Abstract:
Methods for detecting G-protein coupled receptor (GPCR) activity; methods for assaying GPCR activity; and methods for screening for GPCR ligands, G-protein-coupled receptor kinase (GRK) activity, and compounds that interact with components of the GPCR regulatory process are described. Included are methods for expanding ICAST technologies for assaying GPCR activity with applications for ligand fishing, and agonist or antagonist screening. These methods include: engineering seronine/threonine phosphorylation sites into known or orphan GPCR open reading frames in order to increase the affinity of arrestin for the activated form of the GPCR or to increase the reside time of arrestin on the activated GPCR; engineering mutant arrestin proteins that bind to activated GPCRs in the absence of G-protein coupled receptor kinases which may be limiting; and engineering mutant super arrestin proteins that have an increased affinity for activated GPCRs with or without phosphorylation. These methods are intended to increase the robustness of the GPCR/ICAST technology in situations in which G-protein coupled receptor kinases are absent or limiting, or in which the GPCR is not efficiently down-regulated or is rapidly resensitized (thus having a labile interaction with arrestin). Included are also more specific methods for using ICAST complementary enzyme fragments to monitor GPCR homo- and hetero-dimerization with applications for drug lead discovery and ligand and function discovery for orphan GPCRs.

Description:
[0001]    This application is a continuation-in-part of U.S. application Ser. No. 09/654,499, filed Sep. 1, 2000, which claims the benefit from Provisional Application Ser. No. 60/180,669, filed Feb. 7, 2000. The entirety of U.S. application Ser. No. 09/654,499 and Provisional Application Ser. No. 60/180,669 are incorporated herein by reference. 
     
    
     
       BACKGROUND OF THE INVENTION  
         [0002]    1. Field of the Invention  
           [0003]    The present invention relates to methods of detecting G-protein-coup led receptor (GPCR) activity, and provides methods of assaying GPCR activity, methods for screening for GPCR ligands, agonists and/or antagonists, methods for screening natural and surrogate ligands for orphan GPCRs, and methods for screening compounds that interact with components of the GPCR regulatory process.  
         BACKGROUND OF THE TECHNOLOGY  
         [0004]    The actions of many extracellular signals are mediated by the interaction of G-protein-coupled receptors (GPCRs) and guanine nucleotide-binding regulatory proteins (G-proteins). G-protein-mediated signaling systems have been identified in many divergent organisms, such as mammals and yeast. The GPCRs represent a large super family of proteins which have divergent amino acid sequences, but share common structural features, in particular, the presence of seven transmembrane helical domains. GPCRs respond to, among other extracellular signals, neurotransmitters, hormones, odorants and light. Individual GPCR types activate a particular signal transduction pathway; at least ten different signal transduction pathways are known to be activated via GPCRs. For example, the beta 2-adrenergic receptor (β2AR) is a prototype mammalian GPCR. In response to agonist binding, β2AR receptors activate a G-protein (Gs) which in turn stimulates adenylate cyclase activity and results in increased cyclic adenosine monophosphate (cAMP) production in the cell.  
           [0005]    The signaling pathway and final cellular response that result from GPCR stimulation depends on the specific class of G-protein with which the particular receptor is coupled (Hamm, “The Many Faces of G-Protein Signaling.” J. Biol.  
           [0006]    Chem., 273:669-672 (1998)). For instance, coupling to the Gs class of G-proteins stimulates cAMP production and activation of the Protein Kinase A and C pathways, whereas coupling to the Gi class of G-proteins down regulates cAMP. Other second messenger systems such as calcium, phospholipase C, and phosphatidylinositol 3 may also be utilized. As a consequence, GPCR signaling events have predominantly been measured via quantification of these second messenger products.  
           [0007]    The decrease of a response to a persistent stimulus is a widespread biological phenomenon. Signaling by diverse GPCRs is believed to be terminated by a uniform two-step mechanism. Activated receptor is first phosphorylated by a GPCR kinase (GRK). An arrestin protein binds to the activated and phosphorylated receptor, thus blocking G-protein interaction. This process is commonly referred to as desensitization, a general mechanism that has been demonstrated in a variety of functionally diverse GPCRs. Arrestin also plays a part in regulating GPCR internalization and resensitization, processes that are heterogenous among different GPCRs (Oakley, et al., J. Biol. Chem., 274:32248-32257 (1999)). The interaction between an arrestin and GPCR in processes of internalization and resensitization is dictated by the specific sequence motif in the carboxyl terminus of a given GPCR. Only a subset of GPCRs, which possess clusters of three serine or threonine residues at the carboxyl termini, were found to co-traffick with the arrestins into the endocytic vesicles after ligand stimulation. The number of receptor kinases and arrestins involved in desensitization of GPCRs is rather limited.  
           [0008]    A common feature of GPCR physiology is desensitization and recycling of the receptor through the processes of receptor phosphorylation, endocytosis and dephosphorylation (Ferguson, et al., “G-protein-coupled receptor regulation: role of G-protein-coupled receptor kinases and arrestins.” Can. J. Physiol. Pharmacol., 74:1095-1110 (1996)). Ligand-occupied GPCRs can be phosphorylated by two families of serine/threonine kinases, the G-protein-coupled receptor kinases (GRKs) and the second messenger-dependent protein kinases such as protein kinase A and protein kinase C. Phosphorylation by either class of kinases serves to down-regulate the receptor by uncoupling it from its corresponding G-protein. GRK-phosphorylation also serves to down-regulate the receptor by recruitment of a class of proteins known as the arrestins that bind the cytoplasmic domain of the receptor and promote clustering of the receptor into endocytic vescicles. Once the receptor is endocytosed, it will either be degraded in lysosomes or dephosphorylated and recycled back to the plasma membrane as a fully-functional receptor.  
           [0009]    Binding of an arrestin protein to an activated receptor has been documented as a common phenomenon of a variety of GPCRs ranging from rhodopsin to β2AR to the neurotensin receptor (Barak, et al., “A β-arrestin/Green Fluorescent Fusion Protein Biosensor for Detecting G-Protein-Coupled Receptor Activation,” J. Biol. Chem., 272:27497-500 (1997)). Consequently, monitoring arrestin interaction with a specific GPCR can be utilized as a generic tool for measuring GPCR activation. Similarly, a single G-protein and GRK also partner with a variety of receptors (Hamm, et al. (1998) and Pitcher et al., “G-Protein-Coupled Receptor Kinases,” Annu. Rev. Biochem., 67:653-92 (1998)), such that these protein/protein interactions may also be monitored to determine receptor activity.  
           [0010]    Many therapeutic drugs in use today target GPCRs, as they regulate vital physiological responses, including vasodilation, heart rate, bronchodilation, endocrine secretion and gut peristalsis. See, e.g., Lefkowitz et al., Annu. Rev. Biochem., 52:159 (1983). Some of these drugs mimic the ligand for this receptor. Other drugs act to antagonize the receptor in cases when disease arises from spontaneous activity of the receptor.  
           [0011]    Efforts such as the Human Genome Project are identifying new GPCRs (“orphan” receptors) whose physiological roles and ligands are unknown. It is estimated that several thousand GPCRs exist in the human genome.  
           [0012]    Various approaches have been used to monitor intracellular activity in response to a stimulant, e.g., enzyme-linked immunosorbent assay (ELISA); Fluorescense Imaging Plate Reader assay (FLIPR™, Molecular Devices Corp., Sunnyvale, Calif.); EVOscreen™, EVOTEC™, Evotec Biosystems Gmbh, Hamburg, Germany; and techniques developed by CELLOMICS™, Cellomics, Inc., Pittsburgh, Pa.  
           [0013]    Germino et al., “Screening for in vivo protein-protein interactions.” Proc. Natl. Acad. Sci., 90(3):933-937 (1993), discloses an in vivo approach for the isolation of proteins interacting with a protein of interest.  
           [0014]    Phizicky et al., “Protein-protein interactions: methods for detection and analysis.” Microbiol. Rev., 59(1): 94-123 (1995), discloses a review of biochemical, molecular biological and genetic methods used to study protein-protein interactions.  
           [0015]    Offermanns et al., “Gα 15  and Gα 16  Couple a Wide Variety of Receptors to Phospholipase C.” J. Biol. Chem., 270(25):15175-15180 (1995), discloses that Gα 15  and Gα 16  can be activated by a wide variety of G-protein-coupled receptors. The selective coupling of an activated receptor to a distinct pattern of G-proteins is regarded as an important requirement to achieve accurate signal transduction. Id.  
           [0016]    Barak et al., “A β-arrestin/Green Fluorescent Protein Biosensor for Detecting G Protein-coupled Receptor Activation.” J. Biol. Chem., 272(44):27497-27500 (1997) and U.S. Pat. Nos. 5,891,646 and 6,110,693 disclose the use of a β-arrestin/green fluorescent fusion protein (GFP) for imaging protein translocation upon stimulation of GPCR with optical devices.  
           [0017]    Each of the references described above has drawbacks. For example,  
           [0018]    The prior art methodologies require over-expression of the proteins, which could cause artifact and tip the balance of cellular regulatory machineries.  
           [0019]    The prior art visualization or imaging assays are low throughput and lack thorough quantification. Therefore, they are not suitable for high throughput pharmacological and kinetic assays.  
           [0020]    In addition, many of the prior art assays require isolation of the GPCR rather than observation of the GPCR in a cell. There thus exists a need for improved methods for monitoring GPCR function.  
         SUMMARY OF THE INVENTION  
         [0021]    The present invention provides modifications to the disclosure in U.S. application Ser. No. 09/654,499. In particular, the present invention is directed to modifications of the below aspects of the invention to further enhance assay sensitivity. The modifications include the use of genetically modified arrestins that exhibit enhanced binding to activated GPCR regardless of whether the GPCR is phosphorylated or non-phosphorylated; the use of a serine/threonine cluster strategy to facilitate screening assays for orphan receptors that do not possess this structural motif on their own; and the use of a combination of the above modifications to achieve even more enhanced detection.  
           [0022]    A first aspect of the present invention is a method that monitors GPCR function proximally at the site of receptor activation, thus providing more information for drug discovery purposes due to fewer competing mechanisms. Activation of the GPCR is measured by a read-out for interaction of the receptor with a regulatory component such as arrestin, G-protein, GRK or other kinases, the binding of which to the receptor is dependent upon agonist occupation of the receptor. The present invention involves the detection of protein/protein interaction by complementation of mutant reporter enzymes.  
           [0023]    Binding of arrestin to activated GPCR is a common process in the first step of desensitization that has been demonstrated for most, if not all, GPCRs studied so far. Measurement of GPCR interaction with arrestin via mutant enzyme complementation (Le., ICAST) provides a more generic assay technology applicable for a wide variety of GPCRs and orphan receptors.  
           [0024]    A further aspect of the present invention is a method of assessing GPCR pathway activity under test conditions by providing a test cell that expresses a GPCR, e.g., muscarinic, adrenergic, dopamine, angiotensin or endothelin, as a fusion protein to a mutant reporter enzyme and interacting a protein in the GPCR pathway, e.g., G-protein, arrestin or GRK, as a fusion protein with a complementing mutant reporter enzyme. When test cells are exposed to a known agonist to the target GPCR under test conditions, activation of the GPCR will be monitored by complementation of the reporter enzyme. Increased reporter enzyme activity reflects interaction of the GPCR with its interacting protein partner.  
           [0025]    A further aspect of the present invention is a method of assessing GPCR pathway activity in the presence of a test arrestin, e.g., β-arrestin.  
           [0026]    A further aspect of the present invention is a method of assessing GPCR pathway activity in the presence of a test G-protein.  
           [0027]    A further aspect of the present invention is a method of assessing GPCR pathway activity upon exposure of the test cell to a test ligand.  
           [0028]    A further aspect of the present invention is a method of assessing GPCR activity upon co-expression in the test cell of a second receptor. The second receptor could be the same GPCR or orphan receptor (i.e., homo-dimerization), a different GPCR or orphan receptor (i.e., hetero-dimerization) or could be a receptor of another type.  
           [0029]    A further aspect of the present invention is a method for screening for a ligand or agonist to an orphan GPCR. The ligand or agonist could be contained in natural or synthetic libraries or mixtures or could be a physical stimulus. A test cell is provided that expresses the orphan GPCR as a fusion protein with a mutant reporter enzyme, e.g., a β-galactosidase mutant, and, for example, an arrestin or mutant form of arrestin as a fusion protein with a complementing mutant reporter enzyme, e.g., another β-galactosidase mutant. The interaction of the arrestin with the orphan GPCR upon receptor activation is measured by enzymatic activity of the complemented reporter enzyme. The test cell is exposed to a test compound, and an increase in reporter enzyme activity indicates the presence of a ligand or agonist.  
           [0030]    A further aspect of the present invention is a method for screening a protein of interest, for example, an arrestin protein (or mutant form of the arrestin protein) for the ability to bind to a phosphorylated, or activated, GPCR. A test cell is provided that expresses a GPCR as a fusion protein with a mutant reporter enzyme, e.g., a β-galactosidase mutant, and contains arrestin (or a mutant form of arrestin) as a fusion protein with a complementing mutant reporter enzyme, e.g., another β-galactosidase mutant. The interaction of arrestin with the GPCR upon receptor activation is measured by enzymatic activity of the complemented reporter enzyme. The test cell is exposed to a known GPCR agonist and then reporter enzyme activity is detected. Increased reporter enzyme activity indicates that the β-arrestin molecule can bind to phosphorylated, or activated, GPCR in the test cell.  
           [0031]    A further aspect of the present invention is a method to screen for an agonist to a specific GPCR. The agonist could be contained in natural or synthetic libraries or could be a physical stimulus. A test cell is provided that expresses a GPCR as a fusion protein with a mutant reporter enzyme, e.g., a β-galactosidase mutant, and, for example, an arrestin as a fusion protein with a complementing mutant reporter enzyme, e.g., another β-galactosidase mutant. The interaction of arrestin with the GPCR upon receptor activation is measured by enzymatic activity of the complemented reporter enzyme. The test cell is exposed to a test compound, and an increase in reporter enzyme activity indicates the presence of an agonist. The test cell may express a known GPCR or a variety of known GPCRs, or may express an unknown GPCR or a variety of unknown GPCRs. The GPCR may be, for example, an odorant GPCR or a βAR GPCR.  
           [0032]    A further aspect of the present invention is a method for screening a test compound for GPCR antagonist activity. A test cell is provided that expresses a GPCR as a fusion protein with a mutant reporter enzyme, e.g., a β-galactosidase mutant, and, for example, an arrestin as a fusion protein with a complementing mutant reporter enzyme, e.g., another β-galactosidase mutant. The interaction of arrestin with the GPCR upon receptor activation is measured by enzymatic activity of the complemented reporter enzyme. The test cell is exposed to a test compound, and an increase in reporter enzyme activity indicates the presence of an agonist. The cell is exposed to a test compound and to a GPCR agonist, and reporter enzyme activity is detected. When exposure to the agonist occurs at the same time as or subsequent to exposure to the test compound, a decrease in reporter enzyme activity after exposure to the test compound indicates that the test compound has antagonist activity to the GPCR.  
           [0033]    A further aspect of the present invention is a method of screening a sample solution for the presence of an agonist, antagonist or ligand to a GPCR. A test cell is provided that expresses GPCR as a fusion protein with a mutant reporter enzyme, e.g., a β-galactosidase mutant, and contains, for example, a β-arrestin as a fusion protein with a complementing reporter, e.g., another β-galactosidase mutant. The test cell is exposed to a sample solution, and reporter enzyme activity is assessed. Changed reporter enzyme activity after exposure to the sample solution indicates the sample solution contains an agonist, antagonist or ligand for a GPCR expressed in the cell.  
           [0034]    A further aspect of the present invention is a method of screening a cell for the presence of a GPCR. According to this aspect, an arrestin fusion protein with a mutant reporter enzyme and a GPCR downstream signaling fusion protein with a mutant reporter enzyme are employed to detect GPCR action. A modification of this aspect of the invention can be employed to provide a method of screening a plurality of cells for those cells which contain a GPCR. According to this aspect, a plurality of cells containing a conjugate comprising a β-arrestin protein as a fusion protein with a reporter enzyme are provided; the plurality of cells are exposed to a GPCR agonist; and activity of reporter enzyme activity is detected. An increase in reporter enzymatic activity after exposure to the GPCR agonist indicates β-arrestin protein binding to a GPCR, thereby indicating that the cell contains a GPCR responsive to the GPCR agonist.  
           [0035]    A further aspect of the invention is a method for mapping GPCR-mediated signaling pathways. For instance, the system could be utilized to monitor interaction of c-src with β-arrestin-1 upon GPCR activation. Additionally, the system could be used to monitor protein/protein interactions involved in cross-talk between GPCR signaling pathways and other pathways such as that of the receptor tyrosine kinases or Ras/Raf. According to this aspect, a test cell is provided that expresses a GPCR or other related protein with a mutant reporter enzyme, e.g., a β-galactosidase mutant, and contains a protein from another pathway as a fusion protein with a complementing mutant reporter enzyme, e.g., another β-galactosidase mutant. Increased reporter enzymatic activity indicates protein/protein interaction.  
           [0036]    A further aspect of the invention is a method for monitoring homo- or hetero-dimerization of GPCRs upon agonist or antagonist stimulation. Increasing evidence indicates that GPCR dimerization is important for biological activity (AbdAlla, et al., “AT1-receptor heterodimers show enhanced G-protein activation and altered receptor sequestration.” Nature, 407:94-98 (2000); Bockaert, et al., “Molecular tinkering of G protein-coupled receptors: an evolutionary success.” EMBO J. 18:1723-29 (1999)). Jordan, et al., “G-protein-coupled receptor heterodimerization modulates receptor function.” Nature, 399:697-700 (1999), demonstrated that two non-functional opioid receptors, K and 6, heterodimerize to form a functional receptor. Gordon et al., “Dopamine D2 receptor dimers and receptor blocking peptides.” Bioch. Biophys. Res. Commun. 227:200-204 (1996), showed different pharmacological properties associated with the monomeric and dimeric forms of Dopamine receptor D2. The D2 receptors exist either as monomers that are selective targets for spiperone or as dimer forms that are targets for nemonapride. Herbert, et al., “A peptide derived from a β2-adrenergic receptor transmembrane domain inhibits both receptor dimerization and activation.” J.B.C. 271:16384-92 (1996), demonstrated that the agonist stimulation was found to stabilize the dimeric state of the receptor, whereas inverse agonists favored the monomeric form. Indeed, the same study showed that a peptide corresponding to the sixth transmembrane domain of the β2-adrenergic receptor inhibited both receptor dimerization and activation. Further, Angers et al., Detection of beta-2-adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer, Proc. Natl. Acad. Sci. USA, 97(7):3684-3689, discloses the use of β2-adrenergic receptor fusion proteins (i.e., β2-adrenergic receptor fused to luciferase and β2-adrenergic receptor fused to an enhanced red-shifted green fluorescent protein) to study β2-adrenergic receptor dimerization.  
           [0037]    GPCR dimerization in the context of cellular physiology and pharmacology can be monitored in accordance with the invention. For example, β-galactosidase complementation can be measured in test cells that co-express GPCR fusion proteins of β-galactosidase mutant enzymes, e.g., GPCR 1 Δα and GPCR 2 Δω (FIG. 27). According to this aspect, the interconversion between monomeric to dimeric forms of the GPCRs or orphan receptors can be measured by mutant reporter enzyme complementation. FIG. 27 illustrates a test cell co-expressing GPCR or an orphan receptor as a fusion protein with Δα form of β-galactosidase mutant (e.g., GPCR 1 Δα), and the same GPCR or orphan receptor as a fusion protein with Δω form of β-galactosidase mutant (e.g. GPCR 1 Δω). Formation of the GPCR homodimer is reflected by formation of an active enzyme, which can be measured by enzyme activity assays, such as the Gal-Screen™ assay. Similarly, hetero-dimerization between two distinct GPCRs, or two distinct orphan receptors, or between one known GPCR and one orphan receptor can be analyzed in test cells co-expressing two fusion proteins, e.g., GPCR 1 Δα and GPCR 2 Δω. The increased β-galactosidase activity indicates that the two receptors can form a heterodimer.  
           [0038]    A further aspect of the invention is a method of monitoring the interconversion between the monomeric and dimeric form of GPCRs under the influence of agonist or antagonist treatment. The test receptor(s) can be between the same GPCR or orphan receptor (homodimer), or between two distinct GPCRs or orphan receptors (heterodimer). The increased β-galactosidase activity after treatment with a compound means that the compound binds to and/or stabilizes the dimeric form of the receptor. The decreased β-galactosidase activity after treatment with a compound means that the compound binds to and/or stabilizes the monomeric form of the receptor.  
           [0039]    A further aspect of the invention is a method of screening a cell for the presence of a GPCR responsive to a GPCR agonist. A cell is provided that contains protein partners that interact downstream in the GPCR&#39;s pathway. The protein partners are expressed as fusion proteins to the mutant, complementing enzyme and are used to monitor activation of the GPCR. The cell is exposed to a GPCR agonist and then enzymatic activity of the reporter enzyme is detected. Increased reporter enzyme activity indicates that the cell contains a GPCR responsive to the agonist.  
           [0040]    The present invention involves the use of a combination of proprietary technologies (including ICAST™, Intercistronic Complementation Analysis Screening Technology, Gal-Screen™, etc.) to monitor protein/protein interactions in GPCR signaling. As disclosed in U.S. application Ser. No. 09/654,499, the method of the invention in part involves using ICAST™, which in turn involves the use of two inactive β-galactosidase mutants, each of which is fused with one of two interacting target protein pairs, such as a GPCR and an arrestin. The formation of an active β-galactosidase complex is driven by interaction of the target proteins. In this system, β-galactosidase activity can be detected using, e.g., the Gal-Screen™ assay system, wherein direct cell lysis is combined with rapid ultrasensitive chemiluminescent detection of β-galactosidase reporter enzyme. This system uses, e.g., a Galacton-Star® chemiluminescent substrate for measurement in a luminometer as a read out of GPCR activity.  
           [0041]    [0041]FIG. 23 is a schematic depicting the use of the complementation technology in the method of the present invention. FIG. 23 shows two inactive β-galactosidase mutants that become active when they are forced together by specific interactions between the fusion partners of an arrestin molecule and an activated GPCR or orphan receptor. This assay technology will be especially useful in high throughput screening assays for ligand fishing for orphan receptors, a process called de-orphaning. As illustrated in FIG. 28, a β-galactosidase fusion protein of an orphan receptor (e.g., GPCR orphan Δα) is co-expressed in the test cell with a fusion protein of β-arrestin (e.g., β-ArrΔω). When the test cell is subjected to compounds, which could be natural or synthetic, the increased β-galactosidase activity means the compound is either a natural or surrogate ligand for this GPCR. The same assay system can be used to find drug leads for the new GPCRs. The increased β-galactosidase activity in the test cell after treatment indicates the agonist activity of the compound. The decreased β-galactosidase activity in the test cell indicates antagonist activity or inverse agonist activity of the compound. In addition, the method of the invention could be used to monitor GPCR-mediated signaling pathways via other downstream signaling components such as G-proteins, GRKs or the proto-oncogene c-Src.  
           [0042]    The invention is achieved in part by using ICAST™ protein/protein interaction screening to map signaling pathways. This technology is applicable to a variety of known and unknown GPCRs with diverse functions. They include, but are not limited to, the following sub-families of GPCRs:  
           [0043]    (a) receptors that bind to amine-like ligands-Acetylcholine muscarinic receptor (M1 to M5), alpha and beta Adrenoceptors, Dopamine receptors (D1, D2, D3 and D4), Histamine receptors (H1 and H2), Octopamine receptor and Serotonin receptors (5HT1, 5HT2, 5HT4, 5HT5, 5HT6, 5HT7);  
           [0044]    (b) receptors that bind to a peptide ligand-Angiotensin receptor, Bombesin receptor, Bradykinin receptor, C-C chemokine receptors (CCR1 to CCR8, and CCR10), C-X-C type Chemokine receptors (CXC-R5), Cholecystokinin type A receptor, CCK type receptors, Endothelin receptor, Neurotesin receptor, FMLP-related receptors, Somatostatin receptors (type 1 to type 5) and Opioid receptors (type D, K, M, X);  
           [0045]    (c) receptors that bind to hormone proteins-Follic stimulating hormone receptor, Thyrotrophin receptor and Lutropin-choriogonadotropic hormone receptor;  
           [0046]    (d) receptors that bind to neurotransmitters-substance P receptor, Substance K receptor and neuropeptide Y receptor;  
           [0047]    (e) Olfactory receptors-Olfactory type 1 to type 11, Gustatory and odorant receptors;  
           [0048]    (f) Prostanoid receptors-Prostaglandin E2 (EP1 to EP4 subtypes), Prostacyclin and Thromboxane;  
           [0049]    (g) receptors that bind to metabotropic substances-Metabotropic glutamate group I to group III receptors;  
           [0050]    (h) receptors that respond to physical stimuli, such as light, or to chemical stimuli, such as taste and smell; and  
           [0051]    (i) orphan GPCRs-the natural ligand to the receptor is undefined.  
           [0052]    Use of the ICAST™ technology in combination with the invention provides many benefits to the GPCR screening process, including the ability to monitor protein interactions in any sub-cellular compartment-membrane, cytosol and nucleus; the ability to achieve a more physiologically relevant model without requiring protein overexpression; and the ability to achieve a functional assay for receptor binding allowing high information content. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0053]    [0053]FIG. 1. Cellular expression levels of β2 adrenergic receptor (β2AR) and β-arrestin-2 (βArr2) in C2 clones. Quantification of β-galactosidase (β-gal) fusion protein was performed using antibodies against β-gal and purified β-gal protein in a titration curve by a standardized ELISA assay.  
         [0054]    [0054]FIG. 1A shows expression levels of β32AR-βgalΔα clones (in expression vector pICAST ALC).  
         [0055]    [0055]FIG. 1B shows expression levels of 3Arr2-βgalΔω in expression vector pICAST OMC4 for clones 9-3,-7,-9,-10,-19 and -24, or in expression vector pICAST OMN4 for clones 12-4,-9,-16,-18,-22 and -24.  
         [0056]    [0056]FIG. 2. Receptor β2AR activation was measured by agonist-stimulated cAMP production. C2 cells expressing pICAST ALC β2AR (clone 5) or parental cells were treated with increasing concentrations of (−)isoproterenol and 0.1 mM IBMX. The quantification of cAMP level was expressed as pmol/well.  
         [0057]    [0057]FIG. 3. Interaction of activated receptor β2AR and arrestin can be measured by β-galactosidase complementation.  
         [0058]    [0058]FIG. 3A shows a time course of β-galactosidase activity in response to agonist (−)isoproterenol stimulation in C2 expressing β2AR-βgalΔα (β2AR alone, in expression vector pICAST ALC), or a pool of doubly transduced C2 co-expressing β2AR-βgalΔα and βArr2-βgalΔω (in expression vectors pICAST ALC and pICAST OMC and clones isolated from the same pod (43-1, 43-2, 43-7 and 43-8)).  
         [0059]    [0059]FIG. 3B shows a time course of β-galactosidase activity in response to agonist (−)isoproterenol stimulation in C2 cells expressing β2AR-βgalΔα alone (in expression vector pICAST ALC) and C2 clones co-expressing β2AR-βgalΔα and βArr1-βgalΔω (in expression vectors ICAST ALC and pICAST OMC).  
         [0060]    [0060]FIG. 4. Agonist dose response for interaction of β2AR and arrestin can be measured by β-galactosidase complementation.  
         [0061]    [0061]FIG. 4A shows a dose response to agonists (−)isoproterenol and procaterol in C2 cells co-expressing β2AR-βgalΔα and βArr2-βgalΔω fusion constructs.  
         [0062]    [0062]FIG. 4B shows a dose response to agonists (−)isoproterenol and procaterol in C2 cells co-expressing β2AR-βgalΔα and βArr1-βgalΔω fusion constructs.  
         [0063]    [0063]FIG. 5. Antagonist mediated inhibition of receptor activity can be measured by β-galactosidase complementation in cells co-expressing β2AR-βgalΔα and βArr-βgalΔω.  
         [0064]    [0064]FIG. 5A shows specific inhibition with adrenergic antagonists ICI-118,551 and propranolol of β-galactosidase activity in C2 clones co-expressing β2AR-βgalΔα and βArr2-βgalΔω fusion constructs after incubation with agonist (−)isoproterenol.  
         [0065]    [0065]FIG. 5B shows specific inhibition of β-galactosidase activity with adrenergic antagonists ICI-118,551 and propranolol in C2 clones co-expressing β2AR-βgalΔα and βArr1-βgalΔω fusion constructs in the presence of agonist (−)isoproterenol.  
         [0066]    [0066]FIG. 6. C2 cells expressing adenosine receptor A2a show cAMP induction in response to agonist (CGS-21680) treatment. C2 parental cells and C2 cells co-expressing A2aR-βgalΔα and βArr1-βgalΔω as a pool or as selected clones (47-2 and 47-13) were measured for agonist-induced cAMP response (pmol/well).  
         [0067]    [0067]FIG. 7. Agonist stimulated cAMP response in C2 cells co-expressing Dopamine receptor D1 (D1-βgalΔα) and β-arrestin-2 (βArr2-βgalΔω). The clone expressing βArr2-βgalΔω (Arr2 alone) was used as a negative control in the assay. Cells expressing D1-βgalΔα in addition to βArr2-βgalΔω responded agonist treatment (3-hydroxytyramine hydrochloride at 3 μM). D1(PIC2) or D1(PIC3) designate D1 in expression vector pICAST ALC2 or pICAST ALC4, respectively.  
         [0068]    [0068]FIG. 8. Variety of mammalian cell lines can be used to generate stable cells for monitoring GPCR and arrestin interactions.  
         [0069]    [0069]FIG. 8A, FIG. 8B and FIG. 8C show the examples of HEK 293, CHO and CHW cell lines co-expressing adrenergic receptor β2AR and arrestin fusion proteins of β-galactosidase mutants. The β-galactosidase activity was used to monitor agonist-induced interaction of β32AR and arrestin proteins.  
         [0070]    [0070]FIG. 9. Beta-gal complementation can be used to monitor β2 adrenergic receptor homo-dimerization.  
         [0071]    [0071]FIG. 9A shows β-galactosidase activity in HEK 293 clones co-expressing β2AR-βgalΔα and β2AR-βgalΔω.  
         [0072]    [0072]FIG. 9B shows a cAMP response to agonist (−)isoproterenol in HEK 293 clones co-expressing β2AR-βgalΔα and β2AR-βgalΔω. HEK293 parental cells were included in the assays as negative controls.  
         [0073]    [0073]FIG. 10A. pICAST ALC: Vector for expression of β-galΔα as a C-terminal fusion to the target protein. This construct contains the following features: MCS, multiple cloning site for cloning the target protein in frame with the -galΔα; GS Linker, (GGGGS)n; NeoR, neomycin resistance gene; IRES, internal ribosome entry site; ColE1ori, origin of replication for growth in  E. coli;  5′MoMuLV LTR and 3′MoMuLV LTR, viral promoter and polyadenylation signals from the Moloney Murine leukemia virus.  
         [0074]    [0074]FIG. 10B. Nucleotide sequence for pICAST ALC.  
         [0075]    [0075]FIG. 11A. pICAST ALN: Vector for expression of β-galΔα as an N-terminal fusion to the target protein. This construct contains the following features: MCS, multiple cloning site for cloning the target protein in frame with the β-galΔα; GS Linker, (GGGGS)n; NeoR, neomycin resistance gene; IRES, internal ribosome entry site; ColE1ori, origin of replication for growth in  E. coli;  5′MoMuLV LTR and 3′MoMuLV LTR, viral promoter and polyadenylation signals from the Moloney Murine leukemia virus.  
         [0076]    [0076]FIG. 11B. Nucleotide sequence for pICAST ALN.  
         [0077]    [0077]FIG. 12A. pICAST OMC: Vector for expression of β-galΔω as a C-terminal fusion to the target protein. This construct contains the following features: MCS, multiple cloning site for cloning the target protein in frame with the β-galΔω; GS Linker, (GGGGS)n; Hygro, hygromycin resistance gene; IRES, internal ribosome entry site; ColE1ori, origin of replication for growth in  E. coli;  5′MoMuLV LTR and 3′MoMuLV LTR, viral promoter and polyadenylation signals from the Moloney Murine leukemia virus.  
         [0078]    [0078]FIG. 12B. Nucleotide sequence for pICAST OMC.  
         [0079]    [0079]FIG. 13A. pICAST OMN: Vector for expression of β-galΔω as an N-terminal fusion to the target protein. This construct contains the following features: MCS, multiple cloning site for cloning the target protein in frame with the β-galΔω; GS Linker, (GGGGS)n; Hygro, hygromycin resistance gene; IRES, internal ribosome entry site; ColE1ori, origin of replication for growth in  E. coli;  5′MoMuLV LTR and 3′MoMuLV LTR, viral promoter and polyadenylation signals from the Moloney Murine leukemia virus.  
         [0080]    [0080]FIG. 13B. Nucleotide sequence for pICAST OMN.  
         [0081]    [0081]FIG. 14. pICAST ALC βArr2: Vector for expression of β-galΔα as a C-terminal fusion to β-arrestin-2. The coding sequence of human β-arrestin-2 (Genebank Accession Number: NM — 004313) was cloned in frame to β-galΔα in a pICAST ALC vector.  
         [0082]    [0082]FIG. 15. pICAST OMC βArr2: Vector for expression of β-galΔω as a C-terminal fusion to β-arrestin-2. The coding sequence of human β-arrestin-2 (Genebank Accession Number: NM — 004313) was cloned in frame to β-galΔω in a pICAST OMC vector.  
         [0083]    [0083]FIG. 16. pICAST ALC βArr1: Vector for expression of β-galΔα as a C-terminal fusion to β-arrestin-l. The coding sequence of human β-arrestin-1 (Genebank Accession Number: NM — 004041) was cloned in frame to β-galΔα in a pICAST ALC vector.  
         [0084]    [0084]FIG. 17. pICAST OMC βArr1: Vector for expression of β-galΔω as a C-terminal fusion to β-arrestin-1. The coding sequence of human β-arrestin-l (Genebank Accession Number: NM — 004041) was cloned in frame to β-galΔω in a pICAST OMC vector.  
         [0085]    [0085]FIG. 18. pICAST ALC β2AR: Vector for expression of β-galΔ(x as a C-terminal fusion to β2 Adrenergic Receptor. The coding sequence of human β2 Adrenergic Receptor (Genebank Accession Number: NM — 000024) was cloned in frame to β-galΔα in a pICAST ALC vector.  
         [0086]    [0086]FIG. 19. pICAST OMC β2AR: Vector for expression of β-galΔω as a C-terminal fusion β2 Adrenergic Receptor. The coding sequence of human β2 Adrenergic Receptor (Genebank Accession Number: NM — 000024) was cloned in frame to β-galΔω in a pICAST OMC vector.  
         [0087]    [0087]FIG. 20. pICAST ALC A2aR: Vector for expression of β-galΔα as a C-terminal fusion to Adenosine 2a Receptor. The coding sequence of human Adenosine 2a Receptor (Genebank Accession Number: NM — 000675) was cloned in frame to β-galΔα in a pICAST ALC vector.  
         [0088]    [0088]FIG. 21. pICAST OMC A2aR: Vector for expression of β-galΔω as a C-terminal fusion to Adenosine 2a Receptor. The coding sequence of human Adenosine 2a Receptor (Genebank Accession Number: NM — 000675) was cloned in frame to β-galΔω in a pICAST OMC vector.  
         [0089]    [0089]FIG. 22. pICAST ALC D1: Vector for expression of β-galΔα as a C-terminal fusion to Dopamine D 1  Receptor. The coding sequence of human Dopamine D1 Receptor (Genebank Accession Number: X58987) was cloned in frame to β-galΔα in a pICAST ALC vector.  
         [0090]    [0090]FIG. 23. A schematic depicting use of the complementation technology in the method of the invention. FIG. 23 shows two inactive mutant reporter enzymes that become active when the corresponding fusion partners, GPCR and β-arrestin interact.  
         [0091]    [0091]FIG. 24. Vector for expression of a GPCR with inserted seronine/threonine amino acid sequences as a fusion with β-galΔα. The open reading frame of a known or orphan GPCR is engineered to contain additional seronine/threonine sequences, such as SSS (seronine, seronine, seronine), within the C-terminal tail. The engineered GPCR is cloned in frame with β-galΔα in a pICAST ALC vector. The pICAST ALC vector contains the following features: MCS, multiple cloning site for cloning the target protein in frame with the β-galΔα; GS Linker, (GGGGS)n; NeoR, neomycin resistance gene; IRES, internal ribosome entry site; ColE1ori, origin of replication for growth in  E. coli;  5′MoMuLV LTR and 3′MoMuLV LTR, viral promotor and polyadenylation signals from the Moloney Murine leukemia virus.  
         [0092]    [0092]FIG. 25. Vector for expression of mutant (R170E) β-arrestin2 as a fusion with β-galΔω. The open reading frame of β-arrestin2 is engineered to contain a point mutation that converts arginine 170 to a glutamate. The mutant β-arrestin2 is cloned in frame with β-galΔω in a pICAST OMC vector. The pICAST OMC vector contains the following features: MCS, multiple cloning site for cloning the target protein in frame with the β-galΔα; GS Linker, (GGGGS)n; Hygro, hygromycin resistance gene; IRES, internal ribosome entry site; ColE1 ori, origin of replication for growth in  E. coli;  5′MoMuLV LTR and 3′MoMuLV LTR, viral promotor and polyadenylation signals from the Moloney Murine leukemia virus.  
         [0093]    [0093]FIG. 26. Phosphorylation insensitive Mutant R170E β-Arrestin2Δω binds to β2ARΔα in Response to Agonist Activation. A parental β2ARΔα C2 cell line was tranduced with the Mutant R170E β-Arrestin2Δω construct. Clonal populations co-expressing the two constructions were plated at 10,000 cells/well in 96 well plates and treated with 10 μM (−)isoproterenol, 0.3 mM ascorbic acid for the indicated time period. β-galactosidase activity was measured by addition of Tropix Gal-Screen™ assay system substrate (Applied Biosystems) and luminescence was measured using a Tropix TR717™ luminometer (Applied Biosystems). Treatments were performed in triplicate. For comparison, a clonal cell line (43-8) co-expressing β2ARΔα and wild-type β-Arrestin2Δω was also plated at 10,000 cells/well and given the same agonist treatment regimen. Minutes of (−)isoproterenol treatment is shown on the X-axis and β-galactosidase activity indicated by relative light units (RLU) is shown on the Y-axis.  
         [0094]    [0094]FIG. 27. GPCR dimerization measured by β-galactosidase complementation. A schematic depicting the utilization of the invention for monitoring GPCR homo- or hetero-dimerization. One GPCR is fused to one complement enzyme fragment, while the second GPCR is fused to the second complement enzyme fragment. Interaction of the two GPCRs is monitored by complementation of the enzyme fragments to produce an active enzyme complex (i.e., β-galactosidase activity). GPCR homo- or hetero-dimerization can be monitored in the absence or presence of ligand, agonists, inverse agonists or antagonists.  
         [0095]    [0095]FIG. 28. Ligand fishing for orphan receptors by β-galactosidase mutant complementation in ICAST™ system. A schematic depicting the utilization of the invention for ligand fishing and agonist/antagonist screening for orphan GPCRs. As an example, a test cell expressing two β-gal fission proteins, GPCR orphan- Δα and Arrestin-Δω, is subjected to treatments with samples from natural or synthetic compound libraries, or from tissue extracts, or from conditioned media of cultured cells. An increased β-gal activity after treatment indicates the activation of the orphan receptor by a ligand in the testing sample. The readout of increased β-gal activity reflects the interaction of an activated GPCR orphan receptor with a β-arrestin. Therefore, a cognate or a surrogate ligand for the testing receptor is identified. 
     
    
     DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS  
       [0096]    The present invention provides a method to interrogate GPCR function and pathways. The G-protein-coupled superfamily continues to expand rapidly as new receptors are discovered through automated sequencing of cDNA libraries or genomic DNA. It is estimated that several thousand GPCRs may exist in the human genome. Only a portion have been cloned and even fewer have been associated with ligands. The means by which these, or newly discovered orphan receptors, will be associated with their cognate ligands and physiological functions represents a major challenge to biological and biomedical research. The identification of an orphan receptor generally requires an individualized assay and a guess as to its function. The present invention involves the interrogation of GPCR function by monitoring the activation of the receptor using activation dependent protein-protein interactions between the test GPCR or orphan receptor and a β-arrestin. The specific protein-protein interactions are measured using the mutant enzyme complementation technology disclosed herein. This assay system eliminates the prerequisite guessing because it can be performed with and without prior knowledge of other signaling events. It is sensitive, rapid and easily performed and is applicable to nearly all GPCRs because the majority of these receptors desensitize by a common mechanism.  
         [0097]    The present invention provides a complete assay system for monitoring protein-protein interactions in GPCR pathways. The invention employs the complementation technology, ICAST™ (Intercistronic Complementation Analysis Screening Technology as disclosed in pending U.S. patent application Ser. No. 053,614, filed Apr. 1, 1998, the entire contents of which are incorporated herein by reference). The ICAST™ technology involves the use of two mutant forms of a reporter enzyme fused to proteins of interest. When the proteins of interest do not interact, the reporter enzyme remains inactive. When the proteins of interest do interact, the reporter enzyme mutants come together and form an active enzyme. According to an embodiment of the invention, the activity of β-galactosidase may be detected with the Gal-Screen™ assay system developed by Advanced Discovery Sciences™, which involves the use of Galacton-Star®, an ultrasensitive chemiluminescent substrate. The Gal-Screen™ assay system and the Galacton-Star® chemiluminescent substrate are disclosed in U.S. Pat. Nos. 5,851,771; 5,538,847; 5,326,882; 5,145,772; 4,978,614; and 4,931,569, the contents of which are incorporated herein by reference in their entirety. The invention provides an array of assays, including GPCR binding assays, that can be achieved directly within the cellular environment in a rapid, non-radioactive assay format. The methods of the invention are an advancement over the invention disclosed in U.S. Pat. Nos. 5,891,646 and 6,110,693 and the method disclosed in Angers et al., supra., which rely on microscopic imaging or spectrometry of GPCR components as fusion with Green-fluorescent-protein. The imaging technique disclosed in U.S. Pat. Nos. 5,891,646 and 6,110,693 and spectrometry-based technique in Angers et al. are limited by low-throughput and lack of thorough quantification.  
         [0098]    The assay system of the invention combined with Advanced Discovery Sciences™ technologies provide highly sensitive cell-based methods for interrogating GPCR pathways which are amenable to high-throughput screening (HTS). Among some of the technologies developed by Advanced Discovery Sciences™ that may be used with the present invention are the Gal-Screen™ assay system (discussed above) and the cAMP-Screen™ immunoassay system. The cAMP-Screen™ immunoassay system provides ultrasensitive determination of cAMP levels in cell lysates. The cAMP-Screen™ assay utilizes the high-sensitivity chemiluminescent alkaline phosphatase (AP) substrate CSPD® (disodium 3-(4-methoxyspiro {1,2-dioxetane-3,2′-(5′-chloro) tricyclo 3.3.1.1. 3,7 } decan-4-yl phenyl phosphate) with Sapphire-II™ luminescence enhancer.  
         [0099]    Unlike yeast-based-two-hybrid assays used to monitor protein/protein interactions in high-throughput assays, the present invention (1) is applicable to a variety of cells including mammalian cells, plant cells, protozoa cells such as  E. coli  and cells of invertebrate origin such as yeast, slime mold (Dictyostelium) and insects; (2) detects interactions at the membrane at the site of the receptor target or in the cytosol at the site of downstream target proteins rather than a limited cellular localization, i.e., nucleus; and (3) does not rely on indirect read-outs such as transcriptional activation. The present invention thus provides assays with greater physiological relevance and fewer false positives.  
         [0100]    The present inventors have developed modifications to the embodiment disclosed in U.S. patent application Ser. No. 053,614 described above in order to enhance the sensitivity of the inventive GPCR assay. According to an embodiment, the invention incorporates the use of serine/threonine clusters to enhance and prolong the interaction of GPCR with arrestin in order to make the detection more robust. The clusters can be utilized for orphan receptors or known GPCRs, which do not have this sequence motif. By adding this sequence to the C-terminal tail of the receptor, the activation of the receptor can be detected more readily by readouts of arrestin binding to GPCR, i.e., β-galactosidase complementation from fusion proteins of target proteins with β-galactosidase mutants.  
         [0101]    According to another embodiment, the invention incorporates the use of arrestin point mutations to bypass the requirement of phosphorylation, by the action of specific GRK, on the C-terminal tail or intracellular loops of GPCR upon activation. The applications include i) wherein the cognate GRK for a particular GPCR or orphan receptor is unknown; and ii) wherein the specific GRK for the receptor of interest (or under test) may not be present or may have low activity in the host cell that is used for receptor activation assay.  
         [0102]    According to another embodiment, the invention incorporates the use of a super arrestin to increase the binding efficiency of arrestin to an activated GPCR and to stabilize the GPCR/arrestin complex during GPCR desensitization. This application can be used to increase the robustness of ICAST/GPCR applications in cases where the GPCR is normally resensitized rapidly post desensitization.  
         [0103]    Each of these methodologies is discussed below.  
         [0104]    The invention will now be described in the following non-limiting examples.  
       EXAMPLE  
       [0105]    According to an embodiment of the invention, GPCR activation is measured through monitoring the binding of arrestin to ligand-activated GPCR. In this assay system, a GPCR, e.g., β-adrenergic receptor (β2AR), and an arrestin, e.g., β-arrestin, are co-expressed in the same cell as fusion proteins with mutant forms of a reporter enzyme, e.g., β-galactosidase (β-gal). As illustrated in FIG. 23, the β2AR is expressed as a fusion protein with Δω form of β-gal mutant (β2ARΔα) and the β-arrestin as a fusion protein with the Aco form of β-gal mutant (β-ArrΔω). The two fusion proteins, which at first exist in a resting (or un-stimulated) cell in separate compartments, i.e., the membrane for GPCR and the cytosol for arrestin, cannot form an active β-galactosidase enzyme. When such a cell is treated with an agonist or a ligand, the ligand-occupied and activated receptor becomes a high affinity binding site for arrestin. The interaction between an activated GPCR, β2ARΔα, and arrestin, β-ArrΔω, drives the β-gal mutant complementation. The enzyme activity can be measured by using an enzyme substrate, which upon cleavage releases a product measurable by colorimetry, fluorescence, or chemiluminescence (e.g., the Gal-Screen™ assay system).  
         [0106]    Experiment Protocol  
         [0107]    1. In the first step, the expression vectors for β2ARΔα and β3Arr2Δω were engineered in selectable retroviral vectors pICAST ALC, as described in FIG. 18 and pICAST OMC, as described in FIG. 15.  
         [0108]    2. In the second step, the two expression constructs were transduced into either C2C12 myoblast cells, or other mammalian cell lines, such as COS-7, CHO, A431, HEK 293, and CHW. Following selection with antibiotic drugs, stable clones expressing both fusion proteins at appropriate levels were selected.  
         [0109]    3. In the last step, the cells expressing both 2ARΔα and βArr2Δω were tested for response by agonist/ligand stimulated β-galactosidase activity. Triplicate samples of cells were plated at 10,000 cells in 100 microliter volume into a well of 96-well culture plate. Cells were cultured for 24 hours before assay. For agonist assay (FIGS. 3 and 4), cells were treated with variable concentrations of agonist, for example, (−)isoproterenol, procaterol, dobutamine, terbutaline or L-L-phenylephrine for 60 min at 37° C. The induced β-galactosidase activity was measured by addition of Tropix Gal-Screen™ assay system substrate (Applied Biosystems) and luminescence measured in a Tropix TR717™ luminometer (Applied Biosystems). For antagonist assay (FIG. 5), cells were pre-incubated for 10 min in fresh medium without serum in the presence of ICI-118,551 or propranolol followed by addition of 10 micro molar (−)isoproterenol.  
         [0110]    Serine/Threonine Cluster Strategy  
         [0111]    Background  
         [0112]    Based on structure-function relationship studies on β-arrestins, a large region within the amino-terminal half of β-arrestins (termed the activation-recognition domain) recognizes the agonist-activated state of GPCRs. This region of β-arrestin also contains a small positively charged domain (approximately 20 amino acids with net charge +7) called the phosphorylation-recognition domain, which appears to interact with the GRK-phosphorylated carboxyl termini of GPCRs.  
         [0113]    GPCRs can be divided into two classes based on their affinities for β-arresting. Oakley et al., “Association of β-Arrestin with G Protein-Coupled Receptors During Clathrin-Mediated Endocytosis Dictates the Profile of Receptor Resensitization.” J. Biol. Chem., 274(45):32248-32257 (1999). The molecular determinants underlying this classification appear to reside in specific serine or threonine residues located in the carboxyl-terminal tail of the receptor. The receptor class that contains serine/threonine clusters (defined as serine or threonine residues occupying three consecutive or three out of four positions) in the carboxyl-termini binds β-arrestin with high affinity upon activation and phosphorylation and remains bound with β-arrestin even after receptor internalization, whereas the receptor class that contains only scattered serine and threonine residues in the carboxy-terminal tail binds β-arrestins with less affinity and disassociates from the β-arrestin upon internalization. Several known GPCRs, such as vasopressin V2 receptor (Oakley, et al.), neurotensin receptor 1 and angiotensin II receptor type 1A (Zhang, et al., “Cellular Trafficking of G Protein-Coupled Receptor/β-Arrestin Endocytic Complexes.” J. Biol. Chem., 274(16):10999-11006 (1999)), which possess one or more of such serine/threonine clusters in their carboxyl-termini, were shown to bind β-arrestins with high affinity.  
       EXAMPLE  
       [0114]    According to an embodiment of the invention, a serine/threonine cluster strategy is used to facilitate screening assays for orphan receptors that do not possess this structural motif of their own. The orphan receptors are easily classified by sequence alignment. Orphan receptors lacking the serine/threonine clusters are each cloned into an expression vector that is modified to introduce one or more serine/threonine cluster(s) to the carboxyl-terminal tail of the receptor (FIG. 24). The serine/threonine clusters enhance the receptor activation dependent interaction between the activated and phosphorylated receptor (negative charges) and β-arrestin (positive charges in the phosphorylation-recognition domain) through strong ionic interactions, thus prolonging interaction between the receptor and arrestin. The modification of the orphan receptor tail thus makes detection of receptor activation more robust.  
         [0115]    Experiment Protocol  
         [0116]    1. In a first step, the open-reading-frame (ORF) of an orphan receptor, which lacks the serine/threonine clusters, is cloned into a modified expression vector such as pICAST ALC described in FIG. 10A. The modified pICAST ALC includes coding sequences for one or more sets of serine/threonine clusters (for example, SSS or SST) located downstream from the insert of the ORF of an orphan receptor (FIG. 24).  
         [0117]    2. In a second step, chimeric orphan receptor, ORF orphan R -(SSS) n -Δα, is co-expressed in a mammalian cell with a β-arrestin chimera, such as βArr2Δω described in FIG. 15.  
         [0118]    3. In a third step, the cell is treated with an agonist or a ligand and the activated receptor with phosphorylated serine cluster(s) binds the β-arrestin with high affinity producing strong signals in readouts of β-gal complementation.  
         [0119]    This assay, which provides a means for sensitive measurement of functional activation of the orphan receptors, can be used to screen for natural or surrogate ligands for orphan receptors, a process called de-orphaning or target discovery for new GPCRs (FIG. 28). Furthermore, this assay is also useful in screening for potential agonists and antagonists for lead discovery of GPCRs.  
         [0120]    Enhanced Binding of Arrestin in the Presence and in the Absence of GPCR Phosphorylation  
         [0121]    Background  
         [0122]    Six different classes of G-protein coupled receptor kinases (GRKs) have been identified and each of these has been reported to be expressed as multiple splice variants. Krupnick et al., “The role of receptor kinases and arrestins in G protein-coupled receptor regulation.” Ann. Rev. Pharmacol. Toxicol., 38:289-319 (1998). Although many cell lines express a variety of GRKs, the specific GRK required for phosphorylation of a given GPCR may not always be present in the cell line used for recombinant GPCR and arrestin expression. This is particularly an issue for applications using orphan receptors, in which case the cognate GRK will likely be unknown. In other cases, the cell line used for recombinant expression work may have the required GRK, but may express the GRK at low levels. In order to bypass such caveats, genetically modified arrestins that bind specifically to activated GPCRs, but without the requirement of GRK phosphorylation are employed.  
         [0123]    Mutagenesis studies on arrestins demonstrate that point mutations in the phosphorylation-recognition domain, particularly mutations converting Arg175 (of visual arrestin) to an oppositely charged residue such as glutamate (R175E mutation), result in an arrestin which specifically binds to activated GPCRs, but does so without the requirement for phosphorylation.  
         [0124]    Numerous observations have led to the hypothesis that arrestin exists in an inactive state that has a low affinity for GPCRs. Once a GPCR is both activated and phosphorylated, the phosphorylated region of the GPCR C-terminus interacts with the phosphorylation-recognition domain of arrestin causing the arrestin to change conformations allowing the activation-recognition region to be exposed for binding to the activated/phosphorylated receptor. Vishnivetskiv et al., “How does arrestin respond to the phosphorylated state of rhodopsin?” J. Biol. Chem., 274(17):11451-11454 (1999); Gurevich et al., “Arrestin interactions with G protein-coupled receptors. Direct binding studies of wild-type and mutant arrestins with rhodopsin, beta 2-adrenergic and m2 muscarinic cholinergic receptors.” J. Biol. Chem., 270(2):720-731, (1995); Gurevich et al., “Mechanism of phosphorylation-recognition by visual arrestin and the transition of arrestin into a high affinity binding site.” Mol. Pharnacol., 51(1):161-169 (1997); Kovoor et al., “Targeted construction of phosphorylation-independent beta-arrestin mutants with constitutive activity in cells.” J. Biol. Chem., 274(11):6831-6834 (1999). In summary, binding studies of single mutation, double mutation, deletion, and chimerical arrestins with inactive, inactive and phosphorylated, activated but not phosphorylated, or activated and phosphorylated visual or non-visual GPCRs all support this model.  
       EXAMPLE  
       [0125]    A phosphorylation insensitive mutant of arrestin fused to mutant reporter protein can be produced that will bind to activated GPCRs in a phosphorylation independent manner. As proof of concept, a point mutation for β-arrestin2, R170E β-arrestin2, has been produced and its interaction with β2AR has been analyzed in accordance with the invention.  
         [0126]    Experimental Protocol:  
         [0127]    1) In the first step, β-arrestin2 was mutated such that Arg170 was converted to Glu. This mutation is equivalent to the R175E mutation of visual arrestin. The mutant β-arrestin2 open reading frame was cloned in frame with Δω-β-galactosidase in the pICAST OMC expression vector to produce a modified expression vector R170E β-arrestin2 (FIG. 25).  
         [0128]    2) In the second step, the R170E β-arrestin2 expression construct was transduced into a C2C12 myoblast cell line that had been engineered to express β2AR as a fusion to Δα-β-galactosidase as described in FIG. 18 of U.S. application Ser. No. 09/654,499. Following selection with antibiotic drugs, a population of clones expressing both fusion proteins was obtained.  
         [0129]    3) In the last step, this population of cells expressing both R170E β-arrestin2Δω and β2Δα were tested for response by agonist/ligand stimulated β-galactosidase activity as demonstrated in FIG. 26. The C2C12 clone 43-8 co-expressing β2ARΔα and wild-type 1β-arrestin2Δω (FIG. 26) was used as reference control. Triplicate samples of cells were plated at 10,000 cells in 100 microliter volume into wells of a 96-well culture plate. Cells were cultured for 24 hours before assay. For agonist assay as in FIG. 26, cells were treated with 10 μm (−)isoproterenol stabilized with 0.3 mM ascorbic acid 37° C. for 0, 5, 10, 15, 30, 45 or 60 minutes. The induced β-galactosidase activity was measured by addition of Tropix Gal-Screen™ assay system substrate (Applied Biosystems) and luminescence measured in a Tropix TR717™ luminometer (Applied Biosystems). As shown in FIG. 26, the mutant arrestin interacts with β2AR in an agonist-dependent manner and was comparable with that of wild-type arrestin.  
         [0130]    4) To expand the application of phosphorylation-insensitive arrestin, cell lines such as C2C12, CHO or HEK 293, are developed that express the R170E β-arrestin2Δω construction. These cell lines can be used to transduce orphan or known GPCRs as fusions with Δα-β-galactosidase in order to develop cell lines for agonist and antagonist screening and  
         [0131]    Development of Super Arrestins:  
         [0132]    Background  
         [0133]    Attenuation of GPCR signaling by the arrestin pathway serves to ensure that a cell or organism does not over-react to a stimulus. At the same time, the arrestin pathway often serves to recycle the GPCR such that it can be temporarily inactivated but then quickly resensitized to allow for sensitivity to new stimuli. The down-regulation process involves phosphorylation of the receptor, binding to arrestin and endocytosis. Following endocytosis of the desensitized receptor, the receptor is either degraded in lysosomes or resensitized and sent back to the membrane. Resensitization involves release of arrestin from the receptor, dephosphorylation and cycling back to the membrane. The actual route a GPCR follows upon activation depends on its biological function and the needs of the organism. Because of these diverse pathways that may be required of the down-regulation pathway, arrestin affinities for activated GPCRs vary from receptor to receptor. It would thus be very advantageous to engineer super arrestins that have a higher affinity and avidity for activated GPCRs than what nature has provided.  
         [0134]    Although mutational, deletion and chimerical studies of arrestins have focused on understanding regulatory switches in the molecule that respond to GPCR phosphorylation states, several of these altered recombinant forms of arrestin have resulted in molecules with enhanced binding to activated, phosphorylated GPCRs. Conversion of Arg175 to histidine, tyrosine, phenylalanine or threonine results in significantly higher amounts of binding to phosphorylated, activated rhodopsin than wild-type arrestin or R175E arrestin, although these mutations result in less binding to activated, non-phosphorylated receptor. Gurevich et al. (1997). In addition, conversion of Valine 170 to alanine increased the constitutive affect of the R175E mutation, but also nearly doubled the amount of interaction of wild-type arrestin with activated, phosphorylated rhodopsin. Gurevich et al. (1997).  
         [0135]    Truncation of β-arrestin1 at amino acid 382 has been reported to enhance binding of both R169E (equivalent to arrestin R175E) and wild-type β-arrestin1 to activated or activated and phosphorylated receptor, respectively. Kovoor et al. Chimerical arrestins in which functional regions of visual arrestin were swapped with those of β-arrestin1 have been reported to be altered in binding affinity to activated, phosphorylated GPCRs. Gurevich et al. (1995). Several of these chimeras, such as β-arrestin1 containing the visual arrestin extreme N-terminus, show increased specific binding to phosphorylated activated GPCRs compared to wild-type β-arrestin1 (Gurevich et al. (1995)). Modifications that enhance arrestin affinity for the activated GPCR such as described above, whether phosphorylated or non-phosphorylated, could also enhance signal to noise of β-galactosidase activity since the arrestin/GPCR complex is stabilized and/or more long-lived. The use of mutant arrestins with higher activated-GPCR affinity would improve the inventive technology for GPCR targets, without compromising receptor/ligand biology.  
         [0136]    In addition, this “super arrestin” approach can be combined with the use of arrestin point mutations to provide a stronger signal to noise with or without GRK requirements.  
       EXAMPLE  
       [0137]    An arrestin mutant fused to mutant reporter protein can be produced to enhance binding of the arrestin to an activated GPCR to enhance sensitivity of detection.  
         [0138]    Experiment Protocol  
         [0139]    1) In the first step, mutant β-arrestin2 constructions will be generated which include R170E/T/Y/or H, V165A, substitution of a.a. 1-43 with a.a. 1-47 of visual arrestin, or deletion of the C-terminal and combinations of these alterations. The mutant β-arrestin2 open reading frames will be cloned in frame with Δω-β-galactosidase in the pICAST OMC expression vector similar to cloning of the R170E β-arrestin2 mutation shown in FIG. 25.  
         [0140]    2) In the second step, mutant expression constructs will be transduced into a C2C12 myoblast cell line that has been engineered to express β2AR as a fusion to Δα-β-galactosidase. Following selection with antibiotic drugs, a population of clones expressing both fusion proteins will be obtained. Wild type and R117E β-arrestin2 constructions will be transduced to generate control, reference clonal populations.  
         [0141]    3) In the third step, populations of cells expressing both β-arrestin2Δω (mutant or wild type) and β2ARΔα will be tested for response by agonist/ligand stimulated β-galactosidase activity.  
         [0142]    4) In the next step, mutant (super) β-arrestin2Δω constructions that show a significantly higher signal to noise ratio in the agonist assay compared with wild-type β-arrestin2Δω will be chosen. These constructions will be used to develop stable cell lines expressing the “super” β-arrestin2Δω that can be used for transducing in known or orphan GPCRs. Use of a super β-arrestin2Δω could increase the signal to noise of ICAST/GPCR applications allowing improved screening capabilities for lead and ligand discovery.  
         [0143]    Super Arrestin is used to increase the binding efficiency of arrestin to an activated GPCR and to stabilize the GPCR/arrestin complex during GPCR desensitization. This application can be used to increase the robustness of ICAST/GPCR applications in cases where the GPCR is normally resensitized rapidly post desensitization.  
         [0144]    The assays of this invention, and their application and preparation have been described both generically, and by specific example. The examples are not intended as limiting. Other substituent identities, characteristics and assays will occur to those of ordinary skill in the art, without the exercise of inventive faculty. Such modifications remain within the scope of the invention, unless excluded by the express recitation of the claims advanced below.  
     
       
       
         1 
         
           
             9  
           
           
             1  
             6700  
             DNA  
             Unknown  
             
               pICAST ALC.  
             
           
            1 

ctgcagcctg aatatgggcc aaacaggata tctgtggtaa gcagttcctg ccccggctca     60 

gggccaagaa cagatggaac agctgaatat gggccaaaca ggatatctgt ggtaagcagt    120 

tcctgccccg gctcagggcc aagaacagat ggtccccaga tgcggtccag ccctcagcag    180 

tttctagaga accatcagat gtttccaggg tgccccaagg acctgaaatg accctgtgcc    240 

ttatttgaac taaccaatca gttcgcttct cgcttctgtt cgcgcgcttc tgctccccga    300 

gctcaataaa agagcccaca acccctcact cggggcgcca gtcctccgat tgactgagtc    360 

gcccgggtac ccgtgtatcc aataaaccct cttgcagttg catccgactt gtggtctcgc    420 

tgttccttgg gagggtctcc tctgagtgat tgactacccg tcagcggggg tctttcattt    480 

gggggctcgt ccgggatcgg gagacccctg cccagggacc accgacccac caccgggagg    540 

caagctggcc agcaacttat ctgtgtctgt ccgattgtct agtgtctatg actgatttta    600 

tgcgcctgcg tcggtactag ttagctaact agctctgtat ctggcggacc cgtggtggaa    660 

ctgacgagtt ctgaacaccc ggccgcaacc ctgggagacg tcccagggac tttgggggcc    720 

gtttttgtgg cccgacctga ggaagggagt cgatgtggaa tccgaccccg tcaggatatg    780 

tggttctggt aggagacgag aacctaaaac agttcccgcc tccgtctgaa tttttgcttt    840 

cggtttggaa ccgaagccgc gcgtcttgtc tgctgcagca tcgttctgtg ttgtctctgt    900 

ccgactgtgt ttctgtattt gtctgaaaat tagggccaga ctgttaccac tcccttaagt    960 

ttgaccttag gtaactggaa agatgtcgag cggctcgctc acaaccagtc ggtagatgtc   1020 

aagaagagac gttgggttac cttctgctct gcagaatggc caacctttaa cgtcggatgg   1080 

ccgcgagacg gcacctttaa ccgagacctc atcacccagg ttaagatcaa ggtcttttca   1140 

cctggcccgc atggacaccc agaccaggtc ccctacatcg tgacctggga agccttggct   1200 

tttgaccccc ctccctgggt caagcccttt gtacacccta agcctccgcc tcctcttcct   1260 

ccatccgccc cgtctctccc ccttgaacct cctcgttcga ccccgcctcg atcctccctt   1320 

tatccagccc tcactccttc tctaggcgcc ggccgctcta gcccattaat acgactcact   1380 

atagggcgat tcgaatcagg ccttggcgcg ccggatcctt aattaagcgc aattgggagg   1440 

tggcggtagc ctcgag atg ggc gtg att acg gat tca ctg gcc gtc gtg gcc   1492 
                  Met Gly Val Ile Thr Asp Ser Leu Ala Val Val Ala 
                  1               5                   10 

cgc acc gat cgc cct tcc caa cag tta cgc agc ctg aat ggc gaa tgg     1540 
Arg Thr Asp Arg Pro Ser Gln Gln Leu Arg Ser Leu Asn Gly Glu Trp 
        15                  20                  25 

cgc ttt gcc tgg ttt ccg gca cca gaa gcg gtg ccg gaa agc tgg ctg     1588 
Arg Phe Ala Trp Phe Pro Ala Pro Glu Ala Val Pro Glu Ser Trp Leu 
    30                  35                  40 

gag tgc gat ctt cct gag gcc gat act gtc gtc gtc ccc tca aac tgg     1636 
Glu Cys Asp Leu Pro Glu Ala Asp Thr Val Val Val Pro Ser Asn Trp 
45                  50                  55                  60 

cag atg cac ggt tac gat gcg ccc atc tac acc aac gtg acc tat ccc     1684 
Gln Met His Gly Tyr Asp Ala Pro Ile Tyr Thr Asn Val Thr Tyr Pro 
                65                  70                  75 

att acg gtc aat ccg ccg ttt gtt ccc acg gag aat ccg acg ggt tgt     1732 
Ile Thr Val Asn Pro Pro Phe Val Pro Thr Glu Asn Pro Thr Gly Cys 
            80                  85                  90 

tac tcg ctc aca ttt aat gtt gat gaa agc tgg cta cag gaa ggc cag     1780 
Tyr Ser Leu Thr Phe Asn Val Asp Glu Ser Trp Leu Gln Glu Gly Gln 
        95                  100                 105 

acg cga att att ttt gat ggc gtt aac tcg gcg ttt cat ctg tgg tgc     1828 
Thr Arg Ile Ile Phe Asp Gly Val Asn Ser Ala Phe His Leu Trp Cys 
    110                 115                 120 

aac ggg cgc tgg gtc ggt tac ggc cag gac agt cgt ttg ccg tct gaa     1876 
Asn Gly Arg Trp Val Gly Tyr Gly Gln Asp Ser Arg Leu Pro Ser Glu 
125                 130                 135                 140 

ttt gac ctg agc gca ttt tta cgc gcc gga gaa aac cgc ctc gcg gtg     1924 
Phe Asp Leu Ser Ala Phe Leu Arg Ala Gly Glu Asn Arg Leu Ala Val 
                145                 150                 155 

atg gtg ctg cgc tgg agt gac ggc agt tat ctg gaa gat cag gat atg     1972 
Met Val Leu Arg Trp Ser Asp Gly Ser Tyr Leu Glu Asp Gln Asp Met 
            160                 165                 170 

tgg cgg atg agc ggc att ttc cgt gac gtc tcg ttg ctg cat aaa ccg     2020 
Trp Arg Met Ser Gly Ile Phe Arg Asp Val Ser Leu Leu His Lys Pro 
        175                 180                 185 

act aca caa atc agc gat ttc cat gtt gcc act cgc ttt aat gat gat     2068 
Thr Thr Gln Ile Ser Asp Phe His Val Ala Thr Arg Phe Asn Asp Asp 
    190                 195                 200 

ttc agc cgc gct gta ctg gag gct gaa gtt cag atg tgc ggc gag ttg     2116 
Phe Ser Arg Ala Val Leu Glu Ala Glu Val Gln Met Cys Gly Glu Leu 
205                 210                 215                 220 

cgt gac tac cta cgg gta aca gtt tct tta tgg cag ggt gaa acg cag     2164 
Arg Asp Tyr Leu Arg Val Thr Val Ser Leu Trp Gln Gly Glu Thr Gln 
                225                 230                 235 

gtc gcc agc ggc acc gcg cct ttc ggc ggt gaa att atc gat gag cgt     2212 
Val Ala Ser Gly Thr Ala Pro Phe Gly Gly Glu Ile Ile Asp Glu Arg 
            240                 245                 250 

ggt ggt tat gcc gat cgc gtc aca cta cgt ctg aac gtc gaa aac ccg     2260 
Gly Gly Tyr Ala Asp Arg Val Thr Leu Arg Leu Asn Val Glu Asn Pro 
        255                 260                 265 

aaa ctg tgg agc gcc gaa atc ccg aat ctc tat cgt gcg gtg gtt gaa     2308 
Lys Leu Trp Ser Ala Glu Ile Pro Asn Leu Tyr Arg Ala Val Val Glu 
    270                 275                 280 

ctg cac acc gcc gac ggc acg ctg att gaa gca gaa gcc tgc gat gtc     2356 
Leu His Thr Ala Asp Gly Thr Leu Ile Glu Ala Glu Ala Cys Asp Val 
285                 290                 295                 300 

ggt ttc cgc gag gtg cgg att gaa aat ggt ctg ctg ctg ctg aac ggc     2404 
Gly Phe Arg Glu Val Arg Ile Glu Asn Gly Leu Leu Leu Leu Asn Gly 
                305                 310                 315 

aag ccg ttg ctg att cga ggc gtt aac cgt cac gag cat cat cct ctg     2452 
Lys Pro Leu Leu Ile Arg Gly Val Asn Arg His Glu His His Pro Leu 
            320                 325                 330 

cat ggt cag gtc atg gat gag cag acg atg gtg cag gat atc ctg ctg     2500 
His Gly Gln Val Met Asp Glu Gln Thr Met Val Gln Asp Ile Leu Leu 
        335                 340                 345 

atg aag cag aac aac ttt aac gcc gtg cgc tgt tcg cat tat ccg aac     2548 
Met Lys Gln Asn Asn Phe Asn Ala Val Arg Cys Ser His Tyr Pro Asn 
    350                 355                 360 

cat ccg ctg tgg tac acg ctg tgc gac cgc tac ggc ctg tat gtg gtg     2596 
His Pro Leu Trp Tyr Thr Leu Cys Asp Arg Tyr Gly Leu Tyr Val Val 
365                 370                 375                 380 

gat gaa gcc aat att gaa acc cac ggc atg gtg cca atg aat cgt ctg     2644 
Asp Glu Ala Asn Ile Glu Thr His Gly Met Val Pro Met Asn Arg Leu 
                385                 390                 395 

acc gat gat ccg cgc tgg cta ccg gcg atg agc gaa cgc gta acg cga     2692 
Thr Asp Asp Pro Arg Trp Leu Pro Ala Met Ser Glu Arg Val Thr Arg 
            400                 405                 410 

atg gtg cag cgc gat cgt aat cac ccg agt gtg atc atc tgg tcg ctg     2740 
Met Val Gln Arg Asp Arg Asn His Pro Ser Val Ile Ile Trp Ser Leu 
        415                 420                 425 

ggg aat gaa tca ggc cac ggc gct aat cac gac gcg ctg tat cgc tgg     2788 
Gly Asn Glu Ser Gly His Gly Ala Asn His Asp Ala Leu Tyr Arg Trp 
    430                 435                 440 

atc aaa tct gtc gat cct tcc cgc ccg gtg cag tat gaa ggc ggc gga     2836 
Ile Lys Ser Val Asp Pro Ser Arg Pro Val Gln Tyr Glu Gly Gly Gly 
445                 450                 455                 460 

gcc gac acc acg gcc acc gat att att tgc ccg atg tac gcg cgc gtg     2884 
Ala Asp Thr Thr Ala Thr Asp Ile Ile Cys Pro Met Tyr Ala Arg Val 
                465                 470                 475 

gat gaa gac cag ccc ttc ccg gct gtg ccg aaa tgg tcc atc aaa aaa     2932 
Asp Glu Asp Gln Pro Phe Pro Ala Val Pro Lys Trp Ser Ile Lys Lys 
            480                 485                 490 

tgg ctt tcg cta cct gga gag acg cgc ccg ctg atc ctt tgc gaa tac     2980 
Trp Leu Ser Leu Pro Gly Glu Thr Arg Pro Leu Ile Leu Cys Glu Tyr 
        495                 500                 505 

gcc cac gcg atg ggt aac agt ctt ggc ggt ttc gct aaa tac tgg cag     3028 
Ala His Ala Met Gly Asn Ser Leu Gly Gly Phe Ala Lys Tyr Trp Gln 
    510                 515                 520 

gcg ttt cgt cag tat ccc cgt tta cag ggc ggc ttc gtc tgg gac tgg     3076 
Ala Phe Arg Gln Tyr Pro Arg Leu Gln Gly Gly Phe Val Trp Asp Trp 
525                 530                 535                 540 

gtg gat cag tcg ctg att aaa tat gat gaa aac ggc aac ccg tgg tcg     3124 
Val Asp Gln Ser Leu Ile Lys Tyr Asp Glu Asn Gly Asn Pro Trp Ser 
                545                 550                 555 

gct tac ggc ggt gat ttt ggc gat acg ccg aac gat cgc cag ttc tgt     3172 
Ala Tyr Gly Gly Asp Phe Gly Asp Thr Pro Asn Asp Arg Gln Phe Cys 
            560                 565                 570 

atg aac ggt ctg gtc ttt gcc gac cgc acg ccg cat cca gcg ctg acg     3220 
Met Asn Gly Leu Val Phe Ala Asp Arg Thr Pro His Pro Ala Leu Thr 
        575                 580                 585 

gaa gca aaa cac cag cag cag ttt ttc cag ttc cgt tta tcc ggg caa     3268 
Glu Ala Lys His Gln Gln Gln Phe Phe Gln Phe Arg Leu Ser Gly Gln 
    590                 595                 600 

acc atc gaa gtg acc agc gaa tac ctg ttc cgt cat agc gat aac gag     3316 
Thr Ile Glu Val Thr Ser Glu Tyr Leu Phe Arg His Ser Asp Asn Glu 
605                 610                 615                 620 

ctc ctg cac tgg atg gtg gcg ctg gat ggt aag ccg ctg gca agc ggt     3364 
Leu Leu His Trp Met Val Ala Leu Asp Gly Lys Pro Leu Ala Ser Gly 
                625                 630                 635 

gaa gtg cct ctg gat gtc gct cca caa ggt aaa cag ttg att gaa ctg     3412 
Glu Val Pro Leu Asp Val Ala Pro Gln Gly Lys Gln Leu Ile Glu Leu 
            640                 645                 650 

cct gaa cta ccg cag ccg gag agc gcc ggg caa ctc tgg ctc aca gta     3460 
Pro Glu Leu Pro Gln Pro Glu Ser Ala Gly Gln Leu Trp Leu Thr Val 
        655                 660                 665 

cgc gta gtg caa ccg aac gcg acc gca tgg tca gaa gcc ggg cac atc     3508 
Arg Val Val Gln Pro Asn Ala Thr Ala Trp Ser Glu Ala Gly His Ile 
    670                 675                 680 

agc gcc tgg cag cag tgg cgt ctg gcg gaa aac ctc agt gtg acg ctc     3556 
Ser Ala Trp Gln Gln Trp Arg Leu Ala Glu Asn Leu Ser Val Thr Leu 
685                 690                 695                 700 

ccc gcc gcg tcc cac gcc atc ccg cat ctg acc acc agc gaa atg gat     3604 
Pro Ala Ala Ser His Ala Ile Pro His Leu Thr Thr Ser Glu Met Asp 
                705                 710                 715 

ttt tgc atc gag ctg ggt aat aag cgt tgg caa ttt aac cgc cag tca     3652 
Phe Cys Ile Glu Leu Gly Asn Lys Arg Trp Gln Phe Asn Arg Gln Ser 
            720                 725                 730 

ggc ttt ctt tca cag atg tgg att ggc gat aaa aaa caa ctg ctg acg     3700 
Gly Phe Leu Ser Gln Met Trp Ile Gly Asp Lys Lys Gln Leu Leu Thr 
        735                 740                 745 

ccg ctg cgc gat cag ttc acc cgt gca ccg ctg gat aac gac att ggc     3748 
Pro Leu Arg Asp Gln Phe Thr Arg Ala Pro Leu Asp Asn Asp Ile Gly 
    750                 755                 760 

gta agt gaa gcg acc cgc att gac cct aac gcc tgg gtc gaa cgc tgg     3796 
Val Ser Glu Ala Thr Arg Ile Asp Pro Asn Ala Trp Val Glu Arg Trp 
765                 770                 775                 780 

aag gcg gcg ggc cat tac cag gcc gaa gca gcg ttg ttg cag tgc acg     3844 
Lys Ala Ala Gly His Tyr Gln Ala Glu Ala Ala Leu Leu Gln Cys Thr 
                785                 790                 795 

gca gat aca ctt gct gat gcg gtg ctg att acg acc gct cac gcg tgg     3892 
Ala Asp Thr Leu Ala Asp Ala Val Leu Ile Thr Thr Ala His Ala Trp 
            800                 805                 810 

cag cat cag ggg aaa acc tta ttt atc agc cgg aaa acc tac cgg att     3940 
Gln His Gln Gly Lys Thr Leu Phe Ile Ser Arg Lys Thr Tyr Arg Ile 
        815                 820                 825 

gat ggt agt ggt caa atg gcg att acc gtt gat gtt gaa gtg gcg agc     3988 
Asp Gly Ser Gly Gln Met Ala Ile Thr Val Asp Val Glu Val Ala Ser 
    830                 835                 840 

gat aca ccg cat ccg gcg cgg att ggc ctg aac tgc cag ctg gcg cag     4036 
Asp Thr Pro His Pro Ala Arg Ile Gly Leu Asn Cys Gln Leu Ala Gln 
845                 850                 855                 860 

gta gca gag cgg gta aac tgg ctc gga tta ggg ccg caa gaa aac tat     4084 
Val Ala Glu Arg Val Asn Trp Leu Gly Leu Gly Pro Gln Glu Asn Tyr 
                865                 870                 875 

ccc gac cgc ctt act gcc gcc tgt ttt gac cgc tgg gat ctg cca ttg     4132 
Pro Asp Arg Leu Thr Ala Ala Cys Phe Asp Arg Trp Asp Leu Pro Leu 
            880                 885                 890 

tca gac atg tat acc ccg tac gtc ttc ccg agc gaa aac ggt ctg cgc     4180 
Ser Asp Met Tyr Thr Pro Tyr Val Phe Pro Ser Glu Asn Gly Leu Arg 
        895                 900                 905 

tgc ggg acg cgc gaa ttg aat tat ggc cca cac cag tgg cgc ggc gac     4228 
Cys Gly Thr Arg Glu Leu Asn Tyr Gly Pro His Gln Trp Arg Gly Asp 
    910                 915                 920 

ttc cag ttc aac atc agc cgc tac agt caa cag caa ctg atg gaa acc     4276 
Phe Gln Phe Asn Ile Ser Arg Tyr Ser Gln Gln Gln Leu Met Glu Thr 
925                 930                 935                 940 

agc cat cgc cat ctg ctg cac gcg gaa gaa ggc aca tgg ctg aat atc     4324 
Ser His Arg His Leu Leu His Ala Glu Glu Gly Thr Trp Leu Asn Ile 
                945                 950                 955 

gac ggt ttc cat atg ggg att ggt ggc gac gac tcc tgg agc ccg tca     4372 
Asp Gly Phe His Met Gly Ile Gly Gly Asp Asp Ser Trp Ser Pro Ser 
            960                 965                 970 

gta tcg gcg gaa ttc cag ctg agc gcc ggt cgc tac cat tac cag ttg     4420 
Val Ser Ala Glu Phe Gln Leu Ser Ala Gly Arg Tyr His Tyr Gln Leu 
        975                 980                 985 

gtc tgg tgt caa aaa aga tct gac tat aaa gat gag  gac ctc gac cat    4468 
Val Trp Cys Gln Lys Arg Ser Asp Tyr Lys Asp Glu  Asp Leu Asp His 
    990                 995                 1000 

cat  cat cat cat cac cgg  taataatagg tagataagtg actgattaga          4516 
His  His His His His Arg 
1005                 1010 

tgcattgatc cctcgaccaa ttccggttat tttccaccat attgccgtct tttggcaatg   4576 

tgagggcccg gaaacctggc cctgtcttct tgacgagcat tcctaggggt ctttcccctc   4636 

tcgccaaagg aatgcaaggt ctgttgaatg tcgtgaagga agcagttcct ctggaagctt   4696 

cttgaagaca aacaacgtct gtagcgaccc tttgcaggca gcggaacccc ccacctggcg   4756 

acaggtgcct ctgcggccaa aagccacgtg tataagatac acctgcaaag gcggcacaac   4816 

cccagtgcca cgttgtgagt tggatagttg tggaaagagt caaatggctc tcctcaagcg   4876 

tattcaacaa ggggctgaag gatgcccaga aggtacccca ttgtatggga tctgatctgg   4936 

ggcctcggtg cacatgcttt acatgtgttt agtcgaggtt aaaaaacgtc taggcccccc   4996 

gaaccacggg gacgtggttt tcctttgaaa aacacgatga taataccatg attgaacaag   5056 

atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg   5116 

cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc   5176 

cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag   5236 

cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca   5296 

ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat   5356 

ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata   5416 

cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac   5476 

gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc   5536 

tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg   5596 

tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg   5656 

gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta   5716 

cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg   5776 

gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct   5836 

gagcgggact ctggggttcg catcgataaa ataaaagatt ttatttagtc tccagaaaaa   5896 

ggggggaatg aaagacccca cctgtaggtt tggcaagcta gcttaagtaa cgccattttg   5956 

caaggcatgg aaaaatacat aactgagaat agagaagttc agatcaaggt caggaacaga   6016 

tggaacagct gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc   6076 

agggccaaga acagatggaa cagctgaata tgggccaaac aggatatctg tggtaagcag   6136 

ttcctgcccc ggctcagggc caagaacaga tggtccccag atgcggtcca gccctcagca   6196 

gtttctagag aaccatcaga tgtttccagg gtgccccaag gacctgaaat gaccctgtgc   6256 

cttatttgaa ctaaccaatc agttcgcttc tcgcttctgt tcgcgcgctt ctgctccccg   6316 

agctcaataa aagagcccac aacccctcac tcggggcgcc agtcctccga ttgactgagt   6376 

cgcccgggta cccgtgtatc caataaaccc tcttgcagtt gcatccgact tgtggtctcg   6436 

ctgttccttg ggagggtctc ctctgagtga ttgactaccc gtcagcgggg gtctttcatt   6496 

catgcagcat gtatcaaaat taatttggtt ttttttctta agtatttaca ttaaatggcc   6556 

atagttgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttggcgctct   6616 

tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca   6676 

gctcactcaa aggcggtaat acgg                                          6700 

 
           
             2  
             1010  
             PRT  
             Unknown  
             
               pICAST ALC.  
             
           
            2 

Met Gly Val Ile Thr Asp Ser Leu Ala Val Val Ala Arg Thr Asp Arg 
1               5                   10                  15 

Pro Ser Gln Gln Leu Arg Ser Leu Asn Gly Glu Trp Arg Phe Ala Trp 
            20                  25                  30 

Phe Pro Ala Pro Glu Ala Val Pro Glu Ser Trp Leu Glu Cys Asp Leu 
        35                  40                  45 

Pro Glu Ala Asp Thr Val Val Val Pro Ser Asn Trp Gln Met His Gly 
    50                  55                  60 

Tyr Asp Ala Pro Ile Tyr Thr Asn Val Thr Tyr Pro Ile Thr Val Asn 
65                  70                  75                  80 

Pro Pro Phe Val Pro Thr Glu Asn Pro Thr Gly Cys Tyr Ser Leu Thr 
                85                  90                  95 

Phe Asn Val Asp Glu Ser Trp Leu Gln Glu Gly Gln Thr Arg Ile Ile 
            100                 105                 110 

Phe Asp Gly Val Asn Ser Ala Phe His Leu Trp Cys Asn Gly Arg Trp 
        115                 120                 125 

Val Gly Tyr Gly Gln Asp Ser Arg Leu Pro Ser Glu Phe Asp Leu Ser 
    130                 135                 140 

Ala Phe Leu Arg Ala Gly Glu Asn Arg Leu Ala Val Met Val Leu Arg 
145                 150                 155                 160 

Trp Ser Asp Gly Ser Tyr Leu Glu Asp Gln Asp Met Trp Arg Met Ser 
                165                 170                 175 

Gly Ile Phe Arg Asp Val Ser Leu Leu His Lys Pro Thr Thr Gln Ile 
            180                 185                 190 

Ser Asp Phe His Val Ala Thr Arg Phe Asn Asp Asp Phe Ser Arg Ala 
        195                 200                 205 

Val Leu Glu Ala Glu Val Gln Met Cys Gly Glu Leu Arg Asp Tyr Leu 
    210                 215                 220 

Arg Val Thr Val Ser Leu Trp Gln Gly Glu Thr Gln Val Ala Ser Gly 
225                 230                 235                 240 

Thr Ala Pro Phe Gly Gly Glu Ile Ile Asp Glu Arg Gly Gly Tyr Ala 
                245                 250                 255 

Asp Arg Val Thr Leu Arg Leu Asn Val Glu Asn Pro Lys Leu Trp Ser 
            260                 265                 270 

Ala Glu Ile Pro Asn Leu Tyr Arg Ala Val Val Glu Leu His Thr Ala 
        275                 280                 285 

Asp Gly Thr Leu Ile Glu Ala Glu Ala Cys Asp Val Gly Phe Arg Glu 
    290                 295                 300 

Val Arg Ile Glu Asn Gly Leu Leu Leu Leu Asn Gly Lys Pro Leu Leu 
305                 310                 315                 320 

Ile Arg Gly Val Asn Arg His Glu His His Pro Leu His Gly Gln Val 
                325                 330                 335 

Met Asp Glu Gln Thr Met Val Gln Asp Ile Leu Leu Met Lys Gln Asn 
            340                 345                 350 

Asn Phe Asn Ala Val Arg Cys Ser His Tyr Pro Asn His Pro Leu Trp 
        355                 360                 365 

Tyr Thr Leu Cys Asp Arg Tyr Gly Leu Tyr Val Val Asp Glu Ala Asn 
    370                 375                 380 

Ile Glu Thr His Gly Met Val Pro Met Asn Arg Leu Thr Asp Asp Pro 
385                 390                 395                 400 

Arg Trp Leu Pro Ala Met Ser Glu Arg Val Thr Arg Met Val Gln Arg 
                405                 410                 415 

Asp Arg Asn His Pro Ser Val Ile Ile Trp Ser Leu Gly Asn Glu Ser 
            420                 425                 430 

Gly His Gly Ala Asn His Asp Ala Leu Tyr Arg Trp Ile Lys Ser Val 
        435                 440                 445 

Asp Pro Ser Arg Pro Val Gln Tyr Glu Gly Gly Gly Ala Asp Thr Thr 
    450                 455                 460 

Ala Thr Asp Ile Ile Cys Pro Met Tyr Ala Arg Val Asp Glu Asp Gln 
465                 470                 475                 480 

Pro Phe Pro Ala Val Pro Lys Trp Ser Ile Lys Lys Trp Leu Ser Leu 
                485                 490                 495 

Pro Gly Glu Thr Arg Pro Leu Ile Leu Cys Glu Tyr Ala His Ala Met 
            500                 505                 510 

Gly Asn Ser Leu Gly Gly Phe Ala Lys Tyr Trp Gln Ala Phe Arg Gln 
        515                 520                 525 

Tyr Pro Arg Leu Gln Gly Gly Phe Val Trp Asp Trp Val Asp Gln Ser 
    530                 535                 540 

Leu Ile Lys Tyr Asp Glu Asn Gly Asn Pro Trp Ser Ala Tyr Gly Gly 
545                 550                 555                 560 

Asp Phe Gly Asp Thr Pro Asn Asp Arg Gln Phe Cys Met Asn Gly Leu 
                565                 570                 575 

Val Phe Ala Asp Arg Thr Pro His Pro Ala Leu Thr Glu Ala Lys His 
            580                 585                 590 

Gln Gln Gln Phe Phe Gln Phe Arg Leu Ser Gly Gln Thr Ile Glu Val 
        595                 600                 605 

Thr Ser Glu Tyr Leu Phe Arg His Ser Asp Asn Glu Leu Leu His Trp 
    610                 615                 620 

Met Val Ala Leu Asp Gly Lys Pro Leu Ala Ser Gly Glu Val Pro Leu 
625                 630                 635                 640 

Asp Val Ala Pro Gln Gly Lys Gln Leu Ile Glu Leu Pro Glu Leu Pro 
                645                 650                 655 

Gln Pro Glu Ser Ala Gly Gln Leu Trp Leu Thr Val Arg Val Val Gln 
            660                 665                 670 

Pro Asn Ala Thr Ala Trp Ser Glu Ala Gly His Ile Ser Ala Trp Gln 
        675                 680                 685 

Gln Trp Arg Leu Ala Glu Asn Leu Ser Val Thr Leu Pro Ala Ala Ser 
    690                 695                 700 

His Ala Ile Pro His Leu Thr Thr Ser Glu Met Asp Phe Cys Ile Glu 
705                 710                 715                 720 

Leu Gly Asn Lys Arg Trp Gln Phe Asn Arg Gln Ser Gly Phe Leu Ser 
                725                 730                 735 

Gln Met Trp Ile Gly Asp Lys Lys Gln Leu Leu Thr Pro Leu Arg Asp 
            740                 745                 750 

Gln Phe Thr Arg Ala Pro Leu Asp Asn Asp Ile Gly Val Ser Glu Ala 
        755                 760                 765 

Thr Arg Ile Asp Pro Asn Ala Trp Val Glu Arg Trp Lys Ala Ala Gly 
    770                 775                 780 

His Tyr Gln Ala Glu Ala Ala Leu Leu Gln Cys Thr Ala Asp Thr Leu 
785                 790                 795                 800 

Ala Asp Ala Val Leu Ile Thr Thr Ala His Ala Trp Gln His Gln Gly 
                805                 810                 815 

Lys Thr Leu Phe Ile Ser Arg Lys Thr Tyr Arg Ile Asp Gly Ser Gly 
            820                 825                 830 

Gln Met Ala Ile Thr Val Asp Val Glu Val Ala Ser Asp Thr Pro His 
        835                 840                 845 

Pro Ala Arg Ile Gly Leu Asn Cys Gln Leu Ala Gln Val Ala Glu Arg 
    850                 855                 860 

Val Asn Trp Leu Gly Leu Gly Pro Gln Glu Asn Tyr Pro Asp Arg Leu 
865                 870                 875                 880 

Thr Ala Ala Cys Phe Asp Arg Trp Asp Leu Pro Leu Ser Asp Met Tyr 
                885                 890                 895 

Thr Pro Tyr Val Phe Pro Ser Glu Asn Gly Leu Arg Cys Gly Thr Arg 
            900                 905                 910 

Glu Leu Asn Tyr Gly Pro His Gln Trp Arg Gly Asp Phe Gln Phe Asn 
        915                 920                 925 

Ile Ser Arg Tyr Ser Gln Gln Gln Leu Met Glu Thr Ser His Arg His 
    930                 935                 940 

Leu Leu His Ala Glu Glu Gly Thr Trp Leu Asn Ile Asp Gly Phe His 
945                 950                 955                 960 

Met Gly Ile Gly Gly Asp Asp Ser Trp Ser Pro Ser Val Ser Ala Glu 
                965                 970                 975 

Phe Gln Leu Ser Ala Gly Arg Tyr His Tyr Gln Leu Val Trp Cys Gln 
            980                 985                 990 

Lys Arg Ser Asp Tyr Lys Asp Glu  Asp Leu Asp His His  His His His 
        995                 1000                 1005 

His Arg 
    1010 

 
           
             3  
             6700  
             DNA  
             Unknown  
             
               pICAST ALC.  
             
           
            3 

gacgtcggac ttatacccgg tttgtcctat agacaccatt cgtcaaggac ggggccgagt     60 

cccggttctt gtctaccttg tcgacttata cccggtttgt cctatagaca ccattcgtca    120 

aggacggggc cgagtcccgg ttcttgtcta ccaggggtct acgccaggtc gggagtcgtc    180 

aaagatctct tggtagtcta caaaggtccc acggggttcc tggactttac tgggacacgg    240 

aataaacttg attggttagt caagcgaaga gcgaagacaa gcgcgcgaag acgaggggct    300 

cgagttattt tctcgggtgt tggggagtga gccccgcggt caggaggcta actgactcag    360 

cgggcccatg ggcacatagg ttatttggga gaacgtcaac gtaggctgaa caccagagcg    420 

acaaggaacc ctcccagagg agactcacta actgatgggc agtcgccccc agaaagtaaa    480 

cccccgagca ggccctagcc ctctggggac gggtccctgg tggctgggtg gtggccctcc    540 

gttcgaccgg tcgttgaata gacacagaca ggctaacaga tcacagatac tgactaaaat    600 

acgcggacgc agccatgatc aatcgattga tcgagacata gaccgcctgg gcaccacctt    660 

gactgctcaa gacttgtggg ccggcgttgg gaccctctgc agggtccctg aaacccccgg    720 

caaaaacacc gggctggact ccttccctca gctacacctt aggctggggc agtcctatac    780 

accaagacca tcctctgctc ttggattttg tcaagggcgg aggcagactt aaaaacgaaa    840 

gccaaacctt ggcttcggcg cgcagaacag acgacgtcgt agcaagacac aacagagaca    900 

gactgacaca aagacataaa cagactttta atcccggtct gacaatggtg agggaattca    960 

aactggaatc cattgacctt tctacagctc gccgagcgag tgttggtcag ccatctacag   1020 

ttcttctctg caacccaatg gaagacgaga cgtcttaccg gttggaaatt gcagcctacc   1080 

ggcgctctgc cgtggaaatt ggctctggag tagtgggtcc aattctagtt ccagaaaagt   1140 

ggaccgggcg tacctgtggg tctggtccag gggatgtagc actggaccct tcggaaccga   1200 

aaactggggg gagggaccca gttcgggaaa catgtgggat tcggaggcgg aggagaagga   1260 

ggtaggcggg gcagagaggg ggaacttgga ggagcaagct ggggcggagc taggagggaa   1320 

ataggtcggg agtgaggaag agatccgcgg ccggcgagat cgggtaatta tgctgagtga   1380 

tatcccgcta agcttagtcc ggaaccgcgc ggcctaggaa ttaattcgcg ttaaccctcc   1440 

accgccatcg gagctctacc cgcactaatg cctaagtgac cggcagcacc gggcgtggct   1500 

agcgggaagg gttgtcaatg cgtcggactt accgcttacc gcgaaacgga ccaaaggccg   1560 

tggtcttcgc cacggccttt cgaccgacct cacgctagaa ggactccggc tatgacagca   1620 

gcaggggagt ttgaccgtct acgtgccaat gctacgcggg tagatgtggt tgcactggat   1680 

agggtaatgc cagttaggcg gcaaacaagg gtgcctctta ggctgcccaa caatgagcga   1740 

gtgtaaatta caactacttt cgaccgatgt ccttccggtc tgcgcttaat aaaaactacc   1800 

gcaattgagc cgcaaagtag acaccacgtt gcccgcgacc cagccaatgc cggtcctgtc   1860 

agcaaacggc agacttaaac tggactcgcg taaaaatgcg cggcctcttt tggcggagcg   1920 

ccactaccac gacgcgacct cactgccgtc aatagacctt ctagtcctat acaccgccta   1980 

ctcgccgtaa aaggcactgc agagcaacga cgtatttggc tgatgtgttt agtcgctaaa   2040 

ggtacaacgg tgagcgaaat tactactaaa gtcggcgcga catgacctcc gacttcaagt   2100 

ctacacgccg ctcaacgcac tgatggatgc ccattgtcaa agaaataccg tcccactttg   2160 

cgtccagcgg tcgccgtggc gcggaaagcc gccactttaa tagctactcg caccaccaat   2220 

acggctagcg cagtgtgatg cagacttgca gcttttgggc tttgacacct cgcggcttta   2280 

gggcttagag atagcacgcc accaacttga cgtgtggcgg ctgccgtgcg actaacttcg   2340 

tcttcggacg ctacagccaa aggcgctcca cgcctaactt ttaccagacg acgacgactt   2400 

gccgttcggc aacgactaag ctccgcaatt ggcagtgctc gtagtaggag acgtaccagt   2460 

ccagtaccta ctcgtctgct accacgtcct ataggacgac tacttcgtct tgttgaaatt   2520 

gcggcacgcg acaagcgtaa taggcttggt aggcgacacc atgtgcgaca cgctggcgat   2580 

gccggacata caccacctac ttcggttata actttgggtg ccgtaccacg gttacttagc   2640 

agactggcta ctaggcgcga ccgatggccg ctactcgctt gcgcattgcg cttaccacgt   2700 

cgcgctagca ttagtgggct cacactagta gaccagcgac cccttactta gtccggtgcc   2760 

gcgattagtg ctgcgcgaca tagcgaccta gtttagacag ctaggaaggg cgggccacgt   2820 

catacttccg ccgcctcggc tgtggtgccg gtggctataa taaacgggct acatgcgcgc   2880 

gcacctactt ctggtcggga agggccgaca cggctttacc aggtagtttt ttaccgaaag   2940 

cgatggacct ctctgcgcgg gcgactagga aacgcttatg cgggtgcgct acccattgtc   3000 

agaaccgcca aagcgattta tgaccgtccg caaagcagtc ataggggcaa atgtcccgcc   3060 

gaagcagacc ctgacccacc tagtcagcga ctaatttata ctacttttgc cgttgggcac   3120 

cagccgaatg ccgccactaa aaccgctatg cggcttgcta gcggtcaaga catacttgcc   3180 

agaccagaaa cggctggcgt gcggcgtagg tcgcgactgc cttcgttttg tggtcgtcgt   3240 

caaaaaggtc aaggcaaata ggcccgtttg gtagcttcac tggtcgctta tggacaaggc   3300 

agtatcgcta ttgctcgagg acgtgaccta ccaccgcgac ctaccattcg gcgaccgttc   3360 

gccacttcac ggagacctac agcgaggtgt tccatttgtc aactaacttg acggacttga   3420 

tggcgtcggc ctctcgcggc ccgttgagac cgagtgtcat gcgcatcacg ttggcttgcg   3480 

ctggcgtacc agtcttcggc ccgtgtagtc gcggaccgtc gtcaccgcag accgcctttt   3540 

ggagtcacac tgcgaggggc ggcgcagggt gcggtagggc gtagactggt ggtcgcttta   3600 

cctaaaaacg tagctcgacc cattattcgc aaccgttaaa ttggcggtca gtccgaaaga   3660 

aagtgtctac acctaaccgc tattttttgt tgacgactgc ggcgacgcgc tagtcaagtg   3720 

ggcacgtggc gacctattgc tgtaaccgca ttcacttcgc tgggcgtaac tgggattgcg   3780 

gacccagctt gcgaccttcc gccgcccggt aatggtccgg cttcgtcgca acaacgtcac   3840 

gtgccgtcta tgtgaacgac tacgccacga ctaatgctgg cgagtgcgca ccgtcgtagt   3900 

ccccttttgg aataaatagt cggccttttg gatggcctaa ctaccatcac cagtttaccg   3960 

ctaatggcaa ctacaacttc accgctcgct atgtggcgta ggccgcgcct aaccggactt   4020 

gacggtcgac cgcgtccatc gtctcgccca tttgaccgag cctaatcccg gcgttctttt   4080 

gatagggctg gcggaatgac ggcggacaaa actggcgacc ctagacggta acagtctgta   4140 

catatggggc atgcagaagg gctcgctttt gccagacgcg acgccctgcg cgcttaactt   4200 

aataccgggt gtggtcaccg cgccgctgaa ggtcaagttg tagtcggcga tgtcagttgt   4260 

cgttgactac ctttggtcgg tagcggtaga cgacgtgcgc cttcttccgt gtaccgactt   4320 

atagctggca aaggtatacc cctaaccacc gctgctgagg acctcgggca gtcatagccg   4380 

ccttaaggtc gactcgcggc cagcgatggt aatggtcaac cagaccacag ttttttctag   4440 

actgatattt ctactcctgg agctggtagt agtagtagta gtggccatta ttatccatct   4500 

attcactgac taatctacgt aactagggag ctggttaagg ccaataaaag gtggtataac   4560 

ggcagaaaac cgttacactc ccgggccttt ggaccgggac agaagaactg ctcgtaagga   4620 

tccccagaaa ggggagagcg gtttccttac gttccagaca acttacagca cttccttcgt   4680 

caaggagacc ttcgaagaac ttctgtttgt tgcagacatc gctgggaaac gtccgtcgcc   4740 

ttggggggtg gaccgctgtc cacggagacg ccggttttcg gtgcacatat tctatgtgga   4800 

cgtttccgcc gtgttggggt cacggtgcaa cactcaacct atcaacacct ttctcagttt   4860 

accgagagga gttcgcataa gttgttcccc gacttcctac gggtcttcca tggggtaaca   4920 

taccctagac tagaccccgg agccacgtgt acgaaatgta cacaaatcag ctccaatttt   4980 

ttgcagatcc ggggggcttg gtgcccctgc accaaaagga aactttttgt gctactatta   5040 

tggtactaac ttgttctacc taacgtgcgt ccaagaggcc ggcgaaccca cctctccgat   5100 

aagccgatac tgacccgtgt tgtctgttag ccgacgagac tacggcggca caaggccgac   5160 

agtcgcgtcc ccgcgggcca agaaaaacag ttctggctgg acaggccacg ggacttactt   5220 

gacgtcctgc tccgtcgcgc cgatagcacc gaccggtgct gcccgcaagg aacgcgtcga   5280 

cacgagctgc aacagtgact tcgcccttcc ctgaccgacg ataacccgct tcacggcccc   5340 

gtcctagagg acagtagagt ggaacgagga cggctctttc ataggtagta ccgactacgt   5400 

tacgccgccg acgtatgcga actaggccga tggacgggta agctggtggt tcgctttgta   5460 

gcgtagctcg ctcgtgcatg agcctacctt cggccagaac agctagtcct actagacctg   5520 

cttctcgtag tccccgagcg cggtcggctt gacaagcggt ccgagttccg cgcgtacggg   5580 

ctgccgctcc tagagcagca ctgggtaccg ctacggacga acggcttata gtaccacctt   5640 

ttaccggcga aaagacctaa gtagctgaca ccggccgacc cacaccgcct ggcgatagtc   5700 

ctgtatcgca accgatgggc actataacga cttctcgaac cgccgcttac ccgactggcg   5760 

aaggagcacg aaatgccata gcggcgaggg ctaagcgtcg cgtagcggaa gatagcggaa   5820 

gaactgctca agaagactcg ccctgagacc ccaagcgtag ctattttatt ttctaaaata   5880 

aatcagaggt ctttttcccc ccttactttc tggggtggac atccaaaccg ttcgatcgaa   5940 

ttcattgcgg taaaacgttc cgtacctttt tatgtattga ctcttatctc ttcaagtcta   6000 

gttccagtcc ttgtctacct tgtcgactta tacccggttt gtcctataga caccattcgt   6060 

caaggacggg gccgagtccc ggttcttgtc taccttgtcg acttataccc ggtttgtcct   6120 

atagacacca ttcgtcaagg acggggccga gtcccggttc ttgtctacca ggggtctacg   6180 

ccaggtcggg agtcgtcaaa gatctcttgg tagtctacaa aggtcccacg gggttcctgg   6240 

actttactgg gacacggaat aaacttgatt ggttagtcaa gcgaagagcg aagacaagcg   6300 

cgcgaagacg aggggctcga gttattttct cgggtgttgg ggagtgagcc ccgcggtcag   6360 

gaggctaact gactcagcgg gcccatgggc acataggtta tttgggagaa cgtcaacgta   6420 

ggctgaacac cagagcgaca aggaaccctc ccagaggaga ctcactaact gatgggcagt   6480 

cgcccccaga aagtaagtac gtcgtacata gttttaatta aaccaaaaaa aagaattcat   6540 

aaatgtaatt taccggtatc aacgtaatta cttagccggt tgcgcgcccc tctccgccaa   6600 

acgcataacc gcgagaaggc gaaggagcga gtgactgagc gacgcgagcc agcaagccga   6660 

cgccgctcgc catagtcgag tgagtttccg ccattatgcc                         6700 

 
           
             4  
             8518  
             DNA  
             Unknown  
             
               pICAST ALN.  
             
           
            4 

ctgcagcctg aatatgggcc aaacaggata tctgtggtaa gcagttcctg ccccggctca     60 

gggccaagaa cagatggaac agctgaatat gggccaaaca ggatatctgt ggtaagcagt    120 

tcctgccccg gctcagggcc aagaacagat ggtccccaga tgcggtccag ccctcagcag    180 

tttctagaga accatcagat gtttccaggg tgccccaagg acctgaaatg accctgtgcc    240 

ttatttgaac taaccaatca gttcgcttct cgcttctgtt cgcgcgcttc tgctccccga    300 

gctcaataaa agagcccaca acccgtcact cggggcgcca gtcctccgat tgactgagtc    360 

gcccgggtac ccgtgtatcc aataaaccct cttgcagttg catccgactt gtggtctcgc    420 

tgttccttgg gagggtctcc tctgagtgat tgactacccg tcagcggggg tctttcattt    480 

gggggctcgt ccgggatcgg gagacccctg cccagggacc accgacccac caccgggagg    540 

caagctggcc agcaacttat ctgtgtctgt ccgattgtct agtgtctatg actgatttta    600 

tgcgcctgcg tcggtactag ttagctaact agctctgtat ctggcggacc cgtggtggaa    660 

ctgacgagtt ctgaacaccc ggccgcaacc ctgggagacg tcccagggac tttgggggcc    720 

gtttttgtgg cccgacctga ggaagggagt cgatgtggaa tccgaccccg tcaggatatg    780 

tggttctggt aggagacgag aacctaaaac agttcccgcc tccgtctgaa tttttgcttt    840 

cggtttggaa ccgaagccgc gcgtcttgtc tgctgcagca tcgttctgtg ttgtctctgt    900 

ctgactgtgt ttctgtattt gtctgaaaat tagggccaga ctgttaccac tcccttaagt    960 

ttgaccttag gtaactggaa agatgtcgag cggctcgctc acaaccagtc ggtagatgtc   1020 

aagaagagac gttgggttac cttctgctct gcagaatggc caacctttaa cgtcggatgg   1080 

ccgcgagacg gcacctttaa ccgagacctc atcacccagg ttaagatcaa ggtcttttca   1140 

cctggcccgc atggacaccc agaccaggtc ccctacatcg tgacctggga agccttggct   1200 

tttgaccccc ctccctgggt caagcccttt gtacacccta agcctccgcc tcctcttcct   1260 

ccatccgccc cgtctctccc ccttgaacct cctcgttcga ccccgcctcg atcctccctt   1320 

tatccagccc tcactccttc tctaggcgcc ggccgctcta gcccattaat acgactcact   1380 

atagggcgat tcgaacacca tgcaccatca tcatcatcac gtcgactata aagatgagga   1440 

cctcgagatg ggcgtgatta cggattcact ggccgtcgtg gcccgcaccg atcgcccttc   1500 

ccaacagtta cgcagcctga atggcgaatg gcgctttgcc tggtttccgg caccagaagc   1560 

ggtgccggaa agctggctgg agtgcgatct tcctgaggcc gatactgtcg tcgtcccctc   1620 

aaactggcag atgcacggtt acgatgcgcc catctacacc aacgtgacct atcccattac   1680 

ggtcaatccg ccgtttgttc ccacggagaa tccgacgggt tgttactcgc tcacatttaa   1740 

tgttgatgaa agctggctac aggaaggcca gacgcgaatt atttttgatg gcgttaactc   1800 

ggcgtttcat ctgtggtgca acgggcgctg ggtcggttac ggccaggaca gtcgtttgcc   1860 

gtctgaattt gacctgagcg catttttacg cgccggagaa aaccgcctcg cggtgatggt   1920 

gctgggctgg agtgacggca gttatctgga agatcaggat atgtggcgga tgagcggcat   1980 

tttccgtgac gtctcgttgc tgcataaacc gactacacaa atcagcgatt tccatgttgc   2040 

cactcgcttt aatgatgatt rcagccgcgc tgtactggag gctgaagttc agatgtgcgg   2100 

cgagttgcgt gactacctac gggtaacagt ttctttatgg cagggtgaaa cgcaggtcgc   2160 

cagcggcacc gcgcctttcg gcggtgaaat tatcgatgag cgtggtggtt atgccgatcg   2220 

cgtcacacta cgtctgaacg tcgaaaaccc gaaactgtgg agcgccgaaa tcccgaatct   2280 

ctatcgtgcg gtggttgaac tgcacaccgc cgacggcacg ctgattgaag cagaagcctg   2340 

cgatgtcggt ttccgcgagg tgcggattga aaatggtctg ctgctgctga acggcaagcc   2400 

gttgctgatt cgaggcgtta accgtcacga gcatcatcct ctgcatggtc aggtcatgga   2460 

tgagcagacg atggtgcagg atatcctgct gatgaagcag aacaacttta acgccgtgcg   2520 

ctgttcgcat tatccgaacc atccgctgtg gtacacgctg tgcgaccgct acggcctgta   2580 

tgtggtggat gaagccaata ttgaaaccca cggcatggtg ccaatgaatc gtctgaccga   2640 

tgatccgcgc tggctaccgg cgatgagcga acgcgtaacg cgaatggtgc agcgcgatcg   2700 

taatcacccg agtgtgatca tctggtcgct ggggaatgaa tcaggccacg gcgctaatca   2760 

cgacgcgctg tatcgctgga tcaaatctgt cgatccttcc cgcccggtgc agtatgaagg   2820 

cggcggagcc gacaccacgg ccaccgatat tatttgcccg atgtacgcgc gcgtggatga   2880 

agaccagccc ttcccggctg tgccgaaatg gtccatcaaa aaatggcttt cgctacctgg   2940 

agagacgcgc ccgctgatcc tttgcgaata cgcccacgcg atgggtaaca gtcttggcgg   3000 

tttcgctaaa tactggcagg cgtttcgtca gtatccccgt ttacagggcg gcttcgtctg   3060 

ggactgggtg gatcagtcgc tgattaaata tgatgaaaac ggcaacccgt ggtcggctta   3120 

cggcggtgat tttggcgata cgccgaacga tcgccagttc tgtatgaacg gtctggtctt   3180 

tgccgaccgc acgccgcatc cagcgctgac ggaagcaaaa caccagcagc agtttttcca   3240 

gttccgttta tccgggcaaa ccatcgaagt gaccagcgaa tacctgttcc gtcatagcga   3300 

taacgagctc ctgcactgga tggtggcgct ggatggtaag ccgctggcaa gcggtgaagt   3360 

gcctctggat gtcgctccac aaggtaaaca gttgattgaa ctgcctgaac taccgcagcc   3420 

ggagagcgcc gggcaactct ggctcacagt acgcgtagtg caaccgaacg cgaccgcatg   3480 

gtcagaagcc gggcacatca gcgcctggca gcagtggcgt ctggcggaaa acctcagtgt   3540 

gacgctcccc gccgcgtccc acgccatccc gcatctgacc accagcgaaa tggatttttg   3600 

catcgagctg ggtaataagc gttggcaatt taaccgccag tcaggctttc tttcacagat   3660 

gtggattggc gataaaaaac aactgctgac gccgctgcgc gatcagttca cccgtgcacc   3720 

gctggataac gacattggcg taagtgaagc gacccgcatt gaccctaacg cctgggtcga   3780 

acgctggaag gcggcgggcc attaccaggc cgaagcagcg ttgttgcagt gcacggcaga   3840 

tacacttgct gatgcggtgc tgattacgac cgctcacgcg tggcagcatc aggggaaaac   3900 

cttatttatc agccggaaaa cctaccggat tgatggtagt ggtcaaatgg cgattaccgt   3960 

tgatgttgaa gtggcgagcg atacaccgca tccggcgcgg attggcctga actgccagct   4020 

ggcgcaggta gcagagcggg taaactggct cggattaggg ccgcaagaaa actatcccga   4080 

ccgccttact gccgcctgtt ttgaccgctg ggatctgcca ttgtcagaca tgtatacccc   4140 

gtacgtcttc ccgagcgaaa acggtctgcg ctgcgggacg cgcgaattga attatggccc   4200 

acaccagtgg cgcggcgact tccagttcaa catcagccgc tacagtcaac agcaactgat   4260 

ggaaaccagc catcgccatc tgctgcacgc ggaagaaggc acatggctga atatcgacgg   4320 

tttccatatg gggattggtg gcgacgactc ctggagcccg tcagtatcgg cggaattcca   4380 

gctgagcgcc ggtcgctacc attaccagtt ggtctggtgt caaaaaagat ctggaggtgg   4440 

tggcagcagg ccttggcgcg ccggatcctt aattaacaat tgaccggtaa taataggtag   4500 

ataagtgact gattagatgc attgatccct cgaccaattc cggttatttt ccaccatatt   4560 

gccgtctttt ggcaatgtga gggcccggaa acctggccct gtcttcttga cgagcattcc   4620 

taggggtctt tcccctctcg ccaaaggaat gcaaggtctg ttgaatgtcg tgaaggaagc   4680 

agttcctctg gaagcttctt gaagacaaac aacgtctgta gcgacccttt gcaggcagcg   4740 

gaacccccca cctggcgaca ggtgcctctg cggccaaaag ccacgtgtat aagatacacc   4800 

tgcaaaggcg gcacaacccc agtgccacgt tgtgagttgg atagttgtgg aaagagtcaa   4860 

atggctctcc tcaagcgtat tcaacaaggg gctgaaggat gcccagaagg taccccattg   4920 

tatgggatct gatctggggc ctcggtgcac atgctttaca tgtgtttagt cgaggttaaa   4980 

aaacgtctag gccccccgaa ccacggggac gtggttttcc tttgaaaaac acgatgataa   5040 

taccatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct   5100 

attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct   5160 

gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga   5220 

actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc   5280 

tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg   5340 

gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc   5400 

aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca   5460 

tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga   5520 

cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc   5580 

cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga   5640 

aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca   5700 

ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg   5760 

cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct   5820 

tcttgacgag ttcttctgag cgggactctg gggttcgcat cgataaaata aaagatttta   5880 

tttagtctcc agaaaaaggg gggaatgaaa gaccccacct gtaggtttgg caagctagct   5940 

taagtaacgc cattttgcaa ggcatggaaa aatacataac tgagaataga gaagttcaga   6000 

tcaaggtcag gaacagatgg aacagctgaa tatgggccaa acaggatatc tgtggtaagc   6060 

agttcctgcc ccggctcagg gccaagaaca gatggaacag ctgaatatgg gccaaacagg   6120 

atatctgtgg taagcagttc ctgccccggc tcagggccaa gaacagatgg tccccagatg   6180 

cggtccagcc ctcagcagtt tctagagaac catcagatgt ttccagggtg ccccaaggac   6240 

ctgaaatgac cctgtgcctt atttgaacta accaatcagt tcgcttctcg cttctgttcg   6300 

cgcgcttctg ctccccgagc tcaataaaag agcccacaac ccctcactcg gggcgccagt   6360 

cctccgattg actgagtcgc ccgggtaccc gtgtatccaa taaaccctct tgcagttgca   6420 

tccgacttgt ggtctcgctg ttccttggga gggtctcctc tgagtgattg actacccgtc   6480 

agcgggggtc tttcattcat gcagcatgta tcaaaattaa tttggttttt tttcttaagt   6540 

atttacatta aatggccata gttgcattaa tgaatcggcc aacgcgcggg gagaggcggt   6600 

ttgcgtattg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc   6660 

tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg   6720 

ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg   6780 

ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac   6840 

gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg   6900 

gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct   6960 

ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg   7020 

tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct   7080 

gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac   7140 

tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt   7200 

tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc   7260 

tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca   7320 

ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat   7380 

ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac   7440 

gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttgcggc   7500 

cgcaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc   7560 

agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc   7620 

gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata   7680 

ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg   7740 

gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc   7800 

cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct   7860 

acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa   7920 

cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt   7980 

cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca   8040 

ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac   8100 

tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca   8160 

atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt   8220 

tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc   8280 

actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca   8340 

aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata   8400 

ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc   8460 

ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttc     8518 

 
           
             5  
             8518  
             DNA  
             Unknown  
             
               pICAST ALN.  
             
           
            5 

gacgtcggac ttatacccgg tttgtcctat agacaccatt cgtcaaggac ggggccgagt     60 

cccggttctt gtctaccttg tcgacttata cccggtttgt cctatagaca ccattcgtca    120 

aggacggggc cgagtcccgg ttcttgtcta ccaggggtct acgccaggtc gggagtcgtc    180 

aaagatctct tggtagtcta caaaggtccc acggggttcc tggactttac tgggacacgg    240 

aataaacttg attggttagt caagcgaaga gcgaagacaa gcgcgcgaag acgaggggct    300 

cgagttattt tctcgggtgt tggggagtga gccccgcggt caggaggcta actgactcag    360 

cgggcccatg ggcacatagg ttatttggga gaacgtcaac gtaggctgaa caccagagcg    420 

acaaggaacc ctcccagagg agactcacta actgatgggc agtcgccccc agaaagtaaa    480 

cccccgagca ggccctagcc ctctggggac gggtccctgg tggctgggtg gtggccctcc    540 

gttcgaccgg tcgttgaata gacacagaca ggctaacaga tcacagatac tgactaaaat    600 

acgcggacgc agccatgatc aatcgattga tcgagacata gaccgcctgg gcaccacctt    660 

gactgctcaa gacttgtggg ccggcgttgg gaccctctgc agggtccctg aaacccccgg    720 

caaaaacacc gggctggact ccttccctca gctacacctt aggctggggc agtcctatac    780 

accaagacca tcctctgctc ttggattttg tcaagggcgg aggcagactt aaaaacgaaa    840 

gccaaacctt ggcttcggcg cgcagaacag acgacgtcgt agcaagacac aacagagaca    900 

gactgacaca aagacataaa cagactttta atcccggtct gacaatggtg agggaattca    960 

aactggaatc cattgacctt tctacagctc gccgagcgag tgttggtcag ccatctacag   1020 

ttcttctctg caacccaatg gaagacgaga cgtcttaccg gttggaaatt gcagcctacc   1080 

ggcgctctgc cgtggaaatt ggctctggag tagtgggtcc aattctagtt ccagaaaagt   1140 

ggaccgggcg tacctgtggg tctggtccag gggatgtagc actggaccct tcggaaccga   1200 

aaactggggg gagggaccca gttcgggaaa catgtgggat tcggaggcgg aggagaagga   1260 

ggtaggcggg gcagagaggg ggaacttgga ggagcaagct ggggcggagc taggagggaa   1320 

ataggtcggg agtgaggaag agatccgcgg ccggcgagat cgggtaatta tgctgagtga   1380 

tatcccgcta agcttgtggt acgtggtagt agtagtagtg cagctgatat ttctactcct   1440 

ggagctctac ccgcactaat gcctaagtga ccggcagcac cgggcgtggc tagcgggaag   1500 

ggttgtcaat gcgtcggact taccgcttac cgcgaaacgg accaaaggcc gtggtcttcg   1560 

ccacggcctt tcgaccgacc tcacgctaga aggactccgg ctatgacagc agcaggggag   1620 

tttgaccgtc tacgtgccaa tgctacgcgg gtagatgtgg ttgcactgga tagggtaatg   1680 

ccagttaggc ggcaaacaag ggtgcctctt aggctgccca acaatgagcg agtgtaaatt   1740 

acaactactt tcgaccgatg tccttccggt ctgcgcttaa taaaaactac cgcaattgag   1800 

ccgcaaagta gacaccacgt tgcccgcgac ccagccaatg ccggtcctgt cagcaaacgg   1860 

cagacttaaa ctggactcgc gtaaaaatgc gcggcctctt ttggcggagc gccactacca   1920 

cgacgcgacc tcactgccgt caatagacct tctagtccta tacaccgcct actcgccgta   1980 

aaaggcactg cagagcaacg acgtatttgg ctgatgtgtt tagtcgctaa aggtacaacg   2040 

gtgagcgaaa ttactactaa agtcggcgcg acatgacctc cgacttcaag tctacacgcc   2100 

gctcaacgca ctgatggatg cccattgtca aagaaatacc gtcccacttt gcgtccagcg   2160 

gtcgccgtgg cgcggaaagc cgccacttta atagctactc gcaccaccaa tacggctagc   2220 

gcagtgtgat gcagacttgc agcttttggg ctttgacacc tcgcggcttt agggcttaga   2280 

gatagcacgc caccaacttg acgtgtggcg gctgccgtgc gactaacttc gtcttcggac   2340 

gctacagcca aaggcgctcc acgcctaact tttaccagac gacgacgact tgccgttcgg   2400 

caacgactaa gctccgcaat tggcagtgct cgtagtagga gacgtaccag tccagtacct   2460 

actcgtctgc taccacgtcc tataggacga ctacttcgtc ttgttgaaat tgcggcacgc   2520 

gacaagcgta ataggcttgg taggcgacac catgtgcgac acgctggcga tgccggacat   2580 

acaccaccta cttcggttat aactttgggt gccgtaccac ggttacttag cagactggct   2640 

actaggcgcg accgatggcc gctactcgct tgcgcattgc gcttaccacg tcgcgctagc   2700 

attagtgggc tcacactagt agaccagcga ccccttactt agtccggtgc cgcgattagt   2760 

gctgcgcgac atagcgacct agtttagaca gctaggaagg gcgggccacg tcatacttcc   2820 

gccgcctcgg ctgtggtgcc ggtggctata ataaacgggc tacatgcgcg cgcacctact   2880 

tctggtcggg aagggccgac acggctttac caggtagttt tttaccgaaa gcgatggacc   2940 

tctctgcgcg ggcgactagg aaacgcttat gcgggtgcgc tacccattgt cagaaccgcc   3000 

aaagcgattt atgaccgtcc gcaaagcagt cataggggca aatgtcccgc cgaagcagac   3060 

cctgacccac ctagtcagcg actaatttat actacttttg ccgttgggca ccagccgaat   3120 

gccgccacta aaaccgctat gcggcttgct agcggtcaag acatacttgc cagaccagaa   3180 

acggctggcg tgcggcgtag gtcgcgactg ccttcgtttt gtggtcgtcg tcaaaaaggt   3240 

caaggcaaat aggcccgttt ggtagcttca ctggtcgctt atggacaagg cagtatcgct   3300 

attgctcgag gacgtgacct accaccgcga cctaccattc ggcgaccgtt cgccacttca   3360 

cggagaccta cagcgaggtg ttccatttgt caactaactt gacggacttg atggcgtcgg   3420 

cctctcgcgg cccgttgaga ccgagtgtca tgcgcatcac gttggcttgc gctggcgtac   3480 

cagtcttcgg cccgtgtagt cgcggaccgt cgtcaccgca gaccgccttt tggagtcaca   3540 

ctgcgagggg cggcgcaggg tgcggtaggg cgtagactgg tggtcgcttt acctaaaaac   3600 

gtagctcgac ccattattcg caaccgttaa attggcggtc agtccgaaag aaagtgtcta   3660 

cacctaaccg ctattttttg ttgacgactg cggcgacgcg ctagtcaagt gggcacgtgg   3720 

cgacctattg ctgtaaccgc attcacttcg ctgggcgtaa ctgggattgc ggacccagct   3780 

tgcgaccttc cgccgcccgg taatggtccg gcttcgtcgc aacaacgtca cgtgccgtct   3840 

atgtgaacga ctacgccacg actaatgctg gcgagtgcgc accgtcgtag tccccttttg   3900 

gaataaatag tcggcctttt ggatggccta actaccatca ccagtttacc gctaatggca   3960 

actacaactt caccgctcgc tatgtggcgt aggccgcgcc taaccggact tgacggtcga   4020 

ccgcgtccat cgtctcgccc atttgaccga gcctaatccc ggcgttcttt tgatagggct   4080 

ggcggaatga cggcggacaa aactggcgac cctagacggt aacagtctgt acatatgggg   4140 

catgcagaag ggctcgcttt tgccagacgc gacgccctgc gcgcttaact taataccggg   4200 

tgtggtcacc gcgccgctga aggtcaagtt gtagtcggcg atgtcagttg tcgttgacta   4260 

cctttggtcg gtagcggtag acgacgtgcg ccttcttccg tgtaccgact tatagctgcc   4320 

aaaggtatac ccctaaccac cgctgctgag gacctcgggc agtcatagcc gccttaaggt   4380 

cgactcgcgg ccagcgatgg taatggtcaa ccagaccaca gttttttcta gacctccacc   4440 

accgtcgtcc ggaaccgcgc ggcctaggaa ttaattgtta actggccatt attatccatc   4500 

tattcactga ctaatctacg taactaggga gctggttaag gccaataaaa ggtggtataa   4560 

cggcagaaaa ccgttacact cccgggcctt tggaccggga cagaagaact gctcgtaagg   4620 

atccccagaa aggggagagc ggtttcctta cgttccagac aacttacagc acttccttcg   4680 

tcaaggagac cttcgaagaa cttctgtttg ttgcagacat cgctgggaaa cgtccgtcgc   4740 

cttggggggt ggaccgctgt ccacggagac gccggttttc ggtgcacata ttctatgtgg   4800 

acgtttccgc cgtgttgggg tcacggtgca acactcaacc tatcaacacc tttctcagtt   4860 

taccgagagg agttcgcata agttgttccc cgacttccta cgggtcttcc atggggtaac   4920 

ataccctaga ctagaccccg gagccacgtg tacgaaatgt acacaaatca gctccaattt   4980 

tttgcagatc cggggggctt ggtgcccctg caccaaaagg aaactttttg tgctactatt   5040 

atggtactaa cttgttctac ctaacgtgcg tccaagaggc cggcgaaccc acctctccga   5100 

taagccgata ctgacccgtg ttgtctgtta gccgacgaga ctacggcggc acaaggccga   5160 

cagtcgcgtc cccgcgggcc aagaaaaaca gttctggctg gacaggccac gggacttact   5220 

tgacgtcctg ctccgtcgcg ccgatagcac cgaccggtgc tgcccgcaag gaacgcgtcg   5280 

acacgagctg caacagtgac ttcgcccttc cctgaccgac gataacccgc ttcacggccc   5340 

cgtcctagag gacagtagag tggaacgagg acggctcttt cataggtagt accgactacg   5400 

ttacgccgcc gacgtatgcg aactaggccg atggacgggt aagctggtgg ttcgctttgt   5460 

agcgtagctc gctcgtgcat gagcctacct tcggccagaa cagctagtcc tactagacct   5520 

gcttctcgta gtccccgagc gcggtcggct tgacaagcgg tccgagttcc gcgcgtacgg   5580 

gctgccgctc ctagagcagc actgggtacc gctacggacg aacggcttat agtaccacct   5640 

tttaccggcg aaaagaccta agtagctgac accggccgac ccacaccgcc tggcgatagt   5700 

cctgtatcgc aaccgatggg cactataacg acttctcgaa ccgccgctta cccgactggc   5760 

gaaggagcac gaaatgccat agcggcgagg gctaagcgtc gcgtagcgga agatagcgga   5820 

agaactgctc aagaagactc gccctgagac cccaagcgta gctattttat tttctaaaat   5880 

aaatcagagg tctttttccc cccttacttt ctggggtgga catccaaacc gttcgatcga   5940 

attcattgcg gtaaaacgtt ccgtaccttt ttatgtattg actcttatct cttcaagtct   6000 

agttccagtc cttgtctacc ttgtcgactt atacccggtt tgtcctatag acaccattcg   6060 

tcaaggacgg ggccgagtcc cggttcttgt ctaccttgtc gacttatacc cggtttgtcc   6120 

tatagacacc attcgtcaag gacggggccg agtcccggtt cttgtctacc aggggtctac   6180 

gccaggtcgg gagtcgtcaa agatctcttg gtagtctaca aaggtcccac ggggttcctg   6240 

gactttactg ggacacggaa taaacttgat tggttagtca agcgaagagc gaagacaagc   6300 

gcgcgaagac gaggggctcg agttattttc tcgggtgttg gggagtgagc cccgcggtca   6360 

ggaggctaac tgactcagcg ggcccatggg cacataggtt atttgggaga acgtcaacgt   6420 

aggctgaaca ccagagcgac aaggaaccct cccagaggag actcactaac tgatgggcag   6480 

tcgcccccag aaagtaagta cgtcgtacat agttttaatt aaaccaaaaa aaagaattca   6540 

taaatgtaat ttaccggtat caacgtaatt acttagccgg ttgcgcgccc ctctccgcca   6600 

aacgcataac cgcgagaagg cgaaggagcg agtgactgag cgacgcgagc cagcaagccg   6660 

acgccgctcg ccatagtcga gtgagtttcc gccattatgc caataggtgt cttagtcccc   6720 

tattgcgtcc tttcttgtac actcgttttc cggtcgtttt ccggtccttg gcatttttcc   6780 

ggcgcaacga ccgcaaaaag gtatccgagg cggggggact gctcgtagtg tttttagctg   6840 

cgagttcagt ctccaccgct ttgggctgtc ctgatatttc tatggtccgc aaagggggac   6900 

cttcgaggga gcacgcgaga ggacaaggct gggacggcga atggcctatg gacaggcgga   6960 

aagagggaag cccttcgcac cgcgaaagag tatcgagtgc gacatccata gagtcaagcc   7020 

acatccagca agcgaggttc gacccgacac acgtgcttgg ggggcaagtc gggctggcga   7080 

cgcggaatag gccattgata gcagaactca ggttgggcca ttctgtgctg aatagcggtg   7140 

accgtcgtcg gtgaccattg tcctaatcgt ctcgctccat acatccgcca cgatgtctca   7200 

agaacttcac caccggattg atgccgatgt gatcttcttg tcataaacca tagacgcgag   7260 

acgacttcgg tcaatggaag cctttttctc aaccatcgag aactaggccg tttgtttggt   7320 

ggcgaccatc gccaccaaaa aaacaaacgt tcgtcgtcta atgcgcgtct ttttttccta   7380 

gagttcttct aggaaactag aaaagatgcc ccagactgcg agtcaccttg cttttgagtg   7440 

caattcccta aaaccagtac tctaatagtt tttcctagaa gtggatctag gaaaacgccg   7500 

gcgtttagtt agatttcata tatactcatt tgaaccagac tgtcaatggt tacgaattag   7560 

tcactccgtg gatagagtcg ctagacagat aaagcaagta ggtatcaacg gactgagggg   7620 

cagcacatct attgatgcta tgccctcccg aatggtagac cggggtcacg acgttactat   7680 

ggcgctctgg gtgcgagtgg ccgaggtcta aatagtcgtt atttggtcgg tcggccttcc   7740 

cggctcgcgt cttcaccagg acgttgaaat aggcggaggt aggtcagata attaacaacg   7800 

gcccttcgat ctcattcatc aagcggtcaa ttatcaaacg cgttgcaaca acggtaacga   7860 

tgtccgtagc accacagtgc gagcagcaaa ccataccgaa gtaagtcgag gccaagggtt   7920 

gctagttccg ctcaatgtac tagggggtac aacacgtttt ttcgccaatc gaggaagcca   7980 

ggaggctagc aacagtcttc attcaaccgg cgtcacaata gtgagtacca ataccgtcgt   8040 

gacgtattaa gagaatgaca gtacggtagg cattctacga aaagacactg accactcatg   8100 

agttggttca gtaagactct tatcacatac gccgctggct caacgagaac gggccgcagt   8160 

tatgccctat tatggcgcgg tgtatcgtct tgaaattttc acgagtagta accttttgca   8220 

agaagccccg cttttgagag ttcctagaat ggcgacaact ctaggtcaag ctacattggg   8280 

tgagcacgtg ggttgactag aagtcgtaga aaatgaaagt ggtcgcaaag acccactcgt   8340 

ttttgtcctt ccgttttacg gcgttttttc ccttattccc gctgtgcctt tacaacttat   8400 

gagtatgaga aggaaaaagt tataataact tcgtaaatag tcccaataac agagtactcg   8460 

cctatgtata aacttacata aatcttttta tttgtttatc cccaaggcgc gtgtaaag     8518 

 
           
             6  
             8175  
             DNA  
             Unknown  
             
               pICAST OMC.  
             
           
            6 

ctgcagcctg aatatgggcc aaacaggata tctgtggtaa gcagttcctg ccccggctca     60 

gggccaagaa cagatggaac agctgaatat gggccaaaca ggatatctgt ggtaagcagt    120 

tcctgccccg gctcagggcc aagaacagat ggtccccaga tgcggtccag ccctcagcag    180 

tttctagaga accatcagat gtttccaggg tgccccaagg acctgaaatg accctgtgcc    240 

ttatttgaac taaccaatca gttcgcttct cgcttctgtt cgcgcgcttc tgctccccga    300 

gctcaataaa agagcccaca acccctcact cggggcgcca gtcctccgat tgactgagtc    360 

gcccgggtac ccgtgtatcc aataaaccct cttgcagttg catccgactt gtggtctcgc    420 

tgttccttgg gaggytctcc tctgagtgat tgactacccg tcagcggggg tctttcattt    480 

gggggctcgt ccgggatcgg gagacccctg cccagggacc accgacccac caccgggagg    540 

caagctggcc agcaacttat ctgtgtctgt ccgattgtct agtgtctatg actgatttta    600 

tgcgcctgcg tcggtactag ttagctaact agctctgtat ctggcggacc cgtggtggaa    660 

ctgacgagtt ctgaacaccc ggccgcaacc ctgggagacg tcccagggac tttgggggcc    720 

gtttttgtgg cccgacctga ggaagggagt cgatgtggaa tccgaccccg tcaggatatg    780 

tggttctggt aggagacgag aacctaaaac agttcccgcc tccgtctgaa tttttgcttt    840 

cggtttggaa ccgaagccgc gcgtcttgtc tgctgcagca tcgttctgtg ttgtctctgt    900 

ctgactgtgt ttctgtattt gtctgaaaat tagggccaga ctgttaccac tcccttaagt    960 

ttgaccttag gtaactggaa agatgtcgag cggctcgctc acaaccagtc ggtagatgtc   1020 

aagaagagac gttgggttac cttctgctct gcagaatggc caacctttaa cgtcggatgg   1080 

ccgcgagacg gcacctttaa ccgagacctc atcacccagg ttaagatcaa ggtcttttca   1140 

cctggcccgc atggacaccc agaccaggtc ccctacatcg tgacctggga agccttggct   1200 

tttgaccccc ctccctgggt caagcccttt gtacacccta agcctccgcc tcctcttcct   1260 

ccatccgccc cgtctctccc ccttgaacct cctcgttcga ccccgcctcg atcctccctt   1320 

tatccagccc tcactccttc tctaggcgcc ggccgctcta gcccattaat acgactcact   1380 

atagggcgat tcgaatcagg ccttggcgcg ccggatcctt aattaagcgc aattgggagg   1440 

tggcggtagc ctcgagatgg gcgtgattac ggattcactg gccgtcgttt tacaacgtcg   1500 

tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc   1560 

cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt tacgcagcct   1620 

gaatggcgaa tggcgctttg cctggtttcc ggcaccagaa gcggtgccgg aaagctggct   1680 

ggagtgcgat cttcctgagg ccgatactgt cgtcgtcccc tcaaactggc agatgcacgg   1740 

ttacgatgcg cccatctaca ccaacgtgac ctatcccatt acggtcaatc cgccgtttgt   1800 

tcccacggag aatccgacgg gttgttactc gctcacattt aatgttgatg aaagctggct   1860 

acaggaaggc cagacgcgaa ttatttttga tggcgttaac tcggcgtttc atctgtggtg   1920 

caacgggcgc tgggtcggtt acggccagga cagtcgtttg ccgtctgaat ttgacctgag   1980 

cgcattttta cgcgccggag aaaaccgcct cgcggtgatg gtgctgcgct ggagtgacgg   2040 

cagttatctg gaagatcagg atatgtggcg gatgagcggc attttccgtg acgtctcgtt   2100 

gctgcataaa ccgactacac aaatcagcga tttccatgtt gccactcgct ttaatgatga   2160 

tttcagccgc gctgtactgg aggctgaagt tcagatgtgc ggcgagttgc gtgactacct   2220 

acgggtaaca gtttctttat ggcagggtga aacgcaggtc gccagcggca ccgcgccttt   2280 

cggcggtgaa attatcgatg agcgtggtgg ttatgccgat cgcgtcacac tacgtctgaa   2340 

cgtcgaaaac ccgaaactgt ggagcgccga aatcccgaat ctctatcgtg cggtggttga   2400 

actgcacacc gccgacggca cgctgattga agcagaagcc tgcgatgtcg gtttccgcga   2460 

ggtgcggatt gaaaatggtc tgctgctgct gaacggcaag ccgttgctga ttcgaggcgt   2520 

taaccgtcac gagcatcatc ctctgcatgg tcaggtcatg gatgagcaga cgatggtgca   2580 

ggatatcctg ctgatgaagc agaacaactt taacgccgtg cgctgttcgc attatccgaa   2640 

ccatccgctg tggtacacgc tgtgcgaccg ctacggcctg tatgtggtgg atgaagccaa   2700 

tattgaaacc cacggcatgg tgccaatgaa tcgtctgacc gatgatccgc gctggctacc   2760 

ggcgatgagc gaacgcgtaa cgcgaatggt gcagcgcgat cgtaatcacc cgagtgtgat   2820 

catctggtcg ctggggaatg aatcaggcca cggcgctaat cacgacgcgc tgtatcgctg   2880 

gatcaaatct gtcgatcctt cccgcccggt gcagtatgaa ggcggcggag ccgacaccac   2940 

ggccaccgat attatttgcc cgatgtacgc gcgcgtggat gaagaccagc ccttcccggc   3000 

tgtgccgaaa tggtccatca aaaaatggct ttcgctacct ggagagacgc gcccgctgat   3060 

cctttgcgaa tacgcccacg cgatgggtaa cagtcttggc ggtttcgcta aatactggca   3120 

ggcgtttcgt cagtatcccc gtttacaggg cggcttcgtc tgggactggg tggatcagtc   3180 

gctgattaaa tatgatgaaa acggcaaccc gtggtcggct tacggcggtg attttggcga   3240 

tacgccgaac gatcgccagt tctgtatgaa cggtctggtc tttgccgacc gcacgccgca   3300 

tccagcgctg acggaagcaa aacaccagca gcagtttttc cagttccgtt tatccgggca   3360 

aaccatcgaa gtgaccagcg aatacctgtt ccgtcatagc gataacgagc tcctgcactg   3420 

gatggtggcg ctggatggta agccgctggc aagcggtgaa gtgcctctgg atgtcgctcc   3480 

acaaggtaaa cagttgattg aactgcctga actaccgcag ccggagagcg ccgggcaact   3540 

ctggctcaca gtacgcgtag tgcaaccgaa cgcgaccgca tggtcagaag ccgggcacat   3600 

cagcgcctgg cagcagtggc gtctggcgga aaacctcagt gtgacgctcc ccgccgcgtc   3660 

ccacgccatc ccgcatctga ccaccagcga aatggatttt tgcatcgagc tgggtaataa   3720 

gcgttggcaa tttaaccgcc agtcaggctt tctttcacag atgtggattg gcgataaaaa   3780 

acaactgctg acgccgctgc gcgatcagtt cacccgtgtc gatagatctg aacagaaact   3840 

catttccgaa gaagacctag tcgaccatca tcatcatcat caccggtaat aataggtaga   3900 

taagtgactg attagatgca tttcgactag atccctcgac caattccggt tattttccac   3960 

catattgccg tcttttggca atgtgagggc ccggaaacct ggccctgtct tcttgacgag   4020 

cattcctagg ggtctttccc ctctcgccaa aggaatgcaa ggtctgttga atgtcgtgaa   4080 

ggaagcagtt cctctggaag cttcttgaag acaaacaacg tctgtagcga ccctttgcag   4140 

gcagcggaac cccccacctg gcgacaggtg cctctgcggc caaaagccac gtgtataaga   4200 

tacacctgca aaggcggcac aaccccagtg ccacgttgtg agttggatag ttgtggaaag   4260 

agtcaaatgg ctctcctcaa gcgtattcaa caaggggctg aaggatgccc agaaggtacc   4320 

ccattgtatg ggatctgatc tggggcctcg gtgcacatgc tttacatgtg tttagtcgag   4380 

gttaaaaaac gtctaggccc cccgaaccac ggggacgtgg ttttcctttg aaaaacacga   4440 

tgataatacc atgaaaaagc ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga   4500 

aaagttcgac agcgtctccg acctgatgca gctctcggag ggcgaagaat ctcgtgcttt   4560 

cagcttcgat gtaggagggc gtggatatgt cctgcgggta aatagctgcg ccgatggttt   4620 

ctacaaagat cgttatgttt atcggcactt tgcatcggcc gcgctcccga ttccggaagt   4680 

gcttgacatt ggggaattta gcgagagcct gacctattgc atctcccgcc gtgcacaggg   4740 

tgtcacgttg caagacctgc ctgaaaccga actgcccgct gttctgcagc cggtcgcgga   4800 

ggccatggat gcgatcgctg cggccgatct tagccagacg agcgggttcg gcccattcgg   4860 

accgcaagga atcggtcaat acactacatg gcgtgatttc atatgcgcga ttgctgatcc   4920 

ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc   4980 

tctcgatgag ctgatgcttt gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc   5040 

ggatttcggc tccaacaatg tcctgacgga caatggccgc ataacagcgg tcattgactg   5100 

gagcgaggcg atgttcgggg attcccaata cgaggtcgcc aacatcttct tctggaggcc   5160 

gtggttggct tgtatggagc agcagacgcg ctacttcgag cggaggcatc cggagcttgc   5220 

aggatcgccg cggctccggg cgtatatgct ccgcattggt cttgaccaac tctatcagag   5280 

cttggttgac ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt   5340 

ccgatccgga gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg   5400 

gaccgatggc tgtgtagaag tactcgccga tagtggaaac cgacgcccca gcactcgtcc   5460 

gagggcaaag gaatagagta gatgccgacc gggatctatc gataaaataa aagattttat   5520 

ttagtctcca gaaaaagggg ggaatgaaag accccacctg taggtttggc aagctagctt   5580 

aagtaacgcc attttgcaag gcatggaaaa atacataact gagaatagag aagttcagat   5640 

caaggtcagg aacagatgga acagctgaat atgggccaaa caggatatct gtggtaagca   5700 

gttcctgccc cggctcaggg ccaagaacag atggaacagc tgaatatggg ccaaacagga   5760 

tatctgtggt aagcagttcc tgccccggct cagggccaag aacagatggt ccccagatgc   5820 

ggtccagccc tcagcagttt ctagagaacc atcagatgtt tccagggtgc cccaaggacc   5880 

tgaaatgacc ctgtgcctta tttgaactaa ccaatcagtt cgcttctcgc ttctgttcgc   5940 

gcgcttctgc tccccgagct caataaaaga gcccacaacc cctcactcgg ggcgccagtc   6000 

ctccgattga ctgagtcgcc cgggtacccg tgtatccaat aaaccctctt gcagttgcat   6060 

ccgacttgtg gtctcgctgt tccttgggag ggtctcctct gagtgattga ctacccgtca   6120 

gcgggggtct ttcattcatg cagcatgtat caaaattaat ttggtttttt ttcttaagta   6180 

tttacattaa atggccatag ttgcattaat gaatcggcca acgcgcgggg agaggcggtt   6240 

tgcgtattgg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct   6300 

gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga   6360 

taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc   6420 

cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg   6480 

ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg   6540 

aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt   6600 

tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt   6660 

gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg   6720 

cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact   6780 

ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt   6840 

cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct   6900 

gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac   6960 

cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc   7020 

tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg   7080 

ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta   7140 

aaaatgaagt ttgcggccgc aaatcaatct aaagtatata tgagtaaact tggtctgaca   7200 

gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca   7260 

tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc   7320 

ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa   7380 

accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc   7440 

agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca   7500 

acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat   7560 

tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag   7620 

cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac   7680 

tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt   7740 

ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt   7800 

gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc   7860 

tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat   7920 

ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca   7980 

gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga   8040 

cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg   8100 

gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg   8160 

ttccgcgcac atttc                                                    8175 

 
           
             7  
             8175  
             DNA  
             Unknown  
             
               pICAST OMC.  
             
           
            7 

gacgtcggac ttatacccgg tttgtcctat agacaccatt cgtcaaggac ggggccgagt     60 

cccggttctt gtctaccttg tcgacttata cccggtttgt cctatagaca ccattcgtca    120 

aggacggggc cgagtcccgg ttcttgtcta ccaggggtct acgccaggtc gggagtcgtc    180 

aaagatctct tggtagtcta caaaggtccc acggggttcc tggactttac tgggacacgg    240 

aataaacttg attggttagt caagcgaaga gcgaagacaa gcgcgcgaag acgaggggct    300 

cgagttattt tctcgggtgt tggggagtga gccccgcggt caggaggcta actgactcag    360 

cgggcccatg ggcacatagg ttatttggga gaacgtcaac gtaggctgaa caccagagcg    420 

acaaggaacc ctcccagagg agactcacta actgatgggc agtcgccccc agaaagtaaa    480 

cccccgagca ggccctagcc ctctggggac gggtccctgg tggctgggtg gtggccctcc    540 

gttcgaccgg tcgttgaata gacacagaca ggctaacaga tcacagatac tgactaaaat    600 

acgcggacgc agccatgatc aatcgattga tcgagacata gaccgcctgg gcaccacctt    660 

gactgctcaa gacttgtggg ccggcgttgg gaccctctgc agggtccctg aaacccccgg    720 

caaaaacacc gggctggact ccttccctca gctacacctt aggctggggc agtcctatac    780 

accaagacca tcctctgctc ttggattttg tcaagggcgg aggcagactt aaaaacgaaa    840 

gccaaacctt ggcttcggcg cgcagaacag acgacgtcgt agcaagacac aacagagaca    900 

gactgacaca aagacataaa cagactttta atcccggtct gacaatggtg agggaattca    960 

aactggaatc cattgacctt tctacagctc gccgagcgag tgttggtcag ccatctacag   1020 

ttcttctctg caacccaatg gaagacgaga cgtcttaccg gttggaaatt gcagcctacc   1080 

ggcgctctgc cgtggaaatt ggctctggag tagtgggtcc aattctagtt ccagaaaagt   1140 

ggaccgggcg tacctgtggg tctggtccag gggatgtagc actggaccct tcggaaccga   1200 

aaactggggg gagggaccca gttcgggaaa catgtgggat tcggaggcgg aggagaagga   1260 

ggtaggcggg gcagagaggg ggaacttgga ggagcaagct ggggcggagc taggagggaa   1320 

ataggtcggg agtgaggaag agatccgcgg ccggcgagat cgggtaatta tgctgagtga   1380 

tatcccgcta agcttagtcc ggaaccgcgc ggcctaggaa ttaattcgcg ttaaccctcc   1440 

accgccatcg gagctctacc cgcactaatg cctaagtgac cggcagcaaa atgttgcagc   1500 

actgaccctt ttgggaccgc aatgggttga attagcggaa cgtcgtgtag ggggaaagcg   1560 

gtcgaccgca ttatcgcttc tccgggcgtg gctagcggga agggttgtca atgcgtcgga   1620 

cttaccgctt accgcgaaac ggaccaaagg ccgtggtctt cgccacggcc tttcgaccga   1680 

cctcacgcta gaaggactcc ggctatgaca gcagcagggg agtttgaccg tctacgtgcc   1740 

aatgctacgc gggtagatgt ggttgcactg gatagggtaa tgccagttag gcggcaaaca   1800 

agggtgcctc ttaggctgcc caacaatgag cgagtgtaaa ttacaactac tttcgaccga   1860 

tgtccttccg gtctgcgctt aataaaaact accgcaattg agccgcaaag tagacaccac   1920 

gttgcccgcg acccagccaa tgccggtcct gtcagcaaac ggcagactta aactggactc   1980 

gcgtaaaaat gcgcggcctc ttttggcgga gcgccactac cacgacgcga cctcactgcc   2040 

gtcaatagac cttctagtcc tatacaccgc ctactcgccg taaaaggcac tgcagagcaa   2100 

cgacgtattt ggctgatgtg tttagtcgct aaaggtacaa cggtgagcga aattactact   2160 

aaagtcggcg cgacatgacc tccgacttca agtctacacg ccgctcaacg cactgatgga   2220 

tgcccattgt caaagaaata ccgtcccact ttgcgtccag cggtcgccgt ggcgcggaaa   2280 

gccgccactt taatagctac tcgcaccacc aatacggcta gcgcagtgtg atgcagactt   2340 

gcagcttttg ggctttgaca cctcgcggct ttagggctta gagatagcac gccaccaact   2400 

tgacgtgtgg cggctgccgt gcgactaact tcgtcttcgg acgctacagc caaaggcgct   2460 

ccacgcctaa cttttaccag acgacgacga cttgccgttc ggcaacgact aagctccgca   2520 

attggcagtg ctcgtagtag gagacgtacc agtccagtac ctactcgtct gctaccacgt   2580 

cctataggac gactacttcg tcttgttgaa attgcggcac gcgacaagcg taataggctt   2640 

ggtaggcgac accatgtgcg acacgctggc gatgccggac atacaccacc tacttcggtt   2700 

ataactttgg gtgccgtacc acggttactt agcagactgg ctactaggcg cgaccgatgg   2760 

ccgctactcg cttgcgcatt gcgcttacca cgtcgcgcta gcattagtgg gctcacacta   2820 

gtagaccagc gaccccttac ttagtccggt gccgcgatta gtgctgcgcg acatagcgac   2880 

ctagtttaga cagctaggaa gggcgggcca cgtcatactt ccgccgcctc ggctgtggtg   2940 

ccggtggcta taataaacgg gctacatgcg cgcgcaccta cttctggtcg ggaagggccg   3000 

acacggcttt accaggtagt tttttaccga aagcgatgga cctctctgcg cgggcgacta   3060 

ggaaacgctt atgcgggtgc gctacccatt gtcagaaccg ccaaagcgat ttatgaccgt   3120 

ccgcaaagca gtcatagggg caaatgtccc gccgaagcag accctgaccc acctagtcag   3180 

cgactaattt atactacttt tgccgttggg caccagccga atgccgccac taaaaccgct   3240 

atgcggcttg ctagcggtca agacatactt gccagaccag aaacggctgg cgtgcggcgt   3300 

aggtcgcgac tgccttcgtt ttgtggtcgt cgtcaaaaag gtcaaggcaa ataggcccgt   3360 

ttggtagctt cactggtcgc ttatggacaa ggcagtatcg ctattgctcg aggacgtgac   3420 

ctaccaccgc gacctaccat tcggcgaccg ttcgccactt cacggagacc tacagcgagg   3480 

tgttccattt gtcaactaac ttgacggact tgatggcgtc ggcctctcgc ggcccgttga   3540 

gaccgagtgt catgcgcatc acgttggctt gcgctggcgt accagtcttc ggcccgtgta   3600 

gtcgcggacc gtcgtcaccg cagaccgcct tttggagtca cactgcgagg ggcggcgcag   3660 

ggtgcggtag ggcgtagact ggtggtcgct ttacctaaaa acgtagctcg acccattatt   3720 

cgcaaccgtt aaattggcgg tcagtccgaa agaaagtgtc tacacctaac cgctattttt   3780 

tgttgacgac tgcggcgacg cgctagtcaa gtgggcacag ctatctagac ttgtctttga   3840 

gtaaaggctt cttctggatc agctggtagt agtagtagta gtggccatta ttatccatct   3900 

attcactgac taatctacgt aaagctgatc tagggagctg gttaaggcca ataaaaggtg   3960 

gtataacggc agaaaaccgt tacactcccg ggcctttgga ccgggacaga agaactgctc   4020 

gtaaggatcc ccagaaaggg gagagcggtt tccttacgtt ccagacaact tacagcactt   4080 

ccttcgtcaa ggagaccttc gaagaacttc tgtttgttgc agacatcgct gggaaacgtc   4140 

cgtcgccttg gggggtggac cgctgtccac ggagacgccg gttttcggtg cacatattct   4200 

atgtggacgt ttccgccgtg ttggggtcac ggtgcaacac tcaacctatc aacacctttc   4260 

tcagtttacc gagaggagtt cgcataagtt gttccccgac ttcctacggg tcttccatgg   4320 

ggtaacatac cctagactag accccggagc cacgtgtacg aaatgtacac aaatcagctc   4380 

caattttttg cagatccggg gggcttggtg cccctgcacc aaaaggaaac tttttgtgct   4440 

actattatgg tactttttcg gacttgagtg gcgctgcaga cagctcttca aagactagct   4500 

tttcaagctg tcgcagaggc tggactacgt cgagagcctc ccgcttctta gagcacgaaa   4560 

gtcgaagcta catcctcccg cacctataca ggacgcccat ttatcgacgc ggctaccaaa   4620 

gatgtttcta gcaatacaaa tagccgtgaa acgtagccgg cgcgagggct aaggccttca   4680 

cgaactgtaa ccccttaaat cgcrctcgga ctggataacg tagagggcgg cacgtgtccc   4740 

acagtgcaac gttctggacg gactttggct tgacgggcga caagacgtcg gccagcgcct   4800 

ccggtaccta cgctagcgac gccggctaga atcggtctgc tcgcccaagc cgggtaagcc   4860 

tggcgttcct tagccagtta tgtgatgtac cgcactaaag tatacgcgct aacgactagg   4920 

ggtacacata gtgaccgttt gacactacct gctgtggcag tcacgcaggc agcgcgtccg   4980 

agagctactc gactacgaaa cccggctcct gacggggctt caggccgtgg agcacgtgcg   5040 

cctaaagccg aggttgttac aggactgcct gttaccggcg tattgtcgcc agtaactgac   5100 

ctcgctccgc tacaagcccc taagggttat gctccagcgg ttgtagaaga agacctccgg   5160 

caccaaccga acatacctcg tcgtctgcgc gatgaagctc gcctccgtag gcctcgaacg   5220 

tcctagcggc gccgaggccc gcatatacga ggcgtaacca gaactgcttg agatagtctc   5280 

gaaccaactg ccgttaaagc tactacgtcg aacccgcgtc ccagctacgc tgcgttagca   5340 

ggctaggcct cggccctgac agcccgcatg tgtttagcgg gcgtcttcgc gccggcagac   5400 

ctggctaccg acacatcttc atgagcggct atcacctttg gctgcggggt cgtgagcagg   5460 

ctcccgtttc cttatctcat ctacggctgg ccctagatag ctattttatt ttctaaaata   5520 

aatcagaggt ctttttcccc ccttactttc tggggtggac atccaaaccg ttcgatcgaa   5580 

ttcattgcgg taaaacgttc cgtacctttt tatgtattga ctcttatctc ttcaagtcta   5640 

gttccagtcc ttgtctacct tgtcgactta tacccggttt gtcctataga caccattcgt   5700 

caaggacggg gccgagtccc ggttcttgtc taccttgtcg acttataccc ggtttgtcct   5760 

atagacacca ttcgtcaagg acggggccga gtcccggttc ttgtctacca ggggtctacg   5820 

ccaggtcggg agtcgtcaaa gatctcttgg tagtctacaa aggtcccacg gggttcctgg   5880 

actttactgg gacacggaat aaacttgatt ggttagtcaa gcgaagagcg aagacaagcg   5940 

cgcgaagacg aggggctcga gttattttct cgggtgttgg ggagtgagcc ccgcggtcag   6000 

gaggctaact gactcagcgg gcccatgggc acataggtta tttgggagaa cgtcaacgta   6060 

ggctgaacac cagagcgaca aggaaccctc ccagaggaga ctcactaact gatgggcagt   6120 

cgcccccaga aagtaagtac gtcgtacata gttttaatta aaccaaaaaa aagaattcat   6180 

aaatgtaatt taccggtatc aacgtaatta cttagccggt tgcgcgcccc tctccgccaa   6240 

acgcataacc gcgagaaggc gaaggagcga gtgactgagc gacgcgagcc agcaagccga   6300 

cgccgctcgc catagtcgag tgagtttccg ccattatgcc aataggtgtc ttagtcccct   6360 

attgcgtcct ttcttgtaca ctcgttttcc ggtcgttttc cggtccttgg catttttccg   6420 

gcgcaacgac cgcaaaaagg tatccgaggc ggggggactg ctcgtagtgt ttttagctgc   6480 

gagttcagtc tccaccgctt tgggctgtcc tgatatttct atggtccgca aagggggacc   6540 

ttcgagggag cacgcgagag gacaaggctg ggacggcgaa tggcctatgg acaggcggaa   6600 

agagggaagc ccttcgcacc gcgaaagagt atcgagtgcg acatccatag agtcaagcca   6660 

catccagcaa gcgaggttcg acccgacaca cgtgcttggg gggcaagtcg ggctggcgac   6720 

gcggaatagg ccattgatag cagaactcag gttgggccat tctgtgctga atagcggtga   6780 

ccgtcgtcgg tgaccattgt cctaatcgtc tcgctccata catccgccac gatgtctcaa   6840 

gaacttcacc accggattga tgccgatgtg atcttcttgt cataaaccat agacgcgaga   6900 

cgacttcggt caatggaagc ctttttctca accatcgaga actaggccgt ttgtttggtg   6960 

gcgaccatcg ccaccaaaaa aacaaacgtt cgtcgtctaa tgcgcgtctt tttttcctag   7020 

agttcttcta ggaaactaga aaagatgccc cagactgcga gtcaccttgc ttttgagtgc   7080 

aattccctaa aaccagtact ctaatagttt ttcctagaag tggatctagg aaaatttaat   7140 

ttttacttca aacgccggcg tttagttaga tttcatatat actcatttga accagactgt   7200 

caatggttac gaattagtca ctccgtggat agagtcgcta gacagataaa gcaagtaggt   7260 

atcaacggac tgaggggcag cacatctatt gatgctatgc cctcccgaat ggtagaccgg   7320 

ggtcacgacg ttactatggc gctctgggtg cgagtggccg aggtctaaat agtcgttatt   7380 

tggtcggtcg gccttcccgg ctcgcgtctt caccaggacg ttgaaatagg cggaggtagg   7440 

tcagataatt aacaacggcc cttcgatctc attcatcaag cggtcaatta tcaaacgcgt   7500 

tgcaacaacg gtaacgatgt ccgtagcacc acagtgcgag cagcaaacca taccgaagta   7560 

agtcgaggcc aagggttgct agttccgctc aatgtactag ggggtacaac acgttttttc   7620 

gccaatcgag gaagccagga ggctagcaac agtcttcatt caaccggcgt cacaatagtg   7680 

agtaccaata ccgtcgtgac gtattaagag aatgacagta cggtaggcat tctacgaaaa   7740 

gacactgacc actcatgagt tggttcagta agactcttat cacatacgcc gctggctcaa   7800 

cgagaacggg ccgcagttat gccctattat ggcgcggtgt atcgtcttga aattttcacg   7860 

agtagtaacc ttttgcaaga agccccgctt ttgagagttc ctagaatggc gacaactcta   7920 

ggtcaagcta cattgggtga gcacgtgggt tgactagaag tcgtagaaaa tgaaagtggt   7980 

cgcaaagacc cactcgtttt tgtccttccg ttttacggcg ttttttccct tattcccgct   8040 

gtgcctttac aacttatgag tatgagaagg aaaaagttat aataacttcg taaatagtcc   8100 

caataacaga gtactcgcct atgtataaac ttacataaat ctttttattt gtttatcccc   8160 

aaggcgcgtg taaag                                                    8175 

 
           
             8  
             8161  
             DNA  
             Unknown  
             
               pICAST OMN.  
             
           
            8 

ctgcagcctg aatatgggcc aaacaggata tctgtggtaa gcagttcctg ccccggctca     60 

gggccaagaa cagatggaac agctgaatat gggccaaaca ggatatctgt ggtaagcagt    120 

tcctgccccg gctcagggcc aagaacagat ggtccccaga tgcggtccag ccctcagcag    180 

tttctagaga accatcagat gtttccaggg tgccccaagg acctgaaatg accctgtgcc    240 

ttatttgaac taaccaatca gttcgcttct cgcttctgtt cgcgcgcttc tgctccccga    300 

gctcaataaa agagcccaca acccctcact cggggcgcca gtcctccgat tgactgagtc    360 

gcccgggtac ccgtgtatcc aataaaccct cttgcagttg catccgactt gtggtctcgc    420 

tgttccttgg gagggtctcc tctgagtgat tgactacccg tcagcggggg tctttcattt    480 

gggggctcgt ccgggatcgg gagacccctg cccagggacc accgacccac caccgggagg    540 

caagctggcc agcaacttat ctgtgtctgt ccgattgtct agtgtctatg actgatttta    600 

tgcgcctgcg tcggtactag ttagctaact agctctgtat ctggcggacc cgtggtggaa    660 

ctgacgagtt ctgaacaccc ggccgcaacc ctgggagacg tcccagggac tttgggggcc    720 

gtttttgtgg cccgacctga ggaagggagt cgatgtggaa tccgaccccg tcaggatatg    780 

tggttctggt aggagacgag aacctaaaac agttcccgcc tccgtctgaa tttttgcttt    840 

cggtttggaa ccgaagccgc gcgtcttgtc tgctgcagca tcgttctgtg ttgtctctgt    900 

ctgactgtgt ttctgtattt gtctgaaaat tagggccaga ctgttaccac tcccttaagt    960 

ttgaccttag gtaactggaa agatgtcgag cggctcgctc acaaccagtc ggtagatgtc   1020 

aagaagagac gttgggttac cttctgctct gcagaatggc caacctttaa cgtcggatgg   1080 

ccgcgagacg gcacctttaa ccgagacctc atcacccagg ttaagatcaa ggtcttttca   1140 

cctggcccgc atggacaccc agaccaggtc ccctacatcg tgacctggga agccttggct   1200 

tttgaccccc ctccctgggt caagcccttt gtacacccta agcctccgcc tcctcttcct   1260 

ccatccgccc cgtctctccc ccttgaacct cctcgttcga ccccgcctcg atcctccctt   1320 

tatccagccc tcactccttc tctaggcgcc ggccgctcta gcccattaat acgactcact   1380 

atagggcgat tcgaacacca tgcaccatca tcatcatcac gtcgacgaac agaaactcat   1440 

ttccgaagaa gacctactcg agatgggcgt gattacggat tcactggccg tcgttttaca   1500 

acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc   1560 

tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttacg   1620 

cagcctgaat ggcgaatggc gctttgcctg gtttccggca ccagaagcgg tgccggaaag   1680 

ctggctggag tgcgatcttc ctgaggccga tactgtcgtc gtcccctcaa actggcagat   1740 

gcacggttac gatgcgccca tctacaccaa cgtgacctat cccattacgg tcaatccgcc   1800 

gtttgttccc acggagaatc cgacgggttg ttactcgctc acatttaatg ttgatgaaag   1860 

ctggctacag gaaggccaga cgcgaattat ttttgatggc gttaactcgg cgtttcatct   1920 

gtggtgcaac gggcgctggg tcggttacgg ccaggacagt cgtttgccgt ctgaatttga   1980 

cctgagcgca tttttacgcg ccggagaaaa ccgcctcgcg gtgatggtgc tgcgctggag   2040 

tgacggcagt tatctggaag atcaggatat gtggcggatg agcggcattt tccgtgacgt   2100 

ctcgttgctg cataaaccga ctacacaaat cagcgatttc catgttgcca ctcgctttaa   2160 

tgatgatttc agccgcgctg tactggaggc tgaagttcag atgtgcggcg agttgcgtga   2220 

ctacctacgg gtaacagttt ctttatggca gggtgaaacg caggtcgcca gcggcaccgc   2280 

gcctttcggc ggtgaaatta tcgatgagcg tggtggttat gccgatcgcg tcacactacg   2340 

tctgaacgtc gaaaacccga aactgtggag cgccgaaatc ccgaatctct atcgtgcggt   2400 

ggttgaactg cacaccgccg acggcacgct gattgaagca gaagcctgcg atgtcggttt   2460 

ccgcgaggtg cggattgaaa atggtctgct gctgctgaac ggcaagccgt tgctgattcg   2520 

aggcgttaac cgtcacgagc atcatcctct gcatggtcag gtcatggatg agcagacgat   2580 

ggtgcaggat atcctgctga tgaagcagaa caactttaac gccgtgcgct gttcgcatta   2640 

tccgaaccat ccgctgtggt acacgctgtg cgaccgctac ggcctgtatg tggtggatga   2700 

agccaatatt gaaacccacg gcatggtgcc aatgaatcgt ctgaccgatg atccgcgctg   2760 

gctaccggcg atgagcgaac gcgtaacgcg aatggtgcag cgcgatcgta atcacccgag   2820 

tgtgatcatc tggtcgctgg ggaatgaatc aggccacggc gctaatcacg acgcgctgta   2880 

tcgctggatc aaatctgtcg atccttcccg cccggtgcag tatgaaggcg gcggagccga   2940 

caccacggcc accgatatta tttgcccgat gtacgcgcgc gtggatgaag accagccctt   3000 

cccggctgtg ccgaaatggt ccatcaaaaa atggctttcg ctacctggag agacgcgccc   3060 

gctgatcctt tgcgaatacg cccacgcgat gggtaacagt cttggcggtt tcgctaaata   3120 

ctggcaggcg tttcgtcagt atccccgttt acagggcggc ttcgtctggg actgggtgga   3180 

tcagtcgctg attaaatatg atgaaaacgg caacccgtgg tcggcttacg gcggtgattt   3240 

tggcgatacg ccgaacgatc gccagttctg tatgaacggt ctggtctttg ccgaccgcac   3300 

gccgcatcca gcgctgacgg aagcaaaaca ccagcagcag tttttccagt tccgtttatc   3360 

cgggcaaacc atcgaagtga ccagcgaata cctgttccgt catagcgata acgagctcct   3420 

gcactggatg gtggcgctgg atggtaagcc gctggcaagc ggtgaagtgc ctctggatgt   3480 

cgctccacaa ggtaaacagt tgattgaact gcctgaacta ccgcagccgg agagcgccgg   3540 

gcaactctgg ctcacagtac gcgtagtgca accgaacgcg accgcatggt cagaagccgg   3600 

gcacatcagc gcctggcagc agtggcgtct ggcggaaaac ctcagtgtga cgctccccgc   3660 

cgcgtcccac gccatcccgc atctgaccac cagcgaaatg gatttttgca tcgagctggg   3720 

taataagcgt tggcaattta accgccagtc aggctttctt tcacagatgt ggattggcga   3780 

taaaaaacaa ctgctgacgc cgctgcgcga tcagttcacc cgtgtcgata gatctggagg   3840 

tggtggcagc aggccttggc gcgccggatc cttaattaac aattgaccgg taataatagg   3900 

tagataagtg actgattaga tgcatttcga ctagatccct cgaccaattc cggttatttt   3960 

ccaccatatt gccgtctttt ggcaatgtga gggcccggaa acctggccct gtcttcttga   4020 

cgagcattcc taggggtctt tcccctctcg ccaaaggaat gcaaggtctg ttgaatgtcg   4080 

tgaaggaagc agttcctctg gaagcttctt gaagacaaac aacgtctgta gcgacccttt   4140 

gcaggcagcg gaacccccca cctggcgaca ggtgcctctg cggccaaaag ccacgtgtat   4200 

aagatacacc tgcaaaggcg gcacaacccc agtgccacgt tgtgagttgg atagttgtgg   4260 

aaagagtcaa atggctctcc tcaagcgtat tcaacaaggg gctgaaggat gcccagaagg   4320 

taccccattg tatgggatct gatctggggc ctcggtgcac atgctttaca tgtgtttagt   4380 

cgaggttaaa aaacgtctag gccccccgaa ccacggggac gtggttttcc tttgaaaaac   4440 

acgatgataa taccatgaaa aagcctgaac tcaccgcgac gtctgtcgag aagtttctga   4500 

tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc ggagggcgaa gaatctcgtg   4560 

ctttcagctt cgatgtagga gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg   4620 

gtttctacaa agatcgttat gtttatcggc actttgcatc ggccgcgctc ccgattccgg   4680 

aagtgcttga cattggggaa tttagcgaga gcctgaccta ttgcatctcc cgccgtgcac   4740 

agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg   4800 

cggaggccat ggatgcgatc gctgcggccg atcttagcca gacgagcggg ttcggcccat   4860 

tcggaccgca aggaatcggt caatacacta catggcgtga tttcatatgc gcgattgctg   4920 

atccccatgt gtatcactgg caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc   4980 

aggctctcga tgagctgatg ctttgggccg aggactgccc cgaagtccgg cacctcgtgc   5040 

acgcggattt cggctccaac aatgtcctga cggacaatgg ccgcataaca gcggtcattg   5100 

actggagcga ggcgatgttc ggggattccc aatacgaggt cgccaacatc ttcttctgga   5160 

ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt cgagcggagg catccggagc   5220 

ttgcaggatc gccgcggctc cgggcgtata tgctccgcat tggtcttgac caactctatc   5280 

agagcttggt tgacggcaat ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa   5340 

tcgtccgatc cggagccggg actgtcgggc gtacacaaat cgcccgcaga agcgcggccg   5400 

tctggaccga tggctgtgta gaagtactcg ccgatagtgg aaaccgacgc cccagcactc   5460 

gtccgagggc aaaggaatag agtagatgcc gaccgggatc tatcgataaa ataaaagatt   5520 

ttatttagtc tccagaaaaa ggggggaatg aagaccccaa cctgtaggtt tggcaagcta   5580 

gcttaagtaa cgccattttg caaggcatgg aaaaatacat aactgagaat agagaagttc   5640 

agatcaaggt caggaacaga tggaacagct gaatatgggc caaacaggat atctgtggta   5700 

agcagttcct gccccggctc agggccaaga acagatggaa cagctgaata tgggccaaac   5760 

aggatatctg tggtaagcag ttcctgcccc ggctcagggc caagaacaga tggtccccag   5820 

atgcggtcca gccctcagca gtttctagag aaccatcaga tgtttccagg gtgccccaag   5880 

gacctgaaat gaccctgtgc cttatttgaa ctaaccaatc agttcgcttc tcgcttctgt   5940 

tcgcgcgctt ctgctccccg agctcaataa aagagcccac aacccctcac tcggggcgcc   6000 

agtcctccga ttgactgagt cgcccgggta cccgtgtatc caataaaccc tcttgcagtt   6060 

gcatccgact tgtggtctcg ctgttccttg ggagggtctc ctctgagtga ttgactaccc   6120 

gtcagcgggg gtctttcatt catgcagcat gtatcaaaat taatttggtt ttttttctta   6180 

agtatttaca ttaaatggcc atagttgcat taatgaatcg gccaacgcgc ggggagaggc   6240 

ggtttgcgta ttggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc   6300 

ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag   6360 

gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa   6420 

aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc   6480 

gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc   6540 

ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg   6600 

cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt   6660 

cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc   6720 

gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc   6780 

cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag   6840 

agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg   6900 

ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa   6960 

ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag   7020 

gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact   7080 

cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttgc   7140 

ggccgcaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta   7200 

atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc   7260 

cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg   7320 

ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga   7380 

agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt   7440 

tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt   7500 

gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc   7560 

caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc   7620 

ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca   7680 

gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag   7740 

tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg   7800 

tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa   7860 

cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa   7920 

cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga   7980 

gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga   8040 

atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg   8100 

agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt   8160 

c                                                                   8161 

 
           
             9  
             8161  
             DNA  
             Unknown  
             
               pICAST OMN.  
             
           
            9 

gacgtcggac ttatacccgg tttgtcctat agacaccatt cgtcaaggac ggggccgagt     60 

cccggttctt gtctaccttg tcgacttata cccggtttgt cctatagaca ccattcgtca    120 

aggacggggc cgagtcccgg ttcttgtcta ccaggggtct acgccaggtc gggagtcgtc    180 

aaagatctct tggtagtcta caaaggtccc acggggttcc tggactttac tgggacacgg    240 

aataaacttg attggttagt caagcgaaga gcgaagacaa gcgcgcgaag acgaggggct    300 

cgagttattt tctcgggtgt tggggagtga gccccgcggt caggaggcta actgactcag    360 

cgggcccatg ggcacatagg ttatttggga gaacgtcaac gtaggctgaa caccagagcg    420 

acaaggaacc ctcccagagg agactcacta actgatgggc agtcgccccc agaaagtaaa    480 

cccccgagca ggccctagcc ctctggggac gggtccctgg tggctgggtg gtggccctcc    540 

gttcgaccgg tcgttgaata gacacagaca ggctaacaga tcacagatac tgactaaaat    600 

acgcggacgc agccatgatc aatcgattga tcgagacata gaccgcctgg gcaccacctt    660 

gactgctcaa gacttgtggg ccggcgttgg gaccctctgc agggtccctg aaacccccgg    720 

caaaaacacc gggctggact ccttccctca gctacacctt aggctggggc agtcctatac    780 

accaagacca tcctctgctc ttggattttg tcaagggcgg aggcagactt aaaaacgaaa    840 

gccaaacctt ggcttcggcg cgcagaacag acgacgtcgt agcaagacac aacagagaca    900 

gactgacaca aagacataaa cagactttta atcccggtct gacaatggtg agggaattca    960 

aactggaatc cattgacctt tctacagctc gccgagcgag tgttggtcag ccatctacag   1020 

ttcttctctg caacccaatg gaagacgaga cgtcttaccg gttggaaatt gcagcctacc   1080 

ggcgctctgc cgtggaaatt ggctctggag tagtgggtcc aattctagtt ccagaaaagt   1140 

ggaccgggcg tacctgtggg tctggtccag gggatgtagc actggaccct tcggaaccga   1200 

aaactggggg gagggaccca gttcgggaaa catgtgggat tcggaggcgg aggagaagga   1260 

ggtaggcggg gcagagaggg ggaacttgga ggagcaagct ggggcggagc taggagggaa   1320 

ataggtcggg agtgaggaag agatccgcgg ccggcgagat cgggtaatta tgctgagtga   1380 

tatcccgcta agcttgtggt acgtggtagt agtagtagtg cagctgcttg tctttgagta   1440 

aaggcttctt ctggatgagc tctacccgca ctaatgccta agtgaccggc agcaaaatgt   1500 

tgcagcactg acccttttgg gaccgcaatg ggttgaatta gcggaacgtc gtgtaggggg   1560 

aaagcggtcg accgcattat cgcttctccg ggcgtggcta gcgggaaggg ttgtcaatgc   1620 

gtcggactta ccgcttaccg cgaaacggac caaaggccgt ggtcttcgcc acggcctttc   1680 

gaccgacctc acgctagaag gactccggct atgacagcag caggggagtt tgaccgtcta   1740 

cgtgccaatg ctacgcgggt agatgtggtt gcactggata gggtaatgcc agttaggcgg   1800 

caaacaaggg tgcctcttag gctgcccaac aatgagcgag tgtaaattac aactactttc   1860 

gaccgatgtc cttccggtct gcgcttaata aaaactaccg caattgagcc gcaaagtaga   1920 

caccacgttg cccgcgaccc agccaatgcc ggtcctgtca gcaaacggca gacttaaact   1980 

ggactcgcgt aaaaatgcgc ggcctctttt ggcggagcgc cactaccacg acgcgacctc   2040 

actgccgtca atagaccttc tagtcctata caccgcctac tcgccgtaaa aggcactgca   2100 

gagcaacgac gtatttggct gatgtgttta gtcgctaaag gtacaacggt gagcgaaatt   2160 

actactaaag tcggcgcgac atgacctccg acttcaagtc tacacgccgc tcaacgcact   2220 

gatggatgcc cattgtcaaa gaaataccgt cccactttgc gtccagcggt cgccgtggcg   2280 

cggaaagccg ccactttaat agctactcgc accaccaata cggctagcgc agtgtgatgc   2340 

agacttgcag cttttgggct ttgacacctc gcggctttag ggcttagaga tagcacgcca   2400 

ccaacttgac gtgtggcggc tgccgtgcga ctaacttcgt cttcggacgc tacagccaaa   2460 

ggcgctccac gcctaacttt taccagacga cgacgacttg ccgttcggca acgactaagc   2520 

tccgcaattg gcagtgctcg tagtaggaga cgtaccagtc cagtacctac tcgtctgcta   2580 

ccacgtccta taggacgact acttcgtctt gttgaaattg cggcacgcga caagcgtaat   2640 

aggcttggta ggcgacacca tgtgcgacac gctggcgatg ccggacatac accacctact   2700 

tcggttataa ctttgggtgc cgtaccacgg ttacttagca gactggctac taggcgcgac   2760 

cgatggccgc tactcgcttg cgcattgcgc ttaccacgtc gcgctagcat tagtgggctc   2820 

acactagtag accagcgacc ccttacttag tccggtgccg cgattagtgc tgcgcgacat   2880 

agcgacctag tttagacagc taggaagggc gggccacgtc atacttccgc cgcctcggct   2940 

gtggtgccgg tggctataat aaacgggcta catgcgcgcg cacctacttc tggtcgggaa   3000 

gggccgacac ggctttacca ggtagttttt taccgaaagc gatggacctc tctgcgcggg   3060 

cgactaggaa acgcttatgc gggtgcgcta cccattgtca gaaccgccaa agcgatttat   3120 

gaccgtccgc aaagcagtca taggggcaaa tgtcccgccg aagcagaccc tgacccacct   3180 

agtcagcgac taatttatac tacttttgcc gttgggcacc agccgaatgc cgccactaaa   3240 

accgctatgc ggcttgctag cggtcaagac atacttgcca gaccagaaac ggctggcgtg   3300 

cggcgtaggt cgcgactgcc ttcgttttgt ggtcgtcgtc aaaaaggtca aggcaaatag   3360 

gcccgtttgg tagcttcact ggtcgcttat ggacaaggca gtatcgctat tgctcgagga   3420 

cgtgacctac caccgcgacc taccattcgg cgaccgttcg ccacttcacg gagacctaca   3480 

gcgaggtgtt ccatttgtca actaacttga cggacttgat ggcgtcggcc tctcgcggcc   3540 

cgttgagacc gagtgtcatg cgcatcacgt tggcttgcgc tggcgtacca gtcttcggcc   3600 

cgtgtagtcg cggaccgtcg tcaccgcaga ccgccttttg gagtcacact gcgaggggcg   3660 

gcgcagggtg cggtagggcg tagactggtg gtcgctttac ctaaaaacgt agctcgaccc   3720 

attattcgca accgttaaat tggcggtcag tccgaaagaa agtgtctaca cctaaccgct   3780 

attttttgtt gacgactgcg gcgacgcgct agtcaagtgg gcacagctat ctagacctcc   3840 

accaccgtcg tccggaaccg cgcggcctag gaattaattg ttaactggcc attattatcc   3900 

atctattcac tgactaatct acgtaaagct gatctaggga gctggttaag gccaataaaa   3960 

ggtggtataa cggcagaaaa ccgttacact cccgggcctt tggaccggga cagaagaact   4020 

gctcgtaagg atccccagaa aggggagagc ggtttcctta cgttccagac aacttacagc   4080 

acttccttcg tcaaggagac cttcgaagaa cttctgtttg ttgcagacat cgctgggaaa   4140 

cgtccgtcgc cttggggggt ggaccgctgt ccacggagac gccggttttc ggtgcacata   4200 

ttctatgtgg acgtttccgc cgtgttgggg tcacggtgca acactcaacc tatcaacacc   4260 

tttctcagtt taccgagagg agttcgcata agttgttccc cgacttccta cgggtcttcc   4320 

atggggtaac ataccctaga ctagaccccg gagccacgtg tacgaaatgt acacaaatca   4380 

gctccaattt tttgcagatc cggggggctt ggtgcccctg caccaaaagg aaactttttg   4440 

tgctactatt atggtacttt ttcggacttg agtggcgctg cagacagctc ttcaaagact   4500 

agcttttcaa gctgtcgcag aggctggact acgtcgagag cctcccgctt cttagagcac   4560 

gaaagtcgaa gctacatcct cccgcaccta tacaggacgc ccatttatcg acgcggctac   4620 

caaagatgtt tctagcaata caaatagccg tgaaacgtag ccggcgcgag ggctaaggcc   4680 

ttcacgaact gtaacccctt aaatcgctct cggactggat aacgtagagg gcggcacgtg   4740 

tcccacagtg caacgttctg gacggacttt ggcttgacgg gcgacaagac gtcggccagc   4800 

gcctccggta cctacgctag cgacgccggc tagaatcggt ctgctcgccc aagccgggta   4860 

agcctggcgt tccttagcca gttatgtgat gtaccgcact aaagtatacg cgctaacgac   4920 

taggggtaca catagtgacc gtttgacact acctgctgtg gcagtcacgc aggcagcgcg   4980 

tccgagagct actcgactac gaaacccggc tcctgacggg gcttcaggcc gtggagcacg   5040 

tgcgcctaaa gccgaggttg ttacaggact gcctgttacc ggcgtattgt cgccagtaac   5100 

tgacctcgct ccgctacaag cccctaaggg ttatgctcca gcggttgtag aagaagacct   5160 

ccggcaccaa ccgaacatac ctcgtcgtct gcgcgatgaa gctcgcctcc gtaggcctcg   5220 

aacgtcctag cggcgccgag gcccgcatat acgaggcgta accagaactg gttgagatag   5280 

tctcgaacca actgccgtta aagctactac gtcgaacccg cgtcccagct acgctgcgtt   5340 

agcaggctag gcctcggccc tgacagcccg catgtgttta gcgggcgtct tcgcgccggc   5400 

agacctggct accgacacat cttcatgagc ggctatcacc tttggctgcg gggtcgtgag   5460 

caggctcccg tttccttatc tcatctacgg ctggccctag atagctattt tattttctaa   5520 

aataaatcag aggtcttttt ccccccttac tttctggggt ggacatccaa accgttcgat   5580 

cgaattcatt gcggtaaaac gttccgtacc tttttatgta ttgactctta tctcttcaag   5640 

tctagttcca gtccttgtct accttgtcga cttatacccg gtttgtccta tagacaccat   5700 

tcgtcaagga cggggccgag tcccggttct tgtctacctt gtcgacttat acccggtttg   5760 

tcctatagac accattcgtc aaggacgggg ccgagtcccg gttcttgtct accaggggtc   5820 

tacgccaggt cgggagtcgt caaagatctc ttggtagtct acaaaggtcc cacggggttc   5880 

ctggacttta ctgggacacg gaataaactt gattggttag tcaagcgaag agcgaagaca   5940 

agcgcgcgaa gacgaggggc tcgagttatt ttctcgggtg ttggggagtg agccccgcgg   6000 

tcaggaggct aactgactca gcgggcccat gggcacatag gttatttggg agaacgtcaa   6060 

cgtaggctga acaccagagc gacaaggaac cctcccagag gagactcact aactgatggg   6120 

cagtcgcccc cagaaagtaa gtacgtcgta catagtttta attaaaccaa aaaaaagaat   6180 

tcataaatgt aatttaccgg tatcaacgta attacttagc cggttgcgcg cccctctccg   6240 

ccaaacgcat aaccgcgaga aggcgaagga gcgagtgact gagcgacgcg agccagcaag   6300 

ccgacgccgc tcgccatagt cgagtgagtt tccgccatta tgccaatagg tgtcttagtc   6360 

ccctattgcg tcctttcttg tacactcgtt ttccggtcgt tttccggtcc ttggcatttt   6420 

tccggcgcaa cgaccgcaaa aaggtatccg aggcgggggg actgctcgta gtgtttttag   6480 

ctgcgagttc agtctccacc gctttgggct gtcctgatat ttctatggtc cgcaaagggg   6540 

gaccttcgag ggagcacgcg agaggacaag gctgggacgg cgaatggcct atggacaggc   6600 

ggaaagaggg aagcccttcg caccgcgaaa gagtatcgag tgcgacatcc atagagtcaa   6660 

gccacatcca gcaagcgagg ttcgacccga cacacgtgct tggggggcaa gtcgggctgg   6720 

cgacgcggaa taggccattg atagcagaac tcaggttggg ccattctgtg ctgaatagcg   6780 

gtgaccgtcg tcggtgacca ttgtcctaat cgtctcgctc catacatccg ccacgatgtc   6840 

tcaagaactt caccaccgga ttgatgccga tgtgatcttc ttgtcataaa ccatagacgc   6900 

gagacgactt cggtcaatgg aagccttttt ctcaaccatc gagaactagg ccgtttgttt   6960 

ggtggcgacc atcgccacca aaaaaacaaa cgttcgtcgt ctaatgcgcg tctttttttc   7020 

ctagagttct tctaggaaac tagaaaagat gccccagact gcgagtcacc ttgcttttga   7080 

gtgcaattcc ctaaaaccag tactctaata gtttttccta gaagtggatc taggaaaacg   7140 

ccggcgttta gttagatttc atatatactc atttgaacca gactgtcaat ggttacgaat   7200 

tagtcactcc gtggatagag tcgctagaca gataaagcaa gtaggtatca acggactgag   7260 

gggcagcaca tctattgatg ctatgccctc ccgaatggta gaccggggtc acgacgttac   7320 

tatggcgctc tgggtgcgag tggccgaggt ctaaatagtc gttatttggt cggtcggcct   7380 

tcccggctcg cgtcttcacc aggacgttga aataggcgga ggtaggtcag ataattaaca   7440 

acggcccttc gatctcattc atcaagcggt caattatcaa acgcgttgca acaacggtaa   7500 

cgatgtccgt agcaccacag tgcgagcagc aaaccatacc gaagtaagtc gaggccaagg   7560 

gttgctagtt ccgctcaatg tactaggggg tacaacacgt tttttcgcca atcgaggaag   7620 

ccaggaggct agcaacagtc ttcattcaac cggcgtcaca atagtgagta ccaataccgt   7680 

cgtgacgtat taagagaatg acagtacggt aggcattcta cgaaaagaca ctgaccactc   7740 

atgagttggt tcagtaagac tcttatcaca tacgccgctg gctcaacgag aacgggccgc   7800 

agttatgccc tattatggcg cggtgtatcg tcttgaaatt ttcacgagta gtaacctttt   7860 

gcaagaagcc ccgcttttga gagttcctag aatggcgaca actctaggtc aagctacatt   7920 

gggtgagcac gtgggttgac tagaagtcgt agaaaatgaa agtggtcgca aagacccact   7980 

cgtttttgtc cttccgtttt acggcgtttt ttcccttatt cccgctgtgc ctttacaact   8040 

tatgagtatg agaaggaaaa agttataata acttcgtaaa tagtcccaat aacagagtac   8100 

tcgcctatgt ataaacttac ataaatcttt ttatttgttt atccccaagg cgcgtgtaaa   8160 

g                                                                   8161