CN101440379B - Obtaining method and use of novel oncolytic adenovirus construct with selective tumor blockage STAT3 - Google Patents
Obtaining method and use of novel oncolytic adenovirus construct with selective tumor blockage STAT3 Download PDFInfo
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Abstract
The invention discloses a proposal for constructing novel oncocytic adenovirus of selectively closed tumor STAT3 obtained by manual reconstruction of human 5 type adenovirus (Ad5), and particular application of a recombined adenovirus constructor in tumor treatment, and belongs to the technical field of medical genetic engineering. By techniques of PCR enlarging fixed point deletion, enzyme cutting, connection, cloning, homologous recombination, transfection, single cloning purification of adenovirus and the like, the recombination adenovirus constructor is obtained. The technical characteristics comprise that 27 basic groups are deleted in an E1A conservative sequence 2(CR2) zone of Ad5 genome; 29477 to 29714nt in an E3 zone of ADP gene are deleted; and partial STAT cDNA segments are oppositely inserted in the deletion zones. The constructor is a novel oncocytic adenovirus vector with higher tumor selective copy capability. The constructor has unique practical values in biological treatment of tumor, and also provides a reasonable gene target point specificity treatment mode for the genetic treatment of tumor.
Description
One, technical field
The present invention relates to a kind of human 5 type adenovirus (Human adenovirus type 5, Ad5) constructing plan of recombinant chou, its technical characterictic is: the genomic 920-946nt of directed disappearance Ad5, be GAT CTTACC TGC CAC GAG GCTGGC TTT, this sequence encoding E1A protein 12 1 to 129 amino acids, on this basis, further lack Ad5 E3 district 29477-29714nt, introduce a ClaI restriction enzyme site in above-mentioned disappearance zone; In the ClaI restriction enzyme site, oppositely insert the exogenous STAT3 cDNA fragment of 770bp, be equivalent to the 960-190nt of STAT3 mRNA, thereby obtain the recombination adenovirus construction body Ad5/ Δ E1/ Δ ADP/ASSTAT3 that tumour is had therapeutic action.Summary of the invention belongs to the cma gene field of engineering technology.
Two, background technology
Malignant tumour is becoming the primary disease of harm humans health.Show according to latest domestic epidemiology survey data: China has cancer patient more than 300 ten thousand people now, the patient who dies from malignant tumour every year is about 1,300,000 people, the malignant tumour case that 1,600,000 to 2,000,000 New Developments are arranged every year, and, cause grave danger for China people's life and health with 3% speed increase.Conventional at present oncotherapy means all can't fundamentally be removed malignant tumour, and the treatment of most kinds of tumor still is faced with stern challenge, and the thorough treatment of malignant tumour needs the innovation of tumor therapeuticing method.
In tumor invasion under the new historical conditions that molecular mechanism is understood day by day, tumour molecular medicine theory and methodological breakthrough have caused the trial of a series of development novel tumors treatment patterns, a large amount of molecular target specificity anticancer compounds enter clinic trial, the weight break point of a key that indicated breaking through to of oncotherapy, and phasic results have been obtained, tentatively highlight the elementary contour that following tumour problem solves, its the most one of the solution of core to from the complicated gene regulatory network of tumour, decomposite the most key molecular target exactly, implement molecular targeted treatment.By at the bottom of calendar year 2001, obtain drugs approved by FDA and enter the molecular therapy kind in clinical experiment stage above 3,000 kinds, comprise cell conduction block agent, angiogenesis inhibitor, tumor vaccine and monoclonal antibody, for the final solution of tumour problem has brought new hope, we can say the development that effectively being controlled at of following human tumor fundamentally relies on molecular therapy and perfect.
The main means of molecular therapy mainly are divided three classes: monoclonal antibody, micromolecular compound and gene therapy, wherein, gene therapy is as having most one of part of vitality in the medical field development in recent years, be widely used in the clinical trial treatment of kinds of tumors, demonstrated good prospects for application.From 1989 to the end of the year 2000, U.S.'s center for biologic evaluation and research (Center for Biologics Evaluations and Research, CBER) Pi Zhun clinical gene therapy test subject is above 350, it is therapy of tumor that 70% project is wherein arranged approximately, part has entered the III clinical trial phase, obtain better curative effect, under the promotion of huge medical requirement, the gene therapy preparation might become the clinical treatment medicine at official listing in recent years, become an indispensable integral part of the existing treatment system of tumour.
One of carrier that reorganization Ad5 carrier is most widely used as therapy of tumor, its Clinical feasibility and security have obtained generally acknowledged, and adenoviral gene treatment carrier has following outstanding biology advantage and clinical application advantage: pattern of infection is wide; Unconformability is gone into host genome, no mutagenesis, carcinogenic danger; Clinical application safety, side effect is slight; Clinical application is convenient; Adenovirus continuous expression time in cell paste is short, especially is fit to oncotherapy; The virus of clinical grade quantity, quality is easy to produce, purifying.
Clinical tumor most carriers on probation are the replication defective sexual gland virus that has lacked E1 and E3 district at present, after entering tumor focus, the killing effect of tumour is depended on the input of external high density treatment virus, the killing effect of the focus far away apart from injection point a little less than.For this reason, in recent years, occurred from the new development trend of replication defective sexual gland virus carrier to condition replicability adenovirus carrier (conditionally replicative adenovirus) conversion, so-called conditionality is duplicated adenovirus carrier and is meant that carrier can optionally duplicate propagation in tumour cell, with the place of tumour cell as production virus, finally reach cracking and kill the therapeutic purpose of tumour cell, after the tumour cell cracking discharges the virus of high density, can further infect the focus tumour cell far away apart from injection point, form the infection ripple of a new round, going round and beginning again forms a kind of positive regeeration, reaches maximum treatment effect.Examples of such carriers another very the ideal advantage be that they can not be finished in normal cell and duplicate, therefore normal cell is not had any lethal effect.In the 21st U.S.'s tumor research annual meeting, a research report from Houston Anderson tumor center shows: behind the local injection, the distribution of replication defective sexual gland virus in human malignant glioma focus be limitation, influenced the performance of curative effect to a certain extent, form a sharp contrast therewith, the adenovirus ONYX-015 that can duplicate at specifically inside tumor cell is by the propagation at tumor by local, its distribution spreads all over full knurl kitchen range, obtains significant curative effect.ONYX-015 be U.S. ONYX Pharma Biotech develop in recent years can be in tumour cell the adenovirus of copy choice, its structure characteristics are on the genome of wt-Ad5, the encoding sequence of excalation E1B 55 kDa, and add the translation termination signal, but keep the encoding sequence of 19kDa E1B.
Since entering clinical trial in 1996, obtained very challenging clinical efficacy as the ONYX-015 of condition replicability adenovirus carrier representative products.At present, Pfizer (pifzer) pharmaceutical companies has been bought from ONYX biotech company and has been used patent, and carried out clinical phase test in kinds of tumors, wherein, to recurrent, the treatment of intractable tumor of head and neck is just being carried out the clinical III phase and is being tested, and has obtained very satisfied curative effect, and the clinical I-II phase of other tumours such as transitivity colorectal carcinoma, lung cancer, carcinoma of the pancreas, oral leukoplakia (oral leukoplakia) and glioblastoma is tested well afoot.The patent application of domestic existing 4 examples and ONYX-015 similar principles, wherein patent application (a kind of defective virus and construction process thereof) 99124030.8 is positioned at the 2809-3329bp encoding sequence by disappearance E1B 55 kDa, and obtains the construct of E1B 55 kDa excalations in the method for disappearance position insertion stop code.Patent application (structure of disappearance apoptosis suppressor virus and the application in therapy of tumor) 96013494.7 is by excalation E1B 55 kDa encoding sequences, and at this disappearance position insertion marker gene LacZ, obtain E1B 55 kDa excalations and carry the virus formulation body of marker gene simultaneously, another characteristics of this construct are disappearances that the E3 district is arranged.Other two applications for research group's submission of applicant leader, the patent No. 01144628.5 " a kind of acquisition and purposes that lacks the recombination adenovirus construction body of E1A encoding sequence ", disappearance E1A 382-1630nt sequence, filter out the recombination adenovirus construction body that to express the E1A functional protein, the patent No. 01144629.3 " a kind of structure and purposes of uniting disappearance 19kDa, 55kDa E1B encoding sequence recombination adenovirus construction body " filters out the recombination adenovirus construction body that can not express the E1B functional protein.
Domestic and international existing condition replicability adenovirus carrier invention is compared with first-generation Ad5 gene therapy vector, no matter treating on the principle, still on the actual therapeutic effect, all have tangible improvement, and make a breakthrough, be called as s-generation Ad5 gene therapy vector.Yet, s-generation Ad5 gene therapy vector all has some common shortcomings:＜1〉though, possess the treatment advantage of tumour condition replicability based on the carrier of adenovirus Basic of Biology design, because all deficiencies in the design, the effectiveness of the adenovirus of obtaining duplicating in tumour cell, cracking tumour cell is weaker than wt-Ad5 far away, as single only be 0-14% with the anticancer efficient of ONYX-015.＜2 〉, the application of s-generation Ad5 gene therapy vector is still based on locally injected into tumor, in application, be subjected to great restriction, under most situations, tumour is a kind of systemic disease, needs the approach through intravenous administration, carries out systemic treatment, to control former and metastatic lesion, therefore, the selectivity of s-generation Ad5 gene therapy vector and must further strengthen the killing-efficiency of tumour cell is to adapt to the needs that improve route of administration.＜3 〉, s-generation Ad5 gene therapy vector all do not carry effective therapeutic goal gene, and the performance of treatment effect is restricted.＜4 〉, because the existing molecular target overwhelming majority lacks tumour-specific, though have relative tumor-selective with s-generation Ad5 gene therapy vector in conjunction with the strategy of exogenous promotor, therapeutic gene, but still can not avoid to normal histiocytic toxic action.＜5 〉, behind the existing Ad5 carrier target cell infection that carries therapeutic gene, the therapeutic gene that carries promptly obtains the ability expressed, can not produce with avoiding and align normal histiocytic toxic action, especially the bigger foreign gene of toxicity or lethality.＜6 〉, the activity of the exogenous promotor of s-generation Ad5 gene therapy vector bonded in cell is unstable or lower.＜7〉even existing Ad5 gene therapy vector has powerful oncolytic effect, but too early cracking causes the therapeutic gene that carries can not effective expression to tumour cell, adds that immunity makes that to the removing of virus viral result of treatment is lower the upper body in.
On the other hand, many tumour molecule medicine targets have been determined in recent years, at these molecule medicine target design monoclonal antibodies, micromolecular compound, become the oncotherapy main development tendency, wherein part has become prescription drugs, a large amount of molecular therapy means are still arranged just in clinic trial evaluation stage, obtained PRELIMINARY RESULTS preferably.However, because these new molecular targets and molecule thereof check means, the overwhelming majority lacks tumour-specific, consequent dose-limiting toxicity, remain the key issue that these therapies generally face and need to be resolved hurrily at present, greatly limited the solution of tumour problem.
Three, summary of the invention
Based on above technical background and great medical requirement, the present invention will disclose a kind of constructing plan of new third generation AD5 gene therapy vector, the construct that obtains by this scheme has that tumour-specific duplicates, height tumour-specific mass expressing external insertion gene, powerful remarkable advantages such as tumour-specific bystander effect, and possess the height therapeutic index, be suitable for intravenous administration, can solve crucial weak point in present oncotherapy technology and the practice.
Theoretical basis of the present invention is based on the deep understanding of existing research to the Ad5 viral biology.After AD5 enters cell in 1 hour, Ad5 will give expression to the E1A functional protein, its biological effect mainly comprises two aspects, at first, E1A protein binding retinoblastoma mutator gene (Rb) albumen, host cell endogenous transcription factor E2F is discharged from the mixture of Rb-E2F, meanwhile, E1A albumen will activate Ad5 and give expression to the E1B functional protein, drive kinase whose inhibition with this deactivation host cell endogenous transcription factor P53 cell cycle, at E1A, under the common driving of E1B and E2F, host cell enters the DNA synthesis phase, for virus genomic self-replacation provides precondition; Secondly, Ad5 relies on the early gene (E1B that E1A albumen activates Ad5, E2, E3, E4) and the expression of late gene (L1-6), for the packing of virus genomic self-replacation, virion, the immune clearance of escaping host cell, lysing cell, releasing virus particle, complete life cycle such as infection provides the permission environment again.This shows that it is synthetic that Ad5 relies on other proteic sequential property of E1A protein promoter, finish the life cycle of virus, final cracking host cell.
The encoding sequence of functional protein E1A is positioned at Ad5 genome 560-1112nt, and 1229-1545nt comprises three important sequences, is respectively 677-799nt, and this sequence is E1A conserved sequence 1 (CR1), is used in conjunction with transcribing cofactor P
300, 917-979nt is E1A conserved sequence 2 (CR2), is used in conjunction with retinoblastoma mutator gene (Rb), 1007-1115nt is conserved sequence 3 (CR3), is the genetic transcription active region.Because all there is the defective of Rb regulation and control pathway in nearly all tumour, if the disappearance transformation different in addition to the CR2 district of E1A917-979nt, the effect that keeps the E1A transcripting activating characteristic when reaching the proteic RB binding characteristic of deactivation E1A as far as possible, the adenovirus recombinant chou that obtains thus might effectively duplicate in tumour cell, finally kill tumour cell, on the other hand, the Rb regulation and control pathway that normal cell has, can effectively check the life cycle of its sequential property of adenovirus recombinant chou dependence E1A protein promoter, finally only form abortive infection.This is theoretical infer by recent two independently the work of study group confirmed, (Oncogene such as Fueyo, 2000,19:2-12) made up the adenovirus recombinant chou that lacks E1A923-946nt, observing this adenovirus recombinant chou optionally duplicates in tumour cell, its duplication characteristic is better than wt-Ad5, and normal cell is not had any toxic action, and list can significantly suppress tumor growth with this adenovirus recombinant chou; Heise etc. (Nature Medicine, 2000,10:1134-1139) confirm that more in depth this construct has high selectivity to tumour cell, can significantly suppress tumor growth and suppress metastases with this adenovirus recombinant chou from the vein list.
Although the previous work of Fueyo and Heise etc. provides crucial theoretical clue for development third generation Ad5 gene therapy vector, their work still has tangible weak point, they develop the Ad5 gene therapy vector that and do not comprise the exogenous therapeutic gene of any insertion, have limited the performance of anti-tumour effect.For this reason, the present invention makes up a kind of novel Ad5 recombinant chou on this basis, its technical characterictic is: the genomic 920-946nt sequence of directed disappearance Ad5, and further lack Ad5 E3 district 29477-29714nt, introduce a ClaI restriction enzyme site in this disappearance zone; Oppositely insert the exogenous STAT3 cDNA fragment of 770bp in the ClaI restriction enzyme site, this fragment is equivalent to the 960-190nt of STAT3 mRNA, thereby obtains the recombination adenovirus construction body Ad5/ Δ E1/ Δ ADP/ASSTAT3 that tumour is had therapeutic action.
STAT3 gene full name is signal transduction and transcriptional activators 3 (signal transducer and activator oftranscription 3), unconventionality expression or be the continuous activation state in most of tumour cell, its function is to promote the tumour cell increment to suppress its apoptosis, promote tumor-blood-vessel growth, make tumour cell obtain the ability of immune evasion, and apart from normal cell and do not rely on these functions.Therefore, be chosen as molecule medicine target in the present invention.
Compare with existing recombination adenovirus construction body, the present invention has reached following effect:
1, the proteic 920-946nt sequence of this recombination adenovirus construction body disappearance E1A is theoretical and in fact reached the effect that keeps the E1A transcripting activating characteristic in the proteic RB binding characteristic of deactivation E1A as far as possible.What is more important, we oppositely insert the exogenous STAT3 cDNA fragment of 770bp in the E3 ADP district of Δ 920-946Ad5 carrier, this fragment is equivalent to the 960-190nt of STAT3 mRNA, make the endogenous promotor of expression dependence Ad5 self the E3 district transcription unit of antisense STAT 3 cDNA, this promotor has the high reactivity of similar CMV promotor, the opening of the promotor virus genom DNA that places one's entire reliance upon duplicates intracellular, virus is not as duplicating, and exogenous antisense STAT 3 cDNA will not express; And because the disappearance of ADP gene, the ability of recombinant adenovirus lysing cell reduces relatively, but the amplification amount of intracellular virus significantly increases, thereby is just become the source mill of therapeutic gene by the cell of virus infection.Because this brand-new design, make this new construct can reach the therapeutic purpose of selectively killing tumour cell by triple mechanism, that is: construct only duplicates in tumour cell and the time of lysing cell relatively lags behind, and exogenous antisense STAT 3 cDNA copy number is increased greatly; The antisense STAT 3 cDNA that construct carries only expresses in tumour cell, this expression virus genom DNA that places one's entire reliance upon duplicates intracellular, therefore the STAT3 genetic expression of energy specificity sealing tumour cell reaches the purpose of killing tumour cell, has avoided Normocellular attack; Construct for duplicating the host, can produce high concentration virus at tumor by local with tumour cell, forms potent bystander effect.The immune modulator telotism of construct has been avoided the removing of body to recombinant adenovirus to a certain extent.In addition, the present invention also will provide a kind of new Design Mode for follow-up other molecular therapies.
2, advance year over a large amount of studies show that, STAT3 unconventionality expression or be the continuous activation state in most of tumour cell, and tumour cell more depends on this activation and survive, and this activatory is suppressed also more responsive.Thereby for of short duration partially enclosed of STAT3 being provided comparatively ideal treatment window.
3, tumor cell line and normal control cells in vitro experimental result are shown, this recombination adenovirus construction body does not have obvious influence to normal cell STAT3 expression of gene, the obviously influence of the active nothing of pair cell, on the contrary, but STAT3 expression of gene in its specificity sealing tumour cell has the effect of killing tumour cell significantly.
4, the body inner model to animal model for tumour studies confirm that, by locally injected into tumor or intravenous route of administration, all animal models for tumour equal tool significant therapeutic action of this recombination adenovirus construction body to being tried, and can effectively suppress metastases, and it is xicity related that animal is not had obvious treatment.
Four, description of drawings
Accompanying drawing 1:Ad5 E3 plot structure figure, 7 kinds of albumen of this regional code, the zone that lack is 29477-29714bp, is E3 ADP gene, oppositely insert the exogenous STAT3 cDNA fragment of 770bp in this zone, this fragment is equivalent to the 960-190nt of STAT3mRNA.
Accompanying drawing 2: papova construct Ad5/ Δ E1/ Δ ADP/ASSTAT3 structural representation.
Appendix 1: recombination adenovirus construction body Ad5/ Δ E1/ Δ ADP/ASSTAT3 sequence and structure explanation.
Five, specific embodiment
In the following example, except that indicating especially, used enzyme and PCR primer are all available from U.S. Gibco company in the techniqueflow.
The structure of example 1, pXC1 series mutation body (Δ 920-946pXC1)
PXC1 purchase in Microbix Biosystem Inc. (Toronato, Ontario, Canada, catalog number (Cat.No.): PD-01-03), this plasmid comprises human 5 type adenovirus (Ad5) 21-5790nt sequences.
Adopt 3 PCR methods disappearance 920-946nt, the obtaining of fragment 1: primer 1=5 '-CG GGA TCC GGG CCCCCATTT CC-3 ', be equivalent to 9883-9902nt, underscore partly is the BamHI restriction enzyme site; Primer 2=5 '-GTC
ACT GGG TGG ATC GAT CAC CTCCGG TAC-3 ' is equivalent to 922-905nt, and underscore partly is and primer 3 complementary portions;
With pXC1 is template, the performing PCR reaction, and the reaction system cumulative volume is that 100 μ l comprise:
Contain MgCl
210 * PCR damping fluid, 10 μ l
2mM?dNTP 10μl
10 μ M primers, 11 μ l
10 μ M primer 2s, 1 μ l
pXC1?10ng/μl 1μl
Pfu high-fidelity Taq enzyme 2.5u
Add water to 100 μ l;
Reaction conditions is: 95 ℃ 30 seconds; 95 ℃ 45 seconds, 60 ℃ 1 minute, 72 ℃ totally 28 circulations in 2 minutes; 72 ℃ were extended 10 minutes.The long 940bp of PCR product forms fragment 1, and behind the conventional electrophoretic separation purifying, detectable level is used for follow-up PCR reaction.
Obtaining of fragment 2: primer 3=5 '-
GAG GTG ATC GAT CCA CCC AGT GACGAC GAG-3 ' is equivalent to 911-947nt, and underscore partly is and the primer 2 complementary portion; Primer 4=5 '-TGC
TCT AGACAC AGGTGA TGT CG-3 ' is equivalent to 1344-1325nt, and underscore partly is the XbaI enzyme cutting site;
With pXC1 is template, the performing PCR reaction, and reaction conditions is the same, and the long 400bp of product forms fragment 2, and behind the conventional electrophoretic separation purifying, detectable level is used for follow-up PCR reaction.
Obtaining of fragment 3: 50ng/2 μ l fragment 1 is mixed with 25ng/1 μ l fragment 2, and as the reaction of template performing PCR, upstream primer is a primer 1, downstream primer is a primer 4, reaction conditions is the same, and product is about 1400bp, forms fragment 3, with QIAquick 8 PCR product purification test kit (QIAGEN, German Cat:28142) behind the purifying, uses BamHI, the XbaI double digestion spends the night, and enzyme is cut product recovery endonuclease bamhi after 1% agarose gel electrophoresis separates and is used for the clone.
With pXC1 BamHI, the XbaI double digestion spends the night, and enzyme is cut product and separated back generation 2 bands at 1% agarose gel electrophoresis, is about 1400bp and 8500bp, reclaims the 8500bp endonuclease bamhi and is used for the clone.
Get 8500bp pXC1 endonuclease bamhi and the 90ng fragment 3 of 40ng, use DNAT
4Ligase enzyme is done ligation, get 1.5 μ l and transform 100 μ l DH5 α competence bacteriums, shop ware incubated overnight, second day single colony clone of picking, extract the plasmid of amplification in it, the dna sequencing evaluation and screening obtains lacking pXC1 plasmid encoding mutant body-Δ 920-946pXC1 of 121-129AA 920-946nt, is used for the clone of recombinant adenovirus.
The structure of example 2, Δ 920-946Ad5 recombinant adenovirus
PBHGE3 purchase in Microbix Biosystem Inc. (Toronato, Ontario, Canada, catalog number (Cat.No.): PD-01-12), this plasmid comprises the full gene group sequence except that ADd5 packaging signal (194-358nt).
When pBHGE3 obtained from Microbix Biosystem Inc., total amount was 10 μ g, and first electricity changes the competence bacterium over to, and the picking positive colony extracts plasmid, the plasmid CsCl that obtains
2-EB ultracentrifugation purifying.
Homologous recombination method obtains Δ 920-946Ad5 recombination adenovirus construction body, and method is as follows:
In the 15cm culture dish, plant 7.5 * 10
5293 cells, nutrient solution are 10%FBS DMEM, and by second day, cell should be 1-1.5 * 10
6, about 70% cytogamy; Preceding 3-4 hour of transfection changes fresh medium into.
Preparation cotransfection DNA-calcium phosphate solution: with 2 * HBS (280mM NaCl, 43mMHEPES, 10mM KCl, the 10mM Na of 1600 μ l sterilization
2HPO
4.7H
2O, 2%dextrose, pH 7.05-7.1 5)/pBHGE3 and each 42 μ g/ of Δ 920-946pXC1 add distilled water to the 2840 μ l mixing of sterilization, slowly add the CaCl of 50 μ l2.5M
2, put upside down mixing, at room temperature allow DNA/CaCl
2Precipitate 45-60 minute, form slightly turbid precipitation.
Add the above-mentioned mixed solution of 500 μ l to 293 cells that contain 5ml 60mm culture dish, in 37 ℃, 5%CO2, hatched 4-6 hour, inhale and remove aforesaid liquid, wash once with PBS.
Handle 1-2 minute to promote transfection efficiency with the glycerine/DMEM that contains 15%, wash once, be changed to complete culture solution with PBS.
Prepare 1.8% LMP agarose, autoclaving is distributed into 5ml, melts in boiling water with preceding, and insulation is at 45 ℃, and the time spent adds equivalent 4%FBS DMEM, and the shop is gone in the culture dish at once.
Nutrient solution is removed in suction, adds the 5ml aforesaid liquid.Every 4-5 days, add the 3ml aforesaid liquid.
14-21 days, plaque occurred, selected 6-12 plaque.Plaque is transferred in the EP pipe of the 1.5ml that contains serum-free DMEM substratum, hatched 24 hours at 37 ℃.
In 24 orifice plate culture dish, plant 1 * 10
5293 cells, nutrient solution are 10%FBS DMEM, and by second day, cell should be 2 * 10
5, about 70% cytogamy is inhaled and is removed liquid, gets 100 μ l from above-mentioned Incubating Solution and (is approximately 10
3Virus) add, rock liquid gently 3 times, at 37 ℃, 5%CO
2In hatched 90 minutes.
Add perfect medium to 1ml, cell is positioned over 37 ℃, hatched among the 5%CO2 5-10 days, occur until CPE completely, so-called CPE, i.e. cellulotoxic effect, cell show as and become round, floating, cell based on kernel.If after 10 days, CPE does not occur completely, tiring of then prompting virus is too low, need carry out second amplification of taking turns.
Culture plate is carried out the circulation of freezing/thawing of three-wheel, discharge virus, lysate is collected in the 15ml test tube, maximum velocity centrifugation 10 minutes is collected supernatant liquor, freezes in-80 ℃, and this liquid is called s-generation virus, is approximately 5 * 10
7/ ml virus.
Above virus is increased once more, at 75cm
2Plant 5 * 10 in the culture dish
6293 cells, nutrient solution are 10ml10%FBS DMEM, and by second day, cell should be 1 * 10
7, about 70% cytogamy;
Preceding 3-4 hour of transfection changes fresh medium into;
Get 1 ml s-generation virus storage liquid and add perfect medium, be used for transfection to 1ml; This MOI is about 5; Remove the liquid of 75cm2 culture dish, add above liquid, shake gently three times; In 37 ℃, 5%CO2, hatched 90 minutes; Add 9ml 2%FBS DMEM, in 37 ℃, 5%CO2, hatched 4-7 days, be used to extract the screening that viral DNA is used for positive-virus.
Because 293 cellular genome comprise complete e1a gene, pollute 293 cell DNAs easily during extraction positive-virus DNA, cause and identify failure, Δ 920-946 Ad5 is increased once in tumour cell Hela more for this reason, be used for identifying that step is as follows:
In 6 orifice plate culture dish, plant 1 * 10
5Hela cell, nutrient solution are 10%FBS DMEM, and by second day, cell should be 2 * 10
5, about 70% cytogamy is inhaled and is removed liquid, gets 100 μ l from above-mentioned filtered solution and (is approximately 10
3Virus) add, rock liquid gently 3 times, at 37 ℃, 5%CO
2In hatched 90 minutes.
Add perfect medium to 1ml, cell is positioned in 37 ℃, 5%CO2 hatched 5-10 days, occur until CPE completely, cell is scraped, be collected in 1.5ml EP pipe, the centrifugal supernatant of abandoning, add 300 μ l PBS solution, carry out the circulation of freezing/thawing of three-wheel, discharge virus, with lysate maximum velocity centrifugation 10 minutes, collect supernatant liquor, freeze in-80 ℃, with the Qiagen test kit mini DNA isolation kit of company, DNA is extracted in the explanation of reference reagent box.With the viral DNA is template, the performing PCR reaction, upstream primer=5 '-CG GGA TCC GGG CCC CCA TTT CC-3 ', downstream primer=5 '-TGC TCT AGA CAC AGG TGA TGT CG-3 ', the reaction system cumulative volume is that 100 μ l comprise: contain 10 * PCR damping fluid, 10 μ l/2mM dNTP, 10 μ l/10 μ M upstream primers, the 1 μ l of MgCl2,10 μ M downstream primers, 1 μ l/ viral DNA 10ng/pfu high-fidelity Taq enzyme 2.5u/ adds water to 100 μ l.Reaction conditions is: 95 ℃ 30 seconds/95 ℃ 45 seconds, 60 ℃ 1 minute, 72 ℃ 2 minutes totally 28 circulations extended 10 minutes for/72 ℃.The PCR product is 1400bp, and behind the conventional electrophoretic separation purifying, detectable level is used for dna sequencing, and sequencing primer is: 5 '-AGCCGGAGCAGAGAGCCTTG-3 ', choose correct clone and be used to make up Ad5/ Δ E1/ Δ ADP/ASSTAT3 recombinant adenovirus.
The structure of the subcloning vector pCDNA3.1-Δ ADP in example 3, Ad5 E3 district
Ad5 E3 district full name is an Ad5 early region 3, this zone under the driving of endogenous promotor, the 7 kinds of albumen of encoding, sequence, structure, function be referring to accompanying drawing 1:12.5k, 27858-28179nt, function is not bright; 6.7k 27547-28736nt and RID complex body suppress 1,2 expression at cell surface of TRAIL acceptor together; Gp19k, 28735-29215nt in conjunction with MHC I class antigen, suppresses it to the presenting of cell surface, and escapes the removing of CTL; ADP, 29419-29770nt, lysing cell, releasing virus; RID α, 29784-30057nt forms mixture with RID β, prevents the cracking of TNF, removes FAS antigen; RID β, 30062-30458nt and 14.7k, 30453-30837 suppresses the cracking of TNF.
The purpose of this experiment is to lack 29477-29714nt zone, E3 district, and inserts exogenous therapeutic gene in the E3 of recombinant adenovirus ADP zone.
Adopt PCR method disappearance Ad5 E3 district 29477-29714nt 3 times:
Obtaining of fragment 1: primer 1=5 '-ATACGCGCCCACCGAAAC-3 ' is equivalent to 27306-27323nt; Primer 2=5 '-
AATCTATGGATATCGAT AGGGTGGGTCGCTGTAGTT-3 ' is equivalent to 29477-29495nt, (underscore partly is and primer 3 complementary portions that ATCGAT is the ClaI restriction enzyme site);
With Ad5 DNA is template, the performing PCR reaction, and the reaction system cumulative volume is that 100 μ l comprise:
Contain MgCl
210 * PCR damping fluid, 10 μ l
2mM?dNTP 10μl
10 μ M primers, 11 μ l
10 μ M primer 2s, 1 μ l
Ad5?DNA?200ng/μl 1μl
Pfu high-fidelity Taq enzyme 2.5 u
Add water to 100 μ l;
Reaction conditions is:
94 ℃ 30 seconds;
94 ℃ 30 seconds, 46 ℃ 1 minute, 72 ℃ totally 30 circulations in 1 minute;
72 ℃ were extended 10 minutes.
The PCR product is 2207bp (fragment 1), and behind the conventional electrophoretic separation purifying, detectable level is used for follow-up PCR reaction.
Obtaining of fragment 2:
Primer 3=5 '-
CGACCCACCCTATCGATATCCATAGATTGGACGGACTG-3 ' (being equivalent to 29714-29734n) (underscore partly is and the primer 2 complementary portion that ATC GAT is a Cla I restriction enzyme site) primer 4=5 '-ATGTCTTTGAGGCTTGGAGG-3 ' (being equivalent to 30137-30118nt); The PCR reaction conditions is the same, and product is 422bp (fragment 2), and behind the conventional electrophoretic separation purifying, detectable level is used for follow-up PCR reaction.
Obtaining of fragment 3: with fragment 1 and fragment 2 balanced mix, as the reaction of template performing PCR, upstream primer is a primer 1, and downstream primer is a primer 4, reaction conditions is the same, product is about 2612bp (fragment 3), with QIAquick 8 PCR product purification test kit (QIAGEN, German, Cat:28142) behind the purifying, cut with the EcoRI enzyme and to spend the night, enzyme is cut product and is separated the back at 1% agarose gel electrophoresis and reclaim, and endonuclease bamhi is used for follow-up ligation.
(Invitrogen, U.S.A. Cat:V79020) cut with the EcoRI enzyme and spend the night, and enzyme is cut product and separated the back at 1% agarose gel electrophoresis and reclaim endonuclease bamhi, is used for follow-up ligation behind the dephosphorylation with pCDNA3.1.
With PCR reaction product fragment 3 cut with enzyme, pCDNA3.1 is connected behind the dephosphorylation, get 1.5 μ l and change 100 μ l DH5 α competence bacteriums over to, the picking positive colony, plasmid is carried for a short time, the dna sequencing evaluation and screening obtains lacking pCDNA3.1 plasmid encoding mutant body-pCDNA3.1-Δ ADP of 29477-29714nt, is used to insert the subclone of exogenous therapeutic gene.
Example 4, in carrier pCDNA3.1-Δ ADP, oppositely insert the exogenous STAT3 cDNA fragment of 770bp
STAT3 cDNA fragment increases from the RNA of breast cancer cell line MDA-MB-231, the reverse transcription condition:
5 * MMLV damping fluid, 4 μ l
10mM?dNTP 1μl
OligdT(50ng/ml) 1μl
Total RNA 2 μ l
Rnasin 0.5μl
MMLV reversed transcriptive enzyme 1 μ l
Add no RNA enzyme water to 20 μ l;
The reverse transcription reaction product is used for the PCR reaction.Upstream primer is 5 '-CC
ATCGATGGCCTCTCGGCCTCTGCC-3 ', this fragment is equivalent to the 190-1207nt (underscore partly is Cla I restriction enzyme site) of STAT3 cDNA encoding sequence; Downstream primer is 5 '-CC
ATCGATTTGCCGCCTCTTCCAGTCAGC-3 ', this fragment is equivalent to the 960-961nt (underscore partly is Cla I restriction enzyme site) of STAT3cDNA encoding sequence, and amplified fragments comprises the 960-190nt of STAT3 cDNA encoding sequence, and total length is about 770bp.
The reaction system cumulative volume is that 100 μ l comprise:
Contain MgCl
210 * PCR damping fluid, 10 μ l
2mM?dNTP 10μl
10 μ M upstream primers, 1 μ l
10 μ M downstream primers, 1 μ l
Reverse transcription reaction product 2 μ l
Pfu high-fidelity Taq enzyme 2.5u
Add water to 100 μ l;
Reaction conditions is:
94 ℃ 30 seconds;
94 ℃ 30 seconds, 52 ℃ 1 minute, 72 ℃ totally 30 circulations in 1 minute;
72 ℃ were extended 10 minutes.
The PCR product is 776bp, and after Cla I enzyme was cut, conventional electrophoretic separation purifying, detectable level were used for follow-up PCR reaction.After carrier pCDNA3.1-Δ ADP uses Cla I enzyme tangent line shapeization, conventional electrophoretic separation purifying, be connected with the exogenous STAT3 cDNA fragment of 770bp behind the dephosphorylation, get 1.5 μ l and change 100 μ l DH5 α competence bacteriums over to, the picking positive colony, plasmid is carried for a short time, and the dna sequencing evaluation and screening obtains oppositely inserting at Cla I restriction enzyme site the segmental plasmid encoding mutant body of exogenous STAT3 cDNA-pCDNA3.1-Δ ADP/ASSTAT3 of 770bp, is used for the clone of follow-up recombinant adenovirus.
Example 5, recombination adenovirus construction body Ad5/ Δ E1/ Δ ADP/ASSTAT3 obtain
The connection method of setting up according to reference " Adenovirus methodsand protocol; edited by William S.M.Wold; construction of mutations in the adenovirus earlyregion 3 (E3) transcription units; 11-24 " (ligation protocol) of obtaining of recombination adenovirus construction body Ad5/ Δ E1/ Δ ADP/ASSTAT3 is carried out, and key step comprises the extraction of TPDNA; Be used for the preparation of recombination adenovirus construction E3 district DNA; Ligation; Cotransfection 293 cell homologous recombination; The picking of plaque and evaluation; The amplification of positive-virus and purifying.Detailed method is referring to the document.Obtain recombination adenovirus construction body Ad5/ Δ E1/ Δ ADP/ASSTAT3 by above experimental procedure, this adenovirus has lacked Ad5 920-946nt sequence (GATCTT ACC TGC CAC GAG GCT GGC TTT), this sequence encoding E1A protein 12 1 to 129 amino acids, and disappearance Ad5 E3 district 29477-29714nt, in the ClaI restriction enzyme site, oppositely insert the exogenous STAT3 cDNA fragment of 770bp, this fragment is equivalent to the 960-190nt of STAT3 mRNA, other genome structures and wt-Ad5 are identical, and its sequence and structure are seen accompanying drawing 2 and appendix 1.
Example 6, recombination adenovirus construction body Ad5/ Δ E1/ Δ ADP/ASSTAT3 characterized
1, the external tumour-specific of the recombination adenovirus construction body evaluation of duplicating
The purpose of this experiment is to identify the duplicate characteristics of Ad5/ Δ E1/ Δ ADP/ASSTAT3 recombinant adenovirus in a series of tumours and normal cell, and experiment will be with the positive contrast of wt-Ad5, with the negative contrast of replication-defective virus Ad5CMV-GFP.Selecting clone for use is human lung cancer cell line A549 (P53+, RB-), the high metastatic prostate cancer clone of people PC3M-1E8 (P53-, RB-), human osteoblast cell's oncocyte be U-2OS (P53+, RB-), CCL188 HCT-8 (P53+, RB+), and MCF-7 MCF-7 (P53+, RB+).The clone of selecting for use has different P53, the RB gene phenotype; Have different tissues-derivedly, so experimental result can be represented different types of tumors.The normal cell of selecting for use is lung tracheole epithelial cell, prostate epithelial cell, the BMNC in the vascular endothelial cell in former generation, former generation, selects for use principle to be: the normal tissue cell that will contact a large amount of adenovirus after the intravenous administration.
In 24 orifice plate culture dish, plant 1 * 10
5Tumour or normal bone marrow mononuclearcell, nutrient solution are 10%FBS DMEM or RIPM1640, to second day about 70% cytogamy, inhale and remove liquid, and adding reaches 70% required MOI and is diluted to 100 μ l adding, rocks liquid gently 3 times, at 37 ℃, 5%CO
2In hatched 90 minutes.
Add and to contain 2%FBS DMEM or RIPM1640 substratum to 1ml, cell is positioned among 37 ℃ of 5%CO2 hatched 3-10 days, the required time of complete CPE appears in observation of cell.
Experimental result shows, positive control wt-Ad5 non-selectivity ground cracking tumour and normal cell; Replication-defective adenoviral Ad5CMV-GFP can not any cell of cracking, recombination adenovirus construction body Ad5/ Δ E1/ Δ ADP/ASSTAT3 is killing tumor cell optionally, its effect significantly is better than the cracking effect of wt-Ad5 pair cell, and this recombination adenovirus construction body is to the obviously influence of normal control cells system's nothing simultaneously.The result fully shows recombination adenovirus construction body Ad5/ Δ E1/ Δ ADP/ASSTAT3 selectively killing tumour cell, and concrete data see the following form:
| wt-Ad5 | Ad5CMV-GFP | Ad5/ΔE1/ΔADP/ASSTAT3 | |
| A549 | |||
| The 3rd day | 48％ | 1％ | 80％ |
| The 6th day | 100％ | 2％ | 100％ |
| The 10th day | 4％ | ||
| PC3M-1E8 | |||
| The 3rd day | 62％ | 1％ | 90％ |
| The 6th day | 100％ | 2％ | 100％ |
| The 10th day | 4％ | ||
| U-2OS | |||
| The 3rd day | 61％ | 1％ | 89％ |
| The 6th day | 100％ | 2％ | 100％ |
| The 10th day | 4％ | ||
| HCT-8 | |||
| The 3rd day | 63％ | 1％ | 91％ |
| The 6th day | 100％ | 2％ | 100％ |
| The 10th day | 4％ | ||
| PGBE1 | |||
| The 3rd day | 60％ | 1％ | 89％ |
| The 6th day | 100％ | 2％ | 100％ |
| The 10th day | 4％ | ||
| MCF-7 | |||
| The 3rd day | 55％ | 1％ | 82％ |
| The 6th day | 100％ | 1％ | 100％ |
| The 10th day | 1％ | ||
| Vascular endothelial cell | |||
| The 3rd day | 58％ | 1％ | 1％ |
| The 6th day | 100％ | 2％ | 2％ |
| The 10th day | 4％ | 3％ | |
| Lung tracheole epithelial cell | |||
| The 3rd day | 61％ | 1％ | 1％ |
| The 6th day | 100％ | 2％ | 2％ |
| The 10th day | 4％ | 3％ | |
| Prostate epithelial cell | |||
| The 3rd day | 63％ | 1％ | 2％ |
| The 6th day | 100％ | 2％ | 3％ |
| The 10th day | 4％ | 3％ | |
| BMNC | |||
| The 3rd day | 52％ | 1％ | 1％ |
| The 6th day | 100％ | 2％ | 3％ |
| The 10th day | 4％ | 4％ |
For further carrying out detection by quantitative Ad5/ Δ E1/ Δ ADP/ASSTAT3 recombination adenovirus construction body duplicating efficiency in tumour cell, carried out following experiment:
Variously in 24 orifice plate culture dish go into 1 * 10
5Various tumour cells or former generation normal cell, nutrient solution was 10%FBSDMEM or RIPM1640, by second day, about 70% cytogamy is inhaled and is removed liquid, adds Ad5/ Δ E1/ Δ ADP/ASSTAT3 10 MOI and is diluted to 100 μ l adding, rock liquid gently 3 times, at 37 ℃, 5%CO
2In hatched 90 minutes.
Add and to contain 2%FBS DMEM substratum, cell is positioned in 37 ℃, 5%CO2 hatched 3 days to 1ml.
Add 300 μ l PBS solution, carry out the circulation of freezing/thawing of three-wheel, discharge virus,, collect supernatant liquor, freeze in-80 ℃ TCID lysate maximum velocity centrifugation 10 minutes
50Method detects the titre of virus.
The result shows and duplicates the back by Ad5/ Δ E1/ Δ ADP/ASSTAT3 in tumour cell it is tired and is better than initial 3000-5000 doubly, and infectious titer does not have considerable change in normal cell, and the result shows that fully this recombination adenovirus construction body has the characteristic of duplicating at specifically inside tumor cell.
2, the evaluation of the external selective inactivation tumour cell STAT3 expression characterization of recombination adenovirus construction body
The purpose of this experiment is to identify that Ad5/ Δ E1/ Δ ADP/ASSTAT3 recombinant adenovirus is in the influence to a series of tumours and normal cell STAT3 expression, it is human lung cancer cell line A549 (P53+ that clone is selected in experiment for use, RB-), human osteoblast cell's oncocyte be U-2OS (P53+, RB-), metastatic human prostate cancer cell line PC3M-1E8 (P53-, RB-), and CCL188 HCT-8 (P53+, RB+), MCF-7 MCF-7 (P53+, RB+).The clone of selecting for use has different P53, the RB gene phenotype; Have different tissues-derivedly, so experimental result can be represented different types of tumors.The normal cell of selecting for use is lung tracheole epithelial cell, prostate epithelial cell, the BMNC in the vascular endothelial cell in former generation, former generation, selects for use principle to be: the normal tissue cell that will contact a large amount of adenovirus after the intravenous administration.
In 24 orifice plate culture dish, plant 1 * 10
5Tumour or normal cell, nutrient solution are 10%FBS DMEM or RIPM1640, to second day about 70% cytogamy, inhale and remove liquid, and adding reaches 70% required MOI and is diluted to 100 μ l adding, rocks liquid gently 3 times, at 37 ℃, 5%CO
2In hatched 90 minutes.
Add and to contain 2%FBS DMEM or RIPM1640 substratum, cell is positioned in 37 ℃, 5%CO2 hatched 3-10 days, extract the variation that total protein of cell detects the STAT3 protein expression level to 1ml.
Experimental result shows that recombination adenovirus construction body Ad5/ Δ E1/ Δ ADP/ASSTAT3 can selectivity seal the proteic expression of tumour cell STAT3, and this recombination adenovirus construction body is that the STAT3 protein expression does not have obvious influence to normal control cells simultaneously.The result fully shows recombination adenovirus construction body Ad5/ Δ E1/ Δ ADP/ASSTAT3 selective inactivation tumour cell STAT3 expression of gene.
3, the evaluation of recombination adenovirus construction body Ad5/ Δ E1/ Δ ADP/ASSTAT3 anti-tumor in vivo effect
The tumour cell animal model: select the BALB/c nude mice in 4～6 ages in week, the stomach cancer cell in the vegetative period of taking the logarithm is the MKN-45 cell, adjusts cell density 1 * 10
6/ 100 μ l, every nude mice right side back subcutaneous vaccination 200 μ l, when treating that tumor average diameter reaches 0.4-0.6cm, be divided into 4 groups at random by the tumour size, promptly medium control group, wt-Ad5 group, Ad5CMV-GFP group and Ad5/ Δ E1/ Δ ADP/ASSTAT3 organize every group of 10 nude mices, difference intratumor injection PBS, wt-Ad5, Ad5CMV-GFP and Ad5/ Δ E1/ Δ ADP/ASSTAT3, every 2 * 10
8Pfu/ (only inferior), once a day, continuous 5 days.Observe and measure gross tumor volume weekly 2 times, (100 days or gross tumor volume are greater than 1cm until the experiment terminal point
3).
Experimental result shows that when the experiment terminal point, wt-Ad5 group average tumor inhibiting rate is 40% ± 2%; Ad5CMV-GFP group tumor control rate is-2% ± 3%; Three nude mice tumours of Ad5/ Δ E1/ Δ ADP/ASSTAT3 group disappear fully, and the average tumor inhibiting rate is 90% ± 3%, and the result shows that fully recombination adenovirus construction body Ad5/ Δ E1/ Δ ADP/ASSTAT3 has definite anti-tumor in vivo effect.
The vena systemica medication is to the treatment of stomach carcinoma in situ model and to the inhibition of tumour micrometastasis: when the cancer of the stomach Subcutaneous tumor grows to 0.7-0.8cm, select the better tumor animal of overall health of patients, the cervical vertebra dislocation is put to death, aseptic condition takes out the knurl piece down, cut into the knurl piece of 1-2mm with scalpel, be transplanted under the lesser gastric curvature mucous membrane.Animal B ultrasonic monitoring is divided into 4 groups at random when tumor growth during to 3mm, and promptly PBS control group, wt-Ad5 group, Ad5CMV-GFP group and Ad5/ Δ E1/ Δ ADP/ASSTAT3 organize, and every group of 10 nude mices are with 2 * 10
8The intravenous injection of pfu virus was treated 5 days continuously.Animal B ultrasonic monitoring gross tumor volume.(30 days or gross tumor volume are greater than 1.2cm to testing terminal point
3), take out lung, liver, sky, colon and peritonaeum after, portal vein and hilar lymph node, the pathology detection micrometastasis.Experimental result shows that when the experiment terminal point, the PBS control group all has many organs, tissue tumor to shift, and wt-Ad5 group tumor control rate is 8 many organ metastasis of generation tumour in 25% ± 2%, 10 nude mices; The Ad5CMV-GFP tumor control rate is-3% ± 4%; The incidence of metastases is 100%; 3 nude mice tumours of Δ AD5/ Δ E1/ Δ ADP/ASSTAT31 group disappear fully, and tumor control rate is 85% ± 4%, 1 tumour sky, colon implantation metastasis take place.Above result shows that fully recombination adenovirus construction body AD5/ Δ E1/ Δ ADP/ASSTAT3 intravenous administration has the effect of significant anti-tumor in vivo and inhibition metastases.
Annex dna sequence dna table
＜110〉AoNiKeSi gene Technology Co., Ltd., Shenzhen City
＜110〉Martin Zhou Jianfeng king generation a surname Lu Yun duckweed Han Zhi is strong
＜120〉acquisition of the novel oncolytic adenovirus construct of selective tumor blockage STAT 3 and purposes
<140>
<141>
<160>1
<170>
<210>1
<211>
<212>DNA
＜213〉artificial sequence
<220>
<223>misc_feature
<400>
catcatcaat?aatatacctt?attttggatt?gaagccaata?tgataatgag?ggggtggagt 60
ttgtgacgtg?gcgcggggcg?tgggaacggg?gcgggtgacg?tagtagtgtg?gcggaagtgt 120
gatgttgcaa?gtgtggcgga?acacatgtaa?gcgacggatg?tggcaaaagt?gacgtttttg 180
gtgtgcgccg?gtgtacacag?gaagtgacaa?ttttcgcgcg?gttttaggcg?gatgttgtag 240
taaatttggg?cgtaaccgag?taagatttgg?ccattttcgc?gggaaaactg?aataagagga 300
agtgaaatct?gaataatttt?gtgttactca?tagcgcgtaa?tatttgtcta?gggccgcggg 360
gactttgacc?gtttacgtgg?agactcgccc?aggtgttttt?ctcaggtgtt?ttccgcgttc 420
cgggtcaaag?ttggcgtttt?attattatag?tcagctgacg?tgtagtgtat?ttatacccgg 480
tgagttcctc?aagaggccac?tcttgagtgc?cagcgagtag?agttttctcc?tccgagccgc 540
tccgacaccg?ggactgaaaa?tgagacatat?tatctgccac?ggaggtgtta?ttaccgaaga 600
aatggccgcc?agtcttttgg?accagctgat?cgaagaggta?ctggctgata?atcttccacc 660
tcctagccat?tttgaaccac?ctacccttca?cgaactgtat?gatttagacg?tgacggcccc 720
cgaagatccc?aacgaggagg?cggtttcgca?gatttttccc?gactctgtaa?tgttggcggt 780
gcaggaaggg?attgacttac?tcacttttcc?gccggcgccc?ggttctccgg?agccgcctca 840
cctttcccgg?cagcccgagc?agccggagca?gagagccttg?ggtccggttt?ctatgccaaa 900
ccttgtaccg?gaggtgatcg?atcacgaggc?tggctttcca?cccagtgacg?acgaggatga 960
agagggtgag?gagtttgtgt?tagattatgt?ggagcacccc?gggcacggtt?gcaggtcttg 1020
tcattatcac?cggaggaata?cgggggaccc?agatattatg?tgttcgcttt?gctatatgag 1080
gacctgtggc?atgtttgtct?acagtaagtg?aaaattatgg?gcagtgggtg?atagagtggt 1140
gggtttggtg?tggtaatttt?ttttttaatt?tttacagttt?tgtggtttaa?agaattttgt 1200
attgtgattt?ttttaaaagg?tcctgtgtct?gaacctgagc?ctgagcccga?gccagaaccg 1260
gagcctgcaa?gacctacccg?ccgtcctaaa?atggcgcctg?ctatcctgag?acgcccgaca 1320
tcacctgtgt?ctagagaatg?caatagtagt?acggatagct?gtgactccgg?tccttctaac 1380
acacctcctg?agatacaccc?ggtggtcccg?ctgtgcccca?ttaaaccagt?tgccgtgaga 1440
gttggtgggc?gtcgccaggc?tgtggaatgt?atcgaggact?tgcttaacga?gcctgggcaa 1500
cctttggact?tgagctgtaa?acgccccagg?ccataaggtg?taaacctgtg?attgcgtgtg 1560
tggttaacgc?ctttgtttgc?tgaatgagtt?gatgtaagtt?taataaaggg?tgagataatg 1620
tttaacttgc?atggcgtgtt?aaatggggcg?gggcttaaag?ggtatataat?gcgccgtggg 1680
ctaatcttgg?ttacatctga?cctcatggag?gcttgggagt?gtttggaaga?tttttctgct 1740
gtgcgtaact?tgctggaaca?gagctctaac?agtacctctt?ggttttggag?gtttctgtgg 1800
ggctcatccc?aggcaaagtt?agtctgcaga?attaaggagg?attacaagtg?ggaatttgaa 1860
gagcttttga?aatcctgtgg?tgagctgttt?gattctttga?atctgggtca?ccaggcgctt 1920
ttccaagaga?aggtcatcaa?gactttggat?ttttccacac?cggggcgcgc?tgcggctgct 1980
gttgcttttt?tgagttttat?aaaggataaa?tggagcgaag?aaacccatct?gagcgggggg 2040
tacctgctgg?attttctggc?catgcatctg?tggagagcgg?ttgtgagaca?caagaatcgc 2100
ctgctactgt?tgtcttccgt?ccgcccggcg?ataataccga?cggaggagca?gcagcagcag 2160
caggaggaag?ccaggcggcg?gcggcaggag?cagagcccat?ggaacccgag?agccggcctg 2220
gaccctcggg?aatgaatgtt?gtacaggtgg?ctgaactgta?tccagaactg?agacgcattt 2280
tgacaattac?agaggatggg?caggggctaa?agggggtaaa?gagggagcgg?ggggcttgtg 2340
aggctacaga?ggaggctagg?aatctagctt?ttagcttaat?gaccagacac?cgtcctgagt 2400
gtattacttt?tcaacagatc?aaggataatt?gcgctaatga?gcttgatctg?ctggcgcaga 2460
agtattccat?agagcagctg?accacttact?ggctgcagcc?aggggatgat?tttgaggagg 2520
ctattagggt?atatgcaaag?gtggcactta?ggccagattg?caagtacaag?atcagcaaac 2580
ttgtaaatat?caggaattgt?tgctacattt?ctgggaacgg?ggccgaggtg?gagatagata 2640
cggaggatag?ggtggccttt?agatgtagca?tgataaatat?gtggccgggg?gtgcttggca 2700
tggacggggt?ggttattatg?aatgtaaggt?ttactggccc?caattttagc?ggtacggttt 2760
tcctggccaa?taccaacctt?atcctacacg?gtgtaagctt?ctatgggttt?aacaatacct 2820
gtgtggaagc?ctggaccgat?gtaagggttc?ggggctgtgc?cttttactgc?tgctggaagg 2880
gggtggtgtg?tcgccccaaa?agcagggctt?caattaagaa?atgcctcttt?gaaaggtgta 2940
ccttgggtat?cctgtctgag?ggtaactcca?gggtgcgcca?caatgtggcc?tccgactgtg 3000
gttgcttcat?gctagtgaaa?agcgtggctg?tgattaagca?taacatggta?tgtggcaact 3060
gcgaggacag?ggcctctcag?atgctgacct?gctcggacgg?caactgtcac?ctgctgaaga 3120
ccattcacgt?agccagccac?tctcgcaagg?cctggccagt?gtttgagcat?aacatactga 3180
cccgctgttc?cttgcatttg?ggtaacagga?ggggggtgtt?cctaccttac?caatgcaatt 3240
tgagtcacac?taagatattg?cttgagcccg?agagcatgtc?caaggtgaac?ctgaacgggg 3300
tgtttgacat?gaccatgaag?atctggaagg?tgctgaggta?cgatgagacc?cgcaccaggt 3360
gcagaccctg?cgagtgtggc?ggtaaacata?ttaggaacca?gcctgtgatg?ctggatgtga 3420
ccgaggagct?gaggcccgat?cacttggtgc?tggcctgcac?ccgcgctgag?tttggctcta 3480
gcgatgaaga?tacagattga?ggtactgaaa?tgtgtgggcg?tggcttaagg?gtgggaaaga 3540
atatataagg?tgggggtctt?atgtagtttt?gtatctgttt?tgcagcagcc?gccgccgcca 3600
tgagcaccaa?ctcgtttgat?ggaagcattg?tgagctcata?tttgacaacg?cgcatgcccc 3660
catgggccgg?ggtgcgtcag?aatgtgatgg?gctccagcat?tgatggtcgc?cccgtcctgc 3720
ccgcaaactc?tactaccttg?acctacgaga?ccgtgtctgg?aacgccgttg?gagactgcag 3780
cctccgccgc?cgcttcagcc?gctgcagcca?ccgcccgcgg?gattgtgact?gactttgctt 3840
tcctgagccc?gcttgcaagc?agtgcagctt?cccgttcatc?cgcccgcgat?gacaagttga 3900
cggctctttt?ggcacaattg?gattctttga?cccgggaact?taatgtcgtt?tctcagcagc 3960
tgttggatct?gcgccagcag?gtttctgccc?tgaaggcttc?ctcccctccc?aatgcggttt 4020
aaaacataaa?taaaaaacca?gactctgttt?ggatttggat?caagcaagtg?tcttgctgtc 4080
tttatttagg?ggttttgcgc?gcgcggtagg?cccgggacca?gcggtctcgg?tcgttgaggg 4140
tcctgtgtat?tttttccagg?acgtggtaaa?ggtgactctg?gatgttcaga?tacatgggca 4200
taagcccgtc?tctggggtgg?aggtagcacc?actgcagagc?ttcatgctgc?ggggtggtgt 4260
tgtagatgat?ccagtcgtag?caggagcgct?gggcgtggtg?cctaaaaatg?tctttcagta 4320
gcaagctgat?tgccaggggc?aggcccttgg?tgtaagtgtt?tacaaagcgg?ttaagctggg 4380
atgggtgcat?acgtggggat?atgagatgca?tcttggactg?tatttttagg?ttggctatgt 4440
tcccagccat?atccctccgg?ggattcatgt?tgtgcagaac?caccagcaca?gtgtatccgg 4500
tgcacttggg?aaatttgtca?tgtagcttag?aaggaaatgc?gtggaagaac?ttggagacgc 4560
ccttgtgacc?tccaagattt?tccatgcatt?cgtccataat?gatggcaatg?ggcccacggg 4620
cggcggcctg?ggcgaagata?tttctgggat?cactaacgtc?atagttgtgt?tccaggatga 4680
gatcgtcata?ggccattttt?acaaagcgcg?ggcggagggt?gccagactgc?ggtataatgg 4740
ttccatccgg?cccaggggcg?tagttaccct?cacagatttg?catttcccac?gctttgagtt 4800
cagatggggg?gatcatgtct?acctgcgggg?cgatgaagaa?aacggtttcc?ggggtagggg 4860
agatcagctg?ggaagaaagc?aggttcctga?gcagctgcga?cttaccgcag?ccggtgggcc 4920
cgtaaatcac?acctattacc?gggtgcaact?ggtagttaag?agagctgcag?ctgccgtcat 4980
ccctgagcag?gggggccact?tcgttaagca?tgtccctgac?tcgcatgttt?tccctgacca 5040
aatccgccag?aaggcgctcg?ccgcccagcg?atagcagttc?ttgcaaggaa?gcaaagtttt 5100
tcaacggttt?gagaccgtcc?gccgtaggca?tgcttttgag?cgtttgacca?agcagttcca 5160
ggcggtccca?cagctcggtc?acctgctcta?cggcatctcg?atccagcata?tctcctcgtt 5220
tcgcgggttg?gggcggcttt?cgctgtacgg?cagtagtcgg?tgctcgtcca?gacgggccag 5280
ggtcatgtct?ttccacgggc?gcagggtcct?cgtcagcgta?gtctgggtca?cggtgaaggg 5340
gtgcgctccg?ggctgcgcgc?tggccagggt?gcgcttgagg?ctggtcctgc?tggtgctgaa 5400
gcgctgccgg?tcttcgccct?gcgcgtcggc?caggtagcat?ttgaccatgg?tgtcatagtc 5460
cagcccctcc?gcggcgtggc?ccttggcgcg?cagcttgccc?ttggaggagg?cgccgcacga 5520
ggggcagtgc?agacttttga?gggcgtagag?cttgggcgcg?agaaataccg?attccgggga 5580
gtaggcatcc?gcgccgcagg?ccccgcagac?ggtctcgcat?tccacgagcc?aggtgagctc 5640
tggccgttcg?gggtcaaaaa?ccaggtttcc?cccatgcttt?ttgatgcgtt?tcttacctct 5700
ggtttccatg?agccggtgtc?cacgctcggt?gacgaaaagg?ctgtccgtgt?ccccgtatac 5760
agacttgaga?ggcctgtcct?cgagcggtgt?tccgcggtcc?tcctcgtata?gaaactcgga 5820
ccactctgag?acaaaggctc?gcgtccaggc?cagcacgaag?gaggctaagt?gggaggggta 5880
gcggtcgttg?tccactaggg?ggtccactcg?ctccagggtg?tgaagacaca?tgtcgccctc 5940
ttcggcatca?aggaaggtga?ttggtttgta?ggtgtaggcc?acgtgaccgg?gtgttcctga 6000
aggggggcta?taaaaggggg?tgggggcgcg?ttcgtcctca?ctctcttccg?catcgctgtc 6060
tgcgagggcc?agctgttggg?gtgagtactc?cctctgaaaa?gcgggcatga?cttctgcgct 6120
aagattgtca?gtttccaaaa?acgaggagga?tttgatattc?acctggcccg?cggtgatgcc 6180
tttgagggtg?gccgcatcca?tctggtcaga?aaagacaatc?tttttgttgt?caagcttggt 6240
ggcaaacgac?ccgtagaggg?cgttggacag?caacttggcg?atggagcgca?gggtttggtt 6300
tttgtcgcga?tcggcgcgct?ccttggccgc?gatgtttagc?tgcacgtatt?cgcgcgcaac 6360
gcaccgccat?tcgggaaaga?cggtggtgcg?ctcgtcgggc?accaggtgca?cgcgccaacc 6420
gcggttgtgc?agggtgacaa?ggtcaacgct?ggtggctacc?tctccgcgta?ggcgctcgtt 6480
ggtccagcag?aggcggccgc?ccttgcgcga?gcagaatggc?ggtagggggt?ctagctgcgt 6540
ctcgtccggg?gggtctgcgt?ccacggtaaa?gaccccgggc?agcaggcgcg?cgtcgaagta 6600
gtctatcttg?catccttgca?agtctagcgc?ctgctgccat?gcgcgggcgg?caagcgcgcg 6660
ctcgtatggg?ttgagtgggg?gaccccatgg?catggggtgg?gtgagcgcgg?aggcgtacat 6720
gccgcaaatg?tcgtaaacgt?agaggggctc?tctgagtatt?ccaagatatg?tagggtagca 6780
tcttccaccg?cggatgctgg?cgcgcacgta?atcgtatagt?tcgtgcgagg?gagcgaggag 6840
gtcgggaccg?aggttgctac?gggcgggctg?ctctgctcgg?aagactatct?gcctgaagat 6900
ggcatgtgag?ttggatgata?tggttggacg?ctggaagacg?ttgaagctgg?cgtctgtgag 6960
acctaccgcg?tcacgcacga?aggaggcgta?ggagtcgcgc?agcttgttga?ccagctcggc 7020
ggtgacctgc?acgtctaggg?cgcagtagtc?cagggtttcc?ttgatgatgt?catacttatc 7080
ctgtcccttt?tttttccaca?gctcgcggtt?gaggacaaac?tcttcgcggt?ctttccagta 7140
ctcttggatc?ggaaacccgt?cggcctccga?acggtaagag?cctagcatgt?agaactggtt 7200
gacggcctgg?taggcgcagc?atcccttttc?tacgggtagc?gcgtatgcct?gcgcggcctt 7260
ccggagcgag?gtgtgggtga?gcgcaaaggt?gtccctgacc?atgactttga?ggtactggta 7320
tttgaagtca?gtgtcgtcgc?atccgccctg?ctcccagagc?aaaaagtccg?tgcgcttttt 7380
ggaacgcgga?tttggcaggg?cgaaggtgac?atcgttgaag?agtatctttc?ccgcgcgagg 7440
cataaagttg?cgtgtgatgc?ggaagggtcc?cggcacctcg?gaacggttgt?taattacctg 7500
ggcggcgagc?acgatctcgt?caaagccgtt?gatgttgtgg?cccacaatgt?aaagttccaa 7560
gaagcgcggg?atgcccttga?tggaaggcaa?ttttttaagt?tcctcgtagg?tgagctcttc 7620
aggggagctg?agcccgtgct?ctgaaagggc?ccagtctgca?agatgagggt?tggaagcgac 7680
gaatgagctc?cacaggtcac?gggccattag?catttgcagg?tggtcgcgaa?aggtcctaaa 7740
ctggcgacct?atggccattt?tttctggggt?gatgcagtag?aaggtaagcg?ggtcttgttc 7800
ccagcggtcc?catccaaggt?tcgcggctag?gtctcgcgcg?gcagtcacta?gaggctcatc 7860
tccgccgaac?ttcatgacca?gcatgaaggg?cacgagctgc?ttcccaaagg?cccccatcca 7920
agtataggtc?tctacatcgt?aggtgacaaa?gagacgctcg?gtgcgaggat?gcgagccgat 7980
cgggaagaac?tggatctccc?gccaccaatt?ggaggagtgg?ctattgatgt?ggtgaaagta 8040
gaagtccctg?cgacgggccg?aacactcgtg?ctggcttttg?taaaaacgtg?cgcagtactg 8100
gcagcggtgc?acgggctgta?catcctgcac?gaggttgacc?tgacgaccgc?gcacaaggaa 8160
gcagagtggg?aatttgagcc?cctcgcctgg?cgggtttggc?tggtggtctt?ctacttcggc 8220
tgcttgtcct?tgaccgtctg?gctgctcgag?gggagttacg?gtggatcgga?ccaccacgcc 8280
gcgcgagccc?aaagtccaga?tgtccgcgcg?cggcggtcgg?agcttgatga?caacatcgcg 8340
cagatgggag?ctgtccatgg?tctggagctc?ccgcggcgtc?aggtcaggcg?ggagctcctg 8400
caggtttacc?tcgcatagac?gggtcagggc?gcgggctaga?tccaggtgat?acctaatttc 8460
caggggctgg?ttggtggcgg?cgtcgatggc?ttgcaagagg?ccgcatcccc?gcggcgcgac 8520
tacggtaccg?cgcggcgggc?ggtgggccgc?gggggtgtcc?ttggatgatg?catctaaaag 8580
cggtgacgcg?ggcgagcccc?cggaggtagg?gggggctccg?gacccgccgg?gagagggggc 8640
aggggcacgt?cggcgccgcg?cgcgggcagg?agctggtgct?gcgcgcgtag?gttgctggcg 8700
aacgcgacga?cgcggcggtt?gatctcctga?atctggcgcc?tctgcgtgaa?gacgacgggc 8760
ccggtgagct?tgagcctgaa?agagagttcg?acagaatcaa?tttcggtgtc?gttgacggcg 8820
gcctggcgca?aaatctcctg?cacgtctcct?gagttgtctt?gataggcgat?ctcggccatg 8880
aactgctcga?tctcttcctc?ctggagatct?ccgcgtccgg?ctcgctccac?ggtggcggcg 8940
aggtcgttgg?aaatgcgggc?catgagctgc?gagaaggcgt?tgaggcctcc?ctcgttccag 9000
acgcggctgt?agaccacgcc?cccttcggca?tcgcgggcgc?gcatgaccac?ctgcgcgaga 9060
ttgagctcca?cgtgccgggc?gaagacggcg?tagtttcgca?ggcgctgaaa?gaggtagttg 9120
agggtggtgg?cggtgtgttc?tgccacgaag?aagtacataa?cccagcgtcg?caacgtggat 9180
tcgttgatat?cccccaaggc?ctcaaggcgc?tccatggcct?cgtagaagtc?cacggcgaag 9240
ttgaaaaact?gggagttgcg?cgccgacacg?gttaactcct?cctccagaag?acggatgagc 9300
tcggcgacag?tgtcgcgcac?ctcgcgctca?aaggctacag?gggcctcttc?ttcttcttca 9360
atctcctctt?ccataagggc?ctccccttct?tcttcttctg?gcggcggtgg?gggagggggg 9420
acacggcggc?gacgacggcg?caccgggagg?cggtcgacaa?agcgctcgat?catctccccg 9480
cggcgacggc?gcatggtctc?ggtgacggcg?cggccgttct?cgcgggggcg?cagttggaag 9540
acgccgcccg?tcatgtcccg?gttatgggtt?ggcggggggc?tgccatgcgg?cagggatacg 9600
gcgctaacga?tgcatctcaa?caattgttgt?gtaggtactc?cgccgccgag?ggacctgagc 9660
gagtccgcat?cgaccggatc?ggaaaacctc?tcgagaaagg?cgtctaacca?gtcacagtcg 9720
caaggtaggc?tgagcaccgt?ggcgggcggc?agcgggcggc?ggtcggggtt?gtttctggcg 9780
gaggtgctgc?tgatgatgta?attaaagtag?gcggtcttga?gacggcggat?ggtcgacaga 9840
agcaccatgt?ccttgggtcc?ggcctgctga?atgcgcaggc?ggtcggccat?gccccaggct 9900
tcgttttgac?atcggcgcag?gtctttgtag?tagtcttgca?tgagcctttc?taccggcact 9960
tcttcttctc?cttcctcttg?tcctgcatct?cttgcatcta?tcgctgcggc?ggcggcggag 10020
tttggccgta?ggtggcgccc?tcttcctccc?atgcgtgtga?ccccgaagcc?cctcatcggc 10080
tgaagcaggg?ctaggtcggc?gacaacgcgc?tcggctaata?tggcctgctg?cacctgcgtg 10140
agggtagact?ggaagtcatc?catgtccaca?aagcggtggt?atgcgcccgt?gttgatggtg 10200
taagtgcagt?tggccataac?ggaccagtta?acggtctggt?gacccggctg?cgagagctcg 10260
gtgtacctga?gacgcgagta?agccctcgag?tcaaatacgt?agtcgttgca?agtccgcacc 10320
aggtactggt?atcccaccaa?aaagtgcggc?ggcggctggc?ggtagagggg?ccagcgtagg 10380
gtggccgggg?ctccgggggc?gagatcttcc?aacataaggc?gatgatatcc?gtagatgtac 10440
ctggacatcc?aggtgatgcc?ggcggcggtg?gtggaggcgc?gcggaaagtc?gcggacgcgg 10500
ttccagatgt?tgcgcagcgg?caaaaagtgc?tccatggtcg?ggacgctctg?gccggtcagg 10560
cgcgcgcaat?cgttgacgct?ctagaccgtg?caaaaggaga?gcctgtaagc?gggcactctt 10620
ccgtggtctg?gtggataaat?tcgcaagggt?atcatggcgg?acgaccgggg?ttcgagcccc 10680
gtatccggcc?gtccgccgtg?atccatgcgg?ttaccgcccg?cgtgtcgaac?ccaggtgtgc 10740
gacgtcagac?aacgggggag?tgctcctttt?ggcttccttc?caggcgcggc?ggctgctgcg 10800
ctagcttttt?tggccactgg?ccgcgcgcag?cgtaagcggt?taggctggaa?agcgaaagca 10860
ttaagtggct?cgctccctgt?agccggaggg?ttattttcca?agggttgagt?cgcgggaccc 10920
ccggttcgag?tctcggaccg?gccggactgc?ggcgaacggg?ggtttgcctc?cccgtcatgc 10980
aagaccccgc?ttgcaaattc?ctccggaaac?agggacgagc?cccttttttg?cttttcccag 11040
atgcatccgg?tgctgcggca?gatgcgcccc?cctcctcagc?agcggcaaga?gcaagagcag 11100
cggcagacat?gcagggcacc?ctcccctcct?cctaccgcgt?caggaggggc?gacatccgcg 11160
gttgacgcgg?cagcagatgg?tgattacgaa?cccccgcggc?gccgggcccg?gcactacctg 11220
gacttggagg?agggcgaggg?cctggcgcgg?ctaggagcgc?cctctcctga?gcggtaccca 11280
agggtgcagc?tgaagcgtga?tacgcgtgag?gcgtacgtgc?cgcggcagaa?cctgtttcgc 11340
gaccgcgagg?gagaggagcc?cgaggagatg?cgggatcgaa?agttccacgc?agggcgcgag 11400
ctgcggcatg?gcctgaatcg?cgagcggttg?ctgcgcgagg?aggactttga?gcccgacgcg 11460
cgaaccggga?ttagtcccgc?gcgcgcacac?gtggcggccg?ccgacctggt?aaccgcatac 11520
gagcagacgg?tgaaccagga?gattaacttt?caaaaaagct?ttaacaacca?cgtgcgtacg 11580
cttgtggcgc?gcgaggaggt?ggctatagga?ctgatgcatc?tgtgggactt?tgtaagcgcg 11640
ctggagcaaa?acccaaatag?caagccgctc?atggcgcagc?tgttccttat?agtgcagcac 11700
agcagggaca?acgaggcatt?cagggatgcg?ctgctaaaca?tagtagagcc?cgagggccgc 11760
tggctgctcg?atttgataaa?catcctgcag?agcatagtgg?tgcaggagcg?cagcttgagc 11820
ctggctgaca?aggtggccgc?catcaactat?tccatgctta?gcctgggcaa?gttttacgcc 11880
cgcaagatat?accatacccc?ttacgttccc?atagacaagg?aggtaaagat?cgaggggttc 11940
tacatgcgca?tggcgctgaa?ggtgcttacc?ttgagcgacg?acctgggcgt?ttatcgcaac 12000
gagcgcatcc?acaaggccgt?gagcgtgagc?cggcggcgcg?agctcagcga?ccgcgagctg 12060
atgcacagcc?tgcaaagggc?cctggctggc?acgggcagcg?gcgatagaga?ggccgagtcc 12120
tactttgacg?cgggcgctga?cctgcgctgg?gccccaagcc?gacgcgccct?ggaggcagct 12180
ggggccggac?ctgggctggc?ggtggcaccc?gcgcgcgctg?gcaacgtcgg?cggcgtggag 12240
gaatatgacg?aggacgatga?gtacgagcca?gaggacggcg?agtactaagc?ggtgatgttt 12300
ctgatcagat?gatgcaagac?gcaacggacc?cggcggtgcg?ggcggcgctg?cagagccagc 12360
cgtccggcct?taactccacg?gacgactggc?gccaggtcat?ggaccgcatc?atgtcgctga 12420
ctgcgcgcaa?tcctgacgcg?ttccggcagc?agccgcaggc?caaccggctc?tccgcaattc 12480
tggaagcggt?ggtcccggcg?cgcgcaaacc?ccacgcacga?gaaggtgctg?gcgatcgtaa 12540
acgcgctggc?cgaaaacagg?gccatccggc?ccgacgaggc?cggcctggtc?tacgacgcgc 12600
tgcttcagcg?cgtggctcgt?tacaacagcg?gcaacgtgca?gaccaacctg?gaccggctgg 12660
tgggggatgt?gcgcgaggcc?gtggcgcagc?gtgagcgcgc?gcagcagcag?ggcaacctgg 12720
gctccatggt?tgcactaaac?gccttcctga?gtacacagcc?cgccaacgtg?ccgcggggac 12780
aggaggacta?caccaacttt?gtgagcgcac?tgcggctaat?ggtgactgag?acaccgcaaa 12840
gtgaggtgta?ccagtctggg?ccagactatt?ttttccagac?cagtagacaa?ggcctgcaga 12900
ccgtaaacct?gagccaggct?ttcaaaaact?tgcaggggct?gtggggggtg?cgggctccca 12960
caggcgaccg?cgcgaccgtg?tctagcttgc?tgacgcccaa?ctcgcgcctg?ttgctgctgc 13020
taatagcgcc?cttcacggac?agtggcagcg?tgtcccggga?cacataccta?ggtcacttgc 13080
tgacactgta?ccgcgaggcc?ataggtcagg?cgcatgtgga?cgagcatact?ttccaggaga 13140
ttacaagtgt?cagccgcgcg?ctggggcagg?aggacacggg?cagcctggag?gcaaccctaa 13200
actacctgct?gaccaaccgg?cggcagaaga?tcccctcgtt?gcacagttta?aacagcgagg 13260
aggagcgcat?tttgcgctac?gtgcagcaga?gcgtgagcct?taacctgatg?cgcgacgggg 13320
taacgcccag?cgtggcgctg?gacatgaccg?cgcgcaacat?ggaaccgggc?atgtatgcct 13380
caaaccggcc?gtttatcaac?cgcctaatgg?actacttgca?tcgcgcggcc?gccgtgaacc 13440
ccgagtattt?caccaatgcc?atcttgaacc?cgcactggct?accgccccct?ggtttctaca 13500
ccgggggatt?cgaggtgccc?gagggtaacg?atggattcct?ctgggacgac?atagacgaca 13560
gcgtgttttc?cccgcaaccg?cagaccctgc?tagagttgca?acagcgcgag?caggcagagg 13620
cggcgctgcg?aaaggaaagc?ttccgcaggc?caagcagctt?gtccgatcta?ggcgctgcgg 13680
ccccgcggtc?agatgctagt?agcccatttc?caagcttgat?agggtctctt?accagcactc 13740
gcaccacccg?cccgcgcctg?ctgggcgagg?aggagtacct?aaacaactcg?ctgctgcagc 13800
cgcagcgcga?aaaaaacctg?cctccggcat?ttcccaacaa?cgggatagag?agcctagtgg 13860
acaagatgag?tagatggaag?acgtacgcgc?aggagcacag?ggacgtgcca?ggcccgcgcc 13920
cgcccacccg?tcgtcaaagg?cacgaccgtc?agcggggtct?ggtgtgggag?gacgatgact 13980
cggcagacga?cagcagcgtc?ctggatttgg?gagggagtgg?caacccgttt?gcgcaccttc 14040
gccccaggct?ggggagaatg?ttttaaaaaa?aaaaaagcat?gatgcaaaat?aaaaaactca 14100
ccaaggccat?ggcaccgagc?gttggttttc?ttgtattccc?cttagtatgc?ggcgcgcggc 14160
gatgtatgag?gaaggtcctc?ctccctccta?cgagagtgtg?gtgagcgcgg?cgccagtggc 14220
ggcggcgctg?ggttctccct?tcgatgctcc?cctggacccg?ccgtttgtgc?ctccgcggta 14280
cctgcggcct?accgggggga?gaaacagcat?ccgttactct?gagttggcac?ccctattcga 14340
caccacccgt?gtgtacctgg?tggacaacaa?gtcaacggat?gtggcatccc?tgaactacca 14400
gaacgaccac?agcaactttc?tgaccacggt?cattcaaaac?aatgactaca?gcccggggga 14460
ggcaagcaca?cagaccatca?atcttgacga?ccggtcgcac?tggggcggcg?acctgaaaac 14520
catcctgcat?accaacatgc?caaatgtgaa?cgagttcatg?tttaccaata?agtttaaggc 14580
gcgggtgatg?gtgtcgcgct?tgcctactaa?ggacaatcag?gtggagctga?aatacgagtg 14640
ggtggagttc?acgctgcccg?agggcaacta?ctccgagacc?atgaccatag?accttatgaa 14700
caacgcgatc?gtggagcact?acttgaaagt?gggcagacag?aacggggttc?tggaaagcga 14760
catcggggta?aagtttgaca?cccgcaactt?cagactgggg?tttgaccccg?tcactggtct 14820
tgtcatgcct?ggggtatata?caaacgaagc?cttccatcca?gacatcattt?tgctgccagg 14880
atgcggggtg?gacttcaccc?acagccgcct?gagcaacttg?ttgggcatcc?gcaagcggca 14940
acccttccag?gagggcttta?ggatcaccta?cgatgatctg?gagggtggta?acattcccgc 15000
actgttggat?gtggacgcct?accaggcgag?cttgaaagat?gacaccgaac?agggcggggg 15060
tggcgcaggc?ggcagcaaca?gcagtggcag?cggcgcggaa?gagaactcca?acgcggcagc 15120
cgcggcaatg?cagccggtgg?aggacatgaa?cgatcatgcc?attcgcggcg?acacctttgc 15180
cacacgggct?gaggagaagc?gcgctgaggc?cgaagcagcg?gccgaagctg?ccgcccccgc 15240
tgcgcaaccc?gaggtcgaga?agcctcagaa?gaaaccggtg?atcaaacccc?tgacagagga 15300
cagcaagaaa?cgcagttaca?acctaataag?caatgacagc?accttcaccc?agtaccgcag 15360
ctggtacctt?gcatacaact?acggcgaccc?tcagaccgga?atccgctcat?ggaccctgct 15420
ttgcactcct?gacgtaacct?gcggctcgga?gcaggtctac?tggtcgttgc?cagacatgat 15480
gcaagacccc?gtgaccttcc?gctccacgcg?ccagatcagc?aactttccgg?tggtgggcgc 15540
cgagctgttg?cccgtgcact?ccaagagctt?ctacaacgac?caggccgtct?actcccaact 15600
catccgccag?tttacctctc?tgacccacgt?gttcaatcgc?tttcccgaga?accagatttt 15660
ggcgcgcccg?ccagccccca?ccatcaccac?cgtcagtgaa?aacgttcctg?ctctcacaga 15720
tcacgggacg?ctaccgctgc?gcaacagcat?cggaggagtc?cagcgagtga?ccattactga 15780
cgccagacgc?cgcacctgcc?cctacgttta?caaggccctg?ggcatagtct?cgccgcgcgt 15840
cctatcgagc?cgcacttttt?gagcaagcat?gtccatcctt?atatcgccca?gcaataacac 15900
aggctggggc?ctgcgcttcc?caagcaagat?gtttggcggg?gccaagaagc?gctccgacca 15960
acacccagtg?cgcgtgcgcg?ggcactaccg?cgcgccctgg?ggcgcgcaca?aacgcggccg 16020
cactgggcgc?accaccgtcg?atgacgccat?cgacgcggtg?gtggaggagg?cgcgcaacta 16080
cacgcccacg?ccgccaccag?tgtccacagt?ggacgcggcc?attcagaccg?tggtgcgcgg 16140
agcccggcgc?tatgctaaaa?tgaagagacg?gcggaggcgc?gtagcacgtc?gccaccgccg 16200
ccgacccggc?actgccgccc?aacgcgcggc?ggcggccctg?cttaaccgcg?cacgtcgcac 16260
cggccgacgg?gcggccatgc?gggccgctcg?aaggctggcc?gcgggtattg?tcactgtgcc 16320
ccccaggtcc?aggcgacgag?cggccgccgc?agcagccgcg?gccattagtg?ctatgactca 16380
gggtcgcagg?ggcaacgtgt?attgggtgcg?cgactcggtt?agcggcctgc?gcgtgcccgt 16440
gcgcacccgc?cccccgcgca?actagattgc?aagaaaaaac?tacttagact?cgtactgttg 16500
tatgtatcca?gcggcggcgg?cgcgcaacga?agctatgtcc?aagcgcaaaa?tcaaagaaga 16560
gatgctccag?gtcatcgcgc?cggagatcta?tggccccccg?aagaaggaag?agcaggatta 16620
caagccccga?aagctaaagc?gggtcaaaaa?gaaaaagaaa?gatgatgatg?atgaacttga 16680
cgacgaggtg?gaactgctgc?acgctaccgc?gcccaggcga?cgggtacagt?ggaaaggtcg 16740
acgcgtaaaa?cgtgttttgc?gacccggcac?caccgtagtc?tttacgcccg?gtgagcgctc 16800
cacccgcacc?tacaagcgcg?tgtatgatga?ggtgtacggc?gacgaggacc?tgcttgagca 16860
ggccaacgag?cgcctcgggg?agtttgccta?cggaaagcgg?cataaggaca?tgctggcgtt 16920
gccgctggac?gagggcaacc?caacacctag?cctaaagccc?gtaacactgc?agcaggtgct 16980
gcccgcgctt?gcaccgtccg?aagaaaagcg?cggcctaaag?cgcgagtctg?gtgacttggc 17040
acccaccgtg?cagctgatgg?tacccaagcg?ccagcgactg?gaagatgtct?tggaaaaaat 17100
gaccgtggaa?cctgggctgg?agcccgaggt?ccgcgtgcgg?ccaatcaagc?aggtggcgcc 17160
gggactgggc?gtgcagaccg?tggacgttca?gatacccact?accagtagca?ccagtattgc 17220
caccgccaca?gagggcatgg?agacacaaac?gtccccggtt?gcctcagcgg?tggcggatgc 17280
cgcggtgcag?gcggtcgctg?cggccgcgtc?caagacctct?acggaggtgc?aaacggaccc 17340
gtggatgttt?cgcgtttcag?ccccccggcg?cccgcgcggt?tcgaggaagt?acggcgccgc 17400
cagcgcgcta?ctgcccgaat?atgccctaca?tccttccatt?gcgcctaccc?ccggctatcg 17460
tggctacacc?taccgcccca?gaagacgagc?aactacccga?cgccgaacca?ccactggaac 17520
ccgccgccgc?cgtcgccgtc?gccagcccgt?gctggccccg?atttccgtgc?gcagggtggc 17580
tcgcgaagga?ggcaggaccc?tggtgctgcc?aacagcgcgc?taccacccca?gcatcgttta 17640
aaagccggtc?tttgtggttc?ttgcagatat?ggccctcacc?tgccgcctcc?gtttcccggt 17700
gccgggattc?cgaggaagaa?tgcaccgtag?gaggggcatg?gccggccacg?gcctgacggg 17760
cggcatgcgt?cgtgcgcacc?accggcggcg?gcgcgcgtcg?caccgtcgca?tgcgcggcgg 17820
tatcctgccc?ctccttattc?cactgatcgc?cgcggcgatt?ggcgccgtgc?ccggaattgc 17880
atccgtggcc?ttgcaggcgc?agagacactg?attaaaaaca?agttgcatgt?ggaaaaatca 17940
aaataaaaag?tctggactct?cacgctcgct?tggtcctgta?actattttgt?agaatggaag 18000
acatcaactt?tgcgtctctg?gccccgcgac?acggctcgcg?cccgttcatg?ggaaactggc 18060
aagatatcgg?caccagcaat?atgagcggtg?gcgccttcag?ctggggctcg?ctgtggagcg 18120
gcattaaaaa?tttcggttcc?accgttaaga?actatggcag?caaggcctgg?aacagcagca 18180
caggccagat?gctgagggat?aagttgaaag?agcaaaattt?ccaacaaaag?gtggtagatg 18240
gcctggcctc?tggcattagc?ggggtggtgg?acctggccaa?ccaggcagtg?caaaataaga 18300
ttaacagtaa?gcttgatccc?cgccctcccg?tagaggagcc?tccaccggcc?gtggagacag 18360
tgtctccaga?ggggcgtggc?gaaaagcgtc?cgcgccccga?cagggaagaa?actctggtga 18420
cgcaaataga?cgagcctccc?tcgtacgagg?aggcactaaa?gcaaggcctg?cccaccaccc 18480
gtcccatcgc?gcccatggct?accggagtgc?tgggccagca?cacacccgta?acgctggacc 18540
tgcctccccc?cgccgacacc?cagcagaaac?ctgtgctgcc?aggcccgacc?gccgttgttg 18600
taacccgtcc?tagccgcgcg?tccctgcgcc?gcgccgccag?cggtccgcga?tcgttgcggc 18660
ccgtagccag?tggcaactgg?caaagcacac?tgaacagcat?cgtgggtctg?ggggtgcaat 18720
ccctgaagcg?ccgacgatgc?ttctgaatag?ctaacgtgtc?gtatgtgtgt?catgtatgcg 18780
tccatgtcgc?cgccagagga?gctgctgagc?cgccgcgcgc?ccgctttcca?agatggctac 18840
cccttcgatg?atgccgcagt?ggtcttacat?gcacatctcg?ggccaggacg?cctcggagta 18900
cctgagcccc?gggctggtgc?agtttgcccg?cgccaccgag?acgtacttca?gcctgaataa 18960
caagtttaga?aaccccacgg?tggcgcctac?gcacgacgtg?accacagacc?ggtcccagcg 19020
tttgacgctg?cggttcatcc?ctgtggaccg?tgaggatact?gcgtactcgt?acaaggcgcg 19080
gttcacccta?gctgtgggtg?ataaccgtgt?gctggacatg?gcttccacgt?actttgacat 19140
ccgcggcgtg?ctggacaggg?gccctacttt?taagccctac?tctggcactg?cctacaacgc 19200
cctggctccc?aagggtgccc?caaatccttg?cgaatgggat?gaagctgcta?ctgctcttga 19260
aataaaccta?gaagaagagg?acgatgacaa?cgaagacgaa?gtagacgagc?aagctgagca 19320
gcaaaaaact?cacgtatttg?ggcaggcgcc?ttattctggt?ataaatatta?caaaggaggg 19380
tattcaaata?ggtgtcgaag?gtcaaacacc?taaatatgcc?gataaaacat?ttcaacctga 19440
acctcaaata?ggagaatctc?agtggtacga?aactgaaatt?aatcatgcag?ctgggagagt 19500
ccttaaaaag?actaccccaa?tgaaaccatg?ttacggttca?tatgcaaaac?ccacaaatga 19560
aaatggaggg?caaggcattc?ttgtaaagca?acaaaatgga?aagctagaaa?gtcaagtgga 19620
aatgcaattt?ttctcaacta?ctgaggcgac?cgcaggcaat?ggtgataact?tgactcctaa 19680
agtggtattg?tacagtgaag?atgtagatat?agaaacccca?gacactcata?tttcttacat 19740
gcccactatt?aaggaaggta?actcacgaga?actaatgggc?caacaatcta?tgcccaacag 19800
gcctaattac?attgctttta?gggacaattt?tattggtcta?atgtattaca?acagcacggg 19860
taatatgggt?gttctggcgg?gccaagcatc?gcagttgaat?gctgttgtag?atttgcaaga 19920
cagaaacaca?gagctttcat?accagctttt?gcttgattcc?attggtgata?gaaccaggta 19980
cttttctatg?tggaatcagg?ctgttgacag?ctatgatcca?gatgttagaa?ttattgaaaa 20040
tcatggaact?gaagatgaac?ttccaaatta?ctgctttcca?ctgggaggtg?tgattaatac 20100
agagactctt?accaaggtaa?aacctaaaac?aggtcaggaa?aatggatggg?aaaaagatgc 20160
tacagaattt?tcagataaaa?atgaaataag?agttggaaat?aattttgcca?tggaaatcaa 20220
tctaaatgcc?aacctgtgga?gaaatttcct?gtactccaac?atagcgctgt?atttgcccga 20280
caagctaaag?tacagtcctt?ccaacgtaaa?aatttctgat?aacccaaaca?cctacgacta 20340
catgaacaag?cgagtggtgg?ctcccgggtt?agtggactgc?tacattaacc?ttggagcacg 20400
ctggtccctt?gactatatgg?acaacgtcaa?cccatttaac?caccaccgca?atgctggcct 20460
gcgctaccgc?tcaatgttgc?tgggcaatgg?tcgctatgtg?cccttccaca?tccaggtgcc 20520
tcagaagttc?tttgccatta?aaaacctcct?tctcctgccg?ggctcataca?cctacgagtg 20580
gaacttcagg?aaggatgtta?acatggttct?gcagagctcc?ctaggaaatg?acctaagggt 20640
tgacggagcc?agcattaagt?ttgatagcat?ttgcctttac?gccaccttct?tccccatggc 20700
ccacaacacc?gcctccacgc?ttgaggccat?gcttagaaac?gacaccaacg?accagtcctt 20760
taacgactat?ctctccgccg?ccaacatgct?ctaccctata?cccgccaacg?ctaccaacgt 20820
gcccatatcc?atcccctccc?gcaactgggc?ggctttccgc?ggctgggcct?tcacgcgcct 20880
taagactaag?gaaaccccat?cactgggctc?gggctacgac?ccttattaca?cctactctgg 20940
ctctataccc?tacctagatg?gaacctttta?cctcaaccac?acctttaaga?aggtggccat 21000
tacctttgac?tcttctgtca?gctggcctgg?caatgaccgc?ctgcttaccc?ccaacgagtt 21060
tgaaattaag?cgctcagttg?acggggaggg?ttacaacgtt?gcccagtgta?acatgaccaa 21120
agactggttc?ctggtacaaa?tgctagctaa?ctacaacatt?ggctaccagg?gcttctatat 21180
cccagagagc?tacaaggacc?gcatgtactc?cttctttaga?aacttccagc?ccatgagccg 21240
tcaggtggtg?gatgatacta?aatacaagga?ctaccaacag?gtgggcatcc?tacaccaaca 21300
caacaactct?ggatttgttg?gctaccttgc?ccccaccatg?cgcgaaggac?aggcctaccc 21360
tgctaacttc?ccctatccgc?ttataggcaa?gaccgcagtt?gacagcatta?cccagaaaaa 21420
gtttctttgc?gatcgcaccc?tttggcgcat?cccattctcc?agtaacttta?tgtccatggg 21480
cgcactcaca?gacctgggcc?aaaaccttct?ctacgccaac?tccgcccacg?cgctagacat 21540
gacttttgag?gtggatccca?tggacgagcc?cacccttctt?tatgttttgt?ttgaagtctt 21600
tgacgtggtc?cgtgtgcacc?ggccgcaccg?cggcgtcatc?gaaaccgtgt?acctgcgcac 21660
gcccttctcg?gccggcaacg?ccacaacata?aagaagcaag?caacatcaac?aacagctgcc 21720
gccatgggct?ccagtgagca?ggaactgaaa?gccattgtca?aagatcttgg?ttgtgggcca 21780
tattttttgg?gcacctatga?caagcgcttt?ccaggctttg?tttctccaca?caagctcgcc 21840
tgcgccatag?tcaatacggc?cggtcgcgag?actgggggcg?tacactggat?ggcctttgcc 21900
tggaacccgc?actcaaaaac?atgctacctc?tttgagccct?ttggcttttc?tgaccagcga 21960
ctcaagcagg?tttaccagtt?tgagtacgag?tcactcctgc?gccgtagcgc?cattgcttct 22020
tcccccgacc?gctgtataac?gctggaaaag?tccacccaaa?gcgtacaggg?gcccaactcg 22080
gccgcctgtg?gactattctg?ctgcatgttt?ctccacgcct?ttgccaactg?gccccaaact 22140
cccatggatc?acaaccccac?catgaacctt?attaccgggg?tacccaactc?catgctcaac 22200
agtccccagg?tacagcccac?cctgcgtcgc?aaccaggaac?agctctacag?cttcctggag 22260
cgccactcgc?cctacttccg?cagccacagt?gcgcagatta?ggagcgccac?ttctttttgt 22320
cacttgaaaa?acatgtaaaa?ataatgtact?agagacactt?tcaataaagg?caaatgcttt 22380
tatttgtaca?ctctcgggtg?attatttacc?cccacccttg?ccgtctgcgc?cgtttaaaaa 22440
tcaaaggggt?tctgccgcgc?atcgctatgc?gccactggca?gggacacgtt?gcgatactgg 22500
tgtttagtgc?tccacttaaa?ctcaggcaca?accatccgcg?gcagctcggt?gaagttttca 22560
ctccacaggc?tgcgcaccat?caccaacgcg?tttagcaggt?cgggcgccga?tatcttgaag 22620
tcgcagttgg?ggcctccgcc?ctgcgcgcgc?gagttgcgat?acacagggtt?gcagcactgg 22680
aacactatca?gcgccgggtg?gtgcacgctg?gccagcacgc?tcttgtcgga?gatcagatcc 22740
gcgtccaggt?cctccgcgtt?gctcagggcg?aacggagtca?actttggtag?ctgccttccc 22800
aaaaagggcg?cgtgcccagg?ctttgagttg?cactcgcacc?gtagtggcat?caaaaggtga 22860
ccgtgcccgg?tctgggcgtt?aggatacagc?gcctgcataa?aagccttgat?ctgcttaaaa 22920
gccacctgag?cctttgcgcc?ttcagagaag?aacatgccgc?aagacttgcc?ggaaaactga 22980
ttggccggac?aggccgcgtc?gtgcacgcag?caccttgcgt?cggtgttgga?gatctgcacc 23040
acatttcggc?cccaccggtt?cttcacgatc?ttggccttgc?tagactgctc?cttcagcgcg 23100
cgctgcccgt?tttcgctcgt?cacatccatt?tcaatcacgt?gctccttatt?tatcataatg 23160
cttccgtgta?gacacttaag?ctcgccttcg?atctcagcgc?agcggtgcag?ccacaacgcg 23220
cagcccgtgg?gctcgtgatg?cttgtaggtc?acctctgcaa?acgactgcag?gtacgcctgc 23280
aggaatcgcc?ccatcatcgt?cacaaaggtc?ttgttgctgg?tgaaggtcag?ctgcaacccg 23340
cggtgctcct?cgttcagcca?ggtcttgcat?acggccgcca?gagcttccac?ttggtcaggc 23400
agtagtttga?agttcgcctt?tagatcgtta?tccacgtggt?acttgtccat?cagcgcgcgc 23460
gcagcctcca?tgcccttctc?ccacgcagac?acgatcggca?cactcagcgg?gttcatcacc 23520
gtaatttcac?tttccgcttc?gctgggctct?tcctcttcct?cttgcgtccg?cataccacgc 23580
gccactgggt?cgtcttcatt?cagccgccgc?actgtgcgct?tacctccttt?gccatgcttg 23640
attagcaccg?gtgggttgct?gaaacccacc?atttgtagcg?ccacatcttc?tctttcttcc 23700
tcgctgtcca?cgattacctc?tggtgatggc?gggcgctcgg?gcttgggaga?agggcgcttc 23760
tttttcttct?tgggcgcaat?ggccaaatcc?gccgccgagg?tcgatggccg?cgggctgggt 23820
gtgcgcggca?ccagcgcgtc?ttgtgatgag?tcttcctcgt?cctcggactc?gatacgccgc 23880
ctcatccgct?tttttggggg?cgcccgggga?ggcggcggcg?acggggacgg?ggacgacacg 23940
tcctccatgg?ttgggggacg?tcgcgccgca?ccgcgtccgc?gctcgggggt?ggtttcgcgc 24000
tgctcctctt?cccgactggc?catttccttc?tcctataggc?agaaaaagat?catggagtca 24060
gtcgagaaga?aggacagcct?aaccgccccc?tctgagttcg?ccaccaccgc?ctccaccgat 24120
gccgccaacg?cgcctaccac?cttccccgtc?gaggcacccc?cgcttgagga?ggaggaagtg 24180
attatcgagc?aggacccagg?ttttgtaagc?gaagacgacg?aggaccgctc?agtaccaaca 24240
gaggataaaa?agcaagacca?ggacaacgca?gaggcaaacg?aggaacaagt?cgggcggggg 24300
gacgaaaggc?atggcgacta?cctagatgtg?ggagacgacg?tgctgttgaa?gcatctgcag 24360
cgccagtgcg?ccattatctg?cgacgcgttg?caagagcgca?gcgatgtgcc?cctcgccata 24420
gcggatgtca?gccttgccta?cgaacgccac?ctattctcac?cgcgcgtacc?ccccaaacgc 24480
caagaaaacg?gcacatgcga?gcccaacccg?cgcctcaact?tctaccccgt?atttgccgtg 24540
ccagaggtgc?ttgccaccta?tcacatcttt?ttccaaaact?gcaagatacc?cctatcctgc 24600
cgtgccaacc?gcagccgagc?ggacaagcag?ctggccttgc?ggcagggcgc?tgtcatacct 24660
gatatcgcct?cgctcaacga?agtgccaaaa?atctttgagg?gtcttggacg?cgacgagaag 24720
cgcgcggcaa?acgctctgca?acaggaaaac?agcgaaaatg?aaagtcactc?tggagtgttg 24780
gtggaactcg?agggtgacaa?cgcgcgccta?gccgtactaa?aacgcagcat?cgaggtcacc 24840
cactttgcct?acccggcact?taacctaccc?cccaaggtca?tgagcacagt?catgagtgag 24900
ctgatcgtgc?gccgtgcgca?gcccctggag?agggatgcaa?atttgcaaga?acaaacagag 24960
gagggcctac?ccgcagttgg?cgacgagcag?ctagcgcgct?ggcttcaaac?gcgcgagcct 25020
gccgacttgg?aggagcgacg?caaactaatg?atggccgcag?tgctcgttac?cgtggagctt 25080
gagtgcatgc?agcggttctt?tgctgacccg?gagatgcagc?gcaagctaga?ggaaacattg 25140
cactacacct?ttcgacaggg?ctacgtacgc?caggcctgca?agatctccaa?cgtggagctc 25200
tgcaacctgg?tctcctacct?tggaattttg?cacgaaaacc?gccttgggca?aaacgtgctt 25260
cattccacgc?tcaagggcga?ggcgcgccgc?gactacgtcc?gcgactgcgt?ttacttattt 25320
ctatgctaca?cctggcagac?ggccatgggc?gtttggcagc?agtgcttgga?ggagtgcaac 25380
ctcaaggagc?tgcagaaact?gctaaagcaa?aacttgaagg?acctatggac?ggccttcaac 25440
gagcgctccg?tggccgcgca?cctggcggac?atcattttcc?ccgaacgcct?gcttaaaacc 25500
ctgcaacagg?gtctgccaga?cttcaccagt?caaagcatgt?tgcagaactt?taggaacttt 25560
atcctagagc?gctcaggaat?cttgcccgcc?acctgctgtg?cacttcctag?cgactttgtg 25620
cccattaagt?accgcgaatg?ccctccgccg?ctttggggcc?actgctacct?tctgcagcta 25680
gccaactacc?ttgcctacca?ctctgacata?atggaagacg?tgagcggtga?cggtctactg 25740
gagtgtcact?gtcgctgcaa?cctatgcacc?ccgcaccgct?ccctggtttg?caattcgcag 25800
ctgcttaacg?aaagtcaaat?tatcggtacc?tttgagctgc?agggtccctc?gcctgacgaa 25860
aagtccgcgg?ctccggggtt?gaaactcact?ccggggctgt?ggacgtcggc?ttaccttcgc 25920
aaatttgtac?ctgaggacta?ccacgcccac?gagattaggt?tctacgaaga?ccaatcccgc 25980
ccgccaaatg?cggagcttac?cgcctgcgtc?attacccagg?gccacattct?tggccaattg 26040
caagccatca?acaaagcccg?ccaagagttt?ctgctacgaa?agggacgggg?ggtttacttg 26100
gacccccagt?ccggcgagga?gctcaaccca?atccccccgc?cgccgcagcc?ctatcagcag 26160
cagccgcggg?cccttgcttc?ccaggatggc?acccaaaaag?aagctgcagc?tgccgccgcc 26220
acccacggac?gaggaggaat?actgggacag?tcaggcagag?gaggttttgg?acgaggagga 26280
ggaggacatg?atggaagact?gggagagcct?agacgaggaa?gcttccgagg?tcgaagaggt 26340
gtcagacgaa?acaccgtcac?cctcggtcgc?attcccctcg?ccggcgcccc?agaaatcggc 26400
aaccggttcc?agcatggcta?caacctccgc?tcctcaggcg?ccgccggcac?tgcccgttcg 26460
ccgacccaac?cgtagatggg?acaccactgg?aaccagggcc?ggtaagtcca?agcagccgcc 26520
gccgttagcc?caagagcaac?aacagcgcca?aggctaccgc?tcatggcgcg?ggcacaagaa 26580
cgccatagtt?gcttgcttgc?aagactgtgg?gggcaacatc?tccttcgccc?gccgctttct 26640
tctctaccat?cacggcgtgg?ccttcccccg?taacatcctg?cattactacc?gtcatctcta 26700
cagcccatac?tgcaccggcg?gcagcggcag?cggcagcaac?agcagcggcc?acacagaagc 26760
aaaggcgacc?ggatagcaag?actctgacaa?agcccaagaa?atccacagcg?gcggcagcag 26820
caggaggagg?agcgctgcgt?ctggcgccca?acgaacccgt?atcgacccgc?gagcttagaa 26880
acaggatttt?tcccactctg?tatgctatat?ttcaacagag?caggggccaa?gaacaagagc 26940
tgaaaataaa?aaacaggtct?ctgcgatccc?tcacccgcag?ctgcctgtat?cacaaaagcg 27000
aagatcagct?tcggcgcacg?ctggaagacg?cggaggctct?cttcagtaaa?tactgcgcgc 27060
tgactcttaa?ggactagttt?cgcgcccttt?ctcaaattta?agcgcgaaaa?ctacgtcatc 27120
tccagcggcc?acacccggcg?ccagcacctg?tcgtcagcgc?cattatgagc?aaggaaattc 27180
ccacgcccta?catgtggagt?taccagccac?aaatgggact?tgcggctgga?gctgcccaag 27240
actactcaac?ccgaataaac?tacatgagcg?cgggacccca?catgatatcc?cgggtcaacg 27300
gaatccgcgc?ccaccgaaac?cgaattctct?tggaacaggc?ggctattacc?accacacctc 27360
gtaataacct?taatccccgt?agttggcccg?ctgccctggt?gtaccaggaa?agtcccgctc 27420
ccaccactgt?ggtacttccc?agagacgccc?aggccgaagt?tcagatgact?aactcagggg 27480
cgcagcttgc?gggcggcttt?cgtcacaggg?tgcggtcgcc?cgggcagggt?ataactcacc 27540
tgacaatcag?agggcgaggt?attcagctca?acgacgagtc?ggtgagctcc?tcgcttggtc 27600
tccgtccgga?cgggacattt?cagatcggcg?gcgccggccg?tccttcattc?acgcctcgtc 27660
aggcaatcct?aactctgcag?acctcgtcct?ctgagccgcg?ctctggaggc?attggaactc 27720
tgcaatttat?tgaggagttt?gtgccatcgg?tctactttaa?ccccttctcg?ggacctcccg 27780
gccactatcc?ggatcaattt?attcctaact?ttgacgcggt?aaaggactcg?gcggacggct 27840
acgactgaat?gttaagtgga?gaggcagagc?aactgcgcct?gaaacacctg?gtccactgtc 27900
gccgccacaa?gtgctttgcc?cgcgactccg?gtgagttttg?ctactttgaa?ttgcccgagg 27960
atcatatcga?gggcccggcg?cacggcgtcc?ggcttaccgc?ccagggagag?cttgcccgta 28020
gcctgattcg?ggagtttacc?cagcgccccc?tgctagttga?gcgggacagg?ggaccctgtg 28080
ttctcactgt?gatttgcaac?tgtcctaacc?ttggattaca?tcaagatctt?tgttgccatc 28140
tctgtgctga?gtataataaa?tacagaaatt?aaaatatact?ggggctccta?tcgccatcct 28200
gtaaacgcca?ccgtcttcac?ccgcccaagc?aaaccaaggc?gaaccttacc?tggtactttt 28260
aacatctctc?cctctgtgat?ttacaacagt?ttcaacccag?acggagtgag?tctacgagag 28320
aacctctccg?agctcagcta?ctccatcaga?aaaaacacca?ccctccttac?ctgccgggaa 28380
cgtacgagtg?cgtcaccggc?cgctgcacca?cacctaccgc?ctgaccgtaa?accagacttt 28440
ttccggacag?acctcaataa?ctctgtttac?cagaacagga?ggtgagctta?gaaaaccctt 28500
agggtattag?gccaaaggcg?caatcgatgc?agctcgttaa?tggttgggcg?ggcgatggga 28560
tctgtctgaa?gcatctcttg?gatgagggag?gcggccacgg?ggttgatgtg?cttgggaata 28620
ctgtattcat?tcttcttgat?ccggaggtag?gtctctttta?ggcaagaagt?ctcaaaaggt 28680
ggtttgccca?ctaacaaggt?atacatgata?cacccaatgg?accacacatc?cacctcgaaa 28740
ctgtgccctt?tcttgctcag?cacctcggga?gctatgtaat?taggagtccc?acacagggtc 28800
ttcttcctct?ccccgtcata?ttcgactttg?gttgccagtc?caaaatcccc?tattttcacc 28860
tccagatctt?cattcaggaa?aaggttgccc?agcttgaggt?ctcgatgaat?aactcggttt 28920
cggtgcaggt?actggcagcc?aagcacaatt?tgccgtaggt?agtatcgggc?ctcaggctca 28980
gtcagggctt?tcctcctctt?gtgcagctcc?aggagagacc?tccggcggca?gagctccaac 29040
accacgaaca?cgaagtcgtt?gtcctcgaaa?aagccgtgga?atcctacgac?gtgctggtgg 29100
gcgaggctgc?ggtgaatgga?tatttccatg?gacatcttct?ccctctggtg?cggcttgagc 29160
agcagagact?taggcacaat?cttgcccgcg?aacacctcct?tggtgtccgc?gtccgagatc 29220
tcgaagcact?tggcaaagcc?gcccttgccc?aaaaagcggc?cccgcacata?gcgccgccgg 29280
ctgcgtgggt?ccactaggac?ctccgggatc?tctttcgccg?gtggagccat?cgatactcta 29340
tgtgggatat?gctccagcgc?tacaaccttg?aagtcaggct?tcctggatgt?cagcatctga 29400
ctttggccag?cacctgtccc?gcggatttgt?tccagtccaa?ctacagcgac?ccaccctaac 29460
agagatgacc?aacacaacca?acgcggccgc?cgctaccgga?cttacatcta?ccacaaatac 29520
accccaagtt?tctgcctttg?tcaataactg?ggataacttg?ggcatgtggt?ggttctccat 29580
agcgcttatg?tttgtatgcc?ttattattat?gtggctcatc?tgctgcctaa?agcgcaaacg 29640
cgcccgacca?cccatctata?gtcccatcat?tgtgctacac?ccaaacaatg?atggaatcca 29700
tagattggac?ggactgaaac?acatgttctt?ttctcttaca?gtatgattaa?atgagacatg 29760
attcctcgag?tttttatatt?actgaccctt?gttgcgcttt?tttgtgcgtg?ctccacattg 29820
gctgcggttt?ctcacatcga?agtagactgc?attccagcct?tcacagtcta?tttgctttac 29880
ggatttgtca?ccctcacgct?catctgcagc?ctcatcactg?tggtcatcgc?ctttatccag 29940
tgcattgact?gggtctgtgt?gcgctttgca?tatctcagac?accatcccca?gtacagggac 30000
aggactatag?ctgagcttct?tagaattctt?taattatgaa?atttactgtg?acttttctgc 30060
tgattatttg?caccctatct?gcgttttgtt?ccccgacctc?caagcctcaa?agacatatat 30120
catgcagatt?cactcgtata?tggaatattc?caagttgcta?caatgaaaaa?agcgatcttt 30180
ccgaagcctg?gttatatgca?atcatctctg?ttatggtgtt?ctgcagtacc?atcttagccc 30240
tagctatata?tccctacctt?gacattggct?ggaaacgaat?agatgccatg?aaccacccaa 30300
ctttccccgc?gcccgctatg?cttccactgc?aacaagttgt?tgccggcggc?tttgtcccag 30360
ccaatcagcc?tcgccccact?tctcccaccc?ccactgaaat?cagctacttt?aatctaacag 30420
gaggagatga?ctgacaccct?agatctagaa?atggacggaa?ttattacaga?gcagcgcctg 30480
ctagaaagac?gcagggcagc?ggccgagcaa?cagcgcatga?atcaagagct?ccaagacatg 30540
gttaacttgc?accagtgcaa?aaggggtatc?ttttgtctgg?taaagcaggc?caaagtcacc 30600
tacgacagta?ataccaccgg?acaccgcctt?agctacaagt?tgccaaccaa?gcgtcagaaa 30660
ttggtggtca?tggtgggaga?aaagcccatt?accataactc?agcactcggt?agaaaccgaa 30720
ggctgcattc?actcaccttg?tcaaggacct?gaggatctct?gcacccttat?taagaccctg 30780
tgcggtctca?aagatcttat?tccctttaac?taataaaaaa?aaataataaa?gcatcactta 30840
cttaaaatca?gttagcaaat?ttctgtccag?tttattcagc?agcacctcct?tgccctcctc 30900
ccagctctgg?tattgcagct?tcctcctggc?tgcaaacttt?ctccacaatc?taaatggaat 30960
gtcagtttcc?tcctgttcct?gtccatccgc?acccactatc?ttcatgttgt?tgcagatgaa 31020
gcgcgcaaga?ccgtctgaag?ataccttcaa?ccccgtgtat?ccatatgaca?cggaaaccgg 31080
tcctccaact?gtgccttttc?ttactcctcc?ctttgtatcc?cccaatgggt?ttcaagagag 31140
tccccctggg?gtactctctt?tgcgcctatc?cgaacctcta?gttacctcca?atggcatgct 31200
tgcgctcaaa?atgggcaacg?gcctctctct?ggacgaggcc?ggcaacctta?cctcccaaaa 31260
tgtaaccact?gtgagcccac?ctctcaaaaa?aaccaagtca?aacataaacc?tggaaatatc 31320
tgcacccctc?acagttacct?cagaagccct?aactgtggct?gccgccgcac?ctctaatggt 31380
cgcgggcaac?acactcacca?tgcaatcaca?ggccccgcta?accgtgcacg?actccaaact 31440
tagcattgcc?acccaaggac?ccctcacagt?gtcagaagga?aagctagccc?tgcaaacatc 31500
aggccccctc?accaccaccg?atagcagtac?ccttactatc?actgcctcac?cccctctaac 31560
tactgccact?ggtagcttgg?gcattgactt?gaaagagccc?atttatacac?aaaatggaaa 31620
actaggacta?aagtacgggg?ctcctttgca?tgtaacagac?gacctaaaca?ctttgaccgt 31680
agcaactggt?ccaggtgtga?ctattaataa?tacttccttg?caaactaaag?ttactggagc 31740
cttgggtttt?gattcacaag?gcaatatgca?acttaatgta?gcaggaggac?taaggattga 31800
ttctcaaaac?agacgcctta?tacttgatgt?tagttatccg?tttgatgctc?aaaaccaact 31860
aaatctaaga?ctaggacagg?gccctctttt?tataaactca?gcccacaact?tggatattaa 31920
ctacaacaaa?ggcctttact?tgtttacagc?ttcaaacaat?tccaaaaagc?ttgaggttaa 31980
cctaagcact?gccaaggggt?tgatgtttga?cgctacagcc?atagccatta?atgcaggaga 32040
tgggcttgaa?tttggttcac?ctaatgcacc?aaacacaaat?cccctcaaaa?caaaaattgg 32100
ccatggccta?gaatttgatt?caaacaaggc?tatggttcct?aaactaggaa?ctggccttag 32160
ttttgacagc?acaggtgcca?ttacagtagg?aaacaaaaat?aatgataagc?taactttgtg 32220
gaccacacca?gctccatctc?ctaactgtag?actaaatgca?gagaaagatg?ctaaactcac 32280
tttggtctta?acaaaatgtg?gcagtcaaat?acttgctaca?gtttcagttt?tggctgttaa 32340
aggcagtttg?gctccaatat?ctggaacagt?tcaaagtgct?catcttatta?taagatttga 32400
cgaaaatgga?gtgctactaa?acaattcctt?cctggaccca?gaatattgga?actttagaaa 32460
tggagatctt?actgaaggca?cagcctatac?aaacgctgtt?ggatttatgc?ctaacctatc 32520
agcttatcca?aaatctcacg?gtaaaactgc?caaaagtaac?attgtcagtc?aagtttactt 32580
aaacggagac?aaaactaaac?ctgtaacact?aaccattaca?ctaaacggta?cacaggaaac 32640
aggagacaca?actccaagtg?catactctat?gtcattttca?tgggactggt?ctggccacaa 32700
ctacattaat?gaaatatttg?ccacatcctc?ttacactttt?tcatacattg?cccaagaata 32760
aagaatcgtt?tgtgttatgt?ttcaacgtgt?ttatttttca?attgcagaaa?atttcaagtc 32820
atttttcatt?cagtagtata?gccccaccac?cacatagctt?atacagatca?ccgtacctta 32880
atcaaactca?cagaacccta?gtattcaacc?tgccacctcc?ctcccaacac?acagagtaca 32940
cagtcctttc?tccccggctg?gccttaaaaa?gcatcatatc?atgggtaaca?gacatattct 33000
taggtgttat?attccacacg?gtttcctgtc?gagccaaacg?ctcatcagtg?atattaataa 33060
actccccggg?cagctcactt?aagttcatgt?cgctgtccag?ctgctgagcc?acaggctgct 33120
gtccaacttg?cggttgctta?acgggcggcg?aaggagaagt?ccacgcctac?atgggggtag 33180
agtcataatc?gtgcatcagg?atagggcggt?ggtgctgcag?cagcgcgcga?ataaactgct 33240
gccgccgccg?ctccgtcctg?caggaataca?acatggcagt?ggtctcctca?gcgatgattc 33300
gcaccgcccg?cagcataagg?cgccttgtcc?tccgggcaca?gcagcgcacc?ctgatctcac 33360
ttaaatcagc?acagtaactg?cagcacagca?ccacaatatt?gttcaaaatc?ccacagtgca 33420
aggcgctgta?tccaaagctc?atggcgggga?ccacagaacc?cacgtggcca?tcataccaca 33480
agcgcaggta?gattaagtgg?cgacccctca?taaacacgct?ggacataaac?attacctctt 33540
ttggcatgtt?gtaattcacc?acctcccggt?accatataaa?cctctgatta?aacatggcgc 33600
catccaccac?catcctaaac?cagctggcca?aaacctgccc?gccggctata?cactgcaggg 33660
aaccgggact?ggaacaatga?cagtggagag?cccaggactc?gtaaccatgg?atcatcatgc 33720
tcgtcatgat?atcaatgttg?gcacaacaca?ggcacacgtg?catacacttc?ctcaggatta 33780
caagctcctc?ccgcgttaga?accatatccc?agggaacaac?ccattcctga?atcagcgtaa 33840
atcccacact?gcagggaaga?cctcgcacgt?aactcacgtt?gtgcattgtc?aaagtgttac 33900
attcgggcag?cagcggatga?tcctccagta?tggtagcgcg?ggtttctgtc?tcaaaaggag 33960
gtagacgatc?cctactgtac?ggagtgcgcc?gagacaaccg?agatcgtgtt?ggtcgtagtg 34020
tcatgccaaa?tggaacgccg?gacgtagtca?tatttcctga?agcaaaacca?ggtgcgggcg 34080
tgacaaacag?atctgcgtct?ccggtctcgc?cgcttagatc?gctctgtgta?gtagttgtag 34140
tatatccact?ctctcaaagc?atccaggcgc?cccctggctt?cgggttctat?gtaaactcct 34200
tcatgcgccg?ctgccctgat?aacatccacc?accgcagaat?aagccacacc?cagccaacct 34260
acacattcgt?tctgcgagtc?acacacggga?ggagcgggaa?gagctggaag?aaccatgttt 34320
ttttttttat?tccaaaagat?tatccaaaac?ctcaaaatga?agatctatta?agtgaacgcg 34380
ctcccctccg?gtggcgtggt?caaactctac?agccaaagaa?cagataatgg?catttgtaag 34440
atgttgcaca?atggcttcca?aaaggcaaac?ggccctcacg?tccaagtgga?cgtaaaggct 34500
aaacccttca?gggtgaatct?cctctataaa?cattccagca?ccttcaacca?tgcccaaata 34560
attctcatct?cgccaccttc?tcaatatatc?tctaagcaaa?tcccgaatat?taagtccggc 34620
cattgtaaaa?atctgctcca?gagcgccctc?caccttcagc?ctcaagcagc?gaatcatgat 34680
tgcaaaaatt?caggttcctc?acagacctgt?ataagattca?aaagcggaac?attaacaaaa 34740
ataccgcgat?cccgtaggtc?ccttcgcagg?gccagctgaa?cataatcgtg?caggtctgca 34800
cggaccagcg?cggccacttc?cccgccagga?accttgacaa?aagaacccac?actgattatg 34860
acacgcatac?tcggagctat?gctaaccagc?gtagccccga?tgtaagcttt?gttgcatggg 34920
cggcgatata?aaatgcaagg?tgctgctcaa?aaaatcaggc?aaagcctcgc?gcaaaaaaga 34980
aagcacatcg?tagtcatgct?catgcagata?aaggcaggta?agctccggaa?ccaccacaga 35040
aaaagacacc?atttctctct?caaacatgtc?tgcgggtttc?tgcataaaca?caaaataaaa 35100
taacaaaaaa?acatttaaac?attagaagcc?tgtcttacaa?caggaaaaac?aacccttata 35160
agcataagac?ggactacggc?catgccggcg?tgaccgtaaa?aaaactggtc?accgtgatta 35220
aaaagcacca?ccgacagctc?ctcggtcatg?tccggagtca?taatgtaaga?ctcggtaaac 35280
acatcaggtt?gattcatcgg?tcagtgctaa?aaagcgaccg?aaatagcccg?ggggaataca 35340
tacccgcagg?cgtagagaca?acattacagc?ccccatagga?ggtataacaa?aattaatagg 35400
agagaaaaac?acataaacac?ctgaaaaacc?ctcctgccta?ggcaaaatag?caccctcccg 35460
ctccagaaca?acatacagcg?cttcacagcg?gcagcctaac?agtcagcctt?accagtaaaa 35520
aagaaaacct?attaaaaaaa?caccactcga?cacggcacca?gctcaatcag?tcacagtgta 35580
aaaaagggcc?aagtgcagag?cgagtatata?taggactaaa?aaatgacgta?acggttaaag 35640
tccacaaaaa?acacccagaa?aaccgcacgc?gaacctacgc?ccagaaacga?aagccaaaaa 35700
acccacaact?tcctcaaatc?gtcacttccg?ttttcccacg?ttacgtaact?tcccatttta 35760
agaaaactac?aattcccaac?acatacaagt?tactccgccc?taaaacctac?gtcacccgcc 35820
ccgttcccac?gccccgcgcc?acgtcacaaa?ctccaccccc?tcattatcat?attggcttca 35880
atccaaaata?aggtatatta?ttgatgatg 35909
Appendix 1: sequence and the structure explanation of recombination adenovirus construction body Ad5/ Δ E1/ Δ ADP/ASSTAT3
1?catcatcaat?aatatacctt?attttggatt?gaagccaata?tgataatgag?ggggtggagt
61?ttgtgacgtg?gcgcggggcg?tgggaacggg?gcgggtgacg?tagtagtgtg?gcggaagtgt
121?gatgttgcaa?gtgtggcgga?acacatgtaa?gcgacggatg?tggcaaaagt?gacgtttttg
181?gtgtgcgccg?gtgtacacag?gaagtgacaa?ttttcgcgcg?gttttaggcg?gatgttgtag
241?taaatttggg?cgtaaccgag?taagatttgg?ccattttcgc?gggaaaactg?aataagagga
301?agtgaaatct?gaataatttt?gtgttactca?tagcgcgtaa?tatttgtcta?gggccgcggg
361?gactttgacc?gtttacgtgg?agactcgccc?aggtgttttt?ctcaggtgtt?ttccgcgttc
421?cgggtcaaag?ttggcgtttt?attattatag?tcagctgacg?tgtagtgtat?ttatacccgg
481?tgagttcctc?aagaggccac?tcttgagtgc?cagcgagtag?agttttctcc?tccgagccgc
541?tccgacaccg?ggactgaaaa?tgagacatat?tatctgccac?ggaggtgtta?ttaccgaaga
601?aatggccgcc?agtcttttgg?accagctgat?cgaagaggta?ctggctgata?atcttccacc
661?tcctagccat?tttgaaccac?ctacccttca?cgaactgtat?gatttagacg?tgacggcccc
721?cgaagatccc?aacgaggagg?cggtttcgca?gatttttccc?gactctgtaa?tgttggcggt
781?gcaggaaggg?attgacttac?tcacttttcc?gccggcgccc?ggttctccgg?agccgcctca
841?cctttcccgg?cagcccgagc?agccggagca?gagagccttg?ggtccggttt?ctatgccaaa
901?ccttgtaccg?gaggtgatcg?at ccac?ccagtgacga
Vacancy partly is the disappearance part
961?cgaggatgaa?gagggtgagg?agtttgtgtt?agattatgtg?gagcaccccg?ggcacggttg
1021?caggtcttgt?cattatcacc?ggaggaatac?gggggaccca?gatattatgt?gttcgctttg
1081?ctatatgagg?acctgtggca?tgtttgtcta?cagtaagtga?aaattatggg?cagtgggtga
1141?tagagtggtg?ggtttggtgt?ggtaattttt?tttttaattt?ttacagtttt?gtggtttaaa
1201?gaattttgta?ttgtgatttt?tttaaaaggt?cctgtgtctg?aacctgagcc?tgagcccgag
1261?ccagaaccgg?agcctgcaag?acctacccgc?cgtcctaaaa?tggcgcctgc?tatcctgaga
1321?cgcccgacat?cacctgtgtc?tagagaatgc?aatagtagta?cggatagctg?tgactccggt
1381?ccttctaaca?cacctcctga?gatacacccg?gtggtcccgc?tgtgccccat?taaaccagtt
1441?gccgtgagag?ttggtgggcg?tcgccaggct?gtggaatgta?tcgaggactt?gcttaacgag
1501?cctgggcaac?ctttggactt?gagctgtaaa?cgccccaggc?cataaggtgt?aaacctgtga
1561?ttgcgtgtgt?ggttaacgcc?tttgtttgct?gaatgagttg?atgtaagttt?aataaagggt
1621?gagataatgt?ttaacttgca?tggcgtgtta?aatggggcgg?ggcttaaagg?gtatataatg
1681?cgccgtgggc?taatcttggt?tacatctgac?ctcatggagg?cttgggagtg?tttggaagat
1741?ttttctgctg?tgcgtaactt?gctggaacag?agctctaaca?gtacctcttg?gttttggagg
1801?tttctgtggg?gctcatccca?ggcaaagtta?gtctgcagaa?ttaaggagga?ttacaagtgg
1861?gaatttgaag?agcttttgaa?atcctgtggt?gagctgtttg?attctttgaa?tctgggtcac
1921?caggcgcttt?tccaagagaa?ggtcatcaag?actttggatt?tttccacacc?ggggcgcgct
1981?gcggctgctg?ttgctttttt?gagttttata?aaggataaat?ggagcgaaga?aacccatctg
2041?agcggggggt?acctgctgga?ttttctggcc?atgcatctgt?ggagagcggt?tgtgagacac
2101?aagaatcgcc?tgctactgtt?gtcttccgtc?cgcccggcga?taataccgac?ggaggagcag
2161?cagcagcagc?aggaggaagc?caggcggcgg?cggcaggagc?agagcccatg?gaacccgaga
2221?gccggcctgg?accctcggga?atgaatgttg?tacaggtggc?tgaactgtat?ccagaactga
2281?gacgcatttt?gacaattaca?gaggatgggc?aggggctaaa?gggggtaaag?agggagcggg
2341?gggcttgtga?ggctacagag?gaggctagga?atctagcttt?tagcttaatg?accagacacc
2401?gtcctgagtg?tattactttt?caacagatca?aggataattg?cgctaatgag?cttgatctgc
2461?tggcgcagaa?gtattccata?gagcagctga?ccacttactg?gctgcagcca?ggggatgatt
2521?ttgaggaggc?tattagggta?tatgcaaagg?tggcacttag?gccagattgc?aagtacaaga
2581?tcagcaaact?tgtaaatatc?aggaattgtt?gctacatttc?tgggaacggg?gccgaggtgg
2641?agatagatac?ggaggatagg?gtggccttta?gatgtagcat?gataaatatg?tggccggggg
2701?tgcttggcat?ggacggggtg?gttattatga?atgtaaggtt?tactggcccc?aattttagcg
2761?gtacggtttt?cctggccaat?accaacctta?tcctacacgg?tgtaagcttc?tatgggttta
2821?acaatacctg?tgtggaagcc?tggaccgatg?taagggttcg?gggctgtgcc?ttttactgct
2881?gctggaaggg?ggtggtgtgt?cgccccaaaa?gcagggcttc?aattaagaaa?tgcctctttg
2941?aaaggtgtac?cttgggtatc?ctgtctgagg?gtaactccag?ggtgcgccac?aatgtggcct
3001?ccgactgtgg?ttgcttcatg?ctagtgaaaa?gcgtggctgt?gattaagcat?aacatggtat
3061?gtggcaactg?cgaggacagg?gcctctcaga?tgctgacctg?ctcggacggc?aactgtcacc
3121?tgctgaagac?cattcacgta?gccagccact?ctcgcaaggc?ctggccagtg?tttgagcata
3181?acatactgac?ccgctgttcc?ttgcatttgg?gtaacaggag?gggggtgttc?ctaccttacc
3241?aatgcaattt?gagtcacact?aagatattgc?ttgagcccga?gagcatgtcc?aaggtgaacc
3301?tgaacggggt?gtttgacatg?accatgaaga?tctggaaggt?gctgaggtac?gatgagaccc
3361?gcaccaggtg?cagaccctgc?gagtgtggcg?gtaaacatat?taggaaccag?cctgtgatgc
3421?tggatgtgac?cgaggagctg?aggcccgatc?acttggtgct?ggcctgcacc?cgcgctgagt
3481?ttggctctag?cgatgaagat?acagattgag?gtactgaaat?gtgtgggcgt?ggcttaaggg
3541?tgggaaagaa?tatataaggt?gggggtctta?tgtagttttg?tatctgtttt?gcagcagccg
3601?ccgccgccat?gagcaccaac?tcgtttgatg?gaagcattgt?gagctcatat?ttgacaacgc
3661?gcatgccccc?atgggccggg?gtgcgtcaga?atgtgatggg?ctccagcatt?gatggtcgcc
3721?ccgtcctgcc?cgcaaactct?actaccttga?cctacgagac?cgtgtctgga?acgccgttgg
3781?agactgcagc?ctccgccgcc?gcttcagccg?ctgcagccac?cgcccgcggg?attgtgactg
3841?actttgcttt?cctgagcccg?cttgcaagca?gtgcagcttc?ccgttcatcc?gcccgcgatg
3901?acaagttgac?ggctcttttg?gcacaattgg?attctttgac?ccgggaactt?aatgtcgttt
3961?ctcagcagct?gttggatctg?cgccagcagg?tttctgccct?gaaggcttcc?tcccctccca
4021?atgcggttta?aaacataaat?aaaaaaccag?actctgtttg?gatttggatc?aagcaagtgt
4081?cttgctgtct?ttatttaggg?gttttgcgcg?cgcggtaggc?ccgggaccag?cggtctcggt
4141?cgttgagggt?cctgtgtatt?ttttccagga?cgtggtaaag?gtgactctgg?atgttcagat
4201?acatgggcat?aagcccgtct?ctggggtgga?ggtagcacca?ctgcagagct?tcatgctgcg
4261?gggtggtgtt?gtagatgatc?cagtcgtagc?aggagcgctg?ggcgtggtgc?ctaaaaatgt
4321?ctttcagtag?caagctgatt?gccaggggca?ggcccttggt?gtaagtgttt?acaaagcggt
4381?taagctggga?tgggtgcata?cgtggggata?tgagatgcat?cttggactgt?atttttaggt
4441?tggctatgtt?cccagccata?tccctccggg?gattcatgtt?gtgcagaacc?accagcacag
4501?tgtatccggt?gcacttggga?aatttgtcat?gtagcttaga?aggaaatgcg?tggaagaact
4561?tggagacgcc?cttgtgacct?ccaagatttt?ccatgcattc?gtccataatg?atggcaatgg
4621?gcccacgggc?ggcggcctgg?gcgaagatat?ttctgggatc?actaacgtca?tagttgtgtt
4681?ccaggatgag?atcgtcatag?gccattttta?caaagcgcgg?gcggagggtg?ccagactgcg
4741?gtataatggt?tccatccggc?ccaggggcgt?agttaccctc?acagatttgc?atttcccacg
4801?ctttgagttc?agatgggggg?atcatgtcta?cctgcggggc?gatgaagaaa?acggtttccg
4861?gggtagggga?gatcagctgg?gaagaaagca?ggttcctgag?cagctgcgac?ttaccgcagc
4921?cggtgggccc?gtaaatcaca?cctattaccg?ggtgcaactg?gtagttaaga?gagctgcagc
4981?tgccgtcatc?cctgagcagg?ggggccactt?cgttaagcat?gtccctgact?cgcatgtttt
5041?ccctgaccaa?atccgccaga?aggcgctcgc?cgcccagcga?tagcagttct?tgcaaggaag
5101?caaagttttt?caacggtttg?agaccgtccg?ccgtaggcat?gcttttgagc?gtttgaccaa
5161?gcagttccag?gcggtcccac?agctcggtca?cctgctctac?ggcatctcga?tccagcatat
5221?ctcctcgttt?cgcgggttgg?ggcggctttc?gctgtacggc?agtagtcggt?gctcgtccag
5281?acgggccagg?gtcatgtctt?tccacgggcg?cagggtcctc?gtcagcgtag?tctgggtcac
5341?ggtgaagggg?tgcgctccgg?gctgcgcgct?ggccagggtg?cgcttgaggc?tggtcctgct
5401?ggtgctgaag?cgctgccggt?cttcgccctg?cgcgtcggcc?aggtagcatt?tgaccatggt
5461?gtcatagtcc?agcccctccg?cggcgtggcc?cttggcgcgc?agcttgccct?tggaggaggc
5521?gccgcacgag?gggcagtgca?gacttttgag?ggcgtagagc?ttgggcgcga?gaaataccga
5581?ttccggggag?taggcatccg?cgccgcaggc?cccgcagacg?gtctcgcatt?ccacgagcca
5641?ggtgagctct?ggccgttcgg?ggtcaaaaac?caggtttccc?ccatgctttt?tgatgcgttt
5701?cttacctctg?gtttccatga?gccggtgtcc?acgctcggtg?acgaaaaggc?tgtccgtgtc
5761?cccgtataca?gacttgagag?gcctgtcctc?gagcggtgtt?ccgcggtcct?cctcgtatag
5821?aaactcggac?cactctgaga?caaaggctcg?cgtccaggcc?agcacgaagg?aggctaagtg
5881?ggaggggtag?cggtcgttgt?ccactagggg?gtccactcgc?tccagggtgt?gaagacacat
5941?gtcgccctct?tcggcatcaa?ggaaggtgat?tggtttgtag?gtgtaggcca?cgtgaccggg
6001?tgttcctgaa?ggggggctat?aaaagggggt?gggggcgcgt?tcgtcctcac?tctcttccgc
6061?atcgctgtct?gcgagggcca?gctgttgggg?tgagtactcc?ctctgaaaag?cgggcatgac
6121?ttctgcgcta?agattgtcag?tttccaaaaa?cgaggaggat?ttgatattca?cctggcccgc
6181?ggtgatgcct?ttgagggtgg?ccgcatccat?ctggtcagaa?aagacaatct?ttttgttgtc
6241?aagcttggtg?gcaaacgacc?cgtagagggc?gttggacagc?aacttggcga?tggagcgcag
6301?ggtttggttt?ttgtcgcgat?cggcgcgctc?cttggccgcg?atgtttagct?gcacgtattc
6361?gcgcgcaacg?caccgccatt?cgggaaagac?ggtggtgcgc?tcgtcgggca?ccaggtgcac
6421?gcgccaaccg?cggttgtgca?gggtgacaag?gtcaacgctg?gtggctacct?ctccgcgtag
6481?gcgctcgttg?gtccagcaga?ggcggccgcc?cttgcgcgag?cagaatggcg?gtagggggtc
6541?tagctgcgtc?tcgtccgggg?ggtctgcgtc?cacggtaaag?accccgggca?gcaggcgcgc
6601?gtcgaagtag?tctatcttgc?atccttgcaa?gtctagcgcc?tgctgccatg?cgcgggcggc
6661?aagcgcgcgc?tcgtatgggt?tgagtggggg?accccatggc?atggggtggg?tgagcgcgga
6721?ggcgtacatg?ccgcaaatgt?cgtaaacgta?gaggggctct?ctgagtattc?caagatatgt
6781?agggtagcat?cttccaccgc?ggatgctggc?gcgcacgtaa?tcgtatagtt?cgtgcgaggg
6841?agcgaggagg?tcgggaccga?ggttgctacg?ggcgggctgc?tctgctcgga?agactatctg
6901?cctgaagatg?gcatgtgagt?tggatgatat?ggttggacgc?tggaagacgt?tgaagctggc
6961?gtctgtgaga?cctaccgcgt?cacgcacgaa?ggaggcgtag?gagtcgcgca?gcttgttgac
7021?cagctcggcg?gtgacctgca?cgtctagggc?gcagtagtcc?agggtttcct?tgatgatgtc
7081?atacttatcc?tgtccctttt?ttttccacag?ctcgcggttg?aggacaaact?cttcgcggtc
7141?tttccagtac?tcttggatcg?gaaacccgtc?ggcctccgaa?cggtaagagc?ctagcatgta
7201?gaactggttg?acggcctggt?aggcgcagca?tcccttttct?acgggtagcg?cgtatgcctg
7261?cgcggccttc?cggagcgagg?tgtgggtgag?cgcaaaggtg?tccctgacca?tgactttgag
7321?gtactggtat?ttgaagtcag?tgtcgtcgca?tccgccctgc?tcccagagca?aaaagtccgt
7381?gcgctttttg?gaacgcggat?ttggcagggc?gaaggtgaca?tcgttgaaga?gtatctttcc
7441?cgcgcgaggc?ataaagttgc?gtgtgatgcg?gaagggtccc?ggcacctcgg?aacggttgtt
7501?aattacctgg?gcggcgagca?cgatctcgtc?aaagccgttg?atgttgtggc?ccacaatgta
7561?aagttccaag?aagcgcggga?tgcccttgat?ggaaggcaat?tttttaagtt?cctcgtaggt
7621?gagctcttca?ggggagctga?gcccgtgctc?tgaaagggcc?cagtctgcaa?gatgagggtt
7681?ggaagcgacg?aatgagctcc?acaggtcacg?ggccattagc?atttgcaggt?ggtcgcgaaa
7741?ggtcctaaac?tggcgaccta?tggccatttt?ttctggggtg?atgcagtaga?aggtaagcgg
7801 gtcttgttcc?cagcggtccc?atccaaggtt?cgcggctagg?tctcgcgcgg?cagtcactag
7861 aggctcatct?ccgccgaact?tcatgaccag?catgaagggc?acgagctgct?tcccaaaggc
7921 ccccatccaa?gtataggtct?ctacatcgta?ggtgacaaag?agacgctcgg?tgcgaggatg
7981 cgagccgatc?gggaagaact?ggatctcccg?ccaccaattg?gaggagtggc?tattgatgtg
8041 gtgaaagtag?aagtccctgc?gacgggccga?acactcgtgc?tggcttttgt?aaaaacgtgc
8101 gcagtactgg?cagcggtgca?cgggctgtac?atcctgcacg?aggttgacct?gacgaccgcg
8161 cacaaggaag?cagagtggga?atttgagccc?ctcgcctggc?gggtttggct?ggtggtcttc
8221 tacttcggct?gcttgtcctt?gaccgtctgg?ctgctcgagg?ggagttacgg?tggatcggac
8281 caccacgccg?cgcgagccca?aagtccagat?gtccgcgcgc?ggcggtcgga?gcttgatgac
8341 aacatcgcgc?agatgggagc?tgtccatggt?ctggagctcc?cgcggcgtca?ggtcaggcgg
8401 gagctcctgc?aggtttacct?cgcatagacg?ggtcagggcg?cgggctagat?ccaggtgata
8461 cctaatttcc?aggggctggt?tggtggcggc?gtcgatggct?tgcaagaggc?cgcatccccg
8521 cggcgcgact?acggtaccgc?gcggcgggcg?gtgggccgcg?ggggtgtcct?tggatgatgc
8581 atctaaaagc?ggtgacgcgg?gcgagccccc?ggaggtaggg?ggggctccgg?acccgccggg
8641 agagggggca?ggggcacgtc?ggcgccgcgc?gcgggcagga?gctggtgctg?cgcgcgtagg
8701 ttgctggcga?acgcgacgac?gcggcggttg?atctcctgaa?tctggcgcct?ctgcgtgaag
8761 acgacgggcc?cggtgagctt?gagcctgaaa?gagagttcga?cagaatcaat?ttcggtgtcg
8821 ttgacggcgg?cctggcgcaa?aatctcctgc?acgtctcctg?agttgtcttg?ataggcgatc
8881 tcggccatga?actgctcgat?ctcttcctcc?tggagatctc?cgcgtccggc?tcgctccacg
8941 gtggcggcga?ggtcgttgga?aatgcgggcc?atgagctgcg?agaaggcgtt?gaggcctccc
9001 tcgttccaga?cgcggctgta?gaccacgccc?ccttcggcat?cgcgggcgcg?catgaccacc
9061 tgcgcgagat?tgagctccac?gtgccgggcg?aagacggcgt?agtttcgcag?gcgctgaaag
9121 aggtagttga?gggtggtggc?ggtgtgttct?gccacgaaga?agtacataac?ccagcgtcgc
9181 aacgtggatt?cgttgatatc?ccccaaggcc?tcaaggcgct?ccatggcctc?gtagaagtcc
9241 acggcgaagt?tgaaaaactg?ggagttgcgc?gccgacacgg?ttaactcctc?ctccagaaga
9301 cggatgagct?cggcgacagt?gtcgcgcacc?tcgcgctcaa?aggctacagg?ggcctcttct
9361 tcttcttcaa?tctcctcttc?cataagggcc?tccccttctt?cttcttctgg?cggcggtggg
9421 ggagggggga?cacggcggcg?acgacggcgc?accgggaggc?ggtcgacaaa?gcgctcgatc
9481 atctccccgc?ggcgacggcg?catggtctcg?gtgacggcgc?ggccgttctc?gcgggggcgc
9541 agttggaaga?cgccgcccgt?catgtcccgg?ttatgggttg?gcggggggct?gccatgcggc
9601 agggatacgg?cgctaacgat?gcatctcaac?aattgttgtg?taggtactcc?gccgccgagg
9661 gacctgagcg?agtccgcatc?gaccggatcg?gaaaacctct?cgagaaaggc?gtctaaccag
9721 tcacagtcgc?aaggtaggct?gagcaccgtg?gcgggcggca?gcgggcggcg?gtcggggttg
9781 tttctggcgg?aggtgctgct?gatgatgtaa?ttaaagtagg?cggtcttgag?acggcggatg
9841 gtcgacagaa?gcaccatgtc?cttgggtccg?gcctgctgaa?tgcgcaggcg?gtcggccatg
9901 ccccaggctt?cgttttgaca?tcggcgcagg?tctttgtagt?agtcttgcat?gagcctttct
9961 accggcactt?cttcttctcc?ttcctcttgt?cctgcatctc?ttgcatctat?cgctgcggcg
10021?gcggcggagt?ttggccgtag?gtggcgccct?cttcctccca?tgcgtgtgac?cccgaagccc
10081?ctcatcggct?gaagcagggc?taggtcggcg?acaacgcgct?cggctaatat?ggcctgctgc
10141?acctgcgtga?gggtagactg?gaagtcatcc?atgtccacaa?agcggtggta?tgcgcccgtg
10201?ttgatggtgt?aagtgcagtt?ggccataacg?gaccagttaa?cggtctggtg?acccggctgc
10261?gagagctcgg?tgtacctgag?acgcgagtaa?gccctcgagt?caaatacgta?gtcgttgcaa
10321?gtccgcacca?ggtactggta?tcccaccaaa?aagtgcggcg?gcggctggcg?gtagaggggc
10381?cagcgtaggg?tggccggggc?tccgggggcg?agatcttcca?acataaggcg?atgatatccg
10441?tagatgtacc?tggacatcca?ggtgatgccg?gcggcggtgg?tggaggcgcg?cggaaagtcg
10501?cggacgcggt?tccagatgtt?gcgcagcggc?aaaaagtgct?ccatggtcgg?gacgctctgg
10561?ccggtcaggc?gcgcgcaatc?gttgacgctc?tagaccgtgc?aaaaggagag?cctgtaagcg
10621?ggcactcttc?cgtggtctgg?tggataaatt?cgcaagggta?tcatggcgga?cgaccggggt
10681?tcgagccccg?tatccggccg?tccgccgtga?tccatgcggt?taccgcccgc?gtgtcgaacc
10741?caggtgtgcg?acgtcagaca?acgggggagt?gctccttttg?gcttccttcc?aggcgcggcg
10801?gctgctgcgc?tagctttttt?ggccactggc?cgcgcgcagc?gtaagcggtt?aggctggaaa
10861?gcgaaagcat?taagtggctc?gctccctgta?gccggagggt?tattttccaa?gggttgagtc
10921?gcgggacccc?cggttcgagt?ctcggaccgg?ccggactgcg?gcgaacgggg?gtttgcctcc
10981?ccgtcatgca?agaccccgct?tgcaaattcc?tccggaaaca?gggacgagcc?ccttttttgc
11041?ttttcccaga?tgcatccggt?gctgcggcag?atgcgccccc?ctcctcagca?gcggcaagag
11101?caagagcagc?ggcagacatg?cagggcaccc?tcccctcctc?ctaccgcgtc?aggaggggcg
11161?acatccgcgg?ttgacgcggc?agcagatggt?gattacgaac?ccccgcggcg?ccgggcccgg
11221?cactacctgg?acttggagga?gggcgagggc?ctggcgcggc?taggagcgcc?ctctcctgag
11281?cggtacccaa?gggtgcagct?gaagcgtgat?acgcgtgagg?cgtacgtgcc?gcggcagaac
11341?ctgtttcgcg?accgcgaggg?agaggagccc?gaggagatgc?gggatcgaaa?gttccacgca
11401?gggcgcgagc?tgcggcatgg?cctgaatcgc?gagcggttgc?tgcgcgagga?ggactttgag
11461?cccgacgcgc?gaaccgggat?tagtcccgcg?cgcgcacacg?tggcggccgc?cgacctggta
11521?accgcatacg?agcagacggt?gaaccaggag?attaactttc?aaaaaagctt?taacaaccac
11581?gtgcgtacgc?ttgtggcgcg?cgaggaggtg?gctataggac?tgatgcatct?gtgggacttt
11641?gtaagcgcgc?tggagcaaaa?cccaaatagc?aagccgctca?tggcgcagct?gttccttata
11701?gtgcagcaca?gcagggacaa?cgaggcattc?agggatgcgc?tgctaaacat?agtagagccc
11761?gagggccgct?ggctgctcga?tttgataaac?atcctgcaga?gcatagtggt?gcaggagcgc
11821?agcttgagcc?tggctgacaa?ggtggccgcc?atcaactatt?ccatgcttag?cctgggcaag
11881?ttttacgccc?gcaagatata?ccatacccct?tacgttccca?tagacaagga?ggtaaagatc
11941?gaggggttct?acatgcgcat?ggcgctgaag?gtgcttacct?tgagcgacga?cctgggcgtt
12001?tatcgcaacg?agcgcatcca?caaggccgtg?agcgtgagcc?ggcggcgcga?gctcagcgac
12061?cgcgagctga?tgcacagcct?gcaaagggcc?ctggctggca?cgggcagcgg?cgatagagag
12121?gccgagtcct?actttgacgc?gggcgctgac?ctgcgctggg?ccccaagccg?acgcgccctg
12181?gaggcagctg?gggccggacc?tgggctggcg?gtggcacccg?cgcgcgctgg?caacgtcggc
12241?ggcgtggagg?aatatgacga?ggacgatgag?tacgagccag?aggacggcga?gtactaagcg
12301?gtgatgtttc?tgatcagatg?atgcaagacg?caacggaccc?ggcggtgcgg?gcggcgctgc
12361?agagccagcc?gtccggcctt?aactccacgg?acgactggcg?ccaggtcatg?gaccgcatca
12421?tgtcgctgac?tgcgcgcaat?cctgacgcgt?tccggcagca?gccgcaggcc?aaccggctct
12481?ccgcaattct?ggaagcggtg?gtcccggcgc?gcgcaaaccc?cacgcacgag?aaggtgctgg
12541?cgatcgtaaa?cgcgctggcc?gaaaacaggg?ccatccggcc?cgacgaggcc?ggcctggtct
12601?acgacgcgct?gcttcagcgc?gtggctcgtt?acaacagcgg?caacgtgcag?accaacctgg
12661?accggctggt?gggggatgtg?cgcgaggccg?tggcgcagcg?tgagcgcgcg?cagcagcagg
12721?gcaacctggg?ctccatggtt?gcactaaacg?ccttcctgag?tacacagccc?gccaacgtgc
12781?cgcggggaca?ggaggactac?accaactttg?tgagcgcact?gcggctaatg?gtgactgaga
12841?caccgcaaag?tgaggtgtac?cagtctgggc?cagactattt?tttccagacc?agtagacaag
12901?gcctgcagac?cgtaaacctg?agccaggctt?tcaaaaactt?gcaggggctg?tggggggtgc
12961?gggctcccac?aggcgaccgc?gcgaccgtgt?ctagcttgct?gacgcccaac?tcgcgcctgt
13021?tgctgctgct?aatagcgccc?ttcacggaca?gtggcagcgt?gtcccgggac?acatacctag
13081?gtcacttgct?gacactgtac?cgcgaggcca?taggtcaggc?gcatgtggac?gagcatactt
13141?tccaggagat?tacaagtgtc?agccgcgcgc?tggggcagga?ggacacgggc?agcctggagg
13201?caaccctaaa?ctacctgctg?accaaccggc?ggcagaagat?cccctcgttg?cacagtttaa
13261?acagcgagga?ggagcgcatt?ttgcgctacg?tgcagcagag?cgtgagcctt?aacctgatgc
13321?gcgacggggt?aacgcccagc?gtggcgctgg?acatgaccgc?gcgcaacatg?gaaccgggca
13381?tgtatgcctc?aaaccggccg?tttatcaacc?gcctaatgga?ctacttgcat?cgcgcggccg
13441?ccgtgaaccc?cgagtatttc?accaatgcca?tcttgaaccc?gcactggcta?ccgccccctg
13501?gtttctacac?cgggggattc?gaggtgcccg?agggtaacga?tggattcctc?tgggacgaca
13561?tagacgacag?cgtgttttcc?ccgcaaccgc?agaccctgct?agagttgcaa?cagcgcgagc
13621?aggcagaggc?ggcgctgcga?aaggaaagct?tccgcaggcc?aagcagcttg?tccgatctag
13681?gcgctgcggc?cccgcggtca?gatgctagta?gcccatttcc?aagcttgata?gggtctctta
13741?ccagcactcg?caccacccgc?ccgcgcctgc?tgggcgagga?ggagtaccta?aacaactcgc
13801?tgctgcagcc?gcagcgcgaa?aaaaacctgc?ctccggcatt?tcccaacaac?gggatagaga
13861?gcctagtgga?caagatgagt?agatggaaga?cgtacgcgca?ggagcacagg?gacgtgccag
13921?gcccgcgccc?gcccacccgt?cgtcaaaggc?acgaccgtca?gcggggtctg?gtgtgggagg
13981?acgatgactc?ggcagacgac?agcagcgtcc?tggatttggg?agggagtggc?aacccgtttg
14041?cgcaccttcg?ccccaggctg?gggagaatgt?tttaaaaaaa?aaaaagcatg?atgcaaaata
14101?aaaaactcac?caaggccatg?gcaccgagcg?ttggttttct?tgtattcccc?ttagtatgcg
14161?gcgcgcggcg?atgtatgagg?aaggtcctcc?tccctcctac?gagagtgtgg?tgagcgcggc
14221?gccagtggcg?gcggcgctgg?gttctccctt?cgatgctccc?ctggacccgc?cgtttgtgcc
14281?tccgcggtac?ctgcggccta?ccggggggag?aaacagcatc?cgttactctg?agttggcacc
14341?cctattcgac?accacccgtg?tgtacctggt?ggacaacaag?tcaacggatg?tggcatccct
14401?gaactaccag?aacgaccaca?gcaactttct?gaccacggtc?attcaaaaca?atgactacag
14461?cccgggggag?gcaagcacac?agaccatcaa?tcttgacgac?cggtcgcact?ggggcggcga
14521?cctgaaaacc?atcctgcata?ccaacatgcc?aaatgtgaac?gagttcatgt?ttaccaataa
14581?gtttaaggcg?cgggtgatgg?tgtcgcgctt?gcctactaag?gacaatcagg?tggagctgaa
14641?atacgagtgg?gtggagttca?cgctgcccga?gggcaactac?tccgagacca?tgaccataga
14701?ccttatgaac?aacgcgatcg?tggagcacta?cttgaaagtg?ggcagacaga?acggggttct
14761?ggaaagcgac?atcggggtaa?agtttgacac?ccgcaacttc?agactggggt?ttgaccccgt
14821?cactggtctt?gtcatgcctg?gggtatatac?aaacgaagcc?ttccatccag?acatcatttt
14881?gctgccagga?tgcggggtgg?acttcaccca?cagccgcctg?agcaacttgt?tgggcatccg
14941?caagcggcaa?cccttccagg?agggctttag?gatcacctac?gatgatctgg?agggtggtaa
15001?cattcccgca?ctgttggatg?tggacgccta?ccaggcgagc?ttgaaagatg?acaccgaaca
15061?gggcgggggt?ggcgcaggcg?gcagcaacag?cagtggcagc?ggcgcggaag?agaactccaa
15121?cgcggcagcc?gcggcaatgc?agccggtgga?ggacatgaac?gatcatgcca?ttcgcggcga
15181?cacctttgcc?acacgggctg?aggagaagcg?cgctgaggcc?gaagcagcgg?ccgaagctgc
15241?cgcccccgct?gcgcaacccg?aggtcgagaa?gcctcagaag?aaaccggtga?tcaaacccct
15301?gacagaggac?agcaagaaac?gcagttacaa?cctaataagc?aatgacagca?ccttcaccca
15361?gtaccgcagc?tggtaccttg?catacaacta?cggcgaccct?cagaccggaa?tccgctcatg
15421?gaccctgctt?tgcactcctg?acgtaacctg?cggctcggag?caggtctact?ggtcgttgcc
15481?agacatgatg?caagaccccg?tgaccttccg?ctccacgcgc?cagatcagca?actttccggt
15541?ggtgggcgcc?gagctgttgc?ccgtgcactc?caagagcttc?tacaacgacc?aggccgtcta
15601?ctcccaactc?atccgccagt?ttacctctct?gacccacgtg?ttcaatcgct?ttcccgagaa
15661?ccagattttg?gcgcgcccgc?cagcccccac?catcaccacc?gtcagtgaaa?acgttcctgc
15721?tctcacagat?cacgggacgc?taccgctgcg?caacagcatc?ggaggagtcc?agcgagtgac
15781?cattactgac?gccagacgcc?gcacctgccc?ctacgtttac?aaggccctgg?gcatagtctc
15841?gccgcgcgtc?ctatcgagcc?gcactttttg?agcaagcatg?tccatcctta?tatcgcccag
15901?caataacaca?ggctggggcc?tgcgcttccc?aagcaagatg?tttggcgggg?ccaagaagcg
15961?ctccgaccaa?cacccagtgc?gcgtgcgcgg?gcactaccgc?gcgccctggg?gcgcgcacaa
16021?acgcggccgc?actgggcgca?ccaccgtcga?tgacgccatc?gacgcggtgg?tggaggaggc
16081?gcgcaactac?acgcccacgc?cgccaccagt?gtccacagtg?gacgcggcca?ttcagaccgt
16141?ggtgcgcgga?gcccggcgct?atgctaaaat?gaagagacgg?cggaggcgcg?tagcacgtcg
16201?ccaccgccgc?cgacccggca?ctgccgccca?acgcgcggcg?gcggccctgc?ttaaccgcgc
16261?acgtcgcacc?ggccgacggg?cggccatgcg?ggccgctcga?aggctggccg?cgggtattgt
16321?cactgtgccc?cccaggtcca?ggcgacgagc?ggccgccgca?gcagccgcgg?ccattagtgc
16381?tatgactcag?ggtcgcaggg?gcaacgtgta?ttgggtgcgc?gactcggtta?gcggcctgcg
16441?cgtgcccgtg?cgcacccgcc?ccccgcgcaa?ctagattgca?agaaaaaact?acttagactc
16501?gtactgttgt?atgtatccag?cggcggcggc?gcgcaacgaa?gctatgtcca?agcgcaaaat
16561?caaagaagag?atgctccagg?tcatcgcgcc?ggagatctat?ggccccccga?agaaggaaga
16621?gcaggattac?aagccccgaa?agctaaagcg?ggtcaaaaag?aaaaagaaag?atgatgatga
16681?tgaacttgac?gacgaggtgg?aactgctgca?cgctaccgcg?cccaggcgac?gggtacagtg
16741?gaaaggtcga?cgcgtaaaac?gtgttttgcg?acccggcacc?accgtagtct?ttacgcccgg
16801?tgagcgctcc?acccgcacct?acaagcgcgt?gtatgatgag?gtgtacggcg?acgaggacct
16861?gcttgagcag?gccaacgagc?gcctcgggga?gtttgcctac?ggaaagcggc?ataaggacat
16921?gctggcgttg?ccgctggacg?agggcaaccc?aacacctagc?ctaaagcccg?taacactgca
16981?gcaggtgctg?cccgcgcttg?caccgtccga?agaaaagcgc?ggcctaaagc?gcgagtctgg
17041?tgacttggca?cccaccgtgc?agctgatggt?acccaagcgc?cagcgactgg?aagatgtctt
17101?ggaaaaaatg?accgtggaac?ctgggctgga?gcccgaggtc?cgcgtgcggc?caatcaagca
17161?ggtggcgccg?ggactgggcg?tgcagaccgt?ggacgttcag?atacccacta?ccagtagcac
17221?cagtattgcc?accgccacag?agggcatgga?gacacaaacg?tccccggttg?cctcagcggt
17281?ggcggatgcc?gcggtgcagg?cggtcgctgc?ggccgcgtcc?aagacctcta?cggaggtgca
17341?aacggacccg?tggatgtttc?gcgtttcagc?cccccggcgc?ccgcgcggtt?cgaggaagta
17401?cggcgccgcc?agcgcgctac?tgcccgaata?tgccctacat?ccttccattg?cgcctacccc
17461?cggctatcgt?ggctacacct?accgccccag?aagacgagca?actacccgac?gccgaaccac
17521?cactggaacc?cgccgccgcc?gtcgccgtcg?ccagcccgtg?ctggccccga?tttccgtgcg
17581?cagggtggct?cgcgaaggag?gcaggaccct?ggtgctgcca?acagcgcgct?accaccccag
17641?catcgtttaa?aagccggtct?ttgtggttct?tgcagatatg?gccctcacct?gccgcctccg
17701?tttcccggtg?ccgggattcc?gaggaagaat?gcaccgtagg?aggggcatgg?ccggccacgg
17761?cctgacgggc?ggcatgcgtc?gtgcgcacca?ccggcggcgg?cgcgcgtcgc?accgtcgcat
17821?gcgcggcggt?atcctgcccc?tccttattcc?actgatcgcc?gcggcgattg?gcgccgtgcc
17881?cggaattgca?tccgtggcct?tgcaggcgca?gagacactga?ttaaaaacaa?gttgcatgtg
17941?gaaaaatcaa?aataaaaagt?ctggactctc?acgctcgctt?ggtcctgtaa?ctattttgta
18001?gaatggaaga?catcaacttt?gcgtctctgg?ccccgcgaca?cggctcgcgc?ccgttcatgg
18061?gaaactggca?agatatcggc?accagcaata?tgagcggtgg?cgccttcagc?tggggctcgc
18121?tgtggagcgg?cattaaaaat?ttcggttcca?ccgttaagaa?ctatggcagc?aaggcctgga
18181?acagcagcac?aggccagatg?ctgagggata?agttgaaaga?gcaaaatttc?caacaaaagg
18241?tggtagatgg?cctggcctct?ggcattagcg?gggtggtgga?cctggccaac?caggcagtgc
18301?aaaataagat?taacagtaag?cttgatcccc?gccctcccgt?agaggagcct?ccaccggccg
18361?tggagacagt?gtctccagag?gggcgtggcg?aaaagcgtcc?gcgccccgac?agggaagaaa
18421?ctctggtgac?gcaaatagac?gagcctccct?cgtacgagga?ggcactaaag?caaggcctgc
18481?ccaccacccg?tcccatcgcg?cccatggcta?ccggagtgct?gggccagcac?acacccgtaa
18541?cgctggacct?gcctcccccc?gccgacaccc?agcagaaacc?tgtgctgcca?ggcccgaccg
18601?ccgttgttgt?aacccgtcct?agccgcgcgt?ccctgcgccg?cgccgccagc?ggtccgcgat
18661?cgttgcggcc?cgtagccagt?ggcaactggc?aaagcacact?gaacagcatc?gtgggtctgg
18721?gggtgcaatc?cctgaagcgc?cgacgatgct?tctgaatagc?taacgtgtcg?tatgtgtgtc
18781?atgtatgcgt?ccatgtcgcc?gccagaggag?ctgctgagcc?gccgcgcgcc?cgctttccaa
18841?gatggctacc?ccttcgatga?tgccgcagtg?gtcttacatg?cacatctcgg?gccaggacgc
18901?ctcggagtac?ctgagccccg?ggctggtgca?gtttgcccgc?gccaccgaga?cgtacttcag
18961?cctgaataac?aagtttagaa?accccacggt?ggcgcctacg?cacgacgtga?ccacagaccg
19021?gtcccagcgt?ttgacgctgc?ggttcatccc?tgtggaccgt?gaggatactg?cgtactcgta
19081?caaggcgcgg?ttcaccctag?ctgtgggtga?taaccgtgtg?ctggacatgg?cttccacgta
19141?ctttgacatc?cgcggcgtgc?tggacagggg?ccctactttt?aagccctact?ctggcactgc
19201?ctacaacgcc?ctggctccca?agggtgcccc?aaatccttgc?gaatgggatg?aagctgctac
19261?tgctcttgaa?ataaacctag?aagaagagga?cgatgacaac?gaagacgaag?tagacgagca
19321?agctgagcag?caaaaaactc?acgtatttgg?gcaggcgcct?tattctggta?taaatattac
19381?aaaggagggt?attcaaatag?gtgtcgaagg?tcaaacacct?aaatatgccg?ataaaacatt
19441?tcaacctgaa?cctcaaatag?gagaatctca?gtggtacgaa?actgaaatta?atcatgcagc
19501?tgggagagtc?cttaaaaaga?ctaccccaat?gaaaccatgt?tacggttcat?atgcaaaacc
19561?cacaaatgaa?aatggagggc?aaggcattct?tgtaaagcaa?caaaatggaa?agctagaaag
19621?tcaagtggaa?atgcaatttt?tctcaactac?tgaggcgacc?gcaggcaatg?gtgataactt
19681?gactcctaaa?gtggtattgt?acagtgaaga?tgtagatata?gaaaccccag?acactcatat
19741?ttcttacatg?cccactatta?aggaaggtaa?ctcacgagaa?ctaatgggcc?aacaatctat
19801?gcccaacagg?cctaattaca?ttgcttttag?ggacaatttt?attggtctaa?tgtattacaa
19861?cagcacgggt?aatatgggtg?ttctggcggg?ccaagcatcg?cagttgaatg?ctgttgtaga
19921?tttgcaagac?agaaacacag?agctttcata?ccagcttttg?cttgattcca?ttggtgatag
19981?aaccaggtac?ttttctatgt?ggaatcaggc?tgttgacagc?tatgatccag?atgttagaat
20041?tattgaaaat?catggaactg?aagatgaact?tccaaattac?tgctttccac?tgggaggtgt
20101?gattaataca?gagactctta?ccaaggtaaa?acctaaaaca?ggtcaggaaa?atggatggga
20161?aaaagatgct?acagaatttt?cagataaaaa?tgaaataaga?gttggaaata?attttgccat
20221?ggaaatcaat?ctaaatgcca?acctgtggag?aaatttcctg?tactccaaca?tagcgctgta
20281?tttgcccgac?aagctaaagt?acagtccttc?caacgtaaaa?atttctgata?acccaaacac
20341?ctacgactac?atgaacaagc?gagtggtggc?tcccgggtta?gtggactgct?acattaacct
20401?tggagcacgc?tggtcccttg?actatatgga?caacgtcaac?ccatttaacc?accaccgcaa
20461?tgctggcctg?cgctaccgct?caatgttgct?gggcaatggt?cgctatgtgc?ccttccacat
20521?ccaggtgcct?cagaagttct?ttgccattaa?aaacctcctt?ctcctgccgg?gctcatacac
20581?ctacgagtgg?aacttcagga?aggatgttaa?catggttctg?cagagctccc?taggaaatga
20641?cctaagggtt?gacggagcca?gcattaagtt?tgatagcatt?tgcctttacg?ccaccttctt
20701?ccccatggcc?cacaacaccg?cctccacgct?tgaggccatg?cttagaaacg?acaccaacga
20761?ccagtccttt?aacgactatc?tctccgccgc?caacatgctc?taccctatac?ccgccaacgc
20821?taccaacgtg?cccatatcca?tcccctcccg?caactgggcg?gctttccgcg?gctgggcctt
20881?cacgcgcctt?aagactaagg?aaaccccatc?actgggctcg?ggctacgacc?cttattacac
20941?ctactctggc?tctataccct?acctagatgg?aaccttttac?ctcaaccaca?cctttaagaa
21001?ggtggccatt?acctttgact?cttctgtcag?ctggcctggc?aatgaccgcc?tgcttacccc
21061?caacgagttt?gaaattaagc?gctcagttga?cggggagggt?tacaacgttg?cccagtgtaa
21121?catgaccaaa?gactggttcc?tggtacaaat?gctagctaac?tacaacattg?gctaccaggg
21181?cttctatatc?ccagagagct?acaaggaccg?catgtactcc?ttctttagaa?acttccagcc
21241?catgagccgt?caggtggtgg?atgatactaa?atacaaggac?taccaacagg?tgggcatcct
21301?acaccaacac?aacaactctg?gatttgttgg?ctaccttgcc?cccaccatgc?gcgaaggaca
21361?ggcctaccct?gctaacttcc?cctatccgct?tataggcaag?accgcagttg?acagcattac
21421?ccagaaaaag?tttctttgcg?atcgcaccct?ttggcgcatc?ccattctcca?gtaactttat
21481?gtccatgggc?gcactcacag?acctgggcca?aaaccttctc?tacgccaact?ccgcccacgc
21541?gctagacatg?acttttgagg?tggatcccat?ggacgagccc?acccttcttt?atgttttgtt
21601?tgaagtcttt?gacgtggtcc?gtgtgcaccg?gccgcaccgc?ggcgtcatcg?aaaccgtgta
21661?cctgcgcacg?cccttctcgg?ccggcaacgc?cacaacataa?agaagcaagc?aacatcaaca
21721?acagctgccg?ccatgggctc?cagtgagcag?gaactgaaag?ccattgtcaa?agatcttggt
21781?tgtgggccat?attttttggg?cacctatgac?aagcgctttc?caggctttgt?ttctccacac
21841?aagctcgcct?gcgccatagt?caatacggcc?ggtcgcgaga?ctgggggcgt?acactggatg
21901?gcctttgcct?ggaacccgca?ctcaaaaaca?tgctacctct?ttgagccctt?tggcttttct
21961?gaccagcgac?tcaagcaggt?ttaccagttt?gagtacgagt?cactcctgcg?ccgtagcgcc
22021?attgcttctt?cccccgaccg?ctgtataacg?ctggaaaagt?ccacccaaag?cgtacagggg
22081?cccaactcgg?ccgcctgtgg?actattctgc?tgcatgtttc?tccacgcctt?tgccaactgg
22141?ccccaaactc?ccatggatca?caaccccacc?atgaacctta?ttaccggggt?acccaactcc
22201?atgctcaaca?gtccccaggt?acagcccacc?ctgcgtcgca?accaggaaca?gctctacagc
22261?ttcctggagc?gccactcgcc?ctacttccgc?agccacagtg?cgcagattag?gagcgccact
22321?tctttttgtc?acttgaaaaa?catgtaaaaa?taatgtacta?gagacacttt?caataaaggc
22381?aaatgctttt?atttgtacac?tctcgggtga?ttatttaccc?ccacccttgc?cgtctgcgcc
22441?gtttaaaaat?caaaggggtt?ctgccgcgca?tcgctatgcg?ccactggcag?ggacacgttg
22501?cgatactggt?gtttagtgct?ccacttaaac?tcaggcacaa?ccatccgcgg?cagctcggtg
22561?aagttttcac?tccacaggct?gcgcaccatc?accaacgcgt?ttagcaggtc?gggcgccgat
22621?atcttgaagt?cgcagttggg?gcctccgccc?tgcgcgcgcg?agttgcgata?cacagggttg
22681?cagcactgga?acactatcag?cgccgggtgg?tgcacgctgg?ccagcacgct?cttgtcggag
22741?atcagatccg?cgtccaggtc?ctccgcgttg?ctcagggcga?acggagtcaa?ctttggtagc
22801?tgccttccca?aaaagggcgc?gtgcccaggc?tttgagttgc?actcgcaccg?tagtggcatc
22861?aaaaggtgac?cgtgcccggt?ctgggcgtta?ggatacagcg?cctgcataaa?agccttgatc
22921?tgcttaaaag?ccacctgagc?ctttgcgcct?tcagagaaga?acatgccgca?agacttgccg
22981?gaaaactgat?tggccggaca?ggccgcgtcg?tgcacgcagc?accttgcgtc?ggtgttggag
23041?atctgcacca?catttcggcc?ccaccggttc?ttcacgatct?tggccttgct?agactgctcc
23101?ttcagcgcgc?gctgcccgtt?ttcgctcgtc?acatccattt?caatcacgtg?ctccttattt
23161?atcataatgc?ttccgtgtag?acacttaagc?tcgccttcga?tctcagcgca?gcggtgcagc
23221?cacaacgcgc?agcccgtggg?ctcgtgatgc?ttgtaggtca?cctctgcaaa?cgactgcagg
23281?tacgcctgca?ggaatcgccc?catcatcgtc?acaaaggtct?tgttgctggt?gaaggtcagc
23341?tgcaacccgc?ggtgctcctc?gttcagccag?gtcttgcata?cggccgccag?agcttccact
23401?tggtcaggca?gtagtttgaa?gttcgccttt?agatcgttat?ccacgtggta?cttgtccatc
23461?agcgcgcgcg?cagcctccat?gcccttctcc?cacgcagaca?cgatcggcac?actcagcggg
23521?ttcatcaccg?taatttcact?ttccgcttcg?ctgggctctt?cctcttcctc?ttgcgtccgc
23581?ataccacgcg?ccactgggtc?gtcttcattc?agccgccgca?ctgtgcgctt?acctcctttg
23641?ccatgcttga?ttagcaccgg?tgggttgctg?aaacccacca?tttgtagcgc?cacatcttct
23701?ctttcttcct?cgctgtccac?gattacctct?ggtgatggcg?ggcgctcggg?cttgggagaa
23761?gggcgcttct?ttttcttctt?gggcgcaatg?gccaaatccg?ccgccgaggt?cgatggccgc
23821?gggctgggtg?tgcgcggcac?cagcgcgtct?tgtgatgagt?cttcctcgtc?ctcggactcg
23881?atacgccgcc?tcatccgctt?ttttgggggc?gcccggggag?gcggcggcga?cggggacggg
23941?gacgacacgt?cctccatggt?tgggggacgt?cgcgccgcac?cgcgtccgcg?ctcgggggtg
24001?gtttcgcgct?gctcctcttc?ccgactggcc?atttccttct?cctataggca?gaaaaagatc
24061?atggagtcag?tcgagaagaa?ggacagccta?accgccccct?ctgagttcgc?caccaccgcc
24121?tccaccgatg?ccgccaacgc?gcctaccacc?ttccccgtcg?aggcaccccc?gcttgaggag
24181?gaggaagtga?ttatcgagca?ggacccaggt?tttgtaagcg?aagacgacga?ggaccgctca
24241?gtaccaacag?aggataaaaa?gcaagaccag?gacaacgcag?aggcaaacga?ggaacaagtc
24301?gggcgggggg?acgaaaggca?tggcgactac?ctagatgtgg?gagacgacgt?gctgttgaag
24361?catctgcagc?gccagtgcgc?cattatctgc?gacgcgttgc?aagagcgcag?cgatgtgccc
24421?ctcgccatag?cggatgtcag?ccttgcctac?gaacgccacc?tattctcacc?gcgcgtaccc
24481?cccaaacgcc?aagaaaacgg?cacatgcgag?cccaacccgc?gcctcaactt?ctaccccgta
24541?tttgccgtgc?cagaggtgct?tgccacctat?cacatctttt?tccaaaactg?caagataccc
24601?ctatcctgcc?gtgccaaccg?cagccgagcg?gacaagcagc?tggccttgcg?gcagggcgct
24661?gtcatacctg?atatcgcctc?gctcaacgaa?gtgccaaaaa?tctttgaggg?tcttggacgc
24721?gacgagaagc?gcgcggcaaa?cgctctgcaa?caggaaaaca?gcgaaaatga?aagtcactct
24781?ggagtgttgg?tggaactcga?gggtgacaac?gcgcgcctag?ccgtactaaa?acgcagcatc
24841?gaggtcaccc?actttgccta?cccggcactt?aacctacccc?ccaaggtcat?gagcacagtc
24901?atgagtgagc?tgatcgtgcg?ccgtgcgcag?cccctggaga?gggatgcaaa?tttgcaagaa
24961?caaacagagg?agggcctacc?cgcagttggc?gacgagcagc?tagcgcgctg?gcttcaaacg
25021?cgcgagcctg?ccgacttgga?ggagcgacgc?aaactaatga?tggccgcagt?gctcgttacc
25081?gtggagcttg?agtgcatgca?gcggttcttt?gctgacccgg?agatgcagcg?caagctagag
25141?gaaacattgc?actacacctt?tcgacagggc?tacgtacgcc?aggcctgcaa?gatctccaac
25201?gtggagctct?gcaacctggt?ctcctacctt?ggaattttgc?acgaaaaccg?ccttgggcaa
25261?aacgtgcttc?attccacgct?caagggcgag?gcgcgccgcg?actacgtccg?cgactgcgtt
25321?tacttatttc?tatgctacac?ctggcagacg?gccatgggcg?tttggcagca?gtgcttggag
25381?gagtgcaacc?tcaaggagct?gcagaaactg?ctaaagcaaa?acttgaagga?cctatggacg
25441?gccttcaacg?agcgctccgt?ggccgcgcac?ctggcggaca?tcattttccc?cgaacgcctg
25501?cttaaaaccc?tgcaacaggg?tctgccagac?ttcaccagtc?aaagcatgtt?gcagaacttt
25561?aggaacttta?tcctagagcg?ctcaggaatc?ttgcccgcca?cctgctgtgc?acttcctagc
25621?gactttgtgc?ccattaagta?ccgcgaatgc?cctccgccgc?tttggggcca?ctgctacctt
25681?ctgcagctag?ccaactacct?tgcctaccac?tctgacataa?tggaagacgt?gagcggtgac
25741?ggtctactgg?agtgtcactg?tcgctgcaac?ctatgcaccc?cgcaccgctc?cctggtttgc
25801?aattcgcagc?tgcttaacga?aagtcaaatt?atcggtacct?ttgagctgca?gggtccctcg
25861?cctgacgaaa?agtccgcggc?tccggggttg?aaactcactc?cggggctgtg?gacgtcggct
25921?taccttcgca?aatttgtacc?tgaggactac?cacgcccacg?agattaggtt?ctacgaagac
25981?caatcccgcc?cgccaaatgc?ggagcttacc?gcctgcgtca?ttacccaggg?ccacattctt
26041?ggccaattgc?aagccatcaa?caaagcccgc?caagagtttc?tgctacgaaa?gggacggggg
26101?gtttacttgg?acccccagtc?cggcgaggag?ctcaacccaa?tccccccgcc?gccgcagccc
26161?tatcagcagc?agccgcgggc?ccttgcttcc?caggatggca?cccaaaaaga?agctgcagct
26221?gccgccgcca?cccacggacg?aggaggaata?ctgggacagt?caggcagagg?aggttttgga
26281?cgaggaggag?gaggacatga?tggaagactg?ggagagccta?gacgaggaag?cttccgaggt
26341?cgaagaggtg?tcagacgaaa?caccgtcacc?ctcggtcgca?ttcccctcgc?cggcgcccca
26401?gaaatcggca?accggttcca?gcatggctac?aacctccgct?cctcaggcgc?cgccggcact
26461?gcccgttcgc?cgacccaacc?gtagatggga?caccactgga?accagggccg?gtaagtccaa
26521?gcagccgccg?ccgttagccc?aagagcaaca?acagcgccaa?ggctaccgct?catggcgcgg
26581?gcacaagaac?gccatagttg?cttgcttgca?agactgtggg?ggcaacatct?ccttcgcccg
26641?ccgctttctt?ctctaccatc?acggcgtggc?cttcccccgt?aacatcctgc?attactaccg
26701?tcatctctac?agcccatact?gcaccggcgg?cagcggcagc?ggcagcaaca?gcagcggcca
26761?cacagaagca?aaggcgaccg?gatagcaaga?ctctgacaaa?gcccaagaaa?tccacagcgg
26821?cggcagcagc?aggaggagga?gcgctgcgtc?tggcgcccaa?cgaacccgta?tcgacccgcg
26881?agcttagaaa?caggattttt?cccactctgt?atgctatatt?tcaacagagc?aggggccaag
26941?aacaagagct?gaaaataaaa?aacaggtctc?tgcgatccct?cacccgcagc?tgcctgtatc
27001?acaaaagcga?agatcagctt?cggcgcacgc?tggaagacgc?ggaggctctc?ttcagtaaat
27061?actgcgcgct?gactcttaag?gactagtttc?gcgccctttc?tcaaatttaa?gcgcgaaaac
27121?tacgtcatct?ccagcggcca?cacccggcgc?cagcacctgt?cgtcagcgcc?attatgagca
27181?aggaaattcc?cacgccctac?atgtggagtt?accagccaca?aatgggactt?gcggctggag
27241?ctgcccaaga?ctactcaacc?cgaataaact?acatgagcgc?gggaccccac?atgatatccc
27301?gggtcaacgg?aatccgcgcc?caccgaaacc?gaattctctt?ggaacaggcg?gctattacca
27361?ccacacctcg?taataacctt?aatccccgta?gttggcccgc?tgccctggtg?taccaggaaa
27421?gtcccgctcc?caccactgtg?gtacttccca?gagacgccca?ggccgaagtt?cagatgacta
27481?actcaggggc?gcagcttgcg?ggcggctttc?gtcacagggt?gcggtcgccc?gggcagggta
27541?taactcacct?gacaatcaga?gggcgaggta?ttcagctcaa?cgacgagtcg?gtgagctcct
27601?cgcttggtct?ccgtccggac?gggacatttc?agatcggcgg?cgccggccgt?ccttcattca
27661?cgcctcgtca?ggcaatccta?actctgcaga?cctcgtcctc?tgagccgcgc?tctggaggca
27721?ttggaactct?gcaatttatt?gaggagtttg?tgccatcggt?ctactttaac?cccttctcgg
27781?gacctcccgg?ccactatccg?gatcaattta?ttcctaactt?tgacgcggta?aaggactcgg
27841?cggacggcta?cgactgaatg?ttaagtggag?aggcagagca?actgcgcctg?aaacacctgg
27901?tccactgtcg?ccgccacaag?tgctttgccc?gcgactccgg?tgagttttgc?tactttgaat
27961?tgcccgagga?tcatatcgag?ggcccggcgc?acggcgtccg?gcttaccgcc?cagggagagc
28021?ttgcccgtag?cctgattcgg?gagtttaccc?agcgccccct?gctagttgag?cgggacaggg
28081?gaccctgtgt?tctcactgtg?atttgcaact?gtcctaacct?tggattacat?caagatcttt
28141?gttgccatct?ctgtgctgag?tataataaat?acagaaatta?aaatatactg?gggctcctat
28201?cgccatcctg?taaacgccac?cgtcttcacc?cgcccaagca?aaccaaggcg?aaccttacct
28261?ggtactttta?acatctctcc?ctctgtgatt?tacaacagtt?tcaacccaga?cggagtgagt
28321?ctacgagaga?acctctccga?gctcagctac?tccatcagaa?aaaacaccac?cctccttacc
28381?tgccgggaac?gtacgagtgc?gtcaccggcc?gctgcaccac?acctaccgcc?tgaccgtaaa
28441?ccagactttt?tccggacaga?cctcaataac?tctgtttacc?agaacaggag?gtgagcttag
28501?aaaaccctta?gggtattagg?ccaaaggcgc?agctactgtg?gggtttatga?acaattcaag
28561?caactctacg?ggctattcta?attcaggttt?ctctagaatc?ggggttgggg?ttattctctg
28621?tcttgtgatt?ctctttattc?ttatactaac?gcttctctgc?ctaaggctcg?ccgcctgctg
28681?tgtgcacatt?tgcatttatt?gtcagctttt?taaacgctgg?ggtcgccacc?caagatgatt
28741?aggtacataa?tcctaggttt?actcaccctt?gcgtcagccc?acggtaccac?ccaaaaggtg
28801?gattttaagg?agccagcctg?taatgttaca?ttcgcagctg?aagctaatga?gtgcaccact
28861?cttataaaat?gcaccacaga?acatgaaaag?ctgcttattc?gccacaaaaa?caaaattggc
28921?aagtatgctg?tttatgctat?ttggcagcca?ggtgacacta?cagagtataa?tgttacagtt
28981?ttccagggta?aaagtcataa?aacttttatg?tatacttttc?cattttatga?aatgtgcgac
29041?attaccatgt?acatgagcaa?acagtataag?ttgtggcccc?cacaaaattg?tgtggaaaac
29101?actggcactt?tctgctgcac?tgctatgcta?attacagtgc?tcgctttggt?ctgtacccta
29161?ctctatatta?aatacaaaag?cagacgcagc?tttattgagg?aaaagaaaat?gccttaattt
29221?actaagttac?aaagctaatg?tcaccactaa?ctgctttact?cgctgcttgc?aaaacaaatt
29281?caaaaagtta?gcattataat?tagaatagga?tttaaacccc?ccggtcattt?cctgctcaat
29341?accattcccc?tgaacaattg?actctatgtg?ggatatgctc?cagcgctaca?accttgaagt
29401?caggcttcct?ggatgtcagc?atctgacttt?ggccagcacc?tgtcccgcgg?atttgttcca
29461 gtccaactac agcgac (lacking 29477-29714nt herein)
ATC GAT (the artificial ClaI restriction enzyme site that adds) (following capitalization part is the antisense STAT 3 sequence)
TTGCCGCCTCTTCCAGTCAGCCAGCTCCTCGTCCGTGAGAGTTTTCTGCACGTACTCCATCGCTG
ACAAAAGCCCCGCCAGCTCACTCACGATGCTTCTCCGCATCTGGTCCAGCGCAGTGAGCATCTGT
TCCAGCTGCTGCATCTTCTGCCTGGTCACTGACTGGTTGTTTCCATTCAGATCTTGCATGTCTCC
TTGACTCTTGAGGGTTTTATAGTTGAAATCAAAGTCATCCTGGAGATTCTCTACCACTTTCATTT
TCTGTTCTAGATCCTGCACTCTCTTCCGGACATCCTGAAGGTGCTGCTCCAGCATCTGCTGCT
TCTCCGTCACCACGGCTGCTGTGGGGTGGTTGGCCTGGCCCCCTTGCTGGGCCGCAGTGGCTG
CAGTCTGTAGAAGGCGTGATTCTTCCCACAGGCACCGGGCCACAATCCGGGCAATCTCCATTG
GCTTCTCAAGATACCTGCTCTGAAGAAACTGCTTGATTCTTCGTAGATTGTGCTGATAGAGAA
CATTCGACTCTTGCAGGAAGCGGCTATACTGCTGGTCAATCTCTCCCAGGAGATTATGAAACA
CCAAAGTGGCATGTGATTCTTTGCTGGCCGCATATGCCCAATCTTGACTCTCAATCCAAGGGG
CCAGAAACTGCCGCAGCTCCATTGGGAAGCTGTCACTGTAGAGCTGATGGAGCTGCTCCAGGT
ACCGTGTGTCAAGCTGCTGTAGCTGATTCCATTGGGCCATCCTGTTTCTCCGGCAGAGGCCGA
GAGGCC ATC GAT (the artificial ClaI restriction enzyme site that adds)
29701 atgatgg?aatccataga?ttggacggac?tgaaacacat?gttcttttct
29761?cttacagtat?gattaaatga?gacatgattc?ctcgagtttt?tatattactg?acccttgttg
29821?cgcttttttg?tgcgtgctcc?acattggctg?cggtttctca?catcgaagta?gactgcattc
29881?cagccttcac?agtctatttg?ctttacggat?ttgtcaccct?cacgctcatc?tgcagcctca
29941?tcactgtggt?catcgccttt?atccagtgca?ttgactgggt?ctgtgtgcgc?tttgcatatc
30001?tcagacacca?tccccagtac?agggacagga?ctatagctga?gcttcttaga?attctttaat
30061?tatgaaattt?actgtgactt?ttctgctgat?tatttgcacc?ctatctgcgt?tttgttcccc
30121?gacctccaag?cctcaaagac?atatatcatg?cagattcact?cgtatatgga?atattccaag
30181?ttgctacaat?gaaaaaagcg?atctttccga?agcctggtta?tatgcaatca?tctctgttat
30241?ggtgttctgc?agtaccatct?tagccctagc?tatatatccc?taccttgaca?ttggctggaa
30301?acgaatagat?gccatgaacc?acccaacttt?ccccgcgccc?gctatgcttc?cactgcaaca
30361?agttgttgcc?ggcggctttg?tcccagccaa?tcagcctcgc?cccacttctc?ccacccccac
30421?tgaaatcagc?tactttaatc?taacaggagg?agatgactga?caccctagat?ctagaaatgg
30481?acggaattat?tacagagcag?cgcctgctag?aaagacgcag?ggcagcggcc?gagcaacagc
30541?gcatgaatca?agagctccaa?gacatggtta?acttgcacca?gtgcaaaagg?ggtatctttt
30601?gtctggtaaa?gcaggccaaa?gtcacctacg?acagtaatac?caccggacac?cgccttagct
30661?acaagttgcc?aaccaagcgt?cagaaattgg?tggtcatggt?gggagaaaag?cccattacca
30721?taactcagca?ctcggtagaa?accgaaggct?gcattcactc?accttgtcaa?ggacctgagg
30781?atctctgcac?ccttattaag?accctgtgcg?gtctcaaaga?tcttattccc?tttaactaat
30841?aaaaaaaaat?aataaagcat?cacttactta?aaatcagtta?gcaaatttct?gtccagttta
30901?ttcagcagca?cctccttgcc?ctcctcccag?ctctggtatt?gcagcttcct?cctggctgca
30961?aactttctcc?acaatctaaa?tggaatgtca?gtttcctcct?gttcctgtcc?atccgcaccc
31021?actatcttca?tgttgttgca?gatgaagcgc?gcaagaccgt?ctgaagatac?cttcaacccc
31081?gtgtatccat?atgacacgga?aaccggtcct?ccaactgtgc?cttttcttac?tcctcccttt
31141?gtatccccca?atgggtttca?agagagtccc?cctggggtac?tctctttgcg?cctatccgaa
31201?cctctagtta?cctccaatgg?catgcttgcg?ctcaaaatgg?gcaacggcct?ctctctggac
31261?gaggccggca?accttacctc?ccaaaatgta?accactgtga?gcccacctct?caaaaaaacc
31321?aagtcaaaca?taaacctgga?aatatctgca?cccctcacag?ttacctcaga?agccctaact
31381?gtggctgccg?ccgcacctct?aatggtcgcg?ggcaacacac?tcaccatgca?atcacaggcc
31441?ccgctaaccg?tgcacgactc?caaacttagc?attgccaccc?aaggacccct?cacagtgtca
31501?gaaggaaagc?tagccctgca?aacatcaggc?cccctcacca?ccaccgatag?cagtaccctt
31561?actatcactg?cctcaccccc?tctaactact?gccactggta?gcttgggcat?tgacttgaaa
31621?gagcccattt?atacacaaaa?tggaaaacta?ggactaaagt?acggggctcc?tttgcatgta
31681?acagacgacc?taaacacttt?gaccgtagca?actggtccag?gtgtgactat?taataatact
31741?tccttgcaaa?ctaaagttac?tggagccttg?ggttttgatt?cacaaggcaa?tatgcaactt
31801?aatgtagcag?gaggactaag?gattgattct?caaaacagac?gccttatact?tgatgttagt
31861?tatccgtttg?atgctcaaaa?ccaactaaat?ctaagactag?gacagggccc?tctttttata
31921?aactcagccc?acaacttgga?tattaactac?aacaaaggcc?tttacttgtt?tacagcttca
31981?aacaattcca?aaaagcttga?ggttaaccta?agcactgcca?aggggttgat?gtttgacgct
32041?acagccatag?ccattaatgc?aggagatggg?cttgaatttg?gttcacctaa?tgcaccaaac
32101?acaaatcccc?tcaaaacaaa?aattggccat?ggcctagaat?ttgattcaaa?caaggctatg
32161?gttcctaaac?taggaactgg?ccttagtttt?gacagcacag?gtgccattac?agtaggaaac
32221?aaaaataatg?ataagctaac?tttgtggacc?acaccagctc?catctcctaa?ctgtagacta
32281?aatgcagaga?aagatgctaa?actcactttg?gtcttaacaa?aatgtggcag?tcaaatactt
32341?gctacagttt?cagttttggc?tgttaaaggc?agtttggctc?caatatctgg?aacagttcaa
32401?agtgctcatc?ttattataag?atttgacgaa?aatggagtgc?tactaaacaa?ttccttcctg
32461?gacccagaat?attggaactt?tagaaatgga?gatcttactg?aaggcacagc?ctatacaaac
32521?gctgttggat?ttatgcctaa?cctatcagct?tatccaaaat?ctcacggtaa?aactgccaaa
32581?agtaacattg?tcagtcaagt?ttacttaaac?ggagacaaaa?ctaaacctgt?aacactaacc
32641?attacactaa?acggtacaca?ggaaacagga?gacacaactc?caagtgcata?ctctatgtca
32701?ttttcatggg?actggtctgg?ccacaactac?attaatgaaa?tatttgccac?atcctcttac
32761?actttttcat?acattgccca?agaataaaga?atcgtttgtg?ttatgtttca?acgtgtttat
32821?ttttcaattg?cagaaaattt?caagtcattt?ttcattcagt?agtatagccc?caccaccaca
32881?tagcttatac?agatcaccgt?accttaatca?aactcacaga?accctagtat?tcaacctgcc
32941?acctccctcc?caacacacag?agtacacagt?cctttctccc?cggctggcct?taaaaagcat
33001?catatcatgg?gtaacagaca?tattcttagg?tgttatattc?cacacggttt?cctgtcgagc
33061?caaacgctca?tcagtgatat?taataaactc?cccgggcagc?tcacttaagt?tcatgtcgct
33121?gtccagctgc?tgagccacag?gctgctgtcc?aacttgcggt?tgcttaacgg?gcggcgaagg
33181?agaagtccac?gcctacatgg?gggtagagtc?ataatcgtgc?atcaggatag?ggcggtggtg
33241?ctgcagcagc?gcgcgaataa?actgctgccg?ccgccgctcc?gtcctgcagg?aatacaacat
33301?ggcagtggtc?tcctcagcga?tgattcgcac?cgcccgcagc?ataaggcgcc?ttgtcctccg
33361?ggcacagcag?cgcaccctga?tctcacttaa?atcagcacag?taactgcagc?acagcaccac
33421?aatattgttc?aaaatcccac?agtgcaaggc?gctgtatcca?aagctcatgg?cggggaccac
33481?agaacccacg?tggccatcat?accacaagcg?caggtagatt?aagtggcgac?ccctcataaa
33541?cacgctggac?ataaacatta?cctcttttgg?catgttgtaa?ttcaccacct?cccggtacca
33601?tataaacctc?tgattaaaca?tggcgccatc?caccaccatc?ctaaaccagc?tggccaaaac
33661?ctgcccgccg?gctatacact?gcagggaacc?gggactggaa?caatgacagt?ggagagccca
33721?ggactcgtaa?ccatggatca?tcatgctcgt?catgatatca?atgttggcac?aacacaggca
33781?cacgtgcata?cacttcctca?ggattacaag?ctcctcccgc?gttagaacca?tatcccaggg
33841?aacaacccat?tcctgaatca?gcgtaaatcc?cacactgcag?ggaagacctc?gcacgtaact
33901?cacgttgtgc?attgtcaaag?tgttacattc?gggcagcagc?ggatgatcct?ccagtatggt
33961?agcgcgggtt?tctgtctcaa?aaggaggtag?acgatcccta?ctgtacggag?tgcgccgaga
34021?caaccgagat?cgtgttggtc?gtagtgtcat?gccaaatgga?acgccggacg?tagtcatatt
34081?tcctgaagca?aaaccaggtg?cgggcgtgac?aaacagatct?gcgtctccgg?tctcgccgct
34141?tagatcgctc?tgtgtagtag?ttgtagtata?tccactctct?caaagcatcc?aggcgccccc
34201?tggcttcggg?ttctatgtaa?actccttcat?gcgccgctgc?cctgataaca?tccaccaccg
34261?cagaataagc?cacacccagc?caacctacac?attcgttctg?cgagtcacac?acgggaggag
34321?cgggaagagc?tggaagaacc?atgttttttt?ttttattcca?aaagattatc?caaaacctca
34381?aaatgaagat?ctattaagtg?aacgcgctcc?cctccggtgg?cgtggtcaaa?ctctacagcc
34441?aaagaacaga?taatggcatt?tgtaagatgt?tgcacaatgg?cttccaaaag?gcaaacggcc
34501?ctcacgtcca?agtggacgta?aaggctaaac?ccttcagggt?gaatctcctc?tataaacatt
34561?ccagcacctt?caaccatgcc?caaataattc?tcatctcgcc?accttctcaa?tatatctcta
34621?agcaaatccc?gaatattaag?tccggccatt?gtaaaaatct?gctccagagc?gccctccacc
34681?ttcagcctca?agcagcgaat?catgattgca?aaaattcagg?ttcctcacag?acctgtataa
34741?gattcaaaag?cggaacatta?acaaaaatac?cgcgatcccg?taggtccctt?cgcagggcca
34801?gctgaacata?atcgtgcagg?tctgcacgga?ccagcgcggc?cacttccccg?ccaggaacct
34861?tgacaaaaga?acccacactg?attatgacac?gcatactcgg?agctatgcta?accagcgtag
34921?ccccgatgta?agctttgttg?catgggcggc?gatataaaat?gcaaggtgct?gctcaaaaaa
34981?tcaggcaaag?cctcgcgcaa?aaaagaaagc?acatcgtagt?catgctcatg?cagataaagg
35041?caggtaagct?ccggaaccac?cacagaaaaa?gacaccattt?ttctctcaaa?catgtctgcg
35101?ggtttctgca?taaacacaaa?ataaaataac?aaaaaaacat?ttaaacatta?gaagcctgtc
35161?ttacaacagg?aaaaacaacc?cttataagca?taagacggac?tacggccatg?ccggcgtgac
35221?cgtaaaaaaa?ctggtcaccg?tgattaaaaa?gcaccaccga?cagctcctcg?gtcatgtccg
35281?gagtcataat?gtaagactcg?gtaaacacat?caggttgatt?catcggtcag?tgctaaaaag
35341?cgaccgaaat?agcccggggg?aatacatacc?cgcaggcgta?gagacaacat?tacagccccc
35401?ataggaggta?taacaaaatt?aataggagag?aaaaacacat?aaacacctga?aaaaccctcc
35461?tgcctaggca?aaatagcacc?ctcccgctcc?agaacaacat?acagcgcttc?acagcggcag
35521?cctaacagtc?agccttacca?gtaaaaaaga?aaacctatta?aaaaaacacc?actcgacacg
35581?gcaccagctc?aatcagtcac?agtgtaaaaa?agggccaagt?gcagagcgag?tatatatagg
35641?actaaaaaat?gacgtaacgg?ttaaagtcca?caaaaaacac?ccagaaaacc?gcacgcgaac
35701?ctacgcccag?aaacgaaagc?caaaaaaccc?acaacttcct?caaatcgtca?cttccgtttt
35761?cccacgttac?gtaacttccc?attttaagaa?aactacaatt?cccaacacat?acaagttact
35821?ccgccctaaa?acctacgtca?cccgccccgt?tcccacgccc?cgcgccacgt?cacaaactcc
35881?accccctcat?tatcatattg?gcttcaatcc?aaaataaggt?atattattga?tgatg
Claims (4)
1. recombination adenovirus construction body, its technical characterictic is:
This recombination adenovirus construction body is called Ad5/ Δ E1/ Δ ADP/ASSTAT3, this construct is to have lacked its 920-946 nt GAT CTT ACC TGC CAC GAG GCT GGC TTT on the basis of wild-type Ad5, proteic 121 to 129 amino acids of this sequence encoding Ad5E1A, lack the 29477-29714nt that the E3 district is arranged in the ADP gene simultaneously, introduce a ClaI restriction enzyme site in this disappearance zone; Utilize this restriction enzyme site oppositely to insert the exogenous STAT3 cDNA fragment of 770bp, this fragment is equivalent to the 960-190nt of STAT3 mRNA, and other genome structure and wild-type Ad5 are identical.
2. prepare the method for the described recombination adenovirus construction body of claim 1, it is characterized in that:
Lack the coding region 920-946nt of E1A in the pXC1 plasmid that comprises adenovirus 21nt to 5790nt sequence respectively with pcr amplification fixed point deletion technology, sequence is GAT CTT ACC TGC CAC GAG GCT GGC TTT, form new carrier Δ 920-946pXC1, Δ 920-946pXC1 plasmid and shuttle vectors pBHGE3 cotransfection 293 cells filter out the recombination adenovirus construction body Δ 920-946Ad5 that expresses E1A mutant functional protein; Adopt pcr amplification fixed point deletion techno-absence Ad5 E3 district 29477-29714nt, and at ClaI restriction enzyme site of above-mentioned disappearance zone introducing, form new carrier pCDNA3.1-Δ ADP, oppositely insert the exogenous STAT3 cDNA fragment of 770bp at ClaI restriction enzyme site place, this fragment is equivalent to the 960-190nt of STAT3 mRNA, forms new carrier pCDNA3.1-Δ ADP/ASSTAT3; Extract Δ 920-946 Ad5 TP-DNA, cut,, filter out the recombination adenovirus construction body Ad5/ Δ E1/ Δ ADP/ASSTAT3 that expresses the E1A mutant with carrier pCDNA3.1-Δ ADP/ASSTAT3 fragment cotransfection 293 cells that cut out with the EcoRI enzyme with the EcoRI enzyme.
3. the described recombination adenovirus construction body of claim 1 is used for the application of the medicine of lung cancer, mammary cancer, cancer of the stomach, colorectal carcinoma, prostate cancer and osteoblastoma treatment in preparation.
4. the described recombination adenovirus construction body of claim 1 is in the purposes of preparation in the gene therapy vector.
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| 卢运萍等.腺病毒-单纯疱疹病毒胸苷激酶基因治疗载体的构建及鉴定.《同济医科大学学报》.1999,第28卷(第3期),198-200，203. * |
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