KR102110600B1 - Frrs1l gene knockout Autism Spectrum Disorder animal model and using thereof - Google Patents

Abstract

The present invention relates to an autism spectrum disorder animal model and uses thereof, and the Frrs1l gene knockout autism spectrum disorder animal model according to the present invention can be used in various ways, such as identifying mechanisms of disease, exploring therapeutic substances, and developing diagnostic methods. .

Description

Frrs1l gene knockout Autism Spectrum Disorder animal model and using thereof}

The present invention relates to autism spectrum disorder animal models and uses thereof.

Autism Spectrum Disorder (ASD) refers to a mental illness that causes difficulties in establishing and communicating with others due to various developmental conditions. Narrow interest-limited editing and repetitive behavior are symptoms of typical autism spectrum disorders. Genetic, neurobiological, and developmental research on autism spectrum disorders has been conducted, but the mechanisms that cause autism spectrum disorders have not been precisely identified, and the treatment is not yet complete.

For the treatment of autism spectrum disorder, it is essential to understand the biological mechanisms that cause autism spectrum disorder, so for the biological research on this, several behavioral experiments were developed to test social, communication, and repetitive behaviors, which are typical symptoms of autism spectrum disorder. Became. The 3-chamber test is a well-known behavioral test paradigm that tests social and social novelty perception.

On the other hand, Frrs1l (Ferric Chelate Reductase 1 Like) protein is a protein expressed in a number of brain regions, and corresponds to an auxiliary protein of the AMPA receptor. According to a recent study, when knocking down the Frrs1l gene, it is possible to reduce the amount of current mediated by the AMPA receptor. However, the effect of Frrs1l protein on the treatment of autism spectrum disorder has not been known.

Korean Registered Patent No. 10-1167945

The present inventors have made extensive efforts to develop an animal behavior experiment model that can identify autism spectrum disorders. As a result, the present invention was completed by identifying the autism spectrum disorder specific behavioral disorder and social novel cognitive disorder in the knock-out mouse of the Frrs1l gene.

Therefore, the object of the present invention is to provide an animal model for autism spectrum disorder (Autism Spectrum Disorder) in which the Frrs1l (Ferric Chelate Reductase 1 Like) gene is knocked out.

Another object of the present invention is to provide a method for preparing an animal model for autism spectrum disorder comprising knocking out the Frrs1l gene.

Another object of the present invention is to provide a method for improving autism spectrum disorder or screening a therapeutic agent.

Another object of the present invention is to provide an information providing method for diagnosing autism spectrum disorder.

According to one aspect of the present invention, the present invention provides an animal model for autism spectrum disorder (knock-out) in which the Frrs1l (Ferric Chelate Reductase 1 Like) gene is knocked out.

The present inventors have made extensive efforts to develop an animal behavior experiment model that can identify autism spectrum disorders. As a result, it was investigated that a novel autism spectrum disorder-specific behavioral disorder and social novel cognitive disorder appeared in the knock-out mouse of the Frrs1l gene.

The Frrs1l protein expressing the Frrs1l (Ferric Chelate Reductase 1 Like) gene, which is the target gene of the present invention, is a protein expressed in a number of brain regions and is known as an auxiliary protein of the AMPA receptor, but suppresses the expression or activity of Frrs1l Social novelty through cognitive disability has not been reported.

The nucleotide sequence of the gene is shown in SEQ ID NO: 1, and the amino acid sequence of the protein is shown in SEQ ID NO: 2. In addition, since the nucleotide sequence of the gene of the present invention is registered in the gene bank, it will be readily available to those skilled in the art.

In the present invention, "animal model" refers to an animal having a disease in a form very similar to a human disease. The significance of disease model animals in human disease studies is due to physiological or genetic similarities between humans and animals. In disease research, biomedical disease model animals provide research materials for various causes of disease, pathogenesis, and diagnosis, and through disease model animals research, they can identify genes related to diseases and understand interactions between genes. In addition, it is possible to obtain basic data to judge the possibility of practical use through the actual efficacy and toxicity test of the developed new drug candidate.

In the present invention, "animal" or "experimental animal" means any mammalian animal other than human. Such animals include animals of all ages, including embryos, fetuses, newborns and adults. Animals for use in the present invention are available, for example, from commercial sources. These animals include rabbits, rodents (e.g. mice, rats, hamsters, gerbils, and guinea pigs), cows, sheep, pigs, goats, horses, dogs, cats, and birds (e.g. chickens, turkeys, ducks, Geese), primates (eg, chimpanzees, monkeys, rhesus monkeys), preferably mice, but are not limited thereto.

According to another aspect of the present invention, the present invention provides a method for preparing an animal model for autism spectrum disorder comprising knocking out the Frrs1l gene in animals other than humans.

In the present invention, "step of knocking out the Frs1l gene" can be performed by any method used to knock out the Frrs1l gene in the art, for example, heterozygous for the Frrs1l gene homologue. It can be performed by a method of performing knockout, specifically, inserting a LoxP site before and after the third exon of the Frrs1l gene, and deleting the third exon after Cre recombinase action.

According to another aspect of the present invention, the present invention provides a method for screening for an autism spectrum disorder improvement or therapeutic agent comprising the following steps.

(a) administering an autism spectrum disorder improvement or therapeutic candidate to the animal model of claim 1;

(b) measuring and comparing autism spectrum disorders with a control animal model not administered with the animal model administered with the candidate substance; And

(C) selecting a candidate substance that significantly recovered autism spectrum disorder compared to the control group.

In the present invention, "candidate" means a substance to be tested as an autism spectrum disorder improvement and therapeutic agent, and includes any molecule such as, for example, extracts, proteins, oligopeptides, small organic molecules, polysaccharides, polynucleotides, and a wide range of compounds. can do. Such candidates also include natural as well as synthetic materials.

In the present invention, "improvement" and "treatment" means all actions in which the symptoms of autism spectrum disorder in the animal model are improved or beneficially changed by administration of the candidate substance.

In the present invention, "control group" refers to a group that does not apply any manipulation or conditions to determine whether or not an experimental result has been properly derived, and unlike the experimental group, which is a group set to achieve the direct purpose of the experiment, the control group has some results of the experimental group. It is a group established to make it more certain. The control group in the present invention preferably refers to a group not treated with a candidate substance in the autism spectrum disorder animal model.

In the present invention, "selecting a candidate material" may include a step of confirming that a tendency to favor a new animal in social novelty recognition is significantly reduced compared to a control group.

In the present invention, the animal model of autism spectrum disorder is a disease model for autism spectrum disorder, and the material obtained by the screening method is variously utilized for the exploration of therapeutic substances for the patients with autism spectrum disorder, identification of side effects, development of diagnostic methods, etc. Improvements or treatments for spectrum disorders can be developed.

According to another aspect of the present invention, the present invention provides a method for providing information for autism spectrum disorder diagnosis comprising the following steps.

(a) measuring the level of expression or activity of Frrs1l present in the biological sample; And

 (b) comparing the measured expression or activity level of Frrs1l with that of a normal control.

In the present invention, when the level of expression or activity of Frrs1l present in a biological sample is measured, and the measurement result is compared with the level of expression or activity of a normal control sample, when the level of expression or activity of Frrs1l decreases, it is referred to as autism spectrum disorder. Can be judged.

The methods of the invention include mammals, especially humans. At this time, the human subject includes a person suspected of having autism spectrum disorder or a person who is not suspected and needs to be diagnosed with autism spectrum disorder.

In the present invention, "biological sample" may be composed of tissue, cells, blood, serum, plasma, saliva, and urine, but is not limited thereto.

The sample may be pre-treated prior to use for detection, and may include, for example, filtration, distillation, extraction, concentration, inactivation of interference components, addition of reagents, and the like. In addition, nucleic acids and proteins can be separated from the sample and used for detection.

In the present invention, measuring the level of expression or activity of Frrs1l includes polymerase reaction (PCR), reverse transcriptase reaction (RT-PCR), competitive reverse transcriptase reaction (Competitive RT-PCR), and real-time reverse transcriptase reaction ( Realtime RT-PCR), RNase protection assay (RPA), Northern blotting, DNA chip, Western blotting, Enzyme linked immunosorbent assay (ELISA), Radioimmunoassay, RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunoprecipitation assay, complement fixation assay, protein chip, and In It can be measured by one or more methods selected from the group consisting of in vitro kinase assay, but is not limited thereto.

Duplicate contents are omitted in consideration of the complexity of the present specification, and terms that are not otherwise defined herein have meanings commonly used in the technical field to which the present invention pertains.

The present invention relates to an autism spectrum disorder animal model and uses thereof, and the Frrs1l gene knockout autism spectrum disorder animal model according to the present invention can be used in various ways, such as identifying mechanisms of disease, exploring therapeutic substances, and developing diagnostic methods. .

1 shows a gene schematic of Frrs1l-knockout mice.
FIG. 2 shows the results of a sociability experiment of a 3-chamber test for Frrs1l-knockout mice (Frrs1l ^+/- ) and WT (Wild Type) mice, a) Representative heatmap image. b) Bonferroni's post hoc multiple comparison test results (n = 16, WT; n = 17, Frrs1l ^+/- ; chamber F (1,62) = 44.04, P <0.0001, two-way ANOVA; ^**** P < 0.0001), and c) differential index (P = 0.725, unpaired two-tailed t-test).
FIG. 3 shows the results of a social novelty recognition experiment of a 3-chamber test for Frrs1l-knockout mice (Frrs1l ^+/- ) and WT (Wild Type) mice, a) Heat map image. b) Bonferroni's post hoc multiple comparison test results (n = 16, WT; n = 17, Frrs1l ^+/- ; chamberХgenotype F (1,62) = 5.563, P = 0.0215, two-way ANOVA; ^* P <0.05), And c) differential index (P = 0.0008, unpaired two-tailed t-test).
FIG. 4 shows the results of an Open Field Test for Frrs1l-knockout mice (Frrs1l ^+/- ) and Wild Type (WT) mice, a) Total distance traveled in Open filed (n = 16, WT ; n = 17, Frrs1l ^+/- ; P = 0.9599, unpaired two-tailed t-test), and b) time spent in the center zone (P = 2645, unpaired two-tailed t-test) .
FIG. 5 shows the results of an object location memory experiment for Frrs1l-knockout mice (Frrs1l ^+/- ) and WT (Wild Type) mice, a) a discrimination index (n = 8, WT ; n = 12, Frrs1l ^+/- ; P = 0.6917, unpaired two-tailed t-test), and b) total exploration time (P = 0.3913, unpaired two-tailed t-test).
FIG. 6 shows the results of a contextual fear memory experiment for Frrs1l-knockout mice (Frrs1l ^+/- ) and Wild Type (WT) mice (n = 8, WT; n = 12, Frrs1l ^+/- ; P = 0.2935, unpaired two-tailed t-test).

Hereinafter, the present invention will be described in more detail through examples. These examples are only intended to illustrate the present invention more specifically, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Ready. Experimental animals

C57BL / 6 mouse-based Frrs1l heterozygous knockout mice (Medical Research Council (MRC) Harwell) were used.

The Frrs1l-knockout mouse was prepared by inserting the LoxP site back and forth in the third exon of the Frrs1l gene, and deleting the third exon after Cre recombinase action, and the knockout effect was generated by generating a truncated protein due to the frameshift mutation according to the exon deletion. Occurs (see FIG. 1).

The produced Frrs1l-knockout mice were lined with wild-type (WT) C57B / 6 mice to maintain the line, and adult (2-3 months old) animals were used without sex. In this case, a littermate heterozygous mouse and a WT mouse were used as an experimental group and a control group, respectively. The experimental animals were kept in a 12-hour light / dark cycle, constant-humidity (22 ± 1 ℃, 40-60%) conditions, and all animals used in the experiment were handled by the experimenter for 5 days before the experiment was conducted. .

Experimental Example 1. 3-Chamber Test

In order to examine the effect of the Frrs1l-knockout effect, a 3-chamber test was performed as follows using Frrs1l ^+/- mice and littermate WT mice.

1-1. Check sociality

First, in order to confirm sociality, the experimental animal was subjected to a habituation process by allowing three equally sized chambers to freely enter and exit through the door. Thereafter, an empty wire cup was placed in one of the two chambers, and a stranger mouse of the same species, which had no contact with the experimental animal, was placed in the wire cup of the other chamber. The time to interact with the empty cup and the cup containing the unfamiliar animals was measured.

As a result, as shown in Figure 2, Frrs1l ^+/- and littermate WT mice, it was confirmed that the interaction time for the cup containing the unfamiliar animals was statistically significantly higher than the interaction time for the empty cup. That is, Frrs1l ^+/- mice show normal sociality.

1-2. Check social novelty recognition

After the sociality experiment of Experimental Example 1-1, the social novelty cognition experiment was conducted. This is an experiment using the tendency that the mouse prefers a novel mouse over a familiar mouse.

More specifically, the animal (familiar mouse) used in Experimental Example 1 was put into a wire cup in one of the two chambers, and a new animal (novel mouse) of the same species that had no contact with the experimental animal in the wire cup in the other chamber. ), The time at which the experimental animals interacted with the cup containing each familiar animal and the cup containing the unfamiliar animals was measured.

As a result, as shown in FIG. 3, a tendency to more favor new animals as predicted in WT mice was observed, whereas in Frrs1l ^+/- mice, there was no difference in preference between familiar and new mice, and Frrs1l ^{+ /-The} discrimination index of the mouse (see Experimental Example 3 below) was also confirmed to be significantly lower than that of the WT mouse. That is, it means that Frrs1l ^+/- mouse does not recognize and recognize familiar animals and new animals, so it may be difficult to recognize social novelty.

Experimental Example 2. Open Field Test

Based on the results of Experimental Example 1, the locomotor activity and anxiety level of Frrs1l-knockout mice were confirmed.

More specifically, the total distance traveled in the Open Filed (motor activity) and the time in the center zone (anxiety level) were measured and compared using Frrs1l ^+/- and littermate WT mice, respectively.

As a result, as shown in FIG. 4, no statistically significant difference was observed in the total distance traveled in the Open Filed and the time stayed in the central zone in both groups. That is, the motor activity and anxiety level of Frrs1l ^+/- mice are normal.

Experimental Example 3. Checking object location memory (OLM)

Based on the results of Experimental Example 1, the object location memory level of Frrs1l-knockout mice was confirmed.

More specifically, Frrs1l +/- and littermate WT mice were used to learn the location of two identical objects. The next day, after changing the position of one object to a new position, the mouse was searched again, the object exploration time of each mouse was measured, and the discrimination index was calculated. At this time, the discrimination index uses the characteristic that the mouse exhibits a higher search activity when the object is in the new position rather than the learned position, as shown below, the search time for the object in the new position and the position in the learned position. The difference in search time for an object was calculated by dividing by the sum.

As a result, as shown in FIG. 5, similar levels of discrimination index and total search time were observed in both groups. That is, there is no abnormality in Frrs1l ^+/- mouse's object location memory.

Experimental Example 4. Confirmation of contextual fear memory

Based on the results of Experimental Example 1, the contextual fear memory level of Frrs1l-knockout mice was confirmed. Contextual fear memory corresponds to hippocampus-dependent brain function.

First, after learning the contextual fear memory with the context of the conditioning chamber as the conditioned stimulus (CS) and the foot shock as the unconditioned stimulus (US), To verify memory formation, after 24 hours, exposure to the same stimulus and freezing behavior were measured.

As a result, as shown in FIG. 6, it was confirmed that similar levels of freezing were induced in the two groups. This means that Frrs1l ^+/- mice have no abnormalities in contextual fear memory.

Sintering

Taken together, Frrs1l ^+/- mice of the present invention are normal for motor activity, anxiety level and hippocampal dependent memory (object location memory and contextual fear memory), while one of the key symptoms of autism spectrum disorder (ASD) It can be seen that there are specific obstacles only to recognition of social novelty.

That is, the deletion of the heterozygous deletion of the Frrs1l gene causes social novelty cognitive-specific disorders, rather than a wide range of social behavioral disorders including sociality, social novelty cognition, and socially transmitted fear, so Frrs1l gene is autism spectrum disorder ( ASD) can be usefully used for diagnosis, treatment, and prevention.

<110> Korea Advanced Institute of Science and Technology <120> Frrs1l gene knockout Autism Spectrum Disorder animal model and          using thereof <130> PN180048 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 36999 <212> DNA <213> Human FRRS1L genome <220> <223> Human FRRS1L genome <400> 1 atgcgcagac cgaggcaggg aggcgggggt gcgggaggct ccgcggctgc gcgggcgcgt 60 gctggtggcc tcggcggagg ctcagtcccg gcccgcgccc gcggcgcccc cgctgcggcc 120 cgagcggcct ggctgcggga tctgtgcgca cggatggcgc ggccgccccg gcagcacccg 180 ggggtctggg cgtcgctgct cctgctgcta ctgacggggc ccgccgcctg cgcagccagc 240 cccgcggacg acggtgcggg cccggggggc cggggacccc ggggacgcgc gcggggggac 300 acgggcgccg acgaggcggt gccgcgccac gactcctcct acggcacctt cgcgggggag 360 ttctacgacc tgcgctacct gtcggaggag ggtgaggctg cgggagggcg aggggttgcg 420 ggaggggaga ccgggggtgg gggaccgagg atcgggggaa cgaggagagg gggcgaggga 480 gggggagggg gtcggaaaag atgtccgtgg aggcagctct tgcagaggcg aggccaggat 540 ggggagcgca gtgcccaggc ctccgccggc ctggaggaaa ggcccaaaga gctgtcaatg 600 cctgccgccc 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acagctagaa agtagcagag aactgtgtgg atttgttgat 4860 ggacttaatg gataaagtgg tagaaggtgt cctgcccatc tggaatggag ggtccctgtt 4920 gacttgcact aggaaatgga ttggacaggt agaattgctt tttcgttttt gtttttgaga 4980 cagaatctcg ctctgtcatc caggctggag tgcagtagtg cgatctcagc tcactgcaac 5040 ctccacctcc taggttcaaa tgatgctcat gcctcagcct cccaagtagc tgggattaca 5100 ggcatgagcc accacacctg gctaattttt gtatttttag tagagacagg gtttcaccct 5160 gttgaccggg ctgatcttga actcctgacc tcaagagatc tgcctgcctt ggccccccaa 5220 agtgctggaa ttacatgcgt gagccaccat gtctggccta gagttgggta tttttgtgag 5280 tgaacttgag aggaagagtt tggaactgat atggtagaca gttagggact acagtttcct 5340 aagcagtgga gtgccaccat gaaggctgag tttgcttctg tgtggaactt tacaccttat 5400 aaaacccctc gatactttta cttcataata cgaccctgaa aagagaagga acaaaaattg 5460 aggccgtcgg gaaagagact gatgcagtag ttctcagatg aaactgaatg gtgtggccag 5520 acaattaaaa cttaaacttc aaggaatcaa gtactaaatt aatagctaaa ggctgtgagc 5580 caagccatta attagaagca ataaatagca aaccagaagc cattaaccac ccagtagagt 5640 gtggtaaaag ggattgcaaa aaaggaacag atagtttctt gaattagaca agaatggaaa 5700 ccacaaagcc caattgagaa cctaaaaaat caaccaagaa aataagcacc tttcatggtt 5760 cagcaataaa tgaagggatt ataaaataat agagaggaaa ggagggagag aaaaaaatgg 5820 ctcgaattgt tctccaaaac agaatttact gaaagagctt atagagtcca tgtcagcaga 5880 aggaaagaaa aggaaggaga agaattgata cacccttttt taaaaaaccc ccaagccaat 5940 attaatgtag tctcattcag gttcttattt tagagggaaa aaaatagcat caaaatggaa 6000 aatggagaga aaggaacaag ccaaaacaat ttaagtgatg aaaaatgggg gaacggacag 6060 agcgaaggga agagggaaaa gctggttgtg tctgggtggt tcaacacata tgtctcaaga 6120 ctaaggagga caggtaggtg taggggagat ggaatgctgg ggagtagtgt caggagcagt 6180 gggatgacag aattagattt ctgcttcagg ccgggtgagg cggcctgtaa tcccagcacg 6240 ttgggaagcc gaggcggcag atcacctgag gtcaggagtt caagaccaac ctggccaaca 6300 tggtgaaacc tgtctctaca aaaatacaaa aattagccgg gcatgatggc gggtgcctgt 6360 aatcccagct actcgggagg ctgaggcagg agaaccactt gaacctggca ggcggagctt 6420 gcagtgagcc aagatcatgc cattgcactc cagcttgggc gacagagcga gactctgtct 6480 aaaaaaaaaa aaaaaaaaaa gatttctttc tcagaaagat ggcggcaggg cagaaggagt 6540 gagagagaag ccagagaacc agataagaga cagaactaca gccaaaagtc aggctctggt 6600 ttctgaggta ggaataacaa gaagtggtca ggtgcagtgg ctcatgcctg taatcccagc 6660 actttgagag gagcccagga gtttgagatc agcttgggca agaaagtgat accccatctc 6720 tacaaaaaga taaaaaagtt agccagtcat ggtggcacac agctgtggtc ccagctacac 6780 gagaggctga ggcaggagga tcacttgagc ctaggaggtc gaggctgtgc tccagtctgg 6840 gtgacagagt gagaccctgt ctcaaaataa ataaataaaa ggaatagcag gaagtgatgg 6900 ctgagtaaat gtgccaggtt ggggaaaggg agaaacttag gactgaggtt tcaggattgt 6960 gagactgtag gtggctagtg ggattatcca ctaagataag aaacatagga cgatagcttt 7020 tgggaggcgg aggaactcat ctggaattga tttgcttgtg gaatatccag ggagaaaccc 7080 ctggaggcag ttggctgttg ggtttaaact gatagaagaa aagaagaaag gccgaaaact 7140 gtgaagtgac agaaagaagg gaataaatct aaagatagta gaaggaaaca aataacaaaa 7200 taataaagat atagcagaat taatgaaata gaaaccaaat attcagtaaa ggggatccac 7260 aaagcctaaa gtgattcttt gaaaatatta ataagacaga taagtgtata gtcagggttc 7320 tccagagaaa caaaagcaat aggatatgta tctatgtaga cagatttatt ttaaggaact 7380 ggttcacatg attatgcagg ctggcaggtt ccacagtctg caggttgggt cggcaggctg 7440 gagacccaga gatgagctga tgtttgcaag tcaggtccaa aggccatctg ctgctacact 7500 ggttggtgga ggtcactctt tttgttctct tcaggccttt gactgattgg atgaggccaa 7560 cttgcattat ggagggcaat ctactttgct caaaggccac ctgcttaaat gtttttttgg 7620 ttgttgttgt tgttgagaca gagtctcgct ctgtcaccca ggctggaatg caatggcgcg 7680 atcttggctc actacacttc ccaggttcaa gcgattctcc tgcctcagct tcccaagtag 7740 ctgggattac aggtgggtcc caccatgcct ggcaaatttt tgtattttta gtaaagacag 7800 ggtttcacca tgttggtcag gctggtctcg aacccctgac ctcgtgatcc acccaccttg 7860 gccagagtgc tgggattaca ggtgtgagcc accacgcccg gcccacccac ttaaatgtta 7920 atccactcct aaaacacgct taaagaaaca ttcaggttct ttaaaccaca tatctgggca 7980 ctttggccag ccaaattgac atataaaatt aaccatctgg caaaattaat caagaaaaaa 8040 agaaataaga atgacaaagg gggaacctta gacatagcag agattaaaaa gctaattaaa 8100 ttgtaattat tacaacttaa taaaaagata atccaattta aaaacagaaa aaagctttaa 8160 aaagacattt ttccaaagaa gacatacaaa ttgccaacaa gctcatgaaa agatgcccaa 8220 catcatgggc tgttaaggaa atgcaaatcc aaatcataaa gagataccat ttcacaccca 8280 ctaggatggt tgattttttt aaaaggaaaa taacaagtcc tggcaaggag gtgaagaaat 8340 tggacctctc atacattgtt ggtaaggatg tgaaatggtg cagctacttt ggaaaatagt 8400 ttggcagttt ctcaaaaagt taaacacaga attaccatat gatgcagcat ttctactcca 8460 agggaattgg aaacatatgt tcacacaaaa acttgtccat agcagcagca tttgtaatag 8520 ccaaaaagtg gaaacaaccc aaatgtctat caaactgatg aatggatata cacatgtggt 8580 tgatccatac aatggaatat tatttagcca gaaaaaggag tgaagttcca atacatgcta 8640 ccatgtaatg aaccttgaaa atattatgct aagtgaagga ggtcagacac aaaagaccac 8700 atgttgtatg attccattga tataaaatgt ccagaataag caaatccata gaggtagaaa 8760 gtggattcat ggttgccagg ggctgagagg agggggaatt gggagtgact gctaacaggt 8820 acaggggttc ttcttggaat catggaagtg tttgtgaatt agatagtggt gatggttgca 8880 taaccttatg aatatattaa aaaacactga tctgtatact tgaaaaaggt gaattttacg 8940 gtatgtgaat tagagcttac taaaaatgaa agcagataag ctaataagaa atacaaggaa 9000 cagttttatg ccaatacact cgaaaattta aatggacaaa ttcctagaaa aaaaatgcaa 9060 cttaccaaaa actgactcaa gaaaaattag aaaaactgaa aaatttttta cccatcaaag 9120 aaattgaatc agttaaaaaa atcttccaac caagaaagct ccaggctcag ttctaccaga 9180 cactcaagtg acagctgatg ctaaccttaa acacattatt tcaaaggacc ctcaaagaag 9240 gaatagtttc cagcttgttt atgaggataa cataacctta ttagcaaaac cagacaagga 9300 cacaatgaga aaggaacatt acagaagagt tttgatcacg tacatatgca taagtactat 9360 acaaaatatt tctgaaggaa tggaaaatga tattttaggc aaagatgaac cataagaaaa 9420 ccaagatgac agtaacatcg gacaaaatat agaagataaa acataaggag caaattatgt 9480 cgtatatcga tagaagaaac taatcaggaa tgtttacctg taacagtata gtatccaaat 9540 aaaagcagta actgatggaa atggtgtaca gcaaaatata aaccaacatt tgtaattgga 9600 cataccaccc ttggtattga aaaatcaaac acatttttaa aataaaagat atagggagac 9660 caaggcagga ggatagcttt gagaccagga gtttgagacc agcctgggca acatattccc 9720 tacaaaaaaa tttaaaaatt agccaggcat ggtggcacct gcctgtggtc ccagctgctc 9780 agggtgctga ggcaggagga tggcttgagt ccaggaattt gaggctgcaa tgagccatga 9840 ttgtgccact gcactctagc ctgggtgaca cagtgaggcc ctgtctccaa aaaataaaaa 9900 taaaatatat agatagtttc aattatatga atgataagtt caattaaata gatatgtaaa 9960 acccacaaat acagaatcat actctttttg agcacataag atccactaaa atcagccata 10020 taccagaata taagaaagtc aacaaattac aaaacagtct acctcataca gatgtcttct 10080 gattacaatg ctgtaagatt tgaaattaac aaaagtttag gggaaaaaat cacctatgtc 10140 tgtaaataaa aacaatatta acctatatgt taaggagaaa aatcataaag aaaattaaga 10200 tatgcatata atgaccatca aagcactgca tgtcaaaata tctggcgtga atctaaaaga 10260 gagcttagag ggaaatttac agttttgaat acatttatca gaaaataaga aagattagac 10320 gctgggcgtg gtggcttaca actgtaattt aagcactttg gaaggctgag aggggcagat 10380 agcttgagcc tgggagttgg agagcagcct ggtcaacgtg gcaaaaccct gtctctacaa 10440 aaaatttaaa aaaagttgct gggtgtggtg gctcatgcct gtagtcccgg ggtgaggtag 10500 gaggatcacc tgagccgggg aggtggaggt tacagtcatc gtgccagtgc actccagcct 10560 gggtgagaca gcaagacccc gtctcaaaaa aaaaaaaaaa aacatccagg tgcagtggct 10620 catgcctgta atcccagcac tttgggaggc cgaggcgggc ggatcacgag gtcaggggat 10680 tgagaccatc ctggctaaca tggtgaaacc ccgtctctac taaaagtaca aaaaaattag 10740 ccgggcgtgg tggtccaccc ctgtagttcc agctactcgg gaggctgagg caggagaatc 10800 gcttgaaccc gggaggtgga ggttgcagtg aggcgagatc gtgccactgc actccagcct 10860 gggcaacaga gtgagactcc atctcaaaaa aaagaaagta agctaagcat tcaattaaaa 10920 atggaaacaa aaaggaagga aataacagat ctgggtggaa ataaatgaaa tggaaaacac 10980 atgtgaaaat gagtgatgac ccaaactgaa tgtggattgt tttgaaaact ctcataagat 11040 acacaaattt ctggcatgac caatcaaggg aaagagtgaa aatacagata aactgtgtat 11100 gaaattaaaa aggagaaatt tgacaaatac ggtgaatgaa ctccctctgc atctatccca 11160 gactctctag tgtgccggcc cctgatccgg aggctagatt attaccggga atcctgagtt 11220 ctggatacaa ttttgattct gtgaatcatg tacatgacac tggagtcaga actgggttat 11280 gtgaagggag ggaatgggat gcaagcatcc attttttttc tgcaacagtt ttattgagat 11340 gtaattcaca taccatgcaa ttcacccaat taaaatgtgc aatgcagtgg gttttagtat 11400 attcacaggg ttgtgcatct gtcaccacaa tcaattttag aacatttgca tcatccccca 11460 aagaaatcct atacctttag ctactactct gcagtccctc atctccctcc tccccacccc 11520 agctcgagac aaccacgaat ctttctgatt caatatttgt gtatcctgga cttttttttt 11580 ttttttttga gatggagtct tgctctgtcg cccaggctgg agtgcagtgg tgcaatttta 11640 gctcactgca acctccacct cccaggttca agcaattctc ctgtctcagc ctcttgagta 11700 gttaggacta caggtgtgct ttttttattt ttgtattttt agtagaaaca gggtttcacc 11760 atgttggcca ggctggtctc gaactcctga cctcaagtga tctgcctgcc tcagcctcct 11820 aaagtgctgg gattacaggc atgagccact gtgcccagcc ttatcctaga catttcatgt 11880 aaatgagatt atataatcaa ggtacgtcca tgttgtaaca tgtatcagta cttcattttt 11940 tatgactgaa taatattcag tcttacatat gtggatagat ttccaaaggg aaataataaa 12000 cagaatgaga tgtactgcac agtgtcattt atgcaaatta aaacatacac aagtgatata 12060 ttaagagcac acctatgtaa aaataaacat agaggtgaat ggtaagtttc agtgggccct 12120 cactggtcag ctctgtttgg gcatttccag gctttgtgcc tggccatgaa caggctaatg 12180 cagtggagct gtgggcagca tggcaagggg tgctgttggc agagtgagaa attgtgcctg 12240 gaacctgaac cttagatcca ggatctgctg aaggcagttt ggtaattatc gtgtaaggta 12300 atcatgcaag agtttcagga tgataatctt gggaatatga aaaacttttt gcaacctggc 12360 aggactttac cctggacagg cagaattcca tgctacatag aatggactga gatgagaacc 12420 ttaccccacc accaaattgt ctgttatcca ttatcgctaa tgtgtccttt cagtttatca 12480 tagtaaaata ttgcttttct aagacgtatg tgtcagagtg ttcatttttt gttaaaactt 12540 ccatccttgg gagaaggtta tgcatgaaat atgctggacc tcgtgtctaa ggtggggaag 12600 aaaatggagt tagtgatgga ttggtggaaa aatataagat tggctttgta tagactgata 12660 atgagtgttc cataaatcaa gaaataccaa tttaattctg tgtacctagg gtacttagaa 12720 ttgtacaatt aaaataattt cccattagaa ttgtctaatg catgtaaaac atagatccta 12780 ttgaatgtct ttattttcta gccttcggat aagtactaat aagctacagt tttggcaaac 12840 acattgtatc agtattgaaa ggttaatcca tttagtctgc atcaatcaag ttccagtaag 12900 aaagatggaa atcatactag ctgtttaaac aaaagaccag cccagggaat tgtctaagca 12960 agaagttgcg ggactggaga agcaaaaagg gagtaccaag atagcatgca gaaaataact 13020 tcaggaaagt taccatcctt ggggctggag ttgcaaagag aagggcttga gattataaaa 13080 aggtcccaga ccaggacctc gcagagctgt gacccatcct ttgagaatga ggctgtgctc 13140 agctgctgct gatgcctcag agcagcatgg gatgaggttg gttctgggag cctagcaccg 13200 aactgggaac tcaggccaac tgccaggatg aagtagagtt gctgagaact tgcttattgg 13260 aacagcaatc acacaggaag gaacagtcct ctttccctcc tcctgccttc ctctaatgct 13320 tgctattggc agagtctaag cacctggcaa aggagaaatg gtttgcagag tactcagcct 13380 atagaaggct gggttgtagc tgagagacaa gagcttaaat attggcacat ggtccaaact 13440 tattttatat caaatagaga tcatttaatt tcaggaaaaa aatctaattc tgctctgctt 13500 atattccagt atcaactccc gactggtgta ctgcaattca gttcctacac taatgaccca 13560 gagttagtgc agatcccaca agtgaaaggg cacagttcac aataagattg tccacacctc 13620 agaccaactg gctataaatg tggggtcttc ccacaagata gaagaagctt cccacttcag 13680 cttctattat ttgctagaat gtttcacaga actcagggaa aaaaaagtgc tatttttact 13740 atgacagttt tattataaag gatacaactc aggaacagtc aatgaaaaga cacatagggt 13800 gaggtctgag aggctcccaa aggcagagtt tccatgcctt ctcctcgtgg gattatcaca 13860 ttgccctccc agcacatcga tgtgttcaac caggaagctg cactgggccg cagtgtccag 13920 agtttttatt ggggtctcac tatgttggaa tgattgattt aatcatttgg ccacatggtt 13980 aaactcaatc tccagcccca caaccctccc tataggtagg tctagcacaa agccccaacc 14040 ttctaattac atggtttttc cagtgaccag cctctattct tagtcatccc gttgctcagc 14100 ataaactcag gtgtaatcca aggggctcat gaagaacaaa aacactccca ttacttggga 14160 aattccaagg ttttagagtt tgtctcccag aaaccaggga caaaggccaa attctttatt 14220 tttcaacagt ttgcaatata agaggaaaat gttattaaag gcatgtccaa gtgcatttga 14280 cagaacacaa gttcctatta caagttttag aagcatatat gataatagaa aattagtgct 14340 agaaaagtcc tgagaagcca tttgccaaca tgaataaact agttcagaac atttatgagc 14400 tagctttgtt ccaaaatttg ctttcatttg gagtttagaa acctgttgtt cttttttttt 14460 tttttttttt tttttttttt ttttttttga gatggagtct ctctctgtca cccaggctgg 14520 agtgcagtgg tgcgatcttg gctcactgca cgctccacct cccgggttca tgccattctc 14580 ctgcctcagc ctcctgagta gctgggacta caggtgccca ccaccacgcc cagctaattt 14640 tttgtatttt ttagtagaga cgggtttcac tgtgttagcc aggatggtct cgatctcctg 14700 acctcatgat ctgcccacct tggcctccca aagtgctggg attacaggtg tgagccacca 14760 tgcccagctg aaacttgttg ttcttaaaga gacaatcgta cccaatggtt attacttctc 14820 aggccagttt acaaaattta atagggccag gcgcagtggc tcatgcctgt aatcccagca 14880 ctttgggagg ctgaggcagg cggatcacga ggtcagcagt tcgagaccag cctgaccaac 14940 atggtgaaac cccgtctcta caaaaaatac aaaaattagc cgggcgtggt cgtgggtgcc 15000 tgtaatccca gctactcagg aggctgaggc aggagaattg cttgaaccca agaggcagag 15060 gttacagtga gctgagatta tgccacttca ctccagcctg ggcaacagag caaggctctg 15120 tctcaaaaaa aaaatagtga taataatcca gtagtaatag taactggtat ttattgagct 15180 ggaaatctgc tgtatgctaa cgtattattc tagatgccag gaagtagtga ggatgatatg 15240 tttgaaaatg gaagaagatg gggaacctga gtcagatgca acattaaaag agattgtctt 15300 tgtcagtgaa ctctagaatt tcaaaaacat gtaagtgaac aatcattgaa ttcctggata 15360 aatattctga gacaaatggc ctcaggactg tagtgagaag taacaggaag aagatatatg 15420 aagctgctta acatacagta gtttgtagtg gctcaataac tattcagaaa aaaacaaaac 15480 tcgttcaagt caggactcag tctatagcag tgaaccccag cctttttgat accaaggact 15540 ggtttagtgg aagacgattt ttccatggac tgggtcaagg ggaaggtttt tgggtgattc 15600 aagtacatta catttattgt gcactttatt tctattatta tgacattgta atatataatg 15660 aaataattat acaactcacc ataatgtaga atcagtggga gccctgagct tgttttcctg 15720 caactagaag gtctcatttg ggggtgatgg gagacagtgg cagatcatca ggcattagat 15780 tctcataagg agcgtacaac ctacatcctt cacatatgca gttcacaata gggactgcac 15840 tcctatgaga atctaatgcc actgctgatc taagaggagg tggagctcag gcagttatgc 15900 aagccatggg gagtggctat aaatacaggt gaagcttcgc ttgcttgcct gccactcacc 15960 tcctgctgtg cggcttagtt cctaacaggc catggactgg tactggttag tggcccaggg 16020 attggggatc cctggtctct agtactgaag cagatatagg acttagcata aaataagcat 16080 aaaactagaa aaagtaatgc atttgatcca actactttgg tggtaaaaag ttaggattgt 16140 actttatgat gtgatcaagg tccaggatag taaaaatgga ctattcagag tgcacagaag 16200 tgtgccagaa aagtgtgacc tggtggcttt tttaaagtat aataaatcta atggacaaaa 16260 ttagcaaagc ctcactttaa aaaaagtagc attacaaatc ccaaagaaaa aacttaaaaa 16320 taacaagaaa aaaactcata aaatagaatg atttttaaaa gtttgtactg atttacattt 16380 cctaatgtct gtgggtgatt ttatttattc ctttgacaat catttattca gtgtccagtg 16440 ccaagcacta tgctgggcag agaatacaag ataagtctca catgattcct gtgtacgtgg 16500 agtaaacagc ctaggagaaa agatttctct cacaaagtgg aggcataatg cagccttacc 16560 cttcaatgta tggtttgatg cattatggtg gtaagtaatc ttataagaaa gatagacaac 16620 tgttagaaat tatgtgttta aagaatttag cgacaagttg ggtatggtat ggtttgatgc 16680 attatggtgt atggtttgat gcattatggt ggtaagtaat cttataagaa agatagacaa 16740 ctgttagaaa ttatgtgttt aaagaattta gcgacaagtt gggtgcggtg gcttatacct 16800 gtaaccctag cactttggga ggctgaggac tgtggatcac ttgaggtcag gaattcgaga 16860 ccagcctagg caatatggtg aaaccctgtc tctactaaaa atacaaaaat tagccgggtt 16920 tgatggccgg cgcctgtaat cccagctact cgggaggctg agacaagaga atcccttgaa 16980 cctaggagac aggttgcagt gagccaggat ggcaccactg cactccaacc tgggcaacag 17040 agggagactc tgtctcaaaa acaacaaaaa acaaaaacaa agaatttagt gacctaagaa 17100 aatgttacct acaggttaac gcaaaatgga agttgaaaat ggaatggaaa aatcctgtta 17160 tcaagtatat aaatggaaaa atcccactat caagtatata aatgtatcac tgttaacttt 17220 tttctcttag aacatgtata ttgtattaaa cgtgtgattc tcaaactccc aggggacagc 17280 ggtaagcccg tggttatatg aaattgtgcc tctctcagtg ccccaggttc ctctcctgta 17340 aagtgagaaa aggacacacc tactctcagt tcacatgcaa gattaggtcc aaaggtgtgt 17400 gtcctacacc ctcccccatg aaggggatga ttagccttca tcaaagccat tttctgagtc 17460 cccaaacatt tctcgatatg tgtgagaaaa aacagtcaac acatttttaa aaattctcat 17520 tggaacagtt gttactggat tttctaaccc ttctttattc tcactgccca ggttacccct 17580 tccctactgc tcctcctgtg gatccatttg ccaaaatcaa agtggacgac tgtggaaaaa 17640 ctaagggatg ctttaggttg gtggaactgc aggttggata actttaaggc taagactgat 17700 ttacttctca gggtagtatt taaattgcat gcagttcctt ctaatcaggg ggcctgagct 17760 ccttgatcac ctcagcaggg ttatgaagtt tcgtaacggc catcccgctt ttcagtctta 17820 aaggcctttt aactttagta tgtcctgggc ccgggaccat tcttccatgg ttcttttcta 17880 gctgtaccac cattgctcca ggtgtctcct actcaccaat tgctgatatg cagaagactc 17940 atgtgacaca gaacgtgtct gtgcttattc agcaggggct gtgggcagga caggcaatga 18000 tagacatgtt tagtacccaa ttcctttttt agtaaatagt cctgacctat ggatatctgt 18060 atgattctaa ctagtcatta agcattcatt gttgctagct tgttatctca ggcatggtga 18120 gtctctacat gattattagt agtatcacga catgacgggc tgggctagaa aacagtcatg 18180 tggacaatgg gcctcccatc tagacagctc agcttctttg ggaaagcctc ctcctttgaa 18240 ccctttactt cctaaaacaa attgcccctg aataatttga tggtcagggg caaagttttg 18300 atggtcactg tactgaaggt tctgattttg ctgttgttca gtacttaagg tctaagaagg 18360 aaaaattgaa tacaggggct ttgagctttc tttggattac aaggccactt ctaaggaatg 18420 aataaaaata gccaaaaatg ttcttattga ggaagatggt cagcaatgta taaacctggt 18480 gattggggga tattttgaga agggagggaa ttttagtact ctactggctt caaagtgact 18540 tgttttcttg tccatttact ccttttttta acttcccttg ctccttttca tctttttcac 18600 cccagtgccc tgttgccttg atcctattat atctcttcct gactgctcgg gtatttctgc 18660 cagcatagta gtacatttct gtcccctcct caacacttag cacccctgtg ccctgttcct 18720 ttagatacct tccatctgcg cgtatattgt cccacccatt gcttcaactt tctaccattc 18780 tgcaaatggt gtaatacagt gttcctcaaa ccagggcttg ccagttctga aaggatccac 18840 aagtgctctg taagaaaatt taaatttgtg ttttcatttc agtggtgctc tcaaaaaaac 18900 atgaatgtct gcataatctc actatctgga taatcacctt atattgttgt atgttttagt 18960 gtctgaattt gtaattatat tagcagcaag tagcccaaca gaacaaaggc aaattttata 19020 aataaggcca agcacagtgg cttgactgta atcccagcac tttgggaggc tgaggtgggt 19080 ggatcacctg aggtcaggag ttcaagatca gcctggccaa caaggtgaaa tcccgtctct 19140 actaaaaata caaaaattag ccaggtgtgg tgacccatgc ctgtagtccc aactacttgg 19200 gaggctgagg caggagaact gcttgaaccc aagaggtgga ggttgcagtg agccaagatc 19260 ccgccattgc actccagcct gggcaacaga gtgagactgt ctcaaaaaaa aaaaaagaaa 19320 aaaaaattgt aaataaatat tgcattcatt catggttcat gagtaccaga tgggggtatt 19380 catttggtgt atcgcataaa taaggatttt gcagcagttc aaacagataa aagtgaagag 19440 ttgagccatc atgctataga gagtagtaag aacatgctct caactccaaa gaccctgcac 19500 gatctaatcg gataccacat gcacataagc agaaatttac tttcagtaaa gccccattat 19560 ctgttgcatg agtgctgtga atttcaatgt tcctggttta ttagtttaat ttatattgaa 19620 tttgtaaatt tgtgttagtt tgattatatg aaaatttgta agttaaaaag agttggtata 19680 catttaatta acagaaacaa tttaaatggt taacttggag aatctttttc cattaaaagg 19740 gattcagaaa taattcagta gtaactaaca tgtaaaagac tctaatacta atataatgta 19800 gagaacactg gctgggagtt tagataccca gattcaagtc ctgttgcaac cgctgacttc 19860 actatacaac ctcgggcaag ccccttccca tctcaggact gcagttcttg tgtacaaagt 19920 ggggcagatc atctctaagg ttcctcctaa gagagtgaaa ctgtgttttt aagattccag 19980 gtagcaaaaa tccaatgtgt ttaataatat tgcttagcat cccacatgat acatggtaga 20040 tgctctctta atgtcttttg tgtaagtaga agcagtaaag tctctttcga tatctcttct 20100 agatatggca aaccaggctg taatgcagag acctgtgact atttcctcag ctaccggatg 20160 ataggggctg atgtagaatt tgagctgagt gcagacacag atggttgggt agcagttgga 20220 ttctcttcag acaagaaaat ggtaagatgc aaaacatgat agaagctgat tttttctctt 20280 tagttggctg tgctattttg attttagaag caagttaatt gatcaaagca gtataatgat 20340 caaattagtt atggctctct gattgcaagt taaaaaaaat tcaggctagc ttaaatgaaa 20400 gggttggcta tcagaatcca agctgtcttg tggcacctct tttcatcagc tgtttccaca 20460 gtaaggctgc aagaagaggc ccccatgtta taagccttat atggcttata acaatgagca 20520 tttattgctt gctcacatgt tcatggattg actggggtgc agctcatttt aggtggggtt 20580 catctggact tgacgcctgt ccatgagttg gattcaggtt ttctctgtgt tttcactagt 20640 catccaaggc atattttttt catggcagag gcagaagtga aagaaccact tataagaggg 20700 aaaaacaact tttttccgtc tctactcata ttctcactac agaaagaata cttctgtgac 20760 cagatgtgtg ggggtgttcc cccacacccc aagcatttct ccagtggaca gctaggtgtc 20820 ctctaattca ctttgtttct gacactacct acctggagat agcattagat cccacaggtt 20880 aagggttcaa aaccacaaga ctgcccccat ttcagacacc cgtcacaagc cccaggttgt 20940 gacctgtatt tctgaccagc cagctataaa tctggggtgc caccaccctc ttctgttcag 21000 ttaatctcct ggagtggctc acagaactca gagaaacact ttactcattt atttatttat 21060 ttatttaatt tatttgtttg tttgtttgtt tgttgagata gagtttcgct cttgttgccc 21120 aggctggagt gcaatggtgt gaccttggct cattgcagcc tgcacctccc aggttcaagc 21180 gattctcctg cctcagcatc ccaagtagct gggattacgg gcacctgcca ccacgccctg 21240 ctaatttttt gtatttttag tagagacgag gtttcaccgt gttggccagg ctgttctcaa 21300 actcctgacc tcagatgatc cacccacctc gacctctcag agtcctggga ttacaggcat 21360 gagccaccgt atcccaccta ctttactcat ttattatgaa ggctattttg aaggatacag 21420 atgaacagtt agatggaaga gacacttagg ggaaggcatg gggaagggag tgaggagctt 21480 caacaccctg ttgggtgtgc tacccttcag gcacctccat ctgttcagca atccagaagc 21540 tcatccgaac cctgtccttt tgggtttttt ttatttttta gacagagtct cgctctgttg 21600 cccaggctgg agtgcaatgg catgatgtcg gctcactgca acctttgcct cctgggttca 21660 agtttgattt ttctgcctcg ggctcccaac atagctgaga ttacaggtgt aatcccacct 21720 gtaatacaaa aaacacccag ctaatttttg taattttagt agagatgggg tttcacacgt 21780 tggccaggct ggtctcgaac tcctggcctc aagcaatctg cccaccccag cctcccaaat 21840 tgccgggatt acaggcactt ccgtgcctgg cctctttttg aatatttgtg gaggcttcat 21900 tataaaaggc atgttggatt acatcaggga agttacgaat ttgtttcaac agttttatct 21960 tcctctgtac ccaggggcaa gaaaacctct ggacctcaga caggactgtg gccaggcaca 22020 ggcggggcct caggtgcaca ggcagctgtc atcactggcc cctgctcttc cctgtgcctt 22080 gctgcgtttt tctctctttc ctctggagcc ccattctttt gccacttcac tgggccaggc 22140 ttgttcccaa actatgagac tttctcagtc ctctgtgctt ttgcccttgt ggctgtttct 22200 gcctagagca ccccgccctt ctcttcacct ggcctattct gaggcttgtg tggggctaca 22260 ggggagagag gagggcggct cagaccaaag ttccagcagt gacagtgctg agggctcaga 22320 ttttgaatgt tttgaaagta gcactaactg aggccaaggc aggtggatcg cctgaggtca 22380 ggagttcgag aacagcctgg ccaacatagt gaaaccccgt ctctactaaa aatacaaaaa 22440 attagctggg tgtggtggcg ggcgcctgta atcccagcta ctcgagaggc tgaggcggga 22500 gaatcgcttg aacccaggag gcggaggttg cagcgagctg agatcgtgcc attgtactcc 22560 agcctgggca acgagaatga aactccgtct caaaaaaaag aaaaagaaaa agaaaatagc 22620 actaactggc tgggcatggt ggctcacacc tataatccca gcactttggg aggctgaagc 22680 aggtggattg cttgaggtcg ggagttcgag accagcctgg ccaacatggt gaaaccctgt 22740 ctctactaaa aacacaaaac ttagccgggt atggtggcag gtgcctgtaa tcccagctac 22800 tcagaaggca gaggcatgag aattgcttag gaggcagagg ttgcagtgag ctgagatggc 22860 gccattgcac tccaccctga gcaacagagc gagactgtct caaaaaaaga aaaaaagtag 22920 cactgactga caggatttgc aagtggattg gatatatggg ttgtaggtgt gtcgtaggaa 22980 aagactgata agcagtctaa gcagtggctg gtctggtggc tttattcttg gagaggtcac 23040 caggagaata gccttcaatg agttccagtc caagtagtaa gacaatgggt accactggta 23100 agactgcatg gatagatatt agtctactta tctcctgccc tagaggcagc aggagaaaca 23160 gcagggcatg ccacacccag aagctggctt gcattgaaag tgaccacttc ctgtcctaaa 23220 tgcatttgca ctcgtggtag ggctgttaag attaggggag tgaggcagag tgaggagcat 23280 tcaaattttg tttgatattt tggtatctaa aggcctataa agtatctact gtcatattat 23340 ctgtgaaagt agaggtctct ttttttcttg tattattcca agaaaaagca cagattaatt 23400 ttttttgaga acctaatgta taggaaaaac aaaatggaac ccctttttaa aagcgtttaa 23460 ctttttaaaa atggtacatt attagccggg cgcagtggct cacaatccca gaactttggg 23520 aggccgaggc aggcggatcc cctgaggtca ggagtttgag accagcctgg ccaacatagt 23580 gaagccctgt ctctactaaa aatacaaaaa ttagctgggc atggtggcgg gtgcctgtag 23640 tcccagccac tcgggaggct gaggcaggag aattgcttga acccgagagg cggaggttgc 23700 agtgagccga gatcgcgcca ctgcactcca gcctgagcaa cagtgagact ctgtcgggga 23760 aaaaaaaaaa aaaaaggtgc attattgtgt gcgtgtgtgt gtgttttaat tagactttac 23820 gttttacagc agttttaggt tcacagtgaa atggagccga aggtacagat ttcccatata 23880 ccccctgggg agcctccccc attatcaaca tcctgcacag gatggtacat gcattacaac 23940 tgattaacac ttttttttgt aactttttat ttttttgtga tggagtctta ctgtattgcc 24000 caggcttgga gttcagtggc actatttcgg ctcactgcag cctccacctc tcaggttcaa 24060 gcgactctcc tgcctcagcc tcccaagtag ctgggattac aggcacctgc caccacacct 24120 agctaatttt tgtattttaa gtagagacga ggtttcacca tgttggccag gctggtcttg 24180 aactcctgac ctcaagtggt ctgcctcagc tttccacagt gctgggatta taggcgtgag 24240 ccatcacacc cgaccaaact cttttaactt ttttaaactt gttttgaaaa aaatgtaaac 24300 tgtaggcaag caggaaggat agtaaaggtt taagtgaccc tcccacctca ggctcccaag 24360 tagctgggac tacagctact tagattaaca aaataataac ttttttattg tggtaaaacg 24420 cacataacaa aattaaccat tttatttttt gttgttgtgt attttttttt ttatttttat 24480 tttttgagac agggtctcac cctattgccc aagctgcagt gcagtggcac aaacaagact 24540 cactgcagcc tcaacctccc cagcgatcct cctacctcag cctcctgagt agctaggact 24600 acaggcatga gccccatgcc cagctaattt tttaattttt tgtagagata gtgtctcact 24660 gtattgccta gactggtctt gaacttctgg gctcaaatga tcctcctgtc ttgacttccc 24720 acattgttgg gatgacaggc actagtcacc acacctggcc agaattgacc attttatttt 24780 atttttttga gaaagggtct cactctgtca tccgggctgg agtgcagtag caccaaaatt 24840 tatgatttta aagtgtcaat gcagtggtat caaccacatt cagtgttctg caaccatcac 24900 tactccctag ttccagaact ttttcataaa taatgacatt agccacacat gctttattct 24960 ctgtactgct tgctgctact gctactgcta ctgctactac tactgattat tatttttttg 25020 attgttattt tctggaattt tggcatttaa atagcaaata tcataatcct ttaaccctaa 25080 atacttaagt aggttatctc ctaaaatcaa gaatgttctc ttttattata tagtttatta 25140 aattcaggaa gtttaacatt gatatgatgg tattattaaa tatattgttt gttttcatat 25200 gttgctactt gtcccaatac tgtccttagg gaaattttgg gtttatagcc caaatttcta 25260 tgtaggaaca cctagaactg cattttgttg tcatgtatat tttgaaatag tttcttagcc 25320 tttttttttt ttttcttttt ttggtctttt atgacactga ctccttgaag attataggct 25380 aggtattttt ttaatttttt ttgtagaatg accttcagtt tgagtttgtc tgattgtttt 25440 ttcttgatta ggttggtttg tgttttgggc aggattacca cataggtggt aatgtcttct 25500 ccaaacatca gtcaggaggc actgaatgtc acttattgtg atggtaactt tggtctcctg 25560 gttgagactt tttaatggca cttaaaagaa tttttttggt gggatttcaa attgcagaga 25620 tgatggaatg tatatttcta gtgaccagtg tgcaaaagat gaacctttga cttttttttt 25680 tctcaatcat ttctcatgtt agggtggtga tgatgtcatg gcctgcgtcc atgatgacaa 25740 tggcagggtc cgcatacagc acttctataa tgtaggccag tgggcaaagg agattcagag 25800 aaaccctgcc agagatgaag aaggagtttt tgagaacaat cgcgtcacct gcagatttaa 25860 acgccctgtg aatgttccca gagatgaaac aattgttgat ctgcatttga gttggtatta 25920 tctgtttgct tggggtccag ccattcaggg taagcttaca tttcagcatc tggattaaac 25980 gccagacacc actgtgcttg tgttcattgt taattgaagt cacactgtgt gcacaagaga 26040 gcgataagca ctctggatca tttaaaggaa aaggagatgc gactcctacc ttcagaaggt 26100 tataatggac ttattataat ggacttatta tacggacaaa caaatgaaca gttacacaca 26160 tgcaaagcta cagcagtaca agatagtatg tcatgagtga tgtgcttaat gaaacagatc 26220 atgtatctgc tgtatgtagg gctctgttca attctcacag cattcttatt tactcctcac 26280 catgtccttg agaggtatag gtattattgt cctgatttta gagagattaa gaaacttggt 26340 caaggctata caactaatta gtagaagaat taaaattcaa tcctaagtct gtctgacccc 26400 aaagcccatg aatactctta actcctatgc tgtaaatata aaaagactga acgggggcca 26460 gacgtggtgg ctcatgcctg taatcccagc actttgggag gatggtttga gcccaggagt 26520 tcaagaccag cctgggcact atagtgagac cctgtctcca ttcaaaaaaa aaaaagaaga 26580 agaaagaaat aataaaaagt actgaacagg aagggcttct ggagacttct ccagagattg 26640 acaccaagca tttcattagg gcccactttg tgactattct gttagtcaca aatctaccaa 26700 attatcccat agtttaaccc attactcctt aaatatttat gtgtatagga attacctggc 26760 tatattgtta aagtgcagtt ttctgtaggt cttcccctct ctcctcccct ctactggtct 26820 cccctcccct tccctcctct cccagggtct tgctctgtca cccaggctgg agtgcaatga 26880 aacaatcaga gctcactgca gcctccacct cccaggttta agtgaccctc ccacctcagg 26940 ctcccaagta gctgggacta cagtcatgcg ccactatgcc tggctaactt ttttgtattt 27000 ttagtagaga cagggtttca ccatattgcc caggctggtc ttgaactcct gggctcaacc 27060 gatccaccag ctttggcctc cctaagtgct gggattacag gtgtgagcta ccatgcctgg 27120 caagattctg catttctaat aagctccctg gtcatgctgg ttcattgatg acctcatttt 27180 gggtggcaag tctctgcagc atttgcccag caaagaccca aggatatcat gagacctttt 27240 agagactgct aaaatcaagt tacactgagt cttgagcatt ccttctggtt gattagccta 27300 agggcctgct caagaatgga agtgagtgtg gtttgcagtc cttgcactga gaattcattc 27360 tggctcctgg taattgcatc tttctttact gtgtgctcat caactatttg ctgcctaatc 27420 tgatctggtt gtttttttcc ttcaattcgt ttccaccaat cagcccagag atgacataat 27480 ctacctttta ttttgcctgt tgccaattgg gaagacactt gactgtctcc agattggtga 27540 caccttttcc atttgctaat ttcccagatt accaacagtg gctctgcagt tataacttca 27600 attactttcg gtaccctggg aaataaatta tatctcagcc tggagaaatg gagtaatcta 27660 aaatagtggg atttgaagtc taaaaatttg agtttgagta ttggttctgc cccttggtag 27720 ctgtataata tttagcctat tatttaattt ctgaatttca tttccttata tctaaagtgg 27780 ggataataat acttcctatt ccatggaaat attgtaagga ttaagtgaaa acaagtattt 27840 gaaattgctt taccaattgg aaaaagctgt attttcagaa ggtcatcttt ttacatatat 27900 ggcagactag cctaaagttc aaagccttct ccaaatctgg tcctgcttta cctgataagc 27960 tctctcaaaa cttctgttct tctcactaac tctaagcaaa acaggcacat tcctacccca 28020 tatactggct tctgcttttc ttgtttcctg aatgcccttc tatttttttt atgtttttat 28080 ttttattttt ttttaaacgg agtctcactc tgtcgcccag gctggagtgc aatggcgcga 28140 tcttggctca ctgcaacctc agcctcccag gttcaagcag ttctcctgcc tcagcctccc 28200 gagtagctgg gattacagat gcccaccacc acacccagat aacttttgta tttttagtag 28260 agacgctgtg tcaccatgtt tgccaggctg gtctcaaact cctgatcttg tgatccgccc 28320 acctcagcct cccaaagtgc tgggattaca ggcatgagcc accacacccg gcctcctgaa 28380 tgccctttta attgattata tcctacctat tctcctaagg tccctgtaag ggggaaaact 28440 ttgaactaag gcttgaaagg ggtatgaggt tggatttcac tcaaagaagg catgaactgt 28500 aggcgcatag ttaagttgag cagcccttgc aggtgaccag tgagctgagg ttaggtagga 28560 gaggtgactg ggatctattg agagaaaagt ctgctctgtt acccagtctg gagtgcagtg 28620 aaacaatcac agctcactgc agcctccacc tcccaggttt aagtgatcct cccacctcag 28680 cctcccaagt gtctgaaact acaggcatac aaacctgaga agtttgattt ttgctaactg 28740 gggccagcac ggacactaag cagggacctg acatgtcaag tttggcaggg cttccctagc 28800 agataggacc ttaaaaaccc tcactctgag ccctattcct aaggagacct ttgcactgaa 28860 tgtatatgcc ttagtctccc taacccaagt ttcaaatgga ttattcagga ggtcagacac 28920 ttttaatcaa ctgaagatta ttaaatatat tttacacaca gtctgtactt ctttccactt 28980 gtaacaaaat taaaagaaag tttcccatcc aaagactagt ttggtacttt aatcatccca 29040 ctttctatac cattctaccc taatctgcca gacacacaga cacacacaga cacacatcca 29100 gtataccaca ctccctctgg catggcctgg gcaggctact aagtgtccca cagacagtag 29160 aggagggctt ccatctgtaa gggagacgtt agtagtgttg tcaggagttg ttgatgatcc 29220 atatatatac aagatagatt tgagggaaaa agttagtagt ctctgctgtt tgaaataaaa 29280 atatagtttg cccatttggg tattaataag acaaaactca tagagccgtt ttaggaggca 29340 atcatacaat aatcacacaa aattatgtaa ctagtagttt taaaattgag atggcaggaa 29400 acagatcaac agcattgttc tgatggggga gtgcagatta ataagaacta gtctttttgg 29460 ataaatttaa ctaatttcta tgttctttat tatatgtaca atgatagaga attctgataa 29520 ctcaaatacc tgaatccaga tctttgtaac cactgcttac gtattcaaca ctaaatagac 29580 ttttgatttt tccattgttt gcctatacca ttataaatct gttctttttt caattgcccc 29640 tgctcttctc tttttttctt aaaggctcta tcactcgaca tgatatagac tcaccgccgg 29700 cttcagagcg tgttgtcagt atttacaagt atgaagacat ttttatgcca tcagctgcct 29760 atcaaacctt ctcatctcca ttttgtttgc ttctgattgt tgctctgacc ttctacctat 29820 tgatgggaac cccctaacca cagctgcagg gccaacagat tacatggatt gggaagtctt 29880 tagtataaat atatttttta aagaatatcc agtataattt tagcttcaat tatttaagaa 29940 aaaaaacctc atataatttc agctttttgg aagaaagaac aagcttcttt tgtagtcaaa 30000 gaagattgtt taataatgac cctatacttt tggaacatac ttacaccttt tctaagcact 30060 ttcacatatg ttttcttatt taacttcaca acatctctga gttggattga tgaaaattgt 30120 gattgccatc ttcctgaatg ggatttgaga cccaaaaaga ctgccttttt atgatccaca 30180 taccatataa ctttggctag aaatgacaaa tagaaaggca gagaaggaac taagtataga 30240 aatcatgtct caaaacagtc tttactatga gtgctatagc acagagagtt attttgaata 30300 ttactaggga gaactttact gaatgtcata agttcacagt cagccttttg ggcagaagaa 30360 atttcaaatt tccgtaaagc agaagtgctt ttggatattt acacagccaa tggggagatt 30420 ggaggaaaat acaatgatat gaaaatagtt catatttatt tgaaattttg gccgagcatg 30480 gtggctcacg cctgtgatcc cagcacttta ggaggccaag acgggcaggt cacctgaggt 30540 caggagttcg agaccagcct ggccaatgtg gtgaaacccc cgtctctatt aaaaagtaca 30600 aaaatgagcc aggcgtggtg gtgcacacct ataatcccag ctattcggga ggctgaggca 30660 ggagaattgc tggaacccag gaggcggagg ttgcagtgag ccgagattgt gccagtgcac 30720 tccagcctag gtgacagagc cagactctgt ctcaaaaata actaactaaa taaataatat 30780 ttatttgaaa tttcttctca gttagagagc agctatggga aaacattttt actgctattt 30840 gcaaaggttg gagaccaaac tgtgagcatt cctgaagatg taaagttgtg ttaatgatat 30900 taaaaaaaaa aaaaaaggga attcttagca gttagaattt gaacttttct accactgaat 30960 tttataaaac taaggtattc atctgtggtt acagtgaatt ctcttgtcag agccagcata 31020 ccttaagtgt gattaaaaat gtattttttt ttttttttgc gatgaatttt ccctcttgtc 31080 gcccaggctg gagtacaatg gtgtgatctc agcttcactg caacctccgc ctcccaggtt 31140 caagtgattc tcctgcctca gcctcctgag tagatgggat tacaggcatg tgccactatg 31200 cctggctaat ttttttgtat ttttagtaga gatggagttt caccatttta gtcaggctgg 31260 tctcgaactc ctgacctcag gtgatccacc tgctttggcc tcccagagtg ctaggattac 31320 aggtgtgagc caccgtgcct ggcctaaaaa tgtgttatat cagacaaaac atcttagtaa 31380 aaatttaggg cattttaata ataagccaac agtttttgag actgtagaga ttttgagtag 31440 aatttttttt atgaggtggc tttttaaaaa ataattgaaa atgggaaaat gacccataaa 31500 gtgaattcac ctagaatatc catttaaatg ttcagacttg tggctgagcg cggtggctca 31560 cacctgtaat cccagcactt tgggaggcca aggcaggcgg atcacctgag gtcaggagtt 31620 cgagaccagt ctggccaaca tggcaaaacc ttgtctccac taaaaataca aaaattagcc 31680 gggtgtggtg gcacatgcct gtaatcccag ccacttggga ggctgaggca cgagaatcac 31740 ttgaaccttg gaggcagagc ttgcagtgag ccaaaattgt gccacttgca ctccagcctg 31800 ggtgacagag ggagaccctg tctcaaaaaa ataagtaaaa taaaatgttc agacttataa 31860 actagacagg tagtcagcat aattcatagt aatgccttga cattaggcgc cccaaaggac 31920 tcctttgttc tttgttccaa atgtctgtgc taactgccca ttttgaggag agaagtctgc 31980 aggtatagtt agtgctgctg agacatttcc actctggcat tctagagttc tctggcttgc 32040 ccttgttgct ttaaagctgg ttccaacttc gtgggcttta tgattgagca acactggtga 32100 ttgtgatttc taataccacc ctccagattc atgtcacagg tgaaagtctg cctgtgggga 32160 gagctttcac attctcctgg agttgaacca aaataaggaa aacatcaaca aacacagtta 32220 ccaaaagctt ccaaggaggg atggaggaga aattctgttc attgcagatg gggagaaatt 32280 aaatgacatc aagtgcataa tggaactggg taggattcaa actcaagtcc cctggttcaa 32340 gtccttgact ccactctggg catggctaag tctggcctgt gtctcatctt ctcagctcta 32400 gtggctgttt agtagtgaag atactgatat gcaggcagcc atgttgtaaa taagaagcaa 32460 atatttttat atttctctct cttatgtgta taatatttga gctacaaaca ggttaaaaca 32520 aaagagagac ttcagcttag tagcaggcag ggaaggaaga agtgaatgca gcttccttcc 32580 gcttacattt ctaatcaagc taattatttc actagtgatt tctcatttga ttaccgattt 32640 tgcttattta tgcttagaac tttcaatgac ttctcagtgc cattaaggta aaatgcaagc 32700 catttctgtg acctgcaata tcatcttttg ccactcctcc tcacttgcta tgtcttagca 32760 agactgagag gctctcagtt cttcagacct gctctatctc acctctcatt ttgttcatat 32820 tctccacctc ttgtgcttga tgaactccta ttcacctatc aggtcacaac ttaaatattt 32880 tttcctccaa atctctggac tacataggtc caggccccca tcaaatgctc ccatagcaca 32940 ttgggcttcc tcatcaacat gctcgttata cttttctaga attgctggct tgtcctcctt 33000 gttatctccc taatgcagag accattactc atcattgtag cagatattta tccatatcag 33060 gtactcagca aacatttgtg gaatgaatct atatgtgaaa ctatacttct atggcaagtt 33120 tgtttggttc agaaggcttg catttcatgt atattgggag actggccaac atttttatat 33180 tcttcctcct atgctcaaac cataaataat tatgttagca aacactgaac tttgtctttt 33240 gaaacataaa tagaaacatc cttagtaaca gtactaacag taatcatttt tcagaaataa 33300 gaattcacac cagtgataca gaatggcctt tgaccattat ctcagagaac tagcagtcga 33360 tagacatgct gaagtgataa ttccaaaaac ttcagtttca ttcatcccta cttaaatata 33420 agagaatcaa cttctatttt atgtctgtat tcccattttc taatatacta cttgtcacag 33480 aggagatgcc caaccagtgt ttttcgtaaa gtacctgaat agattaataa taatgctagt 33540 gaaatatatt tagtgctgtt ctaagcactt tacatgaatt aactcattta atcctcaccg 33600 caaccatagg aagtagattc tattatttat ccaattttac agatgaaaaa actgaggctg 33660 gtcacttgac ataaattacc agtgctggcc aggactttag agaccattta atccaatgct 33720 cttattttac agaggtgcag ctgaagccta aggaggtgat atgatttcct acacattcat 33780 gcaccttcgt gcacatccat taaaactttt attaaatgga acccactatc catactccaa 33840 ttagctaggt gccttttttc ccccatttag taatttacaa ttaattggag gactttttat 33900 agttatattt agaggaaagg agcatgtttt actctatatc aactttcttt tctgcttaaa 33960 agcattttga ttctttattt taccagtcat tttagttctt ttaataggtt taatacacag 34020 atcaaacaag acttaaatac atttttaaca atgtaacaat ttaaaagaaa ttgcctaaaa 34080 cctaggtctt ctaacttgga gcccagtgct ttttttattg ctccgctgag tcaaaaggtg 34140 aatatataaa catgatttca gatagaatgc agaagctgta tttaattaag ttggcttttg 34200 tcattaaaga aatcagaaag tttggacaac tcaggaatgg atttccatcc tagatggtat 34260 ttactactat ttctggaatt aagtttactg tctatattca ggattttgtg cataagagac 34320 gtgaccactg aggttgtgtg tcattcttat ctctactcca ggggtcagca aactttctct 34380 gttaagggtc agatagtagt tttttggctt ttcagtctct gtttggctct gctataaaca 34440 atacataaat gaatgagcat ggctatgttc caataaaact taattgacac tgaactttga 34500 atttcatgta cttttcaaat gtcacaaatg ttttcctttt gatttttttc agaaagccat 34560 taaaaaaatc caaagaccat tcttagcttg caggctgcac aaaaacagga agcaggcggg 34620 gtttgcttat ggactgtggc tttcccaccc ttgttcttag atagggacac accaatatat 34680 catgattagt tgtaaaggat actgccagta acatattatt actggttagt tgtaagaata 34740 tcaaagttag tgaataattt gcttttaaaa taaattattg ttgatttatt tttataaata 34800 aatggcacag ggcatctcat gctcttgcat tctaatatat tttctggaat tgatgcaaaa 34860 gtggtggcag taaagtacag agccggctaa gcagcagttt tgtcttaagt aactgtggtg 34920 atcatggaca accattgcgc tgaactgctc ttcatggtaa ggatttcagt gtttcagagt 34980 aattcattaa cctagagatg ttttaaaaag ttgtatcggc cgggcgcggt ggctcaagcc 35040 tgtaatccca gcacttcggg aggccgaggt gggcggatca cgagatcagg agatcgagac 35100 catcctggct gacacggtga aaccccatct ctactaaaaa tacaaaaaaa attagctggg 35160 cgtggtggcg ggagcctgta gtcccagcta ctcaggaggc tgaggcaaga gaatggcgtg 35220 aacccgggag gtggagcttt caatgagccg agattgcccc tctgcactcc agcctgggta 35280 acagagcaag actccgtcta aaaaataaat aaataaaata aaataaaata aaataaaagt 35340 tgtatcaaaa tctgctgcac catgggagtg ttttacacat gtgatgtggc atctacctta 35400 tgtaccataa tggaaaaatg tttgagaacc actgcactag gccaaaggct gggatgtgta 35460 tactttgctg ccgtgtataa ttgtatgtcc ttgttctcta aacggaattg gttatgttta 35520 gtgaacagtt aggtggttct ccatgtgttt atatacacag acgatccccg aatttggatg 35580 gttcaactta agagttttca aacttacgat gatatgaaag tgatatacat ttgatacatt 35640 cctacaactt acaatggggt tatgtcccat aattgttttg acttatgata ttttcaactt 35700 acaattgatg ggtttactga gacataaccc tcttgtaact tgaggagcat gtgtatattt 35760 gtacatacat gatggtgtgt gtttatacac aaaggcctga atatacacac catgagcatt 35820 tgaaggtctt tgtcaaggtt gtttctgttt ctgatctctt gcctacttta aaacccaaga 35880 ggtagaacat tatgcatatt acaggcaaaa ctatgcttga gtttatagca atgtagtttg 35940 tagaaaggat tcctggatgt tttactaagc ctttcctcct ccattcaaca ttatatgaat 36000 tgttaagaac atataaagta ccactgttga atgtgacttt tctatactgt tagttaagaa 36060 accaagacta gtgttagctg agtaactggc atgttgatgc aacctataca ggttttgtat 36120 ttaattaggt tccttattca gttattcaag tatccagtta tttagtttca aggtagattt 36180 ctttgttttt atatttaata aaagacttgc atataagttc ctcttcatga aaatagtggc 36240 taggctagtg tgattttaag tatgtctggc aactcaaaat gtaatggatt catcaagcta 36300 tttgggattt ttaacataaa acacactgct gcttgattaa tgtgttcagt tttcccctgg 36360 atatttgcta ttaggctgat agccatttgg ttggattctt ctggagagta attgattctc 36420 taaggtaatg gggtgctgca gtgggtgtag cattcaccca ggtcactaga atcctacctg 36480 tttcatcaca tagaaggccc tggccatggg gaagagtgaa tgcagcttct ccggatgcct 36540 tatttccaca gacatgctga tatgctggaa caatctggct ccaggagtat taatatactt 36600 ttttgagact gtctacaaaa tactattttg taaaaactat gcctttacat agacattgat 36660 aggttatatt tgttttataa caattgtgta cctaatgttt gtgattaatt ttgagattta 36720 tgtatattta taagcctatc ttatgtaaat ttttactcta agatgtgtgg cacattacta 36780 ataaagacgt ggatttctat ttatgtaaag taattgtagc aatattccta gatgctacct 36840 aaatgattgt tctgtccatt tgccatcaca aatactttta tctttggatg attatgttag 36900 tcagaatata ttaattagat ttaaacttta aaataagaat tacaacatag tttgaaattc 36960 ttcctgttga tgtaattaaa gttagtttat aattcctta 36999 <210> 2 <211> 344 <212> PRT <213> Human FRRS1L protein <220> <223> Human FRRS1L protein <400> 2 Met Arg Arg Pro Arg Gln Gly Gly Gly Gly Ala Gly Gly Ser Ala Ala   1 5 10 15 Ala Arg Ala Arg Ala Gly Gly Leu Gly Gly Gly Ser Val Pro Ala Arg              20 25 30 Ala Arg Gly Ala Pro Ala Ala Ala Arg Ala Ala Trp Leu Arg Asp Leu          35 40 45 Cys Ala Arg Met Ala Arg Pro Pro Arg Gln His Pro Gly Val Trp Ala      50 55 60 Ser Leu Leu Leu Leu Leu Leu Thr Gly Pro Ala Ala Cys Ala Ala Ser  65 70 75 80 Pro Ala Asp Asp Gly Ala Gly Pro Gly Gly Arg Gly Pro Arg Gly Arg                  85 90 95 Ala Arg Gly Asp Thr Gly Ala Asp Glu Ala Val Pro Arg His Asp Ser             100 105 110 Ser Tyr Gly Thr Phe Ala Gly Glu Phe Tyr Asp Leu Arg Tyr Leu Ser         115 120 125 Glu Glu Gly Tyr Pro Phe Pro Thr Ala Pro Pro Val Asp Pro Phe Ala     130 135 140 Lys Ile Lys Val Asp Asp Cys Gly Lys Thr Lys Gly Cys Phe Arg Tyr 145 150 155 160 Gly Lys Pro Gly Cys Asn Ala Glu Thr Cys Asp Tyr Phe Leu Ser Tyr                 165 170 175 Arg Met Ile Gly Ala Asp Val Glu Phe Glu Leu Ser Ala Asp Thr Asp             180 185 190 Gly Trp Val Ala Val Gly Phe Ser Ser Asp Lys Lys Met Gly Gly Asp         195 200 205 Asp Val Met Ala Cys Val His Asp Asp Asn Gly Arg Val Arg Ile Gln     210 215 220 His Phe Tyr Asn Val Gly Gln Trp Ala Lys Glu Ile Gln Arg Asn Pro 225 230 235 240 Ala Arg Asp Glu Glu Gly Val Phe Glu Asn Asn Arg Val Thr Cys Arg                 245 250 255 Phe Lys Arg Pro Val Asn Val Pro Arg Asp Glu Thr Ile Val Asp Leu             260 265 270 His Leu Ser Trp Tyr Tyr Leu Phe Ala Trp Gly Pro Ala Ile Gln Gly         275 280 285 Ser Ile Thr Arg His Asp Ile Asp Ser Pro Pro Ala Ser Glu Arg Val     290 295 300 Val Ser Ile Tyr Lys Tyr Glu Asp Ile Phe Met Pro Ser Ala Ala Tyr 305 310 315 320 Gln Thr Phe Ser Ser Pro Phe Cys Leu Leu Leu Ile Val Ala Leu Thr                 325 330 335 Phe Tyr Leu Leu Met Gly Thr Pro             340

Claims (8)

delete delete delete (a) administering a candidate substance for improving or treating autism spectrum disorder to an animal model for autism spectrum disorder in which the Frrs1l gene is knocked out;
(b) measuring and comparing autism spectrum disorders with a control animal model not administered with the animal model administered with the candidate substance; And
(C) selecting a candidate material that significantly recovered autism spectrum disorder compared to the control group; Autism spectrum disorder improvement or screening method of the therapeutic agent comprising,
The animal is a mouse, monkey, dog, cat, rabbit, mormote, rat, cow, sheep, pig and goat, at least one member selected from the group consisting of.
The method of claim 4, wherein the step of selecting the candidate substance comprises a step of confirming that the tendency to favor a new animal in social novelty recognition is significantly reduced compared to a control group. Methods for improving spectrum disorders or screening therapeutics.
The method of claim 4, wherein the candidate substance is one or more selected from the group consisting of peptides, proteins, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, plasma, vectors and RNA. , Autism spectrum disorder improvement or therapeutic screening method.
A method for providing information for the diagnosis of autism spectrum disorder including the following steps:
(a) measuring the level of expression or activity of Frrs1l present in the biological sample; And
(b) comparing the measured expression or activity level of Frrs1l with that of a normal control.
The method of claim 7, wherein the biological sample is selected from the group consisting of tissue, cells, blood, serum, plasma, saliva, and urine.

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