AU2019445928A1

AU2019445928A1 - Construction method for mutant GABRG2 transgenic zebrafish epilepsy model and applications

Info

Publication number: AU2019445928A1
Application number: AU2019445928A
Authority: AU
Inventors: Juan Chen; Shu'an KE; Dong Liu; Dingding SHEN; Qi Zhang
Original assignee: Nantong University
Current assignee: Nantong University
Priority date: 2019-05-14
Filing date: 2019-12-05
Publication date: 2021-08-26
Anticipated expiration: 2039-12-05
Also published as: AU2019445928B2; CN111944835A; WO2020228305A1; CN111944835B

Abstract

Disclosed is a method constructing a mutant GABRG2 transgene-induced epileptic zebrafish model, with zebrafish serving as a model animal, a mutated human GABRG2 gene is transferred to the zebrafish for same to become a zebrafish system having an epileptic phenotype. The zebrafish system is applicable in researching relations between GABRG2 mutations and the pathogenesis of epilepsy and applicable in screening an antiepileptic medicament.

Description

CONSTRUCTION METHOD AND USE OF MUTANT GABRG2 TRANSGENIC ZEBRAFISH EPILEPSY MODEL

TECHNICAL FIELD

The present invention relates to the field of animal model preparation technologies in biology and basic medical research, and specifically, to a transgenic zebrafish epilepsy model constructed by using an epilepsy-related gene GABRG2 mutant, a construction method of the epilepsy zebrafish model, and use thereof in research work such as epilepsy pathogenesis, drug screening, and antiepileptic drug target identification.

BACKGROUND

Epilepsy is a common chronic neurological disease characterized by spontaneous unprovoked recurrent epileptic seizures. Epilepsy is one of the most common disabling neurological diseases, affecting more than 50 million people worldwide. Approximately 2.4 million people are diagnosed with epilepsy each year. The increase in the frequency, duration, and severity of epileptic seizures may aggravate the cognitive and emotional disorders of patients and lead to the occurrence of epileptic encephalopathy, causing a series of sequelae such as cognitive impairment and emotional anxiety. Epilepsy is divided into primary and secondary epilepsies according to its etiology, where primary epilepsy is mostly related to genetic defects. In the past two decades, with the development of next-generation sequencing and other genome technologies, nearly 1000 gene mutations have been identified that are related to the occurrence of epilepsy, including many gene mutations in ion channels and non ion channels. The y-aminobutyric acid type A receptor (GABAAR) is a major mediator of rapid inhibitory synaptic transmission in the central nervous system, which has been repeatedly shown to play a key role in epileptic seizures. GABAAR is an isopentyl dimer formed by the assembly of a plurality of subunits. It has been detected so far that mutations in subunits al, I1, P2, P3, y2, and 6 of GABAAR are closely related to the onset of epilepsy, and these mutations usually have autosomal dominant inheritance. GABAAR subunit y2 (GABRG2) has important significance for GABAAR transportation and synapse functions. At present, more than 10 GABRG2 mutations related to the onset of epilepsy have been found, including missense mutations, nonsense mutations, frameshift mutations, and truncating mutations. These mutations reduce the function of ion channels to

I varying degrees through different mechanisms, such as mRNA and endoplasmic reticulum associated protein degradation, affecting the normal function of receptors, and aggregation of mutant proteins. The resulting clinical phenotypes vary from simple febrile seizures to more complex genetic epilepsy with febrile seizures plus, and even severe Dravet syndrome. It has been found from studies that there is a group of missense GABRG2 mutations highly related to epileptic encephalopathy in clinical child patients, where the F343L mutation is located in the transmembrane domain of GABRG2, which reduces the GABA current by about 30%, and the child patients with this mutation are basically insensitive to existing antiepileptic drugs. As a vertebrate, the zebrafish has a complex nervous system and a genetic structure similar to human. About 70% of zebrafish genes are shared with human, and about 84% of existing genes related to human diseases are also expressed in the zebrafish. Compared with rodents such as mice, the zebrafish has simple genetic manipulation, and is more suitable for preparing an epilepsy model and for high-throughput drug screening and new molecular target identification of antiepileptic drugs. GABAAR in the zebrafish usually exists in the form of alp2y2, alp26, a2bp3y2, a2bp36, a4p22, a4p26, a6bp22, and a6bp26. Although there is a knockout model of GABAAR subunit al (GABRA1), and the zebrafish juveniles in this model show epileptic phenotypes such as increased activity, brain electrical activity, and increased mortality, there is still no report of a GABRG2 mutant epileptic zebrafish line.

SUMMARY

An objective of the present invention is to provide a construction method of an epilepsy zebrafish model induced by mutant GABRG2 transgene. The method is to transfer mutant GABRG2 genes into zebrafish to convert the zebrafish into a zebrafish line with epileptic phenotypes using the zebrafish as a model animal. The zebrafish line can be used to study the relationship between GABRG2 mutations and epilepsy pathogenesis, and be used for antiepileptic drug screening. To achieve the foregoing objective, the present invention provides the following technical solution: A construction method of a mutant GABRG2 transgenic zebrafish epilepsy model includes transferring mutant human GABRG2 genes into zebrafish to convert the zebrafish into a zebrafish line with epileptic phenotypes. The mutant zebrafish is a zebrafish into which missense mutant GABRG2 genes are transferred.

The epilepsy zebrafish model is caused by GABRG2 missense mutation. The GABRG2 missense mutation is a mutation of base T to C in the codon of Phe at amino acid position 343 in human GABRG2 gene (F343L). The sequence of the transferred mutant human GABRG2 genes is as set forth in SEQ ID No: 1. Preferably, the construction method of the present invention uses the expression vector pDestTol2CG2 (as set forth in SEQ ID No: 2). The expression vector pDestTol2CG2 is to excise coding genes of transposase in the Tol2 transposon and replace the genes with the expression sequence of CMLC2-EGFP-polyA. The plasmid and transposase mRNA are microinjected into zebrafish embryos at the single-cell phase, and the specific expression of green fluorescent protein in zebrafish heart tissues can be observed. (Kwan KM, Fujimoto E, Grabher C, et al. The Tol2 kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs. Dev Dyn, 2007, 236(11): 3088 3099). Preferably, in the construction method of the present invention, the expression vector is constructed by using the gateway technology, which includes the following steps: (1) the construction of the expression vector of the GABRG2 mutant: synthesizing the mutant human GABRG2 genes, and cloning it into the pME-MCS plasmid vector, to construct 27 middle entry clone: pME-GABRG21 1>c (the sequence is as set forth in SEQ ID No: 3); cloning the sequence of the HuC-containing neuron-specific promoter region to construct vectors 5' entry clone: p5E-HuC (the sequence is as set forth in SEQ ID No: 4) and 3' entry clone: p3E-polyA (the sequence is as set forth in SEQ ID No: 5); and recombining the vectors 5' entry clone, middle entry clone, and 3' entry clone, and cloning the recombined vectors into the expression vector pDestTol2CG2 by Gateway LR reaction, to obtain the expression vector: pDestTol2CG2;HuC:GABRG21o 27 T>CpolyA (SEQ ID No: 6); and (2) microinjection of zebrafish embryos: microinjecting the foregoing expression vector of the GABRG2 mutant and Tol2 transposase/transposase mRNA into wild-type zebrafish zygotes at the 1-2 cell phase, and after the embryos are developed for 24 hpf, screening the embryos with green fluorescence expressed in the heart, to obtain positive zebrafish containing the sequence of the GABRG2 mutant. The foregoing construction method further includes the steps of identifying genotypes of the transgenic zebrafish and obtaining a stable genetic strain: identifying the genotypes of the positive zebrafish embryos containing the sequence of the GABRG2 mutant through PCR after matured, selecting the positive zebrafish containing the sequence of the GABRG2 mutant to be crossbred with a wild-type zebrafish to obtain an F1 generation zebrafish, and finally obtaining the GABRG2 mutant zebrafish line through the green fluorescence expressed in the heart and genomic DNA detection. In the present invention, the epileptic phenotypes of the GABRG2 mutant zebrafish line are observed and verified by related experiments: By using a high-throughput behavior screening box (DanioVision Chamber) of zebrafish juveniles and an animal motion trajectory tracking system (EthoVision XT), the trajectories of wild-type zebrafish and the mutant zebrafish line are recorded, swimming distances and average swimming speeds are compared, and video tracking analysis is conducted on zebrafish excitement. Then, epileptic seizures are induced by using PTZ and light stimulation respectively to analyze the susceptibility of the GABRG2 mutant zebrafish line to the related stimulation that induces epileptic seizures. Further, the expression of the c-fos gene in the brain of the GABRG2 mutant zebrafish line is detected through real-time quantitative PCR to prove from the perspective of molecular biology that the mutant zebrafish line has the characteristics of the epilepsy model. Another objective of the present invention is to provide use of the zebrafish epilepsy model that can be used in basic research and clinical application research. The zebrafish epilepsy model based on the GABRG2 mutant can be used to study the epilepsy pathogenesis in biochemistry, molecular biology, and genetics, and can also be used for screening and research on clinical antiepileptic drugs. That is, clinically common antiepileptic drugs such as valproic acid, carbamazepine, clonazepam, and levetiracetam are directly added into a zebrafish culture medium, then effective drugs for the treatment of epilepsy are screened through zebrafish swimming behavior analysis, and the mechanism of the drugs is studied. The present invention provides an effective method basis for the establishment of a zebrafish epilepsy model, and provides an important tool for epilepsy pathological research and antiepileptic drug screening research, which has great basic and clinical research application value.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of expression plasmids required to construct a transgenic zebrafish model by using the gateway technology. Using an F343L mutant as an example, 5' entry clone (p5E-HuC), 3' entry clone (p3E-polyA), and middle entry clone (pME GABRG21o 27T>C) are respectively constructed, and the plasmid pDestTol2CG2;HuC:GABRG21 27 >polyA that expresses mutant GABRG2 is obtained through the LR reaction. FIG. 2 is an image of agarose gel electrophoresis of the constructed GABRG2 (F343L) mutant plasmid. M is DNA molecular weight Marker. FIG. 3 shows an obtained Fl-generation positive mutant zebrafish. WT is a wild-type zebrafish, and F343L is a mutant zebrafish, where green fluorescence expressed in the heart can be seen. FIG. 4 shows zebrafish genomic DNA sequencing to identify genotypes. FIG. 5 shows swimming behavior analysis of GABRG2 (F343L) mutant zebrafish induced by PTZ. Panel A is a visualization of swimming trajectories of wild-type and mutant zebrafish in a normal culture medium or a culture medium containing 15mM PTZ (1 min). Panel B is analysis of movement distances of wild-type and mutant zebrafish in a normal culture medium or a culture medium containing 15mM PTZ (30 min). Panel C is a statistical analysis graph (*P<0.05, **P<0.01). FIG. 6 shows swimming behavior analysis of GABRG2 (F343L) mutant zebrafish induced by light stimulation. Panel A is a visualization of swimming trajectories of wild-type and mutant zebrafish. Panel B is analysis of swimming behaviors of wild-type and mutant zebrafish in different time periods with lights on or lights off. Panel C is a statistical analysis graph

(**P<0.01). FIG. 7 shows c-fos gene expression of wild-type and GABRG2 (F343L) mutant zebrafish at different times (24 h, 48 h, 72 h, 96 h, and 120 h) after birth detected by real-time fluorescent quantitative PCR (**P<0.01, compared with WT). FIG. 8 shows swimming behavior analysis of GABRG2 (F343L) mutant zebrafish in different antiepileptic drugs. 50 M, 100 M, 50 M, and 30 mM of valproic acid, carbamazepine, clonazepam, and levetiracetam are respectively added into a culture medium, a video analysis system is used to record for 30 min after 24 h, and parameters are collected every minute to analyze movement distances of the zebrafish (*P<0.05, **P<0.01). FIG. 9 shows changes in circadian rhythm of swimming of GABRG2 (R177G) mutant zebrafish. Under the condition of normal light time, the swimming behavior of 5dpf zebrafish juveniles is recorded by the motion trajectory tracking system. The recording starts at 4 pm for a total of 24 h (14 h of light, and 10 h of darkness). Behavioral parameters are recorded every 1 h, and differences in the total movement distances of wild-type and transgenic zebrafish in each time period are compared, to analyze the changes in circadian rhythm.

DETAILED DESCRIPTION

The present invention is further described below in detail with reference to examples, but implementations of the present invention are not limited thereto. The terms "wild-type" and "WT" used in the present invention all refer to wild-type zebrafish. Unless otherwise stated, other related terms generally have the meanings commonly understood by those of ordinary skill in the art. In the following examples, various processes and methods that are not described in detail are conventional methods known in the art. Example 1 Construction of GABRG2 (F343L)transgenic zebrafish epilepsy model strain In Example 1, a solution to transfer mutant GABRG2 (F343L) into zebrafish zygotes by transgenic means and construct an epilepsy zebrafish model is provided. The solution specifically includes the following steps: Step 1. The construction of the transgenic expression vector: The Pubmed database was utilized to search for the human GABRG2 gene sequence, the GABRG2 open reading frame sequence was synthesized, the base T at position 1027 was mutated to C (SEQ ID No: 1), and SEQ ID No: 1 was cloned into the pME-MCS plasmid vector with the 5'cloning site of EcoR and the 3' cloning site of XbaI, to construct the intermediate vector middle entry clone: pME 27 GABRG21 T> (SEQ ID No: 3). The sequence of the HuC-containing neuron-specific promoter region was cloned to construct the vector of 5' entry clone: p5E-HuC (SEQ ID No: 4), and the vector of 3' entry clone: p3E-polyA (SEQ ID No: 5) was cloned. The vectors of 5' entry clone, middle entry clone, and 3' entry clone were recombined and cloned into the expression vector pDestTol2CG2 by LR reaction, to construct the transgenic mutant expression 27 vector pDestTol2CG2;HuC:GABRG21 T>CpolyA (SEQ ID No: 6) (as shown in FIG. 1) with 27 the reaction system as follows: TE buffer 4 L, p5E-HuC (30 ng) 1 L, pME-GABRG21 >c (30 ng) 1 L, p3E-polyA (30 ng) 1 L, pDestTol2CG2 (60 ng) 1 L, LR clonase II2 L, and a total volume of 10 pL. The reaction was carried out at 25°C for 20 h. The constructed plasmid was identified by agarose gel electrophoresis, and the result was shown in FIG. 2. A positive band was detected near 20 kb, which is the required expression vector of GABRG2 gene mutation. Preparation of Tol2 transposase mRNA (pCS2FA-transposase, the DNA sequence is as set forth in SEQ ID No: 7): the enzyme cutting system was as follows: l0xBuffer 5 L, DNA 1

[tg, NotI-HF restriction endonuclease 1 L, and a total volume of 50 pL with the addition of ddH20. The system was kept at 37C for 15 min, and heat inactivated at 65°C for 15 min. The enzyme-cut product was subjected to agarose gel electrophoresis, and the Tol2 transposase mRNA was transcribed using an RNA in vitro transcription kit after the gel was recovered.

Step 2. Microinjection of zebrafish embryos: The foregoing transgenic mutant expression 27 vector pDestTol2CG2;HuC:GABRG2o 1>c-polyA and Tol2 transposase mRNA were microinjected into wild-type zebrafish zygotes at the 1-2 cell phase simultaneously, after the embryos were developed for 24 hpf, the embryos with green fluorescence expressed in the heart were screened for subsequent experiments, and the genotypes of the zebrafish were identified by PCR and genome sequencing after the zebrafish matured. As shown in FIG. 3, the embryos of mutant zebrafish with green fluorescence expressed in the heart were obtained. Step 3. Identifying genotypes of the transgenic zebrafish and obtaining a stable genetic strain: Zebrafish genomic DNA extraction: the zebrafish genomic DNA was extracted in one step using a genomic DNA extraction kit (All-DNA-Out), 20 L of the All-DNA-Out solution was added, and heating was performed at 85°C for 5 min. Identifying genotypes: The genotypes of the zebrafish were identified through the PCR reaction, and the reaction system was as follows: 2xKOD FX Buffer 10 L, dNTP 4 L, a forward primer (Primer F) 0.6 [L, a reverse primer (Primer R) 0.6 L, KOD FX enzyme 0.4

[tL, a DNA template 2 L, and a total volume of 20 L. The reaction condition was as follows: 95°C for 5 min; 98°C for 10s, 50°C for 30s, 68°C for 30s (35 cycles); 68°C for 5 min. The sequences of the primers were as follows: F: 5'-GCACAGGAAGCTCAGTCTAC-3'(SEQ ID No: 8), R: 5'-GGAGTCCATTTTGGCAATGC-3'(SEQ ID No: 9). Obtaining a stable genetic strain of the transgenic zebrafish: The zebrafish with the positive result of the foregoing gene identification were selected to be crossbred with a wild-type zebrafish to obtain an F1 generation zebrafish, and the mutant GABRG2 transgenic zebrafish line was obtained through the green fluorescence expressed in the heart and genomic DNA detection. After genomic DNA sequencing, the result was shown in FIG. 4. It can be seen that the base at position 1027 is mutated from T to C, which confirms that the zebrafish contains GABRG2 (F343L) mutation. Step 4. Observation of epileptic phenotypes of the GABRG2(F343L) mutant zebrafish line and related experimental verification: Observation of epilepsy behavior phenotypes: 5dpf zebrafish juveniles were selected, by using a high-throughput behavior screening box (DanioVision Chamber) of zebrafish juveniles and an animal motion trajectory tracking system (EthoVision XT 13), the trajectories of wild-type zebrafish and the mutant zebrafish line under normal conditions were recorded for 30 min. The excitement of zebrafish juveniles was analyzed through swimming distances and average swimming speeds, and the results were shown in FIG. 5. It is found that the excitement of the mutant zebrafish increases under normal culture conditions, and the swimming distance increases significantly.

Change of the swimming behavior of the zebrafish in PTZ: An epileptic seizure inducer PTZ with a final concentration of 15 mM was added into the zebrafish culture medium, and the swimming behavior of the zebrafish was recorded by using the zebrafish behavior video analysis system EthoVision XT 13 with one parameter every 1 min for a total of 30 min. The behavioral differences between wild-type and transgenic zebrafish were compared. The result was shown in FIG. 5. PTZ can cause a significant increase in excitement of wild-type zebrafish and transgenic zebrafish, but the swimming distance of the transgenic zebrafish is still significantly greater than that of the wild-type zebrafish. Change of the swimming behavior of the zebrafish in light stimulation: Under alternating light and dark conditions (5s), the movement behavior of the zebrafish in 100 cycles was recorded by using the zebrafish behavior video analysis system with one parameter every 5s, and the difference in average travel distance between the wild-type zebrafish and the transgenic zebrafish every 5s was recorded. The result was shown in FIG. 6. The movement distance of the transgenic zebrafish under normal light is significantly greater than that of the wild-type zebrafish, but light and dark stimulation have no significant effect on the wild-type and transgenic zebrafish. Phenotype verification of epilepsy in molecular biology: c-fos gene expression detected by real-time fluorescent quantitative PCR: The total RNA of wild-type and transgenic zebrafish at 24 h, 48 h, 72 h, 96 h, and 120 h after birth was extracted and reverse transcribed into cDNA, and real-time fluorescence quantitative PCR analysis was performed using the SYBR green dye method with the reaction condition as follows: 95°C for 6 min; 95°C for 1Os, 60°C for 30s, 72°C for 1Os (41 cycles); gradient heating from 65°C to 95°C for 5s every 0.5°C. The sequences of the primers were as follows: F: 5'-AACTGTCACGGCGATCTCTT-3'(SEQ ID No: 10), R: 5-CTTGCAGATGGGTTTGTGTG-3'(SEQ ID No: 11). The result was shown in FIG. 7. At 72 h after birth, the expression of c-fos in the transgenic zebrafish reaches the highest peak, which is significantly higher than that of the wild-type zebrafish, and then gradually decreases. Example 2 Use of the GABRG2 (F343L)transgenic zebrafish epilepsy model in antiepileptic drug screening The clinically common antiepileptic drugs such as valproic acid, carbamazepine, clonazepam, and levetiracetam were selected and added into the zebrafish culture medium with concentrations of 50 M, 100 M, 50 M, and 30 mM respectively, and recording was performed for 30 min with one parameter every minute by using the video analysis system 24 h after the drugs were added. The drugs with therapeutic effects on the epileptic phenotypes of the transgenic zebrafish were screened through swimming behavior analysis and other methods.

The result was shown in FIG. 8. The foregoing four drugs can significantly reduce the swimming distance of the transgenic zebrafish and reduce the excitement thereof, which proves that the foregoing drugs have inhibitory effects on the epileptic seizures of the GABRG2 (F343L) transgenic zebrafish. Example 3 Construction of GABRG2 (R177G)transgenic zebrafish strain According to the method in Example 1, different mutant GABRG2 gene expression sequences were designed, and the mutation site was a mutation of base C to G at position 529 in the human GABRG2 gene (SEQ ID No: 12). The GABRG2 mutant expression vector 529 pDestTol2CG2;HuC:GABRG2 C>G-polyA was constructed, and the transgenic zebrafish gene strain was obtained through the microinjection of zebrafish embryos. Phenotype analysis of GABRG2 (R177G) transgenic zebrafish strain: 5dpf zebrafish juveniles were selected, by using a high-throughput behavior screening box (DanioVision Chamber) of zebrafish juveniles and an animal motion trajectory tracking system (EthoVision XT 13), the trajectories of wild-type zebrafish and the mutant zebrafish line under normal conditions were recorded. The recording started at 4 pm for a total of 24 h (14 h of light, and 10 h of darkness). Behavioral parameters were recorded every 1 h, and differences in the total movement distances of wild-type and transgenic zebrafish in each time period were compared, to analyze the changes in circadian rhythm. The excitement of zebrafish juveniles was analyzed through swimming distances, and the results were shown in FIG. 9. It was found that, under normal culture conditions, the GABRG2 (R177G) transgenic zebrafish has no significant increase in swimming distance compared with the wild-type zebrafish. The results show that: the GABRG2 (R177G) transgenic zebrafish has no epileptic phenotype. The foregoing examples are preferred implementations of the present invention. However, the implementation of the present invention is not limited by the above examples, and any alternation, modification, substitution, combination, and simplification without departing from the spiritual essence and principles of the present invention should all be equivalent replacements, and all fall within the scope of protection of the present invention.

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<210> 1 <211> 1425 <212> DNA <213> ÈË¹¤ÐòÁÐ(Artificial Sequence)

<400> 1 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 atgatggtcg 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 caaggtctcc tatgtcacag cgatggatct ctttgtatct 1020 gtttgtctca tctttgtctt ctctgctctg gtggagtatg gcaccttgca ttattttgtc 1080 agcaaccgga aaccaagcaa ggacaaagat aaaaagaaga aaaaccctct tcttcggatg 1140 ttttccttca aggcccctac cattgatatc cgcccaagat cagcaaccat tcaaatgaat 1200 aatgctacac accttcaaga gagagatgaa gagtacggct atgagtgtct ggacggcaag 1260 gactgtgcca gttttttctg ctgttttgaa gattgtcgaa caggagcttg gagacatggg 1320 aggatacata tccgcattgc caaaatggac tcctatgctc ggatcttctt ccccactgcc 1380 ttctgcctgt ttaatctggt ctattgggtc tcctacctct acctg 1425

<210> 2 <211> 6693 <212> DNA <213> ÈË¹¤ÐòÁÐ(Artificial Sequence)

<400> 2 aaatagcagg aaacgtgagc agagactccc tggtgtctga aacacaggcc agatgggccc 60 tcgagcagga aacagctatg accatgatta cgccaagcta tcaactttgt atagaaaagt 120 tgaacgagaa acgtaaaatg atataaatat caatatatta aattagattt tgcataaaaa 180 acagactaca taatactgta aaacacaaca tatccagtca ctatggtcga cctgcagact 240 ggctgtgtat aagggagcct gacatttata ttccccagaa catcaggtta atggcgtttt 300 Page 1

Sequence Listing as published tgatgtcatt ttcgcggtgg ctgagatcag ccacttcttc cccgataacg gagaccggca 360 cactggccat atcggtggtc atcatgcgcc agctttcatc cccgatatgc accaccgggt 420 aaagttcacg ggggacttta tctgacagca gacgtgcact ggccaggggg atcaccatcc 480 gtcgcccggg cgtgtcaata atatcactct gtacatccac aaacagacga taacggctct 540 ctcttttata ggtgtaaacc ttaaactgca tttcaccagc ccctgttctc gtcggcaaaa 600 gagccgttca tttcaataaa ccgggcgacc tcagccatcc cttcctgatt ttccgctttc 660 cagcgttcgg cacgcagacg acgggcttca ttctgcatgg ttgtgcttac cgaaccggag 720 atattgacat catatatgcc ttgagcaact gatagctgtc gctgtcaact gtcactgtaa 780 tacgctgctt catagcatac ctctttttga catacttcgg gtatacatat cagtatatat 840 tcttataccg caaaaatcag cgcgcaaata cgcatactgt tatctggctt ttagtaagcc 900 ggatcctcta gattacgccc cgccctgcca ctcatcgcag tactgttgta attcattaag 960 cattctgccg acatggaagc catcacaaac ggcatgatga acctgaatcg ccagcggcat 1020 cagcaccttg tcgccttgcg tataatattt gcccatggtg aaaacggggg cgaagaagtt 1080 gtccatattg gccacgttta aatcaaaact ggtgaaactc acccagggat tggctgagac 1140 gaaaaacata ttctcaataa accctttagg gaaataggcc aggttttcac cgtaacacgc 1200 cacatcttgc gaatatatgt gtagaaactg ccggaaatcg tcgtggtatt cactccagag 1260 cgatgaaaac gtttcagttt gctcatggaa aacggtgtaa caagggtgaa cactatccca 1320 tatcaccagc tcaccgtctt tcattgccat acggaattcc ggatgagcat tcatcaggcg 1380 ggcaagaatg tgaataaagg ccggataaaa cttgtgctta tttttcttta cggtctttaa 1440 aaaggccgta atatccagct gaacggtctg gttataggta cattgagcaa ctgactgaaa 1500 tgcctcaaaa tgttctttac gatgccattg ggatatatca acggtggtat atccagtgat 1560 ttttttctcc attttagctt ccttagctcc tgaaaatctc gacggatcct aactcaaaat 1620 ccacacatta tacgagccgg aagcataaag tgtaaagcct gggggtgcct aatgcggccg 1680 ccatagtgac tggatatgtt gtgttttaca gtattatgta gtctgttttt tatgcaaaat 1740 ctaatttaat atattgatat ttatatcatt ttacgtttct cgttcaactt tattatacat 1800 agttgataat tcactggccg tcgttttacg gtaccatcga tgatgatcca gacatgataa 1860 gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt 1920 gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta 1980 acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt 2040 aaagcaagta aaacctctac aaatgtggta tggctgatta tgatcctcta gatcagatct 2100 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa 2160 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta 2220 tcatgtctgg atctacgtaa tacgactcac tatagttcta gaggctcgag aggggccgct 2280 ttacttgtac agctcgtcca tgccgagagt gatcccggcg gcggtcacga actccagcag 2340 gaccatgtga tcgcgcttct cgttggggtc tttgctcagg gcggactggg tgctcaggta 2400 gtggttgtcg ggcagcagca cggggccgtc gccgatgggg gtgttctgct ggtagtggtc 2460 ggcgagctgc acgctgccgt cctcgatgtt gtggcggatc ttgaagttca ccttgatgcc 2520 gttcttctgc ttgtcggcca tgatatagac gttgtggctg ttgtagttgt actccagctt 2580 gtgccccagg atgttgccgt cctccttgaa gtcgatgccc ttcagctcga tgcggttcac 2640 cagggtgtcg ccctcgaact tcacctcggc gcgggtcttg tagttgccgt cgtccttgaa 2700 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 Page 2

Sequence Listing as published 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 Page 3

Sequence Listing as published 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> ÈË¹¤ÐòÁÐ(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 Page 4

Sequence Listing as published 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> ÈË¹¤ÐòÁÐ(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 Page 5

Sequence Listing as published 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 Page 6

Sequence Listing as published 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 Page 7

Sequence Listing as published 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 Page 8

Sequence Listing as published 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> ÈË¹¤ÐòÁÐ(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 Page 9

Sequence Listing as published 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> ÈË¹¤ÐòÁÐ(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 Page 10

Sequence Listing as published 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 Page 11

Sequence Listing as published 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 Page 12

Sequence Listing as published 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 Page 13

Sequence Listing as published 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 Page 14

Sequence Listing as published 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 Page 15

Sequence Listing as published tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag 16920 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtg 16965

<210> 7 <211> 6034 <212> DNA <213> ÈË¹¤ÐòÁÐ(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 Page 16

Sequence Listing as published 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 Page 17

Sequence Listing as published 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> ÈË¹¤ÐòÁÐ(Artificial Sequence)

<400> 8 gcacaggaag ctcagtctac 20

<210> 9 <211> 20 <212> DNA <213> ÈË¹¤ÐòÁÐ(Artificial Sequence)

<400> 9 ggagtccatt ttggcaatgc 20

<210> 10 <211> 20 <212> DNA <213> ÈË¹¤ÐòÁÐ(Artificial Sequence)

<400> 10 aactgtcacg gcgatctctt 20

<210> 11 <211> 20 <212> DNA <213> ÈË¹¤ÐòÁÐ(Artificial Sequence)

<400> 11 cttgcagatg ggtttgtgtg 20

<210> 12 <211> 1401 <212> DNA <213> ÈË¹¤ÐòÁÐ(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 Page 18

Sequence Listing as published 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

Page 19

Claims

CLAIMS What is claimed is:

1. A construction method of a mutant GABRG2 transgenic zebrafish epilepsy model, comprising transferring mutant human GABRG2 genes into zebrafish to convert the zebrafish into a zebrafish line with epileptic phenotypes.

2. The construction method according to claim 1, wherein the mutant human GABRG2 gene is obtained from a mutation of base T to C in the codon of Phe at amino acid position 343 in human GABRG2.

3. The construction method according to claim 1, wherein the sequence of the transferred mutant human GABRG2 genes is as set forth in SEQ ID No: 1.

4. The construction method according to claim 3, wherein the expression vector is pDestTol2CG2.

5. The construction method according to claim 4, wherein the expression vector is constructed by using the gateway technology.

6. The construction method according to claim 5, comprising the following steps: (1) the construction of the expression vector of the GABRG2 mutant: synthesizing the mutant human GABRG2 genes, and cloning it into the pME-MCS plasmid vector, to construct middle entry clone: pME-GABRG21o 2 1>C; 7 cloning the sequence of the HuC-containing neuron-specific promoter region to construct vectors 5'entry clone: p5E-HuC and 3'entry clone: p3E-polyA; and recombining the vectors 5'entry clone, middle entry clone, and 3'entry clone, and cloning the recombined vectors into the expression vector pDestTol2CG2 by Gateway LR 72 reaction, to obtain the expression vector: pDestTol2CG2;HuC:GABRG21 >c-polyA; and (2) microinjection of zebrafish embryos: microinjecting the foregoing expression vector of the GABRG2 mutant and Tol2 transposase/transposase mRNA into wild-type zebrafish zygotes at the 1-2 cell phase, and after the embryos are developed for 24 hpf, screening the embryos with green fluorescence expressed in the heart, to obtain positive zebrafish containing the sequence of the GABRG2 mutant.

7. The construction method according to claim 6, wherein the sequence of the 27 pDestTol2CG2;HuC:GABRG21 T>CpolyA is as set forth in SEQ ID No: 6.

8. The construction method according to any one of claims I to 7, further comprising the steps of identifying genotypes of the transgenic zebrafish and obtaining a stable genetic strain.

9. The construction method according to claim 8, comprising identifying the genotypes of the positive zebrafish embryos containing the sequence of the GABRG2 mutant through PCR after matured, selecting the positive zebrafish containing the sequence of the GABRG2 mutant to be crossbred with a wild-type zebrafish to obtain an F1 generation zebrafish, and finally obtaining the GABRG2 mutant zebrafish line through the green fluorescence expressed in the heart and genomic DNA detection.

10. Use of the mutant GABRG2 transgenic zebrafish epilepsy model constructed by using the method according to any one of claims 1 to 9 in epilepsy pathology research and antiepileptic drug screening.

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FIG. 9