Abstract:
The present invention relates to novel hepatitis B virus vectors for use in gene therapy which can deliver therapeutic genes to liver cells. The invention also provides methods for the production of novel recombinant hepatitis B viruses. The recombinant viruses produced by this invention can deliver therapeutic genes specifically to liver cells either through in vivo or ex vivo therapy protocols. This vector can be used not only to treat liver diseases but also genetic diseases.

Description:
FIELD OF THE INVENTION  
         [0001]    The present invention relates to recombinant hepatitis B viral vectors useful for the expression of heterologous genes in liver cells. The invention also provides methods for the production of novel recombinant hepatitis B viruses. Liver-specific targeting ability of these HBV vectors extend its use for in vivo gene therapy protocol as well as for ex vivo therapy protocol. The recombinant viruses produced by this invention can deliver therapeutic genes specifically to liver cells either in vivo therapy protocol or ex vivo therapy protocol. This vector can be used not only to treat liver diseases but also genetic diseases.  
         BACKGROUND OF THE INVENTION  
         [0002]    Gene therapy is considered as a new healer of modem medicine since genome sequencing is nearly completed (Anderson, 1992). Numerous methods for gene therapy have been developed in recent years (Mulligan, 1993). Gene therapy vectors used in current clinical trials can be divided into two groups: viral vectors such as retroviruses, adenoviruses or adeno-associated viruses (AAV) and nonviral vectors such as liposomes or naked DNAs (Friedmann, 1999). The most critical parameter of gene therapy is the efficiency of delivery of therapeutic genes to the recipient cells. To meet this goal, vectors need to not only specifically target recipient cells but also stably express therapeutic genes so that the therapeutic effect can be achieved. Lack of tissue-specificity and lack of long-term stable expression are serious drawbacks of current gene therapy vectors (Crystal, 1995).  
           [0003]    To obtain efficient delivery of transgenes to target cells, viral vectors are frequently employed for gene therapy protocols. In particular, vectors that are used most often are those derived from retroviruses, adenoviruses or adeno-associated viruses (Crystal, 1995). These viral vectors are nonpathogenic and are designed to be replication-incompetent in recipient cells.  
           [0004]    Most attempts to use viral vectors for gene therapy have relied on either retrovirus vectors or adenovirus vectors. Retroviral vectors are capable of maintaining stable gene expression because of their ability to integrate into the cellular genome. However, the disadvantages of retroviral vectors are becoming increasingly clear, including their tropism for dividing cells only, the possibility of insertional mutagenesis upon integration into the cell genome, decreased expression of the transgene over time and the possibility of generation of replication-competent retroviruses. On the other hand adenoviruses can infect nondividing cells, but can induce only transient expression of therapeutic genes. Further, repetitive administration of adenoviral vector to obtain long-term expression frequently induces severe inflammation (Yang et al., 1995). Evidently, these viral vectors need significant improvement before clinical use.  
           [0005]    Although these viral vectors are most frequently used, they have a few unacceptable drawbacks. To improve the lack of tissue specificity, targeted viral vectors have been studied in laboratories (Douglas et al., 1999). However, it is not clear whether targeted viral vectors can be clinically used in the near future.  
           [0006]    Regarding liver-directed gene therapy, the protocol for these viral vectors are by and large limited to ex vivo therapy, since these vectors lack tissue-specificity (i. e., hepatocyte-specificity). Ex vivo liver-directed therapy involves the surgical removal of liver cells, transduction of the liver cells in vitro (e. g., infection of the explanted cells with recombinant viral vectors) followed by injection of the genetically modified liver cells into the liver or spleen of the patient. A serious drawback for ex vivo liver- directed gene therapy is the fact that hepatocytes (i. e., liver cells) cannot be maintained and expanded in culture. Besides the technical difficulties and complexities, costs involved in each protocol are evidently astronomical.  
           [0007]    Ideally, liver-directed gene therapy would be achieved by in vivo transfer of vectors which specifically target hepatocytes. Vectors derived from hepatotropic viruses, such as hepatitis B viruses (HBV), can be administered via circulation and target hepatocytes using the same receptor as the wild-type virus. However, the hepatitis B viruses have not been explored as a gene therapy vector due to lack of information on cis-acting elements essential for HBV genome replication.  
           [0008]    Hepatitis B virus (HBV) is the prototype of the hepadnaviridae, a family of a small enveloped DNA virus with pronounced host and tissue specificity (Ganem, 1996). Hepadnaviruses have been found in mammals, e.g., human (HBV), woodchuck (WHV) and ground squirrels (GSHV), as well as in birds, e. g., Pekins ducks (DHBV) and grey herons (HHBV).  
           [0009]    One of the bottlenecks in developing an HBV-derived gene therapy vector was a lack of information on cis-acting elements that are essential for viral genome replication. Thus, it is prerequisite to map cis-acting elements across the entire HBV genome.  
         SUMMARY OF THE INVENTION  
         [0010]    The present invention comprises a novel hepatitis B virus vector and methods for making and using such vectors in liver-targeting gene therapy. The recombinant hepatitis B virus particles will specifically target hepatocytes of liver tissue. It is thought that the HBV vector will be particularly useful in gene transfer to liver tissue. Further, it is contemplated that the tissue specificity of the HBV vector will enable the HBV vector to be suitable even for in vivo therapy as well as ex vivo therapy. These novel HBV vectors may be used to deliver genes to liver in vivo by a variety of means including infection via circulation or direct injection of DNA into liver tissue.  
           [0011]    The tropism of hepadnaviruses for hepatocytes has particular relevance to the use of HBV in gene therapy for diseases, which are caused by lack of gene expression in liver tissue. These diseases include numerous metabolic diseases, such as hemophilia lacking factor VIII or IV expression in liver. In addition, the HBV vector will be very useful to treat patients with chronic HBV infection. Since most hepatocytes of these chronic patients are equipped with packaging function (i. e., core, polymerase, and surface antigen expression), administration of the vector DNA could lead to packaging of the recombinant HBV particles, which could then infect neighboring hepatocytes. Thus, the vector DNA encoding various antiviral functions could induce therapeutic benefit. The vector DNA could be administered via direct intrahepatic injection or via circulation.  
           [0012]    The invention provides information on two novel cis-acting elements of the hepatitis B virus genome that are essential for viral genome replication: α element and β element. The invention also provides a nucleotide sequence of the α element and β element.  
           [0013]    The present invention is illustrated using recombinant HBV genome; however, the invention contemplates the use of other hepadnaviruses, including but not limited to woodchuck hepatitis virus (WHV), ground squirrel hepatitis virus (GSHV), and duck hepatitis B virus (DHBV). The art is well aware that the genomic organization of these hepadnaviruses is similar and that the teachings of the present invention can be translated to other hepadnaviruses.  
           [0014]    In one embodiment of the present invention, the HBV vectors retain all cis-acting elements essential for viral genome replication. However, the present invention does not limit the position of the two novel cis-acting elements (i. e., α element and βelement) at the indicated position on the map. In one embodiment, it is contemplated that in order to accomodate a larger insertion without exceeding the packaging size limit, the position of novel cis-acting elements could be changed without compromising vector function.  
           [0015]    The present invention also delineates methods for the encapsidation of a recombinant hepatitis B virus genome, comprising the steps of providing: i) a recombinant HBV vector encoding at least one heterologous gene sequence inserted; ii) a helper plasmid capable of providing in trans hepatitis B virus gene products to complement the HBV vector for encapsidation and viral genome replication; and iii) introducing the recombinant HBV vector plasmid and a helper plasmid into the liver cell under conditions such that the recombinant HBV genome is encapsidated into the viral particles. It is contemplated that the liver cells of the present invention be selected from the group consisting of human liver cells including HepG2 cells, Huh7 cells, Chang liver cells, and rodent cells.  
           [0016]    In one embodiment of the method, a heterologous gene sequences is inserted between the α element and DR2 of the prototype HBV vector (FIG. 10). In another embodiment of the method, it is contemplated that a heterologous gene sequences is inserted between 5′ epsilon and the α element of the prototype HBV vector (FIG. 8).  
           [0017]    A few attempts were made to generate recombinant HBV viruses, in which a subset of the HBV genome was substituted by heterologous genes (Chiang et al., 1992; Chaisomchit et al., 1997; Protzer et al., 1999). The present invention significantly differs from reported U.S. Pat. No. 5,981,274 as follows (Chaisomchit, et al., 1997). First of all, a heterologous sequence was inserted into the spacer (or tether) domain of the HBV polymerase ORF as a fusion protein in the patent above. Contrary to this, the present invention indicates that this insertion site overlaps with the α element found to be essential for the viral genome replication in this invention. Thus, the 50-fold reduction of the viral genome replication as indicated in the patent is a consequence of disruption of the α element in the vector. Further, the size of insert (267 bp or 374 bp) and its expression as a fusion protein limits its use as a vector. In conclusion, the recombinant HBV vector claimed in the patent above is defective or not capable of packaging a recombinant HBV genome encoding a heterologous gene sequence.  
           [0018]    In addition, it has been claimed that two HBV mutants, in which a part of the HBV genome was substituted by the 0.7 K bp luciferase gene fragment, could produce virion particles in culture medium (Chiang et al., 1992). Further, the first successful production of recombinant hepadnaviruses (i. e., DHBV and HBV) encoding either GFP or interferon alpha were recently reported (Protzer et al., 1999). In both of these studies, without knowing the cis-elements essential for genome replication, they succeed in making recombinant hepadnaviruses when they substituted S ORF of DHBV or HBV with GFP or interferon-alpha in their recombinant vectors. The present invention significantly differs from the published reports in that this invention completely mapped cis-elements essential for viral genome replication. Based on the mapping data, this invention provides the prototype recombinant HBV vector in which heterologous sequences can be inserted into two different sites with an insert size of up to 0.90 K bp or 1.7 K bp, respectively. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0019]    [0019]FIG. 1. A schematic representation of the pregenomic RNA of hepatitis B virus. cis-elements are indicated: DR1 (direct repeat 1), DR2 (direct repeat 2), epsilon (encapsidation signal), r (repeat) element, PRE (posttranscriptional RNA processing element). Four open reading frames of HBV are represented as arrowed open boxes: precore region (nt. 1816-1902), core region (nt. 1903-2454); pre-S1 region (nt. 2850-3173), pre-S2 region (nt. 3174-156), S region (nt. 157-837); P region (nt. 2310-1625), X region (nt. 1376-1840).  
         [0020]    [0020]FIG. 2. Life cycle of hepadnaviruses.  
         [0021]    [0021]FIG. 3. Model for the synthesis of hepadnaviral DNA through reverse transcription of the pregenomic RNA as a template for the viral polymerase. (see text for explanation).  
         [0022]    [0022]FIG. 4. A schematic representation of gene therapy procedure using the hepatitis B virus vector to deliver a heterologous gene (e. g., GFP) to liver cells. A recombinant HBV vector DNA encoding GFP gene is transfected into a packaging cell line that expresses viral proteins necessary for packaging the recombinant HBV genome. The produced recombinant HBV particles then infect hepatocyctes. Upon the entry of the HBV particles into cells, the viral DNA is repaired to CCC (covalently closed circular) form DNA in the nucleus and induces the expression of GFP, as the wild-type HBV does. The packaging cell line can be replaced by a helper plasmid that provides core and the viral polymerase.  
         [0023]    [0023]FIG. 5 a.  A schematic representation of the subcloning procedure of R015 plasmid.  
         [0024]    [0024]FIG. 5 b.  Map of R015 plasmid. Nucleotide sequence of R015 plasmid is attached in SEQ ID 3.  
         [0025]    [0025]FIG. 6. Map of a series of small deletion mutants. The sequence deleted in each mutant was indicated by a solid line. The plasmid names are indicated by prefix R followed by numbers. The nucleotide position and the size of the deletions are indicated.  
         [0026]    [0026]FIG. 7. An autoradiograph of Southern blot analysis of a series of deletion mutants to determine if each deletion mutant is replication-competent. RC, relaxed circular DNA; DL, double-stranded linear DNA; SS, single-stranded DNA.  
         [0027]    [0027]FIG. 8. Map of the prototype HBV gene therapy vector. Sequence elements of the HBV vector are drawn on the pregenomic RNA. Two novel cis-elements; α element, βelement. Two proposed insertion sites are indicated by open boxes. Appropriately located viral promoters are indicated; core promoter and pre-S2/S promoter, respectively.  
         [0028]    [0028]FIG. 9. A schematic representation of subcloning procedure of R7 11 plasmid.  
         [0029]    [0029]FIG. 10. A schematic representation of R711 plasmid with cis-acting elements.  
         [0030]    [0030]FIG. 11 a.  An autoradiograph of Southern blot analysis of R7 11 plasmid. HBV probe was used to detect the viral replication-intermedaites. RC, relaxed circular DNA; DL, double-stranded linear DNA; SS, single-stranded DNA.  
         [0031]    [0031]FIG. 11 b.  An autoradiograph of Southern blot analysis of R711 plasmid. GFP probe was used to detect the viral replication-intermediates. RC, relaxed circular DNA; DL, double-stranded linear DNA; SS, single-stranded DNA.  
         [0032]    Table 1. Summary of data obtained from a series of deletion mutants. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0033]    A. DEFINITIONS  
         [0034]    To facilitate understanding of the invention, a number of terms are defined below.  
         [0035]    As used herein, the term “cis” is used in reference to the presence of genes on the same chromosome.  
         [0036]    The term “trans-acting” is used in reference to the controlling effect of a regulatory gene on a gene present on a different chromosome.  
         [0037]    As used herein, the term “in trans” is used in reference to indicate the complementation effect of a gene product on a gene present on a different chromosome.  
         [0038]    The term “cis-acting” is used in reference to the controlling effect of a regulatory gene on a gene present on the same chromosome.  
         [0039]    Nucleotide sequences of HBV genome were numbered according to Galibert et al. (Galibert et al., 1979), unless otherwise indicated. In this numbering system, the 5′-end of the pregenomic RNA is at nt. 1820 (Nassal et al., 1990). On the other hand, nucleotide sequences of the plasmids included in SEQ were numbered from the 5′-end of the pregenomic RNA.  
         [0040]    B. OVERVIEW  
         [0041]    1. Hepatitis B Viruses  
         [0042]    Hepatitis B virus (HBV), the causative agent of chronic hepatitis in man, is the prototype member of the hepadnaviridae (Ganem, 1996). Related members of the hepadnavirus family include woodchuck hepatitis virus (WHV), ground squirrel hepatitis virus (GSHV), and duck hepatitis B virus (DHBV). HBV genome is a circular DNA of only 3.2 K bp in length. The viral genome is a partially duplex circular DNA, possessing a single-stranded gap region in plus-strand DNA. Although HBV has a DNA genome, it replicates through reverse transcription of an RNA intermediate, the pregenomic RNA (pgRNA), within the subviral core particle. There are four major open reading frames (ORFs), all encoded in same strand (FIG. 1). Inspection of the sequence led to the recognition of conserved repeat elements that play important roles in the genome replication. These direct repeats (denoted DR1 and DR2) are located near the 5′ end of the minus and plus DNA strands (FIG. 1).  
         [0043]    The hepadnaviral life cycle is outlined in FIG. 2 (Ganem et al., 1987). Hepadnaviruses are thought to enter the hepatocytes through receptor-mediated endocytosis. Upon entry, a partial duplex genome is repaired to a covalently closed circular DNA (CCC), which is the template for transcription. Four viral transcripts are synthesized and transported to cytoplasm. The 3.5 K bp RNA, also called pregenomic RNA, serves as a template for reverse transcription as well as for translation of the core (C) and polymerase (P).  
         [0044]    II. Reverse transcription  
         [0045]    Despite of the general similarities to retroviruses, many steps in its replication are distinct (Nassal et al., 1996). The first step of HBV genome replication is the encapsidation of the pregenomic RNA into core particles. Core particle assembly involves the interactions of the structural proteins, core (C) and polymerase (P) with the pregenomic RNA. Incorporation of P protein as well as the pregenomic RNA into assembling core particles is essential for viral DNA synthesis. The cis-acting element for encapsidation, termed ε, has been defined within 85 nucleotides (nt) near the 5′ end of pgRNA, which is necessary and sufficient to direct encapsidation of heterologous RNA sequences into viral core particle (Junker-Niepmann et al., 1990; Hirsch et al., 1991). The epsilon element can fold into a stem-loop structure, which is highly conserved among hepadnaviruses.  
         [0046]    Reverse transcription mechanism of HBV polymerase is quite complicated, as expected from its peculiar genome structure. A process described as template switching is required for the successful synthesis of a double-stranded DNA product (FIG. 3). First, minus-strand DNA synthesis is initiated near the 5′-end of its template, the pregenomic RNA (Loeb et al., 1995). The viral polymerase is both the primer and polymerase for minus-strand DNA synthesis (Wang et al., 1992). Following template switching to an acceptor site near the 3′ end of the pregenomic RNA, minus-strand DNA synthesis resumes at this position, resulting in a genome-length, minus-strand DNA. Upon completion of the synthesis of minus-strand DNA, the final RNase H cleavage product, the 18-nt RNA fragment, serves as a primer for the initiation of plus-strand DNA synthesis (Loeb et al., 1991). Upon translocation to DR2, the RNA primer is used for the initiation of plus-strand DNA synthesis. For the plus-strand DNA initiated at DR2, a third template switch, termed circularization, is required to generate a mature relaxed circular DNA.  
         [0047]    IV. Cis-acting elements essential for HBV genome replication  
         [0048]    Molecular analysis revealed that several elements play a role in the viral DNA synthesis (Nassal et al., 1996). The list of cis-acting elements includes: 5′ epsilon, encapsidation signal (Junker-Niepmann et al., 1990; Hirsch et al., 1991); DR1 and DR2, primer acceptor sites for primer translocation step during viral DNA synthesis (Nassal et al., 1996); r (repeat) for circularization (Loeb et al., 1997); and PRE, posttranscriptional RNA processing element (Huang et al., 1995). All of these known elements are located to either side of the HBV pregenome (FIG. 1). In the case of DHBV, three additional elements, termed 3E, M and 5E, have been reported to be essential for template switching during plus-strand DNA synthesis (Havert et al., 1997). However, it is not known whether any other elements in the middle of the viral genome are essential for HBV genome replication. HBV genome has not been explored as a gene therapy vector, primarily due to the lack of information on its cis-acting elements.  
         [0049]    V. Design of the prototype HBV vector  
         [0050]    Having identified all cis-acting elements required for HBV genome replication, it is possible to design a gene therapy vector that can accomodate a heterologous gene sequence without compromising its ability to replicate, if trans-acting factors are provided in trans. Briefly, the HBV vector encodes all cis-acting elements that are essential for the viral genome replication, but lacks expression of the viral proteins. Nonetheless, the recombinant virus can be produced if the viral proteins (i. e., core, polymerase, surface antigens) are provided in trans via a helper plasmid or packaging cell lines (FIG. 4).  
         [0051]    Several issues need to be considered for the design of a gene therapy vector including insertion site, size of insert(s), promoter to drive transgene transcription. First of all, two insertion sites were selected; one between 5′ epsilon and the a element, the other site located between the a element and DR2 (FIG. 8). These two insertion sites were selected since these sequences are dispensable for viral genome replication. Regarding insert size, fragments of up to 0.90 K bp and 1.7 K bp, respectively, can be inserted into these two insertion sites without significantly exceeding the wild-type genome size. Two endogenous viral promoters (i. e., core promoter and pre-S2/S promoter), conveniently located just upstream of these two insertion sites, are employed to drive transcription. Further, this HBV vector can be used as a bicistronic expression vector, if two insertion sites are used simultaneously (FIG. 8).  
         [0052]    C. EXPERIMENTAL  
         [0053]    Most of the techniques used for vector construction and cell transfection are widely practiced in the art, and most practitioners are familiar with standard resource materials describing specific conditions and procedures.  
         [0054]    Construction of the vectors of the invention employs standard ligation and restriction techniques which are well understood in the art (see Sambrook et al., 2001).  
         [0055]    In this experimental disclosure, the following abbreviations are applied: M (molar), mM (millimolar), ml (milliliters), μg (micrograms), mg (milligrams), PEG (polyethylene glycol), ORF (open reading frame),  
         [0056]    The following examples are presented to illustrate the present invention and to assist one of ordinary skill in making and using the same. The examples are not intended in any way to otherwise limit the scope of the disclosure.  
       EXAMPLE 1  
       [0057]    Construction of a replication-competent plasmid for wild-type HBV  
         [0058]    To design a HBV gene therapy vector, it is prerequisite to map all the cis-acting elements that are essential for the viral genome replication. To achieve this, a replication-competent plasmid that can lead to the production of infectious HBV particles upon transfection was constructed. The fact that a heterologous promoter driven RNA transcript analogous to the pregenomic RNA can lead to the production of infectious viral particles are well understood in the art (Nassal et al., 1990). Thus, a pregenomic RNA expression plasmid was designed such that the 5′-end of the transcripts would be identical to that of wild-type HBV. Specifically, the position of 5′-epsilon element is 30 nucleotides away from the 5′-end (Jeong et al., 2000).  
         [0059]    The nucleotide sequence of the HBV genome was numbered starting at the unique Eco RI site of HBV ayw subtype, according to the method of Galibert et al. (Galibert et al., 1979). Nucleotide numbers (nt.) indicate the HBV sequence number, unless otherwise indicated. In this number system, the 5′ end of the pregenomic RNA is at nt. 1820 (Nassal et al., 1990).  
         [0060]    1-1. Construction of R402 plasmid (pCMV-HBV/164):  
         [0061]    To generate a replication-competent HBV construct, the greater-than-genome-length viral genome should be inserted downstream of the promoter element to maintain terminal redundancy of the pregenomic RNA (see FIG. 1; Ganem et al., 1987). The genome of hepatitis B virus was derived from pSV2A-Neo(HBV)2 plasmid that contains a dimer of HBV ayw subtype (Shih et al., 1989). The greater-than-genome-length Fsp I(nt. 1804)-to-Xba I(nt. 1992) fragment (3354 nt) of HBV ayw subtype (Galibert et al., 1979) was inserted into Eco RV and Xba I sites in the multiple cloning site of pcDNA1/Amp plasmid (Invitrogen, U.S.A.): R402 plasmid (pCMV-HBV/164). The HBV transcript made from this plasmid (pCMV-HBV/164) has a vector-derived 134 nt at the 5′ end relative to that of wild-type pregenomic RNA.  
         [0062]    1-2. Construction of R015 plasmid (pCMV-HBV/30):  
         [0063]    To make a RNA expression plasmid that can transcribe the HBV pregenomic RNA that is almost identical to the wild-type pregenomic RNA with respect to the position of the epsilon element, a small deletion was introduced into R402 plasmid (pCMV-HBV/164). Thus, R015 plasmid (pCMV-HBV/30) was made by removing this pcDNA1/Amp plasmid-derived 134 nucleotides by a PCR-mediated method (Jeong et al., 2000).  
         [0064]    Briefly, a fragment was made by polymerase chain reaction using a forward primer of the sequence 5 -CCCGAGCTCTCTGGCTAACTAACTTTTTCACCTCTGCC-3 (SacI site underlined) and a reverse primer of the sequence 5 -CCCAAGCTTCTATTGTTCCCAAGAATATGG-3 (nt 2839 to 2822) with R402 (pCMV-HBV/164) as a template. The resulting PCR fragment was digested by SacI and BspEI and then inserted between the SacI (nt. 2894 of pcDNAl/amp) and BspEI (nt. 2331) site of R402 (pCMV-HBV/164).  
         [0065]    1-3. Construction of R063 (pCMV-CPS) helper plasmid  
         [0066]    Briefly, PCR was carried out using a forward primer with EcoR I site and a reverse primer with Xho I site to generate the EcoR I-to-Xho I fragment (nt. 1903-to-2454) Then, the 0.5 K bp PCR product was inserted into pcDNA3 (Invitrogen, U. S. A) via EcoR I, Xho I restriction sites to make R062 plasmid. Next, the BspE I (nt. 233 1)-to-Apa I of R062 plasmid was substituted by 2.6 K bp BspE I (nt. 233 1)-to-Apa I of R015 plasmid. This R063 plasmid lacking encapsidation signal, epsilon, was employed as helper plasmid to provide the viral proteins (i. e., core, polymerase, surface antigen) essential for the viral replication and assembly.  
         [0067]    Forward primer: 5′-CATGGAATTCATGGACATCGACCCT-3 (EcoR I site underlined)  
         [0068]    Reverse primer: 5′-CCGCTCGAGCTAACATTGAGATTCCCGAGA-3′ (Xho I site underlined)  
         [0069]    Forward primer: 5′-CATGGAATTCATGGACATCGACCCT-3 (EcoR I site underlined)  
         [0070]    Reverse primer: 5′-CCGCTCGAGCTAACATTGAGATTCCCGAGA-3′ (Xho I site underlined)  
       EXAMPLE 2  
       [0071]    Demonstration of replication-competency of wild-type pregenomic RNA expression plasmid, R015 plasmid (pCMV-HBV/30).  
         [0072]    2-1. Cell growth, transfection of heptoma cell lines  
         [0073]    Human hepatoma cells, designated Huh7 cells were grown in DMEM (Gibco-BRL) supplemented with 10% fetal bovine serum (Gibco-BRL) and 10 □ of gentamicin per mL and were split every third day. The day before transfection, cells were plated at a confluency of 75%. On the following day, cells were washed twice with phosphate- buffered saline (PBS) and given fresh media. After 2 hours, cells were transfected with 10 □ of supercoiled plasmid DNA per 60 mm plate by the CaPO4 coprecipitation technique.  
         [0074]    2-2. Southern blot analysis of the viral replication-intermediate from cytoplasmic core particles.  
         [0075]    Three days after transfection, viral DNAs were extracted from intracellular core particles by PEG precipitation as described previously (Staprans et al., 1991). Briefly, transfected cells from a 100-mm plate were lysed in lysis buffer [10 mM Tris (pH 7.5), 1 mM EDTA, 50 mM NaCl, 8% sucrose, 0.25% Nonidet P-40]. Nuclei were removed by centrifugation for 3 min in a microcentrifuge, and the cytoplasmic extract was adjusted to 6 mM MgCl 2  and digested with DNase I (50 □/□) for 30 min at 37° C. Cores were precipitated by centrifugation for 4 min after adding 4× PNE buffer [26% PEG, 1.4 M NaCl, 25 mM EDTA], and incubating at 4° C. for 30 min.. Core particles resuspended in buffer [10 mM Tris (pH 7.5), 6 mM MgCl 2 ] were then digested with DNase I for an additional 15 min at 37° C., followed by the addition of 5 mM EDTA, 1% SDS, and 500 □ of proteinase K per □ and were incubated for 1 h at 37° C. Core nucleic acid was extracted twice with phenol/CHCl 3  (1:1) and precipitated with ethanol, then resuspended in 50 □ of TE [10 mM Tris(pH 7.5), 1 mM EDTA].  
         [0076]    Extracted viral DNA were subjected to agarose gel electrophoresis, followed by Southern blot analysis, which are well known to those skilled in the art ( Current Protocols in Molecular Biology,  Ausubel, F. et al., eds., Wiley and Sons, New York, 1995).  
       EXAMPLE 3  
       [0077]    Deletion mutants of R015 plasmid (pCMV-HBV/30).  
         [0078]    A series of small deletion mutants were generated by standard recombinant DNA technology (Sambrook et al., 2001).  
         [0079]    3-1. R060(pCMV-ayw Δ1910-1992) plasmid  
         [0080]    Plasmid R059(pBS+Δ1910-1992) was made in which the Sac I-to-EcoR I(nt. 3182) fragment of HBV ayw subtype, but lacking the nt. 1910-1992 fragment (Galibert et al., 1979) was subcloned into pBluescript SK(+) plasmid (Stratagene, USA). Subsequently, the Sac I-to-EcoR I fragment of R015 was replaced by the Sac I-to-EcoR I fragment of R059 to generate R060 plasmid.  
         [0081]    3-2. R048(pCMV-ayw Δ1884-2459) plasmid  
         [0082]    First, plasmid R046 was made in which the Sac I-to-EcoR I(nt. 3182) fragment of HBV ayw subtype (Galibert et al., 1979) was subcloned into pCH110 (Pharmacia). Then, R047 plasmid was generated by deleting the 151 bp Xba I fragment (nt. 1992-2143). Subsequently, the Sac I-to-EcoR I fragment of R015 plasmid was replaced by the Sac I-to-EcoR I fragment of R047 to generate R048 plasmid.  
         [0083]    3-3. R056(pCMV-ayw Δ2143-2459) plasmid  
         [0084]    First, plasmid R049 was made in which the Sac I-to-EcoR I(nt. 3182) fragment of HBV ayw subtype (Galibert et al., 1979) was subcloned into pBluescript II KS(+) (Stratagene, U. S. A). The Sty I(nt. 1884)-to-Sty I(nt. 2459) fragment of R049 was replaced by a PCR product of Sty I(nt. 1884)-to-Xba I(nt. 2143) fragment encoding Sty I restriction site at the 5′-end of the reverse primer to make R051 plasmid. Subsequently, the Sac I-to-EcoR I fragment of R015 was replaced by the Sac I-to-EcoR I fragment of R051 plasmid to generate the R056 deletion mutant.  
         [0085]    Forward primer: 5 -CCCGAGCTCTCTGGCTAACTAACTTTTCACCTCTGCC-3 (Sac I site underlined)  
         [0086]    Reverse primer: 5′-CCCCCCAAGGCGCTGGATCTTCCAAATT-3′ (Sty I site underlined)  
         [0087]    3-4. R021(pCMV-ayw Δ2459-2817) plasmid  
         [0088]    First, plasmid R407 was made in which the Sac I-to-Xho I(nt. 129) fragment of R015 plasmid was subcloned into pBlueBacHis2 plasmid (Invitrogen, U. S. A.). Then, the Sty I(nt. 2459)-to-BstE II(nt. 2817) fragment of R407 was deleted and filled in by Klenow fragment to make R018 plasmid. Subsequently, the BspE I (nt. 2331)-to-EcoR I (nt. 3182) fragment of R015 was replaced by the BspE I-to-EcoR I fragment of R018 to generate R021 deletion mutant.  
         [0089]    3-5. R022(pCMV-ayw Δ2662-3182) plasmid  
         [0090]    To make R022, the BstE II(nt. 2662)-to-EcoR I(nt. 3182) fragment of R015 plasmid was deleted and filled in by Klenow fragment to make R022 plasmid.  
         [0091]    3-6. R045(pCMV-ayw Δ2839-3182) plasmid  
         [0092]    First, plasmid R701 was made in which the BstE II(nt. 2817)-to-Sph I(nt. 1239) fragment of R015 plasmid was subcloned into pGEM-4Z plasmid (Promega, U.S.A). Then, the Bg1 II(nt. 2839)-to-EcoR I(nt. 3182) fragment of R701 was deleted and filled in by Klenow fragment to make R043 plasmid. Subsequently, the BstX I (nt. 2817)-to-BstX I(nt. 620) fragment of R015 was replaced by the corresponding 642 bp BstX I fragment of R043 to generate R045 deletion mutant.  
         [0093]    3-7. R044(pCMV-ayw Δ3052-3182) plasmid  
         [0094]    The Bsu36 I(nt. 3052)-to-EcoR I(nt. 3182) fragment of R701 was deleted and filled in by Klenow fragment to make R042 plasmid. Subsequently, the BstX I (nt. 2817) -to-BstX I(nt. 620) fragment of R015 was replaced by the corresponding 855 bp BstX I fragment of R042 plasmid to generate R044 deletion mutant.  
         [0095]    3-8. R023(pCMV-ayw Δ3182-129) plasmid  
         [0096]    To make R023, the EcoR I(nt. 3182)-to-Xho I(nt. 129) fragment of R015 was deleted and filled in by Klenow fragment to make R023 plasmid.  
         [0097]    3-9. R040(pCMV-ayw Δ129-490) plasmid  
         [0098]    First, plasmid R037 was made in which the EcoR I(nt. 3182)-to-Sph I(nt. 1239) fragment of R015 plasmid was subcloned into pGEM-4Z plasmid (Promega, U.S.A). The Xho I(nt. 129)-to-BamH I(nt. 490) fragment of R037 plasmid was deleted and filled in by Klenow fragment to make R038 plasmid. Subsequently, the EcoR I (nt. 3182)-to-Sph I(nt. 1238) fragment of R015 plasmid was replaced by the corresponding 877 bp EcoR I-to-Sph I fragment of R038 plasmid to generate R040 deletion mutant.  
         [0099]    3-10. R041(pCMV-ayw Δ490-827) plasmid  
         [0100]    The BamH I(nt. 490)-to-Acc I(nt. 827) fragment of R037 was deleted and filled in by Klenow fragment to make R039 plasmid. Subsequently, the EcoR I (nt. 3182)-to-Sph I(nt. 1238) fragment of R015 plasmid was replaced by the corresponding 897 bp EcoR I-to-Sph I fragment of R039 to generate R041 deletion mutant.  
         [0101]    3-11. R025(pCMV-ayw Δ827-1238) plasmid  
         [0102]    First, a plasmid R050 was made in which the EcoR I(nt. 3182)-to-Apa I fragment of R015 plasmid was subcloned into pBluescript II KS(+) (Stratagene, U. S. A). The Acc I(nt. 827)-to-Sph I(nt. 1238) fragment of R050 plasmid was deleted and filled in by T4 DNA polymerase to make R008 plasmid. Subsequently, the EcoR I (nt. 3182)-to-Apa I fragment of R015 was replaced by the corresponding 1591 bp EcoR I-to-Apa I fragment of R008 to generate R025 deletion mutant.  
         [0103]    3-12. R026(pCMV-ayw Δ1238-1374) plasmid  
         [0104]    The Sph I(nt. 1238)-to-Nco I(nt. 1374) fragment of R050 was deleted and filled in by T4 DNA polymerase to make R009 plasmid. Subsequently, the EcoR I (nt. 3182)-to-Apa I fragment of R015 plasmid was replaced by the corresponding 1866 bp EcoR I-to-Apa I fragment of R009 plasmid to generate R026 deletion mutant.  
         [0105]    3-13. R027(pCMV-ayw Δ1374-1419) plasmid  
         [0106]    The Nco I(nt. 1374)-to-Aat II(nt. 1419) fragment of R050 plasmid was deleted and filled in by T4 DNA polymerase to make R012 plasmid. Subsequently, the EcoR I (nt. 3182)-to-Apa I fragment of R015 was replaced by the corresponding 1957 bp EcoR I-to-Apa I fragment of R012 to generate R027 deletion mutant.  
         [0107]    3-14. R028(pCMV-ayw Δ1419-1804) plasmid  
         [0108]    The Aat II(nt. 1419)-to-Fsp I(nt. 1804) fragment of R050 was deleted and filled in by T4 DNA polymerase to make R013 plasmid. Subsequently, the EcoR I (nt. 3182) -to-Apa I fragment of R015 was replaced by the corresponding 1617 bp EcoR I-to-Apa I fragment of R013 to generate R028 deletion mutant.  
         [0109]    3-15. R053(pCMV-ayw Δ1419-1592) plasmid  
         [0110]    The Aat II(nt. 1419)-to-Apa I fragment of R050 was replaced by the PCR product of Aat II(nt. 1592)-to-Apa I fragment encoding Aat II restriction site at the 5′-end of the forward primer to make R052 plasmid. Subsequently, the EcoR I-to-Apa I fragment of R015 was replaced by the EcoR I-to-Apa I fragment of R052 to generate R053 deletion mutant.  
         [0111]    3-16. R035(pCMV-ayw Δ1607-1804) plasmid  
         [0112]    The EcoR I(nt. 3182)-to-Bsa I(nt. 1607) blunted fragment of R050 plasmid was ligated with the EcoR I(nt. 3182)-to-Fsp I(nt. 1804) of R015 plasmid to make R035 plasmid.  
         [0113]    3-17. R029(pCMV-ayw Δ1804-1884) plasmid  
         [0114]    The Fsp I(nt. 1804)-to-Sty I(nt. 1884) fragment of R050 was deleted and filled in by Klenow polymerase to make R010 plasmid. Subsequently, the EcoR I (nt. 3182)-to-Apa I fragment of R015 was replaced by the corresponding 1922 bp EcoR I-to-Apa I fragment of R010 to generate R029 deletion mutant.  
       EXAMPLE 4  
       [0115]    Analysis of cis-elements essential for HBV genome replication  
         [0116]    4-1. Extraction of core-associated DNA and Southern blot analysis  
         [0117]    Transfection, DNA extraction and Southern blots were performed as described in EXAMPLE 2-2. To complement trans-acting viral proteins, a helper plasmid (pCMV-CPS) that provides core protein and polymerase was cotransfected along with each deletion mutant during transfection. FIG. 11 showed a typical Southern blot result. As described above, three species of the HBV replication-intermediates can be seen in this Southern blot of core-associated viral DNA: SS (single-stranded DNA), DL (double-stranded linear DNA), and RC (relaxed circular DNA). RC form is the mature product of viral genome replication found in virions. Thus, lack of the RC form DNA in Southern blots would indicate that a cis-acting element essential for the viral genome synthesis is deleted in the mutants.  
         [0118]    4-2. Analysis of cis-elements essential for HBV genome replication.  
         [0119]    A series of deletion mutants was generated that encompass the entire HBV genome. Each deletion lacks a fragment of between 0.05-0.52 K bp. Southern analysis indicated that only SS DNA was detected from cells transfected by the R022 mutant (pCMV-ayw Δ2662-3182/0). To delineate the region deleted in mutant R022, two addition mutants were made: R045 (pCMV-ayw Δ2839-3182/0) and R044 (pCMV-ayw Δ3052-3182/0). As a result, RC DNA as well as SS DNA and DL DNA were detected in cells transfected by R044 mutants. Thus, a sequence deleted in the R044 mutant is not essential for viral genome synthesis. On the other hand, only trace amount of SS DNA was detected in Southern blots of cells transfected by mutant R045. Thus, a sequence deleted in R045 is essential for the viral genome replication. Taken together, a novel cis-element essential for HBV genome replication, termed ct element (nt 2662-3052), was identified.  
         [0120]    In addition, mutant R028 (pCMV-ayw Δ1419-1804) lacking the DR2 element (nt. 1592-1602) was made. Southern blot analysis of cells transfected by R028 (pCMV-ayw Δ1419-1804) showed the detection of only SS DNA, but not RC DNA. This result is consistent with published reports on the role of DR2 on minus-strand DNA synthesis in DHBV (Loeb et al., 1996; Condreay et al., 1992). To delineate this region further, mutant R053 (pCMV-ayw Δ1419-1592) lacking sequence upstream of DR2 element was made. Southern blot analysis indicated detection of RC DNA from cells transfected by mutant R053. This result indicated that the sequence lacking in the R053 mutant is dispensable for HBV genome replication. To further delineate this region, R035 mutant (pCMV-ayw Δ1607-1804) lacking a sequence between DR2 and DR1 elements was made. Even SS DNA was not detected from cells transfected by the R035 mutant. Thus, the sequence between DR2 and DR1 element, termed β element, is essential for the minus-strand DNA synthesis. In addition, R029 mutant (pCMV-ayw Δ1804-1884) lacking DR1 element was made. Consistent with published data, no SS DNA was detected (Condreay et al., 1992).  
         [0121]    In contrast, sequences deleted in some of the deletion mutants turned out to be dispensable. These mutants included R060 mutant (pCMV-ayw Δ1910-1992), R048 mutant (pCMV-ayw Δ1992-2143), R056 mutant (pCMV-ayw Δ2143-2459), R021 mutant (pCMV-ayw Δ2459-2817), R044 mutant (pCMV-ayw Δ3052-3182), R023 mutant (pCMV-ayw Δ3182-129), R040 mutant (pCMV-ayw Δ120-490), R041 (pCMV-ayw Δ490-827), R025 mutant (pCMV-ayw Δ827-1238), R026 mutant (pCMV-ayw Δ1238-1374), R027 mutant (pCMV-ayw Δ1374-1419), and R053 (pCMV-ayw Δ1419-1592).  
         [0122]    In summary, the present invention reveals two novel cis-acting elements that are essential for the HBV genome replication. The complete mapping of cis-acting elements that are essential for HBV genome replication allowed us to design a prototype HBV vector.  
       EXAMPLE 5  
       [0123]    Design of prototype HBV gene therapy vectors  
         [0124]    The present invention reveals two novel cis-acting elements that are essential for the HBV genome replication. In literature, a half dozen elements have been reported to be essential in various stages of HBV genome synthesis. Among these are: 5′-epsilon for encapsidation (Hirsh et al., 1991; Junker-Niepmann et al., 1990), DR2 element (Condreay et al., 1992; Loeb et al., 1996), DR1 element (Seeger et al., 1991), r (repeat) element for circularization (Loeb et al., 1997), and PRE element for post-transcriptional RNA processing (Yen, 1998). In addition to these, the two elements identified in the invention, termed α and β, complete the mapping of cis-acting elements essential for HBV viral genome replication (FIG. 6).  
         [0125]    Based on the information on these cis-acting elements, a prototype HBV gene therapy vector was designed (FIG. 8). The critical parameters of this gene therapy vector are the size and position of the inserts. In this prototype vector, two insertion sites were identified. First, the sequence (nt 1909-2816) between 5′ epsilon and the α element could be substituted by a heterologous gene of interest. At the very least, the 0.9 K bp fragment can be substituted in this site. Since this insertion site overlaps the core open reading frame, the core promoter can then be used to drive the heterologous gene inserted. Secondly, the sequence (nt. 3052-1592) between a element and DR2 element can be substituted by a heterologous gene. A fragment up to 1.7 K bp can be substituted in this site. Similarly, this insertion site overlaps the pre-S2/S gene. Thus, the pre-S2/S promoter can be used to derive the heterologous gene inserted.  
       EXAMPLE 6  
       [0126]    Construction of HBV vector containing a heterologous gene  
         [0127]    To test feasibility of the prototype vector described in EXAMPLE 5, a HBV vector was made by the insertion of GFP (green fluorescent protein) gene at the site proposed: R711 (pCMV-HBV/GFP) (FIG. 9).  
         [0128]    6-1. The insertion site and the promoter  
         [0129]    The insertion site was determined by considering following points: (i) all cis-acting elements essential for HBV genome replication should be kept intact, (ii) the endogenous viral promoter needs to be employed to maximize the coding capacity of the vector without exceeding the maximal packaging limit.  
         [0130]    6-2. Insertion of GFP (green fluorescent protein) gene into the HBV vector  
         [0131]    Insertion of the 0.7 K bp GFP fragment was facilitated by polymerase chain reaction (PCR). Restriction sites were created at the end of the PCR fragment by an appropriately designed PCR primer. First of all, R709 (pCMV-HBV/ΔPs2GFP) construct was made by substitution of Bsu36 I(nt. 3052)-to-EcoR I(nt. 3182) fragment of R015 with the 0.7 K bp Bsu36 I-to-EcoR I fragment of PCR product encoding the GFP (green fluorescent protein) gene. The primers used for PCR were:  
         [0132]    GFPBsuFII; 5-GTCACTCCTCAGGCCATGAGTAAAGGAGAAG-3 Bsu36I  
         [0133]    GFPEcoRII; 5-GGAATTCCTTATTTGTATAGTTCATC-3 EcoRI  
         [0134]    In this subcloning process, a subset of the pre-S2/S promoter was deleted. To make up this deletion, the Bsu36 I(nt. 3052)-to-Bsu36 I(nt. 3166) fragment was inserted into R709 plasmid to create R710 (pCMV-preHBV/GFP). Subsequently, to construct R711 (pCMV-HBV/GPF), the EcoR I(nt. 3182)-to-Sph I(nt. 1238) fragment was deleted by restriction digestion. Taken together, the 1.3 K bp fragment (EcoR I-to-Sph I) of HBV genome was substituted by the 0.7 K bp GFP fragment. Thus, the size of the genome is approximately 0.6 K bp smaller than the wild-type.  
       EXAMPLE 7  
       [0135]    Confirmation of replication competency of the recombinant HBV vector  
         [0136]    Feasibility of the recombinant HBV vector was examined by testing replication competency of the HBV vector. Huh7 cells were transfected by R711 plasmid, along with a helper, pCMV-CPS. DNA extraction and Southern blots were performed as described in EXAMPLE 2-2. DNA extracted from HepG2 2.2.15 cells was included as a control (Sells et al., 1988). FIG. 10 a  indicated that RC DNA was detected from cells transfected by R711. Further, the amount of RC DNA and the relative amount of three species of replication-intermediate DNA was comparable to that of R015, the wild-type HBV clone. In addition, the replication of the HBV-GFP vector was further confirmed by using GFP probe (FIG. 10 b ).  
         [0137]    All publications and patent applications cited in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not to be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.  
         [0138]    OTHER PUBLICATIONS  
         [0139]    Anderson, W. F, Science 256:808-813(1992).  
         [0140]    Ausubel, F. et al. eds.,  Current Protocols in Molecular Biology,  Wiley and Sons, New York, 1995.  
         [0141]    Chaisomchit, S., D. L. J. Tyrrell, and L. J. Chang. Gene Therapy 4:1330-1340(1997).  
         [0142]    Chiang. P. W., K. S. Jeng, C. P. Hu, and C. M. Chang. Virology 186:701-711(1992).  
         [0143]    Condreay, L. D., T. T. Wu, C. E. Aldrich, M. A. Delaney, J. Summers, C. Seeger, and W. S. Mason. Virology 188:208-216(1992).  
         [0144]    Crystal, R. G. Science 270:404-410(1995).  
         [0145]    Douglas, J. T., R. C. Miller, M. Kim, I. Dmitriev, G. Mikheeva, V. Krasnykh, and D. T. Curiel. Nature Biotechnology 17: 470-475(1999).  
         [0146]    Friedmann, T. ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1999.  
         [0147]    Galibert, F., E. Mandart., F. Fitoussi, P. Tiollais, and P. Charnay. Nature 28: 646-650(1979).  
         [0148]    Ganem, D., and H. E. Varmus. Annu. Rev. Biochem. 56:651-593(1987).  
         [0149]    Ganem, D., “Hepadnaviridae and Their Replication,” in  Fundamental Virology,  3rd edition, Fields, B. N., et al., eds, Lippincott-Raven Press, Philadelphia, 1996.  
         [0150]    Havert, M., and D. L. Loeb. J. Virol. 71: 5336-5344(1997).  
         [0151]    Hirsch, R. C., D. D. Loeb, J. R. Pollack, and D. Ganem. J. Virol. 65: 3309-3316(1991).  
         [0152]    Ho, T.-C., K.-S. Jeng, C.-P. Hu and C. Chang. J. Virol. 74: 9010-9018(2000).  
         [0153]    Huang, Z., and T. S. B. Yen. Mol. Cell. Biol. 15:3864-3869(1995).  
         [0154]    Jeong, J.-K. , G.-S. Yoon, and W.-S. Ryu. J. Virol. 74:5502-5508(2000)  
         [0155]    Junker-Niepmann, M., R. Bartenschlager, and H. Schaller. EMBO J. 9:3389-3396(1990).  
         [0156]    Loeb, D. D., R. C. Hirsh, and D. Ganem. EMBO J. 10:3533-3540(1991).  
         [0157]    Loeb, D. D., and R. Tian. J. Virol. 69: 686-6891(1995).  
         [0158]    Loeb., D. D., R. Tian, and K. Gulya. J. Virol. 70: 8684-8690(1996).  
         [0159]    Loeb., D. D., K. Gulya. and R. Tian J. Virol. 71: 152-160(1997).  
         [0160]    Mulligan, R. C., Science 260: 926-932(1993).  
         [0161]    Nassal, M., Junker-Niepmann, and H. Schaller. Cell 63: 1357-1363(1990).  
         [0162]    Nassal, M., and A. Rieger. J. Virol. 70:2764-2773(1996).  
         [0163]    Nassal, M., H. Schaller. J. Viral Hepatitis 3: 217-226(1996).  
         [0164]    Pollack, J. R., and D. Ganem. J. Virol. 68:5579-5587(1994).  
         [0165]    Protzer, U., M. Nassal., P.-W. Chiang, M. Kirschfink, and H. Schaller. Proc. Natl. Acad. Sci. USA 96:10818-10823(1999)  
         [0166]    Sambrook et al., in  Molecular Cloning: A Laboratory Manual,  Cold Spring Harbor Laboratory Press, 3rd Ed, Cold Spring Harbor (2001).  
         [0167]    Sells, M. A., A. Z. Zelent, M. Shvartsman, and G. Acs. J. Virol. 62: 2836-2844(1988).  
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         [0170]    Staprans, S., D. D. Loeb., and D. Ganem. J. Virol. 65: 1255-1262(1991).  
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         1 
         
           
             5  
           
           
             1  
             235  
             DNA  
             HBV  
             
               gene  
               (1)..(235)  
               Alpha-element of HBV  
             
           
            1 

gtcaccatat tcttgggaac aagatctaca gcatggggca gaatctttcc accagcaatc     60 

ctctgggatt ctttcccgac caccagttgg atccagcctt cagagcaaac accgcaaatc    120 

cagattggga cttcaatccc aacaaggaca cctggccaga cgccaacaag gtaggagctg    180 

gagcattcgg gctgggtttc accccaccgc acggaggcct tttggggtgg agccc         235 

 
           
             2  
             197  
             DNA  
             HBV  
             
               gene  
               (1)..(197)  
               Beta- element of HBV  
             
           
            2 

gcatggagac caccgtgaac gcccaccaaa tattgcccaa ggtcttacat aagaggactc     60 

ttggactctc agcaatgtca acgaccgacc ttgaggcata cttcaaagac tgtttgttta    120 

aagactggga ggagttgggg gaggagatta ggttaaaggt ctttgtacta ggaggctgta    180 

ggcataaatt ggtctgc                                                   197 

 
           
             3  
             8007  
             DNA  
             HBV  
             
               gene  
               (1)..(8007)  
               Prototype vector of HBV  
             
           
            3 

aactttttca cctctgccta atcatctctt gttcatgtcc tactgttcaa gcctccaagc     60 

tgtgccttgg gtggctttgg ggcatggaca tcgaccctta taaagaattt ggagctactg    120 

tggagttact ctcgtttttg ccttctgact tctttccttc agtacgagat cttctagata    180 

ccgcctcagc tctgtatcgg gaagccttag agtctcctga gcattgttca cctcaccata    240 

ctgcactcag gcaagcaatt ctttgctggg gggaactaat gactctagct acctgggtgg    300 

gtgttaattt ggaagatcca gcgtctagag acctagtagt cagttatgtc aacactaata    360 

tgggcctaaa gttcaggcaa ctcttgtggt ttcacatttc ttgtctcact tttggaagag    420 

aaacagttat agagtatttg gtgtctttcg gagtgtggat tcgcactcct ccagcttata    480 

gaccaccaaa tgcccctatc ctatcaacac ttccggagac tactgttgtt agacgacgag    540 

gcaggtcccc tagaagaaga actccctcgc ctcgcagacg aaggtctcaa tcgccgcgtc    600 

gcagaagatc tcaatctcgg gaatctcaat gttagtattc cttggactca taaggtgggg    660 

aactttactg ggctttattc ttctactgta cctgtcttta atcctcattg gaaaacacca    720 

tcttttccta atatacattt acaccaagac attatcaaaa aatgtgaaca gtttgtaggc    780 

ccactcacag ttaatgagaa aagaagattg caattgatta tgcctgccag gttttatcca    840 

aaggttacca aatatttacc attggataag ggtattaaac cttattatcc agaacatcta    900 

gttaatcatt acttccaaac tagacactat ttacacactc tatggaaggc gggtatatta    960 

tataagagag aaacaacaca tagcgcctca ttttgtgggt caccatattc ttgggaacaa   1020 

gatctacagc atggggcaga atctttccac cagcaatcct ctgggattct ttcccgacca   1080 

ccagttggat ccagccttca gagcaaacac cgcaaatcca gattgggact tcaatcccaa   1140 

caaggacacc tggccagacg ccaacaaggt aggagctgga gcattcgggc tgggtttcac   1200 

cccaccgcac ggaggccttt tggggtggag ccctcaggct cagggcatac tacaaacttt   1260 

gccagcaaat ccgcctcctg cctccaccaa tcgccagtca ggaaggcagc ctaccccgct   1320 

gtctccacct ttgagaaaca ctcatcctca ggccatgcag tggaattcca caaccttcca   1380 

ccaaactctg caagatccca gagtgagagg cctgtatttc cctgctggtg gctccagttc   1440 

aggaacagta aaccctgttc tgactactgc ctctccctta tcgtcaatct tctcgaggat   1500 

tggggaccct gcgctgaaca tggagaacat cacatcagga ttcctaggac cccttctcgt   1560 

gttacaggcg gggtttttct tgttgacaag aatcctcaca ataccgcaga gtctagactc   1620 

gtggtggact tctctcaatt ttctaggggg aactaccgtg tgtcttggcc aaaattcgca   1680 

gtccccaacc tccaatcact caccaacctc ttgtcctcca acttgtcctg gttatcgctg   1740 

gatgtgtctg cggcgtttta tcatcttcct cttcatcctg ctgctatgcc tcatcttctt   1800 

gttggttctt ctggactatc aaggtatgtt gcccgtttgt cctctaattc caggatcctc   1860 

aacaaccagc acgggaccat gccggacctg catgactact gctcaaggaa cctctatgta   1920 

tccctcctgt tgctgtacca aaccttcgga cggaaattgc acctgtattc ccatcccatc   1980 

atcctgggct ttcggaaaat tcctatggga gtgggcctca gcccgtttct cctggctcag   2040 

tttactagtg ccatttgttc agtggttcgt agggctttcc cccactgttt ggctttcagt   2100 

tatatggatg atgtggtatt gggggccaag tctgtacagc atcttgagtc cctttttacc   2160 

gctgttacca attttctttt gtctttgggt atacatttaa accctaacaa aacaaagaga   2220 

tggggttact ctctaaattt tatgggttat gtcattggat gttatgggtc cttgccacaa   2280 

gaacacatca tacaaaaaat caaagaatgt tttagaaaac ttcctattaa caggcctatt   2340 

gattggaaag tatgtcaacg aattgtgggt cttttgggtt ttgctgcccc ttttacacaa   2400 

tgtggttatc ctgcgttgat gcctttgtat gcatgtattc aatctaagca ggctttcact   2460 

ttctcgccaa cttacaaggc ctttctgtgt aaacaatacc tgaaccttta ccccgttgcc   2520 

cggcaacggc caggtctgtg ccaagtgttt gctgacgcaa cccccactgg ctggggcttg   2580 

gtcatgggcc atcagcgcat gcgtggaacc ttttcggctc ctctgccgat ccatactgcg   2640 

gaactcctag ccgcttgttt tgctcgcagc aggtctggag caaacattat cgggactgat   2700 

aactctgttg tcctatcccg caaatataca tcgtttccat ggctgctagg ctgtgctgcc   2760 

aactggatcc tgcgcgggac gtcctttgtt tacgtcccgt cggcgctgaa tcctgcggac   2820 

gacccttctc ggggtcgctt gggactctct cgtccccttc tccgtctgcc gttccgaccg   2880 

accacggggc gcacctctct ttacgcggac tccccgtctg tgccttctca tctgccggac   2940 

cgtgtgcact tcgcttcacc tctgcacgtc gcatggagac caccgtgaac gcccaccaaa   3000 

tattgcccaa ggtcttacat aagaggactc ttggactctc agcaatgtca acgaccgacc   3060 

ttgaggcata cttcaaagac tgtttgttta aagactggga ggagttgggg gaggagatta   3120 

ggttaaaggt ctttgtacta ggaggctgta ggcataaatt ggtctgcgca ccagcaccat   3180 

gcaacttttt cacctctgcc taatcatctc ttgttcatgt cctactgttc aagcctccaa   3240 

gctgtgcctt gggtggcttt ggggcatgga catcgaccct tataaagaat ttggagctac   3300 

tgtggagtta ctctcgtttt tgccttctga cttctttcct tcagtacgag atcttctaga   3360 

gggccctatt ctatagtgtc acctaaatgc tagaggatct ttgtgaagga accttacttc   3420 

tgtggtgtga cataattgga caaactacct acagagattt aaagctctaa ggtaaatata   3480 

aaatttttaa gtgtataatg tgttaaacta ctgattctaa ttgtttgtgt attttagatt   3540 

ccaacctatg gaactgatga atgggagcag tggtggaatg cctttaatga ggaaaacctg   3600 

ttttgctcag aagaaatgcc atctagtgat gatgaggcta ctgctgactc tcaacattct   3660 

actcctccaa aaaagaagag aaaggtagaa gaccccaagg actttccttc agaattgcta   3720 

agttttttga gtcatgctgt gtttagtaat agaactcttg cttgctttgc tatttacacc   3780 

acaaaggaaa aagctgcact gctatacaag aaaattatgg aaaaatattt gatgtatagt   3840 

gccttgacta gagatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa   3900 

aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttaa   3960 

cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa   4020 

taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta   4080 

tcatgtctgg atcatcccgc catggtatca acgccatatt tctatttaca gtagggacct   4140 

cttcgttgtg taggtaccgc tgtattccta gggaaatagt agaggcacct tgaactgtct   4200 

gcatcagcca tatagccccc gctgttcgac ttacaaacac aggcacagta ctgacaaacc   4260 

catacacctc ctctgaaata cccatagttg ctagggctgt ctccgaactc attacaccct   4320 

ccaaagtcag agctgtaatt tcgccatcaa gggcagcgag ggcttctcca gataaaatag   4380 

cttctgccga gagtcccgta agggtagaca cttcagctaa tccctcgatg aggtctacta   4440 

gaatagtcag tgcggctccc attttgaaaa ttcacttact tgatcagctt cagaagatgg   4500 

cggagggcct ccaacacagt aattttcctc ccgactctta aaatagaaaa tgtcaagtca   4560 

gttaagcagg aagtggacta actgacgcag ctggccgtgc gacatcctct tttaattagt   4620 

tgctaggcaa cgccctccag agggcgtgtg gttttgcaag aggaagcaaa agcctctcca   4680 

cccaggccta gaatgtttcc acccaatcat tactatgaca acagctgttt tttttagtat   4740 

taagcagagg ccggggaccc ctgggccggc ccgcttactc tggagaaaaa gaagagaggc   4800 

attgtagagg cttccagagg caacttgtca aaacaggact gcttctattt ctgtcacact   4860 

gtctggccct gtcacaaggt ccagcacctc cataccccct ttaataagca gtttgggaac   4920 

gggtgcgggt cttactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc   4980 

gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga   5040 

gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctaat   5100 

tcggcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc   5160 

cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac   5220 

caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac   5280 

cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt   5340 

cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct   5400 

gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat   5460 

acctacagcg tgagcattga gaaagcgcca cgcttcccga agggagaaag gcggacaggt   5520 

atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg   5580 

cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt   5640 

gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcaagc tagcttctag   5700 

ctagaaattg taaacgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc   5760 

tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagccc   5820 

gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac   5880 

tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gccgcccact acgtgaacca   5940 

tcacccaaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa   6000 

gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg   6060 

aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta   6120 

accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtactatgg ttgctttgac   6180 

gagaccgtat aacgtgcttt cctcgttgga atcagagcgg gagctaaaca ggaggccgat   6240 

taaagggatt ttagacagga acggtacgcc agctggatta ccaaagggcc tcgtgatacg   6300 

cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt   6360 

tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta   6420 

tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat   6480 

gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt   6540 

ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg   6600 

agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga   6660 

agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg   6720 

tgttgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt   6780 

tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg   6840 

cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg   6900 

aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga   6960 

tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc   7020 

tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc   7080 

ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc   7140 

ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg   7200 

cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac   7260 

gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc   7320 

actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt   7380 

aaaacttcat ttttaatttc tctagcgcgt tgacattgat tattgactag ttattaatag   7440 

taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt   7500 

acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg   7560 

acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggactat   7620 

ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct   7680 

attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg   7740 

gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg   7800 

ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc   7860 

caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa   7920 

tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc   7980 

tatataagca gagctctctg gctaact                                       8007 

 
           
             4  
             8717  
             DNA  
             Artificial Sequence  
             
               R711 pCMV-HBV/GFP Full Sequence  
             
           
            4 

aactttttca cctctgccta atcatctctt gttcatgtcc tactgttcaa gcctccaagc     60 

tgtgccttgg gtggctttgg ggcatggaca tcgaccctta taaagaattt ggagctactg    120 

tggagttact ctcgtttttg ccttctgact tctttccttc agtacgagat cttctagata    180 

ccgcctcagc tctgtatcgg gaagccttag agtctcctga gcattgttca cctcaccata    240 

ctgcactcag gcaagcaatt ctttgctggg gggaactaat gactctagct acctgggtgg    300 

gtgttaattt ggaagatcca gcgtctagag acctagtagt cagttatgtc aacactaata    360 

tgggcctaaa gttcaggcaa ctcttgtggt ttcacatttc ttgtctcact tttggaagag    420 

aaacagttat agagtatttg gtgtctttcg gagtgtggat tcgcactcct ccagcttata    480 

gaccaccaaa tgcccctatc ctatcaacac ttccggagac tactgttgtt agacgacgag    540 

gcaggtcccc tagaagaaga actccctcgc ctcgcagacg aaggtctcaa tcgccgcgtc    600 

gcagaagatc tcaatctcgg gaatctcaat gttagtattc cttggactca taaggtgggg    660 

aactttactg ggctttattc ttctactgta cctgtcttta atcctcattg gaaaacacca    720 

tcttttccta atatacattt acaccaagac attatcaaaa aatgtgaaca gtttgtaggc    780 

ccactcacag ttaatgagaa aagaagattg caattgatta tgcctgccag gttttatcca    840 

aaggttacca aatatttacc attggataag ggtattaaac cttattatcc agaacatcta    900 

gttaatcatt acttccaaac tagacactat ttacacactc tatggaaggc gggtatatta    960 

tataagagag aaacaacaca tagcgcctca ttttgtgggt caccatattc ttgggaacaa   1020 

gatctacagc atggggcaga atctttccac cagcaatcct ctgggattct ttcccgacca   1080 

ccagttggat ccagccttca gagcaaacac cgcaaatcca gattgggact tcaatcccaa   1140 

caaggacacc tggccagacg ccaacaaggt aggagctgga gcattcgggc tgggtttcac   1200 

cccaccgcac ggaggccttt tggggtggag ccctcaggct cagggcatac tacaaacttt   1260 

gccagcaaat ccgcctcctg cctccaccaa tcgccagtca ggaaggcagc ctaccccgct   1320 

gtctccacct ttgagaaaca ctcatcctca ggagatgagt aaaggagaag aacttttcac   1380 

tggagttgtc ccaattcttg ttgaattaga tggtgatgtt aatgggcaca aattttctgt   1440 

cagtggagag ggtgaaggtg atgcaacata cggaaaactt acccttaaat ttatttgcac   1500 

tactggaaaa ctacctgttc catggccaac acttgtcact actttctctt atggtgttca   1560 

atgcttttca agatacccag atcatatgaa acagcatgac tttttcaaga gtgccatgcc   1620 

cgaaggttat gtacaggaaa gaactatatt tttcaaagat gacgggaact acaagacacg   1680 

tgctgaagtc aagtttgaag gtgataccct tgttaataga atcgagttaa aaggtattga   1740 

ttttaaagaa gatggaaaca ttcttggaca caaattggaa tacaactata actcacacaa   1800 

tgtatacatc atggcagaca aacaaaagaa tggaatcaaa gttaacttca aaattagaca   1860 

caacattgaa gatggaagcg ttcaactagc agaccattat caacaaaata ctccaattgg   1920 

cgatggccct gtccttttac cagacaacca ttacctgtcc acacaatctg ccctttcgaa   1980 

agatcccaac gaaaagagag accacatggt ccttcttgag tttgtaacag ctgctgggat   2040 

tacacatggc atggatgaac tatacaaata aggaattcca caaccttcca ccaaactctg   2100 

caagatccca gagtgagagg cctgtatttc cctgctggtg gctccagttc aggaacagta   2160 

aaccctgttc tgactactgc ctctccctta tcgtcaatct tctcgaggat tggggaccct   2220 

gcgctgaaca tggagaacat cacatcagga ttcctaggac cccttctcgt gttacaggcg   2280 

gggtttttct tgttgacaag aatcctcaca ataccgcaga gtctagactc gtggtggact   2340 

tctctcaatt ttctaggggg aactaccgtg tgtcttggcc aaaattcgca gtccccaacc   2400 

tccaatcact caccaacctc ttgtcctcca acttgtcctg gttatcgctg gatgtgtctg   2460 

cggcgtttta tcatcttcct cttcatcctg ctgctatgcc tcatcttctt gttggttctt   2520 

ctggactatc aaggtatgtt gcccgtttgt cctctaattc caggatcctc aacaaccagc   2580 

acgggaccat gccggacctg catgactact gctcaaggaa cctctatgta tccctcctgt   2640 

tgctgtacca aaccttcgga cggaaattgc acctgtattc ccatcccatc atcctgggct   2700 

ttcggaaaat tcctatggga gtgggcctca gcccgtttct cctggctcag tttactagtg   2760 

ccatttgttc agtggttcgt agggctttcc cccactgttt ggctttcagt tatatggatg   2820 

atgtggtatt gggggccaag tctgtacagc atcttgagtc cctttttacc gctgttacca   2880 

attttctttt gtctttgggt atacatttaa accctaacaa aacaaagaga tggggttact   2940 

ctctaaattt tatgggttat gtcattggat gttatgggtc cttgccacaa gaacacatca   3000 

tacaaaaaat caaagaatgt tttagaaaac ttcctattaa caggcctatt gattggaaag   3060 

tatgtcaacg aattgtgggt cttttgggtt ttgctgcccc ttttacacaa tgtggttatc   3120 

ctgcgttgat gcctttgtat gcatgtattc aatctaagca ggctttcact ttctcgccaa   3180 

cttacaaggc ctttctgtgt aaacaatacc tgaaccttta ccccgttgcc cggcaacggc   3240 

caggtctgtg ccaagtgttt gctgacgcaa cccccactgg ctggggcttg gtcatgggcc   3300 

atcagcgcat gcgtggaacc ttttcggctc ctctgccgat ccatactgcg gaactcctag   3360 

ccgcttgttt tgctcgcagc aggtctggag caaacattat cgggactgat aactctgttg   3420 

tcctatcccg caaatataca tcgtttccat ggctgctagg ctgtgctgcc aactggatcc   3480 

tgcgcgggac gtcctttgtt tacgtcccgt cggcgctgaa tcctgcggac gacccttctc   3540 

ggggtcgctt gggactctct cgtccccttc tccgtctgcc gttccgaccg accacggggc   3600 

gcacctctct ttacgcggac tccccgtctg tgccttctca tctgccggac cgtgtgcact   3660 

tcgcttcacc tctgcacgtc gcatggagac caccgtgaac gcccaccaaa tattgcccaa   3720 

ggtcttacat aagaggactc ttggactctc agcaatgtca acgaccgacc ttgaggcata   3780 

cttcaaagac tgtttgttta aagactggga ggagttgggg gaggagatta ggttaaaggt   3840 

ctttgtacta ggaggctgta ggcataaatt ggtctgcgca ccagcaccat gcaacttttt   3900 

cacctctgcc taatcatctc ttgttcatgt cctactgttc aagcctccaa gctgtgcctt   3960 

gggtggcttt ggggcatgga catcgaccct tataaagaat ttggagctac tgtggagtta   4020 

ctctcgtttt tgccttctga cttctttcct tcagtacgag atcttctaga gggccctatt   4080 

ctatagtgtc acctaaatgc tagaggatct ttgtgaagga accttacttc tgtggtgtga   4140 

cataattgga caaactacct acagagattt aaagctctaa ggtaaatata aaatttttaa   4200 

gtgtataatg tgttaaacta ctgattctaa ttgtttgtgt attttagatt ccaacctatg   4260 

gaactgatga atgggagcag tggtggaatg cctttaatga ggaaaacctg ttttgctcag   4320 

aagaaatgcc atctagtgat gatgaggcta ctgctgactc tcaacattct actcctccaa   4380 

aaaagaagag aaaggtagaa gaccccaagg actttccttc agaattgcta agttttttga   4440 

gtcatgctgt gtttagtaat agaactcttg cttgctttgc tatttacacc acaaaggaaa   4500 

aagctgcact gctatacaag aaaattatgg aaaaatattt gatgtatagt gccttgacta   4560 

gagatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca   4620 

cacctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt   4680 

gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt   4740 

ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg   4800 

atcatcccgc catggtatca acgccatatt tctatttaca gtagggacct cttcgttgtg   4860 

taggtaccgc tgtattccta gggaaatagt agaggcacct tgaactgtct gcatcagcca   4920 

tatagccccc gctgttcgac ttacaaacac aggcacagta ctgacaaacc catacacctc   4980 

ctctgaaata cccatagttg ctagggctgt ctccgaactc attacaccct ccaaagtcag   5040 

agctgtaatt tcgccatcaa gggcagcgag ggcttctcca gataaaatag cttctgccga   5100 

gagtcccgta agggtagaca cttcagctaa tccctcgatg aggtctacta gaatagtcag   5160 

tgcggctccc attttgaaaa ttcacttact tgatcagctt cagaagatgg cggagggcct   5220 

ccaacacagt aattttcctc ccgactctta aaatagaaaa tgtcaagtca gttaagcagg   5280 

aagtggacta actgacgcag ctggccgtgc gacatcctct tttaattagt tgctaggcaa   5340 

cgccctccag agggcgtgtg gttttgcaag aggaagcaaa agcctctcca cccaggccta   5400 

gaatgtttcc acccaatcat tactatgaca acagctgttt tttttagtat taagcagagg   5460 

ccggggaccc ctgggccggc ccgcttactc tggagaaaaa gaagagaggc attgtagagg   5520 

cttccagagg caacttgtca aaacaggact gcttctattt ctgtcacact gtctggccct   5580 

gtcacaaggt ccagcacctc cataccccct ttaataagca gtttgggaac gggtgcgggt   5640 

cttactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc   5700 

tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag   5760 

aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctaat tcggcgtaat   5820 

ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga   5880 

gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt   5940 

ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata   6000 

cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac   6060 

cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg   6120 

ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg   6180 

tgagcattga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag   6240 

cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct   6300 

ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc   6360 

aggggggcgg agcctatgga aaaacgccag caacgcaagc tagcttctag ctagaaattg   6420 

taaacgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta   6480 

accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagccc gagatagggt   6540 

tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca   6600 

aagggcgaaa aaccgtctat cagggcgatg gccgcccact acgtgaacca tcacccaaat   6660 

caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc   6720 

gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga   6780 

aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac   6840 

ccgccgcgct taatgcgccg ctacagggcg cgtactatgg ttgctttgac gagaccgtat   6900 

aacgtgcttt cctcgttgga atcagagcgg gagctaaaca ggaggccgat taaagggatt   6960 

ttagacagga acggtacgcc agctggatta ccaaagggcc tcgtgatacg cctattttta   7020 

taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat   7080 

gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg   7140 

agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa   7200 

catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac   7260 

ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac   7320 

atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt   7380 

ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tgttgacgcc   7440 

gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca   7500 

ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc   7560 

ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag   7620 

gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa   7680 

ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg   7740 

gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa   7800 

ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg   7860 

gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt   7920 

gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt   7980 

caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag   8040 

cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat   8100 

ttttaatttc tctagcgcgt tgacattgat tattgactag ttattaatag taatcaatta   8160 

cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg   8220 

gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc   8280 

ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggactat ttacggtaaa   8340 

ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca   8400 

atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta   8460 

cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt   8520 

acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg   8580 

acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca   8640 

actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca   8700 

gagctctctg gctaact                                                  8717 

 
           
             5  
             7991  
             DNA  
             Artificial Sequence  
             
               R712 pCMV-HBV/GFP3.2 Full Sequence  
             
           
            5 

aactttttca cctctgccta atcatctctt gttcatgtcc tactgttcaa gcctccaagc     60 

tgtgccttgg gtggctttgg ggcatggaca tcgaccctta taaagaattt ggagctactg    120 

tggagttact ctcgtttttg ccttctgact tctttccttc agtacgagat cttctagata    180 

ccgcctcagc tctgtatcgg gaagccttag agtctcctga gcattgttca cctcaccata    240 

ctgcactcag gcaagcaatt ctttgctggg gggaactaat gactctagct acctgggtgg    300 

gtgttaattt ggaagatcca gcgtctagag acctagtagt cagttatgtc aacactaata    360 

tgggcctaaa gttcaggcaa ctcttgtggt ttcacatttc ttgtctcact tttggaagag    420 

aaacagttat agagtatttg gtgtctttcg gagtgtggat tcgcactcct ccagcttata    480 

gaccaccaaa tgcccctatc ctatcaacac ttccggagac tactgttgtt agacgacgag    540 

gcaggtcccc tagaagaaga actccctcgc ctcgcagacg aaggtctcaa tcgccgcgtc    600 

gcagaagatc tcaatctcgg gaatctcaat gttagtattc cttggactca taaggtgggg    660 

aactttactg ggctttattc ttctactgta cctgtcttta atcctcattg gaaaacacca    720 

tcttttccta atatacattt acaccaagac attatcaaaa aatgtgaaca gtttgtaggc    780 

ccactcacag ttaatgagaa aagaagattg caattgatta tgcctgccag gttttatcca    840 

aaggttacca aatatttacc attggataag ggtattaaac cttattatcc agaacatcta    900 

gttaatcatt acttccaaac tagacactat ttacacactc tatggaaggc gggtatatta    960 

tataagagag aaacaacaca tagcgcctca ttttgtgggt caccatattc ttgggaacaa   1020 

gatctacagc atggggcaga atctttccac cagcaatcct ctgggattct ttcccgacca   1080 

ccagttggat ccagccttca gagcaaacac cgcaaatcca gattgggact tcaatcccaa   1140 

caaggacacc tggccagacg ccaacaaggt aggagctgga gcattcgggc tgggtttcac   1200 

cccaccgcac ggaggccttt tggggtggag ccctcaggct cagggcatac tacaaacttt   1260 

gccagcaaat ccgcctcctg cctccaccaa tcgccagtca ggaaggcagc ctaccccgct   1320 

gtctccacct ttgagaaaca ctcatcctca ggagatgagt aaaggagaag aacttttcac   1380 

tggagttgtc ccaattcttg ttgaattaga tggtgatgtt aatgggcaca aattttctgt   1440 

cagtggagag ggtgaaggtg atgcaacata cggaaaactt acccttaaat ttatttgcac   1500 

tactggaaaa ctacctgttc catggccaac acttgtcact actttctctt atggtgttca   1560 

atgcttttca agatacccag atcatatgaa acagcatgac tttttcaaga gtgccatgcc   1620 

cgaaggttat gtacaggaaa gaactatatt tttcaaagat gacgggaact acaagacacg   1680 

tgctgaagtc aagtttgaag gtgataccct tgttaataga atcgagttaa aaggtattga   1740 

ttttaaagaa gatggaaaca ttcttggaca caaattggaa tacaactata actcacacaa   1800 

tgtatacatc atggcagaca aacaaaagaa tggaatcaaa gttaacttca aaattagaca   1860 

caacattgaa gatggaagcg ttcaactagc agaccattat caacaaaata ctccaattgg   1920 

cgatggccct gtccttttac cagacaacca ttacctgtcc acacaatctg ccctttcgaa   1980 

agatcccaac gaaaagagag accacatggt ccttcttgag tttgtaacag ctgctgggat   2040 

tacacatggc atggatgaac tatacaaata aggaattctt cagttatatg gatgatgtgg   2100 

tattgggggc caagtctgta cagcatcttg agtccctttt taccgctgtt accaattttc   2160 

ttttgtcttt gggtatacat ttaaacccta acaaaacaaa gagatggggt tactctctaa   2220 

attttatggg ttatgtcatt ggatgttatg ggtccttgcc acaagaacac atcatacaaa   2280 

aaatcaaaga atgttttaga aaacttccta ttaacaggcc tattgattgg aaagtatgtc   2340 

aacgaattgt gggtcttttg ggttttgctg ccccttttac acaatgtggt tatcctgcgt   2400 

tgatgccttt gtatgcatgt attcaatcta agcaggcttt cactttctcg ccaacttaca   2460 

aggcctttct gtgtaaacaa tacctgaacc tttaccccgt tgcccggcaa cggccaggtc   2520 

tgtgccaagt gtttgctgac gcaaccccca ctggctgggg cttggtcatg ggccatcagc   2580 

gcatgcgtgg aaccttttcg gctcctctgc cgatccatac tgcggaactc ctagccgctt   2640 

gttttgctcg cagcaggtct ggagcaaaca ttatcgggac tgataactct gttgtcctat   2700 

cccgcaaata tacatcgttt ccatggctgc taggctgtgc tgccaactgg atcctgcgcg   2760 

ggacgtcctt tgtttacgtc ccgtcggcgc tgaatcctgc ggacgaccct tctcggggtc   2820 

gcttgggact ctctcgtccc cttctccgtc tgccgttccg accgaccacg gggcgcacct   2880 

ctctttacgc ggactccccg tctgtgcctt ctcatctgcc ggaccgtgtg cacttcgctt   2940 

cacctctgca cgtcgcatgg agaccaccgt gaacgcccac caaatattgc ccaaggtctt   3000 

acataagagg actcttggac tctcagcaat gtcaacgacc gaccttgagg catacttcaa   3060 

agactgtttg tttaaagact gggaggagtt gggggaggag attaggttaa aggtctttgt   3120 

actaggaggc tgtaggcata aattggtctg cgcaccagca ccatgcaact ttttcacctc   3180 

tgcctaatca tctcttgttc atgtcctact gttcaagcct ccaagctgtg ccttgggtgg   3240 

ctttggggca tggacatcga cccttataaa gaatttggag ctactgtgga gttactctcg   3300 

tttttgcctt ctgacttctt tccttcagta cgagatcttc tagagggccc tattctatag   3360 

tgtcacctaa atgctagagg atctttgtga aggaacctta cttctgtggt gtgacataat   3420 

tggacaaact acctacagag atttaaagct ctaaggtaaa tataaaattt ttaagtgtat   3480 

aatgtgttaa actactgatt ctaattgttt gtgtatttta gattccaacc tatggaactg   3540 

atgaatggga gcagtggtgg aatgccttta atgaggaaaa cctgttttgc tcagaagaaa   3600 

tgccatctag tgatgatgag gctactgctg actctcaaca ttctactcct ccaaaaaaga   3660 

agagaaaggt agaagacccc aaggactttc cttcagaatt gctaagtttt ttgagtcatg   3720 

ctgtgtttag taatagaact cttgcttgct ttgctattta caccacaaag gaaaaagctg   3780 

cactgctata caagaaaatt atggaaaaat atttgatgta tagtgccttg actagagatc   3840 

ataatcagcc ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc   3900 

cccctgaacc tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct   3960 

tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca   4020 

ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggatcatc   4080 

ccgccatggt atcaacgcca tatttctatt tacagtaggg acctcttcgt tgtgtaggta   4140 

ccgctgtatt cctagggaaa tagtagaggc accttgaact gtctgcatca gccatatagc   4200 

ccccgctgtt cgacttacaa acacaggcac agtactgaca aacccataca cctcctctga   4260 

aatacccata gttgctaggg ctgtctccga actcattaca ccctccaaag tcagagctgt   4320 

aatttcgcca tcaagggcag cgagggcttc tccagataaa atagcttctg ccgagagtcc   4380 

cgtaagggta gacacttcag ctaatccctc gatgaggtct actagaatag tcagtgcggc   4440 

tcccattttg aaaattcact tacttgatca gcttcagaag atggcggagg gcctccaaca   4500 

cagtaatttt cctcccgact cttaaaatag aaaatgtcaa gtcagttaag caggaagtgg   4560 

actaactgac gcagctggcc gtgcgacatc ctcttttaat tagttgctag gcaacgccct   4620 

ccagagggcg tgtggttttg caagaggaag caaaagcctc tccacccagg cctagaatgt   4680 

ttccacccaa tcattactat gacaacagct gtttttttta gtattaagca gaggccgggg   4740 

acccctgggc cggcccgctt actctggaga aaaagaagag aggcattgta gaggcttcca   4800 

gaggcaactt gtcaaaacag gactgcttct atttctgtca cactgtctgg ccctgtcaca   4860 

aggtccagca cctccatacc ccctttaata agcagtttgg gaacgggtgc gggtcttact   4920 

ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta   4980 

atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag   5040 

tgaggaggct tttttggagg cctaggcttt tgcaaaaagc taattcggcg taatctgctg   5100 

cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc   5160 

aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct   5220 

agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc   5280 

tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt   5340 

ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg   5400 

cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagca   5460 

ttgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag   5520 

ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag   5580 

tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg   5640 

gcggagccta tggaaaaacg ccagcaacgc aagctagctt ctagctagaa attgtaaacg   5700 

ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat   5760 

aggccgaaat cggcaaaatc ccttataaat caaaagaata gcccgagata gggttgagtg   5820 

ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc   5880 

gaaaaaccgt ctatcagggc gatggccgcc cactacgtga accatcaccc aaatcaagtt   5940 

ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta   6000 

gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag   6060 

cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg   6120 

cgcttaatgc gccgctacag ggcgcgtact atggttgctt tgacgagacc gtataacgtg   6180 

ctttcctcgt tggaatcaga gcgggagcta aacaggaggc cgattaaagg gattttagac   6240 

aggaacggta cgccagctgg attaccaaag ggcctcgtga tacgcctatt tttataggtt   6300 

aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc   6360 

ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa   6420 

taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc   6480 

cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa   6540 

acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa   6600 

ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg   6660 

atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtgttga cgccgggcaa   6720 

gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc   6780 

acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc   6840 

atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta   6900 

accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag   6960 

ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgcagc aatggcaaca   7020 

acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata   7080 

gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc   7140 

tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca   7200 

ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca   7260 

actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg   7320 

taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa   7380 

tttctctagc gcgttgacat tgattattga ctagttatta atagtaatca attacggggt   7440 

cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc   7500 

ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag   7560 

taacgccaat agggactttc cattgacgtc aatgggtgga ctatttacgg taaactgccc   7620 

acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg   7680 

gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc   7740 

agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca   7800 

atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca   7860 

atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg   7920 

ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctc   7980 

tctggctaac t