CN110042124A - Genome base editor increases the kit of fetal hemoglobin level and application in human red blood cells - Google Patents

Abstract

The present invention provides a kind of genome base editors to increase the kit of fetal hemoglobin level and application in human red blood cells.The kit includes base editing system and the sgRNA for gamma globulin gene promoter site.The present invention utilizes base editing technique, pass through C to T and/or A to G base mutation, the Binding site for transcription factor for changing gamma globulin gene promoter region provides the method and kit of highly effective and safe to improve the expression of gamma globulin to treat such hemoglobinopathy.

Description

Genome base editor increases the kit of fetal hemoglobin level in human red blood cells And application

Technical field

Fetal hemoglobin (HbF) level in human red blood cells is increased by genome base editor the present invention relates to a kind of Kit and method.

Background technique

Hemoglobinopathy includes that genetic hemoglobin construct or expression change caused a variety of anaemias, including blood red egg The change (such as sickle-cell anemia) of white chain molecular structure and the synthesis of wherein one or more chains reduce or lack (such as Thalassemia).Disease relevant to beta-globin is commonly known as β-hemoglobinopathy.For example, β-thalassemia be by Hemoglobin A (HbA) is caused to lack or be not present in the partially or completely defect of beta-globin gene expression；Drepanocytosis It (SCD) is caused by the mutation in beta-globin structural gene (HBB).It is still at present supportive to the treatment of Most patients , it is intended to alleviate symptom and complication.Unique cure method is allogeneic stem cells transplanting, but deficient due to donor Weary and high risk severe complication, currently, significantly changing clear therapy (such as periodic transfusions or exchange of the natural history of disease Blood transfusion and HSC transplanting) it is limited.

Therefore, researchers always strive to treat by way of gene repair.Especially new gene editing skill The application of art CRISPR/Cas9 technology, so that attempting both at home and abroad poor using the CRISPRCas9 gene editing technology treatment Mediterranean β Blood is more and more concerned.The Yuet Wai Kan professor of University of California-San Francisco in 2014 and its colleague utilize CRISPR/ Cas9 and iPS technology realize repaired in Human cell line cause β-thalassemia mutated gene (Xie et al., 2014), 2016 they apply the technology to attempt to have knocked out one section of pearl containing δ, β in source of people hematopoietic stem/progenitor cells (HSPC) again The segment of the 13kb of protein gene makes the expression of γ globin raise (Ye et al., 2016).2016, Guangzhou medical university Attached Sun Xiaofang team, third hospital is successfully corrected in the poor iPSCs cell in β-ground by CRISPR/Cas9 gene editing technology Beta globin genes mutation (Niu et al., 2016).

Although the above method achieves significant breakthrough, if these strategies are for clinic, there are certain offices It is sex-limited: 1. β its beta globin genes mutational site of poor patient it is different, it is presently found there are about hundreds of mutant form, The gene editing site that treatment must be different to each patients design is carried out in aforementioned manners, causes treatment cost high, program It is complicated；2. iPSCs has certain canceration possible, risk is still had for clinic；3. by iPSCs induction at HSPC technology also It is immature；4. DNA double chain fracture can be generated with CRISPR/Cas9 gene editing technology, induce cell apoptosis, and generates safety Property hidden danger, such as the missing (Adikusuma et al., 2018) of large fragment etc..Therefore, find it is a kind of it is significantly more efficient, have Universality, the lower Innovative therapeutic method of canceration risk are the critical issues for treating hemoglobinopathy.

In order to improve the efficiency of base mutation, and avoid generating safety issue caused by DNA double chain is broken, Yi Zhongtong It crosses cytosine deaminase fusion on dCas9 albumen, base fixed point is efficiently obtained in the case where not causing double-strand break The tool of mutation is developed.This new tool be referred to as base edit tool (Chadwick et al., 2017；Hess et al.,2016；Kim et al.,2017；Komor et al.,2016；Nishida et al.,2016).Although base is compiled The technology of collecting just occurs in recent years, but has been widely used for many fields.For example it is successfully repaired using BE3 system with base edit mode Answered with a base mutation on breast cancer related gene TP53, and with one on Alzheimer's disease related gene APOE4 A base mutation (Komor et al., 2016).It is avoided that using base edit methods and generates excessive DNA double in height copy site Chain fracture (DSB) so cause cell death (Billon et al., 2017；Kuscu et al., 2017), also can increase volume The efficiency and accuracy collected.Therefore, it is this based on CRISPR-Cas9 system generate base edit tool in clinical application more Safety is more effective, with greater advantage.Keith Joung use for laboratory base edit tool A3A-BE3 is more accurately corrected People β-thalassemia promoter mutation (Gehrke et al., 2018).

In general, humanβglobin gene includes 4 β class globin function bases successively expressed that put in order by it Cause, respectively ε, γ, δ, β.Wherein ε globin is mainly expressed in embryonic period, embryonic phase, and γ globin is mainly expressed in foetal period, β pearl egg It is white to be mainly expressed in adulthood.When foetal period, γ globin combines to be formed fetal hemoglobin (HbF) with alpha globin, adult Beta globin combines to form adult gemoglobin (HbA) with alpha globin when the phase.The mankind undergo after birth one from fetus to Adult globin switch conversion mechanism is completed one and is converted by the expression of γ → beta globin, therefore γ-in normal adult Opposite silence state is presented in globin gene.Studies have shown that making it if reactivating the expression of γ globin gene in adult It combines to form HbF with superfluous alpha globin, can effectively mitigate caused by synthesizing reduction or dysfunction because of beta globin β-thalassemia clinical symptoms (Bank, 2006).Illustrate this method not only and can eliminate that superfluous alpha globin accumulation causes Adverse effect, while HbF can play the role of substitute HbA driving functions.It 2018, delivers on Nature genetics Research report two and inhibit the factor B CL11A and ZBTB7A of γ-globins expression in the combination of γ-globin promoter Region is respectively -115 regions and -200 regions (Martyn et al., 2018).Since the range of BE3 effect only has 5nt, 2018, Jiang etc. reported a kind of expansion sphere of action to the base edit tool BE-PLUS of 14nt, can more effectively lead to The mode for crossing base editor changes the sequence of functional domain, to have the function that change and adjust gene function (Jiang et al.,2018)。

The present invention combines corresponding positions using base edit tool (BE3, xBE3, ABE, BE-PLUS or BE-PLUS (AID)) The sgRNA set destroys candidate stem cell (HSC) BCL11A, ZBTB7A and other inhibiting factors in γ-globin promoter (- 114bp, -117bp, -158bp, -175bp, -196bp, -198/199bp (are O with transcription initiation site TSS for bond area Point, 5 ' ends are negative, and 3 ' ends are positive)), it is T base by C base mutation in the site in the case where not generating DNA double chain fracture Or A base mutation is that G base is provided with improving γ-globin expression in human red blood cells for clinical treatment hemoglobinopathy Reliable reagent and method.

Bibliography

Adikusuma,F.,Piltz,S.,Corbett,M.A.,Turvey,M.,McColl,S.R.,Helbig,K.J., Beard,M.R.,Hughes,J.,Pomerantz,R.T.,and Thomas,P.Q.(2018).Large deletions induced by Cas9cleavage.Nature 560,E8-E9.

Billon,P.,Bryant,E.E.,Joseph,S.A.,Nambiar,T.S.,Hayward,S.B., Rothstein,R.,and Ciccia,A.(2017).CRISPR-Mediated Base Editing Enables Efficient Disruption of Eukaryotic Genes through Induction of STOP Codons.Molecular cell 67,1068-1079e1064.

Chadwick,A.C.,Wang,X.,and Musunuru,K.(2017).In Vivo Base Editing of PCSK9(Proprotein Convertase Subtilisin/Kexin Type 9)as a Therapeutic Alternative to Genome Editing.Arteriosclerosis,thrombosis,and vascular biology 37,1741-1747.

Gehrke,J.M.,Cervantes,O.,Clement,M.K.,Wu,Y.,Zeng,J.,Bauer,D.E., Pinello,L.,and Joung,J.K.(2018).An APOBEC3A-Cas9base editor with minimized bystander and off-target activities.Nature biotechnology.

Hess,G.T.,Fresard,L.,Han,K.,Lee,C.H.,Li,A.,Cimprich,K.A.,Montgomery, S.B.,and Bassik,M.C.(2016).Directed evolution using dCas9-targeted somatic hypermutation in mammalian cells.Nature methods 13,1036-1042.

Jiang,W.,Feng,S.,Huang,S.,Yu,W.,Li,G.,Yang,G.,Liu,Y.,Zhang,Y.,Zhang, L.,Hou,Y.,et al.(2018).BE-PLUS:a new base editing tool with broadened editing window and enhanced fidelity.Cell research.

Kim,D.,Lim,K.,Kim,S.T.,Yoon,S.H.,Kim,K.,Ryu,S.M.,and Kim,J.S.(2017) .Genome-wide target specificities of CRISPR RNA-guided programmable deaminases.Nat Biotechnol 35,475-480.

Komor,A.C.,Kim,Y.B.,Packer,M.S.,Zuris,J.A.,and Liu,D.R.(2016) .Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.Nature 533,420-424.

Kuscu,C.,Parlak,M.,Tufan,T.,Yang,J.,Szlachta,K.,Wei,X.,Mammadov,R., and Adli,M.(2017).CRISPR-STOP:gene silencing through base-editing-induced nonsense mutations.Nature methods 14,710-712.

Martyn,G.E.,Wienert,B.,Yang,L.,Shah,M.,Norton,L.J.,Burdach,J.,Kurita, R.,Nakamura,Y.,Pearson,R.C.M.,Funnell,A.P.W.,et al.(2018).Natural regulatory mutations elevate the fetal globin gene via disruption of BCL11A or ZBTB7A binding.Nature genetics 50,498-503.

Nishida,K.,Arazoe,T.,Yachie,N.,Banno,S.,Kakimoto,M.,Tabata,M., Mochizuki,M.,Miyabe,A.,Araki,M.,Hara,K.Y.,et al.(2016).Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems.Science 353.

Niu,X.,He,W.,Song,B.,Ou,Z.,Fan,D.,Chen,Y.,Fan,Y.,and Sun,X.(2016) .Combining Single Strand Oligodeoxynucleotides and CRISPR/Cas9to Correct Gene Mutations in beta-Thalassemia-induced Pluripotent Stem Cells.The Journal of biological chemistry 291,16576-16585.

Xie,F.,Ye,L.,Chang,J.C.,Beyer,A.I.,Wang,J.,Muench,M.O.,and Kan,Y.W. (2014).Seamless gene correction of beta-thalassemia mutations in patient- specific iPSCs using CRISPR/Cas9and piggyBac.Genome research 24,1526-1533.

Ye,L.,Wang,J.,Tan,Y.,Beyer,A.I.,Xie,F.,Muench,M.O.,and Kan,Y.W. (2016).Genome editing using CRISPR-Cas9to create the HPFH genotype in HSPCs: An approach for treating sickle cell disease and beta-thalassemia.Proceedings of the National Academy of Sciences of the United States of America 113, 10661-10665.

Summary of the invention

The object of the present invention is to provide one kind to increase fetal hemoglobin in human red blood cells by genome base editor (HbF；Its two polypeptide chains are from gamma globulin gene expression) horizontal method, it can be used for treating hemoglobinopathy such as β- Thalassemia and drepanocytosis.And provide component, kit and composition, the Yi Jiyou for carrying out such method The cell that they are generated, the self CD34+ artificial blood that can be including but not limited to applied to the patient with hemoglobinopathy are dry thin Born of the same parents (hHSC).

In order to achieve the above object, present invention employs a kind of base editors for efficiently improving hemoglobin in human red blood cells Kit, which is characterized in that including base editing system and for the sgRNA in gamma globulin gene promoter site.

Preferably, the base editing system is BE3, xBE3, ABE, BE-PLUS or BE-PLUS (AID).

Preferably, the base editing system can be plasmid, mRNA or protein form, preferentially select protein form.

Preferably, the sgRNA for gamma globulin gene promoter site, it is corresponding if being located at -114bp SgRNA is SEQ ID NO.1, if being located at -117bp, corresponding sgRNA is SEQ ID NO.2, right if being located at -158bp The sgRNA answered is SEQ ID NO.3, if being located at -175bp, corresponding sgRNA is SEQ ID NO.4, if being located at -196bp, Its corresponding sgRNA is SEQ ID NO.5, if being located at -198/199bp, corresponding sgRNA is SEQ ID NO.6,

Preferably, used sgRNA can be plasmid form, is also possible to rna form, preferentially selects rna form.

The present invention provides one kind to break cyclase protein binding site by multiple base mutations, to change gene expression dose Method, which is characterized in that including designing sgRNA and base editing system according to gamma globulin gene promoter different zones At least one of.

The present invention provides a kind of methods that base editor treats hemoglobinopathy, comprising: is containing beta-globin gene In the HSC of the patient of mutation, using for gamma globulin gene promoter region sgRNA guidance base editing system arrive this Site carries out base editor reparation, the cell after collecting transfection, identifies mutation rate, the expression of gamma globulin is identified after HSC differentiation Variation.

Preferably, it is described according to gamma globulin gene promoter region design sgRNA, by building U6 starting and/ Or the expression vector of T7 starting obtains.

Preferably, the method that the base editor treats hemoglobinopathy further include: Sanger sequencing detection editor's effect Rate；The efficiency of high-flux sequence on-target, indel and off-target.

The reduction of beta-globin or missing be the main reason for causing β-hemoglobinopathy, but its mutation type hundreds of, It is very time-consuming and laborious to repair mutation, and is not readily reachable by, however the expression for improving gamma globulin is a kind of such disease for the treatment of The universal method of disease, base editing system provide a kind of safely and effectively general therapeutic cure.

Inventor will combine suitable sgRNA using based on base edit tool, cause gamma globulin gene promoter area The base mutation in domain, thus destroy inhibit gamma globulin expression regulatory factor (BCL11A, ZBTB7A and other inhibit because Son) binding site, improve the expression of gamma globulin gene, remediation efficiency detected in the way of deep sequencing and is missed the target feelings Condition.The method of highly effective and safe is provided to treat such disease.

Detailed description of the invention

Fig. 1 is the composition schematic diagram of BE3, xBE3, ABE, BE-PLUS or BE-PLUS (AID) 5 kinds of base edit tools.

Fig. 2 is to use 114/117/158/175/196/198/199sgRNA with corresponding base edit tool to gene respectively The sequencing result of PCR fragment after group DNA is edited.Arrow show corresponding position.

Fig. 3 is that the ratio of the CD34+ candidate stem cell obtained after airflow classification is 88.6%.

Fig. 4 is the increment curve in the culture of CD34+ candidate stem cell 7 days after airflow classification.

When Fig. 5 is culture 4 days, the ratio of CD34+ candidate stem cell is 98.6%.

Fig. 6 is that CD34+ candidate stem cell increases to what red system mark CD235 and CD71 in erythroid differentiation 0-21 days was expressed.

Fig. 7 is that red is presented in the cell precipitation after erythroid differentiation, and white is presented in undifferentiated control cell.

Fig. 8 is the variation of HBG expression after qPCR detection carries out base editor with different sgRNA.

Specific embodiment

Detailed description and explanation is carried out to embodiment of the present invention below in conjunction with embodiment, it is clear that described Embodiment is merely to illustrate a part of the embodiments of the present invention, and is not construed as limiting the scope of the invention.It is not infused in embodiment Bright actual conditions person, carries out according to conventional conditions or manufacturer's recommended conditions.Production firm is not specified in agents useful for same or instrument Person is considered as the conventional products that can be obtained by commercially available purchase.

Different types of albumen is purified first, and initial carrier sequence used is BE3 (SEQ ID NO.7), xBE3 (SEQ ID NO.8), ABE (SEQ ID NO.9), BE-PLUS (GCN4-D10A (SEQ ID NO.10) and scFv-APOBEC (SEQ ID )) or BE-PLUS (AID) (GCN4-D10A (SEQ ID NO.10) and scFv-AID (SEQ ID NO.12)) NO.11.

1. the code area of above-mentioned four kinds of plasmids is building up to respectively in carrier pET28a (excellent precious biology, VT1207).

2. shaking bacterium, after about shaking bacterium 4 hours (OD=0.8), IPTG 1mM is added, 16 DEG C are shaken bacterium 48h in 8L LB, Kana.

3. heavy bacterium is centrifuged 5000g, 20min.

4. being resuspended, all heavy bacterium are resuspended with bufferA, bacterium must be broken up completely, blocking when preventing broken below Instrument.

5. broken, bacterium solution is crossed into broken instruments, until solution is limpid, generally at least it is broken twice.Instrument prepares, and needs clear It washes 3-4 times, high pressure part metals pipe needs ice bath, needs to clean 3-4 times after instrument use.

6. collecting the 10 full product of cell lysis of μ L, subsequent western detection.

7. pyrolysis product is placed in 50ml centrifuge tube, 80000g, 40min.

8. collecting supernatant, 7 are repeated, until granule foreign removal is clean.

9.0.45um filter filters supernatant, and 10 μ L is taken to detect for subsequent western, prepares to start the affine layer of solid metallic Analyse (Immobilized Metal Affinity Chromatography (IMAC) (cobalt column).

After 10. cobalt column washes one time with ddH2O, with bufferA rinse several times.

11. protein sample is crossed cobalt column (this time with two pillars), and collect efflux.

12. repeating step 11, and take 10 μ L samples for subsequent western.

13. decontamination crosses pillar added with the bufferA of 5mM imidazoles with 40mL, to remove the lower impurity of affinity. Efflux is collected, and takes 10 μ L samples for subsequent western.

14. elution crosses pillar added with the bufferA of 500mM imidazoles with 30ml, displaces destination protein.Collect purpose Albumen, and take 10 μ L samples for subsequent western.Cobalt column after elution is cleaned with ddH2O, to remove imidazoles, is used again later BufferA balance.

15.western, destination protein about 160KD configure suitable SDS-PAGE glue, 210V electrophoresis according to albumen size.

After electrophoresis, glue is cut off, is placed in Coomassie brilliant blue, micro-wave oven is heated at high temperature 1min.Later, ddH2O is used Cleaning, microwave stove heating 20min.After being rinsed with water, take pictures.

16. protein concentration: the destination protein eluted being added in protein concentration column, 3900rpm, 20min.

17. the albumen after concentration carries out ion-exchange chromatography (Ion exchange chromatography (IEC)), with It removes and protein bound nucleic acid.Under principle, that is, high level salt solution of ion-exchange chromatography, this ionic bond will be destroyed, thus Release destination protein.

18. chromatographing the destination protein collected, digestion is carried out after concentrated, to remove His-tag.

Embodiment 1

In the present embodiment, base edit tool combination sgRNA is utilized on cell strain, this method will utilize base editor work The plasmid form for having (BE3, xBE3, ABE, BE-PLUS or BE-PLUS (AID)) and sgRNA realizes (Fig. 1).

1.1 plasmid construction

In γ-globin (HBG) gene promoter -114bp, -117bp, -158bp, -175bp, -196bp, -198/ 199bp (with transcription initiation site TSS for O point, 5 ' ends are negative, and 3 ' ends are positive) design at position sgRNA (SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3) synthesis oligos.

114sgRNA:

Upstream sequence are as follows: 5 '-ACCGcttgaccaatagccttgaca-3 ' (SEQ ID NO. (13))

Downstream sequence are as follows: 5 '-AAACtgtcaaggctattggtcaag-3 ' (SEQ ID NO. (14))

117sgRNA:

Upstream sequence are as follows: 5 '-ACCGgctattggtcaaggcaaggc-3 ' (SEQ ID NO. (15))

Downstream sequence are as follows: 5 '-AAACgccttgccttgaccaatagc-3 ' (SEQ ID NO. (16))

158sgRNA:

Upstream sequence are as follows: 5 '-ACCGccctggctaaactccaccca-3 ' (SEQ ID NO. (17))

Downstream sequence are as follows: 5 '-AAACtgggtggagtttagccaggg-3 ' (SEQ ID NO. (18))

175sgRNA (ABE):

Upstream sequence are as follows: 5 '-ACCGatatttgcattgagatagtg-3 ' (SEQ ID NO. (19))

Downstream sequence are as follows: 5 '-AAACcactatctcaatgcaaatat-3 ' (SEQ ID NO. (20))

196sgRNA (xBE3):

Upstream sequence are as follows: 5 '-ACCGccttccccacactatctcaa-3 ' (SEQ ID NO. (21))

Downstream sequence are as follows: 5 '-AAACttgagatagtgtggggaagg-3 ' (SEQ ID NO. (22))

198/199sgRNA (ABE):

Upstream sequence are as follows: 5 '-ACCGgtggggaaggggcccccaag-3 ' (SEQ ID NO. (23))

Downstream sequence are as follows: 5 '-AAACcttgggggccccttccccac-3 ' (SEQ ID NO. (24))

Oligo annealing

Fast anneal system:

Fast anneal condition:

95° 5min

95° 30s

85 ° of 30s (- 2 °/s, be down to 25 °)

25° 1min(0.1°/s)

4° ∞

Upstream and downstream sequence is connected to the pGL3-U6sgRNA carrier linearized by BsaI (NEB:R0539L) by annealing On upper (addgene:51133).

Digestion system:

pGL3-U6sgRNA:3μg

Cutsmart(NEB#B7204S):5μL

BsaI(NEB#R0535L) 1μL

ddH₂O to 50μL

Condition: 37 DEG C, overnight.

Linked system:

Condition: 16 ° of connection 30min

The carrier of connection chooses bacterium by conversion, identifies, identification primer upstream sequence: 5 '- Cgattagtgaacggatctcgacg-3 ' (SEQ ID NO.25), downstream sequence are the downstream sequence of corresponding oligo.To the positive It is spare that clone shakes bacterium extraction plasmid (Axygene:AP-MN-P-250G) measurement concentration.The mutant plasmid of acquisition is named as HBG- U6-114/117/158/175/196/198/199sgRNA。

The culture of 1.2 cells and electricity turn

(1) by taking HEK293T cell (being purchased from ATCC) as an example, the present invention carries out the culture and transfection of eukaryotic cells: HEK293T cell inoculation is incubated in the sugared culture solution of DMEM high of addition 10%FBS (HyClone, SH30022.01B), wherein Containing penicillin (100U/ml) and streptomycin (100 μ g/ml).

(2) transfection the first two hour changes the culture medium of antibiotic-free into, utilizes 2000 transfection reagent of lipofectamin (invitrogen 11668-019) is transfected to specifications, and cell is by counting to obtain 1X10⁵It is a.By base editing system BE3, XBE3, ABE, BE-PLUS or BE-PLUS (AID) and sgRNA according to 1 μ g and 0.3 μ g mass mixing (wherein BE-PLUS/BE- The ratio of two plasmids contained by PLUS (AID) is GCN4-D10A:scFv-APOBEC/scFv-AID=0.4:0.6).After transfection 8h is changed containing dual anti-fresh culture, and puromicin to final concentration 2ng/ml, drug screening 2d is added for 24 hours after transfection.

(3) after medicine sieve 2d, lytic cell identifies genotype, and the ingredient of lysate is 50mM KCl, 1.5mM MgCl₂, 10mM Tris pH 8.0,0.5%Nonidet P-40,0.5%Tween 20,100g/ml protease K.

The detection of 1.3 mutation efficiencies

It is that C sports T, and its PAM sequence in view of the mutation purpose site in 114sgRNA, 117sgRNA, 158sgRNA It is NGG, it is preferable to use BE3, BE-PLUS and BE-PLUS (AID)；And the mutation purpose position in 175sgRNA, 198/199sgRNA Point is that A sports G, it is preferable to use ABE；The PAM sequence of 196sgRNA is NG, it is preferable to use xBE3 (Fig. 2).

Embodiment 2

In the present embodiment, in the isolated candidate stem cell of normal person (HSC), the mRNA of base editing system is utilized Base editor, detection HBG expression variation are realized with the rna form of sgRNA.

2.1sgRNA plasmid construction

In γ-globin (HBG) gene promoter -114bp, -117bp, -158bp, -175bp, -196bp, -198/ Design sgRNA synthesis oligos at 199bp (with transcription initiation site TSS for O point, 5 ' ends are negative, and 3 ' ends are positive) position.

114sgRNA:

Upstream sequence are as follows: 5 '-TAGGcttgaccaatagccttgaca-3 ' (SEQ ID NO. (26))

Downstream sequence are as follows: 5 '-AAACtgtcaaggctattggtcaag-3 ' (SEQ ID NO. (27))

117sgRNA:

Upstream sequence are as follows: 5 '-TAGGgctattggtcaaggcaaggc-3 ' (SEQ ID NO. (28))

Downstream sequence are as follows: 5 '-AAACgccttgccttgaccaatagc-3 ' (SEQ ID NO. (29))

158sgRNA:

Upstream sequence are as follows: 5 '-TAGGccctggctaaactccaccca-3 ' (SEQ ID NO. (30))

Downstream sequence are as follows: 5 '-AAACtgggtggagtttagccaggg-3 ' (SEQ ID NO. (31))

175sgRNA (ABE):

Upstream sequence are as follows: 5 '-TAGGatatttgcattgagatagtg-3 ' (SEQ ID NO. (32))

Downstream sequence are as follows: 5 '-AAACcactatctcaatgcaaatat-3 ' (SEQ ID NO. (33))

196sgRNA (xBE3):

Upstream sequence are as follows: 5 '-TAGGccttccccacactatctcaa-3 ' (SEQ ID NO. (34))

Downstream sequence are as follows: 5 '-AAACttgagatagtgtggggaagg-3 ' (SEQ ID NO. (35))

198/199sgRNA (ABE):

Upstream sequence are as follows: 5 '-TAGGgtggggaaggggcccccaag-3 ' (SEQ ID NO. (36))

Downstream sequence are as follows: 5 '-AAACcttgggggccccttccccac-3 ' (SEQ ID NO. (37))

Oligo annealing

Fast anneal system:

Fast anneal condition:

95° 5min

95° 30s

85 ° of 30s (- 2 °/s, be down to 25 °)

25° 1min(0.1°/s)

4° ∞

Upstream and downstream sequence is connected to the pUC57-T7sgRNA by BsaI (NEB:R0539L) linearisation and is carried by annealing On body on (addgene:51132).

Digestion system:

pUC57-T7sgRNA:3μg

Cutsmart(NEB#B7204S):5μL

BsaI(NEB#R0535L) 1μL

ddH₂O to 50μL

Condition: 37 DEG C, overnight.

Linked system:

Condition: 16 ° of connection 30min

The carrier of connection chooses bacterium by conversion, identifies, identification primer upstream sequence: 5 '- Cgattagtgaacggatctcgacg-3 ' (SEQ ID NO.25), downstream sequence are the downstream sequence of corresponding oligo.To the positive It is spare that clone shakes bacterium extraction plasmid (Axygene:AP-MN-P-250G) measurement concentration.The mutant plasmid of acquisition is named as HBG- T7-114/117/158/175/196/198/199sgRNA。

The in-vitro transcription of 2.2sgRNA

Using the HBG-T7-114/117/158/175/196/198/199sgRNA of building as template, amplification contains sgRNA's Segment, the primer are as follows:

FOR:5’-TCTCGCGCGTTTCGGTGATGACGG-3’(SEQ ID NO.38)

REV:5’-AAAAAAAGCACCGACTCGGTGCCACTTTTTC-3’(SEQ ID NO.39)

Amplification system is as follows:

25 μ L of 2Xbuffer (promise is only praised: P505)

dNTP 1μL；F(10pmol/μL) 2μL

R(10pmol/μL) 2μL

Template 1ng

0.5 μ L of archaeal dna polymerase (promise is only praised: P505)；

DdH2O polishing is to 50 μ L.

PCR product purifying:

1. every 100 μ L volume adds 4 μ L RNAsecure (Life:AM7005), and 60 DEG C, 15 minutes；

2. the PCR-A (Axygen:AP-PCR-250G) that three times volume is added crosses column, 12000 revs/min, it is centrifuged 1 minute；

3. abandoning waste liquid, 500 μ L W2 are added, 12000 revs/min, are centrifuged 1 minute；

4. abandoning waste liquid, 12000 revs/min, dally 1 minute；

5. changing collecting pipe, 20 μ L are added without RNAase water elution, 12000 revs/min, 1 minute.

Transcription step is as follows:

It utilizes in-vitro transcription kit (Ambion, Life Technologies, AM1354)

Reaction system are as follows:

Reaction condition: 37 DEG C, 5h.It is added 1 μ L DNase, 37 DEG C, 15 minutes.

Recycle sgRNA step:

It utilizes QIAquick Gel Extraction Kit (Ambion, Life Technologies, AM1908)

1. upper step reaction volume is added 90 μ L Elution solution and moves to 1.5mlEP pipe；

2. 350 μ L Binding solution mixing is added；

3. the mixing of 250 μ L dehydrated alcohols is added；Upper prop；It 10000 revs/min, is centrifuged 30 seconds, outwells waste liquid；

4. 500 μ L Washing solution are added, 10000 revs/min, it is centrifuged 30 seconds, outwells waste liquid；

5. 10000 revs/min, dallying 1 minute；

6. changing collecting pipe, 100 μ L Elution solution elution is added；

7. it is mixed that 10 μ L ammonium acetates (Ambion, Life Technologies, AM1908) is added in the liquid afforded It is even；

8. the mixing of 275 μ L dehydrated alcohols is added；

9. -20 DEG C are placed 30 minutes, while being prepared 70% ethyl alcohol and being placed -20 DEG C；

10. lower 13000 revs/min of 4 DEG C of environment are centrifuged 15 minutes.

Supernatant is abandoned, 500 μ L, 70% ethyl alcohol is added；

It 10000 revs/min, is centrifuged 5 minutes, exhausts waste liquid, dry 5 minutes；

The Rnase-free water dissolution of 20 μ L is added；1 μ L is taken to survey concentration.

The in-vitro transcription of 2.3 base editing systems

(this step is to linearize plasmid) is recycled into the code area digestion of base editing system.

System is as follows:

Condition: 37 DEG C, overnight.

The recycling of linearization plasmid:

1. 4 μ L RNAsecure (Life:AM7005) are added in digestion products, 60 DEG C are reacted 10 minutes；

2. carrying out operating remaining step using QIAquick Gel Extraction Kit (QIAGEN:28004), 5 times of volume buffer PB are added, Cross column；

3. 750 μ L buffer PE centrifugation is added；Idle running 1 minute；

4. measuring concentration with 10 μ L water elutions.

It is transcribed in vitro: sequentially adding system according to the requirement of kit (Invitrogen:AM1345):

Linearized vector 1ug；

2XNTP/ARCA 10μL；

T7ezyme mix2μL；

10xreaction buffer 2μL；

Water polishing is to 20 μ L.

Condition: 37 DEG C, 2 hours.Then 1 μ L DNasea is added to react 15 minutes.

Tailing: transcription product carries out the stability that tailing processing guarantees transcript mRNA.

System is as follows:

Condition: 37 DEG C, 30 minutes.

Recycling: (QIAGEN:74104) is carried out using QIAquick Gel Extraction Kit.

Steps are as follows:

1. up walking in reaction product and 350 μ L buffer RLT being added；

2. 250 μ L dehydrated alcohols are added, column, centrifugation are crossed；

3. 500 μ L RPE are added, it is centrifuged；

4. dallying；

5. 30 μ L water elutions are added.- 80 DEG C of preservations after measurement concentration.

Being separately cultured for 2.4 people HSC turns with electricity

With lymphocyte separation medium separation mononuclearcell, steps are as follows:

1. preparing 4ml sample of bone marrow, PBS is added with 1:1 ratio and is diluted；

2. isometric lymphocyte separation medium (GE 17-1440-03) is added (to note: making ficoll under when addition, edge Slowly sample is added for tube wall, not destroy the layering of ficoll and blood)；

3. 1700 revs/min, being centrifuged 30 minutes；

4. slowly carefully drawing one layer of middle white, i.e. mononuclear cell layer, 1500 revs/min, it is centrifuged 5 minutes；

5. 3ml erythrocyte cracked liquid is added in neat supernatant liquid, 10 minutes are stood, 1500 revs/min, is centrifuged 5 minutes, Abandon supernatant liquid；

It is thin that employment CD34+ sorts kit (STEMCELL 18056) isolated CD34+ Hematopoietic Stem from sample of bone marrow Born of the same parents, steps are as follows:

1. using the 14mL polystyrene tube (e.g.Corning Catalog#352057) of round bottom, the monokaryon that will be collected into Cell is resuspended with EasySepTM Buffer (Catalog#20144) to 0.25mL；

2. the antibody of 25ul is added, piping and druming uniformly, is incubated at room temperature 15min；

3. magnetic pole is turned upside down after mixing, 12.5ul magnetic pole is added in test tube, is incubated at room temperature 10min after blowing and beating uniformly；

4. total volume is resuspended to 5mL using buffer, gently piping and druming is mixed；

5. removing test tube cap, test tube is put into magnetic pole, is incubated at room temperature 5min；

6. test tube not taken out to magnetic pole, magnetic pole and test tube are picked up together, by the liquid in test tube down in sewer pipe；

7. repeating step 4-6 tri- to four times；

8. the cell on test tube wall is washed with buffer, it is ensured that collect the cell on test tube wall complete；It is transferred to In centrifuge tube, 1000rpm is centrifuged 5min；

9. discarding supernatant, cell is resuspended to 1*105/ml using culture medium, is cultivated in 48 orifice plates.

Obtained cell flow cytometer detection CD34+ cell is sorted, steps are as follows:

Buffer: the PBS containing 2%FBS；Antibody: CD34-FITC (eBiosicience 11034941)

1. collecting 5*105 cell, it is resuspended to 100ul volume；

2. 5ul antibody is added, mixes gently, be protected from light, be incubated for 30min on ice；

3. 1000rpm is centrifuged 5min, discards supernatant, 1ml buffer is added, gently uniformly, 1000rpm is centrifuged for piping and druming 5min is discarded supernatant；

4. cell is resuspended to 300ul with buffer, upper machine, flow cytometer detection, positive rate about 80~90% (Fig. 3).

Isolated CD34+ candidate stem cell is with the concentration culture of 6 × 104/ml in StemSpan SFEM culture medium In, cell factor 100ul/ml:StemSpanTM CD34+Expansion Supplement (10X) is added in culture medium (STEMCELL 02691).Cell culture is in 37 DEG C of incubators, 5%CO2 condition, and pre-stimulation 24 hours.By corresponding base editor System mRNA is incubated for 10 minutes with corresponding sgRNA at 37 DEG C, is carried out electricity using LONZA Nucleofector 2b and is turned, program Select U-008.Cell after electricity turns continues culture 2 days.

The differentiation and the detection of expression of HbF of 2.5 people HSC

Steps are as follows for erythroid differentiation:

StemSpan SFEMⅡ(stem cell,09655)StemSpan^TM Erythroid Expansion Supplement (100X) (STEMCELL 02692) antibody CD71-FITC (Biolegend 334104) CD235a-PE (Biolegend 349106)

Cell is equably layered in 24 orifice plates according to 1*104/ml, equal bodies are added when third day or the 4th day Long-pending new culture medium at the 7th day, collects cell, and 200G is centrifuged 5-10min, is then uniformly layered on 24 with new culture medium In orifice plate, cell density < 1*105/ml at this time.At the 10th day, isometric new culture medium is added.At the 14th day, collect thin Born of the same parents do flow cytometer detection (Fig. 4,5,6,7).

Erythroid differentiation flow cytometer detection

CD71-FITC(Biolegend 334104)CD235a-PE(Biolegend 349106)

Buffer: PBS, antibody: the CD34-FITC (eBiosicience 11034941) containing 2%FBS

Blank control group；The mono- dye pipe of CD71；The mono- dye pipe of CD235a；The bis- dye pipes of CD71 and CD235a

1. 5*105 cell of every group of collection is resuspended to 100ul volume；

2. antibody is not added in blank control group, 5ul CD71-FITC antibody is added in the mono- dye pipe of CD71；The mono- dye pipe of CD235a is added 5ul CD235a-PE antibody；5ul CD71-FITC and 5ul CD235a-PE antibody is added in the bis- dye pipes of CD71 and CD235a；

3. being protected from light, being incubated for 30min on ice；

4. 1000rpm is centrifuged 5min, discards supernatant, 1ml buffer is added, gently uniformly, 1000rpm is centrifuged for piping and druming 5min is discarded supernatant；

5. cell is resuspended to 300ul with buffer, upper machine, flow cytometer detection.

The mRNA level in-site of fluorescence quantitative PCR detection HBG:

1. extracting the RNA of cell after differentiation；

2. taking 500ng RNA as reverse transcription template, according to reverse transcription reagent box (Toyobo FSK-100CH) reverse transcription RNA；

3. dissolving cDNA with 10ul H2O, then 100ul is diluted to as Q-PCR template.

Q-PCR system:

The mRNA level in-site of HBG significantly increases (Fig. 8) in the cell of obtained differentiation.

Embodiment 3

In the present embodiment, in the isolated candidate stem cell of patient (HSC), using base editing system albumen with The RNA of sgRNA forms RNP form and realizes base editor, detection HBG expression variation.

3.1sgRNA plasmid construction and in-vitro transcription

With embodiment 2.

The acquisition of 3.2 patient HSC

Well known technology can be used and carry out marrow harvest candidate stem cell (HSC) from patient.

Being separately cultured for 3.3 patient HSC turns with electricity

With embodiment 2.

Isolated CD34+ candidate stem cell is with the concentration culture of 6 × 104/ml in StemSpan SFEM culture medium In, cell factor 100ul/ml:StemSpanTM CD34+Expansion Supplement (10X) is added in culture medium (STEMCELL 02691).Cell culture is in 37 DEG C of incubators, 5%CO2 condition, and pre-stimulation 24 hours.By corresponding base editor Systematic protein is incubated for 10 minutes with corresponding sgRNA at 37 DEG C, is carried out electricity using LONZA Nucleofector 2b and is turned, program Select U-008.Cell after electricity turns continues culture 2 days.

The feedback of 3.4 patient HSC

Resulting edited HSC venous re-transfusion is returned in patient body.

The foregoing is merely present pre-ferred embodiments, are not intended to limit the invention, all in spirit of the invention Within principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.

Sequence table

<110>national health health committee Institute Of Science And Technology

<120>genome base editor increases the kit of fetal hemoglobin level and application in human red blood cells

<160> 39

<170> SIPOSequenceListing 1.0

<210> 1

<211> 23

<212> DNA

<213>artificial sequence ()

<400> 1

cttgaccaat agccttgaca agg 23

<210> 2

<211> 23

<212> DNA

<213>artificial sequence ()

<400> 2

gctattggtc aaggcaaggc tgg 23

<210> 3

<211> 23

<212> DNA

<213>artificial sequence ()

<400> 3

ccctggctaa actccaccca tgg 23

<210> 4

<211> 23

<212> DNA

<213>artificial sequence ()

<400> 4

atatttgcat tgagatagtg tgg 23

<210> 5

<211> 22

<212> DNA

<213>artificial sequence ()

<400> 5

ccttccccac actatctcaa tg 22

<210> 6

<211> 23

<212> DNA

<213>artificial sequence ()

<400> 6

gtggggaagg ggcccccaag agg 23

<210> 7

<211> 8532

<212> DNA

<213>artificial sequence ()

<400> 7

gatcccctag ggtcgactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat 60

ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca 120

acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag gcgttttgcg 180

ctgcttcgcg atgtacgggc cagatatacg cgttgacatt gattattgac tagttattaa 240

tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg cgttacataa 300

cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata 360

atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag 420

tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc 480

cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta 540

tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg 600

cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt 660

ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca 720

aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag 780

gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagagatc cgcggccgct 840

aatacgactc actataggga gagccgccac catgagctca gagactggcc cagtggctgt 900

ggaccccaca ttgagacggc ggatcgagcc ccatgagttt gaggtattct tcgatccgag 960

agagctccgc aaggagacct gcctgcttta cgaaattaat tgggggggcc ggcactccat 1020

ttggcgacat acatcacaga acactaacaa gcacgtcgaa gtcaacttca tcgagaagtt 1080

cacgacagaa agatatttct gtccgaacac aaggtgcagc attacctggt ttctcagctg 1140

gagcccatgc ggcgaatgta gtagggccat cactgaattc ctgtcaaggt atccccacgt 1200

cactctgttt atttacatcg caaggctgta ccaccacgct gacccccgca atcgacaagg 1260

cctgcgggat ttgatctctt caggtgtgac tatccaaatt atgactgagc aggagtcagg 1320

atactgctgg agaaactttg tgaattatag cccgagtaat gaagcccact ggcctaggta 1380

tccccatctg tgggtacgac tgtacgttct tgaactgtac tgcatcatac tgggcctgcc 1440

tccttgtctc aacattctga gaaggaagca gccacagctg acattcttta ccatcgctct 1500

tcagtcttgt cattaccagc gactgccccc acacattctc tgggccaccg ggttgaaaag 1560

cggcagcgag actcccggga cctcagagtc cgccacaccc gaaagtgata aaaagtattc 1620

tattggttta gccatcggca ctaattccgt tggatgggct gtcataaccg atgaatacaa 1680

agtaccttca aagaaattta aggtgttggg gaacacagac cgtcattcga ttaaaaagaa 1740

tcttatcggt gccctcctat tcgatagtgg cgaaacggca gaggcgactc gcctgaaacg 1800

aaccgctcgg agaaggtata cacgtcgcaa gaaccgaata tgttacttac aagaaatttt 1860

tagcaatgag atggccaaag ttgacgattc tttctttcac cgtttggaag agtccttcct 1920

tgtcgaagag gacaagaaac atgaacggca ccccatcttt ggaaacatag tagatgaggt 1980

ggcatatcat gaaaagtacc caacgattta tcacctcaga aaaaagctag ttgactcaac 2040

tgataaagcg gacctgaggt taatctactt ggctcttgcc catatgataa agttccgtgg 2100

gcactttctc attgagggtg atctaaatcc ggacaactcg gatgtcgaca aactgttcat 2160

ccagttagta caaacctata atcagttgtt tgaagagaac cctataaatg caagtggcgt 2220

ggatgcgaag gctattctta gcgcccgcct ctctaaatcc cgacggctag aaaacctgat 2280

cgcacaatta cccggagaga agaaaaatgg gttgttcggt aaccttatag cgctctcact 2340

aggcctgaca ccaaatttta agtcgaactt cgacttagct gaagatgcca aattgcagct 2400

tagtaaggac acgtacgatg acgatctcga caatctactg gcacaaattg gagatcagta 2460

tgcggactta tttttggctg ccaaaaacct tagcgatgca atcctcctat ctgacatact 2520

gagagttaat actgagatta ccaaggcgcc gttatccgct tcaatgatca aaaggtacga 2580

tgaacatcac caagacttga cacttctcaa ggccctagtc cgtcagcaac tgcctgagaa 2640

atataaggaa atattctttg atcagtcgaa aaacgggtac gcaggttata ttgacggcgg 2700

agcgagtcaa gaggaattct acaagtttat caaacccata ttagagaaga tggatgggac 2760

ggaagagttg cttgtaaaac tcaatcgcga agatctactg cgaaagcagc ggactttcga 2820

caacggtagc attccacatc aaatccactt aggcgaattg catgctatac ttagaaggca 2880

ggaggatttt tatccgttcc tcaaagacaa tcgtgaaaag attgagaaaa tcctaacctt 2940

tcgcatacct tactatgtgg gacccctggc ccgagggaac tctcggttcg catggatgac 3000

aagaaagtcc gaagaaacga ttactccatg gaattttgag gaagttgtcg ataaaggtgc 3060

gtcagctcaa tcgttcatcg agaggatgac caactttgac aagaatttac cgaacgaaaa 3120

agtattgcct aagcacagtt tactttacga gtatttcaca gtgtacaatg aactcacgaa 3180

agttaagtat gtcactgagg gcatgcgtaa acccgccttt ctaagcggag aacagaagaa 3240

agcaatagta gatctgttat tcaagaccaa ccgcaaagtg acagttaagc aattgaaaga 3300

ggactacttt aagaaaattg aatgcttcga ttctgtcgag atctccgggg tagaagatcg 3360

atttaatgcg tcacttggta cgtatcatga cctcctaaag ataattaaag ataaggactt 3420

cctggataac gaagagaatg aagatatctt agaagatata gtgttgactc ttaccctctt 3480

tgaagatcgg gaaatgattg aggaaagact aaaaacatac gctcacctgt tcgacgataa 3540

ggttatgaaa cagttaaaga ggcgtcgcta tacgggctgg ggacgattgt cgcggaaact 3600

tatcaacggg ataagagaca agcaaagtgg taaaactatt ctcgattttc taaagagcga 3660

cggcttcgcc aataggaact ttatgcagct gatccatgat gactctttaa ccttcaaaga 3720

ggatatacaa aaggcacagg tttccggaca aggggactca ttgcacgaac atattgcgaa 3780

tcttgctggt tcgccagcca tcaaaaaggg catactccag acagtcaaag tagtggatga 3840

gctagttaag gtcatgggac gtcacaaacc ggaaaacatt gtaatcgaga tggcacgcga 3900

aaatcaaacg actcagaagg ggcaaaaaaa cagtcgagag cggatgaaga gaatagaaga 3960

gggtattaaa gaactgggca gccagatctt aaaggagcat cctgtggaaa atacccaatt 4020

gcagaacgag aaactttacc tctattacct acaaaatgga agggacatgt atgttgatca 4080

ggaactggac ataaaccgtt tatctgatta cgacgtcgat cacattgtac cccaatcctt 4140

tttgaaggac gattcaatcg acaataaagt gcttacacgc tcggataaga accgagggaa 4200

aagtgacaat gttccaagcg aggaagtcgt aaagaaaatg aagaactatt ggcggcagct 4260

cctaaatgcg aaactgataa cgcaaagaaa gttcgataac ttaactaaag ctgagagggg 4320

tggcttgtct gaacttgaca aggccggatt tattaaacgt cagctcgtgg aaacccgcca 4380

aatcacaaag catgttgcac agatactaga ttcccgaatg aatacgaaat acgacgagaa 4440

cgataagctg attcgggaag tcaaagtaat cactttaaag tcaaaattgg tgtcggactt 4500

cagaaaggat tttcaattct ataaagttag ggagataaat aactaccacc atgcgcacga 4560

cgcttatctt aatgccgtcg tagggaccgc actcattaag aaatacccga agctagaaag 4620

tgagtttgtg tatggtgatt acaaagttta tgacgtccgt aagatgatcg cgaaaagcga 4680

acaggagata ggcaaggcta cagccaaata cttcttttat tctaacatta tgaatttctt 4740

taagacggaa atcactctgg caaacggaga gatacgcaaa cgacctttaa ttgaaaccaa 4800

tggggagaca ggtgaaatcg tatgggataa gggccgggac ttcgcgacgg tgagaaaagt 4860

tttgtccatg ccccaagtca acatagtaaa gaaaactgag gtgcagaccg gagggttttc 4920

aaaggaatcg attcttccaa aaaggaatag tgataagctc atcgctcgta aaaaggactg 4980

ggacccgaaa aagtacggtg gcttcgatag ccctacagtt gcctattctg tcctagtagt 5040

ggcaaaagtt gagaagggaa aatccaagaa actgaagtca gtcaaagaat tattggggat 5100

aacgattatg gagcgctcgt cttttgaaaa gaaccccatc gacttccttg aggcgaaagg 5160

ttacaaggaa gtaaaaaagg atctcataat taaactacca aagtatagtc tgtttgagtt 5220

agaaaatggc cgaaaacgga tgttggctag cgccggagag cttcaaaagg ggaacgaact 5280

cgcactaccg tctaaatacg tgaatttcct gtatttagcg tcccattacg agaagttgaa 5340

aggttcacct gaagataacg aacagaagca actttttgtt gagcagcaca aacattatct 5400

cgacgaaatc atagagcaaa tttcggaatt cagtaagaga gtcatcctag ctgatgccaa 5460

tctggacaaa gtattaagcg catacaacaa gcacagggat aaacccatac gtgagcaggc 5520

ggaaaatatt atccatttgt ttactcttac caacctcggc gctccagccg cattcaagta 5580

ttttgacaca acgatagatc gcaaacgata cacttctacc aaggaggtgc tagacgcgac 5640

actgattcac caatccatca cgggattata tgaaactcgg atagatttgt cacagcttgg 5700

gggtgactct ggtggttcta ctaatctgtc agatattatt gaaaaggaga ccggtaagca 5760

actggttatc caggaatcca tcctcatgct cccagaggag gtggaagaag tcattgggaa 5820

caagccggaa agcgatatac tcgtgcacac cgcctacgac gagagcaccg acgagaatgt 5880

catgcttctg actagcgacg cccctgaata caagccttgg gctctggtca tacaggatag 5940

caacggtgag aacaagatta agatgctctc tggtggttct cccaagaaga agaggaaagt 6000

ctaaccggtc atcatcacca tcaccattga gtttaaaccc gctgatcagc ctcgactgtg 6060

ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa 6120

ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt 6180

aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa 6240

gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc 6300

agctggggct cgataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg 6360

tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata 6420

aagtgtaaag cctagggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca 6480

ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc 6540

gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg 6600

cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta 6660

tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc 6720

aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag 6780

catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac 6840

caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc 6900

ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt 6960

aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc 7020

gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga 7080

cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta 7140

ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta 7200

tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga 7260

tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg 7320

cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag 7380

tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc 7440

tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact 7500

tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt 7560

cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta 7620

ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta 7680

tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc 7740

gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat 7800

agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt 7860

atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg 7920

tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca 7980

gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta 8040

agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg 8100

cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact 8160

ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg 8220

ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt 8280

actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga 8340

ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc 8400

atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa 8460

caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga 8520

gatcgatctc cc 8532

<210> 8

<211> 8580

<212> DNA

<213>artificial sequence ()

<400> 8

cctgtcaagg tatccccacg tcactctgtt tatttacatc gcaaggctgt accaccacgc 60

tgacccccgc aatcgacaag gcctgcggga tttgatctct tcaggtgtga ctatccaaat 120

tatgactgag caggagtcag gatactgctg gagaaacttt gtgaattata gcccgagtaa 180

tgaagcccac tggcctaggt atccccatct gtgggtacga ctgtacgttc ttgaactgta 240

ctgcatcata ctgggcctgc ctccttgtct caacattctg agaaggaagc agccacagct 300

gacattcttt accatcgctc ttcagtcttg tcattaccag cgactgcccc cacacattct 360

ctgggccacc gggttgaaat ctggtggttc ttctggtggt tctagcggca gcgagactcc 420

cgggacctca gagtccgcca cacccgaaag ttctggtggt tcttctggtg gttctgataa 480

aaagtattct attggtttag ccatcggcac taattccgtg ggctgggccg tgatcaccga 540

cgagtacaag gtgcccagca agaaattcaa ggtgctgggc aacaccgacc ggcacagcat 600

caagaagaac ctgatcggag ccctgctgtt cgacagcggc gaaacagccg aggccacccg 660

gctgaagaga accgccagaa gaagatacac cagacggaag aaccggatct gctatctgca 720

agagatcttc agcaacgaga tggccaaggt ggacgacagc ttcttccaca gactggaaga 780

gtccttcctg gtggaagagg ataagaagca cgagcggcac cccatcttcg gcaacatcgt 840

ggacgaggtg gcctaccacg agaagtaccc caccatctac cacctgagaa agaaactggt 900

ggacagcacc gacaaggccg acctgcggct gatctatctg gccctggccc acatgatcaa 960

gttccggggc cacttcctga tcgagggcga cctgaacccc gacaacagcg acgtggacaa 1020

gctgttcatc cagctggtgc agacctacaa ccagctgttc gaggaaaacc ccatcaacgc 1080

cagcggcgtg gacgccaagg ccatcctgtc tgccagactg agcaagagca gacggctgga 1140

aaatctgatc gcccagctgc ccggcgagaa gaagaatggc ctgttcggaa acctgattgc 1200

cctgagcctg ggcctgaccc ccaacttcaa gagcaacttc gacctggccg aggataccaa 1260

actgcagctg agcaaggaca cctacgacga cgacctggac aacctgctgg cccagatcgg 1320

cgaccagtac gccgacctgt ttctggccgc caagaacctg tccgacgcca tcctgctgag 1380

cgacatcctg agagtgaaca ccgagatcac caaggccccc ctgagcgcct ctatgatcaa 1440

gctgtacgac gagcaccacc aggacctgac cctgctgaaa gctctcgtgc ggcagcagct 1500

gcctgagaag tacaaagaga ttttcttcga ccagagcaag aacggctacg ccggctacat 1560

tgacggcgga gccagccagg aagagttcta caagttcatc aagcccatcc tggaaaagat 1620

ggacggcacc gaggaactgc tcgtgaagct gaacagagag gacctgctgc ggaagcagcg 1680

gaccttcgac aacggcatca tcccccacca gatccacctg ggagagctgc acgccattct 1740

gcggcggcag gaagattttt acccattcct gaaggacaac cgggaaaaga tcgagaagat 1800

cctgaccttc cgcatcccct actacgtggg ccctctggcc aggggaaaca gcagattcgc 1860

ctggatgacc agaaagagcg aggaaaccat caccccctgg aacttcgaga aggtggtgga 1920

caagggcgct tccgcccaga gcttcatcga gcggatgacc aacttcgata agaacctgcc 1980

caacgagaag gtgctgccca agcacagcct gctgtacgag tacttcaccg tgtataacga 2040

gctgaccaaa gtgaaatacg tgaccgaggg aatgagaaag cccgccttcc tgagcggcga 2100

ccagaaaaag gccatcgtgg acctgctgtt caagaccaac cggaaagtga ccgtgaagca 2160

gctgaaagag gactacttca agaaaatcga gtgcttcgac tccgtggaaa tctccggcgt 2220

ggaagatcgg ttcaacgcct ccctgggcac ataccacgat ctgctgaaaa ttatcaagga 2280

caaggacttc ctggacaatg aggaaaacga ggacattctg gaagatatcg tgctgaccct 2340

gacactgttt gaggacagag agatgatcga ggaacggctg aaaacctatg cccacctgtt 2400

cgacgacaaa gtgatgaagc agctgaagcg gcggagatac accggctggg gcaggctgag 2460

ccggaagctg atcaacggca tccgggacaa gcagtccggc aagacaatcc tggatttcct 2520

gaagtccgac ggcttcgcca acagaaactt catccagctg atccacgacg acagcctgac 2580

ctttaaagag gacatccaga aagcccaggt gtccggccag ggcgatagcc tgcacgagca 2640

cattgccaat ctggccggca gccccgccat taagaagggc atcctgcaga cagtgaaggt 2700

ggtggacgag ctcgtgaaag tgatgggccg gcacaagccc gagaacatcg tgatcgaaat 2760

ggccagagag aaccagacca cccagaaggg acagaagaac agccgcgaga gaatgaagcg 2820

gatcgaagag ggcatcaaag agctgggcag ccagatcctg aaagaacacc ccgtggaaaa 2880

cacccagctg cagaacgaga agctgtacct gtactacctg cagaatgggc gggatatgta 2940

cgtggaccag gaactggaca tcaaccggct gtccgactac gatgtggacc atatcgtgcc 3000

tcagagcttt ctgaaggacg actccatcga caacaaggtg ctgaccagaa gcgacaagaa 3060

ccggggcaag agcgacaacg tgccctccga agaggtcgtg aagaagatga agaactactg 3120

gcggcagctg ctgaacgcca agctgattac ccagagaaag ttcgacaatc tgaccaaggc 3180

cgagagaggc ggcctgagcg aactggataa ggccggcttc atcaagagac agctggtgga 3240

aacccggcag atcacaaagc acgtggcaca gatcctggac tcccggatga acactaagta 3300

cgacgagaat gacaagctga tccgggaagt gaaagtgatc accctgaagt ccaagctggt 3360

gtccgatttc cggaaggatt tccagtttta caaagtgcgc gagatcaaca actaccacca 3420

cgcccacgac gcctacctga acgccgtcgt gggaaccgcc ctgatcaaaa agtaccctaa 3480

gctggaaagc gagttcgtgt acggcgacta caaggtgtac gacgtgcgga agatgatcgc 3540

caagagcgag caggaaatcg gcaaggctac cgccaagtac ttcttctaca gcaacatcat 3600

gaactttttc aagaccgaga ttaccctggc caacggcgag atccggaagc ggcctctgat 3660

cgagacaaac ggcgaaaccg gggagatcgt gtgggataag ggccgggatt ttgccaccgt 3720

gcggaaagtg ctgagcatgc cccaagtgaa tatcgtgaaa aagaccgagg tgcagacagg 3780

cggcttcagc aaagagtcta tcctgcccaa gaggaacagc gataagctga tcgccagaaa 3840

gaaggactgg gaccctaaga agtacggcgg cttcgacagc cccaccgtgg cctattctgt 3900

gctggtggtg gccaaagtgg aaaagggcaa gtccaagaaa ctgaagagtg tgaaagagct 3960

gctggggatc accatcatgg aaagaagcag cttcgagaag aatcccatcg actttctgga 4020

agccaagggc tacaaagaag tgaaaaagga cctgatcatc aagctgccta agtactccct 4080

gttcgagctg gaaaacggcc ggaagagaat gctggcctct gccggcgtgc tgcagaaggg 4140

aaacgaactg gccctgccct ccaaatatgt gaacttcctg tacctggcca gccactatga 4200

gaagctgaag ggctcccccg aggataatga gcagaaacag ctgtttgtgg aacagcacaa 4260

gcactacctg gacgagatca tcgagcagat cagcgagttc tccaagagag tgatcctggc 4320

cgacgctaat ctggacaaag tgctgtccgc ctacaacaag caccgggata agcccatcag 4380

agagcaggcc gagaatatca tccacctgtt taccctgacc aatctgggag cccctgccgc 4440

cttcaagtac tttgacacca ccatcgaccg gaagaggtac accagcacca aagaggtgct 4500

ggacgccacc ctgatccacc agagcatcac cggcctgtac gagacacgga tcgacctgtc 4560

tcagctggga ggcgactctg gtggttctac taatctgtca gatattattg aaaaggagac 4620

cggtaagcaa ctggttatcc aggaatccat cctcatgctc ccagaggagg tggaagaagt 4680

cattgggaac aagccggaaa gcgatatact cgtgcacacc gcctacgacg agagcaccga 4740

cgagaatgtc atgcttctga ctagcgacgc ccctgaatac aagccttggg ctctggtcat 4800

acaggatagc aacggtgaga acaagattaa gatgctctct ggtggttctc ccaagaagaa 4860

gaggaaagtc taaccggtca tcatcaccat caccattgag tttaaacccg ctgatcagcc 4920

tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg 4980

accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat 5040

tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag 5100

gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg 5160

gaaagaacca gctggggctc gataccgtcg acctctagct agagcttggc gtaatcatgg 5220

tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc 5280

ggaagcataa agtgtaaagc ctagggtgcc taatgagtga gctaactcac attaattgcg 5340

ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc 5400

ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact 5460

gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta 5520

atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag 5580

caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc 5640

cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta 5700

taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg 5760

ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc 5820

tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac 5880

gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac 5940

ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg 6000

aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga 6060

agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt 6120

agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag 6180

cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct 6240

gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg 6300

atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat 6360

gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc 6420

tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg 6480

gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct 6540

ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca 6600

actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg 6660

ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg 6720

tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc 6780

cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag 6840

ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg 6900

ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag 6960

tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat 7020

agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg 7080

atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca 7140

gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca 7200

aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat 7260

tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag 7320

aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac 7380

ggatcgggag atcgatctcc cgatccccta gggtcgactc tcagtacaat ctgctctgat 7440

gccgcatagt taagccagta tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc 7500

gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg acaattgcat gaagaatctg 7560

cttagggtta ggcgttttgc gctgcttcgc gatgtacggg ccagatatac gcgttgacat 7620

tgattattga ctagttatta atagtaatca attacggggt cattagttca tagcccatat 7680

atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac 7740

ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc 7800

cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg 7860

tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat 7920

tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc 7980

atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt 8040

gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac 8100

caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc 8160

ggtaggcgtg tacggtggga ggtctatata agcagagctg gtttagtgaa ccgtcagatc 8220

cgctagagat ccgcggccgc taatacgact cactataggg agagccgcca ccatgagctc 8280

agagactggc ccagtggctg tggaccccac attgagacgg cggatcgagc cccatgagtt 8340

tgaggtattc ttcgatccga gagagctccg caaggagacc tgcctgcttt acgaaattaa 8400

ttgggggggc cggcactcca tttggcgaca tacatcacag aacactaaca agcacgtcga 8460

agtcaacttc atcgagaagt tcacgacaga aagatatttc tgtccgaaca caaggtgcag 8520

cattacctgg tttctcagct ggagcccatg cggcgaatgt agtagggcca tcactgaatt 8580

<210> 9

<211> 8792

<212> DNA

<213>artificial sequence ()

<400> 9

tacacgtcgc aagaaccgaa tatgttactt acaagaaatt tttagcaatg agatggccaa 60

agttgacgat tctttctttc accgtttgga agagtccttc cttgtcgaag aggacaagaa 120

acatgaacgg caccccatct ttggaaacat agtagatgag gtggcatatc atgaaaagta 180

cccaacgatt tatcacctca gaaaaaagct agttgactca actgataaag cggacctgag 240

gttaatctac ttggctcttg cccatatgat aaagttccgt gggcactttc tcattgaggg 300

tgatctaaat ccggacaact cggatgtcga caaactgttc atccagttag tacaaaccta 360

taatcagttg tttgaagaga accctataaa tgcaagtggc gtggatgcga aggctattct 420

tagcgcccgc ctctctaaat cccgacggct agaaaacctg atcgcacaat tacccggaga 480

gaagaaaaat gggttgttcg gtaaccttat agcgctctca ctaggcctga caccaaattt 540

taagtcgaac ttcgacttag ctgaagatgc caaattgcag cttagtaagg acacgtacga 600

tgacgatctc gacaatctac tggcacaaat tggagatcag tatgcggact tatttttggc 660

tgccaaaaac cttagcgatg caatcctcct atctgacata ctgagagtta atactgagat 720

taccaaggcg ccgttatccg cttcaatgat caaaaggtac gatgaacatc accaagactt 780

gacacttctc aaggccctag tccgtcagca actgcctgag aaatataagg aaatattctt 840

tgatcagtcg aaaaacgggt acgcaggtta tattgacggc ggagcgagtc aagaggaatt 900

ctacaagttt atcaaaccca tattagagaa gatggatggg acggaagagt tgcttgtaaa 960

actcaatcgc gaagatctac tgcgaaagca gcggactttc gacaacggta gcattccaca 1020

tcaaatccac ttaggcgaat tgcatgctat acttagaagg caggaggatt tttatccgtt 1080

cctcaaagac aatcgtgaaa agattgagaa aatcctaacc tttcgcatac cttactatgt 1140

gggacccctg gcccgaggga actctcggtt cgcatggatg acaagaaagt ccgaagaaac 1200

gattactcca tggaattttg aggaagttgt cgataaaggt gcgtcagctc aatcgttcat 1260

cgagaggatg accaactttg acaagaattt accgaacgaa aaagtattgc ctaagcacag 1320

tttactttac gagtatttca cagtgtacaa tgaactcacg aaagttaagt atgtcactga 1380

gggcatgcgt aaacccgcct ttctaagcgg agaacagaag aaagcaatag tagatctgtt 1440

attcaagacc aaccgcaaag tgacagttaa gcaattgaaa gaggactact ttaagaaaat 1500

tgaatgcttc gattctgtcg agatctccgg ggtagaagat cgatttaatg cgtcacttgg 1560

tacgtatcat gacctcctaa agataattaa agataaggac ttcctggata acgaagagaa 1620

tgaagatatc ttagaagata tagtgttgac tcttaccctc tttgaagatc gggaaatgat 1680

tgaggaaaga ctaaaaacat acgctcacct gttcgacgat aaggttatga aacagttaaa 1740

gaggcgtcgc tatacgggct ggggacgatt gtcgcggaaa cttatcaacg ggataagaga 1800

caagcaaagt ggtaaaacta ttctcgattt tctaaagagc gacggcttcg ccaataggaa 1860

ctttatgcag ctgatccatg atgactcttt aaccttcaaa gaggatatac aaaaggcaca 1920

ggtttccgga caaggggact cattgcacga acatattgcg aatcttgctg gttcgccagc 1980

catcaaaaag ggcatactcc agacagtcaa agtagtggat gagctagtta aggtcatggg 2040

acgtcacaaa ccggaaaaca ttgtaatcga gatggcacgc gaaaatcaaa cgactcagaa 2100

ggggcaaaaa aacagtcgag agcggatgaa gagaatagaa gagggtatta aagaactggg 2160

cagccagatc ttaaaggagc atcctgtgga aaatacccaa ttgcagaacg agaaacttta 2220

cctctattac ctacaaaatg gaagggacat gtatgttgat caggaactgg acataaaccg 2280

tttatctgat tacgacgtcg atcacattgt accccaatcc tttttgaagg acgattcaat 2340

cgacaataaa gtgcttacac gctcggataa gaaccgaggg aaaagtgaca atgttccaag 2400

cgaggaagtc gtaaagaaaa tgaagaacta ttggcggcag ctcctaaatg cgaaactgat 2460

aacgcaaaga aagttcgata acttaactaa agctgagagg ggtggcttgt ctgaacttga 2520

caaggccgga tttattaaac gtcagctcgt ggaaacccgc caaatcacaa agcatgttgc 2580

acagatacta gattcccgaa tgaatacgaa atacgacgag aacgataagc tgattcggga 2640

agtcaaagta atcactttaa agtcaaaatt ggtgtcggac ttcagaaagg attttcaatt 2700

ctataaagtt agggagataa ataactacca ccatgcgcac gacgcttatc ttaatgccgt 2760

cgtagggacc gcactcatta agaaataccc gaagctagaa agtgagtttg tgtatggtga 2820

ttacaaagtt tatgacgtcc gtaagatgat cgcgaaaagc gaacaggaga taggcaaggc 2880

tacagccaaa tacttctttt attctaacat tatgaatttc tttaagacgg aaatcactct 2940

ggcaaacgga gagatacgca aacgaccttt aattgaaacc aatggggaga caggtgaaat 3000

cgtatgggat aagggccggg acttcgcgac ggtgagaaaa gttttgtcca tgccccaagt 3060

caacatagta aagaaaactg aggtgcagac cggagggttt tcaaaggaat cgattcttcc 3120

aaaaaggaat agtgataagc tcatcgctcg taaaaaggac tgggacccga aaaagtacgg 3180

tggcttcgat agccctacag ttgcctattc tgtcctagta gtggcaaaag ttgagaaggg 3240

aaaatccaag aaactgaagt cagtcaaaga attattgggg ataacgatta tggagcgctc 3300

gtcttttgaa aagaacccca tcgacttcct tgaggcgaaa ggttacaagg aagtaaaaaa 3360

ggatctcata attaaactac caaagtatag tctgtttgag ttagaaaatg gccgaaaacg 3420

gatgttggct agcgccggag agcttcaaaa ggggaacgaa ctcgcactac cgtctaaata 3480

cgtgaatttc ctgtatttag cgtcccatta cgagaagttg aaaggttcac ctgaagataa 3540

cgaacagaag caactttttg ttgagcagca caaacattat ctcgacgaaa tcatagagca 3600

aatttcggaa ttcagtaaga gagtcatcct agctgatgcc aatctggaca aagtattaag 3660

cgcatacaac aagcacaggg ataaacccat acgtgagcag gcggaaaata ttatccattt 3720

gtttactctt accaacctcg gcgctccagc cgcattcaag tattttgaca caacgataga 3780

tcgcaaacga tacacttcta ccaaggaggt gctagacgcg acactgattc accaatccat 3840

cacgggatta tatgaaactc ggatagattt gtcacagctt gggggtgact ctggtggttc 3900

tcccaagaag aagaggaaag tctaaccggt catcatcacc atcaccattg agtttaaacc 3960

cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc 4020

gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa 4080

attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac 4140

agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg 4200

gcttctgagg cggaaagaac cagctggggc tcgataccgt cgacctctag ctagagcttg 4260

gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac 4320

aacatacgag ccggaagcat aaagtgtaaa gcctagggtg cctaatgagt gagctaactc 4380

acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg 4440

cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct 4500

tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac 4560

tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga 4620

gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat 4680

aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac 4740

ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct 4800

gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg 4860

ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg 4920

ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt 4980

cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg 5040

attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac 5100

ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga 5160

aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt 5220

gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt 5280

tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga 5340

ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc 5400

taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct 5460

atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata 5520

actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca 5580

cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga 5640

agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga 5700

gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg 5760

gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga 5820

gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt 5880

gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct 5940

cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca 6000

ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat 6060

accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga 6120

aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc 6180

aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg 6240

caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc 6300

ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt 6360

gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca 6420

cctgacgtcg acggatcggg agatcgatct cccgatcccc tagggtcgac tctcagtaca 6480

atctgctctg atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc 6540

gctgagtagt gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc 6600

atgaagaatc tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat 6660

acgcgttgac attgattatt gactagttat taatagtaat caattacggg gtcattagtt 6720

catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga 6780

ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca 6840

atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca 6900

gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg 6960

cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc 7020

tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt 7080

ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt 7140

ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg 7200

acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc tggtttagtg 7260

aaccgtcaga tccgctagag atccgcggcc gctaatacga ctcactatag ggccaccatg 7320

aagcgcaccg ccgatggttc cgagttcgaa agccccaaaa aaaagcgcaa ggtcccgaga 7380

gccgccacca tgtccgaagt cgagttttcc catgagtact ggatgagaca cgcattgact 7440

ctcgcaaaga gggcttggga tgaacgcgag gtgcccgtgg gggcagtact cgtgcataac 7500

aatcgcgtaa tcggcgaagg ttggaatagg ccgatcggac gccacgaccc cactgcacat 7560

gcggaaatca tggcccttcg acagggaggg cttgtgatgc agaattatcg acttatcgat 7620

gcgacgctgt acgtcacgct tgaaccttgc gtaatgtgcg cgggagctat gattcactcc 7680

cgcattggac gagttgtatt cggtgcccgc gacgccaaga cgggtgccgc aggttcactg 7740

atggacgtgc tgcatcaccc aggcatgaac caccgggtag aaatcacaga aggcatattg 7800

gcggacgaat gtgcggcgct gttgtccgac ttttttcgca tgcggaggca ggagatcaag 7860

gcccagaaaa aagcacaatc ctctactgac agcggcggca gcagcggcgg cagcagcggc 7920

agcgagactc ccgggacctc agagtccgcc acacccgaaa gtagcggcgg cagcagcggc 7980

ggcagctccg aagtcgagtt ttcccatgag tactggatga gacacgcatt gactctcgca 8040

aagagggctc gggatgaacg cgaggtgccc gtgggggcag tactcgtgct taacaatcgc 8100

gtaatcggcg aaggttggaa tagggcgatc ggactccacg accccactgc acatgcggaa 8160

atcatggccc ttcgacaggg agggcttgtg atgcagaatt atcgacttat cgatgcgacg 8220

ctgtacgtca cgtttgaacc ttgcgtaatg tgcgcgggag ctatgattca ctcccgcatt 8280

ggacgagttg tattcggtgt ccgcaacgcc aagacgggtg ccgcaggttc actgatggac 8340

gtgctgcatt acccaggcat gaaccaccgg gtagaaatca cagaaggcat attggcggac 8400

gaatgtgcgg cgctgttgtg ctactttttt cgcatgccga ggcaggtgtt caatgcccag 8460

aaaaaagcac aatcctctac tgacagcggc ggcagcagcg gcggcagcag cggcagcgag 8520

actcccggga cctcagagtc cgccacaccc gaaagtagcg gcggcagcag cggcggcagc 8580

gataaaaagt attctattgg tttagccatc ggcactaatt ccgttggatg ggctgtcata 8640

accgatgaat acaaagtacc ttcaaagaaa tttaaggtgt tggggaacac agaccgtcat 8700

tcgattaaaa agaatcttat cggtgccctc ctattcgata gtggcgaaac ggcagaggcg 8760

actcgcctga aacgaaccgc tcggagaagg ta 8792

<210> 10

<211> 10601

<212> DNA

<213>artificial sequence ()

<400> 10

gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 60

catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 120

acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 180

ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 240

aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 300

ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 360

tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc 420

ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt 480

ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa 540

tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag 600

aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg 660

gctagcacca tgggacccaa gaaaaaacgc aaggtggaag atcctaagaa aaagcggaaa 720

gtggacggca ttggtagtgg gagcaacggc agcagcggag aggaactgct gagcaagaac 780

taccacctgg aaaacgaggt ggccagactg aagaaaggct ctggctctgg cggaagcggt 840

tctggcggat caggatctgg aagtggcggc tctggaagcg gaggttctgg atctggcgaa 900

gaactgctgt ctaagaatta tcacctcgag aacgaagtgg ctcggctcaa gaaaggcagt 960

ggcagcggag gaagtggatc cggcggtagt ggtagtggaa gcggcggatc cggctcaggc 1020

ggatctggtt caggggaaga actcctctcc aaaaactacc atctcgagaa tgaggtcgcc 1080

cgcctgaaaa aaggatcagg ttccggtggt tctggcagcg gtggttcagg ctcaggcagc 1140

ggcggtagcg gtagcggtgg aagcggaagc ggagaagaac ttctcagcaa aaattaccac 1200

ttggagaatg aagttgcaag actcaaaaaa ggttccggca gtggcggcag cggcagcggc 1260

ggatctggta gtggatctgg tggcagtggt tcaggcggaa gtggtagcgg agaggaattg 1320

ctctcaaaga actatcattt ggagaacgag gttgcacgct tgaagaaagg cagcggatca 1380

ggcggatctg gcagcggtgg atctggttct ggatccggcg gctccggtag tggtggaagt 1440

ggctctgggg aagaattgct tagcaagaat tatcatcttg aaaatgaggt tgccaggctt 1500

aaaaaaggca gtggctccgg cggatccgga agcggaggca gcggatctgg atctggtggt 1560

tcaggatctg gcggttctgg tagcggggaa gaactgttga gtaaaaacta tcaccttgag 1620

aacgaggtcg caaggttgaa aaaaggatcc ggctctggcg gctccggaag tggcggatct 1680

ggctccggta gcggaggatc aggatccggc ggaagcggat caggcgagga actgctttcc 1740

aaaaattacc accttgaaaa cgaagtcgcc cgcctcaaga aaggttctgg cagcggaggc 1800

tctggcagtg gtggtagcgg aagtggaagt ggtggcagtg gtagcggtgg atctggaagc 1860

ggcgaggaac tcctgtcaaa gaattaccat ctcgaaaacg aggtcgcaag gctcaagaaa 1920

ggctcaggat caggcggctc tggatccggc ggttctggtt ccggctcagg tggaagtgga 1980

tctggcggct caggttccgg cgaagaattg ctttccaaga actaccattt ggaaaatgaa 2040

gtcgctcgtt tgaagaaagg ttcaggctcc ggccagcggc cgcaaggtgg aggtggaccc 2100

aagaagaagc gcaaggtgtc tagagacaag aaatactcta ttggactggc tatcgggaca 2160

aactccgttg gctgggccgt cataaccgac gagtataagg tgccaagcaa gaaattcaag 2220

gtgctgggta atactgaccg ccattcaatc aagaagaacc tgatcggagc actcctcttc 2280

gactccggtg aaaccgctga agctactcgg ctgaagcgga ccgcaaggcg gagatacacc 2340

cgccgcaaga atcggatatg ttatctgcaa gagatcttta gcaacgaaat ggctaaggtg 2400

gacgactcct tctttcaccg cctggaagag agctttctgg tggaggagga taagaaacac 2460

gagaggcacc ctatattcgg aaatatcgtg gatgaggtgg cttaccatga aaagtatcct 2520

acaatctacc atctgaggaa gaagctggtg gacagcaccg ataaagcaga cctgaggctc 2580

atctatctgg ccctggctca tatgataaag tttagaggac actttctgat cgagggcgac 2640

ctgaatcccg ataattccga tgtggataaa ctcttcattc aactggtgca gacatataac 2700

caactgttcg aggagaatcc cataaacgct tctggtgtgg atgccaaggc tattctgtcc 2760

gctcggctgt ccaagtcacg cagactggag aatctgattg cccaactgcc aggagaaaag 2820

aagaacggcc tgtttgggaa cctcatcgcc ctgagcctgg gcctgacacc taacttcaag 2880

tccaattttg atctggccga agatgctaaa ctccagctct ccaaggacac ctatgacgat 2940

gatctggaca acctgctcgc acagataggc gaccagtacg ccgatctctt tctggctgct 3000

aagaatctct ccgacgccat tctgctgagc gacatactcc gggtcaacac tgagatcacc 3060

aaagcacctc tgagcgcctc catgataaaa cgctatgatg aacaccatca agacctgact 3120

ctgctcaaag ccctcgtgag gcaacagctg ccagagaagt acaaagagat attcttcgac 3180

cagagcaaga atggatatgc cggatacatc gatggcggag catcacagga agaattttac 3240

aagttcatca aaccaatcct cgagaagatg gacggtactg aagagctgct ggtgaagctg 3300

aacagggagg acctgctgag gaagcagagg acctttgata atggctccat tccacatcag 3360

atacacctgg gagagctgca tgcaatcctc cgcaggcagg aggatttcta tcctttcctg 3420

aaggataacc gggagaagat agagaagatc ctgaccttca ggatccctta ttacgtcggc 3480

cctctggcta gaggcaactc ccgcttcgct tggatgacca ggaaatctga ggagacaatt 3540

actccttgga acttcgaaga ggtcgtggat aagggcgcaa gcgcccagtc attcatcgaa 3600

cggatgacca atttcgataa gaacctgccc aacgagaagg tcctgcccaa acattcactc 3660

ctgtacgagt atttcaccgt ctataacgag ctgactaaag tgaagtacgt gaccgagggc 3720

atgaggaagc ctgccttcct gtccggagag cagaagaagg ctatcgttga tctgctcttc 3780

aagactaata gaaaggtgac agtgaagcag ctcaaggagg attactttaa gaagatcgaa 3840

tgctttgact cagtggaaat ctctggcgtg gaggaccgct ttaatgccag cctgggcact 3900

taccatgatc tgctgaagat aatcaaagac aaagatttcc tcgataatga ggagaacgag 3960

gacatcctgg aagatatcgt gctgaccctg actctgttcg aggatagaga gatgatcgaa 4020

gagcgcctga agacctatgc ccatctgttt gacgataaag tcatgaaaca gctcaagcgg 4080

cggcgctaca ctgggtgggg tagactctcc aggaaactca taaacggcat ccgcgacaaa 4140

cagagcggaa agaccatcct ggatttcctg aaatccgacg gattcgctaa caggaacttc 4200

atgcaactga ttcacgatga ctctctgaca tttaaagagg acatccagaa ggcacaggtg 4260

agcggtcaag gcgacagcct gcacgagcac atcgccaacc tcgctggatc acccgccata 4320

aagaagggaa tactgcagac agtcaaggtc gtggacgaac tcgtcaaagt gatgggtcgg 4380

cacaagccag agaatatcgt tatcgaaatg gcaagggaga accaaaccac ccagaagggc 4440

cagaagaact ctcgggaacg gatgaaaaga atcgaagagg gaattaagga gctgggatct 4500

cagatactga aggagcaccc tgtggagaat acacagctcc agaacgagaa actctacctg 4560

tactacctcc agaacgggcg ggacatgtac gttgaccagg aactcgacat caaccggctg 4620

tccgattatg acgtggacca tattgttcca cagtccttcc tcaaagatga ctccattgac 4680

aacaaggtgc tgaccagatc cgataagaat cgcggtaagt ctgacaatgt tccatcagaa 4740

gaggtggtca agaagatgaa gaattactgg cggcagctcc tcaacgccaa actgatcacc 4800

cagcggaagt ttgacaatct gactaaggca gaaagaggag gtctgagcga actcgacaag 4860

gccggcttta ttaagaggca actggtcgaa acacgccaga ttaccaaaca cgtggcacaa 4920

atcctcgact ctaggatgaa cactaagtac gatgagaacg ataagctgat cagggaagtg 4980

aaagtgataa ctctgaagag caagctggtg tctgacttcc ggaaggactt tcaattctac 5040

aaagttcgcg aaataaacaa ttaccatcat gctcacgatg cctatctcaa tgctgtcgtt 5100

ggcaccgccc tgatcaagaa ataccctaaa ctggagtctg agttcgtgta cggtgactat 5160

aaagtctacg atgtgaggaa gatgatagca aagtctgagc aagagattgg caaagccacc 5220

gccaagtact tcttctactc taatatcatg aatttcttta agactgagat aaccctggct 5280

aacggcgaaa tccggaagcg cccactgatc gaaacaaacg gagaaacagg agaaatcgtg 5340

tgggataaag gcagggactt cgcaactgtg cggaaggtgc tgtccatgcc acaagtcaat 5400

atcgtgaaga agaccgaagt gcagaccggc ggattctcaa aggagagcat cctgccaaag 5460

cggaactctg acaagctgat cgccaggaag aaagattggg acccaaagaa gtatggcggt 5520

ttcgattccc ctacagtggc ttattccgtt ctggtcgtgg caaaagtgga gaaaggcaag 5580

tccaagaaac tcaagtctgt taaggagctg ctcggaatta ctattatgga gagatccagc 5640

ttcgagaaga atccaatcga tttcctggaa gctaagggct ataaagaagt gaagaaagat 5700

ctcatcatca aactgcccaa gtactctctc tttgagctgg agaatggtag gaagcggatg 5760

ctggcctccg ccggagagct gcagaaagga aacgagctgg ctctgccctc caaatacgtg 5820

aacttcctgt atctggcctc ccactacgag aaactcaaag gtagccctga agacaatgag 5880

cagaagcaac tctttgttga gcaacataaa cactacctgg acgaaatcat tgaacagatt 5940

agcgagttca gcaagcgggt tattctggcc gatgcaaacc tcgataaagt gctgagcgca 6000

tataataagc acagggacaa gccaattcgc gaacaagcag agaatattat ccacctcttt 6060

actctgacta atctgggcgc tcctgctgcc ttcaagtatt tcgatacaac tattgacagg 6120

aagcggtaca cctctaccaa agaagttctc gatgccaccc tgatacacca gtcaattacc 6180

ggactgtacg agactcgcat cgacctgtct cagctcggcg gcgacggttc tgaattcagc 6240

ctgggcagcg gctcccccaa gaagaagcgc aaggtgaccg gtcatcatca ccatcaccat 6300

tgagtttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt 6360

tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa 6420

taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg 6480

gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg 6540

gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac 6600

gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct 6660

acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg 6720

ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt 6780

gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca 6840

tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga 6900

ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa 6960

gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac 7020

gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag 7080

caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc 7140

caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag 7200

tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc 7260

cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct gcctctgagc 7320

tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctcccgg 7380

gagcttgtat atccattttc ggatctgatc agcacgtgtt gacaattaat catcggcata 7440

gtatatcggc atagtataat acgacaaggt gaggaactaa accatggcca agcctttgtc 7500

tcaagaagaa tccaccctca ttgaaagagc aacggctaca atcaacagca tccccatctc 7560

tgaagactac agcgtcgcca gcgcagctct ctctagcgac ggccgcatct tcactggtgt 7620

caatgtatat cattttactg ggggaccttg tgcagaactc gtggtgctgg gcactgctgc 7680

tgctgcggca gctggcaacc tgacttgtat cgtcgcgatc ggaaatgaga acaggggcat 7740

cttgagcccc tgcggacggt gccgacaggt gcttctcgat ctgcatcctg ggatcaaagc 7800

catagtgaag gacagtgatg gacagccgac ggcagttggg attcgtgaat tgctgccctc 7860

tggttatgtg tgggagggct aagcacttcg tggccgagga gcaggactga cacgtgctac 7920

gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg 7980

acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca 8040

acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa 8100

ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt 8160

atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt 8220

ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa 8280

agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac 8340

tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg 8400

cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc 8460

gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat 8520

ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca 8580

ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc 8640

atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc 8700

aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg 8760

gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta 8820

ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg 8880

ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac 8940

acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag 9000

gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat 9060

ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat 9120

ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc 9180

gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt 9240

ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct 9300

agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt 9360

ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc 9420

gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac 9480

catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat 9540

cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg 9600

cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata 9660

gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta 9720

tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt 9780

gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag 9840

tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa 9900

gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc 9960

gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt 10020

taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc 10080

tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta 10140

ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa 10200

taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca 10260

tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac 10320

aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac ggatcgggag 10380

atctcccgat cccctatggt gcactctcag tacaatctgc tctgatgccg catagttaag 10440

ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta 10500

agctacaaca aggcaaggct tgaccgacaa ttgcatgaag aatctgctta gggttaggcg 10560

ttttgcgctg cttcgcgatg tacgggccag atatacgcgt t 10601

<210> 11

<211> 7236

<212> DNA

<213>artificial sequence ()

<400> 11

gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60

ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120

cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180

ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240

gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300

tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360

cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420

attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480

atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540

atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600

tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660

actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720

aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780

gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840

ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900

accatgggac ctaagaaaaa gaggaaggtg gcggccgctg actacaagga tgacgacgat 960

aaatctagaa tgggtcccga catcgtgatg acccagagcc ccagcagcct gagcgccagc 1020

gtgggcgacc gcgtgaccat cacctgccgc agcagcaccg gcgccgtgac caccagcaac 1080

tacgccagct gggtgcagga gaagcccggc aagctgttca agggcctgat cggcggcacc 1140

aacaaccgcg cccccggcgt gcccagccgc ttcagcggca gcctgatcgg cgacaaggcc 1200

accctgacca tcagcagcct gcagcccgag gacttcgcca cctacttctg cgccctgtgg 1260

tacagcaacc actgggtgtt cggccagggc accaaggtgg agctgaagcg cggcggcggc 1320

ggcagcggcg gcggcggcag cggcggcggc ggcagcagcg gcggcggcag cgaggtgaag 1380

ctgctggaga gcggcggcgg cctggtgcag cccggcggca gcctgaagct gagctgcgcc 1440

gtgagcggct tcagcctgac cgactacggc gtgaactggg tgcgccaggc ccccggccgc 1500

ggcctggagt ggatcggcgt gatctggggc gacggcatca ccgactacaa cagcgccctg 1560

aaggaccgct tcatcatcag caaggacaac ggcaagaaca ccgtgtacct gcagatgagc 1620

aaggtgcgca gcgacgacac cgccctgtac tactgcgtga ccggcctgtt cgactactgg 1680

ggccagggca ccctggtgac cgtgagcagc tacccatacg atgttccaga ttacgctggt 1740

ggaggcggag gttctggggg aggaggtagt ggcggtggtg gttcaggagg cggcggatcc 1800

agcggcagcg agactcccgg gacctcagag tccgccacac ccgaaagtat gagctcagag 1860

actggcccag tggctgtgga ccccacattg agacggcgga tcgagcccca tgagtttgag 1920

gtattcttcg atccgagaga gctccgcaag gagacctgcc tgctttacga aattaattgg 1980

gggggccggc actccatttg gcgacataca tcacagaaca ctaacaagca cgtcgaagtc 2040

aacttcatcg agaagttcac gacagaaaga tatttctgtc cgaacacaag gtgcagcatt 2100

acctggtttc tcagctggag cccatgcggc gaatgtagta gggccatcac tgaattcctg 2160

tcaaggtatc cccacgtcac tctgtttatt tacatcgcaa ggctgtacca ccacgctgac 2220

ccccgcaatc gacaaggcct gcgggatttg atctcttcag gtgtgactat ccaaattatg 2280

actgagcagg agtcaggata ctgctggaga aactttgtga attatagccc gagtaatgaa 2340

gcccactggc ctaggtatcc ccatctgtgg gtacgactgt acgttcttga actgtactgc 2400

atcatactgg gcctgcctcc ttgtctcaac attctgagaa ggaagcagcc acagctgaca 2460

ttctttacca tcgctcttca gtcttgtcat taccagcgac tgcccccaca cattctctgg 2520

gccaccgggt tgaaaggcgg aggtggaagc actaatctgt cagatattat tgaaaaggag 2580

accggaaagc aactggttat ccaggaatcc atcctcatgc tcccagagga ggtggaagaa 2640

gtcattggga acaagccgga aagcgatata ctcgtgcaca ccgcctacga cgagagcacc 2700

gacgagaatg tcatgcttct gactagcgac gcccctgaat acaagccttg ggctctggtc 2760

atacaggata gcaacggtga gaacaagatt aagatgctcg gaggaggagg aagcggagga 2820

ggaggtagcg gaggaggtgg aagccggacc gaagagtaca agcttatcct gaacggtaaa 2880

accctgaaag gtgaaaccac caccgaagct gttgacgctg ctaccgcgga aaaagttttc 2940

aaacagtacg ctaacgacaa cggtgttgac ggtgaatgga cctacgacga cgctaccaaa 3000

accttcacgg taaccgaagg tggtggtagc ggtggtggtg gtagtcccaa gaagaagagg 3060

aaagtctcga gcggtggagc tgcaggaggg cccttcgaag gtaagcctat ccctaaccct 3120

ctcctcggtc tcgattctac gcgtaccggt catcatcacc atcaccattg agtttaaacc 3180

cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc 3240

gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa 3300

attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac 3360

agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg 3420

gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc 3480

ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc 3540

gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt 3600

ccccgtcaag ctctaaatcg gggcatccct ttagggttcc gatttagtgc tttacggcac 3660

ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag 3720

acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa 3780

actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgggg 3840

atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc 3900

tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccaggc aggcagaagt 3960

atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca 4020

gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta 4080

actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga 4140

ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag 4200

tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata 4260

tccattttcg gatctgatca gcacgtgttg acaattaatc atcggcatag tatatcggca 4320

tagtataata cgacaaggtg aggaactaaa ccatggccaa gcctttgtct caagaagaat 4380

ccaccctcat tgaaagagca acggctacaa tcaacagcat ccccatctct gaagactaca 4440

gcgtcgccag cgcagctctc tctagcgacg gccgcatctt cactggtgtc aatgtatatc 4500

attttactgg gggaccttgt gcagaactcg tggtgctggg cactgctgct gctgcggcag 4560

ctggcaacct gacttgtatc gtcgcgatcg gaaatgagaa caggggcatc ttgagcccct 4620

gcggacggtg tcgacaggtg cttctcgatc tgcatcctgg gatcaaagcg atagtgaagg 4680

acagtgatgg acagccgacg gcagttggga ttcgtgaatt gctgccctct ggttatgtgt 4740

gggagggcta agcacttcgt ggccgaggag caggactgac acgtgctacg agatttcgat 4800

tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg 4860

atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt 4920

gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt 4980

ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt 5040

ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga 5100

aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc 5160

tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc 5220

cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc 5280

ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt 5340

cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca 5400

ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa 5460

aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat 5520

cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc 5580

cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc 5640

gcctttctcc cttcgggaag cgtggcgctt tctcaatgct cacgctgtag gtatctcagt 5700

tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac 5760

cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg 5820

ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca 5880

gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc 5940

gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa 6000

accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa 6060

ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac 6120

tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta 6180

aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt 6240

taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata 6300

gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc 6360

agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac 6420

cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag 6480

tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac 6540

gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc 6600

agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg 6660

gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc 6720

atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct 6780

gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc 6840

tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc 6900

atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc 6960

agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc 7020

gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca 7080

cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt 7140

tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt 7200

ccgcgcacat ttccccgaaa agtgccacct gacgtc 7236

<210> 12

<211> 7098

<212> DNA

<213>artificial sequence ()

<400> 12

gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60

ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120

cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180

ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240

gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300

tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360

cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420

attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480

atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540

atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600

tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660

actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720

aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780

gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840

ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900

accatgggac ctaagaaaaa gaggaaggtg gcggccgctg actacaagga tgacgacgat 960

aaatctagaa tgggtcccga catcgtgatg acccagagcc ccagcagcct gagcgccagc 1020

gtgggcgacc gcgtgaccat cacctgccgc agcagcaccg gcgccgtgac caccagcaac 1080

tacgccagct gggtgcagga gaagcccggc aagctgttca agggcctgat cggcggcacc 1140

aacaaccgcg cccccggcgt gcccagccgc ttcagcggca gcctgatcgg cgacaaggcc 1200

accctgacca tcagcagcct gcagcccgag gacttcgcca cctacttctg cgccctgtgg 1260

tacagcaacc actgggtgtt cggccagggc accaaggtgg agctgaagcg cggcggcggc 1320

ggcagcggcg gcggcggcag cggcggcggc ggcagcagcg gcggcggcag cgaggtgaag 1380

ctgctggaga gcggcggcgg cctggtgcag cccggcggca gcctgaagct gagctgcgcc 1440

gtgagcggct tcagcctgac cgactacggc gtgaactggg tgcgccaggc ccccggccgc 1500

ggcctggagt ggatcggcgt gatctggggc gacggcatca ccgactacaa cagcgccctg 1560

aaggaccgct tcatcatcag caaggacaac ggcaagaaca ccgtgtacct gcagatgagc 1620

aaggtgcgca gcgacgacac cgccctgtac tactgcgtga ccggcctgtt cgactactgg 1680

ggccagggca ccctggtgac cgtgagcagc tacccatacg atgttccaga ttacgctggt 1740

ggaggcggag gttctggggg aggaggtagt ggcggtggtg gttcaggagg cggcggatcc 1800

ggaggtagcg gcagcgagac tcccgggacc tcagagtccg ccacacccga aagtatggac 1860

agcctgctga tgaacaggag ggagttcctg taccagttca agaacgtcag atgggccaag 1920

ggcaggaggg agacctacct ctgctacgtg gtgaagagaa gggacagcgc cacctccttc 1980

tccctggact tcggatacct gaggaacaag aacggctgcc acgtggagct gctgttcctg 2040

aggtatatca gcgactggga cctggacccc ggcagatgtt acagggtgac ctggttcatc 2100

tcctggagcc cctgctacga ctgcgctagg cacgtggccg acttcctgag gggcaaccct 2160

aacctgagcc tgaggatctt caccgccagg ctgtacttct gcgaggacag gaaggccgaa 2220

cccgagggcc tgaggagact gcacagagcc ggagtgcaga tcgccatcat gaccttcaag 2280

gactattttt actgctggaa caccttcgtg gagaaccacg gcaggacctt caaagcctgg 2340

gagggcctgc acgagaacag cgtgaggctg tccagacagc tgaggcgtat tttactgggc 2400

ggaggtggaa gcactaatct gtcagatatt attgaaaagg agaccggaaa gcaactggtt 2460

atccaggaat ccatcctcat gctcccagag gaggtggaag aagtcattgg gaacaagccg 2520

gaaagcgata tactcgtgca caccgcctac gacgagagca ccgacgagaa tgtcatgctt 2580

ctgactagcg acgcccctga atacaagcct tgggctctgg tcatacagga tagcaacggt 2640

gagaacaaga ttaagatgct cggaggagga ggaagcggag gaggaggtag cggaggaggt 2700

ggaagccgga ccgaagagta caagcttatc ctgaacggta aaaccctgaa aggtgaaacc 2760

accaccgaag ctgttgacgc tgctaccgcg gaaaaagttt tcaaacagta cgctaacgac 2820

aacggtgttg acggtgaatg gacctacgac gacgctacca aaaccttcac ggtaaccgaa 2880

ggtggtggta gcggtggtgg tggtagtccc aagaagaaga ggaaagtctc gagcggtgga 2940

gctgcaggag ggcccttcga aggtaagcct atccctaacc ctctcctcgg tctcgattct 3000

acgcgtaccg gtcatcatca ccatcaccat tgagtttaaa cccgctgatc agcctcgact 3060

gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg 3120

gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg 3180

agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg 3240

gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga 3300

accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg 3360

ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct 3420

ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat 3480

cggggcatcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt 3540

gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg 3600

acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac 3660

cctatctcgg tctattcttt tgatttataa gggattttgg ggatttcggc ctattggtta 3720

aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt 3780

tagggtgtgg aaagtcccca ggctccccag gcaggcagaa gtatgcaaag catgcatctc 3840

aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa 3900

agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc 3960

ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat 4020

gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt 4080

ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat 4140

cagcacgtgt tgacaattaa tcatcggcat agtatatcgg catagtataa tacgacaagg 4200

tgaggaacta aaccatggcc aagcctttgt ctcaagaaga atccaccctc attgaaagag 4260

caacggctac aatcaacagc atccccatct ctgaagacta cagcgtcgcc agcgcagctc 4320

tctctagcga cggccgcatc ttcactggtg tcaatgtata tcattttact gggggacctt 4380

gtgcagaact cgtggtgctg ggcactgctg ctgctgcggc agctggcaac ctgacttgta 4440

tcgtcgcgat cggaaatgag aacaggggca tcttgagccc ctgcggacgg tgtcgacagg 4500

tgcttctcga tctgcatcct gggatcaaag cgatagtgaa ggacagtgat ggacagccga 4560

cggcagttgg gattcgtgaa ttgctgccct ctggttatgt gtgggagggc taagcacttc 4620

gtggccgagg agcaggactg acacgtgcta cgagatttcg attccaccgc cgccttctat 4680

gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg 4740

gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac 4800

aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt 4860

tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc 4920

tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca 4980

attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg 5040

agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg 5100

tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc 5160

tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta 5220

tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag 5280

aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg 5340

tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg 5400

tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg 5460

cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga 5520

agcgtggcgc tttctcaatg ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc 5580

tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt 5640

aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact 5700

ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg 5760

cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt 5820

accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt 5880

ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct 5940

ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg 6000

gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt 6060

aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt 6120

gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc 6180

gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg 6240

cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc 6300

gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg 6360

gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca 6420

ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga 6480

tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct 6540

ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg 6600

cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca 6660

accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata 6720

cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct 6780

tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact 6840

cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa 6900

acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc 6960

atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga 7020

tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga 7080

aaagtgccac ctgacgtc 7098

<210> 13

<211> 24

<212> DNA

<213>artificial sequence ()

<400> 13

accgcttgac caatagcctt gaca 24

<210> 14

<211> 24

<212> DNA

<213>artificial sequence ()

<400> 14

aaactgtcaa ggctattggt caag 24

<210> 15

<211> 24

<212> DNA

<213>artificial sequence ()

<400> 15

accggctatt ggtcaaggca aggc 24

<210> 16

<211> 24

<212> DNA

<213>artificial sequence ()

<400> 16

aaacgccttg ccttgaccaa tagc 24

<210> 17

<211> 24

<212> DNA

<213>artificial sequence ()

<400> 17

accgccctgg ctaaactcca ccca 24

<210> 18

<211> 24

<212> DNA

<213>artificial sequence ()

<400> 18

aaactgggtg gagtttagcc aggg 24

<210> 19

<211> 24

<212> DNA

<213>artificial sequence ()

<400> 19

accgatattt gcattgagat agtg 24

<210> 20

<211> 24

<212> DNA

<213>artificial sequence ()

<400> 20

aaaccactat ctcaatgcaa atat 24

<210> 21

<211> 24

<212> DNA

<213>artificial sequence ()

<400> 21

accgccttcc ccacactatc tcaa 24

<210> 22

<211> 24

<212> DNA

<213>artificial sequence ()

<400> 22

aaacttgaga tagtgtgggg aagg 24

<210> 23

<211> 24

<212> DNA

<213>artificial sequence ()

<400> 23

accggtgggg aaggggcccc caag 24

<210> 24

<211> 24

<212> DNA

<213>artificial sequence ()

<400> 24

aaaccttggg ggccccttcc ccac 24

<210> 25

<211> 23

<212> DNA

<213>artificial sequence ()

<400> 25

cgattagtga acggatctcg acg 23

<210> 26

<211> 24

<212> DNA

<213>artificial sequence ()

<400> 26

taggcttgac caatagcctt gaca 24

<210> 27

<211> 24

<212> DNA

<213>artificial sequence ()

<400> 27

aaactgtcaa ggctattggt caag 24

<210> 28

<211> 24

<212> DNA

<213>artificial sequence ()

<400> 28

tagggctatt ggtcaaggca aggc 24

<210> 29

<211> 24

<212> DNA

<213>artificial sequence ()

<400> 29

aaacgccttg ccttgaccaa tagc 24

<210> 30

<211> 24

<212> DNA

<213>artificial sequence ()

<400> 30

taggccctgg ctaaactcca ccca 24

<210> 31

<211> 24

<212> DNA

<213>artificial sequence ()

<400> 31

aaactgggtg gagtttagcc aggg 24

<210> 32

<211> 24

<212> DNA

<213>artificial sequence ()

<400> 32

taggatattt gcattgagat agtg 24

<210> 33

<211> 24

<212> DNA

<213>artificial sequence ()

<400> 33

aaaccactat ctcaatgcaa atat 24

<210> 34

<211> 24

<212> DNA

<213>artificial sequence ()

<400> 34

taggccttcc ccacactatc tcaa 24

<210> 35

<211> 24

<212> DNA

<213>artificial sequence ()

<400> 35

aaacttgaga tagtgtgggg aagg 24

<210> 36

<211> 24

<212> DNA

<213>artificial sequence ()

<400> 36

tagggtgggg aaggggcccc caag 24

<210> 37

<211> 24

<212> DNA

<213>artificial sequence ()

<400> 37

aaaccttggg ggccccttcc ccac 24

<210> 38

<211> 24

<212> DNA

<213>artificial sequence ()

<400> 38

tctcgcgcgt ttcggtgatg acgg 24

<210> 39

<211> 31

<212> DNA

<213>artificial sequence ()

<400> 39

aaaaaaagca ccgactcggt gccacttttt c 31

Claims (8)

1. the kit that a kind of genome base editor increases fetal hemoglobin level in human red blood cells, which is characterized in that packet Include base editing system and the sgRNA for gamma globulin gene promoter site.

2. kit as described in claim 1, which is characterized in that the base editing system is BE3, xBE3, ABE, BE- One of PLUS or BE-PLUS (AID).

3. kit as described in claim 1, which is characterized in that the base editing system is plasmid, mRNA or albumen Form.

4. kit as described in claim 1, which is characterized in that the sgRNA is plasmid form or rna form.

5. kit as described in claim 1, which is characterized in that the sgRNA for gamma globulin gene promoter Sequence be SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO.6。

6. a kind of method that base editor changes destination gene expression level, which is characterized in that in candidate stem cell, utilize needle It guides base editing system to carry out base editor to mutational site the sgRNA in gamma globulin gene promoter site, collects and turn Cell after dye.

7. the method that base editor as claimed in claim 6 changes destination gene expression level, which is characterized in that the needle To the sgRNA in gamma globulin gene promoter site by designing according to the site, and construct U6 promoter and/or T7 starting The expression vector of son obtains.

8. a kind of method that base editor treats hemoglobinopathy, comprising: in the HSC of the patient containing beta-globin gene mutation In, guide base editing system to carry out base editor to the site using the sgRNA for gamma globulin gene promoter region It repairs, the cell after collecting transfection, identifies mutation rate, the expression variation of gamma globulin is identified after HSC differentiation.