Disclosure of Invention
The glycosyltransferase mutants with different regioselectivity can effectively catalyze flavonoid glycosylation reaction, and have better regioselectivity compared with wild type glycosyltransferase mutant enzymes.
The invention provides the following technical scheme:
the present invention provides a glycosyltransferase mutant having an amino acid sequence that has the mutation site in the amino acid sequence shown in SEQ ID NO. 1 and has 80% or more homology, preferably 90% or more, 95% or more, or 98% or more homology with the mutated amino acid sequence.
The amino acid sequence is shown as SEQ ID NO. 1, and at least one amino acid residue in the amino acid positions G11, P12, F88, M119, Y145, T149, L200, V190 and Y392 is mutated. In one embodiment of the present invention, mutation is a substitution of an original amino acid in the sequence with 1 or more other amino acid residues.
The other amino acid residue is one of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.
The glycosyltransferase mutant comprises a substitution corresponding to SEQ ID NO 1 at the following positions: any one or a combination of more than two of G11Y, Y145F, P12Y, P12G, F88S, M119L, M196L, M119W, L200W, L200F, L200Y, L200M, Y145W, Y392T, V190I and V190W. Preferred combinatorial mutations include: V190I/M119Q, V190I/L200M, V190I/Y392S, G11Y/Y145F, P12Y/L200W, Y392T/L43A, Y392T/F88M, Y392T/T149V, Y392T/M196N, Y392T/L200M and Y392T/K287Y.
In one embodiment, the glycosyltransferase mutant comprises mutations corresponding to 11 th glycine and 145 th tyrosine of SEQ ID NO. 1, such as mutation of glycine G at the 11 th position into tyrosine Y and mutation of tyrosine Y at the 145 th position into phenylalanine F, the mutant can simultaneously carry out glycosylation modification on C3 hydroxyl and C7 hydroxyl of silybin to form a double glycosylation product, and the proportion of the double glycosylation product can reach more than 99%.
In one embodiment, the glycosyltransferase mutant comprises at least a mutation of proline P at position 12 to tyrosine, leucine L at position 200 to phenylalanine F or tryptophan W or tyrosine Y at position 145 to tryptophan W corresponding to SEQ ID No. 1; preferably, the glycosyltransferase mutant comprises mutations at the following positions: the mutant can selectively glycosylate the hydroxyl at C7 position of silybin, and the selectivity of the mutant can reach more than 99%.
In one embodiment, the glycosyltransferase mutant also includes a mutation corresponding to tyrosine 392 or valine V190 of SEQ ID NO. 1, e.g., a mutation of tyrosine Y at 392 to threonine T, or valine V190 to isoleucine I. Preferably, the glycosyltransferase mutant comprises a mutation at: Y392T and T149V, the mutant can selectively glycosylate the C3 hydroxyl of the silybin, and the selection performance reaches more than 94 percent.
The invention also provides a nucleic acid sequence for encoding the glycosyltransferase mutant, wherein the nucleic acid sequence can be obtained according to the amino acid sequence of the glycosyltransferase mutant.
The invention also relates to an expression cassette comprising said nucleic acid sequence, an expression vector comprising said nucleic acid or expression cassette. Further, the present invention also provides a host cell comprising said nucleic acid sequence, expression cassette or said vector, e.g. an original host cell transformed or transfected with said nucleic acid sequence, expression cassette or vector to form a transformant. The expression cassette of the present invention may optionally further comprise an enhancer or other necessary elements.
In a specific embodiment, the expression cassette comprises all elements for expressing the variant, including elements necessary for transcription and translation in a host cell, for example, the expression cassette comprises a promoter and a terminator, which are not particularly limited and may be promoters and terminators known in the art to enable expression of the variant.
The invention also provides application of the glycosyltransferase mutant, wherein the mutant is used for catalyzing glycosyl acceptor, namely catalyzing glycosyl transferred from glycosyl donor to glycosyl acceptor to carry out glycosylation reaction, such as glycosylation reaction of flavonoids. Preferably, the glycosyl acceptor comprises a flavonoid compound, a nitrogen or sulfur atom containing compound.
The glycosyl receptor is selected from flavonoid compounds and compounds containing nitrogen and sulfur atoms; preferably, the mutant catalyzes any one or more of the following glycosylation reactions:
(1) specifically catalyzing the glycosylation reaction of C3 and C7 hydroxyl groups for generating flavonoid compounds such as silybin;
(2) specifically catalyzing the glycosylation reaction of C3 hydroxyl of flavonoid compounds such as silybin;
(3) specifically catalyzing the glycosylation reaction of C7 hydroxyl of flavonoid compounds such as silybin;
(4) Catalyzing the glycosylation reaction of the compound containing nitrogen and sulfur atoms.
The glycosyl receptor is selected from any one of silybin, kaempferol, chrysin, hesperetin, daidzein, calycosin and biochanin; nitrogen and sulfur atom-containing compounds such as any one of 4-aminobenzophenone, 3, 4-dichloroaniline, 3, 4-dichlorothiophenol and 2, 6-dichlorothiophenol; preferably, the glycosyl donor is selected from the group consisting of UDP-glucose, ADP-glucose, TDP-glucose, CDP-glucose, GDP-glucose, UDP-galacturonic acid, ADP-galacturonic acid, TDP-galacturonic acid, CDP-galacturonic acid, GDP-galacturonic acid, UDP-galactose, ADP-galactose, TDP-galactose, CDP-galactose, GDP-galactose, UDP-arabinose, ADP-arabinose, TDP-arabinose, CDP-arabinose, GDP-arabinose, UDP-rhamnose, ADP-rhamnose, TDP-rhamnose, CDP-rhamnose, GDP-rhamnose, UDP-nitryl glucose, ADP-nitryl glucose, TDP-nitryl acetyl glucose, CDP-azaacetylglucose, or any one or more of other nucleoside hexose diphosphate or nucleoside pentose diphosphate.
In one embodiment, the mutant catalyzes the transfer of a glycosyl group from a glycosyl donor to a hydroxyl group of a flavonoid, which differs in glycosylation site from flavonoid species, e.g., the mutant catalyzes glycosylation at kaempferol C7.
In one embodiment, the mutant can catalyze glycosylation of flavonoids such as silybin, kaempferol, chrysin, hesperetin, daidzein, calycosin, biochanin, and the like.
The present invention also provides a glycosylation reaction method, which catalyzes the glycosyl group of glycosyl group donor to be transformed into glycosyl group acceptor by using the glycosylation transferase mutant, transformant or culture thereof, and the glycosylation reaction is carried out.
In one embodiment, the method comprises: adding the glycosyltransferase mutant into a glycosylation reaction system, and contacting the glycosyltransferase mutant with a glycosyl acceptor and a glycosyl donor in the glycosylation reaction system to carry out glycosylation reaction; or adding the transformant to a glycosylation reaction system, culturing, allowing the transformant to produce the glycosyltransferase mutant, and contacting the glycosylacceptor or glycosyl donor in the glycosylation reaction system to allow glycosylation reaction.
The glycosyl acceptor and glycosyl donor have the meanings as described above.
In one embodiment, the above-described transformant is cultured according to a known method for culturing a microorganism to obtain a culture of the transformant.
Has the advantages that:
the invention provides a series of glycosyltransferases with specific region selectivity and mutants thereof, which improve the glycosylation region selectivity of flavonoids compounds such as silybin and the like. Meanwhile, the mutants have obvious improvement on the regioselectivity of various flavonoids, reduce the generation of non-specific products, improve the practical application potential of the enzyme in the specific preparation of important rare compounds, and solve the problem of the specific catalytic preparation of specified glycosylation products by the glycosyltransferase. The flavonoid compound is modified by glycosylation of a designated site, the water solubility is obviously enhanced, and a special physiological activity function is shown, so the enzyme and the mutant thereof have stronger application value.
Detailed Description
Definitions and explanations
Amino acids in the present invention are represented by a single or three letter code and have the following meanings: a: ala (alanine); r: arg (arginine); n: asn (asparagine); d: asp (aspartic acid); c: cys (cysteine); q: gln (glutamine); e: glu (glutamic acid); g: gly (glycine); h: his (histidine); i: ile (isoleucine); l: leu (leucine); k: lys (lysine); m: met (methionine); f: phe (phenylalanine); p: pro (proline); s: ser (serine); t: thr (threonine); w: trp (tryptophan); y: tyr (tyrosine); v: val (valine).
In the present invention, "glycosyltransferase" catalyzes an enzyme in which a glycosyl moiety is transferred from a UDP-sugar to a wide range of substrates. The glycosyltransferase is an enzyme that catalyzes the transfer of a sugar group from an activated glycosyl donor to a glycosyl acceptor molecule, and in particular, it refers to an enzyme that utilizes UDP-sugar as a glycosyl donor. The "glycosyl acceptor" and "substrate" as used herein are used interchangeably and include, but are not limited to, flavonoids which may be selected from any of silybin, kaempferol, chrysin, hesperetin, daidzein, calycosin, biochanin. Nitrogen and sulfur atom-containing compounds such as 4-aminobenzophenone, 3, 4-dichloroaniline, 3, 4-dichlorothiophenol and 2, 6-dichlorothiophenol. The term "flavonoid" is generally considered in the present invention to be a 15-carbon structure having two benzene rings and a heterocyclic ring.
As used herein, a "glycosyl donor" is a compound that provides a glycosyl group, including nucleoside diphosphate sugars. For example, the glycosyl donor is selected from the group consisting of: UDP-glucose, ADP-glucose, TDP-glucose, CDP-glucose, GDP-glucose, UDP-galacturonic acid, ADP-galacturonic acid, TDP-galacturonic acid, CDP-galacturonic acid, GDP-galacturonic acid, UDP-galactose, ADP-galactose, TDP-galactose, CDP-galactose, GDP-galactose, UDP-arabinose, ADP-arabinose, TDP-arabinose, CDP-arabinose, GDP-arabinose, UDP-rhamnose, ADP-rhamnose, TDP-rhamnose, CDP-rhamnose, GDP-rhamnose, UDP-N-acetyl glucose, ADP-N acetyl glucose, TDP-N acetyl glucose, CDP-N acetyl glucose, or other nucleoside hexose diphosphate or nucleoside pentose diphosphate, or combinations thereof.
In the present invention, "homology" has the conventional meaning in the art and refers to "identity" between two nucleic acid or amino acid sequences, the percentage of which represents the statistically significant percentage of identical nucleotides or amino acid residues between the two sequences to be compared, obtained after optimal alignment (best alignment), the differences between the two sequences being randomly distributed over their entire length.
Within the context of the present invention, the variants are described by their mutation at a specific residue, the position of which is determined by alignment with the wild-type enzyme sequence SEQ ID NO 1 or by reference to the enzyme sequence SEQ ID NO 1. In the context of the present invention, it also relates to any variant carrying these same mutations at functionally equivalent residues.
In the present invention, the terms "primer" and "primer strand" are used interchangeably and refer to an initial nucleic acid fragment, typically an RNA oligonucleotide, a DNA oligonucleotide or a chimeric sequence that is complementary to a primer binding site comprised by all or part of a target nucleic acid molecule. The primer strand may comprise natural, synthetic or modified nucleotides. The lower limit of the primer length is the minimum length required to form a stable duplex under the conditions of the nucleic acid amplification reaction.
In the present invention, the terms "mutant" and "variant" are used interchangeably, "substitution" or "mutation" refers to an amino acid relative to the wild-type protein, such as the wild-type sequence of SEQ ID NO:1 UGT74AC2 polypeptide, or a sequence derived from such a polypeptide, that comprises an alteration, i.e. substitution, insertion and/or deletion, at one or more positions and still retains its activity. Variants may be obtained by various techniques known in the art, e.g., may be the product of chemical synthesis, or may be produced from prokaryotic or eukaryotic hosts using recombinant techniques. In particular, exemplary techniques for modifying a DNA sequence encoding a wild-type protein for use in recombinant techniques include, but are not limited to, site-directed mutagenesis, random mutagenesis, and construction of synthetic oligonucleotides.
The term "substitution" with respect to an amino acid position or residue means that the amino acid at a particular position has been replaced with another amino acid. Substitutions may be conservative or non-conservative.
The form "XaY" is used herein to denote a mutation or substitution of an amino acid, wherein a denotes the position of the amino acid in SEQ ID NO. 1, X denotes the wild-type amino acid species at position a in SEQ ID NO. 1, and Y denotes the amino acid species after mutation at position a in SEQ ID NO. 1. For example, "P12Y" indicates that proline P at the position corresponding to position 12 of SEQ ID NO:1 is substituted with tyrosine Y in alignment with SEQ ID NO: 1.
The term "promoter" is a DNA sequence located in the 5' upstream region of the structural gene and capable of activating RNA polymerase to bind the template DNA precisely and with transcription initiation specificity, and the promoter sequence of the present invention may be any known promoter sequence in the art, and may be prokaryotic or eukaryotic, and may be selected from, for example, Lacl, LacZ, pLacT, ptac, T3 or T7 bacteriophage RNA polymerase promoter, CMV promoter, HSV thymidine kinase promoter, SV40 promoter, mouse metallothionein-L promoter, etc.
The host cell of the invention may be a prokaryote, such as E.coli, or a eukaryote. The eukaryote may be a lower eukaryote such as a yeast (e.g. pichia pastoris or kluyveromyces lactis) or a fungus (e.g. Aspergillus) or a higher eukaryote such as an insect cell (e.g. Sf9 or Sf21), a mammalian cell or a plant cell. The cell may be a mammalian cell, such as COS (green monkey cell line), CHO (Chinese hamster ovary cell line), mouse cell, human cell, and the like.
The vector of the present invention may be a plasmid, phage, phagemid, cosmid, virus, YAC, BAC, Agrobacterium (Agrobacterium) pTi plasmid or the like. The vector may preferably comprise one or more elements selected from the group consisting of: an origin of replication, a multiple cloning site and an optional gene. Preferably, the vector is a plasmid. Some non-exhaustive examples of prokaryotic vectors are as follows: pQE70, pQE60, pQE-9 (Qiagen), pbs, pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16A, pNH18A, pNH 46A; ptrc99a, pKK 223-3, pKK 233-3, pDR540, pBR322, pRIT5, pET-32a, pET-28 a. Preferably, the vector is an expression vector, preferably pET-28a and pET32 a.
As used herein, the term "transformation" refers to the introduction of DNA into a host cell such that the DNA may be replicated as an extrachromosomal element or by chromosomal integration. That is, transformation refers to the alteration in the synthesis of a gene caused by the introduction of foreign DNA into a cell. The term "culture of the transformant" refers to a product obtained by culturing the transformant according to a known method of culturing a microorganism.
The present invention is further illustrated in the following examples, which are not intended to limit the scope of the invention. The details of the partial molecular cloning method vary depending on the reagents, enzymes or kits provided by the supplier, and should be conducted according to the product instructions, and will not be described in detail in the examples.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 expression and Activity evaluation of glycosyltransferase
The glycosyltransferase UGT74AC2 wild type and mutant genes are connected to a pET32a vector, transformed into an escherichia coli expression strain BL21(DE3), and induced expression of heterologous proteins is carried out by LB culture medium. And (3) centrifugally collecting the strain, suspending the strain by using a Tris hydrochloric acid buffer solution, then carrying out ultrasonic crushing, centrifuging to obtain a crushed supernatant, purifying by adopting a Ni column affinity chromatography method, and further carrying out ultrafiltration by using a 30kDa ultrafiltration tube to obtain concentrated and purified wild type and mutant proteins.
The enzyme activity of glycosyltransferase is determined as follows: tris & HCl (pH 8.0), 0.2mM Silybin, 3mM UDP-glucose, 10mM MgCl 2100. mu.L of the crude enzyme solution was reacted at 40 ℃ for 16 hours. After the glycosylation reaction is finished, adding methanol with the same volume to stop the reaction, and measuring by using a liquid phase and a mass spectrum. The HPLC analysis was performed under the following conditions: the instrument is an agilent high performance liquid chromatograph 1200, the chromatographic column is a C18 chromatographic column (4.6mm × 250mm, 5 μm particles, shanghai xu science and technology (shanghai) ltd, china), mobile phase: water + 0.1% formic acid, acetonitrile + 0.1% formic acid, flow rate: 1mL/min, and the loading amount is 20. mu.L.
Example 2 design of mutation sites
Firstly, the glycosyltransferase UGT74AC2 from momordica grosvenori is subjected to homology comparison analysis with the glycosyltransferase amino acid sequences reported in a Genbank database, then the glycosyltransferase UGT74AC2 is subjected to structure prediction, the UGT74AC2 wild-type protein is subjected to homologous modeling by using software such as Swiss-Model, Phyre2 and Discovery Studio, molecular docking is carried out by using substrates such as silybin, and therefore the catalytic site and the substrate binding site of the wild-type enzyme are predicted, and the action of amino acid residues near the sites is analyzed, and the amino acid sequence of the mutant is designed.
Example 3 site-directed mutagenesis of predicted sites
Selecting the nucleotide sequence shown in SEQ ID NO: 1, taking 9 sites which are closely related to regioselectivity as an example, the 11 th, 12 th, 88 th, 119 th, 145 th, 149 th, 190 th, 200 th and 392 th sites are subjected to site-directed mutagenesis, and the specific operation is as follows: using the recombinant plasmid pET32-UGT74AC2 as a template and a pair of primers with mutation sites (see Table 3), whole plasmid PCR amplification is carried out with high fidelity enzyme (see Table 4), and the recombinant plasmid with the appointed mutation sites is obtained. The amplification product was digested with DpnI enzyme at 37 ℃ for 2h, degrading the initial template. Coli BL21, spread on LB agar plates containing 100. mu.g/mL ampicillin, incubated overnight at 37 ℃ and 2-3 clones were picked up for each site and sent to sequencing for confirmation of the target mutation. The points at which the regioselectivity changes and the conversion are shown in table 1.
TABLE 1
Example 4 combinatorial mutagenesis
Based on the site-directed mutagenesis described above, a combination of two sites was performed. The results of these double mutants were tested. Table 2 lists the combinatorial mutants with significantly improved regioselectivity and the cases of transformation rates.
TABLE 2
TABLE 3 primer information
TABLE 4 PCR amplification conditions
Example 5 formation of a glycosylated product Using a glycosyltransferase mutant with Silibinin as substrate and UDP-glucose as glycosyl Donor
Through combined mutation, 3 glycosyltransferase UGT74AC2 mutants, M20, M21 and M24 are obtained, and the regioselectivity of the mutants is obviously improved compared with that of a wild type. The transformation experiment is carried out by taking silybin as a substrate, and the glycosylation reaction system is as follows: Tris/HCl (pH 7.0), 0.2mM silybin, 1mM UDP-glucose, 10mM MgCl2100uL of the crude enzyme solution was reacted at 40 ℃ for 16 hours. After the glycosylation reaction is finished, adding methanol with the same volume to stop the reaction, and measuring by using a liquid phase and a mass spectrum. The chromatographic column is Yuehao C18 chromatographic column (4.6mm × 250mm, 5 μm particles, Shanghai Yuehao science and technology (Shanghai) Co., Ltd., China), and the detection conditions for silibinin are as follows: the gradient elution condition is 0-25min, the flow rate of 25% -85% acetonitrile (0.1% formic acid) is 1mL/min, and the ultraviolet detection wavelength is 288 nm. The mass spectrometry conditions were positive ion mode, ESI ion source. As shown in figure 1, by using silybin as a substrate, the UGT74AC2 mutant M20 can specifically catalyze the silybin to generate a glycosylated product (silybin-3, 7-O-2Glc) at positions C3 and C7, the M21 mutant can catalyze the silybin to generate a glycosylated product (silybin-7-O-Glc) at position C7, the selectivity is over 99%, and the M24 mutant can catalyze the silybin to generate a glycosylated product (silybin-3-O-Glc) at position C7, the selectivity is 94%. The wild glycosyltransferase generates silibinin-3-O-Glc, silibinin-7-O-Glc and silibinin-3, 7-O-2Glc in a proportion of 22%: 39%: 39 percent. Through further identification of the generated product by liquid chromatography-mass spectrometry, the molecular weights of the two products of the silybin-3-O-Glc and the silybin-7-O-Glc are 645.18 and 645.18[ M + H ] (M + H) ]+Silybin-3, 7-O-2Glc with molecular weight 807.23[ M + H ]]+。
EXAMPLE 6 glycosylation of other Flavonoids Using glycosyltransferase mutants
Glycosyltransferase UGT74AC1 mutants M20, M21 and M24 with obviously improved regioselectivity are selected, and kaempferol (5), chrysin (9), hesperetin (11), daidzein (15), calycosin (18) and biochanin (22) are respectively used as substrates for glycosylation reaction. Glycosylation reaction system: tris HCl (pH 8.0), 0.2mM yellowKetone Compound, 3mM UDP-glucose, 10mM MgCl 2100. mu.L of the crude enzyme solution was reacted at 40 ℃ for 16 hours. After the glycosylation reaction is finished, methanol with the same volume is added to stop the reaction, and the catalytic activity of the mutant on different substrates is measured by using a liquid phase. The results are shown in fig. 2, and the mutants have obvious differences in the conversion rate and the type of the six flavonoids.
The transformation product of kaempferol (5) is further identified by a liquid chromatography-mass spectrometry method. The chromatographic column is Yuehang C18 chromatographic column (4.6mm × 250mm, 5 μm particle, Shanghai Yuehang science and technology (Shanghai) Co., Ltd., China), and the detection conditions for kaempferol are as follows: gradient elution conditions are 0-25min, 25% -85% acetonitrile (0.1% formic acid), flow rate is 1mL/min, and ultraviolet detection wavelength is 254 nm. The mass spectrometry conditions were positive ion mode, ESI ion source. As shown in FIG. 3, kaempferol-7-O-Glc is produced from wild type UGT74AC2 with kaempferol as substrate and molecular weight of 449.11[ M + H ] ]+And kaempferol-3, 7-O-Glc with molecular weight of 611.16[ M + H ]]+When the mutants are M20 and M24, the mutants can generate kaempferol-3-O-Glc in addition to the two products, and the molecular weight of the mutants is 449.11[ M + H ]]+。
Example 7 glycosylation reaction of Nitrogen-or Sulfur-containing Compounds Using glycosyltransferase mutants
Glycosylation reaction system: tris HCl (pH 8.0), 0.2mM of nitrogen or sulfur containing compounds such as 4-aminobenzophenone (24), 3-aminobenzophenone (25), 3,4 dichloroaniline (26), 3,4 dichlorothiophenol (27) and 2,6 dichlorothiophenol (28), 1mM UDP-glucose, 10mM MgCl (MgCl) (pH 8.0), 1mM UDP-glucose 2100. mu.L of the crude enzyme solution was reacted at 35 ℃ for 16 hours. After the glycosylation reaction is finished, adding methanol with the same volume to stop the reaction, and measuring by using a liquid phase and a mass spectrum. The substrate to product structure and conversion are shown in FIG. 4, where mutant M20 was able to convert substrate 24 at 95%. Taking the substrate as an example, the conversion product of the substrate 5 is further identified by a liquid chromatography-mass spectrometry method. The chromatographic column is Yuehao C18 chromatographic column (4.6mm × 250mm, 5 μm particle, Shanghai Yuehao science and technology (Shanghai) Co., Ltd., China), and the detection conditions for the above substances are as follows: gradient elution conditions are 0-25min, 25% -85% acetonitrile (0.1% formic acid) ) The flow rate is 1mL/min, and the ultraviolet detection wavelength is 254 nm. The mass spectrometry conditions were positive ion mode, ESI ion source. As shown in FIG. 5, UGT74AC2 mutant M20 catalyzes 4-aminobenzophenone (24) to produce a 4-aminobenzophenone monoglycosylated product with a molecular weight of 360.145[ M + H ]]+While the wild type does not have this function.
The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
<110> Guilin Rhine Biotechnology Ltd
<120> momordica grosvenori-derived glycosyltransferase mutant and application thereof
<150> 202110023003.7
<151> 2021-01-08
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<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
ggtcctcgat tggttctgcg taaccatggt ggacgtggc 39
<210> 17
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
ttacgcagaa ccaatcgagg accaagccgg cgagtatgg 39
<210> 18
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
cagtgctggg tggctttctt tcacctccca tcttcaaga 39
<210> 19
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
tgaaagaaag ccacccagca ctggaagtga agaatacat 39
<210> 20
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
ttcacttcca gtgctgggtt tctttctttc acctcccat 39
<210> 21
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
tgaaagaaag aaacccagca ctggaagtga agaatacat 39
<210> 22
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
cattccgggc atttatttca acaaaaacat ggccgagtg 39
<210> 23
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
tgttgaaata aatgcccgga atgaccttgc cgggaaccg 39
<210> 24
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
cattccgggc tggtatttca acaaaaacat ggccgagtg 39
<210> 25
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
tgttgaaata ccagcccgga atgaccttgc cgggaaccg 39
<210> 26
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
ggtcctcgat cagttctgcg taaccatggt ggacgtggc 39
<210> 27
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
ttacgcagaa ctgatcgagg accaagccgg cgagtatgg 39
<210> 28
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
ggccgagtgg atgcacgact gcgcgaggag gttcagaga 39
<210> 29
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
cgcagtcgtg catccactcg gccatgtttt tgttgaaat 39
<210> 30
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
ggccgagtgg tttcacgact gcgcgaggag gttcagaga 39
<210> 31
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
cgcagtcgtg aaaccactcg gccatgtttt tgttgaaat 39
<210> 32
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
ggccgagtgg tatcacgact gcgcgaggag gttcagaga 39
<210> 33
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
cgcagtcgtg ataccactcg gccatgtttt tgttgaaat 39
<210> 34
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
ggccgagtgg tggcacgact gcgcgaggag gttcagaga 39
<210> 35
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
cgcagtcgtg ccaccactcg gccatgtttt tgttgaaat 39
<210> 36
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
atggccgatg accgcggagc aacatttcaa tgcgttcga 39
<210> 37
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
gttgctccgc ggtcatcggc catgtggcaa tgggcacgc 39
<210> 38
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
caacaaaaac ctggccgagt ggttacacga ctgcgcgag 39
<210> 39
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
accactcggc caggtttttg ttgaaataga cgcccggaa 39
<210> 40
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
cttcgggagc agcggaagct taaatccgga tcaagcgcg 39
<210> 41
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
ttaagcttcc gctgctcccg aagcaaagaa ataccaccg 39
<210> 42
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
ggtcctcgat gaattctgcg taaccatggt ggacgtggc 39
<210> 43
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
ttacgcagaa ttcatcgagg accaagccgg cgagtatgg 39
<210> 44
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
tctttctttc gtgtcccatc ttcaagacct ttccgatcg 39
<210> 45
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
gaagatggga cacgaaagaa agatacccag cactggaag 39
<210> 46
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 46
caacaaaaac aatgccgagt ggttacacga ctgcgcgag 39
<210> 47
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 47
accactcggc attgtttttg ttgaaataga cgcccggaa 39
<210> 48
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 48
gccctttcaa atgcaagctg ttttcgaaac ccagaaaca 39
<210> 49
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 49
aaacagcttg catttgaaag ggccctttgg cgtcgtcgg 39
<210> 50
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 50
attgccacat ggccgatgag cgcggagcaa catttcaat 39
<210> 51
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 51
attgaaatgt tgctccgcgc tcatcggcca tgtggcaat 39