Patent Publication Number: US-2022213458-A1

Title: Mad nucleases

Description:
RELATED CASES 
     This application is a continuation of U.S. Ser. No. 17/463,498, filed 31 Aug. 2021, now allowed; which claims priority to U.S. Ser. No. 63/133,502, filed 4 Jan. 2021, entitled “MAD NUCLEASES”, which is incorporated herein in its entirety. 
     INCORPORATION BY REFERENCE 
     Submitted with the present application is an electronically filed sequence listing via EFS-Web as an ASCII formatted sequence listing, entitled “INSC083US2_SEQLIST_20220309”, created Mar. 9, 2022, and 359,000 bytes in size. The sequence listing is part of the specification filed Mar. 9, 2022 and is incorporated by reference in its entirety. 
    
    
     FIELD OF THE INVENTION 
     The present disclosure provides new RNA-guided nuclease systems and engineered nickases for making rational, direct edits to nucleic acids in live cells. 
     BACKGROUND OF THE INVENTION 
     In the following discussion certain articles and methods will be described for background and introductory purposes. Nothing contained herein is to be construed as an “admission” of prior art. Applicant expressly reserves the right to demonstrate, where appropriate, that the methods referenced herein do not constitute prior art under the applicable statutory provisions. 
     The ability to make precise, targeted changes to the genome of living cells has been a long-standing goal in biomedical research and development. Recently, various nucleases have been identified that allow manipulation of gene sequence: hence, gene function. These nucleases include nucleic acid-guided nucleases. The range of target sequences that nucleic acid-guided nucleases can recognize, however, is constrained by the need for a specific PAM to be located near the desired target sequence. PAMs are short nucleotide sequences recognized by a gRNA/nuclease complex where this complex directs editing of the target sequence. The precise PAM sequence and PAM length requirements for different nucleic acid-guided nucleases vary; however, PAMs typically are 2-7 base-pair sequences adjacent or in proximity to the target sequence and, depending on the nuclease, can be 5′ or 3′ to the target sequence. Engineering nucleic acid-guided nucleases or mining for new nucleic acid-guided nucleases may provide nucleases with altered PAM preferences and/or altered activity or fidelity; all changes that may increase the versatility of a nucleic acid-guided nuclease for certain editing tasks. 
     There is thus a need in the art of nucleic acid-guided nuclease gene editing for novel nucleases with varied PAM preferences, varied activity in cells from different organisms such as mammals and/or altered enzyme fidelity. The novel MAD nucleases described herein satisfy this need. 
     SUMMARY OF THE INVENTION 
     This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter. Other features, details, utilities, and advantages of the claimed subject matter will be apparent from the following written Detailed Description including those aspects illustrated in the accompanying drawings and defined in the appended claims. 
     The present disclosure provides Type II MAD nucleases (e.g., RNA-guided nucleases or RGNs) with varied PAM preferences, and/or varied activity in mammalian cells. 
     Thus, in one embodiment there are provided MAD nuclease systems that perform nucleic acid-guided nuclease editing including a MAD2015 system comprising SEQ ID Nos. 1 (MAD2015 nuclease), 2 (CRISPR RNA) and 3 (trans-activating crispr RNA); a MAD2016 system comprising SEQ ID Nos. 4 (MAD2016 nuclease), 5 (CRISPR RNA) and 6 (trans-activating crispr RNA); a MAD2017 system comprising SEQ ID Nos. 7 (MAD2017 nuclease), 8 (CRISPR RNA) and 9 (trans-activating crispr RNA); a MAD2019 system comprising SEQ ID Nos. 10 (MAD2019 nuclease), 11 (CRISPR RNA) and 12 (trans-activating crispr RNA); a MAD2020 system comprising SEQ ID Nos. 13 (MAD2020 nuclease), 14 (CRISPR RNA) and 15 (trans-activating crispr RNA); a MAD2021 system comprising SEQ ID Nos. 16 (MAD2021 nuclease), 17 (CRISPR RNA) and 18 (trans-activating crispr RNA); a MAD2022 system comprising SEQ ID Nos. 19 (MAD2022 nuclease), 20 (CRISPR RNA) and 21 (trans-activating crispr RNA); a MAD2023 system comprising SEQ ID Nos. 22 (MAD2023 nuclease), 23 (CRISPR RNA) and 24 (trans-activating crispr RNA); a MAD2024 system comprising SEQ ID Nos. 25 (MAD2024 nuclease), 26 (CRISPR RNA) and 27 (trans-activating crispr RNA); a MAD2025 system comprising SEQ ID Nos. 28 (MAD2025 nuclease), 29 (CRISPR RNA) and 30 (trans-activating crispr RNA); a MAD2026 system comprising SEQ ID Nos. 31 (MAD2026 nuclease), 32 (CRISPR RNA) and 33 (trans-activating crispr RNA); a MAD2027 system comprising SEQ ID Nos. 34 (MAD2034 nuclease), 35 (CRISPR RNA) and 36 (trans-activating crispr RNA); a MAD2028 system comprising SEQ ID Nos. 37 (MAD2028 nuclease), 38 (CRISPR RNA) and 39 (trans-activating crispr RNA); a MAD2029 system comprising SEQ ID Nos. 40 (MAD2029 nuclease), 41 (CRISPR RNA) and 42 (trans-activating crispr RNA); a MAD2030 system comprising SEQ ID Nos. 43 (MAD2030 nuclease), 44 (CRISPR RNA) and 45 (trans-activating crispr RNA); a MAD2031 system comprising SEQ ID Nos. 46 (MAD2031 nuclease), 47 (CRISPR RNA) and 48 (trans-activating crispr RNA); a MAD2032 system comprising SEQ ID Nos. 49 (MAD2032 nuclease), 50 (CRISPR RNA) and 51 (trans-activating crispr RNA); a MAD2033 system comprising SEQ ID Nos. 52 (MAD2033 nuclease), 53 (CRISPR RNA) and 54 (trans-activating crispr RNA); a MAD2034 system comprising SEQ ID Nos. 55 (MAD2034 nuclease), 56 (CRISPR RNA) and 57 (trans-activating crispr RNA); a MAD2035 system comprising SEQ ID Nos. 58 (MAD2035 nuclease), 59 (CRISPR RNA) and 60 (trans-activating crispr RNA); a MAD2036 system comprising SEQ ID Nos. 61 (MAD2036 nuclease), 62 (CRISPR RNA) and 63 (trans-activating crispr RNA); a MAD2037 system comprising SEQ ID Nos. 64 (MAD2031 nuclease), 65 (CRISPR RNA) and 66 (trans-activating crispr RNA); a MAD2038 system comprising SEQ ID Nos. 67 (MAD2038 nuclease), 68 (CRISPR RNA) and 69 (trans-activating crispr RNA); a MAD2039 system comprising SEQ ID Nos. 70 (MAD2039 nuclease), 71 (CRISPR RNA) and 72 (trans-activating crispr RNA); and a MAD2040 system comprising SEQ ID Nos. 73 (MAD2040 nuclease), 74 (CRISPR RNA) and 75 (trans-activating crispr RNA). In some aspects, the MAD system components are delivered as sequences to be transcribed (in the case of the gRNA components) and transcribed and translated (in the case of the MAD nuclease), and in some aspects, the coding sequence for the MAD nuclease and the gRNA component sequences are on the same vector. In other aspects, the coding sequence for the MAD nuclease and the gRNA component sequences are on a different vector and in some aspects, the gRNA component sequences are located in an editing cassette which also comprises a donor DNA (e.g., homology arm). In other aspects, the MAD nuclease is delivered to the cells as a peptide or the MAD nuclease and gRNA components are delivered to the cells as a ribonuclease complex. 
     Additionally there is provided engineered nickases derived from the nucleases from the above-referenced systems, including MAD2016-H851A (SEQ ID NO: 178); MAD2016-N874A (SEQ ID NO: 179); MAD2032-H590A (SEQ ID NO: 180); MAD2039-H587A (SEQ ID NO: 181); MAD2039-N610A (SEQ ID NO: 182). 
     These aspects and other features and advantages of the invention are described below in more detail. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         FIG. 1  is an exemplary workflow for creating and screening mined MAD nucleases or RGNs. 
         FIG. 2  is a simplified depiction of an in vitro test conducted on candidate enzymes. 
         FIG. 3  is a list of novel Type II MADzymes that have been identified. 
         FIG. 4  is a map of Type II MADzymes in cluster  59 . 
         FIG. 5  is a map of Type II MADzymes in cluster  55 ,  56 ,  57  and  58 . 
         FIG. 6  is a map of Type II MADzymes in cluster  141 . 
         FIG. 7  is a reproduction of a gel showing nicked plasmid formation with different MADzyme nickases compared to corresponding MADzyme nucleases. 
     
    
    
     It should be understood that the drawings are not necessarily to scale. 
     DETAILED DESCRIPTION 
     The description set forth below in connection with the appended drawings is intended to be a description of various, illustrative embodiments of the disclosed subject matter. Specific features and functionalities are described in connection with each illustrative embodiment; however, it will be apparent to those skilled in the art that the disclosed embodiments may be practiced without each of those specific features and functionalities. Moreover, all of the functionalities described in connection with one embodiment are intended to be applicable to the additional embodiments described herein except where expressly stated or where the feature or function is incompatible with the additional embodiments. For example, where a given feature or function is expressly described in connection with one embodiment but not expressly mentioned in connection with an alternative embodiment, it should be understood that the feature or function may be deployed, utilized, or implemented in connection with the alternative embodiment unless the feature or function is incompatible with the alternative embodiment. 
     The practice of the techniques described herein may employ, unless otherwise indicated, conventional techniques and descriptions of organic chemistry, polymer technology, molecular biology (including recombinant techniques), cell biology, biochemistry, biological emulsion generation, and sequencing technology, which are within the skill of those who practice in the art. Such conventional techniques include polymer array synthesis, hybridization and ligation of polynucleotides, and detection of hybridization using a label. Specific illustrations of suitable techniques can be had by reference to the examples herein. However, other equivalent conventional procedures can, of course, also be used. Such conventional techniques and descriptions can be found in standard laboratory manuals such as Green, et al., Eds. (1999),  Genome Analysis: A Laboratory Manual Series  (Vols. I-IV); Weiner, Gabriel, Stephens, Eds. (2007),  Genetic Variation: A Laboratory Manual;  Dieffenbach, Dveksler, Eds. (2003),  PCR Primer:  A  Laboratory Manual;  Bowtell and Sambrook (2003),  DNA Microarrays: A Molecular Cloning Manual;  Mount (2004),  Bioinformatics: Sequence and Genome Analysis;  Sambrook and Russell (2006),  Condensed Protocols from Molecular Cloning: A Laboratory Manual;  and Sambrook and Russell (2002),  Molecular Cloning: A Laboratory Manual  (all from Cold Spring Harbor Laboratory Press); Stryer, L. (1995)  Biochemistry  (4th Ed.) W.H. Freeman, New York N.Y.; Gait, “ Oligonucleotide Synthesis: A Practical Approach”  1984, IRL Press, London; Nelson and Cox (2000),  Lehninger, Principles of Biochemistry  3 rd  Ed., W. H. Freeman Pub., New York, N.Y.; Berg et al. (2002)  Biochemistry,  5 th  Ed., W.H. Freeman Pub., New York, N.Y.;  Cell and Tissue Culture: Laboratory Procedures in Biotechnology  (Doyle &amp; Griffiths, eds., John Wiley &amp; Sons 1998), all of which are herein incorporated in their entirety by reference for all purposes. Nuclease-specific techniques can be found in, e.g.,  Genome Editing and Engineering From TALENs and CRISPRs to Molecular Surgery,  Appasani and Church, 2018; and  CRISPR: Methods and Protocols,  Lindgren and Charpentier, 2015; both of which are herein incorporated in their entirety by reference for all purposes. Basic methods for enzyme engineering may be found in,  Enzyme Engineering Methods and Protocols,  Samuelson, ed., 2013;  Protein Engineering,  Kaumaya, ed., (2012); and Kaur and Sharma, “ Directed Evolution: An Approach to Engineer Enzymes”,  Crit. Rev. Biotechnology, 26:165-69 (2006). 
     Note that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “an oligonucleotide” refers to one or more oligonucleotides. Terms such as “first,” “second,” “third,” etc., merely identify one of a number of portions, components, steps, operations, functions, and/or points of reference as disclosed herein, and likewise do not necessarily limit embodiments of the present disclosure to any particular configuration or orientation. 
     Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications mentioned herein are incorporated by reference for the purpose of describing and disclosing devices, methods and cell populations that may be used in connection with the presently described invention. 
     Where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention. 
     In the following description, numerous specific details are set forth to provide a more thorough understanding of the present invention. However, it will be apparent to one of ordinary skill in the art that the present invention may be practiced without one or more of these specific details. In other instances, well-known features and procedures well known to those skilled in the art have not been described in order to avoid obscuring the invention. 
     The term “complementary” as used herein refers to Watson-Crick base pairing between nucleotides and specifically refers to nucleotides hydrogen bonded to one another with thymine or uracil residues linked to adenine residues by two hydrogen bonds and cytosine and guanine residues linked by three hydrogen bonds. In general, a nucleic acid includes a nucleotide sequence described as having a “percent complementarity” or “percent homology” to a specified second nucleotide sequence. For example, a nucleotide sequence may have 80%, 90%, or 100% complementarity to a specified second nucleotide sequence, indicating that 8 of 10, 9 of 10 or 10 of 10 nucleotides of a sequence are complementary to the specified second nucleotide sequence. For instance, the nucleotide sequence 3′-TCGA-5′ is 100% complementary to the nucleotide sequence 5′-AGCT-3′; and the nucleotide sequence 3′-TCGA-5′ is 100% complementary to a region of the nucleotide sequence 5′-TAGCTG-3′. 
     The term DNA “control sequences” refers collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites, nuclear localization sequences, enhancers, and the like, which collectively provide for the replication, transcription and translation of a coding sequence in a recipient cell. Not all of these types of control sequences need to be present so long as a selected coding sequence is capable of being replicated, transcribed and—for some components—translated in an appropriate host cell. 
     As used herein the term “donor DNA” or “donor nucleic acid” refers to nucleic acid that is designed to introduce a DNA sequence modification (insertion, deletion, substitution) into a locus by homologous recombination using nucleic acid-guided nucleases. For homology-directed repair, the donor DNA must have sufficient homology to the regions flanking the “cut site” or site to be edited in the genomic target sequence. The length of the homology arm(s) will depend on, e.g., the type and size of the modification being made. In many instances and preferably, the donor DNA will have two regions of sequence homology (e.g., two homology arms) to the genomic target locus. Preferably, an “insert” region or “DNA sequence modification” region—the nucleic acid modification that one desires to be introduced into a genome target locus in a cell—will be located between two regions of homology. The DNA sequence modification may change one or more bases of the target genomic DNA sequence at one specific site or multiple specific sites. A change may include changing 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, or 500 or more base pairs of the target sequence. A deletion or insertion may be a deletion or insertion of 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 75, 100, 150, 200, 300, 400, or 500 or more base pairs of the target sequence. 
     The terms “guide nucleic acid” or “guide RNA” or “gRNA” refer to a polynucleotide comprising 1) a guide sequence capable of hybridizing to a genomic target locus, and 2) a scaffold sequence capable of interacting or complexing with a nucleic acid-guided nuclease. 
     “Homology” or “identity” or “similarity” refers to sequence similarity between two peptides or, more often in the context of the present disclosure, between two nucleic acid molecules. The term “homologous region” or “homology arm” refers to a region on the donor DNA with a certain degree of homology with the target genomic DNA sequence. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. 
     “Operably linked” refers to an arrangement of elements where the components so described are configured so as to perform their usual function. Thus, control sequences operably linked to a coding sequence are capable of effecting the transcription, and in some cases, the translation, of a coding sequence. The control sequences need not be contiguous with the coding sequence so long as they function to direct the expression of the coding sequence. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked” to the coding sequence. In fact, such sequences need not reside on the same contiguous DNA molecule (i.e. chromosome) and may still have interactions resulting in altered regulation. 
     A “promoter” or “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase and initiating transcription of a polynucleotide or polypeptide coding sequence such as messenger RNA, ribosomal RNA, small nuclear or nucleolar RNA, guide RNA, or any kind of RNA transcribed by any class of any RNA polymerase I, II or III. Promoters may be constitutive or inducible and, in some embodiments—particularly many embodiments in which selection is employed—the transcription of at least one component of the nucleic acid-guided nuclease editing system is under the control of an inducible promoter. 
     As used herein the term “selectable marker” refers to a gene introduced into a cell, which confers a trait suitable for artificial selection. General use selectable markers are well-known to those of ordinary skill in the art. Drug selectable markers such as ampicillin/carbenicillin, kanamycin, chloramphenicol, erythromycin, tetracycline, gentamicin, bleomycin, streptomycin, rhamnose, puromycin, hygromycin, blasticidin, and G418 may be employed. In other embodiments, selectable markers include, but are not limited to human nerve growth factor receptor (detected with a MAb, such as described in U.S. Pat. No. 6,365,373); truncated human growth factor receptor (detected with MAb); mutant human dihydrofolate reductase (DHFR; fluorescent MTX substrate available); secreted alkaline phosphatase (SEAP; fluorescent substrate available); human thymidylate synthase (TS; confers resistance to anti-cancer agent fluorodeoxyuridine); human glutathione S-transferase alpha (GSTA1; conjugates glutathione to the stem cell selective alkylator busulfan; chemoprotective selectable marker in CD34+cells); CD24 cell surface antigen in hematopoietic stem cells; human CAD gene to confer resistance to N-phosphonacetyl-L-aspartate (PALA); human multi-drug resistance-1 (MDR-1; P-glycoprotein surface protein selectable by increased drug resistance or enriched by FACS); human CD25 (IL-2α; detectable by Mab-FITC); Methylguanine-DNA methyltransferase (MGMT; selectable by carmustine); and Cytidine deaminase (CD; selectable by Ara-C). “Selective medium” as used herein refers to cell growth medium to which has been added a chemical compound or biological moiety that selects for or against selectable markers. 
     The terms “target genomic DNA sequence”, “target sequence”, or “genomic target locus” refer to any locus in vitro or in vivo, or in a nucleic acid (e.g., genome) of a cell or population of cells, in which a change of at least one nucleotide is desired using a nucleic acid-guided nuclease editing system. The target sequence can be a genomic locus or extrachromosomal locus. 
     A “vector” is any of a variety of nucleic acids that comprise a desired sequence or sequences to be delivered to and/or expressed in a cell. Vectors are typically composed of DNA, although RNA vectors are also available. Vectors include, but are not limited to, plasmids, fosmids, phagemids, virus genomes, synthetic chromosomes, and the like. As used herein, the phrase “engine vector” comprises a coding sequence for a nuclease to be used in the nucleic acid-guided nuclease systems and methods of the present disclosure. The engine vector may also comprise, in a bacterial system, the λ Red recombineering system or an equivalent thereto. Engine vectors also typically comprise a selectable marker. As used herein the phrase “editing vector” comprises a donor nucleic acid, optionally including an alteration to the target sequence that prevents nuclease binding at a PAM or spacer in the target sequence after editing has taken place, and a coding sequence for a gRNA. The editing vector may also comprise a selectable marker and/or a barcode. In some embodiments, the engine vector and editing vector may be combined; that is, the contents of the engine vector may be found on the editing vector. Further, the engine and editing vectors comprise control sequences operably linked to, e.g., the nuclease coding sequence, recombineering system coding sequences (if present), donor nucleic acid, guide nucleic acid, and selectable marker(s). 
     Editing in Nucleic Acid-Guided Nuclease Genome Systems 
     RNA-guided nucleases (RGNs) have rapidly become the foundational tools for genome engineering of prokaryotes and eukaryotes. Clustered Rapidly Interspaced Short Palindromic Repeats (CRISPR) systems are an adaptive immunity system which protect prokaryotes against mobile genetic elements (MGEs). RGNs are a major part of this defense system because they identify and destroy MGEs. RGNs can be repurposed for genome editing in various organisms by reprogramming the CRISPR RNA (crRNA) that guides the RGN to a specific target DNA. A number of different RGNs have been identified to date for various applications; however, there are various properties that make some RGNs more desirable than others for specific applications. RGNs can be used for creating specific double strand breaks (DSBs), specific nicks of one strand of DNA, or guide another moiety to a specific DNA sequence. 
     The ability of an RGN to specifically target any genomic sequence is perhaps the most desirable feature of RGNs; however, RGNs can only access their desired target if the target DNA also contains a short motif called PAM (protospacer adjacent motif) that is specific for every RGN. Type V RGNs such as MAD7, AsCas12a and LbCas12a tend to access DNA targets that contain YTTN/TTTN on the 5′ end whereas type II RGNs—such as the MADzymes disclosed herein—target DNA sequences containing a specific short motif on the 3′ end. An example well known in the art for a type II RGN is SpCas9 which requires an NGG on the 3′ end of the target DNA. Type II RGNs, unlike type V RGNS, require a transactivating RNA (tracrRNA) in addition to a crRNA for optimal function. Compared to type V RGNs, the type II RGNs create a double-strand break closer to the PAM sequence, which is highly desirable for precise genome editing applications. 
     A number of type II RGNs have been discovered so far; however, their use in widespread applications is limited by restrictive PAMs. For example, the PAM of SpCas9 occurs less frequently in AT-rich regions of the genome. New type II RGNs with new and less restrictive PAMs are beneficial for the field. Further, not all type II nucleases are active in multiple organisms. For example, a number of RGNs have been discussed in the scientific literature but only a few have been demonstrated to be active in vitro and fewer still are active in cells, particularly in mammalian cells. The present disclosure identifies multiple type II RGNs that have novel PAMs and are active in mammalian cells. 
     In performing nucleic acid-guided nuclease editing, the type II RGNs or MADzymes may be delivered to cells to be edited as a polypeptide; alternatively, a polynucleotide sequence encoding the MADzyme are transformed or transfected into the cells to be edited. The polynucleotide sequence encoding the MADzyme may be codon optimized for expression in particular cells, such as archaeal, prokaryotic or eukaryotic cells. Eukaryotic cells can be yeast, fungi, algae, plant, animal, or human cells. Eukaryotic cells may be those of or derived from a particular organism, such as a mammal, including but not limited to human, mouse, rat, rabbit, dog, or non-human mammals including non-human primates. The choice of the MADzyme to be employed depends on many factors, such as what type of edit is to be made in the target sequence and whether an appropriate PAM is located close to the desired target sequence. The MADzyme may be encoded by a DNA sequence on a vector (e.g., the engine vector) and be under the control of a constitutive or inducible promoter. In some embodiments, the sequence encoding the nuclease is under the control of an inducible promoter, and the inducible promoter may be separate from but the same as an inducible promoter controlling transcription of the guide nucleic acid; that is, a separate inducible promoter may drive the transcription of the nuclease and guide nucleic acid sequences but the two inducible promoters may be the same type of inducible promoter (e.g., both are pL promoters). Alternatively, the inducible promoter controlling expression of the nuclease may be different from the inducible promoter controlling transcription of the guide nucleic acid; that is, e.g., the nuclease may be under the control of the pBAD inducible promoter, and the guide nucleic acid may be under the control of the pL inducible promoter. 
     In general, a guide nucleic acid (e.g., gRNA) complexes with a compatible nucleic acid-guided nuclease and can then hybridize with a target sequence, thereby directing the nuclease to the target sequence. With the type II MADzymes described herein, the nucleic acid-guided nuclease editing system uses two separate guide nucleic acid components that combine and function as a guide nucleic acid; that is, a CRISPR RNA (crRNA) and a transactivating CRISPR RNA (tracrRNA). The gRNA may be encoded by a DNA sequence on a polynucleotide molecule such as a plasmid, linear construct, or the coding sequence may reside within an editing cassette and is under the control of a constitutive promoter, or, in some embodiments, an inducible promoter as described below. 
     A guide nucleic acid comprises a guide polynucleotide sequence having sufficient complementarity with a target sequence to hybridize with the target sequence and direct sequence-specific binding of a complexed nucleic acid-guided nuclease to the target sequence. The degree of complementarity between a guide sequence and the corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences. In some embodiments, a guide sequence is about or more than about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In some embodiments, a guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20 nucleotides in length. Preferably the guide sequence is 10-30 or 15-20 nucleotides long, or 15, 16, 17, 18, 19, or 20 nucleotides in length. 
     In the present methods and compositions, the components of the guide nucleic acid is provided as a sequence to be expressed from a plasmid or vector and comprises both the guide sequence and the scaffold sequence as a single transcript under the control of a promoter, and in some embodiments, an inducible promoter. In general, to generate an edit in a target sequence, the gRNA/nuclease complex binds to a target sequence as determined by the guide RNA, and the nuclease recognizes a protospacer adjacent motif PAM) sequence adjacent to the target sequence. The target sequence can be any polynucleotide endogenous or exogenous to a prokaryotic or eukaryotic cell, or in vitro. For example, the target sequence can be a polynucleotide residing in the nucleus of a eukaryotic cell. A target sequence can be a sequence encoding a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory polynucleotide, an intron, a PAM, or “junk” DNA). 
     The guide nucleic acid may be part of an editing cassette that encodes the donor nucleic acid. Alternatively, the guide nucleic acid may not be part of the editing cassette and instead may be encoded on the engine or editing vector backbone. For example, a sequence coding for a guide nucleic acid can be assembled or inserted into a vector backbone first, followed by insertion of the donor nucleic acid in, e.g., the editing cassette. In other cases, the donor nucleic acid in, e.g., an editing cassette can be inserted or assembled into a vector backbone first, followed by insertion of the sequence coding for the guide nucleic acid. In yet other cases, the sequence encoding the guide nucleic acid and the donor nucleic acid (inserted, for example, in an editing cassette) are simultaneously but separately inserted or assembled into a vector. In yet other embodiments, the sequence encoding the guide nucleic acid and the sequence encoding the donor nucleic acid are both included in the editing cassette. 
     The target sequence is associated with a PAM, which is a short nucleotide sequence recognized by the gRNA/nuclease complex. The precise PAM sequence and length requirements for different nucleic acid-guided nucleases vary; however, PAMs typically are 2-7 base-pair sequences adjacent or in proximity to the target sequence and, depending on the nuclease, can be 5′ or 3′ to the target sequence. Engineering of the PAM-interacting domain of a nucleic acid-guided nuclease may allow for alteration of PAM specificity, improve fidelity, or decrease fidelity. In certain embodiments, the genome editing of a target sequence both introduces a desired DNA change to a target sequence, e.g., the genomic DNA of a cell, and removes, mutates, or renders inactive a proto-spacer mutation (PAM) region in the target sequence. Rendering the PAM at the target sequence inactive precludes additional editing of the cell genome at that target sequence, e.g., upon subsequent exposure to a nucleic acid-guided nuclease complexed with a synthetic guide nucleic acid in later rounds of editing. Thus, cells having the desired target sequence edit and an altered PAM can be selected using a nucleic acid-guided nuclease complexed with a synthetic guide nucleic acid complementary to the target sequence. Cells that did not undergo the first editing event will be cut rendering a double-stranded DNA break, and thus will not continue to be viable. The cells containing the desired target sequence edit and PAM alteration will not be cut, as these edited cells no longer contain the necessary PAM site and will continue to grow and propagate. 
     As mentioned previously, the range of target sequences that nucleic acid-guided nucleases can recognize is constrained by the need for a specific PAM to be located near the desired target sequence. As a result, it often can be difficult to target edits with the precision that is necessary for genome editing. It has been found that nucleases can recognize some PAMs very well (e.g., canonical PAMs), and other PAMs less well or poorly (e.g., non-canonical PAMs). Because the mined MAD nucleases disclosed herein may recognize different PAMs, the mined MAD nucleases increase the number of target sequences that can be targeted for editing; that is, mined MAD nucleases decrease the regions of “PAM deserts” in the genome. Thus, the mined MAD nucleases expand the scope of target sequences that may be edited by increasing the number (variety) of PAM sequences recognized. Moreover, cocktails of mined MAD nucleases may be delivered to cells such that target sequences adjacent to several different PAMs may be edited in a single editing run. 
     Another component of the nucleic acid-guided nuclease system is the donor nucleic acid. In some embodiments, the donor nucleic acid is on the same polynucleotide (e.g., editing vector or editing cassette) as the guide nucleic acid and may be (but not necessarily) under the control of the same promoter as the guide nucleic acid (e.g., a single promoter driving the transcription of both the guide nucleic acid and the donor nucleic acid). For cassettes of this type, see U.S. Pat. Nos. 10,240,167; 10,266,849; 9,982,278; 10,351,877; 10,364,442; 10,435,715; and 10,465,207. The donor nucleic acid is designed to serve as a template for homologous recombination with a target sequence nicked or cleaved by the nucleic acid-guided nuclease as a part of the gRNA/nuclease complex. A donor nucleic acid polynucleotide may be of any suitable length, such as about or more than about 20, 25, 50, 75, 100, 150, 200, 500, or 1000 nucleotides in length. In certain preferred aspects, the donor nucleic acid can be provided as an oligonucleotide of between 20-300 nucleotides, more preferably between 50-250 nucleotides. The donor nucleic acid comprises a region that is complementary to a portion of the target sequence (e.g., a homology arm). When optimally aligned, the donor nucleic acid overlaps with (is complementary to) the target sequence by, e.g., about 20, 25, 30, 35, 40, 50, 60, 70, 80, 90 or more nucleotides. In many embodiments, the donor nucleic acid comprises two homology arms (regions complementary to the target sequence) flanking the mutation or difference between the donor nucleic acid and the target template. The donor nucleic acid comprises at least one mutation or alteration compared to the target sequence, such as an insertion, deletion, modification, or any combination thereof compared to the target sequence. 
     Often the donor nucleic acid is provided as an editing cassette, which is inserted into a vector backbone where the vector backbone may comprise a promoter driving transcription of the gRNA and the coding sequence of the gRNA, or the vector backbone may comprise a promoter driving the transcription of the gRNA but not the gRNA itself. Moreover, there may be more than one, e.g., two, three, four, or more guide nucleic acid/donor nucleic acid cassettes inserted into an engine vector, where each guide nucleic acid is under the control of separate different promoters, separate like promoters, or where all guide nucleic acid/donor nucleic acid pairs are under the control of a single promoter. In some embodiments the promoter driving transcription of the gRNA and the donor nucleic acid (or driving more than one gRNA/donor nucleic acid pair) is an inducible promoter. Inducible editing is advantageous in that isolated cells can be grown for several to many cell doublings to establish colonies before editing is initiated, which increases the likelihood that cells with edits will survive, as the double-strand cuts caused by active editing are largely toxic to the cells. This toxicity results both in cell death in the edited colonies, as well as a lag in growth for the edited cells that do survive but must repair and recover following editing. However, once the edited cells have a chance to recover, the size of the colonies of the edited cells will eventually catch up to the size of the colonies of unedited cells. See, e.g., U.S. Pat. Nos. 10,533,152; 10,550,363; 10,532,324; 10,550,363; 10,633,626; 10,633,627; 10,647,958; 10,760,043; 10,723,995; 10,801,008; and 10,851,339. Further, a guide nucleic acid may be efficacious directing the edit of more than one donor nucleic acid in an editing cassette; e.g., if the desired edits are close to one another in a target sequence. 
     In addition to the donor nucleic acid, an editing cassette may comprise one or more primer sites. The primer sites can be used to amplify the editing cassette by using oligonucleotide primers; for example, if the primer sites flank one or more of the other components of the editing cassette. 
     In addition, the editing cassette may comprise a barcode. A barcode is a unique DNA sequence that corresponds to the donor DNA sequence such that the barcode can identify the edit made to the corresponding target sequence. The barcode typically comprises four or more nucleotides. In some embodiments, the editing cassettes comprise a collection of donor nucleic acids representing, e.g., gene-wide or genome-wide libraries of donor nucleic acids. The library of editing cassettes is cloned into vector backbones where, e.g., each different donor nucleic acid is associated with a different barcode. 
     Additionally, in some embodiments, an expression vector or cassette encoding components of the nucleic acid-guided nuclease system further encodes one or more nuclear localization sequences (NLSs), such as about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs. In some embodiments, the nuclease comprises NLSs at or near the amino-terminus of the MADzyme, NLSs at or near the carboxy-terminus of the MADzyme, or a combination. 
     The engine and editing vectors comprise control sequences operably linked to the component sequences to be transcribed. As stated above, the promoters driving transcription of one or more components of the mined MAD nuclease editing system may be inducible, and an inducible system is likely employed if selection is to be performed. A number of gene regulation control systems have been developed for the controlled expression of genes in plant, microbe, and animal cells, including mammalian cells, including the pL promoter (induced by heat inactivation of the CI857 repressor), the pBAD promoter (induced by the addition of arabinose to the cell growth medium), and the rhamnose inducible promoter (induced by the addition of rhamnose to the cell growth medium). Other systems include the tetracycline-controlled transcriptional activation system (Tet-On/Tet-Off, Clontech, Inc. (Palo Alto, Calif.); Bujard and Gossen, PNAS, 89(12):5547-5551 (1992)), the Lac Switch Inducible system (Wyborski et al., Environ Mol Mutagen, 28(4):447-58 (1996); DuCoeur et al., Strategies 5(3):70-72 (1992); U.S. Pat. No. 4,833,080), the ecdysone-inducible gene expression system (No et al., PNAS, 93(8):3346-3351 (1996)), the cumate gene-switch system (Mullick et al., BMC Biotechnology, 6:43 (2006)), and the tamoxifen-inducible gene expression (Zhang et al., Nucleic Acids Research, 24:543-548 (1996)) as well as others. 
     Typically, performing genome editing in live cells entails transforming cells with the components necessary to perform nucleic acid-guided nuclease editing. For example, the cells may be transformed simultaneously with separate engine and editing vectors; the cells may already be expressing the mined MAD nuclease (e.g., the cells may have already been transformed with an engine vector or the coding sequence for the mined MAD nuclease may be stably integrated into the cellular genome) such that only the editing vector needs to be transformed into the cells; or the cells may be transformed with a single vector comprising all components required to perform nucleic acid-guided nuclease genome editing. 
     A variety of delivery systems can be used to introduce (e.g., transform or transfect) nucleic acid-guided nuclease editing system components into a host cell. These delivery systems include the use of yeast systems, lipofection systems, microinjection systems, biolistic systems, virosomes, liposomes, immunoliposomes, polycations, lipid:nucleic acid conjugates, virions, artificial virions, viral vectors, electroporation, cell permeable peptides, nanoparticles, nanowires, exosomes. Alternatively, molecular trojan horse liposomes may be used to deliver nucleic acid-guided nuclease components across the blood brain barrier. Of particular interest is the use of electroporation, particularly flow-through electroporation (either as a stand-alone instrument or as a module in an automated multi-module system) as described in, e.g., U.S. Pat. Nos. 10,435,713; 10,443,074; 10,323,258; and 10,415,058. 
     After the cells are transformed with the components necessary to perform nucleic acid-guided nuclease editing, the cells are cultured under conditions that promote editing. For example, if constitutive promoters are used to drive transcription of the mined MAD nucleases and/or gRNA, the transformed cells need only be cultured in a typical culture medium under typical conditions (e.g., temperature, CO 2  atmosphere, etc.) Alternatively, if editing is inducible—by, e.g., activating inducible promoters that control transcription of one or more of the components needed for nucleic acid-guided nuclease editing, such as, e.g., transcription of the gRNA, donor DNA, nuclease, or, in the case of bacteria, a recombineering system—the cells are subjected to inducing conditions. 
     EXAMPLES 
     The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent or imply that the experiments below are all of or the only experiments performed. It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific aspects without departing from the spirit or scope of the invention as broadly described. The present aspects are, therefore, to be considered in all respects as illustrative and not restrictive. 
     Example 1 
     Exemplary Workflow Overview 
     The disclosed MADzyme Type II CRISPR enzymes were identified by the method depicted in  FIG. 1 .  FIG. 1  shows an exemplary workflow for creating and for in vitro screening of MADzymes, including those in untapped clusters. In a first step, metagenome mining was performed to identify putative RGNs of interest based on, e.g., sequence (HMMER profile) and a search for CRISPR arrays. Once putative RGNs of interest were identified in silico, candidate pools were created and each MADzyme was identified by cluster, the tracrRNA was identified, and the sgRNA structure was predicted. Final candidates were identified, then the genes were synthesized. An in vitro depletion test was performed (see  FIG. 2 ), where a synthetic target library was constructed in which to test target depletion for each of the candidate MADzymes. After target depletion, amplicons were produced for analysis for in vivo analysis.  FIG. 2  depicts the in vitro depletion test in more detail. 
     Example 2 
     Metagenome Mining 
     The NCBI Metagenome database was used to search for novel, putative CRISPR nucleases using HMMER hidden Markov model searches. Hundreds of potential nucleases were identified. For each potential nuclease candidate, putative CRISPR arrays were identified and CRISPR repeat and anti-repeats were identified. Thirteen nucleases ( FIG. 3 ) were chosen for in vitro validation and 11 active MADzymes were identified and assigned to clusters. There was less than 40% sequence identity between clusters. Cluster  59  shown in  FIG. 4  presents two unique subclusters with distinct sgRNA architecture. Clusters  55 - 57  are shown in  FIG. 5 . These new MADzymes have diverse PAM preferences and distinct sgRNA structure. Cluster  141  ( FIG. 6 ) is a distant cluster from  55 ,  56 ,  57  and  59  and shows diverse Cas protein structure and smaller-sized enzymes (e.g., approximately 200 amino acids shorter than the counterparts from the  55 ,  56 ,  57  and  59  clusters). Table 1 lists the identified MADzymes, including amino acid sequences, origin, and nucleic acid sequences of the CRISPR RNA and the trans-activating crispr RNA. 
     
       
         
           
               
               
               
               
               
               
               
               
             
               
                 TABLE 1 
               
               
                   
               
               
                   
                   
                   
                 Organism 
                   
                   
                   
                   
               
               
                 MAD 
                 Clus- 
                   
                 (meta- 
                   
                   
                 CRISPR 
                   
               
               
                 name 
                 ter 
                 Contig_id 
                 genome) 
                 Source 
                 aa_seq 
                 repeat 
                 tracrRNA 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 MAD2015 
                 59 
                 DPZI01000013.1 
                 
                   Vagococcus 
                 
                   
                 MGKNYTIGLDIGTNSVGWSVVTENQQLVKKRMKIRGDS 
                 GTTTT 
                 TGTTGGT 
               
               
                   
                   
                   
                 sp. 
                   
                 EKKQVKKNFWGVRLFDEGETAEATRLKRTTRRRYTRRR 
                 AGAGC 
                 AGCATTC 
               
               
                   
                   
                   
                   
                   
                 NRVVDLQNIFKDEINQKDSNFFNRLNESFLVVEDKKQP 
                 TATGC 
                 AAAACAA 
               
               
                   
                   
                   
                   
                   
                 KQMIFGTVEEEASYHESFPTIYHLRKELVDNKDQADIR 
                 TGTTT 
                 CATAGCA 
               
               
                   
                   
                   
                   
                   
                 LVYLAMAHMIKYRGHFLIEGQLSTENTSVEEKFHLFLK 
                 TGAAT 
                 AGTTAAA 
               
               
                   
                   
                   
                   
                   
                 EYNSTFCKQEDGSLVNPVNEDINGEEILMGTLSRSKKA 
                 GCTTC 
                 ATAAGGC 
               
               
                   
                   
                   
                   
                   
                 EQIMKSFEGEKSNGVFSQFLKMIVGNQGNFKKAFNLEE 
                 CAAAA 
                 TTTGTCC 
               
               
                   
                   
                   
                   
                   
                 DAKIQFAKEEYDEDLTTLLSNIGDEYANVFSLAKETYE 
                 C 
                 GTTCTCA 
               
               
                   
                   
                   
                   
                   
                 AIELSGILSTKDKETYAKLSSSMTERYEDHEKDLASLK 
                 [SEQ 
                 ACTTTTA 
               
               
                   
                   
                   
                   
                   
                 SFFREHLPEKYAVMFKDVSKNGYAGYIENSNKISQEEF 
                 ID NO. 
                 GTGACGC 
               
               
                   
                   
                   
                   
                   
                 YKYTKKLIGQIEGADYFIKKMEQEAFLRKQRTYDNGVI 
                 2] 
                 TGTTTCG 
               
               
                   
                   
                   
                   
                   
                 PYQVHLSELTHIINNQKKYYPFLLEKEEEIKSILTFKI 
                   
                 GCG 
               
               
                   
                   
                   
                   
                   
                 PYYIGPLAKGNSDFAWLIRNSNDKITPSNFNEVLDIEN 
                   
                 [SEQ ID 
               
               
                   
                   
                   
                   
                   
                 SASQFIERMTNNDVYLPEEKVLPKNSMLYQKYIVFNEL 
                   
                 NO. 3] 
               
               
                   
                   
                   
                   
                   
                 TKVRYINDRGTECNFSGEEKLQIFERFFKDSSTKVKKV 
                   
                   
               
               
                   
                   
                   
                   
                   
                 SLENYLNKEYMIESPTIKGIEDDFNASFRTYHDFIKLG 
                   
                   
               
               
                   
                   
                   
                   
                   
                 VSREMLDDIDNEEMFEDIVKILTIFEDRQMIKKQLEKY 
                   
                   
               
               
                   
                   
                   
                   
                   
                 KDVFDSDILKKMVRRHYTGWGRLSKKLLHEMKDDNSGK 
                   
                   
               
               
                   
                   
                   
                   
                   
                 TILDYLIEDDRLPKHINRNFMQLINDSNLSFKEKIEKA 
                   
                   
               
               
                   
                   
                   
                   
                   
                 QLTDGTEDIDSVVKNLIGSPAIKKGISQSLKIVEELVS 
                   
                   
               
               
                   
                   
                   
                   
                   
                 IMGYQPTSIVVEMARENQTTSKGKRQSIQRYKRLEAAI 
                   
                   
               
               
                   
                   
                   
                   
                   
                 NELGSDLLKVCPTDNHALKDDRLYLYYLQNGRDMYTGL 
                   
                   
               
               
                   
                   
                   
                   
                   
                 ELDIHNLSQYDIDHIVPRSFITDNSIDNRVLVSSKKNR 
                   
                   
               
               
                   
                   
                   
                   
                   
                 GKLDNVPSKEIVQKNKLLWMNLKKSKLMSEKKYANLIK 
                   
                   
               
               
                   
                   
                   
                   
                   
                 GETGGLTEDDKAKFLNRQLVETRQITKNVAQILDQRFN 
                   
                   
               
               
                   
                   
                   
                   
                   
                 TQKDEKGNIIREVKVITLKSALVSQFRQNFEFYKVREV 
                   
                   
               
               
                   
                   
                   
                   
                   
                 NDFHHANDAYLNAVVANTLLKVYPKLTPDFVYGEYRKG 
                   
                   
               
               
                   
                   
                   
                   
                   
                 NPFKNTKATAKKHYYSNIMENLCHETTIIDDETGEILW 
                   
                   
               
               
                   
                   
                   
                   
                   
                 DKKCIGTIKQVLNYHQVNVVKKVETQTGRFSEETLVPR 
                   
                   
               
               
                   
                   
                   
                   
                   
                 GSTKNPIALKSHLDPQKYGGFKSPTIAYTIVIEYKKGK 
                   
                   
               
               
                   
                   
                   
                   
                   
                 KDILIKELLGISIMNRGAFEKNNKEYLEKLNYKEPRVL 
                   
                   
               
               
                   
                   
                   
                   
                   
                 MVLPKYSLFELENGRRRLLASDKESQKGNQMAVPSYLN 
                   
                   
               
               
                   
                   
                   
                   
                   
                 NLLYHTNKSLSKNAKSLEYVNEHRQQFEELLEEIIDFA 
                   
                   
               
               
                   
                   
                   
                   
                   
                 NQFTLAEKNTLLIADLYESNKEADIELLASSFINLLRF 
                   
                   
               
               
                   
                   
                   
                   
                   
                 NQMGAPAEFSFFEKPIPRKRYSSTFELLKGKVIHQSIT 
                   
                   
               
               
                   
                   
                   
                   
                   
                 GLYETHQKV [SEQ ID NO. 1] 
                   
                   
               
               
                   
               
               
                 MAD2016 
                 59 
                 DGLK01000042.1 
                 
                   Entero- 
                 
                 New 
                 MKKDYVIGLDIGTNSVGWAVMTEDYQLVKKKMPIYGNT 
                 GTTTT 
                 TCTTTTG 
               
               
                   
                   
                   
                 
                   coccus 
                 
                 York 
                 EKKKIKKNFWGVRLFEEGHTAEDRRLKRTARRIISRRR 
                 AGAGT 
                 GGACTAT 
               
               
                   
                   
                   
                 
                   faecalis 
                 
                 City 
                 NRLRYLQAFFEEAMTDLDENFFARLQESFLVPEDKKWH 
                 CATGT 
                 TCTAAAC 
               
               
                   
                   
                   
                   
                 MTA 
                 RHPIFAKLEDEVAYHETYPTIYHLRKKLADSSEQADLR 
                 TGTTT 
                 AACATAG 
               
               
                   
                   
                   
                   
                 subway 
                 LIYLALAHIVKYRGHFLIEGKLSTENISVKEQFQQFMI 
                 AGAAT 
                 CAAGTTA 
               
               
                   
                   
                   
                   
                   
                 IYNQTFVNGESRLVSAPLPESVLIEEELTEKASRTKKS 
                 GGTAC 
                 AAATAAG 
               
               
                   
                   
                   
                   
                   
                 EKVLQQFPQEKANGLFGQFLKLMVGNKADFKKVFGLEE 
                 CAAAA 
                 GTTTTAA 
               
               
                   
                   
                   
                   
                   
                 EAKITYASESYEEDLEGILAKVGDEYSDVFLAAKNVYD 
                 C 
                 CCGTAAT 
               
               
                   
                   
                   
                   
                   
                 AVELSTILADSDKKSHAKLSSSMIVRFTEHQEDLKKFK 
                 [SEQ 
                 CAACTGT 
               
               
                   
                   
                   
                   
                   
                 RFIRENCPDEYDNLFKNEQKDGYAGYIAHAGKVSQLKF 
                 ID NO. 
                 AAAGTGG 
               
               
                   
                   
                   
                   
                   
                 YQYVKKIIQDIAGAEYFLEKIAQENFLRKQRTFDNGVI 
                 5] 
                 CGCTGTT 
               
               
                   
                   
                   
                   
                   
                 PHQIHLAELQAIIHRQAAYYPFLKENQEKIEQLVTFRI 
                   
                 TCGGCGC 
               
               
                   
                   
                   
                   
                   
                 PYYVGPLSKGDASTFAWLKRQSEEPIRPWNLQETVDLD 
                   
                 [SEQ ID 
               
               
                   
                   
                   
                   
                   
                 QSATAFIERMTNFDTYLPSEKVLPKHSLLYEKFMVFNE 
                   
                 NO. 6] 
               
               
                   
                   
                   
                   
                   
                 LTKISYTDDRGIKANFSGKEKEKIFDYLFKTRRKVKKK 
                   
                   
               
               
                   
                   
                   
                   
                   
                 DIIQFYRNEYNTEIVTLSGLEEDQFNASFSTYQDLLKC 
                   
                   
               
               
                   
                   
                   
                   
                   
                 GLTRAELDHPDNAEKLEDIIKILTIFEDRQRIRTQLST 
                   
                   
               
               
                   
                   
                   
                   
                   
                 FKGQFSAEVLKKLERKHYTGWGRLSKKLINGIYDKESG 
                   
                   
               
               
                   
                   
                   
                   
                   
                 KTILGYLIKDDGVSKHYNRNFMQLINDSQLSFKNAIQK 
                   
                   
               
               
                   
                   
                   
                   
                   
                 AQSSEHEETLSETVNELAGSPAIKKGIYQSLKIVDELV 
                   
                   
               
               
                   
                   
                   
                   
                   
                 AIMGYAPKRIVVEMARENQTTSTGKRRSIQRLKIVEKA 
                   
                   
               
               
                   
                   
                   
                   
                   
                 MAEIGSNLLKEQPTTNEQLRDTRLFLYYMQNGKDMYTG 
                   
                   
               
               
                   
                   
                   
                   
                   
                 DELSLHRLSHYDIDHIIPQSFMKDDSLDNLVLVGSTEN 
                   
                   
               
               
                   
                   
                   
                   
                   
                 RGKSDDVPSKEVVKDMKAYWEKLYAAGLISQRKFQRLT 
                   
                   
               
               
                   
                   
                   
                   
                   
                 KGEQGGLTLEDKAHFIQRQLVETRQITKNVAGILDQRY 
                   
                   
               
               
                   
                   
                   
                   
                   
                 NANSKEKKVQIITLKASLTSQFRSIFGLYKVREVNDYH 
                   
                   
               
               
                   
                   
                   
                   
                   
                 HGQDAYLNCVVATTLLKVYPNLAPEFVYGEYPKFQTFK 
                   
                   
               
               
                   
                   
                   
                   
                   
                 ENKATAKAIIYTNLLRFFTEDEPRFTKDGEILWSNSYL 
                   
                   
               
               
                   
                   
                   
                   
                   
                 KTIKKELNYHQMNIVKKVEVQKGGFSKESIKPKGPSNK 
                   
                   
               
               
                   
                   
                   
                   
                   
                 LIPVKNGLDPQKYGGFDSPIVAYTVLFTHEKGKKPLIK 
                   
                   
               
               
                   
                   
                   
                   
                   
                 QEILGITIMEKTRFEQNPILFLEEKGFLRPRVLMKLPK 
                   
                   
               
               
                   
                   
                   
                   
                   
                 YTLYEFPEGRRRLLASAKEAQKGNQMVLPEHLLTLLYH 
                   
                   
               
               
                   
                   
                   
                   
                   
                 AKQCLLPNQSESLTYVEQHQPEFQEILERVVDFAEVHT 
                   
                   
               
               
                   
                   
                   
                   
                   
                 LAKSKVQQIVKLFEANQTADVKEIAASFIQLMQFNAMG 
                   
                   
               
               
                   
                   
                   
                   
                   
                 APSTFKFFQKDIERARYTSIKEIFDATIIYQSTTGLYE 
                   
                   
               
               
                   
                   
                   
                   
                   
                 TRRKVVD [SEQ ID NO. 4] 
                   
                   
               
               
                   
               
               
                 MAD2017 
                 59 
                 DMKA01000006.1 
                 
                   Strepto- 
                 
                   
                 MKKPYSIGLDIGTNSVGWAVITDDYKVPAKKMKVLGNT 
                 GTTTT 
                 TGTTGGA 
               
               
                   
                   
                   
                 
                   coccus 
                 
                   
                 DKKYIKKNLLGALLFDSGETAEVTRLKRTARRRYTRRK 
                 AGAGC 
                 ACTATTC 
               
               
                   
                   
                   
                 sp. 
                   
                 NRLRYLQEIFAKEMTKVDESFFQRLEESFLTDDDKTFD 
                 TGTGC 
                 GAAACAA 
               
               
                   
                   
                   
                 ( firmi-   
                   
                 SHPIFGNKAEEDAYHQKFPTIYHLRKYLADSQEKADLR 
                 TGTTT 
                 CACAGCG 
               
               
                   
                   
                   
                   cutes ) 
                   
                 LVYLALAHMIKYRGHFLIEGELNAENTDVQKLFNVFVE 
                 CGAAT 
                 AGTTAAA 
               
               
                   
                   
                   
                   
                   
                 TYDKIVDESHLSEIEVDASSILTEKVSKSRRLENLIKQ 
                 GGTTC 
                 ATAAGGC 
               
               
                   
                   
                   
                   
                   
                 YPTEKKNTLFGNLIALALGLQPNFKTNFKLSEDAKLQF 
                 CAAAA 
                 TTTGTCC 
               
               
                   
                   
                   
                   
                   
                 SKDTYEEDLEELLGKVGDDYADLFISAKNLYDAILLSG 
                 C 
                 GTACACA 
               
               
                   
                   
                   
                   
                   
                 ILTVDDNSTKAPLSASMIKRYVEHHEDLEKLKEFIKIN 
                 [SEQ 
                 ACTTGTA 
               
               
                   
                   
                   
                   
                   
                 KLKLYHDIFKDKTKNGYAGYIDNGVKQDEFYKYLKTIL 
                 ID NO. 
                 AAAGGGG 
               
               
                   
                   
                   
                   
                   
                 TKIDDSDYFLDKIERDDFLRKQRTFDNGSIPHQIHLQE 
                 8] 
                 CACCCGA 
               
               
                   
                   
                   
                   
                   
                 MHSILRRQGEYYPFLKENQAKIEKILTFRIPYYVGPLA 
                   
                 TTCGGGT 
               
               
                   
                   
                   
                   
                   
                 RKDSRFAWANYHSDEPITPWNFDEVVDKEKSAEKFITR 
                   
                 GCA 
               
               
                   
                   
                   
                   
                   
                 MTLNDLYLPEEKVLPKHSHVYETFTVYNELTKIKYVNE 
                   
                 [SEQ ID 
               
               
                   
                   
                   
                   
                   
                 QGESFFFDANMKQEIFDHVFKENRKVTKAKLLSYLNNE 
                   
                 NO. 9] 
               
               
                   
                   
                   
                   
                   
                 FEEFRINDLIGLDKDSKSFNASLGTYHDLKKILDKSFL 
                   
                   
               
               
                   
                   
                   
                   
                   
                 DDKTNEQIIEDIVLTLTLFEDRDMIHERLQKYSDFFTS 
                   
                   
               
               
                   
                   
                   
                   
                   
                 QQLKKLERRHYTGWGRLSYKLINGIRNKENNKTILDFL 
                   
                   
               
               
                   
                   
                   
                   
                   
                 IDDGHANRNFMQLINDESLSFKTIIQEAQVVGDVDDIE 
                   
                   
               
               
                   
                   
                   
                   
                   
                 AVVHDLPGSPAIKKGILQSVKIVDELVKVMGDNPDNIV 
                   
                   
               
               
                   
                   
                   
                   
                   
                 IEMARENQTTGYGRNKSNQRLKRLQDSLKEFGSDILSK 
                   
                   
               
               
                   
                   
                   
                   
                   
                 KKPSYVDSKVENSHLQNDRLFLYYIQNGKDMYTGEELD 
                   
                   
               
               
                   
                   
                   
                   
                   
                 IDRLSDYDIDHIIPQAFIKDNSIDNKVLTSSAKNRGKS 
                   
                   
               
               
                   
                   
                   
                   
                   
                 DDVPSIEIVRNRRSYWYKLYKSGLISKRKFDNLTKAER 
                   
                   
               
               
                   
                   
                   
                   
                   
                 GGLTEADKAGFIKRQLVETRQITKHVAQILDARFNTKR 
                   
                   
               
               
                   
                   
                   
                   
                   
                 DENDKVIRDVKVITLKSNLVSQFRKEFKFYKVREINDY 
                   
                   
               
               
                   
                   
                   
                   
                   
                 HHANDAYLNAVVGTALLKKYPKLTPEFVYGEYKKYDVR 
                   
                   
               
               
                   
                   
                   
                   
                   
                 KLIAKSSDDYSEMGKATAKYFFYSNLMNFFKTEVKYAD 
                   
                   
               
               
                   
                   
                   
                   
                   
                 GRVFERPDIETNADGEVVWNKQKDFDIVRKVLSYPQVN 
                   
                   
               
               
                   
                   
                   
                   
                   
                 IVKKVEAQTGGFSKESILSKGDSDKLIPRKTKKVYWNT 
                   
                   
               
               
                   
                   
                   
                   
                   
                 KKYGGFDSPTVAYSVLVVADIEKGKAKKLKTVKELVGI 
                   
                   
               
               
                   
                   
                   
                   
                   
                 SIMERSFFEENPVSFLEKKGYHNVQEDKLIKLPKYSLF 
                   
                   
               
               
                   
                   
                   
                   
                   
                 EFEGGRRRLLASATELQKGNEVMLPAHLVELLYHAHRI 
                   
                   
               
               
                   
                   
                   
                   
                   
                 DSFNSTEHLKYVSEHKKEFEKVLSCVENFSNLYVDVEK 
                   
                   
               
               
                   
                   
                   
                   
                   
                 NLSKVRAAAESMTNFSLEEISASFINLLTLTALGAPAD 
                   
                   
               
               
                   
                   
                   
                   
                   
                 FNFLGEKIPRKRYTSTKECLSATLIHQSVTGLYETRID 
                   
                   
               
               
                   
                   
                   
                   
                   
                 LSKLGEE [SEQ ID NO. 7] 
                   
                   
               
               
                   
               
               
                 MAD2019 
                 59 
                 DOTL01000042.1 
                 
                   Strepto- 
                 
                   
                 MTKPYSIGLDIGTNSVGWAVITDDYKVPSKKMKVLGNT 
                 GTTTT 
                 GGTTTGA 
               
               
                   
                   
                   
                 
                   coccus 
                 
                   
                 SKKYIKKNLLGALLFDSGITAEGRRLKRTARRRYTRRR 
                 AGAGC 
                 AACCATT 
               
               
                   
                   
                   
                 sp. 
                   
                 NRILYLQEIFSTEMATLDDAFFQRLDDSFLVPDDKRDS 
                 TGTGT 
                 CGAAACA 
               
               
                   
                   
                   
                 ( firmi-   
                   
                 KYPIFGNLVEEKAYHDEFPTIYHLRKYLADSTKKADLR 
                 TGTTT 
                 ATACAGC 
               
               
                   
                   
                   
                   cutes ) 
                   
                 LVYLALAHMIKYRGHFLIEGEFNSKNNDIQKNFQDFLD 
                 CGAAT 
                 AAAGTTA 
               
               
                   
                   
                   
                   
                   
                 TYNAIFESDLSLENSKQLEEIVKDKISKLEKKDRILKL 
                 GGTTC 
                 AAATAAG 
               
               
                   
                   
                   
                   
                   
                 FPGEKNSGIFSEFLKLIVGNQADFKKYFNLDEKASLHF 
                 CAAAA 
                 GCTAGTC 
               
               
                   
                   
                   
                   
                   
                 SKESYDEDLETLLGYIGDDYSDVFLKAKKLYDAILLSG 
                 C 
                 CGTATAC 
               
               
                   
                   
                   
                   
                   
                 ILTVTDNGTETPLSSAMIMRYKEHEEDLGLLKAYIRNI 
                 [SEQ 
                 AACGTGA 
               
               
                   
                   
                   
                   
                   
                 SLKTYNEVFNDDTKNGYAGYIDGKTNQEDFYVYLKKLL 
                 ID NO. 
                 AAACACG 
               
               
                   
                   
                   
                   
                   
                 AKFEGADYFLEKIDREDFLRKQRTFDNGSIPYQIHLQE 
                 11] 
                 TGGCACC 
               
               
                   
                   
                   
                   
                   
                 MRAILDKQAKFYPFLAKNKERIEKILTFRIPYYVGPLA 
                   
                 GATTCGG 
               
               
                   
                   
                   
                   
                   
                 RGNSDFAWSIRKRNEKITPWNFEDVIDKESSAEAFINR 
                   
                 TGC 
               
               
                   
                   
                   
                   
                   
                 MTSFDLYLPEEKVLPKHSLLYETFTVYNELTKVRFIAE 
                   
                 [SEQ ID 
               
               
                   
                   
                   
                   
                   
                 GMSDYQFLDSKQKKDIVRLYFKGKRKVKVTDKDIIEYL 
                   
                 NO. 12] 
               
               
                   
                   
                   
                   
                   
                 HAIDGYDGIELKGIEKQFNSSLSTYHDLLNIINDKEFL 
                   
                   
               
               
                   
                   
                   
                   
                   
                 DDSSNEAIIEEIIHTLTIFEDREMIKQRLSKFENIFDK 
                   
                   
               
               
                   
                   
                   
                   
                   
                 SVLKKLSRRHYTGWGKLSAKLINGIRDEKSGNTILDYL 
                   
                   
               
               
                   
                   
                   
                   
                   
                 IDDGISNRNFMQLIHDDALSFKKKIQKAQIIGDKDKDN 
                   
                   
               
               
                   
                   
                   
                   
                   
                 IKEVVKSLPGSPAIKKGILQSIKIVDELVKVMGRKPES 
                   
                   
               
               
                   
                   
                   
                   
                   
                 IVVEMARENQYTNQGKSNSQQRLKRLEESLEELGSKIL 
                   
                   
               
               
                   
                   
                   
                   
                   
                 KENIPAKLSKIDNNSLQNDRLYLYYLQNGKDMYTGDDL 
                   
                   
               
               
                   
                   
                   
                   
                   
                 DIDRLSNYDIDHIIPQAFLKDNSIDNKVLVSSASNRGK 
                   
                   
               
               
                   
                   
                   
                   
                   
                 SDDVPSLEVVKKRKTLWYQLLKSKLISQRKFDNLTKAE 
                   
                   
               
               
                   
                   
                   
                   
                   
                 RGGLSPEDKAGFIQRQLVETRQITKHVARLLDEKFNNK 
                   
                   
               
               
                   
                   
                   
                   
                   
                 KDENNRAVRTVKIITLKSTLVSQFRKDFELYKVREIND 
                   
                   
               
               
                   
                   
                   
                   
                   
                 FHHAHDAYLNAVVASALLKKYPKLEPEFVYGDYPKYNS 
                   
                   
               
               
                   
                   
                   
                   
                   
                 FRERKSATEKVYFYSNIMNIFKKSISLADGRVIERPLI 
                   
                   
               
               
                   
                   
                   
                   
                   
                 EVNEETGESVWNKESDLATVRRVLSYPQVNVVKKVEVQ 
                   
                   
               
               
                   
                   
                   
                   
                   
                 SGGFSKELVQPHGNSDKLIPRKTKKMIWDTKKYGGFDS 
                   
                   
               
               
                   
                   
                   
                   
                   
                 PIVAYSVLVMAEREKGKSKKLKPVKELVRITIMEKESF 
                   
                   
               
               
                   
                   
                   
                   
                   
                 KENTIDFLERRGLRNIQDENIILLPKFSLFELENGRRR 
                   
                   
               
               
                   
                   
                   
                   
                   
                 LLASAKELQKGNEFILPNKLVKLLYHAKNIHNTLEPEH 
                   
                   
               
               
                   
                   
                   
                   
                   
                 LEYVESHRADFGKILDVVSVFSEKYILAEAKLEKIKEI 
                   
                   
               
               
                   
                   
                   
                   
                   
                 YRKNMNTEIHEMATAFINLLTFTSIGAPATFKFFGHNI 
                   
                   
               
               
                   
                   
                   
                   
                   
                 ERKRYSSVAEILNATLIHQSVTGLYETRIDLGKLGED 
                   
                   
               
               
                   
                   
                   
                   
                   
                 [SEQ ID NO. 10] 
                   
                   
               
               
                   
               
               
                 MAD2020 
                 55 
                 DQFW01000027.1 
                 
                   Achole- 
                 
                 human 
                 MKNNEETLKKLRLGLDIGTNSVGYALLDENNKLIKKNG 
                 GTTTG 
                 TGTAAAT 
               
               
                   
                   
                   
                 
                   plasmatales 
                 
                 gut 
                 HTFWGVRMFDEAETAKDRGSYRKSRRRLLRRKERMEIL 
                 CTAGT 
                 AACATAA 
               
               
                   
                   
                   
                 bacterium 
                   
                 RSFFTKEICDIDPTFFERLDDSFYYKEDKKNKNTYNLF 
                 TATGT 
                 CGAGTGC 
               
               
                   
                   
                   
                   
                   
                 TSEYTDKDFYLEYPTIYHLRKAMQEEDKKFDIRMVYLA 
                 TATTT 
                 AAATAAG 
               
               
                   
                   
                   
                   
                   
                 IAHIIKYRGNFLYPGEEFSTSEYTSIKQFFLDFNDILD 
                 ATAGT 
                 CGTTTCG 
               
               
                   
                   
                   
                   
                   
                 ELSNELEDNEDYSAEYFDKIENINDDFLEKLKVILMEI 
                 ATTAA 
                 CGAAAAT 
               
               
                   
                   
                   
                   
                   
                 KGISNKKKELLDLFNVNKKSIYNELVIPFISGSAKVNI 
                 GCAAA 
                 TTACAGT 
               
               
                   
                   
                   
                   
                   
                 SSLSVIKNSKYPKTEISLGSEELEGQVEEAISVAPEIK 
                 C 
                 GGCCCTG 
               
               
                   
                   
                   
                   
                   
                 SVLEMIIKIKEISDFYFINKILSDSKTISESMVKMYDE 
                 [SEQ 
                 CTGTGGG 
               
               
                   
                   
                   
                   
                   
                 HNEDLKKLKGFFKKYAEDQYNEIFKIRDEKLANYVAYV 
                 ID NO. 
                 GCCTTTT 
               
               
                   
                   
                   
                   
                   
                 GFNKLRKNKVERFKHASREEFYGYLKQKLNNIKYAEAQ 
                 14] 
                 TTATTTA 
               
               
                   
                   
                   
                   
                   
                 EEIKYFIDKIDNNEFLLKQNSNQNGAFPMQLHLKELKT 
                   
                 TCAAA 
               
               
                   
                   
                   
                   
                   
                 ILNNQEKYYPFLSEGNDGYSIKEKIILTFKYKIPYYVG 
                   
                 [SEQ ID 
               
               
                   
                   
                   
                   
                   
                 PLNKESKYSWVVREDEKIYPWNFDKVVKLDETAEKFIL 
                   
                 NO. 15] 
               
               
                   
                   
                   
                   
                   
                 RMQNKCTYLKGDNDYCLPKNSLIFSEYSCLSYLNKLSI 
                   
                   
               
               
                   
                   
                   
                   
                   
                 NGKPIDPIMKSKIFNEVFLIKKQPTKKDIIEFIKTNYN 
                   
                   
               
               
                   
                   
                   
                   
                   
                 ADALTTTEKELPEATCNMASYIKMKEIFGKDFNDNKEM 
                   
                   
               
               
                   
                   
                   
                   
                   
                 IENIIKDITIFEDKSILGNRLKELYKLNNDRIKQIKGL 
                   
                   
               
               
                   
                   
                   
                   
                   
                 NYKGYSRLSKNLLVGLQIVDNQTGEIKGNVIEVMRKTN 
                   
                   
               
               
                   
                   
                   
                   
                   
                 LNLQEILYLDGYRLIDAIDEYNRKNSLNDSYLCARDYI 
                   
                   
               
               
                   
                   
                   
                   
                   
                 AENLVISPSFKRALIQTCSIIQEIERIFHKKIDEFYVE 
                   
                   
               
               
                   
                   
                   
                   
                   
                 VTRTNKDKNKGKTTSSRYDKIKKIYSSCQELAMAYNFD 
                   
                   
               
               
                   
                   
                   
                   
                   
                 MKRLKNELESNKDNLKSDILYFYFTQLGKCMYSLEDID 
                   
                   
               
               
                   
                   
                   
                   
                   
                 ISDLTNNYHYDIDHIYPQSIIKDDSLSNRVLVDKKKNA 
                   
                   
               
               
                   
                   
                   
                   
                   
                 AKTDKFLFEAKVLNPKAQQFYKKLLSLELISKEKYRRL 
                   
                   
               
               
                   
                   
                   
                   
                   
                 TQKEISKDELEGFVNRQLVSTNQSVMGLIKLLKEYYKV 
                   
                   
               
               
                   
                   
                   
                   
                   
                 DEKNIIYSKGENVSDFRHTFDLVKSRTANNFHHANDAY 
                   
                   
               
               
                   
                   
                   
                   
                   
                 LNVVVGGILNKYYTSRRFYQFSDIARIENEGESLNPSR 
                   
                   
               
               
                   
                   
                   
                   
                   
                 IFTKRDILKANGKVIWDKKEDIKRIEKDLYHRFDITET 
                   
                   
               
               
                   
                   
                   
                   
                   
                 IRTYNPNKMYSKVTILPKGEGESAVPFQTTTPRVDVEK 
                   
                   
               
               
                   
                   
                   
                   
                   
                 YGGITSNKFSRYVIIEAHGKKGLDTILEAIPKTACGDN 
                   
                   
               
               
                   
                   
                   
                   
                   
                 NKIEKDIDNYIASLDEYQKYTSYKVVNYNIKANVVIQE 
                   
                   
               
               
                   
                   
                   
                   
                   
                 GSFKYIITGKSGNQYVLQNVQDRFFSKKAMITIKNIDK 
                   
                   
               
               
                   
                   
                   
                   
                   
                 YLNNKKLGIIMAKDNEKIIVSPARGKNNEEIFFEKTEL 
                   
                   
               
               
                   
                   
                   
                   
                   
                 VNLLKEIKTMYSKDIYSFSAIQNIVNNIDCSIDYSIDD 
                   
                   
               
               
                   
                   
                   
                   
                   
                 FIIICNNLLQILKTNERKNADLRLIHLSGNSGTLYLGK 
                   
                   
               
               
                   
                   
                   
                   
                   
                 KLKSGMKFIWQSITGYYEEILYEVK [SEQ ID NO. 
                   
                   
               
               
                   
                   
                   
                   
                   
                 13] 
                   
                   
               
               
                   
               
               
                 MAD2021 
                 57 
                 DEED01000018.1 
                 
                   Lachno- 
                 
                   
                 MSEKYFVGLDMGTSSVGWAVTDEHYHLLRRKGKDLWGA 
                 GTTTG 
                 GATAATG 
               
               
                   
                   
                   
                 
                   spiraceae 
                 
                   
                 RLFDEAETAAGRRTNRVSRRRLARQRARIGWLKELFRP 
                 AGAGC 
                 TTTTACA 
               
               
                   
                   
                   
                 bacterium 
                   
                 YLEEKDAGFLQRLEESRFFLEDKTVKQPYALFSDKEFT 
                 CTTGT 
                 AGGCGAG 
               
               
                   
                   
                   
                   
                   
                 DKDYYQKYPTIFHLRKELLESKAPHDVRLVFLAVLNMY 
                 AAAAC 
                 TTCAAAT 
               
               
                   
                   
                   
                   
                   
                 AHRGHFLNPELQEGTLGDIHDLLSRLDAYIQDLFEDQG 
                 CGTAT 
                 AAGGATT 
               
               
                   
                   
                   
                   
                   
                 WSILENVEEQQKVLAEKNISNTVRLEKILSAIGTSPKD 
                 ATCTC 
                 TATCCGA 
               
               
                   
                   
                   
                   
                   
                 KEKKPLIEIYKLICGLKGSLSLAFSGVEMNETDAQMKF 
                 TCAAG 
                 AATCGCT 
               
               
                   
                   
                   
                   
                   
                 SFSDSNLEENEPEIERILGERYFEMYSILKEIHAWGLL 
                 C 
                 TGCGTGC 
               
               
                   
                   
                   
                   
                   
                 SEIMSDDSGKTYPYISYAKVDLYQKHHEQLRMLKKIIR 
                 [SEQ 
                 ATTGGCA 
               
               
                   
                   
                   
                   
                   
                 TYAPDEYHRMFRSMEDNTYSAYVGSVNSKNKKQRRGAK 
                 ID NO. 
                 CCATCTA 
               
               
                   
                   
                   
                   
                   
                 STDFFKEVKRIIEKIEKEHGELPECEEILDLIARDSFL 
                 17] 
                 TCTTTTA 
               
               
                   
                   
                   
                   
                   
                 PKQLTTANGVIPNQVYATELRQIVTNAAAYLPFLNDKD 
                   
                   
               
               
                   
                   
                   
                   
                   
                 DTGLTNAEKIVEMFKFHIPYYIGPLKNDGNGTAWVVRK 
                   
                 AGACTTT 
               
               
                   
                   
                   
                   
                   
                 QQGTVYPWNIDEKVDMAKTRDQFILNLVRKCSYLNDET 
                   
                 CTTTGAA 
               
               
                   
                   
                   
                   
                   
                 VLPASSLLYEKFKVLNELNNLTINGQKISVELKQDIFR 
                   
                 AGTCTT 
               
               
                   
                   
                   
                   
                   
                 DLFRATGKRVTTRKLMGYLRRKAVIDADADETCLEGFD 
                   
                 [SEQ ID 
               
               
                   
                   
                   
                   
                   
                 KTQGGFVSTLSSYHKFMEIFSTDVLTDRQREIAEGAIY 
                   
                 NO. 18] 
               
               
                   
                   
                   
                   
                   
                 FATVYGEDKSFLKKVLRDKFSPAELSQAQIDRLSGIRF 
                   
                   
               
               
                   
                   
                   
                   
                   
                 KDWSHLSREFLLLEEADHSTGEIMTIIDRLWNTNENLM 
                   
                   
               
               
                   
                   
                   
                   
                   
                 QIIHSDEYTYKQAIEERTARLEKSLSEVSFEDIEDSYM 
                   
                   
               
               
                   
                   
                   
                   
                   
                 SAPVRRMVWQTIRILQEIEEVMGSEPARVFVEMTRSEG 
                   
                   
               
               
                   
                   
                   
                   
                   
                 EKGDKGRKDSRKKKLKELYKKCKDDDQGLLSDIEGRDE 
                   
                   
               
               
                   
                   
                   
                   
                   
                 RDFRIRKLYLYYMQKGLCMYSGHPIDFGKLFDDSYYDI 
                   
                   
               
               
                   
                   
                   
                   
                   
                 DHIYPRHYVKDDSIENNLVLVESKLNRDKKDTLLCPDI 
                   
                   
               
               
                   
                   
                   
                   
                   
                 QERMHPVWEMLHRQGFMNDEKFKRLMRKEPFSEEEFAH 
                   
                   
               
               
                   
                   
                   
                   
                   
                 FIERQLVETGQGTKEIARILNDVLGNKDENNKVIYVKA 
                   
                   
               
               
                   
                   
                   
                   
                   
                 GNVSSFRNDNKKNPEFVKCRVINDHHHAKDAYLNIVVG 
                   
                   
               
               
                   
                   
                   
                   
                   
                 NTYYTKFTLHPANFIRELRNKSHPTLEDQYNMDKLFAR 
                   
                   
               
               
                   
                   
                   
                   
                   
                 RVERNGYTAWNPDTDFQTVKQVLRKNSVLISRRSFIEH 
                   
                   
               
               
                   
                   
                   
                   
                   
                 GQIADLQLVSGRKISEVNGKGYLPIKASDIRLSGPSGT 
                   
                   
               
               
                   
                   
                   
                   
                   
                 MKYGGYNKASGAYFFLVEHELKGKLVRTIEPVYVYMMA 
                   
                   
               
               
                   
                   
                   
                   
                   
                 SIHGKEDLEKYCQEELGYIHPRICLKKIPMYSHIRING 
                   
                   
               
               
                   
                   
                   
                   
                   
                 FDYYLTGRSNDRLFICNAVQLTLSSEWSAYIKALSKAV 
                   
                   
               
               
                   
                   
                   
                   
                   
                 DEKWDAAYIEQQASRIQDSLKSEEVFISKERNDQLYKV 
                   
                   
               
               
                   
                   
                   
                   
                   
                 LLQKHLEGFFNNRINSIGTIMKEGYDSFRALPVNEQAE 
                   
                   
               
               
                   
                   
                   
                   
                   
                 TLMEILKISQLVNIGANLVSIGGKSRSGVATVSKKISD 
                   
                   
               
               
                   
                   
                   
                   
                   
                 SKSFQLISDSVTGIFQRATDLLTI [SEQ ID NO. 
                   
                   
               
               
                   
                   
                   
                   
                   
                 16] 
                   
                   
               
               
                   
               
               
                 MAD2022 
                 57 
                 CACYWR010000004.1 
                 uncultured 
                 Cattle 
                 MEKEYYLGLDMGTSSVGWAVTDKEYRLLRAKGKDMWGI 
                 GTTTG 
                 GAGAATT 
               
               
                   
                   
                   
                 
                   Lachno- 
                 
                 rumen 
                 REFEEAQTAVERRTHRLSKRRRARQLVRIGLLKDYFHD 
                 AGAGT 
                 AACAAGA 
               
               
                   
                   
                   
                 
                   spiraceae 
                 
                   
                 EIMKIDPNFYIRLENSKYYLEDKDVRLASSNGIFDDKN 
                 CTTGT 
                 CGAGTGC 
               
               
                   
                   
                   
                 bacterium 
                   
                 YTDKDYYEQYKTIFHLRSELIHNSQKHDVRLVYLALLN 
                 TAATT 
                 AAATAAG 
               
               
                   
                   
                   
                   
                   
                 MFKHRGHFLFEGDAYVQGNIGDIYKEFIQLLKNEYYED 
                 CTTAA 
                 GTTTATC 
               
               
                   
                   
                   
                   
                   
                 ENVKLTDQIDYFKLKEILSNSEFSRTAKAEKINSLVHI 
                 AGGTG 
                 CGGAATC 
               
               
                   
                   
                   
                   
                   
                 DKKNKLENTYIRLLCGLEIELKILFPEIDEKIKICFAK 
                 TAAAA 
                 GTCAATA 
               
               
                   
                   
                   
                   
                   
                 GYDEKLVEITEILTDNQLQILENLKKIHDIAALDKIRK 
                 C 
                 TGACCTG 
               
               
                   
                   
                   
                   
                   
                 GKEYLSDARVAEYEKHREDLALLKKIYREYMTKQDYDR 
                 [SEQ 
                 CATTGTG 
               
               
                   
                   
                   
                   
                   
                 MFREGEDGSYSAYVNSYNTSKKQRRNMKHRKIDEFYGT 
                 ID NO. 
                 CAGAATC 
               
               
                   
                   
                   
                   
                   
                 IRKDLKLLLKQGIQDDNIERILEEIDGNNDNKFMPKQL 
                 20] 
                 TTTAAAA 
               
               
                   
                   
                   
                   
                   
                 SFANGVIPNSLHKAEMKAILRNAETYLPFLLETDESGL 
                   
                 TCATATG 
               
               
                   
                   
                   
                   
                   
                 TVSERILQLFSFHIPYYIGPVSVNSEKNNGNGWVVRRE 
                   
                 ATTTCAT 
               
               
                   
                   
                   
                   
                   
                 DGEVLPWNIEQKIDYGETSKRFIEKMVRRCTYISGEQV 
                   
                 ATGGTTT 
               
               
                   
                   
                   
                   
                   
                 LPKNSFIYEKYCVLNEINNIKIDGERITVELKQNIYND 
                   
                 TA 
               
               
                   
                   
                   
                   
                   
                 LYLHGKRVTKKQLINYLNNRGMIEDENQVSGIDINLNN 
                   
                 [SEQ ID 
               
               
                   
                   
                   
                   
                   
                 YLGSYGKFLPIFEEKLKEDNYIKIAEDIIYLASIYGDS 
                   
                 NO. 21] 
               
               
                   
                   
                   
                   
                   
                 KKMLKSQIKSKYGDILDDKQIKRILGLKFKDWGRISRR 
                   
                   
               
               
                   
                   
                   
                   
                   
                 FLELEGLDKETGEITTIIKAMWDYNLNFMEIIHSDAFD 
                   
                   
               
               
                   
                   
                   
                   
                   
                 FKDKIEELHANSIKPLAEIEVEDLDDMYFSAPVKRMIW 
                   
                   
               
               
                   
                   
                   
                   
                   
                 QTFKVIKEIEKVMGCPPKKVFIEMTRINDKKSKGKRTN 
                   
                   
               
               
                   
                   
                   
                   
                   
                 SRKEKFLSLYKNIHDELVDWKQLIISSDESGKLNSKKM 
                   
                   
               
               
                   
                   
                   
                   
                   
                 YLYLTQQGICMYTGRRINLEELFDDNKYDIDHIYPRHF 
                   
                   
               
               
                   
                   
                   
                   
                   
                 VKDDNLENNLVLVEKQSNSRKSDTYPIDKSIRNNSQVY 
                   
                   
               
               
                   
                   
                   
                   
                   
                 KHWKSLREGNFISKEKYDRLTGKNEFTDEQKAGFIARQ 
                   
                   
               
               
                   
                   
                   
                   
                   
                 MVETSQGTKGVADIIKQALPQSRIIYSKASNVSEFRRK 
                   
                   
               
               
                   
                   
                   
                   
                   
                 YDILKSRTVNEFHHANDAYLNIVVGNVYDTKFTSNPLN 
                   
                   
               
               
                   
                   
                   
                   
                   
                 FIKKQYNVDRKANNYNLDKMFVYDVKRGNEIAWIGWNP 
                   
                   
               
               
                   
                   
                   
                   
                   
                 KKSEDSSEMSKRGTIVTVKKMLSKNTPLMTRMSFVGHG 
                   
                   
               
               
                   
                   
                   
                   
                   
                 GIAEDNLSSHFVAKNKGYMPNGKESDVTKYGGYKKAKT 
                   
                   
               
               
                   
                   
                   
                   
                   
                 AYFFVVEHGQTNNRIRTIETLPIYRRREVEKYEDGLIK 
                   
                   
               
               
                   
                   
                   
                   
                   
                 YCEQSLSLLNPIIIYKKIKIQSLMKINGYYAYISGKSN 
                   
                   
               
               
                   
                   
                   
                   
                   
                 EVYTFRNGVNMCLSQEWINYVKKLENYIEKDRQDRMIT 
                   
                   
               
               
                   
                   
                   
                   
                   
                 YEKNIELYEIILRKYSTTILNKRLSKMDKKLINAKDRF 
                   
                   
               
               
                   
                   
                   
                   
                   
                 CILNVKEQSQVLINVFVLSRIGDNQTDLSKIGIGKQSG 
                   
                   
               
               
                   
                   
                   
                   
                   
                 QITQNKKITGCKEFKLVNQSVTGLYENEIDLLTV 
                   
                   
               
               
                   
                   
                   
                   
                   
                 [SEQ ID NO. 19] 
                   
                   
               
               
                   
               
               
                 MAD2023 
                 56 
                 DCGJ01000048.1 
                 
                   Lachno- 
                 
                 Feces 
                 MEKNNYLLGLDIGTDSVGYAVTNDKYDILKFHGEPAWG 
                 GTTTG 
                 AGACCCC 
               
               
                   
                   
                   
                 
                   clostridium 
                 
                 of six- 
                 VTIFDEASLSTEKRSFRVSRRRLDRRQQRVLLVQELFA 
                 AGAGT 
                 TATGGAT 
               
               
                   
                   
                   
                 sp. 
                 years 
                 SEVAKVDKDFFKRIQESNLYRSDAENQAGLFIGEDYCD 
                 AGTGT 
                 TTACATT 
               
               
                   
                   
                   
                   
                 old 
                 REYYGQYPTIHHLISDLMNGTSPHDVRLVYLACAWLVA 
                 AAATC 
                 GCGAGTT 
               
               
                   
                   
                   
                   
                 elephant 
                 HRGHFLSNIDKDNLSGLKDFSSVYEGLMQYFSDNGYER 
                 CATAG 
                 CAAATAA 
               
               
                   
                   
                   
                   
                   
                 PWNANVDVKALGDALKKKQGVTAKTKELLALLLDSAKA 
                 GGGTC 
                 AAGTTTA 
               
               
                   
                   
                   
                   
                   
                 EKLPREEFPFSQDGIIKLLAGGTYKLSELFGNEEYKDF 
                 TCAAA 
                 CTCAAAT 
               
               
                   
                   
                   
                   
                   
                 GSVKLSMDDEKLGEIMSNIGEDYELIASLRIVSDWAVL 
                 C 
                 CGTTGGC 
               
               
                   
                   
                   
                   
                   
                 VDVLGESATISEAKVGIYNQHKADLEVLKKIIRKYTGK 
                 [SEQ 
                 TTGACCA 
               
               
                   
                   
                   
                   
                   
                 EGYKKVFRQVDSKENYVAYSQHESDGKAPKEKGIDIAT 
                 ID NO. 
                 ACCGCAC 
               
               
                   
                   
                   
                   
                   
                 FSKFILNIVRLLDVEPEDKEVYEDMVARLELNSFLPKQ 
                 23] 
                 AGCGTGT 
               
               
                   
                   
                   
                   
                   
                 VNTDNRVIPYQLYWFELHKILENASIYLPMLTEKDSNG 
                   
                   
               
               
                   
                   
                   
                   
                   
                 ISVMEKLESVFMFRIPYFVGPLNKHSKYAWLERKEGKI 
                   
                 GCTTAAA 
               
               
                   
                   
                   
                   
                   
                 YPWNFENMVDLDASEANFIKRMTNTCTYLPGQNVLPKD 
                   
                 GATCTCT 
               
               
                   
                   
                   
                   
                   
                 SLRYHRFMVLNEINNLRINNERISVELKQKIYSELFLN 
                   
                 TCAGTGA 
               
               
                   
                   
                   
                   
                   
                 VKKVTRKRLVDFLISNGELRKGEESSLTGIDVEIKANL 
                   
                 GGTC 
               
               
                   
                   
                   
                   
                   
                 APQIAFKKLMESGQLTEEDVESIIERASYAEDKARLAH 
                   
                 [SEQ ID 
               
               
                   
                   
                   
                   
                   
                 WLEAKYSKLSEIDRKYICGIKIKDFGRLSKMFLSELEG 
                   
                 NO. 24] 
               
               
                   
                   
                   
                   
                   
                 VDKTTGEMTTILGAMWNSQLNLMELINSELYSFREAIC 
                   
                   
               
               
                   
                   
                   
                   
                   
                 AYQTDYYSTHSSSLEERMNEMYLSNAVKRPVYRTLDIV 
                   
                   
               
               
                   
                   
                   
                   
                   
                 KDVKKAFGEPKKIFVEMTRGASEEQKGKRTKSRKEQIL 
                   
                   
               
               
                   
                   
                   
                   
                   
                 ELYKQCKDEDVRILQQQLEEMGDLADNKLQGDKLFLYY 
                   
                   
               
               
                   
                   
                   
                   
                   
                 MQKGKCMYTGTPIVLEQLGSKAYDIDHIYPQAYVKDDS 
                   
                   
               
               
                   
                   
                   
                   
                   
                 ILNNRVLVLSEANGKKKDIYPIEKETRDKMHGFWTYLN 
                   
                   
               
               
                   
                   
                   
                   
                   
                 DKGMITEEKYKRLTRTTGFTEEEKWSFINRQLTETSQA 
                   
                   
               
               
                   
                   
                   
                   
                   
                 TKAVATLLGELFPNAEIVYSKARLTSEFRQEFNLLKCR 
                   
                   
               
               
                   
                   
                   
                   
                   
                 SYNDLHHAVDAYLNIVCGNVYNMKFTKRWFNINKDYSI 
                   
                   
               
               
                   
                   
                   
                   
                   
                 KTKTVFTHPVVCGGQVVWDGQEMLNKVIRNAKKNTAHF 
                   
                   
               
               
                   
                   
                   
                   
                   
                 TKYAYIRKGGFFDQMPVKAAEGLTPLKKDMPTAVYGGY 
                   
                   
               
               
                   
                   
                   
                   
                   
                 NKPSVAFLIPTRYKAGKKTEIIILSVEHLFGERFLRDE 
                   
                   
               
               
                   
                   
                   
                   
                   
                 AYAKEYAAERLKKILGKQVDEVSFPMGMRPWKINTVLS 
                   
                   
               
               
                   
                   
                   
                   
                   
                 LDGFLICISGIGSGGKCLRAQSIMQFSSDYRWTIYLKR 
                   
                   
               
               
                   
                   
                   
                   
                   
                 LERLVEKITVNAKYVYSEEFDKVSTIENIELYDLYIEK 
                   
                   
               
               
                   
                   
                   
                   
                   
                 YKATIFSKRVNSPEEIIESGRDKFVKLDVLSQARALLC 
                   
                   
               
               
                   
                   
                   
                   
                   
                 IHQTFGRIVGGCDLGLIGGKKNSAATGNFSSTISNWAK 
                   
                   
               
               
                   
                   
                   
                   
                   
                 YYKDVRIIDQSTSGLWVRKSENLLELV 
                   
                   
               
               
                   
                   
                   
                   
                   
                 [SEQ ID NO. 22] 
                   
                   
               
               
                 MAD2024 
                 56 
                 CADAKQ010000027.1 
                 uncultured 
                 Cattle 
                 MNFDGEYFLGLDIGTDSVGYAVTDQRYNLVKFKGEPMW 
                 GTTTG 
                 GAGCCCT 
               
               
                   
                   
                   
                 
                   Lachno- 
                 
                 rumen 
                 GSHLFDAANQCAERRGFRTARRRLDRRQQRVKLVDEIF 
                 AGAGT 
                 CTGGATT 
               
               
                   
                   
                   
                 
                   spiraceae 
                 
                   
                 APEVAKVDPNFYIRKMESALYPEDKSNKGDLYLYFNKQ 
                 AGTGT 
                 TACACTA 
               
               
                   
                   
                   
                 bacterium 
                   
                 EYDEKHYYKDYPTIHHLICALMNDEKTKFDIRLINIAI 
                 AAATC 
                 CGAGTTC 
               
               
                   
                   
                   
                   
                   
                 DWLVAHRGHFLSEVGTDSVDKVLDFRKIYDEFMALFSD 
                 CAGAG 
                 AAATAAA 
               
               
                   
                   
                   
                   
                   
                 EDDAVSSKPWENINPDELGKVLKIHGKNAKRNELKKLL 
                 GGCTC 
                 AATTATT 
               
               
                   
                   
                   
                   
                   
                 YGGKIPTDEDSFIDRKLLIDFIAGTSVQCNKLFRNSEY 
                 CAAAA 
                 TCAAATC 
               
               
                   
                   
                   
                   
                   
                 EDDLKITISNSDEREVVLPQLEDFHADIIAKLSSMYDW 
                 C 
                 GCCGCTA 
               
               
                   
                   
                   
                   
                   
                 SVLSDILSGSTYISESKVKVYEQHKKDLKELKEFVRKY 
                 [SEQ 
                 TGTCGGC 
               
               
                   
                   
                   
                   
                   
                 APEKYNDIFRLASKETYNYTAYSYNLKSVKDEKDLPKG 
                 ID NO. 
                 CGCACAG 
               
               
                   
                   
                   
                   
                   
                 KASKEDFYSYLKKTLKLDKAENYNFVNDADTRFFDDMV 
                 26] 
                 TGTGTGC 
               
               
                   
                   
                   
                   
                   
                 ERISSGTFLPKQVNSDNRVIPYQVYYIELKKILENAKK 
                   
                 ATTAAGA 
               
               
                   
                   
                   
                   
                   
                 HYAFFEEKDEDGYSNVEKIMSVFTFRIPYYVGPLRNDD 
                   
                 AAAGTCC 
               
               
                   
                   
                   
                   
                   
                 KSPYAWIRRKADGKIYPWDFEEKVDLDASENAFIDRMT 
                   
                 GAAAGGG 
               
               
                   
                   
                   
                   
                   
                 NSCTYIPGADVLPKWSLLYTKYMVLNEINNIKVNNIGI 
                   
                 C 
               
               
                   
                   
                   
                   
                   
                 SVEAKQGIYNELFCKKAKVSLKAIREYLISNGFMQKDD 
                   
                 [SEQ ID 
               
               
                   
                   
                   
                   
                   
                 EMSGIDITVKSSLKSRYDFRHLLEKNELTTDDVEAIIS 
                   
                 NO. 27] 
               
               
                   
                   
                   
                   
                   
                 RSTYAEDKARFKKWLKKEFPQLSDEDYKYVSKLKYKDF 
                   
                   
               
               
                   
                   
                   
                   
                   
                 GRLSRSLLNGLEGASKETGEIGTIMHFLWETNDNLMQL 
                   
                   
               
               
                   
                   
                   
                   
                   
                 LSDRYTFMEEINKKRQDYYIEHKLTLNEQMEELGISNA 
                   
                   
               
               
                   
                   
                   
                   
                   
                 VKRPVTRTLAVVKDVVSAIGYAPQKIFVEMARQEDEKK 
                   
                   
               
               
                   
                   
                   
                   
                   
                 KRSVTRKEQILELYKNVEEDTKELERQLKKMGDTANNE 
                   
                   
               
               
                   
                   
                   
                   
                   
                 LQSDALFLYYLQLGKCMYSGKPIDLTQIKTTKKYDIDH 
                   
                   
               
               
                   
                   
                   
                   
                   
                 IWPQSMVKDDSLLNNRVLVLSEINGDKKDVYPIDESIR 
                   
                   
               
               
                   
                   
                   
                   
                   
                 SKMHSYWKMLLDKNLITKEKYSRLTRPTPFTESEKLGF 
                   
                   
               
               
                   
                   
                   
                   
                   
                 INRQLVETRQSMKAVTQLLNNMYPDSEIVYVKAKLAAD 
                   
                   
               
               
                   
                   
                   
                   
                   
                 FKQDFKLAPKSRIINDLHHAKDTYLNVVAGNVYNERFT 
                   
                   
               
               
                   
                   
                   
                   
                   
                 KKWFNVNEKYSMKTKVLFGHDVKIGDRLIWDSKKDLQT 
                   
                   
               
               
                   
                   
                   
                   
                   
                 VKNTYEKNNIHLTRYAYCQKGGLFDQMPVKKGQGQIQL 
                   
                   
               
               
                   
                   
                   
                   
                   
                 KKGMDIDRYGGYNKATASFFIIARYLRGGKKEVSFVPV 
                   
                   
               
               
                   
                   
                   
                   
                   
                 ELMVSEKFLNDDNFAIEYITNVLTGMNTKKIENVELPL 
                   
                   
               
               
                   
                   
                   
                   
                   
                 GKRVIKIKTVLLLDGYKVWVNGKASGGTRVMLTSAESL 
                   
                   
               
               
                   
                   
                   
                   
                   
                 RMPKEYVEYLKKMENYSEKKKSNRNFMHDSENDGLSEE 
                   
                   
               
               
                   
                   
                   
                   
                   
                 KNILLYDKLLEKLDENHFKKMPGNQCETMKSGRVKFIE 
                   
                   
               
               
                   
                   
                   
                   
                   
                 LDFDVQISTLLNCIDLLKSGRTGGCDLKNIGGKSASGV 
                   
                   
               
               
                   
                   
                   
                   
                   
                 VYISANLSACKYNDVHIIDISPAGLHENISCNLMELFE 
                   
                   
               
               
                   
                   
                   
                   
                   
                 [SEQ ID NO. 25] 
                   
                   
               
               
                   
               
               
                 MAD2025 
                 56 
                 DOQG01000053.1 
                 
                   Rumino- 
                 
                 human 
                 MSFKENSKFYFGLDIGTDSVGWAVTDNLYKLYKYKNNL 
                 GTTTG 
                 TTTTACT 
               
               
                   
                   
                   
                 
                   coccaceae 
                 
                 gut 
                 MWGVSLFEAASPAEDRRNHRTARRRLDRRQQRVALLRE 
                 AGAGT 
                 ACCCTAT 
               
               
                   
                   
                   
                 bacterium 
                   
                 LFAKEILKTDPDFFLRLKESSLYPEDRTNKNVNTYFDD 
                 AGTGT 
                 AAATTTA 
               
               
                   
                   
                   
                   
                   
                 ADFKDSDYFKMYPTVHHLIKELSESDKPHDVRLVYLAC 
                 AAATT 
                 CACTACG 
               
               
                   
                   
                   
                   
                   
                 AFIVAHRGHFLNGADENNVQEVLDFNSSYCEFTDWFKS 
                 TATAG 
                 AGTTCAA 
               
               
                   
                   
                   
                   
                   
                 NDIEDNPFSESTENEFSVILRKKIGITAKEKEIKNLLF 
                 GGTAG 
                 ATAAAAA 
               
               
                   
                   
                   
                   
                   
                 GTTKTPDCYKDEEYPIDIDVLIKFISGGKTNLAKLFRN 
                 TAAAA 
                 TTATTTC 
               
               
                   
                   
                   
                   
                   
                 PAYDELDIQTVEVGKADFADTIDLLASSMEDTDVPLLS 
                 C 
                 AAATCGT 
               
               
                   
                   
                   
                   
                   
                 AVKAMYDWSLLIDVLKGQKTISDAKVCEYEQHKSDLKA 
                 [SEQ 
                 ACTTTTT 
               
               
                   
                   
                   
                   
                   
                 LKHIVRKYLDKAQYDEIFRTAGEKPNYVSYSYNVTDVK 
                 ID NO. 
                 AGTACCT 
               
               
                   
                   
                   
                   
                   
                 LKQLPSNFKKKYSEEFCKYINSKLEKIKPEPDDEAVYN 
                 29] 
                 TCACAAG 
               
               
                   
                   
                   
                   
                   
                 ELIEKCNSKTLCPKQVTDENRVIPYQLYYHELSMILDK 
                   
                 TGTTGTG 
               
               
                   
                   
                   
                   
                   
                 ASAYLDFLNETEDGISVKQKILTLMKFRIPYFVGPSVK 
                   
                 AATATTA 
               
               
                   
                   
                   
                   
                   
                 RNETDNVWIVRKAEGRIYPWNFENMVDYDKSEDGFIRR 
                   
                 ACTCACC 
               
               
                   
                   
                   
                   
                   
                 MTCKCTYLAGEDVLPKYSLLYSRYTVLNEINNIKVKDV 
                   
                 TTCGGGT 
               
               
                   
                   
                   
                   
                   
                 KISPELKQDIFNELFMKTSRVTVKKITELLKRKGAFSE 
                   
                 GAG 
               
               
                   
                   
                   
                   
                   
                 ENGDSLSGVDINIKSSLKSYLDFRRLLENGSLSESDVE 
                   
                 [SEQ ID 
               
               
                   
                   
                   
                   
                   
                 RIIERITVTTDKPRLISWLKTEYPALPAEDIRYISRLS 
                   
                 NO. 30] 
               
               
                   
                   
                   
                   
                   
                 YKDYGRLSAKMLTGCYELDMDTGEIGGRSIIDLMWAEN 
                   
                   
               
               
                   
                   
                   
                   
                   
                 INLMQIMSDSYGYKSFIEEENKKYYAINPTGSIAQTLR 
                   
                   
               
               
                   
                   
                   
                   
                   
                 EMYVSPSASRAIIRTMDIVKELRKIIKRDPDKIFVEMA 
                   
                   
               
               
                   
                   
                   
                   
                   
                 RGSKPEDKGKRTSSRREQIEKLFASAKEFVSDEEISHL 
                   
                   
               
               
                   
                   
                   
                   
                   
                 RSQLGSLSDEQLRSEKYYLYFTQFGKCVYSGEAIDFSR 
                   
                   
               
               
                   
                   
                   
                   
                   
                 LGDNHCYDIDHIYPQSKVKDDSLHNKVLVKSQLNGEKS 
                   
                   
               
               
                   
                   
                   
                   
                   
                 DDYPIKEQIRNKMHPIWKNLFYRDPKNPTDKIKYERLT 
                   
                   
               
               
                   
                   
                   
                   
                   
                 RSTPFTEDELAGFIERQLVETRQSTKAVATLLKEMFPD 
                   
                   
               
               
                   
                   
                   
                   
                   
                 SKIVYVKAGQVSKFRHDFDMLKCREINDLHHAKDAYLN 
                   
                   
               
               
                   
                   
                   
                   
                   
                 VVVGNVHDVKFTSNPLNFVKNADKHYTIKIKETLKHKV 
                   
                   
               
               
                   
                   
                   
                   
                   
                 ARNGETAWNPETDFDTVKRMMSKNSVRYVRYCYKRKGE 
                   
                   
               
               
                   
                   
                   
                   
                   
                 LFKQQPKKAGNPDLAWLKKNLDPVKYGGYNSKSISCFS 
                   
                   
               
               
                   
                   
                   
                   
                   
                 LIKCTGVGVVIIPVELLCEKRYFSDDSFASEYAYSVLK 
                   
                   
               
               
                   
                   
                   
                   
                   
                 NALPAKNIAKISIDDISFPLKRRPIKINTLFEFDGYRV 
                   
                   
               
               
                   
                   
                   
                   
                   
                 NIRSKDSYSVFRISSAMAAIYSKDTSDYIKAISSYIDK 
                   
                   
               
               
                   
                   
                   
                   
                   
                 SDKGSKFKPGEAFDVLSNLKAYDEIAKKCISEPFCKIS 
                   
                   
               
               
                   
                   
                   
                   
                   
                 KLAEAGKKMEEGRNKFAELSIIEQMKTLLLLVDVLKTG 
                   
                   
               
               
                   
                   
                   
                   
                   
                 RVDKCNLKPVGGVDNFHTERMSAILKNTKYSDIRIIDQ 
                   
                   
               
               
                   
                   
                   
                   
                   
                 SPTGLYENKSDNLLEL [SEQ ID NO. 28] 
                   
                   
               
               
                   
               
               
                 MAD2026 
                 65 
                 CADBQN010000053.1 
                 uncultured 
                 Cattle 
                 MEQKDYYIGLDIGTNSVGWAVVDEGYQLCRFKKYDMWG 
                 GTTTG 
                 GACTACC 
               
               
                   
                   
                   
                 
                   Firmicutes 
                 
                 rumen 
                 VRLFDSAETAAERRMNRVNRRRNRRKKQRIDLLQGLFA 
                 AGAGT 
                 ATATGAG 
               
               
                   
                   
                   
                 bacterium 
                   
                 EEIAKIDRTFFVRLNESRLHPEDKSTAFRHPLFNDPNY 
                 AGTGT 
                 ATTACAC 
               
               
                   
                   
                   
                   
                   
                 TDVDYYKEYPTIYHLRKELMDSAEPHDIRLVYLALHHI 
                 AATTT 
                 TACACGG 
               
               
                   
                   
                   
                   
                   
                 LKNRGHFLIEGGFEDSKKFEPTFRQLLEVLTEELGLKM 
                 CATAT 
                 TTCAAAT 
               
               
                   
                   
                   
                   
                   
                 DGADAALAESVLKDRGMKKTEKVKRLKNVFTLNTTDMD 
                 GGTAG 
                 AAAGAAT 
               
               
                   
                   
                   
                   
                   
                 QESQKKQKAQIDAVCKFLAGSKGDFKKLVADEALNELK 
                 TCAAA 
                 GTTCGAA 
               
               
                   
                   
                   
                   
                   
                 LDTFALGTSKAEDIGLEIEKSAPQYCVVFESVKSVFDW 
                 C 
                 ACCGCCC 
               
               
                   
                   
                   
                   
                   
                 KIMTQILGDESTFSSAKVKEYEKHHENLIILRELIRKY 
                 [SEQ 
                 TTTGGGG 
               
               
                   
                   
                   
                   
                   
                 CDKETYRHFFNNVNGGYSRYIGSLKKNGKKYYVAGCTQ 
                 ID NO. 
                 CCCGCTT 
               
               
                   
                   
                   
                   
                   
                 EEFYKELKGLLKSIDQRVDPEDRPVYQRVLAETEDETF 
                 32] 
                 GTTGCGG 
               
               
                   
                   
                   
                   
                   
                 LPLLRSKANSAIPRQIHQKELDDILQNASVYLPFLNDV 
                   
                 ATTTACA 
               
               
                   
                   
                   
                   
                   
                 DEDGLSAAEKIRSIFTFRIPYYVGPLSLRHKDKGAHVW 
                   
                 GACTTGA 
               
               
                   
                   
                   
                   
                   
                 IKRKEEGYIYPWNYEKKIDREKSNEEFIRRLINQCTYL 
                   
                 TATCAAG 
               
               
                   
                   
                   
                   
                   
                 KDEKVLPKKSLLYSEFMVLNELNNLRIRGKRLSEEQVE 
                   
                 TCTG 
               
               
                   
                   
                   
                   
                   
                 LKQRIYRDLFMTKTRVTKKTLLNYLRKEDSDLTEEDLS 
                   
                 [SEQ ID 
               
               
                   
                   
                   
                   
                   
                 GFDNDFKASLSSCLELKNKVFGDRIEEDRVRKIAEDLI 
                   
                 NO. 33] 
               
               
                   
                   
                   
                   
                   
                 RWLTIYDDDKKMIKEVIRAEYPNEFTNEQLDVICRLKF 
                   
                   
               
               
                   
                   
                   
                   
                   
                 SGWGNLSEAFLCGVEGADKDTGEVFTIIEALRNTNHNL 
                   
                   
               
               
                   
                   
                   
                   
                   
                 MELLSGNYTFTEKIREHNAALSSEIKAKDYESLVRDLY 
                   
                   
               
               
                   
                   
                   
                   
                   
                 VSPACKRGIWQTIRITEEIKKIMGHEPKKIFVEMTREH 
                   
                   
               
               
                   
                   
                   
                   
                   
                 RDSGRTTSRKDQLLALYQKCEEDARDWVKEIEDREERD 
                   
                   
               
               
                   
                   
                   
                   
                   
                 FSSIKLFLYYLQQGKCMYSGEAIDLDELMSKNSRWDRD 
                   
                   
               
               
                   
                   
                   
                   
                   
                 HIYPQSKIKDDSLDNLVLVKKELNAVKDNGEIAPDIQK 
                   
                   
               
               
                   
                   
                   
                   
                   
                 RMKGFWLSLLRQGFLSKKKFDRLTRTGPFTSEELAGFI 
                   
                   
               
               
                   
                   
                   
                   
                   
                 SRQLVETSQMSKAVAELLNQLYEDSRVVYVKAGLVSQF 
                   
                   
               
               
                   
                   
                   
                   
                   
                 RQKDLGVLKSRSVNDYHHAKDAYLNVVVGDMFDRKFTS 
                   
                   
               
               
                   
                   
                   
                   
                   
                 DPARWFKKNKKVNYSINQVFRRDYEENGKLIWKGIDRG 
                   
                   
               
               
                   
                   
                   
                   
                   
                 EDGKPLFRDGLIHGGTIDLVRAIAKRNTNIRYTEYTYC 
                   
                   
               
               
                   
                   
                   
                   
                   
                 ETGQLYNLTLLPKTDTAITIPLKKELPAAKYGGFKGAG 
                   
                   
               
               
                   
                   
                   
                   
                   
                 TSYFSLIEFDDKKGHHHKQIVGVPIYVANMLEHNENAF 
                   
                   
               
               
                   
                   
                   
                   
                   
                 IEYLETVCSFRNITVLCEKIKKNALISVNGYPMRIRGE 
                   
                   
               
               
                   
                   
                   
                   
                   
                 NEILNMLKNNLQLVLSQEGEETLRHIEKYFNKKPGFEP 
                   
                   
               
               
                   
                   
                   
                   
                   
                 DKEHDGIDRDAMAALYDEMTEKLCTVYKKRPTNQGELL 
                   
                   
               
               
                   
                   
                   
                   
                   
                 KNNRGLFLNLEKRSEMAKVLSETAKMFGTTAQTTADLS 
                   
                   
               
               
                   
                   
                   
                   
                   
                 LIKGSKYAGKIVINKNTLGAAKLILIHQSVTGLFETRV 
                   
                   
               
               
                   
                   
                   
                   
                   
                 EL [SEQ ID NO. 31] 
                   
                   
               
               
                   
               
               
                 MAD2027 
                 65 
                 CACWRN010000001.1 
                 uncultured 
                 Cattle 
                 MSKKFAGEYYLGLDIGTDSVGWAVTDNQYNVLKFNGKS 
                 GTTTG 
                 TTTACCA 
               
               
                   
                   
                   
                 
                   Succini- 
                 
                 rumen 
                 MWGIRLFDAAQTAAERRMFRTARRRVERRRWRLELLQE 
                 AGAGT 
                 TCCAGTG 
               
               
                   
                   
                   
                 
                   clasticum 
                 
                   
                 LFQNEIEKKDPDFFQRMKDSALYPEDSKTGKPFALFCD 
                 AATGT 
                 AGTTTAC 
               
               
                   
                   
                   
                 sp. 
                   
                 KDLNDKLYYKQYPTIYHLRKALLTENSKFDIRLVYLAI 
                 AAATT 
                 ATTACAA 
               
               
                   
                   
                   
                   
                   
                 HHILKHRGHFLFNGDFSNVTRFSFAFEQLQTCLCNELD 
                 CATAG 
                 GTTCAAA 
               
               
                   
                   
                   
                   
                   
                 MDFECNNVQKLSEILKDTHMSKNDKVKASVALFENSGD 
                 GATGG 
                 TAAAAAT 
               
               
                   
                   
                   
                   
                   
                 KKQLQAVIGLFCGAKKKLADVFLDETLNDTEMPSISIA 
                 TAAAA 
                 TTATTCA 
               
               
                   
                   
                   
                   
                   
                 DKPYEELRPELESILAEKCCVIDYIKAVYDWAILADML 
                 C 
                 ACCCGTT 
               
               
                   
                   
                   
                   
                   
                 DGGEYGNRTYISVARVRQYEKHHDDLKKLKKLVRRYCK 
                 [SEQ 
                 CTTCGGA 
               
               
                   
                   
                   
                   
                   
                 SEYKSFFSVAGTDNYCAYIGDDIETDDRKSVKKCKQED 
                 ID NO. 
                 ACCTCCA 
               
               
                   
                   
                   
                   
                   
                 FYKRIKGLLKKAIENGCPKDEVVEIIKDIDAQVFLPLQ 
                 35] 
                 CCGTGTG 
               
               
                   
                   
                   
                   
                   
                 VTKDNGVIPHQVHEMELKQILKNAEKYYPFLCKKDEEG 
                   
                 GAACATT 
               
               
                   
                   
                   
                   
                   
                 IVTSNKILQLFKFRIPYYVGPLNSRIGKNSWIVRRAEG 
                   
                 AAGGTCT 
               
               
                   
                   
                   
                   
                   
                 KIYPWNFEEKVDFDKSEEGFIRRMTNPCTYMAGADVLP 
                   
                 GCTTTGC 
               
               
                   
                   
                   
                   
                   
                 KYSLLYSEFMVLNELNNVRICGDKLSVEIKQTIIKDLF 
                   
                 AGGCC 
               
               
                   
                   
                   
                   
                   
                 QRTRRVTVRKLCDKLKAEGVISRNSNQKDIDIKGIDQD 
                   
                 [SEQ ID 
               
               
                   
                   
                   
                   
                   
                 LKSSMVSYVDFKNIFGKEIEKYSVQQMCERIIFLLTIH 
                   
                 NO. 36] 
               
               
                   
                   
                   
                   
                   
                 HDDKRRLQKRIRAEFTEAQITDDQLQKVLRLNYQGWGR 
                   
                   
               
               
                   
                   
                   
                   
                   
                 FSAEFLKELKGVDTETGEVFSIINALRETDDNLMQLLS 
                   
                   
               
               
                   
                   
                   
                   
                   
                 NRYTFAEELEKYNSNKRKKIEALTYDNIMEGIVASPAI 
                   
                   
               
               
                   
                   
                   
                   
                   
                 KRSAWQAISIVMELSKIMGREPKRIFVEMARGPEEKKH 
                   
                   
               
               
                   
                   
                   
                   
                   
                 TISRKNQLLELYKSVKDESRDWKTELETKTESDFRSIK 
                   
                   
               
               
                   
                   
                   
                   
                   
                 LFLYYTQMGRCMYTGEPIDLDQLANTTIYDRDHIYPQS 
                   
                   
               
               
                   
                   
                   
                   
                   
                 LTKDDSLNNLVLVKKVENANKGNGLISADIQKKMRGFW 
                   
                   
               
               
                   
                   
                   
                   
                   
                 AELKKKGLISDEKFSRLTRTTPLSDDELAGFINRQLVE 
                   
                   
               
               
                   
                   
                   
                   
                   
                 TRQSSKIVADLFHQLYPTTQVVYVKAKIVSDFRHETLD 
                   
                   
               
               
                   
                   
                   
                   
                   
                 MVKVRSLNDLHHAKDAYLNIVTGNVYYEKFSGNPLTWL 
                   
                   
               
               
                   
                   
                   
                   
                   
                 RKNPDRNYSLNQMFNYDIVKKTKEGTSYVWKKGKDGSI 
                   
                   
               
               
                   
                   
                   
                   
                   
                 AVVRRTMERNDILYTRQATENKNGGLFDQNIVSSKNKP 
                   
                   
               
               
                   
                   
                   
                   
                   
                 FIPVKKGLDVNKYGGYKGITPAYFALIEFTDKKGSRQR 
                   
                   
               
               
                   
                   
                   
                   
                   
                 LLEAVPLYLRADIDNDSNVLRDFYKNVLGLENPVVILN 
                   
                   
               
               
                   
                   
                   
                   
                   
                 RIKKNSLLKINGFLIHLRGTTGFSASQLKVQNAVEFSL 
                   
                   
               
               
                   
                   
                   
                   
                   
                 PHHMEDYVKKLENYEKHIIAERGSTKNSQIKITEWDGI 
                   
                   
               
               
                   
                   
                   
                   
                   
                 SKEKNLQLYDMFINKMENTIYKFRPANQVSNLKENREV 
                   
                   
               
               
                   
                   
                   
                   
                   
                 FNSLAVEDQCSVLNQVLMLFVCKPVTANLSLIKGSKNA 
                   
                   
               
               
                   
                   
                   
                   
                   
                 GNMALSKIISNMRSAYLIHQSVTGLFEQKIDLLKVSSQ 
                   
                   
               
               
                   
                   
                   
                   
                   
                 KD [SEQ ID NO. 34] 
                   
                   
               
               
                   
               
               
                 MAD2028 
                 66 
                 DHKP01000031.1 
                 
                   Bacillales 
                 
                 gut 
                 MANKLFIGLDVGSDSVGWAATDENFHLYRLKGKTAWGA 
                 GTTTG 
                 GCATTGT 
               
               
                   
                   
                   
                 bacterium 
                 meta- 
                 RIFSEASDAKGRRGFRVAGRRLARRKERIRLLNTLFDP 
                 AGAGC 
                 AAGACAA 
               
               
                   
                   
                   
                   
                 genome 
                 LLKEKDPTFLLRLENSAIQNDDPNKPAQAVTDCLLFAN 
                 AGTGT 
                 CACTGCT 
               
               
                   
                   
                   
                   
                   
                 KQEEKGFYKRYPTIWHLRKALMDNEDCAFSDIRFLYLA 
                 TGTCT 
                 ACGTTCA 
               
               
                   
                   
                   
                   
                   
                 IHHIIKYRGNFLRDGEIKIGQFDYSVFDKLNETLSVLF 
                 TATAT 
                 AATAAGC 
               
               
                   
                   
                   
                   
                   
                 DLQSEDEDSQEGHFVGLPKSQYEAFITTANDRNLPKQT 
                 AGCTC 
                 ATATTGC 
               
               
                   
                   
                   
                   
                   
                 KKTKLLSMFEKDEESKSFLEMFCTLCAGGEFSTKKLNK 
                 GAAAA 
                 TACAAGG 
               
               
                   
                   
                   
                   
                   
                 KGEETFDDTKISFNASYDQNEPNYQEILGDAFDLVDIA 
                 C 
                 TTCTCCC 
               
               
                   
                   
                   
                   
                   
                 KAVFDYCDLSDILNGNDNLSNAFVELYDSHKSQLSALK 
                 [SEQ 
                 TCGGAGA 
               
               
                   
                   
                   
                   
                   
                 AICKQIDNQSNLKGDASVYVKLFNDPNDKSNYPAFTHN 
                 ID NO. 
                 ATGACCA 
               
               
                   
                   
                   
                   
                   
                 KTLVDKRCDIHTFDKYVIDTVLPYEPLLMGQDATNWQM 
                 38] 
                 TTAGGTC 
               
               
                   
                   
                   
                   
                   
                 LKSLAEQDRLLQTIALRSTSVIPMQLHQKELKIILKNA 
                   
                 ACTTAGA 
               
               
                   
                   
                   
                   
                   
                 ISRNVKGIAEIEEKILKLFQYKIPYYCGPLTTKSAYSN 
                   
                 TAGCCGG 
               
               
                   
                   
                   
                   
                   
                 VVFKNNEYRPLKPWDYEEAIDWDETKKKFMEGLTNKCT 
                   
                 TTCTTCT 
               
               
                   
                   
                   
                   
                   
                 YLKDKNVLPKQSILYQDFDAWNKLNNLKVNGSKPSLKE 
                   
                 GGCTA 
               
               
                   
                   
                   
                   
                   
                 LKDLFSFVSQRPKTTMKDIQRHFKSDTNSKDKDVVVSG 
                   
                 [SEQ ID 
               
               
                   
                   
                   
                   
                   
                 WNPEDYICCSSRASFGKNGVFDLNNPDSSDPKDLSKCE 
                   
                 NO. 39] 
               
               
                   
                   
                   
                   
                   
                 RMIFLKTIYADSPKDADVAILKEFPDLTNDQKSLLKTI 
                   
                   
               
               
                   
                   
                   
                   
                   
                 KCKEWSPLSKEFLELRYADKYGEIRESIINLLRSGEGN 
                   
                   
               
               
                   
                   
                   
                   
                   
                 LMQILAKYDYQERIDAYNADSFQTKSKSQIVSDLIEEM 
                   
                   
               
               
                   
                   
                   
                   
                   
                 PPKMRRPVIQAVRIVHEVVKVAKKEPDQISIEVTRENN 
                   
                   
               
               
                   
                   
                   
                   
                   
                 NKEKKQQLTKKAKSRSAQIQTFLKNLVKIDTFEEKRVD 
                   
                   
               
               
                   
                   
                   
                   
                   
                 EVLEELKKYSDRSINGKHLYLYFLQNGKDAYTGKPINI 
                   
                   
               
               
                   
                   
                   
                   
                   
                 DDVLSGNKYDTDHVIPQSKMKDDSIDNLVLVERSINQH 
                   
                   
               
               
                   
                   
                   
                   
                   
                 RSNEYPLPESIRKNPANVAFWSKLKKAGMMSEKKFNNL 
                   
                   
               
               
                   
                   
                   
                   
                   
                 TRANPLTEEELSAFVAAQINVVNRSNIVIRDVLKVLYP 
                   
                   
               
               
                   
                   
                   
                   
                   
                 NAKLIFSKAQYPSQIRKELNIPKLRDLNDTHHAVDAYL 
                   
                   
               
               
                   
                   
                   
                   
                   
                 NIVSGVSLTERYGNLSFIKAAQKNENQTDYSLNMERYI 
                   
                   
               
               
                   
                   
                   
                   
                   
                 SSLIQTKEGEKTSLGKLIDQTSRRHDFLLTYRFSYQDS 
                   
                   
               
               
                   
                   
                   
                   
                   
                 AFYNQTIYKKNAGLIPVHEKLPPERYGGYNSMSTEVNC 
                   
                   
               
               
                   
                   
                   
                   
                   
                 VVTIKGKKERRYLVGVPHLLLEKGNKVADINKEIANSV 
                   
                   
               
               
                   
                   
                   
                   
                   
                 PHKENETIAVSLKDIIQLDSMVKKDGLVYLCTTQNKDL 
                   
                   
               
               
                   
                   
                   
                   
                   
                 VKLKPFGPIFLSRESEVYLSNLNKFVEKYPNIADGNEN 
                   
                   
               
               
                   
                   
                   
                   
                   
                 YSLKTNRYGEKSIDFLQEKTGNVLKELVDLSNQKRFDY 
                   
                   
               
               
                   
                   
                   
                   
                   
                 CPMICKLRTIDYRKGVEGKTLTEQLILIRSFVGVFTRK 
                   
                   
               
               
                   
                   
                   
                   
                   
                 SEALSNGSNFRKARGLVLQDGLVLCSDSITGLYHTERK 
                   
                   
               
               
                   
                   
                   
                   
                   
                 L [SEQ ID NO. 37] 
                   
                   
               
               
                   
               
               
                 MAD2029 
                 66 
                 DBKT01000013.1 
                 
                   Bacillales 
                 
                 gut 
                 MADKLFIGLDVGSESVGWAATDENFHLYRLKGKTAWGA 
                 GTTTG 
                 GCATTGT 
               
               
                   
                   
                   
                 bacterium 
                 meta- 
                 RIFSEANDAKTRRGFRVAGRRLARRKERIRLLNTLFDP 
                 AGAGC 
                 AAGACAA 
               
               
                   
                   
                   
                   
                 genome 
                 LLKKDPAFLLRLENSAIQNDDPNKPIQAIADCPLLVNK 
                 AGTGT 
                 CACTGCT 
               
               
                   
                   
                   
                   
                   
                 QEEKDYYKRYPTIWHLRKALMENDDHAFSDIRFLYLAI 
                 TGTCT 
                 ACGTTCA 
               
               
                   
                   
                   
                   
                   
                 HHIIKYRGNFLREGDIKIGQFDYSIFDKLNETLAVLFD 
                 TATAT 
                 AATAAGC 
               
               
                   
                   
                   
                   
                   
                 LQNEDGENEEGRFIGLPKSQYEAFITCANDRNLPKQPK 
                 AGCTC 
                 ATATTGC 
               
               
                   
                   
                   
                   
                   
                 KAKLLSMFEKTEESKAFLEMFCTLCSGGEFSTKKLNAK 
                 GAAAA 
                 TACAAGG 
               
               
                   
                   
                   
                   
                   
                 GEETYQDAKISFNSSYDENEGAYQEILGDFFDLVDIAK 
                 C 
                 TTCTCCA 
               
               
                   
                   
                   
                   
                   
                 AVFDYCDLSDILNGNDNLSSAFVELYDSHKSQLSALKS 
                 [SEQ 
                 TTGGAGA 
               
               
                   
                   
                   
                   
                   
                 ICKRIDNQNGFIGEKSIYVKLFNDPNDKSNYPAFTNNK 
                 ID NO. 
                 ATGACCA 
               
               
                   
                   
                   
                   
                   
                 TLVDKRCDIHTFDKYVKETILPYESSLTGRDAVNWQML 
                 41] 
                 TTAGGTC 
               
               
                   
                   
                   
                   
                   
                 KSLAEQDRLLQTIALRSTSVIPMQLHQKELKIILKNAV 
                   
                 GCTTAGA 
               
               
                   
                   
                   
                   
                   
                 SRNIKGVAEIEEKILKLFQYKIPYYCGPLTTKSDYSNV 
                   
                 TAGCCAG 
               
               
                   
                   
                   
                   
                   
                 VFKNNEYRPLKPWDYEEAIDWDGTKQKFMEGLTNKCTY 
                   
                 TTCTTCT 
               
               
                   
                   
                   
                   
                   
                 LKDKNVLPKQSVLYQDFDTWNKLNNLKVNGNKPSLEDL 
                   
                 GGCTA 
               
               
                   
                   
                   
                   
                   
                 NDLFSFVSQRSKTTMRDIQRYLKSKTNSKENDVVVSGW 
                   
                 [SEQ ID 
               
               
                   
                   
                   
                   
                   
                 NSEDYICCSSRASFNKNGIFNLNNSEVLKECERIIFLK 
                   
                 NO. 42] 
               
               
                   
                   
                   
                   
                   
                 TIYTDSPKDADAAVLKEFPDLTNNQKTLLKTIKCKEWS 
                   
                   
               
               
                   
                   
                   
                   
                   
                 PLSKEFLELRYSDKYGElRQSIIDLLRNGEGNLMQILA 
                   
                   
               
               
                   
                   
                   
                   
                   
                 KYDYQEVIDACNAASFQTKSKSQIVSDLIEEMPPKMRR 
                   
                   
               
               
                   
                   
                   
                   
                   
                 PVIQAVRIVQEVAKVAKKEPDEISIEVTRENNDKEKKQ 
                   
                   
               
               
                   
                   
                   
                   
                   
                 QLTKKAKSRSTQIQNFLKNLVKIDASEKKQANEVLEEL 
                   
                   
               
               
                   
                   
                   
                   
                   
                 KKYSDQSINGKHLYLYFLQNGKDAYTGKPINIDDVLSG 
                   
                   
               
               
                   
                   
                   
                   
                   
                 NKYDTDHIIPQSKMKDDSIDNLVLVEREINQHRSNEYP 
                   
                   
               
               
                   
                   
                   
                   
                   
                 LPESIRKNPANVAFWRKLKKAGMMSEKKFNNLTRSNPL 
                   
                   
               
               
                   
                   
                   
                   
                   
                 TEEELGAFVAAQINVVNRSNVVIRDVLKILYPNAKLIF 
                   
                   
               
               
                   
                   
                   
                   
                   
                 SKAQYPSQIRKELNIPKLRDLNDTHHAVDAYLNIVSGV 
                   
                   
               
               
                   
                   
                   
                   
                   
                 TLTDRYGNMRFIKASQDEEKHSLNMERYISSLIQTKEG 
                   
                   
               
               
                   
                   
                   
                   
                   
                 QRTELGELIDQTSRRHDFLLTYRFSYQDSAFYKQTIYK 
                   
                   
               
               
                   
                   
                   
                   
                   
                 KNAGLIPAHDNLPPERYGGYDSMSTEVNCVATIIGKKT 
                   
                   
               
               
                   
                   
                   
                   
                   
                 TRYLVGVPHLLIKKAKDGIDVNDELIKLVPHKENEVVK 
                   
                   
               
               
                   
                   
                   
                   
                   
                 VDLNTTLQLDCTVKKDGFMYLCTSNNIALVKLKPFSPI 
                   
                   
               
               
                   
                   
                   
                   
                   
                 FLSRESEIYLSNLMKYVEKYPNISDENSEYEFKINREN 
                   
                   
               
               
                   
                   
                   
                   
                   
                 VDPIKFTEKQSIEVVQDLIIKAKQDRFSYCSMISKLRD 
                   
                   
               
               
                   
                   
                   
                   
                   
                 INAEEMIHSKSLTEQLKIIKSLIGVFTRKSEILSDKNN 
                   
                   
               
               
                   
                   
                   
                   
                   
                 FRKSRGAILQEDLFLCSDSITGLYHTERKL [SEQ ID 
                   
                   
               
               
                   
                   
                   
                   
                   
                 NO. 40] 
                   
                   
               
               
                   
               
               
                 MAD2030 
                 66 
                 DBLD01000015.1 
                 
                   Bacillales 
                 
                 gut 
                 MEQNTKKLFIGLDVGTDSVGWAATDEYFNLYRLKGKTA 
                 GTTTG 
                 GCATTGT 
               
               
                   
                   
                   
                 bacterium 
                 meta- 
                 WGARLFLDAANAKDRRQHRVSGRRLARRKERIRLLNAL 
                 AGAGC 
                 AAGACAA 
               
               
                   
                   
                   
                   
                 genome 
                 FDPLLKKVDPTFLLRLESSTLQNDDPNKDQRAVSDALL 
                 AGTGT 
                 CACTGCA 
               
               
                   
                   
                   
                   
                   
                 FGNKKHEKAYYAAFPTIWHLRKALIENDDKAFSDIRYL 
                 TGTCT 
                 CGTTCAA 
               
               
                   
                   
                   
                   
                   
                 YLAIHHIIKYRGNFLRQGEIKIGEFDFSCFDKLNQFFD 
                 TAAAT 
                 ATAAGCA 
               
               
                   
                   
                   
                   
                   
                 IYFSKEDEEEVEFIGLPNENYQRFIDCAADKNLGKGKK 
                 AGCTC 
                 GATTGCT 
               
               
                   
                   
                   
                   
                   
                 KGDLLKLMSFSEDEKPFCEMFCSLCAGLAFSTKKLNKK 
                 GAAAA 
                 ACAAGGT 
               
               
                   
                   
                   
                   
                   
                 DETVFEDIKVEFNGKFDDKQEEIKSVLGDAYDLVELAK 
                 C 
                 TCCCGTA 
               
               
                   
                   
                   
                   
                   
                 FIFDYCDLKDILGASTNRLSEAFAGIYDSHKEELKALK 
                 [SEQ 
                 AGGGAAT 
               
               
                   
                   
                   
                   
                   
                 GICREIDRSLGNESKNSLYREVFNDKGIPNNYAAFIHH 
                 ID NO. 
                 GACCATC 
               
               
                   
                   
                   
                   
                   
                 ETNSSRCGIADFNNYVLQKIEPLENLLSKQNYKNWIQL 
                 44] 
                 TGGTCAC 
               
               
                   
                   
                   
                   
                   
                 KQLASQGRLLQTIAIRSTSIIPMQLHLKDLKLILANAE 
                   
                 ATGAATA 
               
               
                   
                   
                   
                   
                   
                 KRDIPGIKDIKEKILLLFQFKVPYYCGPLTDRSQYSNV 
                   
                 GCCCCCG 
               
               
                   
                   
                   
                   
                   
                 VLKAGTREKITPWNFADQVDLEETKKKFMEGLTNKCTY 
                   
                 GCAACGG 
               
               
                   
                   
                   
                   
                   
                 LKDCNVLPRQSLMFQEYDAWNKLNNLSINGNKPSPEEM 
                   
                 TGGCTG 
               
               
                   
                   
                   
                   
                   
                 NALFDFASKRRKTTMSDIKKFEKRATMSKENDVTVSGW 
                   
                 [SEQ ID 
               
               
                   
                   
                   
                   
                   
                 NENDFIDLSSFVSLSGFFDLGEIHSADYMACEEAILLK 
                   
                 NO. 45] 
               
               
                   
                   
                   
                   
                   
                 TIFTDAPQDADPIIAEKFPNLKPNQLAALKKMSCKGWA 
                   
                   
               
               
                   
                   
                   
                   
                   
                 TLSREFLTLKAVDADGEVMNETLLGLMKEGKGNLMQLL 
                   
                   
               
               
                   
                   
                   
                   
                   
                 HSSLYNFQDVIDSHNRAVFGDKSPKQIANDLIEEMPPQ 
                   
                   
               
               
                   
                   
                   
                   
                   
                 MRRPVIQALRIVREVSKVAKKQPDVISIEVTRESNDKK 
                   
                   
               
               
                   
                   
                   
                   
                   
                 KKEEWSKKATDRKKQIDLFLKNLKKTEDVKQTESELDG 
                   
                   
               
               
                   
                   
                   
                   
                   
                 QAINDIDSIRGKHLYLYFLQNGKDAYTGLPIDINDVLN 
                   
                   
               
               
                   
                   
                   
                   
                   
                 GTKYDTDHIIPQSLMKDDSIDNLVLVNREKNQHKSNEF 
                   
                   
               
               
                   
                   
                   
                   
                   
                 PLPRDIQTKANIERWRALKKAGGMSEKKFNNLTRTTPL 
                   
                   
               
               
                   
                   
                   
                   
                   
                 TEEELSAFVAAQINVVNRSNVVIRDVLKILYPNAKLIF 
                   
                   
               
               
                   
                   
                   
                   
                   
                 SKAQYPSQIRRDLEIPKLRDLNDTHHAVDAFLNIVSGV 
                   
                   
               
               
                   
                   
                   
                   
                   
                 ELTKQFGRMDVIKAAAKGDKDHSLNMTRYLERLLKKVD 
                   
                   
               
               
                   
                   
                   
                   
                   
                 ENKNETMTELGNHVFVTSQRHDFLLTYRFDYQDSAFYN 
                   
                   
               
               
                   
                   
                   
                   
                   
                 ATIYSPDKNLIPMHDGMDPERYGGYSSLNIEYNCIATI 
                   
                   
               
               
                   
                   
                   
                   
                   
                 KGKKKTTRYLLGVPHLLALKFKNDGIDITSDLIKLVPH 
                   
                   
               
               
                   
                   
                   
                   
                   
                 KGDEEVSIDWKNPIPLRITVKKDGVEYLLAPFNAQVME 
                   
                   
               
               
                   
                   
                   
                   
                   
                 LKPVSPVFLPREAAEYLARLKKAVDQKKQFIYQNSAEI 
                   
                   
               
               
                   
                   
                   
                   
                   
                 FQSKDKNNALQFGPEQSKNVALKIYALADAKKYDYCAM 
                   
                   
               
               
                   
                   
                   
                   
                   
                 ISKLRDAALRAEMLDSLSSEALFKQYNDLISLLSQLTR 
                   
                   
               
               
                   
                   
                   
                   
                   
                 RSKKISSKYFSKSRGALLQDGLKIVSKSITGLYETERN 
                   
                   
               
               
                   
                   
                   
                   
                   
                 L [SEQ ID NO. 43] 
                   
                   
               
               
                   
               
               
                 MAD2031 
                 141 
                 CACVOG010000001.1 
                 uncultured 
                 Cattle 
                 MNYILGLDIGIASVGWAAVALDANDEPCKILDLNARIF 
                 ATTGT 
                 TTGTAAT 
               
               
                   
                   
                   
                 
                   Seleno- 
                 
                 rumen 
                 EAAEQPKTGASLAAPRREARGSRRRTRRRRHRMERLRH 
                 ACCAT 
                 AACCTAT 
               
               
                   
                   
                   
                 
                   monadaceae 
                 
                   
                 LFAREELISAENIAALFEAPADVYRLRAEGLSRRLDEG 
                 AGCGA 
                 TTTACCT 
               
               
                   
                   
                   
                 bacterium 
                   
                 EWARVLYHIAKRRGFKSNRKGAASDADEGKVLEAVKEN 
                 GTTAA 
                 CGCTATG 
               
               
                   
                   
                   
                   
                   
                 EALLKNYKTVGEMMFRDEKFQTAKRNKGGSYTFCVSRG 
                 ATTAG 
                 GCACAAT 
               
               
                   
                   
                   
                   
                   
                 MLAEEIGELFAAQREQGNPHASETFETAYSKIFADQRS 
                 GGAAT 
                 TTGTTAT 
               
               
                   
                   
                   
                   
                   
                 FDDGPDANSRSPYAGNQIEKMIGTCSLETDPPEKRAAK 
                 TACAA 
                 TACATGG 
               
               
                   
                   
                   
                   
                   
                 ASYSFMRFSLLQKINHLRLKDAKGEERPLTDEERAAVE 
                 C 
                 ACATTAT 
               
               
                   
                   
                   
                   
                   
                 ALAWKSPSLTYGAIRKALPLPDELRFTDLYYRWDKKPE 
                 [SEQ 
                 ACTAAAC 
               
               
                   
                   
                   
                   
                   
                 EIEKKKLPFAAPYHEIRKALDKREKGRIQSLTPDALDA 
                 ID NO. 
                 ATTTCCT 
               
               
                   
                   
                   
                   
                   
                 VGYAFTVFKNDAKIEAALSAAGIDGEDAVALMAAGLTF 
                 47] 
                 AAAAAAG 
               
               
                   
                   
                   
                   
                   
                 RGFGHISVKACRKLIPHLEKGMTYDKACKEAGYDLQKT 
                   
                 CAACGAA 
               
               
                   
                   
                   
                   
                   
                 GGEKTKLLSGNLDEIREIPNPVVRRAIAQTVKVVNAVI 
                   
                 AAACGTG 
               
               
                   
                   
                   
                   
                   
                 RRYGSPVAVNVELAREMGRTFQERRDMMKSMEDNNAEN 
                   
                 CTGGCAG 
               
               
                   
                   
                   
                   
                   
                 EKRKEELKGYGVVHPSGLDIVKLKLYKEQGGVCAYSLA 
                   
                 CAA 
               
               
                   
                   
                   
                   
                   
                 AMPIEKVLKDHDYAEVDHILPYSRSFDDSYANKVLVLS 
                   
                 [SEQ ID 
               
               
                   
                   
                   
                   
                   
                 KENRDKGNRTPMEYMANMPGRRHDFITWVKSAVRNPRK 
                   
                 NO. 48] 
               
               
                   
                   
                   
                   
                   
                 RDNLLLEKFGEDKEAAWKERHLTDTKYIGSFIANLLRD 
                   
                   
               
               
                   
                   
                   
                   
                   
                 HLEFAPWLNGKKKQHVLAVNGAVTDYTRKRLGIRKIRE 
                   
                   
               
               
                   
                   
                   
                   
                   
                 DGDLHHAVDAAVIATVTQGNIQKLTDYSKQIERAFVKN 
                   
                   
               
               
                   
                   
                   
                   
                   
                 RDGRYVNPDTGEVLKKDEWIVQRSRHFPEPWPGFRHEL 
                   
                   
               
               
                   
                   
                   
                   
                   
                 EARVSDHPKEMIESLRLPTYTPEEIDGLKPPFVSRMPT 
                   
                   
               
               
                   
                   
                   
                   
                   
                 RKVRGAAHLETVVSPRLKDEGMIVKKVSLDALKLTKDK 
                   
                   
               
               
                   
                   
                   
                   
                   
                 DAIENYYAPESDHLLYEALLHRLQAFGGDGEKAFAESF 
                   
                   
               
               
                   
                   
                   
                   
                   
                 HKPKADGTPGPVVKKVKIAEKSTLSVPVHHGRGLAANG 
                   
                   
               
               
                   
                   
                   
                   
                   
                 GMVRVDVFFIPEGKDRGYYLVPVYTSDVVRGELPMRAV 
                   
                   
               
               
                   
                   
                   
                   
                   
                 VQGKSYAEWKLMREEDFIFSLYPNDLVYIEHEKGVKVK 
                   
                   
               
               
                   
                   
                   
                   
                   
                 IQKKLREISTLPREKTMTSGLFYYRTMGIAVASIHIYA 
                   
                   
               
               
                   
                   
                   
                   
                   
                 PDGVYVQESLGVKTLKEFKKWTIDILGGEPHPVQKEKR 
                   
                   
               
               
                   
                   
                   
                   
                   
                 QDFASVKRDPHAAKSTSSG [SEQ ID NO. 46] 
                   
                   
               
               
                   
               
               
                 MAD2032 
                 141 
                 CACVWE010000020.1 
                 uncultured 
                 Cattle 
                 MKYIIGLDMGITSVGFATMMLDDKDEPCRIIRMGSRIF 
                 GTTGT 
                 ATTGTAT 
               
               
                   
                   
                   
                 
                   Rumino- 
                 
                 rumen 
                 EAAEHPKDGSSLAAPRRINRGMRRRLRRKSHRKERIKD 
                 AGTTC 
                 CATACCA 
               
               
                   
                   
                   
                 
                   coccus 
                 
                   
                 LIIKNELMTADEISAIYSTGKQLSDIYQIRAEALDRKL 
                 CCTAA 
                 AGAACAA 
               
               
                   
                   
                   
                 sp. 
                   
                 NTEEFVRLLIHLSQRRGFKSNRKVDAKEKGSDAGKLLS 
                 TTATT 
                 TTAGGTT 
               
               
                   
                   
                   
                   
                   
                 AVNSNKELMIEKNYRTIGEMLYKDEKFSEYKRNKADDY 
                 CTTGG 
                 ACTATGA 
               
               
                   
                   
                   
                   
                   
                 SNTFARSEYEDEIRQIFSAQQEHGNPYATDELKESYLD 
                 TATGG 
                 TAAGGTA 
               
               
                   
                   
                   
                   
                   
                 IYLSQRSFDEGPGGSSPYGGNQIEKMIGNCTLEPEEKR 
                 TATAA 
                 GTATACC 
               
               
                   
                   
                   
                   
                   
                 AAKATFSFEYFNLLSKVNSIKIVSSSGKRALNNDERQS 
                 T 
                 GCAAAGC 
               
               
                   
                   
                   
                   
                   
                 VIRLAFAKNAISYTSLRKELNMEYSERFNISYSQSDKS 
                 [SEQ 
                 TCTAACA 
               
               
                   
                   
                   
                   
                   
                 IEEIEKKTKFTYLTAYHTFKKAYGSVFVEWSADKKNSL 
                 ID NO. 
                 CCTCATC 
               
               
                   
                   
                   
                   
                   
                 AYALTAYKNDTKIIEYLTQKGFDAAETDIALTLPSFSK 
                 50] 
                 TTCGGAT 
               
               
                   
                   
                   
                   
                   
                 WGNLSEKALNNIIPYLEQGMLYHDACTAAGYNFKADDT 
                   
                 GAGGTGT 
               
               
                   
                   
                   
                   
                   
                 DKRMYLPAHEKEAPELDDITNPVVRRAISQTIKVINAL 
                   
                 TATCT 
               
               
                   
                   
                   
                   
                   
                 IREMGESPCFVNIELARELSKNKAERSKIEKGQKENQV 
                   
                 [SEQ ID 
               
               
                   
                   
                   
                   
                   
                 RNDRIMERLRNEFGLLSPTGQDLIKLKLWEEQDGICPY 
                   
                 NO. 51] 
               
               
                   
                   
                   
                   
                   
                 SLKPIKIEKLFDVGYTDIDHIIPYSLSFDDTYNNKVLV 
                   
                   
               
               
                   
                   
                   
                   
                   
                 MSSENRQKGNRIPMQYLEGKRQDDFWLWVDNSNLSRRK 
                   
                   
               
               
                   
                   
                   
                   
                   
                 KQNLTKETLSEDDLSGFKKRNLQDTQYLSRFMMNYLKK 
                   
                   
               
               
                   
                   
                   
                   
                   
                 YLALAPNTTGRKNTIQAVNGAVTSYLRKRWGIQKVREN 
                   
                   
               
               
                   
                   
                   
                   
                   
                 GDTHHAVDAVVISCVTAGMTKRVSEYAKYKETEFQNPQ 
                   
                   
               
               
                   
                   
                   
                   
                   
                 TGEFFDVDIRTGEVINRFPLPYARFRNELLMRCSENPS 
                   
                   
               
               
                   
                   
                   
                   
                   
                 RILHEMPLPTYAADEKVAPIFVSRMPKHKVKGSAHKET 
                   
                   
               
               
                   
                   
                   
                   
                   
                 IRRAFEEDGKKYTVSKVPLTDLKLKNGEIENYYNPESD 
                   
                   
               
               
                   
                   
                   
                   
                   
                 GLLYNALKEQUAFGGDAAKAFEQPFYKPKSDGSEGPLV 
                   
                   
               
               
                   
                   
                   
                   
                   
                 KKVKLINKATLTVPVLNNTAVADNGSMVRVDVFFVEGE 
                   
                   
               
               
                   
                   
                   
                   
                   
                 GYYLVPIYVADTVKKELPNKAIIANKPYEEWKEMREEN 
                   
                   
               
               
                   
                   
                   
                   
                   
                 FVFSLYPNDLIKISSRKDMKFNLVNKESTLAPNCQSKE 
                   
                   
               
               
                   
                   
                   
                   
                   
                 ALVYYKGSDISTAAVTAINHDNTYKLRGLGVKTLLKIE 
                   
                   
               
               
                   
                   
                   
                   
                   
                 KYQVDVLGNVFKVGKEKRVRFK [SEQ ID NO. 49] 
                   
                   
               
               
                   
               
               
                 MAD2033 
                 141 
                 DCJP01000021.1 
                 un- 
                 Feces 
                 MKNTLYGIGLDIGVASVGWAVVGLNGTGEPVGLHRLGV 
                 GTTGT 
                 TTATACC 
               
               
                   
                   
                   
                 cultivated 
                 of 
                 RIFDKAEQPKTGESLAAPRRMARGMRRRLRRKALRRAD 
                 AGTTC 
                 ATACCAA 
               
               
                   
                   
                   
                 
                   Faecali 
                 
                 three- 
                 VYALLERSGLSTREALAQMFEAGGLEDIYALRTRALDE 
                 CCTAA 
                 GAACTGT 
               
               
                   
                   
                   
                 bacterium 
                 weeks 
                 PVGKAEFSRILLHLAQRRGFKSNRRTASDGEDGRLLAA 
                 CAGTT 
                 TATGGTT 
               
               
                   
                   
                   
                 sp. 
                 old 
                 VNENRRRMAQGGWRTVGEMLYRHEAFALRKRNKADEYL 
                 CTTGG 
                 GCTATGA 
               
               
                   
                   
                   
                   
                 elephant 
                 STVGRDMVAEEASLLFQRQRELGCAWATPELQAEYLSI 
                 TATGG 
                 TAAGGTC 
               
               
                   
                   
                   
                   
                   
                 LLRQRSFDEGPGGNSPYGGNQVEKMVGRCTFEPDEPRA 
                 TATAA 
                 TTAGCAC 
               
               
                   
                   
                   
                   
                   
                 AKAAYSFEYFSLLQKLNHIRLAENGETRPLTQPQRQQL 
                 T 
                 CGTAAAG 
               
               
                   
                   
                   
                   
                   
                 LSLAHKTPDVSLARIRKELALPETVQFNGVRCRANETL 
                 [SEQ 
                 CTCTGAC 
               
               
                   
                   
                   
                   
                   
                 EESEKKEKFACLPAYHKMRKALDGVVKGRISSLSISQR 
                 ID NO. 
                 GCCTCGC 
               
               
                   
                   
                   
                   
                   
                 DAAATALSLYKNEDTLRAKLTEAGFQAPEIDALAGLTG 
                 53] 
                 TTTCAGC 
               
               
                   
                   
                   
                   
                   
                 FSKFGHLSLKACRKLIPHLEQGLTYDQACSAAGYDFKG 
                   
                 GGGGCGT 
               
               
                   
                   
                   
                   
                   
                 HGAGERAFTLPAAAPEMEQITSPVVRRAVAQTIKVVNG 
                   
                 CATCTTT 
               
               
                   
                   
                   
                   
                   
                 IIREMDASPAWVRIELARELSKTFGERQEMDRSMRENA 
                   
                 TTTGCCC 
               
               
                   
                   
                   
                   
                   
                 AQNERLMQELRDTFHLLSPTGQDLVKYRLWKEQDGVCA 
                   
                 AAAAGAC 
               
               
                   
                   
                   
                   
                   
                 YSLRRLDVERLFEPGYVDVDHIVPYSLSFDDRRSNKVL 
                   
                 ACGGATA 
               
               
                   
                   
                   
                   
                   
                 VLSSENRQKGNRLPLQYLQGKRREDFIVWTNSSVRDYR 
                   
                 TTTTT 
               
               
                   
                   
                   
                   
                   
                 KRQNLLREKFSGDEAEGFRQRNLQDTQHMARFLYNYIS 
                   
                 [SEQ ID 
               
               
                   
                   
                   
                   
                   
                 DHLAFAQSEALGKKRVFAVSGAVTSHLRKRWGLSKVRA 
                   
                 NO. 54] 
               
               
                   
                   
                   
                   
                   
                 DGDLHHALDAAVIACTTDGMIRRISGYYGHIEGEYLQD 
                   
                   
               
               
                   
                   
                   
                   
                   
                 ADGAGSQHARTKERFPAPWPRFRDELIVRLSEQPGEHL 
                   
                   
               
               
                   
                   
                   
                   
                   
                 LDINPAFYCEYGTEHICPVFVSRMPRRKVTGPGHKETI 
                   
                   
               
               
                   
                   
                   
                   
                   
                 KGAAAADEGLLTVRKALTELKLDKDGEIKDYYMPSSDT 
                   
                   
               
               
                   
                   
                   
                   
                   
                 LLYEALKAQLRRFGGDGKKAFAEPFYKPKADGTPGPLV 
                   
                   
               
               
                   
                   
                   
                   
                   
                 RKVKTIEKATLTVPVHGGAASNDTMVRVDVFLVPGDGY 
                   
                   
               
               
                   
                   
                   
                   
                   
                 YWVPVYVADTLKPELPNRAVVAFKPYSEWKEMREEDFI 
                   
                   
               
               
                   
                   
                   
                   
                   
                 FSLYPNDLVYVEHKSGLKFTLQNADSTLEKTWVPKASF 
                   
                   
               
               
                   
                   
                   
                   
                   
                 AYFVGGDISTAAISLRTHDNAYGLRGLGIKTLKVLKKY 
                   
                   
               
               
                   
                   
                   
                   
                   
                 QVDVLGNISPVHRETRQRFR [SEQ ID NO. 52] 
                   
                   
               
               
                   
               
               
                 MAD2034 
                 141 
                 CACXAV010000001.1 
                 uncultured 
                 Cattle 
                 MAYGIGLDIGIASVGFATVALNEQDEPCGILRMGSRIF 
                 GTTGT 
                 TTATACC 
               
               
                   
                   
                   
                 
                   Clostri- 
                 
                 rumen 
                 DAAEHPKNGASLAAPRREARSARRRLRRHRHRLERIRN 
                 AGTTC 
                 ATACCAA 
               
               
                   
                   
                   
                 
                   diales 
                 
                   
                 LLVESCLISQDGLGSLFEGRLEDIYALRTRALDERLTD 
                 CCTAA 
                 GAACTGT 
               
               
                   
                   
                   
                 bacterium 
                   
                 AELCRVLIHLAQRRGFRSNRKADAADKEAGKLLKAVSE 
                 CGGTT 
                 TGGGTTA 
               
               
                   
                   
                   
                   
                   
                 NDRRMEENGYRTVGEMLYKDPLFAEHRRNKGEAYLSTV 
                 CTTGG 
                 CTACAAT 
               
               
                   
                   
                   
                   
                   
                 TRTAVEQEARLVLSTQREKGNAAITEDFVEKYLDILLS 
                 TATGG 
                 AAGGTAG 
               
               
                   
                   
                   
                   
                   
                 QRPFDVGPGGNSPYGGNMIEKMIGRCTFEPDELRAPKA 
                 TATAA 
                 TAAACCG 
               
               
                   
                   
                   
                   
                   
                 SYSFEYFQLLQKVNHIRLLRDGRSEPLSEEQRRAIIDL 
                 T 
                 AAAAGCT 
               
               
                   
                   
                   
                   
                   
                 ALASADVTFAKIRKALSLPDSVRFNDVYYRESAEEAEK 
                 [SEQ 
                 CTGACGT 
               
               
                   
                   
                   
                   
                   
                 KKKLGCMDAYHEMRKALDKVAKGRICAIPVEQRNAIAY 
                 ID NO. 
                 CTTGTTT 
               
               
                   
                   
                   
                   
                   
                 VLTVHKTDERILTELQNINLERSDIDQLMQMKGFSKFG 
                 56] 
                 GCGCAGG 
               
               
                   
                   
                   
                   
                   
                 HLSIKACDRIIPYLEQGMTYSDACTAAGYAFRGHEGGE 
                   
                 ACGTCAT 
               
               
                   
                   
                   
                   
                   
                 HSLYLPAQTPEMDEITSPVVRRAVSQTIKVVNALIREQ 
                   
                 CTTTATA 
               
               
                   
                   
                   
                   
                   
                 GESPTFVNIELAREMSKDFAERNDIRRENEKNAKANEA 
                   
                 TCAGACG 
               
               
                   
                   
                   
                   
                   
                 VMNELRRTFGLVNPSGQDLVKYKLFLEQGGVCPYTQRP 
                   
                 GATG 
               
               
                   
                   
                   
                   
                   
                 MEPGRLFEAGYADVDHIVPYSISFDDRYCNKVLTFASV 
                   
                 [SEQ ID 
               
               
                   
                   
                   
                   
                   
                 NRKEKGNRLPLQFLKGERRESFIVYVKANVRDYRKQRL 
                   
                 NO. 57] 
               
               
                   
                   
                   
                   
                   
                 LLKETVTEEDRKGFRDRNLQDTKHMAAFLHSYINDHLQ 
                   
                   
               
               
                   
                   
                   
                   
                   
                 FAPFQTDRKRHVTAVNGAVTAYLRKRWGIRKVRAEGDL 
                   
                   
               
               
                   
                   
                   
                   
                   
                 HHASDALVIACTTPGMIQRLSRYAELREAEYMQTEDGA 
                   
                   
               
               
                   
                   
                   
                   
                   
                 VRFDPATGEVLEKFPYPWPCFRQEWTARVSDDPQAMLQ 
                   
                   
               
               
                   
                   
                   
                   
                   
                 DMKLTDYRGLPLEQVKPVFVSRMPKHKVTGAAHKDTVK 
                   
                   
               
               
                   
                   
                   
                   
                   
                 SAKALDRGVVLVKRALTDLKLKDGEIENYYDPASDRLL 
                   
                   
               
               
                   
                   
                   
                   
                   
                 YEALKERLIAFGGDAQKAFAEPFHKPKRDGTPGPLVKK 
                   
                   
               
               
                   
                   
                   
                   
                   
                 VKLMEKSSLTVPVHDGKGVADNDSMVRIDVFFVAGEGY 
                   
                   
               
               
                   
                   
                   
                   
                   
                 YFVPIYVADTVKPELPNRAVVANKPYAEWKEMKDEDFL 
                   
                   
               
               
                   
                   
                   
                   
                   
                 FSLYPSDLMRVTQKKGIKLSLINKESTLKKEEMAQSIL 
                   
                   
               
               
                   
                   
                   
                   
                   
                 LYYVKGSISTGSITAENHDRTYAINSLGIKTLEKLEKY 
                   
                   
               
               
                   
                   
                   
                   
                   
                 QVDVLGNVSPVGKEKRLTFC [SEQ ID NO. 55] 
                   
                   
               
               
                   
               
               
                 MAD2035 
                 141 
                 CADATZ010000012.1 
                 uncultured 
                 Cattle 
                 MLPYAIGLDIGIASVGWAVVGLDTNERPFCILGMGSRI 
                 GTTGT 
                 TTATACC 
               
               
                   
                   
                   
                 
                   Chloroflexi 
                 
                 rumen 
                 FDKAEQPKTGASLALPRREARSLRRRLRRHRHRNERIR 
                 AGTCC 
                 ATTCCAG 
               
               
                   
                   
                   
                 bacterium 
                   
                 NLLLREKIISESELQDLFSGTLSDIYQLRVEALDRKLD 
                 CCTGA 
                 AAACTAT 
               
               
                   
                   
                   
                   
                   
                 DKEFSRVLIHIAQRRGFKSNRKNAAASQEDGKLLSAVT 
                 TGGTT 
                 TATGGTC 
               
               
                   
                   
                   
                   
                   
                 ENQQRMNDKGYRTVSEMLLRDDKFKDHKRNKGGEYLTT 
                 TCTGG 
                 ACTACAA 
               
               
                   
                   
                   
                   
                   
                 VTRTMVEDEVHKIFSAQRTHGNLKADNQLESEYLEILL 
                 AATGG 
                 TAAGGTA 
               
               
                   
                   
                   
                   
                   
                 SQRSFDEGPGGDSPYGGSQIEKMIGKCTFFPEEKRAAK 
                 TATAA 
                 TTAGACC 
               
               
                   
                   
                   
                   
                   
                 ATYTFEYFNLLEKINHIRLVSKDNLPEPLSDFQRRSLI 
                 T 
                 GTAGAGC 
               
               
                   
                   
                   
                   
                   
                 ELAYKVENLTYDRIRKELHISPELKFNTIRYESDDLPE 
                 [SEQ 
                 ACTAACA 
               
               
                   
                   
                   
                   
                   
                 NEKKQKLNCLKAYHElRKALDKLGKGTINTLSKEQLNT 
                 ID NO. 
                 CCCCATT 
               
               
                   
                   
                   
                   
                   
                 IGTVLSMYKTSEIIKNKMEQIPAEIVDKLDEEGINFSK 
                 59] 
                 TGGGGTG 
               
               
                   
                   
                   
                   
                   
                 FGHLSIKACELIIPGLEKGLNYNDACEEAGLNFKAHNN 
                   
                 TTATCTC 
               
               
                   
                   
                   
                   
                   
                 EEKSFLLHPTEDDYADITSPVVKRAASQTIKVINAIIR 
                   
                 TTTAAAC 
               
               
                   
                   
                   
                   
                   
                 KQGCSPTYINIEVARELSKDFYERDKINKRNEANRAEN 
                   
                 TGTCCAA 
               
               
                   
                   
                   
                   
                   
                 ERSLEQIRKEYGKSNASGLDLVKFKLYQKQDGVCAYSQ 
                   
                 AATTTAG 
               
               
                   
                   
                   
                   
                   
                 KQISFERLFEPNYVEVDHIIPYSKCFDDRESNKVLVFA 
                   
                 TATTGCA 
               
               
                   
                   
                   
                   
                   
                 KENREKGNRLPLEYLDGKKRESFIVWVNSKVKDYRKKQ 
                   
                 ATTATTG 
               
               
                   
                   
                   
                   
                   
                 NLLKESLSEEEEKQFKERNLQDTKTVSKFLMNYINDNL 
                   
                 A 
               
               
                   
                   
                   
                   
                   
                 IFSSSNKRKKHVTAVSGGVTSYMRKRWGISKVREDGDQ 
                   
                 [SEQ ID 
               
               
                   
                   
                   
                   
                   
                 HHAVDALVIVCTTDGMIQQVSKYVEYKECQYIQTDAGS 
                   
                 NO. 60] 
               
               
                   
                   
                   
                   
                   
                 LAVDPYTGEVLRSFPYPWARFHEDAVTWTEKIFVSRMP 
                   
                   
               
               
                   
                   
                   
                   
                   
                 MRKVTGPAHKETIKSPKALGEGLLIVRKPLTELKLKNG 
                   
                   
               
               
                   
                   
                   
                   
                   
                 EIENYYKPEADLLLYNGLKERLMEFGGDAKKAFAEPFP 
                   
                   
               
               
                   
                   
                   
                   
                   
                 KPGNPQKIVKKVRLTEKSTLNVPVLKGEGRADNDSMVR 
                   
                   
               
               
                   
                   
                   
                   
                   
                 VDVFLKDGKYYLVPIYVADTLKPELPNKACIAHKPYDE 
                   
                   
               
               
                   
                   
                   
                   
                   
                 WATMDDGDFLFSLYPNDLIYIKHKKGIKLTKINKNSTL 
                   
                   
               
               
                   
                   
                   
                   
                   
                 ADSIEGKEFFLFYKTMGISSAVLTCTNHDNTYYIESLG 
                   
                   
               
               
                   
                   
                   
                   
                   
                 VKTLESLEKCVVGVLGEIHKVRKEKRTGFSGN [SEQ 
                   
                   
               
               
                   
                   
                   
                   
                   
                 ID NO. 58] 
                   
                   
               
               
                   
               
               
                 MAD2036 
                 141 
                 CADAWQ010000026.1 
                 
                   Ruminoe- 
                 
                 Cattle 
                 MLPYAIGLDIGISSVGWASVALDEEDKPCGIIGMGSRI 
                 GTTAT 
                 TTATACC 
               
               
                   
                   
                   
                 
                   coccacea 
                 
                 rumen 
                 FDAAEQPKTGDSLAAPRRAARSARRRLRRRRHRNERIR 
                 AGTTC 
                 ATACCAA 
               
               
                   
                   
                   
                 bacterium 
                   
                 ALMLREGLLSEAELAALFDGRLEDICALRVRALDEAVT 
                 CCTGT 
                 GAACGAA 
               
               
                   
                   
                   
                   
                   
                 NDELARILLHLSQRRGFRSNRKTAATQEDGELLAAVSA 
                 TCGTT 
                 GCAGGTT 
               
               
                   
                   
                   
                   
                   
                 NRALMQERGYRTVAEMLLRDERYRDHRRNKGGAYIATV 
                 CTTGG 
                 ACTATGA 
               
               
                   
                   
                   
                   
                   
                 GRDMVEDEVRQIFAAQRALGSTAASETLETAYLEILLS 
                 TATGG 
                 TAAGGTA 
               
               
                   
                   
                   
                   
                   
                 QRSFDAGPGEPSPYAGGQIERMIGRCTFEPDEPRAARA 
                 TATAA 
                 GTATACC 
               
               
                   
                   
                   
                   
                   
                 TYSFEYFSLLEAVNHIRLTEAGESVPLTKEQREKLIAL 
                 T 
                 GCAGAGC 
               
               
                   
                   
                   
                   
                   
                 AHRTADLSYAKIRKELGVPESQRFNMVTYGKTDSADEA 
                 [SEQ 
                 TCCAACG 
               
               
                   
                   
                   
                   
                   
                 EKKTKLKQLRAYHQMRAAFEKAAKGSFVLLTKEQRNAV 
                 ID NO. 
                 CCTCGCT 
               
               
                   
                   
                   
                   
                   
                 GQTLSIYKTSDNIRPRLREAGLTEAEIDVAEGLSFSKF 
                 62] 
                 TTTGCGG 
               
               
                   
                   
                   
                   
                   
                 GHLSVKACDKIIPFLEQGMKYSEACVAAGYAFRGHEGQ 
                   
                 GGCGTTG 
               
               
                   
                   
                   
                   
                   
                 DKQRLLPPLDNDAKDTITSPVVLRAVSQTIKVVNAIIR 
                   
                 TCTCT 
               
               
                   
                   
                   
                   
                   
                 ERGGSPTFINIELAREMAKDFSERSQIKREQDSNRARN 
                   
                 [SEQ ID 
               
               
                   
                   
                   
                   
                   
                 ERMMERIKTEYGKSSPTGLDLVKLKLYEEQAGVCAYSL 
                   
                 NO. 63] 
               
               
                   
                   
                   
                   
                   
                 KQMSLEHLFDPNYAEIDHIIPYSISFDDGYKNKVLVLA 
                   
                   
               
               
                   
                   
                   
                   
                   
                 KENRDKGNRLPLEYLNGKRREDFIVWVNSSVRDWRKKQ 
                   
                   
               
               
                   
                   
                   
                   
                   
                 NLLKEHVTPEDEAKFKERNLQDTKTASRFLLNYIADNL 
                   
                   
               
               
                   
                   
                   
                   
                   
                 AFAPFQTERKKRVTAVNGSVTAYLRKRWGIAKVRANGD 
                   
                   
               
               
                   
                   
                   
                   
                   
                 LHHAVDALVIACTTDGLIQKVSRYACYQENRYSEAGGV 
                   
                   
               
               
                   
                   
                   
                   
                   
                 IVDSATGEVVAQFPEPWPRFRHELEARLSDDPARAVLG 
                   
                   
               
               
                   
                   
                   
                   
                   
                 LGLAHYMTGEIRPRPLFVSRMPRRKVTGAAHKETVKSP 
                   
                   
               
               
                   
                   
                   
                   
                   
                 RALDEGQLVTKTPLSALKLGKDGEIPGYYKPESDRLLY 
                   
                   
               
               
                   
                   
                   
                   
                   
                 EALKARLRQFGGDGKKAFAEPFHKPKHDGTPGPVVTKV 
                   
                   
               
               
                   
                   
                   
                   
                   
                 KLCEPATLSVPVHGGLGAANNDSMVRIDVFHVEGDGYY 
                   
                   
               
               
                   
                   
                   
                   
                   
                 FVPIYIADTLKLELPNKACVKIKKISEWKHMKPQDFMF 
                   
                   
               
               
                   
                   
                   
                   
                   
                 SLYPNDLFRIVSKKGITLNLVSKESTLPTSVNVSDTLL 
                   
                   
               
               
                   
                   
                   
                   
                   
                 YFVSAGIASACLTCRNHDNTYQIESLGIKTLEKLEKYT 
                   
                   
               
               
                   
                   
                   
                   
                   
                 VDVLGNVHRVEKEPRMSFSQKGD [SEQ ID NO. 
                   
                   
               
               
                   
                   
                   
                   
                   
                 61] 
                   
                   
               
               
                   
               
               
                 MAD2037 
                 141 
                 DGSQ01000028.1 
                 
                   Clostri- 
                 
                 low 
                 MLPYGIGLDIGITSVGWATVALDENDRPYGIIGMGSRI 
                 GTTAT 
                 TTATACC 
               
               
                   
                   
                   
                 
                   diales 
                 
                 methane 
                 FDAAEQPKTGESLAAPRRAARSARRRLRRHRHRNERIR 
                 AGTTC 
                 ATACCAA 
               
               
                   
                   
                   
                 bacterium 
                 producing 
                 ALILRENLLSEGQLLHLYDGQLSDVYSLRVKALDERVS 
                 CCTGA 
                 GAACTAT 
               
               
                   
                   
                   
                   
                 sheep 
                 NEEFARILIHISQRRGFKSNRKGASSKEDSELLAAISA 
                 TAGTT 
                 GAGGTTG 
               
               
                   
                   
                   
                   
                   
                 NQVRMQQQGYRTVAEMYLKDPIYQEHRRNKGGNYIATV 
                 CTTGG 
                 CTATAAT 
               
               
                   
                   
                   
                   
                   
                 SRAMVEDEVHQIFTGQRACGNPAATKELEEAYVEILLS 
                 TATGG 
                 AAGGTAG 
               
               
                   
                   
                   
                   
                   
                 QRSFDDGPGDGSPYAGSQIERMIGKCQLEKEAGEPRAA 
                 TATAA 
                 TAAACCG 
               
               
                   
                   
                   
                   
                   
                 KATYSFEYFSLLAAINNISIISNGQLSPLTKEQREMLI 
                 T 
                 CAGAGCT 
               
               
                   
                   
                   
                   
                   
                 ALAHKTSELNYARIRKELGLSEAQRFNTVSYGKMEIAE 
                 [SEQ 
                 CTAACGC 
               
               
                   
                   
                   
                   
                   
                 AEKKTKFEHLKAYHKMRREFERIAKGHFASITIEQRNA 
                 ID NO. 
                 CTCACAT 
               
               
                   
                   
                   
                   
                   
                 IGDVLSKYKTDAKIRPALREAGLTELDIDAAEALNFSK 
                 65] 
                 TTGTGGG 
               
               
                   
                   
                   
                   
                   
                 FGHISIKACKKIIPWLEQGMKYSEACNAAGYNFKGHDG 
                   
                 GCGTTAT 
               
               
                   
                   
                   
                   
                   
                 QEKSHLLPPLDEESRNVITSPVALRAISQTIKVVNAII 
                   
                 CTCT 
               
               
                   
                   
                   
                   
                   
                 RERGCSPTFINIELAREMSKDFYERIEIKKEQDGNRAK 
                   
                   
               
               
                   
                   
                   
                   
                   
                 NERMMERIRTEYGKASPTGQDLVKFKLYEEQGGVCAYS 
                   
                 [SEQ ID 
               
               
                   
                   
                   
                   
                   
                 LKQMSLAHLFEPDYAEVDHIVPYSISFDDGYKNKVLVL 
                   
                 NO. 66] 
               
               
                   
                   
                   
                   
                   
                 AKENRDKGNRLPLQYLQGKRREDFIAWVNSCVRDYKKR 
                   
                   
               
               
                   
                   
                   
                   
                   
                 QRLLKESISEDDLRAFKERNLQDTKTASRFLLNYISDH 
                   
                   
               
               
                   
                   
                   
                   
                   
                 LEFTQFATERKKHVTAVNGSVTAYLRKRWGITKIRENG 
                   
                   
               
               
                   
                   
                   
                   
                   
                 DLHHAVDALVIACTTDGMIQQVSRFAQHRENQYSLAED 
                   
                   
               
               
                   
                   
                   
                   
                   
                 SRFIIDPETGEVIKEFPYPWPRFRQELEARLSSNPGLA 
                   
                   
               
               
                   
                   
                   
                   
                   
                 VRDRGFLLYMAESIPVHPLFVSRMPRRKVTGAAHKETI 
                   
                   
               
               
                   
                   
                   
                   
                   
                 KSGKAQKDGLLIVKKPLTDLKLDKEGEIANYYNPMSDR 
                   
                   
               
               
                   
                   
                   
                   
                   
                 LLYEALKKRLTAFNGDGKKAFADPFYKPKSDGTQGPLV 
                   
                   
               
               
                   
                   
                   
                   
                   
                 NKVKLCEPSTLNVSVIGGKGVAENDSMVRIDVFRVEGD 
                   
                   
               
               
                   
                   
                   
                   
                   
                 GYYFVPVYVADTVKPELPNKACVANKPYTDWKEMRESD 
                   
                   
               
               
                   
                   
                   
                   
                   
                 FLFSLYPNDLLKVTHKKALILTKAQKDSDLPDCKETKS 
                   
                   
               
               
                   
                   
                   
                   
                   
                 EMLYFVSASISTASLACRTHDNSYRINSLGIKTLEALE 
                   
                   
               
               
                   
                   
                   
                   
                   
                 KYTVDVLGEYHPVRRETRQTFTGRESSGHSGIS [SEQ 
                   
                   
               
               
                   
                   
                   
                   
                   
                 ID NO. 64] 
                   
                   
               
               
                   
               
               
                 MAD2038 
                 141 
                 CACWHR010000008.1 
                 
                   Rumino- 
                 
                 Cattle 
                 MRPYGIGLDIGISSVGWAAIALDHQDSPCGILDMGARI 
                 GTTGT 
                 TTATACC 
               
               
                   
                   
                   
                 
                   coccaceae 
                 
                 rumen 
                 FDAAENPKDGASLAAPRREKRSQRRRLRRHRHRNERIR 
                 AGTTC 
                 ATACCAA 
               
               
                   
                   
                   
                 bacterium 
                   
                 RMLLKEGLLTEAELTGLFDGALEDIYALRTRALDEALT 
                 CCTGA 
                 GAACGAT 
               
               
                   
                   
                   
                   
                   
                 KQEFARVLLHLSQRRGFRSNRRATAAQEDGKLLDAVSE 
                 TCGTT 
                 CAGGTTG 
               
               
                   
                   
                   
                   
                   
                 NAKRMADCGYRTVGEMLCRDATFAKHKRNKGGEYLTTV 
                 CTTGG 
                 CTACAAT 
               
               
                   
                   
                   
                   
                   
                 SRAMIEDEVKLVFASQRRLGSAFASEALEQGYLDILLS 
                 TATGG 
                 AAGGTAG 
               
               
                   
                   
                   
                   
                   
                 QRSFDEGPGGNSPYGGAQIERMIGKCTFYPEEPRAARA 
                 TATAA 
                 TAAACCG 
               
               
                   
                   
                   
                   
                   
                 CYSFEYFSLLQKVNHIRLQKDGESTPLTSEQRLQLIEL 
                 T 
                 AAGAGCT 
               
               
                   
                   
                   
                   
                   
                 ANKTENLDYARIRRALQIPDAYRFNTVSYRIESDPAAA 
                 [SEQ 
                 CTAACGC 
               
               
                   
                   
                   
                   
                   
                 EKKEKFQYLRAYHTMRKAIDGASKGRFALLSQEQRDQI 
                 ID NO. 
                 CCCGTTT 
               
               
                   
                   
                   
                   
                   
                 GTVLTLYKSQERISEKLTEAGIEPCDIAALESVSGFSK 
                 68] 
                 CTTTACG 
               
               
                   
                   
                   
                   
                   
                 TGHISLRACKELIPYLEQGMNYNEACAAAGIEFHGHSG 
                   
                 GGGCGTT 
               
               
                   
                   
                   
                   
                   
                 TERTVVLHPTPDDLADITSPVVRRAVAQTVKVINAVIR 
                   
                 ATCTCT 
               
               
                   
                   
                   
                   
                   
                 RYGSPVFVNIELARELAKDFTERKKLEKDNKTNRAENE 
                   
                 [SEQ ID 
               
               
                   
                   
                   
                   
                   
                 RLMRRIREEYGKMNPTGLDLVKLRLYEEQAGVCPYSQK 
                   
                 NO. 69] 
               
               
                   
                   
                   
                   
                   
                 QMSLQRLFEPNYAEVDHIIPYSISFDDSRRNKVLVLAE 
                   
                   
               
               
                   
                   
                   
                   
                   
                 ENRNKGNRLPLQYLTGERRDNFIVWVNSSVRDYRKKQK 
                   
                   
               
               
                   
                   
                   
                   
                   
                 LLKPTVTDEDKQQFKERNLQDTKTMSRFLMNYINDHLQ 
                   
                   
               
               
                   
                   
                   
                   
                   
                 FGVSAKERKKRVTAVNGIVTSYLRKRWGITKIRGDGDL 
                   
                   
               
               
                   
                   
                   
                   
                   
                 HHAVDALVIACATDGMIRQITRYAQYRECRYMQTDTGS 
                   
                   
               
               
                   
                   
                   
                   
                   
                 AAIDEATGEVLRIFPYPWEHFRKELEARLSSDPARAVN 
                   
                   
               
               
                   
                   
                   
                   
                   
                 ALRLPFYLDSGEPLPKPLFVSRMPRRKVSGAAHKDTVK 
                   
                   
               
               
                   
                   
                   
                   
                   
                 SPKAMAEGKVIVRRALTDLKLKNGEIENYFDPGSDRLL 
                   
                   
               
               
                   
                   
                   
                   
                   
                 YDALKARLAAFGGDGAKAFREPFYKPRHDGTPGPLVKK 
                   
                   
               
               
                   
                   
                   
                   
                   
                 VKLCEPTTLNVAVHGGKGVADNDSMVRIDVFRVEGDGY 
                   
                   
               
               
                   
                   
                   
                   
                   
                 YFVPIYIADTLKPVLPNKACVAFKPYSEWRTMDDRDFI 
                   
                   
               
               
                   
                   
                   
                   
                   
                 FSLYPNDLIRVTHKSALKLSRVSKESTLPESIESKTAL 
                   
                   
               
               
                   
                   
                   
                   
                   
                 LYYVSAGISGAAVSCRNHDNSYEIKSMGIKTLEKLEKY 
                   
                   
               
               
                   
                   
                   
                   
                   
                 TVDVLGEYHKVEKERRMPFTGKRS [SEQ ID NO. 
                   
                   
               
               
                   
                   
                   
                   
                   
                 67] 
                   
                   
               
               
                   
               
               
                 MAD2039 
                 141 
                 CACZLL010000017.1 
                 
                   Rumino- 
                 
                 Cattle 
                 MRPYAIGLDIGITSVGWATVALDADESPCGIIGLGSRI 
                 GTTAT 
                 TTATACC 
               
               
                   
                   
                   
                 
                   coccaceae 
                 
                 rumen 
                 FDAAEQPKTGESLAAPRRAARGSRRRLRRHRHRNERIR 
                 AGTTC 
                 ATACCAA 
               
               
                   
                   
                   
                 bacterium 
                   
                 SLMLEERLISQDELETLFDGRLEDIYALRVKALDEIVS 
                 CCTGA 
                 GAACTAT 
               
               
                   
                   
                   
                   
                   
                 RTDFARILLHISQRRGFKSNRKNPTTKEDGVLLAAVNE 
                 TAGTT 
                 TTAGGTT 
               
               
                   
                   
                   
                   
                   
                 NKQRMSEHGYRTVGEMFLLDETFKDHKRNKGGNYITTV 
                 CTTGG 
                 ACTATGA 
               
               
                   
                   
                   
                   
                   
                 ARDMVADEVRAIFSAQRELGASFASEEFEERYLEILLS 
                 TATGG 
                 TAAGGTT 
               
               
                   
                   
                   
                   
                   
                 QRSFDEGPGGNSPYGGSQIERMVGRCTFFPDEPRAAKA 
                 TATAA 
                 TAGTACA 
               
               
                   
                   
                   
                   
                   
                 TYSFEYFTLLQKVNHIRIVENGVASKLTDEQRRIIIEL 
                 T 
                 CCTTAGA 
               
               
                   
                   
                   
                   
                   
                 AHTTKDVSYAKIRKVLKLSDKQLFNIRYSDNSPAEDSE 
                 [SEQ 
                 GCTCTGA 
               
               
                   
                   
                   
                   
                   
                 KKEKLGIMKAYHQMRSAIDRVSKGRFAMMPRAQRNAIG 
                 ID NO. 
                 CGCCTCG 
               
               
                   
                   
                   
                   
                   
                 TALSLYKTSDKIRKYLTDAGLDEIDINSADSIGSFSKF 
                 71] 
                 CTTTTGC 
               
               
                   
                   
                   
                   
                   
                 GHISVKACDMLIPFLEQGMNYNEACAAAGLNFKGHDAG 
                   
                 GAGGCGT 
               
               
                   
                   
                   
                   
                   
                 EKSKLLHPKEEDYEDITSPVVRRAIAQTIKVINAIIRR 
                   
                 TATCTCT 
               
               
                   
                   
                   
                   
                   
                 EGCSPTFINIELAREMAKDFRERNRIKKENDDNRAKNE 
                   
                 TTATATT 
               
               
                   
                   
                   
                   
                   
                 RLLERIRTEYGKNNPTGLDLVKLRLYEEQSGVCMYSLK 
                   
                 GCCAAAA 
               
               
                   
                   
                   
                   
                   
                 QMSLEKLFEPNYAEVDHIVPYSISFDDSRKNKVLVLTE 
                   
                 ATGCAAA 
               
               
                   
                   
                   
                   
                   
                 ENRNKGNRLPLQYLKGRRREDFIVWVNNNVKDYRKRRL 
                   
                 TATATCG 
               
               
                   
                   
                   
                   
                   
                 LLKEELTAEDESGFKERNLQDTKTMSRFLLNYIADNLE 
                   
                 TACAATG 
               
               
                   
                   
                   
                   
                   
                 FAESTRGRKKKVTAVNGAVTAYMRKRWGITKIREDGDC 
                   
                 GTGGC 
               
               
                   
                   
                   
                   
                   
                 HHAVDAVVIACTTDAMIRQVSRYAQFRECEYMQTESGS 
                   
                 [SEQ ID 
               
               
                   
                   
                   
                   
                   
                 VAVDTGTGEVLRTFPYPWPDFRKELEARLANDPAKVIN 
                   
                 NO. 72] 
               
               
                   
                   
                   
                   
                   
                 DLHLPFYMSAGRPLPEPVFVSRMPRRKVTGAAHKDTIK 
                   
                   
               
               
                   
                   
                   
                   
                   
                 SARELDNGYLIVKRPLTDLKLKNGEIENYYNPQSDKCL 
                   
                   
               
               
                   
                   
                   
                   
                   
                 YDALKNALIEHGGDAKKAFAGEFRKPKRDGTPGPIVKK 
                   
                   
               
               
                   
                   
                   
                   
                   
                 VKLLEPTTMCVPVHGGKGAADNDSMVRVDVFLSGGKYY 
                   
                   
               
               
                   
                   
                   
                   
                   
                 LVPIYVADTLKPELPNKAVTRGKKYSEWLEMADEDFIF 
                   
                   
               
               
                   
                   
                   
                   
                   
                 SLYPNDLICATSKNGITLSVCRKDSTLPPTVESKSFML 
                   
                   
               
               
                   
                   
                   
                   
                   
                 YYRGTDISTGSISCITHDNAYKLRGLGVKTLEKLEKYT 
                   
                   
               
               
                   
                   
                   
                   
                   
                 VDVLGEYHKVGKEVRQPFNIKRRKACPSEML [SEQ 
                   
                   
               
               
                   
                   
                   
                   
                   
                 ID NO. 70] 
                   
                   
               
               
                   
               
               
                 MAD2040 
                 141 
                 DHKF01000115.1 
                 
                   Clostri- 
                 
                 Feces 
                 MHRYAIGLDIGITSVGWAAIALDAEENPCGMLDFGSRI 
                 GTTGT 
                 TTATACC 
               
               
                   
                   
                   
                 
                   diales 
                 
                   
                 FTGAEHPKTGASLAAPRREARGARRRLRRHRHRNERIR 
                 AGTTC 
                 ATACCAA 
               
               
                   
                   
                   
                 bacterium 
                   
                 RLMVSGGLISQEQLESLFAGQLEDIYALRTRALDEQVA 
                 CCTGA 
                 GAACTGC 
               
               
                   
                   
                   
                 UBA4701 
                   
                 REELARIMLHLSQRRGFRSNRKGGADAEDGKLLEAVGD 
                 TGGTT 
                 TCAGGTT 
               
               
                   
                   
                   
                   
                   
                 NKRRMDEKGYRTAGEMFFKDEAFAAHKRNKGGNYIATV 
                 CTTGG 
                 ACTATGA 
               
               
                   
                   
                   
                   
                   
                 TRAMTEDEVHRIFAAQRGFGAEYANEKLEAAYLDILLS 
                 TATGG 
                 TAAGGTA 
               
               
                   
                   
                   
                   
                   
                 QRSFDEGPGGDSPYGGSQIERMIGTCAFEPDQPRAAKA 
                 TATAA 
                 GTAAACC 
               
               
                   
                   
                   
                   
                   
                 AYSFEYFSLLEKLNHIRLVSGGKSEPLTDAQRKKLIEL 
                 T 
                 GAAGAGC 
               
               
                   
                   
                   
                   
                   
                 AHKQDTLSYAKIRKELELNEAVRFNSVRYTDDATFEEQ 
                 [SEQ 
                 TCTAATG 
               
               
                   
                   
                   
                   
                   
                 EKKEKIVCMKAYHAMRKAVDKNAKGRFAYLTIPQRNEI 
                 ID NO. 
                 CCCCGTC 
               
               
                   
                   
                   
                   
                   
                 GRVLSTYKTSAKIEPALAAAGIEPCDIAALEGLSFSKF 
                 74] 
                 TCGCACG 
               
               
                   
                   
                   
                   
                   
                 GHLSIKACDKLIPFLEKAMNYNDACAAAGYDFRGHSRD 
                   
                 GGGCATT 
               
               
                   
                   
                   
                   
                   
                 GRQMYLPPLGGDCTEITSPVVRRAVSQTIKVINAIIRR 
                   
                 ATCTCTA 
               
               
                   
                   
                   
                   
                   
                 YGTSPVYVNIELAREMSKDFAERNKIKKQNDDNRSKNE 
                   
                 ACAGCGA 
               
               
                   
                   
                   
                   
                   
                 KIKEQVAEYKHGAATGLDIVKMKLFNEQGGICAYSQRQ 
                   
                 AAAGGCA 
               
               
                   
                   
                   
                   
                   
                 MSLERLFDPNYAEVDHIVPYSISFDDRYKNKVLVLTEE 
                   
                 AA 
               
               
                   
                   
                   
                   
                   
                 NRNKGNRLPLQYLTGERRDRFIVWVNNSVRDFQKRKLL 
                   
                 [SEQ ID 
               
               
                   
                   
                   
                   
                   
                 LKEALTPEEENDWKERNLQDTKFVSSFLLNYINDNLLF 
                   
                 NO. 75] 
               
               
                   
                   
                   
                   
                   
                 APSVRRKKRVTAVNGAVTDYMRKRWGISKVREDGDRHH 
                   
                   
               
               
                   
                   
                   
                   
                   
                 AVDAVVIACTNDALIQKVSRYESWHERHYMPTENGSIL 
                   
                   
               
               
                   
                   
                   
                   
                   
                 VDPATGEIKQTFPYPWAMFRKELEARLSNDPSRAVADL 
                   
                   
               
               
                   
                   
                   
                   
                   
                 KLPFYMDADAPPVKPLFVSRMPTRKVTGAAHKDTVKSA 
                   
                   
               
               
                   
                   
                   
                   
                   
                 RALADGLAIVRRPLTALKLDKDGEIAGYYNKDSDRLLY 
                   
                   
               
               
                   
                   
                   
                   
                   
                 DALKARLTEYGGNAAKAFAEPFYKPKSDGTPGPVVNKV 
                   
                   
               
               
                   
                   
                   
                   
                   
                 KLTEPTTLSVPVQDGTGIADNDSMVRIDVFRVVGDGYY 
                   
                   
               
               
                   
                   
                   
                   
                   
                 FVPVYVADTLKQELPDRAVVAFKAHSEWKVMSDGDFVF 
                   
                   
               
               
                   
                   
                   
                   
                   
                 SLYPNDLVKVTRKKDVILKRSFDNSTLPETIASNECLL 
                   
                   
               
               
                   
                   
                   
                   
                   
                 YYAGADISTGAISCVTNDNAYSIRGLGIKTLVSMEKYT 
                   
                   
               
               
                   
                   
                   
                   
                   
                 VDILGEYHPVRKEERQRFNTKR [SEQ ID NO. 73] 
               
               
                   
               
            
           
         
       
     
     Example 3 
     Vector Cloning, MADZYME Library Construction and PCR 
     The MADzyme coding sequences were cloned into a pUC57 vector with T7-promoter sequence attached to the 5′-end of the coding sequence and a T7-terminator sequence attached to the 3′-end of the coding sequence. 
     First, Q5 Hot Start 2× master mix reagent (NEB, Ipswich, MA) was used to amplify the MADzyme sequences cloned in the pUC57 vector. The forward primer 5′-TTGGGTAACGCCAGGGTTTT [SEQ ID No. 172] and reverse primer 5′-TGTGTGGAATTGTGAGCGGA [SEQ ID No. 173] amplified the sequences flanking the MADzyme in the pUC57 vector including the T7-promoter and T7-terminator components at the 5′- and 3′-end of the MADzymes, respectively. 1 μM primers were used in a 10 μL PCR reaction using 3.3 μL boiled cell samples as templates in 96 well PCR plates. The PCR conditions shown in Table 2 were used: 
     
       
         
           
               
               
               
               
             
               
                   
                 TABLE 2 
               
               
                   
                   
               
               
                   
                 STEP 
                 TEMPERATURE 
                 TIME 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 DENATURATION 
                 98° C. 
                 30 
                 SEC 
               
               
                   
                 30 CYCLES 
                 98° C. 
                 10 
                 SEC 
               
               
                   
                   
                 66° C. 
                 30 
                 SEC 
               
               
                   
                   
                 72° C. 
                 3 
                 MIN 
               
               
                   
                 FINAL EXTENSION 
                 72° C. 
                 2 
                 MIN 
               
               
                   
                 HOLD 
                 12° C. 
               
               
                   
                   
               
            
           
         
       
     
     Example 4 
     gRNA Construction 
     Several functional gRNAs associated with each MADzyme was designed by truncating the 5′ region, the 3′ region and the repeat/anti-repeat duplex (see Table 3). 
     
       
         
           
               
               
               
               
               
               
             
               
                 TABLE 3 
               
               
                   
               
               
                 gRNA  
                   
                   
                   
                   
                   
               
               
                 name 
                 sgRNAv1 
                 sgRNAv2 
                 sgRNAv3 
                 sgRNAv4 
                 sgRNAv5 
               
               
                   
               
             
            
               
                 sgM 
                 GTTTTAGAGCTATGC 
                 GTTTTAGAGCTATGC 
                 GTTTTAGAGCTATGC 
                 GTTTTAGAGCT 
                 NONE 
               
               
                 2015 
                 TGTTTTGAATGCTTC 
                 TGTTTTGAATGCTTC 
                 TGTTAACAACATAGC 
                 ATGCAAACATA 
                   
               
               
                   
                 CAAAACGAAATGTTG 
                 GTAGCATTCAAAACA 
                 AAGTTAAAATAAGGC 
                 GCAAGTTAAAA 
                   
               
               
                   
                 GTAGCATTCAAAACA 
                 ACATAGCAAGTTAAA 
                 TTTGTCCGTTCTCAA 
                 TAAGGCTTTGT 
                   
               
               
                   
                 ACATAGCAAGTTAAA 
                 ATAAGGCTTTGTCCG 
                 CTTTTAGTGACGCTG 
                 CCGTTCTCAAC 
                   
               
               
                   
                 ATAAGGCTTTGTCCG 
                 TTCTCAACTTTTAGT 
                 TTTCGGCG 
                 TTTTAGTGACG 
                   
               
               
                   
                 TTCTCAACTTTTAGT 
                 GACGCTGTTTCGGCG 
                 [SEQ ID NO. 78] 
                 CTGTTTCGGCG 
                   
               
               
                   
                 GACGCTGTTTCGGCG 
                 [SEQ ID NO. 77] 
                   
                 [SEQ ID NO. 
                   
               
               
                   
                 [SEQ ID NO. 76] 
                   
                   
                 79] 
                   
               
               
                   
               
               
                 sgM 
                 GTTTTAGAGTCATGT 
                 GTTTTAGAGTCATGT 
                 GTTTTAGAGTCATGT 
                 NONE 
                 NONE 
               
               
                 2016 
                 TGTTTAGAATGGTAC 
                 TGTAAAAACAACATA 
                 TGTAAAAACAACATA 
                   
                   
               
               
                   
                 CAAAACATCTTTTGG 
                 GCAAGTTAAAATAAG 
                 GCAAGTTAAAATAAG 
                   
                   
               
               
                   
                 GACTATTCTAAACAA 
                 GTTTTAACCGTAATC 
                 CGTAATCAACTGTAA 
                   
                   
               
               
                   
                 CATAGCAAGTTAAAA 
                 AACTGTAAAGTGGCG 
                 AGTGGCGCTGTTTCG 
                   
                   
               
               
                   
                 TAAGGTTTTAACCGT 
                 CTGTTTCGGCGC 
                 GCGC 
                   
                   
               
               
                   
                 AATCAACTGTAAAGT 
                 [SEQ ID NO. 81] 
                 [SEQ ID NO. 82] 
                   
                   
               
               
                   
                 GGCGCTGTTTCGGCG 
                   
                   
                   
                   
               
               
                   
                 C [SEQ ID NO. 
                   
                   
                   
                   
               
               
                   
                 80] 
                   
                   
                   
                   
               
               
                   
               
               
                 sgM 
                 GTTTTAGAGCTGTGC 
                 GTTTTAGAGCTGTGC 
                 GTTTTAGAGCTGTGC 
                 GTTTTAGAGCT 
                 NONE 
               
               
                 2017 
                 TGTTTCGAATGGTTC 
                 TGTTTCGAAAAATCG 
                 TGTAAAAACAACACA 
                 GTGCAAACACA 
                   
               
               
                   
                 CAAAACGAAATGTTG 
                 AAACAACACAGCGAG 
                 GCGAGTTAAAATAAG 
                 GCGAGTTAAAA 
                   
               
               
                   
                 GAACTATTCGAAACA 
                 TTAAAATAAGGCTTT 
                 GCTTTGTCCGTACAC 
                 TAAGGCTTTGT 
                   
               
               
                   
                 ACACAGCGAGTTAAA 
                 GTCCGTACACAACTT 
                 AACTTGTAAAAGGGG 
                 CCGTACACAAC 
                   
               
               
                   
                 ATAAGGCTTTGTCCG 
                 GTAAAAGGGGCACCC 
                 CACCCGATTCGGGTG 
                 TTGTAAAAGGG 
                   
               
               
                   
                 TACACAACTTGTAAA 
                 GATTCGGGTGC 
                 C 
                 GCACCCGATTC 
                   
               
               
                   
                 AGGGGCACCCGATTC 
                 [SEQ ID NO. 84] 
                 [SEQ ID NO. 85] 
                 GGGTGC 
                   
               
               
                   
                 GGGTGCA 
                   
                   
                 [SEQ ID NO. 
                   
               
               
                   
                 [SEQ ID NO. 83] 
                   
                   
                 86] 
                   
               
               
                   
               
               
                 sgM 
                 GTTTTAGAGCTGTGT 
                 GTTTTAGAGCTGTGT 
                 GTTTTAGAGCTGTGT 
                 NONE 
                 NONE 
               
               
                 2019 
                 TGTTTCGAATGGTTC 
                 TGTAAAAACAATACA 
                 TGTAAAAACAATACA 
                   
                   
               
               
                   
                 CAAAACGGTTTGAAA 
                 GCAAAGTTAAAATAA 
                 GCAAGTTAAAATAAG 
                   
                   
               
               
                   
                 CCATTCGAAACAATA 
                 GGCTAGTCCGTATAC 
                 GCTAGTCCGTATACA 
                   
                   
               
               
                   
                 CAGCAAAGTTAAAAT 
                 AACGTGAAAACACGT 
                 ACGTGAAAACACGTG 
                   
                   
               
               
                   
                 AAGGCTAGTCCGTAT 
                 GGCACCGATTCGGTG 
                 GCACCGATTCGGTGC 
                   
                   
               
               
                   
                 ACAACGTGAAAACAC 
                 C 
                 [SEQ ID NO. 89 
                   
                   
               
               
                   
                 GTGGCACCGATTCGG 
                 [SEQ ID NO. 88] 
                   
                   
                   
               
               
                   
                 TGC [SEQ ID NO. 
                   
                   
                   
                   
               
               
                   
                 87] 
                   
                   
                   
                   
               
               
                   
               
               
                 sgM 
                 GTTTGCTAGTTATGT 
                 GTTTGCTAGTTATGT 
                 GTTTGCTAGTTATGT 
                 NONE 
                 NONE 
               
               
                 2020 
                 TATTTATAGTATTAA 
                 TATAAAAATAACATA 
                 TATAAAAATAACATA 
                   
                   
               
               
                   
                 GCAAACTGTAAATAA 
                 ACGAGTGCAAATAAG 
                 ACGAGTGCAAATAAG 
                   
                   
               
               
                   
                 CATAACGAGTGCAAA 
                 CGTTTCGCGAAAATT 
                 CGTTTCGCGAAAATT 
                   
                   
               
               
                   
                 TAAGCGTTTCGCGAA 
                 TACAGTGGCCCTGCT 
                 TACAGTGGCCCTGCT 
                   
                   
               
               
                   
                 AATTTACAGTGGCCC 
                 GTGGGGCCTTTTTTA 
                 GTGGGGCC 
                   
                   
               
               
                   
                 TGCTGTGGGGCCTTT 
                 TTTATCAAA 
                 [SEQ ID NO. 92] 
                   
                   
               
               
                   
                 TTTATTTATCAAA 
                 [SEQ ID NO. 91] 
                   
                   
                   
               
               
                   
                 [SEQ ID NO. 90] 
                   
                   
                   
                   
               
               
                   
               
               
                 sgM 
                 GTTTGAGAGCCTTGT 
                 NONE 
                 NONE 
                 NONE 
                 NONE 
               
               
                 2021 
                 AAAACCGTATATCTC 
                   
                   
                   
                   
               
               
                   
                 TCAAGCGAAAGATAA 
                   
                   
                   
                   
               
               
                   
                 TGTTTTACAAGGCGA 
                   
                   
                   
                   
               
               
                   
                 GTTCAAATAAGGATT 
                   
                   
                   
                   
               
               
                   
                 TATCCGAAATCGCTT 
                   
                   
                   
                   
               
               
                   
                 GCGTGCATTGGCACC 
                   
                   
                   
                   
               
               
                   
                 ATCTATCTTTTAAGA 
                   
                   
                   
                   
               
               
                   
                 CTTTCTTTGAAAGTC 
                   
                   
                   
                   
               
               
                   
                 TT [SEQ ID NO. 
                   
                   
                   
                   
               
               
                   
                 93] 
                   
                   
                   
                   
               
               
                   
               
               
                 sgM 
                 GTTTGAGAGTCTTGT 
                 GTTTGAGAGTCTTGT 
                 GTTTGAGAGTCTTGT 
                 GTTTGAGAGTC 
                 NONE 
               
               
                 2022 
                 TAATTCTTAAAGGTG 
                 AAAAACAAGACGAGT 
                 AAAAACAAGACGAGT 
                 TTGTTAATTCA 
                   
               
               
                   
                 TAAAACGAGAATTAA 
                 GCAAATAAGGTTTAT 
                 GCAAATAAGGTTTAT 
                 AAAGAATTAAC 
                   
               
               
                   
                 CAAGACGAGTGCAAA 
                 CCGGAATCGTCAATA 
                 CCGGAATCGTCAATA 
                 AAGACGAGTGC 
                   
               
               
                   
                 TAAGGTTTATCCGGA 
                 TGACCTGCATTGTGC 
                 TGACCTGCATTGTGC 
                 AAATAAGGTTT 
                   
               
               
                   
                 ATCGTCAATATGACC 
                 AGAATCTTTAAAATC 
                 AG [SEQ ID NO. 
                 ATCCGGAATCG 
                   
               
               
                   
                 TGCATTGTGCAGAAT 
                 ATATGATTTCATATG 
                 96] 
                 TCAATATGACC 
                   
               
               
                   
                 CTTTAAAATCATATG 
                 GTTTTA [SEQ ID 
                   
                 TGCATTGTGCA 
                   
               
               
                   
                 ATTTCATATGGTTTT 
                 NO. 95] 
                   
                 GAATCTTTAAA 
                   
               
               
                   
                 A [SEQ ID NO. 
                   
                   
                 ATCATATGATT 
                   
               
               
                   
                 94] 
                   
                   
                 TCATATGGTTT 
                   
               
               
                   
                   
                   
                   
                 TA [SEQ ID 
                   
               
               
                   
                   
                   
                   
                 NO. 97] 
                   
               
               
                   
               
               
                 sgM 
                 GTTTGAGAGTAGTGT 
                 NONE 
                 NONE 
                 NONE 
                 NONE 
               
               
                 2023 
                 AAATCCATAGGGGTC 
                   
                   
                   
                   
               
               
                   
                 TCAAACGAAAAGACC 
                   
                   
                   
                   
               
               
                   
                 CCTATGGATTTACAT 
                   
                   
                   
                   
               
               
                   
                 TGCGAGTTCAAATAA 
                   
                   
                   
                   
               
               
                   
                 AAGTTTACTCAAATC 
                   
                   
                   
                   
               
               
                   
                 GTTGGCTTGACCAAC 
                   
                   
                   
                   
               
               
                   
                 CGCACAGCGTGTGCT 
                   
                   
                   
                   
               
               
                   
                 TAAAGATCTCTTCAG 
                   
                   
                   
                   
               
               
                   
                 TGAGGTC [SEQ ID 
                   
                   
                   
                   
               
               
                   
                 NO. 98] 
                   
                   
                   
                   
               
               
                   
               
               
                 sgM 
                 GTTTGAGAGTAGTGT 
                 NONE 
                 NONE 
                 NONE 
                 NONE 
               
               
                 2024 
                 AAATCCAGAGGGCTC 
                   
                   
                   
                   
               
               
                   
                 CAAAACGAGCCCTCT 
                   
                   
                   
                   
               
               
                   
                 GGATTTACACTACGA 
                   
                   
                   
                   
               
               
                   
                 GTTCAAATAAAAATT 
                   
                   
                   
                   
               
               
                   
                 ATTTCAAATCGCCGC 
                   
                   
                   
                   
               
               
                   
                 TATGTCGGCCGCACA 
                   
                   
                   
                   
               
               
                   
                 GTGTGTGCATTAAGA 
                   
                   
                   
                   
               
               
                   
                 AAAGTCCGAAAGGGC 
                   
                   
                   
                   
               
               
                   
                 [SEQ ID NO. 99] 
                   
                   
                   
                   
               
               
                   
               
               
                 sgM 
                 GTTTGAGAGTAGTGT 
                 GTTTGAGAGTAGTGT 
                 GTTTGAGAGTAGTGT 
                 GTTTGAGAGTA 
                 NONE 
               
               
                 2025 
                 AAATTTATAGGGTAG 
                 AAAAATACACTACGA 
                 AAAAATACACTACGA 
                 GTGTAAATTTA 
                   
               
               
                   
                 TAAAACAAATTTTAC 
                 GTTCAAATAAAAATT 
                 GTTCAAATAAAAATT 
                 TAGGAAAACCT 
                   
               
               
                   
                 TACCCTATAAATTTA 
                 ATTTCAAATCGTACT 
                 ATTTCAAATCGTACT 
                 ATAAATTTACA 
                   
               
               
                   
                 CACTACGAGTTCAAA 
                 TTTTAGTACCTTCAC 
                 TTTTAGTACCTTCAC 
                 CTACGAGTTCA 
                   
               
               
                   
                 TAAAAATTATTTCAA 
                 AAGTGTTGTGAATAT 
                 AAGTGTTGTGAA 
                 AATAAAAATTA 
                   
               
               
                   
                 ATCGTACTTTTTAGT 
                 TAACTCACCTTCGGG 
                 [SEQ ID NO. 
                 TTTCAAATCGT 
                   
               
               
                   
                 ACCTTCACAAGTGTT 
                 TGAG [SEQ ID 
                 102] 
                 ACTTTTTAGTA 
                   
               
               
                   
                 GTGAATATTAACTCA 
                 NO. 101] 
                   
                 CCTTCACAAGT 
                   
               
               
                   
                 CCTTCGGGTGAG 
                   
                   
                 GTTGTGAATAT 
                   
               
               
                   
                 [SEQ ID NO. 
                   
                   
                 TAACTCACCTT 
                   
               
               
                   
                 100] 
                   
                   
                 CGGGTGAG 
                   
               
               
                   
                   
                   
                   
                 [SEQ ID NO.  
                   
               
               
                   
                   
                   
                   
                 103] 
                   
               
               
                   
               
               
                 sgM 
                 GTTTGAGAGTAGTGT 
                 NONE 
                 NONE 
                 NONE 
                 NONE 
               
               
                 2026 
                 AATTTCATATGGTAG 
                   
                   
                   
                   
               
               
                   
                 TCAAACGACTACCAT 
                   
                   
                   
                   
               
               
                   
                 ATGAGATTACACTAC 
                   
                   
                   
                   
               
               
                   
                 ACGGTTCAAATAAAG 
                   
                   
                   
                   
               
               
                   
                 AATGTTCGAAACCGC 
                   
                   
                   
                   
               
               
                   
                 CCTTTGGGGCCCGCT 
                   
                   
                   
                   
               
               
                   
                 TGTTGCGGATTTACA 
                   
                   
                   
                   
               
               
                   
                 GACTTGATATCAAGT 
                   
                   
                   
                   
               
               
                   
                 CTG [SEQ ID NO. 
                   
                   
                   
                   
               
               
                   
                 104] 
                   
                   
                   
                   
               
               
                   
               
               
                 sgM 
                 GTTTGAGAGTAATGT 
                 GTTTGAGAGTAATGT 
                 GTTTGAGAGTAATGT 
                 GTTTGAGAGTA 
                 NONE 
               
               
                 2027 
                 AAATTCATAGGATGG 
                 AAAAATACATTACAA 
                 AAAAATACATTACAA 
                 ATGTAAATTCA 
                   
               
               
                   
                 TAAAACGAAATTTAC 
                 GTTCAAATAAAAATT 
                 GTTCAAATAAAAATT 
                 TAAAAGTGAGT 
                   
               
               
                   
                 CATCCAGTGAGTTTA 
                 TATTCAACCCGTTCT 
                 TATTCAACCCGTTCT 
                 TTACATTACAA 
                   
               
               
                   
                 CATTACAAGTTCAAA 
                 TCGGAACCTCCACCG 
                 TCGGAACCTCCACCG 
                 GTTCAAATAAA 
                   
               
               
                   
                 TAAAAATTTATTCAA 
                 TGTGGAACATTAAGG 
                 TGTGGA [SEQ ID 
                 AATTTATTCAA 
                   
               
               
                   
                 CCCGTTCTTCGGAAC 
                 TCTGCTTTGCAGGCC 
                 NO. 107] 
                 CCCGTTCTTCG 
                   
               
               
                   
                 CTCCACCGTGTGGAA 
                 [SEQ ID NO. 
                   
                 GAACCTCCACC 
                   
               
               
                   
                 C [SEQ ID NO. 
                 106] 
                   
                 GTGTGGAACAT 
                   
               
               
                   
                 105] 
                   
                   
                 TAAG [SEQ 
                   
               
               
                   
                   
                   
                   
                 ID NO. 108] 
                   
               
               
                   
               
               
                 sgM 
                 GTTTGAGAGCAGTGT 
                 NONE 
                 NONE 
                 NONE 
                 NONE 
               
               
                 2028 
                 TGTCTTATATAGCTC 
                   
                   
                   
                   
               
               
                   
                 GAAAACGCATTGTAA 
                   
                   
                   
                   
               
               
                   
                 GACAACACTGCTACG 
                   
                   
                   
                   
               
               
                   
                 TTCAAATAAGCATAT 
                   
                   
                   
                   
               
               
                   
                 TGCTACAAGGTTCTC 
                   
                   
                   
                   
               
               
                   
                 CCTCGGAGAATGACC 
                   
                   
                   
                   
               
               
                   
                 ATTAGGTCACTTAGA 
                   
                   
                   
                   
               
               
                   
                 TAGCCGGTTCTTCTG 
                   
                   
                   
                   
               
               
                   
                 GCTA [SEQ ID 
                   
                   
                   
                   
               
               
                   
                 NO. 109] 
                   
                   
                   
                   
               
               
                   
               
               
                 sgM 
                 GTTTGAGAGCAGTGT 
                 GTTTGAGAGCAGTGT 
                 GTTTGAGAGCAGTGT 
                 GTTTGAGAGCA 
                 NONE 
               
               
                 2029 
                 TGTCTTATATAGCTC 
                 AAAAACACTGCTACG 
                 AAAAACACTGCTACG 
                 GTGTTGTCAAA 
                   
               
               
                   
                 GAAAACGCATTGTAA 
                 TTCAAATAAGCATAT 
                 TTCAAATAAGCATAT 
                 AGACAACACTG 
                   
               
               
                   
                 GACAACACTGCTACG 
                 TGCTACAAGGTTCTC 
                 TGCTACAAGGTTCTC 
                 CTACGTTCAAA 
                   
               
               
                   
                 TTCAAATAAGCATAT 
                 CATTGGAGAATGACC 
                 CATTGGAGAATGACC 
                 TAAGCATATTG 
                   
               
               
                   
                 TGCTACAAGGTTCTC 
                 ATTAGGTCGCTTAGA 
                 ATTAGGTC [SEQ 
                 CTACAAGGTTC 
                   
               
               
                   
                 CATTGGAGAATGACC 
                 TAGCCAGTTCTTCTG 
                 ID NO. 112] 
                 TCCATTGGAGA 
                   
               
               
                   
                 ATTAGGTCGCTTAGA 
                 GCTA [SEQ ID 
                   
                 ATGACCATTAG 
                   
               
               
                   
                 TAGCCAGTTCTTCTG 
                 NO. 111] 
                   
                 GTCGCTTAGAT 
                   
               
               
                   
                 GCTA [SEQ ID 
                   
                   
                 AGCCAGTTCTT 
                   
               
               
                   
                 NO. 110] 
                   
                   
                 CTGGCTA 
                   
               
               
                   
                   
                   
                   
                 [SEQ ID NO.  
                   
               
               
                   
                   
                   
                   
                 113] 
                   
               
               
                   
               
               
                 sgM 
                 GTTTGAGAGCAGTGT 
                 NONE 
                 NONE 
                 NONE 
                 NONE 
               
               
                 2030 
                 TGTCTTAAATAGCTC 
                   
                   
                   
                   
               
               
                   
                 GAAAACGCATTGTAA 
                   
                   
                   
                   
               
               
                   
                 GACAACACTGCACGT 
                   
                   
                   
                   
               
               
                   
                 TCAAATAAGCAGATT 
                   
                   
                   
                   
               
               
                   
                 GCTACAAGGTTCCCG 
                   
                   
                   
                   
               
               
                   
                 TAAGGGAATGACCAT 
                   
                   
                   
                   
               
               
                   
                 CTGGTCACATGAATA 
                   
                   
                   
                   
               
               
                   
                 GCCCCCGGCAACGGT 
                   
                   
                   
                   
               
               
                   
                 GGCTG [SEQ ID 
                   
                   
                   
                   
               
               
                   
                 NO. 114] 
                   
                   
                   
                   
               
               
                   
               
               
                 sgM 
                 ATTGTACCATAGCGA 
                 NONE 
                 NONE 
                 NONE 
                 NONE 
               
               
                 2031 
                 GTTAAATTAGGGAAT 
                   
                   
                   
                   
               
               
                   
                 TACAACGAAATTGTA 
                   
                   
                   
                   
               
               
                   
                 ATAACCTATTTTACC 
                   
                   
                   
                   
               
               
                   
                 TCGCTATGGCACAAT 
                   
                   
                   
                   
               
               
                   
                 TTGTTATTACATGGA 
                   
                   
                   
                   
               
               
                   
                 CATTATACTAAACAT 
                   
                   
                   
                   
               
               
                   
                 TTCCTAAAAAAGCAA 
                   
                   
                   
                   
               
               
                   
                 CGAAAAACGTGCT 
                   
                   
                   
                   
               
               
                   
                 [SEQ ID NO. 
                   
                   
                   
                   
               
               
                   
                 115] 
                   
                   
                   
                   
               
               
                   
               
               
                 sgM 
                 GTTGTAGTTCCCTAA 
                 GTTGTAGTTCCCTAA 
                 GTTGTAGTTCCCTAA 
                 GTTGTAGTTCC 
                 NONE 
               
               
                 2032 
                 TTATTCTTGGTATGG 
                 TTATTCTTGGTAAAA 
                 TTATTCTTGGTAAAA 
                 CTAATTATTCT 
                   
               
               
                   
                 TATAATGAAAATTGT 
                 ACCAAGAACAATTAG 
                 ACCAAGAACAATTAG 
                 TGGTATGGTAA 
                   
               
               
                   
                 ATCATACCAAGAACA 
                 GTTACTATGATAAGG 
                 GTTACTATGATAAGG 
                 AAATATCATAC 
                   
               
               
                   
                 ATTAGGTTACTATGA 
                 TAGTATACCGCAAAG 
                 TAGTATACCGCAAAG 
                 CAAGAACAATA 
                   
               
               
                   
                 TAAGGTAGTATACCG 
                 CTCTAACACCTCATC 
                 CTCTAACACCTCATC 
                 GGTTACTATGA 
                   
               
               
                   
                 CAAAGCTCTAACACC 
                 TTCGGATGAGGTGTT 
                 TTCGGATGAG [SEQ 
                 TAAGGTAGTAT 
                   
               
               
                   
                 TCATCTTCGGATGAG 
                 A [SEQ ID NO. 
                 ID NO. 118] 
                 ACCGCAAAGCT 
                   
               
               
                   
                 GTGTTATCT [SEQ 
                 117] 
                   
                 CTAACACCTCA 
                   
               
               
                   
                 ID NO. 116] 
                   
                   
                 TCTTCGGATGA 
                   
               
               
                   
                   
                   
                   
                 GGTGTTATCT 
                   
               
               
                   
                   
                   
                   
                 [SEQ ID NO.  
                   
               
               
                   
                   
                   
                   
                 119] 
                   
               
               
                   
               
               
                 sgM 
                 GTTGTAGTTCCCTAA 
                 GTTGTAGTTCCCTAA 
                 GTTGTAGTTCCCTAA 
                 GTTGTAGTTCC 
                 NONE 
               
               
                 2033 
                 CAGTTCTTGGTATGG 
                 CAGTTCTAAAAAGAA 
                 CAGTTCTAAAAAGAA 
                 CTAACAGTAAA 
                   
               
               
                   
                 TATAATAAAAATTAT 
                 CTGTTATGGTTGCTA 
                 CTGTTATGGTTGCTA 
                 AACTGTTATGG 
                   
               
               
                   
                 ACCATACCAAGAACT 
                 TGATAAGGTCTTAGC 
                 TGATAAGGTCTTAGC 
                 TTGCTATGATA 
                   
               
               
                   
                 GTTATGGTTGCTATG 
                 ACCGTAAAGCTCTGA 
                 ACCGTAAAGCTCTGA 
                 AGGTCTTAGCA 
                   
               
               
                   
                 ATAAGGTCTTAGCAC 
                 CGCCTCGCTTTCAGC 
                 CGCCTCGCTTTCAGC 
                 CCGTAAAGCTC 
                   
               
               
                   
                 CGTAAAGCTCTGACG 
                 GGGGCGTCA [SEQ 
                 GGGG [SEQ ID 
                 TGACGCCTCGC 
                   
               
               
                   
                 CCTCGCTTTCAGCGG 
                 ID NO. 121] 
                 NO. 122] 
                 TTTCAGCGGGG 
                   
               
               
                   
                 GGCGTCATCTTTTTT 
                   
                   
                 CGTCA 
                   
               
               
                   
                 GCCCAAAAGACACGG 
                   
                   
                 [SEQ ID NO.  
                   
               
               
                   
                 ATATTTTT [SEQ 
                   
                   
                 123] 
                   
               
               
                   
                 ID NO. 120] 
                   
                   
                   
                   
               
               
                   
               
               
                 sgM 
                 GTTGTAGTTCCCTAA 
                 GTTGTAGTTCCCTAA 
                 GTTGTAGTTCCCTAA 
                 GTTGTAGTTCC 
                 NONE 
               
               
                 2034 
                 CGGTTCTTGGTATGG 
                 CGGTACTGTTGGGTT 
                 CGGTACTGTTGGGTT 
                 CTAACGGTTCT 
                   
               
               
                   
                 TATAATGAATTATAC 
                 ACTACAATAAGGTAG 
                 ACTACAATAAGGTAG 
                 TGAAAACAAGA 
                   
               
               
                   
                 CATACCAAGAACTGT 
                 TAAACCGAAAAGCTC 
                 TAAACCGAAAAGCTC 
                 ACTGTTGGGTT 
                   
               
               
                   
                 TGGGTTACTACAATA 
                 TGACGTCTTGTTTGC 
                 TGACGTCTTGTTTGC 
                 ACTACAATAAG 
                   
               
               
                   
                 AGGTAGTAAACCGAA 
                 GCAGGACGTCATCTT 
                 GCAGGACGTCATCTT 
                 GTAGTAAACCG 
                   
               
               
                   
                 AAGCTCTGACGTCTT 
                 TATATCAGACGGATG 
                 T [SEQ ID NO. 
                 AAAAGCTCTGA 
                   
               
               
                   
                 GTTTGCGCAGGACGT 
                 [SEQ ID NO. 
                 126] 
                 CGTCTTGTTTG 
                   
               
               
                   
                 CATCTTTATATCAGA 
                 125] 
                   
                 CGCAGGACGTC 
                   
               
               
                   
                 CGGATG [SEQ ID 
                   
                   
                 ATCTTTATATC 
                   
               
               
                   
                 NO. 124] 
                   
                   
                 AGACGGATG 
                   
               
               
                   
                   
                   
                   
                 [SEQ ID NO.  
                   
               
               
                   
                   
                   
                   
                 127] 
                   
               
               
                   
               
               
                 sgM 
                 GTTGTAGTCCCCTGA 
                 NONE 
                 NONE 
                 NONE 
                 NONE 
               
               
                 2035 
                 TGGTTTCTGGAATGG 
                   
                   
                   
                   
               
               
                   
                 TATAATGAAATTATA 
                   
                   
                   
                   
               
               
                   
                 CCATTCCAGAAACTA 
                   
                   
                   
                   
               
               
                   
                 TTATGGTCACTACAA 
                   
                   
                   
                   
               
               
                   
                 TAAGGTATTAGACCG 
                   
                   
                   
                   
               
               
                   
                 TAGAGCACTAACACC 
                   
                   
                   
                   
               
               
                   
                 CCATTTGGGGTGTTA 
                   
                   
                   
                   
               
               
                   
                 TCTCTTTAAACTGTC 
                   
                   
                   
                   
               
               
                   
                 CAAAATTTAGTATTG 
                   
                   
                   
                   
               
               
                   
                 CAATTATTGA [SEQ 
                   
                   
                   
                   
               
               
                   
                 ID NO. 128] 
                   
                   
                   
                   
               
               
                   
               
               
                 sgM 
                 GTTATAGTTCCCTGT 
                 NONE 
                 NONE 
                 NONE 
                 NONE 
               
               
                 2036 
                 TCGTTCTTGGTATGG 
                   
                   
                   
                   
               
               
                   
                 TATAATGAAATTATA 
                   
                   
                   
                   
               
               
                   
                 CCATACCAAGAACGA 
                   
                   
                   
                   
               
               
                   
                 AGCAGGTTACTATGA 
                   
                   
                   
                   
               
               
                   
                 TAAGGTAGTATACCG 
                   
                   
                   
                   
               
               
                   
                 CAGAGCTCCAACGCC 
                   
                   
                   
                   
               
               
                   
                 TCGCTTTTGCGGGGC 
                   
                   
                   
                   
               
               
                   
                 GTTGTCTCT [SEQ 
                   
                   
                   
                   
               
               
                   
                 ID NO. 128] 
                   
                   
                   
                   
               
               
                   
               
               
                 sgM 
                 GTTATAGTTCCCTGA 
                 NONE 
                 NONE 
                 NONE 
                 NONE 
               
               
                 2037 
                 TAGTTCTTGGTATGG 
                   
                   
                   
                   
               
               
                   
                 TATAATGAAATTATA 
                   
                   
                   
                   
               
               
                   
                 CCATACCAAGAACTA 
                   
                   
                   
                   
               
               
                   
                 TGAGGTTGCTATAAT 
                   
                   
                   
                   
               
               
                   
                 AAGGTAGTAAACCGC 
                   
                   
                   
                   
               
               
                   
                 AGAGCTCTAACGCCT 
                   
                   
                   
                   
               
               
                   
                 CACATTTGTGGGGCG 
                   
                   
                   
                   
               
               
                   
                 TTATCTCT [SEQ 
                   
                   
                   
                   
               
               
                   
                 ID NO. 129] 
                   
                   
                   
                   
               
               
                   
               
               
                 sgM 
                 GTTGTAGTTCCCTGA 
                 NONE 
                 NONE 
                 NONE 
                 NONE 
               
               
                 2038 
                 TCGTTCTTGGTATGG 
                   
                   
                   
                   
               
               
                   
                 TATAATGAAATTATA 
                   
                   
                   
                   
               
               
                   
                 CCATACCAAGAACGA 
                   
                   
                   
                   
               
               
                   
                 TCAGGTTGCTACAAT 
                   
                   
                   
                   
               
               
                   
                 AAGGTAGTAAACCGA 
                   
                   
                   
                   
               
               
                   
                 AGAGCTCTAACGCCC 
                   
                   
                   
                   
               
               
                   
                 CGTTTCTTTACGGGG 
                   
                   
                   
                   
               
               
                   
                 CGTTATCTCT [SEQ 
                   
                   
                   
                   
               
               
                   
                 ID NO. 130] 
                   
                   
                   
                   
               
               
                   
               
               
                 sgM 
                 GTTATAGTTCCCTGA 
                 GTTATAGTTCCCTGA 
                 GTTATAGTTCCCTGA 
                 GTTATAGTTCC 
                 GTTATAGTTC 
               
               
                 2039 
                 TAGTTCTTGGTATGG 
                 TAGTTCTTGGTATGG 
                 TAGTTCTTAACCAAG 
                 CTGATAGTTCT 
                 CCTGATAGTT 
               
               
                   
                 TATAATGAATTATAC 
                 TATAATGAATTATAC 
                 AACTATTTAGGTTAC 
                 TGCAAGAACTA 
                 CTTGCAAGAA 
               
               
                   
                 CATACCAAGAACTAT 
                 CATACCAAGAACTAT 
                 TATGATAAGGTTTAG 
                 TTTAGGTTACT 
                 CTATTTAGGT 
               
               
                   
                 TTAGGTTACTATGAT 
                 TTAGGTTACTATGAT 
                 TACACCTTAGAGCTC 
                 ATGATAAGGTT 
                 TACTATGATA 
               
               
                   
                 AAGGTTTAGTACACC 
                 AAGGTTTAGTACACC 
                 TGACGCCTCGCTTTT 
                 TAGTACACCTT 
                 AGGTTTAGTA 
               
               
                   
                 TTAGAGCTCTGACGC 
                 TTAGAGCTCTGACGC 
                 GCGAGGCGTTATCTC 
                 AGAGCTCTGAC 
                 CACCTTAGAG 
               
               
                   
                 CTCGCTTTTGCGAGG 
                 CTCGCTTTTGCGAGG 
                 T [SEQ ID NO. 
                 GCCTCGCTTTT 
                 CTCTGACGCC 
               
               
                   
                 CGTTATCTCTTTATA 
                 CGTTATCTCT [SEQ 
                 133] 
                 GCGAGGCGTTA 
                 AAAAGGCGTT 
               
               
                   
                 TTGCCAAAAATGCAA 
                 ID NO. 132] 
                   
                 TCTCT 
                 ATCTCT 
               
               
                   
                 ATATATCGTACAATG 
                   
                   
                 [SEQ ID 
                 [SEQ ID 
               
               
                   
                 GTGGC [SEQ ID 
                   
                   
                 NO. 134]  
                 NO. 135] 
               
               
                   
                 NO. 131] 
                   
                   
                   
                   
               
               
                   
               
               
                 sgM 
                 GTTGTAGTTCCCTGA 
                 NONE 
                 GTTGTAGTTCCCTGA 
                 GTTGTAGTTCC 
                 NONE 
               
               
                 2040 
                 TGGTTCTTGGTATGG 
                   
                 TGGTTCTTGAAAAAG 
                 CTGATGGTTCT 
                   
               
               
                   
                 TATAATAAATTATAC 
                   
                 AACTGCTCAGGTTAC 
                 TGAAAAAGAAC 
                   
               
               
                   
                 CATACCAAGAACTGC 
                   
                 TATGATAAGGTAGTA 
                 TGCTCAGGTTA 
                   
               
               
                   
                 TCAGGTTACTATGAT 
                   
                 AACCGAAGAGCTCTA 
                 CTATGATAAGG 
                   
               
               
                   
                 AAGGTAGTAAACCGA 
                   
                 ATGCCCCGTCTCGCA 
                 TAGTAAACCGA 
                   
               
               
                   
                 AGAGCTCTAATGCCC 
                   
                 CGGGGCATTATCTCT 
                 AGAGCTCTAAT 
                   
               
               
                   
                 CGTCTCGCACGGGGC 
                   
                 [SEQ ID NO. 
                 GCCAAAGGGCA 
                   
               
               
                   
                 ATTATCTCT [SEQ 
                   
                 137] 
                 TTATCTCT 
                   
               
               
                   
                 ID NO. 136] 
                   
                   
                 [SEQ ID NO.  
                   
               
               
                   
                   
                   
                   
                 138] 
               
               
                   
               
            
           
         
       
     
     To find the optimal gRNA length, different lengths of spacer, repeat:anti-repeat duplex and 3′ end of the tracrRNA were included. These gRNAs were then synthesized as a single stranded DNA downstream of the T7 promoter (see Table 4). These sgRNAs were amplified using two primers (5′-AAACCCCTCCGTTTAGAGAG [SEQ ID NO. 174] and 5′-AAGCTAATACGACTCACTATAGGCCAGTC [SEQ ID NO. 175]) and 1 uL of 10 uM diluted single stranded DNA as a template in 25 uL PCR reactions for each sgRNA according to the conditions of Table 5. 
     
       
         
           
               
               
             
               
                 TABLE 4 
               
               
                   
               
               
                 Name 
                 Sequence 
               
               
                   
               
             
            
               
                 sg M201 
                 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTAGCGCCGAAACAGCGCCACTTTACAGTTGATTACGGT 
               
               
                 6v1 
                 TAAAACCTTATTTTAACTTGCTATGTTGTTTAGAATAGTCCCAAAAGATGTTTTGGTACCATTCTAAACAA 
               
               
                   
                 CATGACTCTAAAACCCAGTAACATTACTGACTGGCCTATAGTGAGTCGTATTA [SEQ ID NO. 139] 
               
               
                   
               
               
                 sg M201 
                 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTAGCGCCGAAACAGCGCCACTTTACAGTTGATTACGGT 
               
               
                 6v2 
                 TAAAACCTTATTTTAACTTGCTATGTTGTTTTTACAACATGACTCTAAAACCCAGTAACATTACTGACTGG 
               
               
                   
                 CCTATAGTGAGTCGTATTA [SEQ ID NO. 140] 
               
               
                   
               
               
                 sg M201 
                 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTAGCGCCGAAACAGCGCCACTTTACAGTTGATTACGCT 
               
               
                 6v3 
                 TATTTTAACTTGCTATGTTGTTTTTACAACATGACTCTAAAACCCAGTAACATTACTGACTGGCCTATAGT 
               
               
                   
                 GAGTCGTATTA [SEQ ID NO. 141] 
               
               
                   
               
               
                 sg M201 
                 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTAGCACCGAATCGGTGCCACGTGTTTTCACGTTGTATA 
               
               
                 9v1 
                 CGGACTAGCCTTATTTTAACTTTGCTGTATTGTTTCGAATGGTTTCAAACCGTTTTGGAACCATTCGAAAC 
               
               
                   
                 AACACAGCTCTAAAACCCAGTAACATTACTGACTGGCCTATAGTGAGTCGTATTA [SEQ ID NO. 
               
               
                   
                 142] 
               
               
                   
               
               
                 sg M201 
                 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTAGCACCGAATCGGTGCCACGTGTTTTCACGTTGTATA 
               
               
                 9v2 
                 CGGACTAGCCTTATTTTAACTTTGCTGTATTGTTTTTACAACACAGCTCTAAAACCCAGTAACATTACTGA 
               
               
                   
                 CTGGCCTATAGTGAGTCGTATTA [SEQ ID NO. 143] 
               
               
                   
               
               
                 sg M201 
                 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTAGCACCGAATCGGTGCCACGTGTTTTCACGTTGTATA 
               
               
                 9v3 
                 CGGACTAGCCTTATTTTAACTTGCTGTATTGTTTTTACAACACAGCTCTAAAACCCAGTAACATTACTGAC 
               
               
                   
                 TGGCCTATAGTGAGTCGTATTA [SEQ ID NO. 144] 
               
               
                   
               
               
                 sg M202 
                 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTATTTGATAAATAAAAAAGGCCCCACAGCAGGGCCACT 
               
               
                 0v1 
                 GTAAATTTTCGCGAAACGCTTATTTGCACTCGTTATGTTATTTACAGTTTGCTTAATACTATAAATAACAT 
               
               
                   
                 AACTAGCAAACCCAGTAACATTACTGACTGGCCTATAGTGAGTCGTATTA [SEQ ID NO. 145] 
               
               
                   
               
               
                 sg M202 
                 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTATTTGATAAATAAAAAAGGCCCCACAGCAGGGCCACT 
               
               
                 0v2 
                 GTAAATTTTCGCGAAACGCTTATTTGCACTCGTTATGTTATTTTTATAACATAACTAGCAAACCCAGTAAC 
               
               
                   
                 ATTACTGACTGGCCTATAGTGAGTCGTATTA [SEQ ID NO. 146] 
               
               
                   
               
               
                 sg M202 
                 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTAGGCCCCACAGCAGGGCCACTGTAAATTTTCGCGAAA 
               
               
                 0v3 
                 CGCTTATTTGCACTCGTTATGTTATTTTTATAACATAACTAGCAAACCCAGTAACATTACTGACTGGCCTA 
               
               
                   
                 TAGTGAGTCGTATTA [SEQ ID NO. 147] 
               
               
                   
               
               
                 sg M202 
                 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTATAAAACCATATGAAATCATATGATTTTAAAGATTCT 
               
               
                 2v1 
                 GCACAATGCAGGTCATATTGACGATTCCGGATAAACCTTATTTGCACTCGTCTTGTTAATTCTTTTGAATT 
               
               
                   
                 AACAAGACTCTCAAACCCAGTAACATTACTGACTGGCCTATAGTGAGTCGTATTA [SEQ ID NO. 
               
               
                   
                 148] 
               
               
                   
               
               
                 sg M202 
                 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTATAAAACCATATGAAATCATATGATTTTAAAGATTCT 
               
               
                 2v2 
                 GCACAATGCAGGTCATATTGACGATTCCGGATAAACCTTATTTGCACTCGTCTTGTTTTTACAAGACTCTC 
               
               
                   
                 AAACCCAGTAACATTACTGACTGGCCTATAGTGAGTCGTATTA [SEQ ID NO. 149] 
               
               
                   
               
               
                 sg M202 
                 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTACTGCACAATGCAGGTCATATTGACGATTCCGGATAA 
               
               
                 2v3 
                 ACCTTATTTGCACTCGTCTTGTTTTTACAAGACTCTCAAACCCAGTAACATTACTGACTGGCCTATAGTGA 
               
               
                   
                 GTCGTATTA [SEQ ID NO. 150] 
               
               
                   
               
               
                 sg M202 
                 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGCTCACCCGAAGGTGAGTTAATATTCACAACACTTGTGAA 
               
               
                 5v1 
                 GGTACTAAAAAGTACGATTTGAAATAATTTTTATTTGAACTCGTAGTGTAAATTTATAGGTTTTCCTATAA 
               
               
                   
                 ATTTACACTACTCTCAAACCCAGTAACATTACTGACTGGCCTATAGTGAGTCGTATTA [SEQ ID NO. 
               
               
                   
                 151] 
               
               
                   
               
               
                 sg M202 
                 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTACTCACCCGAAGGTGAGTTAATATTCACAACACTTGT 
               
               
                 5v2 
                 GAAGGTACTAAAAAGTACGATTTGAAATAATTTTTATTTGAACTCGTAGTGTATTTTTACACTACTCTCAA 
               
               
                   
                 ACCCAGTAACATTACTGACTGGCCTATAGTGAGTCGTATTA [SEQ ID NO. 152] 
               
               
                   
               
               
                 sg M202 
                 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTATTCACAACACTTGTGAAGGTACTAAAAAGTACGATT 
               
               
                 5v3 
                 TGAAATAATTTTTATTTGAACTCGTAGTGTATTTTTACACTACTCTCAAACCCAGTAACATTACTGACTGG 
               
               
                   
                 CCTATAGTGAGTCGTATTA [SEQ ID NO. 153] 
               
               
                   
               
               
                 sg M202 
                 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTAGGCCTGCAAAGCAGACCTTAATGTTCCACACGGTGG 
               
               
                 7v1 
                 AGGTTCCGAAGAACGGGTTGAATAAATTTTTATTTGAACTTGTAATGTAAACTCACTTTTATGAATTTACA 
               
               
                   
                 TTACTCTCAAACCCAGTAACATTACTGACTGGCCTATAGTGAGTCGTATTA [SEQ ID NO. 154] 
               
               
                   
               
               
                 sg M202 
                 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTAGGCCTGCAAAGCAGACCTTAATGTTCCACACGGTGG 
               
               
                 7v2 
                 AGGTTCCGAAGAACGGGTTGAATAAATTTTTATTTGAACTTGTAATGTATTTTTACATTACTCTCAAACCC 
               
               
                   
                 AGTAACATTACTGACTGGCCTATAGTGAGTCGTATTA [SEQ ID NO. 155] 
               
               
                   
               
               
                 sg M202 
                 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTATCCACACGGTGGAGGTTCCGAAGAACGGGTTGAATA 
               
               
                 7v3 
                 AATTTTTATTTGAACTTGTAATGTATTTTTACATTACTCTCAAACCCAGTAACATTACTGACTGGCCTATA 
               
               
                   
                 GTGAGTCGTATTA [SEQ ID NO. 156] 
               
               
                   
               
               
                 sg M202 
                 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTATAGCCAGAAGAACTGGCTATCTAAGCGACCTAATGG 
               
               
                 9v1 
                 TCATTCTCCAATGGAGAACCTTGTAGCAATATGCTTATTTGAACGTAGCAGTGTTGTCTTTTGACAACACT 
               
               
                   
                 GCTCTCAAACCCAGTAACATTACTGACTGGCCTATAGTGAGTCGTATTA [SEQ ID NO. 157] 
               
               
                   
               
               
                 sg M202 
                 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTATAGCCAGAAGAACTGGCTATCTAAGCGACCTAATGG 
               
               
                 9v2 
                 TCATTCTCCAATGGAGAACCTTGTAGCAATATGCTTATTTGAACGTAGCAGTGTTTTTACACTGCTCTCAA 
               
               
                   
                 ACCCAGTAACATTACTGACTGGCCTATAGTGAGTCGTATTA [SEQ ID NO. 158] 
               
               
                   
               
               
                 sg M202 
                 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTAGACCTAATGGTCATTCTCCAATGGAGAACCTTGTAG 
               
               
                 9v3 
                 CAATATGCTTATTTGAACGTAGCAGTGTTTTTACACTGCTCTCAAACCCAGTAACATTACTGACTGGCCTA 
               
               
                   
                 TAGTGAGTCGTATTA [SEQ ID NO. 159] 
               
               
                   
               
               
                 sg M203 
                 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTAAGATAACACCTCATCCGAAGATGAGGTGTTAGAGCT 
               
               
                 2v1 
                 TTGCGGTATACTACCTTATCATAGTAACCTAATTGTTCTTGGTATGATATTTTTACCATACCAAGAATAAT 
               
               
                   
                 TAGGGAACTACAACCCAGTAACATTACTGACTGGCCTATAGTGAGTCGTATTA [SEQ ID NO. 160] 
               
               
                   
               
               
                 sg M203 
                 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTATAACACCTCATCCGAAGATGAGGTGTTAGAGCTTTG 
               
               
                 2v2 
                 CGGTATACTACCTTATCATAGTAACCTAATTGTTCTTGGTTTTTACCAAGAATAATTAGGGAACTACAACC 
               
               
                   
                 CAGTAACATTACTGACTGGCCTATAGTGAGTCGTATTA [SEQ ID NO. 161] 
               
               
                   
               
               
                 sg M203 
                 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTACTCATCCGAAGATGAGGTGTTAGAGCTTTGCGGTAT 
               
               
                 2v3 
                 ACTACCTTATCATAGTAACCTAATTGTTCTTGGTTTTTACCAAGAATAATTAGGGAACTACAACCCAGTAA 
               
               
                   
                 CATTACTGACTGGCCTATAGTGAGTCGTATTA [SEQ ID NO. 162] 
               
               
                   
               
               
                 sg M203 
                 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTATGACGCCCCGCTGAAAGCGAGGCGTCAGAGCTTTAC 
               
               
                 3v1 
                 GGTGCTAAGACCTTATCATAGCAACCATAACAGTTTTTACTGTTAGGGAACTACAACCCAGTAACATTACT 
               
               
                   
                 GACTGGCCTATAGTGAGTCGTATTA [SEQ ID NO. 163] 
               
               
                   
               
               
                 sg M203 
                 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTATGACGCCCCGCTGAAAGCGAGGCGTCAGAGCTTTAC 
               
               
                 3v2 
                 GGTGCTAAGACCTTATCATAGCAACCATAACAGTTCTTTTTAGAACTGTTAGGGAACTACAACCCAGTAAC 
               
               
                   
                 ATTACTGACTGGCCTATAGTGAGTCGTATTA [SEQ ID NO. 164] 
               
               
                   
               
               
                 sg M203 
                 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTACCCCGCTGAAAGCGAGGCGTCAGAGCTTTACGGTGC 
               
               
                 3v3 
                 TAAGACCTTATCATAGCAACCATAACAGTTCTTTTTAGAACTGTTAGGGAACTACAACCCAGTAACATTAC 
               
               
                   
                 TGACTGGCCTATAGTGAGTCGTATTA [SEQ ID NO. 165] 
               
               
                   
               
               
                 sg M203 
                 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTACATCCGTCTGATATAAAGATGACGTCCTGCGCAAAC 
               
               
                 4v1 
                 AAGACGTCAGAGCTTTTCGGTTTACTACCTTATTGTAGTAACCCAACAGTTCTTGTTTTCAAGAACCGTTA 
               
               
                   
                 GGGAACTACAACCCAGTAACATTACTGACTGGCCTATAGTGAGTCGTATTA [SEQ ID NO. 166] 
               
               
                   
               
               
                 sg M203 
                 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTACATCCGTCTGATATAAAGATGACGTCCTGCGCAAAC 
               
               
                 4v2 
                 AAGACGTCAGAGCTTTTCGGTTTACTACCTTATTGTAGTAACCCAACAGTACCGTTAGGGAACTACAACCC 
               
               
                   
                 AGTAACATTACTGACTGGCCTATAGTGAGTCGTATTA [SEQ ID NO. 167] 
               
               
                   
               
               
                 sg M203 
                 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTAAAAGATGACGTCCTGCGCAAACAAGACGTCAGAGCT 
               
               
                 4v3 
                 TTTCGGTTTACTACCTTATTGTAGTAACCCAACAGTACCGTTAGGGAACTACAACCCAGTAACATTACTGA 
               
               
                   
                 CTGGCCTATAGTGAGTCGTATTA [SEQ ID NO. 168] 
               
               
                   
               
               
                 sg M203 
                 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTAAGAGATAACGCCTCGCAAAAGCGAGGCGTCAGAGCT 
               
               
                 9v1 
                 CTAAGGTGTACTAAACCTTATCATAGTAACCTAAATAGTTCTTGCAAGAACTATCAGGGAACTATAACCCA 
               
               
                   
                 GTAACATTACTGACTGGCCTATAGTGAGTCGTATTA [SEQ ID NO. 169] 
               
               
                   
               
               
                 sg M203 
                 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTAAGAGATAACGCCTTTTGGCGTCAGAGCTCTAAGGTG 
               
               
                 9v2 
                 TACTAAACCTTATCATAGTAACCTAAATAGTTCTTGCAAGAACTATCAGGGAACTATAACCCAGTAACATT 
               
               
                   
                 ACTGACTGGCCTATAGTGAGTCGTATTA [SEQ ID NO. 170] 
               
               
                   
               
               
                 sg M203 
                 AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTAAGAGATAACGCCTCGCAAAAGCGAGGCGTCAGAGCT 
               
               
                 9v3 
                 CTAAGGTGTACTAAACCTTATCATAGTAACCTAAATAGTTCTTGGTTAAGAACTATCAGGGAACTATAACC 
               
               
                   
                 CAGTAACATTACTGACTGGCCTATAGTGAGTCGTATTA [SEQ ID NO. 171] 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
             
               
                   
                 TABLE 5 
               
               
                   
                   
               
               
                   
                 STEP 
                 TEMPERATURE 
                 TIME 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 DENATURATION 
                 98° C. 
                 30 
                 SEC 
               
               
                   
                 12 CYCLES 
                 98° C. 
                 10 
                 SEC 
               
               
                   
                   
                 66° C. 
                 30 
                 SEC 
               
               
                   
                   
                 72° C. 
                 2 
                 MIN 
               
               
                   
                 FINAL EXTENSION 
                 72° C. 
                 2 
                 MIN 
               
               
                   
                 HOLD 
                 12° C. 
               
               
                   
                   
               
            
           
         
       
     
     The target library was designed based on an assumption that the eight randomized NNNNNNNN [SEQ ID NO. 176] PAMs of these nucleases reside on the 3′ end of the target sequence (5′-CCAGTCAGTAATGTTACTGG [SEQ ID NO. 177]). 
     Example 5 
     In Vitro Transcription and Translation for Production of MAD Nucleases and gRNAs 
     The MADZYMEs were tested for activity by in vitro transcription and translation (txtl). Both the gRNA plasmid and nuclease plasmid were included in each txtl reaction. A PURExpress® In Vitro Protein Synthesis Kit (NEB, Ipswich, Mass.) was used to produce MADzymes from the PCR-amplified MADZYME library and also to produce the gRNA libraries. In each well in a 96-well plate, the reagents listed in Table 6 were mixed to start the production of MADzymes and gRNAs: 
     
       
         
           
               
               
               
             
               
                   
                 TABLE 6 
               
               
                   
                   
               
               
                   
                 REAGENTS 
                 VOLUME (μl) 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                   
                 1 
                 SolA (NEB kit) 
                 10 
               
               
                   
                 2 
                 SolB (NEB kit) 
                 7.5 
               
               
                   
                 3 
                 PCR amplified gRNA 
                 0.4 
               
               
                   
                 4 
                 Murine RNase inhibitor (NEB) 
                 0.5 
               
               
                   
                 5 
                 Water 
                 3.0 
               
               
                   
                 6 
                 PCR amplified T7 MADZYMEs 
                 3.6 
               
               
                   
                   
               
            
           
         
       
     
     A master mix with all reagents was mixed on ice with the exception of the PCR-amplified T7-MADZYMEs to cover enough 96-well plates for the assay. After 21 μL of the master mix was distributed in each well in 96 well plates, 4 μL of the mixture of PCR amplified MADZYMEs and gRNA under the control of T7 promoter was added. The 96-well plates were sealed and incubated for 4 hrs at 37° C. in a thermal cycler. The plates were kept at room temperature until the target pool was added to perform the target depletion reaction. 
     After 4 hours incubation to allow production of the MADzymes and gRNAs, 4 μL of the target library pool (10 ng/μL) was added to the 10 μL aliquots of in vitro transcription/translation reaction mixture and allowed to deplete for 30 min, 3 hrs or overnight at 37° C. and 48° C. The target depletion reaction mixtures were diluted into PCR-grade water that contains RNAse A incubated for 5 min at room temperature. Proteinase K was then added and the mixtures were incubated for 5 min at 55° C. RNAseA/Proteinase K treated samples were purified with DNA purification kits and the purified DNA samples were then amplified and sequenced. The PCR conditions are shown in Table 7: 
     
       
         
           
               
               
               
               
             
               
                   
                 TABLE 7 
               
               
                   
                   
               
               
                   
                 STEP 
                 TEMPERATURE 
                 TIME 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 DENATURATION 
                 98° C. 
                 30 
                 SEC 
               
               
                   
                  4 CYCLES 
                 98° C. 
                 10 
                 SEC 
               
               
                   
                   
                 66° C. 
                 30 
                 SEC 
               
               
                   
                   
                 72° C. 
                 20 
                 SEC 
               
               
                   
                 12 CYCLES 
                 98° C. 
                 10 
                 SEC 
               
               
                   
                   
                 72° C. 
                 20 
                 SEC 
               
               
                   
                 FINAL EXTENSION 
                 72° C. 
                 2 
                 MINUTES 
               
               
                   
                 HOLD 
                 12° C. 
               
               
                   
                   
               
            
           
         
       
     
     Example 6 
     Measurement of Nicked Plasmid with Nickase RNP Complexes 
     Proteins were produced in vitro under a PURExpress® In Vitro Protein Synthesis Kit (NEB, Ipswich, Mass.). Guide RNAs that target the target plasmid were also produced under a T7 promoter in the same mixture. The MADzyme Nickase or Nuclease and guide complexes (RNP complex) formed as they were produced in the in vitro transcription and translation reagent. Supercoiled plasmid target was diluted into the digestion buffer, then the RNP complex was added to the same digestion buffer to initiate the plasmid digestion. After incubation at 37° C. to allow digestion of the plasmid, the resulting mixtures were treated with RNAase and Proteinase K, then the target plasmid was purified with a PCR cleanup kit, and run on TAE-agarose gel to observe the formation of nicked or double stand cut plasmid. The results are shown in  FIG. 7 . Table 8 lists the identified MADzyme nickases, including the variations from the nuclease sequence in Table 1 and the amino acid sequence. 
     
       
         
           
               
               
               
             
               
                 TABLE 8 
               
               
                   
               
               
                 MAD 
                   
                   
               
               
                 zyme 
                 SEQ 
                   
               
               
                 Nickase 
                 ID 
                   
               
               
                 Name 
                 NO 
                 Amino Acid Sequence 
               
               
                   
               
             
            
               
                 MAD2016- 
                 178 
                 MKKDYVIGLDIGTNSVGWAVMTEDYQLVKKKMPIYGNTEKKKIKKNFWGVRLFEEGHTAEDRR 
               
               
                 H851A 
                   
                 LKRTARRIISRRRNRLRYLQAFFEEAMTDLDENFFARLQESFLVPEDKKWHRHPIFAKLEDEV 
               
               
                   
                   
                 AYHETYPTIYHLRKKLADSSEQADLRLIYLALAHIVKYRGHFLIEGKLSTENISVKEQFQQFM 
               
               
                   
                   
                 IIYNQTFVNGESRLVSAPLPESVLIEEELTEKASRTKKSEKVLQQFPQEKANGLFGQFLKLMV 
               
               
                   
                   
                 GNKADFKKVFGLEEEAKITYASESYEEDLEGILAKVGDEYSDVFLAAKNVYDAVELSTILADS 
               
               
                   
                   
                 DKKSHAKLSSSMIVRFTEHQEDLKKFKRFIRENCPDEYDNLFKNEQKDGYAGYIAHAGKVSQL 
               
               
                   
                   
                 KFYQYVKKIIQDIAGAEYFLEKIAQENFLRKQRTFDNGVIPHQIHLAELQAIIHRQAAYYPFL 
               
               
                   
                   
                 KENQEKIEQLVTFRIPYYVGPLSKGDASTFAWLKRQSEEPIRPWNLQETVDLDQSATAFIERM 
               
               
                   
                   
                 TNFDTYLPSEKVLPKHSLLYEKFMVFNELTKISYTDDRGIKANFSGKEKEKIFDYLFKTRRKV 
               
               
                   
                   
                 KKKDIIQFYRNEYNTEIVTLSGLEEDQFNASFSTYQDLLKCGLTRAELDHPDNAEKLEDIIKI 
               
               
                   
                   
                 LTIFEDRQRIRTQLSTFKGQFSAEVLKKLERKHYTGWGRLSKKLINGIYDKESGKTILGYLIK 
               
               
                   
                   
                 DDGVSKHYNRNFMQLINDSQLSFKNAIQKAQSSEHEETLSETVNELAGSPAIKKGIYQSLKIV 
               
               
                   
                   
                 DELVAIMGYAPKRIVVEMARENQTTSTGKRRSIQRLKIVEKAMAEIGSNLLKEQPTTNEQLRD 
               
               
                   
                   
                 TRLFLYYMQNGKDMYTGDELSLHRLSHYDIDAIIPQSFMKDDSLDNLVLVGSTENRGKSDDVP 
               
               
                   
                   
                 SKEVVKDMKAYWEKLYAAGLISQRKFQRLTKGEQGGLTLEDKAHFIQRQLVETRQITKNVAGI 
               
               
                   
                   
                 LDQRYNANSKEKKVQIITLKASLTSQFRSIFGLYKVREVNDYHHGQDAYLNCVVATTLLKVYP 
               
               
                   
                   
                 NLAPEFVYGEYPKFQTFKENKATAKAIIYTNLLRFFTEDEPRFTKDGEILWSNSYLKTIKKEL 
               
               
                   
                   
                 NYHQMNIVKKVEVQKGGFSKESIKPKGPSNKLIPVKNGLDPQKYGGFDSPIVAYTVLFTHEKG 
               
               
                   
                   
                 KKPLIKQEILGITIMEKTRFEQNPILFLEEKGFLRPRVLMKLPKYTLYEFPEGRRRLLASAKE 
               
               
                   
                   
                 AQKGNQMVLPEHLLTLLYHAKQCLLPNQSESLTYVEQHQPEFQEILERVVDFAEVHTLAKSKV 
               
               
                   
                   
                 QQIVKLFEANQTADVKEIAASFIQLMQFNAMGAPSTFKFFQKDIERARYTSIKEIFDATIIYQ 
               
               
                   
                   
                 STTGLYETRRKVVD 
               
               
                   
               
               
                 MAD2016- 
                 179 
                 MKKDYVIGLDIGTNSVGWAVMTEDYQLVKKKMPIYGNTEKKKIKKNFWGVRLFEEGHTAEDRR 
               
               
                 N874A 
                   
                 LKRTARRIISRRRNRLRYLQAFFEEAMTDLDENFFARLQESFLVPEDKKWHRHPIFAKLEDEV 
               
               
                   
                   
                 AYHETYPTIYHLRKKLADSSEQADLRLIYLALAHIVKYRGHFLIEGKLSTENISVKEQFQQFM 
               
               
                   
                   
                 IIYNQTFVNGESRLVSAPLPESVLIEEELTEKASRTKKSEKVLQQFPQEKANGLFGQFLKLMV 
               
               
                   
                   
                 GNKADFKKVFGLEEEAKITYASESYEEDLEGILAKVGDEYSDVFLAAKNVYDAVELSTILADS 
               
               
                   
                   
                 DKKSHAKLSSSMIVRFTEHQEDLKKFKRFIRENCPDEYDNLFKNEQKDGYAGYIAHAGKVSQL 
               
               
                   
                   
                 KFYQYVKKIIQDIAGAEYFLEKIAQENFLRKQRTFDNGVIPHQIHLAELQAIIHRQAAYYPFL 
               
               
                   
                   
                 KENQEKIEQLVTFRIPYYVGPLSKGDASTFAWLKRQSEEPIRPWNLQETVDLDQSATAFIERM 
               
               
                   
                   
                 TNFDTYLPSEKVLPKHSLLYEKFMVFNELTKISYTDDRGIKANFSGKEKEKIFDYLFKTRRKV 
               
               
                   
                   
                 KKKDIIQFYRNEYNTEIVTLSGLEEDQFNASFSTYQDLLKCGLTRAELDHPDNAEKLEDIIKI 
               
               
                   
                   
                 LTIFEDRQRIRTQLSTFKGQFSAEVLKKLERKHYTGWGRLSKKLINGIYDKESGKTILGYLIK 
               
               
                   
                   
                 DDGVSKHYNRNFMQLINDSQLSFKNAIQKAQSSEHEETLSETVNELAGSPAIKKGIYQSLKIV 
               
               
                   
                   
                 DELVAIMGYAPKRIVVEMARENQTTSTGKRRSIQRLKIVEKAMAEIGSNLLKEQPTTNEQLRD 
               
               
                   
                   
                 TRLFLYYMQNGKDMYTGDELSLHRLSHYDIDHIIPQSFMKDDSLDNLVLVGSTEARGKSDDVP 
               
               
                   
                   
                 SKEVVKDMKAYWEKLYAAGLISQRKFQRLTKGEQGGLTLEDKAHFIQRQLVETRQITKNVAGI 
               
               
                   
                   
                 LDQRYNANSKEKKVQIITLKASLTSQFRSIFGLYKVREVNDYHHGQDAYLNCVVATTLLKVYP 
               
               
                   
                   
                 NLAPEFVYGEYPKFQTFKENKATAKAIIYTNLLRFFTEDEPRFTKDGEILWSNSYLKTIKKEL 
               
               
                   
                   
                 NYHQMNIVKKVEVQKGGFSKESIKPKGPSNKLIPVKNGLDPQKYGGFDSPIVAYTVLFTHEKG 
               
               
                   
                   
                 KKPLIKQEILGITIMEKTRFEQNPILFLEEKGFLRPRVLMKLPKYTLYEFPEGRRRLLASAKE 
               
               
                   
                   
                 AQKGNQMVLPEHLLTLLYHAKQCLLPNQSESLTYVEQHQPEFQEILERVVDFAEVHTLAKSKV 
               
               
                   
                   
                 QQIVKLFEANQTADVKEIAASFIQLMQFNAMGAPSTFKFFQKDIERARYTSIKEIFDATIIYQ 
               
               
                   
                   
                 STTGLYETRRKVVD 
               
               
                   
               
               
                 MAD2032- 
                 180 
                 MKYIIGLDMGITSVGFATMMLDDKDEPCRIIRMGSRIFEAAEHPKDGSSLAAPRRINRGMRRR 
               
               
                 H590A 
                   
                 LRRKSHRKERIKDLIIKNELMTADEISAIYSTGKQLSDIYQIRAEALDRKLNTEEFVRLLIHL 
               
               
                   
                   
                 SQRRGFKSNRKVDAKEKGSDAGKLLSAVNSNKELMIEKNYRTIGEMLYKDEKFSEYKRNKADD 
               
               
                   
                   
                 YSNTFARSEYEDEIRQIFSAQQEHGNPYATDELKESYLDIYLSQRSFDEGPGGSSPYGGNQIE 
               
               
                   
                   
                 KMIGNCTLEPEEKRAAKATFSFEYFNLLSKVNSIKIVSSSGKRALNNDERQSVIRLAFAKNAI 
               
               
                   
                   
                 SYTSLRKELNMEYSERFNISYSQSDKSIEEIEKKTKFTYLTAYHTFKKAYGSVFVEWSADKKN 
               
               
                   
                   
                 SLAYALTAYKNDTKIIEYLTQKGFDAAETDIALTLPSFSKWGNLSEKALNNIIPYLEQGMLYH 
               
               
                   
                   
                 DACTAAGYNFKADDTDKRMYLPAHEKEAPELDDITNPVVRRAISQTIKVINALIREMGESPCF 
               
               
                   
                   
                 VNIELARELSKNKAERSKIEKGQKENQVRNDRIMERLRNEFGLLSPTGQDLIKLKLWEEQDGI 
               
               
                   
                   
                 CPYSLKPIKIEKLFDVGYTDIDAIIPYSLSFDDTYNNKVLVMSSENRQKGNRIPMQYLEGKRQ 
               
               
                   
                   
                 DDFWLWVDNSNLSRRKKQNLTKETLSEDDLSGFKKRNLQDTQYLSRFMMNYLKKYLALAPNTT 
               
               
                   
                   
                 GRKNTIQAVNGAVTSYLRKRWGIQKVRENGDTHHAVDAVVISCVTAGMTKRVSEYAKYKETEF 
               
               
                   
                   
                 QNPQTGEFFDVDIRTGEVINRFPLPYARFRNELLMRCSENPSRILHEMPLPTYAADEKVAPIF 
               
               
                   
                   
                 VSRMPKHKVKGSAHKETIRRAFEEDGKKYTVSKVPLTDLKLKNGEIENYYNPESDGLLYNALK 
               
               
                   
                   
                 EQUAFGGDAAKAFEQPFYKPKSDGSEGPLVKKVKLINKATLTVPVLNNTAVADNGSMVRVDVF 
               
               
                   
                   
                 FVEGEGYYLVPIYVADTVKKELPNKAIIANKPYEEWKEMREENFVFSLYPNDLIKISSRKDMK 
               
               
                   
                   
                 FNLVNKESTLAPNCQSKEALVYYKGSDISTAAVTAINHDNTYKLRGLGVKTLLKIEKYQVDVL 
               
               
                   
                   
                 GNVFKVGKEKRVRFK 
               
               
                   
               
               
                 MAD2039- 
                 181 
                 MRPYAIGLDIGITSVGWATVALDADESPCGIIGLGSRIFDAAEQPKTGESLAAPRRAARGSRR 
               
               
                 H587A 
                   
                 RLRRHRHRNERIRSLMLEERLISQDELETLFDGRLEDIYALRVKALDEIVSRTDFARILLHIS 
               
               
                   
                   
                 QRRGFKSNRKNPTTKEDGVLLAAVNENKQRMSEHGYRTVGEMFLLDETFKDHKRNKGGNYITT 
               
               
                   
                   
                 VARDMVADEVRAIFSAQRELGASFASEEFEERYLEILLSQRSFDEGPGGNSPYGGSQIERMVG 
               
               
                   
                   
                 RCTFFPDEPRAAKATYSFEYFTLLQKVNHIRIVENGVASKLTDEQRRIIIELAHTTKDVSYAK 
               
               
                   
                   
                 IRKVLKLSDKQLFNIRYSDNSPAEDSEKKEKLGIMKAYHQMRSAIDRVSKGRFAMMPRAQRNA 
               
               
                   
                   
                 IGTALSLYKTSDKIRKYLTDAGLDEIDINSADSIGSFSKFGHISVKACDMLIPFLEQGMNYNE 
               
               
                   
                   
                 ACAAAGLNFKGHDAGEKSKLLHPKEEDYEDITSPVVRRAIAQTIKVINAIIRREGCSPTFINI 
               
               
                   
                   
                 ELAREMAKDFRERNRIKKENDDNRAKNERLLERIRTEYGKNNPTGLDLVKLRLYEEQSGVCMY 
               
               
                   
                   
                 SLKQMSLEKLFEPNYAEVDAIVPYSISFDDSRKNKVLVLTEENRNKGNRLPLQYLKGRRREDF 
               
               
                   
                   
                 IVWVNNNVKDYRKRRLLLKEELTAEDESGFKERNLQDTKTMSRFLLNYIADNLEFAESTRGRK 
               
               
                   
                   
                 KKVTAVNGAVTAYMRKRWGITKIREDGDCHHAVDAVVIACTTDAMIRQVSRYAQFRECEYMQT 
               
               
                   
                   
                 ESGSVAVDTGTGEVLRTFPYPWPDFRKELEARLANDPAKVINDLHLPFYMSAGRPLPEPVFVS 
               
               
                   
                   
                 RMPRRKVTGAAHKDTIKSARELDNGYLIVKRPLTDLKLKNGEIENYYNPQSDKCLYDALKNAL 
               
               
                   
                   
                 IEHGGDAKKAFAGEFRKPKRDGTPGPIVKKVKLLEPTTMCVPVHGGKGAADNDSMVRVDVFLS 
               
               
                   
                   
                 GGKYYLVPIYVADTLKPELPNKAVTRGKKYSEWLEMADEDFIFSLYPNDLICATSKNGITLSV 
               
               
                   
                   
                 CRKDSTLPPTVESKSFMLYYRGTDISTGSISCITHDNAYKLRGLGVKTLEKLEKYTVDVLGEY 
               
               
                   
                   
                 HKVGKEVRQPFNIKRRKACPSEML 
               
               
                   
               
               
                 MAD2039- 
                 182 
                 MRPYAIGLDIGITSVGWATVALDADESPCGIIGLGSRIFDAAEQPKTGESLAAPRRAARGSRR 
               
               
                 N610A 
                   
                 RLRRHRHRNERIRSLMLEERLISQDELETLFDGRLEDIYALRVKALDEIVSRTDFARILLHIS 
               
               
                   
                   
                 QRRGFKSNRKNPTTKEDGVLLAAVNENKQRMSEHGYRTVGEMFLLDETFKDHKRNKGGNYITT 
               
               
                   
                   
                 VARDMVADEVRAIFSAQRELGASFASEEFEERYLEILLSQRSFDEGPGGNSPYGGSQIERMVG 
               
               
                   
                   
                 RCTFFPDEPRAAKATYSFEYFTLLQKVNHIRIVENGVASKLTDEQRRIIIELAHTTKDVSYAK 
               
               
                   
                   
                 IRKVLKLSDKQLFNIRYSDNSPAEDSEKKEKLGIMKAYHQMRSAIDRVSKGRFAMMPRAQRNA 
               
               
                   
                   
                 IGTALSLYKTSDKIRKYLTDAGLDEIDINSADSIGSFSKFGHISVKACDMLIPFLEQGMNYNE 
               
               
                   
                   
                 ACAAAGLNFKGHDAGEKSKLLHPKEEDYEDITSPVVRRAIAQTIKVINAIIRREGCSPTFINI 
               
               
                   
                   
                 ELAREMAKDFRERNRIKKENDDNRAKNERLLERIRTEYGKNNPTGLDLVKLRLYEEQSGVCMY 
               
               
                   
                   
                 SLKQMSLEKLFEPNYAEVDHIVPYSISFDDSRKNKVLVLTEENRNKGNRLPLQYLKGRRREDF 
               
               
                   
                   
                 IVWVNNNVKDYRKRRLLLKEELTAEDESGFKERNLQDTKTMSRFLLNYIADNLEFAESTRGRK 
               
               
                   
                   
                 KKVTAVNGAVTAYMRKRWGITKIREDGDCHHAVDAVVIACTTDAMIRQVSRYAQFRECEYMQT 
               
               
                   
                   
                 ESGSVAVDTGTGEVLRTFPYPWPDFRKELEARLANDPAKVINDLHLPFYMSAGRPLPEPVFVS 
               
               
                   
                   
                 RMPRRKVTGAAHKDTIKSARELDNGYLIVKRPLTDLKLKNGEIENYYNPQSDKCLYDALKNAL 
               
               
                   
                   
                 IEHGGDAKKAFAGEFRKPKRDGTPGPIVKKVKLLEPTTMCVPVHGGKGAADNDSMVRVDVFLS 
               
               
                   
                   
                 GGKYYLVPIYVADTLKPELPAKAVTRGKKYSEWLEMADEDFIFSLYPNDLICATSKNGITLSV 
               
               
                   
                   
                 CRKDSTLPPTVESKSFMLYYRGTDISTGSISCITHDNAYKLRGLGVKTLEKLEKYTVDVLGEY 
               
               
                   
                   
                 HKVGKEVRQPFNIKRRKACPSEML 
               
               
                   
               
            
           
         
       
     
     While this invention is satisfied by embodiments in many different forms, as described in detail in connection with preferred embodiments of the invention, it is understood that the present disclosure is to be considered as exemplary of the principles of the invention and is not intended to limit the invention to the specific embodiments illustrated and described herein. Numerous variations may be made by persons skilled in the art without departure from the spirit of the invention. The scope of the invention will be measured by the appended claims and their equivalents. The abstract and the title are not to be construed as limiting the scope of the present invention, as their purpose is to enable the appropriate authorities, as well as the general public, to quickly determine the general nature of the invention. In the claims that follow, unless the term “means” is used, none of the features or elements recited therein should be construed as means-plus-function limitations pursuant to 35 U.S.C. § 112, ¶6.