KR20200021320A - Method for inducing reactive oxygen species-mediated base mutation of target gene - Google Patents

Abstract

The present invention relates to a method for inducing reactive oxygen species-mediated base mutation of a target gene. In the present invention, an inactive dCas9 can be combined with an enzyme component that produces free radicals to induce various kinds of base changes in a specific gene, thereby inducing various mutations in an in vivo state to produce many alleles. Therefore, the present invention may be used for various purposes in the fields of biotechnology and agricultural biotechnology.

Description

Method for inducing reactive oxygen species-mediated base mutation of target gene}

The present invention relates to a method for inducing reactive oxygen species mediated base mutation of a target gene.

Sugars (orthosaccharides) and nitrogenous base moieties of DNA are sensitive to oxidation by reactive oxygen species (ROS). The average dispersion distance before ROS reacts with the components of the cell is only 3 nm, which is the average diameter level of a typical protein. The half-life, reactivity and proliferation of various types of ROS have a great influence on the potential of each ROS to damage cells and DNA. Thus, ROS generated in very close regions of DNA can oxidize DNA. In prokaryotes and eukaryotes, ROS-induced DNA damage is endogenous DNA, such as base-excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), direct repair, or repair of single- and double-stranded cleavage. It is recovered by the recovery path.

Transformation of various microorganisms and plants is based on mutations. Classical mutagenesis methods use chemical treatment, irradiation or transposon, which randomly induce mutations in the whole genome. The present invention relates to a system that uses a CRISPR system (dCAS9, a mutant of Cas9 without endonuclease activity; or dCpf1) to send an enzyme that produces ROS to a specific gene to cause a single base mutation in the target gene.

Meanwhile, Korean Patent Laid-Open Publication No. 2001-0022652 discloses the use of hybrid helix oligonucleotides to induce local genetic modification in plants, and Korean Patent Publication No. 2016-0128306 discloses nuclease interactions. Although a 'mutagenic method' using a chimeric spliced RNA molecule comprising a transcribed exon spliced into an RNA fragment is disclosed, a method for inducing reactive oxygen species mediated base mutation of a target gene of the present invention has been described. none.

The present invention is derived from the above requirements, and the present inventors are transformed into a plasmid (Hygro-linker2-mRuby2) artificially inserted with a stop codon in a hygromycin phosphotransferase, which is resistant to hygromycin. For yeasts that do not have, glycolylate oxidase; Inactive dCas9; And a guide RNA for the hygromycin resistance gene of Hygro-linker2-mRuby2. As a result, the growth of the yeast showing resistance to hygromycin was confirmed. In addition, as a result of culturing the hygromycin-resistant yeast and extracting the plasmid to analyze the sequencing of the hygromycin resistance gene, mutations occurred at the artificially inserted stop codon positions, resulting in the yeast having hygromycin resistance 29 The present invention was completed by confirming that the genome editing of the target gene based on the glycol oxidase-dCas9 system was confirmed at the% level.

In order to solve the above problems, the present invention is a polynucleotide encoding an enzyme that produces reactive oxygen species; Polynucleotides encoding an inactivated Cas9 (CRISPR associated protein 9) protein or a functional analog thereof; And a recombinant vector comprising a guide RNA coding sequence.

The present invention also provides a host cell transformed with the recombinant vector.

The present invention also provides a method of inducing a single base mutation of a target gene comprising the step of transforming a host cell with the recombinant vector.

In addition, the present invention provides a composition for inducing a single base mutation comprising the recombinant vector as an active ingredient.

The present invention combines enzyme components that produce free radicals with inactive dCas9 to induce various kinds of base changes in specific genes, thereby inducing various mutations in vivo to produce many alleles, and DNA bases. Since only one target is converted to another base directly to another base, there is an advantage that can be corrected without DNA cleavage, it can be used for a variety of applications in the field of biotechnology and agricultural biotechnology.

1 is a schematic diagram for selecting mutagenic yeast cells.
2 is a schematic diagram of a GOX3-dCas9-guide RNA construct prepared for mutagenesis of the target gene (Hygro-linker2-mRuby2) using the Arabidopsis-derived glycolic acid oxidase gene ( AtGOX3 ).
Figure 3 shows the results of analyzing the mutagenesis results of the GOX3-dCas9-guide RNA system performed on yeast cells.
4 is a graph showing the window of mutations induced in a system using AtGOX3.
5 is a schematic diagram of a GOX3-dCas9-guide RNA construct prepared for mutagenesis of a target gene (Hygro-linker3-mTurqiose2) using the AtGOX3 gene.
FIG. 6 is a schematic diagram of a CRY1-dCas9-guide RNA construct prepared for mutagenesis of a target gene (Hygro-linker3-mTurqiose2) using the Arabidopsis-derived Cryptochrome gene ( AtRYRY ).
7 is a schematic diagram of a CRY1ΔCCE-dCas9-guide RNA construct prepared for mutagenesis of a target gene (Hygro-linker3-mTurqiose2) using the AtCRY1 gene from which the CRY C-terminal Extension (CCE) domain was removed. to be.
8 is a schematic diagram of a CRY2-dCas9-guide RNA construct prepared for mutagenesis of a target gene (Hygro-linker3-mTurqiose2) using the Arabidopsis-derived cryptochromium gene ( AtRYRY ).
9 is a schematic diagram of the CRY2ΔCCE-dCas9-guide RNA construct prepared for mutagenesis of the target gene (Hygro-linker3-mTurqiose2) using the AtCRY2 gene from which the CCE domain was removed.
Figure 10 shows the nucleotide sequence of AtGOX3-ydCas9-6XHis-2XNLS, the underlined sequence of the yeast codon optimized AtGOX3 ; Lowercase blue letters show the SGGS ₂ -XTEN-SGGS ₂ linker sequence; Gray shaded portions include Hig tag sequences (including ATCCCG sequences to avoid frame shifts in 2XNLS); Double underlines are 2XNLS sequences; And the boxed portion shows the mutant codon position for inactivation of Cas9 protein.
Figure 11 shows the nucleotide sequence of Hygro-linker2-mRuby2.
12 shows the nucleotide sequence of Hygro-linker3-mTurqiose2.

In order to achieve the object of the present invention, the present invention is a polynucleotide encoding an enzyme that produces a reactive oxygen species; Polynucleotides encoding an inactivated Cas9 (CRISPR associated protein 9) protein or a functional analog thereof; And a recombinant vector comprising a guide RNA coding sequence.

As used herein, the term 'active oxygen species' refers to a chemically reactive molecule including an oxygen atom, which is more reactive and rich in oxygen species than the tribasic oxygen ( ³ O ₂ ) which is usually present. . Superoxide (O ₂ −), hydrogen peroxide (H ₂ O ₂ ), hydroxy radical (· OH), singlet oxygen ( ¹ O ₂ ), alkoxy radical (RO ·). It contains highly reactive oxygen compounds such as peroxy radicals (ROO ·), ozone (O ₃ ) and nitrogen dioxide (NO ₂ ), and is highly reactive due to unpaired electrons. Free radicals occur naturally due to the normal metabolism of oxygen, and the concentration of free radicals can increase very rapidly due to environmental stresses such as exposure to ultraviolet rays or high heat, and increased free radical species can damage cell structure. This is oxidative stress.

In the recombinant vector according to the present invention, the enzyme producing the active oxygen species may be glycolic acid oxidase (Glycolate oxidase, GOX) or cryptochrome (Cryrochrome, CRY), more specifically Arabidopsis thaliana Origin AtGOX3 (At4g18360), AtCRY1 (At4g08920) or AtCRY2 (At1g04400), but is not limited thereto. The AtGOX3, AtCRY1 or AtCRY2 protein may be composed of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5, respectively.

Polynucleotide encoding the AtGOX3, AtCRY1 or AtCRY2 protein according to an embodiment of the present invention may be composed of the nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6, but is not limited thereto, the host cell to be transformed The base sequence may be optimized for codon of. In addition, the polynucleotide encoding the AtGOX3 protein was used for cloning except for the base portion (1069 to 1,104th in the nucleotide sequence of SEQ ID NO: 2) encoding the peroxisome targeting signal (PTS) portion of the carboxy terminus, and the AtCRY1 or AtCRY2 protein. The CCE domain coding sequence of corresponds to the 1,504-2,043, or 1,507-1,836 th in the nucleotide sequence of SEQ ID NO: 4 or SEQ ID NO: 6, respectively.

Glycolic acid oxidase is an enzyme that catalyzes the reaction of glycolic acid by oxygen to produce glyoxylic acid and hydrogen peroxide.Cryptochrome is a chromophore of flavin and terin in plants and animals. As a flavo protein that senses blue light to be used, hydrogen peroxide solution can be produced under blue light conditions. The hydrogen peroxide solution thus produced can act on DNA to induce transition and transversion mutations.

In addition, in the recombinant vector according to the present invention, the inactivated Cas9 (catallytically-dead Cas9, dCas9) is a loss of endonuclease activity through mutations in the endonuclease domain, D10A and H840A point mutation Is known as an important residue for endonuclease inactivation. dCas9 has lost endonuclease activity but can bind to the DNA sequence of the target gene. In addition, the functional analog of the inactivated Cas9 protein may be, but not limited to, dCpf1 (dead centromere and promoter factor 1).

Cas9 (CRISPR associated protein 9) is one of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) type II RNA-guided DNA endonuclease and is RNA-guided endonuclease (RGEN). Cas9 protein according to the present invention may be derived from Streptococcus pyogenes , but is not limited thereto.

As used herein, the term 'guide RNA' refers to a single-stranded RNA that serves to find a DNA position to be corrected. In the composition for inducing base mutation of the present invention, the guide RNA is adjacent to the PAM site. It may include a sequence complementary to the base sequence of ~ 20bp, but is not limited thereto.

As used herein, the term 'protospacer adjacent motif (PAM) site' refers to a sequence necessary for the Cas9 protein to bind to the target DNA, and is positioned after the target sequence. Bacteria store part of the sequence of invading viruses in parts of the bacterial genome, which is called the protospacer. Since the protospacer sequence is part, its contiguous sequence is the bacterial sequence. The role of the PAM site is that many sequences within the bacterium can match the protospacer, but if the PAM is not attached next to it, the sequence will not be cut. The PAM of Cas9-derived Streptococcus pneumoniae is an NGG sequence, and considering the double-stranded DNA structure, the target sequence can be set inside the target gene every 8bp.

The term 'recombinant' as used herein refers to a cell replicating a heterologous nucleic acid, expressing the nucleic acid or expressing a protein encoded by a peptide, a heterologous peptide or a heterologous nucleic acid. Recombinant cells can express genes or gene fragments that are not found in the natural form of the cell in either the sense or antisense form. Recombinant cells may also express genes found in natural cells, but the genes are modified and reintroduced into cells by artificial means.

The term 'vector' is used herein to refer to DNA fragment (s), nucleic acid molecules that are delivered into a cell. Vectors can replicate DNA and be reproduced independently in host cells. The term "carrier" is often used interchangeably with "vector". By recombinant vector is meant bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell viral vector, or other vector. In principle, any plasmid and vector can be used as long as it can replicate and stabilize in the host. An important feature of the expression vector is that it has a origin of replication, a promoter, a marker gene and a translation control element.

The present invention also provides a host cell transformed with the recombinant vector.

Method for transforming the recombinant vector to the flora and fauna cells and individuals according to the present invention is polyethylene glycol (PEG) -based protoplast transformation, microinjection, electroporation, gene gun, Particle bombardment), liposome mediated transfection and Agrobacterium mediated transformation may be used, but are not limited thereto, and such transformation or transduction methods are well known in the art.

The host cell according to the present invention may be plant cells, bacteria or fungi, but is not limited thereto.

The present invention also provides a method of inducing base mutation of a target gene comprising the step of transforming a host cell with the recombinant vector.

In the method of inducing base mutation of a target gene according to the present invention, the host cell transformation method is as described above.

In one embodiment of the present invention, in the case of yeast, the base mutation induction module complex is cloned through a golden gate assembly based on an E. coli-yeast shuttle vector, and a yeast through a chemical shock or electroporation method. Transformed into cells.

The method of inducing base mutation of a target gene according to the present invention comprises an enzyme producing a reactive oxygen species that is transcribed and translated from a recombinant vector introduced into a host cell; Inactivated Cas9 (dCas9) protein; And mutations may be induced in the base of the target gene by a ribonucleoprotein complex (RNP) fused with a guide RNA. Specifically, oxidative stress is induced in the target gene by the active oxygen species generated from the enzyme that produces the active oxygen species fused to the dCas9 is moved to the DNA sequence position of the target gene through the guide RNA sequence Base mutations are induced.

The method of inducing base mutations of the present invention is characterized by random base mutations, preferably random single base mutations, of reactive oxygen species mediated target genes, the mutations of which are not only a base transition but also a transversion. ) May be included. The reactive oxygen species may be derived from an active oxygen species producing enzyme such as glycolic acid oxidase (GOX) or cryptochrome (CRY), but is not limited thereto.

The base mutation induction method according to the present invention can induce a variety of base changes in the target gene to induce a variety of mutations in vivo to produce a large number of alleles (allele), and because dCas9 is used without DNA cleavage Mutations can be induced in the target gene.

The present invention also provides a composition for inducing base mutations comprising the recombinant vector as an active ingredient. Compositions of the present invention include polynucleotides encoding enzymes that produce reactive oxygen species; Polynucleotides encoding an inactivated Cas9 (CRISPR associated protein 9) protein or a functional analog thereof; And a recombinant vector comprising a guide RNA coding sequence as an active ingredient, which can induce random base mutations of reactive oxygen species-mediated target genes. Mutations can be induced in the base of the gene of interest.

The base mutation induction composition of the present invention can be used to mutate DNA in the mitochondria or plastids of eukaryotic cells, or in vitro and in vivo , prokaryotic cells / living cells and eukaryotic cells / living chromosomes.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Materials and methods

1. Yeast Strains and Culture Conditions

Saccharomyces cerevisiae strains used in all experiments were haploid PJ69-4A (genotype MATa trp1-901 leu2-3, 112 ura3-52, his3-200, gal4Δ gal80Δ, Met2 :: GAL7-lacZ, LYS2: : GAL1-HIS3 GAL2-ADE2), which was sold by Professor Han Chang-deok of Gyeongsang National University. Wild-type yeast was cultured in YPD (10 g Bacto Yeast extract, 20 g peptone and 20 g D-glucose for 1 L.) medium, and the transformed yeast was synthesized in a restriction medium (26.7 g Minimal SD Base with 2% glucose and without amino acids). (Clontech); 0.77g appropriate (-Ura) drop-out supplement (Clontech, USA)) and selected using an axoxotrophic marker (URA3). Yeast transformants were cultured in different combinations of carbon sources (eg, D-glucose, L-lactate, glycolate).

2. Plasmid Construct

PCR reactions were performed using a Phusion Taq PCR master mix (Thermo Scientific, USA). All starting gene templates were either bacterial or yeast codon optimized using an online tool from Integrated DNA Technologies (USA). Primer information used in the PCR is shown in Table 1 and Table 2.

Golden Gate assembly reactants were transformed by heat-shock method into 10-β competent E. coli cells (New England Biolabs, USA), and the transformed cells were selected by culturing in LB medium containing antibiotics. All bacterial constructs were transformed into competent cells and stored at −80 ° C. using a glycerol cryopreservative, or stored at −20 ° C. in the form of isolated and purified plasmids. Ultrapure water (ultrapure water, Arioso, 18.2MΩcm, Human Corporation, Korea) was used for all experiments, and all PCR by-products and plasmids were used with HiGene Gel and PCR purification kit and HiGene Plasmid mini prep kit (Biofact, Korea).

Yeast expression cassettes were designed using the Golden Gate assembly described in this report (ACS Synth Biol 2015; 4 (9): 975-986) (using BsmB I and Bsa I restriction sites). All yeast constructs included CEN6 / ARS4 (Chromosome VI centromere / Autonomously Replicating Sequence 4) as the origin of replication, resulting in low and 2-5 copies per haploid cell and episomal replication in yeast cells. The yeast codon-optimized cas9 gene from S. pyogenes was prepared using PCR specific primer sets (D10A and H840A) to inactivate enzyme activity. Cas9 of the enzyme-inactivated form was expressed as dCas9, and a heath-tag and two copies of a nuclear location signal (NLS) were connected to the carboxy terminus of dCas9 (dCas9-6XHis-2XNLS). Arabidopsis derived glycolic acid oxidase ( AtGOX3 ) was cloned except for the peroxisome targeting signal (PTS). Genes encoding enzymes that produce reactive oxygen species ( AtGOX3 , AtCRY1 , AtCRY2 ) were linked to the amino terminus of dCas9-6XHis-2XNLS using (SGGS) ₂ -XTEN- (SGGS) ₂ linker. The expression cassette contains the coding sequence for the guide RNA and the guide RNA-scaffold sequence for the 20 bp size of the target gene following the SNR52 promoter and includes a SUP4 terminator.

3. Yeast Transformation

Yeast cells were inoculated in YPD plate medium and incubated at 30 ° C. for 2 days. Single yeast colonies were inoculated in 3 ml of YPD medium and incubated overnight at 30 ° C. with stirring. The cell density (OD ₆₀₀ ) was measured by diluting the culture solution 1/10 in sterile distilled water, and the amount of yeast culture required to inoculate 50 ml culture medium at a cell density of 5 × 10 ⁶ cells / mL (OD ₆₀₀ = 0.1). The amount was calculated, and the yeast culture was incubated with stirring at a speed of 180-250 rpm at 30 ° C. until the OD ₆₀₀ value was 0.6-0.8.

The cultured cells were recovered by centrifugation at 3,000 rpm, 10 minutes, and 4 ° C., the cell pellets were suspended in cold water, and then recentrifuged at 3,000 rpm, 8 minutes, and 4 ° C. to discard the supernatant. Only recovered. The cell pellet was resuspended using sterile distilled water, and then recentrifuged at 3,000 rpm, 5 minutes, and 4 ° C. to recover the cell pellet. The cell pellet was then suspended using 2 ml of cold 1M sorbitol and centrifuged at 3,000 rpm for 8 minutes at 5 ° C. Cell pellets obtained through the centrifugation were treated with 2 ml of a buffer consisting of 100 mM LiAc (Lithium Acetate), 1X TE buffer and 25 mM DTT (dithiothreitol), followed by centrifugation at 3,000 rpm, 5 minutes, and 4 ° C. 2 ml of cold 1M sorbitol was added to the obtained cell pellet and the cells were suspended. The supernatant was removed by centrifugation at 3,000 rpm for 5 minutes at 4 ° C. Thereafter, 1.5 ml of cold 1M sorbitol was added thereto, and the cells were suspended and aliquoted with 60 µl. For transformation, 5 μl of DNA (200 ng) was mixed in the cell suspension, and the yeast cell / DNA mixture was transferred to a 0.2 cm cooled cuvette and the bottom of the cuvette was lightly tapped. Electroporation was performed with a pulse charge of 1.5 kV, 200 mA, and 25 μF (pulse time 5 ms), and immediately added 1 ml of YPF medium. After the cells were allowed to recover by stirring at 30 ° C. at a speed of 180 rpm for 1 hour, the supernatant was removed by centrifugation at 3,000 rpm for 10 minutes at room temperature. Cells suspended with 1 ml of 1M sorbitol were applied onto a dropout plate corresponding to the trophic marker (URA3) and cultured to confirm colony growth. Colonies grown on the plates were used for further analysis.

4. Free radical species-mediated mutagenesis and base sequencing

Yeast transformant colonies containing the desired plasmids also grew under conditions of varying concentrations and combinations of glucose, lactate and glycolate. Glycolate and lactate were used as substrates for glycolic acid oxidase (GOX3). Since GOX3 has been reported in a multimeric form, in the present invention, GOX3 is linked to the amino terminal position of dCas9-6XHis-2XNLS using a specific linker, and expresses free form of GOX3 with 2XNLS sequence. A plasmid was prepared to make.

Plasmids were isolated from the yeast colonies grown on YPD containing hygromycin using the Easy Yeast Plasmid Isolation Kit (Clontech). The isolated plasmid was retransformed into 10-β competent E. coli cells, and the plasmid isolated from the transformed E. coli cells was used for sequencing analysis (Solgent, Korea).

Example 1. Analysis of reactive oxygen species-mediated mutagenesis efficiency

For 70 plasmids, approximately 500 bp of the sequence of the hygromycin resistance gene region artificially added with a stop codon was found in 31 (44%) no mutations were found in the hygromycin resistance gene. Twenty (29%) mutations occurred in artificially inserted stop codons, indicating that the expression of hygromycin resistance protein was possible. In addition, the remaining 19 (27%) was found to be 8 mutations 22-26bp upstream of the stop codon and 19 mutations around the PAM site (-2-2bp), but in other locations No mutation was found, indicating that the mutation was specific for GOX3-dCas9 induced in the guide RNA. That is, out of a total of 70 plasmids surveyed, 19 mutated at positions not related to hygromycin resistance, so that GOX3-dCas9-based systems had up to 29% mutagenesis efficiency for target genes (or sequences). It was found that (Fig. 3).

<110> INDUSTRY-ACADEMIC COOPERATION FOUNDATION GYEONGSANG NATIONAL UNIVERSITY <120> Method for inducing reactive oxygen species-mediated base          mutation of target gene <130> PN18188 <160> 57 <170> KoPatentIn 3.0 <210> 1 <211> 368 <212> PRT <213> Arabidopsis thaliana <400> 1 Met Glu Ile Thr Asn Val Met Glu Tyr Glu Lys Ile Ala Lys Glu Lys   1 5 10 15 Leu Pro Lys Met Val Tyr Asp Tyr Tyr Ala Ser Gly Ala Glu Asp Gln              20 25 30 Trp Thr Leu Gln Glu Asn Arg Asn Ala Phe Ser Arg Ile Leu Phe Arg          35 40 45 Pro Arg Ile Leu Ile Asp Val Ser Lys Ile Asp Val Ser Thr Thr Val      50 55 60 Leu Gly Phe Asn Ile Ser Met Pro Ile Met Ile Ala Pro Thr Ala Met  65 70 75 80 Gln Lys Met Ala His Pro Asp Gly Glu Leu Ala Thr Ala Arg Ala Thr                  85 90 95 Ser Ala Ala Gly Thr Ile Met Thr Leu Ser Ser Trp Ala Thr Cys Ser             100 105 110 Val Glu Glu Val Ala Ser Thr Gly Pro Gly Ile Arg Phe Phe Gln Leu         115 120 125 Tyr Val Tyr Lys Asp Arg Asn Val Val Ile Gln Leu Val Lys Arg Ala     130 135 140 Glu Glu Ala Gly Phe Lys Ala Ile Ala Leu Thr Val Asp Thr Pro Arg 145 150 155 160 Leu Gly Arg Arg Glu Ser Asp Ile Lys Asn Arg Phe Ala Leu Pro Arg                 165 170 175 Gly Leu Thr Leu Lys Asn Phe Glu Gly Leu Asp Leu Gly Lys Ile Asp             180 185 190 Lys Thr Asn Asp Ser Gly Leu Ala Ser Tyr Val Ala Gly Gln Val Asp         195 200 205 Gln Ser Leu Ser Trp Lys Asp Ile Lys Trp Leu Gln Ser Ile Thr Ser     210 215 220 Leu Pro Ile Leu Val Lys Gly Val Ile Thr Ala Glu Asp Ala Arg Ile 225 230 235 240 Ala Val Glu Tyr Gly Ala Ala Gly Ile Ile Val Ser Asn His Gly Ala                 245 250 255 Arg Gln Leu Asp Tyr Val Pro Ala Thr Ile Val Ala Leu Glu Glu Val             260 265 270 Val Lys Ala Val Glu Gly Arg Ile Pro Val Phe Leu Asp Gly Gly Val         275 280 285 Arg Arg Gly Thr Asp Val Phe Lys Ala Leu Ala Leu Gly Ala Ser Gly     290 295 300 Val Phe Val Gly Arg Pro Ser Leu Phe Ser Leu Ala Ala Asp Gly Glu 305 310 315 320 Ala Gly Val Arg Lys Met Leu Gln Met Leu Arg Asp Glu Phe Glu Leu                 325 330 335 Thr Met Ala Leu Ser Gly Cys Arg Ser Leu Arg Glu Ile Ser Arg Thr             340 345 350 His Ile Lys Thr Asp Trp Asp Thr Pro His Tyr Leu Ser Ala Lys Leu         355 360 365 <210> 2 <211> 1107 <212> DNA <213> Arabidopsis thaliana <400> 2 atggagataa caaacgtgat ggaatatgag aagatcgcaa aggagaaatt accaaagatg 60 gtttatgatt actatgcatc tggtgctgaa gatcaatgga ctcttcaaga gaatcgaaac 120 gctttctcta ggattctatt taggcctcgg attcttatcg atgtaagcaa gattgatgtg 180 agcacaacgg ttttagggtt taatatttct atgccaatta tgattgctcc tactgcaatg 240 cagaaaatgg ctcatcctga tggcgagctt gcaaccgcga gagctacttc tgctgctgga 300 acaatcatga ctttatcttc atgggctact tgtagtgttg aggaagttgc ttcaaccgga 360 ccagggattc gttttttcca actttatgtt tataaggata gaaatgtggt tatacagctt 420 gtgaaacgag ctgaagaagc tggattcaaa gctattgctc ttactgtaga tactccaagg 480 cttggacgca gagaatctga catcaaaaac agattcgcgc ttcctcgagg tctaacgttg 540 aagaacttcg aagggttgga tcttgggaaa atagacaaga cgaatgactc agggctagct 600 tcatatgttg ctggtcaagt tgatcaatca cttagctgga aggatataaa atggctccaa 660 tctatcacaa gcttgccaat tcttgtcaag ggtgttatta cagctgagga tgcaagaatt 720 gctgttgaat atggagctgc agggataata gtctctaacc acggagctcg tcagctagat 780 tatgttcctg caactatagt ggccttagaa gaggtggtta aagccgtgga gggtcggatt 840 ccggtttttc ttgacggtgg ggttcgccgt ggaaccgatg tctttaaggc attggctctt 900 ggcgcttcag gtgtctttgt cgggaggccg agcttgttct cgcttgcagc ggatggagaa 960 gcgggagtta ggaagatgct acaaatgcta agagatgagt ttgagcttac aatggcacta 1020 agtggttgcc gttcactgag agagatcagc cgaacccaca tcaagactga ttgggacact 1080 cctcattacc tctcggccaa gctgtag 1107 <210> 3 <211> 681 <212> PRT <213> Arabidopsis thaliana <400> 3 Met Ser Gly Ser Val Ser Gly Cys Gly Ser Gly Gly Cys Ser Ile Val   1 5 10 15 Trp Phe Arg Arg Asp Leu Arg Val Glu Asp Asn Pro Ala Leu Ala Ala              20 25 30 Ala Val Arg Ala Gly Pro Val Ile Ala Leu Phe Val Trp Ala Pro Glu          35 40 45 Glu Glu Gly His Tyr His Pro Gly Arg Val Ser Arg Trp Trp Leu Lys      50 55 60 Asn Ser Leu Ala Gln Leu Asp Ser Ser Leu Arg Ser Leu Gly Thr Cys  65 70 75 80 Leu Ile Thr Lys Arg Ser Thr Asp Ser Val Ala Ser Leu Leu Asp Val                  85 90 95 Val Lys Ser Thr Gly Ala Ser Gln Ile Phe Phe Asn His Leu Tyr Asp             100 105 110 Pro Leu Ser Leu Val Arg Asp His Arg Ala Lys Asp Val Leu Thr Ala         115 120 125 Gln Gly Ile Ala Val Arg Ser Phe Asn Ala Asp Leu Leu Tyr Glu Pro     130 135 140 Trp Glu Val Thr Asp Glu Leu Gly Arg Pro Phe Ser Met Phe Ala Ala 145 150 155 160 Phe Trp Glu Arg Cys Leu Ser Met Pro Tyr Asp Pro Glu Ser Pro Leu                 165 170 175 Leu Pro Pro Lys Lys Ile Ile Ser Gly Asp Val Ser Lys Cys Val Ala             180 185 190 Asp Pro Leu Val Phe Glu Asp Asp Ser Glu Lys Gly Ser Asn Ala Leu         195 200 205 Leu Ala Arg Ala Trp Ser Pro Gly Trp Ser Asn Gly Asp Lys Ala Leu     210 215 220 Thr Thr Phe Ile Asn Gly Pro Leu Leu Glu Tyr Ser Lys Asn Arg Arg 225 230 235 240 Lys Ala Asp Ser Ala Thr Thr Ser Phe Leu Ser Pro His Leu His Phe                 245 250 255 Gly Glu Val Ser Val Arg Lys Val Phe His Leu Val Arg Ile Lys Gln             260 265 270 Val Ala Trp Ala Asn Glu Gly Asn Glu Ala Gly Glu Glu Ser Val Asn         275 280 285 Leu Phe Leu Lys Ser Ile Gly Leu Arg Glu Tyr Ser Arg Tyr Ile Ser     290 295 300 Phe Asn His Pro Tyr Ser His Glu Arg Pro Leu Leu Gly His Leu Lys 305 310 315 320 Phe Phe Pro Trp Ala Val Asp Glu Asn Tyr Phe Lys Ala Trp Arg Gln                 325 330 335 Gly Arg Thr Gly Tyr Pro Leu Val Asp Ala Gly Met Arg Glu Leu Trp             340 345 350 Ala Thr Gly Trp Leu His Asp Arg Ile Arg Val Val Val Ser Ser Phe         355 360 365 Phe Val Lys Val Leu Gln Leu Pro Trp Arg Trp Gly Met Lys Tyr Phe     370 375 380 Trp Asp Thr Leu Leu Asp Ala Asp Leu Glu Ser Asp Ala Leu Gly Trp 385 390 395 400 Gln Tyr Ile Thr Gly Thr Leu Pro Asp Ser Arg Glu Phe Asp Arg Ile                 405 410 415 Asp Asn Pro Gln Phe Glu Gly Tyr Lys Phe Asp Pro Asn Gly Glu Tyr             420 425 430 Val Arg Arg Trp Leu Pro Glu Leu Ser Arg Leu Pro Thr Asp Trp Ile         435 440 445 His His Pro Trp Asn Ala Pro Glu Ser Val Leu Gln Ala Ala Gly Ile     450 455 460 Glu Leu Gly Ser Asn Tyr Pro Leu Pro Ile Val Gly Leu Asp Glu Ala 465 470 475 480 Lys Ala Arg Leu His Glu Ala Leu Ser Gln Met Trp Gln Leu Glu Ala                 485 490 495 Ala Ser Arg Ala Ala Ile Glu Asn Gly Ser Glu Glu Gly Leu Gly Asp             500 505 510 Ser Ala Glu Val Glu Glu Ala Pro Ile Glu Phe Pro Arg Asp Ile Thr         515 520 525 Met Glu Glu Thr Glu Pro Thr Arg Leu Asn Pro Asn Arg Arg Tyr Glu     530 535 540 Asp Gln Met Val Pro Ser Ile Thr Ser Ser Leu Ile Arg Pro Glu Glu 545 550 555 560 Asp Glu Glu Ser Ser Leu Asn Leu Arg Asn Ser Val Gly Asp Ser Arg                 565 570 575 Ala Glu Val Pro Arg Asn Met Val Asn Thr Asn Gln Ala Gln Gln Arg             580 585 590 Arg Ala Glu Pro Ala Ser Asn Gln Val Thr Ala Met Ile Pro Glu Phe         595 600 605 Asn Ile Arg Ile Val Ala Glu Ser Thr Glu Asp Ser Thr Ala Glu Ser     610 615 620 Ser Ser Ser Gly Arg Arg Glu Arg Ser Gly Gly Ile Val Pro Glu Trp 625 630 635 640 Ser Pro Gly Tyr Ser Glu Gln Phe Pro Ser Glu Glu Asn Gly Ile Gly                 645 650 655 Gly Gly Ser Thr Thr Ser Ser Tyr Leu Gln Asn His His Glu Ile Leu             660 665 670 Asn Trp Arg Arg Leu Ser Gln Thr Gly         675 680 <210> 4 <211> 2046 <212> DNA <213> Arabidopsis thaliana <400> 4 atgtctggtt ctgtatctgg ttgtggttct ggtggttgta gtattgtatg gtttagaaga 60 gatcttaggg ttgaagataa tccagcttta gcagcagcag taagagctgg tccagtgatt 120 gctctgtttg tttgggcacc agaagaagaa ggacactatc atccaggtag ggtttctagg 180 tggtggctca agaacagttt ggctcagctt gattcttctc ttagaagtct tggtacttgt 240 cttatcacca agagatctac tgatagtgtt gcttctcttc ttgatgttgt taaatccact 300 ggtgcttctc agatcttctt caaccatttg tatgatccat tgtctttggt gcgtgatcac 360 cgagctaaag atgttttgac ggcgcaaggc atagcggttc gatcattcaa cgcagacttg 420 ctttatgagc catgggaagt gactgatgaa ttaggccgtc ctttctctat gtttgctgcg 480 ttttgggaga gatgtcttag tatgccttat gaccctgagt ctcctcttct tccacctaag 540 aagatcattt caggggatgt gtctaaatgt gttgcggatc cattggtgtt tgaggatgac 600 tctgagaaag gaagcaatgc acttctggct cgtgcttggt ctcctggatg gagtaatggt 660 gataaagctc tcacaacgtt tataaacggt ccattgcttg aatactctaa gaaccgcaga 720 aaagccgata gtgctacaac ctcgtttctt tctccacact tgcattttgg ggaagtgagt 780 gtgagaaaag tttttcatct tgttcggatc aaacaggtcg cgtgggcaaa cgaaggaaac 840 gaggccgggg aagaaagcgt gaatcttttc ctgaaatcta ttggtctcag ggagtattct 900 aggtacataa gttttaacca tccatattcc catgaaagac cacttcttgg ccatctaaag 960 ttcttccctt gggctgtgga tgagaactat ttcaaggcat ggaggcaagg ccggactgga 1020 tatccgttgg tcgatgccgg gatgagagag ttatgggcta ctggttggtt gcatgatcgc 1080 ataagagtag ttgtttcaag cttctttgtt aaagtgcttc aattaccatg gagatggggg 1140 atgaagtatt tctgggacac acttcttgat gcggatttag aaagcgatgc tcttggttgg 1200 caatacatta ccggtactct cccggatagc cgggagtttg atcgcataga taaccctcag 1260 tttgaagggt acaagtttga tccaaatggt gaatacgtaa ggcgatggct tcctgaactc 1320 tctagactcc cgacagactg gatacatcat ccgtggaacg cacctgagtc cgttcttcaa 1380 gctgctggta tcgagcttgg atcaaactat cctctaccaa ttgtaggatt agacgaagca 1440 aaagcacggc ttcatgaagc gctttcacag atgtggcaac tagaagctgc ttcaagagct 1500 gcaatagaga acggatccga agaaggactt ggagattctg ctgaggtaga ggaagctcct 1560 atagagttcc caagggacat tacaatggaa gagactgaac caaccagact caacccaaac 1620 aggagatatg aggatcagat ggttccaagc attacttctt ctttgatcag acctgaagaa 1680 gacgaagagt cgtctcttaa tttgagaaat tcagtaggag atagcagagc agaggttcca 1740 aggaacatgg ttaacaccaa ccaagctcag cagcggagag cagaaccggc ttcaaaccaa 1800 gtcactgcta tgattccaga atttaatatc agaattgttg cagagagcac tgaagactca 1860 acagcggaat cttccagcag cggaaggaga gaaagaagcg gaggcatagt ccccgagtgg 1920 tctccagggt actcagagca gttccctagt gaagaaaatg gtattggagg aggaagtaca 1980 acgtctagct acttgcagaa tcaccatgaa atactgaact ggagacggct ttcacaaacc 2040 gggtga 2046 <210> 5 <211> 612 <212> PRT <213> Arabidopsis thaliana <400> 5 Met Lys Met Asp Lys Lys Thr Ile Val Trp Phe Arg Arg Asp Leu Arg   1 5 10 15 Ile Glu Asp Asn Pro Ala Leu Ala Ala Ala Ala His Glu Gly Ser Val              20 25 30 Phe Pro Val Phe Ile Trp Cys Pro Glu Glu Glu Gly Gln Phe Tyr Pro          35 40 45 Gly Arg Ala Ser Arg Trp Trp Met Lys Gln Ser Leu Ala His Leu Ser      50 55 60 Gln Ser Leu Lys Ala Leu Gly Ser Asp Leu Thr Leu Ile Lys Thr His  65 70 75 80 Asn Thr Ile Ser Ala Ile Leu Asp Cys Ile Arg Val Thr Gly Ala Thr                  85 90 95 Lys Val Val Phe Asn His Leu Tyr Asp Pro Val Ser Leu Val Arg Asp             100 105 110 His Thr Val Lys Glu Lys Leu Val Glu Arg Gly Ile Ser Val Gln Ser         115 120 125 Tyr Asn Gly Asp Leu Leu Tyr Glu Pro Trp Glu Ile Tyr Cys Glu Lys     130 135 140 Gly Lys Pro Phe Thr Ser Phe Asn Ser Tyr Trp Lys Lys Cys Leu Asp 145 150 155 160 Met Ser Ile Glu Ser Val Met Leu Pro Pro Pro Trp Arg Leu Met Pro                 165 170 175 Ile Thr Ala Ala Ala Glu Ala Ile Trp Ala Cys Ser Ile Glu Glu Leu             180 185 190 Gly Leu Glu Asn Glu Ala Glu Lys Pro Ser Asn Ala Leu Leu Thr Arg         195 200 205 Ala Trp Ser Pro Gly Trp Ser Asn Ala Asp Lys Leu Leu Asn Glu Phe     210 215 220 Ile Glu Lys Gln Leu Ile Asp Tyr Ala Lys Asn Ser Lys Lys Val Val 225 230 235 240 Gly Asn Ser Thr Ser Leu Leu Ser Pro Tyr Leu His Phe Gly Glu Ile                 245 250 255 Ser Val Arg His Val Phe Gln Cys Ala Arg Met Lys Gln Ile Ile Trp             260 265 270 Ala Arg Asp Lys Asn Ser Glu Gly Glu Glu Ser Ala Asp Leu Phe Leu         275 280 285 Arg Gly Ile Gly Leu Arg Glu Tyr Ser Arg Tyr Ile Cys Phe Asn Phe     290 295 300 Pro Phe Thr His Glu Gln Ser Leu Leu Ser His Leu Arg Phe Phe Pro 305 310 315 320 Trp Asp Ala Asp Val Asp Lys Phe Lys Ala Trp Arg Gln Gly Arg Thr                 325 330 335 Gly Tyr Pro Leu Val Asp Ala Gly Met Arg Glu Leu Trp Ala Thr Gly             340 345 350 Trp Met His Asn Arg Ile Arg Val Ile Val Ser Ser Phe Ala Val Lys         355 360 365 Phe Leu Leu Leu Pro Trp Lys Trp Gly Met Lys Tyr Phe Trp Asp Thr     370 375 380 Leu Leu Asp Ala Asp Leu Glu Cys Asp Ile Leu Gly Trp Gln Tyr Ile 385 390 395 400 Ser Gly Ser Ile Pro Asp Gly His Glu Leu Asp Arg Leu Asp Asn Pro                 405 410 415 Ala Leu Gln Gly Ala Lys Tyr Asp Pro Glu Gly Glu Tyr Ile Arg Gln             420 425 430 Trp Leu Pro Glu Leu Ala Arg Leu Pro Thr Glu Trp Ile His His Pro         435 440 445 Trp Asp Ala Pro Leu Thr Val Leu Lys Ala Ser Gly Val Glu Leu Gly     450 455 460 Thr Asn Tyr Ala Lys Pro Ile Val Asp Ile Asp Thr Ala Arg Glu Leu 465 470 475 480 Leu Ala Lys Ala Ile Ser Arg Thr Arg Glu Ala Gln Ile Met Ile Gly                 485 490 495 Ala Ala Pro Asp Glu Ile Val Ala Asp Ser Phe Glu Ala Leu Gly Ala             500 505 510 Asn Thr Ile Lys Glu Pro Gly Leu Cys Pro Ser Val Ser Ser Asn Asp         515 520 525 Gln Gln Val Pro Ser Ala Val Arg Tyr Asn Gly Ser Lys Arg Val Lys     530 535 540 Pro Glu Glu Glu Glu Glu Arg Asp Met Lys Lys Ser Arg Gly Phe Asp 545 550 555 560 Glu Arg Glu Leu Phe Ser Thr Ala Glu Ser Ser Ser Ser Ser Ser Val                 565 570 575 Phe Phe Val Ser Gln Ser Cys Ser Leu Ala Ser Glu Gly Lys Asn Leu             580 585 590 Glu Gly Ile Gln Asp Ser Ser Asp Gln Ile Thr Thr Ser Leu Gly Lys         595 600 605 Asn Gly Cys Lys     610 <210> 6 <211> 1839 <212> DNA <213> Arabidopsis thaliana <400> 6 atgaagatgg acaaaaagac tatagtttgg tttagaagag acctaaggat tgaggataat 60 cctgcattag cagcagctgc tcacgaagga tctgtttttc ctgtcttcat ttggtgtcct 120 gaagaagaag gacagtttta tcctggaaga gcttcaagat ggtggatgaa acaatcactt 180 gctcacttat ctcaatcctt gaaggctctt ggatctgacc tcactttaat caaaacccac 240 aacacgattt cagcgatctt ggattgtatc cgcgttaccg gtgctacaaa agtcgtcttt 300 aaccacctct atgatcctgt ttcgttagtt cgggaccata ccgtaaagga gaagctggtg 360 gaacgtggga tctctgtgca aagctacaat ggagatctat tgtatgaacc gtgggagata 420 tactgcgaaa agggcaaacc ttttacgagt ttcaattctt actggaagaa atgcttagat 480 atgtcgattg aatccgttat gcttcctcct ccttggcggt tgatgccaat aactgcagcg 540 gctgaagcga tttgggcgtg ttcgattgaa gaactagggc tggagaatga ggccgagaaa 600 ccgagcaatg cgttgttaac tagagcttgg tctccaggat ggagcaatgc tgataagtta 660 ctaaatgagt tcatcgagaa gcagttgata gattatgcaa agaacagcaa gaaagttgtt 720 gggaattcta cttcactact ttctccgtat ctccatttcg gggaaataag cgtcagacac 780 gttttccagt gtgcccggat gaaacaaatt atatgggcaa gagataagaa cagtgaagga 840 gaagaaagtg cagatctttt tcttagggga atcggtttaa gagagtattc tcggtatata 900 tgtttcaact tcccgtttac tcacgagcaa tcgttgttga gtcatcttcg gtttttccct 960 tgggatgctg atgttgataa gttcaaggcc tggagacaag gcaggaccgg ttatccgttg 1020 gtggatgccg gaatgagaga gctttgggct accggatgga tgcataacag aataagagtg 1080 attgtttcaa gctttgctgt gaagtttctt ctccttccat ggaaatgggg aatgaagtat 1140 ttctgggata cacttttgga tgctgatttg gaatgtgaca tccttggctg gcagtatatc 1200 tctgggagta tccccgatgg ccacgagctt gatcgcttgg acaatcccgc gttacaaggc 1260 gccaaatatg acccagaagg tgagtacata aggcaatggc ttcccgagct tgcgagattg 1320 ccaactgaat ggatccatca tccatgggac gctcctttaa ccgtactcaa agcttctggt 1380 gtggaactcg gaacaaacta tgcgaaaccc attgtagaca tcgacacagc tcgtgagcta 1440 ctagctaaag ctatttcaag aacccgtgaa gcacagatca tgatcggagc agcacctgat 1500 gagattgtag cagatagctt cgaggcctta ggggctaata ccattaaaga acctggtctt 1560 tgcccatctg tgtcttctaa tgaccaacaa gtaccttcgg ctgttcgtta caacgggtca 1620 aagagagtga aacctgagga agaagaagag agagacatga agaaatctag gggattcgat 1680 gaaagggagt tgttttcgac tgctgaatct tcttcttctt cgagtgtgtt tttcgtttcg 1740 cagtcttgct cgttggcatc agaagggaag aatctggaag gtattcaaga ttcatctgat 1800 cagattacta caagtttggg aaaaaatggt tgcaaatga 1839 <210> 7 <211> 1815 <212> DNA <213> Artificial Sequence <220> <223> dHygro-Linker2- mRuby <400> 7 atgtcttaga aatacaccaa atcttggggt aaaaagcctg aactcaccgc gacgtctgtc 60 gagaagtttc tgatcgaaaa gttcgacagc gtgtccgacc tgatgcagct ctcggagggc 120 gaagaatctc gtgctttcag cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat 180 agctgcgccg atggtttcta caaagatcgt tatgtttatc ggcactttgc atcggccgcg 240 ctcccgattc cggaagtgct tgacattggg gaatttagcg agagcctgac ctattgcatc 300 tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt 360 ctgcaaccgg tcgcggaggc catggatgcg atcgctgcgg ccgatcttag ccagacgagc 420 gggttcggcc cattcggacc gcaaggaatc ggtcaataca ctacatggcg tgatttcata 480 tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg tgatggacga caccgtcagt 540 gcgtccgtcg cgcaggctct cgatgagctg atgctttggg ccgaggactg ccccgaagtc 600 cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc tgacggacaa tggccgcata 660 acagcggtca ttgactggag cgaggcgatg ttcggggatt cccaatacga ggtcgccaac 720 atcttcttct ggaggccgtg gttggcttgt atggagcagc agacgcgcta cttcgagcgg 780 aggcatccgg agcttgcagg atcgccgcgg ctccgggcgt atatgctccg cattggtctt 840 gaccaactct atcagagctt ggttgacggc aatttcgatg atgcagcttg ggcgcagggt 900 cgatgcgacg caatcgtccg atccggagcc gggactgtcg ggcgtacaca aatcgcccgc 960 agaagcgcgg ccgtctggac cgatggctgt gtagaagtac tcgccgatag tggaaaccga 1020 cgccccagca ctcgtccgag ggcaaaggaa ttctcaggag gatctgaaga agaagaagga 1080 tcaggatcgg caggaggatc atccgtgtcc aaaggagagg agttaatcaa ggaaaacatg 1140 agaatgaaag ttgtcatgga gggctccgtt aatggtcacc aattcaagtg tacaggggaa 1200 ggtgaaggta atccttacat gggtacacaa actatgagaa ttaaagtaat tgaaggcgga 1260 ccactaccat ttgcatttga cattctggca acgtcattca tgtacggatc acgaactttc 1320 atcaagtacc ctaaaggtat accagacttt ttcaagcaat cttttccaga gggttttaca 1380 tgggaaaggg ttacaagata cgaagatggg ggtgtcgtca cagttatgca agatacttca 1440 ttagaagatg gctgccttgt ctatcatgtg caagtaagag gggtgaattt tccttctaac 1500 ggacctgtga tgcagaaaaa gaccaaaggt tgggaaccaa atactgaaat gatgtaccca 1560 gctgatggag gtttgagagg ctacacacac atggcgctta aagttgatgg tggaggtcat 1620 ttgtcttgta gttttgttac cacttatcgt tctaaaaaga ctgttggcaa tatcaaaatg 1680 ccaggaatac atgctgtaga ccacagacta gaaagactcg aagagagcga taacgaaatg 1740 ttcgttgtac agagagagca tgccgtagcc aaatttgctg gcttaggcgg tggtatggat 1800 gaattgtata agtaa 1815 <210> 8 <211> 1815 <212> DNA <213> Artificial Sequence <220> <223> Hygro-Linker2- mRuby <400> 8 atgtcttata aatacaccaa atcttggggt aaaaagcctg aactcaccgc gacgtctgtc 60 gagaagtttc tgatcgaaaa gttcgacagc gtgtccgacc tgatgcagct ctcggagggc 120 gaagaatctc gtgctttcag cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat 180 agctgcgccg atggtttcta caaagatcgt tatgtttatc ggcactttgc atcggccgcg 240 ctcccgattc cggaagtgct tgacattggg gaatttagcg agagcctgac ctattgcatc 300 tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt 360 ctgcaaccgg tcgcggaggc catggatgcg atcgctgcgg ccgatcttag ccagacgagc 420 gggttcggcc cattcggacc gcaaggaatc ggtcaataca ctacatggcg tgatttcata 480 tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg tgatggacga caccgtcagt 540 gcgtccgtcg cgcaggctct cgatgagctg atgctttggg ccgaggactg ccccgaagtc 600 cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc tgacggacaa tggccgcata 660 acagcggtca ttgactggag cgaggcgatg ttcggggatt cccaatacga ggtcgccaac 720 atcttcttct ggaggccgtg gttggcttgt atggagcagc agacgcgcta cttcgagcgg 780 aggcatccgg agcttgcagg atcgccgcgg ctccgggcgt atatgctccg cattggtctt 840 gaccaactct atcagagctt ggttgacggc aatttcgatg atgcagcttg ggcgcagggt 900 cgatgcgacg caatcgtccg atccggagcc gggactgtcg ggcgtacaca aatcgcccgc 960 agaagcgcgg ccgtctggac cgatggctgt gtagaagtac tcgccgatag tggaaaccga 1020 cgccccagca ctcgtccgag ggcaaaggaa ttctcaggag gatctgaaga agaagaagga 1080 tcaggatcgg caggaggatc atccgtgtcc aaaggagagg agttaatcaa ggaaaacatg 1140 agaatgaaag ttgtcatgga gggctccgtt aatggtcacc aattcaagtg tacaggggaa 1200 ggtgaaggta atccttacat gggtacacaa actatgagaa ttaaagtaat tgaaggcgga 1260 ccactaccat ttgcatttga cattctggca acgtcattca tgtacggatc acgaactttc 1320 atcaagtacc ctaaaggtat accagacttt ttcaagcaat cttttccaga gggttttaca 1380 tgggaaaggg ttacaagata cgaagatggg ggtgtcgtca cagttatgca agatacttca 1440 ttagaagatg gctgccttgt ctatcatgtg caagtaagag gggtgaattt tccttctaac 1500 ggacctgtga tgcagaaaaa gaccaaaggt tgggaaccaa atactgaaat gatgtaccca 1560 gctgatggag gtttgagagg ctacacacac atggcgctta aagttgatgg tggaggtcat 1620 ttgtcttgta gttttgttac cacttatcgt tctaaaaaga ctgttggcaa tatcaaaatg 1680 ccaggaatac atgctgtaga ccacagacta gaaagactcg aagagagcga taacgaaatg 1740 ttcgttgtac agagagagca tgccgtagcc aaatttgctg gcttaggcgg tggtatggat 1800 gaattgtata agtaa 1815 <210> 9 <211> 1812 <212> DNA <213> Artificial Sequence <220> <223> dHygro-Linker3- mTurquiose2 <400> 9 atgtcttaga aatacaccaa atcttggggt aaaaagcctg aactcaccgc gacgtctgtc 60 gagaagtttc tgatcgaaaa gttcgacagc gtgtccgacc tgatgcagct ctcggagggc 120 gaagaatctc gtgctttcag cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat 180 agctgcgccg atggtttcta caaagatcgt tatgtttatc ggcactttgc atcggccgcg 240 ctcccgattc cggaagtgct tgacattggg gaatttagcg agagcctgac ctattgcatc 300 tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt 360 ctgcaaccgg tcgcggaggc catggatgcg atcgctgcgg ccgatcttag ccagacgagc 420 gggttcggcc cattcggacc gcaaggaatc ggtcaataca ctacatggcg tgatttcata 480 tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg tgatggacga caccgtcagt 540 gcgtccgtcg cgcaggctct cgatgagctg atgctttggg ccgaggactg ccccgaagtc 600 cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc tgacggacaa tggccgcata 660 acagcggtca ttgactggag cgaggcgatg ttcggggatt cccaatacga ggtcgccaac 720 atcttcttct ggaggccgtg gttggcttgt atggagcagc agacgcgcta cttcgagcgg 780 aggcatccgg agcttgcagg atcgccgcgg ctccgggcgt atatgctccg cattggtctt 840 gaccaactct atcagagctt ggttgacggc aatttcgatg atgcagcttg ggcgcagggt 900 cgatgcgacg caatcgtccg atccggagcc gggactgtcg ggcgtacaca aatcgcccgc 960 agaagcgcgg ccgtctggac cgatggctgt gtagaagtac tcgccgatag tggaaaccga 1020 cgccccagca ctcgtccgag ggcaaaggaa ttctcaggtg gaggcgggtc tggaggcggg 1080 ggtagtggat catccgtttc taaaggtgaa gaattattca ctggtgttgt cccaattttg 1140 gttgaattag atggtgatgt taatggtcac aaattttctg tctccggtga aggtgaaggt 1200 gatgctactt acggtaaatt gaccttaaaa tttatttgta ctactggtaa attgccagtt 1260 ccatggccaa ccttagtcac tactttatct tggggtgttc aatgttttgc aagataccca 1320 gatcatatga aacaacatga ctttttcaag tctgccatgc cagaaggtta tgttcaagaa 1380 agaactattt ttttcaaaga tgacggtaac tacaagacca gagctgaagt caagtttgaa 1440 ggtgatacct tagttaatag aatcgaatta aaaggtattg attttaaaga agatggtaac 1500 attttaggtc acaaattgga atacaattat ttctctgaca atgtttacat cactgctgac 1560 aaacaaaaga atggtatcaa agctaacttc aaaattagac acaacattga agatggtggt 1620 gttcaattag ctgaccatta tcaacaaaat actccaattg gtgatggtcc agtcttgtta 1680 ccagacaacc attacttatc cactcaatct aagttatcca aagatccaaa cgaaaagagg 1740 gaccacatgg tcttgttaga atttgttact gctgctggta ttaccttggg tatggatgaa 1800 ttgtacaaat aa 1812 <210> 10 <211> 1812 <212> DNA <213> Artificial Sequence <220> <223> Hygro-Linker3- mTurquiose2 <400> 10 atgtcttata aatacaccaa atcttggggt aaaaagcctg aactcaccgc gacgtctgtc 60 gagaagtttc tgatcgaaaa gttcgacagc gtgtccgacc tgatgcagct ctcggagggc 120 gaagaatctc gtgctttcag cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat 180 agctgcgccg atggtttcta caaagatcgt tatgtttatc ggcactttgc atcggccgcg 240 ctcccgattc cggaagtgct tgacattggg gaatttagcg agagcctgac ctattgcatc 300 tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt 360 ctgcaaccgg tcgcggaggc catggatgcg atcgctgcgg ccgatcttag ccagacgagc 420 gggttcggcc cattcggacc gcaaggaatc ggtcaataca ctacatggcg tgatttcata 480 tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg tgatggacga caccgtcagt 540 gcgtccgtcg cgcaggctct cgatgagctg atgctttggg ccgaggactg ccccgaagtc 600 cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc tgacggacaa tggccgcata 660 acagcggtca ttgactggag cgaggcgatg ttcggggatt cccaatacga ggtcgccaac 720 atcttcttct ggaggccgtg gttggcttgt atggagcagc agacgcgcta cttcgagcgg 780 aggcatccgg agcttgcagg atcgccgcgg ctccgggcgt atatgctccg cattggtctt 840 gaccaactct atcagagctt ggttgacggc aatttcgatg atgcagcttg ggcgcagggt 900 cgatgcgacg caatcgtccg atccggagcc gggactgtcg ggcgtacaca aatcgcccgc 960 agaagcgcgg ccgtctggac cgatggctgt gtagaagtac tcgccgatag tggaaaccga 1020 cgccccagca ctcgtccgag ggcaaaggaa ttctcaggtg gaggcgggtc tggaggcggg 1080 ggtagtggat catccgtttc taaaggtgaa gaattattca ctggtgttgt cccaattttg 1140 gttgaattag atggtgatgt taatggtcac aaattttctg tctccggtga aggtgaaggt 1200 gatgctactt acggtaaatt gaccttaaaa tttatttgta ctactggtaa attgccagtt 1260 ccatggccaa ccttagtcac tactttatct tggggtgttc aatgttttgc aagataccca 1320 gatcatatga aacaacatga ctttttcaag tctgccatgc cagaaggtta tgttcaagaa 1380 agaactattt ttttcaaaga tgacggtaac tacaagacca gagctgaagt caagtttgaa 1440 ggtgatacct tagttaatag aatcgaatta aaaggtattg attttaaaga agatggtaac 1500 attttaggtc acaaattgga atacaattat ttctctgaca atgtttacat cactgctgac 1560 aaacaaaaga atggtatcaa agctaacttc aaaattagac acaacattga agatggtggt 1620 gttcaattag ctgaccatta tcaacaaaat actccaattg gtgatggtcc agtcttgtta 1680 ccagacaacc attacttatc cactcaatct aagttatcca aagatccaaa cgaaaagagg 1740 gaccacatgg tcttgttaga atttgttact gctgctggta ttaccttggg tatggatgaa 1800 ttgtacaaat aa 1812 <210> 11 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> AtGOX3-N1-Fwd <400> 11 gcatcgtctc atcggtctca tatggagata acaaacgtga tgga 44 <210> 12 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> AtGOX3-N1-Rev <400> 12 atgccgtctc attgtagcat cttcctaact cccgcttc 38 <210> 13 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> AtGOX3-C1-Fwd <400> 13 gactcgtctc aacaaatgct aagagatgag tttgagct 38 <210> 14 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> AtGOX3-C1-Rev <400> 14 atgccgtctc aggtctcagg atagtcttga tgtgggttcg gctgat 46 <210> 15 <211> 96 <212> DNA <213> Artificial Sequence <220> <223> NLS-2x-4a <400> 15 gcatcgtctc atcggtctca atcccgccaa aaaagaagag aaaggtagat ccaaaaaaga 60 agagaaaggt ataatgcctg agacctgaga cggcat 96 <210> 16 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> NLS-4a-Fwd1 <400> 16 agtgcatcgt ctcatcggtc tcaatcccg 29 <210> 17 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> NLS-4a-Rev1 <400> 17 gacatgccgt ctcaggtctc aggcatta 28 <210> 18 <211> 56 <212> DNA <213> Artificial Sequence <220> <223> ydCas9-N1-Fwd <400> 18 cagtccgtct catcggtctc atatggacaa gaagtattct atcggactgg cgatcg 56 <210> 19 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> ydCas9-N1-Rev <400> 19 cagtccgtct cacgtagtct gagagcctgt tgatatcaag ctcttggtcc 50 <210> 20 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> ydCas9-C1-Fwd <400> 20 cagtccgtct cttacgacgt ggacgcgatc gtccctcaga gcttcctc 48 <210> 21 <211> 66 <212> DNA <213> Artificial Sequence <220> <223> ydCas9-C1-Rev <400> 21 cagtccgtct caggtctcag gatgtgatga tgatgatgat gaccatcccc tccgagctgt 60 gagagg 66 <210> 22 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> NLS-4a-Fwd2 <400> 22 agtgcatcgt ctcatcggtc tcaatcccgc ca 32 <210> 23 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> NLS-4a-Rev2 <400> 23 gacatgccgt ctcaggtctc agccatta 28 <210> 24 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> ydHygro-Fwd1 <400> 24 cagtccgtct catcggtctc atatgtctta gaaatacacc aaatcttggg gtaaaaagcc 60 tgaactcacc gcgac 75 <210> 25 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> yHygro-Fwd1 <400> 25 cagtccgtct catcggtctc atatgtctta taaatacacc aaatcttggg gtaaaaagcc 60 tgaactcacc gcgac 75 <210> 26 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> ydHygro-Rev1 <400> 26 cagtccgtct caggtctcaa gaattccttt gccctcggac gagtgctgg 49 <210> 27 <211> 93 <212> DNA <213> Artificial Sequence <220> <223> Linker2 <400> 27 cagtccgtct catcggtctc attctcagga ggatctgaag aagaagaagg atcaggatcg 60 gcaggaggat catcctgaga cctgagacgg cat 93 <210> 28 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Linker2-Fwd1 <400> 28 agtgcatcgt ctcatcggtc tcattctc 28 <210> 29 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Linker2-Rev1 <400> 29 gacatgccgt ctcaggtctc aggatgatc 29 <210> 30 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Hygro-gRNA -Fwd1 <400> 30 gcatgaagac ttgatcctta gaaatacacc aaatctgttt atgtcttcac t 51 <210> 31 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Hygro-gRNA-Rev1 <400> 31 agtgaagaca taaacagatt tggtgtattt ctaaggatca agtcttcatg c 51 <210> 32 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> mRuby2-gRNA -Fwd1 <400> 32 gcatgaagac ttgatcagaa gaagaaggat caggatgttt atgtcttcac t 51 <210> 33 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> mRUby2-gRNA -Fwd1 <400> 33 agtgaagaca taaacatcct gatccttctt cttctgatca agtcttcatg c 51 <210> 34 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> AtGOX3-Rev1 <400> 34 atgccgtctc aggtctcacg gaagtcttga tgtgggttcg gctgat 46 <210> 35 <211> 99 <212> DNA <213> Artificial Sequence <220> <223> Linker1 <400> 35 tccggcgggt catcaggggg gagtagcgga tctgagactc cggggaccag cgaatccgct 60 accccagagt caagcggtgg tagttctggg ggaagttct 99 <210> 36 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> Linker1 Fwd <400> 36 agtgcatcgt ctcatcggtc tcatccggcg ggtcatcagg ggggagt 47 <210> 37 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Linker1 Rev <400> 37 gacatgccgt ctcaggtctc aagaacttcc cccagaacta ccaccgct 48 <210> 38 <211> 56 <212> DNA <213> Artificial Sequence <220> <223> ydCas9-Fwd1 <400> 38 cagtccgtct catcggtctc attctgacaa gaagtattct atcggactgg cgatcg 56 <210> 39 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> ScTDH3 Pro-Fwd <400> 39 cagtccgtct catcggtctc atgatgacac aaggcaattg acccacgcat g 51 <210> 40 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> ScENO1 Ter-Rev <400> 40 cagtccgtct caggtctcaa tacatgggtg accaaaagag cgg 43 <210> 41 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> mRuby-Fwd1 <400> 41 gtgtccaaag gagaggagtt aatc 24 <210> 42 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> mRuby-Rev1 <400> 42 ttacttatac aattcatcca taccaccgcc 30 <210> 43 <211> 84 <212> DNA <213> Artificial Sequence <220> <223> Linker3 <400> 43 cagtccgtct catcggtctc attctcaggt ggaggcgggt ctggaggcgg gggtagtgga 60 tcatcctgag acctgagacg gcat 84 <210> 44 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> CRY1-Fwd1 <400> 44 gcatcgtctc atcggtctca tatgtctggt tctgtatctg gttgtggttc tggtggt 57 <210> 45 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> CRY1-Rev1 <400> 45 atgccgtctc accagaagtg cattgcttcc tttctcagag tc 42 <210> 46 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> CRY1-Fwd2 <400> 46 gactcgtctc actggctcgt gcttggtcac ctggatggag taatggtgat aaag 54 <210> 47 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> CRY1-Rev2 <400> 47 atgccgtctc acaggaaaag attcacgctt tcttccccgg cct 43 <210> 48 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> CRY1-Fwd3 <400> 48 gactcgtctc acctgaaatc tattggcctc agggagt 37 <210> 49 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> CRY1-Rev3 <400> 49 atgccgtctc aggtctgatc aaagaagaag taatgcttgg aacca 45 <210> 50 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> CRY1-Fwd4 <400> 50 gactcgtctc agacctgaag aagacgaaga gtcgtcactt aatttg 46 <210> 51 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> CRY1-Rev4 <400> 51 atgccgtctc aatgcctccg cttctttctc tccttccgct 40 <210> 52 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> CRY1-Fwd5 <400> 52 gactcgtctc agcatagtcc ccgagtggtc accagggtac tcagagca 48 <210> 53 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> CRY1-Rev5 <400> 53 atgccgtctc aggtctcagg atttacccgg tttgtgaaag ccgcctcca 49 <210> 54 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> CRY2-Fwd1 <400> 54 gcatcgtctc atcggtctca tatgaagatg gacaaaaaga ctatagtttg gtttagaagg 60 gacctaagga 70 <210> 55 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> CRY2-Rev1 <400> 55 atgccgtctc acaacgcatt gctcggtttc tcggcctcat tc 42 <210> 56 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> CRY2-Fwd2 <400> 56 gactcgtctc agttgttaac tagagcttgg tcaccaggat ggagcaat 48 <210> 57 <211> 66 <212> DNA <213> Artificial Sequence <220> <223> CRY2-Rev2 <400> 57 atgccgtctc aggtctcagg attcatttgc aaccattttt tcccaaactt gtagtaatct 60 gatcag 66

Claims (6)

Polynucleotides encoding enzymes that produce reactive oxygen species; Polynucleotides encoding an inactivated Cas9 (CRISPR associated protein 9) protein or a functional analog thereof; And a recombinant vector comprising a guide RNA coding sequence. The recombinant vector according to claim 1, wherein the enzyme producing the reactive oxygen species is glycolic oxidase or cryptochrome. A host cell transformed with the recombinant vector of claim 1. A method of inducing mutation of a base of a target gene comprising transforming a host cell with the recombinant vector of claim 1. 5. The method of claim 4, wherein said method of inducing mutations of the bases is characterized by random base mutations of reactive oxygen species mediated target genes. Base mutation inducing composition comprising the recombinant vector of claim 1 or 2 as an active ingredient.

Images