pore monomer is modified to increase the distance between the first constriction region and the second constriction region.

[0009] The invention also provides a pore monomer conjugate capable of forming a pore complex comprising a channel, wherein the pore monomer conjugate comprises (1) a pore monomer comprising a first constriction region which forms part of a first constriction in the channel and (2) an accessory polypeptide comprising a second constriction region which forms part a second constriction in the channel, and wherein the pore monomer is modified to increase the distance between the first constriction region and the second constriction region.

[0010] The invention also provides:

[0011] - a construct comprising two or more covalently attached pore monomers or pore monomer conjugates of the invention; a pore comprising at least one pore monomer of the invention; a pore complex comprising at least one pore monomer conjugate of the invention; a pore complex comprising at least one construct of the invention; an oligomeric pore formed from pore monomers surrounding a channel, wherein the channel comprises a first constriction and a second constriction, and wherein each pore monomer is modified to increase the distance between the first constriction and the second constriction; an oligomeric pore complex formed from pore monomer conjugates surrounding a channel, wherein each pore monomer conjugate comprises a pore monomer and an accessory polypeptide, wherein the channel comprises a first constriction formed by the pore monomers and a second constriction formed by the accessory polypeptide, and wherein each pore monomer conjugate is modified to increase the distance between the first constriction and the second constriction; a membrane comprising a pore of the invention, a pore complex of the invention, an oligomeric pore of the invention or an oligomeric pore complex of the invention; a method for determining the presence, absence or one or more characteristics of a target analyte, comprising the steps of: (i) contacting the target analyte with a pore of the invention, a pore complex of the invention, an oligomeric pore of the invention or an oligomeric pore complex of the invention, such that the target analyte moves with respect to the pore, pore complex, oligomeric pore or oligomeric pore complex; and (ii) taking one or more measurements as the analyte moves with respect to the pore, pore complex, oligomeric pore or oligomeric pore complex and thereby determining the presence, absence or one or more characteristics of the analyte; use of a pore of the invention, a pore complex of the invention, an oligomeric pore of the invention or an oligomeric pore complex of the invention to determine the presence, absence or one or more characteristics of a target analyte; a polynucleotide which encodes a pore monomer or pore monomer conjugate of the invention or a construct of the invention;

[0012] - a kit for characterising a target analyte comprising (a) a pore of the invention, a pore complex of the invention, an oligomeric pore of the invention or an oligomeric pore complex of the invention and (b) the components of a membrane;

[0013] - a kit for characterising a target polynucleotide or a target polypeptide comprising (a) a pore of the invention, a pore complex of the invention, an oligomeric pore of the invention or an oligomeric pore complex of the invention and (b) a polynucleotide binding protein or a polypeptide handling enzyme;

[0014] - an apparatus for characterising a target polynucleotide or a target polypeptide in a sample, comprising (a) a plurality of a pores of the invention, a plurality of pore complexes of the invention, a plurality of oligomeric pores of the invention or a plurality of oligomeric pore complexes of the invention and (b) a plurality of polynucleotide binding proteins or a plurality of polypeptide handling enzymes; an array comprising a plurality of membranes of the invention; a system comprising (a) a membrane of the invention or an array of the invention, (b) means for applying a potential across the membrane(s) and (c) means for detecting electrical or optical signals across the membrane(s); an apparatus comprising a pore of the invention, a pore complex of the invention, an oligomeric pore of the invention or an oligomeric pore complex of the invention inserted into an in vitro membrane;

[0015] - an apparatus produced by a method comprising (i) obtaining a pore of the invention, a pore complex of the invention, an oligomeric pore of the invention or an oligomeric pore complex of the invention and (ii) contacting the pore, pore complex, oligomeric pore or oligomeric pore complex with an in vitro membrane such that the pore, pore complex, oligomeric pore or oligomeric pore complex is inserted in the in vitro membrane; and a method of improving the ability of an oligomeric pore formed from pore monomers surrounding a channel to characterise an analyte, wherein the channel comprises a first constriction and a second constriction, the method comprising modifying at least one of the pore monomers to increase the distance between the first constriction and the second constriction.

[0016] DESCRIPTION OF THE FIGURES

[0017] Figure 1: CsgG / CsgF pore complex, monomer and cross section of the CsgG / CsgF wild type pore (protein databank accession code 6SI7), and the 2aa, 4aa, 6aa, 8aa and lOaa predicted 3-dimensional pore structures according to the AlphaFold algorithm (Hassabis D et al, Nature, 596, 583-589, 2021; incorporated herein by reference in its entirety).

[0018] Figure 2: Distance between the CsgG and CsgF constrictions for the wild type pore (protein databank accession code 6SI7), and the 2aa, 4aa, 6aa, 8aa and lOaa predicted 3- dimensional pore structures according to the AlphaFold algorithm (Hassabis D et al, supra). The distance was measured between the C-alpha atoms of residue 55 in CsgG and residue 17 in CsgF.

[0019] Figure 3: Ionic current (pA) versus time (s) traces as single stranded DNA translocates through CsgG / CsgF complex pores. The pore monomers used to form the pores are shown in Table 3. Similar data were obtained for 2aa_2, 2aa_3, 2aa_4, 4aa_2 and 4aa_3 (data not shown).

[0020] Figure 4: Ionic current (pA) versus time (s) traces as single stranded DNA translocates through CsgG / CsgF complex pores. For each pore, the top row shows the full DNA current trace, whilst the bottom row shows a zoomed in view of the current trace. The pore monomers used to form the pores are shown in Table 4. Similar data were obtained for ONT-4aa_2, ONT-4aa_3, ONT-4aa_4, ONT-4aa_5, ONT-4aa_6, ONT-4aa_7, ONT-4aa_8, ONT-4aa_9, ONT-4aa_10, ONT-4aa_ll, ONT-6aa_2, ONT-8aa_2, and ONT_10aa_2 (data not shown).

[0021] Figure 5: Profiles demonstrating positions within the pore and their contribution to overall changes in ionic current level (Discrimination) when a DNA molecule is translocated through the pore. Distances within the pore are measured in nucleotide steps relative to the major constriction. Negative values correspond to positions below the main constriction and positive values correspond to positions above the main constriction (CsgG). The dashed line indicates the position of the CsgF constriction, which is at position -6, -7 to -8 and -9 for the non-extended control, +2 aa and +4 aa pores respectively. The pore monomers used to form the pores are shown in Table 3.

[0022] Figure 6: Profiles demonstrating positions within the pore and their contribution to overall changes in ionic current level (Discrimination) when a DNA molecule is translocated through the pore. Distances within the pore are measured in nucleotide steps relative to the major constriction. Negative values correspond to positions below the main constriction and positive values correspond to positions above the main constriction (CsgG). The dashed line indicates the position of the CsgF constriction, which is at position -5, -6, -7 to -8, -9, -11 to -12 and -12 for the non-extended control, +2 aa, +4 aa, +6 aa, +8 aa and +10 aa pores respectively. The pore monomers used to form the pores are shown in Table 4.

[0023] Figure 7: Homopolymer length versus percentage of correctly basecalled homopolymers for CsgG I CsgF complex pores. The analyte used for these measurements was from DNA extracted from the human cell line GM24385 (obtained from the Coriell Institute BioBank), where reads above 98.4% accuracy were used for assessment. The "dashed", "dotdash", "dot" and "solid" lines represent the non-extended control, +4 aa, +6 aa and +10 aa pores respectively.

[0024] Figure 8: The structure and size of a conjugate between the wild-type CsgG pore from Escherichia coli strain K12 (the databank accession code for this structure is 4UV3) and residues 1-29 of wild-type CsgF. This represents a conjugate before it is modified in accordance with the invention. The distances shown are measured from backbone to backbone of the amino acids forming the pore structure. The CsgG pore is a tightly interconnected symmetrical nonameric pore that resembles a crown. The overall height is 91 A, and the largest outer diameter is 115 A. It defines a central channel and consists of three parts: (A) the cap region, (B) the constriction region and (C) the transmembrane beta barrel region. Cap axial length, or height, is 45 A. It has an inner diameter of 39 A and a 63 A mouth. The beta barrel has 36 strands, an axial length of 21 A and inner diameter of 47 A. Transition between the pore cap and beta barrel is sharp, being the constriction located among them, at the level of the predicted lipid-aqueous interface. The constriction is approximately 19.2 A in diameter and exhibits a length of 25 A along the axis of the channel. CsgF is also a tightly interconnected symmetrical nonameric structure that resembles a crown. It sits in the channel of the CsgG pore and adds a second constriction approximately at the boundary between the constriction region (B) and transmembrane beta barrel region (C). The CsgF constriction is approximately 19 A in diameter

[0025] Figure 9: Structure and dimensions of PorARc (cryoEM structure). This represents a PorARc before it is modified in accordance with the invention. The distances are measured from backbone to backbone of the amino acids forming the pore structure. The PorARc pore is a symmetrical octameric pore. The overall height is 90.4 A, and the largest outer dimension is 90.7 A. The PorARc pore consists of the cap region (A) and the transmembrane beta barrel region (B) which together make the cap region (or scaffold) (C), and the constriction region (D). The cap region (or scaffold) has a height of 73.6 A corresponding to height of the cap region (A), 44.7 A, and the transmembrane beta barrel region (B), 26.2 A. The constriction region (C) has a height of 19.7 A. The PorARc pore has an overall funnel shape with an entry width of 49 A which narrows to 41.5 A at the bottom of the cap region (A) and narrows further to 39.8 A at the transmembrane beta barrel region (B). The constriction region (C) has a sharp narrowing to 27.4 A and then widens to 36.1 A at the base of the pore structure.

[0026] Figure 10: Left: Cartoon depicting how a pore might be modified to extend the distance between constrictions. Right: Cartoon depicting how a pore with an accessory protein might be modified to extend the distance between constrictions. The pore or both the pore and accessory protein might be modified.

[0027] Figure 11: CsgG / CsgF pore complex, monomer and cross section of the 12aa and 14aa predicted 3-dimensional pore structures according to the AlphaFold algorithm (Hassabis D et al, supra).

[0028] Figure 12: Distance between the CsgG and CsgF constrictions for the 12aa and 14aa predicted 3-dimensional pore structures according to the AlphaFold algorithm (Hassabis D et al, supra). The distance was measured between the C-alpha atoms of residue 55 in CsgG and residue 17 in CsgF.

[0029] Figure 13: Ionic current (pA) versus time (s) traces as single stranded DNA translocates through CsgG / CsgF complex pores. For each pore, the top row shows the full DNA current trace, whilst the bottom row shows a zoomed in view of the current trace. The pore monomers used to form the pores are shown in Table 5. Similar data were obtained for ONT-14aa_2, and ONT-14aa_3 (data not shown).

[0030] Figure 14: Profiles demonstrating positions within the pore and their contribution to overall changes in ionic current level (Discrimination) when a DNA molecule is translocated through the pore. Distances within the pore are measured in nucleotide steps relative to the major constriction. Negative values correspond to positions below the main constriction and positive values correspond to positions above the main constriction (CsgG). The dashed line indicates the position of the CsgF constriction, which is at position -5, -14, and -15 for the non-extended control, +12 aa and +14 aa pores respectively. The pore monomers used to form the pores are shown in Table 5. Figure 15: Lysenin pore and cross section of the background sequence (protein databank accession code 5EC5) and 4aa predicted 3-dimensional pore structures according to the AlphaFold algorithm (Hassabis D et al, supra).

[0031] Figure 16: Ionic current (pA) versus time (s) traces as single stranded DNA translocates through lysenin pores. For each pore, the top row shows the full DNA current trace, whilst the bottom row shows a zoomed in view of the current trace. The pore monomers used to form the pores are shown in Table 6.

[0032] Figure 17: Profiles demonstrating positions within the pore and their contribution to overall changes in ionic current level (Discrimination) when a DNA molecule is translocated through the pore. Distances within the pore are measured in nucleotide steps relative to the major constriction. Negative values correspond to positions below the main constriction and positive values correspond to positions above the main constriction. The dashed line indicates the position of the upper constriction, which is at position 5 and 7 for the nonextended control, and +4 aa pores respectively. The pore monomers used to form the pores are shown in Table 6.

[0033] Figure 18: Alpha-hemolysin pore and cross section of the WT sequence (protein databank accession code 7AHL) and 4aa predicted 3-dimensional pore structures according to the AlphaFold algorithm (Hassabis D et al, supra).

[0034] Figure 19: Ionic current (pA) versus time (s) traces as single stranded DNA translocates through alpha-hemolysin pores. For each pore, the top row shows the full DNA current trace, whilst the bottom row shows a zoomed in view of the current trace. The pore monomers used to form the pores are shown in Table 7.

[0035] Figure 20: Profiles demonstrating positions within the pore and their contribution to overall changes in ionic current level (Discrimination) when a DNA molecule is translocated through the pore. Distances within the pore are measured in nucleotide steps relative to the major constriction. Negative values correspond to positions below the main constriction and positive values correspond to positions above the main constriction. The dashed line indicates the position of the upper and lower constrictions, which are 8 and 9 positions apart for the non-extended control, and +4 aa pores respectively. The pore monomers used to form the pores are shown in Table 7.

[0036] DESCRIPTION OF THE SEQUENCE LISTING

[0037] SEQ ID NO: 1 shows the polynucleotide sequence of wild-type E. coli CsgG from strain K12, including signal sequence (Gene ID: 945619).

[0038] SEQ ID NO: 2 shows the amino acid sequence of wild-type E. coli CsgG including signal sequence (Uniprot accession number P0AEA2). SEQ ID NO: 3 shows the amino acid sequence of wild-type E. coli CsgG as a mature protein (Uniprot accession number P0AEA2).

[0039] SEQ ID NO: 4 shows the polynucleotide sequence of wild-type E. coli CsgF from strain K12, including signal sequence (Gene ID: 945622).

[0040] SEQ ID NO: 5 shows the amino acid sequence of wild-type E. coli CsgF including signal sequence (Uniprot accession number P0AE98).

[0041] SEQ ID NO: 6 shows the amino acid sequence of wild-type E. coli CsgF as a mature protein (Uniprot accession number P0AE98).

[0042] SEQ ID NO: 7 shows the amino acid sequence of wild-type lysenin.

[0043] SEQ ID NO: 8 shows the amino acid sequence of wild-type alpha-hemolysin.

[0044] SEQ ID NO: 9 shows the amino acid sequence of wild-type CytK.

[0045] DETAILED DESCRIPTION

[0046] All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety. All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the invention contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.

[0047] The present invention will be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims. Any reference signs in the claims shall not be construed as limiting the scope. Of course, it is to be understood that not necessarily all aspects or advantages may be achieved in accordance with any particular embodiment of the invention. Thus, for example those skilled in the art will recognize that the invention may be embodied or carried out in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other aspects or advantages as may be taught or suggested herein.

[0048] In addition, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a pore monomer" includes two or more pore monomers, reference to "a conjugate" includes two or more conjugates, reference to "an accessory polypeptide" includes two or more accessory polypeptides, reference to "a pore" includes two or more pores, reference to "a polynucleotide" includes two or more polynucleotides, reference to "a polynucleotide binding protein" includes two or more such proteins, reference to "a helicase" includes two or more helicases, and the like. In all instances here, the terms "comprises" or "comprising" cover and can be replaced by "consists of" or "consisting of".

[0049] In all of the discussion herein, the standard one letter codes for amino acids are used. These are as follows: alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine (C), glutamic acid (E), glutamine (Q), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y) and valine (V). Standard substitution notation is also used, i.e., Q42R means that Q at position 42 is replaced with R.

[0050] In the paragraphs herein where different amino acids at a specific position are separated by the I symbol, the I symbol means "or". For instance, F56I / L means F56I or F56L. In the paragraphs herein where different positions are separated by the I symbol, the I symbol means "and" such that Y51 / N55 is Y51 and N55.

[0051] A "pore" in the context of the invention is a transmembrane protein structure defining a channel or hole that allows the translocation of molecules and ions from one side of the membrane to the other. The translocation of ionic species through the pore may be driven by an electrical potential difference applied to either side of the pore. A "nanopore" is a biological pore in which the minimum diameter of the channel through which molecules or ions pass is in the order of nanometres (IO-9nanometres). In some embodiments, the pore can be a transmembrane protein pore. The transmembrane protein structure of a biological pore may be monomeric or oligomeric in nature. Typically, the pore comprises a plurality of polypeptide monomers or subunits arranged around a central axis thereby forming a protein-lined channel that extends substantially perpendicular to the membrane in which the pore resides. The number of polypeptide monomers or subunits is not limited. Typically, the number of monomers or subunits is from 5 to up to 30, suitably the number of monomers or subunits is from 6 to 10. The portions of the protein monomers or subunits within the pore that form protein-lined channel typically comprise secondary structural motifs that may include one or more trans-membrane [3-barrel, and / or o-helix sections.

[0052] In its broadest context, the term "pore" covers a pore complex or a pore multimer. A "pore complex" of the invention typically comprises pore monomers and accessory polypeptides, such as a modified CsgG-CsgF complex.

[0053] "About" as used herein when referring to a measurable value such as a percentage or an amount and the like, is meant to encompass variations of ± 20 % or ± 10 %, more preferably ± 5 %, even more preferably ± 1 %, and still more preferably ± 0.1 % from the specified value, as such variations are appropriate to perform the disclosed methods. Any statement herein including the term "about" includes the same feature without the term. For instance, at least "about" 20% includes at least 20%. A "variant" of a polypeptide or protein encompasses peptides, oligopeptides, polypeptides, proteins, and enzymes having structural similarity with the unmodified or wild-type polypeptide or protein. Structural variants are defined below with reference to root mean square deviation (RMSD). Standard methods in the art can be used to determine structural similarity, including RMSD. Suitable methods include, but are not limited to, AlphaFold, PSIPRED, TM-Align, US-Align or FATCAT. In all instances herein, RMSD is preferably measured over all alpha carbon atoms (Co atoms). RMSD may be measured over all heavy atoms.

[0054] A "variant" of a polypeptide or protein encompasses peptides, oligopeptides, polypeptides, proteins, and enzymes having amino acid substitutions, deletions and / or insertions relative to the unmodified or wild-type polypeptide or protein in question and having similar biological and functional activity as the unmodified polypeptide or protein from which they are derived. The term "amino acid identity" as used herein refers to the extent that sequences are identical on an amino acid-by-amino acid basis over a window of comparison. Thus, a "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Vai, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison ( / .e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.

[0055] Standard methods in the art may be used to determine identity and / or homology. For example, the UWGCG Package provides the BESTFIT program which can be used to calculate homology, for example used on its default settings (Devereux et al (1984) Nucleic Acids Research 12, p387-395). The PILEUP and BLAST algorithms can be used to calculate homology or line up sequences (such as identifying equivalent residues or corresponding sequences (typically on their default settings)), for example as described in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S.F et al (1990) J Mol Biol 215:403-10. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). In all instances herein, identity or homology is typically measured over the entire length of the reference sequence.

[0056] Corresponding positions or regions may be determined by standard techniques in the art. For example, the PILEUP and BLAST algorithms mentioned below can be used to align the sequence of the invention, such as a pore monomer, with the reference sequence, e.g., SEQ ID NO: 3, and identify corresponding positions or regions. The general definitions in WO 2019 / 002893 are incorporated by reference herein in their entirety.

[0057] Pore monomers

[0058] The invention provides a pore monomer capable of forming a pore comprising a channel. The ability of the pore monomer to form a channel can be measured using routine methods, including any of those described in WO 2016 / 034591, WO 2017 / 149316, WO 2017 / 149317, WO 2017 / 149318, WO 2018 / 211241, WO 2019 / 002893, WO 2023 / 118404, WO 2023 / 198911, WO 2024 / 033421, WO 2024 / 033422, WO 2024 / 033443, and WO 2024 / 089270 (all incorporated by reference herein in their entirety) and in the Example.

[0059] The pore monomer comprises a first constriction region which forms part of a first constriction in the channel. The pore monomer also comprises a second constriction region which forms part of a second constriction in the channel. The first and second constriction regions "form part" of the first and second constrictions in the channel in the sense they form first and second constrictions, respectively, in a pore formed by the pore monomer. The first and second constriction regions "form part" of the first and second constrictions in the channel, respectively, with the first and second constriction regions from the other pore monomers used to form the channel. As explained in more detail below, the pore monomers of the invention may be used to form the pores of the invention.

[0060] The pore monomer comprises two or more constriction regions. These regions are capable of forming a channel. These regions are capable of forming constrictions in the channel. The pore monomer may comprise any number of two or more constriction regions, such as 2, 3, 4, 5, 6 or more. The pore monomer may comprise a first constriction region which forms part of a first constriction in the channel, a second constriction region which forms part of a second constriction in the channel and a third constriction region which forms part of a third constriction in the channel.

[0061] The term "constriction", "orifice", "channel constriction", or "constriction site", as used interchangeably herein, refers to an aperture defined by a luminal surface of a pore, which acts to allow the passage of ions and target analytes through the pore channel. Constrictions and constriction regions are known in the art. The two or more constrictions formed by the two or more constriction regions are typically the narrowest apertures within a pore or within the channel defined by the pore. The two or more constrictions formed by the two or more constriction regions may be defined functionally. For instance, the two or more constrictions may be identified using the level of discrimination of a target analyte, such as a target polynucleotide. Peak discrimination levels may identified two or more constrictions. Discrimination is the contribution of the two or more constrictions to overall changes in ionic current level when target analyte, such as a DNA molecule, is translocated through the pore. A skilled person is capable of doing this, for instance as described in the Example and shown in Figures 5 and 6.

[0062] The two or more constrictions may serve to limit the passage of molecules through the pore. The size of the constrictions is typically a key factor in determining suitability of a pore for target analyte characterisation. If the constrictions are too small, the target analyte to be characterised will not be able to pass through. However, to achieve a maximal effect on ion flow through the channel, the constrictions should not be too large. For example, the constrictions should not be wider than the solvent-accessible transverse diameter of a target analyte. Ideally, any constriction should be as close as possible in diameter to the transverse diameter of the analyte passing through.

[0063] The narrowing of a central cavity or aperture typically forms a constriction in the channel. In some embodiments, the diameter of a constriction is calculated by measuring the distance between the alpha-carbons (Ca) of the amino acid residues that extend furthest into the lumen of pore or pore complex to form the constriction. In some embodiments, the diameter of a constriction is calculated by measuring the distance between the Van der Waals radii of the atoms extending furthest into the lumen of the pore to form the constriction. In some embodiments, the minimum diameter of a constriction, including the first constriction and / or the second constriction, ranges from about 0.5 nanometres (nm) to about 4.0 nm (e.g., as measured by the distance between Van der Waals radii). In some embodiments, the minimum diameter of the constriction ranges from about 0.5 to about 3.0 nm or from about 0.5 to about 2.0 nm, preferably from about 0.7 to about 1.8 nm, from about 0.8 to about 1.7 nm, from about 0.9 to about 1.6 nm, or from about 1.0 to about 1.5 nm, such as about 1.1, about 1.2, about 1.3 or about 1.4 nm. In some embodiments, the minimum diameter of a constriction, including the first constriction and / or the second constriction, ranges from about 10 A to about 30 A, for example about 10 A, about 11 A, about 12 A, about 13 A, about 14 A, about 15 A, about 16 A, about 17 A, about 18 A, about 19 A, about 19.2 A, about 20 A, about 21 A, about 22 A, about 23 A, about 24 A, about 25 A, about 26 A, about 27 A, about 28 A, about 29 A, or about 30 A (e.g., as measured by Ca to Ca). In some embodiments, the minimum diameter of a constriction, including the first constriction and / or the second constriction, ranges from about 10 A to about 30 A (e.g., as measured by Ca to Ca). In some embodiments, the minimum diameter of a constriction, including the first constriction and / or the second constriction, ranges from about 15 A to about 25 A (e.g., as measured by Ca to Ca).

[0064] The first constriction region and / or the second constriction region is / are preferably from about 5 to about 114 amino acids in length. The first constriction region and / or the second constriction region is preferably about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about

[0065] 100, about 101, about 102, about 103, about 104, about 105, about 106, about 107, about 108, about 109, about 110, about 111, about 112, about 113, or about 114 amino acids in length. The first constriction region and / or the second constriction region is preferably from about 15 to about 94 amino acids in length. The first constriction region and / or the second constriction region is preferably about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about

[0066] 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about

[0067] 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about

[0068] 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about

[0069] 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about

[0070] 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about

[0071] 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about

[0072] 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about

[0073] 92, about 93, about or 94 amino acids in length. The first constriction region and / or the second constriction region is preferably about 15, about 16, about 17, about 18, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 39, about 50, about 52, about 86, about 94 amino acids in length. The third or further constriction region may be any of these lengths.

[0074] The first constriction region and / or the second constriction region is preferably from about 6 to about 46 amino acids in length. The first constriction region and / or the second constriction region is preferably about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45 or 46 amino acids in length. The first constriction region and / or the second constriction region is preferably from about 16 to about 36 amino acids in length. The first constriction region and / or the second constriction region is preferably about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27 , about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35 or 36 amino acids in length. The first constriction region and / or the second constriction region is preferably about 26 amino acids in length. The third or further constriction region may be any of these lengths.

[0075] Before modification in accordance with the invention, the distance, such as vertical distance, between the first constriction region and the second constriction region may be about 5 A to about 55 A, such as from about 10 A to about 50 A, from about 15 A to about 45 A, from about 20 A to about 40 A or from about 25 A to about 35 A. This can be measured as the distance between the alpha-carbons (Ca) of the amino acid residue extending furthest into the lumen of the channel forming the first constriction and the amino acid residue extending furthest into the lumen of the channel forming the second constriction.

[0076] Before modification in accordance with the invention, the distance between the first constriction region and the second constriction region is about 5 A, about 6 A, about 7 A, about 8 A, about 9 A, about 10 A, about 11 A, about 12 A, about 13 A, about 14 A, about 15 A, about 16 A, about 17 A, about 18 A, about 19 A, about 20 A, about 21 A, about 22 A, about 23 A, about 24 A, about 24.8 A, about 25 A, about 26 A, about 27 A, about 28 A, about 29 A, about 30 A, about 31 A, about 32 A, about 33 A, about 34 A, about 35 A, about 36 A, about 37 A, about 38 A, about 39 A, about 40 A, about 41 A, about 42 A, about 43 A, about 44 A, about 45 A, about 46 A, about 47 A, about 48 A, about 49 A, about 50 A, about 51 A, about 52 A, about 53 A, about 54 A, or about 55 A. In some embodiments, the distance between the first constriction region and the second constriction region is more than about 55 A in length, such as about 56 A, about 57 A, about 58 A, about 59 A, about 60 A, about 61 A, about 62 A, about 63 A, about 64 A, about 65 A, about 66 A, about 67 A, about 68 A, about 69 A, about 70 A, about 71 A, about 72 A, about 73 A, about 74 A, about 75 A, about 76 A, about 77 A, about 78 A, about 79 A, about 80 A, about 81 A, about 82 A, about 83 A, about 84 A, about 85 A, about 86 A, about 87 A, about 88 A, about 89 A, about 90 A, about 91 A, about 92 A, about 93 A, about 94 A, about 95 A, about 96 A, about 97 A, about 98 A, about 99 A, or about 100 A in length or more. Before modification in accordance with the invention, the distance between the first constriction region and the second constriction region is about 24.8 A.

[0077] The pore monomer typically further comprises a cap region and / or a transmembrane region. Both of these regions are capable of forming a channel. The pore monomer may comprise a first constriction region, a second constriction region and a cap region. The pore monomer may comprise a first constriction region, a second constriction region and a transmembrane region. The pore monomer may comprise a first constriction region, a second constriction region, a cap region, and a transmembrane region. The transmembrane region may be a transmembrane beta barrel region or a transmembrane alpha helical region. The transmembrane region is preferably a transmembrane beta barrel region.

[0078] The cap region and / or the transmembrane region may have any of the dimensions and / or lengths discussed below. The cap region and / or the transmembrane region may have any of the dimensions and / or lengths described in WO 2024 / 089270 (incorporated by reference herein in its entirety).

[0079] The pore monomer is preferably from or derived from Wza, Iota toxin, Anthrax protective antigen, Vibrio cholerae cytolysin, Cytotoxin K (CytK), CELIII, CsgG, CsgF, CsgG-CsgF, Aerolysin, alpha hemolysin, MspA, MspB, MspC, PorARr, PorBRr, PorARc, PilQ, necrotic enteritis B-like toxin (NetB), FraC, portal proteins including G20c, P23_45, T4, SPP1, P22 and Phi29, gamma hemolysin, Monalysin, Lysenin, ClyA, an actinoporin, Clostridium perfringens beta toxin, parasporin-2, epsilon toxin, lectin from the parasitic mushroom Laetiporus sulphureus (LSL), volvatoxin, Cry toxins, CytlAa, Cyt2Aa, Complement component 9 (C9), Perfringolysin O, Pleurotolysin, Listeriolysin, Perforin-2, Gasdermin-A3, L-, P- and M-ring protein, Type II secretion system protein D, GspD, InvG, VirB7, SpoIIIAG, Cag8, Cag3, Cag or other proteins in the Type IV secretion system apparatus protein CagY, WzzB, Pentraxin, Afp2, Major vault protein, Thioredoxin-dependent peroxidase reductase, Arf-GAP, Respiratory syncytial virus ribonucleoprotein, Chikungunya virus nonstructural protein 1, PRC, YaxA, XaxA, HfaB, NfpAB, leukocidin or PrgH. Suitable actinoporins include, but are not limited to, actinoporins from or derived from Orbicella faveolata, such as those described in PCT / EP2024 / 060202.

[0080] The pore monomer is preferably from or derived from Wza, Iota toxin, Anthrax protective antigen, Vibrio cholerae cytolysin, Cytotoxin K (CytK), CELIII, CsgG-CsgF, Aerolysin, alpha hemolysin, PilQ, necrotic enteritis B-like toxin (NetB), portal proteins including G20c, P23_45, T4, SPP1, P22, gamma hemolysin, Monalysin, Lysenin, actinoporin, Clostridium perfringens beta toxin, parasporin-2, epsilon toxin, lectin from the parasitic mushroom Laetiporus sulphureus (LSL), Cry toxins, CytlAa, Cyt2Aa , Listeriolysin, Perforin-2, L-, P-ring protein, GspD, Pentraxin, Afp2, or Respiratory syncytial virus ribonucleoprotein.

[0081] The pore monomer is preferably from or derived from Iota toxin, Anthrax protective antigen, Vibrio cholerae cytolysin, Cytotoxin K (CytK), CELIII, CsgG-CsgF, Aerolysin, alpha hemolysin, PilQ, necrotic enteritis B-like toxin (NetB), gamma hemolysin, Monalysin, Lysenin, Clostridium perfringens beta toxin, parasporin-2, epsilon toxin, lectin from the parasitic mushroom Laetiporus sulphureus (LSL), Cry toxins, CytlAa, Cyt2Aa , Listeriolysin, or Perforin-2. The pore monomer is preferably from or derived from Cytotoxin K (CytK), CsgG-CsgF, alpha hemolysin, or Lysenin.

[0082] A pore monomer is "from" or "derived from" a pore if it shares structural similarity with a monomer from the pore. A pore monomer is "from" or "derived from" a pore if it shares significant homology / identity with a monomer from the pore. The pore monomer preferably comprises a sequence having at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% or more preferably at least about 95%, at least about 97%, at least about 98% or at least about 99% homology and / or identity to the sequence of the pore monomer from the pore. The pore monomer preferably comprises a sequence having 100% homology to the sequence of pore monomer from the pore. Homology and / or identity is typically measured over the entire length of the region.

[0083] These regions, such as the constriction regions, cap region, and / or transmembrane region, may be from or derived from any of the pores listed above. A region is "from" or "derived from" a pore if it shares significant homology / identity with a region from the pore. The region, preferably the constriction regions, cap region, or transmembrane region, preferably comprises a sequence having at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% or more preferably at least about 95%, at least about 97%, at least about 98% or at least about 99% homology or identity to the sequence of the corresponding region from the pore. The region preferably comprises a sequence having 100% homology to the sequence of the corresponding region from the pore. Homology and / or identity is typically measured over the entire length of the region.

[0084] Pore monomer conjugates

[0085] The invention also provides a pore monomer conjugate capable of forming a pore complex comprising a channel. The ability of the pore monomer conjugate can be measured as discussed above with reference to the pore monomers of the invention.

[0086] Pore monomer

[0087] The pore monomer conjugate comprises a pore monomer comprising a first constriction region which forms part of a first constriction in the channel. The first constriction region "forms part" of a first constriction in the channel in the sense it forms a first constriction in a pore complex formed from the pore monomer conjugate. The first constriction region "forms part" of a first constriction in the channel with the first constriction regions from the other pore monomer conjugates used to form the channel. As explained in more detail below, the pore monomer conjugates of the invention may be used to form the pore complexes of the invention.

[0088] The pore monomer may comprise any number of constriction regions, such as 2, 3, 4, 5, 6 or more. The pore monomer may comprise any number of constrictions, such as 2, 3, 4, 5, 6 or more.

[0089] Constrictions are defined above with reference to the pore monomers of the invention and the same applies to the pore monomer conjugates of the invention except any discussion of a pore instead applies to a pore complex. The first constriction region may be any of those described above with reference to the pore monomers of the invention. The first constriction may have any of the diameters discussed above. The first constriction region may have any of the lengths discussed above.

[0090] The pore monomer typically further comprises a cap region and / or a transmembrane region. Both of these regions are capable of forming a channel. The pore monomer may comprise a first constriction region and a transmembrane region. The pore monomer may comprise a first constriction region and a cap region. The pore monomer may comprise a first constriction region, a cap region, and a transmembrane region. The transmembrane region may be a transmembrane beta barrel region or a transmembrane alpha helical region. The transmembrane region is preferably a transmembrane beta barrel region.

[0091] The cap region and / or the transmembrane region may have any of the dimensions and / or lengths discussed below. The cap region and / or the transmembrane region may have any of the dimensions and / or lengths described in WO 2024 / 089270 (incorporated by reference herein in its entirety).

[0092] The pore monomer is preferably from or derived from Wza, Iota toxin, Anthrax protective antigen, Vibrio cholerae cytolysin, Cytotoxin K (CytK), CELIII, CsgG, CsgF, CsgG-CsgF, Aerolysin, alpha hemolysin, MspA, MspB, MspC, PorARr, PorBRr, PorARc, PilQ, necrotic enteritis B-like toxin (NetB), FraC, portal proteins including G20c, P23_45, T4, SPP1, P22 and Phi29, gamma hemolysin, Monalysin, Lysenin, ClyA, an actinoporin, Clostridium perfringens beta toxin, parasporin-2, epsilon toxin, lectin from the parasitic mushroom Laetiporus sulphureus (LSL), volvatoxin, Cry toxins, CytlAa, Cyt2Aa, Complement component 9 (C9), Perfringolysin O, Pleurotolysin, Listeriolysin, Perforin-2, Gasdermin-A3, L-, P- and M-ring protein, Type II secretion system protein D, GspD, InvG, VirB7, SpoIIIAG, Cag8, Cag3, Cag or other proteins in the Type IV secretion system apparatus protein CagY, WzzB, Pentraxin, Afp2, Major vault protein, Thioredoxin-dependent peroxidase reductase, Arf-GAP, Respiratory syncytial virus ribonucleoprotein, Chikungunya virus nonstructural protein 1, PRC, YaxA, XaxA, HfaB, NfpAB, leukocidin or PrgH. Suitable actinoporins include, but are not limited to, actinoporins from or derived from Orbicella faveolata, such as those described in PCT / EP2024 / 060202.

[0093] The terms "from" and "derived from" are defined above and these definitions equally apply to the any of the pore monomers, first constriction region, cap region and transmembrane region in the pore monomer conjugates of the invention.

[0094] The pore monomer in the pore monomer conjugate of the invention may be a chimeric pore monomer comprising two or more regions, wherein at least two of the two or more regions are from at least two different pores. The chimeric pore monomer may comprise any number of regions, such as three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more or ten or more regions, from different pores. The chimeric pore monomer may comprise two or three regions. The regions may be any of those discussed above. The regions are preferably selected from a cap region, a constriction region, and a transmembrane region. The regions may be a cap region and a constriction region. The regions may be a cap region, a constriction region, and a transmembrane region. The at least two different pores are typically at least two different pores that appear in nature. The at least two different pores are typically at least two different wild-type or naturally occurring pores. The at least two different pores are preferably different before any artificial or synthetic modifications, such as additions, deletions and / or substitutions, are made to them. The at least two different pores are preferably homologues, for example structural homologues. A structural homologue refers to a protein or molecule that shares a similar three-dimensional structure with another protein or molecule. This can be determined using standard methods in the art (e.g., AlphaFold or PSIPRED). Structural homologues typically have similar sequences. Structural homologues are normally identified in similar species. The at least two different pores may be selected from any of the pores listed above. The at least two different pores may be two different PorARc pores or three different PorARc pores. The at least two different pores may be two different CsgG pores or three different CsgG pores. The chimeric pore monomer may be any of those described in WO 2024 / 089270 (incorporated by reference herein in its entirety).

[0095] The pore monomer in the pore monomer conjugate is preferably a CsgG pore monomer. These are also discussed in more detail below. The CsgG monomer may comprise one or more of a cap region, a constriction region, and a transmembrane beta barrel region. All of these regions are capable of forming a channel, i.e., are channel-forming.

[0096] The pore monomer in the pore monomer conjugate may be any of the pore monomers described in WO 2023 / 123370, WO 2024 / 138470, WO 2024 / 138472, WO 2024 / 138424, WO 2024 / 138425, WO 2024 / 138512 and WO 2024 / 138565 (all incorporated herein by reference in their entireties) or a variant thereof. Accessory polypeptide

[0097] The pore monomer conjugate also comprises an accessory polypeptide comprising a second constriction region which forms part a second constriction in the channel. The second constriction region "forms part" of a second constriction in the channel in the sense it forms a second constriction in a pore complex formed by the pore monomer conjugate. The second constriction region "forms part" of a second constriction in the channel with the second constriction regions from the other pore monomer conjugates used to form the channel. As explained in more detail below, the pore monomer conjugates of the invention may be used to form the pore complexes of the invention.

[0098] The accessory polypeptide may comprise any number of constriction regions, such as 2, 3, 4, 5, 6 or more. The accessory polypeptide may comprise any number of constrictions, such as 2, 3, 4, 5, 6 or more.

[0099] Constrictions are defined above with reference to the pore monomers of the invention and the same applies to the pore monomer conjugates of the invention except any discussion of a pore instead applies to a pore complex. The second constriction region may be any of those described above with reference to the pore monomers of the invention. The second constriction may have any of the diameters discussed above. The second constriction region may have any of the lengths discussed above.

[0100] The accessory polypeptide may be any accessory polypeptide for the pore monomer which comprises the second constriction region. The accessory polypeptide may be a native accessory polypeptide or a non-native, engineered or de novo accessory polypeptide. The accessory polypeptide may be selected from a CsgF peptide, CsgA peptide, CsgB peptide, CsgE peptide, or any peptide that binds to or is designed to bind to CsgG. CsgF peptides are discussed in more detail below.

[0101] The accessory polypeptide is preferably further modified to comprise at least a third constriction region which forms part of a third constriction in the channel. Specific examples of this are discussed below and in WO 2024 / 033447 (incorporated by reference herein in its entirety).

[0102] CsoG pore monomers

[0103] A CsgG pore monomer is a monomer that is capable of forming a CsgG pore. Such monomers are known in the art, especially from WO 2019 / 002893 (incorporated by reference herein in its entirety). The CsgG pore preferably comprises one or more of (a) a cap region, (b) a constriction region, and (c) a transmembrane beta barrel region, such as (a), (b), (c), (a) and (b), (a) and (c), (b) and (c), or (a), (b) and (c). The CsgG pore monomer preferably comprises one or more of (a) a cap forming region, (b) a constriction forming region, and (c) a transmembrane beta barrel forming region, such as (a), (b), (c), (a) and (b), (a) and (c), (b) and (c), or (a), (b) and (c). The residues of SEQ ID NO: 3 which form these regions are defined below. The CsgG pore formed by the monomer may have any structure but preferably has or comprises the structure of the wild-type CsgG pore (Figure 8). The protein structure of CsgG defines a channel or hole that allows the translocation of molecules and ions from one side of the membrane to the other.

[0104] The CsgG pore may be any size but preferably has the dimensions of the wild-type CsgG pore (Figure 8). The CsgG pore preferably has an external diameter of from about 100 to about 150 A at its widest point, such as from about 105 to about 140 A or from about 110 to about 125 A at its widest point. The CsgG pore preferably has an external diameter of about 115 A at its widest point. The CsgG pore preferably has a total length of from about 75 to about 120 A, such as from about 80 to about 110 A or from about 85 to about 105 A. The CsgG pore preferably has a total length of about 91 A. References to "total length" and "length" relate to the length of the pore or pore region when viewed from the side (see, e.g., the side view in Figure 8).

[0105] The cap region preferably has a length of from about 20 to about 60 A, such as from about 30 to about 55 A or from about 35 to about 50 A. The cap region preferably has a length of about 45 A. The channel defined by the cap region preferably has an opening of from about 45 to about 85 A in diameter, such as from about 55 to about 75 A or from about 60 to about 70 A in diameter. The channel defined by the cap region preferably has an opening of about 63 A in diameter. The channel defined by the cap region is preferably from about 25 to about 70 A in diameter at its narrowest point, such as from about 30 to about 60 A or from about 35 to about 50 A in diameter at its narrowest point. The channel defined by the cap region is preferably about 39 A in diameter at its narrowest point.

[0106] The constriction region preferably has a length of from about 5 to about 40 A, such as from about 10 to about 35 A or from about 15 to about 30 A. The constriction region preferably has a length of about 25 A. The channel defined by the constriction region is preferably from about 2 to about 40 A in diameter at its narrowest point, such as from about 5 to about 35 A, from about 8 to about 25 A or from about 10 to about 20 A in diameter at its narrowest point. The channel defined by the constriction region is preferably about 9 A, 12 A or 18.5 A in diameter. The channel defined by the constriction region is preferably about 19.2 A in diameter. The constriction is preferably from about 2 to about 40 A in diameter, such as from about 5 to about 35 A, from about 8 to about 25 A or from about 10 to about 20 A in diameter. The constriction is preferably about 9 A, 12 A or 18.5 A in diameter. The constriction is preferably about 19.2 A in diameter. In the context of a protein conjugate of the invention, the constriction region may be the first constriction region and the constriction may be the first constriction. The transmembrane beta barrel region preferably has a length of from about 5 to about 50 A, such as from about 10 to about 40 A or from about 15 to about 30 A. The transmembrane beta barrel preferably has a length of about 21 A. The channel defined by the transmembrane beta barrel region is preferably from about 30 to about 75 A in diameter at its narrowest point, such as from about 40 to about 65 A or from about 45 to about 60 A in diameter at its narrowest point. The channel defined by the transmembrane beta barrel region is preferably about 47 A in diameter at its narrowest point. All of the measurements above are based on measuring from backbone to backbone of the amino acids forming the different regions (as shown in Figure 8). All of the measurements typically apply to the CsgG pore monomer or CsgG pore before it is modified in accordance with the invention. The dimensions of the CsgF accessory polypeptide are discussed below.

[0107] SEQ ID NO: 3 shows the sequence of wild-type E. coli CsgG as a mature protein. Residues 1 to 41, 64 to 131, 156 to 180 and 212 to 262 of SEQ ID NO: 3 form the cap region. Residues 42 to 63 of SEQ ID NO: 3 form the constriction region. Residues 132 to 155 and 181 to 211 of SEQ ID NO: 3 form the transmembrane beta barrel region.

[0108] The CsgG pore monomer is preferably a variant of SEQ ID NO: 3. The variant CsgG momomer may also be referred to as a modified CsgG pore monomer or a mutant CsgG pore monomer. The modifications, or mutations, in the variant include but are not limited to any one or more of the modifications disclosed herein, or combinations of said modifications. The CsgG pore monomer may be a CsgG homologue monomer. A CsgG homologue monomer is a polypeptide that has at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 99% complete sequence identity to wildtype E. coli CsgG as shown in SEQ ID NO: 3. A CsgG homologue is also referred to as a polypeptide that contains the PFAM domain PF03783, which is characteristic for CsgG-like proteins. A list of presently known CsgG homologues and CsgG architectures can be found at http: / / pfam.xfam.Org / / family / PF03783.

[0109] The variant of SEQ ID NO: 3 may be a structural variant. The structural variant may have a root mean square deviation (RMSD) of less than about 4.0 Angstroms (A), such as less than about 3.5 A, less than about 3.0 A, less than about 2.5 A, less than about 2.0 A, less than about 1.5 A, less than about 1.0 A or less than about 0.5 A, when compared with a protein having the sequence of SEQ ID NO: 3. RMSD may be calculated as discussed above.

[0110] Over the entire length of the amino acid sequence of SEQ ID NO: 3, a variant will preferably be at least 40% homologous to that sequence based on amino acid identity. More preferably, the variant may be at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% and more preferably at least 95%, 97% or 99% homologous based on amino acid identity to the amino acid sequence of SEQ ID NO: 3 over the entire sequence. Over the entire length of the amino acid sequence of SEQ ID NO: 3, a variant will preferably be at least 40% identical to that sequence. More preferably, the variant may be at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% and more preferably at least 95%, 97% or 99% identical to SEQ ID NO: 3 over the entire sequence.

[0111] Sequence identity can also relate to a fragment or portion of the CsgG pore monomer. Hence, a sequence may have less than 40% overall sequence homology / identity with SEQ ID NO: 3, but the sequence of a particular region, domain or subunit could share at least about 80%, at least about 90%, or as much as about 99% sequence homology / identity with the corresponding region of SEQ ID NO: 3. There may be at least about 80%, for example at least 85%, at least about 90% or at least about 95%, amino acid identity over a stretch of 100 or more, for example 125, 150, 175 or 200 or more, contiguous amino acids ("hard homology"). The CsgG pore monomer is preferably a variant of SEQ ID NO: 3 comprising a sequence that is at least 40% homologous to the cap region of SEQ ID NO: 3 (residues 1 to 41, 64 to 131, 156 to 180 and 212 to 262). More preferably, the variant may comprise a sequence that is at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% and more preferably at least about 95%, about 97% or about 99% homologous based on amino acid identity to residues 1 to 41, 64 to 131, 156 to 180 and 212 to 262 of SEQ ID NO: 3. The variant preferably comprises a sequence that is at least about 40% identical to residues 1 to 41, 64 to 131, 156 to 180 and 212 to 262 of SEQ ID NO: 3. More preferably, the variant may comprise a sequence that is at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% and more preferably at least about 95%, about 97% or about 99% identical to residues of 1 to 41, 64 to 131, 156 to 180 and 212 to 262 of SEQ ID NO: 3. Homology and / or identity is typically measured over the entire length of the cap region.

[0112] The CsgG pore monomer is preferably a variant of SEQ ID NO: 3 comprising a sequence that is at least about 40% homologous to the constriction region of SEQ ID NO: 3 (residues 42 to 63). More preferably, the variant may comprise a sequence that is at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% and more preferably at least about 95%, 97% or 99% homologous based on amino acid identity to residues 42 to 63 of SEQ ID NO: 3. The variant preferably comprises a sequence that is at least about 40% identical to residues 42 to 63 of SEQ ID NO: 3. More preferably, the variant may comprise a sequence that is at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% and more preferably at least about 95%, 97% or 99% identical to residues of 42 to 63 of SEQ ID NO: 3. Homology and / or identity is typically measured over the entire length of the constriction region.

[0113] The CsgG pore monomer is preferably a variant of SEQ ID NO: 3 comprising a sequence that is at least about 40% homologous to the transmembrane beta barrel region of SEQ ID NO: 3 (residues 132 to 155 and 181 to 211). More preferably, the variant may comprise a sequence that is at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% and more preferably at least about 95%, 97% or 99% homologous based on amino acid identity to residues 132 to 155 and 181 to 211 of SEQ ID NO: 3. The variant preferably comprises a sequence that is at least about 40% identical to residues 132 to 155 and 181 to 211 of SEQ ID NO: 3. More preferably, the variant may comprise a sequence that is at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% and more preferably at least about 95%, 97% or 99% identical to residues of 132 to 155 and 181 to 211 of SEQ ID NO: 3. Homology and / or identity is typically measured over the entire length of the transmembrane beta barrel region.

[0114] CsgG pore monomers are highly conserved (as can be readily appreciated from Figures 45 to 47 of WO 2017 / 149317). Furthermore, from knowledge of the mutations in relation to SEQ ID NO: 3 it is possible to determine the equivalent positions for mutations of CsgG pore monomers other than that of SEQ ID NO: 3.

[0115] Thus, reference to a mutant CsgG pore monomer comprising a variant of the sequence as shown in SEQ ID NO: 3 and specific amino-acid mutations thereof as set out in the claims and elsewhere in the specification also encompasses a mutant CsgG pore monomer comprising a variant of any of the sequences shown in SEQ ID NOs: 68 to 88 of WO 2019 / 002893 (incorporated by reference herein in its entirety) and corresponding aminoacid mutations thereof.

[0116] The CsgG pore monomer may also be any of the sequences described in WO 2023 / 060420, WO 2023 / 60418, WO 2023 / 60422, WO 2023 / 060421, WO 2023 / 019470, CN114957412, WO 2023 / 019471, WO 2023 / 060419 and WO 2023 / 050031 (all incorporated herein by reference in their entireties) or a variant thereof. The CsgG pore monomer may also be any of the sequences described in WO 2023 / 123370, WO 2024 / 138470, WO 2024 / 138472, WO 2024 / 138424, WO 2024 / 138425, WO 2024 / 138512 and WO 2024 / 138565 (all incorporated herein by reference in their entireties) or a variant thereof.

[0117] It will further be appreciated that the invention extends to other variant CsgG pore monomers not expressly identified in the specification that show highly conserved regions.

[0118] Standard methods in the art may be used to determine homology. For example, the UWGCG Package provides the BESTFIT program which can be used to calculate homology, for example used on its default settings (Devereux et al (1984) Nucleic Acids Research 12, p387-395). The PILEUP and BLAST algorithms can be used to calculate homology or line up sequences (such as identifying equivalent residues or corresponding sequences (typically on their default settings)), for example as described in Altschul S. F. (1993) J Mol Evol 36:290- 300; Altschul, S.F et al (1990) J Mol Biol 215:403-10. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ).

[0119] SEQ ID NO: 3 is the wild-type CsgG pore monomer from Escherichia coli Str. K-12 substr. MC4100. A variant of SEQ ID NO: 3 may comprise any of the substitutions present in another CsgG homologue. Preferred CsgG homologues are shown in SEQ ID NOs: 68 to 88 of WO 2019 / 002893 (incorporated by reference herein in its entirety). The variant may comprise combinations of one or more of the substitutions present in SEQ ID NOs: 68 to 88 WO 2019 / 002893 (incorporated by reference herein in its entirety) compared with SEQ ID NO: 3, including one or more substitutions, one or more conservative mutations, one or more deletions or one or more insertion mutations, such as deletion or insertion of 1 to 10 amino acids, such as of 2 to 8 or 3 to 6 amino acids.

[0120] The CsgG pore monomer in the pore monomer conjugate of the invention typically retains the ability to form the same 3D structure as the wild-type CsgG pore monomer, such as the same 3D structure as a CsgG pore monomer having the sequence of SEQ ID NO: 3. The 3D structure of CsgG is known in the art and is disclosed, for example, in Goyal et al (2014) Nature 516(7530):250-3. Any number of mutations may be made in the wild-type CsgG sequence in addition to the mutations described herein provided that the CsgG pore monomer retains the improved properties imparted on it by the mutations of the present invention.

[0121] Typically, the CsgG pore monomer will retain the ability to form a structure comprising five alpha-helices and five beta-strands. Therefore, it is envisaged that further mutations may be made in any of these regions in any CsgG pore monomer without affecting the ability of the monomer to form a pore that can translocate analytes, such as polynucleotides or polypeptides. It is also expected that deletions of one or more amino acids can be made in any of the loop regions linking the alpha helices and beta-strands and / or in the N-terminal and / or C-terminal regions of the CsgG pore monomer without affecting the ability of the monomer to form a pore that can translocate polynucleotides.

[0122] Amino acid substitutions may be made to the amino acid sequence of SEQ ID NO: 3 in addition to those discussed above, for example up to 1, 2, 3, 4, 5, 10, 20 or 30 substitutions. Conservative substitutions replace amino acids with other amino acids of similar chemical structure, similar chemical properties, or similar side-chain volume. The amino acids introduced may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality, or charge to the amino acids they replace. Alternatively, the conservative substitution may introduce another amino acid that is aromatic or aliphatic in the place of a pre-existing aromatic or aliphatic amino acid. Conservative amino acid changes are well- known in the art.

[0123] The CsgG pore monomer may be modified to introduce one or more cysteines, one or more hydrophobic amino acids, one or more charged amino acids, one or more non-native amino acids, or one or more polar amino acids. Any number and combination of such introductions may be made. The introduction is preferably by substitution or addition.

[0124] One or more amino acid residues of the amino acid sequence of SEQ ID NO: 3 may additionally be deleted from the polypeptides described above. Up to 1, 2, 3, 4, 5, 10, 20 or 30 or more residues may be deleted.

[0125] Variants may include fragments of SEQ ID NO: 3. Such fragments retain pore forming activity. Fragments may be at least about 50, at least about 100, at least about 150, at least about 200 or at least about 250 amino acids in length. Such fragments may be used to produce the pores. A fragment preferably comprises the transmembrane beta barrel region of SEQ ID NO: 3, namely residues 132 to 155 and 181 to 211, or a variant thereof as discussed above.

[0126] One or more amino acids may be alternatively or additionally added to the polypeptides described above. An extension may be provided at the amino terminal or carboxy terminal of the amino acid sequence of SEQ ID NO: 3 or polypeptide variant or fragment thereof. The extension may be quite short, for example from 1 to 10 amino acids in length. Alternatively, the extension may be longer, for example up to 50 or 100 amino acids. A carrier protein may be fused to an amino acid sequence according to the invention. Other fusion proteins are discussed in more detail below.

[0127] A variant of SEQ ID NO: 3 is a polypeptide that has an amino acid sequence which varies from that of SEQ ID NO: 3 and which retains its ability to form a pore. A variant typically contains the regions of SEQ ID NO: 3 that are responsible for pore formation. The pore forming ability of CsgG, which contains a beta-barrel (p-barrel), is provided by p-strands in the transmembrane beta barrel region of each monomer. A variant of SEQ ID NO: 3 typically comprises the region in SEQ ID NO: 3 that forms p-strands, namely residues 132 to 155 and 181 to 211, or a variant thereof as discussed above. One or more modifications can be made to the region of SEQ ID NO: 3 that form p-strands as long as the resulting variant retains its ability to form a pore.

[0128] The one or more modifications in the CsgG pore monomer preferably improve the ability of a pore complex comprising the pore monomer to characterise an analyte. For example, modifications / mutations / substitutions are contemplated to alter the number, size, shape, placement, or orientation of the constriction within a channel from the pore monomer conjugate of the invention. The CsgG pore monomer or the variant of SEQ ID NO: 3 may have any of the particular modifications or substitutions disclosed in WO 2016 / 034591, WO 2017 / 149316, WO 2017 / 149317, WO 2017 / 149318, WO 2018 / 211241, WO 2019 / 002893, WO 2023 / 198911, WO 2024 / 033421, WO 2024 / 033422, WO 2024 / 033443, and WO 2024 / 089270 (all incorporated by reference herein in their entirety).

[0129] CsgF peptide

[0130] The accessory polypeptide may be a CsgF peptide. The term "CsgF peptide" preferably defines a CsgF peptide that has been truncated from its C-terminal end (i.e., is an N- terminal fragment). The CsgF peptide may be a fragment of wild-type E. coli CsgF (SEQ ID NO: 5 or SEQ ID NO: 6), or of a wild-type homologue of E. coli CsgF, such as for example, a peptide comprising any one of the amino acid sequences shown in WO 2019 / 002893 (incorporated by reference herein in its entirety). A CsgF homologue is referred to as a polypeptide that has at least about 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% complete sequence identity to wild-type E. coli CsgF as shown in SEQ ID NO: 6. A CsgF homologue may also referred to as a polypeptide that contains the PFAM domain PF10614, which is characteristic for CsgF-like proteins. A list of presently known CsgF homologues and CsgF architectures can be found at . Mature CsgF

[0131] (shown in SEQ ID NO:6) can be divided into three main regions: a "CsgF constriction peptide" (FCP), a "neck" region and a "head" region. The "head" region of the CsgF peptide is distinct from a constriction of a pore as described herein. The "head" region of the CsgF peptide may also be referred to as the "C-terminal head domain". The structure of CsgF is discussed in detail in WO 2019 / 002893 (incorporated by reference herein in its entirety).

[0132] The CsgF peptide used in the pore monomer conjugate of the invention is preferably a truncated CsgF peptide lacking the C-terminal head; lacking the C-terminal head and a part of the neck domain of CsgF (e.g., the truncated CsgF peptide may comprise only a portion of the neck domain of CsgF); or lacking the C-terminal head and neck domains of CsgF. The CsgF peptide may lack part of the CsgF neck domain, e.g. the CsgF peptide may comprise a portion of the neck domain, such as for example, from amino acid residue 36 at the N- terminal end of the neck domain (see SEQ ID:NO:6) (e.g. residues 36-40, 36-41, 36-42, 36-43, 36-45,36-46 up to residues 36-50 or 36-60 of SEQ ID NO: 6). The CsgF peptide preferably comprises a CsgG-binding region and a region that forms a constriction in the pore. The CsgG-binding region typically comprises residues 1 to 11 and / or 29 to 32 of the CsgF protein (SEQ ID NO: 6 or a homologue from another species) and may include one or more modifications. The region that forms a constriction in the pore typically comprises residues 9 to 28 of the CsgF protein (SEQ ID NO: 6 or a homologue from another species) and may include one or more modifications. Residues 9 to 17 comprise the conserved motif N9PXFGGXXX17 and form a turn region. Residues 9 to 28 form an alpha-helix. Xi7(N17 in SEQ ID NO: 6) forms the apex of the constriction region, corresponding to the narrowest part of the CsgF constriction in the pore. The CsgF constriction region also makes stabilising contacts with the CsgG beta-barrel, primarily at residues 8, 9, 10, 11, 12, 18, 21, 22, 29 and 30 of SEQ ID NO: 6.

[0133] The CsgF peptide typically has a length of from 5 to 50 amino acids, such as 10 to 49, 20 to 47, 30 to 45 or 32 to 40 amino acids. Preferably the CsgF peptide comprises from 29 to 35 amino acids, or 29 to 45 amino acids. The CsgF peptide comprises all or part of the FCP, which corresponds to residues 1 to 35 of SEQ ID NO: 6. Where the CsgF peptide is shorter that the FCP, the truncation is preferably made at the C-terminal end.

[0134] The CsgF peptide may have a length of about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about

[0135] 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about

[0136] 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about

[0137] 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about

[0138] 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54 or about 55 amino acids.

[0139] The CsgF peptide may be produced by cleavage of a longer protein, such as full-length CsgF using an enzyme. Cleavage at a particular site may be directed by modifying the longer protein, such as full-length CsgF, to include an enzyme cleavage site at an appropriate position. Examples of CsgF amino acid sequences that have been modified to include such enzyme cleavage sites are shown in SEQ ID NOs: 56 to 67 of WO 2019 / 002893 (incorporated by reference herein in its entirety). Following cleavage all or part of the added enzyme cleavage site may be present in the CsgF peptide that associates with CsgG to form a pore. Thus, the CsgF peptide may further comprise all or part of an enzyme cleavage site at its C-terminal end.

[0140] Some examples of suitable CsgF peptides are shown in Table 3 of WO 2019 / 002893 (incorporated by reference herein in its entirety). The CsgF peptide may be any size but preferably has the dimensions of residues 1-29 of wild-type CsgF (Figure 8). The channel defined by the bottom of the CsgF peptide is preferably from about 10 to about 55 A in diameter, such as from about 20 to about 50 A or from about 30 to about 40 A in diameter. The channel defined by the bottom of the CsgF peptide region is preferably about 34 A in diameter.

[0141] The channel defined by the CsgF peptide is preferably from about 2 to about 40 A in diameter at its narrowest point, such as from about 5 to about 35 A, from about 8 to about 25 A or from about 10 to about 20 A in diameter at its narrowest point. The channel defined by the constriction region is preferably about 19.0 A in diameter at its narrowest point. The constriction in the CsgF peptide is preferably from about 2 to about 40 A in diameter, such as from about 5 to about 35 A, from about 8 to about 25 A or from about 10 to about 20 A in diameter. The constriction in the CsgF peptide is preferably about 19.0 A in diameter. In the context of a protein conjugate of the invention, the CsgF peptide constriction may be the second constriction. All of the measurements above are based on measuring from backbone to backbone of the amino acids forming the different regions (as shown in Figure 8). All of the measurements typically apply to the CsgF peptide before it is modified in accordance with the invention.

[0142] The CsgF peptide is preferably a variant of any of the CsgF sequences discussed above, including SEQ ID NO: 6, comprising one or more modifications compared with the comparative sequence. Over the entire length of the amino acid sequence of SEQ ID NO: 6, a variant will preferably be at least about 40% homologous to that sequence based on amino acid identity. More preferably, the variant may be at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% and more preferably at least about 95%, 97% or 99% homologous based on amino acid identity to the amino acid sequence of SEQ ID NO: 6 over the entire sequence. Over the entire length of the amino acid sequence of SEQ ID NO: 6, a variant will preferably be at least about 40% identical to that sequence. More preferably, the variant may be at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% and more preferably at least about 95%, 97% or 99% identical to SEQ ID NO: 6 over the entire sequence. There may be at least about 80%, for example at least about 85%, at least about 90% or at least about 95%, amino acid identity over a stretch of 100 or more, for example 125, 150, 175 or 200 or more, contiguous amino acids ("hard homology"). These levels of homology / identity equally apply to any of the other CsgF peptides described above.

[0143] Any number of the CsgF peptides in the pore or pore complex of the invention, such as 6, 7, 8, 9 or 10, may contain one or more substitutions compared with SEQ ID NO: 6. All six to ten monomers in the pore or pore complex preferably contain one or more substitutions compared with SEQ ID NO: 6. The CsgF peptides in the pore complex may be the same or different. The CsgF peptides are preferably identical in each pore monomer conjugate in the pore complex of the invention.

[0144] Stabilisation and other mutations

[0145] In the conjugates or pore complexes of the invention, the interaction between the CsgF peptide and the CsgG pore may, for example, be stabilised by hydrophobic interactions and / or electrostatic interactions. These may be interactions between one or more of the following pairs of positions of SEQ ID NO: 6 and SEQ ID NO: 3, respectively: 1 and 153, 4 and 133, 5 and 136, 8 and 187, 8 and 203, 9 and 203, 11 and 142, 11 and 201, 12 and 149, 12 and 203, 26 and 191, and 29 and 144.

[0146] The residues in the CsgF peptide and / or the CsgG pore monomer at one or more of the positions listed above may be modified in order to enhance the interaction between CsgG and CsgF in the pore complex. Although the CsgG:CsgF complex is very stable, when CsgF is truncated, the stability of CsgG:CsgF complexes decrease compared to a complex comprising full length CsgF. Therefore, disulfide bonds can be made between CsgG and CsgF to make the complex more stable, for example following introduction of cysteine residues at the positions identified herein. The pore complex can be made in any of the previously mentioned methods and disulfide bond formation can be induced by using oxidising agents (eg: Copper-orthophenanthroline). Other interactions (eg: hydrophobic interactions, charge-charge interactions / electrostatic interactions) can also be used in those positions instead of cysteine interactions.

[0147] Unnatural amino acids can also be incorporated in those positions. Covalent bonds may be by via click chemistry. For example, unnatural amino acids with azide or alkyne or with a di benzocyclooctyne (DBCO) group and / or a bicyclo[6.1.0]nonyne (BCN) group may be introduced at one or more of these positions.

[0148] Such stabilising mutations can be combined with any other modifications to CsgG and / or CsgF, for example the modifications disclosed herein.

[0149] To facilitate such interactions, one or more non-native reactive amino acids may be included / substituted in the CsgG pore monomer at one or more positions corresponding to one or more of positions 132, 133, 136, 138, 140, 142, 144, 145, 147, 149, 151, 153, 155, 183, 185, 187, 189, 191, 201, 203, 205, 207 and 209 of SEQ ID NO: 3.

[0150] To facilitate such interactions, one or more non-native reactive amino acids may be included / substituted at one or more positions corresponding to one or more of positions 1, 4, 5, 8, 9, 11, 12, 26 or 29 of SEQ ID NO: 6. PorARc pores

[0151] A PorARc pore monomer is a monomer that is capable of forming a PorARc pore. Such monomers are known in the art, especially from WO 2023 / 118404 and WO 2024 / 089270 (incorporated by reference herein in their entireties).

[0152] The PorARc pore monomer preferably comprises a cap region (or scaffold) (e.g., C in Figure 9) and a constriction region (e.g., D is Figure 9). The PorARc pore monomer preferably comprises one or more of (a) a cap region (e.g. A in Figure 9), (b) a constriction region (e.g., D in Figure 9), and (c) a transmembrane beta barrel region (e.g., B in Figure 9), such as (a), (b), (c), (a) and (b), (a) and (c), (b) and (c), or (a), (b) and (c). The chimeric pore monomer preferably comprises (a)-(c). The PorARc pore may have any structure but preferably has or comprises the structure of the wild-type PorARc pore (Figure 9). The protein structure of PorARc defines a channel or hole that allows the translocation of molecules and ions from one side of the membrane to the other.

[0153] The PorARc pore may be any size but preferably has the dimensions of the wild-type PorARc_Rco (Figure 9). The PorARc pore preferably has an external diameter of from about 70 to about 110 A at its widest point, such as from about 80 to about 100 A or from about 85 to about 95 A at its widest point. The PorARc pore preferably has an external diameter of about 90.7 A at its widest point. The PorARc pore preferably has a total length of from about 70 to about 110 A, such as from about 80 to about 100 A or from about 85 to about 95 A. The PorARc pore preferably has a total length of about 90.4 A. References to "total length" and "length" relate to the length of the pore or pore region when viewed from the side (see, e.g., the side view in Figure 9).

[0154] The cap region (A in Figure 9) preferably has a length of from about 25 to about 65 A, such as from about 35 to about 55 A or from about 40 to about 50 A. The cap region preferably has a length of about 44.7 A. The channel defined by the cap region preferably has an opening of from about 30 to about 70 A in diameter, such as from about 40 to about 60 A or from about 45 to about 55 A in diameter. The channel defined by the cap region preferably has an opening of about 49 A in diameter. The channel defined by the cap region is preferably from about 20 to about 60 A in diameter at its narrowest point, such as from about 30 to about 50 A or from about 35 to about 45 A in diameter at its narrowest point. The channel defined by the cap region is preferably about 41.5 A in diameter at its narrowest point.

[0155] The transmembrane beta barrel region (B in Figure 9) preferably has a length of from about 5 to about 45 A, such as from about 15 to about 35 A or from about 20 to about 30 A. The transmembrane beta barrel preferably has a length of about 26.2 A. The channel defined by the transmembrane beta barrel region is preferably from about 20 to about 60 A in diameter at its narrowest point, such as from about 30 to about 50 A or from about 35 to about 45 A in diameter at its narrowest point. The channel defined by the transmembrane beta barrel region is preferably about 39.8 A in diameter at its narrowest point.

[0156] The cap region (or scaffold) (C in Figure 9 and formed from A and B) preferably has a length of from about 55 to about 95 A, such as from about 65 to about 85 A or from about 70 to about 80 A. The cap region (or scaffold) preferably has a length of about 73.6 A. The channel defined by the cap region (or scaffold) (C) is preferably from about 20 to about 60 A in diameter at its narrowest point, such as from about 30 to about 50 A or from about 35 to about 45 A in diameter at its narrowest point. The channel defined by the cap region (or scaffold) (C) is preferably about 39.8 A in diameter at its narrowest point.

[0157] The constriction region (D in Figure 9) preferably has a length of from about 5 to about 40 A, such as from about 10 to about 30 A or from about 15 to about 25 A. The constriction region preferably has a length of about 19.7 A. The channel defined by the constriction region is preferably from about 10 to about 50 A in diameter at its narrowest point, such as from about 20 to about 40 A, from about 22 to about 32 A or from about 25 to about 35 A in diameter at its narrowest point. The channel defined by the constriction region is preferably about 27.4 A in diameter at its narrowest point. The channel defined by the constriction region is preferably from about 10 to about 50 A in diameter at its narrowest point, such as from about 15 to about 55 A, from about 25 to about 45 A or from about 30 to about 40 A in diameter at the base of the pore structure. The channel defined by the constriction region is preferably about 36.1 A in diameter at the base of the pore structure. The constriction region is preferably from about 20 to about 60 A in diameter, such as from about 30 to about 50 A or from about 35 to about 45 A. The constriction region is preferably about 41.9 A in diameter.

[0158] All of the measurements above are based on measuring from backbone to backbone of the amino acids forming the different regions (as shown in Figure 9). All of the measurements typically apply to the PorARc pore monomer or PorARc pore before it is modified in accordance with the invention.

[0159] The regions in PorARc may have any of the amino acid lengths described in WO 2024 / 089270 (incorporated by reference herein in its entirety). The PorARc pore may be any of those in WO 2023 / 118404 or Table 2 of WO 2024 / 089270 (incorporated by reference herein in their entireties).

[0160] Third constriction region

[0161] The accessory polypeptide is preferably further modified to comprise at least a third constriction region which forms part of a third constriction in the channel. The addition of the third constriction region in these embodiments does not increase the distance between the first constriction region and the second constriction region.

[0162] The accessory polypeptide may be a fusion polypeptide comprising a first portion comprising a CsgF peptide and a second portion comprising a helix-forming auxiliary protein, wherein the fusion protein is attached to the pore monomer. The auxiliary protein can be designed de novo using computer-based structural analysis tools to confer certain desirable features to the CsgG monomer (e.g., modulation of pore width, lengthening of pore lumen, formation of one or more additional constrictions, etc.). The de novo designed auxiliary protein may form one or more additional constrictions in the lumen of a CsgG pore and improve discrimination of polymer units as an analyte moves through the pore. The auxiliary protein may be any of the pore monomers described in WO 2024 / 033447 (incorporated by reference herein in its entirety).

[0163] As used herein "a portion" refers to 2 or more amino acids. In some embodiments, a portion comprises at least about 5, about 10, about 20, about 30, about 50, about or about 100 amino acids (e.g., about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about

[0164] 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about

[0165] 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about

[0166] 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about

[0167] 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about

[0168] 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about

[0169] 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about

[0170] 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about

[0171] 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about

[0172] 93, about 94, about 95, about 96, about 97, about 98, about 99, or about 100 amino acids), either consecutive or with gaps, of the complete amino acid sequence of the polypeptide / protein, or the full amino acid sequence of the polypeptide / protein.

[0173] The first portion typically forms the second constriction region. The second portion typically forms a third constriction region which forms part of a third constriction in the channel.

[0174] In some embodiments, the distance (e.g., vertical distance) between the second constriction region and third constriction region ranges from about 5 A to about 80 A or from about 20 A to about 80 A. This can be measured as the distance between the alphacarbons (Ca) of the amino acid residue extending furthest into the lumen of the nanopore forming the first constriction and the amino acid residue extending furthest into the lumen of the nanopore forming the second constriction. In some embodiments, the pore monomer conjugate or the complex has an axial length greater than about 90 A, optionally wherein the axial length ranges from about 95 A to about 160 A.

[0175] In some embodiments, the distance between the second constriction region and the third constriction region is about 5 A, about 6 A, about 7 A, about 8 A, about 9 A, about 10 A, about 11 A, about 12 A, about 13 A, about 14 A, about 15 A, about 16 A, about 17 A, about 18 A, about 19 A, about 20 A, about 21 A, about 22 A, about 23 A, about 24 A, about 25 A, about 26 A, about 27 A, about 28 A, about 29 A, about 30 A, about 31 A, about 32 A, about 33 A, about 34 A, about 35 A, about 36 A, about 37 A, about 38 A, about 39 A, about 40 A, about 41 A, about 42 A, about 43 A, about 44 A, about 45 A, about 46 A, about 47 A, about 48 A, about 49 A, about 50 A, about 51 A, about 52 A, about 53 A, about 54 A, about 55 A, about 56 A, about 57 A, about 58 A, about 59 A, about 60 A, about 61 A, about 62 A, about 63 A, about 64 A, about 65 A, about 66 A, about 67 A, about 68 A, about 69 A, about 70 A, about 71 A, about 72 A, about 73 A, about 74 A, about 75 A, about 76 A, about 77 A, about 78 A, about 79 A, about or 80 A in length. In some embodiments, the distance between the second constriction region and the third constriction region is more than about 80 A in length, such as about 90 A or about 100 A in length or more.

[0176] In some embodiments, the distance between the first constriction region and the third constriction region ranges from about 10 A to about 160 A. In some embodiments, the distance between the first constriction region and the third constriction region is about 10 A, about 11 A, about 12 A, about 13 A, about 14 A, about 15 A, about 16 A, about 17 A, about 18 A, about 19 A, about 20 A, about 21 A, about 22 A, about 23 A, about 24 A, about 25 A, about 26 A, about 27 A, about 28 A, about 29 A, about 30 A, about 31 A, about 32 A, about 33 A, about 34 A, about 35 A, about 36 A, about 37 A, about 38 A, about 39 A, about 40 A, about 41 A, about 42 A, about 43 A, about 44 A, about 45 A, about 46 A, about 47 A, about 48 A, about 49 A, about 50 A, about 51 A, about 52 A, about 53 A, about 54 A, about 55 A, about 56 A, about 57 A, about 58 A, about 59 A, about 60 A, about 61 A, about 62 A, about 63 A, about 64 A, about 65 A, about 66 A, about 67 A, about 68 A, about 69 A, about 70 A, about 71 A, about 72 A, about 73 A, about 74 A, about 75 A, about 76 A, about 77 A, about 78 A, about 79 A, about 80 A, about 81 A, about 82 A, about 83 A, about 84 A, about 85 A, about 86 A, about 87 A, about 88 A, about 89 A, about 90 A, about 91 A, about 92 A, about 93 A, about 94 A, about 95 A, about 96 A, about 97 A, about 98 A, about 99 A, about 100 A, about 101 A, about 102 A, about 103 A, about 104 A, about 105 A, about 106 A, about 107 A, about 108 A, about 109 A, about 110 A, about 111 A, about 112 A, about 113 A, about 114 A, about 115 A, about 116 A, about 117 A, about 118 A, about 119 A, about 120 A, about 121 A, about 122 A, about 123 A, about 124 A, about 125 A, about 126 A, about 127 A, about 128 A, about 129 A, about 130 A, about 131 A, about 132 A, about 133 A, about 134 A, about 135 A, about 136 A, about 137 A, about 138 A, about 139 A, about 140 A, about 141 A, about 142 A, about 143 A, about 144 A, about 145 A, about 146 A, about 147 A, about 148 A, about 149 A, about 150 A, about 151 A, about 152 A, about 153 A, about 154 A, about 155 A, about 156 A, about 157 A, about 158 A, about 159 A, about or 160 A in length. In some embodiments, about the distance between the first constriction region and the third constriction region is more than about 160 A in length, such as about 190 A or about 200 A in length or more.

[0177] Lvsenin pore monomers

[0178] A lysenin pore monomer is a monomer that is capable of forming a lysenin pore. The lysenin pore formed by the monomer may have any structure but preferably has or comprises the structure of the wild-type lysenin pore (Figure 15). The protein structure of lysenin defines a channel or hole that allows the translocation of molecules and ions from one side of the membrane to the other.

[0179] SEQ ID NO: 7 shows the sequence of wild-type lysenin pore monomer. The lysenin pore monomer is preferably a variant of SEQ ID NO: 7. The variant lysenin momomer may also be referred to as a modified lysenin pore monomer or a mutant lysenin pore monomer. The modifications, or mutations, in the variant include but are not limited to any one or more of the modifications disclosed herein, or combinations of said modifications. The lysenin pore monomer may be a lysenin homologue monomer. A lysenin homologue monomer is a polypeptide that has at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 99% complete sequence identity to SEQ ID NO: 7.

[0180] The variant of SEQ ID NO: 7 may be a structural variant. The structural variant may have a root mean square deviation (RMSD) of less than about 4.0 Angstroms (A), such as less than about 3.5 A, less than about 3.0 A, less than about 2.5 A, less than about 2.0 A, less than about 1.5 A, less than about 1.0 A or less than about 0.5 A, when compared with a protein having the sequence of SEQ ID NO: 7. RMSD may be calculated as discussed above.

[0181] Over the entire length of the amino acid sequence of SEQ ID NO: 7, a variant will preferably be at least 40% homologous to that sequence based on amino acid identity. More preferably, the variant may be at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% and more preferably at least 95%, 97% or 99% homologous based on amino acid identity to the amino acid sequence of SEQ ID NO: 7 over the entire sequence. Over the entire length of the amino acid sequence of SEQ ID NO: 7, a variant will preferably be at least 40% identical to that sequence. More preferably, the variant may be at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% and more preferably at least 95%, 97% or 99% identical to SEQ ID NO: 7 over the entire sequence.

[0182] Sequence identity can also relate to a fragment or portion of the lysenin pore monomer. Hence, a sequence may have less than 40% overall sequence homology / identity with SEQ ID NO: 7, but the sequence of a particular region, domain or subunit could share at least about 80%, at least about 90%, or as much as about 99% sequence homology / identity with the corresponding region of SEQ ID NO: 7. There may be at least about 80%, for example at least 85%, at least about 90% or at least about 95%, amino acid identity over a stretch of 100 or more, for example 125, 150, 175 or 200 or more, contiguous amino acids ("hard homology"). Homology and / or identity is typically measured over the entire length of the cap region.

[0183] The variant may include any of the substitutions, additions and / or deletions discussed above with reference to CsgG pore monomers.

[0184] The pore monomer may be any of those described in WO 2013 / 153359 and WO 2017 / 174990 (incorporated by reference herein in their entireties).

[0185] Alpha-hemolysin pore monomers

[0186] An alpha-hemolysin pore monomer is a monomer that is capable of forming an alphahemolysin pore. The alpha-hemolysin pore formed by the monomer may have any structure but preferably has or comprises the structure of the wild-type alpha-hemolysin pore (Figure 15). The protein structure of alpha-hemolysin defines a channel or hole that allows the translocation of molecules and ions from one side of the membrane to the other.

[0187] SEQ ID NO: 8 shows the sequence of wild-type alpha-hemolysin pore monomer. The alphahemolysin pore monomer is preferably a variant of SEQ ID NO: 8. The variant alphahemolysin momomer may also be referred to as a modified alpha-hemolysin pore monomer or a mutant alpha-hemolysin pore monomer. The modifications, or mutations, in the variant include but are not limited to any one or more of the modifications disclosed herein, or combinations of said modifications. The alpha-hemolysin pore monomer may be an alphahemolysin homologue monomer. An alpha-hemolysin homologue monomer is a polypeptide that has at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 99% complete sequence identity to SEQ ID NO: 8.

[0188] The variant of SEQ ID NO: 8 may be a structural variant. The structural variant may have a root mean square deviation (RMSD) of less than about 4.0 Angstroms (A), such as less than about 3.5 A, less than about 3.0 A, less than about 2.5 A, less than about 2.0 A, less than about 1.5 A, less than about 1.0 A or less than about 0.5 A, when compared with a protein having the sequence of SEQ ID NO: 8. RMSD may be calculated as discussed above.

[0189] Over the entire length of the amino acid sequence of SEQ ID NO: 8, a variant will preferably be at least 40% homologous to that sequence based on amino acid identity. More preferably, the variant may be at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% and more preferably at least 95%, 97% or 99% homologous based on amino acid identity to the amino acid sequence of SEQ ID NO: 8 over the entire sequence. Over the entire length of the amino acid sequence of SEQ ID NO: 8, a variant will preferably be at least 40% identical to that sequence. More preferably, the variant may be at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% and more preferably at least 95%, 97% or 99% identical to SEQ ID NO: 8 over the entire sequence.

[0190] Sequence identity can also relate to a fragment or portion of the alpha-hemolysin pore monomer. Hence, a sequence may have less than 40% overall sequence homology / identity with SEQ ID NO: 8, but the sequence of a particular region, domain or subunit could share at least about 80%, at least about 90%, or as much as about 99% sequence homology / identity with the corresponding region of SEQ ID NO: 8. There may be at least about 80%, for example at least 85%, at least about 90% or at least about 95%, amino acid identity over a stretch of 100 or more, for example 125, 150, 175 or 200 or more, contiguous amino acids ("hard homology"). Homology and / or identity is typically measured over the entire length of the cap region.

[0191] The variant may include any of the substitutions, additions and / or deletions discussed above with reference to CsgG pore monomers.

[0192] A variant of SEQ ID NO: 8 may lack Ml (i.e., may lack the methionine at the N-terminus).

[0193] The pore monomer may be any of those described in WO 2010 / 004273 (incorporated by reference herein in its entirety).

[0194] CvtK pore monomers

[0195] A CytK pore monomer is a monomer that is capable of forming a CytK pore. The CytK pore formed by the monomer may have any structure but preferably has or comprises the structure of the wild-type CytK pore. The protein structure of CytK defines a channel or hole that allows the translocation of molecules and ions from one side of the membrane to the other. SEQ ID NO: 9 shows the sequence of wild-type CytK pore monomer. The CytK pore monomer is preferably a variant of SEQ ID NO: 9. The variant CytK momomer may also be referred to as a modified CytK pore monomer or a mutant CytK pore monomer. The modifications, or mutations, in the variant include but are not limited to any one or more of the modifications disclosed herein, or combinations of said modifications. The CytK pore monomer may be a CytK homologue monomer. A CytK homologue monomer is a polypeptide that has at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 99% complete sequence identity to SEQ ID NO: 9.

[0196] The variant of SEQ ID NO: 9 may be a structural variant. The structural variant may have a root mean square deviation (RMSD) of less than about 4.0 Angstroms (A), such as less than about 3.5 A, less than about 3.0 A, less than about 2.5 A, less than about 2.0 A, less than about 1.5 A, less than about 1.0 A or less than about 0.5 A, when compared with a protein having the sequence of SEQ ID NO: 9. RMSD may be calculated as discussed above.

[0197] Over the entire length of the amino acid sequence of SEQ ID NO: 9, a variant will preferably be at least 40% homologous to that sequence based on amino acid identity. More preferably, the variant may be at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% and more preferably at least 95%, 97% or 99% homologous based on amino acid identity to the amino acid sequence of SEQ ID NO: 9 over the entire sequence. Over the entire length of the amino acid sequence of SEQ ID NO: 9, a variant will preferably be at least 40% identical to that sequence. More preferably, the variant may be at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% and more preferably at least 95%, 97% or 99% identical to SEQ ID NO: 9 over the entire sequence.

[0198] Sequence identity can also relate to a fragment or portion of the CytK pore monomer. Hence, a sequence may have less than 40% overall sequence homology / identity with SEQ ID NO: 9, but the sequence of a particular region, domain or subunit could share at least about 80%, at least about 90%, or as much as about 99% sequence homology / identity with the corresponding region of SEQ ID NO: 9. There may be at least about 80%, for example at least 85%, at least about 90% or at least about 95%, amino acid identity over a stretch of 100 or more, for example 125, 150, 175 or 200 or more, contiguous amino acids ("hard homology"). Homology and / or identity is typically measured over the entire length of the cap region.

[0199] The variant may include any of the substitutions, additions and / or deletions discussed above with reference to CsgG pore monomers. The pore monomer may be any of those described in WO 2023 / 026056 (incorporated by reference herein in its entirety).

[0200] Increasing the distance between the first and second constrictions

[0201] In both embodiments of the invention, i.e. in the pore monomers of the invention and in the pore monomer conjugates of the invention, the pore monomer is modified to increase the distance between the first constriction region and the second constriction region. The pore monomer may be elongated to increase the distance between the first constriction region and the second constriction region. The length (such as the vertical length) of the pore monomer may be increased to increase the distance between the first constriction region and the second constriction region. The skilled person is capable of determining whether or not a pore monomer has been modified or elongated in accordance with the invention. For instance, the skilled person can compare the pore monomer with the natural or wild-type sequence, such as SEQ ID NO: 3.

[0202] The distance between the first constriction region and the second constriction region may be increased by any amount. For instance, the distance may be increased by at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 43.5%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 69.7%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97.1%, at least about 100%, at least about 110%, at least about 113%, at least about 120%, at least about 130%, at least about 140%, at least about 149%, or at least about 150%. The at least one property is preferably increased by at least about 1.1 fold, at least about 1.49 fold, at least about 1.5 fold, at least about 2-fold, such as by at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7- fold, at least about 8-fold, at least about 9-fold, or at least about 10-fold. This is compared to any of the distances between constrictions set out above.

[0203] The distance between the first constriction region and the second constriction region is preferably increased by about 5 A or more. The distance is preferably increased by from about 5 A to about 100 A, such as from about 5.5 A to about 90 A, from about 6 A to about 80 A, from about 7 A to about 70 A, from about 8 A to about 60 A, from about 9 A to about 50 A or from about 10 A to about 40 A.

[0204] The distance may be increased by about 5 A, about 5.5 A, about 6 A, about 6.5 A, about 7 A, about 7.5 A, about 8 A, about 8.5 A, about 9 A, about 9.5 A, about 10 A, about 10.5 A, about 10.8 A, about 11 A, about 11.5 A, about 12 A, about 12.5 A, about 13 A, about 13.5 A, about 14 A, about 14.5 A, about 15 A, about 15.5 A, about 16 A, about 16.5 A, about 17 A, about 17.3 A, about 17.5 A, about 18 A, about 18.5 A, about 19 A, about 19.5 A, about 20 A, about 20.5 A, about 21 A, about 21.5 A, about 22 A, about 22.5 A, about 23 A, about

[0205] 23.5 A, about 24 A, about 24.1 A, about 24.5 A, about 25 A, about 25.5 A, about 26 A, about 26.5 A, about 27 A, about 27.5 A, about 28 A, about 28.1 A, about 28.5 A, about 29 A, about 29.5 A, about 30 A, about 30.5 A, about 31 A, about 31.5 A, about 32 A, about

[0206] 32.5 A, about 33 A, about 33.5 A, about 34 A, about 34.5 A, about 35 A, about 35.5 A, about 36 A, about 36.5 A, about 37 A, about 37.1 A, about 37.5 A, about 38 A, about 38.5 A, about 39 A, about 39.5 A, about 40 A, about 40.5 A, about 41 A, about 41.5 A, about 42 A, about 42.5 A, about 43 A, about 43.5 A, about 44 A, about 44.5 A, about 45 A, about

[0207] 45.5 A, about 46 A, about 46.5 A, about 47 A, about 47.5 A, about 48 A, about 48.5 A, about 49 A, about 49.5 A, about 50 A, about 50.5 A, about 51 A, about 51.5 A, about 52 A, about 52.5 A, about 53 A, about 53.5 A, about 54 A, about 54.5 A, about 55 A, about 55.5 A, about 56 A, about 56.5 A, about 57 A, about 57.5 A, about 58 A, about 58.5 A, about 59 A, about 59.5 A, about 60 A, about 60.5 A, about 61 A, about 61.5 A, about 62 A, about

[0208] 62.5 A, about 63 A, about 63.5 A, about 64 A, about 64.5 A, about 65 A, about 65.5 A, about 66 A, about 66.5 A, about 67 A, about 67.5 A, about 68 A, about 68.5 A, about 69 A, about 69.5 A, about 70 A, about 70.5 A, about 71 A, about 71.5 A, about 72 A, about 72.5 A, about 73 A, about 73.5 A, about 74 A, about 74.5 A, about 75 A, about 75.5 A, about 76 A, about 76.5 A, about 77 A, about 77.5 A, about 78 A, about 78.5 A, about 79 A, about

[0209] 79.5 A, about 80 A, about 80.5 A, about 81 A, about 81.5 A, about 82 A, about 82.5 A, about 83 A, about 83.5 A, about 84 A, about 84.5 A, about 85 A, about 85.5 A, about 86 A, about 86.5 A, about 87 A, about 87.5 A, about 88 A, about 88.5 A, about 89 A, about 89.5 A, about 90 A, about 90.5 A, about 91 A, about 91.5 A, about 92 A, about 92.5 A, about 93 A, about 93.5 A, about 94 A, about 94.5 A, about 95 A, about 95.5 A, about 96 A, about

[0210] 96.5 A, about 97 A, about 97.5 A, about 98 A, about 98.5 A, about 99 A, about 99.5 A, or about 100 A. The distance may be increased by about 41.7 A or by about 48.3 A. These distances are compared with any of the distances between constrictions set out above.

[0211] These distances are compared with pore monomer that has not been modified in accordance with the invention.

[0212] The distance between the first constriction region and the second constriction region is preferably increased by the length equivalent to about 1 or more nucleotides. The distance is preferably increased by the length equivalent to from about 1 nucleotide to about 50 nucleotides, such as from about 2 nucleotides to about 45 nucleotides, from about 3 nucleotides to about 40 nucleotides, from about 4 nucleotides to about 35 nucleotides or from about 5 nucleotides to about 30 nucleotides. The distance may be increased by the length equivalent to about 1 nucleotide, about 2 nucleotides, about 3 nucleotides, about 4 nucleotides, about 5 nucleotides, about 6 nucleotides, about 7 nucleotides, about 8 nucleotides , about 9 nucleotides, about 10 nucleotides, about 11 nucleotides, about 12 nucleotides, about 13 nucleotides, about 14 nucleotides, about 15 nucleotides, about 16 nucleotides, about 17 nucleotides, about 18 nucleotides, about 19 nucleotides, about 20 nucleotides, about 21 nucleotides, about 22 nucleotides, about 23 nucleotides, about 24 nucleotides, about 25 nucleotides, about 26 nucleotides, about 27 nucleotides, about 28 nucleotides, about 29 nucleotides, about 30 nucleotides, about 31 nucleotides, about 32 nucleotides, about 33 nucleotides, about 34 nucleotides, about 35 nucleotides, about 36 nucleotides, about 37 nucleotides, about 38 nucleotides, about 39 nucleotides, about 40 nucleotides, about 41 nucleotides, about 42 nucleotides, about 43 nucleotides, about 44 nucleotides, about 45 nucleotides, about 46 nucleotides, about 47 nucleotides, about 48 nucleotides, about 49 nucleotides, or about 50 nucleotides. These distances relate to the number of nucleotides equivalent to the length between and including the first constriction region and the second constriction region when a polynucleotide analyte passes through a pore or pore complex formed from the pore monomer or pore monomer conjugate. These distances are compared with any of the distances between constrictions set out above. These distances are compared with pore monomer that has not been modified in accordance with the invention.

[0213] In pore monomer conjugates comprising a CsgG pore monomer and a CsgF peptide, the distance between the first constriction region and the second constriction region is preferably increased to about 30 A or more. The distance is preferably increased to from about 30 A to about 100 A, such as from about 31 A to about 90 A, from about 32 A to about 80 A, from about 33 A to about 75 A from about 34 A to about 70 A or from about 35 A to about 65 A.

[0214] The distance may be increased to about 30 A, about 31 A, about 32 A, about 33 A, about 34 A, about 35 A, about 35.6 A, about 36 A, about 37 A, about 38 A, about 39 A, about 40 A, about 41 A, about 42 A, about 42.1 A, about 43 A, about 44 A, about 45 A, about 46 A, about 47 A, about 48 A, about 48.9 A, about 49 A, about 50 A, about 51 A, about 52 A, about 52.9 A, about 53 A, about 54 A, about 55 A, about 56 A, about 57 A, about 58 A, about 59 A, about 60 A, about 61 A, about 61.9 A, about 62 A, about 63 A, about 64 A, about 65 A, about 66 A, about 67 A, about 68 A, about 69 A, about 70 A, about 71 A, about 72 A, about 73 A, about 74 A, about 75 A, about 76 A, about 77 A, about 78 A, about 79 A, or about 80 A. The distance may be increased to about 66.5 A or about 73.1 A.

[0215] In pore monomer conjugates comprising a CsgG pore monomer and a CsgF peptide, the distance between the first constriction region and the second constriction region is preferably increased to about 6 or more nucleotides. The distance is preferably increased by from about 6 nucleotides to about 50 nucleotides, such as from about 7 nucleotides to about 45 nucleotides, from about 8 nucleotides to about 40 nucleotides, from about 9 nucleotides to about 35 nucleotides or from about 10 nucleotides to about 30 nucleotides. The distance may be increased to about 6 nucleotides, about 7 nucleotides, about 8 nucleotides , about 9 nucleotides, about 10 nucleotides, about 11 nucleotides, about 12 nucleotides, about 13 nucleotides, about 14 nucleotides, about 15 nucleotides, about 16 nucleotides, about 17 nucleotides, about 18 nucleotides, about 19 nucleotides, about 20 nucleotides, about 21 nucleotides, about 22 nucleotides, about 23 nucleotides, about 24 nucleotides, about 25 nucleotides, about 26 nucleotides, about 27 nucleotides, about 28 nucleotides, about 29 nucleotides, about 30 nucleotides, about 31 nucleotides, about 32 nucleotides, about 33 nucleotides, about 34 nucleotides, about 35 nucleotides, about 36 nucleotides, about 37 nucleotides, about 38 nucleotides, about 39 nucleotides, about 40 nucleotides, about 41 nucleotides, about 42 nucleotides, about 43 nucleotides, about 44 nucleotides, about 45 nucleotides, about 46 nucleotides, about 47 nucleotides, about 48 nucleotides, about 49 nucleotides, or about 50 nucleotides. These distances relate to the number of nucleotides equivalent to the length between and including the first constriction region and the second constriction region when a polynucleotide analyte passes through a pore or pore complex formed from the pore monomer or pore monomer conjugate.

[0216] The channel forming region in the pore monomer or the pore monomer conjugate may comprise or consist of a beta-sheet and / or one or more alpha helices. The channel in the pore may comprise or consist of a beta-barrel and / or an alpha-helix barrel. The channel may comprise or consist of a beta-barrel. The channel may comprise or consist of an alphahelix barrel. The channel may comprise or consist of a beta-barrel and an alpha-helix barrel.

[0217] The pore monomer may comprise a beta-sheet and / or one or more alpha helices. The pore monomer may comprise or consist of a beta-sheet. The pore monomer may comprise or consist of one or more alpha helices. The channel may comprise or consist of a beta-sheet and one or more alpha helices.

[0218] The beta-sheet in the pore monomer typically comprises one or more beta-strands. The beta-sheet may comprise any number of one or more beta-strands, such one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more or ten or more beta-strands. The beta-sheet may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 beta-strands. The beta-sheet may comprise 4 beta-strands. Multiple betasheets in multiple pore monomers may form a pore comprising a beta-barrel channel as discussed in more detail below.

[0219] The one or more alpha helices may comprise any number of one or more alpha helices, such one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more or ten or more alpha helices. The pore monomer may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 alpha helices. In some embodiments, one or more beta-strands and / or one or more alpha helices are modified to increase the distance between the first constriction region and the second constriction region. One or more beta-strands may be modified to increase the distance between the first constriction region and the second constriction region. One or more alpha helices may be modified to increase the distance between the first constriction region and the second constriction region. One or more beta-strands and one or more alpha helices may modified to increase the distance between the first constriction region and the second constriction region. The one or more beta-strands and / or one or more alpha helices are in the pore monomer.

[0220] Any number of one or more beta-strands and / or one or more alpha helices may be modified, such one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more or ten or more. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 may be modified. These numbers relate to the number of one or more beta-strands and / or one or more alpha helices modified in each pore monomer. Preferably, all of the one or more beta-strands and / or one or more alpha helices.

[0221] Any number of one or more beta-strands may be modified, such one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more or ten or more beta-strands. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 beta-strands may be modified. Preferably, all of the one or more beta-strands are modified. All 4 beta-strands may be modified.

[0222] The one or more beta-strands and / or one or more alpha helices are typically elongated. In any of the paragraphs above discussing modifying one or more beta-strands and / or one or more alpha helices, the term "modified" may be replaced with "elongated". Specific methods of doing this are discussed in more detail below.

[0223] When two or more beta-strands and / or one or more alpha helices are modified or elongated, one or more of the beta-strands and / or one or more alpha helices may be modified or elongated. All of the two or more beta-strands and / or two or more alpha helices may modified or elongated. The two or more beta-strands and / or two or more alpha helices may be modified or elongated to different degrees. The two or more beta-strands and / or two or more alpha helices are preferably modified or elongated to the same degree. In this context, the term "degree" typically relates to the number of amino acids added to the two or more beta-strands and / or two or more alpha helices. For instance, one amino acid may be added to one beta-strand and / or alpha helix and two amino acids may be added to a different beta-strand and / or alpha helix. Alternatively, two amino acids can be added to both beta-strands and / or alpha helices. Even if two or more beta-strands and / or one or more alpha helices are modified or elongated to the same degree, they may be modified in different ways. For instance, two amino acids may be added to one beta-strand and / or alpha helix and two different amino acids may be added to a different beta-strand and / or alpha helix. Alternatively, four consecutive amino acids may be added to one beta-strand and / or alpha helix and two separate stretches of two amino acids may be added to a different beta-strand and / or alpha helix. The identities of the four consecutive amino acids and the two separate stretches of two amino acids may be the same or different. The skilled person is capable of designing suitable modifications and elongations.

[0224] Since different beta-strands and / or alpha helices in the pore monomer may be different lengths and they may be modified or elongated to the same degree or different degrees, the two or more beta-strands and / or two or more alpha helices may be the same length or different lengths once they are modified or elongated. As discussed above, the skilled person is capable of recognising when one or more beta-strands and / or one or more alpha helices are modified or elongated, for instance with reference to the natural or wild-type sequence.

[0225] The one or more beta-strands and / or one or more alpha helices typically comprise one or more preferred positions for modifications or elongations. Each beta-strand and / or alpha helix may comprise at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9 or at least about 10 preferred positions for modifications or elongations. In any of these embodiments, preferred positions typically represent positions between amino acids in the sequence of the one or more beta-strands and / or one or more alpha helices at which one or more amino acids may be inserted. For two or more beta-sheets, two or more preferred positions typically line up approximately horizontally in the beta-sheet.

[0226] The one or more beta-strands and / or one or more alpha helices may be elongated by any amount. The one or more beta-strands and / or one or more alpha helices are preferably elongated by from about 1 to about 12 amino acids. The one or more beta-strands and / or one or more alpha helices are preferably elongated by from about 1 to about 12 amino acids. The one or more beta-strands and / or one or more alpha helices are preferably elongated by about 1 amino acid, about 2 amino acids, about 3 amino acids, about 4 amino acids, about 5 amino acids, about 6 amino acids, about 7 amino acids, about 8 amino acids, about 9 amino acids, about 10 amino acids, about 11 amino acids or about 12 amino acids. The one or more beta-strands and / or one or more alpha helices are preferably elongated by about 2 amino acids, about 4 amino acids, about 6 amino acids, about 8 amino acids, about 10 amino acids, or about 12 amino acids. The one or more beta-strands and / or one or more alpha helices may be elongated by inserting these numbers of amino acids. The one or more beta-strands and / or one or more alpha helices are preferably elongated by from about 1 to about 14 amino acids. The one or more beta-strands and / or one or more alpha helices are preferably elongated by about 1 amino acid, about 2 amino acids, about 3 amino acids, about 4 amino acids, about 5 amino acids, about 6 amino acids, about 7 amino acids, about 8 amino acids, about 9 amino acids, about 10 amino acids, about 11 amino acids, about 12 amino acids, about 13 amino acids or about 14 amino acids. The one or more beta-strands and / or one or more alpha helices are preferably elongated by about 2 amino acids, about 4 amino acids, about 6 amino acids, about 8 amino acids, about 10 amino acids, about 12 amino acids, or about 14 amino acids. The one or more beta-strands and / or one or more alpha helices may be elongated by inserting these numbers of amino acids.

[0227] The one or more beta-strands and / or one or more alpha helices may be elongated using any amino acid(s). The amino(s) may be natural and / or non-natural amino acids. The amino acid(s) is / are preferably selected from tyrosine (Y), arginine (R), lysine (K), phenylalanine (F), tryptophan (W), threonine (T), valine (V), alanine (A), isoleucine (I), leucine (L), glycine (G), glutamic acid (E), aspartic acid (D), histidine (H), methionine (M), cysteine (C), proline (P), serine (S), asparagine (N) and glutamine (Q).

[0228] The amino acid(s) is / are preferably selected from tyrosine (Y), threonine (T), valine (V), isoleucine (I), leucine (L), glycine (G), serine (S), glutamic acid (E), lysine (K), arginine (R), asparagine (N), glutamine (Q), aspartic acid (D) and alanine (A).

[0229] For 2 amino acid elongations or insertions, the amino acids may be selected from any amino acids. The amino acids may be natural and / or non-natural amino acids. The 2 amino acid elongations or insertions are preferably selected from tyrosine (Y), arginine (R), lysine (K), phenylalanine (F), tryptophan (W), threonine (T), valine (V), alanine (A), isoleucine (I), leucine (L), glycine (G), glutamic acid (E), aspartic acid (D), histidine (H), methionine (M), cysteine (C), proline (P), serine (S), asparagine (N) and glutamine (Q). For 2 amino acid elongations or insertions, the amino acids are preferably selected from G, V, R, K, S, T, I, E, Q, D, N, H, L, A, Y, F, M, and C.

[0230] For 4 amino acid elongations or insertions, the amino acids may be selected from any amino acids. The amino acids may be natural and / or non-natural amino acids. The 4 amino acid elongations or insertions are preferably selected from tyrosine (Y), arginine (R), lysine (K), phenylalanine (F), tryptophan (W), threonine (T), valine (V), alanine (A), isoleucine (I), leucine (L), glycine (G), glutamic acid (E), aspartic acid (D), histidine (H), methionine (M), cysteine (C), proline (P), serine (S), asparagine (N) and glutamine (Q). For 4 amino acid elongations or insertions, the amino acids are preferably selected from T, V, K, L, R, Y, S, I, Q, E, N, H, A, D, F, M, W, G, C, and P. For 6 amino acid elongations or insertions, the amino acids may be selected from any amino acids. The amino acids may be natural and / or non-natural amino acids. The 6 amino acid elongations or insertions are preferably selected from tyrosine (Y), arginine (R), lysine (K), phenylalanine (F), tryptophan (W), threonine (T), valine (V), alanine (A), isoleucine (I), leucine (L), glycine (G), glutamic acid (E), aspartic acid (D), histidine (H), methionine (M), cysteine (C), proline (P), serine (S), asparagine (N) and glutamine (Q). For 6 amino acid elongations or insertions, the amino acids are preferably selected from T, V, K, S, N, I, R, L, Y, E, Q, A, D, H, F, G, M, W, P, and C.

[0231] For 8 amino acid elongations or insertions, the amino acids may be selected from any amino acids. The amino acids may be natural and / or non-natural amino acids. The 8 amino acid elongations or insertions are preferably selected from tyrosine (Y), arginine (R), lysine (K), phenylalanine (F), tryptophan (W), threonine (T), valine (V), alanine (A), isoleucine (I), leucine (L), glycine (G), glutamic acid (E), aspartic acid (D), histidine (H), methionine (M), cysteine (C), proline (P), serine (S), asparagine (N) and glutamine (Q). For 8 amino acid elongations or insertions, the amino acids are preferably selected from T, V, S, N, K, E, I, Y, R, L, Q, D, A, H, F, M, G, W, P, and C.

[0232] For 10 amino acid elongations or insertions, the amino acids may be selected from any amino acids. The amino acids may be natural and / or non-natural amino acids. The 10 amino acid elongations or insertions are preferably selected from tyrosine (Y), arginine (R), lysine (K), phenylalanine (F), tryptophan (W), threonine (T), valine (V), alanine (A), isoleucine (I), leucine (L), glycine (G), glutamic acid (E), aspartic acid (D), histidine (H), methionine (M), cysteine (C), proline (P), serine (S), asparagine (N) and glutamine (Q). For 10 amino acid elongations or insertions, the amino acids are preferably selected from T, V, S, N, E, K, I, Y, Q, R, L, D, A, H, F, G, M, W, P, and C.

[0233] For 12 amino acid elongations or insertions, the amino acids may be selected from any amino acids. The amino acids may be natural and / or non-natural amino acids. The 12 amino acid elongations or insertions are preferably selected from tyrosine (Y), arginine (R), lysine (K), phenylalanine (F), tryptophan (W), threonine (T), valine (V), alanine (A), isoleucine (I), leucine (L), glycine (G), glutamic acid (E), aspartic acid (D), histidine (H), methionine (M), cysteine (C), proline (P), serine (S), asparagine (N) and glutamine (Q). For 12 amino acid elongations or insertions, the amino acids are preferably selected from T, V, S, N, E, K, I, Y, Q, R, L, D, A, H, F, G, M, W, P, and C.

[0234] For 14 amino acid elongations or insertions, the amino acids may be selected from any amino acids. The amino acids may be natural and / or non-natural amino acids. The 14 amino acid elongations or insertions are preferably selected from tyrosine (Y), arginine (R), lysine (K), phenylalanine (F), tryptophan (W), threonine (T), valine (V), alanine (A), isoleucine (I), leucine (L), glycine (G), glutamic acid (E), aspartic acid (D), histidine (H), methionine (M), cysteine (C), proline (P), serine (S), asparagine (N) and glutamine (Q). For 14 amino acid elongations or insertions, the amino acids are preferably selected from T, V, S, N, E, K, I, Y, Q, R, L, D, A, H, F, G, M, W, P, and C.

[0235] The one or more beta strands and / or one or more alpha helices may be elongated by one or more of EE, LS, FS, LE, NT, SH, CV, FD, SQ, QA, AN, AK, QD, VH, DV, LD, KL, QQ, QV, AL, RQ, Al, TI, II, HS, RI, ED, KI, YD, MV, KN, SD, HQ, KY, IG, GV, VG, GI, NL, GL, LG, NA, GA, GF, PG, GG, SA, GT, DL, NI, GY, TL, NG, NF, AG, PV, SF, GW, DF, IP, TH, QP, NR, IL, LV, RY, AE, DG, RE, RK, GH, QI, YE, NY, OS, VR, RY, LC, LI, LP, EL, IK, MW, VC, AW, PR, EP, HI, FW, HE, ML, HM, WE, MI, QG, VY, GR, YA, GM, CP, TH, LR, YL, MT, YW, MG, EM, IQ, FE, WY, PC, TS, GC, CM, MN, SM, NE, IF, TA, VP, WA, NH, ES, SL, DN, KR, MK, VV, ER, HW, LP, RS, FC, DQ, TG, NS, GD, TV, QL, KT, KM, CG, and LM. The one or more beta strands and / or one or more alpha helices may comprise one or more of the listed insertions. Different combination of these two amino acid elongations / insertions may be used in the same beta strand or alpha helix or different combinations of two or more beta strands and / or two or more alpha helices. The two amino acid elongations / insertions may be used in the one or more beta strands and / or one or more alpha helices consecutively or separated by existing sequences.

[0236] The one or more beta strands and / or one or more alpha helices may be elongated by one or more of TVTV, VTVT, VKVD, NTYK, TYTN, DTKK, VLNI, VTYI, VTPG, VTVP, GVQA, NLSI, INYR, VLQL, LEYK, SQTV, LQVK, VVQV, VTYT, QQLV, VQVK, VEYE, LERI, STLL, EYKK, KVNL, LSYT, SYLL, LNYK, VMNL, LTYK, TQTF, LTQR, VYTY, LTYT, TYTL, YTVK, KTIL, LEYN, LIFK, VVTV, ITYK, TQTV, VTTK, ILTL, LTYR, TRLV, LLER, VYTL, LTFK, LTKK, VIAV, SYLA, VSQK, VWNV, LTFT, SYTF, VSRR, VVQL, LTYV, GYQY, LQNK, VETT, HTLT, SSTT, TTVK, IEKD, QDTV, SQTT, DKVR, VVRV, VDYT, SRRV, VFHK, TMNV, VINH, RRNI, HLER, EQTD, ITRK, SQTL, DTVR, VTTV, QTYT, VSVK, VVEV, HYSV, VEKK, VLRL, TKTL, LTVK, VVKI, LEYQ, SYTA, IKYK, QHNH, LNYR, TSTY, HTVR, VVRI, AEYT, SMNV, IRQK, VERT, VAKK, VENE, ITRT, SVLR, EATR, VLSI, FTYN, SYSA, FSYK, VVTL, TYTV, LTVR, VQTV, LSTT, TQTY, VTIR, LNYT, SYNF, VSYA, TYQL, LTTK, ERRV, LTDR, SREA, VRTR, ITYS, TTTV, LQQR, VERE, LTRT, TQEV, ERRR, VVNI, SMLC, IFFK, KVQV, LEYV, KTTV, VQKK, VLKV, TYTF, VSYK, VLTV, FTYT, SYNV, VATK, IVSV, TYTT, VTVK, QVSI, IEYK, ISKK, VISI, SYTL, ISYR, VITI, KQTV, ITKR, VHQH, RMEL, HQVK, LTYN, TYLA, ITTR, VEQR, QTRV, QQTQ, RQQR, VVDI, VTYS, KRTV, ISER, YEYK, SQLL, LAKK, LNRT, KMTI, YTHK, IVRV, TKTV, VRVR, VSVH, TQTL, HTRK, VTRT, LERR, TTLT, TLVR, LTYS, SYGA, VQRK, VVSV, LTRV, STTV, VSRK, VTQD, NTRV, DQVK, IYNL, TDYT, NQTV, INKK, VLTL, LTQK, AQYS, SFTL, VQRR, VLNL, LQYT, SKTV, LSYR, VLSL, LEYR, LKFK, HTYT, SYQF, VHNH, SQLQ, HSQK, IINV, YTRK, TQEL, VNTR, VQNV, YTMT, SQLV, VLRK, VTLK, IVRL, TEYT, SYTV, LRLR, VLTY, AEYS, YTEK, LEYS, VRQK, IVQV, VQVR, IALL, HYLL, LATR, VINL, FTYQ, SYNL, QLRV, LDYI, SKTT, VRER, VENI, YTRT, DDLI, IYSR, VVRL, KKLL, LLEK, VTRE, IETK, DKTV, ETVK, VYVH, SMTV, HVYR, STTA, VYML, HTFI, TKQI, LMIR, VIQN, VQYN, SLTY, NQTK, LEYT, SKTL, IKEK, IISN, TYEL, NSTK, IEQV, IQRQ, TETI, VQQK, NVNI, LNYS, AQTL, INIK, TWHL, QEYR, EYLL, LHTK, VVSL, SYQL, LRYR, TLRV, SYTI, LYEK, VIEV, VLYR, VIRV, FEYQ, VAVK, VIQI, NYLV, IQYK, VVNF, SMTL, FSQK, VYSY, NQTL, YSYR, VNIH, LIRN, HIER, VSTD, TQTR, DVQR, LTEK, LVQI, LTYQ, KQQV, IQQR, VLTI, QTYK, ITQR, VLKL, AEYV, LKKK, RLSV, SYSL, VYRD, AYNR, DQKR, VLSY, SKNV, YSVR, VESE, VDRT, KSTS, ESTR, AEYK, KEQI, LEKT, KYTL, IEVK, FEYK, AMTL, LSVK, VEYK, VTYK, VTYR, VLNV, ITVR, ILNL, TQNV, LLTK, VEAT, HTRT, SDSS, TATK, VDKH, TERK, HKIK, VNED, VKTV, SQTK, DAQK, RIQI, ILTF, FTYK, IISV, LTYI, QTTL, VTKR, SISL, VEYN, SQSV, LSSK, TINV, FLYN, SQLI, VFKK, IIKV, SYLG, VKYR, IEQE, YTYK, EQNK, IQNK, NVNV, VNVR, TVSV, FQYV, SYLM, INRK, LEYI, TQKV, VMVR, VIQV, LKYT, SYQV, VLQK, VYSL, INYT, SYLV, LYRK, VLNN, TRTV, NTTR, KVNV, VIVV, VKYT, SRTI, VYEK, LTSR, TRTF, VTER, VTQI, AEYI, IQTR, VSTH, ITTK, SRLT, HTKR, VSEI, IDTT, IQKK, TQLY, ILQR, SLNL, SKNL, LALR, IERE, HERK, TELK, ELKK, VENL, QRYT, SKST, LNEK, LDYT, IRRK, VINV, YEYR, EQTV, VNRR, IYRN, SKLL, NHRK, TIQL, IERI, STTL, LSNK, VLEV, LRYV, TYEA, VIVK, VQNN, HTYK, TRTY, NQQK, VSYR, RVWV, IEYV, VYSK, INNN, LNKV, VQAQ, TNYT, TSTQ, QTKK, YAKK, IIEV, TYTA, TIYI, STNV, IYTK, TVTH, SQTY, ITDK, VINI, ITWR, IQNI, QTYI, IMRK, IEQA, VTRK, SYQS, AQNR, LNYV, VTVR, AYEV, VIEI, ATYR, VQKH, LTQT, TTEA, HKEK, IKKD, NEYK, SKTN, VHNV, LDRK, VNEK, INRH, LEMT, SQTQ, HRSK, QINV, SYNA, VKQR, IEYT, YYNV, ILVK, SKLV, VSKK, VVSI, ISYK, SMTF, ISVK, TTLV, KVSV, ITYT, SLTV, VESV, YNLT, RRTI, VTQK, THTV, LTKR, IVRI, WEYK, KITI, NMTI, ITRR, SYDL, TDIR, TRSL, RIER, IVSI, FNYK, INKT, VIRY, YARR, ILSI, ISVR, TMTV, VTSR, INKR, VAIT, VEYR, ERTV, TIEK, INAL, LERK, STTI, LAER, HTQR, YQYT, SRLV, LQVR, VSRS, SRSK, VEYT, SQMV, LRIR, QTRI, TNTD, TTSK, DNLR, SMSQ, KVKV, SKTI, VTEK, TVYV, SYEL, VYWK, EYNA, VKSV, VEYV, STSV, VSEK, VANV, YEYT, TNLF, VQQR, VLYL, RQAV, VVNV, VNQR, VKTH, LTTA, SRTA, HSSR, VLTT, TTYV, TKTT, TTEK, VTNL, LTYE, SQSA, LSQR, VTYV, VSTR, VVIV, VLQR, KALL, SQLA, QDIT, RQLN, ELKE, VNQH, RNYT, SRLD, HQER, VVQI, SMLL, LSYS, SYTG, ISWK, ISSI, YERT, HQLL, ISSR, IVNV, QKYI, VFQR, VISV, LTYC, VYTF, FEYT, SYSF, FTKK, INVK, VQTH, LDYR, HTSR, VTRH, IERV, HAVR, TLSL, TVDI, IDYT, VHSH, VTEI, EVTI, ITVK, SLTA, VRQE, LTET, SRLF, TYNL, HNYT, SKSI, LNQR, IVNI, QKNV, VSVR, VYIL, TYGL, LIYR, KICI, YNYI, SQNY, MNYT, ILKK, IENH, LSRT, SYTY, HSKK, VIHK, KHRK, TVRV, VRTK, VIHI, ISYI, IHWK, IYRY, LDTQ, YRQK, VLVL, LTLK, KVLE, IEYI, EQKR, VFKI, TQEN, ITQK, TITI, VNYN, TVNL, NTYV, TQTN, LSEK, VTNV, YNYS, TREA, VRRR, VVTI, ISQD, TQTQ, DQKA, ITTL, ATYN, VLRD, SMLV, DKYK, VYNY, RSYV, KQTD, YTLR, VYNV, STLV, VNKK, TLTL, RVSV, VSQR, FSYT, SMTI, ISTR, LTWV, TGSV, VTRR, VLSV, RKTV, NLTI, LNYN, LTKT, NQNF, ESKR, IQKQ, SKNE, QQVK, VQSQ, NTYT, SQQT, QSTR, VEEV, LTRR, SQAT, VLVR, TVNI, NYLL, SQLH, LNKK, IYYR, LRER, VQEV, YTRI, KQLI, VAQS, ITYQ, TINT, HTYL, TQNY, TYTK, SEYR, SQSG, VQEK, VIRL, KQSV, IENI, LTRK, IKVK, TQTT, TTNK, VKRL, TKEL, LRQR, NTYN, SYTT, IYVK, TQNL, VYQY, DTQV, YQVR, VQSN, AKTV, NSQR, QVNI, INQR, VETE, ETKK, VLML, RQQI, LMTK, TVSI, VNTN, NTVK, TYTY, VETI, ITEK, KKTV, ITES, QWNL, LSYK, TLLV, AYTL, VAYK, VLWR, SYVA, VHKK, KYRI, VDSV, VFNT, YTYQ, SANY, TTKK, VLYK, KVQI, SGTV, ITKK, VAYR, TYQA, YRYR, HNYK, TSTA, VNVK, IDED, IKRI, RLVL, KTTA, LEEK, IIQV, SQQY, YSKK, TASL, YTYR, LERT, SQTA, VTSK, LETR, YAYR, VQYR, LVHV, LEYG, VYYK, VVNT, QMTV, TTIK, LNVR, TWKE, EKTK, LTVT, NTRT, NQTN, VLRY, YSYK, SNMF, ISIR, VISH, HSVK, VRKA, VYSH, FTYV, HSYK, TMTA, NQLK, TMTT, VGTL, WTQK, VAMA, SEYV, RKLN, AEER, IENL, FTRK, SQTF, VITV, FTYR, VSEE, TRSV, LNKR, VTRI, VWKH, QNYT, AQTF, VNAR, SQTI, ISTK, VSTL, EKTV, LTER, KLSL, LSTR, HLSL, QDYT, SYSN, VISK, KTVK, IFHL, LHVK, VEVR, YTYT, VFTI, STYT, ISQK, QVNV, SQSI, VKRK, IQRT, LETK, IHME, VERK, EMTR, VVSH, RESH, QDRT, VVEL, LLYT, SFTV, LSQK, VEKH, EYLF, HKYK, TVNV, TIQV, QTVT, ITTV, QNHT, KSTT, VTDK, VLDI, TYNM, ITYR, QRSQ, TKTY, IVKV, SMLF, VKKK, AQYK, SRTT, IHEK, KVAL, YEYL, SQNV, LYFK, IEHE, SLTE, EHVR, TVHV, ARNV, VHVR, TVAL, LAYK, ITSK, LSKK, TLEN, QQYT, NTKK, FQQK, VNYK, VLER, VDTN, QTNK, SQTD, NSVK, ISIK, QVDI, AQTV, ISKR, INQH, LEGV, KQTF, HVQK, IVSL, QQYR, LKYR, ILNI, ITYV, AQTI, IIVK, VRRE, TRNL, ERER, VVHL, SRSA, LAQK, KVLI, QVYV, RQLL, VYRK, IEKQ, HQNV, ELKL, LKYK, ATRT, VLQS, SHNL, SQVK, IETT, KQTY, VTKK, VTSN, ATYT, SSTQ, NSTR, VAIL, KKTL, LSER, AQNV, VEER, FTRR, SYLY, REER, LETT, IFYK, IEEE, VTRQ, SKTY, EEQK, LKQK, LTTR, SRLI, VMDR, IATL, LPQK, VDRN, TERT, VNTT, TSYK, TLNV, ISFK, LDYK, VVTY, YTKR, ILRL, YTYI, LSVR, HYTL, VQTN, SQTS, INTV, KQTL, VTHK, RLSA, ANYN, SYDV, ASFR, IYSV, TQTI, IESE, HTTT, SQLN, ESKK, AYTV, YRVR, KVNI, LDYV, EIRV, SREV, KQVE, TRTT, ETVR, TVHF, FYKK, VNYQ, KQNV, ILTR, SYSV, TYQF, YTTK, KEYT, TNLD, NYKK, VSAH, VYER, VLRI, FDYR, TFLF, ILTK, VEET, QERK, QQKT, TQTK, VQED, SYTK, DAKK, LSSR, VLEN, YYTF, NSKK, KENH, DERV, SQTH, HNEK, LTYF, TYTI, VQIK, VTTS, TQEF, SSVR, SYSY, VTFR, TRTL, VYKW, TREV, WKTK, RLRL, ISYT, TQLQ, LLKK, VQVV, QERT, SQLT, VFSR, EYLV, VQYK, TTYT, VSIK, TFEY, EYTL, EMTI, IQRR, VNNH, SYTE, HSLK, HTYV, SYTS, ISRK, VETR, RTVR, TLQL, IITV, DRLV, VSWR, IVNH, HTTI, VRSD, NETS, DSRK, IVDV, YTYA, VFAL, LAVK, LNRE, HSEK, VEAK, INRT, NRSE, KAVK, QISV, VYTK, SYSG, VNQK, VNRK, RIHF, IDYV, FNKR, VERR, KRLD, RREK, VVTQ, TTYK, TRTD, QTER, IIRI, ILKR, GYVV, VRYK, TITY, TQLF, VLSF, FSFK, EIDI, SQAV, IAQR, TINI, INRR, VIRI, IRYK, WSYR, LQYV, IQYR, VVQT, DTYT, TQVR, DYSV, LLYR, LLNL, FSYS, RQTL, ISEK, ILQH, HLTR, VHAH, HSHR, WMV, NMML, VMSK, SRTF, TNYR, SQQV, VTSD, SQST, DSKK, SMTA, LQQK, TVTI, ITRD, VDKR, AQRV, DYQK, FEYR, SMTY, YSEK, VEQI, YNFT, SMLM, TVTF, VGVG, GVNF, IEVE, VEVG, ISVG, IGVG, VTVG, VSVG, ITVT, VEVE, VNVG, LGVG, VDVD, GVNI, IEVG, GVNG, LEVE, VSVS, LSVG, LEVG, VTVV, VSVT, VDVV, IDVD, VGIG, ESVS, VEVV, KEVG, VQVG, ITVG, VSVN, VAVV, VEIG, LRVG, LQVG, ISVS, GVGF, VAVA, IGIG, GKNF, LEVT, LDVD, VQVQ, VTVI, IAVA, VNVD, INVN, IIVI, RSVG, GYNI, IQVQ, VNVN, LSVS, GLNF, ISVT, IVVV, VTVE, LTVG, VNVT, IEVN, AEVG, GEVG, VVVV, VSVE, ISVL, AGVG, INEN, GVTF, VHVH, IEVV, VSIG, LGFG, VVQY, VNSN, KVRI, HEYA, IEMR, PVTV, HVQV, LEMV, VNNY, NTKI, VLLL, TSTE, LTDT, VVLN, HERR, VFTF, TLYL, FQYT, VNYS, VVDV, IIYT, VLRF, VVVH, TISV, EVNV, KVRV, LEYD, EVSV, IVTI, KETV, IISL, IRYT, RENV, LNYI, VLEL, IETH, LDRR, EEHV, QYQY, INYN, VITH, VQQN, IINY, ITRV, WAI, RIAS, VVYT, EVKV, IHDV, TITV, TEYK, VVVL, VVHV, TVQI, TYVV, LTQN, SIYI, DARN, VMQH, LERS, VWEV, EYTY, YTYN, VFTL, LLRY, FEYD, VFQH, VISL, VQYV, HEYR, VVTT, ATYV, VKKI, QTYQ, VIDV, VDQH, IERK, EVTV, ILRT, FEYV, VYDH, VNTS, ITNR, RINI, IQSD, YERR, MIQI, ITTT, IYTY, ILVL, LVYT, EITI, KTNA, LTRE, RVNV, LTYL, VFSI, VSEV, YTET, VVKV, TNTN, NTNT, YTGG, ATGT, RTRT, ERTR, GTGS, TGTL, EGTF, GTLK, TLTG, LTGT, TDEI, TLKG, TDVI, TNTL, NGLT, GTYT, TGTQ, RENS, TSGE, EGKN, HTES, TRTN, LTGE, YEVG, and VTGS. The one or more beta strands and / or one or more alpha helices may comprise one or more of the listed insertions. Different combination of these four amino acid elongations / insertions may be used in the same beta strand or alpha helix or in different combinations of two or more beta strands and / or two or more alpha helices. The four amino acid elongations / insertions may be used in the one or more beta strands and / or one or more alpha helices consecutively or separated by existing sequences.

[0237] The one or more beta strands and / or one or more alpha helices may be elongated by one or more of TVTVTV, VTVTVT, VDVYTV, YTYNEK, TKTYNV, YTVTVK, ILTLNV, STLTVK, SVTVTV, LTLTVK, VSTIRV, VEITVT, SVTITV, ITITVK, VLNVRL, SEVTVR, TVTVTL, VTYTRR, VVEVKV, VEVKQT, TKTVKV, VVVTQR, RDIHTV, HTRDVV, TTIIDV, HKSTVK, VDTDTV, YTSTRT, TNTDTV, WTKTTK, VVTITV, STITVK, TTTITV, ITITRK, ISSNTI, NSNTVV, TTTNTI, NTVSVK, KYNYKV, YDYELT, TEKYEV, YIYEQR, VLTVRV, VTVTVK, VHTQNV, STQQQT, TQTQQV, QTQTTR, VDTNTV, QTSTTK, SKTNTV, QTTTIK, VYEVRI, LTARRT, STEVRI, VTLTQK, EVNYQV, SGENLT, YTETVK, ITNYKA, YTNTVV, KTTYTA, YTVSRR, VLSLKL, SDANTT, TRTLNL, LSLTVK, IDTVSV, VEKKTV, TVTVKV, VTKKTK, VTKSSV, STNTIQ, TTTSTV, SVLTQK, VYTYTV, VTYTQS, TTTYTV, YTYTSK, VRTNTV, TENNTV, TKTTNV, NTVTEK, VGTYTV, GTYTQT, KNTYTV, YTYTTK, VENSLV, YEKTRT, TEKITV, KTKTRK, ELTLSV, STLTTR, TTTLTV, LTLTVR, TVTTTI, NTTTLT, I I I I I I, TTVSVK, TLTYDV, YNYTQN, NRTVTV, YTYSRK, VVNVSV, LNVNTT, TKTVNV, VTVSVK, TLNVRV, VTYNQK, STTVNV, VQWSVK, VVTVRL, AEVRVQ, TTTVRL, VTVLQR, TVNVNL, SQVLTT, TVQVLL, VNVTVK, IYTLSV, STITIK, SVSVNV, LSLSVK, KVNINL, SSIEEV, SKRIEL, ITVEVK, TVTTTL, STVVIT, TVTTVL, TTVTRK, IVQHTV, HTHTTQ, TRTHTV, HQHTER, VLSVIV, ISVTVT, VIVRVR, KNTVTV, ITENRV, SQTVNV, VTTSVR, STINLK, TTTINV, ITITLR, VNVKVV, TVTVEV, VKVTRR, VVTVYV, YRHNRV, RNTVNV, VTVTVR, VNTYTV, HVYNEK, TQTYNV, YTYTRK, TKRVTV, VNVTRT, TKTVTV, VTYTSR, RLTISV, STITVV, SVKITV, TLTITV, LTINVV, TKTINV, ITISTK, VLNYTV, VNVNVT, SVTYNV, VVTVTV, LTIRVA, INQIQV, SRTEQT, TNRIEV, IQYTSR, VVNVNV, TVTVSV, VVVTVR, IDVKTV, STSTQN, TTTKTV, KTQTVK, VTTLTV, YTDTDI, LTTTVK, IDIITV, STLART, SDTIAV, ITRSVK, VTYNDT, SKTINI, ITVSEK, IDETTI, STSKTT, TKKTDI, VSNTVR, VTTYSL, FTNQQV, TNTYQL, YTNTIK, ITENKV, STNSVR, TTSNSV, NKNTRR, IVNVTV, VNVTVT, SFTVTV, VTLSVK, VVTYTV, TTYTTT, SKTYTI, YTYSEK, IRKDTV, SSHTTV, SRTDTV, QVNSTK, IVEVQV, VRVEIR, SVEVEV, VKVAVK, VNTETR, TVSTQQ, TRTETR, STETQK, VDNVNV, SNHTRT, SDTVTI, STRSDK, VNTVTV, VNTNNT, SKTVNV, VTTSTK, VVTGTV, STGTLV, TVTGTV, GTGTVK, LTYTVS, TKTITV, ITTTVK, TVTITL, VTVTLT, SLTLTL, ITVSVK, TTNLDL, SSLKVV, SNTLKL, LTISVK, ISTYKL, YESKVT, DVEVKL, YTLKVK, VRKERK, SEEVVQ, SRTEVK, ETESRK, IVTVTV, VTIDQR, SVTVDV, ITVTVK, VTKITV, STTNTT, TRTINV, VTVKLR, EVTVKV, VTVTER, VTTVQA, VTSTVT, TTTVTA, VTNTVR, MVNISV, SNITIT, SVEITV, ITIKTR, VIEVTV, SKITIT, ISTYQV, YTYTTT, TRTVTV, YTYTTR, TVTITV, STLNVQ, TVTINV, VLNVRV, SSYTLT, VTYTTK, TVTISV, STIVTN, TKTIVI, ITITTR, VGTYSV, STTKQV, TETYKV, YTVETK, VYKVTV, VTVNLT, TVKVNV, VTINRK, VVTLRL, STVTVV, TVTLTL, LTVIVK, STVTVT, SKTVTV, VTITVK, TQVSTD, STKTER, TKTETD, KTKTEK, TLLVNV, SNLNVT, SVTVNV, LILSVK, ITTVTV, STITRT, TNTVTV, ITVKVR, VTNVTV, SNVTVT, TTTVTV, IVTVNV, VTVTTT, VTVTSK, VVNLTA, LNYTQK, SQTLTA, LTYSVK, TINVNV, VNVTVN, VNVEVK, VRLYTV, STYTEK, TRTYTV, YTLTVK, KVEKSR, SNENQV, TKTTNR, KKETVK, ILNVEA, LKGKRI, SKEVKA, VSFYTR, VETHTV, STSTRK, HTKQER, VNTLTV, VTKTLK, ARTLTV, LALSVK, KQTYTV, STATTT, TNTYTV, YTATTK, VYTFTV, VTTTLT, SSTFMV, IIISTR, STVTIS, GVTVTV, VTISVR, VREYKV, YTSTQT, TDDYTV, YKYTVR, EVTVTV, VTVNTK, TVTVNV, VNENTV, KTNTVT, TEKNTV, NTVTTK, TLNVTL, VTYNLV, TNTLNL, VTYTVR, VYTLDV, VTSTYI, QRTLTV, LTYTVR, INNATV, SSVTVK, TRTATV, ATVTVK, VNTETV, SKATDV, TNTETV, ETVQVK, LTVSTK, AKTVSV, VRTYTA, AEYVRK, TTTLVA, YTYSER, INNITV, YSNNTT, TKKINV, ITSEVK, INDRTV, SSSKTV, TNTRKV, SESTTK, VVTYKA, ATLTVT, TVSYTA, VLNTNV, SNDRRT, TDSTRV, TNVTVR, TVNITV, STINTT, TVSINV, IQITVR, IRTYTV, VTYTLT, KKTYTV, YTLTEK, TVVVTV, VNVQVV, EVKVQV, LTVAVK, VRQYSV, FTYTYT, YTYTVR, QVSVTV, VTVSRS, VTITER, LTATIT, ITVTVS, VTTVTL, STLSVN, SVTVTL, VTTVKV, VTITTT, KKTVTV, VTISVK, STVTET, TKKVTL, ILNVNV, SNINAR, SVDVNV, VNVSTK, VSTYAV, FTYTQT, SNTYTV, YTYSQK, IKNINV, ISNNIN, KKTINV, ITNTRS, VLNLNV, LNYTRT, SDTLNV, LTYTVK, VVTIDV, SRVTVV, QVTITV, ITVSVR, VTVTTA, LTLETI, TVTTEA, LTLRRR, VLTYTV, YTYTVT, TKLITV, YAHQVK, STETVV, NRTYTV, YTVSVK, VATVKI, VTSTKT, TTTVTI, VTYTIS, VQVWTV, STLVLV, TVTWVV, WTLTTK, VRNYTA, YNWNVK, NQTYNA, YTLSVK, IRTVTV, VTSVTT, SQKVVV, VTVQVR, INNYTV, STRVET, SNTYVV, YSRSTK, KDTVTI, VTNTTI, TKTVTI, VTNTVK, ITSNKK, HDHNDI, TRSDNK, HAVTVK, VTTNTV, NTNTTT, NETRTV, NTNTVK, VQEIRV, LEYKQT, TKEIKV, YTYRQK, VLNVSI, LSYNTK, AKTVNI, VTYSQK, STNTQT, RQTVTV, NTNTIK, VPTVTA, TDTVTG, YTNTQK, SNTVTG, YTNQTK, VTTVNA, VTTTVT, TVTVTA, VITTVR, STNTTI, ITITTK, VNTSIV, STSTVK, TNTSTI, SISTVK, VLTYTL, YSNNRT, TNTYNL, YTNTTR, VDNYMV, YTNNQV, STTSNA, YTSTRR, VVTVKV, IEHSRT, TDTVSV, VVTVTI, TTVVVV, TITVVI, LTSTVV, VLEVIV, SSVKIT, SVTVKV, ITYTQK, EEKNIV, NENTTI, TKENTV, NINRTR, VVSVSL, ANINVK, QVSVNL, VSITVR, QLVVKV, SQVETR, SVTVEV, VKVTVR, VNINTR, LTNNVQ, TTTNNR, NTNTRK, VVNLTV, SSLTIT, TSTLTV, LNLTSR, VTATTL, SSVTTK, ERTTTL, VTTTVK, ELTVKV, TTTVSV, VTMTNR, ERKYTV, YTYVEN, TKTYVV, YTLKER, LTNNTT, RTTVNV, LTNTVK, KAKYTV, VNVEVT, KRKYEV, YSLEVR, VGTITL, SQITIT, TVKITL, ISITVK, IDTNNV, NTNNVT, TVTNNV, NTNTVR, KDTTTV, SSNTRT, TQKTNV, TTSEVK, VDTITV, STKTTT, SETITV, DTRSVK, INTYTV, STITTK, TVTYTV, YTITVK, LTSVVK, SVTVVV, VISSVK, TVNVTV, VIVSTK, VKNNTT, SKNTNT, TKNNTT, NANTVK, VRTYNL, VTVTRK, TTTSTL, VVRVTV, STAVLT, TTTVVV, VEVTVK, ILTVNL, VTSNVK, TLTVNL, VTLTVK, VSNITV, IESNVI, KRTINV, LTLQVI, QKTVQA, VTLTVR, VNLKLT, VTYTAK, IATVQV, CRRDIV, SEDVVT, TRTDVV, DIVTIR, KVTVRV, VETNLK, SKKVNV, VTVEVK, QINITV, VSVNTR, TLSINV, VVTTTV, STVTVK, TVTTTV, TTVTVK, IYTGTL, GTGKLI, STSAKL, GTGSQR, VVTITI, TNTITI, ITISVK, LTVVVN, TVTVVL, VGTIDV, STISRR, SKKISV, ITIEVK, EITYTV, STYNNI, TTTYNV, INNYNV, YNYQKT, SRTYQV, YQYSIK, VDTSTV, TTVTRT, STSSVK, YEYKQT, TKENKV, YTVKEK, IVNVRV, VSVTVK, TVKVTV, VRNINV, VNSNDT, RKNINV, INDTTR, VDTYQT, NTRTRT, TDTYTT, YTRTVK, VLSYTV, STYTLR, YIYTTK, VKEYTV, STDKEI, TEEYKV, YTDRNK, VENIAV, VTTTTV, RKKITV, IAYQSR, VSTKTV, STETTN, TETKTV, KTETVK, IRTYKV, IEVTVR, TKTYTV, YTVTEK, IVSVSV, TLTVNV, VNVYKV, YTQETK, TKTHEV, YSQTQK, VVNVEV, SNVTRT, KTNVTV, VNVTVQ, IVTVRV, ATVTRT, SVTVTI, VTVSEK, VRTDKK, DTKTST, TKRDTK, DSVTER, VDNVTL, SNYTQT, SNTVTL, YTQSVK, STITVI, TVKITV, ITIEVR, VNTNNV, STNTVT, TRTNTV, NTNTTR, VDTHNV, HTNNRT, NKTHNV, HTSSLR, VDNYNV, VSNNQV, TRTYNV, YNHTDR, KETKTD, STKTVT, TVKITD, KTIEVR, VTIVQD, SEVKIT, TTEVKD, VIVKVK, VFEIEV, STIKTT, RKEIKV, IEISIK, ITEVTV, ISIKVT, SVEVKV, VRTYTV, YTYTQT, AETYTV, YTYKTR, VNTYTL, YTHNEK, TKTDNL, YTKTEK, VTVETK, TSIFTV, ITFVRE, KNTFDV, FVFTEK, VLNLSD, STQLKT, TRTELD, RNQTER, THTRTV, STRTEV, TRTDTV, RTVTTK, VVKVTA, LEVEVK, TTKVEA, VVVSVK, VNTHTV, HTHTTT, TNEHTV, HTERVK, TVQVQL, TTVKQT, STSVNL, VQVSEK, ILTITV, LTITVT, VLVLSV, LTWTVT, SRTLTV, LEVSVK, IDTSTV, STSTKI, SQTSTV, NTIVQV, STVNQT, TTTVNV, VIVQQR, VNTYRV, SETNTV, RRTYNV, YTTTVR, VLTLTL, AELTVR, SVTLTL, LTLSVR, VVNVSL, SNVNVV, TVSVNL, VQTATK, STQTQT, TVNATK, YTDQVK, RNTHTV, HTNTIV, TQTHTV, HTHSLK, STSTAV, TNTTTV, TTITVR, IYTVTL, VTVEKR, TTRVEL, VTVELR, VEEEIV, NQSTVK, TKSETV, ELRSQK, VATKTV, HTYTQR, SETKTV, KTYTVK, VDESTL, STSTKT, TETSTL, STKTVK, VYELKL, VTLNVT, SVTLNL, LKVSTR, VHNFTV, FSNTTT, RNNFTV, FNFDTR, VTKMTV, VTEEEI, SKTMEV, VKETTK, VSAQSQ, HNDTRT, SKRNDQ, KTDTSR, VLNVTV, SNVTTR, SVNVTV, VVTLRV, STLTIA, TSTLLV, LTVKVK, ITVRKL, IEINRI, ETTINL, ITVRVK, QRTVTV, VTNNLV, VTVTEK, VVNISV, VNAKYT, TTNIKV, INYTRK, TLTYRV, STLTLT, KTTISV, VTNSKT, TQTISV, ITNTVK, VATFSV, STYTLT, TQTVTV, FTLTVR, VVTTTL, STFNVV, TETTNL, VTTVTA, VTQQQT, TNTVQA, VTQTQK, VKNYTL, ASNNEI, TKKYNL, VTVEEK, ILTLTV, STVTTT, TLTKTV, LTVTTR, VVRTTV, SVTTTV, VTTQTV, SSQTRV, SDTQSV, QTQSVK, VYTLTV, STLTIT, TVTLTV, LTFTVK, VTKTSV, STNNRT, TGTTNV, NLNQTK, VVTVRV, VTVQVA, SVTVQV, TTVAVK, ILNINL, SEIKVV, SVTIKL, TETRRV, SEHTRV, TETRTV, HTRTVR, TRTVTL, VTVTLV, KRTVTV, ISVFTV, STGIVI, TVAFIV, FLYIVR, VINVTV, SRNVSV, VEVSVK, VKINTV, NTNNRS, KQTNNV, NLLSSR, IERRAE, SEDVTT, DNRRVE, RSVQVK, VTNIKV, SETKTT, TQEIKV, ILTTTK, VYNVKA, FTYKET, TRTLKA, FQYTVR, VRTYSV, FTYTET, YTYTER, ITEVSV, SELRVT, TVEVRV, VGVTVS, ISVSKV, SESKTV, TKTSKV, SIVTQK, VTTYNV, FTSNRT, SEKYNV, YSIVRR, VKVNTL, NSNRES, TKENRL, NTVTVK, VVRVSV, STVEQK, STTVEV, IVNITV, ITVTVR, TVTVQV, LTLTVV, TTVTIK, VDTYSV, SELKET, TKEQKV, YLRREK, VYNVTV, TTVNVT, TVNVNV, VTVKVK, TVVITI, VTSKLV, SSTIKI, IVSYDV, SSYTVT, VHTITV, STITVT, ITITVA, VESSSV, VSNKTM, SKSSKV, SSVEVK, IVNVNV, VTVKLT, TATVKV, VTVTDK, VTVTVN, TTTVTL, VNRYTL, ATYNEK, SKTYNL, YTKHER, IAEYKV, FKYNVS, SVEYYV, YLYKVK, IREYNL, GTYKRS, TSEYNL, YTYMVR, STGTVT, TNQVTV, VENYTV, YAYTQV, ITTLSV, TTSTTS, SKTLTV, LTSSTK, IEMYKV, QEYTKK, TETYTV, YTYRRK, VDTVTA, VSVTVV, RKTVSA, VTVTTR, QVRLTV, STLNVT, TN ELN V, STNTTR, SVLSTV, STRQVR, VVEITV, STVKVT, SVEIKV, ISVSIR, HAKVRV, VEVGVT, TVGVGV, VDVTTK, IYTVKV, VVVKVV, VTVTTK, TTSFTV, STEVTV, VSVSVK, KVVISV, STITTV, SRTITV, ITISQK, VWTHTA, HTHTVT, TVTHTA, HTVSVK, IDKNTV, SNIETT, TTKNEV, TTQTVK, VSSYKV, YNNNIS, TNTYNV, YTNRVK, YTNTET, SDTYNV, YTNATK, TLNQRV, SESNLT, TQTQSV, QLSKVK, YTHTRT, SETYTV, YTQTTR, KYSYNV, GNINVT, SITYNV, YSLSVK, VTQYSA, STTTVT, SKQYTA, YTLRVR, VLTVSV, ATVNTT, IYTFTV, STFTLT, SKTFTV, FTVSIK, IVTTTV, SSSTTT, TKTTTV, TVTTVK, VDTYTV, DRSKRV, TDTYVV, LTAKVT, SKTIKV, ITLSER, VLEIDV, STLVIK, EVTIVV, LKLTVK, VEVEVI, TN KIN V, VIVQIK, LTKVTV, SATTVK, VTTYSV, SSSTRV, LTLSVK, VATYTV, STYKIV, TRTYKV, VLLIKV, LEGVVV, SVKLVV, ILAQVR, LTFERR, TRSYEV, FTLTER, VKNENV, STKNVK, TDTENV, ELITVK, IQKNTN, STSEEK, EKKNEN, SSNKVK, LTTTMT, I I I I I V, I I I I I R, STTYTV, YTYSTK, VRNDDV, SEDVET, SKQDVV, DSDAVR, VVTVSV, SVLLTV, VTISVT, VGNLKT, SQLNVV, SVSLNT, VGSATA, LTGTVT, TVTATA, ATGTTR, VTTVTV, VTYRRV, TDTVRV, VTYSTK, VVSIRV, SEIIVK, SVTVIV, ITISIK, VKIYTV, KVTYTV, YTVTVR, VTYNRT, VTYSVK, VVKIIV, SEVTYV, VVTLTV, TRTLTV, LTITVK, TVTVTT, TTVQTV, TKTVQT, VYTYTL, YTYTVA, YTITER, LTTTNR, FTTTVR, VRTVTV, FNVTQT, VGSYRV, SEYTSV, SKTYTV, YTYSRR, KVTVTL, VTVEVV, GVTVEL, VTVSAK, VFTYAV, VLNLTL, TVKLTL, LTVTQR, IDTKSV, KKETTI, RRTKTV, KTFDQR, TLTIRV, STLNIT, ITATVK, VLTISV, STITLK, VEGKKV, ESKTTS, KTTRKK, STYNRT, NVTYNV, YTYSVK, VKTLKV, STNTET, TKTLTV, LTNTTR, FTVTLI, SVSVTV, VTVSVS, TTVVTL, ATVEVK, SKTVEL, VKVSVR, VLTITV, ITYTVK, IFSVEL, AKATIR, ASATVR, RNTIEV, STIDVN, TTKIDV, IENENK, VGIYRV, SEVKLT, NLEYKV, YIYAEK, STYTTT, ITESVK, QITSTV, STSAVI, TNTSAV, STVMVR, ATFTVK, TKTYIV, YAFTER, KGTYKV, SEYTLN, YTYSIK, STGTTI, RKTYTV, YTYSQR, ILTLDV, ATLTAR, LTLTQR, TQTYTV, YTYTQN, YIYSSK, VTTLTL, LTLNRE, TTTVNL, LTLKVK, VSTVTL, STVTVN, VERYRA, YEYERT, NTLYEA, YRYQTK, IVRVEV, VKVETI, RKSVEV, LKVTVR, VINYRV, YNYERT, TNNYNV, YNVTVK, FSYTVT, TKKITV, YTLEVR, IDTFTV, SITITV, VTRSVR, VETKTV, KTKTVR, SETTTV, KTVSVK, VLENKQ, STNKVT, TQENKQ, NKITTR, GTVTVK, SSTVTV, VQTYTL, TNTYTL, VAEVTV, STTVTV, VTVSVA, VEKRED, STREVT, SDKRED, RERKVR, VRTETV, STKTQT, TDTETV, ETTTVK, RVTYKV, STYEYR, KLAYEV, YKYELQ, IVKVSV, VNTTLT, EKTVTV, VTYTEK, KLNLTL, STSNVV, TTTLNL, LTITVR, IRTTKV, SEDTEV, YTDTVR, QVNITV, SETKRV, STEIKV, VTVSTK, VVNTTV, YTLVVK, SVRTVV, YEVDVR, VYTVTV, ITVTLT, TYTVTV, VVNVKV, IDVTVK, TVSVTV, VLEVTL, VSINVR, VTITQR, VVTIKA, LEVNVT, TVTINA, ITVTHK, VSFTQT, ITVSTK, VSTINV, SNDTVT, TNTITV, KVDTVK, ALTISV, SNLTVT, SVVITV, ITLKTR, KVTVTV, VTVSIK, VTEITA, LTNTQK, TNTITA, ITNTQR, VFTVKL, STVTVS, VNTQNT, NTQTYK, TKTQTT, QTETVT, VENYTA, FSYTRI, SEKYTA, YTVERK, SEVKTT, ERTVKV, VTVKTK, ATVTVT, INTVND, TTNTVT, TNTVTD, VTNQIK, VTKTII, VTVKVT, VVNINV, SKAIQK, TQNIIV, INVTVK, KISYTV, FNSVLS, SSTYVV, LTQSQK, KVTAEL, ATAVVV, TKTVVL, VVNATV, VSLTVK, TVTATV, QVQVSL, SSLTVT, TQQVTL, IQVSTR, EVTLTV, STLTIK, LTISSR, TVNVTQ, SEKTVV, TVRVTQ, EVVVTL, TEVKVI, TVEVKL, VDTVTV, STHTVE, YTHTER, STVTIR, YTVITK, NTLYSL, YSYNQT, TDEVNL, YTLRVK, TLQIYV, SAKTLN, VLISVR, VTRYQH, SEYSLT, TKTYSH, YIVTQK, IVRVRV, LDTNVT, TLVVNV, LRYLVR, VTNRTN, ESETRI, TQTRTN, ETETDR, INTITV, VTHTNS, YTMKSK, IYNFNA, LNSTLR, TRSFTA, FQYVSR, VETNTS, STTTTK, TRTNTS, SVTLNV, LTVSVR, VRNYRV, VNKTEV, YTKTER, VDTQTV, SEQTRV, TTTQTV, QTRTVR, VVKIKL, SEGTRQ, TETITL, ITVTRK, VYVHTD, HTHEKT, TNKHED, HVHTEK, ITNINI, FEHKTT, TQVIKI, INVEVK, INTKSV, STSNTT, KTTKNV, VTVVSV, STSTRV, N ETVTV, ITTVSV, LTTNLT, KVTVNV, VTQTVR, VTNKTV, KNTTEI, TKTRTV, KTTTVK, VDNVSV, SNKTTT, RDTVTV, KNKTTR, VQTNTV, TTNTNA, SITTTV, NTNNEK, ITNLTV, SNITQT, STTLTV, LTISEK, TTLVKV, VESNVT, ITTSVK, ISVTVK, VNTNTV, VTNNRV, SETNNV, NTNSVK, TLTITE, STITTE, TKTITE, ITLTQR, VLTVTL, VRRITV, VTINTT, ITITIR, TVTTKV, NTSTVT, TRTTTV, NTVTRR, STVNVV, TKTTNL, TTVNVR, ATTTTV, SNNTVV, IETHSV, HTVTEK, EVTITA, LTTTTI, TRTITA, IKTTVK, KLIIIL, LKYTRI, TLTITL, IIYTEK, VDHVRV, TEVNRT, KTTVNV, VRVEVR, VVIVQI, STVQIT, TVEVQI, VIVKER, VDNINN, SNQTTT, TKTITN, QTQTQK, STRQQV, SETVQL, VTQSQR, VKVYTY, YKYELR, TRTEEI, VNTTTV, STSTQT, TQTTTV, STTAVR, FTTTRT, STTVTL, VTVNVV, TVKVNL, VVTIAV, ATVTIK, ILKLSV, LSLTLT, TVKLTV, LTLTTK, VSKRRK, SEEETK, TKTREK, RKETTK, VVNVND, SRVIVT, SVTVID, VAVATV, STTTVI, AVFTVR, STHTLL, VVRVRV, VTSETV, SRKVEV, VEVQTR, VVTVSG, VTVTIT, TVTVTG, ITYTEK, TNTFTV, VNTLSL, STYNVK, YTYTQS, VVNVRV, VDVKVT, TVSVKV, VSVVER, ERTETE, SKSTRV, TETETE, ETVTVK, IVQITV, VSVTVT, STSTVV, STTTVK, KTTARL, SELAVT, EVTAAL, ATVLVR, IITYDV, SSYNEQ, TRAYNV, YTYTQR, VEDYND, YNYNRT, TEDYTA, YKLTVK, KNVSTV, SQTSKV, SVSTVK, SEVTVT, VETITV, STLTRT, TTTIVV, VTVTNT, YTQQTR, ILVINV, VRVNLI, VNNVNV, TANNTK, NREVNV, SKSSRK, VDSKQV, TKTKTV, KTKTIK, VVNITN, SELKVT, SLTIKI, IILSSK, IDTQTV, SAQTRS, KQTQTV, QTRTIK, IKTYEV, YRYREI, TKEYRV, YEYLVK, INTTAV, ATYNQV, TKKTNV, TTIEQK, VTIVSV, SILTKT, VTTTVR, VDRYTL, TKTHTL, YTWTEK, IYSYEV, FRSNQR, SNTYNV, YRVFER, TTTVTD, SVTVTD, IDNNTI, STQTTK, STTNTI, QTTSTK, LTVTTK, TITILV, APVAVT, TVTIAV, ILVTTK, STVETR, TNTVEV, VVTVTL, GTLTTT, VFNVTV, STVKTT, VTVTVH, VYRVNV, VTLEVT, TKKVEV, VNVTVR, VSTVRV, VEITRR, TETVTV, VKNVKV, VSVRER, VIEINV, VNITVT, IKITVK, RVTISS, STNSVT, TRTISS, TITVTV, SAVSTT, SVTVSV, VITVTV, VVTLQL, SVKLLL, LEISEK, IIKIEV, STNNTK, TKEINV, ITIKES, STINVT, ITISVR, VGNVTV, VTDTIV, SQTVTV, VIDNVK, VDLQLS, SENKVV, TNTQKS, QLNTRR, IVQVTV, VTVLVR, TVQVLV, VTVIER, VTVTQK, VVNITV, VTVNTT, VTVEGD, TGEVTV, FVFVFT, TLVFVF, VEVKVT, TLEVEV, WWW, TVTVVV, ITITVT, TVKITI, ITITIT, TVEITI, TLEVVV, RTRTRE, KKTETR, ATATVT, TATATA, DTDTDI, TRRDRD, DTDTDT, TDTDTD, ITITVE, TAKITI, ITITFK, VTVTVV, ATATIT, TLTATA, LTLTVT, TLTLTL, FVFVVV, KVEFVF, TVTITI, LSLSVL, TISISL, ATATAT, TTTRQV, TNTQTQ, VIVIVI, TVSVIV, LTLTLT, LTLTYT, AVAVAV, TVQLVA, SVSVSV, FVFVLV, RLEFVF, VTVTIV, EVETEV, KKTKVK, TTTTVT, TVTKTL, IKIEIV, TIKIKI, LRLELT, TNNTGQ, TLRLRL, TITYTY, ITITIV, TIKLTI, KTKTKV, TETETK, LTLTIV, IITITL, NTNTTE, TTKNTN, TLTVTV, ATATVV, LTLTLV, TATLTL, VTVTVL, QTQTQT, RDTDTQ, TTVTVT, YTYTYT, TLTLTY, VVTLTL, VTLTIV, TYTITI, TLVVVV, DTDTIT, TVTDTD, TVKVEV, ETETRT, TNKETE, LTLTLE, TATITI, DVDVDV, TKVNVD, TLTITI, KVQDTK, TLRVTV, LVLVIT, TVTLVL, IQITII, TVVIQI, LELKVV, KIELEL, RVTVTV, TATVTV, FTFTVT, TVTFTF, ITITVI, DTDTVV, TKTATD, IVIVIV, TVEIVI, VVVVVT, TVEVVV, RVTITI, KVTLTL, IQITIT, NVQVQI, TSKIVI, ITNTNT, TKTITI, HTHTHV, TKTLTH, IVIKLT, KLEIVI, IKIKIT, KVEIKI, KIKKKV, KDEKIK, ITITVV, VKVKVE, TLKVKV, VSVPVP, ITITID, TVRIRI, STSTVT, TTTNTN, FTFTLT, TATFTF, VTVTVI, IVIVVV, TMVIVI, RTRTRT, TEKRTR, ETETVV, TRTKTK, HTHTHT, TATYTH, KVEITI, LVLTAV, IQIQIT, TVQIQI, VEVEVV, TVKVKV, ITITLT, KLTITI, YTYTYV, VISYTY, TVELTL, LILSLT, TVSLIL, TVTYTY, QTQSTT, TTSQTQ, TKTKTD, GTVTVT, TFTGTG, LILILT, TIKLIL, ITVTVT, TTTHTH, TLTLVL, TITITV, TTTTLV, TGLTTT, VTVTMV, TGTVTV, LTLTLI, LVTLTL, ITIEIT, TASATA, TLKATA, RVEVTV, TAVLTL, KGTITI, KVTITI, ITITIN, VVKLTL, NVTVTI, LTLTIT, IEIKVI, KIEIEI, TSVIVI, SIKTKT, ATATLT, LTLTLQ, RTRTET, TKTRTR, EVEVVV, TKVEVE, DTDTDV, TTTDTD, EEETEV, IREEEE, RLTTTT, GTGTVT, TLTITG, TVTATG, TTTLTL, ITVTVV, YTLTLT, TTTYTY, KITLTL, TLVLVV, TVKVVV, TVVVVV, TVTYTL, DTDTVT, TTRDTD, ITFTFT, RLEVTV, TKTNTD, TAVTTV, I I I I I I , TVTATT, NTNTNV, TSTNTN, KVKVKV, TKSKVK, AKAEIT, VVKAKA, TITLTL, TAKATA, RTRTRV, TETRTR, DIDILI, TRVRVD, VVETET, VIVIVE, TIKVIV, TVEVKV, TVTNTN, TITITI, KLTVTV, VTVTSV, TLTYTY, TKTKTK, TLMLTL, VTLTLT, TVQVTV, VDVDVK, TKDVDV, ATATAV, TLSATA, SVSQST, TKQEVE, NTNTKT, TNTNTN, TLQLTL, NTNTNT, TKTNTN, ETETEV, MTMTVV, TVTMTM, TLVITI, HEHRTR, VKEKEK, TNTTTT, KTKTKT, TEQKTK, HTHTKV, TQTTTT, LVLVIV, AVATAT, TSTAVA, DEDEID, TTNTEN, TDEDED, NTNTVV, TRTNTN, TVVIVI, TATLTV, VTVTRV, TDTRTV, LELRVV, TLELEL, IVIVAT, VITIVI, HTHTVV, TRTHTH, AVATLV, TATAVA, YEYKIT, KSEIEY, TIKVTI, VVVTVT, TVQVVV, TTTIVI, FTLTIT, LKKETE, TATVTI, TIKVTV, TKTLTL, KTKTIT, LKTNTK, VRVEKT, EERVRV, TVTWTA, YVYVYT, TATYVY, KVELTL, VQVQVT, TVKVQV, VVVVVR, STSTSR, TVTSTS, TIEIEI, LTLTFT, TGTLTL, ITITIK, LTLSLC, TLSVTL, VTVQTV, VTVTVD, QGQVTV, VIVTVV, LELELE, RLKLKL, TKTNTH, TLQYTP, QRTRTR, TVVAVA, HIHTLV, TVTHTH, GTGTLT, TLTGTG, LELTLT, TLTAEL, TLTLVI, NTNTAV, TVTVTN, EVETKT, KQTVVE, RLELEL, TVRVTI, TLTLIL, LELTVI, TVKLEL, YTYTIT, ETETEI, QIQEQE, TVTVTI, KITITI, TLTVTL, LVRVVT, TEVRVR, IRIEVD, GLRIRI, TVSFVI, VTVTAT, TVVLVI, ETETET, TQTRTR, TLQLQL, QVQTTV, TVTQTQ, AATLTL, YSYSIV, TASYSY, VTVTIE, AVAVIV, IVIVVT, YTYTAT, TLKVTY, AIATAT, TITATA, KKKKKT, SGGVGA, TKEKKK, HIHIHT, TTIHIH, NTNTVT, KATVTV, VSVSVT, TCSVSV, VKVTVT, LTVTVT, TVELTV, IVTITI, TTATAT, IQIQIV, KVEATA, IVITIT, QVTIVI, KIKIKI, TKIKIK, KYEITI, TTNTER, TVLITI, AEAKVV, TVTAEA, TVLVTV, NKNTNT, KTKTKI, TETKTK, VKVELT, TMVVVV, VEVEVL, TLKVEV, TLTAVA, TVEVEV, KTKTQV, TDTDTK, ATATVR, TVEVTA, ITITIE, TLTLTI, LTLTAV, ITISIV, TVKIKI, VKVEVV, KLKTKV, TGTATA, AIAIAK, TVSAIA, TTTSTS, LVLVVV, TLELVL, IEITTT, TVKIEI, TLVIVV, VEVTVV, TETKAV, TLKTET, TGKVTV, TTTTKV, KVTTTT, LILILI, KIEIIL, VEVKIV, TTTKTK, AQAQAT, TLQLQA, FTFTIT, EQEQVT, TKQKQK, LILIVV, TIILIL, VTVEVT, AKAEAT, TIKIKA, IEIEII, LVLVVR, TVELVL, NVNVNV, TTTRVN, ETETRR, TLLETE, VVTVTA, VEVTVR, TVLVEV, TAQLTL, DLDQRV, ETRDLD, FKFKIE, TVKIKF, KVTVTA, VTVSVV, VEVTII, KVEVEV, NVNVTV, TKVNVN, TAMLTL, LVLVLT, KLTLVL, LILTIT, TQTQVN, SYEVQA, AEAEVI, VVEAEA, LKLELD, EMKLKL, TKTDTD, KTTVTV, LTATAT, QKQTQE, TKKKKQ, TGKLTL, IEITIT, EVKIEI, MSMSML, TISASM, YQYTYN, QSQYQY, TTTTVV, TVTLTT, KVKVVI, IKTETE, VLTITI, STSQSV, TTTETS, TIRITI, VKVTMT, EIKVKV, YTYTLT, TKEYTY, RMRTIL, TRMRMR, QTQTTV, THTQTQ, VKVEIE, TGKVKV, GTGTVV, TLTLTV, VIVIII, TIIVIV, VQVTVT, TLAVQV, TAEVVV, QTQTTT, TKTETQ, KIKIKT, KKEKIK, TATLTI, KLTLTV, IEIEIE, VVKIKI, VEVKVI, KVTVTI, YTYTID, TKKYTY, IQIQVL, YTYTYE, TLKYTY, WTWTVV, TATWTW, LILELT, NTLTLT, AIAIVI, TIIAIA, TVVLVL, IEIEVR, VGYTSN, TVSIEI, TLTATI, VQVQIT, TVQVQV, DRDEKT, RDERRD, TITVVV, TTTITI, LQLQLV, TAQLQL, TTTTET, TKTTTT, AIATVV, TKTATA, KKEKTY, RVRTRV, TETRVR, IKIEIT, TVTIKI, TLKITI, DTDTET, TGTRTR, TVSIVI, HVHTQT, KKTQVQ, TVTVLV, TLKIKI, TLKVTV, HQHTIT, EKTHQH, TNTNNG, VTVTLQ, TLSVTV, TATTTT, IEIKIV, RVEIEI, ATLTLV, TTTATA, LTLSLT, ATATAR, ILELTA, TVQVNV, TVKIVI, ITITFT, ATATIV, TQTITI, NVTLTL, STSTTT, TKTHTS, YTYTIV, KTKTHT, TDTKTK, ITITVN, AEAKAV, TIEAEA, TVQSTS, TVQLTL, TKTQTN, YTYTYQ, VKTYTY, KVKVKT, TKQKVK, RKTKTK, TIKLTL, VVVVFV, TNQITI, TSKVTV, TETDTD, TSTLTA, DITLTL, LVKLTL, VVVTVE, TNINVN, TVEVEL, ITLTVT, RTRTYN, RTRTDT, AVATVV, RREDTD, VTVTVE, FTFTVV, RVEFTF, EERETE, VVVTLT, TATVVV, TLQVQV, KIKITI, KALRTR, ITITLV, TDTVTV, KEEETK, VTVQVV, ATATLV, TATVTA, TVSLTL, IVIVLV, VSTIVI, AVATVI, LTITVV, ITITAI, TVTITT, TIKITI, KVKTTV, TKTDVD, TLELVA, SNGTGL, TVTETI, SVSVVT, TVTVVT, HVHTHV, TTVHVH, IQITVT, TVTVQI, AMAMIV, TVMAMA, LVLVII, KIKVKV, AKAELT, TVKLKL, KRELTY, LQLQLT, TLTLQL, VTATAT, VGTVTV, VVVVVI, TLVLVL, TLQLTA, VTLTIT, KTKTTV, KKEKTK, VDVKVK, TITVDV, KIEVEV, ITITIQ, KKELTL, QTQTRV, TDTQTQ, TVRLTL, AVATLL, TVYAYA, TLQVTV, TVTAVS, VIVIIK, EVEVIV, AIAIAV, NVIAIA, EKTKTK, LILTLT, KTTNTN, VVVSVV, KVEVVV, TAIVIV, VTVTLL, LTLTAT, RVTVTI, TVKVKI, TITTTT, TTTTIV, TITITT, ATATYT, LILILV, TAILIL, YIYKVK, VKEYEY, TVTTTT, NININI, TKININ, KQKQKL, TKQDQK, TKTFTI, VLTVTV, TNNTGT, IIIIIV, TVTVII, RSEATI, LQLQVT, TVQLQL, KTTDTD, ALTLTL, LTLTVI, TVTVTR, TVTITA, TVILIA, LTLELI, ELRLRL, VTVTRT, TLNVNV, AKAKVT, TVKLKA, TVTLQL, GVGVLV, TAELEL, RIDIDV, TRIRIR, ITIKIT, TETYTY, RIDLTL, TVQLTI, TLVIVI, AVITIT, ATAEAD, TVRARA, ITITAV, KTEITI, ITITHT, TLKLTL, KTKTKK, TILKTK, DTDTTV, FIFSIV, KVVYVY, LTLTTT, TVTATL, VIVTLT, AIKIIV, NVNVVV, VTVNIN, SDSQTC, IVAVAV, IVIEVE, TIRLRI, KTKTLV, TFIKTK, TVKNTN, TATATY, AVAIAI, TAKYVA, VNTTTL, ITIQIV, TAQITI, TVSISI, DTDTRT, TQTTTD, LELEQT, TTELEL, ATATAI, RVEATA, TATFVF, ITITMT, VTVTST, LLTLTL, VVVVVK, ELEVVV, SNETQL, VTVKVE, TKTYTY, IKITLV, TLTIKI, HEHKVV, TREHEH, ATATAE, LVLTLV, TLRLVL, TEKKEK, TKTETE, TATITL, KLTLTL, VTVTIK, LTLSLV, AEAKVT, TAEAEA, TVIVTV, TISVTV, TRVYTY, TVELTF, QIKLEL, YQYQYT, TYQYQY, VTLTVT, NTNTYT, VTVTKV, STTSVK, LKLEIV, TVTLKL, STSTVA, GTATIT, TITGTG, SISISV, TAISIS, KVKIKI, VVVKVT, TAEITI, VLQLQL, INITIK, TITIEI, EVKVKV, LVGGKP, ANANGV, GGPSGG, SRSSGG, LVNGVP, ADASGN, SPATGG, LVNGKP, AGASGK, GGGYQS, TVDGLP, SAASGF, ATPEEG, GPGGGG, LVDGRP, GGGPFT, RVNGEP, VVGGKP, ANEKGI, GGPNGG, LVNGEP, IVGGKP, HDGLGG, GGAGGG, TVDGRV, VVDGTL, ADATLR, GVSAGG, STLKVQ, VNSGTA, GSGTGK, SGSGGG, SVNGVR, VNNNTT, VNNGTV, TNNTTT, SGSFNG, NTNNTK, IVNRVN, SDELIR, SVKRGN, SVDEVQ, VINGTA, GNGTGK, SGSGSG, LTNDVR, VTTGTA, TTTGGG, S I I I I I , VTNGVA, SNTTNL, SDETLS, SGTQGG, SVESVK, VNNNVV, NNDLLK, TGGNGV, NVRGES, INNNNN, SNNTGS, GNTNNG, NTDNVA, VTSGTN, SNNELK, TGTTNN, SGTTVN, VVNGNV, ADATGK, TGTGTG, ATAGVQ, VTENKV, VNVKLT, GGEVGA, VKVGEK, VSSGVV, SNDNLT, SGSGNG, SVDGVK, VVNGTV, ANVTVK, TVTGTG, ATVTVQ, VTSGTA, SNNTLV, SGSVNG, STDGVK, VTNGTA, ATGNGV, GGTSNG, ATGQVQ, TVNGTT, GNGNGK, GGTGNG, GSGGVK, LSGTGK, TGTGGG, IVNNTA, TSSTLK, and SVSVSA. The one or more beta strands and / or one or more alpha helices may comprise one or more of the listed insertions. Different combination of these six amino acid elongations / insertions may be used in the same beta strand or alpha helix or in different combinations of two or more beta strands and / or two or more alpha helices. The six amino acid elongations / insertions may be used in the one or more beta strands and / or one or more alpha helices consecutively or separated by existing sequences.

[0238] The one or more beta strands and / or one or more alpha helices may be elongated by one or more of TVTVTVTV, VTVTVTVT, VRIHVYTD, STSEFTER, TQTHEYND, FIYVETVK, INTSSYTV, SSSTYTLK, SETSTYTV, STYSYSSK, VVTSTYSV, HTSTITQT, SRTSTYTV, HTYTVTEK, VTTSKVKA, SESEVEVT, STKSEVVA, NTSKVSVK, RTTSTYAV, STSKVDVQ, TVTTKYTV, STYTVTVR, VSTVSINI, SAVQINVT, SKTVQINI, VTVSNSVK, IRTITYNV, VSSEITER, SKSVKIEV, ITYTETIK, VATTTVNH, STQTVTTT, SKTWTVNH, VTVTLTVR, VVKFNVEA, VSVEVTVK, SVKVEVEA, FKVNVEVK, VNINTFRV, SENSFTLS, TEKNSFNV, NIFTFTER, TLISTYTV, SKSKVNVK, TKESKVNV, YIYTLSIR, VATATVNV, VSSTVTTI, SYTATVNV, VTATTQYK, TVTSSVRV, SDSTVTVS, SRTSTVNV, VTVSVTER, VDTSTKTV, STSTRTVT, SETITVNI, STKTVSTK, ELNININT, SNNLINVK, KTTILINT, ININYTVK, VSSYEYRI, STYKYKLT, SKSYKYNI, YKYEFSSK, MQTSTVTL, SSSDVTVT, TVKSSVNL, STMTVIVK, VVTVTVNL, STVTVTVN, SVTVTVNL, VTVTISRK, KTTVTVTD, SGLTVTVT, SKTVTVND, VTVTVSVR, IESVLATV, LTLQAKTT, NREVQATV, VSALSRTR, ITTVTITI, STSTVEQK, TKTVTVNI, VTITQTQK, IYTSTVNV, ISSQVTET, TTTSQVTV, ITVTVQEK, TLTVTVTV, STVTVVVD, TTTVTVTV, VTVTVTEK, VVEVTYTI, STVKVTVK, STTVKYNI, VEVTVSQK, VVTVTLAA, LNLTVTTT, TVTTTLNA, VTLTVTVR, VINVNVSV, VGVEVKLT, TSSLEVNV, VNVNVTLR, ISSSEYNL, SNSKLLTR, TVSSKYNL, SSYELTER, KVTVTVSA, STVTVVVN, SVTITVNA, VTVTVSVK, VETSNITI, SNSSLTRT, TEEYSITI, STINQRVK, VLSITVYY, VKSLVTLK, TRTILVNY, VSITVSEK, IRSIFYSV, SSISYTQK, SLTISYNV, ISYLDSVK, TRTVNKTL, SNSSKTET, HRSVSKEL, VTVNEDVK, VVEKKITI, STNVVTET, SKTKVINI, NEIKVTTR, VNTNTIKV, STNTKTIT, TNTNTIEV, NTITSTVK, SIKTEVDN, SKKEVIVN, TVKTEVDN, VKVEVRVK, VLSIKISV, SSIEITYT, SVKIEINV, VSIKYLVK, VDTNTLNL, STSTLTTK, TRTNTLNL, ITITKTVK, TNTVTVSI, STSTVTVQ, TVTRTVNI, VNTSTYQH, STSTVTET, RKTSTYTH, STYTVSEK, VYTSTYTV, STSTVTTR, SKTSTYTV, VTYTVTVR, TDVSLITI, STSQKQTV, TESSQINI, SLHLQTER, VGTVTISI, VNVTVNVV, TVTVTITI, VSITVTEQ, VVTSVVKH, SESQVEKR, TVKTQVTH, STVVVESR, IEEDTKTL, STEKKTRK, SETEKKNL, DEKTKSVK, TTTLTYRL, SELTVTRT, TTTVTYNL, LTLTVTQK, VLTIRASL, SQSVATTT, SKEIVANL, ITIRTLTR, VDANNNTH, SNSNNTVT, SKKNNNNH, SNNNNEQK, VRTLSYRI, VGYTYTET, TTNKTYNI, VTYSLSEK, VVTSTVTV, STSEVTVV, TVKSEVNV, STVTVTVK, VLVVVVNV, VTVEVRQQ, TRTVEVNV, VVVVLQQK, VDISEYTV, NSSRRTLT, TRTDRYNT, NIYERTVK, VLTVSQNV, STITQTQT, SRKDTQKV, VTISQDVK, VNTSIYTV, STSTYTET, SRKSTYNV, STYIKSQK, VATTTYSV, STSKYTLR, SVETKYTV, FTYTLLQK, VATYTYEV, SKSTYTET, TRTYTYQV, YTYTLQER, VTVYTYRD, STYQHQIT, TTTYQYSD, YHYTHTVK, KVSVTVTV, STVTVTVT, SETVTVNV, VSVTYSQR, VNIYTYTV, YTYTYKQT, SKTYTYNV, YIYTYEQK, VLTSTVTV, STSTVKQQ, TQTITVTV, STVTVEQK, VDASTYRV, NNESITRV, TTTTSYNV, NAYTRIVK, KTTNTYTL, NTNTREET, STTNTYTL, NTNTESLK, VRTVTYKV, SEVVYTET, STTVVYNV, VTYTESVK, TNTSTVRV, STSTVTTV, TNTSTVTV, S I I I I I ER, VDMINVSV, SSVTVRLI, STTITVSV, IMVNRSIK, VATVTYKV, SEVEVTRR, SVTAEYMV, VTYTVSER, VLTITVTV, STVTVTVS, SVTITVNV, VTVTYRSK, VNIHQTTY, SSHTTKKE, STTHTTNY, HITQKKEK, VGTQTDTR, STRTVTTT, TRTETDNR, QTVTNKVK, NDVTSINI, VSNDNNLR, TNKNDINI, VLKSNEVR, INTLTNTL, STLLSKTV, NKETLNNL, LTNTSNVR, VDTFTYTL, STFRYTTT, TKTFRYNL, FTYTNTTK, QLTVTVTV, VTVTVTVV, VTVTVTVK, TDNSTYSY, SSSVNTTT, TNTSVYNY, STYTRSIK, VDTNTNNV, NNNTNTTT, SKTNTNNV, NTNTTTTK, VSIVSVTL, ANVSVTTK, TRTVSVNL, VIVSTTEK, TKSYTYRV, SEYQYTQT, TKTYQYNV, YSYTYTSK, EVTVKVHV, ISSTVTRK, TDTVTVLV, ITVKVTVK, VDVTTNTN, STNTNTLT, NKTQTNNN, NVNTNSVK, VFTSTVNI, STVINQKQ, KETSIVNI, STVTKEQK, VIKETKTN, STEEKTRT, TEKEEKTN, EKKTIQRK, RESTRTNT, SNSDQERV, SEVTDQTT, SSTRVEVK, VDTSKNTV, STSSNTTK, TKTSSNNV, STNKRTSK, VDTVTVRV, LNVTVVNS, SKTVTINV, LTVTVKVA, VTTLTLTL, STLTLTRR, STTVTLTL, LTLTRSRR, VYSYTYNA, ITSTITVK, TVTYTINA, YSYTVTVK, VYVVRYTV, STSEYERV, NNTVEYNV, VVYRYSVK, VVTSTISL, VNVQITQQ, TTTSQITL, VTVTITQK, VSTSINRA, SEATQTRV, TSTTTNRA, STNILTVK, VLTTTLTL, STSTLTTE, KKTTTLNL, LTLTLLER, VNEQLVSV, SNQEVKET, SRSDEVSV, QEVLNSVK, KYSYNYNV, SNSEHTES, KDKYEYNV, YSYNNETR, VNMSTHRI, STSTHKTV, TVTLTHNI, SMHEKTLR, VKSYSERR, YEYEEKEV, TRSYEENR, YSESETVK, VITDTVNV, SESSVTTT, TRTDSVNV, STVTVTTR, VDVSTYSV, SVSTNTQV, RNKSTYNV, SVYTTEVR, KDSLLYNV, SSSNYTTT, TNSHNYNV, ITYLSTTR, IQESNRSE, STSRRITT, SKASRRNE, STSNTATK, TEAYRYSV, SESEYTRT, TETVEYNV, YTYRYTRR, VLKLTLKI, STSSLTRR, SETKSLTI, LKLTRAEK, TLTSTIYV, SSSEITEK, TNTVEINV, STITLTVK, VRSFTYRV, STYSINLK, SRTFSYNV, FSYTIAEK, VVTVTVNV, STVTVTVV, SVTLTVNV, VTVTVSMK, VSTLTYNV, SNLTYTLT, SRTLTYNV, LTYTYSEK, VDNHTYTV, HTHSNTTT, STTNSYNV, HTHTSAVK, INIHTQSN, STSEVKER, TIEREQSN, EIYTNRTR, VKTYEYTV, STSKVTQV, TKTYKYNV, YNYEVTEK, IDTKTKSE, STSTDTTQ, KRTKTDTE, SSKTTSVR, VDKFTYRF, SESEYLEN, KRTIEYNF, FKYTYTER, VVTTSYRD, STHTYTVT, GVKTTYND, ITYSYESK, VVTVTLNL, STLTITTT, SNTVTINL, YTLTSSTR, IDIIEVNV, SSVKVEVK, SETIKVNV, VLVEHSVK, VATYEYSL, YTYGYVDR, ESKYGYNL, YTYEVEMK, VNTFKYQH, SNHEYQET, SKKFEYNH, FTYKDETR, VDISRLNV, DESRLVTR, TRESRLNV, DIYRSRTR, VQVSTYRV, STSTYELK, TQTSTYTV, SVYTETRK, VVAVTLAL, STSELTLI, TVRLELNL, VALTLRVR, VLEVKVSV, SEVTTVVK, SVIVTTSV, VEVKLELK, KVVITINV, SSSAITTN, TKTIAINV, VTITITEK, TATVTVRV, VEVLVTKT, TTTVLVNV, VQNYSYTL, SNYTVTQT, NTNYTYNL, YNYSLSTK, KVTVEYKL, STVKVEVT, NVTVKYTL, VTVEVSVK, VNTSEYTV, FTSTYQQE, SKKSTYTV, FTYEQSEK, VLISKVSV, SDSEVKLT, SQESEVNV, SIVKLSTK, IARSEVNL, STSTVTVT, SRTSTVNL, YRVEVAEK, VTTFTYSL, SNFTYTVT, TRENTYIL, FTYTVTEK, VATSKYTV, STSEYEVK, TRTTEYTV, STYKYTEK, VVVVKVTR, SESEVTLK, SVLVEVTR, VVVKVEQK, VNKNTVNI, NSNSVTVS, TVTKSVNI, NKVTDTIK, WTVKVSI, SNSTVTVT, RVEVTVEI, VTVKYQSK, IFTNEYSV, TTATMTVT, RRKLTYMV, VTYEAEVR, NQTISVTV, SSITITNN, TKTVTINV, ITVSNTVK, VETVKYSL, FSYENTKR, TERQEYNL, VTYKELVR, VVTITINI, STSTIKVV, ERTVTINI, ITITIKVK, VKSSTESL, STSTETTV, SRSNTENL, SSETESVK, VESYTYSV, STSVITKT, SDNYVINV, YSYTLYTK, VETYTYNL, SNYENKNI, TKTYEYNL, YTYTNTIR, RQLTMKKK, DTKVNDES, KQTNVQTK, TLKMNSVK, VVTVTVTL, VTSTVTVT, SETLTVNL, VTVTVSCK, QTSIKITL, SESSIKVT, TVEISITL, ISIKVKVK, VDQSSHRV, STTTHRRT, TDSSTHNV, SQHSRTVK, RISVTYNV, STVSYTDT, SRTISYNV, VTYTYASK, VNTYTYTV, YTYTYTTT, DTTYTLTV, YTYTTSVK, TISITVNV, STVSVTVT, TVTISVNV, ISVTVRMR, VSTSTYTL, YTYTYKLR, TEESTYNL, YTYTYKRK, NQNYTWSL, SSSTLWRT, TTTYTLTL, VNWTETVK, VSTSLTSS, STSETNNS, SNSSETNS, TTSLNSER, IYEKKKKT, SEKEFKVN, SKEKEKVT, KEKKFKEK, VLEHRVTL, HTHSNVVV, SITNSSTL, HEVRVSVK, IEASEYNV, STSKYVVT, TKEIKYNV, SAYEHTTK, VIVVTISA, VTITVTVT, TKKVTISA, VVITVLEK, LNMSRYSA, SSSELTTR, TDESEYNA, SMYRQKRR, VDITTTSH, SSTTTTVQ, STSVTTTH, TTTTNSRR, VTTTTHTA, LAHTVVVT, TVTTTVTA, LTHTVTVR, VQSTTYTL, STSTNNST, RNDTTYNL, NSYKNSNR, ILTIVISL, STIVIRVT, SVTVVINL, ITIVISVK, IEKYEYSL, STYIEEKK, SETYEYNL, YKYVESRK, QVTLDYSV, SNLVVVLT, TVDLVYNV, LTYDVRTK, VVTVRVSV, SEVTVTVT, TVEVTVNV, VTVRVTVR, VYTTVYNV, STSTYTYR, TRTTTYTV, NTYVYTVR, VNKVTINV, PKSEITVT, SVKVEINV, VKITIERR, RNTIEYRK, STIRYEMV, TKRERYTK, ISYEMEQR, VNTETKTT, NTTTLTKR, QETETKTT, NTKTLTVR, VVEVKTNV, VSTVNVEK, KKTVVTSV, VETKHEVR, TTSVSYAI, TNITVTVN, TKAVTYNI, VSYSVTVK, KDTTTVTV, STSEVTTL, TRKTEVNV, TTVTVEQK, VYTFEFSV, STFTYERT, TTKVTFNV, FTFEYQLK, VNERTENV, SSISETEK, TKTRSENV, YEQTESVK, VERSEYSV, STSVNTRK, TETDVYTV, SRYENTRK, IQESKYRV, SESTQKQI, TNESTYTV, SEYKQKEK, VNTYEYRQ, SNYKYEEK, TKKRKYTQ, YTYEETTK, VVTVTYKL, STVTITVK, TVTRTYNL, VTYTVTVK, VDSSKKNN, SESEKTVR, SKKSIKNN, STKKKQER, YTYTAEQQ, RTTYTYTV, YTYTQTQK, VLTVTVTV, TTVTVTLT, TKRLTVNV, VTVTHIEK, VNQSVNQL, SQSQNLQK, SKTSQNDL, SQTVSQEK, LETSTYRT, SESTVEMR, TKRETYNT, STYTVQHR, VRENKTTV, SSNKEKRS, SLEKKTTV, NENKEAEK, ISTDTRTH, DTDKVEVV, TKKIKRTH, DTDTELKK, VDTTTVTL, STITVTRT, RVTTTVTL, VTVTVARR, VETITYTV, SNYTYTRT, TETITYTV, ITYTYTQK, IVMSTTQI, STSTVNLK, TSTATTVI, SMTTVTKK, VDTSTVSV, STSDVTVE, RTSSDVTV, STVTVTRR, IVRVEVTV, VTVTVEQR, TTKVTVTV, VTVERSVS, VVTVTVNA, VTVVVTIK, SKTVVVNA, VTVTYSSK, IANYTYTV, YTYEYTTV, TQAYEYNV, YTYTLLVK, VDTSTYTL, SNSTYTRT, SVTSTYTL, STYTRNER, VNVNTNSV, NTNTNNVK, TTTNTNNV, NVNTNTVK, VVTVSIKD, STIEITVK, SVTVEIED, VTISVSVK, VTVSTVTL, SSVTVTVT, SKTSTVTL, VVVTVQSK, VLVSTYRV, SESSYKET, TRTLSYTV, YVYTYTQK, VNTSINKH, STSGNTIV, TQTTGNNH, DTSINTVR, VNTSTYTD, STSTNNVQ, STTSTNND, STYTNSVK, VKTLAKKN, SQSVKTTT, TKEVVKTN, ETQADKTV, VVTVTVTV, SRTATVNV, VTVTVTQR, VRTYTYSV, STYTYTET, RVTYTYNV, YTYTESVK, IEKIEYTI, STSEVEVI, TKKIEYVI, IKYELEIR, VVTYNYHL, ATVTITVS, TVNKTYTL, VTYNISVR, VFTVIVSV, STVQVTVT, RTTVQTNV, VTVIVTIR, VERSEYTV, SNSEYELV, TERSEYNV, SRYEVRER, QEINENSQ, NRSRRQLT, TRRERNEQ, NIEERLEK, TLLIEISV, SKIKIVRK, TVEIKINV, ILIEARVR, VVTVSVNV, SNVTVTVQ, SVTVTVNV, VTVSVSVR, VTTITVTI, SNVTVTTK, TVSVTVNI, ITVTTTTK, VVNITINA, FNISITVT, TVTVSINA, INITISIK, VTTVTVSI, SNVEVTTR, STTVEVNI, VTVTVSLK, VKTSQESL, FESTETVT, TKKSTETL, FTEQLEEK, VYTSSYKA, SESTYTRI, TTSITYTA, STSSYTIK, VKIIRYTV, FESSYTMI, TNEISYNV, FIYRSRVK, VLNSTINI, STSTITNT, SRTITINI, SNITISQK, VVSTNYNL, SNSTVVTT, SVSVTYSL, TTYNVQTK, RIRSTTTV, STSETLEE, KKRSETNV, SRSTHVER, VLTTEVRV, STSTVTLK, I l l i I VNV, VTVELTVK, IYTSVYKD, SESTYTIT, TKEVTYND, STYVIIQK, VQTSTANA, VTVTATTT, SETKTANA, VTATQHTT, VVTVNVNL, SNVTVKVT, SVNVTVNL, VTVNVDVK, VYVSKLRL, SESTLTVV, TVTSTLNL, VVLKRTVK, TTVSTHNL, SAHMVTTT, TTTSMHTL, VTHTVTTR, VETSTYTV, STYKYTRT, TETEKYTV, STYTVTTK, VVKSKTPI, SESTTITK, TNTSTTNI, SKSKVTKK, VNQSTNTL, STSNNKNV, NRTNNNTL, SQITNSEK, VNNLVYTA, LTVTYAKT, TNTLTYTA, LNIVQTTK, VATVTISV, STSTITTT, AKTVTINV, ITITYYAR, VGTFTYNA, LTSTFTLT, SKKFTYLA, FTYTLRVK, KKEVNYTI, SNVKYSTT, TKEVKYNI, VEYNIRQK, VESSISTT, HTHQHQRE, TETSQSTT, HSVIRRSK, VVTVTIKV, STVTIKVT, NVTVTINV, VTITISTK, VETVIYSV, SNSTYVRT, SETVTYNV, VTYIATVK, IVTVGVSL, SPVIVTVT, SVTVIVNL, VTVGVTVK, VQVTKTKV, SESKSIVR, STTTKTTV, SVTKNSVK, VDTKTYHV, AEKTLNVN, TVTKTYNV, KTYTHTVR, VVKSTLKD, STSKLTTK, TKTEKLTD, SELTITTK, QNTNIDTR, SSNVDVVT, TKTNVDSR, NTNIVTEK, VNTTTVTE, STSTVTTT, SNTTTVTE, LTVTTSTK, ITSIKVSV, IKVEVTTK, SREIEVDV, ISVKTKVK, VNTNTNTN, STNENTVN, EVRTEITN, NTNTNLVK, IRSSSINV, SNSKRTLT, SETSKINV, SSISESTK, VALSTYQH, SSSTYTVT, TVTSTYSH, SLYTLTVK, VLKIENSV, SNIINERR, ENTVINNV, IKTERRVR, VDKSSYNI, SQSEYTIV, GKKTEYNI, YKYSISEK, VITVVVQV, VTVVVSQK, KKTTTASV, STITQQQQ, TQTTTANV, VTVTQSTK, VKTSTLTL, SSVEVKVI, SVESELTL, VTVTVLVK, VVKVTVNL, SSTTVTQQ, TKTVTVNL, VKVTVTQK, RVEVSVDV, VNVSVVET, TRSVSVSV, VLVSVTER, VLEIKIDL, SSSEVKVQ, TVTVEVNL, VEIKVTVK, VVTISISV, LTSSIIVK, QVTISINV, ITISISVK, IKTSTVTL, SSVTVTTR, TRTSTVSL, KEVFEYKV, TDKFTYNV, FVYEYQVK, IFEKKYTV, STNIIKLT, TTEKIYNV, NEYKIRLR, VNKYSYNL, SNVSYKVT, TEEYSYNL, YKYSTTSR, VVTTTTSA, LTSTTVRR, SVTTTTSA, TTTTNRRK, TLKIEINV, VSVSITRT, TVEISINV, VKIELTRK, VATTTVTL, STTTVTTT, TTTKTVNL, TLVTYTSR, VDIFTYRN, STFTYKRT, SRTVTYNL, FIYTQSEK, VVTVTYSH, SNSQYKVE, TNTVQYTH, VTYTVTEK, VVTVKVTH, SNVTVTVT, KVPVTVTH, VTVKVEVK, VLLLTYNL, STTTYQYT, TKTLTYNL, LLYTYQQR, VNQSTSTV, STSSQGVK, SKNSSQNV, QQSTGSTR, LLTSTITA, VTITITTK, TEESTITA, ITITVRTQ, RREIVRDL, SRIERTED, TVREERNL, IEIVDTVR, IEVIKEKV, SNSEEKRT, RDEREEDV, IVEKRITR, VVSSTVTI, SSSTVVVT, TVGSTVNI, VSTTVITK, ITTSEYRQ, SKNIQIQK, TTKSIYEQ, STYEQEIR, TVTTTISV, SNSTITTK, STTATINV, TTITVSVK, VVTITVSV, STVTVTKT, TVRITVTV, VTITVTVK, VRVTNDDL, SNSTDTVK, TETTTDNL, DVDNDTKK, VQTSTYTV, STSTVTLV, TKTETYNV, STYTVQTK, VSSSTYQV, SNSTYTVQ, TQTSTYNV, SSYTYTQR, KNTSDNKL, SRSNNNTV, SQTSNNQL, SASDNSTK, VLEVNNTV, VSSNNKSV, TRSVNNNV, VENNLTEK, VVVSRYTF, STSTYTLT, SVTSTYNF, SVYRYTVR, ISTVDVSV, SDLEVVDQ, TRTVEVTV, VTVDVLEK, VLSVRVNV, LSSTLTIT, TVSVTLNV, VSVRLEVR, RVEITVTV, ITITLTLV, TTTLTVNV, IEVTISLK, SKVKVTER, SREVKVDV, VIVTYYSR, VVEIKIRV, SSSEITVK, STKIEISV, IEIKVEVK, VQTYTYSV, STYTLTTT, TRQYTLRV, YTYTTQTK, INTVNYQV, VSKEDTQV, SQKVEYTV, VTYNNQVR, VNSVTISV, STVDNREN, SRQVDNNV, VSVTNRNR, VDTITIST, SKSTITRV, TTTITINT, ITITKRVK, VTTSTVRL, SSSTVTKT, TTSSTVNL, KTSTVTEK, INVYTYTV, YTYEVTTT, TTKYEVNV, YVYTTTTR, VELLQYNV, STSTYRET, TRTVTYNV, VLYQRTEK, VVTVTARV, ITLTATTT, SNTVTANV, VTLTVQTK, VREYKYTV, YTYTIKQQ, TTQYTINV, YTYKELQR, VSSSTHIV, HSSTHTLK, STTSTHNV, HSHTHSEK, TVTVTVNV, INTITYSV, STSTQTTT, KNSITQTV, ETYTRTER, VRTSTVNV, STSVNNQT, TNTSVVNV, STVTNTTK, VQRYIYTD, STYEYEKI, TRRYEYND, YRYILEEK, VNVSTITE, STSSITVT, TKTSSINE, NVNTVTVK, VVTSVRND, STSTVTLT, TLQLTRND, STSVTIVK, VKINKHRS, STNLHEMR, TNKTLHTS, NIHKLEVK, VDTSIYKV, SDSTYVRK, SLTSTYNV, YTYVATQR, VTVIELKV, STSEYTET, SRKIELNV, IVLEYTVK, TVNVTLKV, SQLTVART, TTSVTLEV, VRLTVQTK, VFESKYTV, STSEYKVT, NESSEYTV, STYKYDRK, KRENIQRV, SESSQKEI, TRENSQRV, SEIIETEK, VVTVEYTA, VTVVVTVV, SVTVVYSA, VTVEVSVR, VVASEVNV, STSIVTVK, TVTSIVNV, NEVEVTVK, IDENKNDA, NTNKNKKT, TN EN KN NA, NENKRKEK, TDEIKYSV, SSISYKKT, SNTISYNV, IPYKRQVR, VVTVEVNL, STIIIKVK, ITVEITVK, VEEYTYRV, STYSYKRV, RETRSYNV, YEYTHARK, IVGVTTTV, KATVTTEV, VKVTVSVK, RIESSNTV, STSTNTTK, EKNTTNTV, SEVSVTTK, IKNVTVKV, SSVEVKVT, TKEVEVTV, VTVTVKTK, KMQYTYTV, STNTYNQN, SNTKTYNV, YTYTYSEK, KVTSTYTY, SSSTYNRT, TETLTYNY, ITNTNTTK, VNIITYNV, SKSEKRTT, SVKNEYNV, IIYTNSTK, KVKLEVNI, VSSVVETT, KRKVVVNI, LKVEITTQ, VSNLSYEV, SEYDYTRK, TKTLDYDV, YNYSYTVK, TDIYEYKV, YEYKYTRT, TQSLKYTV, YTYEYTSK, TVTSSVTV, SSSTVTVV, TVNSTVNV, STSSVTVR, KLQFKYSN, STSVYDVD, TKTFVYNN, FQYKYTEK, VKTISYNL, SSSTYTEQ, TKTITYNL, ITYSYTEK, VNTVHVNL, VTVTVKTT, SRDVTVNL, VTVHVSEK, VLTVTLNV, STLTLNQV, SQTVTLNV, VTLTQTQR, VVISTYNL, SDSEIEVT, TVKSEYNL, SIYTIQVK, VITATVSV, SNVTVTLT, TVTVTLNV, ATVTSTTR, INKVTVQV, VTSTVTNN, TSTYTVNV, VKVTNKEK, VDKVTYSL, STSEVERT, SDKVEYTL, LKYTQLRR, VNESNDRD, SESTKTTT, KQRSTDND, SESNKTTS, VDTITVSV, STVTITVT, SVTITINV, ITVTISRR, VTAQQYRH, SSSNQQQT, STKQTYNH, QTYQQQQK, VGTFVYTV, STYEVTVT, TVGFEYSV, FTYVVVTR, VVTVSYRV, SESVVKLT, TVDVVYNV, VTVSVRRR, IVSISITL, STISITVS, SVTISISL, ISISYSQK, KVTVSISV, VTVTIEVT, SVKVTINV, VTVSFLSK, VTTYEYYN, SNSKYTQV, TTKYKYQN, STYEDERK, IDKSVHRI, STSEITYK, SDENEIDI, SKHVHKER, VDKKTKTV, STSVTERT, TVKEVTTV, SKKTEIEK, IVVSTSTV, STSTVKVV, TVESTSNV, SVSTVKVK, VGTTQYNV, STYSYQRI, SETTSYNV, VTYQQSRK, TITSRISL, SDSEITLT, TLQSEINL, ITIRIKIR, VDTSRYSL, VNSTVERR, TTKNTYTL, VTYREERR, ENLSNVKA, LQVKVKNI, KNTSKVNA, VTVNNTQQ, VQENRKRV, SDNRKLNK, TTTNRKNV, NENRDTRK, VTSSNYRL, LESTYTTT, SKSSTYVL, YSYNTDVK, VN I I I I I L, STSTTTET, TKTETTTL, TTYTSTEK, QRTVTVRV, VTVTVELR, SVKVTVNV, VTVTYEVR, IRTYTYNV, STYTYTQD, TRRVTYNV, YTYTYTER, TNEVKVRV, SEVTSEKT, KETTTSTV, VTVKRSVK, VVTITYQY, SNITITVV, SVTVTYNY, ITITITVK, TVVVTVSL, STVVVTVT, TVTTVVNL, VVVTVTLK, VLTSTLNV, ITSTLTTT, TQTSTLNV, LTLTTTTK, KITVSVTA, STVTVNHI, TEEVTVNA, VTVSYLDR, VHTVEYNA, LTVTITVT, TKRVTYNA, VTYEITER, VVTSTVTH, TVKSTVTH, VDTNSKRH, SQTNTKTH, STKSTSIK, TVTITISI, SSSSITVR, STRISINI, ITITIEVR, VLVNQYTD, STSQYTRR, TTTVQYAD, NVYQETVR, ILTSQVSV, VSVTVNLT, TTNSTVNV, VTVQNTTR, VLRINITV, STSEIEVV, STKVEITV, IRINLSVR, VNTNQYTN, NQNMNNQT, SQTNMNNN, NTNQNSQK, INISTNND, SKSVNNEK, TNTRVNND, SIITNTEK, VKEIRIEV, SRIEYKLI, SKEIEINV, IEIRYSKK, VLTITIDV, STITITKR, TVTITINV, ITITIKVR, VETSVKTL, SNWKKTTT, KETSKKTL, KTKVKSVK, VLSVNYSL, ADVHYENT, TRSVHYNL, VSYNFTEK, VNTVTIKV, SQSVITNK, SDTVVINV, ITVTSSVR, VINVIIKD, SEIIINRT, TVEVIIND, INIIYKRK, QLRANVNV, VSYDKLES, STTQDVTV, VTVLSQSR, VTSSQKTV, SSSTKQQQ, NETVTKNV, STKQQSQK, TLTVEYTV, STSKVTVT, TLTVKYEV, VTYEVTSS, VDITTVTI, VSTTVNNK, TKTTTVTI, VIKTNSTR, TLKVEVRV, SNVVLEVT, KTTLVVNV, VSVSLTVK, VAVSKYSF, YSYELNVV, TLTSEYNF, YVYKLTVK, IVNIIVNV, VTINVNTQ, SVTINVNV, INVIVKQR, VQTYSYSV, SSSRYTQK, TTNTQYNV, YAYSETVK, LLTWRYTI, SSVEFQQK, SLTWEYNI, LTYTFAQK, KLTTTVTL, SVTTTVTL, VSVTYSVK, IETNSNQN, SSNTNTLV, SRSNTNDN, NSNSTKEN, VKESEARL, VSSLDVET, SKEELANL, VEVKDQTR, VVTSTYTH, STSTNTTR, STTSTNNH, STYTNSRR, VEQSTISN, TRQSTVTN, SQSTVTVK, VTTSNVSL, SSSSVEQR, TTTSSVNL, STSNVTRK, VTTITITS, STIVNKQT, TKTIVINS, ITIKQTTK, VHTSTYSL, STSKNTVN, SKTDKNTL, STYTNSTK, VLESTIRL, SESTIRRV, SETSTISL, SEITVSVR, VRTSRVTI, NTSEDTET, TRTSEVNI, NIVRDTVR, VVSVTYTL, SQVVVTLR, SVTVVYTL, VSYTFEDR, VTSYTYNA, TLYTVTTT, STTYTYNA, YTYTVTVK, KRLYTYTL, SNYTYEET, SRKYTYNL, YLYTYEEK, VTTIEITV, SSVSIKVT, KVAISINV, ITIEIEVK, TTTVTGNV, STSTGTTN, SKTVTGNV, VTGTNSEK, VVEVRINH, SSILISTT, KKEVLINH, IEIRVAVK, IMESRYSV, STSMKKET, STEEMYSV, KEYRKKER, VVNVTVNV, ITSEVKTE, TKTVEVNV, LTVTVTTR, RRINMNTV, STNSEEVT, TKSNSNTV, MINMEVTK, VEENKYKV, SENEYKRT, TQLNEYKV, NEYKVLEK, IQTNTNSL, SSNTNKQN, TNEYTNNL, NTNTNKNK, VDEVEERV, SEVREERI, SKRVRENV, VEVERFER, VYTYNYDV, YSYTYTQT, TRTETYNV, YTYNYTTR, IVSVSVTL, VNVQVTLT, TVSVQVNL, VSVSVLVK, KASSETRR, SNSHVEVV, SVKSHVNR, SSTEVEVK, VITVTINV, STVTITVQ, STQVTINV, VTITITLK, VVTVTNTL, STSTNNNV, AKTVTNNL, VTITNSVR, VVTVAISV, TTVIITVT, SVKVIISV, VTVAVHEK, VRTIKVSL, ISITVTEK, TRTVTVTL, VNSVSYTR, SSVNVQTT, TNQVNYTR, VSYSVISK, TVTITIKV, FNITITQT, TTKITINI, ITVTITTK, VLTTTVTV, SSSTVTVK, SVTTTVNV, VKTSTVNV, SNSTVTVK, TRTTTVNV, STITYTEK, VLAVDYQI, SNLEVKVT, TTKVEYNI, VAYIVQRK, VVTVTVRA, VTVEVTVQ, SVAVEVTA, VTVTVTQK, INTSTNRA, STSTNTVV, SNTSTNTA, NTSTNSVR, VMTDTKKN, NESTKITT, SKTETKTN, NTNTTSVR, VTTVTHTA, SENELKQT, TTTQEHNA, VTHTLTQK, VLTVTVNV, STSMVTLT, RRNVMVNV, VTVTTSVK, RIVVRVKV, VEVEVEIV, TVRVEVNV, VVVRVEVR, VLNITISV, STSQIVES, TRKIQINV, VNITVEEK, VNTSSENV, SASRERVV, TRESRETV, STSSEEVK, VQVSTYSV, STSVVTEK, TRTSVYNV, SVYTETTK, VETSTLTL, SSSTLTRV, TTTSTLNL, STLTRTRR, VSTFKYNL, FTSEWKQE, TTTFEYNL, FNYKETVR, TNSSSVND, SSSTVTRK, SVSVTVND, STVSVTRK, TVKVTVSA, STSEATIT, TRTVELNA, VKVTVTVK, IVDVTVNV, SSIKVTKT, TTTNKVNV, IDVTITEK, IDTSTYNL, STDTYNTT, KKTSTYNL, STYTHTTK, VETKIFSL, SNSKDTKT, SNEKKDNL, TTKITKEK, VNTSNYSI, SSSNNTVR, STTSNYNI, ETYSVSRR, KSEVTVSV, SKVTFTLT, RTKVTVNV, VKVTFELK, IDTSTYTE, STSTNTRR, TDTSTNNE, STYTNQRR, LSVTVTTT, TVTVTVTL, VTVTTSSK, TVQSRYTV, YTSEYVTR, TKTSEYTV, YQYRYTDK, TVMTTVSV, KTTTTVAV, IMVTVTIT, VTILKLNL, SSLELKVS, SVELELNL, LILKYQEK, TVQLQLQL, TTLQYNTT, TKTLQAGL, LQLQYSTK, VTISTVNV, TVTTTVNV, SITTVTRS, VDTVTVSL, VNVTVTTT, TNTVTVNL, VTVTVTSK, VLTVALTL, VSLELKVQ, TVNVELNL, LTVAFTQR, KVSSSYSV, SSSAVEKI, NKKSAYNV, SSYSKDIR, RYRYEYKH, SESVYTET, SQKYVYTH, YRYEEETK, VVENKNNS, STNTNTVK, TVTNTNTS, NENKETSR, VN EITAN V, STSTATLR, TQTITANV, VEATLLRK, VTTNTITV, STNANTTQ, TNTNAINV, NTNTNQVK, RVMVMVSL, SNVVVELT, TLTVVVNL, VMVMVTTK, TVTVTISV, STSTIEVT, TRKVTINV, ITITVEER, VVTVKVQV, VTVKVSVK, VVKSSVTV, SNSNVEVK, AVTTRVNV, FSVEVNTR, VTTVTVTV, ITSTITRV, SETVTIEV, VTVTIHET, VNNSEYQL, SESKYINK, TNTSKYNL, SNYENQTK, VVQVKVNV, LTVEVKTT, RKEVEVNV, LQVKVSTK, VNVSTSTL, SSSQSQNN, SETSQSNL, TISTSSNK, TSTIGIKV, LESELEIK, SKKVELTV, ITIGVSEK, RVVITVTL, STITVSVN, SVSITVTL, IVITVSVK, VETKTYQA, STKTETEK, TTTKTYDA, KTKTETVK, VEIVQVTL, LTVQVKRT, TLTVQVNL, LIVQKSVK, TAVVTYNI, SSVTVTVV, TITTTYNI, VVTTRYQT, STSEVVDT, SRVEEYNT, STYRVAIK, VVTSTVSV, STSTVTQR, TTTSTVNV, TTVTVSQR, RVQIDYTL, SSVRVTVV, HVEIRYSL, IQYMVSVK, VLQLTLTL, STSTLLVR, TTTLTLNL, LQLTLTVR, VFKVENRI, SLKENERV, TRRVENNI, VKIERTEK, VVTVTVSL, QTSEVTVT, SVSVEVTL, TTVTVSVK, TMTVKINL, SVITVTKV, SVTNTINL, VTIKVHVK, KTTTTITV, VNISITLT, KLTTSITV, VTITVEQK, VVTSSISV, VTVTITTR, SRSSTINV, VTISTSVK, VSTSNYNV, YNSTLTIN, TTTSTQTV, YTFNITVR, VDESSISV, STIKITRR, TLTSKITV, IEISRSVK, KETTEYTV, TTGKYTQI, TTDTKYTV, TTYEQTSK, VAVININI, SNIVNNKT, NENIVINI, IVINNTTK, I l l i I NNN, STSTNTLT, STTTTNNN, STITIHVK, IRSVTVSL, AQLTVTTV, SQTVTVNL, VSVTISVR, VLTTNVQV, VNTTVTRK, TTVNKTRK, VFTSTNSL, SESVNTTK, TTTSVNTL, STNTSTER, VVRVSVNS, SRVMVVVT, TVEVMVDS, VRVSIKVS, TRSYTLTL, STYSLTEQ, RRTHSLDL, YSYTETQR, ILKVTVSV, VSTEVREV, VKVTVKVK, SINVSVSI, TRVNVSVN, TVSVNVTI, VNVSVTNK, TLTVTVQL, VTVTLSVK, VDSVTVKA, ISVTVVTQ, SNSVTVNA, VSVTVHQK, LQQHTYEV, HNHEITTT, TVQHEYTV, HQYTIQTR, VDTINYNV, SNIQITNN, TQTIQYNV, ITYNNNRK, VVISTITV, STSIINLT, TLTSIINV, SIITITLK, ILEVSLKN, SSATLKLT, SQEVTLNN, VEVSLKVK, VTTNTNRN, SNSTNTLS, KQTNTNNN, NTNTNTMK, VLKVEVSL, STSEVTYE, SVKVEVNL, VKVEYQER, VTTVTVNI, VTSTVTRA, TETVTVNI, VTVTVTVR, SKTVTVNV, VTVSVSRK, VDTKTESA, ATKTETKT, TETKTETA, VTKTRTTK, VESHSLKN, HESQLKKV, SDTHQLTN, HSLSKQQR, KDVYSYNV, YSYNYERV, RVKVEYNV, YVYRREEK, RTTTNYNV, STSKYDKV, SENTKYTV, YTYNYSEK, VKTLTYNV, SSSTYQVT, KESLTYTV, LTYNYEDK, VVVVTVTV, STSTVVVR, VVVTVSRR, VFTYSYRL, SEYTYTKQ, TETVTYNL, YTYTYTRK, VNTVRINI, SDTKITIK, TKTVKITI, TTVRITEK, VTTVKLTL, ATLEAKTT, TKTVKLNL, ATLETTER, IEINTYTV, STSIYTTR, TKTNIYNV, YIYTTTTR, VYTVNVNL, VNVTIEVR, TTTVTVNL, VTVNITVK, ITPITVRV, LSIEVTVT, TVQAEVNV, ITVTVTSK, TNVTKSTL, STFEEKEV, SRSTESTL, VVSKESVK, VETVTVSA, GKVTVTRN, TETTTVEA, VTVTESQK, VNESVYSV, STSKVKTT, TTESKYNV, SQHVTTTK, VDESKSRV, SDSVVKVV, TKTSVSNV, SESKVTQK, VDTFTYQV, SEFTYTKV, SDTFTYQV, FTYTRSVK, IVTVTVNL, STATVTVV, SVKVTVNL, VTVTISVK, VEKSKVKV, SESEVTRT, RNESEVNV, VKKKTTVK, VTVGVQTV, NIGTGVQA, MTMSITIT, RTEVSITM, KSKTKTKT, TDTKTKNK, ITISITIT, TITISITI, ITITITIT, DAKITITI, TVTISITI, VEVSVEVK, VNEVSVEV, LITITITI, ITISITIK, VITISITI, VTVSVTVT, TINVSVTV, ATATATAT, VATATATA, VTVSVTVK, DVTVSVSV, VVVSVTLT, ILTVSVTV, KVTVTVTV, TVEVSVEV, ENKSKTKE, KETESKTE, ITYSYTVT, TLSISYTY, TVTVSVTV, TVKVSVTV, IEITAEAT, KITITIRI, VITVSVTV, IVIEIVVK, ITEIEIVI, KLTISITI, IEINIEIT, NLNINILI, ISISISVT, TLKISISI, VTVKVEVE, NIKVKVEV, VTITITVT, VTVAVTAT, VVTVAVTV, AQYSYQYT, VNSISYQY, ITISITVT, TISISITI, VVVSVVVV, TLKVSVVV, VTFSFTFT, NATVSFDV, VTVAVTVK, RVEVAVTV, DTDSSTTE, ERKDSRTY, YSYSYSYT, VQQSSYQY, KITITITI, VTVSVTIV, VISVSVTV, YTYVYSEK, VTVKVTVE, RVTVKVTV, NTISITIK, INENSIIN, TITVSVTV, VEVRVEVV, ERKVRVEV, IVISLVLT, LLVISIVI, VTVKVTVT, TVKVKVTV, KEKSVEVT, ISKKSKEK, FVFSFVFT, TLTFSFVF, ATATITIT, DSKVTVTA, VLMVSVTV, RVTVSVTV, VTVAVTIT, TVTVAVTV, NTNKNTNK, EKTNKNTN, ITYSYTYT, KKDYSYKI, TITITITI, NLNSNLNL, TSSNSNLV, VVVSVVVT, VLQVSVVV, VLVSILIK, ELSVSVLV, VTVSATAT, TLVVSVTV, VTVSVQVT, DVTVSVTV, VKEKSITI, DVTVSITV, IVISNVNT, INSISIVI, DIDSYTYT, TVTDSITD, ATASATIT, TITISATA, VTASVSIT, KLTVSVSV, VTVSVTVV, VVTVSVTV, VTTITITI, QTQTQKQT, VKTQTQEQ, TTVSVTVT, VGRASVEV, KSKSKSVT, REEKSKSK, DKDEDKDT, VDEEEDKK, VKVSVKIT, VVTVSVEV, EKRSRTRT, VDTTVRTQ, ITISITIE, VTVTVTLT, TITVTVTV, YVYKYEVE, IIKYKYEY, VDVTLILV, VLTVTVIV, QVSVSKNV, DTDSRTRT, TDTDSRTD, ITITITIK, EITITITI, VERYVYSL, VEVKVEVE, VVKVKVEV, EKKESVTI, RTRTRTRT, RDTRTRTR, NQNSNQIK, VTENSNQN, VRVEVRVI, RKEVEVEV, VTVTVTIT, ITITKTKT, IDTITITI, VSVSVSVT, TVSVSVSV, GTGRGTVE, RAVGRGTG, TILITITI, VEVSVSVR, TAEVSVTV, EKKKTVTV, VTVTVSVT, ELTVTVTA, KVKSKKKI, VKEKSKEK, QTKTVTVT, KETKTKTK, NTNSVTVT, TKTNSNTN, YTYTITIT, TITYTYTY, VLVSVLIT, TVTVSVLV, FTFSFTFT, TATFSFTF, DTDADTDE, RETDADTD, NRNTNRIT, VNTSTNEN, YQYSYQYI, IRKYSYQY, NTNSKTKT, TSTNSNTN, VVVTVVVQ, NLVVTVVV, TATATVLV, RVEVSVLV, VITVTVTV, KKTVSVKV, EITISITI, IKVTVEVT, NVKVTVKV, VATISITI, IVLSLVLT, TLTISIVI, ATAAVTVR, RVEAAATA, IEISIEIT, LVKISIKI, VAVAVAVV, VAAVAVAV, ITISLTLK, IIEISITI, VKLKVTVD, VGRVKVEV, VTITITIT, NVTVTITS, VTVSITIT, QTQTKTKT, NKEQTYTQ, ATASATVT, RATASATA, HIHSHQIT, IEQHSHTH, ITVSVTVT, VLSVSVTV, YEYAYEYE, VEKEAYKI, VIVSVTVT, VLTVSVTV, MTMSMTVK, VVEMSMLM, KVTVTVSV, LITISITI, IEIKIEII, ILKIKIII, VTVSVTVQ, KKTVSVTV, LTLSLTLT, TLTLSLTL, VVVTVVVV, VLTVTVVV, ITITITDT, NSAITISI, VTVDVTVR, RVDVDVTV, NITITITS, YTYTYVIK, EYTYTYMY, TINISINI, VTVTITIT, KITVTVKV, QTQSQTQI, QNTQSQTQ, LTLTLTLT, TLTLTLTL, VTLSLTLS, NATVSLTV, VVVTVVVT, VVTVTVVV, NVNTIVIT, VTTNTNAN, ITISFTFT, YLTISITI, DSDSVSVV, LTSRSHSD, KTKTKTIT, VKKETETE, ETEAKTKT, TNIEAKTE, LTLTLTVT, TVTLTLTL, STKSKTKT, QTTSSKTS, FTFTFTIT, TTTFTFTF, NITVSVTV, ITISITIV, TITISIKI, STDTDTDV, EDTDTDTD, KTKAVTVT, LKVKAVTK, KVTLTVVV, YTVSVTVT, TLTYSVTY, VTVSVSVT, KVTKSVDV, VTVAVTVT, LVTVAVHV, LLLQLNLV, LVQVQLNL, VTVSLNLR, ELEVSLNV, ITIKFTFE, TAKIKITI, TLTVSVTV, IKITIKIT, IITITIEI, VVVSVVVE, VLKVSVVV, ILTLSLKL, RITITITK, IAISIAIT, DLTISIAI, VIVSVNVT, LLTVSVIV, ITISLTLE, TLRISITI, VAQVSVTV, DTDTITIT, TTTDTDTD, KLTKTVTV, YTYTYTVV, SRRIEYNL, TMTYTYTY, ENESENIT, NKVESKNE, ITITITIV, ITISIKIE, SVSTSVST, NSTSTSVS, EQTVSVVV, ITITITLT, TVTITITI, ATASLTLT, TLTASATA, ASASISIT, VISASVSA, LTVSVTVT, HTHAHTTT, RATHAHTH, FTFTFTFT, VVTFTFSF, VTIEITIT, KMEVEIKV, VVSVSVTV, HLHTHLVQ, VETHTHLH, VTVTVTVA, DDTRTITI, HTHTHTHT, TITNTHTD, NLTISITI, VTTATVVV, ITISITII, VIVTVIVT, TLTVTVIV, GVGSGVVV, KTTGSGVG, EGTVTVEV, ATASATAV, VAVASAEA, DRDEDRDR, VDEDEDRD, NVTVSVTV, EITITITA, QTQSQTQR, RQDQSQTQ, VLVSVQVT, TVKVSVQV, VTVQVTVT, KATVQVVV, NLTVTVTV, ISIKISIT, RIEIKIEI, VSVDVSVR, EVDVDVSV, ETTVTVEV, KTKVKTKT, DDEDVDTK, ITIAITIR, DIEIAITI, TLEISIRI, KVTVKVTV, GVGSIKIV, KVEGSGIG, ETETYTYT, TKTETETE, VTTITIVI, ITITITVT, KITITISI, VNVSVNKT, NVTVSVNV, VVVSVVVK, VVEVSVVV, AQASANAT, TLTASANA, NLTITIQI, VVVSYVYT, TLTVSVKV, TVTVTVVV, EVKTKTKV, IDTETKTK, VTVQVTVE, VLRVQVTV, VTVSVSVK, TTTVSVTV, VTLTLTLT, TVTVTLTC, TATVTVTV, VLVSVLVT, VISVSVLV, ATASITIT, TVEVSATA, VVTVKVEV, I I I I I I IT, TVTKTKTK, TNTVSVTV, VTVAITIE, RLAVAVTV, LTLTFTVT, VATLTLTL, ITITLTLT, LVTITITI, NTNTNTNT, VVVEVVVK, LIEVEVVV, KKTKTNTN, YVYSYVYT, VEQYSYVY, ITVTVTVT, VVTVTVLI, HVHTHVVV, EKRHRHVH, VTVSLTLT, RVSVSVAV, ATARVTVE, VTRARATA, HTHSHTHT, TSEHSHTH, VVKVAVNV, YTYEYEVT, VITSSVTN, VKVSLKLK, EVEVSVKV, IVTVTVTV, VKVSVKVE, KVTVTVEV, VKVSVKVT, VVKVSVKV, VTVEVKVK, VVEVEVKV, KITISITI, VVVTLVLV, VLTVTRVV, VTTTTVTH, YTYTYTVT, DKTETYTY, ELESELET, LKEISELN, VSISISIE, RVTVSVSV, VTVRVEVE, HTRVRVEV, I I I I I I I I , TA I I I I I I , YTYDVTVR, DVDVDVTY, VEVTFKFT, TLTVTVNV, TFTVSVTV, YTYSRNRV, RRSESEVY, VVVSIVIV, VVSVSVVV, TLLVSVIV, ITIQVTVT, TLQIQITI, VIKISITI, DVDTDVTV, KDTRTRER, KVKTRVET, VKSKTKVK, DVVVSVVV, LKLQIKIK, ELLLQLEL, ITIKITIT, ELKIKITI, TKTKSVTV, VEVSVEVT, QVSVSVRV, VTLQVTVE, KVTVQVTV, VTVSKEKT, DVKVSVKV, NVNTNVNK, TNTNTNVN, NTVTVTVT, VNTNTVEN, TTTISIEI, RGTVSVTV, VTIQITIT, TLQVQITV, VTVSVLVT, LLLVSVEV, VTVSVTVE, TVRASVTV, QMGQGKVT, VTQQQGVQ, KTKSKTKT, NKTKSKTK, VTVTVTNT, TSKATVTV, ATATVTVT, VGTATVLV, IEITIEIT, DKTETIRI, NTYSYKYE, KNTNSYEY, ITISINKT, EATVSITV, DIVVSVFV, KITVSVEV, VVVEVVVT, TVEVEVVV, TLTATATA, ATASATVK, VSEASATA, VRVSVVVK, VNVQIQIT, TVQVQVQV, KQKSIQIK, VKEKSEQK, I I I I I I NT, TS I I I I I I , VTLTVTIT, TITIAVTV, ISISISIT, NISISILI, VSVSVSVK, ELSVSVKV, NTNSNTNK, EITNSNTN, NKNSNKKV, VNVNSNEN, KTVSVTVT, KLTKSVTK, ITISITID, KVTVSITI, EKKSKLKT, TDKKSKEK, NVNSVLVK, VNENSNLN, TVEVTVTV, ITVSVTVE, KVTVSVKI, YTYSYTYT, NKSHSYTS, VTVSVNIT, TKTVSVTV, TVEVSVTV, ITISITVI, NVTISIQI, VEVSVEIE, TVKVSVKV, FQFKFEVT, WVKFKFEL, EVTVTVTV, MRMEMRMR, EVEMEMRM, VTVAVEVT, TVRVAVEV, VSVEVSVK, EVEVEVSV, VTVEVTVT, ELRVEVTV, TAEVEVTV, KSKSKSKT, LDTDSKSE, FVFTFVFT, TLTFTFVF, VTVVSVVV, VVVSVVLT, TLSVSVVV, VVTVTVSV, KKTKAVTV, RTRSRTRT, TETESRTR, KDKSEDEV, VKTKSEKK, EVRVSVTV, TATASATA, LVTVTVTV, ITISVTVT, NITISITA, HKHKHKIT, TTVHKHEH, ITIQITII, VLTIQITI, ITIAITIV, ETKVAVTI, VEVTVTVT, VQTETVTV, IIISIIIK, SISISIII, VSVKVEVE, LIKHKVLV, TQSHSLTL, LELKLELT, VTELKVEL, KTKSKTET, IKEKSKTK, LTLSVTVT, VLTLSLTL, KTKTNTVT, KKTKTKTK, AVASAVVV, DVTVSAVA, FSFSFSFT, TLSFSFSF, TKTATAKA, VVVSVVVI, IVTVSITI, NTNSNTNT, TNTNSNTN, VTVSVVVT, VDKESVTV, LTLSLTVT, LITLSLTL, VTISITIE, TARVSITI, KQTISITI, QVTVSVTV, ATATATVT, IVTATATA, TVSVSVLV, VLVSILIL, VVWVSVLV, VTVSVTAT, TLTVSVYA, KVEVKVTL, KTKSITIT, VKAKSKTK, VVEVSVSV, VAVAVAVE, VTKVAVAV, TLTGSLSL, TITVSVKV, ATASFTFT, KATASATA, VTVSYTYT, NKSTSVIV, TLKVKVTV, KTKSKTIT, KKTKSKTK, LTLSLTLL, VRILSLTL, TVTVSVVV , TASVTVTV, DVDSDRVV, RREDSDVD, VTVAYTYT, ERRVAVTV, EEDSDEDT, DTIHSAKT, LLKVKVEV, VKVSEKEE, LKKESVKV, YTYSYTYK, VKEYSYTY, AIFTFIFT, IATATFMA, ITVSVVVT, VTVTDTDT, ERKVSVKA, VVVTIIIT, EVTVTVVV, HTHTHTLT, VNTHTHTH, VVVSVTVT, VITISVTV, NTKSKTKT, VDVNSKTN, TVSVSVTV, ITISITVQ, TTTISITI, TKTVTVTV, NNNKNEVE, NYKQKNIN, VTVKFTFT, RAEVKVTV, VTLTLTLV, TGTLTLTL, TAVTTVTI, RVEEIVIR, EREREEVR, KTDSDTVT, KKQKSKTK, KTTITLTY, VTVMVTVV, VKMVMVTV, DDTKSKTK, KKTKTVTV, ISISISIV, VVSVSISI, ILKVSVTV, VTATATAT, STSQSTVT, TTQSQSTS, HTHSNTNT, THTHSHTH, TVTATATA, KTTVSVTV, VYKVSVSV, NIVNKSKT, VENNNVSN, KTKSYKYT, NKQKSKEK, VQVSVQVT, ILQVSVQV, IEISIEIK, LITISIKI, NTNENTLT, TAENENTN, VATASATA, IVITITIT, SATITITI, KTKTITIT, IKEKTKTK, ETETVRVT, VLTLTFER, ITISRTRT, VNSISITI, NITVTVTV, ITIQITIT, TTQVQITI, VTVSITVV, VVVVSVTV, VISISITI, IITITITI, TTTSTTTT, TLTTSTTT, EIKISIKI, TEVSVSVT, VLEVSVSV, STSSVTVT, TLTSSITS, VTVTVEVK, VVTVTVKV, VVTVTITI, TVTVSITI, VTVQVTET, TRTVQVTV, ATASIEIK, EVTASVRA, HTHSHTVE, LSTHSHTH, IDISIDIT, VITISIKQ, TLTVTITI, VTVSVTVI, VFVVSVTV, TTTSITIT, TVTTSTTT, TVVTVVVK, EVTTTVVT, LVEVEVTV, IVTVSVTV, FTFSITIT, VKTISFTF, VSVSVSLK, TLEVSVSV, IEISIELE, KLKISIEI, TVNVSVTV, DTTVTVVV, VLVSILIT, TTSASVLV, TVTVTVKV, AVASARAT, TVEASAEA, ITITIDIT, TSTITIQI, IVISIWT, KIEISIVI, VAVSAVAT, RLSLSVEV, TTTLTLTL, TVTQTVTA, VKVSVKVK, TIEVEVKV, ETEAETET, ENTEVEDE, FTFSFTVT, TLTFTFTF, IRIAIRVE, IIRIAIDI, TGEVKVTV, VKIEIKIK, IKEVEIKI, KLTVSVTV, VTVAVLVT, VVTVAVLV, AVTVTVTV, KTKTKTKT, IDTDTDTD, ESESKSKT, TTTESESE, TETSTETE, DLRTSTET, TVTVTVTA, NLNSNLNT, VTQNSNLN, TFSLSLTL, TTTSVSVT, TISTSTET, VTVSVTLT, TGAVSVTV, VTVRVTIT, DVEVRVTV, QITVTVTV, VEVTIEIT, VIKVTVKV, LVLTIVIE, RLTLTLVL, VITITITI, VLVSVLIL, VVSVSVLV, NNTNTVDV, VLTVTVVY, VNVTVTVT, RATVTVTV, NLVVSVTV, TETTTEVT, TLTTTTKT, ITVMVTLV, VAMVMVTV, VGTVTVTV, ELTISITI, NDSDSVIV, RITVSVTV, VGTASVEV, KKKSLKLT, KKEKSKEK, VTVQITIT, VLKVQVTV, IKISIKVT, EIKISIEI, VTVSRTRR, VDEVSVTV, ITITVTVT, TITITIII, NTNTNTTT, MKKNTNKN, KVTVSVTV, TETRSRTR, KVTTSVNV, RAKVSVTV, INITINIK, KLTVAVTV, TVTRSVTV, KSKTKSKT, TLTKTKSK, TTTVTVVA, VEVRVVVT, RLEVRVEV, VISISIQI, VTVTVIVV, DVDSTKIE, VVTDSVVD, TVTTLVLT, TLTTTTVT, EIIVSVSV, ETETETIV, TKIKTETE, RLTVSVEV, RVNVSVTV, YVYRYEYT, RVTYRYEY, QTQSQTQT, TETQSQTQ, VTVTVTAT, TFTVTVTV, INININIT, KINININI, VVVQVVVV, VAFVQVVV, VTVTVTQT, NNTVTVTV, HTHKHTHE, LKKEKHIH, KTKAKTKT, KKEEAKTK, VTLTLTLK, KTKEKTET, TKKKEKTK, EVTLTLTL, FTFTFTVT, VLTFTFTF, DISISITI, EATVSVVV, NTNSNTQT, TLTISITI, VSVAISIT, VVTVAVSV, IKIEIKIK, EKKIEIKI, NVNTYVVV, VKVNTYVN, NTNTNTVT, TVTTTNTN, TTVTVTVT, NTTTTVTT, VSVISITI, PTKTKTKT, VKTDTDTD, ATA I I I I I , TATATATA, YTYTYNVT, VKTKTYNY, TVTTSVTV, VKEVSVTV, SVTVSVVV, VATVTVTV, DIDSHIVK, VDVDSDID, RVEVTLTV, TETKTKTK, IINISITI, LVTVSVTV, KTKEITIE, VAKKKVTK, ITISVLVE, KLIISIKI, KLTVTLVV, VVSVSVSV, VIVSVIVT, VVSVSVIV, RLTVSVKV, ETYTYTYL, VKTETYTE, KITVSVTV, QTQSQEQR, IQDQSQTQ, TTTNTNTN, NVTVTVTV, VTSSVTVT, VTISIKIE, KVSVSVLV, ITITIKIT, DITITITI, VVVSVEVR, ELSVSVEV, EVKEKVKL, DRREEEVE, AIASAIIT, TASASAIA, VEVDVEIR, DLDVDVEV, VVVVSVVV, ISISISQT, TLTLSISI, ATYTYTVT, TLTLTYEY, KLTVTVTV, IGTGSITV, TLTVTVDV, RDEVSVLV, VTLSLTLT, VVEVSLTV, NVKVSVTV, DVNSNINT, ENVNSNVN, YVYSYVVV, IILYSYVY, ATAAVTVT, VVTLAVTA, IKITVVVK, EQTITVEI, VVVKVVVE, KVTVKVVV, TMVVTVTV, EIETEIET, TETETEIE, VQVSVVVT, RIQVSVVV, IEIKIEIT, VAKIKLEI, IKIEIKIE, RVKIEIKI, DTDTYTYT, TETDTDTD, DTDSKTKV, TDTDSDFD, VTVSITIK, EVTVSVTV, STSQVTVT, TSQSQSKS, VSVQVTVT, TITVQVTV, NTNSNTVT, RSTNSNIN, GTGEGTGR, ELEGEGKG, QTQTVTVT, VTTQTQTQ, TVTKSITI, ETKVSVKV, VTVSITIE, TLTVSVDV, VKVEVKVT, VKEVEVKV, FTFTVTVT, TVTFTFTF, VVVQVKVT, TVEVQVVV, SLSASAAA, ITISISVT, VDVRVQVV, VVTVRVDV, IKKESVSV, VTVAITVT, IRIEIRIL, RIEIEIRI, TVVVSVTV, ITVKVTVT, VVKVKVTV, ITIEVRVR, NIEIEIRI, YTYTYTYT, IATYTYTY, TLTITITI, TLTASVTV, RGTVTVTV, VTVSVTIT, ATATATLT, ATAQATAK, TGVAQATA, ATASATVE, RAIASATA, NTNTNTKT, TTVNTNTN, ELSVSIEV, AVASASLL, NAVASADA, VLTVSVKV, VVVSVTVE, ELKISVTI, ETESTTTG, EKKESENE, VTEVSVSV, VEVRVEVE, TVRVRVEV, ATASATAT, VLTASATA, VTSVSISI, LTLTLVLV, I I I I I LVL, TTSTSTTT, ITITITIA, RVTVTVTV, DVSVSVTV, VLTVSLTL, VTVSIQIK, VIEVSVTV, RVQVSVLV, VTVTLTLT, NTNSNQNT, VNRNSNLN, VVVVVVVV, LVVVVVVV, IEISIEIS, VLVSVLVA, TVLVSVLV, LQLSLQVT, RAQLSLQL, IEIAIEIK, EIVIAIEI, STSTSTSV, TTISTSTS, VTVVVTVT, VTMVVVHV, ASAQISIT, ALTAQASA, YVYSYVVT, RREESYVY, VAVSVSVT, VTLTSVSV, DTKTKTKT, VDTETKTK, RVVVSVEV, VVVVSVNV, TKTNTYTY, ATASGTGT, LAEASATA, ITITITVV, TVQVSVTV, VKVKVKVE, VTVTVIVT, VTVSITVT, AKAEAKAT, VVEVEAKA, GKGSGKVK, VLEGEGKG, AVASVVVR, DLTASVEA, EIKVKVEV, VTDSDTLT, TQKVSVIV, NENSNNLT, NAENSNNN, VTITIVIT, VVQVTVTV, RTEREVTV, KTKSIMIK, VKEKSKLK, LTLKLTLE, VLKLKLKA, TTTVTVRV, ITIKIEIT, TIKIKIEI, RTVVSVTV, VNVSVNVT, VVNGSVNV, VTVAVTVV, VVAVAVTV, LTVNVTVE, NLNVNVEV, EVKVSVVV, ITISITMT, TITVSITI, ATASVTVT, TVTASATA, IKTKSITI, HTHQHTHV, VKEKQHIH, ETFTFTFT, TGTETFIE, VRVAVRIE, RTTIAVRV, VVEVSVQV, QEQRQESV, IQTQRQEQ, IKISIKIE, NIKISIKI, VTSVSVNV, REVSVEVT, VRTESVEV, TVPVSVTV, KTKSKTVT, TKTKSGTK, DTDSDTVT, DNSNSDTD, YSYSISIR, EIIYSYSY, VKVEVKVK, RFEVEVEV, ITISILAT, KFTISILI, DLTVSVTV, TVTVTVIV, FTFSFTFR, ELTYSFTF, RTETETET, VQTRTEVR, VKVSVKVN, DLEVSVEV, ETESVTVL, IKSESVTE, TDTKTKTK, ITISITIR, DNTISITI, VQVQVQVT, RVQVQVQV, IKVSVKVK, VASASITI, VITVSVIV, TNTNTKTN, VVTVTLTV, VITTTVTG, KTKEKTLQ, TEKKEKTK, VLELSIKI, NVTVSVKV, NISDSVTL, ELTVTVTV, ATAAATVT, TATAAATA, VTVSATAV, VLAVSVTV, VEVSVEVE, TIHVSVEH, AVASAYAL, VVLASALA, KKEKSVTV, IKISIKIT, KVEISIKI, KNTNTNTN, LVLSLVLT, EIKVSLVL, HTHAHTHI, NKEKAHTH, VTLSLALK, DEDDDTIT, EKRDDDVD, YVYTYVYV, ERTYTYVY, FQFTFQFT, TATFTFQL, VTVEVTVR, EVDVEVRV, ETRARTRR, LDDEARTE, VGTGAVTV, RLTVTVTV, EKEEYKYV, KEEEEKKK, ITIKITIE, KVTIKIKI, VTVTVLVT, ALTVTVTV, EVTVTVKV, ITIEITIV, VVVIEIKI, KEEESKTK, MTMTMTVE, VVKVTMTM, VTVAVAVT, EVKVAVLV, GTGTGTGT, TLTGTGTG, DVDEVVVR, HTDDEVVD, ITIEITIR, ELEIEILI, VTVSVRVT, TVEVSVIV, RVKVKVIV, NVDSNVIT, VATVSDVD, TVTVQVTV, TLSVTVTV, VTVTITIE, TVKVTVTV, ITTISITI, AVATITIT, VVTATALA, ATASVTIT, TLTASVTA, KVTYTYTY, ITITIIIV, VVISIVIK, DVEVIIVV, ATTNSVTV, TESHSVEV, NIKVKVEA, KTKLKQEE, TKQKLKQK, TATVSVTV, VTLSVTVT, ALSVSVIV, TVTVSVEV, VSVSVTVT, RITVTVTV, VTVTFTFT, DKCKSYKY, ITISIEIT, TLTITIKI, TMKISITI, KVTVAVEV, ATLTLTLT, TATATLLA, IIISITIT, KIEISITI, TTTVAVAV, YTYTYTYE, KNTYTYTY, TTTTSITI, ELNVSVTV, VKVSIEIK, EATNSVSV, VWISIKI, TTTKTTTT, EVKTKTTT, ATATATAV, RVTATANA, TQNKTYLL, RISVSVTV, VVVSLVLT, TLEVSVVV, ITIEITIK, ELTIEIII, ETESHTHT, DDTESEKE, DVTVTVTV, VIQVQLTV, YVYTYVVT, VVTYTYVY, TVTVTVLV, VTINVTVT, LVNVNVTV, VEVTVEVK, TVTVTVEV, LVLMLVLE, KLMLMLVL, EEETVLVR, ERTETELE, LQLSLQVE, RLTLSLQL, IKISIKII, VWVSIKI, NLTSSVSV, LTVSVTVR, TLELSVRL, VAVSVAVT, AIATVIVT, TVTATAIA, LTLSLTVE, LFKLSLTL, VTISITIT, RLSVSITV, TTTSTTVT, VKTTSTLT, NLEISITI, TLTVSITV, VTVKITIT, TVEVKVVV, ELTASVTV, KVEVQVTV, VEVSVLVT, VLEVSVLV, TKTKTVTV, DVDSEVEV, VDEDSDVD, IATLTLTL, LSVSVSVT, RITISVTV, VEVNVEVK, EVNVNVEV, TTTSVTVT, VTTTSTTT, VQVSVSVR, EVSVSVDV, RVEVSVQV, VKVTVKVT, LVTVTVKV, DVEVEVKV, DVVSVTVE, KKVKSKLD, LTETELEV, TSTETETL, VQLSLQLT, TLTVSVQV, QTRTRTRT, VDTQTQTQ, VATVQVTV, ITISILIE, IVKASALA, KVSVSVNV, VTVTVTVE, RLTVTVIV, LLEVSVTV, KTLTEVET, VTVGVTVT, TVTVGVTV, ETETVTVV, VKTETKTE, TVTVSVQV, KEKTKKVT, IKEKTKEK, HITVSVTV, AEASAEAV, RAEASAEA, VGTISISI, VITDSDKD, IVISIVIT, LLTISIKI, IDIAIDIT, VIKIAIDI, VQVAVQVV, RVEVAVQV, HEHRKTKT, RSESESIH, VEVAVLVL, VVKVAVLV, EKKDSVTV, YTYTATVT, LSLSISII, TLILSLNL, VEVSVEIT, TIIVSVEV, VATVSVTV, SSSSVSVT, TLTSSSSS, EAKVSVTV, ISVSVSVT, HTHSHTLV, DIKHSHTH, LALSLSLT, VLTLSLSL, LIKISITI, VKVEVKVE, ELKVEVKV, HKYSYDYK, VKTHSYHH, VTVAVTLE, VLRAAVIV, KIKSKVKV, VDVKSQIK, VLTVSITI, VEVSLELT, QLQVSVKV, ITISITLV, DITISITI, TTESETET, TTTTSLTG, VQTNSVTV, VTVSVTVR, NVDVSVTV, ELTASATA, VRVSKLTV, SNSELTET, TRRSELMV, AVLKLEER, TSVSTHKI, SESMHTQT, TEARM HGI, SVHTELTR, VLTNTYSI, STSTYTRT, TVKNTYTI, NTYTESVS, VDTLTYQN, STFTYTRK, TKTLTYSN, YTYTKTVK, VTKNQEAR, STSSEQKT, TEKKSETR, SSEQKLVK, VLTYTWTV, STSTWTLK, SETYTWNV, FTVTIAVK, ITEKRYAV, SEYQYVEK, TQTKQVNV, YTYRESTK, VVSVTVNL, ISSNVTIK, TVTYNVNL, VSVTVTVK, VLSYTFNV, YTYTFTVV, TNTITFNV, YSFTLTVR, VVTVTYNV, QVTVTYYD, STSTVTTK, TKTVTYSD, VTTTVTEK, TTTSTSSL, STTSTSDL, STSTVSRK, VVESTYNQ, TSSTKKVV, TEESTYNQ, KTYTKKQS, VVTVGVTV, TVKVTVNV, VTVGVSVK, INMTKVSV, SSTTVTNT, ANKTTVNV, TMVKNLVK, IVTNNYNN, SSNINEQK, SHTNIYNN, KNNKSSQK, VDTSTHTI, STSQVTRT, TETVQHSI, STHTRTVK, VETYTYSV, YNYTVSVV, SKTYTYTV, YTYTVHVK, VASFTYVV, KVTVTYVV, FNYTVTVK, VDKSEINI, HTSRVEQT, SVKSRINI, HKIEQFEK, VENVTYNV, VNVKITNT, RKTVKYNV, VSYTYTVR, KVQLQVTL, and STLKFERT. The one or more beta strands and / or one or more alpha helices may comprise one or more of the listed insertions. Different combination of these eight amino acid elongations / insertions may be used in the same beta strand or alpha helix or in different combinations of two or more beta strands and / or two or more alpha helices. The eight amino acid elongations / insertions may be used in the one or more beta strands and / or one or more alpha helices consecutively or separated by existing sequences.

[0239] The one or more beta strands and / or one or more alpha helices may be elongated by one or more of TVTVTVTVTV, VTVTVTVTVT, KVTFTYTVTL, SEFEVEVTVT, SVTFKYEVSL, FTFTYTVSVT, TLNVGVGVTS, SSSSVNVLVV, SVTVNVNVTS, NNVGVGFSVT, VNTVTVEGRG, VNVRVEVTRR, SVEVRVEGEG, VTVEVTVRRT, TEVTENNYTA, TTNSYKVETT, TKRNENQYSA, TTNNYKVTTT, VLTATVTNTQ, STSTTTVTRT, GETVTVTNTQ, ATATVTRVVT, ETSITITYSL, STTTITINVK, STTTEITYNL, ISITYTYKRT, RVRLIVTYSI, SEVTVEVLRQ, SEELEVEYNI, LRVTVLVQQT, VLSISVSYTA, SSINYTVTNT, TTTITVTYNA, ISISVTVTQT, RVVVTVVYSV, STITYEVNVQ, TVTVEVEYNV, VTVVYKVTVT, TNTNTNTFNS, NNNTNTNTTT, TNVNTNTFNS, NTNTNSNRET, IDLVEIEVTL, SNVRVRQNTK, SRSVEIRVNL, VLVEIMHSVT, VATSETTVSL, STSTQKQTYR, SRRLETKVEL, STIEQKYERT, VFNNKYEYTQ, SNSEYEYTER, SRSEKYEYEQ, NKVHYEYAET, VTTVMNKNNT, SNTELEVTTR, TRTVENENNT, VTNKYTVQET, VLTVRVEYRV, STVAVEVTIE, TLTVEVEYNV, VTVEVLVSQT, VQINENKNRN, SNNTNEYELV, SRSNEMENNN, SINKNEYKVT, VTTVTVTVTS, STVTVTVTRK, STKVTVTVDS, VTVTVTTKVT, TLTIEITISL, STSEIEITTT, TVTISAEINL, ITITITTQVT, KISVTVTYTL, SNVTVTVTVV, SVTVTVTYKL, VNVTVTITVT, VTKNTNTDNH, SQSTDTNEKK, STTNTNTDNH, NTNTVTETVT, VVEVTVSVKL, SEVTVTVKTT, TVSVNVTVKL, VEVSVKVSVT, VENVKIKYNV, STSEYEYNLS, RKTHEIEYNV, VNIKYKYTQT, VVEIEVEYMV, IKQRIKVKKK, SNEIRVKYVV, IEVEYKVKET, VTEVETSKTA, STSSKSTRTI, TKSVETSKTA, VTTSRRTTVT, TTTLKLTYNA, GTLSYEYNKT, SDTLELEYNA, VSLKYTNATT, VTQTVVTYTD, SSSSVSVVNK, STTTSVSYTD, TITTVVVNTT, IREVVYEYTY, VSVKVEEKEV, TREVKVEYTY, VKVEYKETVT, VDKRISIITD, STIQIQMRTV, SKTRQSQIND, ITIISISSTT, VLTVTITISN, STSTITKTVK, STTVTITINN, VTITITKKVT, VTEVNVRYTH, SSSEYRVIVR, TRTVTVRYQH, VEVDVNVTVT, VLQVTVTRTR, STLTITNNTT, KGTVTVTRNR, VQVTVTNTTT, INTNTTTVNV, VTTEVEYKQV, SQSNETEVNV, VNTTVNESTT, VANVSVEYRN, STSRVTVTTK, TVTVTVTYNN, VNVEVTVTVT, VLTLQIQINV, SSITIIVTVR, TKTLIIIINV, ITIQITVTVT, TGMVTASDND, SNSTDAATVT, TTKVAAADND, STASATLSTT, VVTVTVTVTV, STVTITVKKT, RVTVTVTINV, VTVTVTKSVT, TSTITITYTK, SSITITTTVK, SVTIEITYTK, ITITITTKVT, RQTILVNITV, SNSTITFSRT, SRKIIVTISV, ITITVSFSET, RIEITVTYTN, SGIVIVVTNV, RREIVVVYTN, ITITLSVTMT, VKTVTTTYTV, STRSYSRTVK, SETKSISYTV, VTVTYTESRT, TVSVKVLVRN, SEVEVEVKIT, SVTVEVEVNN, VSVKVKISVT, LTKHTSIYQD, HTSTKEVEEK, SQKNESEYND, HTEIKIESVT, VTTVTVTKTN, STSTATVTRK, TVTVTVTKSN, VTVTKTVKST, VKSTLVAVRV, SGVEVEDTTI, RTKREVEVRV, TSVAVTDSVT, VSEVTTTANV, SDVVVKNKTK, SKEVTTKATV, VEVTATNSAT, VFTITITYSN, STSTITITTK, TRTRTDTYTN, ITITITITRT, VYTTQTQVNQ, STSSVEVKET, SVETETEVNQ, SNTQVKVKET, VNKVSQTYSN, SSVEQKEELT, SQRVKVKYNN, VEVTQEEQVT, VTTQTQTIRT, STSTISQTTQ, TNTQSQSIST, STQTITQQST, TTTVTVTIKV, SESTITVTVN, SVTVEVTITV, VTVTIKVSVT, VKTVTTNDIK, STRSDEETEK, SRTVTTSDNK, VTTNDTESET, ITSVTVTYTL, STSSVSVTRQ, SETVSVSYTL, VSVTVTVSQT, KETVLTVVNV, LNTQVANNTV, SKTVTTAVNV, VTTVATNTTT, VNTVTVTYSN, SNSSVTYTQT, KETVTVTYTN, VTVTVTHSVT, TTLNTYTYQL, SKASYSLERT, SERNEYSYNL, NLATYTQLRT, VNTNTNTNKN, NESTNTNTQT, I l l i I NTNKN, NVNTNTNKVT, VKQYQYRATT, SNYEYQYTQV, RQTYVYQANT, YQYQYKQQVT, VVKTTITDTD, SSSRITVTVE, STRTEVTDTD, TKTTISVSVT, ITTYTYTYSN, SNYVYTLTTK, TLTVTVVYNN, YTYTYTLSVT, TRTVSVNNSQ, SSYSYNVTQT, TTKTSVNNNQ, KSVNVTVQST, IKEITFTYNQ, STSTYTEKII, SKEITFTYNQ, VTFTYKEKIT, VTTVSVNYSL, SNSKVEVTVT, TKKVEVEYSL, VTINVSVSTT, IEKITNTNTV, YTSTNTNTTN, KVTKTETNNV, YKITNLDTST, VQIIVYVYSN, SKIEYEKKVT, KETIIYEYDN, ITIVYKKTVT, VDVTEKEVSN, TKNETEKRNV, TDTVEKEVEN, TVKEKRKQVT, VLKVTINVSN, SNITVKVEVT, TVKVKIKVNN, LTITVELQVT, VLEVTVTVSL, STVSFTYRKV, SNEVTVTVDL, VTVTVRYKVT, IDKSISIRTD, SNSERTDITR, SRESTTTRND, SKSIRIDKRT, ISTVTISYTV, VSVTYTVTTV, RKTVSITYNV, VTASY I I I I I , VQEVTYSYST, QNTTYTYREK, TRTETYTYNT, VNYTYTYTVT, NVTIKIEVNV, IGSTIEVKVN, RVTIEIEINV, ITIEVKVTVT, TVTILISYSV, STIEYKITLK, SVKIEIKYNV, ITISITIKET, VLTLTVTATL, SSITVSVRTT, TKTVEVSATL, LTVTVTMTVT, VRKNTNTYTL, STNKTENVRK, STRNENEYTL, NTNTDVVTQT, VSSNVNTVRN, SENTVTENEV, TKTRTETVNN, NTNTNTETVT, VNKKEDTDTH, DTNEDIDETT, KQEKRDIDTH, DKDEDESEVT, VDSSTSTSTL, STSTSTNTRN, SETSTNTSTL, SSSTSTRAVT, VTTVTVIYNV, STSIYTVNET, SKSVTVTYNV, YTVIYNVKST, QQLLLLTYSV, STIQIQQQQQ, TQWLQLQYNV, LRLVYQQQVT, VARHTYTKNN, STSTKEVETR, TTRTEYEKNN, HTYTYVVETT, VSKITNEYTA, SNSTYTNNVT, KNTITNTYNA, YKIENTNTVT, KVTISVTLNL, STITFTFTVI, SRTITVTLNL, ITITFTSSVT, VTTISLNLSL, SNSELELTRR, SEKITLELNL, ITLNLSTHQT, TTINININTV, NNSKNKVKVT, SVTNENKNLV, NININKVSVT, QTVITIAVNK, SDITVTVEQK, TNTVTITVNK, ITITVTVQVT, VQQNSNKDTV, SSSENRNELQ, TTRNENRDTV, NDNKNLNQQT, VTTVVVTYNY, TLVTVVNTVV, TTTVVVVYSY, VTVTYTNQVT, ELTVEIRVSV, VEVEVVINVK, SVTVVIVVNV, VTIRIEVKVT, KVTVVNVNTI, STVSNAVTVT, SVTVANANTI, VTNVNTVSVT, VDTETKTINV, ATVTVVKTVT, SEKEVKVINV, VTVTKTKQVT, VNVDERVLSL, SNNQKQQEQV, RTTDRRQLSL, DVPVQEQTVT, VIKITITVKL, SESKIEIEKK, STKVKIEVSL, SKITIEIQRT, VDKKEERYND, STHEYIDENT, SEKKKEIYND, KKKEYEDINT, IKKKEVAKKL, SEIEKEVEET, TKKKEVEKEL, KEIKKEETVT, VKTVTINITL, SNVTIVATTS, SQNVVIVINL, VTVNISERTT, VLTLTVTLSA, TTVTLTVTTT, TRQVSVTLTA, LTVSVTVRLT, VVEVTVNYNV, SNVTVTTKLT, TLEVEVTYNV, VTVNVTTRVT, TVTVTVTYTE, STVTVTVIIR, STTVTVTYTE, VTVTVKVSVT, EVSVTTTVTV, VTVTVTQTDK, SQTLTTTVNV, VSVTTTLTVT, VAEIEININV, SNSKIRIKVT, TRSIEIRINV, IETNIKVTVT, VQVIRVTVDV, SKVSVTVVQR, TQEITVTVNV, IVVTVRQQRT, VTIVTVTVNN, STVTVTHRVT, TRTETVTVNN, VTVTVERSVT, VLSISISINV, SNISISIKKV, TVTISVSINV, ISISITKTVT, VITEEKSNTV, SSSTNKITIK, SKTEEKENNV, ETKKNKISET, VRRNTNTNTI, STNENEEEVR, TVENENENII, SRNTNKEQRT, VFVSSYSYTN, SSSTYTYEVK, SATSSYTYDN, SVYSYSLSVT, VDTSKKTITY, STSTIDTTRV, SVTSTKDISY, STKTIQTTVT, TSTVTVTVTV, STVTVTVTVS, SVTVTVTVTV, LTVTVEVSVT, VTVSEFNYNL, STSRYRYETV, SRTSRFRYNL, STFEYEYRST, KEENNTNYTH, SNSNQNNVVN, SVTNNTNYNH, NNTNNKNSVT, VATIVVATTN, SNITIKVQRK, STTIKVKTSN, ITVVFQVSRT, SATYVYVRKL, STSEYEYTVN, TKTYTYERTL, YKYVYTYTST, VRETKTEISL, STSTTTNKEV, SNTATNTINI, TEIETKESVT, VTVVTITYTN, TQVTVTVKVV, AVTVVVTYNN, VVVTIKLQVT, KRNTEIKISA, SNSEIELETK, TKKTRIEINA, TNIKIELNET, VETETATYTD, STSTYEVTRK, TTKEEVEYTD, ETVTYTVEST, IEEISFNYNG, SSSEIENTRR, TEKIEFEYNG, FDFRYRNQRT, VETRRVTTRE, RESEIEETTT, TRTREVETTE, RTVTVRESVT, VLEVRTKVRD, SETEVEYKVT, TKEVETEVDD, VETKVKYKVT, EVQLQYQYTL, STSTVTIQVK, SVTLTNTYTL, LQYQVQVRVT, VTTITITITI, SGLNITHTRK, TETITITINI, ITITITRTKT, VLTDTVTYTD, STSTVTRTVR, KETDTVTYND, HDVTVTRSVT, VLVVKVDYKR, SNVVVEVTVT, SVRVEVEYDR, VVVKVTVQVT, VNTTIINYNV, SSSTYEDTRV, TNRTEIEYNQ, TTIKYTNRVT, VDTVRVEVRV, SSTVVVTTKK, STTKVVVVNV, VNVEVTSHVT, VDTVVVKVSV, VSVVVVQTKR, SDTVVVVVNV, VTVKVVQKVT, VATVQVTTTS, STVTTIVSLR, SLSVIVTTNS, VTVQYSVSRT, VNTFTFTYEL, SKSTFTNEVT, SKTFTTTYTL, FTFTFERSET, IQVHTNTYKV, SNTTVTNETV, SNTVTNTYNV, HTNTYTNSVT, TTEKSYSVKV, SNYTVSEKRT, TENKEESVNV, SEYSVKEKRT, VLKVIVTVTV, VTATLTTTVR, TVTVTVTLDV, VKVTVTTTVT, VRTNANAVSV, STSDVKVTVR, TETNKNKVEV, NTNAVTVTRT, IEKIEYSYSI, SSFSYSNEVK, SVKISYSYNI, IKINYERSQT, VATVNYNYNV, SLFTYQLQTR, SNTVQYQYNV, VTYNYTLSTT, VYTNTTTVTI, STTTVSFTLR, SYTNSTSVNI, NTTTVTFSST, VNKNVKESRE, SQSKDEKETK, SQTNEKESNE, NVKEKKVSVT, IYKVEIKISN, SEVEIEIEIT, TIKVVIEINN, VKIKFEIKRT, TVKVTVTVKL, VESSVTVTET, TRSVTYTVNL, VKVTVTVTET, VDKHIHTYKV, TEHTYTREQR, TERDTHTYTV, HTHTHEREVT, IYRYKFEYSV, YTYEYEYKTK, SVTLEVEYNV, YRYEFKYSET, KSTIQITVNV, LTIISINDQT, STKTIIIVNV, ITIQNDNQVT, QVTVTVTYTL, STSTVTVTEN, TKTVTATYTL, VTVTVTVKET, INSVSVEVSD, VKVEVKVEVR, SVPVKVKVSI, VSVEVETQIT, IETVTITVNN, SNITVTVTKK, TETVTITVNN, VTITVTKKVT, QNTQESLTTY, QSSETKNNRV, KQTKKSKTNY, QTSLSENSVT, STETETTTNV, SKTKIKNNIT, TNETKTKTNV, TETTTKNKVT, VERNTNTDKV, HNNTDINERV, TTTNTNIDTV, NTNTITSTET, VEEYEYKHTV, STSKHKKKLK, TEEDKYKHTV, YEYEYEKSVT, VVTTTVVQTH, STRTQTVMTA, TRSTTVTQTH, TTVVQTVTQT, I I I I I NTFTN, STSTNTVTRK, S I I I I I I FNN, TTNTNTVSVT, TVVVTNTNTV, STSTNTVEVQ, SVTVTVTNNV, SVVTNTIKVT, VVTVTVTVNL, STVTVTVTQT, SKSYTVTVDL, VTVTVKVQRT, VVVETVTYND, SESRVTVKTT, TRTVEVTYND, EVITVKVTET, VEEEKKEYTV, SSNKEEKKKV, TDEEEKEYTV, EKKEKKKKVT, KNTITLKVSV, STITVENTET, TKKVELEVNV, ITLKLTEEVT, KSKYEYEYTI, YTFKYQYEIT, TTKYQYEYNI, YNYKYEYEVT, VNSNLNIRQN, SQNENRNTIT, TAKNENRRDN, TVNINTVQTT, VSTVTVTYDA, SNVEYEVTKK, STKVEVEYDA, VTVTYTLSVT, VDQTQVNIRI, STTIIIVTQT, ATRTEVIINI, AQVNIVSQTT, VKTVENSYNK, VNEKNEVVTS, TLTVENEYNK, VLESNKVTRT, RAQFVFTVTV, STFTFTFVVR, SETRTFTVTV, FQFNFVLAVT, TQQLTLTLTN, SNLSLTLTRT, KTTTTLTLNN, LTLTLTLSVT, QEERSYRVRV, YSREVKHHRR, ADTREVKVDV, REYSYRHSVT, VLSTEVNVTV, SNSQVQYTVK, TRTVQVQVNV, SSVEVVNKIT, KKTNTNTSTL, SSKTNTNTTT, NKTETNTSDL, NTNTNINSTT, KREVEVTYTF, STLSVKVNIT, KVSVKVKLNF, VEISVNVTVT, VTTNSNSYSI, VNLTDVNTIT, TESKVNVYSI, VTNSNSNTTT, VVTQTQDYNN, SNQTVTQTTR, TRTEVQTYNN, QTQTQTVEET, VFLSTYTYTD, STSTYTVRKS, SVTVTYTYDD, SIYTYTVSVT, VNTTISTNNQ, SASTNKKTVV, KETAVSKNNQ, TTVTKINTVT, VNQFTYQYTI, STSQVQYTKK, TNTFTVQYTI, FTFQYTYTTT, IDEVEVEYMA, ITSSYSAVTT, TKRVNVSYGA, VEVEYTHVTT, TRRGVITYKA, SESEIENVEV, KRTKRIEYTA, GTIVYVETVT, VVTTTITITF, SSVTITVTVK, SVSTTITINF, TTVTISVSVT, VSTVTSSYQV, YNATYTITQI, KNTVLSTYDV, YTLSSTITVT, IVEVKVTVTN, STVTVEVKQV, TTTVEVEVNN, VTVTVKVQET, VYKSQVQVRI, SDVSTEVEQT, TKKTVVSVGI, SKYQVEVNTT, VVAVTITISI, TTSSISITRQ, TTKVTISITI, VAITITIQQT, VTNKTNTNRQ, SNSQNQIQQT, TTVKQNQNMQ, KNNQNQQRTT, VEEYISSSRI, SNSTNRSRRR, TVETRSSSNI, YTSIHISKRT, VNTVTTTVTI, ITSTVTNTQK, TNTVTTTVNI, V I I I I I NQIT, VNTYSYSYSR, SSAEYRKEAT, TTKESYRYNR, YTYSYEKLQT, TVKTELTLAY, TTSTLQYTQK, TQTTRLQLTY, TTLSLEYTKT, VKTKKYNYSL, SNKEYENTTR, CKRKENEYNL, KTLNYTNTTT, TTTVTVTVNV, VNVTVTNTVR, AKTVTVTVNV, VTVTVTVTTT, IERNEHKKTE, SNSQHVKEKR, SEKQVNEKNE, NINKHEKQVT, VANHIHRVQN, SESEHRHIQQ, NETEEHRVTN, HEHTHIQSTT, TVTVTVTYSE, STVQVQVTLT, SQSIQVQYNE, VTVTVTVQVT, TFNNEVSVTV, VNSNVNVTVK, RNTNNVNVNV, NNVSVTVTVT, VTTKTETKNV, STNTKVVNVK, SVTQVEVKNV, KTNEEKVSLT, TVTVTTTVTV, STSTQTVTVR, TVTVTTTVSV, V I I I I I VTRT, VATVTVTVNV, VNVTVTVTTK, TRTTTVTVNV, VTITVTTTET, RDMVKSKVQI, SESEVETVTT, NVKTESEVNI, VYVKKKTETT, TVTVTVTVSV, STVSNTVTRN, SVNVNVTVTV, VTVTVIVSVT, VNRVQYNYNL, SDYSVSVTVV, HTTVENSYNL, VRYQYTVSVT, QVTVEIRYSA, FSLTVTVNQQ, HTTVTITYTA, VTIRVNIDQT, VKTITVTVTN, STTTVTYTEK, TLTTTSTVTN, ITVTVIYKET, VRTVIVQVEY, STSQVVVTKT, TVETVVVVNY, VTVTVKVRVT, VRTITYTLKD, STTTLTQSEK, TKTETYTLQD, STVTYTDSVT, TSTNSNSNTN, SNSNNENTVT, SIAKENENNN, NTNSNSQRLT, VVKVKFQLNV, SDSTLEVEVK, SVKVEKTLNV, VKVQFAVELT, VNEYKYTYTV, YTYEYKYQQK, TVDHKYKYSV, YEYTYALSQT, VLQINININV, SSISISITLT, TRNISISIGV, IQINISLTVT, NTNIKQTIRV, SESEIEINRT, SENIEQEINV, INQTQKISRT, KKNINPNISI, SSININNNIT, NKNTNVNITI, ITININNSVT, INTVTVTVNV, STLTVEVNKK, KNTKNVEVNV, VTVTVTNTET, IVINRNTYRD, SENTYKNSKT, TNKNQNKYTD, NSNEYEVQVT, ERSLRLTITI, SNLDLEVSVT, RVVLELELTI, LSLTLRVTET, KTEKTETVTV, STQTVSKTVV, TESKSKSVSV, KEKTVSVTVT, VVEFEYSYNI, STSSYKWVVT, KQEFRYSYNI, FEFSYEVRVT, VRVDENNKSN, SNSSKKTTES, SKSDKDKKNN, DIDEKSTSVT, VDRVKVTYTV, STIEYEVTRT, TEEVEVEYTV, VRVTKLVKRT, RLNTKVTYKV, SDVVVEVIVT, TVETKVEYTV, TTVTYELKVT, VTTYTNTITV, YRYENENRVV, STESENEITV, YTYTNTHRVT, VEKVTVSVNN, VNVTVTEETT, SRTVTVTVSN, VTVTVEERTT, VLTVPVDVNV, SNVVVVVTRV, STTNVVVVNV, VKVDVTEKTT, VNCVTSTNTN, SNSTNTKTQQ, SKNVTSTNTN, STVTNTQSVT, VVTQTKTYSD, SNVTQTLTLT, KKTIKLTYSD, QTVTKTLSTT, NLTVTINYNV, SDVTVTVTLT, KVTVTITYNV, VTVNVTLSVT, VVTVNSSLSL, SSVTLTVTEA, RLSVQSTLNL, VTVSVSVTRT, VETVTVTVTT, NRVEVVKTRT, KETVVKTVTT, VTVTVTTQRT, TITVTNTSQV, VRRTVTNTVK, SVTVTNTSSV, VTNTVTVSVT, TVLSQYQNDL, SNSVNVHQEV, TRTSVYVNDL, SLYQNQLTVT, VNDASDLDSD, SSQEDHQRRQ, SETATQHDSD, QSALDTQSRT, VVEIEVTVNL, STIIVIVKAK, TKEIIVIVNL, IEVTVKVTVT, IVSVQVIVNV, ITVNVNVTIT, TVSVNVNVDV, VSVQVTVVTT, VTQISQSQNV, SSSSQSQTQQ, TTEINQSQNV, IQISQKQKQT, VNENVNTYKN, SESTNTVKII, TTTNTNTYKN, NENTYTVKVT, VNENTNTNTN, HTNNNSVTKK, TETNSQSNTN, NTNTNTVKVT, VVTFSFTYKQ, STSTYTYVER, KRTIKFTYDQ, LTFTYVETRT, TIESTYTYTN, STSTFTLVVV, SVSLTYTYTN, STYTYKLGVT, INKITSNITI, SSSKIENNNT, NTKIESEINI, ISINNTNEVT, KLTITVTVNN, SNITVTVTQT, STKITVTVNN, ITVTVELQET, TDTVSVSVST, SSVTVTVTRR, SESVTVTVST, VTVSTSRSTT, VNTYEYEVNV, SNFKVKEEVK, TKTVEYKVNV, YTYEYKETLT, VVTVTYNYRT, STVTYTVTVK, STTVTVTYNT, VSVNYTVSVT, IRKISYKYTL, SSSSYEYRET, SRRIEYEYSL, ISIKYSYSET, VVTVEVEVTE, SSKKVKVEVR, HVSVKVKVTE, VTQEVHVEVT, KVEVITWINV, SNVNINVAVN, NVKVETNINV, VNTNTAVETT, VVKVVVVVNL, AQVKVEVEVT, STKVKVEVNL, VKVVVEVQQT, ETTNTVTVTV, SQTTITNTVK, SETHTNTVNV, ITTTVTRKET, VVTVSYNQRQ, SQSNQNVTVQ, TVTVNYNQNQ, VTVNQTVTVT, VVTVTQETQQ, SNSSTSITVK, SVTVSQSTTQ, VTLETTVSVT, IDEHLKEVRV, SLSKVELRRR, KETHIDKVSV, HISEKRLTRT, VSTSTYTYSI, SNSSYSKTHR, TQTSVYSYNI, STSTYTRTVT, VQKQENNVSV, SNQEQENTTV, STTQGNEVNV, QKQNNETSVT, VLKIPVSYNT, SKSSYSNVNT, SKTISVSYTT, IKVGYVNSTT, VDKVKYVYNL, SRSEYEEERR, TETVEYEYNL, VKVVYEHTRT, VIRVEVDITL, SNVEIEVERT, SVRVEVEIDL, TRVEVEVSTT, VEKVSVEVEV, FKSKVSVQQN, TKKKSVSVEV, VKVEVSQSIT, TEKNTNTGSN, STNTNSKTKT, STSNTTSGGN, NTNTLSKTVT, TDRSVSMNTN, SSSLNNKTKV, SETSNKDNEN, SRSCNVQSVT, TVTVSVSFTA, STATVTVTVT, TQTVTVTFTA, VTVSVTVKVT, TVTVVVTYNE, SVTVNVTYTE, VTVVVTVKFT, INRYTYNYQN, SKSSYTNEVR, STAYTYTYQN, YTYNYTNQVT, VIKEEDTTIV, VTEETKKETV, KTKEVDETGV, EKDKKEQEVT, VLTITVTYTD, SSITVTVTRR, STSITVTYTD, ITITVHIQRT, VTTTSHSNKN, SNSTNSNTLT, SKTTSHSNNN, SDTSYTLSET, VTTVTVTYTF, SSVTVTVTTK, TLTVTVTYTF, VTVTVTVKKT, MNTTTITYTV, SQFNYNNNVR, SNTTTINYTV, FTITYTNSTT, VLTVTVTVNT, NTVSVTVTKT, TTTYTNTVTT, VTVTVTKTVT, VNTQTQTNNV, STQTNSVTVT, SVTQSQSNNV, SHQTQTVTTT, VVRSEFTSRV, SESESRTEVT, STTVRFRSNV, SEFTFTTSIT, TRTTEVTYNN, SNNTVTVTEK, SRTTTVTYNN, TTVTVTVSVT, VDKSSHSLDD, STSTLTHKVV, SETTEHTLRD, STHSYSHTTT, KKQVTITYSV, SRVEYEVTIT, SVRVEVEYNV, VQVTITVSTT, IDDYTFTLSN, STYELERRVT, RETYEFELNN, YDFTLKRTVT, VEKFSREDTQ, FNSTDTDTQT, TRKTSRTDTQ, FTSEDTVQET, VDTVTVTVSV, STVTVTHTEK, SKTVTVTVTV, VTVTVTHSET, VYRYSVKYSV, FTYSYEVTIL, TVTIEVEYNV, YAYKYTVQRT, VLTTTYSYQN, SNVTVTNTNK, TNTTTVTYNN, TTVSYTNTVT, VSREEIIISV, STVEIEIEDR, SKEEEIEINV, ISIEIEIKET, VEKKRKETEK, SKSQYEQKTE, KETKEKETEK, KKEEYKLTKT, ILTVTVTVTL, STVTVTVTTK, SKTVTLTVNL, VTVTVTTKLT, VTRSEFSYTE, SNSTFTYVQN, SQKRTLTYTE, SRFEFEYQST, VETVTVEYSN, STVTVKITRK, SETVKVKYNN, VTVEYTHQRT, VDVHTHTISL, HTHTHTHTEV, KETHAHTINI, HTHTHTHTVT, VSENTDTDTD, NTNTDTDKIK, SKENTDTDTD, NNDTDKDKVT, VVETKHEYSV, STNENTHKVV, RKDNKHTYTV, TKHEEKVTET, VEKSRNSNIN, SNDENENTTT, TNSSENENNN, SKNSNKITTT, VKTNTNTYTN, STSKVKNTTV, SDTKKNKYNN, NTNTNEVKVT, LKKVTTTVNV, SNVTVTEEET, MKKKTTTVSV, VKVTTTEKTT, ILTLKLTLSV, SELELELTVT, TVDLQLELAV, LTLTLKLQVT, IETLVYIAPA, LKVTVEYTLR, TKKLEYEVDA, LRVIYVLSST, VTEIVITYRH, SESTYKIKRK, STSIEIKYKH, IEITYVNHET, KKLVEQQYNH, NSSSQRQNSK, KTTTEQRYDH, NLQQQTNSET, IDRHNYTYTN, STSEHEIEVK, TVKVEYEYNN, HNETYIETVT, VNTFTYNYTV, SNYTYTQTTT, SQTVTYTYNL, FTYTYTQSTT, VNEVRVEYRG, STVRNRVVTK, SKTVEVRYNG, SEVENRITTT, VNNNNNNYNN, SSGTYNNEVR, STKNNNNYNN, NNVNNTESTT, VDRITKTHTN, STSEHEKTVT, HTTIKKEHNN, SRITKTKSVT, VLTVTVTVTV, VTVTVTDTVR, SRTVTVTVTV, VTVTTTDQVT, QVEVTVTYNV, STSTYKITQT, RKTVEVKYNV, VEVTYTITQT, VTTVSTDNIF, SSSTNTTTTI, SKNVSTTNNF, VSTDTNVSVT, VQTYEFKLRV, SELQLTYTET, SKEYEFTLNV, LTFKFRYKET, TTTVSVSATL, QDVSVTITEK, TKTVTVSATL, VTVSVTMTTT, IVTTKNTYKV, SESSYENTKR, SETSEN EYNV, TTVKNKTKVT, IDRYQYEYSN, STYKYEREVR, SVTNRYEYSN, YTYQYERTVT, ERVKKNTQTQ, STHEHTREVT, SQEEKNTQNQ, KVTTHLVRTT, ITTRTSTRTL, STSTDIVTQQ, SVTRISTRTL, STQTDTVSQT, VQTITVTVRV, TESTVTVTRR, TETITVTVNV, ITVTVTDSVT, VEEVTVTYNT, STVEVRKKKI, TEKTEVRYNT, VEVTYKKIVT, VEKQVQKYTT, SNQTNDNTTT, KKSQNQDYNT, QTQKNATTRT, VLTNTNTTTN, STSTNTNTTT, KKTVTNTTNN, SSNTTTNTET, VVQVTAKVNV, SNAEVTVTTR, TNTVEVTVNV, VQTKVTVRDT, IERHEYTYRV, SESEYEKKEK, SQTEKAEYNV, YSHKYERSVT, EAEIVGVVTL, STSQVQVKVV, TVEIEGQVTL, IEGVGKVMVT, VHTVVVTVTV, STVQVKVEVT, SKTVKVQVNV, VTVTVEVKVT, ITEISISISI, SNIDINIKSE, KNKINININI, VEISISIEVT, VNTNTNTDTE, NTNTNTNKVI, KETNVNTDTE, NTNTNKNQRT, VDKHEHIIRI, ATINITNVQT, KQSKTVTISI, ITHIHHNTVT, IDTNTNNITI, SNSTNTKTTI, TQNNTVTINI, NTNTNTSRVT, VKRVTVIVRV, VEVKVEVRVV, TTAVEVEVNV, VTVIVEVSVT, VETVTVTYQV, SQSTYTYKKT, GEEETVTYNV, VTVTYTKKVT, KVTVTITTSL, STITIEITTT, TVKVEIETNL, VTITITTQIT, TLNVLVTLRN, SNISVEYKIT, SRSVEVELNN, VNVTVKYSET, TTTVTVTVRV, SEVTVTVTTT, KKTVVVTVNV, VTVTVTTTQT, VLEVTVTVTV, STVTVTFNLV, SKKVTVTVTV, VTVTVNFSVT, VESKSSTKVQ, YNSEKVSKEE, KRTKKSVKNQ, YSSTSSIKET, VVNLTLTYTV, FNLELTVVVT, KVNLTLTYNV, LNLTLTVSVT, VEVYTYTVTV, YTYTYTDTVV, SEKKEYTVNV, YTYTYSVSQT, QITVNVTVSL, STVTVTNNVT, KVNVTVTVNL, VTVTVNNLRT, INKVVVVVEL, VAVNVELTKT, KETKEVEVEL, VKVVVRQTVT, TETNINVYTN, NTNVVVVERT, KTKVKNVYTN, NTVVYIVDVT, VLKINVTYTQ, SNSTYTVEVV, SVKIIVTYTQ, ITVNVEVQVT, VREVTVSVDV, SNVSVSDKET, SREVSVSVDV, VKVSVIDKET, VDRVEVYYKN, SSYLYRHTRT, TTRVEVRYDN, YSVYYSIQTT, VLTVTVTVNV, VTVTVNHTET, RVSDTHNVNV, VTVTVSRTVT, VETTVYTYTH, SQSTVTYTRV, TSTTTYTYNH, TTTTYVYTRT, VENVIVTYTA, VTVKVEVTRK, SEEVEVEYQA, VNVYYKRHQT, VDTVTVTYTV, STTTVTYTQT, TKTVTVTYTV, VTVTYTHTTT, TVTVTVTVTS, STVTVTVTVT, TKTVTVTVNS, VTVTVTVTQT, VHVITRNDTR, SNSQDQQRTV, SVTIQRQDNR, SVINEQYTTT, KVNIDVKFKA, LSVEVEVTFN, SVTITVEFNA, INVKVEVSVT, VDKVTVTVTQ, STVSVEVTTE, RKKVKVEVTQ, VKVTVEVQET, VNTFTYEYTL, STFTVKNTQK, SNTTKYKYNL, FTYEYTNKET, TETDTKTVTV, HNYTVTKTTK, SKTDSKTVTV, DTYTKTVSET, VQMITVTYTV, STSTYTVTVT, RNTITVTYTV, ITVTYTLTVT, VNTTTVTYTD, STSKTTVTNV, TTTTEVTYTD, STVTVTVRMT, TLTYTYTHTI, STYTHTNTQI, SNTYIYTHNI, DSYTTTNSVT, VLQSRVLVEL, TRVSVEEKKV, NVLNEVEVEL, TRVLVKESVT, VDTLTLTYNL, STSTLSITKT, TTTTSVSYNL, LTLTLTNQVT, KVTVNTNYTN, SNISVEVTVQ, STTVTTEYNN, VTTNVTVSTT, KDKNKNTQTV, DSKDIDQEQT, TNKKKNDQTV, NKETN EQTIT, RVTVTVTVTT, STVTVTNVTT, TNEVTITVTT, VTVTVKNTTT, VRTFTSTNTT, STSTNTYKET, TKEYTSTNTT, FTSTNNERVT, ILRVTVSVTL, STVEVEVTQQ, STTVEVEVDL, VNVSVTHAQT, KSTNTNTNTN, NTNGNTNEQV, KSKNVTTNNN, NTQTNENQVT, VR I I I I I ITY, STSAITISVS, TV I I I I I ISY, TTTTITIKVT, IRKVKYEYSV, SNYEYEYEVK, SKRVEYEYNV, VKYEYVKEET, VTAVQTQLNL, SNVTVQIQQT, KTTQQTQLDL, VQVQVQQTTT, VDRVEYTYTL, SSTSYRTERR, SDRVLYRYNL, VTVTYESSRT, VVNITVSYSN, SNSTVTVTTK, STTITVTYSN, ITVSVTVSVT, VNERIIEDKD, SNRKKEENTR, TRDREEEDVD, REELKDETVT, VVTNTYTYRL, STSTYTHTVT, TERNTYTYDL, NTNTYTVQVT, QHTVTVTVSV, SSVQVQNTKT, SQTVQVQVNV, VTVTVTDKTT, TLTLVLSSSV, MSLSSSLTIT, TKTLSLSSNV, LTLSSTLSVT, VTTLTQVNVN, STSVQTQTVT, TKTLTNVNNN, STQVQTVKVT, VLTISISVTV, SEITVTVTLQ, RQSITTTVTV, ITISVSVSVT, TNNVSVSYKV, VEDSVSVNNK, TTTVVVSYNV, VNVSVQHNTT, INEVTVTVTA, LTVTVTVKNE, TKTKTVTVNA, VEVTVKNTVT, TLVVTVTYTN, SSSTVKVTLK, SVTVKVTYTN, VTVTVTVNVT, VNRFMFNANG, STFAIYIEQR, STKVSFAANG, FRFNAEHRET, VETYTGTSSI, YSYTSVETVR, TQTKVGVSVI, YTGTSSEQRT, VTKVEVTYTL, LTVSVEFETK, SEKVEVNYTL, VNVKVEFQST, ITRVRVTASI, YTVTIEVQRE, KQTVKVEAEI, VRVTIERTET, VVNVQVTVTV, STVVVVVQVR, SRTVVVVVNV, VNVTVQVHST, VSEKEIRVEV, STSEIEYTEV, AEEKEIEVEV, IEIRITYKRT, TLSININVDA, IGITVTVNTN, SVNINITVDA, ISINVNVSET, VQTNTNTYTV, NTSTIQNTQA, SQTNQITYTV, NTNVNTNQQT, SYTVTSTTKN, STVTTTYNAN, SRTVTYTTNN, VTVTSTYSET, KDTVIITVTD, SSVTVTINTT, KKTVNITVND, VTVTKINSTT, VNNVQYTYSV, STSTYTFQAT, TLTVEYTYNV, VSVQYKAQVT, VLTATFNYTV, SSSTYNYTVT, TRRVEFNYNV, ATFNYTTTET, VRVSVDTDND, SNSEVEINVK, TRTAEIEDVD, SVNTDKSTTT, VQIKETNTSL, SKSKTKVKVK, TTTKETKTNL, KETNTKVQIT, VTLVTVTYTI, STVTYTNNTR, SNRTTVTYNI, VTVTYTNHVT, KTIVNVDYSA, FNFSYTVTRV, STTTKITYNA, YNVDYTVSVT, TTSIQKQVSL, QQSSVQVTVT, SKTIQKQVNL, ISIQKTVQTT, IRRENSESRN, SNTRNEETRK, SRERRVESQN, SNTEVLESET, VLEVKVNYTN, SSVTVEVKVT, TKEVEVEYNN, VTVNVKVTVT, VVNVTVTVSL, SNSEVEVTVN, SVKVEVEVNL, VNVTVTVSVT, VLEVIVNVKL, SSSTVEVRTT, TKEVEVEVNL, VSVIVRVRVT, VREIEYTVRD, SESRVENKET, SNETVYEVND, IQYTYKTKET, VNKVEINKTD, STIRKEEEEV, RKKIEIEKND, VSIEEEEEVT, IWEDKKTKTK, DTQTDTEKRT, TRTDEATDTK, DTVTEKTQVT, KVTVKVKITI, STSEIEVTVT, SVKVEIEITI, VTVKVVVQTT, VDNVNVNVTV, VTVQYSYTLT, SKQVSVSVTV, VAVNYNYKET, VERRTHTDTV, RTNTTTNTRK, TVSTVITDDV, RRSTHSLTET, VNSVTYTYSL, STESVSVTVA, KTTVSISYNL, VSVTYTVSVT, VSTQSISVTI, SSSNINNTEK, SKTQNINVNI, ITISITNKST, SEEHEHEVNV, SESEEEEEVK, SREHEHEVNV, HKHEEEEKVT, VVNVDVTISL, STTSVTVTVT, KVKVTVRINL, VDVKVEVTQT, ASSVLVSYRN, SNVTLSVYTE, KRLVKVSYTN, VTVTVTVKTT, VNKVTVQYNL, STSEVENTTA, SQTSTVEYNL, VKQQVTNSTT, INKVTITVNL, GNVTVTQQQK, SRTVIITVNL, VTITVQQQVT, VNLVINIYNV, SQSTVVNVKT, KTLVVNVYNV, VQVINVVAKT, QYSVSYNYSV, ASVSVTVNVT, KVTVTVTYTV, VSVSYSVTVT, VVTVTVTLTN, SSLTVTVVVT, KRTVTLTLNN, VTVTVNVTVT, TAKNNFKNTN, SSSTNEVTVT, SVKVEFENNN, NTFKFSVSVT, VDSNVNKASV, SNSEAENTTK, SKEIENEAQV, NSNTNKNAVT, VNIVTVTIKL, SEVSISVKVV, RVTVIVSITL, VTVTVKVTVT, VRYQRLRYTQ, STSEYELEET, SRSQQLEYNQ, QTHRYELRTT, VANVKSEYKV, SESEVEVVVR, SEEVKSEYKV, VLVEYQVSVT, TLSITVTNSV, SNSTINVTVT, KVSINVNNNV, ISITVAVTTT, VNSLSINYSN, SSSNYNVVKT, TDTLNINYNN, LQINYVVKVT, RLEVSVSVNV, KSVSVSKTVV, TVRVEVSVNV, VEVSVSKQVT, VDRSEITYNH, STSRYEIERQ, KTRSEIEYNH, SRITIEKTET, VREVKVTYKN, SESTYTVKTT, STERIVTYNN, VTVTYKVKET, RREVKVKITV, VSVEIEVVSI, TNEVEVEITV, VTVKVKVQVT, TITTTYTYRD, SESTYTQQQQ, SNTTTYTYSD, TTYTYTQKQT, VDTNTNTYSY, SSITNTNIQR, SNTSTQTYNY, NTNTYTNSVT, KRTVITTYNN, STKTVTDQNT, SEKVETTYNN, VTVTVLDQQT, IYTAQTTDTV, STSTDTHQIT, STTKIITDTV, ATNKDQRSET, LKVNKIQIQL, SVSEIEQQVK, SVKNEIEIQL, NVIQIEQKIT, KNKKEVTGRG, ATSTVTVERT, SERKTVTGNG, LTVTVEVQQT, RVVVEVELTV, VNVSLRVETR, TRAVRVRLTV, VTVELEVQET, TTTQTQTVTI, STQTVTVTTT, SQKQTQTVNI, QTQTVTVSQT, KVVVKVVYTI, SSVSYDVTIT, KVTVDVDYTI, VTVVYTVSVT, KLRLELELTA, LTLNLEVEVT, TKKLKLELTA, LRVELEVQVT, INTVTVVVTV, YTNVVVVTVI, TVTVVVVVNV, VTNVVTVTVT, IDESEYKVSV, SNYTVSYTAT, TKESSYSVNV, SEYKYTLKWT, VVKVEVEVTD, SSSKVTHEIR, SEKITVTVND, VEVEVETQVT, VTQTTVTESQ, SHSTISVLTT, KTTTTVSENQ, TTTTETVTTT, VNTVTYTVAV, STSTVSHTQV, RTTVSVTVNV, VTVTYTHTET, VNRSVNKNSN, SSSTVEVVTK, TNSVEVENNN, SRSVNVVKTT, IQIVNTTYNA, LTINDNVERK, STKVITNYSA, LNVSYKDEVT, VVTVTTTYTV, SNSTIVTTVK, SVSVVTVYTV, LTTTTVTSVT, VESVTVTVTI, SNVTVTVKVT, TKEVTVTVTI, VSVTVLVKTT, VVTITVTYTL, SKITVTVTNV, KKTITVTYNL, ITVTKTVSVT, VLTNSNSNTN, STNININTVK, TTSQININNN, NTNSNTVRTT, IENNMYRYTD, SNYSNQQTVT, TNDNQYQYND, NNYRYTKRVT, VQSNTETKTH, NTSQKSVTKT, TETNSVSKSH, TTITETVKTT, VTVNTIIVTD, SNIRVEIRVS, SVENEIEVTD, INIIHREYVT, ISNNTNTKEV, STNSNSNTTT, SNTNSNSKEV, NNNTNTNKVT, INSNTSTTSL, NSNTTSSTRK, TKSNSSTTSL, NTSTSSKRVT, VTTVQVTYNI, SSVTYTEEER, TKTVTVTYNI, VTVTYQEQVT, TLTTTVTVTN, STNTVTVTVK, AVTKTVTVTN, TTVTVNVSMT, INQNENKYTL, STYEYKNKLT, TKQNINKYQL, NQYENENKIT, IIKIKITITV, STSEIEIIVK, SVKIKIEITV, IKITISIEVT, VTTSLNNYNN, SDSSITNNTK, STTSTNTYNN, STNQINNSVT, QLNNNYNYNL, SNSNYNFSLK, SKTNNFNYNL, NTSNYSWTNT, VVSLTLNLEV, SSFTYTITVT, RVTLSLTLSV, YTLNLTVSVT, VYKIEIKISV, VTIEIKIEKQ, STKIKIKINV, ITIKIEASVT, VRTWEVALTN, SSVKFKYIDR, SRTWIVKLNN, STVAFKYSLT, TVTTTVSVSN, SNTTVNYSVT, RKTTNVNVNN, TTVSVTYITT, VVENLTQVSV, FSNTVNSKQT, RQENNHNASV, NSNQTKSLQT, TETSTSIYTA, STSITKKETT, RKNTKSKYNA, STSIYTKKVT, VDTNTNTNTQ, STNTNTTTTK, TNTKTNTNTQ, NTNTNTITVT, VDTKTVTVNL, STSTETVTTK, STTKTVTVNL, KTKTVTRKET, VRTTTVTYSV, STSTYSDTET, SKTTSVSYTV, TTVTYTDTTT, KRSVTTNYQI, SLSTVEVEQT, SRKVTVEYNI, VSVKVTEQET, VNRVVYNYNL, VSVEVENINR, SQRVEIEYNL, VEYRYVNEQT, VETVSVSVNI, SSVKVEDTVT, KKEVKVEVNI, VNVSVTSLTT, VNHNENTNTA, STSVNNNTNV, KNDNVNNNTA, NTNTNRNSVT, NLSSTNTNTN, STSNNNVDVT, TTTDEVNNTN, STTTNKVTIT, IVTNINTNTT, STNTNLNTVI, RRTNLNLNNT, NTNTNLLTVT, VLEVEVTLSQ, STSSVKVKVT, QKEVEVKLNQ, VEVKVKYTRT, VWKNENEIKL, SESKNKNEKK, TVTEKNKINL, TKNENEQKVT, VTRNEQEYTN, STIEREEEEV, TIKNKQEYTN, ITQEQEERKT, VATVTVTYTV, STSTVEVTTT, TTRVEVEYTV, VTVTYTVKTT, KNNNYNGATI, LTNNNNNNTT, TNTNNNNNNI, NNNYFTNTQT, RKTIITRYSL, SNIEVEYDEQ, SRKIRTEYNL, ISIRYDYQTT, VDSYQYQYEL, STYSYTDTKV, TVKEEYTYTL, YTYQYDRQVT, VLYVSVTATL, SSVTVTYEET, RKKVTVTATL, VSVTVSYEST, KLSWDYTYHV, SEWDVDYLQQ, TKTWDIDYAI, WSWNYTYTQT, VDIIVHTDRV, STSIDVQRIS, SERRVHIDNV, SSHTHESHRT, VNTVTVTLQN, STDTVTNTQT, TNTVTVTLTN, VTVTVTNQVT, EARVQVEYTL, SSVKVENLTK, TRTNEVEYNL, VNVKYQNTVT, TEKTKNVNTK, SNSTNTHETT, TKTTTNTNNK, TKEVNKVETT, VDVRTNTNSV, STSTNKDNVT, SNSRKNTNNV, SNNTNENRVT, VVTVSVTVTL, STSSVSVTVT, SVTVSVSVSL, VTSTVTVTTT, TLTVTISYTT, STSIYEKTIK, SVTVKGEYTT, VTKSITKSVT, IKRVEVKVQV, VNVEVEVEET, KKQVEVEVNV, VTVRVEVLET, VNEYEYHLTV, STSRYTNRNV, SKSYTNTLTV, YEYHYRNSVT, VISVKVIDRD, STSEVEVKTV, TVKVEVEDTD, VSVIVKVEVT, VQTRTSINKH, SESTNTVEVK, TTKNTKTNAH, RTSINSVEVT, TLETTVSESH, SSVKENRTEV, STTTEVNEVH, REVSEKRSVT, IQTTTVTLTL, STSVVVQTQR, TTTTVVVLNL, TTVTVTQKRT, VVETSLTEQD, SESNETQKER, SVETSQTEQD, TTLTLNQRRT, VLSVTHTNTN, STSTITYTTR, SRTVTHTNNN, VSHTNTYSVT, VLTVIVTVSV, VTSTVEVKVT, SVTVEVEVSV, VAVTVKYSVT, TLYVVVVVTV, NTSTVVVTVT, KVTRVVVVTV, VTAVVTATRT, VDTTLVTVNN, STSTTTHTTS, TKTNEVTVNN, TTVTVKVTET, VRTETFTARL, SSYTATFSQT, SRTETFTASL, ETFTFTFSVT, TVTLILTVTV, STSSISVTVT, KVTLVLSITV, LTLTVTVTVT, VVSVSISVSV, VSSTVTISVK, TTTVTITVNV, VSISISIKQT, TVTVTVSATL, STSTATVTIS, SRTVEVTARL, VTVSARVQET, SVNNNNNNNL, NTSNNNNSTQ, TERNENNNNL, NNNNNTIQRT, VSKRSYTYTN, STDSYSIEQK, SKTTSISYNN, RDATYEETQT, TTEVTVTVTL, STVTVTIKNV, SKTVTVTVTL, VEVTVTDSVT, ILKVVVDYRN, SNVSVSVKVT, SVEVSVSYDN, VKVDVTLKLT, ITKTKVVYNN, SNSDYDVEVR, TVKTTVDYNN, TKVVVEVEVT, TRTVTVTYTA, STSTVVHTER, SETTVVTYNA, VSVTYTHQVT, IDEISYTYKI, STITYNYKKI, SKNITVNYTI, IEITYSKHET, VRMTMTTVQV, STSSVSTVVF, SVTTSTSVSV, TMTTVVVSVT, VASTTTTYTL, SKSTNTITLT, TVETETTYTL, TSTTYKLTTT, VTTYTHTTRT, RETYTHTTST, YTHTYTNTRT, VDTNRNTNTN, STNENENTKT, STKNENENTN, NTNVNTRAVT, KTTNTNTNTT, NTTTNTQVLQ, SKNSTNTNTT, NTNTNNQQTT, VTVEVTVESG, RGTVRVTVRV, ITITITITVT, TVRLRITITI, TV, DTDTDTDTTT, TTTDTDTDTD, VVVVVVVVVS , TVTVTVVVVV , ATATATATVT, TVTYTATATA, VTVTVTVTVD, KISVSVTVTS, ITITITITIT, NVTITITITI, ATATLTATAT, TV I I I I I ATA, VTVTVTVTLT, TSTVTVTLTV, LTLTLTLTLT, TATLTLTLTL, NTNTNTNTTT, KKTNTNTNTN, LVLVLVLVLT, TVTLVLVLVL, TVTVTLTVTV, VVEITITITI, ATATATATAT, NLSVTATATA, AEAEAEAEAK, VAEAEAEAEA, TVTITITITI, STNTNTNTIT, TKLVTNTNTN, TLTVTVTVTV, VVVVVVVVIK, SLEVEVVVVV, TVRVTVTVTV, 11 I I I I I I LT, TA I I I I I I I I , FTFTFTFTFE, QLSFTFTFTF, DVQIVITITI, VVVVVVVTVT, TVKVVVVVVV , IVHVHVHVHT, KIEIVIVHVH, SISIHISIST, VKQSISISIS, TRTDTDTDTT, TVKITITITI, YTVTVTVTVK, IVEVTVTVTV, TITITVTVTV, YTYTYTYTVT, TVTYTYTLTY, TIKVTITITI, ITITFTITLT, TSTITITITI, TVTVTITITI, QQQQQQQQQT, TVTDQQQQQQ, SLTVTATATA, ITITITVTVT, VEVEVEKTKK, EEEEVVEVEV, VEVEVEVEVE, KAEVVVEVEV, TITITITITI, TGSTITITIT, LDLDLDLDLD, EFDADLDLDL, ITITITITIE, KITITITITI, TISITLTLTL, KIEITITITI, HTHTHTHTHT, TTRTTATKTH, SVTITITITI, TITITITIVI, IVVVVTITIT, DTDTDTDTDT, VDVDTDTDTD, TIKVTVTVTV, TIIITITITI, NVNVNVNVNT, TGVNVNVNVN, TTTITITITI, ITITITISIS, NVKITITLTI, LVLVLVLVLK, TVEVVLVLVL, LKLKLKLTLT, TATLKLKLKL, VEVEVEVTVT, TKKKEVEVTV, NTNTNTNTNT, KTTNTNTNTN, VQVQVQVQVT, QARVQVQVQV, ITITIVITIR, TAEVTVTITI, TTLTLTQTQT, TSS I I I I I I Q, KIELTVTVTV, TVTFTLTLTL, IVIVIVIKIH, KVEIRIVIVI, VTATATATAT, KLDATATATA, LTLTLTLTVT, TVQLTLTLTL, VVVVVVVVVK, TVTVVVVVVV, TAVVTVTVTV, VTLTVVVTVT, TETVVVVVTV, KKSNTNTNTN, FEFEFEFEVT, TVKFKFEFEF, TIKIKITITI, KIEITIIIII, TAELTLTLTL, IKIKIKIEIE, EVKIKIKIKI, VEVEVEVVVT, EARVEVEVEV, VTVTVTVTVV, ELKVTVTVTV, ITITITITIQ, KVSVTITITI, KVSITITITI, TKVNVNVNTN, TITVTVTVTV, TTDTQTQTQT, TN I I I I I QTQ, VTVTVTVTVK, TVVVTVTVTV, ISISISIKIK, TLELSISISI, LTQTQTQTQT, KLTQTQTQTQ, TVEVEVEVTV, TVNITITITI, LTVTVTLTVT, EGKLTLTVTV, QIQIQVQIQT, TQKQIQIQIQ, TLTITITVTV, LVLTVTVTVT, TLKIKLTNTN, GTGTGTGTGE, LGRVTGTGTG, YVYVYVYVYT, KKENVYVYVY, SVSVSVSVVV, SVKSVSVSVS, TTEKTVTVTV, TVTHTHTHTH, ITITITITIK, TLTVTITITI, FTFTFTFTFK, TITFTFTFTF, NTNTNTHTHT, TNTNTNTNTH, AVAVAVAVAT, KVSISIVAVA, ITFTFTFTFT, TVTVTITFTF, I I I I I I I I I I , TAAATATTTT, KIEIVVVVVV, ITVTVTVTAI, KSEVTVTVTV, VTVTVTVTIT, TIEVTVTVTI, NTTVTVTVTV, LTLTITITIK, ITITITLTIT, TVKVVIVIVI, VTVTVIVTVT, TKTVTVTVTV, EVEIVVVVVV, MVMVMVIVIT, KAQMVMVMVM, YTYTYVLTLK, DIEEEYTYTL, TVSVTVTVTV, TLRATATVTV, VTVTLTVTVT, TLSVTVTITV, TVKVTITITI, DVNVNVNVVT, TKTKTNVNVN, ITITIIITIT, NINVTITITI, VIVIVIVIVK, TVEVIVIVIV, LTLTLTLTIT, KVELTLTLTL, AIAIAIAIAT, TVELIAIAIA, TVKVTVTVTV, KLTYTYTYTY, HI HI HI HI HR, QEEAIHIHIH, ITVTITITIT, KVEITITITI, DVKVTVTVTV, ETETEKEKEK, TETETETEKE, LTLTLTLKLK, VEEQELELTL, TVKITITVTI, TVTVTVTVTT, TVTTVTVTVT, VVIVIVIVIT, KITVVVVIVI, ATATATATRE, TVQETETATA, ITVTVTVTVT, TVTITITVTV, TVTITLTLTL, KVTVTVTVTV, TVTVTITVTV, TLEATATATA, VVEVTVTVTV, STSTSISTVT, TATSTSISTS, HIYTYTYTTT, TRTHTHTHTH, TLVVTATATA, TININITITI, LTITITITIT, TTKITITITI, YKYKYKYEQK, EKKYKYKYKQ, TITLTLVVTV, NTNTNTNTST, INTNTNTNTN, TIKVKVVVTV, QTVTVTVTVT, KKTQTQTVTV, YTITITITIT, TIEVTITITI, VAEIVITITI, KATAIAIEIA, NTNENENTNT, DVKNKNENTN, FTFTFTFTFT, TVSVTFTFTF, TETNTNVNVN, SNSNYNNETT, SREHTHTHTH, VVVVVVVVVT, TVVVVVVVVV, VTATVTVTVT, TISVTVTVTV, ATATATATIT, TVTATATATA, NVKSTITITI, NTNTNTNTNN, ETWNTNTNTN, VTVTITITVK, TVEVEVTVTV, ITITIMITIT, IGKIMIMITI, TVNFTFTFTF, STSTSTSTVT, TVTSTSTSTS, VVRVVVVVTV, TVTFTFTFTF, TVSVTITITI, VTITITITIT, TVAVTVTVTI, LTLTLTLTAT, TAILTLTLTL, VTNQNTNTNT, ILEITITNTN, GTGTG I I I I I , TAQGTGTGTT, DVDVDVDVDT, TDVDVDVDVD, KVQITITITI, ATLTLTLTVT, TATATATLTL, VTVTITVTVT, AVKVTVIVTV, STTNTNTNTN, KKEIEHTHTH, VIVIVEVVVT, TEKVEVEVIV, TATLTLTVTV, VTVTVTLTVT, TVKVTVTTTT, EVKSTSTVTS, TVTVTVTLTV, VTITITITIK, IEIEIEIMIK, TVEVEVEIEI, YTNTNTNTNT, VQKKTKTNTN, QATVTVTVTV, TLVLTITITI, KVKVKVTVTV, TLR I I I I I I I , DVEVTVTVTI, TLTLTLTLTL, IKIKIKITIT, VKEVKIKIKI, LTLTLTITIT, IVELTLTLTI, ITITIVITIT, NINITITITI, KVTITITITI, LILILILIVT, TAKLILILIL, TIEVTVTVTV, QTQTQTQTQT, TNTQTQTQTQ, TKQKTHTHTH, IEIEIEIEIE, EVKVEIEIEI, TKKNTNTNTN, TTRVTVTATA, GTGTGTGTGT, NIRVTVTGTG, YTYTYTYTYT, TRKYTYTYTY, TIKITITITI, KVSVTVTVTI, TIKLTFTFTF, TAQLTLTLTL, LTLTLTYTYT, TITLTLTLTY, KIKIKIKTKT, TATAIAIKIK, TKEYVYTYTY, HVHTHTHTVT, EIKKKHEHTH, EVKVVLVVTV, TAKITITITI, KKEKTDTDTD, TATVTITITI, VTVTLTVTVK, KVDVTVTVTV, TAQATLTLTI, ITTNTNTNTN, RLELVITITI, TVKVTYTYTY, TALLTVTVTV, TVTNTNTNTN, FTFTFTFTVT, RVEFTFTFTV, ETETETETVT, TNTNTETETE, TVSNTNTNTN, ATVTVTVTVK, TVSATATVTV, SIKVTVTVTV, VATHTHTHTH, AVATATATVT, TAEVKVTVTV, VTVVVTVTVT, KVEVTVTVTV, ININNENKTT, TLTATATATA, DVRAVAVAVA, NLNLNVNLVT, TVTVLNLNLN, NTNTNTNTVT, TTKNKNTNTN, IEIQIQIQIK, VITIQIQIQI, YTYTGVGTGT, TVTVTYTYTY, TLQITITITI, TVTLTLTLTL, VVVVVVVEVT, EAKVVVVVVV, I I I I I I I I I I , TVTTN I I I I I , VTVVVVVVVT, TVEVVVVVVV , TVTATAAATV, LVVVVVVVVT, TVETVVVVVV, YTYTYTYTYS, TLTLTLTYTY, TVKVTVTITI, TVTVTVTVTL, TATYTITITI, NVVVTVTVTV, VTETETETET, TTTVTVTETE, DIEATATATA, TLEIKITITI, VVRVTVTVTV, KTKTKTSTKT, TVTKTKTKTK, FVYVYVYVYT, EIRFVFVYVY, NTNTNTNTIT, VKKKTNTNTN, SKTINTNYNA, TISSSSTITI, I I I I I I I I KT, TTTTV I I I I I , ITATATATAT, KTAETAVATA, TKQKTQTQTQ, YTYTYTVTVT, TYTYTYTYTV, VTLTLTLTFT, SVRVTVTLTL, VVGVVVTVTV, ITKTKTKTKT, VVTETKTKTK, ETKITITITI, FTFVFVFTFT, TVKVKFVFTF, TGTLTLTLTL, ISISISISIS, TLLVSISISI, I I I I I VTSTS, TV I I I I I I I I , DGKVTVTVTV, LTITITITVT, TKKITITITI, TQTVTVTVTV, TLTVVLVLVL, LTVTVTVTVT, TVVATATATA, QTQTQTQSQS, DKKHTQTQTQ, TKEVIVIVIV, NIKITITVTV, VKVKVKVTVT, TVEVKVKVKV, ATSTSTSTVT, IVTATATSTS, IEITITITVT, IIKITITITI, NLNLNLNLNT, KNNNQNLNLN, VIVIVIVIVT, TIQVIVIVIV, IVIVIVIVIK, EVEIEIVIVI, DIDIDIDIDT, KRERSDIDID, VSVSVSVSVS, TLTVSVSVSV, TVTVTVTVTK, EEQTVTVTVT, IVIVIVITIT, TAKIVIVIVI, QIQIQIQIVE, NKKQIQIQIQ, ITITIVITIK, EIEITIVITI, NININININT, TKTNINININ, ETETYTGTGT, VREETETETG, TVEITITITI, NITVTVTVTV, VTVTVTVEVL, VVKVKVKVEV, TLTATVTVTV, TLTLTVTATV, NLTVTATATA, ITTTNENSVS, TVKVKTTTST, VTNTVTVTVT, TVRVRVTNTN, ITITITITKT, TLTITITITI, VTVTRTRTRT, TEKRTRTRTR, TNTNTNTNTN, TTTDTVTVTV, ITQTQTQTQN, TNTSTSTQTQ, TKMSTNTNTN, TVQVQVTVTV, VTVTVTVTFT, TNTLTVTVTV, VVKITITITV, AIAIAIAIAK, VVEAIAIAIA, TTTNTNTNTN, VTVTVTATAT, TLTVTVTATA, QTTVTVTITV, TVTVTVSVTV, VTLTVTVTVT, TLELKLTLTL, VTVSVSVSVQ, KETVTVSVSV, LILILILILT, AATLILILIL, RVEYTYTYTV, ITITITITVK, EIEIEITITI, EVEVEVTVTV, ITITYTYTKK, TKEITITITY, ITITIKITVK, KVEIEIKITI, TVLVVATATA, NTNTNTNKNT, TKVTDNDNDN, ATGTGTGTGT, TKTATATGTG, TVTVTFTFTF, TVSITITITI, IVVVVVVTVT, KKEEVVVVVV, NTNTNTNTKT, SVQITITITI, KRSDTDTVTV, VTVTATATAS, VANVTVTVTA, AVEVTVTVTV, TVKVTVTNTN, VTNTNTNTNT, LTQSTNENTN, TPTGTGTGTM, VTITITVTVN, VTQVTVTVTV, NTNINININI, KKQNINININ, TVKLTARATA, TAEVTYTYTY, FTFTFTFTIT, KIEFVFTFTF, TAKATATATA, TVELILILIV, VTVTVTVTYT, VKKEKQTQTQ, AVAVAVAVAK, NVELVAVAVA, IVVVVVVVVV, TVFVVVVVVV, TIGVVVVVVV, EVTVTVTVTV, IVTVTVTITI, EVKITITITI, VTVTVTVTVE, TAKVTVTVTV, IVIVIDNVNK, ETEIVIVIVN, IQETITITIT, VAEFKFTFTF, IVTLTLTITI, TVEVTVTITI, TALVTVTVTV, IEIEIKIEVK, TITIAIAIEI, TVTAVVVVVV, TVKITITLTI, TITI I I I I I I , TISITITITI, TSTVTVTITI, LVLVLVLTVT, VLTLVLVLVL, NVSITITITV, IEIVIVIVIS, TVSAVAVIVI, KTKTKTKTKT, TASVTVTVTK, VTTTVTVTVT, KATVVVTVTV, EKEVTVTVTV, TIKLTLTLTL, STSTSVSTVT, TVTSTSVSTS, NTNTNTNTNK, TETDTDTDTD, HTHTHTHTIK, TITQTHTHTH, NTNTNTNTLT, TAVNVNTNTN, QIQIQIQTQT, EKKQIQIQIQ, VIVIVIVTVT, KVEVIVIVIV, YAKITITVTV, TAKVVVVVVV, KNKIETNYTA, VTVTVTITVT, KATVTVTVTV, VRTNTNTNTN, FTITITITIT, TATLVLVLVL, ITITIVITIE, TIKKVKTITI, TARSTATATA, HVHTHTHTHT, VRTETHTHTH, TFEIEITVTV, VTVTNTNTNT, TKSVTVTVTN, VTVTVTVTAT, NVTITITVTV, VTVTITITVT, TVKVTVIVTV, RTRTRTRTVT, EKKDTRTRTR, IEIEIEITIT, TLKIEIEIEI, NININVNINT, TKKNINININ, TQNVTLTLTL, VVVVVVVVAT, EERVVVVVVA, RTATATATAT, QVKVTVTVTV, LELELELESE, TRKIKLELEL, QTQTQTQTTT, TKTETQTQTQ, DTRTDTHTDT, TRVDTDTDTD, VTGTVTVTIT, NTNVNKNSVT, TVTV I I I I I I , DKKNTNTNTN, HIHIHIHIVK, VREEIHIHIH, LTATATATAT, GTGTGTGTVT, TVVGVGTGTG, ETETETETET, VREVTVTETE, YIS I I I I I I I , INITVVITIT, TRTITIVITI, ITI, ATATATATVK, TATATATATV, TITVTVIVIV, AIAILILILT, TVTATAIAIL, VTVTVCVTVT, YELELRLRLT, GLTLTLTLRL, KVEVIITITI, I I I I I I HTHT, SESESETHTH, VQVQVQVTVT, TVNVQVQVQV, ITITISITIT, TIEITITITI, VVTLTLTLTL, TTNVTVTVTV, MVMVMVMVMT, TVEMVMVMVM, TKRVTVTNTN, DTDTDTDTVT, TVRVVVVDTD, TVTKTVTVTV, VTVTVTVVVK, I I I I I I I I VT, TVK I I I I I I I , VVVVVVVVVE, TVRVVVVVVV, STSTSTSTST, TISSTSTSTS, TKENTNTNTN, TKTATNTNTN, QVTITITITI, STSTSTSVAE, TVRSTSTSTS, NVKITITITI, LTLTLTLTLK, VVELTLTLTL, TETVTVTVTV, ITVTVEVTVT, I I I I I NTNTN, LTGTGTGTGT, KLTITGTGTG, NVNVNVNVNK, DKSNVNVNVN, IVIVIVIVIV, VSLVVIVIVI, TIEVNVTVTV, VAQFTFTFTF, NLKATATATK, KKTVTVTNTN, RIRIRIRIHT, TDKRIRIRIH, IVVVVVVVVT, TVEAVIVVVV, IEIEIEIKIK, TVEIEIEIEI, AVQVTVTVTV, ITITIEITIT, ELKIKIEIEI, AAAAAAAAVT, TSTATAAAAA, TATVTLTLTL, TQTYTYTYTY, VIKITITITI, TLGLTLTLTL, LTLTLTLTVK, TVELTLILTL, IEIEITITVT, TVKVEVEVEV, EETETKTKTK, TGKVTVTVTV, SVSVSVSVTV, TVRIVSVSVS, QVQVQVQVQT, TTTQVQVQVQ, DTDTDTDTKT, TVKTK I I I I I , TITVTVVVTV, KINVTITITF, TVTLTLVLVL, TQENTNTNTN, KTETETETQT, RTERRERETE, HQHQHQHQTQ, TRVHQHQHQH, TVEVTITITI, LVRVTVTVTV, HSHTHTHTCT, TVTVTHTHTC, VVIVIVIVIK, ELSVWVIVI, TEEATDTDTD, DVNVNVNVTV, IVKIKIKVIV, SVIVIVITVT, TAELVIVIVI, TFEVTFTFTF, TKTDTDTDTD, V I I I I I I I I I , TVTVTVTTTT, GVGVGVGVVT, TVEVVGVGVG, TIEVTVTITI, LELELELEVT, VVKLKLKLEL, TVTITIEIEI, ATATATATAV, ITEEEATATA, YTFTFTFTFT, DSTFTFTFTF, TVKIKITATI, KTKTKTKTKK, TEEKEKTKTK, QVKVKVTVTV, TVQVTITITI, VTITVTVTVT, TFEITITITI, ATATAIATAT, AVKDTAIATA, TKTNTNTNTN, KVTVTITITI, ITITITITFT, TYLGTITITI, TAQVTVTVTV, SNTNENENSV, AEAEAVAKAK, TAEAEAEAKA, KLTVTITITI, TATITIQITI, ITLTLTLTLT, TLNLNLTLTL, ITNTNTNTIT, TKTITITITI, TVKITVTVTV, ATITITITVS, TTTVTITITI, EVKVKVKVKT, EERETEVKVK, EIEIEVTVTV, LELELELELT, TATAELELEL, YTATATATVK, TVELTATATA, VKEIEIEITI, NISVTVTVTV, HQHTHTHTHT, TLSHSHTHTH, TVTITITFTI, LELELELELR, VLEVEVELEL, NININININA, INLNINININ, ITITLTITIT, VDTNKNTNSV, TVKATITITI, VEVTVTVTVT, TITITITVTV, NNTNTNTSTS, KTKTKTKTVT, TKTETKTKTK, ITVTTTVTVK, EIEIEIEIEI, KIQEIEIEIE, SVSVSVSVSV, TGTVTVVSVS, TVTATFTFTF, TKVNVNTNTN, VTVTITVTLT, TATVTVTITV, VTVTVVVEVD, VARVRVTVTV, ITIIITITIT, YTYTVTVVVA, TVAAIAIDTV, VIVIVIIIIT, TGKVIVIVII, AVAVAVATAT, TLEVVVVAVA, VTVTVTVTVS, TVKVTATATA, TVRVRVTVTV, LTLTLTLTLQ, TVTVTLTLTL, EVEITITITI, TVTDTDTDTD, QKTYTYTVTV, LSLSLSLTLT, TATLTLSLSL, NISISISISI, QSSSISISIS, LTLTLTNTNS, TSKLTLTLTN, RAQLQLQLTL, TLKVTVTVTV, TVTLTVTVTV, KTVTVTVTVT, FTFTLTLTLT, TATFTFTFTF, ATATLTATIA, IVVTITITIT, VVTITITITI, EKKNTNTNTN, LTLTLVLTLT, TLRLTLTLTL, AVAVAVAVAR, TVEVVAVAVA, TIRVTITITI, VVTFTFTFTF, KDHVHVHTHT, TTVKVKVHVH, VTLTLTLTIT, TVTVTVTLTL, LVLVLVLVVT, EIEINITITI, TVKVTKTKTK, KTKTDTKTKT, KKSKTKTKTK, KTKTKTETET, KKIKTKTKTK, NIKITITITI, VIKVTVTVTV, IVIVIVIVVT, EVEIVIVIVI, ELRVTITITI, VVDVVVVVAK, KLEVVVVVVV, TVVVTVTATA, TLTLTATATA, STNTNENTTK, NVTLTLTLTL, KLEIEITITI, VVVVVVVVVR, TATGTGTGTG, GVSVTVTVTV, TETKTKTKTK, NVNVNVNTNT, TKENVNVNVN, KVKVTVTVTV, VVEVVVVVVV, TAQIVIVIVI, TVQVTVTVTV, NVTLTVTVTV, VVIVIVIEIT, EIRVRVVIVI, TVELTLTLTL, EETKTKTKTK, SISISISIST, TSSSISISIS, TLVATITITI, ITITITITVS, FKFKFQFTVT, DVEVKFKFKF, TTETTNINTN, ATVTVTVTVT, TNTITITTTT, YTITITITVT, IMITITITIK, TATTTITITI, MTMTMTMKIK, TIEMTMTMTM, VTVTYTVTVT, NVTVTVTVTV, TIRITITITI, FTLTLTLTIT, DVTLTLTLTL, VTVTFTVTVT, TITVTVTLTV, ERRYTYTYTY, NLNITITITI, VTVTVVVVVV, FIFIFIFIFT, TGEFIFIFIF, VVVVVEVVVS, ILSITIIITI, TVKVIVIVIV, AVAVAVATVT, TTQEVAVAVA, VVTATVTLTV, TITVVIVIVI, NTNTNTITIT, TAKNTNTNTN, TTTVTVTVTV, EVKVEVTVTV, VTVTVVVTVK, EVEVEVVVTV, LTGTGTGTVT, EVEAVATGTG, TLSVSVVVVV, TNKITITITI, ITITITISLS, PLELTLTLTI, KKKYTYTYTY, TVKIEITITI, EIKITITITI, KEKEKVKKIK, TKEKEKVKKI, TLTYTYTYTY, TYTLTLTLTV, EVKVVVVVVV, TATLTLTLTV, NSNTNTNTNT, ETETETETKT, KETETETETE, VQVTVTVTVT, KITVTVVVTV, VEVEVEVKVK, EIEVEVEVEV, TIKG I I I I I I , NTQVTSTSTS, KVEIEITITI, KTKTETETEE, TKKKTKTKTK, VTVTITITIT, TITVTVTVTI, VTVTNTVTVT, TKTNTNTVTV, ITETETETET, VKEIVITETE, LTVTVTVTVM, TVLVTVTVTV, TITATGTGTG, IDIDIDIDIT, QLSVTVIVTV, VKEKTHTHTH, IIEITITITI, ILKITITITI, KEEKTKTKTK, TLTQTQTQTQ, LTFTFTFTFT, NTRSTSTSTS, STTETKTKTK, IEEETETETE, VVVTVTVTVT, KNKNKVKNVN, EAERTKNKNK, DVSV I I I I I I , TFEVTVTVTV, FEFEFEFEFK, KVEFEFEFEF, ITITITIIIK, TATITITITI, KITITITATA, TVKVDADATA, TVKVVVTVTV, TVNVVVVNVV, NTITITITVT, TVNVTVTITV, KTEITITITI, IEIEIKIKII, TIEIEIEIKI, YTYTYVYTIT, TVEYVYVYTY, IQILILISIS, QIKILILILI, TLTLVLVLVL, VTATFTATAT, VAKATATATA, EVELTVTVTV, TVTITTTLTT, I I I I I I I I DT, TTTL I I I I I I , VQVQIQVQVQ, TITITIQVQV, KVEVKATATA, TLTLTLSLTL, TTLTLTLTLT, IIANININLN, TASINININI, VKEDIKIKIK, KITVTVTVTV, VTLTLTVTVT, TVQVTVIVTV, AVAVAVAVVT, TVELVAVAVA, TITLTLTVTV, TVTLTLTITI, STNTNTNTNT, NTSNTNTNTN, RVTVTFTFTF, TKEETETETE, ITITVTVTVT, TITGTVTVTV, VVVVVVVVVV, DVKVVVVVVV, DVKITITITI, EVEVVVVVVV, KTKTITKTKK, ITITITATAT, TAKVTVTVTA,

[0240] TETETNTNTN, KVKVTITITI, ITITITITIN, NISITITITL, IKEVVIVIVI, KVTLTLTVTV, VIEITVTVTV. IARVTITITI, QVQVQVQTQT, TTKTVTVQVQ, TVEITITITV, KDKTKTKTKT, TTTDTDTDTK, EVKVTVTLTV, TDRSVSLNTN, SRSANVQSVT, VTTIVLVVNL, SNSTVTLQQT, STTIVLTVNL, ITLVLVQSTT, VVKVTTTTKV, MSVTTTVVVS, SVTVVATTTV, VTVTTVVQST, TEKIKKEYAD, SNSEKEVVKT, TEKTEKEYED, NGIEKVVNVT, QIRRSYKYKN, RSSESERQTT, TSTEEYEYNN, RRIKYTRTTT, QLRVEVTVSV, STSVVRVERR, SRRVVVRVNV, VTVTVERRET, ILRVEINETD, SQSRIEIEYV, KVRVEIEEND, VRINIEYELT, VLTNTNTNKE, SSNTNTNTIV, KRTNTNTNNE, NTNTNTNSTT, ALKSVKVDTN, STSKDEKEVK, TVTQVKEDDN, SKKVDEKTIT, VVRVAVAVTV, SSITVEVAVA, TVTIHVEVNV, VDVAVAAQVT, KNTSTNTYSL, SKSENKNTTT, SQTSENKYSL, STSTNTNQTT, VDKVEIEVKV, SEITVKNEHN, SNKKKIKVTV, VTIENENKVT, VDTNNNNYTI, SNSNYRNTNN, TRTDENRYNI, YDNNYTNTRT, VK I I I I I VSV, STVTVTTTQQ, SS I I I I I VNV, V I I I I I QKQT, VDKIEVDHSL, SNSSHETTKT, KTTKKEEHDL, IKIDVETTVT, VNEVTITVNI, SNSEVKIRKV, TETVVIKVNI, VEITITTTVT, VLSVSVSARV, VDVQVEVQQT, TTRTEVEANV, VEVSVSQQVT, VARTSVSLTA, STLEVEEERA, QRRTTIELNA, TTVSVEESVT, VVTVTVTYTD, SSVTVTVTTT, STTVSVTYND, VTVTVTVSTT, VDTVSISYNV, LNVSVSQQQK, SQTVSISYNV, VTVSIQQTVT, TTRVQVQVTN, STTTVQDEDQ, SKRVQVQVSN, VTVQNEDLTT, VLEVEVEVKV, VEIVVVVKNT, KSEVKVVVNV, VSIEVNKQVT, VHSINVNVTV, STINVNVTNT, RSTININVNV, ISVNVTVSVT, KNTVTKTVDL, AREVVVKTTT, TKTETKVVDL, and VTKTVTTTQT. The one or more beta strands and / or one or more alpha helices may comprise one or more of the listed insertions. Different combination of these ten amino acid elongations / insertions may be used in the same beta strand or alpha helix or in different combinations of two or more beta strands and / or two or more alpha helices. The ten amino acid elongations / insertions may be used in the one or more beta strands and / or one or more alpha helices consecutively or separated by existing sequences.

[0241] The one or more beta strands and / or one or more alpha helices may be elongated by one or more of TNVNTSTSTYNI, TTSTSTTTFEVT, SQRNSITSTYNI, STNTSSSTFMQR, TSQTRTN ITYSV, GSTDVRIEQKQQ, SNTTETRIRYTV, VTTEVNIKQTQK, TTTQTQTQSTN L, SNAENKQKQTQN, TQTQEQKQKTNL, ATQTSTQTQRQQ, TQTNVNVTGNTN, SSSTNMNTNQLT, TQTNTNTNNNGN, NEIVNVNQNQIK, VQTQTNTNSVSV, SNSTTTNVNTTS, TNSQVNVNSVNV, STNTNTNTNITR, VQKVSNSNTNTN, SNSKNETEHTMI, TTTVTNENENTN, NSNSNSIMHTVK, KTVIKQEQDQLV, SNQVQRVEQENV, SNKVKQRQVQSV, QIQKQEQEILVK, QNTQTNTYTYTG, STNTSTYKQEIK, SQKQENKYTYSG, NTTTYTYEQSVR, VVTLTVTTTVTL, STVSVTTTVNTT, SVTLTVTTTVTL, VTL I I I I I VTTR, VNTITVTVTLTV, STSSTSVKVNQQ, SNNITVKVSLTV, VNITVTVNNQQR, VQTHAYQLTHTV, SNSRVELIQQTV, SQTTTYKLEHTV, YKHTYQLQQTVK, KVEIKINIDTNV, SDIDIIYQTSTT, SVKTEIQIDTNV, INIDINVKTSTK, KVSNKVKVKITA, SNIGVTHEVTTT, TNRNTVEVDINA, VNIKVKHTVETR, TNTITVTYTYTN, SSVTVTNQVTVV, STTIQVTYTYNN, VTVTYTQTVSTK, KTTIIVVVEVEN, SKVEVEVETTTI, SNTINVEVEVEN, VKIIVVVTTQVK, TQKVAIAVNVHV, SNVEQVLENTNT, TNTVEIELVVNV, VTVAVAITNTTK, VSSITVTSTVEL, SKVTVTVTNVTT, SQSNTVTVTVEL, VTVTVTSVNTVR, TNTTNVTINVTL, ATTIVIYNYTTT, TVTTNVNIIVNL, VITNVNYTNTTK, IVTVTVTVNNTV, SSSTVTVTAAVS, TTTVTTTVTN DV, and VTTTVTVTATVK. The one or more beta strands and / or one or more alpha helices may comprise one or more of the listed insertions. Different combination of these twelve amino acid elongations / insertions may be used in the same beta strand or alpha helix or different combinations of two or more beta strands and / or two or more alpha helices. The twelve amino acid elongations / insertions may be used in the one or more beta strands and / or one or more alpha helices consecutively or separated by existing sequences.

[0242] The one or more beta strands and / or one or more alpha helices may be elongated by one or more of INRNVNVNVNVYET, SKSENKNEIENVTR, TQKNKNEIENKYET, SRNVNVNVNTNEVK, VVINENQNVNNYNV, SNNLNQNQNRVETT, TTKNENQNQNEYNV, NNNKNVNRNIVTVK, VNTNTNTNSNDYSV, SNSNNTNTNTLTTS, SQTNINTATNDYDV, INNTNTNTNTHTVK, QNTIVTVTVTVTSV, SKSVTVVTTQNTNP, RNSTQTTTVTVTSV, SVTVTVTVTQNSTR, KNSNTNTNTVVYTA, STLTVTTTNANNNS, SNKTASANTVTYTA, VTITNTNTNNNETK, VITNTNTNENTYKN, SESNNTNTNTVTVK, SVENTNTNTNNYTN, SKNTNTNENTVIVR, IVQTETETEVTVLV, STTSTSNETSVTIR, STRTSTETEVSVSV, TNTETSVETSVGVK, VVTTKVIVLTTYAN, NRNEVKAKVEVTQR, TTKTTVSVKTKYSN, NTVITLVIVEVEQR, ILKTQQKQKVNINI, SNYTVEVEQEQQVT, KVSTSQEQEVRINI, IEVQVEQKQQQEVR, VLSVEVSTSNDYNI, SNSKVDNDVRVKTT, TNEVIVDVDNKYNI, SSNSVSTSVRVKTK, LVLIQNTQTHTVSV, SSYTYTTTQTVLLQ, TTAITNTQTHTVSV, YTYTQTTTQLVVQR, VAKVTNTVTNTYSI, SKISITNTVTSTLN, RQTVTHTVTNTYSI, ISNTVTNTVTVSVK, VVEISVNNNVSYRN, SNSTNSVSNTVKVT, SVSIKVTNSVSYSN, and STVSNNVNVSVSVK. The one or more beta strands and / or one or more alpha helices may comprise one or more of the listed insertions. Different combination of these fourteen amino acid elongations / insertions may be used in the same beta strand or alpha helix or different combinations of two or more beta strands and / or two or more alpha helices. The fourteen amino acid elongations / insertions may be used in the one or more beta strands and / or one or more alpha helices consecutively or separated by existing sequences.

[0243] All of the lists of preferred amino acids above are particularly preferred for modifications, elongations or insertions in one or more beta-strands.

[0244] The pore monomer preferably comprise four beta-strands. At least two of the four betstrands may be modified or elongated. At least three of the four bet-strands may be modified or elongated. Preferably, all of the four beta-strands are modified or elongated. The four beta-strands may be modified or elongated to different degrees. The four betastrands are preferably modified or elongated to the same degree. In this context, the term "degree" typically relates to the number of amino acids added to the two or more betastrands. For instance, two amino acids may be added to each of the four beta-strands. Even if the four beta-strands are modified or elongated to the same degree, they may be modified in different ways. For instance, the two amino acids added to each of the four beta-strands may be different. The two amino acids may be selected from the list above. Alternatively, four consecutive amino acids may be added to two of the beta-strands and two separate stretches of two amino acids may be added to the other two beta-strands. The identities of the four consecutive amino acids and the two separate stretches of two amino acids may be the same or different. The skilled person is capable of designing suitable modifications and elongations / insertions.

[0245] Each beta-strand may comprise one or more preferred positions for modifications, elongations or insertions. Each beta-strand may comprise at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9 or at least about 10 preferred positions for modifications, elongations or insertions. The preferred positions in each of the four beta-strands typically line up approximately horizontally in the beta-sheet formed from the four beta-strands.

[0246] Table 1 below shows a pore monomer comprising four beta-strands capable of forming a pore comprising a beta-barrel channel. Each strand comprises four preferred positions for modifications, elongations or insertions (labelled A-D, E-H, I-L and M-P). The preferred positions in each row, e.g., A, E, I and M, typically line up approximately horizontally in the beta-sheet formed by the four beta-strands.

[0247] The pore monomer may comprise any of the following modifications, elongations or insertions based on Table 1 :

[0248] • The insertion of the same number of consecutive amino acids, such as 2, 4, 6, 8, or 10 consecutive amino acids, in beta-strands 1-4 at the positions in the same row, such as (i) A, E, I and M, (ii) B, F, J and N, (iii) C, G, K and O or (iv) D, H, L and P.

[0249] • The insertion of the same number of consecutive amino acids, such as 2, 4, 6, 8, or 10 consecutive amino acids, at any one of positions A-D in beta-strand 1, at any one of positions E-H in beta-strand 2, at any one of positions I-L in beta-strand 3, and at any one of positions M-P in beta-strand 4.

[0250] • The insertion of the same number of amino acids, such as 2, 4, 6, 8, or 10 amino acids, in each of beta-strands 1-4, wherein the same number of amino acids are consecutive amino acids inserted at any one position in a strand and / or the same number of amino acids are two or more stretches of one or more amino acids inserted at two or more positions in a strand. The two or more stretches may be 2, 3, 4, 5 or more stretches. The two or more stretches may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids.

[0251] In any of the embodiments, the inserted amino acids, consecutive amino acids and / or two or more stretches of one or more amino acids may be selected any of the individual amino acids or stretches of 2, 4, 6, 8 or 10 amino acids listed above.

[0252] The pore monomer or the pore monomer conjugate is preferably not modified to alter the length of the first constriction region and / or the length of the second constriction region. The pore monomer or the pore monomer conjugate is preferably not modified to alter the length of the first constriction region. The pore monomer or the pore monomer conjugate is preferably not modified to alter the length of the second constriction region. The pore monomer or the pore monomer conjugate is preferably not modified to alter the length of the first constriction region and is preferably not modified to alter the length of the second constriction region.

[0253] In some embodiments, modifying the pore monomer to increase the distance between the first constriction region and the second constriction region does not comprise adding a third constriction region. In these embodiment, the accessory polypeptide may be further modified to comprise at least a third constriction region which forms part of a third constriction in the channel. The accessory polypeptide may be modified in any of the ways discussed above. However, the addition of the third constriction region in these embodiments does not increase the distance between the first constriction region and the second constriction region.

[0254] The pore monomer or the pore monomer conjugate preferably does not comprise two or more pore monomers. The pore monomer or the pore monomer conjugate is preferably not a double pore monomer. The pore monomer or the pore monomer conjugate is preferably not a double pore monomer described in WO 2018 / 211241 (herein incorporated by reference in its entirety). In particular, the pore monomer or the pore monomer conjugate preferably does not comprise a first pore monomer from a first CsgG pore, or a homologue thereof, and a second pore monomer from a second CsgG pore, or a homologue thereof. Insertion positions in CsqG (SEO ID NO: 3)

[0255] CsgG pore monomers, such as those based on SEQ ID NO: 3, comprise four barrel forming beta-strands. The transmembrane region of beta-strand 1 corresponds to V134-Y143 in SEQ ID NO: 3. The transmembrane region of beta-strand 2 corresponds to I146-D155 in SEQ ID NO: 3. The transmembrane region of beta-strand 3 corresponds to I181-V190 in SEQ ID NO: 3. The transmembrane region of beta-strand 4 corresponds to L200-E210. A pore monomer based on SEQ ID NO: 3, including any of the variants discussed above, typically comprises beta-strands 1-4 or variants thereof.

[0256] A variant of SEQ ID NO: 3 preferably comprises a sequence that is at least about 40% homologous to V134-Y143 of SEQ ID NO: 3. More preferably, the variant may comprise a sequence that is at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% and more preferably at least about 95%, 97% or 99% homologous based on amino acid identity (i.e., identical to) to residues V134-Y143 of SEQ ID NO: 3.

[0257] A variant of SEQ ID NO: 3 preferably comprises a sequence that is at least about 40% homologous to U46-D155 of SEQ ID NO: 3. More preferably, the variant may comprise a sequence that is at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% and more preferably at least about 95%, 97% or 99% homologous based on amino acid identity (i.e., identical to) to residues U46-D155 of SEQ ID NO: 3.

[0258] A variant of SEQ ID NO: 3 preferably comprises a sequence that is at least about 40% homologous to U81-V190 of SEQ ID NO: 3. More preferably, the variant may comprise a sequence that is at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% and more preferably at least about 95%, 97% or 99% homologous based on amino acid identity (i.e., identical to) to residues U81-V190 of SEQ ID NO: 3.

[0259] A variant of SEQ ID NO: 3 preferably comprises a sequence that is at least about 40% homologous to L200-E210 of SEQ ID NO: 3. More preferably, the variant may comprise a sequence that is at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% and more preferably at least about 95%, 97% or 99% homologous based on amino acid identity (i.e., identical to) to residues L200-E210 of SEQ ID NO: 3. A variant of SEQ ID NO: 3 preferably comprises all of the sequences in the preceding four paragraphs.

[0260] As discussed above, one or more of, such as 1, 2, 3 or 4, of beta-strands 1-4 may comprise one or more modifications, elongations or insertions. In a pore monomer comprising or consisting of the sequence shown in SEQ ID NO: 3 or a variant thereof, including any of the variants described above, the one or more modifications, elongations or insertions may be made between any of V134-Ins-K135, K135-Ins-S136, S136-Ins-G137, G137-Ins-G138, G138-Ins-V139-Ins-G140, G140-Ins-A141, A141-Ins-R142 and R142-Ins-Y143 or any positions which correspond to any of these positions.

[0261] In a pore monomer comprising or consisting of the sequence shown in SEQ ID NO: 3 or a variant thereof, including any of the variants described above, the one or more modifications, elongations or insertions may be made between any of G147-Ins-A148, A148-D159, D149-T150, T150-Q151, Q151-Y152, Y152-Q153, Q153-L154, L154-D155 or any positions which correspond to any of these positions.

[0262] In a pore monomer comprising or consisting of the sequence shown in SEQ ID NO: 3 or a variant thereof, including any of the variants described above, the one or more modifications, elongations or insertions may be made between any of I181-L182, L182- S183, S183-Y184, Y184-E185, E185-V186, V186-Q187, Q187-A188, A188-G189 and G189- V190 or any positions which correspond to any of these positions.

[0263] In a pore monomer comprising or consisting of the sequence shown in SEQ ID NO: 3 or a variant thereof, including any of the variants described above, the one or more modifications, elongations or insertions may be made between any of L200-E201, E201- G202, G202-E203, E203-V204, V204-G205, G205-Y206, Y206-T207, T207-S208, S208- N209 and N209-E210 or any positions which correspond to any of these positions.

[0264] In these embodiments, the is the position where the modification, elongation or insertion is made.

[0265] Table 2 below shows summarises the preferred positions for modifications, elongations or insertions in the four beta-strands in a pore monomer comprising or consisting of the sequence shown in SEQ ID NO: 3 or a variant thereof, including any of the variants described above,. "V134-K135" means between V134 and K135 or the positions corresponding to V134 and K135. The preferred positions in each row typically line up approximately horizontally in the beta-sheet formed by the four beta-strands.

[0266] A pore monomer comprising or consisting of the sequence shown in SEQ ID NO: 3 or a variant thereof, including any of the variants described above, may comprise any of the following modifications, elongations or insertions:

[0267] • The insertion of the same number of consecutive amino acids, such as 2, 4, 6, 8, or 10 consecutive amino acids, in beta-strands 1-4 at the positions in the same row.

[0268] • The insertion of the same number of consecutive amino acids, such as 2, 4, 6, 8, or 10 consecutive amino acids, at any one of the positions in beta-strand 1, at any one of the positions in beta-strand 2, at any one of the positions in beta-strand 3, and at any one of positions in beta-strand 4.

[0269] • The insertion of the same number of amino acids, such as 2, 4, 6, 8, or 10 amino acids, in each of beta-strands 1-4, wherein the same number of amino acids are consecutive amino acids inserted at any one position in a strand and / or the same number of amino acids are two or more stretches of one or more amino acids inserted at two or more positions in a strand. The two or more stretches may be 2, 3, 4, 5 or more stretches. The two or more stretches may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids.

[0270] A pore monomer "based on" SEQ ID NO: 3 may be any of the variants, fragments, etc. of SEQ ID NO: 3 disclosed above. In any of the embodiments, the inserted amino acids, consecutive amino acids and / or two or more stretches of one or more amino acids may be selected any of the individual amino acids or stretches of 2, 4, 6, 8 or 10 amino acids listed above.

[0271] Preferred pore monomers of the invention comprise or consist of any of the insertions shown in Tables 3 and 4, especially the insertions shown in a single row.

[0272] Elongating the accessory polypeptide

[0273] In the pore monomer conjugates of the invention, the accessory polypeptide may also be elongated. The accessory polypeptide is typically elongated when the pore monomer is modified or elongated in the region which binds to the accessory polypeptide.

[0274] Residues within the range Y130 to N209 of the CsgG pore monomer typically bind to CsgF peptides. The accessory polypeptide, such as the CsgF peptide, is typically elongated when the CsgG pore monomer is modified or elongated between residues Y130 and N209. Residues Y130, E131, N133, S136, G137, G138, V139, G140, A141, R142, Y143, F144, G145, A148, D149, Q151, Y152, Q153, D155, 1181, S183, Y184, E185, Q187, F191, F193, 1194, D195, Y196, Q197, R198, L199, E201, G202, E203, G205, Y206, T207 and N209 of the CsgG pore monomer typically bind CsgF. The accessory polypeptide, such as the CsgF peptide, is typically elongated when the CsgG pore monomer is modified or elongated at or adjacent to any of these residues. Residues Y130, E131, N133, S136, G137, G138, V139, G140, A141, R142, A148, D149, Q151, Y152, Q153, D155, 1181, S183, Y184, E185, Q187, F191, E201, G202, E203, G205, Y206, T207 and N209 of the CsgG pore monomer preferably bind CsgF. The accessory polypeptide, such as the CsgF peptide, is typically elongated when the CsgG pore monomer is modified or elongated at or adjacent to any of these residues.

[0275] The skilled person is capable of determining whether or not an accessory polypeptide has been elongated in accordance with the invention. For instance, the skilled person can compare the accessory polypeptide with the natural or wild-type sequence, such as SEQ ID NO: 6 or positions 1-29 of SEQ ID NO: 6.

[0276] The accessory polypeptide may be any of those described above, including any of the CsgF peptides discussed above. The accessory polypeptide is typically elongated by about the same distance as the pore monomer in the conjugate. The accessory polypeptide, such as the CsgF peptide, may be elongated by any of the percentages or any of the distances in A or nucleotides discussed above for pore monomers.

[0277] In pore monomer conjugates comprising a CsgG pore monomer and a CsgF peptide, the distance between the first constriction region and the second constriction region is preferably increased to and the CsgF peptide is preferably elongated by about 30 A or more. The distance is preferably increased to and the CsgF peptide is preferably elongated by from about 30 A to about 100 A, such as from about 31 A to about 90 A, from about 32 A to about 80 A, from about 33 A to about 75 A from about 34 A to about 70 A or from about 35 A to about 65 A. In pore monomer conjugates comprising a CsgG pore monomer and a CsgF peptide, the distance between the first constriction region and the second constriction region is preferably increased to and the CsgF peptide may be elongated by about 6 or more nucleotides. The distance is preferably increased by and the CsgF peptide is preferably elongated by from about 6 nucleotides to about 50 nucleotides, such as from about 7 nucleotides to about 45 nucleotides, from about 8 nucleotides to about 40 nucleotides, from about 9 nucleotides to about 35 nucleotides or from about 10 nucleotides to about 30 nucleotides. The distance may be increased to and the CsgF peptide may be elongated by any of the specific distances discussed above for the pore monomers of the invention.

[0278] The pore monomer and accessory polypeptide may be elongated at corresponding positions. The pore monomer and accessory polypeptide may be elongated at positions which align in the pore monomer conjugate. Alignment is typically vertical alignment. The pore monomer and accessory polypeptide may be elongated at positions such that the elongations align or vertically align in the pore monomer conjugate. This is shown schematically in Figure 10 (second from the right).

[0279] The pore monomer and accessory polypeptide may be elongated at different positions. The pore monomer and accessory polypeptide may be elongated at positions which do not align or are off set in the pore monomer conjugate. Alignment is typically vertical alignment. The pore monomer and accessory polypeptide may be elongated at positions such that the elongations do not align, do not vertically align or are off set in the pore monomer conjugate. This is shown schematically in Figure 10 (far right).

[0280] The accessory polypeptide, such as the CsgF peptide, may be elongated by insertion or addition. The accessory polypeptide, such as the CsgF peptide, may be elongated by the insertion of one or more amino acids or the addition of one or more amino acids. One or more amino acids may be inserted into or added to any of the accessory polypeptides or CsgF peptides described above. The one or more amino acids may be added to the N- terminus and / or C-terminus of the accessory polypeptide. The one or more amino acids may be inserted at any position in the accessory polypeptide. The one or more amino acids are typically not inserted in the second constriction region.

[0281] The accessory polypeptide, such as the CsgF peptide, is preferably elongated by from about 1 to about 17 amino acids. The accessory polypeptide, such as the CsgF peptide, is preferably elongated by about 1 amino acid, about 2 amino acids, about 3 amino acids, about 4 amino acids, about 5 amino acids, about 6 amino acids, about 7 amino acids, about 8 amino acids, about 9 amino acids, about 10 amino acids, about 11 amino acids, about 12 amino acids, about 13 amino acids, about 14 amino acids, about 15 amino acids, about 16 amino acids or about 17 amino acids. The accessory polypeptide, such as the CsgF peptide, is preferably elongated by from about 1 to about 10 amino acids. The accessory polypeptide, such as the CsgF peptide, is preferably elongated by about 1 amino acid, about 2 amino acids, about 3 amino acids, about 4 amino acids, about 5 amino acids, about 6 amino acids, about 7 amino acids, about 8 amino acids, about 9 amino acids, or about 10 amino acids.

[0282] The accessory polypeptide, such as the CsgF peptide, is preferably elongated by about 2 amino acids, about 4 amino acids, about 6 amino acids, about 8 amino acids, about 10 amino acids, about 12 amino acids, or about 14 amino acids.

[0283] The accessory polypeptide, such as the CsgF peptide, may be elongated using any amino acid(s). The amino(s) may be natural and / or non-natural amino acids. The amino acid(s) is / are preferably selected from tyrosine (Y), arginine (R), lysine (K), phenylalanine (F), tryptophan (W), threonine (T), valine (V), alanine (A), isoleucine (I), leucine (L), glycine (G), glutamic acid (E), aspartic acid (D), histidine (H), methionine (M), cysteine (C), proline (P), serine (S), asparagine (N) and glutamine (Q). The amino acid(s) is / are preferably selected from tyrosine (Y), threonine (T), valine (V), isoleucine (I), leucine (L), glycine (G), serine (S), glutamic acid (E), lysine (K), arginine (R), asparagine (N), glutamine (Q), aspartic acid (D) and alanine (A). The amino acid(s) may replicate existing stretches of amino acid(s) in the accessory polypeptide. The amino acid(s) may differ from those already in the accessory polypeptide. The accessory polypeptide may be elongated using any of the specific sequences listed above. The pore monomer and accessory polypeptide may be elongated using the same sequences, including any of the specific sequences listed above.

[0284] Elongation of the accessory polypeptide, such as the CsgF peptide, may improve the function of the pore complex formed from the pore monomer conjugate. Elongation of the accessory polypeptide, such as the CsgF peptide, may increase the range and / or decrease the noise when characterising an analyte using the pore complex. A decreased noise can result in an increased signal-to-noise ratio (SNR).

[0285] Attachment

[0286] In the pore monomer conjugate, the pore monomer is preferably attached to the accessory polypeptide. The pore monomer and the accessory polypeptide may be attached in any manner. Suitable attachment chemistries and linkers are described with reference to CsgG and CsgF in WO 2024 / 033421, WO 2024 / 033422, and WO 2024 / 033443 (all incorporated by reference herein in their entirety). Any of these may be used. The pore monomer and the accessory polypeptide are preferably attached by a linker or two or more linkers. SEQ ID NO: 3 shows the amino acid sequence of wild-type E. coli CsgG as a mature protein. The CsgF peptide is preferably covalently attached to a cysteine residue in the CsgG pore monomer at a position corresponding to position 153 or 133 in SEQ ID NO: 3 by the linker. The linker preferably covalently attaches the CsgF peptide to a cysteine residue in the CsgG pore monomer at a position corresponding to position 153 or 133 in SEQ ID NO: 3. The cysteine residue at either of these positions has been introduced into the pore monomer, for instance by substitution, i.e., Q153C or N133C, or addition. The cysteine residue at either of these positions may be introduced into the pore monomer by addition. If a maleimide- containing linker or a linker comprising a thiol reactive group is used, the maleimide group or thiol reactive group in the linker is capable of covalently coupling with the cysteine residue. The CsgF peptide is preferably covalently attached to a cysteine residue in the CsgG pore monomer at the position corresponding to position 153 in SEQ ID NO: 3 by the linker. The linker preferably covalently attaches the CsgF peptide to a cysteine residue in the CsgG pore monomer at the position corresponding to position 153 in SEQ ID NO: 3. The CsgF peptide is preferably covalently attached to a cysteine residue in the CsgG pore monomer at the position corresponding to position 133 in SEQ ID NO: 3 by the linker. The linker preferably covalently attaches the CsgF peptide to a cysteine residue in the CsgG pore monomer at the position corresponding to position 133 in SEQ ID NO: 3. As explained in more detail below, the CsgG pore monomer may be a modified or mutated version of SEQ ID NO: 3, including truncated versions or fragments. The skilled person is capable of determining the position corresponding to position 153 or 133 in SEQ ID NO: 3 using routine sequence alignments, including those discussed below.

[0287] SEQ ID NO: 6 shows the amino acid sequence of wild-type E. coli CsgF as a mature protein. The N terminus of the CsgF peptide or the residue at the position in the CsgF peptide corresponding to position 4 of SEQ ID NO: 6 is preferably covalently attached to the CsgG pore monomer by the linker. The linker preferably covalently attaches the N terminus of the CsgF peptide or the residue at the position in the CsgF peptide corresponding to position 4 of SEQ ID NO: 6 to the CsgG pore monomer. The N terminus of the CsgF peptide is preferably covalently attached to the CsgG pore monomer by the linker. The linker preferably covalently attaches the N terminus of the CsgF peptide to the CsgG pore monomer. The linker may be attached to the N terminus of the CsgF peptide by an amide bond formed between the amine group on the N terminal amino acid and a carboxyl group at one end of the linker. The residue at the position in the CsgF peptide corresponding to position 4 of SEQ ID NO: 6 is preferably covalently attached to the CsgG pore monomer by the linker. The linker preferably covalently attaches the residue at the position in the CsgF peptide corresponding to position 4 of SEQ ID NO: 6 to the CsgG pore monomer. As explained in more detail below, the CsgF peptide may be a modified or mutated version of SEQ ID NO: 6, including truncated versions or fragments. The skilled person is capable of determining the position corresponding to position 4 in SEQ ID NO: 3 using routine sequence alignments, including those discussed below.

[0288] The N terminus of the CsgF peptide is preferably covalently attached to a cysteine residue in the CsgG pore monomer at the position corresponding to position 153 in SEQ ID NO: 3 by the linker. The linker preferably covalently attaches the N terminus of the CsgF peptide to a cysteine residue in the CsgG pore monomer at the position corresponding to position 153 in SEQ ID NO: 3. The position in the CsgF peptide corresponding to position 4 of SEQ ID NO: 6 is preferably covalently attached to a cysteine residue in the CsgG pore monomer at the position corresponding to position 133 in SEQ ID NO: 3 by the linker. The linker preferably covalently attaches the position in the CsgF peptide corresponding to position 4 of SEQ ID NO: 6 to a cysteine residue in the CsgG pore monomer at the position corresponding to position 133 in SEQ ID NO: 3. The position in the CsgF peptide corresponding to position 4 of SEQ ID NO: 6 is preferably covalently attached to a cysteine residue in the CsgG pore monomer at the position corresponding to position 153 in SEQ ID NO: 3 by the linker. The linker preferably covalently attaches the position in the CsgF peptide corresponding to position 4 of SEQ ID NO: 6 to a cysteine residue in the CsgG pore monomer at the position corresponding to position 153 in SEQ ID NO: 3. The N terminus of the CsgF peptide is preferably covalently attached to a cysteine residue in the CsgG pore monomer at the position corresponding to position 133 in SEQ ID NO: 3 by the linker. The linker preferably covalently attaches the N terminus of the CsgF peptide to a cysteine residue in the CsgG pore monomer at the position corresponding to position 133 in SEQ ID NO: 3.

[0289] The CsgF peptide may be attached to the CsgG pore monomer at two or more positions, such as 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more or 10 or more positions. The CsgF peptide may be covalently attached to the CsgG pore monomer at two or more positions, such as 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more or 10 or more positions.

[0290] In the context of the invention, attachment at two or more positions mean two or more pairs of residues in the CsgF peptide and CsgG pore monomer are attached, preferably covalently attached, to each another. For instance, residue 1 in the CsgF peptide may be attached to residue 153 in the CsgG pore monomer (one pair of residues = 1 and 153) and residue 30 in the CsgF peptide may be attached to residue 196 in the CsgG pore monomer (a second pair of residues = 30 and 196).

[0291] The two or more positions in the CsgG pore monomer are preferably selected from residues corresponding to positions 47-54, 57, 59, 60, 130-134, 136, 137, 138, 140, 142-145, 147, 149, 151, 153, 155, 181, 183, 185, 187, 189, 191, 193, 195-199, 201, 203, 205, 207, 209 and 211-212 in SEQ ID NO: 3. The two or more positions in the CsgF peptide are preferably selected from the N terminus and residues corresponding to positions 1-35 in SEQ ID NO: 6. The N terminus is the amino group of the first residue in the CsgF peptide (i.e., residue 1). In this context, residue 1 refers to the side chain of residue 1 or the residue corresponding to position 1 in SEQ ID NO: 6. The two or more positions are preferably the following positions / residues in the CsgF peptide or the positions / residues in the CsgF peptide which correspond to the following positions in SEQ ID NO: 6: the N terminus and any one of 1-35, 1 and any of the N terminus and 2-35, 2 and any of the N terminus, 1 and 3-35, 3 and any of the N terminus, 1-2 and 4-35, 4 and any of the N terminus, 1-3 and 5-35, 5 and any of the N terminus, 1-4 and 6-35, 6 and any of the N terminus, 1-5 and 7-35, 7 and any of the N terminus, 1-6 and 8-35, 8 and any of the N terminus, 1-7 and 9-35, 9 and any of the N terminus, 1-8 and 10-35, 10 and any of the N terminus, 1-9 and 11-35, 11 and any of the N terminus, 1-10 and 12-35, 12 and any of the N terminus, 1-11 and 13-35, 13 and any of the N terminus, 1-12 and 14-35, 14 and any of the N terminus, 1-13 and 15-35, 15 and any of the N terminus, 1-14 and 16-35, 16 and any of the N terminus, 1-15 and 17-35, 17 and any of the N terminus, 1-16 and 18-35, 18 and any of the N terminus, 1-17 and 19-35, 19 and any of the N terminus, 1-18 and 20-35, 20 and any of the N terminus, 1-19 and 21-35, 21 and any of the N terminus, 1-20 and 22-35, 22 and any of the N terminus, 1-21 and 23- 35, 23 and any of the N terminus, 1-22 and 24-35, 24 and any of the N terminus, 1-23 and 25-35, 25 and any of the N terminus, 1-24 and 26-35, 26 and any of the N terminus, 1-25 and 27-35, 27 and any of the N terminus, 1-26 and 28-35, 28 and any one of the N terminus, 1-27 and 29-35, 29 and any of the N terminus, 1-28 and 30-35, 30 and any of the N terminus, 1-29 and 31-35, 31 and any of the N terminus, 1-30 and 32-35, 32 and any of the N terminus, 1-31 and 33-35, 33 and any of the N terminus, 1-32 and 34-35, 34 and any of the N terminus, 1-33 and 35, and 35 and any of the N terminus and 1-34.

[0292] Preferred combinations of two or more positions are described in WO 2024 / 033422 (incorporated herein by reference in its entirety). One of the two or more attachments preferably comprises the N terminus of the CsgF peptide attached, preferably covalently attached, to a cysteine residue in the CsgG pore monomer corresponding to position 153 in SEQ ID NO: 3. Corresponding positions may be determined as described above.

[0293] The attachment preferably comprises one or more reactive groups which react with lysine, cysteine, tyrosine, serine, threonine, proline, tryptophan, arginine, histidine, methionine, or phenylalanine in the pore monomer. The attachment preferably comprises a reaction between a position, residue, or linker in the accessory polypeptide with lysine, cysteine, tyrosine, serine, threonine, proline, tryptophan, arginine, histidine, methionine, or phenylalanine in the pore monomer.

[0294] The lysine, cysteine, tyrosine, serine, threonine, proline, tryptophan, arginine, histidine, methionine, or phenylalanine may be native to the pore monomer. The lysine, cysteine, tyrosine, serine, threonine, proline, tryptophan, arginine, histidine, methionine, or phenylalanine may be introduced into the pore monomer, preferably by substitution or addition.

[0295] Reactive groups which react with lysine include, but are not limited to, maleimide, activated esters, anhydrides, carbonates, isocyanates, isothiocyanates, a range of other acylating and alkylating agents, oxidative coupling O-aminophenols, aldehydes, activated carbodiimides, ketenes, sulfonyl halides, fluorosulfates, and sulfonyl triazoles. Positions, residues, or linkers may also be attached to lysine using periodate oxidation, reductive amination, transamination, aniline / arylamine conjugation via oxidative coupling, azaelectrocyclization, iminoboronate formation, or conjugation of arene diazonium salts.

[0296] Reactive groups which react with cysteine include, but are not limited to, haloacetamides and other alpha-halocarbonyls, maleimides, acrylates, vinyl sulfones, vinylpyridines, epoxides, oxanorbornadienes, methylsulfonyl functioanlised heteroaromatic, allenes, allyl selenosulfate salts, perfluoroaromatic, thiol-ene and thiol-yne click chemistry, pyridyl dithiol, vinylsulfones, sulfonyl halides, fluorosulfates, and sulfonyl triazoles. Positions, residues, or linkers may also be attached to cysteine using strain-release alkylation, nickel(II)-catalyzed oxidative coupling, oxidative coupling with aminophenols, conjugation with allenes (in the presence of gold catalyst, or allyl selenosulfate salts), native chemical ligation, Pd-catalysed arylation / alkynylation, or allylation followed by cross-metathesis.

[0297] Reactive groups which react with tyrosine include, but are not limited to, sulfonyl halides, fluorosulfates, and sulfonyl triazoles. Positions, residues, or linkers may also be attached to tyrosine using oxidative conjugation of tyrosines including O-alkylation, hydrazone and oxime condensations, addition reactions with electron deficient alkynes such as alkynones, alkynoate, amide or esters, cyclic diazodicarboxamides, Pd catalysed alkylation, diazonium salts, or Mannich reaction with imines formed from aldehydes, cyclic diazodicarboxamides, modification with Rhodium carbenoids.

[0298] Reactive groups which react with serine or threonine include, but are not limited to, sulfonyl halides, fluorosulfates, and sulfonyl triazoles. Positions, residues, or linkers may also be attached to serine or threonine using periodate oxidation and subsequent transimination reactions of ketones / aldehydes with hydrazides / alkoxyamines. Resultant aldehydes / ketones may also modified through aldol ligation.

[0299] Positions, residues, or linkers may be attached to proline using oxidative coupling with O- aminophenols at N-terminus.

[0300] Reactive groups which react with tryptophan include, but are not limited to, aldehydes, ketones, and tetrazoles. Positions, residues, or linkers may be attached to tryptophan using a condensation reaction, modification with Rhodium carbenoids, conjugation with N / O centred radicals, and N-terminal Trp modification using Pictet-Spengler reaction.

[0301] Positions, residues, or linkers may be attached to arginine using condensation with a, [3- dicarbonyl compounds.

[0302] Reactive groups which react with histidine include, but are not limited to, vinylsulfones, sulfonyl halides, fluorosulfates, and sulfonyl triazoles. Positions, residues, or linkers may be attached to hisitidine using C2 alkylation and N3 alkylation / thiophosphorylation.

[0303] Positions, residues, or linkers may be attached to methionine using S-alkylation / imidation.

[0304] Positions, residues, or linkers may be attached to phenylalanine using modification with Rhodium carbenoids.

[0305] The attachment preferably comprises one or more reactive groups which react with any amino acid in the CsgG pore monomer. Reactive groups which react with any amino acid include, but are not limited to, activated esters, anhydrides, carbonates, isocyanates, isothiocyanates, and a range of other acylating and alkylating agents, oxidative coupling O- aminophenols, aldehydes, activated carbodiimides, ketenes, transamination, and vinylboronic acids.

[0306] The attachment preferably comprises reacting a position, residue, or linker in the CsgF peptide with any amino acid in the CsgG pore monomer. Positions, residues, or linkers may be attached to any amino acid using periodate oxidation, or reductive amination.

[0307] The attachment preferably comprises one or more reactive groups which undergo click chemistry. Suitable click chemistries include, but are not limited to, CuAAC Azide / alkyne, staudinger ligation, strain-promoted azide-alkyne cycloaddition, inverse-electron demand Diels-Alder reaction between 1,2,4,5-tetrazines and strained alkenes.

[0308] All of the discussion above with reference to reactive groups and reactions for attaching the accessory polypeptide to residues / amino acids in the pore monomer equally applies to attaching the pore monomer to the accessory polypeptide. Any of the reactive groups or reactions may be used for attachment in the accessory polypeptide. Specific residues in the accessory polypeptide may be native to the protein. Specific residues may also be introduced into the accessory polypeptide, preferably by substitution or addition. The skilled person is capable of attaching, preferably covalently attaching, two proteins.

[0309] The attachment at two or more positions preferably comprises two or more versions of the same or similar reactive groups, such as maleimide. The attachment at two or more positions preferably comprises two or more versions of the same or similar reaction. The attachment at two or more positions preferably comprises two or more maleimide- containing linkers. The attachment at two or more positions preferably comprises two or more maleimide reactions. Any of the maleimide groups and linkers discussed above may be used.

[0310] The attachment at two or more positions preferably comprises two or more different reactive groups. The attachment at two or more positions preferably comprises two or more different reactions. The two or more reactive groups or reactions may be any of those discussed above in relation to the accessory polypeptide and / or the pore monomer.

[0311] The accessory polypeptide is preferably attached to the CsgG pore monomer using one or more linkers. The accessory polypeptide is preferably attached to the pore monomer using two or more linkers, such as 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more or 10 or more linkers. The two or more linkers may be the same. The two or more linkers may be different. The skilled person is capable of designing two or more linkers for use in the invention. The one or more linkers may be any of the linkers discussed below with reference to the conjugates of the invention.

[0312] The one or more linkers preferably comprise or consist of a linear carbon chain of 2, 3, 4, 5, 6 or more carbon atoms and / or cyclic groups containing 3, 5 or 6 carbon atoms. The linker is preferably a maleimide-containing linker. One or more of, such as all of the, two or more linkers are preferably a maleimide-containing linker. The maleimide group may be used to react with cysteine in the accessory polypeptide and / or the pore monomer. The maleimide- containing linker preferably comprises or consists of a maleimide group and a linear carbon chain of 2, 3, 4, 5, 6 or more carbon atoms. The linear carbon chain is typically attached to the nitrogen atom in the maleimide group. The linear carbon chain also preferably comprises a terminal carboxyl group. This carboxyl group is capable of forming an amide bond with an amino acid in the accessory polypeptide. The maleimide-containing linker is preferably maleimidoacetic acid, maleimidopropionic acid, maleimidobutyric acid, maleimidopentanoic acid or maleimidohexanonic acid. Any combination of these linkers may be used in the two or more linkers. The maleimide-containing linker is most preferably maleimidopropionic acid.

[0313] The distance between the pore monomer and the accessory polypeptide in the conjugate and / or the length of the linker is preferably less than about 2.00 nm, such as less than about 1.90 nm, less than about 1.80 nm, less than about 1.70 nm, less than about 1.60 nm, less than about 1.50 nm, less than about 1.40 nm, less than about 1.30 nm, less than about 1.20 nm, less than about 1.10 nm, less than about 1.00 nm, less than about 0.90 nm, less than about 0.80 nm, less than about 0.70 nm, less than about 0.60 nm, less than about 0.50 nm, or less than about 0.40 nm. The distance between the pore monomer and the accessory polypeptide in the conjugate and / or the length of the linker is preferably less than about 1.20 nm. The distance between the pore monomer and the accessory polypeptide in the conjugate and / or the length of the linker is preferably less than about 0.8 nm.

[0314] The distance between the pore monomer and the accessory polypeptide in the conjugate and / or the length of the linker is preferably from about 0.40 nm to about 2.0 nm, such as about 0.45 nm to about 1.90 nm, from about 0.50 nm to about 1.80 nm, from about 0.55 nm to about 1.7 nm, from about 0.60 nm to about 1.6 nm, from about 0.65 nm to about 1.5 nm, from about 0.7 nm to about 1.4 nm, from about 0.75 nm to about 1.3 nm, from about 0.80 nm to about 1.2 nm, from about 0.85 nm to about 1.1 nm and from about 0.90 nm to about 1.00 nm. The distance between the pore monomer and the accessory polypeptide in the conjugate and / or the length of the linker is preferably from about 0.50 nm to about 1.50 nm. The distance between the pore monomer and the accessory polypeptide in the conjugate and / or the length of the linker is preferably from about 0.60 nm to about 1.2 nm. This distance / length can be achieved using any of specific maleimide- containing linkers discussed above, including maleimidoacetic acid, maleimidopropionic acid, maleimidobutyric acid, maleimidopentanoic acid or maleimidohexanonic acid. The linker is most preferably maleimidopropionic acid.

[0315] Constructs

[0316] The invention also provides a construct comprising two or more covalently attached pore monomers of the invention or two or more pore monomer conjugates of the invention. The invention also provides a construct comprising two or more covalently attached pore monomers of the invention. The invention also provides a construct comprising two or more pore monomer conjugates of the invention.

[0317] The construct may comprise 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more or 10 or more pore monomers of the invention or pore monomer conjugates of the invention. The construct may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 pore monomers of the invention or pore monomer conjugates of the invention.

[0318] The two or more pore monomers of the invention or two or more pore monomer conjugates of the invention may be the same or different. The two or more pore monomers or two or more pore monomer conjugates may differ based on one or more of (a) the sequence of the pore monomer, (b) the accessory polypeptide, (c) any linker, (d) the attachment position on the pore monomer, and (e) the attachment position on the accessory polypeptide. The pore monomers or pore monomer conjugates may differ based on (a); (b); (c); (d); (e); (a) and (b); (a) and (c); (a) and (d); (a) and (e); (b) and (c); (b) and (d); (b) and (e); (c) and (d); (c) and (e); (d) and (e); (a), (b) and (c); (a), (b) and (d); (a), (b) and (e); (a), (c) and (d); (a), (c) and (e); (a), (d) and (e); (b), (c) and (d); (b), (c) and (e); (b), (d) and (e); (c), (d) and (e); (a), (b), (c) and (d); (a), (b), (c) and (e); (a), (b), (d) and (e); (a), (c), (d) and (e); (b), (c), (d) and (e); and (a), (b), (c), (d) and (e). The two or more pore monomers or two or more pore monomer conjugates are preferably the same (i.e., identical).

[0319] The construct preferably comprises two pore monomers or two pore monomer conjugates. The two pore monomers or two pore monomer conjugates may be the same or different. The two pore monomers or two pore monomer conjugates are preferably the same (i.e., identical).

[0320] The pore monomers or pore monomer conjugates may be genetically fused, optionally via a linker, or chemically fused, for instance via a chemical crosslinker. Methods for covalently attaching monomers are disclosed in WO 2017 / 149316, WO 2017 / 149317, and WO 2017 / 149318 (incorporated herein by reference in their entirety). The pore monomers or pore monomer conjugates may be genetically fused.

[0321] The linker is preferably an amino acid sequence and / or a chemical crosslinker. Suitable amino acid linkers, such as peptide linkers, are known in the art. The length, flexibility and hydrophilicity of the amino acid or peptide linker are typically designed such that the CsgF peptide forms a constriction or alters at least one property of a constriction in the pore complex of the invention. Preferred flexible peptide linkers are stretches of 2 to 20, such as 4, 6, 8, 10 or 16, serine and / or glycine amino acids. More preferred flexible linkers include (SG)i, (SG)2, (SG)3, (SG)4, (SG)5, (SG)8, (SG)IO, (SG)i5or (SG)2o wherein S is serine and G is glycine. Preferred rigid linkers are stretches of 2 to 30, such as 4, 6, 8, 16 or 24, proline amino acids. More preferred rigid linkers include (P)i2wherein P is proline.

[0322] Suitable chemical crosslinkers are well-known in the art. Suitable chemical crosslinkers include, but are not limited to, those including the following functional groups: maleimide, active esters, succinimide, azide, alkyne (such as dibenzocyclooctynol (DIBO or DBCO), difluoro cycloalkynes and linear alkynes), phosphine (such as those used in traceless and non-traceless Staudinger ligations), haloacetyl (such as iodoacetamide), phosgene type reagents, sulfonyl chloride reagents, isothiocyanates, acyl halides, hydrazines, disulfides, vinyl sulfones, aziridines and photoreactive reagents (such as aryl azides, diaziridines).

[0323] Reactions between amino acids and functional groups may be spontaneous, such as cysteine / maleimide, or may require external reagents, such as Cu(I) for linking azide and linear alkynes.

[0324] Linkers can comprise any molecule that stretches across the distance required. Linkers can vary in length from one carbon (phosgene-type linkers) to many Angstroms. Examples of linker molecules, include but are not limited to, are polyethyleneglycols (PEGs), polypeptides, polysaccharides, deoxyribonucleic acid (DNA), peptide nucleic acid (PNA), threose nucleic acid (TNA), glycerol nucleic acid (GNA), saturated and unsaturated hydrocarbons, polyamides. These linkers may be inert or reactive, in particular they may be chemically cleavable at a defined position, or may be themselves modified with a fluorophore or ligand. The linker is preferably resistant to reducing agents, such as dithiothreitol (DTT), following the covalent attachment of the CsgF peptide to the CsgG pore monomer.

[0325] Preferred crosslinkers include 2,5-dioxopyrrolidin-l-yl 3-(pyridin-2-yldisulfanyl)propanoate, 2,5-dioxopyrrolidin-l-yl 4-(pyridin-2-yldisulfanyl)butanoate and 2,5-dioxopyrrolidin-l-yl 8- (pyridin-2-yldisulfanyl)octananoate, di-maleimide PEG Ik, di-maleimide PEG 3.4k, di- maleimide PEG 5k, di-maleimide PEG 10k, bis(maleimido)ethane (BMOE), bis- maleimidohexane (BMH), 1,4-bis-maleimidobutane (BMB), 1,4 bis-maleimidyl-2,3- di hydroxybutane (BMDB), BM[PEO]2 (1,8-bis-maleimidodiethyleneglycol), BM[PEO]3 (1,11- bis-maleimidotriethylene glycol), tris[2-maleimidoethyl]amine (TMEA), DTME dithiobismaleimidoethane, bis-maleimide PEG3, bis-maleimide PEGU, DBCO-maleimide, DBCO-PEG4-maleimide, DBCO-PEG4-NH2, DBCO-PEG4-NHS, DBCO-NHS, DBCO-PEG-DBCO 2.8kDa, DBCO-PEG-DBCO 4.0kDa, DBCO-15 atoms-DBCO, DBCO-26 atoms-DBCO, DBCO- 35 atoms-DBCO, DBCO-PEG4-S-S-PEG3-biotin, DBCO-S-S-PEG3-biotin, DBCO-S-S-PEG11- biotin, (succinimidyl 3-(2-pyridyldithio)propionate (SPDP) and maleimide-PEG(2kDa)- maleimide (ALPHA, OMEGA-BIS-MALEIMIDO POLYETHYLENE GLYCOL)). The most preferred crosslinker is maleimide-propyl-SRDFWRS-(l,2-diaminoethane)-propyl-maleimide.

[0326] The linker is preferably resistant to dithiothreitol (DTT). Suitable linkers include, but are not limited to, iodoacetamide-based and maleimide-based linkers.

[0327] The two or more conjugates may be connected using two or more linkers each comprising a hybridizable region and a group capable of forming a covalent bond. The hybridizable regions in the linkers hybridize and link the pore monomer and accessory polypeptide. The linked pore monomer and accessory polypeptide are then coupled via the formation of covalent bonds between the groups. Any of the specific linkers disclosed in WO 2010 / 086602 (incorporated herein by reference in its entirety) may be used in accordance with the invention.

[0328] The linkers may be labelled. Suitable labels include, but are not limited to, fluorescent molecules (such as Cy3 or AlexaFluor®555), radioisotopes, e.g.125I,35S,32P, enzymes, antibodies, antigens, polynucleotides, and ligands such as biotin. Such labels allow the amount of linker to be quantified. The label could also be a cleavable purification tag, such as biotin, or a specific sequence to show up in an identification method, such as a peptide that is not present in the protein itself, but that is released by trypsin digestion.

[0329] A preferred method of connecting the two or more conjugates is via cysteine linkage. This can be mediated by a bi-functional chemical crosslinker or by an amino acid linker with a terminal presented cysteine residue.

[0330] Another preferred method of attachment via 4-azidophenylalanine or Faz linkage. This can be mediated by a bi-functional chemical linker or by a polypeptide linker with a terminal presented 4-azidophenylalanine or Faz residue. Additional suitable linkers are discussed in more detail below.

[0331] Pores and pore complexes of the invention

[0332] The invention provides a pore comprising at least one pore monomer of the invention. The pore monomer of the invention may be any of those described above.

[0333] The invention also provides an oligomeric pore formed from pore monomers surrounding a channel, wherein the channel comprises a first constriction and a second constriction, and wherein each pore monomer is modified to increase the distance between the first constriction and the second constriction. Any of the embodiments discussed above with reference to the pore monomers of the invention, especially in relation to distances between constrictions, equally apply to this embodiment. Any embodiments above relating to a first constriction region and a second constriction region apply to the first constriction and second constriction of the oligomeric pore of the invention.

[0334] Pores are defined above. The pore or oligomeric pore is preferably a homooligomer comprising from about 6 to about 24 pore monomers of the invention. The pore or oligomeric pore is preferably a homooligomer comprising about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23 or about 24 pore monomers of the invention. The pore monomers are typically identical. The pore or oligomeric pore preferably comprises 8 or 9 identical pore monomers of the invention. The pore monomers of the invention may be any of those discussed above.

[0335] The invention also provides a pore complex comprising at least one pore monomer conjugate of the invention. The pore monomer conjugate may be any of those described above.

[0336] The invention also provides an oligomeric pore complex formed from pore monomer conjugates surrounding a channel, wherein each pore monomer conjugate comprises a pore monomer and an accessory polypeptide, wherein the channel comprises a first constriction formed by the pore monomers and a second constriction formed by the accessory polypeptide, and wherein each pore monomer conjugate is modified to increase the distance between the first constriction and the second constriction. Any of the embodiments discussed above with reference to the pore monomer conjugates of the invention, especially in relation to distances between constrictions, equally apply to this embodiment. Any embodiments above relating to a first constriction region and a second constriction region apply to the first constriction and second constriction of the oligomeric pore complex of the invention.

[0337] The pore monomer part of the pore complex or oligomeric pore complex of the invention preferably has or comprises any of the structures and / or dimensions of the pores discussed above. The accessory polypeptide forms at least a second constriction in the pore complex or oligomeric pore complex. The accessory polypeptide is preferably inserted into the lumen of the pore complex.

[0338] Pore complexes and oligomeric pore complexes of the invention can improve the characterisation of analytes, such as polynucleotides, providing a more discriminating direct relationship between the observed current as the analyte moves through the pore. In particular, by having two stacked constrictions spaced at a defined distance, the pore complex or oligomeric pore complex may facilitate characterization of polynucleotides that contain at least one homopolymeric stretch, e.g., several consecutive copies of the same nucleotide that otherwise exceed the interaction length of a single constriction. Additionally, by having two stacked constrictions at a defined distance, small molecule analytes including organic or inorganic drugs and pollutants passing through the pore complex or oligomeric pore complex will consecutively pass the two constrictions. The chemical nature of either constriction can be independently modified, each giving unique interaction properties with the analyte, thus providing additional discriminating power during analyte detection.

[0339] The pore complex or oligomeric pore complex is preferably a homooligomer comprising from about 6 to about 24 pore monomer conjugates of the invention. The pore complex or oligomeric pore complex is preferably a homooligomer comprising about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23 or about 24 pore monomer conjugates of the invention. The pore monomer conjugates are typically identical. The pore complex or oligomeric pore complex preferably comprises 8 or 9 identical pore conjugates of the invention. The pore monomer conjugates of the invention may be any of those discussed above.

[0340] The invention also provides a pore complex comprising at least one construct of the invention. The pore complex typically comprises at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11 or about 12 constructs of the invention. The pore complex typically comprises sufficient pore monomers to form a pore complex. For instance, an octameric pore complex may comprise (a) four constructs each comprising two pore monomers or pore monomer conjugates, (b) two constructs each comprising four pore monomers or pore monomer conjugates, (c) one construct comprising two pore monomers or pore monomer conjugates and six pore monomers or pore monomer conjugates that do not form part of a construct, (d) three constructs and two pore monomers or pore monomer conjugates that do not form part of a construct, and (e) combinations thereof. Same and additional possibilities are provided for a nonameric pore for instance. Other combinations of constructs and pore monomers or pore monomer conjugates can be envisaged by the skilled person. One or more constructs of the invention may be used to form a pore complex for characterising, such as sequencing, polynucleotides. The pore preferably comprises about 4 constructs of the invention each of which comprises two pore monomers or pore monomer conjugates. The constructs are typically the same (i.e., identical).

[0341] The pore complex is preferably a homooligomer comprising from about 1 to about 12 constructs of the invention. The pore complex may comprise about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11 or about 12 constructs of the invention. The constructs are typically the same (i.e., identical). The pore complex preferably comprises 4 identical constructs of the invention each of which comprises two pore monomers or pore monomer conjugates. The constructs may be any of those discussed above.

[0342] The pore monomers or pore monomer conjugates in the pore, pore complex, oligomeric pore or oligomeric pore complex of the invention are preferably all approximately the same length or are the same length. The channels of the pore monomers or pore monomer conjugates in the pore, pore complex, oligomeric pore or oligomeric pore complex of the invention are preferably approximately the same length or are the same length. Length may be measured in number of amino acids and / or units of length.

[0343] The the pore, pore complex, oligomeric pore or oligomeric pore complex of the invention may be isolated, substantially isolated, purified or substantially purified. A pore, pore complex, oligomeric pore or oligomeric pore complex of the invention is isolated or purified if it is completely free of any other components, such as lipids or other pores. A pore, pore complex, oligomeric pore or oligomeric pore complex is substantially isolated if it is mixed with carriers or diluents which will not interfere with its intended use. For instance, a pore, pore complex, oligomeric pore or oligomeric pore complex is substantially isolated or substantially purified if it is present in a form that comprises less than 10%, less than 5%, less than 2% or less than 1% of other components, such as block copolymers, lipids, or other pores. Alternatively, a pore, pore complex, oligomeric pore or oligomeric pore complex of the invention may be present in a membrane. Suitable membranes are discussed below.

[0344] A pore, pore complex, oligomeric pore or oligomeric pore complex of the invention may be present as an individual or single pore, pore complex, oligomeric pore or oligomeric pore complex. Alternatively, a pore, pore complex, oligomeric pore or oligomeric pore complex of the invention may be present in a homologous or heterologous population of two or more pores, pore complexes, oligomeric pores or oligomeric pore complexes. Other formats involving the pores, pore complex, oligomeric pores or oligomeric pore complexes of the invention are discussed in more detail below.

[0345] The pore, pore complex, oligomeric pore or oligomeric pore complex preferably does not comprise two or more pores. The pore, pore complex, oligomeric pore or oligomeric pore complex is preferably not a double pore. The pore, pore complex, oligomeric pore or oligomeric pore complex is preferably not a double pore described in WO 2018 / 211241 (herein incorporated by reference in its entirety). In particular, the pore, pore complex, oligomeric pore or oligomeric pore complex preferably does not comprise a double pore comprising a first CsgG pore, or a homologue thereof, and a second CsgG pore, or a homologue thereof.

[0346] Membrane embodiments

[0347] The invention also provides a pore of the invention, a pore complex of the invention, a oligomeric pore of the invention or an oligomeric pore complex of the invention separately or in combination which is comprised in a membrane.

[0348] The invention also provides a membrane comprising...

Claims

1. CLAIMS1. A pore monomer capable of forming a pore comprising a channel, wherein the pore monomer comprises a first constriction region which forms part of a first constriction in the channel and a second constriction region which forms part of a second constriction in the channel, and wherein the pore monomer is modified to increase the distance between the first constriction region and the second constriction region.

2. A pore monomer conjugate capable of forming a pore complex comprising a channel, wherein the pore monomer conjugate comprises (1) a pore monomer comprising a first constriction region which forms part of a first constriction in the channel and (2) an accessory polypeptide comprising a second constriction region which forms part a second constriction in the channel, and wherein the pore monomer is modified to increase the distance between the first constriction region and the second constriction region.

3. A pore monomer conjugate according to claim 2, wherein the accessory polypeptide is further modified to increase the distance between the first constriction region and the second constriction region.

4. A pore monomer conjugate according to claim 2 or 3, wherein the accessory polypeptide is further modified to comprise at least a third constriction region which forms part of a third constriction in the channel.

5. A pore monomer according to claim 1 or a pore monomer conjugate according to any one of claims 2-4, wherein the channel comprises a beta-barrel and / or an alpha-helix.

6. A pore monomer or pore monomer conjugate according to claim 5, wherein one or more beta-strands and / or one or more alpha helices are modified to increase the distance between the first constriction region and the second constriction region.

7. A pore monomer or pore monomer conjugate according to claim 6, wherein the one or more beta-strands and / or one or more alpha helices are elongated.

8. A pore monomer or pore monomer conjugate according to claim 7, wherein the one or more beta-strands and / or one or more alpha helices are elongated by the addition of from about 1 to about 12 amino acids.

9. A pore monomer or pore monomer conjugate according to claim 8, wherein the amino acid(s) is / are selected from tyrosine (Y), arginine (R), lysine (K), phenylalanine (F), tryptophan (W), threonine (T), valine (V), alanine (A), isoleucine (I), leucine (L), glycine (G), glutamic acid (E), aspartic acid (D), histidine (H), methionine (M), cysteine (C), proline (P), serine (S), asparagine (N) and glutamine (Q).

10. A pore monomer or pore monomer conjugate according to any one of the preceding claims, wherein the distance between the first constriction region and the second constriction region is increased by about 5.5 or more angstroms and / or by the length of about 1 or more nucleotides.

11. A pore monomer or pore monomer conjugate according to any one of the preceding claims, wherein the pore monomer or the pore monomer conjugate is not modified to alter the length of the first constriction region and / or the length of the second constriction region.

12. A pore monomer or pore monomer conjugate according to any one of the preceding claims, wherein the pore monomer is from Wza, Iota toxin, Anthrax protective antigen, Vibrio cholerae cytolysin, Cytotoxin K (CytK), CELIII, CsgG, CsgF, CsgG-CsgF, Aerolysin, alpha hemolysin, MspA, MspB, MspC, PorARr, PorBRr, PorARc, PilQ, necrotic enteritis B- like toxin (NetB), FraC, portal proteins including G20c, P23_45, T4, SPP1, P22 and Phi29, gamma hemolysin, Monalysin, Lysenin, ClyA, an actinoporin, Clostridium perfringens beta toxin, parasporin-2, epsilon toxin, lectin from the parasitic mushroom Laetiporus sulphureus (LSL), volvatoxin, Cry toxins, CytlAa, Cyt2Aa, Complement component 9 (C9), Perfringolysin O, Pleurotolysin, Listeriolysin, Perforin-2, Gasdermin-A3, L-, P- and M-ring protein, Type II secretion system protein D, GspD, InvG, VirB7, SpoIIIAG, Cag8, Cag3, Cag or other proteins in the Type IV secretion system apparatus protein CagY, WzzB, Pentraxin, Afp2, Major vault protein, Thioredoxin-dependent peroxidase reductase, Arf-GAP, Respiratory syncytial virus ribonucleoprotein, Chikungunya virus nonstructural protein 1, PRC, YaxA, XaxA, HfaB, NfpAB, leukocidin or PrgH.

13. A pore monomer or pore monomer conjugate according to any one of the preceding claims, wherein the pore monomer is from CsgG.

14. A pore monomer conjugate according to any one of claims 2-13, wherein the accessory polypeptide is a CsgF peptide, CsgA peptide, CsgB peptide, CsgE peptide, or any peptide that binds to or is designed to bind to CsgG.

15. A construct comprising two or more covalently attached pore monomers or pore monomer conjugates according to any one of the preceding claims.

16. A pore comprising at least one pore monomer according to any one of claims 1 and 5-13.

17. A pore complex comprising at least one pore monomer conjugate according to any one of claims 2-14.

18. A pore complex comprising at least one construct according to claim 15.

19. An oligomeric pore formed from pore monomers surrounding a channel, wherein the channel comprises a first constriction and a second constriction, and wherein each pore monomer is modified to increase the distance between the first constriction and the second constriction.

20. An oligomeric pore complex formed from pore monomer conjugates surrounding a channel, wherein each pore monomer conjugate comprises a pore monomer and an accessory polypeptide, wherein the channel comprises a first constriction formed by the pore monomers and a second constriction formed by the accessory polypeptide, and wherein each pore monomer conjugate is modified to increase the distance between the first constriction and the second constriction.

21. A pore according to claim 16, a pore complex according to claim 17 or 18, an oligomeric pore according to claim 19 or an oligomeric pore complex according to claim 20, wherein the pore, pore complex, oligomeric pore or oligomeric pore complex is a homooligomer comprising 6 to 24 pore monomers or pore monomer conjugates.

22. A membrane comprising a pore according to claim 16, a pore complex according to claim 17 or 18, an oligomeric pore according to claim 19 or an oligomeric pore complex according to claim 20.

23. A method for determining the presence, absence or one or more characteristics of a target analyte, comprising the steps of: (i) contacting the target analyte with a pore according to claim 16, a pore complex according to claim 17 or 18, an oligomeric pore according to claim 19 or an oligomeric pore complex according to claim 20, such that the target analyte moves with respect to the pore, pore complex, oligomeric pore or oligomeric pore complex; and (ii) taking one or more measurements as the analyte moves with respect to the pore, pore complex, oligomeric pore or oligomeric pore complex and thereby determining the presence, absence or one or more characteristics of the analyte.

24. A method according to claim 23, wherein the target analyte is a polymer, an amino acid, a peptide, a polypeptide, a protein, a nucleotide, an oligonucleotide, a polynucleotide, a polynucleotide-polypeptide conjugate, a monosaccharide, an oligosaccharide, or a polysaccharide.

25. A method according to claim 23 or 24, wherein the target analyte comprises at least one homopolymeric region.

26. A method according to any one of claims 23-25, wherein the method comprises determining one or more characteristics selected from (i) the length of the targetanalyte, (ii) the identity of the target analyte, (iii) the sequence of the target analyte, (iv) the secondary structure of the target analyte and (v) whether or not the target analyte is modified.

27. Use of a pore according to claim 16, a pore complex according to claim 17 or 18, an oligomeric pore according to claim 19 or an oligomeric pore complex according to claim 20 to determine the presence, absence or one or more characteristics of a target analyte.

28. A polynucleotide which encodes a pore monomer or pore monomer conjugate according to any one of claims 1-14 or a construct according to claim 15.

29. A kit for characterising a target analyte comprising (a) a pore according to claim 16, a pore complex according to claim 17 or 18, an oligomeric pore according to claim 19 or an oligomeric pore complex according to claim 20 and (b) the components of a membrane.

30. A kit for characterising a target polynucleotide or a target polypeptide comprising (a) a pore according to claim 16, a pore complex according to claim 17 or 18, an oligomeric pore according to claim 19 or an oligomeric pore complex according to claim 20 and (b) a polynucleotide binding protein or a polypeptide handling enzyme.

31. An apparatus for characterising a target polynucleotide or a target polypeptide in a sample, comprising (a) a plurality of a pores according to claim 16, a plurality of pore complexes according to claim 17 or 18, a plurality of oligomeric pores according to claim 19 or a plurality of oligomeric pore complexes according to claim 20 and (b) a plurality of polynucleotide binding proteins or a plurality of polypeptide handling enzymes.

32. An array comprising a plurality of membranes according to claim 22.

33. A system comprising (a) a membrane according to claim 22 or an array according to claim 32, (b) means for applying a potential across the membrane(s) and (c) means for detecting electrical or optical signals across the membrane(s).

34. An apparatus comprising a pore according to claim 16, a pore complex according to claim 17 or 18, an oligomeric pore according to claim 19 or an oligomeric pore complex according to claim 20 inserted into an in vitro membrane.

35. An apparatus produced by a method comprising (i) obtaining a pore according to claim 16, a pore complex according to claim 17 or 18, an oligomeric pore according to claim 19 or an oligomeric pore complex according to claim 20 and (ii) contacting the pore, pore complex, oligomeric pore or oligomeric pore complex with an in vitromembrane such that the pore, pore complex, oligomeric pore or oligomeric pore complex is inserted in the in vitro membrane.

36. A method of improving the ability of an oligomeric pore formed from pore monomers surrounding a channel to characterise an analyte, wherein the channel comprises a first constriction and a second constriction, the method comprising modifying at least one of the pore monomers to increase the distance between the first constriction and the second constriction.