Synthon formation

Abstract

The invention is entitled "Formation of Synthons". The present disclosure provides, among other things, a composition comprising: a 5'-3' exonuclease; a strand displacement polymerase; and optionally, a single-stranded DNA binding protein and / or a ligase. Also described are polynucleotide assembly methods for forming synthons and kits for performing the methods.

Description

[0001] This application is a divisional application. The application date of the original application is August 27, 2015, the application number is 201580058526.8, and the name of the invention is “Formation of Syntheton”. Background Art

[0002] Synthetic biology relies on the ability to construct new DNA from component parts. Double-stranded (ds) DNA molecules have been assembled by producing staggered ends at the two ends of a first DNA double-strand. This has been achieved using restriction endonucleases, or by digesting with exonucleases, or by T4 DNA polymerase, followed by hybridization, and optionally connecting a second DNA double-strand to the first double-strand. The technology of using exonucleases and ligases in a reaction mixture also specifies the use of non-strand displacement polymerases. Summary of the Invention

[0003] The present disclosure provides, among other things, a composition comprising: i. a 5'-3' exonuclease; ii. a strand-displacing polymerase; iii. a single-stranded (ss) DNA binding protein; and iv. a non-naturally occurring buffer, wherein the composition does not include a crowding agent and / or a non-strand-displacing polymerase. The composition can be used to assemble polynucleotides into synthons. Depending on whether the assembly is performed for the purpose of cloning in bacterial cells that contain their own ligase or for the purpose of excluding the cloning step in bacteria, embodiments of the composition optionally include a ligase.

[0004] Previously described assembly methods require non-strand displacing polymerases and further additionally require crowding agents (e.g., see US 8,968,999). In contrast to the prior art, embodiments of the present invention demonstrate that strand displacing polymerases have advantages over non-strand displacing polymerases when used with 5'-3' exonucleases. This combination favors the inclusion of ss-binding proteins over crowding agents, contrary to prior art teachings that claim the use of crowding agents is four times more effective than alternatives including the use of single-strand binding proteins. Embodiments of the present invention provide compositions, methods, and kits that provide for increased efficiency in the assembly and cloning of functional genes and synthons from oligonucleotides and polynucleotides comprising ds and / or ss nucleic acid molecules in a single-step process and / or in a single reaction vessel. These embodiments do not rely on crowding agents and do not require a non-strand displacing polymerase to fill the gap that exists after the two molecules have annealed. For example, when a 5'-3' exonuclease acting on dsDNA produces a 3'ssDNA overhang that can effectively anneal to a 3'ssDNA overhang from another molecule, a strand-displacing polymerase can fill the gap left after the molecules anneal. The combination of strand-displacing DNA polymerase and 5'-3' exonuclease activity produces a double-stranded synthon that contains a nick at or near the junction site. This nick can be sealed in vitro by a ligase or in vivo by an endogenous cellular ligase. In addition, including ssDNA binding proteins in the reaction mixture enables the effective assembly of relatively low concentrations of nucleic acid fragments, thereby saving costs without losing efficiency or accuracy of the junction.

[0005] In some composition embodiments, the strand-displacing polymerase is a B-family polymerase. The strand-displacing polymerase should preferably be used under the same reaction conditions (e.g., using a Figures 1A to 1E The strand displacement activity was greater than that of Polymerase (Thermo Fisher, Waltham, MA) (usually described as non-strand displacement) observed. In the compositions, methods and kits of the present invention, the strand displacement activity of the strand displacement polymerase is mainly used. In some embodiments, the strand displacement polymerase can be non-natural, for example, the strand displacement polymerase can be a mutant. The example of a mutant includes a polymerase with one or more amino acid replacements, and the non-natural polymerase can or can be a fusion protein with a portion of an unrelated amino acid sequence, wherein the fusion polymerase does not exist in nature. Preferably, the strand displacement polymerase is stable at 50°C or higher, and therefore can be referred to as a thermostable strand displacement polymerase. In some cases, the strand displacement polymerase is a fusion polymerase with an unrelated or heterologous DNA binding domain. In some embodiments, the amino acid sequence of the polymerase portion can be at least 90% or 95% or 98% or 99% identical to SEQ ID NO: 102. In another embodiment, the amino acid sequence of the polymerase may be at least 90% or 95% or 98% or 99% or 100% identical to SEQ ID NO: 1, preferably at least 90%. In another embodiment, the amino acid sequence of the polymerase may be at least 90% or 95% or 98% or 99% or 100% identical to any of SEQ ID NO: 33 to SEQ ID NO: 55, preferably at least 90%. In some embodiments, the amino acid sequence of the DNA binding domain portion may be at least 90% or 95% or 98% or 99% identical to SEQ ID NO: 2. In another embodiment, the amino acid sequence of the polymerase may be at least 90% or 95% or 98% or 99% or 100% identical to any of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 56 to SEQ ID NO: 96, or SEQ ID NO: 102, preferably at least 90%. In another embodiment, any polymerase domain portion described herein can be combined with any DNA binding domain described herein, provided that the polymerase portion and the DNA binding domain are heterologous. For example, in other embodiments, the amino acid sequence of the fusion protein can be at least 90% or 95% or 99% or 100% identical to SEQ ID NO: 1 and SEQ ID NO: 2, preferably at least 90%. In other embodiments, the sequence identity of the fusion protein to SEQ ID NO: 3 can be at least 90% or 95% or 98% or 99% or 100%, preferably at least 90%. The strand displacement polymerase may or may not have 3'-5' exonuclease activity. When the strand displacement polymerase has 3'-5' exonuclease activity, polynucleotide ligation can be optimized by balancing 3'-5' exonuclease activity, 5'-3' polymerization activity, and strand displacement activity using conditions including those exemplified herein.The efficiency and accuracy of assembly can be confirmed using the assays described herein (e.g., see. Figure 3A and 3B In some embodiments, the polymerase is not Phusion, 9°N, Pfu, or Vent, or has an amino acid sequence that is at least 90% identical to Phusion or wild-type 9°N, Pfu, or Vent. In some embodiments, the polymerase is thermostable, i.e., active at temperatures of at least 40°C or at least 50°C. In contrast to strand-displacing polymerases, some polymerases, such as Taq DNA polymerase, degrade the downstream strand encountered via 5'→3' exonuclease activity. This activity is used in nick translation protocols. Therefore, Taq DNA polymerase is not included in the definition of a strand-displacing polymerase.

[0006] Assays to determine the efficiency and accuracy of synthon formation are described in the Examples and are shown in Figure 3A ~B. The designed assembly fragments encode lacI and lacZ proteins, which produce blue colonies if the DNA fragments are assembled correctly. Therefore, the number of "blue" colonies on the overnight plate indicates the efficiency and accuracy of the assembly. In the absence of blue, effective assembly may occur, but errors in the junction / extension region prevent expression. When the synthon is assembled and then cloned into the host cell, the efficiency and accuracy of synthon formation are converted into confidence that each clone will contain a correctly assembled synthon. With this confidence, only one or more replicated clones need to be sequenced to confirm the presence of the synthon. This reduces the cost and inconvenience of sequencing clones that may contain errors. In one embodiment, at least 80% or alternatively at least 90% of the clones will contain correctly assembled synthons.

[0007] In some embodiments, the methods using the compositions described herein are capable of achieving yields that substantially exceed the minimum requirements. For example, up to 5,000 or 10,000 clones can be prepared in a single transformation event. If the purpose of the assembly is not to prepare a library of synthons, but to prepare a single synthon, lower starting amounts of nucleic acid fragments and reagents can be used, even lower than the ranges provided herein. Examples of concentration ranges suitable for assembling mixtures include the following: 0.02 nM to 100 nM of DNA fragments or, for example, 0.2 nM to 10 nM DNA can be added to the reagent mixture in the reaction vessel. In one embodiment, although higher or lower ratios can be used, vector DNA and DNA fragments are included in a 1:1 ratio. Higher ss DNA concentrations may be preferred compared to the concentrations selected for ds DNA. The reagent mixture in the reaction vessel can also include 0.0004 U / μl to 0.064 U / μl of 5'-3' exonuclease (e.g., 0.0004 U / μl to 0.01 U / μl); 0.5 U / μl to 32 U / μl of optional ligase (e.g., 1 U / μl to 10 U / μl); 0.0025 U / μl to 0.25 U / μl of strand-displacing polymerase (e.g., 0.005 U / μl to 0.1 U / μl); and 0.001 μg / μl to 0.1 μg / μl of ss-binding protein (e.g., 0.01 μg / μl to 0.5 μg / μl) (units corresponding to those specified by the manufacturer (New England Biolabs, Ipswich, MA)).

[0008] The amount of 5'-3' exonuclease can be further optimized based on the overlap length of the nucleic acid fragments and the size of each fragment. For example, the amount of 5'-3' exonuclease can be increased within the range where the nucleic acid fragment length is greater than 80 nucleotides. The absolute concentration of the strand-displacing polymerase within the specified range is not critical.

[0009] Although many other ssDNA binding proteins are known and can be used in the composition, the ssDNA binding protein used in the composition can be Escherichia coli (E. coli) recA, the T7 gene 2.5 product, RedB (from phage lambda) or RecT (from the Rac prophage), ET SSB (extremely thermostable single-stranded DNA binding protein), or an ss-binding protein with 90% sequence identity to SEQ ID NO: 100. Including the ss-binding protein improves assembly efficiency, as measured by colony number, particularly for nucleic acid fragments with longer overlapping sequences (e.g., at least 20 nucleotides), compared to what would otherwise occur in the absence of the ss-binding protein.

[0010] The optional ligase may be NAD-dependent +However, for PCR, since ATP can inhibit the Taq polymerase used in the subsequent synthon amplification, it is better to use an NAD-dependent ligase. + Examples of suitable ligases include proteins having a sequence identity of at least 90% to SEQ ID NO: 101.

[0011] The 5'-3' exonuclease used herein can be an enzyme having 5'-3' exonuclease activity and ss endonuclease activity (see, e.g., Garforth et al., PNAS, 96, 38-43 (1999)). Examples of 5'-3' exonucleases having exonuclease and ss endonuclease activity include T5 exonuclease, and homologs and variants thereof. In one embodiment, the 5'-3' exonuclease has at least 90% amino acid sequence identity to SEQ ID NO: 98. There is no requirement to denature the 5'-3' exonuclease prior to joining the polynucleotide with a strand displacement polymerase. Therefore, the use of a thermostable 5'-3' exonuclease is described in the Examples.

[0012] In some embodiments, the composition may further include dNTPs (i.e., dGTP, dATP, a mixture of dGTP and dTTP), and, in some embodiments, when T5 5'-3' exonuclease is used, the composition may further include a potassium salt such as KCl (e.g., in a concentration range of 7 mM to 150 mM).

[0013] In summary, a method for preparing a synthon is provided. In some embodiments, the method may comprise incubating an embodiment of a composition described herein with a set of polynucleotides and / or oligonucleotides under suitable reaction conditions, the embodiment of the composition comprising a strand displacement polymerase and a 5'-3' exonuclease as described herein and optionally a ligase (if the reaction is in vitro or in vivo in a cell or organism without a ligase), and may further comprise an ss-binding protein, wherein at least one or some of the members of the set have a sequence that overlaps with one or some other members of the set. In some embodiments, the polynucleotides or oligonucleotides may be dsDNA, e.g., overlapping PCR products or overlapping restriction fragments. In other embodiments, the polynucleotides may be ssDNA or RNA. In some embodiments, the set of polynucleotides may comprise ssDNA or RNA. In some embodiments, the set of polynucleotides may comprise dspolynucleotides. In some embodiments, the set of polynucleotides may comprise at least one dspolynucleotide and at least one sspolynucleotide. In some embodiments, the set of polynucleotides may comprise subpopulations of polynucleotides having identical sequences except for subsequences that vary between subpopulation members. In other embodiments, the set of polynucleotides may include ss or ds polynucleotides, or polynucleotides having overlapping regions at their ends for joining purposes but differing in the internal sequence that forms the synthon. Thus, in one embodiment of the method of the present invention, the polynucleotides in the set of polynucleotides are ds; for example, wherein the ds polynucleotides are overlapping PCR products or overlapping restriction fragments or are assembled from ss polynucleotides. In an alternative embodiment of the method of the present invention, the synthon is assembled from ss polynucleotides in the set of polynucleotides. In another alternative embodiment of the method of the present invention, the synthon is assembled from a set of polynucleotides comprising a mixture of at least one ds polynucleotide and at least one ss oligonucleotide. In an embodiment of the method of the present invention, the synthon is assembled from a set of polynucleotides comprising subpopulations of polynucleotides that are identical to each other except for subsequences that vary between members of the subpopulations.

[0014] Embodiments of the methods can be used to prepare various synthons, including coding sequences, vectors, guide molecules for genetic engineering and expression components.

[0015] Before assembly, the length of the initial ds polynucleotide can be in the range of 100 bases to 30kb, although polynucleotides outside this range can also be used in some cases. For example, in some embodiments, the size of a single fragment can be up to 20kb to 30kb or longer, or as short as 30 bases to 500 bases. Moreover, in some embodiments, fragments of different sizes can be joined in the assembly reaction. In one embodiment, long polynucleotides (e.g., fragments of 5kb to 20kb in length) are joined with short nucleotides (e.g., fragments of 100 bases to 500 bases in length). The newly assembled synthons can be sequenced directly using single molecule sequencing methods, or sequenced after cloning or amplification.

[0016] In one embodiment, the group members may comprise overlapping sequences less than 2 kb in length, for example, in the range of 15 to 200 nucleotides, such as 20 to 100 nucleotides.

[0017] In one embodiment, a composition is provided, wherein the composition has a 5'-3' exonuclease; a strand-displacing polymerase; and a buffer comprising a potassium salt (such as KCl) in a concentration range of 7mM to 150mM (e.g., 20mM to 50mM). In addition to potassium salts, sodium salts (such as sodium chloride) in a range of 10mM to 100mM (e.g., 20mM) can also be used. The composition can include an ss-binding protein. In some embodiments, the composition does not contain a crowding agent, such as polyethylene glycol (PEG), Ficoll, or dextran. In some embodiments, the composition does not contain a non-strand-displacing polymerase. In another embodiment, the composition for forming a synthon includes polynucleotides and / or oligonucleotide fragments.

[0018] In another embodiment of the method, a set of oligonucleotides can be joined in the absence of a non-strand-displacing polymerase using a composition comprising a crowding agent such as polyethylene glycol (PEG), Ficoll, or dextran in addition to or in place of an ss-binding protein, and at least 7 mM potassium salt such as KCl, along with a strand-displacing polymerase and a 5'-3' exonuclease. In one embodiment, the potassium salt concentration is less than 150 mM, for example, 20 mM to 50 mM.

[0019] Additionally provided is a kit for polynucleotide assembly comprising: i. a 5'-3' exonuclease; ii. an optional ligase; iii. a strand-displacing polymerase; and iv. an ss DNA binding protein. In certain embodiments, for example, the kit may further comprise dNTPs and / or a buffer. The components of the kit may be in separate containers (e.g., one or more different reaction tubes), or the components of the kit may be in a single container. The components may be lyophilized, or in solution, or a portion may be lyophilized and a portion may be in solution. The components may be partially or completely immobilized on a solid surface, such as a bead, or on the surface of a reaction chamber, or in solution. The components may be added to a partially or completely immobilized or solution target polynucleotide. In some embodiments, the kit may comprise one or more mixtures of kit components. In some embodiments, the kit does not comprise a non-strand-displacing polymerase or a crowding agent.

[0020] In one embodiment, a polymerase having at least 80%, 85%, 90%, or 95% sequence identity to SEQ ID: 1 is provided for use in assembling a mixture. In another embodiment, a polymerase having a binding domain having at least 80%, 85%, 90%, or 95% sequence identity to SEQ ID: 2 is provided for use in assembling a composition. In another embodiment, a polymerase having at least 80%, 85%, 90%, or 95% sequence identity to SEQ ID: 3 is provided for use in assembling a mixture. These compositions can be used under reaction conditions in which the polymerase is strand-displacing. The compositions can be used under reaction conditions in which any 3' exonuclease activity associated with the polymerase activity is active.

[0021] The assembly reaction can occur using ss or ds nucleic acids. Any number of fragments can be assembled, for example 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more fragments. Chemically synthesized ss polynucleotides can be assembled into ds overlapping fragments and hybridized and ligated to the ends of the linearized vector to form synthons suitable for cloning. Alternatively, double-stranded fragments can be assembled into synthons and inserted into vectors for cloning or PCR or isothermal amplification (see, e.g., Figures 2A to 2C ss nucleic acid fragments can also be directly inserted into a vector by hybridizing the nucleic acid fragment to the 3'ss end of a linearized ds vector (see, e.g., Figure 7). The ss nucleic acid can be assembled by overlapping complementary ends at the same time, before or after the ss nucleic acid fragment is hybridized with the 3'ss end of the linearized vector. Before being inserted into the vector for cloning, the assembled fragment can be amplified by PCR or a constant temperature method. The nucleic acid fragment may contain a randomized nucleotide sequence or a degenerate code so that a library of representative variants at each nucleotide position in the variable region can be formed. The random sequence can be located between a given sequence at each end. In one embodiment, the random sequence located between the specified sequences can be used to hybridize with a second nucleic acid fragment (e.g., a second ss genomic polynucleotide) or the end of a linearized vector. In one embodiment, the random sequence hybridizes to a target genomic sequence for guiding the Cas9 protein to the target nucleic acid for gene editing (see, for example, Figures 9A to 9C ). Thus, according to this aspect of the invention, the method can be used to hybridize at least one member of a set of polynucleotides to a second nucleic acid fragment (e.g., a second ss genomic polynucleotide) or to a linearized vector end. For example, the method can be used to hybridize at least one member of the set of polynucleotides to a target genomic sequence to guide a Cas endonuclease, such as Cas9, to a target genomic nucleic acid in a gene editing method.

[0022] In some embodiments, a method for assembling a plurality of polynucleotides into a synthon is provided, comprising: combining the plurality of polynucleotides with a composition comprising a 5'-3' exonuclease, a strand-displacing polymerase, an optional ligase, an ss-binding protein, and a buffer, wherein each polynucleotide has a 3'ss terminal polynucleotide sequence on one strand that can hybridize to a complementary ss-complementary sequence on a second polynucleotide and can optionally be joined in a single vessel under constant temperature conditions to form a continuous double-stranded polynucleotide. The synthon can further be joined at its terminus to the terminus of a linearized plasmid for amplification and / or cloning.

[0023] In some embodiments, the entire assembly method can be performed as a "one-step" reaction (in a single tube that does not need to be opened during the reaction). In one embodiment, the components are mixed together in a reaction vessel and incubated at a temperature of 40° C. to 60° C. for a period of time, for example, 5 minutes to 12 hours, to prepare the synthon.

[0024] In one aspect, the method comprises a step of strand displacement in the polynucleotides to be joined by a polymerase having at least 90% amino acid sequence identity to SEQ ID NO: 1 or SEQ ID NO: 102 and / or SEQ ID NO: 2 or SEQ ID NO: 3. In embodiments of the method, an additional 3'-5' exonuclease chewback step is not required. In another aspect, the efficiency of the assembly reaction is enhanced by including a potassium salt in the reaction mixture at a minimum concentration of 7 mM, wherein an example of a potassium salt is KCl.

[0025] In one aspect, a method is provided in which a polynucleotide comprises a random sequence between the ends of a given sequence. In another aspect, the method further comprises screening for random sequences having hybridization activity with genomic DNA and identifying random sequences having hybridization activity. In another aspect, the method further comprises performing gene editing by transcribing the random sequence having hybridization activity to form RNA, and using the RNA for gene editing in the presence of a Cas endonuclease.

[0026] In one embodiment of the compositions, kits, or methods of the present invention, the strand-displacing polymerase used in the compositions, kits, or methods of the present invention may be non-naturally occurring, such as a mutant or fusion protein. In the compositions, kits, or methods of the present invention, the non-naturally occurring strand-displacing fusion protease may be characterized by a polymerase portion having an amino acid composition that is at least 90%, 95%, 99%, or 100% identical to any of SEQ ID NO:33 to SEQ ID NO:55, or SEQ ID NO:1, or a DNA-binding portion having an amino acid composition that is at least 90%, 95%, 99%, or 100% identical to any of SEQ ID NO:56 to SEQ ID NO:98, or SEQ ID NO:2. In one embodiment, the polymerase portion having an amino acid sequence that is 90%, 95%, 98%, or 99% identical to SEQ ID NO:102 is fused to a heterologous DNA-binding portion selected from a polypeptide having an amino acid sequence that is at least 90%, 95%, 99%, or 100% identical to any of SEQ ID NO:56 to SEQ ID NO:98.

[0027] In summary, in one aspect, a formulation is provided comprising a composition having an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 3. The formulation may further comprise a 5'-3' exonuclease, such as a T5 exonuclease. The formulation may further comprise an ss DNA binding protein, such as ET SSB, E. coli recA, a product of the T7 gene 2.5, phage lambda RedB, or Rac prophage RecT, and more specifically, a thermostable ss binding protein, such as ET SSB. The formulation may further comprise a ligase. In one aspect, the formulation comprising the composition may further comprise an ss binding domain and a 5'-3' exonuclease, wherein the formulation does not comprise a crowding agent and / or a non-strand-displacing polymerase. The formulation may further comprise a potassium salt.

[0028] In one aspect, a formulation comprising a composition can further comprise a plurality of polynucleotides from a group, wherein the sequence of at least one polynucleotide from the group overlaps with another polynucleotide from the group; and wherein the polynucleotides are selected from: (i) a ds polynucleotide; (ii) an ss oligonucleotide; (iii) at least one ds polynucleotide and at least one ss oligonucleotide; and (iv) a subgroup of polynucleotides that are identical to one another except for sequences that vary between members of the subgroup. In one aspect, the group of polynucleotides has at least 3 members, or at least 4 members, or at least 5 members.

[0029] Generally, a method for preparing a synthon comprises: incubating the composition of claim 1 further comprising a 5'-3' exonuclease and, optionally, a ligase and an ss DNA binding protein with a plurality of polynucleotides forming a set under suitable reaction conditions, wherein members of the set have overlapping sequences; and ligating at least two polynucleotides to prepare the synthon. In one aspect, the set of polynucleotides comprises at least 3 members, or at least 4 members, or at least 5 members. In one aspect, the formulation further comprises a ligase. In one aspect, the formulation further comprises an ss DNA binding protein. In one aspect, the polynucleotide is a ds, and the ds polynucleotide is an overlapping PCR product, an overlapping restriction fragment, or assembled from ss oligonucleotides. In one aspect, the polynucleotide is an ss oligonucleotide. In one aspect, the set of polynucleotides comprises at least one ds polynucleotide and at least one ss oligonucleotide.

[0030] In summary, the present invention provides a kit comprising a formulation according to claim 1 and a 5'-3' exonuclease, such as, for example, a T5 exonuclease. In one aspect, the kit can further comprise an ss-binding protein. In another aspect, the kit can comprise a ligase. In another aspect, the kit can comprise a buffer. In one aspect, the kit does not comprise a crowding agent. In one aspect, the composition and the 5'-3' exonuclease are in the same container. In another aspect, the composition and the 5'-3' exonuclease are in different containers, optionally in a buffer suitable for combination in a single container.

[0031] In summary, the present invention provides a composition for assembling a synthon, comprising: a 5'-3' exonuclease, which in one aspect has ss endonuclease activity, e.g., 90% sequence identity to SEQ ID NO: 98; a strand-displacing polymerase, including a family B strand-displacing polymerase, which is preferably non-naturally occurring, such as a mutant or fusion protein derived from a naturally occurring polymerase, which may additionally be thermostable; an optional ss DNA binding protein, e.g., the ss DNA binding protein is ET SSB, E. coli recA, the product of T7 gene 2.5, phage lambda RedB, or Rac prophage RecT; and a non-naturally occurring buffer, wherein the composition does not include a crowding agent and / or a non-strand-displacing polymerase. In one aspect, the composition further includes a ligase and / or an ss binding domain. In one aspect, the composition includes a set of at least two polynucleotides (a plurality of polynucleotides). In one aspect, the composition does not include a non-strand-displacing polymerase. In another aspect, the composition does not include 9°N, Phusion, Vent, or Pfu DNA polymerase.

[0032] In one aspect, the strand-displacing polymerase in the composition is a fusion protein, wherein the amino acid sequence of the polymerase portion is at least 90% identical to SEQ ID NO: 1 or SEQ ID NO: 102 or any of SEQ ID NO: 33 to SEQ ID NO: 55. For example, the amino acid sequence of the fusion protein can be at least 90% identical to SEQ ID NO: 1 or SEQ ID NO: 102 and SEQ ID NO: 2. For example, the strand-displacing polymerase can be at least 90% identical to the sequence of SEQ ID NO: 3. In one aspect, the composition can include potassium salt at a concentration of at least 7 mM. In one aspect, the composition can include a group of polynucleotides, wherein at least one polynucleotide in the group has a sequence that overlaps with another polynucleotide in the group, and wherein the polynucleotides are selected from: (i) a ds polynucleotide; (ii) an ss oligonucleotide; (iii) at least one ds polynucleotide and at least one ss oligonucleotide; and (iv) a subgroup of polynucleotides that are identical to each other except for sequences that vary between members of the subgroup. In one aspect, at least one member of the set of polynucleotides comprises a random sequence located between a given sequence at each end for hybridizing to a second ss genomic polynucleotide, wherein, for example, the random sequence is ss and is capable of hybridizing to a target genomic sequence for guiding a Cas protein to a target genomic nucleic acid for gene editing.

[0033] In summary, the present invention provides a method for forming a synthon, comprising incubating any of the above-described compositions comprising a set of polynucleotides having overlapping sequences under suitable reaction conditions; and joining at least some of the polynucleotides to other polynucleotides to produce a synthon. In one aspect of the method, all or a portion of the polynucleotides in the set are ds. In another aspect, the ds polynucleotides are overlapping PCR products; overlapping restriction fragments or synthetic ds molecules assembled from complementary ss oligonucleotides, wherein these oligonucleotides can be produced in a synthesizer. In one aspect, all or a portion of the polynucleotides in the set are ss oligonucleotides. In one aspect, the set of polynucleotides comprises at least one ds polynucleotide and at least one ss oligonucleotide. In one aspect, the set of polynucleotides comprises subpopulations of polynucleotides that are identical to one another except for sequences that vary between members of the subpopulation. In one aspect, the overlapping sequences of the polynucleotides are less than 2 kilobases in length. In one aspect of the method, the strand displacement polymerase comprises an amino acid sequence that is at least 90% identical to any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 33 to SEQ ID NO: 96 or SEQ ID NO: 102. In one aspect of the method, at least one member of the polynucleotide set comprises a random sequence between the ends of a given sequence. Another aspect of the method includes screening for random sequences that have activity in hybridizing with genomic DNA and identifying random sequences that have hybridization activity. Another aspect of the method includes performing gene editing by transcribing a random sequence with hybridization activity to form RNA, and using the RNA for gene editing in the presence of a Cas protein.

[0034] In summary, the present invention provides a kit for polynucleotide assembly comprising: a 5'-3' exonuclease; a strand-displacing polymerase; and optionally, a ss DNA-binding protein, wherein the kit optionally excludes a crowding agent and / or a non-strand-displacing polymerase. In one aspect, the kit includes a ligase. In another aspect, the kit includes dNTPs. In another aspect, the kit includes a buffer. In another aspect, the individual components of the kit can be in the same container or in separate containers, such as one or more different storage or reaction vessels.

[0035] In summary, the present invention provides a composition comprising a polymerase fusion protein, wherein the polymerase fusion protein comprises an amino acid sequence at least 90% identical to any of SEQ ID NO: 2, SEQ ID NO: 56 to SEQ ID NO: 96, and a heterologous polymerase domain. In one aspect, the polymerase fusion protein comprises an amino acid sequence at least 90% identical to any of SEQ ID NO: 2; and a heterologous polymerase domain.

[0036] In summary, the present invention provides a composition comprising a polymerase fusion protein, wherein the polymerase fusion protein comprises a polymerase domain having an amino acid sequence at least 90% identical to any of SEQ ID NO: 1, SEQ ID NO: 33 to SEQ ID NO: 55, or SEQ ID NO: 102; and a heterologous DNA binding domain. In one aspect, the polymerase fusion protein comprises a polymerase domain having an amino acid sequence at least 90% identical to SEQ ID NO: 1; and a heterologous DNA binding domain. In one aspect, the polymerase fusion protein comprises a polymerase domain having an amino acid sequence at least 90% identical to SEQ ID NO: 102; and a heterologous DNA binding domain. In one aspect, the polymerase fusion protein has an amino acid sequence at least 90% identical to SEQ ID NO: 3.

[0037] In one aspect, the composition further comprises a 5'-3' exonuclease, such as T5 exonuclease. In one aspect, the composition further comprises a single-stranded DNA binding protein, such as a single-stranded binding protein selected from ET SSB, Escherichia coli recA, T7 gene 2.5 product, phage λ RedB or Rac prophage RecT. In one aspect, the composition may include a ligase. In one aspect, the ligase is thermostable. In one aspect, the composition does not include a crowding agent and / or a non-strand displacement polymerase. In another aspect, the composition further comprises dNTPs. In another aspect, the composition further comprises a potassium salt at a concentration of at least 7 mM. One aspect of the composition comprises a group of polynucleotides, wherein the sequence of at least one polynucleotide in the group overlaps with another polynucleotide in the group; and wherein the polynucleotides are selected from: (i) ds polynucleotides; (ii) ss oligonucleotides; (iii) at least one ds polynucleotide and at least one ss oligonucleotide; and (iv) a subgroup of polynucleotides that are identical to each other except for sequences that vary between subgroup members.

[0038] In summary, the present invention provides a method for preparing a synthon, comprising incubating a set of polynucleotides with a composition characterized as described above comprising a polymerase, wherein individual polynucleotides comprise sequences that overlap with sequences in other polynucleotides, wherein overlapping sequences of different polynucleotides are capable of cross-hybridizing under suitable reaction conditions, and, for example, wherein the overlapping region is less than 2 kilobases, wherein the composition further comprises a 5'-3' exonuclease and optionally a ligase and an ss DNA binding protein; and ligating the polynucleotides to prepare the synthon.

[0039] In various aspects, the composition comprises a ligase; and / or an ss DNA binding protein. In another aspect, one or more polynucleotides in the set are ds, wherein the ds polynucleotides are PCR products, overlapping restriction fragments, or assembled from ss oligonucleotides; and / or one or more polynucleotides are ss oligonucleotides; and / or the set of polynucleotides comprises at least one ds polynucleotide and at least one ss oligonucleotide.

[0040] In summary, the present invention provides a kit for polynucleotide assembly, comprising the polymerase fusion protein described above, a 5'-3' exonuclease, and a ss DNA binding protein. In one aspect, the kit may include any or all of the ligase, dNTPs, and a buffer, wherein the kit components may be in the same container or in different containers. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Those skilled in the art will appreciate that the following drawings are for illustration purposes only and are not intended to limit the scope of the present teachings in any way.

[0042] Figures 1A to 1E The assay described in Example 1 shows how to distinguish between strand-displacing and non-strand-displacing polymerases. This assay confirms that T4 DNA polymerase is non-strand-displacing and terminates template DNA synthesis when the blocking oligonucleotide (2) is 44 nucleotides in length ( Figure 1C ), while Bst polymerase ( Figure 1D ) and non-natural polymerases ( Figure 1E ) is strand-displacing and can continue DNA synthesis of the FAM primer (1) by displacing the blocking oligonucleotide (27 nucleotides in length).

[0043] Figure 1A Shown are the sequences and positions of primers (1) and blocking oligonucleotides (2) on the DNA template used in the following enzyme assay.

[0044] Figure 1B Shown is the fluorescence observed in a sample obtained after capillary electrophoresis, in which no enzyme was added and the starting material formed a peak at a position corresponding to a 24-nucleotide FAM primer.

[0045] Figure 1C Results for the addition of T4 DNA polymerase are shown. The primer is extended to a final length of 44 nucleotides (24 nucleotides plus 20 nucleotides) but is terminated by a blocking oligonucleotide.

[0046] Figure 1DBst DNA polymerase (large fragment) is shown to strand displace the blocking oligonucleotide and copy the template by extending the primer to a total length of 72 nucleotides (24+20+27+dA).

[0047] Figure 1E A family B strand-displacing DNA polymerase, acting as a proofreading polymerase, is shown to strand-displace the blocking oligonucleotide and copy the template by extending the primer by 71 nucleotides (24+20+27).

[0048] Figures 2A to 2C Shown are the steps in the DNA assembly method.

[0049] Figure 2A The amplicons of each of the five fragments are shown added to five plasmids with an ampicillin resistance marker. The five fragments were initially amplified using primers that produced amplicons with overlapping regions and flanked by NotI restriction sites. NotI cleavage produces sticky ends. The NotI restriction site (3) allows each amplicon to be released from the vector. The restriction enzyme fragments have an 80 base pair overlap (4) with the adjacent fragments. Figure 2C For convenience and cost reduction, the overlap between the first fragment and the adjacent reagent carrier end and the last fragment and the adjacent reagent carrier end is 15 to 25 nucleotides, for example, 20 nucleotides, but this is not intended to be limiting.

[0050] Figure 2B The optionally sequenced fragments (5) (amplicons recovered from the vector) cleaved with NotI are shown and then treated in a single reaction vessel with an enzyme cocktail including T5 / 5'-3' exonuclease, a DNA polymerase with 3'-5' exonuclease activity, the ss binding protein ET SSB (New England Biolabs, Ipswich, MA), and DNA ligase (6)-(8). Although NotI is used here, other restriction endonucleases may be used for cleavage depending on convenience. Double digestion can be performed with two or more restriction endonucleases. For example, it has been found that double digestion of vector DNA with two restriction endonucleases reduces background from uncut vector. Overlapping ss DNA sequences are hybridized to adjacent fragments. T5 exonuclease chews back the DNA strands from 5' to 3' on each fragment to expose the 3'ss region (6), which causes the fragments to anneal together in the presence of the ss binding protein (7). Removal of the 2-base flap is achieved by means of the 3'-5' exonuclease activity associated with a strand-displacing polymerase, followed by extension by a strand-displacing polymerase to fill the gap in the assembled product (8). Any remaining nick or 5' flap can be repaired by ligase and / or T5 exonuclease.

[0051] Figure 2C Shown are the five fragments (Fragment 1 to Fragment 5) now joined in sequence and inserted into a second plasmid carrying the chloramphenicol resistance gene (Cam) for transformation into a bacterial host.

[0052] Figure 3A and 3B As shown, chloramphenicol plates were used to select for colonies that grew on the plates, and in the presence of IPTG and X-Gal, those colonies containing the lac1Z gene produced blue colonies. The assay provides a quantitative assessment of clones in which the gene was efficiently assembled in a functional form.

[0053] Figure 3A Only chloramphenicol is shown.

[0054] Figure 3B Chloramphenicol + IPTG + Xgal are shown.

[0055] Figure 4 This shows that the plasmid does in fact contain the entire gene. Figure 2B The assembly products shown were PCR amplified to confirm that all fragments were joined and ligated before transformation. Bands 1 and 2 are duplicate PCR results. Band M is a 2-log DNA ladder from New England Biolabs, Ipswich, MA.

[0056] Figure 5 The effect of KCl in the assembly mixture as determined by colony count is shown. Increasing the concentration of KCl used in the buffer shows an increase in the accuracy / efficiency of assembly using a strand-displacing polymerase in the assembly mixture. The histogram on the left (T26) contains no KCl, while the histogram on the right (T26K) contains 25 mM KCl, which shows a 1.5-fold increase in efficiency. This increase occurs regardless of the assembly conditions. Similar relative efficiency increases are expected if PEG or other crowding agents are used in the absence of ss-binding proteins.

[0057] Figure 6 The mixture described in Example 2 (strand displacement polymerase / ss binding protein / 5'-3' exonuclease / ligase) (Mix 1) and commercially available Gibson Comparison between Mix (GAMM) (non-strand-displacing polymerase and polyethylene glycol) (Synthetic Genomics, La Jolla, CA / New England Biolabs, Ipswich, MA). Mix 1 produced significantly higher DNA assembly and transformation efficiencies.

[0058] Figure 7 A schematic diagram of DNA assembly between an ss DNA oligonucleotide and a ds DNA fragment is shown. The ss target DNA oligonucleotide is inserted into a DNA vector. The ss target DNA oligonucleotide has been synthesized to have a 20-30 nucleotide overlap region with the 3' vector end at each end. However, the oligonucleotide can be sized to have an overlap region of less than 20 nucleotides, such as less than 15 nucleotides or less than 10 nucleotides, or alternatively greater than 30 nucleotides, such as at least 40 or 50 or 60 nucleotides or more. Outside of the overlap region, the oligonucleotide preferably has 1 or more non-overlapping nucleotides between the ends. An assembly master mix comprising a 5'-3' exonuclease, a strand displacement polymerase, a ligase and an ss binding protein is added to the mixture of the ss oligonucleotide and the vector so that the 5' end of the ds DNA vector is chewed back to produce an ss overhang (9). The 3' end of the ss DNA can then anneal to the 5' end of the vector, and DNA polymerase then replicates the ss template to fill the gap and produce blunt-ended ds DNA. The cut is sealed by ligase (10). Again, an exonuclease (here, T5 exonuclease) cuts back the 5' end of the blunt end of the target DNA, creating a 3'ss region to allow the complementary sequence to anneal and complete the integration of the target DNA ds into the DNA vector (11). The fragments can be annealed with the help of DNA polymerase to fill the gap and ligase to seal the cut (12) to produce synthons.

[0059] Figures 8A to 8C The workflow for bridging ds DNA by short ss oligonucleotides is shown. (The protocol is described in Figure 7 middle).

[0060] Figure 8A Examples of short ss oligonucleotide sequences are provided for integration into a ds DNA vector, shown here as a CRISPR nuclease vector with an OFP reporter.

[0061] Figure 8B The workflow shown starts with ss oligonucleotides and ds CRISPR nuclease vector (9424 bp), which are treated with 5'-3' exonuclease, strand displacement polymerase, ligase, and ss binding protein (13) to generate complete ds circular DNA. This DNA is transformed into competent cells (14). After overnight incubation, colonies are analyzed by miniprep and then plasmid sequencing (15). Figure 8CThe inserted sequence and adjacent sequences, U6 promoter sequence (vector), sequence of the designed ss oligonucleotide (71 mer), and scaffold template-specific sequence (vector) are shown. The ss oligonucleotide (71 mer) including a 25-nucleotide overlap region at each end (21 nucleotides of the target DNA shown in bold) is appropriately integrated into the vector in the host cell.

[0062] Figures 9A to 9C As shown, a similar workflow to that in Figure 8 can be used for ss oligonucleotides with degenerate bases between overlapping ends. Figure 9A The starting sequence in the workflow is shown in ( Figure 9B ) above, and Sanger sequencing results of colonies from the assembled library are shown below ( Figure 9C ), the solid line refers to the sgRNA target sequence library. The sgRNA target sequence contains 21 variable nucleotide positions, providing 4 21 The library contains every possible variant, and each variant is suitable for cloning reflecting the degeneracy of the sequence between the overlapping ends and the vector. Figure 9A The sequence of the ss oligonucleotide containing degenerate bases is shown. Figure 9A The sgRNA target sequence was inserted between the U6 promoter sequence and the backbone template-specific sequence of the vector (16), transformed into host cells (17), and synthon analysis was performed by miniprep and sequencing (18), as described above and herein. Clones from the assembled library were subjected to Sanger sequencing. Figure 9C Provides an instance of a sequence.

[0063] Figure 10 The result of 187 bacterium colonies selected from a plate after assembly reaction product is transformed into Escherichia coli is shown. Each bacterium colony is carried out pcr amplification and order-checking, to confirm the insertion of ss DNA, and the distribution of degenerate bases is analyzed. The result shown here confirms that different bacterium colonies do comprise different degenerate sequences. No bias is detected. First, by converting the sequence into fastq file, then using the fastx_quality_stats tool from the fastx toolkit on github, analyze. Use the weblogo from Berkeley to produce sequence logo.

[0064] Terminology

[0065] Unless otherwise indicated, 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. Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULARBIOLOGY, 2nd ed., John Wiley and Sons, New York (1994) and Hale & Markham, THE HARPERCOLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide ordinary meanings for many of the terms used herein. For clarity and ease of reference, certain terms are defined below.

[0066] As used herein, the term "synthon" as used in the field of gene synthesis refers to a polynucleotide assembly. Polynucleotide assembly can include assembling overlapping fragments of a certain size, which can be prepared on an oligonucleotide synthesizer, currently typically 2000 to 3000 bases for each synthesized polynucleotide. Alternatively, overlapping fragments can be obtained by PCR from naturally occurring nucleic acids connected with adapters to provide overlapping sequences. For assembly purposes, there is no limit on the size of each fragment. Depending on the overlapping sequences at the ends, many fragments can be assembled end-to-end, making it possible to accurately and efficiently produce constructs with any desired length. Preferably, a synthon is a longer continuous polynucleotide formed by an assembly of shorter polynucleotides without gaps and cuts. However, the length of the synthon produced by the nucleic acid fragment assembly is not limited to any specific size.

[0067] As used herein, the term "5'-3' exonuclease" refers to an exonuclease that degrades DNA from the 5' end, i.e., in a 5' to 3' direction. 5'-3' exonucleases of interest can remove nucleotides from the 5' end of a dsDNA strand at the blunt end and, in certain embodiments, at the 3' and / or 5' overhangs. T5 exonuclease, lambda exonuclease, and T7 exonuclease are examples of 5'-3' exonucleases. In certain embodiments, T5 exonuclease is preferred. T5 exonuclease additionally has ss endonuclease activity.

[0068] As used herein, the term "ligase" refers to an enzyme that can covalently join the 3' end of a DNA molecule to the 5' end of another DNA molecule, specifically at the nick. Although many other ligases are known and can be used herein, examples of ligases include T7 ligase, T4 DNA ligase, E. coli DNA ligase, and Taq ligase.

[0069] As used herein, the term "strand displacement polymerase" refers to a polymerase that is capable of displacing one or more nucleotides, such as at least 10 or 100 or more nucleotides, downstream of the enzyme. A strand displacement polymerase can be distinguished from Phusion, which is a non-strand displacement polymerase as defined in the art. In some embodiments, the strand displacement polymerase is stable and active (including strand displacement activity) at a temperature of at least 50° C. or at least 55° C. Taq polymerase is a nick translation polymerase and, therefore, is not a strand displacement polymerase.

[0070] As used herein, the term "single-stranded (ss) DNA binding protein" refers to a protein that binds to ss DNA, prevents premature annealing, protects ss DNA from digestion by nucleases and polymerases, and / or removes DNA secondary structure to allow other enzymes to function effectively therewith. It is preferred to include an ss-binding protein in the compositions described herein to optimize the efficiency of synthon formation. Examples of ss-DNA binding proteins include T4 gene 32 protein, E. coli SSB, T7 gp2.5 SSB, and phage phi29 SSB, and ET SSB, although many others are known and can be used herein, such as lambda phage RedB, RecT of the Rac prophage, and the sequences listed below. In some cases, thermostable ss-DNA binding proteins that are stable at 50°C can be used. Thus, in one embodiment of the composition, kit, or method of the present invention, the ssDNA binding protein is T4 gene 32 protein, E. coli SSB, T7 gp2.5SSB, phage phi29 SSB, ET SSB, lambda phage RedB, or Rac prophage RecT. In one embodiment, the ssDNA binding protein is ET SSB. In one embodiment of the composition, kit, or method of the present invention, the ssDNA binding protein is thermostable (i.e., stable at 40°C to 60°C).

[0071] As used herein, the term "buffer" refers to an agent that makes a solution resistant to changes in pH when an acid or base is added to the solution. Examples of suitable non-naturally occurring buffers that can be used in the compositions, kits and methods of the invention include, for example, Tris, HEPES, TAPS, MOPS, tricine or MES.

[0072] The term "non-naturally occurring" means that the composition does not exist in nature.

[0073] Any protein described herein may be non-naturally occurring, wherein the term "non-naturally occurring" refers to a protein having an amino acid sequence and / or post-translational modification pattern that is different from that in its natural state. For example, a non-naturally occurring protein may have one or more amino acid substitutions, deletions, or insertions at the N-terminus, C-terminus, and / or between the N-terminus and the C-terminus of the protein. The amino acid sequence of a "non-naturally occurring" protein may be different from a naturally occurring amino acid sequence (i.e., less than 100% identity to the amino acid sequence of a naturally occurring protein), but may be at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to a naturally occurring amino acid sequence. In some cases, if a non-naturally occurring protein is prepared by a different (e.g., bacterial) cell, it may contain an N-terminal methionine or may lack one or more post-translational modifications (e.g., glycosylation, phosphorylation, etc.). A "mutant" protein may have one or more amino acid substitutions relative to a wild-type protein and may include a "fusion" protein. The term "fusion protein" refers to a protein composed of multiple polypeptide components that are not joined in their natural state. Fusion proteins can be combinations of 2, 3 or even 4 or more different proteins. The term polypeptide includes fusion proteins, including, but not limited to, the fusion of two or more heterologous amino acid sequences, polypeptides and the following fusions: heterologous target sequences, linkers, immunological tags, detectable fusion partners such as fluorescent proteins, beta-galactosidase, luciferase, etc., etc. The fusion protein can have one or more heterologous domains added to the N-terminus, C-terminus and / or middle part of the protein. If the two parts of the fusion protein are "heterologous", they are not part of the same protein in their native state.

[0074] The term "non-naturally occurring" when referring to a nucleic acid means that the nucleic acid comprises: a) a nucleotide sequence that is different from that found in nature (i.e., the sequence identity to a naturally occurring nucleic acid sequence is less than 100%), b) one or more non-naturally occurring nucleotide monomers (which may produce a non-natural backbone or sugar that is not G, A, T or C), and / or c) may comprise one or more other modifications (i.e., added tags or other moieties) at the 5'-terminus, the 3'-terminus and / or between the 5'- and 3'-termini of the nucleic acid.

[0075] When referring to a formulation in this context, the term "non-naturally occurring" refers to: a) a composition of components that are not found in nature, for example, because the components are in a different location, in a different cell, or in a different cellular compartment; b) a composition of components whose relative concentrations are not found in nature; c) a composition that lacks certain components that are normally associated with one of the components in nature; d) a composition that is not found in nature in a form such as a dried, lyophilized, crystalline, or aqueous form; and / or e) a composition that contains components that are not found in nature. For example, a formulation can contain a "non-naturally occurring" buffer (e.g., Tris, HEPES, TAPS, MOPS, a zwitterionic buffer, or MES), a detergent, a dye, a reaction accelerator or inhibitor, an oxidizing agent, a reducing agent, a solvent, or a preservative that is not found in nature.

[0076] It may be necessary to use a strand-displacing polymerase with 3' exonuclease activity. Although not intending to be bound by theory, a 3' exonuclease is required to remove flanking sequences at the 3' end of the double strand, where the flanking sequences may be products of enzymatic cleavage, in order to extract the target polynucleotide from the plasmid in which they are placed. This is the case when using NotI as described in the Examples. However, if a restriction endonuclease is used that produces blunt ends on the excised fragment, 3' exonuclease activity may not be required.

[0077] 3' exonuclease activity can be routinely assayed using a standard DNA template and primers with or without non-hybridizing 3' nucleotides. If the polymerase has 3' exonuclease activity, then amplicons will be detected using either primer pair. If the polymerase lacks 3' exonuclease activity, then no amplicons will be detected using primers with non-hybridizing 3' nucleotides.

[0078] As used herein, the term "potassium salt" refers to potassium salts including, but not limited to, KCl. The term "sodium salt" refers to sodium salts including, but not limited to, NaCl.

[0079] As used herein, the term "polynucleotide" includes oligonucleotides and refers to nucleic acids of any length. A polynucleotide can be DNA or RNA. Unless otherwise specified, a polynucleotide can be ss or ds. A polynucleotide can be synthetic, e.g., synthesized in a DNA synthesizer, or naturally occurring, e.g., extracted from a natural source or derived from cloned or amplified material. The polynucleotides referred to herein may contain modified bases.

[0080] As used herein, the term "polynucleotide set" or "a group of polynucleotides" refers to a collection of at least two polynucleotides. In some embodiments, a group of polynucleotides can include at least 5, at least 10, at least 12, or at least 15 or more polynucleotides.

[0081] As used herein, the term "overlapping sequence" refers to sequences that are complementary in two polynucleotides, and wherein the overlapping sequence is ss, which on one polynucleotide can hybridize to another overlapping complementary ss region on another polynucleotide. For example, in a set of polynucleotides, overlapping sequences can be complementary in at least 5, 10, 15 or more polynucleotides. Overlapping sequences can be at the 3' end of two different molecules (e.g., the 3' end of two ss oligonucleotides, or the 3' end of the top strand of a first ds polynucleotide and the 3' end of the bottom strand of a second ds molecule) or close thereto (e.g., within about 5, 10, 20 nucleotides), wherein, if non-overlapping sequences are at the 3' end, the non-overlapping sequences can be removed using the 3'-5' exonuclease activity of a polymerase. The length of the overlapping sequences can vary, and in some cases, can be at least 12 nucleotides in length (e.g., at least 15, 20, or more nucleotides in length), and / or can be up to 100 nucleotides in length (e.g., up to 50, up to 30, up to 20, or up to 15 nucleotides in length). Alternatively, the overlapping sequences in the set of polynucleotides can be 2 kb or less, or 1 kb or less, or less than 900 bases, 800 bases, 700 bases, 600 bases, 500 bases, 400 bases, 300 bases, 200 bases, or 100 bases. Preferably, the overlapping sequence length is in the range of 15 nucleotides to 80 nucleotides, for example, at most 20, at most 25, at most 30, at most 35, at most 40, at most 45, at most 50, at most 55, at most 60, at most 65, at most 70, at most 75 or at most 80 nucleotides. The minimum length of the overlap can be defined by the Tm, which is preferably equal to or greater than 48°C.

[0082] As used herein, the term "polynucleotide assembly" refers to a reaction in which two or more, four or more, six or more, eight or more, ten or more, twelve or more, fifteen or more polynucleotides, e.g., four or more polynucleotides, are joined to each other to produce a longer polynucleotide. In many embodiments, the product of a polynucleotide assembly reaction, i.e., an "assembled polynucleotide" or "synthon," will contain one copy of each overlapping sequence.

[0083] As used herein, the term "incubating under suitable reaction conditions" refers to maintaining the reaction at a suitable temperature and time to achieve the desired result, i.e., polynucleotide assembly. The reaction conditions for enzymes and reagents suitable for use in the methods of the present invention are known (e.g., as described in the Examples herein), and therefore, suitable reaction conditions for the methods of the present invention can be readily determined. These reaction conditions can vary depending on the enzyme used (e.g., depending on its optimal temperature, etc.).

[0084] As used herein, the term "isothermal" refers to temperature conditions that do not require active regulation of the temperature for assembly to proceed. Insignificant variations in the temperature of a water bath or heating block are within the meaning of the term isothermal. For example, the term "isothermal" may refer to reaction conditions that do not require a thermal denaturation step after the start of the reaction. More specifically, isothermal methods do not involve thermal cycling, i.e., cycling between a denaturation temperature and an annealing / extension temperature that is greater than 90°C. Isothermal conditions typically involve incubation at a temperature below 90°C for a period of time (e.g., 5 minutes to 12 hours or longer). In one embodiment, the isothermal amplification reaction is carried out at a temperature in the range of 30°C to 75°C, e.g., 40°C to 60°C.

[0085] As used herein, the term "conjugate" refers to the creation of a covalent link between two sequences.

[0086] As used herein, the term "composition" refers to a combination of reagents that, in addition to those listed, may include other reagents such as glycerol, salts, dNTPs, etc. The composition can be in any form, for example, aqueous or lyophilized, and can be in any state (for example, frozen or liquid form).

[0087] As used herein, a "vector" is a suitable DNA into which a fragment or synthon can be incorporated so that the transformed vector can be replicated in a host cell. Linearized vectors can be produced by restriction endonuclease digestion of circular vectors or by PCR. The concentration of fragments and / or linearized vectors can be determined by gel electrophoresis or other means.

[0088] Any one or more proteins used herein (e.g., ligase, SSBP, 5'-3' exonuclease or polymerase, etc.) can be temperature-sensitive or thermostable, wherein, as used herein, the term "temperature-sensitive" means that the enzyme loses at least 95% of its activity after 10 minutes at a temperature of 65°C, and the term "thermostable" means that the enzyme retains at least 95% of its activity after 10 minutes at a temperature of 65°C. DETAILED DESCRIPTION

[0089] Before describing various embodiments in more detail, it should be understood that the teachings of the present disclosure are not limited to the specific embodiments described, and therefore, they may vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting, as the scope of the present teachings will be limited only by the claims.

[0090] Although the teaching of the present invention is described in conjunction with various embodiments, it is not intended to limit the teaching of the present invention to these embodiments. On the contrary, as will be appreciated by those skilled in the art, the teaching of the present invention includes various alternatives, modifications and equivalents.

[0091] When a range of values ​​is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range, and any other intervening value stated or within its stated range, is encompassed within the disclosure.

[0092] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present teachings, some exemplary methods and materials are now described.

[0093] The citation of any publication for its disclosure prior to the filing date should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publications provided may be different from the actual publication dates which need to be independently confirmed.

[0094] It must be noted that, as used herein and in the claims hereof, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It is also noted that the claims can be drafted to exclude any optional elements. As such, this statement is intended to serve as antecedent basis for using such exclusive terminology as "solely," "only," and the like in connection with the recitation of claim elements, or for using a "negative" limitation.

[0095] As will be apparent to those skilled in the art upon reading this disclosure, each individual embodiment described and illustrated herein has independent components and features that can be readily separated or combined with the features of any other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be implemented in the order of events recited or in any other logically possible order.

[0096] The overlapping sequences in the polynucleotide set can be any suitable length, for example, 2 kb or less, or 1 kb or less, or less than 900 bases, 800 bases, 700 bases, 600 bases, 500 bases, 400 bases, 300 bases, 200 bases or 100 bases. The overlapping region can be as few as 8 nucleotides. Preferably, the overlapping sequence length is in the range of 15 nucleotides to 80 nucleotides, for example, at most 20, at most 25, at most 30, at most 35, at most 40, at most 45, at most 50, at most 55, at most 60, at most 65, at most 70, at most 75 or at most 80 nucleotides. For example, the minimum overlap length can be defined by Tm, which is preferably equal to or greater than 48°C.

[0097] The synthesized oligonucleotides and polynucleotides may contain errors generated during their synthesis before they are used for synthon assembly. In order to correct these errors before assembly, it is desirable to perform a mismatch repair step. In this regard, a variety of methods for achieving mismatch repair of synthetic nucleic acids before assembly have been described. The population of synthetic nucleic acids can have random errors so that denaturation and renaturation of the preparation can reveal mismatches. Proteins that have been isolated from nature, such as mutHLS, cel-1 nuclease, T7 endo 1, uvrD, T4 EndoVII, E. coli EndoV (see US 7,851,192 and US 8,048,664), can selectively bind to double-stranded DNA containing mismatches; cut nucleic acids at mismatch bases; and optionally replace with correct bases based on the template nucleotide sequence.

[0098] Although the art teaches that non-strand-displacing polymerases must be used in conjunction with ss-binding proteins, 5'-3' exonucleases, and ligases to assemble DNA fragments, it is unexpectedly shown herein that strand-displacing polymerases can be used under conditions where strand displacement occurs and are effective at unexpectedly low concentrations of starting polynucleotide fragments to efficiently generate a single nucleic acid from multiple fragments.

[0099] Examples of strand-displacing polymerases that can be used in embodiments of the assembly mixtures, compositions, kits, or methods of the invention include members of the B family polymerases, such as, but not limited to, any of those identified in Table 1 (SEQ ID NO: 33 to SEQ ID NO: 55). In addition, fusions of these polymerases can be used, for example, fusions between various polymerase and / or ss binding domains (e.g., as shown in Table 2) (SEQ ID NO: 56 to SEQ ID NO: 97). In embodiments, any polymerase portion of Table 1, or a protein having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% amino acid sequence identity with any of these protein portions in Table 1, can be fused at the N-terminus or C-terminus to any DNA binding domain described in Table 2, or a portion of a protein having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% amino acid sequence identity with any of the DNA binding portions in Table 2, to form a strand-displacing fusion polymerase for use herein. The DNA binding domain can optionally be fused to the N-terminus or C-terminus of the polymerase.

[0100] Assays such as those provided herein (see, e.g. Figures 1A to 1Eand Example 1) to identify additional polymerase variants or novel isolates that exhibit strand displacement. The sequences of polymerases discovered from these sources are readily available through GenBank. Due to the high conservation of strand displacement sequences, any variant that has an amino acid sequence identity of 80%, 85%, 90%, or 95% to these wild-type polymerases can be expected to have strand displacement properties, which can be quickly and easily verified in the assay provided in Example 1 in a preselected buffer without undue experimentation.

[0101] In one embodiment, a reaction mixture, composition, kit, or method of the invention comprises or uses a strand-displacing polymerase that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical (e.g., 100% identical) to SEQ ID NO: 1 or SEQ ID NO: 102. In another embodiment, a reaction mixture, composition, kit, or method of the invention comprises or uses a polymerase that has a binding domain that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical (e.g., 100% identical) to SEQ ID NO: 2. In another embodiment, the reaction mixtures, compositions, kits, or methods of the invention include or utilize a polymerase that has at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 102 and to any of SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 33 to SEQ ID NO: 97 (e.g., 100% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 102 and to any of SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 33 to SEQ ID NO: 97). These compositions can be used under reaction conditions in which the polymerase is strand-displacing. The compositions can be used under reaction conditions in which any 3'-5' exonuclease activity associated with the polymerase activity is active. This can be helpful when a restriction enzyme such as NotI is used in the reaction. In this case, the 3'-5' exonuclease can remove flanking sequences at the 3'-end of the double strand. However, if a restriction endonuclease is used that produces blunt ends on the excised fragments, 3'-5' exonuclease activity may not be required. The assembly reaction may be performed under isothermal conditions. In one embodiment, the isothermal conditions are 50°C.

[0102] Table 1. List of polymerases

[0103] Methanocaldococcus vulcanius M7 SP-13 gi|502573182 SEQ ID NO:33 Archaeoglobus fulgidus DSM 4304 SP-16 gi|499180464 SEQ ID NO:34 Archaeoglobus profundus DSM 5631 SP-17 gi|502704426 SEQ ID NO:35 Caldicellulosiruptor hydrothermalis 108 SP-19 gi|503168530 SEQ ID NO:36 Desulfurococcus mucosus DSM 2162 SP-27 gi|503328138 SEQ ID NO:37 Pyrolobus fumarii SP-29 gi|503791850 SEQ ID NO:38 Pyrobaculum oguniense CH SP-30 gi|379003208 SEQ ID NO:39 Staphylothermus marinus F1 SP-33 gi|500164563 SEQ ID NO:40 Pyrococcus yayaosii CH1 SP-42 gi|503672202 SEQ ID NO:41 Thermococcus sp.AM4-del SP-43 gi|503888003 SEQ ID NO:42 Thermococcus hydrothermalis SP-44 gi|17375628 SEQ ID NO:43 Thermococcus thioreducens SP-45 gi|117958105 SEQ ID NO:44 Thermococcus waiotapuensis SP-46 gi|378813034 SEQ ID NO:45 Thermococcus sibiricus MM 739 SP-47 gi|506329477 SEQ ID NO:46 Pyrococcus glycovorans SP-48 gi|7288074 SEQ ID NO:47 Pyrococcus sp.NA2 SP-49 gi|503513858 SEQ ID NO:48 Ferroglobus placidus DSM 10642 SP-61 gi|502730992 SEQ ID NO:49 Palaeococcus ferrophilus DSM 13482 SP-5 gi|851288004 SEQ ID NO:50 Thermococcus gammatolerans EJ3 SP-50 gi|506339349 SEQ ID NO:51 Thermococcus celericrescens SP-51 gi|332308985 SEQ ID NO:52 Vulcanisaeta distributa DSM 14429 SP-60 gi|503101260 SEQ ID NO:53 Methanopyrus kandleri AV19 SP-7 gi|20094475 SEQ ID NO:54 Thermoproteus neutrophilus V24Sta SP-9 gi|171185774 SEQ ID NO:55

[0104] Table 2. DNA binding proteins

[0105]

[0106]

[0107] In embodiments of the present invention, the reaction mixture, composition, kit or method may include or use a 5'-3' exonuclease, such as T5 / 5'-3' exonuclease, which is temperature sensitive and can be inactivated by raising the temperature above 50°C. In one embodiment, the 5'-3' exonuclease has exonuclease activity as well as ss endonuclease activity. In some embodiments, the reaction mixture may also include a ligase, such as one that requires NAD + The reaction mixture may include a ligase and / or a thermostable ligase, such as Taq ligase. In a preferred embodiment, the reaction mixture may include an SS binding protein. The SS binding protein may be thermostable, such as ET SSB. The assembly reaction may be performed under isothermal conditions.

[0108] In certain embodiments, the use of a ligase is optional. For example, when the assembled fragments are directly introduced into a vector for host cell transformation, a ligase is not required because host cells such as E. coli are able to repair the nicks in vivo. However, if the assembled fragments are amplified for the purpose of confirming correct assembly prior to transformation, it is desirable to use a ligase to seal the nicks and enable the polymerase to amplify the entire target DNA.

[0109] The cloning of a single fragment can use chemically synthesized polynucleotide fragments whose sequences are obtained from any database or publication, wherein the polynucleotide fragments have overlapping sequences. These fragments can be cloned in a plasmid by inserting the polynucleotide into a site in the plasmid adjacent to a restriction endonuclease site suitable for cutting the inserted polynucleotide.

[0110] Any plasmid can be used. The present embodiment uses the commercially available pACYC184 comprising the chloramphenicol gene as a selective marker. Any selective marker can be used to replace the chloramphenicol resistance gene. Similarly, the specific recognition site of any cutting enzyme can be selected, as long as the cutting enzyme can specifically cut to produce staggered ends or blunt ends at the oligonucleotide end, wherein except for the position of the through engineering approaches of the end of the adjacent fragment being paid attention to, the specific cutting site does not occur in the fragment being paid attention to. In the present embodiment, the recognition site (CGCCGGCG) of the 8 base cutters NotI that will produce staggered ends by the method for DNA synthesis is introduced adjacent to the polynucleotide being paid attention to. However, this site can be present in the selected plasmid, or is added on the synthetic oligonucleotide being paid attention to by an amplification primer. The example of specific cutting enzyme includes restriction endonuclease and homing endonuclease.

[0111] Once the oligonucleotide or DNA fragment of interest has been chemically synthesized or cloned or amplified from existing DNA and cloned into a vector with a selectable marker, it is preferably excised by enzymatic cleavage. The fragments or oligonucleotides that have been synthesized or amplified so as to include overlapping sequences with the adjacent fragments or oligonucleotides to be joined are then assembled in an assembly reaction.

[0112] Under the selected hybridization conditions, the 5'-3' exonuclease in the reaction mixture (e.g., at a concentration range of 0.004-0.016 U / μl) chews back any ss region at the 5' end of the fragment or oligonucleotide and continues to chew back through the overlapping sequence region and may further continue for a limited distance (e.g., at least 100 bases) to provide a 3'ss region (see, e.g., Figures 2A-2C and Figure 7 ). At the same time, through Figures 1A-1E The remaining gap between the hybridized ds region and any remaining ss region is repaired by a strand-displacing polymerase (e.g., at a concentration range of 0.005 U / μl-0.5 U / μl) as determined experimentally in Example 1. Because the polymerase is strand-displacing, it can displace additional downstream sequence to form an ss flank. However, the ss endonuclease activity of T5 exonuclease will remove this flank, and any associated nicks can be repaired by a ligase (e.g., at a concentration range of 0.001 U / μl-20 U / μl).

[0113] Once the fragments are assembled into a larger DNA, they are cloned in host cell colonies under selective pressure. DNA from these colonies can be released from the vector and assembled again with other fragments and transformed into host cells, thereby extending the size of the DNA multiple times. The host cell can be a competent bacterial cell, or it can be a yeast cell or other eukaryotic cell.

[0114] The assembly methods described herein have been found to be very effective. For example, larger fragments can be assembled using 0.02nM-100nM oligonucleotides (ss) or DNA fragments (ds), wherein the concentration of the ss oligonucleotides used in the reaction can be up to about 50 times higher than the amount of the ds DNA fragments used in similar assembly reactions. Similarly, equimolar concentrations of plasmids comprising a single fragment and a selection marker and similar amounts of vectors with different selection markers comprising assembly fragments can be used. These amounts are intended to be used for guidance, but regardless of whether the assembly efficiency is improved, these amounts can be reduced. For example, as measured by the colony number of the assembly using lac1Z as an indicator, the addition of potassium salt KCl can increase the efficiency of the assembly by 1.5 times (see, e.g., Figure 5 ).

[0115] The assembly process of ss target oligonucleotides for use between two ds DNA molecules or for introduction into linearized vectors is also very efficient. This article provides an example, which is not intended to be limiting, that uses specific / random sequences to identify guide RNAs for CRISPR-Cas gene editing protocols that can be introduced into cells to determine phenotypic changes. Initially, it may not be known what sequences may be suitable for achieving this purpose. The generation of libraries containing degenerate sequences makes this type of analysis possible. Gene editing based on CRISPR / Cas-9 has rapidly gained popularity in the field of genome editing. Due to the size of the most commonly used plasmids containing Cas9, it is cumbersome to construct sgRNA or sgRNA libraries into Cas9 / sgRNA expression vectors. Using ss DNA oligonucleotides, this method solves this problem.

[0116] In separate embodiments, any of the DNA binding domains of Table 2 can be fused to Bst polymerase, Bst large fragment, or a mutant thereof (see, e.g., US 8,993,298 and US 2015 / 0152396, which include all Bst variants described and claimed herein).

[0117] Reagent test kit

[0118] The present disclosure further provides a kit for implementing the above-mentioned method. In certain embodiments, the kit may comprise: i. 5'-3' exonuclease, ii. optional ligase, iii. strand-displacing polymerase; and iv. ss DNA binding protein. The kit components may be combined in one container, or each component may be in its own container. For example, the kit components may be combined in a single reaction tube, or in one or more different reaction tubes. Further details of the kit components are described above. Depending on how the method is implemented, the kit may also comprise other reagents described above and below that can be used in the method, for example, mismatch repair enzymes such as mutHLS, cel-1 nuclease, T7 endo 1, uvrD, T4EndoVII, E. coli EndoV, buffers, dNTPs, plasmids into which synthons are inserted and / or competent cells for receiving the plasmids, controls, etc. In some embodiments, the kit does not contain a non-strand-displacing polymerase and / or a crowding agent.

[0119] In addition to the above components, the test kit also includes the use of the test kit components to implement the instructions for use of the method. The instructions for use of the method are typically recorded on a suitable recording medium. For example, the instructions can be printed on a medium such as paper or plastic. Like this, the instructions can be present in the test kit as a package insert, in the container label of the test kit or its components (i.e., associated with packaging or sub-packaging), etc. In other embodiments, the instructions exist as an electronic storage data file, in a suitable computer-readable storage medium, such as a CD-ROM, a floppy disk, etc. In another other embodiment, actual instructions are not present in the test kit, but are provided for, for example, a method obtained via the Internet from a remote resource. The example of this embodiment is a test kit comprising a network address from which instructions can be browsed and / or instructions can be downloaded. With regard to instructions, this method for obtaining instructions is recorded on a suitable substrate.

[0120] The compositions, kits, and methods described herein for assembling fragments and forming synthons produce products of ds-completely sealed DNA that can be used as templates for PCR, RCA, or a variety of other molecular biology applications, including direct transformation of competent bacteria or transfection of eukaryotic host cells.

[0121] In order to further illustrate the present invention, the following specific examples are provided. It should be understood that they are provided for the purpose of illustrating the present invention and should not be understood as limiting the scope of the present invention in any way.

[0122] All references cited herein, including U.S. Provisional Application Nos. 62 / 042,527, filed August 27, 2014, 62 / 189,599, filed July 7, 2015, and 62 / 193,168, filed July 16, 2015, are incorporated by reference.

[0123] Example

[0124] Example 1: Assays to determine the strand displacement properties of polymerases

[0125] Develop an assay to distinguish between strand-displacing and non-strand-displacing polymerases - Prepare a 10 nM FAM-primer / template / blocking oligonucleotide, Buffer (New England Biolabs, Ipswich, MA) Figure 1A ) and 0.1 mM dNTP in a 10 μl reaction. Figure 1B As a control, a FAM-labeled primer was used without any polymerase. Strand-displacing DNA polymerase was added to the reaction and incubated with 1 μl of a 10-fold diluted sample at 50°C for 30 minutes. The reaction was analyzed by capillary electrophoresis, and the FAM primer was extended by the displaced blocking oligonucleotide. The results are shown in Figures 1D-1E middle. Figure 1D The peak position of the strand-displacing polymerase Bst polymerase identified in the assay corresponds to the peak observed for the non-native polymerase SPB49F. The shift in migration is attributed to the blunt ends generated by SPB49F due to its 3'-5' exonuclease activity, which are not present in Bst polymerase. This results in the products replicated by Bst polymerase having 3'dA. Figure 1C The product of the non-strand-displacing polymerase, T4 DNA polymerase, is shown, where synthesis terminates at the blocking primer.

[0126] Example 2: Synthesis of a large DNA molecule from 6 fragments using a strand-displacing polymerase and confirmation using a strand-displacing polymerase Assembly is effective

[0127] use Plasmids A, B, C, D, and E were constructed from PCR products (fragments 1, 2, 3, 4, and 5, which together cover the Lac1- and LacZ gene regions) using a PCR cloning kit (New England Biolabs, Ipswich, MA).

[0128] In this experiment, the following concentrations of five different fragments integrated into separate plasmids were used: 50 ng of each PCR ("fragment" source), and 25 ng of the ampicillin-containing plasmid pMiniT TMVector (NEB #E1202). First, PCR was used to amplify the five fragments used for assembly. The primers used in the preparation of the Lacl-lacZ DNA fragment assembly system are as follows:

[0129] 5PLaclZ-pACYC184VF1 ttggtctggtgtcaaaaatgaATCGTCACGGCGATTTATG (SEQ ID NO: 4) 5PLaclZ-pACYC184VR1 gggtcattttcggcgaggacTGCATCAACGCATATAGCG (SEQ ID NO: 5) Not-IZ F1 GCGGCCGCgtcctcgccgaaaatgacccag (SEQ ID NO: 6) Not-IZ R1 GCGGCCGCtggtgtcgatggtagaacgaagcg (SEQ ID NO: 7) Not-IZ F2 GCGGCCGCcccactgacgcgttgcgcgagaag (SEQ ID NO: 8) Not-IZ R2 GCGGCCGCggctgcgcaactgttgggaagggc (SEQ ID NO: 9) Not-IZ F3 GCGGCCGCtgcagcacatccccctttcgccag (SEQ ID NO: 10) Not-IZ R3 GCGGCCGCatgatgctcgtgacggttaacgcc (SEQ ID NO: 11) Not-IZ F4 GCGGCCGCaggtgcggattgaaaatggtctgc (SEQ ID NO: 12) Not-IZ R4 GCGGCCGCtcaccgcttgccagcggcttacca (SEQ ID NO: 13) Not-IZ F5 GCGGCCGCgaatacctgttccgtcatagcgat (SEQ ID NO: 14) Not-IZ R5 GCGGCCGCtcatttttgacaccagaccaactgg (SEQ ID NO: 15)

[0130] The amplified fragments were cloned and sequenced to confirm that no errors were introduced during the amplification process.

[0131] Sequence of PCR amplified fragment 1 (SEQ ID NO: 16):

[0132]

[0133] Sequence of PCR amplified fragment 2 (SEQ ID NO: 17):

[0134]

[0135] Sequence of PCR fragment 3 (SEQ ID NO: 18):

[0136]

[0137] Sequence of PCR fragment 4 (SEQ ID NO: 19):

[0138]

[0139] Sequence of PCR fragment 5 (SEQ ID NO: 20):

[0140]

[0141] Each of the five fragments has an 80 bp overlap with the adjacent fragment in the order of the final assembly (between fragments 1 and 2, between 2 and 3, between 3 and 4, and between 4 and 5). Fragments 1 and 5 also share a 20 bp overlap with the ends of the vector. Any available vector can be used, for example, pACYC184 (New England Biolabs, Ipswich, MA). The pACYC184 vector was prepared by inverse PCR, which allows for the use of After treatment with ELISA (New England Biolabs, Ipswich, MA) and heat inactivation, assembly of fragments 1-5 was performed in the presence of the above assembly mix (see Figures 2A-2C ).

[0142] During assembly, nucleotides extending from the shaded region are degraded by T5 exonuclease, while nucleotides shown in gray are removed by polymerase. After fragment assembly and transformation into E. coli, productive assembly is recorded on plates with IPTG and X-Gal, as determined by blue / white selection.

[0143] T5 exonuclease, Taq ligase, strand displacement DNA polymerase and ss binding protein (ET SSB) are combined in the reaction mixture in a buffer to form Mix 1. These enzymes are all available from New England Biolabs, Ipswich, MA. Five 150ng NotI-HF-digested plasmids (plasmids A, B, C, D and E) are mixed with 105ng carrier and Mix 1 or GAMM to a total volume of 20 μl. The reactants are incubated at 50°C for 60 minutes. The product assembled using 2 μl is transformed into NEB 5-α (New England Biolabs, Ipswich, MA) competent cells. The cells are then plated on a flat plate containing chloramphenicol. Positive assemblies can be identified as blue colonies on a flat plate with chloramphenicol+IPTG+X-Gal and incubated overnight at 37°C.

[0144] PCR of the assembled product to confirm that all fragments were joined and connected before transformation included the following steps: 1 μl of the assembled product was used in a PCR to ensure that the 5 fragments and the vector were connected together. Paired PCR primers annealed on the vector were used to amplify the entire assembled LaclZ gene (5.3 kb). Bands 1 and 2 are duplicate PCR results. Band M is a 2-log DNA ladder from New England Biolabs, Ipswich, MA (see Figure 4 ).

[0145] Sequencing results were obtained from eight selected colonies, and plasmid DNA was purified for Sanger sequencing. Six primers were used to sequence the 4.8 kb fragment. The junction sequences between the fragments and the extension regions from the overlap regions showed less than 2% sequence errors.

[0146] Primers used to sequence assembled DNA:

[0147] Seq Not-IZ-MO aaaaccacctggcgcccaatacg(SEQ ID NO:21)

[0148] Seq Not-IZ-Ml cccggactcggtaatggcgcgcat(SEQ ID NO:22)

[0149] Seq Not-IZ-M2 ggaagcagcccagtagtaggttga (SEQ ID NO:23)

[0150] Seq Not-IZ-M3 ggtgctgcgctggagtgacggcag(SEQ ID NO:24)

[0151] Seq Not-IZ-M4 cggccaccgatattatttgcccga(SEQ ID NO:25)

[0152] Seq Not-IZ-M5 gattaggggccgcaagaaaactatc(SEQ ID NO:26)

[0153] Example 3: Assemble single-stranded oligonucleotides into a linearized vector or two different ds DNA

[0154] Oligonucleotides corresponding to sgRNAs for targeting genes in H. sapiens were designed as follows:

[0155] 1. Screen for PAM sequences with the desired target sequence. For example, NGG

[0156] 5'GCGAAGAACCTCTTCCCAAGANGG3'(SEQ ID NO:27)

[0157] 2. Design a 71-base ss DNA oligonucleotide containing a 21-nucleotide target sequence flanked by partial U6 promoter sequence and backbone RNA sequence.

[0158] See e.g. Figure 8A -C, where the ss oligonucleotide is identified as:

[0159] 5'ATTCTTGTGGAAAGGACGAAACACCGGCGAAGAACCTCTTCCCAAGAGTTTTAGAGCTAGAAATAGCAAGTT3' (SEQ ID NO: 28)

[0160] or Figure 9A -C, where the ss oligonucleotides were designed to generate the following random library:

[0161] 5'ATCTTGTGGAAAGGACGAAACACCGN 21 GTTTTAGAGCTAGAAATAGCAAGTT3'(SEQ ID NO:29)

[0162] 3. Prepare ss DNA oligonucleotides in 1× NEBuffer 2 (New England Biolabs, Ipswich, MA) to a final concentration of 0.2 μM.

[0163] 4. A 10 μl reaction mixture was prepared containing 5 μl of ss DNA oligonucleotide (0.2 μM), 30 ng of restriction enzyme-linearized vector and ddH2O.

[0164] 5. A vector suitable for use in the above method is the ds vector from Life Technology ( CRISPR Nuclease Vector Kit with OFP Reporter, Catalog Number: A21174). Other vectors, such as plasmid #42230 (pX330-U6-Chimeric_BB-CBh-hSpCas9), are provided by Addgene (see https: / / www.addgene.org / 42230 / for details). Alternatively, any plasmid containing an sgRNA backbone under the control of the U6 promoter can be used.

[0165] 6. Add 10 μl of the master mix containing ss-binding protein, ligase, exonuclease, and polymerase to the reaction mixture and incubate the assembly reaction at 50°C for 1 hour.

[0166] 7. Transform NEB 10-β competent E. coli with 2 μl of the assembly product according to the manufacturer's protocol (New England Biolabs).

[0167] 8. Spread 100 μl of transformed cells on plates with ampicillin antibiotic and incubate at 37°C overnight.

[0168] 9. Pick 10 colonies, grow them, and purify the plasmid DNA for sequencing.

[0169] Unlike conventional cloning methods that require the synthesis of two oligonucleotides and their reannealing, this embodiment provides a simple way to design oligonucleotides and assemble them with the desired vector, which represents a significant improvement over conventional methods, especially in terms of time saving, ease of use and cost.

[0170] SEQ ID NO: 1

[0171] MILDADYITEDGKPIIRLFKKENGRFKVEYDRNFRPYIYALLKDDSAIDDVRKITSERHGKVVRVIDVEKVKKKFLGRPIEVWKLYFEHPQDVPAMRDKIREHPAVIDIFEYDIPFAKRYLIDKGLIPMEGNEELTFLAVDIETLYHEGEEFGKGPIIMISYADEEGAKVITWKKIDLPYVEVVANEREMIKR LIKVIREKDPDVIITYNGDNFDFPYLLKRAEKLGMKLPLGRDNSEPKMQRLGDSLAVEIKGRIHFDLFPVIRRTINLPTYTLEAVYEAIFGKQKEKVYPHEIAEAWETGKGLERVAKYSMEDAKVTYELGKEFFPMEAQLARLVGQPLWDVSRSSTGNLVEWYLLRKAYERNELAPNKPDEREYERRLRESYEG GYVKEPERGLWEGIVSLDFRSLYPSIIITHNVSPDTLNKEGCGEYDEAPEVGHRFCKDFPGFIPSLLGSLLEERQKIKKRMKESKDPVERKLLDYRQRAIKILANSFYGYYGYAKARWYCKECAESVTAWGRQYIELVRRELEERGFKVLYIDTDGLYATIPGEKNWEEIKRRALEFVNYINSKLPGILELEYE GFYTRGFFVTKKKYALIDEEGKIVTRGLEIVRRDWSEIAKETQAKVLEAILKHGNVEEAVKIVKEVTEKLSNYEIPVEKLVIYEQITRPLNEYKAIG PHVAVAKRLAAKGIKIKPGMVIGYVVLRGDGPISKRAIAIEEFDGKKHKYDAEYYIENQVLPAVERILKAFGYKREDLRWQKTKQVGLGAWLKVKKS

[0172] SEQ ID NO:2

[0173] IINPQARLTPLELEILEIIKQKKSITITEIKEILSRRKSEYPLSLVSEYSRLERKGYVKKIAKGRKKFVEALI

[0174] SEQ ID NO:3

[0175] MILDADYITEDGKPIIRLFKKENGRFKVEYDRNFRPYIYALLKDDSAIDDVRKITSERHGKVVRVIDVEKVKKKFLGRPIEVWKLYFEHPQDVPAMRDKIREHPAVIDIFEYDIPFAKRYLIDKGLIPMEGNEELTFLAVDIETLYHEGEEFGKGPIIMISYADEEGAKVITWKKIDLPYVEVVANEREMIKRLIKVIREKDPDVIITYNGDNFDFPYLLKRAEKLGMKLPLGRDNSEPKMQRLGDSLAVEIKGRIHFDLFPVIRRTINLPTYTLEAVYEAIFGKQKEKVYPHEIAEWETGKGLERVAKYSMEDAKVTYELGKEFFPMEAQLARLVGQPLWDVSRSSTGNLVEWYLLRKAYERNELAPNKPDEREYERRLRESYEGGYVKEPERGLWEGIVSLDFRSLYPSIITHNVSPDTLNKEG CGEYDEAPEVGHRFCKDFPGFIPSLLGSLLEERQKIKKRMKESKDPVERKLLDYRQRAIKILANSFYGYYYAKAARWYCKECAESVTAWGRQYIELVRRELEERGFKVLYIDTDGLYATIPGEKNWEEIKRRALEFVNYINSKLPGILELEYEGFYTRGFFVTKKKYALIDEEGKIVTRGLEIVRRDWSEIAKETQAKVLEAILKHGNVEEAVKIVKEVTEKLSNYEIPVEKLVIYEQITRPLNEYKAIGPHVAKRLAAKGIKIKPGMVIGYVVLRGGPISKRAIAIEEEFDGKKHKYDAEYYYIENQVLPAVERILKAFGYKREDLRWQKTKQVGLGAWLKVKKSGTGGGGIINPQARLTPLELEILEIIIKQKKSITITEIKEILSERRKSEYPLSLVSEYISRLERKGYVKKIAKGRKKFVEALI

[0176] SEQ ID NO:33

[0177]

[0178] MERVEGWLIDADYETIGGKAVVRLWCKDDQGIFVAYDYNFDPYFYVIGVDEDILKNAATSTRREVIKLKSFEKAQLKTLGREVEGYIVYAHHPQHVPKLRDYLSQFGDVREADIPFAYRYLIDKDLACMDGIAIEGEKQGGVIRSYKIEKVERIPRMEFPELKMLVFDCEMLSSFGMPEPEKDPIIVISVKTNDDDEIILTGDERKIISDFVKLIKSYDPDIIVGYNQDAFDWPYLRKRAERWNIPLDVGRDGSNVVFRGGRPKITGRLNVDLYDIAMRISDIKIKKLENVAEFLGTKIEIADIEAKDIYRYWSRGEKEKVLNYARQDAINTYLIAKELLPMHYELSKMIRLPVDDVTRMGRGKQVDWLLLSEAKKIGEIAPNPPEHAESYEGAFVLEPERGLHENVACLDFASMYPSIMIAFNISPDTYGCRDDCYEAPEVGHKFRKSPDGFFKRILRMLIEKRRELKVELKNLSPESSEYKLLDIKQQTLKVLTNSFYGYMGWNLARWYCHPCAEATTAWGRHFIRTSAKIAESMGFKVLYGDTDSIFVTKAGMTKEDVDRLIDKLHEELPIQIEVDEYYSAIFFVEKKRYAGLTEDGRLVVKGLEVRRGDWCELAKKVQREVIEVILKEKNPEKALSLVKDVILRIKEGKVSLEEVVIYKGLTKKPSKYESMQAHVKAALKAREMGIIYPVSSKIGYVIVKGSGNIGDRAYPIDLIEDFDGENLRIKTKSGIEIKKLDKDYYIDNQIIPSVLRILERFGYTEASLKGSSQMSLDSFFS

[0179] SEQ ID NO:35

[0180] MIKAWLLDVDYVTENDRAVIRLWCKDDKGVFVAYDRNFLPYFYVIGCKAEDVMKVKVRTNEGIITPLKVEEIEAKSLGKPIKALKVYTRHPQHVPKLREEIKKFAEVREADIPFAYRYLIDKDLACMDGIEIEPIAVKEGVLRAYEVRSVRRVEKKGFPDLKILAFDCEMLAQFMPDPEKDPIIAIAVKCGDFEEVLHGDERDILRRFVSIIKEQDPDIIVGYNQDNFDWPYVKKRAEKFGIRLDIGRDRSEISFRGGRPKIAGRLNVDLYDIALKIPDVKIKTLKKVAEFLGAKVEEEDIEGRDIYKCWMRGEKEKVFKHVLNDVLTTYRLALELLPMHYELSRMIRLPLDDVARLGRGKQVDYFLLSEAKKINEIAPNPPEIEESYEGAFVLEPARGLHENVACLDFASMYPSIMINFNISPDTLVKGECEDCYVAPEVGHKFRKSPDGFFKRILKMLIEKRREMKRQMKELDPDSEDYKLLDIKQQTLKVLTNSFYGYTGWNLARWYCRECAEATTAWGRYFIKRAVKIAESMGFEVLYGDTDSLFIKKNKLNLKDLEKECLKLIDVISKELPIQLEIDEFYKAIFFVEKKRYAGLTDDDRIVVKGLEVRRGDWCELAKRVQREVIEIILRERNPDKALKFVKNVIEEIKEGKFKLEDYVIYKGLTKKPDKYESKQAHVKAALRAMEMGIYYPIGTKVGFVIVKGGGSISDRAYPIELIEEFDGENLKIRTPSGIMVKKIDKDYYIDHQIIPAVMRILERFGYTEASLKTTIQKTLFDFT

[0181] SEQ ID NO:36

[0182] MKLVIFDGNSILYRAFFALPELTTSSNIPTNAIYGFINVILKYLEQEKPDYIAVAFDKRGREARKSEYQEYKANRKPMPDNLQVQIPYVREILYALNIPIVEFEGYEADDVIGSLVNKFKNTGLDIVIITGDRDTLQLLDKNVVVKIVSTKFDRTMEDLYTIENIKEKYGVWANQVPDYKALVGDQSDNIPGVKGIGEKSAQKLLEEYSSLEEIYQNLDKIKGSIREKLEAGKDMAFLSKRLATIVCDLPLNVNLEDLRTKEWNKERLYEILVQLEFKSIIKRLGSENIQFEFVQQRTDIPDVEQRELESISRIRSKEIPLMFVQDEKCFYLYDQESNTVFVTRDRHLVEEILKSDTVKIVYDLKNIFHQLNLEDTDNIKNCEDVMIASYVLDSTRSSYELETLFVSYLNTDIEAVKKDKKMV SVVLLKRLWDDLRLIDLNSCQFLYENIERPLIPVLYEMEKTGFKVDRDALLQYTKEIENKILKLETQIYQIAGEWFNINSPKQLSYILFEKLKLPVIKKTKTGYSTDAEVLEEFDKHEIVPLILDYRMYTKILTYCQGLLQAINPSSGRVHTTFIQTGTATGRLASSDPNLQNIPVKYDEGKLIRKVFVPEEGHVLIDADYSQIELRILAHISEDERLINAFKNNIDIHSQTAAEVFGVDIADVTPEMRSQAKAVNFGIVYGISDYGLARDIKISRKEAAEFINKYFERYPKVKEYLDNIVRFARENGYVLTLFNRKRYVKDIKSANRNARSYAERIAMNSPIQGSAADIMKLAMIKVYQKLKENNLKSKIILQVHDELLIEAPYEEKDIVKRIVKREMENAVALKVPLVVEVKEGLNWYETK

[0183] SEQ ID NO:37

[0184] MEKRVYLVDITYGLVGNSPEIRMFGVDENGEKVVILDRGFRPYFYVIPEEGFEDQVARVIGKMQNVIKADVTERRLFGKPIKVVKVTVTVPDKVRELRDRVKSIQHVKEVLEADIRFYIRYMIDNDIRPGWLMFSNLKPVDNKIGGVSNVYLTETPPTSLDLGIMPRLNYMALDIEVYNPRGTPDPKRDPIIIALANSNGDVKLLTLDNYKHEREMLNDMMSVIKEWDPDVLFGYNSNKFDMPYLVNRADALNVKLQLSKYGTPPEQSVYGHWSIIGRAHIDLYNFIEDMTDVKRKSLDYVAEYFGVMKRSERVNIPGHRIYQYWDDEGKRSQLIKYARDDVLSTLGLKGILLPYAMQLASVSGLPLDQVGPASVGSRVEMMIMHEAYK MGELAPNRVERPYETYKGAIVLEPKPGIHYNIAVLDFSSMYPNIMLKYNISPDTLVLDSSEGDYYTAPEVGYRFRKSPRGLYASLLQKLIEARREARDEMRNYPEGSFEWVLLNERQRALKIMANAMYGYCGWLGARWYIREVAESVTAWGRYLLKTAMSMAKERGLTVIYGDTDSLFVTYDKDKVADIISRINEMGFEVKIDKVYSKLIFTESKKRYIGLTADGEVDIVGFEAVRGDWSELARNVQERVAELVLRESVDEAVKYVKSVIDDLRNYRFTIDDVIIWKTLDKDINEYKAIQPHVVAARRLMEKGYVVNKGDTVGFVIVKDSGDKLTQRAYPYVFINDVKEIDVDYYVEKQVIPAALRILEVFGVNEAALLGKTGKSILDYFH

[0185] SEQ ID NO:38

[0186] MTEVVFTVLDSSYEVVGKEPQVIIWGIAENGERVVLIDRSFRPYFYALLAPGADPKQVAQRIRALSRPKSPIIGVEDDKRKYFGRPRRVLRIRTVLPEAVREYRELVKNVDGVEDVLEADIRFAMRYLIDHLDLPFTWYRVEAEPLENKMGFRVDKVYLVKSRPEPLYGEALAPTKLPDLRILAFDIEVYSKQGSPRPERDPVIVIAVKTDDGDEVLFIAEGKDDRKPIREFVEYVKRYDPDIIVGYNNNHFDWPYLLRRARILGIKLDVTRRVGAEPTTSVHGHVSVPGRLNVDLYDYAEEMPEIKIKSLEEVAEYLGVMKKSERVIINWWEIPDYWDDPKKRPLLLQYARDDVRATYGLAEKILPFAIQLSYVTGLPLDQVGAMSVGFRLEWYLIRAAF KMKELVPNRVERPEETYRGAIVLEPLRGVHENIAVLDFSSMYPNIMIKYNVGPDTLVRPGEECGECGCWEAPEVKHRFRRCPPGFFKTVLERLLELKRVRRAEMKKYPPDSPEYRLLDERQKALKVLANASYGYMGWSGARWYCRECAEAVTAWGRHLIRTAINIARKLGLKVIYGDTDSLFVTYDPEKVEKFIKIIEELLGFEIKLEKVYKRVFFTEAKRYAGLLEDGRIDIVGFEAVRGDWCELAKEVQTKVVEIVLKTSDVNKAVEYVRKIVKELEEGKVPIEKLVIWKTLSKRLEEYTTEAPHVVAAKRMLSAGYRVSPGDKIGYVIVKGGGRISQRAWPYFMVKDPSQIDVTYYVDHQIIPAALRILGYFGITEKKLKASATGQKTLFDFLAKKSK

[0187] SEQ ID NO:39

[0188] MEIRVWPLDVTYIVVGGVPEVRVFGIAEGGERVVLADRSFRPYFYVDCAGCDPHAVKTHLGRTAPVEGVELVERRFLGRPRQFLKVVAKIPEDVRRLREAASTIPGVRGVYEADIRFYMRYVIDMGVVPCSWNVAEVEVADEKLGSLPVYRVVKWGGAVEGFPPPLRVLAFDIEVYNERGTPDPARDPIVMIAVQSSDGRLEVFEASGRDDRGVLRSFVEYVRSFDPDVVVGYNSNNFDWPYLAERAKAVGVPLRVDRLGGAPQQSVYGHWSVLGRANVDLYNIVDEFPEIKLKTLDRVAEYFGVMRRDERVLIPGHKIYEYWRDPSKRPLLRQYVIDDVRSTYGLAERLLPFLIQLSSVSGLPLDQVAAASVGNRVEWMLLYAYGLGEVAP NREEREYEPYKGAIVLEPKPGLYSDVLVLDFSSMYPNVMMRYNLSPDTYLEPGEPDPPEGVYVAPEVGHRFRKEPPGFIPQVLRRLVALRRAVREEMKKYQPDTPEYRVLDERQKALKIMANAMYGYTGWVGARWYKKEVAESVTAFARAILKDVIDYARRLGIVVIYGDTDSLFVKKGGDLEKLARYVDEKYGIEIKVDKDYEKVLFTEAKRYAGLLRDGRIDIVGFEVVRGDWSELAKEVQLKVIELILKARDLSEARQRVIKYVKDVIERLKSGKFDLDDLIIWKTLDKDLGEYKAYPPHVRAALILKKGYKVGRGTTIGYVVVKGGEGVSERSLPYILVDDLAKIDVDYYIEKQVIPAALRIAEVIGVKEGDLRAGRSEKSLLDFFE

[0189] SEQ ID NO:40

[0190] MSEKINLEFYFLDNSYEVIGNEPHIIIWGITRDGRRVLLRDRRFRPYFYAILKDKVNIEDLARKIRTYSDPKSPIIGVEVEKKYFGRKVSALKIITMIPEYVRKYREKIKSLPEVLEVVEADIRFSIRYIIDHDLRPCGWHVAEVVEVPKKPIYRVDAEYEIIGDIKPLEQTLQPDLRIIAFDIEVYNKSGTPRPQTDP IIIIGIMNNNGDIKQFLANKYDDKISVEEFVNYVKTFDPDIIVGYNTDGFDWPYLIERSKYIGVKLDVTRRVGATPRTSTYGHISVPGRNLDTLYHFAEIPEVKVKSLENVAEYLGVMKKSERVIIEYIDIPKYWDDEKLRPKLLQYNIDDVKSTYGLAEKFLFPAMQLSNITGLPLDQVGAASVGFRLEWYLMREAFR YGELVPNRVERAAESYRGAVVLKPVKGVHENIAVLDFSSMYPNIMIKYNVGPDTIVRNEKCNPDKHNIAPEVGHCFRKEPPGFFKRVLETLLRLRKQIKSEMKKYPPTSYEYRLLDERQKAVKVLANATYGYMGWIHARWYCRECAEAVTAWGRQTIKSAIELARKLGLKVIYGDTDSLFVTYDKDKVEKLIELIQTKLGFEIKIDKIYKRVFFTEAKRYAGLLEDGRIDIVGFEAVRGDWAEIAKEVQEKVTEILLKENSIDKAIEYVRQVIADLKAGKIPLDKLIIWKTLSKRIEEYSVDAPHVVAAKKLIKAGIKVSTNDKIGYVILKGGGKISSRAEPYIFVKDPKLIDTEYYVDHQIVPAALRILNYFGVTETQLKRAAASAGQKSLFDFFGGKK

[0191] SEQ ID NO:41

[0192] MILDADYITENGKPVVRIFKKENGEFKVEYDRSFRPYIYALLRDDSAIEDIKKITAERHGKVVRVVEAEKVRKKFLGRPIEVWKLYFEHPQDVPAIREKIREHPAVIDIFEYDIPFAKRYLIDKGLIPMEGNEELKLLAFDIETLYHEGDEFGSGPIIMISYADEKGAVITWKGVITDLPYVEVVSSEREMIKRFLRVIREKDPDVIITYNGDNFDFPYLLKRAEKLGMKLPIGRDGSEPKMQRMGDGFAVEVKGRIHFDIYPVIRRTINLPTYTLEAVYEAVFGRPKEKVYPNEIARAWENCKGLERVAKYSMEDAKVTYELGREFFPMEAQLARLVGQPVWDVSRSSTGNLVEWFLLRKAYERNELAPNRPDEREYERRLRESYEGG YVKEPEKGLWEGIIYLDFRSLYPSIIITHNISPDTLNKEGCNSYDVAPKVGHRFCKDFPGFIPSLLGQLLDERQKIKRKMKATIDPIERKLLDYRQRAIKILANSYYGYYYAKAKARWYCKECAESVTAWGREYIELVSRELEKRGFKVLYIDTDGLYATIPGSREWDKIKERALEFVKYINARLPGLLELEYEGFYKRGFFVTKKKYALIDEEGKIITRGLEIVRRDWSEIAKETQARVLEAILKEGNLEKAVKIVKEVTEKLSKYEVPPEKLVIYEQITRDLKDYKAVGPHVAVAKRLAARGIKVRPGMVIGYLVLRGGPISRRAIPAEEFDPSRHKYDAEYYYIENQVLPAVLRILEAFGYRKEDLRYQKTRQAGLDAWLKRKASL

[0193] SEQ ID NO:42

[0194] MILDTDYITENGKPIVIRVFKKENGEFKIEYDRTFEPYFYALLKDDSAIEDVKKVTAKRHGTVVRVKRAEKVQRKFLGRPIEVWKLYFTHPQDVPAIRDKIRAHPAVIDIYEYDIPFAKRYLIDKGLIPMEGDEELTMLAFDIETLYHEGEEFGTGPILMISYADGSEARVITWKKIDLPYVDVVSTEKEMIKRFLRIVKEKDPDVLITYNGDNFDFAYLKRKCEKLGIKFTLGRDGSEPKIQRMGDRFAVEVKGRIHFDLYPVIRRTINLPTYTLEAVYEAVFGKPKEKVYAEEIAQAWESGEGLERVARYSMEDAKVTYELGREFFMEAQLSRLIGQSLWDVSRSSTGNLVEWFLLRKAYERNELAPNKPDEKELARRRGGYAG GYVKEPERGLWDNIVYLDFRSLYPSIITHNVSPDTLNREGCKEYDVAPEVGHKFCKDFPGFIPSLLGDLLEERQKIKRKMKATVDPLEKKLLDYRQRAIKILANSYYGYYAKAARWYCRECAESVTAWGREYIETTIREIEEKFGFKVLYADTDGFFATIPGADAETVKKKAKEFLKYINAKLPGLLELEYEGFYVRGFFVTKKKYAVIDEEGKITTRGLEIVRRDWSEIAKETQARVLEAILKHGDVEEAVRIVKEVTEKLSKYEVPPEKLVIHEQITRDLRDYKATGPHVAKRLAARGVKIRPGTVISYIVLKGSGRIGDRAIPFDEFDPTKHRYDAEYYYIENQVLPAVERILKAFGYRKEDLRYQKTKQVGLGAWLKVKK

[0195] SEQ ID NO:43

[0196] MILDTDYITEDGKPVIRIFKKENGEFKIEYDREFEPYIYALLKDDSAIEEVKKITAGRHGRVVKVKRAEKVKKKFLGRPIEVWKLYFTHPQDVPAIRDEIRRHSAVVDIYEYDIPFAKRYLIDKGLIPMEGDEELKMMSFDIETLYHEGEEFGTGPILMISYADEGEARVITWKKIDLPYVEVVSTEKEMIKRFLKVVKEKDPDVLITYNGDNFDFAYLKKRCEKIGIKFTLRRDGSEPKIQRMGDRFAVEVKGRIHFDLYPVIRRTINLPTYTLEAVYEAVFGTPKEKVYPEEITTAWETGEGLERVARYSMEDAKVTYELGREFFPMEAQLSRLIGQSLWDVSRSSTGNLVEWFLLRKAYERNEIAPNKPDERELARRRGGYAGG YVKEPERGLWDNIVYLDFMSLYPSIITHNVSPDTFNREGCKEYDTAPQVGHKFCKDVQGFIPSLLGALLDERQKIKKRMKASIDPLEKKLLDYRQKAIKILANSYYGYYARARWYCKECAESVTAWGRDYIETTIHEIEERFGFKVLYADTDGFFATIPGADAETVKKKAKEFLKYINAKLPGLLELEYEGFYVRGFFVTKKKYAVIDEEGKITTRGLEIVRRDWSEIAKETQARVLEAILRHGDVEEAVRIVKDVTEKLSKYEVPPEKLVIHEQITRELKDYKATGPHVAIAKRLAARGIKIRPGTVISYIVLKGSGRIGDRAIPFDEPTKHRYDAEYYYIENQVLPAVERILKAFGYKKEELRYQKTRQVGLGAWLKLKKK

[0197] SEQ ID NO:44

[0198] MILDADYITEDGKPVVRIFKKENGEFKIEYDREFEPYIYALLRDDSAIEEIKKITADRHGKVVKVKRAEKVQKKFLGRPIEVWKLYFTHPQDVPAIRDEIRKHPAVVDIYEYDIPFAKRYLIDKGLIPMEGDEELKMLAFDIETLYHEGEEFGTGPILMISYADEDGARVITWKKIDLPYVDVVSTEKEMIKRFLKVVKEKDPDVLITYNGDNFDFAYLKRKCEKLGIKFTLGRDGSEPKIQRMGDRFAVEVKGRIHFDLYPLIRRTINLPTYTLEAVYEAVFGKPKEKVYAEEIALAWESGEGLERVARYSMEDAKVTFELGREFFPMEAQLSRLIGQSLWDVSRSSTGNLVEWFLLRKAYERNELAPNKPNERELARRRGGYAGG YVKEPERGLWDNIVYLDFRSLYPSIITHNVSPDTLNREGCKEYDRAPQVGHKFCKDVPGFIPSLLGSLLDERQKIKRKMKATIDPIEKKLLDYRQRAIKILANSYYGYYYGYARARWYCRECAESVTAWGREYIEMAIREELEEKFGFKVLYADTDGLHATIPGADAETVKKKAMEFLKYINPKLPGLLELEYEGFYARGFFVTKKKYAVIDEEGKITTRGLEIVRRDWSEIAKETQARVLEAILRHGDVEEAVRIVKEVTEKLSKYEVPPEKLVIHEQITRELKDYRATGPHVAIAKRLAKRGIKIRPGTVISYIVLKGSGRIGDRAIPFDEPTKHKYDAEYYYIENQVLPAVERVLKAFGYRKDDLRYQKTRQVGLGAWLKVKKR

[0199] SEQ ID NO:45

[0200] MILDADYITEDGKPVIRVFKKEKGEFKINYDRDFEPYIYALLKDDSAIEDIKKITAERHGTTVRVTRAERVKKKFLGRPVEVWKLYFTHPQDVPAIRDKIREHPAVVDIYEYDIPFAKRYLIDKGLIPMEGNEELRMLAFDIETLYHEGEFGEGPILMISYADEEGARVITWKNIDLPYVESVSTEKEMIKRFLKVIQEKDPDVLITYNGDNFDFAYLKKRSETLGVKFILGRDGSEPKIQRMGDRFAVEVKGRIHFDLYPVIRRTINLPTYTLETVYEAIFGQPKEKVYAEEIAQAWESGEGLERVARYSMEDAKATYELGKEFFPMEAQLSRLVGQSLWDVSRSSTGNLVEWFLLRKAYERNELAPNKPDERELARRAESYAG GYVKEPEKGLWENIVYLDYKSLYPSIITHNVSPDTLNREGCREYDVAPQVGHRFCKDFPGFIPSLLGDLLEERQKVKKKMKATVDPIERKLLDYRQRAIKILANSYYGYYANARWYCRECAESVTAWGRQYIETTMREIEEKFGFKVLYADTDGFFATIPGADAETVKKKTKEFLNYINPRLPGLLELEYEGFYRRGFFVTKKKYAVIDEEDKITTRGLEIVRRDWSEIAKETQARVLEAILKHGDVEEAVRIVKEVTEKLSRYEVPPEKLVIYEQITRNLRDYRATGPHVAKRLAARGIKIRPGTVISYIVLKGPGRVGDRAIPFDEFDPAKHRYDAEYYYIENQVLPAVERILRAFGYRKEDLRYQKTKQAGLGAWLKPKT

[0201] SEQ ID NO:46

[0202] MILGADYITKDGKPIVRIFKKENGEFKIELDPHFQPYIYALLSEDSAIDEIKQIKGERHGKTVRIVDAVKVEKKFLKKPVKVKWKLILEHPQDVPAIRNKIREHPAVQDIYEYDIPFAKRYLIDNGLIPMEGDEELKMLAFDIETFYHEGDEFGKGEIIMISYADEEGARVITWKNIDLPYVDVVSNEREMIKRFIQIIKEKDPDVIITYNGDNFDLPYLIKRAEKGLRLILSRDNENPVPKIQRMGNSFAVEIKGRIHFDLFPVVKRAVNLPTYTLEAVYETVLGKHKSKLGAEEIAAIWETEEGLKKLAQYSMEDAKATYELGREFFPMEVELAKLIGQSVWDVSRSSTGNLVEWYMLRVAYERNELAPNRPSDEEYKRRLRTTY LGGYVKEPERGLWENIIYLDFRSLYPSIIVTHNVSPDTLERKGCQNYDVAPIVGYKFCKDFSGFIPSILEDLIETRQKIKKEMKSTIDPIKKKMLDYRQRAVKLLANSYYGYMGYPKARWYSKECAESVTAWGRHYIEMTIREIEEKFGFKVLYADTDGFYATIPGADPETIKKKAKEFLNYINSKLPGLLELEYEGFYLRGFFVTKKRYAVIDEEEKITTRGLEVVRRDWSEIAKETQARVLEAILREGSVEKAVEIVKEVVEAITKYKVPLEKLIIHEQITRELRDYKAVGPHVAIAKRLAAKGIKIKPGTIISYIVLRGSGKISDRVVLLTEYDPRKHKYDPDYIENQVLPAVLRILEAFGYRKEDLKYQSSKQTGLESWLKK

[0203] SEQ ID NO:47

[0204] MILDADYITEDGKPIIRIFKKENGEFKVEYDRNFRPYIYALLKDDSQIDEVKKITAERHGKIVRIVDVEKVKKKFLGRPIEVWKLYFEHPQDVPAIRDKIREHPAVVDIFEYDIPFAKRYLIDKGLIPMEGDEELKLLAFDIETLYHEGEEFAKGPIIMISYADEEGAKVITWKKVDLPYVEVVSSEREMIKRFLKVIREKDPDVIITYNGDSFDLPYLVKRAEKGIKLPLGRDGSEPKMQRLGDMTAVEIKGRIHFDLYHVIRRTINLPTYTLEAVYEAIFGKPKEKVYAHEIAEWETGKGLERVAKYSMEDAKVTYELGREFFPMEAQLSRLVGQPLWDVSRSSTGNLVEWYLLRKAYERNELAPNKPDEREYERRLRESYAG GYVKEPEKGLWEGLVSLDFRSLYPSIITHNVSPDTLNREGCMEYDVAPEVKHKFCKDFPGFIPSLLKRLLDERQEIKRRMKASKDPIEKKMLDYRQRAIKILANSYYGYYYAKAARWYCKECAESVTAWGREYIEFVRKELEEKFGFKVLYIDTDGLYATIPGAKPEEIKRKALEFVEYINAKLPGLLELEYEGFYVRGFFVTKKKYALIDEEGKIITRGLEIVRRDWSEIAKETQAKVLEAILKHGNVEEAVKIVKEVTEKLSKYEIPPEKLVIYEQITRPLHEYKAIGPHVAKAKRLAARGVKVRPGMVIGYIVLRGDGPISKRAILEEFDPRKHKYDAEYYYIENQVLPAVLRILEAFGYRKEDLRWQKTKQTGLTAWLNVKKK

[0205] SEQ ID NO:48

[0206] MILDADYITEDGKPIIRLFKKENGRFKVEYDRNFRPYIYALLKDDSAIDDVRKITSERHGKVVRVIDVEKVKKKFLGRPIEVWKLYFEHPQDVPAMRDKIREHPAVIDIFEYDIPFAKRYLIDKGLIPMEGNEELTFLAVDIETLYHEGEEFGKGPIIMISYADEEGAKVITWKKIDLPYVEVVANEREMIKRLIKVIREKDPDVIITYNGDNFDFPYLLKRAEKLGMKLPLGRDNSEPKMQRLGDSLAVEIKGRIHFDLFPVIRRTINLPTYTLEAVYEAIFGKQKEKVYPHEIAEWETGKGLERVAKYSMEDAKVTYELGKEFFPMEAQLARLVGQPLWDVSRSSTGNLVEWYLLRKAYERNELAPNKPDEREYERRLRESYEG GYVKEPERGLWEGIVSLDFRSLYPSIITHNVSPDTLNKEGCGEYDEAPEVGHRFCKDFPGFIPSLLGSLLEERQKIKKRMKESKDPVERKLLDYRQRAIKILANSFYGYYAKAARWYCKECAESVTAWGRQYIELVRRELEERGFKVLYIDTDGLYATIPGEKNWEEIKRRALEFVNYINSKLPGILELEYEGFYTRGFFVTKKKYALIDEEGKIVTRGLEIVRRDWSEIAKETQAKVLEAILKHGNVEEAVKIVKEVTEKLSNYEIPVEKLVIYEQITRPLNEYKAIGPHVAKARLAAKGIKIKPGMVIGYVVLRGDPISKRAIAIEEEFDGKKHKYDAEYYYIENQVLPAVERILKAFGYKREDLRWQKTKQVGLGAWLKVKKS

[0207] SEQ ID NO:49

[0208] MEGWLLDADYITAEDGRAVVRLWCKDFDGNTFVVYDRNFQPYFYAFKNGLSKEDIEKIVVKSREGVIKPFKVEEVRRKVFGKEVEVFKIYAYHPQHVPKLREELKITEVREADIPFAYRYLIDKDLACMDGIRVEGKVREERGLKVIDAEHVERFEIPLPEPPKVLAFDCEMLTELGMPDPEKDKIIIIGVKCGDFEEIITGNEREILLRFVEIIKEQDPDVIVGYNQDNFDWPYIRKRAEKLSVKLNIGRDGSEISFRGGRPKIAGRLNVDLYDIAMKLDVKVKTLENVAEFLGRKVELADIAKDIYKRWTSGDKESVLKYSKQDVLNTYFIAEELLPMHYELSRMIRIPTDDVARIGRGKQVDWFLLSEAYKIGEIANPPAEVEESYEG AFVLEPSRGLHKNVVCLDFASMYPSIMIAYNISPDTYVFGKCDDCYVAPEVGHKFRKHPDGFFKRILKMLIEKRREIKNQMKSLDRNSREYLLLNIKQQTLKILTNSFYGYTGWSGARWYCRQCAEATTAWGRHLIKSAVEIAKKLGFEVLYGDTDSIFVKKGNLSLEKIRGEVEKLIEEISEKFPVQIEVDEYYKTIFFVEKKRYAGLTEDGILVVKGLEVRRGDWCELAKEVQKKVIEIILKEENPEKAAEYVRKVINDIKSGKVKLEDVVIYKGLTKRPDKYESKQAHVKAALRAMELGIVYNVGSKVGFVVVEGAGNVGDRAYPIDLIEEFDGENLVIRTRSGSIVKKLDKDYYYINHQIIPSVLRILERFGYNEASLKATQKTLDAFW

[0209] SEQ ID NO:50

[0210] MILDADYITENGKPVVRIFKKENGEFKVEYDRNFEPYIYALLKDDSAIEEIKKITAERHGTVVRITKAEKVERKFLGRPVEVWKLYFTHPQDVPAIRDKIRSHPAVVDIYEYDIPFAKRYLIDKGLVPMEGDEELKMLAFDIETLYHEGEEFAEGPILMISYADESEARVITWKKVDLPYVDAVSTEKDMIKAFLRVVKEKDPDVLITYNGDNFDFAYLKKRCEKLGVKFILGRDGSEPKIQRMGDRFAVDVKGRIHFDLYPVIRRTINLPTYTLEAVYEAIFGRPKEKVYAEEIAQAWETNEGLERVARYSMEDAKVTYELGKEFFPMEAQLSRLIGQPLWDVSRSSTGNLVEWFLLRKAYERNELAPNKPSGREYDERRGGYAGG YVKEPEKGLWENIVYLDYKSLYPSIITHNVSPDTLNREGCKEYDVAPQVGHRFCKDFPGFIPSLLGDLLEERQKIKRKMKATIDPIERRLDYRQRAIKILANSYYGYYYGYARARWYCKECAESVTAWGREYIEMSIREIEEKYGFKVLYADTDGFHATIPGEDAETIKKKAMEFLKYINSKLPGALELEYEGFYRRGFFVTKKKYAVIDEEGKITTRGLEIVRRDWSEIAKETQARVLEALLKDGNVEEAVSIVKEVTEKLSKYEVPPEKLVIHEQITRELKDYKATGPHVAIAKRLAARGVKIRPGTVISYIVLKGSGRIGDRAIPFDEFDPAKHRYDAEYYYIENQVLPAVERILKAFGYRKEDLRYQKTRQVGLGAWLKPKGKK

[0211] SEQ ID NO:51

[0212] MILDTDYITENGKPIVIRVFKKENGEFKIEYDRTFEPYFYALLKDDSAIEDVKKVTAKRHGAVVKVKRAEKVQRKFLGRPIEVWKLYFTHPQDVPAIRDKIRAHPAVVDIYEYDIPFAKRYLIDKGLIPMEGDEELRMLAFDIETLYHEGEEFGTGPILMISYADENEARVITWKKIDLPYVDVVSTEKEMIKRFLRVVKEKDPDVLITYNGDNFDFAYLKRKCEKLGIKFTLGRDGSEPKIQRMGDRFAVEVKGRIHFDLYPVIRRTINLPTYTLEAVYEAVFGKPKEKVYAEEITEAWESGEGLERVARYSMEDAKVTYELGREFFPMEAQLSRLIGQSLWDVSRSSTGNLVEWFLLRKAYERNELAPNKPDERELARRRESYAGG YVKEPERGLWDNIVYLDFRSLYPSIITHNVSPDTLNREGCKEYDVAPEVGHKFCKDFPGFIPSLLGDLLEERQKIKRKMKATIDPLEKKLLDYRQRAIKILANSFYGYYAKAARWYCKECAESVTAWGREYIETTIREIEEKFGFKVLYADTDGFFATIPGADAETVKKKAKEFLKYINAKLPGLLELEYEGFYVRGFFVTKKKYAVIDEEGKITTRGLEIVRRDWSEIAKETQARVLEAILKHGDVEEAVRIVKEVTEKLSKYEVPPEKLVIHEQITRDLRDYKATGPHVAKRLAAKGVKIRPGTVISYIVLKGSGRIGDRAIPADEFDPTKHRYDAEYYYIENQVLPAVERILKAFGYRKEDLRYQKTKQVGLGAWLKVKGKK

[0213] SEQ ID NO:52

[0214] MILDTDYITEDGKPVIRIFKKDNGEFKIEYDRNFEPYIYALLRDDSAIEDVKKITAERHGRVVKVKRAEKVKKKFLGRPVEVWKLYFTRPQDVPAIRDRIRAHPAVVDIYEYDIPFAKRYLIDKGIIPMEGDEELKMLAFDIETLYHEGEEFAEGPILMISYADENEARVITWKKIDLPFVDVVSTEKEMIKRFLKVIKEKDPDVLITYNGDNFDFAYLKRKCEKFGIKFTLGRDGSDPKIQRMGDRFAVEVKGRIHFDLYPVILRTVNLPTYTLEAVYEAIFGTPKEKVYPEEITTAWETGEGLERVARYSMEDAKVTFELGREFFPMEAQLSRLVGQSFWDVSRSSTGNLVEWFLLRKAYERNELAPNKPDERELVRRRNSYTGG YVKEPERGLWDNIVYLDFRSLYPSIITHNVSPDTLNREGCKEYDEAPQVGHKFCKDFPGFIPSLLGNLLDERQKIKKRMKATIDPLEKKLLDYRQRAIKILANSYYGYYAYARARWYCKECAESVTAWGREYIEMSIREIEEKYGFKVLYADTDGFHATIPGADAETVKKKAMEFLKYINAKLPGALELEYEGFYKRGFFVTKKKYAVIDEEGKITTRGLEIVRRDWSEIAKETQARVLEALLKDGNVEEAVSIVKEVTEKLGKYEVPPEKLVIHEQITRELKDYKATGPHVAIAKRLAARGVKIRPGTVISYIVLKGSGRIGDRAIPFDEFDPTKHRYDAEYYYIENQVLPAVERILKAFGYRAEDLRYQKTRQVGLGVWLQPKGKK

[0215] SEQ ID NO:53

[0216] MELAFWLLDITYGVIGNTPELRLFGITDDGKRVLVLDRSFRPYFYVIPSGDVNAVFNNVKRKLEGKVLNVEVIKRKMFGNEVDAIRVTATIPEKVRELRELAAEVPGVEDVLEADIRFSQRYLLDMGVKPSNWIVVDQCEEVKGNYQVDLVCLAKSRPRMIEEHKLPSFRVLAFDIEVYNPRGMPNPDRDPVIIISTMTKEDGVKMFVVDDNKNDAKIIREFLDYFRKYDPDIVVGYNNNGFDWPYLVNRSSRVGVRLALSRMGNPPEPSVYGHWSIIGRANVDLYNFIEEISEIKVKSLDRAAEFFGIMKRSERVLIPGHRIHEYWDDKNKRDLLLKYARDDVVSTYGLAEKLLPFAIQLSSISGLPLDQVGAASVGARVEWMIFYEAVKR GELAPNREERPYETYKGAVVLEPRGLHENAVIDFSSMYPSIMMKYNVSPDTLVLDGDCGDCYVAPEVNYKFRRSPEGLYPGLRIVESRRRRVRDLMKKYPENSPEWVLLNERQRALKVMANAMYGYCGWLGARWIRREVAEAVTAWGRNLLRTVIEKARSLGLPIIYGDTDSLFVRNISDKVDALINYVNNELG FEVKVDKVYRRVLFTEAKKRYVGLTVEGEVDIGFEAVRGDWAEIAKDVQENVAEIVLTTGDVGKAISYVKSVIDKVKAYQFDIDDVIIWKTLDKSLNEYKVLTPHVAAAKQLVEAGYKVGKGDMIGYVVVKGGGAKLAYKVKPYILIKDIREVDVDYYVEKQIVPAAMRILEVLGVKESQLMEGKAGKSILDYFS

[0217] SEQ ID NO:54

[0218] MLRTVWVDYARKGEPDVILVGRREDGNPAALVVKGFRPYFYAEVEDGFDPSEVERLSGVVEVEEVLLEHPYGGDRVELLRIVATYPKVVPKLREQVKKLDGVKEVYEADIPFVRRAAVDLNLPPASEVDVSDLDTGSWSGLPAYFADVEDARELDHRPYPIEDLVVASFDLEVLAEPGTTIKGASGPIIAISFAYSTPDGERRNYVITWKGEDESFEVDGVETEVIVCRSEAAALRRFFDEFRRVDPDVVFTYNGDEFDLPYLQHRAGKLGIDVSPLARPAGKRGIILKHGGGRYASDIFGRAHVDLYHTARKNLKLERFTLEEAVKDVLGVEKEEMELADINEAWKRGNLDELMRYSAEDAHYTLELGLELAQVELELSYLTRLPLPDATRFSFGQLAEWRAIYKARQEDILVPNKPTRDEYKRRRRKAYKGAIVFEPEIGLHENVVCVDFASLYPNVMVAHNISPDTFDCDCCPRVTVEEVDDPTDATVAPDVGHKFCKRRKGFFPRLVEGLIERRRELKRRLRKLDTESHPHEAKILDVRQQAYKVLANSYYGYMGWANARWFCRECAESVTAWGRYYISEVRRIAEEKYGLKVVYGDTDSLFVKLPDADLEETIERVKEFLKEVNGRLPVELELEDAYKRILFVTKKKYAGYTEDGKIVTKGLEVVRRDWAPIARETQRRVLKRILADNDPEAALKEIHEVLERLKSGDVDIDELAVTSQLTKKPSEYVQKGPHVRAALRLARHLGVEPEPGTIVRYVIVRGPGSVSDKAYPVELVREEGKEPDVDYYIEHQILPAVERIMRAIGYSRGQIVGETASQKTLDQFFGSEQ ID NO:55

[0219] MELKIWPLDVTYAVVGGYPEVRVFGLTEGGGRVVLVDRSFKPYFYVDCPTCEVGVVKSSLSRVAPVDEVSAAERRFLGRPRRFLMVVARVPEDVRRLREAAAQIPGVAGVYEADIRFYMRYMIDVGLLPCSWNRAEVEGGGKVGGLPQYTVVQWLGPAGGFPPPLRVLAFDIEVYNERGTPDPPARDVMIAVKTD DGREEVFEAEGRDDRGVLRSFVEFVKSYDPDVVVGYNSNGFDWPYLAGRARAIGVPLRVDRLGGLPQQSVYGHWSIVGRANVDLYGIVEEFPEIKLKTLDRVAEYFGVMRREERVLIPGHKIYEYWRDPGKRPLLRQYVLDDVRSTLGLADKLLPFLIQLSSVSGLPLDQVAASVGNRVEWMLLRYAYRLGEVAP NREEREYEPYKGAIVLEPKPGMYEDVLVLDFSSMYPNIMMKYNLSPDTYLEPGEPDPPEGVNAAPEVGHRFRRSPLGFVPQVLKSLVELRKAVREEAKRYPPDSPEFRILDERQRALKVMANAMYGYLGWVGARWYKREVAESVTAFARAILKDVIEQARRLGIVVVYGDTDSLFVKKHVNVDKLIQYVEEKYGIEIKVDKDYAKVLFTEAKRYAGLLRDGRIDIVGFEVVRGDWSELAKEVQLKVVEIILNSRDVAEARRRVTQYVREIIERLREYKFNVDDLIIWKTLDKELGEYKAYPPHVHAALILKRHGYKVGGNMVGYVVVKGGGGKISEKALPYILLDDVKKIDVEYYIERQIIPAALRIAEVIGVKEADLKTGKSERSLLDFF

[0220] SEQ ID NO:56

[0221] MKTFLTEQQIKVLMLRAKGYKQSEIAKILGTSRANVSILEKRAMEKIEKARNTLLLWEQINSKVIVEIKAGEDIFSIPEKFFKKADKVGVKVPYSTAEIITFLVEHAPVEDRLAKRDFVLFLDSKNKLRIGDCLVIEEIKED

[0222] SEQ ID NO:57

[0223] MPITKVTRNYQITIPAEIRKALGIKEGELLEVRLENGKIIIERLKKERKTLKLGKKLTLEEIEKAIEEGMKQCMQ

[0224] SEQ ID NO:58

[0225] TKIEILRLLKEREMYAYEIWSLLGKPLKYQAVHQHIKELLELGLVEQAYRKGKRVYYKITEKGLRILQNFEDLENI

[0226] SEQ ID NO:59

[0227] MNTGAQGVSEMSRMKIISVQLPQSLIHGLDALVKRGIYPNRSEAIRVAIRELLKKELYKEEIQEEIPEYVVK

[0228] SEQ ID NO:60

[0229] VIIPRPIDPRDIRRIRKELGITQEELARKAGVTQAYIAKLEAGKVDPRLSTFNKILRALIECQKAKI

[0230] SEQ ID NO:61

[0231] NNCECMVVKEKLYTVKQASEILGVHPKTIQKWDREGKIKTVRTPGGRRRIPESEIKRLLGISEEK

[0232] SEQ ID NO:62

[0233] MLKDSAPKRKILEELRKGETVSGDYLASKLGVSRVAIWKHIRELKELGYGIIADKKGYKLVYEPKKPYPWE

[0234] SEQ ID NO:63

[0235] MIDERDKIILEILEKDARTPFTEIAKKLGISETAVRKRVKALEEKGIIEGYTIKINPKKLGYSLVTITGVDTKPEKLFEVAEKLKE

[0236] SEQ ID NO:64

[0237] MEIDDLDRKILSLLIEDSRLSYREIAKKLNVAVGTIYNRIKKLEDMGVIQGFTVKLNYEKLGYELTAIIGIKAQGKK

[0238] SEQ ID NO:65

[0239] EMLWMYILKLLKDRPMAYEYIRNELKKRFGFEPATVSSYVVLYRLEEGGYVSSEWHESEAGRPSRKYYRLTEKGEKKLEKGIETIVNLMLKS

[0240] SEQ ID NO:66

[0241] MKVSKATASKVLRSLINKGIVERSRGKTYLVRLTNKGLLEEISKAGKELDEKIFAEMSVDERIVL

[0242] SEQ ID NO:67

[0243] SEDYMLQNRRKVLAKVLELLNYNPKALNISELARMFGVSRDTIYNDIQQIIKNVEV

[0244] SEQ ID NO:68

[0245] SKEISRFLKVISNPIRYGILKMLNDRWMCVCLISEALEIDQTLVSHHIRILKELLDLLEERKEGKLRFYRTNKEKLREYLEKVLEDFNHGTSKGS

[0246] SEQ ID NO:69

[0247] MCRKDVMIISDPKQIKALSDPTRVKILELLRYHPMTVSEISRVIGKDKSTIYRHIKALEEAGLVEEVEKIGNETVYGR

[0248] SEQ ID NO:70

[0249] MEPVEFKLNQKGIKSILPTMEAEIMEYMWEIKEATAGEVEYYMKTKYPEIRRSTVSILMNRLCERGLLKRRMEKKGKGGIRYVYSITTTREEFERKVVEKIIESLMMNFREATFAYLSKINKK

[0250] SEQ ID NO:71

[0251] MKKSNLDLILLAKAGGIEKEILTTSRELSKMLNVSPQTIVRWLEDLEKDGLIKKSESRKGTLVTITEEGVKFLEKLHEELSDALYR

[0252] SEQ ID NO:72

[0253] MEIPPEISHALSEIGFTKYEILTYWTLLVYGPSAKEISTKSGIPYNRVYDTISSLKLRGFVTEIEGTPKVYAAYSPRIAFFRFKKELEDIMKKLEIELNNVKK

[0254] SEQ ID NO:73

[0255] IINPQARLTPLELEILEIIKQKKSITITEIELSERRKSEYPLSLVSEYISRLLERKGYVKKIAKGRKKFVEALI

[0256] SEQ ID NO:74

[0257] GIDVVIPEIKHDPIARDIVKILFDLRRANVSQIARELKGRRGKASRNTVRKKLKELEKLGVVKEVPGERGSVYTLSREVVKKWLDLIGIPINLL

[0258] SEQ ID NO:75

[0259] MTKRVKVITDPPEVIKVMLEDTRRKILQLLRNREMTISQLSEILGKMPQTIYHHIEKLKEAGLVEVKR

[0260] SEQ ID NO:76

[0261] MEEIKEIMKSHTLGNPVRLGIMIYLFPRRRAPFSHIQKALDLTPGNLDSHIKVLEKHGFVRTYKVIADRPRTMVEITDYGMEETRKFLSHLKTVIDAIHF

[0262] SEQ ID NO:77

[0263] MGEELNRLLDVLGNETRRRILFLLTKRPYFVSELSRELGVGQKAVLEHLRILEEAGLIESRVEKIPRGRPRKYIMIKGLRLEILLTPTLFGSEMYEAK

[0264] SEQ ID NO:78

[0265] MRRMDKVDLQLIKILSQNSRLTYRELAAMLGTTRQRVARKVDKLKKLGIIRKFTIIPNLEK

[0266] SEQ ID NO:79

[0267] GRKVRTQQNEILNLLNKEKAVLRAILEHGGEIKQEDLPELVGYSRPTISKVIQELENKGLIKREKSGKTFVVKIERKIKLD

[0268] SEQ ID NO:80

[0269] KSLQRFLRRNTTSIKHLSEITGVARNRLSDILNGKTQKIRGETLRKIAKAFEKSNILSF

[0270] SEQ ID NO:81

[0271] DVIQRIKEKYDEFTNAEKKIADTILSDPKGIIESSISDLSEKAGVKSEASVVKFYKKLGLNSFQQFKVLLAQSISRAPLEIVYEDVSSEDDTKTITEKIFKATVRAI

[0272] SEQ ID NO:82

[0273] KIRDKILNVYTQFSPAERKVADYVLERPDDVIHYSITEFAKIVGVSETTIHRMIKKLDFEGYQAFKIALARELSGLEETIERRDFIDEEIDILRRLKDTLD

[0274] SEQ ID NO:83

[0275] KRRPTINDVAKLAGVSISTVSRYLKDPSQVSEKLGERIREAIKKLGYKPNKIAQGLRTGD

[0276] SEQ ID NO:84

[0277] MASIKDVAKLAGVSIATVSRVINGYNNVSEETRKKVIDAIRKLNYHPVYAVKGAVLKR

[0278] SEQ ID NO:85

[0279] MKKKYVTIRDIAEKAGVSINTVSRALNNKPDISEETRRKILKIAQELGYVKNATASSLRSK

[0280] SEQ ID NO:86

[0281] MPTIEDVAKLAGVSIATVSRVINGSGYVSEKTRYKVWKAIEELGYKPEISAKLLASKG

[0282] SEQ ID NO:87

[0283] MRIGEKLRKLRLSRGLTQEELAERTDLSRSFISQLESDKTSPSIDTLERILEALGTDLKHF

[0284] SEQ ID NO:88

[0285] MHMKTVRQERLKSIVRILERSKEPVSGAQLAEELSVSRQVIVQDIAYLRSLGYNIVATPRGYVLAGG

[0286] SEQ ID NO:89

[0287] MNTLKKAFEILDFIVKNPGDVSVSEIAEKFNMSVSNAYKYMVVLEEKGFVLRKKDKRYVPGYKLIEYGSFVLRRF

[0288] SEQ ID NO:90

[0289] MKISKKRRQELIRKIIHEKKISNQFQIVEELKKYGIKAVQPTVARDLKEIGAVKIMDESGNYVYKLLDETPVIDPWKELKR

[0290] SEQ ID NO:91

[0291] MHKKLNPKSMKRENKKMVLRYLIESGPHSRVEIARKTGLAQSAIWRIIEELVNEGLVEEKGTATGRRRKAVTYGPTRSFITS

[0292] SEQ ID NO:92

[0293] MPSPLLRRENKIKILRYILKNGKTTRNQLASNLNLAHSTLSYIIDELLDEGFLVFEEIKKKRGRPYQILSVNPEKFTAI

[0294] SEQ ID NO:93

[0295] MKEERLKEILDIVDRNGFISMKDLQEQLGVSMITVRRDVAELVKRNLVKKVHGGIRKVNYFEKETDFMKRLSINREAKE

[0296] SEQ ID NO:94

[0297] MFTMRSEYALRLMIVMAKEYGNYLSMTEILEKAKQSVPREFAEKILYTLKKAGLVKTRRGKSGGYMLSRPPKEIKVSEIVFLLDRKSKVFFDMPGCPDELDCVIRALWKRVENEIEKILSGVTLEDLVREQEEKMKQ

[0298] SEQ ID NO:95

[0299] MRDTKGHLKFLVLHIISQQPSHGYYIMKKISQIIGAEPPSPGALYPILSSLRKQKYIETYNEGKRKVYRLTDKGRKYLEEHKEEIKKALDFAERF

[0300] SEQ ID NO:96

[0301] MRHRGGRGFRGWWLASTILLLVAEKPSHGYELAERLAEFGIEIPGIGHMGNIYRVLADLEESGFLSTEWDTTVSPPRKIYRITPQGKLYLREILRSLEDMKRRIETLEERIKRVLQEE

[0302] SEQ ID NO:97

[0303] MLSKRDAILKAAVEVFGKKGYDRATTDEIAEKAGVAKGLIFHYFKNKEELYYQAYMSVTEKLQKEFENFL

[0304] SEQ ID NO:98

[0305] MSKSWGKFIEEEEAEMASRRNLMIVDGTNLGFRFKHNNSKKPFASSYVSTIQSLAKSYSARTTIVLGDKGKSVFRLEHLPEYKGNRDEKYAQRTEEEKALDEQFFEYLKDAFELCKTTFPTFTIRGVEADDMAAYIVKLIGHLYDHVWLISTDGDWDTLLTDKVSRFSFTTRREYHLRDMYEHHNVDDVEQFISLKAIMGDLGDNIRGVEGIGAKRGYNIIREFGNVLDIIDQLPLPGKQKYIQNLNASEELLFRNLILVDLPTYCVDAIAAVGQDVLDKFTKDILEIAEQ

[0306] SEQ ID NO:100

[0307] MEEKVGNLKPNMESVNVTVRVLEASEARQIQTKNGVRTISEAIVGDETGRVKLTLWGKHAGSIKEGQVVKIENAWTTAFKGQVQLNAGSKTKIAEASEDGFPESSQIPENTPTAPQQMRGGGRGFRGGGRRYGRRGGRRQENEEGEEE

[0308] SEQ ID NO:101

[0309] MTLEEARKRVNELRDLIRYHNYRYYVLADPISDAEYDRLLRELELEERFPELKSPDSPTLQVGARPLEATFRPVRHPTRMYSLDNAFNLDELKAFEERIERALGRKGPFAYTVEHKVDGLSVNLYYEEGVLVYGATRGDGEVGEEVTQNLLTIPTIPRRLKGVPERL EVRGEMPIAFLRLNEELEERGERIFKNPRNAAAGSLRQKDPRITAKRGLRATFYAGLGLEEVEREGVATQFALLHWLKEKGFPVEHGYARAVGAEGVEAVYQDWLKKRRALPFEADGVVVKLDELALWRELGYTARAPRFAIAYKFPAEEKETRLLDVVFQVGRTG RVTPVGILEPVFLEGSEVSRVTLHNESYIELDIRIGDWVLVHKAGGVIPEVLRVLKERRTGEERPIRWPETCPECGHRLLKEGKVHRCPNPLCPAKRFEAIRHFASRKAMDIQGLGEKLIERLLEKLVKDVADLYRLRKEDLVGLERMGEKSAQNLLRQIEESKKRGLERLLYALGLPGVGEVLARNLAARFGNMDRLLEASLEELLEVEEVGELTARAILETLKDPAFRDLVRRLKEAGVEMEAKEKGGEALKGLTFVITGELSRPREEVKALLRRLGAKVTDSVSRKTSYLVVGENPGSKLEKARALGVPTLTEEELYRLLEARTGKKAEELV

[0310] SEQ ID NO:102

[0311] MILDADYITEDGKPIIRIFKKENGEFKVEYDRNFRPYIYALLKDDSAIDDVKKITAERHGKVVRVVDVEKVKKKFLGRPIEVWKLYFEHPQDVPAIRDKIREHPAVIDIFEYDIPFAKRYLIDKGLIPMEGNEELKLLAFDIETLYHEGEEFGKGPIIMISYADEEGAKVITWKKVDLPYVEVVSSEREMIKRFLKVIREKDPDVIITYNGDNFDFPYLLKRAEKLGMKLPLGRDGSEPKMQRLGDSLAVEIKGRIHFDLYPVIRRTINLPTYTLEAVYEAIFGKPKEKVYPHEIAEAEWETGKGLERVAKYSMEDAKVTYELGREFFPMEAQLARLVGQPLWDVSRSSTGNLVEWYLLRKAYERNELAPNKPDEREYERRLRESYEG GYVKEPEKGLWEGIVSLDFRSLYPSIITHNVSPDTLNKEGCGEYDVAPEVGHRFCKDFPGFIPSLLGSLLDERQKIKRRMKASKDPIERKLLDYRQRAIKILANSYYGYYAKAARWYCKECAESVTAWGREYIELVRRELEERGFKVLYIDTDGLYATIPGEKNWEEIKRRALEFVNYINAKLPGLLELEYEGFYTRGFFVTKKKYALIDEEGKIITRGLEIVRRDWSEIAKETQAKVLEAILKHGNVEEAVKIVKEVTEKLSKYEIPPEKLVIYEQITRPLNEYKAIGPHVAKRLAARGIKVRPGMVIGYVVLRGDPISKRAIAAEEFDPKKHKYDAEYYYIENQVLPAVLRILEAFGYRKEDLRWQKTKQVGLGAWLKVKKS sequence <110> New England Biological Laboratory Company <120> the formation of the composite <130> NEB-380-PCT <150> 62 / 042,527 <151> 2014-08-27 <150> 62 / 189,599 <151> 2015-07-07 <150> 62 / 193,168 <151> 2015-07-16 <160> 102 <170> PatentIn version 3.5 <210> 1 <211> 775 <212> PRT <213> Artificial sequence <220> <223> Synthetic construct <400> 1 Met Ile Leu Asp Ala Asp Tyr Ile Thr Glu Asp Gly Lys Pro Ile Ile 1 5 10 15 Arg Leu Phe Lys Lys Glu Asn Gly Arg Phe Lys Val Glu Tyr Asp Arg 20 25 30 Asn Phe Arg Pro Tyr Ile Tyr Ala Leu Leu Lys Asp Asp Ser Ala Ile 35 40 45 Asp Asp Val Arg Lys Ile Thr Ser Glu Arg His Gly Lys Val Val Arg 50 55 60 Val Ile Asp Val Glu Lys Val Lys Lys Lys Phe Leu Gly Arg Pro Ile 65 70 75 80 Glu Val Trp Lys Leu Tyr Phe Glu His Pro Gln Asp Val Pro Ala Met 85 90 95 Arg Asp Lys Ile Arg Glu His Pro Ala Val Ile Asp Ile Phe Glu Tyr 100 105 110 Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Lys Gly Leu Ile Pro 115 120 125 Met Glu Gly Asn Glu Glu Leu Thr Phe Leu Ala Val Asp Ile Glu Thr 130 135 140 Leu Tyr His Glu Gly Glu Glu Phe Gly Lys Gly Pro Ile Ile Met Ile 145 150 155 160 Ser Tyr Ala Asp Glu Glu Gly Ala Lys Val Ile Thr Trp Lys Lys Ile 165 170 175 Asp Leu Pro Tyr Val Glu Val Val Ala Asn Glu Arg Glu Met Ile Lys 180 185 190 Arg Leu Ile Lys Val Ile Arg Glu Lys Asp Pro Asp Val Ile Ile Thr 195 200 205 Tyr Asn Gly Asp Asn Phe Asp Phe Pro Tyr Leu Leu Lys Arg Ala Glu 210 215 220 Lys Leu Gly Met Lys Leu Pro Leu Gly Arg Asp Asn Ser Glu Pro Lys 225 230 235 240 Met Gln Arg Leu Gly Asp Ser Leu Ala Val Glu Ile Lys Gly Arg Ile 245 250 255 His Phe Asp Leu Phe Pro Val Ile Arg Arg Thr Ile Asn Leu Pro Thr 260 265 270 Tyr Thr Leu Glu Ala Val Tyr Glu Ala Ile Phe Gly Lys Gln Lys Glu 275 280 285 Lys Val Tyr Pro His Glu Ile Ala Glu Ala Trp Glu Thr Gly Lys Gly 290 295 300 Leu Glu Arg Val Ala Lys Tyr Ser Met Glu Asp Ala Lys Val Thr Tyr 305 310 315 320 Glu Leu Gly Lys Glu Phe Phe Pro Met Glu Ala Gln Leu Ala Arg Leu 325 330 335 Val Gly Gln Pro Leu Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu 340 345 350 Val Glu Trp Tyr Leu Leu Arg Lys Ala Tyr Glu Arg Asn Glu Leu Ala 355 360 365 Pro Asn Lys Pro Asp Glu Arg Glu Tyr Glu Arg Arg Leu Arg Glu Ser 370 375 380 Tyr Glu Gly Gly Tyr Val Lys Glu Pro Glu Arg Gly Leu Trp Glu Gly 385 390 395 400 Ile Val Ser Leu Asp Phe Arg Ser Leu Tyr Pro Ser Ile Ile Ile Thr 405 410 415 His Asn Val Ser Pro Asp Thr Leu Asn Lys Glu Gly Cys Gly Glu Tyr 420 425 430 Asp Glu Ala Pro Glu Val Gly His Arg Phe Cys Lys Asp Phe Pro Gly 435 440 445 Phe Ile Pro Ser Leu Leu Gly Ser Leu Leu Glu Glu Arg Gln Lys Ile 450 455 460 Lys Lys Arg Met Lys Glu Ser Lys Asp Pro Val Glu Arg Lys Leu Leu 465 470 475 480 Asp Tyr Arg Gln Arg Ala Ile Lys Ile Leu Ala Asn Ser Phe Tyr Gly 485 490 495 Tyr Tyr Gly Tyr Ala Lys Ala Arg Trp Tyr Cys Lys Glu Cys Ala Glu 500 505 510 Ser Val Thr Ala Trp Gly Arg Gln Tyr Ile Glu Leu Val Arg Arg Glu 515 520 525 Leu Glu Glu Arg Gly Phe Lys Val Leu Tyr Ile Asp Thr Asp Gly Leu 530 535 540 Tyr Ala Thr Ile Pro Gly Glu Lys Asn Trp Glu Glu Ile Lys Arg Arg 545 550 555 560 Ala Leu Glu Phe Val Asn Tyr Ile Asn Ser Lys Leu Pro Gly Ile Leu 565 570 575 Glu Leu Glu Tyr Glu Gly Phe Tyr Thr Arg Gly Phe Phe Val Thr Lys 580 585 590 Lys Lys Tyr Ala Leu Ile Asp Glu Glu Gly Lys Ile Val Thr Arg Gly 595 600 605 Leu Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu Thr Gln 610 615 620 Ala Lys Val Leu Glu Ala Ile Leu Lys His Gly Asn Val Glu Glu Ala 625 630 635 640 Val Lys Ile Val Lys Glu Val Thr Glu Lys Leu Ser Asn Tyr Glu Ile 645 650 655 Pro Val Glu Lys Leu Val Ile Tyr Glu Gln Ile Thr Arg Pro Leu Asn 660 665 670 Glu Tyr Lys Ala Ile Gly Pro His Val Ala Val Ala Lys Arg Leu Ala 675 680 685 Ala Lys Gly Ile Lys Ile Lys Pro Gly Met Val Ile Gly Tyr Val Val 690 695 700 Leu Arg Gly Asp Gly Pro Ile Ser Lys Arg Ala Ile Ala Ile Glu Glu 705 710 715 720 Phe Asp Gly Lys Lys His Lys Tyr Asp Ala Glu Tyr Tyr Ile Glu Asn 725 730 735 Gln Val Leu Pro Ala Val Glu Arg Ile Leu Lys Ala Phe Gly Tyr Lys 740 745 750 Arg Glu Asp Leu Arg Trp Gln Lys Thr Lys Gln Val Gly Leu Gly Ala 755 760 765 Trp Leu Lys Val Lys Lys Ser 770 775 <210> 2 <211> 75 <212> PRT <213> Pyrococcus furiosus <400> 2 Ile Ile Asn Pro Gln Ala Arg Leu Thr Pro Leu Glu Leu Glu Ile Leu 1 5 10 15 Glu Ile Ile Lys Gln Lys Lys Ser Ile Thr Ile Thr Glu Ile Lys Glu 20 25 30 Ile Leu Ser Glu Arg Arg Lys Ser Glu Tyr Pro Leu Ser Leu Val Ser 35 40 45 Glu Tyr Ile Ser Arg Leu Glu Arg Lys Gly Tyr Val Lys Lys Ile Ala 50 55 60 Lys Gly Arg Lys Lys Phe Val Glu Ala Leu Ile 65 70 75 <210> 3 <211> 856 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 3 Met Ile Leu Asp Ala Asp Tyr Ile Thr Glu Asp Gly Lys Pro Ile Ile 1 5 10 15 Arg Leu Phe Lys Lys Glu Asn Gly Arg Phe Lys Val Glu Tyr Asp Arg 20 25 30 Asn Phe Arg Pro Tyr Ile Tyr Ala Leu Leu Lys Asp Asp Ser Ala Ile 35 40 45 Asp Asp Val Arg Lys Ile Thr Ser Glu Arg His Gly Lys Val Val Arg 50 55 60 Val Ile Asp Val Glu Lys Val Lys Lys Lys Phe Leu Gly Arg Pro Ile 65 70 75 80 Glu Val Trp Lys Leu Tyr Phe Glu His Pro Gln Asp Val Pro Ala Met 85 90 95 Arg Asp Lys Ile Arg Glu His Pro Ala Val Ile Asp Ile Phe Glu Tyr 100 105 110 Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Lys Gly Leu Ile Pro 115 120 125 Met Glu Gly Asn Glu Glu Leu Thr Phe Leu Ala Val Asp Ile Glu Thr 130 135 140 Leu Tyr His Glu Gly Glu Glu Phe Gly Lys Gly Pro Ile Ile Met Ile 145 150 155 160 Ser Tyr Ala Asp Glu Glu Gly Ala Lys Val Ile Thr Trp Lys Lys Ile 165 170 175 Asp Leu Pro Tyr Val Glu Val Val Ala Asn Glu Arg Glu Met Ile Lys 180 185 190 Arg Leu Ile Lys Val Ile Arg Glu Lys Asp Pro Asp Val Ile Ile Thr 195 200 205 Tyr Asn Gly Asp Asn Phe Asp Phe Pro Tyr Leu Leu Lys Arg Ala Glu 210 215 220 Lys Leu Gly Met Lys Leu Pro Leu Gly Arg Asp Asn Ser Glu Pro Lys 225 230 235 240 Met Gln Arg Leu Gly Asp Ser Leu Ala Val Glu Ile Lys Gly Arg Ile 245 250 255 His Phe Asp Leu Phe Pro Val Ile Arg Arg Thr Ile Asn Leu Pro Thr 260 265 270 Tyr Thr Leu Glu Ala Val Tyr Glu Ala Ile Phe Gly Lys Gln Lys Glu 275 280 285 Lys Val Tyr Pro His Glu Ile Ala Glu Ala Trp Glu Thr Gly Lys Gly 290 295 300 Leu Glu Arg Val Ala Lys Tyr Ser Met Glu Asp Ala Lys Val Thr Tyr 305 310 315 320 Glu Leu Gly Lys Glu Phe Phe Pro Met Glu Ala Gln Leu Ala Arg Leu 325 330 335 Val Gly Gln Pro Leu Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu 340 345 350 Val Glu Trp Tyr Leu Leu Arg Lys Ala Tyr Glu Arg Asn Glu Leu Ala 355 360 365 Pro Asn Lys Pro Asp Glu Arg Glu Tyr Glu Arg Arg Leu Arg Glu Ser 370 375 380 Tyr Glu Gly Gly Tyr Val Lys Glu Pro Glu Arg Gly Leu Trp Glu Gly 385 390 395 400 Ile Val Ser Leu Asp Phe Arg Ser Leu Tyr Pro Ser Ile Ile Ile Thr 405 410 415 His Asn Val Ser Pro Asp Thr Leu Asn Lys Glu Gly Cys Gly Glu Tyr 420 425 430 Asp Glu Ala Pro Glu Val Gly His Arg Phe Cys Lys Asp Phe Pro Gly 435 440 445 Phe Ile Pro Ser Leu Leu Gly Ser Leu Leu Glu Glu Arg Gln Lys Ile 450 455 460 Lys Lys Arg Met Lys Glu Ser Lys Asp Pro Val Glu Arg Lys Leu Leu 465 470 475 480 Asp Tyr Arg Gln Arg Ala Ile Lys Ile Leu Ala Asn Ser Phe Tyr Gly 485 490 495 Tyr Tyr Gly Tyr Ala Lys Ala Arg Trp Tyr Cys Lys Glu Cys Ala Glu 500 505 510 Ser Val Thr Ala Trp Gly Arg Gln Tyr Ile Glu Leu Val Arg Arg Glu 515 520 525 Leu Glu Glu Arg Gly Phe Lys Val Leu Tyr Ile Asp Thr Asp Gly Leu 530 535 540 Tyr Ala Thr Ile Pro Gly Glu Lys Asn Trp Glu Glu Ile Lys Arg Arg 545 550 555 560 Ala Leu Glu Phe Val Asn Tyr Ile Asn Ser Lys Leu Pro Gly Ile Leu 565 570 575 Glu Leu Glu Tyr Glu Gly Phe Tyr Thr Arg Gly Phe Phe Val Thr Lys 580 585 590 Lys Lys Tyr Ala Leu Ile Asp Glu Glu Gly Lys Ile Val Thr Arg Gly 595 600 605 Leu Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu Thr Gln 610 615 620 Ala Lys Val Leu Glu Ala Ile Leu Lys His Gly Asn Val Glu Glu Ala 625 630 635 640 Val Lys Ile Val Lys Glu Val Thr Glu Lys Leu Ser Asn Tyr Glu Ile 645 650 655 Pro Val Glu Lys Leu Val Ile Tyr Glu Gln Ile Thr Arg Pro Leu Asn 660 665 670 Glu Tyr Lys Ala Ile Gly Pro His Val Ala Val Ala Lys Arg Leu Ala 675 680 685 Ala Lys Gly Ile Lys Ile Lys Pro Gly Met Val Ile Gly Tyr Val Val 690 695 700 Leu Arg Gly Asp Gly Pro Ile Ser Lys Arg Ala Ile Ala Ile Glu Glu 705 710 715 720 Phe Asp Gly Lys Lys His Lys Tyr Asp Ala Glu Tyr Tyr Ile Glu Asn 725 730 735 Gln Val Leu Pro Ala Val Glu Arg Ile Leu Lys Ala Phe Gly Tyr Lys 740 745 750 Arg Glu Asp Leu Arg Trp Gln Lys Thr Lys Gln Val Gly Leu Gly Ala 755 760 765 Trp Leu Lys Val Lys Lys Ser Gly Thr Gly Gly Gly Gly Ile Ile Asn 770 775 780 Pro Gln Ala Arg Leu Thr Pro Leu Glu Leu Glu Ile Leu Glu Ile Ile 785 790 795 800 Lys Gln Lys Lys Ser Ile Thr Ile Thr Glu Ile Lys Glu Ile Leu Ser 805 810 815 Glu Arg Arg Lys Ser Glu Tyr Pro Leu Ser Leu Val Ser Glu Tyr Ile 820 825 830 Ser Arg Leu Glu Arg Lys Gly Tyr Val Lys Lys Ile Ala Lys Gly Arg 835 840 845 Lys Lys Phe Val Glu Ala Leu Ile 850 855 <210> 4 <211> 40 <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <400> 4 ttggtctggt gtcaaaaatg aatcgtcacg gcgatttatg 40 <210> 5 <211> 39 <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <400> 5 gggtcatttt cggcgaggac tgcatcaacg catatagcg 39 <210> 6 <211> 32 <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <400> 6 gcggccgcgt cctcgccgaa aatgacccag ag 32 <210> 7 <211> 32 <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <400> 7 gcggccgctg gtgtcgatgg tagaacgaag cg 32 <210> 8 <211> 32 <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <400> 8 gcggccgccc cactgacgcg ttgcgcgaga ag 32 <210> 9 <211> 32 <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <400> 9 gcggccgcgg ctgcgcaact gttggggaagg gc 32 <210> 10 <211> 32 <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <400> 10 gcggccgctg cagcacatcc ccctttcgcc ag 32 <210> 11 <211> 32 <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <400> 11 gcggccgcat gatgctcgtg acggttaacg cc 32 <210> 12 <211> 32 <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <400> 12 gcggccgcag gtgcggattg aaaatggtct gc 32 <210> 13 <211> 32 <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <400> 13 gcggccgctc accgcttgcc agcggcttac ca 32 <210> 14 <211> 32 <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <400> 14 gcggccgcga atacctgttc cgtcatagcg at 32 <210> 15 <211> 33 <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <400> 15 gcggccgctc atttttgaca ccagaccaac tgg 33 <210> 16 <211> 1016 <212> DNA <213> Artificial sequence <220> <223> Synthesized construct <400> 16 gcggccgcgt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc 60 atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag 120 gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg 180 agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg 240 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc 300 cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg 360 gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg 420 tttgatggtg gttaacggcg ggatataacc aacgcgcagc ccggactcgg taatatccca 480 ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca 540 gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt 600 gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct 660 gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag 720 aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca 780 cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag 840 agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct 900 ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca 960 ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccagc ggccgc 1016 <210> 17 <211> 1016 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Construct <400> 17 gcggccgccc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg 60 acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat 120 ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca 180 atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc 240 tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc 300 accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt 360 actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg 420 cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc 480 ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa 540 tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc 600 cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat 660 gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg 720 tccggcgtag aggatcgaga tctcgatccc gcgaaattaa tacgactcac tataggggaa 780 ttgtgagcgg ataacaattc ccctctagaa ataattttgt ttaactttaa gaaggagata 840 tacatatgac catgattacg gattcactgg ccgtcgtttt acaacgtcgt gactgggaaa 900 accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta 960 atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagccgc ggccgc 1016 <210> 18 <211> 1016 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Construct <400> 18 gcggccgctg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc 60 gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcgctttgc ctggtttccg 120 gcaccagaag cggtgccgga aagctggctg gagtgcgatc ttcctgaggc cgatactgtc 180 gtcgtcccct caaactggca gatgcacggt tacgatgcgc ccatctacac caacgtgacc 240 tatcccatta cggtcaatcc gccgtttgtt cccacggaga atccgacggg ttgttactcg 300 ctcacattta atgttgatga aagctggcta caggaaggcc agacgcgaat tatttttgat 360 ggcgttaact cggcgtttca tctgtggtgc aacgggcgct gggtcggtta cggccaggac 420 agtcgtttgc cgtctgaatt tgacctgagc gcatttttac gcgccggaga aaaccgcctc 480 gcggtgatgg tgctgcgctg gagtgacggc agttatctgg aagatcagga tatgtggcgg 540 atgagcggca ttttccgtga cgtctcgttg ctgcataaac cgactacaca aatcagcgat 600 ttccatgttg ccactcgctt taatgatgat ttcagccgcg ctgtactgga ggctgaagtt 660 cagatgtgcg gcgagttgcg tgactaccta cgggtaacag tttctttatg gcagggtgaa 720 acgcaggtcg ccagcggcac cgcgcctttc ggcggtgaaa ttatcgatga gcgtggtggt 780 tatgccgatc gcgtcacact acgtctgaac gtcgaaaacc cgaaactgtg gagcgccgaa 840 atcccgaatc tctatcgtgc ggtggttgaa ctgcacaccg ccgacggcac gctgattgaa 900 gcagaagcct gcgatgtcgg tttccgcgag gtgcggattg aaaatggtct gctgctgctg 960 aacggcaagc cgttgctgat tcgaggcgtt aaccgtcacg agcatcatgc ggccgc 1016 <210> 19 <211> 1016 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Construct <400> 19 gcggccgcag gtgcggattg aaaatggtct gctgctgctg aacggcaagc cgttgctgat 60 tcgaggcgtt aaccgtcacg agcatcatcc tctgcatggt caggtcatgg atgagcagac 120 gatggtgcag gatatcctgc tgatgaagca gaacaacttt aacgccgtgc gctgttcgca 180 ttatccgaac catccgctgt ggtacacgct gtgcgaccgc tacggcctgt atgtggtgga 240 tgaagccaat attgaaaccc acggcatggt gccaatgaat cgtctgaccg atgatccgcg 300 ctggctaccg gcgatgagcg aacgcgtaac gcgaatggtg cagcgcgatc gtaatcaccc 360 gagtgtgatc atctggtcgc tggggaatga atcaggccac ggcgctaatc acgacgcgct 420 gtatcgctgg atcaaatctg tcgatccttc ccgcccggtg cagtatgaag gcggcggagc 480 cgacaccacg gccaccgata ttatttgccc gatgtacgcg cgcgtggatg aagaccagcc 540 cttcccggct gtgccgaaat ggtccatcaa aaaatggctt tcgctacctg gagagacgcg 600 cccgctgatc ctttgcgaat acgcccacgc gatgggtaac agtcttggcg gtttcgctaa 660 atactggcag gcgtttcgtc agtatccccg tttacagggc ggcttcgtct gggactgggt 720 ggatcagtcg ctgattaaat atgatgaaaa cggcaacccg tggtcggctt acggcggtga 780 ttttggcgat acgccgaacg atcgccagtt ctgtatgaac ggtctggtct ttgccgaccg 840 cacgccgcat ccagcgctga cggaagcaaa acaccagcag cagtttttcc agttccgttt 900 atccgggcaa accatcgaag tgaccagcga atacctgttc cgtcatagcg ataacgagct 960 cctgcactgg atggtggcgc tggatggtaa gccgctggca agcggtgagc ggccgc 1016 <210> 20 <211> 1168 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Construct <400> 20 gcggccgcga atacctgttc cgtcatagcg ataacgagct cctgcactgg atggtggcgc 60 tggatggtaa gccgctggca agcggtgaag tgcctctgga tgtcgctcca caaggtaaac 120 agttgattga actgcctgaa ctaccgcagc cggagagcgc cgggcaactc tggctcacag 180 tacgcgtagt gcaaccgaac gcgaccgcat ggtcagaagc cgggcacatc agcgcctggc 240 agcagtggcg tctggcggaa aacctcagtg tgacgctccc cgccgcgtcc cacgccatcc 300 cgcatctgac caccagcgaa atggattttt gcatcgagct gggtaataag cgttggcaat 360 ttaaccgcca gtcaggcttt ctttcacaga tgtggattgg cgataaaaaa caactgctga 420 cgccgctgcg cgatcagttc acccgtgcac cgctggataa cgacattggc gtaagtgaag 480 cgacccgcat tgaccctaac gcctgggtcg aacgctggaa ggcggcgggc cattaccagg 540 ccgaagcagc gttgttgcag tgcacggcag atacacttgc tgatgcggtg ctgattacga 600 ccgctcacgc gtggcagcat caggggaaaa ccttatttat cagccggaaa acctaccgga 660 ttgatggtag tggtcaaatg gcgattaccg ttgatgttga agtggcgagc gatacaccgc 720 atccggcgcg gattggcctg aactgccagc tggcgcaggt agcagagcgg gtaaactggc 780 tcggattagg gccgcaagaa aactatcccg accgccttac tgccgcctgt tttgaccgct 840 gggatctgcc attgtcagac atgtataccc cgtacgtctt cccgagcgaa aacggtctgc 900 gctgcgggac gcgcgaattg aattatggcc cacaccagtg gcgcggcgac ttccagttca 960 acatcagccg ctacagtcaa cagcaactga tggaaaccag ccatcgccat ctgctgcacg 1020 cggaagaagg cacatggctg aatatcgacg gtttccatat ggggattggt ggcgacgact 1080 cctggagccc gtcagtatcg gcggaattcc agctgagcgc cggtcgctac cattaccagt 1140 tggtctggtg tcaaaaatga gcggccgc 1168 <210> twenty one <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <400> twenty one aaaaccaccc tggcgcccaa tacg 24 <210> twenty two <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <400> twenty two cccggactcg gtaatggcgc gcat 24 <210> twenty three <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <400> twenty three ggaagcagcc cagtagtagg ttga 24 <210> twenty four <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <400> twenty four ggtgctgcgc tggagtgacg gcag 24 <210> 25 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <400> 25 cggccaccga tattatttgc ccga 24 <210> 26 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <400> 26 gattagggcc gcaagaaaac tatc 24 <210> 27 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> Features not yet classified <222> (22)..(22) <223> n is a, c, g or t <400> 27 gcgaagaacc tcttcccaag angg 24 <210> 28 <211> 71 <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <400> 28 atcttgtgga aaggacgaaa caccggcgaa gaacctcttc ccaagagttt tagagctaga 60 aatagcaagt t 71 <210> 29 <211> 71 <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> Features not yet classified <222> (26)..(46) <223> n is a, c, g or t <400> 29 atcttgtgga aaggacgaaa caccgnnnnn nnnnnnnnnn nnnnnngttt tagagctaga 60 aatagcaagt t 71 <210> 30 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <400> 30 cgccagggtt ttcccagtca cgac 24 <210> 31 <211> 27 <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <400> 31 gccaagcttg catgcctgca ggtcgac 27 <210> 32 <211> 71 <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <400> 32 gcggtcccaa aagggtcagt gctgcaacat tttgctgccg gtcacggttc gaacgtacgg 60 acgtccagct g 71 <210> 33 <211> 1184 <212> PRT <213> Methanocaldococcus vulcanius <400> 33 Met Lys Glu Lys Ala Pro Lys Ile Asp Ala Leu Ile Asp Cys Thr Tyr 1 5 10 15 Lys Thr Glu Asp Asn Arg Ala Val Ile Tyr Leu Tyr Leu Leu Glu Asn 20 25 30 Ile Leu Lys Asp Arg Glu Phe Ser Pro Tyr Phe Tyr Val Glu Met Leu 35 40 45 Lys Asp Arg Ile Glu Lys Glu Asp Ile Asp Lys Ile Lys Glu Phe Leu 50 55 60 Leu Lys Glu Asp Leu Leu Lys Phe Val Glu Asn Leu Glu Val Val Asn 65 70 75 80 Lys Thr Ile Leu Lys Lys Glu Lys Glu Ile Val Lys Ile Ile Ala Thr 85 90 95 His Pro Gln Arg Val Pro Lys Leu Arg Lys Ile Lys Glu Cys Asp Ile 100 105 110 Val Lys Glu Ile Tyr Glu His Asp Ile Pro Phe Ala Lys Arg Tyr Leu 115 120 125 Ile Asp Ser Asp Ile Val Pro Met Thr Tyr Trp Asp Phe Glu Asn Arg 130 135 140 Lys Gln Val Ser Ile Glu Ile Pro Lys Leu Lys Thr Val Ser Phe Asp 145 150 155 160 Met Glu Val Tyr Asn Arg Asp Thr Glu Pro Asp Pro Glu Lys Asp Pro 165 170 175 Ile Leu Met Ala Ser Phe Trp Asp Asp Asn Gly Gly Lys Val Ile Thr 180 185 190 Tyr Lys His Phe Asp His Ser Asn Ile Glu Val Val Asn Ser Glu Lys 195 200 205 Asp Leu Ile Lys Lys Ile Val Glu Met Leu Arg Gln Tyr Asp Val Ile 210 215 220 Phe Thr Tyr Asn Gly Asp Asn Phe Asp Phe Pro Tyr Leu Lys Ala Arg 225 230 235 240 Ala Lys Ile Tyr Gly Ile Asp Ile Lys Leu Gly Arg Asp Gly Glu Glu 245 250 255 Leu Lys Ile Lys Arg Gly Gly Met Glu Phe Arg Ser Tyr Ile Pro Gly 260 265 270 Arg Val His Ile Asp Leu Tyr Pro Ile Ser Arg Arg Leu Leu Lys Leu 275 280 285 Thr Lys Tyr Thr Leu Glu Asp Val Val Tyr Asn Leu Phe Gly Ile Glu 290 295 300 Lys Leu Lys Ile Pro His Thr Lys Ile Val Asp Tyr Trp Ala Asn Asn 305 310 315 320 Asp Lys Ile Leu Ile Glu Tyr Ser Leu Gln Asp Ala Lys Tyr Thr His 325 330 335 Lys Ile Gly Lys Tyr Phe Phe Pro Leu Glu Val Met Phe Ser Arg Ile 340 345 350 Val Asn Gln Thr Pro Phe Glu Ile Thr Arg Met Ser Ser Gly Gln Met 355 360 365 Val Glu Tyr Leu Leu Met Lys Asn Ala Phe Lys Glu Asn Met Ile Val 370 375 380 Pro Asn Lys Pro Asp Glu Lys Glu Tyr Arg Lys Arg Leu Leu Thr Ser 385 390 395 400 Tyr Glu Gly Gly Tyr Val Lys Glu Pro Glu Lys Gly Met Phe Glu Asp 405 410 415 Ile Ile Ser Met Asp Phe Arg Cys His Pro Arg Gly Thr Lys Val Ile 420 425 430 Val Lys Asn Asn Gly Leu Thr Asp Ile Glu Asn Val Lys Val Gly Asp 435 440 445 Tyr Val Leu Gly Ile Asp Gly Trp Gln Lys Val Lys Arg Val Trp Lys 450 455 460 Tyr Pro Tyr Asn Gly Phe Leu Val Asn Val Asn Gly Leu Lys Ser Thr 465 470 475 480 Pro Asn His Lys Ile Pro Val Ile Lys Lys Glu Asn Gly Lys Asp Arg 485 490 495 Val Ile Asp Val Ser Ser Ile Tyr Leu Leu Asn Leu Lys Gly Cys Lys 500 505 510 Ile Leu Lys Ile Lys Asn Phe Glu Ser Ile Gly Met Phe Gly Lys Ile 515 520 525 Phe Lys Lys Asp Thr Lys Ile Lys Lys Val Lys Gly Leu Leu Glu Lys 530 535 540 Ile Ala Tyr Ile Asp Pro Arg Glu Gly Leu Val Ile Lys Val Lys Asn 545 550 555 560 Glu Lys Glu Asp Ile Phe Lys Thr Val Ile Pro Ile Leu Lys Glu Leu 565 570 575 Asn Ile Leu Tyr Lys Gln Val Asp Glu Lys Thr Ile Ile Ile Asp Ser 580 585 590 Ile Asp Gly Leu Leu Lys Tyr Ile Val Thr Ile Gly Phe Asn Asp Lys 595 600 605 Asn Glu Glu Lys Ile Lys Glu Ile Ile Lys Glu Lys Ser Phe Leu Glu 610 615 620 Phe Lys Glu Leu Glu Asp Ile Lys Ile Ser Ile Glu Glu Tyr Glu Gly 625 630 635 640 Tyr Val Tyr Asp Leu Thr Leu Glu Gly Arg Pro Tyr Tyr Phe Ala Asn 645,650,655 Gly With Leo Thr His Asn To Be Leu Tyr Pro To Be With Lew With Only Tyr 660,665,670 Asn Served Pro Glu Thr Leu Asp Cys Glu Cys Cys Lys Asp Ser 675,680,685 Glu Lys Ile Leu Gly His Trp Phe Cys Lys Arg Glu Gly Leu Ile 690,695,700 Pro Lys Thr Leu Arg Gly Leu And Glu Arg Arg With Asn And Lys Asn 705 710 715 720 Lys Met Lys Lys Met Glu Ser Glu Lys Glu Ile Asn Glu Glu Tyr Asn 725 730 735 Leu Asp Tyr Glu Gln Arg Served Lys Ile Leu Ala Asn Served Val 740,745,750 Tyr Gly Tyr Leu Ala Phe Pro Arg Ala Arg Phe Tyr Ser Arg Glu Cys 755,760,765 Only Glu Val Ile Thr Tyr With Gly Arg Lys Tyr Ile With Glu Thr Ile 770,775,780 Glu Glu Ala Glu Lys Phe Gly Phe Lys Val Ile Tyr Ala Asp Ser Val 785,790,795,800 Val Lys Asp Ala Lys Val Ile Ile Lys Glu Asp Gly Lys Ile Lys Glu 805 810 815 Ile Lys Ile Glu Asp Leu Phe Lys Lys Val Asp Tyr Thr Ile Gly Asp 820 825 830 Lys Glu Tyr Cys Ile Leu Asn Asn Val Glu Thr Leu Thr Ile Glu Asp 835 840 845 Thr Lys Leu Val Trp Arg Lys Val Pro Tyr Ile Met Arg His Arg Thr 850 855 860 Asn Lys Lys Ile Tyr Arg Val Lys Val Lys Asp Arg Tyr Val Asp Ile 865 870 875 880 Thr Glu Asp His Ser Ile Ile Gly Val Lys Asn Asn Lys Leu Val Glu 885 890 895 Leu Lys Pro Thr Glu Ile Lys Asp Asp Glu Thr Lys Leu Ile Ile Leu 900 905 910 Asn Lys Asp Leu Lys Ser Tyr Asn Phe Ala Ser Val Glu Glu Ile Asn 915 920 925 Cys Ile Lys Tyr Ser Asp Tyr Val Tyr Asp Ile Glu Val Glu Asn Thr 930 935 940 His Arg Phe Phe Ala Asn Gly Ile Leu Val His Asn Thr Asp Gly Phe 945 950 955 960 Tyr Ala Val Trp Lys Glu Lys Ile Ser Lys Asp Asp Leu Ile Lys Lys 965 970 975 Ala Leu Glu Phe Val Lys Tyr Ile Asn Ser Lys Leu Pro Gly Thr Met 980 985 990 Glu Leu Glu Phe Glu Gly Tyr Phe Lys Arg Gly Ile Phe Ile Thr Lys 995 1000 1005 Lys Arg Tyr Ala Leu Ile Asp Glu Asn Gly Arg Val Ile Val Lys 1010 1015 1020 Gly Leu Glu Phe Val Arg Arg Asp Trp Ser Asn Leu Ala Arg Ile 1025 1030 1035 Thr Gln Arg Arg Val Leu Glu Ala Leu Leu Leu Glu Gly Asp Ile 1040 1045 1050 Asn Lys Ala Lys Lys Ala Ile Gln Asp Val Ile Lys Asp Leu Arg 1055 1060 1065 Glu Lys Lys Ile Lys Lys Glu Asp Leu Ile Ile Tyr Thr Gln Leu 1070 1075 1080 Thr Lys Asn Pro Asn Glu Tyr Lys Thr Thr Ala Pro His Val Glu 1085 1090 1095 Ile Ala Lys Lys Met Met Arg Glu Gly Lys Lys Ile Lys Ile Gly 1100 1105 1110 Asp Val Ile Gly Tyr Ile Ile Val Lys Gly Ser Lys Ser Ile Ser 1115 1120 1125 Glu Arg Ala Lys Leu Pro Glu Glu Val Ser Ile Glu Glu Ile Asp 1130 1135 1140 Val Asn Tyr Tyr Ile Asp Asn Gln Ile Leu Pro Pro Val Leu Arg 1145 1150 1155 Ile Met Glu Ala Val Gly Val Ser Lys Asn Glu Leu Lys Lys Glu 1160 1165 1170 Gly Thr Gln Leu Thr Leu Asp Arg Phe Leu Lys 1175 1180 <210> 34 <211> 781 <212> PRT <213> Archaeoglobus fulgidus <400> 34 Met Glu Arg Val Glu Gly Trp Leu Ile Asp Ala Asp Tyr Glu Thr Ile 1 5 10 15 Gly Gly Lys Ala Val Val Arg Leu Trp Cys Lys Asp Asp Gln Gly Ile 20 25 30 Phe Val Ala Tyr Asp Tyr Asn Phe Asp Pro Tyr Phe Tyr Val Ile Gly 35 40 45 Val Asp Glu Asp Ile Leu Lys Asn Ala Ala Thr Ser Thr Arg Arg Glu 50 55 60 Val Ile Lys Leu Lys Ser Phe Glu Lys Ala Gln Leu Lys Thr Leu Gly 65 70 75 80 Arg Glu Val Glu Gly Tyr Ile Val Tyr Ala His His Pro Gln His Val 85 90 95 Pro Lys Leu Arg Asp Tyr Leu Ser Gln Phe Gly Asp Val Arg Glu Ala 100 105 110 Asp Ile Pro Phe Ala Tyr Arg Tyr Leu Ile Asp Lys Asp Leu Ala Cys 115 120 125 Met Asp Gly Ile Ala Ile Glu Gly Glu Lys Gln Gly Gly Val Ile Arg 130 135 140 Ser Tyr Lys Ile Glu Lys Val Glu Arg Ile Pro Arg Met Glu Phe Pro 145 150 155 160 Glu Leu Lys Met Leu Val Phe Asp Cys Glu Met Leu Ser Ser Phe Gly 165 170 175 Met Pro Glu Pro Glu Lys Asp Pro Ile Ile Val Ile Ser Val Lys Thr 180 185 190 Asn Asp Asp Asp Glu Ile Ile Leu Thr Gly Asp Glu Arg Lys Ile Ile 195 200 205 Ser Asp Phe Val Lys Leu Ile Lys Ser Tyr Asp Pro Asp Ile Ile Val 210 215 220 Gly Tyr Asn Gln Asp Ala Phe Asp Trp Pro Tyr Leu Arg Lys Arg Ala 225 230 235 240 Glu Arg Trp Asn Ile Pro Leu Asp Val Gly Arg Asp Gly Ser Asn Val 245 250 255 Val Phe Arg Gly Gly Arg Pro Lys Ile Thr Gly Arg Leu Asn Val Asp 260 265 270 Leu Tyr Asp Ile Ala Met Arg Ile Ser Asp Ile Lys Ile Lys Lys Leu 275 280 285 Glu Asn Val Ala Glu Phe Leu Gly Thr Lys Ile Glu Ile Ala Asp Ile 290 295 300 Glu Ala Lys Asp Ile Tyr Arg Tyr Trp Ser Arg Gly Glu Lys Glu Lys 305 310 315 320 Val Leu Asn Tyr Ala Arg Gln Asp Ala Ile Asn Thr Tyr Leu Ile Ala 325 330 335 Lys Glu Leu Leu Pro Met His Tyr Glu Leu Ser Lys Met Ile Arg Leu 340 345 350 Pro Val Asp Asp Val Thr Arg Met Gly Arg Gly Lys Gln Val Asp Trp 355 360 365 Leu Leu Leu Ser Glu Ala Lys Lys Ile Gly Glu Ile Ala Pro Asn Pro 370 375 380 Pro Glu His Ala Glu Ser Tyr Glu Gly Ala Phe Val Leu Glu Pro Glu 385 390 395 400 Arg Gly Leu His Glu Asn Val Ala Cys Leu Asp Phe Ala Ser Met Tyr 405 410 415 Pro Ser Ile Met Ile Ala Phe Asn Ile Ser Pro Asp Thr Tyr Gly Cys 420 425 430 Arg Asp Asp Cys Tyr Glu Ala Pro Glu Val Gly His Lys Phe Arg Lys 435 440 445 Ser Pro Asp Gly Phe Phe Lys Arg Ile Leu Arg Met Leu Ile Glu Lys 450 455 460 Arg Arg Glu Leu Lys Val Glu Leu Lys Asn Leu Ser Pro Glu Ser Ser 465 470 475 480 Glu Tyr Lys Leu Leu Asp Ile Lys Gln Gln Thr Leu Lys Val Leu Thr 485 490 495 Asn Ser Phe Tyr Gly Tyr Met Gly Trp Asn Leu Ala Arg Trp Tyr Cys 500 505 510 His Pro Cys Ala Glu Ala Thr Thr Ala Trp Gly Arg His Phe Ile Arg 515 520 525 Thr Ser Ala Lys Ile Ala Glu Ser Met Gly Phe Lys Val Leu Tyr Gly 530 535 540 Asp Thr Asp Ser Ile Phe Val Thr Lys Ala Gly Met Thr Lys Glu Asp 545 550 555 560 Val Asp Arg Leu Ile Asp Lys Leu His Glu Glu Leu Pro Ile Gln Ile 565 570 575 Glu Val Asp Glu Tyr Tyr Ser Ala Ile Phe Phe Val Glu Lys Lys Arg 580 585 590 Tyr Ala Gly Leu Thr Glu Asp Gly Arg Leu Val Val Lys Gly Leu Glu 595 600 605 Val Arg Arg Gly Asp Trp Cys Glu Leu Ala Lys Lys Val Gln Arg Glu 610 615 620 Val Ile Glu Val Ile Leu Lys Glu Lys Asn Pro Glu Lys Ala Leu Ser 625 630 635 640 Leu Val Lys Asp Val Ile Leu Arg Ile Lys Glu Gly Lys Val Ser Leu 645 650 655 Glu Glu Val Val Ile Tyr Lys Gly Leu Thr Lys Lys Pro Ser Lys Tyr 660 665 670 Glu Ser Met Gln Ala His Val Lys Ala Ala Leu Lys Ala Arg Glu Met 675 680 685 Gly Ile Ile Tyr Pro Val Ser Ser Lys Ile Gly Tyr Val Ile Val Lys 690,695,700 Gly Ser Gly Asn With Gly Asp Arg Ala Tyr Pro With Asp Leu With Glu 705 710 715 720 Asp Phe Asp Gly Glu Asn Leu Arg Ile Lys Thr Lys Ser Gly Ile Glu 725 730 735 Ile Lys Lys Leu Asp Lys Asp Tyr Tyr Ile Asp Gln Ile Ile Pro 740,745,750 Ser Val Leu Arg Ile Leu Glu Arg Phe Gly Tyr Thr Glu Ala Ser Leu 755,760,765 Lys Gly Ser Gln Met Ser Leu Asp Ser PhE Ser 770,775,780 <210> 35 <211> 783 <212> PRT <213> Deep Archaeoglobus <400> 35 With Lys Ala Trp Leu Leu Asp Val Asp Tyr Val Thr Glu Asn Asp 1 5 10 15 Arg Ala Val Ile Arg Leu Trp Cys Lys Asp Asp Lys Gly Val Phe Val 20 25 30 Ala Tyr Asp Arg Asn Phe Leu Pro Tyr Phe Tyr Val Ile Gly Cys Lys 35 40 45 Ala Glu Asp Val Met Lys Val Lys Val Arg Thr Asn Glu Gly Ile Ile 50 55 60 Thr Pro Leu Lys Val Glu Glu Ile Glu Ala Lys Ser Leu Gly Lys Pro 65 70 75 80 Ile Lys Ala Leu Lys Val Tyr Thr Arg His Pro Gln His Val Pro Lys 85 90 95 Leu Arg Glu Glu Ile Lys Lys Phe Ala Glu Val Arg Glu Ala Asp Ile 100 105 110 Pro Phe Ala Tyr Arg Tyr Leu Ile Asp Lys Asp Leu Ala Cys Met Asp 115 120 125 Gly Ile Glu Ile Glu Pro Ile Ala Val Lys Glu Gly Val Leu Arg Ala 130 135 140 Tyr Glu Val Arg Ser Val Arg Arg Val Glu Lys Lys Gly Phe Pro Asp 145 150 155 160 Leu Lys Ile Leu Ala Phe Asp Cys Glu Met Leu Ala Gln Phe Met Pro 165 170 175 Asp Pro Glu Lys Asp Pro Ile Ile Ala Ile Ala Val Lys Cys Gly Asp 180 185 190 Phe Glu Glu Val Leu His Gly Asp Glu Arg Asp Ile Leu Arg Arg Phe 195 200 205 Val Ser Ile Ile Lys Glu Gln Asp Pro Asp Ile Ile Val Gly Tyr Asn 210 215 220 Gln Asp Asn Phe Asp Trp Pro Tyr Val Lys Lys Arg Ala Glu Lys Phe 225 230 235 240 Gly Ile Arg Leu Asp Ile Gly Arg Asp Arg Ser Glu Ile Ser Phe Arg 245 250 255 Gly Gly Arg Pro Lys Ile Ala Gly Arg Leu Asn Val Asp Leu Tyr Asp 260 265 270 Ile Ala Leu Lys Ile Pro Asp Val Lys Ile Lys Thr Leu Lys Lys Val 275 280 285 Ala Glu Phe Leu Gly Ala Lys Val Glu Glu Glu Asp Ile Glu Gly Arg 290 295 300 Asp Ile Tyr Lys Cys Trp Met Arg Gly Glu Lys Glu Lys Val Phe Lys 305 310 315 320 His Val Leu Asn Asp Val Leu Thr Thr Tyr Arg Leu Ala Leu Glu Leu 325 330 335 Leu Pro Met His Tyr Glu Leu Ser Arg Met Ile Arg Leu Pro Leu Asp 340 345 350 Asp Val Ala Arg Leu Gly Arg Gly Lys Gln Val Asp Tyr Phe Leu Leu 355 360 365 Ser Glu Ala Lys Lys Ile Asn Glu Ile Ala Pro Asn Pro Pro Glu Ile 370 375 380 Glu Glu Ser Tyr Glu Gly Ala Phe Val Leu Glu Pro Ala Arg Gly Leu 385 390 395 400 His Glu Asn Val Ala Cys Leu Asp Phe Ala Ser Met Tyr Pro Ser Ile 405 410 415 Met Ile Asn Phe Asn Ile Ser Pro Asp Thr Leu Val Lys Gly Glu Cys 420 425 430 Glu Asp Cys Tyr Val Ala Pro Glu Val Gly His Lys Phe Arg Lys Ser 435 440 445 Pro Asp Gly Phe Phe Lys Arg Ile Leu Lys Met Leu Ile Glu Lys Arg 450 455 460 Arg Glu Met Lys Arg Gln Met Lys Glu Leu Asp Pro Asp Ser Glu Asp 465 470 475 480 Tyr Lys Leu Leu Asp Ile Lys Gln Gln Thr Leu Lys Val Leu Thr Asn 485 490 495 Ser Phe Tyr Gly Tyr Thr Gly Trp Asn Leu Ala Arg Trp Tyr Cys Arg 500 505 510 Glu Cys Ala Glu Ala Thr Thr Ala Trp Gly Arg Tyr Phe Ile Lys Arg 515,520,525 Ala Val Lys Ile Ala Glu Ser Met Gly Phe Glu Val Leu Tyr Gly Asp 530 535 540 Thr Asp Ser Leu Phe Ile Lys Lys Asn Lys Leu Asn Leu Lys Asp Leu 545 550 555 560 Glu Lys Glu Cys Liver Glue Asp Val Ile Ser Lys Glu Lion Pro 565,570,575 Ile Gln Leu Glu Ile Asp Glu Phe Tyr Lys Ala Ile Phe Phe Val Glu 580,585,590 Lys Lys Arg Tyr Ala Gly Leu Thr Asp Asp Asp Arg Ile Val Val Lys 595,600,605 Gly Leu Glu Val Arg Arg Gly Asp Trp Cys Glu Leu Ala Lys Arg Val 610 615 620 Gln Arg Glue Val With Glue Ile Leu Arg Glue Arg Asn Pro Asp Lys 625 630 635 640 Ala Leu Lys Phe Val Lys Asn Val Ile Glu Glu Ile Lys Glu Gly Lys 645,650,655 Phe Lys Leu Glu Asp Tyr Val Ile Tyr Gly Leu Thr Lys Pro 660,665,670 Asp Lys Tyr Glu Ser Lys Gln Ala His Val Lys Ala Ala Leu Arg Ala 675 680 685 Met Glu Met Gly Ile Tyr Tyr Pro Ile Gly Thr Lys Val Gly Phe Val 690 695 700 Ile Val Lys Gly Gly Gly Ser Ile Ser Asp Arg Ala Tyr Pro Ile Glu 705 710 715 720 Leu Ile Glu Glu Phe Asp Gly Glu Asn Leu Lys Ile Arg Thr Pro Ser 725 730 735 Gly Ile Met Val Lys Lys Ile Asp Lys Asp Tyr Tyr Ile Asp His Gln 740 745 750 Ile Ile Pro Ala Val Met Arg Ile Leu Glu Arg Phe Gly Tyr Thr Glu 755 760 765 Ala Ser Leu Lys Thr Thr Ile Gln Lys Thr Leu Phe Asp Phe Thr 770 775 780 <210> 36 <211> 849 <212> PRT <213> Caldicellulosiruptor hydrothermalis <400> 36 Met Lys Leu Val Ile Phe Asp Gly Asn Ser Ile Leu Tyr Arg Ala Phe 1 5 10 15 Phe Ala Leu Pro Glu Leu Thr Thr Ser Ser Asn Ile Pro Thr Asn Ala 20 25 30 Ile Tyr Gly Phe Ile Asn Val Ile Leu Lys Tyr Leu Glu Gln Glu Lys 35 40 45 Pro Asp Tyr Ile Ala Val Ala Phe Asp Lys Arg Gly Arg Glu Ala Arg 50 55 60 Lys Ser Glu Tyr Gln Glu Tyr Lys Ala Asn Arg Lys Pro Met Pro Asp 65 70 75 80 Asn Leu Gln Val Gln Ile Pro Tyr Val Arg Glu Ile Leu Tyr Ala Leu 85 90 95 Asn Ile Pro Ile Val Glu Phe Glu Gly Tyr Glu Ala Asp Asp Val Ile 100 105 110 Gly Ser Leu Val Asn Lys Phe Lys Asn Thr Gly Leu Asp Ile Val Ile 115 120 125 Ile Thr Gly Asp Arg Asp Thr Leu Gln Leu Leu Asp Lys Asn Val Val 130 135 140 Val Lys Ile Val Ser Thr Lys Phe Asp Arg Thr Met Glu Asp Leu Tyr 145 150 155 160 Thr Ile Glu Asn Ile Lys Glu Lys Tyr Gly Val Trp Ala Asn Gln Val 165 170 175 Pro Asp Tyr Lys Ala Leu Val Gly Asp Gln Ser Asp Asn Ile Pro Gly 180 185 190 Val Lys Gly Ile Gly Glu Lys Ser Ala Gln Lys Leu Leu Glu Glu Tyr 195 200 205 Ser Ser Leu Glu Glu Ile Tyr Gln Asn Leu Asp Lys Ile Lys Gly Ser 210 215 220 Ile Arg Glu Lys Leu Glu Ala Gly Lys Asp Met Ala Phe Leu Ser Lys 225 230 235 240 Arg Leu Ala Thr Ile Val Cys Asp Leu Pro Leu Asn Val Asn Leu Glu 245 250 255 Asp Leu Arg Thr Lys Glu Trp Asn Lys Glu Arg Leu Tyr Glu Ile Leu 260 265 270 Val Gln Leu Glu Phe Lys Ser Ile Ile Lys Arg Leu Gly Leu Ser Glu 275 280 285 Asn Ile Gln Phe Glu Phe Val Gln Gln Arg Thr Asp Ile Pro Asp Val 290 295 300 Glu Gln Arg Glu Leu Glu Ser Ile Ser Arg Ile Arg Ser Lys Glu Ile 305 310 315 320 Pro Leu Met Phe Val Gln Asp Glu Lys Cys Phe Tyr Leu Tyr Asp Gln 325 330 335 Glu Ser Asn Thr Val Phe Val Thr Arg Asp Arg His Leu Val Glu Glu 340 345 350 Ile Leu Lys Ser Asp Thr Val Lys Ile Val Tyr Asp Leu Lys Asn Ile 355 360 365 Phe His Gln Leu Asn Leu Glu Asp Thr Asp Asn Ile Lys Asn Cys Glu 370 375 380 Asp Val Met Ile Ala Ser Tyr Val Leu Asp Ser Thr Arg Ser Ser Tyr 385 390 395 400 Glu Leu Glu Thr Leu Phe Val Ser Tyr Leu Asn Thr Asp Ile Glu Ala 405 410 415 Val Lys Lys Asp Lys Lys Met Val Ser Val Val Leu Leu Lys Arg Leu 420 425 430 Trp Asp Asp Leu Leu Arg Leu Ile Asp Leu Asn Ser Cys Gln Phe Leu 435 440 445 Tyr Glu Asn Ile Glu Arg Pro Leu Ile Pro Val Leu Tyr Glu Met Glu 450 455 460 Lys Thr Gly Phe Lys Val Asp Arg Asp Ala Leu Leu Gln Tyr Thr Lys 465 470 475 480 Glu Ile Glu Asn Lys Ile Leu Lys Leu Glu Thr Gln Ile Tyr Gln Ile 485 490 495 Ala Gly Glu Trp Phe Asn Ile Asn Ser Pro Lys Gln Leu Ser Tyr Ile 500 505 510 Leu Phe Glu Lys Leu Lys Leu Pro Val Ile Lys Lys Thr Lys Thr Gly 515 520 525 Tyr Ser Thr Asp Ala Glu Val Leu Glu Glu Phe Asp Lys His Glu Ile 530 535 540 Val Pro Leu Ile Leu Asp Tyr Arg Met Tyr Thr Lys Ile Leu Thr Thr 545 550 555 560 Tyr Cys Gln Gly Leu Leu Gln Ala Ile Asn Pro Ser Ser Gly Arg Val 565 570 575 His Thr Thr Phe Ile Gln Thr Gly Thr Ala Thr Gly Arg Leu Ala Ser 580 585 590 Ser Asp Pro Asn Leu Gln Asn Ile Pro Val Lys Tyr Asp Glu Gly Lys 595 600 605 Leu Ile Arg Lys Val Phe Val Pro Glu Glu Gly His Val Leu Ile Asp 610 615 620 Ala Asp Tyr Ser Gln Ile Glu Leu Arg Ile Leu Ala His Ile Ser Glu 625 630 635 640 Asp Glu Arg Leu Ile Asn Ala Phe Lys Asn Asn Ile Asp Ile His Ser 645 650 655 Gln Thr Ala Ala Glu Val Phe Gly Val Asp Ile Ala Asp Val Thr Pro 660,665,670 Glu Met Arg Ser Gln Ala Lys Ala Val Asn Phe Gly Ile Val Tyr Gly 675,680,685 Asp Ser Tyr Gly Leu Has Arg Asp Lys Ser and Arg Lys Glu 690,695,700 Ala Ala Glu Phe Ile Asn Lys Tyr Phe Glu Arg Tyr Pro Lys Val Lys 705 710 715 720 Glu Tyr Leu Asp Asn Ile Val Arg Phe Ala Arg Glu Asn Gly Tyr Val 725 730 735 Leu Thr Leu Phe Asn Arg Lys Arg Tyr Val Lys Asp Ile Lys Ser Ala 740,745,750 Asn Arg Asn Ala Arg Ser Tyr Ala Glu Arg Ile Ala Met Asn Ser Pro 755,760,765 Gln Gly Ser Only Asp Only Met Only Lys Only Only Only Only Only Only on Lys Val 770,775,780 Tyr Gln Lys with Lys Glu Asn Asn Lys with Lys Ile with Gln 785,790,795,800 Val His Asp Glu Leo Leo Ile Glu Wing Pro Tyr Glu Lys Asp Ile 805 810 815 Val Lys Arg Ile Val Lys Arg Glu Met Glu Asn Ala Val Ala Leu Lys 820 825 830 Val Pro Leu Val Val Glu Val Lys Glu Gly Leu Asn Trp Tyr Glu Thr 835 840 845 Lys <210> 37 <211> 781 <212> PRT <213> Desulfurococcus mucosus <400> 37 Met Glu Lys Arg Val Tyr Leu Val Asp Ile Thr Tyr Gly Leu Val Gly 1 5 10 15 Asn Ser Pro Glu Ile Arg Met Phe Gly Val Asp Glu Asn Gly Glu Lys 20 25 30 Val Val Ile Leu Asp Arg Gly Phe Arg Pro Tyr Phe Tyr Val Ile Pro 35 40 45 Glu Glu Gly Phe Glu Asp Gln Val Ala Arg Val Ile Gly Lys Met Gln 50 55 60 Asn Val Ile Lys Ala Asp Val Thr Glu Arg Arg Leu Phe Gly Lys Pro 65 70 75 80 Ile Lys Val Val Lys Val Thr Val Thr Val Pro Asp Lys Val Arg Glu 85 90 95 Leu Arg Asp Arg Val Lys Ser Ile Gln His Val Lys Glu Val Leu Glu 100 105 110 Ala Asp Ile Arg Phe Tyr Ile Arg Tyr Met Ile Asp Asn Asp Ile Arg 115 120 125 Pro Gly Trp Leu Met Phe Ser Asn Leu Lys Pro Val Asp Asn Lys Ile 130 135 140 Gly Gly Val Ser Asn Val Tyr Leu Thr Glu Thr Pro Pro Thr Ser Leu 145 150 155 160 Asp Leu Gly Ile Met Pro Arg Leu Asn Tyr Met Ala Leu Asp Ile Glu 165 170 175 Val Tyr Asn Pro Arg Gly Thr Pro Asp Pro Lys Arg Asp Pro Ile Ile 180 185 190 Ile Ile Ala Leu Ala Asn Ser Asn Gly Asp Val Lys Leu Leu Thr Leu 195 200 205 Asp Asn Tyr Lys His Glu Arg Glu Met Leu Asn Asp Met Met Ser Val 210 215 220 Ile Lys Glu Trp Asp Pro Asp Val Leu Phe Gly Tyr Asn Ser Asn Lys 225 230 235 240 Phe Asp Met Pro Tyr Leu Val Asn Arg Ala Asp Ala Leu Asn Val Lys 245 250 255 Leu Gln Leu Ser Lys Tyr Gly Thr Pro Pro Glu Gln Ser Val Tyr Gly 260 265 270 His Trp Ser Ile Ile Gly Arg Ala His Ile Asp Leu Tyr Asn Phe Ile 275 280 285 Glu Asp Met Thr Asp Val Lys Arg Lys Ser Leu Asp Tyr Val Ala Glu 290 295 300 Tyr Phe Gly Val Met Lys Arg Ser Glu Arg Val Asn Ile Pro Gly His 305 310 315 320 Arg Ile Tyr Gln Tyr Trp Asp Asp Glu Gly Lys Arg Ser Gln Leu Ile 325 330 335 Lys Tyr Ala Arg Asp Asp Val Leu Ser Thr Leu Gly Leu Gly Lys Ile 340 345 350 Leu Leu Pro Tyr Ala Met Gln Leu Ala Ser Val Ser Gly Leu Pro Leu 355 360 365 Asp Gln Val Gly Pro Ala Ser Val Gly Ser Arg Val Glu Met Met Ile 370 375 380 Met His Glu Ala Tyr Lys Met Gly Glu Leu Ala Pro Asn Arg Val Glu 385 390 395 400 Arg Pro Tyr Glu Thr Tyr Lys Gly Ala Ile Val Leu Glu Pro Lys Pro 405 410 415 Gly Ile His Tyr Asn Ile Ala Val Leu Asp Phe Ser Ser Met Tyr Pro 420 425 430 Asn Ile Met Leu Lys Tyr Asn Ile Ser Pro Asp Thr Leu Val Leu Asp 435 440 445 Ser Ser Glu Gly Asp Tyr Tyr Thr Ala Pro Glu Val Gly Tyr Arg Phe 450 455 460 Arg Lys Ser Pro Arg Gly Leu Tyr Ala Ser Leu Leu Gln Lys Leu Ile 465 470 475 480 Glu Ala Arg Arg Glu Ala Arg Asp Glu Met Arg Asn Tyr Pro Glu Gly 485 490 495 Ser Phe Glu Trp Val Leu Leu Asn Glu Arg Gln Arg Ala Leu Lys Ile 500 505 510 Met Ala Asn Ala Met Tyr Gly Tyr Cys Gly Trp Leu Gly Ala Arg Trp 515 520 525 Tyr Ile Arg Glu Val Ala Glu Ser Val Thr Ala Trp Gly Arg Tyr Leu 530 535 540 Leu Lys Thr Ala Met Ser Met Ala Lys Glu Arg Gly Leu Thr Val Ile 545 550 555 560 Tyr Gly Asp Thr Asp Ser Leu Phe Val Thr Tyr Asp Lys Asp Lys Val 565 570 575 Ala Asp Ile Ile Ser Arg Ile Asn Glu Met Gly Phe Glu Val Lys Ile 580 585 590 Asp Lys Val Tyr Ser Lys Leu Ile Phe Thr Glu Ser Lys Lys Arg Tyr 595 600 605 Ile Gly Leu Thr Ala Asp Gly Glu Val Asp Ile Val Gly Phe Glu Ala 610 615 620 Val Arg Gly Asp Trp Ser Glu Leu Ala Arg Asn Val Gln Glu Arg Val 625 630 635 640 Ala Glu Leu Val Leu Arg Glu Ser Val Asp Glu Ala Val Lys Tyr Val 645 650 655 Lys Ser Val Ile Asp Asp Leu Arg Asn Tyr Arg Phe Thr Ile Asp Asp 660 665 670 Val Ile Ile Trp Lys Thr Leu Asp Lys Asp Ile Asn Glu Tyr Lys Ala 675 680 685 Ile Gln Pro His Val Val Ala Ala Arg Arg Leu Met Glu Lys Gly Tyr 690 695 700 Val Val Asn Lys Gly Asp Thr Val Gly Phe Val Ile Val Lys Asp Ser 705 710 715 720 Gly Asp Lys Leu Thr Gln Arg Ala Tyr Pro Tyr Val Phe Ile Asn Asp 725 730 735 Val Lys Glu Ile Asp Val Asp Tyr Tyr Val Glu Lys Gln Val Ile Pro 740 745 750 Ala Ala Leu Arg Ile Leu Glu Val Phe Gly Val Asn Glu Ala Ala Leu 755 760 765 Leu Gly Lys Thr Gly Lys Ser Ile Leu Asp Tyr Phe His 770 775 780 <210> 38 <211> 803 <212> PRT <213> Pyrolobus fumarii <400> 38 Met Thr Glu Val Val Phe Thr Val Leu Asp Ser Ser Tyr Glu Val Val 1 5 10 15 Gly Lys Glu Pro Gln Val Ile Ile Trp Gly Ile Ala Glu Asn Gly Glu 20 25 30 Arg Val Val Leu Ile Asp Arg Ser Phe Arg Pro Tyr Phe Tyr Ala Leu 35 40 45 Leu Ala Pro Gly Ala Asp Pro Lys Gln Val Ala Gln Arg Ile Arg Ala 50 55 60 Leu Ser Arg Pro Lys Ser Pro Ile Ile Gly Val Glu Asp Asp Lys Arg 65 70 75 80 Lys Tyr Phe Gly Arg Pro Arg Arg Val Leu Arg Ile Arg Thr Val Leu 85 90 95 Pro Glu Ala Val Arg Glu Tyr Arg Glu Leu Val Lys Asn Val Asp Gly 100 105 110 Val Glu Asp Val Leu Glu Ala Asp Ile Arg Phe Ala Met Arg Tyr Leu 115 120 125 Ile Asp His Asp Leu Phe Pro Phe Thr Trp Tyr Arg Val Glu Ala Glu 130 135 140 Pro Leu Glu Asn Lys Met Gly Phe Arg Val Asp Lys Val Tyr Leu Val 145 150 155 160 Lys Ser Arg Pro Glu Pro Leu Tyr Gly Glu Ala Leu Ala Pro Thr Lys 165 170 175 Leu Pro Asp Leu Arg Ile Leu Ala Phe Asp Ile Glu Val Tyr Ser Lys 180 185 190 Gln Gly Ser Pro Arg Pro Glu Arg Asp Pro Val Ile Val Ile Ala Val 195 200 205 Lys Thr Asp Asp Gly Asp Glu Val Leu Phe Ile Ala Glu Gly Lys Asp 210 215 220 Asp Arg Lys Pro Ile Arg Glu Phe Val Glu Tyr Val Lys Arg Tyr Asp 225 230 235 240 Pro Asp Ile Ile Val Gly Tyr Asn Asn Asn His Phe Asp Trp Pro Tyr 245 250 255 Leu Leu Arg Arg Ala Arg Ile Leu Gly Ile Lys Leu Asp Val Thr Arg 260 265 270 Arg Val Gly Ala Glu Pro Thr Thr Ser Val His Gly His Val Ser Val 275 280 285 Pro Gly Arg Leu Asn Val Asp Leu Tyr Asp Tyr Ala Glu Glu Met Pro 290 295 300 Glu Ile Lys Ile Lys Ser Leu Glu Glu Val Ala Glu Tyr Leu Gly Val 305 310 315 320 Met Lys Lys Ser Glu Arg Val Ile Ile Asn Trp Trp Glu Ile Pro Asp 325 330 335 Tyr Trp Asp Asp Pro Lys Lys Arg Pro Leu Leu Leu Gln Tyr Ala Arg 340 345 350 Asp Asp Val Arg Ala Thr Tyr Gly Leu Ala Glu Lys Ile Leu Pro Phe 355 360 365 Ala Ile Gln Leu Ser Tyr Val Thr Gly Leu Pro Leu Asp Gln Val Gly 370 375 380 Ala Met Ser Val Gly Phe Arg Leu Glu Trp Tyr Leu Ile Arg Ala Ala 385 390 395 400 Phe Lys Met Lys Glu Leu Val Pro Asn Arg Val Glu Arg Pro Glu Glu 405 410 415 Thr Tyr Arg Gly Ala Ile Val Leu Glu Pro Leu Arg Gly Val His Glu 420 425 430 Asn Ile Ala Val Leu Asp Phe Ser Ser Met Tyr Pro Asn Ile Met Ile 435 440 445 Lys Tyr Asn Val Gly Pro Asp Thr Leu Val Arg Pro Gly Glu Glu Cys 450 455 460 Gly Glu Cys Gly Cys Trp Glu Ala Pro Glu Val Lys His Arg Phe Arg 465 470 475 480 Arg Cys Pro Pro Gly Phe Phe Lys Thr Val Leu Glu Arg Leu Leu Glu 485 490 495 Leu Arg Lys Arg Val Arg Ala Glu Met Lys Lys Tyr Pro Pro Asp Ser 500 505 510 Pro Glu Tyr Arg Leu Leu Asp Glu Arg Gln Lys Ala Leu Lys Val Leu 515 520 525 Ala Asn Ala Ser Tyr Gly Tyr Met Gly Trp Ser Gly Ala Arg Trp Tyr 530 535 540 Cys Arg Glu Cys Ala Glu Ala Val Thr Ala Trp Gly Arg His Leu Ile 545 550 555 560 Arg Thr Ala Ile Asn Ile Ala Arg Lys Leu Gly Leu Lys Val Ile Tyr 565 570 575 Gly Asp Thr Asp Ser Leu Phe Val Thr Tyr Asp Pro Glu Lys Val Glu 580 585 590 Lys Phe Ile Lys Ile Ile Glu Glu Glu Leu Gly Phe Glu Ile Lys Leu 595 600 605 Glu Lys Val Tyr Lys Arg Val Phe Phe Thr Glu Ala Lys Lys Arg Tyr 610 615 620 Ala Gly Leu Leu Glu Asp Gly Arg Ile Asp Ile Val Gly Phe Glu Ala 625 630 635 640 Val Arg Gly Asp Trp Cys Glu Leu Ala Lys Glu Val Gln Thr Lys Val 645 650 655 Val Glu Ile Val Leu Lys Thr Ser Asp Val Asn Lys Ala Val Glu Tyr 660 665 670 Val Arg Lys Ile Val Lys Glu Leu Glu Glu Gly Lys Val Pro Ile Glu 675 680 685 Lys Leu Val Ile Trp Lys Thr Leu Ser Lys Arg Leu Glu Glu Tyr Thr 690 695 700 Thr Glu Ala Pro His Val Val Ala Ala Lys Arg Met Leu Ser Ala Gly 705 710 715 720 Tyr Arg Val Ser Pro Gly Asp Lys Ile Gly Tyr Val Ile Val Lys Gly 725 730 735 Gly Gly Arg Ile Ser Gln Arg Ala Trp Pro Tyr Phe Met Val Lys Asp 740 745 750 Pro Ser Gln Ile Asp Val Thr Tyr Tyr Val Asp His Gln Ile Ile Pro 755 760 765 Ala Ala Leu Arg Ile Leu Gly Tyr Phe Gly Ile Thr Glu Lys Lys Leu 770 775 780 Lys Ala Ser Ala Thr Gly Gln Lys Thr Leu Phe Asp Phe Leu Ala Lys 785 790 795 800 Lys Ser Lys <210> 39 <211> 786 <212> PRT <213> Pyrobaculum oguniense <400> 39 Met Glu Ile Arg Val Trp Pro Leu Asp Val Thr Tyr Ile Val Val Gly 1 5 10 15 Gly Val Pro Glu Val Arg Val Phe Gly Ile Ala Glu Gly Gly Glu Arg 20 25 30 Val Val Leu Ala Asp Arg Ser Phe Arg Pro Tyr Phe Tyr Val Asp Cys 35 40 45 Ala Gly Cys Asp Pro His Ala Val Lys Thr His Leu Gly Arg Thr Ala 50 55 60 Pro Val Glu Gly Val Glu Leu Val Glu Arg Arg Phe Leu Gly Arg Pro 65 70 75 80 Arg Gln Phe Leu Lys Val Val Ala Lys Ile Pro Glu Asp Val Arg Arg 85 90 95 Leu Arg Glu Ala Ala Ser Thr Ile Pro Gly Val Arg Gly Val Tyr Glu 100 105 110 Ala Asp Ile Arg Phe Tyr Met Arg Tyr Val Ile Asp Met Gly Val Val 115 120 125 Pro Cys Ser Trp Asn Val Ala Glu Val Glu Val Ala Asp Glu Lys Leu 130 135 140 Gly Ser Leu Pro Val Tyr Arg Val Val Lys Trp Gly Gly Ala Val Glu 145 150 155 160 Gly Phe Pro Pro Pro Leu Arg Val Leu Ala Phe Asp Ile Glu Val Tyr 165 170 175 Asn Glu Arg Gly Thr Pro Asp Pro Ala Arg Asp Pro Ile Val Met Ile 180 185 190 Ala Val Gln Ser Ser Asp Gly Arg Leu Glu Val Phe Glu Ala Ser Gly 195 200 205 Arg Asp Asp Arg Gly Val Leu Arg Ser Phe Val Glu Tyr Val Arg Ser 210 215 220 Phe Asp Pro Asp Val Val Val Gly Tyr Asn Ser Asn Asn Phe Asp Trp 225 230 235 240 Pro Tyr Leu Ala Glu Arg Ala Lys Ala Val Gly Val Pro Leu Arg Val 245 250 255 Asp Arg Leu Gly Gly Ala Pro Gln Gln Ser Val Tyr Gly His Trp Ser 260 265 270 Val Leu Gly Arg Ala Asn Val Asp Leu Tyr Asn Ile Val Asp Glu Phe 275 280 285 Pro Glu Ile Lys Leu Lys Thr Leu Asp Arg Val Ala Glu Tyr Phe Gly 290 295 300 Val Met Arg Arg Asp Glu Arg Val Leu Ile Pro Gly His Lys Ile Tyr 305 310 315 320 Glu Tyr Trp Arg Asp Pro Ser Lys Arg Pro Leu Leu Arg Gln Tyr Val 325 330 335 Ile Asp Asp Val Arg Ser Thr Tyr Gly Leu Ala Glu Arg Leu Leu Pro 340 345 350 Phe Leu Ile Gln Leu Ser Ser Val Ser Gly Leu Pro Leu Asp Gln Val 355 360 365 Ala Ala Ala Ser Val Gly Asn Arg Val Glu Trp Met Leu Leu Arg Tyr 370 375 380 Ala Tyr Gly Leu Gly Glu Val Ala Pro Asn Arg Glu Glu Arg Glu Tyr 385 390 395 400 Glu Pro Tyr Lys Gly Ala Ile Val Leu Glu Pro Lys Pro Gly Leu Tyr 405 410 415 Ser Asp Val Leu Val Leu Asp Phe Ser Ser Met Tyr Pro Asn Val Met 420 425 430 Met Arg Tyr Asn Leu Ser Pro Asp Thr Tyr Leu Glu Pro Gly Glu Pro 435 440 445 Asp Pro Pro Glu Gly Val Tyr Val Ala Pro Glu Val Gly His Arg Phe 450 455 460 Arg Lys Glu Pro Pro Gly Phe Ile Pro Gln Val Leu Arg Arg Leu Val 465 470 475 480 Ala Leu Arg Arg Ala Val Arg Glu Glu Met Lys Lys Tyr Gln Pro Asp 485 490 495 Thr Pro Glu Tyr Arg Val Leu Asp Glu Arg Gln Lys Ala Leu Lys Ile 500 505 510 Met Ala Asn Ala Met Tyr Gly Tyr Thr Gly Trp Val Gly Ala Arg Trp 515 520 525 Tyr Lys Lys Glu Val Ala Glu Ser Val Thr Ala Phe Ala Arg Ala Ile 530 535 540 Leu Lys Asp Val Ile Asp Tyr Ala Arg Arg Leu Gly Ile Val Val Ile 545 550 555 560 Tyr Gly Asp Thr Asp Ser Leu Phe Val Lys Lys Gly Gly Asp Leu Glu 565 570 575 Lys Leu Ala Arg Tyr Val Asp Glu Lys Tyr Gly Ile Glu Ile Lys Val 580 585 590 Asp Lys Asp Tyr Glu Lys Val Leu Phe Thr Glu Ala Lys Lys Arg Tyr 595 600 605 Ala Gly Leu Leu Arg Asp Gly Arg Ile Asp Ile Val Gly Phe Glu Val 610 615 620 Val Arg Gly Asp Trp Ser Glu Leu Ala Lys Glu Val Gln Leu Lys Val 625 630 635 640 Ile Glu Leu Ile Leu Lys Ala Arg Asp Leu Ser Glu Ala Arg Gln Arg 645 650 655 Val Ile Lys Tyr Val Lys Asp Val Ile Glu Arg Leu Lys Ser Gly Lys 660 665 670 Phe Asp Leu Asp Asp Leu Ile Ile Trp Lys Thr Leu Asp Lys Asp Leu 675 680 685 Gly Glu Tyr Lys Ala Tyr Pro Pro His Val Arg Ala Ala Leu Ile Leu 690 695 700 Lys Lys Lys Gly Tyr Lys Val Gly Arg Gly Thr Thr Ile Gly Tyr Val 705 710 715 720 Val Val Lys Gly Gly Glu Lys Val Ser Glu Arg Ser Leu Pro Tyr Ile 725 730 735 Leu Val Asp Asp Leu Ala Lys Ile Asp Val Asp Tyr Tyr Ile Glu Lys 740 745 750 Gln Val Ile Pro Ala Ala Leu Arg Ile Ala Glu Val Ile Gly Val Lys 755 760 765 Glu Gly Asp Leu Arg Ala Gly Arg Ser Glu Lys Ser Leu Leu Asp Phe 770 775 780 Phe Glu 785 <210> 40 <211> 801 <212> PRT <213> Staphylothermus marinus <400> 40 Met Ser Glu Lys Ile Asn Leu Glu Phe Tyr Phe Leu Asp Asn Ser Tyr 1 5 10 15 Glu Val Ile Gly Asn Glu Pro His Ile Ile Ile Trp Gly Ile Thr Arg 20 25 30 Asp Gly Arg Arg Val Leu Leu Arg Asp Arg Arg Phe Arg Pro Tyr Phe 35 40 45 Tyr Ala Ile Leu Lys Asp Lys Val Asn Ile Glu Asp Leu Ala Arg Lys 50 55 60 Ile Arg Thr Tyr Ser Asp Pro Lys Ser Pro Ile Ile Gly Val Glu Pro 65 70 75 80 Val Glu Lys Lys Tyr Phe Gly Arg Lys Val Ser Ala Leu Lys Ile Ile 85 90 95 Thr Met Ile Pro Glu Tyr Val Arg Lys Tyr Arg Glu Lys Ile Lys Ser 100 105 110 Leu Pro Glu Val Leu Glu Val Val Glu Ala Asp Ile Arg Phe Ser Ile 115 120 125 Arg Tyr Ile Ile Asp His Asp Leu Arg Pro Cys Gly Trp His Val Ala 130 135 140 Glu Val Val Glu Val Pro Lys Lys Pro Ile Tyr Arg Val Asp Ala Glu 145 150 155 160 Tyr Glu Ile Ile Gly Asp Ile Lys Pro Leu Glu Gln Thr Leu Gln Pro 165 170 175 Asp Leu Arg Ile Ile Ala Phe Asp Ile Glu Val Tyr Asn Lys Ser Gly 180 185 190 Thr Pro Arg Pro Gln Thr Asp Pro Ile Ile Ile Ile Gly Ile Met Asn 195 200 205 Asn Asn Gly Asp Ile Lys Gln Phe Leu Ala Asn Lys Tyr Asp Asp Lys 210 215 220 Ile Ser Val Glu Glu Phe Val Asn Tyr Val Lys Thr Phe Asp Pro Asp 225 230 235 240 Ile Ile Val Gly Tyr Asn Thr Asp Gly Phe Asp Trp Pro Tyr Leu Ile 245 250 255 Glu Arg Ser Lys Tyr Ile Gly Val Lys Leu Asp Val Thr Arg Arg Val 260 265 270 Gly Ala Thr Pro Arg Thr Ser Thr Tyr Gly His Ile Ser Val Pro Gly 275 280 285 Arg Leu Asn Thr Asp Leu Tyr His Phe Ala Glu Glu Ile Pro Glu Val 290 295 300 Lys Val Lys Ser Leu Glu Asn Val Ala Glu Tyr Leu Gly Val Met Lys 305 310 315 320 Lys Ser Glu Arg Val Ile Ile Glu Tyr Ile Asp Ile Pro Lys Tyr Trp 325 330 335 Asp Asp Glu Lys Leu Arg Pro Lys Leu Leu Gln Tyr Asn Ile Asp Asp 340 345 350 Val Lys Ser Thr Tyr Gly Leu Ala Glu Lys Phe Leu Pro Phe Ala Met 355 360 365 Gln Leu Ser Asn Ile Thr Gly Leu Pro Leu Asp Gln Val Gly Ala Ala 370 375 380 Ser Val Gly Phe Arg Leu Glu Trp Tyr Leu Met Arg Glu Ala Phe Arg 385 390 395 400 Tyr Gly Glu Leu Val Pro Asn Arg Val Glu Arg Ala Ala Glu Ser Tyr 405 410 415 Arg Gly Ala Val Val Leu Lys Pro Val Lys Gly Val His Glu Asn Ile 420 425 430 Ala Val Leu Asp Phe Ser Ser Met Tyr Pro Asn Ile Met Ile Lys Tyr 435 440 445 Asn Val Gly Pro Asp Thr Ile Val Arg Asn Glu Lys Cys Asn Pro Asp 450 455 460 Lys His Asn Ile Ala Pro Glu Val Gly His Cys Phe Arg Lys Glu Pro 465 470 475 480 Pro Gly Phe Phe Lys Arg Val Leu Glu Thr Leu Leu Arg Leu Arg Lys 485 490 495 Gln Ile Lys Ser Glu Met Lys Lys Tyr Pro Pro Thr Ser Tyr Glu Tyr 500 505 510 Arg Leu Leu Asp Glu Arg Gln Lys Ala Val Lys Val Leu Ala Asn Ala 515 520 525 Thr Tyr Gly Tyr Met Gly Trp Ile His Ala Arg Trp Tyr Cys Arg Glu 530 535 540 Cys Ala Glu Ala Val Thr Ala Trp Gly Arg Gln Thr Ile Lys Ser Ala 545 550 555 560 Ile Glu Leu Ala Arg Lys Leu Gly Leu Lys Val Ile Tyr Gly Asp Thr 565 570 575 Asp Ser Leu Phe Val Thr Tyr Asp Lys Asp Lys Val Glu Lys Leu Ile 580 585 590 Glu Leu Ile Gln Thr Lys Leu Gly Phe Glu Ile Lys Ile Asp Lys Ile 595 600 605 Tyr Lys Arg Val Phe Phe Thr Glu Ala Lys Lys Arg Tyr Ala Gly Leu 610 615 620 Leu Glu Asp Gly Arg Ile Asp Ile Val Gly Phe Glu Ala Val Arg Gly 625 630 635 640 Asp Trp Ala Glu Ile Ala Lys Glu Val Gln Glu Lys Val Thr Glu Ile 645 650 655 Leu Leu Lys Glu Asn Ser Ile Asp Lys Ala Ile Glu Tyr Val Arg Gln 660,665,670 Val Ile Ala Asp Leu Lys Ala Gly Lys Ile Pro Leu Asp Lys Leu Ile 675,680,685 Trp Lys Thr Leu Ser Lys Arg Ile Glu Glu Tyr Ser Val Asp Ala 690,695,700 Pro His Val Val Ala Lys Lys Leu Ile Lys Ala Gly Ile Lys Val 705 710 715 720 The Three Asn Asp Lys Ile Gly Tyr Val Ile Leu Lys Gly Gly Gly Lys 725 730 735 Ile Ser Ser Arg Ala Glu Pro Tyr Ile Phe Val Lys Asp Pro Lys Leu 740,745,750 Ile Asp Thr Glu Tyr Tyr Val Asp His Gln Ile Val Pro Ala Ala Leu 755,760,765 Arg Has Tyr Asn PhE Gly Val Thr Glu Thr Gln Leu Lys Arg Ala 770,775,780 Ala Ala Ser Ala Gly Gln Lys Ser Leu Phe Asp Phe Phe Gly Gly Lys 785,790,795,800 Lys <210> 41 <211> 776 <212> PRT <213> Pyrococcus yayaoii <400> 41 Met Ile Leu Asp Ala Asp Tyr Ile Thr Glu Asn Gly Lys Pro Val Val 1 5 10 15 Arg Ile Phe Lys Lys Glu Asn Gly Glu Phe Lys Val Glu Tyr Asp Arg 20 25 30 Ser Phe Arg Pro Tyr Ile Tyr Ala Leu Leu Arg Asp Asp Ser Ala Ile 35 40 45 Glu Asp Ile Lys Lys Ile Thr Ala Glu Arg His Gly Lys Val Val Arg 50 55 60 Val Val Glu Ala Glu Lys Val Arg Lys Lys Phe Leu Gly Arg Pro Ile 65 70 75 80 Glu Val Trp Lys Leu Tyr Phe Glu His Pro Gln Asp Val Pro Ala Ile 85 90 95 Arg Glu Lys Ile Arg Glu His Pro Ala Val Ile Asp Ile Phe Glu Tyr 100 105 110 Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Lys Gly Leu Ile Pro 115 120 125 Met Glu Gly Asn Glu Glu Leu Lys Leu Leu Ala Phe Asp Ile Glu Thr 130 135 140 Leu Tyr His Glu Gly Asp Glu Phe Gly Ser Gly Pro Ile Ile Met Ile 145 150 155 160 Ser Tyr Ala Asp Glu Lys Gly Ala Lys Val Ile Thr Trp Lys Gly Val 165 170 175 Asp Leu Pro Tyr Val Glu Val Val Ser Ser Glu Arg Glu Met Ile Lys 180 185 190 Arg Phe Leu Arg Val Ile Arg Glu Lys Asp Pro Asp Val Ile Ile Thr 195 200 205 Tyr Asn Gly Asp Asn Phe Asp Phe Pro Tyr Leu Leu Lys Arg Ala Glu 210 215 220 Lys Leu Gly Met Lys Leu Pro Ile Gly Arg Asp Gly Ser Glu Pro Lys 225 230 235 240 Met Gln Arg Met Gly Asp Gly Phe Ala Val Glu Val Lys Gly Arg Ile 245 250 255 His Phe Asp Ile Tyr Pro Val Ile Arg Arg Thr Ile Asn Leu Pro Thr 260 265 270 Tyr Thr Leu Glu Ala Val Tyr Glu Ala Val Phe Gly Arg Pro Lys Glu 275 280 285 Lys Val Tyr Pro Asn Glu Ile Ala Arg Ala Trp Glu Asn Cys Lys Gly 290 295 300 Leu Glu Arg Val Ala Lys Tyr Ser Met Glu Asp Ala Lys Val Thr Tyr 305 310 315 320 Glu Leu Gly Arg Glu Phe Phe Pro Met Glu Ala Gln Leu Ala Arg Leu 325 330 335 Val Gly Gln Pro Val Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu 340 345 350 Val Glu Trp Phe Leu Leu Arg Lys Ala Tyr Glu Arg Asn Glu Leu Ala 355 360 365 Pro Asn Arg Pro Asp Glu Arg Glu Tyr Glu Arg Arg Leu Arg Glu Ser 370 375 380 Tyr Glu Gly Gly Tyr Val Lys Glu Pro Glu Lys Gly Leu Trp Glu Gly 385 390 395 400 Ile Ile Tyr Leu Asp Phe Arg Ser Leu Tyr Pro Ser Ile Ile Ile Thr 405 410 415 His Asn Ile Ser Pro Asp Thr Leu Asn Lys Glu Gly Cys Asn Ser Tyr 420 425 430 Asp Val Ala Pro Lys Val Gly His Arg Phe Cys Lys Asp Phe Pro Gly 435 440 445 Phe Ile Pro Ser Leu Leu Gly Gln Leu Leu Asp Glu Arg Gln Lys Ile 450 455 460 Lys Arg Lys Met Lys Ala Thr Ile Asp Pro Ile Glu Arg Lys Leu Leu 465 470 475 480 Asp Tyr Arg Gln Arg Ala Ile Lys Ile Leu Ala Asn Ser Tyr Tyr Gly 485 490 495 Tyr Tyr Gly Tyr Ala Lys Ala Arg Trp Tyr Cys Lys Glu Cys Ala Glu 500 505 510 Ser Val Thr Ala Trp Gly Arg Glu Tyr Ile Glu Leu Val Ser Arg Glu 515 520 525 Leu Glu Lys Arg Gly Phe Lys Val Leu Tyr Ile Asp Thr Asp Gly Leu 530 535 540 Tyr Ala Thr Ile Pro Gly Ser Arg Glu Trp Asp Lys Ile Lys Glu Arg 545 550 555 560 Ala Leu Glu Phe Val Lys Tyr Ile Asn Ala Arg Leu Pro Gly Leu Leu 565 570 575 Glu Leu Glu Tyr Glu Gly Phe Tyr Lys Arg Gly Phe Phe Val Thr Lys 580 585 590 Lys Lys Tyr Ala Leu Ile Asp Glu Glu Gly Lys Ile Ile Thr Arg Gly 595 600 605 Leu Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu Thr Gln 610 615 620 Ala Arg Val Leu Glu Ala Ile Leu Lys Glu Gly Asn Leu Glu Lys Ala 625 630 635 640 Val Lys Ile Val Lys Glu Val Thr Glu Lys Leu Ser Lys Tyr Glu Val 645 650 655 Pro Pro Glu Lys Leu Val Ile Tyr Glu Gln Ile Thr Arg Asp Leu Lys 660 665 670 Asp Tyr Lys Ala Val Gly Pro His Val Ala Val Ala Lys Arg Leu Ala 675 680 685 Ala Arg Gly Ile Lys Val Arg Pro Gly Met Val Ile Gly Tyr Leu Val 690 695 700 Leu Arg Gly Asp Gly Pro Ile Ser Arg Arg Ala Ile Pro Ala Glu Glu 705 710 715 720 Phe Asp Pro Ser Arg His Lys Tyr Asp Ala Glu Tyr Tyr Ile Glu Asn 725 730 735 Gln Val Leu Pro Ala Val Leu Arg Ile Leu Glu Ala Phe Gly Tyr Arg 740 745 750 Lys Glu Asp Leu Arg Tyr Gln Lys Thr Arg Gln Ala Gly Leu Asp Ala 755 760 765 Trp Leu Lys Arg Lys Ala Ser Leu 770 775 <210> 42 <211> 773 <212> PRT <213> Thermococcus sp.AM4 part <400> 42 Met Ile Leu Asp Thr Asp Tyr Ile Thr Glu Asn Gly Lys Pro Val Ile 1 5 10 15 Arg Val Phe Lys Lys Glu Asn Gly Glu Phe Lys Ile Glu Tyr Asp Arg 20 25 30 Thr Phe Glu Pro Tyr Phe Tyr Ala Leu Leu Lys Asp Asp Ser Ala Ile 35 40 45 Glu Asp Val Lys Lys Val Thr Ala Lys Arg His Gly Thr Val Val Arg 50 55 60 Val Lys Arg Ala Glu Lys Val Gln Arg Lys Phe Leu Gly Arg Pro Ile 65 70 75 80 Glu Val Trp Lys Leu Tyr Phe Thr His Pro Gln Asp Val Pro Ala Ile 85 90 95 Arg Asp Lys Ile Arg Ala His Pro Ala Val Ile Asp Ile Tyr Glu Tyr 100 105 110 Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Lys Gly Leu Ile Pro 115 120 125 Met Glu Gly Asp Glu Glu Leu Thr Met Leu Ala Phe Asp Ile Glu Thr 130 135 140 Leu Tyr His Glu Gly Glu Glu Phe Gly Thr Gly Pro Ile Leu Met Ile 145 150 155 160 Ser Tyr Ala Asp Gly Ser Glu Ala Arg Val Ile Thr Trp Lys Lys Ile 165 170 175 Asp Leu Pro Tyr Val Asp Val Val Ser Thr Glu Lys Glu Met Ile Lys 180 185 190 Arg Phe Leu Arg Ile Val Lys Glu Lys Asp Pro Asp Val Leu Ile Thr 195 200 205 Tyr Asn Gly Asp Asn Phe Asp Phe Ala Tyr Leu Lys Lys Arg Cys Glu 210 215 220 Lys Leu Gly Ile Lys Phe Thr Leu Gly Arg Asp Gly Ser Glu Pro Lys 225 230 235 240 Ile Gln Arg Met Gly Asp Arg Phe Ala Val Glu Val Lys Gly Arg Ile 245 250 255 His Phe Asp Leu Tyr Pro Val Ile Arg Arg Thr Ile Asn Leu Pro Thr 260 265 270 Tyr Thr Leu Glu Ala Val Tyr Glu Ala Val Phe Gly Lys Pro Lys Glu 275 280 285 Lys Val Tyr Ala Glu Glu Ile Ala Gln Ala Trp Glu Ser Gly Glu Gly 290 295 300 Leu Glu Arg Val Ala Arg Tyr Ser Met Glu Asp Ala Lys Val Thr Tyr 305 310 315 320 Glu Leu Gly Arg Glu Phe Phe Pro Met Glu Ala Gln Leu Ser Arg Leu 325 330 335 Ile Gly Gln Ser Leu Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu 340 345 350 Val Glu Trp Phe Leu Leu Arg Lys Ala Tyr Glu Arg Asn Glu Leu Ala 355 360 365 Pro Asn Lys Pro Asp Glu Lys Glu Leu Ala Arg Arg Arg Gly Gly Tyr 370 375 380 Ala Gly Gly Tyr Val Lys Glu Pro Glu Arg Gly Leu Trp Asp Asn Ile 385 390 395 400 Val Tyr Leu Asp Phe Arg Ser Leu Tyr Pro Ser Ile Ile Ile Thr His 405 410 415 Asn Val Ser Pro Asp Thr Leu Asn Arg Glu Gly Cys Lys Glu Tyr Asp 420 425 430 Val Ala Pro Glu Val Gly His Lys Phe Cys Lys Asp Phe Pro Gly Phe 435 440 445 Ile Pro Ser Leu Leu Gly Asp Leu Leu Glu Glu Arg Gln Lys Ile Lys 450 455 460 Arg Lys Met Lys Ala Thr Val Asp Pro Leu Glu Lys Lys Leu Leu Asp 465 470 475 480 Tyr Arg Gln Arg Only With Lys And Leu Only Asn Ser Tyr Gly Tyr 485,490,495 Tyr Gly Tyr Ala Lys Ala Arg Trp Tyr Cys Arg Glu Cys Ala Glu Ser 500 505 510 Val Thr Only Trp Gly Arg Glu Ile Tyr Glu Ile Thr Thr Ile Arg Glu Ile 515,520,525 Glu Glu Lys Phe Gly Phe Lys Val Leu Tyr Ala Asp Thr Asp Gly Phe 530 535 540 Phe Ala Thr Ile Pro Gly Ala Asp Ala Glu Thr Val Lys Lys Ala 545 550 555 560 Lys Glu Phe Leu Lys Tyr Ile Asn Ala Lys Leu Pro Gly Leu Leu Glu 565,570,575 Leu Glu Tyr Glu Gly Phe Tyr Val Arg Gly Phe Phe Val Thr Lys Lys 580,585,590 Lys Tyr Ala Val Ile Asp Glu Glu Gly Lys Ile Thr Thr Arg Gly Leu 595,600,605 Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu Thr Gln Ala 610 615 620 Arg Val Leu Glu Ala Ile Leu Lys His Gly Asp Val Glu Glu Ala Val 625 630 635 640 Arg Ile Val Lys Glu Val Thr Glu Lys Leu Ser Lys Tyr Glu Val Pro 645 650 655 Pro Glu Lys Leu Val Ile His Glu Gln Ile Thr Arg Asp Leu Arg Asp 660 665 670 Tyr Lys Ala Thr Gly Pro His Val Ala Val Ala Lys Arg Leu Ala Ala 675 680 685 Arg Gly Val Lys Ile Arg Pro Gly Thr Val Ile Ser Tyr Ile Val Leu 690 695 700 Lys Gly Ser Gly Arg Ile Gly Asp Arg Ala Ile Pro Phe Asp Glu Phe 705 710 715 720 Asp Pro Thr Lys His Arg Tyr Asp Ala Glu Tyr Tyr Ile Glu Asn Gln 725 730 735 Val Leu Pro Ala Val Glu Arg Ile Leu Lys Ala Phe Gly Tyr Arg Lys 740 745 750 Glu Asp Leu Arg Tyr Gln Lys Thr Lys Gln Val Gly Leu Gly Ala Trp 755 760 765 Leu Light Val Light Light 770 <210> 43 <211> 775 <212> PRT <213> Thermococcus hydrothermalis <400> 43 Met Ile Leu Asp Thr Asp Tyr Ile Thr Glu Asp Gly Lys Pro Val Ile 1 5 10 15 Arg Ile Phe Lys Lys Glu Asn Gly Glu Phe Lys Ile Glu Tyr Asp Arg 20 25 30 Glu Phe Glu Pro Tyr Ile Tyr Ala Leu Leu Lys Asp Asp Ser Ala Ile 35 40 45 Glu Glu Val Lys Lys Ile Thr Ala Gly Arg His Gly Arg Val Val Lys 50 55 60 Val Lys Arg Ala Glu Lys Val Lys Lys Lys Phe Leu Gly Arg Pro Ile 65 70 75 80 Glu Val Trp Lys Leu Tyr Phe Thr His Pro Gln Asp Val Pro Ala Ile 85 90 95 Arg Asp Glu Ile Arg Arg His Ser Ala Val Val Asp Ile Tyr Glu Tyr 100 105 110 Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Lys Gly Leu Ile Pro 115 120 125 Met Glu Gly Asp Glu Glu Leu Lys Met Met Ser Phe Asp Ile Glu Thr 130 135 140 Leu Tyr His Glu Gly Glu Glu Phe Gly Thr Gly Pro Ile Leu Met Ile 145 150 155 160 Ser Tyr Ala Asp Glu Gly Glu Ala Arg Val Ile Thr Trp Lys Lys Ile 165 170 175 Asp Leu Pro Tyr Val Glu Val Val Ser Thr Glu Lys Glu Met Ile Lys 180 185 190 Arg Phe Leu Lys Val Val Lys Glu Lys Asp Pro Asp Val Leu Ile Thr 195 200 205 Tyr Asn Gly Asp Asn Phe Asp Phe Ala Tyr Leu Lys Lys Arg Cys Glu 210 215 220 Lys Ile Gly Ile Lys Phe Thr Leu Arg Arg Asp Gly Ser Glu Pro Lys 225 230 235 240 Ile Gln Arg Met Gly Asp Arg Phe Ala Val Glu Val Lys Gly Arg Ile 245 250 255 His Phe Asp Leu Tyr Pro Val Ile Arg Arg Thr Ile Asn Leu Pro Thr 260 265 270 Tyr Thr Leu Glu Ala Val Tyr Glu Ala Val Phe Gly Thr Pro Lys Glu 275 280 285 Lys Val Tyr Pro Glu Glu Ile Thr Thr Ala Trp Glu Thr Gly Glu Gly 290 295 300 Leu Glu Arg Val Ala Arg Tyr Ser Met Glu Asp Ala Lys Val Thr Tyr 305 310 315 320 Glu Leu Gly Arg Glu Phe Phe Pro Met Glu Ala Gln Leu Ser Arg Leu 325 330 335 Ile Gly Gln Ser Leu Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu 340 345 350 Val Glu Trp Phe Leu Leu Arg Lys Ala Tyr Glu Arg Asn Glu Ile Ala 355 360 365 Pro Asn Lys Pro Asp Glu Arg Glu Leu Ala Arg Arg Arg Gly Gly Tyr 370 375 380 Ala Gly Gly Tyr Val Lys Glu Pro Glu Arg Gly Leu Trp Asp Asn Ile 385 390 395 400 Val Tyr Leu Asp Phe Met Ser Leu Tyr Pro Ser Ile Ile Ile Thr His 405 410 415 Asn Val Ser Pro Asp Thr Phe Asn Arg Glu Gly Cys Lys Glu Tyr Asp 420 425 430 Thr Ala Pro Gln Val Gly His Lys Phe Cys Lys Asp Val Gln Gly Phe 435 440 445 Ile Pro Ser Leu Leu Gly Ala Leu Leu Asp Glu Arg Gln Lys Ile Lys 450 455 460 Lys Arg Met Lys Ala Ser Ile Asp Pro Leu Glu Lys Leu Leu Asp 465 470 475 480 Tyr Arg Gln Lys Only With Lys Only Leu Only Asn Ser Tyr Tyr Gly Tyr 485,490,495 Tyr Gly Tyr Ala Arg Ala Arg Trp Tyr Cys Lys Glu Cys Ala Glu Ser 500 505 510 Val Thr Only Trp Gly Arg Asp Tyr With Glu Thr Thr With His Glu 515,520,525 Glu Glu Arg Phe Gly Phe Lys Val Leu Tyr Ala Asp Thr Asp Gly Phe 530 535 540 Phe Ala Thr Ile Pro Gly Ala Asp Ala Glu Thr Val Lys Lys Ala 545 550 555 560 Lys Glu Phe Leu Lys Tyr Ile Asn Ala Lys Leu Pro Gly Leu Leu Glu 565,570,575 Leu Glu Tyr Glu Gly Phe Tyr Val Arg Gly Phe Phe Val Thr Lys Lys 580,585,590 Lys Tyr Ala Val Ile Asp Glu Glu Gly Lys Ile Thr Thr Arg Gly Leu 595,600,605 Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu Thr Gln Ala 610 615 620 Arg Val Leu Glu Ala Ile Leu Arg His Gly Asp Val Glu Glu Ala Val 625 630 635 640 Arg Ile Val Lys Asp Val Thr Glu Lys Leu Ser Lys Tyr Glu Val Pro 645 650 655 Pro Glu Lys Leu Val Ile His Glu Gln Ile Thr Arg Glu Leu Lys Asp 660 665 670 Tyr Lys Ala Thr Gly Pro His Val Ala Ile Ala Lys Arg Leu Ala Ala 675 680 685 Arg Gly Ile Lys Ile Arg Pro Gly Thr Val Ile Ser Tyr Ile Val Leu 690 695 700 Lys Gly Ser Gly Arg Ile Gly Asp Arg Ala Ile Pro Phe Asp Glu Phe 705 710 715 720 Asp Pro Thr Lys His Arg Tyr Asp Ala Glu Tyr Tyr Ile Glu Asn Gln 725 730 735 Val Leu Pro Ala Val Glu Arg Ile Leu Lys Ala Phe Gly Tyr Lys Lys 740 745 750 Glu Glu Leu Arg Tyr Gln Lys Thr Arg Gln Val Gly Leu Gly Ala Trp 755 760 765 Leu Lys Leu Lys Gly Lys Lys 770 775 <210> 44 <211> 774 <212> PRT <213> Thermococcus thioreducens <400> 44 Met Ile Leu Asp Ala Asp Tyr Ile Thr Glu Asp Gly Lys Pro Val Val 1 5 10 15 Arg Ile Phe Lys Lys Glu Asn Gly Glu Phe Lys Ile Glu Tyr Asp Arg 20 25 30 Glu Phe Glu Pro Tyr Ile Tyr Ala Leu Leu Arg Asp Asp Ser Ala Ile 35 40 45 Glu Glu Ile Lys Lys Ile Thr Ala Asp Arg His Gly Lys Val Val Lys 50 55 60 Val Lys Arg Ala Glu Lys Val Gln Lys Lys Phe Leu Gly Arg Pro Ile 65 70 75 80 Glu Val Trp Lys Leu Tyr Phe Thr His Pro Gln Asp Val Pro Ala Ile 85 90 95 Arg Asp Glu Ile Arg Lys His Pro Ala Val Val Asp Ile Tyr Glu Tyr 100 105 110 Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Lys Gly Leu Ile Pro 115 120 125 Met Glu Gly Asp Glu Glu Leu Lys Met Leu Ala Phe Asp Ile Glu Thr 130 135 140 Leu Tyr His Glu Gly Glu Glu Phe Gly Thr Gly Pro Ile Leu Met Ile 145 150 155 160 Ser Tyr Ala Asp Glu Asp Gly Ala Arg Val Ile Thr Trp Lys Lys Ile 165 170 175 Asp Leu Pro Tyr Val Asp Val Val Ser Thr Glu Lys Glu Met Ile Lys 180 185 190 Arg Phe Leu Lys Val Val Lys Glu Lys Asp Pro Asp Val Leu Ile Thr 195 200 205 Tyr Asn Gly Asp Asn Phe Asp Phe Ala Tyr Leu Lys Lys Arg Cys Glu 210 215 220 Lys Leu Gly Ile Lys Phe Thr Leu Gly Arg Asp Gly Ser Glu Pro Lys 225 230 235 240 Ile Gln Arg Met Gly Asp Arg Phe Ala Val Glu Val Lys Gly Arg Ile 245 250 255 His Phe Asp Leu Tyr Pro Leu Ile Arg Arg Thr Ile Asn Leu Pro Thr 260 265 270 Tyr Thr Leu Glu Ala Val Tyr Glu Ala Val Phe Gly Lys Pro Lys Glu 275 280 285 Lys Val Tyr Ala Glu Glu Ile Ala Leu Ala Trp Glu Ser Gly Glu Gly 290 295 300 Leu Glu Arg Val Ala Arg Tyr Ser Met Glu Asp Ala Lys Val Thr Phe 305 310 315 320 Glu Leu Gly Arg Glu Phe Phe Pro Met Glu Ala Gln Leu Ser Arg Leu 325 330 335 Ile Gly Gln Ser Leu Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu 340 345 350 Val Glu Trp Phe Leu Leu Arg Lys Ala Tyr Glu Arg Asn Glu Leu Ala 355 360 365 Pro Asn Lys Pro Asn Glu Arg Glu Leu Ala Arg Arg Arg Gly Gly Tyr 370 375 380 Ala Gly Gly Tyr Val Lys Glu Pro Glu Arg Gly Leu Trp Asp Asn Ile 385 390 395 400 Val Tyr Leu Asp Phe Arg Ser Leu Tyr Pro Ser Ile Ile Ile Thr His 405 410 415 Asn Val Ser Pro Asp Thr Leu Asn Arg Glu Gly Cys Lys Glu Tyr Asp 420 425 430 Arg Ala Pro Gln Val Gly His Lys Phe Cys Lys Asp Val Pro Gly Phe 435 440 445 Pro Ile Lean Gly Ser Lean Asp Glu Arg Gln Lys Ile Lys 450 455 460 Arg Lys Met Lys Ala Thr Ile Asp Pro Ile Glu Lys Lys Leu Leu Asp 465 470 475 480 Tyr Arg Gln Arg Only With Lys And Leu Only Asn Ser Tyr Gly Tyr 485,490,495 Tyr Gly Tyr Ala Arg Ala Arg Trp Tyr Cys Arg Glu Cys Ala Glu Ser 500 505 510 Val Thr Ala Trp Gly Arg Glu Tyr Ile Glu Met Ala Ile Arg Glu Leu 515,520,525 Glu Glu Lys Phe Gly Phe Lys Val Leu Tyr Ala Asp Thr Asp Gly Leu 530 535 540 His Ala Thr Ile Pro Gly Ala Asp Ala Glu Thr Val Lys Lys Ala 545 550 555 560 Met Glu Phe Leu Tyr Ile Asn Pro Lys Leu Pro Gly Leu Leu Glu 565,570,575 Leu Glu Tyr Glu Gly Phe Tyr Ala Arg Gly Phe Phe Val Thr Lys Lys 580,585,590 Lys Tyr Ala Val Ile Asp Glu Glu Gly Lys Ile Thr Thr Arg Gly Leu 595,600,605 Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu Thr Gln Ala 610 615 620 Arg Val Leu Glu Ala Ile Leu Arg His Gly Asp Val Glu Glu Ala Val 625 630 635 640 Arg Ile Val Lys Glu Val Thr Glu Lys Leu Ser Lys Tyr Glu Val Pro 645 650 655 Pro Glu Lys Leu Val Ile His Glu Gln Ile Thr Arg Glu Leu Lys Asp 660 665 670 Tyr Arg Ala Thr Gly Pro His Val Ala Ile Ala Lys Arg Leu Ala Lys 675 680 685 Arg Gly Ile Lys Ile Arg Pro Gly Thr Val Ile Ser Tyr Ile Val Leu 690 695 700 Lys Gly Ser Gly Arg Ile Gly Asp Arg Ala Ile Pro Phe Asp Glu Phe 705 710 715 720 Asp Pro Thr Lys His Lys Tyr Asp Ala Glu Tyr Tyr Ile Glu Asn Gln 725 730 735 Val Leu Pro Ala Val Glu Arg Val Leu Lys Ala Phe Gly Tyr Arg Lys 740 745 750 Asp Asp Leu Arg Tyr Gln Lys Thr Arg Gln Val Gly Leu Gly Ala Trp 755,760,765 Leu Lys Val Lys Arg 770 <210> 45 <211> 773 <212> PRT <213> Thermococcus waiotapuensis <400> 45 Met Leu Asp Ala Asp Tyr Ile Thr Glu Asp Gly Lys Pro Val Ile 1 5 10 15 Arg Val Phe Lys Glu Lys Gly Glu Phe Lys Ile Asn Tyr Asp Arg 20 25 30 Asp Phe Glu Pro Tyr Ile Tyr Ala Leu Leu Lys Asp Ser Ala Ile 35 40 45 Glu Asp With Lys Lys With Thr Ala Glu Arg His Gly Thr Thr Val Arg 50 55 60 Val Thr Arg Ala Glu Arg Val Lys Lys Lys Phe Leu Gly Arg Pro Val 65 70 75 80 Glu Val Trp Lys Leu Tyr Phe Thr His Pro Gln Asp Val Pro Ala Ile 85 90 95 Arg Asp Lys Ile Arg Glu His Pro Ala Val Val Asp Ile Tyr Glu Tyr 100 105 110 Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Lys Gly Leu Ile Pro 115 120 125 Met Glu Gly Asn Glu Glu Leu Arg Met Leu Ala Phe Asp Ile Glu Thr 130 135 140 Leu Tyr His Glu Gly Glu Glu Phe Gly Glu Gly Pro Ile Leu Met Ile 145 150 155 160 Ser Tyr Ala Asp Glu Glu Gly Ala Arg Val Ile Thr Trp Lys Asn Ile 165 170 175 Asp Leu Pro Tyr Val Glu Ser Val Ser Thr Glu Lys Glu Met Ile Lys 180 185 190 Arg Phe Leu Lys Val Ile Gln Glu Lys Asp Pro Asp Val Leu Ile Thr 195 200 205 Tyr Asn Gly Asp Asn Phe Asp Phe Ala Tyr Leu Lys Lys Arg Ser Glu 210 215 220 Thr Leu Gly Val Lys Phe Ile Leu Gly Arg Asp Gly Ser Glu Pro Lys 225 230 235 240 Ile Gln Arg Met Gly Asp Arg Phe Ala Val Glu Val Lys Gly Arg Ile 245 250 255 His Phe Asp Leu Tyr Pro Val Ile Arg Arg Thr Ile Asn Leu Pro Thr 260 265 270 Tyr Thr Leu Glu Thr Val Tyr Glu Ala Ile Phe Gly Gln Pro Lys Glu 275 280 285 Lys Val Tyr Ala Glu Glu Ile Ala Gln Ala Trp Glu Ser Gly Glu Gly 290 295 300 Leu Glu Arg Val Ala Arg Tyr Ser Met Glu Asp Ala Lys Ala Thr Tyr 305 310 315 320 Glu Leu Gly Lys Glu Phe Phe Pro Met Glu Ala Gln Leu Ser Arg Leu 325 330 335 Val Gly Gln Ser Leu Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu 340 345 350 Val Glu Trp Phe Leu Leu Arg Lys Ala Tyr Glu Arg Asn Glu Leu Ala 355 360 365 Pro Asn Lys Pro Asp Glu Arg Glu Leu Ala Arg Arg Ala Glu Ser Tyr 370 375 380 Ala Gly Gly Tyr Val Lys Glu Pro Glu Lys Gly Leu Trp Glu Asn Ile 385 390 395 400 Val Tyr Leu Asp Tyr Lys Ser Leu Tyr Pro Ser Ile Ile Ile Thr His 405 410 415 Asn Val Ser Pro Asp Thr Leu Asn Arg Glu Gly Cys Arg Glu Tyr Asp 420 425 430 Val Ala Pro Gln Val Gly His Arg Phe Cys Lys Asp Phe Pro Gly Phe 435 440 445 Pro Ile Ser Wolf Gly Asp Wolf Glu Glu Arg Gln Lys Val Lys 450 455 460 Lys Lys Met Lys Ala Thr Val Asp Pro Ile Glu Arg Lys Leu Leu Asp 465 470 475 480 Tyr Arg Gln Arg Only With Lys And Leu Only Asn Ser Tyr Gly Tyr 485,490,495 Tyr Gly Tyr Ala Asn Ala Arg Trp Tyr Cys Arg Glu Cys Ala Glu Ser 500 505 510 Val Thr Only Trp Gly Arg Gln Tyr With Glu Thr Thr With Arg Glu 515,520,525 Glu Glu Lys Phe Gly Phe Lys Val Leu Tyr Ala Asp Thr Asp Gly Phe 530 535 540 Phe Ala Thr Ile Pro Gly Ala Asp Ala Glu Thr Val Lys Lys Thr 545 550 555 560 Lys Glu Phe Leu Asn Tyr Ile Asn Pro Arg Leu Pro Gly Leu Leu Glu 565,570,575 Leu Glu Tyr Glu Gly Phe Tyr Arg Arg Gly Phe Phe Val Thr Lys Lys 580,585,590 Lys Tyr Ala Val Ile Asp Glu Glu Asp Lys Ile Thr Thr Arg Gly Leu 595 600 605 Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu Thr Gln Ala 610 615 620 Arg Val Leu Glu Ala Ile Leu Lys His Gly Asp Val Glu Glu Ala Val 625 630 635 640 Arg Ile Val Lys Glu Val Thr Glu Lys Leu Ser Arg Tyr Glu Val Pro 645 650 655 Pro Glu Lys Leu Val Ile Tyr Glu Gln Ile Thr Arg Asn Leu Arg Asp 660 665 670 Tyr Arg Ala Thr Gly Pro His Val Ala Val Ala Lys Arg Leu Ala Ala 675 680 685 Arg Gly Ile Lys Ile Arg Pro Gly Thr Val Ile Ser Tyr Ile Val Leu 690 695 700 Lys Gly Pro Gly Arg Val Gly Asp Arg Ala Ile Pro Phe Asp Glu Phe 705 710 715 720 Asp Pro Ala Lys His Arg Tyr Asp Ala Glu Tyr Tyr Ile Glu Asn Gln 725 730 735 Val Leu Pro Ala Val Glu Arg Ile Leu Arg Ala Phe Gly Tyr Arg Lys 740 745 750 Glu Asp Leu Arg Tyr Gln Lys Thr Lys Gln Ala Gly Leu Gly Ala Trp 755 760 765 Leu Lys Pro Lys Thr 770 <210> 46 <211> 774 <212> PRT <213> Thermococcus sibiricus <400> 46 Met Ile Leu Gly Ala Asp Tyr Ile Thr Lys Asp Gly Lys Pro Ile Val 1 5 10 15 Arg Ile Phe Lys Lys Glu Asn Gly Glu Phe Lys Ile Glu Leu Asp Pro 20 25 30 His Phe Gln Pro Tyr Ile Tyr Ala Leu Leu Ser Glu Asp Ser Ala Ile 35 40 45 Asp Glu Ile Lys Gln Ile Lys Gly Glu Arg His Gly Lys Thr Val Arg 50 55 60 Ile Val Asp Ala Val Lys Val Glu Lys Lys Phe Leu Lys Lys Pro Val 65 70 75 80 Lys Val Trp Lys Leu Ile Leu Glu His Pro Gln Asp Val Pro Ala Ile 85 90 95 Arg Asn Lys Ile Arg Glu His Pro Ala Val Gln Asp Ile Tyr Glu Tyr 100 105 110 Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Asn Gly Leu Ile Pro 115 120 125 Met Glu Gly Asp Glu Glu Leu Lys Met Leu Ala Phe Asp Ile Glu Thr 130 135 140 Phe Tyr His Glu Gly Asp Glu Phe Gly Lys Gly Glu Ile Ile Met Ile 145 150 155 160 Ser Tyr Ala Asp Glu Glu Gly Ala Arg Val Ile Thr Trp Lys Asn Ile 165 170 175 Asp Leu Pro Tyr Val Asp Val Val Ser Asn Glu Arg Glu Met Ile Lys 180 185 190 Arg Phe Ile Gln Ile Ile Lys Glu Lys Asp Pro Asp Val Ile Ile Thr 195 200 205 Tyr Asn Gly Asp Asn Phe Asp Leu Pro Tyr Leu Ile Lys Arg Ala Glu 210 215 220 Lys Leu Gly Leu Arg Leu Ile Leu Ser Arg Asp Asn Glu Asn Pro Val 225 230 235 240 Pro Lys Ile Gln Arg Met Gly Asn Ser Phe Ala Val Glu Ile Lys Gly 245 250 255 Arg Ile His Phe Asp Leu Phe Pro Val Val Lys Arg Ala Val Asn Leu 260 265 270 Pro Thr Tyr Thr Leu Glu Ala Val Tyr Glu Thr Val Leu Gly Lys His 275 280 285 Lys Ser Lys Leu Gly Ala Glu Glu Ile Ala Ala Ile Trp Glu Thr Glu 290 295 300 Glu Gly Leu Lys Lys Leu Ala Gln Tyr Ser Met Glu Asp Ala Lys Ala 305 310 315 320 Thr Tyr Glu Leu Gly Arg Glu Phe Phe Pro Met Glu Val Glu Leu Ala 325 330 335 Lys Leu Ile Gly Gln Ser Val Trp Asp Val Ser Arg Ser Ser Thr Gly 340 345 350 Asn Leu Val Glu Trp Tyr Met Leu Arg Val Ala Tyr Glu Arg Asn Glu 355 360 365 Leu Ala Pro Asn Arg Pro Ser Asp Glu Glu Tyr Lys Arg Arg Leu Arg 370 375 380 Thr Thr Tyr Leu Gly Gly Tyr Val Lys Glu Pro Glu Arg Gly Leu Trp 385 390 395 400 Glu Asn Ile Ile Tyr Leu Asp Phe Arg Ser Leu Tyr Pro Ser Ile Ile 405 410 415 Val Thr His Asn Val Ser Pro Asp Thr Leu Glu Arg Lys Gly Cys Gln 420 425 430 Asn Tyr Asp Val Ala Pro Ile Val Gly Tyr Lys Phe Cys Lys Asp Phe 435 440 445 Ser Gly Phe and Pro Ser and Glu Asp and Glu Thr Arg Gln 450 455 460 Lys Ile Lys Glu Met Lys Ser Thr Ile Asp Pro Ile Lys Lys Lys 465 470 475 480 Met Leo Asp Tyr Arg Gln Arg Ala Val Lys Leo Ala Asn Ser Tyr 485,490,495 Tyr Gly Tyr Met Gly Tyr Pro Lys Ala Arg Trp Tyr Ser Lys Glu Cys 500 505 510 Only Glu Ser Val Thr Only Trp Gly Arg His Tyr Ile Glu Met Thr Ile 515,520,525 Arg Glu Ile Glu Glu Lys Phe Gly Phe Lys Val Leu Tyr Ala Asp Thr 530 535 540 Asp Gly Phe Tyr Ala Thr Ile Pro Gly Ala Asp Pro Glu Thr Ile Lys 545 550 555 560 Lys Lys Ala Lys Glu Phe Leu Asn Tyr Ile Asn Ser Lys Leu Pro Gly 565,570,575 Leu Leu Glu Leu Glu Tyr Glu Gly Phe Tyr Leu Arg Gly Phe Phe Val 580 585 590 Thr Lys Lys Arg Tyr Ala Val Ile Asp Glu Glu Glu Lys Ile Thr Thr 595 600 605 Arg Gly Leu Glu Val Val Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu 610 615 620 Thr Gln Ala Arg Val Leu Glu Ala Ile Leu Arg Glu Gly Ser Val Glu 625 630 635 640 Lys Ala Val Glu Ile Val Lys Glu Val Val Glu Ala Ile Thr Lys Tyr 645 650 655 Lys Val Pro Leu Glu Lys Leu Ile Ile His Glu Gln Ile Thr Arg Glu 660 665 670 Leu Arg Asp Tyr Lys Ala Val Gly Pro His Val Ala Ile Ala Lys Arg 675 680 685 Leu Ala Ala Lys Gly Ile Lys Ile Lys Pro Gly Thr Ile Ile Ser Tyr 690 695 700 Ile Val Leu Arg Gly Ser Gly Lys Ile Ser Asp Arg Val Val Leu Leu 705 710 715 720 Thr Glu Tyr Asp Pro Arg Lys His Lys Tyr Asp Pro Asp Tyr Tyr Ile 725 730 735 Glu Asn Gln Val Leu Pro Ala Val Leu Arg Ile Leu Glu Ala Phe Gly 740 745 750 Tyr Arg Lys Glu Asp Leu Lys Tyr Gln Ser Ser Lys Gln Thr Gly Leu 755 760 765 Glu Ser Trp Leu Lys Lys 770 <210> 47 <211> 775 <212> PRT <213> Pyrococcus glycovorans <400> 47 Met Ile Leu Asp Ala Asp Tyr Ile Thr Glu Asp Gly Lys Pro Ile Ile 1 5 10 15 Arg Ile Phe Lys Lys Glu Asn Gly Glu Phe Lys Val Glu Tyr Asp Arg 20 25 30 Asn Phe Arg Pro Tyr Ile Tyr Ala Leu Leu Lys Asp Asp Ser Gln Ile 35 40 45 Asp Glu Val Lys Lys Ile Thr Ala Glu Arg His Gly Lys Ile Val Arg 50 55 60 Ile Val Asp Val Glu Lys Val Lys Lys Lys Phe Leu Gly Arg Pro Ile 65 70 75 80 Glu Val Trp Lys Leu Tyr Phe Glu His Pro Gln Asp Val Pro Ala Ile 85 90 95 Arg Asp Lys Ile Arg Glu His Pro Ala Val Val Asp Ile Phe Glu Tyr 100 105 110 Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Lys Gly Leu Ile Pro 115 120 125 Met Glu Gly Asp Glu Glu Leu Lys Leu Leu Ala Phe Asp Ile Glu Thr 130 135 140 Leu Tyr His Glu Gly Glu Glu Phe Ala Lys Gly Pro Ile Ile Met Ile 145 150 155 160 Ser Tyr Ala Asp Glu Glu Gly Ala Lys Val Ile Thr Trp Lys Lys Val 165 170 175 Asp Leu Pro Tyr Val Glu Val Val Ser Ser Glu Arg Glu Met Ile Lys 180 185 190 Arg Phe Leu Lys Val Ile Arg Glu Lys Asp Pro Asp Val Ile Ile Thr 195 200 205 Tyr Asn Gly Asp Ser Phe Asp Leu Pro Tyr Leu Val Lys Arg Ala Glu 210 215 220 Lys Leu Gly Ile Lys Leu Pro Leu Gly Arg Asp Gly Ser Glu Pro Lys 225 230 235 240 Met Gln Arg Leu Gly Asp Met Thr Ala Val Glu Ile Lys Gly Arg Ile 245 250 255 His Phe Asp Leu Tyr His Val Ile Arg Arg Thr Ile Asn Leu Pro Thr 260 265 270 Tyr Thr Leu Glu Ala Val Tyr Glu Ala Ile Phe Gly Lys Pro Lys Glu 275 280 285 Lys Val Tyr Ala His Glu Ile Ala Glu Ala Trp Glu Thr Gly Lys Gly 290 295 300 Leu Glu Arg Val Ala Lys Tyr Ser Met Glu Asp Ala Lys Val Thr Tyr 305 310 315 320 Glu Leu Gly Arg Glu Phe Phe Pro Met Glu Ala Gln Leu Ser Arg Leu 325 330 335 Val Gly Gln Pro Leu Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu 340 345 350 Val Glu Trp Tyr Leu Leu Arg Lys Ala Tyr Glu Arg Asn Glu Leu Ala 355 360 365 Pro Asn Lys Pro Asp Glu Arg Glu Tyr Glu Arg Arg Leu Arg Glu Ser 370 375 380 Tyr Ala Gly Gly Tyr Val Lys Glu Pro Glu Lys Gly Leu Trp Glu Gly 385 390 395 400 Leu Val Ser Leu Asp Phe Arg Ser Leu Tyr Pro Ser Ile Ile Ile Thr 405 410 415 His Asn Will Be Pro Asp Thr Leu Asn Arg Glu Gly Cys Met Glu Tyr 420 425 430 Asp Val Ala Pro Glu Val Lys His Lys Phe Cys Lys Asp Phe Pro Gly 435 440 445 Phe Ile Pro Ser Leu Leu Lys Arg Leu Leu Leu Asp Glu Arg Gln Glu Ile 450 455 460 Lys Arg Arg Met Lys Ala Ser Lys Asp Pro Ile Glu Lys Met Leu 465 470 475 480 Asp Tyr Arg Gln Arg Only With Lys And Leu With Asn Ser Tyr Gly 485,490,495 Tyr Tyr Gly Tyr Ala Lys Ala Arg Trp Tyr Cys Lys Glu Cys Ala Glu 500 505 510 Ser Val Thr Ala Trp Gly Arg Glu Tyr Ile Glu Phe Val Arg Lys Glu 515,520,525 Leu Glu Glu Lys Phe Gly Phe Lys Val Leu Tyr Ile Asp Thr Asp Gly 530 535 540 Leu Tyr Ala Thr Ile Pro Gly Ala Lys Pro Glu Glu Ile Lys Arg Lys 545 550 555 560 Leu Glu Phe Val Glu Tyr Ile Asn Leu Leu Pro Gly Leu 565 570 575 Glu Leu Glu Tyr Glu Gly Phe Tyr Val Arg Gly Phe Phe Val Thr Lys 580 585 590 Lys Lys Tyr Ala Leu Ile Asp Glu Glu Gly Lys Ile Ile Thr Arg Gly 595 600 605 Leu Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu Thr Gln 610 615 620 Ala Lys Val Leu Glu Ala Ile Leu Lys His Gly Asn Val Glu Glu Ala 625 630 635 640 Val Lys Ile Val Lys Glu Val Thr Glu Lys Leu Ser Lys Tyr Glu Ile 645 650 655 Pro Pro Glu Lys Leu Val Ile Tyr Glu Gln Ile Thr Arg Pro Leu His 660 665 670 Glu Tyr Lys Ala Ile Gly Pro His Val Ala Val Ala Lys Arg Leu Ala 675 680 685 Ala Arg Gly Val Lys Val Arg Pro Gly Met Val Ile Gly Tyr Ile Val 690 695 700 Leu Arg Gly Asp Gly Pro Ile Ser Lys Arg Ala Ile Leu Ala Glu Glu 705 710 715 720 Phe Asp Pro Arg Lys His Lys Tyr Asp Ala Glu Tyr Ile Glu Asn 725 730 735 Gln Val Leu Pro Ala Val Leu Arg Ile Leu Glu Ala Phe Gly Tyr Arg 740,745,750 Lys Glu Asp Leu Arg Trp Gln Lys Thr Lys Gln Thr Gly Leu Thr Ala 755,760,765 Trp Leu Asn Val Lys Lys Lys 770,775 <210> 48 <211> 775 <212> PRT <213> Pyrococcus sp. NA2 <400> 48 With Leu Asp Alone Asp Tyr Isolation Thr Glu Asp Gly Lys Pro Isolation 1 5 10 15 Arg Leu Phe Lys Glu Asn Gly Arg Phe Lys Val Glu Tyr Asp Arg 20 25 30 Asn Phe Arg Pro Tyr Ile Tyr Ala Leu Leu Lys Asp Asp Ser Ala Ile 35 40 45 Asp Asp Val Arg Lys Ile Thr Ser Glu Arg His Gly Lys Val Val Arg 50 55 60 Val Ile Asp Val Glu Lys Val Lys Lys Lys Phe Leu Gly Arg Pro Ile 65 70 75 80 Glu Val Trp Lys Leu Tyr Phe Glu His Pro Gln Asp Val Pro Ala Met 85 90 95 Arg Asp Lys Ile Arg Glu His Pro Ala Val Ile Asp Ile Phe Glu Tyr 100 105 110 Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Lys Gly Leu Ile Pro 115 120 125 Met Glu Gly Asn Glu Glu Leu Thr Phe Leu Ala Val Asp Ile Glu Thr 130 135 140 Leu Tyr His Glu Gly Glu Glu Phe Gly Lys Gly Pro Ile Ile Met Ile 145 150 155 160 Ser Tyr Ala Asp Glu Glu Gly Ala Lys Val Ile Thr Trp Lys Lys Ile 165 170 175 Asp Leu Pro Tyr Val Glu Val Val Ala Asn Glu Arg Glu Met Ile Lys 180 185 190 Arg Leu Ile Lys Val Ile Arg Glu Lys Asp Pro Asp Val Ile Ile Thr 195 200 205 Tyr Asn Gly Asp Asn Phe Asp Phe Pro Tyr Leu Leu Lys Arg Ala Glu 210 215 220 Lys Leu Gly Met Lys Leu Pro Leu Gly Arg Asp Asn Ser Glu Pro Lys 225 230 235 240 Met Gln Arg Leu Gly Asp Ser Leu Ala Val Glu Ile Lys Gly Arg Ile 245 250 255 His Phe Asp Leu Phe Pro Val Ile Arg Arg Thr Ile Asn Leu Pro Thr 260 265 270 Tyr Thr Leu Glu Ala Val Tyr Glu Ala Ile Phe Gly Lys Gln Lys Glu 275 280 285 Lys Val Tyr Pro His Glu Ile Ala Glu Ala Trp Glu Thr Gly Lys Gly 290 295 300 Leu Glu Arg Val Ala Lys Tyr Ser Met Glu Asp Ala Lys Val Thr Tyr 305 310 315 320 Glu Leu Gly Lys Glu Phe Phe Pro Met Glu Ala Gln Leu Ala Arg Leu 325 330 335 Val Gly Gln Pro Leu Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu 340 345 350 Val Glu Trp Tyr Leu Leu Arg Lys Ala Tyr Glu Arg Asn Glu Leu Ala 355 360 365 Pro Asn Lys Pro Asp Glu Arg Glu Tyr Glu Arg Arg Leu Arg Glu Ser 370 375 380 Tyr Glu Gly Gly Tyr Val Lys Glu Pro Glu Arg Gly Leu Trp Glu Gly 385 390 395 400 Ile Val Ser Leu Asp Phe Arg Ser Leu Tyr Pro Ser Ile Ile Ile Thr 405 410 415 His Asn Val Ser Pro Asp Thr Leu Asn Lys Glu Gly Cys Gly Glu Tyr 420 425 430 Asp Glu Ala Pro Glu Val Gly His Arg Phe Cys Lys Asp Phe Pro Gly 435 440 445 Phe Ile Pro Ser Leu Leu Gly Ser Leu Leu Glu Glu Arg Gln Lys Ile 450 455 460 Lys Lys Arg Met Lys Glu Ser Lys Asp Pro Val Glu Arg Lys Leu Leu 465 470 475 480 Asp Tyr Arg Gln Arg Ala Ile Lys Ile Leu Ala Asn Ser Phe Tyr Gly 485 490 495 Tyr Tyr Gly Tyr Ala Lys Ala Arg Trp Tyr Cys Lys Glu Cys Ala Glu 500 505 510 Ser Val Thr Ala Trp Gly Arg Gln Tyr Ile Glu Leu Val Arg Arg Glu 515 520 525 Leu Glu Glu Arg Gly Phe Lys Val Leu Tyr Ile Asp Thr Asp Gly Leu 530 535 540 Tyr Ala Thr Ile Pro Gly Glu Lys Asn Trp Glu Glu Ile Lys Arg Arg 545 550 555 560 Ala Leu Glu Phe Val Asn Tyr Ile Asn Ser Lys Leu Pro Gly Ile Leu 565 570 575 Glu Leu Glu Tyr Glu Gly Phe Tyr Thr Arg Gly Phe Phe Val Thr Lys 580 585 590 Lys Lys Tyr Ala Leu Ile Asp Glu Glu Gly Lys Ile Val Thr Arg Gly 595 600 605 Leu Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu Thr Gln 610 615 620 Ala Lys Val Leu Glu Ala Ile Leu Lys His Gly Asn Val Glu Glu Ala 625 630 635 640 Val Lys Ile Val Lys Glu Val Thr Glu Lys Leu Ser Asn Tyr Glu Ile 645 650 655 Pro Val Glu Lys Leu Val Ile Tyr Glu Gln Ile Thr Arg Pro Leu Asn 660 665 670 Glu Tyr Lys Ala Ile Gly Pro His Val Ala Val Ala Lys Arg Leu Ala 675 680 685 Ala Lys Gly Ile Lys Ile Lys Pro Gly Met Val Ile Gly Tyr Val Val 690 695 700 Leu Arg Gly Asp Gly Pro Isolation Ser Lys Arg Ala Ile Ile Ala Glu Glu 705 710 715 720 Phe Asp Gly Lys His Lys Tyr Asp Ala Glu Tyr Tyr Ile Glu Asn 725 730 735 Gln Val Leu Pro Ala Val Glu Arg Ile Leu Lys Ala Phe Gly Tyr Lys 740,745,750 Arg Glu Asp Leu Arg Trp Gln Lys Thr Lys Gln Val Gly Leu Gly Ala 755,760,765 About Leu Lys Val Lys Ser 770,775 <210> 49 <211> 785 <212> PRT <213> Ferroglobus placidus <400> 49 Met Glu Gly Trp Leu Leu Asp Ala Asp Tyr Ile Thr Ala Glu Asp Gly 1 5 10 15 Arg Ala Val Val Arg Leu Trp Cys Lys Asp Phe Asp Gly Asn Thr Phe 20 25 30 Val Val Tyr Asp Arg Asn Phe Gln Pro Tyr Phe Tyr Ala Phe Lys Asn 35 40 45 Gly Leu Ser Lys Glu Asp Ile Glu Lys Ile Val Val Lys Ser Arg Glu 50 55 60 Gly Val Ile Lys Pro Phe Lys Val Glu Glu Val Arg Arg Lys Val Phe 65 70 75 80 Gly Lys Glu Val Glu Val Phe Lys Ile Tyr Ala Tyr His Pro Gln His 85 90 95 Val Pro Lys Leu Arg Glu Glu Leu Lys Lys Ile Thr Glu Val Arg Glu 100 105 110 Ala Asp Ile Pro Phe Ala Tyr Arg Tyr Leu Ile Asp Lys Asp Leu Ala 115 120 125 Cys Met Asp Gly Ile Arg Val Glu Gly Lys Val Arg Glu Glu Arg Gly 130 135 140 Leu Lys Val Ile Asp Ala Glu His Val Glu Arg Phe Glu Ile Pro Leu 145 150 155 160 Pro Glu Pro Lys Val Leu Ala Phe Asp Cys Glu Met Leu Thr Glu Leu 165 170 175 Gly Met Pro Asp Pro Glu Lys Asp Lys Ile Ile Ile Ile Gly Val Lys 180 185 190 Cys Gly Asp Phe Glu Glu Ile Ile Thr Gly Asn Glu Arg Glu Ile Leu 195 200 205 Leu Arg Phe Val Glu Ile Ile Lys Glu Gln Asp Pro Asp Val Ile Val 210 215 220 Gly Tyr Asn Gln Asp Asn Phe Asp Trp Pro Tyr Ile Arg Lys Arg Ala 225 230 235 240 Glu Lys Leu Ser Val Lys Leu Asn Ile Gly Arg Asp Gly Ser Glu Ile 245 250 255 Ser Phe Arg Gly Gly Arg Pro Lys Ile Ala Gly Arg Leu Asn Val Asp 260 265 270 Leu Tyr Asp Ile Ala Met Lys Leu Asp Val Lys Val Lys Thr Leu Glu 275 280 285 Asn Val Ala Glu Phe Leu Gly Arg Lys Val Glu Leu Ala Asp Ile Glu 290 295 300 Ala Lys Asp Ile Tyr Lys Arg Trp Thr Ser Gly Asp Lys Glu Ser Val 305 310 315 320 Leu Lys Tyr Ser Lys Gln Asp Val Leu Asn Thr Tyr Phe Ile Ala Glu 325 330 335 Glu Leu Leu Pro Met His Tyr Glu Leu Ser Arg Met Ile Arg Ile Pro 340 345 350 Thr Asp Asp Val Ala Arg Ile Gly Arg Gly Lys Gln Val Asp Trp Phe 355 360 365 Leu Leu Ser Glu Ala Tyr Lys Ile Gly Glu Ile Ala Pro Asn Pro Ala 370 375 380 Glu Val Glu Glu Ser Tyr Glu Gly Ala Phe Val Leu Glu Pro Ser Arg 385 390 395 400 Gly Leu His Lys Asn Val Val Cys Leu Asp Phe Ala Ser Met Tyr Pro 405 410 415 Ser Ile Met Ile Ala Tyr Asn Ile Ser Pro Asp Thr Tyr Val Phe Gly 420 425 430 Lys Cys Asp Asp Cys Tyr Val Ala Pro Glu Val Gly His Lys Phe Arg 435 440 445 Lys His Pro Asp Gly Phe Phe Lys Arg Ile Leu Lys Met Leu Ile Glu 450 455 460 Lys Arg Arg Glu Ile Lys Asn Gln Met Lys Ser Leu Asp Arg Asn Ser 465 470 475 480 Arg Glu Tyr Leu Leu Leu Asn Ile Lys Gln Gln Thr Leu Lys Ile Leu 485 490 495 Thr Asn Ser Phe Tyr Gly Tyr Thr Gly Trp Ser Gly Ala Arg Trp Tyr 500 505 510 Cys Arg Gln Cys Ala Glu Ala Thr Thr Ala Trp Gly Arg His Leu Ile 515 520 525 Lys Ser Ala Val Glu Ile Ala Lys Lys Leu Gly Phe Glu Val Leu Tyr 530 535 540 Gly Asp Thr Asp Ser Ile Phe Val Lys Lys Gly Asn Leu Ser Leu Glu 545 550 555 560 Lys Ile Arg Gly Glu Val Glu Lys Leu Ile Glu Glu Ile Ser Glu Lys 565 570 575 Phe Pro Val Gln Ile Glu Val Asp Glu Tyr Tyr Lys Thr Ile Phe Phe 580 585 590 Val Glu Lys Lys Arg Tyr Ala Gly Leu Thr Glu Asp Gly Ile Leu Val 595 600 605 Val Lys Gly Leu Glu Val Arg Arg Gly Asp Trp Cys Glu Leu Ala Lys 610 615 620 Glu Val Gln Lys Lys Val Ile Glu Ile Ile Leu Lys Glu Glu Asn Pro 625 630 635 640 Glu Lys Ala Ala Glu Tyr Val Arg Lys Val Ile Asn Asp Ile Lys Ser 645 650 655 Gly Lys Val Lys Leu Glu Asp Val Val Ile Tyr Lys Gly Leu Thr Lys 660 665 670 Arg Pro Asp Lys Tyr Glu Ser Lys Gln Ala His Val Lys Ala Ala Leu 675 680 685 Arg Ala Met Glu Leu Gly Ile Val Tyr Asn Val Gly Ser Lys Val Gly 690 695 700 Phe Val Val Val Glu Gly Ala Gly Asn Val Gly Asp Arg Ala Tyr Pro 705 710 715 720 Ile Asp Leu Ile Glu Glu Phe Asp Gly Glu Asn Leu Val Ile Arg Thr 725 730 735 Arg Ser Gly Ser Ile Val Lys Lys Leu Asp Lys Asp Tyr Tyr Ile Asn 740 745 750 His Gln Ile Ile Pro Ser Val Leu Arg Ile Leu Glu Arg Phe Gly Tyr 755 760 765 Asn Glu Ala Ser Leu Lys Gly Ala Thr Gln Lys Thr Leu Asp Ala Phe 770 775 780 Trp 785 <210> 50 <211> 775 <212> PRT <213> Palaeococcus ferrophilus <400> 50 Met Ile Leu Asp Ala Asp Tyr Ile Thr Glu Asn Gly Lys Pro Val Val 1 5 10 15 Arg Ile Phe Lys Lys Glu Asn Gly Glu Phe Lys Val Glu Tyr Asp Arg 20 25 30 Asn Phe Glu Pro Tyr Ile Tyr Ala Leu Leu Lys Asp Asp Ser Ala Ile 35 40 45 Glu Glu Ile Lys Lys Ile Thr Ala Glu Arg His Gly Thr Val Val Arg 50 55 60 Ile Thr Lys Ala Glu Lys Val Glu Arg Lys Phe Leu Gly Arg Pro Val 65 70 75 80 Glu Val Trp Lys Leu Tyr Phe Thr His Pro Gln Asp Val Pro Ala Ile 85 90 95 Arg Asp Lys Ile Arg Ser His Pro Ala Val Val Asp Ile Tyr Glu Tyr 100 105 110 Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Lys Gly Leu Val Pro 115 120 125 Met Glu Gly Asp Glu Glu Leu Lys Met Leu Ala Phe Asp Ile Glu Thr 130 135 140 Leu Tyr His Glu Gly Glu Glu Phe Ala Glu Gly Pro Ile Leu Met Ile 145 150 155 160 Ser Tyr Ala Asp Glu Ser Glu Ala Arg Val Ile Thr Trp Lys Lys Val 165 170 175 Asp Leu Pro Tyr Val Asp Ala Val Ser Thr Glu Lys Asp Met Ile Lys 180 185 190 Ala Phe Leu Arg Val Val Lys Glu Lys Asp Pro Asp Val Leu Ile Thr 195 200 205 Tyr Asn Gly Asp Asn Phe Asp Phe Ala Tyr Leu Lys Lys Arg Cys Glu 210 215 220 Lys Leu Gly Val Lys Phe Ile Leu Gly Arg Asp Gly Ser Glu Pro Lys 225 230 235 240 Ile Gln Arg Met Gly Asp Arg Phe Ala Val Asp Val Lys Gly Arg Ile 245 250 255 His Phe Asp Leu Tyr Pro Val Ile Arg Arg Thr Ile Asn Leu Pro Thr 260 265 270 Tyr Thr Leu Glu Ala Val Tyr Glu Ala Ile Phe Gly Arg Pro Lys Glu 275 280 285 Lys Val Tyr Ala Glu Glu Ile Ala Gln Ala Trp Glu Thr Asn Glu Gly 290 295 300 Leu Glu Arg Val Ala Arg Tyr Ser Met Glu Asp Ala Lys Val Thr Tyr 305 310 315 320 Glu Leu Gly Lys Glu Phe Phe Pro Met Glu Ala Gln Leu Ser Arg Leu 325 330 335 Ile Gly Gln Pro Leu Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu 340 345 350 Val Glu Trp Phe Leu Leu Arg Lys Ala Tyr Glu Arg Asn Glu Leu Ala 355 360 365 Pro Asn Lys Pro Ser Gly Arg Glu Tyr Asp Glu Arg Arg Gly Gly Tyr 370 375 380 Ala Gly Gly Tyr Val Lys Glu Pro Glu Lys Gly Leu Trp Glu Asn Ile 385 390 395 400 Val Tyr Leu Asp Tyr Lys Ser Leu Tyr Pro Ser Ile Ile Ile Thr His 405 410 415 Asn Val Ser Pro Asp Thr Leu Asn Arg Glu Gly Cys Lys Glu Tyr Asp 420 425 430 Val Ala Pro Gln Val Gly His Arg Phe Cys Lys Asp Phe Pro Gly Phe 435 440 445 Ile Pro Ser Leu Leu Gly Asp Leu Leu Glu Glu Arg Gln Lys Ile Lys 450 455 460 Arg Lys Met Lys Ala Thr Ile Asp Pro Ile Glu Arg Arg Leu Leu Asp 465 470 475 480 Tyr Arg Gln Arg Ala Ile Lys Ile Leu Ala Asn Ser Tyr Tyr Gly Tyr 485 490 495 Tyr Gly Tyr Ala Arg Ala Arg Trp Tyr Cys Lys Glu Cys Ala Glu Ser 500 505 510 Val Thr Ala Trp Gly Arg Glu Tyr Ile Glu Met Ser Ile Arg Glu Ile 515 520 525 Glu Glu Lys Tyr Gly Phe Lys Val Leu Tyr Ala Asp Thr Asp Gly Phe 530 535 540 His Ala Thr Ile Pro Gly Glu Asp Ala Glu Thr Ile Lys Lys Lys Ala 545 550 555 560 Met Glu Phe Leu Lys Tyr Ile Asn Ser Lys Leu Pro Gly Ala Leu Glu 565 570 575 Leu Glu Tyr Glu Gly Phe Tyr Arg Arg Gly Phe Phe Val Thr Lys Lys 580 585 590 Lys Tyr Ala Val Ile Asp Glu Glu Gly Lys Ile Thr Thr Arg Gly Leu 595 600 605 Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu Thr Gln Ala 610 615 620 Arg Val Leu Glu Ala Leu Leu Lys Asp Gly Asn Val Glu Glu Ala Val 625 630 635 640 Ser Ile Val Lys Glu Val Thr Glu Lys Leu Ser Lys Tyr Glu Val Pro 645 650 655 Pro Glu Lys Leu Val Ile His Glu Gln Ile Thr Arg Glu Leu Lys Asp 660 665 670 Tyr Lys Ala Thr Gly Pro His Val Ala Ile Ala Lys Arg Leu Ala Ala 675 680 685 Arg Gly Val Lys Ile Arg Pro Gly Thr Val Ile Ser Tyr Ile Val Leu 690 695 700 Lys Gly Ser Gly Arg Ile Gly Asp Arg Ala Ile Pro Phe Asp Glu Phe 705 710 715 720 Asp Pro Ala Lys His Arg Tyr Asp Ala Glu Tyr Tyr Ile Glu Asn Gln 725 730 735 Val Leu Pro Ala Val Glu Arg Ile Leu Lys Ala Phe Gly Tyr Arg Lys 740 745 750 Glu Asp Leu Arg Tyr Gln Lys Thr Arg Gln Val Gly Leu Gly Ala Trp 755 760 765 Leu Lys Pro Lys Gly Lys Lys 770 775 <210> 51 <211> 775 <212> PRT <213> Thermococcus gammatolerans <400> 51 Met Ile Leu Asp Thr Asp Tyr Ile Thr Glu Asn Gly Lys Pro Val Ile 1 5 10 15 Arg Val Phe Lys Lys Glu Asn Gly Glu Phe Lys Ile Glu Tyr Asp Arg 20 25 30 Thr Phe Glu Pro Tyr Phe Tyr Ala Leu Leu Lys Asp Asp Ser Ala Ile 35 40 45 Glu Asp Val Lys Lys Val Thr Ala Lys Arg His Gly Ala Val Val Lys 50 55 60 Val Lys Arg Ala Glu Lys Val Gln Arg Lys Phe Leu Gly Arg Pro Ile 65 70 75 80 Glu Val Trp Lys Leu Tyr Phe Thr His Pro Gln Asp Val Pro Ala Ile 85 90 95 Arg Asp Lys Ile Arg Ala His Pro Ala Val Val Asp Ile Tyr Glu Tyr 100 105 110 Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Lys Gly Leu Ile Pro 115 120 125 Met Glu Gly Asp Glu Glu Leu Arg Met Leu Ala Phe Asp Ile Glu Thr 130 135 140 Leu Tyr His Glu Gly Glu Glu Phe Gly Thr Gly Pro Ile Leu Met Ile 145 150 155 160 Ser Tyr Ala Asp Glu Asn Glu Ala Arg Val Ile Thr Trp Lys Lys Ile 165 170 175 Asp Leu Pro Tyr Val Asp Val Val Ser Thr Glu Lys Glu Met Ile Lys 180 185 190 Arg Phe Leu Arg Val Val Lys Glu Lys Asp Pro Asp Val Leu Ile Thr 195 200 205 Tyr Asn Gly Asp Asn Phe Asp Phe Ala Tyr Leu Lys Lys Arg Cys Glu 210 215 220 Lys Leu Gly Ile Lys Phe Thr Leu Gly Arg Asp Gly Ser Glu Pro Lys 225 230 235 240 Ile Gln Arg Met Gly Asp Arg Phe Ala Val Glu Val Lys Gly Arg Ile 245 250 255 His Phe Asp Leu Tyr Pro Val Ile Arg Arg Thr Ile Asn Leu Pro Thr 260 265 270 Tyr Thr Leu Glu Ala Val Tyr Glu Ala Val Phe Gly Lys Pro Lys Glu 275 280 285 Lys Val Tyr Ala Glu Glu Ile Thr Glu Ala Trp Glu Ser Gly Glu Gly 290 295 300 Leu Glu Arg Val Ala Arg Tyr Ser Met Glu Asp Ala Lys Val Thr Tyr 305 310 315 320 Glu Leu Gly Arg Glu Phe Phe Pro Met Glu Ala Gln Leu Ser Arg Leu 325 330 335 Ile Gly Gln Ser Leu Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu 340 345 350 Val Glu Trp Phe Leu Leu Arg Lys Ala Tyr Glu Arg Asn Glu Leu Ala 355 360 365 Pro Asn Lys Pro Asp Glu Arg Glu Leu Ala Arg Arg Arg Glu Ser Tyr 370 375 380 Ala Gly Gly Tyr Val Lys Glu Pro Glu Arg Gly Leu Trp Asp Asn Ile 385 390 395 400 Val Tyr Leu Asp Phe Arg Ser Leu Tyr Pro Ser Ile Ile Thr His 405 410 415 Asn Will Be Pro Asp Thr To Asn Arg Glu Gly Cys Lys Glu Tyr Asp 420 425 430 Val Ala Pro Glu Val Gly His Lys Phe Cys Lys Asp Phe Pro Gly Phe 435 440 445 Pro Serr Ile Leu Gly Asp Leu Glu Glu Arg Gln Lys Ile Lys 450 455 460 Arg Lys Met Lys Ala Thr Ile Asp Pro Leu Glu Lys Lys Leu Leu Asp 465 470 475 480 Tyr Arg Gln Arg Only With Lys And Leu With Asn Ser Phe Tyr Gly Tyr 485,490,495 Tyr Gly Tyr Ala Lys Ala Arg Trp Tyr Cys Lys Glu Cys Ala Glu Ser 500 505 510 Val Thr Ala Trp Gly Arg Glu Tyr Ile Glu Thr Thr Ile Arg Glu Ile 515 520 525 Glu Glu Lys Phe Gly Phe Lys Val Leu Tyr Ala Asp Thr Asp Gly Phe 530 535 540 Phe Ala Thr Ile Pro Gly Ala Asp Ala Glu Thr Val Lys Lys Lys Ala 545 550 555 560 Lys Glu Phe Leu Lys Tyr Ile Asn Ala Lys Leu Pro Gly Leu Leu Glu 565 570 575 Leu Glu Tyr Glu Gly Phe Tyr Val Arg Gly Phe Phe Val Thr Lys Lys 580 585 590 Lys Tyr Ala Val Ile Asp Glu Glu Gly Lys Ile Thr Thr Arg Gly Leu 595 600 605 Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu Thr Gln Ala 610 615 620 Arg Val Leu Glu Ala Ile Leu Lys His Gly Asp Val Glu Glu Ala Val 625 630 635 640 Arg Ile Val Lys Glu Val Thr Glu Lys Leu Ser Lys Tyr Glu Val Pro 645 650 655 Pro Glu Lys Leu Val Ile His Glu Gln Ile Thr Arg Asp Leu Arg Asp 660 665 670 Tyr Lys Ala Thr Gly Pro His Val Ala Val Ala Lys Arg Leu Ala Ala 675 680 685 Lys Gly Val Lys Ile Arg Pro Gly Thr Val Ile Ser Tyr Ile Val Leu 690 695 700 Lys Gly Ser Gly Arg Ile Gly Asp Arg Ala Ile Pro Ala Asp Glu Phe 705 710 715 720 Asp Pro Thr Lys His Arg Tyr Asp Ala Glu Tyr Tyr Ile Glu Asn Gln 725 730 735 Val Leu Pro Ala Val Glu Arg Ile Leu Lys Ala Phe Gly Tyr Arg Lys 740 745 750 Glu Asp Leu Arg Tyr Gln Lys Thr Lys Gln Val Gly Leu Gly Ala Trp 755 760 765 Leu Lys Val Lys Gly Lys Lys 770 775 <210> 52 <211> 775 <212> PRT <213> Thermococcus celericrescens <400> 52 Met Ile Leu Asp Thr Asp Tyr Ile Thr Glu Asp Gly Lys Pro Val Ile 1 5 10 15 Arg Ile Phe Lys Lys Asp Asn Gly Glu Phe Lys Ile Glu Tyr Asp Arg 20 25 30 Asn Phe Glu Pro Tyr Ile Tyr Ala Leu Leu Arg Asp Asp Ser Ala Ile 35 40 45 Glu Asp Val Lys Lys Ile Thr Ala Glu Arg His Gly Arg Val Val Lys 50 55 60 Val Lys Arg Ala Glu Lys Val Lys Lys Lys Phe Leu Gly Arg Pro Val 65 70 75 80 Glu Val Trp Lys Leu Tyr Phe Thr Arg Pro Gln Asp Val Pro Ala Ile 85 90 95 Arg Asp Arg Ile Arg Ala His Pro Ala Val Val Asp Ile Tyr Glu Tyr 100 105 110 Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Lys Gly Ile Ile Pro 115 120 125 Met Glu Gly Asp Glu Glu Leu Lys Met Leu Ala Phe Asp Ile Glu Thr 130 135 140 Leu Tyr His Glu Gly Glu Glu Phe Ala Glu Gly Pro Ile Leu Met Ile 145 150 155 160 Ser Tyr Ala Asp Glu Asn Glu Ala Arg Val Ile Thr Trp Lys Lys Ile 165 170 175 Asp Leu Pro Phe Val Asp Val Val Ser Thr Glu Lys Glu Met Ile Lys 180 185 190 Arg Phe Leu Lys Val Ile Lys Glu Lys Asp Pro Asp Val Leu Ile Thr 195 200 205 Tyr Asn Gly Asp Asn Phe Asp Phe Ala Tyr Leu Lys Lys Arg Cys Glu 210 215 220 Lys Phe Gly Ile Lys Phe Thr Leu Gly Arg Asp Gly Ser Asp Pro Lys 225 230 235 240 Ile Gln Arg Met Gly Asp Arg Phe Ala Val Glu Val Lys Gly Arg Ile 245 250 255 His Phe Asp Leu Tyr Pro Val Ile Leu Arg Thr Val Asn Leu Pro Thr 260 265 270 Tyr Thr Leu Glu Ala Val Tyr Glu Ala Ile Phe Gly Thr Pro Lys Glu 275 280 285 Lys Val Tyr Pro Glu Glu Ile Thr Thr Ala Trp Glu Thr Gly Glu Gly 290 295 300 Leu Glu Arg Val Ala Arg Tyr Ser Met Glu Asp Ala Lys Val Thr Phe 305 310 315 320 Glu Leu Gly Arg Glu Phe Phe Pro Met Glu Ala Gln Leu Ser Arg Leu 325 330 335 Val Gly Gln Ser Phe Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu 340 345 350 Val Glu Trp Phe Leu Leu Arg Lys Ala Tyr Glu Arg Asn Glu Leu Ala 355 360 365 Pro Asn Lys Pro Asp Glu Arg Glu Leu Val Arg Arg Arg Asn Ser Tyr 370 375 380 Thr Gly Gly Tyr Val Lys Glu Pro Glu Arg Gly Leu Trp Asp Asn Ile 385 390 395 400 Val Tyr Leu Asp Phe Arg Ser Leu Tyr Pro Ser Ile Ile Thr His 405 410 415 Asn Will Be Pro Asp Thr To Asn Arg Glu Gly Cys Lys Glu Tyr Asp 420 425 430 Glu Ala Pro Gln Val Gly His Lys Phe Cys Lys Asp Phe Pro Gly Phe 435 440 445 Ile Pro Ser Let Gly Asn Let Asp Glu Arg Gln Lys Ile Lys 450 455 460 Lys Arg Met Lys Ala Thr Ile Asp Pro Leu Glu Lys Leu Leu Asp 465 470 475 480 Tyr Arg Gln Arg Only With Lys And Leu Only Asn Ser Tyr Gly Tyr 485,490,495 Tyr Ala Tyr Ala Arg Ala Arg Trp Tyr Cys Lys Glu Cys Ala Glu Ser 500 505 510 Val Thr Ala Trp Gly Arg Glu Tyr Ile Glu Met Ser Ile Arg Glu Ile 515 520 525 Glu Glu Lys Tyr Gly Phe Lys Val Leu Tyr Ala Asp Thr Asp Gly Phe 530 535 540 His Ala Thr Ile Pro Gly Ala Asp Ala Glu Thr Val Lys Lys Lys Ala 545 550 555 560 Met Glu Phe Leu Lys Tyr Ile Asn Ala Lys Leu Pro Gly Ala Leu Glu 565 570 575 Leu Glu Tyr Glu Gly Phe Tyr Lys Arg Gly Phe Phe Val Thr Lys Lys 580 585 590 Lys Tyr Ala Val Ile Asp Glu Glu Gly Lys Ile Thr Thr Arg Gly Leu 595 600 605 Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu Thr Gln Ala 610 615 620 Arg Val Leu Glu Ala Leu Leu Lys Asp Gly Asn Val Glu Glu Ala Val 625 630 635 640 Ser Ile Val Lys Glu Val Thr Glu Lys Leu Gly Lys Tyr Glu Val Pro 645 650 655 Pro Glu Lys Leu Val Ile His Glu Gln Ile Thr Arg Glu Leu Lys Asp 660 665 670 Tyr Lys Ala Thr Gly Pro His Val Ala Ile Ala Lys Arg Leu Ala Ala 675 680 685 Arg Gly Val Lys Ile Arg Pro Gly Thr Val Ile Ser Tyr Ile Val Leu 690 695 700 Lys Gly Ser Gly Arg Ile Gly Asp Arg Ala Ile Pro Phe Asp Glu Phe 705 710 715 720 Asp Pro Thr Lys His Arg Tyr Asp Ala Glu Tyr Tyr Ile Glu Asn Gln 725 730 735 Val Leu Pro Ala Val Glu Arg Ile Leu Lys Ala Phe Gly Tyr Arg Ala 740 745 750 Glu Asp Leu Arg Tyr Gln Lys Thr Arg Gln Val Gly Leu Gly Val Trp 755 760 765 Leu Gln Pro Lys Gly Lys Lys 770 775 <210> 53 <211> 784 <212> PRT <213> Vulcanisaeta distributa <400> 53 Met Glu Leu Ala Phe Trp Leu Leu Asp Ile Thr Tyr Gly Val Ile Gly 1 5 10 15 Asn Thr Pro Glu Leu Arg Leu Phe Gly Ile Thr Asp Asp Gly Lys Arg 20 25 30 Val Leu Val Leu Asp Arg Ser Phe Arg Pro Tyr Phe Tyr Val Ile Pro 35 40 45 Ser Gly Asp Val Asn Ala Val Phe Asn Asn Val Lys Arg Lys Leu Glu 50 55 60 Gly Lys Val Leu Asn Val Glu Val Ile Lys Arg Lys Met Phe Gly Asn 65 70 75 80 Glu Val Asp Ala Ile Arg Val Thr Ala Thr Ile Pro Glu Lys Val Arg 85 90 95 Glu Leu Arg Glu Leu Ala Ala Glu Val Pro Gly Val Glu Asp Val Leu 100 105 110 Glu Ala Asp Ile Arg Phe Ser Gln Arg Tyr Leu Leu Asp Met Gly Val 115 120 125 Lys Pro Ser Asn Trp Ile Val Val Asp Gln Cys Glu Glu Val Lys Gly 130 135 140 Asn Tyr Gln Val Asp Leu Val Cys Leu Ala Lys Ser Arg Pro Arg Met 145 150 155 160 Ile Glu Glu His Lys Leu Pro Ser Phe Arg Val Leu Ala Phe Asp Ile 165 170 175 Glu Val Tyr Asn Pro Arg Gly Met Pro Asn Pro Asp Arg Asp Pro Val 180 185 190 Ile Ile Ile Ser Thr Met Thr Lys Glu Asp Gly Val Lys Met Phe Val 195 200 205 Val Asp Asp Asn Lys Asn Asp Ala Lys Ile Ile Arg Glu Phe Leu Asp 210 215 220 Tyr Phe Arg Lys Tyr Asp Pro Asp Ile Val Val Gly Tyr Asn Asn Asn 225 230 235 240 Gly Phe Asp Trp Pro Tyr Leu Val Asn Arg Ser Ser Arg Val Gly Val 245 250 255 Arg Leu Ala Leu Ser Arg Met Gly Asn Pro Pro Glu Pro Ser Val Tyr 260 265 270 Gly His Trp Ser Ile Ile Gly Arg Ala Asn Val Asp Leu Tyr Asn Phe 275 280 285 Ile Glu Glu Ile Ser Glu Ile Lys Val Lys Ser Leu Asp Arg Ala Ala 290 295 300 Glu Phe Phe Gly Ile Met Lys Arg Ser Glu Arg Val Leu Ile Pro Gly 305 310 315 320 His Arg Ile His Glu Tyr Trp Asp Asp Lys Asn Lys Arg Asp Leu Leu 325 330 335 Leu Lys Tyr Ala Arg Asp Asp Val Val Ser Thr Tyr Gly Leu Ala Glu 340 345 350 Lys Leu Leu Pro Phe Ala Ile Gln Leu Ser Ser Ile Ser Gly Leu Pro 355 360 365 Leu Asp Gln Val Gly Ala Ala Ser Val Gly Ala Arg Val Glu Trp Met 370 375 380 Ile Phe Tyr Glu Ala Val Lys Arg Gly Glu Leu Ala Pro Asn Arg Glu 385 390 395 400 Glu Arg Pro Tyr Glu Thr Tyr Lys Gly Ala Val Val Leu Glu Pro Arg 405 410 415 Pro Gly Leu His Glu Asn Ile Ala Val Ile Asp Phe Ser Ser Met Tyr 420 425 430 Pro Ser Ile Met Met Lys Tyr Asn Val Ser Pro Asp Thr Leu Val Leu 435 440 445 Gly Asp Cys Gly Asp Cys Tyr Val Ala Pro Glu Val Asn Tyr Lys Phe 450 455 460 Arg Arg Ser Pro Glu Gly Leu Tyr Pro Gly Leu Leu Arg Ile Leu Val 465 470 475 480 Glu Ser Arg Arg Arg Val Arg Asp Leu Met Lys Lys Tyr Pro Glu Asn 485 490 495 Ser Pro Glu Trp Val Leu Leu Asn Glu Arg Gln Arg Ala Leu Lys Val 500 505 510 Met Ala Asn Ala Met Tyr Gly Tyr Cys Gly Trp Leu Gly Ala Arg Trp 515 520 525 Tyr Arg Arg Glu Val Ala Glu Ala Val Thr Ala Trp Gly Arg Asn Leu 530 535 540 Leu Arg Thr Val Ile Glu Lys Ala Arg Ser Leu Gly Leu Pro Ile Ile 545 550 555 560 Tyr Gly Asp Thr Asp Ser Leu Phe Val Arg Asn Ile Ser Asp Lys Val 565 570 575 Asp Ala Leu Ile Asn Tyr Val Asn Asn Glu Leu Gly Phe Glu Val Lys 580 585 590 Val Asp Lys Val Tyr Arg Arg Val Leu Phe Thr Glu Ala Lys Lys Arg 595 600 605 Tyr Val Gly Leu Thr Val Glu Gly Glu Val Asp Ile Val Gly Phe Glu 610 615 620 Ala Val Arg Gly Asp Trp Ala Glu Ile Ala Lys Asp Val Gln Glu Asn 625 630 635 640 Val Ala Glu Ile Val Leu Thr Thr Gly Asp Val Gly Lys Ala Ile Ser 645 650 655 Tyr Val Lys Ser Val Ile Asp Lys Val Lys Ala Tyr Gln Phe Asp Ile 660 665 670 Asp Asp Val Ile Ile Trp Lys Thr Leu Asp Lys Ser Leu Asn Glu Tyr 675 680 685 Lys Val Leu Thr Pro His Val Ala Ala Ala Lys Gln Leu Val Glu Ala 690 695 700 Gly Tyr Lys Val Gly Lys Gly Asp Met Ile Gly Tyr Val Val Val Lys 705 710 715 720 Gly Gly Gly Ala Lys Leu Ala Tyr Lys Val Lys Pro Tyr Ile Leu Ile 725 730 735 Lys Asp Ile Arg Glu Val Asp Val Asp Tyr Tyr Val Glu Lys Gln Ile 740 745 750 Val Pro Ala Ala Met Arg Ile Leu Glu Val Leu Gly Val Lys Glu Ser 755 760 765 Gln Leu Met Glu Gly Lys Ala Gly Lys Ser Ile Leu Asp Tyr Phe Ser 770 775 780 <210> 54 <211> 830 <212> PRT <213> Methanopyrus kandleri <400> 54 Met Leu Arg Thr Val Trp Val Asp Tyr Ala Arg Lys Gly Glu Pro Asp 1 5 10 15 Val Ile Leu Val Gly Arg Arg Glu Asp Gly Asn Pro Ala Ala Leu Val 20 25 30 Val Lys Gly Phe Arg Pro Tyr Phe Tyr Ala Glu Val Glu Asp Gly Phe 35 40 45 Asp Pro Ser Glu Val Glu Arg Leu Ser Gly Val Val Glu Val Glu Glu 50 55 60 Val Leu Leu Glu His Pro Tyr Gly Gly Asp Arg Val Glu Leu Leu Arg 65 70 75 80 Ile Val Ala Thr Tyr Pro Lys Val Val Pro Lys Leu Arg Glu Gln Val 85 90 95 Lys Lys Leu Asp Gly Val Lys Glu Val Tyr Glu Ala Asp Ile Pro Phe 100 105 110 Val Arg Arg Ala Ala Val Asp Leu Asn Leu Pro Pro Ala Ser Glu Val 115 120 125 Asp Val Ser Asp Leu Asp Thr Gly Ser Trp Ser Gly Leu Pro Ala Tyr 130 135 140 Phe Ala Asp Val Glu Asp Ala Arg Glu Leu Asp His Arg Pro Tyr Pro 145 150 155 160 Ile Glu Asp Leu Val Val Ala Ser Phe Asp Leu Glu Val Leu Ala Glu 165 170 175 Pro Gly Thr Thr Ile Lys Gly Ala Ser Gly Pro Ile Ile Ala Ile Ser 180 185 190 Phe Ala Tyr Ser Thr Pro Asp Gly Glu Arg Arg Asn Tyr Val Ile Thr 195 200 205 Trp Lys Gly Glu Asp Glu Ser Phe Glu Val Asp Gly Val Glu Thr Glu 210 215 220 Val Ile Val Cys Arg Ser Glu Ala Ala Ala Leu Arg Arg Phe Phe Asp 225 230 235 240 Glu Phe Arg Arg Val Asp Pro Asp Val Val Phe Thr Tyr Asn Gly Asp 245 250 255 Glu Phe Asp Leu Pro Tyr Leu Gln His Arg Ala Gly Lys Leu Gly Ile 260 265 270 Asp Val Ser Pro Leu Ala Arg Pro Ala Gly Lys Arg Gly Ile Ile Leu 275 280 285 Lys His Gly Gly Gly Arg Tyr Ala Ser Asp Ile Phe Gly Arg Ala His 290 295 300 Val Asp Leu Tyr His Thr Ala Arg Lys Asn Leu Lys Leu Glu Arg Phe 305 310 315 320 Thr Leu Glu Glu Ala Val Lys Asp Val Leu Gly Val Glu Lys Glu Glu 325 330 335 Met Glu Leu Ala Asp Ile Asn Glu Ala Trp Lys Arg Gly Asn Leu Asp 340 345 350 Glu Leu Met Arg Tyr Ser Ala Glu Asp Ala His Tyr Thr Leu Glu Leu 355 360 365 Gly Leu Glu Leu Ala Gln Val Glu Leu Glu Leu Ser Tyr Leu Thr Arg 370 375 380 Leu Pro Leu Pro Asp Ala Thr Arg Phe Ser Phe Gly Gln Leu Ala Glu 385 390 395 400 Trp Arg Ala Ile Tyr Lys Ala Arg Gln Glu Asp Ile Leu Val Pro Asn 405 410 415 Lys Pro Thr Arg Asp Glu Tyr Lys Arg Arg Arg Arg Lys Ala Tyr Lys 420 425 430 Gly Ala Ile Val Phe Glu Pro Glu Ile Gly Leu His Glu Asn Val Val 435 440 445 Cys Val Asp Phe Ala Ser Leu Tyr Pro Asn Val Met Val Ala His Asn 450 455 460 Ile Ser Pro Asp Thr Phe Asp Cys Asp Cys Cys Pro Arg Val Thr Val 465 470 475 480 Glu Glu Val Asp Asp Pro Thr Asp Ala Thr Val Ala Pro Asp Val Gly 485 490 495 His Lys Phe Cys Lys Arg Arg Lys Gly Phe Phe Pro Arg Leu Val Glu 500 505 510 Gly Leu Ile Glu Arg Arg Arg Glu Leu Lys Arg Arg Leu Arg Lys Leu 515 520 525 Asp Thr Glu Ser His Pro His Glu Ala Lys Ile Leu Asp Val Arg Gln 530 535 540 Gln Ala Tyr Lys Val Leu Ala Asn Ser Tyr Tyr Gly Tyr Met Gly Trp 545 550 555 560 Ala Asn Ala Arg Trp Phe Cys Arg Glu Cys Ala Glu Ser Val Thr Ala 565 570 575 Trp Gly Arg Tyr Tyr Ile Ser Glu Val Arg Arg Ile Ala Glu Glu Lys 580 585 590 Tyr Gly Leu Lys Val Val Tyr Gly Asp Thr Asp Ser Leu Phe Val Lys 595 600 605 Leu Pro Asp Ala Asp Leu Glu Glu Thr Ile Glu Arg Val Lys Glu Phe 610 615 620 Leu Lys Glu Val Asn Gly Arg Leu Pro Val Glu Leu Glu Leu Glu Asp 625 630 635 640 Ala Tyr Lys Arg Ile Leu Phe Val Thr Lys Lys Lys Tyr Ala Gly Tyr 645 650 655 Thr Glu Asp Gly Lys Ile Val Thr Lys Gly Leu Glu Val Val Arg Arg 660 665 670 Asp Trp Ala Pro Ile Ala Arg Glu Thr Gln Arg Arg Val Leu Lys Arg 675 680 685 Ile Leu Ala Asp Asn Asp Pro Glu Ala Ala Leu Lys Glu Ile His Glu 690 695 700 Val Leu Glu Arg Leu Lys Ser Gly Asp Val Asp Ile Asp Glu Leu Ala 705 710 715 720 Val Thr Ser Gln Leu Thr Lys Lys Pro Ser Glu Tyr Val Gln Lys Gly 725 730 735 Pro His Val Arg Ala Ala Leu Arg Leu Ala Arg His Leu Gly Val Glu 740 745 750 Pro Glu Pro Gly Thr Ile Val Arg Tyr Val Ile Val Arg Gly Pro Gly 755 760 765 Ser Val Ser Asp Lys Ala Tyr Pro Val Glu Leu Val Arg Glu Glu Gly 770 775 780 Lys Glu Pro Asp Val Asp Tyr Tyr Ile Glu His Gln Ile Leu Pro Ala 785 790 795 800 Val Glu Arg Ile Met Arg Ala Ile Gly Tyr Ser Arg Gly Gln Ile Val 805 810 815 Gly Glu Thr Ala Ser Gln Lys Thr Leu Asp Gln Phe Phe Gly 820 825 830 <210> 55 <211> 784 <212> PRT <213> Thermoproteus neutrophilus <400> 55 Met Glu Leu Lys Ile Trp Pro Leu Asp Val Thr Tyr Ala Val Val Gly 1 5 10 15 Gly Tyr Pro Glu Val Arg Val Phe Gly Leu Thr Glu Gly Gly Gly Arg 20 25 30 Val Val Leu Val Asp Arg Ser Phe Lys Pro Tyr Phe Tyr Val Asp Cys 35 40 45 Pro Thr Cys Glu Val Gly Val Val Lys Ser Ser Leu Ser Arg Val Ala 50 55 60 Pro Val Asp Glu Val Ser Ala Ala Glu Arg Arg Phe Leu Gly Arg Pro 65 70 75 80 Arg Arg Phe Leu Met Val Val Ala Arg Val Pro Glu Asp Val Arg Arg 85 90 95 Leu Arg Glu Ala Ala Ala Gln Ile Pro Gly Val Ala Gly Val Tyr Glu 100 105 110 Ala Asp Ile Arg Phe Tyr Met Arg Tyr Met Ile Asp Val Gly Leu Leu 115 120 125 Pro Cys Ser Trp Asn Arg Ala Glu Val Glu Gly Gly Gly Lys Val Gly 130 135 140 Gly Leu Pro Gln Tyr Thr Val Val Gln Trp Leu Gly Pro Ala Gly Gly 145 150 155 160 Phe Pro Pro Pro Leu Arg Val Leu Ala Phe Asp Ile Glu Val Tyr Asn 165 170 175 Glu Arg Gly Thr Pro Asp Pro Ala Arg Asp Pro Val Val Met Ile Ala 180 185 190 Val Lys Thr Asp Asp Gly Arg Glu Glu Val Phe Glu Ala Glu Gly Arg 195 200 205 Asp Asp Arg Gly Val Leu Arg Ser Phe Val Glu Phe Val Lys Ser Tyr 210 215 220 Asp Pro Asp Val Val Val Gly Tyr Asn Ser Asn Gly Phe Asp Trp Pro 225 230 235 240 Tyr Leu Ala Gly Arg Ala Arg Ala Ile Gly Val Pro Leu Arg Val Asp 245 250 255 Arg Leu Gly Gly Leu Pro Gln Gln Ser Val Tyr Gly His Trp Ser Ile 260 265 270 Val Gly Arg Ala Asn Val Asp Leu Tyr Gly Ile Val Glu Glu Phe Pro 275 280 285 Glu Ile Lys Leu Lys Thr Leu Asp Arg Val Ala Glu Tyr Phe Gly Val 290 295 300 Met Arg Arg Glu Glu Arg Val Leu Ile Pro Gly His Lys Ile Tyr Glu 305 310 315 320 Tyr Trp Arg Asp Pro Gly Lys Arg Pro Leu Leu Arg Gln Tyr Val Leu 325 330 335 Asp Asp Val Arg Ser Thr Leu Gly Leu Ala Asp Lys Leu Leu Pro Phe 340 345 350 Leu Ile Gln Leu Ser Ser Val Ser Gly Leu Pro Leu Asp Gln Val Ala 355 360 365 Ala Ala Ser Val Gly Asn Arg Val Glu Trp Met Leu Leu Arg Tyr Ala 370 375 380 Tyr Arg Leu Gly Glu Val Ala Pro Asn Arg Glu Glu Arg Glu Tyr Glu 385 390 395 400 Pro Tyr Lys Gly Ala Ile Val Leu Glu Pro Lys Pro Gly Met Tyr Glu 405 410 415 Asp Val Leu Val Leu Asp Phe Ser Ser Met Tyr Pro Asn Ile Met Met 420 425 430 Lys Tyr Asn Leu Ser Pro Asp Thr Tyr Leu Glu Pro Gly Glu Pro Asp 435 440 445 Pro Pro Glu Gly Val Asn Ala Ala Pro Glu Val Gly His Arg Phe Arg 450 455 460 Arg Ser Pro Leu Gly Phe Val Pro Gln Val Leu Lys Ser Leu Val Glu 465 470 475 480 Leu Arg Lys Ala Val Arg Glu Glu Ala Lys Arg Tyr Pro Pro Asp Ser 485 490 495 Pro Glu Phe Arg Ile Leu Asp Glu Arg Gln Arg Ala Leu Lys Val Met 500 505 510 Ala Asn Ala Met Tyr Gly Tyr Leu Gly Trp Val Gly Ala Arg Trp Tyr 515 520 525 Lys Arg Glu Val Ala Glu Ser Val Thr Ala Phe Ala Arg Ala Ile Leu 530 535 540 Lys Asp Val Ile Glu Gln Ala Arg Arg Leu Gly Ile Val Val Val Tyr 545 550 555 560 Gly Asp Thr Asp Ser Leu Phe Val Lys Lys His Val Asn Val Asp Lys 565 570 575 Leu Ile Gln Tyr Val Glu Glu Lys Tyr Gly Ile Glu Ile Lys Val Asp 580 585 590 Lys Asp Tyr Ala Lys Val Leu Phe Thr Glu Ala Lys Lys Arg Tyr Ala 595 600 605 Gly Leu Leu Arg Asp Gly Arg Ile Asp Ile Val Gly Phe Glu Val Val 610 615 620 Arg Gly Asp Trp Ser Glu Leu Ala Lys Glu Val Gln Leu Lys Val Val 625 630 635 640 Glu Ile Ile Leu Asn Ser Arg Asp Val Ala Glu Ala Arg Arg Arg Val 645 650 655 Thr Gln Tyr Val Arg Glu Ile Ile Glu Arg Leu Arg Glu Tyr Lys Phe 660 665 670 Asn Val Asp Asp Leu Ile Ile Trp Lys Thr Leu Asp Lys Glu Leu Gly 675 680 685 Glu Tyr Lys Ala Tyr Pro Pro His Val His Ala Ala Leu Ile Leu Lys 690 695 700 Arg His Gly Tyr Lys Val Gly Lys Gly Asn Met Val Gly Tyr Val Val 705 710 715 720 Val Lys Gly Gly Gly Lys Ile Ser Glu Lys Ala Leu Pro Tyr Ile Leu 725 730 735 Leu Asp Asp Val Lys Lys Ile Asp Val Glu Tyr Tyr Ile Glu Arg Gln 740 745 750 Ile Ile Pro Ala Ala Leu Arg Ile Ala Glu Val Ile Gly Val Lys Glu 755 760 765 Ala Asp Leu Lys Thr Gly Lys Ser Glu Arg Ser Leu Leu Asp Phe Phe 770 775 780 <210> 56 <211> 142 <212> PRT <213> Pyrococcus furiosus <400> 56 Met Lys Thr Phe Leu Thr Glu Gln Gln Ile Lys Val Leu Met Leu Arg 1 5 10 15 Ala Lys Gly Tyr Lys Gln Ser Glu Ile Ala Lys Ile Leu Gly Thr Ser 20 25 30 Arg Ala Asn Val Ser Ile Leu Glu Lys Arg Ala Met Glu Lys Ile Glu 35 40 45 Lys Ala Arg Asn Thr Leu Leu Leu Trp Glu Gln Ile Asn Ser Lys Val 50 55 60 Ile Val Glu Ile Lys Ala Gly Glu Asp Ile Phe Ser Ile Pro Glu Lys 65 70 75 80 Phe Phe Lys Lys Ala Asp Lys Val Gly Val Lys Val Pro Tyr Ser Thr 85 90 95 Ala Glu Ile Ile Thr Phe Leu Val Glu His Ala Pro Val Glu Asp Arg 100 105 110 Leu Ala Lys Arg Asp Phe Val Leu Phe Leu Asp Ser Lys Asn Lys Leu 115 120 125 Arg Ile Gly Asp Cys Leu Val Ile Glu Glu Ile Lys Glu Asp 130 135 140 <210> 57 <211> 75 <212> PRT <213> Pyrococcus furiosus <400> 57 Met Pro Ile Thr Lys Val Thr Arg Asn Tyr Gln Ile Thr Ile Pro Ala 1 5 10 15 Glu Ile Arg Lys Ala Leu Gly Ile Lys Glu Gly Glu Leu Leu Glu Val 20 25 30 Arg Leu Glu Asn Gly Lys Ile Ile Ile Glu Arg Leu Lys Lys Glu Arg 35 40 45 Lys Thr Leu Lys Leu Gly Lys Lys Leu Thr Leu Glu Glu Ile Glu Lys 50 55 60 Ala Ile Glu Glu Gly Met Lys Gln Cys Met Gln 65 70 75 <210> 58 <211> 76 <212> PRT <213> Pyrococcus furiosus <400> 58 Thr Lys Ile Glu Ile Leu Arg Leu Leu Lys Glu Arg Glu Met Tyr Ala 1 5 10 15 Tyr Glu Ile Trp Ser Leu Leu Gly Lys Pro Leu Lys Tyr Gln Ala Val 20 25 30 His Gln His Ile Lys Glu Leu Leu Glu Leu Gly Leu Val Glu Gln Ala 35 40 45 Tyr Arg Lys Gly Lys Arg Val Tyr Tyr Lys Ile Thr Glu Lys Gly Leu 50 55 60 Arg Ile Leu Gln Asn Phe Glu Asp Leu Glu Asn Ile 65 70 75 <210> 59 <211> 72 <212> PRT <213> Pyrococcus furiosus <400> 59 Met Asn Thr Gly Ala Gln Gly Val Ser Glu Met Ser Arg Met Lys Ile 1 5 10 15 Ile Ser Val Gln Leu Pro Gln Ser Leu Ile His Gly Leu Asp Ala Leu 20 25 30 Val Lys Arg Gly Ile Tyr Pro Asn Arg Ser Glu Ala Ile Arg Val Ala 35 40 45 Ile Arg Glu Leu Leu Lys Lys Glu Leu Tyr Lys Glu Glu Ile Gln Glu 50 55 60 Glu Ile Pro Glu Tyr Val Val Lys 65 70 <210> 60 <211> 67 <212> PRT <213> Pyrococcus furiosus <400> 60 Val Ile Ile Pro Arg Pro Ile Asp Pro Arg Asp Ile Arg Arg Ile Arg 1 5 10 15 Lys Glu Leu Gly Ile Thr Gln Glu Glu Leu Ala Arg Lys Ala Gly Val 20 25 30 Thr Gln Ala Tyr Ile Ala Lys Leu Glu Ala Gly Lys Val Asp Pro Arg 35 40 45 Leu Ser Thr Phe Asn Lys Ile Leu Arg Ala Leu Ile Glu Cys Gln Lys 50 55 60 Ala Lys Ile 65 <210> 61 <211> 65 <212> PRT <213> Pyrococcus furiosus <400> 61 Asn Asn Cys Glu Cys Met Val Val Lys Glu Lys Leu Tyr Thr Val Lys 1 5 10 15 Gln Ala Ser Glu Ile Leu Gly Val His Pro Lys Thr Ile Gln Lys Trp 20 25 30 Asp Arg Glu Gly Lys Ile Lys Thr Val Arg Thr Pro Gly Gly Arg Arg 35 40 45 Arg Ile Pro Glu Ser Glu Ile Lys Arg Leu Leu Gly Ile Ser Glu Glu 50 55 60 Lys 65 <210> 62 <211> 71 <212> PRT <213> Pyrococcus furiosus <400> 62 Met Leu Lys Asp Ser Ala Pro Lys Arg Lys Ile Leu Glu Glu Leu Arg 1 5 10 15 Lys Gly Glu Thr Val Ser Gly Asp Tyr Leu Ala Ser Lys Leu Gly Val 20 25 30 Ser Arg Val Ala Ile Trp Lys His Ile Arg Glu Leu Lys Glu Leu Gly 35 40 45 Tyr Gly Ile Ile Ala Asp Lys Lys Gly Tyr Lys Leu Val Tyr Glu Pro 50 55 60 Lys Lys Pro Tyr Pro Trp Glu 65 70 <210> 63 <211> 86 <212> PRT <213> Pyrococcus furiosus <400> 63 Met Ile Asp Glu Arg Asp Lys Ile Ile Leu Glu Ile Leu Glu Lys Asp 1 5 10 15 Ala Arg Thr Pro Phe Thr Glu Ile Ala Lys Lys Leu Gly Ile Ser Glu 20 25 30 Thr Ala Val Arg Lys Arg Val Lys Ala Leu Glu Glu Lys Gly Ile Ile 35 40 45 Glu Gly Tyr Thr Ile Lys Ile Asn Pro Lys Lys Leu Gly Tyr Ser Leu 50 55 60 Val Thr Ile Thr Gly Val Asp Thr Lys Pro Glu Lys Leu Phe Glu Val 65 70 75 80 Ala Glu Lys Leu Lys Glu 85 <210> 64 <211> 77 <212> PRT <213> Pyrococcus furiosus <400> 64 Met Glu Ile Asp Asp Leu Asp Arg Lys Ile Leu Ser Leu Leu Ile Glu 1 5 10 15 Asp Ser Arg Leu Ser Tyr Arg Glu Ile Ala Lys Lys Leu Asn Val Ala 20 25 30 Val Gly Thr Ile Tyr Asn Arg Ile Lys Lys Leu Glu Asp Met Gly Val 35 40 45 Ile Gln Gly Phe Thr Val Lys Leu Asn Tyr Glu Lys Leu Gly Tyr Glu 50 55 60 Leu Thr Ala Ile Ile Gly Ile Lys Ala Gln Gly Lys Lys 65 70 75 <210> 65 <211> 94 <212> PRT <213> Pyrococcus furiosus <400> 65 Glu Met Leu Trp Met Tyr Ile Leu Lys Leu Leu Lys Asp Arg Pro Met 1 5 10 15 Tyr Ala Tyr Glu Ile Arg Asn Glu Leu Lys Lys Arg Phe Gly Phe Glu 20 25 30 Pro Ala Thr Val Ser Ser Tyr Val Val Leu Tyr Arg Leu Glu Glu Gly 35 40 45 Gly Tyr Val Ser Ser Glu Trp His Glu Ser Glu Ala Gly Arg Pro Ser 50 55 60 Arg Lys Tyr Tyr Arg Leu Thr Glu Lys Gly Glu Lys Leu Leu Glu Lys 65 70 75 80 Gly Ile Glu Thr Ile Glu Asp Val Leu Asn Met Leu Lys Ser 85 90 <210> 66 <211> 68 <212> PRT <213> Pyrococcus furiosus <400> 66 Met Lys Val Ser Lys Ala Thr Ala Ser Lys Val Leu Arg Ser Leu Glu 1 5 10 15 Asn Lys Gly Ile Val Glu Arg Glu Arg Arg Gly Lys Thr Tyr Leu Val 20 25 30 Arg Leu Thr Asn Lys Gly Leu Glu Leu Leu Glu Glu Ile Ser Lys Ala 35 40 45 Gly Lys Glu Leu Asp Glu Lys Ile Phe Ala Glu Met Ser Val Asp Glu 50 55 60 Arg Ile Val Leu 65 <210> 67 <211> 56 <212> PRT <213> Pyrococcus furiosus <400> 67 Ser Glu Asp Tyr Met Leu Gln Asn Arg Arg Lys Val Leu Ala Lys Val 1 5 10 15 Leu Glu Leu Leu Asn Tyr Asn Pro Lys Ala Leu Asn Ile Ser Glu Leu 20 25 30 Ala Arg Met Phe Gly Val Ser Arg Asp Thr Ile Tyr Asn Asp Ile Gln 35 40 45 Gln Ile Ile Lys Asn Val Glu Val 50 55 <210> 68 <211> 94 <212> PRT <213> Pyrococcus furiosus <400> 68 Ser Lys Glu Ile Ser Arg Phe Leu Lys Val Ile Ser Asn Pro Ile Arg 1 5 10 15 Tyr Gly Ile Leu Lys Met Leu Asn Asp Arg Trp Met Cys Val Cys Leu 20 25 30 Ile Ser Glu Ala Leu Glu Ile Asp Gln Thr Leu Val Ser His His Ile 35 40 45 Arg Ile Leu Lys Glu Leu Asp Leu Leu Glu Glu Arg Lys Glu Gly Lys 50 55 60 Leu Arg Phe Tyr Arg Thr Asn Lys Glu Lys Leu Arg Glu Tyr Leu Glu 65 70 75 80 Lys Val Leu Glu Asp Phe Asn His Gly Thr Ser Lys Gly Ser 85 90 <210> 69 <211> 78 <212> PRT <213> Pyrococcus furiosus <400> 69 Met Cys Arg Lys Asp Val Met Ile Ile Ser Asp Pro Lys Gln Ile Lys 1 5 10 15 Ala Leu Ser Asp Pro Thr Arg Val Lys Ile Leu Glu Leu Leu Arg Tyr 20 25 30 His Pro Met Thr Val Ser Glu Ile Ser Arg Val Ile Gly Lys Asp Lys 35 40 45 Ser Thr Ile Tyr Arg His Ile Lys Ala Leu Glu Glu Ala Gly Leu Val 50 55 60 Glu Glu Val Glu Lys Ile Gly Asn Glu Thr Val Tyr Gly Arg 65 70 75 <210> 70 <211> 122 <212> PRT <213> Pyrococcus furiosus <400> 70 Met Glu Pro Val Glu Phe Lys Leu Asn Gln Lys Gly Ile Lys Ser Ile 1 5 10 15 Leu Pro Thr Met Glu Ala Glu Ile Met Glu Tyr Met Trp Glu Ile Lys 20 25 30 Glu Ala Thr Ala Gly Glu Val Tyr Glu Tyr Met Lys Thr Lys Tyr Pro 35 40 45 Glu Ile Arg Arg Ser Thr Val Ser Ile Leu Met Asn Arg Leu Cys Glu 50 55 60 Arg Gly Leu Leu Lys Arg Arg Met Glu Lys Gly Lys Gly Gly Ile Arg 65 70 75 80 Tyr Val Tyr Ser Ile Thr Thr Thr Arg Glu Glu Phe Glu Arg Lys Val 85 90 95 Val Glu Lys Ile Ile Glu Ser Leu Met Met Asn Phe Arg Glu Ala Thr 100 105 110 Phe Ala Tyr Leu Ser Lys Ile Asn Lys Lys 115 120 <210> 71 <211> 87 <212> PRT <213> Pyrococcus furiosus <400> 71 Met Lys Lys Ser Asn Leu Asp Leu Leu Ile Leu Leu Ala Lys Ala Gly 1 5 10 15 Gly Ile Glu Lys Glu Ile Leu Thr Thr Ser Arg Glu Leu Ser Lys Met 20 25 30 Leu Asn Val Ser Pro Gln Thr Ile Val Arg Trp Leu Glu Asp Leu Glu 35 40 45 Lys Asp Gly Leu Ile Lys Lys Ser Glu Ser Arg Lys Gly Thr Leu Val 50 55 60 Thr Ile Thr Glu Glu Gly Val Lys Phe Leu Glu Lys Leu His Glu Glu 65 70 75 80 Leu Ser Asp Ala Leu Tyr Arg 85 <210> 72 <211> 104 <212> PRT <213> Thermococcaceae <400> 72 Met Glu Ile Pro Pro Glu Ile Ser His Ala Leu Ser Glu Ile Gly Phe 1 5 10 15 Thr Lys Tyr Glu Ile Leu Thr Tyr Trp Thr Leu Leu Val Tyr Gly Pro 20 25 30 Ser Thr Ala Lys Glu Ile Ser Thr Lys Ser Gly Ile Pro Tyr Asn Arg 35 40 45 Val Tyr Asp Thr Ile Ser Ser Leu Lys Leu Arg Gly Phe Val Thr Glu 50 55 60 Ile Glu Gly Thr Pro Lys Val Tyr Ala Ala Tyr Ser Pro Arg Ile Ala 65 70 75 80 Phe Phe Arg Phe Lys Lys Glu Leu Glu Asp Ile Met Lys Lys Leu Glu 85 90 95 Ile Glu Leu Asn Asn Val Lys Lys 100 <210> 73 <211> 75 <212> PRT <213> Pyrococcus furiosus <400> 73 Ile Ile Asn Pro Gln Ala Arg Leu Thr Pro Leu Glu Leu Glu Ile Leu 1 5 10 15 Glu Ile Ile Lys Gln Lys Lys Ser Ile Thr Ile Thr Glu Ile Lys Glu 20 25 30 Ile Leu Ser Glu Arg Arg Lys Ser Glu Tyr Pro Leu Ser Leu Val Ser 35 40 45 Glu Tyr Ile Ser Arg Leu Glu Arg Lys Gly Tyr Val Lys Lys Ile Ala 50 55 60 Lys Gly Arg Lys Lys Phe Val Glu Ala Leu Ile 65 70 75 <210> 74 <211> 94 <212> PRT <213> Pyrococcus furiosus <400> 74 Gly Ile Asp Val Val Ile Pro Glu Ile Lys His Asp Pro Ile Ala Arg 1 5 10 15 Asp Ile Val Lys Ile Leu Phe Asp Leu Arg Arg Ala Asn Val Ser Gln 20 25 30 Ile Ala Arg Glu Leu Lys Gly Arg Arg Gly Lys Ala Ser Arg Asn Thr 35 40 45 Val Arg Lys Lys Leu Lys Glu Leu Glu Lys Leu Gly Val Val Lys Glu 50 55 60 Val Pro Gly Glu Arg Gly Ser Val Tyr Thr Leu Ser Arg Glu Val Val 65 70 75 80 Lys Lys Trp Leu Asp Leu Ile Gly Ile Pro Ile Asn Leu Leu 85 90 <210> 75 <211> 67 <212> PRT <213> Pyrococcus furiosus <400> 75 Met Thr Lys Arg Val Lys Val Ile Thr Asp Pro Glu Val Ile Lys Val 1 5 10 15 Met Leu Glu Asp Thr Arg Arg Lys Ile Leu Gln Leu Leu Arg Asn Arg 20 25 30 Glu Met Thr Ile Ser Gln Leu Ser Glu Ile Leu Gly Lys Met Pro Gln 35 40 45 Thr Ile Tyr His His Ile Glu Lys Leu Lys Glu Ala Gly Leu Val Glu 50 55 60 Val Lys Arg 65 <210> 76 <211> 100 <212> PRT <213> Pyrococcus furiosus <400> 76 Met Glu Glu Ile Lys Glu Ile Met Lys Ser His Thr Leu Gly Asn Pro 1 5 10 15 Val Arg Leu Gly Ile Met Ile Tyr Leu Phe Pro Arg Arg Arg Ala Pro 20 25 30 Phe Ser His Ile Gln Lys Ala Leu Asp Leu Thr Pro Gly Asn Leu Asp 35 40 45 Ser His Ile Lys Val Leu Glu Lys His Gly Phe Val Arg Thr Tyr Lys 50 55 60 Val Ile Ala Asp Arg Pro Arg Thr Met Val Glu Ile Thr Asp Tyr Gly 65 70 75 80 Met Glu Glu Thr Arg Lys Phe Leu Ser His Leu Lys Thr Val Ile Asp 85 90 95 Ala Ile His Phe 100 <210> 77 <211> 99 <212> PRT <213> Pyrococcus furiosus <400> 77 Met Gly Glu Glu Leu Asn Arg Leu Leu Asp Val Leu Gly Asn Glu Thr 1 5 10 15 Arg Arg Arg Ile Leu Phe Leu Leu Thr Lys Arg Pro Tyr Phe Val Ser 20 25 30 Glu Leu Ser Arg Glu Leu Gly Val Gly Gln Lys Ala Val Leu Glu His 35 40 45 Leu Arg Ile Leu Glu Glu Ala Gly Leu Ile Glu Ser Arg Val Glu Lys 50 55 60 Ile Pro Arg Gly Arg Pro Arg Lys Tyr Tyr Met Ile Lys Lys Gly Leu 65 70 75 80 Arg Leu Glu Ile Leu Leu Thr Pro Thr Leu Phe Gly Ser Glu Met Tyr 85 90 95 Glu Ala Lys <210> 78 <211> 61 <212> PRT <213> Pyrococcus furiosus <400> 78 Met Arg Arg Met Asp Lys Val Asp Leu Gln Leu Ile Lys Ile Leu Ser 1 5 10 15 Gln Asn Ser Arg Leu Thr Tyr Arg Glu Leu Ala Glu Met Leu Gly Thr 20 25 30 Thr Arg Gln Arg Val Ala Arg Lys Val Asp Lys Leu Lys Lys Leu Gly 35 40 45 Ile Ile Arg Lys Phe Thr Ile Ile Pro Asn Leu Glu Lys 50 55 60 <210> 79 <211> 82 <212> PRT <213> Pyrococcus furiosus <400> 79 Gly Arg Lys Val Arg Thr Gln Gln Asn Glu Ile Leu Asn Leu Leu Asn 1 5 10 15 Glu Lys Glu Lys Ala Val Leu Arg Ala Ile Leu Glu His Gly Gly Glu 20 25 30 Ile Lys Gln Glu Asp Leu Pro Glu Leu Val Gly Tyr Ser Arg Pro Thr 35 40 45 Ile Ser Lys Val Ile Gln Glu Leu Glu Asn Lys Gly Leu Ile Lys Arg 50 55 60 Glu Lys Ser Gly Lys Thr Phe Val Val Lys Ile Glu Arg Lys Ile Lys 65 70 75 80 Leu Asp <210> 80 <211> 59 <212> PRT <213> Thermotoga maritima <400> 80 Lys Ser Leu Gln Arg Phe Leu Arg Arg Asn Thr Thr Ser Ile Lys His 1 5 10 15 Leu Ser Glu Ile Thr Gly Val Ala Arg Asn Arg Leu Ser Asp Ile Leu 20 25 30 Asn Gly Lys Thr Gln Lys Ile Arg Gly Glu Thr Leu Arg Lys Ile Ala 35 40 45 Lys Ala Phe Glu Lys Ser Asn Ile Leu Ser Phe 50 55 <210> 81 <211> 107 <212> PRT <213> Thermotoga <400> 81 Asp Val Ile Gln Arg Ile Lys Glu Lys Tyr Asp Glu Phe Thr Asn Ala 1 5 10 15 Glu Lys Lys Ile Ala Asp Thr Ile Leu Ser Asp Pro Lys Gly Ile Ile 20 25 30 Glu Ser Ser Ile Ser Asp Leu Ser Glu Lys Ala Gly Val Lys Ser Glu 35 40 45 Ala Ser Val Val Lys Phe Tyr Lys Lys Leu Gly Leu Asn Ser Phe Gln 50 55 60 Gln Phe Lys Val Leu Leu Ala Gln Ser Ile Ser Arg Ala Pro Leu Glu 65 70 75 80 Ile Val Tyr Glu Asp Val Ser Ser Glu Asp Asp Thr Lys Thr Ile Thr 85 90 95 Glu Lys Ile Phe Lys Ala Thr Val Arg Ala Ile 100 105 <210> 82 <211> 101 <212> PRT <213> Thermotoga maritima <400> 82 Lys Ile Arg Asp Lys Ile Leu Asn Val Tyr Thr Gln Phe Ser Pro Ala 1 5 10 15 Glu Arg Lys Val Ala Asp Tyr Val Leu Glu Arg Pro Asp Asp Val Ile 20 25 30 His Tyr Ser Ile Thr Glu Phe Ala Lys Ile Val Gly Val Ser Glu Thr 35 40 45 Thr Ile His Arg Met Ile Lys Lys Leu Asp Phe Glu Gly Tyr Gln Ala 50 55 60 Phe Lys Ile Ala Leu Ala Arg Glu Leu Ser Gly Leu Glu Glu Thr Ile 65 70 75 80 Glu Arg Arg Asp Phe Ile Asp Glu Glu Ile Asp Ile Leu Arg Arg Leu 85 90 95 Lys Asp Thr Leu Asp 100 <210> 83 <211> 60 <212> PRT <213> Thermotoga maritima <400> 83 Lys Arg Arg Pro Thr Ile Asn Asp Val Ala Lys Leu Ala Gly Val Ser 1 5 10 15 Ile Ser Thr Val Ser Arg Tyr Leu Lys Asp Pro Ser Gln Val Ser Glu 20 25 30 Lys Leu Gly Glu Arg Ile Arg Glu Ala Ile Lys Lys Leu Gly Tyr Lys 35 40 45 Pro Asn Lys Ile Ala Gln Gly Leu Arg Thr Gly Asp 50 55 60 <210> 84 <211> 58 <212> PRT <213> Thermotoga maritima <400> 84 Met Ala Ser Ile Lys Asp Val Ala Lys Leu Ala Gly Val Ser Ile Ala 1 5 10 15 Thr Val Ser Arg Val Ile Asn Gly Tyr Asn Asn Val Ser Glu Glu Thr 20 25 30 Arg Lys Lys Val Ile Asp Ala Ile Arg Lys Leu Asn Tyr His Pro Val 35 40 45 Tyr Ala Val Lys Gly Ala Val Leu Lys Arg 50 55 <210> 85 <211> 61 <212> PRT <213> Thermotoga maritima <400> 85 Met Lys Lys Lys Tyr Val Thr Ile Arg Asp Ile Ala Glu Lys Ala Gly 1 5 10 15 Val Ser Ile Asn Thr Val Ser Arg Ala Leu Asn Asn Lys Pro Asp Ile 20 25 30 Ser Glu Glu Thr Arg Arg Lys Ile Leu Lys Ile Ala Gln Glu Leu Gly 35 40 45 Tyr Val Lys Asn Ala Thr Ala Ser Ser Leu Arg Ser Lys 50 55 60 <210> 86 <211> 58 <212> PRT <213> Thermotoga maritima <400> 86 Met Pro Thr Ile Glu Asp Val Ala Lys Leu Ala Gly Val Ser Ile Ala 1 5 10 15 Thr Val Ser Arg Val Ile Asn Gly Ser Gly Tyr Val Ser Glu Lys Thr 20 25 30 Arg Tyr Lys Val Trp Lys Ala Ile Glu Glu Leu Gly Tyr Lys Pro Glu 35 40 45 Ile Ser Ala Lys Leu Leu Ala Ser Lys Gly 50 55 <210> 87 <211> 61 <212> PRT <213> Thermotoga neapolitana <400> 87 Met Arg Ile Gly Glu Lys Leu Arg Lys Leu Arg Leu Ser Arg Gly Leu 1 5 10 15 It should be noted that in the original text, the name "栖热袍菌" was translated as "Thermotoga maritima" which is the scientific name of a thermophilic bacterium. However, there is an error in the original text where "海栖热袍菌" should be "嗜热栖热菌" (Thermotoga neapolitana). The corrected translation is provided above.Thr Gln Glu Glu Leu Ala Glu Arg Thr Asp Leu Ser Arg Ser Phe Ile 20 25 30 Ser Gln Leu Glu Ser Asp Lys Thr Ser Pro Ser Ile Asp Thr Leu Glu 35 40 45 Arg Ile Leu Glu Ala Leu Gly Thr Asp Leu Lys His Phe 50 55 60 <210> 88 <211> 67 <212> PRT <213> Thermotoga maritima <400> 88 Met His Met Lys Thr Val Arg Gln Glu Arg Leu Lys Ser Ile Val Arg 1 5 10 15 Ile Leu Glu Arg Ser Lys Glu Pro Val Ser Gly Ala Gln Leu Ala Glu 20 25 30 Glu Leu Ser Val Ser Arg Gln Val Ile Val Gln Asp Ile Ala Tyr Leu 35 40 45 Arg Ser Leu Gly Tyr Asn Ile Val Ala Thr Pro Arg Gly Tyr Val Leu 50 55 60 Ala Gly Gly 65 <210> 89 <211> 75 <212> PRT <213> Thermotoga maritima <400> 89 Met Asn Thr Leu Lys Lys Ala Phe Glu Ile Leu Asp Phe Ile Val Lys 1 5 10 15 Asn Pro Gly Asp Val Ser Val Ser Glu Ile Ala Glu Lys Phe Asn Met 20 25 30 Ser Val Ser Asn Ala Tyr Lys Tyr Met Val Val Leu Glu Glu Lys Gly 35 40 45 Phe Val Leu Arg Lys Lys Asp Lys Arg Tyr Val Pro Gly Tyr Lys Leu 50 55 60 Ile Glu Tyr Gly Ser Phe Val Leu Arg Arg Phe 65 70 75 <210> 90 <211> 81 <212> PRT <213> Thermotoga maritima <400> 90 Met Lys Ile Ser Lys Lys Arg Arg Gln Glu Leu Ile Arg Lys Ile Ile 1 5 10 15 His Glu Lys Lys Ile Ser Asn Gln Phe Gln Ile Val Glu Glu Leu Lys 20 25 30 Lys Tyr Gly Ile Lys Ala Val Gln Pro Thr Val Ala Arg Asp Leu Lys 35 40 45 Glu Ile Gly Ala Val Lys Ile Met Asp Glu Ser Gly Asn Tyr Val Tyr 50 55 60 Lys Leu Leu Asp Glu Thr Pro Val Ile Asp Pro Trp Lys Glu Leu Lys 65 70 75 80 Arg <210> 91 <211> 82 <212> PRT <213> Thermotoga maritima <400> 91 Met His Lys Lys Leu Asn Pro Lys Ser Met Lys Arg Glu Asn Lys Lys 1 5 10 15 Met Val Leu Arg Tyr Leu Ile Glu Ser Gly Pro His Ser Arg Val Glu 20 25 30 Ile Ala Arg Lys Thr Gly Leu Ala Gln Ser Ala Ile Trp Arg Ile Ile 35 40 45 Glu Glu Leu Val Asn Glu Gly Leu Val Glu Glu Lys Gly Thr Ala Thr GCGGCCGCatgatgctcgtgacggttaacgcc (SEQ ID NO: 11) 6>50 55 60 Gly Arg Arg Arg Lys Ala Val Thr Tyr Gly Pro Thr Arg Ser Phe Ile 65 70 75 80 Thr Ser <210> 92 <211> 79 <212> PRT <213> Thermotoga maritima <400> 92 Met Pro Ser Pro Leu Leu Arg Arg Glu Asn Lys Ile Lys Ile Leu Arg 1 5 10 15 Tyr Ile Leu Lys Asn Gly Lys Thr Thr Arg Asn Gln Leu Ala Ser Asn 20 25 30 Leu Asn Leu Ala His Ser Thr Leu Ser Tyr Ile Ile Asp Glu Leu Leu 35 40 45 Asp Glu Gly Phe Leu Val Phe Glu Glu Ile Lys Lys Lys Arg Gly Arg 50 55 60 Pro Tyr Gln Ile Leu Ser Val Asn Pro Glu Lys Phe Thr Ala Ile 65 70 75 <210> 93 <211> 79 <212> PRT <213> Thermotoga maritima <400> 93 Met Lys Glu Glu Arg Leu Lys Glu Ile Leu Asp Ile Val Asp Arg Asn 1 5 10 15 Gly Phe Ile Ser Met Lys Asp Leu Gln Glu Gln Leu Gly Val Ser Met 20 25 30 Ile Thr Val Arg Arg Asp Val Ala Glu Leu Val Lys Arg Asn Leu Val 35 40 45 Lys Lys Val His Gly Gly Ile Arg Lys Val Asn Tyr Phe Glu Lys Glu 50 55 60 Thr Asp Phe Met Lys Arg Leu Ser Ile Asn Arg Glu Ala Lys Glu 65 70 75 <210> 94 <211> 137 <212> PRT <213> Thermotoga maritima <400> 94 Met Phe Thr Met Arg Ser Glu Tyr Ala Leu Arg Leu Met Ile Val Met 1 5 10 15 Ala Lys Glu Tyr Gly Asn Tyr Leu Ser Met Thr Glu Ile Leu Glu Lys 20 25 30 Ala Lys Gln Ser Val Pro Arg Glu Phe Ala Glu Lys Ile Leu Tyr Thr 35 40 45 Leu Lys Lys Ala Gly Leu Val Lys Thr Arg Arg Gly Lys Ser Gly Gly 50 55 60 Tyr Met Leu Ser Arg Pro Pro Lys Glu Ile Lys Val Ser Glu Ile Val 65 70 75 80 Phe Leu Leu Asp Arg Lys Ser Lys Val Phe Phe Asp Met Pro Gly Cys 85 90 95 Pro Asp Glu Leu Asp Cys Val Ile Arg Ala Leu Trp Lys Arg Val Glu 100 105 110 Asn Glu Ile Glu Lys Ile Leu Ser Gly Val Thr Leu Glu Asp Leu Val 115 120 125 Arg Glu Gln Glu Glu Lys Met Lys Gln 130 135 <210> 95 <211> 95 <212> PRT <213> Thermotoga naphthophila <400> 95 Met Arg Asp Thr Lys Gly His Leu Lys Phe Leu Val Leu His Ile Ile 1 5 10 15 Ser Gln Gln Pro Ser His Gly Tyr Tyr Ile Met Lys Lys Ile Ser Gln 20 25 30 Ile Ile Gly Ala Glu Pro Pro Ser Pro Gly Ala Leu Tyr Pro Ile Leu 35 40 45 Ser Ser Leu Arg Lys Gln Lys Tyr Ile Glu Thr Tyr Asn Glu Gly Lys 50 55 60 Arg Lys Val Tyr Arg Leu Thr Asp Lys Gly Arg Lys Tyr Leu Glu Glu 65 70 75 80 His Lys Glu Glu Ile Lys Lys Ala Leu Asp Phe Ala Glu Arg Phe 85 90 95 <210> 96 <211> 118 <212> PRT <213> Thermotoga maritima <400> 96 Met Arg His Arg Gly Gly Arg Gly Phe Arg Gly Trp Trp Leu Ala Ser 1 5 10 15 Thr Ile Leu Leu Leu Val Ala Glu Lys Pro Ser His Gly Tyr Glu Leu 20 25 30 Ala Glu Arg Leu Ala Glu Phe Gly Ile Glu Ile Pro Gly Ile Gly His 35 40 45 Met Gly Asn Ile Tyr Arg Val Leu Ala Asp Leu Glu Glu Ser Gly Phe 50 55 60 Leu Ser Thr Glu Trp Asp Thr Thr Val Ser Pro Pro Arg Lys Ile Tyr 65 70 75 80 Arg Ile Thr Pro Gln Gly Lys Leu Tyr Leu Arg Glu Ile Leu Arg Ser 85 90 95 Leu Glu Asp Met Lys Arg Arg Ile Glu Thr Leu Glu Glu Arg Ile Lys 100 105 110 Arg Val Leu Gln Glu Glu 115 <210> 97 <211> 70 <212> PRT <213> Thermotoga maritima <400> 97 Met Leu Ser Lys Arg Asp Ala Ile Leu Lys Ala Ala Val Glu Val Phe 1 5 10 15 Gly Lys Lys Gly Tyr Asp Arg Ala Thr Thr Asp Glu Ile Ala Glu Lys 20 25 30 Ala Gly Val Ala Lys Gly Leu Ile Phe His Tyr Phe Lys Asn Lys Glu 35 40 45 Glu Leu Tyr Tyr Gln Ala Tyr Met Ser Val Thr Glu Lys Leu Gln Lys 50 55 60 Glu Phe Glu Asn Phe Leu 65 70 <210> 98 <211> 291 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 98 Met Ser Lys Ser Trp Gly Lys Phe Ile Glu Glu Glu Glu Ala Glu Met 1 5 10 15 Ala Ser Arg Arg Asn Leu Met Ile Val Asp Gly Thr Asn Leu Gly Phe 20 25 30 Arg Phe Lys His Asn Asn Ser Lys Lys Pro Phe Ala Ser Ser Tyr Val 35 40 45 Ser Thr Ile Gln Ser Leu Ala Lys Ser Tyr Ser Ala Arg Thr Thr Ile 50 55 60 Val Leu Gly Asp Lys Gly Lys Ser Val Phe Arg Leu Glu His Leu Pro 65 70 75 80 Glu Tyr Lys Gly Asn Arg Asp Glu Lys Tyr Ala Gln Arg Thr Glu Glu 85 90 95 Glu Lys Ala Leu Asp Glu Gln Phe Phe Glu Tyr Leu Lys Asp Ala Phe 100 105 110 Glu Leu Cys Lys Thr Thr Phe Pro Thr Phe Thr Ile Arg Gly Val Glu 115 120 125 Ala Asp Asp Met Ala Ala Tyr Ile Val Lys Leu Ile Gly His Leu Tyr 130 135 140 Asp His Val Trp Leu Ile Ser Thr Asp Gly Asp Trp Asp Thr Leu Leu 145 150 155 160 Thr Asp Lys Val Ser Arg Phe Ser Phe Thr Thr Arg Arg Glu Tyr His 165 170 175 Leu Arg Asp Met Tyr Glu His His Asn Val Asp Asp Val Glu Gln Phe 180 185 190 Ile Ser Leu Lys Ala Ile Met Gly Asp Leu Gly Asp Asn Ile Arg Gly 195 200 205 Val Glu Gly Ile Gly Ala Lys Arg Gly Tyr Asn Ile Ile Arg Glu Phe 210 215 220 Gly Asn Val Leu Asp Ile Ile Asp Gln Leu Pro Leu Pro Gly Lys Gln 225 230 235 240 Lys Tyr Ile Gln Asn Leu Asn Ala Ser Glu Glu Leu Leu Phe Arg Asn 245 250 255 Leu Ile Leu Val Asp Leu Pro Thr Tyr Cys Val Asp Ala Ile Ala Ala 260 265 270 Val Gly Gln Asp Val Leu Asp Lys Phe Thr Lys Asp Ile Leu Glu Ile 275 280 285 Ala Glu Gln 290 <210> 99 <211> 53 <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> Features not yet classified <222> (17)..(37) <223> n is a, c, g or t <400> 99 tttctagctc taaaacnnnn nnnnnnnnnn nnnnnnncgg tgtttcgtcc ttt 53 <210> 100 <211> 148 <212> PRT <213> Sulfolobus solfataricus <400> 100 Met Glu Glu Lys Val Gly Asn Leu Lys Pro Asn Met Glu Ser Val Asn 1 5 10 15 Val Thr Val Arg Val Leu Glu Ala Ser Glu Ala Arg Gln Ile Gln Thr 20 25 30 Lys Asn Gly Val Arg Thr Ile Ser Glu Ala Ile Val Gly Asp Glu Thr 35 40 45 Gly Arg Val Lys Leu Thr Leu Trp Gly Lys His Ala Gly Ser Ile Lys 50 55 60 Glu Gly Gln Val Val Lys Ile Glu Asn Ala Trp Thr Thr Ala Phe Lys 65 70 75 80 Gly Gln Val Gln Leu Asn Ala Gly Ser Lys Thr Lys Ile Ala Glu Ala 85 90 95 Ser Glu Asp Gly Phe Pro Glu Ser Ser Gln Ile Pro Glu Asn Thr Pro 100 105 110 Thr Ala Pro Gln Gln Met Arg Gly Gly Gly Arg Gly Phe Arg Gly Gly 115 120 125 Gly Arg Arg Tyr Gly Arg Arg Gly Gly Arg Arg Gln Glu Asn Glu Glu 130 135 140 Gly Glu Glu Glu 145 <210> 101 <211> 673 <212> PRT <213> Thermus thermophilus ) <400> 101 Met Thr Leu Glu Glu Ala Arg Lys Arg Val Asn Glu Leu Arg Asp Leu 1 5 10 15 Ile Arg Tyr His Asn Tyr Arg Tyr Tyr Val Leu Ala Asp Pro Ile Ser 20 25 30 Asp Ala Glu Tyr Asp Arg Leu Leu Arg Glu Leu Lys Glu Leu Glu Glu 35 40 45 Arg Phe Pro Glu Leu Lys Ser Pro Asp Ser Pro Thr Leu Gln Val Gly 50 55 60 Ala Arg Pro Leu Glu Ala Thr Phe Arg Pro Val Arg His Pro Thr Arg 65 70 75 80 Met Tyr Ser Leu Asp Asn Ala Phe Asn Leu Asp Glu Leu Lys Ala Phe 85 90 95 Glu Glu Arg Ile Glu Arg Ala Leu Gly Arg Lys Gly Pro Phe Ala Tyr 100 105 110 Thr Val Glu His Lys Val Asp Gly Leu Ser Val Asn Leu Tyr Tyr Glu 115 120 125 Glu Gly Val Leu Val Tyr Gly Ala Thr Arg Gly Asp Gly Glu Val Gly 130 135 140 Glu Glu Val Thr Gln Asn Leu Leu Thr Ile Pro Thr Ile Pro Arg Arg 145 150 155 160 Leu Lys Gly Val Pro Glu Arg Leu Glu Val Arg Gly Glu Met Pro Ile 165 170 175 Glu Ala Phe Leu Arg Leu Asn Glu Glu Leu Glu Glu Arg Gly Glu Arg 180 185 190 Ile Phe Lys Asn Pro Arg Asn Ala Ala Ala Gly Ser Leu Arg Gln Lys 195 200 205 Asp Pro Arg Ile Thr Ala Lys Arg Gly Leu Arg Ala Thr Phe Tyr Ala 210 215 220 Leu Gly Leu Gly Leu Glu Glu Val Glu Arg Glu Gly Val Ala Thr Gln 225 230 235 240 Phe Ala Leu Leu His Trp Leu Lys Glu Lys Gly Phe Pro Val Glu His 245 250 255 Gly Tyr Ala Arg Ala Val Gly Ala Glu Gly Val Glu Ala Val Tyr Gln 260 265 270 Asp Trp Leu Lys Lys Arg Arg Ala Leu Pro Phe Glu Ala Asp Gly Val 275 280 285 Val Val Lys Leu Asp Glu Leu Ala Leu Trp Arg Glu Leu Gly Tyr Thr 290 295 300 Ala Arg Ala Pro Arg Phe Ala Ile Ala Tyr Lys Phe Pro Ala Glu Glu 305 310 315 320 Lys Glu Thr Arg Leu Leu Asp Val Val Phe Gln Val Gly Arg Thr Gly 325 330 335 Arg Val Thr Pro Val Gly Ile Leu Glu Pro Val Phe Leu Glu Gly Ser 340 345 350 Glu Val Ser Arg Val Thr Leu His Asn Glu Ser Tyr Ile Glu Glu Leu 355 360 365 Asp Ile Arg Ile Gly Asp Trp Val Leu Val His Lys Ala Gly Gly Val 370 375 380 Ile Pro Glu Val Leu Arg Val Leu Lys Glu Arg Arg Thr Gly Glu Glu 385 390 395 400 Arg Pro Ile Arg Trp Pro Glu Thr Cys Pro Glu Cys Gly His Arg Leu 405 410 415 Leu Lys Glu Gly Lys Val His Arg Cys Pro Asn Pro Leu Cys Pro Ala 420 425 430 Lys Arg Phe Glu Path To Arg His Phe Path Ser Arg Lys Path Met Asp 435 440 445 Ile Gln Gly Leu Gly Glu Lys Leu Ile Glu Arg Leu Leu Glu Lys Gly 450 455 460 Leu Val Lys Asp Val Ala Asp Leu Tyr Arg Leu Arg Lys Glu Asp Leu 465 470 475 480 Val Gly Leu Glu Arg Met Gly Glu Lys Ser Ala Gln Asn Leu Leu Arg 485 490 495 Gln Ile Glu Glu Ser Lys Lys Arg Gly Leu Glu Arg Leu Leu Tyr Ala 500 505 510 Leu Gly Leu Pro Gly Val Gly Glu Val Leu Ala Arg Asn Leu Ala Ala 515 520 525 Arg Phe Gly Asn Met Asp Arg Leu Leu Glu Ala Ser Leu Glu Glu Leu 530 535 540 Leu Glu Val Glu Glu Val Gly Glu Leu Thr Pathway Arg Pathway To Leu Glu 545 550 555 560 Thr Leu Lys Asp Pro Ala Phe Arg Asp Leu Val Arg Arg Leu Lys Glu 565 570 575 Ala Gly Val Glu Met Glu Ala Lys Glu Lys Gly Gly Glu Ala Leu Lys 580 585 590 Gly Leu Thr Phe Val Ile Thr Gly Glu Leu Ser Arg Pro Arg Glu Glu 595 600 605 Val Lys Ala Leu Leu Arg Arg Leu Gly Ala Lys Val Thr Asp Ser Val 610 615 620 Ser Arg Lys Thr Ser Tyr Leu Val Val Gly Glu Asn Pro Gly Ser Lys 625 630 635 640 Leu Glu Lys Ala Arg Ala Leu Gly Val Pro Thr Leu Thr Glu Glu Glu 645 650 655 Leu Tyr Arg Leu Leu Glu Ala Arg Thr Gly Lys Lys Ala Glu Glu Leu 660 665 670 Val <210> 102 <211> 775 <212> PRT <213> artificial sequence <220> <223> Synthesis <400> 102 Met Ile Leu Asp Ala Asp Tyr Ile Thr Glu Asp Gly Lys Pro Ile Ile 1 5 10 15 Arg Ile Phe Lys Lys Glu Asn Gly Glu Phe Lys Val Glu Tyr Asp Arg 20 25 30 Asn Phe Arg Pro Tyr Ile Tyr Ala Leu Leu Lys Asp Asp Ser Ala Ile 35 40 45 Asp Asp Val Lys Lys Ile Thr Ala Glu Arg His Gly Lys Val Val Arg 50 55 60 Val Val Asp Val Glu Lys Val Lys Lys Lys Phe Leu Gly Arg Pro Ile 65 70 75 80 Glu Val Trp Lys Leu Tyr Phe Glu His Pro Gln Asp Val Pro Ala Ile 85 90 95 Arg Asp Lys Ile Arg Glu His Pro Ala Val Ile Asp Ile Phe Glu Tyr 100 105 110 Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Lys Gly Leu Ile Pro 115 120 125 Met Glu Gly Asn Glu Glu Leu Lys Leu Leu Ala Phe Asp Ile Glu Thr 130 135 140 Leu Tyr His Glu Gly Glu Glu Phe Gly Lys Gly Pro Ile Ile Met Ile 145 150 155 160 Ser Tyr Ala Asp Glu Glu Gly Ala Lys Val Ile Thr Trp Lys Lys Val 165 170 175 Asp Leu Pro Tyr Val Glu Val Val Ser Ser Glu Arg Glu Met Ile Lys 180 185 190 Arg Phe Leu Lys Val Ile Arg Glu Lys Asp Pro Asp Val Ile Ile Thr 195 200 205 Tyr Asn Gly Asp Asn Phe Asp Phe Pro Tyr Leu Leu Lys Arg Ala Glu 210 215 220 Lys Leu Gly Met Lys Leu Pro Leu Gly Arg Asp Gly Ser Glu Pro Lys 225 230 235 240 Met Gln Arg Leu Gly Asp Ser Leu Ala Val Glu Ile Lys Gly Arg Ile 245 250 255 His Phe Asp Leu Tyr Pro Val Ile Arg Arg Thr Ile Asn Leu Pro Thr 260 265 270 Tyr Thr Leu Glu Ala Val Tyr Glu Ala Ile Phe Gly Lys Pro Lys Glu 275 280 285 Lys Val Tyr Pro His Glu Ile Ala Glu Ala Trp Glu Thr Gly Lys Gly 290 295 300 Leu Glu Arg Val Ala Lys Tyr Ser Met Glu Asp Ala Lys Val Thr Tyr 305 310 315 320 Glu Leu Gly Arg Glu Phe Phe Pro Met Glu Ala Gln Leu Ala Arg Leu 325 330 335 Val Gly Gln Pro Leu Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu 340 345 350 Val Glu Trp Tyr Leu Leu Arg Lys Ala Tyr Glu Arg Asn Glu Leu Ala 355 360 365 Pro Asn Lys Pro Asp Glu Arg Glu Tyr Glu Arg Arg Leu Arg Glu Ser 370 375 380 Tyr Glu Gly Gly Tyr Val Lys Glu Pro Glu Lys Gly Leu Trp Glu Gly 385 390 395 400 Ile Val Ser Leu Asp Phe Arg Ser Leu Tyr Pro Ser Ile Ile Ile Thr 405 410 415 His Asn Val Ser Pro Asp Thr Leu Asn Lys Glu Gly Cys Gly Glu Tyr 420 425 430 Asp Val Ala Pro Glu Val Gly His Arg Phe Cys Lys Asp Phe Pro Gly 435 440 445 Phe Ile Pro Ser Leu Leu Gly Ser Leu Leu Asp Glu Arg Gln Lys Ile 450 455 460 Lys Arg Arg Met Lys Ala Ser Lys Asp Pro Ile Glu Arg Lys Leu Leu 465 470 475 480 Asp Tyr Arg Gln Arg Ala Ile Lys Ile Leu Ala Asn Ser Tyr Tyr Gly 485 490 495 Tyr Tyr Gly Tyr Ala Lys Ala Arg Trp Tyr Cys Lys Glu Cys Ala Glu 500 505 510 Ser Val Thr Ala Trp Gly Arg Glu Tyr Ile Glu Leu Val Arg Arg Glu 515 520 525 Leu Glu Glu Arg Gly Phe Lys Val Leu Tyr Ile Asp Thr Asp Gly Leu 530 535 540 Tyr Ala Thr Ile Pro Gly Glu Lys Asn Trp Glu Glu Ile Lys Arg Arg 545 550 555 560 Ala Leu Glu Phe Val Asn Tyr Ile Asn Ala Lys Leu Pro Gly Leu Leu 565 570 575 Glu Leu Glu Tyr Glu Gly Phe Tyr Thr Arg Gly Phe Phe Val Thr Lys 580 585 590 Lys Lys Tyr Ala Leu Ile Asp Glu Glu Gly Lys Ile Ile Thr Arg Gly 595 600 605 Leu Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu Thr Gln 610 615 620 Ala Lys Val Leu Glu Ala Ile Leu Lys His Gly Asn Val Glu Glu Ala 625 630 635 640 Val Lys Ile Val Lys Glu Val Thr Glu Lys Leu Ser Lys Tyr Glu Ile 645 650 655 Pro Pro Glu Lys Leu Val Ile Tyr Glu Gln Ile Thr Arg Pro Leu Asn 660 665 670 Glu Tyr Lys Ala Ile Gly Pro His Val Ala Val Ala Lys Arg Leu Ala 675 680 685 Ala Arg Gly Ile Lys Val Arg Pro Gly Met Val Ile Gly Tyr Val Val 690 695 700 Leu Arg Gly Asp Gly Pro Ile Ser Lys Arg Ala Ile Ala Ala Glu Glu 705 710 715 720 Phe Asp Pro Lys Lys His Lys Tyr Asp Ala Glu Tyr Tyr Ile Glu Asn 725 730 735 Gln Val Leu Pro Ala Val Leu Arg Ile Leu Glu Ala Phe Gly Tyr Arg 740 745 750 Lys Glu Asp Leu Arg Trp Gln Lys Thr Lys Gln Val Gly Leu Gly Ala 755 760 765 Trp Leu Lys Val Lys Lys Ser 770 775

Claims

1. A composition for assembling a synthon, comprising: (a) 5'-3' exonuclease; (b) a strand displacement polymerase comprising an amino acid sequence selected from any of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 33 to SEQ ID NO: 55, or SEQ ID NO: 102; (c) optionally, a single-stranded DNA binding protein comprising an amino acid sequence selected from SEQ ID NO: 2 or any of SEQ ID NO: 56 to SEQ ID NO: 97; and (d) non-naturally occurring buffers, wherein the composition does not include a crowding agent and / or a non-strand displacing polymerase.

2. The composition according to claim 1, further comprising a ligase and / or a single-stranded binding domain.

3. The composition according to any one of claims 1-2, further comprising a set of at least two polynucleotides.

4. The composition of any one of claims 1-2, wherein the strand-displacing polymerase is non-naturally occurring.

5. The composition of claim 1 or 2, wherein the strand-displacing polymerase is thermostable.

6. The composition of claim 1 or 2, wherein the strand-displacing polymerase is a B-family polymerase.

7. The composition of claim 1 or 2, which does not include 9°N, Phusion, Vent or Pfu DNA polymerase.

8. The composition of claim 1 or 2, wherein the strand displacement polymerase is SEQ ID NO: 1 and the single-stranded DNA binding protein is SEQ ID NO:

2.

9. The composition of claim 1 or 2, wherein the strand displacement polymerase has the amino acid sequence of SEQ ID NO:

3.

10. The composition according to claim 1 or 2, wherein the single-stranded DNA binding protein is an extremely thermostable single-stranded DNA binding protein, Escherichia coli recA, T7 gene 2.5 product, bacteriophage λRedB or Rac prophage RecT.

11. The composition according to claim 1 or 2, wherein the 5'-3' exonuclease has single-stranded endonuclease activity. 12 . The composition of claim 1 , wherein the 5′-3′ exonuclease has an amino acid sequence of SEQ ID NO:

98.

13. The composition of claim 1 or 2, further comprising a potassium salt at a concentration of at least 7 mM.

14. A composition according to claim 1 or 2, further comprising a group of polynucleotides, wherein the sequence of at least one polynucleotide in the group overlaps with another polynucleotide in the group; and wherein the polynucleotides in the group are selected from: (i) double-stranded polynucleotides; (ii) single-stranded oligonucleotides; (iii) at least one double-stranded polynucleotide and at least one single-stranded oligonucleotide; and (iv) a subgroup of polynucleotides that are identical to each other except for sequences that vary between members of the subgroup.

15. The composition of claim 14, wherein at least one member of the set of polynucleotides comprises a random sequence located between given sequences at each end for hybridization to a second single-stranded genomic polynucleotide.

16. The composition of claim 15, wherein the random sequence is single-stranded and is capable of hybridizing with a target genomic sequence for guiding the Cas protein to a target genomic nucleic acid for gene editing.

17. A method for forming a synthon, comprising: The composition according to any one of claims 1-13 is incubated with a group of polynucleotides having overlapping sequences capable of hybridizing under suitable reaction conditions, wherein the polynucleotides in the group are selected from: (i) double-stranded polynucleotides; (ii) single-stranded oligonucleotides; (iii) at least one double-stranded polynucleotide and at least one single-stranded oligonucleotide; and (iv) a subpopulation of polynucleotides that are identical to each other except for sequences that vary between members of the subpopulation; and at least some of the polynucleotides in the group are joined with other polynucleotides in the group to prepare synthons.

18. The method of claim 17, wherein at least one member of the set of polynucleotides comprises a random sequence located between given sequences at each end for hybridization to a second single-stranded genomic polynucleotide.

19. The method of claim 18, wherein the random sequence is single-stranded and is capable of hybridizing with a target genomic sequence for directing a Cas protein to a target genomic nucleic acid for gene editing.

20. The method of any one of claims 17-19, wherein all or part of the polynucleotides in the set are double-stranded.

21. The method according to any one of claims 17 to 19, wherein the double-stranded polynucleotide is an overlapping PCR product; overlapping restriction fragments or a synthetic double-stranded molecule assembled from complementary single-stranded oligonucleotides.

22. The method of any one of claims 17-19, wherein all or part of the polynucleotides in the set are single-stranded oligonucleotides.

23. The method of any one of claims 17-19, wherein the set of polynucleotides comprises at least one double-stranded polynucleotide and at least one single-stranded oligonucleotide.

24. The method of any one of claims 17-19, wherein the set of polynucleotides comprises subpopulations of polynucleotides that are identical to one another except for sequences that vary between members of the subpopulation.

25. The method of any one of claims 17-19, wherein the length of overlapping sequences of the polynucleotides in the group is less than 2 kilobases.

26. The method of any one of claims 17-19, wherein the strand-displacing polymerase comprises an amino acid sequence selected from any of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 33 to SEQ ID NO: 55, or SEQ ID NO:

102.

27. The method of any one of claims 17-19, wherein at least one member of the set of polynucleotides comprises a random sequence between the ends of a given sequence.

28. The method according to claim 27, further comprising screening random sequences having activity of hybridizing with genomic DNA and identifying random sequences having hybridization activity.

29. The method according to claim 27, further comprising performing gene editing by transcribing a random sequence having hybridization activity to form RNA, and using the RNA for gene editing in the presence of a Cas protein.

30. A kit for polynucleotide assembly, comprising: (a) 5'-3' exonuclease; (b) a strand displacement polymerase comprising an amino acid sequence selected from any of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 33 to SEQ ID NO: 55, or SEQ ID NO: 102; and (c) optionally, a single-stranded DNA binding protein comprising an amino acid sequence selected from SEQ ID NO: 2 or any of SEQ ID NO: 56 to SEQ ID NO: 97; wherein the kit optionally does not include a crowding agent and / or a non-strand displacing polymerase.

31. The kit of claim 30, further comprising a ligase.

32. The kit according to claim 30 or 31 further comprises dNTPs.

33. The kit according to any one of claims 30-31, further comprising a buffer.

34. The kit of any one of claims 30-31, wherein (a) to (c) are in one or more different containers.

35. The kit of claim 34, wherein at least one of the containers is a storage container or a reaction container.

Images