Patent Publication Number: US-2003224513-A1

Title: Antisense modulation of Notch1 expression

Description:
FIELD OF THE INVENTION  
       [0001] The present invention provides compositions and methods for modulating the expression of Notch1. In particular, this invention relates to compounds, particularly oligonucleotides, specifically hybridizable with nucleic acids encoding Notch1. Such compounds have been shown to modulate the expression of Notch1.  
       BACKGROUND OF THE INVENTION  
       [0002] Intrinsic, cell-autonomous factors as well as non-autonomous, short-range and long-range signals guide cells through distinct developmental paths. An organism frequently uses the same signaling pathway within different cellular contexts to achieve unique developmental goals.  
       [0003] Notch signaling is an evolutionarily conserved mechanism used to control cell fates through local cell interactions. The gene encoding the original Notch receptor was discovered in  Drosophila melanogaster  due to the fact that partial loss of function of the gene results in notches at the wing margin (Artavanis-Tsakonas et al.,  Science,  1999, 284, 770-776). Signals transmitted through the Notch receptor, in combination with other cellular factors, influence differentiation, proliferation and apoptotic events at all stages of development (Artavanis-Tsakonas et al.,  Science,  1999, 284, 770-776).  
       [0004] Mature Notch proteins are heterodimeric receptors derived from the cleavage of Notch pre-proteins into an extracellular subunit containing multiple EGF-like repeats and a transmembrane subunit including the intracellular region (Blaumueller et al.,  Cell,  1997, 90, 281-291). Notch activation results from the binding of ligands expressed by neighboring cells or soluble ligands and signaling from activated Notch involves networks of transcription regulators (Artavanis-Tsakonas et al.,  Science,  1995, 268, 225-232).  
       [0005] In context of experimental cancer immunotherapy, the Notch signaling network is acquiring increasing importance for its possible roles in neoplastic cells and the immune system (Jang et al.,  Curr. Opin. Mol. Ther.,  2000, 2, 55-65).  
       [0006] Four mammalian Notch homologs have been identified and are designated Notch1, Notch2, Notch3 and Notch4. Human Notch1 (also known as Notch gene homolog 1 and TAN-1) was first identified in 1991 and later mapped to chromosome 9q34, a region associated with neoplasia-associated translocations (Ellisen et al.,  Cell,  1991, 66, 649-661; Larsson et al.,  Genomics,  1994, 24, 253-258). Larsson et al. predicted that the human Notch genes are proto-oncogenes and candidates for sites of chromosome breakage in neoplasia-associated translocations (Larsson et al.,  Genomics,  1994, 24, 253-258). Notch1 is expressed in many human tissues but is particularly abundant in lymphoid tissues (Ellisen et al.,  Cell,  1991, 66, 649-661).  
       [0007] An expressed sequence tag has been identified which represents a possible variant of Notch1, herein designated Notch1-B which starts in exon 27 and continues into intron 28.  
       [0008] Disclosed and claimed in U.S. Pat. No. 5,789,195 are nucleic acid sequences encoding Notch genes. Antibodies to human Notch proteins are additionally provided (Artavanis-Tsakonas et al., 1998). Amino acid sequences of Notch genes and antibodies against Notch proteins are also disclosed and claimed in U.S. Pat. No. 6,090,922 (Artavanis-Tsakonas et al., 2000).  
       [0009] Modulation of expression of Notch genes may prove to be a useful point for therapeutic intervention in developmental, hyperproliferative or autoimmune disorders or disorders arising from aberrant apoptosis.  
       [0010] Methods for producing allergen- or antigen-tolerant T-cells employing compositions capable of upregulating expression of an endogenous Notch protein are disclosed and claimed in PCT publication WO 00/36089 (Lamb et al., 2000).  
       [0011] Disclosed and claimed in U.S. Pat. No. 6,149,902 is a method for cell transplantation which includes contacting a precursor cell with an agonist of Notch1 function effective to inhibit differentiation of the cell wherein said agonist is a Delta protein, a Serrate protein or an antibody to a Notch protein (Artavanis-Tsakonas et al., 2000).  
       [0012] Disclosed in U.S. Pat. No. 6,083,904 and PCT publication WO 94/07474 are therapeutic and diagnostic methods and compositions based on Notch proteins and nucleic acids, wherein antisense methods are generally disclosed (Artavanis-Tsakonas, 2000; Artavanis-Tsakonas et al., 1994).  
       [0013] Disclosed and claimed in U.S. Pat. No. 5,786,158 are methods and compositions for the detection of malignancy or nervous system disorders based on the level of Notch proteins or nucleic acids (Artavanis-Tsakonas et al., 1998).  
       [0014] A Notch1 antisense transgenic mouse has been engineered and employed in investigations of regulation of NF-kappa-B activity by Notch1 (Cheng et al.,  J. Immunol.,  2001, 167, 4458-4467).  
       [0015] Transfections of antisense Notch1 RNA have been carried out in 3T3-L1 cells and in K562 erythroleukemic cells in investigations of the roles of Notch1 in adipogenesis, ras signaling and megakaryocytic differentiation (Garces et al.,  J. Biol. Chem.,  1997, 272, 29729-29734; Lam et al.,  J. Biol. Chem.,  2000, 275, 19676-19684; Ruiz-Hidalgo et al.,  Int. J. Oncol.,  1999, 14, 777-783).  
       [0016] A Notch1 antisense oligonucleotide has been employed in a study of the role of Notch1 in tumor necrosis factor-alpha-induced proliferation of human synoviocytes (Nakazawa et al.,  Arthritis Rheum.,  2001, 44, 1545-1554).  
       [0017] Austin et al. have investigated the role of Notch1 in development of retinal ganglion cells by employing Notch1 antisense phosphorothioate oligonucleotides designed against three distinct regions of the chicken Notch1 sequence: the EGF repeat region, the lin12/notch region and the cdc10/ankyrin repeat region (Austin et al., Development, 1995, 121, 3637-3650). These same oligonucleotides have subsequently been employed in further investigations of the role of Notch1 in retinal cell development and in auditory hair cell and neuronal differentiation (Faux et al.,  J. Neurosci.,  2001, 21, 5587-5596; Waid and McLoon,  Development,  1998, 125, 1059-1066; Zine et al.,  Development,  2000, 127, 3373-3383.).  
       [0018] Disclosed and claimed in PCT publication WO 01/25422 and Japanese Patent JP13122787 are antisense oligonucleotides directed to translational start codon and exon 28 of human Notch1, respectively (Bartelmez and Iversen, 2001; Nakajima and Nishioka, 2001).  
       [0019] Disclosed and claimed in PCT publication WO 00/20576 are methods for inducing differentiation and apoptosis in human cells that over express Notch proteins wherein Notch function is disrupted using antisense oligonucleotides that target the EGF repeat region, the lin/notch region and the ankyrin region (Miele et al., 2000). These same oligonucleotides have also been employed in an investigation of the role of Notch1 in murine erythroleukemia cell apoptosis (Shelly et al.,  J. Cell. Biochem.,  1999, 73, 164-175).  
       [0020] Currently, there are no known therapeutic agents that effectively inhibit the synthesis of Notch1. To date, investigative strategies aimed at modulating Notch1 expression have involved the use of antibodies and Notch-regulating proteins as well as antisense RNA and oligonucleotides. Consequently, there remains a long felt need for additional agents capable of effectively inhibiting Notch1 function.  
       [0021] Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of expression of Notch1.  
       [0022] The present invention provides compositions and methods for modulating expression of Notch1, including expression of variants of Notch1.  
       SUMMARY OF THE INVENTION  
       [0023] The present invention is directed to compounds, particularly antisense oligonucleotides, which are targeted to a nucleic acid encoding Notch1, and which modulate the expression of Notch1. Pharmaceutical and other compositions comprising the compounds of the invention are also provided. Further provided are methods of modulating the expression of Notch1 in cells or tissues comprising contacting said cells or tissues with one or more of the antisense compounds or compositions of the invention. Further provided are methods of treating an animal, particularly a human, suspected of having or being prone to a disease or condition associated with expression of Notch1 by administering a therapeutically or prophylactically effective amount of one or more of the antisense compounds or compositions of the invention.  
       DETAILED DESCRIPTION OF THE INVENTION  
       [0024] The present invention employs oligomeric compounds, particularly antisense oligonucleotides, for use in modulating the function of nucleic acid molecules encoding Notch1, ultimately modulating the amount of Notch1 produced. This is accomplished by providing antisense compounds which specifically hybridize with one or more nucleic acids encoding Notch1. As used herein, the terms “target nucleic acid” and “nucleic acid encoding Notch1” encompass DNA encoding Notch1, RNA (including pre-mRNA and mRNA) transcribed from such DNA, and also cDNA derived from such RNA. The specific hybridization of an oligomeric compound with its target nucleic acid interferes with the normal function of the nucleic acid. This modulation of function of a target nucleic acid by compounds which specifically hybridize to it is generally referred to as “antisense”. The functions of DNA to be interfered with include replication and transcription. The functions of RNA to be interfered with include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translocation of the RNA to sites within the cell which are distant from the site of RNA synthesis, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in or facilitated by the RNA. The overall effect of such interference with target nucleic acid function is modulation of the expression of Notch1. In the context of the present invention, “modulation” means either an increase (stimulation) or a decrease (inhibition) in the expression of a gene. In the context of the present invention, inhibition is the preferred form of modulation of-gene expression and mRNA is a preferred target.  
       [0025] It is preferred to target specific nucleic acids for antisense. “Targeting” an antisense compound to a particular nucleic acid, in the context of this invention, is a multistep process. The process usually begins with the identification of a nucleic acid sequence whose function is to be modulated. This may be, for example, a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent. In the present invention, the target is a nucleic acid molecule encoding Notch1. The targeting process also includes determination of a site or sites within this gene for the antisense interaction to occur such that the desired effect, e.g., detection or modulation of expression of the protein, will result. Within the context of the present invention, a preferred intragenic site is the region encompassing the translation initiation or termination codon of the open reading frame (ORF) of the gene. Since, as is known in the art, the translation initiation codon is typically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in the corresponding DNA molecule), the translation initiation codon is also referred to as the “AUG codon,” the “start codon” or the “AUG start codon”. A minority of genes have a translation initiation codon having the RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo. Thus, the terms “translation initiation codon” and “start codon” can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine (in eukaryotes) or formylmethionine (in prokaryotes). It is also known in the art that eukaryotic and prokaryotic genes may have two or more alternative start codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions. In the context of the invention, “-start-codon” and “translation initiation codon” refer to the codon or codons that are used in vivo to initiate translation of an mRNA molecule transcribed from a gene encoding Notch1, regardless of the sequence(s) of such codons.  
       [0026] It is also known in the art that a translation termination codon (or “stop codon”) of a gene may have one of three sequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA, 5′-TAG and 5′-TGA, respectively). The terms “start codon region” and “translation initiation codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation initiation codon. Similarly, the terms “stop codon region” and “translation termination codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation termination codon.  
       [0027] The open reading frame (ORF) or “coding region,” which is known in the art to refer to the region between the translation initiation codon and the translation termination codon, is also a region which may be targeted effectively. Other target regions include the 5′ untranslated region (5′UTR), known in the art to refer to the portion of an mRNA in the 5′ direction from the translation initiation codon, and thus including nucleotides between the 5′ cap site and the translation initiation codon of an mRNA or corresponding nucleotides on the gene, and the 3′ untranslated region (3′UTR), known in the art to refer to the portion of an mRNA in the 3′ direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3′ end of an mRNA or corresponding nucleotides on the gene. The 5′ cap of an mRNA comprises an N7-methylated guanosine residue joined to the 5′-most residue of the mRNA via a 5′-5′ triphosphate linkage. The 5′ cap region of an mRNA is considered to include the 5′ cap—structure itself as well as the first 50 nucleotides adjacent to the cap. The 5′ cap region may also be a preferred target region.  
       [0028] Although some eukaryotic mRNA transcripts are directly translated, many contain one or more regions, known as “introns,” which are excised from a transcript before it is translated. The remaining (and therefore translated) regions are known as “exons” and are spliced together to form a continuous mRNA sequence. mRNA splice sites, i.e., intron-exon junctions, may also be preferred target regions, and are particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular mRNA splice product is implicated in disease. Aberrant fusion junctions due to rearrangements or deletions are also preferred targets. mRNA transcripts produced via the process of splicing of two (or more) mRNAs from different gene sources are known as “fusion transcripts”. It has also been found that introns can be effective, and therefore preferred, target regions for antisense compounds targeted, for example, to DNA or pre-mRNA.  
       [0029] It is also known in the art that alternative RNA transcripts can be produced from the same genomic region of DNA. These alternative transcripts are generally known as “variants”. More specifically, “pre-mRNA variants” are transcripts produced from the same genomic DNA that differ from other transcripts produced from the same genomic DNA in either their start or stop position and contain both intronic and extronic regions.  
       [0030] Upon excision of one or more exon or intron regions or portions thereof during splicing, pre-mRNA variants produce smaller “mRNA variants”. Consequently, mRNA variants are processed pre-mRNA variants and each unique pre-mRNA variant must always produce a unique mRNA variant as a result of splicing. These mRNA variants are also known as “alternative splice variants”. If no splicing of the pre-mRNA variant occurs then the pre-mRNA variant is identical to the mRNA variant.  
       [0031] It is also known in the art that variants can be produced through the use of alternative signals to start or stop transcription and that pre-mRNAs and mRNAs can possess more that one start codon or stop codon. Variants that originate from a pre-mRNA or mRNA that use alternative start codons are known as “alternative start variants” of that pre-mRNA or mRNA. Those transcripts that use an alternative stop codon are known as “alternative stop variants” of that pre-mRNA or mRNA. One specific type of alternative stop variant is the “polyA variant” in which the multiple transcripts produced result from the alternative selection of one of the “polyA stop signals” by the transcription machinery, thereby producing transcripts that terminate at unique polyA sites.  
       [0032] Once one or more target sites have been identified, oligonucleotides are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect.  
       [0033] In the context of this invention, “hybridization” means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases. For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds. “Complementary,” as used herein, refers to the capacity for precise pairing between two nucleotides. For example, if a nucleotide at a certain position of an oligonucleotide is capable of hydrogen bonding with a nucleotide at the same position of a DNA or RNA molecule, then the oligonucleotide and the DNA or RNA are considered to be complementary to each other at that position. The oligonucleotide and the DNA or RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can hydrogen bond with each other. Thus, “specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the oligonucleotide and the DNA or RNA target. It is understood in the art that the sequence of an antisense compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable.  
       [0034] An antisense compound is specifically hybridizable when binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA to cause a loss of activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed. It is preferred that the antisense compounds of the present invention comprise at least 80% sequence complementarity to a target region within the target nucleic acid, moreover that they comprise 90% sequence complementarity and even more comprise 95% sequence complementarity to the target region within the target nucleic acid sequence to which they are targeted. For example, an antisense compound in which 18 of 20 nucleobases of the antisense compound are complementary, and would therefore specifically hybridize, to a target region would represent 90 percent complementarity. Percent complementarity of an antisense compound with a region of a target nucleic acid can be determined routinely using basic local alignment search tools (BLAST programs) (Altschul et al.,  J. Mol. Biol.,  1990, 215, 403-410; Zhang and Madden,  Genome Res.,  1997, 7, 649-656).  
       [0035] Antisense and other compounds of the invention, which hybridize to the target and inhibit expression of the target, are identified through experimentation, and representative sequences of these compounds are hereinbelow identified as preferred embodiments of the invention. The sites to which these preferred antisense compounds are specifically hybridizable are hereinbelow referred to as “preferred target regions”and are therefore preferred sites for targeting. As used herein the term “preferred target region” is defined as at least an 8-nucleobase portion of a target region to which an active antisense compound is targeted. While not wishing to be bound by theory, it is presently believed that these target regions represent regions of the target nucleic acid which are accessible for hybridization.  
       [0036] While the specific sequences of particular preferred target regions are set forth below, one of skill in the art will recognize that these serve to illustrate and describe particular embodiments within the scope of the present invention. Additional preferred target regions may be identified by one having ordinary skill.  
       [0037] Target regions 8-80 nucleobases in length comprising a stretch of at least eight (8) consecutive nucleobases selected from within the illustrative preferred target regions are considered to be suitable preferred target regions as well.  
       [0038] Exemplary good preferred target regions include DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 5′-terminus of one of the illustrative preferred target regions (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately upstream of the 5′-terminus of the target region and continuing until the DNA or RNA contains about 8 to about 80 nucleobases). Similarly good preferred target regions are represented by DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 3′-terminus of one of the illustrative preferred target regions (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately downstream of the 3′-terminus of the target region and continuing until the DNA or RNA contains about 8 to about 80 nucleobases). One having skill in the art, once armed with the empirically-derived preferred target regions illustrated herein will be able, without undue experimentation, to identify further preferred target regions. In addition, one having ordinary skill in the art will also be able to identify additional compounds, including oligonucleotide probes and primers, that specifically hybridize to these preferred target regions using techniques available to the ordinary practitioner in the art.  
       [0039] Antisense compounds are commonly used as research reagents and diagnostics. For example, antisense oligonucleotides, which are able to inhibit gene expression with exquisite specificity, are often used by those of ordinary skill to elucidate the function of particular genes. Antisense compounds are also used, for example, to distinguish between functions of various members of a biological pathway. Antisense modulation has, therefore, been harnessed for research use.  
       [0040] For use in kits and diagnostics, the antisense compounds of the present invention, either alone or in combination with other antisense compounds or therapeutics, can be used as tools in differential and/or combinatorial analyses to elucidate expression patterns of a portion or the entire complement of genes expressed within cells and tissues.  
       [0041] Expression patterns within cells or tissues treated with one or more antisense compounds are compared to control cells or tissues not treated with antisense compounds and the patterns produced are analyzed for differential levels of gene expression as they pertain, for example, to disease association, signaling pathway, cellular localization, expression level, size, structure or function of the genes examined. These analyses can be performed on stimulated or unstimulated cells and in the presence or absence of other compounds which affect expression patterns.  
       [0042] Examples of methods of gene expression analysis known in the art include DNA arrays or microarrays (Brazma and Vilo,  FEBS Lett.,  2000, 480, 17-24; Celis, et al.,  FEBS Lett.,  2000, 480, 2-16), SAGE (serial analysis of gene expression) (Madden, et al.,  Drug Discov. Today,  2000, 5, 415-425), READS (restriction enzyme amplification of digested cDNAs) (Prashar and Weissman,  Methods Enzymol.,  1999, 303, 258-72), TOGA (total gene expression analysis) (Sutcliffe, et al.,  Proc. Natl. Acad. Sci. U.S.A.,  2000, 97, 1976-81), protein arrays and proteomics (Celis, et al.,  FEBS Lett.,  2000, 480, 2-16; Jungblut, et al.,  Electrophoresis,  1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, et al.,  FEBS Lett.,  2000, 480, 2-16; Larsson, et al.,  J. Biotechnol.,  2000, 80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al.,  Anal. Biochem.,  2000, 286, 91-98; Larson, et al.,  Cytometry,  2000, 41, 203-208), subtractive cloning, differential display (DD) (Jurecic and Belmont,  Curr. Opin. Microbiol.,  2000, 3, 316-21), comparative genomic hybridization (Carulli, et al.,  J. Cell Biochem. Suppl.,  1998, 31, 286-96), FISH (fluorescent in situ hybridization) techniques (Going and Gusterson, Eur.  J. Cancer,  1999, 35, 1895-904) and mass spectrometry methods (reviewed in To,  Comb. Chem. High Throughput Screen,  2000, 3, 235-41).  
       [0043] The specificity and sensitivity of antisense is also harnessed by those of skill in the art for therapeutic uses. Antisense oligonucleotides have been employed as therapeutic moieties in the treatment of disease states in animals and man. Antisense oligonucleotide drugs, including ribozymes, have been safely and effectively administered to humans and numerous clinical trials are presently underway. It is thus established that oligonucleotides can be useful therapeutic modalities that can be configured to be useful in treatment regimes for treatment of cells, tissues and animals, especially humans.  
       [0044] In the context of this invention, the term “oligonucleotide” refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term includes oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally-occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced-cellular uptake, enhanced affinity for nucleic acid target and increased stability in the presence of nucleases.  
       [0045] While antisense oligonucleotides are a preferred form of antisense compound, the present invention comprehends other oligomeric antisense compounds, including but not limited to oligonucleotide mimetics such as are described below. The antisense compounds in accordance with this invention preferably comprise from about 8 to about 80 nucleobases (i.e. from about 8 to about 80 linked nucleosides). Particularly preferred antisense compounds are antisense oligonucleotides from about 8 to about 50 nucleobases, even more preferably those comprising from about 12 to about 30 nucleobases. Antisense compounds include ribozymes, external guide sequence (EGS) oligonucleotides (oligozymes), and other short catalytic RNAs or catalytic oligonucleotides which hybridize to the target nucleic acid and modulate its expression.  
       [0046] Antisense compounds 8-80 nucleobases in length comprising a stretch of at least eight (8) consecutive nucleobases selected from within the illustrative antisense compounds are considered to be suitable antisense compounds as well.  
       [0047] Exemplary preferred antisense compounds include DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 5′-terminus of one of the illustrative preferred antisense compounds (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately upstream of the 5′-terminus of the antisense compound which is specifically hybridizable to the target nucleic acid and continuing until the DNA or RNA contains about 8 to about 80 nucleobases). Similarly preferred antisense compounds are represented by DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 3′-terminus of one of the illustrative preferred antisense compounds (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately downstream of the 3′-terminus of the antisense compound which is specifically hybridizable to the target nucleic acid and continuing until the DNA or RNA contains about 8 to about 80 nucleobases). One having skill in the art, once armed with the empirically-derived preferred antisense compounds illustrated herein will be able, without undue experimentation, to identify further preferred antisense compounds. Antisense and other compounds of the invention, which hybridize to the target and inhibit expression of the target, are identified through experimentation, and representative sequences of these compounds are herein identified as preferred embodiments of the invention. While specific sequences of the antisense compounds are set forth herein, one of skill in the art will recognize that these serve to illustrate and describe particular embodiments within the scope of the present invention. Additional preferred antisense compounds may be identified by one having ordinary skill.  
       [0048] As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn, the respective ends of this linear polymeric structure can be further joined to form a circular structure, however, open linear structures are generally preferred. In addition, linear structures may also have internal nucleobase complementarity and may therefore fold in a manner as to produce a double stranded structure. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiester linkage.  
       [0049] Specific examples of preferred antisense compounds useful in this invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. As defined in this specification, oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.  
       [0050] Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage. Preferred oligonucleotides having inverted polarity comprise a single 3′ to 3′ linkage at the 3′-most internucleotide linkage i.e. a single inverted nucleoside residue which may be a basic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included.  
       [0051] Representative United States patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and 5,625,050, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.  
       [0052] Preferred modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH 2  component parts.  
       [0053] Representative United States patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.  
       [0054] In other preferred oligonucleotide mimetics, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to-have-excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al.,  Science,  1991, 254, 1497-1500.  
       [0055] Most preferred embodiments of the invention are oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH 2 —NH—O—CH 2 —, —CH2—N(CH 3 )—O—CH 2 -[known as a methylene (methylimino) or MMI backbone], —CH 2 —O—N(CH 3 )—CH 2 —, —CH 2 —N(CH 3 )—N(CH 3 )—CH 2 — and —O—N(CH 3 )—CH 2 —CH 2 -[wherein the native phosphodiester backbone is represented as —O—P—O—CH 2 -] of the above referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above referenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotides having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.  
       [0056] Modified oligonucleotides may also contain one or more substituted sugar moieties. Preferred oligonucleotides comprise one of the following at the 2′ position: OH; F; O—, S—, or N-alkyl; O—, S—, or N-alkenyl; O—, S— or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C 1  to C 10  alkyl or C 2  to C 10  alkenyl and alkynyl. Particularly preferred are O[(CH 2 ) n O] m CH 3 , O(CH 2 ) n OCH 3 , O(CH 2 )NH 2 , O(CH 2 ) n CH 3 , O(CH 2 ) n ONH 2 , and O(CH 2 ) n ON[(CH 2 ) n CH 3 ] 2 , where n and m are from 1 to about 10. Other preferred oligonucleotides comprise one of the following at the 2′ position: C 1  to C 10  lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 31  OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , SO 2 CH 3 , ONO 2 , NO 2 , N 3 , NH 2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A preferred modification includes 2′-methoxyethoxy (2′-O—CH 2 CH 2 OCH 3 , also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al.,  Helv. Chim. Acta,  1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further preferred modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH 2 ) 2 ON(CH 3 ) 2  group, also known as 2′-DMAOE, as described in examples hereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e., 2′-O—CH 2 —O—CH 2 —N(CH 3 ) 2 , also described in examples hereinbelow.  
       [0057] Other preferred modifications include 2′-methoxy (2′-O—CH 3 ), 2′-aminopropoxy (2′-OCH 2 CH 2 CH 2 NH 2 ), 2′-allyl (2′-CH 2 —CH═CH 2 ), 2′-O-allyl (2′-O—CH 2 —CH═CH 2 ) and 2′-fluoro (2′-F). The 2′-modification may be in the arabino (up) position or ribo (down) position. A preferred 2′-arabino modification is 2′-F. Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.  
       [0058] A further preferred modification includes Locked Nucleic Acids (LNAs) in which the 2′-hydroxyl group is linked to the 3′ or 4′ carbon atom of the sugar ring thereby forming a bicyclic sugar moiety. The linkage is preferably a methelyne (—CH 2 —) n  group bridging the 2′ oxygen atom and the 4′ carbon atom wherein n is 1 or 2. LNAs and preparation thereof are described in WO 98/39352 and WO 99/14226.  
       [0059] Oligonucleotides may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C≡C—CH 3 ) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido [5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in  The Concise Encyclopedia Of Polymer Science And Engineering , pages 858-859, Kroschwitz, J. I., ed. John Wiley &amp; Sons, 1990, those disclosed by Englisch et al.,  Angewandte Chemie , International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15 , Antisense Research and Applications , pages 289-302, Crooke, S. T. and Lebleu, B. ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds.,  Antisense Research and Applications, CRC Press, Boca Raton,  1993, pp. 276-278) and are presently preferred base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.  
       [0060] Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and 5,681,941, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference, and U.S. Pat. No. 5,750,692, which is commonly owned with the instant application and also herein incorporated by reference.  
       [0061] Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. The compounds of the invention can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugate groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve oligomer uptake, enhance oligomer resistance to degradation, and/or strengthen sequence-specific hybridization with RNA. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve oligomer uptake, distribution, metabolism or excretion. Representative conjugate groups are disclosed in International Patent Application PCT/US92/09196, filed Oct. 23, 1992 the entire disclosure of which is incorporated herein by reference. Conjugate moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al.,  Proc. Natl. Acad. Sci. USA,  1989, 86, 6553-6556), cholic acid (Manoharan et al.,  Bioorg. Med. Chem. Let.,  1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al.,  Ann. N.Y. Acad. Sci.,  1992, 660, 306-309; Manoharan et al.,  Bioorg. Med. Chem. Let.,  1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al.,  Nucl. Acids Res.,  1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al.,  EMBO J.,  1991, 10, 1111-1118; Kabanov et al.,  FEBS Lett.,  1990, 259, 327-330; Svinarchuk et al.,  Biochimie,  1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,  Tetrahedron Lett.,  1995, 36, 3651-3654; Shea et al.,  Nucl. Acids Res.,  1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al.,  Nucleosides  &amp;  Nucleotides,  1995, 14, 969-973), or adamantane acetic acid (Manoharan et al.,  Tetrahedron Lett.,  1995, 36, 3651-3654), a palmityl moiety (Mishra et al.,  Biochim. Biophys. Acta,  1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J.  Pharmacol. Exp. Ther.,  1996, 277, 923-937). Oligonucleotides of the invention may also be conjugated to active drug substances, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drug conjugates and their preparation are described in U.S. patent application Ser. No. 09/334,130 (filed Jun. 15, 1999) which is incorporated herein by reference in its entirety.  
       [0062] Representative United States patents that teach the preparation of such oligonucleotide conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference.  
       [0063] It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an oligonucleotide. The present invention also includes antisense compounds which are chimeric compounds. “Chimeric” antisense compounds or “chimeras,” in the context of this invention, are antisense compounds, particularly oligonucleotides, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound. These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, increased stability and/or increased binding affinity for the target nucleic acid. An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNAse H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide inhibition of gene expression. The cleavage of RNA:RNA hybrids can, in like fashion, be accomplished through the actions of endoribonucleases, such as interferon-induced RNAseL which cleaves both cellular and viral RNA. Consequently, comparable results can often be obtained with shorter oligonucleotides when chimeric oligonucleotides are used, compared to phosphorothioate deoxyoligonucleotides hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.  
       [0064] Chimeric antisense compounds of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as hybrids or gapmers. Representative United States patents that teach the preparation of such hybrid structures include, but are not limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.  
       [0065] The antisense compounds used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives.  
       [0066] The compounds of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor-targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption. Representative United States patents that teach the preparation of such uptake, distribution and/or absorption-assisting formulations include, but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721; 4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170; 5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854; 5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948; 5,580,575; and 5,595,756, each of which is herein incorporated by reference.  
       [0067] The antisense compounds of the invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of the compounds of the invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents.  
       [0068] The term “prodrug” indicates a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions. In particular, prodrug versions of the oligonucleotides of the invention are prepared as SATE [(S-acetyl-2-thioethyl) phosphate] derivatives according to the methods disclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 or in WO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach et al.  
       [0069] The term “pharmaceutically acceptable salts” refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.  
       [0070] Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Examples of metals used as cations are sodium, potassium, magnesium, calcium, and the like. Examples of suitable amines are N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine (see, for example, Berge et al., “Pharmaceutical Salts,” J. of Pharma Sci.,  1977, 66, 1-19). The base addition salts of said acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in the conventional manner. The free acid form may be regenerated by contacting the salt form with an acid and isolating the free acid in the conventional manner. The free acid forms differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free acid for purposes of the present invention. As used herein, a “pharmaceutical addition salt” includes a pharmaceutically acceptable salt of an acid form of one of the components of the compositions of the invention. These include organic or inorganic acid salts of the amines. Preferred acid salts are the hydrochlorides, acetates, salicylates, nitrates and phosphates. Other suitable pharmaceutically acceptable salts are well known to those skilled in the art and include basic salts of a variety of inorganic and organic acids, such as, for example, with inorganic acids, such as for example hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid; with organic carboxylic, sulfonic, sulfo or phospho acids or N-substituted sulfamic acids, for example acetic acid, propionic acid, glycolic acid, succinic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid, oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, salicylic acid, 4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, embonic acid, nicotinic acid or isonicotinic acid; and with amino acids, such as the 20 alpha-amino acids involved in the synthesis of proteins in nature, for example glutamic acid or aspartic acid, and also with phenylacetic acid, methanesulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid, benzenesulfonic acid, 4-methylbenzenesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or 3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (with the formation of cyclamates), or with other acid organic compounds, such as ascorbic acid. Pharmaceutically acceptable salts of compounds may also be prepared with a pharmaceutically acceptable cation. Suitable pharmaceutically acceptable cations are well known to those skilled in the art and include alkaline, alkaline earth, ammonium and quaternary ammonium cations. Carbonates or hydrogen carbonates are also possible.  
       [0071] For oligonucleotides, preferred examples of pharmaceutically acceptable salts include but-are not limited to (a) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, etc.; (b) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; (c) salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d) salts formed from elemental anions such as chlorine, bromine, and iodine.  
       [0072] The antisense compounds of the present invention can be utilized for diagnostics, therapeutics, prophylaxis and as research reagents and kits. For therapeutics, an animal, preferably a human, suspected of having a disease or disorder which can be treated by modulating the expression of Notch1 is treated by administering antisense compounds in accordance with this invention. The compounds of the invention can be utilized in pharmaceutical compositions by adding an effective amount of an antisense compound to a suitable pharmaceutically acceptable diluent or carrier. Use of the antisense compounds and methods of the invention may also be useful prophylactically, e.g., to prevent or delay infection, inflammation or tumor formation, for example.  
       [0073] The antisense compounds of the invention are useful for research and diagnostics, because these compounds hybridize to nucleic acids encoding Notch1, enabling sandwich and other assays to easily be constructed to exploit this fact. Hybridization of the antisense oligonucleotides of the invention with a nucleic acid encoding Notch1 can be detected by means known in the art. Such means may include conjugation of an enzyme to the oligonucleotide, radiolabelling of the oligonucleotide or any other suitable detection means. Kits using such detection means for detecting the level of Notch1 in a sample may also be prepared.  
       [0074] The present invention also includes pharmaceutical compositions and formulations which include the antisense compounds of the invention. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Oligonucleotides with at least one 2′-O-methoxyethyl modification are believed to be particularly useful for oral administration.  
       [0075] Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful. Preferred topical formulations include those in which the oligonucleotides of the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Preferred lipids and liposomes include neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g. dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA). Oligonucleotides of the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, oligonucleotides may be complexed to lipids, in particular to cationic lipids. Preferred fatty acids and esters include but are not limited arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C 1-10  alkyl ester (e.g. isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof. Topical formulations are described in detail in U.S. patent application Ser. No. 09/315,298 filed on May 20, 1999 which is incorporated herein by reference in its entirety.  
       [0076] Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. Preferred oral formulations are those in which oligonucleotides of the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators. Preferred surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Preferred bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Preferred fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an-acylcholine, or a-monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g. sodium). Also preferred are combinations of penetration enhancers, for example, fatty acids/salts in combination with bile acids/salts. A particularly preferred combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. Oligonucleotides of the invention may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. Oligonucleotide complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches. Particularly preferred complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylamino-methylethylene P(TDAE), polyaminostyrene (e.g. p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for oligonucleotides and their preparation are described in detail in U.S. application Ser. No. 08/886,829 (filed Jul. 1, 1997), Ser. No. 09/108,673 (filed Jul. 1, 1998), Ser. No. 09/256,515 (filed Feb. 23, 1999), Ser. No. 09/082,624 (filed May 21, 1998) and Ser. No. 09/315,298 (filed May 20, 1999), each of which is incorporated herein by reference in their entirety.  
       [0077] Compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.  
       [0078] Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.  
       [0079] The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.  
       [0080] The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.  
       [0081] In one embodiment of the present invention the pharmaceutical compositions may be formulated and used as foams. Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically similar in nature these formulations vary in the components and the consistency of the final product. The preparation of such compositions and formulations is generally known to those skilled in the pharmaceutical and formulation arts and may be applied to the formulation of the compositions of the present invention.  
       [0082] Emulsions  
       [0083] The compositions of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogenous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter (Idson, in  Pharmaceutical Dosage Forms , Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in  Pharmaceutical Dosage Forms , Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in  Pharmaceutical Dosage Forms , Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in  Remington&#39;s Pharmaceutical Sciences , Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions may be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions may contain additional components in addition to the dispersed phases, and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants may also be present in emulsions as needed. Pharmaceutical emulsions may also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion.  
       [0084] Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion may be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that may be incorporated into either phase of the emulsion. Emulsifiers may broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (Idson, in  Pharmaceutical Dosage Forms , Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).  
       [0085] Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (Rieger, in  Pharmaceutical Dosage Forms , Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in  Pharmaceutical Dosage Forms , Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants may be classified into different classes based on the nature of the hydrophilic group: nonionic, —anionic, cationic and amphoteric (Rieger, in  Pharmaceutical Dosage Forms , Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).  
       [0086] Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.  
       [0087] A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in  Pharmaceutical Dosage Forms , Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in  Pharmaceutical Dosage Forms , Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).  
       [0088] Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.  
       [0089] Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that may readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used may be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.  
       [0090] The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (Idson, in  Pharmaceutical Dosage Forms , Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of ease of formulation, as well as efficacy from an absorption and bioavailability standpoint (Rosoff, in  Pharmaceutical Dosage Forms , Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in  Pharmaceutical Dosage Forms , Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.  
       [0091] In one embodiment of the present invention, the compositions of oligonucleotides and nucleic acids are formulated as microemulsions. A microemulsion may be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (Rosoff, in  Pharmaceutical Dosage Forms , Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in:  Controlled Release of Drugs: Polymers and Aggregate Systems , Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in  Remington&#39;s Pharmaceutical Sciences , Mack Publishing Co., Easton, Pa., 1985, p. 271).  
       [0092] The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (Rosoff, in  Pharmaceutical Dosage Forms , Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in  Pharmaceutical Dosage Forms , Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.  
       [0093] Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DA0750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions may, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and triglycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.  
       [0094] Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (Constantinides et al.,  Pharmaceutical Research,  1994, 11, 1385-1390; Ritschel,  Meth. Find. Exp. Clin. Pharmacol.,  1993, 13, 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (Constantinides et al.,  Pharmaceutical Research,  1994, 11, 1385; Ho et al.,  J. Pharm. Sci.,  1996, 85, 138-143). Often microemulsions may form spontaneously when their components are brought together at ambient temperature. This may be particularly advantageous when formulating thermolabile drugs, peptides or oligonucleotides. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of oligonucleotides and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of oligonucleotides and nucleic acids within the gastrointestinal tract, vagina, buccal cavity and other areas of administration.  
       [0095] Microemulsions of the present invention may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the oligonucleotides and nucleic acids of the present invention. Penetration enhancers used in the microemulsions of the present invention may be classified as belonging to one of five broad categories—surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al.,  Critical Reviews in Therapeutic Drug Carrier Systems,  1991, p. 92). Each of these classes has been discussed above.  
       [0096] Liposomes  
       [0097] There are many organized surfactant structures besides microemulsions that have been studied and used for the formulation of drugs. These include monolayers, micelles, bilayers and vesicles. Vesicles, such as liposomes, have attracted great interest because of their specificity and the duration of action they offer from the standpoint of drug delivery. As used in the present invention, the term “liposome” means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers.  
       [0098] Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo.  
       [0099] In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. Therefore, it is desirable to use a liposome which is highly deformable and able to pass through such fine pores.  
       [0100] Further advantages of liposomes include; liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated drugs in their internal compartments from metabolism and degradation (Rosoff, in  Pharmaceutical Dosage Forms , Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.  
       [0101] Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the cellular membranes and as the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act.  
       [0102] Liposomal formulations have been the focus of extensive investigation as the mode of delivery for many drugs. There is growing evidence that for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side-effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to-administer a wide variety of drugs, both hydrophilic and hydrophobic, into the skin.  
       [0103] Several reports have detailed the ability of liposomes to deliver agents including high-molecular weight DNA into the skin. Compounds including analgesics, antibodies, hormones and high-molecular weight DNAs have been administered to the skin. The majority of applications resulted in the targeting of the upper epidermis.  
       [0104] Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged DNA molecules to form a stable complex The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al.,  Biochem. Biophys. Res. Commun.,  1987, 147, 980-985).  
       [0105] Liposomes which are pH-sensitive or negatively-charged, entrap DNA rather than complex with it. Since both the DNA and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some DNA is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al.,  Journal of Controlled Release,  1992, 19, 269-274).  
       [0106] One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.  
       [0107] Several studies have assessed the topical delivery of liposomal drug formulations to the skin. Application of liposomes containing interferon to guinea pig skin resulted in a reduction of skin herpes sores while delivery of interferon via other means (e.g. as a solution or as an emulsion) were ineffective (Weiner et al.,  Journal of Drug Targeting,  1992, 2, 405-410). Further, an additional study tested the efficacy of interferon administered as part of a liposomal formulation to the administration of interferon using an aqueous system, and concluded that the liposomal formulation was superior to aqueous administration (du Plessis et al.,  Antiviral Research,  1992, 18, 259-265).  
       [0108] Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome™ I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporin-A into different layers of the skin (Hu et al.  S.T.P. Pharma. Sci.,  1994, 4, 6, 466).  
       [0109] Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more-glycolipids, such as monosialoganglioside G M1 , or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al.,  FEBS Letters,  1987, 223, 42; Wu et al.,  Cancer Research,  1993, 53, 3765).  
       [0110] Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y.  Acad. Sci.,  1987, 507, 64) reported the ability of monosialoganglioside G M1 , galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. ( Proc. Natl. Acad. Sci. U.S.A.,  1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside G M1  or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al.).  
       [0111] Many liposomes comprising lipids derivatized with one or more hydrophilic polymers, and methods of preparation thereof, are known in the art. Sunamoto et al. ( Bull. Chem. Soc. Jpn.,  1980, 53, 2778) described liposomes comprising a nonionic detergent, 2C 12 15G, that contains a PEG moiety. Ilium et al. ( FEBS Lett.,  1984, 167, 79) noted that hydrophilic coating of polystyrene particles with polymeric glycols results in significantly enhanced blood half-lives. Synthetic phospholipids modified by the attachment of carboxylic groups of polyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899). Klibanov et al. ( FEBS Lett.,  1990, 268, 235) described experiments demonstrating that liposomes comprising phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have significant increases in blood circulation half-lives. Blume et al. ( Biochimica et Biophysica Acta,  1990, 1029, 91) extended such observations to other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from the combination of distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes having covalently bound PEG moieties on their external surface are described in European Patent No. EP 0 445 131 B1 and WO 90/04384 to Fisher. Liposome compositions containing 1-20 mole percent of PE derivatized with PEG, and methods of use thereof, are described by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496 813 B1). Liposomes comprising a number of other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprising PEG-modified ceramide lipids are described in WO 96/10391 (Choi et al.). U.S. Pat. Nos. 5,540,935 (Miyazaki et al.) and 5,556,948 (Tagawa et al.) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces.  
       [0112] A limited number of liposomes comprising nucleic acids are known in the art. WO 96/40062 to Thierry et al. discloses methods for encapsulating high molecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomes and asserts that the contents of such liposomes may include an antisense RNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methods of encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. discloses liposomes comprising antisense oligonucleotides targeted to the raf gene.  
       [0113] Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive—candidates for drug delivery vehicles. Transfersomes may be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g. they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.  
       [0114] Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the “head”) provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in  Pharmaceutical Dosage Forms , Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).  
       [0115] If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.  
       [0116] If the surfactant molecule carries a-negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.  
       [0117] If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.  
       [0118] If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.  
       [0119] The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in  Pharmaceutical Dosage Forms , Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).  
       [0120] Penetration Enhancers  
       [0121] In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly oligonucleotides, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs may cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.  
       [0122] Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al.,  Critical Reviews in Therapeutic Drug Carrier Systems,  1991, p.92). Each of the above mentioned classes of penetration enhancers are described below in greater detail.  
       [0123] Surfactants: In connection with the present invention, surfactants (or “surface-active agents”) are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of oligonucleotides through the mucosa is enhanced. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Lee et al.,  Critical Reviews in Therapeutic Drug Carrier Systems,  1991, p.92); and perfluorochemical emulsions, such as FC-43. Takahashi et al.,  J. Pharm. Pharmacol.,  1988, 40, 252).  
       [0124] Fatty acids: Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C 1-10  alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al., Critical Reviews in  Therapeutic Drug Carrier Systems,  1991, p.92; Muranishi,  Critical Reviews in Therapeutic Drug Carrier Systems,  1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).  
       [0125] Bile salts: The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38 in: Goodman &amp; Gilman&#39;s  The Pharmacological Basis of Therapeutics,  9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus the term “bile salts” includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. The bile salts of the invention include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee et al.,  Critical Reviews in Therapeutic Drug Carrier Systems,  1991, page 92; Swinyard, Chapter 39 In: Remington&#39;s Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi,  Critical Reviews in Therapeutic Drug Carrier Systems,  1990, 7, 1-33; Yamamoto et al.,  J. Pharm. Exp. Ther.,  1992, 263, 25; Yamashita et al.,  J. Pharm. Sci.,  1990, 79, 579-583).  
       [0126] Chelating Agents: Chelating agents, as used in connection with the present invention, can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of oligonucleotides through the mucosa is enhanced. With regards to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett,  J. Chromatogr.,  1993, 618, 315-339). Chelating agents of the invention include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(Lee et al., Critical Reviews in  Therapeutic Drug Carrier Systems,  1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al.,  J. Control Rel.,  1990, 14, 43-51).  
       [0127] Non-chelating non-surfactants: As used herein, non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of oligonucleotides through the alimentary mucosa (Muranishi,  Critical Reviews in Therapeutic Drug Carrier Systems,  1990, 7, 1-33). This class of penetration enhancers include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al.,  Critical Reviews in Therapeutic Drug Carrier Systems,  1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al.,  J. Pharm. Pharmacol.,  1987, 39, 621-626).  
       [0128] Agents that enhance uptake of oligonucleotides at the cellular level may also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97/30731), are also known to enhance the cellular uptake of oligonucleotides.  
       [0129] Other agents may be utilized to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone.  
       [0130] Carriers  
       [0131] Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, “carrier compound” or “carrier” can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioate oligonucleotide in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al.,  Antisense Res. Dev.,  1995, 5, 115-121; Takakura et al.,  Antisense  &amp;  Nucl. Acid Drug Dev.,  1996, 6, 177-183).  
       [0132] Excipients  
       [0133] In contrast to a carrier compound, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc.).  
       [0134] Pharmaceutically acceptable organic or inorganic excipient suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.  
       [0135] Formulations for topical administration of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions may also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.  
       [0136] Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose,-polyvinylpyrrolidone-and the like.  
       [0137] Other Components  
       [0138] The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.  
       [0139] Aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.  
       [0140] Certain embodiments of the invention provide pharmaceutical compositions containing (a) one or more antisense compounds and (b) one or more other chemotherapeutic agents which function by a non-antisense mechanism. Examples of such chemotherapeutic agents include but are not limited to daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan, topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol (DES). See, generally,  The Merck Manual of Diagnosis and Therapy,  15th Ed. 1987, pp. 1206-1228, Berkow et al., eds., Rahway, N.J. When used with the compounds of the invention, such chemotherapeutic agents may be used individually (e.g., 5-FU and oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for a period of time followed by MTX and oligonucleotide), or in combination with one or more other such chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU, radiotherapy and oligonucleotide). Anti-inflammatory drugs, including but not limited to nonsteroidal anti-inflammatory drugs and corticosteroids, and antiviral drugs, including but not limited to ribivirin, vidarabine, acyclovir and ganciclovir, may also be combined in compositions of the invention. See, generally,  The Merck Manual of Diagnosis and Therapy,  15th Ed., Berkow et al., eds., 1987, Rahway, N.J., pages 2499-2506 and 46-49, respectively). Other non-antisense chemotherapeutic agents are also within the scope of this invention. Two or more combined compounds may be used together or sequentially.  
       [0141] In another related embodiment, compositions of the invention may contain one or more antisense compounds, particularly oligonucleotides, targeted to a first nucleic acid and one or more additional antisense compounds targeted to a second nucleic acid target. Numerous examples of antisense compounds are known in the art. Two or more combined compounds may be used together or sequentially.  
       [0142] The formulation of therapeutic compositions and their subsequent administration is believed to be within the skill of those in the art. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC 50 s found to be effective in in vitro and in vivo animal models. In general, dosage is from 0.01 ug to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.01 ug to 100 g per kg of body weight, once or more daily, to once every 20 years.  
       [0143] While the present invention has been described with specificity in accordance with certain of its preferred embodiments, the following examples serve only to illustrate the invention and are not intended to limit the same.  
     
    
    
     EXAMPLES  
     Example 1  
     [0144] Nucleoside Phosphoramidites for Oligonucleotide Synthesis Deoxy and 2′-alkoxy Amidites  
     [0145] 2′-Deoxy and 2′-methoxy beta-cyanoethyldiisopropyl phosphoramidites were purchased from commercial sources (e.g. Chemgenes, Needham Mass. or Glen Research, Inc. Sterling Va.). Other 2′-O-alkoxy substituted nucleoside amidites are prepared as described in U.S. Pat. No. 5,506,351, herein incorporated by reference. For oligonucleotides synthesized using 2′-alkoxy amidites, optimized synthesis cycles were developed that incorporate multiple—steps coupling longer wait times relative to standard synthesis cycles.  
     [0146] The following abbreviations are used in the text: thin layer chromatography (TLC), melting point (MP), high pressure liquid chromatography (HPLC), Nuclear Magnetic Resonance (NMR), argon (Ar), methanol (MeOH), dichloromethane (CH 2 Cl 2 ), triethylamine (TEA), dimethyl formamide (DMF), ethyl acetate (EtOAc), dimethyl sulfoxide (DMSO), tetrahydrofuran (THF).  
     [0147] Oligonucleotides containing 5-methyl-2′-deoxycytidine (5-Me-dC) nucleotides were synthesized according to published methods (Sanghvi, et. al.,  Nucleic Acids Research,  1993, 21, 3197-3203) using commercially available phosphoramidites (Glen Research, Sterling Va. or ChemGenes, Needham Mass.) or prepared as follows:  
     [0148] Preparation of 5′-O-Dimethoxytrityl-thymidine Intermediate for 5-methyl dC Amidite  
     [0149] To a 50 L glass reactor equipped with air stirrer and Ar gas line was added thymidine (1.00 kg, 4.13 mol) in anhydrous pyridine (6 L) at ambient temperature. Dimethoxytrityl (DMT) chloride (1.47 kg, 4.34 mol, 1.05 eq) was added as a solid in four portions over 1 h. After 30 min, TLC indicated approx. 95% product, 2% thymidine, 5% DMT reagent and by-products and 2% 3′,5′-bis DMT product (Rf in EtOAc 0.45, 0.05, 0.98, 0.95 respectively). Saturated sodium bicarbonate (4 L) and CH 2 Cl 2  were added with stirring (pH of the aqueous layer 7.5). An additional 18 L of water was added, the mixture was stirred, the phases were separated, and the organic layer was transferred to a second 50 L vessel. The aqueous layer was extracted with additional CH 2 Cl 2  (2×2 L). The combined organic layer was washed with water (10 L) and then concentrated in a rotary evaporator to approx. 3.6 kg total weight. This was redissolved in CH 2 Cl 2  (3.5 L), added to the reactor followed by water (6 L) and hexanes (13 L). The mixture was vigorously stirred and seeded to give a fine white suspended solid starting at the interface. After stirring for 1 h, the suspension was removed by suction through a ½″ diameter teflon tube into a 20 L suction flask, poured onto a 25 cm Coors Buchner funnel, washed with water (2×3 L) and a mixture of hexanes—CH 2 Cl 2  (4:1, 2×3 L) and allowed to air dry overnight in pans (1″ deep). This was further dried in a vacuum oven (75° C., 0.1 mm Hg, 48 h) to a constant weight of 2072 g (93%) of a white solid, (mp 122-124° C.). TLC indicated a trace contamination of the bis DMT product. NMR spectroscopy also indicated that 1-2 mole percent pyridine and about 5 mole percent of hexanes was still present.  
     [0150] Preparation of 5′-O-Dimethoxytrityl-2′-deoxy-5-methylcytidine Intermediate for 5-methyl-dC Amidite  
     [0151] To a 50 L Schott glass-lined steel reactor equipped with an electric stirrer, reagent addition pump (connected to an addition funnel), heating/cooling system, internal thermometer and an Ar gas line was added 5′-O-dimethoxytrityl-thymidine (3.00 kg, 5.51 mol), anhydrous acetonitrile (25 L) and TEA (12.3 L, 88.4 mol, 16 eq). The mixture was chilled with stirring to −10° C. internal temperature (external −20° C.). Trimethylsilylchloride (2.1 L, 16.5 mol, 3.0 eq) was added over 30 minutes while maintaining the internal temperature below −5° C., followed by a wash of anhydrous acetonitrile (1 L). Note: the reaction is mildly exothermic and copious hydrochloric acid fumes form over the course of the addition. The reaction was allowed to warm to 0° C. and the reaction progress was confirmed by TLC (EtOAc-hexanes 4:1; R f  0.43 to 0.84 of starting material and silyl product, respectively). Upon completion, triazole (3.05 kg, 44 mol, 8.0 eq) was added the reaction was cooled to −20° C. internal temperature (external −30° C.). Phosphorous oxychloride (1035 mL, 11.1 mol, 2.01 eq) was added over 60 min so as to maintain the temperature between −20° C. and −10° C. during the strongly exothermic process, followed by a wash of anhydrous acetonitrile (1 L). The reaction was warmed to 0° C. and stirred for 1 h. TLC indicated a complete conversion to the triazole product (Rf 0.83 to 0.34 with the product spot glowing in long wavelength UV light). The reaction mixture was a peach-colored thick suspension, which turned darker red upon warming without apparent decomposition. The reaction was cooled to −15° C. internal temperature and water (5 L) was slowly added at a rate to maintain the temperature below +10° C. in order to quench the reaction and to form a homogenous solution. (Caution: this reaction is initially very strongly exothermic). Approximately one-half of the reaction volume (22 L) was transferred by air pump to another vessel, diluted with EtOAc (12 L) and extracted with water (2×8 L). The combined water layers were back-extracted with EtOAc (6 L). The water layer was discarded and the organic layers were concentrated in a 20 L rotary evaporator to an oily foam. The foam was coevaporated with anhydrous acetonitrile (4 L) to remove EtOAc. (note: dioxane may be used instead of anhydrous acetonitrile if dried to a hard foam). The second half of the reaction was treated in the same way. Each residue was dissolved in dioxane (3 L) and concentrated ammonium hydroxide (750 mL) was added. A homogenous solution formed in a few minutes and the reaction was allowed to stand overnight (although the reaction is complete within 1 h).  
     [0152] TLC indicated a complete reaction (product R f  0.35 in EtOAc-MeOH 4:1). The reaction solution was concentrated on a rotary evaporator to a dense foam. Each foam was slowly redissolved in warm EtOAc (4 L; 50° C.), combined in a 50 L glass reactor vessel, and extracted with water (2×4L) to remove the triazole by-product. The water was back-extracted with EtOAc (2 L). The organic layers were combined and concentrated to about 8 kg total weight, cooled to 0° C. and seeded with crystalline product. After 24 hours, the first crop was collected on a 25 cm Coors Buchner funnel and washed repeatedly with EtOAc (3×3L) until a white powder was left and then washed with ethyl ether (2×3L). The solid was put in pans (1″ deep) and allowed to air dry overnight. The filtrate was concentrated to an oil, then redissolved in EtOAc (2 L), cooled and seeded as before. The second crop was collected and-washed as before (with proportional solvents) and the filtrate was first extracted with water (2×1L) and then concentrated to an oil. The residue was dissolved in EtOAc (1 L) and yielded a third crop which was treated as above except that more washing was required to remove a yellow oily layer.  
     [0153] After air-drying, the three crops were dried in a vacuum oven (50° C., 0.1 mm Hg, 24 h) to a constant weight (1750, 600 and 200 g, respectively) and combined to afford 2550 g (85%) of a white crystalline product (MP 215-217° C.) when TLC and NMR spectroscopy indicated purity. The mother liquor still contained mostly product (as determined by TLC) and a small amount of triazole (as determined by NMR spectroscopy), bis DMT product and unidentified minor impurities. If desired, the mother liquor can be purified by silica gel chromatography using a gradient of MeOH (0-25%) in EtOAc to further increase the yield.  
     [0154] Preparation of 5′-O-Dimethoxytrityl-2′-deoxy-N-4-benzoyl-5-methylcytidine Penultimate Intermediate for 5-methyl dC Amidite  
     [0155] Crystalline 5′-O-dimethoxytrityl-5-methyl-2′-deoxycytidine (2000 g, 3.68 mol) was dissolved in anhydrous DMF (6.0 kg) at ambient temperature in a 50 L glass reactor vessel equipped with an air stirrer and argon line. Benzoic anhydride (Chem Impex not Aldrich, 874 g, 3.86 mol, 1.05 eq) was added and the reaction was stirred at ambient temperature for 8 h. TLC (CH 2 Cl 2 -EtOAc; CH 2 Cl 2 -EtOAc 4:1; R f  0.25) indicated approx. 92% complete reaction. An additional amount of benzoic anhydride (44 g, 0.19 mol) was added. After a total of 18 h, TLC indicated approx. 96% reaction completion. The solution was diluted with EtOAc (20 L), TEA (1020 mL, 7.36 mol, ca 2.0 eq) was added with stirring, and the mixture was extracted with water (15 L, then 2×10 L). The aqueous layer was removed (no back-extraction was needed) and the organic layer was concentrated in 2×20 L rotary evaporator flasks until a foam began to form. The residues were coevaporated with acetonitrile (1.5 L each) and dried (0.1 mm Hg, 25° C., 24 h) to 2520 g of a dense foam. High pressure liquid chromatography (HPLC) revealed a contamination of 6.3% of N4, 3′-O-dibenzoyl product, but very little other impurities.  
     [0156] THe product was purified by Biotage column chromatography (5 kg Biotage) prepared with 65:35:1 hexanes-EtOAc-TEA (4L). The crude product (800 g),dissolved in CH 2 Cl 2  (2 L), was applied to the column. The column was washed with the 65:35:1 solvent mixture (20 kg), then 20:80:1 solvent mixture (10 kg), then 99:1 EtOAc:TEA (17 kg). The fractions containing the product were collected, and any fractions containing the product and impurities were retained to be resubjected to column chromatography. The column was reequilibrated with the original 65:35:1 solvent mixture (17 kg). A second batch of crude product (840 g) was applied to the column as before. The column was washed with the following solvent gradients: 65:35:1 (9 kg), 55:45:1 (20 kg), 20:80:1 (10 kg), and 99:1 EtOAc:TEA(15 kg). The column was reequilibrated as above, and a third batch of the crude product (850 g) plus impure fractions recycled from the two previous columns (28 g) was purified following the procedure for the second batch. The fractions containing pure product combined and concentrated on a 20L rotary evaporator, co-evaportated with acetontirile (3 L) and dried (0.1 mm Hg, 48 h, 25° C.) to a constant weight of 2023 g (85%) of white foam and 20 g of slightly contaminated product from the third run. HPLC indicated a purity of 99.8% with the balance as the diBenzoyl product.  
     [0157] [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N 4 -benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (5-methyl dC Amidite)  
     [0158] 5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N 4 -benzoyl-5-methylcytidine (998 g, 1.5 mol) was dissolved in anhydrous DMF (2 L). The solution was co-evaporated with toluene (300 ml) at 50° C. under reduced pressure, then cooled to room temperature and 2-cyanoethyl tetraisopropylphosphorodiamidite (680 g, 2.26 mol) and tetrazole (52.5 g, 0.75 mol) were added. The mixture was shaken until all tetrazole was dissolved, N-methylimidazole (15 ml) was added and the mixture was left at room temperature for 5 hours. TEA (300 ml) was added, the mixture was diluted with DMF (2.5 L) and water (600 ml), and extracted with hexane (3×3 L). The mixture was diluted with water (1.2 L) and extracted with a mixture of toluene (7.5 L) and hexane (6 L). The two layers were separated, the upper layer was washed with DMF-water (7:3 v/v, 3×2 L) and water (3×2 L), and the phases were separated. The organic layer was dried (Na 2 SO 4 ), filtered and rotary evaporated. The residue was co-evaporated with acetonitrile (2×2 L) under reduced pressure and dried to a constant weight (25° C., 0.1 mm Hg, 40 h) to afford 1250 g an off-white foam solid (96%).  
     [0159] 2′-Fluoro Amidites  
     [0160] 2′-Fluorodeoxyadenosine Amidites  
     [0161] 2′-fluoro oligonucleotides were synthesized as described previously [Kawasaki, et. al.,  J. Med. Chem.,  1993, 36, 831-841] and U.S. Pat. No. 5,670,633, herein incorporated by reference. The preparation of 2′-fluoropyrimidines containing a 5-methyl substitution are described in U.S. Pat. No. 5,861,493. Briefly, the protected nucleoside N6-benzoyl-2′-deoxy-2′-fluoroadenosine was synthesized utilizing commercially available 9-beta-D-arabinofuranosyladenine as starting material and whereby the 2′-alpha-fluoro atom is introduced by a S N 2-displacement of a 2′-beta-triflate group. Thus N6-benzoyl-9-beta-D-arabinofuranosyladenine was selectively protected in moderate yield as the 3′,5′-ditetrahydropyranyl (THP) intermediate. Deprotection of the THP and N6-benzoyl groups was accomplished using standard methodologies to obtain the 5′-dimethoxytrityl-(DMT) and 5′-DMT-3′-phosphoramidite intermediates.  
     [0162] 2′-Fluorodeoxyguanosine  
     [0163] The synthesis of 2′-deoxy-2′-fluoroguanosine was accomplished using tetraisopropyldisiloxanyl (TPDS) protected 9-beta-D-arabinofuranosylguanine as starting material, and conversion to the intermediate isobutyrylarabinofuranosylguanosine. Alternatively, isobutyrylarabinofuranosylguanosine was prepared as described by Ross et al., (Nucleosides &amp; Nucleosides, 16, 1645, 1997). Deprotection of the TPDS group was followed by protection of the hydroxyl group with THP to give isobutyryl di-THP protected arabinofuranosylguanine. Selective O-deacylation and triflation was followed by treatment of the crude product with fluoride, then deprotection of the THP groups. Standard methodologies were used to obtain the 5′-DMT- and 5′-DMT-3′-phosphoramidites.  
     [0164] 2′-Fluorouridine  
     [0165] Synthesis of 2′-deoxy-2′-fluorouridine was accomplished by the modification of a literature procedure in which 2,2′-anhydro-1-beta-D-arabinofuranosyluracil was treated with 70% hydrogen fluoride-pyridine. Standard procedures were used to obtain the 5′-DMT and 5′-DMT-3′phosphoramidites.  
     [0166] 2′-Fluorodeoxycytidine  
     [0167] 2′-deoxy-2′-fluorocytidine was synthesized via amination of 2′-deoxy-2′-fluorouridine, followed by selective protection to give N4-benzoyl-2′-deoxy-2′-fluorocytidine. Standard procedures were used to obtain the 5′-DMT and 5′-DMT-3′phosphoramidites.  
     [0168] 2′-O-(2-Methoxyethyl) modified Amidites  
     [0169] 2′-O-Methoxyethyl-substituted nucleoside amidites (otherwise known as MOE amidites) are prepared as follows, or alternatively, as per the methods of Martin, P., (Helvetica Chimica Acta, 1995, 78, 486-504).  
     [0170] Preparation of 2′-O-(2-methoxyethyl)-5-methyluridine intermediate  
     [0171] 2,2′-Anhydro-5-methyl-uridine (2000 g, 8.32 mol), tris(2-methoxyethyl)borate (2504 g, 10.60 mol), sodium bicarbonate (60 g, 0.70 mol) and anhydrous 2-methoxyethanol (5 L) were combined in a 12 L three necked flask and heated to 130° C. (internal temp) at atmospheric pressure, under an argon atmosphere with stirring for 21 h. TLC indicated a complete reaction. The solvent was removed under reduced pressure until a sticky gum formed (50-85° C. bath temp and 100-11 mm Hg) and the residue was redissolved in water (3 L) and heated to boiling for 30 min in order the hydrolyze the borate esters. The water was removed under reduced pressure until a foam began to form and then the process was repeated. HPLC indicated about 77% product, 15% dimer (5′ of product attached to 2′ of starting material) and unknown derivatives, and the balance was a single unresolved early eluting peak.  
     [0172] The gum was redissolved in brine (3 L), and the flask was rinsed with additional brine (3 L). The combined aqueous solutions were extracted with chloroform (20 L) in a heavier-than continuous extractor for 70 h. The chloroform layer was concentrated by rotary evaporation in a 20 L flask to a sticky foam (2400 g). This was coevaporated with MeOH (400 mL) and EtOAc (8 L) at 75° C. and 0.65 atm until the foam dissolved at which point the vacuum was lowered to about 0.5 atm. After 2.5 L of distillate was collected a precipitate began to form and the flask was removed from the rotary evaporator and stirred until the suspension reached ambient temperature. EtOAc (2 L) was added and the slurry was filtered on a 25 cm table top Buchner funnel and the product was washed with EtOAc (3×2 L). The bright white solid was air dried in pans for 24 h then further dried in a vacuum oven (50° C., 0.1 mm Hg, 24 h) to afford 1649 g of a white crystalline solid (mp 115.5-116.5° C.).  
     [0173] The brine layer in the 20 L continuous extractor was further extracted for 72 h with recycled chloroform. The chloroform was concentrated to 120 g of oil and this was combined with the mother liquor from the above filtration (225 g), dissolved in brine (250 mL) and extracted once with chloroform (250 mL). The brine solution was continuously extracted and the product was crystallized as described above to afford an additional 178 g of crystalline product containing about 2% of thymine. The combined yield was 1827 g (69.4%). HPLC indicated about 99.5% purity with the balance being the dimer.  
     [0174] Preparation of 5′-O-DMT-2′-O-(2-methoxyethyl)-5-methyluridine Penultimate Intermediate  
     [0175] In a 50 L glass-lined steel reactor, 2′-O-(2-methoxyethyl)-5-methyl-uridine (MOE-T, 1500 g, 4.738 mol), lutidine (1015 g, 9.476 mol) were dissolved in anhydrous acetonitrile (15 L). The solution was stirred rapidly and chilled to −10° C. (internal temperature). Dimethoxytriphenylmethyl chloride (1765.7 g, 5.21 mol) was added as a solid in one portion. The reaction was allowed to warm to −2° C. over 1 h. (Note: The reaction was monitored closely by TLC (EtOAc) to determine when to stop the reaction so as to not generate the undesired bis-DMT substituted side product). The reaction was allowed to warm from −2 to 3° C. over 25 min. then quenched by adding MeOH (300 mL) followed after 10 min by toluene (16 L) and water (16 L). The solution was transferred to a clear 50 L vessel with a bottom outlet, vigorously stirred for 1 minute, and the layers separated. The aqueous layer was removed and the organic layer was washed successively with 10% aqueous citric acid (8 L) and water (12 L). The product was then extracted into the aqueous phase by washing the toluene solution with aqueous sodium hydroxide (0.5N, 16 L and 8 L). The combined aqueous layer was overlayed with toluene (12 L) and solid citric acid (8 moles, 1270 g) was added with vigorous stirring to lower the pH of the aqueous layer to 5.5 and extract the product into the toluene. The organic layer was washed with water (10 L) and TLC of the organic layer indicated a trace of DMT-O-Me, bis DMT and dimer DMT.  
     [0176] The toluene solution was applied to a silica gel column (6 L sintered glass funnel containing approx. 2 kg of silica gel slurried with toluene (2 L) and TEA(25 mL)) and the fractions were eluted with toluene (12 L) and EtOAc (3×4 L) using vacuum applied to a filter flask placed below the column. The first EtOAc fraction containing both the desired product and impurities were resubjected to column chromatography as above. The clean fractions were combined, rotary evaporated to a foam, coevaporated with acetonitrile (6 L) and dried in a vacuum oven (0.1 mm Hg, 40 h, 40° C.) to afford 2850 g of a white crisp foam. NMR spectroscopy indicated a 0.25 mole % remainder of acetonitrile (calculates to be approx. 47 g) to give a true dry weight of 2803 g (96%). HPLC indicated that the product was 99.41% pure, with the remainder being 0.06 DMT-O-Me, 0.10 unknown, 0.44 bis DMT, and no detectable dimer DMT or 3′-O-DMT.  
     [0177] Preparation of [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE T Amidite)  
     [0178] 5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridine (1237 g, 2.0 mol) was dissolved in anhydrous DMF (2.5 L). The solution was co-evaporated with toluene (200 ml) at 50° C. under reduced pressure, then cooled to room temperature and 2-cyanoethyl tetraisopropylphosphorodiamidite (900 g, 3.0 mol) and tetrazole (70 g, 1.0 mol) were added. The mixture was shaken until all tetrazole was dissolved, N-methylimidazole (20 ml) was added and the solution was left at room temperature for 5 hours. TEA (300 ml) was added, the mixture was diluted with DMF (3.5 L) and water (600 ml) and extracted with hexane (3×3L). The mixture was diluted with water (1.6 L) and extracted with the mixture of toluene (12 L) and hexanes (9 L). The upper layer was washed with DMF-water (7:3 v/v, 3×3 L) and water (3×3 L). The organic layer was dried (Na 2 SO 4 ), filtered and evaporated. The residue was co-evaporated with acetonitrile (2×2 L) under reduced pressure and dried in a vacuum oven (25° C., 0.1 mm Hg, 40 h) to afford 1526 g of an off-white foamy solid (95%).  
     [0179] Preparation of 5′-O-Dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methylcytidine Intermediate  
     [0180] To a 50 L Schott glass-lined steel reactor equipped with an electric stirrer, reagent addition pump (connected to an addition funnel), heating/cooling system, internal thermometer and argon gas line was added 5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methyl-uridine (2.616 kg, 4.23 mol, purified by base extraction only and no scrub column), anhydrous acetonitrile (20 L), and TEA (9.5 L, 67.7 mol, 16 eq). The mixture was chilled with stirring to −10° C. internal temperature (external −20° C.). Trimethylsilylchloride (1.60 L, 12.7 mol, 3.0 eq) was added over 30 min. while maintaining the internal temperature below −5° C., followed by a wash of anhydrous acetonitrile (1 L). (Note: the reaction is mildly exothermic and copious hydrochloric acid fumes form over the course of the addition). The reaction was allowed to warm to 0° C. and the reaction progress was confirmed by TLC (EtOAc, R f  0.68 and 0.87 for starting material and silyl product, respectively). Upon completion, triazole (2.34 kg, 33.8 mol, 8.0 eq) was added the reaction was cooled to −20° C. internal temperature (external −30° C.). Phosphorous oxychloride (793 mL, 8.51 mol, 2.01 eq) was added slowly over 60 min so as to maintain the temperature between −20° C. and −10° C. (note: strongly exothermic), followed by a wash of anhydrous acetonitrile (1 L). The reaction was warmed to 0° C. and stirred for 1 h, at which point it was an off-white thick suspension. TLC indicated a complete conversion to the triazole product (EtOAc, R f  0.87 to 0.75 with the product spot glowing in long wavelength UV light). The reaction was cooled to −15° C. and water (5 L) was slowly added at a rate to maintain the temperature below +10° C. in order to quench the reaction and to form a homogenous solution. (Caution: this reaction is initially very strongly exothermic). Approximately one-half of the reaction volume (22 L) was transferred by air pump to another vessel, diluted with EtOAc (12 L) and extracted with water (2×8 L). The second half of the reaction was treated in the same way. The combined aqueous layers were back-extracted with EtOAc (8 L) The organic layers were combined and concentrated in a 20 L rotary evaporator to an oily foam. The foam was coevaporated with anhydrous acetonitrile (4 L) to remove EtOAc. (note: dioxane may be used instead of anhydrous acetonitrile if dried to a hard foam). The residue was dissolved in dioxane (2 L) and concentrated ammonium hydroxide (750 mL) was added. A homogenous solution formed in a few minutes and the reaction was allowed to stand overnight  
     [0181] TLC indicated a complete reaction (CH 2 Cl 2 -acetone-MeOH, 20:5:3, R f  0.51). The reaction solution was concentrated on a rotary evaporator to a dense foam and slowly redissolved in warm CH 2 Cl 2  (4 L, 40° C.) and transferred to a 20 L glass extraction vessel equipped with a air-powered stirrer. The organic layer was extracted with water (2×6 L) to remove the triazole by-product. (Note: In the first extraction an emulsion formed which took about 2 h to resolve). The water layer was back-extracted with CH 2 Cl 2  (2×2 L), which in turn was washed with water (3 L). The combined organic layer was concentrated in 2×20 L flasks to a gum and then recrystallized from EtOAc seeded with crystalline product. After sitting overnight, the first crop was collected on a 25 cm Coors Buchner funnel and washed repeatedly with EtOAc until a white free-flowing powder was left (about 3×3 L). The filtrate was concentrated to an oil recrystallized from EtOAc, and collected as above. The solid was air-dried in pans for 48 h, then further dried in a vacuum oven (50° C., 0.1 mm Hg, 17 h) to afford 2248 g of a bright white, dense solid (86%). An HPLC analysis indicated both crops to be 99.4% pure and NMR spectroscopy indicated only a faint trace of EtOAc remained.  
     [0182] Preparation of 5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-N-4-benzoyl-5-methyl-cytidine Penultimate Intermediate:  
     [0183] Crystalline 5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methyl-cytidine (1000 g, 1.62 mol) waS suspended in anhydrous DMF (3 kg) at ambient temperature and stirred under an Ar atmosphere. Benzoic anhydride (439.3 g, 1.94 mol) was added in one portion. The solution clarified after 5 hours and was stirred for 16 h. HPLC indicated 0.45% starting material remained (as well as 0.32% N4, 3′-O-bis Benzoyl). An additional amount of benzoic anhydride (6.0 g, 0.0265 mol) was added and after 17 h, HPLC indicated no starting material was present. TEA (450 mL, 3.24 mol) and toluene (6 L) were added with stirring for 1 minute. The solution was washed with water (4×4 L), and brine (2×4 L). The organic layer was partially evaporated on a 20 L rotary evaporator to remove 4 L of toluene and traces of water. HPLC indicated that the bis benzoyl side product was present as a 6% impurity. The residue was diluted with toluene (7 L) and anhydrous DMSO (200 mL, 2.82 mol) and sodium hydride (60% in oil, 70 g, 1.75 mol) was added in one portion with stirring at ambient temperature over 1 h. The reaction was quenched by slowly adding then washing with aqueous citric acid (10%, 100 mL over 10 min, then 2×4 L), followed by aqueous sodium bicarbonate (2%, 2 L), water (2×4 L) and brine (4 L). The organic layer was concentrated on a 20 L rotary evaporator to about 2 L total volume. The residue was purified by silica gel column chromatography (6 L Buchner funnel containing 1.5 kg of silica gel wetted with a solution of EtOAc-hexanes-TEA(70:29:1)). The product was eluted with the same solvent (30 L) followed by straight EtOAc (6 L). The fractions containing the product were combined, concentrated on a rotary evaporator to a foam and then dried in a vacuum oven (50° C., 0.2 mm Hg, 8 h) to afford 1155 g of a crisp, white foam (98%). HPLC indicated a purity of &gt;99.7%.  
     [0184] Preparation of [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N 4 -benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE 5-Me-C Amidite)  
     [0185] 5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N 4 -benzoyl-5-methylcytidine (1082 g, 1.5 mol) was dissolved in anhydrous DMF (2 L) and co-evaporated with toluene (300 ml) at 50° C. under reduced pressure. The mixture was cooled to room temperature and 2-cyanoethyl tetraisopropylphosphorodiamidite (680 g, 2.26 mol) and tetrazole (52.5 g, 0.75 mol) were added. The mixture was shaken until all tetrazole was dissolved, N-methylimidazole (30 ml) was added, and the mixture was left at room temperature for 5 hours. TEA (300 ml) was added, the mixture was diluted with DMF (1 L) and water (400 ml) and extracted with hexane (3×3 L). The mixture was diluted with water (1.2 L) and extracted with a mixture of toluene (9 L) and hexanes (6 L). The two layers were separated and the upper layer was washed with DMF-water (60:40 v/v, 3×3 L) and water (3×2 L). The organic layer was dried (Na 2 SO 4 ), filtered and evaporated. The residue was co-evaporated with acetonitrile (2×2 L) under reduced pressure and dried in a vacuum oven (25° C., 0.1 mm Hg, 40 h) to afford 1336 g of an off-white foam (97%).  
     [0186] Preparation of [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N 6 -benzoyladenosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE A Amdite)  
     [0187] 5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N 6 -benzoyladenosine (purchased from Reliable Biopharmaceutical, St. Lois, Mo.), 1098 g, 1.5 mol) was dissolved in anhydrous DMF (3 L) and co-evaporated with toluene (300 ml) at 50° C. The mixture was cooled to room temperature and 2-cyanoethyl tetraisopropylphosphorodiamidite (680 g, 2.26 mol) and tetrazole (78.8 g, 1.24 mol) were added. The mixture was shaken until all tetrazole was dissolved, N-methylimidazole (30 ml) was added, and mixture was left at room temperature for 5 hours. TEA (300 ml) was added, the mixture was diluted with DMF (1 L) and water (400 ml) and extracted with hexanes (3×3 L). The mixture was diluted with water (1.4 L) and extracted with the mixture of toluene (9 L) and hexanes (6 L). The two layers were separated and the upper layer was washed with DMF-water (60:40, v/v, 3×3 L)-and water (3×2 L). The organic layer was dried (Na 2 SO 4 ), filtered and evaporated to a sticky foam. The residue was co-evaporated with acetonitrile (2.5 L) under reduced pressure and dried in a vacuum oven (25° C., 0.1 mm Hg, 0.40 h) to afford 1350 g of an off-white foam solid (96%).  
     [0188] Prepartion of [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N 4 -isobutyrylguanosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE G Amidite)  
     [0189] 5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N-isobutyrlguanosine (purchased from Reliable Biopharmaceutical, St. Louis, Mo., 1426 g, 2.0 mol) was dissolved in anhydrous DMF (2 L). The solution was co-evaporated with toluene (200 ml) at 50° C., cooled to room temperature and 2-cyanoethyl tetraisopropylphosphorodiamidite (900 g, 3.0 mol) and tetrazole (68 g, 0.97 mol) were added. The mixture was shaken until all tetrazole was dissolved, N-methylimidazole (30 ml) was added, and the mixture was left at room temperature for 5 hours. TEA (300 ml) was added, the mixture was diluted with DMF (2 L) and water (600 ml) and extracted with hexanes (3×3 L). The mixture was diluted with water (2 L) and extracted with a mixture of toluene (10 L) and hexanes (5 L). The two layers were separated and the upper layer was washed with DMF-water (60:40, v/v, 3×3 L). EtOAc (4 L) was added and the solution was washed with water (3×4 L). The organic layer was dried (Na 2 SO 4 ), filtered and evaporated to approx. 4 kg. Hexane (4 L) was added, the mixture was shaken for 10 min, and the supernatant liquid was decanted. The residue was co-evaporated with acetonitrile (2×2 L) under reduced pressure and dried in a vacuum oven (25° C., 0.1 mm Hg, 40 h) to afford 1660 g of an off-white foamy solid (91%).  
     [0190] 2′-O-(Aminooxyethyl) nucleoside amidites and 2′-O-(dimethylaminooxyethyl) Nucleoside Amidites  
     [0191] 2′-(Dimethylaminooxyethoxy) Nucleoside Amidites  
     [0192] 2′-(Dimethylaminooxyethoxy) nucleoside amidites (also known in-the art-as 2°-O-(dimethylaminooxyethyl) nucleoside amidites) are prepared as described in the following paragraphs. Adenosine, cytidine and guanosine nucleoside amidites are prepared similarly to the thymidine (5-methyluridine) except the exocyclic amines are protected with a benzoyl moiety in the case of adenosine and cytidine and with isobutyryl in the case of guanosine.  
     [0193] 5′-O-tert-Butyldiphenylsilyl-0 2 -2′-anhydro-5-methyluridine  
     [0194] O 2 -2′-anhydro-5-methyluridine (Pro. Bio. Sint., Varese, Italy, 100.0 g, 0.416 mmol), dimethylaminopyridine (0.66 g, 0.013 eq, 0.0054 mmol) were dissolved in dry pyridine (500 ml) at ambient temperature under an argon atmosphere and with mechanical stirring. tert-Butyldiphenylchlorosilane (125.8 g, 119.0 mL, l.leq, 0.458 mmol) was added in one portion. The reaction was stirred for 16 h at ambient temperature. TLC (Rf 0.22, EtOAc) indicated a complete reaction. The solution was concentrated under reduced pressure to a thick oil. This was partitioned between CH 2 Cl 2  (1 L) and saturated sodium bicarbonate (2×1 L) and brine (1 L). The organic layer was dried over sodium sulfate, filtered, and concentrated under reduced pressure to a thick oil. The oil was dissolved in a 1:1 mixture of EtOAc and ethyl ether (600 mL) and cooling the solution to −10° C. afforded a white crystalline solid which was collected by filtration, washed with ethyl ether (3×2 00 mL) and dried (40° C., 1 mm Hg, 24 h) to afford 149 g of white solid (74.8%). TLC and NMR spectroscopy were consistent with pure product.  
     [0195] 5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine  
     [0196] In the fume hood, ethylene glycol (350 mL, excess) was added cautiously with manual stirring to a 2 L stainless steel pressure reactor containing borane in tetrahydrofuran (1.0 M, 2.0 eq, 622 mL). (Caution: evolves hydrogen gas). 5′-O-tert-Butyldiphenylsilyl-0 2 -2′-anhydro-5-methyluridine (149 g, 0.311 mol) and sodium bicarbonate (0.074 g, 0.003 eq) were added with manual stirring. The reactor was sealed and heated in an oil bath until an internal temperature of 160° C. was reached and then maintained for 16 h (pressure &lt;100 psig). The reaction vessel was cooled to ambient temperature and opened. TLC (EtOAc, R f  0.67 for desired product and R f  0.82 for ara-T side product) indicated about 70% conversion to the product. The solution was concentrated under reduced pressure (10 to 1 mm Hg) in a warm water bath (40-100° C.) with the more extreme conditions used to remove the ethylene glycol. (Alternatively, once the THF has evaporated the solution can be diluted with water and the product extracted into EtOAc). The residue was purified by column chromatography (2 kg silica gel, EtOAc-hexanes gradient 1:1 to 4:1). The appropriate fractions were combined, evaporated and dried to afford 84 g of a white crisp foam (50%), contaminated starting material (17.4 g, 12% recovery) and pure reusable starting material (20 g, 13% recovery). TLC and NMR spectroscopy were consistent with 99% pure product.  
     [0197] 2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine  
     [0198] 5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine (20 g, 36.98 mmol) was mixed with triphenylphosphine (11.63 g, 44.36 mmol) and N-hydroxyphthalimide (7.24 g, 44.36 mmol) and dried over P2O under high vacuum for two days at 40° C. The reaction mixture was flushed with argon and dissolved in dry THF (369.8 mL, Aldrich, sure seal bottle). Diethyl-azodicarboxylate (6.98 mL, 44.36 mmol) was added dropwise to the reaction mixture with the rate of addition maintained such that the resulting deep red coloration is just discharged before adding the next drop. The reaction mixture was stirred for 4 hrs., after which time TLC (EtOAc:hexane, 60:40) indicated that the reaction was complete. The solvent was evaporated in vacuuo and the residue purified by flash column chromatography (eluted with 60:40 EtOAc:hexane), to yield 2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine as white foam (21.819 g, 86%) upon rotary evaporation.  
     [0199] 5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine  
     [0200] 2-o-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine (3.1 g, 4.5 mmol) was dissolved in dry CH 2 Cl 2  (4.5 mL) and methylhydrazine (300 mL, 4.64 mmol) was added dropwise at −10° C. to 0° C. After 1 h the mixture was filtered, the filtrate washed with ice cold CH 2 Cl 2 , and the combined organic phase was washed with water and brine and dried (anhydrous Na 2 SO 4 ). The solution was filtered and evaporated to afford 2′-O-(aminooxyethyl) thymidine, which was then dissolved in MeOH (67.5 mL). Formaldehyde (20% aqueous solution, w/w, 1.1 eq.) was added and the resulting mixture was stirred for 1 h. The solvent was removed under vacuum and the residue was purified by column chromatography to yield 5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy) ethyl]-5-methyluridine as white foam (1.95 g, 78%) upon rotary evaporation. 5′-O-tert-Butyldiphenylsilyl-2′-O-[N,N dimethylaminooxyethyl]-5-methyluridine 5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine (1.77 g, 3.12 mmol) was dissolved in a solution of 1M pyridinium p-toluenesulfonate (PPTS) in dry MeOH (30.6 mL) and cooled to 10° C. under inert atmosphere. Sodium cyanoborohydride (0.39 g, 6.13 mmol) was added and the reaction mixture was stirred. After 10 minutes the reaction was warmed to room temperature and stirred for 2 h. while the progress of the reaction was monitored by TLC (5% MeOH in CH 2 Cl 2 ). Aqueous NaHCO 3  solution (5%, 10 mL) was added and the product was extracted with EtOAc (2×20 mL). The organic phase was dried over anhydrous Na 2 SO 4 , filtered, and evaporated to dryness. This entire procedure was repeated with the resulting residue, with the exception that formaldehyde (20% w/w, 30 mL, 3.37 mol) was added upon dissolution of the residue in the PPTS/MeOH solution. After the extraction and evaporation, the residue was purified by flash column chromatography and (eluted with 5% MeOH in CH 2 Cl 2 ) to afford 5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine as a white foam (14.6 g, 80%) upon rotary evaporation.  
     [0201] 2′-O-(dimethylaminooxyethyl)-5-methyluridine  
     [0202] Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) was dissolved in dry THF and TEA (1.67 mL, 12 mmol, dry, stored over KOH) and added to 5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine (1.40 g, 2.4 mmol). The reaction was stirred at room temperature for 24 hrs and monitored by TLC (5% MeOH in CH 2 Cl 2 ). The solvent was removed under vacuum and the residue purified by flash column chromatography (eluted with 10% MeOH in CH 2 Cl 2 ) to afford 2′-O-(dimethylaminooxyethyl)-5-methyluridine (766 mg, 92.5%) upon rotary evaporation of the solvent.  
     [0203] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine  
     [0204] 2′-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17 mmol) was dried over P 2 O 5  under high vacuum overnight at 40° C., co-evaporated with anhydrous pyridine (20 mL), and dissolved in pyridine (11 mL) under argon atmosphere. 4-dimethylaminopyridine (26.5 mg, 2.60 mmol) and 4,4′-dimethoxytrityl chloride (880 mg, 2.60 mmol) were added to the pyridine solution and the reaction mixture was stirred at room temperature until all of the starting material had reacted. Pyridine was removed under vacuum and the residue was purified by column chromatography (eluted with 10% MeOH in CH 2 Cl 2  containing a few drops of pyridine) to yield 5′-O-DMT-2′-O-(dimethylamino-oxyethyl)-5-methyluridine (1.13 g, 80%) upon rotary evaporation. 5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite] 
     [0205] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine (1.08 g, 1.67 mmol) was co-evaporated with toluene (20 mL), N,N-diisopropylamine tetrazonide (0.29 g, 1.67 mmol) was added and the mixture was dried over P 2 O 5  under high vacuum overnight at 40° C. This was dissolved in anhydrous acetonitrile (8.4 mL) and 2-cyanoethyl-N,N,N 1 ,N 1 -tetraisopropylphosphoramidite (2.12 mL, 6.08 mmol) was added. The reaction mixture was stirred at ambient temperature for 4 h under inert atmosphere. The progress of the reaction was monitored by TLC (hexane:EtOAc 1:1). The solvent was evaporated, then the residue was dissolved in EtOAc (70 mL) and washed with 5% aqueous NaHCO 3  (40 mL). The EtOAc layer was dried over anhydrous Na 2 SO 4 , filtered, and concentrated. The residue obtained was purified by column chromatography (EtOAc as eluent) to afford 5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite] as a foam (1.04 g, 74.9%) upon rotary evaporation.  
     [0206] 2′-(Aminooxyethoxy) nucleoside Amidites  
     [0207] 2′-(Aminooxyethoxy) nucleoside amidites (also known in the art as 2′-O-(aminooxyethyl) nucleoside amidites) are prepared as described in the following paragraphs. Adenosine, cytidine and thymidine nucleoside amidites are prepared similarly.  
     [0208] N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite] 
     [0209] The 2′-O-aminooxyethyl guanosine analog may be obtained by selective 2′-O-alkylation of diaminopurine riboside. Multigram quantities of diaminopurine riboside may be purchased from Schering AG (Berlin) to provide 2′-O-(2-ethylacetyl) diaminopurine riboside along with aminor amount of the 3′-O-isomer. 2′-O-(2-ethylacetyl) diaminopurine riboside may be resolved and converted to 2′-O-(2-ethylacetyl)guanosine by treatment with adenosine deaminase. (McGee, D. P. C., Cook, P. D., Guinosso, C. J., WO 94/02501 A1 940203.) Standard protection procedures should afford 2′-O-(2-ethylacetyl)-5′-O-(4′,4′-dimethoxytrityl)guanosine and 2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine which may be reduced to provide 2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-hydroxyethyl)-5′-O-(4,4′-dimethoxytrityl)guanosine. As before the hydroxyl group may be displaced by N-hydroxyphthalimide via a Mitsunobu reaction, and the protected nucleoside may be phosphitylated as usual to yield 2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-([2-phthalmidoxy]ethyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite].  
     [0210] 2′-dimethylaminoethoxyethoxy (2′-DMAEOE) Nucleoside Amidites  
     [0211] 2′-dimethylaminoethoxyethoxy nucleoside amidites (also known in the art as 2′-O-dimethylaminoethoxyethyl, i.e., 2′-O—CH 2 —O—CH 2 —N(CH 2 ) 2 , or 2′-DMAEOE nucleoside amidites) are prepared as follows. Other nucleoside amidites are prepared similarly.  
     [0212] 2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine  
     [0213] 2[2-(Dimethylamino)ethoxy]ethanol (Aldrich, 6.66 g, 50 mmol) was slowly added to a solution of borane in tetra-hydrofuran (1 M, 10 mL, 10 mmol) with stirring in a 100 ML bomb. (Caution: Hydrogen gas evolves as the solid dissolves). O 2 —,2′-anhydro-5-methyluridine (1.2 g, 5 mmol), and sodium bicarbonate (2.5 mg) were added and the bomb was sealed, placed in an oil bath and heated to 155° C. for 26 h. then cooled to room temperature. The crude solution was concentrated, the residue was diluted with water (200 mL) and extracted with hexanes (200 mL). The product was extracted from the aqueous layer with EtOAc (3×200 mL) and the combined organic layers were washed once with water, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by silica gel column chromatography (eluted with 5:100:2 MeOH/CH 2 Cl 2 /TEA) as the eluent. The appropriate fractions were combined and evaporated to afford the product as a white solid.  
     [0214] 5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy) ethyl)]-5-methyl Uridine  
     [0215] To 0.5 g (1.3 mmol) of 2′-O-[2(2-N,N-dimethylamino-ethoxy)ethyl)]-5-methyl uridine in anhydrous pyridine (8 mL), was added TEA (0.36 mL) and dimethoxytrityl chloride (DMT-Cl, 0.87 g, 2 eq.) and the reaction was stirred for 1 h. The reaction mixture was poured into water (200 mL) and extracted with CH 2 Cl 2  (2×200 mL). The combined CH 2 Cl 2  layers were washed with saturated NaHCO 3  solution, followed by saturated NaCl solution, dried over anhydrous sodium sulfate, filtered and evaporated. The residue was purified by silica gel column chromatography (eluted with 5:100:1 MeOH/CH 2 Cl 2 /TEA) to afford the product.  
     [0216] 5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyl uridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite  
     [0217] Diisopropylaminotetrazolide (0.6 g) and 2-cyanoethoxy-N,N-diisopropyl phosphoramidite (1.1 mL, 2 eq.) were added to a solution of 5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyluridine (2.17 g, 3 mmol) dissolved in CH 2 c1 2  (20 mL) under an atmosphere of argon. The reaction mixture was stirred overnight and the solvent evaporated. The resulting residue was purified by silica gel column chromatography with EtOAc as the eluent to afford the title compound.  
     Example 2  
     [0218] Oligonucleotide Synthesis  
     [0219] Unsubstituted and substituted phosphodiester (P═O) oligonucleotides are synthesized on an automated DNA synthesizer (Applied Biosystems model 394) using standard phosphoramidite chemistry with oxidation by iodine.  
     [0220] Phosphorothioates (P═S) are synthesized similar to phosphodiester oligonucleotides with the following exceptions: thiation was effected by utilizing a 10% w/v solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the oxidation of the phosphite linkages. The thiation reaction step time was increased to 180 sec and preceded by the normal capping step. After cleavage from the CPG column and deblocking in concentrated ammonium hydroxide at 55° C. (12-16 hr), the oligonucleotides were recovered by precipitating with &gt;3 volumes of ethanol from a 1 M NH 4 OAc solution. Phosphinate oligonucleotides are prepared as described in U.S. Pat. No. 5,508,270, herein incorporated by reference.  
     [0221] Alkyl phosphonate oligonucleotides are prepared as described in U.S. Pat. No. 4,469,863, herein incorporated by reference. 3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared as described in U.S. Pat. Nos. 5,610,289 or 5,625,050, herein incorporated by reference.  
     [0222] Phosphoramidite oligonucleotides are prepared as described in U.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated by reference.  
     [0223] Alkylphosphonothioate oligonucleotides are prepared as described in published PCT applications PCT/US94/00902 and PCT/US93/06976 (published as WO 94/17093 and WO 94/02499, respectively), herein incorporated by reference.  
     [0224] 3′-Deoxy-3′-amino phosphoramidate oligonucleotides are prepared as described in U.S. Pat. No. 5,476,925, herein incorporated by reference.  
     [0225] Phosphotriester oligonucleotides are prepared as described in U.S. Pat. No. 5,023,243, herein incorporated by reference.  
     [0226] Borano phosphate oligonucleotides are prepared as described in U.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.  
     Example 3  
     [0227] Oligonucleoside Synthesis  
     [0228] Methylenemethylimino linked oligonucleosides, also identified as MMI linked oligonucleosides, methylenedimethylhydrazo linked oligonucleosides, also identified as MDH linked oligonucleosides, and methylenecarbonylamino linked oligonucleosides, also identified as amide-3 linked oligonucleosides, and methyleneaminocarbonyl linked oligonucleosides, also identified as amide-4 linked oligonucleosides, as well as mixed backbone compounds having, for instance, alternating MMI and P═O or P═S linkages are prepared as described in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of which are herein incorporated by reference.  
     [0229] Formacetal and thioformacetal linked oligonucleosides are prepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporated by reference.  
     [0230] Ethylene oxide linked oligonucleosides are prepared as described in U.S. Pat. No. 5,223,618, herein incorporated by reference.  
     Example 4  
     [0231] PNA Synthesis  
     [0232] Peptide nucleic acids (PNAs) are prepared in accordance with any of the various procedures referred to in Peptide Nucleic Acids (PNA): Synthesis, Properties and Potential Applications,  Bioorganic  &amp;  Medicinal Chemistry,  1996, 4, 5-23. They may also be prepared in accordance with U.S. Pat. Nos. 5,539,082, 5,700,922, and 5,719,262, herein incorporated by reference.  
     Example 5  
     [0233] Synthesis of Chimeric Oligonucleotides  
     [0234] Chimeric oligonucleotides, oligonucleosides or mixed oligonucleotides/oligonucleosides of the invention can be of several different types. These include a first type wherein the “gap” segment of linked nucleosides is positioned between 5′ and 3′ “wing” segments of linked nucleosides and a second “open end” type wherein the “gap” segment is located at either the 3′ or the 5′ terminus of the oligomeric compound. Oligonucleotides of the first type are also known in the art as “gapmers” or gapped oligonucleotides. Oligonucleotides of the second type are also known in the art as “hemimers” or “wingmers”.  
     [0235] [2′-O-Me]—[2′-deoxy]—[2′-O-Me] Chimeric Phosphorothioate Oligonucleotides  
     [0236] Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and 2′-deoxy phosphorothioate oligonucleotide segments are synthesized using an Applied Biosystems automated DNA synthesizer Model 394, as above. Oligonucleotides are synthesized using the automated synthesizer and 2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and 5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings. The standard synthesis cycle is modified by incorporating coupling steps with increased reaction times for the 5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite. The fully protected oligonucleotide is cleaved from the support and deprotected in concentrated ammonia (NH 4 OH) for 12-16 hr at 55° C. The deprotected oligo is then recovered by an appropriate method (precipitation, column chromatography, volume reduced in vacuo and analyzed spetrophotometrically for yield and for purity by capillary electrophoresis and by mass spectrometry.  
     [0237] [2′-O-(2-Methoxyethyl)]—[2′-deoxy]—[2′-O-(Methoxyethyl)] Chimeric Phosphorothioate Oligonucleotides  
     [0238] [2′-O-(2-methoxyethyl)]—[2′-deoxy]—[-2′-O-(methoxyethyl)] chimeric phosphorothioate oligonucleotides were prepared as per the procedure above for the 2′-O-methyl chimeric oligonucleotide, with the substitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites.  
     [0239] [2′-O-(2-Methoxyethyl)Phosphodiester]—[2′-deoxy Phosphorothioate]—[2′-O-(2-Methoxyethyl) Phosphodiester] Chimeric Oligonucleotides  
     [0240] [2′-O-(2-methoxyethyl phosphodiester]—[2′-deoxy phosphorothioate]—[2′-O-(methoxyethyl) phosphodiester] chimeric oligonucleotides are prepared as per the above procedure for the 2′-O-methyl chimeric oligonucleotide with the substitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidation with iodine to generate the phosphodiester internucleotide linkages within the wing portions of the chimeric structures and sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) to generate the phosphorothioate internucleotide linkages for the center gap.  
     [0241] Other chimeric oligonucleotides, chimeric oligonucleosides and mixed chimeric oligonucleotides/oligonucleosides are synthesized according to U.S. Pat. No. 5,623,065, herein incorporated by reference.  
     Example 6  
     [0242] Oligonucleotide Isolation  
     [0243] After cleavage from the controlled pore glass solid support and deblocking in concentrated ammonium hydroxide at 55° C. for 12-16 hours, the oligonucleotides or oligonucleosides are recovered by precipitation out of 1 M NH 4 OAc with &gt;3 volumes of ethanol. Synthesized oligonucleotides were analyzed by electrospray mass spectroscopy (molecular weight determination) and by capillary gel electrophoresis and judged to be at least 70% full length material. The relative amounts of phosphorothioate and phosphodiester linkages obtained in the synthesis was determined by the ratio of correct molecular weight relative to the −16 amu product (+/−32+/−48). For some studies oligonucleotides were purified by HPLC, as described by Chiang et al.,  J. Biol. Chem.  1991, 266, 18162-18171. Results obtained with HPLC-purified material were similar to those obtained with non-HPLC purified material.  
     Example 7  
     [0244] Oligonucleotide Synthesis—96 Well Plate Format  
     [0245] Oligonucleotides were synthesized via solid phase P(III) phosphoramidite chemistry on an automated synthesizer capable of assembling 96 sequences simultaneously in a 96-well format. Phosphodiester internucleotide linkages were afforded by oxidation with aqueous iodine. Phosphorothioate internucleotide linkages were generated by sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) in anhydrous acetonitrile. Standard base-protected beta-cyanoethyl-diiso-propyl phosphoramidites were purchased from commercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., or Pharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesized as per standard or patented methods. They are utilized as base protected beta-cyanoethyldiisopropyl phosphoramidites.  
     [0246] Oligonucleotides were cleaved from support and deprotected with concentrated NH 4 OH at elevated temperature (55-60° C.) for 12-16 hours and the released product then dried in vacuo. The dried product was then re-suspended in sterile water to afford a master plate from which all analytical and test plate samples are then diluted utilizing robotic pipettors.  
     Example 8  
     [0247] Oligonucleotide Analysis—96-Well Plate Format  
     [0248] The concentration of oligonucleotide in each well was assessed by dilution of samples and UV absorption spectroscopy. The full-length integrity of the individual products was evaluated by capillary electrophoresis (CE) in either the 96-well format (Beckman P/ACETM MDQ) or, for individually prepared samples, on a commercial CE apparatus (e.g., Beckman P/ACETM 5000, ABI 270). Base and backbone composition was confirmed by mass analysis of the compounds utilizing electrospray-mass spectroscopy. All assay test plates were diluted from the master plate using single and multi-channel robotic pipettors. Plates were judged to be acceptable if at least 85% of the compounds on the plate were at least 85% full length.  
     Example 9  
     [0249] Cell Culture and Oligonucleotide Treatment  
     [0250] The effect of antisense compounds on target nucleic acid expression can be tested in any of a variety of cell types provided that the target nucleic acid is present at measurable levels. This can be routinely determined using, for example, PCR or Northern blot analysis. The following cell types are provided for illustrative purposes, but other cell types can be routinely used, provided that the target is expressed in the cell type chosen. This can be readily determined by methods routine in the art, for example Northern blot analysis, ribonuclease protection assays, or RT-PCR.  
     [0251] T-24 Cells:  
     [0252] The human transitional cell bladder carcinoma cell line T-24 was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). T-24 cells were routinely cultured in complete McCoy&#39;s 5A basal media (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10% fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. Cells were seeded into 96-well plates (Falcon-Primaria #3872) at a density of 7000 cells/well for use in RT-PCR analysis.  
     [0253] For Northern blotting or other analysis, cells may be seeded onto 100 mm or other standard tissue culture plates and treated similarly, using appropriate volumes of medium and oligonucleotide.  
     [0254] A549 cells:  
     [0255] The human lung carcinoma cell line A549 was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). A549 cells were routinely cultured in DMEM basal media (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10% fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence.  
     [0256] NHDF cells:  
     [0257] Human neonatal dermal fibroblast (NHDF) were obtained from the Clonetics Corporation (Walkersville, Md.). NHDFs were routinely maintained in FibrQblast GrQwth Medium (Clonetics Corporation, Walkersville, Md.) supplemented as recommended by the supplier. Cells were maintained for up to 10 passages as recommended by the supplier.  
     [0258] HEK cells:  
     [0259] Human embryonic keratinocytes (HEK) were obtained from the Clonetics Corporation (Walkersville, Md.). HEKs were routinely maintained in Keratinocyte Growth Medium (Clonetics Corporation, Walkersville, Md.) formulated as recommended by the supplier. Cells were routinely maintained for up to 10 passages as recommended by the supplier.  
     [0260] Treatment with Antisense Compounds:  
     [0261] When cells reached 70% confluency, they were treated with oligonucleotide. For cells grown in 96-well plates, wells were washed once with 100 μL OPTI-MEM™-1 reduced-serum medium (Invitrogen Corporation, Carlsbad, Calif.) and then treated with 130 μL of OPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTIN™ (Invitrogen Corporation, Carlsbad, Calif.) and the desired concentration of oligonucleotide. After 4-7 hours of treatment, the medium was replaced with fresh medium. Cells were harvested 16-24 hours after oligonucleotide treatment.  
     [0262] The concentration of oligonucleotide used varies from cell line to cell line. To determine the optimal oligonucleotide concentration for a particular cell line, the cells are treated with a positive control oligonucleotide at a range of concentrations. For human cells the positive control oligonucleotide is selected from either ISIS 13920 (TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1) which is targeted to human H-ras, or ISIS 18078, (GTGCGCGCGAGCCCGAAATC, SEQ ID NO: 2) which is targeted to human Jun-N-terminal kinase-2 (JNK2). Both controls are 2′-O-methoxyethyl gapmers (2′-O-methoxyethyls shown in bold) with a phosphorothioate backbone. For mouse or rat cells the positive control oligonucleotide is ISIS 15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 3, a 2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown in bold) with a phosphorothioate backbone which is targeted to both mouse and rat c-raf. The concentration of positive control oligonucleotide that results in 80% inhibition of c-Ha-ras (for ISIS 13920) or c-raf (for ISIS 15770) mRNA is then utilized as the screening concentration for new oligonucleotides in subsequent experiments for that cell line. If 80% inhibition is not achieved, the lowest concentration of positive control oligonucleotide that results in 60% inhibition of H-ras or c-raf mRNA is then utilized as the oligonucleotide screening concentration in subsequent experiments for that cell line. If 60% inhibition is not achieved, that particular cell line is deemed as unsuitable for oligonucleotide transfection experiments.  
     Example 10  
     [0263] Analysis of Oligonucleotide Inhibition of Notch1 Expression  
     [0264] Antisense modulation of Notch1 expression can be assayed in a variety of ways known in the art. For example, Notch1 mRNA levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or real-time PCR (RT-PCR). Real-time quantitative PCR is presently preferred. RNA analysis can be performed on total cellular RNA or poly(A)+mRNA.-The preferred method of RNA analysis of the present invention is the use of total cellular RNA as described in other examples herein. Methods of RNA isolation are taught in, for example, Ausubel, F. M. et al.,  Current Protocols in Molecular Biology , Volume 1, pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley &amp; Sons, Inc., 1993. Northern blot analysis is routine in the art and is taught in, for example, Ausubel, F. M. et al.,  Current Protocols in Molecular Biology, Volume  1, pp. 4.2.1-4.2.9, John Wiley &amp; Sons, Inc., 1996. Real-time quantitative (PCR) can be conveniently accomplished using the commercially available ABI PRISM™ 7700 Sequence Detection System, available from PE-Applied Biosystems, Foster City, Calif. and used according to manufacturer&#39;s instructions.  
     [0265] Protein levels of Notch1 can be quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), ELISA or fluorescence-activated cell sorting (FACS). Antibodies directed to Notch1 can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.), or can be prepared via conventional antibody generation methods. Methods for preparation of polyclonal antisera are taught in, for example, Ausubel, F. M. et al., ( Current Protocols in Molecular Biology , Volume 2, pp. 11.12.1-11.12.9, John Wiley &amp; Sons, Inc., 1997). Preparation of monoclonal antibodies is taught in, for example, Ausubel, F. M. et al., ( Current Protocols in Molecular Biology , Volume 2, pp. 11.4.1-11.11.5, John Wiley &amp; Sons, Inc., 1997).  
     [0266] Immunoprecipitation methods are standard in the art and can be found at, for example, Ausubel, F. M. et al., ( Current Protocols in Molecular Biology , Volume 2, pp. 10.16.1-10.16.11, John Wiley &amp; Sons, Inc., 1998). Western blot (immunoblot) analysis is standard in the art and can be found at, for example, Ausubel, F. M. et al., (Current Protocols in Molecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley &amp; Sons, Inc., 1997). Enzyme-linked immunosorbent assays (ELISA) are standard in the art and can be found at, for example, Ausubel, F. M. et al., ( Current Protocols in Molecular Biology , Volume 2, pp. 11.2.1-11.2.22, John Wiley &amp; Sons, Inc., 1991).  
     Example 11  
     [0267] Poly(A)+ mRNA Isolation  
     [0268] Poly(A)+ mRNA was isolated according to Miura et al., (Clin. Chem., 1996, 42, 1758-1764). Other methods for poly(A)+ mRNA isolation are taught in, for example, Ausubel, F. M. et al., ( Current Protocols in Molecular Biology , Volume 1, pp. 4.5.1-4.5.3, John Wiley &amp; Sons, Inc., 1993). Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 60 μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) was added to each well, the plate was gently agitated and then incubated at room temperature for five minutes. 55 μL of lysate was transferred to Oligo d(T) coated 96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated for 60 minutes at room temperature, washed 3 times with 200 μL of wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the plate was blotted on paper towels to remove excess wash buffer and then air-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH 7.6), preheated to 70° C., was added to each well, the plate was incubated on a 90° C. hot plate for 5 minutes, and the eluate was then transferred to a fresh 96-well plate.  
     [0269] Cells grown on 100 mm or other standard plates may be treated similarly, using appropriate volumes of all solutions.  
     Example 12  
     [0270] Total RNA Isolation  
     [0271] Total RNA was isolated using an RNEASY 96 TM kit and buffers purchased from Qiagen Inc. (Valencia, Calif.) following the manufacturer&#39;s recommended procedures. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 150 μL Buffer RLT was added to each well and the plate vigorously agitated for 20 seconds. 150 μL of 70% ethanol was then added to each well and the contents mixed by pipetting three times up and down. The samples were then transferred to the RNEASY 96 ™ well plate attached to a QIAVACTM manifold fitted with a waste collection tray and attached to a vacuum source. Vacuum was applied for 1 minute. 500 μL of Buffer RW1 was added to each well of the RNEASY 96 ™ plate and incubated for 15 minutes and the vacuum was again applied for 1 minute. An additional 500 μL of Buffer RW1 was added to each well of the RNEASY 96 ™ plate and the vacuum was applied for 2 minutes. 1 mL of Buffer RPE was then added to each well of the RNEASY 96™ plate and the vacuum applied for a period of 90 seconds. The Buffer RPE wash was then repeated and the vacuum was applied for an additional 3 minutes. The plate was then removed from the QIAVAC™ manifold and blotted dry on paper towels. The plate was then re-attached to the QIAVAC™ manifold fitted with a collection tube rack containing 1.2 mL collection tubes. RNA was then eluted by pipetting 170 μL water into each well, incubating 1 minute, and then applying the vacuum for 3 minutes.  
     [0272] The repetitive pipetting and elution steps may be automated using a QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially, after lysing of the cells on the culture plate, the plate is transferred to the robot deck where the pipetting, DNase treatment and elution steps are carried out.  
     Example 13  
     [0273] Real-time Quantitative PCR Analysis of Notch1 mRNA Levels  
     [0274] Quantitation of Notch1 mRNA levels was determined by real-time quantitative PCR using the ABI PRISM™ 7700 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to manufacturer&#39;s instructions. This is a closed-tube, non-gel-based, fluorescence detection system which allows high-throughput quantitation of polymerase chain reaction (PCR) products in real-time. As opposed to standard PCR in which amplification products are quantitated after the PCR is completed, products in real-time quantitative PCR are quantitated as they accumulate. This is accomplished by including in the PCR reaction an oligonucleotide probe that anneals specifically between the forward and reverse PCR primers, and contains two fluorescent dyes. A reporter dye (e.g., FAM or JOE, obtained from either PE-Applied Biosystems, Foster City, Calif., Operon Technologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) is attached to the 5′ end of the probe and a quencher dye (e.g., TAMRA, obtained from either PE-Applied Biosystems, Foster City, Calif., Operon Technologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) is attached to the 3′ end of the probe. When the probe and dyes are intact, reporter dye emission is quenched by the proximity of the 3′ quencher dye. During amplification, annealing of the probe to the target sequence creates a substrate that can be cleaved by the 5′-exonuclease activity of Taq polymerase. During the extension phase of the PCR amplification cycle, cleavage of the probe by Taq polymerase releases the reporter dye from the remainder of the probe (and hence from the quencher moiety) and a sequence-specific fluorescent signal is generated. With each cycle, additional reporter dye molecules are cleaved from their respective probes, and the fluorescence intensity is monitored at regular intervals by laser optics built into the ABI PRISM™ 7700 Sequence Detection System. In each assay, a series of parallel reactions containing serial dilutions of mRNA from untreated control samples generates a standard curve that is used to quantitate the percent inhibition after antisense oligonucleotide treatment of test samples.  
     [0275] Prior to quantitative PCR analysis, primer-probe sets specific to the target gene being measured are evaluated for their ability to be “multiplexed” with a GAPDH amplification reaction. In multiplexing, both the target gene and the internal standard gene GAPDH are amplified concurrently in a single sample. In this analysis, mRNA isolated from untreated cells is serially diluted. Each dilution is amplified in the presence of primer-probe sets specific for GAPDH only, target gene only (“single-plexing”), or both (multiplexing). Following PCR amplification, standard curves of GAPDH and target mRNA signal as a function of dilution are generated from both the single-plexed and multiplexed samples. If both the slope and correlation coefficient of the GAPDH and target signals generated from the multiplexed samples fall within 10% of their corresponding values generated from the single-plexed samples, the primer-probe set specific for that target is deemed multiplexable. Other methods of PCR are also known in the art.  
     [0276] PCR reagents were obtained from Invitrogen Corporation, (Carlsbad, Calif.). RT-PCR reactions were carried out by adding 20 μL PCR cocktail (2.5×PCR buffer (—MgCl2), 6.6 mM MgCl2, 375 μM each of dATP, dCTP, dCTP and dGTP, 375 nM each of forward primer and reverse primer, 125 nM of probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM® Taq, 5 Units MULV reverse transcriptase, and 2.5×ROX dye) to 96-well plates containing 30 μL total RNA solution. The RT reaction was carried out by incubation for 30 minutes at 48° C. Following a 10 minute incubation at 95° C. to activate the PLATINUM® Taq, 40 cycles of a two-step PCR protocol were carried out: 95° C. for 15 seconds (denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).  
     [0277] Gene target quantities obtained by real time RT-PCR are normalized using either the expression level of GAPDH, a gene whose expression is constant, or by quantifying total RNA using RiboGreenTM (Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression is-quantified by real time RT-PCR, by being run simultaneously with the target, multiplexing, or separately. Total RNA is quantified using RiboGreenTM RNA quantification reagent from Molecular Probes. Methods of RNA quantification by RiboGreenTM are taught in Jones, L. J., et al, (Analytical Biochemistry, 1998, 265, 368-374).  
     [0278] In this assay, 170 μL of RiboGreenTM working reagent (RiboGreenTM reagent diluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a 96-well plate containing 30 μL purified, cellular RNA. The plate is read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at 480 nm and emission at 520 nm.  
     [0279] Probes and primers to human Notch1 were designed to hybridize to a human Notch1 sequence, using published sequence information (the complement of residues 322000-377000 of GenBank accession number NT — 024000.7, representing a genomic sequence of Notch1, incorporated herein as SEQ ID NO:4). For human Notch1 the PCR primers were: forward primer: CGGGTCCACCAGTTTGAAT (SEQ ID NO: 5) reverse primer: TTGTATTGGTTCGGCACCAT (SEQ ID NO: 6) and the PCR probe was: FAM-TCCCGGCTGCAGAGCGG-TAMRA (SEQ ID NO: 7) where FAM is the fluorescent dye and TAMRA is the quencher dye. For human GAPDH the PCR primers were: forward primer: GAAGGTGAAGGTCGGAGTC(SEQ ID NO:8) reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO:9) and the PCR probe was: 5′ JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3′ (SEQ ID NO: 10) where JOE is the fluorescent reporter dye and TAMRA is the quencher dye.  
     Example 14  
     [0280] Northern Blot Analysis of Notch1 mRNA Levels  
     [0281] Eighteen hours after antisense treatment, cell monolayers were washed twice with cold PBS and lysed in 1 mL RNAZOLTM (TEL-TEST “B” Inc., Friendswood, Tex.). Total RNA was prepared following manufacturer&#39;s recommended protocols. Twenty micrograms of total RNA was fractionated by electrophoresis through 1.2% agarose gels containing 1.1% formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNA was transferred from the gel to HYBONDTM-N+nylon membranes (Amersham Pharmacia Biotech, Piscataway, N.J.) by overnight capillary transfer using a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc., Friendswood, Tex.). RNA transfer was confirmed by UV visualization. Membranes were fixed by UV cross-linking using a STRATALINKER™ UV Crosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then probed using QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.) using manufacturer&#39;s recommendations for stringent conditions.  
     [0282] To detect human Notch1, a human Notch1 specific probe was prepared by PCR using the forward primer CGGGTCCACCAGTTTGAAT (SEQ ID NO: 5) and the reverse primer TTGTATTGGTTCGGCACCAT (SEQ ID NO: 6). To normalize for variations in loading and transfer efficiency membranes were stripped and probed for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).  
     [0283] Hybridized membranes were visualized and quantitated using a PHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics, Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreated controls.  
     Example 15  
     [0284] Antisense Inhibition of Human Notch1 Expression by Chimeric Phosphorothioate Oligonucleotides Having 2′-MOE Wings and a Deoxy Gap  
     [0285] In accordance with the present invention, a series of oligonucleotides were designed to target different regions of the human Notch1 RNA, using published sequences (the complement of residues 322000-377000 of GenBank accession number NT — 024000.7, representing a genomic sequence of Notch1, incorporated herein as SEQ ID NO: 4; GenBank accession number AF308602.1, incorporated herein as SEQ ID NO: 11, GenBank accession number AI802214.1, the complement of which is incorporated herein as SEQ ID NO: 12, and GenBank accession number BC013208.1, incorporated herein as SEQ ID NO: 13). The oligonucleotides are shown in Table 1. “Target site” indicates the first (5′-most) nucleotide number on the particular target sequence to which the oligonucleotide binds. All compounds in Table 1 are chimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”. The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. The compounds were analyzed for their effect on human Notch1 mRNA levels by quantitative real-time PCR as described in other examples herein. Data are averages from two experiments in which A549 cells were treated with the antisense oligonucleotides of the present invention. The positive control for each datapoint is identified in the table by sequence ID number. If present, “N.D.” indicates “no data”.  
                   TABLE 1                          Inhibition of human Notch1 mRNA levels by chimeric           phosphorothioate oligonucleotides having 2′-MOE wings and a       deoxy gap                                                         TARGET                                       SEQ ID   TARGET       %   SEQ   CONTROL       ISIS #   REGION   NO   SITE   SEQUENCE   INHIB   ID NO   SEQ ID NO                                                         226818   Intron   4   20230   cgtgcgtccctcttagggtc   38   14   1           226819   Intron:   4   29652   cacagcagacctgggcaggc   40   15   1           Exon           Junction               226820   Intron   4   40111   cagccctcccctaatgagac   6   16   1               226821   Intron:   4   40500   cggccacgcactgtgcaggc   70   17   1           Exon           Junction               226822   Intron:   4   45449   acggtctcacctgcgggcac   19   18   1           Exon           Junction               226823   Exon:   4   45660   tcacttgaggcccacggagt   46   19   1           Intron           Junction               226824   Exon:   4   47322   cactgcctacctggaagaca   36   20   1           Intron           Junction               226825   Intron   4   49376   accacctgcgtcaccacatt   55   21   1               226826   Coding   11   394   cacgatttccctgaccagcc   13   22   1               226827   Coding   11   454   aagggcaggcactggccacc   66   23   1               226828   Coding   11   781   ttgcagttgtttcctggaca   66   24   1               226829   Coding   11   904   ttctggcaggcatttggcat   59   25   1               226830   Coding   11   1093   tggcacagcagacctgtgcg   30   26   1               226831   Coding   11   1098   tgaggtggcacagcagacct   52   27   1               226832   Coding   11   1222   tccacgtcctggctgcaggc   83   28   1               226833   Coding   11   1399   tccccaatctggtccaggca   77   29   1               226834   Coding   11   1404   ggaactccccaatctggtcc   18   30   1               226835   Coding   11   1963   ccatcgatcttgtccagaca   78   31   1               226836   Coding   11   2246   gttgttgttgatgtcacagt   67   32   1               226837   Coding   11   2251   cactcgttgttgttgatgtc   68   33   1               226838   Coding   11   2788   ggcaggcagtcgcagaaggc   64   34   1               226839   Coding   11   2794   aagccgggcaggcagtcgca   76   35   1               226840   Coding   11   2887   gtgtagctgtccacgcagtc   8   36   1               226841   Coding   11   2897   gcaggtgcacgtgtagctgt   22   37   1               226842   Coding   11   3165   gcacaaggttctggcagttg   47   38   1               226843   Coding   11   3298   gcagccacctcacaggacac   0   39   1               226844   Coding   11   3345   gccctccatgctggcacagg   93   40   1               226845   Coding   11   3350   acagagccctccatgctggc   64   41   1               226846   Coding   11   3613   gggcaggagcacttgtaggt   38   42   1               226847   Coding   11   3870   ggcactcgcagtggaagtca   65   43   1               226848   Coding   11   4008   cctcgaagcccgcagggcac   28   44   1               226849   Coding   11   4207   tcggatgtgggctcacaggt   64   45   1               226850   Coding   11   4274   gtagtccaggatgtggcaca   66   46   1               226851   Coding   11   4279   aagctgtagtccaggatgtg   55   47   1               226852   Coding   11   4435   ttgaagttgagggagcagtc   26   48   1               226853   Coding   11   4440   ggtcattgaagttgagggag   33   49   1               226854   Coding   11   4459   tgcgtgcagttcttccaggg   40   50   1               226855   Coding   11   4464   gagactgcgtgcagttcttc   40   51   1               226856   Coding   11   4507   tggctgtcacagtggccgtc   53   52   1               226857   Coding   11   4512   tgcactggctgtcacagtgg   71   53   1               226858   Coding   11   4600   tggtccttgcagtactggtc   70   54   1               226859   Coding   11   4605   tgaagtggtccttgcagtac   40   55   1               226860   Coding   11   4797   ccacgttggtgtgcagcacg   70   56   1               226861   Coding   11   4802   gaagaccacgttggtgtgca   49   57   1               226862   Coding   11   4807   cgcttgaagaccacgttggt   64   58   1               226863   Coding   11   4837   gggaagatcatctgctggcc   20   59   1               226864   Coding   11   5068   ctctggaagcactgcgagga   77   60   1               226865   Coding   11   5073   tggcactctggaagcactgc   75   61   1               226866   Coding   11   5260   ttgcgggacagcagcacccc   66   62   1               226867   Coding   11   5290   aaccagagctggccatgctg   60   63   1               226868   Coding   11   5295   cagggaaccagagctggcca   44   64   1               226869   Coding   11   5452   aacttcttggtctccaggtc   56   65   1               226870   Coding   11   5457   accggaacttcttggtctcc   58   66   1               226871   Coding   11   5554   gcagacatgcgcaggtcagc   63   67   1               226872   Coding   11   5559   ccatggcagacatgcgcagg   69   68   1               226873   Coding   11   5762   gcggtctgtctggttgtgca   38   69   1               226874   Coding   11   5848   ttggcatctgcgctggcctc   60   70   1               226875   Coding   11   6030   tgatgaggtcctccagcatg   50   71   1               226876   Coding   11   6035   tgagttgatgaggtcctcca   73   72   1               226877   Coding   11   6076   gcggacttgcccaggtcatc   55   73   1               226878   Coding   11   6136   ccgttcttcaggagcacaac   66   74   1               226879   Coding   11   6257   atccgtgatgtcccggttgg   75   75   1               226880   Coding   11   6418   tagccgttgggcgagcagag   73   76   1               226881   Coding   11   6523   ctccgtgccttgaggtcctt   88   77   1               226882   Coding   11   6528   tcttcctccgtgccttgagg   82   78   1               226883   Coding   11   6550   cagcccttgccatcctggga   84   79   1               226884   Coding   11   7129   gtctgcaccaggtgaggctg   62   80   1               226885   Coding   11   7135   tgctgggtctgcaccaggtg   53   81   1               226886   Coding   11   7140   gcacctgctgggtctgcacc   44   82   1               226887   Coding   11   7145   tggctgcacctgctgggtct   77   83   1               226888   Stop   11   7660   tcgagctattacttgaacgc   0   84   1           Codon               226889   3′UTR   11   7672   gctgctggcacctcgagcta   72   85   1               226890   5′UTR   12   17   ttcacatgacaccgatcaat   69   86   1               226891   5′UTR   12   117   ctttaaggacggagggatag   0   87   1               226892   3′UTR   13   2011   tctgtgtaaaataaaagtac   57   88   1               226893   3′UTR   13   2249   tgctcgttcaacttcccttc   69   89   1               226894   3′UTR   13   2823   ctggagcatcttcttcggaa   57   90   1               226895   3′UTR   13   3186   cccgagctgagccaagtctg   64   91   1                  
 
     [0286] As shown in Table 1, SEQ ID NOs 17, 19, 21, 23, 24, 25, 27, 28, 29, 31, 32, 33, 34, 35, 38, 40, 41, 43, 45, 46, 47, 52, 53, 54, 56, 57, 58, 60, 61, 62, 63, 65, 66, 67, 68, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 83, 85, 86, 88, 89, 90 and 91 demonstrated at least 45% inhibition of human Notch1 expression in this assay and are therefore preferred. The target sites to which these preferred sequences are complementary are herein referred to as “preferred target regions” and are therefore preferred sites for targeting by compounds of the present invention. These preferred target regions are shown in Table 2. The sequences represent the reverse complement of the preferred antisense compounds shown in Table 1. “Target site” indicates the first (5′-most) nucleotide number of the corresponding target nucleic acid. Also shown in Table 2 is the species in which each of the preferred target regions was found.  
                   TABLE 2                           Sequence and position of preferred target regions identified           in Notch1.                                                 TARGET   TARGET       REV COMP       SEQ ID           SITEID   SEQ ID NO   SITE   SEQUENCE   OF SEQ ID   ACTIVE IN   NO                                                     143473   4   40500   gcctgcacagtgcgtggccg   17     H. sapiens     92                   143475   4   45660   actccgtgggcctcaagtga   19     H. sapiens     93               143477   4   49376   aatgtggtgacgcaggtggt   21     H. sapiens     94               143479   11   454   ggtggccagtgcctgccctt   23     H. sapiens     95               143480   11   781   tgtccaggaaacaactgcaa   24     H. sapiens     96               143481   11   904   atgccaaatgcctgccagaa   25     H. sapiens     97               143483   11   1098   aggtctgctgtgccacctca   27     H. sapiens     98               143484   11   1222   gcctgcagccaggacgtgga   28     H. sapiens     99               143485   11   1399   tgcctggaccagattgggga   29     H. sapiens     100               143487   11   1963   tgtctggacaagatcgatgg   31     H. sapiens     101               143488   11   2246   actgtgacatcaacaacaac   32     H. sapiens     102               143489   11   2251   gacatcaacaacaacgagtg   33     H. sapiens     103               143490   11   2788   gccttctgcgactgcctgcc   34     H. sapiens     104               143491   11   2794   tgcgactgcctgcccggctt   35     H. sapiens     105               143494   11   3165   caactgccagaaccttgtgc   38     H. sapiens     106               143496   11   3345   cctgtgccagcatggagggc   40     H. sapiens     107               143497   11   3350   gccagcatggagggctctgt   41     H. sapiens     108               143499   11   3870   tgacttccactgcgagtgcc   43     H. sapiens     109               143501   11   4207   acctgtgagcccacatccga   45     H. sapiens     110               143502   11   4274   tgtgccacatcctggactac   46     H. sapiens     111               143503   11   4279   cacatcctggactacagctt   47     H. sapiens     112               143508   11   4507   gacggccactgtgacagcca   52     H. sapiens     113               143509   11   4512   ccactgtgacagccagtgca   53     H. sapiens     114               143510   11   4600   gaccagtactgcaaggacca   54     H. sapiens     115               143512   11   4797   cgtgctgcacaccaacgtgg   56     H. sapiens     116               143513   11   4802   tgcacaccaacgtggtcttc   57     H. sapiens     117               143514   11   4807   accaacgtggtcttcaagcg   58     H. sapiens     118               143516   11   5068   tcctcgcagtgcttccagag   60     H. sapiens     119               143517   11   5073   gcagtgcttccagagtgcca   61     H. sapiens     120               143518   11   5260   ggggtgctgctgtcccgcaa   62     H. sapiens     121               143519   11   5290   cagcatggccagctctggtt   63     H. sapiens     122               143521   11   5452   gacctggagaccaagaagtt   65     H. sapiens     123               143522   11   5457   ggagaccaagaagttccggt   66     H. sapiens     124               143523   11   5554   gctgacctgcgcatgtctgc   67     H. sapiens     125               143524   11   5559   cctgcgcatgtctgccatgg   68     H. sapiens     126               143526   11   5848   gaggccagcgcagatgccaa   70     H. sapiens     127               143527   11   6030   catgctggaggacctcatca   71     H. sapiens     128               143528   11   6035   tggaggacctcatcaactca   72     H. sapiens     129               143529   11   6076   gatgacctgggcaagtccgc   73     H. sapiens     130               143530   11   6136   gttgtgctcctgaagaacgg   74     H. sapiens     131               143531   11   6257   ccaaccgggacatcacggat   75     H. sapiens     132               143532   11   6418   ctctgctcgcccaacggcta   76     H. sapiens     133               143533   11   6523   aaggacctcaaggcacggag   77     H. sapiens     134               143534   11   6528   cctcaaggcacggaggaaga   78     H. sapiens     135               143535   11   6550   tcccaggatggcaagggctg   79     H. sapiens     136               143536   11   7129   cagcctcacctggtgcagac   80     H. sapiens     137               143537   11   7135   cacctggtgcagacccagca   81     H. sapiens     138               143539   11   7145   agacccagcaggtgcagcca   83     H. sapiens     139               143541   11   7672   tagctcgaggtgccagcagc   85     H. sapiens     140               143542   12   17   attgatcggtgtcatgtgaa   86     H. sapiens     141               143544   13   2011   gtacttttattttacacaga   88     H. sapiens     142               143545   13   2249   gaagggaagttgaacgagca   89     H. sapiens     143               143546   13   2823   ttccgaagaagatgctccag   90     H. sapiens     144               143547   13   3186   cagacttggctcagctcggg   91     H. sapiens     145                  
 
     [0287] As these “preferred target regions” have been found by experimentation to be open to, and accessible for, hybridization with the antisense compounds of the present invention, one of skill in the art will recognize or be able to ascertain, using no more than routine experimentation, further embodiments of the invention that encompass other compounds that specifically hybridize to these sites and consequently inhibit the expression of Notch1.  
     Example 16  
     [0288] Western blot analysis of Notch1 Protein Levels  
     [0289] Western blot analysis (immunoblot analysis) is carried out using standard methods. Cells are harvested 16-20 h after oligonucleotide treatment, washed once with PBS, suspended in Laemmli buffer (100 ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gels are run for 1.5 hours at 150 V, and transferred to membrane for western blotting. Appropriate primary antibody directed to Notch1 is used, with a radiolabeled or fluorescently labeled secondary antibody directed against the primary antibody species. Bands are visualized using a PHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.).  
    
     
       
         1 
         
           
             145  
           
           
             1  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            1 

tccgtcatcg ctcctcaggg                                                 20 

 
           
             2  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            2 

gtgcgcgcga gcccgaaatc                                                 20 

 
           
             3  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            3 

atgcattctg cccccaagga                                                 20 

 
           
             4  
             55001  
             DNA  
             Homo sapiens  
             
 
           
            4 

tcctctcctg gggcgctgac cccaagatgt taccccaggc ctgcagaagt gaggccagat     60 

tctgggagga cgcaggcgag ggaggctccc agcagcagct acaggggcgg gacaggccgc    120 

ctgcactggc tgtttccaga gtgctcagca tttggcaaga ggtgtgccag gaaaggcttg    180 

ctggggtcag gtgtgagatt tgtcctgctt tctgtgcctg cctctgccac catgctctgg    240 

ggtttctgca gccctccccc caggccccca cagtcctagg tccctcccag cctttcggtc    300 

tccccgcagg gcaggcttag ccctcctact ccaggagggg cgccgggaca cgcgccttct    360 

gccatcgcac tcaccaccct gtgttggggt ggggaggggg cgctgtctgt ccctggcacg    420 

aggccgtgaa ctttgcgcag ggactatggc aggcattttg gactcctggc gcttaaccag    480 

gcttggcaca gggtgcccgt gcctggcatc gtggtggaga aataaatcag ccggaaggag    540 

cacgtgggaa gggctgcgcc ggcgccagcg gcagatccgc ccgacccgtt tgtgctttct    600 

ggggccacct taggcaggcg gcgcccgggc aggaggagga cggtgaccga ggagcgtgtc    660 

gacgcgggtc ccccttgggg gagcggggca caatccgtcc gcgaggcact ggtcccggct    720 

gctccctcgg ggcgcccgga gtcccctgcc cagaggccgc ggcgccccca tccctcgcgg    780 

tcaagtgctc gggcaatcac ggccagggat gtctggcggc gatcgatcct ctggacgcct    840 

aaagccgcgg ccacggggcc ctcgggaggg agtgaagccg ctgggctagg ggcgcacaca    900 

cggctaggcc actctcccag agccctgccc cgggcccggg gtcccccaac gtggcctcag    960 

ctgctccccg cccggcccag cgcacggtgc acacggctgt ccgcggcctc gccctcccca   1020 

ttccgccccg ggctcctccg cttattcaca tgcaaatttc agtcgccagt tgtcgccgag   1080 

cgcggcaacc gccagagccg gatccttccg cagccccggc tcaaactttt ggcctctgaa   1140 

aactttcaaa cgagaagtag tcccaggcgc ccgctcccga cccacgccgc gccgacgggt   1200 

ccctcctccc cggagaggct gggctcggga cgcgcggctc agctcggaga ggcgcaaagg   1260 

cggacggtgc gtgcgggagg aggtggtcgc cgagggggcc gagggaccgg cggtggtggg   1320 

gccgggcgga gcggggccgg cggtggcgga gcgcacctcg actctgagcc tcactagtgc   1380 

ctcggccgcg ggagggagcg caagggcgcg gggcgcgggg cgcgggcgcg ggcgcgagcg   1440 

cagcgaagga acgagccggg cgcggagccg ggcccggggg cccgcgagag cacagcgccg   1500 

ccagccagcc ggggaagaga gggcgggacc gtccgccgcc gccccgggac cgtacgccgc   1560 

gcgtgtgcgt cccagccccg ccggccagcg caggaggccg ccgcccgggc gcagagggca   1620 

gccggtgggg aggcatgccg ccgctcctgg cgcccctgct ctgcctggcg ctgctgcccg   1680 

cgctcgccgc acgaggtagg cgcccaccca cccgcgagcc cccactttcc gcgccctttg   1740 

gaaactttgg cggcgcccgg cgcgcgcgcc ccacggctgg gagcgggcgg cggggaggcc   1800 

agcatggaga gggaaaagcg ggcggcccgg ggcgtggggt tctggagtcc cgggatcagg   1860 

gaggaccgac cttccccctc gatccccccg tggaggcgga ctcgcgccgc ccgtgcctgg   1920 

agccgagtta ggaggccggt gtggggtgct ggggccccgg aggccctact ccgggcccgc   1980 

ccttcacccg ccgcgcgtgg ggcttgccgc cggtcggccg ggcgggcggg ctgcctacta   2040 

tttttcgatt tgaatagagt cggttttggt ttcctgttgc ttctccgggc catttatctt   2100 

ctttcttctt cgcctctggc ccacgccggg gcggatgttg gggcgcggag tgtgggctct   2160 

gcggcgccgc gttcgccttc actgacccgc gcggccgggc tgggtccccg ggctcccggt   2220 

cgccccgccc gccggtgccc cccagcccgg ctctcagttt gggggagggg ttgcgtaaga   2280 

agccgccgcg cccgggggga ctgaactttc cttttgcttt gcggagttga agtttggaaa   2340 

gcttgggggc ggagagcggg acgcgggtgg ggggctctta catttctccc cgccgcacag   2400 

cgagcggggt ctctggggaa tcgagtgatt aatccactct ttctccgaga gttggaggcg   2460 

agaattatct gtcctcttcc agaaagtgcg gctctgtgtc acacccccct cccccgtttc   2520 

tcagccccgg taagatgggg agggaggggc ttgagtaatt gatcccttct cgagatgggg   2580 

tcgaattcct tccgaatggg ggaccttcat ccccctcctg tgggtgtatg ggggctgccc   2640 

tggagatgcg cgcccgcgga ggcaggtatt gggtgtcggc ggaggcgggg ccgcgtcccc   2700 

agggtgctgt cccggtgccc ctggaggcgg ccccgactcc acaatgggcc gctctgattc   2760 

tgaggcggag gccggcgctt tgttggcggg cggggtagcg ggcgagcagc tgtcgcattt   2820 

tcccacgggc gggagctgag tgtggccacc ccccctcccc ccgtccagct ctgccccctt   2880 

gcagaacggg tttgaacaga ggcaatctcg gcggctggat ggggggccct gccctgccag   2940 

actcctcagt gagcctccag ggtggggggc aacgctttgg agagtagccc actttgtctc   3000 

tgcttttccc ccactgcgtc caggcggcac atcaggccac cccaccctgc ttcaagcagg   3060 

acgtgttccc tgctgctccc cttcccccca tttctgacta cagaactctg ggcagaatgt   3120 

tgacgcccct ttggtttcat ggaggggctt cctgcggacc cgtgccccag caacccatga   3180 

tactgagcag agtgcgtccg gggtaggggg cggacgatgc cccttggctg ggtgcaggtc   3240 

cccgccccca gggtaaccag ggccttcggt ggtggtggtg cgggtgagac tgacctctct   3300 

tctcctgccc ctgcctaggc ccgcgatgct cccagcccgg tgagacctgc ctgaatggcg   3360 

ggaagtgtga agcggccaat ggcacggagg cctgcgtgtg agtaccaccc ctgcgggacc   3420 

tgttgctttg tcgagggcag agcccctgcc ttctgcagcc gcgcagggga cagaacacta   3480 

ggccattgtc ttctggagat ggcccagagt ctgggcacgg tcacgtgctg acttttattg   3540 

gacaaagtct ggcaattact taatcaccag caattatgct gcgtgtggag ctggtctggc   3600 

tgctgagggc ttgggcactc ctccgtgggc cacctcaggc tccggggtca ctaagtggga   3660 

gtcccagtac cccaattctt ctttaagtcc ccaagaaaca gttgatctgc caggggaagc   3720 

tctggaccat ttactgagag gcccagcccc caccaccctg ccaacttggg gcgcctcctc   3780 

tgggcgggtg atgcctcctg ggcacaggtg atgcctcctc tcgggggtga tgcctcctag   3840 

gtgggtggtg atgcctcttg ggctggtagt gatgcctcct ctggcacagg tgatgccacc   3900 

tcctgggcgg gtgatgcctc ctctgggtgc gtgatgcctc ctgggctggc ggcaatgcct   3960 

cctctgggca caggtgatgc cacctcctgg gcgggtgatg cctcctctgg gtgcgtgatg   4020 

cctcctctgg gcacgggtga tgccacctcc tgggtgggtg atgcctcctc tgggtggtga   4080 

tacctcctct aggtgggtga tgcctcctct gggtggtgat acctcctcta ggcgggtgat   4140 

gcctcctcta ggcacaggtg atacttcctg ggcaggtgat gccacctccg ggtgggtgat   4200 

gcctcttcaa aggaaattga ctcttcaaaa tcggacccgc ctttgggctc tctgtttgcc   4260 

ccagctccct cttcccggcc tcaggctggg gctggggaag agaaggtgga actcatgttt   4320 

tggcgtgggg tgtggggtaa ttattgagtc tggtttctgt cctggcatct gcaaactctg   4380 

acctcaaaat cccgtggccc ttctggtctg ccagcctcag atgtaaaatg ggcactgagg   4440 

gggccgggaa gtcgtgcgga accgctccct cctgcctgcc tgtgagtgcc catcagccgt   4500 

ggggaactgt tctgctgtgg ccgatgccct tcaacttgag tccatccctg atgagaataa   4560 

tgaggaggtg tttggtgtct ggaaaaagag ttccctccgc cccccaggag ctacagatga   4620 

agaaatggct ccccagccag tctgcgcgcc ctcattccaa ctgcagaagg tcccattccc   4680 

ttgacaaccc cagcccggct cctgcctgtg ccgactgccc agcctgtggg cagcaggccc   4740 

cctccctccc cggcccccac cctccccaaa ctgaagccaa ttaagcaggt ctgtgagcag   4800 

tttaatggac aaagccgatt gtgtctttgt cagacactaa tgaatgcgca ctactttttt   4860 

tttttttttt ttttttgctt tttggggccc ccttctccct ctgcagaagc cgcacggacg   4920 

cttgatgccc taaatcttgt ttcctttcat tcagagaccg acagctggga tccaggggaa   4980 

gtgcagcagc tattgttggg ggaggcggat taatgccgtg tgattaatta tgcgtggctt   5040 

cccagaatgt ataaccccct tccccgccac tgcgctcccg gctcggactt tgcctctcac   5100 

ccattcgggg ctctgggaga agcttcttgc tcctgcagag gactttggga gggtgggagg   5160 

aacaccgtgc tgggaaaatt gggggcctgt tgcttcttct cagtggcacc agggagtagg   5220 

cacagcctgg cccacagggg tctgggactg aggtctccct gggccagagc gctgtgcttt   5280 

ggggtacaga ttgctgcagc tccctggtgt ctgggttaaa accctgccac agggccgcaa   5340 

aacactggtg tcatgggccc actttcccat tccccaaacc tgggatcccg gtgagctgtt   5400 

tccagcccaa ggcaggccgc cgaccatggc tttggtgcct ggccttgtcc cctttgctgg   5460 

ggcctggcgc cccctccttc ttggcgggaa gcctgcttca gtttggcctg acaggcagcc   5520 

gagctcagcc cacagagacc ccattgtgca caccccacaa gtccaacgag aagatgggac   5580 

ggaagcgcgt cctcaggctc ctgcttctgg gccgcagagt tggaggccag ggccacagcc   5640 

gagcccggct tgtggaggca caggtcaggg ctcacacagc agtctggggg acattgcagc   5700 

cgcttcggag aaggccccgg gctccctccc tgggcttgag ccccacccaa catacacccc   5760 

tcaggtgcct ctgccttccc gttcctgccc atccttctca gaaggcccag gaggtctgtc   5820 

caggagttag ccagggcacc tgcaggaggc ctggctcccc cagcaagggg agaaacccac   5880 

actgtttctc gaaagagtct ggggtgcagg ctcttggagc cctgccagcc tgagtgggaa   5940 

tcctggcccc gtggtatcca gctgtgaccc cggaaagagc tttggctttt ggtgccattt   6000 

gctgcctccg ctgtagaagg ggcatggtca tgatgggcgt cacagtgcct ggctcactag   6060 

tactgggtga atcatttctt tccatcccaa agagaaggaa ccttcaccgg aagctgccaa   6120 

tcaggtggtt gcagggcccg ggattggaaa aagtggtttt gttggtgcaa aatgccttcc   6180 

caggaggtca gagtgtcgcc gtggcctggt cagccagtgg ccaggtagaa agcacctggc   6240 

agcacatgct ggtgggcgga ggatggttgg tctctgggca gctgctgtgc gaggtgtggc   6300 

cgtggtggct gtggtcggag atggtggcct tctgagcagg cccagggggt cactgtggcc   6360 

cttggctctt ctctagcccc ctcatcagct ccgtcagtgt gagcaagggg tactgaagag   6420 

ggctccacct tttttctctt tgttttgagg agagctggga agagctgcag ggaccctatc   6480 

tgtctgggtg agactacgtc cttggtctga aacgctctcc cagggctgcc tgcgtctggg   6540 

ctgggcgttg aggcaggggg cagctaagat gttggctgct gacggcagag tcagaatggc   6600 

acggggcgtc ttccaggagc tgggagaaat gggggtaggc tgtcacaccc agcacctcca   6660 

ggaggatgtc cagctgcctg ctgggtggtg cgctttagct ttggacaacc tcgtgtctga   6720 

ggcctggcct ggccactagc tctgtgacct tgggtcatca ggtcatgcaa gcctcagttt   6780 

ccccagcatg tcagccgggc tgatacacgc tggatctctt ggtggtgtgt gaggattgca   6840 

tgagaaccgt gtggagaggg cacctggtca gcgctccttt cttccccgcc ttctccccgt   6900 

tggctgacag tgctggtttc cagagcctcc ttcctgaatg tttaattgat tgacgagtga   6960 

attcagagaa tcctaaaggt gctgcctggt gggctgggcc tggcactgcc tggggaggga   7020 

cttgccggct ctggggaagt ccatctccca ggatggctcc agctccgggg aagtccatct   7080 

cccagggtgg ctccagttca cagacacgtt tgagcgcctg ctgtgtgctg cgttctgctc   7140 

caggccaggc ccccggtggg gacagggcag acatggcccc tgctcctgtg gagttcactt   7200 

cccagcaagg cgtgagagag gcacctagct cacatgtggg acatgttggg ggtgtgccgg   7260 

cccggtggtg gcctggcata gctcccggca gccccgtaaa gctgtctgga tcaggcaccg   7320 

ataagtgaga agaagagacc cagagaagtc gccatcagcc ccagggtcac acagcagtgg   7380 

cagaattcct actagccctg cccctctcct tctcccaagc gaatgtccct aaacacagcc   7440 

ccagccagcc tgagctgccc cgtcatttcc cgactacaag cggactgggg gcgtggcttc   7500 

cccttaaaag aagaggaagg aggctcaggc gggaagtgac ttggccctgc agccggcctg   7560 

ggaggctggg gagggacggg gtagctcctg tcacccggtc tggctctttc cattgagtca   7620 

cctgcctcgt cttgggcgtg gccaggggag gaacaggttg attctcctcc tcatgctgag   7680 

ctgagcggaa aggcctgtga caggacctcc tgtttatgca gaacctggtc ttcaagggcg   7740 

ccagggttag aaaaacatgt ttaaaaacat gcagcatccg aggtgggcag atcacctgag   7800 

gtcaggagtt caagaccagc ctggccaaca tggcgaaacc cccgtctcta ctaaaaatac   7860 

aaaaattacc caggcgcagt ggcgtgtgcc tgtaatccca gctacttggg aagctgaggc   7920 

aggaggatcc tttgaaccca ggagacggag gctgcagtga gctgagatcg tgccactgca   7980 

ctccagcctg ggtgacaaag tgagactctg tctcaaaaaa acaaaaaaca acaaaaaaaa   8040 

ccccacacag tgtcaaatag aaagtctcac ctactcagac tggggagaca tcaggaacag   8100 

gaaggcctgg ggcggagact ggggtgggag cctggagtgc tctgaaggaa gctggctcct   8160 

tggggcaggc cctgcctctg aacagcagcc aacatgtttt ccattgaaaa gaataataat   8220 

agaaaaagag agagggagaa agaaaacagc aaccctggca ggaagagggt agggcagggc   8280 

agacagtgca ccaggggaag tgcttccaca gggcagagct gggtgcgggg acccccagat   8340 

gcgggcggca aactggagac cccacaggag ccgggactgg cttcattcct cattccctgc   8400 

acctgtgatg ggcagcagcg gccaccggtg cccactgggc agttccaggc cgggccgagg   8460 

gactgcaccc ttttgctgtg gggaggggac ctttcctgtt tcatgtctcg tctataaata   8520 

gtgaaaggat gtggctttgc agaatcgatt gggcgtctag cttgccttcc tcaataatgc   8580 

agcgatgaca gctccacact caggacgccg ctgttaccca gcctcgtaaa agccctggca   8640 

gacggccgcc caccctccag gtgtgccagt gcagttttca tccccagcgc acaggtggga   8700 

aagctgtggc tgcgagggcg tggggatctg aggcaggctg gcgtcagctt tcaccttcat   8760 

gtgcaggcat atccagaact ttccggatct ttccccgggc tgaggaggaa ataccagcag   8820 

ctttaacgag cggctggctt tggggcatca cacctgtggg ccgggctgcg tttgacggcc   8880 

tcgagtttcc caggcagcgc tggcgctttc tcctgtgggg cccggagctc cgaggggctg   8940 

tgaagcaggg ctggctctga tttgctgtgc tccccacact gtcccctccc tgggcctcag   9000 

tttccccatc tgtatgtgat tccccttcca actacagcca gcgggaccct ggggcgggct   9060 

gaaggcgtgg tggtggctgt gggccctgcg gcgaggcctg cgctgggctc ggtggtcgcc   9120 

ccagctgggg aaggggcagt ggctgcaggg gtgggagggt ggagggaagc ccttttccaa   9180 

agttgcctgg ttgggttctc cttgtggccc tgccccaccc caccctgctc cctgggagca   9240 

aagggagcta aggactggtt tggggatgga tatgtatatg gagggattct gggtgatgac   9300 

ttattcatat tacaaaattt gccttttttt gtttgttttt tgagatggag tcttgctttg   9360 

tcacccagac tggattagag tgacgcagtc ttggctcact gtaacctctg cctcccagat   9420 

tcacgcgatg ctcctgagta gctgggacta caggcacata ccaccactcc cagctaattt   9480 

ttgtattctt agtaggggca gggtttcacc atattggtca ggctggtctc aaactcctaa   9540 

cctcaggtga tccacctgcc ttggcctctc aaagtgctgg gattacaggc gtgagccact   9600 

gtgcatggcc acttttttgt tttttgagat ggggtctcac cctgtcacac aggctggagt   9660 

gaagtggtgg gatcacggct gactgcagcc tcaaactcct gggctcaagc gatcctcctg   9720 

cctccgcctc cagagtagct gggaccacag gtgtgcacca ccatgcctag ctaattttaa   9780 

atttttttgc agaagcgggg tcttgctatg ttgtccaggc tgaaaatgtg acttttgaac   9840 

taaagcatgc tgttgcttga catctagtcc attcacaagt tcattcaacc actatctagt   9900 

tccgggactt tctcactgca caaaacagaa actgtaccca aatactgtca ctctgccctg   9960 

actgccccag cccctggcaa ccactaaccc gctttctgcc tctgaattcg cctcttctgg  10020 

acgttgccta tgaatggaat tgtgccatag gtggtctttt gcccctggct tctttcactg  10080 

agcgtgatgt gtttaaggtt catccatgtc acgcgtggac tagaactgca ttcttctgag  10140 

gactacattt tagcctccag aagcatcagg gactggccag aggtggaccc cagcatccca  10200 

gctcccagac agtccagagg aggcaggtgt tgggagggcg tccggggtgg cttcctggaa  10260 

gagatggcgc tcactctggg ccggtgcagc cacccgacag ccacagagcc ccctagaggg  10320 

agtcctggca tattcccagg tgaacctgta ggatggactg gcgtgcccgc ggcccctgtc  10380 

ctgctcagga ctccgagaca ggctcacaac ctgtccctgg gcctcactca tgccagccgc  10440 

caagctgttc agcatccctt agcccccagg aggcacacag tggggccgcg gtcactgagc  10500 

gtccacacgc ctggtgctgt aagaatgcaa agtagagggt ctttattgag catctactac  10560 

atgctgaacc ctggggtatg ggtgggcacg gcagatgcag gcccccctgg gcaggggagg  10620 

agcatgcccc ttaagaacca ggccacagga tggacgggct tggccacaag ctaggggtag  10680 

tcactgcagc cttcatggcc tcctgtgtgc aaagtcagtg ctgtgggacc ccatctgggt  10740 

gcacctgctg tggctttcaa gtgaggccca ccacggagca gcttccacgc tggctgggac  10800 

agggtcagcg gcagggagct ccagaccaaa ggagcggcgg tggctggggc agcggggggc  10860 

agtgaggctt tggacacatc catggggcgg gcaggcatct cagcccccac ccgcccagtc  10920 

tgcctgctcc ggggccttcc cttccctgga cagagctctg tgtgattggc tccaggctgc  10980 

ttctccccct ggaccctcag ggtcagctta gagtgactgg ctatgccctg gcctagatcc  11040 

aggcccgtgt gacaggagag agctaggaac ctggcccctc actctgagac acagatggtg  11100 

tcctctccaa gttgcccttg ggacggggaa gcgccatgtc ccctgcaggg caccaggcca  11160 

gccagtggcc cttgacagcg aggccctgtc tgcaggcagc tgggggactt ctccctgtcg  11220 

ggaggatgcg tgcgtgttag catcggggtg cctggttgcc ccaggggtgg tgcagggcgg  11280 

ttataggcgg tcctagcatt ttggggttgc tttcagtgta tctccgagac ctgtggaatt  11340 

ctgggaggag aaactcccca gcagtgtcct cagtgcgccc ccctaccctg tacccgttgc  11400 

tcctgggagg gatcctgagt gctccccccc acccaccatg cacccattgc cccaggaggg  11460 

atcctgagtg ctcccctctc cacccaccct gcactcattg ccccgggagg gatcctgagt  11520 

gctcccccct ccacccactc ccaccctgca cccattgccc ctgggaggga tcctgagtga  11580 

tcccccctcc acccaccctg cacccattgc ccctgggagg gatcctgagt gctcccctct  11640 

ccacccaccc tgcactcatt gccccgggag ggatcctgag tgctcccccc tccacccact  11700 

cccaccctgc acccattgcc cctgggaggg atcctgagtg ctccccccga cccaccctgc  11760 

acccattgcc ccaggaggga tcctgagtgc tcccctctcc acccaccctg cacccattgc  11820 

ccctgggagg gatcctgagt gctcccccct ccacccacca tgcacccatt gccccgggag  11880 

ggatcctgag tgctcccccc tccacccact cccaccctgc acccattgcc cctgggaggg  11940 

atcctgagtg ctccccgcct accctgcacc cattgcccct gggagggaga cttctgtaga  12000 

ggcctcacct ctgaaccccc cactcacagg ctcaggggcc cagggctggg ggcgtcctgc  12060 

cgtctacact ccagattgag cccgcacggt taaacagggc cctctccgca cgccctgcct  12120 

aatgcaaagc ggtggcttcg ggtgggtccc caaggggcct tgtgaaccct gggggcctgg  12180 

cccctcccac ctggctgcac ccgctgggcg ggagatccca tcctccgagt cctcagcccc  12240 

tcagcctcag ctgcagccca gacaccaggg ctctgtgggg cgggggtggg gcagcggttc  12300 

cctcaggtct gtggggctgc cctgctgtgg ttcctgcaat ttcctggaag gaggtctggg  12360 

gcctggtgtc cagtggggcc tgtggggacg gactcggggg ctctgtgggg agggactggg  12420 

ggctctgtgg ggacggactt gggggctctg tggggaggga ctggggggct ctgtggggag  12480 

ggactggggg ctctgtgggg agggactcgg gggctctgtg aggagggact ggggggctct  12540 

gtggggaggg actgggggct ctgtggggag ggactggagc ctggtgtcca gtggggcctg  12600 

tggggacaga ctcgggggct ctgtggggag ggactcgggg gctctgtggg gagggactgg  12660 

agcctggtgt ccagtggggc ctgtggggac agactcgggg gctctgtggg gggggactgg  12720 

ggggctctgt ggggagggac tgggggcctg tggggacgga cttgggggct ctgtggggag  12780 

ggagtggggg ctctgtgggg acggactcgg gctctgtggg gagggactcg ggggctctgt  12840 

ggggagggac tgggggctct gtggggaggg actcgggggc tctgtgggga gggactgggg  12900 

ggctctgtgg ggagggactg gggggctctg gggagggact ggggggctct gtggggaggg  12960 

actggggggg ctctgtgggg acggactcgg gggctctgtg gggagggact ggggggctct  13020 

gtggggaggg actggggggg gggctctgtg gggagggact gggggctctg tggggaggga  13080 

ctgggggctc tgtgagaggg aactgggggg ctctgatgtg ctgggcgtgc acaggggagg  13140 

ctttgcctct gacatccaca cctgcagttc ccaggcacac acagcggccc catcggggtc  13200 

ctcgcaatca cagtctgtga gtggccacta ccaagggcgt ctccggatca ggccaggccc  13260 

agatgctgcc cggacccaac ccccacagcg cctgccgcca cagtagggac caatggccac  13320 

agaacactca cccggcctca cttgcacggg gctgttcagt ctggtgggca ccaggtgggc  13380 

gctgttctcg cccagtggga ggcctgccag ctccttggtt gctagttgag atttttcccc  13440 

aagaaatagt cgtgttgttc cttctgctcc gcctggcact gaggttggtg acacgccccg  13500 

accttgctct aattggcaga tgagaatttg tcatcagatg tcgaccctgt ctacgcagtc  13560 

ccctggcctc ggagtccatt gtgcatctct gaagggcttg aattggttgt ttaaagctgg  13620 

gtaaatgccc ccttgacatt ctgttgacac tgtcaatttg ctgaacaaac tcttccacag  13680 

gtagcacagg aggccagtct cgccccagcc tgagccaagg cccagggagg caggtcctgt  13740 

gtggagtcag cctttttgtg agtgcagcgg ccctgcagga tgcaccgggt ttgagaagtg  13800 

gactcccccg tctcccgcca catcccaaaa gccctcagcc atgagggcag gacgaagccg  13860 

acggtcgccc tcgtgccgag atgacaaccg ggcacagagg cggtggctcc cagcttcctg  13920 

ccctcttccc cgtagtgggg ttacccagcg tgactcatgg cccgagccac aggcacccgc  13980 

ccagggagcc acggcgggga gacctggctt tattagagat gcgttcgtgc ctcatcaagt  14040 

ccaaaggagg aaactggcgc gtccctcact ttccctgaaa caggctcccc cacagcccaa  14100 

gcggtcaggt taaggtgcat ttgcagacag atgccctagt aggaggctgg agcttttggc  14160 

ccagaatttt ccctgctgta ggcccatggt caccgagcct gcccgggggc tgaggccctg  14220 

aggaggtgcc attgccccac ccaagcctca gacaagtgtc ctgccctcga ccctgcggga  14280 

ggcagcagct caggccctga ccctggccag agagggcagc tctctgaggc tgctgtggcc  14340 

gtggcaggca ggggctgggc ttgccgggcc gtcgagtggg cacagagact atctgggaag  14400 

gaggtgggcg gtgagagctg actccccgca gccgggggac agatgagccc cgtgccgcac  14460 

agcagggccg cgtggtgggg ccccccaagt tccgctcggt ttcagctgtt gacgagcgga  14520 

aacacacagt tactggaaat gagaggtttg ggggagccgc caggggttga acatgtgtgg  14580 

tttccctgac ggcttttcag taagagatgc ttgagctcag gctgggcact ggctggtgcg  14640 

cggagggggc tgggccagag tccccccctt gggcctccac tctccatcct tatagaggga  14700 

cctgggttgg tcccagctcc cggggtgcag cccctgggtg gcccctccta cttccccttc  14760 

agtggtcaga aggaggctgg cctcattcct gtctgacgga gagccaggtg gggaggccgg  14820 

gcggggctga gtgtccaggg tccacatggt ggcggggctc aggccgtggc gaggtcctgt  14880 

cctgggcctc cagggtctgg cccagcacag agctctgcag ctctctggtg gacgggcctg  14940 

gctgggctgt cggcagccct ggggccaggt gggcagtggg aatggaggtg gaggcaggtg  15000 

ggtggctgca ggtgactgga aggcttggct ccagcctgca ggggctgcag tccaggccgg  15060 

ccacccccac gagaccttcc cccttccagg tgtggggtgg cactgggtca acaacacagg  15120 

ccacggactt ctttccccca cgtgggccgt gcgaccccgg gccgccacca cctctctgag  15180 

ctcccctcct ccttaggaga tgggaagcgg ctggggccat gaagtgtccc ccagggctcc  15240 

tgacagcccc agctgccgtc ctcgtggctg ggcgactgcc gcgaattgtt gcgccttctt  15300 

ttgagttggc tacggagtca tggcggctgt gggaggtgta gctgggtctc gggcggcacg  15360 

cccggctccc ccggggatgt gtcatctgtg tcatccaccc tggagcccct tcctggtgct  15420 

gtgggggccc ctccaggaag gtctctccca gaggccagcc ccagccacag gctctgggga  15480 

gactgtgtgg gatttgcatg cagttgtgtg ctatttgatc gaggtcatgt gtgggatttg  15540 

ggcgaggtcc tgcgggattg ggtgaggtag tgcgagatct ttgggcgagg ccgcgaggca  15600 

ctgggcgagg ctgggcatta ttcagtgcct tggaggggca gaagttttag gacctgccaa  15660 

gtccagccca agaatcccca actttcccca cagggaaagg taccccagac agtccccagc  15720 

ctgtgccagc tgtcttggga caccctggtg tcccccaggt tgtggcactg caggcccctc  15780 

ccccaggtgc ccctccttat gggcccctcc cagggctgca ggcacctgcc tggatcccac  15840 

ttcccaagtg tggggtaatc ccaggcgccc ggtgatcacg ctgggcccgg gcagagtgag  15900 

gctctgaggg ggtgccaggg gcacagtggg ccagtatgtg agccgggctg ggcgctggga  15960 

ctgggccggc aggcgagcgg tgttccgaga ggaggattcc tgggctcgga ggcccctctg  16020 

tgcctcgtgt ggtcagagag cagggagccg gcgggtgtga tggggatgag gtgacctctg  16080 

gggccaccac tgaagccgcc gtccatctgc ggccgaatcg ggaggcacac agaggtgtcc  16140 

tggtggtttc ccgggacagg cgggaggagg cgcaggaggg cggcgtctgc ccccgggatg  16200 

ccaggagtgc tcctccggca gcgtgggctt cggcttcgtc cctctttcac cagtgccgac  16260 

gccccgcggg tgctgtgggt gaaggggcat cggtgccctc ctgctgggcg ggaggaagga  16320 

aggggagcgg gaggctgggt ctctctcgcc cagggctctg cccccaccac cctagggttt  16380 

tgtgtttcgg tggagctgta agaatgcttt gtctgctcca gacttcccgg tcctgggaga  16440 

tcaggtggtc aggcaaaaca tccagaccgt ctgggtgggg ctgggggtgc gggggacacg  16500 

cgccttcctc ctctttccca gggagcttct gccactcctg tgccaccctg gggagctgcc  16560 

ttgccctgcc ccgctctggt ccccatcagc aggtgtggtt ctgagctgcc ttcagatccg  16620 

ggtggtgtgc tgggcatagc tgccccgttt tctcacctgt gaaatgggcc cattacacgc  16680 

ccccagggcg gttgtggggg tcccatgaga cagagcttgg aaagcccgag tgtaggtcag  16740 

gccaggaggg ttgtgggctc agtttcttca ctgctgggct ggtgtgtgcc ggacagatgt  16800 

ctcgggatcc cttccctccc tgcagcattc ttaacaagga cgctggacat ggagtcagca  16860 

cctccgggcg actgccctgc tggctgtggg gctcactcca gaggagaaca ggagcccctc  16920 

cggggagccc cttcctgcca cccccagcga caggatctgt tcaggagcag tggtgggagg  16980 

ttgccagaag catgtctggc gctggcccgg agcgcagcct gtgatgcccg acttcattca  17040 

tggagtgtgg tttgagccct ccagcagcgc tgctgtgggc ctgtccattg ccttccctgc  17100 

ctctgagcca tcgtggggtg cagggctggg ctgggtcttc ttgggagcag agccccggcg  17160 

tgtcatgacc atcttggcct ctccaactca agaggtttgt ccacatgggt ccctaggggc  17220 

cgttggggtg acccagggca ggagacacat gcctgctttt gggggatccg cgtgggaaat  17280 

tcccgcagtg gagcagcagg tggggctcca gcaggcgttt ttcacactcc aaacagcctg  17340 

cgggggcact ttggtgacac ctagttcctg gggtctcaga gcctgctggg ctgtgtcttg  17400 

gcattttggg gaggggctgg ccacatgccc ccagagtggc caccccagct ccgccatctc  17460 

cacgggcctt ctgtcggtca gatgcagaca gccgttccca tgtcgggtga gggcattgtt  17520 

ttcccggctg cgggaaaggt agcttcccgg gaggacaggt tctgtcccca tcccgcctcc  17580 

cacacaaagg gtttccccgg agccagagga ggaaacctgg ccttcctgca cttcctcctt  17640 

cgcacatttt aaaccatccg aggatgcaac tgggggaaaa gatttgcaga caaaagagct  17700 

gggggtgccg gcaacagctg tttgggccgg aaagcctggc cccggctcag accccggctg  17760 

ggtccctgcg ctgtggcggg ccggccgtga cccccgcccc acccaactcc gccccctgct  17820 

gcccgctgtc tccggagcgg cggctgtttg gggctggctc cttttctggg cccccacctc  17880 

cctgaagccc ccactgcccc tttcctcctt cctggggccc tgggagccag ggcagggccc  17940 

ccgtgcagct ttcagcagct ccagcccaac ggctgcaggc gctggggaag accacagctc  18000 

aggtgggacc gcaggtggtg gctggaggca gccatcccac atcgcagccc ccaatctcgt  18060 

ggttttgtgc cccaacggtg aacaggcctg gtggtgctgg gctgtcgtgg gccccttggc  18120 

cccctcggtg cctgcatccc tctctcctgt ccaagatggg gaaggaagcc gggagcagaa  18180 

agggaagaaa gacccagcgg ctgcagggcg cctttcaggg cctcctagcc accgcagagg  18240 

ctccagccag ctgtttgaac aggggttggg acacagggtc ctggcaggtt tgaggagccc  18300 

ggggccttcc ttgctatagc tctttgttct gggggatttg gggaggccag ggaaggggct  18360 

gtgagcaacg aggttccagc atcttcccaa gcctcctcca tcccccaggg ctgggctgct  18420 

tggccgttcc agtgggagag gacccgaggt gggaccccga gccccctgcc cagctcattt  18480 

cttagcagcc ggtgcctcct ggaaggtggg tggtgcccac tgggattggg gaaacttggt  18540 

ctagcctcta cccaagggag gggctgaggt cccaggactc cccctgcccc agaagccttg  18600 

gtccgggcta gagctgggct cgttggcctc atggaagcct gccttgggaa gcatccccac  18660 

ccaggtctgg ccttgtctcc ccgtctccag tagggggtca cagtgtcccc ccagccagtc  18720 

cacatatggc cccatttgct tgggcaggcc gatgggtggg cctgtctgtg tggcccaggt  18780 

cacctggtgg ggcccactgt cagtggcgta ggtagaaggt ggtcagcctt gctgggtctt  18840 

agcagagcac ggcaggatct gggtctgggg agggcgtggg ggtgcccctg ccatcacagg  18900 

agcaggcaga gtgggagccc tagactcccc tctgggcaga aagccccttg aggctggggg  18960 

caggtcctgc tggtgaggga ggggtcccag gggcaggggc accctcaggc tggccgccta  19020 

gcgattggct cctgcaggac gtggccacgg gctccctggg agcagcagct gttgggggtg  19080 

tctggggaga agacccccat ttattgccct gtgagggacc acactgctgc caggggaccc  19140 

ttgggcttct gtggcaggac agtgggccgg gattggcagt gaggcccaag gagggcaggt  19200 

ggggctggac ctgtggctct gctggggagc aggtgggatg tgttaagatg ctgatttcag  19260 

ccgggcagct cctccccttc ccttcccagg gagccggggt gtgcagggcc gttccttccc  19320 

ctctgggaag tgcagctcct gtgcaccgga gaggcccccg cactgtccct gcccgtccca  19380 

ccaactgtcc ctgcccgtcc ctccactgtc cctgcccatc cccctcactg tccctgccca  19440 

ttccccgcac tgtccctgct catccccaac actgtccctg cccatccccc cctcaccgtc  19500 

cctgcccatc ccccccgcac cgtccctgcc catccccccc cactgtccct gcccaccccc  19560 

cactgtccct gcccatcctt gccttgggag gtgctgtcac ctggtggagc catgggaagc  19620 

acctctccca cccagggtct ctggttcctg tggcgggtgc agagccagcc acatcactga  19680 

caccccagct gggttgggtc cagctgaccc cacaccccca cccctctgct cggcctctgc  19740 

ctgtgccccc atccctggcc ctgcatgacc ataaactcct tgagggcagg aagtgagtga  19800 

gtcctacccg cactgagtcc ctctcacccc tccttctcat tttggggcgg agggaactgt  19860 

cttctgtccc ctgggggtgg cagatggccg cttagcttag aaaggaaaac agcacaattt  19920 

agaattcgtc ctgggtgaaa ggtctcggca gcatctgcct cctgctgctt tctggagtcg  19980 

catttccatg gagcggccct ggcagggagg gtgggagtgg cacagtggaa atactgaggc  20040 

gggaacaaag tggtggcggt agaggtggcg ggagcggcac agcaggcgcc ggggccactt  20100 

ggcaccctcc ccccgcagcc ctcctggagt ccccgcccgc cgctcacctg cggggggccc  20160 

agataggaca ggttacccca gcgccgccag gccactgagc gtgggggatg ggacaggcag  20220 

gtgccgggtg accctaagag ggacgcacgg aaggggagtc cacgggggca cacctggcgt  20280 

gggttgggtc tcctccgtgg gagcccgaga gctgggtggc ttggtggaca gggcagctct  20340 

ctcgtccagt cgggggagac tggcgttccc tatctcaagc ctcacttttt agccccagag  20400 

gggtctcccc accgcccctg ataggaggag atgcggggac ggtggaaggt ggggtgcggg  20460 

tcactcatgg aagccctgag gatccccgtg ggcgcggcag ctgttgggtg ccacctcact  20520 

cggccaagcc ctggcgccca cctcgcggcc cacacccacc tggccgccgg gacggcattg  20580 

aagacagagt ggcccgggta ccgggggtgt ggccagcgcc aaagctgtga aggatttgct  20640 

cacaggctgg tggtcctggc catggccatg ctgcaggcct gaggtccctc ccaccttctc  20700 

agggacgtct gggtgctgtg cggagggcag gtgttcgccg tctgtagggt gctccagctc  20760 

tgtggccatg tgggagagag gtacagactg gggcgggcac aggctggagg ctgcccacag  20820 

cagcagcggg agactgggcc caaagctgcc ccccacacac agacccccac acctggctca  20880 

aaactttctc caggctcggc ccggttcccc ccagctagcg cgtctcacgg aacctccaag  20940 

ccgtggctgt ggggccgtgg caggggcctt ccctgtggtg tccgatgccc aggctgggag  21000 

tgggggcgga caatgggccc tctcagcctg cattccccca cgggagcctc ctcaggcctc  21060 

cggctggggc tcctgcccca cccgctggga gcagctgccg ccacgtctga cacctgctcc  21120 

cagccgccct ttgtccgagc accgacgcct gatttatggg gcgtgttttc ctcttgctgg  21180 

cccggcgtga tacagcctcg ttaactggga tgcgtccggt tgggcagatt ctcccacagt  21240 

cctccacgtt ggggtccgcg ctcagggtgg ggagtggggc ccggtgtccg ggcagagtgt  21300 

cgtggtccat tcagggacag ccaatgtcct gtgacctcag ggcacttggt gggagactgc  21360 

tctaggggct cggacctcca gcgtctcccc tctggggtcc cctaaagctg tgggatgggc  21420 

ctcccctgcc cctcctcccc gtcttgccca gcaggtgagc ttttcccgct ccttggagcc  21480 

tcgaggggcc gggaggcagg ggaccagctt gcatgggcca ggctgaggag cctccagccc  21540 

cggctttgat gaccgagcgc cctctgtcct gtacccaccc cctgccgact tcagcggctg  21600 

ggaaagcaaa aagggccttt tctccaagca ggagctgtgg ccaactcccg cctgacaatg  21660 

gggcatttat gtggagggag ggcgcccctc ctgtggccac agcccagccc agcccagcct  21720 

gtgcactcca cgggttccca gcggccgctc cacctcgggg accggccagc ctgtaactgg  21780 

atctgcatgc agtgggaacc acgtctgccc tcccaacagg tgcccgcaga ggcaggtctg  21840 

tcgctgcagc cccggagtga ccggccacat gggatggagg agggggagcc tcattccggg  21900 

gctgtgcccg tcgctcactg ggctggcagg agccgtagca aggcctgagt gaattcccca  21960 

gagccccttt ggaaggggtg acatgtgttt tctttaagcc agttggtttg ggacttttgg  22020 

cttctggcaa cagatggtcg gccagagtgg cccttttgcg ttctctctac ccaagccgtg  22080 

gtcacgggta aaagccttcg gggaagacag atgcctcccg ggcctttggg acaggggcgt  22140 

ggctggctgc tgagaaacgc acaggaaccc ccacccccag cctccacaag cccctcgggg  22200 

taaggccccc ccaggccact gccacacata gcacaggttt gattggagac actggggtgg  22260 

gaataagcgg gagaggaggc gaggacttgg ggggccggtg ggggtcctgt gggaagccgg  22320 

cagcttcagg gccggtcctg gggcaggcgg ccgccgcagc cccgcaccca ggtgggctct  22380 

ggaaggcggg acggttccct ctgtacatcc catctggagg aggccatgcc cacccagggc  22440 

ctgagccgtg aggccgctgc cttcccgggc cgctggggcc gctgaggctg gtggcatgtt  22500 

ctgcttcaga ggcttcacca ctgggaagag cggcgcccgg cgggtcccag accttttccc  22560 

ggagtgcaag gccaggaggt gactggggcc ggagatttat gaccacagca ggtggctggg  22620 

gggggcaggc tctgcgcctg ttgccgcagg gaacaggtga gaccaggcct gggtggagat  22680 

gcaggacccc tcccctggct ccccttgttc tctctgagcc cagactgtgt gtccggggtg  22740 

aggggcagcc tttcacccac cttgacgggg ttgcagagac ctggcagagg gacctcctcc  22800 

tggcaccttg gtttccccgg ctgtaatgtg ggtgcttctg ctcctggctg atgtgcctgg  22860 

atgtgagtta tgaagactca atgccgaaag ggaaacgtgt ctgggggctg agcgtgggca  22920 

gggaacgggc cagccttcat ccccagtgcg atcttgacat gagatgtccc atccccattg  22980 

cagtctcagc ctgaggtgtc ccatcagaaa ggcttcctgg ccgggcgcgg tggcttacgc  23040 

ctgtaatccc agcactttgg gaggccgagg cgggcagatc acgaggtcaa gagatcaaga  23100 

ccagcctggc caacatggtg aaaccctgtc tctactaaaa atacaaaaat tagctgggca  23160 

tggtgtcagg tgcctgtagt cccagctact cgggaggctg aggcaggaga atcgcttgaa  23220 

cctgggaggc agaggttgca gtgagcctag atcgcgcccc tgcactccag cctgggcaac  23280 

agagcaagac tcaaaaaaaa aaaaaaaaaa aaaagaaaag aaaaagaaag gcttccacgt  23340 

aggaaggggc tgatccacac tttccgtggc tgccatcatg ttgacacctg aaggttggaa  23400 

aaaccacggc ctttgttact gcccaagcaa tgcctgttcg ctttagcaaa gctgggaaga  23460 

aaagcagata ggcctgcaga cccggaggct ctacggagct aatgcacccg gctcctctcc  23520 

atggctctcc gtgggtgtag acccagaccc ctgcccgggc tgtggagtcc aagctgggtt  23580 

ctgctggaac atcctagtct ctagcttgtt ccccaagtta gcagtcattt gtcttcttct  23640 

ttctttcttt ttttgagtct tgctctgtca cccaggctgg agtgcagtgg cgtgatctcg  23700 

gctcactgca acctctgcct cctgggttca agtgattctc ctgcctcagc ctcattcagt  23760 

ggcattcagt acattcccaa agttgtgcaa tcgcggcatg tgtctggttc cagaacattt  23820 

tatcagccct aagggaaccc cgctaagcag ccgttgtgcc cgtgcccggg gtgtgggggg  23880 

aggcggctgt gcccatgaca ggttcccggt gctgagtggc gggggcgctg gggaagcagg  23940 

tgggaactaa ctgccctggc acatctgcca acagctgtgg cggggccttc gtgggcccgc  24000 

gatgccagga ccccaacccg tgcctcagca ccccctgcaa gaacgccggg acatgccacg  24060 

tggtggaccg cagaggcgtg gcagactatg cctgcagctg tgccctgggc ttctctgggc  24120 

ccctctgcct gacacccctg gacaacgcct gcctcaccaa cccctgccgc aacgggggca  24180 

cctgcgacct gctcacgctg acggagtaca agtgccgctg cccgcccggc tggtcaggtg  24240 

agggaggacc cacagcctga ggggtgggca gacccaggcc cgccgccagc tgcccaggca  24300 

acttgaggta cttggcggga tccaggcccg ggtctctgac ccggaagtaa ttgggagccc  24360 

ctgccccagg gtccctgccc ccctcccaga cctggaatcg cctcgttttc agctgcttgc  24420 

tctggcccaa ctcttgctgt ccccgcttct gcgagacaag gggagcctct gtgtccaggc  24480 

cgcccctgcc cccttcctcc tgccgtgcac tggccctcac ctgttagggt gaagggtgtc  24540 

tggaggtgtc agctctggga agagacggcc caggctcagg cctcttggga gctgtggtcc  24600 

ttcatctgcc aaaggcgacc ggtctcacgg caggtcctga ggatcaaatg tgatagcacc  24660 

taaggtggag cccaggctgg cacctccagg tggcctgaag ggagggaagg cccggcctcg  24720 

gggagcagtg aggccagcac gaccctcttg tccccttgtc tccagggaaa tcgtgccagc  24780 

aggctgaccc gtgcgcctcc aacccctgcg ccaacggtgg ccagtgcctg cccttcgagg  24840 

cctcctacat ctgccactgc ccacccagct tccatggccc cacctgccgg caggatgtca  24900 

acgagtgtgg ccagaagccc gggctttgcc gccacggagg cacctgccac aacgaggtcg  24960 

gctcctaccg ctgcgtctgc cgcgccaccc acactggccc caactgcgag cggccctacg  25020 

tgccctgcag cccctcgccc tgccagaacg ggggcacctg ccgccccacg ggcgacgtca  25080 

cccacgagtg tgcctgcctg ccaggtagtg ctgcccgctg gccggggtgc acgagcccct  25140 

ccccggctgc caggcccagg gggtgggaac ggggcgcctg gtgtaggggc agccctggga  25200 

gggcccatgg cggggagttg ggaaggcggg atggcgagtt ccccagactg cgtgtggtgt  25260 

cggggggacg cgtctggagc ggggtgtgcg ctgggaggag gcttgcacag ggtccaggtg  25320 

ccagcccgtg tctctggagt ttcccggaac tcacattcga gctgtaggac gccctgcctc  25380 

cccccagtgc aggcaccctg acttgcgcaa cccccagggt ttcccctgca gtggggctgg  25440 

ggccggagag ggcgtgggag ctgcgggggc accatcactt cccaagccat ggcccttcct  25500 

gccatcctgc tggtctcctg cgggtgtggg tgacaggtag gcgcagccgc ctgcccgtgt  25560 

ccccagtgct ccccgatccc cgtgttgtac ataggagcct ctctgctgta gttcttgttt  25620 

gagtaaattt tgcagcagct ctaaaaataa ggaagagttt tgaaaacctc cagtctcggc  25680 

cgcgcgtggt ggctcacgcc tgtactccca gcactttggg aggccgaggt gggcagatca  25740 

caaggtcagg agatggagac catcctggct aacacggcga aaccccgtct ctactaaaaa  25800 

tacaaaaaat tagccaggca tggtggcacg cgcctgtagt cccagctact caggaggctg  25860 

agggaggaga atcacttgaa cccgggaggt ggaggttgca gtgagccgag atcccgccac  25920 

tgcactccag cctgggcgac agagcgagac tccgtctcaa aaaaaaaaaa aaaaaagtga  25980 

agagaaaaaa gaaaacctcc aatctcagct gcacagaatg gctgttaggt gggaatgggg  26040 

agagggcatt caggcagggg tggggctgga gggtgcacag ggtcctggct gacccacggg  26100 

tggcccaagg gctgccccac ccccttggaa gccccaagct ggcctggccc ctgcaaaggg  26160 

gaagctccct ggctgcccag tcagctgagc ccagccacct ctgcgtgcag acaccagacg  26220 

tgtgcgtttc ctgcaagtca ctgggcgagc gtggggtggg gacagacact ccctggccag  26280 

cttggatctc tgcagggtgg ggcgcagagc ccttctgctg gtgggggctc agatgccttc  26340 

ctagcagcac gggaccctac ctccagtcac acggattgag gggccaagct cttggggggc  26400 

cgtggttcag gctagggcag gtggggtgtg tgcggccttg tccaaggcca cctgtgtgca  26460 

gagctgatgc ccacccgcct tgggcagggt gggtgaggac ctcctggtga ggcccagcgg  26520 

cagagatgcc aggggtgctg ggccggctca gagccactgc ccgtgcaggg gcagctcagg  26580 

gccctgggga gtgggcaggg agggaagggg aggggctggt ggggttcact cacttgagtg  26640 

tgggtagatc tcagaggctg gggtgctggg ggctgggtca cccgtccggt gaggggtgtg  26700 

ctcactgcct gctgtggagt ctggccgttt cagggtgaga tgaccatccg ggggcctccc  26760 

caaatgactc aggcctgcca gaccccagct tcagactgtc cccggcggcc cctgcaccca  26820 

ggccccctga agggccctca gccacctctg ggtggtcagg agcccaggag acagctgtgt  26880 

gtggagtcac ggggaaccga gatgggggcg gccctgagct ctgcaggccc gggaactggc  26940 

tgggatttgg ggtcgcacag cctgcctggc acgtcagcac tgtctcctcg tggggtcgct  27000 

ggccgaggct acgtcgcaga gggtccagct ggggtccacg gggctctcgc tggcttcccg  27060 

ccctctcccc cactgtgtgc ctcagccagt gctttgcagg gcagccctgg ggtcgcaggc  27120 

ccagccaggg gccccatctg cctaggagcc ggggtgcagg gccccacaca tgagtgggac  27180 

cgggactcag gctccaggct ctgaattttt tttttttttt tgagacaggt ctctgtcact  27240 

caggctggag tacagtgacg tgccctcagc tcactgcagc cttgacctcc caggctaaag  27300 

cgatccttct gcctcagccc ctcaagtagc ttgggaccac aggccggcgc caccacgctt  27360 

tgctacttct tgtatttttt tttgtagaga cggggtttcg ccatgttgtc caggctggtc  27420 

ttgaactcct gggcgcaagc gatccaccca cttcagcctc ccaaagtgcc gggattccag  27480 

gcgtgagcca ccgcgccagg gctggggtct ggttttgggt gctccccaag ctgatttgca  27540 

ttttggattt tggttgtctc tggctgtctg cttttgctcc ggtccagcag acagacagaa  27600 

cggacggggt ggtggcggtg gggcagtgtc tcagagggag gggccgagca ggtgggagct  27660 

ccaagggcaa cgggggttca aaggcatctc gggagggcgg caattagtaa ccggctgcag  27720 

gtttggctgt tgccgaacgc gcctttctgg gttgatcatt gaaccagcgg gagttgagtg  27780 

gattaatagc taactggctg ccttggaggc ctgggtgggg ccatacctcg ggggaggggg  27840 

ccctggcagc ctgtggagga ggagggggag gaggaggtgg gcaccagagc tctgccccag  27900 

ggaggatggg ccatctgaat ctctgcagct tccctggggc tgcgaggggc ccaccctgcc  27960 

ccagccagca gcctgctggc cccatgtccc cctgctgctc agagcccagg ccccagtgtc  28020 

agcccctgaa gatggggtat gcggcccccc tgtggagaca ttgtagggag ctctgagcat  28080 

gcaggcggca cccaggtgca ggagctgccg gaacctctta aaagagaccc aagggttttt  28140 

gtgacagaat cttctggaac cctgtgttcc tgagtccctg tttagccagt cctgtccaaa  28200 

atgattcttt caagcagggc gtgtccctct gggcactgga gtgaggcagg ggacgggcac  28260 

agaccccggg ggccccaggg cagggccgtg gaggccaggc tcttgtgtcc agagcagtgt  28320 

gtcgggggtc caggcaggag ccggactgga cctcagggaa gaggctgacc cggcccctct  28380 

tgcggcaggc ttcaccggcc agaactgtga ggaaaatatc gacgattgtc caggaaacaa  28440 

ctgcaagaac gggggtgcct gtgtggacgg cgtgaacacc tacaactgcc gctgcccgcc  28500 

agagtggaca ggcgcgtata cgggtcgccg cagggcgggg tagccggggc ggggctgcta  28560 

cccacttact aaactgcact cacaggcata gcggggagca gccgagaccc tgtcccaggc  28620 

caggcagact agggttggcc gagaaggaca acatgatggg ggccccactt cccgtggcca  28680 

gggtctggtg cccctcacct cgggggtcag cacagacagg gccctggttg gtctgtgtgg  28740 

gccgtttgca acctggctcc aggcagcccc tgtgcccagt ggggtgtcct ggcattgagg  28800 

gccttcagca ccccactcag gccacgcttc ctgggccagt cacagggatg cctggatgaa  28860 

cagacaggcc ctatgttcgg gaattaccct ctggtggggt gacgggcaaa ggtgcacccc  28920 

gacgcagcag tgtccacaga gcacaaagag gaggcaagac tccatggtgc tgggcctgca  28980 

gaggaaggag tgatttaggc tggcggtggg tggaggagtc agcccaggaa ggcttcctgg  29040 

agggggcggc attgccaccc tgcgtcttag gggacaggga gctcagggag tggcagctgc  29100 

ccggggccga cagctcctgt tccctgcagg tcagtactgt accgaggatg tggacgagtg  29160 

ccagctgatg ccaaatgcct gccagaacgg cgggacctgc cacaacaccc acggtggcta  29220 

caactgcgtg tgtgtcaacg gctggactgg tgaggactgc agcgagaaca ttgatgactg  29280 

tgccagcgcc gcctgcttcc acggcgccac ctgccatgac cgtgtggcct ccttctactg  29340 

cgagtgtccc catggccgca caggtgagtg ccttgaggcc caggtgggcg cagggggctc  29400 

acatgggcca ggcctgagct gtgtgacctc acccagggac tgtggccctc ctgcacccag  29460 

gataacctga aagggccttt ctggtcaggg aggccggtgg tcgctgtgag tcccccggaa  29520 

catcccaagt gtcacgggat gcccgacggg gtcacggcgg gcagctcctc ctggggtgtc  29580 

agtggggttg gaccccagcc gtgggtggtg tgccatgcct ggcccagggg ccgtgcccac  29640 

tgaccgccgc tgcctgccca ggtctgctgt gccacctcaa cgacgcatgc atcagcaacc  29700 

cctgtaacga gggctccaac tgcgacacca accctgtcaa tggcaaggcc atctgcacct  29760 

gcccctcggg gtacacgggc ccggcctgca gccaggacgt ggatgagtgc tcgctgggta  29820 

ggtgccagca cagggggtgc ggccaggtgg gggtggcagc ctggccccag aaccgatgag  29880 

aagtcgattc tggcttcagg gggtgcccag ttagatgggg gatgggaccc tgccagtccg  29940 

atgggggtgg tgtgcagtga ggtgttgcag gggccagggt gcctggagca gcctctcacc  30000 

cgtgtgccct cgccaggtgc caacccctgc gagcatgcgg gcaagtgcat caacacgctg  30060 

ggctccttcg agtgccagtg tctgcagggc tacacgggcc cccgatgcga gatcgacgtc  30120 

aacgagtgcg tctcgaaccc gtgccagaac gacgccacct gcctggacca gattggggag  30180 

ttccagtgca tctgcatgcc cggtgcgttg gccggggcca gggcgggaaa ccgagtcgag  30240 

gctgggcagc cttggagggg cagccccggg aacatctgtg ggttgcttcc gctttcccca  30300 

gcctccatgc cttctgggcc cacagcctgc acggggcatg gggaaactga ggccaggcca  30360 

cagggccagc agagcacggg ggcagtgcta ggcagatgag ggtgccgggc caggggccac  30420 

gggctgcagg ggagcgggtg cgcagggggc ccgtggtggc tgggacactg ggctggaggc  30480 

agggttcgtt tctgtcccaa gtccacagct gtgcccagtg gggcattggg gccgcgcgtg  30540 

cccccctcac tgttgcccca ccccacaggc tacgagggtg tgcactgcga ggtcaacaca  30600 

gacgagtgtg ccagcagccc ctgcctgcac aatggccgct gcctggacaa gatcaatgag  30660 

ttccagtgcg agtgccccac gggtgagggc cgcccccgcc ccctgccccc gggtcgtctg  30720 

caccctggcc tcctgagggg tgcctgggcg tgggttgtgt cccctgcccc cgggccatct  30780 

gcaccccggc ctcctgaggg gtgagggccg cccccacccc ctgccctcgg gccgtccgca  30840 

ccccggcctc ctgaggggtg agggccgtgg gttgtgtccc ctgctcctgg gccgtctgca  30900 

ccccggcctc ctgagggggg cctggctgtg gattgtgtcc cctgcccctg ggctgtctgc  30960 

accccggcct cctgaggggt gcctggccgt aggttgtgtc ccctgcccct gggccgtctg  31020 

caccccggcc tcctgagggg ggcctggcca ttggttgtgt tcagattccc aggagagcga  31080 

gggttttgcc tcatgagtgg atgggagtgt tttcagactt ccccgaagga agggcagggc  31140 

ccagtgggga gtgggagctt gcccaggggt cgcggtggag cccagggcca ggagatcctc  31200 

cttgatctgg gtgcagcccc ttctgcctgg agagaagggt ataccaggag ctgcagtgcc  31260 

cagacaggga ggaggctcca gcctggcttt ctgagggctc gaggctggga gggaggccag  31320 

cctgccctgt cctgccatgt cccctgccca acggcacgcc agcgacaagg gtatgcagag  31380 

aaccacgtgg ggcaggttgg cctgcaatgg agatgatggc tgcccggggc ggacttggag  31440 

gaactctcag gggcctgggt gaaaggtgtt tccatctgtc ccagagctgg gggccggggc  31500 

gcagagccca gggaggcgtt gccagcccaa gcctaggctc ccaagacata gattacccgt  31560 

cccagacact ggagcgaggg gagccccatt ctcagcccgg ctccactgta gccatagcaa  31620 

cccagtcggt ttgggaaaaa ggccccttct gtgggagcga gggcgtgtgg tgctggggga  31680 

ggggctttga tccggggagc gggcagcggg aggcagggag agctggggct ggctgagctc  31740 

aggctgagtg tgccctgggg agcccagcca ggcgctcact gctggggtct ggccaggggt  31800 

ccctgaaggg ccatagcgct gttgcacatg actccctccc ctcccctccc cggccccagg  31860 

cttcactggg catctgtgcc agtacgatgt ggacgagtgt gccagcaccc cctgcaagaa  31920 

tggtgccaag tgcctggacg gacccaacac ttacacctgt gtgtgcacgg aaggtgcggg  31980 

ccggcgccca ccagcgggga gggactgggg acgggacagg gcactcaggg cagtgaaact  32040 

gacaaggtca tggacacctt ggtctgggcc acctggtcca gaggccgaga gcacagcaat  32100 

cctgagtagg tgggaatgcc acgctcggag ccctcctcag gttggcacag gtgaccccag  32160 

gtctggtcat gggtgtcccg ggggccgcca gtcctaagtc ttcctgtgcc cgcccctccc  32220 

gcggtccagg gtacacgggg acgcactgcg aggtggacat cgatgagtgc gaccccgacc  32280 

cctgccacta cggctcctgc aaggacggcg tcgccacctt cacctgcctc tgccgcccag  32340 

gctacacggg ccaccactgc gagaccaaca tcaacgagtg ctccagccag ccctgccgcc  32400 

acgggggcac ctgccaggac cgcgacaacg cctacctctg cttctgcctg aaggggacca  32460 

caggtggccg gccaggcggg tggccggcgg ggggccagtg ggcagggcgg gcctgaggac  32520 

tgaccgacac gtgccacccc ttcaggaccc aactgcgaga tcaacctgga tgactgtgcc  32580 

agcagcccct gcgactcggg cacctgtctg gacaagatcg atggctacga gtgtgcctgt  32640 

gagccgggct acacaggtga gcggccctgc acgtgggggc tgactgcact gtgctcagag  32700 

gtcaaggtca gccactctgc cctggggctg ctgaggggtt gggtgagggc tttggtgctg  32760 

cccacctggg cagaccgtgg tctgcagcag agattgcagc gggaccaggg ctataactgg  32820 

cagcacgatg ggtccccccg acccctcttg tctccggtca gagctgtccc aggccgtgtt  32880 

ggcagagtgg cccagcaggc caagagggag cagccagccc tgaggccagg cttaagtacc  32940 

ttctgcctct gtggctcacc agcacctttg agcaggtccc tctgcctctc tgggcctcag  33000 

attctcatca gtcagggctg cagtagcccc tgcccctggg gcccctggga ggatgacttg  33060 

agtgggcact ggtgcccggg gaggctggca catggcacat tgtgatccaa gatggtttct  33120 

gacacctgga aggatgtggc cagaagggtg atcgccccgg ctcaacagac agggaaatcg  33180 

aggttgacgt gggtgggacc ccctgggcgc tgggcctcgg agtctgaccc gccctgccct  33240 

tagggagcat gtgtaacatc aacatcgatg agtgtgcggg caacccctgc cacaacgggg  33300 

gcacctgcga ggacggcatc aatggcttca cctgccgctg ccccgagggc taccacgacc  33360 

ccacctgcct gtctgaggtc aatgagtgca acagcaaccc ctgcgtccac ggggcctgcc  33420 

gggacagcct caacgggtat gcggcggggc cgatcatggg gacacatcag tcctaaaccc  33480 

tgggagctct gctgccagga gggtggcacc tgcacagagc ttgagatggg ccagaaacgg  33540 

gcctcggacg gggctgggtg gcagggagct cccctcgggg acgcacatgc ctctgggtcc  33600 

tcagtcagac tacagtccac acccagcagc tgtgtgctgc gcgtccttgt gggctgcatg  33660 

gtgtgctcag gacacaggcc cacagtgacc ctggaacacg tttccatggt aacagccgca  33720 

agtgtttctg atgcccatga tgtgcctggc agcactctgc ctggatggga ccatttaaat  33780 

cagcagggac cccattgccc tcatttggcg cagagaaggc acgtgctttg ttcggggcca  33840 

cacagcaggc aagtggtggg gggcttttcg cagcacccta ctgccctgca cttggcatgg  33900 

tcccctcgct aatgaaccaa acccctgccg cagtcttggg tatgggaagc gctgcggccc  33960 

ccactttttt ctttattttg agacagagcc ttgctctgtt gtccaggctg gagtgcaatg  34020 

gcatgatctc agctcactgc aacctccacc cctcaggttc aagtgattcc cctgcctcag  34080 

cctcccgagt agctgagatt acaggcatgt gccaccacac ctggctaatt tttgtatttt  34140 

tagtatatat ggggtggggg agtttcgcca tgttggccag gctgatcttg aactcctggc  34200 

ctcaagtgac tcagccaccc ccgcctccca aagtgctggg attacaggtg tgagccacag  34260 

cgcccagcct gcccccacta aaggactctg cgagtctgag tggatgggca cctccgccag  34320 

cccatagggc attgcagacc cgggagtgcc caggccccgg ccgtgctgct cgggcctccc  34380 

tcgacctgca gtgtggtccc ccttgcaggt acaagtgcga ctgtgaccct gggtggagtg  34440 

ggaccaactg tgacatcaac aacaacgagt gtgaatccaa cccttgtgtc aacggcggca  34500 

cctgcaaaga catgaccagt ggctacgtgt gcacctgccg ggagggcttc agcggtgagt  34560 

gggctgcgcg tttctcagtg cagagggccg ccctcgagtc tggggagctg gagacacagg  34620 

atcgggaccc aggtccagcc agcaccatgc atggtgtctc cctcccgtgg caaagcctta  34680 

gctccaggcc tctggctctt ggagatgagg aggggcctgg ggtggggagc aggcccaggg  34740 

ctgctgttgt ggggccctgc caaggtgcct gggagtgctg gacatgccga gtgctgtccc  34800 

cttccctcca ggtcccaact gccagaccaa catcaacgag tgtgcgtcca acccatgtct  34860 

gaaccagggc acgtgtattg acgacgttgc cgggtacaag tgcaactgcc tgctgcccta  34920 

cacaggtgag gggtgggtgg ggcctatgct ggagggggcg gggcctatgt gggaggggcg  34980 

ggacctgtgc tggagagggc ggagcctgtg ctggagaggg tggggccagg ggtgggggcg  35040 

gcctgggaag ccgtgactgg agggtaaagt ggtggctttc ttggggcggg gccttccaga  35100 

aggttctgcg cctcactgag tgccttaggc ccctgcagta tgtgggcctt ctcccggggg  35160 

tggggttggc agattagcaa agatacagga tgcctgggag aaaattttca tgcaagcatg  35220 

tgttttacct ggcaagcctg ccgcaagaca caggtaagat gcaggtgaat ccttagcagg  35280 

gccgggcctg ggtcactggg ctccgtgctc ccggcgccca ccctgctggg gtctctgggg  35340 

gccgggcttc cccacccccg ccagcaggac ctgagggatt cctttgatgg cgggctctct  35400 

ttgtgtgagc ggaacaagga gccctctgtt tgggctgagc ggggaggggg atgttccccc  35460 

aactgtccag cccctgctat gaacccagcc aaggggatca caggaaatga tctcctctag  35520 

aaaagacaaa gaagagtcca caagcaccgg ggtcctggcg ctccccagga gcgggcagag  35580 

ggtgctgcca cggggcctgg tcagggaggg cctgcctggg ccaggcagcc gggctggagg  35640 

tcccacgcct gcggttccca tgacatccag cagcctgtgg ccagaacgct gttttctttg  35700 

accgaagtta aggacctggg ttttctctcc tgtgtcagtc agtgaggacc ctccctccac  35760 

ggctgggcgc ccctgtccct ttcagacacg tcgatcgggc cggcaggagg agcggcggtg  35820 

ctgacccgct tcctatctgc ccgcttcctg cttcctttcg cacaattgtc ggaaactctg  35880 

gcgcagttcc tgcctgcccg ggtcactgtg gaaaccaata ggaagagagg ccctggaaag  35940 

tcgccccagc aggccaggga gataatgggg gacagggaag ataacgcagt ccccacggct  36000 

ataaacagga agctcggcca tgggcttgtg cagaaaaagg cccttggctg tgcacgtggt  36060 

tcctacatag cctttgccag acacgctgcg tggccctgga ggtctccagg ccgtgccccg  36120 

tgccccgaca cgcaggatgt gctgggggcc tgggcctgtc ccctccatcg cagcccttgt  36180 

ttgttggcct cccgtgatgg gcgtttgctg cctgtgacac ggagccccag gattagtttc  36240 

aggcctcggt ctcaggccag ggcctttcct cgtcacccag agatgagggg gcttgcgtcc  36300 

ggggtgggcc caggtccctg cagtgtcgtg gagcccaccc gactgaggcc gcctctcctg  36360 

gtgcagtcct tgttcctggg cacgcctggt ggggtgaggc ctaggctgtg ccccgctgcc  36420 

aaccgggaat gaggggtggc ttctgagcgt gacatttgtg cagcttgtct tcagcgtccc  36480 

cccatctgtg ctccactcct ccctgggtca gggtcctggc acagccggga gcgctcaggg  36540 

gtctcggtgc acatttgcct cccggcggga ccagcagacg gcactctgat ggcggaaaga  36600 

ccagcaggcg gtggccgatt tgggagatcc ctctgggtga ggctcccggg gtcacgtgtg  36660 

tctccttccc gcaggtgcca cgtgtgaggt ggtgctggcc ccgtgtgccc ccagcccctg  36720 

cagaaacggc ggggagtgca ggcaatccga ggactatgag agcttctcct gtgtctgccc  36780 

cacgggctgg caaggtgagg ctggccaggg cccggtgagg gctgggatgg gaggtcagga  36840 

tgtctgcggg acacaggcag ctcccaggca ggctagatga gtctttgaag aggagccggt  36900 

gggtgctgag gaggccctgg tcagagaagt tctggaatca ggaattgacc tgggagcacc  36960 

gttcccaact ccagttcctg tgaccttctt aggccaaaat taggggagag gggatggtcc  37020 

tggggtcacg gaagcccact cctgggtcgg gagaggcact gtaggtgggt gggccagcct  37080 

gggaagggcc tggagggcca ggggccgctg gtgaccaacc ggcctcctcc tgccccacag  37140 

ggcagacctg tgaggtcgac atcaacgagt gcgttctgag cccgtgccgg cacggcgcat  37200 

cctgccagaa cacccacggc ggctaccgct gccactgcca ggccggctac agtgggcgca  37260 

actgcgagac cgacatcgac gactgccggc ccagtgagta gccccgcggc tctggcctcc  37320 

tccaggaagc tctcaggcct cagttccccc gggcagggtt ggtgcgcatg gtgctggcca  37380 

taagcaccca gggaggccgg agtgtggccg gggagggttg ggtcaggtgt gggggatgtg  37440 

ctgagggatg gccagggtgg ttcaggaggg ccccagagcc ggcttgctcc tccctgcact  37500 

tcgcggaaga gtcacgagag cccctcccac ggctcttcac tgagccgagg atggctcggg  37560 

cccggcctgc actcccgcct ggctcatcgg gccccgctgc aggccagggc tctcaggcct  37620 

cccagccctg cttcacagag gttgaggttg gcagaagccg ggggtcttgc tggcatcacc  37680 

tggcaggcag aggcaagaac tgtctctcct cccctgctcg gtctgtgaag cctgcaaagc  37740 

tgccccctgc cctggagctt gaggacagga gcaggtgggg agagagaccc caagcacagg  37800 

agacgggtgt gacgcagcct gtgggtgctg gggctcccca ccagacacct ttgtcacagg  37860 

gctgcttgcc gcagcctcgg caacgaaagg ctgggctgtc cccagccatg cagccttccc  37920 

ggccccctcc cgcaggtgtg ggtttgtgcc tgcccctcac actcaccctt ccgtcctctc  37980 

ccagacccgt gtcacaacgg gggctcctgc acagacggca tcaacacggc cttctgcgac  38040 

tgcctgcccg gcttccgggg cactttctgt gaggaggaca tcaacgagtg tgccagtgac  38100 

ccctgccgca acggggccaa ctgcacggac tgcgtggaca gctacacgtg cacctgcccc  38160 

gcaggcttca gcgggatcca ctgtgagaac aacacgcctg actgcacaga gaggtgtgcg  38220 

gggctcgagt gaggccgtgg aagggaacgg gcggtgcggg ccacacgcag tagctggcag  38280 

cctggcacac catgcaggcg tccgcctagg ccggctgggc acttagcgct ggctgcattg  38340 

agtccagaca ttgttcattg taccgacctg tccctcctga gagggccatt gtgacctcct  38400 

cctgtgtccc gcaccaggag cgccggtagt ctgtcctgaa gaactggtgc gcttccttct  38460 

attagacaac gagaactctg tccgttcttg tttccttttt gccgtgtttg ccaggaagct  38520 

catttggtaa acgtcagtct cgtccctggt ttctgacgta attacctccc gtgttaccag  38580 

gacggtttct ggtttctccc tcatttactt taaccaccct atgcccacgc gtttggtaaa  38640 

accactctcg acctgactta taaagaaagc aggggaggga ggtgtgacgt ggtgtgagag  38700 

ccgggccccg ggttcccact ggcctccctg ggtcagccag gctgccctgg gcctgtcctg  38760 

gccatcaggc ccctagggtt gagcagaagg ggaggtgctg gcgaggcaga atctgctgga  38820 

gcggggaccc accaatgccc tccgctcagc ccccgcctgc ccaccccctt gcagctcctg  38880 

cttcaacggt ggcacctgcg tggacggcat caactcgttc acctgcctgt gtccacccgg  38940 

cttcacgggc agctactgcc agcacgatgt caatgagtgc gactcacagc cctgcctgca  39000 

tggcggcacc tgtcaggacg gctgcggctc ctacaggtgc acctgccccc agggctacac  39060 

tggccccaac tgccaggtga gtgcgccggc cacagaggtg cccgaaggag gggccctggg  39120 

tgggtgcctg cctgcgggag gtgggaacgg tcacccccag gtcccactgt gtcggctcgg  39180 

ggtcaccccc acacccccgg gcagagggtt tctggggatc tgagcagccg gtgaaggaac  39240 

ctgatgcgga agcagcaggc accttcgttt caatcccagg tttctggagc cggggcagga  39300 

gctcaggaat tggggaattg aggaaaagtg ttccttctag caaaggccga gggtggtctg  39360 

gacctgctga gggccctgag ggagacccag cccaggctgt tcctggtgtg ggggtggtgg  39420 

ggagtccagg ggcggcagtt tccacttctg tagaatgggt tgcagcctgg gttggagtag  39480 

gccccttggc agatgtgcgt tctgagctac cggggaaatg gccggggcgc gggcacccag  39540 

ctgaccccaa tctgtcccca gaaccttgtg cactggtgtg actcctcgcc ctgcaagaac  39600 

ggcggcaaat gctggcagac ccacacccag taccgctgcg agtgccccag cggctggacc  39660 

ggcctttact gcgacgtgcc cagcgtgtcc tgtgaggtgg ctgcgcagcg acaaggtaac  39720 

ctgctgtgcc cacccggctc gggtcccagc ccatcaaggt cctctgtggg cctgggcctc  39780 

acctgtctac caccccatcc cccgcaggtg ttgacgttgc ccgcctgtgc cagcatggag  39840 

ggctctgtgt ggacgcgggc aacacgcacc actgccgctg ccaggcgggc tacacaggca  39900 

gctactgtga ggacctggtg gacgagtgct cacccagccc ctgccagaac ggggccacct  39960 

gcacggacta cctgggcggc tactcctgca aggtgggggt ccctcctagg gtaagggttg  40020 

tggccggcac gagtgttgcc acacaccagg ccctggctgg gagctggccc agtggagaaa  40080 

actgaacctg ataggcccat gcactgttca gtctcattag gggagggctg gggtaatcag  40140 

ggtagactgc ctggaagagg tggcctgtgg aaagcctgaa ggagggtacc tatactgaga  40200 

agtgggatgg ggttttccct tcccctagat tgtgtctggg cttggccaac acctaccctg  40260 

aggccctcac ctctatccta tgggacgggg tccacccacc cccaacaggc agtgactccg  40320 

gtcaccgagg ccccgccagg gtctgttggg ctgggtctct ctccaggtct gacaggagcg  40380 

aggggcccgt ggcttcgctg gacctgaggg cagctcatgt ggccctgtca gccctcacag  40440 

tggggtgtgg gagcactgca tcctggcgcc ggctgagccg aagggcccct cgttctgtcg  40500 

cctgcacagt gcgtggccgg ctaccacggg gtgaactgct ctgaggagat cgacgagtgc  40560 

ctctcccacc cctgccagaa cgggggcacc tgcctcgacc tccccaacac ctacaagtgc  40620 

tcctgcccac ggggcactca gggtaagggc cgctgcacgg agggctggtg ttggccatcc  40680 

atggccaggg caggggcagg gcaggcaccc cgggaccggc cagaggcatc caggaaccag  40740 

ccagaaactc ccattttcct gtgtgaggcc aaggccgact cacagctgcc cagggaaagg  40800 

gcccagagcg ggggtcccag tcggaagggc gtctccacgg cacccttgac acctgcctct  40860 

cccgagtgtc cgtgcagccc cagacctgag cgcttgtctt ccgggacgga cacgcggcac  40920 

ggcagggccg gggtgtggcg ggcttgggcc actgacgaaa cctggccccg caggtgtgca  40980 

ctgtgagatc aacgtggacg actgcaatcc ccccgttgac cccgtgtccc ggagccccaa  41040 

gtgctttaac aacggcacct gcgtggacca ggtgggcggc tacagctgca cctgcccgcc  41100 

gggcttcgtg ggtgagcgct gtgaggggga tgtcaacgag tgcctgtcca atccctgcga  41160 

cgcccgtggc acccagaact gcgtgcagcg cgtcaatgac ttccactgcg agtgccgtgc  41220 

tggtcacacc ggtgggtgcc gcgcccaggc gggtggggcg tgtggggcag cagggtgagc  41280 

ctctcactgc cctgctctta cccctagggc gccgctgcga gtccgtcatc aatggctgca  41340 

aaggcaagcc ctgcaagaat gggggcacct gcgccgtggc ctccaacacc gcccgcgggt  41400 

tcatctgcaa gtgccctgcg gtaggtgcag gggtgcaggg aggcaggggc ccgccagggg  41460 

agacacctgg agaggtccac gtgggggcct cgggacgcag accgggcagt gatcctcccg  41520 

gccttcatcc tcctcctcac cctgatgtct tttttttttt tttagtttca ataaatgatt  41580 

ttagagacat tttagattta tagaaaaatg gagcaggaag tagactccct ggggtggctg  41640 

tcccctgcac gcagttcccc tggttagcag cttgcatcaa tttcactctg gatttcagtg  41700 

atacattgtt accaacagca gcccttcctg tacctggggc ttgcccttgg ctttgtggtt  41760 

gtgggtttgg gctgaggtat gacatgcctg cccaccctcg caggatcacg gcagagagtc  41820 

cctgccctaa agtcctcagc actccaccag tttacccctt cctccatctc ccgaccccct  41880 

ggcacccccg atctttctct gttggtagaa tgtgtgtaaa gggttttgct gctgggggca  41940 

aggttgcagg ccgcctccca ggttagagga gagcggtggc actgctggcc gagggctggg  42000 

tgtgaggtgg cgggggggcg gggggtggcc caccccgaca ccgtcctgtc ttccctctcg  42060 

ggcagggctt cgagggcgcc acgtgtgaga atgacgctcg tacctgcggc agcctgcgct  42120 

gcctcaacgg cggcacatgc atctccggcc cgcgcagccc cacctgcctg tgcctgggcc  42180 

ccttcacggg ccccgaatgc cagttcccgg ccagcagccc ctgcctgggc ggcaacccct  42240 

gctacaacca ggggacctgt gagcccacat ccgagagccc cttctaccgt tgcctgtgcc  42300 

ccgccaaatt caacgggctc ttgtgccaca tcctggacta cagcttcggg ggtggggccg  42360 

ggcgcgacat ccccccgccg ctgatcgagg aggcgtgcga gctgcccgag tgccaggagg  42420 

acgcgggcaa caaggtctgc agcctgcagt gcaacaacca cgcgtgcggc tgggacggcg  42480 

gtgactgctc cctcaacttc aatgacccct ggaagaactg cacgcagtct ctgcagtgct  42540 

ggaagtactt cagtgacggc cactgtgaca gccagtgcaa ctcagccggc tgcctcttcg  42600 

acggctttga ctgccagcgt gcggaaggcc agtgcaagta aggctgcggg gctcatgggg  42660 

ctgagggagg acctgaactt ggatgtggcc tggcttgggc ccggaggcca gcatgcagtt  42720 

ctaaggctct gctcaggggg tgcagggacg tcccccgcgg ctggccagtg ggctggaggc  42780 

accggacggc gggtgcgagg ccccccgagg aaggcggcct gagcgtgtcc cgccccccac  42840 

agccccctgt acgaccagta ctgcaaggac cacttcagcg acgggcactg cgaccagggc  42900 

tgcaacagcg cggagtgcga gtgggacggg ctggactgtg cggagcatgt acccgagagg  42960 

ctggcggccg gcacgctggt ggtggtggtg ctgatgccgc cggagcagct gcgcaacagc  43020 

tccttccact tcctgcggga gctcagccgc gtgctgcaca ccaacgtggt cttcaagcgt  43080 

gacgcacacg gccagcagat gatcttcccc tactacggcc gcgaggagga gctgcgcaag  43140 

caccccatca agcgtgccgc cgagggctgg gccgcacctg acgccctgct gggccaggtg  43200 

aaggcctcgc tgctccctgg tggcagcgag ggtgggcggc ggcggaggga gctggacccc  43260 

atggacgtcc gcgggtgagt gagacccggc gcccacggtc aatccccgca actctcctgg  43320 

gccctccccg acggcctccc tgcccctcac ggccggcgcc atggcaagca gtactctccc  43380 

cactttatgg taaaagagac ggaggttccg agaggacctg ggacttcagg gcctgcgcag  43440 

gcaaggagta gaggcagcat tccagctcag ggccctgacc ccacagccac acccttttcc  43500 

tgggccactg ccttcccctg gacaggcggc actcctgtgc ccagtaggtg attttgagat  43560 

tgagctgtgc cttaggcact ggatactaca ctgattaaaa ctcagccctc tgccaggcga  43620 

ggaggctcac acctgtaatc ccagcacttt gggaggctga ggcaggcgga gcccttgagc  43680 

ccaggagttc gagaccagtc tgggcaacat agggagacct tgtctctgtt tttttaaaaa  43740 

aagtattaaa agaagtaaaa aacaaaacac tcagctctcc aggggttccc acagggctga  43800 

acagccccac cccagacaag aatgccgctt ggtcatggcg tctgcctggc ttgggctgga  43860 

gaggaggcag gtggaggtcc tgggaggggg cattgctggg ccttgcagtt ggaggaggag  43920 

ctggtggggg tggggggtcc cacggtggga gaacacaggc aggagcagct tgagtgcagg  43980 

gggcacccca caaggctccc gcccatgcct actcgatctg gggcagctgg acccaggagc  44040 

caggttggtt gtgcccttcg tgtgcctgac cctggtgggt ttgcctgtca gttctgctgg  44100 

cttggagcaa tcctggaggt cagaagcatc atctcacagg ctggatggga tcctgctcac  44160 

gggaacccag tcctggggag cagagctcac ccccagccag cctcaccaca cagcccacca  44220 

ccgctgcacc cacccccacc tcacgcctgt gcttcctgca gggcctgggg atggctcctg  44280 

ggggaggact gggctcctgg cacatactct gtcctgagat gaggaaacgt gtctgtggca  44340 

ccagcaggag ccagagaggg tgtcagggcg ggccagggag agcgcgttgg tgggtatctg  44400 

ggatgagccg tgatcagcac tggccggagt cggggggctg gcaccagtcc cctgcagggt  44460 

agctgctgtc agacctggct tcccaccacc ccaggctgcc tcaccatgtc ctgactgtgg  44520 

cgtcatgggc ctcagtgtcc tgcggcagca tccctggccg gtgggcgggg gaggaggaag  44580 

cctcgggtcc cagcccctct ctgattgtcc gcccagctcc atcgtctacc tggagattga  44640 

caaccggcag tgtgtgcagg cctcctcgca gtgcttccag agtgccaccg atgtggccgc  44700 

attcctggga gcgctcgcct cgctgggcag cctcaacatc ccctacaaga tcgaggccgt  44760 

gcagagtaag tgtggcccca tcccgggaac aggctctgcc tgcagggggt gccatccccc  44820 

cgtgccccag acacgctggc tgtttgtgcc agttgctacc cacgggtgtg agcgttgccg  44880 

tccgagttgg ggtagggctt ttctggaatt ttctgaatgg cactccgccc ccacctgcgg  44940 

cggtcacagc tgccggtgga gccacctggg aacgagtcca gccacgggaa agtgggtgcc  45000 

tgcttctctc cccacccttt cctcctgaat tttctttgtt gggtattatt tcaaaatcat  45060 

tacggctttt tttaaagaaa aaaaaagaga gagagagaag aattgatcgg tgtcatgtga  45120 

agtgttgaag tttgtatctt gaaaatccct ctaaatcctt tgtcttaaca gctcagtgcg  45180 

agtgcagcga tttgaagttg actaatcctc cttccttaaa ggagaaaaaa gtaaaagccg  45240 

tctccagata gagtcggctg gtgcaggaga gaatttagcg atagtttgca attctgatta  45300 

atcgcgtaga aaatgacctt attttggagg gcgggatgga ggagagtggg tgaggaggcg  45360 

cccggacgcg gagccagtcc gccgcccccc ggccaccagc ctgctgcgta gccgctgcct  45420 

gatgtccggg cacctgcccc tggcccccgt gcccgcaggt gagaccgtgg agccgccccc  45480 

gccggcgcag ctgcacttca tgtacgtggc ggcggccgcc tttgtgcttc tgttcttcgt  45540 

gggctgcggg gtgctgctgt cccgcaagcg ccggcggcag catggccagc tctggttccc  45600 

tgagggcttc aaagtgtctg aggccagcaa gaagaagcgg cgggagcccc tcggcgagga  45660 

ctccgtgggc ctcaagtgag cggacgccgc ccctgcttct gggtccccag tgggaggcca  45720 

ggcccgggcc aggggtctct gggggcttcc tagggagctc gctcagcctc acttctcgac  45780 

ccctcacccc ccaggcccct gaagaacgct tcagacggtg ccctcatgga cgacaaccag  45840 

aatgagtggg gggacgagga cctggagacc aagaagttcc gggtgagtcg cgaggctccc  45900 

gggctcctgg gctcccgggc acctgccgcc gggctgccct gacaggctct gctcactccc  45960 

tctatgtagt tcgaggagcc cgtggttctg cctgacctgg acgaccagac agaccaccgg  46020 

cagtggactc agcagcacct ggatgccgct gacctgcgca tgtctgccat ggcccccaca  46080 

ccgccccagg gtgaggttga cgccgactgc atggacgtca atgtccgcgg gcctggtaag  46140 

ggtgccagca gccagggctt ccctagcccc gtggcccacc tgcctctctc ccctaagccc  46200 

cgaggctggg gtacagttga tactctggaa acttagaatt gggggtgaga gctttcatcc  46260 

ttggggtgtt tcccattcag agtagacgtg ggggggtcct ggagtcctcc tgttcttcca  46320 

ccaacccctt cctggggtga taccgcaggg ccactctgtc cctgtaaatt actcttttct  46380 

gaaaccttct tgagacatgg aaagcgttga ttttcttttt cttttttttt tttctttttt  46440 

tttgtttttg agatggagtc tcgctccgtc acccaggctg gagtgcagtg gcacgatctc  46500 

ggctccctgc aacctccacc tctcgggttc aagcaattct cctgcctcag cctccccagt  46560 

agctgggact acaggcgcct gccaccacac ctggctaatt tttgtatttt tagcagcgat  46620 

ggggtttcac catgttggcc aggctggtct cgaactcctg acctcaggtg atccgcccac  46680 

ctcggcctcc cccagtgcta ggatgacagc gtgagcctcc gcatcctgct gaagcattaa  46740 

ttttctaact gcatgcttct ggggtcctct ttttcctggg tggattttgg gtgccaggtc  46800 

ttgggcaggt caggaagcag ttgcccgcca ggagtttaag ctggattcgg ctctgtccac  46860 

taagcagccc aggcggactt cagctgcccc ttggtggctg tgtgcacggg gccaccaagt  46920 

gctgggtggt gcatccacac cgtggcccct tgagcttggg gctgctgccc ccctccccct  46980 

gggctgcagc tggggaccgg ccctccagac tgagcacccg tctctgcctc tgcagatggc  47040 

ttcaccccgc tcatgatcgc ctcctgcagc gggggcggcc tggagacggg caacagcgag  47100 

gaagaggagg acgcgccggc cgtcatctcc gacttcatct accagggcgc cagcctgcac  47160 

aaccagacag accgcacggg cgagaccgcc ttgcacctgg ccgcccgcta ctcacgctct  47220 

gatgccgcca agcgcctgct ggaggccagc gcagatgcca acatccagga caacatgggc  47280 

cgcaccccgc tgcatgcggc tgtgtctgcc gacgcacaag gtgtcttcca ggtaggcagt  47340 

ggctgcctgt gtgcccacct gccctcctca gggccgcctg gtggtctggg gcagtggcca  47400 

ggcttacgtg gccctgggag cctgaccccg agcacagctg agtccgggac aactggtgcc  47460 

tccacctggg accttcgcag tcagcgaggt gcgaggggga gggcgtcggg cccatctgtg  47520 

ttctccaggg aagtcaggca gaggcgggtc tggcaggagg cctgggggat ctgctgagtg  47580 

aggcagcacc tccccacccc cagcaaaaca ggctccatca gggttgtggg ccttgctcaa  47640 

ggtccaggtt ccactgctgc agcccctcgc agccccgccc ctccctcaac cttggctgcc  47700 

ggcgtagcct gtggcagtga gaagcagggt ttagaggctg ccgctcggtg cctgcagacc  47760 

tcgggctcag cttgccggtg agctcgtggc aagaatggat ttagggattt ggatgcctgg  47820 

gtctccaggg agtgtccctg gcaggggctg cctttgcagt cacccctgct gcgagtcccc  47880 

aggctccagg cggccctgga gcaagcaggt tcagatgggc acagcccggg gagttaccac  47940 

agagcttctc atttcctgat ttcttagctc aggtgacaca ttgtcatctc gcagaaataa  48000 

atgtaggtga gcagaaagag cgtggaaggg agccccgtgc gggtttggtg tgtgcctctc  48060 

tcagctgcct tctttgcaca gatgtggaag ttcccaggtg ctttgcagga atcaggccaa  48120 

gttgcctttt gcacccgcca gtgagcaggg gccattcctc ctgccagatg ccaagacccg  48180 

gctgcattac aagggctgcc caccccctct ctgggcagag ccggacacta gctcggcggt  48240 

tctgagacgg ctgtagggcc gtgagcccgg cttcctgagt gccagctgta accgccgttg  48300 

ggggcaggga cttgacctct ctgttttgta ggtggtcatg gcagctagga ccacagtgag  48360 

gattaaatga ggccacacgt ggtcacgatg gatcccactg tcaccaaggg gcctgtctgt  48420 

gggcagcaca gcccctgccc ccatccgggc ccctcctcag gggcagcccc atggcgtttc  48480 

gtcttgcccg gccgtgccga tctcaggagg gtctcgtctg tgtccggaag acagtggggc  48540 

ttcccgtcca ggcgttcgtt ctgggcagga ggcatcggtg tacgtctgcc cagcacccgc  48600 

ctgagcctct ccctgttgcc cagatcctga tccggaaccg agccacagac ctggatgccc  48660 

gcatgcatga tggcacgacg ccactgatcc tggctgcccg cctggccgtg gagggcatgc  48720 

tggaggacct catcaactca cacgccgacg tcaacgccgt agatgacctg ggtgagccca  48780 

cgggggcacg gctgctctgt cgtggggcgg gaccgccaca gggacgggtg ggactgggtt  48840 

gcctccagct ggtctccaac ctaccccatc tgcttctttc acgcaggcaa gtccgccctg  48900 

cactgggccg ccgccgtgaa caatgtggat gccgcagttg tgctcctgaa gaacggggct  48960 

aacaaagata tgcagaacaa cagggtgagc gcgaggctgg gatgccaggg gagacgtgag  49020 

ggctgaatcc acagagaagt gagggccaaa cccacggggc gcagggacac aagggcctga  49080 

cccacagggt ctcgagggtc gctgggcccg catgtgggcc ccacccgcat gatgcgggca  49140 

cctcgttagt gctcctgccc tccttcagcc cctgtttatg gagcccttcc aagtgcaggt  49200 

ccagctgtca ggacacggcg gctcgcccct cagacccagc cctaccctcc tgggcggcag  49260 

tcatggcagg gcacacagcc tggcgtgggg aacgctgctg ctgccactgc tgtgtcaccg  49320 

tcacctggac ctcaccttct gtccccagct gtcaccaggc gggggtgggg atgggaatgt  49380 

ggtgacgcag gtggtgcaga ttgagccaga acacgcgtgg cggcagctcc ctgcggggcg  49440 

gggcctcttg gtgtgttcac caaggccaag gacctcaagg ctcagaggaa gaagtctcag  49500 

gactatatcc aaggggagcc acccccagcc ctcatcctgg ccctgcagct cctggccctg  49560 

cagctgctgt tggtttcccc tgggtgctca gggcacaggt gcagacaccc ccacctccct  49620 

gccgccagaa cccccacccc cgcccccaac tcctgctgcc cccttggcat gtcaggctca  49680 

ggcgtctctc cctcctgggt gagggcacac agcgggcctg ggcaccgggg catgttggcc  49740 

caggcgctcc ccagtccgtg gcagcatggc cccacagaga ctgggcccca gagggcatca  49800 

aggcctggga agccccttcc cactcccaca cagcagcctc acccagacct gtgacgtgtc  49860 

caccgcacag aggagacccc aggaaggagc ttggtggcca ccagggtggc ttgatggccg  49920 

tccagatgac gagctccctc cctggtgccc tcgccggtgc ctctgaaccg ctccaggaat  49980 

ttccttttgt gccttattgg gggcagggaa gagggcctgc agttggttag attttcagtg  50040 

gggtctgtga cccccccata gaggtggagc cccgctgatc tagggtagag gactgcacag  50100 

atcccctctc tgggtgggtt tcagaagatg tatcaaagcc ttaacattta acaagagtca  50160 

ggctaggtgg ttgcaggacg ctggggtggg gtcctgagga gcagcctgcc tgcccccacc  50220 

ccgcggagga ggttgtactg ctgcttcctc tggtgatgga accttgggga gggtccccac  50280 

gcctggcctg gcccccctca ccggcccccg ccctcatccc ccaggaggag acacccctgt  50340 

ttctggccgc ccgggagggc agctacgaga ccgccaaggt gctgctggac cactttgcca  50400 

accgggacat cacggatcat atggaccgcc tgccgcgcga catcgcacag gagcgcatgc  50460 

atcacgacat cgtgaggctg ctggacgagt acaacctggt gcgcagcccg cagctgcacg  50520 

gagccccgct ggggggcacg cccaccctgt cgcccccgct ctgctcgccc aacggctacc  50580 

tgggcagcct caagcccggc gtgcagggca agaaggtccg caagcccagc agcaaaggcc  50640 

tggcctgtgg aagcaaggag gccaaggacc tcaaggcacg gaggaagaag tcccaggatg  50700 

gcaagggctg cctgctggac agctccggca tgctctcgcc cgtggactcc ctggagtcac  50760 

cccatggcta cctgtcagac gtggcctcgc cgccactgct gccctccccg ttccagcagt  50820 

ctccgtccgt gcccctcaac cacctgcctg ggatgcccga cacccacctg ggcatcgggc  50880 

acctgaacgt ggcggccaag cccgagatgg cggcgctggg tgggggcggc cggctggcct  50940 

ttgagactgg cccacctcgt ctctcccacc tgcctgtggc ctctggcacc agcaccgtcc  51000 

tgggctccag cagcggaggg gccctgaatt tcactgtggg cgggtccacc agtttgaatg  51060 

gtcaatgcga gtggctgtcc cggctgcaga gcggcatggt gccgaaccaa tacaaccctc  51120 

tgcgggggag tgtggcacca ggccccctga gcacacaggc cccctccctg cagcatggca  51180 

tggtaggccc gctgcacagt agccttgctg ccagcgccct gtcccagatg atgagctacc  51240 

agggcctgcc cagcacccgg ctggccaccc agcctcacct ggtgcagacc cagcaggtgc  51300 

agccacaaaa cttacagatg cagcagcaga acctgcagcc agcaaacatc cagcagcagc  51360 

aaagcctgca gccgccacca ccaccaccac agccgcacct tggcgtgagc tcagcagcca  51420 

gcggccacct gggccggagc ttcctgagtg gagagccgag ccaggcagac gtgcagccac  51480 

tgggccccag cagcctggcg gtgcacacta ttctgcccca ggagagcccc gccctgccca  51540 

cgtcgctgcc atcctcgctg gtcccacccg tgaccgcagc ccagttcctg acgcccccct  51600 

cgcagcacag ctactcctcg cctgtggaca acacccccag ccaccagcta caggtgcctg  51660 

agcacccctt cctcaccccg tcccctgagt cccctgacca gtggtccagc tcgtccccgc  51720 

attccaacgt ctccgactgg tccgagggcg tctccagccc tcccaccagc atgcagtccc  51780 

agatcgcccg cattccggag gccttcaagt aaacggcgcg ccccacgaga ccccggcttc  51840 

ctttcccaag ccttcgggcg tctgtgtgcg ctctgtggat gccagggccg accagaggag  51900 

cctttttaaa acacatgttt ttatacaaaa taagaacaag gattttaatt ttttttagta  51960 

tttatttatg tacttttatt ttacacagaa acactgcctt tttatttata tgtactgttt  52020 

tatctggccc caggtagaaa cttttatcta ttctgagaaa acaagcaagt tctgagagcc  52080 

agggttttcc tacgtaggat gaaaagattc ttctgtgttt ataaaatata aacaaagatt  52140 

catgatttat aaatgccatt tatttattga ttcctttttt caaaatccaa aaagaaatga  52200 

tgttggagaa gggaagttga acgagcatag tccaaaaagc tcctggggcg tccaggccgc  52260 

gccctttccc cgacgcccac ccaaccccaa gccagcccgg ccgctccacc agcatcacct  52320 

gcctgttagg agaagctgca tccagaggca aacggaggca aagctggctc accttccgca  52380 

cgcggattaa tttgcatctg aaataggaaa caagtgaaag catatgggtt agatgttgcc  52440 

atgtgtttta gatggtttct tgcaagcatg cttgtgaaaa tgtgttctcg gagtgtgtat  52500 

gccaagagtg cacccatggt accaatcatg aatctttgtt tcaggttcag tattatgtag  52560 

ttgttcgttg gttatacaag ttcttggtcc ctccagaacc accccggccc cctgcccgtt  52620 

cttgaaatgt aggcatcatg catgtcaaac atgagatgtg tggactgtgg cacttgcctg  52680 

ggtcacacac ggaggcatcc tacccttttc tggggaaaga cactgcctgg gctgaccccg  52740 

gtggcggccc cagcacctca gcctgcacag tgtcccccag gttccgaaga agatgctcca  52800 

gcaacacagc ctgggcccca gctcgcggga cccgaccccc cgtgggctcc cgtgttttgt  52860 

aggagacttg ccagagccgg gcacattgag ctgtgcaacg ccatgggctg cgtcctttgg  52920 

tcctgtcccc gcagccctgg cagggggcat gcggtcgggc aggggctgga gggaggcggg  52980 

ggctgccctt gggccacccc tcctagtttg ggaggagcag atttttgcaa taccaagtat  53040 

agcctatggc agaaaaaatg tctgtaaata tgtttttaaa ggtggatttt gtttaaaaaa  53100 

tcttaatgaa tgagtctgtt gtgtgtcatg ccagtgaggg acgtcagact cggctcagct  53160 

cggggagcct tagccgccca tgcactgggg acgctccgct gccgtgccgc ctgcactcct  53220 

cagggcagcc tcccccggct ctacgggggc cgcgtggtgc catccccagg gggcatgacc  53280 

agatgcgtcc caagatgttg atttttactg tgttttataa aatagagtgt agtttacaga  53340 

aaaagacttt aaaagtgatc tacatgagga actgtagatg atgtattttt ttcatctttt  53400 

ttgttaactg atttgcaata aaaatgatac tgatggtgat ctggcttcca ctcccctctg  53460 

ctctggcctt tggctccctt tctgggaggg aggcagggct gctatgctct gagggagcca  53520 

ggagtcgagg gccccttctg ctgggagagt gacggtgagg ctgcctagtc ctggcccacg  53580 

ggggtgtggg gaccacgctg cctccaggga ctccatggtg tctgcagcct gcctggtcca  53640 

ggccctttgt agggagatgg acacacagca gcaagggggt tgcagcccta tgggaggtgg  53700 

ggctcgtgcc tggggtgaca cggctcccag gacaccatgc gtgagtctgg ccctcctggc  53760 

agctcggggc tcttctcctc tcagcctcgg agggatgaag gtcccaccca gccatcctgg  53820 

ggacagcccc tcagggagct ctgcagcagg cagggggctg catagggagg gccttgaggc  53880 

agtggcatga cccctctacg ggctggagac cacagggagg actctggccc ctataggagg  53940 

gcaaagggag ctgtcgaagc ctatgagcag ggcagcatgg gtcgtggcag agtctggaaa  54000 

gttgtgtgat cctaaggaac tggctgagcg aggcagaggc ggcctggtcc tggggccctg  54060 

cccctgaata gagctgccag cctcacacaa gggtgggccc cttctctccc cactgcctgg  54120 

gcctctgccc agccccagac cttcagggca ggccagtggc ttcaaaccag agcggtgggg  54180 

agtctgagat ccctctttgg attgcaaagc actgcctgcc ctgggcccag tctctccaag  54240 

gagggatgtg agcccgaggc ctctactatg gctgggggct gcgtctgcca gccagcgctg  54300 

ggcaccagga ccaggagggg ccaccgtgga actgcagtga gtggcctgac tcttgtcttc  54360 

aaagggggtg acccagccgg agtcctgccc ataaaactcc cagcaccctg aaattccact  54420 

cctgggggtc tgtccgaaag aagtgaaaac agggactcaa acaaacgcac gtggccactt  54480 

gctgtcccag catcactcag tacagccaca gacagcctga gcgtccactg ccaacgacgg  54540 

gtcagcaaaa ccgtctgctg tgacggtgaa ccttagtgtc agcttggccg ggccatttcg  54600 

ccaaacggag tgtgtctctg aggtgttctg gatgaggttc gcatttgcat ccacagcccg  54660 

ggtaaggcag tccgccctcc ccagtgtggg tgggccccgt gcggtccgct gaggcccggg  54720 

gagaacaata ggccgagtgc caggggtcct cccacccaac cgcttgcact ggacattggt  54780 

cttttcctgc cttcagactt ggactgaaaa cgtgggctct tcctgggcct ggagcccacc  54840 

ggccttcgga ctcgcaacgc catcaggcct cctgcctgca actgcagacc ctgggaactg  54900 

caggcctcga ggtcgtgtgg ccaattccct gcaataacct ctccagtggc acgtcttatg  54960 

ggctccgctt gccggaagaa ccctgacgaa tgcgcacgag g                      55001 

 
           
             5  
             19  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            5 

cgggtccacc agtttgaat                                                  19 

 
           
             6  
             20  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            6 

ttgtattggt tcggcaccat                                                 20 

 
           
             7  
             17  
             DNA  
             Artificial Sequence  
             
               PCR Probe  
             
           
            7 

tcccggctgc agagcgg                                                    17 

 
           
             8  
             19  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            8 

gaaggtgaag gtcggagtc                                                  19 

 
           
             9  
             20  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            9 

gaagatggtg atgggatttc                                                 20 

 
           
             10  
             20  
             DNA  
             Artificial Sequence  
             
               PCR Probe  
             
           
            10 

caagcttccc gttctcagcc                                                 20 

 
           
             11  
             7693  
             DNA  
             H. sapiens  
             
               CDS  
               (1)...(7671)  
             
             
               misc_feature  
               2672, 2673  
               n = A,T,C or G  
             
           
            11 

atg ccg ccg ctc ctg gcg ccc ctg ctc tgc ctg gcg ctg ctg ccc gcg       48 
Met Pro Pro Leu Leu Ala Pro Leu Leu Cys Leu Ala Leu Leu Pro Ala 
  1               5                  10                  15 

ctc gcc gca cga ggc ccg cga tgc tcc cag ccc ggt gag acc tgc ctg       96 
Leu Ala Ala Arg Gly Pro Arg Cys Ser Gln Pro Gly Glu Thr Cys Leu 
             20                  25                  30 

aat ggc ggg aag tgt gaa gcg gcc aat ggc acg gag gcc tgc gtc tgt      144 
Asn Gly Gly Lys Cys Glu Ala Ala Asn Gly Thr Glu Ala Cys Val Cys 
         35                  40                  45 

ggc ggg gcc ttc gtg ggc ccg cga tgc cag gac ccc aac ccg tgc ctc      192 
Gly Gly Ala Phe Val Gly Pro Arg Cys Gln Asp Pro Asn Pro Cys Leu 
     50                  55                  60 

agc acc ccc tgc aag aac gcc ggg aca tgc cac gtg gtg gac cgc aga      240 
Ser Thr Pro Cys Lys Asn Ala Gly Thr Cys His Val Val Asp Arg Arg 
 65                  70                  75                  80 

ggc gtg gca gac tat gcc tgc agc tgt gcc ctg ggc ttc tct ggg ccc      288 
Gly Val Ala Asp Tyr Ala Cys Ser Cys Ala Leu Gly Phe Ser Gly Pro 
                 85                  90                  95 

ctc tgc ctg aca ccc ctg gac aac gcc tgc ctc acc aac ccc tgc cgc      336 
Leu Cys Leu Thr Pro Leu Asp Asn Ala Cys Leu Thr Asn Pro Cys Arg 
            100                 105                 110 

aac ggg ggc acc tgc gac ctg ctc acg ctg acg gag tac aag tgc cgc      384 
Asn Gly Gly Thr Cys Asp Leu Leu Thr Leu Thr Glu Tyr Lys Cys Arg 
        115                 120                 125 

tgc ccg ccc ggc tgg tca ggg aaa tcg tgc cag cag gct gac ccg tgc      432 
Cys Pro Pro Gly Trp Ser Gly Lys Ser Cys Gln Gln Ala Asp Pro Cys 
    130                 135                 140 

gcc tcc aac ccc tgc gcc aac ggt ggc cag tgc ctg ccc ttc gag gcc      480 
Ala Ser Asn Pro Cys Ala Asn Gly Gly Gln Cys Leu Pro Phe Glu Ala 
145                 150                 155                 160 

tcc tac atc tgc cac tgc cca ccc agc ttc cat ggc ccc acc tgc cgg      528 
Ser Tyr Ile Cys His Cys Pro Pro Ser Phe His Gly Pro Thr Cys Arg 
                165                 170                 175 

cag gat gtc aac gag tgt ggc cag aag ccc agg ctt tgc cgc cac gga      576 
Gln Asp Val Asn Glu Cys Gly Gln Lys Pro Arg Leu Cys Arg His Gly 
            180                 185                 190 

ggc acc tgc cac aac gag gtc ggc tcc tac cgc tgc gtc tgc cgc gcc      624 
Gly Thr Cys His Asn Glu Val Gly Ser Tyr Arg Cys Val Cys Arg Ala 
        195                 200                 205 

acc cac act ggc ccc aac tgc gag cgg ccc tac gtg ccc tgc agc ccc      672 
Thr His Thr Gly Pro Asn Cys Glu Arg Pro Tyr Val Pro Cys Ser Pro 
    210                 215                 220 

tcg ccc tgc cag aac ggg ggc acc tgc cgc ccc acg ggc gac gtc acc      720 
Ser Pro Cys Gln Asn Gly Gly Thr Cys Arg Pro Thr Gly Asp Val Thr 
225                 230                 235                 240 

cac gag tgt gcc tgc ctg cca ggc ttc acc ggc cag aac tgt gag gaa      768 
His Glu Cys Ala Cys Leu Pro Gly Phe Thr Gly Gln Asn Cys Glu Glu 
                245                 250                 255 

aat atc gac gat tgt cca gga aac aac tgc aag aac ggg ggt gcc tgt      816 
Asn Ile Asp Asp Cys Pro Gly Asn Asn Cys Lys Asn Gly Gly Ala Cys 
            260                 265                 270 

gtg gac ggc gtg aac acc tac aac tgc ccg tgc ccg cca gag tgg aca      864 
Val Asp Gly Val Asn Thr Tyr Asn Cys Pro Cys Pro Pro Glu Trp Thr 
        275                 280                 285 

ggt cag tac tgt acc gag gat gtg gac gag tgc cag ctg atg cca aat      912 
Gly Gln Tyr Cys Thr Glu Asp Val Asp Glu Cys Gln Leu Met Pro Asn 
    290                 295                 300 

gcc tgc cag aac ggc ggg acc tgc cac aac acc cac ggt ggc tac aac      960 
Ala Cys Gln Asn Gly Gly Thr Cys His Asn Thr His Gly Gly Tyr Asn 
305                 310                 315                 320 

tgc gtg tgt gtc aac ggc tgg act ggt gag gac tgc agc gag aac att     1008 
Cys Val Cys Val Asn Gly Trp Thr Gly Glu Asp Cys Ser Glu Asn Ile 
                325                 330                 335 

gat gac tgt gcc agc gcc gcc tgc ttc cac ggc gcc acc tgc cat gac     1056 
Asp Asp Cys Ala Ser Ala Ala Cys Phe His Gly Ala Thr Cys His Asp 
            340                 345                 350 

cgt gtg gcc tcc ttt tac tgc gag tgt ccc cat ggc cgc aca ggt ctg     1104 
Arg Val Ala Ser Phe Tyr Cys Glu Cys Pro His Gly Arg Thr Gly Leu 
        355                 360                 365 

ctg tgc cac ctc aac gac gca tgc atc agc aac ccc tgt aac gag ggc     1152 
Leu Cys His Leu Asn Asp Ala Cys Ile Ser Asn Pro Cys Asn Glu Gly 
    370                 375                 380 

tcc aac tgc gac acc aac cct gtc aat ggc aag gcc atc tgc acc tgc     1200 
Ser Asn Cys Asp Thr Asn Pro Val Asn Gly Lys Ala Ile Cys Thr Cys 
385                 390                 395                 400 

ccc tcg ggg tac acg ggc ccg gcc tgc agc cag gac gtg gat gag tgc     1248 
Pro Ser Gly Tyr Thr Gly Pro Ala Cys Ser Gln Asp Val Asp Glu Cys 
                405                 410                 415 

tcg ctg ggt gcc aac ccc tgc gag cat gcg ggc aag tgc atc aac acg     1296 
Ser Leu Gly Ala Asn Pro Cys Glu His Ala Gly Lys Cys Ile Asn Thr 
            420                 425                 430 

ctg ggc tcc ttc gag tgc cag tgt ctg cag ggc tac acg ggc ccc cga     1344 
Leu Gly Ser Phe Glu Cys Gln Cys Leu Gln Gly Tyr Thr Gly Pro Arg 
        435                 440                 445 

tgc gag atc gac gtc aac gag tgc gtc tcg aac ccg tgc cag aac gac     1392 
Cys Glu Ile Asp Val Asn Glu Cys Val Ser Asn Pro Cys Gln Asn Asp 
    450                 455                 460 

gcc acc tgc ctg gac cag att ggg gag ttc cag tgc atg tgc atg ccc     1440 
Ala Thr Cys Leu Asp Gln Ile Gly Glu Phe Gln Cys Met Cys Met Pro 
465                 470                 475                 480 

ggc tac gag ggt gtg cac tgc gag gtc aac aca gac gag tgt gcc agc     1488 
Gly Tyr Glu Gly Val His Cys Glu Val Asn Thr Asp Glu Cys Ala Ser 
                485                 490                 495 

agc ccc tgc ctg cac aat ggc cgc tgc ctg gac aag atc aat gag ttc     1536 
Ser Pro Cys Leu His Asn Gly Arg Cys Leu Asp Lys Ile Asn Glu Phe 
            500                 505                 510 

cag tgc gag tgc ccc acg ggc ttc act ggg cat ctg tgc cag tac gat     1584 
Gln Cys Glu Cys Pro Thr Gly Phe Thr Gly His Leu Cys Gln Tyr Asp 
        515                 520                 525 

gtg gac gag tgt gcc agc acc ccc tgc aag aat ggt gcc aag tgc ctg     1632 
Val Asp Glu Cys Ala Ser Thr Pro Cys Lys Asn Gly Ala Lys Cys Leu 
    530                 535                 540 

gac gga ccc aac act tac acc tgt gtg tgc acg gaa ggg tac acg ggg     1680 
Asp Gly Pro Asn Thr Tyr Thr Cys Val Cys Thr Glu Gly Tyr Thr Gly 
545                 550                 555                 560 

acg cac tgc gag gtg gac atc gat gag tgc gac ccc gac ccc tgc cac     1728 
Thr His Cys Glu Val Asp Ile Asp Glu Cys Asp Pro Asp Pro Cys His 
                565                 570                 575 

tac ggc tcc tgc aag gac ggc gtc gcc acc ttc acc tgc ctc tgc cgc     1776 
Tyr Gly Ser Cys Lys Asp Gly Val Ala Thr Phe Thr Cys Leu Cys Arg 
            580                 585                 590 

cca ggc tac acg ggc cac cac tgc gag acc aac atc aac gag tgc tcc     1824 
Pro Gly Tyr Thr Gly His His Cys Glu Thr Asn Ile Asn Glu Cys Ser 
        595                 600                 605 

agc cag ccc tgc cgc cta cgg ggc acc tgc cag gac ccg gac aac gcc     1872 
Ser Gln Pro Cys Arg Leu Arg Gly Thr Cys Gln Asp Pro Asp Asn Ala 
    610                 615                 620 

tac ctc tgc ttc tgc ctg aag ggg acc aca gga ccc aac tgc gag atc     1920 
Tyr Leu Cys Phe Cys Leu Lys Gly Thr Thr Gly Pro Asn Cys Glu Ile 
625                 630                 635                 640 

aac ctg gat gac tgt gcc agc agc ccc tgc gac tcg ggc acc tgt ctg     1968 
Asn Leu Asp Asp Cys Ala Ser Ser Pro Cys Asp Ser Gly Thr Cys Leu 
                645                 650                 655 

gac aag atc gat ggc tac gag tgt gcc tgt gag ccg ggc tac aca ggg     2016 
Asp Lys Ile Asp Gly Tyr Glu Cys Ala Cys Glu Pro Gly Tyr Thr Gly 
            660                 665                 670 

agc atg tgt aac agc aac atc gat gag tgt gcg ggc aac ccc tgc cac     2064 
Ser Met Cys Asn Ser Asn Ile Asp Glu Cys Ala Gly Asn Pro Cys His 
        675                 680                 685 

aac ggg ggc acc tgc gag gac ggc atc aat ggc ttc acc tgc cgc tgc     2112 
Asn Gly Gly Thr Cys Glu Asp Gly Ile Asn Gly Phe Thr Cys Arg Cys 
    690                 695                 700 

ccc gag ggc tac cac gac ccc acc tgc ctg tct gag gtc aat gag tgc     2160 
Pro Glu Gly Tyr His Asp Pro Thr Cys Leu Ser Glu Val Asn Glu Cys 
705                 710                 715                 720 

aac agc aac ccc tgc gtc cac ggg gcc tgc cgg gac agc ctc aac ggg     2208 
Asn Ser Asn Pro Cys Val His Gly Ala Cys Arg Asp Ser Leu Asn Gly 
                725                 730                 735 

tac aag tgc gac tgt gac cct ggg tgg agt ggg acc aac tgt gac atc     2256 
Tyr Lys Cys Asp Cys Asp Pro Gly Trp Ser Gly Thr Asn Cys Asp Ile 
            740                 745                 750 

aac aac aac gag tgt gaa tcc aac cct tgt gtc aac ggc ggc acc tgc     2304 
Asn Asn Asn Glu Cys Glu Ser Asn Pro Cys Val Asn Gly Gly Thr Cys 
        755                 760                 765 

aaa gac atg acc agt ggc atc gtg tgc acc tgc cgg gag ggc ttc agc     2352 
Lys Asp Met Thr Ser Gly Ile Val Cys Thr Cys Arg Glu Gly Phe Ser 
    770                 775                 780 

ggt ccc aac tgc cag acc aac atc aac gag tgt gcg tcc aac cca tgt     2400 
Gly Pro Asn Cys Gln Thr Asn Ile Asn Glu Cys Ala Ser Asn Pro Cys 
785                 790                 795                 800 

ctg aac aag ggc acg tgt att gac gac gtt gcc ggg tac aag tgc aac     2448 
Leu Asn Lys Gly Thr Cys Ile Asp Asp Val Ala Gly Tyr Lys Cys Asn 
                805                 810                 815 

tgc ctg ctg ccc tac aca ggt gcc acg tgt gag gtg gtg ctg gcc ccg     2496 
Cys Leu Leu Pro Tyr Thr Gly Ala Thr Cys Glu Val Val Leu Ala Pro 
            820                 825                 830 

tgt gcc ccc agc ccc tgc aga aac ggc ggg gag tgc agg caa tcc gag     2544 
Cys Ala Pro Ser Pro Cys Arg Asn Gly Gly Glu Cys Arg Gln Ser Glu 
        835                 840                 845 

gac tat gag agc ttc tcc tgt gtc tgc ccc acg gct ggg gcc aaa ggg     2592 
Asp Tyr Glu Ser Phe Ser Cys Val Cys Pro Thr Ala Gly Ala Lys Gly 
    850                 855                 860 

cag acc tgt gag gtc gac atc aac gag tgc gtt ctg agc ccg tgc cgg     2640 
Gln Thr Cys Glu Val Asp Ile Asn Glu Cys Val Leu Ser Pro Cys Arg 
865                 870                 875                 880 

cac ggc gca tcc tgc cag aac acc cac ggc gnn tac cgc tgc cac tgc     2688 
His Gly Ala Ser Cys Gln Asn Thr His Gly Xaa Tyr Arg Cys His Cys 
                885                 890                 895 

cag gcc ggc tac agt ggg cgc aac tgc gag acc gac atc gac gac tgc     2736 
Gln Ala Gly Tyr Ser Gly Arg Asn Cys Glu Thr Asp Ile Asp Asp Cys 
            900                 905                 910 

cgg ccc aac ccg tgt cac aac ggg ggc tcc tgc aca gac ggc atc aac     2784 
Arg Pro Asn Pro Cys His Asn Gly Gly Ser Cys Thr Asp Gly Ile Asn 
        915                 920                 925 

acg gcc ttc tgc gac tgc ctg ccc ggc ttc cgg ggc act ttc tgt gag     2832 
Thr Ala Phe Cys Asp Cys Leu Pro Gly Phe Arg Gly Thr Phe Cys Glu 
    930                 935                 940 

gag gac atc aac gag tgt gcc agt gac ccc tgc cgc aac ggg gcc aac     2880 
Glu Asp Ile Asn Glu Cys Ala Ser Asp Pro Cys Arg Asn Gly Ala Asn 
945                 950                 955                 960 

tgc acg gac tgc gtg gac agc tac acg tgc acc tgc ccc gca ggc ttc     2928 
Cys Thr Asp Cys Val Asp Ser Tyr Thr Cys Thr Cys Pro Ala Gly Phe 
                965                 970                 975 

agc ggg atc cac tgt gag aac aac acg cct gac tgc aca gag agc tcc     2976 
Ser Gly Ile His Cys Glu Asn Asn Thr Pro Asp Cys Thr Glu Ser Ser 
            980                 985                 990 

tgc ttc aac ggt ggc acc tgc gtg gac ggc atc aac tcg ttc acc tgc     3024 
Cys Phe Asn Gly Gly Thr Cys Val Asp Gly Ile Asn Ser Phe Thr Cys 
        995                 1000                1005 

ctg tgt cca ccc ggc ttc acg ggc agc tac tgc cag cac gta gtc aat     3072 
Leu Cys Pro Pro Gly Phe Thr Gly Ser Tyr Cys Gln His Val Val Asn 
    1010                1015                1020 

gag tgc gac tca cga ccc tgc ctg cta ggc ggc acc tgt cag gac ggt     3120 
Glu Cys Asp Ser Arg Pro Cys Leu Leu Gly Gly Thr Cys Gln Asp Gly 
1025                1030                1035                1040 

cgc ggt ctc cac agg tgc acc tgc ccc cag ggc tac act ggc ccc aac     3168 
Arg Gly Leu His Arg Cys Thr Cys Pro Gln Gly Tyr Thr Gly Pro Asn 
                1045                1050                1055 

tgc cag aac ctt gtg cac tgg tgt gac tcc tcg ccc tgc aag aac ggc     3216 
Cys Gln Asn Leu Val His Trp Cys Asp Ser Ser Pro Cys Lys Asn Gly 
            1060                1065                1070 

ggc aaa tgc tgg cag acc cac acc cag tac cgc tgc gag tgc ccc agc     3264 
Gly Lys Cys Trp Gln Thr His Thr Gln Tyr Arg Cys Glu Cys Pro Ser 
        1075                1080                1085 

ggc tgg acc ggc ctt tac tgc gac gtg ccc agc gtg tcc tgt gag gtg     3312 
Gly Trp Thr Gly Leu Tyr Cys Asp Val Pro Ser Val Ser Cys Glu Val 
    1090                1095                1100 

gct gcg cag cga caa ggt gtt gac gtt gcc cgc ctg tgc cag cat gga     3360 
Ala Ala Gln Arg Gln Gly Val Asp Val Ala Arg Leu Cys Gln His Gly 
1105                1110                1115                1120 

ggg ctc tgt gtg gac gcg ggc aac acg cac cac tgc cgc tgc cag gcg     3408 
Gly Leu Cys Val Asp Ala Gly Asn Thr His His Cys Arg Cys Gln Ala 
                1125                1130                1135 

ggc tac aca ggc agc tac tgt gag gac ctg gtg gac gag tgc tca ccc     3456 
Gly Tyr Thr Gly Ser Tyr Cys Glu Asp Leu Val Asp Glu Cys Ser Pro 
            1140                1145                1150 

agc ccc tgc cag aac ggg gcc acc tgc acg gac tac ctg ggc ggc tac     3504 
Ser Pro Cys Gln Asn Gly Ala Thr Cys Thr Asp Tyr Leu Gly Gly Tyr 
        1155                1160                1165 

tcc tgc aag tgc gtg gcc ggc tac cac ggg gtg aac tgc tct gag gag     3552 
Ser Cys Lys Cys Val Ala Gly Tyr His Gly Val Asn Cys Ser Glu Glu 
    1170                1175                1180 

atc gac gag tgc ctc tcc cac ccc tgc cag aac ggg ggc acc tgc ctc     3600 
Ile Asp Glu Cys Leu Ser His Pro Cys Gln Asn Gly Gly Thr Cys Leu 
1185                1190                1195                1200 

gac ctc ccc aac acc tac aag tgc tcc tgc cca cgg ggc act cag ggt     3648 
Asp Leu Pro Asn Thr Tyr Lys Cys Ser Cys Pro Arg Gly Thr Gln Gly 
                1205                1210                1215 

gtg cac tgt gag atc aac gtg gac gac tgc aat ccc ccc gtt gac ccc     3696 
Val His Cys Glu Ile Asn Val Asp Asp Cys Asn Pro Pro Val Asp Pro 
            1220                1225                1230 

gtg tcc cgg agc ccc aag tgc ttt aac aac ggc acc tgc gtg gac cag     3744 
Val Ser Arg Ser Pro Lys Cys Phe Asn Asn Gly Thr Cys Val Asp Gln 
        1235                1240                1245 

gtg ggc ggc tac agc tgc acc tgc ccg ccg ggc ttc gtg ggt gag cgc     3792 
Val Gly Gly Tyr Ser Cys Thr Cys Pro Pro Gly Phe Val Gly Glu Arg 
    1250                1255                1260 

tgt gag ggg gat gtc aac gag tgc ctg tcc aat ccc tgc gac gcc cgt     3840 
Cys Glu Gly Asp Val Asn Glu Cys Leu Ser Asn Pro Cys Asp Ala Arg 
1265                1270                1275                1280 

ggc acc cag aac tgc gtg cag cgc gtc aat gac ttc cac tgc gag tgc     3888 
Gly Thr Gln Asn Cys Val Gln Arg Val Asn Asp Phe His Cys Glu Cys 
                1285                1290                1295 

cgt gct ggt cac acc ggg cgc cgc tgc gag tcc gtc atc aat ggc tgc     3936 
Arg Ala Gly His Thr Gly Arg Arg Cys Glu Ser Val Ile Asn Gly Cys 
            1300                1305                1310 

aaa ggc aag ccc tgc aag aat ggg ggc acc tgc gcc gtg gcc tcc aac     3984 
Lys Gly Lys Pro Cys Lys Asn Gly Gly Thr Cys Ala Val Ala Ser Asn 
        1315                1320                1325 

acc gcc cgc ggg ttc atc tgc aag tgc cct gcg ggc ttc gag ggc gcc     4032 
Thr Ala Arg Gly Phe Ile Cys Lys Cys Pro Ala Gly Phe Glu Gly Ala 
    1330                1335                1340 

acg tgt gag aat gac gct cgt acc tgc ggc agc ctg cgc tgc ctc aac     4080 
Thr Cys Glu Asn Asp Ala Arg Thr Cys Gly Ser Leu Arg Cys Leu Asn 
1345                1350                1355                1360 

ggc ggc aca tgc atc tcc ggc ccg cgc agc ccc acc tgc ctg tgc ctg     4128 
Gly Gly Thr Cys Ile Ser Gly Pro Arg Ser Pro Thr Cys Leu Cys Leu 
                1365                1370                1375 

ggc ccc ttc acg ggc ccc gaa tgc cag ttc ccg gcc agc agc ccc tgc     4176 
Gly Pro Phe Thr Gly Pro Glu Cys Gln Phe Pro Ala Ser Ser Pro Cys 
            1380                1385                1390 

ctg ggc ggc aac ccc tgc tac aac cag ggg acc tgt gag ccc aca tcc     4224 
Leu Gly Gly Asn Pro Cys Tyr Asn Gln Gly Thr Cys Glu Pro Thr Ser 
        1395                1400                1405 

gag agc ccc ttc tac cgt tgc ctg tgc ccc gcc aaa ttc aac ggg ctc     4272 
Glu Ser Pro Phe Tyr Arg Cys Leu Cys Pro Ala Lys Phe Asn Gly Leu 
    1410                1415                1420 

ttg tgc cac atc ctg gac tac agc ttc ggg ggt ggg gcc ggg cgc gac     4320 
Leu Cys His Ile Leu Asp Tyr Ser Phe Gly Gly Gly Ala Gly Arg Asp 
1425                1430                1435                1440 

atc ccc ccg ccg ctg atc gag gag gcg tgc gag ctg ccc gag tgc cag     4368 
Ile Pro Pro Pro Leu Ile Glu Glu Ala Cys Glu Leu Pro Glu Cys Gln 
                1445                1450                1455 

gag gac gcg ggc aac aag gtc tgc agc ctg cag tgc aac aac cac gcg     4416 
Glu Asp Ala Gly Asn Lys Val Cys Ser Leu Gln Cys Asn Asn His Ala 
            1460                1465                1470 

tgc ggc tgg gac ggc ggt gac tgc tcc ctc aac ttc aat gac ccc tgg     4464 
Cys Gly Trp Asp Gly Gly Asp Cys Ser Leu Asn Phe Asn Asp Pro Trp 
        1475                1480                1485 

aag aac tgc acg cag tct ctg cag tgc tgg aag tac ttc agt gac ggc     4512 
Lys Asn Cys Thr Gln Ser Leu Gln Cys Trp Lys Tyr Phe Ser Asp Gly 
    1490                1495                1500 

cac tgt gac agc cag tgc aac tca gcc ggc tgc ctc ttc gac ggc ttt     4560 
His Cys Asp Ser Gln Cys Asn Ser Ala Gly Cys Leu Phe Asp Gly Phe 
1505                1510                1515                1520 

gac tgc cag cgt gcg gaa ggc cag tgc aac ccc ctg tac gac cag tac     4608 
Asp Cys Gln Arg Ala Glu Gly Gln Cys Asn Pro Leu Tyr Asp Gln Tyr 
                1525                1530                1535 

tgc aag gac cac ttc agc gac ggg cac tgc gac cag ggc tgc aac agc     4656 
Cys Lys Asp His Phe Ser Asp Gly His Cys Asp Gln Gly Cys Asn Ser 
            1540                1545                1550 

gcg gag tgc gag tgg gac ggg ctg gac tgt gcg gag cat gta ccc gag     4704 
Ala Glu Cys Glu Trp Asp Gly Leu Asp Cys Ala Glu His Val Pro Glu 
        1555                1560                1565 

agg ctg gcg gcc ggc acg ctg gtg gtg gtg gtg ctg atg ccg ccg gag     4752 
Arg Leu Ala Ala Gly Thr Leu Val Val Val Val Leu Met Pro Pro Glu 
    1570                1575                1580 

cag ctg cgc aac agc tcc ttc cac ttc ctg cgg gag ctc agc cgc gtg     4800 
Gln Leu Arg Asn Ser Ser Phe His Phe Leu Arg Glu Leu Ser Arg Val 
1585                1590                1595                1600 

ctg cac acc aac gtg gtc ttc aag cgt gac gca cac ggc cag cag atg     4848 
Leu His Thr Asn Val Val Phe Lys Arg Asp Ala His Gly Gln Gln Met 
                1605                1610                1615 

atc ttc ccc tac tac ggc cgc gag gag gag ctg cgc aag cac ccc atc     4896 
Ile Phe Pro Tyr Tyr Gly Arg Glu Glu Glu Leu Arg Lys His Pro Ile 
            1620                1625                1630 

aag cgt gcc gcc gag ggc tgg gcc gca cct gac gcc ctg ctg ggc cag     4944 
Lys Arg Ala Ala Glu Gly Trp Ala Ala Pro Asp Ala Leu Leu Gly Gln 
        1635                1640                1645 

gtg aag gcc tcg ctg ctc cct ggt ggc agc gag ggt ggg cgg cgg cgg     4992 
Val Lys Ala Ser Leu Leu Pro Gly Gly Ser Glu Gly Gly Arg Arg Arg 
    1650                1655                1660 

agg gag ctg gac ccc atg gac gtc cgc ggc tcc atc gtc tac ctg gag     5040 
Arg Glu Leu Asp Pro Met Asp Val Arg Gly Ser Ile Val Tyr Leu Glu 
1665                1670                1675                1680 

att gac aac cgg cag tgt gtg cag gcc tcc tcg cag tgc ttc cag agt     5088 
Ile Asp Asn Arg Gln Cys Val Gln Ala Ser Ser Gln Cys Phe Gln Ser 
                1685                1690                1695 

gcc acc gac gtg gcc gca ttc ctg gga gcg ctc gcc tcg ctg ggc agc     5136 
Ala Thr Asp Val Ala Ala Phe Leu Gly Ala Leu Ala Ser Leu Gly Ser 
            1700                1705                1710 

ctc aac atc ccc tac aag atc gag gcc gtg cag agt gag acc gtg gag     5184 
Leu Asn Ile Pro Tyr Lys Ile Glu Ala Val Gln Ser Glu Thr Val Glu 
        1715                1720                1725 

ccg ccc ccg ccg gcg cag ctg cac ttc atg tac gtg gcg gcg gcc gcc     5232 
Pro Pro Pro Pro Ala Gln Leu His Phe Met Tyr Val Ala Ala Ala Ala 
    1730                1735                1740 

ttt gtg ctt ctg ttc ttc gtg ggc tgc ggg gtg ctg ctg tcc cgc aag     5280 
Phe Val Leu Leu Phe Phe Val Gly Cys Gly Val Leu Leu Ser Arg Lys 
1745                1750                1755                1760 

cgc cgg cgg cag cat ggc cag ctc tgg ttc cct gag ggc ttc aaa gtg     5328 
Arg Arg Arg Gln His Gly Gln Leu Trp Phe Pro Glu Gly Phe Lys Val 
                1765                1770                1775 

tct gag gcc agc aag aag aag cgg cgg gag ccc ctc ggc gag gac tcc     5376 
Ser Glu Ala Ser Lys Lys Lys Arg Arg Glu Pro Leu Gly Glu Asp Ser 
            1780                1785                1790 

gtg ggc ctc aag ccc ctg aag aac gct tca gac ggt gcc ctc atg gac     5424 
Val Gly Leu Lys Pro Leu Lys Asn Ala Ser Asp Gly Ala Leu Met Asp 
        1795                1800                1805 

gac aac cag aat gag tgg ggg gac gag gac ctg gag acc aag aag ttc     5472 
Asp Asn Gln Asn Glu Trp Gly Asp Glu Asp Leu Glu Thr Lys Lys Phe 
    1810                1815                1820 

cgg ttc gag gag ccc gtg gtt ctg cct gac ctg gac gac cag aca gac     5520 
Arg Phe Glu Glu Pro Val Val Leu Pro Asp Leu Asp Asp Gln Thr Asp 
1825                1830                1835                1840 

cac cgg cag tgg act cag cag cac ctg gat gcc gct gac ctg cgc atg     5568 
His Arg Gln Trp Thr Gln Gln His Leu Asp Ala Ala Asp Leu Arg Met 
                1845                1850                1855 

tct gcc atg gcc ccc aca ccg ccc cag ggt gag gtt gac gcc gac tgc     5616 
Ser Ala Met Ala Pro Thr Pro Pro Gln Gly Glu Val Asp Ala Asp Cys 
            1860                1865                1870 

atg gac gtc aat gtc cgc ggg cct gat ggc ttc acc ccg ctc atg atc     5664 
Met Asp Val Asn Val Arg Gly Pro Asp Gly Phe Thr Pro Leu Met Ile 
        1875                1880                1885 

gcc tcc tgc agc ggg ggc ggc ctg gag acg ggc aac agc gag gaa gag     5712 
Ala Ser Cys Ser Gly Gly Gly Leu Glu Thr Gly Asn Ser Glu Glu Glu 
    1890                1895                1900 

gag gac gcg ccg gcc gtc atc tcc gac ttc atc tac cag ggc gcc agc     5760 
Glu Asp Ala Pro Ala Val Ile Ser Asp Phe Ile Tyr Gln Gly Ala Ser 
1905                1910                1915                1920 

ctg cac aac cag aca gac cgc acg ggc gag acc gcc ttg cac ctg gcc     5808 
Leu His Asn Gln Thr Asp Arg Thr Gly Glu Thr Ala Leu His Leu Ala 
                1925                1930                1935 

gcc cgc tac tca cgc tct gat gcc gcc aag cgc ctg ctg gag gcc agc     5856 
Ala Arg Tyr Ser Arg Ser Asp Ala Ala Lys Arg Leu Leu Glu Ala Ser 
            1940                1945                1950 

gca gat gcc aac atc cag gac aac atg ggc cgc acc ccg ctg cat gcg     5904 
Ala Asp Ala Asn Ile Gln Asp Asn Met Gly Arg Thr Pro Leu His Ala 
        1955                1960                1965 

gct gtg tct gcc gac gca caa ggt gtc ttc cag atc ctg atc cgg aac     5952 
Ala Val Ser Ala Asp Ala Gln Gly Val Phe Gln Ile Leu Ile Arg Asn 
    1970                1975                1980 

cga gcc aca gac ctg gat gcc cgc atg cat gat ggc acg acg cca ctg     6000 
Arg Ala Thr Asp Leu Asp Ala Arg Met His Asp Gly Thr Thr Pro Leu 
1985                1990                1995                2000 

atc ctg gct gcc cgc ctg gcc gtg gag ggc atg ctg gag gac ctc atc     6048 
Ile Leu Ala Ala Arg Leu Ala Val Glu Gly Met Leu Glu Asp Leu Ile 
                2005                2010                2015 

aac tca cac gcc gac gtc aac gcc gta gat gac ctg ggc aag tcc gcc     6096 
Asn Ser His Ala Asp Val Asn Ala Val Asp Asp Leu Gly Lys Ser Ala 
            2020                2025                2030 

ctg cac tgg gcc gcc gcc gtg aac aat gtg gat gcc gca gtt gtg ctc     6144 
Leu His Trp Ala Ala Ala Val Asn Asn Val Asp Ala Ala Val Val Leu 
        2035                2040                2045 

ctg aag aac ggg gct aac aaa gat atg cag aac aac agg gag gag aca     6192 
Leu Lys Asn Gly Ala Asn Lys Asp Met Gln Asn Asn Arg Glu Glu Thr 
    2050                2055                2060 

ccc ctg ttt ctg gcc gcc cgg gag ggc agc tac gag acc gcc aag gtg     6240 
Pro Leu Phe Leu Ala Ala Arg Glu Gly Ser Tyr Glu Thr Ala Lys Val 
2065                2070                2075                2080 

ctg ctg gac cac ttt gcc aac cgg gac atc acg gat cat atg gac cgc     6288 
Leu Leu Asp His Phe Ala Asn Arg Asp Ile Thr Asp His Met Asp Arg 
                2085                2090                2095 

ctg ccg cgc gac atc gca cag gag cgc atg cat cac gac atc gtg agg     6336 
Leu Pro Arg Asp Ile Ala Gln Glu Arg Met His His Asp Ile Val Arg 
            2100                2105                2110 

ctg ctg gac gag tac aac ctg gtg cgc agc ccg cag ctg cac gga gcc     6384 
Leu Leu Asp Glu Tyr Asn Leu Val Arg Ser Pro Gln Leu His Gly Ala 
        2115                2120                2125 

ccg ctg ggg ggc acg ccc acc ctg tcg ccc ccg ctc tgc tcg ccc aac     6432 
Pro Leu Gly Gly Thr Pro Thr Leu Ser Pro Pro Leu Cys Ser Pro Asn 
    2130                2135                2140 

ggc tac ctg ggc agc ctc aag ccc ggc gtg cag ggc aag aag gtc cgc     6480 
Gly Tyr Leu Gly Ser Leu Lys Pro Gly Val Gln Gly Lys Lys Val Arg 
2145                2150                2155                2160 

aag ccc agc agc aaa ggc ctg gcc tgt gga agc aag gag gcc aag gac     6528 
Lys Pro Ser Ser Lys Gly Leu Ala Cys Gly Ser Lys Glu Ala Lys Asp 
                2165                2170                2175 

ctc aag gca cgg agg aag aag tcc cag gat ggc aag ggc tgc ctg ctg     6576 
Leu Lys Ala Arg Arg Lys Lys Ser Gln Asp Gly Lys Gly Cys Leu Leu 
            2180                2185                2190 

gac agc tcc ggc atg ctc tcg ccc gtg gac tcc ctg gag tca ccc cat     6624 
Asp Ser Ser Gly Met Leu Ser Pro Val Asp Ser Leu Glu Ser Pro His 
        2195                2200                2205 

ggc tac ctg tca gac gtg gcc tcg ccg cca ctg ctg ccc tcc ccg ttc     6672 
Gly Tyr Leu Ser Asp Val Ala Ser Pro Pro Leu Leu Pro Ser Pro Phe 
    2210                2215                2220 

cag cag tct ccg tcc gtg ccc ctc aac cac ctg cct ggg atg ccc gac     6720 
Gln Gln Ser Pro Ser Val Pro Leu Asn His Leu Pro Gly Met Pro Asp 
2225                2230                2235                2240 

acc cac ctg ggc atc ggg cac ctg aac gtg gcg gcc aag ccc gag atg     6768 
Thr His Leu Gly Ile Gly His Leu Asn Val Ala Ala Lys Pro Glu Met 
                2245                2250                2255 

gcg gcg ctg ggt ggg ggc ggc cgg ctg gcc ttt gag act ggc cca cct     6816 
Ala Ala Leu Gly Gly Gly Gly Arg Leu Ala Phe Glu Thr Gly Pro Pro 
            2260                2265                2270 

cgt ctc tcc cac ctg cct gtg gcc tct ggc acc agc acc gtc ctg ggc     6864 
Arg Leu Ser His Leu Pro Val Ala Ser Gly Thr Ser Thr Val Leu Gly 
        2275                2280                2285 

tcc agc agc gga ggg gcc ctg aat ttc act gtg ggc ggg tcc acc agt     6912 
Ser Ser Ser Gly Gly Ala Leu Asn Phe Thr Val Gly Gly Ser Thr Ser 
    2290                2295                2300 

ttg aat ggt caa tgc gag tgg ctg tcc cgg ctg cag agc ggc atg gtg     6960 
Leu Asn Gly Gln Cys Glu Trp Leu Ser Arg Leu Gln Ser Gly Met Val 
2305                2310                2315                2320 

ccg aac caa tac aac cct ctg cgg ggg agt gtg gca cca ggc ccc ctg     7008 
Pro Asn Gln Tyr Asn Pro Leu Arg Gly Ser Val Ala Pro Gly Pro Leu 
                2325                2330                2335 

agc aca cag gcc ccc tcc ctg cag cat ggc atg gta ggc ccg ctg cac     7056 
Ser Thr Gln Ala Pro Ser Leu Gln His Gly Met Val Gly Pro Leu His 
            2340                2345                2350 

agt agc ctt gct gcc agc gcc ctg tcc cag atg atg agc tac cag ggc     7104 
Ser Ser Leu Ala Ala Ser Ala Leu Ser Gln Met Met Ser Tyr Gln Gly 
        2355                2360                2365 

ctg ccc agc acc cgg ctg gcc acc cag cct cac ctg gtg cag acc cag     7152 
Leu Pro Ser Thr Arg Leu Ala Thr Gln Pro His Leu Val Gln Thr Gln 
    2370                2375                2380 

cag gtg cag cca caa aac tta cag atg cag cag cag aac ctg cag cca     7200 
Gln Val Gln Pro Gln Asn Leu Gln Met Gln Gln Gln Asn Leu Gln Pro 
2385                2390                2395                2400 

gca aac atc cag cag cag caa agc ctg cag ccg cca cca cca cca cca     7248 
Ala Asn Ile Gln Gln Gln Gln Ser Leu Gln Pro Pro Pro Pro Pro Pro 
                2405                2410                2415 

cag ccg cac ctt ggc gtg agc tca gca gcc agc ggc cac ctg ggc cgg     7296 
Gln Pro His Leu Gly Val Ser Ser Ala Ala Ser Gly His Leu Gly Arg 
            2420                2425                2430 

agc ttc ctg agt gga gag ccg agc cag gca gac gtg cag cca ctg ggc     7344 
Ser Phe Leu Ser Gly Glu Pro Ser Gln Ala Asp Val Gln Pro Leu Gly 
        2435                2440                2445 

ccc agc agc ctg gcg gtg cac act att ctg ccc cag gag agc ccc gcc     7392 
Pro Ser Ser Leu Ala Val His Thr Ile Leu Pro Gln Glu Ser Pro Ala 
    2450                2455                2460 

ctg ccc acg tcg ctg cca tcc tcg ctg gtc cca ccc gtg acc gca gcc     7440 
Leu Pro Thr Ser Leu Pro Ser Ser Leu Val Pro Pro Val Thr Ala Ala 
2465                2470                2475                2480 

cag ttc ctg acg ccc ccc tcg cag cac agc tac tcc tcg cct gtg gac     7488 
Gln Phe Leu Thr Pro Pro Ser Gln His Ser Tyr Ser Ser Pro Val Asp 
                2485                2490                2495 

aac acc ccc agc cac cag cta cag gtg cct gag cac ccc ttc ctg acc     7536 

cct tcg ccg gag tcg ccc gac caa tgg tcg tcc tcg tcg ccg cac tct     7584 
Pro Ser Pro Glu Ser Pro Asp Gln Trp Ser Ser Ser Ser Pro His Ser 
            2500                2505                2510 

aat gtg tct gac tgg tct gag ggc gtg tcg tcg ccc ccg acc tcc atg     7632 
Asn Val Ser Asp Trp Ser Glu Gly Val Ser Ser Pro Pro Thr Ser Met 
        2515                2520                2525 

cag tcc cag atc gcg cgc atc ccg gag gcg ttc aag taa tagctcgagg      7681 
Gln Ser Gln Ile Ala Arg Ile Pro Glu Ala Phe Lys 
    2530                2535                2540 

tgccagcagc tc                                                       7693 

 
           
             12  
             423  
             DNA  
             Homo sapiens  
             
               CDS  
               (374)...(423)  
             
             
               misc_feature  
               149, 150  
               n = A,T,C or G  
             
           
            12 

agagagagag agaagaattg atcggtgtca tgtgaagtgt tgaagtttgt atcttgaaaa     60 

tccctctaaa tcctttgtct taacagctca gtgcgagtgc agcgatttga agttgactat    120 

ccctccgtcc ttaaaggaga aaaaagtann agccgtctcc agatagagtc ggctggtgca    180 

ggagagaatt tagcgatagt ttgcaattct gattaatcgc gtagaaaatg accttatttt    240 

ggagggcggg atggaggaga gtgggtgagg aggcgcccgg acgcggagcc agtccgccgc    300 

cccccggcca ccagcctgct gcgtagccgc tgcctgatgt ccgggcacct gcccctggcc    360 

cccgtgcccg cag gtg aga ccg tgg agc cgc ccc cgc cgg cgc agc tgc       409 
               Val Arg Pro Trp Ser Arg Pro Arg Arg Arg Ser Cys 
                1               5                   10 

act tca tgt acg tg                                                   423 
Thr Ser Cys Thr 
         15 

 
           
             13  
             3494  
             DNA  
             H. sapiens  
             
 
           
            13 

cacgctctga tgccgccaag cgcctgctgg aggccagcgc agatgccaac atccaggaca     60 

acatgggccg caccccgctg catgcggctg tgtctgccga cgcacaaggt gtcttccaga    120 

tcctgatccg gaaccgagcc acagacctgg atgcccgcat gcatgatggc acgacgccac    180 

tgatcctggc tgcccgcctg gccgtggagg gcatgctgga ggacctcatc aactcacacg    240 

ccgacgtcaa cgccgtagat gacctgggca agtccgccct gcactgggcc gccgccgtga    300 

acaatgtgga tgccgcagtt gtgctcctga agaacggggc taacaaagat atgcagaaca    360 

acagggagga gacacccctg tttctggccg cccgggaggg cagctacgag accgccaagg    420 

tgctgctgga ccactttgcc aaccgggaca tcacggatca tatggaccgc ctgccgcgcg    480 

acatcgcaca ggagcgcatg catcacgaca tcgtgaggct gctggacgag tacaacctgg    540 

tgcgcagccc gcagctgcac ggagccccgc tggggggcac gcccaccctg tcgcccccgc    600 

tctgctcgcc caacggctac ctgggcagcc tcaagcccgg cgtgcagggc aagaaggtcc    660 

gcaagcccag cagcaaaggc ctggcctgtg gaagcaagga ggccaaggac ctcaaggcac    720 

ggaggaagaa gtcccaggat ggcaagggct gcctgctgga cagctccggc atgctctcgc    780 

ccgtggactc cctggagtca ccccatggct acctgtcaga cgtggcctcg ccgccactgc    840 

tgccctcccc gttccagcag tctccgtccg tgcccctcaa ccacctgcct gggatgcccg    900 

acacccacct gggcatcggg cacctgaacg tggcggccaa gcccgagatg gcggcgctgg    960 

gtgggggcgg ccggctggcc tttgagactg gcccacctcg tctctcccac ctgcctgtgg   1020 

cctctggcac cagcaccgtc ctgggctcca gcagcggagg ggccctgaat ttcactgtgg   1080 

gcgggtccac cagtttgaat ggtcaatgcg agtggctgtc ccggctgcag agcggcatgg   1140 

tgccgaacca atacaaccct ctgcggggga gtgtggcacc aggccccctg agcacacagg   1200 

ccccctccct gcagcatggc atggtaggcc cgctgcacag tagccttgct gccagcgccc   1260 

tgtcccagat gatgagctac cagggcctgc ccagcacccg gctggccacc cagcctcacc   1320 

tggtgcagac ccagcaggtg cagccacaaa acttacagat gcagcagcag aacctgcagc   1380 

cagcaaacat ccagcagcag caaagcctgc agccgccacc accaccacca cagccgcacc   1440 

ttggcgtgag ctcagcagcc agcggccacc tgggccggag cttcctgagt ggagagccga   1500 

gccaggcaga cgtgcagcca ctgggcccca gcagcctggc ggtgcacact attctgcccc   1560 

aggagagccc cgccctgccc acgtcgctgc catcctcgct ggtcccaccc gtgaccgcag   1620 

cccagttcct gacgcccccc tcgcagcaca gctactcctc gcctgtggac aacaccccca   1680 

gccaccagct acaggtgcct gagcacccct tcctcacccc gtcccctgag tcccctgacc   1740 

agtggtccag ctcgtccccg cattccaacg tctccgactg gtccgagggc gtctccagcc   1800 

ctcccaccag catgcagtcc cagatcgccc gcattccgga ggccttcaag taaacggcgc   1860 

gccccacgag accccggctt cctttcccaa gccttcgggc gtctgtgtgc gctctgtgga   1920 

tgccagggcc gaccagagga gcctttttaa aacacatgtt tttatacaaa ataagaacaa   1980 

ggattttaat tttttttagt atttatttat gtacttttat tttacacaga aacactgcct   2040 

ttttatttat atgtactgtt ttatctggcc ccaggtagaa acttttatct attctgagaa   2100 

aacaagcaag ttctgagagc cagggttttc ctacgtagga tgaaaagatt cttctgtgtt   2160 

tataaaatat aaacaaagat tcatgattta taaatgccat ttatttattg attccttttt   2220 

tcaaaatcca aaaagaaatg atgttggaga agggaagttg aacgagcata gtccaaaaag   2280 

ctcctggggc gtccaggccg cgccctttcc ccgacgccca cccaacccca agccagcccg   2340 

gccgctccac cagcatcacc tgcctgttag gagaagctgc atccagaggc aaacggaggc   2400 

aaagctggct caccttccgc acgcggatta atttgcatct gaaataggaa acaagtgaaa   2460 

gcatatgggt tagatgttgc catgtgtttt agatggtttc ttgccagcat gcttgtgaaa   2520 

atgtgttctc ggagtgtgta tgccaagagt gcacccatgg taccaatcat gaatctttgt   2580 

ttcaggttca gtattatgta gttgttcgtt ggttatacaa gttcttggtc cctccagaac   2640 

caccccggcc ccctgcccgt tcttgaaatg taggcatcat gcatgtcaaa catgagatgt   2700 

gtggactgtg gcacttgcct gggtcacaca cggaggcatc ctaccctttt ctggggaaag   2760 

acactgcctg ggctgacccc ggtggcggcc ccagcacctc agcctgcaca gtgtccccca   2820 

ggttccgaag aagatgctcc agcaacacag cctgggcccc agctcgcggg acccgacccc   2880 

ccgtgggctc ccgtgttttg taggagactt gccagagccg ggcacattga gctgtgcaac   2940 

gccgtgggct gcgtcctttg gtcctgtccc cgcagccctg gcagggggca tgcggtcggg   3000 

caggggctgg agggaggcgg gggctgccct tgggccaccc ctcctagttt gggaggagca   3060 

gatttttgca ataccaagta tagcctatgg cagaaaaaat gtctgtaaat atgtttttaa   3120 

aggtggattt tgtttaaaaa atcttaatga atgagtctgt tgtgtgtcat gccagtgagg   3180 

gacgtcagac ttggctcagc tcggggagcc ttagccgccc atgcactggg gacgctccgc   3240 

tgccgtgccg cctgcactcc tcagggcagc ctcccccggc tctacggggg ccgcgtggtg   3300 

ccatccccag ggggcatgac cagatgcgtc ccaagatgtt gatttttact gtgttttata   3360 

aaatagagtg tagtttacag aaaaagactt taaaagtgat ctacatgagg aactgtagat   3420 

gatgtatttt tttcatcttt tttgttaact gatttgcaat aaaaatgata ctgatggtga   3480 

aaaaaaaaaa aaaa                                                     3494 

 
           
             14  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            14 

cgtgcgtccc tcttagggtc                                                 20 

 
           
             15  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            15 

cacagcagac ctgggcaggc                                                 20 

 
           
             16  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            16 

cagccctccc ctaatgagac                                                 20 

 
           
             17  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            17 

cggccacgca ctgtgcaggc                                                 20 

 
           
             18  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            18 

acggtctcac ctgcgggcac                                                 20 

 
           
             19  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            19 

tcacttgagg cccacggagt                                                 20 

 
           
             20  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            20 

cactgcctac ctggaagaca                                                 20 

 
           
             21  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            21 

accacctgcg tcaccacatt                                                 20 

 
           
             22  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            22 

cacgatttcc ctgaccagcc                                                 20 

 
           
             23  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            23 

aagggcaggc actggccacc                                                 20 

 
           
             24  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            24 

ttgcagttgt ttcctggaca                                                 20 

 
           
             25  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            25 

ttctggcagg catttggcat                                                 20 

 
           
             26  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            26 

tggcacagca gacctgtgcg                                                 20 

 
           
             27  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            27 

tgaggtggca cagcagacct                                                 20 

 
           
             28  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            28 

tccacgtcct ggctgcaggc                                                 20 

 
           
             29  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            29 

tccccaatct ggtccaggca                                                 20 

 
           
             30  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            30 

ggaactcccc aatctggtcc                                                 20 

 
           
             31  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            31 

ccatcgatct tgtccagaca                                                 20 

 
           
             32  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            32 

gttgttgttg atgtcacagt                                                 20 

 
           
             33  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            33 

cactcgttgt tgttgatgtc                                                 20 

 
           
             34  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            34 

ggcaggcagt cgcagaaggc                                                 20 

 
           
             35  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            35 

aagccgggca ggcagtcgca                                                 20 

 
           
             36  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            36 

gtgtagctgt ccacgcagtc                                                 20 

 
           
             37  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            37 

gcaggtgcac gtgtagctgt                                                 20 

 
           
             38  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            38 

gcacaaggtt ctggcagttg                                                 20 

 
           
             39  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            39 

gcagccacct cacaggacac                                                 20 

 
           
             40  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            40 

gccctccatg ctggcacagg                                                 20 

 
           
             41  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            41 

acagagccct ccatgctggc                                                 20 

 
           
             42  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            42 

gggcaggagc acttgtaggt                                                 20 

 
           
             43  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            43 

ggcactcgca gtggaagtca                                                 20 

 
           
             44  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            44 

cctcgaagcc cgcagggcac                                                 20 

 
           
             45  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            45 

tcggatgtgg gctcacaggt                                                 20 

 
           
             46  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            46 

gtagtccagg atgtggcaca                                                 20 

 
           
             47  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            47 

aagctgtagt ccaggatgtg                                                 20 

 
           
             48  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            48 

ttgaagttga gggagcagtc                                                 20 

 
           
             49  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            49 

ggtcattgaa gttgagggag                                                 20 

 
           
             50  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            50 

tgcgtgcagt tcttccaggg                                                 20 

 
           
             51  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            51 

gagactgcgt gcagttcttc                                                 20 

 
           
             52  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            52 

tggctgtcac agtggccgtc                                                 20 

 
           
             53  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            53 

tgcactggct gtcacagtgg                                                 20 

 
           
             54  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            54 

tggtccttgc agtactggtc                                                 20 

 
           
             55  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            55 

tgaagtggtc cttgcagtac                                                 20 

 
           
             56  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            56 

ccacgttggt gtgcagcacg                                                 20 

 
           
             57  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            57 

gaagaccacg ttggtgtgca                                                 20 

 
           
             58  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            58 

cgcttgaaga ccacgttggt                                                 20 

 
           
             59  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            59 

gggaagatca tctgctggcc                                                 20 

 
           
             60  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            60 

ctctggaagc actgcgagga                                                 20 

 
           
             61  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            61 

tggcactctg gaagcactgc                                                 20 

 
           
             62  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            62 

ttgcgggaca gcagcacccc                                                 20 

 
           
             63  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            63 

aaccagagct ggccatgctg                                                 20 

 
           
             64  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            64 

cagggaacca gagctggcca                                                 20 

 
           
             65  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            65 

aacttcttgg tctccaggtc                                                 20 

 
           
             66  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            66 

accggaactt cttggtctcc                                                 20 

 
           
             67  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            67 

gcagacatgc gcaggtcagc                                                 20 

 
           
             68  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            68 

ccatggcaga catgcgcagg                                                 20 

 
           
             69  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            69 

gcggtctgtc tggttgtgca                                                 20 

 
           
             70  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            70 

ttggcatctg cgctggcctc                                                 20 

 
           
             71  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            71 

tgatgaggtc ctccagcatg                                                 20 

 
           
             72  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            72 

tgagttgatg aggtcctcca                                                 20 

 
           
             73  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            73 

gcggacttgc ccaggtcatc                                                 20 

 
           
             74  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            74 

ccgttcttca ggagcacaac                                                 20 

 
           
             75  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            75 

atccgtgatg tcccggttgg                                                 20 

 
           
             76  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            76 

tagccgttgg gcgagcagag                                                 20 

 
           
             77  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            77 

ctccgtgcct tgaggtcctt                                                 20 

 
           
             78  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            78 

tcttcctccg tgccttgagg                                                 20 

 
           
             79  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            79 

cagcccttgc catcctggga                                                 20 

 
           
             80  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            80 

gtctgcacca ggtgaggctg                                                 20 

 
           
             81  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            81 

tgctgggtct gcaccaggtg                                                 20 

 
           
             82  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            82 

gcacctgctg ggtctgcacc                                                 20 

 
           
             83  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            83 

tggctgcacc tgctgggtct                                                 20 

 
           
             84  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            84 

tcgagctatt acttgaacgc                                                 20 

 
           
             85  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            85 

gctgctggca cctcgagcta                                                 20 

 
           
             86  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            86 

ttcacatgac accgatcaat                                                 20 

 
           
             87  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            87 

ctttaaggac ggagggatag                                                 20 

 
           
             88  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            88 

tctgtgtaaa ataaaagtac                                                 20 

 
           
             89  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            89 

tgctcgttca acttcccttc                                                 20 

 
           
             90  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            90 

ctggagcatc ttcttcggaa                                                 20 

 
           
             91  
             20  
             DNA  
             Artificial Sequence  
             
               Antisense Oligonucleotide  
             
           
            91 

cccgagctga gccaagtctg                                                 20 

 
           
             92  
             20  
             DNA  
             H. sapiens  
             
 
           
            92 

gcctgcacag tgcgtggccg                                                 20 

 
           
             93  
             20  
             DNA  
             H. sapiens  
             
 
           
            93 

actccgtggg cctcaagtga                                                 20 

 
           
             94  
             20  
             DNA  
             H. sapiens  
             
 
           
            94 

aatgtggtga cgcaggtggt                                                 20 

 
           
             95  
             20  
             DNA  
             H. sapiens  
             
 
           
            95 

ggtggccagt gcctgccctt                                                 20 

 
           
             96  
             20  
             DNA  
             H. sapiens  
             
 
           
            96 

tgtccaggaa acaactgcaa                                                 20 

 
           
             97  
             20  
             DNA  
             H. sapiens  
             
 
           
            97 

atgccaaatg cctgccagaa                                                 20 

 
           
             98  
             20  
             DNA  
             H. sapiens  
             
 
           
            98 

aggtctgctg tgccacctca                                                 20 

 
           
             99  
             20  
             DNA  
             H. sapiens  
             
 
           
            99 

gcctgcagcc aggacgtgga                                                 20 

 
           
             100  
             20  
             DNA  
             H. sapiens  
             
 
           
            100 

tgcctggacc agattgggga                                                 20 

 
           
             101  
             20  
             DNA  
             H. sapiens  
             
 
           
            101 

tgtctggaca agatcgatgg                                                 20 

 
           
             102  
             20  
             DNA  
             H. sapiens  
             
 
           
            102 

actgtgacat caacaacaac                                                 20 

 
           
             103  
             20  
             DNA  
             H. sapiens  
             
 
           
            103 

gacatcaaca acaacgagtg                                                 20 

 
           
             104  
             20  
             DNA  
             H. sapiens  
             
 
           
            104 

gccttctgcg actgcctgcc                                                 20 

 
           
             105  
             20  
             DNA  
             H. sapiens  
             
 
           
            105 

tgcgactgcc tgcccggctt                                                 20 

 
           
             106  
             20  
             DNA  
             H. sapiens  
             
 
           
            106 

caactgccag aaccttgtgc                                                 20 

 
           
             107  
             20  
             DNA  
             H. sapiens  
             
 
           
            107 

cctgtgccag catggagggc                                                 20 

 
           
             108  
             20  
             DNA  
             H. sapiens  
             
 
           
            108 

gccagcatgg agggctctgt                                                 20 

 
           
             109  
             20  
             DNA  
             H. sapiens  
             
 
           
            109 

tgacttccac tgcgagtgcc                                                 20 

 
           
             110  
             20  
             DNA  
             H. sapiens  
             
 
           
            110 

acctgtgagc ccacatccga                                                 20 

 
           
             111  
             20  
             DNA  
             H. sapiens  
             
 
           
            111 

tgtgccacat cctggactac                                                 20 

 
           
             112  
             20  
             DNA  
             H. sapiens  
             
 
           
            112 

cacatcctgg actacagctt                                                 20 

 
           
             113  
             20  
             DNA  
             H. sapiens  
             
 
           
            113 

gacggccact gtgacagcca                                                 20 

 
           
             114  
             20  
             DNA  
             H. sapiens  
             
 
           
            114 

ccactgtgac agccagtgca                                                 20 

 
           
             115  
             20  
             DNA  
             H. sapiens  
             
 
           
            115 

gaccagtact gcaaggacca                                                 20 

 
           
             116  
             20  
             DNA  
             H. sapiens  
             
 
           
            116 

cgtgctgcac accaacgtgg                                                 20 

 
           
             117  
             20  
             DNA  
             H. sapiens  
             
 
           
            117 

tgcacaccaa cgtggtcttc                                                 20 

 
           
             118  
             20  
             DNA  
             H. sapiens  
             
 
           
            118 

accaacgtgg tcttcaagcg                                                 20 

 
           
             119  
             20  
             DNA  
             H. sapiens  
             
 
           
            119 

tcctcgcagt gcttccagag                                                 20 

 
           
             120  
             20  
             DNA  
             H. sapiens  
             
 
           
            120 

gcagtgcttc cagagtgcca                                                 20 

 
           
             121  
             20  
             DNA  
             H. sapiens  
             
 
           
            121 

ggggtgctgc tgtcccgcaa                                                 20 

 
           
             122  
             20  
             DNA  
             H. sapiens  
             
 
           
            122 

cagcatggcc agctctggtt                                                 20 

 
           
             123  
             20  
             DNA  
             H. sapiens  
             
 
           
            123 

gacctggaga ccaagaagtt                                                 20 

 
           
             124  
             20  
             DNA  
             H. sapiens  
             
 
           
            124 

ggagaccaag aagttccggt                                                 20 

 
           
             125  
             20  
             DNA  
             H. sapiens  
             
 
           
            125 

gctgacctgc gcatgtctgc                                                 20 

 
           
             126  
             20  
             DNA  
             H. sapiens  
             
 
           
            126 

cctgcgcatg tctgccatgg                                                 20 

 
           
             127  
             20  
             DNA  
             H. sapiens  
             
 
           
            127 

gaggccagcg cagatgccaa                                                 20 

 
           
             128  
             20  
             DNA  
             H. sapiens  
             
 
           
            128 

catgctggag gacctcatca                                                 20 

 
           
             129  
             20  
             DNA  
             H. sapiens  
             
 
           
            129 

tggaggacct catcaactca                                                 20 

 
           
             130  
             20  
             DNA  
             H. sapiens  
             
 
           
            130 

gatgacctgg gcaagtccgc                                                 20 

 
           
             131  
             20  
             DNA  
             H. sapiens  
             
 
           
            131 

gttgtgctcc tgaagaacgg                                                 20 

 
           
             132  
             20  
             DNA  
             H. sapiens  
             
 
           
            132 

ccaaccggga catcacggat                                                 20 

 
           
             133  
             20  
             DNA  
             H. sapiens  
             
 
           
            133 

ctctgctcgc ccaacggcta                                                 20 

 
           
             134  
             20  
             DNA  
             H. sapiens  
             
 
           
            134 

aaggacctca aggcacggag                                                 20 

 
           
             135  
             20  
             DNA  
             H. sapiens  
             
 
           
            135 

cctcaaggca cggaggaaga                                                 20 

 
           
             136  
             20  
             DNA  
             H. sapiens  
             
 
           
            136 

tcccaggatg gcaagggctg                                                 20 

 
           
             137  
             20  
             DNA  
             H. sapiens  
             
 
           
            137 

cagcctcacc tggtgcagac                                                 20 

 
           
             138  
             20  
             DNA  
             H. sapiens  
             
 
           
            138 

cacctggtgc agacccagca                                                 20 

 
           
             139  
             20  
             DNA  
             H. sapiens  
             
 
           
            139 

agacccagca ggtgcagcca                                                 20 

 
           
             140  
             20  
             DNA  
             H. sapiens  
             
 
           
            140 

tagctcgagg tgccagcagc                                                 20 

 
           
             141  
             20  
             DNA  
             H. sapiens  
             
 
           
            141 

attgatcggt gtcatgtgaa                                                 20 

 
           
             142  
             20  
             DNA  
             H. sapiens  
             
 
           
            142 

gtacttttat tttacacaga                                                 20 

 
           
             143  
             20  
             DNA  
             H. sapiens  
             
 
           
            143 

gaagggaagt tgaacgagca                                                 20 

 
           
             144  
             20  
             DNA  
             H. sapiens  
             
 
           
            144 

ttccgaagaa gatgctccag                                                 20 

 
           
             145  
             20  
             DNA  
             H. sapiens  
             
 
           
            145 

cagacttggc tcagctcggg                                                 20