Abstract:
Antisense oligonucleotides are provided which are complementary to at least a portion of HCV RNA and specifically hybridizable therewith. These oligonucleotides can be administered to inhibit the replication of Hepatitis C virus in vivo or in vitro and to treat Hepatitis C virus-associated disease. These compounds can be used either prophylactically or therapeutically to reduce the severity of diseases associated with Hepatitis C virus.

Description:
This is a continuation of application Ser. No. 08/453,085, filed May 30, 1995, now abandoned which is a continuation of application Ser. No. 07/945,289, filed Sep. 10, 1972, now abandoned. 
    
    
     FIELD OF THE INVENTION 
     This invention relates to the design and synthesis of antisense oligonucleotides which can be administered to inhibit the replication of Hepatitis C virus in vivo or in vitro and to treat Hepatitis C virus-associated disease. These compounds can be used either prophylactically or therapeutically to reduce the severity of diseases associated with Hepatitis C virus. Oligonucleotides which are specifically hybridizable with RNA targets are disclosed. 
     BACKGROUND OF THE INVENTION 
     The predominant form of hepatitis currently resulting from transfusions is not related to the previously characterized Hepatitis A virus or Hepatitis B virus and has been referred to as Non-A, Non-B Hepatitis (NANBH). NANBH currently accounts for over 90% of cases of post-transfusion hepatitis. Estimates of the frequency of NANBH in transfusion recipients range from 5%-13% for those receiving volunteer blood, or 25-54% for those receiving blood from commercial sources. 
     Acute NANBH, while often less severe than acute disease caused by Hepatitis A or Hepatitis B viruses, occasionally leads to severe or fulminant hepatitis. Of greater concern, progression to chronic hepatitis is much more common after NANBH than after either Hepatitis A or Hepatitis B infection. Chronic NANBH has been reported in 10%-70% of infected individuals. This form of hepatitis can be transmitted even by asymptomatic patients, and frequently progresses to malignant disease such as cirrhosis and hepatocellular carcinoma. Chronic active hepatitis, with or without cirrhosis, is seen in 44%-90% of posttransfusion hepatitis cases. Of those patients who developed cirrhosis, approximately one-fourth died of liver failure. 
     Chronic active NANBH is a significant problem to hemophiliacs who are dependent on blood products; 5%-11% of hemophiliacs die of chronic end-stage liver disease. Cases of NANBH other than those traceable to blood or blood products are frequently associated with hospital exposure, accidental needle stick, or tattooing. Transmission through close personal contact also occurs, though this is less common for NANBH than for Hepatitis B. 
     The causative agent of the majority of NANBH has recently been identified and is now referred to as Hepatitis C Virus (HCV). Houghton et al., EP Publication 318,216; Choo et al.,  Science  1989, 244, 359-362. Based on serological studies using recombinant DNA-generated antigens it is now clear that HCV is the causative agent of most cases of post-transfusion NANBH. Clones of cDNA prepared from nucleic acid isolated from concentrated virus particles were originally isolated based on their ability to encode polypeptides which reacted with sera from NANBH patients. These clones hybridized with RNA, but not DNA, isolated from infected liver tissue, indicating the presence of an RNA genome. Hybridization analyses and sequencing of the cDNA clones revealed that RNA present in infected liver and particles was the same polarity as that of the coding strand of the cDNAs; in other words, the virus genome is a positive or plus-strand RNA genome. EP Publication 318,216 (Houghton et al.) disclose partial genomic sequences of HCV-1, and teach recombinant DNA methods of cloning and expressing HCV sequences and HCV polypeptides, techniques of HCV immunodiagnostics, HCV probe diagnostic techniques, anti-HCV antibodies, and methods of isolating new HCV sequences. Houghton et al. also disclose additional HCV sequences and teach application of these sequences and polypeptides in immunodiagnostics, probe diagnostics, anti-HCV antibody production, PCR technology and recombinant DNA technology. The concept of using antisense polynucleotides as inhibitors of viral replication is disclosed, but no specific targets are taught. Oligomer probes and primers based on the sequences disclosed are also provided. EP Publication 419,182 (Miyamura et al.) discloses new HCV isolates J1 and J7 and use of sequences distinct from HCV-1 sequences for screens and diagnostics. 
     The only treatment regimen shown to be effective for the treatment of chronic NANBH is interferon-α. Most NANBH patients show an improvement of clinical symptoms during interferon treatment, but relapse is observed in at least half of patients when treatment is interrupted. Significant improvements in antiviral therapy are therefore greatly desired. 
     OBJECTS OF THE INVENTION 
     It is an object of this invention to provide oligonucleotides which are capable of hybridizing with RNA of HCV to inhibit the synthesis or function of said RNA. 
     It is another object of this invention to provide oligonucleotides which are capable of hybridizing with RNA of HCV to inhibit replication of the virus. 
     It is a further object to provide oligonucleotides which can modulate the expression of HCV through antisense interaction with viral RNA. 
     Yet another object of this invention is to provide methods of prophylaxis, diagnostics and therapeutics for acute or chronic HCV infection. 
     A further object of this invention is to provide methods of prophylaxis, diagnostics and therapeutics for HCV-associated diseases. 
     Methods, materials and kits for detecting the presence or absence of HCV or HCV RNA in a sample suspected of containing it are further objects of the invention. 
     These and other objects will become apparent to persons of ordinary skill in the art from a review of the instant specification and appended claims. 
     SUMMARY OF THE INVENTION 
     In accordance with the present invention, compositions and methods for modulating the effects of HCV infection are provided. Oligonucleotides complementary to, and specifically hybridizable with, selected sequences of HCV RNA are provided. The HCV 5′ end hairpin loop, 5′ end 6-base-pair repeats, 5′ end untranslated region, polyprotein translation initiation codon, ORF 3 translation initiation codon, 3′-untranslated region, 3′ end palindrome region, R2 sequence and 3′ end hairpin loop are preferred targets. Methods for diagnosing or treating disease states by administering oligonucleotides, either alone or in combination with a pharmaceutically acceptable carrier, to animals suspected of having HCV-associated diseases are also provided. 
     The relationship between the target RNA and oligonucleotides complementary to at least a portion of the target, and specifically hybridizable with it, is commonly denoted as “antisense”. The oligonucleotides are able to inhibit the function of viral RNA by interfering with its replication, transcription into mRNA, translation into protein, packaging into viral particles or any other activity necessary to its overall biological function. The failure of the RNA to perform all or part of its function results in failure of all or a portion of the normal life cycle of the virus. 
     It has been found that antisense oligonucleotides designed to target viruses can be effective in diminishing viral infection. It is preferred that oligonucleotides have between about 5 and about 50 nucleotide units. It is also preferred that the oligonucleotides be specifically hybridizable with the HCV 5′ end hairpin loop, 5′ end 6-base-pair repeats, 5′ end untranslated region, polyprotein translation initiation codon, ORF 3 translation initiation codon, 3′-untranslated region, 3′ end palindrome region, R2 sequence or 3′ end hairpin loop. The oligonucleotide may be modified to increase nuclease resistance and to increase its efficacy. 
     In accordance with preferred embodiments, the viral RNA is interfered with to an extent sufficient to inhibit HCV infection and/or HCV replication. Thus, oligonucleotides which are capable of interacting with portions of HCV RNA are comprehended. Animals suspected of having HCV-associated disease are contacted with an oligonucleotide made in accordance with this invention. In particular, the present invention is believed to be effective in the treatment of acute and chronic HCV infections and HCV-associated disease, either prophylactically or therapeutically. 
     It is to be expected that differences in the RNA of HCV from different strains and from different types within a strain exist. Thus, it is believed, for example, that the regions of the various HCV strains serve essentially the same function for the respective strains and that interference with expression of the genetic information will afford similar results in the various strains. This is believed to be so even though differences in the nucleotide sequences among the strains exist. 
     Accordingly, nucleotide sequences set forth in the present specification will be understood to be representational for the particular strain being described. Homologous or analogous sequences for different strains of HCV are specifically contemplated as being within the scope of this invention. 
     DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
     Antisense oligonucleotides hold great promise as therapeutic agents for the treatment of many human diseases. In most cases, oligonucleotides complementary to specific RNA target sequences bind by Watson-Crick base pairing to pre-mRNA or mature mRNA, inhibiting the flow of genetic information from DNA to protein. In the case of RNA viruses such as HCV, oligonucleotides are designed to specifically hybridize to viral genomic RNA, mRNA, or replicative intermediate RNA, interfering with the function of the RNA such that viral replication or protein expression is modulated. 
     Numerous recent studies have documented the utility of antisense oligonucleotides as biochemical tools for studying target proteins. Rothenberg et al.,  J. Natl. Cancer Inst.  1989, 81, 1539-1544; Zon, G.  Pharmaceutical Res.  1987, 5, 539-549. Because of recent advances in oligonucleotide chemistry, synthesis of nuclease-resistant oligonucleotides, and availability of types of oligonucleotides which exhibit enhanced cell uptake, it is now possible to consider the use of antisense oligonucleotides as a novel form of therapeutics. 
     For therapeutics, an animal suspected of having an HCV infection or HCV-associated disease is treated by administering oligonucleotides in accordance with this invention. Oligonucleotides may be formulated in a pharmaceutical composition, which may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the oligonucleotide. Pharmaceutical compositions may also include one or more active ingredients such as, for example, antimicrobial agents, antiinflammatory agents, anesthetics, and the like in addition to oligonucleotide. 
     The pharmaceutical composition may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Administration may be topically (including ophthalmically, vaginally, rectally, intranasally), orally, by inhalation, or parenterally, for example by intravenous drip, subcutaneous, intraperitoneal or intramuscular injection. 
     Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms may also be useful. 
     Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable. 
     Formulations for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives. 
     Dosing is dependent on severity and responsiveness of the condition to be treated, but will normally be one or more doses per day, with course of treatment lasting from several days to several months or until a cure is effected or a diminution of disease state is achieved. Dosage and frequency will vary depending on, for example, body weight of patient and means of administration. Individual doses will normally range from about 0.001 mg to 500 mg, but may be higher or lower. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. 
     The present invention employs oligonucleotides complementary to specific regions of HCV. RNA for antisense inhibition of HCV. In the context of this invention, the term “oligonucleotide” refers to an oligomer or polymer of ribonucleic acid or deoxyribonucleic acid. This term includes oligomers consisting of naturally occurring bases, sugars and intersugar (backbone) linkages as well as oligomers having non-naturally occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of properties such as, for example, enhanced cellular uptake and increased stability in the presence of nucleases. 
     Specific examples of some preferred oligonucleotides envisioned for this invention may contain phosphorothioates, phosphotriesters, methyl phosphonates, chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. Most preferred are those with CH 2 —NH—O—CH 2 , CH 2 —N(CH 3 )—O—CH 2 , CH 2 —O—N(CH 3 )—CH 2 , CH 2 —N(CH 3 )—N(CH 3 )—CH 2  and O—N(CH 3 )—CH 2 —CH 2  backbones (where phosphodiester is O—P—O—CH 2 ). Also preferred are oligonucleotides having morpholino backbone structures. Summerton, J. E. and Weller, D. D. U.S. Pat. No. 5,034,506. In other preferred embodiments, such as the protein-nucleic acid (PNA) backbone, the phosphodiester backbone of the oligonucleotide may be replaced with a polyamide backbone, the bases being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone. P. E. Nielsen, M. Egholm, R. H. Berg, O. Buchardt,  Science  1991, 254, 1497. Other preferred oligonucleotides may contain alkyl and halogen-substituted sugar moieties comprising one of the following at the 2′ position: OH, SH, SCH 3 , F, OCN, O(CH 2 ) n NH 2  or O(CH 2 ) n CH 3  where n is from 1 to about 10; C 1  to C 10  lower alkyl, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF 3 ; OCF 3 ; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; SOCH 3 ; SO 2 CH 3 ; ONO 2 ; NO 2 ; N 3 ; NH 2 ; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a conjugate; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an oligonucleotide; or a group for improving the pharmacodynamic properties of an oligonucleotide and other substituents having similar properties. Oligonucleotides may also have sugar mimetics such as cyclobutyls in place of the pentofuranosyl group. 
     All such oligonucleotides are comprehended by this invention so long as they function effectively to hybridize with HCV RNA. The oligonucleotides in accordance with this invention preferably comprise from about 5 to about 50 nucleic acid base units. It is more preferred that such oligonucleotides comprise from about 8 to 30 nucleic acid base units, and still more preferred to have from about 12 to 25 nucleic acid base units. As will be appreciated, a nucleic acid base unit is a base-sugar combination suitably bound to adjacent nucleic acid base unit through phosphodiester or other bonds. 
     The oligonucleotides used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including Applied Biosystems. Any other means for such synthesis may also be employed, however the actual synthesis of the oligonucleotides are well within the talents of the routineer. It is also well known to use similar techniques to prepare other oligonucleotides such as the phosphorothioates and alkylated derivatives. 
     In accordance with this invention, persons of ordinary skill in the art will understand that messenger RNA includes not only the sequence information to encode a protein using the three letter genetic code, but also associated ribonucleotides which form regions known to such persons as the 5′-untranslated region, the 3′-untranslated region, and the 5′ cap region, as well as ribonucleotides which form various secondary structures. Thus, oligonucleotides may be formulated in accordance with this invention which are targeted wholly or in part to these associated ribonucleotides as well as to the coding ribonucleotides. In preferred embodiments, the oligonucleotide is specifically hybridizable with the HCV 5′ end hairpin loop, 5′ end 6-base-pair repeats, ORF 3 translation initiation codon (all of which are contained in the 5′-untranslated region), polyprotein translation initiation codon, 3′-untranslated region, R2 region, 3′ hairpin loop or 3′ end palindrome region. 
     The size of the HCV genome is approximately 9400 nucleotides, with a single translational reading frame encoding a polyprotein which is subsequently processed to several structural and non-structural proteins. 
     Several regions of the HCV genome have been identified as antisense targets in the present invention. It should be noted that sequence availability and nucleotide numbering schemes vary from strain to strain. The 5′ untranslated region of HCV consists of approximately 350 nucleotides upstream of the polyprotein translation initiation codon. A hairpin loop present at nucleotides 1-22 at the 5′ end of the genome (HCV-1) identified herein as the “5′ end hairpin loop” is believed to serve as a recognition signal for the viral replicase or nucleocapsid proteins. Han et al.,  Proc. Natl. Acad. Sci.  1991, 88, 1711-1715. 
     Three small (12-16 amino acids each) open reading frames (ORFs) are located in the 5′-untranslated region of HCV RNA. These ORFs may be involved in control of translation. The ORF 3 translation initiation codon as denominated herein is found at nucleotides 315-317 of HCV-1 according to the scheme of Han et al.,  Proc. Natl. Acad. Sci.  1991, 88, 1711-1715; and at nucleotides-127 to -125 according to the scheme of Choo et al.,  Proc. Natl. Acad. Sci.  1991, 88, 2451-2455 and depicted in SEQ ID NO: 25. 
     The polyprotein translation initiation codon as denominated herein is an AUG sequence located at nucleotides 342-344 of HCV-1 according to Han et al.,  Proc. Natl. Acad. Sci.  1991, 88, 1711-1715 or at nucleotide 1-3 according to the HCV-1 numbering scheme of Choo et al.,  Proc. Natl. Acad. Sci.  1991, 88, 2451-2455 and SEQ ID NO: 25. 
     The 3′ untranslated region, as denominated herein, consists of nucleotides downstream of the polyprotein translation termination site (ending at nt 9037 according to Choo et al. and SEQ ID NO: 25; nt 9377 according to schemes of Han and Inchauspe as depicted in SEQ ID NO: 26). Nucleotides 9697-9716 (numbering scheme of Inchauspe for HCV-H which is depicted in SEQ ID NO: 26) at the 3′ terminus of the genome within the 3′ untranslated region can be organized into a stable hairpin loop structure identified herein as the 3′ hairpin loop. A short nucleotide stretch (R2) immediately upstream (nt 9691-9696 of HCV-H depicted in SEQ ID NO: 26) of the 3′ hairpin, and denominated herein “the R2 sequence”, is thought to play a role in cyclization of the viral RNA, possibly in combination with a set of 5′ end 6-base-pair repeats of the same sequence at nt 23-28 and 38-43. (Inchauspe et al.,  Proc. Natl. Acad. Sci.  1991, 88, 10292-10296) is identified herein as “5′ end 6-base-pair repeat”. Palindrome sequences present near the 3′ end of the genome (nucleotides 9312-9342 according to the scheme of Takamizawa et al.,  J. Virol.  1991, 65, 1105-1113 depicted in SEQ ID NO: 27) are capable of forming a stable secondary structure. This is referred to herein as the 3′ end palindrome region. 
     Oligonucleotides useful in the invention are complementary to HCV RNA. Thus, the oligonucleotides in accordance with the invention preferably have one of the sequences shown in Table 1, or an effective portion thereof. It is preferred to employ any of these oligonucleotides as set forth above or any of the similar oligonucleotides, which persons of ordinary skill in the art can prepare from knowledge of the preferred antisense targets for the modulation of HCV infection. 
     
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 RNA SEQUENCE TARGETS AND ANTISENSE 
               
               
                 OLIGONUCLEOTIDES FOR HCV 
               
               
                 [Sequences are from HCV-1 (US) and HCV-J (Japan)] 
               
             
          
           
               
                 SEQ ID 
                 Antisense oligo 
                 Target 
                 Target 
               
               
                 NO: 
                 sequence: 
                 description: 
                 strand: 
               
               
                   
               
             
          
           
               
                 1 
                 5′-ATG GTG GAG TGT CGC CCC GTC-3′ 
                 5′ end hairpin 
                 + 
               
               
                 2 
                 5′-GGA GTG ATC TAT GGT GGA GTG-3′ 
                 5′ end 6-bp repeat 
                 + 
               
               
                 3 
                 5′-GAT TCG TGC TCA TGG TGC ACG-3′ 
                 Polyprotein AUG 
                 + 
               
               
                 4 
                 5′-TCC AGG CAT TGA GCG GGT TGA-3′ 
                 ORF 3 AUG 
                 + 
               
               
                 5 
                 5′-TGG CCT GGA GTG TTT ATC TCC-3′ 
                 3′-untranslated 
                 + 
               
               
                 6 
                 5′-GGG GTA GGC ATC TAC CTG CTC-3′ 
                 3′ palindrome 
                 − 
               
               
                 7 
                 5′-CGC CCC CAT CAG GGG GCT GGC-3′ 
                 5′ end hairpin 
                 + 
               
               
                 8 
                 5′-TTC ATG GTG GAG TGT CGC CCC-3′ 
                 5′ end hairpin 
                 + 
               
               
                 9 
                 5′-GTT CCT CAC AGG GGA GTG ATT-3′ 
                 5′ untranslated 
                 + 
               
               
                 10 
                 5′-TAC TAA CGC CAT GGC TAG ACG-3′ 
                 5′ untranslated 
                 + 
               
               
                 11 
                 5′-CTA TGG CTC TCC CGG GAG GGG-3′ 
                 5′ untranslated 
                 + 
               
               
                 12 
                 5′-CCA CTA TGG CTC TCC CGG GAG-3′ 
                 5′ untranslated 
                 + 
               
               
                 13 
                 5′-CGG TGT ACT CAC CGG TTC CGC-3′ 
                 5′ untranslated 
                 + 
               
               
                 14 
                 5′-CTG GCA ATT CCG GTG TAC TCA-3′ 
                 5′ untranslated 
                 + 
               
               
                 15 
                 5′-GGG GCA CGC CCA AAT CTC CAG-3′ 
                 5′ untranslated 
                 + 
               
               
                 16 
                 5′-CCT TTC GCG ACC CAA CAC TAC-3′ 
                 5′ untranslated 
                 + 
               
               
                 17 
                 5′-CCC TAT CAG GCA GTA CCA CAA-3′ 
                 5′ untranslated 
                 + 
               
               
                 18 
                 5′-CTC CCG GGG CAC TCG CAA GCA-3′ 
                 5′ untranstated 
                 + 
               
               
                 19 
                 5′-CAT GGT GCA CGG TCT ACG AGA-3′ 
                 Polyprotein AUG 
                 + 
               
               
                 20 
                 5′-GAT TCG TGC TCA TGG TGC ACG-3′ 
                 Polyprotein AUG 
                 + 
               
               
                 21 
                 5′-TTT AGG ATT CGT GCT CAT GGT-3′ 
                 Polyprotein AUG 
                 + 
               
               
                 22 
                 5′-GAG TGG TTA GCC CAA TCT TCA-3′ 
                 3′ untranslated 
                 + 
               
               
                 23 
                 5′-TAT TGG CCT GGA GTG GTT AGC-3′ 
                 R2 
                 + 
               
               
                 24 
                 5′-AGG GAA TGG CCT ATT GGC CTG-3′ 
                 R2/3′ hairpin 
                 + 
               
               
                   
               
             
          
         
       
     
     The oligonucleotides of this invention can be used in diagnostics, therapeutics and as research reagents and kits. Since the oligonucleotides of this invention hybridize to RNA from HCV, sandwich and other assays can easily be constructed to exploit this fact. Provision of means for detecting hybridization of oligonucleotide with HCV or HCV RNA present in a sample suspected of containing it can routinely be accomplished. Such provision may include enzyme conjugation, radiolabelling or any other suitable detection systems. Kits for detecting the presence or absence of HCV may also be prepared. 
    
    
     The following specific examples are given for illustrative purposes only and are not intended to limit the invention. 
     EXAMPLES 
     Example 1 
     Oligonucleotide Synthesis 
     Unmodified DNA oligonucleotides are synthesized on an automated DNA synthesizer (Applied Biosystems model 380B) using standard phosphoramidite chemistry with oxidation by iodine. β-cyanoethyldiisopropyl-phosphoramidites are purchased from Applied Biosystems (Foster City, Calif.). For phosphorothioate oligonucleotides, the standard oxidation bottle is replaced by a 0.2 M solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the stepwise thiation of the phosphite linkages. The thiation cycle wait step is increased to 68 seconds and is followed by the capping step. 
     After cleavage from the controlled pore glass column (Applied Biosystems) and deblocking in concentrated ammonium hydroxide at 55° C. for 18 hours, the oligonucleotides are purified by precipitation twice out of 0.5 M NaCl with 2.5 volumes ethanol. Analytical gel electrophoresis is accomplished in 20% acrylamide, 8 M urea, 45 mM Tris-borate buffer, pH 7.0. 
     Example 2 
     Transcription and Translation of HCV RNA in Genetically Engineered Cells 
     A recombinant DNA vector capable of expressing HCV genes in mammalian cells is constructed using standard genetic engineering methods. A cDNA fragment representing the HCV mRNA or genomic transcript is placed behind an inducible eukaryotic promotor such as the LTR from mouse mammary tumor virus in such a way that transcription of the HCV cDNA begins at the appropriate nucleotide position. At the 3′ end of the gene, a polyadenylation signal is incorporated to ensure termination at the appropriate nucleotide position. It may be advantageous to modify the coding sequence by insertion of an in-frame reporter domain (e.g., the enzymatically active domain of the firefly luciferase gene) which can simplify detection procedures for expression of the fusion protein. The vector also contains one or more selectable genetic markers such as neomycin resistance. 
     The described vector is introduced into mammalian cells using a standard calcium chloride transfection procedure. Cells containing transfected DNA are identified by growth in the presence of selective agents such as neomycin, and cloned by limiting dilution. Expression of HCV RNA in cloned transfectants can be verified using any one of a number of assays such as northern blots, RNA polymerase chain reaction, or nuclease protection. Protein expression can be verified using western blotting or immune precipitation with specific HCV antibodies, or by monitoring for the presence of detectable enzymatic activity resulting from the incorporation of an assayable reporter domain. If an inducible promotor such as the MMTV LTR is used in construction of the vector, a glucocorticoid inducer such as dexamethasone should be added to the transfected cells prior to assays in order to induce gene expression. 
     Example 3 
     Evaluation of Antisense Oligonucleotide Inhibition of HCV Gene Expression from Genetically Engineered Cells 
     Mammalian cells transfected with expression vectors such as those described in Example 2 are incubated overnight in medium containing antisense oligonucleotides. After oligonucleotide treatment, cells are treated with dexamethasone in order to induce expression of HCV gene products. After a suitable incubation period (4-24 hours) cells are harvested, and expression of specific HCV polypeptide can be detected immunologically using specific antisera in a western blot or immunoprecipitation assay. If the cells contain a vector containing a reporter domain, such as that for firefly luciferase, fused in-frame with the HCV polyprotein, cell extracts can be harvested and evaluated for enzymatic activity of the reporter domain. 
     Example 4 
     Transcription and Translation of HCV RNA from Cytoplasmic Virus Vectors 
     A cDNA fragment representing the HCV mRNA or genomic transcript is placed behind a Vaccinia virus promotor in such a way that transcription of the HCV cDNA begins at the appropriate nucleotide position. At the 3′ end of the gene, a polyadenylation signal is incorporated to ensure termination at the appropriate nucleotide position. It may be advantageous in some instances to modify the coding sequence by insertion of an in-frame reporter domain (e.g., the enzymatically active domain of the firefly luciferase gene) which can simplify detection procedures for expression of the fusion protein. 
     Incorporation of the expression unit into the genome of a cytoplasmic replicating DNA virus such as Vaccinia is facilitated by inclusion of sequences upstream and downstream of the expression unit which are homologous to the Vaccinia virus genome. Co-transfection of vector into Vaccinia virus-infected mammalian cells can result in homologous recombination of vector with Vaccinia. If a suitable enzymatic marker such as β-galactosidase is present at the appropriate recombination site in the virus, then recombinant plaques can be identified by a lack of color under appropriate substrate conditions. Cloned virus can be propagated in appropriate host mammalian cell lines and expression of HCV gene products verified as described in Example 2. 
     Example 5 
     Evaluation of Antisense Oligonucleotide Inhibition of HCV Gene Expression from Cytoplasmic Virus Vectors in Mammalian Cells 
     Mammalian cells are incubated overnight in medium containing antisense oligonucleotides. After oligonucleotide treatment, cells are infected with recombinant Vaccinia virus expressing HCV gene products. After a suitable incubation period (4-24 hours) cells are harvested, and expression of specific HCV polypeptide can be detected immunologically using specific antisera in a western blot or immunoprecipitation assay. If the cells contain a vector containing a reporter domain, such as that for firefly luciferase, fused in-frame with the HCV polyprotein, cell extracts can be harvested and evaluated for enzymatic activity of the reporter domain. 
     Example 6 
     Evaluation of Antisense Oligonucleotide Inhibition of HCV Particle Assembly in Cells Transfected with HCV Genes or Infected with Cytoplasmic Virus Vectors Expressing HCV Genes 
     HCV genomic RNA and protein are expressed in cells transfected with HCV cDNA expression vectors, or in cells infected with Vaccinia virus vectors expressing the HCV cDNA. It is likely that the RNA genomes and proteins will associate to form HCV-like particles. The presence of these particles can be verified using electron microscopy. To evaluate the effects of oligonucleotides complementary to presumed packaging signals of the viral RNA on particle assembly, specific biochemical assays can be developed to measure the appearance of extracellular particles containing both HCV nucleic acid and proteins. 
     Mammalian cells transfected with expression vectors such as those described in Example 2 are incubated overnight in medium containing antisense oligonucleotides. After oligonucleotide treatment, cells are treated with dexamethasone in order to induce expression of HCV gene products. After a suitable incubation period (4-24 hours) extracellular fluid from treated cells is harvested, and particles are concentrated by pelleting in the ultracentrifuge. Proteins and nucleic acids are extracted from the pellet and quantitated by northern blot and western blot analysis respectively as described in Examples 4 and 5. A similar procedure could be used to monitor effects of oligonucleotide treatment on virus particle assembly resulting from infection of cells with recombinant Vaccinia virus expressing the HCV polyprotein. 
     
       
         
           
             27 
           
           
             
               21 
               Nucleic 
               Single 
               Linear 
             
             Yes 
              1
ATGGTGGAGT GTCGCCCCGT C                                               21
 
           
           
             
               21 
               Nucleic 
               Single 
               Linear 
             
             Yes 
              2
GGAGTGATCT ATGGTGGAGT G                                               21
 
           
           
             
               21 
               Nucleic 
               Single 
               Linear 
             
             Yes 
              3
GATTCGTGCT CATGGTGCAC G                                               21
 
           
           
             
               21 
               Nucleic 
               Single 
               Linear 
             
             Yes 
              4
TCCAGGCATT GAGCGGGTTG A                                               21
 
           
           
             
               21 
               Nucleic 
               Single 
               Linear 
             
             Yes 
              5
TGGCCTGGAG TGTTTATCTC C                                               21
 
           
           
             
               21 
               Nucleic 
               Single 
               Linear 
             
             Yes 
              6
GGGGTAGGCA TCTACCTGCT C                                               21
 
           
           
             
               21 
               Nucleic 
               Single 
               Linear 
             
             Yes 
              7
CGCCCCCATC AGGGGGCTGG C                                               21
 
           
           
             
               21 
               Nucleic 
               Single 
               Linear 
             
             Yes 
              8
TTCATGGTGG AGTGTCGCCC C                                               21
 
           
           
             
               21 
               Nucleic 
               Single 
               Linear 
             
             Yes 
              9
GTTCCTCACA GGGGAGTGAT T                                               21
 
           
           
             
               21 
               Nucleic 
               Single 
               Linear 
             
             Yes 
              10
TACTAACGCC ATGGCTAGAC G                                               21
 
           
           
             
               21 
               Nucleic 
               Single 
               Linear 
             
             Yes 
              11
CTATGGCTCT CCCGGGAGGG G                                               21
 
           
           
             
               21 
               Nucleic 
               Single 
               Linear 
             
             Yes 
              12
CCACTATGGC TCTCCCGGGA G                                               21
 
           
           
             
               21 
               Nucleic 
               Single 
               Linear 
             
             Yes 
              13
CGGTGTACTC ACCGGTTCCG C                                               21
 
           
           
             
               21 
               Nucleic 
               Single 
               Linear 
             
             Yes 
              14
CTGGCAATTC CGGTGTACTC A                                               21
 
           
           
             
               21 
               Nucleic 
               Single 
               Linear 
             
             Yes 
              15
GGGGCACGCC CAAATCTCCA G                                               21
 
           
           
             
               21 
               Nucleic 
               Single 
               Linear 
             
             Yes 
              16
CCTTTCGCGA CCCAACACTA C                                               21
 
           
           
             
               21 
               Nucleic 
               Single 
               Linear 
             
             Yes 
              17
CCCTATCAGG CAGTACCACA A                                               21
 
           
           
             
               21 
               Nucleic 
               Single 
               Linear 
             
             Yes 
              18
CTCCCGGGGC ACTCGCAAGC A                                               21
 
           
           
             
               21 
               Nucleic 
               Single 
               Linear 
             
             Yes 
              19
CATGGTGCAC GGTCTACGAG A                                               21
 
           
           
             
               21 
               Nucleic 
               Single 
               Linear 
             
             Yes 
              20
GATTCGTGCT CATGGTGCAC G                                               21
 
           
           
             
               21 
               Nucleic 
               Single 
               Linear 
             
             Yes 
              21
TTTAGGATTC GTGCTCATGG T                                               21
 
           
           
             
               21 
               Nucleic 
               Single 
               Linear 
             
             Yes 
              22
GAGTGGTTAG CCCAATCTTC A                                               21
 
           
           
             
               21 
               Nucleic 
               Single 
               Linear 
             
             Yes 
              23
TATTGGCCTG GAGTGGTTAG C                                               21
 
           
           
             
               21 
               Nucleic 
               Single 
               Linear 
             
             Yes 
              24
AGGGAATGGC CTATTGGCCT G                                               21
 
           
           
             
               9401 
               Nucleic 
               Single 
               Linear 
             
             NO 
              25
gccagccccc tgatgggggc gacactccac catgaatcac tcccctgtga                50
ggaactactg tcttcacgca gaaagcgtct agccatggcg ttagtatgag               100
tgtcgtgcag cctccaggac cccccctccc gggagagcca tagtggtctg               150
cggaaccggt gagtacaccg gaattgccag gacgaccggg tcctttcttg               200
gatcaacccg ctcaatgcct ggagatttgg gcgtgccccc gcaagactgc               250
tagccgagta gtgttgggtc gcgaaaggcc ttgtggtact gcctgatagg               300
gtgcttgcga gtgccccggg aggtctcgta gaccgtgcac catgagcacg               350
aatcctaaac ctcaaaaaaa aaacaaacgt aacaccaacc gtcgcccaca               400
ggacgtcaag ttcccgggtg gcggtcagat cgttggtgga gtttacttgt               450
tgccgcgcag gggccctaga ttgggtgtgc gcgcgacgag aaagacttcc               500
gagcggtcgc aacctcgagg tagacgtcag cctatcccca aggctcgtcg               550
gcccgagggc aggacctggg ctcagcccgg gtacccttgg cccctctatg               600
gcaatgaggg ctgcgggtgg gcgggatggc tcctgtctcc ccgtggctct               650
cggcctagct ggggccccac agacccccgg cgtaggtcgc gcaatttggg               700
taaggtcatc gataccctta cgtgcggctt cgccgacctc atggggtaca               750
taccgctcgt cggcgcccct cttggaggcg ctgccagggc cctggcgcat               800
ggcgtccggg ttctggaaga cggcgtgaac tatgcaacag ggaaccttcc               850
tggttgctct ttctctatct tccttctggc cctgctctct tgcttgactg               900
tgcccgcttc ggcctaccaa gtgcgcaact ccacggggct ttaccacgtc               950
accaatgatt gccctaactc gagtattgtg tacgaggcgg ccgatgccat              1000
cctgcacact ccggggtgcg tcccttgcgt tcgtgagggc aacgcctcga              1050
ggtgttgggt ggcgatgacc cctacggtgg ccaccaggga tggcaaactc              1100
cccgcgacgc agcttcgacg tcacatcgat ctgcttgtcg ggagcgccac              1150
cctctgttcg gccctctacg tgggggacct atgcgggtct gtctttcttg              1200
tcggccaact gttcaccttc tctcccaggc gccactggac gacgcaaggt              1250
tgcaattgct ctatctatcc cggccatata acgggtcacc gcatggcatg              1300
ggatatgatg atgaactggt cccctacgac ggcgttggta atggctcagc              1350
tgctccggat cccacaagcc atcttggaca tgatcgctgg tgctcactgg              1400
ggagtcctgg cgggcatagc gtatttctcc atggtgggga actgggcgaa              1450
ggtcctggta gtgctgctgc tatttgccgg cgtcgacgcg gaaacccacg              1500
tcaccggggg aagtgccggc cacactgtgt ctggatttgt tagcctcctc              1550
gcaccaggcg ccaagcagaa cgtccagctg atcaacacca acggcagttg              1600
gcacctcaat agcacggccc tgaactgcaa tgatagcctc aacaccggct              1650
ggttggcagg gcttttctat caccacaagt tcaactcttc aggctgtcct              1700
gagaggctag ccagctgccg accccttacc gattttgacc agggctgggg              1750
ccctatcagt tatgccaacg gaagcggccc cgaccagcgc ccctactgct              1800
ggcactaccc cccaaaacct tgcggtattg tgcccgcgaa gagtgtgtgt              1850
ggtccggtat attgcttcac tcccagcccc gtggtggtgg gaacgaccga              1900
caggtcgggc gcgcccacct acagctgggg tgaaaatgat acggacgtct              1950
tcgtccttaa caataccagg ccaccgctgg gcaattggtt cggttgtacc              2000
tggatgaact caactggatt caccaaagtg tgcggagcgc ctccttgtgt              2050
catcggaggg gcgggcaaca acaccctgca ctgccccact gattgcttcc              2100
gcaagcatcc ggacgccaca tactctcggt gcggctccgg tccctggatc              2150
acacccaggt gcctggtcga ctacccgtat aggctttggc attatccttg              2200
taccatcaac tacaccatat ttaaaatcag gatgtacgtg ggaggggtcg              2250
aacacaggct ggaagctgcc tgcaactgga cgcggggcga acgttgcgat              2300
ctggaagaca gggacaggtc cgagctcagc ccgttactgc tgaccactac              2350
acagtggcag gtcctcccgt gttccttcac aaccctacca gccttgtcca              2400
ccggcctcat ccacctccac cagaacattg tggacgtgca gtacttgtac              2450
ggggtggggt caagcatcgc gtcctgggcc attaagtggg agtacgtcgt              2500
tctcctgttc cttctgcttg cagacgcgcg cgtctgctcc tgcttgtgga              2550
tgatgctact catatcccaa gcggaggcgg ctttggagaa cctcgtaata              2600
cttaatgcag catccctggc cgggacgcac ggtcttgtat ccttcctcgt              2650
gttcttctgc tttgcatggt atttgaaggg taagtgggtg cccggagcgg              2700
tctacacctt ctacgggatg tggcctctcc tcctgctcct gttggcgttg              2750
ccccagcggg cgtacgcgct ggacacggag gtggccgcgt cgtgtggcgg              2800
tgttgttctc gtcgggttga tggcgctgac tctgtcacca tattacaagc              2850
gctatatcag ctggtgcttg tggtggcttc agtattttct gaccagagtg              2900
gaagcgcaac tgcacgtgtg gattcccccc ctcaacgtcc gaggggggcg              2950
cgacgccgtc atcttactca tgtgtgctgt acacccgact ctggtatttg              3000
acatcaccaa attgctgctg gccgtcttcg gacccctttg gattcttcaa              3050
gccagtttgc ttaaagtacc ctactttgtg cgcgtccaag gccttctccg              3100
gttctgcgcg ttagcgcgga agatgatcgg aggccattac gtgcaaatgg              3150
tcatcattaa gttaggggcg cttactggca cctatgttta taaccatctc              3200
actcctcttc gggactgggc gcacaacggc ttgcgagatc tggccgtggc              3250
tgtagagcca gtcgtcttct cccaaatgga gaccaagctc atcacgtggg              3300
gggcagatac cgccgcgtgc ggtgacatca tcaacggctt gcctgtttcc              3350
gcccgcaggg gccgggagat actgctcggg ccagccgatg gaatggtctc              3400
caaggggtgg aggttgctgg cgcccatcac ggcgtacgcc cagcagacaa              3450
ggggcctcct agggtgcata atcaccagcc taactggccg ggacaaaaac              3500
caagtggagg gtgaggtcca gattgtgtca actgctgccc aaaccttcct              3550
ggcaacgtgc atcaatgggg tgtgctggac tgtctaccac ggggccggaa              3600
cgaggaccat cgcgtcaccc aagggtcctg tcatccagat gtataccaat              3650
gtagaccaag accttgtggg ctggcccgct ccgcaaggta gccgctcatt              3700
gacaccctgc acttgcggct cctcggacct ttacctggtc acgaggcacg              3750
ccgatgtcat tcccgtgcgc cggcggggtg atagcagggg cagcctgctg              3800
tcgccccggc ccatttccta cttgaaaggc tcctcggggg gtccgctgtt              3850
gtgccccgcg gggcacgccg tgggcatatt tagggccgcg gtgtgcaccc              3900
gtggagtggc taaggcggtg gactttatcc ctgtggagaa cctagagaca              3950
accatgaggt ccccggtgtt cacggataac tcctctccac cagtagtgcc              4000
ccagagcttc caggtggctc acctccatgc tcccacaggc agcggcaaaa              4050
gcaccaaggt cccggctgca tatgcagctc agggctataa ggtgctagta              4100
ctcaacccct ctgttgctgc aacactgggc tttggtgctt acatgtccaa              4150
ggctcatggg atcgatccta acatcaggac cggggtgaga acaattacca              4200
ctggcagccc catcacgtac tccacctacg gcaagttcct tgccgacggc              4250
gggtgctcgg ggggcgctta tgacataata atttgtgacg agtgccactc              4300
cacggatgcc acatccatct tgggcatcgg cactgtcctt gaccaagcag              4350
agactgcggg ggcgagactg gttgtgctcg ccaccgccac ccctccgggc              4400
tccgtcactg tgccccatcc caacatcgag gaggttgctc tgtccaccac              4450
cggagagatc cctttttacg gcaaggctat ccccctcgaa gtaatcaagg              4500
gggggagaca tctcatcttc tgtcattcaa agaagaagtg cgacgaactc              4550
gccgcaaagc tggtcgcatt gggcatcaat gccgtggcct actaccgcgg              4600
tcttgacgtg tccgtcatcc cgaccagcgg cgatgttgtc gtcgtggcaa              4650
ccgatgccct catgaccggc tataccggcg acttcgactc ggtgatagac              4700
tgcaatacgt gtgtcaccca gacagtcgat ttcagccttg accctacctt              4750
caccattgag acaatcacgc tcccccagga tgctgtctcc cgcactcaac              4800
gtcggggcag gactggcagg gggaagccag gcatctacag atttgtggca              4850
ccgggggagc gcccctccgg catgttcgac tcgtccgtcc tctgtgagtg              4900
ctatgacgca ggctgtgctt ggtatgagct cacgcccgcc gagactacag              4950
ttaggctacg agcgtacatg aacaccccgg ggcttcccgt gtgccaggac              5000
catcttgaat tttgggaggg cgtctttaca ggcctcactc atatagatgc              5050
ccactttcta tcccagacaa agcagagtgg ggagaacctt ccttacctgg              5100
tagcgtacca agccaccgtg tgcgctaggg ctcaagcccc tcccccatcg              5150
tgggaccaga tgtggaagtg tttgattcgc ctcaagccca ccctccatgg              5200
gccaacaccc ctgctataca gactgggcgc tgttcagaat gaaatcaccc              5250
tgacgcaccc agtcaccaaa tacatcatga catgcatgtc ggccgacctg              5300
gaggtcgtca cgagcacctg ggtgctcgtt ggcggcgtcc tggctgcttt              5350
ggccgcgtat tgcctgtcaa caggctgcgt ggtcatagtg ggcagggtcg              5400
tcttgtccgg gaagccggca atcatacctg acagggaagt cctctaccga              5450
gagttcgatg agatggaaga gtgctctcag cacttaccgt acatcgagca              5500
agggatgatg ctcgccgagc agttcaagca gaaggccctc ggcctcctgc              5550
agaccgcgtc ccgtcaggca gaggttatcg cccctgctgt ccagaccaac              5600
tggcaaaaac tcgagacctt ctgggcgaag catatgtgga acttcatcag              5650
tgggatacaa tacttggcgg gcttgtcaac gctgcctggt aaccccgcca              5700
ttgcttcatt gatggctttt acagctgctg tcaccagccc actaaccact              5750
agccaaaccc tcctcttcaa catattgggg gggtgggtgg ctgcccagct              5800
cgccgccccc ggtgccgcta ctgcctttgt gggcgctggc ttagctggcg              5850
ccgccatcgg cagtgttgga ctggggaagg tcctcataga catccttgca              5900
gggtatggcg cgggcgtggc gggagctctt gtggcattca agatcatgag              5950
cggtgaggtc ccctccacgg aggacctggt caatctactg cccgccatcc              6000
tctcgcccgg agccctcgta gtcggcgtgg tctgtgcagc aatactgcgc              6050
cggcacgttg gcccgggcga gggggcagtg cagtggatga accggctgat              6100
agccttcgcc tcccggggga accatgtttc ccccacgcac tacgtgccgg              6150
agagcgatgc agctgcccgc gtcactgcca tactcagcag cctcactgta              6200
acccagctcc tgaggcgact gcaccagtgg ataagctcgg agtgtaccac              6250
tccatgctcc ggttcctggc taagggacat ctgggactgg atatgcgagg              6300
tgttgagcga ctttaagacc tggctaaaag ctaagctcat gccacagctg              6350
cctgggatcc cctttgtgtc ctgccagcgc gggtataagg gggtctggcg              6400
agtggacggc atcatgcaca ctcgctgcca ctgtggagct gagatcactg              6450
gacatgtcaa aaacgggacg atgaggatcg tcggtcctag gacctgcagg              6500
aacatgtgga gtgggacctt ccccattaat gcctacacca cgggcccctg              6550
tacccccctt cctgcgccga actacacgtt cgcgctatgg agggtgtctg              6600
cagaggaata tgtggagata aggcaggtgg gggacttcca ctacgtgacg              6650
ggtatgacta ctgacaatct caaatgcccg tgccaggtcc catcgcccga              6700
atttttcaca gaattggacg gggtgcgcct acataggttt gcgcccccct              6750
gcaagccctt gctgcgggag gaggtatcat tcagagtagg actccacgaa              6800
tacccggtag ggtcgcaatt accttgcgag cccgaaccgg acgtggccgt              6850
gttgacgtcc atgctcactg atccctccca tataacagca gaggcggccg              6900
ggcgaaggtt ggcgagggga tcacccccct ctgtggccag ctcctcggct              6950
agccagctat ccgctccatc tctcaaggca acttgcaccg ctaaccatga              7000
ctcccctgat gctgagctca tagaggccaa cctcctatgg aggcaggaga              7050
tgggcggcaa catcaccagg gttgagtcag aaaacaaagt ggtgattctg              7100
gactccttcg atccgcttgt ggcggaggag gacgagcggg agatctccgt              7150
acccgcagaa atcctgcgga agtctcggag attcgcccag gccctgcccg              7200
tttgggcgcg gccggactat aaccccccgc tagtggagac gtggaaaaag              7250
cccgactacg aaccacctgt ggtccatggc tgtccgcttc cacctccaaa              7300
gtcccctcct gtgcctccgc ctcggaagaa gcggacggtg gtcctcactg              7350
aatcaaccct atctactgcc ttggccgagc tcgccaccag aagctttggc              7400
agctcctcaa cttccggcat tacgggcgac aatacgacaa catcctctga              7450
gcccgcccct tctggctgcc cccccgactc cgacgctgag tcctattcct              7500
ccatgccccc cctggagggg gagcctgggg atccggatct tagcgacggg              7550
tcatggtcaa cggtcagtag tgaggccaac gcggaggatg tcgtgtgctg              7600
ctcaatgtct tactcttgga caggcgcact cgtcaccccg tgcgccgcgg              7650
aagaacagaa actgcccatc aatgcactaa gcaactcgtt gctacgtcac              7700
cacaatttgg tgtattccac cacctcacgc agtgcttgcc aaaggcagaa              7750
gaaagtcaca tttgacagac tgcaagttct ggacagccat taccaggacg              7800
tactcaagga ggttaaagca gcggcgtcaa aagtgaaggc taacttgcta              7850
tccgtagagg aagcttgcag cctgacgccc ccacactcag ccaaatccaa              7900
gtttggttat ggggcaaaag acgtccgttg ccatgccaga aaggccgtaa              7950
cccacatcaa ctccgtgtgg aaagaccttc tggaagacaa tgtaacacca              8000
atagacacta ccatcatggc taagaacgag gttttctgcg ttcagcctga              8050
gaaggggggt cgtaagccag ctcgtctcat cgtgttcccc gatctgggcg              8100
tgcgcgtgtg cgaaaagatg gctttgtacg acgtggttac aaagctcccc              8150
ttggccgtga tgggaagctc ctacggattc caatactcac caggacagcg              8200
ggttgaattc ctcgtgcaag cgtggaagtc caagaaaacc ccaatggggt              8250
tctcgtatga tacccgctgc tttgactcca cagtcactga gagcgacatc              8300
cgtacggagg aggcaatcta ccaatgttgt gacctcgacc cccaagcccg              8350
cgtggccatc aagtccctca ccgagaggct ttatgttggg ggccctctta              8400
ccaattcaag gggggagaac tgcggctatc gcaggtgccg cgcgagcggc              8450
gtactgacaa ctagctgtgg taacaccctc acttgctaca tcaaggcccg              8500
ggcagcctgt cgagccgcag ggctccagga ctgcaccatg ctcgtgtgtg              8550
gcgacgactt agtcgttatc tgtgaaagcg cgggggtcca ggaggacgcg              8600
gcgagcctga gagccttcac ggaggctatg accaggtact ccgccccccc              8650
tggggacccc ccacaaccag aatacgactt ggagctcata acatcatgct              8700
cctccaacgt gtcagtcgcc cacgacggcg ctggaaagag ggtctactac              8750
ctcacccgtg accctacaac ccccctcgcg agagctgcgt gggagacagc              8800
aagacacact ccagtcaatt cctggctagg caacataatc atgtttgccc              8850
ccacactgtg ggcgaggatg atactgatga cccatttctt tagcgtcctt              8900
atagccaggg accagcttga acaggccctc gattgcgaga tctacggggc              8950
ctgctactcc atagaaccac ttgatctacc tccaatcatt caaagactcc              9000
atggcctcag cgcattttca ctccacagtt actctccagg tgaaattaat              9050
agggtggccg catgcctcag aaaacttggg gtaccgccct tgcgagcttg              9100
gagacaccgg gcccggagcg tccgcgctag gcttctggcc agaggaggca              9150
gggctgccat atgtggcaag tacctcttca actgggcagt aagaacaaag              9200
ctcaaactca ctccaatagc ggccgctggc cagctggact tgtccggctg              9250
gttcacggct ggctacagcg ggggagacat ttatcacagc gtgtctcatg              9300
cccggccccg ctggatctgg ttttgcctac tcctgcttgc tgcaggggta              9350
ggcatctacc tcctccccaa ccgatgaagg ttggggtaaa cactccggcc              9400
t                                                                   9401
 
           
           
             
               9416 
               Nucleic 
               Single 
               Linear 
             
             NO 
              26
gccagccccc tgatgggggc gacactccac catgaatcac tcccctgtga                50
ggaactactg tcttcacgca gaaagcgtct agccatggcg ttagtatgag               100
tgtcgtgcag cctccaggac cccccctccc gggagagcca tagtggtctg               150
cggaaccggt gagtacaccg gaattgccag gacgaccggg tcctttcttg               200
gataaacccg ctcaatgcct ggagatttgg gcgtgccccc gcaagactgc               250
tagccgagta gtgttgggtc gcgaaaggcc ttgtggtact gcctgatagg               300
gtgcttgcga gtgccccggg aggtctcgta gaccgtgcac catgagcacg               350
aatcctaaac ctcaaagaaa aaccaaacgt aacaccaacc gtcgcccaca               400
ggacgtcaag ttcccgggtg gcggtcagat cgttggtgga gtttacttgt               450
tgccgcgcag gggccctaga ttgggtgtgc gcgcgacgag gaagacttcc               500
gagcggtcgc aacctcgagg tagacgtcag cctatcccca aggcacgtcg               550
gcccgagggc aggacctggg ctcagcccgg gtacccttgg cccctctatg               600
gcaatgaggg ttgcgggtgg gcgggatggc tcctgtctcc ccgtggctct               650
cggcctagct ggggccccac agacccccgg cgtaggtcgc gcaatttggg               700
taaggtcatc gataccctta cgtgcggctt cgccgacctc atggggtaca               750
taccgctcgt cggcgcccct cttggaggcg ctgccagggc cctggcgcat               800
ggcgtccggg ttctggaaga cggcgtgaac tatgcaacag ggaaccttcc               850
tggttgctct ttctctatct tccttctggc cctgctctct tgcctgactg               900
tgcccgcttc agcctaccaa gtgcgcaatt cctcggggct ttaccatgtc               950
accaatgatt gccctaactc gagtgttgtg tacgaggcgg ccgatgccat              1000
cctgcacact ccggggtgtg tcccttgcgt tcgcgagggt aacgcctcga              1050
ggtgttgggt ggcggtgacc cccacggtgg ccaccaggga cggcaaactc              1100
cccacaacgc agcttcgacg tcatatcgat ctgcttgtcg ggagcgccac              1150
cctctgctcg gccctctacg tgggggacct gtgcgggtct gtctttcttg              1200
ttggtcaact gtttaccttc tctcccaggc accactggac gacgcaagac              1250
tgcaattgtt ctatctatcc cggccatata acgggtcatc gcatggcatg              1300
gaatatgatg atgaactggt cccctacggc agcgttggtg gtagctcagc              1350
tgctccgaat cccacaagcc atcatggaca tgatcgctgg cgcccactgg              1400
ggagtcctgg cgggcataaa gtatttctcc atggtgggga actgggcgaa              1450
ggtcctggta gtgctgctgc tatttgccgg cgtcgacgcg gaaacccacg              1500
tcaccggggg aaatgccggc cgcaccacgg ctgggcttgt tggtctcctt              1550
acaccaggcg ccaagcagaa catccaactg atcaacacca acggcagttg              1600
gcacatcaat agcacggcct tgaactgcaa tgaaagcctt aacaccggct              1650
ggttagcagg gctcttctat cagcacaaat tcaactcttc aggctgtcct              1700
gagaggttgg ccagctgccg acgccttacc gattttgccc agggctgggg              1750
tcctatcagt tatgccaacg gaagcggcct cgacgaacgc ccctactgct              1800
ggcactaccc tccaagacct tgtggcattg tgcccgcaaa gagcgtgtgt              1850
ggcccggtat attgcttcac tcccagcccc gtggtggtgg gaacgaccga              1900
caggtcgggc gcgcctacct acagctgggg tgcaaatgat acggatgtct              1950
tcgtccttaa caacaccagg ccaccgctgg gcaattggtt cggttgtacc              2000
tggatgaact caactggatt caccaaagtg tgcggagcgc ccccttgtgt              2050
catcggaggg gtgggcaaca acaccttgct ctgccccact gattgcttcc              2100
gcaaatatcc ggaagccaca tactctcggt gcggctccgg tcccaggatt              2150
acacccaggt gcatggtcga ctacccgtat aggctttggc actatccttg              2200
taccatcaat tacaccatat tcaaagtcag gatgtacgtg ggaggggtcg              2250
agcacaggct ggaagcggcc tgcaactgga cgcggggcga acgctgtgat              2300
ctggaagaca gggacaggtc cgagctcagc ccgttgctgc tgtccaccac              2350
acagtggcag gtccttccgt gttctttcac gaccctgcca gccttgtcca              2400
ccggcctcat ccacctccac cagaacattg tggacgtgca gtacttgtac              2450
ggggtagggt caagcatcgc gtcctgggcc attaagtggg agtacgtcgt              2500
tctcctgttc cttctgcttg cagacgcgcg cgtctgttcc tgcttgtgga              2550
tgatgttact catatcccaa gcggaggcgg ctttggagaa cctcgtaata              2600
ctcaatgcag catccctggc cgggacgcat ggtcttgtgt ccttcctcgt              2650
gttcttctgc tttgcgtggt atctgaaggg taggtgggtg cccggagcgg              2700
tctacgccct ctacgggatg tggcctctcc tcctgctcct gctggcgttg              2750
cctcagcggg catacgcact ggacacggag gtggccgcgt cgtgtggcgg              2800
cgttgttctt gtcgggttaa tggcgctgac tctgtcgcca tattacaagc              2850
gctatatcag ctggtgcatg tggtggcttc agtattttct gaccagagta              2900
gaagcgcaac tgcacgtgtg ggttcccccc ctcaacgtcc ggggggggcg              2950
cgatgccgtc atcttactca cgtgtgtagt acacccggcc ctggtatttg              3000
acatcaccaa actactcctg gccatcttcg gacccctttg gattcttcaa              3050
gccagtttgc ttaaagtccc ctacttcgtg cgcgttcaag gccttctccg              3100
gatctgcgcg ctagcgcgga agatagccgg aggtcattac gtgcaaatgg              3150
ccatcatcaa gttaggggcg cttactggca cctgtgtgta taaccatctc              3200
gctcctcttc gagactgggc gcacaacggc ctgcgagatc tggccgtggc              3250
tgtggaacca gtcgtcttct cccgaatgga gaccaagctc atcacgtggg              3300
gggcagatac cgccgcgtgc ggtgacatca tcaacggctt gcccgtctct              3350
gcccgtaggg gccaggagat actgcttggg ccagccgacg gaatggtctc              3400
caaggggtgg aggttgctgg cgcccatcac ggcgtacgcc cagcagacga              3450
gaggcctcct agggtgtata atcaccagcc tgactggccg ggacaaaaac              3500
caagtggagg gtgaggtcca gatcgtgtca actgctaccc agaccttcct              3550
ggcaacgtgc atcaatgggg tatgctggac tgtctaccac ggggccggaa              3600
cgaggaccat cgcatcaccc aagggtcctg tcatccagac gtataccaat              3650
gtggatcaag acctcgtggg ctggcccgct cctcaaggtt cccgctcatt              3700
gacaccctgc acctgcggct cctcggacct ttacctggtc acgaggcacg              3750
ccgatgtcat tcccgtgcgc cggcgaggtg atagcagggg tagcctgctt              3800
tcgccccggc ccatttccta cttgaaaggc tcctcggggg gtccgctgtt              3850
gtgccccacg ggacacgccg tgggcctatt cagggccgcg gtgtgcaccc              3900
gtggagtggc taaggcggtg gactttatcc ctgtggagaa cctagagaca              3950
accatgagat ccccggtgtt cacggacaac tcctctccac cagcagtgcc              4000
ccagagcttc caggtggccc acctgcatgc tcccaccggc agcggtaaga              4050
gcaccaaggt cccggctgcg tacgcagcca agggctacaa ggtgttggtg              4100
ctcaacccct ctgttgctgc aacactgggc tttggtgctt acatgtccaa              4150
ggcccatggg gttgatccta atatcaggac cggggtgaga acaattacca              4200
ctggcagccc catcacgtac tccacctacg gcaagttcct tgccgacgcc              4250
gggtgctcag gaggtgctta tgacataata atttgtgacg agtgccactc              4300
cacggatgcc acatccatct cgggcatcgg cactgtcctt gaccaagcag              4350
agactgcggg ggcgagactg gttgtgctcg ccactgctac ccctccgggc              4400
tccgtcactg tgtcccatcc taacatcgag gaggttgctc tgtccaccac              4450
cggagagatc cccttttacg gcaaggctat ccccctcgag gtgatcaagg              4500
ggggaagaca tctcatcttc tgccactcaa agaagaagtg cgacgagctc              4550
gccgcgaagc tggtcgcatt gggcatcaat gccgtggcct actaccgcgg              4600
tcttgacgtg tctgtcatcc cgaccagcgg cgatgttgtc gtcgtgtcga              4650
ccgatgctct catgactggc tttaccggcg acttcgactc tgtgatagac              4700
tgcaacacgt gtgtcactca gacagtcgat tttagccttg accctacctt              4750
taccattgag acaaccacgc tcccccagga tgctgtctcc aggactcaac              4800
gccggggcag gactggcagg gggaagccag gcatctatag atttgtggca              4850
ccgggggagc gcccctccgg catgttcgac tcgtccgtcc tctgtgagtg              4900
ctatgacgcg ggctgtgctt ggtatgagct cacgcccgcc gagactacag              4950
ttaggctacg agcgtacatg aacaccccgg ggcttcccgt gtgccaggac              5000
catcttggat tttgggaggg cgtctttacg ggcctcactc atatagatgc              5050
ccactttcta tcccagacaa agcagagtgg ggagaacttt ccttacctgg              5100
tagcgtacca agccaccgtg tgcgctaggg ctcaagcccc tcccccatcg              5150
tgggaccaga tgcggaagtg tttgatccgc cttaaaccca ccctccatgg              5200
gccaacaccc ctgctataca gactgggcgc tgttcagaat gaagtcaccc              5250
tgacgcaccc aatcaccaaa tacatcatga catgcatgtc ggccgacctg              5300
gaggtcgtca cgagcacctg ggtgctcgtt ggcggcgtcc tggctgctct              5350
ggccgcgtat tgcctgtcaa caggctgcgt ggtcatagtg ggcaggatcg              5400
tcttgtccgg gaagccggca attatacctg acagggaggt tctctaccag              5450
gagttcgatg agatggaaga gtgctctcag cacttaccgt acatcgagca              5500
agggatgatg ctcgctgagc agttcaagca gaaggccctc ggcctcctgc              5550
agaccgcgtc ccgccatgca gaggttatca cccctgctgt ccagaccaac              5600
tggcagaaac tcgaggtctt ttgggcgaag cacatgtgga atttcatcag              5650
tgggatacaa tacttggcgg gcctgtcaac gctgcctggt aaccccgcca              5700
ttgcttcatt gatggctttt acagctgccg tcaccagccc actaaccact              5750
ggccaaaccc tcctcttcaa catattgggg gggtgggtgg ctgcccagct              5800
cgccgccccc ggtgccgcta ccgcctttgt gggcgctggc ttagctggcg              5850
ccgcactcga cagcgttgga ctggggaagg tcctcgtgga cattcttgca              5900
ggctatggcg cgggcgtggc gggagctctt gtggcattca agatcatgag              5950
cggtgaggtc ccctccacgg aggacctggt caatctgctg cccgccatcc              6000
tctcacctgg agcccttgca gtcggtgtgg tctttgcatc aatactgcgc              6050
cggcgtgttg gcccgggcga gggggcagtg caatggatga accggctaat              6100
agccttcgcc tcccggggga accatgtttc ccccacacac tacgtgccgg              6150
agagcgatgc agccgcccgc gtcactgcca tactcagcag cctcactgta              6200
acccagctcc tgaggcgact gcatcagtgg ataagctcgg agtgtaccac              6250
tccatgctcc ggttcctggc taagggacat ctgggactgg atatgcgagg              6300
tgctgagcga ctttaagacc tggctgaaag ccaagctcat gccacaactg              6350
cctgggattc cctttgtgtc ctgccagcgc gggtataggg gggtctggcg              6400
aggagacggc attatgcaca ctcgctgcca ctgtggagct gagatcactg              6450
gacatgtcaa aaacgggacg atgaggatcg tcggtcctag gacctgcaag              6500
aacatgtgga gtgggacgtt cttcattaat gcctacacca cgggcccctg              6550
tactcccctt cctgcgccga actataagtt cgcgctgtgg agggtgtctg              6600
cagaggaata cgtggagata aggcgggtgg gggacttcca ctacgtatcg              6650
ggcatgacta ctgacaatct caaatgcccg tgccagatcc catcgcccga              6700
atttttcaca gaattggacg gggtgcgcct acataggttt gcgccccctt              6750
gcaagccctt gctgcgggag gaggtatcat tcagagtagg actccacgag              6800
tacccggtgg ggtcgcaatt accttgcgag cccgaaccgg acgtagccgt              6850
gttgacgtcc atgctcactg atccctccca tataacagca gaggcggccg              6900
ggagaaggtt ggcgagaggg tcaccccctt ctatggccag ctcctcggct              6950
agccagctgt ccgctccatc tctcaaggca acttgcaccg ccaaccatga              7000
ctcccctgac gccgagctca tagaggctaa cctcctgtgg aggcaggaga              7050
tgggcggcaa catcaccagg gttgagtcag agaacaaagt ggtgattctg              7100
gactccttcg atccgcttgt ggcagaggag gatgagcggg aggtctccgt              7150
acccgcagaa attctgcgga agtctcggag attcgcccca gccctgcccg              7200
tctgggcgcg gccggactac aaccccctgc tagtagagac gtggaaaaag              7250
cctgactacg aaccacctgt ggtccatggc tgcccgctac cacctccacg              7300
gtcccctcct gtgcctccgc ctcggaaaaa gcgtacggtg gtcctcaccg              7350
aatcaaccct acctactgcc ttggccgagc ttgccaccaa aagttttggc              7400
agctcctcaa cttccggcat tacgggcgac aatacgacaa catcctctga              7450
gcccgcccct tctggctgcc cccccgactc cgacgttgag tcctattctt              7500
ccatgccccc cctggagggg gagcctgggg atccggatct cagcgacggg              7550
tcatggtcga cggtcagtag tggggccgac acggaagatg tcgtgtgctg              7600
ctcaatgtct tattcctgga caggcgcact cgtcaccccg tgcgctgcgg              7650
aggaacaaaa actgcccatc aacgcactga gcaactcgtt gctacgccat              7700
cacaatctgg tgtattccac cacttcacgc agtgcttgcc aaaggaagaa              7750
gaaagtcaca tttgacagac tgcaagttct ggacagccat taccaggacg              7800
tgctcaagga ggtcaaagca gcggcgtcaa aagtgaaggc taacttgcta              7850
tccgtagagg aagcttgcag cctggcgccc ccacattcag ccaaatccaa              7900
gtttggctat ggggcaaaag acgtccgttg ccatgccaga aaggccgtag              7950
cccacatcaa ctccgtgtgg aaagaccttc tggaagacag tgtaacacca              8000
atagacacta ccatcatggc caagaacgag gttttctgcg ttcagcctga              8050
gaaggggggt cgtaagccag ctcgtctcat cgtgttcccc gacctgggcg              8100
tgcgcgtgtg cgagaagatg gccctgtacg acgtggttag caagctcccc              8150
ttggccgtga tgggaagctc ctacggattc caatactcac caggacagcg              8200
ggttgaattc ctcgtgcaag cgtggaagtc caagaagacc ccgatggggc              8250
tctcgtatga tacccgctgt tttgactcca cagtcactga gagcgacatc              8300
cgtacggagg aggcaattta ccaatgttgt gacctggacc cccaagcccg              8350
cgtggccatc aagtccctca ctgagaggct ttatgttggg ggccctctta              8400
ctaattcaag gggggaaaac tgcggctacc gcaggtgccg cgcgagcaga              8450
gtactgacaa ctagctgtgg taacaccctc actcgctaca tcaaggcccg              8500
ggcagcctgt cgagccgcag ggctccagga ctgcaccatg ctcgtgtgtg              8550
gcgacgactt agtcgttatc tgtgaaagtg cgggggtcca ggaggacgcg              8600
gcgagcctga gagccttcac ggaggctatg accaggtact ccgccccccc              8650
cggggacccc ccacaaccag aatacgactt ggagcttata acatcatgct              8700
cctccaacgt gtcagtcgcc cacgacggcg ctggaaagag ggtctactac              8750
cttacccgtg accctacaac ccccctcgcg agagccgcgt gggagacagc              8800
aagacacact ccagtcaatt cctggctagg caacataatc atgtttgccc              8850
ccacactgtg ggcgaggatg atactgatga cccacttctt tagcgtcctc              8900
atagccaggg atcagcttga acaggctctc aactgcgaga tctacggagc              8950
ctgctactcc atagaaccac tggatctacc tccaatcatt caaagactcc              9000
atggcctcag cgcattttca ctccacagtt actctccagg tgaaattaat              9050
agggtggccg catgcctcag aaaacttggg gtcccgccct tgcgagcttg              9100
gagacaccgg gcctggagcg tccgcgctag gcttctggcc agaggaggca              9150
aggctgccat atgtggcaag tacctcttca actgggcagt aagaacaaag              9200
ctcaaactca ctccgataac ggccgctggc cggctggact tgtccggctg              9250
gttcacggct ggctacagcg ggggagacat ttatcacagc gtgtctcatg              9300
cccggccccg ctggttctgg ttttgcctac tcctgcttgc tgcaggggta              9350
ggcatctacc tcctccccaa ccgatgaaga ttgggctaac cactccaggc              9400
caataggcca ttccct                                                   9416
 
           
           
             
               9416 
               Nucleic 
               Single 
               Linear 
             
             NO 
              27
cgattggggg cgacactcca ccatagatca ctcccctgtg aggaactact                50
gtcttcacgc agaaagcgtc tagccatggc gttagtatga gtgtcgtgca               100
gcctccagga ccccccctcc cgggagagcc atagtggtct gcggaaccgg               150
tgagtacacc ggaattgcca ggacgaccgg gtcctttctt ggatcaaccc               200
gctcaatgcc tggagatttg ggcgtgcccc cgcgagactg ctagccgagt               250
agtgttgggt cgcgaaaggc cttgtggtac tgcctgatag ggtgcttgcg               300
agtgccccgg gaggtctcgt agaccgtgca ccatgagcac gaatcctaaa               350
cctcaaagaa aaaccaaacg taacaccaac cgccgcccac aggacgtcaa               400
gttcccgggc ggtggtcaga tcgttggtgg agtttacctg ttgccgcgca               450
ggggccccag gttgggtgtg cgcgcgccca ggaagacttc cgagcggtcg               500
caacctcgtg gaaggcgaca acctatcccc aaggctcgcc ggcccgaggg               550
caggacctgg gctcagcccg ggtacccttg gcctctctat ggcaatgagg               600
gcttagggtg ggcaggatgg ctcctgtcac cccgcggctc ccggcctagt               650
tggggcccca cggacccccg gcgtaggtcg cgtaatttgg gtaaggtcat               700
cgataccctc acatgcggct tcgccgatct catggggtac attccgctcg               750
tcggcgcccc cctggggggc gctgccaggg ccctggcaca tggtgtccgg               800
gttctggagg acggcgtgaa ctatgcaaca gggaatctgc ccggttgctc               850
tttttctatc ttcctcttgg ctctgctgtc ctgcctgacc accccagctt               900
ccgcttacga agtgcacaac gtgtccggga tatatcatgt cacgaacgac               950
tgctccaacg caagcattgt gtatgaggca gcggacttga tcatgcatac              1000
tcctgggtgc gtgccctgcg ttcgggaagg caactcctcc cgctgctggg              1050
tagcgctcac tcccacgctc gcagccagga acgtcaccat ccccaccacg              1100
acgatacgac gccacgtcga tctgctcgtt ggggcggctg ctttctgttc              1150
cgctatgtac gtgggggacc tctgcggatc tgttttcctc gtctctcagc              1200
tgttcacctt ctcgcctcgc cggcatgtga cattacagga ctgtaactgc              1250
tcaatttatc ccggccatgt gtcgggtcac cgtatggctt gggacatgat              1300
gatgaactgg tcgcccacaa cagccctagt ggtgtcgcag ttactccgga              1350
tcccacaagc cgtcgtggac atggtggcgg gggcccactg gggagtcctg              1400
gcgggccttg cctactattc catggcgggg aactgggcta aggttctgat              1450
tgtgatgcta ctttttgctg gcgttgacgg ggatacccac gtgacagggg              1500
gggcgcaagc caaaaccacc aacaggctcg tgtccatgtt cgcaagtggg              1550
ccgtctcaga aaatccagct tataaacacc aatgggagtt ggcacatcaa              1600
caggactgcc ctgaactgca atgactctct ccagactggg tttcttgccg              1650
cgctgttcta cacacatagt ttcaactcgt ccgggtgccc agagcgcatg              1700
gcccagtgcc gcaccattga caagttcgac cagggatggg gtcccattac              1750
ttatgctgag tctagcagat cagaccagag gccatattgc tggcactacc              1800
cacctccaca atgtaccatc gtacctgcgt cggaggtgtg cggcccagtg              1850
tactgcttca ccccaagccc tgtcgtcgtg gggacgaccg atcgtttcgg              1900
tgtccctacg tatagatggg gggagaacga gactgacgtg ctgctgctca              1950
acaacacgcg gccgccgcaa ggcaactggt tcggctgcac atggatgaat              2000
agcaccgggt tcaccaagac atgtgggggg cccccgtgta acatcggggg              2050
ggtcggcaac aacaccctga cctgccccac ggactgcttc cggaagcacc              2100
ccgaggctac ctacacaaaa tgtggttcgg ggccttggct gacacctagg              2150
tgcatggttg actatccata caggctctgg cattacccct gcactgttaa              2200
ctttaccatc ttcaaggtta ggatgtatgt ggggggggtg gagcacaggc              2250
tcaatgctgc atgcaattgg acccgaggag agcgttgtga cttggaggac              2300
agggataggc cggagctcag cccgctgctg ctgtctacaa cagagtggca              2350
ggtactgccc tgttccttca ccaccctacc agctctgtcc actggcttga              2400
ttcacctcca tcagaacatc gtggacgtgc aatacctata cggtataggg              2450
tcagcggttg tctcctttgc aatcaaatgg gagtatgtcc tgttgctttt              2500
ccttctccta gcggacgcac gtgtctgtgc ctgcttgtgg atgatgctgc              2550
tgatagccca ggccgaggcc gccttggaga acctggtggt cctcaattcg              2600
gcgtctgtgg ccggcgcaca tggcatcctc tccttccttg tgttcttctg              2650
tgccgcctgg tacatcaaag gcaggctggt ccctggggcg acatatgctc              2700
tttatggcgt gtggccgctg ctcctgctct tgctggcatt accaccgcga              2750
gcttacgcca tggaccggga gatggctgca tcgtgcggag gcgcggtttt              2800
tgtgggtctg gtactcctga ctttgtcacc atactacaag gtgttcctcg              2850
ctaggctcat atggtggtta caatatttta ccaccagagc cgaggcggac              2900
ttacatgtgt ggatcccccc cctcaacgct cggggaggcc gcgatgccat              2950
catcctcctc atgtgcgcag tccatccaga gctaatcttt gacatcacca              3000
aacttctaat tgccatactc ggtccgctca tggtgctcca agctggcata              3050
accagagtgc cgtacttcgt gcgcgctcaa gggctcattc atgcatgcat              3100
gttagtgcgg aaggtcgctg ggggtcatta tgtccaaatg gccttcatga              3150
agctgggcgc gctgacaggc acgtacattt acaaccatct taccccgcta              3200
cgggattggc cacgcgcggg cctacgagac cttgcggtgg cagtggagcc              3250
cgtcgtcttc tccgacatgg agaccaagat catcacctgg ggagcagaca              3300
ccgcggcgtg tggggacatc atcttgggtc tgcccgtctc cgcccgaagg              3350
ggaaaggaga tactcctggg cccggccgat agtcttgaag ggcgggggtt              3400
gcgactcctc gcgcccatca cggcctactc ccaacagacg cggggcctac              3450
ttggttgcat catcactagc cttacaggcc gggacaagaa ccaggtcgag              3500
ggagaggttc aggtggtttc caccgcaaca caatccttcc tggcgacctg              3550
cgtcaacggc gtgtgttgga ccgtttacca tggtgctggc tcaaagacct              3600
tagccgcgcc aaaggggcca atcacccaga tgtacactaa tgtggaccag              3650
gacctcgtcg gctggcccaa gccccccggg gcgcgttcct tgacaccatg              3700
cacctgtggc agctcagacc tttacttggt cacgagacat gctgacgtca              3750
ttccggtgcg ccggcggggc gacagtaggg ggagcctgct ctcccccagg              3800
cctgtctcct acttgaaggg ctcttcgggt ggtccactgc tctgcccctt              3850
cgggcacgct gtgggcatct tccgggctgc cgtatgcacc cggggggttg              3900
cgaaggcggt ggactttgtg cccgtagagt ccatggaaac tactatgcgg              3950
tctccggtct tcacggacaa ctcatccccc ccggccgtac cgcagtcatt              4000
tcaagtggcc cacctacacg ctcccactgg cagcggcaag agtactaaag              4050
tgccggctgc atatgcagcc caagggtaca aggtgctcgt cctcaatccg              4100
tccgttgccg ctaccttagg gtttggggcg tatatgtcta aggcacacgg              4150
tattgacccc aacatcagaa ctggggtaag gaccattacc acaggcgccc              4200
ccgtcacata ctctacctat ggcaagtttc ttgccgatgg tggttgctct              4250
gggggcgctt atgacatcat aatatgtgat gagtgccatt caactgactc              4300
gactacaatc ttgggcatcg gcacagtcct ggaccaagcg gagacggctg              4350
gagcgcggct tgtcgtgctc gccaccgcta cgcctccggg atcggtcacc              4400
gtgccacacc caaacatcga ggaggtggcc ctgtctaata ctggagagat              4450
ccccttctat ggcaaagcca tccccattga agccatcagg gggggaaggc              4500
atctcatttt ctgtcattcc aagaagaagt gcgacgagct cgccgcaaag              4550
ctgtcaggcc tcggaatcaa cgctgtggcg tattaccggg ggctcgatgt              4600
gtccgtcata ccaactatcg gagacgtcgt tgtcgtggca acagacgctc              4650
tgatgacggg ctatacgggc gactttgact cagtgatcga ctgtaacaca              4700
tgtgtcaccc agacagtcga cttcagcttg gatcccacct tcaccattga              4750
gacgacgacc gtgcctcaag acgcagtgtc gcgctcgcag cggcggggta              4800
ggactggcag gggtaggaga ggcatctaca ggtttgtgac tccgggagaa              4850
cggccctcgg gcatgttcga ttcctcggtc ctgtgtgagt gctatgacgc              4900
gggctgtgct tggtacgagc tcaccccggc cgagacctcg gttaggttgc              4950
gggcctacct gaacacacca gggttgcccg tttgccagga ccacctggag              5000
ttctgggaga gtgtcttcac aggcctcacc catatagatg cacacttctt              5050
gtcccagacc aagcaggcag gagacaactt cccctacctg gtagcatacc              5100
aagccacggt gtgcgccagg gctcaggccc cacctccatc atgggatcaa              5150
atgtggaagt gtctcatacg gctgaaacct acgctgcacg ggccaacacc              5200
cttgctgtac aggctgggag ccgtccagaa tgaggtcacc ctcacccacc              5250
ccataaccaa atacatcatg gcatgcatgt cggctgacct ggaggtcgtc              5300
actagcacct gggtgctggt gggcggagtc cttgcagctc tggccgcgta              5350
ttgcctgaca acaggcagtg tggtcattgt gggtaggatt atcttgtccg              5400
ggaggccggc cattgttccc gacagggagc ttctctacca ggagttcgat              5450
gaaatggaag agtgcgcctc gcacctccct tacatcgagc agggaatgca              5500
gctcgccgag caattcaagc agaaagcgct cgggttactg caaacagcca              5550
ccaaacaagc ggaggctgct gctcccgtgg tggagtccaa gtggcgagcc              5600
cttgagacat tctgggcgaa gcacatgtgg aatttcatca gcgggataca              5650
gtacttagca ggcttatcca ctctgcctgg gaaccccgca atagcatcat              5700
tgatggcatt cacagcctct atcaccagcc cgctcaccac ccaaagtacc              5750
ctcctgttta acatcttggg ggggtgggtg gctgcccaac tcgccccccc              5800
cagcgccgct tcggctttcg tgggcgccgg catcgccggt gcggctgttg              5850
gcagcatagg ccttgggaag gtgcttgtgg acattctggc gggttatgga              5900
gcaggagtgg ccggcgcgct cgtggccttt aaggtcatga gcggcgagat              5950
gccctccacc gaggacctgg tcaatctact tcctgccatc ctctctcctg              6000
gcgccctggt cgtcggggtc gtgtgtgcag caatactgcg tcgacacgtg              6050
ggtccgggag agggggctgt gcagtggatg aaccggctga tagcgttcgc              6100
ctcgcggggt aatcatgttt cccccacgca ctatgtgcct gagagcgacg              6150
ccgcagcgcg tgttactcag atcctctcca gccttaccat cactcagctg              6200
ctgaaaaggc tccaccagtg gattaatgaa gactgctcca caccgtgttc              6250
cggctcgtgg ctaagggatg tttgggactg gatatgcacg gtgttgactg              6300
acttcaagac ctggctccag tccaagctcc tgccgcagct acctggagtc              6350
ccttttttct cgtgccaacg cgggtacaag ggagtctggc ggggagacgg              6400
catcatgcaa accacctgcc catgtggagc acagatcacc ggacatgtca              6450
aaaacggttc catgaggatc gtcgggccta agacctgcag caacacgtgg              6500
catggaacat tccccatcaa cgcatacacc acgggcccct gcacaccctc              6550
tccagcgcca aactattcta gggcgctgtg gcgggtggcc gctgaggagt              6600
acgtggaggt cacgcgggtg ggggatttcc actacgtgac gggcatgacc              6650
actgacaacg taaagtgccc atgccaggtt ccggctcctg aattcttctc              6700
ggaggtggac ggagtgcggt tgcacaggta cgctccggcg tgcaggcctc              6750
tcctacggga ggaggttaca ttccaggtcg ggctcaacca atacctggtt              6800
gggtcacagc taccatgcga gcccgaaccg gatgtagcag tgctcacttc              6850
catgctcacc gacccctccc acatcacagc agaaacggct aagcgtaggt              6900
tggccagggg gtctcccccc tccttggcca gctcttcagc tagccagttg              6950
tctgcgcctt ccttgaaggc gacatgcact acccaccatg tctctccgga              7000
cgctgacctc atcgaggcca acctcctgtg gcggcaggag atgggcggga              7050
acatcacccg cgtggagtcg gagaacaagg tggtagtcct ggactctttc              7100
gacccgcttc gagcggagga ggatgagagg gaagtatccg ttccggcgga              7150
gatcctgcgg aaatccaaga agttccccgc agcgatgccc atctgggcgc              7200
gcccggatta caaccctcca ctgttagagt cctggaagga cccggactac              7250
gtccctccgg tggtgcacgg gtgcccgttg ccacctatca aggcccctcc              7300
aataccacct ccacggagaa agaggacggt tgtcctaaca gagtcctccg              7350
tgtcttctgc cttagcggag ctcgctacta agaccttcgg cagctccgaa              7400
tcatcggccg tcgacagcgg cacggcgacc gcccttcctg accaggcctc              7450
cgacgacggt gacaaaggat ccgacgttga gtcgtactcc tccatgcccc              7500
cccttgaggg ggaaccgggg gaccccgatc tcagtgacgg gtcttggtct              7550
accgtgagcg aggaagctag tgaggatgtc gtctgctgct caatgtccta              7600
cacatggaca ggcgccttga tcacgccatg cgctgcggag gaaagcaagc              7650
tgcccatcaa cgcgttgagc aactctttgc tgcgccacca taacatggtt              7700
tatgccacaa catctcgcag cgcaggcctg cggcagaaga aggtcacctt              7750
tgacagactg caagtcctgg acgaccacta ccgggacgtg ctcaaggaga              7800
tgaaggcgaa ggcgtccaca gttaaggcta aactcctatc cgtagaggaa              7850
gcctgcaagc tgacgccccc acattcggcc aaatccaagt ttggctatgg              7900
ggcaaaggac gtccggaacc tatccagcaa ggccgttaac cacatccact              7950
ccgtgtggaa ggacttgctg gaagacactg tgacaccaat tgacaccacc              8000
atcatggcaa aaaatgaggt tttctgtgtc caaccagaga aaggaggccg              8050
taagccagcc cgccttatcg tattcccaga tctgggagtc cgtgtatgcg              8100
agaagatggc cctctatgat gtggtctcca cccttcctca ggtcgtgatg              8150
ggctcctcat acggattcca gtactctcct gggcagcgag tcgagttcct              8200
ggtgaatacc tggaaatcaa agaaaaaccc catgggcttt tcatatgaca              8250
ctcgctgttt cgactcaacg gtcaccgaga acgacatccg tgttgaggag              8300
tcaatttacc aatgttgtga cttggccccc gaagccagac aggccataaa              8350
atcgctcaca gagcggcttt atatcggggg tcctctgact aattcaaaag              8400
ggcagaactg cggttatcgc cggtgccgcg cgagcggcgt gctgacgact              8450
agctgcggta acaccctcac atgttacttg aaggcctctg cagcctgtcg              8500
agctgcgaag ctccaggact gcacgatgct cgtgaacgga gacgacctcg              8550
tcgttatctg tgaaagcgcg ggaacccaag aggacgcggc gagcctacga              8600
gtcttcacgg aggctatgac taggtactcc gccccccccg gggacccgcc              8650
ccaaccagaa tacgacttgg agctgataac atcatgttcc tccaatgtgt              8700
cggtcgccca cgatgcatca ggcaaaaggg tgtactacct cacccgtgat              8750
cccaccaccc ccctagcacg ggctgcgtgg gagacagcta gacacactcc              8800
agttaactcc tggctaggca acattattat gtatgcgccc actttgtggg              8850
caaggatgat tctgatgact cacttcttct ccatccttct agcgcaggag              8900
caacttgaaa aagccctgga ctgccagatc tacggggcct gttactccat              8950
tgagccactt gacctacctc agatcattga acgactccat ggccttagcg              9000
cattttcact ccatagttac tctccaggtg agatcaatag ggtggcttca              9050
tgcctcagga aacttggggt accacccttg cgagtctgga gacatcgggc              9100
caggagcgtc cgcgctaggc tactgtccca gggagggagg gccgccactt              9150
gtggcaaata cctcttcaac tgggcagtaa aaaccaaact taaactcact              9200
ccaatcccgg ctgcgtcccg gctggacttg tccggctggt tcgttgctgg              9250
ttacagcggg ggagacatat atcacagcct gtctcgtgcc cgaccccgtt              9300
ggttcatgct gtgcctactc ctactttctg taggggtagg catctacctg              9350
ctccccaacc gatgaacggg gagataaaca ctccaggcca ataggccatc              9400
cccctttttt tttttt                                                   9416