CN111944835B - Construction method and application of mutant GABRG2 transgenic zebra fish epilepsy model - Google Patents

Abstract

The invention discloses a method for constructing an epileptic zebra fish model induced by mutant GABRG2 transgenosis, which takes zebra fish as a model animal, and transfers mutant human GABRG2 gene into zebra fish to enable the zebra fish to become a zebra fish line with epileptic phenotype, and the zebra fish line can be used for researching the relation between GABRG2 mutation and epileptic pathogenesis and screening antiepileptic drugs.

Description

Construction method and application of mutant GABRG2 transgenic zebra fish epilepsy model

Technical Field

The invention belongs to the technical field of animal model preparation in biology and basic medicine research, and particularly relates to a transgenic zebra fish epilepsy model constructed by using an epilepsy-related gene GABRG2 mutant, a construction method of the epilepsy zebra fish model and application of the epilepsy zebra fish model in research works such as epilepsy pathogenesis, drug screening and antiepileptic drug target identification.

Background

Epilepsy is a common chronic neurological disorder characterized by the presence of spontaneous, unproductive recurrent seizures, one of the most common disabling neurological disorders, affecting over 5000 million people worldwide, with about 240 million people diagnosed with epilepsy each year. The frequency, duration and severity of epileptic seizure increase, which may increase cognitive and emotional disorders of patients, lead to epileptic encephalopathy, and cause a series of sequelae such as cognitive disorder and emotional anxiety of patients. Epilepsy is divided into primary epilepsy and secondary epilepsy according to etiology, wherein the primary epilepsy is mostly related to gene defect. Over the past two decades, with the development of genomic technologies such as next generation sequencing, mutations in nearly 1000 genes have been identified as being associated with the occurrence of epilepsy, including mutations in genes for many ion channels and non-ion channels.

Gamma-aminobutyric acid A receptor (GABA)_AR) is the major mediator of rapidly inhibitory synaptic transmission in the central nervous system, which has been repeatedly shown to have a key role in seizures. GABA_AThe receptor is an isoprene dimer formed by assembly of multiple subunits, and GABA has been detected_AMutations in the receptor subunits α 1, β 1, β 2, β 3, γ 2 and δ are all closely associated with epileptic onset, and these mutations often have autosomal dominant inheritance.

GABA_AReceptor gamma 2 subunit (GABRG2) for GABA_AThe receptor transport and synaptic function have important significance, more than ten GABRG2 mutations related to epileptic onset are discovered at present, including missense mutation, nonsense mutation, frameshift mutation and truncation mutation, and the mutations reduce ion channel function to different degrees through different mechanisms, such as mRNA and endoplasmic reticulum related protein degradation, influence on normal function of a receptor, aggregation of mutant protein and the like, and the clinical phenotypes caused by the mutations are different from simple heat convulsion to more complicated hereditary epilepsy heat-accompanied convulsion additional symptoms, even serious Dravet syndrome and the like. A group of missense type GABRG2 mutations highly related to epileptic encephalopathy are found in clinical children, wherein the F343L mutation is positioned in the transmembrane region of GABRG2, so that GABA current is reduced by about 30%, and the children with the mutation are basically insensitive to the existing antiepileptic drugs.

Zebrafish, as a vertebrate, have a complex nervous system, are similar in genetic structure to humans, and share approximately 70% of genes with humans, and about 84% of genes related to human diseases are known to be expressed in zebrafish. Compared with rodents such as mice and the like, the zebra fish is simple in genetic operation, and is more suitable for preparing an epileptic model and used for researches such as high-throughput drug screening and new molecular target identification of antiepileptic drugs. GABA in zebra fish_AReceptors are typically present in α 1 β 2 γ 2, β 01 β 12 β 4, β 22b β 33 γ 2, β 52b β 63 β 9, β 74 β 82 γ 2, α 4 β 2 δ, α 6b β 2 γ 2, and α 6b β 2 δ, among other constituent forms. At present, although GABA has been already available_AReceptor alpha 1 subunit (GABRA1) knock-out model, in which zebra fish juvenile fish show epileptic phenotypes such as increased activity, brain electrical activity and increased mortality, but there is no report of GABRG2 mutant epileptic zebra fish line.

Disclosure of Invention

The invention aims to provide a method for constructing an epileptic zebra fish model induced by mutant GABRG2 transgenosis. The method takes zebra fish as a model animal, and the mutant GABRG2 gene is transferred into the zebra fish to enable the zebra fish to become a zebra fish line with epileptic phenotype, and the zebra fish line can be used for researching the relation between GABRG2 mutation and epileptic pathogenesis and screening antiepileptic drugs.

In order to achieve the purpose, the technical scheme of the invention is as follows:

a method for constructing a mutant GABRG2 transgenic zebra fish epilepsy model is characterized in that a mutant human GABRG2 gene is transferred into zebra fish to enable the zebra fish to become a zebra fish line with an epilepsy phenotype.

The mutant zebra fish is the zebra fish with missense mutation GABRG2 gene.

The epilepsy zebra fish model is caused by GABRG2 missense mutation.

The GABRG2 missense mutation is that the base T in the 343 rd amino acid Phe codon of the human GABRG2 gene is mutated into C (F343L).

The sequence of the transferred mutant human GABRG2 gene is shown as SEQ ID No: 1 is shown.

Preferably, the construction method of the invention uses a pDesttol2CG2 expression vector (shown in SEQ ID No: 2).

The pDesttol2CG2 expression vector is obtained by removing a transposase coding gene in a Tol2transposon, replacing the gene with a CMLC2-EGFP-polyA expression sequence, microinjecting the plasmid and transposase mRNA into a zebra fish single cell stage embryo, and observing the specific expression of green fluorescent protein in the heart tissue of the zebra fish. (KWan KM, Fujimoto E, Grabher C, et al. the Tol2kit: a multisite gateway-based construction kit for Tol2transposon transformations. Dev Dyn,2007,236(11): 3088-.

Preferably, the construction method of the invention adopts gateway technology to construct the expression vector, and comprises the following steps:

(1) construction of GABRG2 mutant expression vector: synthesizing mutant human GABRG2 gene, cloning it to pME-MCS plasmid vector, constructing midle entry clone pME-GABRG2^1027T>C(the sequence is shown as SEQ ID No: 3); cloning a sequence containing a HuC neuron specific promoter region, and constructing a 5' entry clone: p5E-HuC (SEQ ID NO: 4) and 3' entry clone: p3E-polyA (SEQ ID NO: 5); the 5 'entry clone, the midle entry clone and the 3' entry clone vectors are recombined and cloned into an expression vector pDestTol2CG2 through Gateway LR reaction to obtain an expression vector: pDestTol2CG2; HuC GABRG2^1027T>C-polyA(SEQ ID No：6)。

(2) Microinjection of zebrafish embryos: microinjecting the GABRG2 mutant expression vector and Tol2 transposase/transposase mRNA into wild zebra fish fertilized eggs at 1-2 cell stage, and screening embryos expressing green fluorescence by hearts after the embryos develop to 24hpf to obtain positive zebra fish containing GABRG2 mutant sequences.

The construction method also comprises the following steps of further identifying the genotype of the transgenic zebra fish and obtaining a stable genetic strain: after the positive zebra fish embryo containing the GABRG2 mutant sequence is matured, the genotype of the positive zebra fish is identified through PCR, the positive zebra fish containing the GABRG2 mutant sequence is selected to be hybridized with wild zebra fish to obtain F1 generation zebra fish, and finally, a GABRG2 mutant zebra fish line is obtained through screening by means of green fluorescence expressed by the heart and genomic DNA detection.

The invention carries out observation and related experimental verification on the epilepsy phenotype of the GABRG2 mutant zebra fish system: the method comprises the steps of recording the movement tracks of wild zebra fish and mutant zebra fish under normal conditions by adopting a zebra fish juvenile high-throughput behavior screening box (DanioVision Chamber) and an animal movement track tracking system (EthoVision XT), comparing the swimming distance and the average swimming speed, and carrying out video tracking analysis on the excitation degree of the zebra fish. And then, PTZ and light stimulation are respectively adopted to induce the epileptic seizure, and the susceptibility of the GABRG2 mutant fish line to the related stimulation for inducing the epileptic seizure is analyzed. And detecting the expression of the c-fos gene in the brain of the GABRG2 mutant zebra fish line by real-time quantitative PCR, and proving that the mutant zebra fish line has the characteristics of an epilepsy model from the aspect of molecular biology.

The invention also aims to provide application of the zebra fish epilepsy model for basic scientific research and clinical application research. Based on the GABRG2 mutant zebra fish epilepsy model, the method can be used for researching mechanisms such as biochemistry, molecular biology, genetics and the like related to epilepsy attack, and can also be used for screening and researching clinical antiepileptic drugs, namely, clinically commonly used antiepileptic drugs such as sodium valproate (valproic acid), carbamazepine (carbamazepine), clonazepam (clonazepam), Levetiracetam (Levetiracetam) and the like are directly added into a zebra fish culture solution, then effective drugs for epilepsy treatment are screened by means of zebra fish swimming behavior analysis and the like, and the action mechanism of the drugs is researched.

The invention provides an effective method basis for establishing a zebra fish epilepsy model, and also provides an important tool for epilepsy pathological research and antiepileptic drug screening research, thereby having larger basis and clinical research and application values.

Drawings

FIG. 1 is a schematic diagram of expression plasmids required for constructing transgenic fish by using Gateway technology. Using F343L mutant as an example, 5 'entry clone (p5E-HuC), 3' entry clone (p3E-polyA) and midle entry clone (pME-GABRG 2) were constructed respectively^1027T>C) Obtaining a plasmid pDesstTol 2CG2 expressing the mutant GABRG2 by LR reaction; HuC GABRG2^1027T>C-polyA。

FIG. 2 is an agarose gel electrophoresis of the constructed GABRG2(F343L) mutant plasmid. M is DNA molecular weight Marker.

FIG. 3 shows the obtained F1 generation positive mutant zebra fish. Wherein WT is wild type zebra fish, F343L is mutant zebra fish, and it can be seen that heart expresses green fluorescence.

FIG. 4 shows the identification of genotypes by sequencing of zebra fish genomic DNA.

FIG. 5 is a swimming behavior analysis of GABRG2(F343L) mutant zebrafish induced by PTZ. A is a visual graph of swimming traces of wild type zebra fish and mutant zebra fish in a normal culture medium or a culture medium containing 15mM PTZ respectively (1 min); panel B shows the distance of movement analysis (30min) of wild type and mutant zebra fish in normal medium or medium containing 15mM PTZ, respectively; c is the statistical analysis plot (. P <0.05,. P < 0.01).

FIG. 6 is an analysis of the swimming behavior of the mutant zebrafish GABRG2(F343L) induced by light stimulation. A is a visual graph of swimming tracks of wild type zebra fish and mutant zebra fish; b is the swimming behavior analysis of wild type and mutant type in different time periods under the condition of turning on or turning off the light; c is the statistical analysis plot (× P < 0.01).

FIG. 7 shows real-time fluorescent quantitative PCR detection of c-fos gene expression (P <0.01, compared with WT) at different times (24h, 48h, 72h, 96h, 120h) after the postnatal period of wild type and GABRG2(F343L) mutant zebrafish.

FIG. 8 shows the analysis of the swimming behavior of the mutant zebrafish GABRG2(F343L) in different antiepileptic drugs. Respectively adding 50 mu M, 100 mu M, 50 mu M and 30mM sodium valproate, carbamazepine, clonazepam and levetiracetam into the culture medium, recording for 30min by using a video analysis system after 24h, collecting parameters once per minute, and analyzing the moving distance of the zebrafish (P <0.05 and P < 0.01).

FIG. 9 shows the change in the swimming circadian rhythm of mutant zebrafish GABRG2 (R177G). Under the condition of normal illumination time, a motion trail tracking system is adopted to record the swimming behavior of 5dpf zebra fish juvenile fish, 24 hours (illumination 14 hours and darkness 10 hours) are recorded in total from 4 pm, behavior parameters are recorded every 1 hour, the total motion distance difference of wild type zebra fish and transgenic zebra fish in each time period is compared, and the circadian rhythm change is analyzed.

Detailed Description

The present invention will be described in further detail with reference to examples, but the method of carrying out the present invention is not limited thereto.

The terms "wild type" or "WT" as used herein both refer to wild type zebrafish. Other related terms, unless otherwise specified, generally have the meanings that are commonly understood by those of ordinary skill in the art.

In the following examples, various procedures and methods not described in detail are conventional methods well known in the art.

Example 1 construction of GABRG2(F343L) transgenic zebrafish model line for epilepsy.

This example 1 provides a protocol for transferring a mutant GABRG2(F343L) into fertilized eggs of zebra fish by transgenic means and constructing a zebra fish model of epilepsy. The method specifically comprises the following steps:

firstly, constructing a transgenic expression vector: searching human GABRG2 gene sequence by using Pubmed database, synthesizing GABRG2 open reading frame sequence, mutating 1027 th base T to C (SEQ ID No: 1), cloning into pME-MCS plasmid vector, 5 'cloning site to EcoRI, 3' cloning site to XbaI, constructing intermediate vector middle entry clone pME-GABRG2^1027T>C(SEQ ID No: 3). Cloning the sequence containing the HuC neuron specific promoter region, and constructing a 5' entry clone: p5E-HuC vector (SEQ ID No: 4). Clone 3' entry clone: p3E-polyA vector (SEQ ID No: 5). Recombining and cloning the 5 'entry clone, the midle entry clone and the 3' entry clone vectors into an expression vector pDestTol2CG2 through LR reaction to construct a transgenic mutation expression vector pDestTol2CG2; HuC GABRG2^1027T>CpolyA (SEQ ID No: 6) (shown in FIG. 1), the reaction system of which is as follows: TE buffer 4. mu.L, p5E-HuC (30ng) 1. mu.L, pME-GABRG2^1027T>C1 μ L (30ng), 1 μ L p3E-polyA (30ng), 1 μ L pDesttol2CG2(60ng), 2 μ L LR clonase II, in a total volume of 10 μ L. The reaction is carried out for 20h at 25 ℃. The constructed plasmid is identified by agarose gel electrophoresis, and the result is shown in figure 2, and a positive band is detected near 20kb, namely the GABRG2 gene mutation expression vector.

Preparation of Tol2 transposase mRNA (pCS2FA-transposase, DNA sequence shown in SEQ ID No: 7): the enzyme digestion system is as follows: 10 XBuffer 5. mu.L, DNA 1. mu.g, NotI-HF restriction enzyme 1. mu.L, add ddH2O to a total volume of 50. mu.L. 15min at 37 ℃ and 15min at 65 ℃. The enzyme products were electrophoresed on agarose gel, and the gel was recovered and then transcribed into Tol2 transposase mRNA using an RNA in vitro transcription kit.

And secondly, microinjecting zebrafish embryos: the transgenic mutation expression vector pDesttol2CG2; HuC GABRG2^1027T>CAnd-simultaneously microinjecting polyA and Tol2 transposase mRNA into wild zebra fish fertilized eggs at a 1-2 cell stage, after the embryos develop to 24hpf, screening embryos with green fluorescence expressed by hearts for subsequent experiments, and after the zebra fish mature, identifying the genotypes of the zebra fish through PCR and genome sequencing. As shown in FIG. 3, mutant zebrafish embryos were obtained whose hearts expressed green fluorescence.

Thirdly, identifying the genotype of the transgenic zebra fish and obtaining a stable genetic strain: extracting zebra fish genome DNA: extracting zebra fish genome DNA by using a genome DNA extraction kit (All-DNA-Out) one-step method, adding 20ul of All-DNA-Out solution, and heating at 85 ℃ for 5 min.

And (3) genotype identification: the PCR reaction is adopted to identify the genotype of the zebra fish, and the reaction system is as follows: 2 XKOD FX Buffer 10. mu.L, dNTP 4. mu.L, forward Primer (Primer F) 0.6. mu.L, reverse Primer (Primer R) 0.6. mu.L, KOD FX enzyme 0.4. mu.L, DNA template 2. mu.L, total volume 20. mu.L. The reaction conditions are as follows: 5min at 95 ℃; 98 ℃ 10s, 50 ℃ 30s, 68 ℃ 30s (35 cycles); 5min at 68 ℃. Wherein the primer sequence is as follows: f: 5'-GCACAGGAAGCTCAGTCTAC-3' (SEQ ID No: 8), R: 5'-GGAGTCCATTTTGGCAATGC-3' (SEQ ID No: 9).

Obtaining a stable genetic line of the transgenic zebra fish: and (3) selecting the zebra fish with positive gene identification result to be hybridized with wild zebra fish to obtain F1 generation zebra fish, and obtaining the mutant GABRG2 transgenic zebra fish line by heart expression green fluorescence and genomic DNA detection screening. As a result of genomic DNA sequencing, as shown in FIG. 4, it was confirmed that the mutation of the 1027 th base from T to C resulted in the determination that the zebrafish contained the mutation GABRG2 (F343L).

Fourthly, the observation of the epilepsy phenotype of the GABRG2(F343L) mutant zebra fish line and the verification of related experiments: observation of epilepsy behavioral phenotype: selecting zebra fish juvenile fish with 5dpf, adopting a zebra fish juvenile fish high-throughput behavior screening box (Danio Vision Chamber) and an animal motion trajectory tracking system (EthoVision XT 13), recording the motion trajectories of the wild zebra fish and the mutant zebra fish under normal conditions, recording for 30min totally, analyzing the excitation degree of the zebra fish juvenile fish through the swimming distance and the average swimming speed, and finding that the excitation of the mutant fish is increased under normal culture conditions and the swimming distance is remarkably increased as shown in a result of figure 5.

Swimming behavior changes in zebrafish in PTZ: adding a seizure inducer PTZ with the final concentration of 15mM into the zebra fish culture solution, and recording the swimming movement behavior of the zebra fish by adopting an EthoVision XT 13 zebra fish behavior video analysis system, wherein the swimming movement behavior is recorded once every 1min for 30 min. Comparing the behavior difference of the wild type and the transgenic zebra fish. Results as shown in fig. 5, PTZ can cause significant increase in excitability of wild-type zebrafish and transgenic zebrafish, but swimming distance of transgenic zebrafish is still significantly higher than wild-type.

Swimming behavior change of zebra fish in light change stimulation: under the condition of alternating illumination and darkness (5s), a zebra fish behavior video analysis system is adopted to record the movement behavior of the zebra fish in 100 cycles, every 5s is a parameter, and the average moving distance difference of every 5s between the wild zebra fish and the transgenic zebra fish is counted. The results are shown in fig. 6, the normal light movement distance of the transgenic zebra fish is significantly higher than that of the wild type, but the light and dark stimuli have no significant effect on the wild type and the transgenic zebra fish.

Molecular biological phenotype verification of epilepsy: and (3) detecting c-fos gene expression by real-time fluorescent quantitative PCR: respectively extracting wild type zebra fish total RNA and transgenic zebra fish total RNA of 24h, 48h, 72h, 96h and 120h after the birth, performing reverse transcription to obtain cDNA, and performing real-time fluorescent quantitative PCR analysis by adopting a SYBR green dye method, wherein the reaction conditions are as follows: 6min at 95 ℃; 95 ℃ 10s, 60 ℃ 30s, 72 ℃ 10s (41 cycles); the temperature is increased to 95 ℃ in a gradient way at 65 ℃ and is maintained for 5s every 0.5 ℃. Wherein the primer sequence is as follows: f: 5'-AACTGTCACGGCGATCTCTT-3' (SEQ ID No: 10), R: 5-CTTGCAGATGGGTTTGTGTG-3' (SEQ ID No: 11). The results are shown in fig. 7, and at 72h after birth, the expression of the c-fos of the transgenic zebrafish reaches the highest peak and is obviously higher than that of the wild type, and then the expression is gradually reduced.

Example 2 application of GABRG2(F343L) transgenic zebrafish epilepsy model in antiepileptic drug screening.

Selecting clinically common antiepileptic drugs, such as sodium valproate, carbamazepine, clonazepam, levetiracetam and the like, adding the drugs into a zebrafish culture solution, wherein the use concentrations are respectively 50 mu M, 100 mu M, 50 mu M and 30mM, recording for 30min by adopting a video analysis system after 24h of adding the drugs, and collecting parameters once per minute. The medicine with the treatment effect on the epilepsy phenotype of the transgenic zebra fish is screened by means of swimming behavior analysis and the like. The results are shown in fig. 8, the four drugs can significantly reduce the swimming distance of the transgenic zebra fish, so that the excitability of the transgenic zebra fish is reduced, and the drugs have an inhibitory effect on the seizures of the transgenic zebra fish of GABRG2 (F343L).

Example 3 construction of transgenic Zebra fish line of GABRG2(R177G)

As described in example 1, different mutant GABRG2 gene expression sequences were designed, the mutation site is G (SEQ ID No: 12) from 529 th base C of human GABRG2 gene. Constructing a GABRG2 mutation expression vector pDesttol2CG2; HuC GABRG2^529C>G-polyA, by microinjection of zebrafish embryos, obtaining transgenic zebrafish gene lines.

Phenotypic analysis of GABRG2(R177G) transgenic zebrafish lines:

selecting zebra fish juvenile fish with 5dpf, adopting a zebra fish juvenile fish high-throughput behavior screening box (Danio Vision Chamber) and an animal motion trail tracking system (EthoVision XT 13), recording the motion trail of the wild zebra fish and the mutant zebra fish under normal conditions, recording 24h (illumination 14h and darkness 10h) from 4 o/m, recording behavior parameters once every 1h, comparing the total motion distance difference of the wild zebra fish and the transgenic zebra fish in each time period, and analyzing the circadian rhythm change. The excitation degree of the zebra fish juvenile fish was analyzed by the swimming distance, and as a result, as shown in fig. 9, it was found that under normal culture conditions, GABRG2(R177G) transgenic zebra fish had no significant increase in the swimming distance compared to wild zebra fish. The results show that: GABRG2(R177G) transgenic zebrafish did not have an epileptic phenotype.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

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gaagatggtg cgctcctgga cgtagccttc gggcatggcg gacttgaaga agtcgtgctg 2760

cttcatgtgg tcggggtagc ggctgaagca ctgcacgccg taggtcaggg tggtcacgag 2820

ggtgggccag ggcacgggca gcttgccggt ggtgcagatg aacttcaggg tcagcttgcc 2880

gtaggtggca tcgccctcgc cctcgccgga cacgctgaac ttgtggccgt ttacgtcgcc 2940

gtccagctcg accaggatgg gcaccacccc ggtgaacagc tcctcgccct tgctcaccat 3000

ggtcactgtc tgctttgctg ttggtctggg ctcctgggtc actggcttac taatggagtc 3060

tttatgtatg aggactctta tcaattgttc ttctataaag gtctgcagtg tttctgttcg 3120

tcccctacat ggacacccag agcctcctaa atacaggagc cctgataact gcacaagtgc 3180

tcagattcca gcagggtgga aaatgagata aagtgtgcag atggggaggg ggacgtgaat 3240

gagagatttg agggatgaaa aggatggatg aacgcattga aaatagcccc tttcacaaaa 3300

taataccagt aaattgtcat ataattaatg accagacctt tacctgtaaa tgatgtgatg 3360

ttcatataca gaagaatgcc aacaatttac agatgatttt acaaattctt aattttcctt 3420

ttctaaatgg atttgttcac acattatctc ttcaaagcaa ttgaattttc tggaaaagat 3480

tggctgtgtg tgtaaaaggt gctactgagg aatttaatgt gacatggaat gaagcaaaca 3540

gcagcctaag acaggatggc aggaaaaatg tcacatgttt aaaataagag tgaaaccaac 3600

gacctggact tatagagact gtgtgtcctt aggcaaagct cttgtacaaa cagtgcttgt 3660

gactgtgaaa ttcggggttt gcctggattg tgttaaatgt gtgcttggat gtatcataaa 3720

aaagaatcgg ttctctgtat ttgacatttt ctgtaattta tctaaagtta caactggcta 3780

tgcctgattt ttatcactat tgaatcaatc attttaaaga atgttaagtc tgctgatttg 3840

gagagctcat ttacatttat tttgaatgtc tcttatttaa cacaactgat ttaagcttta 3900

gatctgcgaa gatacggcca cgggtgctct tgatcctgtg gctgattttg gactgtgctg 3960

ctcgcagctg ctgatgaatc acatacttcc tccattttct tccactgatt gactgttata 4020

atttccctaa tttccaggtc aaggtgctgt gcattgtggt aatagatgtg acatgacgtc 4080

acttccaaag gaccaatgaa catgtctgac caatttcata taatgtgaaa acgattttca 4140

taggcagaat aaataacatt taaattaaac tgggcatcag cgcaattcaa ttggtttggt 4200

aatagcaagg gaaaatagaa tgaagtgatc tccaaaaaat aagtactttt tgactgtaaa 4260

taaaattgta aggagtaaaa agtacttttt tttctaaaaa aatgtaatta agtaaaagta 4320

aaagtattga tttttaattg tactcaagta aagtaaaaat ccccaaaaat aatacttaag 4380

tacagtaatc aagtaaaatt actcaagtac tttacacctc tggttcttga ccccctacct 4440

tcagcaagcc cagcagatcc actagttcta gagcggccgc caccgcggtg gagctccagc 4500

ttttgttccc tttagtgagg gttaattgcg cgcttggcgt aatcatggtc atagctgttt 4560

cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag 4620

tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg 4680

cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg 4740

gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc 4800

tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc 4860

acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg 4920

aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat 4980

cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag 5040

gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga 5100

tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg 5160

tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt 5220

cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac 5280

gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc 5340

ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt 5400

ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc 5460

ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc 5520

agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg 5580

aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag 5640

atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg 5700

tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt 5760

tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca 5820

tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca 5880

gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc 5940

tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt 6000

ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg 6060

gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc 6120

aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg 6180

ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga 6240

tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga 6300

ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta 6360

aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg 6420

ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact 6480

ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata 6540

agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt 6600

tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa 6660

ataggggttc cgcgcacatt tccccgaaaa gtg 6693

<210> 3

<211> 4175

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 60

taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 120

gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 180

cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc 240

tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta 300

gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc 360

acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa 420

caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg 480

gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa 540

aacgacggcc agtcttaagc tcgggcccca aataatgatt ttattttgac tgatagtgac 600

ctgttcgttg caacacattg atgagcaatg cttttttata atgccaactt tgtacaaaaa 660

agcaggcttg taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat 720

tgggtaccgg gccccccctc gaggtcgacg gtatcgataa gcttgatatc gaattcgcca 780

ccatgagttc gccaaatata tggagcacag gaagctcagt ctactcgact cctgtatttt 840

cacagaaaat gacggtgtgg attctgctcc tgctgtcgct ctaccctggc ttcactagcc 900

agaaatctga tgatgactat gaagattatg cttctaacaa aacatgggtc ttgactccaa 960

aagttcctga gggtgatgtc actgtcatct taaacaacct gctggaagga tatgacaata 1020

aacttcggcc tgatatagga gtgaagccaa cgttaattca cacagacatg tatgtgaata 1080

gcattggtcc agtgaacgct atcaatatgg aatacactat tgatatattt tttgcgcaaa 1140

cgtggtatga cagacgtttg aaatttaaca gcaccattaa agtcctccga ttgaacagca 1200

acatggtggg gaaaatctgg attccagaca ctttcttcag aaattccaaa aaagctgatg 1260

cacactggat caccaccccc aacaggatgc tgagaatttg gaatgatggt cgagtgctct 1320

acaccctaag gttgacaatt gatgctgagt gccaattaca attgcacaac tttccaatgg 1380

atgaacactc ctgccccttg gagttctcca gttatggcta tccacgtgaa gaaattgttt 1440

atcaatggaa gcgaagttct gttgaagtgg gcgacacaag atcctggagg ctttatcaat 1500

tctcatttgt tggtctaaga aataccaccg aagtagtgaa gacaacttcc ggagattatg 1560

tggtcatgtc tgtctacttt gatctgagca gaagaatggg atactttacc atccagacct 1620

atatcccctg cacactcatt gtcgtcctat cctgggtgtc tttctggatc aataaggatg 1680

ctgttccagc cagaacatct ttaggtatca ccactgtcct gacaatgacc accctcagca 1740

ccattgcccg gaaatcgctc cccaaggtct cctatgtcac agcgatggat ctctttgtat 1800

ctgtttgtct catctttgtc ttctctgctc tggtggagta tggcaccttg cattattttg 1860

tcagcaaccg gaaaccaagc aaggacaaag ataaaaagaa gaaaaaccct cttcttcgga 1920

tgttttcctt caaggcccct accattgata tccgcccaag atcagcaacc attcaaatga 1980

ataatgctac acaccttcaa gagagagatg aagagtacgg ctatgagtgt ctggacggca 2040

aggactgtgc cagttttttc tgctgttttg aagattgtcg aacaggagct tggagacatg 2100

ggaggataca tatccgcatt gccaaaatgg actcctatgc tcggatcttc ttccccactg 2160

ccttctgcct gtttaatctg gtctattggg tctcctacct ctacctgtga tctagagcgg 2220

ccgccaccgc ggtggagctc cagcttttgt tccctttagt gagggttaat tgcgcgcttg 2280

gcgtaatcat ggtcatagca cccagctttc ttgtacaaag ttggcattat aagaaagcat 2340

tgcttatcaa tttgttgcaa cgaacaggtc actatcagtc aaaataaaat cattatttgc 2400

catccagctg atatccccta tagtgagtcg tattacatgg tcatagctgt ttcctggcag 2460

ctctggcccg tgtctcaaaa tctctgatgt tacattgcac aagataaaat aatatcatca 2520

tgaacaataa aactgtctgc ttacataaac agtaatacaa ggggtgttat gagccatatt 2580

caacgggaaa cgtcgaggcc gcgattaaat tccaacatgg atgctgattt atatgggtat 2640

aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa tctatcgctt gtatgggaag 2700

cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca 2760

gatgagatgg tcagactaaa ctggctgacg gaatttatgc ctcttccgac catcaagcat 2820

tttatccgta ctcctgatga tgcatggtta ctcaccactg cgatccccgg aaaaacagca 2880

ttccaggtat tagaagaata tcctgattca ggtgaaaata ttgttgatgc gctggcagtg 2940

ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc cttttaacag cgatcgcgta 3000

tttcgtctcg ctcaggcgca atcacgaatg aataacggtt tggttgatgc gagtgatttt 3060

gatgacgagc gtaatggctg gcctgttgaa caagtctgga aagaaatgca taaacttttg 3120

ccattctcac cggattcagt cgtcactcat ggtgatttct cacttgataa ccttattttt 3180

gacgagggga aattaatagg ttgtattgat gttggacgag tcggaatcgc agaccgatac 3240

caggatcttg ccatcctatg gaactgcctc ggtgagtttt ctccttcatt acagaaacgg 3300

ctttttcaaa aatatggtat tgataatcct gatatgaata aattgcagtt tcatttgatg 3360

ctcgatgagt ttttctaatc agaattggtt aattggttgt aacactggca gagcattacg 3420

ctgacttgac gggacggcgc aagctcatga ccaaaatccc ttaacgtgag ttacgcgtcg 3480

ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt 3540

ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg 3600

ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata 3660

ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca 3720

ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag 3780

tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc 3840

tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga 3900

tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg 3960

tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac 4020

gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg 4080

tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg 4140

ttcctggcct tttgctggcc ttttgctcac atgtt 4175

<210> 4

<211> 11596

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 60

taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 120

gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 180

cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc 240

tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta 300

gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc 360

acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa 420

caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg 480

gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa 540

aacgacggcc agtcttaagc tcgggcccga gttaacgcta ccatggagct ccaaataatg 600

attttatttt gactgatagt gacctgttcg ttgcaacaaa ttgataagca atgctttttt 660

ataatgccaa ctttgtatag aaaagttggt aatacgactc actatagggc gaattgggta 720

ccgggccccc cctcgacgaa ttcccggccg cgtaccgggc cccccctcga aattcactaa 780

tttgaattta aatgcattat ctttctattc ctaaagacct tgggtgacca aaatcttatt 840

ttaataaata aaactgttta ttaaaacttt tttgtttcaa agaaccatat gtatagtgaa 900

atttataaaa atatcaattt ttaaaaagct ggtgtactca tttatgttat gaactctaaa 960

accatatact gactgcaagt gatgatgtat agagtgatgt ttacgagtaa acatatttag 1020

ttgtatacat cctactgagc acattttgat gtatgaaata acattacaag ctttatccaa 1080

attaagccat tttaaaacac tgccaattga aaatacaaat cctggaaaaa atcgtcttta 1140

gcgcagtcat ttgagccatc ctaatccgtt acctcagacc ataataagaa gggataacac 1200

tagctgtagc aatggaacac atctgtttca cacaatcata tctcctgcgc cggtgctaag 1260

cagattcagc gtgatcataa catgctttcc actcataaat gtaaatttac aatttgcaca 1320

tgtaaaacag acacttttga gatattggat aaaaaaacaa gagtatattg cttagtttca 1380

tccaccagtc atccccacag cgtttggaag gccataaaaa gtgtctaaaa tcaatgatca 1440

ttgaaagagc acaagagaga ctcttacgct gtaatgccac tggggacaaa agtgacagtc 1500

tcttaatggg ctcttctgga ggggctcctg aacattaaaa attatcagcg aaattaccga 1560

aagagcttca agcaactggc atgcttgatc ctctgcgtcg gggcggtgaa taggtgcttc 1620

agatgccctc ttacccacgg gctggattca gctgccccgc taccagcgga gaccccctaa 1680

tgagcctctg caattaagtt tattcatgtt aagtgtgaac ggggtgcgtg cggaactgtg 1740

ggcagctaac agacctgggt tctttgtgcc acaagtgctg cctttattcg gctcacaaag 1800

cagaaaacaa cacccgcacc tataatggcg ccctcggctg ggtctaagaa acgtggcgag 1860

ttgacagagc agagtgggcg gggttaagac agactgacag cgggacccat ctccatcctc 1920

ttattaacgc ttaacgagtg ccttcctcat gcaatattca tcgccactaa tatcatccaa 1980

gctctgagct gagctggcca cttatgtaag gcaattatgt aaaatatcag acagggccca 2040

cactcagaat ctgactgggg tagagacgcg ggacgagaac cgagagcaag aactgaaagt 2100

gaaagtgacc actaaaggga ggagaggaca gaggggcagg atgtgtcaag attaccagag 2160

aacacttggc cagaaatgcg caaccattgg agctctccgg attacccaaa ggttaacgag 2220

tttgaacgcc tctgcccact cgccatctct gatggtttcc caagaactcc tcaagcaaaa 2280

tatatataat tgtgtgtatt atgcacagac acgagaaaat gctgtttttc tgatctgcat 2340

tacagcacat ttgcccgcca acgacaatac cacccactcg gtacctcgct gactcctgat 2400

gcctgatacc tgcgcggtga ctgtctacaa tctgcataat caagagaagt tgtgttgaag 2460

acgagcgcca cacaaccgtt tccacaaggt cacccaaggc cggtgcagat gtaggtgagg 2520

tctccataaa cagactgaaa taaacacatc ctccgctggg aacaacaacc ccctcacgcc 2580

tcatgcattc cataagccta catgcatctc ttccaactta tggagactcg cacctaccaa 2640

catccgcaca acaaagatat acagagcgcg ctccctcagg tcaaggcctg tgggggtctg 2700

tgcagaaata ggtcatttgt cacacatcaa gtcctggggc aggagatgca ttatagatga 2760

gaccaaacag cctgtctcgg tgagctctac ccactccctg agactagaaa tgggggaagg 2820

gagcttgaga taacaaccgc tgcaatcact gtgtcgatgt taatatcagc accaaccggg 2880

aacaataagg agatggatgc attcatgttc acatcttacc agtcaagtat catcgaaccg 2940

gcttgataac cacacctcgt gtaatagctg agcagatagt tgtcatttta aagcgttggc 3000

ctttgtcgat tatgtaatgc gcacattcaa cacatggtaa tatagaaacg gttatgtcga 3060

ggttgttttg tccagagatg accttcacac agttacagcc gctctgcatc cacacaaatg 3120

gaggacttaa tcgtggactg cattcttaga aatgatctac aaagacaaat aatgtgaaat 3180

caagaaagga caaaatttaa gtaaggggat gagggagaga gagaacgagg ggcaaggaga 3240

aagcatggct cctgtctttt tctgcaccca tctgttcgga gtgcaggtgg agctctattc 3300

actcagctct gcatgtgtgt ttgggggggg caggaagaaa gggagggcaa aaggaagagt 3360

ggagagatgg tgggggctgg agggatgggg ggttctcggt gatctctcct gaaggggata 3420

atgggagagc agcgctttgc aatggctgcc atgtagtacc ctccctgcac aattagccaa 3480

tcagcagcaa ctctgccagc cagaaggaca cataaagaag aacattgcag cagaggcaca 3540

gaaggagcct gcgaggagct gggaaataca cacacaacag cagaaccaca acaccctccc 3600

ctggacacac cctactgggg atcactgctt ttcttttttt ctgaaccatc gcccacgcca 3660

cacggagaga aatctctctc tcatcatcat cctgaagaaa acccccttat cctcattttc 3720

acactgctga ggaaaaccta caatcgcacg ggctgagatt tcctggcgaa gactgtcctt 3780

tttccttttt cttttttttt cttttccttt ggaaactgac atttgcattt ctccattcca 3840

agccacggcg taataatatc tgcaatccag cctgaagacc tgcaaatcga aggacctaga 3900

tcactatcat ctttgtacgt caagagttac tgtacgtata acctttcttt cttttgctct 3960

gaccaatatg aaacactaaa atcgattcga gcagcctctc agcatcaatt acagtgcgtg 4020

aaaaacattc aaatagaggc agcaaatatt acatgtgaaa atacaggctg gctaaatcca 4080

agctaattta gaaatgtggt caaaacgcat actggcacgt ctaatcgcgg attcagtaaa 4140

caagattaac gattagccca gtgtataagt catatgatac aggcatgcgc gagagcatcg 4200

ctacacccga gctggcttca ttttcggagg aaaatcaaaa cattgctttc tcctgccgtg 4260

cgaaccattc gtcataaacc gtaatacgca acatacattt attactacat ccgttaatta 4320

gcgataatta gccgttatta acaaagagcg ctgaggaatt cctctcaaat agcggaggtg 4380

cggcggaggc agaggggcgt aaaagggcac atgcgtgcct ggctcaaaaa aggatgctcc 4440

agactgaaac tcagagacaa aacacgatgc ctcggaggct gagagcccat cgagaggaaa 4500

ggcaaagaaa agggtcgagt cactcagagg gggaggggag atattgtgca tccattggtt 4560

tgaattgaag caggcagaat aaaaatcctt gcgattatgc ttcgtgagag atcgcgagag 4620

aggaaaaggc taaatgccac gtatgcaaat ggataaatgt catctatttc ttcgcgagta 4680

gacattttgt ggacaacgag agtgtaaata agcgatatga ggggaataca aaggccttga 4740

gggaaagcgt tttgggcgtg tcttgtcgaa aagaagacag cgatggcacg cgctcagaac 4800

caccatcttc acgtagataa tccgcgtgaa aacaataaca aaactgctca ttttaacacg 4860

agcaggtgtt tgaaaccacc caaaaaagat tgtctaaaat attaagatta attaatgacg 4920

tttaaatgtc caggcctgat ataaatgaag cgtgtagtaa gagtaatcgc ctttgtttct 4980

tttcagagat acgttttcag agattcaggg aattatatct tcaataaaat aatgtttgat 5040

ttcattattt taaagctttt taagaagtaa aaatacacat tattcattta cattttattt 5100

tatttaaagg cctatggtct tgaaaataat aagtggaata tataggctat atattcgtct 5160

aaggacagcc ggtgttattt tatagtgacc tcagacaaaa gccgagacaa aacgaggcgc 5220

ttgcctttca ttttaatcct taatagacgg tgtgatgaat gaatgaatga atgaataaat 5280

gaatgaatga cgggcccttc ggcatgttgt tccgcagtcc tccgtgagca cgagtctcta 5340

ccgcagtgcc aagcacataa aaccgttgag tcagataacc ttctccaggc gcgtatacac 5400

aattcaagga caagcacatc acaactatac acaacacaac acaaccgcct aaacgcacga 5460

gctcatcaat cgcaatttaa aggcatgtca gtcaaagaaa aaggcggcgt tatttaaaac 5520

caatgttcaa caatatttgt atgctgacta gcccgggcgc gcacgctata agatcacaag 5580

gccttgtttt ttccctttat tcaaaatatc attaattatt acaggtcata aaacaaaaca 5640

atgcaaatta caatgattta aagactattg atttaacaat aatcagttaa ttatagaata 5700

atattataat attttacata caaatgaaag atgacattat ttgatgacat tgtaattaaa 5760

atatatgcaa tatattttta cgtttttatt aattaggcta ttactattat tataattatc 5820

attattttta ttattattat tattattaaa tagtcttgcg aaataagagt gaaggcagtt 5880

gcaatgttca ttgttcccgg agagtcccgc agcctctggc ctcaacacaa agcgctactg 5940

tttacattaa tattcatagt gctgccatgg tctctgtaga ggaggaacgg gtggctcttt 6000

tgttcgctag tgtcaatacg tttattcagc aaaaagcagc tcggatttgt gaatgcccaa 6060

caagcacacg cgcactccaa tcaaaggacg agaggataac gattttaaac gattcgcaaa 6120

cgtgtttatt tgtaaagacg acatcaagag cttaatgtcc acttgaaaag aaataaatat 6180

gcccgcggcg ccaaggatat tttgaatgta gtcgttcatg gtgagccact cccgaattca 6240

gcactctgga gagcgaccaa agaggaatag cggaatagca actacttgag ccatttctct 6300

ccgtttgatg gcgttcatga ttaaaaatat aatcacgtga ttttttatac atatataaat 6360

atatacataa agtaacgctt tccttttgag aatacataaa tatttattcg cgtgataaat 6420

caatgatctt atatttattt gcacgaccga aattagccaa attcaccaat taaaaaaaaa 6480

ataagccaac aaaaaagaag ggtttatccg ttcagttttg acttgtgtcc tgttgtttta 6540

cagctgccac tgtgatccct taagctgcag tgagagtgtg gctaatgcct tttgtttaag 6600

ataatcctgt aatctgttac cgaaacggcc tattgacaag ccggcattca catttcagtc 6660

aggaggcccg tccagacatg tacatttatg aattacgaat gataaaatta agatctgcat 6720

taggcgtcag aaatgtcacg aacacccatt catttcccac agccaataat ggatcatgct 6780

gggaagcgct atcctcgcag tctcaaaatt aatgttgaca tgttgcgtag agctacagat 6840

tagatcagcc atatgcttta tgtgtttacc tcagcggatc ttcagcaaga tgtgtttatt 6900

ttaagaaaat ggcttcctcg ctgcctgcac aggggcatta aggaagtgac gtgagcgtcc 6960

tcacagtcaa attactttat ctcatgctgc tttcaggcct ctgcttattt tattattatt 7020

attttaaatt agggggaatc acggtggcgt tgtgggtagg gtagcgcgat caccttacag 7080

caagaaggtc gctggttcga gctctggctg ggtcagttgg catttctgtg tggagtttaa 7140

attgaattga aattgaataa actaaattgg ccctagtgta tgtgtgtgaa tgtaagtgtg 7200

catgggtgtt tcccagtgtt gggtttgact ggaagagcat ccgcttcgta aaacatatgc 7260

tggataagtt ggcggttcat ttcactgtgg tgaccgctga ttaataaagg gactaagctg 7320

aaaatgaaat gaattaatac attttaaatg agcttttgct gacatgttta tgattacaaa 7380

tagtgaaatt gtatgttaat ttacgataag tgcattgttc taaattcatt tttatgtagc 7440

agctggtttt tcttccattt gtgtcattca acagaacatt tttctaaaaa agtgaactta 7500

aattttctcc tccctctgac aaggcttgat ggtaatttca gccgactcta attacagact 7560

cactaaaagg aatcggtgca gtcatttatc tttatcagcg cgctggtggg aaggtaatag 7620

gttttgctta tgagttgttt gcgtgcgagt ctgggtcctt gtgtaggcga tgctgatgca 7680

ctgcattaaa tattgaggga gaagtcgctc aaaaatagca aagtacaggc tggggcagga 7740

acgtcaaagc ccgctcagtg gccacgtttc ttgtttcgta agtcctattt ttaatagcga 7800

taattcaggc tcagtagaag gaactccagc actataagca gtctctcgcc tgtctcggcg 7860

agttatgtat tttgggatga agtgctcttc cacctctacc tctgctgtgc tggcgccagt 7920

atctgggctg taagatagtg tacgtgtcag tctgtgtggc cgaagagcct gtgtgttgtg 7980

taactcacta cattcgtcgc tgtggatggc tgacttgcca tggggaggtt tacttcagtt 8040

cacgaatcac attcgccacg atcttgtcac tttgtgtcaa gactcgctct ctgtgtgtca 8100

acagctctgc tgtttgaggt tgaaattctg aacttgagat atatcccagg atatattagg 8160

gctactgcca acggaaaaag caaatggtgc catggccatt tgattgatca aacaaacacc 8220

cttttgcttg ggtgacagtg aaatgtcaga tccatgtttg tactgcttta ataacttgcc 8280

tctctgagct ttctccttgt cagtgtcaat ttgcctgaga tataaattcc ggttgccccg 8340

aaagcttatt ttattaaaaa aaagctaaca tgcactcata caacgtgcgc acatacatgt 8400

gaggcttgct aaaagtcaag aagcaagcca atagcaagag tcgtcagact gctgttacct 8460

gggcaacctg gttagaggta aggctcaagg caagaataaa tcattccact gaagagacac 8520

agcgttacag cgagatgaga gcaagaggcg atagtgaagg gagaacaaaa gaacagccta 8580

cccacactag tgcagatcta cctgtttgag ctactgattg ttcagtttga ttagatatca 8640

gacaattctg ctatctccaa ttgcattgct gcatgaattc ctagaaatca ttttaatcac 8700

caaaaactag tagggacact tgtggcgtat atgattctgc ctgttgattg tggggatcca 8760

acagaaagat catgaatgca ttgttaatta agtcggtgag acaccctgtt ccaccctagg 8820

ggccgttctg gggaaagagt gctttcagtc agctaaagat gactaatatg tgaacatata 8880

ttactacaca tgccaatttg tacattctgg atagttacca gccctgggaa aacatcgaaa 8940

aaaaataaat caacatagat aaaattggca aatcccttct ggtacgatgc agagtgcttg 9000

ctctgagaac ttgtgcattg tgacagaaag cgaccgttaa aaactgaaag tagcatctac 9060

aacctgcata catctgttct ttgaaaatcc cctgtaggcc aaattaagca tcacgctgtg 9120

ctccacagca acacggatga accgaaaacc cagacacaaa cgcacacact tttcacataa 9180

acaaggttta gttatgctag ctatgctttt tcgtcctccc tttgtcatca gcggcgagtg 9240

acgtaacaca gtttgaccct ggacagcgta aagggggggt tgggaatgag tgaaaggact 9300

gtgacatctg cagggccagg ggctgatgga gagagctgaa gtgagggagg tcagaggagg 9360

aggaagggtg ggatattgtc ctcctcaggc cctatccctc tcagacgcat tgatttttaa 9420

cctctcagcg agaatgccaa gcattacacc cctcaaatta tggttcctca cccaacctgt 9480

tatattttcc acctgcagat aattagcacc attgaaactc gaggtcgacg gtatcgataa 9540

gcttgatatc gaattcctgc agcccggggg atccactagt tctagagcgg ccgccaccgc 9600

ggtggagctc cagcttttgt tccctttagt gagggttaat tcaagtttgt acaaaaaagt 9660

tgaacgagaa acgtaaaatg atataaatat caatatatta aattagattt tgcataaaaa 9720

acagactaca taatactgta aaacacaaca tatgcagtca ctatgaacca actacttaga 9780

tggtattagt gacctgtaga attcgagctc tagagctgca gggcggccgc gatatcccct 9840

atagtgagtc gtattacatg gtcatagctg tttcctggca gctctggccc gtgtctcaaa 9900

atctctgatg ttacattgca caagataaaa atatatcatc atgaacaata aaactgtctg 9960

cttacataaa cagtaataca aggggtgtta tgagccatat tcaacgggaa acgtcgaggc 10020

cgcgattaaa ttccaacatg gatgctgatt tatatgggta taaatgggct cgcgataatg 10080

tcgggcaatc aggtgcgaca atctatcgct tgtatgggaa gcccgatgcg ccagagttgt 10140

ttctgaaaca tggcaaaggt agcgttgcca atgatgttac agatgagatg gtcagactaa 10200

actggctgac ggaatttatg cctcttccga ccatcaagca ttttatccgt actcctgatg 10260

atgcatggtt actcaccact gcgatccccg gaaaaacagc attccaggta ttagaagaat 10320

atcctgattc aggtgaaaat attgttgatg cgctggcagt gttcctgcgc cggttgcatt 10380

cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc 10440

aatcacgaat gaataacggt ttggttgatg cgagtgattt tgatgacgag cgtaatggct 10500

ggcctgttga acaagtctgg aaagaaatgc ataaactttt gccattctca ccggattcag 10560

tcgtcactca tggtgatttc tcacttgata accttatttt tgacgagggg aaattaatag 10620

gttgtattga tgttggacga gtcggaatcg cagaccgata ccaggatctt gccatcctat 10680

ggaactgcct cggtgagttt tctccttcat tacagaaacg gctttttcaa aaatatggta 10740

ttgataatcc tgatatgaat aaattgcagt ttcatttgat gctcgatgag tttttctaat 10800

cagaattggt taattggttg taacactggc agagcattac gctgacttga cgggacggcg 10860

caagctcatg accaaaatcc cttaacgtga gttacgcgtc gttccactga gcgtcagacc 10920

ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct 10980

tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa 11040

ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag 11100

tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc 11160

tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg 11220

actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca 11280

cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagcatt 11340

gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg 11400

tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc 11460

ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc 11520

ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc 11580

cttttgctca catgtt 11596

<210> 5

<211> 2838

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 60

taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 120

gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 180

cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc 240

tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta 300

gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc 360

acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa 420

caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg 480

gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa 540

aacgacggcc agtcttaagc tcgggccctg cagctctaga gctcgaattc tacaggtcac 600

taataccatc taagtagttg gttcatagtg actgcatatg ttgtgtttta cagtattatg 660

tagtctgttt tttatgcaaa atctaattta atatattgat atttatatca ttttacgttt 720

ctcgttcaac tttcttgtac aaagtggggg atccagacat gataagatac attgatgagt 780

ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg 840

ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca 900

ttcattttat gtttcaggtt cagggggagg tgtgggaggt tttttccaac tttattatac 960

aaagttggca ttataaaaaa gcattgctta tcaatttgtt gcaacgaaca ggtcactatc 1020

agtcaaaata aaatcattat ttggagctcc atggtagcgt taacgcggcc gcgatatccc 1080

ctatagtgag tcgtattaca tggtcatagc tgtttcctgg cagctctggc ccgtgtctca 1140

aaatctctga tgttacattg cacaagataa aaatatatca tcatgaacaa taaaactgtc 1200

tgcttacata aacagtaata caaggggtgt tatgagccat attcaacggg aaacgtcgag 1260

gccgcgatta aattccaaca tggatgctga tttatatggg tataaatggg ctcgcgataa 1320

tgtcgggcaa tcaggtgcga caatctatcg cttgtatggg aagcccgatg cgccagagtt 1380

gtttctgaaa catggcaaag gtagcgttgc caatgatgtt acagatgaga tggtcagact 1440

aaactggctg acggaattta tgcctcttcc gaccatcaag cattttatcc gtactcctga 1500

tgatgcatgg ttactcacca ctgcgatccc cggaaaaaca gcattccagg tattagaaga 1560

atatcctgat tcaggtgaaa atattgttga tgcgctggca gtgttcctgc gccggttgca 1620

ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc tcgctcaggc 1680

gcaatcacga atgaataacg gtttggttga tgcgagtgat tttgatgacg agcgtaatgg 1740

ctggcctgtt gaacaagtct ggaaagaaat gcataaactt ttgccattct caccggattc 1800

agtcgtcact catggtgatt tctcacttga taaccttatt tttgacgagg ggaaattaat 1860

aggttgtatt gatgttggac gagtcggaat cgcagaccga taccaggatc ttgccatcct 1920

atggaactgc ctcggtgagt tttctccttc attacagaaa cggctttttc aaaaatatgg 1980

tattgataat cctgatatga ataaattgca gtttcatttg atgctcgatg agtttttcta 2040

atcagaattg gttaattggt tgtaacactg gcagagcatt acgctgactt gacgggacgg 2100

cgcaagctca tgaccaaaat cccttaacgt gagttacgcg tcgttccact gagcgtcaga 2160

ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg 2220

cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc 2280

aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct 2340

agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc 2400

tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt 2460

ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg 2520

cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagca 2580

ttgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag 2640

ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag 2700

tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg 2760

gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg 2820

gccttttgct cacatgtt 2838

<210> 6

<211> 16965

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ccacctaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca 60

gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga 120

ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg 180

actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat 240

caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag 300

ggagcccccg atttagagct tgacggggaa agccgggctg gctaagaact catcagcctc 360

cccggtccat ctacccacgt accaatgcac caattggcca caatgacggc tactacatgg 420

tgccattcct tcctctttat aggaatggag actacctcct gtccaacaag gctcttggat 480

acgagtacgc ctacctgttg gacccaggtc attgcacaac accagaaatg ccctctgatc 540

tgcaaaagac gtgaatatct gttcagacac ccatatccac tctgttccac acaggtcaga 600

ggtttgtcca ggagttcttg acagaggtgt aaaaagtact caaaaatttt actcaagtga 660

aagtacaagt acttagggaa aattttactc aattaaaagt aaaagtatct ggctagaatc 720

ttacttgagt aaaagtaaaa aagtactcca ttaaaattgt acttgagtat taaggaagta 780

aaagtaaaag caagaaagaa aactagagat tcttgtttaa gcttttaatc tcaaaaaaca 840

ttaaatgaaa tgcatacaag gttttatcct gctttagaac tgtttgtatt taattatcaa 900

actataagac agacaatcta atgccagtac acgctactca aagttgtaaa acctcagatt 960

taacttcagt agaagctgat tctcaaaatt gttagtgtca agcctagctc ttttggggct 1020

gaaaagcaat cctgcagtgc tgaaaagcct ctcacaggca gccgatgcgg gaagaggtgt 1080

attagtcttg atagagaggc tgcaaatagc aggaaacgtg agcagagact ccctggtgtc 1140

tgaaacacag gccagatggg ccctcgagca ggaaacagct atgaccatga ttacgccaag 1200

ctatcaactt tgtatagaaa agttggtaat acgactcact atagggcgaa ttgggtaccg 1260

ggccccccct cgacgaattc ccggccgcgt accgggcccc ccctcgaaat tcactaattt 1320

gaatttaaat gcattatctt tctattccta aagaccttgg gtgaccaaaa tcttatttta 1380

ataaataaaa ctgtttatta aaactttttt gtttcaaaga accatatgta tagtgaaatt 1440

tataaaaata tcaattttta aaaagctggt gtactcattt atgttatgaa ctctaaaacc 1500

atatactgac tgcaagtgat gatgtataga gtgatgttta cgagtaaaca tatttagttg 1560

tatacatcct actgagcaca ttttgatgta tgaaataaca ttacaagctt tatccaaatt 1620

aagccatttt aaaacactgc caattgaaaa tacaaatcct ggaaaaaatc gtctttagcg 1680

cagtcatttg agccatccta atccgttacc tcagaccata ataagaaggg ataacactag 1740

ctgtagcaat ggaacacatc tgtttcacac aatcatatct cctgcgccgg tgctaagcag 1800

attcagcgtg atcataacat gctttccact cataaatgta aatttacaat ttgcacatgt 1860

aaaacagaca cttttgagat attggataaa aaaacaagag tatattgctt agtttcatcc 1920

accagtcatc cccacagcgt ttggaaggcc ataaaaagtg tctaaaatca atgatcattg 1980

aaagagcaca agagagactc ttacgctgta atgccactgg ggacaaaagt gacagtctct 2040

taatgggctc ttctggaggg gctcctgaac attaaaaatt atcagcgaaa ttaccgaaag 2100

agcttcaagc aactggcatg cttgatcctc tgcgtcgggg cggtgaatag gtgcttcaga 2160

tgccctctta cccacgggct ggattcagct gccccgctac cagcggagac cccctaatga 2220

gcctctgcaa ttaagtttat tcatgttaag tgtgaacggg gtgcgtgcgg aactgtgggc 2280

agctaacaga cctgggttct ttgtgccaca agtgctgcct ttattcggct cacaaagcag 2340

aaaacaacac ccgcacctat aatggcgccc tcggctgggt ctaagaaacg tggcgagttg 2400

acagagcaga gtgggcgggg ttaagacaga ctgacagcgg gacccatctc catcctctta 2460

ttaacgctta acgagtgcct tcctcatgca atattcatcg ccactaatat catccaagct 2520

ctgagctgag ctggccactt atgtaaggca attatgtaaa atatcagaca gggcccacac 2580

tcagaatctg actggggtag agacgcggga cgagaaccga gagcaagaac tgaaagtgaa 2640

agtgaccact aaagggagga gaggacagag gggcaggatg tgtcaagatt accagagaac 2700

acttggccag aaatgcgcaa ccattggagc tctccggatt acccaaaggt taacgagttt 2760

gaacgcctct gcccactcgc catctctgat ggtttcccaa gaactcctca agcaaaatat 2820

atataattgt gtgtattatg cacagacacg agaaaatgct gtttttctga tctgcattac 2880

agcacatttg cccgccaacg acaataccac ccactcggta cctcgctgac tcctgatgcc 2940

tgatacctgc gcggtgactg tctacaatct gcataatcaa gagaagttgt gttgaagacg 3000

agcgccacac aaccgtttcc acaaggtcac ccaaggccgg tgcagatgta ggtgaggtct 3060

ccataaacag actgaaataa acacatcctc cgctgggaac aacaaccccc tcacgcctca 3120

tgcattccat aagcctacat gcatctcttc caacttatgg agactcgcac ctaccaacat 3180

ccgcacaaca aagatataca gagcgcgctc cctcaggtca aggcctgtgg gggtctgtgc 3240

agaaataggt catttgtcac acatcaagtc ctggggcagg agatgcatta tagatgagac 3300

caaacagcct gtctcggtga gctctaccca ctccctgaga ctagaaatgg gggaagggag 3360

cttgagataa caaccgctgc aatcactgtg tcgatgttaa tatcagcacc aaccgggaac 3420

aataaggaga tggatgcatt catgttcaca tcttaccagt caagtatcat cgaaccggct 3480

tgataaccac acctcgtgta atagctgagc agatagttgt cattttaaag cgttggcctt 3540

tgtcgattat gtaatgcgca cattcaacac atggtaatat agaaacggtt atgtcgaggt 3600

tgttttgtcc agagatgacc ttcacacagt tacagccgct ctgcatccac acaaatggag 3660

gacttaatcg tggactgcat tcttagaaat gatctacaaa gacaaataat gtgaaatcaa 3720

gaaaggacaa aatttaagta aggggatgag ggagagagag aacgaggggc aaggagaaag 3780

catggctcct gtctttttct gcacccatct gttcggagtg caggtggagc tctattcact 3840

cagctctgca tgtgtgtttg gggggggcag gaagaaaggg agggcaaaag gaagagtgga 3900

gagatggtgg gggctggagg gatggggggt tctcggtgat ctctcctgaa ggggataatg 3960

ggagagcagc gctttgcaat ggctgccatg tagtaccctc cctgcacaat tagccaatca 4020

gcagcaactc tgccagccag aaggacacat aaagaagaac attgcagcag aggcacagaa 4080

ggagcctgcg aggagctggg aaatacacac acaacagcag aaccacaaca ccctcccctg 4140

gacacaccct actggggatc actgcttttc tttttttctg aaccatcgcc cacgccacac 4200

ggagagaaat ctctctctca tcatcatcct gaagaaaacc cccttatcct cattttcaca 4260

ctgctgagga aaacctacaa tcgcacgggc tgagatttcc tggcgaagac tgtccttttt 4320

cctttttctt tttttttctt ttcctttgga aactgacatt tgcatttctc cattccaagc 4380

cacggcgtaa taatatctgc aatccagcct gaagacctgc aaatcgaagg acctagatca 4440

ctatcatctt tgtacgtcaa gagttactgt acgtataacc tttctttctt ttgctctgac 4500

caatatgaaa cactaaaatc gattcgagca gcctctcagc atcaattaca gtgcgtgaaa 4560

aacattcaaa tagaggcagc aaatattaca tgtgaaaata caggctggct aaatccaagc 4620

taatttagaa atgtggtcaa aacgcatact ggcacgtcta atcgcggatt cagtaaacaa 4680

gattaacgat tagcccagtg tataagtcat atgatacagg catgcgcgag agcatcgcta 4740

cacccgagct ggcttcattt tcggaggaaa atcaaaacat tgctttctcc tgccgtgcga 4800

accattcgtc ataaaccgta atacgcaaca tacatttatt actacatccg ttaattagcg 4860

ataattagcc gttattaaca aagagcgctg aggaattcct ctcaaatagc ggaggtgcgg 4920

cggaggcaga ggggcgtaaa agggcacatg cgtgcctggc tcaaaaaagg atgctccaga 4980

ctgaaactca gagacaaaac acgatgcctc ggaggctgag agcccatcga gaggaaaggc 5040

aaagaaaagg gtcgagtcac tcagaggggg aggggagata ttgtgcatcc attggtttga 5100

attgaagcag gcagaataaa aatccttgcg attatgcttc gtgagagatc gcgagagagg 5160

aaaaggctaa atgccacgta tgcaaatgga taaatgtcat ctatttcttc gcgagtagac 5220

attttgtgga caacgagagt gtaaataagc gatatgaggg gaatacaaag gccttgaggg 5280

aaagcgtttt gggcgtgtct tgtcgaaaag aagacagcga tggcacgcgc tcagaaccac 5340

catcttcacg tagataatcc gcgtgaaaac aataacaaaa ctgctcattt taacacgagc 5400

aggtgtttga aaccacccaa aaaagattgt ctaaaatatt aagattaatt aatgacgttt 5460

aaatgtccag gcctgatata aatgaagcgt gtagtaagag taatcgcctt tgtttctttt 5520

cagagatacg ttttcagaga ttcagggaat tatatcttca ataaaataat gtttgatttc 5580

attattttaa agctttttaa gaagtaaaaa tacacattat tcatttacat tttattttat 5640

ttaaaggcct atggtcttga aaataataag tggaatatat aggctatata ttcgtctaag 5700

gacagccggt gttattttat agtgacctca gacaaaagcc gagacaaaac gaggcgcttg 5760

cctttcattt taatccttaa tagacggtgt gatgaatgaa tgaatgaatg aataaatgaa 5820

tgaatgacgg gcccttcggc atgttgttcc gcagtcctcc gtgagcacga gtctctaccg 5880

cagtgccaag cacataaaac cgttgagtca gataaccttc tccaggcgcg tatacacaat 5940

tcaaggacaa gcacatcaca actatacaca acacaacaca accgcctaaa cgcacgagct 6000

catcaatcgc aatttaaagg catgtcagtc aaagaaaaag gcggcgttat ttaaaaccaa 6060

tgttcaacaa tatttgtatg ctgactagcc cgggcgcgca cgctataaga tcacaaggcc 6120

ttgttttttc cctttattca aaatatcatt aattattaca ggtcataaaa caaaacaatg 6180

caaattacaa tgatttaaag actattgatt taacaataat cagttaatta tagaataata 6240

ttataatatt ttacatacaa atgaaagatg acattatttg atgacattgt aattaaaata 6300

tatgcaatat atttttacgt ttttattaat taggctatta ctattattat aattatcatt 6360

atttttatta ttattattat tattaaatag tcttgcgaaa taagagtgaa ggcagttgca 6420

atgttcattg ttcccggaga gtcccgcagc ctctggcctc aacacaaagc gctactgttt 6480

acattaatat tcatagtgct gccatggtct ctgtagagga ggaacgggtg gctcttttgt 6540

tcgctagtgt caatacgttt attcagcaaa aagcagctcg gatttgtgaa tgcccaacaa 6600

gcacacgcgc actccaatca aaggacgaga ggataacgat tttaaacgat tcgcaaacgt 6660

gtttatttgt aaagacgaca tcaagagctt aatgtccact tgaaaagaaa taaatatgcc 6720

cgcggcgcca aggatatttt gaatgtagtc gttcatggtg agccactccc gaattcagca 6780

ctctggagag cgaccaaaga ggaatagcgg aatagcaact acttgagcca tttctctccg 6840

tttgatggcg ttcatgatta aaaatataat cacgtgattt tttatacata tataaatata 6900

tacataaagt aacgctttcc ttttgagaat acataaatat ttattcgcgt gataaatcaa 6960

tgatcttata tttatttgca cgaccgaaat tagccaaatt caccaattaa aaaaaaaata 7020

agccaacaaa aaagaagggt ttatccgttc agttttgact tgtgtcctgt tgttttacag 7080

ctgccactgt gatcccttaa gctgcagtga gagtgtggct aatgcctttt gtttaagata 7140

atcctgtaat ctgttaccga aacggcctat tgacaagccg gcattcacat ttcagtcagg 7200

aggcccgtcc agacatgtac atttatgaat tacgaatgat aaaattaaga tctgcattag 7260

gcgtcagaaa tgtcacgaac acccattcat ttcccacagc caataatgga tcatgctggg 7320

aagcgctatc ctcgcagtct caaaattaat gttgacatgt tgcgtagagc tacagattag 7380

atcagccata tgctttatgt gtttacctca gcggatcttc agcaagatgt gtttatttta 7440

agaaaatggc ttcctcgctg cctgcacagg ggcattaagg aagtgacgtg agcgtcctca 7500

cagtcaaatt actttatctc atgctgcttt caggcctctg cttattttat tattattatt 7560

ttaaattagg gggaatcacg gtggcgttgt gggtagggta gcgcgatcac cttacagcaa 7620

gaaggtcgct ggttcgagct ctggctgggt cagttggcat ttctgtgtgg agtttaaatt 7680

gaattgaaat tgaataaact aaattggccc tagtgtatgt gtgtgaatgt aagtgtgcat 7740

gggtgtttcc cagtgttggg tttgactgga agagcatccg cttcgtaaaa catatgctgg 7800

ataagttggc ggttcatttc actgtggtga ccgctgatta ataaagggac taagctgaaa 7860

atgaaatgaa ttaatacatt ttaaatgagc ttttgctgac atgtttatga ttacaaatag 7920

tgaaattgta tgttaattta cgataagtgc attgttctaa attcattttt atgtagcagc 7980

tggtttttct tccatttgtg tcattcaaca gaacattttt ctaaaaaagt gaacttaaat 8040

tttctcctcc ctctgacaag gcttgatggt aatttcagcc gactctaatt acagactcac 8100

taaaaggaat cggtgcagtc atttatcttt atcagcgcgc tggtgggaag gtaataggtt 8160

ttgcttatga gttgtttgcg tgcgagtctg ggtccttgtg taggcgatgc tgatgcactg 8220

cattaaatat tgagggagaa gtcgctcaaa aatagcaaag tacaggctgg ggcaggaacg 8280

tcaaagcccg ctcagtggcc acgtttcttg tttcgtaagt cctattttta atagcgataa 8340

ttcaggctca gtagaaggaa ctccagcact ataagcagtc tctcgcctgt ctcggcgagt 8400

tatgtatttt gggatgaagt gctcttccac ctctacctct gctgtgctgg cgccagtatc 8460

tgggctgtaa gatagtgtac gtgtcagtct gtgtggccga agagcctgtg tgttgtgtaa 8520

ctcactacat tcgtcgctgt ggatggctga cttgccatgg ggaggtttac ttcagttcac 8580

gaatcacatt cgccacgatc ttgtcacttt gtgtcaagac tcgctctctg tgtgtcaaca 8640

gctctgctgt ttgaggttga aattctgaac ttgagatata tcccaggata tattagggct 8700

actgccaacg gaaaaagcaa atggtgccat ggccatttga ttgatcaaac aaacaccctt 8760

ttgcttgggt gacagtgaaa tgtcagatcc atgtttgtac tgctttaata acttgcctct 8820

ctgagctttc tccttgtcag tgtcaatttg cctgagatat aaattccggt tgccccgaaa 8880

gcttatttta ttaaaaaaaa gctaacatgc actcatacaa cgtgcgcaca tacatgtgag 8940

gcttgctaaa agtcaagaag caagccaata gcaagagtcg tcagactgct gttacctggg 9000

caacctggtt agaggtaagg ctcaaggcaa gaataaatca ttccactgaa gagacacagc 9060

gttacagcga gatgagagca agaggcgata gtgaagggag aacaaaagaa cagcctaccc 9120

acactagtgc agatctacct gtttgagcta ctgattgttc agtttgatta gatatcagac 9180

aattctgcta tctccaattg cattgctgca tgaattccta gaaatcattt taatcaccaa 9240

aaactagtag ggacacttgt ggcgtatatg attctgcctg ttgattgtgg ggatccaaca 9300

gaaagatcat gaatgcattg ttaattaagt cggtgagaca ccctgttcca ccctaggggc 9360

cgttctgggg aaagagtgct ttcagtcagc taaagatgac taatatgtga acatatatta 9420

ctacacatgc caatttgtac attctggata gttaccagcc ctgggaaaac atcgaaaaaa 9480

aataaatcaa catagataaa attggcaaat cccttctggt acgatgcaga gtgcttgctc 9540

tgagaacttg tgcattgtga cagaaagcga ccgttaaaaa ctgaaagtag catctacaac 9600

ctgcatacat ctgttctttg aaaatcccct gtaggccaaa ttaagcatca cgctgtgctc 9660

cacagcaaca cggatgaacc gaaaacccag acacaaacgc acacactttt cacataaaca 9720

aggtttagtt atgctagcta tgctttttcg tcctcccttt gtcatcagcg gcgagtgacg 9780

taacacagtt tgaccctgga cagcgtaaag ggggggttgg gaatgagtga aaggactgtg 9840

acatctgcag ggccaggggc tgatggagag agctgaagtg agggaggtca gaggaggagg 9900

aagggtggga tattgtcctc ctcaggccct atccctctca gacgcattga tttttaacct 9960

ctcagcgaga atgccaagca ttacacccct caaattatgg ttcctcaccc aacctgttat 10020

attttccacc tgcagataat tagcaccatt gaaactcgag gtcgacggta tcgataagct 10080

tgatatcgaa ttcctgcagc ccgggggatc cactagttct agagcggccg ccaccgcggt 10140

ggagctccag cttttgttcc ctttagtgag ggttaattca agtttgtaca aaaaagcagg 10200

cttgtaaaac gacggccagt gagcgcgcgt aatacgactc actatagggc gaattgggta 10260

ccgggccccc cctcgaggtc gacggtatcg ataagcttga tatcgaattc gccaccatga 10320

gttcgccaaa tatatggagc acaggaagct cagtctactc gactcctgta ttttcacaga 10380

aaatgacggt gtggattctg ctcctgctgt cgctctaccc tggcttcact agccagaaat 10440

ctgatgatga ctatgaagat tatgcttcta acaaaacatg ggtcttgact ccaaaagttc 10500

ctgagggtga tgtcactgtc atcttaaaca acctgctgga aggatatgac aataaacttc 10560

ggcctgatat aggagtgaag ccaacgttaa ttcacacaga catgtatgtg aatagcattg 10620

gtccagtgaa cgctatcaat atggaataca ctattgatat attttttgcg caaacgtggt 10680

atgacagacg tttgaaattt aacagcacca ttaaagtcct ccgattgaac agcaacatgg 10740

tggggaaaat ctggattcca gacactttct tcagaaattc caaaaaagct gatgcacact 10800

ggatcaccac ccccaacagg atgctgagaa tttggaatga tggtcgagtg ctctacaccc 10860

taaggttgac aattgatgct gagtgccaat tacaattgca caactttcca atggatgaac 10920

actcctgccc cttggagttc tccagttatg gctatccacg tgaagaaatt gtttatcaat 10980

ggaagcgaag ttctgttgaa gtgggcgaca caagatcctg gaggctttat caattctcat 11040

ttgttggtct aagaaatacc accgaagtag tgaagacaac ttccggagat tatgtggtca 11100

tgtctgtcta ctttgatctg agcagaagaa tgggatactt taccatccag acctatatcc 11160

cctgcacact cattgtcgtc ctatcctggg tgtctttctg gatcaataag gatgctgttc 11220

cagccagaac atctttaggt atcaccactg tcctgacaat gaccaccctc agcaccattg 11280

cccggaaatc gctccccaag gtctcctatg tcacagcgat ggatctcttt gtatctgttt 11340

gtctcatctt tgtcttctct gctctggtgg agtatggcac cttgcattat tttgtcagca 11400

accggaaacc aagcaaggac aaagataaaa agaagaaaaa ccctcttctt cggatgtttt 11460

ccttcaaggc ccctaccatt gatatccgcc caagatcagc aaccattcaa atgaataatg 11520

ctacacacct tcaagagaga gatgaagagt acggctatga gtgtctggac ggcaaggact 11580

gtgccagttt tttctgctgt tttgaagatt gtcgaacagg agcttggaga catgggagga 11640

tacatatccg cattgccaaa atggactcct atgctcggat cttcttcccc actgccttct 11700

gcctgtttaa tctggtctat tgggtctcct acctctacct gtgatctaga gcggccgcca 11760

ccgcggtgga gctccagctt ttgttccctt tagtgagggt taattgcgcg cttggcgtaa 11820

tcatggtcat agcacccagc tttcttgtac aaagtggggg atccagacat gataagatac 11880

attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa 11940

atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac 12000

aacaattgca ttcattttat gtttcaggtt cagggggagg tgtgggaggt tttttccaac 12060

tttattatac atagttgata attcactggc cgtcgtttta cggtaccatc gatgatgatc 12120

cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa 12180

aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca 12240

ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag ggggaggtgt 12300

gggaggtttt ttaaagcaag taaaacctct acaaatgtgg tatggctgat tatgatcctc 12360

tagatcagat ctcttgttta ttgcagctta taatggttac aaataaagca atagcatcac 12420

aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat 12480

caatgtatct tatcatgtct ggatctacgt aatacgactc actatagttc tagaggctcg 12540

agaggggccg ctttacttgt acagctcgtc catgccgaga gtgatcccgg cggcggtcac 12600

gaactccagc aggaccatgt gatcgcgctt ctcgttgggg tctttgctca gggcggactg 12660

ggtgctcagg tagtggttgt cgggcagcag cacggggccg tcgccgatgg gggtgttctg 12720

ctggtagtgg tcggcgagct gcacgctgcc gtcctcgatg ttgtggcgga tcttgaagtt 12780

caccttgatg ccgttcttct gcttgtcggc catgatatag acgttgtggc tgttgtagtt 12840

gtactccagc ttgtgcccca ggatgttgcc gtcctccttg aagtcgatgc ccttcagctc 12900

gatgcggttc accagggtgt cgccctcgaa cttcacctcg gcgcgggtct tgtagttgcc 12960

gtcgtccttg aagaagatgg tgcgctcctg gacgtagcct tcgggcatgg cggacttgaa 13020

gaagtcgtgc tgcttcatgt ggtcggggta gcggctgaag cactgcacgc cgtaggtcag 13080

ggtggtcacg agggtgggcc agggcacggg cagcttgccg gtggtgcaga tgaacttcag 13140

ggtcagcttg ccgtaggtgg catcgccctc gccctcgccg gacacgctga acttgtggcc 13200

gtttacgtcg ccgtccagct cgaccaggat gggcaccacc ccggtgaaca gctcctcgcc 13260

cttgctcacc atggtcactg tctgctttgc tgttggtctg ggctcctggg tcactggctt 13320

actaatggag tctttatgta tgaggactct tatcaattgt tcttctataa aggtctgcag 13380

tgtttctgtt cgtcccctac atggacaccc agagcctcct aaatacagga gccctgataa 13440

ctgcacaagt gctcagattc cagcagggtg gaaaatgaga taaagtgtgc agatggggag 13500

ggggacgtga atgagagatt tgagggatga aaaggatgga tgaacgcatt gaaaatagcc 13560

cctttcacaa aataatacca gtaaattgtc atataattaa tgaccagacc tttacctgta 13620

aatgatgtga tgttcatata cagaagaatg ccaacaattt acagatgatt ttacaaattc 13680

ttaattttcc ttttctaaat ggatttgttc acacattatc tcttcaaagc aattgaattt 13740

tctggaaaag attggctgtg tgtgtaaaag gtgctactga ggaatttaat gtgacatgga 13800

atgaagcaaa cagcagccta agacaggatg gcaggaaaaa tgtcacatgt ttaaaataag 13860

agtgaaacca acgacctgga cttatagaga ctgtgtgtcc ttaggcaaag ctcttgtaca 13920

aacagtgctt gtgactgtga aattcggggt ttgcctggat tgtgttaaat gtgtgcttgg 13980

atgtatcata aaaaagaatc ggttctctgt atttgacatt ttctgtaatt tatctaaagt 14040

tacaactggc tatgcctgat ttttatcact attgaatcaa tcattttaaa gaatgttaag 14100

tctgctgatt tggagagctc atttacattt attttgaatg tctcttattt aacacaactg 14160

atttaagctt tagatctgcg aagatacggc cacgggtgct cttgatcctg tggctgattt 14220

tggactgtgc tgctcgcagc tgctgatgaa tcacatactt cctccatttt cttccactga 14280

ttgactgtta taatttccct aatttccagg tcaaggtgct gtgcattgtg gtaatagatg 14340

tgacatgacg tcacttccaa aggaccaatg aacatgtctg accaatttca tataatgtga 14400

aaacgatttt cataggcaga ataaataaca tttaaattaa actgggcatc agcgcaattc 14460

aattggtttg gtaatagcaa gggaaaatag aatgaagtga tctccaaaaa ataagtactt 14520

tttgactgta aataaaattg taaggagtaa aaagtacttt tttttctaaa aaaatgtaat 14580

taagtaaaag taaaagtatt gatttttaat tgtactcaag taaagtaaaa atccccaaaa 14640

ataatactta agtacagtaa tcaagtaaaa ttactcaagt actttacacc tctggttctt 14700

gaccccctac cttcagcaag cccagcagat ccactagttc tagagcggcc gccaccgcgg 14760

tggagctcca gcttttgttc cctttagtga gggttaattg cgcgcttggc gtaatcatgg 14820

tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc 14880

ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg 14940

ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc 15000

ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact 15060

gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta 15120

atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag 15180

caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc 15240

cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta 15300

taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg 15360

ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc 15420

tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac 15480

gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac 15540

ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg 15600

aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga 15660

aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt 15720

agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag 15780

cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct 15840

gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg 15900

atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat 15960

gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc 16020

tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg 16080

gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct 16140

ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca 16200

actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg 16260

ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg 16320

tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc 16380

cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag 16440

ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg 16500

ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag 16560

tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat 16620

agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg 16680

atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca 16740

gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca 16800

aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat 16860

tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag 16920

aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtg 16965

<210> 7

<211> 6034

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

tcgaccatag ccaattcaat atggcgtata tggactcatg ccaattcaat atggtggatc 60

tggacctgtg ccaattcaat atggcgtata tggactcgtg ccaattcaat atggtggatc 120

tggaccccag ccaattcaat atggcggact tggcaccatg ccaattcaat atggcggact 180

tggcactgtg ccaactgggg aggggtctac ttggcacggt gccaagtttg aggaggggtc 240

ttggccctgt gccaagtccg ccatattgaa ttggcatggt gccaataatg gcggccatat 300

tggctatatg ccaggatcaa tatataggca atatccaata tggccctatg ccaatatggc 360

tattggccag gttcaatact atgtattggc cctatgccat atagtattcc atatatgggt 420

tttcctattg acgtagatag cccctcccaa tgggcggtcc catataccat atatggggct 480

tcctaatacc gcccatagcc actcccccat tgacgtcaat ggtctctata tatggtcttt 540

cctattgacg tcatatgggc ggtcctattg acgtatatgg cgcctccccc attgacgtca 600

attacggtaa atggcccgcc tggctcaatg cccattgacg tcaataggac cacccaccat 660

tgacgtcaat gggatggctc attgcccatt catatccgtt ctcacgcccc ctattgacgt 720

caatgacggt aaatggccca cttggcagta catcaatatc tattaatagt aacttggcaa 780

gtacattact attggaagta cgccagggta cattggcagt actcccattg acgtcaatgg 840

cggtaaatgg cccgcgatgg ctgccaagta catccccatt gacgtcaatg gggaggggca 900

atgacgcaaa tgggcgttcc attgacgtaa atgggcggta ggcgtgccta atgggaggtc 960

tatataagca atgctcgttt agggaaccgc cattctgcct ggggacgtcg gagcaagctt 1020

gatttaggtg acactataga atacaagcta cttgttcttt ttgcaggatc cggccggcca 1080

ccatggagga agtatgtgat tcatcagcag ctgcgagcag cacagtccaa aatcagccac 1140

aggatcaaga gcacccgtgg ccgtatcttc gcgaattctt ttctttaagt ggtgtaaata 1200

aagattcatt caagatgaaa tgtgtcctct gtctcccgct taataaagaa atatcggcct 1260

tcaaaagttc gccatcaaac ctaaggaagc atattgagag aatgcaccca aattacctca 1320

aaaactactc taaattgaca gcacagaaga gaaagatcgg gacctccacc catgcttcca 1380

gcagtaagca actgaaagtt gactcagttt tcccagtcaa acatgtgtct ccagtcactg 1440

tgaacaaagc tatattaagg tacatcattc aaggacttca tcctttcagc actgttgatc 1500

tgccatcatt taaagagctg attagtacac tgcagcctgg catttctgtc attacaaggc 1560

ctactttacg ctccaagata gctgaagctg ctctgatcat gaaacagaaa gtgactgctg 1620

ccatgagtga agttgaatgg attgcaacca caacggattg ttggactgca cgtagaaagt 1680

cattcattgg tgtaactgct cactggatca accctggaag tcttgaaaga cattccgctg 1740

cacttgcctg caaaagatta atgggctctc atacttttga ggtactggcc agtgccatga 1800

atgatatcca ctcagagtat gaaatacgtg acaaggttgt ttgcacaacc acagacagtg 1860

gttccaactt tatgaaggct ttcagagttt ttggtgtgga aaacaatgat atcgagactg 1920

aggcaagaag gtgtgaaagt gatgacactg attctgaagg ctgtggtgag ggaagtgatg 1980

gtgtggaatt ccaagatgcc tcacgagtcc tggaccaaga cgatggcttc gaattccagc 2040

taccaaaaca tcaaaagtgt gcctgtcact tacttaacct agtctcaagc gttgatgccc 2100

aaaaagctct ctcaaatgaa cactacaaga aactctacag atctgtcttt ggcaaatgcc 2160

aagctttatg gaataaaagc agccgatcgg ctctagcagc tgaagctgtt gaatcagaaa 2220

gccggcttca gcttttaagg ccaaaccaaa cgcggtggaa ttcaactttt atggctgttg 2280

acagaattct tcaaatttgc aaagaagcag gagaaggcgc acttcggaat atatgcacct 2340

ctcttgaggt tccaatgttt aatccagcag aaatgctgtt cttgacagag tgggccaaca 2400

caatgcgtcc agttgcaaaa gtactcgaca tcttgcaagc ggaaacgaat acacagctgg 2460

ggtggctgct gcctagtgtc catcagttaa gcttgaaact tcagcgactc caccattctc 2520

tcaggtactg tgacccactt gtggatgccc tacaacaagg aatccaaaca cgattcaagc 2580

atatgtttga agatcctgag atcatagcag ctgccatcct tctccctaaa tttcggacct 2640

cttggacaaa tgatgaaacc atcataaaac gaggcatgga ctacatcaga gtgcatctgg 2700

agcctttgga ccacaagaag gaattggcca acagttcatc tgatgatgaa gattttttcg 2760

cttctttgaa accgacaaca catgaagcca gcaaagagtt ggatggatat ctggcctgtg 2820

tttcagacac cagggagtct ctgctcacgt ttcctgctat ttgcagcctc tctatcaaga 2880

ctaatacacc tcttcccgca tcggctgcct gtgagaggct tttcagcact gcaggattgc 2940

ttttcagccc caaaagagct aggcttgaca ctaacaattt tgagaatcag cttctactga 3000

agttaaatct gaggttttac aactttgagt agggcgcgcc tctagaacta tagtgagtcg 3060

tattacgtag atccagacat gataagatac attgatgagt ttggacaaac cacaactaga 3120

atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc 3180

attataagct gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt 3240

cagggggagg tgtgggaggt tttttaattc gcggccgcgg cgccaatgca ttgggcccgg 3300

tacccagctt ttgttccctt tagtgagggt taattgcgcg cttggcgtaa tcatggtcat 3360

agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa 3420

gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc 3480

gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc 3540

aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact 3600

cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac 3660

ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa 3720

aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg 3780

acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa 3840

gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc 3900

ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac 3960

gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac 4020

cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg 4080

taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt 4140

atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga 4200

cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct 4260

cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga 4320

ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg 4380

ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct 4440

tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt 4500

aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc 4560

tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg 4620

gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag 4680

atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt 4740

tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag 4800

ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt 4860

ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca 4920

tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg 4980

ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat 5040

ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta 5100

tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca 5160

gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct 5220

taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat 5280

cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa 5340

agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt 5400

gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa 5460

ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctaaa ttgtaagcgt 5520

taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata 5580

ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt 5640

tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg 5700

aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt 5760

ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc 5820

ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg 5880

cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct 5940

taatgcgccg ctacagggcg cgtcccattc gccattcagg ctgcgcaact gttgggaagg 6000

gcgatcggtg cgggcctctt cgctattacg ccag 6034

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

gcacaggaag ctcagtctac 20

<210> 9

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

ggagtccatt ttggcaatgc 20

<210> 10

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

aactgtcacg gcgatctctt 20

<210> 11

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

cttgcagatg ggtttgtgtg 20

<210> 12

<211> 1401

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

atgagttcgc caaatatatg gagcacagga agctcagtct actcgactcc tgtattttca 60

cagaaaatga cggtgtggat tctgctcctg ctgtcgctct accctggctt cactagccag 120

aaatctgatg atgactatga agattatgct tctaacaaaa catgggtctt gactccaaaa 180

gttcctgagg gtgatgtcac tgtcatctta aacaacctgc tggaaggata tgacaataaa 240

cttcggcctg atataggagt gaagccaacg ttaattcaca cagacatgta tgtgaatagc 300

attggtccag tgaacgctat caatatggaa tacactattg atatattttt tgcgcaaacg 360

tggtatgaca gacgtttgaa atttaacagc accattaaag tcctccgatt gaacagcaac 420

atggtgggga aaatctggat tccagacact ttcttcagaa attccaaaaa agctgatgca 480

cactggatca ccacccccaa caggatgctg agaatttgga atgatggtgg agtgctctac 540

accctaaggt tgacaattga tgctgagtgc caattacaat tgcacaactt tccaatggat 600

gaacactcct gccccttgga gttctccagt tatggctatc cacgtgaaga aattgtttat 660

caatggaagc gaagttctgt tgaagtgggc gacacaagat cctggaggct ttatcaattc 720

tcatttgttg gtctaagaaa taccaccgaa gtagtgaaga caacttccgg agattatgtg 780

gtcatgtctg tctactttga tctgagcaga agaatgggat actttaccat ccagacctat 840

atcccctgca cactcattgt cgtcctatcc tgggtgtctt tctggatcaa taaggatgct 900

gttccagcca gaacatcttt aggtatcacc actgtcctga caatgaccac cctcagcacc 960

attgcccgga aatcgctccc catggtctcc tatgtcacag cgatggatct ctttgtatct 1020

gtttgtttca tctttgtctt ctctgctctg gtggagtatg gcaccttgca ttattttgtc 1080

agcaaccgga aaccaagcaa ggacaaagat aaaaagaaga aaaaccctgc ccctaccatt 1140

gatatccgcc caagatcagc aaccattcaa atgaataatg ctacacacct tcaagagaga 1200

gatgaagagt acggctatga gtgtctggac ggcaaggact gtgccagttt tttctgctgt 1260

tttgaagatt gtcgaacagg agcttggaga catgggagga tacatatccg cattgccaaa 1320

atggactcct atgctcggat cttcttcccc actgccttct gcctgtttaa tctggtctat 1380

tgggtctcct acctctacct g 1401

Claims (6)

1. A method for constructing a mutant GABRG2 transgenic zebra fish epilepsy model is characterized in that a mutant human GABRG2 gene is transferred into zebra fish to enable the zebra fish to become a zebra fish line with an epilepsy phenotype, the mutant human GABRG2 gene is characterized in that a base T in a 343 rd amino acid Phe codon of human GABRG2 is mutated into C, and the gene sequence is as shown in SEQ ID No: 1, constructing an expression vector by adopting gateway technology, wherein the expression vector is pDesttol2CG 2.

2. The method of construction of claim 1, comprising the steps of:

(1) construction of GABRG2 mutant expression vector: synthesizing mutant human GABRG2 gene, cloning it to pME-MCS plasmid vector, constructing midle entry clone pME-GABRG2^1027T>C(ii) a Cloning promoter region sequence containing HuC neuron specificity, constructing vector 5' entry clone: p5E-HuC and 3' entry clone: p 3E-polyA; the 5 'entry clone, the midle entry clone and the 3' entry clone vectors are recombined and cloned into an expression vector pDestTol2CG2 through Gateway LR reaction to obtain an expression vector: pDesttol2CG2, HuC GABRG2^1027T>C–polyA；

(2) Microinjection of zebrafish embryos: microinjecting the GABRG2 mutant expression vector and Tol2 transposase mRNA into wild type zebra fish fertilized eggs at 1-2 cell stage, and screening embryos expressing green fluorescence by heart after the embryos develop to 24hpf to obtain the positive zebra fish containing the GABRG2 mutant sequence.

3. The method of claim 1, wherein said pDesttol2CG2, HuC GABRG2^1027T>The sequence of C-polyA is shown as SEQ ID No: and 6.

4. The method of any one of claims 1 to 3, further comprising the steps of identifying the genotype of the transgenic zebrafish and obtaining a stable genetic line.

5. The construction method of claim 4, wherein the genotype of the positive zebra fish embryo containing the GABRG2 mutant sequence is identified by PCR after the positive zebra fish embryo is mature, the positive zebra fish containing the GABRG2 mutant sequence is selected to be hybridized with the wild zebra fish to obtain F1 generation zebra fish, and the GABRG2 mutant zebra fish line is finally screened by means of green fluorescence of heart expression and genomic DNA detection.

6. Use of the mutant GABRG2 transgenic zebrafish model for epilepsy constructed by the method of any one of claims 1-5 in the study of epilepsy pathology and in the screening of antiepileptic drugs.

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