Variant of protein and a method for producing L-Threonine using the same

Abstract

The present disclosure relates to a microorganism of the genus Corynebacterium comprising a protein variant, a polynucleotide encoding the protein variant, and one or more selected from the group consisting of the protein variant and the polynucleotide, and a method for producing L-threonine using the microorganism.

Description

Technology Field

[0001] The present disclosure relates to a novel protein variant, a microorganism of the genus Corynebacterium comprising said protein variant, and a method for producing L-threonine using said microorganism. Background Technology

[0003] L-threonine (Thr, T) is classified as an essential amino acid and is used in the manufacture of various products, including animal feed, food additives, and pharmaceuticals. Its use is increasing across diverse fields due to its functions in improving gut health, such as regulating nutritional metabolism (including lipid metabolism), protein synthesis, the proliferation and differentiation of embryonic stem cells, intestinal barrier function, immunological responses, and the dynamic equilibrium of the gut microbiome.

[0004] Based on this trend, various attempts are being made to improve production capacity in the manufacturing method of L-threonine using various microorganisms, including those of the genus Corynebacterium.

[0005] Despite these efforts, the development of technology to improve L-threonine production capacity is still required. Prior art literature

[0007] Republic of Korea Published Patent No. 10-1996-0023072 The problem to be solved

[0008] One objective of the present disclosure is to provide a novel protein variant.

[0009] Another object of the present disclosure is to provide a polynucleotide encoding the protein variant.

[0010] Another object of the present disclosure is a genus of Corynebacterium comprising one or more selected from the group consisting of the protein variant and the polynucleotide encoding the same. Corynebacterium It is to provide microorganisms.

[0011] Another object of the present disclosure is to provide a method for producing L-threonine, comprising the step of culturing the microorganism in a medium. means of solving the problem

[0013] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in this disclosure may be applied to other descriptions and embodiments. That is, all combinations of the various elements disclosed in this disclosure fall within the scope of this disclosure. Furthermore, the scope of this disclosure should not be considered limited by the specific descriptions provided below. Additionally, numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated by reference into this specification in their entirety to more clearly explain the state of the art to which the present invention pertains and the content of the present invention.

[0015] One aspect of the present disclosure provides a protein variant comprising an amino acid sequence in which the amino acid corresponding to the 233rd position from the N-terminus in the amino acid sequence of SEQ ID NO. 304 is substituted with an amino acid different from the original, or an amino acid sequence in which the amino acid corresponding to the 41st position from the N-terminus in the amino acid sequence of SEQ ID NO. 305 is substituted with an amino acid different from the original.

[0016] In the present disclosure, the sequences of SEQ ID NO. 304 and SEQ ID NO. 305 are as shown in Table 1 below.

[0017] 단백질명(gene ID,gene name) 서열 (N-말단 → C-말단) 서열번호 Glutamate dehydrogenase (NCgl1999, gdh) MTVDEQVSNYYDMLLKRNAGEPEFHQAVAEVLESLKIVLEKDPHYADYGLIQRLCEPERQLIFRVPWVDDQGQVHVNRGFRVQFNSALGPYKGGLRFHPSVNLGIVKFLGFEQIFKNSLTGLPIGGGKGGSDFDPKGKSDLEIMRFCQSFMTELHRHIGEYRDVPAGDIGVGGREIGYLFGHYRRMANQHESGVLTGKGLTWGGSLVRTEATGYGCVYFVSEMIKAKGESISGQKIIVSGSGNVATYAIEKAQELGATVIGFSDSSGWVHTPNGVDVAKLREIKEVRRARVSVYADEVEGATYHTDGSIWDLKCDIALPCATQNELNGENAKTLADNGCRFVAEGANMPSTPEAVEVFRERDIRFGPGKAANAGGVATSALEMQQNASRDSWSFEYTDERLQVIMKNIFKTCAETAAEYGHENDYVVGANIAGFKKVADAMLAQGVI 304 DNA-directed RNA polymerase subunit beta(NCgl0472, rpoC) MLDVNVFDELRIGLATADDIRRWSKGEVKKPETINYRTLKPEKDGLFCERIFGPTRDWECACGKYKRVRYKGIICERCGVEVTKSKVRRERMGHIELAAPVTHIWYFKGVPSRLGYLLDLAPKDLDLIIYFGANIITSVDEEARHSDQTTLEAEMLLEKKDVEADAESDIAERAEKLEEDLAELEAAGAKADARRKVQAAADKEMQHIRERAQREIDRLDEVWQTFIKLAPKQMIRDEKLYDELIDRYEDYFTGGMGAESIEALIQNFDLDAEAEELRDIINNGKGQKKMRALKRLKVVAAFQRSGNDPAGMVLNAIPVIPPELRPMVQLDGGRFATSDLNDLYRRVINRNNRLKRMIELGAPEIIVNNEKRMLQESVDALFDNGRRGRPVTGPGNRPLKSLSDLLKGKQGRFRQNLLGKRVDYSGRSVIIVGPQLRLHECGLPKLMALELFKPFVMKRLVENEYAQNIKSAKRMVERQRPEVWDVLEEAISEHPVMLNRAPTLHRLGIQAFEPVLVEGKAIQLHPLACEAFNADFDGDQMAVHLPLSAEAQAEARVLMLASNNILSPASGKPLAMPRLDMVTGLYYLTLEKSSEEFGGQGAYQPADENGPEKGVYSSLAEAIMAYDRGVLGLQAPVRIRLNHLRPPAEVEAEQFPDGWNQGETWLAHTTLGRVMFNEILPWNYPYLEGVMVRKGGGSDKIMLGDVVNDLAAKYPMITVAQTMDKMKDAGFYWSTRSGVTIAMSDVLVLPNKEEMLDRYEESARQIEVKYNRGKLTGRERYDRLVELWKDATDEVGQAVEDLYPDDNPIPMIVKSGAAGNMRQIWTLAGMKGMVVNSKGDYITRPIKTSFREGLTVLEYFNNSHGSRKGLADTALRTADSGYLTRRLVDVAQDVIVRVEDCGTRQGVRVPVAAEVLDATGAVTGYTRHDLIETSVSGRVLAGDATNAAGEVVLAAGTDLTELNIDLLVEAGIKDVKVRSVLTCQTPTGVCAKCYGKSMASGQQVDIGEAVGIVAAQSIGEPGTQLTMRTFHQGGVGGDITGGLPRVQELFEARVPKNCAPIASVEGVIHLEDEGNFYTLTIVPDDGSDNVVYEKLSKRQGLASTRVAMESNAGAFIERTLTEGDRVTVGQRLLRGAADPHDVLEILGRRGVEQHLIDEVQAVYRAQGVAIHDKHIEIIIRQMLRRGTVIESGSTEFLPGSLVDLSEAKLANSEAIGAGGQPAELRSEIMGITKASLATESWLSAASFQETTRVLTDAAINKRSDKLIGLKENVIIGKLIPAGTGISRYRNISIKPTEAARNAAYSIPTYGESIYGDDGFGEFTGASVPLDEAF 305

[0018] In one embodiment, the protein variant of the present disclosure described above may essentially consist of an amino acid sequence in which the amino acid corresponding to the 233rd position from the N-terminus in the amino acid sequence of SEQ ID NO. 304 is substituted with an amino acid different from the original, or an amino acid sequence in which the amino acid corresponding to the 41st position from the N-terminus in the amino acid sequence of SEQ ID NO. 305 is substituted with an amino acid different from the original. In another embodiment, the protein variant of the present disclosure described above may consist of an amino acid sequence in which the amino acid corresponding to the 233rd position from the N-terminus in the amino acid sequence of SEQ ID NO. 304 is substituted with an amino acid different from the original, or an amino acid sequence in which the amino acid corresponding to the 41st position from the N-terminus in the amino acid sequence of SEQ ID NO. 305 is substituted with an amino acid different from the original. In the present disclosure, the amino acid sequence of SEQ ID NO. 304 may be the amino acid sequence of glutamate dehydrogenase. In one embodiment, the glutamate dehydrogenase may be a protein encoded by the NCgl1999 gene (gdh).

[0019] In the present disclosure, the amino acid sequence of SEQ ID NO. 305 may be the amino acid sequence of DNA-directed RNA polymerase subunit beta. In one embodiment, the DNA-directed RNA polymerase subunit beta may be a protein encoded by the NCgl0472 gene (rpoC).

[0020] In one embodiment, the amino acid corresponding to the 233rd position from the N-terminus in the amino acid sequence of SEQ ID NO. 304 may be glycine (Gly, G), but is not limited thereto.

[0021] In one embodiment, the amino acid corresponding to the 41st position from the N-terminus in the amino acid sequence of SEQ ID NO. 305 may be proline (Pro, P), but is not limited thereto.

[0022] In one embodiment, the amino acid corresponding to the 233rd position from the N-terminus in the amino acid sequence of SEQ ID NO. 304 may be substituted with aspartic acid (Asp, D), but is not limited thereto.

[0023] In one embodiment, the amino acid corresponding to the 41st position from the N-terminus in the amino acid sequence of SEQ ID NO. 305 may be substituted with serine (Ser, S), but is not limited thereto.

[0024] The protein variant of the present disclosure may have an activity that increases the L-threonine production capacity compared to the wild-type protein (polypeptide).

[0025] Protein variants of the present disclosure may include amino acid sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.7% or more homology or identity with an amino acid sequence in which the amino acid corresponding to the 233rd position from the N-terminus of the amino acid sequence of SEQ ID NO. 304 is substituted with an amino acid different from the original (e.g., aspartic acid) or an amino acid sequence in which the amino acid corresponding to the 41st position from the N-terminus of the amino acid sequence of SEQ ID NO. 305 is substituted with an amino acid different from the original (e.g., serine). Furthermore, it is obvious that variants having amino acid sequences in which some sequences are deleted, modified, substituted, conservedly substituted, or added are included within the scope of the present disclosure, provided that such amino acid sequences have such homology or identity and exhibit an efficacy (efficacy of increasing L-threonine production) corresponding to the variants of the present disclosure.

[0026] For example, cases where there are sequence additions or deletions, naturally occurring mutations, silent mutations, or conservative substitutions that do not alter the function of the variant of the present disclosure at the N-terminus, C-terminus, and / or within the amino acid sequence.

[0027] The aforementioned "conservative substitution" refers to the substitution of one amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions can generally occur based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. Typically, conservative substitutions have little to no effect on the activity of a protein or polypeptide.

[0028] In this disclosure, the term “variant” refers to a polypeptide in which one or more amino acids are conservatively substituted and / or modified, resulting in a sequence of amino acids different from that of the variant prior to modification, while retaining functions or properties. Such variants can generally be identified by modifying one or more amino acids in the amino acid sequence of the polypeptide and evaluating the properties of the modified polypeptide. That is, the capabilities of the variant may be increased, unchanged, or decreased compared to the polypeptide prior to modification. Additionally, some variants may include variants in which one or more parts, such as an N-terminal leader sequence or a transmembrane domain, have been removed. Other variants may include variants in which a portion of the N- and / or C-terminus of a mature protein has been removed. The term "variant" mentioned above may be used interchangeably with terms such as variant, modification, variant polypeptide, mutated protein, mutation, and variant (in English, modification, modified polypeptide, modified protein, mutant, mutein, divergent, etc.), and is not limited to these terms as long as they are used with the meaning of being mutated.

[0029] Additionally, the variant may include deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, a signal (or leader) sequence involved in co-translational or post-translational protein translocation may be conjugated to the N-terminus of the variant. Additionally, the variant may be conjugated with another sequence or linker to enable identification, purification, or synthesis.

[0030] In one embodiment, the protein variant of the present disclosure described above may have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more and less than 100% homology or identity with the amino acid sequence of SEQ ID NO. 304, or may have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more and less than 100% homology or identity with the amino acid sequence of SEQ ID NO. 305. That is, the protein variant of the present disclosure described above may have amino acid sequence identity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more and less than 100% with respect to the amino acid sequence of SEQ ID NO. 304 or SEQ ID NO. 305, respectively.

[0032] Another aspect of the present disclosure provides a protein variant comprising any one amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs. 36 to 68. In one embodiment, the protein variant may be a protein variant comprising the amino acid sequence of SEQ ID NO. 43 or SEQ ID NO. 47. In another embodiment, the variant of the present disclosure may essentially consist of the amino acid sequence of SEQ ID NO. 43 or SEQ ID NO. 47. In yet another embodiment, the variant of the present disclosure may consist of the amino acid sequence of SEQ ID NO. 43 or SEQ ID NO. 47.

[0033] In the present disclosure, the amino acid sequence of SEQ ID NO. 43 may be the sequence of a protein variant in which the 223rd amino acid of the protein (Glutamate dehydrogenase) encoded by the NCgl1999 gene (gdh) is substituted from glycine (Gly, G) to aspartic acid (Asp, D).

[0034] In the present disclosure, the amino acid sequence of SEQ ID NO. 47 may be the sequence of a protein variant in which the 41st amino acid of the protein (DNA-directed RNA polymerase subunit beta) encoded by the NCgl0472 gene (rpoC) is substituted from proline (Pro, P) to serine (Ser, S).

[0035] Protein variants of the present disclosure may comprise any one amino acid sequence selected from the group consisting of amino acid sequences of SEQ ID NOs. 36 to 68 (e.g., the amino acid sequence of SEQ ID NO. 43 or SEQ ID NO. 47), or may comprise an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% or more homology or identity with said amino acid sequence. Furthermore, it is obvious that variants having an amino acid sequence in which some sequences are deleted, modified, substituted, conservedly substituted, or added are also included within the scope of the present disclosure, provided that such an amino acid sequence has such homology or identity and exhibits an efficacy (efficacy of increasing L-threonine production) corresponding to that of the variants of the present disclosure.

[0036] For example, cases where there are sequence additions or deletions, naturally occurring mutations, silent mutations, or conservative substitutions that do not alter the function of the variant of the present disclosure at the N-terminus, C-terminus, and / or within the amino acid sequence.

[0037] In the above protein variants, conservative substitutions, variants, etc. are as described above.

[0039] In this disclosure, the terms 'homology' or 'identity' refer to the degree of similarity between two given amino acid sequences or base sequences and may be expressed as a percentage. The terms homology and identity may often be used interchangeably.

[0040] Sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard arrangement algorithms, and a default gap penalty established by the program used may be utilized. Practically, homologous or identical sequences can generally be hybridized with the entire sequence or a part thereof under moderate or high stringent conditions. It is evident that hybridization also includes hybridization with polynucleotides containing common codons or codons that account for codon degeneracy.

[0041] Whether any two polynucleotide or polypeptide sequences have homology, similarity, or identity can be determined using a known computer algorithm, such as the “FASTA” program, using default parameters as in, for example, Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), as performed in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later) (GCG program package (Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] (Including Academic Press, San Diego, 1994, and [CARILLO ETA / .](1988) SIAM J Applied Math 48: 1073). For example, homology, similarity, or identity can be determined using BLAST from the National Biotechnology Information Database Center or ClustalW.

[0042] The homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program, such as that described in, for example, Smith and Waterman, Adv. Appl. Math (1981) 2:482, or Needleman et al. (1970), J Mol Biol. 48:443. In summary, a GAP program can be defined as the total number of symbols in the shorter of the two sequences divided by the number of similarly arranged symbols (i.e., nucleotides or amino acids). The default parameters for a GAP program are (1) a binary comparison matrix (containing values ​​of 1 for identity and 0 for non-identity) and, as disclosed by Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979), or Gribskov et al. (1986) Nucl. Acids Res. 14: A weighted comparison matrix of 6745 (or an EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a gap opening penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for terminal gaps.

[0043] As an example of the present disclosure, the variant of the present disclosure described above may have an activity that increases the L-threonine production capacity compared to the wild-type protein (polypeptide).

[0045] In this disclosure, the term “corresponding to” refers to an amino acid residue at a position listed in a polypeptide, or an amino acid residue that is similar, identical, or homologous to a residue listed in a polypeptide. Identifying the amino acid at the corresponding position may involve determining a specific amino acid of a sequence that references a specific sequence. As used in this disclosure, “corresponding region” generally refers to a similar or corresponding position in a related protein or a reference protein.

[0046] For example, any amino acid sequence is aligned with an amino acid sequence in which the amino acid corresponding to the 233rd position from the N-terminus in the amino acid sequence of SEQ ID NO. 304 is substituted with an amino acid different from the original (e.g., aspartic acid) or an amino acid sequence in which the amino acid corresponding to the 41st position from the N-terminus in the amino acid sequence of SEQ ID NO. 305 is substituted with an amino acid different from the original (e.g., serine); or any one amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO. 36 to SEQ ID NO. 68 (e.g., the amino acid sequence of SEQ ID NO. 43 or SEQ ID NO. 47), and based thereon, each amino acid residue of the said amino acid sequence is replaced with an amino acid sequence in which the amino acid corresponding to the 233rd position from the N-terminus in the amino acid sequence of SEQ ID NO. 304 is substituted with an amino acid different from the original (e.g., aspartic acid) or an amino acid sequence in which the amino acid corresponding to the 41st position from the N-terminus in the amino acid sequence of SEQ ID NO. 305 is substituted with an amino acid different from the original (e.g., serine); Alternatively, numbering may be performed by referring to the numerical positions of amino acid residues corresponding to amino acid residues of any one amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs 36 to 68 (e.g., the amino acid sequence of SEQ ID NO. 43 or SEQ ID NO. 47). For example, a sequence alignment algorithm such as that described in the present disclosure can identify the position of an amino acid, or the position where modifications such as substitution, insertion, or deletion occur, by comparing with a query sequence (also referred to as a “reference sequence”).

[0047] For such alignment, examples such as the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000), Trends Genet. 16: 276-277) may be used, but are not limited thereto, and sequence alignment programs and pairwise sequence comparison algorithms known in the art may be appropriately used.

[0049] Another aspect of the present disclosure is to provide a polynucleotide encoding a protein variant of the present disclosure.

[0050] In the present disclosure, the term “polynucleotide” refers to a polymer of nucleotides in which nucleotide monomers are linked together in a long chain by covalent bonds, as a DNA or RNA strand of a certain length or longer, and more specifically, as a polynucleotide fragment encoding the variant.

[0051] In one example, the polynucleotide may be a polynucleotide encoding a protein variant comprising an amino acid sequence in which the amino acid corresponding to the 233rd position from the N-terminus in the amino acid sequence of SEQ ID NO. 304 is substituted with an amino acid different from the original (e.g., aspartic acid) or an amino acid sequence in which the amino acid corresponding to the 41st position from the N-terminus in the amino acid sequence of SEQ ID NO. 305 is substituted with an amino acid different from the original (e.g., serine).

[0052] In one example, the polynucleotide may comprise a polynucleotide comprising any one nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NO. 69 to SEQ ID NO. 101, such as the nucleotide sequence of SEQ ID NO. 76 or SEQ ID NO. 80.

[0053] A polynucleotide encoding a protein variant of the present disclosure may have or include any one nucleotide sequence selected from the group consisting of an amino acid sequence in which the amino acid corresponding to the 233rd position from the N-terminus in the amino acid sequence of SEQ ID NO. 304 is substituted with an amino acid different from the original (e.g., aspartic acid) or an amino acid sequence in which the amino acid corresponding to the 41st position from the N-terminus in the amino acid sequence of SEQ ID NO. 305 is substituted with an amino acid different from the original (e.g., serine), and the nucleotide sequences of SEQ ID NO. 69 to SEQ ID NO. 101, such as the nucleotide sequence of SEQ ID NO. 76 or SEQ ID NO. 80. In another example, the polynucleotide may comprise a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% or more homology or identity with the nucleotide sequence. Furthermore, it is obvious that polynucleotides having nucleotide sequences in which some sequences are deleted, modified, substituted, conservatively substituted, or added, provided that the sequences have such homology or identity and encode a polypeptide or protein that exhibits efficacy (increased L-threonine production) corresponding to the variants of the present disclosure, are also included within the scope of the present disclosure.

[0054] In one example, the nucleotide sequence or amino acid sequence provided in this specification may include one that has been modified by conventional mutagenesis, such as direct evolution and / or site-directed mutagenesis, to the extent that their original or intended function is maintained. In one example, the phrase “a polynucleotide or polypeptide comprises a specific nucleotide sequence or amino acid sequence” may mean that the polynucleotide or polypeptide is composed of or essentially comprises the specific nucleotide sequence or amino acid sequence, or (ii) composed of or essentially comprises an amino acid sequence having 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 99.9% or more homology with the specific nucleotide sequence or amino acid sequence, and maintains its original function and / or intended function.

[0055] The polynucleotide of the present disclosure may have various modifications made to its coding region within a range that does not alter the amino acid sequence of the variant of the present disclosure, taking into account the degeneracy of the codons or the codons preferred by the organism intended to express the variant of the present disclosure. Specifically, the polynucleotide of the present disclosure may have or include a nucleotide sequence having homology or identity of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% with any one nucleotide sequence selected from the group consisting of nucleotide sequences of SEQ ID NO. 69 to 101 (e.g., the nucleotide sequence of SEQ ID NO. 76 or SEQ ID NO. 80), or may be composed of or essentially composed of a nucleotide sequence having homology or identity of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% with said nucleotide sequence, but is not limited thereto.

[0056] Additionally, the polynucleotides of the present disclosure may be included without limitation as long as they are probes that can be prepared from known gene sequences, for example, sequences that can be hybridized under stringent conditions with a sequence complementary to all or part of the polynucleotide sequence of the present disclosure. The term "stringent condition" means a condition that enables specific hybridization between polynucleotides. For example, conditions may be listed in which polynucleotides with high homology or identity are hybridized with each other, polynucleotides with homology or identity of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, and polynucleotides with lower homology or identity are not hybridized with each other, or conditions in which washing is performed once, specifically two to three times, at a salt concentration and temperature equivalent to the washing conditions of conventional southern hybridization, such as 60°C, 1×SSC, 0.1% SDS, specifically 60°C, 0.1×SSC, 0.1% SDS, more specifically 68°C, 0.1×SSC, 0.1% SDS.

[0057] Hybridization requires that two nucleic acids have complementary sequences, even though a mismatch between bases may be possible depending on the degree of hybridization. The term “complementary” is used to describe the relationship between nucleotide bases that can hybridize with each other. For example, with respect to DNA, adenine is complementary to thymine and cytosine is complementary to guanine. Accordingly, the polynucleotides of the present disclosure may also include isolated nucleic acid fragments that are complementary to the entire sequence as well as substantially similar nucleotide sequences.

[0058] Specifically, a polynucleotide having homology or identity with the polynucleotide of the present disclosure can be detected using hybridization conditions including a hybridization step at a Tm value of 55°C and using the conditions described above. Additionally, the Tm value may be 60°C, 63°C, or 65°C, but is not limited thereto and can be appropriately adjusted by a person skilled in the art according to the purpose.

[0059] The appropriate strictness for hybridizing the above polynucleotides depends on the length and degree of complementarity of the polynucleotides, and the variables are well known in the relevant technical field.

[0061] Another aspect of the present disclosure is to provide a vector comprising the polynucleotide of the present disclosure. The vector may be an expression vector for expressing the polynucleotide in a host cell, but is not limited thereto.

[0062] The vector of the present disclosure may comprise a DNA product comprising a sequence of a polynucleotide encoding said target polypeptide, which is operably linked to a suitable expression control region (or expression control sequence) so as to enable the expression of said target polypeptide within a suitable host. The expression control region may comprise a promoter capable of initiating transcription, any operator sequence for regulating such transcription, a sequence coding for a suitable mRNA ribosome binding site, and a sequence regulating the termination of transcription and translation. After being transformed into a suitable host cell, the vector may replicate or function independently of the host genome and may be incorporated into the genome itself.

[0063] The vectors used in this disclosure are not particularly limited, and any vector known in the art may be used. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in their natural or recombinant state. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A may be used as phage vectors or cosmid vectors, and pDZ-based, pBR-based, pUC-based, pBluescriptII-based, pGEM-based, pTZ-based, pCL-based, and pET-based vectors may be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, or pDC24 vectors may be used.

[0064] For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome using a vector for intracellular chromosome insertion. The insertion of the polynucleotide into the chromosome may be achieved by any method known in the art, for example, homologous recombination, but is not limited thereto. A selection marker may be additionally included to confirm whether the chromosome insertion has occurred. The selection marker is intended to select cells transformed by the vector, that is, to confirm whether the target nucleic acid molecule has been inserted, and markers conferring selectable phenotypes such as drug resistance, nutritional requirements, resistance to cytotoxic agents, or expression of a surface polypeptide may be used. Since only cells expressing the selection marker survive or exhibit other phenotypes in an environment treated with a selective agent, the transformed cells can be selected.

[0065] In the present disclosure, the term “transformation” means introducing a vector containing a polynucleotide encoding a target polypeptide into a host cell or microorganism so that the polypeptide encoded by said polynucleotide can be expressed within the host cell. The transformed polynucleotide may include both inserted into and located within the chromosomes of the host cell and extrachromosomally, as long as it can be expressed within the host cell. Additionally, said polynucleotide includes DNA and / or RNA encoding the target polypeptide. said polynucleotide may be introduced in any form that can be introduced into and expressed within the host cell. For example, said polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a genetic structure containing all the elements necessary for self-expression. That expression cassette may typically include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to said polynucleotide. The above expression cassette may be in the form of a self-replicating expression vector. Additionally, the polynucleotide may be introduced into a host cell in its own form and operably linked to a sequence required for expression in the host cell, but is not limited thereto.

[0066] In addition, the term "operably connected" as above means that a promoter sequence and a polynucleotide sequence are functionally connected to initiate and mediate the transcription of a polynucleotide encoding the target variant of the present disclosure.

[0068] Another aspect of the present disclosure comprises one or more selected from the group consisting of protein variants of the present disclosure and polynucleotides of the present disclosure, of the genus Corynebacterium Corynebacterium ) provides microorganisms.

[0069] The microorganism of the present disclosure may include a protein variant of the present disclosure, a polynucleotide encoding said protein variant, or a vector comprising the polynucleotide of the present disclosure.

[0070] In the present disclosure, the term “microorganism (or strain)” includes both wild-type microorganisms and microorganisms that have undergone natural or artificial genetic modification, and may be microorganisms in which specific mechanisms are weakened or strengthened due to causes such as the insertion of external genes or the enhancement or inactivation of the activity of endogenous genes, and may be microorganisms that include genetic modification for the production of a desired polypeptide, protein, or product.

[0071] The microorganism of the present disclosure may be, but is not limited to, a microorganism comprising one or more of the protein variant of the present disclosure, the polynucleotide of the present disclosure, and a vector comprising the polynucleotide of the present disclosure; a microorganism modified to express a variant of the present disclosure or a polynucleotide of the present disclosure; a microorganism expressing a variant of the present disclosure or a polynucleotide of the present disclosure (e.g., a recombinant microorganism); or a microorganism having the activity of a variant of the present disclosure (e.g., a recombinant microorganism).

[0072] The microorganism of the present disclosure may be a microorganism capable of producing L-threonine.

[0073] The microorganism of the present disclosure may have an increased L-threonine production capacity compared to a microorganism of the genus Corynebacterium that does not include one or more selected from the group consisting of the protein variant of the present disclosure described above and the polynucleotide encoding said protein variant (i.e., a protein variant comprising an amino acid sequence in which the amino acid corresponding to the 233rd position from the N-terminus of the amino acid sequence of SEQ ID NO. 304 is substituted with an amino acid different from the original (e.g., aspartic acid) or an amino acid sequence in which the amino acid corresponding to the 41st position from the N-terminus of the amino acid sequence of SEQ ID NO. 305 is substituted with an amino acid different from the original (e.g., serine); or a microorganism that does not express a protein variant comprising any one amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO. 36 to SEQ ID NO. 68, such as the amino acid sequence of SEQ ID NO. 43 or SEQ ID NO. 47, or a microorganism that expresses a wild-type protein corresponding to said protein variant).

[0074] The microorganisms of the present disclosure may be, but are not limited to, microorganisms that naturally possess the ability to produce L-threonine, or microorganisms to which a variant of the present disclosure or a polynucleotide encoding the same (or a vector containing said polynucleotide) is introduced into a parent strain that lacks the ability to produce L-threonine, and / or microorganisms to which the ability to produce L-threonine is conferred.

[0075] For example, the microorganism of the present disclosure is a cell or microorganism that expresses a protein variant of the present disclosure by being transformed with a vector comprising a polynucleotide of the present disclosure or a polynucleotide encoding a variant of the present disclosure, and for the purposes of the present disclosure, the strain of the present disclosure may include all microorganisms capable of producing L-threonine, including the variant of the present disclosure. For example, the microorganism of the present disclosure may be a recombinant microorganism in which the L-threonine production capacity is increased by introducing a polynucleotide encoding a variant of the present disclosure into a natural wild-type microorganism or a microorganism that produces L-threonine.

[0076] In one embodiment, the microorganism of the present disclosure may have increased L-threonine production capacity compared to a microorganism of the genus Corynebacterium that does not include one or more selected from the group consisting of the protein variant of the present disclosure and the polynucleotide encoding said protein variant. The microorganism of the genus Corynebacterium that does not include one or more selected from the group consisting of the protein variant of the present disclosure and the polynucleotide encoding said protein variant may be a natural wild-type microorganism or an unmodified microorganism, and may also be expressed as a microorganism as a parent strain or a host cell.

[0077] In this disclosure, the term "non-mutated microorganism" does not exclude microorganisms containing mutations that may occur naturally in microorganisms, and may refer to wild-type microorganisms or natural-type microorganisms themselves, or microorganisms prior to genetic mutations caused by natural or artificial factors. For example, the non-mutated microorganism may refer to a microorganism in which the protein variant described herein has not been introduced or is prior to being introduced. The term "non-mutated microorganism" may be used interchangeably with "pre-mutation strain," "pre-mutation microorganism," "non-mutated strain," "non-mutated strain," "non-mutated microorganism," or "reference microorganism."

[0078] In the present disclosure, the microorganism as the host cell (non-modified microorganism) may be (1) a microorganism that naturally has the ability to produce L-threonine, (2) a microorganism that naturally has no or significantly low ability to produce L-threonine, and / or (3) a microorganism into which a mutation is introduced (transformed) to the microorganism that naturally has the ability to produce L-threonine or the microorganism that has no or significantly low ability to produce L-threonine, thereby having the ability to produce L-threonine or having an enhanced ability to produce L-threonine. In one embodiment, the microorganism as the host cell may be CJTHR-1, CJTHR-2, or CJTHR-3, but is not limited thereto.

[0079] In one embodiment, the microorganism (a mutant microorganism or a microorganism as a host cell) may be a microorganism that has additionally enhanced the L-threonine biosynthetic pathway to increase the production of L-threonine, but is not limited thereto.

[0080] In another embodiment, the microorganism may have released feedback inhibition of homoserine dehydrogenase.

[0081] In this disclosure, the term “homoserine dehydrogenase (EC:1.1.1.3)” refers to an enzyme encoded by the hom gene that catalyzes the synthesis of homoserine. Homoserrine dehydrogenase is known to be feedback inhibited by threonine or isoleucine. The amino acid sequence of the homoserrine dehydrogenase can be obtained from known databases such as NCBI’s GenBank or US 10982244 B2.

[0082] In one embodiment, the microorganism may have released feedback inhibition of lysine-sensitive aspartokinase.

[0083] In the present disclosure, the term “lysine-sensitive aspartokinase (lysine-sensitive aspartokinase 3, LysC, EC: 2.7.2.4)” is encoded by the lysC gene, and the amino acid sequence of said aspartate kinase can be obtained from known databases such as NCBI’s GenBank or US 10662450 B2.

[0084] In one embodiment, the microorganism is E. coli ( Escherichia coli It may be that the L-threonine biosynthetic gene family (thrABC) derived from ) has been introduced.

[0085] In the present disclosure, “thrABC” is 대장균 It is a group of genes responsible for threonine biosynthesis, consisting of three major genes: thrA, thrB, and thrC.

[0086] In the present disclosure, “thrA” is a gene encoding a bifunctional aspartokinase / homoserine dehydrogenase 1 in Escherichia coli, comprising two domains of homoserine dehydrogenase 1 and aspartokinase. The aspartokinase of the bifunctional aspartokinase / homoserine dehydrogenase 1 catalyzes a reaction that adds a phosphate group to aspartic acid to produce aspartyl-β-phosphate, and homoserine dehydrogenase 1 catalyzes a reaction that converts aspartyl-β-phosphate to homoserine. The homoserine can be used as a precursor for the synthesis of threonine or isoleucine.

[0087] In the present disclosure, the microorganism may have the feedback inhibition of the Bifunctional aspartokinase / homoserine dehydrogenase 1 released.

[0088] In the present disclosure, “thrB” is a gene encoding homoserine dehydrogenase (homoserine kinase) in Escherichia coli. The homoserine dehydrogenase catalyzes the reaction in which L-homoserine is ATP-dependently phosphorylated to L-homoserine phosphate.

[0089] In the present disclosure, “thrC” is a gene encoding threonine synthase in Escherichia coli. The threonine synthase catalyzes the reaction of converting O-phosphohomoserine into threonine.

[0090] The sequences of the above thrA, thrB, and thrC genes and the amino acid sequences of the proteins they code for can be obtained from GenBank of NCBI, a known database.

[0091] In one embodiment, the thrABC may have a promoter replaced with a powerful promoter (e.g., an SPL7 promoter), but is not limited thereto.

[0092] In the present disclosure, the term feedback inhibition release may mean that the activity of a polypeptide, protein, or enzyme is increased or enhanced relative to its intrinsic activity, or that the activity is not inhibited relative to its intrinsic activity.

[0093] In one embodiment, the microorganism may be one into which the gene for L-threonine efflux protein (threonine / homoserine lactone efflux protein, rhtC) derived from E. coli has been introduced.

[0094] In this disclosure, the term L-threonine efflux protein (threonine / homoserine lactone efflux protein, rhtC) is a protein that mediates the efflux of L-threonine out of the cell and is known as an inner membrane protein having five transmembrane domains.

[0095] In one embodiment, the rhtC gene may have its promoter replaced with a strong promoter (e.g., gapA promoter), but is not limited thereto.

[0096] In one embodiment, the microorganism further introduces a genetic variant (G378E and / or R398Q) into the hom gene (Korean Patent No. 10-2183209), further introduces a genetic variant (R407H) into the hom gene (US 10982244 B2), further introduces a genetic variant (S352P) into the thrA gene (J Bacteriol. 1993 Feb;175(4):959-65), further introduces a genetic variant (L62S) into the rhtC gene (Korean Patent No. 10-2183209), further introduces a genetic variant (T381A, F383A and / or V323A) into the ilvA gene (US 2023-0098971 A1, US 10982244 B2), encoding lysine-sensitive aspartokinase It may be a microorganism that additionally includes one or more of the variants that additionally introduce a genetic variant (L377K) (US Registered Publication US 10662450 B2) into the lysC gene, but is not limited thereto.

[0097] In one embodiment, the microorganism with increased L-threonine production capacity (production amount) of the present disclosure may have an L-threonine production capacity (production amount) that is increased by about 1% or more, about 5% or more, about 10% or more, about 20% or more, about 30% or more, about 32.5% or more, about 35% or more, about 37.5% or more, or about 40% or more compared to the parent strain before mutation or the non-mutated microorganism (the upper limit is not specifically limited and may be, for example, about 200% or less, about 150% or less, about 100% or less, or about 50% or less), but is not limited thereto. In another embodiment, the microorganism with increased L-threonine production capacity (production amount) of the present disclosure may have an L-threonine production capacity (production amount) that is increased by about 1.01 times or more, about 1.01 times or more, about 1.05 times or more, about 1.1 times or more, about 1.2 times or more, about 1.3 times or more, about 1.325 times or more, about 1.35 times or more, about 1.375 times or more, or about 1.4 times or more compared to the parent strain before mutation or the non-mutated microorganism (the upper limit is not specifically limited and may be, for example, about 10 times or less, about 5 times or less, about 3 times or less, or about 2 times or less), but is not limited thereto.

[0098] The above term “about” refers to a range that includes ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes, but is not limited to, all numerical values ​​within a range equivalent to or similar to the numerical value following the term “about.”

[0100] In one embodiment, the microorganism of the present disclosure is Corynebacterium glutamicum ( Corynebacterium glutamicum ), Corynebacterium crudilactis( Corynebacterium crudilactis ), Corynebacterium deserti( Corynebacterium deserti ), Corynebacterium epiphysiens( Corynebacterium efficiens ), Corynebacterium calunae( Corynebacterium callunae ), Corynebacterium stationaryis( Corynebacterium stationis ), Corynebacterium singulare( Corynebacterium singulare ), Corynebacterium halotolerans( Corynebacterium halotolerans ), Corynebacterium striatum ( Corynebacterium striatum ), Corynebacterium ammoniagenes( Corynebacterium ammoniagenes ), Corynebacterium pollatisoli ( Corynebacterium pollutisoli ), Corynebacterium imitans( Corynebacterium imitans ), Corynebacterium testudinoris( Corynebacterium testudinoris ), or Corynebacterium flavescens ( Corynebacterium flavescens It could be, and more specifically, it could be Corynebacterium glutamicum.

[0102] In this disclosure, the term "weakening" of a polypeptide is a concept that includes both reduced activity and lack of activity relative to its intrinsic activity. Such weakening may be used interchangeably with terms such as inactivation, deficiency, down-regulation, decrease, reduce, and attenuation.

[0103] The above weakening may include cases where the activity of the polypeptide itself is reduced or eliminated compared to the polypeptide activity originally possessed by the microorganism due to mutations in the polynucleotide encoding the polypeptide, etc.; cases where the overall polypeptide activity and / or concentration (expression amount) within the cell is lower than that of the natural strain due to inhibition of gene expression of the polynucleotide encoding it or inhibition of translation into the polypeptide; cases where the expression of the polynucleotide does not occur at all; and / or cases where the polypeptide is not active even if the polynucleotide is expressed. The above "intrinsic activity" refers to the activity of a specific polypeptide originally possessed by the parent strain, wild-type, or non-modified microorganism prior to the change in trait due to genetic mutation caused by natural or artificial factors. This may be used interchangeably with "activity before modification." The statement that the activity of a polypeptide is "inactivated, deficient, reduced, downregulated, lowered, or attenuated" relative to its intrinsic activity means that the activity of a specific polypeptide has decreased compared to the activity originally possessed by the parent strain or non-transformed microorganism prior to transformation.

[0104] The attenuation of the activity of such polypeptides can be performed by any method known in the art, but is not limited thereto, and can be achieved by the application of various methods well known in the art (e.g., Nakashima N et al., Bacterial cellular engineering by genome editing and gene silencing. Int J Mol Sci. 2014;15(2):2773-2793, Sambrook et al. Molecular Cloning 2012 et al.).

[0105] Specifically, the weakening of the polypeptide of the present disclosure is

[0106] 1) Deletion of all or part of the gene encoding a polypeptide;

[0107] 2) Modification of the expression regulatory region (or expression regulatory sequence) to reduce the expression of the gene encoding the polypeptide;

[0108] 3) Modification of the amino acid sequence constituting the polypeptide so as to remove or weaken the activity of the polypeptide (e.g., deletion / substitution / addition of one or more amino acids in the amino acid sequence);

[0109] 4) Modification of the gene sequence encoding the polypeptide so as to remove or weaken the activity of the polypeptide (e.g., deletion / substitution / addition of one or more nucleotides on the nucleotide sequence of the polypeptide gene to code for a polypeptide modified so as to remove or weaken the activity of the polypeptide);

[0110] 5) A modification of the nucleotide sequence encoding the start codon or the 5'-UTR region of the gene transcript encoding the polypeptide;

[0111] 6) Introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the transcript of the gene encoding the polypeptide;

[0112] 7) Addition of a sequence complementary to the Shine-Dalgarno sequence to the upstream end of the Shine-Dalgarno sequence of a polypeptide-coding gene to form a secondary structure incapable of ribosome attachment;

[0113] 8) Addition of a reverse-transcribed promoter to the 3' end of the open reading frame (ORF) of a gene sequence encoding a polypeptide (Reverse transcription engineering, RTE); or

[0114] 9) Regulation of the cellular localization of proteins (polypeptides); or

[0115] 10) It may be based on two or more combinations selected from 1) to 9) above, but is not specifically limited thereto.

[0116] for example,

[0117] The deletion of part or all of the gene encoding the polypeptide mentioned above 1) may be the removal of the entire polynucleotide encoding the intrinsic target polypeptide within the chromosome, replacement with a polynucleotide in which some nucleotides have been deleted, or replacement with a marker gene.

[0118] Additionally, modification of the expression regulatory region (or expression regulatory sequence) described in 2) above may be a deletion, insertion, non-conservative or conservative substitution, or a combination thereof, resulting in a mutation on the expression regulatory region (or expression regulatory sequence), or replacement with a sequence having weaker activity. The expression regulatory region includes, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating the termination of transcription and translation.

[0119] In addition, the above 3) modification of the nucleotide sequence encoding the start codon or 5'-UTR region of the gene transcript encoding the polypeptide may, for example, be a substitution with a nucleotide sequence encoding another start codon that has a lower polypeptide expression rate compared to the intrinsic start codon, but is not limited thereto.

[0120] In addition, modifications to the amino acid sequences or polynucleotide sequences of 4) and 5) above may involve the occurrence of sequence variations in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, to weaken the activity of the polypeptide, or may involve replacement with an amino acid sequence or polynucleotide sequence modified to have weaker activity or an amino acid sequence or polynucleotide sequence modified to have no activity, but are not limited thereto. For example, gene expression may be inhibited or weakened by introducing a variation within the polynucleotide sequence to form a stop codon, but are not limited thereto.

[0121] For the introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the transcript of the gene encoding the polypeptide 6) above, refer to the literature [Weintraub, H. et al., Antisense-RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol. 1(1) 1986].

[0122] 7) In order to form a secondary structure in which ribosome attachment is impossible, the addition of a sequence complementary to the Shine-Dalgarno sequence to the front of the Shine-Dalgarno sequence of a gene encoding a polypeptide may make mRNA translation impossible or slow it down.

[0123] Reverse transcription engineering (RTE) of a promoter that is transcribed in the opposite direction to the 3' end of the ORF (open reading frame) of the gene sequence encoding the polypeptide above may weaken the activity by creating an antisense nucleotide complementary to the transcript of the gene encoding the polypeptide.

[0124] The above 9) regulation of the intracellular localization of the protein (polypeptide) may involve targeting the protein (polypeptide) to a specific intracellular organelle or a specific intracellular space. For example, it may involve targeting to the periplasm or cytoplasm through the addition or removal of a leader sequence that functions for the targeting of the protein (polypeptide), but is not limited thereto.

[0125] Such weakening of polypeptide activity may involve a reduction in the activity or concentration expression of the corresponding polypeptide relative to the activity or concentration of the polypeptide expressed in the wild-type or pre-modification microbial strain, or an increase in the amount of product produced from said polypeptide, but is not limited thereto.

[0127] In the present disclosure, the term “enhancement” of polypeptide activity means that the activity of the polypeptide is increased compared to its intrinsic activity. Such enhancement may be used interchangeably with terms such as activation, up-regulation, overexpression, and increase. Here, activation, enhancement, up-regulation, overexpression, and increase may include exhibiting activity that was not originally possessed, or exhibiting improved activity compared to the intrinsic activity or activity prior to modification. The “intrinsic activity” refers to the activity of a specific polypeptide originally possessed by the parent strain or the non-modified microorganism prior to the change in traits caused by genetic mutations due to natural or artificial factors. This may be used interchangeably with “activity prior to modification.” "Enhancement," "upregulation," "overexpression," or "increase" of polypeptide activity relative to intrinsic activity means that the activity and / or concentration (expression amount) of a specific polypeptide were originally possessed by the parent strain or non-modified microorganism prior to transformation.

[0128] The above enhancement can be achieved by introducing an exogenous polypeptide or by enhancing the activity and / or concentration (expression amount) of an intrinsic polypeptide. Whether the activity of the polypeptide is enhanced can be confirmed by an increase in the degree of activity, expression amount, or amount of product released from the polypeptide.

[0129] The enhancement of the activity of the above polypeptide may be achieved by applying various methods well known in the art, and is not limited to, as long as the activity of the target polypeptide can be enhanced compared to the microorganism before modification. Specifically, it may utilize, but is not limited to, gene engineering and / or protein engineering known to a person skilled in the art, which are routine methods of molecular biology (e.g., Sitnicka et al. Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2. 1-16, Sambrook et al. Molecular Cloning 2012, etc.).

[0130] Specifically, the reinforcement of the polypeptide of the present disclosure is

[0131] 1) Increase in the intracellular copy number of polynucleotides encoding polypeptides;

[0132] 2) Replace the chromosomal gene expression regulatory region encoding a polypeptide with a potent sequence;

[0133] 3) A modification of the nucleotide sequence encoding the start codon or the 5'-UTR region of the gene transcript encoding the polypeptide;

[0134] 4) Modification of the amino acid sequence of the polypeptide to enhance polypeptide activity;

[0135] 5) Modification of the polynucleotide sequence encoding the polypeptide to enhance polypeptide activity (e.g., modification of the polynucleotide sequence of the polypeptide gene to code for a polypeptide modified to enhance polypeptide activity);

[0136] 6) Introduction of a foreign polypeptide exhibiting polypeptide activity or a foreign polynucleotide encoding the same;

[0137] 7) Codon optimization of polynucleotides encoding polypeptides;

[0138] 8) Analyze the tertiary structure of the polypeptide to select and modify or chemically modify the exposed sites; or

[0139] 9) Regulation of the cellular localization of proteins (polypeptides); or

[0140] 10) It may be based on two or more combinations selected from 1) to 9) above, but is not specifically limited thereto.

[0141] The increase in the intracellular copy number of the polynucleotide encoding the above 1) polypeptide may be achieved by introducing into a host cell a vector to which the polynucleotide encoding the said polypeptide is operably linked, which can replicate and function independently of the host. Alternatively, it may be achieved by introducing one or more copies of the polynucleotide encoding the said polypeptide into the chromosomes within the host cell. The introduction into the chromosomes may be performed by introducing into the host cell a vector capable of inserting said polynucleotide into the chromosomes within the host cell, but is not limited thereto. The said vector is as described above.

[0142] Replacing the gene expression regulatory region (or expression regulatory sequence) on the chromosome encoding the polypeptide 2) above with a sequence having potent activity may, for example, involve deletion, insertion, non-conservative or conservative substitution, or a combination thereof, to further enhance the activity of the expression regulatory region, or may involve a sequence mutation, or replacement with a sequence having stronger activity. The expression regulatory region may include, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating the termination of transcription and translation. As an example, the original promoter may be replaced with a potent promoter, but is not limited thereto.

[0143] Examples of known strong promoters include, but are not limited to, CJ1 to CJ7 promoters (US Patent No. 7662943 B2), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13(sm3) promoter (US Patent No. 10584338 B2), O2 promoter (US Patent No. 10273491 B2), tkt promoter, yccA promoter, etc.

[0144] The above 3) modification of the nucleotide sequence encoding the start codon or 5'-UTR region of the gene transcript encoding the polypeptide may, for example, be a substitution with a nucleotide sequence encoding another start codon that has a higher polypeptide expression rate compared to the intrinsic start codon, but is not limited thereto.

[0145] The modification of the amino acid sequence or polynucleotide sequence of 4) and 5) above may involve the occurrence of sequence variations in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, to enhance the activity of the polypeptide, or may involve replacement with an amino acid sequence or polynucleotide sequence modified to have stronger activity or an amino acid sequence or polynucleotide sequence modified to increase activity, but is not limited thereto. Specifically, the replacement may be performed by inserting the polynucleotide into the chromosome by homologous recombination, but is not limited thereto. The vector used in this case may additionally include a selection marker to confirm whether chromosome insertion has occurred. The selection marker is as described above.

[0146] The introduction of an exogenous polynucleotide exhibiting the activity of the polypeptide described in 6) above may be the introduction into a host cell of an exogenous polynucleotide encoding a polypeptide that exhibits the same or similar activity as the polypeptide. As long as the exogenous polynucleotide exhibits the same or similar activity as the polypeptide, there are no restrictions on its origin or sequence. The method used for the introduction may be performed by a person skilled in the art by appropriately selecting a known transformation method, and the polypeptide may be generated and its activity increased by the expression of the introduced polynucleotide within the host cell.

[0147] The above 7) codon optimization of a polynucleotide encoding a polypeptide may be a codon optimization of the intrinsic polynucleotide such that transcription or translation within the host cell increases, or a codon optimization of the extrinsic polynucleotide such that optimized transcription or translation occurs within the host cell.

[0148] 8) The above method of analyzing the tertiary structure of the polypeptide to select and modify or chemically modify an exposed site may involve, for example, determining a template protein candidate based on the degree of sequence similarity by comparing the sequence information of the polypeptide to be analyzed with a database in which sequence information of known proteins is stored, confirming the structure based on this, and selecting and modifying or modifying an exposed site to be modified or chemically modified.

[0149] The above 9) regulation of the intracellular localization of the protein (polypeptide) may involve targeting the protein (polypeptide) to a specific intracellular organelle or a specific intracellular space. For example, it may involve targeting to the periplasm or cytoplasm through the addition or removal of a leader sequence that functions for the targeting of the protein (polypeptide), but is not limited thereto.

[0150] Such enhancement of polypeptide activity may involve increasing the activity or concentration of the corresponding polypeptide based on the activity or concentration of the polypeptide expressed in the wild-type or pre-modification microbial strain, or increasing the amount of the product produced from said polypeptide, but is not limited thereto.

[0152] Modification of part or all of a polynucleotide in the microorganism of the present disclosure (e.g., modification to code for the protein variant described above) may be induced by (a) homologous recombination using a vector for chromosomal insertion within the microorganism or genome editing using engineered nucleases (e.g., CRISPR-Cas9) and / or (b) treatment by light and / or chemicals such as ultraviolet rays and radiation, but is not limited thereto. The method of modifying part or all of the gene may include methods by DNA recombination technology. For example, deletion of part or all of the gene may be achieved by inducing homologous recombination by injecting a nucleotide sequence or vector containing a nucleotide sequence homologous to the target gene into the microorganism. The injected nucleotide sequence or vector may include a dominant selection marker, but is not limited thereto.

[0154] In the microorganism of the present disclosure, the protein variant, polynucleotide, and L-threonine, etc., are as described in the other embodiments above.

[0156] Another aspect of the present disclosure provides a method for producing L-threonine, comprising the step of culturing a microorganism of the genus Corynebacterium, comprising a protein variant of the present disclosure or a polynucleotide of the present disclosure, in a medium.

[0157] The method for producing L-threonine of the present disclosure may include the step of culturing a microorganism of the genus Corynebacterium in a medium comprising the protein variant of the present disclosure, the polynucleotide of the present disclosure, or the vector of the present disclosure.

[0159] In this disclosure, the term "culture" means growing the microorganisms of the genus Corynebacterium of this disclosure under appropriately controlled environmental conditions. The culture process of this disclosure may be carried out according to suitable media and culture conditions known in the art. Such culture process can be easily adjusted and used by those skilled in the art depending on the strain selected. Specifically, the culture may be batch, continuous, and / or fed-batch, but is not limited thereto.

[0160] In this disclosure, the term "medium" refers to a substance mixed with nutrients as the main component required to culture the microorganisms of the genus Corynebacterium of this disclosure, and supplies nutrients and growth factors, including water, which is indispensable for survival and growth. Specifically, the medium and other culture conditions used for culturing the microorganisms of the genus Corynebacterium of this disclosure may be any medium used for culturing ordinary microorganisms without special limitations; however, the microorganisms of the genus Corynebacterium of this disclosure may be cultured under aerobic conditions while controlling the temperature, pH, etc., in a conventional medium containing a suitable carbon source, nitrogen source, phosphorus, inorganic compounds, amino acids, and / or vitamins.

[0161] Specifically, culture media for strains of the genus Corynebacterium can be found in the literature ["Manual of Methods for General Bacteriology" by the American Society for Bacteriology (Washington DC, USA, 1981)].

[0162] In the present disclosure, the carbon source may include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvate, lactic acid, citric acid, etc.; and amino acids such as glutamic acid, methionine, lysine, etc. Additionally, natural organic nutrient sources such as starch hydrolysate, molasses, blackstrap molasses, rice winter, cassava, sugarcane residue, and corn steeping liquid may be used. Specifically, carbohydrates such as glucose and sterilized pre-treated molasses (i.e., molasses converted into reducing sugars) may be used, and other carbon sources in appropriate amounts may be used without limitation. These carbon sources may be used individually or in combination of two or more types, but are not limited thereto.

[0163] The above nitrogen sources may include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.; and organic nitrogen sources such as amino acids such as glutamic acid, methionine, glutamine, etc., peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquid, casein hydrolysate, fish or its decomposition products, defatted soybean cake or its decomposition products, etc. These nitrogen sources may be used alone or in combination of two or more types, but are not limited thereto.

[0164] The above ingredients may include monopotassium phosphate, dipotassium phosphate, or corresponding sodium-containing salts. Inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc., and may also include amino acids, vitamins, and / or suitable precursors. These components or precursors may be added to the culture medium in a batch or continuous manner. However, they are not limited thereto.

[0165] In addition, during the cultivation of the microorganisms of the genus Corynebacterium disclosed in this disclosure, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc., may be added to the medium in an appropriate manner to adjust the pH of the medium. In addition, during cultivation, an antifoaming agent such as a fatty acid polyglycol ester may be used to suppress the formation of bubbles. Furthermore, to maintain an aerobic state of the medium, oxygen or an oxygen-containing gas may be injected into the medium, or nitrogen, hydrogen, or carbon dioxide gas may be injected without gas injection to maintain an anaerobic and microaerobic state, but is not limited thereto.

[0166] In the culture of the present disclosure, the culture temperature may be 20°C to 45°C, specifically 25°C to 40°C, 25°C to 40°C, 25°C to 37°C, 25°C to 35°C, 27°C to 40°C, 27°C to 37°C, 27°C to 35°C, 30°C to 40°C, 30°C to 37°C, or 30°C to 35°C, but is not limited thereto. In the culture of the present disclosure, the culture time may be about 10 to 160 hours, but is not limited thereto.

[0167] L-threonine produced by the culture of the present disclosure may be secreted into the medium or remain in the cell.

[0168] The method for producing L-threonine of the present disclosure may additionally include, for example, the step of preparing a microorganism of the genus Corynebacterium of the present disclosure, the step of preparing a medium for culturing said strain, or a combination thereof (in any order), prior to said culturing step.

[0169] The method for producing L-threonine according to the present disclosure may further include a step of recovering L-threonine from a culture medium (a culture medium in which the culture is performed) or from a microorganism of the genus Corynebacterium. The recovery step may be further included after the culture step.

[0170] The above recovery may involve collecting the desired L-threonine using a suitable method known in the art according to the culture method of the microorganism disclosed in this disclosure, for example, batch, continuous, or fed-batch culture methods. For example, various chromatographic methods such as centrifugation, filtration, treatment with a crystallizing protein precipitating agent (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC, or a combination thereof may be used, and the desired L-threonine may be recovered from the culture medium or microorganism using a suitable method known in the art.

[0171] Additionally, the method for producing L-threonine of the present disclosure may further include a purification step. The purification may be performed using a suitable method known in the art. In one example, where the method for producing L-threonine of the present disclosure includes both a recovery step and a purification step, the recovery step and the purification step may be performed continuously or discontinuously regardless of the order, or simultaneously or integrated into a single step, but are not limited thereto.

[0172] In the method of the present disclosure, the variant, polynucleotide, vector, strain, etc. are as described in the other embodiments above.

[0174] Another aspect of the present disclosure provides a composition for producing L-threonine comprising one or more microorganisms of the genus Corynebacterium selected from the group consisting of a protein variant of the present disclosure, a polynucleotide encoding said protein variant, and a vector comprising said polynucleotide; a culture medium in which said is cultured; or a combination of two or more of these.

[0175] The composition of the present disclosure may further include any suitable excipients commonly used in compositions for producing amino acids, and such excipients may be, for example, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers or isotonic agents, but are not limited thereto.

[0176] In the composition of the present disclosure, the variant, polynucleotide, vector, strain, medium, and L-threonine, etc. are as described in the other embodiments above.

[0178] According to another aspect of the present disclosure, the present disclosure may provide a method for increasing the L-threonine production capacity of a microorganism, a method for conferring L-threonine production capacity to the microorganism, and / or a method for producing a microorganism with increased L-threonine production capacity, comprising the step of introducing (e.g., transforming) a protein variant of the present disclosure described above, a polynucleotide encoding said variant, and / or a recombinant vector comprising said polynucleotide into said microorganism.

[0179] In the method for increasing the L-threonine production capacity of a microorganism, the method for conferring L-threonine production capacity to a microorganism, and / or the method for producing a microorganism with increased L-threonine production capacity according to the present disclosure, the protein variant, polynucleotide, recombinant vector, and microorganism, etc. are as described above. Effects of the invention

[0181] When culturing a microorganism of the genus Corynebacterium comprising the novel protein variant of the present disclosure, it is possible to produce L-threonine in a high yield compared to a microorganism having a conventional unmodified polypeptide. Specific details for implementing the invention

[0183] The present disclosure is described in more detail below by way of examples. However, the following examples are merely preferred embodiments for illustrating the present disclosure and are therefore not intended to limit the scope of the rights of the present disclosure. Meanwhile, technical matters not described in this specification can be fully understood and easily implemented by a person skilled in the art who is proficient in the technical field of the present disclosure or a similar technical field.

[0185] 실시예

[0186] Example 1. Selection of mutant strains with increased threonine production capacity via artificial mutation method

[0187] Example 1-1. Preparation of Threonine-Producing Strain

[0188] 1-1-1. Production of Homoserine Dehydrogenase (Hom) Mutant Strains

[0189] We intended to introduce a variant (G378E, R398Q) for unlocking feedback inhibition of the hom gene to L-threonine (Korean Registered Patent No. 10-2183209) into a strain of the genus Corynebacterium. A plasmid for this purpose was constructed by the following method.

[0190] To apply the hom mutation (G378E, R398Q), the hom (G378E, R398Q) gene fragments and the hom (R398Q) gene fragments, which involve chromosomal homologous recombination, were obtained. Specifically, using the genomic DNA of the Corynebacterium glutamicum ATCC13032 strain as a template, PCR was performed using the primer pairs SEQ NOs. 1 and 2, SEQ NOs. 3 and 4, and SEQ NOs. 5 and 6 to obtain the hom protein's G378E gene upper fragment, hom (G378E, R398Q) gene fragment, and R398Q gene lower fragment, respectively. The PCR reaction was performed using the polymerase Solg TMThe procedure was performed using Pfu-X DNA polymerase under conditions of denaturation at 95°C for 5 minutes, followed by denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, polymerization at 72°C for 2 minutes, repeated 30 times, and polymerization at 72°C for 5 minutes.

[0191] A recombinant plasmid was obtained by cloning the gene fragments obtained from the above process into the pDC24 vector (SEQ No. 7) cleaved with SmaI restriction enzyme using the Gibson assembly method (DG Gibson et al., NATURE METHODS, VOL.6 NO.5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix). Cloning was performed by mixing the Gibson assembly reagents with each gene fragment in calculated moles and incubating at 50°C for 1 hour. The constructed recombinant plasmid was named pDC24△hom::hom(G378E / R398Q).

[0192] After transforming the pDC24△hom::hom(G378E / R398Q) vector produced above into a wild-type Corynebacterium glutamicum ATCC13032 strain by electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545), a strain was obtained in which the wild-type hom gene on the chromosome was replaced with the mutant hom(G378E, R398Q) gene through a secondary crossing process. The genetic manipulation was confirmed by performing PCR and genome sequencing using primer pairs of SEQ ID NO. 8 and SEQ ID NO. 9, which are capable of amplifying the external regions of the homologous recombination upstream and downstream regions, respectively, into which the gene was inserted.

[0193] The primer sequences used here were as shown in Table 2 below.

[0194] Sequence number Sequence(5'->3') 1 CGAGCTCGGTACCCATGACCTCAGCATCTGCCCC 2 GCCAAAACCTCCACGCGATCTT 3 AAGATCGCGTGGAGGTTTTGGC 4 GCGCTCTTCCTGTTGGATTGTACGC 5 GCGTACAATCCAACAGGAAGAGCGC 6 CTCTAGAGGATCCCCGACTGCGGAATGTTGTTGTG 8 TGGGTAGGTCGAGTTGTTAA 9 CAGCGCAGTCGCACGAATAT

[0195] The transformed strain obtained above was named CJTHR-1.

[0197] 1-1-2. Production of Lysine-Sensitive Aspartokinase 3 (LysC) Mutant Strains

[0198] We intended to introduce a variant (L377K) for unlocking feedback inhibition of L-lysine and L-threonine of the lysC gene (Korean Registered Patent No. 10-2011994) into the strain prepared in Example 1-1-1. A plasmid for this purpose was prepared by the following method.

[0199] To apply the LysC variant (L377K), the upper fragment and lower fragment of the LysC (L377K) gene, in which homologous recombination occurs on the chromosome, were obtained. Specifically, using the genomic DNA of the Corynebacterium glutamicum ATCC13032 strain as a template, PCR was performed in the same manner as above using the primer pairs of SEQ ID NO. 10 and SEQ ID NO. 11 and the primer pairs of SEQ ID NO. 12 and SEQ ID NO. 13, and the upper fragment and lower fragment of the LysC (L377K) gene were obtained, respectively.

[0200] A recombinant plasmid was obtained by cloning the gene fragments obtained through the above process into a pDC24 vector cleaved with SmaI restriction enzyme using the Gibson assembly method. Gibson cloning was performed in the same manner as above. The constructed recombinant plasmid was named pDC24△lysC::lysC(L377K). After transforming the CJTHR-1 strain constructed in Example 1-1-1 with the above-mentioned pDC24△lysC::lysC(L377K) vector by electroporation, a strain was obtained in which the wild-type lysC gene on the chromosome was replaced with the mutant lysC(L377K) gene through a secondary crossover process. The genetic manipulation was confirmed through PCR using primers of SEQ ID NO. 14 and SEQ ID NO. 15, which are capable of amplifying the external regions of the homologous recombination upstream and downstream regions where the gene was inserted, respectively, and through genome sequencing. The primer sequences used here were as shown in Table 3 below.

[0201] Sequence number Sequence(5'->3') 10 CGAGCTCGGTACCCGTGGCCCTGTCGTACAGAA 11 GGTGGAAATCTTTTTCGATGTTCACGTTGAC 12 GAACATCGAAAAGATTTCCACCTCTGAGAT 13 CTCTAGAGGATCCCCGTTCACCTCAGAGACGATTA 14 ACATTCCACCCATTACTGCA 15 TCTTCATCGGTTTCGAAGGT

[0202] The transformed strain obtained above was named CJTHR-2.

[0204] 1-1-3. Production of strains introduced with the Escherichia coli threonine biosynthetic gene family (thrABC) and threonine / homoserine lactone efflux protein (RhtC)

[0205] In order to enhance L-threonine biosynthesis in the strain prepared in Example 1-1-2, the E. coli-derived L-threonine biosynthesis gene group (thrABC) was applied. In particular, the thrA (Bifunctional aspartokinase / homoserine dehydrogenase 1) gene, which possesses dual functionality for aspartokinase and homoserine dehydrogenase 1, was modified into thrA(S352P) (J Bacteriol. 1993 Feb;175(4):959-65) to remove feedback inhibition for L-threonine. Additionally, the thrABC expression was increased by replacing the thrABC promoter with a strong promoter. Furthermore, to enhance L-threonine efflux capacity, the E. coli-derived L-threonine efflux protein (threonine / homoserine lactone efflux protein, rhtC) was applied. In particular, the rhtC gene was applied using a variant (L62S) (Korean Registered Patent No. 10-2183209) that exhibited a high yield increase effect compared to the wild type. Additionally, the rhtC promoter was replaced with a strong promoter to increase the expression of the efflux protein. A plasmid for this purpose was constructed using the following method.

[0206] To insert the thrABC and rhtC genes derived from E. coli into the strain, NCgl0179, known as a gene encoding a transposon in Corynebacterium glutamicum, was used as the insertion site. Specifically, to construct the NCgl0179 deletion and target gene insertion vector, PCR was performed in the same manner as above using the chromosome of Corynebacterium glutamicum ATCC13032 as a template and the primer pairs of SEQ ID NO. 16 and SEQ ID NO. 17 and SEQ ID NO. 18 and SEQ ID NO. 19, and the left homologous cancer region of NCgl0179 and the right homologous cancer region of NCgl0179 were obtained, respectively. Primers of SEQ ID NO. 17 and 18 were designed to include a Sma1 cleavage site between the left and right homologous cancers. A recombinant plasmid was obtained by cloning the obtained gene fragments into a pDC24 vector cleaved with SmaI restriction enzyme using the Gibson assembly method. Gibson cloning was performed in the same manner as above. The recombinant plasmid produced was named pDC24△NCgl0179.

[0207] Next, using the genomic DNA of Escherichia coli w3110 as a template, PCR was performed in the same manner as above using the primer pairs of SEQ ID NO. 20 and SEQ ID NO. 21 and the primer pairs of SEQ ID NO. 22 and SEQ ID NO. 23, and the upper fragment of the thrA(S352P)BC gene and the lower fragment of the thrA(S352P)BC gene were obtained, respectively. In addition, to replace with a strong promoter, PCR was performed in the same manner as above using the primer pairs of SEQ ID NO. 25 and SEQ ID NO. 26 with the synthetic promoter spl7 promoter (SEQ ID NO. 24, Korean Registered Patent No. 10-1783170) as a template, and the Pspl7 promoter fragment was obtained. Using the amplified gene fragments above as templates, PCR was performed in the same manner as above using the primer pair of SEQ ID NO. 25 and SEQ ID NO. 23, and the 'Pspl7_thrA(S352P)BC(E.co)' gene fragment was obtained.

[0208] Next, using the genomic DNA of Escherichia coli w3110 as a template, PCR was performed in the same manner as above using primer pairs of SEQ ID NO. 27 and SEQ ID NO. 28 and SEQ ID NO. 29 and SEQ ID NO. 30, and the upper fragment of the rhtC(L62S) gene and the lower fragment of the rhtC(L62S) gene were obtained, respectively. In addition, to replace with a strong promoter, PCR was performed in the same manner as above using primer pairs of SEQ ID NO. 31 and SEQ ID NO. 32 using Corynebacterium glutamicum ATCC13032 as a template, and the gapA promoter fragment (SEQ ID NO. 33) was obtained. Using the amplified gene fragments as a template, PCR was performed in the same manner as above using primer pairs of SEQ ID NO. 31 and SEQ ID NO. 30, and the 'PgapA_rhtC(L62S, E.co)' gene fragment was obtained.

[0209] After treating the above-described pDC24ΔNCgl0179 vector with the restriction enzyme Sma1, a recombinant plasmid was obtained by cloning the obtained 'Pspl7_thrA(S352P)BC(E.co)' gene fragment and 'PgapA_rhtC(L62S, E.co)' gene fragment using the Gibson assembly method. Gibson cloning was performed in the same manner as above. The constructed recombinant plasmid was named pDC24△NCgl0179:: Pspl7_thrA(S352P)BC(E.co)-PgapA_rhtC(L62S, E.co). The pDC24△NCgl0179:: Pspl7_thrA(S352P)BC(E.co)-PgapA_rhtC(L62S, E.co) vector prepared above was transformed into the CJTHR-2 strain prepared in Example 1-1-2 above by electroporation, and a strain in which the threonine biosynthetic gene group and threonine release protein derived from E. coli were introduced on the chromosomes was obtained through a secondary crossover process. The genetic modification was confirmed through PCR using primers of SEQ ID NO. 34 and SEQ ID NO. 35, which are capable of amplifying the external regions of the homologous recombination upstream and downstream regions where the genes were inserted, and through genome sequencing. The primer sequences used here are as shown in Table 4 below.

[0210] Sequence number Sequence(5'->3') 16 TCGAGCTCGGTACCCTTTTTTCAGGGCTGT 17 AACCTAGGGCGAATCCCGGGGTGATCAGCAA 18 TTGCTGATCACCCGGGATTCGCCCCTAGGTT 19 CTAGAGGATCCCCCACAGGATATGGTAGAA 20 GAGGAGATCAAAACAATGCGAGTGTTGAAG 21 GTATTCGGAAGATGGTTGCGTAATCAGCAC 22 GTGCTGATTACGCAACCATCTTCCGAATAC 23 GAATCCACAAATTAATTACTGATGATTCAT 25 TTTGCTGATCACCCGGCGCTTCATGTCAAC 26 CTTCAACACTCGCATTGTTTTGATCTCCTC 27 GAGGAGACACAACATGTTGATGTTATTTCT 28 TTTTTCGATAATCGAATGCAGGCCAAGCAG 29 CTGCTTGGCCTGCATTCGATTATCGAAAAA 30 CCTAGGGCGAATCCCTCACCGCGAAAATAAT 31 ATGAATCATCAGTAATTAATTTGTGGATTC 32 AGAAATAACATCAACATGTTGTGTCTCCTC 34 GTGCACCGTTGATTACTCCGG 35 TATCAATCGTTCGATAATAA

[0211] The transformed strain obtained above was named CJTHR-3.

[0213] Example 1-2. Random mutagenesis via UV irradiation

[0214] In order to select a mutant strain with increased threonine production capacity, CJTHR-3, a threonine-producing strain prepared in Example 1-1 above, was plated on a nutrient medium containing agar and cultured at 30°C for 16 hours. The colonies obtained in this way were irradiated with UV (Ultraviolet mutation) at room temperature to induce random mutations on the genome within the strain.

[0215] Nutrient medium (pH 7.2)

[0216] Glucose 10g, meat extract 5g, polypeptide 10g, sodium chloride 2.5g, yeast extract 5g, agar 20g, urea 2g (based on 1 liter of distilled water)

[0218] Examples 1-3: Selection of strains with improved L-threonine production capacity

[0219] In order to select mutant strains with increased threonine production capacity compared to the parent strain CJTHR-3, the CJTHR-3 strain and the above mutant strains with randomly induced mutations were cultured in the following manner.

[0220] The above strains were each inoculated into a 96-Deep Well Plate-Dome (Bioneer) containing 400 µl of seed medium and cultured in a plate shaking incubator (TAITEC) at 32°C and 1200 rpm for about 48 hours. The threonine concentrations of approximately 3,000 cultured strains were each analyzed using a near-infrared (NIR) spectrometer, and the top 5 mutant strains with improved threonine production capacity compared to the parent strain CJTHR-3 were selected.

[0221] <Seed medium (pH 7.0)>

[0222] Glucose 20 g, Peptone 10 g, Yeast extract 5 g, Urea 1.5 g, KH2PO4 4 g, K2HPO4 8 g, MgSO4·7H2O 0.5 g, Biotin 100 μg, Thiamine HCl 1000 μg, Calcium-Pantothenic Acid 2000 μg, Nicotinamide 2000 μg (based on 1 liter of distilled water)

[0223] To finally select strains with reproducibly increased L-threonine production capacity from the five selected mutant strains, they were cultured and evaluated using the following method.

[0224] After inoculating the parent strain and the mutant strain into a 250 mL Corner-Barpool flask containing 25 mL of threonine production medium, the culture was incubated at 30°C for 48 hours with shaking at 200 rpm. After the culture was completed, the L-threonine concentration in the culture medium was analyzed using HPLC, and the L-threonine production concentrations of each mutant strain are shown in Table 5 below.

[0225] Production Medium (pH 7.2)

[0226] Glucose 10%, Yeast extract 0.2%, Ammonium sulfate 1.6%, Potassium dihydrogen phosphate 0.1%, Magnesium sulfate heptahydrate 0.1%, Iron heptahydrate 10 mg / ℓ, Manganese sulfate monohydrate 10 mg / ℓ, Biotin 200 μg / ℓ (based on 1 L of distilled water)

[0227] strain name L-Threonine (g / L) CJTHR-3 2.0 CJTHR-3_mt1 3.5 CJTHR-3_mt2 3.0 CJTHR-3_mt3 2.8 CJTHR-3_mt4 2.7 CJTHR-3_mt5 2.5

[0228] Among the five selected mutant strains, CJTHR-3_mt1 was finally selected as the strain with significantly improved L-threonine production capacity.

[0230] Example 2: Derivation of variants via Whole-genome sequencing (WGS)

[0231] Whole-genome sequencing (WGS) was performed on the CJTHR-3_mt1 strain selected in Examples 1-3 above to analyze the sequence, and by comparing it with the parent strain CJTHR-3, locations where mutations occurred in the nucleotide sequence were identified, and each substitution type is indicated in Tables 6 to 9 below.

[0232] Variation in the CDS (Coding Sequence) region Wild-type strain amino acids Protein mutation location Mutant strain amino acids gene ID Gene name expression protein Variant amino acid sequence number Variant nucleic acid sequence number E 27 D NCgl0658 lpdA flavoprotein disulfide reductase 36 69 A 265 T NCgl0865 dld D-LACTATE DEHYDROGENASE 37 70 P 28 L NCgl1063 dapD2 tetrahydrodipicolinate N-succinyltransferase 38 71 D 461 E NCgl1165 atpD ATP synthase F0F1 subunit beta 39 72 S 2 G NCgl1511 ctaB Protoheme IX farnesyltransferase 40 73 G 527 S NCgl1523 ppc Phosphoenolpyruvate carboxylase 41 74 R 134 Q NCgl2139 thrC Threonine synthase 42 75 R 135 P G 233 D NCgl1999 gdh Glutamate dehydrogenase 43 76 H 217 Q NCgl2133 glnA glutamine synthase 44 77 A 410 V NCgl0239 dnaZX DNA polymerase III subunits gamma and tau 45 78 E 363 D NCgl0337 wzz chromosome partitioning ATPase 46 79 P 41 S NCgl0472 rpoC DNA-directed RNA polymerase subunit beta 47 80 A 182 T NCgl0674 wbpC acyltransferase 48 81 L 1112 F NCgl0706 - Type II restriction enzyme, methylase subunits 49 82 G 3 S NCgl0948 - Mycothiol conjugate amidase Mca 50 83 L 48 F F 188 V A 332 V NCgl1120 - DNA repair ATPase 51 84 E 521 O NCgl1142 narG anaerobic dehydrogenase 52 85 L 305 P NCgl1253 thiC thiamine biosynthesis protein ThiC 53 86 S 13 F NCgl1533 ribA bifunctional 3,4-dihydroxy-2-butanone 4-phosphate synthase / GTP cyclohydrolase II protein 54 87 G 75 V NCgl1859 fruR transcriptional regulator of sugar metabolism 55 88 S 164 P NCgl1874 miaB (dimethylallyl)adenosine tRNA methylthiotransferase 56 89 A 184 T NCgl2308 pcaR transcriptional regulator 57 90 A 431 V NCgl2859 - cation transport ATPase 58 91 R 14 W NCgl2863 cgtR9 Two-component system, response regulator 59 92 93G 135 D NCgl2928 trpG anthranilate synthase II 60 93 F 344 L NCgl0569 gcp DNA-binding / iron metalloprotein / AP endonuclease 61 94 G 31 D NCgl1320 - hypothetical protein 62 95 V 81 I NCgl1588 - hypothetical protein 63 96 V 118 G NCgl1760 - hypothetical protein 64 97 V 23 A NCgl0686 - hypothetical protein 65 98 L 180 Q NCgl2707 - hypothetical protein 66 99 R 313 U NCgl2851 - hypothetical protein 67 100 S 312 C C 309 R L 305 F L 304 S R 303 C Y 302 H L 300 F R 299 C R 288 L P 283 S D 146 N NCgl2320 benA benzoate dioxygenase large subunit 68 101

[0233] Variation in the promoter region 프로모터 명 목적 유전자 ID 목적 유전자 명 발현 단백질 변이체 서열번호 Pncgl0247_mt NCgl0247 lysC aspartate kinase 102 Pncgl2528_mt NCgl2528 ddh D-2-hydroxyisocaproate dehydrogenase 103 Pncgl1353_mt ncgl1353 uspA2 universal stress protein UspA 104 Pncgl1501_mt ncgl1501 sufC ABC transporter ATPase 105 Pncgl1836_mt ncgl1836 sigA RNA polymerase sigma factor 106 Pncgl1969_mt ncgl1969 pcaB2 Adenylosuccinate lyase 107 Pncgl2466_mt ncgl2466 - sugar phosphate isomerase / epimerase 108 Pncgl2960_mt ncgl2960 - hypothetical protein 109

[0234] Variations in tRNA and rRNA regions TYPE gene ID Variant sequence number rRNA NCg1r05 110 rRNA NCg1r09 111 rRNA NCg1r13 112 rRNA NCg1r15 113 rRNA NCg1r16 114 tRNA NCg1t06 115

[0235] Sequence number Sequence (Amino acid sequence: N-terminus → C-terminus; Nucleotide sequence: 5'→3') 36 MAKRIVIIGGGPAGYEAALAGAKYGADVTVIEDVGVGGSAVTMDCVPSKSFIAGTGIKTDLRRADDMGLNRGLGKAHLEIDALNIRVKDLAKAQSEDILGQLQRSDVRMINGVGRFD DYNTKQTTHYIKVTHSDGSEETVECDLVLVATGATPRILKGAEPDGERILTWRQVYDIEELPTHLIVVGSGVTGAEFVSAFAELGVKVTMVASRDRILPHDDADAADVLETVLAERG VSLEKHARVESVTRTEDGGVCVRTADGREIYGSHALMTVGSIPNTADLGLENIGVELAPSGHIKVDRVSRTNIPGVYAAGDCTDLFPLASVAAMQGRIAMYHALGEGVSPIRLKTVA TAVFTRPEIAAVGITHAQVDSGEVSARVIVLPLATNPRAKMRSLRHGFVKLFCRRNSGLIIGGVVVAPTASELILPIAVAVTNRLTVADLADTFAVYPSLSGSITEAARQLVQHDDLG 37 MTQPGQTTTTSHEAIDAFKRIVGDEHVLTSERATMPFSKGYRFGGGPVFAVVRPGTLVEMWRALQVSVDNNLIVIPQASNTGLTGGSGPGFQDYDRPIVIISTHRIDEVHLINDAREAISLAGTPLTHLDTALAKHQREPHSVIGSTSIGASVIGGIANNSGGSQIRKGPAFTREAIFARVNDDGKVELVNHLGISLGDDPEVALDRLQRGEWSPEDVTPAPEDSNETEYAEHLRKIVPSPARYNANPEYLFEASGSAGKLMVFTVRTRTFPREVHPTVFYIGTN NTHELEEIRRLFLEADMPLPISGEYMGRSAFDLAEKYGKDTVFLKFMSPALQTRMFSFKTWANGLSKIPGIGPTFADTVSQAMFSVLPNQLPKRMMEYRNRFHEHHLLLTVSESQKAASEKMLKEFFAEPEHTGEFFICTSD EEKSASLNRFGAASAATRYAALKRRHIAGlipIDVALRRDDWNWLEVLPEEIDDQLEVKAYYGHFFCHVMHQDYVAKQGVDLLEALHDRIQHLLEERGAKLPAEHNYGRIYKLPESMEEHFKELDPTNTFNAGIGGTSPHKDWA 38 MSENIRGAQAVGIANIAMDGTILDTWYLEPQIFNPDQWAERYPLEVGTTRLGANELTPRMLQLVKLDQDRLVEQVAVRTVIPDLSQPPVDAHDVYLRLHLLSHRLVRPHEMHMQNTLELLSDVVWTNKGPCLPENFEWVRGALRSRGLIHVYCVDRLP RMVDYVVPPGVRISEAERVRLGAYLAPGTSVLREGFVSFNSGTLGAAKVEGRLSSGVVIGEGSEIGLSSTIQSPRDEQRRRLPLSIGQNCNFGVSSGIIGVSLDGDNCDIGNIVLDGDTPIWFAADEELRTIDSIEGQANWSIKRESGFHEPVARLKA 39 MTTALEEQNAQQAATAGRVVRVIGAVVDVEFPRGELPALYNALTVEVTLESVKKTVVLEVAQHLGDNLIRTIAMAPTDGLVRGAAVTDTARPISVPVGDVVKGHVFNALGDCLDDVSLNNNPEIERWGIHREPPSFDQLEGKTEILETGIKVIDLLTPYVKGGKIGLFGGAGVGKTVLIQMITRIAREFSGTSVFAGVGERTREGTDLFEMEEMGVLQDTALVFGQMDEPPGVRMRVALSGLTMAEYFRDVQNQDVLLFIDNIFRFTQAGSEVSTLLGRMPSAVGYQPTLADEMGVLQERITSTKGRSITSLQAVYVPADDYTDPAPATTFAHLDATTELDSIASKGIYPAVNPLTSTSRILEPAIVGERHYEVSQRVIGILQKNKELQDIIAILGMDELSEEDKITVARARRIERFLGQNFFVAEKFTGLPGSYVPLTDTVDAFERICNGDFDHYPEQAFNGLGGLDDVEAAYKKLTGK 40 MGDLKMQRSGGEPLDTIKAYIALTKPRVIELLLVATIPTMLQAERGENNIVLILLTVFGGWMGAAAANTFNMVADSDIDQRMGRTRARPLVRHTVSNRDASIFAWVLTVASFLWLWLLCDSMLAGIFVLITIFFYIFVYTKWLKRRTHMNIVWGGAAGCMPVLVGWAVIVDQFEPGVPQQWWQAIVLFMVIFFWTPPHTWALAMKYREDYKAAGVPMLPVVRTPVQVTAQIVWYSVATVLTTFLLIPATGWIYAAIAVISGVTFLFMAIKLHLGIKNGGKVKPLKLFILSNNYLAVLFVALSVDAVLGLETIGEMLGWTTTFF 41 MTDFLRDDIRFLGQILGEVIAEQEGQEVYELVEQARLTSFDIAKGNAEMDSLVQVFDGITPAKATPIARAFSHFALLANLAEDLYDEELREQALDAGDTPPDSTLDATWLKLNEGNVGAEAVADVLRNAEVAPVLTAHPTETRRTVFDAQKWITTHMRERHALQSAEPTARTQSKLDEIEKNIRRRITILWQTALIRVARPRIEDEIEVGLRYYKLSLLEEIPRINRD VAVELRERFGEGVPLKPVVKPGSWIGGDHDGNPYVTAETVEYSTHRAAETVLKYYARQLHSLEHELSLSDRMNKVTPQLLALADAGHNDVPSRVDEPYRRAVHGVRGRILATTAELIGEDAVEGVWFKVFTPYASPEEFLNDALTIDHSLRESKDVLIADDRLSVLISAIESFGFNLYALDLRQNSESYEDVLTELFERAQVTANYRELSEAEKLEVLLKELRSPRPLIP HGSDEYSEVTDRELGIFRTASEAVKKFGPRMVPHCIISMASSVTDVLEPMVLLKEFGLIAANGDNPRSTVDVIPLFETIEDLQAGAGILDELWKIDLYRNYLLQRDNVQEVMLGYSDSNKDGGYFSANWALYDAELQLVELCRSAGVKLRLFHGRGGTVGRGGGPSYDAILAQPRGAVQGSVRITEQGEIISAKYGNPETARRNLEALVSATLEASLLDVSELTDHQRAY DIMSEILSLKKYASLVHEDQGFIDYFTQSTPLQEIGSLNIGSRPSSRKQTSSVEDLRAIPWVLSWSQSRVMLPGWFGVGTALEQWIGEGEQATQRIAELQTLNESWPFFTSVLDNMAQVMSKAELRLAKLYADLIPDTEVAERVYSVIREEYFLTKKMFCVITGSDDLLDDNPLLARSVQRRYPYLPLNVIQVEMMRRYKGDQSEQVSRNIQLTMNGLSTALRNSG 42 MDYISTRDASRTPARFSDILLGGLAPDGGLYLPATYPQLDDAQLSKWREVLANEGYAALAAEVISLFVDDIPVEDIKAITARAYTYPKFNSEDIVPVTELEDNIYLGHLSEGPTAAFMQLLGELFEYELQPRNETINILGATSGDTGSSAEYAMRGREGIRVFMLTPAGRMTPFQQAQMFGLDDPNIFNIALDGVFDDCQDVVKAVSADAEFKKDNRIGAVNSINWARLMAQVVYYVSSWIRTTTSNDQKVSFSVPTGNFGDICAGHIARQMGLPIDRLIVATNENDVLDEFFRTGDYRVRSSADTHETSSPSMDISRASNFERFIFDLLGRDATRVNDLFGTQVRQGGFSLADDANFEKAAAEYGFASGRSTHADRVATIADVHSRLDVLIDPHTADGVHVARQWRDEVNTPIIVLETALPVKFADTIVEAIGEAPQTPERFAAIMDAPFKVSDLPNDTDAVKQYIVDAIANTSVK 43 MTVDEQVSNYYDMLLKRNAGEPEFHQAVAEVLESLKIVLEKDPHYADYGLIQRLCEPERQLIFRVPWVDDQGQVHVNRGFRVQFNSALGPYKGGLRFHPSVNLGIVKFLGFEQIFKNSLTGLPIGGGKGGSDFDPKGKSDLEIMRFCQSFMTELHRHIGEYRDVPAGDIGVGGREIGYLFGHYRRMANQHESGVLGLTGKGLTWGGSLVRTEATGGYGCVYFVSEMIKAKGESISDQKIIVSGSGNVATYAIEKAQELGATVIGFSDSSGWVHTPNGVDVAKLREIKEVRRARVSVYADEVEGATYHTDGSIWDLKCDIALPCATQNELNGENAKTLADNGCRFVAEGANMPSTPEAVEVFRERDIRFGPGKAANAGGVATSALEMQQNASRDSWSFEYTDERLQVIMKNIFKTCAETAAEYGHENDYVVGANIAGFKKVADAMLAQGVI 44 MAFETPEEIVKFIKDENVEFVDVRFTDLPGTEQHFSIPAASFDADTIEEGLAFDGSSIRGFTTIDESDMNLLPDLGTATLDPFRKAKTLNVKFFVHDPFTREAFSRDPRNVARKAEQYL ASTGIADTCNFGAEAEFYLFDSVRYSTEMNSGFYEVDTEEGWWNRGKETNLDGTPNLGAKNRVKGGYFPVAPYDQTVDVRDDMVRNLAASGFALERFQHEVGGGQQEINYRFNTMLHAA DDIQTFKYIIKNTARLHGKAATFMPKPLAGDNGSGMHAHQSLWKDGKPLFHDESGYAGLSDIARYYIGGILHHAGAVLAFTNATLNSYHRLVPGFEAPINLVYSQRNRSAAVRIPITGSNPKAKRIEFRAPDPPSGNPYLGFAAMMMAGLDGIKNRIEPHAPVDKDLYELPPEEAASIPQAPTSLEASLKALQEDTDFLTESDVFTEDLIEAYIQYKYDNEISPVRLRPTPQEFELYFDC 45 MALYSKYRPASFGELVGQSQVTDPLSAALDSGRINHAYLFSGPRGCGKTSSARILARSLNCVEGPTSTPCGVCNSCVALAPGGPGTLDVTELDAASNNGVDDMRELRERANYAPAESRYRVFIIDEAHMISTQGFNALLKIVEEPPAHLIFIFATTEPDKMIGTIRSRTHNYPFRLLTPGDMRKVLKNAVDGEGVHVDDSVYPLVIRAGGGSPRDSLSILDQLIAGSGPEGLTYERALPLLGVTSFTLIDDSIHALASKDNASMFTTIDNVIEEGLEPRRFTIDLLDRLRDLMVLQAVPEALNLGLVDAPTDRADALIEQSTLFKGNELANLASMVNSGLDDMRGATSPRLLLEILCARLLLASNTVAGPAVSSSTDAAPAATPGGLTGIAAARAKAREYGQKKAAPAPVPTPAPEPVREQSLAPTPEPTPAAEPTSQPAPEPEPAREPVVEVREASVEKQPASSDPLETIRSRWSELRNIVEKQSVRTSIMLTEARVLGLRGDTLVLGHSTGALAARLNAADHNGILVKVLAEETGLQLKVECIVGTNPAEAGFTAQPAVQKSTWNPNYDSKPATPSAPAQPQTPKQESVPTEPEEPESSAETSGWGQPVKIGGPAPEAQTPTPPPAPVVPATPAAPAAGAAKPAWKERVEQAAANAAQQRQHRQGSSEPFERGVPLPPEPDLPPDPYGYPADEGFPERNQGFQQQPAPPVEQPAPEPATEPVPAVAPEPEVSQLSEEEQLIREADEEPGEMDRRDAKTIAMELLAQELGAKPL 46 MELREYATILMKNWVLIVIASILGIAAGAGFSLLATPEYQSRTQLYVSVRSGAGTTSDMVQGANFSRQIVNSYVDVIKTGVVLEPVVDELGLELTANQLSSHISAASPADTALINITASSPSPQQAAEIANAVGESFKNVIQTELEPDSGDGMSPINLTTTQVALEPSPVSPNVMMNILLGLLVGLAIGVGIAVLRAALDTRIHSLRDIEEVTDKPLLGGIIADSEVEKHPLIIKHKPHSPIAESFRALRTNLQFLNVGGSSSVFVISSANPGEGKSTTSVNLALALAEAGSRVALIEADLRPRVSKYLGVEGNAGLTDILIGKAEVNDVLQRWGRTQLYYLPAGRIPPNPSELLGSAEMDKVIAELEESFDYVIIDAPPALAVTDAAVIGHGKAGILIAVSAGSTKKPELEATLSTLENADANVVGVVATMLPPKSVAGYGYGNYGYGDTSKINAPKPDNTELTTTDASKANNEQ 47 MLDVNVFDELRIGLATADDIRRWSKGEVKKPETINYRTLKSEKDGLFCERIFGPTRDWECACGKYKRVRYKGIICERCGVEVTKSKVRRERMGHIELAAPVTHIWYFKGVPSRLGYLLDLAPKDLDLIIYFGANIITSVDEEARHSDQTTLEAEMLLEKKDVEADAESDIAERAEKLEEDLAELEAAGAKADARRKVQAAADKEMQHIRERAQREIDRLDEVWQTFIKLAPKQMIRDEKLYDELIDRYEDYFTGGMGAESIEALIQNFDLDAEAEELRDIINNGKGQKKMRALKRLKVVAAFQRSGNDPAGMVLNAIPVIPPELRPMVQLDGGRFATSDLNDLYRRVINRNNRLKRMIELGAPEIIVNNEKRMLQESVDALFDNGRRGRPVTGPGNRPLKSLSDLLKGKQGRFRQNLLGKRVDYSGRSVIIVGPQLRLHECGLPKLMALELFKPFVMKRLVENEYAQNIKSAKRMVERQRPEVWDVLEEAISEHPVMLNRAPTLHRLGIQAFEPVLVEGKAIQLHPLACEAFNADFDGDQMAVHLPLSAEAQAEARVLMLASNNILSPASGKPLAMPRLDMVTGLYYLTLEKSSEEFGGQGAYQPADENGPEKGVYSSLAEAIMAYDRGVLGLQAPVRIRLNHLRPPAEVEAEQFPDGWNQGETWLAHTTLGRVMFNEILPWNYPYLEGVMVRKGGGSDKIMLGDVVNDLAAKYPMITVAQTMDKMKDAGFYWSTRSGVTIAMSDVLVLPNKEEMLDRYEESARQIEVKYNRGKLTGRERYDRLVELWKDATDEVGQAVEDLYPDDNPIPMIVKSGAAGNMRQIWTLAGMKGMVVNSKGDYITRPIKTSFREGLTVLEYFNNSHGSRKGLADTALRTADSGYLTRRLVDVAQDVIVRVEDCGTRQGVRVPVAAEVLDATGAVTGYTRHDLIETSVSGRVLAGDATNAAGEVVLAAGTDLTELNIDLLVEAGIKDVKVRSVLTCQTPTGVCAKCYGKSMASGQQVDIGEAVGIVAAQSIGEPGTQLTMRTFHQGGVGGDITGGLPRVQELFEARVPKNCAPIASVEGVIHLEDEGNFYTLTIVPDDGSDNVVYEKLSKRQGLASTRVAMESNAGAFIERTLTEGDRVTVGQRLLRGAADPHDVLEILGRRGVEQHLIDEVQAVYRAQGVAIHDKHIEIIIRQMLRRGTVIESGSTEFLPGSLVDLSEAKLANSEAIGAGGQPAELRSEIMGITKASLATESWLSAASFQETTRVLTDAAINKRSDKLIGLKENVIIGKLIPAGTGISRYRNISIKPTEAARNAAYSIPTYGESIYGDDGFGEFTGASVPLDEAF 48 MGTARGLSQVLKEARQGGKPTSATQKVPRSGYRYDLDGLRGIAIAFVVLFHVFVGKVSGGVDVFLLLSGYFFLGSQLRYADRPDSSINPWWPIWRTLRRLLPALVLVLGVSMVLILAWVPRLQRIEIANQAVASLFYVQNWELASQGAAYGAASAEVSPFQHLWSMAVQGQFYLFAILLSMTIILIRRYRPEYSAVRLATPVLAVLTSVSFFSAILWHFIDQSVNYYSTFTRFWELGLGALLVLHAPRILISAKTKSILAAVGLFMVLSTGFFMDGAETFPGFPALYPILGACLVILGDGKISVFLSRKWMLWLGDIAYPLYLWHWPLLIIFTALFNQEEPSIWLGIAVIMLSLGLAQLTNKYVEKPMQQQGKRPLFRESRSADALQKLRSSRPARMRAYAGIAVVAVGFALTSTPQVLYQRVLDTQMSDLDPEIYPGALSLAGFDAPEVKPKPNPYVLADTVSPAWAKGCMSVFGDDPSELAPDNYDEDYCAFGNPNSPVEVYLVGGSHAEQWMAPLDALGKQHNFKVIPLVRQSCPTFVVELDGIFSDDCIEFNEKVIERLAEVKPDLVVSNSTRPLLEKGRGIDEVPESYITLWDFLEAEDIPFMGLRDNPWFIQPGGEGWMVSQCYEEEQSLVDCSITKDNFYAPVDPAEKELAIRNNMLAVDTSRWFCPDDLCIPVIGNVYVYRDGNHMSDEYAQSLAPFLWSNMRRLLVA 49 MSAFDSILNVEEWISDHYLTNDDAKGASFSKRVRERIKEWKTTEDATQQSGPLTRFSSNRLQLQHALSELDDATTAASLVASALGYGVPSARHAQRGSDTISYSSWVGNAGSVEFLAATPAESFEENFRSLPLEPVAVNDKPQDITAAKLVGQIFLSDTPPAFVVITAGKWVVLAERETWPLGRHLAIDISLVVERNDTKAQGEMQQTVVALARENTERAADGTTWWEETIEQSREHAVKVSGELRSAVRESIEILGNDVLTRYEAKELSTAEIDGGELAKQSLRYLYRILFLLFAEASPELEILPTGTPEYDEGYGLARLRELILNPPVTQHARTGTHLYDSLQLLFTLVDKGHHPTDAKAVAFDAEAGEEGLHFRNLSADLFLPAATELIDRVGLSNEALNKVLENLLLSRVQSGKDRGFISYATLGVTELGQVYEGLMSYTGFIAQEDLFEVAPHGKADKGSWMLPVSKADEVPADSFIEVDQEAPGGGVIKVRKRHPRGSFVFRQSSRDRERSASFYTPQVLTSFTVTQAIEELQASKRITTANDVLSLTICEPAMGSGAFAVEAVRQLAELYLELRQEELEQQIPAEDRAKELQKVKAHIALHQVYGVDLNSTAVELAEISLWLDTMNAEMDAPWYGLHLRNGNSLVGATRSLYAPSLLNKKAWLTATPTRYRLDDIAQAIDENKAEPLFNHGIHHFLLPSTGWGATADAKDLKDLMATEIKELKSWRTSIRASLSKTQIKQLNNLALRVETLWRFVLMRIRIAESQISRSTTLWGQEPAEVSEVVTREQIEQDLFGNIDGAYNRLRLVMDAWCALWFWPLDAVATAEHPERPALPDLDEWLATLTEILGIDLPLKSKNENQIVLGPDTNWLAINDAEATDLGFSGALSFERVSANHPWINVARQVAKQQSFFHWDLDFAHVFAKGGFDLQVGNPPWVRPDVNFEDLLAEHDPWWAVMSKPTQASKKERQKNFHNNPKSLEHVVSGAGETVATSAILGSVTLYPHLKDQRPDLYRGFMEKTWSNASPAGAISLIHPESHFTEKKAAPLRRGAYERLRRHWQFINELILFDVHDLVKYGVHVYGAPQESINFLSAASLYHPQTVLDSFDHDGSGNLPGLKDDNGNWDRRPHKDRIQLVNADTLTVWKSILEDEQTPYLDTRMVYTVNTEAAAVLEKLASAPRIKELGLQFSSGWNETTDKKKGYFDVGWGYPASWSDAILQGPHLGVATPMIKQPNPTMKHNQDWSEIDFEAIPANFIPATAYQPDRQTKPTYDADYGTWTFGDKQVPVADTFRIAWREMAATTGFRTVYPSVIPPGANHVHTVNSAASRSNLKTILVGAQLGAILSDYFARSSGSSHIFNDIVRKIPLPNFTSLEKQFARTYLRLNCLTSAYAPLWEEITGEPWDVQVPLRNAEQRRAAQNDIDAMVALSLGISADELCMIYRTQFPVMRRYDQEDHFDANGRKVPKEIIKLQQKLKDGQELSVEKRTWVHPQSEVSYTFEYPFRVLDREADLRAAYAKFENQLKEP 50 MSSLRLMAIHAHPDDESSKGAATMARYAAEGNQVMVVTCTGGERGDIFNPAMDKPGILDNIFAVRQEEMAKAMEILGTEHRWLGYEDSGLPQGDPLPPLPEGCFALEDSDKVTQDLVKILREFRPHVIITYDENGGYPHPDHLKVHEVSMLAWEKSGDAAYAPELGAPWEPLKLYYTHGFIRQRMEMVHDLLIEQGKPSPYTPMLERWKANEADVMARVTTQVPCERFFDQRDDALRAHATQIDPAGAFFGTPVEVQRRLWPTEEFELAKTRVKTSIPEDDLFAGITPDAE 51 MRIHEIIIDNFRAIEHLELRDIPDQGVIVIHGDNEQGKSSILEAIKTVLNSKHRTTSKTIKAIQPVDRDVPISITLEATVGTVRFRIHKRFLKSTAAELQVIEPRPSNHRGLEAEAALAEILESHLDTSLLDALFMKQGEVEAGISAVGIPTLTSALNAQNGNTEDATEDTALMEAVEKEYLKFYTNSGKANTRFLQFSKQVETLRTDLDEANAEVAKLSSHVDRVKRLEIDRDQATAQLPKAEELAGRKAELEEAQKVKAQATEILAQFSRAEEQLEQATGAQKRRKELRNKLELAQTEVEKAEAGQENLAQEASREAEEFLASEKFEVARAAETLAVEKVKAARQSVAGIKNRDRKEHLTVMLGELDRIGQRLYELRSVQHSSVRVSQRDIDALQKAITEVDIQRTLVEAQQGSITLSASTPTDIQLGDDTVSA TDAGTTVALDRELTVVVGDVTLVINPGKTAAESRTDFESAEAALAELLDQLDVSDLDQLRERFTAQEQRDADIAELVREQQRMSGGTETTVLRAELEGLHVPEDLDPSISVDDAQTQLNEAEESRELAAEAHKHANAALDGLRSRPVDKALTVFNAQLAALQRNLSSAQVELDRAVAETSDDEVDAAVQRCAEALAGVRVQKQEIEQVLAKTNPDMAQRLCDAAEANVRSYKTAVSDATTELVRLEGLIGVAAGAKERLDKVKSALTAAENRFESEQRRAHAAARRLYALMVFYRDESRKRYAAPFADKLSRLAASVFGESADFDLDDELKISSRSIGPRTVDLANLSGGAKEQLAILTRFAIAELVAESSAQGAVPVFIDDALGSTDPERLTRISTLFSDAGKDSQVFVLTCVPDRYNYVEVTQKHSIESLKTANALL 52 MTTTTSSGKSSEKINPLFKLGSFLRKGTVGSEGQQIFLQGGRQADVFYRNRWAFDKVVRSTHGVNCTGSCSWKVYVKDGVITWESQAVDYPTTGADMPDYEPRGCPRGASFSWYTYSPTRIRYPYIRGVLVDMYREAKERLGDPVLAWRDIVETPEKRKAYVSQRGKGGLIRVQYEEAMEIAAAAHVYTIRQYGPDRIHGFTVIPAMSQVSYGAGTRFLQMIGGVALSFYDWYADLPPASPQTFGDQTDVPESGDWYNSSYLMMWGSNIPVTRTPDSHFMVEARYKGTKVVVVSPDFADSTKFADEWARIHPGTDGALAFAMGHVILKEFHVDKKTPYFMDYMRKYTDSPFLVELDEHGDGTYTPGKFLTADRAADISPALAATPNATHRLLVLQKDGSVVDPGGTVADRWGEEGMGKWNLRLDGVDPVMTIADVQTDTETAEVLFPRFDLPATATQEGPIGAGTISRGVPTITLNGRKYTTVFDVLLAHYGVNREELNLPGEWPKDFQDPVMGTPAWQEOLTGVPANQAIRLGREFAQNADDSKGRSQIIMGAGVNHYFHADSIYRTFLALTSMCGTQGVNGGGWAHYVGQEKLRPMNGWAQYAFATDWQRPPRQMITTGFYYLTTDQWRYDNTRANRLASPLANRGTVGDKMTADTLVESMKRGWMPSFPQFNRNPLILSQEAEEKGVSVSDHIVQQLTDGDLQFACEDPDAPENWPRILLNWRTNLMGSSAKGTEFFLRHMLGVDSDASAEENAPEDRPSSIVWRDEAPEGKLDLMLTTDFRNTSTTLVSDIVLPAATWYEKHDLSTTDMHPFIHSFNAAINPPWETRTDWEVFHDLTKEFSSQAATWLGTQTDVITAPIAHDSPDELNMPGGIVPDIDEVGLIPGKTMAKIIPVERDYSKVYEKWTHLGPLTAKAGTGTHGTAFNVTKQTEELALINGTSITESAGERPRLDSAVKAIEMILHLSGVSNGEVAAEGFKNQGARVGTDMMDLIADHEGTHINWDMVKERPAEVITSPEWTGSKKDGRRYTAFSINIEYDKPWHTLSGRMHYYLDHDWFIDYGEQLPIFRPPLDKIHINGEVGPGQSVTGTDGEPEVTVRYLTTHNKWSIHSQYYDNLHVLSISRGGQVIWMSNKDAEKLGIADNDWIEAYNRNGVVSARAIVSHRIPEGTVFMNHAQERTAGTPLNEKSGRRGGTHNSLTRIMIKPVHVAGGYGHLTYGFNYIGPTGNNRDEVTRIRRRSQEVQY 53 MTPTQNEIHPKHSYSPIRKDGLEVPETEIRLDDSPSGPNEPFRIYRTRGPETNPKQGLPRLRESWITARGDVATYQGRERLLIDDGRSAMRRGQASAEWKGQKPAPLKALPGKRVTQMAYARAGVITREMEFVALREHVDAEFVRSEVARGRAIIPNNVNHPESEPMIIGRKFLTKINANIGNSAVTSSIEEVSKLQWATRWGADTVMDLSTGDDIHTTREWIIRNSVPIGTVPIYQALEKVNGVAADLNWEVFRDTIIEQCEQGVDYMTIHAGVLLAYIPLTTRVTGIV SRGGSIMAGWCPAHHRESFLYEHFDELCEIFAQYDVAFSLGDGLRPGSLADANDAAQFAELKTIGELTQRAWEYDVQVMVEGPGHVPLNMIQENNELEQKWAADAPFYTLGPLVTDIAPGYDHITSAIGAAHIAMGGTAMLCYVTPKEHLGLPNRDDVKTGVITYKLAAHAADVAKGHPGARAWDDAMSKARFEFRWNDQFALSLDPDTAIAYHDETLPAEPAKTAHFCSMCGPKFCSMRISQDIRDMFGDQIAELGMPGVGDSSSAVASSGAREGMAEKSREFIAGGAEVYRR 54 MSEHEQAHSQLDFVEEAIADIAAGKAVVVVDDEDRENEGDIIFAAELATPELVAFMVRYSSGYICAPLATKADRALLDLPPMTAHNQDARGTAYTVTVDANTGTTGISATDRAHTLRLLADPEADRTDFTRPGHVVPLRAREGGVLVRAGHTEAAVDLARAAGLRPAGVICEVVSEEDPTGMARVPELRRFCDEHDLKLISIEQLIEWRRKNEILVERQVETVLPTDFGTFKAVGYRSIIDGTELVAIVAGDVASDGGENVLVRVHSECLTGDVFGSRRCDCGQQLHESLRLIQEAGRGVVVYMRGHEGRGIGLLAKLRAYQLQDEGADTVDANLALGLPADAREFGTSAQILYDLGVRSLNISNNPAKKVGLEGHGISIASRTPIPVAVHEDNVRYLKTKRDRMGHDLPDVALWEQEHPEN 55 MVSQTERQHAIASLLAPTGAVSVGDLAEHFHVTTETVRRDLRIMESLGLLQRVHGGAISPEPMGTSPPRLKPALVKGMPPEPRVLELAETAVSLITPLARSIFLDSGLACTAIATVLGDPPEDARWTVVTSSPGAVIALSADTATSTVVLHGQVHGNCSSIIGSTAVDMISQLRADIAFVEVDAIQSDTSLCTFFPETIPIKQAMIKNAAFTVAVLSPRSPQDQELQLLKHPFSTLADFDALVTDDHTLDFPVLPDHNFQVVTP 56 MTQQLNHAKVNQHPGQATLPETAEGQVRTYEVKTYGCQMNVHDSERLSGLLEEAGYVAAPEDTTPDLVVFNTCAVRENADMRLYGTLGNLRSVKEKNPGMQIAVGGCLAQKDKDTVVKKAPWVDVVFGTHNIGSLPTLLQRAEHNAQAEVEIVDSLEQFPSVLPAKRESAYAGWVSVSVGCNNTCTFCIVPSLRGKEQDRRPGDILAEVQALVDQGVTEVTLLGQNVNAYGVNFVDPELERDRSAFSKLLRACGEIEGLERVRFTSPHPAEFTSDVIDAMAETPNICPQLHMPLQSGSDKVLKEMRRSYRSKKFLSILDEVRAKIPHASITTDIIVGFPGETEEDFQATLDVVKKARFTSAYTFQYSPRPGTPAAEYENQLPKEVVQERYERLMVVQEQVCEEENQKLIGTTVELLVQAGGGRKNDATKRMSGRARDGRLVHFAPEGDIDGEIRPGDFVTVTVTEAKPFFLIADSGVQTHRRTKAGDNSAVGQVPTTAPIGVGLGLPQIGAPKVAPATESACCSIN 57 MVEQSPDFVQSFARGLSVIRSFSADNPSQTLSEVASQTGLSRATARRFLHTLTDLGYAVNNDSRFQLTPRVLELGASYLSALSLPAIAQPRLEVLSRQVGESSSMSVLDGTDIIYVCRVPVRRIMTVNITIGTRFPAYATSMGRIMLANLPEEELDEMLAAAPPEQLTTRSLTSIASIREEIITTRERGWSLVDQELEPGLRSLAAPITNAQGEVVASINVSTQSASHSVEDIRKLVLPQLLETAQAISTDLSAL 58 * 59 MADRTPTTATPPGWVLVVDDEQPLAQMVASYLIRAGFDTRQAHTGTQAVDEARRFSPDVVVLDLGLPELDGLEVCRRIRTFSDCYILMLTARGSEDDKISGLTGADDYITKPFSIRELVTRVHAVLRRPRTSTTTPPQVTTPLIVGDLILDPVAHQVWVGETTVELTRTEFELLVALALRPGQVLTRHDLITEVWDTTWVGDERIVDVHIGNLRRKLGTDTRGRGFIDTVRGVGYRVGQP 60 MTHVVLIDNHDSSFVYNLVDAFAVAGYKCTVFRNTVPVETILAANPDLICLSPGPGYPADAGNMMALIERTLGQIPLLGICLGYQALIEYHGGKVEPCGPVHGTTDNMILTADAGVQSPVFAGLATDVEDHPEIPDRKVPIGRYHSLGCVVAPDGIESLGTCSSEIGDVIMAARTTDGKAIGLQFHPESVLSPTGPVILSRCVEQLLAN 61 MIVLGIESSCDETGVGVVKLDGEGNLEILADSVASSMQEHARFGGVVPEIASRAHLESMVPVMREALRQAGVDRPDAVAATVGPGLAGALLVGASAAKAYAAAWGVPFYAVNHLGGHVAVANLEGETLPHAVALLVSGGHTQLLEVDAVGLPMKELGSTLDDAAGEAYDKVSRLLGLGYPGGPIIDKLARRGNPEAIAFPRGLMKKSDSRHDFSFSGLKTSVARYVEAAERNGEVISVEDVCASFQEAVCDVLTFKAVRACRDVGAKVLLLGGGVAANSRLRELAQERCDKADIELRVPRFNLCTDNGVMIAALAAQRIHEGAQESPISVGTDPLSVETPQVL 62 MIRKLARPMLASVYVADGAETVLNTSAHVEDTQVVLDRIRYVLPRKYAKRISRDPELVTRVIGGTKVGAGSLLAIGRAPRTSAATLAILTIPNILARNAFWETQDADEKRNRRNGFLTNIALLGGLFITSVDTEGKPGVKWRATNATKRGKKQLQQALPTKSETEKFGEKASDWFNDTSDKVTEYAYTAQDFVGENKDDWIKSATETAHKVADTVSDYAHKATSYLEENSGDWLEAAQANAKTARKSAVKAAGKAQEKANFALQVAEETSGRANKKATKASYDKLQKQADKAIDRAQKKLKGIEL 63 MKLFSRTSLVALGTAAAITLSGVTAPAFADEDSNAAVSALKTAEDNTEAPGASTPLKLEQPGTITGVPGKAITPVTVKVIAGEAESFTSDNLPSGLLIDNTGKITGTPKKEFTGSAKIIAKNEAGVEAEVYVNFDFNEEPSSEEPSSGSSDTDNIENWIKIITAVIGALTTILTFSTKLDSFLK 64 MDWSIIINVLAVATVVILTLIIAAALYRGFTRNKIKKLEAIREAQQHERDNPTIRIADLRGIMDTHHYIYTDVMVTRAHELMITAPAFFDVTLPEAVLYEDTKRAAHDALNGYKDTTGASAEKIMLVDAVTAAWTLYSTKANTGST 65 MSTAYFIAHGNDTYRPTEHSSGAWRDDELHLAPVAGLVIHHMERWRREVVGDALVFSRFSLEVLGQIARDDVTLRTEIVRPGRTIELIETVAEINGRVTIRARAWLLKTSDLAHISGDTFEALPSMTELADVKSAFQWSGGFMDSIVFVEDAEKIPGKNRAWITGDVQLVDGEDSEPLAEFSKFLDVANGVAVREDPKTWMFPNVDLTFHLFRQPVGTKVGFDTRAAFGPNGIGLTTSVIHDAEGPVGTLNQSLTVRPIAELNK 66 MNDQLWEGDTGTLTFGSRKALVQLLKGPMVNALQHVEVWRAITTDQDALNAVLNNLFLELVLDEDAGVAFTRPANGRQEVLVGNNKTEAMPKVLRTETLSHFDTLIILILRQELTMAPPGERVIVDREEIREQVLLYRVDEERDEAKLAKRFDAAFRRIVDYSLAKKTETPERFEVSPAQRQIFDADTVAGVRAEYEKFNKAAHDGNEEEQK 67 MVHMVFGDMNTDRAAQAYIIVITTIVMVVLFWIVLSYWSLADRARAQRFTASITEIGRKIFLNRLRPRMSRQNTYTDKDISQFHWTNGLPPTDDESPEWIAARDNEWEGYTITLGDDPNGTEKTITLDDLRELPQTSYVAVHTCMQGWSATARWTGVRLRDVLCHDLVHTLDLHHRHSPRLLTIEIIPKPLPPETRCKIIDFDLFAPNSPSIEEFCSRLKISRRSFYNIRNRYQQDASAALHPRSSAQITSRRTYDESITSILLAIRAPPESPRMGIRSDLYSIRRHLHRGTDCTDSICFNHCSFVTRRRSCUK 68 MSTPVSNLASVQKTLDHALEDRPEEGIVRVNRNIFTDPEIFELEMRHIFEGNWIYLAHESQIPNAGDYFTTYIGRQPIMITRSKDGTLNCLINACSHRGAMLCRRKTDNRTTLTCPFHGWTFSNDGALLKVKDEKEGAYPENFRTDGSHDLRRVPKFESYRGFLFGSLNDDVVSLEEHLGDTRTVIDMLVDQSPEGLEVLRGSSTYTYDGNWKLQTENGADGYHVSSTHWNYAATTSRRGTGESANETKAMDAGTWGKQGGGYFSYPYGHMLLWMWWGNPEDRPLFERRDEFKKEFGEEKGEFMVGASRNLCLYPNVYLMDQFSSQIRHIRPISVDQTEVTIYCIAPKGESAEARANRIRQYEDFFNATGMATPDDLEEFRSCQKTYQASAFPWNDMTRGLGHQVQGPNEVAKGLGMNEVLSSGARTEDEGLYPIQHGFWHELMQEAVNKQSIKEKELADDTASSLATVAAAKIREEAKAAAKSDAGEPRRRRRTRG 69 ATGGCAAAGAGGATCGTAATTATCGGCGGTGGACCTGCAGGCTATGAAGCCGCACTCGCAGGCGCTAAATACGGTGCAGACGTTACCGTTATTGAAGATGTCGGAGTTGGCGGATCCGCAGTCACCATGGACTGTGTACCTTCAAAGTCCTTCATCGCTGGTACCGGTATCAAAACCGACCTCCGACGTGCTGATGACATGGGACTTAACCGTGGGCTTGGAAAAGCACACCTAGAAATCGATGCACTGAACATCCGTGTGAAGGACCTTGCGAAAGCACAGTCCGAAGATATCTTGGGCCAGCTGCAGCGCTCAGATGTCCGCATGATTAACGGTGTGGGCCGCTTTGATGATTACAACACCAAGCAAACCACCCACTACATTAAAGTCACCCACAGCGATGGCTCCGAAGAAACCGTTGAGTGCGATCTGGTGCTGGTTGCAACTGGTGCAACCCCCCGCATTCTTAAAGGTGCAGAGCCAGACGGCGAGCGCATCTTGACCTGGCGTCAGGTCTACGACATTGAAGAACTCCCCACCCACCTTATCGTGGTTGGTTCCGGTGTGACCGGTGCGGAATTTGTCTCTGCGTTTGCTGAACTCGGCGTCAAAGTCACCATGGTGGCATCCCGTGACCGCATTTTGCCTCACGATGACGCAGATGCCGCAGACGTGCTGGAAACCGTTCTGGCTGAGCGCGGAGTATCCCTGGAAAAGCATGCCCGCGTGGAGTCTGTCACCCGCACCGAAGACGGTGGCGTGTGTGTTCGCACTGCTGACGGACGAGAAATCTACGGTTCTCACGCGTTGATGACTGTTGGTTCCATTCCAAACACGGCAGATCTTGGCCTGGAGAACATCGGTGTTGAGCTGGCACCATCCGGCCATATCAAGGTTGACCGCGTCTCCCGCACCAACATCCCCGGTGTGTACGCAGCAGGTGACTGTACTGACCTATTCCCACTGGCGTCCGTTGCAGCGATGCAGGGCCGTATCGCCATGTATCACGCACTCGGTGAAGGCGTGAGCCCCATCCGTTTGAAGACTGTTGCCACCGCAGTGTTTACCCGCCCAGAGATCGCAGCAGTAGGTATCACCCATGCACAAGTTGATTCCGGCGAAGTGTCTGCTCGCGTGATTGTGCTTCCTTTGGCTACTAACCCACGCGCCAAGATGCGTTCCCTGCGCCACGGTTTTGTGAAGCTGTTCTGCCGCCGTAACTCTGGCCTGATCATCGGTGGTGTCGTGGTGGCACCGACCGCGTCTGAGCTGATCCTACCGATCGCTGTGGCAGTGACCAACCGTCTGACAGTTGCTGATCTGGCTGATACCTTCGCGGTGTACCCATCATTGTCAGGTTCGATTACTGAAGCAGCACGTCAGCTGGTTCAACATGATGATCTAGGCTAA 70 ATGACGCAACCAGGACAGACCACCACGACTTCGCACGAAGCGATCGATGCGTTCAAGAGAATCGTCGGCGACGAACATGTACTGACCTCTGAGCGTGCCACGATGCCATTCAGCAAAGGCTATCGATTCGGCGGAGGACCAGTCTTCGCCGTGGTGCGCCCCGGCACGCTGGTCGAGATGTGGCGGGCGCTGCAGGTATCCGTCGACAACAACCTCATCGTCATCCCGCAGGCATCGAACACGGGCCTGACTGGTGGATCCGGCCCCGGCTTCCAAGACTACGATCGCCCCATTGTGATCATCTCGACTCACCGCATCGATGAGGTGCACCTCATCAACGACGCGCGCGAGGCGATCTCGCTCGCGGGCACCCCGCTGACACACCTGACCGACGCGCTCGCCAAGCACCAGCGCGAGCCGCACTCGGTGATCGGGTCGACATCAATCGGCGCCTCGGTCATCGGCGGCATCGCGAACAACTCGGGCGGCAGCCAGATTCGCAAGGGTCCGGCATTCACGCGCGAAGCGATCTTCGCCCGCGTCAACGACGACGGCAAGGTCGAGCTGGTCAATCACCTGGGCATCTCGCTCGGAGACGACCCTGAGGTCGCACTCGACCGTCTACAGCGCGGCGAGTGGTCTCCCGAGGATGTCACCCCAGCTCCCGAAGACTCGAACGAGACCGAGTACGCCGAGCACTTGCGCAAGATCGTGCCTTCGCCTGCTCGCTACAATGCGAACCCCGAGTACCTGTTCGAGGCTTCCGGCTCGGCCGGCAAGCTGATGGTGTTCACGGTGCGCACCCGCACCTTCCCTCGCGAAGTGCACCCGACCGTGTTTTACATCGGCACGAACAACACGCACGAGCTCGAAGAGATCCGTCGGTTGTTCCTCGAAGCCGACATGCCGCTGCCTATCTCTGGTGAGTACATGGGCCGCAGTGCCTTCGACTTGGCCGAGAAGTACGGCAAAGACACCTTCGTCTTCCTGAAGTTCATGAGTCCAGCGCTGCAGACGCGCATGTTCTCGTTCAAGACGTGGGCCAACGGCTTGTTCTCGAAGATTCCCGGCATTGGTCCGACCTTCGCCGACACGGTATCGCAAGCCATGTTCAGCGTGCTGCCCAACCAGCTGCCCAAGCGCATGATGGAGTACCGCAACCGTTTCGAGCATCACCTGCTGCTCACCGTCAGCGAGTCGCAGAAGGCCGCGAGCGAGAAGATGCTCAAGGAGTTCTTCGCAGAGCCCGAGCACACTGGTGAGTTCTTCATCTGCACGTCTGATGAAGAAAAGAGCGCGTCGCTCAACCGGTTCGGCGCGGCCAGTGCCGCCACTCGCTACGCCGCGTTGAAGCGCCGGCACATCGCAGGGCTCATCCCCATCGATGTGGCCCTGCGTCGCGACGATTGGAACTGGCTCGAGGTGCTGCCGGAGGAGATCGACGACCAGCTTGAGGTCAAGGCGTATTACGGGCACTTCTTCTGCCATGTGATGCACCAGGACTATGTCGCCAAGCAGGGCGTGGATCTCGAGGCGCTGCACGACCGCATCCAGCACCTGCTGGAGGAGCGCGGCGCGAAGCTGCCCGCCGAGCACAACTACGGTCGCATCTACAAGCTGCCGGAGTCCATGGAAGAGCACTTCAAGGAGCTCGATCCGACGAATACGTTCAACGCCGGTATCGGCGGCACGTCGCCGCACAAGGACTGGGCCTAA 71 ATGAGTGAAAACATTCGCGGAGCCCAAGCAGTTGGAATCGCAAATATCGCCATGGACGGGACCATCCTGGACACGTGGTACCTAGAACCCCAAATTTTCAACCCGGATCAGTGGGCTGAACGCTACCCATTGGAAGTGGGCACCACACGCCTCGGAGCAAACGAACTCACCCCACGGATGCTGCAGTTGGTAAAACTGGACCAAGATCGCCTCGTCGAACAGGTAGCAGTCCGCACCGTTATCCCCGATCTGTCTCAACCTCCAGTAGACGCGCACGATGTTTACCTGCGCCTCCACCTGCTTTCCCACCGCCTGGTCCGCCCCCACGAAATGCACATGCAAAACACCTTGGAGCTGCTGTCCGACGTGGTGTGGACAAACAAGGGCCCTTGCCTTCCTGAAAACTTTGAGTGGGTGCGTGGTGCTCTGCGGTCCCGCGGACTCATCCACGTCTACTGTGTGGACCGTCTTCCCCGCATGGTCGACTATGTGGTTCCCCCTGGAGTCCGCATCTCCGAAGCAGAACGCGTGCGCCTAGGTGCATACCTTGCTCCGGGTACCTCTGTGCTGCGTGAAGGTTTCGTGTCTTTCAACTCCGGCACCTTGGGTGCCGCAAAGGTGGAAGGCCGCCTGAGTTCCGGTGTGGTCATCGGTGAAGGTTCCGAGATTGGACTGTCTTCTACTATTCAGTCCCCGAGAGATGAACAGCGCCGCCGTTTGCCGTTGAGCATCGGCCAAAACTGCAACTTTGGTGTCAGCTCCGGAATCATCGGAGTCAGTCTGGGAGACAATTGCGACATCGGAAATAACATTGTCTTGGATGGAGATACCCCCATTTGGTTCGCAGCCGATGAGGAGTTACGCACTATCGACTCCATCGAAGGCCAAGCAAATTGGTCAATCAAGCGTGAATCCGGCTTCCATGAGCCAGTTGCCCGCCTCAAAGCTTGA 72 ATGACTACAGCTCTTGAAGAGCAGAACGCACAGCAGGCAGCCACTGCCGGCCGTGTCGTGCGTGTCATTGGTGCGGTCGTCGACGTGGAGTTTCCCCGCGGCGAGCTGCCAGCACTGTACAACGCACTTACTGTAGAGGTAACCCTCGAATCAGTTAAGAAGACCGTTGTTCTCGAGGTTGCTCAGCACCTCGGCGACAACCTCATCCGCACCATCGCTATGGCACCAACCGACGGACTTGTCCGCGGTGCTGCTGTAACCGATACTGCACGCCCAATTTCCGTACCAGTGGGCGATGTTGTTAAGGGCCACGTATTCAACGCTTTGGGCGACTGCCTAGACGACGTTTCCCTGAACAACAACCCAGAGATCGAGCGTTGGGGCATCCACCGCGAGCCACCATCATTCGATCAGCTTGAGGGTAAGACCGAGATCCTGGAAACAGGCATCAAGGTTATCGACCTTCTCACCCCTTACGTTAAGGGTGGAAAGATCGGCCTCTTCGGTGGTGCAGGTGTGGGTAAGACCGTTCTTATCCAGGAAATGATCACCCGTATTGCACGTGAGTTCTCCGGTACTTCCGTGTTCGCAGGTGTTGGTGAGCGTACCCGTGAGGGCACCGACCTCTTCCTCGAAATGGAAGAAATGGGCGTTCTCCAGGACACCGCCCTGGTGTTCGGTCAGATGGATGAGCCACCAGGAGTCCGTATGCGCGTGGCTCTGTCCGGCCTGACCATGGCGGAGTACTTCCGCGATGTTCAGAACCAGGACGTGCTGCTGTTCATCGACAACATCTTCCGTTTCACCCAGGCAGGTTCTGAGGTTTCCACCCTTCTGGGTCGTATGCCTTCCGCCGTGGGTTACCAGCCAACCCTGGCTGACGAGATGGGTGTTCTCCAGGAGCGCATTACCTCCACCAAGGGCCGTTCGATTACCTCTCTGCAGGCCGTTTACGTTCCTGCCGATGACTACACCGACCCGGCTCCAGCGACCACCTTCGCTCACTTGGATGCAACCACCGAGCTTGACCGCTCCATTGCTTCCAAGGGTATTTACCCAGCAGTGAACCCACTGACCTCCACCTCTCGTATTCTCGAGCCAGCAATCGTTGGTGAGCGTCACTACGAGGTTTCTCAGCGTGTCATCGGCATTCTGCAGAAGAACAAGGAACTTCAGGACATCATCGCCATCCTTGGTATGGACGAGCTTTCTGAAGAGGACAAGATCACCGTTGCACGTGCGCGTCGCATCGAGCGCTTCCTGGGTCAGAACTTCTTCGTTGCAGAGAAGTTCACCGGTCTTCCTGGCTCCTACGTGCCACTGACCGACACCGTCGACGCTTTCGAGCGTATTTGCAACGGCGACTTCGACCACTACCCAGAGCAGGCTTTCAACGGCCTCGGTGGTTTGGACGATGTCGAAGCTGCATACAAGAAGCTGACCGGAAAGTAA 73 ATGGGTGATTTGAAAATGCAACGTTCTGGAGGAGAACCCTTGGACACGATCAAGGCCTATATTGCGCTAACGAAGCCCAGGGTTATTGAACTCCTCCTTGTCGCCACAATCCCCACAATGCTTCAGGCTGAACGCGGTGAGAACAACATTGTGCTCATCTTGCTGACTGTGTTCGGTGGCTGGATGGGTGCGGCCGCCGCCAACACCTTCAACATGGTGGCAGACTCCGATATTGATCAGCGCATGGGACGCACTAGGGCTCGCCCTTTGGTGCGCCACACCGTGAGTAATCGCGACGCCTCCATTTTTGCGTGGGTCCTGACAGTGGCCAGCTTCTTGTGGCTGTGGCTGCTGTGCGATTCGATGCTCGCCGGCATCTTCGTGTTGATCACGATTTTCTTCTACATTTTTGTCTACACCAAGTGGCTGAAGCGCCGCACGCACATGAATATCGTGTGGGGCGGAGCCGCAGGTTGTATGCCAGTGCTCGTCGGCTGGGCAGTGATCGTTGATCAGTTTGAGCCAGGCGTTCCACAGCAGTGGTGGCAGGCAATTGTCCTGTTCATGGTGATTTTCTTCTGGACCCCACCTCACACCTGGGCTCTGGCCATGAAGTACCGCGAAGACTACAAGGCGGCTGGCGTCCCAATGCTTCCTGTCGTGCGCACCCCAGTCCAGGTCACCGCACAAATCGTGTGGTACTCCGTGGCAACTGTGCTGACCACCTTCTTGCTCATCCCAGCAACTGGTTGGATCTACGCAGCGATCGCCGTCATTTCCGGCGTCACCTTCTTGTTCATGGCCATCAAGCTGCACCTCGGCATCAAAAACGGTGGCAAGGTCAAGCCTCTGAAGCTGTTTATTTTGTCCAACAACTACTTGGCAGTCCTCTTCGTGGCATTGTCCGTCGACGCGGTCCTCGGCCTTGAGACCATCGGCGAGATGCTCGGCTGGACCACCACCTTCTTCTAA 74 ATGACTGATTTTTTACGCGATGACATCAGGTTCCTCGGTCAAATCCTCGGTGAGGTAATTGCGGAACAAGAAGGCCAGGAGGTTTATGAACTGGTCGAACAAGCGCGCCTGACTTCTTTTGATATCGCCAAGGGCAACGCCGAAATGGATAGCCTGGTTCAGGTTTTCGACGGCATTACTCCAGCCAAGGCAACACCGATTGCTCGCGCATTTTCCCACTTCGCTCTGCTGGCTAACCTGGCGGAAGACCTCTACGATGAAGAGCTTCGTGAACAGGCTCTCGATGCAGGCGACACCCCTCCGGACAGCACTCTTGATGCCACCTGGCTGAAACTCAATGAGGGCAATGTTGGCGCAGAAGCTGTGGCCGATGTGCTGCGCAATGCTGAGGTGGCGCCGGTTCTGACTGCGCACCCAACTGAGACTCGCCGCCGCACTGTTTTTGATGCGCAAAAGTGGATCACCACCCACATGCGTGAACGCCACGCTTTGCAGTCTGCGGAGCCTACCGCTCGTACGCAAAGCAAGTTGGATGAGATCGAGAAGAACATCCGCCGTCGCATCACCATTTTGTGGCAGACCGCGTTGATTCGTGTGGCCCGCCCACGTATCGAGGACGAGATCGAAGTAGGGCTGCGCTACTACAAGCTGAGCCTTTTGGAAGAGATTCCACGTATCAACCGTGATGTGGCTGTTGAGCTTCGTGAGCGTTTCGGCGAGGGTGTTCCTTTGAAGCCCGTGGTCAAGCCAGGTTCCTGGATTGGTGGAGACCACGACGGTAACCCTTATGTCACCGCGGAAACAGTTGAGTATTCCACTCACCGCGCTGCGGAAACCGTGCTCAAGTACTATGCACGCCAGCTGCATTCCCTCGAGCATGAGCTCAGCCTGTCGGACCGCATGAATAAGGTCACCCCGCAGCTGCTTGCGCTGGCAGATGCAGGGCACAACGACGTGCCAAGCCGCGTGGATGAGCCTTATCGACGCGCCGTCCATGGCGTTCGCGGACGTATCCTCGCGACGACGGCCGAGCTGATCGGCGAGGACGCCGTTGAGGGCGTGTGGTTCAAGGTCTTTACTCCATACGCATCTCCGGAAGAATTCTTAAACGATGCGTTGACCATTGATCATTCTCTGCGTGAATCCAAGGACGTTCTCATTGCCGATGATCGTTTGTCTGTGCTGATTTCTGCCATCGAGAGCTTTGGATTCAACCTTTACGCACTGGATCTGCGCCAAAACTCCGAAAGCTACGAGGACGTCCTCACCGAGCTTTTCGAACGCGCCCAAGTCACCGCAAACTACCGCGAGCTGTCTGAAGCAGAGAAGCTTGAGGTGCTGCTGAAGGAACTGCGCAGCCCTCGTCCGCTGATCCCGCACGGTTCAGATGAATACAGCGAGGTCACCGACCGCGAGCTCGGCATCTTCCGCACCGCGTCGGAGGCTGTTAAGAAATTCGGGCCACGGATGGTGCCTCACTGCATCATCTCCATGGCATCATCGGTCACCGATGTGCTCGAGCCGATGGTGTTGCTCAAGGAATTCGGACTCATCGCAGCCAACGGCGACAACCCACGCAGCACCGTCGATGTCATCCCACTGTTCGAAACCATCGAAGATCTCCAGGCCGGCGCCGGAATCCTCGACGAACTGTGGAAAATTGATCTCTACCGCAACTACCTCCTGCAGCGCGACAACGTCCAGGAAGTCATGCTCGGTTACTCCGATTCCAACAAGGATGGCGGATATTTCTCCGCAAACTGGGCGCTTTACGACGCGGAACTGCAGCTCGTCGAACTATGCCGATCAGCCGGGGTCAAGCTTCGCCTGTTCCACGGCCGTGGTGGCACCGTCGGCCGCGGTGGCGGACCTTCCTACGACGCGATTCTTGCCCAGCCCAGGGGGGCTGTCCAAGGTTCCGTGCGCATCACCGAGCAGGGCGAGATCATCTCCGCTAAGTACGGCAACCCCGAAACCGCGCGCCGAAACCTCGAAGCCCTGGTCTCAGCCACGCTTGAGGCATCGCTTCTCGACGTCTCCGAACTCACCGATCACCAACGCGCGTACGACATCATGAGTGAGATCTCTGAGCTCAGCTTGAAGAAGTACGCCTCCTTGGTGCACGAGGATCAAGGCTTCATCGATTACTTCACCCAGTCCACGCCGCTGCAGGAGATTGGATCCCTCAACATCGGATCCAGGCCTTCCTCACGCAAGCAGACCTCCTCGGTGGAAGATTTGCGAGCCATCCCATGGGTGCTCAGCTGGTCACAGTCTCGTGTCATGCTGCCAGGCTGGTTTGGTGTCGGAACCGCATTAGAGCAGTGGATTGGCGAAGGGGAGCAGGCCACCCAACGCATTGCCGAGCTGCAAACACTCAATGAGTCCTGGCCATTTTTCACCTCAGTGTTGGATAACATGGCTCAGGTGATGTCCAAGGCAGAGCTGCGTTTGGCAAAGCTCTACGCAGACCTGATCCCAGATACGGAAGTAGCCGAGCGAGTCTATTCCGTCATCCGCGAGGAGTACTTCCTGACCAAGAAGATGTTCTGCGTAATCACCGGCTCTGATGATCTGCTTGATGACAACCCACTTCTCGCACGCTCTGTCCAGCGCCGATACCCCTACCTGCTTCCACTCAACGTGATCCAGGTAGAGATGATGCGACGCTACCGAAAAGGCGACCAAAGCGAGCAAGTGTCCCGCAACATTCAGCTGACCATGAACGGTCTTTCCACTGCGCTGCGCAACTCCGGCTAG 75 GTGGACTACATTTCGACGCGTGATGCCAGCCGTACCCCTGCCCGCTTCAGTGATATTTTGCTGGGCGGTCTAGCACCAGACGGCGGCCTGTACCTGCCTGCAACCTACCCTCAACTAGATGATGCCCAGCTGAGTAAATGGCGTGAGGTATTAGCCAACGAAGGATACGCAGCTTTGGCTGCTGAAGTTATCTCCCTGTTTGTTGATGACATCCCAGTAGAAGACATCAAGGCGATCACCGCACGCGCCTACACCTACCCGAAGTTCAACAGCGAAGACATCGTTCCTGTCACCGAACTCGAGGACAACATTTACCTGGGCCACCTTTCCGAAGGCCCAACCGCTGCATTCAAAGACATGGCCATGCAGCTGCTCGGCGAACTTTTCGAATACGAGCTTCAGCCGCGCAACGAAACCATCAACATCCTGGGCGCTACCTCTGGCGATACCGGCTCCTCTGCGGAATACGCCATGCGCGGCCGCGAGGGAATCCGCGTATTCATGCTGACCCCAGCTGGCCGCATGACCCCATTCCAGCAAGCACAGATGTTTGGCCTTGACGATCCAAACATCTTCAACATCGCCCTCGACGGCGTTTTCGACGATTGCCAAGACGTAGTCAAGGCTGTCTCCGCCGACGCAGAATTCAAAAAAGACAACCGCATCGGTGCCGTGAACTCCATCAACTGGGCACGCCTTATGGCACAGGTTGTGTACTACGTTTCCTCATGGATCCGCACCACAACCAGCAATGACCAAAAGGTCAGCTTCTCCGTACCAACCGGCAACTTCGGTGACATTTGCGCAGGCCACATCGCCCGCCAAATGGGACTTCCCATCGATCGCCTCATCGTGGCCACCAACGAAAACGATGTGCTCGACGAGTTCTTCCGTACCGGCGACTACCGAGTCCGCAGCTCCGCAGACACCCACGAGACCTCCTCACCTTCGATGGATATCTCCCGCGCCTCCAACTTCGAGCGTTTCATCTTCGACCTGCTCGGCCGCGACGCCACCCGCGTCAACGATCTATTTGGTACCCAGGTTCGCCAAGGCGGATTCTCACTGGCTGATGACGCCAACTTTGAGAAGGCTGCAGCAGAATACGGTTTCGCCTCCGGACGATCCACCCATGCTGACCGTGTGGCAACCATCGCTGACGTGCATTCCCGCCTCGACGTACTAATCGATCCCCACACCGCCGACGGCGTTCACGTGGCACGCCAGTGGAGGGACGAGGTCAACACCCCAATCATCGTCCTAGAAACTGCACTCCCAGTGAAATTTGCCGACACCATCGTCGAAGCAATTGGTGAAGCACCTCAAACTCCAGAGCGTTTCGCCGCGATCATGGATGCTCCATTCAAGGTTTCCGACCTACCAAACGACACCGATGCAGTTAAGCAGTACATAGTCGATGCGATTGCAAACACTTCCGTGAAGTAA 76 ATGACAGTTGATGAGCAGGTCTCTAACTATTACGACATGCTTCTGAAGCGCAATGCTGGCGAGCCTGAATTTCACCAGGCAGTGGCAGAGGTTTTGGAATCTTTGAAGATCGTCCTGGAAAAGGACCCTCATTACGCTGATTACGGTCTCATCCAGCGCCTGTGCGAGCCTGAGCGTCAGCTCATCTTCCGTGTGCCTTGGGTTGATGACCAGGGCCAGGTCCACGTCAACCGTGGTTTCCGCGTGCAGTTCAACTCTGCACTTGGACCATACAAGGGCGGCCTGCGCTTCCACCCATCTGTAAACCTGGGCATTGTGAAGTTCCTGGGCTTTGAGCAGATCTTTAAAAACTCCCTAACCGGCCTGCCAATCGGTGGTGGCAAGGGTGGATCCGACTTCGACCCTAAGGGCAAGTCCGATCTGGAAATCATGCGTTTCTGCCAGTCCTTCATGACCGAGCTACACCGCCACATCGGTGAGTACCGCGACGTTCCTGCAGGTGACATCGGAGTTGGTGGCCGCGAGATCGGTTACCTGTTTGGCCACTACCGTCGCATGGCTAACCAGCACGAGTCCGGCGTTTTGACCGGTAAGGGCCTGACCTGGGGTGGATCCCTGGTCCGCACCGAGGCAACTGGCTACGGCTGCGTTTACTTCGTGAGTGAAATGATCAAGGCTAAGGGCGAGAGCATCAGCGACCAGAAGATCATCGTTTCCGGTTCCGGCAACGTAGCAACCTACGCGATTGAAAAGGCTCAGGAACTCGGCGCAACCGTTATTGGTTTCTCCGATTCCAGCGGTTGGGTTCATACCCCTAACGGCGTTGACGTGGCTAAGCTCCGCGAAATCAAGGAAGTTCGTCGCGCACGCGTATCCGTGTACGCCGACGAAGTTGAAGGCGCAACCTACCACACCGACGGTTCCATCTGGGATCTCAAGTGCGATATCGCTCTTCCTTGTGCAACTCAGAACGAGCTCAACGGCGAGAACGCTAAGACTCTTGCAGACAACGGCTGCCGTTTCGTTGCTGAAGGCGCGAACATGCCTTCCACCCCTGAGGCTGTTGAGGTCTTCCGTGAGCGCGACATCCGCTTCGGACCAGGCAAGGCAGCTAACGCTGGTGGCGTTGCAACCTCCGCTCTGGAGATGCAGCAGAACGCTTCGCGCGATTCCTGGAGCTTCGAGTACACCGACGAGCGCCTCCAGGTGATCATGAAGAACATCTTCAAGACCTGTGCAGAGACCGCAGCAGAGTATGGACACGAGAACGATTACGTTGTCGGCGCTAACATTGCTGGCTTCAAGAAGGTAGCTGACGCGATGCTGGCACAGGGCGTCATCTAA 77 GTGGCGTTTGAAACCCCGGAAGAAATTGTCAAGTTCATCAAGGATGAAAACGTCGAGTTCGTTGACGTTCGATTCACCGACCTTCCCGGCACCGAGCAGCACTTCAGCATCCCAGCTGCCAGCTTCGATGCAGATACAATCGAAGAAGGTCTCGCATTCGACGGATCCTCGATCCGTGGCTTCACCACGATCGACGAATCTGACATGAATCTCCTGCCAGACCTCGGAACGGCCACCCTTGATCCATTCCGCAAGGCAAAGACCCTGAACGTTAAGTTCTTCGTTCACGATCCTTTCACCCGCGAGGCATTCTCCCGCGACCCACGCAACGTGGCACGCAAGGCAGAGCAGTACCTGGCATCCACCGGCATTGCAGACACCTGCAACTTCGGCGCCGAGGCTGAGTTCTACCTCTTCGACTCCGTTCGCTACTCCACCGAGATGAACTCCGGCTTCTACGAAGTAGATACCGAAGAAGGCTGGTGGAACCGTGGCAAGGAAACCAACCTCGACGGCACCCCAAACCTGGGCGCAAAGAACCGCGTCAAGGGTGGCTACTTCCCAGTAGCACCATACGACCAAACCGTTGACGTGCGCGATGACATGGTTCGCAACCTCGCAGCTTCCGGCTTCGCTCTTGAGCGTTTCCAGCACGAAGTCGGTGGCGGACAGCAGGAAATCAACTACCGCTTCAACACCATGCTCCACGCGGCAGATGATATCCAGACCTTCAAGTACATCATCAAGAACACCGCTCGCCTCCACGGCAAGGCTGCAACCTTCATGCCTAAGCCACTGGCTGGCGACAACGGTTCCGGCATGCACGCTCACCAGTCCCTCTGGAAGGACGGCAAGCCACTCTTCCACGATGAGTCCGGCTACGCAGGCCTGTCCGACATCGCCCGCTACTACATCGGCGGCATCCTGCACCACGCAGGCGCTGTTCTGGCGTTCACCAACGCAACCCTGAACTCCTACCACCGTCTGGTTCCAGGCTTCGAGGCTCCAATCAACCTGGTGTACTCACAGCGCAACCGTTCCGCTGCTGTCCGTATCCCAATCACCGGATCCAACCCGAAGGCAAAGCGCATCGAATTCCGCGCTCCAGACCCATCAGGCAACCCATACCTGGGCTTTGCAGCGATGATGATGGCCGGCCTCGACGGCATCAAGAACCGCATCGAGCCACACGCTCCAGTGGACAAGGACCTCTACGAACTACCACCAGAGGAAGCTGCATCCATTCCACAGGCACCAACCTCCCTGGAAGCATCCCTGAAGGCACTGCAGGAAGACACCGACTTCCTCACCGAGTCTGACGTCTTCACCGAGGATCTCATCGAGGCGTACATCCAGTACAAGTACGACAACGAGATCTCCCCAGTTCGCCTGCGCCCAACCCCGCAGGAATTCGAATTGTACTTCGACTGCTAA 78 GTGGCTTTGTATAGCAAGTATCGACCGGCAAGTTTTGGTGAACTAGTTGGGCAGTCGCAAGTGACTGACCCTCTGTCCGCAGCTTTGGATAGCGGGCGCATCAACCATGCGTACCTTTTTTCGGGTCCGCGTGGTTGTGGCAAGACGTCGTCGGCACGCATCCTTGCCCGGTCCCTCAACTGCGTGGAAGGCCCAACTTCCACGCCGTGTGGGGTGTGTAATTCTTGCGTAGCGCTGGCCCCGGGTGGCCCTGGAACCCTTGATGTAACAGAGCTCGACGCCGCGAGTAACAATGGTGTCGATGACATGCGTGAACTGCGCGAACGCGCGAACTACGCCCCGGCGGAATCTCGCTACCGCGTGTTCATCATTGACGAAGCCCACATGATCAGTACCCAAGGCTTCAACGCCTTGCTGAAAATCGTTGAAGAGCCACCAGCGCACCTGATTTTCATCTTCGCCACCACCGAGCCGGACAAAATGATCGGTACGATCCGTTCCCGCACGCACAATTACCCATTCCGCCTGCTCACCCCAGGGGATATGCGCAAAGTGCTGAAAAATGCGGTCGATGGCGAAGGCGTCCACGTCGACGATTCCGTTTACCCACTGGTCATCCGCGCCGGCGGCGGCAGCCCCCGCGACAGCCTCTCCATCCTCGACCAGCTCATCGCCGGCTCGGGCCCGGAGGGCTTGACATATGAGCGCGCCTTGCCGCTGCTCGGTGTCACAAGCTTCACGCTTATCGACGATTCGATCCATGCCCTTGCATCTAAAGACAACGCAAGCATGTTCACCACGATCGATAACGTCATCGAAGAAGGCCTCGAACCGCGACGCTTCACGATCGACCTCCTCGACCGCCTCCGCGACTTGATGGTCCTCCAAGCCGTCCCAGAAGCACTAAACCTCGGGCTTGTCGACGCCCCCACCGACCGCGCCGACGCCCTAATCGAACAATCCACCCTGTTCAAAGGCAACGAGCTCGCCAACCTAGCCTCCATGGTGAACTCCGGACTCGACGACATGCGTGGTGCCACCTCACCGCGCCTCCTTCTGGAAATCCTCTGCGCCCGACTGCTCCTGGCAAGCAATACCGTGGCAGGTCCAGCGGTCAGTAGTTCGACTGACGCTGCGCCTGCAGCTACTCCGGGTGGTCTCACTGGTATTGCTGCTGCCCGCGCGAAAGCACGGGAGTATGGACAGAAGAAGGCAGCTCCAGCTCCTGTACCAACTCCTGCGCCCGAGCCAGTGCGCGAACAGTCTCTTGCACCAACGCCTGAACCAACGCCAGCGGCTGAACCTACATCTCAGCCCGCGCCGGAACCGGAACCCGCCAGGGAACCAGTGGTGGAAGTGCGGGAGGCGAGCGTCGAAAAGCAGCCTGCAAGCAGTGATCCCCTCGAAACCATTCGAAGCCGCTGGTCAGAGCTGCGCAACATCGTGGAAAAACAAAGTGTGCGCACCTCAATCATGCTGACCGAAGCGCGAGTTTTGGGACTGCGAGGCGATACCCTCGTGCTCGGTCACAGCACCGGGGCGTTGGCTGCGCGTTTGAACGCTGCTGATCACAACGGAATTTTGGTCAAGGTGTTGGCTGAGGAAACTGGTCTGCAGCTCAAGGTCGAATGCATTGTGGGCACGAACCCAGCCGAAGCTGGATTTACCGCCCAGCCTGCTGTGCAAAAGAGCACGTGGAACCCCAACTACGACTCCAAACCAGCCACTCCAAGTGCGCCTGCGCAGCCTCAGACGCCTAAGCAAGAATCCGTGCCCACCGAGCCAGAGGAGCCTGAGAGTTCGGCTGAAACTTCGGGATGGGGGCAACCGGTAAAAATCGGTGGGCCAGCACCTGAGGCACAGACTCCAACGCCTCCGCCTGCTCCGGTGGTTCCGGCTACTCCTGCTGCTCCGGCTGCGGGTGCTGCGAAACCAGCGTGGAAGGAACGCGTCGAACAAGCCGCGGCAAATGCAGCGCAACAACGTCAGCATCGCCAGGGCAGCTCCGAGCCCTTCGAGCGGGGTGTTCCGCTGCCACCAGAACCTGACCTGCCACCGGACCCCTATGGATACCCAGCTGACGAGGGTTTCCCCGAACGAAACCAAGGGTTCCAGCAACAGCCAGCGCCTCCCGTTGAGCAACCAGCTCCTGAGCCTGCGACGGAGCCAGTTCCAGCAGTTGCGCCTGAGCCGGAGGTCAGCCAGCTATCTGAAGAAGAGCAGCTGATCCGCGAAGCAGACGAAGAGCCAGGTGAAATGGATCGTCGTGATGCGAAAACCATCGCGATGGAACTGCTTGCTCAGGAACTCGGCGCGAAACCTTTGTAA 79 ATGGAATTAAGGGAATACGCGACAATCCTGATGAAGAATTGGGTGTTGATCGTTATCGCATCCATTCTTGGAATTGCAGCGGGCGCTGGTTTTTCCCTTCTAGCTACACCGGAGTATCAGTCACGTACTCAGTTGTATGTATCGGTGCGGTCGGGGGCTGGGACAACCTCTGACATGGTTCAGGGTGCTAACTTTTCGCGTCAGATTGTGAATAGTTATGTTGATGTCATTAAGACGGGTGTTGTTCTAGAGCCGGTTGTTGATGAGCTTGGTCTGGAGTTGACGGCTAACCAGTTGAGTTCTCATATCAGTGCGGCTTCTCCTGCTGATACTGCGTTGATCAACATCACTGCTTCTAGTCCTTCTCCTCAGCAGGCGGCTGAGATCGCCAATGCAGTGGGGGAGAGTTTCAAGAATGTGATTCAGACTGAATTGGAACCAGACTCCGGCGATGGCATGAGCCCGATTAACCTGACTACTACTCAGGTGGCTCTGGAACCATCTTCTCCGGTCAGTCCTAATGTGATGATGAATATCCTCCTCGGCCTACTTGTAGGTCTTGCAATAGGTGTTGGTATTGCAGTGCTTCGCGCGGCTTTGGATACTCGTATTCATTCCTTGCGCGATATTGAAGAGGTTACTGATAAGCCACTTTTGGGCGGAATCATTGCGGATTCTGAAGTTGAGAAGCACCCGTTGATCATTAAGCACAAGCCGCATAGTCCTATTGCGGAGTCCTTTCGTGCGTTACGTACTAACCTGCAGTTCCTTAACGTCGGCGGCTCATCTTCAGTATTTGTTATCTCCTCTGCTAATCCTGGTGAGGGTAAGTCAACCACTTCTGTAAACCTAGCTTTGGCGCTTGCAGAGGCCGGCTCCCGTGTAGCGCTGATCGAAGCTGATCTTCGCTTGCCACGAGTGAGCAAGTACCTCGGAGTTGAGGGCAACGCAGGTCTGACTGACATTCTCATTGGCAAGGCCGAGGTTAATGATGTGTTGCAGCGTTGGGGTAGGACTCAGTTGTACTATCTCCCGGCAGGGCGCATTCCGCCGAACCCGAGTGAGTTGCTTGGTTCAGCTGAGATGGACAAGGTCATTGCGGAGCTTGAGGAAAGCTTTGATTATGTGATCATCGATGCCCCTCCGGCGTTGGCGGTTACCGATGCTGCAGTTATTGGTCATGGCAAGGCTGGCATCCTGATTGCGGTCTCCGCAGGTTCTACAAAGAAGCCTGAGTTGGAAGCTACGCTGTCCACGCTTGAGAATGCGGATGCCAATGTTGTTGGCGTTGTCGCTACGATGCTCCCGCCTAAGTCTGTGGCTGGTTATGGCTACGGAAATTACGGCTACGGCGACACCTCCAAAATCAATGCCCCTAAGCCCGACAACACCGAACTAACCACCACCGATGCTTCCAAGGCCAACAATGAGCAATAG 80 GTGCTCGACGTAAACGTCTTCGATGAGCTCCGCATCGGCCTGGCCACCGCCGACGACATCCGCCGTTGGTCCAAGGGTGAGGTCAAGAAGCCGGAGACCATCAACTACCGAACCCTCAAGTCTGAGAAGGACGGTCTGTTCTGCGAGCGTATCTTCGGTCCAACTCGCGACTGGGAGTGCGCCTGCGGTAAGTACAAGCGTGTCCGCTACAAGGGCATCATCTGTGAACGCTGTGGCGTTGAGGTCACCAAGTCCAAGGTGCGCCGTGAGCGCATGGGACACATTGAGCTCGCTGCACCAGTAACCCACATTTGGTACTTCAAGGGCGTTCCATCACGCCTCGGCTACCTTTTGGACCTTGCTCCAAAGGACCTGGACCTCATCATCTACTTCGGTGCGAACATCATCACCAGCGTGGACGAAGAGGCTCGCCACAGCGACCAGACCACTCTTGAGGCAGAAATGCTTCTGGAGAAGAAGGACGTTGAGGCAGACGCAGAGTCTGACATTGCTGAGCGTGCTGAAAAGCTCGAAGAGGATCTTGCTGAACTTGAGGCAGCTGGCGCTAAGGCCGACGCTCGCCGCAAGGTTCAGGCTGCTGCCGATAAGGAAATGCAGCACATCCGTGAGCGTGCACAGCGCGAAATCGATCGTCTCGATGAGGTCTGGCAGACCTTCATCAAGCTTGCTCCAAAGCAGATGATCCGCGATGAGAAGCTCTACGATGAACTGATCGACCGCTACGAGGATTACTTCACCGGTGGTATGGGTGCAGAGTCCATTGAGGCTTTGATCCAGAACTTCGACCTTGATGCTGAGGCTGAAGAGCTGCGCGACATCATCAACAATGGCAAGGGCCAGAAGAAGATGCGTGCACTGAAGCGCCTGAAGGTTGTTGCAGCCTTCCAGCGTTCCGGCAACGATCCTGCCGGCATGGTTTTGAACGCGATCCCAGTGATCCCACCAGAGCTTCGCCCAATGGTTCAGCTTGACGGTGGTCGCTTCGCTACCTCCGACTTGAACGACCTTTACCGTCGTGTGATCAACCGCAACAACCGTCTGAAGCGCATGATTGAGCTCGGTGCACCTGAGATCATCGTGAACAACGAGAAGCGCATGCTGCAGGAATCTGTGGACGCGCTGTTCGACAACGGTCGTCGTGGTCGCCCAGTTACCGGACCGGGTAACCGTCCGCTGAAGTCTCTGTCTGACTTGCTCAAGGGCAAGCAAGGCCGTTTCCGTCAGAACCTTCTGGGTAAGCGTGTTGACTACTCTGGTCGTTCCGTAATTATCGTTGGTCCTCAGCTGCGCCTCCACGAATGTGGTCTGCCTAAGCTGATGGCTCTCGAGCTCTTCAAGCCTTTCGTCATGAAGCGCTTGGTGGAGAACGAGTACGCACAGAACATCAAGTCTGCAAAGCGCATGGTTGAGCGTCAGCGCCCTGAGGTGTGGGACGTCCTCGAAGAGGCCATCTCTGAGCACCCAGTGATGCTGAACCGTGCACCAACCCTGCACCGCTTGGGCATTCAGGCTTTCGAGCCTGTCCTTGTTGAGGGTAAGGCTATTCAGCTGCACCCACTTGCTTGTGAAGCTTTCAACGCCGACTTCGATGGTGACCAGATGGCAGTTCACCTGCCGCTGTCCGCTGAAGCTCAGGCTGAGGCTCGCGTGCTGATGCTTGCATCCAACAACATTTTGTCCCCAGCTTCCGGTAAGCCTTTGGCTATGCCTCGTCTGGATATGGTGACCGGTCTGTACTACCTGACTCTGGAGAAGTCTTCCGAGGAGTTCGGTGGACAGGGCGCTTACCAGCCTGCAGATGAAAACGGTCCTGAAAAGGGCGTGTATTCCTCACTGGCAGAAGCAATCATGGCTTATGACCGTGGTGTACTTGGCCTGCAGGCCCCAGTTCGCATCCGTTTGAACCACCTGCGCCCACCAGCTGAGGTAGAAGCAGAGCAGTTCCCAGATGGATGGAACCAGGGCGAGACTTGGTTGGCTCACACCACCTTGGGTCGCGTTATGTTCAACGAGATCCTGCCTTGGAACTACCCATACCTTGAGGGCGTTATGGTCCGTAAGGGTGGCGGCTCCGACAAGATCATGCTTGGCGACGTAGTCAATGACCTCGCTGCTAAGTACCCAATGATCACCGTGGCTCAGACCATGGACAAGATGAAGGATGCTGGCTTCTACTGGTCAACCCGTTCCGGTGTGACCATCGCTATGTCTGACGTTTTGGTTCTTCCTAACAAGGAAGAAATGCTGGACCGCTACGAGGAATCTGCACGCCAGATCGAAGTTAAGTACAACCGCGGTAAGCTCACCGGCCGTGAGCGCTACGACCGTCTGGTCGAGCTGTGGAAGGACGCAACTGACGAGGTTGGACAGGCTGTCGAGGATCTGTACCCAGACGACAACCCAATTCCAATGATCGTGAAGTCTGGTGCTGCCGGTAACATGCGTCAGATCTGGACCCTTGCTGGTATGAAGGGCATGGTTGTGAACTCGAAGGGTGACTACATCACCCGCCCGATCAAGACTTCCTTCCGTGAAGGCTTGACCGTTCTCGAGTACTTCAACAACTCCCACGGTTCCCGTAAGGGCCTGGCCGATACCGCGCTGCGTACCGCTGACTCCGGTTACCTGACCCGTCGTCTTGTTGACGTCGCTCAGGACGTCATCGTGCGTGTTGAGGACTGTGGCACCCGCCAGGGTGTTCGCGTTCCTGTCGCTGCTGAGGTTCTGGATGCAACTGGTGCTGTCACCGGCTACACCCGCCATGACCTGATCGAGACTTCTGTCTCCGGTCGTGTTCTGGCTGGCGATGCAACCAACGCTGCAGGCGAGGTTGTGCTTGCTGCTGGTACCGACCTGACCGAGCTCAACATTGACCTTCTGGTCGAGGCTGGCATCAAGGACGTCAAGGTTCGTTCCGTACTTACCTGCCAGACCCCAACCGGTGTTTGTGCTAAGTGCTACGGCAAGTCCATGGCTTCCGGCCAGCAGGTTGATATCGGAGAGGCTGTCGGTATTGTTGCTGCACAGTCCATTGGTGAGCCTGGTACCCAGCTGACCATGCGTACCTTCCACCAGGGTGGTGTCGGTGGCGATATTACCGGCGGTCTGCCTCGTGTTCAGGAGCTGTTTGAGGCACGTGTTCCTAAGAACTGTGCACCAATTGCTTCTGTTGAAGGTGTTATCCACCTTGAGGATGAAGGCAACTTCTACACTCTGACCATCGTTCCTGACGATGGCTCCGACAACGTTGTCTACGAGAAGCTGTCCAAGCGACAGGGTCTTGCATCCACTCGCGTGGCTATGGAGTCCAACGCTGGTGCGTTCATTGAGCGCACCTTGACCGAAGGTGACCGCGTCACCGTTGGTCAGCGTCTGCTCCGTGGTGCAGCTGATCCACACGACGTGCTCGAGATCCTCGGTCGCCGTGGTGTGGAGCAGCACCTCATCGATGAGGTGCAGGCTGTTTACCGTGCACAGGGTGTGGCCATCCACGACAAGCACATCGAAATCATCATTCGTCAGATGCTGCGTCGCGGTACCGTCATTGAGTCCGGTTCCACCGAGTTCCTTCCTGGTTCTTTGGTTGACCTCTCTGAGGCGAAGCTGGCTAACTCTGAGGCAATCGGTGCGGGCGGTCAGCCTGCAGAGCTGCGTTCTGAGATCATGGGTATCACCAAGGCCTCTCTCGCAACTGAGTCTTGGCTGTCTGCAGCGTCCTTCCAGGAGACCACTCGTGTCCTGACTGATGCTGCTATCAACAAGCGCTCCGATAAGCTCATCGGCCTGAAGGAGAACGTGATCATCGGTAAGCTGATCCCAGCTGGTACTGGTATTTCCCGTTACCGCAACATCTCCATCAAGCCAACCGAGGCTGCTCGCAACGCCGCATACTCGATCCCAACTTATGGTGAGTCGATTTACGGTGACGATGGATTCGGTGAGTTCACCGGCGCATCCGTCCCATTGGATGAGGCTTTCTAG 81 GTGGGAACGGCGAGAGGGCTTTCTCAAGTTCTGAAAGAAGCCAGACAAGGTGGAAAACCCACTTCAGCCACCCAGAAAGTTCCACGCTCCGGCTACAGATATGACCTCGATGGGCTCCGCGGCATCGCGATCGCCTTCGTAGTTTTGTTCCATGTTTTCGTCGGAAAAGTCTCCGGCGGTGTGGATGTCTTCCTGCTGCTGTCTGGCTATTTCTTCTTAGGGTCGCAATTGCGTTATGCAGATCGTCCAGATTCTTCCATCAACCCCTGGTGGCCGATTTGGCGCACGCTACGCAGATTACTTCCTGCGTTAGTGCTGGTGTTGGGCGTTTCCATGGTCCTCATCTTGGCGTGGGTGCCCAGACTGCAACGAATAGAAATAGCCAACCAAGCAGTAGCCAGCCTCTTCTATGTCCAAAACTGGGAGCTCGCATCCCAAGGTGCTGCCTACGGAGCAGCCTCTGCAGAAGTCAGTCCTTTCCAGCACTTGTGGTCCATGGCTGTGCAAGGGCAGTTCTACCTCTTTGCCATCTTGTTGAGCATGACGATCATCCTGATTCGTCGATACCGCCCCGAATACTCCGCAGTGCGACTAGCGACTCCTGTGCTGGCAGTGCTCACATCCGTATCATTTTTCAGTGCAATCCTGTGGCATTTCATTGATCAATCAGTCAACTACTATTCCACCTTCACCAGGTTCTGGGAGCTCGGCCTTGGTGCACTATTGGTGCTGCATGCGCCTCGAATTTTGATTTCTGCGAAGACTAAATCAATACTCGCAGCCGTCGGTTTGTTTATGGTGCTATCCACTGGATTCTTCATGGATGGCGCAGAGACTTTCCCTGGATTCCCCGCGCTGTATCCCATCTTGGGTGCTTGCTTAGTCATCCTTGGCGACGGTAAAATCTCGGTCTTTCTCTCCCGAAAATGGATGCTTTGGCTCGGCGATATCGCCTACCCGCTCTACTTGTGGCACTGGCCTCTGCTGATCATTTTCACCGCTTTGTTCAACCAAGAAGAGCCATCCATCTGGCTGGGTATCGCCGTGATTATGCTGTCCCTTGGCTTGGCGCAGCTGACTAACAAATACGTAGAAAAACCAATGCAACAGCAGGGAAAGCGCCCACTATTTAGGGAATCCCGCAGTGCTGATGCTTTGCAAAAGCTGCGTTCCTCCAGGCCTGCCCGAATGCGTGCGTATGCGGGTATCGCAGTAGTCGCAGTCGGTTTCGCCCTGACATCTACTCCGCAGGTGCTCTACCAACGAGTCCTCGATACCCAAATGAGTGATCTCGATCCGGAGATCTACCCCGGTGCACTCTCCCTGGCGGGCTTCGATGCCCCCGAAGTAAAACCCAAACCAAACCCCTACGTGCTGGCCGATACCGTCTCACCCGCCTGGGCAAAAGGCTGCATGTCCGTCTTCGGCGACGATCCATCTGAACTAGCACCAGACAACTACGACGAAGATTACTGCGCTTTCGGCAACCCCAACTCACCCGTCGAGGTCTACCTCGTCGGCGGATCCCACGCCGAACAATGGATGGCACCACTCGACGCCCTAGGCAAGCAACACAACTTCAAAGTGATCCCCCTCGTCCGCCAATCCTGCCCCACCTTCGTGGTGGAACTCGACGGAATTTTCAGCGACGACTGCATCGAATTCAACGAAAAAGTGATCGAACGCCTCGCAGAAGTAAAACCCGACCTCGTGGTCTCCAACTCCACCAGACCGCTCCTTGAAAAAGGCCGCGGCATCGACGAAGTGCCAGAATCATACATCACGCTGTGGGACTTCCTCGAAGCCGAAGACATCCCATTCATGGGACTCCGCGACAACCCGTGGTTCATCCAGCCAGGAGGCGAAGGATGGATGGTGTCCCAATGCTACGAAGAAGAACAATCCTTGGTGGACTGCTCGATCACCAAAGACAACTTCTACGCACCAGTAGACCCCGCTGAAAAAGAACTAGCCATCAGAAACAATATGCTCGCAGTGGATACTTCACGATGGTTCTGCCCAGACGACCTGTGTATCCCAGTGATCGGCAATGTGTATGTATACCGCGACGGCAACCACATGTCCGATGAATACGCACAATCTTTGGCTCCATTCCTGTGGAGCAATATGCGCAGGCTTCTGGTCGCTTAG 82 ATGAGTGCATTTGATTCGATCCTAAATGTTGAAGAGTGGATCAGTGACCACTACCTCACCAATGACGATGCCAAAGGTGCCTCATTTTCCAAGCGGGTGCGCGAGCGCATTAAAGAATGGAAAACCACCGAGGACGCAACCCAGCAGAGTGGCCCTTTAACTCGTTTTTCCAGCAACCGCCTGCAGTTGCAGCATGCTCTTTCTGAGCTTGACGACGCCACCACCGCCGCCAGTTTAGTGGCCTCTGCACTGGGGTATGGTGTCCCCAGCGCGCGCCACGCGCAGCGCGGCTCCGACACAATATCCTATTCCTCTTGGGTGGGAAATGCCGGCAGTGTGGAATTTCTTGCAGCGACTCCCGCTGAAAGCTTTGAAGAGAACTTCCGATCCCTTCCCCTTGAGCCAGTAGCGGTCAATGACAAGCCCCAGGATATCACCGCAGCCAAATTGGTGGGCCAGATTTTCCTTAGTGATACTCCCCCTGCTTTTGTTGTTATCACCGCTGGTAAATGGGTGGTTTTAGCCGAGCGTGAAACCTGGCCTCTAGGCCGCCACCTAGCTATTGATATTTCCCTGGTGGTGGAACGTAATGACACCAAAGCCCAGGGTGAGATGCAGCAGACGGTCGTAGCACTAGCCCGCGAAAATACCGAGCGTGCCGCCGATGGCACCACCTGGTGGGAAGAAACCATTGAGCAATCCCGCGAACATGCTGTCAAGGTTTCTGGCGAGCTACGCAGTGCGGTGCGTGAATCCATTGAAATCCTGGGCAATGACGTGCTCACACGCTATGAAGCTAAAGAGCTCTCCACCGCTGAGATCGACGGTGGCGAGCTAGCTAAGCAATCTTTGCGCTATCTCTACCGCATTTTGTTCCTGCTTTTTGCCGAGGCTTCACCAGAGCTTGAAATCCTGCCAACCGGCACCCCGGAATATGACGAGGGTTATGGCTTGGCACGCCTGCGCGAGCTTATTCTTAATCCCCCAGTTACCCAGCATGCTCGCACGGGCACCCACCTTTATGATTCCCTGCAGCTGCTGTTCACTCTGGTGGATAAAGGCCACCACCCAACAGATGCTAAGGCTGTAGCTTTTGATGCCGAGGCTGGAGAAGAAGGCCTGCACTTCCGCAACCTTTCAGCGGATCTCTTCCTCCCTGCAGCCACAGAACTTATTGATCGAGTTGGTCTTTCCAATGAAGCCCTAAACAAGGTCTTGGAAAACCTCCTGCTCTCCCGGGTGCAATCCGGTAAAGACCGCGGCTTTATCTCCTATGCCACCTTGGGTGTTACCGAGCTTGGCCAAGTTTATGAGGGTCTGATGTCCTATACCGGCTTTATCGCCCAGGAAGATCTTTTTGAGGTTGCACCACATGGCAAAGCCGATAAAGGTTCCTGGATGCTCCCGGTCTCAAAGGCTGATGAAGTCCCTGCCGATAGCTTTATCGAAGTTGATCAAGAAGCCCCTGGTGGCGGCGTAATCAAGGTGCGTAAACGCCACCCGCGCGGATCATTTGTGTTCCGTCAGTCCTCTCGTGACCGCGAACGCTCAGCGTCCTTCTACACCCCACAAGTACTCACCAGCTTTACTGTCACCCAGGCTATTGAAGAACTCCAGGCATCAAAGCGCATCACCACAGCCAATGATGTTCTCAGCCTCACCATCTGTGAACCTGCCATGGGTTCCGGCGCCTTCGCTGTGGAAGCAGTACGCCAATTAGCAGAGCTTTATTTGGAATTGCGCCAAGAAGAACTAGAGCAGCAGATTCCAGCGGAAGACCGTGCCAAGGAACTCCAAAAGGTCAAAGCGCACATTGCGCTGCACCAGGTTTATGGTGTGGACCTTAACAGCACTGCTGTGGAGTTGGCGGAAATCTCGCTGTGGCTAGACACCATGAATGCAGAAATGGACGCACCTTGGTATGGCCTGCACCTGCGTAATGGTAACTCCCTCGTTGGTGCCACCCGTTCGCTGTATGCACCTAGTCTGCTTAATAAAAAAGCCTGGTTAACTGCTACTCCAACCCGCTATCGGCTTGATGATATCGCGCAGGCTATTGATGAAAACAAAGCAGAACCCCTCTTCAACCACGGCATCCACCACTTCCTCTTGCCCTCTACTGGCTGGGGAGCCACTGCAGATGCCAAAGATCTTAAAGATCTTATGGCTACTGAAATCAAGGAGCTTAAATCTTGGCGTACTTCCATCCGTGCGTCTTTGAGTAAAACTCAGATTAAGCAGCTCAATAACCTTGCCCTACGCGTGGAAACACTATGGCGATTTGTGCTGATGCGTATTCGCATTGCAGAATCCCAGATCTCACGTAGCACTACTCTCTGGGGTCAAGAGCCAGCTGAGGTTTCGGAGGTTGTCACACGTGAGCAAATTGAACAAGACCTCTTTGGCAATATTGATGGTGCATATAACCGTCTACGCTTGGTGATGGATGCTTGGTGTGCGCTGTGGTTCTGGCCTTTGGATGCTGTTGCTACCGCTGAGCATCCGGAGCGTCCAGCCCTTCCAGATCTTGATGAGTGGCTAGCCACCCTGACGGAGATTCTGGGTATTGATCTCCCTCTGAAGTCCAAAAACGAAAATCAGATTGTCTTAGGTCCAGATACCAATTGGCTAGCCATTAATGATGCCGAGGCTACTGATCTTGGTTTTTCTGGGGCATTGAGCTTTGAGCGTGTTAGCGCGAATCACCCGTGGATCAATGTTGCCCGCCAAGTGGCTAAACAACAGAGCTTCTTCCACTGGGATCTAGACTTCGCCCACGTTTTTGCCAAGGGTGGATTTGATCTGCAGGTTGGTAATCCACCATGGGTGCGACCAGATGTGAACTTTGAGGATCTGCTTGCTGAACATGATCCGTGGTGGGCTGTAATGAGTAAACCAACCCAGGCATCCAAAAAAGAACGCCAGAAGAATTTTCACAACAATCCTAAGAGCCTCGAACATGTGGTCAGTGGTGCAGGTGAAACCGTGGCTACTTCTGCGATCCTCGGTTCGGTTACTCTCTATCCGCATCTTAAAGATCAACGCCCGGACCTCTACCGGGGCTTTATGGAAAAGACTTGGTCTAATGCCTCCCCGGCAGGTGCGATCTCACTGATCCACCCCGAATCCCACTTCACTGAGAAAAAAGCTGCCCCGCTACGTCGGGGAGCATATGAGCGTCTGCGCCGCCACTGGCAGTTCATTAATGAGTTGATTCTTTTTGACGTCCACGACTTGGTTAAATATGGCGTACATGTCTATGGCGCTCCGCAGGAATCTATTAACTTTTTAAGTGCTGCGTCGCTTTATCACCCACAAACAGTGCTTGATTCATTTGATCATGACGGTTCAGGTAATCTCCCTGGTCTTAAAGACGACAATGGCAACTGGGACCGTCGCCCACACAAGGACCGTATCCAACTGGTCAATGCCGATACTTTGACGGTGTGGAAGTCCATCCTGGAGGATGAACAAACGCCATACTTGGATACCCGCATGGTTTATACCGTCAACACGGAAGCAGCAGCAGTGTTGGAAAAGTTGGCTTCTGCACCTCGTATCAAAGAACTCGGGCTGCAGTTCTCCAGTGGCTGGAATGAAACCACCGATAAGAAAAAGGGATACTTTGACGTTGGTTGGGGCTACCCAGCTTCCTGGTCTGATGCCATTTTGCAGGGGCCGCACCTGGGTGTTGCTACACCAATGATCAAGCAGCCCAATCCGACAATGAAGCATAATCAAGATTGGTCTGAAATTGATTTCGAGGCCATTCCTGCAAACTTCATACCTGCAACGGCGTACCAGCCCGATCGCCAAACAAAGCCCACTTATGATGCTGACTACGGCACCTGGACTTTCGGGGACAAGCAGGTACCAGTTGCAGACACTTTCCGAATTGCATGGAGGGAGATGGCTGCCACCACGGGATTTAGGACTGTCTACCCATCAGTAATTCCACCGGGAGCCAACCATGTGCACACAGTTAATAGCGCTGCATCACGTTCAAACTTAAAAACCATTCTCGTTGGAGCACAGCTTGGTGCAATTCTAAGTGACTATTTTGCTCGGTCCTCGGGTTCAAGCCACATATTTAACGACATTGTTCGCAAGATTCCACTTCCAAATTTCACATCCTTGGAAAAGCAGTTCGCCCGCACATACCTCCGCCTCAACTGCCTGACCTCAGCTTATGCCCCATTGTGGGAAGAGATCACCGGTGAGCCGTGGGATGTTCAGGTGCCTTTGCGCAATGCCGAGCAACGTCGAGCAGCGCAAAACGATATTGATGCCATGGTGGCATTGTCTTTGGGTATTAGTGCTGATGAGCTGTGCATGATTTATCGCACTCAATTCCCAGTGATGCGTAGATATGATCAAGAAGATCATTTTGATGCCAATGGCCGTAAAGTTCCTAAAGAGATCATCAAGCTGCAGCAGAAACTTAAAGATGGCCAAGAGCTCAGCGTGGAAAAGCGCACCTGGGTGCATCCCCAATCAGAAGTGTCCTATACCTTTGAATATCCTTTCCGGGTGTTGGATCGTGAAGCTGATCTGCGTGCTGCATATGCAAAATTTGAAAACCAGCTAAAGGAGCCATAG 83 GTGAGTAGTCTACGCCTAATGGCGATCCACGCCCACCCTGACGACGAGTCAAGCAAGGGCGCAGCAACCATGGCGCGCTATGCAGCTGAGGGCAATCAAGTAATGGTTGTTACCTGCACTGGTGGTGAGCGTGGAGACATTTTCAACCCTGCTATGGATAAGCCAGGAATCCTGGACAATATTTTTGCTGTGCGTCAGGAAGAGATGGCGAAGGCCATGGAAATTCTTGGCACTGAGCACAGATGGCTAGGTTATGAGGACTCTGGGCTGCCCCAAGGTGATCCTTTGCCTCCTCTGCCTGAGGGCTGTTTTGCTTTAGAAGACTCGGATAAAGTCACCCAAGATTTAGTGAAGATTCTGCGCGAGTTCCGCCCACACGTCATCATTACCTATGATGAGAACGGCGGTTACCCACACCCGGATCACCTCAAGGTTCATGAGGTGTCGATGCTTGCATGGGAGAAGTCCGGCGATGCAGCGTATGCACCTGAGTTGGGCGCACCGTGGGAGCCACTGAAGCTTTATTACACCCACGGCTTTATCCGTCAGCGCATGGAAATGGTCCATGATCTGCTCATTGAACAGGGCAAGCCCAGCCCATACACCCCGATGCTTGAGCGTTGGAAGGCAAATGAGGCTGATGTGATGGCTCGAGTTACCACTCAGGTTCCTTGTGAGCGCTTCTTTGATCAGCGTGATGACGCCCTGCGTGCGCACGCAACTCAGATTGATCCTGCGGGTGCTTTCTTTGGAACTCCCGTTGAGGTGCAGCGTCGCCTGTGGCCGACTGAAGAGTTCGAATTAGCTAAAACTCGTGTGAAGACGTCGATCCCAGAAGATGACCTGTTTGCCGGAATCACACCCGATGCAGAATAA 84 ATGCGTATTCATGAAATCATCATTGACAATTTCCGAGCCATCGAACATCTAGAACTTCGTGACATTCCTGACCAGGGCGTTATCGTGATCCACGGTGACAATGAGCAAGGCAAATCTTCAATCCTTGAAGCCATAAAAACAGTTCTGAATAGTAAACACAGAACCACCAGCAAGACGATCAAGGCCATTCAGCCTGTTGACCGTGATGTTCCCATCAGCATCACCCTCGAGGCAACCGTAGGCACGGTTCGATTCCGCATACACAAACGCTTCCTAAAATCCACAGCCGCAGAACTGCAAGTCATCGAGCCACGTCCATCCAACCACCGTGGACTAGAAGCCGAAGCTGCACTGGCAGAAATACTGGAAAGCCATTTGGACACCTCTTTGCTTGACGCATTGTTTATGAAACAGGGTGAAGTAGAAGCAGGTATCAGCGCTGTAGGAATTCCTACCCTGACCAGCGCTTTGAACGCTCAAAATGGCAATACCGAAGATGCCACCGAAGACACCGCACTCATGGAGGCTGTAGAAAAGGAATACCTAAAGTTCTACACCAACTCCGGAAAGGCGAATACGAGGTTCCTACAGTTTTCCAAACAGGTGGAAACCCTTCGTACTGATTTGGATGAGGCTAACGCTGAAGTAGCAAAGCTCTCCTCCCATGTTGACCGAGTTAAACGATTGGAAATTGATCGGGATCAGGCCACAGCACAGTTACCCAAGGCAGAGGAAGAACTCGCAGGGAGGAAAGCAGAACTGGAGGAAGCCCAAAAGGTTAAGGCTCAAGCCACGGAAATCCTTGCGCAGTTTTCCCGGGCGGAAGAACAGTTAGAGCAGGCAACAGGAGCTCAGAAACGACGTAAAGAACTCCGCAATAAGCTGGAATTAGCACAGACAGAAGTCGAAAAGGCTGAAGCAGGGCAAGAGAACCTGGCGCAAGAAGCTTCCCGTGAAGCAGAAGAATTCCTAGCCTTAAGTGAAAAGTTCGAGGTAGCGCGCGCAGCTGAAACTCTGGCCGTTGAAAAGGTGAAAGCAGCACGTCAATCGGTTGCTGGAATCAAAAACCGTGACCGTAAAGAACACCTCACGGTTATGCTTGGTGAACTTGATCGAATTGGGCAGCGCCTCTACGAATTGCGCAGCGTCCAGCACTCATCGGTGCGTGTGAGCCAGCGTGATATTGATGCTCTGCAAAAGGCGATCACCGAGGTCGATATCCAAAGGACGCTCGTTGAAGCGCAGCAGGGAAGTATTACCCTCAGTGCTTCCACACCCACCGACATTCAACTAGGTGATGACACCGTTTCAGCTACCGACGCTGGAACCACAGTTGCCCTCGACCGAGAACTCACCGTCGTGGTCGGCGATGTCACCTTGGTGATTAACCCAGGAAAAACAGCAGCGGAAAGTCGCACCGATTTTGAATCCGCCGAGGCAGCATTGGCGGAGCTGCTTGATCAACTCGACGTCTCAGACTTGGACCAGCTCAGGGAACGTTTCACTGCCCAAGAGCAACGAGACGCGGATATTGCAGAACTGGTTCGCGAACAACAGCGCATGAGCGGTGGGACAGAAACAACAGTTTTACGTGCCGAGCTAGAAGGCCTGCACGTCCCAGAGGATCTAGACCCATCAATTTCTGTGGATGATGCGCAAACTCAGTTGAATGAGGCAGAAGAGTCCAGAGAGCTCGCTGCGGAGGCGCATAAACACGCAAATGCAGCACTTGATGGATTGCGTTCGCGTCCCGTAGATAAAGCTCTGACAGTTTTCAACGCTCAGCTCGCAGCACTGCAGAGAAACCTCTCTTCCGCACAAGTTGAATTAGACCGTGCAGTGGCAGAAACCTCGGATGACGAAGTTGATGCTGCTGTACAGCGGTGCGCTGAAGCTCTAGCTGGGGTGCGGGTACAGAAGCAGGAGATTGAACAGGTCTTGGCAAAAACCAACCCAGACATGGCTCAACGCTTATGTGATGCTGCTGAAGCAAATGTGCGATCGTATAAAACGGCAGTGTCTGATGCCACCACAGAGTTGGTGCGCCTGGAAGGCCTCATCGGTGTAGCTGCAGGTGCGAAAGAACGACTAGATAAAGTTAAGTCTGCGCTGACAGCAGCTGAAAACCGGTTTGAATCAGAACAACGTCGCGCCCACGCTGCACGTCGCCTCTATGCCCTCATGGTGTTCTACCGTGATGAATCCCGAAAGCGCTACGCAGCACCCTTCGCGGACAAACTATCCAGGCTGGCAGCATCCGTATTTGGAGAATCAGCAGACTTTGATCTTGATGATGAGCTGAAGATCTCCAGTCGTTCCATAGGACCACGAACAGTGGACCTTGCCAATCTTTCTGGCGGCGCAAAGGAACAGCTGGCAATCCTCACCCGCTTTGCTATCGCAGAATTGGTAGCAGAATCCTCCGCGCAAGGTGCTGTGCCTGTGTTTATTGATGATGCATTGGGGAGCACAGACCCAGAGCGACTGACCCGCATATCTACTCTGTTTAGTGATGCAGGCAAGGACTCTCAGGTGTTTGTTCTCACCTGTGTTCCAGACCGGTACAACTATGTAGAGGTCACCCAAAAGCACAGCATCGAGTCATTGAAAACTGCCAACGCTCTGCTGTAG 85 ATGACTACAACTACTTCTTCTGGGAAGTCTTCTGAAAAGATCAACCCCCTCTTCAAGCTCGGCAGTTTCCTAAGAAAAGGCACCGTCGGTTCTGAAGGCCAGCAGATTTTCCTTCAGGGCGGACGCCAAGCCGATGTGTTTTATCGCAACCGATGGGCTTTCGATAAAGTCGTGCGCTCCACACATGGCGTGAACTGCACGGGCTCCTGCTCGTGGAAAGTGTATGTAAAAGACGGTGTGATCACCTGGGAATCCCAGGCAGTGGATTACCCAACTACCGGTGCGGATATGCCCGACTATGAACCACGTGGCTGCCCTCGTGGTGCATCATTTTCCTGGTACACCTACTCCCCAACCCGCATCCGCTACCCATACATCCGTGGCGTGCTAGTTGATATGTACCGCGAAGCCAAGGAACGCCTGGGCGATCCGGTGCTGGCGTGGCGCGACATTGTAGAAACCCCAGAAAAGCGCAAAGCATATGTATCCCAGCGGGGCAAAGGTGGCCTCATCCGCGTTCAGTATGAGGAAGCCATGGAGATTGCTGCGGCAGCCCATGTGTACACCATCCGCCAATACGGCCCCGACCGCATTCATGGATTCACCGTTATTCCCGCAATGTCGCAGGTGTCTTACGGTGCTGGTACTCGCTTCTTGCAGATGATCGGCGGAGTGGCGCTGTCCTTCTACGATTGGTACGCCGACCTCCCACCAGCATCACCACAAACTTTCGGCGATCAAACTGACGTTCCGGAATCTGGCGACTGGTACAACTCCAGCTACCTCATGATGTGGGGTTCCAACATTCCGGTGACCCGCACGCCTGACTCCCACTTCATGGTGGAAGCCCGCTACAAGGGCACCAAGGTTGTTGTGGTTTCCCCGGATTTCGCTGACTCCACCAAATTTGCTGATGAATGGGCACGCATCCACCCTGGTACTGACGGCGCACTCGCCTTTGCCATGGGCCATGTGATCTTGAAGGAATTCCATGTTGACAAGAAGACGCCGTACTTCATGGACTACATGCGCAAATACACGGACTCTCCTTTCCTCGTGGAATTAGATGAGCACGGCGATGGCACCTACACCCCAGGTAAATTCCTCACTGCAGACCGCGCAGCTGATATCTCCCCAGCGCTTGCCGCCACTCCAAATGCCACCCACCGTCTCCTTGTGCTGCAAAAAGATGGCTCAGTTGTAGATCCCGGTGGCACTGTCGCGGACCGTTGGGGTGAAGAAGGCATGGGTAAGTGGAATCTGCGCTTAGACGGCGTAGATCCAGTGATGACTATTGCAGATGTACAGACTGACACCGAAACTGCGGAAGTCCTCTTCCCCCGCTTCGATCTCCCAGCAACTGCCACCCAAGAAGGCCCCATTGGTGCTGGCACCATCAGCCGGGGCGTTCCCACCATCACGTTGAATGGCCGAAAGTACACCACTGTCTTTGATGTGTTGCTCGCACACTACGGTGTGAACCGCGAAGAGCTCAACCTTCCTGGTGAGTGGCCTAAGGATTTCCAGGATCCAGTCATGGGTACTCCTGCGTGGCAGGAATAGCTCACGGGTGTTCCTGCTAATCAGGCGATTCGTTTGGGTCGGGAATTTGCTCAGAATGCTGATGATTCCAAGGGCCGTTCCCAGATCATCATGGGTGCTGGTGTGAACCACTACTTCCATGCGGATTCTATTTATCGCACATTCTTGGCGCTGACCTCTATGTGTGGCACCCAAGGTGTTAACGGTGGCGGTTGGGCTCACTACGTTGGTCAGGAGAAACTCCGTCCAATGAATGGTTGGGCACAGTATGCCTTTGCTACAGACTGGCAGCGTCCACCACGTCAGATGATCACCACTGGTTTCTACTACCTCACCACGGATCAGTGGAGGTATGACAACACTCGTGCTAATCGTCTGGCTTCCCCACTGGCTAATCGTGGCACCGTGGGTGACAAAATGACGGCGGATACCTTGGTGGAATCCATGAAACGTGGATGGATGCCGTCATTCCCGCAATTCAACCGCAATCCCCTCATCTTGAGCCAGGAGGCGGAAGAAAAGGGCGTGTCTGTTTCTGACCATATTGTTCAGCAGCTCACCGATGGTGACTTGCAGTTCGCCTGCGAGGATCCGGATGCACCGGAAAACTGGCCACGCATTCTGCTTAACTGGCGCACAAACCTAATGGGCTCTTCAGCTAAGGGCACGGAGTTTTTCTTGCGCCATATGTTGGGTGTGGATTCTGATGCATCTGCTGAAGAAAACGCGCCGGAGGATCGTCCAAGTTCCATTGTGTGGAGGGATGAGGCTCCGGAAGGAAAGCTCGATTTGATGCTGACCACGGATTTCCGCAACACTTCCACCACCTTGGTCTCGGATATCGTGCTGCCGGCAGCCACCTGGTATGAGAAGCATGATTTGTCCACCACGGATATGCACCCCTTCATCCACTCGTTCAATGCTGCGATCAACCCACCGTGGGAGACGCGTACTGACTGGGAGGTCTTCCACGATCTCACCAAAGAATTCTCCTCACAGGCAGCAACCTGGTTGGGCACCCAAACCGATGTGATCACCGCACCGATTGCCCATGACTCCCCGGATGAGCTCAATATGCCTGGCGGTATCGTGCCAGATATTGATGAGGTCGGGCTGATCCCTGGCAAGACGATGGCCAAGATCATCCCGGTGGAACGTGATTACTCCAAGGTGTATGAAAAGTGGACACACTTGGGACCACTCACCGCCAAAGCGGGTACCGGAACCCACGGCACTGCGTTTAACGTGACCAAGCAAACCGAGGAGCTGGCGCTGATCAACGGCACCTCCATCACGGAGTCTGCTGGTGAACGCCCCCGACTTGATTCCGCAGTCAAGGCCATTGAGATGATTCTGCACTTAAGTGGCGTGTCTAATGGTGAGGTTGCCGCCGAAGGTTTCAAGAATCAGGGTGCACGCGTGGGCACCGACATGATGGATCTGATTGCAGACCACGAAGGTACCCACATCAATTGGGACATGGTCAAAGAACGTCCCGCCGAGGTGATCACCTCACCGGAGTGGACTGGTTCCAAGAAGGACGGACGTCGCTACACCGCGTTTTCCATCAACATTGAATACGACAAGCCGTGGCACACCCTGTCTGGTCGCATGCACTACTACCTCGACCACGATTGGTTTATTGATTACGGCGAGCAGTTGCCAATCTTTAGGCCACCGTTGGACAAGATCCACATCAATGGTGAGGTCGGCCCTGGCCAGTCGGTCACAGGCACCGACGGCGAACCAGAAGTAACCGTGCGTTATCTGACCACCCACAACAAGTGGTCGATTCACTCGCAGTACTACGACAATCTGCATGTGCTTTCTATTTCTCGTGGCGGCCAGGTGATCTGGATGTCCAACAAGGATGCAGAGAAACTCGGTATCGCTGACAACGATTGGATCGAGGCTTATAACCGCAACGGCGTTGTTTCTGCTCGTGCGATTGTCTCCCACCGCATTCCTGAAGGCACCGTGTTTATGAACCACGCGCAGGAACGCACCGCTGGCACCCCGCTGAACGAGAAGTCTGGCAGGCGCGGCGGAACTCACAACTCTCTTACTCGAATCATGATTAAGCCGGTCCATGTTGCCGGTGGCTACGGCCACTTAACCTATGGCTTCAACTACATCGGCCCAACCGGAAATAACCGCGATGAGGTCACCAGAATTCGTCGCCGCTCCCAGGAGGTGCAGTACTAA 86 ATGACGCCTACCCAAAATGAGATCCACCCGAAACATAGCTACTCCCCCATCCGCAAGGACGGTCTCGAGGTCCCGGAGACCGAAATCCGCCTCGATGACTCGCCAAGCGGCCCCAACGAACCCTTCCGCATCTACCGCACCCGTGGCCCAGAAACCAACCCCAAGCAGGGACTTCCGCGGCTGCGCGAGTCATGGATCACCGCCCGCGGCGACGTTGCCACCTATCAGGGGCGCGAGCGTTTGCTTATCGACGACGGCCGCTCGGCAATGCGTCGAGGTCAAGCTTCGGCTGAGTGGAAAGGCCAAAAACCAGCTCCTTTGAAGGCGCTACCTGGCAAAAGAGTCACCCAAATGGCCTATGCACGTGCTGGCGTGATTACTCGTGAAATGGAGTTTGTAGCGCTGCGCGAACACGTTGATGCGGAGTTTGTGCGCTCTGAGGTGGCGCGCGGTCGGGCCATTATTCCCAACAACGTCAACCACCCCGAATCTGAACCGATGATTATTGGTCGCAAATTTTTGACCAAAATCAACGCCAATATTGGCAATTCTGCGGTCACCTCTTCAATCGAGGAAGAGGTGTCCAAGCTGCAGTGGGCCACGCGCTGGGGTGCCGATACCGTGATGGATCTATCCACCGGCGATGATATTCACACCACCCGCGAATGGATTATCCGCAACTCCCCCGTTCCTATCGGCACCGTCCCGATCTACCAAGCGCTGGAAAAAGTAAATGGCGTGGCCGCAGACCTTAACTGGGAAGTATTCCGCGATACCATCATTGAGCAGTGTGAACAAGGCGTGGACTATATGACCATCCACGCCGGCGTCCTGCTGGCTTATATCCCACTGACTACCCGTCGTGTCACCGGCATTGTCTCCCGCGGCGGATCCATTATGGCCGGTTGGTGTCCGGCGCATCACCGCGAATCATTCCTCTACGAGCATTTCGACGAGCTGTGCGAAATCTTTGCACAATATGACGTCGCATTCTCCCTCGGTGATGGCCTACGCCCCGGATCGCTTGCCGATGCCAACGACGCCGCGCAATTCGCCGAGCTGAAAACCATTGGTGAGCTCACCCAACGCGCCTGGGAATACGATGTACAAGTAATGGTCGAAGGACCTGGACACGTGCCACTAAACATGATCCAGGAAAACAACGAGCTGGAACAAAAGTGGGCAGCGGACGCACCTTTTTACACTCTTGGACCACTAGTTACCGACATCGCTCCAGGTTATGACCACATCACTTCTGCCATTGGTGCAGCTCACATCGCCATGGGTGGCACCGCCATGCTGTGTTATGTCACCCCGAAAGAACACCTTGGCCTGCCCAACCGTGACGACGTCAAAACCGGCGTAATCACCTACAAGCTCGCTGCCCACGCAGCAGATGTGGCCAAGGGTCATCCCGGCGCGCGTGCCTGGGACGACGCCATGAGTAAAGCGCGTTTTGAATTCCGTTGGAATGATCAGTTTGCGCTCTCCCTCGACCCCGACACTGCAATCGCTTATCACGACGAAACCCTGCCGGCAGAGCCTGCGAAAACCGCACACTTCTGTTCAATGTGTGGCCCGAAGTTCTGCTCCATGCGAATTAGCCAGGACATTCGCGATATGTTTGGCGATCAAATCGCGGAATTGGGGATGCCTGGGGTTGGGGATTCTTCTAGTGCTGTTGCTTCTAGTGGGGCACGGGAGGGGATGGCTGAGAAATCCCGGGAATTTATTGCTGGTGGTGCGGAGGTTTATCGGCGTTAG 87 GTGAGTGAACATGAGCAGGCACACAGCCAATTAGATTTTGTTGAAGAGGCCATCGCTGACATCGCTGCGGGTAAAGCCGTCGTGGTGGTAGATGATGAAGATCGTGAAAATGAAGGCGACATCATCTTTGCCGCCGAATTAGCCACTCCAGAATTAGTCGCTTTCATGGTGCGTTATTCCTCGGGATACATCTGTGCGCCATTAACCGCAAAGGATGCAGATCGTCTTGATCTGCCTCCGATGACCGCGCACAATCAGGATGCCCGCGGCACCGCTTACACCGTGACCGTTGATGCCAACACCGGCACCACAGGCATTTCTGCAACAGACCGCGCCCACACTTTGCGCTTGCTTGCTGATCCAGAAGCCGACCGCACGGATTTCACCCGTCCCGGACACGTTGTGCCACTGCGTGCTCGTGAAGGTGGCGTCTTGGTGCGCGCTGGACACACCGAAGCAGCTGTCGATTTGGCTCGCGCTGCAGGCCTGCGCCCAGCAGGTGTTATCTGCGAAGTGGTCAGTGAAGAGGACCCCACCGGCATGGCTCGGGTTCCTGAGCTGCGCCGCTTCTGCGATGAGCACGATCTGAAGCTGATCTCTATTGAGCAGCTCATTGAGTGGCGTCGCAAGAATGAAATTTTGGTGGAGCGCCAGGTGGAAACTGTGCTGCCTACCGATTTCGGCACGTTCAAGGCTGTTGGTTACCGTTCCATCATCGATGGCACCGAGCTTGTTGCCATTGTTGCCGGCGACGTGGCATCCGACGGTGGCGAAAACGTCCTGGTTCGAGTCCACTCTGAGTGCTTGACTGGTGATGTTTTTGGATCCCGGCGCTGCGACTGTGGACAGCAGCTGCACGAGTCTTTGCGCCTGATCCAGGAAGCTGGTCGGGGAGTAGTGGTGTACATGCGTGGGCATGAGGGACGAGGCATTGGTCTGCTCGCCAAGCTACGCGCCTACCAACTCCAGGATGAAGGTGCCGACACCGTCGATGCCAACCTCGCACTTGGTCTTCCAGCCGATGCCCGCGAATTTGGCACCAGCGCCCAGATTCTCTACGACTTGGGTGTGCGCTCGCTCAACTTGATCAGCAACAACCCAGCCAAGAAGGTGGGACTTGAAGGCCACGGCATTTCCATTGCCAGCCGAACCCCCATCCCTGTTGCTGTTCATGAAGACAATGTTCGATACCTGAAAACCAAGCGTGACCGCATGGGACATGACCTCCCAGATGTCGCACTGTGGGAACAAGAGCACCCAGAAAACTAA 88 ATGGTCAGCCAAACGGAAAGACAGCATGCAATTGCTTCTTTACTGGCACCAACTGGTGCGGTGTCTGTAGGAGATTTAGCCGAGCATTTCCATGTGACAACCGAAACAGTGCGACGTGATCTTCGGATCATGGAGTCACTGGGTTTGTTGCAACGAGTTCACGGTGGCGCCATTAGCCCAGAGCCCATGGGTACGTCTCCCCCTCGGCTGAAACCTGCCTTGGTTAAAGGAATGCCACCGGAACCCCGTGTTTTAGAACTTGCAGAAACTGCAGTTTCCCTCATCACACCTCTAGCACGCAGCATTTTCCTGGATTCAGGTTTAGCGTGCACGGCGATTGCCACGGTGTTGGGGGATCCTCCAGAAGATGCCAGGTGGACTGTTGTTACAAGTTCCCCCGGCGCTGTGATTGCCTTGTCCGCGACAGATGCCACCTCCACGGTGGTGCTGCACGGGCAGGTTCACGGTAATTGTTCTTCAATCATTGGGTCCACGGCAGTAGACATGATTTCGCAGTTGCGCGCTGATATCGCCTTCGTGGAGGTTGATGCGATTCAATCCGATACAAGTCTGTGCACGTTTTTCCCGGAGACGATTCCCATCAAGCAAGCCATGATCAAAAACGCGGCTTTCACAGTTGCTGTTCTCAGCCCGAGATCTCCCCAAGATCAAGAACTTCAACTTTTGAAGCACCCTTTTTCCACCTTGGCTGATTTTGATGCCCTTGTTACCGATGACCACACGCTAGATTTTCCAGTTTTGCCCGACCACAACTTTCAGGTGGTAACCCCATGA 89 GTGACGCAGCAATTGAACCACGCAAAGGTAAATCAACATCCCGGTCAAGCCACCCTCCCAGAAACCGCAGAGGGGCAGGTTCGCACCTACGAGGTAAAAACCTACGGCTGTCAGATGAATGTGCACGATTCTGAGCGCCTTTCGGGCCTGCTCGAGGAGGCTGGATACGTTGCTGCTCCGGAGGACACCACTCCGGATCTTGTCGTATTTAATACGTGCGCCGTGCGTGAAAACGCCGATATGCGCCTCTATGGCACTTTGGGCAACCTGCGCAGCGTGAAAGAAAAGAACCCAGGCATGCAAATCGCTGTCGGTGGTTGTTTGGCTCAAAAAGACAAAGATACCGTGGTGAAAAAAGCACCGTGGGTGGACGTGGTGTTTGGTACCCACAACATTGGTTCCTTGCCAACCTTGCTTCAGCGCGCGGAGCACAATGCCCAAGCGGAAGTCGAAATTGTCGATTCCCTCGAGCAGTTCCCGTCAGTACTTCCTGCAAAGCGCGAGTCTGCTTACGCTGGTTGGGTGTCCGTATCAGTCGGATGTAACAACACCTGTACTTTCTGCATCGTTCCGTCGCTGCGCGGTAAAGAGCAGGACCGTCGACCAGGAGACATCCTCGCAGAGGTACAAGCACTGGTGGATCAGGGAGTTACCGAGGTAACTCTACTTGGCCAAAACGTAAATGCTTACGGCGTGAACTTTGTTGATCCTGAGCTAGAGCGCGATCGCAGTGCATTTTCCAAGCTGCTTCGTGCCTGTGGTGAGATCGAAGGCCTCGAGCGGGTTCGCTTCACCAGCCCTCACCCTGCAGAATTCACCTCTGATGTCATTGACGCCATGGCAGAGACCCCAAACATCTGCCCGCAGCTGCACATGCCACTGCAGTCCGGATCTGACAAGGTGCTCAAAGAGATGCGCCGTTCCTACCGATCCAAGAAGTTCCTCTCCATCTTGGATGAGGTCCGTGCGAAGATCCCTCACGCCTCTATCACCACCGATATTATTGTCGGATTCCCTGGCGAAACAGAGGAGGATTTCCAAGCAACCCTCGACGTTGTCAAGAAGGCACGCTTTACTTCTGCTTACACCTTCCAATACAGCCCACGCCCTGGCACCCCTGCAGCGGAATATGAAAACCAGCTTCCAAAAGAAGTTGTGCAGGAACGCTACGAGCGCCTCATGGTCGTTCAGGAACAAGTCTGCGAAGAAGAAAACCAAAAGCTCATCGGCACCACCGTCGAATTGCTGGTCCAGGCTGGCGGAGGCCGCAAGAACGATGCCACCAAGCGCATGAGTGGTCGCGCACGCGATGGACGCCTCGTGCACTTTGCGCCAGAGGGCGACATTGATGGTGAGATCCGCCCCGGCGATTTTGTCACTGTCACGGTGACTGAGGCCAAGCCTTTCTTCCTCATCGCAGACTCCGGTGTGCAGACCCACCGCCGCACCAAAGCTGGTGACAACTCTGCAGTTGGTCAAGTTCCAACCACAGCACCGATCGGTGTGGGCTTGGGACTGCCACAAATCGGCGCACCAAAGGTGGCTCCTGCCACAGAATCTGCCTGCTGCTCCATTAACTAA 90 ATGGTCGAACAATCGCCAGATTTCGTACAATCATTTGCCCGCGGCTTATCTGTGATCCGAAGTTTCAGCGCAGATAATCCATCGCAAACACTGTCCGAAGTCGCCAGCCAAACTGGACTCTCAAGGGCCACCGCTAGGCGCTTTCTCCACACCTTGACCGACCTTGGATATGCGGTAAACAACGATTCCCGGTTCCAGCTCACACCACGTGTTTTGGAGCTTGGAGCAAGCTACCTTTCCGCATTGTCCCTGCCTGCGATCGCGCAGCCCCGCCTGGAGGTACTCTCCCGCCAGGTCGGCGAATCAAGCTCCATGTCCGTACTCGACGGCACTGACATCATCTACGTTTGCCGCGTTCCGGTGCGCCGCATCATGACGGTGAACATCACCATCGGCACCCGTTTCCCTGCGTACGCCACCTCCATGGGACGCATCATGCTGGCCAACCTTCCCGAAGAAGAATTAGATGAAATGCTGGCGGCGGCACCCCCTGAACAGTTGACCACCCGGTCACTGACCTCCATCGCCTCAATCCGGGAAGAGATCATTACTACCCGCGAAAGGGGGTGGTCATTGGTGGATCAGGAGCTCGAGCCGGGCCTGCGTTCGCTCGCGGCGCCGATCACCAATGCCCAGGGCGAAGTGGTTGCTTCCATCAATGTGTCGACCCAATCGGCATCACATTCGGTGGAAGATATCCGCAAGCTGGTGCTGCCGCAGCTTTTAGAAACGGCTCAAGCAATTTCGACAGATCTCTCTGCACTCTAA 91 ATGAGCACTCCCCACCACCACGGTGATCACCCCGCTCCGGAAACAGACCACACCCACCACCCGAATCATGCCGGTCACGAGCACCATGCGGATGCCGCCACCCACGGCCAGGCCATGCCGCACGATCATCCGCATTCCACTGTCGATGAAGAACATCAGGTCCACAGTCACGGTGAACACGCCGGCCACAGCGCCGCGATGTTCCGGGACCGCTTCTGGTGGTCGCTGATCCTGTCGGTTCCGGTGGTGTTCTTCAGCCCGATGTTCGCCGACCTGCTGGGATATAATATTCCGGAGATTCCGGGAGCCTACTGGATTCCTCCGGTCCTGGGCACGATCATCTTCCTCTACGGCGGCACCCCCTTCCTCAAGGGCGCAATGACCGAGCTGAAATCCCGCCAACCGGGCATGATGCTCCTGATCGCCATGGCGATCACCGTGGCGTTTATCGCCTCCTGGGTCACCACCCTGGGGCTGGGCGGGTTCCACCTAGATTTCTGGTGGGAACTGGCCCTGCTGGTGACCATCATGCTGTTGGGCCACTGGCTGGAGATGCGCGCTCTTGGTGCAGCCTCCTCCGCGCTTGACGCGCTGGCAGCGCTCCTGCCCGATGAGGCCGAGAAGGTCGTCGACGGGACCACCCGCACCGTAGCGATCTCAGAGCTGGCCGTCGACGATGTCGTGCTGGTCCGAGCAGGTGCCCGCGTCCCGGCCGACGGGACCATCATCGACGGAGCGGCCGAATTCGATGAGGCCATGATCACCGGCGAATCCCGACCCGTCTACCGGGATACCGGTGAGACCGTGGTGGCCGGCACCGTGGCCACCGACAACACCGTCCGTATCCGGGTGGAGGCCACCGGTGGGGACACCGCCCTGGCAGGCATCCAGCGCATGGTCGCCGACGCCCAGGCCTCCTCCTCCCGGGCCCAGGCCCTGGCCGATCGAGCCGCAGCCTTCCTGTTCTGGTTCGCCCTGATCACGGCCCTGATCACCGCCGTGGTCTGGACCATCATCGGCAGCCCCGACGATGCCGTGGTCCGCGCGGTGACCGTGCTGATCATCGCCTGCCCGCACGCCCTGGGCCTGGCCATCCCGCTGGTCATCGCGATCTCCTCCGAGCGCGCCGCGAAATCCGGGGTGCTCATCAAGGACCGCATGGCACTCGAGCACATGCGCACCATCGACGTCGTCTTGTTCGATAAGACCGGCACCCTGACCGAAGGCGCACACGCCGTCACCGGCGTGGCTCCGGCCACGGGTATCGCCGAGGGTGAGCTGCTGGCCCTGGTCGCCGCCGCTGAGGCCGATAGTGAGCACCCCGTGGCCCGCGCGATCGTGACTGCCGCGGCCGCACACCCGGAGGCCTCGCAGCGTCAGCTGCGCGCAACCGGTTTCACCGCCGCCTCCGGCCGCGGGATCCGGGCCACCGTCGACGGTGCCGAAATCCTCGTGGGCGGGCCGAACATGCTACGCGAGTTCAATCTGACCACCCCGGGTGAGCTCGCCGACATCACCGGTTCCTGGGCACAGCGAGGTGCCGGAGTGCTCCATGTCGTCCGCGACGGTGAGATCATCGGTGCGGTGGCAGTGGAGGACAAAATCCGCCCCGAATCCCGCGCGGCGGTACGCGCCCTGCAGGCCCGCGGGGTGAAGGTGGCGATGATCACCGGTGACGCCACCCAGGTCGCCCAGGCAGTGGGCAAGGATCTGGGGATCGATGAGGTCTTCGCCGAGGTTCTGCCGCAGGACAAGGACACCAAGGTCACCCAGCTGCAGGAGCGCGGTCTGAGCGTGGCCATGGTCGGCGACGGTGTCAATGACGCCCCGGCCCTGGCCCGGGCCGAGGTCGGTATTGCGATTGGCGCGGGTACAGATGTGGCGATGGAGTCCGCCGGGGTGGTCCTGGCCAGTGATGATCCCCGGGCCGTGCTGTCGATGATCGAGCTCTCCCATGCCAGCTACCGCAAGATGGTCCAGAACCTGGTCTGGGCGACCGGGTACAACATCGTGGCCGTTCCGCTGGCCGCCGGTGTGCTCGCCCCTATCGGTGTGCTGCTTCCCCCGGCGGCGGCCGCCATCTTGATGTCCCTGTCCACGATCATCGTCGCCCTCAACGCCCAGCTGCTACGCCGGATCGACCTGGACCCGGCTCACCTAGCTCCGACCGACGGGAAGGAGGAGAAGGCTGCTGTGAGCTCTGCAGCCCCCGTCCGCTGA 92 ATGGCTGACCGCACACCGACCACCGCCACGCCCCCGGGGTGGGTGCTGGTCGTCGATGATGAACAACCCCTGGCTCAGATGGTGGCCTCCTACCTCATCCGGGCCGGCTTCGACACCCGCCAGGCGCACACCGGCACCCAGGCCGTGGACGAGGCCCGTCGCTTTTCCCCCGATGTTGTGGTGCTGGATCTGGGGCTGCCCGAACTCGACGGCCTGGAGGTGTGCCGACGGATCCGCACCTTCTCGGACTGCTACATCCTCATGCTCACCGCGCGTGGCAGCGAGGACGACAAGATCAGCGGTTTGACCCTGGGGGCGGATGACTACATCACCAAACCTTTTAGCATCCGGGAACTGGTGACCCGGGTGCATGCGGTGCTGCGCCGCCCGCGCACCAGCACCACCCCACCGCAGGTGACCACCCCCTTGATCGTTGGTGACCTCATCCTTGACCCCGTCGCCCATCAGGTGTGGGTGGGGGAGACGACCGTGGAGCTCACCCGCACGGAGTTCGAGCTGCTGGTTGCCCTGGCCCTGCGCCCCGGCCAGGTGCTGACCCGCCACGACCTGATCACCGAGGTCTGGGACACCACCTGGGTCGGCGATGAACGCATCGTCGATGTCCACATCGGCAACTTGCGTCGCAAGCTCGGCACCGACACCCGGGGCCGGGGGTTTATCGACACCGTGCGTGGCGTGGGCTACCGGGTGGGGCAGCCATGA 93 ATGACACACGTTGTTCTCATTGATAATCACGATTCTTTTGTCTACAACCTGGTGGATGCGTTCGCCGTGGCCGGTTATAAGTGCACGGTGTTCCGCAATACGGTGCCAGTGGAAACCATTTTGGCAGCCAACCCGGACCTGATCTGCCTTTCACCTGGACCTGGTTACCCTGCCGATGCGGGCAACATGATGGCGCTGATCGAGCGCACACTCGGCCAGATTCCTTTACTGGGTATTTGCCTCGGCTACCAGGCACTCATCGAATACCACGGCGGCAAGGTTGAGCCTTGTGGCCCTGTGCACGGCACCACCGACAACATGATCCTTACTGATGCAGGTGTGCAGAGCCCTGTTTTTGCAGGTCTTGCCACTGATGTTGAGCCTGATCATCCAGAAATCCCAGACCGCAAGGTTCCAATTGGCCGTTATCACTCACTGGGCTGCGTGGTTGCCCCAGACGGTATTGAATCACTAGGTACCTGTTCCTCGGAGATTGGTGATGTCATCATGGCGGCACGCACCACCGATGGAAAGGCCATTGGCCTGCAGTTTCACCCTGAGTCAGTGCTAAGCCCAACGGGTCCTGTCATTTTGTCCCGCTGTGTCGAACAGCTTCTCGCGAACTAA 94 ATGATCGTTTTGGGAATTGAAAGCTCCTGCGATGAAACAGGCGTAGGCGTAGTCAAACTTGACGGCGAAGGAAACCTAGAGATCCTCGCCGACTCAGTGGCCTCCTCCATGCAAGAACATGCCCGCTTTGGTGGCGTCGTGCCAGAAATCGCCTCCCGGGCGCACCTGGAATCTATGGTCCCCGTGATGCGTGAAGCGTTGAGGCAGGCGGGCGTCGACAGGCCAGATGCTGTGGCTGCAACCGTGGGCCCTGGTTTGGCGGGCGCGCTGCTCGTTGGAGCCAGCGCTGCGAAGGCGTATGCCGCTGCGTGGGGAGTTCCGTTTTACGCGGTCAACCACCTGGGCGGACACGTCGCCGTGGCCAATCTGGAAGGTGAAACTCTTCCACACGCGGTGGCTTTGCTGGTTTCCGGCGGACACACTCAATTGTTGGAAGTCGACGCGGTGGGATTACCCATGAAGGAATTGGGATCCACCCTCGACGATGCCGCTGGCGAAGCCTATGACAAAGTCTCAAGGCTGTTGGGATTGGGCTACCCAGGCGGCCCCATCATTGATAAATTGGCGCGCCGGGGTAATCCAGAGGCCATTGCTTTCCCCCGCGGATTGATGAAAAAGTCGGATTCTCGGCATGATTTCAGCTTTTCCGGTTTGAAAACCTCCGTTGCCCGCTACGTGGAAGCTGCGGAAAGAAACGGTGAAGTTATTTCCGTGGAGGACGTCTGCGCATCATTCCAAGAAGCGGTGTGTGATGTGTTGACGTTTAAGGCCGTGCGTGCGTGCCGCGATGTCGGTGCGAAGGTGCTGCTGTTGGGTGGAGGAGTGGCTGCCAACTCTCGTCTGCGGGAGCTTGCTCAAGAACGTTGCGATAAAGCCGACATCGAACTCCGGGTTCCTCGTTTCAATTTGTGCACCGATAATGGTGTCATGATTGCAGCGTTGGCGGCTCAAAGAATCCACGAAGGTGCCCAAGAATCACCAATTTCGGTCGGAACTGATCCTTCTTTGTCCGTTGAGACCCCACAGGTGCTTTAA 95 ATGATCCGCAAACTTGCTCGACCAATGCTTGCATCGGTCTACGTCGCAGATGGCGCAGAAACAGTATTGAACACCAGCGCACACGTCGAAGACACTCAGGTAGTTCTGGATCGTATCCGTTATGTGCTGCCCCGTAAGTACGCAAAGCGCATTTCCAGAGATCCAGAATTGGTCACCCGCGTCATTGGCGGCACCAAAGTCGGTGCGGGTTCTTTGCTAGCTATTGGTCGTGCACCACGCACCTCTGCAGCTACCCTCGCAATCCTGACTATCCCTAACATCCTGGCTCGCAATGCGTTCTGGGAAACCCAGGATGCGGATGAAAAGCGTAACCGCCGCAACGGTTTCCTCACCAACATTGCCCTGCTTGGTGGCCTGTTTATCACTTCTGTTGATACTGAGGGCAAGCCTGGCGTGAAGTGGCGTGCAACCAATGCTACAAAGCGTGGCAAGAAGCAGCTGCAGCAGGCACTTCCAACCAAATCTGAGACTGAAAAGTTCGGTGAGAAGGCCTCTGATTGGTTCAACGATACTTCTGACAAGGTCACCGAGTACGCGTACACCGCTCAGGATTTTGTCGGTGAGAACAAGGATGACTGGATCAAGTCCGCAACCGAGACTGCTCACAAGGTCGCTGATACTGTGAGCGATTACGCTCACAAGGCTACCTCTTACCTTGAGGAGAACAGCGGTGACTGGCTTGAGGCTGCGCAGGCTAACGCCAAGACTGCTCGTAAGTCTGCAGTGAAGGCTGCCGGCAAGGCTCAGGAAAAGGCTAACTTTGCTCTTCAGGTCGCAGAGGAAACCTCTGGTCGCGCCAACAAGAAGGCAACTAAGAGCTACGACAAGCTTCAGAAGCAGGCTGATAAGGCCATCGATCGTGCACAGAAGAAGCTGAAGGGCATCGAACTTTAA 96 ATGAAGCTTTTCTCCCGCACCTCACTGGTTGCACTTGGTACCGCTGCAGCTATCACTCTCTCCGGCGTAACCGCTCCTGCATTCGCTGATGAGGACTCCAACGCAGCTGTAAGTGCTCTTAAGACCGCTGAAGATAATACCCCTGAGGCGCCAGGGGCTTCTACTCCCCTGAAGCTTGAGCAGCCTGGCACCATCACCGGCGTCCCTGGCAAGGCGATCACCCCGGTCACTGTTAAGGTCATCGCAGGCGAAGCAGAATCCTTCACCTCAGACAACCTTCCTTCTGGTCTCCTTATCGACAACACCGGAAAGATCACCGGTACTCCAAAGAAGGAATTCACTGGTTCCGCAAAGATCATTGCCAAGAACGAAGCAGGCGTTGAAGCAGAAGTTTACGTTAACTTTGATTTCAACGAAGAGCCTTCCAGCGAAGAGCCTTCCAGCGGTTCTTCCGACACTGACAACATCGAAAACTGGATCAAGATCATCACAGCAGTCATCGGTGCACTAACCACCATTTTAACCTTCAGTACCAAGCTGGATTCCTTCCTGAAGTAA 97 ATGGACTGGTCGATCATTATTAATGTGCTTGCCGTTGCTACTGTCGTGATACTCACCCTTATCATCGCAGCTGCATTATATAGGGGTTTCACACGCAACAAAATAAAAAAACTTGAAGCTATACGAGAAGCCCAGCAACACGAACGCGACAATCCCACTATCCGCATTGCTGATCTTCGCGGCATCATGGATACTCACCACTACATCTACACCGACGTCATGGTTACACGTGCACATGAACTCATGATTACTGCACCCGCATTCTTTGATGTCACTCTCCCCGAAGCAGTGCTCTACGAAGATACTAAACGTGCTGCTCATGATGCCCTTAACGGCTATAAAGACACCACTGGCGCTAGTGCAGAGAAGATCATGCTTGTAGATGCAGTCACCGCAGCATGGACTCTCTATTCCACAAAAGCAAACACCGGATCCACGTGA 98 ATGTCAACTGCATATTTTATTGCGCACGGAAACGATACGTACCGCCCAACGGAGCATTCCAGCGGTGCGTGGCGCGATGATGAACTCCACCTTGCACCGGTTGCCGGTTTGGTGATTCACCATATGGAACGTTGGCGCAGGGAAGTGGTTGGCGATGCTTTGGTGTTCAGCCGTTTTAGTTTGGAGGTCTTGGGCCAGATCGCTCGGGATGATGTCACCTTGAGAACAGAGATCGTTCGTCCCGGGCGCACCATTGAACTTATTGAAACCGTGGCTGAAATCAACGGCAGAGTGACGATTCGTGCACGCGCCTGGTTGCTGAAGACCTCTGATCTTGCCCATATTTCTGGCGATACTTTTGAAGCGTTGCCGTCAATGACTGAACTGGCAGATGTTAAGTCAGCGTTCCAGTGGTCTGGTGGATTTATGGACAGCATTGTGTTTGTTGAGGACGCTGAGAAAATTCCGGGCAAAAACCGCGCGTGGATTACCGGCGATGTGCAGTTGGTTGACGGCGAAGATTCTGAGCCTTTGGCAGAGTTTTCCAAATTCTTGGATGTGGCCAATGGCGTGGCGGTTCGGGAAGATCCAAAGACTTGGATGTTCCCGAACGTGGATCTTACTTTCCATCTGTTCCGCCAGCCGGTGGGCACCAAGGTGGGTTTTGATACCCGCGCGGCGTTTGGACCCAACGGCATCGGTTTGACCACGTCGGTGATTCACGATGCCGAAGGTCCTGTGGGCACGCTTAATCAATCATTGACAGTGCGCCCAATTGCTGAGTTAAACAAGTGA 99 ATGAATGATCAGCTGTGGGAAGGCGACACTGGAACGCTGACTTTTGGGTCCAGGAAAGCTCTGGTGCAATTACTCAAAGGTCCCATGGTGAATGCTTTGCAGCATGTTGAAGTGTGGCGGGCTATCACCACAGATCAAGATGCGCTCAATGCTGTGCTTAACAATTTGTTCCTCGAGTTGGTTCTTGATGAGGATGCGGGTGTTGCATTTACTCGGCCTGCCAATGGTAGACAAGAAGTATTGGTTGGAAATAACAAAACTGAAGCGATGCCCAAAGTGCTGCGCACGGAGACGCTGTCGCATTTTGATACGTTGATCATTTTGATTCTGCGCCAAGAACTCACCATGGCGCCACCGGGGGAACGAGTCATTGTGGATCGTGAAGAAATCCGCGAACAAGTGTTGCTCTACCGCGTTGATGAAGAGCGAGATGAAGCCAAACTAGCTAAGCGATTCGACGCTGCATTTAGGCGCATCGTGGATTATTCATTAGCTAAAAAGACAGAGACACCTGAACGTTTTGAGGTCTCACCAGCACAGCGCCAGATTTTTGATGCCGACACTGTGGCAGGTGTGCGCGCTGAGTACGAAAAATTTAACAAAGCAGCCCATGATGGAAATGAAGAGGAACAGAAGTGA 100 ATGGTCCACATGGTCTTCGGCGATATGAACACTGACCGTGCGGCGCAGGCCTACATCATCGTGATCACCACCATCGTCATGGTGGTGTTGTTCTGGATCGTGCTCAGCTATTGGTCGCTGGCTGACCGGGCCCGCGCCCAGCGGTTCACCGCCAGCATCACGGAGATCGGACGCAAAATCTTCCTCAACCGGCTGCGTCCCCGGATGAGCAGGCAGAACACCTACACGGACAAGGACATCTCGCAGTTCCACTGGACCAATGGCCTGCCGCCGACCGATGATGAATCCCCCGAGTGGATCGCCGCCCGCGACAACGAGTGGGAGGGATACACCATCACCCTCGGCGACGATCCCAACGGCACCGAGAAAACCATCACCCTCGACGATCTGCGGGAGCTGCCGCAGACCTCGTATGTCGCCGTCCACACGTGCATGCAGGGCTGGTCAGCTACCGCCCGGTGGACAGGGGTGCGGTTACGTGATGTTTTGTGTCATGACCTTGTGCACACCCTAGACCTTCACCACAGGCATAGTCCTCGGCTGCTCACAATTGAGATCATACCTAAACCCCTGCCCCCAGAAACCCGATGCAAGATCATCGATTTCGATCTGTTCGCACCGAACAGTCCCTCGATCGAAGAGTTCTGCAGTCGGCTTAAAATATCGCGGCGCAGCTTCTACAACATCCGCAACCGATACCAACAAGATGCCAGTGCAGCGCTGCATCCACGCTCCAGCGCCCAGATCACCTCCCGGCGAACATACGATGAATCCATCACCAGTATCTTGTTGGCCATCCGCGCACCGCCTGAAAGCCCAAGGATGGGAATACGGTCCGATCTCTATTCGATTCGAAGGCATCTCCACCGGGGAACTGACTGCACCGATTCCATCTGTTTCAACCATTGCTCGTTTGTTACGCGCCGCAGGAGCTGTTGAAAGTAA 101 ATGTCCACACCAGTTTCAAATTTGGCAAGCGTTCAGAAAACTCTGGACCATGCGCTTGAGGACCGCCCTGAAGAGGGAATCGTCCGCGTCAACCGCAACATCTTCACTGACCCTGAGATCTTCGAGCTGGAGATGCGCCACATCTTCGAAGGCAACTGGATCTACCTGGCTCACGAGTCCCAGATCCCTAACGCTGGCGACTACTTCACCACCTACATTGGCCGCCAGCCGATCATGATCACCCGTTCCAAGGACGGCACACTCAACTGCCTGATCAACGCGTGTTCTCACCGTGGCGCCATGCTCTGCCGTCGCAAGACTGACAACCGCACCACCTTGACCTGCCCATTCCACGGCTGGACATTCAGCAACGACGGCGCACTGCTCAAGGTCAAGGACGAAAAAGAAGGCGCCTACCCAGAGAATTTCCGCACCGACGGCTCCCACGATCTGCGTCGCGTTCCTAAGTTCGAGTCCTACCGTGGCTTCCTCTTCGGCTCCCTCAACGATGATGTCGTTTCTTTGGAAGAGCACCTCGGCGACACCCGTACCGTCATTGACATGCTGGTTGACCAATCCCCAGAAGGCCTCGAAGTACTGCGCGGATCCTCCACCTACACCTACGACGGCAACTGGAAGCTCCAGACCGAAAACGGTGCAGACGGCTACCACGTTTCCTCCACCCACTGGAACTACGCTGCAACCACCTCCCGCCGTGGCACTGGTGAATCCGCGAACGAAACCAAGGCAATGGACGCTGGTACCTGGGGCAAGCAGGGTGGCGGATACTTCTCCTACCCTTACGGCCACATGCTGCTGTGGATGTGGTGGGGCAACCCAGAAGACCGCCCACTGTTCGAGCGCCGCGACGAGTTCAAGAAGGAATTCGGCGAAGAAAAGGGCGAGTTCATGGTTGGTGCTTCCCGCAATCTGTGCCTCTACCCCAATGTTTACCTGATGGATCAGTTCTCCTCACAGATCCGCCACATCCGCCCAATCTCAGTTGATCAGACCGAAGTCACCATCTACTGCATCGCACCTAAGGGCGAGTCCGCGGAAGCACGTGCAAACCGCATCCGCCAGTACGAAGACTTCTTCAACGCAACGGGCATGGCAACCCCAGATGACCTGGAGGAATTCCGCTCCTGCCAGAAGACCTACCAGGCATCTGCCTTCCCATGGAATGACATGACCCGCGGTTTGGGCCACCAGGTACAGGGACCAAACGAGGTTGCCAAGGGCCTAGGCATGAACGAAGTTCTTTCCTCCGGAGCACGCACCGAAGATGAAGGCCTCTACCCAATCCAGCACGGCTTCTGGCATGAACTCATGCAGGAGGCTGTGAATAAGCAGAGCATCAAGGAAAAGGAATTGGCTGACGATACCGCTTCTTCCCTTGCCACCGTAGCTGCAGCCAAAATCCGTGAGGAAGCAAAGGCAGCCGCGAAGTCCGACGCTGGAGAGCCTCGCCGCCGTCGTCGCACCCGCGGTTAG 102 TTGACATATGTTCAGGGTAGTTGACTAAAGAGTTGCTCGCGAAGTAGCACCTGTCACTTTTGTCTCAAATATTAAATCGAATATCAATATATGGTCTGTTTATTGGAACGCGTCCCAGTGGCTGAGACGCATCCGCTAAAGCCCCAGGAACCCTGTGCAGAAAGAAAACACTCCTCTGGCTAGGTAGACACAGTTTATTGTGGTAGAGTTGAGCGGGTAACTGTCAGCACGTAGATCGAAAGGTGCACAAAGCAT 103 GCGGCCGCCGCATCATTCCAATGCTGATCCACCCCATCCGGATAAACCACCATGAACGGCAACGGATCAAAAGTCCTGTTGGTGAAGCTGCGCCCCACAGATCCTGACTGCTGGGAGCCATGAAAATAGATCAGCGCATCCGTGGTGGAACCAAAAGGCTCAACAATACGAAACGTTCGCTTT CGGTCCTGATGAAAGAGATGTCCCTGAATCATCATCTAAGTATGCATTGTGGTAAGCTCGACCAGGACAGTGCCACCACAATTTTGGAGGATTACAAG CATATG 104 GGTTGGAATCCGTCCCATCATTACTGGGATATACGCGTCGCCAACCCCCAAAAAGAGGCTTCCAGTCAAACGCTCGAGAAACTAGAAGTATTCTTCTGAGAGCATTTTTGAACCGAAGACTATGACTAGACATTCGGACAATAGTAAGATGTGAGCTGAAACTCAGTTTCCATCCTTATTCAACCTGGAAGGTAGATACCGCACC 105 TTAAGCCAAAGTAACTAAGCCAAGTGACTAAGCACAGTTACTTGGCCAAGCTGGGCGGCAGAAAAACCGGCCCAGCTAATACTTCAGTTTAAAATTCGCTTCAACCCTGAAAGATTGTGACAG 106 TAGTTGACTCAACGTATTTCGGCATTTAAAAAAGCCGAGAGCAAATGAGACTTTCCAGGAGAAGGCACCAGGGACATGAACAATTGATCGGCTGACCAACTCTATAAGAGATGCACCTCAAGTTTGGGGATACTTATTCGGCGTTTCTGGGGAACAAATACGTTTCCCTATTGTTGTATATAGGTATTCGCACTTAAGAAACATCTCTCATGGAAAGAAGCTAGGCGGAAAGGGCGTTAAGTACTTGCCATTTAATCCTCAGCATCACTCGGATCAGTCGGAGATGTCGATGAAAATGCACCAGGAGCC 107 CGCCGCGTGGGGCAAGTGTGAGTTTTCCTACTTTTCGGCTCCAGGAATAACGATCATCATACCTTTGTGGATACGATTCCACACAAGCCCATAGGTGCTAGGAGCGCGCACAATGTATCCCACTGACAAAGCCCCATTCTCCTAACTTTGGCAGTACGCAGGAAATGACACTCAACTAGAAATCTTTTCTTCAGATGCCACAATCGAAAGCTGG 108 TTAGGTGTGATCTTTTGTGGGGAGGTATTTAATGATCCTACCAATCCTTTAGTTTCGTCTGTAGAGCGATGTGTGACGCAAGTTTTAAGACTCTAAACCCACGTCACAAGAAATGAGGGGTGCTTTGTTTACTATCACTGGTTTTGCAGATGAGATCGCACATGATCTCGACGAACAGATTTCCTTACTTAATAAACTGAAGAACAGCTCGCGGAAGCTAAAGCCAAA 109 GGGGCGAGATGAACCCTCAATGTCACGTGAAACATTGAGGGTTTTACACCCCAATTAAAGAAAAAATGAATTAAACCAAAACTTCTTAATCGCAGGGGTTAAGGTAAGTTCAGCTAATCAATGAAACTTCCCCTTTCAGCCCGTTGAAGAACCAATGAGCCCTGCCCGCACCCAGCCCCGAGCGGCTTTCCTAAGGAAGCTCCGACCCATCACATGTAGCCTTTCC 110 TTGTTGTTTTGTAGGGCACACGGTGGATGCCTTGGCATATCAAGCCGATGAAGGACGTGAGAGGCTGCGTTATGCCTCGGGGAGTTGCCAACTAAGCGTTGATCCGAGGATGTCCGAATGGGGAAACCCAGTCGCAGTGATGTGTGGTTACCTGCCAGTGAATGTATAGCTGGTGTGGAGGTTTACACGGGGAAGTGAAACATCTCAGTACCCGTAGGAGAAGAAAACAATTGTGATTCCGTTAGTAGTGGCGAGCGAACGTGGATGATGGCTAAAACTTATGTGTGTGATACCCGGCAGGGGTTGCATGTAGGTGGTTGTGGGGCAATGACGTTCATATTCTGCCGGATGTGGGCATGTGCTGCGTGGTTAGTGGAAGTGGTGTGGAAACACCTGCCGTAGAAGGTGAGAGTCCTGTACACGAAGATCATGGTGGTGTGTGTGGTTGTTGATACCCCGAGTAGCAGCGGGCTCGTGGAATCTGCTGTGAATTAGCCGGGACCACCCGGTAAGCCTGAATACTTGATATGACCGATAGCGGATTAGTACCGTGAGGGAATGGTGAAAAGTACCCCGGGAGGGGAGTGAAATAGTACCTGAAACCGTGTGCTGTCAATCCGTCAGAGCATCCTTTGTGGTGTGATGGCGTGCCTTTTGAAGAATGAGCCTGCGAGTCAGCGGCATGTCGCGAGGTTAACCCGTGTGGGGTAGCCGTAGGGAAACCGAATCCTAACGAGGGTGATTTTAGTGGCATGTCTTGGACCCGAAGCGGAGTGATCTACCCATGGCCAGTGTGAAGCAGCTGTAAGAGGTTGTGGAGGCGCGAACCCACTTAGGTTGAAAACTGAGGGGATGAGTTGTGGGTAGGGGTGAAAGGCCAATCAAACTCCGTGATAGCTGGTTCTCCCCGAAATGCATTTAGGTGCAGCGTCGTGTGTTTCTTGCCGGAGGTAGAGCTACTGGATGGTTTAGCGGGACCAACATCTTAGCGACATCAGCCAAACTCCGAATGCCGGTAAGTTAGAGCACGGCAGTGAGACTGCGGGGGATAAGCTTCGTAGTCGAGAGGGAAACAGCCCAGATCGCCGGCTAAGGCCCCTAAGGGTGTGCTAAGTGGAAAAGGAGGTGGGGTCGCGAAGACAGCCAGGAGGTTGGCTTAGAAGCAGCCATCCTTGAAAGAGTGCGTAATAGCTCACTGGTCGAGTGATTCCGCGCCGACAATGTAGTGGGGCTTAAGTACACCGCCGAAGCCGCGGCAATGATCTTTATAGGATTGTTGGGTAGGGGAGCGTCGTGCATGCGTTGAAGCTTTGGGGTGACCTTGGGTGGAGTGTGTGCGAGTGAGAATGCAGGCATGAGTAACGAATGATGCGTGAGAAACGTATCCGCCGGATGACTAAGGGTTCCTGGGTCAAGTTAATCTTCCCAGGGTGAGTCGGGGCCTAAGGCGAGGCCGACAGGCGTAGTCGATGGATAACGGGTTGATATTCCCGTACCCGAGTATGAGCGACCATGGTGAATCAGTGATACTAACCACCCATAAGCACCCGCGAAAAGGCTTTGCTTTTTTGTGGTGTGTGGTTGCGTGGGACCTGATCTGGTAGTAGCTAAGTGATGGGGTGACGCAGGGAGGTAGCTCAGCCACTTATTGGATTGTGGTGTAAGCGTGTGGCACGCAGTGTTGGTAAATCCGCACTGTTTTTGTGTGAGGCGTGATGCGGAGCCCGTAAAGGGTGAAGTGGGTGATCCTGTGCTGTCGAGAAAAGCCTCTAGCGATGTTGATATTCGGCCCGTACCCTAAACCGACACAGGTAGTCAGGTAGAGAATACTAAGGCGTTCGGGTGAACTGTGGTTAAGGAACTCGGCAAAATGCCCCCGTAACTTCGGGAGAAGGGGGGCCACGGCGTGTGAACAACTTTTCGTTGGGAGCGTGTTGTGGTCGCAGAGAATAGAGGGAAGCGACTGTTTACTAAAAACACAGGTCCGTGCGAAGACGTTTAAGTTGATGTATACGGACTGACGCCTGCCCGGTGCTGGAAGGTTAAGAGGACCGGTTAGCCGTAAGGCGAAGCTGAGAATTTAAGCCCCAGTAAACGGCGGTGGTAACTATAACCATCCTAAGGTAGCGAAATTCCTTGTCGGGTAAGTTCCGACCTGCACGAATGGCGTAACGACTTCCCTGCTGTCTCAACCACAGGCCCGGTGAAATTGCAGTACGAGTAAAGATGCTCGTTACGCGCGGCAGGACGAAAAGACCCCGGGACCTTCACTATAGCTTGGTATTGGTGTTTGATTCGGTTTGTGTAGGATAGGTGGGAGACTGTGATCATATGACGCTAGTTGTGTGTGAGTCGTTGGTGAAATACCACTCTGATCGGATTGGATGTCTTAACCTTGGCCCATGATCTGGGTTGGGGACAGTGCCTGGTGGGTAGTTTAACTGGGGCGGTTGCCTCCTAAAATGTAACGGAGGCGCCCAAAGGTTTCCTCAGCTTGGTTGGTAATCAGGTGGTGAGTGTAAGTGCACAAGGGAGCTTGACTGTGACACTGACAGGTGGAGCAGGGACGAAAGTCGGGACTAGTGATCCGGCACCTACTTGTGGTTGTGGTGTCGCTCAACGGATAAAAGGTACCCCGGGGATAACAGGCTGATCTTCCCCAAGAGTCCATATCGACGGGATGGTTTGGCACCTCGATGTCGGCTCGTCGCATCCTGGGGCTGGAGTAGGTCCCAAGGGTTGGGCTGTTCGCCCATTAAAGCGGCACGCGAGCTGGGTTTAGAACGTCGTGAGACAGTTCGGTCTCTATCCATCGCGCGCGTTGAAACTTGAAGGAAGGCTGTCCCTAGTACGAGAGGACCGGGACGGACGTACCTCTGGTGTGCCAGTTGTTCCGCCAGGAGCAGGGCTGGTTGGCTACGTACGGGAGGGATAACCGCTGAAAGCATCTAAGCGGGAAGCCTGTTTCGAGATGAGGTTTCTTTTGAGGTTCCCTAGAGATTATGGGGTTGATAGGCCAGATCTGGAAGCACTGTGAGGTGTGGAGGTGACTGGTACTAATTTACCGATAACAACAAACC 111 TTGTTGTTTTGTAGGGCACACGGTGGATGCCTTGGCATATCAAGCCGATGAAGGACGTGAGAGGCTGCGTTATGCCTCGGGGAGTTGCCAACTAAGCGTTGATCCGAGGATGTCCGAATGGGGAAACCCAGCCGCAGTGATGTGTGGTTACCTGCCAGTGAATGTATAGCTGGTGTGGAGGTTTACACGGGGAAGTGAAACATCTCAGTACCCGTAGGAGAAGAAAACAATTGTGATTCCGTTAGTAGTGGCGAGCGAACGTGGATGATGGCTAAAACTTATGTGTGTGATACCCGGCAGGGGTTGCATGTAGGTGGTTGTGGGGCAATAACATCCATATTCTGCCGGATGTGGGCATGTGCTGCGTGGTTAGTGGAAGTGGTGTGGAAACACCTGCCGTAGAAGGTGAGAGTCCTGTACACGAAGATCATGGTGGTGTGTGTGGTTGTTGATACCCCGAGTAGCAGCGGGCTCGTGGAATCTGCTGTGAATTAGCCGGGACCACCCGGTAAGCCTGAATACTTGATATGACCGATAGCGGATTAGTACCGTGAGGGAATGGTGAAAAGTACCCCGGGAGGGGAGTGAAATAGTACCTGAAACCGTGTGCTGTCAATCCGTCAGAGCATCCTTTGTGGTGTGATGGCGTGCCTTTTGAAGAATGAGCCTGCGAGTCAGCGGCATGTCGCGAGGTTAACCCGTGTGGGGTAGCCGTAGGGAAACCGAATCCTAACGAGGGTGTTTTAGTGGCATGTCTTGGACCCGAAGCGGAGTGATCTACCCATGGCCAGTGTGAAGCAGCTGTAAGAGGTTGTGGAGGCGCGAACCCACTTAGGTTGAAAACTGAGGGGATGAGTTGTGGGTAGGGGTGAAAGGCCAATCAAACTCCGTGATAGCTGGTTCTCCCCGAAATGCATTTAGGTGCAGCGTCGTGTGTTTCTTGCCGGAGGTAGAGCTACTGGATGGTTTAGCGGGACCAACATCTTAGCGACATCAGCCAAACTCCGAATGCCGGTAAGTGAGAGCACGGCAGTGAGACTGCGGGGGATAAGCTTCGTAGTCGAGAGGGAAACAGCCCAGATCGCCGGCTAAGGCCCCTAAGGGTGTGCTAAGTGGAAAAGGAGGTGGGGTCGCGAAGACAGCCAGGAGGTTGGCTTAGAAGCAGCCATCCTTGAAAGAGTGCGTAATAGCTCACTGGTCGAGTGATTCCGCGCCGACAATGTAGTGGGGCTTAAGTACACCGCCGAAGCCGCTGGCAATGATCTTTATAGGATTGTTGGGTAGGGGAGCGTCGTGCATGCGTTGAAGCTTTGGGGTGACCTTGGGTGGAGTGTGTGCGAGTGAGAATGCAGGCATGAGTAACGAATGATGCGTGAGAAACGTATCCGCCGGATGACTAAGGGTTCCTGGGTCAAGTTAATCTTCCCAGGGTGAGTCGGGGCCTAAGGCGAGGCCGACAGGCGTAGTCGATGGATAACGGGTTGATATTCCCGTACCCGAGTATGAGCGACCATGGTGAATCAGTGATACTAACCACCCATAAGCACCCGCGAAAAGGCTTTGCTTTTTTGTGGTGTGTGGTTGCGTGGGACCTGATCTGGTAGTAGCTAAGTGATGGGGTGACGCAGGGAGGTAGCTCAGCCACTTATTGGATTGTGGTGTAAGCGTGTGGCACGCAGTGTTGGTAAATCCGCACTGTTTTTGTGTGAGGCGTGATGCGGAGCCCGTAAAGGGTGAAGTGGGTGATCCTGTGCTGTCGAGAAAAGCCTCTAGCGATGTTGATATTCGGCCCGTACCCTAAACCGACACAGGTAGTCAGGTAGAGAATACTAAGGCGTTCGGGTGAACTGTGGTTAAGGAACTCGGCAAAATGCCCCCGTAACTTCGGGAGAAGGGGGGCCACGGCGTGTGAACAACTTTTCGTTGGGAGCGTGTTGTGGTCGCAGAGAATAGAGGGAAGCGACTGTTTACTAAAAACACAGGTCCGTGCGAAGACGTTTAAGTTGATGTATACGGACTGACGCCTGCCCGGTGCTGGAAGGTTAAGAGGACCGGTTAGCCGTAAGGCGAAGCTGAGAATTTAAGCCCCAGTAAACGGCGGTGGTAACTATAACCATCCTAAGGTAGCGAAATTCCTTGTCGGGTAAGTTCCGACCTGCACGAATGGCGTAACGACTTCCCTGCTGTCTCAACCACAGGCCCGGTGAAATTGCAGTACGAGTAAAGATGCTCGTTACGCGCGGCAGGACGAAAAGACCCCGGGACCTTCACTATAGCTTGGTATTGGTGTTTGATTCGGTTTGTGTAGGATAGGTGGGAGACTTCGATCATATGACGCTAGTTGTGTGTGAGTCGTTGGTGAAATACCACTCTGATCGGATTGGATGTCTTAACCTTGGCCCATGATCTGGGTTGGGGACAGTGCCTGGTGGGTAGTTTAACTGGGGCGGTTGCCTCCTAAAATGTAACGGAGGCGCCCAAAGGTTTCCTCAGCTTGGTTGGTAATCAGGTGGTGAGTGTAAGTGCACAAGGGAGCTTGACTGTGACACTGACAGGTGGAGCAGGGACGAAAGTCGGGACTAGTGATCCGGCACCTACTTGTGGTTGTGGTGTCGCTCAACGGATAAAAGGTACCCCGGGGATAACAGGCTGATCTTCCCCAAGAGTCCATATCGACGGGATGGTTTGGCACCTCGATGTCGGCTCGTCGCATCCTGGGGCTGGAGTAGGTCCCAAGGGTTGGGCTGTTCGCCCATTAAAGCGAGCACGCGAGCTGGGTTTAGAACGTCGTGAGACAGTTCGGTCTCTATCCGCCGCGCGCGTTGAAACTTGAAGGAAGGCTGTCCCTAGTACGAGAGGACCGGGACGGACGTACCTCTGGTGTGCCAGTTGTTCCGCCAGGAGCAGGGCTGGTTGGCTACGTACGGGAGGGATAACCGCTGAAAGCATCTAAGCGGGAAGCCTGTTTCGAGATGAGGTTTCTTTTGAGGTTCCCTAGAGATTATGGGGTTGATAGGCCAGATCTGGAAGCACTGTGAGGTGTGGAGGTGACTGGTACTAATTTACCGATAACAACAAACC 112 TGTGGAGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGCTGAAACCGGAGCTTGCTTTGGTGGATGAGTGGCGAACGGGTGAGTAACACGTGGGTGATCTGCCCTACACTTTGGGATAAGCCTGGGAAACTGGGTCTAATACCGAATATTCACACCACCGTAGGGGTGGTGTGGAAAGCTTTATGCGGTGTGGGATGAGCCTGCGGCCTATCAGCTTGTTGGTGGGGTAATGGCCTACCAAGGCGTCGACGGGTAGCCGGCCTGAGAGGGTGTACGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGGGGGATGAAGGCCTTCGGGTTGTAAACTCCTTTCGCTAGGGACGAAGCCTTATGGTGACGGTACCTGGAGAAGAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTGTCCGGAATTACTGGGCGTAAAGAGCTCGTAGGTGGTTTGTCGCGTCGTCTGTGAAATCCCGGGGCTTAACTTCGGGCGTGCAGGCGATACGGGCATAACTTGAGTGCTGTAGGGGAGACTGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCAATGGCGAAGGCAGGTCTCTGGGCAGTAACTGACGCTGAGGAGCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGGTGGGCGCTAGGTGTAGGGGTCTTCCACGACTTCTGTGCCGCAGCTAACGCATTAAGCGCCCCGCCTGGGGAGTACGGTCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGATGGAGCATGTGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGCTTGACATGGACCGGATCGGCGTAGAGATACGTTTTCCCTTGTGGTCGGTTCACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCTTATGTTGCCAGCACATTATGGTGGGTACTCATGAGAGACTGCCGGGGTTAACTCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGTCCAGGGCTTCACACATGCTACAATGGTCGGTACAGCGAGTTGCCACACCGTGAGGTGGAGCTAATCTCTTAAAGCCGGCCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCATGAAAGTTGGTAACACCCGAAGCCAGTGGCCCAACCTTTTAGGGGGGAGCTGTCGAAGGTGGGATCGGCGATTGGGACGAAGTCGTAACAAGGTAGCCGTACCGGAAGGTGCGACTGGATCACCTCCTTTCT 113 TTGTTGTTTTGTAGGGCACACGGTGGATGCCTTGGCATATCAAGCCGATGAAGGACGTGAGAGGCTGCGTTATGCCTCGGGGAGTTGCCAACTAAGCGTTGATCCGAGGATGTCCGAATGGGGAAACCCAGCCGCAGTGATGTGTGGTTACCTGCCAGTGAATGTATAGCTGGTGTGGAGGTTTACACGGGGAAGTGAAACATCTCAGTACCCGTAGGAGAAGAAAACAATTGTGATTCCGTTAGTAGTGGCGAGCGAACGTGGATGATGGCTAAAACTTATGTGTGTGATACCCGGCAGGGGTTGCATGTAGGTGGTTGTGGGGCAATGACGTTCATATTCTGCCGGATGTGGGCATGTGCTGCGTGGTTAGTGGAAGTGGTGTGGAAACACCTGCCGTAGAAGGTGAGAGTCCTGTACACGAAGATCATGGTGGTGTGTGTGGTTGTTGATACCCCGAGTAGCAGCGGGCTCGTGGAATCTGCTGTGAATTAGCCGGGACCACCCGGTAAGCCTGAATACTTGATATGACCGATAGCGGATTAGTACCGTGAGGGAATGGTGAAAAGTACCCCGGGAGGGGAGTGAAATAGTACCTGAAACCGTGTGCTGTCAATCCGTCAGAGCATCCTTTGTGGTGTGATGGCGTGCCTTTTGAAGAATGAGCCTGCGAGTCAGCGGCATGTCGCGAGGTTAACCCGTGTGGGGTAGCCGTAGGGAAACCGAATCCTAACGAGGGTGATTTTAGTGGCATGTCTTGGACCCGAAGCGGAGTGATCTACCCATGGCCAGTGTGAAGCAGCTGTAAGAGGTTGTGGAGGCGCGAACCCACTTAGGTTGAAAACTGAGGGGATGAGTTGTGGGTAGGGGTGAAAGGCCAATCAAACTCCGTGATAGCTGGTTCTCCCCGAAATGCATTTAGGTGCAGCGTCGTGTGTTTCTTGCCGGAGGTAGAGCTACTGGATGGTTTAGCGGGACCAACATCTTAGCGACATCAGCCAAACTCCGAATGCCGGTAAGTTAGAGCACGGCAGTGAGACTGCGGGGGATAAGCTTCGTAGTCGAGAGGGAAACAGCCCAGATCGCCGGCTAAGGCCCCTAAGGGTGTGCTAAGTGGAAAAGGAGGTGGGGTCGCGAAGACAGCCAGGAGGTTGGCTTAGAAGCAGCCATCCTTGAAAGAGTGCGTAATAGCTCACTGGTCGAGTGATTCCGCGCCGACAATGTAGTGGGGCTTAAGTACACCGCCGAAGCCGCTGACAATGATCTTTATAGGATTGTTGGGTAGGGGAGCGTCGTGCATGCGTTGAAGCTTTGGGGTGACCTTGGGTGGAGTGTGTGCGAGTGAGAATGCAGGCATGAGTAACGAATGATGCGTGAGAAACGTATCCGCCGGATGACTAAGGGTTCCTGGGTCAAGTTAATCTTCCCAGGGTGAGTCGGGGCCTAAGGCGAGGCCGACAGGCGTAGTCGATGGATAACGGGTTGATATTCCCGTACCCGAGTATGAGCGACCATGGTGAATCAGTGATACTAACCACCCATAAGCACCCGCGAAAAGGCTTTGCTTTTTTGTGGTGTGTGGTTGCGTGGGACCTGATCTGGTAGTAGCTAAGTGATGGGGTGACGCAGGGAGGTAGCTCAGCCACTTATTGGATTGTGGTGTAAGCGTGTGGCACGCAGTGTTGGTAAATCCGCACTGTTTTTGTGTGAGGCGTGATGCGGAGCCCGTAAAGGGTGAAGTGGGTGATCCTGTGCTGTCGAGAAAAGCCTCTAGCGATGTTGATATTCGGCCCGTACCCTAAACCGACACAGGTAGTCAGGTAGAGAATACTAAGGCGTTCGGGTGAACTGTGGTTAAGGAACTCGGCAAAATGCCCCCGTAACTTCGGGAGAAGGGGGGCCACGGCGTGTGAACAACTTTTCGTTGGGAGCGTGTTGTGGTCGCAGAGAATAGAGGGAAGCGACTGTTTACTAAAAACACAGGTCCGTGCGAAGACGTTTAAGTTGATGTATACGGACTGACGCCTGCCCGGTGCTGGAAGGTTAAGAGGACCGGTTAGGAAAACTTGTTTTTTCGAAGCTGAGAATTTAAGCCCCAGTAAACGGCGGTGGTAACTATAACCATCCTAAGGTAGCGAAATTCCTTGTCGGGTAAGTTCCGACCTGCACGAATGGCGTAACGACTTCCCTGCTGTCTCAACCACAGGCCCGGTGAAATTGCAGTACGAGTAAAGATGCTCGTTACGCGCGGCAGGACGAAAAGACCCCGGGACCTTCACTATAGCTTGGTATTGGTGTTTGATTCGGTTTGTGTAGGATAGGTGGGAGACTTCGATCATATGACGCTAGTTGTGTGTGAGTCGTTGGTGAAATACCACTCTGATCGGATTGGATGTCTTAACCTTGGCCCATGATCTGGGTTGGGGACAGTGCCTGGTGGGTAGTTTAACTGGGGCGGTTGCCTCCTAAAATGTAACGGAGGCGCCCAAAGGTTTCCTCAGCTTGGTTGGTAATCAGGTGGTGAGTGTAAGTGCACAAGGGAGCTTGACTGTGACACTGACAGGTGGAGCAGGGACGAAAGTCGGGACTAGTGATCCGGCACCTACTTGTGGTTGTGGTGTCGCTCAACGGATAAAAGGTACCCCGGGGATAACAGGCTGATCTTCCCCAAGAGTCCATATCGACGGGATGGTTTGGCACCTCGATGTCGGCTCGTCGCATCCTGGGGCTGGAGTAGGTCCCAAGGGTTGGGCTGTTCGCCCATTAAAGCGGCACGCGAGCTGGGTTTAGAACGTCGTGAGACAGTTCGGTCTCTATCCATCGCGCGCGTTGAAACTTGAAGGAAGGCTGTCCCTAGTACGAGAGGACCGGGACGGACGTACCTCTGGTGTGCCAGTTGTTCCGCCAGGAGCAGGGCTGGTTGGCTACGTACGGGAGGGATAACCGCTGAAAGCATCTAAGCGGGAAGCCTGTTTCGAGATGAGGTTTCTTTTGAGGTTCCCTAGAGATTATGGGGTTGATAGGCCAGATCTGGAAGCACTGTGAGGTGTGGAGGTGACTGGTACTAATTTACCGATAACAACAAACC 114 TGTGGAGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGCTGAAACCGGAGCTTGCTTTGGTGGATGAGTGGCGAACGGGTGAGTAACACGTGGGTGATCTGCCCTACACTTTGGGATAAGCCTGGGAAACTGGGTCTAATACCGAATATTCACACCACCGTAGGGGTGGTGTGGAAAGCTTTATGCGGTGTGGGATGAGCCTGCGGCCTATCAGCTTGTTGGTGGGGTAATGGCCTACCAAGGCGTCGACGGGTAGCCGGCCTGAGAGGGTGTACGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACAGCCGCGTGGGGGATGAAGGCCTTCGGGTTGTAAACTCCTTTCGCTAGGGACGAAGCCTTATGGTGACGGTACCTGGAGAAGAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTGTCCGGAATTACTGGGCGTAAAGAGCTCGTAGGTGGTTTGTCGCGTCGTCTGTGAAATCCCGGGGCTTAACTTCGGGCGTGCAGGCGATACGGGCATAACTTGAGTGCTGTAGGGGAGACTGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCAATGGCGAAGGCAGGTCTCTGGGCAGTAACTGACGCTGAGGAGCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGGTGGGCGCTAGGTGTAGGGGTCTTCCACGACTTCTGTGCCGCAGCTAACGCATTAAGCGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGCTTGACATGGACCGGATCGGCGTAGAGATACGTTTTCCCTTGTGGTCGGTTCACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCTTATGTTGCCAGCACATTATGGTGGGTACTCATGAGAGACTGCCGGGGTTAACTCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGTCCAGGGCTTCACACATGCTACAATGGTCGGTACAGCGAGTTGCCACACCGTGAGGTGGAGCTAATCTCTTAAAGCCGGCCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCATGAAAGTTGGTAACACCCGAAGCCAGTGGCCCAACCTTTTAGGGGGGAGCTGTCGAAGGTGGGATCGGCGATTGGGACGAAGTCGTAACAAGGTAGCCGTACCGGAAGGTGCGGCTGGATCACCTCCTTTCT 115 GCCCGGGTGGCGGAATTGGCAGACGCGCTGGCTTCAGGGTGCCAGTGTTCGCAAGGACGTGGGGTTCAAGTCCCCCCCCCGGGCACCA

[0236] In the following examples, the effect of each variant listed in Tables 6 to 9 on the L-threonine production capacity of microorganisms of the genus Corynebacterium was evaluated to identify the effective factors affecting L-threonine production capacity.

[0238] Example 3: Production of an L-threonine-producing strain with introduced variant gene and promoter

[0239] Example 3-1: Construction of a recombinant vector for the introduction of a variant gene and promoter

[0240] To confirm the effect of the variant identified in Example 2 above, a vector capable of introducing it onto a chromosome was constructed.

[0241] Specifically, NCgl0658, NCgl0865, NCgl1063, NCgl1165, NCgl1511, NCgl1523, NCgl2139, NCgl1999, NCgl2133, NCgl0239, NCgl0337, NCgl0472, NCgl0674, NCgl0706, NCgl0948, NCgl1120, NCgl1142, NCgl1253, NCgl1533, NCgl1859, NCgl1874, NCgl2308, NCgl2859, NCgl2863, NCgl2928, NCgl0569, NCgl1320, NCgl1588, NCgl1760, NCgl0686, Vectors containing target variants were constructed to insert the promoters of the NCgl2707, NCgl2851, NCgl2320, NCg1r05, NCg1r09, NCg1r13, NCg1r15, NCg1r16, NCg1t06 gene variants and the variants of the NCgl0247, NCgl2528, NCgl1353, NCgl1501, NCgl1836, NCgl1969, NCgl2466, NCgl2960 genes into CJTHR-3.

[0242] Specifically, the genomic DNA of the above CJTHR-3_mt1 strain was extracted using the G-spin Total DNA Extraction Mini Kit (Intron, Cat. No. 17045) according to the protocol provided in the kit, and PCR was performed using the genomic DNA as a template. Solg™ Pfu-X DNA polymerase was used as the polymerase, and the PCR conditions were as follows: denaturation at 95°C for 4 minutes; 27 cycles of denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and polymerization at 72°C for 1 minute; and polymerization at 72°C for 5 minutes, after which the polymerization reaction was performed to obtain the respective PCR products. The primer sequences used in the above experiment are as shown in Table 10 below.

[0243] Recombinant plasmids were obtained by cloning the above-mentioned mutation-introduced fragments and pDC24 treated with restriction enzyme smaI using the Gibson assembly method, and vectors containing each mutation-introduced fragment PDC24-NCgl0658*, PDC24-NCgl0865*, PDC24-NCgl1063*, PDC24-NCgl1165*, PDC24-NCgl1511*, PDC24-NCgl1523*, PDC24-NCgl2139*, PDC24-NCgl1999*, PDC24-NCgl2133*, PDC24-NCgl0239*, PDC24-NCgl0337*, PDC24-NCgl0472*, PDC24-NCgl0674*, PDC24-NCgl0706*, PDC24-NCgl0948*, PDC24-NCgl1120*, PDC24-NCgl1142*, PDC24-NCgl1253*, PDC24-NCgl1533*, PDC24-NCgl1859*, PDC24-NCgl1874*, PDC24-NCgl2308*, PDC24-NCgl2859*, PDC24-NCgl2863*, PDC24-NCgl2928*, PDC24-NCgl0569*, PDC24-NCgl1320*, PDC24-NCgl1588*, PDC24-NCgl1760*, PDC24-NCgl0686*, PDC24-NCgl2707*, PDC24-NCgl2851*, PDC24-NCgl2320*, PDC24-PNCgl0247*, PDC24-PNCgl2528*, PDC24-PNCgl1353*, PDC24-PNCgl1501*, PDC24-PNCgl1836*, PDC24-PNCgl1969*, PDC24-PNCgl2466*, PDC24-PNCgl2960*, PDC24-NCg1r05*, PDC24-NCg1r09*, PDC24-NCg1r13*, PDC24-NCg1r15*, PDC24-NCg1r16*, and It was named PDC24-NCg1t06*.

[0244] Sequence number designation Sequence (5'->3') 116 NCgl0658*_F tgaattcgagctcggtacccGATCGCACTCAACGGTTTCT 117 NCgl0865*_F tgaattcgagctcggtacccAATCGGCGCCTCGGTCATCG 118 NCgl1063*_F tgaattcgagctcggtacccCCTCGAGGCCAATCCCCTGT 119 NCgl1165*_F tgaattcgagctcggtacccACCTCATTCGCCCAGACGGC 120 NCgl1511*_F tgaattcgagctcggtacccAATAGGCGATAGCATGGCAA 121 NCgl1523*_F tgaattcgagctcggtacccTCTGCGCCAAAACTCCGAAA 122 NCgl2139*_F tgaattcgagctcggtacccTGATATTTTGCTGGGCGGTC 123 NCgl1999*_F tgaattcgagctcggtacccAACTCCCTAACCGGCCTGCC 124 NCgl2133*_F tgaattcgagctcggtacccCGAAGCCTGGAACCAGACGG 125 NCgl0239*_F tgaattcgagctcggtacccTCCGTTGTGATCAGCAGCGT 126 NCgl0337*_F tgaattcgagctcggtacccGCTATTGCTCATTGTTGGCC 127 NCgl0472*_F tgaattcgagctcggtacccAGAAGCATTTCTGCCTCAAG 128 NCgl0674*_F tgaattcgagctcggtacccCCAAGGATGACTAAGCAAGC 129 NCgl0706*_F tgaattcgagctcggtacccCGTGGCTACTTCTGCGATCC 130 NCgl0948*_F tgaattcgagctcggtacccGTGTGATTCCGGCAAACAGG 131 NCgl1120*_F tgaattcgagctcggtacccGTCGAGGGCAACTGTGGTTC 132 NCgl1142*_F tgaattcgagctcggtacccTGGCACTGTCGCGGACCGTT 133 NCgl1253*_F tgaattcgagctcggtacccTCAATCGAGGAAGAGGTGTC 134 NCgl1533*_F tgaattcgagctcggtacccCCAGCTCAGAGAACTGGGAT 135 NCgl1859*_F tgaattcgagctcggtacccCAGACTTGTATCGGATTGAA 136 NCgl1874*_F tgaattcgagctcggtacccTTCGGGCCTGCTCGAGGAGG 137 NCgl2308*_F tgaattcgagctcggtacccATCGCGTTGATCACGGTGGT 138 NCgl2859*_F tgaattcgagctcggtacccTGCAGGGCGCGTACCGCCGC 139 NCgl2863*_F tgaattcgagctcggtacccGGGCGGCGCAGCACCGCATG 140 NCgl2928*_F tgaattcgagctcggtacccGATGCGTTCGCCGTGGCCGG 141 NCgl0569*_F tgaattcgagctcggtacccATGATCTGCCCGCCCAAAGA 142 NCgl1320*_F tgaattcgagctcggtacccCACCCTTGGAGCCGAGCGTC 143 NCgl1588*_F tgaattcgagctcggtacccATTCACAAAGGATTAGTCGC 144 NCgl1760*_F tgaattcgagctcggtacccCAATTGAGAATGCGGAGTTG 145 NCgl0686*_F tgaattcgagctcggtacccAGACCACTGGAACGCTGACT 146 NCgl2707*_F tgaattcgagctcggtacccGAGGATGCGGGTGTTGCATT 147 NCgl2851*_F tgaattcgagctcggtacccAATGCTTAAACACCCCACCG 148 NCgl2320*_F tgaattcgagctcggtacccTCAACCGCAACATCTTCACT 149 PNCgl0247*_F tgaattcgagctcggtacccCCCACGGCTACGGTGAGGAG 150 PNCgl2528*_F tgaattcgagctcggtacccGTAGAGACCAGTGCAACGTT 151 PNCgl1353*_F tgaattcgagctcggtacccGTGTCACCTTGATGTGCTTT 152 PNCgl1501*_F tgaattcgagctcggtacccTCATCCGCTCAAACGCCCAC 153 PNCgl1836*_F tgaattcgagctcggtacccTCTTTTTAGCGACCGTCTTT 154 PNCgl1969*_F tgaattcgagctcggtacccGCCCGCCGTTCTGGCGGAAA 155 PNCgl2466*_F tgaattcgagctcggtacccGGATGTATTTCGCGCCGAAG 156 PNCgl2960*_F tgaattcgagctcggtacccGTCCTTCATTGCCGGGGAAG 157 NCg1r05*_F tgaattcgagctcggtacccTGGTTTGTTGTTATCGGTAA 158 NCg1r09*_F tgaattcgagctcggtacccAAATTAGTACCAGTCACCTC 159 NCg1r13*_F tgaattcgagctcggtacccACTGTCACCTTTGTGTGTGG 160 NCg1r15*_F tgaattcgagctcggtacccGCTACTGGATGGTTTAGCGG 161 NCg1r16*_F tgaattcgagctcggtacccACTGTCACCTTTGTGTGTGG 162 NCg1t06*_F tgaattcgagctcggtacccGTTCCATAAGTGTCTTCTCC 163 NCgl0658*_R gtcgactctagaggatccccACGGACCCAAGCAAGACCTC 164 NCgl0865*_R gtcgactctagaggatccccGCGCTTGGGCAGCTGGTTGG 165 NCgl1063*_R gtcgactctagaggatccccACCGCAGAGCACCACGCACC 166 NCgl1165*_R gtcgactctagaggatccccTCCATTGCTTCCAAGGGTAT 167 NCgl1511*_R gtcgactctagaggatccccCAGCAGCCACAGCCACAAGA 168 NCgl1523*_R gtcgactctagaggatccccGATGCGCACGGAACCTTGGA 169 NCgl2139*_R gtcgactctagaggatccccGCTGGTTGTGGTGCGGATCC 170 NCgl1999*_R gtcgactctagaggatccccAAGGCATGTTCGCGCCTTCA 171 NCgl2133*_R gtcgactctagaggatccccCGCGAGGCATTCTCCCGCGA 172 NCgl0239*_R gtcgactctagaggatccccGGTCCTCCAAGCCGTCCCAG 173 NCgl0337*_R gtcgactctagaggatccccTTTCGTGCGTTACGTACTAA 174 NCgl0472*_R gtcgactctagaggatccccCAGTCCTTGTGCCTGAACGT 175 NCgl0674*_R gtcgactctagaggatccccTGCTGCTGTCTGGCTTATTTC 176 NCgl0706*_R gtcgacttagaggatccccGTGCGGCCCCTGCAAAAATGG 177 NCgl0948*_R gtcgactctagaggatccccCCGGGCGAGGCGAATTGTAT 178 NCgl1120*_R gtcgactctagaggatccccAGTAGCAAAGCTCTCCTCCC 179 NCgl1142*_R gtcgactctagaggatccccAGCACGAGTGTTGTCATACC 180 NCgl1253*_R gtcgactctagaggatccccCAATGGCAGAAGTGATGTGG 181 NCgl1533*_R gtcgactctagaggatccccGGGTGAAATCCGTGCGGTCG 182 NCgl1859*_R gtcgactctagaggatccccCACTTTTAATAACTTCATAC 183 NCgl1874*_R gtcgactctagaggatccccCATGGCGTCAATGACATCAG 184 NCgl2308*_R gtcgactctagaggatccccTCACACCACGTGTTTTGGAG 185 NCgl2859*_R gtcgactctagaggatccccCGATCGAGCCGCAGCCTTCC 186 NCgl2863*_R gtcgactctagaggatccccGAAATCAAAAACCCTGCTCA 187 NCgl2928*_R gtcgactctagaggatccccGTATTCACCCACGGTCAGCG 188 NCgl0569*_R gtcgactctagaggatccccAAGCTGCGGAAAGAAACGGT 189 NCgl1320*_R gtcgactctagaggatccccTTGTAGCATTGGTTGCACGC 190 NCgl1588*_R gtcgactctagaggatccccCTTAAATAACATCTTGCTTG 191 NCgl1760*_R gtcgactctagaggatccccTGGACTGGTCGATCATTATT 192 NCgl0686*_R gtcgacttagaggatccccGCCCACGCATTATTCCGGA 193 NCgl2707*_R gtcgacttagaggatccccCTGCGTCCCTATTCCGCGG 194 NCgl2851*_R gtcgactctagaggatccccACCTTGTGCACACCCTAGAC 195 NCgl2320*_R gtcgactctagaggatccccTATCCGCCACCCTGCTTGCC 196 PNCgl0247*_R gtcgactctagaggatccccGTGACATCAACAATGCGTGC 197 PNCgl2528*_R gtcgactctagaggatccccGACATGCCCACGCCACGGCG 198 PNCgl1353*_R gtcgactctagaggatccccAGACCGCTGGTAAGCATCGGA 199 PNCgl1501*_R gtcgactctagaggatccccGCACGCTCATCAACTTCCAT 200 PNCgl1836*_R gtcgactctagaggatccccAAACTTTTCTCCCCATCCGT 201 PNCgl1969*_R gtcgactctagaggatccccGGATGGGGTAGCCGACATTT 202 PNCgl2466*_R gtcgactctagaggatccccCACCATAAGAGCATCAACAT 203 PNCgl2960*_R gtcgactctagaggatccccCCAAAGCGTTTGCGCTTGGA 204 NCg1r05*_R gtcgactctagaggatccccAACATCGAGCATCGTCAACA 205 NCg1r09*_R gtcgactctagaggatccccAGCTACTGGATGGTTTAGCG 206 NCg1r13*_R gtcgactctagaggatccccCACCCCCACCACAACCAAAG 207 NCg1r15*_R gtcgactctagaggatccccTTTTCGTTACGTTGCGTTAC 208 NCg1r16*_R gtcgactctagaggatccccTCCTCCTGATATCTGCGCAT 209 NCg1t06*_R gtcgactctagaggatccccCTGGAGTTACTTCCTTATGC

[0246] Example 3-2: Production of an L-threonine-producing strain with introduced variant gene and promoter

[0247] The 47 vectors produced in Example 3-1 were transformed into the threonine-producing strain CJTHR-3 by electroporation, and strains in which the vector was inserted onto the chromosome by recombination of homologous sequences were selected using kanamycin medium. Subsequently, strains into which the variant gene was introduced were identified by PCR using the primer pairs in Table 11 on the transformed strains in which secondary recombination was completed. PCR was performed in the same manner as in Example 3-1.

[0248] Sequence number designation Sequence (5'->3') 210 NCgl0658_seq_F ACGATAAGGTGGGTGGGGAG 211 NCgl0865_seq_F AGGTGCACCTCATCAACGAC 212 NCgl1063_seq_F AATACACCGCCAACCCAGAT 213 NCgl1165_seq_F CAGCTAAACGGAGGTTTAGA 214 NCgl1511_seq_F CGAACGGAACGAATCGGACC 215 NCgl1523_seq_F GATGCGTTGACCATTGATCA 216 NCgl2139_seq_F CACCTTTTTCAACATATTTG 217 NCgl1999_seq_F AACCGTGGTTTCCGCGTGCA 218 NCgl2133_seq_F TGCCTGATGGGTCTGGAGCG 219 NCgl0239_seq_F CCACGTGCTCTTTTGCACAG 220 NCgl0337_seq_F CGCCTGAACACCGGCGGAAT 221 NCgl0472_seq_F ACCTTGCGGCGAGCGTCGGC 222 NCgl0674_seq_F TGGTTGAACAAAGCGGTGAA 223 NCgl0706_seq_F ACATGATCCGTGGTGGGCTG 224 NCgl0948_seq_F AGATGATTGCGTTGGTGTCG 225 NCgl1120_seq_F TTGATCAAGCAGCTCCGCCA 226 NCgl1142_seq_F CCCAGGTAAATTCCTCACTG 227 NCgl1253_seq_F GGCGCGCGGTCGGGCCATTA 228 NCgl1533_seq_F TGGGCGCATTATCCACCGGT 229 NCgl1859_seq_F GTGCTTCAAAAGTTGAAGTT 230 NCgl1874_seq_F CGCAAAGGTAAATCAACATC 231 NCgl2308_seq_F TGGCCAAGCTTGGAATCTAG 232 NCgl2859_seq_F GTGTCCTTGTCCTGCGGCAG 233 NCgl2863_seq_F TCCGTGCGGGTGAGCTCCAC 234 NCgl2928_seq_F GCACAAGGCGTATGCCGTGT 235 NCgl0569_seq_F CCTGAAGCGGAAAAAGACTC 236 NCgl1320_seq_F CGGTTTGGGCAAAACCAGCA 237 NCgl1588_seq_F TGCACTTTCACCATTGTGCA 238 NCgl1760_seq_F GTTATCCTAAAGTCCGCAAT 239 NCgl0686_seq_F TGCCAAAGGCTCAGAATCTT 240 NCgl2707_seq_F TTACTCAAAGGTCCCATGGT 241 NCgl2851_seq_F TTCAATATAGTGCGCACATT 242 NCgl2320_seq_F ACCAACTTCTTTTCACTCTC 243 PNCgl0247_seq_F TATTCCGCAAGCAGCCACAT 244 PNCgl2528_seq_F CGCAAAGCATCGGAGTGGCC 245 PNCgl1353_seq_F GTTCTCAAACAACATACGCA 246 PNCgl1501_seq_F GCCTGCTGGTTGACCACGCT 247 PNCgl1836_seq_F CAACAGGGGTTGAGGTTCCA 248 PNCgl1969_seq_F GGGGCGTTGCCTCAGGGGAC 249 PNCgl2466_seq_F TCGTGGAGGAGGGTGATGCC 250 PNCgl2960_seq_F TGATGAGGTTCCTTCTGGAA 251 NCg1r05_seq_F CCACCGACACAACCACACAC 252 NCg1r09_seq_F CACACCAACCAACAGGTTGG 253 NCg1r13_seq_F TCTGCTTGTTCCCCGTCAAG 254 NCg1r15_seq_F GGTTGAAAACTGAGGGGATG 255 NCg1r16_seq_F TCTGCTTGTTCCCCGTCAAG 256 NCg1t06_seq_F AACGCATTATCAAGGCCGCG 257 NCgl0658_seq_R ACGCGACATGGACACACAAT 258 NCgl0865_seq_R TCACCAGTGTGCTCGGGCTC 259 NCgl1063_seq_R AGCAAGGTATGCACCTAGGC 260 NCgl1165_seq_R TCCACCAAGGGCCGTTCGAT 261 NCgl1511_seq_R ACCTGCGGCTCCGCCCCACA 262 NCgl1523_seq_R GGTGAGTTCGGAGACGTCGA 263 NCgl2139_seq_R TCGTTTTCGTTGGTGGCCAC 264 NCgl1999_seq_R CGCGCGAAGCGTTCTGCTGC 265 NCgl2133_seq_R TTCACCACGATCGACGAATC 266 NCgl0239_seq_R CATGCCCTTGCATCTAAAGA 267 NCgl0337_seq_R TTGCGCGATATTGAAGAGGT 268 NCgl0472_seq_R CTGACCATCAAGTCTGATGA 269 NCgl0674_seq_R AGAAAGTTCCACGCTCCGGC 270 NCgl0706_seq_R CTGGTACGCCGTTGCAGGTA 271 NCgl0948_seq_R ACACTGTTTGTGCATCGCGG 272 NCgl1120_seq_R GGCTGTAGAAAAGGAATACC 273 NCgl1142_seq_R ATTGCGGTTGAATTGCGGGA 274 NCgl1253_seq_R AGCGAGCTTGTAGGTGATTA 275 NCgl1533_seq_R AGATAACACCTGCTGGGCGC 276 NCgl1859_seq_R TGATATTCCACCAGCTTATC 277 NCgl1874_seq_R CCTCATCCAAGATGGAGAGG 278 NCgl2308_seq_R CATCGCAAACACTGTCCGAA 279 NCgl2859_seq_R GGCCACCGACAACACCGTCC 280 NCgl2863_seq_R GCGGCGGCTGCGATCGCTGC 281 NCgl2928_seq_R GCCAGTAATCGGGAACGGAC 282 NCgl0569_seq_R AATTGGCGCGCCGGGGTAAT 283 NCgl1320_seq_R GTGTACGCGTACTCGGTGAC 284 NCgl1588_seq_R TACTTTTAAAAAGGAGCCTG 285 NCgl1760_seq_R CATCAGTTATGGTATAGCTT 286 NCgl0686_seq_R CCCGTTGTGGTCAGCGGTTT 287 NCgl2707_seq_R TTGGAATAAGTCAATCCAAC 288 NCgl2851_seq_R CCCTCGACGATCTGCGGGAG 289 NCgl2320_seq_R GAACTCGCCCTTTTCTTCGCC 290 PNCgl0247_seq_R GTGGTGTCAGAACCACCACG 291 PNCgl2528_seq_R CCAAAACTCAACGGGATTTT 292 PNCgl1353_seq_R ACTTCAGCAGGCTGTCCCTC 293 PNCgl1501_seq_R CACTCGCGAAGCTTAGGAGC 294 PNCgl1836_seq_R CACATGATCGTTGACGGCGA 295 PNCgl1969_seq_R AAGAGGCAGTCATTTGCAAC 296 PNCgl2466_seq_R TGATGGATAAAATTGGCGAT 297 PNCgl2960_seq_R CTGGGATGGAATCGGAAGTT 298 NCg1r05_seq_R ATGACCCCTCGGGTGACAAC 299 NCg1r09_seq_R AGGTTGAAAACTGAGGGGAT 300 NCg1r13_seq_R CAAAACAACAAACAAAATAC 301 NCg1r15_seq_R GACCGGGCGTTTCCCTGCTA 302 NCg1r16_seq_R CAAAACAACAAACAAAATAC 303 NCg1t06_seq_R GGGTCATTCCATCGAATCCA

[0249] 상기 재조합 균주를 각각 CJTHR-3△NCgl0658::NCgl0658*, CJTHR-3△NCgl0865::NCgl0865*, CJTHR-3△NCgl1063::NCgl1063*, CJTHR-3△NCgl1165::NCgl1165*, CJTHR-3△NCgl1511::NCgl1511*, CJTHR-3△NCgl1523::NCgl1523*, CJTHR-3△NCgl2139::NCgl2139*, CJTHR-3△NCgl1999::NCgl1999*, CJTHR-3△NCgl2133::NCgl2133*, CJTHR-3△NCgl0239::NCgl0239*, CJTHR-3△NCgl0337::NCgl0337*, CJTHR-3△NCgl0472::NCgl0472*, CJTHR-3△NCgl0674::NCgl0674*, CJTHR-3△NCgl0706::NCgl0706*, CJTHR-3△NCgl0948::NCgl0948*, CJTHR-3△NCgl1120::NCgl1120*, CJTHR-3△NCgl1142::NCgl1142*, CJTHR-3△NCgl1253::NCgl1253*, CJTHR-3△NCgl1533::NCgl1533*, CJTHR-3△NCgl1859::NCgl1859*, CJTHR-3△NCgl1874::NCgl1874*, CJTHR-3△NCgl2308::NCgl2308*, CJTHR-3△NCgl2859::NCgl2859*, CJTHR-3△NCgl2863::NCgl2863*, CJTHR-3△NCgl2928::NCgl2928*, CJTHR-3△NCgl0569::NCgl0569*, CJTHR-3△NCgl1320::NCgl1320*, CJTHR-3△NCgl1588::NCgl1588*, CJTHR-3△NCgl1760::NCgl1760*, CJTHR-3△NCgl0686::NCgl0686*, CJTHR-3△NCgl2707::NCgl2707*, CJTHR-3△NCgl2851::NCgl2851*, CJTHR-3△NCgl2320::NCgl2320*, CJTHR-3△Pn::PNCgl0247*,CJTHR-3△Pn::PNCgl2528*, CJTHR-3△Pn::PNCgl1353*, CJTHR-3△Pn::PNCgl1501*, CJTHR-3△Pn::PNCgl1836*, CJTHR-3△Pn::PNCgl1969*, CJTHR-3△Pn::PNCgl2466*, CJTHR-3△Pn::PNCgl2960*, CJTHR-3△NCg1r05::NCg1r05*, CJTHR-3△NCg1r09::NCg1r09*, CJTHR-3△NCg1r13::NCg1r13*, CJTHR-3△NCg1r15::NCg1r15*, It was named CJTHR-3△NCg1r16::NCg1r16* and CJTHR-3△NCg1t06::NCg1t06*.

[0250] Example 4. Evaluation of Threonine Production Capacity of L-Threonine Producing Strains Introduced with Variant Genes and Promoters

[0251] To confirm the L-threonine production capacity of the strain prepared in Example 3-2 above at the flask level, it was evaluated by culturing in the following manner. The parent strain and the mutant strain were inoculated into a 250 mL Corner-Barpool flask containing 25 mL of threonine production medium, and then cultured at 30°C for 48 hours with shaking at 200 rpm. After the culture was completed, the L-threonine concentration in the culture medium was analyzed using HPLC, and the L-threonine production concentrations of each mutant strain are shown in Table 12 below.

[0252] Production Medium (pH 7.2)

[0253] Glucose 10%, Yeast extract 0.2%, Ammonium sulfate 1.6%, Potassium dihydrogen phosphate 0.1%, Magnesium sulfate heptahydrate 0.1%, Iron heptahydrate 10 mg / ℓ, Manganese sulfate monohydrate 10 mg / ℓ, Biotin 200 μg / ℓ (based on 1 L of distilled water)

[0254] strain name L-Threonine Concentration (g / L) CJTHR-3 2.0 CJTHR-3△NCgl0658::NCgl0658* 2.4 CJTHR-3△NCgl0865::NCgl0865* 2.3 CJTHR-3△NCgl1063::NCgl1063* 2.3 CJTHR-3△NCgl1165::NCgl1165* 2.4 CJTHR-3△NCgl1511::NCgl1511* 2.2 CJTHR-3△NCgl1523::NCgl1523* 2.4 CJTHR-3△NCgl2139::NCgl2139* 2.4 CJTHR-3△NCgl1999::NCgl1999* 2.8 CJTHR-3△NCgl2133::NCgl2133* 2.3 CJTHR-3△NCgl0239::NCgl0239* 2.2 CJTHR-3△NCgl0337::NCgl0337* 2.3 CJTHR-3△NCgl0472::NCgl0472* 2.7 CJTHR-3△NCgl0674::NCgl0674* 2.2 CJTHR-3△NCgl0706::NCgl0706* 2.2 CJTHR-3△NCgl0948::NCgl0948* 2.2 CJTHR-3△NCgl1120::NCgl1120* 2.3 CJTHR-3△NCgl1142::NCgl1142* 2.2 CJTHR-3△NCgl1253::NCgl1253* 2.1 CJTHR-3△NCgl1533::NCgl1533* 2.3 CJTHR-3△NCgl1859::NCgl1859* 2.4 CJTHR-3△NCgl1874::NCgl1874* 2.1 CJTHR-3△NCgl2308::NCgl2308* 2.3 CJTHR-3△NCgl2859::NCgl2859* 2.1 CJTHR-3△NCgl2863::NCgl2863* 2.3 CJTHR-3△NCgl2928::NCgl2928* 2.2 CJTHR-3△NCgl0569::NCgl0569* 2.2 CJTHR-3△NCgl1320::NCgl1320* 2.1 CJTHR-3△NCgl1588::NCgl1588* 2.3 CJTHR-3△NCgl1760::NCgl1760* 2.1 CJTHR-3△NCgl0686::NCgl0686* 2.2 CJTHR-3△NCgl2707::NCgl2707* 2.1 CJTHR-3△NCgl2851::NCgl2851* 2.3 CJTHR-3△NCgl2320::NCgl2320* 2.1 CJTHR-3△Pn::PNCgl0247* 2.5 CJTHR-3△Pn::PNCgl2528* 2.5 CJTHR-3△Pn::PNCgl1353* 2.1 CJTHR-3△Pn::PNCgl1501* 2.0 CJTHR-3△Pn::PNCgl1836* 2.0 CJTHR-3△Pn::PNCgl1969* 2.1 CJTHR-3△Pn::PNCgl2466* 2.0 CJTHR-3△Pn::PNCgl2960* 2.2 CJTHR-3△NCg1r05::NCg1r05* 2.1 CJTHR-3△NCg1r09::NCg1r09* 2.0 CJTHR-3△NCg1r13::NCg1r13* 2.1 CJTHR-3△NCg1r15::NCg1r15* 2.0 CJTHR-3△NCg1r16::NCg1r16* 2.0 CJTHR-3△NCg1t06::NCg1t06* 2.1

[0255] As a result, as shown in Table 7 above, it was confirmed that most of the mutant strains had threonine production capabilities equivalent to or superior to those of the parent strain. In particular, it was confirmed that CJTHR-3△NCgl1999::Ncgl1999* and CJTHR-3△NCgl0472::NCgl0472* showed a significant increase in threonine production capacity compared to the parent strain CJTHR-3. Through this, it was confirmed that L-threonine can be produced more efficiently by introducing a protein variant in which the 233rd amino acid of the protein (Glutamate dehydrogenase) coded by the NCgl1999 gene is substituted from glycine (Gly, G) to aspartic acid (Asp, D), or a protein variant in which the 41st amino acid of the protein (DNA-directed RNA polymerase subunit beta) coded by the NCgl0472 gene is substituted from proline (Pro, P) to serine (Ser, S).

Claims

Claim 1 A protein variant comprising the amino acid sequence of SEQ ID NO.

53. Claim 2 A polynucleotide encoding the protein variant of claim 1. Claim 3 In paragraph 2, a polynucleotide comprising the nucleotide sequence of SEQ ID NO.

86. Claim 4 A genus of Corynebacterium comprising one or more selected from the group consisting of the protein variant of claim 1 and the polynucleotide encoding said protein variant. Corynebacterium ) Microorganisms. Claim 5 In paragraph 4, the microorganism of the genus Corynebacterium is a microorganism that is Corynebacterium glutamicum. Claim 6 In paragraph 4, the microorganism is a microorganism having increased L-threonine production capacity compared to a microorganism of the genus Corynebacterium that does not include one or more selected from the group consisting of the protein variant and the polynucleotide encoding the protein variant. Claim 7 A method for producing L-threonine, comprising the step of culturing a microorganism of any one of claims 4 to 6 in a culture medium. Claim 8 A method for producing L-threonine according to claim 7, wherein the method further comprises the step of recovering L-threonine from a cultured medium or cultured microorganism.