Vaccines against aids comprising cmv/r-nucleic acid constructs

Abstract

ovides compositions for eliciting an immune response, including a prophylactic immune response, against human immunodeficiency virus. The composition includes nucleic acid constructs encoding HIV antigenic polypeptides of multiple clades or strains. Methods for eliciting an immune response by administering the composition to a subject are also provided.

Description

CROSS REFERENCE TO RELATED APPLICATION

[0001]This application claims priority to and benefit of U.S. Provisional Patent Application No. 60/588,378, filed Jul. 16, 2004, and to copending PCT applications PCT/US2004/030284, filed Sep. 15, 2004 and PCT/US2005/12291, filed Apr. 12, 2005. The disclosures of each of these applications is incorporated herein in its entirety.

FIELD

[0003]This application relates to the field of vaccines. More specifically, this application relates to a multi-plasmid vaccine for the prevention of human immunodeficiency virus (HIV).

BACKGROUND

[0004]More than 40 million people are infected worldwide with HIV-1 and an estimated 14,000 new infections occur every day. Over 25 million people have died of HIV/AIDS since the first cases of AIDS were identified in 1981 (CDC, MMWR Morb. Mortal Wkly. Rep., 52:1145-1148, 2003; UNAIDS, 2003 Report on the Global AIDS Epidemic Executive Summary, 2004). Development of a globally relevant HIV-1 vaccine is critical for controlling the HIV/AIDS pandemic.

[0005]The combination of a high transcriptional error rate and frequent recombination results in a remarkable amount of genetic diversity among HIV-1 strains and presents a challenge for selecting viral antigens. The other potential impact of HIV genetic variation is the high rate of mutation within each individual, which creates the opportunity for viral escape from epitope-specific immune responses and poses particular challenges for T cell based vaccine approaches (Altfield et al., J. Virol., 77:12764-12772, 2002; Bhardwaj et al., Nat. Med., 9:13-14, 2003; Brander et al., Curr. Opin. Immunol., 11:451-459, 1999; Letvin et al., Nat. Med., 9:861-866, 2003). A variety of vaccine strategies to elicit effective immunity to HIV-1 have been explored. Among them, immunization by plasmid DNA encoding genes for HIV protein antigens is a promising vaccine approach (Mascola et al., Curr. Opin. Immunol., 13:489-494, 2001; Nabel, G. J., Nature, 410:1002-1007, 2001). Gene-based immunization promotes host cell synthesis and expression of the viral antigen and physiologic post-translational processing and folding in the cell cytoplasm. Therefore, DNA immunization elicits both CD4⁺ and CD8⁺ T lymphocyte responses with a variety of immunogens in animal models (Graham, B. S., Annu. Rev. Med., 53:207-221, 2002; Rollman et al., Gene Ther., 11:1146-1154, 2004; Barouch et al., Science, 290:486-492, 2000; Subbramanian et al., J. Virol., 77:10113-10118, 2003; Mascola et al., J. Virol., 79:771-779, 2005).

[0006]Delivering viral antigens by DNA plasmid vaccine vectors has potential advantages over other vector delivery systems, notably the lack of anti-vector immunity. However, DNA immunization has shown only limited immunogenicity in humans, despite many examples of vaccine-induced protection in mice and nonhuman primates (Rollman et al., Gene Ther., 11:1146-1154, 2004; Donnelly et al., Nat. Med., 1:583-587, 1995). The first DNA vaccine demonstrated to be immunogenic in antigen-naive humans was a construct expressing the circumsporozoite antigen from Plasmodium falciparum delivered by Biojector®. In this study, CD8⁺ CTL responses were detected only after in vitro expansion of effectors (Wang et al., Science, 282:476-480, 1998). Another report described a DNA plasmid expressing the Hepatitis B surface antigen delivered by a different needleless injection device, Powderject®, induced antibody as well as vaccine-specific T cell responses in antigen-naive humans (Roy et al., Vaccine, 19:764-778, 2000). A DNA plasmid vaccine expressing the HIV-1 Env and Rev proteins tested in both HIV-infected and HIV-uninfected subjects (MacGregor et al., J. Infect. Dis., 178:92-100, 1998) was not associated with adverse events, but only sporadic lymphoproliferative and antibody responses were observed (MacGregor et al., J. Infect. Dis., 181:406, 2000; MacGregor et al., AIDS, 16:2137-2143, 2002).

[0007]This disclosure describes vaccine compositions that elicit broad spectrum immunity against HIV, by providing robust expression of HIV antigens corresponding to important immunogenic epitopes of multiple clades and strains of human immunodeficiency virus 1. The foregoing and other objects, features, and advantages of the invention will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

SUMMARY

[0008]This disclosure relates to nucleic acid constructs that encode HIV antigens. These nucleic acid constructs are capable of eliciting an immune response against multiple variants of HIV, and are suitable for therapeutic (for example, prophylactic) administration. In the context of an immunogenic composition, multiple nucleic acids are combined, each of which encodes an HIV antigenic polypeptide, for example different HIV antigenic polypeptides (such as Gag, Pol, and Nef). A single immunogenic composition includes nucleic acid constructs that encode antigenic polypeptides of multiple clades or strains of HIV for example multiple clades or strains of Gag, Pol or Nef, or multiple clades or strains on Gag, Pol and Nef. Thus, when administered to a subject, the composition elicits an immune response against multiple clades or strains prevalent in human populations.

[0009]Methods of using the compositions are also described. Such methods involve administering compositions including the disclosed nucleic acid constructs to a subject, for example, for the purpose of eliciting an immune response against multiple clades or strains of HIV. The compositions can be administered alone or in combination with additional immunogenic compositions.

[0010]The foregoing and other objects, features, and advantages of the invention will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011]FIG. 1 is a schematic representation of a multi-clade, multi-valent HIV vaccine composition.

[0012]FIG. 2 is a schematic representation of the plasmid VRC 4401.

[0013]FIG. 3 is a schematic representation of the plasmid VRC 4409.

[0014]FIG. 4 is a schematic representation of the plasmid VRC 4404.

[0015]FIG. 5 is a schematic representation of the plasmid VRC 5736.

[0016]FIG. 6 is a schematic representation of the plasmid VRC 5737.

[0017]FIG. 7 is a schematic representation of the plasmid VRC 5738.

[0018]FIG. 8A schematically represents antigenic expression constructs with different transcription regulatory sequences. FIG. 8B is an image of a Western blot showing relative expression of the various constructs.

[0019]FIGS. 9A and B are bar graphs illustrating CD4⁺ and CD8⁺ T cell responses in mice immunized with expression plasmids with different transcription regulatory sequences.

[0020]FIGS. 10A, B and C are bar graphs illustrating relative immune responses against HIV Gag, Pol and Nef antigens in mice immunized with nucleic acid constructs having either a CMV/R transcription control sequence or a CMV IE transcription control sequence.

[0021]FIGS. 11A, B and C are bar graphs illustrating relative immune responses against HIV Gag, Pol, Nef and Env antigens in cynomolgous macaques immunized with different vaccine compositions.

[0022]FIGS. 12A, B and C are bar graphs illustrate the time course of development of the immune response against HIV antigens following immunization of cynomolgous macaques with different vaccine compositions.

[0023]FIG. 13 is a series of bar graphs illustrating the cellular immune response measured by intracellular cytokine staining (ICS) in humans immunized with VRC-HIVDNA016-00-VP.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

[0024]SEQ ID NOs:1-6 represent the VRC-HIVDNA016-00-VP plasmids 4401, 4409, 4404, 5736, 5737, and 5738, respectively. Each of these plasmids is a nucleic acid construct for expressing a single HIV antigenic polypeptide.

[0025]SEQ ID NOs:7-15 represent chimeric Env plasmids.

[0026]SEQ ID NOs:16-19 represent adenovirus vectors

[0027]SEQ ID NOs:20-25 represent exemplary Gag, Pol, Nef, clade A Env, clade B Env and clade C Env polypeptides, respectively.

[0028]SEQ ID NO:26 represents the CMV/R transcription regulatory sequence.

DETAILED DESCRIPTION

[0029]The present disclosure relates to a nucleic acid constructs suitable for use as a preventive vaccine for HIV-1. Specific examples of compositions disclosed herein provide two significant advances with respect to prior HIV vaccine candidates. Such compositions exhibit increased expression and immunogenicity, and are capable of eliciting an immune response against multiple divergent strains of HIV. The vaccine includes a mixture of different nucleic acid constructs, and is designed to produce Gag, Pol, Nef and Env HIV-1 proteins to elicit broad immune responses against multiple HIV-1 subtypes isolated in human infections. Most typically, the nucleic acids are incorporated into a plasmid vector. An exemplary clinical embodiment of the multi-plasmid vaccine is designated VRC-HIVDNA016-00-VP.

[0030]The rationale in development of the exemplary vaccine disclosed herein is to separate the gag, pol and nef genes into separate nucleic acid constructs, such as, plasmids, rather than having one construct that produces a fusion protein immunogen, as was the case with previously developed HIV vaccines. In exemplary embodiments, the nucleic acid construct has been modified to increase production of immunogenic protein products in vivo. The modifications include: 1) a change in the promoter incorporated into these plasmids and/or 2) a 68 amino acid addition to the gag gene (for example, in the VRC 4401 (Gag protein only) plasmid). Whereas previous HIV vaccine plasmids have most commonly utilized the cytomegalovirus (CMV) immediate early promoter to regulate transcription of the polynucleotide sequence encoding the antigenic polypeptide, in the nucleic acid constructs disclosed herein, the polynucleotide sequence encoding the immunogenic HIV polypeptides is operably linked to a promoter designated CMV/R. The CMV/R promoter was previously described in published US patent application no. 20040259825, the disclosure of which is incorporated herein in its entirety.

[0031]The nucleic acid constructs disclosed herein can incorporate polynucleotide sequences encoding essentially any HIV antigenic polypeptide, so long as antigens corresponding to multiple clades and/or strains are included. The compositions are described in detail with respect to a specific example of the nucleic acid constructs collectively designated the VRC-HIVDNA016-00-VP vaccine composition. This exemplary embodiment is illustrated in FIG. 1.

[0032]The vaccine composition VRC-HIVDNA016-00-VP includes six closed circular plasmid DNA macromolecules, VRC 4401, VRC 4409, VRC 4404, VRC 5736, VRC 5737 and VRC 5738, which can, for example, be combined in equal concentrations (mg/mL). VRC 4401 encodes the clade B HIV-1 Gag structural core protein that encapsidates the viral RNA and exhibits highly conserved domains. VRC 4409 encodes for clade B polymerase (Pol), which is also highly conserved, and VRC 4404 encodes for clade B Nef, an accessory protein against which a vigorous T-cell response is mounted in natural infection. The DNA plasmid expressing HIV-1 Pol has been modified to reduce potential toxicity through the incorporation of changes in the regions affecting the protease, reverse transcriptase, and integrase activities. Two amino acids in the myristoylation site in the HIV-1 nef gene were deleted to abrogate MHC class I and CD4⁺ down-regulation by the Nef protein. No modifications were made to the amino acid sequence of Gag. The other three plasmids express synthetic versions of modified, truncated envelope glycoproteins (gp145) from three strains of HIV-1: VRC 5736 (clade A), VRC 5737 (clade B) and VRC 5738 (clade C). The sequences used to create the DNA plasmids encoding Env are derived from three HIV-1 CCR5-tropic strains of virus. These genes have been modified to improve immunogenicity, which has been demonstrated in mice and monkeys. The vaccine is designed to elicit immune responses to a broad range of HIV-1 strains.

[0033]In particular examples, plasmids containing Gag, Pol, Nef and Env complementary DNAs (cDNAs) were used to subclone the relevant inserts into plasmid DNA expression vectors that use the CMV/R promoter and the bovine growth hormone polyadenylation sequence. All the plasmids expressing the HIV-1 genes were made synthetically with sequences designed to disrupt viral RNA structures that limit protein expression by using codons typically found in humans, thereby increasing gene expression. The translational enhancer region of the CMV immediate early region 1 enhancer was substituted with the 5'-untranslated HTLV-1 R-U5 region of the human T-cell leukemia virus type 1 (HTLV-1) long terminal repeat (LTR) to further optimize gene expression.

[0034]The DNA plasmids are typically produced in bacterial cell cultures containing a kanamycin selection medium. In all such cases, bacterial cell growth is dependent upon the cellular expression of the kanamycin resistance protein encoded by a portion of the plasmid DNA. Following growth of bacterial cells harboring the plasmid, the plasmid DNA is purified from cellular components. In a particular example, the Gag plasmid (VRC 4401) is 5886 nucleotide pairs in length and has an approximate molecular weight of 3.9 MDa; the Pol plasmid (VRC 4409) is 7344 nucleotide pairs in length and has an approximate molecular weight of 4.8 MDa; the Nef plasmid (VRC 4404) is 5039 nucleotide pairs in length and has an approximate molecular weight of 3.3 MDa; the clade A, B, and C Env plasmids (VRC 5736, 5737, and 5738) are 6305, 6338 and 6298 nucleotides in length, respectively, and have an approximate molecular weight of 4.2 MDa.

[0035]Thus, one aspect of the present disclosure relates to compositions capable of eliciting an immune response against HIV. For example, the compositions can be capable of eliciting a protective immune response against HIV when administered alone or in combination with at least one additional immunogenic compositions. It will be understood by those of skill in the art, the ability to produce an immune response after exposure to an antigen is a function of complex cellular and humoral processes, and that different subjects have varying capacity to respond to an immunological stimulus. Accordingly, the compositions disclosed herein are capable of eliciting an immune response in an immunocompetent subject, that is a subject that is physiologically capable of responding to an immunological stimulus by the production of a substantially normal immune response, e.g., including the production of antibodies that specifically interact with the immunological stimulus, and/or the production of functional T cells (CD4⁺ and/or CD8⁺ T cells) that bear receptors that specifically interact with the immunological stimulus. It will further be understood, that a particular effect of infection with HIV is to render a previously immunocompetent subject immunodeficient. Thus, with respect to therapeutic methods discussed below, it is generally desirable to administer the compositions to a subject prior to exposure to HIV (that is, prophylactically, e.g., as a vaccine) or therapeutically at a time following exposure to HIV during which the subject is nonetheless capable of developing an immune response to a stimulus, such as an antigenic polypeptide.

[0036]The compositions include a plurality of (that is two, three, four, five, six or even more) different nucleic acid constructs. Multiple copies of each of the different nucleic acid constructs are typically present. Each of the different nucleic acid constructs includes a polynucleotide sequence encoding an HIV antigenic polypeptide operably linked to a transcription regulatory sequence capable of directing its expression in the cells of a subject following systemic or localized administration. Included among the nucleic acid constructs are polynucleotide sequences that encode antigenic polypeptides of more than one (multiple) clades or strains of HIV. Thus, the composition includes multiple nucleic acid constructs, at least two of which incorporate polynucleotide sequences that encode HIV antigenic polypeptides from different clades or strains. Frequently, the composition includes nucleic acid constructs that encode HIV antigenic polypeptides from at least three different clades or strains.

[0037]In one embodiment, the composition includes multiple separate nucleic acid constructs, each of which includes a polynucleotide sequence encoding an HIV antigenic polypeptide operably linked to a CMV/R transcription control sequence. In one example, the CMV/R transcription control sequence has the sequence of SEQ ID NO:26. In another embodiment, the composition includes multiple separate nucleic acid constructs, each of which includes a polynucleotide sequence encoding a single HIV antigenic polypeptide. In certain embodiments, the nucleic acid constructs are plasmids.

[0038]The compositions typically include a first nucleic acid construct that includes a polynucleotide sequence that encodes an HIV Gag polypeptide, a second nucleic acid construct that includes a polynucleotide sequence that encodes an HIV Pol polypeptide, a third nucleic acid construct comprising a polynucleotide sequence that encodes an HIV Nef polypeptide, and at least one additional nucleic acid construct that includes a polynucleotide sequence that encodes an HIV Env polypeptide. The composition can also include one or more additional nucleic acid constructs that include a polynucleotide sequence that encodes an Env polypeptide of a different HIV clade or strain.

[0039]For example, the first nucleic acid construct can include a polynucleotide sequence that encodes a clade B Gag polypeptide, the second nucleic acid construct can include a polynucleotide sequence that encodes a clade B Pol polypeptide, and the third nucleic acid construct can include a polynucleotide sequence that encodes a clade B Nef polypeptide. Alternatively, the first, second and third nucleic acid constructs can include polynucleotide sequences that encode Gag, Pol and Nef polypeptides of a different clade, such as clade A or clade C, etc. For example, the composition can include a nucleic acid construct that include a polynucleotide sequence that encodes a Gag polypeptide with at least about 95% sequence identity to SEQ ID NO:20; a nucleic acid construct that includes a polynucleotide sequence that encodes a Pol polypeptide with at least about 95% sequence identity to SEQ ID NO:21 and/or a nucleic acid construct that includes a polynucleotide sequence that encodes a Nef polypeptide with at least about 95% sequence identity to SEQ ID NO:22. In one embodiment, the immunogenic composition includes a first nucleic acid construct with a polynucleotide sequence that encodes the Gag polypeptide of SEQ ID NO:20, a second nucleic acid construct with a polynucleotide sequence that encodes the Pol polypeptide of SEQ ID NO:21; and a third nucleic acid construct with a polynucleotide sequence that encodes the Nef polypeptide of SEQ ID NO:22. For example, the composition can include a nucleic acid construct that include a polynucleotide sequence with at least 95% sequence identity to positions 1375-2883 of SEQ ID NO:1; a nucleic acid construct that include a polynucleotide sequence with at least 95% sequence identity to positions 1349-4357 of SEQ ID NO:2 and/or a nucleic acid construct that include a polynucleotide sequence with at least 95% sequence identity to positions 1392-2006 of SEQ ID NO:3, or differing from the reference sequence by the substitution of one or more degenerate condons. In one embodiment, the composition includes the nucleic acid constructs represented by SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 (plasmids VRC 4401, VRC 4409 and VRC 4404), or constructs having at least 95% sequence identity thereto.

[0040]Additionally, the composition can include multiple nucleic acid constructs that encode Env polypeptides from different clades or strains. For example, the composition can include a first additional nucleic acid construct including a polynucleotide sequence that encodes a clade A Env polypeptide, a second additional nucleic acid construct including a polynucleotide sequence that encodes a clade B Env polypeptide, and a third additional nucleic acid construct including a polynucleofide sequence that encodes a clade C Env polypeptide. Generally, clade A, clade B and clade C Env polypeptides will be utilized as clades A, B and C collectively account for the highest proportion of HIV infections worldwide. However, one of skill in the art will recognize that compositions can be produced that include Env polypeptides from any combination of HIV clades or strains. In certain embodiments, the immunogenic compositions include a first additional nucleic acid construct that includes a polynucleotide sequence that encodes a clade A Env polypeptide with at least 95% sequence identity to SEQ ID NO:23; a second additional nucleic acid construct that includes a polynucleotide sequence that encodes a clade B Env polypeptide with at least 95% sequence identity to SEQ ID NO:24; and/or a third additional nucleic acid construct that includes a polynucleotide sequence that encodes a clade C Env polypeptide with at least 95% sequence identity to SEQ ID NO:25. In one embodiment, the composition includes a first additional nucleic acid construct with a polynucleotide sequence that encodes the clade A Env polypeptide of SEQ ID NO:23; a second additional nucleic acid construct with a polynucleotide sequence that encodes the clade B Env polypeptide of SEQ ID NO:24; and a third additional nucleic acid construct with a polynucleotide sequence that encodes the clade C Env polypeptide of SEQ ID NO:25. For example, the immunogenic composition can include a nucleic acid construct with a polynucleotide sequence that is at least about 95% identical to positions 1392-3272 of SEQ ID NO:4; a nucleic acid construct with a polynucleotide sequence that is at least about 95% identical to positions 1384-3312 of SEQ ID NO:5 and/or a nucleic acid construct with a polynucleotide sequence that is at least about 95% identical to positions 1392-3272 of SEQ ID NO:6. In an embodiment, the immunogenic compositions includes nucleic acid constructs represented by SEQ ID NO:4; SEQ ID NO:5 and SEQ ID NO:6 (plasmids VRC 5736, 5737 and 5738, respectively), or constructs having at least 95% sequence identity thereto, or constructs differing from the reference sequence by the substitution of degenerate codons.

[0041]Thus, in certain embodiments, the immunogenic composition includes a first nucleic construct with a polynucleotide sequence encoding a Gag polypeptide, a second nucleic acid construct with polynucleotide sequence encoding a Pol polypeptide, a third nucleic acid construct with a polynucleotide sequence encoding a Nef polypeptide, a fourth nucleic acid construct with a polynucleotide sequence encoding a clade A Env polypeptide, a fifth nucleic acid construct with a polynucleotide sequence encoding a clade B Env polypeptide, and a sixth nucleic acid construct with a polynucleotide sequence encoding a clade C Env polypeptide. In one such an embodiment, the first nucleic acid construct encodes a polypeptide with at least 95% sequence identity to SEQ ID NO:20, the second nucleic acid construct encodes a polypeptide with at least 95% sequence identity to SEQ ID NO:21; the third nucleic acid construct encodes a polypeptide with at least 95% sequence identity to SEQ ID NO:22; the fourth nucleic acid construct encodes a polypeptide with at least 95% sequence identity to SEQ ID NO:23; the fifth nucleic acid construct encodes a polypeptide with at least 95% sequence identity to SEQ ID NO:24 and the sixth nucleic acid construct encodes a polypeptide with at least 95% sequence identity to SEQ ID NO:25. In an embodiment, the immunogenic composition includes six nucleic acid constructs, of which one or more are at least 95% identical to positions 1375-2883 of SEQ ID NO:1; positions 1349-4357 of SEQ ID NO:2; positions 1392-2006 of SEQ ID NO:3; positions 1392-3272 of SEQ ID NO:4; positions 1384-3312 of SEQ ID NO:5 and positions 1392-3272 of SEQ ID NO:6. For example, the composition can include six nucleic acid constructs with the polynucleotide sequences represented by SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 (plasmids VRC 4401, VRC 4409, VRC 4404, VRC 5736, VRC 5737 and VRC 5738, respectively), or constructs having at least 95% sequence identity thereto, or constructs differing from the reference sequences by the substitution of degenerate codons. When combined in an immunogenic composition, the nucleic acid constructs can be combined in a substantially equal ratio by weight (that is an approximately 1:1:1:1:1:1 ratio).

[0042]In some cases, the compositions include nucleic acid constructs that each encode an HIV antigenic polypeptide of a single clade or strain. In other cases, it can be useful to include nucleic acid constructs that incorporate polynucleotide sequences that encode a chimeric Env polypeptide. Thus, in certain embodiments, the nucleic acid construct can encode a chimeric Env polypeptide with at least 95% identity to a polypeptide encoded by one of SEQ ID NOs:7-15.

[0043]Typically, when formulated for administration to a subject, the compositions also include a pharmaceutically acceptable carrier or excipient, for example, an aqueous carrier, such as phosphate buffered saline (PBS) or another neutral physiological salt solution. The composition can also include an adjuvant or other immunostimulatory molecule. The composition can be administered one or more times to a subject to elicit an immune response. For example, the composition can be administered multiple times at intervals of at least about 28 days, or at different intervals as dictated by logistical or therapeutic concerns.

[0044]Thus, a feature of the disclosure includes pharmaceutical compositions or medicaments for the therapeutic or prophylactic treatment of an HIV infection. The use of the compositions disclosed herein in the production of medicament for the therapeutic or prophylactic treatment of HIV is also expressly contemplated. Any of the limitations or formulations disclosed above with respect to compositions are applicable to their use in or as medicaments for the treatment of an HIV infection.

[0045]Another aspect of the disclosure relates to methods for eliciting an immune response against HIV by administering the compositions described above to a human subject. When administered to an immunocompetent subject, the composition is capable of eliciting an immune response against multiple clades or strains of HIV. For example, in one embodiment the method involves administering a composition that includes multiple different nucleic acid constructs, each of which includes a polynucleotide sequence encoding an HIV antigenic polypeptide operably linked to a CMV/R transcription control sequence. In another embodiment, the method involves administering a composition that includes multiple different nucleic acid constructs, each of which includes a polynucleotide sequence encoding a single HIV antigenic polypeptide. In certain embodiments, the administered nucleic acid constructs are plasmids. Indeed, any of the above described compositions are suitable for administration to human subjects in the methods disclosed herein.

[0046]One dose or multiple doses of the composition can be administered to a subject to elicit an immune response with desired characteristics, including the production of HIV specific antibodies, or the production of functional T cells that react with HIV. In certain embodiments, the composition is administered intramuscularly, for example, using a needleless delivery device. Alternatively, the composition is administered by other routes, such as intravenous, transdermal, intranasal, oral (or via another mucosa).

[0047]In some embodiments, the methods also involve administering viral vectors that encode HIV antigenic polypeptides, instead of, or in combination with one or more of the nucleic acid constructs already described. In some cases, the viral vectors are adenoviral vectors (for example a replication deficient adenoviral vectors). For example, one or more doses of a "primer" composition, such as those disclosed above, can be administered to a subject, followed by administration of one or more doses of a "booster" composition including multiple adenoviral vectors encoding HIV antigenic polypeptides. In certain embodiments, the adenoviral vectors encode one or more HIV antigenic polypeptide that is identical to an HIV antigenic polypeptide previously administered in the primer composition. Exemplary recombinant adenoviral vectors are represented by SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19. Of course, alternative adenoviral vectors, for example, that encode polypeptides with at least about 95% sequence identity to a polypeptide encoded by one of these sequences, or that share at least about 95% sequence identity to one of these sequences, can also be used.

[0048]In another aspect, the disclosure concerns isolated or recombinant nuclei acids that include a polynucleotide sequence that encodes an HIV antigenic polypeptide operably linked to a CMV/R transcription regulatory sequence. For example, such a nucleic acid can be a plasmid or a viral vector. The polynucleotide sequence can encode an HIV Gag polypeptide, an HIV Pol polypeptide, an HIV Nef polypeptide or an HIV Env polypeptide. In some examples, the HIV polypeptide encoded by the nucleic acid construct is the only HIV antigen encoded by the isolated or recombinant nucleic acid. Exemplary polypeptides encoded by these nucleic acids are represented by SEQ ID NOs:20-25, and include sequences that are at least 95% identical to the amino acid sequences of SEQ ID NOs:20-25. For example, such a nucleic acid can include a polynucleotide sequence that is at least 95% identical to: positions 1375-2883 of SEQ ID NO:1; positions 1349-4357 of SEQ ID NO:2; positions 1392-2006 of SEQ ID NO:3; positions 1392-3272 of SEQ ID NO:4; positions 1384-3312 of SEQ ID NO:5 or positions 1392-3272 of SEQ ID NO:6, any of which can be operably linked to a CMV/R transcription regulatory sequence. For example, the CMV/R transcription control sequence can be a polynucleotide sequence with at least 95% sequence identity to SEQ ID NO:26. Exemplary embodiments of such nucleic acids include the plasmids VRC 4401, VRC 4409, VRC 4404, VRC 5736, VRC 5737 and VRC 5738 represented by SEQ ID NOs:1-6, respectively.

[0049]In other embodiments, the nucleic acids include a polynucleotide sequence that encodes a chimeric HIV polypeptide that incorporates at least a subsequence of multiple HIV clades or strains. For example, the chimeric HIV polypeptide can be a chimeric Env polypeptide that includes subsequences of different HIV clades or strains. Examples of such nucleic acids include SEQ ID NOs:7-15, as well as substantially similar polynucleotide sequences, such as those having at least about 95% sequence identity to one of SEQ ID NOs:7-15, or polynucleotide sequences in which one or more degenerate codons have been substituted for each other. Alternatively, the nucleic acids can include a polynucleotide sequence that encodes a chimeric HIV Env polypeptide operably linked to a transcription regulatory sequence other than the CMV/R transcription regulatory region (for example, the CMV immediate early promoter enhance or other promoter and/or enhancer as discussed below). Chimeric Env polypeptides are also a feature of this disclosure.

[0050]Additional technical details are provided under the specific topic headings below. In order to facilitate review of the various embodiments of this disclosure, the following explanations of specific terms are provided:

Terms

[0051]Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

[0052]The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. The term "plurality" refers to two or more. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The term "comprises" means "includes." The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example."

[0053]In order to facilitate review of the various embodiments of this disclosure, the following explanations of specific terms are provided:

[0054]Adjuvant: A vehicle used to enhance antigenicity; such as a suspension of minerals (alum, aluminum hydroxide, aluminum phosphate) on which antigen is adsorbed; or water-in-oil emulsion in which antigen solution is emulsified in oil (MF-59, Freund's incomplete adjuvant), sometimes with the inclusion of killed mycobacteria (Freund's complete adjuvant) to further enhance antigenicity (inhibits degradation of antigen and/or causes influx of macrophages). Adjuvants also include immunostimulatory molecules, such as cytokines, costimulatory molecules, and for example, immunostimulatory DNA or RNA molecules, such as CpG oligonucleotides.

[0055]Antigen: A compound, composition, or substance that can stimulate the production of antibodies or a T cell response in an animal, including compositions that are injected, absorbed or otherwise introduced into an animal. The term "antigen" includes all related antigenic epitopes. An "antigenic polypeptide" is a polypeptide to which an immune response, such as a T cell response or an antibody response, can be stimulated. "Epitope" or "antigenic determinant" refers to a site on an antigen to which B and/or T cells respond. In one embodiment, T cells respond to the epitope when the epitope is presented in conjunction with an MHC molecule. Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of an antigenic polypeptide. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5, about 9, or about 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and multi-dimensional nuclear magnetic resonance spectroscopy.

[0056]Antibody: Immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, that is, molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen. A naturally occurring antibody (for example, IgG, IgM, IgD) includes four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. The phrase "antibody response" refers to an immunological response against an antigen involving the secretion of antibodies specific for the antigen. An antibody response is a B cell mediated immune response initiated through the interaction of an antigen (or epitope) with a B cell receptor (membrane bound IgD) on the surface of a B cell. Following binding of the stimulation of the B cell receptor by its cognate antigen, the B cell differentiates into a plasma cell that secretes antigen specific immunoglobulin to produce an antibody response. "Neutralizing antibodies" are antibodies that bind to an epitope on a virus inhibiting infection and/or replication as measured, for example, in a plaque neutralization assay.

[0057]cDNA (complementary DNA): A piece of DNA lacking internal, non-coding segments (introns) and regulatory sequences that determine transcription. cDNA is typically synthesized in the laboratory by reverse transcription from messenger RNA extracted from cells. In the context of preparing nucleic acid constructs including polynucleotide sequences that encode an HIV antigenic polypeptide, a cDNA can be prepared, for example by reverse transcription or amplification (e.g., by the polymerase chain reaction, PCR) from an HIV RNA genome (or genome segment).

[0058]Host cells: Cells in which a polynucleotide, for example, a polynucleotide vector or a viral vector, can be propagated and its DNA expressed. The cell may be prokaryotic or eukaryotic. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term "host cell" is used. Thus, the nucleic acid constructs described herein can be introduced into host cells where their polynucleotide sequences (including those encoding HIV antigenic polypeptides) can be expressed.

[0059]Immune response: A response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus. In some cases, the response is specific for a particular antigen (that is, an "antigen-specific response"). In some cases, an immune response is a T cell response, such as a CD4⁺ response or a CD8⁺ response. Alternatively, the response is a B cell response, and results in the production of specific antibodies. A "protective immune response" is an immune response that inhibits a detrimental function or activity of a pathogen (such as HIV), reduces infection by the pathogen, or decreases symptoms (including death) that result from infection by the pathogen. A protective immune response can be measured, for example, by the inhibition of viral replication or plaque formation in a plaque reduction assay or ELISA-neutralization assay (NELISA), or by measuring resistance to viral challenge in vivo in an experimental system.

[0060]Immunogenic composition: A composition comprising at least one epitope of a pathogenic organism, that induces a measurable CTL response, or induces a measurable B cell response (for example, production of antibodies that specifically bind the epitope), or both, when administered to an immunocompetent subject. Thus, an immunogenic composition is a composition capable of eliciting an immune response in an immunocompetent subject. For example, an immunogenic composition can include isolated nucleic acid constructs (such as plasmids or viral vectors) that encode one or more immunogenic epitopes of an HIV antigenic polypeptide that can be used to express the epitope(s) (and thus be used to elicit an immune response against this polypeptide or a related polypeptide expressed by the pathogen). For in vitro use, the immunogenic composition can consist of the isolated nucleic acid, protein or peptide. For in vivo use, the immunogenic composition will typically include the nucleic acid or virus that expresses the immunogenic epitope in pharmaceutically acceptable carriers or excipients, and/or other agents, for example, adjuvants. An immunogenic polypeptide (such as an HIV antigen), or nucleic acid encoding the polypeptide, can be readily tested for its ability to induce a CTL or antibody response by art-recognized assays.

[0061]Pharmaceutically acceptable carriers and/or pharmaceutically acceptable excipients: The pharmaceutically acceptable carriers or excipients of use are conventional. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, Pa., 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of the polypeptides and polynucleotides disclosed herein.

[0062]In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (for example, powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

[0063]A "therapeutically effective amount" is a quantity of a composition used to achieve a desired effect in a subject. For instance, this can be the amount of the composition necessary to inhibit viral (or other pathogen) replication or to prevent or measurably alter outward symptoms of viral (or other pathogenic) infection. When administered to a subject, a dosage will generally be used that will achieve target tissue concentrations (for example, in lymphocytes) that has been shown to achieve an in vitro effect.

[0064]Inhibiting or treating a disease: Inhibiting infection by HIV refers to inhibiting the full development of disease caused by exposure to human immunodeficiency virus. For example, inhibiting an HIV infection refers to lessening symptoms resulting from infection by the virus, such as preventing the development of symptoms in a person who is known to have been exposed to the virus, or to reducing virus load or infectivity of a virus in a subject exposed to the virus. "Treatment" refers to a therapeutic or prophylactic intervention that ameliorates or inhibits or otherwise avoids a sign or symptom of a disease or pathological condition related to infection of a subject with a virus.

[0065]Subject: Living multi-cellular vertebrate organisms, a category that includes both human and veterinary subjects, including human and non-human mammals. In a clinical setting with respect to HIV, a subject is usually a human subject. An immunocompetent subject is a subject that is able to produce a substantially normal immune response against an antigenic stimulus.

[0066]T Cell: A white blood cell critical to the immune response. T cells include, but are not limited to, CD4⁺ T cells and CD8⁺ T cells. A CD4⁺ T lymphocyte is an immune cell that carries a marker on its surface known as CD4, for example, a "helper" T cell. These cells, also known as helper T cells, help orchestrate the immune response, including antibody responses as well as killer T cell responses. CD8⁺ T cells carry the CD8 marker, and include T cells with cytotoxic or "killer" effector function.

[0067]Transduced or Transfected: A transduced cell is a cell into which a nucleic acid molecule has been introduced, for example, by molecular biology techniques. As used herein, the term introduction or transduction encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transformation with plasmid vectors, transfection with viral vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration.

[0068]Vaccine: A vaccine is a pharmaceutical composition that elicits a prophylactic or therapeutic immune response in a subject. In some cases, the immune response is a protective immune response. Typically, a vaccine elicits an antigen-specific immune response to an antigen of a pathogen. In the context of this disclosure, the vaccines elicit an immune response against HIV. The vaccines described herein include nucleic acid constructs, for example, plasmids or viral vectors, encoding HIV antigens.

[0069]Vector: A nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell. A vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector may also include one or more selectable marker gene and other genetic elements known in the art. The term vector includes plasmids, linear nucleic acid molecules, and viral vectors, such as adenovirus vectors and adenoviruses. The term adenovirus vector is utilized herein to refer to nucleic acids including one or more components of an adenovirus that generate viral particles in host cells. Such particles may be capable of one or more rounds of infection and replication, or can be replication deficient, e.g., due to a mutation. An adenovirus includes nucleic acids that encode at least a portion of the assembled virus. Thus, in many circumstances, the terms can be used interchangeably.

Nucleic Acid Constructs Encoding HIV Antigens

[0070]The present disclosure concerns nucleic acid constructs including polynucleotide sequences that encode antigenic polypeptides of human immunodeficiency virus-1 ("HIV-1" or simply, "HIV"). The term polynucleotide or nucleic acid sequence refers to a polymeric form of nucleotide at least 10 bases in length. The nucleotides can be ribonucleotides, deoxyribonucleotides, or modified forms of either nucleotide. The term includes single- and double-stranded forms of DNA. In the context of this disclosure, the nucleic acid constructs are "recombinant" nucleic acids. A recombinant nucleic acid is a nucleic acid that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence, for example, a heterologous sequence that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques.

[0071]In some cases, the nucleic acids are "isolated" nucleic acids. An "isolated" nucleic acid (and similarly, an isolated protein) has been substantially separated or purified away from other biological components in the cell of the organism in which the nucleic acid naturally occurs, for example, other chromosomal and extra-chromosomal DNA and RNA, proteins and organelles. Nucleic acids and proteins that have been "isolated" include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.

[0072]An "HIV antigenic polypeptide" or "HIV antigen" can include any proteinaceous HIV molecule or portion thereof that is capable of provoking an immune response in an immunocompetent mammal. An "HIV molecule" is a molecule that is a part of a human immunodeficiency virus, is encoded by a nucleic acid sequence of a human immunodeficiency virus, or is derived from or synthetically based upon any such molecule. Administration of a nucleic acid that encodes an HIV antigen that provokes an immune response preferably leads to protective immunity against HIV. In this regard, an "immune response" to HIV is an immune response to any one or more HIV antigens.

[0073]Examples of suitable HIV antigens include as all or part of the HIV Gag, Pol, Nef or Env proteins. In the virus, Gag proteins are components of the viral capsid. The Pol polyprotein provides reverse transcriptase (RT); integrase (IN) and protease(PR) functions, which reverse transcribe the viral RNA into double stranded DNA, integrated into the chromosome of a host cell, and cleave the gag-pol derived proteins into functional polypeptides, respectively. The Nef polypeptide is a negative regulatory factor involved in determining pathogenicity of the virus following infection. Env proteins are envelope proteins involved in viral attachment and fusion to target cells. One of skill in the art will recognize that functional attributes of the polypeptides can be altered (for example, deleted) without altering antigenic properties of the polypeptides. Immunogenic variants or fragments of each of Gag, Pol, Nef or Env are also HIV antigenic polypeptides that can included in the immunogenic compositions disclosed herein. Immunogenic variants include those, for example, having at least 90%, 95%, or 98% sequence identity to SEQ ID NOs:20-25, or immunogenic fragments thereof. The nucleic acid vaccines disclosed herein can include SEQ ID NOs:1-19 or sequences that encode HIV antigens, such as those represented by SEQ ID NOS:20-25, or HIV antigens that have at least 90%, 95% or 98% sequence identity to SEQ ID NOs:20-25.

[0074]Suitable Env proteins are known in the art and include, for example, gp160, gp120, gp41, and gp140. Any clade of HIV is appropriate for antigen selection, including HIV clades A, B, C, and the like. Thus, it will be appreciated that any one, or a combination, of the following HIV antigens can be used in the inventive method: HIV clade A gp140, Gag, Pol, Nef and/or Env; HIV clade B gp140, Gag, Pol, Nef and/or Env proteins; and HIV clade C gp140, Gag, Pol, Nef and/or Env proteins. While the compositions and methods are described in detail with respect to Gag, Pol, Nef and/or Env proteins, any HIV protein or portion thereof capable of inducing an immune response in a mammal can be used in connection with the inventive method. HIV Gag, Pol, Nef and/or Env proteins from HIV clades A, B, C, as well as nucleic acid sequences encoding such proteins and methods for the manipulation and insertion of such nucleic acid sequences into vectors, are known (see, for example, HIV Sequence Compendium, Division of AIDS, National Institute of Allergy and Infectious Diseases, 2003, HIV Sequence Database (on the world wide web at hiv-web.lanl.gov/content/hiv-db/mainpage.html), Sambrook et al., Molecular Cloning, a Laboratory Manual, 2d edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1989, and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, N.Y., 1994).

[0075]Gag, Pol, Nef and Env polypeptide sequences are known in the art, and numerous amino acid sequences are available from publicly accessible databases, such as GENBANK®. For example, a Gag polypeptide corresponding to the amino acid sequence of the strain HXB2 is represented by the sequence of GENBANK® accession number K03455. Pol and Nef polypeptides corresponding to the amino acid sequence of the strain NL4-3 is represented by the sequence of GENBANK® accession number M19921. Exemplary Env polypeptides, for example, corresponding to clades A, B and C are represented by the sequences of GENBANK® accession numbers U08794, K03455 and AF286227, respectively. Particular exemplary sequences encoded by the nucleic acid constructs disclosed herein are represented by SEQ ID NOs:20-25, corresponding to Gag, Pol, Nef, clade A Env, clade B Env, and clade C Env, respectively. Certain of these exemplary polypeptides have been modified functionally (as indicated in further detail in the Examples) but nonetheless retain important antigenic characteristics of the naturally occurring proteins.

[0076]An entire, intact HIV protein is not required to produce an immune response. Indeed, most antigenic epitopes of HIV proteins are relatively small in size. Thus, fragments (for example, epitopes or other antigenic fragments) of an HIV protein, such as any of the HIV proteins described herein, can be used as an HIV antigen. Antigenic fragments and epitopes of the HIV Gag, Pol, Nef and/or Env proteins, as well as nucleic acid sequences encoding such antigenic fragments and epitopes, are known (see, for example, HIV Immunology and HIV/SIV Vaccine Databases, Vol. 1, Division of AIDS, National Institute of Allergy and Infectious Diseases, 2003).

[0077]A nucleic acid construct is said to "encode" an antigen when a polynucleotide sequence incorporated into the construct includes one or more open reading frames that upon recognition and activity by cellular transcriptional and translational processes gives rise to a sequence of amino acids constituting the antigen.

[0078]HIV antigens are "different" if they comprise a different antigenic amino acid sequence. When referring to a plurality of different HIV antigens, the two or more different HIV antigens can be any HIV antigens, such as two or more (or three, or four, or five, or six, or more) of the HIV antigens described herein. Different HIV antigenic polypeptides can be two or more antigenic polypeptides from different HIV proteins, that is proteins encoded by different genes in the HIV genome (for example, an HIV Gag polypeptide is different from an HIV Pol polypeptide, which is different from an HIV Nef polypeptide, which again is different from an HIV Env polypeptide). Thus, Gag, Pol, Nef and Env are different HIV proteins or antigenic polypeptides. Alternatively, different HIV antigenic polypeptides are different if they are encoded by a homologous genomic segment (or gene) from different strains or clades of HIV. Thus, a clade A Env polypeptide is different from a clade B Env polypeptide, which is different from a clade C Env polypeptide, and the like. In the context of immunogenic (for example, vaccine) compositions described herein, the two or more different HIV antigens include HIV antigens from two or more different HIV clades or strains, such as from three or more different HIV clades (such as clades A, B and C) or from two or more variant HIV strains of the same clade. Exposing the immune system of a mammal to a "cocktail" of different HIV antigens can elicit a broader and more effective immune response than exposing the immune system to only a single HIV antigen.

[0079]Thus, a plurality of separate nucleic acid constructs each including a polynucleotide sequence encoding a single HIV antigenic polypeptide, wherein the plurality of nucleic acid constructs encode a plurality of antigenic polypeptides or a plurality of HIV clades or strains, can include a plurality of encoded polypeptides of the same clade or strain (for example all clade B) or encoded polypeptides of different clades or strains (for example some of clade A and others of clade B).

[0080]In some particularly disclosed embodiments the composition includes a plurality of different nucleic acid constructs. The nucleic acid constructs include a polynucleotide sequence encoding a single (no more than once) HIV antigen operably linked to a transcription control sequence, and the single HIV antigen is different for the different nucleic acid constructs. In particular examples, the different single HIV antigens of the different nucleic acid constructs, are different encoded polypeptides of the same clade or strain, but may further include different encoded polypeptides, expressed from different constructs, of clades or strains that differ from the encoded polypeptides that share the same clade or strain. For example, the different nucleic acid constructs that encode HIV antigens of the same clade or strain can be three separate constructs that respectively encode Gag, Pol, and Nef as the only HIV antigen expressed from each of the constructs, and each of Gag, Pol, and Nef are of the same clade or strain (for example, all clade B). In addition, in some embodiments the composition can further include separate nucleic acid constructs that encode Env antigens of different clades or strains. For example, at least three separate constructs independently encode clade A Env, clade B Env and clade C Env as their only encoded HIV antigen.

[0081]For example, a nucleic acid construct can include a polynucleotide sequence that encodes a single HIV antigenic polypeptide. In specific examples provided herein, the nucleic acid construct encodes a single Gag polypeptide, a single Pol polypeptide, a single Nef polypeptide or a single Env polypeptide. For example, the nucleic acid construct can include a polynucleotide sequence that encodes a single Gag polypeptide, such as a clade B Gag polypeptide (e.g., the amino acid sequence of SEQ ID NO:20); a polynucleotide sequence that encodes a single Pol polypeptide, such as a clade B Pol polypeptide (e.g., SEQ ID NO:21); a polynucleotide sequence that encodes a single Nef polypeptide, such as a clade B Nef polypeptide (e.g., SEQ ID NO:22), or a polynucleotide sequence that encodes a single Env polypeptide, such as a clade A, a clade B or a clade C Env polypeptide (for examples, SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO:25). Exemplary nucleic acid constructs encoding these polypeptides are represented by SEQ ID NOs:1-6, respectively.

[0082]Alternatively, a nucleic acid construct can include a polynucleotide sequence that encodes an HIV antigenic polypeptide that includes subsequences of multiple clades or strains, that is, a "chimeric" HIV polypeptide. A chimeric HIV antigenic polypeptide can include subsequences of two or more clades or strains, such as three or more different clades or strains. For example, a chimeric HIV Env polypeptide can include one or more subsequence of a clade A Env polypeptide in combination with one or more subsequence of a clade B Env polypeptide and/or one or more subsequence of a clade C Env polypeptide, or in combination with one or more subsequences of a different clade A strain (or strains) of HIV with a different amino acid sequence. Similarly, subsequences of clade B and C Env polypeptides can be combined with subsequences of other clades and/or strains. Nucleic acid constructs including chimeric Env polypeptides are represented by SEQ ID NOs:7-15.

[0083]Typically, the nucleic acid constructs encoding the HIV antigenic polypeptides are plasmids. However, other vectors (for example, viral vectors, phage, cosmids, etc.) can be utilized to replicate the nucleic acids. In the context of this disclosure, the nucleic acid constructs typically are expression vectors that contain a promoter sequence which facilitates the efficient transcription of the inserted genetic sequence of the host. The expression vector typically contains an origin of replication, a promoter, as well as specific nucleic acid sequences that allow phenotypic selection of the transformed cells. In exemplary nucleic acid constructs, the coding sequence is operably linked under the transcriptional control of a human cytomegalovirus (CMV) immediate early (IE) enhancer/promoter that has been modified to include a regulatory sequence from the R region of the long terminal repeat (LTR) of human T-cell leukemia virus type 1 (HTLV-1). This transcription regulatory sequence is designated "CMV/R" or "CMV/R promoter." The CMV/R transcription regulatory sequence (alternatively referred to as a "transcription control sequence") contains, in a 5' to 3' direction: the CMV IE enhancer/promoter; the HTLV-1 R region; and a 123 base pair (bp) fragment of the CMV IE 3' intron. The CMV/R transcription regulatory region confers substantially increased expression and improved cellular immune responses to HIV antigens operably linked under its control. An exemplary CMV/R is represented by SEQ ID NO:26. However, transcription control sequences that retain the regulatory properties or have been modified to enhance expression, including transcription regulatory regions that are at least about 90%, or 95% or 98% identical to SEQ ID NO:26, can also be used.

[0084]More generally, polynucleotide sequences encoding HIV antigenic polypeptides can be operably linked to any promoter and/or enhancer that is capable of driving expression of the nucleic acid following introduction into a host cell. A promoter is an array of nucleic acid control sequences that directs transcription of a nucleic acid. A promoter includes necessary nucleic acid sequences (which can be) near the start site of transcription, such as in the case of a polymerase II type promoter (a TATA element). A promoter also can include distal enhancer or repressor elements which can be located as much as several thousand base pairs from the start site of transcription. Both constitutive and inducible promoters are included (see, for example, Bitter et al., Methods in Enzymology 153:516-544, 1987). Specific, non-limiting examples of promoters include promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the cytomegalovirus immediate early gene promoter, the retrovirus long terminal repeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter) may be used. Promoters produced by recombinant DNA or synthetic techniques may also be used. A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.

[0085]To produce such nucleic acid constructs, polynucleotide sequences encoding HIV antigenic polypeptides are inserted into a suitable expression vector, such as a plasmid expression vector that use the CMV/R promoter and the bovine growth hormone polyadenylation sequence to regulate expression. The CMV/R promoter consists of a translational enhancer region of the CMV immediate early region 1 enhancer (CMV-IE) substituted with the 5'-untranslated HTLV-1 R-U5 region of the human T-cell leukemia virus type 1 (HTLV-1) long terminal repeat (LTR) to optimize gene expression. The HIV-1 polynucleotide sequences are typically modified to optimize expression in human cells. The plasmid expression vectors are introduced into bacterial cells, such as, E. coli, which are grown in culture in kanamycin selection medium. In all cases, bacterial cell growth is dependent upon the cellular expression of the kanamycin resistance protein encoded by a portion of the plasmid DNA. Following growth of bacterial cells harboring the plasmid, the plasmid DNA is purified from cellular components. Procedures for producing polynucleotide sequences encoding HIV antigenic polypeptides and for manipulating them in vitro are well known to those of skill in the art, and can be found, e.g., in Sambrook and Ausubel, supra.

[0086]In addition to the polynucleotide sequences encoding the polypeptides represented by SEQ ID NOs:20-25 disclosed herein, such as SEQ ID NOs:1-6 (as well as nucleic acids encoding chimeric Env polypeptides represented by SEQ ID NOs:7-15 and nucleic acids encoding adenoviral vectors represented by SEQ ID NOs:16-19) as disclosed herein, the nucleic acid constructs can include variant polynucleotide sequences that encode polypeptides that are substantially similar to SEQ ID NOs:20-25 (for example, are substantially similar to SEQ ID NOs:1-6 and/or SEQ ID NOs:16-19). Similarly, the nucleic acid constructs can include polynucleotides that encode chimeric polypeptides that are substantially similar to those encoded by SEQ ID NOs:7-15. The similarity between amino acid (and polynucleotide) sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity); the higher the percentage, the more similar are the primary structures of the two sequences. In general, the more similar the primary structures of two amino acid sequences, the more similar are the higher order structures resulting from folding and assembly. Variants of an HIV antigenic polypeptide (for example, of a particular clade) can have one or a small number of amino acid deletions, additions or substitutions but will nonetheless share a very high percentage of their amino acid (and generally their polynucleotide sequence). To the extent that variants of a subtype differ from each other, their overall antigenic characteristics are maintained. In contrast, HIV antigens of different clades share less sequence identity and/or differ from each other such that their antigenic characteristics are no longer identical. Thus, the nucleic acid constructs can include polynucleotides that encode polypeptides that are at least about 90%, or 95%, or 98% identical to one of SEQ ID NOs:20-25 with respect to amino acid sequence, or that have at least about 90%, 95%, or 98% sequence identity to one or more of SEQ ID NOs;1-19 and/or that differ from one of these sequences by the substitution of degenerate codons.

[0087]Methods of determining sequence identity are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; Higgins and Sharp, Gene 73:237, 1988; Higgins and Sharp, CABIOS 5:151, 1989; Corpet et al., Nucleic Acids Research 16:10881, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988. Altschul et al., Nature Genet. 6:119, 1994, presents a detailed consideration of sequence alignment methods and homology calculations. The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, Md.) and on the internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. A description of how to determine sequence identity using this program is available on the NCBI website on the internet.

[0088]Another indicia of sequence similarity between two nucleic acids is the ability to hybridize. The more similar are the sequences of the two nucleic acids, the more stringent the conditions at which they will hybridize. The stringency of hybridization conditions are sequence-dependent and are different under different environmental parameters. Thus, hybridization conditions resulting in particular degrees of stringency will vary depending upon the nature of the hybridization method of choice and the composition and length of the hybridizing nucleic acid sequences. Generally, the temperature of hybridization and the ionic strength (especially the Na⁺ and/or Mg⁺+ concentration) of the hybridization buffer will determine the stringency of hybridization, though wash times also influence stringency. Generally, stringent conditions are selected to be about 5° C. to 20° C. lower than the thermal melting point (T_m) for the specific sequence at a defined ionic strength and pH. The T_m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Conditions for nucleic acid hybridization and calculation of stringencies can be found, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; Tijssen, Hybridization With Nucleic Acid Probes, Part I: Theory and Nucleic Acid Preparation, Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Ltd., NY, N.Y., 1993. and Ausubel et al. Short Protocols in Molecular Biology, 4^th ed., John Wiley & Sons, Inc., 1999.

[0089]For purposes of the present disclosure, "stringent conditions" encompass conditions under which hybridization will only occur if there is less than 25% mismatch between the hybridization molecule and the target sequence. "Stringent conditions" may be broken down into particular levels of stringency for more precise definition. Thus, as used herein, "moderate stringency" conditions are those under which molecules with more than 25% sequence mismatch will not hybridize; conditions of "medium stringency" are those under which molecules with more than 15% mismatch will not hybridize, and conditions of "high stringency" are those under which sequences with more than 10% mismatch will not hybridize. Conditions of "very high stringency" are those under which sequences with more than 6% mismatch will not hybridize. In contrast nucleic acids that hybridize under "low stringency conditions include those with much less sequence identity, or with sequence identity over only short subsequences of the nucleic acid. For example, a nucleic acid construct can include a polynucleotide sequence that hybridizes under high stringency or very high stringency, or even higher stringency conditions to a polynucleotide sequence that encodes any one of SEQ ID NOs:20-25. Similarly, the nucleic acid constructs can hybridize under such conditions to any one of SEQ ID NOs:1-19.

[0090]Thus, in addition to polynucleotides encoding the particular amino acid sequences represented by SEQ ID NOs:20-25, for example those polynucleotides represented by the codon optimized constructs of SEQ ID NO:s1-19, the nucleic acid constructs used in the vaccine compositions can include polynucleotide sequences having a high percentage of sequence identity, for example, that hybridize under high stringency, or very high stringency (or even higher stringency) to one of these sequences. A codon composition at one or more positions that is found in a naturally occurring or mutant strain of HIV are also encompassed within the nucleic acid constructs disclosed herein. One of skill in the art can easily identify numerous HIV polynucleotide sequences, and determine which nucleotides can be varied without substantially altering the amino acid content of the encoded polypeptide. In addition, polynucleotide sequences that encode variants with a small number of amino acid additions, deletions or substitution are also encompassed within the nucleic acid constructs described herein. Typically, any amino acid additions, deletions and/or substitutions are located in positions that do not alter the antigenic epitopes and that do not interfere with folding, or other translational or post-translational processing. Most commonly, any amino acid substitutions are conservative amino acid substitutions. For example, a variant polynucleotide sequence can encode an HIV antigenic polypeptide with one or two or three or four or five, or more amino acid additions, deletions or substitutions.

[0091]Conservative variants of particular amino acids are well known in the art, and can be selected, for example from groupings set forth in Table 1.

TABLE-US-00001 TABLE 1 Conservative amino acid substitutions Original Residue Conservative Substitutions Ala Ser Arg Lys Asn Gln, His Asp Glu Cys Ser Gln Asn Glu Asp His Asn; Gln Ile Leu, Val Leu Ile; Val Lys Arg; Gln; Glu Met Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp; Phe Val Ile; Leu

Immunogenic Compositions

[0092]Used in combination, the nucleic acid constructs, such as those exemplified by SEQ ID NOs:1-6, can be used to provide immunogenic compositions that elicit a broad spectrum immune response against HIV. This specific combination of nucleic acid constructs is referred to herein as VRC-HIVDNA016-00-VP, and includes the plasmids VRC-4401, VRC-4409, VRC-4404, VRC-5736, VRC 5737, and VRC-5738, corresponding respectively to SEQ ID NOs:1-6).

[0093]The composition including two or more nucleic acid construct encoding different HIV antigens is typically provided by a composition including multiple nucleic acid constructs, each of which encodes a single HIV antigen. Collectively, the two or more nucleic acid constructs encode antigens from more than one clade or strain, for example, from two or more clades or strains, or from three or more clades or strains. In some cases, the composition includes polynucleotide sequences that encode a chimeric HIV antigen, with subsequences of more than one clade or strain.

[0094]For clinical purposes, all nucleic acid constructs, such as plasmids and host E. coli strains used in the production of the vaccine are characterized in accordance with the relevant sections of the "Points to Consider in the Production and Testing of New Drugs and Biologicals Produced by Recombinant DNA Technology" (1985), the "Supplement: Nucleic Acid Characterization and Genetic Stability" (1992), and "Points to Consider in Human Somatic Cell Therapy and Gene Therapy" (1991, 1998), "Points to Consider on Plasmid DNA Vaccines for Preventive Infectious Disease Indications" (1996). In addition for clinical testing and use, all compositions are produced in compliance with current Good Manufacturing Practices (cGMP).

[0095]Thus, in one embodiment, the immunogenic composition is VRC-HIVDNA16-00-VP, a six-component multiclade plasmid DNA vaccine, expressing Gag, Pol and Nef proteins from clade B HIV-1 and Env glycoproteins from clades A, B and C. This composition is suitable for the prophylactic treatment of HIV, that is, as a preventive HIV-1 vaccine. The vaccine has been designed to elicit immune responses against several proteins from a variety of HIV-1 strains. This vaccine differs from previous multiclade vaccine compositions in two significant ways. First, previous compositions have relied on a single plasmid encoding a Gag-Pol-Nef fusion protein. In the particular examples described herein, these three proteins are separated into three different plasmids, encoding Gag (VRC 4401), Pol (VRC 4409), and Nef (VRC 4404) individually. Additionally, there is a 68 amino acid addition in the gag gene as compared to the previous fusion protein composition. Second, the promoter is modified to include the 5'-untranslated HTLV-1 R-U5 region of the human T-cell leukemia virus type 1 (HTLV-1) long terminal repeat (LTR) rather than a portion of the translational enhancer region of the CMV immediate early region 1 enhancer used in previous constructs. Vaccination, for example, of non-human primates, with plasmids containing CMV/R transcription regulatory region elicited higher and more consistent HIV-1 specific cellular immune responses than vaccination with plasmids constructed with the unmodified CVM IE promoter/enhancer sequence.

[0096]VRC-HIVDNA016-00-VP is designed to elicit immune responses against several proteins from a variety of HIV-1 strains. This vaccine product has evolved from the initial HIV-1 DNA plasmid product (VRC-4302; BB-IND 9782) that encoded for an HIV-1 clade B Gag-Pol fusion protein. Preclinical studies demonstrated expression of immunogenic protein in small animals, and an ongoing Phase I clinical trial has revealed no safety concerns at the doses tested to date. The VRC-HIVDNA009-00-VP vaccine (BB-IND 10681) expanded upon the product concept to include proteins from multiple subtypes (clades) of HIV-1 and increased the number of vaccine components to include a highly immunogenic regulatory protein (Nef), as well as modified Envelope glycoproteins that have been able to generate immune responses in rhesus macaques.

[0097]The four plasmid product, VRC-HIVDNA009-00-VP, was chosen to advance to clinical testing based upon preclinical immunogenicity studies conducted in rhesus macaques and mice, as well as preclinical safety studies of a vaccine product (VRC-HIVDNA006-00-VP) consisting of the same four plasmids and two additional Gag-Pol-Nef expressing plasmids. Based on biological safety testing of these plasmid products, and the high degree of homology between the candidate vaccines VRC-HIVDNA009-00-VP (BB-IND 10681) and VRC-HIVDNA016-00-VP, it was determined that the six plasmid vaccine was safe for human clinical trials.

Therapeutic Methods

[0098]The nucleic acid constructs encoding HIV antigenic polypeptides described herein are used, for example, in combination, as pharmaceutical compositions (medicaments) for use in therapeutic, for example, prophylactic regimens (e.g., vaccines) and administered to subjects (e.g., human subjects) to elicit an immune response against one or more clade or strain of HIV. For example, the compositions described herein can be administered to a human (or non-human) subject prior to infection with HIV to inhibit infection by or replication of the virus. Thus, the pharmaceutical compositions described above can be administered to a subject to elicit a protective immune response against HIV. To elicit an immune response, a therapeutically effective (e.g., immunologically effective) amount of the nucleic acid constructs are administered to a subject, such as a human (or non-human) subject.

[0099]A "therapeutically effective amount" is a quantity of a chemical composition (such as a nucleic acid construct or vector) used to achieve a desired effect in a subject being treated. For instance, this can be the amount necessary to express an adequate amount of antigen to elicit an antibody or T cell response, or to inhibit or prevent infection by or replication of the virus, or to prevent, lessen or ameliorate symptoms caused by infection with the virus. When administered to a subject, a dosage will generally be used that will achieve target tissue or systemic concentrations that are empirically determined to achieve an in vitro effect. Such dosages can be determined without undue experimentation by those of ordinary skill in the art. Exemplary dosages are described in detail in the Examples.

[0100]A pharmaceutical composition including an HIV encoding nucleic acid construct can be administered by any means known to one of skill in the art (see Banga, A., "Parenteral Controlled Delivery of Therapeutic Peptides and Proteins," in Therapeutic Peptides and Proteins, Technomic Publishing Co., Inc., Lancaster, Pa., 1995; DNA Vaccines: Methods and Protocols (Methods in Molecular Medicine) by Douglas B. Lowrie and Robert G. Whalen (Eds.), Humana Press, 2000) such as by intramuscular, subcutaneous, or intravenous injection, but even oral, nasal, or anal administration is contemplated. In one embodiment, administration is by subcutaneous or intramuscular injection. Actual methods for preparing administrable compositions will be known or apparent to those skilled in the art and are described in more detail in such publications as Remingtons Pharmaceutical Sciences, 19^th Ed., Mack Publishing Company, Easton, Pa., 1995.

[0101]Suitable formulations for the nucleic acid constructs, for example, the primer or booster compositions disclosed herein, include aqueous and non-aqueous solutions, isotonic sterile solutions, which can contain anti-oxidants, buffers, and bacteriostats, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water, immediately prior to use. Extemporaneous solutions and suspensions can be prepared from sterile powders, granules, and tablets. Preferably, the carrier is a buffered saline solution. More preferably, the composition for use in the inventive method is formulated to protect the nucleic acid constructs from damage prior to administration. For example, the composition can be formulated to reduce loss of the adenoviral vectors on devices used to prepare, store, or administer the expression vector, such as glassware, syringes, or needles. The compositions can be formulated to decrease the light sensitivity and/or temperature sensitivity of the components. To this end, the composition preferably comprises a pharmaceutically acceptable liquid carrier, such as, for example, those described above, and a stabilizing agent selected from the group consisting of polysorbate 80, L-arginine, polyvinylpyrrolidone, trehalose, and combinations thereof. Use of such an adenoviral vector composition will extend the shelf life of the vector, facilitate administration, and increase the efficiency of the inventive method. Formulations for adenoviral vector-containing compositions are further described in, for example, U.S. Pat. Nos. 6,225,289, 6,514,943, U.S. Patent Application Publication No. 2003/0153065 A1, and International Patent Application Publication WO 00/34444. An adenoviral vector composition also can be formulated to enhance transduction efficiency. In addition, one of ordinary skill in the art will appreciate that the composition can comprise other therapeutic or biologically-active agents. For example, factors that control inflammation, such as ibuprofen or steroids, can be part of the adenoviral vector composition to reduce swelling and inflammation associated with in vivo administration of the adenoviral vectors. As discussed herein, immune system stimulators can be administered to enhance any immune response to the antigens. Antibiotics, i.e., microbicides and fungicides, can be present to treat existing infection and/or reduce the risk of future infection, such as infection associated with gene transfer procedures.

[0102]The compositions can be administered for therapeutic treatments. In therapeutic applications, a therapeutically effective amount of the composition is administered to a subject prior to or following exposure to or infection by HIV. When administered prior to exposure, the therapeutic application can be referred to as a prophylactic administration (e.g., a vaccine). Single or multiple administrations of the compositions are administered depending on the dosage and frequency as required and tolerated by the subject. In one embodiment, the dosage is administered once as a bolus, but in another embodiment can be applied periodically until a therapeutic result, such as a protective immune response, is achieved. Generally, the dose is sufficient to treat or ameliorate symptoms or signs of disease without producing unacceptable toxicity to the subject. Systemic or local administration can be utilized.

[0103]Controlled release parenteral formulations can be made as implants, oily injections, or as particulate systems. Particulate systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Particles, microspheres, and microcapsules smaller than about 1 μm are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively. Capillaries have a diameter of approximately 5 μm so that only nanoparticles are administered intravenously. Microparticles are typically around 100 μm in diameter and are administered subcutaneously or intramuscularly (see, Kreuter, Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, N.Y., pp. 219-342, 1994; Tice & Tabibi, Treatise on Controlled Drug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, N.Y., pp. 315-339, 1992).

[0104]In certain embodiments, the pharmaceutical composition includes an adjuvant. An adjuvant can be a suspension of minerals, such as alum, aluminum hydroxide, aluminum phosphate, on which antigen is adsorbed; or water-in-oil emulsion in which antigen solution is emulsified in oil (MF-59, Freund's incomplete adjuvant), sometimes with the inclusion of killed mycobacteria (Freund's complete adjuvant) to further enhance antigenicity (inhibits degradation of antigen and/or causes influx of macrophages). In the context of nucleic acid vaccines, naturally occurring or synthetic immunostimulatory compositions that bind to and stimulate receptors involved in innate immunity can be administered along with nucleic acid constructs encoding the HIV antigenic polypeptides. For example, agents that stimulate certain Toll-like receptors (such as TLR7, TLR8 and TLR9) can be administered in combination with the nucleic acid constructs encoding HIV antigenic polypeptides. In some embodiments, the nucleic acid construct is administered in combination with immunostimulatory CpG oligonucleotides.

[0105]Nucleic acid constructs encoding HIV antigenic polypeptides can be introduced in vivo as naked DNA plasmids. DNA vectors can be introduced into the desired host cells by methods known in the art, including but not limited to transfection, electroporation (e.g., transcutaneous electroporation), microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun, or use of a DNA vector transporter (See e.g., Wu et al. J. Biol. Chem., 267:963-967, 1992; Wu and Wu J. Biol. Chem., 263:14621-14624, 1988; and Williams et al. Proc. Natl. Acad. Sci. USA 88:2726-2730, 1991). As described in detail in the Examples, a needleless delivery device, such as a BIOJECTOR® needleless injection device can be utilized to introduce the therapeutic nucleic acid constructs in vivo. Receptor-mediated DNA delivery approaches can also be used (Curiel et al. Hum. Gene Ther., 3:147-154, 1992; and Wu and Wu, J. Biol. Chem., 262:4429-4432, 1987). Methods for formulating and administering naked DNA to mammalian muscle tissue are disclosed in U.S. Pat. Nos. 5,580,859 and 5,589,466, both of which are herein incorporated by reference. Other molecules are also useful for facilitating transfection of a nucleic acid in vivo, such as a cationic oligopeptide (e.g., WO95/21931), peptides derived from DNA binding proteins (e.g., WO96/25508), or a cationic polymer (e.g., WO95/21931).

[0106]Alternatively, electroporation can be utilized conveniently to introduce nucleic acid constructs encoding HIV antigens into cells. Electroporation is well known by those of ordinary skill in the art (see, for example: Lohr et al. Cancer Res. 61:3281-3284, 2001; Nakano et al. Hum Gene Ther. 12:1289-1297, 2001; Kim et al. Gene Ther. 10:1216-1224, 2003; Dean et al. Gene Ther. 10:1608-1615, 2003; and Young et al. Gene Ther 10:1465-1470, 2003). For example, in electroporation, a high concentration of vector DNA is added to a suspension of host cell (such as isolated autologous peripheral blood or bone marrow cells) and the mixture shocked with an electrical field. Transcutaneous electroporation can be utilized in animals and humans to introduce heterologous nucleic acids into cells of solid tissues (such as muscle) in vivo. Typically, the nucleic acid constructs are introduced into tissues in vivo by introducing a solution containing the DNA into a target tissue, for example, using a needle or trochar in conjunction with electrodes for delivering one or more electrical pulses. For example, a series of electrical pulses can be utilized to optimize transfection, for example, between 3 and ten pulses of 100V and 50 msec. In some cases, multiple sessions or administrations are performed.

[0107]Another well known method that can be used to introduce nucleic acid constructs encoding HIV antigens into host cells is particle bombardment (also know as biolistic transformation). Biolistic transformation is commonly accomplished in one of several ways. One common method involves propelling inert or biologically active particles at cells. This technique is disclosed in, e.g., U.S. Pat. Nos. 4,945,050, 5,036,006; and 5,100,792, all to Sanford et al., which are hereby incorporated by reference. Generally, this procedure involves propelling inert or biologically active particles at the cells under conditions effective to penetrate the outer surface of the cell and to be incorporated within the interior thereof. When inert particles are utilized, the plasmid can be introduced into the cell by coating the particles with the plasmid containing the exogenous DNA. Alternatively, the target cell can be surrounded by the plasmid so that the plasmid is carried into the cell by the wake of the particle.

[0108]Alternatively, the vector can be introduced in vivo by lipofection. For the past decade, there has been increasing use of liposomes for encapsulation and transfection of nucleic acids in vitro. Synthetic cationic lipids designed to limit the difficulties and dangers encountered with liposome mediated transfection can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Felgner et. al. Proc. Natl. Acad. Sci. USA 84:7413-7417, 1987; Mackey, et al. Proc. Natl. Acad. Sci. USA 85:8027-8031, 1988; Ulmer et al. Science 259:1745-1748, 1993). The use of cationic lipids can promote encapsulation of negatively charged nucleic acids, and also promote fusion with negatively charged cell membranes (Felgner and Ringold Science 337:387-388, 1989). Particularly useful lipid compounds and compositions for transfer of nucleic acids are described in WO95/18863 and WO96/17823, and in U.S. Pat. No. 5,459,127, herein incorporated by reference.

[0109]In other embodiments, the nucleic acid constructs are viral vectors. Methods for constructing and using viral vectors are known in the art (See e.g., Miller and Rosman, BioTech., 7:980-990, 1992). Preferably, the viral vectors are replication defective, that is, they are unable to replicate autonomously in the target cell. In general, the genome of the replication defective viral vectors that are used within the scope of the present disclosure lack at least one region that is necessary for the replication of the virus in the infected cell. These regions can either be eliminated (in whole or in part), or be rendered non-functional by any technique known to a person skilled in the art. These techniques include the total removal, substitution (by other sequences, in particular by the inserted nucleic acid), partial deletion or addition of one or more bases to an essential (for replication) region. Such techniques can be performed in vitro (for example, on the isolated DNA).

[0110]In some cases, the replication defective virus retains the sequences of its genome that are necessary for encapsidating the viral particles. DNA viral vectors commonly include attenuated or defective DNA viruses, including, but not limited to, herpes simplex virus (HSV), papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV), Moloney leukemia virus (MLV) and human immunodeficiency virus (HIV) and the like. Defective viruses, that entirely or almost entirely lack viral genes, are preferred, as defective virus is not infective after introduction into a cell. Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells. Thus, a specific tissue can be specifically targeted. Examples of particular vectors include, but are not limited to, a defective herpes virus 1 (HSV1) vector (Kaplitt et al. Mol. Cell. Neurosci., 2:320-330, 1991), defective herpes virus vector lacking a glycoprotein L gene (See for example, Patent Publication RD 371005 A), or other defective herpes virus vectors (See e.g., WO 94/21807; and WO 92/05263); an attenuated adenovirus vector, such as the vector described by Stratford-Perricaudet et al. (J. Clin. Invest., 90:626-630 1992; La Salle et al., Science 259:988-990, 1993); and a defective adeno-associated virus vector (Samulski et al., J. Virol., 61:3096-3101, 1987; Samulski et al., J. Virol., 63:3822-3828, 1989; and Lebkowski et al., Mol. Cell. Biol., 8:3988-3996, 1988).

[0111]In one embodiment, the vector is an adenovirus vector. Adenoviruses are eukaryotic DNA viruses that can be modified to efficiently deliver a nucleic acid of the disclosure to a variety of cell types. Various serotypes of adenovirus exist. Of these serotypes, preference is given, within the scope of the present disclosure, to type 2 or type 5 human adenoviruses (Ad 2 or Ad 5), or adenoviruses of animal origin (See e.g., WO94/26914). Those adenoviruses of animal origin that can be used within the scope of the present disclosure include adenoviruses of canine, bovine, murine (e.g., Mav1, Beard et al. Virol., 75-81, 1990), ovine, porcine, avian, and simian (e.g., SAV) origin. In some embodiments, the adenovirus of animal origin is a canine adenovirus, such as a CAV2 adenovirus (e.g. Manhattan or A26/61 strain (ATCC VR-800)).

[0112]The replication defective adenoviral vectors described herein include the ITRs, an encapsidation sequence and the polynucleotide sequence of interest. In some embodiments, at least the E1 region of the adenoviral vector is non-functional. The deletion in the E1 region preferably extends from nucleotides 455 to 3329 in the sequence of the Ad5 adenovirus (PvuII-BglII fragment) or 382 to 3446 (HinfII-Sau3A fragment). Other regions can also be modified, in particular the E3 region (e.g., WO95/02697), the E2 region (e.g., WO94/28938), the E4 region (e.g., WO94/28152, WO94/12649 and WO95/02697), or in any of the late genes L1-L5.

[0113]In other embodiments, the adenoviral vector has a deletion in the E1 region (Ad 1.0). Examples of E1-deleted adenoviruses are disclosed in EP 185,573, the contents of which are incorporated herein by reference. In another embodiment, the adenoviral vector has a deletion in the E1 and E4 regions (Ad 3.0). Examples of E1/E4-deleted adenoviruses are disclosed in WO95/02697 and WO96/22378.

[0114]The replication defective recombinant adenoviruses according to this disclosure can be prepared by any technique known to the person skilled in the art (See e.g., Levrero et al. Gene 101:195, 1991; EP 185 573; and Graham EMBO J., 3:2917, 1984). In particular, they can be prepared by homologous recombination between an adenovirus and a plasmid, which includes, inter alia, the DNA sequence of interest. The homologous recombination is accomplished following co-transfection of the adenovirus and plasmid into an appropriate cell line. The cell line that is employed should preferably (i) be transformable by the elements to be used, and (ii) contain the sequences that are able to complement the part of the genome of the replication defective adenovirus, preferably in integrated form in order to avoid the risks of recombination. Examples of cell lines that can be used are the human embryonic kidney cell line 293 (Graham et al. J. Gen. Virol. 36:59, 1977), which contains the left-hand portion of the genome of an Ad5 adenovirus (12%) integrated into its genome, and cell lines that are able to complement the E1 and E4 functions, as described in applications WO94/26914 and WO95/02697. Recombinant adenoviruses are recovered and purified using standard molecular biological techniques that are well known to one of ordinary skill in the art. Nucleic acids encoding HIV antigens can also be introduced using other viral vectors, such as retroviral vectors, for example, lentivirus vectors or adenovirus-associated viral (AAV) vectors.

[0115]As described in detail in the Examples, in one embodiment, a pharmaceutical composition including nucleic acid constructs encoding HIV antigens that correspond to antigenic polypeptides of multiple clades or strains of HIV are introduced into a subject prior to exposure to HIV to elicit a protective immune response. Typically, the nucleic acid constructs are plasmids. For example, several plasmids including polynucleotide sequences that encode different HIV antigens can be included in a pharmaceutical composition. For example, a set of plasmids that encodes antigenic polypeptides of different HIV clades or strains can be included in the composition to elicit immunity that protects against infection by HIV of multiple clades or strains. In an exemplary embodiment, the composition includes six plasmids. Each of the plasmids includes a polynucleotide sequence encoding a different HIV antigen operably linked to a transcription regulatory sequence that promotes expression of the antigenic polypeptide in vivo. For example, the composition can include different plasmids that encode a Gag polypeptide, a Pol polypeptide, a Nef polypeptide, and optionally, Env polypeptides of different clades or strains (for example, a clade A Env polypeptide, a clade B Env polypeptide and/or a clade C polypeptide. In one specific embodiment, the vaccine composition includes the six plasmids (VRC 4409, VRC 4401, VRC-4404, VRC 5736, VRC 5737 and VRC 5738 represented by SEQ ID NOs:1-6, respectively. This particular embodiment is designated VRC-HIVDNA016-00-VP, and is described in further detail in the Examples.

[0116]Typically, the multi-plasmid composition includes the six plasmids in substantially equal ratio (e.g., approximately 1:1:1:1:1:1). The pharmaceutical composition can be administered to a subject in a single or multiple doses. The dose range can be varied according to the physical, metabolic and immunological characteristics of the subject, however, a dose of at least about 1 mg and no more than about 12 mg is typically administered. For example, a single dose can be at least about 2 mg, or at least about 3 mg, or at least about 4 mg of combined DNA. Typically, a single dose does not exceed about 6 mg, or about 8 mg or about 10 mg of combined DNA. As described in the Examples, a dose of about 4 mg combined plasmid weight is typically effective to elicit a protective immune response in an immunocompetent adult.

[0117]A single dose, or multiple doses separated by a time interval can be administered to elicit an immune response against HIV. For example, two doses, or three doses, or four doses, or five doses, or six doses or more can be administered to a subject over a period of several weeks, several months or even several years, to optimize the immune response.

[0118]In some cases the pharmaceutical composition including the nucleic acid constructs, for example the multi-plasmid vaccine VRC-HIVDNA016-00-VP is included in combination modality regimens using it as a DNA vaccine prime followed by an adenoviral vector boost. Prime-boost regimens have shown promise in non-human primate models of HIV infection. Such regimens have the potential for raising high levels of immune responses. For example, a "primer" composition including one or more nucleic acid constructs that encode at least one HIV antigen that is the same as an HIV antigen encoded by an adenoviral vector of an adenoviral vector composition can be administered to a subject. For example, the primer composition can be administered at least about one week before the administration of the "booster" composition including one or more adenoviral vectors. The one or more nucleic acid sequences of the primer composition (such as VRC-HIVDNA016-00-VP) can be administered as part of a gene transfer vector or as naked DNA. Any gene transfer vector can be employed in the primer composition, including, but not limited to, a plasmid, a retrovirus, an adeno-associated virus, a vaccine virus, a herpesvirus, or an adenovirus. In an exemplary embodiment, the transfer vector is a plasmid.

[0119]Thus, the multi-plasmid composition described above can be used to prime an immune response against HIV, in combination with administration of a composition including one or more adenovirus vectors encoding HIV antigens. For example, the adenoviral vector composition can include (i) a single adenoviral vector that encodes two or more HIV antigens, for example, as a polyprotein or fusion protein, such as a fusion protein encoding a Gag-Pol-Nef polypepetide. Alternatively, the adenoviral vector composition can include (ii) multiple adenoviral vectors each of which encodes a single HIV antigen, such as, two or more, such as three, or four, or more, adenovirus vectors that each encode one HIV antigen, such as an Env polypeptide. Consistent with configuration (i), it is within the scope of the invention to use an adenoviral vector comprising a nucleic acid sequence that encodes more than two different HIV antigens (e.g., three or more, four or more, or even five or more different HIV antigens) or encodes multiple copies of the same antigen, provided that it encodes at least two or more different HIV antigens. Likewise, consistent with configuration (ii), it is within the scope of the invention to use an adenoviral vector comprising several nucleic acid sequences (e.g., three or more, four or more, or even five or more different nucleic acid sequences) each encoding different HIV antigens or multiple copies of the same antigen, provided that the adenoviral vector encodes at least two different HIV antigens. Whether by configuration (i) or (ii), the adenoviral vector composition preferably comprises one or more adenoviral vectors encoding three or more, or even four or more, different HIV antigens (e.g., wherein each vector comprises a nucleic acid sequence that encodes three or more, or four or more different HIV antigens, or wherein each vector comprises three or more, or four or more nucleic acid sequences, and each nucleic acid sequence encodes a different HIV antigen). In certain embodiments, the two or more, three or more, or four or more different HIV antigens are from two or more, three or more, or four or more different HIV clades. There is no upper limit to the number of adenoviral vectors used or the number of different HIV antigens encoded thereby.

[0120]Of course, a combination of the above configurations of adenoviral vectors can be used in a single composition. For example, the adenoviral vector composition used in accordance with the invention can comprise a first adenoviral vector encoding a single HIV antigen and a second adenoviral vector encoding two or more HIV antigens that are different from the HIV antigen encoded by the first adenoviral vector. Other similar combinations and permutations of the adenoviral vector configurations disclosed herein can be readily determined by one of skill in the art.

[0121]In certain embodiments, the booster composition includes multiple adenoviral vectors. For example, the booster can include multiple adenoviral vectors each of which encodes an HIV Env polypeptide, such as Env polypeptide of different clades or strains. In addition, the booster composition can include an adenoviral vector that encodes Gag, Pol and/or Nef polypeptides. In one specific embodiment, designated VRC-HIVDNA014-00VP, the booster composition includes four adenoviral vectors, three of which encode Env polypeptides of different clades (that is, clade A, clade B and clade C), and an adenoviral vector that encodes Gag and Pol antigens (of clade B). Of course, numerous variants can easily be designed by one of skill in the art, incorporating fewer or more adenoviral vectors, and/or encoding antigens of the same or different HIV clades or strains.

[0122]While the HIV antigen encoded by the one or more nucleic acid sequences of the boost composition often is the same as an HIV antigen encoded by the nucleic acid constructs of the primer composition, in some embodiments it may be appropriate to use a primer composition comprising one or more nucleic acid sequences encoding an HIV antigen that is different from the antigen(s) encoded by the adenoviral vector composition. For example, Gag and/or Pol and/or Nef antigens of a different clade or strain, or Env antigens of a different clade or strain.

[0123]The primer composition is administered to the mammal to prime the immune response to HIV. More than one dose of primer composition can be provided in any suitable timeframe (e.g., at least about 1 week, 2 weeks, 4 weeks, 8 weeks, 12 weeks, 16 weeks, or more prior to boosting). Preferably, the primer composition is administered to the mammal at least three months (e.g., three, six, nine, twelve, or more months) before administration of the booster composition. Most preferably, the primer composition is administered to the mammal at least about six months to about nine months before administration of the booster composition. More than one dose of booster composition can be provided in any suitable timeframe to maintain immunity.

[0124]Any route of administration can be used to deliver the adenoviral vector composition and/or the primer composition to the mammal. Indeed, although more than one route can be used to administer the adenoviral vector composition and/or the primer composition, a particular route can provide a more immediate and more effective reaction than another route. Most commonly, the adenoviral vector composition and/or the primer composition is administered via intramuscular injection. The adenoviral vector composition and/or the primer composition also can be applied or instilled into body cavities, absorbed through the skin (for example, via a transdermal patch), inhaled, ingested, topically applied to tissue, or administered parenterally via, for instance, intravenous, peritoneal, or intraarterial administration.

[0125]The adenoviral primer composition and/or the booster composition can be administered in or on a device that allows controlled or sustained release, such as a sponge, biocompatible meshwork, mechanical reservoir, or mechanical implant. Implants (see, e.g., U.S. Pat. No. 5,443,505), devices (see, e.g., U.S. Pat. No. 4,863,457), such as an implantable device, e.g., a mechanical reservoir or an implant or a device comprised of a polymeric composition, are particularly useful for administration of the composition. The adenoviral vector composition and/or the primer composition also can be administered in the form of sustained-release formulations (see, e.g., U.S. Pat. No. 5,378,475) comprising, for example, gel foam, hyaluronic acid, gelatin, chondroitin sulfate, a polyphosphoester, such as bis-2-hydroxyethyl-terephthalate (BHET), and/or a polylactic-glycolic acid.

[0126]A booster composition can include a single dose of adenoviral vector comprising at least about 1×10⁵ particles (which also is referred to as particle units) of adenoviral vector. The dose preferably is at least about 1×10⁶ particles (for example, about 1×10⁶-1×10¹² particles), more preferably at least about 1×10⁹ particles, more preferably at least about 1×10⁸ particles (e.g., about 1×10⁸-1×10¹¹ particles or about 1×10⁸-1×10¹² particles), and most preferably at least about 1×10⁹ particles (e.g., about 1×10⁹-1×10¹⁰ particles or about 1×10⁹-1×10¹² particles), or even at least about 1×10¹⁰ particles (e.g., about 1×10¹⁰-1×10¹² particles) of the adenoviral vector. Alternatively, the dose comprises no more than about 1×10¹⁴ particles, preferably no more than about 1×10¹³ particles, even more preferably no more than about 1×10¹² particles, even more preferably no more than about 1×10¹¹ particles, and most preferably no more than about 1×10¹⁰ particles (e.g., no more than about 1×10⁹ particles). In other words, the adenoviral vector composition can comprise a single dose of adenoviral vector comprising, for example, about 1×10⁶ particle units (pu), 2×10⁶ pu, 4×10⁶ pu, 1×10⁷ pu, 2×10⁷ pu, 4×10⁷ pu, 1×10⁸ pu, 2×10⁸ pu, 4×10⁸ pu, 1×10⁹ pu, 2×10⁹ pu, 4×10⁹ pu, 1×10¹⁰ pu, 2×10¹⁰ pu, 4×10¹⁰ pu, 1×10¹¹ pu, 2×10¹¹ pu, 4×10¹¹ pu, 1×10¹² pu, 2×10¹² pu, or 4×10¹² pu of adenoviral vector.

EXAMPLES

Example 1

Construction of Plasmids

[0127]The nucleic acid constructs were derived from parental 1012 DNA vaccine plasmid containing the human CMV immediate early (IE) enhancer, promoter, and intron. To construct the CMV/R regulatory element, a SacII/HpaI fragment of the 1012 plasmid containing the majority of the CMV IE intron was replaced with a 227 bp EcoRV/HpaI fragment of the HTLV-1 R region (Seiki et al., Proc. Natl. Acad. Sci. USA 80: 3618-3622, 1983). The resulting CMV/R plasmid thus contains the human CMV IE enhancer/promoter, followed by the HTLV-1 R region and a 123 bp fragment of CMV IE 3' intron. The splice donor in the R region and the splice acceptor in the CMV IE 3' intron serve as the pair of splicing signals. RSV/R and mUB/R plasmids were similarly constructed by replacing the CMV enhancer/promoter region of the CMV/R plasmid with a 381 bp AflIII/HindIII fragment of the Rous sarcoma virus (RSV) enhancer/promoter or an 842 bp SpeI/EcoRV fragment of the mouse ubiquitin B(mUB) enhancer/promoter respectively. The mUB enhancer/promoter has been described previously (Yew et al., Mol. Ther. 4:75-82, 2001).

Construction of CMV/R Clade B Gag/h (VRC-4401)

[0128]To construct DNA plasmid VRC-4401, diagrammed in FIG. 1, the protein sequence of the gag polyprotein (Pr55, amino acids 1-432) from HXB2 (GENBANK® accession number K03455) was used to create a synthetic version of the gag gene using codons optimized for expression in human cells. The nucleotide sequence of the synthetic gag gene shows little homology to the HXB2 gene, but the protein encoded is the same. A SalI/BamHI fragment including the synthetic gene encoding Gag (B) was excised from plasmid VRC 3900, which contained the same insert in a pVR1012 backbone, and cloned into the SalI/BamHI sites of the CMV/R backbone described above. A summary of predicted VRC-4401 domains is provided in Table 2. The plasmid is 5886 nucleotide base pairs (bp) in length and has an approximate molecular weight of 3.9 MDa. The sequence of VRC-4401 is provided in SEQ ID NO:1.

TABLE-US-00002 TABLE 2 Description of plasmid VRC-4401 Fragment Size Predicted Fragment Name or Protein Domain (bp) Fragment pUC18 plasmid-derived 247 1-247 CMV-IE Enhancer/Promoter 742 248-989 HTLV-1 R region 231 990-1220 CMV IE Splicing Acceptor 123 1221-1343 Synthetic Linker 31 1344-1374 HIV-1 Gag (Clade B) 1509 1375-2883 Synthetic Linker 23 2884-2906 Bovine Growth Hormone Poly A 548 2907-3454 pUC18 plasmid-derived 1311 3455-4765 Kanamycin Resistance Gene 816 4766-5581 pUC18 plasmid-derived 305 5582-5886

Construction of CMV/R Clade B Pol/h (VRC-4409)

[0129]To construct DNA plasmid VRC-4409 diagrammed in FIG. 2, the protein sequence of the Pol polyprotein (amino acids 3-1003) from NL4-3 (GENBANK® accession number M19921) was used to create a synthetic version of the pol gene using codons optimized for expression in human cells. To initiate translation at the beginning of Pol, a methionine codon was added to the 5'-end of the synthetic polymerase gene to create the Pol/h gene. Additionally, a Protease (PR) mutation was introduced at amino acid 553 (AGG->GGC or amino acids R->G), a Reverse Transcriptase (RT) mutation was introduced at amino acid 771 (GAC->CAC or amino acids D->H), and an Integrase (IN) mutation was introduced at amino acid 1209 (ACT->CAT or amino acids D->A). The gene expressing Pol was inserted into the CMV/R backbone described above. A summary of predicted VRC-4409 domains is provided in Table 3. The plasmid is 7344 nucleotide base pairs (bp) in length and has an approximate molecular weight of 4.8 MDa. The sequence of VRC-4409 is provided in SEQ ID NO:2.

TABLE-US-00003 TABLE 3 Description of Plasmid VRC-4409 Fragment Size Predicted Fragment Name or Protein Domain (bp) Fragment pUC18 plasmid-derived 247 1-247 CMV-IE Enhancer/Promoter 742 248-989 HTLV-1 R region 231 990-1220 CMV IE Splicing Acceptor 123 1221-1343 Synthetic Linker 5 1344-1348 HIV-1 Pol (Clade B) (Pr-, RT-, IN-) 3009 1349-4357 Synthetic Linker 7 4358-4364 Bovine Growth Hormone Poly A 548 4365-4912 pUC18 plasmid-derived 1311 4913-6223 Kanamycin Resistance Gene 816 6224-7039 pUC18 plasmid-derived 305 7040-7344

Construction of CMV/R HIV-1 Nef/h (VRC-4404)

[0130]To construct DNA plasmid VRC-4404, diagrammed in FIG. 3, the protein sequence of the Nef protein from HIV-1 NY5/BRU (LAV-1) clone pNL4-3 (GENBANK® accession number M19921) was used to create a synthetic version of the Nef gene (Nef/h) using codons optimized for expression in human cells. The nucleotide sequence Nef/h shows little homology to the viral gene, but the protein encoded is the same. The Myristol site (GGC-Gly, amino acid 2-3) was deleted. The fragment encoding Nef was digested from the pVR1012 backbone in which it was originally inserted, with XbaI/BamHI, and then cloned into the XbaI/BamHI site of the CMV/R backbone described above. A summary of predicted VRC-4404 domains is provided in Table 4. The plasmid is 5039 nucleotide base pairs (bp) in length and has an approximate molecular weight of 3.3 MDa. The sequence of VRC-4404 is provided in SEQ ID NO:3.

TABLE-US-00004 TABLE 4 Description of plasmid VRC-4404 Fragment Size Predicted Fragment Name or Protein Domain (bp) Fragment pUC18 plasmid-derived 247 1-247 CMV-IE Enhancer/Promoter 742 248-989 HTLV-1 R region 231 990-1220 CMV IE Splicing Acceptor 123 1221-1343 Synthetic Linker 48 1344-1391 HIV-1 Nef (Clade B) (Delta Myr) 615 1392-2006 Synthetic Linker 19 2007-2025 Bovine Growth Hormone Poly A 548 2026-2573 pUC18 plasmid-derived 1345 2574-3918 Kanamycin Resistance Gene 816 3919-4734 pUC18 plasmid-derived 305 4735-5039

CMV/R-HIV-1 Clade A Env/h (VRC-5736)

[0131]To construct DNA plasmid VRC-5736, diagrammed in FIG. 4, the protein sequence of the envelope polyprotein (gp160) from 92rw020 (R5-tropic, GENBANK® accession number U08794) was used to create a synthetic version of the gene (Clade-A gp145delCFI) using codons altered for expression in human cells. Plasmids expressing the HIV-1 genes were made synthetically with sequences designed to disrupt viral RNA structures that limit protein expression by using codons typically found in human cells. The nucleotide sequence R5gp145delCFI shows little homology to the 92rw020 gene, but the protein encoded is the same. The truncated envelope polyprotein contains the entire SU protein and the TM domain, but lacks the fusion domain and cytoplasmic domain. Heptad (H) 1, Heptad 2 and their Interspace (IS) are involved in oligomerization. The Fusion and Cleavage (F/CL) domains, from amino acids 486-519, have been deleted. The Interspace (IS) between Heptad (H) 1 and 2, from amino acids 576-604, has been deleted. The XbaI (18 nt up-stream from ATG) to BamH1 (1912 nt down-stream from ATG) fragment, which contains a polylinker at the 5' end, a Kozak sequence and ATG, was cloned into the XbaI to BamH1 sites of the CMV/R backbone described above. EnvA summary of predicted VRC-5736 domains is provided in Table 5. The plasmid is 6305 nucleotide base pairs (bp) in length and has an approximate molecular weight of 4.2 MDa. The sequence of VRC-5736 is provided in SEQ ID NO:4.

TABLE-US-00005 TABLE 5 Description of plasmid VRC-5736 Fragment Size Predicted Fragment Name or Protein Domain (bp) Fragment pUC18 plasmid-derived 247 1-247 CMV-IE Enhancer/Promoter 742 248-989 HTLV-1 R region 231 990-1220 CMV IE Splicing Acceptor 123 1221-1343 Synthetic Linker 48 1344-1391 HIV-1 Env (Clade A), gp145 (delCFI)/h 1881 1392-3272 Synthetic Linker 19 3273-3291 Bovine Growth Hormone Poly A 548 3292-3839 pUC18 plasmid-derived 1345 3840-5184 Kanamycin Resistance Gene 816 5185-6000 pUC18 plasmid-derived 305 6001-6305

Construction of CMV/R Clade B Env/h (VRC-5737)

[0132]To construct DNA plasmid VRC-5737 diagrammed in FIG. 5, the protein sequence of the envelope polyprotein (gp160) from HXB2 (X4-tropic, GENBANK® accession number K03455) was used to create a synthetic version of the gene (X4gp160/h) using codons optimized for expression in human cells. The nucleotide sequence X4gp160/h shows little homology to the HXB2 gene, but the protein encoded is the same with the following amino acid substitutions: F53L, N94D, K192S, I215N, A224T, A346D, and P470L. To produce an R5-tropic version of the envelope protein (R5gp160/h), the region encoding HIV-1 envelope polyprotein amino acids 275 to 361 from X4gp160/h (VRC3300) were replaced with the corresponding region from the BaL strain of HIV-1 (GENBANK® accession number M68893, again using human preferred codons). The full-length R5-tropic version of the envelope protein gene from pR5gp160/h (VRC3000) was terminated after the codon for amino acid 704. The truncated envelope polyprotein (gp145) contains the entire SU protein and a portion of the TM protein including the fusion domain, the transmembrane domain, and regions important for oligomer formation. Heptad(H) 1, Heptad 2 and their Interspace(IS) are involved in oligomerization. The Fusion and Cleavage (F/CL) domains, from amino acids 503-536, have been deleted. The Interspace (IS) between Heptad (H) 1 and 2, from amino acids 593-620, has been deleted. The expression vector backbone is CMV/R, described above. A summary of predicted VRC-5737 domains is provided in Table 6. The plasmid is 6338 nucleotide base pairs (bp) in length and has an approximate molecular weight of 4.2 MDa. The sequence of VRC-5737 is provided in SEQ ID NO:5.

TABLE-US-00006 TABLE 6 Description of plasmid VRC-5737 Fragment Size Predicted Fragment Name or Protein Domain (bp) Fragment pUC18 plasmid-derived 247 1-247 CMV-IE Enhancer/Promoter 742 248-989 HTLV-1 R region 231 990-1220 CMV IE Splicing Acceptor 123 1221-1343 Synthetic Linker 40 1344-1383 HIV-1 Env (Clade B), gp145 (delCFI)/h 1929 1384-3312 Synthetic Linker 12 3313-3324 Bovine Growth Hormone Poly A 548 3325-3872 pUC18 plasmid-derived 1345 3873-5217 Kanamycin Resistance Gene 816 5218-6033 pUC18 plasmid-derived 305 6034-6338

Construction of CMV/R HIV-1 Clade C Env/h (VRC-5738)

[0133]To construct DNA plasmid VRC-5738, diagrammed in FIG. 6, the protein sequence of the envelope polyprotein (gp145delCFI) from 97ZA012 (R5-tropic, GENBANK® accession number AF286227) was used to create a synthetic version of the gene (Clade-C gp145delCFI) using codons optimized for expression in human cells. The nucleotide sequence R5gp145delCFI shows little homology to the gene 97ZA012, but the protein encoded is the same. The truncated envelope polyprotein contains the entire SU protein and the TM domain, but lacks the fusion domain and cytoplasmic domain. Heptad(H) 1, Heptad 2 and their Interspace (IS) are involved in oligomerization. The Fusion and Cleavage (F/CL) domains, from amino acids 487-520, have been deleted. The Interspace (IS) between Heptad (H) 1 and 2, from amino acids 577-605, has been deleted. The XbaI (18 nt up-stream from ATG) to BamH1 (1914 nt down-stream from ATG) fragment, which contains polylinker at the 5' end, Kozak sequence and ATG, was cloned into the XbaI to BamH1 sites of the CMV/R backbone. A summary of predicted VRC-5738 domains is provided in Table 7. The plasmid is 6298 nucleotide base pairs (bp) in length and has an approximate molecular weight of 4.2 MDa. The sequence of VRC-5738 is provided in SEQ ID NO:6.

TABLE-US-00007 TABLE 7 Description of plasmid VRC-5738 Fragment Size Predicted Fragment Name or Protein Domain (bp) Fragment pUC18 plasmid-derived 247 1-247 CMV-IE Enhancer/Promoter 742 248-989 HTLV-1 R region 231 990-1220 CMV IE Splicing Acceptor 123 1221-1343 Synthetic Linker 48 1344-1391 HIV-1 Env (Clade C), gp145 (delCFI)/h 1881 1392-3272 Synthetic Linker 12 3273-3284 Bovine Growth Hormone Poly A 548 3285-3832 pUC18 plasmid-derived 1345 3833-5177 Kanamycin Resistance Gene 816 5178-5993 pUC18 plasmid-derived 305 5994-6298

Example 2

Increased Expression of HIV Antigenic Polypeptides by CMV/R Transcription Regulatory Sequence

[0134]To assess antigen expression from plasmids containing the CMV/R transcriptional regulatory elements, 3T3 cells were transfected with the above described expression vectors and gp145ΔCFI expression was measured by Western blots. Murine fibroblast 3T3 cells were transfected with 0.5 μg parental 1012 (CMV), CMV/R, RSV, RSV/R, mUB, and mUB/R DNA vaccines expressing HIV-1 Env gp145 ΔCFI (9) in 6-well plates using calcium phosphate. 24 h after transfection, cells were harvested and lysed in 50 mM HEPES, 150 mM NaCl, 1% NP-40 with protease inhibitors. 10 μg total protein was electrophoresed by SDS-PAGE, and gp145 expression was assessed by Western blot analysis. A 1:5000 dilution of human HIV-IgG was utilized as the primary antibody, and a 1:5000 dilution of HRP-conjugated goat anti-human IgG was utilized as the secondary antibody. The blots were developed with the ECL Western blot developing system (Amersham Biosciences, Piscataway, N.J.).

[0135]The expression of gp145ΔCFI from the CMV/R plasmid was 5- to 10-fold higher than expression from the parental 1012 plasmid (FIG. 8). Thus, addition of the HTLV-1 R element substantially increased antigen expression driven by the CMV promoter. Baseline expression from the mUB plasmid was higher than from the 1012 plasmid but was not further enhanced by addition of the R element (FIG. 8), demonstrating that the effects of adding the R element were promoter-dependent. An increase in expression was observed in the RSV/R compared to RSV plasmid (FIG. 8). Expression from RSV plasmids is routinely lower than from the 1012 plasmid.

Example 3

Immunogenicity of CMV/R Multiclade HIV Vaccine

[0136]Non-clinical immunogenicity studies were conducted with plasmid constructs comprising the DNA plasmid vaccine VRC-HIVDNA016-00-VP as well as with DNA plasmid prime/adenoviral vector boost regimens using the recombinant adenoviral vector vaccine VRC-HIVADV014-00-VP in mice and non-human primates. Cellular immune responses were tested in these non-clinical immunogenicity studies by the interferon gamma (IFN-γ) ELISPOT assay which quantitatively measures the production of IFN-γ by peripheral blood mononuclear cells (PBMC) from immunized animals. The cells are exposed in vitro to HIV-1 antigens (a series of short, overlapping peptides that span the length of the protein expressed in the vaccine). The IFN-γ produced by antigen sensitized T-lymphocytes are bound to antibody coating an assay plate and may be counted colorimetrically as spot forming cells (SFC) by using an alkaline phosphatase conjugated read-out system. The results are expressed as SFCs per million PBMC.

[0137]DNA plasmid prime regimens are performed using plasmids expressing HIV-1 genes, identical in composition to clinical grade vaccine VRC-HIVDNA009-00-VP (4 plasmid vaccine, PCT Publication No. WO/05034992) or VRC-HIVDNA016-00-VP. The recombinant adenoviral vector vaccines used in preclinical immunology studies consisted of GMP grade VRC-HIVADV014-00-VP (Lot#026-03017, PCT Application No. PCT/US2005/12291, filed Apr. 12, 2005), composed of four adenoviral vectors that encode clade B gag/pol and clade A, B and C Env, supplied by GenVec, Inc. Gaithersburg, Md.). Table 8 provides a summary of the plasmids.

[0138]A tabulated summary of the immunology studies performed in mice and in non-human primates are summarized in Table 9.

TABLE-US-00008 TABLE 8 Summary of preclinical and clinical studies of VRC DNA vaccines Safety Clinical Plasmid Gag Pol Nef Env (A) Env (B) Env (C) Testing Trial VRC-4302 p10112w/ Gag-Pol (B) Not Not Not + + (1-plasmid) CMV Nef not included included included included promoter VRC- p1012w/ Gag-Pol-Nef (A) 5305 2805 5309 + N/A HIVDNA00 CMV (4413) 6-00-VP promoter Gag-Pol-Nef (B) (6-plasmids) (4306) Gag-Pol-Nef (C) (4311) VRC- p1012w/ Gag-Pol-Nef (B) 5305 2805 5309 + + HIVDNA00 CMV (4306) 9-00-VP promoter (4-plasmids) VRC- p1012w/ Ebola GP's and NP + + EBODNA01 CMV/R 2-00-VP promoter (3-plasmids) VRC- p1012w/ 4401 4409 4404 5736 5737 5738 * In HIVDNA01 CMV/R progress 6-00-VP promoter (6-plasmids)

TABLE-US-00009 TABLE 9 Summary of Vaccine Immunogenicity Studies in Mice and Non-Human Primates Test System Mouse Cynomolgus macaques Study Design Immunogenicity Immunogenicity Route i.m.¹ i.m.² Dose DNA: 50 μg DNA: 8 mg rAd: 1 × 10¹¹ PU Treatments per Animal 1 DNA 3 DNA 1 rAd Treatment Period 0 day 38 Wks Study Duration 21 days 58 Wks Conclusions Vaccination with gag-pol-nef Cynomolgus macaques receiving DNA prime/rAd (CMV/R) elicits higher HIV- boost immunization with the 6-plasmid DNA vaccine 1-specific cellular responses that expresses HIV-1 Gag, Pol, Nef and dade A, B in mice than plasmids and C Env (VRC-HIVDNA016-00-VP), and boosted constructed with the 1012 with rAd expressing HIV-1 Gag/Pol and 3 Env, backbone. elicited cellular immune responses to all viral antigens. References Item (8) Section 2.3.1 Item (8) Section 2.3.2 Study VRC-02-035 PU = Particle Unit ¹DNA plasmid administered intramuscularly (i.m.) by needle and syringe ²DNA Plasmid administered i.m. by Biojector; recombinant adenoviral vector vaccine (rAd) VRC-HIVADV014-00-VP (Lot # 026-03024) delivered i.m. by needle and syringe.

Vaccination with the CMV/R Plasmid Encoding the Gag-Pol-Nef Fusion Protein Elicits Higher HIV-1-Specific Cellular Responses in Mice than the Unmodified 1012 Plasmid Encoding the Same Fusion Protein.

[0139]To explore the possibility that enhanced antigen expression results in improved immunogenicity of these novel DNA vaccines in vivo, Balb/c mice (N=5/group) were immunized with 50 μg of the parental 1012 DNA vaccine or the CMV/R, RSV/R, mUB, or mUB/R DNA vaccines expressing HIV-1 Env gp145 ΔCFI. Mice were immunized three times at weeks 0, 2, and 6. On day 10 following the final immunization, splenocytes were assessed for Env-specific cellular immune responses by IFN-γ and TNF-α intracellular cytokine staining (ICS) assays. The CMV/R DNA vaccine elicited approximately 2-fold higher CD4⁺ (p=0.15) and CD8⁺ (p=0.043) T lymphocyte responses as compared with the parental 1012 DNA vaccine expressing the same antigen (FIG. 9). In contrast, the RSV/R, mUB, and mUB/R DNA vaccines did not elicit enhanced CD8⁺ immune responses, suggesting that the HTLV-1 R element selectively improved immunogenicity in the context of the CMV promoter.

[0140]Immunogenicity of the parental 1012 DNA vaccines and the CMV/R DNA vaccines expressing other antigens were then compared. Mice (N=8/group) were immunized with sham plasmids or with these DNA vaccines expressing the HIV-1 Gag-Pol-Nef fusion protein. Mice were immunized twice at weeks 0 and 6, and cellular immune responses were assessed by IFN-γ ELISPOT assays using splenocytes harvested 3 weeks after the initial or boost immunization. Groups of BALB/c female mice (8 mice per group) were immunized with the following regimens of plasmids diluted in normal saline: [0141]clade B g-p-n (1012): VRC-4306 (50 μg/animal); this plasmid expresses Gag-Pol-Nef as a fusion protein, and is contained in the four-plasmid vaccine VRC-HIVDNA009-00-VP (BB-IND 10681); [0142]clade B g-p-n (CMV/R): VRC-4400 (50 μg/animal); this plasmid expresses Gag-Pol-Nef as a fusion protein.

[0143]Mice were injected with a single intramuscular (i.m.) immunization of 50 μl total DNA in the quadriceps muscles using on day 0. On day 21 following immunization, mice were sacrificed for immunologic assays.

[0144]ICS assays. CD4⁺ and CD8⁺ T lymphocyte responses were evaluated by intracellular cytokine staining (ICS) for interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α). Briefly, splenocytes from immunized mice were harvested and incubated with pools of 15 amino acid peptides overlapping by 11 amino acids (2.5 μg/ml each) covering the entire HIV-1 Env protein, followed by treatment with 110 μg/ml brefeldin A (Sigma, St. Louis, Mo.). Cells were then fixed, permeabilized, and stained using rat anti-mouse CD3, CD4, CD8, IFN-γ and TNF-α monoclonal antibodies (BD Pharmingen, San Diego, Calif.). The IFN-γ and TNF-α positive cells in the CD4⁺ and CD8⁺ cell populations were analyzed with the program FlowJo (Tree Star, Ashland, Oreg.).

[0145]Splenocytes were removed aseptically and homogenized to create a single-cell suspension. IFN-γ ELISPOT assays were then performed using splenocytes from vaccinated mice to assess the magnitude of vaccine-elicited cellular immune responses. Ninety-six-well multiscreen plates (millipore, Bedford, Mass.) coated overnight with 100 μl/well of 10 μg/ml rat anti-mouse IFN-γ (Pharmingen, San Diego, Calif.) in PBS were washed with endotoxin-free Dulbecco's PBS (Life Technologies, Gaithersburg, Md.) containing 0.25% Tween-20 and blocked with PBS containing 5% FBS for 2 h at 37° C. The plates were washed three times with Dulbecco's PBS containing 0.25% Tween-20, rinsed with RPMI 1640 containing 10% FBS, and incubated in triplicate with 5×10⁵ splenocytes per well in a 100 μl reaction volume containing pooled peptides. Responses were measured using the HIV-1 Gag, Pol, and Nef peptide pools (VRC, Bethesda, Md.). Following an 18 h incubation, the plates were washed nine times with Dulbecco's PBS containing 0.25% Tween-20 and once with distilled water. The plates were then incubated for 2 h with 75 μl/well of 5 μg/ml biotinylated rat anti-mouse IFN-γ (Pharmingen, San Diego, Calif.), washed six times with Coulter Wash (Coulter Corporation, Miami, Fla.), and incubated for 2 h with a 1:500 dilution of streptavidin-AP (Southern Biotechnology Associates, Birmingham, Ala.). Following five washes with Coulter Wash and one with PBS, the plates were developed with NBT/BCIP chromogen (Pierce, Rockford, Ill.), stopped by washing with tap water, air dried, and read using an ELISPOT reader (Hitech Instruments, Edgemont, Pa.).

[0146]Immunologic data are presented as means with standard errors. Statistical analyses were performed with GraphPad Prism version 4.01 (GraphPad Software, Inc., 2004). Comparisons of mean cellular immune responses between groups of animals were performed by two-tailed nonparametric Mann-Whitney tests. In all cases, p-values of less than 0.05 were considered significant.

[0147]Consistent with the prior experiment, we observed approximately 2-fold higher Gag- (p=0.038) and Pol-specific (p=0.020) responses elicited by the CMV/R DNA vaccine compared to the parental 1012 DNA vaccine following the initial immunization (FIG. 10A). Following the boost immunization, responses elicited by the CMV/R DNA vaccine remained approximately 2-fold higher than responses elicited by the parental DNA vaccine using both unfractionated splenocytes (FIG. 10B) and CD8-depleted splenocytes (FIG. 10C).

Immunogenicity of DNA Prime/Recombinant Adenoviral Vector Boost Immunization of Cynomolgus Macaques

[0148]Immunogenicity of the parental 1012 DNA vaccines was compared with CMV/R DNA vaccines expressing multiple HIV-1 antigens in cynomolgus monkeys. Two groups of adult cynomolgus monkeys (N=6/group) were immunized with 4-plasmid mixtures of 1012 or CMV/R DNA vaccines expressing HIV-1 Env gp145 ΔCFI from clades A, B, and C and the Gag-Pol-Nef fusion protein from clade B in a 1:1:1:3 ratio. This multiclade, multivalent DNA vaccine has been previously described and is currently being evaluated in clinical trials (VRC-HIVDNA009-00-VP; PCT Publication No. WO/05034992). A third group of monkeys was included to investigate whether separating the Gag-Pol-Nef fusion protein into separate genes encoded on separate plasmids would further increase immune responses to these antigens (VRC-HIVDNA016-00-VP). This third group of monkeys received a 6-plasmid mixture of CMV/R DNA vaccines encoding HIV-1 Env gp145 from clades A, B, and C and separate Gag, Pol, and Nef proteins from clade B in a 1:1:1:1:1:1 ratio. All monkeys received three immunizations of 8 mg total DNA vaccine at weeks 0, 4, and 8.

[0149]Plasmid DNA vectors (Althea Technologies, Inc., San Diego Calif.) expressing HIV-1 Gag, Pol, Nef proteins or Gag-Pol-Nef fusion protein and Clade A, B and C Env were used for the DNA prime immunization. The plasmids expressed the same proteins as those contained in 4-plasmid vaccine VRC-HIVDNA009-00-VP and 6-plasmid vaccine VRC-HIVDNA016-00-VP.

[0150]The 4-plasmid combination was formulated using 1012 plasmids VRC 4306 (clade B Gag-Pol-Nef), VRC 5305 (clade A Env), VRC 2805 (clade B Env), and VRC 5309 (clade C Env). To achieve the required volumes for the three scheduled injections in the animal study, three lots of formulated material were prepared. The three lots were combined in a 50 mL conical tube. Following inversion of the tube several times to mix, 15.6-15.7 mL of the mixture was aliquotted into each of three 50 mL conical tubes. Tubes were labeled with study number, lot number, plasmid numbers, tube number, and date of preparation. Tubes were stored at -20° C. until distributed.

[0151]The 6-plasmid combination was formulated using CMV/R plasmids VRC 4401 (clade B Gag), VRC 4409 (clade B Pol), VRC 4404 (clade B Nef), VRC 5736 (clade A Env), VRC 5737 (clade B Env) and VRC 5738 (clade C Env). To achieve the required volumes for the three scheduled injections of the animal study, three lots of formulated material were prepared. The three lots were combined in a sterile container. Following inversion of the container several times to mix, 16.8 mL of the mixture was aliquotted into each of three 50 mL conical tubes. Tubes were labeled with study number, lot number, plasmid numbers, tube number and date of preparation and stored at -20° C. until distributed.

[0152]VRC-HIVADV014-00-VP (Lot #026-03024) was used as the rAd boost.

[0153]Outbred adult Cynomolgus macaques (6 monkeys per group) were vaccinated with DNA vaccine prime, delivered i.m. at weeks 0, 4, and 8 by Biojector. In each case, plasmid vaccine was delivered as two 0.5 ml injections in the quadriceps muscles using a No. 3 Biojector syringe (BIOJECT). A rAd vaccine boost was delivered i.m. by needle and syringe at week 38 (Group 1) and week 24 (Group 2). The following vaccination regimens were administered: [0154]Group 1: 1012 plasmid DNA prime (4plasmid combination): 8 mg total dose delivered as a combination of clade B Gag-Pol-Nef fusion protein (4 mg), clade A Env (1.3 mg), clade B Env (1.3 mg) and clade C Env (1.3 mg). This is a non-GMP version of the VRC-HIVDNA009-00-VP clinical product (BB-IND 10681). rAd vaccine boost: VRC-HIVADV014-00-VP (10¹¹ PU total dose; GMP lot # 026-03024). [0155]Group 2: CMV/R plasmid DNA (6-plasmid combination): 8 mg total dose delivered as a combination of clade B Gag (1.3 mg), clade B Pol (1.3 mg), clade B Nef (1.3 mg), clade A Env (1.3 mg), clade B Env (1.3 mg) and clade C Env (1.3 mg). This is a non-GMP version of the VRC-HIVDNA016-00-VP clinical product (the subject of this IND submission). rAd vaccine boost: VRC-HIVADV014-00-VP (GMP lot #026-03024). [0156]Group 3: CMV/R plasmid DNA (4 plasmid combination): 8 mg total dose delivered as a combination of clade B Gag-Pol-Nef fusion protein (4 mg), clade A Env (1.3 mg), clade B Env (1.3 mg) and clade C Env (1.3 mg). rAd vaccine boost: VRC-HIVADV014-00-VP (GMP lot #026-03024). [0157]Group 4: 1012 plasmid DNA (6 plasmid combination): 8 mg total dose delivered as a combination of clade B Gag (1.3 mg), clade B Pol (1.3 mg), clade B Nef (1.3 mg), clade A Env (1.3 mg), clade B Env (1.3 mg) and clade C Env (1.3 mg). rAd vaccine boost: VRC-HIVADV014-00-VP (GMP lot # 026-03024).

[0158]Monkeys were bled at various intervals through week 42 post-immunization.

[0159]ELISPOT assays were utilized to monitor the emergence of vaccine-elicited T cell immune responses to multiple viral antigens. Separate assays were performed for each animal using pools of 15 amino acid peptides overlapping by 11 amino acids spanning the HIV-1 Gag, Pol, Nef, clade A Env, clade B Env and clade C Env proteins matching the sequences of the vaccine immunogens. 96-well multiscreen plates were coated overnight with 100 μl/well of 5 μg/ml anti-human IFN-γ (B27; BD Pharmingen) in endotoxin-free Dulbecco's PBS (D-PBS). The plates were then washed three times with D-PBS containing 0.25% Tween-20 (D-PBS/Tween), blocked for 2 h with D-PBS containing 5% FBS at 37° C., washed three times with D-PBS/Tween, rinsed with RPMI 1640 containing 10% FBS to remove the Tween-20, and incubated with peptide pools and 2×10⁵ PBMC in triplicate in 100 μl reaction volumes. Following an 18 h incubation at 37° C., the plates were washed nine times with D-PBS/Tween and once with distilled water. The plates were then incubated with 2 μg/ml biotinylated rabbit anti-human IFN-γ (Biosource) for 2 h at room temperature, washed six times with Coulter Wash (Beckman-Coulter), and incubated for 2.5 h with a 1:500 dilution of streptavidin-AP (Southern Biotechnology). Following five washes with Coulter Wash and one with PBS, the plates were developed with NBT/BCIP chromogen (Pierce), stopped by washing with tap water, air dried, and read using an ELISPOT reader (Hitech Instruments). Spot-forming cells (SFC) per 10⁶ PBMC were calculated. Media backgrounds were consistently <15 spot-forming cells per 10⁶ PBMC.

[0160]Cellular immune responses against Env clade A, Env clade B, Env clade C, and Gag, Pol, and Nef from clade B were compared in monkeys that received the 4-plasmid mixtures under the control of CMV (1012) (Group 1) or CMV/R regulatory elements (Group 3). Monkeys immunized with the parental 1012 DNA vaccines developed low and sporadic IFN-γ ELISPOT responses to Env two weeks following the second immunization at week 6, and no clear responses above background were detected to Gag, Pol, and Nef (FIG. 11A). In contrast, monkeys immunized with the analogous CMV/R DNA vaccines exhibited significantly higher responses to all antigens (FIG. 11B). Compared to the parental 1012 DNA vaccines, the CMV/R DNA vaccines elicited >10-fold higher ELISPOT responses to Gag (p=0.0022), Pol (p=0.0043), and Nef (p=0.041) and 7- to 9-fold higher responses to Env clade A (p=0.026), B (p=0.0087), and C (p=0.030) at this time point. These results demonstrate that the CMV/R DNA vaccines were markedly more immunogenic than the parental 1012 DNA vaccines for multiple HIV-1 antigens in nonhuman primates.

[0161]Separating the Gag-Pol-Nef fusion protein into individual genes encoded on different plasmids further improved these responses. In particular, monkeys that received the 6-plasmid mixture of CMV/R DNA vaccines (Group 2) developed 4-fold higher responses to Gag (p=0.0022), a trend towards 2-fold higher responses to Pol (p=0.19), and 4-fold higher responses to Nef (p=0.049) (FIG. 11C), as compared to animals that received the 4-plasmid mixture of CMV/R DNA vaccines that included the Gag-Pol-Nef fusion protein (FIG. 11B). Env-specific responses between these two groups of monkeys that received the 4-plasmid and 6-plasmid mixtures of CMV/R DNA vaccines were comparable (p=0.48).

[0162]The evolution of mean IFN-γ ELISPOT responses in these groups of monkeys was evaluated at weeks 0, 2, 6, 10, and 12. Following the third DNA immunization at week 8, responses increased in all groups of monkeys (FIG. 12). At week 10, the parental 1012 DNA vaccines elicited Env- and Pol-specific responses in the majority of animals, although Gag- and Nef-specific responses remained low (FIG. 12A). In contrast, the CMV/R DNA vaccines elicited potent and broad responses to all antigens (FIG. 12B-C). At week 10, the 4-plasmid CMV/R DNA vaccines (FIG. 12B) elicited >10-fold higher ELISPOT responses to Gag (p=0.0022) and Nef (p=0.0022), 4-fold higher ELISPOT responses to Pol (p=0.043), and trends toward 1.5- to 4-fold higher responses to Env clade A, B, and C (FIG. 12B), as compared with the 4-plasmid parental 1012 DNA vaccines (FIG. 12A). Gag-, Pol- and Nef-specific responses remained highest in the animals that received the 6-plasmid CMV/R DNA vaccines with these genes encoded on separate plasmids (FIG. 12C). All responses boosted well with rAd. These studies confirm that the CMV/R DNA vaccines elicited substantially higher magnitude and broader cellular immune responses to multiple antigens as compared with the parental 1012 DNA vaccines. Thus, including the HTLV-1 R element and separating the Gag, Pol, and Nef genes significantly enhanced the immunogenicity of HIV-1 DNA vaccines in nonhuman primates.

[0163]In both mice and cynomolgus monkeys, CMV/R DNA vaccines expressing HIV-1 antigens elicited higher cellular immune responses than the parental 1012 DNA vaccines expressing the same antigens. However, the magnitude of the observed effects differed substantially between the two species. While the CMV/R DNA vaccines elicited only 2-fold higher responses in mice (FIG. 10), the CMV/R DNA vaccines elicited >10-fold higher cellular immune responses to Gag, Pol, and Nef and 7- to 9-fold higher responses to Env after two immunizations in cynomolgus monkeys (FIGS. 11,12). This difference reflects the lower baseline immunogenicity of the parental 1012 DNA vaccines in nonhuman primates and indicates that the beneficial effects of the R element is more apparent in limiting situations. Consistent with this observation, the R element had the greatest effect at enhancing the weakest responses elicited by the parental 1012 DNA vaccine against Gag and Nef. However, Env- and Pol-specific cellular immune were also significantly higher when induced by CMV/R DNA vaccines as compared with the parental 1012 DNA vaccines.

[0164]The 6-plasmid mixture of CMV/R DNA vaccines that included Gag, Pol, and Nef on separate plasmids elicited significantly higher cellular immune responses to these antigens as compared to the 4-plasmid mixture of CMV/R DNA vaccines that included the Gag-Pol-Nef fusion protein. These effects are particularly notable since the separate Gag, Pol, and Nef plasmids were each utilized at one-third the dose of the plasmid encoding the Gag-Pol-Nef fusion protein. Without being bound by theory, this increased immunogenicity may reflect enhanced translation or mRNA stability of the shorter genes as compared with the fusion gene, which might potentially affect antigen processing and presentation.

[0165]Accumulating data has confirmed the importance of cellular immune responses in controlling HIV-1 replication in humans and SIV replication in rhesus monkeys. Moreover, vaccines aimed at eliciting virus-specific cellular immune responses have afforded partial control of SHIV and SIV challenges in rhesus monkeys. Thus, the markedly increased magnitude and breadth of HIV-1-specific cellular immune responses afforded by the CMV/R DNA vaccines in nonhuman primates in the present study is believed to be beneficial in the development of second-generation DNA vaccines for both HIV-1 and other pathogens. In particular, incorporating the HTLV-1 R element and utilizing separate genes in place of fusion genes represent simple and practical strategies to improve DNA vaccines, making these vaccines suitable for clinical applications.

Example 4

Preparation of Material for Clinical Use

[0166]The process for manufacturing, filling, and packaging the VRC-HIVDNA016-00-VP drug product involves E. coli fermentation, purification, and formulation as a sterile liquid injectable dosage form for intramuscular injection. This naked DNA product involves no lipid, viral, or cellular vector components.

[0167]The vaccine, VRC-HIVDNA016-00-VP, is composed of a combination of six closed circular plasmid DNA macromolecules (VRC-4401, 4409, 4404, 5736, 5737 and 5738). For preparation of plasmids for clinical use, a master cell band (MCB) was prepared for each source plasmid (VRC-4401, 4409, 4404, 5736, 5737 and 5738). Identity and composition of plasmid DNA samples from each of these MCBs was confirmed by sequence analysis. Restriction enzyme analysis and microbial analysis (including mold and yeast) were also performed to confirm identity and sterility.

[0168]Bulk plasmid preparations are prepared from bacterial cell cultures containing a kanamycin selection medium. In all cases, bacterial cell growth is dependent upon the cellular expression of the kanamycin resistance protein encoded by a portion of the plasmid DNA. Following growth of bacterial cells harboring the plasmid, the plasmid DNA is purified from cellular components.

[0169]Clinical trial vaccines are prepared under cGMP conditions. The vaccines meet lot release specifications prior to administration. The DNA vaccine is manufactured at a 4.0 mg dose in phosphate buffered saline (PBS). Vials are aseptically filled to a volume of 1.2 mL at a ratio of 1:1:1:1:1:1 of the six plasmids. The 4.0 mg plasmid DNA vaccine vials is shipped, unblinded, to the study pharmacist on dry ice, and is stored at or below -20° C. until use. Placebo control vials of 2.4 mL PBS, pH 7.2±0.2, are obtained from Bell-More Labs, Incorporated (Hampstead, Md.).

[0170]Expression testing of the individual plasmids and the final formulated drug product are conducted prior to release of the vaccine product. Qualitative expression of the plasmid proteins is verified by comparing the reactive protein bands on the Western blot with the standards run under the same conditions. Once the plasmids are combined, expression is verified using the same assay procedures. Expression is determined by detecting proteins expressed by transfected 293 human embryonic kidney (HEK) cells. For transfection, 10⁵ to 10⁶ cells are transfected with 1-5 μg of plasmid DNA using the calcium phosphate method. Cells are incubated for 14-20 hours to allow for DNA uptake. Following a medium change, cells are grown for an additional 24-48 hours before harvesting. Transfection efficiency is monitored using a known similar vector in the same backbone. After cell lysis, 10 μg of an appropriate amount of total cellular protein is loaded onto an SDS-PAGE gel to separate the crude lysate proteins.

[0171]Following electrophoresis for approximately 1.5 hours, the proteins are transferred to a nitrocellulose membrane (0.45 μm) for Western blot analysis. The membrane is blocked with skim milk to prevent non-specific binding interaction prior to incubation with the primary antibody for 60 minutes. Following washing, the membrane is incubated for 45 minutes with HRP conjugated second antibody. Visualization of the protein bands is achieved by incubating the membrane with chemiluminescent substrates and exposing to X-ray film for 2 minutes or an appropriate time. Expression of protein produced by transfected cells is determined by observing the intensity of expressed protein on the Western blot. The assay is being further developed to allow for semi-quantitative analysis of protein expression by the vaccine plasmids.

Example 5

Clinical Safety in Humans

[0172]For clinical use, VRC-HIVDNA016-00-VP is composed of 6 closed, circular DNA plasmids that are each 16.67% (by weight) of the vaccine. Each of the 6 plasmids in this vaccine expresses a single gene product. Plasmids VRC 4401, VRC 4409 and VRC 4404 are designed to express clade B HIV-1 Gag, Pol and Nef, respectively. VRC 5736, VRC 5737, and VRC 5738 are designed to express HIV-1 Env glycoprotein from clade A, clade B, and clade C, respectively. Vaccine vials are supplied at 4 mg/mL. Each DNA administration is 1 mL of the vaccine composition delivered intramuscularly (in deltoid muscle) using the Biojector 2000® Needle-Free Injection Management System®.

[0173]Evaluation of the safety of this vaccine includes laboratory studies, medical history, physical assessment by clinicians, and subject self-assessment recorded on a diary card. Potential adverse reactions are further evaluated prior to continuing the immunization schedule. Day 0 is defined as the day of enrollment and first injection. Day 0 evaluations prior to the first injection are the baseline for subsequent safety assessments. The schedule of vaccination is Day 0, Day 28±7, Day 56±7 (with at least 21 days between injection days). All study injections are given by an intramuscular administration of VRC-HIVDNA016-00-VP at a 4 mg dose using a Biojector 2000® needle-free injection system. Study injections are administered into deltoid muscle.

[0174]Following study injections, subjects are observed for a minimum of 30 minutes. Vital signs (temperature, blood pressure, pulse and respiratory rate) are taken at 30-45 minutes post-immunization. The injection site is inspected for evidence of local reaction. Subjects will be given a "Diary Card" on which to record temperature and symptoms daily for 5 days. Follow-up on subject well-being will be performed by telephone on the first or second day following each injection. A clinic visit will occur if indicated by the telephone interview. On each injection day (prior to injection) and at 14±3 days after each injection, study subjects are evaluated by clinical exam and laboratory tests. Long-term follow-up visits are at Week 12±7 days, Week 24±14 days and Week 32±14 days. At intervals throughout the study subjects have blood drawn for immunologic assays. Any cells, serum or plasma not used will be stored for future virological and immunological assays. Subjects are also interviewed at the final clinical visit (Week 32) regarding social harms, including problems with employment, travel, immigration, access to insurance, medical or dental care, and negative reactions from family, friends, and co-workers.

[0175]Assessment of product safety includes clinical observation and monitoring of hematological and chemical parameters. The following parameters will be assessed: local reactogenicity signs and symptoms; systemic reactogenicity signs and symptoms; laboratory measures of safety; and adverse and serious adverse expenences.

[0176]The principal immunogenicity endpoints are measured at Week 0 (baseline) and Weeks 6, 8, 10 and 12 (for cellular immune responses) and consist of HIV-1-specific T cell responses, as measured by intracellular cytokine staining (ICS) assays. ICS at other study timepoints, as well as HIV-1-specific humoral immune responses as measured by HIV-specific antibody assays will be completed as exploratory evaluations.

[0177]Administration of the vaccine composition is performed using a BIOJECTOR 2000® NEEDLE-FREE INJECTION MANAGEMENT SYSTEM® as directed by the company. Neither the material being injected nor the deltoid injection site skin preparation require deviation from standard procedures. In brief, the injection site is disinfected and the area allowed to dry completely. The skin around the injection site is held firmly while the syringe is placed against the injection site at a 90° angle. The actuator is pressed and the material is released into the muscle. Continue to hold firmly for 3 seconds. After the injection, the site is covered with a sterile covering and pressure applied with 3 fingers for 1 minute. BIOJECTOR 2000® utilizes sterile, single-use syringes for variable dose, up to 1.0 mL, medication administration. The study agent is delivered under pressure by a compressed CO₂ gas cartridge that is stored inside the BIOJECTOR®. When the BIOJECTOR®'s actuator is depressed, CO₂ is released, causing the plunger to push the study agent out of the sterile syringe through the skin and into the underlying tissue. The study agent is expelled through a micro-orifice at high velocity in a fraction of a second to pierce the skin. The CO₂ does not come in contact with the injectate and the syringe design prevents any back splatter or contamination of the device by tissue from the subject.

[0178]Fifteen subjects received three 1 mL doses at 4 mg/mL on a 0, 1, 2 month schedule. Vaccinations were administered intramuscularly using the BIOJECTOR 2000®. Fourteen of the 15 subjects received 3 intramuscular injections of a 4 mg dose of vaccine administered by BIOJECTOR 2000®; one subject was lost to follow-up after two vaccinations. No subjects reported fever following vaccination. Reactogenicity was none to mild except that two subjects reported moderate injection site pain and one subject reported moderate nausea and malaise. The only adverse event requiring expedited reporting to the IND sponsor was a grade 3 generalized urticaria. The subject had reported starting an antihistamine about 2 weeks after first vaccination but reported at that time that the reason was latex allergy. While being screened for the rollover booster study, VRC 010, it was learned that the subject had experienced generalized urticaria around the time of the second vaccination when the supply of antihistamine ran out. The subject has chronic urticaria that are well controlled by antihistamine. Evaluation is ongoing. The etiology is unknown but at this time the chronic urticaria is assessed as possibly related to study vaccine. To date, there have been two moderate (grade 2) adverse events possibly attributed to vaccine. These were intermittent dizziness of 2 days duration beginning 13 days after the second vaccination in one subject (this subject received the third vaccination without recurrence of symptoms) and asymptomatic hypoglycemia in another subject, first noted at the follow-up visit that was 14 days after the third vaccination. The last safety evaluation of the subject lost to follow-up was by telephone one day after the second vaccination; at that time the subject reported no side effects from the vaccination.

[0179]An unexpected local injection site reaction for this DNA vaccine has been observed. Mild cutaneous lesions (0.5-1.0 cm diameter) at the vaccination site occurred after 4 of 44 (9%) vaccinations administered; these occurred in 3 of 15 (20%) subjects. Subjects were routinely asked to call if they experience any unusual problem after study vaccinations. The vaccination site cutaneous lesions did not alarm subjects enough to prompt them to contact the VRC Clinic prior to their next regularly-scheduled visit. In retrospect, three subjects reported that they experienced skin lesions that started as a small papule or vesicle within 3 days after vaccination. After a few days the papule or vesicle unroofed and a scab formed. There was surrounding mild erythema and mild induration. After the scab came off, the skin healed without treatment. None of the cutaneous lesions were associated with pustular exudates, fever, rash or urticaria. They did not appear to be either a local infection or an allergic reaction.

[0180]The first three cutaneous lesions were discovered at the first post-vaccination clinic visit (days 14±3 Day); at that time they were largely resolved. The fourth cutaneous lesion was examined in the clinic while still in an active stage and it was biopsied at post-vaccination day 6. This biopsy demonstrated a microscopic subcutaneous and dermal perivascular lymphocytic infiltrate. The infiltrate was composed almost exclusively of CD3 positive cells, including both CD4⁺ and CD8⁺. There were rare eosinophils present and rare giant cells noted. The process appeared to be primarily a subcutaneous and dermal response to vaccination with cutaneous manifestations.

[0181]Whether these reactions correlate with the strength of the vaccine-induced immune response is also not yet known. Eight of the 14 subjects who remained in follow-up have had a vaccine-induced positive HIV ELISA by a commercial test at one or more timepoints; this includes all three subjects who had a cutaneous lesion. Preliminary immunogenicity data with the 6-plasmid DNA indicate that the Env-specific T cell responses are similar to those seen in the 4-plasmid DNA, and the Gag- and Nef-specific responses are also present.

[0182]Cellular responses in subjects were measured by intracellular cytokine staining (ICS) and flow cytometry to detect IFN-γ or IL-2 in both CD4+ and CD8+ T lymphocytes after stimulation with peptide pools representing the viral antigens (FIG. 13). Data for each individual subject is shown in columns. Responses to each peptide pools are shown in rows. Each box represents the entire time course from prevaccination to 12 weeks (4 weeks after the last immunization). The scale for each box is 0-0.2% of the total CD4+ or CD8+ population tested. CD4+ responses are shown in red and CD8+ responses shown in green. Nearly all subject have detectable responses to Env peptides. In contrast to the 4-plasmid product, the majority of subjects have detectable responses to Gag and there are also Nef responders.

Example 6

Immunogenicity of Chimeric Env Proteins

[0183]To demonstrate the role of different genetic sequences in the induction of neutralizing antibodies, nucleic acid constructs expressing chimeric antigenic polypeptides having different regions of the viral envelope from two different clades were produced. Nucleic acid constructs encoding different portions of the clade C Env polypeptide and clade B Env polypeptide were analyzed and compared to the clade C Env polypeptide. The transposition of the proximal 25% of clade C onto the clade B background showed an increase in the potency and breadth of neutralization against a variety of clade β isolates and improved the neutralization of clade C isolates. Replacement of the distal region of clade B Env with the clade C Env resulted in improved neutralization against clade β isolates, demonstrating that the region containing V₃ in clade β isolates contributes to its ability to inhibit a variety of diverse viral isolates. These nucleic acid constructs are represented by SEQ ID NOs:7-15. Thus, certain embodiments of the disclosed compositions can include constructs encoding chimeric Env polypeptides combining multiple clades.

[0184]To demonstrate the roles of V regions in alternative clades, mutations were made both in the V₁V₂ as well as the V₃ regions of clades A, B and C. To demonstrate the role of V₁V₂ in clade A, a clade A prototype was compared to that containing deletions of the V₁ and V₂ regions. Removal of V₁V₂ and/or V₃ enhanced the ability of the clade A Env polypeptide to elicit an immune response that neutralized a variety of clade β isolates, demonstrating that deletion of these regions increases the ability of the antigenic polypeptide to elicit broadly neutralizing antibodies (for example, by increasing accessibility to specific epitopes that elicit cross-reactive antibodies). Accordingly, in certain embodiments disclosed herein, the nucleic acid constructs include deletions of a V₁, V₂ and/or V₃ region.

[0185]To demonstrate the role of V₁V₂ in clade B against a heterologous V₃ from clade C, the V₃ from a South African clade C isolate was inserted in place of the V₃ from a clade B and compared to a stem-shortened version that has been shown to enhance neutralization using clade B V₃ loops. The ability of these plasmid DNA vectors in combination with a recombinant adenovirus boost to elicit neutralizing antibodies was evaluated against the indicated strains. Immunization with both V₃ substitutions allowed neutralization of viral isolates from clades A, B and C, although the magnitude of the response was greater with the stem-shortened 1AB V₃. In addition, the peptide inhibition revealed that the neutralizing antibodies elicited in this response were of greater breadth and intereacted with V₃ regions from diverse clades, A, B and C. Thus, the clade C V₃ loop appeared to elicit broadly reactive V₃ neutralizing antibodies.

[0186]Deletion of the V₁ and V₂ regions of these envelopes improves their ability to elicit neutralizing antibody responses. These responses are directed largely against the V₃ regions in diverse clades. The use of alternative V regions derived from different clades demonstrates that these V regions also display differences in their ability to elicit strain-specific responses. For example, the inclusion of V₃ regions from clade C allowed neutralization of a variety of clade B isolates and greater breadth of neutralization by V₃ peptides from diverse strains. Thus, the elimination of both the V₁ and V₂ regions as well as the presentation of more broadly reactive V₃s can enhance the breadth of neutralization mediated by an Env antigenic polypeptide.

[0187]In addition to the V₃-mediated neutralization, other variable regions contribute to virus neutralization when V₃ is not exposed. Among these, a highly exposed region in V₁ was identified. Although this region is highly likely to show strain-specific variation, there are also conserved subregions within the V₁ that contribute to increased breadth of the immune response to this variable loop.

[0188]The ability to define improved immunogens using genetic information based on viral diversity can improve the ability to design effective HIV vaccines. The results described above demonstrate that genotypic sequence variation can result in neutralization sensitivities that are independent of clade. This finding has important implications for the design of improved HIV immunogens based on genetic sequence.

[0189]In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Sequence CWU 1

2615886DNAArtificial Sequenceplasmid VRC4401 1tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcagattgg 240ctattggcca ttgcatacgt tgtatccata tcataatatg tacatttata ttggctcatg 300tccaacatta ccgccatgtt gacattgatt attgactagt tattaatagt aatcaattac 360ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg 420cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc 480catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac 540tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa 600tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac 660ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta 720catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga 780cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa 840ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag 900agctcgttta gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt ttgacctcca 960tagaagacac cgggaccgat ccagcctcca tcggctcgca tctctccttc acgcgcccgc 1020cgccctacct gaggccgcca tccacgccgg ttgagtcgcg ttctgccgcc tcccgcctgt 1080ggtgcctcct gaactgcgtc cgccgtctag gtaagtttaa agctcaggtc gagaccgggc 1140ctttgtccgg cgctcccttg gagcctacct agactcagcc ggctctccac gctttgcctg 1200accctgcttg ctcaactcta gttaacggtg gagggcagtg tagtctgagc agtactcgtt 1260gctgccgcgc gcgccaccag acataatagc tgacagacta acagactgtt cctttccatg 1320ggtcttttct gcagtcaccg tcgtcgacac gtgtgatcag ataaacttaa gcttatgggc 1380gcccgcgcca gcgtgctgag cggcggcgag ctggaccgct gggagaagat ccgcctgcgc 1440cccggcggca agaagaagta caagctgaag cacatcgtgt gggccagccg cgagctggag 1500cgcttcgccg tgaaccccgg cctgctggag accagcgagg gctgccgcca gatcctgggc 1560cagctgcagc ccagcctgca gaccggcagc gaggagctgc gcagcctgta caacaccgtg 1620gccaccctgt actgcgtgca ccagcgcatc gagatcaagg acaccaagga ggccctggac 1680aagatcgagg aggagcagaa caagagcaag aagaaggccc agcaggccgc cgccgacacc 1740ggccacagca accaggtgag ccagaactac cccatcgtgc agaacatcca gggccagatg 1800gtgcaccagg ccatcagccc ccgcaccctg aacgcctggg tgaaggtggt ggaggagaag 1860gccttcagcc ccgaggtgat ccccatgttc agcgccctga gcgagggcgc caccccccag 1920gacctgaaca ccatgctgaa caccgtgggc ggccaccagg ccgccatgca gatgctgaag 1980gagaccatca acgaggaggc cgccgagtgg gaccgcgtgc accccgtgca cgccggcccc 2040atcgcccccg gccagatgcg cgagccccgc ggcagcgaca tcgccggcac caccagcacc 2100ctgcaggagc agatcggctg gatgaccaac aaccccccca tccccgtggg cgagatctac 2160aagcgctgga tcatcctggg cctgaacaag atcgtgcgca tgtacagccc caccagcatc 2220ctggacatcc gccagggccc caaggagccc ttccgcgact acgtggaccg cttctacaag 2280accctgcgcg ccgagcaggc cagccaggag gtgaagaact ggatgaccga gaccctgctg 2340gtgcagaacg ccaaccccga ctgcaagacc atcctgaagg ccctgggccc cgccgccacc 2400ctggaggaga tgatgaccgc ctgccagggc gtgggcggcc ccggccacaa ggcccgcgtg 2460ctggccgagg ccatgagcca ggtgaccaac agcgccacca tcatgatgca gcgcggcaac 2520ttccgcaacc agcgcaagat cgtgaagtgc ttcaactgcg gcaaggaggg ccacaccgcc 2580cgcaactgcc gcgccccccg caagaagggc tgctggaagt gcggcaagga gggccaccag 2640atgaaggact gcaccgagcg acaggctaat tttttaggga agatctggcc ttcccacaag 2700ggaaggccag ggaattttct tcagagcaga ccagagccaa cagccccacc agaagagagc 2760ttcaggtttg gggaagagac aacaactccc tctcagaagc aggagccgat agacaaggaa 2820ctgtatcctt tagcttccct cagatcactc tttggcagcg acccctcgtc acaataaaga 2880taggtaccga gctcggatcc agatctgctg tgccttctag ttgccagcca tctgttgttt 2940gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat 3000aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg 3060tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg 3120tgggctctat gggtacccag gtgctgaaga attgacccgg ttcctcctgg gccagaaaga 3180agcaggcaca tccccttctc tgtgacacac cctgtccacg cccctggttc ttagttccag 3240ccccactcat aggacactca tagctcagga gggctccgcc ttcaatccca cccgctaaag 3300tacttggagc ggtctctccc tccctcatca gcccaccaaa ccaaacctag cctccaagag 3360tgggaagaaa ttaaagcaag ataggctatt aagtgcagag ggagagaaaa tgcctccaac 3420atgtgaggaa gtaatgagag aaatcataga atttcttccg cttcctcgct cactgactcg 3480ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg 3540ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag 3600gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac 3660gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga 3720taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt 3780accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc 3840tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc 3900cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta 3960agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat 4020gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca 4080gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct 4140tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt 4200acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct 4260cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc 4320acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa 4380acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta 4440tttcgttcat ccatagttgc ctgactcggg gggggggggc gctgaggtct gcctcgtgaa 4500gaaggtgttg ctgactcata ccaggcctga atcgccccat catccagcca gaaagtgagg 4560gagccacggt tgatgagagc tttgttgtag gtggaccagt tggtgatttt gaacttttgc 4620tttgccacgg aacggtctgc gttgtcggga agatgcgtga tctgatcctt caactcagca 4680aaagttcgat ttattcaaca aagccgccgt cccgtcaagt cagcgtaatg ctctgccagt 4740gttacaacca attaaccaat tctgattaga aaaactcatc gagcatcaaa tgaaactgca 4800atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag 4860gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc 4920cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa 4980gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagc ttatgcattt 5040ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa 5100ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga tcgctgttaa 5160aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa 5220caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga 5280tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa 5340gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa 5400cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat 5460agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag 5520catccatgtt ggaatttaat cgcggcctcg agcaagacgt ttcccgttga atatggctca 5580taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat gatgatatat 5640ttttatcttg tgcaatgtaa catcagagat tttgagacac aacgtggctt tccccccccc 5700cccattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta 5760tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg 5820tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct 5880ttcgtc 588627344DNAArtificial Sequenceplasmid VRC4409 2tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcagattgg 240ctattggcca ttgcatacgt tgtatccata tcataatatg tacatttata ttggctcatg 300tccaacatta ccgccatgtt gacattgatt attgactagt tattaatagt aatcaattac 360ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg 420cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc 480catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac 540tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa 600tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac 660ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta 720catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga 780cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa 840ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag 900agctcgttta gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt ttgacctcca 960tagaagacac cgggaccgat ccagcctcca tcggctcgca tctctccttc acgcgcccgc 1020cgccctacct gaggccgcca tccacgccgg ttgagtcgcg ttctgccgcc tcccgcctgt 1080ggtgcctcct gaactgcgtc cgccgtctag gtaagtttaa agctcaggtc gagaccgggc 1140ctttgtccgg cgctcccttg gagcctacct agactcagcc ggctctccac gctttgcctg 1200accctgcttg ctcaactcta gttaacggtg gagggcagtg tagtctgagc agtactcgtt 1260gctgccgcgc gcgccaccag acataatagc tgacagacta acagactgtt cctttccatg 1320ggtcttttct gcagtcaccg tcgtcgacat gagggaagat ctggccttcc cacaagggaa 1380ggccagggaa ttttcttcag agcagaccag agccaacagc cccaccagaa gagagcttca 1440ggtttgggga agagacaaca actccctctc agaagcagga gccgatagac aaggaactgt 1500atcctttagc ttccctcaga tcactctttg gcagcgaccc ctcgtcacaa taaagatagg 1560gggccagctg aaggaggccc tgctggacac cggcgccgac gacaccgtgc tggaggagat 1620gaacctgccc ggccgctgga agcccaagat gatcggcggc atcggcggct tcatcaaggt 1680gggccagtac gaccagatcc tgatcgagat ctgcggccac aaggccatcg gcaccgtgct 1740ggtgggcccc acccccgtga acatcatcgg ccgcaacctg ctgacccaga tcggctgcac 1800cctgaacttc cccatcagcc ccatcgagac cgtgcccgtg aagctgaagc ccggcatgga 1860cggccccaag gtgaagcagt ggcccctgac cgaggagaag atcaaggccc tggtggagat 1920ctgcaccgag atggagaagg agggcaagat cagcaagatc ggccccgaga acccctacaa 1980cacccccgtg ttcgccatca agaagaagga cagcaccaag tggcgcaagc tggtggactt 2040ccgcgagctg aacaagcgca cccaggactt ctgggaggtg cagctgggca tcccccaccc 2100cgccggcctg aagcagaaga agagcgtgac cgtgctggac gtgggcgacg cctacttcag 2160cgtgcccctg gacaaggact tccgcaagta caccgccttc accatcccca gcatcaacaa 2220cgagaccccc ggcatccgct accagtacaa cgtgctgccc cagggctgga agggcagccc 2280cgccatcttc cagtgcagca tgaccaagat cctggagccc ttccgcaagc agaaccccga 2340catcgtgatc taccagtaca tggaccacct gtacgtgggc agcgacctgg agatcggcca 2400gcaccgcacc aagatcgagg agctgcgcca gcacctgctg cgctggggct tcaccacccc 2460cgacaagaag caccagaagg agcccccctt cctgtggatg ggctacgagc tgcaccccga 2520caagtggacc gtgcagccca tcgtgctgcc cgagaaggac agctggaccg tgaacgacat 2580ccagaagctg gtgggcaagc tgaactgggc cagccagatc tacgccggca tcaaggtgcg 2640ccagctgtgc aagctgctgc gcggcaccaa ggccctgacc gaggtggtgc ccctgaccga 2700ggaggccgag ctggagctgg ccgagaaccg cgagatcctg aaggagcccg tgcacggcgt 2760gtactacgac cccagcaagg acctgatcgc cgagatccag aagcagggcc agggccagtg 2820gacctaccag atctaccagg agcccttcaa gaacctgaag accggcaagt acgcccgcat 2880gaagggcgcc cacaccaacg acgtgaagca gctgaccgag gccgtgcaga agatcgccac 2940cgagagcatc gtgatctggg gcaagacccc caagttcaag ctgcccatcc agaaggagac 3000ctgggaggcc tggtggaccg agtactggca ggccacctgg atccccgagt gggagttcgt 3060gaacaccccc cccctggtga agctgtggta ccagctggag aaggagccca tcatcggcgc 3120cgagaccttc tacgtggacg gcgccgccaa ccgcgagacc aagctgggca aggccggcta 3180cgtgaccgac cgcggccgcc agaaggtggt gcccctgacc gacaccacca accagaagac 3240cgagctgcag gccatccacc tggccctgca ggacagcggc ctggaggtga acatcgtgac 3300cgacagccag tacgccctgg gcatcatcca ggcccagccc gacaagagcg agagcgagct 3360ggtgagccag atcatcgagc agctgatcaa gaaggagaag gtgtacctgg cctgggtgcc 3420cgcccacaag ggcatcggcg gcaacgagca ggtggacggc ctggtgagcg ccggcatccg 3480caaggtgctg ttcctggacg gcatcgacaa ggcccaggag gagcacgaga agtaccacag 3540caactggcgc gccatggcca gcgacttcaa cctgcccccc gtggtggcca aggagatcgt 3600ggccagctgc gacaagtgcc agctgaaggg cgaggccatg cacggccagg tggactgcag 3660ccccggcatc tggcagctgg catgcaccca cctggagggc aaggtgatcc tggtggccgt 3720gcacgtggcc agcggctaca tcgaggccga ggtgatcccc gccgagaccg gccaggagac 3780cgcctacttc ctgctgaagc tggccggccg ctggcccgtg aagaccgtgc acaccgacaa 3840cggcagcaac ttcaccagca ccaccgtgaa ggccgcctgc tggtgggccg gcatcaagca 3900ggagttcggc atcccctaca acccccagag ccagggcgtg atcgagagca tgaacaagga 3960gctgaagaag atcatcggcc aggtgcgcga ccaggccgag cacctgaaga ccgccgtgca 4020gatggccgtg ttcatccaca acttcaagcg caagggcggc atcggcggct acagcgccgg 4080cgagcgcatc gtggacatca tcgccaccga catccagacc aaggagctgc agaagcagat 4140caccaagatc cagaacttcc gcgtgtacta ccgcgacagc cgcgaccccg tgtggaaggg 4200ccccgccaag ctgctgtgga agggcgaggg cgccgtggtg atccaggaca acagcgacat 4260caaggtggtg ccccgccgca aggccaagat catccgcgac tacggcaagc agatggccgg 4320cgacgactgc gtggccagcc gccaggacga ggactaggaa ttctgctgtg ccttctagtt 4380gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc 4440ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt 4500ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca 4560ggcatgctgg ggatgcggtg ggctctatgg gtacccaggt gctgaagaat tgacccggtt 4620cctcctgggc cagaaagaag caggcacatc cccttctctg tgacacaccc tgtccacgcc 4680cctggttctt agttccagcc ccactcatag gacactcata gctcaggagg gctccgcctt 4740caatcccacc cgctaaagta cttggagcgg tctctccctc cctcatcagc ccaccaaacc 4800aaacctagcc tccaagagtg ggaagaaatt aaagcaagat aggctattaa gtgcagaggg 4860agagaaaatg cctccaacat gtgaggaagt aatgagagaa atcatagaat ttcttccgct 4920tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac 4980tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga 5040gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat 5100aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac 5160ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct 5220gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg 5280ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg 5340ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt 5400cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg 5460attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac 5520ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga 5580aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt 5640gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt 5700tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga 5760ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc 5820taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct 5880atctcagcga tctgtctatt tcgttcatcc atagttgcct gactcggggg gggggggcgc 5940tgaggtctgc ctcgtgaaga aggtgttgct gactcatacc aggcctgaat cgccccatca 6000tccagccaga aagtgaggga gccacggttg atgagagctt tgttgtaggt ggaccagttg 6060gtgattttga acttttgctt tgccacggaa cggtctgcgt tgtcgggaag atgcgtgatc 6120tgatccttca actcagcaaa agttcgattt attcaacaaa gccgccgtcc cgtcaagtca 6180gcgtaatgct ctgccagtgt tacaaccaat taaccaattc tgattagaaa aactcatcga 6240gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat ttttgaaaaa 6300gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg gcaagatcct 6360ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat ttcccctcgt 6420caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc ggtgagaatg 6480gcaaaagctt atgcatttct ttccagactt gttcaacagg ccagccatta cgctcgtcat 6540caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa 6600atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac cggcgcagga 6660acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct aatacctgga 6720atgctgtttt cccggggatc gcagtggtga gtaaccatgc atcatcagga gtacggataa 6780aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg accatctcat 6840ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct ggcgcatcgg 6900gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg cgagcccatt 6960tatacccata taaatcagca tccatgttgg aatttaatcg cggcctcgag caagacgttt 7020cccgttgaat atggctcata acaccccttg tattactgtt tatgtaagca gacagtttta 7080ttgttcatga tgatatattt ttatcttgtg caatgtaaca tcagagattt tgagacacaa 7140cgtggctttc cccccccccc cattattgaa gcatttatca gggttattgt ctcatgagcg 7200gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc 7260gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata 7320ggcgtatcac gaggcccttt cgtc 734435039DNAArtificial Sequenceplasmid VRC4404 3tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcagattgg 240ctattggcca ttgcatacgt tgtatccata tcataatatg tacatttata ttggctcatg 300tccaacatta ccgccatgtt gacattgatt attgactagt tattaatagt aatcaattac 360ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg 420cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc 480catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac 540tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa 600tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac 660ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta 720catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga 780cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa 840ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag 900agctcgttta gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt ttgacctcca 960tagaagacac cgggaccgat ccagcctcca tcggctcgca tctctccttc acgcgcccgc 1020cgccctacct gaggccgcca tccacgccgg ttgagtcgcg ttctgccgcc tcccgcctgt 1080ggtgcctcct gaactgcgtc cgccgtctag gtaagtttaa agctcaggtc gagaccgggc 1140ctttgtccgg cgctcccttg gagcctacct agactcagcc ggctctccac gctttgcctg 1200accctgcttg ctcaactcta gttaacggtg gagggcagtg tagtctgagc agtactcgtt 1260gctgccgcgc gcgccaccag acataatagc tgacagacta acagactgtt cctttccatg 1320ggtcttttct gcagtcaccg tcgtcgacac gtgtgatcag atatcgcggc cgctctagag 1380atatcgccgc catgaagtgg agcaagagca gcgtgatcgg ctggcccgcc gtgcgcgagc 1440gcatgcgccg cgccgagccc gccgccgacg gcgtgggcgc cgtgagccgc gacctggaga 1500agcacggcgc catcaccagc agcaacaccg ccgccaacaa cgccgcctgc gcctggctgg 1560aggcccagga ggaggaggag gtgggcttcc ccgtgacccc ccaggtgccc ctgcgcccca

1620tgacctacaa ggccgccgtg gacctgagcc acttcctgaa ggagaagggc ggcctggagg 1680gcctgatcca cagccagcgc cgccaggaca tcctggacct gtggatctac cacacccagg 1740gctacttccc cgactggcag aactacaccc ccggccccgg cgtgcgctac cccctgacct 1800tcggctggtg ctacaagctg gtgcccgtgg agcccgacaa ggtggaggag gccaacaagg 1860gcgagaacac cagcctgctg caccccgtga gcctgcacgg catggacgac cccgagcgcg 1920aggtgctgga gtggcgcttc gacagccgcc tggccttcca ccacgtggcc cgcgagctgc 1980accccgagta cttcaagaac tgctgaacac gtgggatcca gatctgctgt gccttctagt 2040tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact 2100cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat 2160tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc 2220aggcatgctg gggatgcggt gggctctatg ggtacccagg tgctgaagaa ttgacccggt 2280tcctcctggg ccagaaagaa gcaggcacat ccccttctct gtgacacacc ctgtccacgc 2340ccctggttct tagttccagc cccactcata ggacactcat agctcaggag ggctccgcct 2400tcaatcccac ccgctaaagt acttggagcg gtctctccct ccctcatcag cccaccaaac 2460caaacctagc ctccaagagt gggaagaaat taaagcaaga taggctatta agtgcagagg 2520gagagaaaat gcctccaaca tgtgaggaag taatgagaga aatcatagaa ttttaaggcc 2580atgatttaag gccatcatgg ccttaatctt ccgcttcctc gctcactgac tcgctgcgct 2640cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca 2700cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga 2760accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc 2820acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg 2880cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat 2940acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt 3000atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc 3060agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg 3120acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg 3180gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg 3240gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg 3300gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca 3360gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga 3420acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga 3480tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt 3540ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt 3600catccatagt tgcctgactc gggggggggg ggcgctgagg tctgcctcgt gaagaaggtg 3660ttgctgactc ataccaggcc tgaatcgccc catcatccag ccagaaagtg agggagccac 3720ggttgatgag agctttgttg taggtggacc agttggtgat tttgaacttt tgctttgcca 3780cggaacggtc tgcgttgtcg ggaagatgcg tgatctgatc cttcaactca gcaaaagttc 3840gatttattca acaaagccgc cgtcccgtca agtcagcgta atgctctgcc agtgttacaa 3900ccaattaacc aattctgatt agaaaaactc atcgagcatc aaatgaaact gcaatttatt 3960catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa 4020ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg 4080tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa 4140atcaccatga gtgacgactg aatccggtga gaatggcaaa agcttatgca tttctttcca 4200gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc 4260gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca 4320attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt 4380ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt 4440ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat 4500aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc 4560tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt 4620cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat 4680gttggaattt aatcgcggcc tcgagcaaga cgtttcccgt tgaatatggc tcataacacc 4740ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgatgata tatttttatc 4800ttgtgcaatg taacatcaga gattttgaga cacaacgtgg ctttcccccc ccccccatta 4860ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa 4920aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga 4980aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtc 503946305DNAArtificial Sequenceplasmid VRC5736 4tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcagattgg 240ctattggcca ttgcatacgt tgtatccata tcataatatg tacatttata ttggctcatg 300tccaacatta ccgccatgtt gacattgatt attgactagt tattaatagt aatcaattac 360ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg 420cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc 480catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac 540tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa 600tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac 660ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta 720catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga 780cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa 840ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag 900agctcgttta gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt ttgacctcca 960tagaagacac cgggaccgat ccagcctcca tcggctcgca tctctccttc acgcgcccgc 1020cgccctacct gaggccgcca tccacgccgg ttgagtcgcg ttctgccgcc tcccgcctgt 1080ggtgcctcct gaactgcgtc cgccgtctag gtaagtttaa agctcaggtc gagaccgggc 1140ctttgtccgg cgctcccttg gagcctacct agactcagcc ggctctccac gctttgcctg 1200accctgcttg ctcaactcta gttaacggtg gagggcagtg tagtctgagc agtactcgtt 1260gctgccgcgc gcgccaccag acataatagc tgacagacta acagactgtt cctttccatg 1320ggtcttttct gcagtcaccg tcgtcgacac gtgtgatcag atatcgcggc cgctctagag 1380atatcgccac catgcgcgtg cgcggcatcc agaccagctg gcagaacctg tggcgctggg 1440gcaccatgat cctgggcatg ctggtgatct acagcgccgc cgagaacctg tgggtggccg 1500tgtactacgg cgtgcccgtg tggaaggacg ccgagaccac cctgttctgc gccagcgacg 1560ccaaggccta cgacaccgag gtgcacaacg tgtgggagac ccacgcctgc gtgcccaccg 1620accccaaccc ccaggagatc cacctggaga acgtgaccga ggacttcaac atgtggcgca 1680acaacatggt ggagcagatg cacaccgaca tcatcagcct gtgggaccag agcctgaagc 1740cctgcgtgaa gctgaccccc ctgtgcgtga ccctggactg caacgccacc gccagcaacg 1800tgaccaacga gatgcgcaac tgtagcttca acatcaccac cgagctgaag gacaagaagc 1860agcaggtgta cagcctgttc tacaagctgg acgtggtgca gatcaacgag aagaacgaga 1920ccgacaagta ccgcctgatc aactgcaaca ccagcgccat cacccaggcc tgccccaagg 1980tgagcttcga gcccatcccc atccactact gcgcccccgc cggcttcgcc atcctgaagt 2040gcaaggacac cgagttcaac ggtaccggcc cctgcaagaa cgtgagcacc gtgcagtgca 2100cccacggcat ccgaccggtg atcagcaccc agctgctgct gaacggcagc ctggccgagg 2160agggcatcca gatccgcagc gagaacatca ccaacaacgc caagaccatc atcgtgcagc 2220tggataaggc cgtgaagatc aactgcaccc gccccaacaa caacacccgc aagggcgtgc 2280gcatcggccc cggccaggcc ttctacgcca ccggcggcat catcggcgac atccgccagg 2340cccactgcca cgtgagccgc gccaagtgga acgacaccct gcgcggcgtg gccaagaagc 2400tgcgcgagca cttcaagaac aagaccatca tcttcgagaa gagcagcggc ggcgacatcg 2460agatcaccac ccacagcttc atctgcggcg gcgagttctt ctactgcaac accagcggcc 2520tgttcaacag cacctgggag agcaacagca ccgagagcaa caacaccacc agcaacgaca 2580ccatcaccct gacctgccgc atcaagcaga tcatcaacat gtggcagaag gtgggccagg 2640ccatgtaccc cccccccatc cagggcgtga tccgctgcga gagcaacatc accggcctgc 2700tgctgacccg cgacggcggc aacaacagca ccaacgagat cttccgcccc ggcggcggca 2760acatgcgcga caactggcgc agcgagctgt acaagtacaa ggtggtgaag atcgagcccc 2820tgggcgtggc ccccagccgc gccaagctta ccgcccaggc ccgccagctg ctgagcggca 2880tcgtgcagca gcagagcaac ctgctgcgcg ccatcgaggc ccagcagcac atgctgaagc 2940tgaccgtgtg gggcatcaag cagctgcagg cccgcgtgct ggccgtggag cgctacctga 3000aggaccagca gctcgagatc tgggacaaca tgacctggct gcagtgggac aaggagatca 3060gcaactacac ccagatcatc tacaacctga tcgaggagag ccagaaccag caggagaaga 3120acgagcagga cctgctggcc ctggacaagt gggccagcct gtggaactgg ttcgacatca 3180gccgctggct gtggtacatc aagatcttca tcatgatcgt gggcggcctg atcggcctgc 3240gcatcgtgtt cgccgtgctg agcgtgatct gaacacgtgg gatccagatc tgctgtgcct 3300tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt 3360gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg 3420tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac 3480aatagcaggc atgctgggga tgcggtgggc tctatgggta cccaggtgct gaagaattga 3540cccggttcct cctgggccag aaagaagcag gcacatcccc ttctctgtga cacaccctgt 3600ccacgcccct ggttcttagt tccagcccca ctcataggac actcatagct caggagggct 3660ccgccttcaa tcccacccgc taaagtactt ggagcggtct ctccctccct catcagccca 3720ccaaaccaaa cctagcctcc aagagtggga agaaattaaa gcaagatagg ctattaagtg 3780cagagggaga gaaaatgcct ccaacatgtg aggaagtaat gagagaaatc atagaatttt 3840aaggccatga tttaaggcca tcatggcctt aatcttccgc ttcctcgctc actgactcgc 3900tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt 3960tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg 4020ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg 4080agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat 4140accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta 4200ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct 4260gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc 4320ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa 4380gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg 4440taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag 4500tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt 4560gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta 4620cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc 4680agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca 4740cctagatccc tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa 4800cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat 4860ttcgttcatc catagttgcc tgactcgggg ggggggggcg ctgaggtctg cctcgtgaag 4920aaggtgttgc tgactcatac caggcctgaa tcgccccatc atccagccag aaagtgaggg 4980agccacggtt gatgagagct ttgttgtagg tggaccagtt ggtgattttg aacttttgct 5040ttgccacgga acggtctgcg ttgtcgggaa gatgcgtgat ctgatccttc aactcagcaa 5100aagttcgatt tattcaacaa agccgccgtc ccgtcaagtc agcgtaatgc tctgccagtg 5160ttacaaccaa ttaaccaatt ctgattagaa aaactcatcg agcatcaaat gaaactgcaa 5220tttattcata tcaggattat caataccata tttttgaaaa agccgtttct gtaatgaagg 5280agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt ctgcgattcc 5340gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa ggttatcaag 5400tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagct tatgcatttc 5460tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac tcgcatcaac 5520caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcgat cgctgttaaa 5580aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac 5640aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt tcccggggat 5700cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga tggtcggaag 5760aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat cattggcaac 5820gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat acaatcgata 5880gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat ataaatcagc 5940atccatgttg gaatttaatc gcggcctcga gcaagacgtt tcccgttgaa tatggctcat 6000aacacccctt gtattactgt ttatgtaagc agacagtttt attgttcatg atgatatatt 6060tttatcttgt gcaatgtaac atcagagatt ttgagacaca acgtggcttt cccccccccc 6120ccattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat 6180ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt 6240ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt 6300tcgtc 630556338DNAartificial sequenceplasmid VRC5737 5tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcagattgg 240ctattggcca ttgcatacgt tgtatccata tcataatatg tacatttata ttggctcatg 300tccaacatta ccgccatgtt gacattgatt attgactagt tattaatagt aatcaattac 360ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg 420cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc 480catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac 540tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa 600tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac 660ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta 720catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga 780cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa 840ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag 900agctcgttta gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt ttgacctcca 960tagaagacac cgggaccgat ccagcctcca tcggctcgca tctctccttc acgcgcccgc 1020cgccctacct gaggccgcca tccacgccgg ttgagtcgcg ttctgccgcc tcccgcctgt 1080ggtgcctcct gaactgcgtc cgccgtctag gtaagtttaa agctcaggtc gagaccgggc 1140ctttgtccgg cgctcccttg gagcctacct agactcagcc ggctctccac gctttgcctg 1200accctgcttg ctcaactcta gttaacggtg gagggcagtg tagtctgagc agtactcgtt 1260gctgccgcgc gcgccaccag acataatagc tgacagacta acagactgtt cctttccatg 1320ggtcttttct gcagtcaccg tcgtcgacac gtgtgatcag atatcgcggc cgctctagac 1380accatgcgcg tgaaggagaa gtaccagcac ctgtggcgct ggggctggcg ctggggcacc 1440atgctgctgg gcatgctgat gatctgcagc gccaccgaga agctgtgggt gaccgtgtac 1500tacggcgtgc ccgtgtggaa ggaggccacc accaccctgc tctgcgccag cgacgccaag 1560gcctacgaca ccgaggtgca caacgtgtgg gccacccacg cctgcgtgcc caccgacccc 1620aacccccagg aggtggtgct ggtgaacgtg accgagaact tcgacatgtg gaagaacgac 1680atggtggagc agatgcacga ggacatcatc agcctgtggg accagagcct gaagccctgc 1740gtgaagctga cccccctgtg cgtgagcctg aagtgcaccg acctgaagaa cgacaccaac 1800accaacagca gcagcggccg catgatcatg gagaagggcg agatcaagaa ctgcagcttc 1860aacatcagca ccagcatccg cggcaaggtg cagaaggagt acgccttctt ctacaagctg 1920gacatcatcc ccatcgacaa cgacaccacc agctacagcc tgaccagctg caacaccagc 1980gtgatcaccc aggcctgccc caaggtgagc ttcgagccca tccccaacca ctactgcgcc 2040cccgccggct tcgccatcct gaagtgcaag gacaagaagt tcaacggcaa gggcccctgc 2100accaacgtga gcaccgtgca gtgcacccac ggcatccgcc ccgtggtgag cacccagctg 2160ctggttacgg gtaacctggc cgaggaggag gtggtgatcc gcagcgctaa cttcgccgac 2220aacgccaagg tgatcatcgt gcagctgaac gagagcgtgg agatcaactg cacccgcccc 2280aacaacaaca cccgcaagag catccacatc ggccccggcc gcgccttcta caccaccggc 2340gagatcatcg gcgacatccg ccaggcccac tgcaacctga gccgcgccaa gtggaacgac 2400accctgaaca agatcgtgat caagctgcgc gagcagttcg gcaacaagac catcgtgttc 2460aagcacagca gcggcggcga ccccgagatc gtgacccaca gcttcaactg cggcggcgag 2520ttcttctact gcaacagcac ccagctgttc aacagcacct ggttcaacag cacctggagc 2580accgagggca gcaacaacac cgagggcagc gacaccatca ccctgccctg ccgcatcaag 2640cagatcatca acatgtggca gaaggtgggc aaggccatgt acgccccccc catcagcggc 2700cagatccgct gcagcagcaa catcaccggc ctgctgctga cccgcgacgg cggcaacagc 2760aacaacgaga gcgagatctt ccgcctgggc ggcggcgaca tgcgcgacaa ctggcgcagc 2820gagctgtaca agtacaaggt ggtgaagatc gagcccctgg gcgtggcccc caccaaggcc 2880aagcttaccg tccaggcccg ccagctgctg agcggcatcg tgcagcagca gaacaacctg 2940ctgcgcgcca tcgaggccca gcagcacctg ctgcagctga ccgtgtgggg catcaagcag 3000ctgcaggccc gcaccctggc cgtggagcgc tacctgaagg accagcagct gctcgagcag 3060atctggaacc acaccacctg gatggagtgg gaccgcgaga tcaacaacta caccagcctg 3120atccacagcc tgatcgagga gagccagaac cagcacgaga agaacgagca ggagctgctg 3180gagctggaca agtgggccag cctgtggaac tggttcaaca tcaccaactg gctgtggtac 3240atcaagctgt tcatcatgat cgtgggcggc ctggtgggcc tgcgcatcgt gttcgccgtg 3300ctgagcatct gaggatccag atctgctgtg ccttctagtt gccagccatc tgttgtttgc 3360ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa 3420aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg 3480gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg 3540ggctctatgg gtacccaggt gctgaagaat tgacccggtt cctcctgggc cagaaagaag 3600caggcacatc cccttctctg tgacacaccc tgtccacgcc cctggttctt agttccagcc 3660ccactcatag gacactcata gctcaggagg gctccgcctt caatcccacc cgctaaagta 3720cttggagcgg tctctccctc cctcatcagc ccaccaaacc aaacctagcc tccaagagtg 3780ggaagaaatt aaagcaagat aggctattaa gtgcagaggg agagaaaatg cctccaacat 3840gtgaggaagt aatgagagaa atcatagaat tttaaggcca tgatttaagg ccatcatggc 3900cttaatcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag 3960cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag 4020gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 4080tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc 4140agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc 4200tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt 4260cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg 4320ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat 4380ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag 4440ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt 4500ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc 4560cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta 4620gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag 4680atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga 4740ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa 4800gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa 4860tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcg 4920gggggggggg gcgctgaggt ctgcctcgtg aagaaggtgt tgctgactca taccaggcct 4980gaatcgcccc atcatccagc cagaaagtga gggagccacg gttgatgaga gctttgttgt 5040aggtggacca gttggtgatt ttgaactttt gctttgccac ggaacggtct gcgttgtcgg 5100gaagatgcgt gatctgatcc ttcaactcag caaaagttcg atttattcaa caaagccgcc 5160gtcccgtcaa gtcagcgtaa tgctctgcca gtgttacaac

caattaacca attctgatta 5220gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc 5280atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag 5340gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat 5400taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga 5460atccggtgag aatggcaaaa gcttatgcat ttctttccag acttgttcaa caggccagcc 5520attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc 5580ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg 5640caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc 5700ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc 5760aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag 5820tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa 5880ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt 5940atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta atcgcggcct 6000cgagcaagac gtttcccgtt gaatatggct cataacaccc cttgtattac tgtttatgta 6060agcagacagt tttattgttc atgatgatat atttttatct tgtgcaatgt aacatcagag 6120attttgagac acaacgtggc tttccccccc cccccattat tgaagcattt atcagggtta 6180ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc 6240gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt 6300aacctataaa aataggcgta tcacgaggcc ctttcgtc 633866298DNAArtificial Sequenceplasmid VRC5738 6tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcagattgg 240ctattggcca ttgcatacgt tgtatccata tcataatatg tacatttata ttggctcatg 300tccaacatta ccgccatgtt gacattgatt attgactagt tattaatagt aatcaattac 360ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg 420cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc 480catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac 540tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa 600tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac 660ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta 720catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga 780cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa 840ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag 900agctcgttta gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt ttgacctcca 960tagaagacac cgggaccgat ccagcctcca tcggctcgca tctctccttc acgcgcccgc 1020cgccctacct gaggccgcca tccacgccgg ttgagtcgcg ttctgccgcc tcccgcctgt 1080ggtgcctcct gaactgcgtc cgccgtctag gtaagtttaa agctcaggtc gagaccgggc 1140ctttgtccgg cgctcccttg gagcctacct agactcagcc ggctctccac gctttgcctg 1200accctgcttg ctcaactcta gttaacggtg gagggcagtg tagtctgagc agtactcgtt 1260gctgccgcgc gcgccaccag acataatagc tgacagacta acagactgtt cctttccatg 1320ggtcttttct gcagtcaccg tcgtcgacac gtgtgatcag atatcgcggc cgctctagag 1380atatcgccac catgcgtgtt cgtggtatcc cgcgtaactg gccgcagtgg tggatgtggg 1440gtatcctggg tttctggatg atcatcatct gccgtgttgt tggtaacatg tgggttaccg 1500tttactacgg tgttccggtt tggaccgacg ctaaaaccac cctgttctgc gcttccgaca 1560ccaaagccta cgaccgtgaa gttcacaacg tttgggctac ccacgcttgc gttccgaccg 1620acccgaaccc gcaggaaatc gttctggaaa acgttaccga aaacttcaac atgtggaaaa 1680acgacatggt tgaccagatg cacgaagaca tcatctccct gtgggaccag tccctgaaac 1740cgtgcgttaa actgaccccg ctgtgcgtta ccctgcactg caccaacgct accttcaaaa 1800acaacgttac caacgacatg aacaaagaaa tccgtaactg ctccttcaac accaccaccg 1860aaatccgtga caaaaaacag cagggttacg ctctgttcta ccgtccggac atcgttctgc 1920tgaaagaaaa ccgtaacaac tccaacaact ccgaatacat cctgatcaac tgcaacgctt 1980ccaccatcac ccaggcttgc ccgaaagtta acttcgaccc gatcccgatc cactactgcg 2040ctccggctgg ttacgctatc ctgaaatgca acaacaaaac cttctccggt aaaggtccgt 2100gcaacaacgt ttccaccgtt cagtgcaccc atggtatcaa accggttgtt tccacccagc 2160tgctgctgaa cggttccctg gctgaaaaag aaatcatcat ccgttccgaa aacctgaccg 2220acaacgttaa aaccatcatc gttcacctga acaaatccgt tgaaatcgtt tgcacccgtc 2280cgaacaacaa cacccgtaaa tccatgcgta tcggtccggg tcagaccttc tacgctaccg 2340gtgacatcat cggtgacatc cgtcaggctt actgcaacat ctccggttcc aaatggaacg 2400aaaccctgaa acgtgttaaa gaaaaactgc aggaaaacta caacaacaac aaaaccatca 2460aattcgctcc gtcctccggt ggtgacctgg aaatcaccac ccactccttc aactgccgtg 2520gtgaattctt ctactgcaac accacccgtc tgttcaacaa caacgctacc gaagacgaaa 2580ccatcaccct gccgtgccgt atcaaacaga tcatcaacat gtggcagggt gttggtcgtg 2640ctatgtacgc tccgccgatc gctggtaaca tcacctgcaa atccaacatc accggtctgc 2700tgctggttcg tgacggtggt gaagacaaca aaaccgaaga aatcttccgt ccgggtggtg 2760gtaacatgaa agacaactgg cgttccgaac tgtacaaata caaagttatc gaactgaaac 2820cgctgggtat cgctccgacc ggtgctaagc ttaccgttca ggctcgtcag ctgctgtcct 2880ccatcgttca gcagcagtcc aacctgctgc gtgctatcga agctcagcag cacatgctgc 2940agctgaccgt ttggggtatc aaacagctgc agacccgtgt tctggctatc gaacgttacc 3000tgaaagacca gcagctcgag atctggaaca acatgacctg gatggaatgg gaccgtgaaa 3060tctccaacta caccgacacc atctaccgtc tgctggaaga ctcccagacc cagcaggaaa 3120aaaacgaaaa agacctgctg gctctggact cctggaaaaa cctgtggtcc tggttcgaca 3180tctccaactg gctgtggtac atcaaaatct tcatcatgat cgttggtggt ctgatcggtc 3240tgcgtatcat cttcgctgtt ctgtccatct gaggatccag atctgctgtg ccttctagtt 3300gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc 3360ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt 3420ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca 3480ggcatgctgg ggatgcggtg ggctctatgg gtacccaggt gctgaagaat tgacccggtt 3540cctcctgggc cagaaagaag caggcacatc cccttctctg tgacacaccc tgtccacgcc 3600cctggttctt agttccagcc ccactcatag gacactcata gctcaggagg gctccgcctt 3660caatcccacc cgctaaagta cttggagcgg tctctccctc cctcatcagc ccaccaaacc 3720aaacctagcc tccaagagtg ggaagaaatt aaagcaagat aggctattaa gtgcagaggg 3780agagaaaatg cctccaacat gtgaggaagt aatgagagaa atcatagaat tttaaggcca 3840tgatttaagg ccatcatggc cttaatcttc cgcttcctcg ctcactgact cgctgcgctc 3900ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac 3960agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa 4020ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca 4080caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc 4140gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata 4200cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta 4260tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca 4320gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga 4380cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg 4440tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg 4500tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg 4560caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag 4620aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa 4680cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat 4740ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc 4800tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc 4860atccatagtt gcctgactcg gggggggggg gcgctgaggt ctgcctcgtg aagaaggtgt 4920tgctgactca taccaggcct gaatcgcccc atcatccagc cagaaagtga gggagccacg 4980gttgatgaga gctttgttgt aggtggacca gttggtgatt ttgaactttt gctttgccac 5040ggaacggtct gcgttgtcgg gaagatgcgt gatctgatcc ttcaactcag caaaagttcg 5100atttattcaa caaagccgcc gtcccgtcaa gtcagcgtaa tgctctgcca gtgttacaac 5160caattaacca attctgatta gaaaaactca tcgagcatca aatgaaactg caatttattc 5220atatcaggat tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac 5280tcaccgaggc agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt 5340ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa 5400tcaccatgag tgacgactga atccggtgag aatggcaaaa gcttatgcat ttctttccag 5460acttgttcaa caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg 5520ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa 5580ttacaaacag gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt 5640tcacctgaat caggatattc ttctaatacc tggaatgctg ttttcccggg gatcgcagtg 5700gtgagtaacc atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata 5760aattccgtca gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct 5820ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc 5880gcacctgatt gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg 5940ttggaattta atcgcggcct cgagcaagac gtttcccgtt gaatatggct cataacaccc 6000cttgtattac tgtttatgta agcagacagt tttattgttc atgatgatat atttttatct 6060tgtgcaatgt aacatcagag attttgagac acaacgtggc tttccccccc cccccattat 6120tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa 6180aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa 6240accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtc 629876298DNAArtificial Sequenceplasmid CMV/R-gp145dCFI(CCCC) 7tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcagattgg 240ctattggcca ttgcatacgt tgtatccata tcataatatg tacatttata ttggctcatg 300tccaacatta ccgccatgtt gacattgatt attgactagt tattaatagt aatcaattac 360ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg 420cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc 480catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac 540tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa 600tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac 660ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta 720catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga 780cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa 840ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag 900agctcgttta gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt ttgacctcca 960tagaagacac cgggaccgat ccagcctcca tcggctcgca tctctccttc acgcgcccgc 1020cgccctacct gaggccgcca tccacgccgg ttgagtcgcg ttctgccgcc tcccgcctgt 1080ggtgcctcct gaactgcgtc cgccgtctag gtaagtttaa agctcaggtc gagaccgggc 1140ctttgtccgg cgctcccttg gagcctacct agactcagcc ggctctccac gctttgcctg 1200accctgcttg ctcaactcta gttaacggtg gagggcagtg tagtctgagc agtactcgtt 1260gctgccgcgc gcgccaccag acataatagc tgacagacta acagactgtt cctttccatg 1320ggtcttttct gcagtcaccg tcgtcgacac gtgtgatcag atatcgcggc cgctctagag 1380atatcgccac catgcgtgtt cgtggtatcc cgcgtaactg gccgcagtgg tggatgtggg 1440gtatcctggg tttctggatg atcatcatct gccgtgttgt tggtaacatg tgggttaccg 1500tttactacgg tgttccggtt tggaccgacg ctaaaaccac cctgttctgc gcttccgaca 1560ccaaagccta cgaccgtgaa gttcacaacg tttgggctac ccacgcttgc gttccgaccg 1620acccgaaccc gcaggaaatc gttctggaaa acgttaccga aaacttcaac atgtggaaaa 1680acgacatggt tgaccagatg cacgaagaca tcatctccct gtgggaccag tccctgaaac 1740cgtgcgttaa actgaccccg ctgtgcgtta ccctgcactg caccaacgct accttcaaaa 1800acaacgttac caacgacatg aacaaagaaa tccgtaactg ctccttcaac accaccaccg 1860aaatccgtga caaaaaacag cagggttacg ctctgttcta ccgtccggac atcgttctgc 1920tgaaagaaaa ccgtaacaac tccaacaact ccgaatacat cctgatcaat tgcaacgctt 1980ccaccatcac ccaggcttgc ccgaaagtta acttcgaccc gatcccgatc cactactgcg 2040ctccggctgg ttacgctatc ctgaaatgca acaacaaaac cttctccggt aaaggtccgt 2100gcaacaacgt ttccaccgtt cagtgcaccc atggtatcaa accggttgtt tccacccagc 2160tgctgctgaa cggttccctg gctgaaaaag aaatcatcat ccgttccgaa aacctgaccg 2220acaacgttaa aaccatcatc gttcacctga acaaatccgt tgaaatcgtt tgcacccgtc 2280cgaacaacaa cacccgtaaa tccatgcgta tcggtccggg tcagaccttc tacgctaccg 2340gtgacatcat cggtgacatc cgtcaggctt actgcaacat ctccggttcc aaatggaacg 2400aaaccctgaa acgtgttaaa gaaaaactgc aggaaaacta caacaacaac aaaaccatca 2460aattcgctcc gtcctccggt ggtgacctgg aaatcaccac ccactccttc aactgccgtg 2520gtgaattctt ctactgcaac accacccgtc tgttcaacaa caacgctacc gaagacgaaa 2580ccatcaccct gccgtgccgt atcaaacaga tcatcaacat gtggcagggt gttggtcgtg 2640ctatgtacgc tccgccgatc gctggtaaca tcacctgcaa atccaacatc accggtctgc 2700tgctggttcg tgacggtggt gaagacaaca aaaccgaaga aatcttccgt ccgggtggtg 2760gtaacatgaa agacaactgg cgttccgaac tgtacaaata caaagttatc gaactgaaac 2820cgctgggtat cgctccgacc ggtgctaagc ttaccgttca ggctcgtcag ctgctgtcct 2880ccatcgttca gcagcagtcc aacctgctgc gtgctatcga agctcagcag cacatgctgc 2940agctgaccgt ttggggtatc aaacagctgc agacccgtgt tctggctatc gaacgttacc 3000tgaaagacca gcagctcgag atctggaaca acatgacctg gatggaatgg gaccgtgaaa 3060tctccaacta caccgacacc atctaccgtc tgctggaaga ctcccagacc cagcaggaaa 3120aaaacgaaaa agacctgctg gctctggact cctggaaaaa cctgtggtcc tggttcgaca 3180tctccaactg gctgtggtac atcaaaatct tcatcatgat cgttggtggt ctgatcggtc 3240tgcgtatcat cttcgctgtt ctgtccatct gaggatccag atctgctgtg ccttctagtt 3300gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc 3360ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt 3420ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca 3480ggcatgctgg ggatgcggtg ggctctatgg gtacccaggt gctgaagaat tgacccggtt 3540cctcctgggc cagaaagaag caggcacatc cccttctctg tgacacaccc tgtccacgcc 3600cctggttctt agttccagcc ccactcatag gacactcata gctcaggagg gctccgcctt 3660caatcccacc cgctaaagta cttggagcgg tctctccctc cctcatcagc ccaccaaacc 3720aaacctagcc tccaagagtg ggaagaaatt aaagcaagat aggctattaa gtgcagaggg 3780agagaaaatg cctccaacat gtgaggaagt aatgagagaa atcatagaat tttaaggcca 3840tgatttaagg ccatcatggc cttaatcttc cgcttcctcg ctcactgact cgctgcgctc 3900ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac 3960agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa 4020ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca 4080caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc 4140gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata 4200cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta 4260tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca 4320gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga 4380cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg 4440tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg 4500tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg 4560caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag 4620aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa 4680cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat 4740ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc 4800tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc 4860atccatagtt gcctgactcg gggggggggg gcgctgaggt ctgcctcgtg aagaaggtgt 4920tgctgactca taccaggcct gaatcgcccc atcatccagc cagaaagtga gggagccacg 4980gttgatgaga gctttgttgt aggtggacca gttggtgatt ttgaactttt gctttgccac 5040ggaacggtct gcgttgtcgg gaagatgcgt gatctgatcc ttcaactcag caaaagttcg 5100atttattcaa caaagccgcc gtcccgtcaa gtcagcgtaa tgctctgcca gtgttacaac 5160caattaacca attctgatta gaaaaactca tcgagcatca aatgaaactg caatttattc 5220atatcaggat tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac 5280tcaccgaggc agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt 5340ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa 5400tcaccatgag tgacgactga atccggtgag aatggcaaaa gcttatgcat ttctttccag 5460acttgttcaa caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg 5520ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa 5580ttacaaacag gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt 5640tcacctgaat caggatattc ttctaatacc tggaatgctg ttttcccggg gatcgcagtg 5700gtgagtaacc atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata 5760aattccgtca gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct 5820ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc 5880gcacctgatt gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg 5940ttggaattta atcgcggcct cgagcaagac gtttcccgtt gaatatggct cataacaccc 6000cttgtattac tgtttatgta agcagacagt tttattgttc atgatgatat atttttatct 6060tgtgcaatgt aacatcagag attttgagac acaacgtggc tttccccccc cccccattat 6120tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa 6180aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa 6240accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtc 629886325DNAArtificial Sequenceplasmid CMV/R-gp145dCFI(BBBB) 8tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcagattgg 240ctattggcca ttgcatacgt tgtatccata tcataatatg tacatttata ttggctcatg 300tccaacatta ccgccatgtt gacattgatt attgactagt tattaatagt aatcaattac 360ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg 420cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc 480catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac 540tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa 600tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac 660ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta 720catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga 780cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa 840ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag 900agctcgttta gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt ttgacctcca 960tagaagacac cgggaccgat ccagcctcca tcggctcgca tctctccttc acgcgcccgc 1020cgccttacct gaggccgcca tccacgccgg ttgagtcgcg ttctgccgcc tcccgcctgt 1080ggtgcctcct gaactacgtc cgccgtctag gtaagtttag agctcaggtc gagaccgggc 1140ctttgtccgg

cgctcccttg gagcctacct agactcagcc ggctctccac gctttgcctg 1200accctgcttg ctcaactcta gttaacggtg gagggcagtg tagtctgagc agtactcgtt 1260gctgccgcgc gcgccaccag acataatagc tgacagacta acagactgtt cctttccatg 1320ggtcttttct gcagtcaccg tcgtcgacac gtgtgatcag atatcgcggc cgctctagac 1380accatgcgcg tgaaggagaa gtaccagcac ctgtggcgct ggggctggcg ctggggcacc 1440atgctgctgg gcatcctgat gatctgcaac gccgaggaga agctgtgggt gaccgtgtac 1500tacggcgtgc ccgtgtggaa ggaggccacc accaccctgt tctgcgccag cgaccgcaag 1560gcctacgaca ccgaggtgca caacgtgtgg gccacccacg cctgcgtgcc caccgacccc 1620aacccccagg aggtggagct gaagaacgtg accgagaact tcaacatgtg gaagaacaac 1680atggtggagc agatgcacga ggacatcatc agcctgtggg accagagcct gaagccctgc 1740gtgaagctga cccccctgtg cgtgaccctg aactgcaccg acctgcgcaa cgccaccaac 1800ggaaacgaca caaacacaac aagcagcagc agaggaatgg tgggaggagg cgagatgaag 1860aactgcagct tcaacatcac caccaacatc cgcggcaagg tgcagaagga gtacgccctg 1920ttctacaagc tggacatcgc ccccatcgac aacaactcca acaacagata tagactgatt 1980agctgcaaca ccagcgtgat cacccaggcc tgccccaagg tgagcttcga gcccatcccc 2040atccactact gcgcccccgc cggcttcgcc atcctgaagt gcaaggacaa gaagttcaac 2100ggcaagggcc cctgcaccaa cgtgagcacc gtgcagtgca cccacggcat ccgccccgtg 2160gtgagcaccc agctgctgct gaacggtagc ctggccgagg aggaggtggt gatccgcagc 2220gctaacttcg ccgacaacgc caaggtgatc atcgtgcagc tgaacgagag cgtggagatc 2280aactgcaccc gccccaacaa caacacccgc aagagcatcc acatcggccc cggccgcgcc 2340ttctacacca ccggcgagat catcggcgac atccgccagg cccactgcaa cctgagccgc 2400gccaagtgga acgacaccct gaacaagatc gtgatcaagc tgcgcgagca gttcggcaac 2460aagaccatcg tgttcaagca cagcagcggc ggcgaccccg agatcgtgac ccacagcttc 2520aactgcggcg gcgaattctt ctactgcaac agcacccagc tgtttaattc cacatggaac 2580gtgaccgagg agagcaacaa caccgtggag aacaacacca tcaccctgcc ctgccgcatc 2640aagcagatca tcaacatgtg gcaggaggtg ggccgcgcca tgtacgcccc ccccatccgc 2700ggccagatcc gctgcagcag caacatcacc ggcctgctgc tgacccgcga cggcggcccc 2760gaggacaaca agaccgaggt gttccgccct ggcggcggcg acatgcgcga caactggcgc 2820agcgagctgt acaagtacaa ggtggtgaag atcgagcccc tgggcgtggc ccccaccaag 2880gccaagctta ccgtccaggc ccgcctgctg ctgagcggca tcgtgcagca gcagaacaac 2940ctgctgcgcg ccatcgaggc ccagcagcac ctgctgcagc tgaccgtgtg gggcatcaag 3000cagctgcagg cccgcgtgct ggccgtggag cgctacctgc gcgaccagca gctcctcaag 3060atctgggaca acatgacctg gatcgagtgg gaccgcgaga tcaacaacta caccagcatc 3120atctacagcc tgatcgagga gagccagaac cagcaggaga agaacgagca ggagctgctg 3180gagctggaca agtgggccag cctgtggaac tggttcgaca tcaccaagtg gctgtggtac 3240atcaagatct tcatcatgat cgtgggcggc ctgatcggcc tgcgcatcgt gttcagcgtg 3300ctgagcatct gaggatccag atctgctgtg ccttctagtt gccagccatc tgttgtttgc 3360ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa 3420aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg 3480gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg 3540ggctctatgg gtacccaggt gctgaagaat tgacccggtt cctcctgggc cagaaagaag 3600caggcacatc cccttctctg tgacacaccc tgtccacgcc cctggttctt agttccagcc 3660ccactcatag gacactcata gctcaggagg gctccgcctt caatcccacc cgctaaagta 3720cttggagcgg tctctccctc cctcatcagc ccaccaaacc aaacctagcc tccaagagtg 3780ggaagaaatt aaagcaagat aggctattaa gtgcagaggg agagaaaatg cctccaacat 3840gtgaggaagt aatgagagaa atcatagaat tttaaggcca tcatggcctt aatcttccgc 3900ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca 3960ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg 4020agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca 4080taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa 4140cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc 4200tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc 4260gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct 4320gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg 4380tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag 4440gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta 4500cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg 4560aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt 4620tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt 4680ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag 4740attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat 4800ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc 4860tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactcgggg ggggggggcg 4920ctgaggtctg cctcgtgaag aaggtgttgc tgactcatac caggcctgaa tcgccccatc 4980atccagccag aaagtgaggg agccacggtt gatgagagct ttgttgtagg tggaccagtt 5040ggtgattttg aacttttgct ttgccacgga acggtctgcg ttgtcgggaa gatgcgtgat 5100ctgatccttc aactcagcaa aagttcgatt tattcaacaa agccgccgtc ccgtcaagtc 5160agcgtaatgc tctgccagtg ttacaaccaa ttaaccaatt ctgattagaa aaactcatcg 5220agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa 5280agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc 5340tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg 5400tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat 5460ggcaaaagct tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca 5520tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga 5580aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg 5640aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg 5700aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata 5760aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca 5820tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg 5880ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat 5940ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctcga gcaagacgtt 6000tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc agacagtttt 6060attgttcatg atgatatatt tttatcttgt gcaatgtaac atcagagatt ttgagacaca 6120acgtggcttt cccccccccc ccattattga agcatttatc agggttattg tctcatgagc 6180ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc 6240cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat 6300aggcgtatca cgaggccctt tcgtc 632596298DNAArtificial sequenceplasmid CMV/R-gp145dCFI(BBCB) 9tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcagattgg 240ctattggcca ttgcatacgt tgtatccata tcataatatg tacatttata ttggctcatg 300tccaacatta ccgccatgtt gacattgatt attgactagt tattaatagt aatcaattac 360ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg 420cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc 480catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac 540tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa 600tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac 660ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta 720catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga 780cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa 840ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag 900agctcgttta gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt ttgacctcca 960tagaagacac cgggaccgat ccagcctcca tcggctcgca tctctccttc acgcgcccgc 1020cgccttacct gaggccgcca tccacgccgg ttgagtcgcg ttctgccgcc tcccgcctgt 1080ggtgcctcct gaactacgtc cgccgtctag gtaagtttag agctcaggtc gagaccgggc 1140ctttgtccgg cgctcccttg gagcctacct agactcagcc ggctctccac gctttgcctg 1200accctgcttg ctcaactcta gttaacggtg gagggcagtg tagtctgagc agtactcgtt 1260gctgccgcgc gcgccaccag acataatagc tgacagacta acagactgtt cctttccatg 1320ggtcttttct gcagtcaccg tcgtcgacac gtgtgatcag atatcgcggc cgctctagac 1380accatgcgcg tgaaggagaa gtaccagcac ctgtggcgct ggggctggcg ctggggcacc 1440atgctgctgg gcatcctgat gatctgcaac gccgaggaga agctgtgggt gaccgtgtac 1500tacggcgtgc ccgtgtggaa ggaggccacc accaccctgt tctgcgccag cgaccgcaag 1560gcctacgaca ccgaggtgca caacgtgtgg gccacccacg cctgcgtgcc caccgacccc 1620aacccccagg aggtggagct gaagaacgtg accgagaact tcaacatgtg gaagaacaac 1680atggtggagc agatgcacga ggacatcatc agcctgtggg accagagcct gaagccctgc 1740gtgaagctga cccccctgtg cgtgaccctg aactgcaccg acctgcgcaa cgccaccaac 1800ggaaacgaca caaacacaac aagcagcagc agaggaatgg tgggaggagg cgagatgaag 1860aactgcagct tcaacatcac caccaacatc cgcggcaagg tgcagaagga gtacgccctg 1920ttctacaagc tggacatcgc ccccatcgac aacaactcca acaacagata tagactgatt 1980agctgcaaca ccagcgtgat cacccaggcc tgccccaagg tgagcttcga gcccatcccc 2040atccactact gcgcccccgc cggcttcgcc atcctgaagt gcaaggacaa gaagttcaac 2100ggcaagggcc cctgcaccaa cgtgagcacc gtgcagtgca cccacggcat ccgccccgtg 2160gtgagcaccc agctgctgct gaacggtagc ctggccgagg aggaggtggt gatccgcagc 2220gctaacttcg ccgacaacgc caaggtgatc atcgtgcagc tgaacgagag cgtggagatc 2280aactgcaccc gccccaacaa caacacccgc aagagcatcc acatcggccc cggccgcgcc 2340ttctacacca ccggcgagat catcggcgac atccgccagg cccactgcaa cctgagccgc 2400gccaagtgga acgacaccct gaacaagatc gtgatcaagc tgcgcgagca gttcggcaac 2460aagaccatcg tgttcaagca cagcagcggc ggcgaccccg agatcgtgac ccacagcttc 2520aactgcggcg gcgaattctt ctactgcaac accacccgtc tgttcaacaa caacgctacc 2580gaagacgaaa ccatcaccct gccgtgccgt atcaaacaga tcatcaacat gtggcagggt 2640gttggtcgtg ctatgtacgc tccgccgatc gctggtaaca tcacctgcaa atccaacatc 2700accggtctgc tgctggttcg tgacggtggt gaagacaaca aaaccgaaga aatcttccgt 2760ccgggtggtg gtaacatgaa agacaactgg cgttccgaac tgtacaaata caaagttatc 2820gaactgaaac cgctgggtat cgctccgacc ggtgctaagc ttaccgtcca ggcccgcctg 2880ctgctgagcg gcatcgtgca gcagcagaac aacctgctgc gcgccatcga ggcccagcag 2940cacctgctgc agctgaccgt gtggggcatc aagcagctgc aggcccgcgt gctggccgtg 3000gagcgctacc tgcgcgacca gcagctcctc aagatctggg acaacatgac ctggatcgag 3060tgggaccgcg agatcaacaa ctacaccagc atcatctaca gcctgatcga ggagagccag 3120aaccagcagg agaagaacga gcaggagctg ctggagctgg acaagtgggc cagcctgtgg 3180aactggttcg acatcaccaa gtggctgtgg tacatcaaga tcttcatcat gatcgtgggc 3240ggcctgatcg gcctgcgcat cgtgttcagc gtgctgagca tctgaggatc cagatctgct 3300gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg 3360gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg 3420agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg 3480gaagacaata gcaggcatgc tggggatgcg gtgggctcta tgggtaccca ggtgctgaag 3540aattgacccg gttcctcctg ggccagaaag aagcaggcac atccccttct ctgtgacaca 3600ccctgtccac gcccctggtt cttagttcca gccccactca taggacactc atagctcagg 3660agggctccgc cttcaatccc acccgctaaa gtacttggag cggtctctcc ctccctcatc 3720agcccaccaa accaaaccta gcctccaaga gtgggaagaa attaaagcaa gataggctat 3780taagtgcaga gggagagaaa atgcctccaa catgtgagga agtaatgaga gaaatcatag 3840aattttaagg ccatcatggc cttaatcttc cgcttcctcg ctcactgact cgctgcgctc 3900ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac 3960agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa 4020ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca 4080caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc 4140gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata 4200cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta 4260tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca 4320gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga 4380cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg 4440tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg 4500tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg 4560caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag 4620aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa 4680cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat 4740ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc 4800tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc 4860atccatagtt gcctgactcg gggggggggg gcgctgaggt ctgcctcgtg aagaaggtgt 4920tgctgactca taccaggcct gaatcgcccc atcatccagc cagaaagtga gggagccacg 4980gttgatgaga gctttgttgt aggtggacca gttggtgatt ttgaactttt gctttgccac 5040ggaacggtct gcgttgtcgg gaagatgcgt gatctgatcc ttcaactcag caaaagttcg 5100atttattcaa caaagccgcc gtcccgtcaa gtcagcgtaa tgctctgcca gtgttacaac 5160caattaacca attctgatta gaaaaactca tcgagcatca aatgaaactg caatttattc 5220atatcaggat tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac 5280tcaccgaggc agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt 5340ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa 5400tcaccatgag tgacgactga atccggtgag aatggcaaaa gcttatgcat ttctttccag 5460acttgttcaa caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg 5520ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa 5580ttacaaacag gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt 5640tcacctgaat caggatattc ttctaatacc tggaatgctg ttttcccggg gatcgcagtg 5700gtgagtaacc atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata 5760aattccgtca gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct 5820ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc 5880gcacctgatt gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg 5940ttggaattta atcgcggcct cgagcaagac gtttcccgtt gaatatggct cataacaccc 6000cttgtattac tgtttatgta agcagacagt tttattgttc atgatgatat atttttatct 6060tgtgcaatgt aacatcagag attttgagac acaacgtggc tttccccccc cccccattat 6120tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa 6180aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa 6240accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtc 6298106328DNAArtificial sequenceplasmid CMV/R-gp145dCFI(BCBB) 10tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcagattgg 240ctattggcca ttgcatacgt tgtatccata tcataatatg tacatttata ttggctcatg 300tccaacatta ccgccatgtt gacattgatt attgactagt tattaatagt aatcaattac 360ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg 420cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc 480catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac 540tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa 600tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac 660ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta 720catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga 780cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa 840ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag 900agctcgttta gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt ttgacctcca 960tagaagacac cgggaccgat ccagcctcca tcggctcgca tctctccttc acgcgcccgc 1020cgccttacct gaggccgcca tccacgccgg ttgagtcgcg ttctgccgcc tcccgcctgt 1080ggtgcctcct gaactacgtc cgccgtctag gtaagtttag agctcaggtc gagaccgggc 1140ctttgtccgg cgctcccttg gagcctacct agactcagcc ggctctccac gctttgcctg 1200accctgcttg ctcaactcta gttaacggtg gagggcagtg tagtctgagc agtactcgtt 1260gctgccgcgc gcgccaccag acataatagc tgacagacta acagactgtt cctttccatg 1320ggtcttttct gcagtcaccg tcgtcgacac gtgtgatcag atatcgcggc cgctctagac 1380accatgcgcg tgaaggagaa gtaccagcac ctgtggcgct ggggctggcg ctggggcacc 1440atgctgctgg gcatcctgat gatctgcaac gccgaggaga agctgtgggt gaccgtgtac 1500tacggcgtgc ccgtgtggaa ggaggccacc accaccctgt tctgcgccag cgaccgcaag 1560gcctacgaca ccgaggtgca caacgtgtgg gccacccacg cctgcgtgcc caccgacccc 1620aacccccagg aggtggagct gaagaacgtg accgagaact tcaacatgtg gaagaacaac 1680atggtggagc agatgcacga ggacatcatc agcctgtggg accagagcct gaagccctgc 1740gtgaagctga cccccctgtg cgtgaccctg aactgcaccg acctgcgcaa cgccaccaac 1800ggaaacgaca caaacacaac aagcagcagc agaggaatgg tgggaggagg cgagatgaag 1860aactgcagct tcaacatcac caccaacatc cgcggcaagg tgcagaagga gtacgccctg 1920ttctacaagc tggacatcgc ccccatcgac aacaactcca acaacagata tagactgatt 1980agctgcaacg cttccaccat cacccaggct tgcccgaaag ttaacttcga cccgatcccg 2040atccactact gcgctccggc tggttacgct atcctgaaat gcaacaacaa aaccttctcc 2100ggtaaaggtc cgtgcaacaa cgtttccacc gttcagtgca cccatggtat caaaccggtt 2160gtttccaccc agctgctgct gaacggttcc ctggctgaaa aagaaatcat catccgttcc 2220gaaaacctga ccgacaacgt taaaaccatc atcgttcacc tgaacaaatc cgttgaaatc 2280gtttgcaccc gtccgaacaa caacacccgt aaatccatgc gtatcggtcc gggtcagacc 2340ttctacgcta ccggtgacat catcggtgac atccgtcagg cttactgcaa catctccggt 2400tccaaatgga acgaaaccct gaaacgtgtt aaagaaaaac tgcaggaaaa ctacaacaac 2460aacaaaacca tcaaattcgc tccgtcctcc ggtggtgacc tggaaatcac cacccactcc 2520ttcaactgcc gtggtgaatt cttctactgc aacagcaccc agctgtttaa ttccacatgg 2580aacgtgaccg aggagagcaa caacaccgtg gagaacaaca ccatcaccct gccctgccgc 2640atcaagcaga tcatcaacat gtggcaggag gtgggccgcg ccatgtacgc cccccccatc 2700cgcggccaga tccgctgcag cagcaacatc accggcctgc tgctgacccg cgacggcggc 2760cccgaggaca acaagaccga ggtgttccgc cctggcggcg gcgacatgcg cgacaactgg 2820cgcagcgagc tgtacaagta caaggtggtg aagatcgagc ccctgggcgt ggcccccacc 2880aaggccaagc ttaccgtcca ggcccgcctg ctgctgagcg gcatcgtgca gcagcagaac 2940aacctgctgc gcgccatcga ggcccagcag cacctgctgc agctgaccgt gtggggcatc 3000aagcagctgc aggcccgcgt gctggccgtg gagcgctacc tgcgcgacca gcagctcctc 3060aagatctggg acaacatgac ctggatcgag tgggaccgcg agatcaacaa ctacaccagc 3120atcatctaca gcctgatcga ggagagccag aaccagcagg agaagaacga gcaggagctg 3180ctggagctgg acaagtgggc cagcctgtgg aactggttcg acatcaccaa gtggctgtgg 3240tacatcaaga tcttcatcat gatcgtgggc ggcctgatcg gcctgcgcat cgtgttcagc 3300gtgctgagca tctgaggatc cagatctgct gtgccttcta gttgccagcc atctgttgtt 3360tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa

3420taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg 3480gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg 3540gtgggctcta tgggtaccca ggtgctgaag aattgacccg gttcctcctg ggccagaaag 3600aagcaggcac atccccttct ctgtgacaca ccctgtccac gcccctggtt cttagttcca 3660gccccactca taggacactc atagctcagg agggctccgc cttcaatccc acccgctaaa 3720gtacttggag cggtctctcc ctccctcatc agcccaccaa accaaaccta gcctccaaga 3780gtgggaagaa attaaagcaa gataggctat taagtgcaga gggagagaaa atgcctccaa 3840catgtgagga agtaatgaga gaaatcatag aattttaagg ccatcatggc cttaatcttc 3900cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc 3960tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat 4020gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt 4080ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg 4140aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc 4200tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt 4260ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa 4320gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta 4380tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa 4440caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa 4500ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt 4560cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt 4620ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat 4680cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat 4740gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc 4800aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc 4860acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcg gggggggggg 4920gcgctgaggt ctgcctcgtg aagaaggtgt tgctgactca taccaggcct gaatcgcccc 4980atcatccagc cagaaagtga gggagccacg gttgatgaga gctttgttgt aggtggacca 5040gttggtgatt ttgaactttt gctttgccac ggaacggtct gcgttgtcgg gaagatgcgt 5100gatctgatcc ttcaactcag caaaagttcg atttattcaa caaagccgcc gtcccgtcaa 5160gtcagcgtaa tgctctgcca gtgttacaac caattaacca attctgatta gaaaaactca 5220tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga 5280aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga 5340tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc 5400tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag 5460aatggcaaaa gcttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg 5520tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga 5580cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc 5640aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc 5700tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg 5760ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc 5820tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca 5880tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc 5940catttatacc catataaatc agcatccatg ttggaattta atcgcggcct cgagcaagac 6000gtttcccgtt gaatatggct cataacaccc cttgtattac tgtttatgta agcagacagt 6060tttattgttc atgatgatat atttttatct tgtgcaatgt aacatcagag attttgagac 6120acaacgtggc tttccccccc cccccattat tgaagcattt atcagggtta ttgtctcatg 6180agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt 6240ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa 6300aataggcgta tcacgaggcc ctttcgtc 6328116311DNAArtificial sequenceplasmid CMV/R-gp145dCFI(BCCC) 11tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcagattgg 240ctattggcca ttgcatacgt tgtatccata tcataatatg tacatttata ttggctcatg 300tccaacatta ccgccatgtt gacattgatt attgactagt tattaatagt aatcaattac 360ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg 420cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc 480catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac 540tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa 600tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac 660ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta 720catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga 780cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa 840ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag 900agctcgttta gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt ttgacctcca 960tagaagacac cgggaccgat ccagcctcca tcggctcgca tctctccttc acgcgcccgc 1020cgccctacct gaggccgcca tccacgccgg ttgagtcgcg ttctgccgcc tcccgcctgt 1080ggtgcctcct gaactgcgtc cgccgtctag gtaagtttaa agctcaggtc gagaccgggc 1140ctttgtccgg cgctcccttg gagcctacct agactcagcc ggctctccac gctttgcctg 1200accctgcttg ctcaactcta gttaacggtg gagggcagtg tagtctgagc agtactcgtt 1260gctgccgcgc gcgccaccag acataatagc tgacagacta acagactgtt cctttccatg 1320ggtcttttct gcagtcaccg tcgtcgacac gtgtgatcag atatcgcggc cgctctagac 1380accatgcgcg tgaaggagaa gtaccagcac ctgtggcgct ggggctggcg ctggggcacc 1440atgctgctgg gcatcctgat gatctgcaac gccgaggaga agctgtgggt gaccgtgtac 1500tacggcgtgc ccgtgtggaa ggaggccacc accaccctgt tctgcgccag cgaccgcaag 1560gcctacgaca ccgaggtgca caacgtgtgg gccacccacg cctgcgtgcc caccgacccc 1620aacccccagg aggtggagct gaagaacgtg accgagaact tcaacatgtg gaagaacaac 1680atggtggagc agatgcacga ggacatcatc agcctgtggg accagagcct gaagccctgc 1740gtgaagctga cccccctgtg cgtgaccctg aactgcaccg acctgcgcaa cgccaccaac 1800ggaaacgaca caaacacaac aagcagcagc agaggaatgg tgggaggagg cgagatgaag 1860aactgcagct tcaacatcac caccaacatc cgcggcaagg tgcagaagga gtacgccctg 1920ttctacaagc tggacatcgc ccccatcgac aacaactcca acaacagata tagactgatt 1980agctgcaacg cttccaccat cacccaggct tgcccgaaag ttaacttcga cccgatcccg 2040atccactact gcgctccggc tggttacgct atcctgaaat gcaacaacaa aaccttctcc 2100ggtaaaggtc cgtgcaacaa cgtttccacc gttcagtgca cccatggtat caaaccggtt 2160gtttccaccc agctgctgct gaacggttcc ctggctgaaa aagaaatcat catccgttcc 2220gaaaacctga ccgacaacgt taaaaccatc atcgttcacc tgaacaaatc cgttgaaatc 2280gtttgcaccc gtccgaacaa caacacccgt aaatccatgc gtatcggtcc gggtcagacc 2340ttctacgcta ccggtgacat catcggtgac atccgtcagg cttactgcaa catctccggt 2400tccaaatgga acgaaaccct gaaacgtgtt aaagaaaaac tgcaggaaaa ctacaacaac 2460aacaaaacca tcaaattcgc tccgtcctcc ggtggtgacc tggaaatcac cacccactcc 2520ttcaactgcc gtggtgaatt cttctactgc aacaccaccc gtctgttcaa caacaacgct 2580accgaagacg aaaccatcac cctgccgtgc cgtatcaaac agatcatcaa catgtggcag 2640ggtgttggtc gtgctatgta cgctccgccg atcgctggta acatcacctg caaatccaac 2700atcaccggtc tgctgctggt tcgtgacggt ggtgaagaca acaaaaccga agaaatcttc 2760cgtccgggtg gtggtaacat gaaagacaac tggcgttccg aactgtacaa atacaaagtt 2820atcgaactga aaccgctggg tatcgctccg accggtgcta agcttaccgt tcaggctcgt 2880cagctgctgt cctccatcgt tcagcagcag tccaacctgc tgcgtgctat cgaagctcag 2940cagcacatgc tgcagctgac cgtttggggt atcaaacagc tgcagacccg tgttctggct 3000atcgaacgtt acctgaaaga ccagcagctc gagatctgga acaacatgac ctggatggaa 3060tgggaccgtg aaatctccaa ctacaccgac accatctacc gtctgctgga agactcccag 3120acccagcagg aaaaaaacga aaaagacctg ctggctctgg actcctggaa aaacctgtgg 3180tcctggttcg acatctccaa ctggctgtgg tacatcaaaa tcttcatcat gatcgttggt 3240ggtctgatcg gtctgcgtat catcttcgct gttctgtcca tctgaggatc cagatctgct 3300gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg 3360gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg 3420agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg 3480gaagacaata gcaggcatgc tggggatgcg gtgggctcta tgggtaccca ggtgctgaag 3540aattgacccg gttcctcctg ggccagaaag aagcaggcac atccccttct ctgtgacaca 3600ccctgtccac gcccctggtt cttagttcca gccccactca taggacactc atagctcagg 3660agggctccgc cttcaatccc acccgctaaa gtacttggag cggtctctcc ctccctcatc 3720agcccaccaa accaaaccta gcctccaaga gtgggaagaa attaaagcaa gataggctat 3780taagtgcaga gggagagaaa atgcctccaa catgtgagga agtaatgaga gaaatcatag 3840aattttaagg ccatgattta aggccatcat ggccttaatc ttccgcttcc tcgctcactg 3900actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa 3960tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc 4020aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc 4080ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat 4140aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc 4200cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct 4260cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg 4320aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc 4380cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga 4440ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa 4500gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta 4560gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc 4620agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg 4680acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga 4740tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg 4800agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct 4860gtctatttcg ttcatccata gttgcctgac tcgggggggg ggggcgctga ggtctgcctc 4920gtgaagaagg tgttgctgac tcataccagg cctgaatcgc cccatcatcc agccagaaag 4980tgagggagcc acggttgatg agagctttgt tgtaggtgga ccagttggtg attttgaact 5040tttgctttgc cacggaacgg tctgcgttgt cgggaagatg cgtgatctga tccttcaact 5100cagcaaaagt tcgatttatt caacaaagcc gccgtcccgt caagtcagcg taatgctctg 5160ccagtgttac aaccaattaa ccaattctga ttagaaaaac tcatcgagca tcaaatgaaa 5220ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa 5280tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc 5340gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt 5400atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagcttatg 5460catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc 5520atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct 5580gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc 5640atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc 5700ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt 5760cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt 5820ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa 5880tcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa 5940atcagcatcc atgttggaat ttaatcgcgg cctcgagcaa gacgtttccc gttgaatatg 6000gctcataaca ccccttgtat tactgtttat gtaagcagac agttttattg ttcatgatga 6060tatattttta tcttgtgcaa tgtaacatca gagattttga gacacaacgt ggctttcccc 6120ccccccccat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga 6180atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc 6240tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag 6300gccctttcgt c 6311126312DNAArtificial sequenceplasmid CMV/R-gp145dCFI(CBBB) 12tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcagattgg 240ctattggcca ttgcatacgt tgtatccata tcataatatg tacatttata ttggctcatg 300tccaacatta ccgccatgtt gacattgatt attgactagt tattaatagt aatcaattac 360ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg 420cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc 480catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac 540tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa 600tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac 660ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta 720catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga 780cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa 840ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag 900agctcgttta gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt ttgacctcca 960tagaagacac cgggaccgat ccagcctcca tcggctcgca tctctccttc acgcgcccgc 1020cgccttacct gaggccgcca tccacgccgg ttgagtcgcg ttctgccgcc tcccgcctgt 1080ggtgcctcct gaactacgtc cgccgtctag gtaagtttag agctcaggtc gagaccgggc 1140ctttgtccgg cgctcccttg gagcctacct agactcagcc ggctctccac gctttgcctg 1200accctgcttg ctcaactcta gttaacggtg gagggcagtg tagtctgagc agtactcgtt 1260gctgccgcgc gcgccaccag acataatagc tgacagacta acagactgtt cctttccatg 1320ggtcttttct gcagtcaccg tcgtcgacac gtgtgatcag atatcgcggc cgctctagag 1380atatcgccac catgcgtgtt cgtggtatcc cgcgtaactg gccgcagtgg tggatgtggg 1440gtatcctggg tttctggatg atcatcatct gccgtgttgt tggtaacatg tgggttaccg 1500tttactacgg tgttccggtt tggaccgacg ctaaaaccac cctgttctgc gcttccgaca 1560ccaaagccta cgaccgtgaa gttcacaacg tttgggctac ccacgcttgc gttccgaccg 1620acccgaaccc gcaggaaatc gttctggaaa acgttaccga aaacttcaac atgtggaaaa 1680acgacatggt tgaccagatg cacgaagaca tcatctccct gtgggaccag tccctgaaac 1740cgtgcgttaa actgaccccg ctgtgcgtta ccctgcactg caccaacgct accttcaaaa 1800acaacgttac caacgacatg aacaaagaaa tccgtaactg ctccttcaac accaccaccg 1860aaatccgtga caaaaaacag cagggttacg ctctgttcta ccgtccggac atcgttctgc 1920tgaaagaaaa ccgtaacaac tccaacaact ccgaatacat cctgatcaat tgcaacacca 1980gcgtgatcac ccaggcctgc cccaaggtga gcttcgagcc catccccatc cactactgcg 2040cccccgccgg cttcgccatc ctgaagtgca aggacaagaa gttcaacggc aagggcccct 2100gcaccaacgt gagcaccgtg cagtgcaccc acggcatccg ccccgtggtg agcacccagc 2160tgctgctgaa cggtagcctg gccgaggagg aggtggtgat ccgcagcgct aacttcgccg 2220acaacgccaa ggtgatcatc gtgcagctga acgagagcgt ggagatcaac tgcacccgcc 2280ccaacaacaa cacccgcaag agcatccaca tcggccccgg ccgcgccttc tacaccaccg 2340gcgagatcat cggcgacatc cgccaggccc actgcaacct gagccgcgcc aagtggaacg 2400acaccctgaa caagatcgtg atcaagctgc gcgagcagtt cggcaacaag accatcgtgt 2460tcaagcacag cagcggcggc gaccccgaga tcgtgaccca cagcttcaac tgcggcggcg 2520aattcttcta ctgcaacagc acccagctgt ttaattccac atggaacgtg accgaggaga 2580gcaacaacac cgtggagaac aacaccatca ccctgccctg ccgcatcaag cagatcatca 2640acatgtggca ggaggtgggc cgcgccatgt acgccccccc catccgcggc cagatccgct 2700gcagcagcaa catcaccggc ctgctgctga cccgcgacgg cggccccgag gacaacaaga 2760ccgaggtgtt ccgccctggc ggcggcgaca tgcgcgacaa ctggcgcagc gagctgtaca 2820agtacaaggt ggtgaagatc gagcccctgg gcgtggcccc caccaaggcc aagcttaccg 2880tccaggcccg cctgctgctg agcggcatcg tgcagcagca gaacaacctg ctgcgcgcca 2940tcgaggccca gcagcacctg ctgcagctga ccgtgtgggg catcaagcag ctgcaggccc 3000gcgtgctggc cgtggagcgc tacctgcgcg accagcagct cctcaagatc tgggacaaca 3060tgacctggat cgagtgggac cgcgagatca acaactacac cagcatcatc tacagcctga 3120tcgaggagag ccagaaccag caggagaaga acgagcagga gctgctggag ctggacaagt 3180gggccagcct gtggaactgg ttcgacatca ccaagtggct gtggtacatc aagatcttca 3240tcatgatcgt gggcggcctg atcggcctgc gcatcgtgtt cagcgtgctg agcatctgag 3300gatccagatc tgctgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc 3360cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg 3420catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca 3480agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatgggta 3540cccaggtgct gaagaattga cccggttcct cctgggccag aaagaagcag gcacatcccc 3600ttctctgtga cacaccctgt ccacgcccct ggttcttagt tccagcccca ctcataggac 3660actcatagct caggagggct ccgccttcaa tcccacccgc taaagtactt ggagcggtct 3720ctccctccct catcagccca ccaaaccaaa cctagcctcc aagagtggga agaaattaaa 3780gcaagatagg ctattaagtg cagagggaga gaaaatgcct ccaacatgtg aggaagtaat 3840gagagaaatc atagaatttt aaggccatca tggccttaat cttccgcttc ctcgctcact 3900gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta 3960atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag 4020caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc 4080cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta 4140taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg 4200ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc 4260tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac 4320gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac 4380ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg 4440aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga 4500agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt 4560agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag 4620cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct 4680gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg 4740atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat 4800gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc 4860tgtctatttc gttcatccat agttgcctga ctcggggggg gggggcgctg aggtctgcct 4920cgtgaagaag gtgttgctga ctcataccag gcctgaatcg ccccatcatc cagccagaaa 4980gtgagggagc cacggttgat gagagctttg ttgtaggtgg accagttggt gattttgaac 5040ttttgctttg ccacggaacg gtctgcgttg tcgggaagat gcgtgatctg atccttcaac 5100tcagcaaaag ttcgatttat tcaacaaagc cgccgtcccg tcaagtcagc gtaatgctct 5160gccagtgtta caaccaatta accaattctg attagaaaaa ctcatcgagc atcaaatgaa 5220actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta 5280atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg 5340cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt 5400tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagcttat 5460gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg 5520catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc 5580tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg

5640catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc 5700cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg 5760tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat 5820tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca 5880atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata 5940aatcagcatc catgttggaa tttaatcgcg gcctcgagca agacgtttcc cgttgaatat 6000ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgatg 6060atatattttt atcttgtgca atgtaacatc agagattttg agacacaacg tggctttccc 6120ccccccccca ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg 6180aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac 6240ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga 6300ggccctttcg tc 6312136295DNAArtificial sequenceplasmid CMV/R-gp145dCFI(CBCC) 13tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcagattgg 240ctattggcca ttgcatacgt tgtatccata tcataatatg tacatttata ttggctcatg 300tccaacatta ccgccatgtt gacattgatt attgactagt tattaatagt aatcaattac 360ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg 420cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc 480catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac 540tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa 600tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac 660ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta 720catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga 780cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa 840ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag 900agctcgttta gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt ttgacctcca 960tagaagacac cgggaccgat ccagcctcca tcggctcgca tctctccttc acgcgcccgc 1020cgccctacct gaggccgcca tccacgccgg ttgagtcgcg ttctgccgcc tcccgcctgt 1080ggtgcctcct gaactgcgtc cgccgtctag gtaagtttaa agctcaggtc gagaccgggc 1140ctttgtccgg cgctcccttg gagcctacct agactcagcc ggctctccac gctttgcctg 1200accctgcttg ctcaactcta gttaacggtg gagggcagtg tagtctgagc agtactcgtt 1260gctgccgcgc gcgccaccag acataatagc tgacagacta acagactgtt cctttccatg 1320ggtcttttct gcagtcaccg tcgtcgacac gtgtgatcag atatcgcggc cgctctagag 1380atatcgccac catgcgtgtt cgtggtatcc cgcgtaactg gccgcagtgg tggatgtggg 1440gtatcctggg tttctggatg atcatcatct gccgtgttgt tggtaacatg tgggttaccg 1500tttactacgg tgttccggtt tggaccgacg ctaaaaccac cctgttctgc gcttccgaca 1560ccaaagccta cgaccgtgaa gttcacaacg tttgggctac ccacgcttgc gttccgaccg 1620acccgaaccc gcaggaaatc gttctggaaa acgttaccga aaacttcaac atgtggaaaa 1680acgacatggt tgaccagatg cacgaagaca tcatctccct gtgggaccag tccctgaaac 1740cgtgcgttaa actgaccccg ctgtgcgtta ccctgcactg caccaacgct accttcaaaa 1800acaacgttac caacgacatg aacaaagaaa tccgtaactg ctccttcaac accaccaccg 1860aaatccgtga caaaaaacag cagggttacg ctctgttcta ccgtccggac atcgttctgc 1920tgaaagaaaa ccgtaacaac tccaacaact ccgaatacat cctgatcaat tgcaacacca 1980gcgtgatcac ccaggcctgc cccaaggtga gcttcgagcc catccccatc cactactgcg 2040cccccgccgg cttcgccatc ctgaagtgca aggacaagaa gttcaacggc aagggcccct 2100gcaccaacgt gagcaccgtg cagtgcaccc acggcatccg ccccgtggtg agcacccagc 2160tgctgctgaa cggtagcctg gccgaggagg aggtggtgat ccgcagcgct aacttcgccg 2220acaacgccaa ggtgatcatc gtgcagctga acgagagcgt ggagatcaac tgcacccgcc 2280ccaacaacaa cacccgcaag agcatccaca tcggccccgg ccgcgccttc tacaccaccg 2340gcgagatcat cggcgacatc cgccaggccc actgcaacct gagccgcgcc aagtggaacg 2400acaccctgaa caagatcgtg atcaagctgc gcgagcagtt cggcaacaag accatcgtgt 2460tcaagcacag cagcggcggc gaccccgaga tcgtgaccca cagcttcaac tgcggcggcg 2520aattcttcta ctgcaacacc acccgtctgt tcaacaacaa cgctaccgaa gacgaaacca 2580tcaccctgcc gtgccgtatc aaacagatca tcaacatgtg gcagggtgtt ggtcgtgcta 2640tgtacgctcc gccgatcgct ggtaacatca cctgcaaatc caacatcacc ggtctgctgc 2700tggttcgtga cggtggtgaa gacaacaaaa ccgaagaaat cttccgtccg ggtggtggta 2760acatgaaaga caactggcgt tccgaactgt acaaatacaa agttatcgaa ctgaaaccgc 2820tgggtatcgc tccgaccggt gctaagctta ccgttcaggc tcgtcagctg ctgtcctcca 2880tcgttcagca gcagtccaac ctgctgcgtg ctatcgaagc tcagcagcac atgctgcagc 2940tgaccgtttg gggtatcaaa cagctgcaga cccgtgttct ggctatcgaa cgttacctga 3000aagaccagca gctcgagatc tggaacaaca tgacctggat ggaatgggac cgtgaaatct 3060ccaactacac cgacaccatc taccgtctgc tggaagactc ccagacccag caggaaaaaa 3120acgaaaaaga cctgctggct ctggactcct ggaaaaacct gtggtcctgg ttcgacatct 3180ccaactggct gtggtacatc aaaatcttca tcatgatcgt tggtggtctg atcggtctgc 3240gtatcatctt cgctgttctg tccatctgag gatccagatc tgctgtgcct tctagttgcc 3300agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca 3360ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta 3420ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc 3480atgctgggga tgcggtgggc tctatgggta cccaggtgct gaagaattga cccggttcct 3540cctgggccag aaagaagcag gcacatcccc ttctctgtga cacaccctgt ccacgcccct 3600ggttcttagt tccagcccca ctcataggac actcatagct caggagggct ccgccttcaa 3660tcccacccgc taaagtactt ggagcggtct ctccctccct catcagccca ccaaaccaaa 3720cctagcctcc aagagtggga agaaattaaa gcaagatagg ctattaagtg cagagggaga 3780gaaaatgcct ccaacatgtg aggaagtaat gagagaaatc atagaatttt aaggccatga 3840tttaaggcca tcatggcctt aatcttccgc ttcctcgctc actgactcgc tgcgctcggt 3900cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga 3960atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg 4020taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa 4080aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt 4140tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct 4200gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct 4260cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc 4320cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt 4380atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc 4440tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat 4500ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa 4560acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa 4620aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga 4680aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct 4740tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga 4800cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc 4860catagttgcc tgactcgggg ggggggggcg ctgaggtctg cctcgtgaag aaggtgttgc 4920tgactcatac caggcctgaa tcgccccatc atccagccag aaagtgaggg agccacggtt 4980gatgagagct ttgttgtagg tggaccagtt ggtgattttg aacttttgct ttgccacgga 5040acggtctgcg ttgtcgggaa gatgcgtgat ctgatccttc aactcagcaa aagttcgatt 5100tattcaacaa agccgccgtc ccgtcaagtc agcgtaatgc tctgccagtg ttacaaccaa 5160ttaaccaatt ctgattagaa aaactcatcg agcatcaaat gaaactgcaa tttattcata 5220tcaggattat caataccata tttttgaaaa agccgtttct gtaatgaagg agaaaactca 5280ccgaggcagt tccataggat ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca 5340acatcaatac aacctattaa tttcccctcg tcaaaaataa ggttatcaag tgagaaatca 5400ccatgagtga cgactgaatc cggtgagaat ggcaaaagct tatgcatttc tttccagact 5460tgttcaacag gccagccatt acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta 5520ttcattcgtg attgcgcctg agcgagacga aatacgcgat cgctgttaaa aggacaatta 5580caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac aatattttca 5640cctgaatcag gatattcttc taatacctgg aatgctgttt tcccggggat cgcagtggtg 5700agtaaccatg catcatcagg agtacggata aaatgcttga tggtcggaag aggcataaat 5760tccgtcagcc agtttagtct gaccatctca tctgtaacat cattggcaac gctacctttg 5820ccatgtttca gaaacaactc tggcgcatcg ggcttcccat acaatcgata gattgtcgca 5880cctgattgcc cgacattatc gcgagcccat ttatacccat ataaatcagc atccatgttg 5940gaatttaatc gcggcctcga gcaagacgtt tcccgttgaa tatggctcat aacacccctt 6000gtattactgt ttatgtaagc agacagtttt attgttcatg atgatatatt tttatcttgt 6060gcaatgtaac atcagagatt ttgagacaca acgtggcttt cccccccccc ccattattga 6120agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat 6180aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc 6240attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtc 6295146325DNAArtificial sequenceplasmid CMV/R-gp145dCFI(CCBC) 14tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcagattgg 240ctattggcca ttgcatacgt tgtatccata tcataatatg tacatttata ttggctcatg 300tccaacatta ccgccatgtt gacattgatt attgactagt tattaatagt aatcaattac 360ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg 420cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc 480catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac 540tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa 600tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac 660ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta 720catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga 780cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa 840ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag 900agctcgttta gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt ttgacctcca 960tagaagacac cgggaccgat ccagcctcca tcggctcgca tctctccttc acgcgcccgc 1020cgccctacct gaggccgcca tccacgccgg ttgagtcgcg ttctgccgcc tcccgcctgt 1080ggtgcctcct gaactgcgtc cgccgtctag gtaagtttaa agctcaggtc gagaccgggc 1140ctttgtccgg cgctcccttg gagcctacct agactcagcc ggctctccac gctttgcctg 1200accctgcttg ctcaactcta gttaacggtg gagggcagtg tagtctgagc agtactcgtt 1260gctgccgcgc gcgccaccag acataatagc tgacagacta acagactgtt cctttccatg 1320ggtcttttct gcagtcaccg tcgtcgacac gtgtgatcag atatcgcggc cgctctagag 1380atatcgccac catgcgtgtt cgtggtatcc cgcgtaactg gccgcagtgg tggatgtggg 1440gtatcctggg tttctggatg atcatcatct gccgtgttgt tggtaacatg tgggttaccg 1500tttactacgg tgttccggtt tggaccgacg ctaaaaccac cctgttctgc gcttccgaca 1560ccaaagccta cgaccgtgaa gttcacaacg tttgggctac ccacgcttgc gttccgaccg 1620acccgaaccc gcaggaaatc gttctggaaa acgttaccga aaacttcaac atgtggaaaa 1680acgacatggt tgaccagatg cacgaagaca tcatctccct gtgggaccag tccctgaaac 1740cgtgcgttaa actgaccccg ctgtgcgtta ccctgcactg caccaacgct accttcaaaa 1800acaacgttac caacgacatg aacaaagaaa tccgtaactg ctccttcaac accaccaccg 1860aaatccgtga caaaaaacag cagggttacg ctctgttcta ccgtccggac atcgttctgc 1920tgaaagaaaa ccgtaacaac tccaacaact ccgaatacat cctgatcaat tgcaacgctt 1980ccaccatcac ccaggcttgc ccgaaagtta acttcgaccc gatcccgatc cactactgcg 2040ctccggctgg ttacgctatc ctgaaatgca acaacaaaac cttctccggt aaaggtccgt 2100gcaacaacgt ttccaccgtt cagtgcaccc atggtatcaa accggttgtt tccacccagc 2160tgctgctgaa cggttccctg gctgaaaaag aaatcatcat ccgttccgaa aacctgaccg 2220acaacgttaa aaccatcatc gttcacctga acaaatccgt tgaaatcgtt tgcacccgtc 2280cgaacaacaa cacccgtaaa tccatgcgta tcggtccggg tcagaccttc tacgctaccg 2340gtgacatcat cggtgacatc cgtcaggctt actgcaacat ctccggttcc aaatggaacg 2400aaaccctgaa acgtgttaaa gaaaaactgc aggaaaacta caacaacaac aaaaccatca 2460aattcgctcc gtcctccggt ggtgacctgg aaatcaccac ccactccttc aactgccgtg 2520gtgaattctt ctactgcaac agcacccagc tgtttaattc cacatggaac gtgaccgagg 2580agagcaacaa caccgtggag aacaacacca tcaccctgcc ctgccgcatc aagcagatca 2640tcaacatgtg gcaggaggtg ggccgcgcca tgtacgcccc ccccatccgc ggccagatcc 2700gctgcagcag caacatcacc ggcctgctgc tgacccgcga cggcggcccc gaggacaaca 2760agaccgaggt gttccgccct ggcggcggcg acatgcgcga caactggcgc agcgagctgt 2820acaagtacaa ggtggtgaag atcgagcccc tgggcgtggc ccccaccaag gccaagctta 2880ccgttcaggc tcgtcagctg ctgtcctcca tcgttcagca gcagtccaac ctgctgcgtg 2940ctatcgaagc tcagcagcac atgctgcagc tgaccgtttg gggtatcaaa cagctgcaga 3000cccgtgttct ggctatcgaa cgttacctga aagaccagca gctcgagatc tggaacaaca 3060tgacctggat ggaatgggac cgtgaaatct ccaactacac cgacaccatc taccgtctgc 3120tggaagactc ccagacccag caggaaaaaa acgaaaaaga cctgctggct ctggactcct 3180ggaaaaacct gtggtcctgg ttcgacatct ccaactggct gtggtacatc aaaatcttca 3240tcatgatcgt tggtggtctg atcggtctgc gtatcatctt cgctgttctg tccatctgag 3300gatccagatc tgctgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc 3360cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg 3420catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca 3480agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatgggta 3540cccaggtgct gaagaattga cccggttcct cctgggccag aaagaagcag gcacatcccc 3600ttctctgtga cacaccctgt ccacgcccct ggttcttagt tccagcccca ctcataggac 3660actcatagct caggagggct ccgccttcaa tcccacccgc taaagtactt ggagcggtct 3720ctccctccct catcagccca ccaaaccaaa cctagcctcc aagagtggga agaaattaaa 3780gcaagatagg ctattaagtg cagagggaga gaaaatgcct ccaacatgtg aggaagtaat 3840gagagaaatc atagaatttt aaggccatga tttaaggcca tcatggcctt aatcttccgc 3900ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca 3960ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg 4020agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca 4080taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa 4140cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc 4200tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc 4260gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct 4320gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg 4380tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag 4440gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta 4500cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg 4560aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt 4620tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt 4680ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag 4740attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat 4800ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc 4860tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactcgggg ggggggggcg 4920ctgaggtctg cctcgtgaag aaggtgttgc tgactcatac caggcctgaa tcgccccatc 4980atccagccag aaagtgaggg agccacggtt gatgagagct ttgttgtagg tggaccagtt 5040ggtgattttg aacttttgct ttgccacgga acggtctgcg ttgtcgggaa gatgcgtgat 5100ctgatccttc aactcagcaa aagttcgatt tattcaacaa agccgccgtc ccgtcaagtc 5160agcgtaatgc tctgccagtg ttacaaccaa ttaaccaatt ctgattagaa aaactcatcg 5220agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa 5280agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc 5340tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg 5400tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat 5460ggcaaaagct tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca 5520tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga 5580aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg 5640aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg 5700aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata 5760aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca 5820tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg 5880ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat 5940ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctcga gcaagacgtt 6000tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc agacagtttt 6060attgttcatg atgatatatt tttatcttgt gcaatgtaac atcagagatt ttgagacaca 6120acgtggcttt cccccccccc ccattattga agcatttatc agggttattg tctcatgagc 6180ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc 6240cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat 6300aggcgtatca cgaggccctt tcgtc 6325156318DNAArtificial sequenceplasmid CMV/Rgp-145dCFI(CN54) 15tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcagattgg 240ctattggcca ttgcatacgt tgtatccata tcataatatg tacatttata ttggctcatg 300tccaacatta ccgccatgtt gacattgatt attgactagt tattaatagt aatcaattac 360ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg 420cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc 480catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac 540tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa 600tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac 660ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta 720catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga 780cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa 840ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag 900agctcgttta gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt ttgacctcca 960tagaagacac cgggaccgat ccagcctcca tcggctcgca tctctccttc acgcgcccgc 1020cgccttacct gaggccgcca tccacgccgg ttgagtcgcg ttctgccgcc tcccgcctgt 1080ggtgcctcct gaactacgtc cgccgtctag gtaagtttag agctcaggtc gagaccgggc 1140ctttgtccgg cgctcccttg gagcctacct agactcagcc ggctctccac gctttgcctg 1200accctgcttg ctcaactcta gttaacggtg gagggcagtg tagtctgagc agtactcgtt 1260gctgccgcgc gcgccaccag acataatagc tgacagacta acagactgtt cctttccatg 1320ggtcttttct gcagtcaccg tcgtcgacac gtgtgatcag atatcgcggc cgctctagag 1380atatcgacac catggacagg gccaagctgc tgctgctgct gctgctgctg ctgctgcccc 1440aggcccaggc cgtgggcaac ctgtgggtga ccgtgtacta cggcgtgccc gtgtggaagg

1500gcgccaccac caccctgttc tgcgccagcg acgccaaggc ctacgacacc gaggtgcaca 1560acgtgtgggc cacccacgcc tgcgtgcccg ccgaccccaa cccccaggag atggtgctgg 1620agaacgtgac cgagaacttc aacatgtgga agaacgagat ggtgaaccag atgcaggagg 1680acgtcatcag cctgtgggac cagagcctga agccctgcgt gaagctgacc cccctgtgcg 1740tgaccctgga gtgcaggaac gtgagcagca acagcaacga cacctaccac gagacctacc 1800acgagagcat gaaggagatg aagaactgca gcttcaacgc caccaccgtg gtgagggaca 1860ggaagcagac cgtgtacgcc ctgttctaca ggctggacat cgtgcccctg accaagaaga 1920actacagcga gaacagcagc gagtactaca ggctgatcaa ctgcaacacc agcgccatca 1980cccaggcctg ccccaaggtg accttcgacc ccatccccat ccactactgc acccccgccg 2040gctacgccat cctgaagtgc aacgacaaga tcttcaacgg caccggcccc tgccacaacg 2100tgagcaccgt gcagtgcacc cacggcatca agcccgtggt gagcacccag ctgctgctga 2160acggcagcct ggccgagggc gagatcatca tcaggagcga gaacctgacc aacaacgtga 2220aaaccatcat cgtgcacctg aaccagagcg tggagatcgt gtgcaccagg cccggcaaca 2280acaccaggaa gagcatcagg atcggccccg gccagacctt ctacgccacc ggcgacatca 2340tcggcgacat caggcaggcc cactgcaaca tcagcgagga caagtggaac gagaccctgc 2400agagggtgag caagaagctg gccgagcact tccagaacaa gaccatcaag ttcgccagca 2460gcagcggcgg cgacctggag gtgaccaccc acagcttcaa ctgcaggggc gagttcttct 2520actgcaacac cagcggcctg ttcaacggcg cctacacccc caacggcacc aagagcaaca 2580gcagcagcat catcaccatc ccctgcagga tcaagcagat catcaacatg tggcaggagg 2640tgggcagggc catgtacgcc cctcccatca agggcaacat cacctgcaag agcaacatca 2700ccggcctgct gctggtgagg gacggcggca ccgagcccaa cgacaccgag accttcaggc 2760ccggcggcgg cgacatgagg aacaactgga ggagcgagct gtacaagtac aaggtggtgg 2820agatcaagcc cctgggcgtg gcccccacca ccaccaagct taccgtgcag gccaggcagc 2880tgctgagcgg catcgtgcag cagcagagca acctgctgag ggccatcgag gcccagcagc 2940acctgctgca gctgaccgtg tggggcatca agcagctgca gaccagggtg ctggccatcg 3000agaggtacct gaaggaccag cagctcgaga tctgggacaa catgacctgg atgcagtggg 3060acaaggagat cagcaactac accaacaccg tgtacaggct gctggaggag agccagaacc 3120agcaggagag gaacgagaag gacctgctgg ccctggacag ctggaagaac ctgtggagct 3180ggttcgacat caccaactgg ctgtggtaca tcaagatctt catcatcatc gtgggcggcc 3240tgatcggcct gaggatcatc ttcgccgtgc tgagcatcgt gaacagggtg aggcagggct 3300actgaggatc cagatctgct gtgccttcta gttgccagcc atctgttgtt tgcccctccc 3360ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg 3420aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg 3480acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta 3540tgggtaccca ggtgctgaag aattgacccg gttcctcctg ggccagaaag aagcaggcac 3600atccccttct ctgtgacaca ccctgtccac gcccctggtt cttagttcca gccccactca 3660taggacactc atagctcagg agggctccgc cttcaatccc acccgctaaa gtacttggag 3720cggtctctcc ctccctcatc agcccaccaa accaaaccta gcctccaaga gtgggaagaa 3780attaaagcaa gataggctat taagtgcaga gggagagaaa atgcctccaa catgtgagga 3840agtaatgaga gaaatcatag aattttaagg ccatcatggc cttaatcttc cgcttcctcg 3900ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag 3960gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa 4020ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 4080cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 4140ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 4200accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 4260catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 4320gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 4380tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 4440agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 4500actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 4560gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc 4620aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg 4680gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca 4740aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt 4800atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca 4860gcgatctgtc tatttcgttc atccatagtt gcctgactcg gggggggggg gcgctgaggt 4920ctgcctcgtg aagaaggtgt tgctgactca taccaggcct gaatcgcccc atcatccagc 4980cagaaagtga gggagccacg gttgatgaga gctttgttgt aggtggacca gttggtgatt 5040ttgaactttt gctttgccac ggaacggtct gcgttgtcgg gaagatgcgt gatctgatcc 5100ttcaactcag caaaagttcg atttattcaa caaagccgcc gtcccgtcaa gtcagcgtaa 5160tgctctgcca gtgttacaac caattaacca attctgatta gaaaaactca tcgagcatca 5220aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga aaaagccgtt 5280tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga tcctggtatc 5340ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa 5400taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag aatggcaaaa 5460gcttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg tcatcaaaat 5520cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc 5580gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc aggaacactg 5640ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc tggaatgctg 5700ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg ataaaatgct 5760tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc tcatctgtaa 5820catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc 5880catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc catttatacc 5940catataaatc agcatccatg ttggaattta atcgcggcct cgagcaagac gtttcccgtt 6000gaatatggct cataacaccc cttgtattac tgtttatgta agcagacagt tttattgttc 6060atgatgatat atttttatct tgtgcaatgt aacatcagag attttgagac acaacgtggc 6120tttccccccc cccccattat tgaagcattt atcagggtta ttgtctcatg agcggataca 6180tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag 6240tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta 6300tcacgaggcc ctttcgtc 63181636066DNAArtificial SequenceAdenoviral vector Adt.GagPol(B).11D 16catcatcaat aatatacctt attttggatt gaagccaata tgataatgag ggggtggagt 60ttgtgacgtg gcgcggggcg tgggaacggg gcgggtgacg tagtagtgtg gcggaagtgt 120gatgttgcaa gtgtggcgga acacatgtaa gcgacggatg tggcaaaagt gacgtttttg 180gtgtgcgccg gtgtacacag gaagtgacaa ttttcgcgcg gttttaggcg gatgttgtag 240taaatttggg cgtaaccgag taagatttgg ccattttcgc gggaaaactg aataagagga 300agtgaaatct gaataatttt gtgttactca tagcgcgtaa tatttgtcta gggcccggga 360tcggtgatca ccgatccaga catgataaga tacattgatg agtttggaca aaccacaact 420agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta 480accattataa gctgcaataa acaagttccc ggatctttct agctagtcta gaattcctag 540tcctcgtcct ggcggctggc cacgcagtcg tcgccggcca tctgcttgcc gtagtcgcgg 600atgatcttgg ccttgcggcg gggcaccacc ttgatgtcgc tgttgtcctg gatcaccacg 660gcgccctcgc ccttccacag cagcttggcg gggcccttcc acacggggtc gcggctgtcg 720cggtagtaca cgcggaagtt ctggatcttg gtgatctgct tctgcagctc cttggtctgg 780atgtcggtgg cgatgatgtc cacgatgcgc tcgccggcgc tgtagccgcc gatgccgccc 840ttgcgcttga agttgtggat gaacacggcc atctgcacgg cggtcttcag gtgctcggcc 900tggtcgcgca cctggccgat gatcttcttc agctccttgt tcatgctctc gatcacgccc 960tggctctggg ggttgtaggg gatgccgaac tcctgcttga tgccggccca ccagcaggcg 1020gccttcacgg tggtgctggt gaagttgctg ccgttgtcgg tgtgcacggt cttcacgggc 1080cagcggccgg ccagcttcag caggaagtag gcggtctcct ggccggtctc ggcggggatc 1140acctcggcct cgatgtagcc gctggccacg tgcacggcca ccaggatcac cttgccctcc 1200aggtgggtgc agtccagctg ccagatgccg gggctgcagt ccacctggcc gtgcatggcc 1260tcgcccttca gctggcactt gtcgcagctg gccacgatct ccttggccac cacggggggc 1320aggttgaagt cgctggccat ggcgcgccag ttgctgtggt acttctcgtg ctcctcctgg 1380gccttgtcga tgccgtccag gaacagcacc ttgcggatgc cggcgctcac caggccgtcc 1440acctgctcgt tgccgccgat gcccttgtgg gcgggcaccc aggccaggta caccttctcc 1500ttcttgatca gctgctcgat gatctggctc accagctcgc tctcgctctt gtcgggctgg 1560gcctggatga tgcccagggc gtactggctg tcggtcacga tgttcacctc caggccgctg 1620tcctgcaggg ccaggtggat ggcctgcagc tcggtcttct ggttggtggt gtcggtcagg 1680ggcaccacct tctggcggcc gcggtcggtc acgtagccgg ccttgcccag cttggtctcg 1740cggttggcgg cgccgtccac gtagaaggtc tcggcgccga tgatgggctc cttctccagc 1800tggtaccaca gcttcaccag ggggggggtg ttcacgaact cccactcggg gatccaggtg 1860gcctgccagt actcggtcca ccaggcctcc caggtctcct tctggatggg cagcttgaac 1920ttgggggtct tgccccagat cacgatgctc tcggtggcga tcttctgcac ggcctcggtc 1980agctgcttca cgtcgttggt gtgggcgccc ttcatgcggg cgtacttgcc ggtcttcagg 2040ttcttgaagg gctcctggta gatctggtag gtccactggc cctggccctg cttctggatc 2100tcggcgatca ggtccttgct ggggtcgtag tacacgccgt gcacgggctc cttcaggatc 2160tcgcggttct cggccagctc cagctcggcc tcctcggtca ggggcaccac ctcggtcagg 2220gccttggtgc cgcgcagcag cttgcacagc tggcgcacct tgatgccggc gtagatctgg 2280ctggcccagt tcagcttgcc caccagcttc tggatgtcgt tcacggtcca gctgtccttc 2340tcgggcagca cgatgggctg cacggtccac ttgtcggggt gcagctcgta gcccatccac 2400aggaaggggg gctccttctg gtgcttcttg tcgggggtgg tgaagcccca gcgcagcagg 2460tgctggcgca gctcctcgat cttggtgcgg tgctggccga tctccaggtc gctgcccacg 2520tacaggtggt ccatgtactg gtagatcacg atgtcggggt tctgcttgcg gaagggctcc 2580aggatcttgg tcatgctgca ctggaagatg gcggggctgc ccttccagcc ctggggcagc 2640acgttgtact ggtagcggat gccgggggtc tcgttgttga tgctggggat ggtgaaggcg 2700gtgtacttgc ggaagtcctt gtccaggggc acgctgaagt aggcgtcgcc cacgtccagc 2760acggtcacgc tcttcttctg cttcaggccg gcggggtggg ggatgcccag ctgcacctcc 2820cagaagtcct gggtgcgctt gttcagctcg cggaagtcca ccagcttgcg ccacttggtg 2880ctgtccttct tcttgatggc gaacacgggg gtgttgtagg ggttctcggg gccgatcttg 2940ctgatcttgc cctccttctc catctcggtg cagatctcca ccagggcctt gatcttctcc 3000tcggtcaggg gccactgctt caccttgggg ccgtccatgc cgggcttcag cttcacgggc 3060acggtctcga tggggctgat ggggaagttc agggtgcagc cgatctgggt cagcaggttg 3120cggccgatga tgttcacggg ggtggggccc accagcacgg tgccgatggc cttgtggccg 3180cagatctcga tcaggatctg gtcgtactgg cccaccttga tgaagccgcc gatgccgccg 3240atcatcttgg gcttccagcg gccgggcagg ttcatctcct ccagcacggt gtcgtcggcg 3300ccggtgtcta gaagggcctc cttcagctgg ccccctatct ttattgtgac gaggggtcgc 3360tgccaaagag tgatctgagg gaagctaaag gatacagttc cttgtctatc ggctcctgct 3420tctgagaggg agttgttgtc tcttccccaa acctgaagct ctcttctggt ggggctgttg 3480gctctggtct gctctgaaga aaattccctg gccttccctt gtgggaaggc cagatcttcc 3540ctattagcct gtcgctcggt gcagtccttc atctggtggc cctccttgcc gcacttccag 3600cagcccttct tgcggggggc gcggcagttg cgggcggtgt ggccctcctt gccgcagttg 3660aagcacttca cgatcttgcg ctggttgcgg aagttgccgc gctgcatcat gatggtggcg 3720ctgttggtca cctggctcat ggcctcggcc agcacgcggg ccttgtggcc ggggccgccc 3780acgccctggc aggcggtcat catctcctcc agggtggcgg cggggcccag ggccttcagg 3840atggtcttgc agtcggggtt ggcgttctgc accagcaggg tctcggtcat ccagttcttc 3900acctcctggc tggcctgctc ggcgcgcagg gtcttgtaga agcggtccac gtagtcgcgg 3960aagggctcct tggggccctg gcggatgtcc aggatgctgg tggggctgta catgcgcacg 4020atcttgttca ggcccaggat gatccagcgc ttgtagatct cgcccacggg gatggggggg 4080ttgttggtca tccagccgat ctgctcctgc agggtgctgg tggtgccggc gatgtcgctg 4140ccgcggggct cgcgcatctg gccgggggcg atggggccgg cgtgcacggg gtgcacgcgg 4200tcccactcgg cggcctcctc gttgatggtc tccttcagca tctgcatggc ggcctggtgg 4260ccgcccacgg tgttcagcat ggtgttcagg tcctgggggg tggcgccctc gctcagggcg 4320ctgaacatgg ggatcacctc ggggctgaag gccttctcct ccaccacctt cacccaggcg 4380ttcagggtgc gggggctgat ggcctggtgc accatctggc cctggatgtt ctgcacgatg 4440gggtagttct ggctcacctg gttgctgtgg ccggtgtcgg cggcggcctg ctgggccttc 4500ttcttgctct tgttctgctc ctcctcgatc ttgtccaggg cctccttggt gtccttgatc 4560tcgatgcgct ggtgcacgca gtacagggtg gccacggtgt tgtacaggct gcgcagctcc 4620tcgctgccgg tctgcaggct gggctgcagc tggcccagga tctggcggca gccctcgctg 4680gtctccagca ggccggggtt cacggcgaag cgctccagct cgcggctggc ccacacgatg 4740tgcttcagct tgtacttctt cttgccgccg gggcgcaggc ggatcttctc ccagcggtcc 4800agctcgccgc cgctcagcac gctggcgcgg gcgcccatgt cgaatcgaat tctgcagtga 4860tcagggatcc gtatagtgag tcgtattagg taccggctgc agttggacct gggagtggac 4920acctgtggag agaaaggcaa agtggatgtc attgtcactc aagtgtatgg ccagatctca 4980agcctgccac acctcaagtg aagccaaggg ggtgggccta tagactctat aggcggtact 5040tacgtcactc ttggcacggg gaatccgcgt tccaatgcac cgttcccggc cgcggaggct 5100ggatcggtcc cggtgtcttc tatggaggtc aaaacagcgt ggatggcgtc tccaggcgat 5160ctgacggttc actaaacgag ctcgtcgacg atctctatca ctgataggga gatctctatc 5220actgataggg agagctctgc ttatatagac ctcccaccgt acacgcctac cgcccatttg 5280cgtcaatggg gcggagttgt tacgacattt tggaaagtcc cgttgatttt ggtgccaaaa 5340caaactccca ttgacgtcaa tggggtggag acttggaaat ccccgtgagt caaaccgcta 5400tccacgccca ttgatgtact gccaaaaccg catcaccatg gtaatagcga tgactaatac 5460gtagatgtac tgccaagtag gaaagtccca taaggtcatg tactgggcat aatgccaggc 5520gggccattta ccgtcattga cgtcaatagg gggcgtactt ggcatatgat acacttgatg 5580tactgccaag tgggcagttt accgtaaata ctccacccat tgacgtcaat ggaaagtccc 5640tattggcgtt actatgggaa catacgtcat tattgacgtc aatgggcggg ggtcgttggg 5700cggtcagcca ggcgggccat ttaccgtaag ttatgtaacg cggaactcca tatatgggct 5760atgaactaat gaccccgtaa ttgattacta ttaataacta gtactgaaat gtgtgggcgt 5820ggcttaaggg tgggaaagaa tatataaggt gggggtctta tgtagttttg tatctgtttt 5880gcagcagccg ccgccgccat gagcaccaac tcgtttgatg gaagcattgt gagctcatat 5940ttgacaacgc gcatgccccc atgggccggg gtgcgtcaga atgtgatggg ctccagcatt 6000gatggtcgcc ccgtcctgcc cgcaaactct actaccttga cctacgagac cgtgtctgga 6060acgccgttgg agactgcagc ctccgccgcc gcttcagccg ctgcagccac cgcccgcggg 6120attgtgactg actttgcttt cctgagcccg cttgcaagca gtgcagcttc ccgttcatcc 6180gcccgcgatg acaagttgac ggctcttttg gcacaattgg attctttgac ccgggaactt 6240aatgtcgttt ctcagcagct gttggatctg cgccagcagg tttctgccct gaaggcttcc 6300tcccctccca atgcggttta aaacataaat aaaaaaccag actctgtttg gatttggatc 6360aagcaagtgt cttgctgtct ttatttaggg gttttgcgcg cgcggtaggc ccgggaccag 6420cggtctcggt cgttgagggt cctgtgtatt ttttccagga cgtggtaaag gtgactctgg 6480atgttcagat acatgggcat aagcccgtct ctggggtgga ggtagcacca ctgcagagct 6540tcatgctgcg gggtggtgtt gtagatgatc cagtcgtagc aggagcgctg ggcgtggtgc 6600ctaaaaatgt ctttcagtag caagctgatt gccaggggca ggcccttggt gtaagtgttt 6660acaaagcggt taagctggga tgggtgcata cgtggggata tgagatgcat cttggactgt 6720atttttaggt tggctatgtt cccagccata tccctccggg gattcatgtt gtgcagaacc 6780accagcacag tgtatccggt gcacttggga aatttgtcat gtagcttaga aggaaatgcg 6840tggaagaact tggagacgcc cttgtgacct ccaagatttt ccatgcattc gtccataatg 6900atggcaatgg gcccacgggc ggcggcctgg gcgaagatat ttctgggatc actaacgtca 6960tagttgtgtt ccaggatgag atcgtcatag gccattttta caaagcgcgg gcggagggtg 7020ccagactgcg gtataatggt tccatccggc ccaggggcgt agttaccctc acagatttgc 7080atttcccacg ctttgagttc agatgggggg atcatgtcta cctgcggggc gatgaagaaa 7140acggtttccg gggtagggga gatcagctgg gaagaaagca ggttcctgag cagctgcgac 7200ttaccgcagc cggtgggccc gtaaatcaca cctattaccg ggtgcaactg gtagttaaga 7260gagctgcagc tgccgtcatc cctgagcagg ggggccactt cgttaagcat gtccctgact 7320cgcatgtttt ccctgaccaa atccgccaga aggcgctcgc cgcccagcga tagcagttct 7380tgcaaggaag caaagttttt caacggtttg agaccgtccg ccgtaggcat gcttttgagc 7440gtttgaccaa gcagttccag gcggtcccac agctcggtca cctgctctac ggcatctcga 7500tccagcatat ctcctcgttt cgcgggttgg ggcggctttc gctgtacggc agtagtcggt 7560gctcgtccag acgggccagg gtcatgtctt tccacgggcg cagggtcctc gtcagcgtag 7620tctgggtcac ggtgaagggg tgcgctccgg gctgcgcgct ggccagggtg cgcttgaggc 7680tggtcctgct ggtgctgaag cgctgccggt cttcgccctg cgcgtcggcc aggtagcatt 7740tgaccatggt gtcatagtcc agcccctccg cggcgtggcc cttggcgcgc agcttgccct 7800tggaggaggc gccgcacgag gggcagtgca gacttttgag ggcgtagagc ttgggcgcga 7860gaaataccga ttccggggag taggcatccg cgccgcaggc cccgcagacg gtctcgcatt 7920ccacgagcca ggtgagctct ggccgttcgg ggtcaaaaac caggtttccc ccatgctttt 7980tgatgcgttt cttacctctg gtttccatga gccggtgtcc acgctcggtg acgaaaaggc 8040tgtccgtgtc cccgtataca gacttgagag gcctgtcctc gagcggtgtt ccgcggtcct 8100cctcgtatag aaactcggac cactctgaga caaaggctcg cgtccaggcc agcacgaagg 8160aggctaagtg ggaggggtag cggtcgttgt ccactagggg gtccactcgc tccagggtgt 8220gaagacacat gtcgccctct tcggcatcaa ggaaggtgat tggtttgtag gtgtaggcca 8280cgtgaccggg tgttcctgaa ggggggctat aaaagggggt gggggcgcgt tcgtcctcac 8340tctcttccgc atcgctgtct gcgagggcca gctgttgggg tgagtactcc ctctgaaaag 8400cgggcatgac ttctgcgcta agattgtcag tttccaaaaa cgaggaggat ttgatattca 8460cctggcccgc ggtgatgcct ttgagggtgg ccgcatccat ctggtcagaa aagacaatct 8520ttttgttgtc aagcttggtg gcaaacgacc cgtagagggc gttggacagc aacttggcga 8580tggagcgcag ggtttggttt ttgtcgcgat cggcgcgctc cttggccgcg atgtttagct 8640gcacgtattc gcgcgcaacg caccgccatt cgggaaagac ggtggtgcgc tcgtcgggca 8700ccaggtgcac gcgccaaccg cggttgtgca gggtgacaag gtcaacgctg gtggctacct 8760ctccgcgtag gcgctcgttg gtccagcaga ggcggccgcc cttgcgcgag cagaatggcg 8820gtagggggtc tagctgcgtc tcgtccgggg ggtctgcgtc cacggtaaag accccgggca 8880gcaggcgcgc gtcgaagtag tctatcttgc atccttgcaa gtctagcgcc tgctgccatg 8940cgcgggcggc aagcgcgcgc tcgtatgggt tgagtggggg accccatggc atggggtggg 9000tgagcgcgga ggcgtacatg ccgcaaatgt cgtaaacgta gaggggctct ctgagtattc 9060caagatatgt agggtagcat cttccaccgc ggatgctggc gcgcacgtaa tcgtatagtt 9120cgtgcgaggg agcgaggagg tcgggaccga ggttgctacg ggcgggctgc tctgctcgga 9180agactatctg cctgaagatg gcatgtgagt tggatgatat ggttggacgc tggaagacgt 9240tgaagctggc gtctgtgaga cctaccgcgt cacgcacgaa ggaggcgtag gagtcgcgca 9300gcttgttgac cagctcggcg gtgacctgca cgtctagggc gcagtagtcc agggtttcct 9360tgatgatgtc atacttatcc tgtccctttt ttttccacag ctcgcggttg aggacaaact 9420cttcgcggtc tttccagtac tcttggatcg gaaacccgtc ggcctccgaa cggtaagagc 9480ctagcatgta gaactggttg acggcctggt aggcgcagca tcccttttct acgggtagcg 9540cgtatgcctg cgcggccttc cggagcgagg tgtgggtgag cgcaaaggtg tccctgacca 9600tgactttgag gtactggtat ttgaagtcag tgtcgtcgca tccgccctgc tcccagagca 9660aaaagtccgt gcgctttttg gaacgcggat ttggcagggc gaaggtgaca tcgttgaaga 9720gtatctttcc cgcgcgaggc ataaagttgc gtgtgatgcg gaagggtccc ggcacctcgg 9780aacggttgtt aattacctgg gcggcgagca cgatctcgtc aaagccgttg atgttgtggc 9840ccacaatgta aagttccaag aagcgcggga tgcccttgat ggaaggcaat tttttaagtt 9900cctcgtaggt gagctcttca ggggagctga gcccgtgctc tgaaagggcc cagtctgcaa 9960gatgagggtt ggaagcgacg aatgagctcc acaggtcacg ggccattagc atttgcaggt 10020ggtcgcgaaa ggtcctaaac tggcgaccta tggccatttt ttctggggtg atgcagtaga 10080aggtaagcgg gtcttgttcc cagcggtccc atccaaggtt cgcggctagg tctcgcgcgg

10140cagtcactag aggctcatct ccgccgaact tcatgaccag catgaagggc acgagctgct 10200tcccaaaggc ccccatccaa gtataggtct ctacatcgta ggtgacaaag agacgctcgg 10260tgcgaggatg cgagccgatc gggaagaact ggatctcccg ccaccaattg gaggagtggc 10320tattgatgtg gtgaaagtag aagtccctgc gacgggccga acactcgtgc tggcttttgt 10380aaaaacgtgc gcagtactgg cagcggtgca cgggctgtac atcctgcacg aggttgacct 10440gacgaccgcg cacaaggaag cagagtggga atttgagccc ctcgcctggc gggtttggct 10500ggtggtcttc tacttcggct gcttgtcctt gaccgtctgg ctgctcgagg ggagttacgg 10560tggatcggac caccacgccg cgcgagccca aagtccagat gtccgcgcgc ggcggtcgga 10620gcttgatgac aacatcgcgc agatgggagc tgtccatggt ctggagctcc cgcggcgtca 10680ggtcaggcgg gagctcctgc aggtttacct cgcatagacg ggtcagggcg cgggctagat 10740ccaggtgata cctaatttcc aggggctggt tggtggcggc gtcgatggct tgcaagaggc 10800cgcatccccg cggcgcgact acggtaccgc gcggcgggcg gtgggccgcg ggggtgtcct 10860tggatgatgc atctaaaagc ggtgacgcgg gcgagccccc ggaggtaggg ggggctccgg 10920acccgccggg agagggggca ggggcacgtc ggcgccgcgc gcgggcagga gctggtgctg 10980cgcgcgtagg ttgctggcga acgcgacgac gcggcggttg atctcctgaa tctggcgcct 11040ctgcgtgaag acgacgggcc cggtgagctt gaacctgaaa gagagttcga cagaatcaat 11100ttcggtgtcg ttgacggcgg cctggcgcaa aatctcctgc acgtctcctg agttgtcttg 11160ataggcgatc tcggccatga actgctcgat ctcttcctcc tggagatctc cgcgtccggc 11220tcgctccacg gtggcggcga ggtcgttgga aatgcgggcc atgagctgcg agaaggcgtt 11280gaggcctccc tcgttccaga cgcggctgta gaccacgccc ccttcggcat cgcgggcgcg 11340catgaccacc tgcgcgagat tgagctccac gtgccgggcg aagacggcgt agtttcgcag 11400gcgctgaaag aggtagttga gggtggtggc ggtgtgttct gccacgaaga agtacataac 11460ccagcgtcgc aacgtggatt cgttgatatc ccccaaggcc tcaaggcgct ccatggcctc 11520gtagaagtcc acggcgaagt tgaaaaactg ggagttgcgc gccgacacgg ttaactcctc 11580ctccagaaga cggatgagct cggcgacagt gtcgcgcacc tcgcgctcaa aggctacagg 11640ggcctcttct tcttcttcaa tctcctcttc cataagggcc tccccttctt cttcttctgg 11700cggcggtggg ggagggggga cacggcggcg acgacggcgc accgggaggc ggtcgacaaa 11760gcgctcgatc atctccccgc ggcgacggcg catggtctcg gtgacggcgc ggccgttctc 11820gcgggggcgc agttggaaga cgccgcccgt catgtcccgg ttatgggttg gcggggggct 11880gccatgcggc agggatacgg cgctaacgat gcatctcaac aattgttgtg taggtactcc 11940gccgccgagg gacctgagcg agtccgcatc gaccggatcg gaaaacctct cgagaaaggc 12000gtctaaccag tcacagtcgc aaggtaggct gagcaccgtg gcgggcggca gcgggcggcg 12060gtcggggttg tttctggcgg aggtgctgct gatgatgtaa ttaaagtagg cggtcttgag 12120acggcggatg gtcgacagaa gcaccatgtc cttgggtccg gcctgctgaa tgcgcaggcg 12180gtcggccatg ccccaggctt cgttttgaca tcggcgcagg tctttgtagt agtcttgcat 12240gagcctttct accggcactt cttcttctcc ttcctcttgt cctgcatctc ttgcatctat 12300cgctgcggcg gcggcggagt ttggccgtag gtggcgccct cttcctccca tgcgtgtgac 12360cccgaagccc ctcatcggct gaagcagggc taggtcggcg acaacgcgct cggctaatat 12420ggcctgctgc acctgcgtga gggtagactg gaagtcatcc atgtccacaa agcggtggta 12480tgcgcccgtg ttgatggtgt aagtgcagtt ggccataacg gaccagttaa cggtctggtg 12540acccggctgc gagagctcgg tgtacctgag acgcgagtaa gccctcgagt caaatacgta 12600gtcgttgcaa gtccgcacca ggtactggta tcccaccaaa aagtgcggcg gcggctggcg 12660gtagaggggc cagcgtaggg tggccggggc tccgggggcg agatcttcca acataaggcg 12720atgatatccg tagatgtacc tggacatcca ggtgatgccg gcggcggtgg tggaggcgcg 12780cggaaagtcg cggacgcggt tccagatgtt gcgcagcggc aaaaagtgct ccatggtcgg 12840gacgctctgg ccggtcaggc gcgcgcaatc gttgacgctc tagcgtgcaa aaggagagcc 12900tgtaagcggg cactcttccg tggtctggtg gataaattcg caagggtatc atggcggacg 12960accggggttc gagccccgta tccggccgtc cgccgtgatc catgcggtta ccgcccgcgt 13020gtcgaaccca ggtgtgcgac gtcagacaac gggggagtgc tccttttggc ttccttccag 13080gcgcggcggc tgctgcgcta gcttttttgg ccactggccg cgcgcagcgt aagcggttag 13140gctggaaagc gaaagcatta agtggctcgc tccctgtagc cggagggtta ttttccaagg 13200gttgagtcgc gggacccccg gttcgagtct cggaccggcc ggactgcggc gaacgggggt 13260ttgcctcccc gtcatgcaag accccgcttg caaattcctc cggaaacagg gacgagcccc 13320ttttttgctt ttcccagatg catccggtgc tgcggcagat gcgcccccct cctcagcagc 13380ggcaagagca agagcagcgg cagacatgca gggcaccctc ccctcctcct accgcgtcag 13440gaggggcgac atccgcggtt gacgcggcag cagatggtga ttacgaaccc ccgcggcgcc 13500gggcccggca ctacctggac ttggaggagg gcgagggcct ggcgcggcta ggagcgccct 13560ctcctgagcg gcacccaagg gtgcagctga agcgtgatac gcgtgaggcg tacgtgccgc 13620ggcagaacct gtttcgcgac cgcgagggag aggagcccga ggagatgcgg gatcgaaagt 13680tccacgcagg gcgcgagctg cggcatggcc tgaatcgcga gcggttgctg cgcgaggagg 13740actttgagcc cgacgcgcga accgggatta gtcccgcgcg cgcacacgtg gcggccgccg 13800acctggtaac cgcatacgag cagacggtga accaggagat taactttcaa aaaagcttta 13860acaaccacgt gcgtacgctt gtggcgcgcg aggaggtggc tataggactg atgcatctgt 13920gggactttgt aagcgcgctg gagcaaaacc caaatagcaa gccgctcatg gcgcagctgt 13980tccttatagt gcagcacagc agggacaacg aggcattcag ggatgcgctg ctaaacatag 14040tagagcccga gggccgctgg ctgctcgatt tgataaacat cctgcagagc atagtggtgc 14100aggagcgcag cttgagcctg gctgacaagg tggccgccat caactattcc atgcttagcc 14160tgggcaagtt ttacgcccgc aagatatacc atacccctta cgttcccata gacaaggagg 14220taaagatcga ggggttctac atgcgcatgg cgctgaaggt gcttaccttg agcgacgacc 14280tgggcgttta tcgcaacgag cgcatccaca aggccgtgag cgtgagccgg cggcgcgagc 14340tcagcgaccg cgagctgatg cacagcctgc aaagggccct ggctggcacg ggcagcggcg 14400atagagaggc cgagtcctac tttgacgcgg gcgctgacct gcgctgggcc ccaagccgac 14460gcgccctgga ggcagctggg gccggacctg ggctggcggt ggcacccgcg cgcgctggca 14520acgtcggcgg cgtggaggaa tatgacgagg acgatgagta cgagccagag gacggcgagt 14580actaagcggt gatgtttctg atcagatgat gcaagacgca acggacccgg cggtgcgggc 14640ggcgctgcag agccagccgt ccggccttaa ctccacggac gactggcgcc aggtcatgga 14700ccgcatcatg tcgctgactg cgcgcaatcc tgacgcgttc cggcagcagc cgcaggccaa 14760ccggctctcc gcaattctgg aagcggtggt cccggcgcgc gcaaacccca cgcacgagaa 14820ggtgctggcg atcgtaaacg cgctggccga aaacagggcc atccggcccg acgaggccgg 14880cctggtctac gacgcgctgc ttcagcgcgt ggctcgttac aacagcggca acgtgcagac 14940caacctggac cggctggtgg gggatgtgcg cgaggccgtg gcgcagcgtg agcgcgcgca 15000gcagcagggc aacctgggct ccatggttgc actaaacgcc ttcctgagta cacagcccgc 15060caacgtgccg cggggacagg aggactacac caactttgtg agcgcactgc ggctaatggt 15120gactgagaca ccgcaaagtg aggtgtacca gtctgggcca gactattttt tccagaccag 15180tagacaaggc ctgcagaccg taaacctgag ccaggctttc aaaaacttgc aggggctgtg 15240gggggtgcgg gctcccacag gcgaccgcgc gaccgtgtct agcttgctga cgcccaactc 15300gcgcctgttg ctgctgctaa tagcgccctt cacggacagt ggcagcgtgt cccgggacac 15360atacctaggt cacttgctga cactgtaccg cgaggccata ggtcaggcgc atgtggacga 15420gcatactttc caggagatta caagtgtcag ccgcgcgctg gggcaggagg acacgggcag 15480cctggaggca accctaaact acctgctgac caaccggcgg cagaagatcc cctcgttgca 15540cagtttaaac agcgaggagg agcgcatttt gcgctacgtg cagcagagcg tgagccttaa 15600cctgatgcgc gacggggtaa cgcccagcgt ggcgctggac atgaccgcgc gcaacatgga 15660accgggcatg tatgcctcaa accggccgtt tatcaaccgc ctaatggact acttgcatcg 15720cgcggccgcc gtgaaccccg agtatttcac caatgccatc ttgaacccgc actggctacc 15780gccccctggt ttctacaccg ggggattcga ggtgcccgag ggtaacgatg gattcctctg 15840ggacgacata gacgacagcg tgttttcccc gcaaccgcag accctgctag agttgcaaca 15900gcgcgagcag gcagaggcgg cgctgcgaaa ggaaagcttc cgcaggccaa gcagcttgtc 15960cgatctaggc gctgcggccc cgcggtcaga tgctagtagc ccatttccaa gcttgatagg 16020gtctcttacc agcactcgca ccacccgccc gcgcctgctg ggcgaggagg agtacctaaa 16080caactcgctg ctgcagccgc agcgcgaaaa aaacctgcct ccggcatttc ccaacaacgg 16140gatagagagc ctagtggaca agatgagtag atggaagacg tacgcgcagg agcacaggga 16200cgtgccaggc ccgcgcccgc ccacccgtcg tcaaaggcac gaccgtcagc ggggtctggt 16260gtgggaggac gatgactcgg cagacgacag cagcgtcctg gatttgggag ggagtggcaa 16320cccgtttgcg caccttcgcc ccaggctggg gagaatgttt taaaaaaaaa aaaagcatga 16380tgcaaaataa aaaactcacc aaggccatgg caccgagcgt tggttttctt gtattcccct 16440tagtatgcgg cgcgcggcga tgtatgagga aggtcctcct ccctcctacg agagtgtggt 16500gagcgcggcg ccagtggcgg cggcgctggg ttctcccttc gatgctcccc tggacccgcc 16560gtttgtgcct ccgcggtacc tgcggcctac cggggggaga aacagcatcc gttactctga 16620gttggcaccc ctattcgaca ccacccgtgt gtacctggtg gacaacaagt caacggatgt 16680ggcatccctg aactaccaga acgaccacag caactttctg accacggtca ttcaaaacaa 16740tgactacagc ccgggggagg caagcacaca gaccatcaat cttgacgacc ggtcgcactg 16800gggcggcgac ctgaaaacca tcctgcatac caacatgcca aatgtgaacg agttcatgtt 16860taccaataag tttaaggcgc gggtgatggt gtcgcgcttg cctactaagg acaatcaggt 16920ggagctgaaa tacgagtggg tggagttcac gctgcccgag ggcaactact ccgagaccat 16980gaccatagac cttatgaaca acgcgatcgt ggagcactac ttgaaagtgg gcagacagaa 17040cggggttctg gaaagcgaca tcggggtaaa gtttgacacc cgcaacttca gactggggtt 17100tgaccccgtc actggtcttg tcatgcctgg ggtatataca aacgaagcct tccatccaga 17160catcattttg ctgccaggat gcggggtgga cttcacccac agccgcctga gcaacttgtt 17220gggcatccgc aagcggcaac ccttccagga gggctttagg atcacctacg atgatctgga 17280gggtggtaac attcccgcac tgttggatgt ggacgcctac caggcgagct tgaaagatga 17340caccgaacag ggcgggggtg gcgcaggcgg cagcaacagc agtggcagcg gcgcggaaga 17400gaactccaac gcggcagccg cggcaatgca gccggtggag gacatgaacg atcatgccat 17460tcgcggcgac acctttgcca cacgggctga ggagaagcgc gctgaggccg aagcagcggc 17520cgaagctgcc gcccccgctg cgcaacccga ggtcgagaag cctcagaaga aaccggtgat 17580caaacccctg acagaggaca gcaagaaacg cagttacaac ctaataagca atgacagcac 17640cttcacccag taccgcagct ggtaccttgc atacaactac ggcgaccctc agaccggaat 17700ccgctcatgg accctgcttt gcactcctga cgtaacctgc ggctcggagc aggtctactg 17760gtcgttgcca gacatgatgc aagaccccgt gaccttccgc tccacgcgcc agatcagcaa 17820ctttccggtg gtgggcgccg agctgttgcc cgtgcactcc aagagcttct acaacgacca 17880ggccgtctac tcccaactca tccgccagtt tacctctctg acccacgtgt tcaatcgctt 17940tcccgagaac cagattttgg cgcgcccgcc agcccccacc atcaccaccg tcagtgaaaa 18000cgttcctgct ctcacagatc acgggacgct accgctgcgc aacagcatcg gaggagtcca 18060gcgagtgacc attactgacg ccagacgccg cacctgcccc tacgtttaca aggccctggg 18120catagtctcg ccgcgcgtcc tatcgagccg cactttttga gcaagcatgt ccatccttat 18180atcgcccagc aataacacag gctggggcct gcgcttccca agcaagatgt ttggcggggc 18240caagaagcgc tccgaccaac acccagtgcg cgtgcgcggg cactaccgcg cgccctgggg 18300cgcgcacaaa cgcggccgca ctgggcgcac caccgtcgat gacgccatcg acgcggtggt 18360ggaggaggcg cgcaactaca cgcccacgcc gccaccagtg tccacagtgg acgcggccat 18420tcagaccgtg gtgcgcggag cccggcgcta tgctaaaatg aagagacggc ggaggcgcgt 18480agcacgtcgc caccgccgcc gacccggcac tgccgcccaa cgcgcggcgg cggccctgct 18540taaccgcgca cgtcgcaccg gccgacgggc ggccatgcgg gccgctcgaa ggctggccgc 18600gggtattgtc actgtgcccc ccaggtccag gcgacgagcg gccgccgcag cagccgcggc 18660cattagtgct atgactcagg gtcgcagggg caacgtgtat tgggtgcgcg actcggttag 18720cggcctgcgc gtgcccgtgc gcacccgccc cccgcgcaac tagattgcaa gaaaaaacta 18780cttagactcg tactgttgta tgtatccagc ggcggcggcg cgcaacgaag ctatgtccaa 18840gcgcaaaatc aaagaagaga tgctccaggt catcgcgccg gagatctatg gccccccgaa 18900gaaggaagag caggattaca agccccgaaa gctaaagcgg gtcaaaaaga aaaagaaaga 18960tgatgatgat gaacttgacg acgaggtgga actgctgcac gctaccgcgc ccaggcgacg 19020ggtacagtgg aaaggtcgac gcgtaaaacg tgttttgcga cccggcacca ccgtagtctt 19080tacgcccggt gagcgctcca cccgcaccta caagcgcgtg tatgatgagg tgtacggcga 19140cgaggacctg cttgagcagg ccaacgagcg cctcggggag tttgcctacg gaaagcggca 19200taaggacatg ctggcgttgc cgctggacga gggcaaccca acacctagcc taaagcccgt 19260aacactgcag caggtgctgc ccgcgcttgc accgtccgaa gaaaagcgcg gcctaaagcg 19320cgagtctggt gacttggcac ccaccgtgca gctgatggta cccaagcgcc agcgactgga 19380agatgtcttg gaaaaaatga ccgtggaacc tgggctggag cccgaggtcc gcgtgcggcc 19440aatcaagcag gtggcgccgg gactgggcgt gcagaccgtg gacgttcaga tacccactac 19500cagtagcacc agtattgcca ccgccacaga gggcatggag acacaaacgt ccccggttgc 19560ctcagcggtg gcggatgccg cggtgcaggc ggtcgctgcg gccgcgtcca agacctctac 19620ggaggtgcaa acggacccgt ggatgtttcg cgtttcagcc ccccggcgcc cgcgccgttc 19680gaggaagtac ggcgccgcca gcgcgctact gcccgaatat gccctacatc cttccattgc 19740gcctaccccc ggctatcgtg gctacaccta ccgccccaga agacgagcaa ctacccgacg 19800ccgaaccacc actggaaccc gccgccgccg tcgccgtcgc cagcccgtgc tggccccgat 19860ttccgtgcgc agggtggctc gcgaaggagg caggaccctg gtgctgccaa cagcgcgcta 19920ccaccccagc atcgtttaaa agccggtctt tgtggttctt gcagatatgg ccctcacctg 19980ccgcctccgt ttcccggtgc cgggattccg aggaagaatg caccgtagga ggggcatggc 20040cggccacggc ctgacgggcg gcatgcgtcg tgcgcaccac cggcggcggc gcgcgtcgca 20100ccgtcgcatg cgcggcggta tcctgcccct ccttattcca ctgatcgccg cggcgattgg 20160cgccgtgccc ggaattgcat ccgtggcctt gcaggcgcag agacactgat taaaaacaag 20220ttgcatgtgg aaaaatcaaa ataaaaagtc tggactctca cgctcgcttg gtcctgtaac 20280tattttgtag aatggaagac atcaactttg cgtctctggc cccgcgacac ggctcgcgcc 20340cgttcatggg aaactggcaa gatatcggca ccagcaatat gagcggtggc gccttcagct 20400ggggctcgct gtggagcggc attaaaaatt tcggttccac cgttaagaac tatggcagca 20460aggcctggaa cagcagcaca ggccagatgc tgagggataa gttgaaagag caaaatttcc 20520aacaaaaggt ggtagatggc ctggcctctg gcattagcgg ggtggtggac ctggccaacc 20580aggcagtgca aaataagatt aacagtaagc ttgatccccg ccctcccgta gaggagcctc 20640caccggccgt ggagacagtg tctccagagg ggcgtggcga aaagcgtccg cgccccgaca 20700gggaagaaac tctggtgacg caaatagacg agcctccctc gtacgaggag gcactaaagc 20760aaggcctgcc caccacccgt cccatcgcgc ccatggctac cggagtgctg ggccagcaca 20820cacccgtaac gctggacctg cctccccccg ccgacaccca gcagaaacct gtgctgccag 20880gcccgaccgc cgttgttgta acccgtccta gccgcgcgtc cctgcgccgc gccgccagcg 20940gtccgcgatc gttgcggccc gtagccagtg gcaactggca aagcacactg aacagcatcg 21000tgggtctggg ggtgcaatcc ctgaagcgcc gacgatgctt ctgatagcta acgtgtcgta 21060tgtgtgtcat gtatgcgtcc atgtcgccgc cagaggagct gctgagccgc cgcgcgcccg 21120ctttccaaga tggctacccc ttcgatgatg ccgcagtggt cttacatgca catctcgggc 21180caggacgcct cggagtacct gagccccggg ctggtgcagt ttgcccgcgc caccgagacg 21240tacttcagcc tgaataacaa gtttagaaac cccacggtgg cgcctacgca cgacgtgacc 21300acagaccggt cccagcgttt gacgctgcgg ttcatccctg tggaccgtga ggatactgcg 21360tactcgtaca aggcgcggtt caccctagct gtgggtgata accgtgtgct ggacatggct 21420tccacgtact ttgacatccg cggcgtgctg gacaggggcc ctacttttaa gccctactct 21480ggcactgcct acaacgccct ggctcccaag ggtgccccaa atccttgcga atgggatgaa 21540gctgctactg ctcttgaaat aaacctagaa gaagaggacg atgacaacga agacgaagta 21600gacgagcaag ctgagcagca aaaaactcac gtatttgggc aggcgcctta ttctggtata 21660aatattacaa aggagggtat tcaaataggt gtcgaaggtc aaacacctaa atatgccgat 21720aaaacatttc aacctgaacc tcaaatagga gaatctcagt ggtacgaaac agaaattaat 21780catgcagctg ggagagtcct aaaaaagact accccaatga aaccatgtta cggttcatat 21840gcaaaaccca caaatgaaaa tggagggcaa ggcattcttg taaagcaaca aaatggaaag 21900ctagaaagtc aagtggaaat gcaatttttc tcaactactg aggcagccgc aggcaatggt 21960gataacttga ctcctaaagt ggtattgtac agtgaagatg tagatataga aaccccagac 22020actcatattt cttacatgcc cactattaag gaaggtaact cacgagaact aatgggccaa 22080caatctatgc ccaacaggcc taattacatt gcttttaggg acaattttat tggtctaatg 22140tattacaaca gcacgggtaa tatgggtgtt ctggcgggcc aagcatcgca gttgaatgct 22200gttgtagatt tgcaagacag aaacacagag ctttcatacc agcttttgct tgattccatt 22260ggtgatagaa ccaggtactt ttctatgtgg aatcaggctg ttgacagcta tgatccagat 22320gttagaatta ttgaaaatca tggaactgaa gatgaacttc caaattactg ctttccactg 22380ggaggtgtga ttaatacaga gactcttacc aaggtaaaac ctaaaacagg tcaggaaaat 22440ggatgggaaa aagatgctac agaattttca gataaaaatg aaataagagt tggaaataat 22500tttgccatgg aaatcaatct aaatgccaac ctgtggagaa atttcctgta ctccaacata 22560gcgctgtatt tgcccgacaa gctaaagtac agtccttcca acgtaaaaat ttctgataac 22620ccaaacacct acgactacat gaacaagcga gtggtggctc ccgggctagt ggactgctac 22680attaaccttg gagcacgctg gtcccttgac tatatggaca acgtcaaccc atttaaccac 22740caccgcaatg ctggcctgcg ctaccgctca atgttgctgg gcaatggtcg ctatgtgccc 22800ttccacatcc aggtgcctca gaagttcttt gccattaaaa acctccttct cctgccgggc 22860tcatacacct acgagtggaa cttcaggaag gatgttaaca tggttctgca gagctcccta 22920ggaaatgacc taagggttga cggagccagc attaagtttg atagcatttg cctttacgcc 22980accttcttcc ccatggccca caacaccgcc tccacgcttg aggccatgct tagaaacgac 23040accaacgacc agtcctttaa cgactatctc tccgccgcca acatgctcta ccctataccc 23100gccaacgcta ccaacgtgcc catatccatc ccctcccgca actgggcggc tttccgcggc 23160tgggccttca cgcgccttaa gactaaggaa accccatcac tgggctcggg ctacgaccct 23220tattacacct actctggctc tataccctac ctagatggaa ccttttacct caaccacacc 23280tttaagaagg tggccattac ctttgactct tctgtcagct ggcctggcaa tgaccgcctg 23340cttaccccca acgagtttga aattaagcgc tcagttgacg gggagggtta caacgttgcc 23400cagtgtaaca tgaccaaaga ctggttcctg gtacaaatgc tagctaacta taacattggc 23460taccagggct tctatatccc agagagctac aaggaccgca tgtactcctt ctttagaaac 23520ttccagccca tgagccgtca ggtggtggat gatactaaat acaaggacta ccaacaggtg 23580ggcatcctac accaacacaa caactctgga tttgttggct accttgcccc caccatgcgc 23640gaaggacagg cctaccctgc taacttcccc tatccgctta taggcaagac cgcagttgac 23700agcattaccc agaaaaagtt tctttgcgat cgcacccttt ggcgcatccc attctccagt 23760aactttatgt ccatgggcgc actcacagac ctgggccaaa accttctcta cgccaactcc 23820gcccacgcgc tagacatgac ttttgaggtg gatcccatgg acgagcccac ccttctttat 23880gttttgtttg aagtctttga cgtggtccgt gtgcaccagc cgcaccgcgg cgtcatcgaa 23940accgtgtacc tgcgcacgcc cttctcggcc ggcaacgcca caacataaag aagcaagcaa 24000catcaacaac agctgccgcc atgggctcca gtgagcagga actgaaagcc attgtcaaag 24060atcttggttg tgggccatat tttttgggca cctatgacaa gcgctttcca ggctttgttt 24120ctccacacaa gctcgcctgc gccatagtca atacggccgg tcgcgagact gggggcgtac 24180actggatggc ctttgcctgg aacccgcact caaaaacatg ctacctcttt gagccctttg 24240gcttttctga ccagcgactc aagcaggttt accagtttga gtacgagtca ctcctgcgcc 24300gtagcgccat tgcttcttcc cccgaccgct gtataacgct ggaaaagtcc acccaaagcg 24360tacaggggcc caactcggcc gcctgtggac tattctgctg catgtttctc cacgcctttg 24420ccaactggcc ccaaactccc atggatcaca accccaccat gaaccttatt accggggtac 24480ccaactccat gctcaacagt ccccaggtac agcccaccct gcgtcgcaac caggaacagc 24540tctacagctt cctggagcgc cactcgccct acttccgcag ccacagtgcg cagattagga 24600gcgccacttc tttttgtcac ttgaaaaaca tgtaaaaata atgtactaga gacactttca 24660ataaaggcaa atgcttttat ttgtacactc tcgggtgatt atttaccccc acccttgccg 24720tctgcgccgt ttaaaaatca aaggggttct gccgcgcatc gctatgcgcc actggcaggg 24780acacgttgcg atactggtgt ttagtgctcc acttaaactc aggcacaacc atccgcggca 24840gctcggtgaa gttttcactc cacaggctgc gcaccatcac caacgcgttt agcaggtcgg 24900gcgccgatat cttgaagtcg cagttggggc ctccgccctg cgcgcgcgag ttgcgataca 24960cagggttgca gcactggaac actatcagcg ccgggtggtg cacgctggcc agcacgctct 25020tgtcggagat cagatccgcg tccaggtcct ccgcgttgct cagggcgaac ggagtcaact 25080ttggtagctg ccttcccaaa aagggcgcgt gcccaggctt tgagttgcac tcgcaccgta 25140gtggcatcaa aaggtgaccg tgcccggtct gggcgttagg atacagcgcc tgcataaaag

25200ccttgatctg cttaaaagcc acctgagcct ttgcgccttc agagaagaac atgccgcaag 25260acttgccgga aaactgattg gccggacagg ccgcgtcgtg cacgcagcac cttgcgtcgg 25320tgttggagat ctgcaccaca tttcggcccc accggttctt cacgatcttg gccttgctag 25380actgctcctt cagcgcgcgc tgcccgtttt cgctcgtcac atccatttca atcacgtgct 25440ccttatttat cataatgctt ccgtgtagac acttaagctc gccttcgatc tcagcgcagc 25500ggtgcagcca caacgcgcag cccgtgggct cgtgatgctt gtaggtcacc tctgcaaacg 25560actgcaggta cgcctgcagg aatcgcccca tcatcgtcac aaaggtcttg ttgctggtga 25620aggtcagctg caacccgcgg tgctcctcgt tcagccaggt cttgcatacg gccgccagag 25680cttccacttg gtcaggcagt agtttgaagt tcgcctttag atcgttatcc acgtggtact 25740tgtccatcag cgcgcgcgca gcctccatgc ccttctccca cgcagacacg atcggcacac 25800tcagcgggtt catcaccgta atttcacttt ccgcttcgct gggctcttcc tcttcctctt 25860gcgtccgcat accacgcgcc actgggtcgt cttcattcag ccgccgcact gtgcgcttac 25920ctcctttgcc atgcttgatt agcaccggtg ggttgctgaa acccaccatt tgtagcgcca 25980catcttctct ttcttcctcg ctgtccacga ttacctctgg tgatggcggg cgctcgggct 26040tgggagaagg gcgcttcttt ttcttcttgg gcgcaatggc caaatccgcc gccgaggtcg 26100atggccgcgg gctgggtgtg cgcggcacca gcgcgtcttg tgatgagtct tcctcgtcct 26160cggactcgat acgccgcctc atccgctttt ttgggggcgc ccggggaggc ggcggcgacg 26220gggacgggga cgacacgtcc tccatggttg ggggacgtcg cgccgcaccg cgtccgcgct 26280cgggggtggt ttcgcgctgc tcctcttccc gactggccat ttccttctcc tataggcaga 26340aaaagatcat ggagtcagtc gagaagaagg acagcctaac cgccccctct gagttcgcca 26400ccaccgcctc caccgatgcc gccaacgcgc ctaccacctt ccccgtcgag gcacccccgc 26460ttgaggagga ggaagtgatt atcgagcagg acccaggttt tgtaagcgaa gacgacgagg 26520accgctcagt accaacagag gataaaaagc aagaccagga caacgcagag gcaaacgagg 26580aacaagtcgg gcggggggac gaaaggcatg gcgactacct agatgtggga gacgacgtgc 26640tgttgaagca tctgcagcgc cagtgcgcca ttatctgcga cgcgttgcaa gagcgcagcg 26700atgtgcccct cgccatagcg gatgtcagcc ttgcctacga acgccaccta ttctcaccgc 26760gcgtaccccc caaacgccaa gaaaacggca catgcgagcc caacccgcgc ctcaacttct 26820accccgtatt tgccgtgcca gaggtgcttg ccacctatca catctttttc caaaactgca 26880agatacccct atcctgccgt gccaaccgca gccgagcgga caagcagctg gccttgcggc 26940agggcgctgt catacctgat atcgcctcgc tcaacgaagt gccaaaaatc tttgagggtc 27000ttggacgcga cgagaagcgc gcggcaaacg ctctgcaaca ggaaaacagc gaaaatgaaa 27060gtcactctgg agtgttggtg gaactcgagg gtgacaacgc gcgcctagcc gtactaaaac 27120gcagcatcga ggtcacccac tttgcctacc cggcacttaa cctacccccc aaggtcatga 27180gcacagtcat gagtgagctg atcgtgcgcc gtgcgcagcc cctggagagg gatgcaaatt 27240tgcaagaaca aacagaggag ggcctacccg cagttggcga cgagcagcta gcgcgctggc 27300ttcaaacgcg cgagcctgcc gacttggagg agcgacgcaa actaatgatg gccgcagtgc 27360tcgttaccgt ggagcttgag tgcatgcagc ggttctttgc tgacccggag atgcagcgca 27420agctagagga aacattgcac tacacctttc gacagggcta cgtacgccag gcctgcaaga 27480tctccaacgt ggagctctgc aacctggtct cctaccttgg aattttgcac gaaaaccgcc 27540ttgggcaaaa cgtgcttcat tccacgctca agggcgaggc gcgccgcgac tacgtccgcg 27600actgcgttta cttatttcta tgctacacct ggcagacggc catgggcgtt tggcagcagt 27660gcttggagga gtgcaacctc aaggagctgc agaaactgct aaagcaaaac ttgaaggacc 27720tatggacggc cttcaacgag cgctccgtgg ccgcgcacct ggcggacatc attttccccg 27780aacgcctgct taaaaccctg caacagggtc tgccagactt caccagtcaa agcatgttgc 27840agaactttag gaactttatc ctagagcgct caggaatctt gcccgccacc tgctgtgcac 27900ttcctagcga ctttgtgccc attaagtacc gcgaatgccc tccgccgctt tggggccact 27960gctaccttct gcagctagcc aactaccttg cctaccactc tgacataatg gaagacgtga 28020gcggtgacgg tctactggag tgtcactgtc gctgcaacct atgcaccccg caccgctccc 28080tggtttgcaa ttcgcagctg cttaacgaaa gtcaaattat cggtaccttt gagctgcagg 28140gtccctcgcc tgacgaaaag tccgcggctc cggggttgaa actcactccg gggctgtgga 28200cgtcggctta ccttcgcaaa tttgtacctg aggactacca cgcccacgag attaggttct 28260acgaagacca atcccgcccg cctaatgcgg agcttaccgc ctgcgtcatt acccagggcc 28320acattcttgg ccaattgcaa gccatcaaca aagcccgcca agagtttctg ctacgaaagg 28380gacggggggt ttacttggac ccccagtccg gcgaggagct caacccaatc cccccgccgc 28440cgcagcccta tcagcagcag ccgcgggccc ttgcttccca ggatggcacc caaaaagaag 28500ctgcagctgc cgccgccacc cacggacgag gaggaatact gggacagtca ggcagaggag 28560gttttggacg aggaggagga ggacatgatg gaagactggg agagcctaga cgaggaagct 28620tccgaggtcg aagaggtgtc agacgaaaca ccgtcaccct cggtcgcatt cccctcgccg 28680gcgccccaga aatcggcaac cggttccagc atggctacaa cctccgctcc tcaggcgccg 28740ccggcactgc ccgttcgccg acccaaccgt agatgggaca ccactggaac cagggccggt 28800aagtccaagc agccgccgcc gttagcccaa gagcaacaac agcgccaagg ctaccgctca 28860tggcgcgggc acaagaacgc catagttgct tgcttgcaag actgtggggg caacatctcc 28920ttcgcccgcc gctttcttct ctaccatcac ggcgtggcct tcccccgtaa catcctgcat 28980tactaccgtc atctctacag cccatactgc accggcggca gcggcagcaa cagcagcggc 29040cacacagaag caaaggcgac cggatagcaa gactctgaca aagcccaaga aatccacagc 29100ggcggcagca gcaggaggag gagcgctgcg tctggcgccc aacgaacccg tatcgacccg 29160cgagcttaga aacaggattt ttcccactct gtatgctata tttcaacaga gcaggggcca 29220agaacaagag ctgaaaataa aaaacaggtc tctgcgatcc ctcacccgca gctgcctgta 29280tcacaaaagc gaagatcagc ttcggcgcac gctggaagac gcggaggctc tcttcagtaa 29340atactgcgcg ctgactctta aggactagtt tcgcgccctt tctcaaattt aagcgcgaaa 29400actacgtcat ctccagcggc cacacccggc gccagcacct gttgtcagcg ccattatgag 29460caaggaaatt cccacgccct acatgtggag ttaccagcca caaatgggac ttgcggctgg 29520agctgcccaa gactactcaa cccgaataaa ctacatgagc gcgggacccc acatgatatc 29580ccgggtcaac ggaatacgcg cccaccgaaa ccgaattctc ctggaacagg cggctattac 29640caccacacct cgtaataacc ttaatccccg tagttggccc gctgccctgg tgtaccagga 29700aagtcccgct cccaccactg tggtacttcc cagagacgcc caggccgaag ttcagatgac 29760taactcaggg gcgcagcttg cgggcggctt tcgtcacagg gtgcggtcgc ccgggcaggg 29820tataactcac ctgacaatca gagggcgagg tattcagctc aacgacgagt cggtgagctc 29880ctcgcttggt ctccgtccgg acgggacatt tcagatcggc ggcgccggcc gctcttcatt 29940cacgcctcgt caggcaatcc taactctgca gacctcgtcc tctgagccgc gctctggagg 30000cattggaact ctgcaattta ttgaggagtt tgtgccatcg gtctacttta accccttctc 30060gggacctccc ggccactatc cggatcaatt tattcctaac tttgacgcgg taaaggactc 30120ggcggacggc tacgactgaa tgttaagtgg agaggcagag caactgcgcc tgaaacacct 30180ggtccactgt cgccgccaca agtgctttgc ccgcgactcc ggtgagtttt gctactttga 30240attgcccgag gatcatatcg agggcccggc gcacggcgtc cggcttaccg cccagggaga 30300gcttgcccgt agcctgattc gggagtttac ccagcgcccc ctgctagttg agcgggacag 30360gggaccctgt gttctcactg tgatttgcaa ctgtcctaac cctggattac atcaagatct 30420ttgttgccat ctctgtgctg agtataataa atacagaaat taaaatatac tggggctcct 30480atcgccatcc tgtaaacgcc accgtcttca cccgcccaag caaaccaagg cgaaccttac 30540ctggtacttt taacatctct ccctctgtga tttacaacag tttcaaccca gacggagtga 30600gtctacgaga gaacctctcc gagctcagct actccatcag aaaaaacacc accctcctta 30660cctgccggga acgtacgagt gcgtcaccgg ccgctgcacc acacctaccg cctgaccgta 30720aaccagactt tttccggaca gacctcaata actctgttta ccagaacagg aggtgagctt 30780agaaaaccct tagggtatta ggccaaaggc gcagctactg tggggtttat gaacaattca 30840agcaactcta cgggctattc taattcaggt ttctctagaa atggacggaa ttattacaga 30900gcagcgcctg ctagaaagac gcagggcagc ggccgagcaa cagcgcatga atcaagagct 30960ccaagacatg gttaacttgc accagtgcaa aaggggtatc ttttgtctgg taaagcaggc 31020caaagtcacc tacgacagta ataccaccgg acaccgcctt agctacaagt tgccaaccaa 31080gcgtcagaaa ttggtggtca tggtgggaga aaagcccatt accataactc agcactcggt 31140agaaaccgaa ggctgcattc actcaccttg tcaaggacct gaggatctct gcacccttat 31200taagaccctg tgcggtctca aagatcttat tccctttaac taataaaaaa aaataataaa 31260gcatcactta cttaaaatca gttagcaaat ttctgtccag tttattcagc agcacctcct 31320tgccctcctc ccagctctgg tattgcagct tcctcctggc tgcaaacttt ctccacaatc 31380taaatggaat gtcagtttcc tcctgttcct gtccatccgc acccactatc ttcatgttgt 31440tgcagatgaa gcgcgcaaga ccgtctgaag ataccttcaa ccccgtgtat ccatatgaca 31500cggaaaccgg tcctccaact gtgccttttc ttactcctcc ctttgtatcc cccaatgggt 31560ttcaagagag tccccctggg gtactctctt tgcgcctatc cgaacctcta gttacctcca 31620atggcatgct tgcgctcaaa atgggcaacg gcctctctct ggacgaggcc ggcaacctta 31680cctcccaaaa tgtaaccact gtgagcccac ctctcaaaaa aaccaagtca aacataaacc 31740tggaaatatc tgcacccctc acagttacct cagaagccct aactgtggct gccgccgcac 31800ctctaatggt cgcgggcaac acactcacca tgcaatcaca ggccccgcta accgtgcacg 31860actccaaact tagcattgcc acccaaggac ccctcacagt gtcagaagga aagctagccc 31920tgcaaacatc aggccccctc accaccaccg atagcagtac ccttactatc actgcctcac 31980cccctctaac tactgccact ggtagcttgg gcattgactt gaaagagccc atttatacac 32040aaaatggaaa actaggacta aagtacgggg ctcctttgca tgtaacagac gacctaaaca 32100ctttgaccgt agcaactggt ccaggtgtga ctattaataa tacttccttg caaactaaag 32160ttactggagc cttgggtttt gattcacaag gcaatatgca acttaatgta gcaggaggac 32220taaggattga ttctcaaaac agacgcctta tacttgatgt tagttatccg tttgatgctc 32280aaaaccaact aaatctaaga ctaggacagg gccctctttt tataaactca gcccacaact 32340tggatattaa ctacaacaaa ggcctttact tgtttacagc ttcaaacaat tccaaaaagc 32400ttgaggttaa cctaagcact gccaaggggt tgatgtttga cgctacagcc atagccatta 32460atgcaggaga tgggcttgaa tttggttcac ctaatgcacc aaacacaaat cccctcaaaa 32520caaaaattgg ccatggccta gaatttgatt caaacaaggc tatggttcct aaactaggaa 32580ctggccttag ttttgacagc acaggtgcca ttacagtagg aaacaaaaat aatgataagc 32640taactttgtg gaccacacca gctccatctc ctaactgtag actaaatgca gagaaagatg 32700ctaaactcac tttggtctta acaaaatgtg gcagtcaaat acttgctaca gtttcagttt 32760tggctgttaa aggcagtttg gctccaatat ctggaacagt tcaaagtgct catcttatta 32820taagatttga cgaaaatgga gtgctactaa acaattcctt cctggaccca gaatattgga 32880actttagaaa tggagatctt actgaaggca cagcctatac aaacgctgtt ggatttatgc 32940ctaacctatc agcttatcca aaatctcacg gtaaaactgc caaaagtaac attgtcagtc 33000aagtttactt aaacggagac aaaactaaac ctgtaacact aaccattaca ctaaacggta 33060cacaggaaac aggagacaca actccaagtg catactctat gtcattttca tgggactggt 33120ctggccacaa ctacattaat gaaatatttg ccacatcctc ttacactttt tcatacattg 33180cccaagaata aagaatcgtt tgtgttatgt ttcaacgtgt ttatttttca attgcccggg 33240atcggtgatc accgatccag acatgataag atacattgat gagtttggac aaaccacaac 33300tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt 33360aaccattata agctgcaata aacaagttcc cggatcgcga tccggcccga ggctgtagcc 33420gacgatggtg cgccaggaga gttgttgatt cattgtttgc ctccctgctg cggtttttca 33480ccgaagttca tgccagtcca gcgtttttgc agcagaaaag ccgccgactt cggtttgcgg 33540tcgcgagtga agatcccttt cttgttaccg ccaacgcgca atatgccttg cgaggtcgca 33600aaatcggcga aattccatac ctgttcaccg acgacggcgc tgacgcgatc aaagacgcgg 33660tgatacatat ccagccatgc acactgatac tcttcactcc acatgtcggt gtacattgag 33720tgcagcccgg ctaacgtatc cacgccgtat tcggtgatga taatcggctg atgcagtttc 33780tcctgccagg ccagaagttc tttttccagt accttctctg ccgtttccaa atcgccgctt 33840tggacatacc atccgtaata acggttcagg cacagcacat caaagagatc gctgatggta 33900tcggtgtgag cgtcgcagaa cattacattg acgcaggtga tcggacgcgt cgggtcgagt 33960ttacgcgttg cttccgccag tggcgcgaaa tattcccgtg caccttgcgg acgggtatcc 34020ggttcgttgg caatactcca catcaccacg cttgggtggt ttttgtcacg cgctatcagc 34080tctttaatcg cctgtaagtg cgcttgctga gtttccccgt tgactgcctc ttcgctgtac 34140agttctttcg gcttgttgcc cgcttcgaaa ccaatgccta aagagaggtt aaagccgaca 34200gcagcagttt catcaatcac cacgatgcca tgttcatctg cccagtcgag catctcttca 34260gcgtaagggt aatgcgaggt acggtaggag ttggccccaa tccagtccat taatgcgtgg 34320tcgtgcacca tcagcacgtt atcgaatcct ttgccacgca agtccgcatc ttcatgacga 34380ccaaagccag taaagtagaa cggtttgtgg ttaatcagga actgttcgcc cttcactgcc 34440actgaccgga tgccgacgcg aagcgggtag atatcacact ctgtctggct tttggctgtg 34500acgcacagtt catagagata accttcaccc ggttgccaga ggtgcggatt caccacttgc 34560aaagtcccgc tagtgccttg tccagttgca accacctgtt gatccgcatc acgcagttca 34620acgctgacat caccattggc caccacctgc cagtcaacag acgcgtggtt acagtcttgc 34680gcgacatgcg tcaccacggt gatatcgtcc acccaggtgt tcggcgtggt gtagagcatt 34740acgctgcgat ggattccggc atagttaaag aaatcatgga agtaagactg ctttttcttg 34800ccgttttcgt cggtaatcac cattcccggc gggatagtct gccagttcag ttcgttgttc 34860acacaaacgg tgatacgtac acttttcccg gcaataacat acggcgtgac atcggcttca 34920aatggcgtat agccgccctg atgctccatc acttcctgat tattgaccca cactttgccg 34980taatgagtga ccgcatcgaa acgcagcacg atacgctggc ctgcccaacc tttcggtata 35040aagacttcgc gctgatacca gacgttgccc gcataattac gaatatctgc atcggcgaac 35100tgatcgttaa aactgcctgg cacagcaatt gcccggcttt cttgtaacgc gctttcccac 35160caacgctgat caattccaca gttttcgcga tccagactga atgcccacag gccgtcgagt 35220tttttgattt cacgggttgg ggtttctaca ggacggacca tgcgttcgac ctttctcttc 35280ttttttgggc ccatgatggc agatccgtat agtgagtcgt attagctggt tctttccgcc 35340tcagaagcca tagagcccac cgcatcccca gcatgcctgc tattgtcttc ccaatcctcc 35400cccttgctgt cctgccccac cccacccccc agaatagaat gacacctact cagacaatgc 35460gatgcaattt cctcatttta ttaggaaagg acagtgggag tggcaccttc cagggtcaag 35520gaaggcacgg gggaggggca aacaacagat ggctggcaac tagaaggcac agtcgaggct 35580gatcagcgag ctctagatgc atgctcgagc ggccgccagt gtgatggata tctgcagaat 35640tccagcacac tggcggccgt tactagtgga tccgagctcg gtacccggcc gttataacac 35700cactcgacac ggcaccagct caatcagtca cagtgtaaaa aagggccaag tgcagagcga 35760gtatatatag gactaaaaaa tgacgtaacg gttaaagtcc acaaaaaaca cccagaaaac 35820cgcacgcgaa cctacgccca gaaacgaaag ccaaaaaacc cacaacttcc tcaaatcgtc 35880acttccgttt tcccacgtta cgtcacttcc cattttaaga aaactacaat tcccaacaca 35940tacaagttac tccgccctaa aacctacgtc acccgccccg ttcccacgcc ccgcgccacg 36000tcacaaactc caccccctca ttatcatatt ggcttcaatc caaaataagg tatattattg 36060atgatg 360661733583DNAArtificial SequenceAdenoviral vector Adgp140(A).11D 17catcatcaat aatatacctt attttggatt gaagccaata tgataatgag ggggtggagt 60ttgtgacgtg gcgcggggcg tgggaacggg gcgggtgacg tagtagtgtg gcggaagtgt 120gatgttgcaa gtgtggcgga acacatgtaa gcgacggatg tggcaaaagt gacgtttttg 180gtgtgcgccg gtgtacacag gaagtgacaa ttttcgcgcg gttttaggcg gatgttgtag 240taaatttggg cgtaaccgag taagatttgg ccattttcgc gggaaaactg aataagagga 300agtgaaatct gaataatttt gtgttactca tagcgcgtaa tatttgtcta gggcccggga 360tcggtgatca ccgatccaga catgataaga tacattgatg agtttggaca aaccacaact 420agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta 480accattataa gctgcaataa acaagttccc ggatctttct agctagtcta gactagctag 540actcgagagc ggccgcaatc gataagcttg atgatcccac gtgttcacca cagccagcgg 600ctgatgtcga accagttcca caggctggcc cacttgtcca gggccagcag gtcctgctcg 660ttcttctcct gctggttctg gctctcctcg atcaggttgt agatgatctg ggtgtagttg 720ctgatctcct tgtcccactg cagccaggtc atgttgtccc agatctcgag ctgctggtcc 780ttcaggtagc gctccacggc cagcacgcgg gcctgcagct gcttgatgcc ccacacggtc 840agcttcagca tgtgctgctg ggcctcgatg gcgcgcagca ggttgctctg ctgctgcacg 900atgccgctca gcagctggcg ggcctgggcg gtaagcttgg cgcggctggg ggccacgccc 960aggggctcga tcttcaccac cttgtacttg tacagctcgc tgcgccagtt gtcgcgcatg 1020ttgccgccgc cggggcggaa gatctcgttg gtgctgttgt tgccgccgtc gcgggtcagc 1080agcaggccgg tgatgttgct ctcgcagcgg atcacgccct ggatgggggg ggggtacatg 1140gcctggccca ccttctgcca catgttgatg atctgcttga tgcggcaggt cagggtgatg 1200gtgtcgttgc tggtggtgtt gttgctctcg gtgctgttgc tctcccaggt gctgttgaac 1260aggccgctgg tgttgcagta gaagaactcg ccgccgcaga tgaagctgtg ggtggtgatc 1320tcgatgtcgc cgccgctgct cttctcgaag atgatggtct tgttcttgaa gtgctcgcgc 1380agcttcttgg ccacgccgcg cagggtgtcg ttccacttgg cgcggctcac gtggcagtgg 1440gcctggcgga tgtcgccgat gatgccgccg gtggcgtaga aggcctggcc ggggccgatg 1500cgcacgccct tgcgggtgtt gttgttgggg cgggtgcagt tgatcttcac ggccttatcc 1560agctgcacga tgatggtctt ggcgttgttg gtgatgttct cgctgcggat ctggatgccc 1620tcctcggcca ggctgccgtt cagcagcagc tgggtgctga tcaccggtcg gatgccgtgg 1680gtgcactgca cggtgctcac gttcttgcag gggccggtac cgttgaactc ggtgtccttg 1740cacttcagga tggcgaagcc ggcgggggcg cagtagtgga tggggatggg ctcgaagctc 1800accttggggc aggcctgggt gatggcgctg gtgttgcagt tgatcaggcg gtacttgtcg 1860gtctcgttct tctcgttgat ctgcaccacg tccagcttgt agaacaggct gtacacctgc 1920tgcttcttgt ccttcagctc ggtggtgatg ttgaagctac agttgcgcat ctcgttggtc 1980acgttgctgg cggtggcgtt gcagtccagg gtcacgcaca ggggggtcag cttcacgcag 2040ggcttcaggc tctggtccca caggctgatg atgtcggtgt gcatctgctc caccatgttg 2100ttgcgccaca tgttgaagtc ctcggtcacg ttctccaggt ggatctcctg ggggttgggg 2160tcggtgggca cgcaggcgtg ggtctcccac acgttgtgca cctcggtgtc gtaggccttg 2220gcgtcgctgg cgcagaacag ggtggtctcg gcgtccttcc acacgggcac gccgtagtac 2280acggccaccc acaggttctc ggcggcgctg tagatcacca gcatgcccag gatcatggtg 2340ccccagcgcc acaggttctg ccagctggtc tggatgccgc gcacgcgcat ggtggcgata 2400tctctagatc gaattctgca gtgatcaggg atcccagatc cgtatagtga gtcgtattag 2460gtaccggctg cagttggacc tgggagtgga cacctgtgga gagaaaggca aagtggatgt 2520cattgtcact caagtgtatg gccagatctc aagcctgcca cacctcaagt gaagccaagg 2580gggtgggcct atagactcta taggcggtac ttacgtcact cttggcacgg ggaatccgcg 2640ttccaatgca ccgttcccgg ccgcggaggc tggatcggtc ccggtgtctt ctatggaggt 2700caaaacagcg tggatggcgt ctccaggcga tctgacggtt cactaaacga gctctgctta 2760tatagacctc ccaccgtaca cgcctaccgc ccatttgcgt caatggggcg gagttgttac 2820gacattttgg aaagtcccgt tgattttggt gccaaaacaa actcccattg acgtcaatgg 2880ggtggagact tggaaatccc cgtgagtcaa accgctatcc acgcccattg atgtactgcc 2940aaaaccgcat caccatggta atagcgatga ctaatacgta gatgtactgc caagtaggaa 3000agtcccataa ggtcatgtac tgggcataat gccaggcggg ccatttaccg tcattgacgt 3060caataggggg cgtacttggc atatgataca cttgatgtac tgccaagtgg gcagtttacc 3120gtaaatactc cacccattga cgtcaatgga aagtccctat tggcgttact atgggaacat 3180acgtcattat tgacgtcaat gggcgggggt cgttgggcgg tcagccaggc gggccattta 3240ccgtaagtta tgtaacgcgg aactccatat atgggctatg aactaatgac cccgtaattg 3300attactatta ataactagta ctgaaatgtg tgggcgtggc ttaagggtgg gaaagaatat 3360ataaggtggg ggtcttatgt agttttgtat ctgttttgca gcagccgccg ccgccatgag 3420caccaactcg tttgatggaa gcattgtgag ctcatatttg acaacgcgca tgcccccatg 3480ggccggggtg cgtcagaatg tgatgggctc cagcattgat ggtcgccccg tcctgcccgc 3540aaactctact accttgacct acgagaccgt gtctggaacg ccgttggaga ctgcagcctc 3600cgccgccgct tcagccgctg cagccaccgc ccgcgggatt gtgactgact ttgctttcct 3660gagcccgctt gcaagcagtg cagcttcccg ttcatccgcc cgcgatgaca agttgacggc 3720tcttttggca caattggatt ctttgacccg ggaacttaat gtcgtttctc agcagctgtt 3780ggatctgcgc cagcaggttt ctgccctgaa ggcttcctcc cctcccaatg cggtttaaaa 3840cataaataaa aaaccagact ctgtttggat ttggatcaag caagtgtctt gctgtcttta 3900tttaggggtt ttgcgcgcgc ggtaggcccg ggaccagcgg tctcggtcgt tgagggtcct 3960gtgtattttt tccaggacgt ggtaaaggtg actctggatg ttcagataca tgggcataag 4020cccgtctctg gggtggaggt agcaccactg cagagcttca tgctgcgggg tggtgttgta

4080gatgatccag tcgtagcagg agcgctgggc gtggtgccta aaaatgtctt tcagtagcaa 4140gctgattgcc aggggcaggc ccttggtgta agtgtttaca aagcggttaa gctgggatgg 4200gtgcatacgt ggggatatga gatgcatctt ggactgtatt tttaggttgg ctatgttccc 4260agccatatcc ctccggggat tcatgttgtg cagaaccacc agcacagtgt atccggtgca 4320cttgggaaat ttgtcatgta gcttagaagg aaatgcgtgg aagaacttgg agacgccctt 4380gtgacctcca agattttcca tgcattcgtc cataatgatg gcaatgggcc cacgggcggc 4440ggcctgggcg aagatatttc tgggatcact aacgtcatag ttgtgttcca ggatgagatc 4500gtcataggcc atttttacaa agcgcgggcg gagggtgcca gactgcggta taatggttcc 4560atccggccca ggggcgtagt taccctcaca gatttgcatt tcccacgctt tgagttcaga 4620tggggggatc atgtctacct gcggggcgat gaagaaaacg gtttccgggg taggggagat 4680cagctgggaa gaaagcaggt tcctgagcag ctgcgactta ccgcagccgg tgggcccgta 4740aatcacacct attaccggct gcaactggta gttaagagag ctgcagctgc cgtcatccct 4800gagcaggggg gccacttcgt taagcatgtc cctgactcgc atgttttccc tgaccaaatc 4860cgccagaagg cgctcgccgc ccagcgatag cagttcttgc aaggaagcaa agtttttcaa 4920cggtttgaga ccgtccgccg taggcatgct tttgagcgtt tgaccaagca gttccaggcg 4980gtcccacagc tcggtcacct gctctacggc atctcgatcc agcatatctc ctcgtttcgc 5040gggttggggc ggctttcgct gtacggcagt agtcggtgct cgtccagacg ggccagggtc 5100atgtctttcc acgggcgcag ggtcctcgtc agcgtagtct gggtcacggt gaaggggtgc 5160gctccgggct gcgcgctggc cagggtgcgc ttgaggctgg tcctgctggt gctgaagcgc 5220tgccggtctt cgccctgcgc gtcggccagg tagcatttga ccatggtgtc atagtccagc 5280ccctccgcgg cgtggccctt ggcgcgcagc ttgcccttgg aggaggcgcc gcacgagggg 5340cagtgcagac ttttgagggc gtagagcttg ggcgcgagaa ataccgattc cggggagtag 5400gcatccgcgc cgcaggcccc gcagacggtc tcgcattcca cgagccaggt gagctctggc 5460cgttcggggt caaaaaccag gtttccccca tgctttttga tgcgtttctt acctctggtt 5520tccatgagcc ggtgtccacg ctcggtgacg aaaaggctgt ccgtgtcccc gtatacagac 5580ttgagaggcc tgtcctcgag cggtgttccg cggtcctcct cgtatagaaa ctcggaccac 5640tctgagacaa aggctcgcgt ccaggccagc acgaaggagg ctaagtggga ggggtagcgg 5700tcgttgtcca ctagggggtc cactcgctcc agggtgtgaa gacacatgtc gccctcttcg 5760gcatcaagga aggtgattgg tttgtaggtg taggccacgt gaccgggtgt tcctgaaggg 5820gggctataaa agggggtggg ggcgcgttcg tcctcactct cttccgcatc gctgtctgcg 5880agggccagct gttggggtga gtactccctc tgaaaagcgg gcatgacttc tgcgctaaga 5940ttgtcagttt ccaaaaacga ggaggatttg atattcacct ggcccgcggt gatgcctttg 6000agggtggccg catccatctg gtcagaaaag acaatctttt tgttgtcaag cttggtggca 6060aacgacccgt agagggcgtt ggacagcaac ttggcgatgg agcgcagggt ttggtttttg 6120tcgcgatcgg cgcgctcctt ggccgcgatg tttagctgca cgtattcgcg cgcaacgcac 6180cgccattcgg gaaagacggt ggtgcgctcg tcgggcacca ggtgcacgcg ccaaccgcgg 6240ttgtgcaggg tgacaaggtc aacgctggtg gctacctctc cgcgtaggcg ctcgttggtc 6300cagcagaggc ggccgccctt gcgcgagcag aatggcggta gggggtctag ctgcgtctcg 6360tccggggggt ctgcgtccac ggtaaagacc ccgggcagca ggcgcgcgtc gaagtagtct 6420atcttgcatc cttgcaagtc tagcgcctgc tgccatgcgc gggcggcaag cgcgcgctcg 6480tatgggttga gtgggggacc ccatggcatg gggtgggtga gcgcggaggc gtacatgccg 6540caaatgtcgt aaacgtagag gggctctctg agtattccaa gatatgtagg gtagcatctt 6600ccaccgcgga tgctggcgcg cacgtaatcg tatagttcgt gcgagggagc gaggaggtcg 6660ggaccgaggt tgctacgggc gggctgctct gctcggaaga ctatctgcct gaagatggca 6720tgtgagttgg atgatatggt tggacgctgg aagacgttga agctggcgtc tgtgagacct 6780accgcgtcac gcacgaagga ggcgtaggag tcgcgcagct tgttgaccag ctcggcggtg 6840acctgcacgt ctagggcgca gtagtccagg gtttccttga tgatgtcata cttatcctgt 6900cccttttttt tccacagctc gcggttgagg acaaactctt cgcggtcttt ccagtactct 6960tggatcggaa acccgtcggc ctccgaacgg taagagccta gcatgtagaa ctggttgacg 7020gcctggtagg cgcagcatcc cttttctacg ggtagcgcgt atgcctgcgc ggccttccgg 7080agcgaggtgt gggtgagcgc aaaggtgtcc ctgaccatga ctttgaggta ctggtatttg 7140aagtcagtgt cgtcgcatcc gccctgctcc cagagcaaaa agtccgtgcg ctttttggaa 7200cgcggatttg gcagggcgaa ggtgacatcg ttgaagagta tctttcccgc gcgaggcata 7260aagttgcgtg tgatgcggaa gggtcccggc acctcggaac ggttgttaat tacctgggcg 7320gcgagcacga tctcgtcaaa gccgttgatg ttgtggccca caatgtaaag ttccaagaag 7380cgcgggatgc ccttgatgga aggcaatttt ttaagttcct cgtaggtgag ctcttcaggg 7440gagctgagcc cgtgctctga aagggcccag tctgcaagat gagggttgga agcgacgaat 7500gagctccaca ggtcacgggc cattagcatt tgcaggtggt cgcgaaaggt cctaaactgg 7560cgacctatgg ccattttttc tggggtgatg cagtagaagg taagcgggtc ttgttcccag 7620cggtcccatc caaggttcgc ggctaggtct cgcgcggcag tcactagagg ctcatctccg 7680ccgaacttca tgaccagcat gaagggcacg agctgcttcc caaaggcccc catccaagta 7740taggtctcta catcgtaggt gacaaagaga cgctcggtgc gaggatgcga gccgatcggg 7800aagaactgga tctcccgcca ccaattggag gagtggctat tgatgtggtg aaagtagaag 7860tccctgcgac gggccgaaca ctcgtgctgg cttttgtaaa aacgtgcgca gtactggcag 7920cggtgcacgg gctgtacatc ctgcacgagg ttgacctgac gaccgcgcac aaggaagcag 7980agtgggaatt tgagcccctc gcctggcggg tttggctggt ggtcttctac ttcggctgct 8040tgtccttgac cgtctggctg ctcgagggga gttacggtgg atcggaccac cacgccgcgc 8100gagcccaaag tccagatgtc cgcgcgcggc ggtcggagct tgatgacaac atcgcgcaga 8160tgggagctgt ccatggtctg gagctcccgc ggcgtcaggt caggcgggag ctcctgcagg 8220tttacctcgc atagacgggt cagggcgcgg gctagatcca ggtgatacct aatttccagg 8280ggctggttgg tggcggcgtc gatggcttgc aagaggccgc atccccgcgg cgcgactacg 8340gtaccgcgcg gcgggcggtg ggccgcgggg gtgtccttgg atgatgcatc taaaagcggt 8400gacgcgggcg agcccccgga ggtagggggg gctccggacc cgccgggaga gggggcaggg 8460gcacgtcggc gccgcgcgcg ggcaggagct ggtgctgcgc gcgtaggttg ctggcgaacg 8520cgacgacgcg gcggttgatc tcctgaatct ggcgcctctg cgtgaagacg acgggcccgg 8580tgagcttgaa cctgaaagag agttcgacag aatcaatttc ggtgtcgttg acggcggcct 8640ggcgcaaaat ctcctgcacg tctcctgagt tgtcttgata ggcgatctcg gccatgaact 8700gctcgatctc ttcctcctgg agatctccgc gtccggctcg ctccacggtg gcggcgaggt 8760cgttggaaat gcgggccatg agctgcgaga aggcgttgag gcctccctcg ttccagacgc 8820ggctgtagac cacgccccct tcggcatcgc gggcgcgcat gaccacctgc gcgagattga 8880gctccacgtg ccgggcgaag acggcgtagt ttcgcaggcg ctgaaagagg tagttgaggg 8940tggtggcggt gtgttctgcc acgaagaagt acataaccca gcgtcgcaac gtggattcgt 9000tgatatcccc caaggcctca aggcgctcca tggcctcgta gaagtccacg gcgaagttga 9060aaaactggga gttgcgcgcc gacacggtta actcctcctc cagaagacgg atgagctcgg 9120cgacagtgtc gcgcacctcg cgctcaaagg ctacaggggc ctcttcttct tcttcaatct 9180cctcttccat aagggcctcc ccttcttctt cttctggcgg cggtggggga ggggggacac 9240ggcggcgacg acggcgcacc gggaggcggt cgacaaagcg ctcgatcatc tccccgcggc 9300gacggcgcat ggtctcggtg acggcgcggc cgttctcgcg ggggcgcagt tggaagacgc 9360cgcccgtcat gtcccggtta tgggttggcg gggggctgcc atgcggcagg gatacggcgc 9420taacgatgca tctcaacaat tgttgtgtag gtactccgcc gccgagggac ctgagcgagt 9480ccgcatcgac cggatcggaa aacctctcga gaaaggcgtc taaccagtca cagtcgcaag 9540gtaggctgag caccgtggcg ggcggcagcg ggcggcggtc ggggttgttt ctggcggagg 9600tgctgctgat gatgtaatta aagtaggcgg tcttgagacg gcggatggtc gacagaagca 9660ccatgtcctt gggtccggcc tgctgaatgc gcaggcggtc ggccatgccc caggcttcgt 9720tttgacatcg gcgcaggtct ttgtagtagt cttgcatgag cctttctacc ggcacttctt 9780cttctccttc ctcttgtcct gcatctcttg catctatcgc tgcggcggcg gcggagtttg 9840gccgtaggtg gcgccctctt cctcccatgc gtgtgacccc gaagcccctc atcggctgaa 9900gcagggctag gtcggcgaca acgcgctcgg ctaatatggc ctgctgcacc tgcgtgaggg 9960tagactggaa gtcatccatg tccacaaagc ggtggtatgc gcccgtgttg atggtgtaag 10020tgcagttggc cataacggac cagttaacgg tctggtgacc cggctgcgag agctcggtgt 10080acctgagacg cgagtaagcc ctcgagtcaa atacgtagtc gttgcaagtc cgcaccaggt 10140actggtatcc caccaaaaag tgcggcggcg gctggcggta gaggggccag cgtagggtgg 10200ccggggctcc gggggcgaga tcttccaaca taaggcgatg atatccgtag atgtacctgg 10260acatccaggt gatgccggcg gcggtggtgg aggcgcgcgg aaagtcgcgg acgcggttcc 10320agatgttgcg cagcggcaaa aagtgctcca tggtcgggac gctctggccg gtcaggcgcg 10380cgcaatcgtt gacgctctag cgtgcaaaag gagagcctgt aagcgggcac tcttccgtgg 10440tctggtggat aaattcgcaa gggtatcatg gcggacgacc ggggttcgag ccccgtatcc 10500ggccgtccgc cgtgatccat gcggttaccg cccgcgtgtc gaacccaggt gtgcgacgtc 10560agacaacggg ggagtgctcc ttttggcttc cttccaggcg cggcggctgc tgcgctagct 10620tttttggcca ctggccgcgc gcagcgtaag cggttaggct ggaaagcgaa agcattaagt 10680ggctcgctcc ctgtagccgg agggttattt tccaagggtt gagtcgcggg acccccggtt 10740cgagtctcgg accggccgga ctgcggcgaa cgggggtttg cctccccgtc atgcaagacc 10800ccgcttgcaa attcctccgg aaacagggac gagccccttt tttgcttttc ccagatgcat 10860ccggtgctgc ggcagatgcg cccccctcct cagcagcggc aagagcaaga gcagcggcag 10920acatgcaggg caccctcccc tcctcctacc gcgtcaggag gggcgacatc cgcggttgac 10980gcggcagcag atggtgatta cgaacccccg cggcgccggg cccggcacta cctggacttg 11040gaggagggcg agggcctggc gcggctagga gcgccctctc ctgagcggca cccaagggtg 11100cagctgaagc gtgatacgcg tgaggcgtac gtgccgcggc agaacctgtt tcgcgaccgc 11160gagggagagg agcccgagga gatgcgggat cgaaagttcc acgcagggcg cgagctgcgg 11220catggcctga atcgcgagcg gttgctgcgc gaggaggact ttgagcccga cgcgcgaacc 11280gggattagtc ccgcgcgcgc acacgtggcg gccgccgacc tggtaaccgc atacgagcag 11340acggtgaacc aggagattaa ctttcaaaaa agctttaaca accacgtgcg tacgcttgtg 11400gcgcgcgagg aggtggctat aggactgatg catctgtggg actttgtaag cgcgctggag 11460caaaacccaa atagcaagcc gctcatggcg cagctgttcc ttatagtgca gcacagcagg 11520gacaacgagg cattcaggga tgcgctgcta aacatagtag agcccgaggg ccgctggctg 11580ctcgatttga taaacatcct gcagagcata gtggtgcagg agcgcagctt gagcctggct 11640gacaaggtgg ccgccatcaa ctattccatg cttagcctgg gcaagtttta cgcccgcaag 11700atataccata ccccttacgt tcccatagac aaggaggtaa agatcgaggg gttctacatg 11760cgcatggcgc tgaaggtgct taccttgagc gacgacctgg gcgtttatcg caacgagcgc 11820atccacaagg ccgtgagcgt gagccggcgg cgcgagctca gcgaccgcga gctgatgcac 11880agcctgcaaa gggccctggc tggcacgggc agcggcgata gagaggccga gtcctacttt 11940gacgcgggcg ctgacctgcg ctgggcccca agccgacgcg ccctggaggc agctggggcc 12000ggacctgggc tggcggtggc acccgcgcgc gctggcaacg tcggcggcgt ggaggaatat 12060gacgaggacg atgagtacga gccagaggac ggcgagtact aagcggtgat gtttctgatc 12120agatgatgca agacgcaacg gacccggcgg tgcgggcggc gctgcagagc cagccgtccg 12180gccttaactc cacggacgac tggcgccagg tcatggaccg catcatgtcg ctgactgcgc 12240gcaatcctga cgcgttccgg cagcagccgc aggccaaccg gctctccgca attctggaag 12300cggtggtccc ggcgcgcgca aaccccacgc acgagaaggt gctggcgatc gtaaacgcgc 12360tggccgaaaa cagggccatc cggcccgacg aggccggcct ggtctacgac gcgctgcttc 12420agcgcgtggc tcgttacaac agcggcaacg tgcagaccaa cctggaccgg ctggtggggg 12480atgtgcgcga ggccgtggcg cagcgtgagc gcgcgcagca gcagggcaac ctgggctcca 12540tggttgcact aaacgccttc ctgagtacac agcccgccaa cgtgccgcgg ggacaggagg 12600actacaccaa ctttgtgagc gcactgcggc taatggtgac tgagacaccg caaagtgagg 12660tgtaccagtc tgggccagac tattttttcc agaccagtag acaaggcctg cagaccgtaa 12720acctgagcca ggctttcaaa aacttgcagg ggctgtgggg ggtgcgggct cccacaggcg 12780accgcgcgac cgtgtctagc ttgctgacgc ccaactcgcg cctgttgctg ctgctaatag 12840cgcccttcac ggacagtggc agcgtgtccc gggacacata cctaggtcac ttgctgacac 12900tgtaccgcga ggccataggt caggcgcatg tggacgagca tactttccag gagattacaa 12960gtgtcagccg cgcgctgggg caggaggaca cgggcagcct ggaggcaacc ctaaactacc 13020tgctgaccaa ccggcggcag aagatcccct cgttgcacag tttaaacagc gaggaggagc 13080gcattttgcg ctacgtgcag cagagcgtga gccttaacct gatgcgcgac ggggtaacgc 13140ccagcgtggc gctggacatg accgcgcgca acatggaacc gggcatgtat gcctcaaacc 13200ggccgtttat caaccgccta atggactact tgcatcgcgc ggccgccgtg aaccccgagt 13260atttcaccaa tgccatcttg aacccgcact ggctaccgcc ccctggtttc tacaccgggg 13320gattcgaggt gcccgagggt aacgatggat tcctctggga cgacatagac gacagcgtgt 13380tttccccgca accgcagacc ctgctagagt tgcaacagcg cgagcaggca gaggcggcgc 13440tgcgaaagga aagcttccgc aggccaagca gcttgtccga tctaggcgct gcggccccgc 13500ggtcagatgc tagtagccca tttccaagct tgatagggtc tcttaccagc actcgcacca 13560cccgcccgcg cctgctgggc gaggaggagt acctaaacaa ctcgctgctg cagccgcagc 13620gcgaaaaaaa cctgcctccg gcatttccca acaacgggat agagagccta gtggacaaga 13680tgagtagatg gaagacgtac gcgcaggagc acagggacgt gccaggcccg cgcccgccca 13740cccgtcgtca aaggcacgac cgtcagcggg gtctggtgtg ggaggacgat gactcggcag 13800acgacagcag cgtcctggat ttgggaggga gtggcaaccc gtttgcgcac cttcgcccca 13860ggctggggag aatgttttaa aaaaaaaaaa agcatgatgc aaaataaaaa actcaccaag 13920gccatggcac cgagcgttgg ttttcttgta ttccccttag tatgcggcgc gcggcgatgt 13980atgaggaagg tcctcctccc tcctacgaga gtgtggtgag cgcggcgcca gtggcggcgg 14040cgctgggttc tcccttcgat gctcccctgg acccgccgtt tgtgcctccg cggtacctgc 14100ggcctaccgg ggggagaaac agcatccgtt actctgagtt ggcaccccta ttcgacacca 14160cccgtgtgta cctggtggac aacaagtcaa cggatgtggc atccctgaac taccagaacg 14220accacagcaa ctttctgacc acggtcattc aaaacaatga ctacagcccg ggggaggcaa 14280gcacacagac catcaatctt gacgaccggt cgcactgggg cggcgacctg aaaaccatcc 14340tgcataccaa catgccaaat gtgaacgagt tcatgtttac caataagttt aaggcgcggg 14400tgatggtgtc gcgcttgcct actaaggaca atcaggtgga gctgaaatac gagtgggtgg 14460agttcacgct gcccgagggc aactactccg agaccatgac catagacctt atgaacaacg 14520cgatcgtgga gcactacttg aaagtgggca gacagaacgg ggttctggaa agcgacatcg 14580gggtaaagtt tgacacccgc aacttcagac tggggtttga ccccgtcact ggtcttgtca 14640tgcctggggt atatacaaac gaagccttcc atccagacat cattttgctg ccaggatgcg 14700gggtggactt cacccacagc cgcctgagca acttgttggg catccgcaag cggcaaccct 14760tccaggaggg ctttaggatc acctacgatg atctggaggg tggtaacatt cccgcactgt 14820tggatgtgga cgcctaccag gcgagcttga aagatgacac cgaacagggc gggggtggcg 14880caggcggcag caacagcagt ggcagcggcg cggaagagaa ctccaacgcg gcagccgcgg 14940caatgcagcc ggtggaggac atgaacgatc atgccattcg cggcgacacc tttgccacac 15000gggctgagga gaagcgcgct gaggccgaag cagcggccga agctgccgcc cccgctgcgc 15060aacccgaggt cgagaagcct cagaagaaac cggtgatcaa acccctgaca gaggacagca 15120agaaacgcag ttacaaccta ataagcaatg acagcacctt cacccagtac cgcagctggt 15180accttgcata caactacggc gaccctcaga ccggaatccg ctcatggacc ctgctttgca 15240ctcctgacgt aacctgcggc tcggagcagg tctactggtc gttgccagac atgatgcaag 15300accccgtgac cttccgctcc acgcgccaga tcagcaactt tccggtggtg ggcgccgagc 15360tgttgcccgt gcactccaag agcttctaca acgaccaggc cgtctactcc caactcatcc 15420gccagtttac ctctctgacc cacgtgttca atcgctttcc cgagaaccag attttggcgc 15480gcccgccagc ccccaccatc accaccgtca gtgaaaacgt tcctgctctc acagatcacg 15540ggacgctacc gctgcgcaac agcatcggag gagtccagcg agtgaccatt actgacgcca 15600gacgccgcac ctgcccctac gtttacaagg ccctgggcat agtctcgccg cgcgtcctat 15660cgagccgcac tttttgagca agcatgtcca tccttatatc gcccagcaat aacacaggct 15720ggggcctgcg cttcccaagc aagatgtttg gcggggccaa gaagcgctcc gaccaacacc 15780cagtgcgcgt gcgcgggcac taccgcgcgc cctggggcgc gcacaaacgc ggccgcactg 15840ggcgcaccac cgtcgatgac gccatcgacg cggtggtgga ggaggcgcgc aactacacgc 15900ccacgccgcc accagtgtcc acagtggacg cggccattca gaccgtggtg cgcggagccc 15960ggcgctatgc taaaatgaag agacggcgga ggcgcgtagc acgtcgccac cgccgccgac 16020ccggcactgc cgcccaacgc gcggcggcgg ccctgcttaa ccgcgcacgt cgcaccggcc 16080gacgggcggc catgcgggcc gctcgaaggc tggccgcggg tattgtcact gtgcccccca 16140ggtccaggcg acgagcggcc gccgcagcag ccgcggccat tagtgctatg actcagggtc 16200gcaggggcaa cgtgtattgg gtgcgcgact cggttagcgg cctgcgcgtg cccgtgcgca 16260cccgcccccc gcgcaactag attgcaagaa aaaactactt agactcgtac tgttgtatgt 16320atccagcggc ggcggcgcgc aacgaagcta tgtccaagcg caaaatcaaa gaagagatgc 16380tccaggtcat cgcgccggag atctatggcc ccccgaagaa ggaagagcag gattacaagc 16440cccgaaagct aaagcgggtc aaaaagaaaa agaaagatga tgatgatgaa cttgacgacg 16500aggtggaact gctgcacgct accgcgccca ggcgacgggt acagtggaaa ggtcgacgcg 16560taaaacgtgt tttgcgaccc ggcaccaccg tagtctttac gcccggtgag cgctccaccc 16620gcacctacaa gcgcgtgtat gatgaggtgt acggcgacga ggacctgctt gagcaggcca 16680acgagcgcct cggggagttt gcctacggaa agcggcataa ggacatgctg gcgttgccgc 16740tggacgaggg caacccaaca cctagcctaa agcccgtaac actgcagcag gtgctgcccg 16800cgcttgcacc gtccgaagaa aagcgcggcc taaagcgcga gtctggtgac ttggcaccca 16860ccgtgcagct gatggtaccc aagcgccagc gactggaaga tgtcttggaa aaaatgaccg 16920tggaacctgg gctggagccc gaggtccgcg tgcggccaat caagcaggtg gcgccgggac 16980tgggcgtgca gaccgtggac gttcagatac ccactaccag tagcaccagt attgccaccg 17040ccacagaggg catggagaca caaacgtccc cggttgcctc agcggtggcg gatgccgcgg 17100tgcaggcggt cgctgcggcc gcgtccaaga cctctacgga ggtgcaaacg gacccgtgga 17160tgtttcgcgt ttcagccccc cggcgcccgc gccgttcgag gaagtacggc gccgccagcg 17220cgctactgcc cgaatatgcc ctacatcctt ccattgcgcc tacccccggc tatcgtggct 17280acacctaccg ccccagaaga cgagcaacta cccgacgccg aaccaccact ggaacccgcc 17340gccgccgtcg ccgtcgccag cccgtgctgg ccccgatttc cgtgcgcagg gtggctcgcg 17400aaggaggcag gaccctggtg ctgccaacag cgcgctacca ccccagcatc gtttaaaagc 17460cggtctttgt ggttcttgca gatatggccc tcacctgccg cctccgtttc ccggtgccgg 17520gattccgagg aagaatgcac cgtaggaggg gcatggccgg ccacggcctg acgggcggca 17580tgcgtcgtgc gcaccaccgg cggcggcgcg cgtcgcaccg tcgcatgcgc ggcggtatcc 17640tgcccctcct tattccactg atcgccgcgg cgattggcgc cgtgcccgga attgcatccg 17700tggccttgca ggcgcagaga cactgattaa aaacaagttg catgtggaaa aatcaaaata 17760aaaagtctgg actctcacgc tcgcttggtc ctgtaactat tttgtagaat ggaagacatc 17820aactttgcgt ctctggcccc gcgacacggc tcgcgcccgt tcatgggaaa ctggcaagat 17880atcggcacca gcaatatgag cggtggcgcc ttcagctggg gctcgctgtg gagcggcatt 17940aaaaatttcg gttccaccgt taagaactat ggcagcaagg cctggaacag cagcacaggc 18000cagatgctga gggataagtt gaaagagcaa aatttccaac aaaaggtggt agatggcctg 18060gcctctggca ttagcggggt ggtggacctg gccaaccagg cagtgcaaaa taagattaac 18120agtaagcttg atccccgccc tcccgtagag gagcctccac cggccgtgga gacagtgtct 18180ccagaggggc gtggcgaaaa gcgtccgcgc cccgacaggg aagaaactct ggtgacgcaa 18240atagacgagc ctccctcgta cgaggaggca ctaaagcaag gcctgcccac cacccgtccc 18300atcgcgccca tggctaccgg agtgctgggc cagcacacac ccgtaacgct ggacctgcct 18360ccccccgccg acacccagca gaaacctgtg ctgccaggcc cgaccgccgt tgttgtaacc 18420cgtcctagcc gcgcgtccct gcgccgcgcc gccagcggtc cgcgatcgtt gcggcccgta 18480gccagtggca actggcaaag cacactgaac agcatcgtgg gtctgggggt gcaatccctg 18540aagcgccgac gatgcttctg atagctaacg tgtcgtatgt gtgtcatgta tgcgtccatg 18600tcgccgccag aggagctgct gagccgccgc gcgcccgctt tccaagatgg ctaccccttc 18660gatgatgccg cagtggtctt acatgcacat ctcgggccag gacgcctcgg agtacctgag 18720ccccgggctg gtgcagtttg cccgcgccac cgagacgtac ttcagcctga ataacaagtt 18780tagaaacccc acggtggcgc ctacgcacga cgtgaccaca gaccggtccc agcgtttgac 18840gctgcggttc atccctgtgg accgtgagga tactgcgtac tcgtacaagg cgcggttcac 18900cctagctgtg ggtgataacc gtgtgctgga catggcttcc acgtactttg acatccgcgg 18960cgtgctggac aggggcccta cttttaagcc ctactctggc actgcctaca acgccctggc 19020tcccaagggt gccccaaatc cttgcgaatg ggatgaagct gctactgctc ttgaaataaa 19080cctagaagaa gaggacgatg acaacgaaga cgaagtagac gagcaagctg agcagcaaaa

19140aactcacgta tttgggcagg cgccttattc tggtataaat attacaaagg agggtattca 19200aataggtgtc gaaggtcaaa cacctaaata tgccgataaa acatttcaac ctgaacctca 19260aataggagaa tctcagtggt acgaaacaga aattaatcat gcagctggga gagtcctaaa 19320aaagactacc ccaatgaaac catgttacgg ttcatatgca aaacccacaa atgaaaatgg 19380agggcaaggc attcttgtaa agcaacaaaa tggaaagcta gaaagtcaag tggaaatgca 19440atttttctca actactgagg cagccgcagg caatggtgat aacttgactc ctaaagtggt 19500attgtacagt gaagatgtag atatagaaac cccagacact catatttctt acatgcccac 19560tattaaggaa ggtaactcac gagaactaat gggccaacaa tctatgccca acaggcctaa 19620ttacattgct tttagggaca attttattgg tctaatgtat tacaacagca cgggtaatat 19680gggtgttctg gcgggccaag catcgcagtt gaatgctgtt gtagatttgc aagacagaaa 19740cacagagctt tcataccagc ttttgcttga ttccattggt gatagaacca ggtacttttc 19800tatgtggaat caggctgttg acagctatga tccagatgtt agaattattg aaaatcatgg 19860aactgaagat gaacttccaa attactgctt tccactggga ggtgtgatta atacagagac 19920tcttaccaag gtaaaaccta aaacaggtca ggaaaatgga tgggaaaaag atgctacaga 19980attttcagat aaaaatgaaa taagagttgg aaataatttt gccatggaaa tcaatctaaa 20040tgccaacctg tggagaaatt tcctgtactc caacatagcg ctgtatttgc ccgacaagct 20100aaagtacagt ccttccaacg taaaaatttc tgataaccca aacacctacg actacatgaa 20160caagcgagtg gtggctcccg ggctagtgga ctgctacatt aaccttggag cacgctggtc 20220ccttgactat atggacaacg tcaacccatt taaccaccac cgcaatgctg gcctgcgcta 20280ccgctcaatg ttgctgggca atggtcgcta tgtgcccttc cacatccagg tgcctcagaa 20340gttctttgcc attaaaaacc tccttctcct gccgggctca tacacctacg agtggaactt 20400caggaaggat gttaacatgg ttctgcagag ctccctagga aatgacctaa gggttgacgg 20460agccagcatt aagtttgata gcatttgcct ttacgccacc ttcttcccca tggcccacaa 20520caccgcctcc acgcttgagg ccatgcttag aaacgacacc aacgaccagt cctttaacga 20580ctatctctcc gccgccaaca tgctctaccc tatacccgcc aacgctacca acgtgcccat 20640atccatcccc tcccgcaact gggcggcttt ccgcggctgg gccttcacgc gccttaagac 20700taaggaaacc ccatcactgg gctcgggcta cgacccttat tacacctact ctggctctat 20760accctaccta gatggaacct tttacctcaa ccacaccttt aagaaggtgg ccattacctt 20820tgactcttct gtcagctggc ctggcaatga ccgcctgctt acccccaacg agtttgaaat 20880taagcgctca gttgacgggg agggttacaa cgttgcccag tgtaacatga ccaaagactg 20940gttcctggta caaatgctag ctaactataa cattggctac cagggcttct atatcccaga 21000gagctacaag gaccgcatgt actccttctt tagaaacttc cagcccatga gccgtcaggt 21060ggtggatgat actaaataca aggactacca acaggtgggc atcctacacc aacacaacaa 21120ctctggattt gttggctacc ttgcccccac catgcgcgaa ggacaggcct accctgctaa 21180cttcccctat ccgcttatag gcaagaccgc agttgacagc attacccaga aaaagtttct 21240ttgcgatcgc accctttggc gcatcccatt ctccagtaac tttatgtcca tgggcgcact 21300cacagacctg ggccaaaacc ttctctacgc caactccgcc cacgcgctag acatgacttt 21360tgaggtggat cccatggacg agcccaccct tctttatgtt ttgtttgaag tctttgacgt 21420ggtccgtgtg caccagccgc accgcggcgt catcgaaacc gtgtacctgc gcacgccctt 21480ctcggccggc aacgccacaa cataaagaag caagcaacat caacaacagc tgccgccatg 21540ggctccagtg agcaggaact gaaagccatt gtcaaagatc ttggttgtgg gccatatttt 21600ttgggcacct atgacaagcg ctttccaggc tttgtttctc cacacaagct cgcctgcgcc 21660atagtcaata cggccggtcg cgagactggg ggcgtacact ggatggcctt tgcctggaac 21720ccgcactcaa aaacatgcta cctctttgag ccctttggct tttctgacca gcgactcaag 21780caggtttacc agtttgagta cgagtcactc ctgcgccgta gcgccattgc ttcttccccc 21840gaccgctgta taacgctgga aaagtccacc caaagcgtac aggggcccaa ctcggccgcc 21900tgtggactat tctgctgcat gtttctccac gcctttgcca actggcccca aactcccatg 21960gatcacaacc ccaccatgaa ccttattacc ggggtaccca actccatgct caacagtccc 22020caggtacagc ccaccctgcg tcgcaaccag gaacagctct acagcttcct ggagcgccac 22080tcgccctact tccgcagcca cagtgcgcag attaggagcg ccacttcttt ttgtcacttg 22140aaaaacatgt aaaaataatg tactagagac actttcaata aaggcaaatg cttttatttg 22200tacactctcg ggtgattatt tacccccacc cttgccgtct gcgccgttta aaaatcaaag 22260gggttctgcc gcgcatcgct atgcgccact ggcagggaca cgttgcgata ctggtgttta 22320gtgctccact taaactcagg cacaaccatc cgcggcagct cggtgaagtt ttcactccac 22380aggctgcgca ccatcaccaa cgcgtttagc aggtcgggcg ccgatatctt gaagtcgcag 22440ttggggcctc cgccctgcgc gcgcgagttg cgatacacag ggttgcagca ctggaacact 22500atcagcgccg ggtggtgcac gctggccagc acgctcttgt cggagatcag atccgcgtcc 22560aggtcctccg cgttgctcag ggcgaacgga gtcaactttg gtagctgcct tcccaaaaag 22620ggcgcgtgcc caggctttga gttgcactcg caccgtagtg gcatcaaaag gtgaccgtgc 22680ccggtctggg cgttaggata cagcgcctgc ataaaagcct tgatctgctt aaaagccacc 22740tgagcctttg cgccttcaga gaagaacatg ccgcaagact tgccggaaaa ctgattggcc 22800ggacaggccg cgtcgtgcac gcagcacctt gcgtcggtgt tggagatctg caccacattt 22860cggccccacc ggttcttcac gatcttggcc ttgctagact gctccttcag cgcgcgctgc 22920ccgttttcgc tcgtcacatc catttcaatc acgtgctcct tatttatcat aatgcttccg 22980tgtagacact taagctcgcc ttcgatctca gcgcagcggt gcagccacaa cgcgcagccc 23040gtgggctcgt gatgcttgta ggtcacctct gcaaacgact gcaggtacgc ctgcaggaat 23100cgccccatca tcgtcacaaa ggtcttgttg ctggtgaagg tcagctgcaa cccgcggtgc 23160tcctcgttca gccaggtctt gcatacggcc gccagagctt ccacttggtc aggcagtagt 23220ttgaagttcg cctttagatc gttatccacg tggtacttgt ccatcagcgc gcgcgcagcc 23280tccatgccct tctcccacgc agacacgatc ggcacactca gcgggttcat caccgtaatt 23340tcactttccg cttcgctggg ctcttcctct tcctcttgcg tccgcatacc acgcgccact 23400gggtcgtctt cattcagccg ccgcactgtg cgcttacctc ctttgccatg cttgattagc 23460accggtgggt tgctgaaacc caccatttgt agcgccacat cttctctttc ttcctcgctg 23520tccacgatta cctctggtga tggcgggcgc tcgggcttgg gagaagggcg cttctttttc 23580ttcttgggcg caatggccaa atccgccgcc gaggtcgatg gccgcgggct gggtgtgcgc 23640ggcaccagcg cgtcttgtga tgagtcttcc tcgtcctcgg actcgatacg ccgcctcatc 23700cgcttttttg ggggcgcccg gggaggcggc ggcgacgggg acggggacga cacgtcctcc 23760atggttgggg gacgtcgcgc cgcaccgcgt ccgcgctcgg gggtggtttc gcgctgctcc 23820tcttcccgac tggccatttc cttctcctat aggcagaaaa agatcatgga gtcagtcgag 23880aagaaggaca gcctaaccgc cccctctgag ttcgccacca ccgcctccac cgatgccgcc 23940aacgcgccta ccaccttccc cgtcgaggca cccccgcttg aggaggagga agtgattatc 24000gagcaggacc caggttttgt aagcgaagac gacgaggacc gctcagtacc aacagaggat 24060aaaaagcaag accaggacaa cgcagaggca aacgaggaac aagtcgggcg gggggacgaa 24120aggcatggcg actacctaga tgtgggagac gacgtgctgt tgaagcatct gcagcgccag 24180tgcgccatta tctgcgacgc gttgcaagag cgcagcgatg tgcccctcgc catagcggat 24240gtcagccttg cctacgaacg ccacctattc tcaccgcgcg taccccccaa acgccaagaa 24300aacggcacat gcgagcccaa cccgcgcctc aacttctacc ccgtatttgc cgtgccagag 24360gtgcttgcca cctatcacat ctttttccaa aactgcaaga tacccctatc ctgccgtgcc 24420aaccgcagcc gagcggacaa gcagctggcc ttgcggcagg gcgctgtcat acctgatatc 24480gcctcgctca acgaagtgcc aaaaatcttt gagggtcttg gacgcgacga gaagcgcgcg 24540gcaaacgctc tgcaacagga aaacagcgaa aatgaaagtc actctggagt gttggtggaa 24600ctcgagggtg acaacgcgcg cctagccgta ctaaaacgca gcatcgaggt cacccacttt 24660gcctacccgg cacttaacct accccccaag gtcatgagca cagtcatgag tgagctgatc 24720gtgcgccgtg cgcagcccct ggagagggat gcaaatttgc aagaacaaac agaggagggc 24780ctacccgcag ttggcgacga gcagctagcg cgctggcttc aaacgcgcga gcctgccgac 24840ttggaggagc gacgcaaact aatgatggcc gcagtgctcg ttaccgtgga gcttgagtgc 24900atgcagcggt tctttgctga cccggagatg cagcgcaagc tagaggaaac attgcactac 24960acctttcgac agggctacgt acgccaggcc tgcaagatct ccaacgtgga gctctgcaac 25020ctggtctcct accttggaat tttgcacgaa aaccgccttg ggcaaaacgt gcttcattcc 25080acgctcaagg gcgaggcgcg ccgcgactac gtccgcgact gcgtttactt atttctatgc 25140tacacctggc agacggccat gggcgtttgg cagcagtgct tggaggagtg caacctcaag 25200gagctgcaga aactgctaaa gcaaaacttg aaggacctat ggacggcctt caacgagcgc 25260tccgtggccg cgcacctggc ggacatcatt ttccccgaac gcctgcttaa aaccctgcaa 25320cagggtctgc cagacttcac cagtcaaagc atgttgcaga actttaggaa ctttatccta 25380gagcgctcag gaatcttgcc cgccacctgc tgtgcacttc ctagcgactt tgtgcccatt 25440aagtaccgcg aatgccctcc gccgctttgg ggccactgct accttctgca gctagccaac 25500taccttgcct accactctga cataatggaa gacgtgagcg gtgacggtct actggagtgt 25560cactgtcgct gcaacctatg caccccgcac cgctccctgg tttgcaattc gcagctgctt 25620aacgaaagtc aaattatcgg tacctttgag ctgcagggtc cctcgcctga cgaaaagtcc 25680gcggctccgg ggttgaaact cactccgggg ctgtggacgt cggcttacct tcgcaaattt 25740gtacctgagg actaccacgc ccacgagatt aggttctacg aagaccaatc ccgcccgcct 25800aatgcggagc ttaccgcctg cgtcattacc cagggccaca ttcttggcca attgcaagcc 25860atcaacaaag cccgccaaga gtttctgcta cgaaagggac ggggggttta cttggacccc 25920cagtccggcg aggagctcaa cccaatcccc ccgccgccgc agccctatca gcagcagccg 25980cgggcccttg cttcccagga tggcacccaa aaagaagctg cagctgccgc cgccacccac 26040ggacgaggag gaatactggg acagtcaggc agaggaggtt ttggacgagg aggaggagga 26100catgatggaa gactgggaga gcctagacga ggaagcttcc gaggtcgaag aggtgtcaga 26160cgaaacaccg tcaccctcgg tcgcattccc ctcgccggcg ccccagaaat cggcaaccgg 26220ttccagcatg gctacaacct ccgctcctca ggcgccgccg gcactgcccg ttcgccgacc 26280caaccgtaga tgggacacca ctggaaccag ggccggtaag tccaagcagc cgccgccgtt 26340agcccaagag caacaacagc gccaaggcta ccgctcatgg cgcgggcaca agaacgccat 26400agttgcttgc ttgcaagact gtgggggcaa catctccttc gcccgccgct ttcttctcta 26460ccatcacggc gtggccttcc cccgtaacat cctgcattac taccgtcatc tctacagccc 26520atactgcacc ggcggcagcg gcagcaacag cagcggccac acagaagcaa aggcgaccgg 26580atagcaagac tctgacaaag cccaagaaat ccacagcggc ggcagcagca ggaggaggag 26640cgctgcgtct ggcgcccaac gaacccgtat cgacccgcga gcttagaaac aggatttttc 26700ccactctgta tgctatattt caacagagca ggggccaaga acaagagctg aaaataaaaa 26760acaggtctct gcgatccctc acccgcagct gcctgtatca caaaagcgaa gatcagcttc 26820ggcgcacgct ggaagacgcg gaggctctct tcagtaaata ctgcgcgctg actcttaagg 26880actagtttcg cgccctttct caaatttaag cgcgaaaact acgtcatctc cagcggccac 26940acccggcgcc agcacctgtt gtcagcgcca ttatgagcaa ggaaattccc acgccctaca 27000tgtggagtta ccagccacaa atgggacttg cggctggagc tgcccaagac tactcaaccc 27060gaataaacta catgagcgcg ggaccccaca tgatatcccg ggtcaacgga atacgcgccc 27120accgaaaccg aattctcctg gaacaggcgg ctattaccac cacacctcgt aataacctta 27180atccccgtag ttggcccgct gccctggtgt accaggaaag tcccgctccc accactgtgg 27240tacttcccag agacgcccag gccgaagttc agatgactaa ctcaggggcg cagcttgcgg 27300gcggctttcg tcacagggtg cggtcgcccg ggcagggtat aactcacctg acaatcagag 27360ggcgaggtat tcagctcaac gacgagtcgg tgagctcctc gcttggtctc cgtccggacg 27420ggacatttca gatcggcggc gccggccgct cttcattcac gcctcgtcag gcaatcctaa 27480ctctgcagac ctcgtcctct gagccgcgct ctggaggcat tggaactctg caatttattg 27540aggagtttgt gccatcggtc tactttaacc ccttctcggg acctcccggc cactatccgg 27600atcaatttat tcctaacttt gacgcggtaa aggactcggc ggacggctac gactgaatgt 27660taagtggaga ggcagagcaa ctgcgcctga aacacctggt ccactgtcgc cgccacaagt 27720gctttgcccg cgactccggt gagttttgct actttgaatt gcccgaggat catatcgagg 27780gcccggcgca cggcgtccgg cttaccgccc agggagagct tgcccgtagc ctgattcggg 27840agtttaccca gcgccccctg ctagttgagc gggacagggg accctgtgtt ctcactgtga 27900tttgcaactg tcctaaccct ggattacatc aagatctttg ttgccatctc tgtgctgagt 27960ataataaata cagaaattaa aatatactgg ggctcctatc gccatcctgt aaacgccacc 28020gtcttcaccc gcccaagcaa accaaggcga accttacctg gtacttttaa catctctccc 28080tctgtgattt acaacagttt caacccagac ggagtgagtc tacgagagaa cctctccgag 28140ctcagctact ccatcagaaa aaacaccacc ctccttacct gccgggaacg tacgagtgcg 28200tcaccggccg ctgcaccaca cctaccgcct gaccgtaaac cagacttttt ccggacagac 28260ctcaataact ctgtttacca gaacaggagg tgagcttaga aaacccttag ggtattaggc 28320caaaggcgca gctactgtgg ggtttatgaa caattcaagc aactctacgg gctattctaa 28380ttcaggtttc tctagaaatg gacggaatta ttacagagca gcgcctgcta gaaagacgca 28440gggcagcggc cgagcaacag cgcatgaatc aagagctcca agacatggtt aacttgcacc 28500agtgcaaaag gggtatcttt tgtctggtaa agcaggccaa agtcacctac gacagtaata 28560ccaccggaca ccgccttagc tacaagttgc caaccaagcg tcagaaattg gtggtcatgg 28620tgggagaaaa gcccattacc ataactcagc actcggtaga aaccgaaggc tgcattcact 28680caccttgtca aggacctgag gatctctgca cccttattaa gaccctgtgc ggtctcaaag 28740atcttattcc ctttaactaa taaaaaaaaa taataaagca tcacttactt aaaatcagtt 28800agcaaatttc tgtccagttt attcagcagc acctccttgc cctcctccca gctctggtat 28860tgcagcttcc tcctggctgc aaactttctc cacaatctaa atggaatgtc agtttcctcc 28920tgttcctgtc catccgcacc cactatcttc atgttgttgc agatgaagcg cgcaagaccg 28980tctgaagata ccttcaaccc cgtgtatcca tatgacacgg aaaccggtcc tccaactgtg 29040ccttttctta ctcctccctt tgtatccccc aatgggtttc aagagagtcc ccctggggta 29100ctctctttgc gcctatccga acctctagtt acctccaatg gcatgcttgc gctcaaaatg 29160ggcaacggcc tctctctgga cgaggccggc aaccttacct cccaaaatgt aaccactgtg 29220agcccacctc tcaaaaaaac caagtcaaac ataaacctgg aaatatctgc acccctcaca 29280gttacctcag aagccctaac tgtggctgcc gccgcacctc taatggtcgc gggcaacaca 29340ctcaccatgc aatcacaggc cccgctaacc gtgcacgact ccaaacttag cattgccacc 29400caaggacccc tcacagtgtc agaaggaaag ctagccctgc aaacatcagg ccccctcacc 29460accaccgata gcagtaccct tactatcact gcctcacccc ctctaactac tgccactggt 29520agcttgggca ttgacttgaa agagcccatt tatacacaaa atggaaaact aggactaaag 29580tacggggctc ctttgcatgt aacagacgac ctaaacactt tgaccgtagc aactggtcca 29640ggtgtgacta ttaataatac ttccttgcaa actaaagtta ctggagcctt gggttttgat 29700tcacaaggca atatgcaact taatgtagca ggaggactaa ggattgattc tcaaaacaga 29760cgccttatac ttgatgttag ttatccgttt gatgctcaaa accaactaaa tctaagacta 29820ggacagggcc ctctttttat aaactcagcc cacaacttgg atattaacta caacaaaggc 29880ctttacttgt ttacagcttc aaacaattcc aaaaagcttg aggttaacct aagcactgcc 29940aaggggttga tgtttgacgc tacagccata gccattaatg caggagatgg gcttgaattt 30000ggttcaccta atgcaccaaa cacaaatccc ctcaaaacaa aaattggcca tggcctagaa 30060tttgattcaa acaaggctat ggttcctaaa ctaggaactg gccttagttt tgacagcaca 30120ggtgccatta cagtaggaaa caaaaataat gataagctaa ctttgtggac cacaccagct 30180ccatctccta actgtagact aaatgcagag aaagatgcta aactcacttt ggtcttaaca 30240aaatgtggca gtcaaatact tgctacagtt tcagttttgg ctgttaaagg cagtttggct 30300ccaatatctg gaacagttca aagtgctcat cttattataa gatttgacga aaatggagtg 30360ctactaaaca attccttcct ggacccagaa tattggaact ttagaaatgg agatcttact 30420gaaggcacag cctatacaaa cgctgttgga tttatgccta acctatcagc ttatccaaaa 30480tctcacggta aaactgccaa aagtaacatt gtcagtcaag tttacttaaa cggagacaaa 30540actaaacctg taacactaac cattacacta aacggtacac aggaaacagg agacacaact 30600ccaagtgcat actctatgtc attttcatgg gactggtctg gccacaacta cattaatgaa 30660atatttgcca catcctctta cactttttca tacattgccc aagaataaag aatcgtttgt 30720gttatgtttc aacgtgttta tttttcaatt gcccgggatc ggtgatcacc gatccagaca 30780tgataagata cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct 30840ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac 30900aagttcccgg atcgcgatcc ggcccgaggc tgtagccgac gatggtgcgc caggagagtt 30960gttgattcat tgtttgcctc cctgctgcgg tttttcaccg aagttcatgc cagtccagcg 31020tttttgcagc agaaaagccg ccgacttcgg tttgcggtcg cgagtgaaga tccctttctt 31080gttaccgcca acgcgcaata tgccttgcga ggtcgcaaaa tcggcgaaat tccatacctg 31140ttcaccgacg acggcgctga cgcgatcaaa gacgcggtga tacatatcca gccatgcaca 31200ctgatactct tcactccaca tgtcggtgta cattgagtgc agcccggcta acgtatccac 31260gccgtattcg gtgatgataa tcggctgatg cagtttctcc tgccaggcca gaagttcttt 31320ttccagtacc ttctctgccg tttccaaatc gccgctttgg acataccatc cgtaataacg 31380gttcaggcac agcacatcaa agagatcgct gatggtatcg gtgtgagcgt cgcagaacat 31440tacattgacg caggtgatcg gacgcgtcgg gtcgagttta cgcgttgctt ccgccagtgg 31500cgcgaaatat tcccgtgcac cttgcggacg ggtatccggt tcgttggcaa tactccacat 31560caccacgctt gggtggtttt tgtcacgcgc tatcagctct ttaatcgcct gtaagtgcgc 31620ttgctgagtt tccccgttga ctgcctcttc gctgtacagt tctttcggct tgttgcccgc 31680ttcgaaacca atgcctaaag agaggttaaa gccgacagca gcagtttcat caatcaccac 31740gatgccatgt tcatctgccc agtcgagcat ctcttcagcg taagggtaat gcgaggtacg 31800gtaggagttg gccccaatcc agtccattaa tgcgtggtcg tgcaccatca gcacgttatc 31860gaatcctttg ccacgcaagt ccgcatcttc atgacgacca aagccagtaa agtagaacgg 31920tttgtggtta atcaggaact gttcgccctt cactgccact gaccggatgc cgacgcgaag 31980cgggtagata tcacactctg tctggctttt ggctgtgacg cacagttcat agagataacc 32040ttcacccggt tgccagaggt gcggattcac cacttgcaaa gtcccgctag tgccttgtcc 32100agttgcaacc acctgttgat ccgcatcacg cagttcaacg ctgacatcac cattggccac 32160cacctgccag tcaacagacg cgtggttaca gtcttgcgcg acatgcgtca ccacggtgat 32220atcgtccacc caggtgttcg gcgtggtgta gagcattacg ctgcgatgga ttccggcata 32280gttaaagaaa tcatggaagt aagactgctt tttcttgccg ttttcgtcgg taatcaccat 32340tcccggcggg atagtctgcc agttcagttc gttgttcaca caaacggtga tacgtacact 32400tttcccggca ataacatacg gcgtgacatc ggcttcaaat ggcgtatagc cgccctgatg 32460ctccatcact tcctgattat tgacccacac tttgccgtaa tgagtgaccg catcgaaacg 32520cagcacgata cgctggcctg cccaaccttt cggtataaag acttcgcgct gataccagac 32580gttgcccgca taattacgaa tatctgcatc ggcgaactga tcgttaaaac tgcctggcac 32640agcaattgcc cggctttctt gtaacgcgct ttcccaccaa cgctgatcaa ttccacagtt 32700ttcgcgatcc agactgaatg cccacaggcc gtcgagtttt ttgatttcac gggttggggt 32760ttctacagga cggaccatgc gttcgacctt tctcttcttt tttgggccca tgatggcaga 32820tccgtatagt gagtcgtatt agctggttct ttccgcctca gaagccatag agcccaccgc 32880atccccagca tgcctgctat tgtcttccca atcctccccc ttgctgtcct gccccacccc 32940accccccaga atagaatgac acctactcag acaatgcgat gcaatttcct cattttatta 33000ggaaaggaca gtgggagtgg caccttccag ggtcaaggaa ggcacggggg aggggcaaac 33060aacagatggc tggcaactag aaggcacagt cgaggctgat cagcgagctc tagatgcatg 33120ctcgagcggc cgccagtgtg atggatatct gcagaattcc agcacactgg cggccgttac 33180tagtggatcc gagctcggta cccggccgtt ataacaccac tcgacacggc accagctcaa 33240tcagtcacag tgtaaaaaag ggccaagtgc agagcgagta tatataggac taaaaaatga 33300cgtaacggtt aaagtccaca aaaaacaccc agaaaaccgc acgcgaacct acgcccagaa 33360acgaaagcca aaaaacccac aacttcctca aatcgtcact tccgttttcc cacgttacgt 33420cacttcccat tttaagaaaa ctacaattcc caacacatac aagttactcc gccctaaaac 33480ctacgtcacc cgccccgttc ccacgccccg cgccacgtca caaactccac cccctcatta 33540tcatattggc ttcaatccaa aataaggtat attattgatg atg 335831833476DNAArtificial SequenceAdenoviral vector Adt.gp140dv12(B).11D 18catcatcaat aatatacctt attttggatt gaagccaata tgataatgag ggggtggagt 60ttgtgacgtg gcgcggggcg tgggaacggg gcgggtgacg tagtagtgtg gcggaagtgt 120gatgttgcaa gtgtggcgga acacatgtaa gcgacggatg tggcaaaagt gacgtttttg 180gtgtgcgccg gtgtacacag gaagtgacaa ttttcgcgcg gttttaggcg gatgttgtag 240taaatttggg cgtaaccgag taagatttgg ccattttcgc gggaaaactg aataagagga 300agtgaaatct gaataatttt gtgttactca tagcgcgtaa tatttgtcta gggcccggga 360tcggtgatca ccgatccaga catgataaga tacattgatg agtttggaca aaccacaact 420agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta 480accattataa gctgcaataa acaagttccc ggatctttct agctagtcta gactagctag

540actcgagagc ggccgcaatc gataagcttg atatcgaatt ctgcagtgat cagggatcct 600caccacagcc agttggtgat gttgaaccag ttccacaggc tggcccactt gtccagctcc 660agcagctcct gctcgttctt ctcgtgctgg ttctggctct cctcgatcag gctgtggatc 720aggctggtgt agttgttgat ctcgcggtcc cactccatcc aggtggtgtg gttccagatc 780tgctcgagca gctgctggtc cttcaggtag cgctccacgg ccagggtgcg ggcctgcagc 840tgcttgatgc cccacacggt cagctgcagc aggtgctgct gggcctcgat ggcgcgcagc 900aggttgttct gctgctgcac gatgccgctc agcagctggc gggcctggac ggtaagcttg 960gccttggtgg gggccacgcc caggggctcg atcttcacca ccttgtactt gtacagctcg 1020ctgcgccagt tgtcgcgcat gtcgccgccg cccaggcgga agatctcgct ctcgttgttg 1080ctgttgccgc cgtcgcgggt cagcagcagg ccggtgatgt tgctgctgca gcggatctgg 1140ccgctgatgg ggggggcgta catggccttg cccaccttct gccacatgtt gatgatctgc 1200ttgatgcggc agggcagggt gatggtgtcg ctgccctcgg tgttgttgct gccctcggtg 1260ctccaggtgc tgttgaacca ggtgctgttg aacagctggg tgctgttgca gtagaagaac 1320tcgccgccgc agttgaagct gtgggtcacg atctcggggt cgccgccgct gctgtgcttg 1380aacacgatgg tcttgttgcc gaactgctcg cgcagcttga tcacgatctt gttcagggtg 1440tcgttccact tggcgcggct caggttgcag tgggcctggc ggatgtcgcc gatgatctcg 1500ccggtggtgt agaaggcgcg gccggggccg atgtggatgc tcttgcgggt gttgttgttg 1560gggcgggtgc agttgatctc cacgctctcg ttcagctgca cgatgatcac cttggcgttg 1620tcggcgaagt tagcgctgcg gatcaccacc tcctcctcgg ccaggttacc cgtaaccagc 1680agctgggtgc tcaccacggg gcggatgccg tgggtgcact gcacggtgct cacgttggtg 1740caggggccct tgccgttgaa cttcttgtcc ttgcacttca ggatggcgaa gccggcgggg 1800gcgcagtagt ggttggggat gggctcgaag ctcaccttgg ggcaggcctg ggtgatcacg 1860ctggtgttgc agctggtgct agcgtcggtg cacttcaggc tcacgcacag gggggtcagc 1920ttcacgcagg gcttcaggct ctggtcccac aggctgatga tgtcctcgtg catctgctcc 1980accatgtcgt tcttccacat gtcgaagttc tcggtcacgt tcaccagcac cacctcctgg 2040gggttggggt cggtgggcac gcaggcgtgg gtggcccaca cgttgtgcac ctcggtgtcg 2100taggccttgg cgtcgctggc gcagagcagg gtggtggtgg cctccttcca cacgggcacg 2160ccgtagtaca cggtcaccca cagcttctcg gtggcgctgc agatcatcag catgcccagc 2220agcatggtgc cccagcgcca gccccagcgc cacaggtgct ggtacttctc cttcacgcgc 2280atggtgtcta gagcggccgc gatcggctgc agttggacct gggagtggac acctgtggag 2340agaaaggcaa agtggatgtc attgtcactc aagtgtatgg ccagatctca agcctgccac 2400acctcaagtg aagccaaggg ggtgggccta tagactctat aggcggtact tacgtcactc 2460ttggcacggg gaatccgcgt tccaatgcac cgttcccggc cgcggaggct ggatcggtcc 2520cggtgtcttc tatggaggtc aaaacagcgt ggatggcgtc tccaggcgat ctgacggttc 2580actaaacgag ctcgtcgacg atctctatca ctgataggga gatctctatc actgataggg 2640agagctctgc ttatatagac ctcccaccgt acacgcctac cgcccatttg cgtcaatggg 2700gcggagttgt tacgacattt tggaaagtcc cgttgatttt ggtgccaaaa caaactccca 2760ttgacgtcaa tggggtggag acttggaaat ccccgtgagt caaaccgcta tccacgccca 2820ttgatgtact gccaaaaccg catcaccatg gtaatagcga tgactaatac gtagatgtac 2880tgccaagtag gaaagtccca taaggtcatg tactgggcat aatgccaggc gggccattta 2940ccgtcattga cgtcaatagg gggcgtactt ggcatatgat acacttgatg tactgccaag 3000tgggcagttt accgtaaata ctccacccat tgacgtcaat ggaaagtccc tattggcgtt 3060actatgggaa catacgtcat tattgacgtc aatgggcggg ggtcgttggg cggtcagcca 3120ggcgggccat ttaccgtaag ttatgtaacg cggaactcca tatatgggct atgaactaat 3180gaccccgtaa ttgattacta ttaataacta gtactgaaat gtgtgggcgt ggcttaaggg 3240tgggaaagaa tatataaggt gggggtctta tgtagttttg tatctgtttt gcagcagccg 3300ccgccgccat gagcaccaac tcgtttgatg gaagcattgt gagctcatat ttgacaacgc 3360gcatgccccc atgggccggg gtgcgtcaga atgtgatggg ctccagcatt gatggtcgcc 3420ccgtcctgcc cgcaaactct actaccttga cctacgagac cgtgtctgga acgccgttgg 3480agactgcagc ctccgccgcc gcttcagccg ctgcagccac cgcccgcggg attgtgactg 3540actttgcttt cctgagcccg cttgcaagca gtgcagcttc ccgttcatcc gcccgcgatg 3600acaagttgac ggctcttttg gcacaattgg attctttgac ccgggaactt aatgtcgttt 3660ctcagcagct gttggatctg cgccagcagg tttctgccct gaaggcttcc tcccctccca 3720atgcggttta aaacataaat aaaaaaccag actctgtttg gatttggatc aagcaagtgt 3780cttgctgtct ttatttaggg gttttgcgcg cgcggtaggc ccgggaccag cggtctcggt 3840cgttgagggt cctgtgtatt ttttccagga cgtggtaaag gtgactctgg atgttcagat 3900acatgggcat aagcccgtct ctggggtgga ggtagcacca ctgcagagct tcatgctgcg 3960gggtggtgtt gtagatgatc cagtcgtagc aggagcgctg ggcgtggtgc ctaaaaatgt 4020ctttcagtag caagctgatt gccaggggca ggcccttggt gtaagtgttt acaaagcggt 4080taagctggga tgggtgcata cgtggggata tgagatgcat cttggactgt atttttaggt 4140tggctatgtt cccagccata tccctccggg gattcatgtt gtgcagaacc accagcacag 4200tgtatccggt gcacttggga aatttgtcat gtagcttaga aggaaatgcg tggaagaact 4260tggagacgcc cttgtgacct ccaagatttt ccatgcattc gtccataatg atggcaatgg 4320gcccacgggc ggcggcctgg gcgaagatat ttctgggatc actaacgtca tagttgtgtt 4380ccaggatgag atcgtcatag gccattttta caaagcgcgg gcggagggtg ccagactgcg 4440gtataatggt tccatccggc ccaggggcgt agttaccctc acagatttgc atttcccacg 4500ctttgagttc agatgggggg atcatgtcta cctgcggggc gatgaagaaa acggtttccg 4560gggtagggga gatcagctgg gaagaaagca ggttcctgag cagctgcgac ttaccgcagc 4620cggtgggccc gtaaatcaca cctattaccg gctgcaactg gtagttaaga gagctgcagc 4680tgccgtcatc cctgagcagg ggggccactt cgttaagcat gtccctgact cgcatgtttt 4740ccctgaccaa atccgccaga aggcgctcgc cgcccagcga tagcagttct tgcaaggaag 4800caaagttttt caacggtttg agaccgtccg ccgtaggcat gcttttgagc gtttgaccaa 4860gcagttccag gcggtcccac agctcggtca cctgctctac ggcatctcga tccagcatat 4920ctcctcgttt cgcgggttgg ggcggctttc gctgtacggc agtagtcggt gctcgtccag 4980acgggccagg gtcatgtctt tccacgggcg cagggtcctc gtcagcgtag tctgggtcac 5040ggtgaagggg tgcgctccgg gctgcgcgct ggccagggtg cgcttgaggc tggtcctgct 5100ggtgctgaag cgctgccggt cttcgccctg cgcgtcggcc aggtagcatt tgaccatggt 5160gtcatagtcc agcccctccg cggcgtggcc cttggcgcgc agcttgccct tggaggaggc 5220gccgcacgag gggcagtgca gacttttgag ggcgtagagc ttgggcgcga gaaataccga 5280ttccggggag taggcatccg cgccgcaggc cccgcagacg gtctcgcatt ccacgagcca 5340ggtgagctct ggccgttcgg ggtcaaaaac caggtttccc ccatgctttt tgatgcgttt 5400cttacctctg gtttccatga gccggtgtcc acgctcggtg acgaaaaggc tgtccgtgtc 5460cccgtataca gacttgagag gcctgtcctc gagcggtgtt ccgcggtcct cctcgtatag 5520aaactcggac cactctgaga caaaggctcg cgtccaggcc agcacgaagg aggctaagtg 5580ggaggggtag cggtcgttgt ccactagggg gtccactcgc tccagggtgt gaagacacat 5640gtcgccctct tcggcatcaa ggaaggtgat tggtttgtag gtgtaggcca cgtgaccggg 5700tgttcctgaa ggggggctat aaaagggggt gggggcgcgt tcgtcctcac tctcttccgc 5760atcgctgtct gcgagggcca gctgttgggg tgagtactcc ctctgaaaag cgggcatgac 5820ttctgcgcta agattgtcag tttccaaaaa cgaggaggat ttgatattca cctggcccgc 5880ggtgatgcct ttgagggtgg ccgcatccat ctggtcagaa aagacaatct ttttgttgtc 5940aagcttggtg gcaaacgacc cgtagagggc gttggacagc aacttggcga tggagcgcag 6000ggtttggttt ttgtcgcgat cggcgcgctc cttggccgcg atgtttagct gcacgtattc 6060gcgcgcaacg caccgccatt cgggaaagac ggtggtgcgc tcgtcgggca ccaggtgcac 6120gcgccaaccg cggttgtgca gggtgacaag gtcaacgctg gtggctacct ctccgcgtag 6180gcgctcgttg gtccagcaga ggcggccgcc cttgcgcgag cagaatggcg gtagggggtc 6240tagctgcgtc tcgtccgggg ggtctgcgtc cacggtaaag accccgggca gcaggcgcgc 6300gtcgaagtag tctatcttgc atccttgcaa gtctagcgcc tgctgccatg cgcgggcggc 6360aagcgcgcgc tcgtatgggt tgagtggggg accccatggc atggggtggg tgagcgcgga 6420ggcgtacatg ccgcaaatgt cgtaaacgta gaggggctct ctgagtattc caagatatgt 6480agggtagcat cttccaccgc ggatgctggc gcgcacgtaa tcgtatagtt cgtgcgaggg 6540agcgaggagg tcgggaccga ggttgctacg ggcgggctgc tctgctcgga agactatctg 6600cctgaagatg gcatgtgagt tggatgatat ggttggacgc tggaagacgt tgaagctggc 6660gtctgtgaga cctaccgcgt cacgcacgaa ggaggcgtag gagtcgcgca gcttgttgac 6720cagctcggcg gtgacctgca cgtctagggc gcagtagtcc agggtttcct tgatgatgtc 6780atacttatcc tgtccctttt ttttccacag ctcgcggttg aggacaaact cttcgcggtc 6840tttccagtac tcttggatcg gaaacccgtc ggcctccgaa cggtaagagc ctagcatgta 6900gaactggttg acggcctggt aggcgcagca tcccttttct acgggtagcg cgtatgcctg 6960cgcggccttc cggagcgagg tgtgggtgag cgcaaaggtg tccctgacca tgactttgag 7020gtactggtat ttgaagtcag tgtcgtcgca tccgccctgc tcccagagca aaaagtccgt 7080gcgctttttg gaacgcggat ttggcagggc gaaggtgaca tcgttgaaga gtatctttcc 7140cgcgcgaggc ataaagttgc gtgtgatgcg gaagggtccc ggcacctcgg aacggttgtt 7200aattacctgg gcggcgagca cgatctcgtc aaagccgttg atgttgtggc ccacaatgta 7260aagttccaag aagcgcggga tgcccttgat ggaaggcaat tttttaagtt cctcgtaggt 7320gagctcttca ggggagctga gcccgtgctc tgaaagggcc cagtctgcaa gatgagggtt 7380ggaagcgacg aatgagctcc acaggtcacg ggccattagc atttgcaggt ggtcgcgaaa 7440ggtcctaaac tggcgaccta tggccatttt ttctggggtg atgcagtaga aggtaagcgg 7500gtcttgttcc cagcggtccc atccaaggtt cgcggctagg tctcgcgcgg cagtcactag 7560aggctcatct ccgccgaact tcatgaccag catgaagggc acgagctgct tcccaaaggc 7620ccccatccaa gtataggtct ctacatcgta ggtgacaaag agacgctcgg tgcgaggatg 7680cgagccgatc gggaagaact ggatctcccg ccaccaattg gaggagtggc tattgatgtg 7740gtgaaagtag aagtccctgc gacgggccga acactcgtgc tggcttttgt aaaaacgtgc 7800gcagtactgg cagcggtgca cgggctgtac atcctgcacg aggttgacct gacgaccgcg 7860cacaaggaag cagagtggga atttgagccc ctcgcctggc gggtttggct ggtggtcttc 7920tacttcggct gcttgtcctt gaccgtctgg ctgctcgagg ggagttacgg tggatcggac 7980caccacgccg cgcgagccca aagtccagat gtccgcgcgc ggcggtcgga gcttgatgac 8040aacatcgcgc agatgggagc tgtccatggt ctggagctcc cgcggcgtca ggtcaggcgg 8100gagctcctgc aggtttacct cgcatagacg ggtcagggcg cgggctagat ccaggtgata 8160cctaatttcc aggggctggt tggtggcggc gtcgatggct tgcaagaggc cgcatccccg 8220cggcgcgact acggtaccgc gcggcgggcg gtgggccgcg ggggtgtcct tggatgatgc 8280atctaaaagc ggtgacgcgg gcgagccccc ggaggtaggg ggggctccgg acccgccggg 8340agagggggca ggggcacgtc ggcgccgcgc gcgggcagga gctggtgctg cgcgcgtagg 8400ttgctggcga acgcgacgac gcggcggttg atctcctgaa tctggcgcct ctgcgtgaag 8460acgacgggcc cggtgagctt gaacctgaaa gagagttcga cagaatcaat ttcggtgtcg 8520ttgacggcgg cctggcgcaa aatctcctgc acgtctcctg agttgtcttg ataggcgatc 8580tcggccatga actgctcgat ctcttcctcc tggagatctc cgcgtccggc tcgctccacg 8640gtggcggcga ggtcgttgga aatgcgggcc atgagctgcg agaaggcgtt gaggcctccc 8700tcgttccaga cgcggctgta gaccacgccc ccttcggcat cgcgggcgcg catgaccacc 8760tgcgcgagat tgagctccac gtgccgggcg aagacggcgt agtttcgcag gcgctgaaag 8820aggtagttga gggtggtggc ggtgtgttct gccacgaaga agtacataac ccagcgtcgc 8880aacgtggatt cgttgatatc ccccaaggcc tcaaggcgct ccatggcctc gtagaagtcc 8940acggcgaagt tgaaaaactg ggagttgcgc gccgacacgg ttaactcctc ctccagaaga 9000cggatgagct cggcgacagt gtcgcgcacc tcgcgctcaa aggctacagg ggcctcttct 9060tcttcttcaa tctcctcttc cataagggcc tccccttctt cttcttctgg cggcggtggg 9120ggagggggga cacggcggcg acgacggcgc accgggaggc ggtcgacaaa gcgctcgatc 9180atctccccgc ggcgacggcg catggtctcg gtgacggcgc ggccgttctc gcgggggcgc 9240agttggaaga cgccgcccgt catgtcccgg ttatgggttg gcggggggct gccatgcggc 9300agggatacgg cgctaacgat gcatctcaac aattgttgtg taggtactcc gccgccgagg 9360gacctgagcg agtccgcatc gaccggatcg gaaaacctct cgagaaaggc gtctaaccag 9420tcacagtcgc aaggtaggct gagcaccgtg gcgggcggca gcgggcggcg gtcggggttg 9480tttctggcgg aggtgctgct gatgatgtaa ttaaagtagg cggtcttgag acggcggatg 9540gtcgacagaa gcaccatgtc cttgggtccg gcctgctgaa tgcgcaggcg gtcggccatg 9600ccccaggctt cgttttgaca tcggcgcagg tctttgtagt agtcttgcat gagcctttct 9660accggcactt cttcttctcc ttcctcttgt cctgcatctc ttgcatctat cgctgcggcg 9720gcggcggagt ttggccgtag gtggcgccct cttcctccca tgcgtgtgac cccgaagccc 9780ctcatcggct gaagcagggc taggtcggcg acaacgcgct cggctaatat ggcctgctgc 9840acctgcgtga gggtagactg gaagtcatcc atgtccacaa agcggtggta tgcgcccgtg 9900ttgatggtgt aagtgcagtt ggccataacg gaccagttaa cggtctggtg acccggctgc 9960gagagctcgg tgtacctgag acgcgagtaa gccctcgagt caaatacgta gtcgttgcaa 10020gtccgcacca ggtactggta tcccaccaaa aagtgcggcg gcggctggcg gtagaggggc 10080cagcgtaggg tggccggggc tccgggggcg agatcttcca acataaggcg atgatatccg 10140tagatgtacc tggacatcca ggtgatgccg gcggcggtgg tggaggcgcg cggaaagtcg 10200cggacgcggt tccagatgtt gcgcagcggc aaaaagtgct ccatggtcgg gacgctctgg 10260ccggtcaggc gcgcgcaatc gttgacgctc tagcgtgcaa aaggagagcc tgtaagcggg 10320cactcttccg tggtctggtg gataaattcg caagggtatc atggcggacg accggggttc 10380gagccccgta tccggccgtc cgccgtgatc catgcggtta ccgcccgcgt gtcgaaccca 10440ggtgtgcgac gtcagacaac gggggagtgc tccttttggc ttccttccag gcgcggcggc 10500tgctgcgcta gcttttttgg ccactggccg cgcgcagcgt aagcggttag gctggaaagc 10560gaaagcatta agtggctcgc tccctgtagc cggagggtta ttttccaagg gttgagtcgc 10620gggacccccg gttcgagtct cggaccggcc ggactgcggc gaacgggggt ttgcctcccc 10680gtcatgcaag accccgcttg caaattcctc cggaaacagg gacgagcccc ttttttgctt 10740ttcccagatg catccggtgc tgcggcagat gcgcccccct cctcagcagc ggcaagagca 10800agagcagcgg cagacatgca gggcaccctc ccctcctcct accgcgtcag gaggggcgac 10860atccgcggtt gacgcggcag cagatggtga ttacgaaccc ccgcggcgcc gggcccggca 10920ctacctggac ttggaggagg gcgagggcct ggcgcggcta ggagcgccct ctcctgagcg 10980gcacccaagg gtgcagctga agcgtgatac gcgtgaggcg tacgtgccgc ggcagaacct 11040gtttcgcgac cgcgagggag aggagcccga ggagatgcgg gatcgaaagt tccacgcagg 11100gcgcgagctg cggcatggcc tgaatcgcga gcggttgctg cgcgaggagg actttgagcc 11160cgacgcgcga accgggatta gtcccgcgcg cgcacacgtg gcggccgccg acctggtaac 11220cgcatacgag cagacggtga accaggagat taactttcaa aaaagcttta acaaccacgt 11280gcgtacgctt gtggcgcgcg aggaggtggc tataggactg atgcatctgt gggactttgt 11340aagcgcgctg gagcaaaacc caaatagcaa gccgctcatg gcgcagctgt tccttatagt 11400gcagcacagc agggacaacg aggcattcag ggatgcgctg ctaaacatag tagagcccga 11460gggccgctgg ctgctcgatt tgataaacat cctgcagagc atagtggtgc aggagcgcag 11520cttgagcctg gctgacaagg tggccgccat caactattcc atgcttagcc tgggcaagtt 11580ttacgcccgc aagatatacc atacccctta cgttcccata gacaaggagg taaagatcga 11640ggggttctac atgcgcatgg cgctgaaggt gcttaccttg agcgacgacc tgggcgttta 11700tcgcaacgag cgcatccaca aggccgtgag cgtgagccgg cggcgcgagc tcagcgaccg 11760cgagctgatg cacagcctgc aaagggccct ggctggcacg ggcagcggcg atagagaggc 11820cgagtcctac tttgacgcgg gcgctgacct gcgctgggcc ccaagccgac gcgccctgga 11880ggcagctggg gccggacctg ggctggcggt ggcacccgcg cgcgctggca acgtcggcgg 11940cgtggaggaa tatgacgagg acgatgagta cgagccagag gacggcgagt actaagcggt 12000gatgtttctg atcagatgat gcaagacgca acggacccgg cggtgcgggc ggcgctgcag 12060agccagccgt ccggccttaa ctccacggac gactggcgcc aggtcatgga ccgcatcatg 12120tcgctgactg cgcgcaatcc tgacgcgttc cggcagcagc cgcaggccaa ccggctctcc 12180gcaattctgg aagcggtggt cccggcgcgc gcaaacccca cgcacgagaa ggtgctggcg 12240atcgtaaacg cgctggccga aaacagggcc atccggcccg acgaggccgg cctggtctac 12300gacgcgctgc ttcagcgcgt ggctcgttac aacagcggca acgtgcagac caacctggac 12360cggctggtgg gggatgtgcg cgaggccgtg gcgcagcgtg agcgcgcgca gcagcagggc 12420aacctgggct ccatggttgc actaaacgcc ttcctgagta cacagcccgc caacgtgccg 12480cggggacagg aggactacac caactttgtg agcgcactgc ggctaatggt gactgagaca 12540ccgcaaagtg aggtgtacca gtctgggcca gactattttt tccagaccag tagacaaggc 12600ctgcagaccg taaacctgag ccaggctttc aaaaacttgc aggggctgtg gggggtgcgg 12660gctcccacag gcgaccgcgc gaccgtgtct agcttgctga cgcccaactc gcgcctgttg 12720ctgctgctaa tagcgccctt cacggacagt ggcagcgtgt cccgggacac atacctaggt 12780cacttgctga cactgtaccg cgaggccata ggtcaggcgc atgtggacga gcatactttc 12840caggagatta caagtgtcag ccgcgcgctg gggcaggagg acacgggcag cctggaggca 12900accctaaact acctgctgac caaccggcgg cagaagatcc cctcgttgca cagtttaaac 12960agcgaggagg agcgcatttt gcgctacgtg cagcagagcg tgagccttaa cctgatgcgc 13020gacggggtaa cgcccagcgt ggcgctggac atgaccgcgc gcaacatgga accgggcatg 13080tatgcctcaa accggccgtt tatcaaccgc ctaatggact acttgcatcg cgcggccgcc 13140gtgaaccccg agtatttcac caatgccatc ttgaacccgc actggctacc gccccctggt 13200ttctacaccg ggggattcga ggtgcccgag ggtaacgatg gattcctctg ggacgacata 13260gacgacagcg tgttttcccc gcaaccgcag accctgctag agttgcaaca gcgcgagcag 13320gcagaggcgg cgctgcgaaa ggaaagcttc cgcaggccaa gcagcttgtc cgatctaggc 13380gctgcggccc cgcggtcaga tgctagtagc ccatttccaa gcttgatagg gtctcttacc 13440agcactcgca ccacccgccc gcgcctgctg ggcgaggagg agtacctaaa caactcgctg 13500ctgcagccgc agcgcgaaaa aaacctgcct ccggcatttc ccaacaacgg gatagagagc 13560ctagtggaca agatgagtag atggaagacg tacgcgcagg agcacaggga cgtgccaggc 13620ccgcgcccgc ccacccgtcg tcaaaggcac gaccgtcagc ggggtctggt gtgggaggac 13680gatgactcgg cagacgacag cagcgtcctg gatttgggag ggagtggcaa cccgtttgcg 13740caccttcgcc ccaggctggg gagaatgttt taaaaaaaaa aaaagcatga tgcaaaataa 13800aaaactcacc aaggccatgg caccgagcgt tggttttctt gtattcccct tagtatgcgg 13860cgcgcggcga tgtatgagga aggtcctcct ccctcctacg agagtgtggt gagcgcggcg 13920ccagtggcgg cggcgctggg ttctcccttc gatgctcccc tggacccgcc gtttgtgcct 13980ccgcggtacc tgcggcctac cggggggaga aacagcatcc gttactctga gttggcaccc 14040ctattcgaca ccacccgtgt gtacctggtg gacaacaagt caacggatgt ggcatccctg 14100aactaccaga acgaccacag caactttctg accacggtca ttcaaaacaa tgactacagc 14160ccgggggagg caagcacaca gaccatcaat cttgacgacc ggtcgcactg gggcggcgac 14220ctgaaaacca tcctgcatac caacatgcca aatgtgaacg agttcatgtt taccaataag 14280tttaaggcgc gggtgatggt gtcgcgcttg cctactaagg acaatcaggt ggagctgaaa 14340tacgagtggg tggagttcac gctgcccgag ggcaactact ccgagaccat gaccatagac 14400cttatgaaca acgcgatcgt ggagcactac ttgaaagtgg gcagacagaa cggggttctg 14460gaaagcgaca tcggggtaaa gtttgacacc cgcaacttca gactggggtt tgaccccgtc 14520actggtcttg tcatgcctgg ggtatataca aacgaagcct tccatccaga catcattttg 14580ctgccaggat gcggggtgga cttcacccac agccgcctga gcaacttgtt gggcatccgc 14640aagcggcaac ccttccagga gggctttagg atcacctacg atgatctgga gggtggtaac 14700attcccgcac tgttggatgt ggacgcctac caggcgagct tgaaagatga caccgaacag 14760ggcgggggtg gcgcaggcgg cagcaacagc agtggcagcg gcgcggaaga gaactccaac 14820gcggcagccg cggcaatgca gccggtggag gacatgaacg atcatgccat tcgcggcgac 14880acctttgcca cacgggctga ggagaagcgc gctgaggccg aagcagcggc cgaagctgcc 14940gcccccgctg cgcaacccga ggtcgagaag cctcagaaga aaccggtgat caaacccctg 15000acagaggaca gcaagaaacg cagttacaac ctaataagca atgacagcac cttcacccag 15060taccgcagct ggtaccttgc atacaactac ggcgaccctc agaccggaat ccgctcatgg 15120accctgcttt gcactcctga cgtaacctgc ggctcggagc aggtctactg gtcgttgcca 15180gacatgatgc aagaccccgt gaccttccgc tccacgcgcc agatcagcaa ctttccggtg 15240gtgggcgccg agctgttgcc cgtgcactcc aagagcttct acaacgacca ggccgtctac 15300tcccaactca tccgccagtt tacctctctg acccacgtgt tcaatcgctt tcccgagaac 15360cagattttgg cgcgcccgcc agcccccacc atcaccaccg tcagtgaaaa cgttcctgct 15420ctcacagatc acgggacgct accgctgcgc aacagcatcg gaggagtcca gcgagtgacc 15480attactgacg ccagacgccg cacctgcccc tacgtttaca aggccctggg catagtctcg 15540ccgcgcgtcc tatcgagccg cactttttga gcaagcatgt ccatccttat atcgcccagc

15600aataacacag gctggggcct gcgcttccca agcaagatgt ttggcggggc caagaagcgc 15660tccgaccaac acccagtgcg cgtgcgcggg cactaccgcg cgccctgggg cgcgcacaaa 15720cgcggccgca ctgggcgcac caccgtcgat gacgccatcg acgcggtggt ggaggaggcg 15780cgcaactaca cgcccacgcc gccaccagtg tccacagtgg acgcggccat tcagaccgtg 15840gtgcgcggag cccggcgcta tgctaaaatg aagagacggc ggaggcgcgt agcacgtcgc 15900caccgccgcc gacccggcac tgccgcccaa cgcgcggcgg cggccctgct taaccgcgca 15960cgtcgcaccg gccgacgggc ggccatgcgg gccgctcgaa ggctggccgc gggtattgtc 16020actgtgcccc ccaggtccag gcgacgagcg gccgccgcag cagccgcggc cattagtgct 16080atgactcagg gtcgcagggg caacgtgtat tgggtgcgcg actcggttag cggcctgcgc 16140gtgcccgtgc gcacccgccc cccgcgcaac tagattgcaa gaaaaaacta cttagactcg 16200tactgttgta tgtatccagc ggcggcggcg cgcaacgaag ctatgtccaa gcgcaaaatc 16260aaagaagaga tgctccaggt catcgcgccg gagatctatg gccccccgaa gaaggaagag 16320caggattaca agccccgaaa gctaaagcgg gtcaaaaaga aaaagaaaga tgatgatgat 16380gaacttgacg acgaggtgga actgctgcac gctaccgcgc ccaggcgacg ggtacagtgg 16440aaaggtcgac gcgtaaaacg tgttttgcga cccggcacca ccgtagtctt tacgcccggt 16500gagcgctcca cccgcaccta caagcgcgtg tatgatgagg tgtacggcga cgaggacctg 16560cttgagcagg ccaacgagcg cctcggggag tttgcctacg gaaagcggca taaggacatg 16620ctggcgttgc cgctggacga gggcaaccca acacctagcc taaagcccgt aacactgcag 16680caggtgctgc ccgcgcttgc accgtccgaa gaaaagcgcg gcctaaagcg cgagtctggt 16740gacttggcac ccaccgtgca gctgatggta cccaagcgcc agcgactgga agatgtcttg 16800gaaaaaatga ccgtggaacc tgggctggag cccgaggtcc gcgtgcggcc aatcaagcag 16860gtggcgccgg gactgggcgt gcagaccgtg gacgttcaga tacccactac cagtagcacc 16920agtattgcca ccgccacaga gggcatggag acacaaacgt ccccggttgc ctcagcggtg 16980gcggatgccg cggtgcaggc ggtcgctgcg gccgcgtcca agacctctac ggaggtgcaa 17040acggacccgt ggatgtttcg cgtttcagcc ccccggcgcc cgcgccgttc gaggaagtac 17100ggcgccgcca gcgcgctact gcccgaatat gccctacatc cttccattgc gcctaccccc 17160ggctatcgtg gctacaccta ccgccccaga agacgagcaa ctacccgacg ccgaaccacc 17220actggaaccc gccgccgccg tcgccgtcgc cagcccgtgc tggccccgat ttccgtgcgc 17280agggtggctc gcgaaggagg caggaccctg gtgctgccaa cagcgcgcta ccaccccagc 17340atcgtttaaa agccggtctt tgtggttctt gcagatatgg ccctcacctg ccgcctccgt 17400ttcccggtgc cgggattccg aggaagaatg caccgtagga ggggcatggc cggccacggc 17460ctgacgggcg gcatgcgtcg tgcgcaccac cggcggcggc gcgcgtcgca ccgtcgcatg 17520cgcggcggta tcctgcccct ccttattcca ctgatcgccg cggcgattgg cgccgtgccc 17580ggaattgcat ccgtggcctt gcaggcgcag agacactgat taaaaacaag ttgcatgtgg 17640aaaaatcaaa ataaaaagtc tggactctca cgctcgcttg gtcctgtaac tattttgtag 17700aatggaagac atcaactttg cgtctctggc cccgcgacac ggctcgcgcc cgttcatggg 17760aaactggcaa gatatcggca ccagcaatat gagcggtggc gccttcagct ggggctcgct 17820gtggagcggc attaaaaatt tcggttccac cgttaagaac tatggcagca aggcctggaa 17880cagcagcaca ggccagatgc tgagggataa gttgaaagag caaaatttcc aacaaaaggt 17940ggtagatggc ctggcctctg gcattagcgg ggtggtggac ctggccaacc aggcagtgca 18000aaataagatt aacagtaagc ttgatccccg ccctcccgta gaggagcctc caccggccgt 18060ggagacagtg tctccagagg ggcgtggcga aaagcgtccg cgccccgaca gggaagaaac 18120tctggtgacg caaatagacg agcctccctc gtacgaggag gcactaaagc aaggcctgcc 18180caccacccgt cccatcgcgc ccatggctac cggagtgctg ggccagcaca cacccgtaac 18240gctggacctg cctccccccg ccgacaccca gcagaaacct gtgctgccag gcccgaccgc 18300cgttgttgta acccgtccta gccgcgcgtc cctgcgccgc gccgccagcg gtccgcgatc 18360gttgcggccc gtagccagtg gcaactggca aagcacactg aacagcatcg tgggtctggg 18420ggtgcaatcc ctgaagcgcc gacgatgctt ctgatagcta acgtgtcgta tgtgtgtcat 18480gtatgcgtcc atgtcgccgc cagaggagct gctgagccgc cgcgcgcccg ctttccaaga 18540tggctacccc ttcgatgatg ccgcagtggt cttacatgca catctcgggc caggacgcct 18600cggagtacct gagccccggg ctggtgcagt ttgcccgcgc caccgagacg tacttcagcc 18660tgaataacaa gtttagaaac cccacggtgg cgcctacgca cgacgtgacc acagaccggt 18720cccagcgttt gacgctgcgg ttcatccctg tggaccgtga ggatactgcg tactcgtaca 18780aggcgcggtt caccctagct gtgggtgata accgtgtgct ggacatggct tccacgtact 18840ttgacatccg cggcgtgctg gacaggggcc ctacttttaa gccctactct ggcactgcct 18900acaacgccct ggctcccaag ggtgccccaa atccttgcga atgggatgaa gctgctactg 18960ctcttgaaat aaacctagaa gaagaggacg atgacaacga agacgaagta gacgagcaag 19020ctgagcagca aaaaactcac gtatttgggc aggcgcctta ttctggtata aatattacaa 19080aggagggtat tcaaataggt gtcgaaggtc aaacacctaa atatgccgat aaaacatttc 19140aacctgaacc tcaaatagga gaatctcagt ggtacgaaac agaaattaat catgcagctg 19200ggagagtcct aaaaaagact accccaatga aaccatgtta cggttcatat gcaaaaccca 19260caaatgaaaa tggagggcaa ggcattcttg taaagcaaca aaatggaaag ctagaaagtc 19320aagtggaaat gcaatttttc tcaactactg aggcagccgc aggcaatggt gataacttga 19380ctcctaaagt ggtattgtac agtgaagatg tagatataga aaccccagac actcatattt 19440cttacatgcc cactattaag gaaggtaact cacgagaact aatgggccaa caatctatgc 19500ccaacaggcc taattacatt gcttttaggg acaattttat tggtctaatg tattacaaca 19560gcacgggtaa tatgggtgtt ctggcgggcc aagcatcgca gttgaatgct gttgtagatt 19620tgcaagacag aaacacagag ctttcatacc agcttttgct tgattccatt ggtgatagaa 19680ccaggtactt ttctatgtgg aatcaggctg ttgacagcta tgatccagat gttagaatta 19740ttgaaaatca tggaactgaa gatgaacttc caaattactg ctttccactg ggaggtgtga 19800ttaatacaga gactcttacc aaggtaaaac ctaaaacagg tcaggaaaat ggatgggaaa 19860aagatgctac agaattttca gataaaaatg aaataagagt tggaaataat tttgccatgg 19920aaatcaatct aaatgccaac ctgtggagaa atttcctgta ctccaacata gcgctgtatt 19980tgcccgacaa gctaaagtac agtccttcca acgtaaaaat ttctgataac ccaaacacct 20040acgactacat gaacaagcga gtggtggctc ccgggctagt ggactgctac attaaccttg 20100gagcacgctg gtcccttgac tatatggaca acgtcaaccc atttaaccac caccgcaatg 20160ctggcctgcg ctaccgctca atgttgctgg gcaatggtcg ctatgtgccc ttccacatcc 20220aggtgcctca gaagttcttt gccattaaaa acctccttct cctgccgggc tcatacacct 20280acgagtggaa cttcaggaag gatgttaaca tggttctgca gagctcccta ggaaatgacc 20340taagggttga cggagccagc attaagtttg atagcatttg cctttacgcc accttcttcc 20400ccatggccca caacaccgcc tccacgcttg aggccatgct tagaaacgac accaacgacc 20460agtcctttaa cgactatctc tccgccgcca acatgctcta ccctataccc gccaacgcta 20520ccaacgtgcc catatccatc ccctcccgca actgggcggc tttccgcggc tgggccttca 20580cgcgccttaa gactaaggaa accccatcac tgggctcggg ctacgaccct tattacacct 20640actctggctc tataccctac ctagatggaa ccttttacct caaccacacc tttaagaagg 20700tggccattac ctttgactct tctgtcagct ggcctggcaa tgaccgcctg cttaccccca 20760acgagtttga aattaagcgc tcagttgacg gggagggtta caacgttgcc cagtgtaaca 20820tgaccaaaga ctggttcctg gtacaaatgc tagctaacta taacattggc taccagggct 20880tctatatccc agagagctac aaggaccgca tgtactcctt ctttagaaac ttccagccca 20940tgagccgtca ggtggtggat gatactaaat acaaggacta ccaacaggtg ggcatcctac 21000accaacacaa caactctgga tttgttggct accttgcccc caccatgcgc gaaggacagg 21060cctaccctgc taacttcccc tatccgctta taggcaagac cgcagttgac agcattaccc 21120agaaaaagtt tctttgcgat cgcacccttt ggcgcatccc attctccagt aactttatgt 21180ccatgggcgc actcacagac ctgggccaaa accttctcta cgccaactcc gcccacgcgc 21240tagacatgac ttttgaggtg gatcccatgg acgagcccac ccttctttat gttttgtttg 21300aagtctttga cgtggtccgt gtgcaccagc cgcaccgcgg cgtcatcgaa accgtgtacc 21360tgcgcacgcc cttctcggcc ggcaacgcca caacataaag aagcaagcaa catcaacaac 21420agctgccgcc atgggctcca gtgagcagga actgaaagcc attgtcaaag atcttggttg 21480tgggccatat tttttgggca cctatgacaa gcgctttcca ggctttgttt ctccacacaa 21540gctcgcctgc gccatagtca atacggccgg tcgcgagact gggggcgtac actggatggc 21600ctttgcctgg aacccgcact caaaaacatg ctacctcttt gagccctttg gcttttctga 21660ccagcgactc aagcaggttt accagtttga gtacgagtca ctcctgcgcc gtagcgccat 21720tgcttcttcc cccgaccgct gtataacgct ggaaaagtcc acccaaagcg tacaggggcc 21780caactcggcc gcctgtggac tattctgctg catgtttctc cacgcctttg ccaactggcc 21840ccaaactccc atggatcaca accccaccat gaaccttatt accggggtac ccaactccat 21900gctcaacagt ccccaggtac agcccaccct gcgtcgcaac caggaacagc tctacagctt 21960cctggagcgc cactcgccct acttccgcag ccacagtgcg cagattagga gcgccacttc 22020tttttgtcac ttgaaaaaca tgtaaaaata atgtactaga gacactttca ataaaggcaa 22080atgcttttat ttgtacactc tcgggtgatt atttaccccc acccttgccg tctgcgccgt 22140ttaaaaatca aaggggttct gccgcgcatc gctatgcgcc actggcaggg acacgttgcg 22200atactggtgt ttagtgctcc acttaaactc aggcacaacc atccgcggca gctcggtgaa 22260gttttcactc cacaggctgc gcaccatcac caacgcgttt agcaggtcgg gcgccgatat 22320cttgaagtcg cagttggggc ctccgccctg cgcgcgcgag ttgcgataca cagggttgca 22380gcactggaac actatcagcg ccgggtggtg cacgctggcc agcacgctct tgtcggagat 22440cagatccgcg tccaggtcct ccgcgttgct cagggcgaac ggagtcaact ttggtagctg 22500ccttcccaaa aagggcgcgt gcccaggctt tgagttgcac tcgcaccgta gtggcatcaa 22560aaggtgaccg tgcccggtct gggcgttagg atacagcgcc tgcataaaag ccttgatctg 22620cttaaaagcc acctgagcct ttgcgccttc agagaagaac atgccgcaag acttgccgga 22680aaactgattg gccggacagg ccgcgtcgtg cacgcagcac cttgcgtcgg tgttggagat 22740ctgcaccaca tttcggcccc accggttctt cacgatcttg gccttgctag actgctcctt 22800cagcgcgcgc tgcccgtttt cgctcgtcac atccatttca atcacgtgct ccttatttat 22860cataatgctt ccgtgtagac acttaagctc gccttcgatc tcagcgcagc ggtgcagcca 22920caacgcgcag cccgtgggct cgtgatgctt gtaggtcacc tctgcaaacg actgcaggta 22980cgcctgcagg aatcgcccca tcatcgtcac aaaggtcttg ttgctggtga aggtcagctg 23040caacccgcgg tgctcctcgt tcagccaggt cttgcatacg gccgccagag cttccacttg 23100gtcaggcagt agtttgaagt tcgcctttag atcgttatcc acgtggtact tgtccatcag 23160cgcgcgcgca gcctccatgc ccttctccca cgcagacacg atcggcacac tcagcgggtt 23220catcaccgta atttcacttt ccgcttcgct gggctcttcc tcttcctctt gcgtccgcat 23280accacgcgcc actgggtcgt cttcattcag ccgccgcact gtgcgcttac ctcctttgcc 23340atgcttgatt agcaccggtg ggttgctgaa acccaccatt tgtagcgcca catcttctct 23400ttcttcctcg ctgtccacga ttacctctgg tgatggcggg cgctcgggct tgggagaagg 23460gcgcttcttt ttcttcttgg gcgcaatggc caaatccgcc gccgaggtcg atggccgcgg 23520gctgggtgtg cgcggcacca gcgcgtcttg tgatgagtct tcctcgtcct cggactcgat 23580acgccgcctc atccgctttt ttgggggcgc ccggggaggc ggcggcgacg gggacgggga 23640cgacacgtcc tccatggttg ggggacgtcg cgccgcaccg cgtccgcgct cgggggtggt 23700ttcgcgctgc tcctcttccc gactggccat ttccttctcc tataggcaga aaaagatcat 23760ggagtcagtc gagaagaagg acagcctaac cgccccctct gagttcgcca ccaccgcctc 23820caccgatgcc gccaacgcgc ctaccacctt ccccgtcgag gcacccccgc ttgaggagga 23880ggaagtgatt atcgagcagg acccaggttt tgtaagcgaa gacgacgagg accgctcagt 23940accaacagag gataaaaagc aagaccagga caacgcagag gcaaacgagg aacaagtcgg 24000gcggggggac gaaaggcatg gcgactacct agatgtggga gacgacgtgc tgttgaagca 24060tctgcagcgc cagtgcgcca ttatctgcga cgcgttgcaa gagcgcagcg atgtgcccct 24120cgccatagcg gatgtcagcc ttgcctacga acgccaccta ttctcaccgc gcgtaccccc 24180caaacgccaa gaaaacggca catgcgagcc caacccgcgc ctcaacttct accccgtatt 24240tgccgtgcca gaggtgcttg ccacctatca catctttttc caaaactgca agatacccct 24300atcctgccgt gccaaccgca gccgagcgga caagcagctg gccttgcggc agggcgctgt 24360catacctgat atcgcctcgc tcaacgaagt gccaaaaatc tttgagggtc ttggacgcga 24420cgagaagcgc gcggcaaacg ctctgcaaca ggaaaacagc gaaaatgaaa gtcactctgg 24480agtgttggtg gaactcgagg gtgacaacgc gcgcctagcc gtactaaaac gcagcatcga 24540ggtcacccac tttgcctacc cggcacttaa cctacccccc aaggtcatga gcacagtcat 24600gagtgagctg atcgtgcgcc gtgcgcagcc cctggagagg gatgcaaatt tgcaagaaca 24660aacagaggag ggcctacccg cagttggcga cgagcagcta gcgcgctggc ttcaaacgcg 24720cgagcctgcc gacttggagg agcgacgcaa actaatgatg gccgcagtgc tcgttaccgt 24780ggagcttgag tgcatgcagc ggttctttgc tgacccggag atgcagcgca agctagagga 24840aacattgcac tacacctttc gacagggcta cgtacgccag gcctgcaaga tctccaacgt 24900ggagctctgc aacctggtct cctaccttgg aattttgcac gaaaaccgcc ttgggcaaaa 24960cgtgcttcat tccacgctca agggcgaggc gcgccgcgac tacgtccgcg actgcgttta 25020cttatttcta tgctacacct ggcagacggc catgggcgtt tggcagcagt gcttggagga 25080gtgcaacctc aaggagctgc agaaactgct aaagcaaaac ttgaaggacc tatggacggc 25140cttcaacgag cgctccgtgg ccgcgcacct ggcggacatc attttccccg aacgcctgct 25200taaaaccctg caacagggtc tgccagactt caccagtcaa agcatgttgc agaactttag 25260gaactttatc ctagagcgct caggaatctt gcccgccacc tgctgtgcac ttcctagcga 25320ctttgtgccc attaagtacc gcgaatgccc tccgccgctt tggggccact gctaccttct 25380gcagctagcc aactaccttg cctaccactc tgacataatg gaagacgtga gcggtgacgg 25440tctactggag tgtcactgtc gctgcaacct atgcaccccg caccgctccc tggtttgcaa 25500ttcgcagctg cttaacgaaa gtcaaattat cggtaccttt gagctgcagg gtccctcgcc 25560tgacgaaaag tccgcggctc cggggttgaa actcactccg gggctgtgga cgtcggctta 25620ccttcgcaaa tttgtacctg aggactacca cgcccacgag attaggttct acgaagacca 25680atcccgcccg cctaatgcgg agcttaccgc ctgcgtcatt acccagggcc acattcttgg 25740ccaattgcaa gccatcaaca aagcccgcca agagtttctg ctacgaaagg gacggggggt 25800ttacttggac ccccagtccg gcgaggagct caacccaatc cccccgccgc cgcagcccta 25860tcagcagcag ccgcgggccc ttgcttccca ggatggcacc caaaaagaag ctgcagctgc 25920cgccgccacc cacggacgag gaggaatact gggacagtca ggcagaggag gttttggacg 25980aggaggagga ggacatgatg gaagactggg agagcctaga cgaggaagct tccgaggtcg 26040aagaggtgtc agacgaaaca ccgtcaccct cggtcgcatt cccctcgccg gcgccccaga 26100aatcggcaac cggttccagc atggctacaa cctccgctcc tcaggcgccg ccggcactgc 26160ccgttcgccg acccaaccgt agatgggaca ccactggaac cagggccggt aagtccaagc 26220agccgccgcc gttagcccaa gagcaacaac agcgccaagg ctaccgctca tggcgcgggc 26280acaagaacgc catagttgct tgcttgcaag actgtggggg caacatctcc ttcgcccgcc 26340gctttcttct ctaccatcac ggcgtggcct tcccccgtaa catcctgcat tactaccgtc 26400atctctacag cccatactgc accggcggca gcggcagcaa cagcagcggc cacacagaag 26460caaaggcgac cggatagcaa gactctgaca aagcccaaga aatccacagc ggcggcagca 26520gcaggaggag gagcgctgcg tctggcgccc aacgaacccg tatcgacccg cgagcttaga 26580aacaggattt ttcccactct gtatgctata tttcaacaga gcaggggcca agaacaagag 26640ctgaaaataa aaaacaggtc tctgcgatcc ctcacccgca gctgcctgta tcacaaaagc 26700gaagatcagc ttcggcgcac gctggaagac gcggaggctc tcttcagtaa atactgcgcg 26760ctgactctta aggactagtt tcgcgccctt tctcaaattt aagcgcgaaa actacgtcat 26820ctccagcggc cacacccggc gccagcacct gttgtcagcg ccattatgag caaggaaatt 26880cccacgccct acatgtggag ttaccagcca caaatgggac ttgcggctgg agctgcccaa 26940gactactcaa cccgaataaa ctacatgagc gcgggacccc acatgatatc ccgggtcaac 27000ggaatacgcg cccaccgaaa ccgaattctc ctggaacagg cggctattac caccacacct 27060cgtaataacc ttaatccccg tagttggccc gctgccctgg tgtaccagga aagtcccgct 27120cccaccactg tggtacttcc cagagacgcc caggccgaag ttcagatgac taactcaggg 27180gcgcagcttg cgggcggctt tcgtcacagg gtgcggtcgc ccgggcaggg tataactcac 27240ctgacaatca gagggcgagg tattcagctc aacgacgagt cggtgagctc ctcgcttggt 27300ctccgtccgg acgggacatt tcagatcggc ggcgccggcc gctcttcatt cacgcctcgt 27360caggcaatcc taactctgca gacctcgtcc tctgagccgc gctctggagg cattggaact 27420ctgcaattta ttgaggagtt tgtgccatcg gtctacttta accccttctc gggacctccc 27480ggccactatc cggatcaatt tattcctaac tttgacgcgg taaaggactc ggcggacggc 27540tacgactgaa tgttaagtgg agaggcagag caactgcgcc tgaaacacct ggtccactgt 27600cgccgccaca agtgctttgc ccgcgactcc ggtgagtttt gctactttga attgcccgag 27660gatcatatcg agggcccggc gcacggcgtc cggcttaccg cccagggaga gcttgcccgt 27720agcctgattc gggagtttac ccagcgcccc ctgctagttg agcgggacag gggaccctgt 27780gttctcactg tgatttgcaa ctgtcctaac cctggattac atcaagatct ttgttgccat 27840ctctgtgctg agtataataa atacagaaat taaaatatac tggggctcct atcgccatcc 27900tgtaaacgcc accgtcttca cccgcccaag caaaccaagg cgaaccttac ctggtacttt 27960taacatctct ccctctgtga tttacaacag tttcaaccca gacggagtga gtctacgaga 28020gaacctctcc gagctcagct actccatcag aaaaaacacc accctcctta cctgccggga 28080acgtacgagt gcgtcaccgg ccgctgcacc acacctaccg cctgaccgta aaccagactt 28140tttccggaca gacctcaata actctgttta ccagaacagg aggtgagctt agaaaaccct 28200tagggtatta ggccaaaggc gcagctactg tggggtttat gaacaattca agcaactcta 28260cgggctattc taattcaggt ttctctagaa atggacggaa ttattacaga gcagcgcctg 28320ctagaaagac gcagggcagc ggccgagcaa cagcgcatga atcaagagct ccaagacatg 28380gttaacttgc accagtgcaa aaggggtatc ttttgtctgg taaagcaggc caaagtcacc 28440tacgacagta ataccaccgg acaccgcctt agctacaagt tgccaaccaa gcgtcagaaa 28500ttggtggtca tggtgggaga aaagcccatt accataactc agcactcggt agaaaccgaa 28560ggctgcattc actcaccttg tcaaggacct gaggatctct gcacccttat taagaccctg 28620tgcggtctca aagatcttat tccctttaac taataaaaaa aaataataaa gcatcactta 28680cttaaaatca gttagcaaat ttctgtccag tttattcagc agcacctcct tgccctcctc 28740ccagctctgg tattgcagct tcctcctggc tgcaaacttt ctccacaatc taaatggaat 28800gtcagtttcc tcctgttcct gtccatccgc acccactatc ttcatgttgt tgcagatgaa 28860gcgcgcaaga ccgtctgaag ataccttcaa ccccgtgtat ccatatgaca cggaaaccgg 28920tcctccaact gtgccttttc ttactcctcc ctttgtatcc cccaatgggt ttcaagagag 28980tccccctggg gtactctctt tgcgcctatc cgaacctcta gttacctcca atggcatgct 29040tgcgctcaaa atgggcaacg gcctctctct ggacgaggcc ggcaacctta cctcccaaaa 29100tgtaaccact gtgagcccac ctctcaaaaa aaccaagtca aacataaacc tggaaatatc 29160tgcacccctc acagttacct cagaagccct aactgtggct gccgccgcac ctctaatggt 29220cgcgggcaac acactcacca tgcaatcaca ggccccgcta accgtgcacg actccaaact 29280tagcattgcc acccaaggac ccctcacagt gtcagaagga aagctagccc tgcaaacatc 29340aggccccctc accaccaccg atagcagtac ccttactatc actgcctcac cccctctaac 29400tactgccact ggtagcttgg gcattgactt gaaagagccc atttatacac aaaatggaaa 29460actaggacta aagtacgggg ctcctttgca tgtaacagac gacctaaaca ctttgaccgt 29520agcaactggt ccaggtgtga ctattaataa tacttccttg caaactaaag ttactggagc 29580cttgggtttt gattcacaag gcaatatgca acttaatgta gcaggaggac taaggattga 29640ttctcaaaac agacgcctta tacttgatgt tagttatccg tttgatgctc aaaaccaact 29700aaatctaaga ctaggacagg gccctctttt tataaactca gcccacaact tggatattaa 29760ctacaacaaa ggcctttact tgtttacagc ttcaaacaat tccaaaaagc ttgaggttaa 29820cctaagcact gccaaggggt tgatgtttga cgctacagcc atagccatta atgcaggaga 29880tgggcttgaa tttggttcac ctaatgcacc aaacacaaat cccctcaaaa caaaaattgg 29940ccatggccta gaatttgatt caaacaaggc tatggttcct aaactaggaa ctggccttag 30000ttttgacagc acaggtgcca ttacagtagg aaacaaaaat aatgataagc taactttgtg 30060gaccacacca gctccatctc ctaactgtag actaaatgca gagaaagatg ctaaactcac 30120tttggtctta acaaaatgtg gcagtcaaat acttgctaca gtttcagttt tggctgttaa 30180aggcagtttg gctccaatat ctggaacagt tcaaagtgct catcttatta taagatttga 30240cgaaaatgga gtgctactaa acaattcctt cctggaccca gaatattgga actttagaaa 30300tggagatctt actgaaggca cagcctatac aaacgctgtt ggatttatgc ctaacctatc 30360agcttatcca aaatctcacg gtaaaactgc caaaagtaac attgtcagtc aagtttactt 30420aaacggagac aaaactaaac ctgtaacact aaccattaca ctaaacggta cacaggaaac 30480aggagacaca actccaagtg catactctat gtcattttca tgggactggt ctggccacaa 30540ctacattaat gaaatatttg ccacatcctc ttacactttt tcatacattg cccaagaata 30600aagaatcgtt tgtgttatgt ttcaacgtgt ttatttttca attgcccggg atcggtgatc

30660accgatccag acatgataag atacattgat gagtttggac aaaccacaac tagaatgcag 30720tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata 30780agctgcaata aacaagttcc cggatcgcga tccggcccga ggctgtagcc gacgatggtg 30840cgccaggaga gttgttgatt cattgtttgc ctccctgctg cggtttttca ccgaagttca 30900tgccagtcca gcgtttttgc agcagaaaag ccgccgactt cggtttgcgg tcgcgagtga 30960agatcccttt cttgttaccg ccaacgcgca atatgccttg cgaggtcgca aaatcggcga 31020aattccatac ctgttcaccg acgacggcgc tgacgcgatc aaagacgcgg tgatacatat 31080ccagccatgc acactgatac tcttcactcc acatgtcggt gtacattgag tgcagcccgg 31140ctaacgtatc cacgccgtat tcggtgatga taatcggctg atgcagtttc tcctgccagg 31200ccagaagttc tttttccagt accttctctg ccgtttccaa atcgccgctt tggacatacc 31260atccgtaata acggttcagg cacagcacat caaagagatc gctgatggta tcggtgtgag 31320cgtcgcagaa cattacattg acgcaggtga tcggacgcgt cgggtcgagt ttacgcgttg 31380cttccgccag tggcgcgaaa tattcccgtg caccttgcgg acgggtatcc ggttcgttgg 31440caatactcca catcaccacg cttgggtggt ttttgtcacg cgctatcagc tctttaatcg 31500cctgtaagtg cgcttgctga gtttccccgt tgactgcctc ttcgctgtac agttctttcg 31560gcttgttgcc cgcttcgaaa ccaatgccta aagagaggtt aaagccgaca gcagcagttt 31620catcaatcac cacgatgcca tgttcatctg cccagtcgag catctcttca gcgtaagggt 31680aatgcgaggt acggtaggag ttggccccaa tccagtccat taatgcgtgg tcgtgcacca 31740tcagcacgtt atcgaatcct ttgccacgca agtccgcatc ttcatgacga ccaaagccag 31800taaagtagaa cggtttgtgg ttaatcagga actgttcgcc cttcactgcc actgaccgga 31860tgccgacgcg aagcgggtag atatcacact ctgtctggct tttggctgtg acgcacagtt 31920catagagata accttcaccc ggttgccaga ggtgcggatt caccacttgc aaagtcccgc 31980tagtgccttg tccagttgca accacctgtt gatccgcatc acgcagttca acgctgacat 32040caccattggc caccacctgc cagtcaacag acgcgtggtt acagtcttgc gcgacatgcg 32100tcaccacggt gatatcgtcc acccaggtgt tcggcgtggt gtagagcatt acgctgcgat 32160ggattccggc atagttaaag aaatcatgga agtaagactg ctttttcttg ccgttttcgt 32220cggtaatcac cattcccggc gggatagtct gccagttcag ttcgttgttc acacaaacgg 32280tgatacgtac acttttcccg gcaataacat acggcgtgac atcggcttca aatggcgtat 32340agccgccctg atgctccatc acttcctgat tattgaccca cactttgccg taatgagtga 32400ccgcatcgaa acgcagcacg atacgctggc ctgcccaacc tttcggtata aagacttcgc 32460gctgatacca gacgttgccc gcataattac gaatatctgc atcggcgaac tgatcgttaa 32520aactgcctgg cacagcaatt gcccggcttt cttgtaacgc gctttcccac caacgctgat 32580caattccaca gttttcgcga tccagactga atgcccacag gccgtcgagt tttttgattt 32640cacgggttgg ggtttctaca ggacggacca tgcgttcgac ctttctcttc ttttttgggc 32700ccatgatggc agatccgtat agtgagtcgt attagctggt tctttccgcc tcagaagcca 32760tagagcccac cgcatcccca gcatgcctgc tattgtcttc ccaatcctcc cccttgctgt 32820cctgccccac cccacccccc agaatagaat gacacctact cagacaatgc gatgcaattt 32880cctcatttta ttaggaaagg acagtgggag tggcaccttc cagggtcaag gaaggcacgg 32940gggaggggca aacaacagat ggctggcaac tagaaggcac agtcgaggct gatcagcgag 33000ctctagatgc atgctcgagc ggccgccagt gtgatggata tctgcagaat tccagcacac 33060tggcggccgt tactagtgga tccgagctcg gtacccggcc gttataacac cactcgacac 33120ggcaccagct caatcagtca cagtgtaaaa aagggccaag tgcagagcga gtatatatag 33180gactaaaaaa tgacgtaacg gttaaagtcc acaaaaaaca cccagaaaac cgcacgcgaa 33240cctacgccca gaaacgaaag ccaaaaaacc cacaacttcc tcaaatcgtc acttccgttt 33300tcccacgtta cgtcacttcc cattttaaga aaactacaat tcccaacaca tacaagttac 33360tccgccctaa aacctacgtc acccgccccg ttcccacgcc ccgcgccacg tcacaaactc 33420caccccctca ttatcatatt ggcttcaatc caaaataagg tatattattg atgatg 334761933589DNAArtificial sequenceAdenoviral vector Adgp140(C).11D 19catcatcaat aatatacctt attttggatt gaagccaata tgataatgag ggggtggagt 60ttgtgacgtg gcgcggggcg tgggaacggg gcgggtgacg tagtagtgtg gcggaagtgt 120gatgttgcaa gtgtggcgga acacatgtaa gcgacggatg tggcaaaagt gacgtttttg 180gtgtgcgccg gtgtacacag gaagtgacaa ttttcgcgcg gttttaggcg gatgttgtag 240taaatttggg cgtaaccgag taagatttgg ccattttcgc gggaaaactg aataagagga 300agtgaaatct gaataatttt gtgttactca tagcgcgtaa tatttgtcta gggcccggga 360tcggtgatca ccgatccaga catgataaga tacattgatg agtttggaca aaccacaact 420agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta 480accattataa gctgcaataa acaagttccc ggatctttct agctagtcta gactagctag 540actcgagagc ggccgcaatc gataagcttg atctagagat gatcctcacc acagccagtt 600ggagatgtcg aaccaggacc acaggttttt ccaggagtcc agagccagca ggtctttttc 660gtttttttcc tgctgggtct gggagtcttc cagcagacgg tagatggtgt cggtgtagtt 720ggagatttca cggtcccatt ccatccaggt catgttgttc cagatctcga gctgctggtc 780tttcaggtaa cgttcgatag ccagaacacg ggtctgcagc tgtttgatac cccaaacggt 840cagctgcagc atgtgctgct gagcttcgat agcacgcagc aggttggact gctgctgaac 900gatggaggac agcagctgac gagcctgaac ggtaagctta gcaccggtcg gagcgatacc 960cagcggtttc agttcgataa ctttgtattt gtacagttcg gaacgccagt tgtctttcat 1020gttaccacca cccggacgga agatttcttc ggttttgttg tcttcaccac cgtcacgaac 1080cagcagcaga ccggtgatgt tggatttgca ggtgatgtta ccagcgatcg gcggagcgta 1140catagcacga ccaacaccct gccacatgtt gatgatctgt ttgatacggc acggcagggt 1200gatggtttcg tcttcggtag cgttgttgtt gaacagacgg gtggtgttgc agtagaagaa 1260ttcaccacgg cagttgaagg agtgggtggt gatttccagg tcaccaccgg aggacggagc 1320gaatttgatg gttttgttgt tgttgtagtt ttcctgcagt ttttctttaa cacgtttcag 1380ggtttcgttc catttggaac cggagatgtt gcagtaagcc tgacggatgt caccgatgat 1440gtcaccggta gcgtagaagg tctgacccgg accgatacgc atggatttac gggtgttgtt 1500gttcggacgg gtgcaaacga tttcaacgga tttgttcagg tgaacgatga tggttttaac 1560gttgtcggtc aggttttcgg aacggatgat gatttctttt tcagccaggg aaccgttcag 1620cagcagctgg gtggaaacaa ccggtttgat accatgggtg cactgaacgg tggaaacgtt 1680gttgcacgga cctttaccgg agaaggtttt gttgttgcat ttcaggatag cgtaaccagc 1740cggagcgcag tagtggatcg ggatcgggtc gaagttaact ttcgggcaag cctgggtgat 1800ggtggaagcg ttgcagttga tcaggatgta ttcggagttg ttggagttgt tacggttttc 1860tttcagcaga acgatgtccg gacggtagaa cagagcgtaa ccctgctgtt ttttgtcacg 1920gatttcggtg gtggtgttga aggagcagtt acggatttct ttgttcatgt cgttggtaac 1980gttgtttttg aaggtagcgt tggtgcagtg cagggtaacg cacagcgggg tcagtttaac 2040gcacggtttc agggactggt cccacaggga gatgatgtct tcgtgcatct ggtcaaccat 2100gtcgtttttc cacatgttga agttttcggt aacgttttcc agaacgattt cctgcgggtt 2160cgggtcggtc ggaacgcaag cgtgggtagc ccaaacgttg tgaacttcac ggtcgtaggc 2220tttggtgtcg gaagcgcaga acagggtggt tttagcgtcg gtccaaaccg gaacaccgta 2280gtaaacggta acccacatgt taccaacaac acggcagatg atgatcatcc agaaacccag 2340gataccccac atccaccact gcggccagtt acgcgggata ccacgaacac gcatggtggc 2400gatatctcta gtcatcgaat tctgcagtga tcagggatcc cagatccgta tagtgagtcg 2460tattaggtac cggctgcagt tggacctggg agtggacacc tgtggagaga aaggcaaagt 2520ggatgtcatt gtcactcaag tgtatggcca gatctcaagc ctgccacacc tcaagtgaag 2580ccaagggggt gggcctatag actctatagg cggtacttac gtcactcttg gcacggggaa 2640tccgcgttcc aatgcaccgt tcccggccgc ggaggctgga tcggtcccgg tgtcttctat 2700ggaggtcaaa acagcgtgga tggcgtctcc aggcgatctg acggttcact aaacgagctc 2760tgcttatata gacctcccac cgtacacgcc taccgcccat ttgcgtcaat ggggcggagt 2820tgttacgaca ttttggaaag tcccgttgat tttggtgcca aaacaaactc ccattgacgt 2880caatggggtg gagacttgga aatccccgtg agtcaaaccg ctatccacgc ccattgatgt 2940actgccaaaa ccgcatcacc atggtaatag cgatgactaa tacgtagatg tactgccaag 3000taggaaagtc ccataaggtc atgtactggg cataatgcca ggcgggccat ttaccgtcat 3060tgacgtcaat agggggcgta cttggcatat gatacacttg atgtactgcc aagtgggcag 3120tttaccgtaa atactccacc cattgacgtc aatggaaagt ccctattggc gttactatgg 3180gaacatacgt cattattgac gtcaatgggc gggggtcgtt gggcggtcag ccaggcgggc 3240catttaccgt aagttatgta acgcggaact ccatatatgg gctatgaact aatgaccccg 3300taattgatta ctattaataa ctagtactga aatgtgtggg cgtggcttaa gggtgggaaa 3360gaatatataa ggtgggggtc ttatgtagtt ttgtatctgt tttgcagcag ccgccgccgc 3420catgagcacc aactcgtttg atggaagcat tgtgagctca tatttgacaa cgcgcatgcc 3480cccatgggcc ggggtgcgtc agaatgtgat gggctccagc attgatggtc gccccgtcct 3540gcccgcaaac tctactacct tgacctacga gaccgtgtct ggaacgccgt tggagactgc 3600agcctccgcc gccgcttcag ccgctgcagc caccgcccgc gggattgtga ctgactttgc 3660tttcctgagc ccgcttgcaa gcagtgcagc ttcccgttca tccgcccgcg atgacaagtt 3720gacggctctt ttggcacaat tggattcttt gacccgggaa cttaatgtcg tttctcagca 3780gctgttggat ctgcgccagc aggtttctgc cctgaaggct tcctcccctc ccaatgcggt 3840ttaaaacata aataaaaaac cagactctgt ttggatttgg atcaagcaag tgtcttgctg 3900tctttattta ggggttttgc gcgcgcggta ggcccgggac cagcggtctc ggtcgttgag 3960ggtcctgtgt attttttcca ggacgtggta aaggtgactc tggatgttca gatacatggg 4020cataagcccg tctctggggt ggaggtagca ccactgcaga gcttcatgct gcggggtggt 4080gttgtagatg atccagtcgt agcaggagcg ctgggcgtgg tgcctaaaaa tgtctttcag 4140tagcaagctg attgccaggg gcaggccctt ggtgtaagtg tttacaaagc ggttaagctg 4200ggatgggtgc atacgtgggg atatgagatg catcttggac tgtattttta ggttggctat 4260gttcccagcc atatccctcc ggggattcat gttgtgcaga accaccagca cagtgtatcc 4320ggtgcacttg ggaaatttgt catgtagctt agaaggaaat gcgtggaaga acttggagac 4380gcccttgtga cctccaagat tttccatgca ttcgtccata atgatggcaa tgggcccacg 4440ggcggcggcc tgggcgaaga tatttctggg atcactaacg tcatagttgt gttccaggat 4500gagatcgtca taggccattt ttacaaagcg cgggcggagg gtgccagact gcggtataat 4560ggttccatcc ggcccagggg cgtagttacc ctcacagatt tgcatttccc acgctttgag 4620ttcagatggg gggatcatgt ctacctgcgg ggcgatgaag aaaacggttt ccggggtagg 4680ggagatcagc tgggaagaaa gcaggttcct gagcagctgc gacttaccgc agccggtggg 4740cccgtaaatc acacctatta ccggctgcaa ctggtagtta agagagctgc agctgccgtc 4800atccctgagc aggggggcca cttcgttaag catgtccctg actcgcatgt tttccctgac 4860caaatccgcc agaaggcgct cgccgcccag cgatagcagt tcttgcaagg aagcaaagtt 4920tttcaacggt ttgagaccgt ccgccgtagg catgcttttg agcgtttgac caagcagttc 4980caggcggtcc cacagctcgg tcacctgctc tacggcatct cgatccagca tatctcctcg 5040tttcgcgggt tggggcggct ttcgctgtac ggcagtagtc ggtgctcgtc cagacgggcc 5100agggtcatgt ctttccacgg gcgcagggtc ctcgtcagcg tagtctgggt cacggtgaag 5160gggtgcgctc cgggctgcgc gctggccagg gtgcgcttga ggctggtcct gctggtgctg 5220aagcgctgcc ggtcttcgcc ctgcgcgtcg gccaggtagc atttgaccat ggtgtcatag 5280tccagcccct ccgcggcgtg gcccttggcg cgcagcttgc ccttggagga ggcgccgcac 5340gaggggcagt gcagactttt gagggcgtag agcttgggcg cgagaaatac cgattccggg 5400gagtaggcat ccgcgccgca ggccccgcag acggtctcgc attccacgag ccaggtgagc 5460tctggccgtt cggggtcaaa aaccaggttt cccccatgct ttttgatgcg tttcttacct 5520ctggtttcca tgagccggtg tccacgctcg gtgacgaaaa ggctgtccgt gtccccgtat 5580acagacttga gaggcctgtc ctcgagcggt gttccgcggt cctcctcgta tagaaactcg 5640gaccactctg agacaaaggc tcgcgtccag gccagcacga aggaggctaa gtgggagggg 5700tagcggtcgt tgtccactag ggggtccact cgctccaggg tgtgaagaca catgtcgccc 5760tcttcggcat caaggaaggt gattggtttg taggtgtagg ccacgtgacc gggtgttcct 5820gaaggggggc tataaaaggg ggtgggggcg cgttcgtcct cactctcttc cgcatcgctg 5880tctgcgaggg ccagctgttg gggtgagtac tccctctgaa aagcgggcat gacttctgcg 5940ctaagattgt cagtttccaa aaacgaggag gatttgatat tcacctggcc cgcggtgatg 6000cctttgaggg tggccgcatc catctggtca gaaaagacaa tctttttgtt gtcaagcttg 6060gtggcaaacg acccgtagag ggcgttggac agcaacttgg cgatggagcg cagggtttgg 6120tttttgtcgc gatcggcgcg ctccttggcc gcgatgttta gctgcacgta ttcgcgcgca 6180acgcaccgcc attcgggaaa gacggtggtg cgctcgtcgg gcaccaggtg cacgcgccaa 6240ccgcggttgt gcagggtgac aaggtcaacg ctggtggcta cctctccgcg taggcgctcg 6300ttggtccagc agaggcggcc gcccttgcgc gagcagaatg gcggtagggg gtctagctgc 6360gtctcgtccg gggggtctgc gtccacggta aagaccccgg gcagcaggcg cgcgtcgaag 6420tagtctatct tgcatccttg caagtctagc gcctgctgcc atgcgcgggc ggcaagcgcg 6480cgctcgtatg ggttgagtgg gggaccccat ggcatggggt gggtgagcgc ggaggcgtac 6540atgccgcaaa tgtcgtaaac gtagaggggc tctctgagta ttccaagata tgtagggtag 6600catcttccac cgcggatgct ggcgcgcacg taatcgtata gttcgtgcga gggagcgagg 6660aggtcgggac cgaggttgct acgggcgggc tgctctgctc ggaagactat ctgcctgaag 6720atggcatgtg agttggatga tatggttgga cgctggaaga cgttgaagct ggcgtctgtg 6780agacctaccg cgtcacgcac gaaggaggcg taggagtcgc gcagcttgtt gaccagctcg 6840gcggtgacct gcacgtctag ggcgcagtag tccagggttt ccttgatgat gtcatactta 6900tcctgtccct tttttttcca cagctcgcgg ttgaggacaa actcttcgcg gtctttccag 6960tactcttgga tcggaaaccc gtcggcctcc gaacggtaag agcctagcat gtagaactgg 7020ttgacggcct ggtaggcgca gcatcccttt tctacgggta gcgcgtatgc ctgcgcggcc 7080ttccggagcg aggtgtgggt gagcgcaaag gtgtccctga ccatgacttt gaggtactgg 7140tatttgaagt cagtgtcgtc gcatccgccc tgctcccaga gcaaaaagtc cgtgcgcttt 7200ttggaacgcg gatttggcag ggcgaaggtg acatcgttga agagtatctt tcccgcgcga 7260ggcataaagt tgcgtgtgat gcggaagggt cccggcacct cggaacggtt gttaattacc 7320tgggcggcga gcacgatctc gtcaaagccg ttgatgttgt ggcccacaat gtaaagttcc 7380aagaagcgcg ggatgccctt gatggaaggc aattttttaa gttcctcgta ggtgagctct 7440tcaggggagc tgagcccgtg ctctgaaagg gcccagtctg caagatgagg gttggaagcg 7500acgaatgagc tccacaggtc acgggccatt agcatttgca ggtggtcgcg aaaggtccta 7560aactggcgac ctatggccat tttttctggg gtgatgcagt agaaggtaag cgggtcttgt 7620tcccagcggt cccatccaag gttcgcggct aggtctcgcg cggcagtcac tagaggctca 7680tctccgccga acttcatgac cagcatgaag ggcacgagct gcttcccaaa ggcccccatc 7740caagtatagg tctctacatc gtaggtgaca aagagacgct cggtgcgagg atgcgagccg 7800atcgggaaga actggatctc ccgccaccaa ttggaggagt ggctattgat gtggtgaaag 7860tagaagtccc tgcgacgggc cgaacactcg tgctggcttt tgtaaaaacg tgcgcagtac 7920tggcagcggt gcacgggctg tacatcctgc acgaggttga cctgacgacc gcgcacaagg 7980aagcagagtg ggaatttgag cccctcgcct ggcgggtttg gctggtggtc ttctacttcg 8040gctgcttgtc cttgaccgtc tggctgctcg aggggagtta cggtggatcg gaccaccacg 8100ccgcgcgagc ccaaagtcca gatgtccgcg cgcggcggtc ggagcttgat gacaacatcg 8160cgcagatggg agctgtccat ggtctggagc tcccgcggcg tcaggtcagg cgggagctcc 8220tgcaggttta cctcgcatag acgggtcagg gcgcgggcta gatccaggtg atacctaatt 8280tccaggggct ggttggtggc ggcgtcgatg gcttgcaaga ggccgcatcc ccgcggcgcg 8340actacggtac cgcgcggcgg gcggtgggcc gcgggggtgt ccttggatga tgcatctaaa 8400agcggtgacg cgggcgagcc cccggaggta gggggggctc cggacccgcc gggagagggg 8460gcaggggcac gtcggcgccg cgcgcgggca ggagctggtg ctgcgcgcgt aggttgctgg 8520cgaacgcgac gacgcggcgg ttgatctcct gaatctggcg cctctgcgtg aagacgacgg 8580gcccggtgag cttgaacctg aaagagagtt cgacagaatc aatttcggtg tcgttgacgg 8640cggcctggcg caaaatctcc tgcacgtctc ctgagttgtc ttgataggcg atctcggcca 8700tgaactgctc gatctcttcc tcctggagat ctccgcgtcc ggctcgctcc acggtggcgg 8760cgaggtcgtt ggaaatgcgg gccatgagct gcgagaaggc gttgaggcct ccctcgttcc 8820agacgcggct gtagaccacg cccccttcgg catcgcgggc gcgcatgacc acctgcgcga 8880gattgagctc cacgtgccgg gcgaagacgg cgtagtttcg caggcgctga aagaggtagt 8940tgagggtggt ggcggtgtgt tctgccacga agaagtacat aacccagcgt cgcaacgtgg 9000attcgttgat atcccccaag gcctcaaggc gctccatggc ctcgtagaag tccacggcga 9060agttgaaaaa ctgggagttg cgcgccgaca cggttaactc ctcctccaga agacggatga 9120gctcggcgac agtgtcgcgc acctcgcgct caaaggctac aggggcctct tcttcttctt 9180caatctcctc ttccataagg gcctcccctt cttcttcttc tggcggcggt gggggagggg 9240ggacacggcg gcgacgacgg cgcaccggga ggcggtcgac aaagcgctcg atcatctccc 9300cgcggcgacg gcgcatggtc tcggtgacgg cgcggccgtt ctcgcggggg cgcagttgga 9360agacgccgcc cgtcatgtcc cggttatggg ttggcggggg gctgccatgc ggcagggata 9420cggcgctaac gatgcatctc aacaattgtt gtgtaggtac tccgccgccg agggacctga 9480gcgagtccgc atcgaccgga tcggaaaacc tctcgagaaa ggcgtctaac cagtcacagt 9540cgcaaggtag gctgagcacc gtggcgggcg gcagcgggcg gcggtcgggg ttgtttctgg 9600cggaggtgct gctgatgatg taattaaagt aggcggtctt gagacggcgg atggtcgaca 9660gaagcaccat gtccttgggt ccggcctgct gaatgcgcag gcggtcggcc atgccccagg 9720cttcgttttg acatcggcgc aggtctttgt agtagtcttg catgagcctt tctaccggca 9780cttcttcttc tccttcctct tgtcctgcat ctcttgcatc tatcgctgcg gcggcggcgg 9840agtttggccg taggtggcgc cctcttcctc ccatgcgtgt gaccccgaag cccctcatcg 9900gctgaagcag ggctaggtcg gcgacaacgc gctcggctaa tatggcctgc tgcacctgcg 9960tgagggtaga ctggaagtca tccatgtcca caaagcggtg gtatgcgccc gtgttgatgg 10020tgtaagtgca gttggccata acggaccagt taacggtctg gtgacccggc tgcgagagct 10080cggtgtacct gagacgcgag taagccctcg agtcaaatac gtagtcgttg caagtccgca 10140ccaggtactg gtatcccacc aaaaagtgcg gcggcggctg gcggtagagg ggccagcgta 10200gggtggccgg ggctccgggg gcgagatctt ccaacataag gcgatgatat ccgtagatgt 10260acctggacat ccaggtgatg ccggcggcgg tggtggaggc gcgcggaaag tcgcggacgc 10320ggttccagat gttgcgcagc ggcaaaaagt gctccatggt cgggacgctc tggccggtca 10380ggcgcgcgca atcgttgacg ctctagcgtg caaaaggaga gcctgtaagc gggcactctt 10440ccgtggtctg gtggataaat tcgcaagggt atcatggcgg acgaccgggg ttcgagcccc 10500gtatccggcc gtccgccgtg atccatgcgg ttaccgcccg cgtgtcgaac ccaggtgtgc 10560gacgtcagac aacgggggag tgctcctttt ggcttccttc caggcgcggc ggctgctgcg 10620ctagcttttt tggccactgg ccgcgcgcag cgtaagcggt taggctggaa agcgaaagca 10680ttaagtggct cgctccctgt agccggaggg ttattttcca agggttgagt cgcgggaccc 10740ccggttcgag tctcggaccg gccggactgc ggcgaacggg ggtttgcctc cccgtcatgc 10800aagaccccgc ttgcaaattc ctccggaaac agggacgagc cccttttttg cttttcccag 10860atgcatccgg tgctgcggca gatgcgcccc cctcctcagc agcggcaaga gcaagagcag 10920cggcagacat gcagggcacc ctcccctcct cctaccgcgt caggaggggc gacatccgcg 10980gttgacgcgg cagcagatgg tgattacgaa cccccgcggc gccgggcccg gcactacctg 11040gacttggagg agggcgaggg cctggcgcgg ctaggagcgc cctctcctga gcggcaccca 11100agggtgcagc tgaagcgtga tacgcgtgag gcgtacgtgc cgcggcagaa cctgtttcgc 11160gaccgcgagg gagaggagcc cgaggagatg cgggatcgaa agttccacgc agggcgcgag 11220ctgcggcatg gcctgaatcg cgagcggttg ctgcgcgagg aggactttga gcccgacgcg 11280cgaaccggga ttagtcccgc gcgcgcacac gtggcggccg ccgacctggt aaccgcatac 11340gagcagacgg tgaaccagga gattaacttt caaaaaagct ttaacaacca cgtgcgtacg 11400cttgtggcgc gcgaggaggt ggctatagga ctgatgcatc tgtgggactt tgtaagcgcg 11460ctggagcaaa acccaaatag caagccgctc atggcgcagc tgttccttat agtgcagcac 11520agcagggaca acgaggcatt cagggatgcg ctgctaaaca tagtagagcc cgagggccgc 11580tggctgctcg atttgataaa catcctgcag agcatagtgg tgcaggagcg cagcttgagc 11640ctggctgaca aggtggccgc catcaactat tccatgctta gcctgggcaa gttttacgcc 11700cgcaagatat accatacccc ttacgttccc atagacaagg aggtaaagat cgaggggttc 11760tacatgcgca tggcgctgaa ggtgcttacc ttgagcgacg acctgggcgt ttatcgcaac 11820gagcgcatcc acaaggccgt gagcgtgagc cggcggcgcg agctcagcga ccgcgagctg 11880atgcacagcc tgcaaagggc cctggctggc acgggcagcg gcgatagaga ggccgagtcc 11940tactttgacg cgggcgctga cctgcgctgg gccccaagcc gacgcgccct ggaggcagct 12000ggggccggac ctgggctggc ggtggcaccc gcgcgcgctg gcaacgtcgg cggcgtggag 12060gaatatgacg aggacgatga gtacgagcca gaggacggcg agtactaagc ggtgatgttt 12120ctgatcagat gatgcaagac gcaacggacc cggcggtgcg ggcggcgctg cagagccagc

12180cgtccggcct taactccacg gacgactggc gccaggtcat ggaccgcatc atgtcgctga 12240ctgcgcgcaa tcctgacgcg ttccggcagc agccgcaggc caaccggctc tccgcaattc 12300tggaagcggt ggtcccggcg cgcgcaaacc ccacgcacga gaaggtgctg gcgatcgtaa 12360acgcgctggc cgaaaacagg gccatccggc ccgacgaggc cggcctggtc tacgacgcgc 12420tgcttcagcg cgtggctcgt tacaacagcg gcaacgtgca gaccaacctg gaccggctgg 12480tgggggatgt gcgcgaggcc gtggcgcagc gtgagcgcgc gcagcagcag ggcaacctgg 12540gctccatggt tgcactaaac gccttcctga gtacacagcc cgccaacgtg ccgcggggac 12600aggaggacta caccaacttt gtgagcgcac tgcggctaat ggtgactgag acaccgcaaa 12660gtgaggtgta ccagtctggg ccagactatt ttttccagac cagtagacaa ggcctgcaga 12720ccgtaaacct gagccaggct ttcaaaaact tgcaggggct gtggggggtg cgggctccca 12780caggcgaccg cgcgaccgtg tctagcttgc tgacgcccaa ctcgcgcctg ttgctgctgc 12840taatagcgcc cttcacggac agtggcagcg tgtcccggga cacataccta ggtcacttgc 12900tgacactgta ccgcgaggcc ataggtcagg cgcatgtgga cgagcatact ttccaggaga 12960ttacaagtgt cagccgcgcg ctggggcagg aggacacggg cagcctggag gcaaccctaa 13020actacctgct gaccaaccgg cggcagaaga tcccctcgtt gcacagttta aacagcgagg 13080aggagcgcat tttgcgctac gtgcagcaga gcgtgagcct taacctgatg cgcgacgggg 13140taacgcccag cgtggcgctg gacatgaccg cgcgcaacat ggaaccgggc atgtatgcct 13200caaaccggcc gtttatcaac cgcctaatgg actacttgca tcgcgcggcc gccgtgaacc 13260ccgagtattt caccaatgcc atcttgaacc cgcactggct accgccccct ggtttctaca 13320ccgggggatt cgaggtgccc gagggtaacg atggattcct ctgggacgac atagacgaca 13380gcgtgttttc cccgcaaccg cagaccctgc tagagttgca acagcgcgag caggcagagg 13440cggcgctgcg aaaggaaagc ttccgcaggc caagcagctt gtccgatcta ggcgctgcgg 13500ccccgcggtc agatgctagt agcccatttc caagcttgat agggtctctt accagcactc 13560gcaccacccg cccgcgcctg ctgggcgagg aggagtacct aaacaactcg ctgctgcagc 13620cgcagcgcga aaaaaacctg cctccggcat ttcccaacaa cgggatagag agcctagtgg 13680acaagatgag tagatggaag acgtacgcgc aggagcacag ggacgtgcca ggcccgcgcc 13740cgcccacccg tcgtcaaagg cacgaccgtc agcggggtct ggtgtgggag gacgatgact 13800cggcagacga cagcagcgtc ctggatttgg gagggagtgg caacccgttt gcgcaccttc 13860gccccaggct ggggagaatg ttttaaaaaa aaaaaaagca tgatgcaaaa taaaaaactc 13920accaaggcca tggcaccgag cgttggtttt cttgtattcc ccttagtatg cggcgcgcgg 13980cgatgtatga ggaaggtcct cctccctcct acgagagtgt ggtgagcgcg gcgccagtgg 14040cggcggcgct gggttctccc ttcgatgctc ccctggaccc gccgtttgtg cctccgcggt 14100acctgcggcc taccgggggg agaaacagca tccgttactc tgagttggca cccctattcg 14160acaccacccg tgtgtacctg gtggacaaca agtcaacgga tgtggcatcc ctgaactacc 14220agaacgacca cagcaacttt ctgaccacgg tcattcaaaa caatgactac agcccggggg 14280aggcaagcac acagaccatc aatcttgacg accggtcgca ctggggcggc gacctgaaaa 14340ccatcctgca taccaacatg ccaaatgtga acgagttcat gtttaccaat aagtttaagg 14400cgcgggtgat ggtgtcgcgc ttgcctacta aggacaatca ggtggagctg aaatacgagt 14460gggtggagtt cacgctgccc gagggcaact actccgagac catgaccata gaccttatga 14520acaacgcgat cgtggagcac tacttgaaag tgggcagaca gaacggggtt ctggaaagcg 14580acatcggggt aaagtttgac acccgcaact tcagactggg gtttgacccc gtcactggtc 14640ttgtcatgcc tggggtatat acaaacgaag ccttccatcc agacatcatt ttgctgccag 14700gatgcggggt ggacttcacc cacagccgcc tgagcaactt gttgggcatc cgcaagcggc 14760aacccttcca ggagggcttt aggatcacct acgatgatct ggagggtggt aacattcccg 14820cactgttgga tgtggacgcc taccaggcga gcttgaaaga tgacaccgaa cagggcgggg 14880gtggcgcagg cggcagcaac agcagtggca gcggcgcgga agagaactcc aacgcggcag 14940ccgcggcaat gcagccggtg gaggacatga acgatcatgc cattcgcggc gacacctttg 15000ccacacgggc tgaggagaag cgcgctgagg ccgaagcagc ggccgaagct gccgcccccg 15060ctgcgcaacc cgaggtcgag aagcctcaga agaaaccggt gatcaaaccc ctgacagagg 15120acagcaagaa acgcagttac aacctaataa gcaatgacag caccttcacc cagtaccgca 15180gctggtacct tgcatacaac tacggcgacc ctcagaccgg aatccgctca tggaccctgc 15240tttgcactcc tgacgtaacc tgcggctcgg agcaggtcta ctggtcgttg ccagacatga 15300tgcaagaccc cgtgaccttc cgctccacgc gccagatcag caactttccg gtggtgggcg 15360ccgagctgtt gcccgtgcac tccaagagct tctacaacga ccaggccgtc tactcccaac 15420tcatccgcca gtttacctct ctgacccacg tgttcaatcg ctttcccgag aaccagattt 15480tggcgcgccc gccagccccc accatcacca ccgtcagtga aaacgttcct gctctcacag 15540atcacgggac gctaccgctg cgcaacagca tcggaggagt ccagcgagtg accattactg 15600acgccagacg ccgcacctgc ccctacgttt acaaggccct gggcatagtc tcgccgcgcg 15660tcctatcgag ccgcactttt tgagcaagca tgtccatcct tatatcgccc agcaataaca 15720caggctgggg cctgcgcttc ccaagcaaga tgtttggcgg ggccaagaag cgctccgacc 15780aacacccagt gcgcgtgcgc gggcactacc gcgcgccctg gggcgcgcac aaacgcggcc 15840gcactgggcg caccaccgtc gatgacgcca tcgacgcggt ggtggaggag gcgcgcaact 15900acacgcccac gccgccacca gtgtccacag tggacgcggc cattcagacc gtggtgcgcg 15960gagcccggcg ctatgctaaa atgaagagac ggcggaggcg cgtagcacgt cgccaccgcc 16020gccgacccgg cactgccgcc caacgcgcgg cggcggccct gcttaaccgc gcacgtcgca 16080ccggccgacg ggcggccatg cgggccgctc gaaggctggc cgcgggtatt gtcactgtgc 16140cccccaggtc caggcgacga gcggccgccg cagcagccgc ggccattagt gctatgactc 16200agggtcgcag gggcaacgtg tattgggtgc gcgactcggt tagcggcctg cgcgtgcccg 16260tgcgcacccg ccccccgcgc aactagattg caagaaaaaa ctacttagac tcgtactgtt 16320gtatgtatcc agcggcggcg gcgcgcaacg aagctatgtc caagcgcaaa atcaaagaag 16380agatgctcca ggtcatcgcg ccggagatct atggcccccc gaagaaggaa gagcaggatt 16440acaagccccg aaagctaaag cgggtcaaaa agaaaaagaa agatgatgat gatgaacttg 16500acgacgaggt ggaactgctg cacgctaccg cgcccaggcg acgggtacag tggaaaggtc 16560gacgcgtaaa acgtgttttg cgacccggca ccaccgtagt ctttacgccc ggtgagcgct 16620ccacccgcac ctacaagcgc gtgtatgatg aggtgtacgg cgacgaggac ctgcttgagc 16680aggccaacga gcgcctcggg gagtttgcct acggaaagcg gcataaggac atgctggcgt 16740tgccgctgga cgagggcaac ccaacaccta gcctaaagcc cgtaacactg cagcaggtgc 16800tgcccgcgct tgcaccgtcc gaagaaaagc gcggcctaaa gcgcgagtct ggtgacttgg 16860cacccaccgt gcagctgatg gtacccaagc gccagcgact ggaagatgtc ttggaaaaaa 16920tgaccgtgga acctgggctg gagcccgagg tccgcgtgcg gccaatcaag caggtggcgc 16980cgggactggg cgtgcagacc gtggacgttc agatacccac taccagtagc accagtattg 17040ccaccgccac agagggcatg gagacacaaa cgtccccggt tgcctcagcg gtggcggatg 17100ccgcggtgca ggcggtcgct gcggccgcgt ccaagacctc tacggaggtg caaacggacc 17160cgtggatgtt tcgcgtttca gccccccggc gcccgcgccg ttcgaggaag tacggcgccg 17220ccagcgcgct actgcccgaa tatgccctac atccttccat tgcgcctacc cccggctatc 17280gtggctacac ctaccgcccc agaagacgag caactacccg acgccgaacc accactggaa 17340cccgccgccg ccgtcgccgt cgccagcccg tgctggcccc gatttccgtg cgcagggtgg 17400ctcgcgaagg aggcaggacc ctggtgctgc caacagcgcg ctaccacccc agcatcgttt 17460aaaagccggt ctttgtggtt cttgcagata tggccctcac ctgccgcctc cgtttcccgg 17520tgccgggatt ccgaggaaga atgcaccgta ggaggggcat ggccggccac ggcctgacgg 17580gcggcatgcg tcgtgcgcac caccggcggc ggcgcgcgtc gcaccgtcgc atgcgcggcg 17640gtatcctgcc cctccttatt ccactgatcg ccgcggcgat tggcgccgtg cccggaattg 17700catccgtggc cttgcaggcg cagagacact gattaaaaac aagttgcatg tggaaaaatc 17760aaaataaaaa gtctggactc tcacgctcgc ttggtcctgt aactattttg tagaatggaa 17820gacatcaact ttgcgtctct ggccccgcga cacggctcgc gcccgttcat gggaaactgg 17880caagatatcg gcaccagcaa tatgagcggt ggcgccttca gctggggctc gctgtggagc 17940ggcattaaaa atttcggttc caccgttaag aactatggca gcaaggcctg gaacagcagc 18000acaggccaga tgctgaggga taagttgaaa gagcaaaatt tccaacaaaa ggtggtagat 18060ggcctggcct ctggcattag cggggtggtg gacctggcca accaggcagt gcaaaataag 18120attaacagta agcttgatcc ccgccctccc gtagaggagc ctccaccggc cgtggagaca 18180gtgtctccag aggggcgtgg cgaaaagcgt ccgcgccccg acagggaaga aactctggtg 18240acgcaaatag acgagcctcc ctcgtacgag gaggcactaa agcaaggcct gcccaccacc 18300cgtcccatcg cgcccatggc taccggagtg ctgggccagc acacacccgt aacgctggac 18360ctgcctcccc ccgccgacac ccagcagaaa cctgtgctgc caggcccgac cgccgttgtt 18420gtaacccgtc ctagccgcgc gtccctgcgc cgcgccgcca gcggtccgcg atcgttgcgg 18480cccgtagcca gtggcaactg gcaaagcaca ctgaacagca tcgtgggtct gggggtgcaa 18540tccctgaagc gccgacgatg cttctgatag ctaacgtgtc gtatgtgtgt catgtatgcg 18600tccatgtcgc cgccagagga gctgctgagc cgccgcgcgc ccgctttcca agatggctac 18660cccttcgatg atgccgcagt ggtcttacat gcacatctcg ggccaggacg cctcggagta 18720cctgagcccc gggctggtgc agtttgcccg cgccaccgag acgtacttca gcctgaataa 18780caagtttaga aaccccacgg tggcgcctac gcacgacgtg accacagacc ggtcccagcg 18840tttgacgctg cggttcatcc ctgtggaccg tgaggatact gcgtactcgt acaaggcgcg 18900gttcacccta gctgtgggtg ataaccgtgt gctggacatg gcttccacgt actttgacat 18960ccgcggcgtg ctggacaggg gccctacttt taagccctac tctggcactg cctacaacgc 19020cctggctccc aagggtgccc caaatccttg cgaatgggat gaagctgcta ctgctcttga 19080aataaaccta gaagaagagg acgatgacaa cgaagacgaa gtagacgagc aagctgagca 19140gcaaaaaact cacgtatttg ggcaggcgcc ttattctggt ataaatatta caaaggaggg 19200tattcaaata ggtgtcgaag gtcaaacacc taaatatgcc gataaaacat ttcaacctga 19260acctcaaata ggagaatctc agtggtacga aacagaaatt aatcatgcag ctgggagagt 19320cctaaaaaag actaccccaa tgaaaccatg ttacggttca tatgcaaaac ccacaaatga 19380aaatggaggg caaggcattc ttgtaaagca acaaaatgga aagctagaaa gtcaagtgga 19440aatgcaattt ttctcaacta ctgaggcagc cgcaggcaat ggtgataact tgactcctaa 19500agtggtattg tacagtgaag atgtagatat agaaacccca gacactcata tttcttacat 19560gcccactatt aaggaaggta actcacgaga actaatgggc caacaatcta tgcccaacag 19620gcctaattac attgctttta gggacaattt tattggtcta atgtattaca acagcacggg 19680taatatgggt gttctggcgg gccaagcatc gcagttgaat gctgttgtag atttgcaaga 19740cagaaacaca gagctttcat accagctttt gcttgattcc attggtgata gaaccaggta 19800cttttctatg tggaatcagg ctgttgacag ctatgatcca gatgttagaa ttattgaaaa 19860tcatggaact gaagatgaac ttccaaatta ctgctttcca ctgggaggtg tgattaatac 19920agagactctt accaaggtaa aacctaaaac aggtcaggaa aatggatggg aaaaagatgc 19980tacagaattt tcagataaaa atgaaataag agttggaaat aattttgcca tggaaatcaa 20040tctaaatgcc aacctgtgga gaaatttcct gtactccaac atagcgctgt atttgcccga 20100caagctaaag tacagtcctt ccaacgtaaa aatttctgat aacccaaaca cctacgacta 20160catgaacaag cgagtggtgg ctcccgggct agtggactgc tacattaacc ttggagcacg 20220ctggtccctt gactatatgg acaacgtcaa cccatttaac caccaccgca atgctggcct 20280gcgctaccgc tcaatgttgc tgggcaatgg tcgctatgtg cccttccaca tccaggtgcc 20340tcagaagttc tttgccatta aaaacctcct tctcctgccg ggctcataca cctacgagtg 20400gaacttcagg aaggatgtta acatggttct gcagagctcc ctaggaaatg acctaagggt 20460tgacggagcc agcattaagt ttgatagcat ttgcctttac gccaccttct tccccatggc 20520ccacaacacc gcctccacgc ttgaggccat gcttagaaac gacaccaacg accagtcctt 20580taacgactat ctctccgccg ccaacatgct ctaccctata cccgccaacg ctaccaacgt 20640gcccatatcc atcccctccc gcaactgggc ggctttccgc ggctgggcct tcacgcgcct 20700taagactaag gaaaccccat cactgggctc gggctacgac ccttattaca cctactctgg 20760ctctataccc tacctagatg gaacctttta cctcaaccac acctttaaga aggtggccat 20820tacctttgac tcttctgtca gctggcctgg caatgaccgc ctgcttaccc ccaacgagtt 20880tgaaattaag cgctcagttg acggggaggg ttacaacgtt gcccagtgta acatgaccaa 20940agactggttc ctggtacaaa tgctagctaa ctataacatt ggctaccagg gcttctatat 21000cccagagagc tacaaggacc gcatgtactc cttctttaga aacttccagc ccatgagccg 21060tcaggtggtg gatgatacta aatacaagga ctaccaacag gtgggcatcc tacaccaaca 21120caacaactct ggatttgttg gctaccttgc ccccaccatg cgcgaaggac aggcctaccc 21180tgctaacttc ccctatccgc ttataggcaa gaccgcagtt gacagcatta cccagaaaaa 21240gtttctttgc gatcgcaccc tttggcgcat cccattctcc agtaacttta tgtccatggg 21300cgcactcaca gacctgggcc aaaaccttct ctacgccaac tccgcccacg cgctagacat 21360gacttttgag gtggatccca tggacgagcc cacccttctt tatgttttgt ttgaagtctt 21420tgacgtggtc cgtgtgcacc agccgcaccg cggcgtcatc gaaaccgtgt acctgcgcac 21480gcccttctcg gccggcaacg ccacaacata aagaagcaag caacatcaac aacagctgcc 21540gccatgggct ccagtgagca ggaactgaaa gccattgtca aagatcttgg ttgtgggcca 21600tattttttgg gcacctatga caagcgcttt ccaggctttg tttctccaca caagctcgcc 21660tgcgccatag tcaatacggc cggtcgcgag actgggggcg tacactggat ggcctttgcc 21720tggaacccgc actcaaaaac atgctacctc tttgagccct ttggcttttc tgaccagcga 21780ctcaagcagg tttaccagtt tgagtacgag tcactcctgc gccgtagcgc cattgcttct 21840tcccccgacc gctgtataac gctggaaaag tccacccaaa gcgtacaggg gcccaactcg 21900gccgcctgtg gactattctg ctgcatgttt ctccacgcct ttgccaactg gccccaaact 21960cccatggatc acaaccccac catgaacctt attaccgggg tacccaactc catgctcaac 22020agtccccagg tacagcccac cctgcgtcgc aaccaggaac agctctacag cttcctggag 22080cgccactcgc cctacttccg cagccacagt gcgcagatta ggagcgccac ttctttttgt 22140cacttgaaaa acatgtaaaa ataatgtact agagacactt tcaataaagg caaatgcttt 22200tatttgtaca ctctcgggtg attatttacc cccacccttg ccgtctgcgc cgtttaaaaa 22260tcaaaggggt tctgccgcgc atcgctatgc gccactggca gggacacgtt gcgatactgg 22320tgtttagtgc tccacttaaa ctcaggcaca accatccgcg gcagctcggt gaagttttca 22380ctccacaggc tgcgcaccat caccaacgcg tttagcaggt cgggcgccga tatcttgaag 22440tcgcagttgg ggcctccgcc ctgcgcgcgc gagttgcgat acacagggtt gcagcactgg 22500aacactatca gcgccgggtg gtgcacgctg gccagcacgc tcttgtcgga gatcagatcc 22560gcgtccaggt cctccgcgtt gctcagggcg aacggagtca actttggtag ctgccttccc 22620aaaaagggcg cgtgcccagg ctttgagttg cactcgcacc gtagtggcat caaaaggtga 22680ccgtgcccgg tctgggcgtt aggatacagc gcctgcataa aagccttgat ctgcttaaaa 22740gccacctgag cctttgcgcc ttcagagaag aacatgccgc aagacttgcc ggaaaactga 22800ttggccggac aggccgcgtc gtgcacgcag caccttgcgt cggtgttgga gatctgcacc 22860acatttcggc cccaccggtt cttcacgatc ttggccttgc tagactgctc cttcagcgcg 22920cgctgcccgt tttcgctcgt cacatccatt tcaatcacgt gctccttatt tatcataatg 22980cttccgtgta gacacttaag ctcgccttcg atctcagcgc agcggtgcag ccacaacgcg 23040cagcccgtgg gctcgtgatg cttgtaggtc acctctgcaa acgactgcag gtacgcctgc 23100aggaatcgcc ccatcatcgt cacaaaggtc ttgttgctgg tgaaggtcag ctgcaacccg 23160cggtgctcct cgttcagcca ggtcttgcat acggccgcca gagcttccac ttggtcaggc 23220agtagtttga agttcgcctt tagatcgtta tccacgtggt acttgtccat cagcgcgcgc 23280gcagcctcca tgcccttctc ccacgcagac acgatcggca cactcagcgg gttcatcacc 23340gtaatttcac tttccgcttc gctgggctct tcctcttcct cttgcgtccg cataccacgc 23400gccactgggt cgtcttcatt cagccgccgc actgtgcgct tacctccttt gccatgcttg 23460attagcaccg gtgggttgct gaaacccacc atttgtagcg ccacatcttc tctttcttcc 23520tcgctgtcca cgattacctc tggtgatggc gggcgctcgg gcttgggaga agggcgcttc 23580tttttcttct tgggcgcaat ggccaaatcc gccgccgagg tcgatggccg cgggctgggt 23640gtgcgcggca ccagcgcgtc ttgtgatgag tcttcctcgt cctcggactc gatacgccgc 23700ctcatccgct tttttggggg cgcccgggga ggcggcggcg acggggacgg ggacgacacg 23760tcctccatgg ttgggggacg tcgcgccgca ccgcgtccgc gctcgggggt ggtttcgcgc 23820tgctcctctt cccgactggc catttccttc tcctataggc agaaaaagat catggagtca 23880gtcgagaaga aggacagcct aaccgccccc tctgagttcg ccaccaccgc ctccaccgat 23940gccgccaacg cgcctaccac cttccccgtc gaggcacccc cgcttgagga ggaggaagtg 24000attatcgagc aggacccagg ttttgtaagc gaagacgacg aggaccgctc agtaccaaca 24060gaggataaaa agcaagacca ggacaacgca gaggcaaacg aggaacaagt cgggcggggg 24120gacgaaaggc atggcgacta cctagatgtg ggagacgacg tgctgttgaa gcatctgcag 24180cgccagtgcg ccattatctg cgacgcgttg caagagcgca gcgatgtgcc cctcgccata 24240gcggatgtca gccttgccta cgaacgccac ctattctcac cgcgcgtacc ccccaaacgc 24300caagaaaacg gcacatgcga gcccaacccg cgcctcaact tctaccccgt atttgccgtg 24360ccagaggtgc ttgccaccta tcacatcttt ttccaaaact gcaagatacc cctatcctgc 24420cgtgccaacc gcagccgagc ggacaagcag ctggccttgc ggcagggcgc tgtcatacct 24480gatatcgcct cgctcaacga agtgccaaaa atctttgagg gtcttggacg cgacgagaag 24540cgcgcggcaa acgctctgca acaggaaaac agcgaaaatg aaagtcactc tggagtgttg 24600gtggaactcg agggtgacaa cgcgcgccta gccgtactaa aacgcagcat cgaggtcacc 24660cactttgcct acccggcact taacctaccc cccaaggtca tgagcacagt catgagtgag 24720ctgatcgtgc gccgtgcgca gcccctggag agggatgcaa atttgcaaga acaaacagag 24780gagggcctac ccgcagttgg cgacgagcag ctagcgcgct ggcttcaaac gcgcgagcct 24840gccgacttgg aggagcgacg caaactaatg atggccgcag tgctcgttac cgtggagctt 24900gagtgcatgc agcggttctt tgctgacccg gagatgcagc gcaagctaga ggaaacattg 24960cactacacct ttcgacaggg ctacgtacgc caggcctgca agatctccaa cgtggagctc 25020tgcaacctgg tctcctacct tggaattttg cacgaaaacc gccttgggca aaacgtgctt 25080cattccacgc tcaagggcga ggcgcgccgc gactacgtcc gcgactgcgt ttacttattt 25140ctatgctaca cctggcagac ggccatgggc gtttggcagc agtgcttgga ggagtgcaac 25200ctcaaggagc tgcagaaact gctaaagcaa aacttgaagg acctatggac ggccttcaac 25260gagcgctccg tggccgcgca cctggcggac atcattttcc ccgaacgcct gcttaaaacc 25320ctgcaacagg gtctgccaga cttcaccagt caaagcatgt tgcagaactt taggaacttt 25380atcctagagc gctcaggaat cttgcccgcc acctgctgtg cacttcctag cgactttgtg 25440cccattaagt accgcgaatg ccctccgccg ctttggggcc actgctacct tctgcagcta 25500gccaactacc ttgcctacca ctctgacata atggaagacg tgagcggtga cggtctactg 25560gagtgtcact gtcgctgcaa cctatgcacc ccgcaccgct ccctggtttg caattcgcag 25620ctgcttaacg aaagtcaaat tatcggtacc tttgagctgc agggtccctc gcctgacgaa 25680aagtccgcgg ctccggggtt gaaactcact ccggggctgt ggacgtcggc ttaccttcgc 25740aaatttgtac ctgaggacta ccacgcccac gagattaggt tctacgaaga ccaatcccgc 25800ccgcctaatg cggagcttac cgcctgcgtc attacccagg gccacattct tggccaattg 25860caagccatca acaaagcccg ccaagagttt ctgctacgaa agggacgggg ggtttacttg 25920gacccccagt ccggcgagga gctcaaccca atccccccgc cgccgcagcc ctatcagcag 25980cagccgcggg cccttgcttc ccaggatggc acccaaaaag aagctgcagc tgccgccgcc 26040acccacggac gaggaggaat actgggacag tcaggcagag gaggttttgg acgaggagga 26100ggaggacatg atggaagact gggagagcct agacgaggaa gcttccgagg tcgaagaggt 26160gtcagacgaa acaccgtcac cctcggtcgc attcccctcg ccggcgcccc agaaatcggc 26220aaccggttcc agcatggcta caacctccgc tcctcaggcg ccgccggcac tgcccgttcg 26280ccgacccaac cgtagatggg acaccactgg aaccagggcc ggtaagtcca agcagccgcc 26340gccgttagcc caagagcaac aacagcgcca aggctaccgc tcatggcgcg ggcacaagaa 26400cgccatagtt gcttgcttgc aagactgtgg gggcaacatc tccttcgccc gccgctttct 26460tctctaccat cacggcgtgg ccttcccccg taacatcctg cattactacc gtcatctcta 26520cagcccatac tgcaccggcg gcagcggcag caacagcagc ggccacacag aagcaaaggc 26580gaccggatag caagactctg acaaagccca agaaatccac agcggcggca gcagcaggag 26640gaggagcgct gcgtctggcg cccaacgaac ccgtatcgac ccgcgagctt agaaacagga 26700tttttcccac tctgtatgct atatttcaac agagcagggg ccaagaacaa gagctgaaaa 26760taaaaaacag gtctctgcga tccctcaccc gcagctgcct gtatcacaaa agcgaagatc 26820agcttcggcg cacgctggaa gacgcggagg ctctcttcag taaatactgc gcgctgactc 26880ttaaggacta gtttcgcgcc ctttctcaaa tttaagcgcg aaaactacgt catctccagc 26940ggccacaccc ggcgccagca cctgttgtca gcgccattat gagcaaggaa attcccacgc 27000cctacatgtg gagttaccag ccacaaatgg gacttgcggc tggagctgcc caagactact 27060caacccgaat aaactacatg agcgcgggac cccacatgat atcccgggtc aacggaatac 27120gcgcccaccg aaaccgaatt ctcctggaac aggcggctat taccaccaca cctcgtaata 27180accttaatcc ccgtagttgg cccgctgccc tggtgtacca ggaaagtccc gctcccacca

27240ctgtggtact tcccagagac gcccaggccg aagttcagat gactaactca ggggcgcagc 27300ttgcgggcgg ctttcgtcac agggtgcggt cgcccgggca gggtataact cacctgacaa 27360tcagagggcg aggtattcag ctcaacgacg agtcggtgag ctcctcgctt ggtctccgtc 27420cggacgggac atttcagatc ggcggcgccg gccgctcttc attcacgcct cgtcaggcaa 27480tcctaactct gcagacctcg tcctctgagc cgcgctctgg aggcattgga actctgcaat 27540ttattgagga gtttgtgcca tcggtctact ttaacccctt ctcgggacct cccggccact 27600atccggatca atttattcct aactttgacg cggtaaagga ctcggcggac ggctacgact 27660gaatgttaag tggagaggca gagcaactgc gcctgaaaca cctggtccac tgtcgccgcc 27720acaagtgctt tgcccgcgac tccggtgagt tttgctactt tgaattgccc gaggatcata 27780tcgagggccc ggcgcacggc gtccggctta ccgcccaggg agagcttgcc cgtagcctga 27840ttcgggagtt tacccagcgc cccctgctag ttgagcggga caggggaccc tgtgttctca 27900ctgtgatttg caactgtcct aaccctggat tacatcaaga tctttgttgc catctctgtg 27960ctgagtataa taaatacaga aattaaaata tactggggct cctatcgcca tcctgtaaac 28020gccaccgtct tcacccgccc aagcaaacca aggcgaacct tacctggtac ttttaacatc 28080tctccctctg tgatttacaa cagtttcaac ccagacggag tgagtctacg agagaacctc 28140tccgagctca gctactccat cagaaaaaac accaccctcc ttacctgccg ggaacgtacg 28200agtgcgtcac cggccgctgc accacaccta ccgcctgacc gtaaaccaga ctttttccgg 28260acagacctca ataactctgt ttaccagaac aggaggtgag cttagaaaac ccttagggta 28320ttaggccaaa ggcgcagcta ctgtggggtt tatgaacaat tcaagcaact ctacgggcta 28380ttctaattca ggtttctcta gaaatggacg gaattattac agagcagcgc ctgctagaaa 28440gacgcagggc agcggccgag caacagcgca tgaatcaaga gctccaagac atggttaact 28500tgcaccagtg caaaaggggt atcttttgtc tggtaaagca ggccaaagtc acctacgaca 28560gtaataccac cggacaccgc cttagctaca agttgccaac caagcgtcag aaattggtgg 28620tcatggtggg agaaaagccc attaccataa ctcagcactc ggtagaaacc gaaggctgca 28680ttcactcacc ttgtcaagga cctgaggatc tctgcaccct tattaagacc ctgtgcggtc 28740tcaaagatct tattcccttt aactaataaa aaaaaataat aaagcatcac ttacttaaaa 28800tcagttagca aatttctgtc cagtttattc agcagcacct ccttgccctc ctcccagctc 28860tggtattgca gcttcctcct ggctgcaaac tttctccaca atctaaatgg aatgtcagtt 28920tcctcctgtt cctgtccatc cgcacccact atcttcatgt tgttgcagat gaagcgcgca 28980agaccgtctg aagatacctt caaccccgtg tatccatatg acacggaaac cggtcctcca 29040actgtgcctt ttcttactcc tccctttgta tcccccaatg ggtttcaaga gagtccccct 29100ggggtactct ctttgcgcct atccgaacct ctagttacct ccaatggcat gcttgcgctc 29160aaaatgggca acggcctctc tctggacgag gccggcaacc ttacctccca aaatgtaacc 29220actgtgagcc cacctctcaa aaaaaccaag tcaaacataa acctggaaat atctgcaccc 29280ctcacagtta cctcagaagc cctaactgtg gctgccgccg cacctctaat ggtcgcgggc 29340aacacactca ccatgcaatc acaggccccg ctaaccgtgc acgactccaa acttagcatt 29400gccacccaag gacccctcac agtgtcagaa ggaaagctag ccctgcaaac atcaggcccc 29460ctcaccacca ccgatagcag tacccttact atcactgcct caccccctct aactactgcc 29520actggtagct tgggcattga cttgaaagag cccatttata cacaaaatgg aaaactagga 29580ctaaagtacg gggctccttt gcatgtaaca gacgacctaa acactttgac cgtagcaact 29640ggtccaggtg tgactattaa taatacttcc ttgcaaacta aagttactgg agccttgggt 29700tttgattcac aaggcaatat gcaacttaat gtagcaggag gactaaggat tgattctcaa 29760aacagacgcc ttatacttga tgttagttat ccgtttgatg ctcaaaacca actaaatcta 29820agactaggac agggccctct ttttataaac tcagcccaca acttggatat taactacaac 29880aaaggccttt acttgtttac agcttcaaac aattccaaaa agcttgaggt taacctaagc 29940actgccaagg ggttgatgtt tgacgctaca gccatagcca ttaatgcagg agatgggctt 30000gaatttggtt cacctaatgc accaaacaca aatcccctca aaacaaaaat tggccatggc 30060ctagaatttg attcaaacaa ggctatggtt cctaaactag gaactggcct tagttttgac 30120agcacaggtg ccattacagt aggaaacaaa aataatgata agctaacttt gtggaccaca 30180ccagctccat ctcctaactg tagactaaat gcagagaaag atgctaaact cactttggtc 30240ttaacaaaat gtggcagtca aatacttgct acagtttcag ttttggctgt taaaggcagt 30300ttggctccaa tatctggaac agttcaaagt gctcatctta ttataagatt tgacgaaaat 30360ggagtgctac taaacaattc cttcctggac ccagaatatt ggaactttag aaatggagat 30420cttactgaag gcacagccta tacaaacgct gttggattta tgcctaacct atcagcttat 30480ccaaaatctc acggtaaaac tgccaaaagt aacattgtca gtcaagttta cttaaacgga 30540gacaaaacta aacctgtaac actaaccatt acactaaacg gtacacagga aacaggagac 30600acaactccaa gtgcatactc tatgtcattt tcatgggact ggtctggcca caactacatt 30660aatgaaatat ttgccacatc ctcttacact ttttcataca ttgcccaaga ataaagaatc 30720gtttgtgtta tgtttcaacg tgtttatttt tcaattgccc gggatcggtg atcaccgatc 30780cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa 30840aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca 30900ataaacaagt tcccggatcg cgatccggcc cgaggctgta gccgacgatg gtgcgccagg 30960agagttgttg attcattgtt tgcctccctg ctgcggtttt tcaccgaagt tcatgccagt 31020ccagcgtttt tgcagcagaa aagccgccga cttcggtttg cggtcgcgag tgaagatccc 31080tttcttgtta ccgccaacgc gcaatatgcc ttgcgaggtc gcaaaatcgg cgaaattcca 31140tacctgttca ccgacgacgg cgctgacgcg atcaaagacg cggtgataca tatccagcca 31200tgcacactga tactcttcac tccacatgtc ggtgtacatt gagtgcagcc cggctaacgt 31260atccacgccg tattcggtga tgataatcgg ctgatgcagt ttctcctgcc aggccagaag 31320ttctttttcc agtaccttct ctgccgtttc caaatcgccg ctttggacat accatccgta 31380ataacggttc aggcacagca catcaaagag atcgctgatg gtatcggtgt gagcgtcgca 31440gaacattaca ttgacgcagg tgatcggacg cgtcgggtcg agtttacgcg ttgcttccgc 31500cagtggcgcg aaatattccc gtgcaccttg cggacgggta tccggttcgt tggcaatact 31560ccacatcacc acgcttgggt ggtttttgtc acgcgctatc agctctttaa tcgcctgtaa 31620gtgcgcttgc tgagtttccc cgttgactgc ctcttcgctg tacagttctt tcggcttgtt 31680gcccgcttcg aaaccaatgc ctaaagagag gttaaagccg acagcagcag tttcatcaat 31740caccacgatg ccatgttcat ctgcccagtc gagcatctct tcagcgtaag ggtaatgcga 31800ggtacggtag gagttggccc caatccagtc cattaatgcg tggtcgtgca ccatcagcac 31860gttatcgaat cctttgccac gcaagtccgc atcttcatga cgaccaaagc cagtaaagta 31920gaacggtttg tggttaatca ggaactgttc gcccttcact gccactgacc ggatgccgac 31980gcgaagcggg tagatatcac actctgtctg gcttttggct gtgacgcaca gttcatagag 32040ataaccttca cccggttgcc agaggtgcgg attcaccact tgcaaagtcc cgctagtgcc 32100ttgtccagtt gcaaccacct gttgatccgc atcacgcagt tcaacgctga catcaccatt 32160ggccaccacc tgccagtcaa cagacgcgtg gttacagtct tgcgcgacat gcgtcaccac 32220ggtgatatcg tccacccagg tgttcggcgt ggtgtagagc attacgctgc gatggattcc 32280ggcatagtta aagaaatcat ggaagtaaga ctgctttttc ttgccgtttt cgtcggtaat 32340caccattccc ggcgggatag tctgccagtt cagttcgttg ttcacacaaa cggtgatacg 32400tacacttttc ccggcaataa catacggcgt gacatcggct tcaaatggcg tatagccgcc 32460ctgatgctcc atcacttcct gattattgac ccacactttg ccgtaatgag tgaccgcatc 32520gaaacgcagc acgatacgct ggcctgccca acctttcggt ataaagactt cgcgctgata 32580ccagacgttg cccgcataat tacgaatatc tgcatcggcg aactgatcgt taaaactgcc 32640tggcacagca attgcccggc tttcttgtaa cgcgctttcc caccaacgct gatcaattcc 32700acagttttcg cgatccagac tgaatgccca caggccgtcg agttttttga tttcacgggt 32760tggggtttct acaggacgga ccatgcgttc gacctttctc ttcttttttg ggcccatgat 32820ggcagatccg tatagtgagt cgtattagct ggttctttcc gcctcagaag ccatagagcc 32880caccgcatcc ccagcatgcc tgctattgtc ttcccaatcc tcccccttgc tgtcctgccc 32940caccccaccc cccagaatag aatgacacct actcagacaa tgcgatgcaa tttcctcatt 33000ttattaggaa aggacagtgg gagtggcacc ttccagggtc aaggaaggca cgggggaggg 33060gcaaacaaca gatggctggc aactagaagg cacagtcgag gctgatcagc gagctctaga 33120tgcatgctcg agcggccgcc agtgtgatgg atatctgcag aattccagca cactggcggc 33180cgttactagt ggatccgagc tcggtacccg gccgttataa caccactcga cacggcacca 33240gctcaatcag tcacagtgta aaaaagggcc aagtgcagag cgagtatata taggactaaa 33300aaatgacgta acggttaaag tccacaaaaa acacccagaa aaccgcacgc gaacctacgc 33360ccagaaacga aagccaaaaa acccacaact tcctcaaatc gtcacttccg ttttcccacg 33420ttacgtcact tcccatttta agaaaactac aattcccaac acatacaagt tactccgccc 33480taaaacctac gtcacccgcc ccgttcccac gccccgcgcc acgtcacaaa ctccaccccc 33540tcattatcat attggcttca atccaaaata aggtatatta ttgatgatg 3358920500PRTHIV 20Met Gly Ala Arg Ala Ser Val Leu Ser Gly Gly Glu Leu Asp Arg Trp1 5 10 15Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Lys Leu Lys 20 25 30His Ile Val Trp Ala Ser Arg Glu Leu Glu Arg Phe Ala Val Asn Pro 35 40 45Gly Leu Leu Glu Thr Ser Glu Gly Cys Arg Gln Ile Leu Gly Gln Leu 50 55 60Gln Pro Ser Leu Gln Thr Gly Ser Glu Glu Leu Arg Ser Leu Tyr Asn65 70 75 80Thr Val Ala Thr Leu Tyr Cys Val His Gln Arg Ile Glu Ile Lys Asp 85 90 95Thr Lys Glu Ala Leu Asp Lys Ile Glu Glu Glu Gln Asn Lys Ser Lys 100 105 110Lys Lys Ala Gln Gln Ala Ala Ala Asp Thr Gly His Ser Asn Gln Val 115 120 125Ser Gln Asn Tyr Pro Ile Val Gln Asn Ile Gln Gly Gln Met Val His 130 135 140Gln Ala Ile Ser Pro Arg Thr Leu Asn Ala Trp Val Lys Val Val Glu145 150 155 160Glu Lys Ala Phe Ser Pro Glu Val Ile Pro Met Phe Ser Ala Leu Ser 165 170 175Glu Gly Ala Thr Pro Gln Asp Leu Asn Thr Met Leu Asn Thr Val Gly 180 185 190Gly His Gln Ala Ala Met Gln Met Leu Lys Glu Thr Ile Asn Glu Glu 195 200 205Ala Ala Glu Trp Asp Arg Val His Pro Val His Ala Gly Pro Ile Ala 210 215 220Pro Gly Gln Met Arg Glu Pro Arg Gly Ser Asp Ile Ala Gly Thr Thr225 230 235 240Ser Thr Leu Gln Glu Gln Ile Gly Trp Met Thr Asn Asn Pro Pro Ile 245 250 255Pro Val Gly Glu Ile Tyr Lys Arg Trp Ile Ile Leu Gly Leu Asn Lys 260 265 270Ile Val Arg Met Tyr Ser Pro Thr Ser Ile Leu Asp Ile Arg Gln Gly 275 280 285Pro Lys Glu Pro Phe Arg Asp Tyr Val Asp Arg Phe Tyr Lys Thr Leu 290 295 300Arg Ala Glu Gln Ala Ser Gln Glu Val Lys Asn Trp Met Thr Glu Thr305 310 315 320Leu Leu Val Gln Asn Ala Asn Pro Asp Cys Lys Thr Ile Leu Lys Ala 325 330 335Leu Gly Pro Ala Ala Thr Leu Glu Glu Met Met Thr Ala Cys Gln Gly 340 345 350Val Gly Gly Pro Gly His Lys Ala Arg Val Leu Ala Glu Ala Met Ser 355 360 365Gln Val Thr Asn Ser Ala Thr Ile Met Met Gln Arg Gly Asn Phe Arg 370 375 380Asn Gln Arg Lys Ile Val Lys Cys Phe Asn Cys Gly Lys Glu Gly His385 390 395 400Thr Ala Arg Asn Cys Arg Ala Pro Arg Lys Lys Gly Cys Trp Lys Cys 405 410 415Gly Lys Glu Gly His Gln Met Lys Asp Cys Thr Glu Arg Gln Ala Asn 420 425 430Phe Leu Gly Lys Ile Trp Pro Ser His Lys Gly Arg Pro Gly Asn Phe 435 440 445Leu Gln Ser Arg Pro Glu Pro Thr Ala Pro Pro Glu Glu Ser Phe Arg 450 455 460Phe Gly Glu Glu Thr Thr Thr Pro Ser Gln Lys Gln Glu Pro Ile Asp465 470 475 480Lys Glu Leu Tyr Pro Leu Ala Ser Leu Arg Ser Leu Phe Gly Ser Asp 485 490 495Pro Ser Ser Gln 500211002PRTHIV 21Met Arg Glu Asp Leu Ala Phe Pro Gln Gly Lys Ala Arg Glu Phe Ser1 5 10 15Ser Glu Gln Thr Arg Ala Asn Ser Pro Thr Arg Arg Glu Leu Gln Val 20 25 30Trp Gly Arg Asp Asn Asn Ser Leu Ser Glu Ala Gly Ala Asp Arg Gln 35 40 45Gly Thr Val Ser Phe Ser Phe Pro Gln Ile Thr Leu Trp Gln Arg Pro 50 55 60Leu Val Thr Ile Lys Ile Gly Gly Gln Leu Lys Glu Ala Leu Leu Asp65 70 75 80Thr Gly Ala Asp Asp Thr Val Leu Glu Glu Met Asn Leu Pro Gly Arg 85 90 95Trp Lys Pro Lys Met Ile Gly Gly Ile Gly Gly Phe Ile Lys Val Gly 100 105 110Gln Tyr Asp Gln Ile Leu Ile Glu Ile Cys Gly His Lys Ala Ile Gly 115 120 125Thr Val Leu Val Gly Pro Thr Pro Val Asn Ile Ile Gly Arg Asn Leu 130 135 140Leu Thr Gln Ile Gly Cys Thr Leu Asn Phe Pro Ile Ser Pro Ile Glu145 150 155 160Thr Val Pro Val Lys Leu Lys Pro Gly Met Asp Gly Pro Lys Val Lys 165 170 175Gln Trp Pro Leu Thr Glu Glu Lys Ile Lys Ala Leu Val Glu Ile Cys 180 185 190Thr Glu Met Glu Lys Glu Gly Lys Ile Ser Lys Ile Gly Pro Glu Asn 195 200 205Pro Tyr Asn Thr Pro Val Phe Ala Ile Lys Lys Lys Asp Ser Thr Lys 210 215 220Trp Arg Lys Leu Val Asp Phe Arg Glu Leu Asn Lys Arg Thr Gln Asp225 230 235 240Phe Trp Glu Val Gln Leu Gly Ile Pro His Pro Ala Gly Leu Lys Gln 245 250 255Lys Lys Ser Val Thr Val Leu Asp Val Gly Asp Ala Tyr Phe Ser Val 260 265 270Pro Leu Asp Lys Asp Phe Arg Lys Tyr Thr Ala Phe Thr Ile Pro Ser 275 280 285Ile Asn Asn Glu Thr Pro Gly Ile Arg Tyr Gln Tyr Asn Val Leu Pro 290 295 300Gln Gly Trp Lys Gly Ser Pro Ala Ile Phe Gln Cys Ser Met Thr Lys305 310 315 320Ile Leu Glu Pro Phe Arg Lys Gln Asn Pro Asp Ile Val Ile Tyr Gln 325 330 335Tyr Met Asp His Leu Tyr Val Gly Ser Asp Leu Glu Ile Gly Gln His 340 345 350Arg Thr Lys Ile Glu Glu Leu Arg Gln His Leu Leu Arg Trp Gly Phe 355 360 365Thr Thr Pro Asp Lys Lys His Gln Lys Glu Pro Pro Phe Leu Trp Met 370 375 380Gly Tyr Glu Leu His Pro Asp Lys Trp Thr Val Gln Pro Ile Val Leu385 390 395 400Pro Glu Lys Asp Ser Trp Thr Val Asn Asp Ile Gln Lys Leu Val Gly 405 410 415Lys Leu Asn Trp Ala Ser Gln Ile Tyr Ala Gly Ile Lys Val Arg Gln 420 425 430Leu Cys Lys Leu Leu Arg Gly Thr Lys Ala Leu Thr Glu Val Val Pro 435 440 445Leu Thr Glu Glu Ala Glu Leu Glu Leu Ala Glu Asn Arg Glu Ile Leu 450 455 460Lys Glu Pro Val His Gly Val Tyr Tyr Asp Pro Ser Lys Asp Leu Ile465 470 475 480Ala Glu Ile Gln Lys Gln Gly Gln Gly Gln Trp Thr Tyr Gln Ile Tyr 485 490 495Gln Glu Pro Phe Lys Asn Leu Lys Thr Gly Lys Tyr Ala Arg Met Lys 500 505 510Gly Ala His Thr Asn Asp Val Lys Gln Leu Thr Glu Ala Val Gln Lys 515 520 525Ile Ala Thr Glu Ser Ile Val Ile Trp Gly Lys Thr Pro Lys Phe Lys 530 535 540Leu Pro Ile Gln Lys Glu Thr Trp Glu Ala Trp Trp Thr Glu Tyr Trp545 550 555 560Gln Ala Thr Trp Ile Pro Glu Trp Glu Phe Val Asn Thr Pro Pro Leu 565 570 575Val Lys Leu Trp Tyr Gln Leu Glu Lys Glu Pro Ile Ile Gly Ala Glu 580 585 590Thr Phe Tyr Val Asp Gly Ala Ala Asn Arg Glu Thr Lys Leu Gly Lys 595 600 605Ala Gly Tyr Val Thr Asp Arg Gly Arg Gln Lys Val Val Pro Leu Thr 610 615 620Asp Thr Thr Asn Gln Lys Thr Glu Leu Gln Ala Ile His Leu Ala Leu625 630 635 640Gln Asp Ser Gly Leu Glu Val Asn Ile Val Thr Asp Ser Gln Tyr Ala 645 650 655Leu Gly Ile Ile Gln Ala Gln Pro Asp Lys Ser Glu Ser Glu Leu Val 660 665 670Ser Gln Ile Ile Glu Gln Leu Ile Lys Lys Glu Lys Val Tyr Leu Ala 675 680 685Trp Val Pro Ala His Lys Gly Ile Gly Gly Asn Glu Gln Val Asp Gly 690 695 700Leu Val Ser Ala Gly Ile Arg Lys Val Leu Phe Leu Asp Gly Ile Asp705 710 715 720Lys Ala Gln Glu Glu His Glu Lys Tyr His Ser Asn Trp Arg Ala Met 725 730 735Ala Ser Asp Phe Asn Leu Pro Pro Val Val Ala Lys Glu Ile Val Ala 740 745 750Ser Cys Asp Lys Cys Gln Leu Lys Gly Glu Ala Met His Gly Gln Val 755 760 765Asp Cys Ser Pro Gly Ile Trp Gln Leu Ala Cys Thr His Leu Glu Gly 770 775 780Lys Val Ile Leu Val Ala Val His Val Ala Ser Gly Tyr Ile Glu Ala785 790 795 800Glu Val Ile Pro Ala Glu Thr Gly Gln Glu Thr Ala Tyr Phe Leu Leu 805 810 815Lys Leu Ala Gly Arg Trp Pro Val Lys Thr Val His Thr Asp Asn Gly 820 825 830Ser Asn Phe Thr Ser Thr Thr Val Lys Ala Ala Cys Trp Trp Ala Gly 835 840 845Ile Lys Gln Glu Phe Gly Ile Pro Tyr Asn Pro Gln Ser Gln Gly Val 850 855 860Ile Glu Ser Met Asn Lys Glu Leu Lys Lys Ile Ile Gly Gln Val Arg865 870 875 880Asp Gln Ala Glu His Leu Lys Thr Ala Val Gln Met Ala Val Phe Ile 885 890 895His Asn Phe Lys Arg Lys Gly Gly Ile Gly Gly Tyr Ser Ala Gly Glu 900 905 910Arg Ile Val Asp Ile Ile Ala Thr Asp Ile Gln Thr Lys Glu Leu Gln 915

920 925Lys Gln Ile Thr Lys Ile Gln Asn Phe Arg Val Tyr Tyr Arg Asp Ser 930 935 940Arg Asp Pro Val Trp Lys Gly Pro Ala Lys Leu Leu Trp Lys Gly Glu945 950 955 960Gly Ala Val Val Ile Gln Asp Asn Ser Asp Ile Lys Val Val Pro Arg 965 970 975Arg Lys Ala Lys Ile Ile Arg Asp Tyr Gly Lys Gln Met Ala Gly Asp 980 985 990Asp Cys Val Ala Ser Arg Gln Asp Glu Asp 995 100022204PRTHIV 22Met Lys Trp Ser Lys Ser Ser Val Ile Gly Trp Pro Ala Val Arg Glu1 5 10 15Arg Met Arg Arg Ala Glu Pro Ala Ala Asp Gly Val Gly Ala Val Ser 20 25 30Arg Asp Leu Glu Lys His Gly Ala Ile Thr Ser Ser Asn Thr Ala Ala 35 40 45Asn Asn Ala Ala Cys Ala Trp Leu Glu Ala Gln Glu Glu Glu Glu Val 50 55 60Gly Phe Pro Val Thr Pro Gln Val Pro Leu Arg Pro Met Thr Tyr Lys65 70 75 80Ala Ala Val Asp Leu Ser His Phe Leu Lys Glu Lys Gly Gly Leu Glu 85 90 95Gly Leu Ile His Ser Gln Arg Arg Gln Asp Ile Leu Asp Leu Trp Ile 100 105 110Tyr His Thr Gln Gly Tyr Phe Pro Asp Trp Gln Asn Tyr Thr Pro Gly 115 120 125Pro Gly Val Arg Tyr Pro Leu Thr Phe Gly Trp Cys Tyr Lys Leu Val 130 135 140Pro Val Glu Pro Asp Lys Val Glu Glu Ala Asn Lys Gly Glu Asn Thr145 150 155 160Ser Leu Leu His Pro Val Ser Leu His Gly Met Asp Asp Pro Glu Arg 165 170 175Glu Val Leu Glu Trp Arg Phe Asp Ser Arg Leu Ala Phe His His Val 180 185 190Ala Arg Glu Leu His Pro Glu Tyr Phe Lys Asn Cys 195 20023626PRTHIV 23Met Arg Val Arg Gly Ile Gln Thr Ser Trp Gln Asn Leu Trp Arg Trp1 5 10 15Gly Thr Met Ile Leu Gly Met Leu Val Ile Tyr Ser Ala Ala Glu Asn 20 25 30Leu Trp Val Ala Val Tyr Tyr Gly Val Pro Val Trp Lys Asp Ala Glu 35 40 45Thr Thr Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Asp Thr Glu Val 50 55 60His Asn Val Trp Glu Thr His Ala Cys Val Pro Thr Asp Pro Asn Pro65 70 75 80Gln Glu Ile His Leu Glu Asn Val Thr Glu Asp Phe Asn Met Trp Arg 85 90 95Asn Asn Met Val Glu Gln Met His Thr Asp Ile Ile Ser Leu Trp Asp 100 105 110Gln Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu 115 120 125Asp Cys Asn Ala Thr Ala Ser Asn Val Thr Asn Glu Met Arg Asn Cys 130 135 140Ser Phe Asn Ile Thr Thr Glu Leu Lys Asp Lys Lys Gln Gln Val Tyr145 150 155 160Ser Leu Phe Tyr Lys Leu Asp Val Val Gln Ile Asn Glu Lys Asn Glu 165 170 175Thr Asp Lys Tyr Arg Leu Ile Asn Cys Asn Thr Ser Ala Ile Thr Gln 180 185 190Ala Cys Pro Lys Val Ser Phe Glu Pro Ile Pro Ile His Tyr Cys Ala 195 200 205Pro Ala Gly Phe Ala Ile Leu Lys Cys Lys Asp Thr Glu Phe Asn Gly 210 215 220Thr Gly Pro Cys Lys Asn Val Ser Thr Val Gln Cys Thr His Gly Ile225 230 235 240Arg Pro Val Ile Ser Thr Gln Leu Leu Leu Asn Gly Ser Leu Ala Glu 245 250 255Glu Gly Ile Gln Ile Arg Ser Glu Asn Ile Thr Asn Asn Ala Lys Thr 260 265 270Ile Ile Val Gln Leu Asp Lys Ala Val Lys Ile Asn Cys Thr Arg Pro 275 280 285Asn Asn Asn Thr Arg Lys Gly Val Arg Ile Gly Pro Gly Gln Ala Phe 290 295 300Tyr Ala Thr Gly Gly Ile Ile Gly Asp Ile Arg Gln Ala His Cys His305 310 315 320Val Ser Arg Ala Lys Trp Asn Asp Thr Leu Arg Gly Val Ala Lys Lys 325 330 335Leu Arg Glu His Phe Lys Asn Lys Thr Ile Ile Phe Glu Lys Ser Ser 340 345 350Gly Gly Asp Ile Glu Ile Thr Thr His Ser Phe Ile Cys Gly Gly Glu 355 360 365Phe Phe Tyr Cys Asn Thr Ser Gly Leu Phe Asn Ser Thr Trp Glu Ser 370 375 380Asn Ser Thr Glu Ser Asn Asn Thr Thr Ser Asn Asp Thr Ile Thr Leu385 390 395 400Thr Cys Arg Ile Lys Gln Ile Ile Asn Met Trp Gln Lys Val Gly Gln 405 410 415Ala Met Tyr Pro Pro Pro Ile Gln Gly Val Ile Arg Cys Glu Ser Asn 420 425 430Ile Thr Gly Leu Leu Leu Thr Arg Asp Gly Gly Asn Asn Ser Thr Asn 435 440 445Glu Ile Phe Arg Pro Gly Gly Gly Asn Met Arg Asp Asn Trp Arg Ser 450 455 460Glu Leu Tyr Lys Tyr Lys Val Val Lys Ile Glu Pro Leu Gly Val Ala465 470 475 480Pro Ser Arg Ala Lys Leu Thr Ala Gln Ala Arg Gln Leu Leu Ser Gly 485 490 495Ile Val Gln Gln Gln Ser Asn Leu Leu Arg Ala Ile Glu Ala Gln Gln 500 505 510His Met Leu Lys Leu Thr Val Trp Gly Ile Lys Gln Leu Gln Ala Arg 515 520 525Val Leu Ala Val Glu Arg Tyr Leu Lys Asp Gln Gln Leu Glu Ile Trp 530 535 540Asp Asn Met Thr Trp Leu Gln Trp Asp Lys Glu Ile Ser Asn Tyr Thr545 550 555 560Gln Ile Ile Tyr Asn Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys 565 570 575Asn Glu Gln Asp Leu Leu Ala Leu Asp Lys Trp Ala Ser Leu Trp Asn 580 585 590Trp Phe Asp Ile Ser Arg Trp Leu Trp Tyr Ile Lys Ile Phe Ile Met 595 600 605Ile Val Gly Gly Leu Ile Gly Leu Arg Ile Val Phe Ala Val Leu Ser 610 615 620Val Ile62524642PRTHIV 24Met Arg Val Lys Glu Lys Tyr Gln His Leu Trp Arg Trp Gly Trp Arg1 5 10 15Trp Gly Thr Met Leu Leu Gly Met Leu Met Ile Cys Ser Ala Thr Glu 20 25 30Lys Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Lys Glu Ala 35 40 45Thr Thr Thr Leu Leu Cys Ala Ser Asp Ala Lys Ala Tyr Asp Thr Glu 50 55 60Val His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro Asn65 70 75 80Pro Gln Glu Val Val Leu Val Asn Val Thr Glu Asn Phe Asp Met Trp 85 90 95Lys Asn Asp Met Val Glu Gln Met His Glu Asp Ile Ile Ser Leu Trp 100 105 110Asp Gln Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Ser 115 120 125Leu Lys Cys Thr Asp Leu Lys Asn Asp Thr Asn Thr Asn Ser Ser Ser 130 135 140Gly Arg Met Ile Met Glu Lys Gly Glu Ile Lys Asn Cys Ser Phe Asn145 150 155 160Ile Ser Thr Ser Ile Arg Gly Lys Val Gln Lys Glu Tyr Ala Phe Phe 165 170 175Tyr Lys Leu Asp Ile Ile Pro Ile Asp Asn Asp Thr Thr Ser Tyr Ser 180 185 190Leu Thr Ser Cys Asn Thr Ser Val Ile Thr Gln Ala Cys Pro Lys Val 195 200 205Ser Phe Glu Pro Ile Pro Asn His Tyr Cys Ala Pro Ala Gly Phe Ala 210 215 220Ile Leu Lys Cys Lys Asp Lys Lys Phe Asn Gly Lys Gly Pro Cys Thr225 230 235 240Asn Val Ser Thr Val Gln Cys Thr His Gly Ile Arg Pro Val Val Ser 245 250 255Thr Gln Leu Leu Val Thr Gly Asn Leu Ala Glu Glu Glu Val Val Ile 260 265 270Arg Ser Ala Asn Phe Ala Asp Asn Ala Lys Val Ile Ile Val Gln Leu 275 280 285Asn Glu Ser Val Glu Ile Asn Cys Thr Arg Pro Asn Asn Asn Thr Arg 290 295 300Lys Ser Ile His Ile Gly Pro Gly Arg Ala Phe Tyr Thr Thr Gly Glu305 310 315 320Ile Ile Gly Asp Ile Arg Gln Ala His Cys Asn Leu Ser Arg Ala Lys 325 330 335Trp Asn Asp Thr Leu Asn Lys Ile Val Ile Lys Leu Arg Glu Gln Phe 340 345 350Gly Asn Lys Thr Ile Val Phe Lys His Ser Ser Gly Gly Asp Pro Glu 355 360 365Ile Val Thr His Ser Phe Asn Cys Gly Gly Glu Phe Phe Tyr Cys Asn 370 375 380Ser Thr Gln Leu Phe Asn Ser Thr Trp Phe Asn Ser Thr Trp Ser Thr385 390 395 400Glu Gly Ser Asn Asn Thr Glu Gly Ser Asp Thr Ile Thr Leu Pro Cys 405 410 415Arg Ile Lys Gln Ile Ile Asn Met Trp Gln Lys Val Gly Lys Ala Met 420 425 430Tyr Ala Pro Pro Ile Ser Gly Gln Ile Arg Cys Ser Ser Asn Ile Thr 435 440 445Gly Leu Leu Leu Thr Arg Asp Gly Gly Asn Ser Asn Asn Glu Ser Glu 450 455 460Ile Phe Arg Leu Gly Gly Gly Asp Met Arg Asp Asn Trp Arg Ser Glu465 470 475 480Leu Tyr Lys Tyr Lys Val Val Lys Ile Glu Pro Leu Gly Val Ala Pro 485 490 495Thr Lys Ala Lys Leu Thr Val Gln Ala Arg Gln Leu Leu Ser Gly Ile 500 505 510Val Gln Gln Gln Asn Asn Leu Leu Arg Ala Ile Glu Ala Gln Gln His 515 520 525Leu Leu Gln Leu Thr Val Trp Gly Ile Lys Gln Leu Gln Ala Arg Thr 530 535 540Leu Ala Val Glu Arg Tyr Leu Lys Asp Gln Gln Leu Leu Glu Gln Ile545 550 555 560Trp Asn His Thr Thr Trp Met Glu Trp Asp Arg Glu Ile Asn Asn Tyr 565 570 575Thr Ser Leu Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln His Glu 580 585 590Lys Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu Trp 595 600 605Asn Trp Phe Asn Ile Thr Asn Trp Leu Trp Tyr Ile Lys Leu Phe Ile 610 615 620Met Ile Val Gly Gly Leu Val Gly Leu Arg Ile Val Phe Ala Val Leu625 630 635 640Ser Ile25626PRTHIV 25Met Arg Val Arg Gly Ile Pro Arg Asn Trp Pro Gln Trp Trp Met Trp1 5 10 15Gly Ile Leu Gly Phe Trp Met Ile Ile Ile Cys Arg Val Val Gly Asn 20 25 30Met Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Thr Asp Ala Lys 35 40 45Thr Thr Leu Phe Cys Ala Ser Asp Thr Lys Ala Tyr Asp Arg Glu Val 50 55 60His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro Asn Pro65 70 75 80Gln Glu Ile Val Leu Glu Asn Val Thr Glu Asn Phe Asn Met Trp Lys 85 90 95Asn Asp Met Val Asp Gln Met His Glu Asp Ile Ile Ser Leu Trp Asp 100 105 110Gln Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu 115 120 125His Cys Thr Asn Ala Thr Phe Lys Asn Asn Val Thr Asn Asp Met Asn 130 135 140Lys Glu Ile Arg Asn Cys Ser Phe Asn Thr Thr Thr Glu Ile Arg Asp145 150 155 160Lys Lys Gln Gln Gly Tyr Ala Leu Phe Tyr Arg Pro Asp Ile Val Leu 165 170 175Leu Lys Glu Asn Arg Asn Asn Ser Asn Asn Ser Glu Tyr Ile Leu Ile 180 185 190Asn Cys Asn Ala Ser Thr Ile Thr Gln Ala Cys Pro Lys Val Asn Phe 195 200 205Asp Pro Ile Pro Ile His Tyr Cys Ala Pro Ala Gly Tyr Ala Ile Leu 210 215 220Lys Cys Asn Asn Lys Thr Phe Ser Gly Lys Gly Pro Cys Asn Asn Val225 230 235 240Ser Thr Val Gln Cys Thr His Gly Ile Lys Pro Val Val Ser Thr Gln 245 250 255Leu Leu Leu Asn Gly Ser Leu Ala Glu Lys Glu Ile Ile Ile Arg Ser 260 265 270Glu Asn Leu Thr Asp Asn Val Lys Thr Ile Ile Val His Leu Asn Lys 275 280 285Ser Val Glu Ile Val Cys Thr Arg Pro Asn Asn Asn Thr Arg Lys Ser 290 295 300Met Arg Ile Gly Pro Gly Gln Thr Phe Tyr Ala Thr Gly Asp Ile Ile305 310 315 320Gly Asp Ile Arg Gln Ala Tyr Cys Asn Ile Ser Gly Ser Lys Trp Asn 325 330 335Glu Thr Leu Lys Arg Val Lys Glu Lys Leu Gln Glu Asn Tyr Asn Asn 340 345 350Asn Lys Thr Ile Lys Phe Ala Pro Ser Ser Gly Gly Asp Leu Glu Ile 355 360 365Thr Thr His Ser Phe Asn Cys Arg Gly Glu Phe Phe Tyr Cys Asn Thr 370 375 380Thr Arg Leu Phe Asn Asn Asn Ala Thr Glu Asp Glu Thr Ile Thr Leu385 390 395 400Pro Cys Arg Ile Lys Gln Ile Ile Asn Met Trp Gln Gly Val Gly Arg 405 410 415Ala Met Tyr Ala Pro Pro Ile Ala Gly Asn Ile Thr Cys Lys Ser Asn 420 425 430Ile Thr Gly Leu Leu Leu Val Arg Asp Gly Gly Glu Asp Asn Lys Thr 435 440 445Glu Glu Ile Phe Arg Pro Gly Gly Gly Asn Met Lys Asp Asn Trp Arg 450 455 460Ser Glu Leu Tyr Lys Tyr Lys Val Ile Glu Leu Lys Pro Leu Gly Ile465 470 475 480Ala Pro Thr Gly Ala Lys Leu Thr Val Gln Ala Arg Gln Leu Leu Ser 485 490 495Ser Ile Val Gln Gln Gln Ser Asn Leu Leu Arg Ala Ile Glu Ala Gln 500 505 510Gln His Met Leu Gln Leu Thr Val Trp Gly Ile Lys Gln Leu Gln Thr 515 520 525Arg Val Leu Ala Ile Glu Arg Tyr Leu Lys Asp Gln Gln Leu Glu Ile 530 535 540Trp Asn Asn Met Thr Trp Met Glu Trp Asp Arg Glu Ile Ser Asn Tyr545 550 555 560Thr Asp Thr Ile Tyr Arg Leu Leu Glu Asp Ser Gln Thr Gln Gln Glu 565 570 575Lys Asn Glu Lys Asp Leu Leu Ala Leu Asp Ser Trp Lys Asn Leu Trp 580 585 590Ser Trp Phe Asp Ile Ser Asn Trp Leu Trp Tyr Ile Lys Ile Phe Ile 595 600 605Met Ile Val Gly Gly Leu Ile Gly Leu Arg Ile Ile Phe Ala Val Leu 610 615 620Ser Ile62526973DNAArtificial sequenceCMV/R promoter 26ccattgcata cgttgtatcc atatcataat atgtacattt atattggctc atgtccaaca 60ttaccgccat gttgacattg attattgact agttattaat agtaatcaat tacggggtca 120ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct 180ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta 240acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac 300ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt 360aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag 420tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat 480gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat 540gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc 600ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcgt 660ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga 720caccgggacc gatccagcct ccatcggctc gcatctctcc ttcacgcgcc cgccgcccta 780cctgaggccg ccatccacgc cggttgagtc gcgttctgcc gcctcccgcc tgtggtgcct 840cctgaactgc gtccgccgtc taggtaagtt taaagctcag gtcgagaccg ggcctttgtc 900cggcgctccc ttggagccta cctagactca gccggctctc cacgctttgc ctgaccctgc 960ttgctcaact cta 973

Claims

1. A composition capable of eliciting an immune response against HIV in an immunocompetent subject, the composition comprising:a plurality of different nucleic acid constructs, each nucleic acid construct comprising a polynucleotide sequence encoding an HIV antigenic polypeptide operably linked to a CMV/R transcription control sequence,wherein the plurality of nucleic acid constructs encode antigenic polypeptides of a plurality of HIV clades or strains.

2. A composition capable of eliciting an immune response against HIV in an immunocompetent subject, the composition comprising:a plurality of different nucleic acid constructs, each nucleic acid construct comprising a polynucleotide sequence encoding a single HIV antigenic polypeptide, wherein the plurality of nucleic acid constructs encode antigenic polypeptides of a plurality of HIV clades or strains.

3. The composition of claim 1 or 2, wherein the plurality of nucleic acid constructs comprises:a first nucleic acid construct comprising a polynucleotide sequence that encodes an HIV Gag polypeptide;a second nucleic acid construct comprising a polynucleotide sequence that encodes an HIV Pol polypeptide;a third nucleic acid construct comprising a polynucleotide sequence that encodes an HIV Nef polypeptide; and,at least one additional nucleic acid construct comprising a polynucleotide sequence that encodes an HIV Env polypeptide.

4. The composition of claim in 3, further comprising a plurality of additional nucleic acid constructs, each of which additional nucleic acid constructs comprises a polynucleotide sequence encoding an Env polypeptide of a different HIV clade or strain.

5. The vaccine composition of claim 3, whereinthe first nucleic acid construct comprises a polynucleotide sequence that encodes a clade B Gag polypeptide;the second nucleic acid construct comprises a polynucleotide sequence that encodes a clade B Pol polypeptide;the third nucleic acid construct comprises a polynucleotide sequence that encodes a clade B Nef polypeptide.

6. The composition of claim 5, whereinthe first nucleic acid construct encodes a polypeptide with at least 95% sequence identity to SEQ ID NO:20;the second nucleic acid construct encodes a polypeptide with at least 95% sequence identity to SEQ ID NO:21; and/orthe third nucleic acid construct encodes a polypeptide with at least 95% sequence identity to SEQ ID NO:22.

7. The composition of claim 5, the composition further comprising a plurality of additional nucleic acid constructs, whereina first additional nucleic acid construct comprises a polynucleotide sequence that encodes a clade A Env polypeptide;a second additional nucleic acid construct comprises a polynucleotide sequence that encodes a clade B Env polypeptide; anda third additional nucleic acid construct comprises a polynucleotide sequence that encodes a clade C Env polypeptide.

8. The composition of claim 7, whereinthe first additional nucleic acid construct encodes a polypeptide with at least 95% sequence identity to SEQ ID NO:23;the second additional nucleic acid construct encodes a polypeptide with at least 95% sequence identity to SEQ ID NO:24; and/orthe third additional nucleic acid construct encodes a polypeptide with at least 95% sequence identity to SEQ ID NO:25.

9. The composition of claim 1, comprising at least one additional nucleic acid construct, which additional nucleic acid construct comprises a polynucleotide sequence encoding a chimeric Env polypeptide comprising at least a subsequence of a plurality of different HIV clades or strains.

10. The composition of claim 1 or 2, wherein the plurality of different nucleic acid constructs comprise:a first nucleic acid construct comprising a polynucleotide sequence encoding a Gag polypeptide;a second nucleic acid construct comprising a polynucleotide sequence encoding a Pol polypeptide;a third nucleic acid construct comprising a polynucleotide sequence encoding a Nef polypeptide;a fourth nucleic acid construct comprising a polynucleotide sequence encoding an Env polypeptide of clade A;a fifth nucleic acid construct comprising a polynucleotide sequence encoding an Env polypeptide of clade B; and,a sixth nucleic acid construct comprising a polynucleotide sequence encoding an Env polypeptide of clade C.

11. The composition of claim 10, wherein:the first nucleic acid construct comprises a polynucleotide sequence with at least 95% sequence identity to SEQ ID NO:1;the second nucleic acid construct comprises the polynucleotide sequence with at least 95% sequence identity to SEQ ID NO:2;the third nucleic acid construct comprises the polynucleotide sequence with at least 95% sequence identity to SEQ ID NO:3;the fourth nucleic acid construct comprises the polynucleotide sequence with at least 95% sequence identity to SEQ ID NO:4;the fifth nucleic acid construct comprises the polynucleotide sequence with at least 95% sequence identity to SEQ ID NO:5; and/or,the sixth nucleic acid construct comprises the polynucleotide sequence with at least 95% sequence identity to SEQ ID NO:6.

12. The composition of claim 11, wherein:the first nucleic acid construct comprises the polynucleotide sequence of SEQ ID NO:1;the second nucleic acid construct comprises the polynucleotide sequence of SEQ ID NO:2;the third nucleic acid construct comprises the polynucleotide sequence of SEQ ID NO:3;the fourth nucleic acid construct comprises the polynucleotide sequence of SEQ ID NO:4;the fifth nucleic acid construct comprises the polynucleotide sequence of SEQ ID NO:5; and,the sixth nucleic acid construct comprises the polynucleotide sequence of SEQ ID NO:6.

13. The composition of claim 10, comprising a substantially equal ratio by weight of each of the six nucleic acid constructs.

14. The composition of claim 1 or 2, wherein the composition is capable of eliciting a protective immune response against HIV in an immunocompetent human subject when administered alone or in combination with at least one additional immunogenic composition.

15. The composition of claim 1 or 2, wherein the plurality of different constructs comprises a plurality of plasmids.

16. The composition of claim 1 or 2, wherein the composition further comprises a pharmaceutically acceptable carrier.

17. The composition of claim 1 or 2, wherein the composition further comprises an adjuvant.

18. The composition of claim 1, wherein the CMV/R transcription control sequence comprises the polynucleotide sequence of SEQ ID NO:26.

19. A composition capable of eliciting an immune response against HIV in an immunocompetent subject, the composition comprising:a plurality of different nucleic acid constructs, each nucleic acid construct comprising a polynucleotide sequence encoding a single HIV antigen operably linked to a transcription control sequence.

20. A method of eliciting an immune response against HIV comprising administering the composition of claim 1, 2 or 19 to a human subject.

21. The method of claim 20, wherein the immune response is a protective immune response against multiple clades or strains of HIV.

22. The method of claim 20, comprising administering the plurality of nucleic acid constructs intramuscularly.

23. The method of claim 20, comprising administering the plurality of nucleic acid constructs using a needleless delivery device.

24. The method of claim 20, further comprising administering a plurality of viral vectors, wherein the plurality of viral vectors comprises at least one polynucleotide sequence encoding an HIV antigenic polypeptide, wherein the at least one polynucleotide encodes an antigenic polypeptide identical to a polypeptide encoded by a nucleic acid of the composition.

25. The method of claim 24, wherein the viral vectors comprise recombinant adenovirus vectors.

26. The method of claim 25, wherein the recombinant adenovirus vectors are replication deficient adenovirus vectors.

27. The method of claim 26, wherein the plurality of adenovirus vectors comprise:a first recombinant adenovirus vector comprising a polynucleotide sequence encoding an HIV Gag polypeptide and an HIV Pol polypeptide;a second recombinant adenovirus vector comprising a polynucleotide sequence encoding an Env polypeptide of clade A;a third recombinant adenovirus vector comprising a polynucleotide sequence encoding an Env polypeptide of clade B; and,a fourth recombinant adenovirus vector comprising a polynucleotide sequence encoding an Env polypeptide of clade C.

28. The method of claim 27, whereinthe first recombinant adenovirus vector comprises SEQ ID NO:16;the second recombinant adenovirus vector comprises SEQ ID NO:17;the third recombinant adenovirus vector comprises SEQ ID NO:18; andthe fourth recombinant adenovirus vector comprises SEQ ID NO:19.

29. An isolated or recombinant nucleic acid comprising a polynucleotide sequence encoding an HIV antigenic polypeptide operably linked to a CMV/R transcription regulatory sequence.

30. The nucleic acid of claim 29, wherein the nucleic acid comprises a plasmid.

31. The nucleic acid of claim 29, wherein the nucleic acid comprises a viral vector.

32. The nucleic acid of claim 29, 30 or 31, wherein the polynucleotide sequence encodes an HIV Gag polypeptide, an HIV Pol polypeptide, an HIV Nef polypeptide, or an HIV Env polypeptide.

33. The nucleic acid of claim 32, wherein the polynucleotide sequence encodes a chimeric HIV polypeptide comprising at least a subsequence of a plurality of HIV clades or strains.

34. The nucleic acid of claim 33, wherein the chimeric HIV polypeptide is an HIV Env polypeptide.

35. The nucleic acid of claim 29, 30 or 31, wherein the polynucleotide sequence is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6; SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ IID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15.

36. A polypeptide encoded by a polynucleotide sequence of claim 35.

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