PATENT ABSTRACT
A method of altering degradation of heterologous proteins in transgenic plants has now been found that utilizes ER-localizing proteins of plant viruses as part of a fusion protein. An engineered fusion protein is protected from degradation by a viral ER-localizing protein, and made more susceptible to degradation by certain mutant viral proteins that fail to localize to the ER.

PATENT DESCRIPTION
CROSS-REFERENCE TO RELATED APPLICATION  
       [0001]    This application claims the benefit of U. S. Provisional Patent Application Serial No. 60/218,504, filed Jul. 15, 2000. 
     
    
     
       TECHNICAL FIELD OF INVENTION  
         [0002]    This invention relates to a method of altering the rate of degradation of proteins in plant cells.  
         BACKGROUND OF THE INVENTION  
         [0003]    Intracellular protein concentration is influenced by many factors, including the rates of transcription, translation, and degradation. When cells are engineered to express a protein from a transgene, robust and stable expression may be desired. In other circumstances, limited accumulation of a protein from a transgene may be desired. Being able to modulate an engineered protein&#39;s rate of degradation has numerous applications.  
           [0004]    One advantage of being able to manipulate a protein&#39;s degradation rate is to increase its intracellular concentration to study its function. After translation, protein levels are controlled by protease activity (Vierstra, 1996) which can limit the accumulation of proteins under study to levels that prevent their biochemical characterization. Another advantage to increasing a selected protein&#39;s intracellular concentration is that it may enhance the accumulation of foreign proteins with beneficial traits in transgenic plants (Vierstra, 1996). As a contrast, there may also be advantages to enhancing a protein&#39;s degradation. The identification of sequences that lead to faster degradation of proteins will benefit researchers interested in repressing accumulation of unwanted endogenous proteins that interfere with important agronomic processes (Vierstra, 1996). Interest in methods to regulate protein accumulation is reflected by the approaches that have been previously reported. One method includes modifying the primary sequence to remove domains conferring instability, and another method is to inhibit proteases (reviewed in Vierstra, 1996.) In another instance, ubiquitin, a stable protein, was fused to a poorly expressed protein to enhance the expression of the latter (Eker et al. 1989).  
           [0005]    Non-host proteins are produced in viral-infected plants. During tobacco mosaic virus (TMV) infection, two such proteins are the 126 kDa protein and the 183 kDa protein, a read-through product containing the 126 kDa protein sequence. Description of these proteins in the prior art indicate that they play a role in replication. Approximately 10% of the 126 kDa protein heterodimerizes with essentially all of the 183 kDa protein in the plant cell, even though the 183 kDa protein alone is capable of replicating the virus in infected cells (Watanabe et al., 1999; Lewandowski and Dawson, 2000). Both proteins are reportedly required for efficient TMV replication in vivo (Osman and Buck, 1996; Watanabe et al., 1999). In fact, the 126 kDa/183 kDa proteins were found with other TMV and host plant factors in the viral replication complex (Heinlein et al., 1998). Additionally, the 126 kDa/183 kDa proteins have putative methyltransferase and helicase domains. Furthermore, the 183 kDa protein contains a carboxy terminal domain required for RNA-dependent RNA polymerase activity.  
           [0006]    Although the role of the 126 kDa protein and/or the 183 kDa protein of TMV is thought in the prior art to be replication, its intracellular localization was unknown. Mas and Beachy (1999) observed that the 126 kDa protein of TMV co-localizes with viral RNA in subcellular bodies and with luminal binding protein (BiP), an endoplasmic reticulum (ER)-specific protein, in infected plants. Although these observations suggested to Mas and Beachy that the 126 kDa protein and/or the 183 kDa protein of TMV localizes to the ER, the localization signal of the proteins was not identified.  
           [0007]    Comparing the 126 kDa protein and/or the 183 kDa protein of TMV to another species suggested in the prior art that the proteins may localize to the ER. Brome mosaic virus BMV), a virus related to Tobacco mosaic virus (TMV), possesses a protein believed to be homologous in function to the 126 kDa protein of TMV, although its overall sequence identity with the TMV protein is 13%. Previous publications determined that the BMV 1a protein localized to the ER during infection of barley cells and that, in the absence of other viral proteins, it localized to the ER in yeast (Restrepo-Hartwig and Ahlquist 1999). Therefore, the BMV 1a protein, like its putative TMV homolog, may localize to specific subcellular locations. In additional to localizing to the endoplasmic reticulum in yeast, the 1a protein also stabilized viral RNA (Sullivan and Ahlquist, 1999) and decreased the viral RNA translation (Janda and Ahlquist, 1998).  
           [0008]    The post-translational regulation of the 126 kDa protein and/or the 183 kDa protein of TMV has also been studied in the prior art, but only with ambiguous results. Previous reports indicated that 26S proteasome inhibitors had no significant effect on 126 kDa or 183 kDa protein accumulation in plant cell suspensions infected with TMV (Reichel and Beachy, 2000). In late stages of TMV infection, what little effect occurred indicated that the protein was more susceptible to degradation in the presence of the 26S proteasome inhibitor. From these results it appeared that induction of 26S proteasome activity had no significant influence on the degradation of the 126 kDa protein. Importantly, the ability of the 126 kDa protein to stabilize its expression in the absence of other viral proteins was not tested in these studies by Reichel and Beachy.  
           [0009]    There is a desire and a need in agronomic biotechnology to modulate the expression level of engineered proteins. Expression in cells engineered to express a protein from a transgene may be robust and stable; in other circumstances, limited accumulation of a protein from a transgene may be desired. To fulfill that need, we have developed a ubiquitin-fusion—independent system in which the degradation—and hence the protein level—of an engineered protein can be modulated in plant cells. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0010]    [0010]FIG. 1 is a partial protein sequence alignment (amino acids 361-370 of SEQ ID NO:2 and SEQ ID NO:4) of the TMV 126/183 kDa protein and its functional analogs from Sindbis—like plant viruses. The conserved “WFP” motif is boxed and the amino acids in bold type are identical to amino acids 365-367 of SEQ ID NO:2 and SEQ ID NO:4. The underlined letters (serine 361 and lysine 368) show the amino acids in the TMV U1 strain that are different from that of the M IC  strain. AMV: alfalfa mosaic virus (SEQ ID NO:9); BMV: brome mosaic virus (SEQ ID NO:10); CiLRV: citrus leaf rugose virus (SEQ ID NO:11); CMV: cucumber mosaic virus (SEQ ID NO:12); SHMV: sunn-hemp mosaic virus (SEQ ID NO:13); TMV: tobacco mosaic virus U1 strain (SEQ ID NO:14); TRV: tobacco rattle virus (SEQ ID NO:15); and TVCV: turnip vein clearing virus (SEQ ID NO:16).  
         [0011]    [0011]FIG. 2A depicts the genome organization of TMV. Three open reading frames (ORFs) which encode the 126 kDa protein (1-3348 of SEQ ID NO:1) and the read-through 183 kDa protein (1-4831 of SEQ ID NO:3), the movement protein (horizontal stripes), and the coat protein (dotted). A black arrowhead indicates the location of the leaky amber stop codon (UAG) within the replicase ORF. The methyltransferase domain of the 126/183 kDa protein is represented with vertical stripes, beginning at nucleotide 142 of SEQ ID NO:1 and ending at nucleotide 900 of SEQ ID NO:1. The helicase domain of the 126/183 kDa protein is represented with diamonds, beginning at nucleotide 2362 of SEQ ID NO:1 and ending at nucleotide 3249 of SEQ ID NO:1. GDD (white) is a motif present in viral RNA-dependent RNA polymerase. Domains I and II of the 126/183 kDa protein each have 4 amino acid mutations that were identified to control the phenotype difference between the TMV U1 strain, which causes severe symptoms, and the cloned Masked strain of TMV (M IC ), which causes mild symptoms. Nucleotide numbers in the figure refer to the entire genome of TMV, Genbank Accession No. AF273221.  
         [0012]    [0012]FIG. 2B depicts the different amino acids present within Domains I and II of the 126/183 kDa protein and the resulting symptoms of the viruses. The following sequences were aligned: TMV-U1 (SEQ ID NO:17), the parental TMV-M IC  (SEQ ID NO:18), and the site-directed mutant viruses studied, TMV-M IC 2 (SEQ ID NO:19), TMV-WAP (SEQ ID NO:20), and TMV-WYP (SEQ ID NO:21). Among all sequences, the amino acids in 8 positions (four mutations in each of two domains) were determined to vary: 325, 360, 367, 416, 587, 601, 668, and 747, referring to the entire genome of TMV, Genbank Accession No. AF273221.  
         [0013]    [0013]FIG. 3A depicts the lesion response (pictorially white spots) on a  N. tabacum  Xanthi “NN” leaf challenged with WFP and WYP viruses. Each half of the leaf was inoculated with either the WFP or WYP virus and the plant grown at 24° C. for ten days. The side of the leaf inoculated with the WFP virus resulted in a slightly larger lesions than the side of the leaf infected with the WYP virus.  
         [0014]    [0014]FIG. 3B depicts the lesion response (pictorially white spots) on a  N. tabacum  Xanthi “NN” leaf challenged with WFP and WYP viruses. Each half of the leaf was inoculated with either the WFP or WYP virus and the plant grown at 32° C. for three days. The temperature was then decreased to 24° C. for another seven days. The size of lesions on the WFP virus—treated side of the leaf increased relative to the side of the leaf treated with the WYP virus.  
         [0015]    FIGS.  4 A-H depict immunolabeling experiments in  N. tabacum  BY-2 protoplasts. All images for FIGS.  4 A-H were captured by confocal laser scanning microscopy using a previously described procedure (Cheng, et al., 2000). Bar=20 μM. FIG. 4A depicts immunolabeling of the 126 kDa protein in an  N. tabacum  BY-2 protoplast infected with the WFP virus. The pictorially light region indicates the presence and location of the 126 kDa protein. FIG. 4B depicts immunolabeling of BiP in an  N. tabacum  BY-2 protoplast infected with the WFP virus. The pictorially light region indicates the presence and location of the BiP protein. FIG. 4C depicts immunolabeling of the 126 kDa protein in an  N. tabacum  BY-2 protoplast infected with the WYP virus. The pictorially light region indicates the presence and location of the 126 kDa protein. FIG. 4D depicts immunolabeling of BiP in an  N. tabacum  BY-2 protoplast infected with the WYP virus. The pictorially light region indicates the presence and location of the BiP protein.  
         [0016]    [0016]FIG. 4E depicts immunolabeling of the 126 kDa protein in an  N. tabacum  BY-2 protoplast infected with the M IC  virus. The pictorially light region indicates the presence and location of the 126 kDa protein. FIG. 4F depicts immunolabeling of BiP in an  N. tabacum  BY-2 protoplast infected with the M IC  virus. The pictorially light region indicates the presence and location of the BiP protein. FIG. 4G depicts immunolabeling of the 126 kDa protein in a mock-inoculated  N. tabacum  BY-2 protoplast. As expected, there is no detection of the 126 kDa protein. FIG. 4H depicts immunolabeling of BiP in a mock-inoculated  N. tabacum  BY-2 protoplast. The pictorially light region indicates the presence and location of the BiP protein that was not localized, unlike when 126 kDa protein from the WFP or M IC  virus was present.  
         [0017]    [0017]FIG. 5A is a diagram of a portion of genetic constructs bombarded into host leaves for transient expression. Open arrows depict the enhanced 35 S promoter; the dotted box represents nucleotides 1-3348 of SEQ ID NO:1 (for construct 126F:GFP) that encodes the 126 kDa protein from TMV; the box with diagonal stripes represents the DNA encoding for GFP (EGFP, Clontech Laboratories, Palo, Alto, Calif.); and the filled arrow represents the mRNA termination sequence. The bolded letter in the sequence depicted in the 126 kDa protein indicates the amino acid differences among the constructs. For construct 126Y:GFP, nucleotides 1-3348 of SEQ ID NO:5 where nucleotides that encode amino acid 366 are “ata” were inserted, and for construct 126A:GFP, nucleotides 1-3348 of SEQ ID NO:5 where nucleotides that encode amino acid 366 are “agc” were inserted. Each genetic element with the exception of the nucleotides encoding the 126 kDa protein from TMV originated in the expression vector pRTL2 (Topfer, et al. 1987 and Restrepo-Hartwig, et al. 1990).  
         [0018]    [0018]FIGS. 5B-5G depict transient expression of the WFP-containing 126 kDa:GFP fusion proteins in  N. tabacum  (N.t) and  N. benthamiana  (N.b) leaves. White color on black background indicates the presence of fused protein. At 16 hours post-bombardment, the WFP-containing 126 kDa:GFP fusion construct bombarded onto  N. tabacum  leaves has similar expression to that of the same construct inoculated on  N. benthamiana  leaves (FIGS. 5B and 5C). This trend continues through the 44 hour and 8 day time points (FIGS. 5D and 5E and FIGS. 5F and 5G, respectively).  
         [0019]    [0019]FIGS. 5H-5M depict transient expression of the WAP-containing 126 kDa:GFP fusion proteins in  N. tabacum  (N.t) and  N. benthamiana  (N.b) leaves. White color on black background indicates the presence of fused protein. The WAP-containing 126 kDa:GFP fusion construct bombarded onto  N. benthamiana  leaves has similar expression 16 hours post-bombardment than the same construct inoculated on  N. tabacum  leaves (FIGS. 5H and 5I). At 44 hours post-bombardment, there is more 126 kDa:GFP fusion expression on  N. benthamiana  leaves than at 16 hours, but far less 126 kDa:GFP fusion expression on  N. tabacum  leaves than the previous time point (FIGS. 5J and 5K). By 8 days there is low expression of the 126 kDa:GFP fusion expression on  N. benthamiana  leaves and no expression on  N. tabacum  leaves (FIGS. 5L and 5M).  
         [0020]    [0020]FIGS. 5N-5S depict transient expression of the WYP containing  126  kDa:GFP fusion proteins in  N. tabacum  (N.t) and  N. benthamiana  (N.b) leaves. White color on black background indicates the presence of fused protein. At 16 hours post-bombardment, there is similar expression of the WYP-containing 126 kDa:GFP fusion constructs in  N. tabacum  and  N. benthamiana  leaves (FIGS. 5N and 5O). At 44 hours post-bombardment, the expression of the WYP-containing 126 kDa:GFP fusion constructs on  N. tabacum  and  N. benthamiana  leaves appears similar and low (FIGS. 5P and 5Q). At 8 days post-bombardment, significant expression of the WYP-containing 126 kDa:GFP fusion constructs on  N. benthamiana  leaves remain, whereas there is little if any expression of the WYP-containing 126 kDa:GFP fusion constructs on  N. tabacum  leaves (FIGS. 5R and 5S).  
         [0021]    [0021]FIGS. 6A-6H depict the resulting expression of the 126F:GFP (WFP-containing construct), 126Y:GFP (WYP-containing construct), and 126A:GFP (WAP-containing construct) in  N. benthamiana  protoplasts. Although fluorescent bodies are detected in all protoplasts electroporated with the fusion constructs (FIGS. 6A-6F), the size of the bodies is smaller in the protoplast electroporated with the 126A:GFP construct (FIG. 6A) than in the other protoplasts (FIGS. 6C and 6E) 7 hours after electroporation. At 24 hours after electroporation, the protoplasts expressing the 126F:GFP and 126Y:GFP constructs appear to have fewer, but larger fluorescent bodies (FIGS. 6D and 6F). The protoplasts expressing free GFP form no punctate bodies even after 24 hours (FIGS. 6G and 6H). Bar=10 μM.  
         [0022]    [0022]FIG. 7A provides the quantities of large (&gt;2 μM) fluorescent bodies per protoplast formed by the transiently expressed WFP-, WYP-, or WAP-containing fusion proteins in  N. benthamiana  protoplasts over time (means±SD). The bars with horizontal stripes represent the expression of the WFP-containing fusion construct. The bars with the vertical stripes represent the expression of the WYP-containing fusion construct. The bars with diagonal stripes represent the expression of the WAP-containing fusion construct. The number of large bodies in  N. benthamiana  protoplasts transiently expressing WFP-, WYP-, or WAP-containing fusion proteins does not significantly differ among treatments at 16-36 hours. However, after 48 hours  N. benthamiana  protoplasts transiently expressing the WFP-containing fusion protein have more large bodies than protoplasts transiently expressing the other fusion proteins and the difference exists at the 72 and 96 hour time points as well.  
         [0023]    [0023]FIG. 7B provides the quantities of small (&lt;2 μM) fluorescent bodies formed by the transiently expressed WFP-, WYP-, or WAP-containing fusion proteins in  N. benthamiana  protoplasts over time (means±SD). Generally, within each treatment the amounts of small fluorescent bodies decrease with time. Although there is no significant difference between treatments at each time point, the  N. benthamiana  protoplasts expressing the WYP-containing fusion protein appear to have a greater number of small bodies than the other treatments until the 96 hour time point.  
         [0024]    [0024]FIG. 7C provides the ratio of small (&lt;2 μM) fluorescent bodies to large (&gt;2 μM) fluorescent bodies formed by the transiently expressed WFP-, WYP-, or WAP-containing fusion proteins in  N. benthamiana  protoplasts over time (means±SD). There does not appear to be significant differences between treatments at every time point, but the smallest ratio of small to large fluorescent bodies in  N. benthamiana  protoplasts have WFP-containing fusion proteins at 48-96 hours post-electroporation.  
         [0025]    [0025]FIGS. 8A-8F depict the transient expression of WFP-containing fusion proteins in  N. tabacum  BY-2 protoplasts in the presence (FIGS. 8B, 8D, and  8 F) or absence (FIGS. 8A, 8C, and  8 E) of a ubiquitin pathway inhibitor, ALLN, over time (12, 24, and 48 hours). There is greater WFP-containing fusion protein expression in protoplasts treated with ALLN than without ALLN at every time point (compare FIG. 8A to FIG. 8B, FIG. 8C to FIG. 8D, and FIG. 8E to FIG. 8F). Bar=10 μM.  
         [0026]    [0026]FIGS. 8G-8L depict the transient expression of WYP-containing fusion proteins in  N. tabacum  BY-2 protoplasts in the presence (FIGS. 8H, 8J, and  8 L) or absence (FIGS. 8G, 8I, and  8 K) of a ubiquitin pathway inhibitor, ALLN, over time (12, 24, and 48 hours). There is greater WFP-containing fusion protein expression in protoplasts treated with ALLN than without ALLN at every time point (compare FIG. 8G to FIG. 8H, FIG. 8I to FIG. 8J, and FIG. 8K to FIG. 8L). However, transient expression in the absence of ALLN peaked 24 hours post-electroporation with little expression at 48 hours post-electroporation. In BY-2 protoplasts expressing the WYP-containing fusion protein and treated with ALLN, the number of small bodies decreased as the large bodies increased in size over time (FIG. 8H, 8J and  8 K). Bar=10 μM.  
         [0027]    [0027]FIGS. 8M-8R depict the transient expression of WAP-containing fusion proteins in  N. tabacum  BY-2 protoplasts in the presence (FIGS. 8N, 8P, and  8 R) or absence (FIGS. 8M, 8O, and  8 Q) of a ubiquitin pathway specific inhibitor, ALLN, over time (12, 24, and 48 hours). At 12 and 24 hours post-electroporation, BY-2 protoplasts in the presence of ALLN transiently express more WAP-containing fusion protein than the time-matched, -ALLN protoplasts (compare FIG. 8M to FIG. 8N and FIG. 8O to FIG. 8P). Also, there was greater WAP-containing fusion protein expression in ALLN treated BY-2 protoplasts at 24 hours than at 12 hours post-electroporation (FIG. 8N and FIG. 8P). However, at 48 hours, there was no detectable WAP-containing fusion protein expression in BY-2 protoplasts in the absence of ALLN (FIG. 8Q) and only very little, but aggregated, expression in the presence of ALLN (FIG. 8R). Bar=10 μM. 
     
    
     SUMMARY OF THE INVENTION  
       [0028]    In one aspect, the invention is a method for decreasing the degradation rate of an engineered protein of interest in a plant cell comprising the steps a) constructing a vector comprising a nucleic acid fragment from position 1 to position 3348 of SEQ ID NO:1 fused to a nucleotide sequence encoding a protein of interest, the vector expressible in said plant cell; and b) introducing and expressing the vector in the plant cell to form a fused protein, wherein the degradation rate of the fused protein is less than the degradation rate of the engineered protein of interest in the plant cell or a plant cell of the same species. The vector may be integrated into the genome of said plant cell. The invention is furthermore a plant cell transformed according to the above method and a plant generated from the transformed plant cell.  
         [0029]    In another aspect, the invention is a method for decreasing the degradation rate of an engineered protein of interest in a plant cell comprising the steps a) constructing a vector comprising a nucleic acid fragment from position 1 to position 4831 of SEQ ID NO:3 fused to a nucleotide sequence encoding a protein of interest, the vector expressible in said plant cell; and b) introducing and expressing the vector in the plant cell to form a fused protein, wherein the degradation rate of the fused protein is less than the degradation rate of the engineered protein of interest in the plant cell or a plant cell of the same species. The vector may be integrated into the genome of said plant cell. The invention is furthermore a plant cell transformed according to the above method and a plant generated from the transformed plant cell.  
         [0030]    In another aspect, the invention is also a method for increasing the degradation rate of an engineered protein of interest in a plant cell comprising the steps a) constructing a vector comprising a nucleic acid fragment from position 1 to position 3348 of SEQ ID NO:5 fused to a nucleotide sequence encoding a protein of interest, the vector expressible in a plant cell; and b) introducing and expressing the vector in the plant cell to form a fused protein, wherein the degradation rate of the fused protein is less than the degradation rate of the engineered protein of interest in the plant cell or a plant cell of the same species. Nucleotides at positions 1096-1098 of SEQ ID NO:5 encode alanine or tyrosine. The vector may be integrated into the genome of said plant cell. The invention is furthermore a plant cell transformed according to the above method and a plant generated from the transformed plant cell.  
         [0031]    In another aspect, the invention is also a method for increasing the degradation rate of an engineered protein of interest in a plant cell comprising the steps a) constructing a vector comprising a nucleic acid fragment from position 1 to position 4831 of SEQ ID NO:7 fused to a nucleotide sequence encoding a protein of interest, the vector expressible in a plant cell; and b) introducing and expressing the vector in the plant cell to form a fused protein, wherein the degradation rate of the fused protein is less than the degradation rate of the engineered protein of interest in the plant cell or a plant cell of the same species. Nucleotides at positions 1096-1098 of SEQ ID NO:7 encode alanine or tyrosine. The vector may be integrated into the genome of said plant cell. The invention is furthermore a plant cell transformed according to the above method and a plant generated from the transformed plant cell.  
         [0032]    In another aspect, the invention is a purified nucleic acid comprising a nucleic acid fragment from position 1 to position 3348 of SEQ ID NO:1 fused to a DNA sequence encoding a protein of interest, wherein expression of said purified nucleic acid in a plant cell results in a fusion protein having increased stability when compared to the stability of said protein of interest engineered without fusion expressed in a plant cell of the same species. The invention is also the resulting fusion protein comprising SEQ ID NO:2 encoded by the purified nucleic acid comprising a nucleic acid fragment from position 1 to position 3348 of SEQ ID NO:1 fused to a DNA sequence encoding a protein of interest. Another embodiment of the invention is the vector comprised of SEQ ID NO:1 encoding SEQ ID NO:2, the plant cell transformed with the vector, and the plant generated with the transformed plant cell.  
         [0033]    In another aspect, the invention is a purified nucleic acid comprising a nucleic acid fragment from position 1 to position 4831 of SEQ ID NO:3 fused to a DNA sequence encoding a protein of interest, wherein expression of said purified nucleic acid in a plant cell results in a fusion protein having increased stability when compared to the stability of said protein of interest engineered without fusion expressed in a plant cell of the same species. The invention is also the resulting fusion protein comprising SEQ ID NO:4 encoded by the purified nucleic acid comprising a nucleic acid fragment from position 1 to position 4831 of SEQ ID NO:3 fused to a DNA sequence encoding a protein of interest. Another embodiment of the invention is the vector comprised of SEQ ID NO:3 encoding SEQ ID NO:4, the plant cell transformed with the vector, and the plant generated with the transformed plant cell.  
         [0034]    In another aspect, the invention is a purified nucleic acid comprising a nucleic acid fragment from position 1 to position 3348 of SEQ ID NO:5 fused to a DNA sequence encoding a protein of interest, wherein expression of said purified nucleic acid in a plant cell results in a fusion protein having increased or decreased stability when compared to the stability of said protein of interest engineered without fusion expressed in a plant cell of the same species. The purified nucleic acid comprising a nucleic acid fragment from position 1 to position 3348 of SEQ ID NO:5 fused to a DNA sequence encoding a protein of interest could also have increased or decreased stability when compared to the stability of the protein of interest fused to a nucleic acid fragment from position 1 to position 3348 of SEQ ID NO:1 expressed in a plant cell of the same species. Nucleotides at positions 1096-1098 of SEQ ID NO:5 encode alanine or tyrosine. The invention is also the resulting fusion protein comprising SEQ ID NO:6 encoded by the purified nucleic acid comprising a nucleic acid fragment from position 1 to position 3348 of SEQ ID NO:5 fused to a DNA sequence encoding a protein of interest. Another embodiment of the invention is the vector comprised of SEQ ID NO:5 encoding SEQ ID NO:6, the plant cell transformed with the vector, and the plant generated with the transformed plant cell.  
         [0035]    In another aspect, the invention is also a purified nucleic acid comprising a nucleic acid fragment from position 1 to position 4831 of SEQ ID NO:7 fused to a DNA sequence encoding a protein of interest, wherein expression of said purified nucleic acid in a plant cell results in a fusion protein having increased or decreased stability when compared to the stability of said protein of interest engineered without fusion expressed in a plant cell of the same species. The purified nucleic acid comprising a nucleic acid fragment from position 1 to position 4831 of SEQ ID NO:7 fused to a DNA sequence encoding a protein of interest could also have increased or decreased stability when compared to the stability of the protein of interest fused to a nucleic acid fragment from position 1 to position 3348 of SEQ ID NO:1 expressed in a plant cell of the same species. Nucleotides at positions 1096-1098 of SEQ ID NO:5 encode alanine or tyrosine. The invention is also the resulting fusion protein comprising SEQ ID NO:8 encoded by the purified nucleic acid comprising a nucleic acid fragment from position 1 to position 4831 of SEQ ID NO:7 fused to a DNA sequence encoding a protein of interest. Another embodiment of the invention is the vector comprised of SEQ ID NO:7 encoding SEQ ID NO:8, the plant cell transformed with the vector, and the plant generated with the transformed plant cell.  
         [0036]    In yet another aspect, the invention is a method for decreasing the degradation rate of an engineered protein of interest in a plant cell comprising the steps a) constructing a vector comprising a nucleic acid sequence that encodes a membrane binding protein from the Sindbis-like plant virus family fused to a nucleotide sequence encoding the protein of interest, the vector expressible in a plant cell; and b) introducing and expressing he vector in the plant cell to form a fused protein, wherein the degradation rate of the fused protein is less than the degradation rate of the engineered protein of interest in the plant cell or a plant cell of the same species. The Sindbis-like plant virus family contains “WFP” motif as depicted at amino acid position 365-367 of SEQ ID NO:2. The vector may be integrated into the genome of said plant cell. The Sindbis-like plant virus is alfalfa mosaic virus, brome mosaic virus, citrus leaf rugose virus, cucumber mosaic virus, sunn-hemp mosaic virus, tobacco mosaic virus, tobacco rattle virus, or turnip vein clearing virus. The invention further embodies a plant cell transformed according to this method and the plant generated from the transformed plant cell.  
         [0037]    In another aspect, the invention also embodies a method for increasing the degradation rate of an engineered protein of interest in a plant cell comprising the steps a) constructing a vector comprising a nucleic acid sequence that encodes a membrane binding protein from the Sindbis-like plant virus family fused to a nucleotide sequence encoding the protein of interest, the vector expressible in a plant cell; and b) introducing and expressing he vector in the plant cell to form a fused protein, wherein the degradation rate of the fused protein is less than the degradation rate of the engineered protein of interest in the plant cell or a plant cell of the same species. The Sindbis-like plant virus family contains a mutation in the “WFP” motif as depicted at amino acid position 365-367 of SEQ ID NO:2. The vector may be integrated into the genome of said plant cell. The Sindbis-like plant virus is alfalfa mosaic virus, brome mosaic virus, citrus leaf rugose virus, cucumber mosaic virus, sunn-hemp mosaic virus, tobacco mosaic virus, tobacco rattle virus, or turnip vein clearing virus. The invention further embodies a plant cell transformed according to this method and the plant generated from the transformed plant cell.  
         [0038]    In another aspect, the invention is furthermore a purified nucleic acid comprising a nucleic acid fragment encoding a membrane binding protein from the Sindbis-like plant virus fused to a DNA sequence encoding a protein of interest. The Sindbis-like plant virus is alfalfa mosaic virus, brome mosaic virus, citrus leaf rugose virus, cucumber mosaic virus, sunn-hemp mosaic virus, tobacco mosaic virus, tobacco rattle virus, and turnip vein clearing virus. The invention is the resulting fusion protein encoded by a purified nucleic acid comprising a nucleic acid fragment encoding a membrane binding protein from the Sindbis-like plant virus fused to a DNA sequence encoding a protein of interest. The resulting fusion protein comprising a membrane binding protein from the Sindbis-like plant virus family containing the “WFP” motif as depicted at amino acid position 365-367 of SEQ ID NO:2 fused to an amino acid sequence of interest has increased stability over the unfused protein of interest expressed in a cell of the same plant species. The invention is also the vector comprising a nucleic acid fragment encoding a membrane binding protein from the Sindbis-like plant virus containing the “WFP” motif as depicted at amino acid position 365-367 of SEQ ID NO:2 fused to a DNA sequence encoding a protein of interest. Additionally, the invention is the plant cell transformed with the vector and the plant generated from the plant cell.  
         [0039]    In another aspect, the invention is a purified nucleic acid comprising a nucleic acid fragment encoding a membrane binding protein from the Sindbis-like plant virus containing a mutation in the “WFP” motif as depicted at amino acid position 365-367 of SEQ ID NO:2 fused to a DNA sequence encoding a protein of interest. The Sindbis-like plant virus is alfalfa mosaic virus, brome mosaic virus, citrus leaf rugose virus, cucumber mosaic virus, sunn-hemp mosaic virus, tobacco mosaic virus, tobacco rattle virus, and turnip vein clearing virus. The invention is also the resulting fusion protein encoded by a purified nucleic acid comprising a nucleic acid fragment encoding a membrane binding protein from the Sindbis-like plant virus fused to a DNA sequence encoding a protein of interest. The resulting fusion protein comprising a membrane binding protein from the Sindbis-like plant virus family containing a mutation in the “WFP” motif as depicted at amino acid position 365-367 of SEQ ID NO:2 fused to an amino acid sequence of interest has increased or decreased stability over the unfused protein of interest expressed in a cell of the same plant species. The invention is also the vector comprising a nucleic acid fragment encoding a membrane binding protein from the Sindbis-like plant virus containing a mutation in the “WFP” motif as depicted at amino acid position 365-367 of SEQ ID NO:2 fused to a DNA sequence encoding a protein of interest. Additionally, the invention is the plant cell transformed with the vector and the plant generated from the plant cell.  
       DETAILED DESCRIPTION  
       [0040]    We have identified an amino acid motif, “WFP”, from the TMV 126 kDa and 183 kDa proteins (amino acid position 365 to 367 of SEQ ID:2 and SEQ ID NO:4) that is conserved among viral membrane—associated proteins. The TMV 126 kDa and 183 kDa proteins localize to the ER in infected  N. tabacum  and  N. benthamiana  cells. Mutating the “WFP” motif to “WYP” or “WAP” resulted in a variety of effects, somewhat dependent upon the host species. Although the “WFP” motif causes a fused protein to resist ubiquitin-mediated degradation, the mutant 126Y:GFP and 126A:GFP resulted in an increased degradation of a fused protein. Thus, we disclose a method to modulate the rate, and therefore the stability, of an engineered protein.  
         [0041]    One method to decrease the rate of degradation of an engineered protein in plant cells includes creating a vector expressible in a plant cell, wherein the vector encodes a fusion protein between the TMV 126 kDa protein and a protein of interest. An exemplary nucleotide sequence for inclusion in this vector is SEQ ID NO:1 which encodes the TMV 126 kDa protein of SEQ ID NO:2. The vector could be designed for transient transfection, or for integration into the plant cell&#39;s genome. After creating the vector expressible in a plant cell, the method includes introducing the vector into one or more plant cells through any currently known methods of the art or other methods that will be known. The resulting plant cell containing the vector expresses the fusion protein, which has a decreased rate of degradation compared to the protein of interest when not expressed as a fusion protein. In addition to the method described for decreasing the rate of degradation of a protein of interest, the invention as disclosed herein also includes the vector created for implementing the disclosed method, the nucleotide sequence that encodes the fusion protein, the fusion protein that results from the expression of the created vector, the plant cell or cells transformed with the created vector, and the plants that are generated from the transformed cells.  
         [0042]    Another method to decrease the rate of degradation of an engineered protein in plant cells includes creating a vector expressible in a plant cell, wherein the vector encodes a fusion protein between the TMV 183 kDa protein and a protein of interest. An exemplary nucleotide sequence for inclusion in this vector is SEQ ID NO:3 which encodes the TMV 186 kDa protein of SEQ ID NO:4. The vector could be designed for transient transfection, or for integration into the plant cell&#39;s genome. After creating the vector expressible in a plant cell, the method includes introducing the vector into one or more plant cells through any currently known methods of the art or other methods that will be known. The resulting plant cell containing the vector expresses the fusion protein, which has a decreased rate of degradation compared to the protein of interest when not expressed as a fusion protein. In addition to the method described for decreasing the rate of degradation of a protein of interest, the invention as disclosed herein also includes the vector created for implementing the disclosed method, the nucleotide sequence that encodes the fusion protein, the fusion protein that results from the expression of the created vector, the plant cell or cells transformed with the created vector, and the plants that are generated from the transformed cells.  
         [0043]    The invention also includes methods to increase the rate of degradation of an engineered protein in plant cells. This method includes creating a vector expressible in a plant cell, wherein the vector encodes a fusion protein between a mutant TMV 126 kDa protein and a protein of interest. An exemplary nucleotide sequence for inclusion in this vector is SEQ ID NO:5 which encodes a mutant TMV 126 kDa protein of SEQ ID NO:6 where amino acid 366 is any amino acid but phenylalanine. Two exemplary amino acid substitutions include tyrosine and alanine. The vector could be designed for transient transfection, or for integration into the plant cell&#39;s genome. After creating the vector expressible in a plant cell, the method includes introducing the vector into one or more plant cells through any currently known methods of the art or other methods that will be known. The resulting plant cell containing the vector expresses the fusion protein, which has an increased rate of degradation compared to the protein of interest when not expressed as a fusion protein. In addition to the method described for increasing the rate of degradation of a protein of interest, the invention as disclosed herein also includes the vector created for implementing the disclosed method, the nucleotide sequence that encodes the fusion protein, the fusion protein that results from the expression of the created vector, the plant cell or cells transformed with the created vector, and the plants that are generated from the transformed cells.  
         [0044]    The invention also includes methods to increase the degradation rate of an engineered protein in plant cells. This method includes creating a vector expressible in a plant cell, wherein the vector encodes a fusion protein between a mutant TMV 183 kDa protein and a protein of interest. An exemplary nucleotide sequence for inclusion in this vector is SEQ ID NO:7 which encodes a mutant TMV 183 kDa protein of SEQ ID NO:8 where amino acid 366 is any amino acid but phenylalanine. Two exemplary amino acid substitutions include tyrosine and alanine. The vector could be designed for transient transfection, or for integration into the plant cell&#39;s genome. After creating the vector expressible in a plant cell, the method includes introducing the vector into one or more plant cells through any currently known methods of the art or other methods that will be known. The resulting plant cell containing the vector expresses the fusion protein, which has an increased rate of degradation compared to the protein of interest when not expressed as a fusion protein. In addition to the method described for increasing the degradation rate of a protein of interest, the invention as disclosed herein also includes the vector created for implementing the disclosed method, the nucleotide sequence that encodes the fusion protein, the fusion protein that results from the expression of the created vector, the plant cell or cells transformed with the created vector, and the plants that are generated from the transformed cells.  
         [0045]    As anyone skilled in the art can recognize, other nucleotide sequences that encode amino acid sequences with analogous function and homologous sequence to TMV&#39;s 126/183 kDa protein may be used to decrease the degradation rate of an engineered protein. This method to decrease the degradation rate of an engineered protein in plant cells includes creating a vector expressible in a plant cell, wherein the vector encodes a fusion protein between a protein with analogous function and homologous sequence to TMV&#39;s 126/183 kDa protein from one of the following Sindbis-like plant viruses: alfalfa mosaic virus, brome mosaic virus, citrus leaf rugose virus, cucumber mosaic virus, sunn-hemp mosaic virus, tobacco mosaic virus, tobacco rattle virus, and turnip vein clearing virus. The vector could be designed for transient transfection, or for integration into the plant cell&#39;s genome. After creating the vector expressible in a plant cell, the method includes introducing the vector into one or more plant cells through any currently known methods of the art or other methods that will be known. The resulting plant cell containing the vector expresses the fusion protein, which has a decreased degradation rate compared to the protein of interest when not expressed as a fusion protein. In addition to the method described for decreasing the degradation rate of a protein of interest, the invention as disclosed herein also includes the vector created for implementing the disclosed method, the nucleotide sequence that encodes the fusion protein, the fusion protein that results from the expression of the created vector, the plant cell or cells transformed with the created vector, and the plants that are generated from the transformed cells.  
         [0046]    Yet another method to increase the degradation rate of an engineered protein in plant cells includes creating a vector expressible in a plant cell, wherein the vector encodes a fusion protein between a mutated protein with analogous function and homologous sequence to TMV&#39;s 126/183 kDa protein from one of the following Sindbis-like plant viruses: alfalfa mosaic virus, brome mosaic virus, citrus leaf rugose virus, cucumber mosaic virus, sunn-hemp mosaic virus, tobacco mosaic virus, tobacco rattle virus, and turnip vein clearing virus. The vector could be designed for transient transfection, or for integration into the plant cell&#39;s genome. After creating the vector expressible in a plant cell, the method includes introducing the vector into one or more plant cells through any currently known methods of the art or other methods that will be known. The resulting plant cell containing the vector expresses the fusion protein, which has an increased degradation rate compared to the protein of interest when not expressed as a fusion protein. In addition to the method described for increasing the degradation rate of a protein of interest, the invention as disclosed herein also includes the vector created for implementing the disclosed method, the nucleotide sequence that encodes the fusion protein, the fusion protein that results from the expression of the created vector, the plant cell or cells transformed with the created vector, and the plants that are generated from the transformed cells.  
         [0047]    Materials and Methods  
         [0048]    Plant Materials  
         [0049]    [0049] Nicotiana benthamiana  and  Nicotiana tabacum  Xanthi “nn” and “NN” were germinated in a tray and individually transplanted into 12 cm pots containing an artificial soil medium (Metro-Mix 350, Grace). Plants were grown in the greenhouse until needed under the following conditions: 16 hour and 25° C. days and 8 hour and 17° C. nights. Supplemental light intensity was 500 μmol photons M −2  s −1 . Plants used for inoculation experiments were six to seven weeks old. Although other conditions may be used, the above growth conditions are preferred.  
         [0050]    Suspension Cells, Protoplasts and Transfection  
         [0051]    The maintenance of suspension cells, preparation of protoplasts and transfection of protoplasts by electroporation were conducted according to Watanabe et al.(1987), modified for electroporating the  N. benthamiana  cells and protoplasts.  N. tabacum  BY-2 (Dr. Richard Cyr, Penn State University) suspension cells were grown in 50 ml of culture media (4.3 g/L M&amp;S salt, 100 mg/L myo-inositol, 1 mg/L thiamine, 0.2 mg/L 2,4-D, 255 mg/L KH 2 PO 4 , 30 g/L sucrose, pH 5.0) at 26° C. constantly shaking at 150 rpm and sub-cultured weekly. Suspension cells of  N. benthamiana  (Dr. Bryce Falk, University  
         [0052]    California—Davis) were grown in culture media (4.3 g/L M&amp;S salt, 0.204 g/L KH 2 PO 4 , 100 mg/L myo-inositol, 0.2 mg/L 2,4-D, 0.1 mg/L Kinetin, 1 mg/L thiamine, 0.5 mg/L pyridoxide, 0.5 mg/L nicotinic acid, 30 g/L sucrose, pH 5.8) at 26° C. constantly shaking at 150 rpm and sub-cultured every 10 days.  
         [0053]    In order to create protoplasts, both BY-2 and  N. benthamiana  cells were digested with 1% Cellulose, R-10; 0.1% Pectolyase Y-23 and 1% Driselase (Karlan) in MMC buffer (13% mannitol, 5 mM MES, 10 mM CaCl 2 , pH 5.8) at room temperature for 3 hours. The digested cells were overlaid on a 20.5% sucrose cushion and spun at 1,100 rpm on an IEC centrifuge for 11 minutes. The protoplasts on top of the cushion were collected and washed twice with MMC buffer. About 1×10 6  protoplasts were resuspended in 0.8 ml of the electroporation buffer (13% mannitol, 70 mM KCl, 5 mM MES, pH 5.8).  
         [0054]    Fifteen μg of plasmid DNA or 5 μg of in vitro transcript viral RNA (see below) were mixed with 0.8 ml of protoplasts in a precooled cuvette and electroporated with the following setting: 250V, 220 μF and 50 mS (ProGenetor II, Hoefer Scientific Instruments, San Francisco, Calif. USA ). After electroporation, protoplasts were incubated on ice for 10 minutes and washed with 2 ml of MMC buffer. The transfected protoplasts were resuspended in 3 ml of culture media with 13% mannitol and incubated at 26° C. in the dark for BY-2 protoplasts or under light for  N. benthamiana  protoplasts. Although alternative methods may be employed, the above methods for maintaining suspension cells, creating protoplasts, and transfecting both are preferred.  
         [0055]    In Vitro Site-Directed Mutagenesis  
         [0056]    To mutate the second amino acid in the “WFP” motif, in vitro site-directed mutagenesis was performed as described before (Bao et al, 1996). The phenylalanine in the “WFP” motif from M IC m2 (an infectious transcript of M IC  TMV altered at a single nucleotide to the UI strain sequence in the 126 kDa protein open reading frame) (Shintaku et al., 1996) was replaced with alanine and tyrosine, respectively. In order to create the “WAP” motif, in vitro site-directed mutagenesis was performed using the following primer complementary to nucleotides 1141-1177: 5′-CTCATTTCGGG AGC CCAGTAATTGACTGATGATGAAT-3′ (SEQ ID NO:22). In order to create the “WYP” motif, in vitro site-directed mutagenesis was performed using the following primer complementary to nucleotides 1141-1173: 5′-TTTCGGG ATA CCAGTAATTGACTGATGATGAAT-3′ (SEQ ID NO:23). The underlined codon indicates the mutated sites. All mutant clones were confirmed to contain the specified alteration by sequence analysis. Although site-directed mutagenesis to the WAP and WYP motifs may be performed using alternative primers, the above methods are preferred. Additionally, other mutations can be made in the place of phenylalanine 366 as numbered in SEQ ID NO:2 in the same way.  
         [0057]    In Vitro Transcription and Inoculation  
         [0058]    Plasmid DNA of infectious TMV cDNA clones was linearized by Acc65 I and gel-purified to act as a template in the in vitro transcription reaction performed as described previously (Shintaku et al., 1996). 5 μg of transcript viral RNA was inoculated on the mature leaves of  N. benthamiana, N. tabacum  Xanthi “NN”, and “nn” which were dusted with the abrasive carborundum. The inoculated plants were kept in the greenhouse to observe local lesions and systemic symptoms. Other method may be utilized for in vitro transcription and inoculation, but the processes described above are preferred.  
         [0059]    Construction of 126 kDa-GFP Fusion Chimeric Vectors  
         [0060]    A cDNA fragment encoding the 126 kDa protein of the M IC  TMV was amplified from plasmid L19 (Shintaku et al., 1996) using the Pfu polymerase (Stratagene) and a pair of primers ST (5′-CCATGCCATGGCGCTCGAG ATGGCATACACACAGACA -3′ (SEQ ID NO:24), where the underlined nucleotides indicate the TMV genome sequence from the position 69 to 86) and GT (5′-CCCTTGCTCACCAT TTGTGTTCCTGCATCG -3′ (SEQ ID NO:25), where the underlined nucleotides indicate the sequence complementary to TMV genome sequence, from the position 3401 to 3416). Green fluorescent protein (GFP) (EGFP, Clontech Laboratories, Inc., Palo Alto, Calif.) was amplified from plasmid pEGFP (Clontech) using the Pfu polymerase and a pair of primers TG (5′-ATGCAGGAACACAAATGGTGAGCAAGGGCG-3′) (SEQ ID NO:26) and 3GFP (5′-CCATGCCATGGCTCGAGTTACTTGTACAGCTCGT-3′) (SEQ ID NO:27). The amplified fragments were gel-purified and mixed as the template for the fusion PCR using the primers 5T and 3GFP (method described by Higuchi, 1990). The PCR product was the fusion of the 126 kDa protein gene and the GFP gene which was purified and digested with Nco I. The digested fragment was purified and ligated with plasmid pRTL2 (Restrepo et al., 1990) previously digested with Nco I. The ligation mixture was transformed into  E. coli  HB 101. The clone containing the insert having the correct orientation was identified by restriction digestion and sequencing, and named p126:GFP. To make the mutated 126K fusion protein construct, the infectious cDNA clones of “WFP”, “WYP” and “WAP” were digested with Mlu I and Dra III, sequentially. The Mlu I-Dra III fragments from each of the clones were inserted into the same site of p126:GFP previously digested with Mlu I and Dra III. Those clones containing wild type “WFP” motif and the mutated motifs (“WYP” and “WAP”) were named p126F:GFP, p126Y:GFP and p126A:GFP, respectively. Although a variety of methods could be utilized to create chimeric vectors, the above methods are preferred. Although only the full length 126 kDa protein was fused to a gene of interest, this application anticipates that truncated portions of the TMV 126 kDa protein or peptides can also be employed in the present invention as long as the amino acid sequence that stabilizes the fusion protein contains the “WFP” motif or elements that act in the same fashion.  
         [0061]    Biolistic Bombardment and Fluorescent Microscopy  
         [0062]    Transient expression of 126 kDa-GFP fusion protein in tobacco leaves by biolistic bombardment was performed according to Itaya, et al. (1997). Five μg of each of p126F:GFP, p126Y:GFP and p126A:GFP was bombarded into the lower epidermis of  N. benthamiana  and  N. tabacum  Xanthi nn leaves using a Biolistic PDS 1000/He System (Bio-Rad) at a pressure of 1,100 psi. The bombarded leaves were incubated in a sealed petri dish with several pieces of water-soaked filter paper at 25° C. with light overnight.  
         [0063]    The leaves were observed under a Nikon Microphot-FX epifluorescent microscope with a filter set B-2A, consisting of a blue excitation filter (450-490 nm), a dichroic mirror (510 nm) and a barrier filter (520 nm). Fluorescent images were photographed with the camera system attached to the microscope using Kodak Royal 400 color film. While biolistic bombardment and fluorescent microscopy could be accomplished in different ways, the above methods are preferred.  
         [0064]    Transient Expression of 126F:GFP, 126Y:GFP and 126A:GFP in Protoplasts  
         [0065]    Fifteen μg of plasmid DNA of the three fusion protein constructs (126F:GFP, 126Y:GFP and 126A:GFP) were transfected into protoplasts of  N. benthamiana  and BY-2 cells by electroporation as described above. The transfected protoplasts were collected at 7, 12, 16, 18, 24, 36, 48, 72, and 96 hours post-incubation and plated on a 12-well slide for a single cell time course observation with a procedure as described previously (Mas and Beachy, 1998). The fluorescent fusion protein expression in the protoplasts was examined by confocal laser scanning microscopy (CLSM) as described below.  
         [0066]    Immunofluorescent Labeling  
         [0067]    Immunofluorescent labeling of TMV 126K protein and host components was conducted according to Heinlein et al. (1995) with a minor modification as follows. First, 0.5 ml of protoplasts of  N. benthamiana  and BY-2 infected with “WFP”, “WYP” and “WAP” viruses were harvested 2 days post-infection. The protoplasts were spun down at 700 rpm in 14 ml tubes (Falcon) at room temperature for 2 minutes and resuspended in fixative buffer (50 mM Na 2 HPO 4 , pH 6.7; 4% paraformadehyde, 0.1% glutaradehyde, 5 mM EGTA, pH 8.0) for 30 minutes at room temperature. The fixed protoplasts were plated on the slides precoated with 0.1% poly-L-lysine and then extracted with cold methanol for 10 minutes. All washes were performed in phosphate-buffered saline (PBS), pH 7.0, containing 0.5% Tween-20 and 5 mM EGTA. Primary antibodies were polyclonal rabbit IgG recognizing the TMV 126K protein (Nelson, et al. 1993) and polyclonal rabbit IgG against BiP, an ER associated protein indicator, kindly provided by Dr. Becky Boston, North Carolina State University. Secondary antibodies were FITC-conjugated goat anti-rabbit IgG and Texas Red-conjugated goat anti-mouse IgG (Molecular Probes, Eugene, Oreg., USA). The samples were mounted with mounting media ( 0.1 M Tris-HCl, pH 9.0; 50% glycerol, 1 mg/ml p-phenylenediamine) and stored at 4° C. before observation. Other methods and materials may be used to visualize fusion protein presence and localization, but the above methods and materials are preferred.  
         [0068]    Proteosome Inhibition  
         [0069]    ALLN (N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal, Sigma Chemical Co. St. Louis, Mo.) was used at a final concentration of 75 μM in dimethyl sulfoxide (DMSO). The BY-2 protoplasts transfected with fusion protein constructs were incubated in the culture media containing 75 μM of ALLN and collected 12, 24, and 48 hours post-transfection. The transient fluorescent protein expression in protoplasts was examined by CLSM as described below. There may be other ways to perform the inhibitor experiment, but the above methods are merely preferred.  
         [0070]    Confocal Microscopy  
         [0071]    Immnunofluorescent labeling signals and transient expression of 126 kDa:GFP fusion protein in protoplasts were examined with CLSM (Cheng et al., 2000). Most images were captured with 3% laser power, but in the inhibitor experiment, 10% laser power was used. The above conditions are merely representative of conditions used to visualize data with confocal microscopy.  
       EXAMPLE 1  
       [0072]    To better understand how the domains within the TMV 126 kDa protein influence pathophysiology, the sequence of the TMV 126 kDa protein was compared to functionally related proteins from other Sindbis-like plant viruses: alfalfa mosaic virus, brome mosaic virus, citrus leaf rugose virus, cucumber mosaic virus, sunn-hemp mosaic virus, tobacco rattle virus, and turnip vein clearing virus. The TMV 126 kDa protein was aligned with its functional analogues from other Sindbis-like plant viruses using the CLUSTAL W program (Thompson et al., 1994) to identify a conserved “WFP” sequence (trypotophan-phenylalanine-proline) (FIG. 1). The “WFP” sequence is contained within Domain I, between the methyltransferase and helicase domains of this protein (FIG. 2A). This “WFP” sequence was also found in several plant proteins, most of which are membrane-associated. A person skilled in the art, understanding concepts of amino acid homology and functionally analogous proteins, will also recognize that the alignment of FIG. 1 identifies parts of other sequences that may be fused to stabilize an engineered protein. Like the TMV 126 kDa protein used herein, some of the proteins in FIG. 1 have a putative ER-colocalizing signal that may be mutated to destabilize a fused engineered protein.  
         [0073]    To create a destabilizing motif, three mutant viruses were constructed that were altered within this motif (FIG. 2B). The WFP virus refers to a virus with a masked (M IC ) genetic background, except for a “Ser” residue, found in the U1 strain, at position 325 (Shintaku et al., 1996). This sequence alteration results in the WFP virus (also referred to as M IC  m 2 ) inducing severe symptoms and accumulating more efficiently in systemic tissue than the parental M IC  virus (Derrick et al., 1997). The WAP and WYP viruses were constructed by replacing “Phe” with “Ala” or “Tyr”, respectively, of the 126 kDa protein (FIG. 2B). Both mutations of the “WFP” motif resulted in a virus unable to cause symptoms of the parental Tobacco mosaic virus. Although only alanine and tyrosine were substituted for phenylalanine in this present example, any substitute amino acid not having phenylalanine characteristics is anticipated in this invention because it acts to destabilize the fused protein.  
         [0074]    Changing the phenylalanine to either alanine or tyrosine in the WFP motif decreased the infectivity of the mutant viruses on tobacco species. The WAP virus did not infect  N. tabacum  plants, but did infect  N. benthamiana  plants (Table 1). The WYP virus induced only mild systemic symptoms on  N. tabacum  plants but severe systemic symptoms on  N. benthamiana . The wild-type WFP virus induced severe symptoms on both Nicotiana species (Table 1). On  N. tabacum  Xanthi “NN” plants, a local lesion host for TMV, the WYP virus induced tiny necrotic lesions at 24° C., whereas the WFP virus induced larger lesions (FIG. 3A). High temperature treatment of 32° C. for three days before returning to 24° C. blocked the necrotic response of Nicotiana, but did not affect the lesion size induced by the WYP virus on “NN” plants (FIG. 3B). The WFP virus, however, induced larger lesions after returning to the lower temperature (FIG. 3B). These data demonstrate that the “WFP” motif within the 126 kDa protein is required for efficient virus replication and infection, and that the necrosis response does not limit the infectivity of the WYP virus.  
                                     TABLE 1                           Summary of biological analyses of the WFP, WYP, and WAP       viruses in  Nicotiana tabacum  and  Nicotiana benthamiana              Host   Phenotypes   WFP   WAP   WYP                 N. tabacum     Replication   +   −   +         N. tabacum     Cell to cell   +   −   +           movement         N. tabacum     Systemic   severe   none   mild a             symptoms         N. benthamiana     Replication   +   −   +         N. benthamiana     Cell to cell   +   −   +           movement         N. benthamiana     Systemic   very severe   mild   severe b             symptoms                                  
 
         [0075]    We immunolabeled  N. tabacum  (cv. BY-2) and infected with the WFP, WYP or WAP viruses using antibodies against the TMV 126 kDa and binding protein (BiP), an ER marker (FIGS.  4 A-H). The TMV 126 kDa protein containing the “WFP” motif (both the WFP and M IC  viruses) localized to subcellular bodies similar to those observed in cells probed with anti-BiP (FIGS. 4A, 4B,  4 E, and  4 F). Both the 126 kDa protein containing the “WYP” motif (FIG. 4C) and BiP (FIG. 4D) failed to localize in  N. tabacum  cells inoculated with WYP virus. Interestingly, the TMV 126 kDa protein was not detected at all in WAP virus-infected cells of  N. tabacum . There was no TMV 126 kDa protein detected in the mock-infected  N. tabacum  protoplast (FIG. 4G). In  N. benthamiana  protoplasts, the 126 kDa proteins of the WYP and WAP viruses localized similarly to the 126 kDa protein from the WFP virus (data not shown). These results indicate that the “WFP” motif within the TMV 126 kDa protein is necessary for the proper interaction of the TMV 126 kDa protein with host factors to localize to the ER, and this association is correlated with the ability of the virus to efficiently infect the host. Altering the “WFP” motif prevents localization to the ER.  
       EXAMPLE 2  
       [0076]    The TMV 126 kDa protein ORFs from the “WFP”, “WYP”, and “WAP” viruses were fused with GFP ORF to yield 126F:GFP (containing the “WFP” motif), 126Y:GFP (containing the “WYP” motif) and 126A:GFP (containing the “WAP” motif) constructs. These constructs were placed behind an enhanced 35S promoter for transient expression in both  N. tabacum  Xanthi nn and  N. benthamiana  leaf cells by biolistic bombardment (FIG. 5A). The fluorescent signal was observed in subcellular bodies as punctate dots and along the periphery of the cells (FIGS. 5B-5S). The fluorescent 126F:GFP was stable for at least 8 days in both Nicotiana species (FIGS. 5B-5G), while the intensity of fluorescence declined rapidly for the 126A:GFP and 126Y:GFP fusions in  N. tabacum  (FIG. 5H, 5J,  5 L,  5 N,  5 P, and  5 R). In  N. benthamiana , however, the fluorescence produced by the 126Y:GFP fusion was not reduced relative to the 126F:GFP fusion over time (FIGS. 5S and 5G). The stability pattern of the various transiently expressed 126 kDa:GFP fusion proteins correlated with the ability of the parental and mutant viruses to efficiently infect the host. This finding also shows that the stabilization of viral replicase complex through the altered 126 kDa protein requires species-specific host factors.  
         [0077]    [0077] N. benthamiana  protoplasts were transfected with 126F:GFP-, 126Y:GFP-, and 126A:GFP-containing plasmids to study the subcellular localization of the 126 kDa:GFP fusion proteins during transient expression. The fusion proteins formed many small irregular bodies within the cytosol (FIGS. 6A, 6C, and  6 E), unlike the non-fused GFP construct which failed to form subcellular bodies 7 hours post-inoculation (FIG. 6G). At 24 hours after inoculation, the protoplasts expressing the 126F:GFP and 126Y:GFP constructs appeared to have fewer, but larger fluorescent bodies (FIGS. 6D and 6F). The protoplasts expressing free GFP formed no punctate bodies even after 24 hours (FIGS. 6G and 6H).  
         [0078]    [0078] N. tabacum  (cv. BY-2) protoplasts were also transfected with 126F:GFP-, 126Y:GFP-, and 126A:GFP-containing plasmids. The irregular fluorescent bodies that resulted could be categorized into two types: small bodies less than 2 μm in diameter which disappeared over time, and large bodies more than 2 μm in diameter which persisted. The wild-type 126F:GFP fusion protein formed both types of bodies in BY-2 cells (FIGS. 7A and 7B). The 126Y:GFP and 126A:GFP fusion proteins formed mostly only small bodies (FIG. 7B). Generally, the 126A:GFP fusion protein produced fewer large bodies than did the 126Y:GFP fusion protein (FIG. 7A). Also, the small bodies produced by the 126A:GFP fusion protein disappeared even more rapidly than did those formed by the 126Y:GFP fusion protein (FIG. 7A). These results indicated that the 126 kDa protein alone, even without other viral proteins, localized to the ER in infected cells. A determinant that controls localization of TMV 126 kDa protein to the ER is the “WFP” motif or the motif affected by the “WFP” motif.  
         [0079]    The previous results indicated that the altered 126 kDa:GFP fusion proteins were less stable than the “WFP” containing fusion protein in BY-2 cells. To determine if the 26S proteosome was responsible for degrading these TMV proteins, we expressed the fusion proteins in BY-2 cells incubated in the presence or absence of Acetyl-Leu-Leu-norleucinal (ALLN), an inhibitor of the 26S proteasome. Cells incubated in ALLN and transfected with either of the mutant 126Y:GFP or 126A:GFP fusion constructs yielded fluorescent signals that were greater and more stable compared to the signals from transfected cells without ALLN (compare FIGS. 8G, 8I,  8 K,  8 M,  8 O, and  8 Q to FIGS. 8B, 8D,  8 F,  8 H,  8 J, and  8 L). In the ALLN-treated cells, the 126Y:GFP fusion protein produced more fluorescent small bodies and also formed the large irregular bodies that localized around the nucleus at late stages, similar to what was observed for the 126F:GFP fusion protein (FIGS. 8G-8L for 126Y:GFP and compare to FIGS. 8B, 8D, and 8E for 126F:GFP). This result demonstrates that the “WYP” fusion protein can form small bodies in the absence of ALLN, but cannot avoid the host degradation machinery in the absence of inhibitor, thereby leading to an inability to form the large stable bodies. Also, the presence of the inhibitor led to greater expression of the 126F: wild-type GFP fusion than in its absence (FIG. 8B, 8D,  8 F, versus  8 A,  8 C, and  8 E). These findings indicate that the instability of the altered 126 kDa:GFP fusion proteins was due to their degradation by the host 26S proteasome. The maintenance of the “WFP” motif within the 126 kDa protein was thus critical to inhibit the degradation of this protein by the host ubiquitin-facilitated pathway. The ability of the altered viral proteins to form bodies in  N. benthamiana  cells and not in  N. tabacum  BY2 cells showed that the ability to degrade the viral protein is controlled by host factors in  N. tabacum  that better recognize structural change in the target than those from  N. benthamiana . Therefore, protein with the WFP motif resists ubiquitin-dependent degradation.  
         [0080]    We have found that the 126 kDa protein stabilizes expression of a fused protein in cells. When the 126 kDa protein was fused with GFP, the expression of the fused protein in the cell cytoplasm, as detected by fluorescence microscopy, was observed for two days longer than unfused GFP. The free GFP was only detectable for up to 5 days, whereas the 126 kDa protein fused with GFP was detectable at 7 days, the last time point collected. Thus, the fusion of the normal 126 kDa protein (i.e. containing the WFP motif) with a foreign protein stabilizes the expression phenotype of the foreign protein.  
         [0081]    In summary, an amino acid motif, “WFP”, was identified in the TMV 126 kDa and 183 kDa proteins (amino acid position 365 to 367 as numbered SEQ ID:2 and SEQ ID NO:4) that was conserved among both viral proteins and host membrane-associated proteins. When the “WFP” motif was mutated to “WYP” or “WAP”, the mutant viruses containing these new motifs were dramatically less capable of infecting and replicating in  N. tabacum , but could infect  N. benthamiana . Immunolabeling of the 126 kDa/183 kDa protein complex in virus-infected cells indicated that the replicase co-localized with binding protein (BiP), a host protein associated with the ER. However, the mutant virus containing WYP failed to localize BiP and the 126 kDa mutant protein to the ER. Transient expression of the 126 kDa protein fused with GFP showed that the mutant 126Y:GFP and 126A:GFP were unstable in plants and protoplasts of  N. tabacum , but stable in plants and protoplasts of  N. benthamiana . Thus, altering the “WFP” motif resulted in an increased degradation of this fusion protein depending on the host cell species. The wild-type 126 kDa:GFP protein fusions formed cytoplasmic bodies in transfected protoplasts and these bodies could be categorized into two types. Small bodies were less than 2 μm in diameter and disappeared in the WYP- and WAP-transfected cells after 48 hours, and large bodies that were more than 2 μm in diameter that persisted for WFP-transfected cells but not for WYP - or WAP-transfected cells. The 126F:GFP fusion maintained expression of large bodies longer than did 126Y:GFP or 126A:GFP. In the presence of the 26S proteasome inhibitor (ALLN), the 126Y:GFP and 126A:GFP fusions appeared more stable than in the absence of the inhibitor. Thus, the ubiquitin degradation pathway is involved in the degradation of the mutant 126 kDa protein. The accumulation of 126F:GFP fusion protein was increased in the presence of a 26S proteosome inhibitor, indicating some resistance of this protein, even in the absence of other viral proteins, to the ubiquitin degradation pathway.  
       EXAMPLE 3  
       [0082]    Anyone skilled in the art of protein biochemistry recognizes that the invention herein disclosed may be combined with known methods and materials to yield embodiments not directly mentioned. Because a three amino acid motif within a larger viral ER-colocalizing protein has been identified to render a fused protein more stable in plant cells, a reasonable embodiment of the current invention is to alter the viral ER-colocalizing protein in positions outside the three amino acid motif. By removing portions of the ER-colocalizing protein, it may be possible to minimize the region that confers stability to a fused engineered protein. Alternatively, amino acid substitutions can be made at regions outside the three amino acid motif that confers stability to a fused engineered protein. Naturally, because the truncations and substitutions that will be successful in the invention disclosed are outside the three amino acid motif, they can be used with a mutated the three amino acid motif to render a fused engineered protein unstable.  
         [0083]    A person skilled in the art that recognizes the possibility of including truncations and substitutions with the invention described herein will also recognize the possibility of fusing a peptide containing within it the three amino acid motif to a gene of interest to confer stability to the engineered protein. Alternatively, the same peptide when identified may contain a mutated three amino acid motif to render a fused engineered protein unstable.  
         [0084]    Literature Cited  
         [0085]    Bao et al. 1996 J. Virol. 70: 6378-6383  
         [0086]    Bao, Y. and Hull, R. 1993, J Gen Virol 74:1611-1616  
         [0087]    Cheng et al., 2000, Plant J. 23: 1-16.  
         [0088]    Cheng et al., 2000, Plant J., in press  
         [0089]    Deom et al. Science, 1987, 237:389-394  
         [0090]    Derrick et al., 1997, Mol. Plant-Microbe Interaction 10: 589-596.  
         [0091]    Ecker et al. 1989 J Biol. Chem. 264:7715-779  
         [0092]    Heinlein et al. 1995 Science 270: 1983-1985  
         [0093]    Heinlein et al., 1998, Plant Cell 10: 1107-1120.  
         [0094]    Holt, et al., 1990, MPMI 3:417-423  
         [0095]    Higuchi, R. (1990) In “PCR Protocols: A guide to methods and applications” (M. A. Innis, D. H. Gelford, J. J. Sninsky and T. J. White, Eds.) p. 177-183 Academic Press, San Diego  
         [0096]    Itaya et al., 1997, Plant J. 12:1223-1230  
         [0097]    Janda and Ahlquist 1998, Proc. Natl. Acad. Sci. USA 95: 2227-2232  
         [0098]    Lewandowski and Dawson 2000, Virology 271: 90-98.  
         [0099]    Laemmli 1970, Nature 227:680-685  
         [0100]    Mas and Beachy 1998, Plant J 15:835-842  
         [0101]    Mas and Beachy 1999, J. Cell Biol. 147: 945-958.  
         [0102]    Nelson, et al. 1993 MPMI 6:45-54  
         [0103]    Osman and Buck 1996, J. Virol. 70: 6227-7234.  
         [0104]    Reichel and Beachy 2000, J. Virol. 74: 3330-3337.  
         [0105]    Restrepo-Hartwig and Ahlquist 1999,J Virol. 73: 10303-10309.  
         [0106]    Restrepo-Hartwig et al., 1990 Plant Cell 2:987-998  
         [0107]    Shintaku et al., 1996, Virology 221: 218-225.  
         [0108]    Sullivan and Ahlquist 1999, J. Virol. 73: 2622-2632  
         [0109]    Szecsi et al., 1999, Mol. Plant-Microbe Interaction. 12: 143-152.  
         [0110]    Thompson et al., 1994, Nucl. Acids Res. 22: 4673-4680.  
         [0111]    Tpfer, et al. 1987, Nucl. Acids Res. 15:5890.  
         [0112]    Vierstra, R. D. 1996 Plant Mol. Biol. 32:275-302  
         [0113]    Watanabe et al. 1987 FEBS Letters 219:65-69  
         [0114]    Watanabe et al., 1999, J. Virol. 73: 2633-2640.  
     
       
       
         1 
         
           
             27  
           
           
             1  
             3351  
             DNA  
             Tobacco mosaic virus  
             
               CDS  
               (1)..(3348)  
             
           
            1 

atg gca tac aca cag aca gct acc aca tca gct ttg ctg gac act gtc       48 
Met Ala Tyr Thr Gln Thr Ala Thr Thr Ser Ala Leu Leu Asp Thr Val 
1               5                   10                  15 

cga gga aac aac tcc ttg gtc aat gat cta gca aag cgt cgt ctt tac       96 
Arg Gly Asn Asn Ser Leu Val Asn Asp Leu Ala Lys Arg Arg Leu Tyr 
            20                  25                  30 

gac aca gcg gtt gaa gag ttt aac gct cgt gac cgc agg ccc aaa gtg      144 
Asp Thr Ala Val Glu Glu Phe Asn Ala Arg Asp Arg Arg Pro Lys Val 
        35                  40                  45 

aac ttt tca aaa gta ata agc gag gag cag acg ctt att gct acc cgg      192 
Asn Phe Ser Lys Val Ile Ser Glu Glu Gln Thr Leu Ile Ala Thr Arg 
    50                  55                  60 

gcg tat cca gaa ttc caa att aca ttt tat aac acg caa aat gcc gtg      240 
Ala Tyr Pro Glu Phe Gln Ile Thr Phe Tyr Asn Thr Gln Asn Ala Val 
65                  70                  75                  80 

cat tcg ctt gca ggt gga ttg cga tct tta gaa ctg gaa tat ctg atg      288 
His Ser Leu Ala Gly Gly Leu Arg Ser Leu Glu Leu Glu Tyr Leu Met 
                85                  90                  95 

atg caa att ccc tac gga tca ttg act tat gac ata ggc ggg aat ttt      336 
Met Gln Ile Pro Tyr Gly Ser Leu Thr Tyr Asp Ile Gly Gly Asn Phe 
            100                 105                 110 

gca tcg cat ctg ttc aag gga cga gca tat gta cac tgc tgc atg ccc      384 
Ala Ser His Leu Phe Lys Gly Arg Ala Tyr Val His Cys Cys Met Pro 
        115                 120                 125 

aac ctg gac gtt cga gac atc atg cgg cat gaa ggc cag aaa gac agt      432 
Asn Leu Asp Val Arg Asp Ile Met Arg His Glu Gly Gln Lys Asp Ser 
    130                 135                 140 

att gaa cta tac ctt tct agg cta gag aga ggg gga aaa aca gtc ccc      480 
Ile Glu Leu Tyr Leu Ser Arg Leu Glu Arg Gly Gly Lys Thr Val Pro 
145                 150                 155                 160 

aac ttc caa aag gaa gca ttt gac aga tac gca gaa att cct gaa gac      528 
Asn Phe Gln Lys Glu Ala Phe Asp Arg Tyr Ala Glu Ile Pro Glu Asp 
                165                 170                 175 

gct gtc tgt cac aat act ttc cag aca tgc gaa cat cag ccg atg caa      576 
Ala Val Cys His Asn Thr Phe Gln Thr Cys Glu His Gln Pro Met Gln 
            180                 185                 190 

caa tca ggc aga gtg tat gcc att gcg cta cac agc ata tat gac ata      624 
Gln Ser Gly Arg Val Tyr Ala Ile Ala Leu His Ser Ile Tyr Asp Ile 
        195                 200                 205 

ccc gct gat gag ttc ggg gca gca ctc ttg agg aaa aat gtc cat acg      672 
Pro Ala Asp Glu Phe Gly Ala Ala Leu Leu Arg Lys Asn Val His Thr 
    210                 215                 220 

tgc tat gcc gct ttc cac ttc tct gag aac ctg ctt ctt gaa gat tca      720 
Cys Tyr Ala Ala Phe His Phe Ser Glu Asn Leu Leu Leu Glu Asp Ser 
225                 230                 235                 240 

tac gtc aat ctg gac gaa atc aac gcg tgt ttt tcg cgc gat gga gac      768 
Tyr Val Asn Leu Asp Glu Ile Asn Ala Cys Phe Ser Arg Asp Gly Asp 
                245                 250                 255 

aag ttg acc ttt tct ttt gca tca gag agt act ctt aat tac tgt cat      816 
Lys Leu Thr Phe Ser Phe Ala Ser Glu Ser Thr Leu Asn Tyr Cys His 
            260                 265                 270 

agt tat tct aat att ctt aag tat gtg tgc aaa act tac ttc ccg gcc      864 
Ser Tyr Ser Asn Ile Leu Lys Tyr Val Cys Lys Thr Tyr Phe Pro Ala 
        275                 280                 285 

tct aat aga gag gtt tac atg aag gag ttt tta gtc acc agg gtt aat      912 
Ser Asn Arg Glu Val Tyr Met Lys Glu Phe Leu Val Thr Arg Val Asn 
    290                 295                 300 

acc tgg ttt tgt aag ttt tct aga ata gat act ttt ctt ttg tac aaa      960 
Thr Trp Phe Cys Lys Phe Ser Arg Ile Asp Thr Phe Leu Leu Tyr Lys 
305                 310                 315                 320 

ggt gtg gcc cat aaa ggt gta gat agt gag cag ttt tat act gca atg     1008 
Gly Val Ala His Lys Gly Val Asp Ser Glu Gln Phe Tyr Thr Ala Met 
                325                 330                 335 

gaa gac gca tgg cat tac aaa aag act ctt gca atg tgc aac agc gag     1056 
Glu Asp Ala Trp His Tyr Lys Lys Thr Leu Ala Met Cys Asn Ser Glu 
            340                 345                 350 

aga atc ctc ctt gag gat tca tca aca gtc aat tac tgg ttt ccc gaa     1104 
Arg Ile Leu Leu Glu Asp Ser Ser Thr Val Asn Tyr Trp Phe Pro Glu 
        355                 360                 365 

atg agg gat atg gtc atc gta cca tta ttc gac att tct ttg gag act     1152 
Met Arg Asp Met Val Ile Val Pro Leu Phe Asp Ile Ser Leu Glu Thr 
    370                 375                 380 

agt aag agg acg cgc aag gaa gtc tta gtg tcc aag gat ttc gtg ttt     1200 
Ser Lys Arg Thr Arg Lys Glu Val Leu Val Ser Lys Asp Phe Val Phe 
385                 390                 395                 400 

aca gtg ctt aac cac att cga aca tac cag gca aaa gct ctt aca tac     1248 
Thr Val Leu Asn His Ile Arg Thr Tyr Gln Ala Lys Ala Leu Thr Tyr 
                405                 410                 415 

gta aat gtt ttg tcc ttc gtc gaa tcg att cga tcg agg gta atc att     1296 
Val Asn Val Leu Ser Phe Val Glu Ser Ile Arg Ser Arg Val Ile Ile 
            420                 425                 430 

aac ggt gtg aca gcg agg tcc gaa tgg gat gtg gac aaa tct ttg tta     1344 
Asn Gly Val Thr Ala Arg Ser Glu Trp Asp Val Asp Lys Ser Leu Leu 
        435                 440                 445 

caa tcc ttg tcc atg acg ttt tac ctg cat act aag ctt gcc gtt cta     1392 
Gln Ser Leu Ser Met Thr Phe Tyr Leu His Thr Lys Leu Ala Val Leu 
    450                 455                 460 

aag gat gac tta ctg att agc aag ttt agt ctc ggt tcg aaa acg gtg     1440 
Lys Asp Asp Leu Leu Ile Ser Lys Phe Ser Leu Gly Ser Lys Thr Val 
465                 470                 475                 480 

tgc cag cat gtg tgg gat gag att tca ctg gcg ttt ggg aac gca ttt     1488 
Cys Gln His Val Trp Asp Glu Ile Ser Leu Ala Phe Gly Asn Ala Phe 
                485                 490                 495 

ccc tcc gtg aaa gag agg ctc ttg aac agg aaa ctt atc aga gtg gca     1536 
Pro Ser Val Lys Glu Arg Leu Leu Asn Arg Lys Leu Ile Arg Val Ala 
            500                 505                 510 

ggc gac gca cta gag atc agg gtg cct gat cta tat gtg acc ttc cac     1584 
Gly Asp Ala Leu Glu Ile Arg Val Pro Asp Leu Tyr Val Thr Phe His 
        515                 520                 525 

gac cga tta gtg act gag tac aag gcc tct gtg gac atg cct gcg ctt     1632 
Asp Arg Leu Val Thr Glu Tyr Lys Ala Ser Val Asp Met Pro Ala Leu 
    530                 535                 540 

gac att agg aag aag atg gaa gaa acg gaa gtg atg tac aat gca ctt     1680 
Asp Ile Arg Lys Lys Met Glu Glu Thr Glu Val Met Tyr Asn Ala Leu 
545                 550                 555                 560 

tca gag tta tcg gtg tta agg gag tct gac aaa ttc gat gtt gat gtt     1728 
Ser Glu Leu Ser Val Leu Arg Glu Ser Asp Lys Phe Asp Val Asp Val 
                565                 570                 575 

ttt tcc cag atg tgc caa tct ttg gaa gtt gac gca atg acg gca gcg     1776 
Phe Ser Gln Met Cys Gln Ser Leu Glu Val Asp Ala Met Thr Ala Ala 
            580                 585                 590 

aag gtt ata gtc gcg gtc atg agc aat aag agc ggt ctg act ctc aca     1824 
Lys Val Ile Val Ala Val Met Ser Asn Lys Ser Gly Leu Thr Leu Thr 
        595                 600                 605 

ttt gaa cga cct act gag gcg aat gtt gcg cta gct tta cag gat caa     1872 
Phe Glu Arg Pro Thr Glu Ala Asn Val Ala Leu Ala Leu Gln Asp Gln 
    610                 615                 620 

gaa aag gct tca gaa ggt gct ttg gta gtt acc tca aga gaa gtt gaa     1920 
Glu Lys Ala Ser Glu Gly Ala Leu Val Val Thr Ser Arg Glu Val Glu 
625                 630                 635                 640 

gaa ccg tcc atg aag ggt tcg atg gcc aga gga gag tta caa tta gct     1968 
Glu Pro Ser Met Lys Gly Ser Met Ala Arg Gly Glu Leu Gln Leu Ala 
                645                 650                 655 

ggt ctt gct gga gat cat ccg gag tcg tcc tat tct agg aac gag gag     2016 
Gly Leu Ala Gly Asp His Pro Glu Ser Ser Tyr Ser Arg Asn Glu Glu 
            660                 665                 670 

ata gag tct tta gag cag ttt cat atg gca acg gca gat tcg tta att     2064 
Ile Glu Ser Leu Glu Gln Phe His Met Ala Thr Ala Asp Ser Leu Ile 
        675                 680                 685 

cgt aag cag atg agc tcg att gtg tac acg ggt ccg att aaa gtt cag     2112 
Arg Lys Gln Met Ser Ser Ile Val Tyr Thr Gly Pro Ile Lys Val Gln 
    690                 695                 700 

caa atg aaa aac ttt atc gat agc ctg gta gca tca cta tct gct gcg     2160 
Gln Met Lys Asn Phe Ile Asp Ser Leu Val Ala Ser Leu Ser Ala Ala 
705                 710                 715                 720 

gtg tcg aat ctc gtc aag atc ctc aaa gat aca gct gct att gac ctt     2208 
Val Ser Asn Leu Val Lys Ile Leu Lys Asp Thr Ala Ala Ile Asp Leu 
                725                 730                 735 

gaa acc cgt caa aag ttt gga gtc ttg gat gtt aca tct agg aag tgg     2256 
Glu Thr Arg Gln Lys Phe Gly Val Leu Asp Val Thr Ser Arg Lys Trp 
            740                 745                 750 

tta att aaa cca acg gcc aag agt cat gca tgg ggt gtt gtt gaa acc     2304 
Leu Ile Lys Pro Thr Ala Lys Ser His Ala Trp Gly Val Val Glu Thr 
        755                 760                 765 

cac gcg agg aag tat cat gtg gcg ctt ctg gaa tat gat gag cag ggt     2352 
His Ala Arg Lys Tyr His Val Ala Leu Leu Glu Tyr Asp Glu Gln Gly 
    770                 775                 780 

gtg gtg aca tgc gat gat tgg aga aga gta gct gtc agc tct gag tct     2400 
Val Val Thr Cys Asp Asp Trp Arg Arg Val Ala Val Ser Ser Glu Ser 
785                 790                 795                 800 

gtt gtt tat tcc gac atg gcg aaa ctc aga act ctg cgc aga ctg ctt     2448 
Val Val Tyr Ser Asp Met Ala Lys Leu Arg Thr Leu Arg Arg Leu Leu 
                805                 810                 815 

cga aac gga gaa ccg cat gtc agt agc gca aag gtt gtt ctt gtg gac     2496 
Arg Asn Gly Glu Pro His Val Ser Ser Ala Lys Val Val Leu Val Asp 
            820                 825                 830 

gga gtt ccg ggc tgt gga aaa acc aaa gaa att ctt tcc agg gtt aat     2544 
Gly Val Pro Gly Cys Gly Lys Thr Lys Glu Ile Leu Ser Arg Val Asn 
        835                 840                 845 

ttt gat gaa gat cta att tta gta cct ggg aag caa gct gct gaa atg     2592 
Phe Asp Glu Asp Leu Ile Leu Val Pro Gly Lys Gln Ala Ala Glu Met 
    850                 855                 860 

atc aga aga cgt gcg aat tcc tca ggg att att gtg gcc acg aag gac     2640 
Ile Arg Arg Arg Ala Asn Ser Ser Gly Ile Ile Val Ala Thr Lys Asp 
865                 870                 875                 880 

aac gtt aaa acc gtt gat tct ttc atg atg aat ttt ggg aaa agc aca     2688 
Asn Val Lys Thr Val Asp Ser Phe Met Met Asn Phe Gly Lys Ser Thr 
                885                 890                 895 

cgc tgt cag ttc aag agg tta ttc att gat gaa ggg ttg atg ttg cat     2736 
Arg Cys Gln Phe Lys Arg Leu Phe Ile Asp Glu Gly Leu Met Leu His 
            900                 905                 910 

act ggt tgt gtt aat ttt ctt gtg gcg atg tca ttg tgc gaa att gca     2784 
Thr Gly Cys Val Asn Phe Leu Val Ala Met Ser Leu Cys Glu Ile Ala 
        915                 920                 925 

tat gtt tac gga gac aca cag cag att cca tac atc aat aga gtt tca     2832 
Tyr Val Tyr Gly Asp Thr Gln Gln Ile Pro Tyr Ile Asn Arg Val Ser 
    930                 935                 940 

gga ttc ccg tac ccc gcc cat ttt gcc aaa ttg gaa gtt gac gag gtg     2880 
Gly Phe Pro Tyr Pro Ala His Phe Ala Lys Leu Glu Val Asp Glu Val 
945                 950                 955                 960 

gag aca cgc aga act act ctc cgt tgt cca gcc gat gtc aca cat tat     2928 
Glu Thr Arg Arg Thr Thr Leu Arg Cys Pro Ala Asp Val Thr His Tyr 
                965                 970                 975 

ctg aac agg aga tat gag ggc ttt gtc atg agc act tct tcg gtt aaa     2976 
Leu Asn Arg Arg Tyr Glu Gly Phe Val Met Ser Thr Ser Ser Val Lys 
            980                 985                 990 

aag tct gtt tcg cag gag atg gtc  ggc gga gcc gcc gtg  atc aat ccg   3024 
Lys Ser Val Ser Gln Glu Met Val  Gly Gly Ala Ala Val  Ile Asn Pro 
        995                 1000                 1005 

atc tca  aaa ccc ttg cat ggc  aag atc ctg act ttt  acc caa tcg      3069 
Ile Ser  Lys Pro Leu His Gly  Lys Ile Leu Thr Phe  Thr Gln Ser 
    1010                 1015                 1020 

gat aaa  gaa gct ctg ctt tca  aga ggg tat tca gat  gtt cac act      3114 
Asp Lys  Glu Ala Leu Leu Ser  Arg Gly Tyr Ser Asp  Val His Thr 
    1025                 1030                 1035 

gtg cat  gaa gtg caa ggc gag  aca tac tct gat gtt  tca cta gtt      3159 
Val His  Glu Val Gln Gly Glu  Thr Tyr Ser Asp Val  Ser Leu Val 
    1040                 1045                 1050 

agg cta  acc cct aca cca gtc  tcc atc att gca gga  gac agc ccg      3204 
Arg Leu  Thr Pro Thr Pro Val  Ser Ile Ile Ala Gly  Asp Ser Pro 
    1055                 1060                 1065 

cat gtt  ttg gtc gca ttg tca  agg cac acc tgt tcg  ctc aag tac      3249 
His Val  Leu Val Ala Leu Ser  Arg His Thr Cys Ser  Leu Lys Tyr 
    1070                 1075                 1080 

tac act  gtt gtt atg gat cct  tta gtt agt atc att  aga gat cta      3294 
Tyr Thr  Val Val Met Asp Pro  Leu Val Ser Ile Ile  Arg Asp Leu 
    1085                 1090                 1095 

gag aaa  ctt agc tcg tac ttg  tta gat atg tat aag  gtc gat gca      3339 
Glu Lys  Leu Ser Ser Tyr Leu  Leu Asp Met Tyr Lys  Val Asp Ala 
    1100                 1105                 1110 

gga aca  caa tag                                                    3351 
Gly Thr  Gln 
    1115 

 
           
             2  
             1116  
             PRT  
             Tobacco mosaic virus  
           
            2 

Met Ala Tyr Thr Gln Thr Ala Thr Thr Ser Ala Leu Leu Asp Thr Val 
1               5                   10                  15 

Arg Gly Asn Asn Ser Leu Val Asn Asp Leu Ala Lys Arg Arg Leu Tyr 
            20                  25                  30 

Asp Thr Ala Val Glu Glu Phe Asn Ala Arg Asp Arg Arg Pro Lys Val 
        35                  40                  45 

Asn Phe Ser Lys Val Ile Ser Glu Glu Gln Thr Leu Ile Ala Thr Arg 
    50                  55                  60 

Ala Tyr Pro Glu Phe Gln Ile Thr Phe Tyr Asn Thr Gln Asn Ala Val 
65                  70                  75                  80 

His Ser Leu Ala Gly Gly Leu Arg Ser Leu Glu Leu Glu Tyr Leu Met 
                85                  90                  95 

Met Gln Ile Pro Tyr Gly Ser Leu Thr Tyr Asp Ile Gly Gly Asn Phe 
            100                 105                 110 

Ala Ser His Leu Phe Lys Gly Arg Ala Tyr Val His Cys Cys Met Pro 
        115                 120                 125 

Asn Leu Asp Val Arg Asp Ile Met Arg His Glu Gly Gln Lys Asp Ser 
    130                 135                 140 

Ile Glu Leu Tyr Leu Ser Arg Leu Glu Arg Gly Gly Lys Thr Val Pro 
145                 150                 155                 160 

Asn Phe Gln Lys Glu Ala Phe Asp Arg Tyr Ala Glu Ile Pro Glu Asp 
                165                 170                 175 

Ala Val Cys His Asn Thr Phe Gln Thr Cys Glu His Gln Pro Met Gln 
            180                 185                 190 

Gln Ser Gly Arg Val Tyr Ala Ile Ala Leu His Ser Ile Tyr Asp Ile 
        195                 200                 205 

Pro Ala Asp Glu Phe Gly Ala Ala Leu Leu Arg Lys Asn Val His Thr 
    210                 215                 220 

Cys Tyr Ala Ala Phe His Phe Ser Glu Asn Leu Leu Leu Glu Asp Ser 
225                 230                 235                 240 

Tyr Val Asn Leu Asp Glu Ile Asn Ala Cys Phe Ser Arg Asp Gly Asp 
                245                 250                 255 

Lys Leu Thr Phe Ser Phe Ala Ser Glu Ser Thr Leu Asn Tyr Cys His 
            260                 265                 270 

Ser Tyr Ser Asn Ile Leu Lys Tyr Val Cys Lys Thr Tyr Phe Pro Ala 
        275                 280                 285 

Ser Asn Arg Glu Val Tyr Met Lys Glu Phe Leu Val Thr Arg Val Asn 
    290                 295                 300 

Thr Trp Phe Cys Lys Phe Ser Arg Ile Asp Thr Phe Leu Leu Tyr Lys 
305                 310                 315                 320 

Gly Val Ala His Lys Gly Val Asp Ser Glu Gln Phe Tyr Thr Ala Met 
                325                 330                 335 

Glu Asp Ala Trp His Tyr Lys Lys Thr Leu Ala Met Cys Asn Ser Glu 
            340                 345                 350 

Arg Ile Leu Leu Glu Asp Ser Ser Thr Val Asn Tyr Trp Phe Pro Glu 
        355                 360                 365 

Met Arg Asp Met Val Ile Val Pro Leu Phe Asp Ile Ser Leu Glu Thr 
    370                 375                 380 

Ser Lys Arg Thr Arg Lys Glu Val Leu Val Ser Lys Asp Phe Val Phe 
385                 390                 395                 400 

Thr Val Leu Asn His Ile Arg Thr Tyr Gln Ala Lys Ala Leu Thr Tyr 
                405                 410                 415 

Val Asn Val Leu Ser Phe Val Glu Ser Ile Arg Ser Arg Val Ile Ile 
            420                 425                 430 

Asn Gly Val Thr Ala Arg Ser Glu Trp Asp Val Asp Lys Ser Leu Leu 
        435                 440                 445 

Gln Ser Leu Ser Met Thr Phe Tyr Leu His Thr Lys Leu Ala Val Leu 
    450                 455                 460 

Lys Asp Asp Leu Leu Ile Ser Lys Phe Ser Leu Gly Ser Lys Thr Val 
465                 470                 475                 480 

Cys Gln His Val Trp Asp Glu Ile Ser Leu Ala Phe Gly Asn Ala Phe 
                485                 490                 495 

Pro Ser Val Lys Glu Arg Leu Leu Asn Arg Lys Leu Ile Arg Val Ala 
            500                 505                 510 

Gly Asp Ala Leu Glu Ile Arg Val Pro Asp Leu Tyr Val Thr Phe His 
        515                 520                 525 

Asp Arg Leu Val Thr Glu Tyr Lys Ala Ser Val Asp Met Pro Ala Leu 
    530                 535                 540 

Asp Ile Arg Lys Lys Met Glu Glu Thr Glu Val Met Tyr Asn Ala Leu 
545                 550                 555                 560 

Ser Glu Leu Ser Val Leu Arg Glu Ser Asp Lys Phe Asp Val Asp Val 
                565                 570                 575 

Phe Ser Gln Met Cys Gln Ser Leu Glu Val Asp Ala Met Thr Ala Ala 
            580                 585                 590 

Lys Val Ile Val Ala Val Met Ser Asn Lys Ser Gly Leu Thr Leu Thr 
        595                 600                 605 

Phe Glu Arg Pro Thr Glu Ala Asn Val Ala Leu Ala Leu Gln Asp Gln 
    610                 615                 620 

Glu Lys Ala Ser Glu Gly Ala Leu Val Val Thr Ser Arg Glu Val Glu 
625                 630                 635                 640 

Glu Pro Ser Met Lys Gly Ser Met Ala Arg Gly Glu Leu Gln Leu Ala 
                645                 650                 655 

Gly Leu Ala Gly Asp His Pro Glu Ser Ser Tyr Ser Arg Asn Glu Glu 
            660                 665                 670 

Ile Glu Ser Leu Glu Gln Phe His Met Ala Thr Ala Asp Ser Leu Ile 
        675                 680                 685 

Arg Lys Gln Met Ser Ser Ile Val Tyr Thr Gly Pro Ile Lys Val Gln 
    690                 695                 700 

Gln Met Lys Asn Phe Ile Asp Ser Leu Val Ala Ser Leu Ser Ala Ala 
705                 710                 715                 720 

Val Ser Asn Leu Val Lys Ile Leu Lys Asp Thr Ala Ala Ile Asp Leu 
                725                 730                 735 

Glu Thr Arg Gln Lys Phe Gly Val Leu Asp Val Thr Ser Arg Lys Trp 
            740                 745                 750 

Leu Ile Lys Pro Thr Ala Lys Ser His Ala Trp Gly Val Val Glu Thr 
        755                 760                 765 

His Ala Arg Lys Tyr His Val Ala Leu Leu Glu Tyr Asp Glu Gln Gly 
    770                 775                 780 

Val Val Thr Cys Asp Asp Trp Arg Arg Val Ala Val Ser Ser Glu Ser 
785                 790                 795                 800 

Val Val Tyr Ser Asp Met Ala Lys Leu Arg Thr Leu Arg Arg Leu Leu 
                805                 810                 815 

Arg Asn Gly Glu Pro His Val Ser Ser Ala Lys Val Val Leu Val Asp 
            820                 825                 830 

Gly Val Pro Gly Cys Gly Lys Thr Lys Glu Ile Leu Ser Arg Val Asn 
        835                 840                 845 

Phe Asp Glu Asp Leu Ile Leu Val Pro Gly Lys Gln Ala Ala Glu Met 
    850                 855                 860 

Ile Arg Arg Arg Ala Asn Ser Ser Gly Ile Ile Val Ala Thr Lys Asp 
865                 870                 875                 880 

Asn Val Lys Thr Val Asp Ser Phe Met Met Asn Phe Gly Lys Ser Thr 
                885                 890                 895 

Arg Cys Gln Phe Lys Arg Leu Phe Ile Asp Glu Gly Leu Met Leu His 
            900                 905                 910 

Thr Gly Cys Val Asn Phe Leu Val Ala Met Ser Leu Cys Glu Ile Ala 
        915                 920                 925 

Tyr Val Tyr Gly Asp Thr Gln Gln Ile Pro Tyr Ile Asn Arg Val Ser 
    930                 935                 940 

Gly Phe Pro Tyr Pro Ala His Phe Ala Lys Leu Glu Val Asp Glu Val 
945                 950                 955                 960 

Glu Thr Arg Arg Thr Thr Leu Arg Cys Pro Ala Asp Val Thr His Tyr 
                965                 970                 975 

Leu Asn Arg Arg Tyr Glu Gly Phe Val Met Ser Thr Ser Ser Val Lys 
            980                 985                 990 

Lys Ser Val Ser Gln Glu Met Val  Gly Gly Ala Ala Val  Ile Asn Pro 
        995                 1000                 1005 

Ile Ser  Lys Pro Leu His Gly  Lys Ile Leu Thr Phe  Thr Gln Ser 
    1010                 1015                 1020 

Asp Lys  Glu Ala Leu Leu Ser  Arg Gly Tyr Ser Asp  Val His Thr 
    1025                 1030                 1035 

Val His  Glu Val Gln Gly Glu  Thr Tyr Ser Asp Val  Ser Leu Val 
    1040                 1045                 1050 

Arg Leu  Thr Pro Thr Pro Val  Ser Ile Ile Ala Gly  Asp Ser Pro 
    1055                 1060                 1065 

His Val  Leu Val Ala Leu Ser  Arg His Thr Cys Ser  Leu Lys Tyr 
    1070                 1075                 1080 

Tyr Thr  Val Val Met Asp Pro  Leu Val Ser Ile Ile  Arg Asp Leu 
    1085                 1090                 1095 

Glu Lys  Leu Ser Ser Tyr Leu  Leu Asp Met Tyr Lys  Val Asp Ala 
    1100                 1105                 1110 

Gly Thr  Gln 
    1115 

 
           
             3  
             4834  
             DNA  
             Tobacco mosaic virus  
             
               gene  
               (1)..(4831)  
             
           
            3 

atggcataca cacagacagc taccacatca gctttgctgg acactgtccg aggaaacaac     60 

tccttggtca atgatctagc aaagcgtcgt ctttacgaca cagcggttga agagtttaac    120 

gctcgtgacc gcaggcccaa agtgaacttt tcaaaagtaa taagcgagga gcagacgctt    180 

attgctaccc gggcgtatcc agaattccaa attacatttt ataacacgca aaatgccgtg    240 

cattcgcttg caggtggatt gcgatcttta gaactggaat atctgatgat gcaaattccc    300 

tacggatcat tgacttatga cataggcggg aattttgcat cgcatctgtt caagggacga    360 

gcatatgtac actgctgcat gcccaacctg gacgttcgag acatcatgcg gcatgaaggc    420 

cagaaagaca gtattgaact atacctttct aggctagaga gagggggaaa aacagtcccc    480 

aacttccaaa aggaagcatt tgacagatac gcagaaattc ctgaagacgc tgtctgtcac    540 

aatactttcc agacatgcga acatcagccg atgcaacaat caggcagagt gtatgccatt    600 

gcgctacaca gcatatatga catacccgct gatgagttcg gggcagcact cttgaggaaa    660 

aatgtccata cgtgctatgc cgctttccac ttctctgaga acctgcttct tgaagattca    720 

tacgtcaatc tggacgaaat caacgcgtgt ttttcgcgcg atggagacaa gttgaccttt    780 

tcttttgcat cagagagtac tcttaattac tgtcatagtt attctaatat tcttaagtat    840 

gtgtgcaaaa cttacttccc ggcctctaat agagaggttt acatgaagga gtttttagtc    900 

accagggtta atacctggtt ttgtaagttt tctagaatag atacttttct tttgtacaaa    960 

ggtgtggccc ataaaggtgt agatagtgag cagttttata ctgcaatgga agacgcatgg   1020 

cattacaaaa agactcttgc aatgtgcaac agcgagagaa tcctccttga ggattcatca   1080 

acagtcaatt actggtttcc cgaaatgagg gatatggtca tcgtaccatt attcgacatt   1140 

tctttggaga ctagtaagag gacgcgcaag gaagtcttag tgtccaagga tttcgtgttt   1200 

acagtgctta accacattcg aacataccag gcaaaagctc ttacatacgt aaatgttttg   1260 

tccttcgtcg aatcgattcg atcgagggta atcattaacg gtgtgacagc gaggtccgaa   1320 

tgggatgtgg acaaatcttt gttacaatcc ttgtccatga cgttttacct gcatactaag   1380 

cttgccgttc taaaggatga cttactgatt agcaagttta gtctcggttc gaaaacggtg   1440 

tgccagcatg tgtgggatga gatttcactg gcgtttggga acgcatttcc ctccgtgaaa   1500 

gagaggctct tgaacaggaa acttatcaga gtggcaggcg acgcactaga gatcagggtg   1560 

cctgatctat atgtgacctt ccacgaccga ttagtgactg agtacaaggc ctctgtggac   1620 

atgcctgcgc ttgacattag gaagaagatg gaagaaacgg aagtgatgta caatgcactt   1680 

tcagagttat cggtgttaag ggagtctgac aaattcgatg ttgatgtttt ttcccagatg   1740 

tgccaatctt tggaagttga cgcaatgacg gcagcgaagg ttatagtcgc ggtcatgagc   1800 

aataagagcg gtctgactct cacatttgaa cgacctactg aggcgaatgt tgcgctagct   1860 

ttacaggatc aagaaaaggc ttcagaaggt gctttggtag ttacctcaag agaagttgaa   1920 

gaaccgtcca tgaagggttc gatggccaga ggagagttac aattagctgg tcttgctgga   1980 

gatcatccgg agtcgtccta ttctaggaac gaggagatag agtctttaga gcagtttcat   2040 

atggcaacgg cagattcgtt aattcgtaag cagatgagct cgattgtgta cacgggtccg   2100 

attaaagttc agcaaatgaa aaactttatc gatagcctgg tagcatcact atctgctgcg   2160 

gtgtcgaatc tcgtcaagat cctcaaagat acagctgcta ttgaccttga aacccgtcaa   2220 

aagtttggag tcttggatgt tacatctagg aagtggttaa ttaaaccaac ggccaagagt   2280 

catgcatggg gtgttgttga aacccacgcg aggaagtatc atgtggcgct tctggaatat   2340 

gatgagcagg gtgtggtgac atgcgatgat tggagaagag tagctgtcag ctctgagtct   2400 

gttgtttatt ccgacatggc gaaactcaga actctgcgca gactgcttcg aaacggagaa   2460 

ccgcatgtca gtagcgcaaa ggttgttctt gtggacggag ttccgggctg tggaaaaacc   2520 

aaagaaattc tttccagggt taattttgat gaagatctaa ttttagtacc tgggaagcaa   2580 

gctgctgaaa tgatcagaag acgtgcgaat tcctcaggga ttattgtggc cacgaaggac   2640 

aacgttaaaa ccgttgattc tttcatgatg aattttggga aaagcacacg ctgtcagttc   2700 

aagaggttat tcattgatga agggttgatg ttgcatactg gttgtgttaa ttttcttgtg   2760 

gcgatgtcat tgtgcgaaat tgcatatgtt tacggagaca cacagcagat tccatacatc   2820 

aatagagttt caggattccc gtaccccgcc cattttgcca aattggaagt tgacgaggtg   2880 

gagacacgca gaactactct ccgttgtcca gccgatgtca cacattatct gaacaggaga   2940 

tatgagggct ttgtcatgag cacttcttcg gttaaaaagt ctgtttcgca ggagatggtc   3000 

ggcggagccg ccgtgatcaa tccgatctca aaacccttgc atggcaagat cctgactttt   3060 

acccaatcgg ataaagaagc tctgctttca agagggtatt cagatgttca cactgtgcat   3120 

gaagtgcaag gcgagacata ctctgatgtt tcactagtta ggctaacccc tacaccagtc   3180 

tccatcattg caggagacag cccgcatgtt ttggtcgcat tgtcaaggca cacctgttcg   3240 

ctcaagtact acactgttgt tatggatcct ttagttagta tcattagaga tctagagaaa   3300 

cttagctcgt acttgttaga tatgtataag gtcgatgcag gaacacaata gcaattacag   3360 

attgactcgg tgttcaaagg ttccaatctt tttgtggcag cgccaaagac tggtgatatt   3420 

tctgatatgc agttttacta tgataagtgt ctcccaggca acagcaccat gatgaataat   3480 

tttgatgctg ttaccatgag gttgactgac atttcattga atgtcaaaga ttgcatattg   3540 

gatatgtcta agtctgttgc tgcgcctaag gatcaaatca aaccactaat acctatggta   3600 

cgaacggcgg cagaaatgcc acgccagact ggactattgg aaaatttagt ggcgatgatt   3660 

aaaaggaact ttaacgcacc cgagttgtct ggcatcattg atattgaaaa tactgcatct   3720 

ttagttgtag ataagttttt cgatagttat ttgcttaaag aaaaaagaaa accaaataaa   3780 

aatgtttctt tgttcagtag agagtctctc aatagatggt tagaaaagca ggaacaggta   3840 

acaataggcc agctcgcaga ttttgatttt gtagatttgc cagcagttga tcagtacaga   3900 

cacatgatca aagcacaacc caagcaaaaa ttggacactt caatccaaac ggagtacccg   3960 

gctttgcaga cgattgtgta ccattcgaaa aagatcaatg caatatttgg cccgttgttt   4020 

agtgagctta ctaggcaatt actggacagt gttgattcga gcagattttt gtttttcaca   4080 

agaaagacac cagcgcagat tgaggatttc ttcggagatc tcgacagtca tgtgccgatg   4140 

gatgtcttgg agctggatat atcaaaatac gacaaatctc agaatgaatt ccactgtgca   4200 

gtagaatacg agatttggcg aagattgggt tttgaagact tcttgggaga agtttggaaa   4260 

caagggcata gaaagaccac cctcaaggat tataccgcag gtatcaaaac ttgcatctgg   4320 

tatcaaagaa agagtgggga cgtcacgaca ttcattggaa acactgtgat cattgctgca   4380 

tgtttggcct cgatgcttcc gatggagaaa ataatcaaag gagccttttg tggtgacgat   4440 

agtctgctgt acttcccaaa gggttgtgag tttccggatg tgcaacactc cgcgaatctt   4500 

atgtggaatt ttgaagcaaa actgtttaaa aaacagtatg gatacttttg cggaagatat   4560 

gtaatacatc acgacagagg atgcattgtg tattacgatc ccctaaagtt gatctcgaaa   4620 

cttggcgcta aacacatcaa ggattgggaa cacttggagg agttcagaag gtctctttgt   4680 

gatgttgctg tttcgttgaa caattgtgcg tattatacac agttggacga cgctgtatgg   4740 

gaggttcata agaccgcccc tccaggttcg tttgtttata aaagtctggt gaagtatttg   4800 

tctgataaag ttctttttag aagtttgttt atag                               4834 

 
           
             4  
             1616  
             PRT  
             Tobacco mosaic virus  
             
               misc_feature  
               (1117)..(1117)  
               Xaa is unknown  
             
           
            4 

Met Ala Tyr Thr Gln Thr Ala Thr Thr Ser Ala Leu Leu Asp Thr Val 
1               5                   10                  15 

Arg Gly Asn Asn Ser Leu Val Asn Asp Leu Ala Lys Arg Arg Leu Tyr 
            20                  25                  30 

Asp Thr Ala Val Glu Glu Phe Asn Ala Arg Asp Arg Arg Pro Lys Val 
        35                  40                  45 

Asn Phe Ser Lys Val Ile Ser Glu Glu Gln Thr Leu Ile Ala Thr Arg 
    50                  55                  60 

Ala Tyr Pro Glu Phe Gln Ile Thr Phe Tyr Asn Thr Gln Asn Ala Val 
65                  70                  75                  80 

His Ser Leu Ala Gly Gly Leu Arg Ser Leu Glu Leu Glu Tyr Leu Met 
                85                  90                  95 

Met Gln Ile Pro Tyr Gly Ser Leu Thr Tyr Asp Ile Gly Gly Asn Phe 
            100                 105                 110 

Ala Ser His Leu Phe Lys Gly Arg Ala Tyr Val His Cys Cys Met Pro 
        115                 120                 125 

Asn Leu Asp Val Arg Asp Ile Met Arg His Glu Gly Gln Lys Asp Ser 
    130                 135                 140 

Ile Glu Leu Tyr Leu Ser Arg Leu Glu Arg Gly Gly Lys Thr Val Pro 
145                 150                 155                 160 

Asn Phe Gln Lys Glu Ala Phe Asp Arg Tyr Ala Glu Ile Pro Glu Asp 
                165                 170                 175 

Ala Val Cys His Asn Thr Phe Gln Thr Cys Glu His Gln Pro Met Gln 
            180                 185                 190 

Gln Ser Gly Arg Val Tyr Ala Ile Ala Leu His Ser Ile Tyr Asp Ile 
        195                 200                 205 

Pro Ala Asp Glu Phe Gly Ala Ala Leu Leu Arg Lys Asn Val His Thr 
    210                 215                 220 

Cys Tyr Ala Ala Phe His Phe Ser Glu Asn Leu Leu Leu Glu Asp Ser 
225                 230                 235                 240 

Tyr Val Asn Leu Asp Glu Ile Asn Ala Cys Phe Ser Arg Asp Gly Asp 
                245                 250                 255 

Lys Leu Thr Phe Ser Phe Ala Ser Glu Ser Thr Leu Asn Tyr Cys His 
            260                 265                 270 

Ser Tyr Ser Asn Ile Leu Lys Tyr Val Cys Lys Thr Tyr Phe Pro Ala 
        275                 280                 285 

Ser Asn Arg Glu Val Tyr Met Lys Glu Phe Leu Val Thr Arg Val Asn 
    290                 295                 300 

Thr Trp Phe Cys Lys Phe Ser Arg Ile Asp Thr Phe Leu Leu Tyr Lys 
305                 310                 315                 320 

Gly Val Ala His Lys Gly Val Asp Ser Glu Gln Phe Tyr Thr Ala Met 
                325                 330                 335 

Glu Asp Ala Trp His Tyr Lys Lys Thr Leu Ala Met Cys Asn Ser Glu 
            340                 345                 350 

Arg Ile Leu Leu Glu Asp Ser Ser Thr Val Asn Tyr Trp Phe Pro Glu 
        355                 360                 365 

Met Arg Asp Met Val Ile Val Pro Leu Phe Asp Ile Ser Leu Glu Thr 
    370                 375                 380 

Ser Lys Arg Thr Arg Lys Glu Val Leu Val Ser Lys Asp Phe Val Phe 
385                 390                 395                 400 

Thr Val Leu Asn His Ile Arg Thr Tyr Gln Ala Lys Ala Leu Thr Tyr 
                405                 410                 415 

Val Asn Val Leu Ser Phe Val Glu Ser Ile Arg Ser Arg Val Ile Ile 
            420                 425                 430 

Asn Gly Val Thr Ala Arg Ser Glu Trp Asp Val Asp Lys Ser Leu Leu 
        435                 440                 445 

Gln Ser Leu Ser Met Thr Phe Tyr Leu His Thr Lys Leu Ala Val Leu 
    450                 455                 460 

Lys Asp Asp Leu Leu Ile Ser Lys Phe Ser Leu Gly Ser Lys Thr Val 
465                 470                 475                 480 

Cys Gln His Val Trp Asp Glu Ile Ser Leu Ala Phe Gly Asn Ala Phe 
                485                 490                 495 

Pro Ser Val Lys Glu Arg Leu Leu Asn Arg Lys Leu Ile Arg Val Ala 
            500                 505                 510 

Gly Asp Ala Leu Glu Ile Arg Val Pro Asp Leu Tyr Val Thr Phe His 
        515                 520                 525 

Asp Arg Leu Val Thr Glu Tyr Lys Ala Ser Val Asp Met Pro Ala Leu 
    530                 535                 540 

Asp Ile Arg Lys Lys Met Glu Glu Thr Glu Val Met Tyr Asn Ala Leu 
545                 550                 555                 560 

Ser Glu Leu Ser Val Leu Arg Glu Ser Asp Lys Phe Asp Val Asp Val 
                565                 570                 575 

Phe Ser Gln Met Cys Gln Ser Leu Glu Val Asp Ala Met Thr Ala Ala 
            580                 585                 590 

Lys Val Ile Val Ala Val Met Ser Asn Lys Ser Gly Leu Thr Leu Thr 
        595                 600                 605 

Phe Glu Arg Pro Thr Glu Ala Asn Val Ala Leu Ala Leu Gln Asp Gln 
    610                 615                 620 

Glu Lys Ala Ser Glu Gly Ala Leu Val Val Thr Ser Arg Glu Val Glu 
625                 630                 635                 640 

Glu Pro Ser Met Lys Gly Ser Met Ala Arg Gly Glu Leu Gln Leu Ala 
                645                 650                 655 

Gly Leu Ala Gly Asp His Pro Glu Ser Ser Tyr Ser Arg Asn Glu Glu 
            660                 665                 670 

Ile Glu Ser Leu Glu Gln Phe His Met Ala Thr Ala Asp Ser Leu Ile 
        675                 680                 685 

Arg Lys Gln Met Ser Ser Ile Val Tyr Thr Gly Pro Ile Lys Val Gln 
    690                 695                 700 

Gln Met Lys Asn Phe Ile Asp Ser Leu Val Ala Ser Leu Ser Ala Ala 
705                 710                 715                 720 

Val Ser Asn Leu Val Lys Ile Leu Lys Asp Thr Ala Ala Ile Asp Leu 
                725                 730                 735 

Glu Thr Arg Gln Lys Phe Gly Val Leu Asp Val Thr Ser Arg Lys Trp 
            740                 745                 750 

Leu Ile Lys Pro Thr Ala Lys Ser His Ala Trp Gly Val Val Glu Thr 
        755                 760                 765 

His Ala Arg Lys Tyr His Val Ala Leu Leu Glu Tyr Asp Glu Gln Gly 
    770                 775                 780 

Val Val Thr Cys Asp Asp Trp Arg Arg Val Ala Val Ser Ser Glu Ser 
785                 790                 795                 800 

Val Val Tyr Ser Asp Met Ala Lys Leu Arg Thr Leu Arg Arg Leu Leu 
                805                 810                 815 

Arg Asn Gly Glu Pro His Val Ser Ser Ala Lys Val Val Leu Val Asp 
            820                 825                 830 

Gly Val Pro Gly Cys Gly Lys Thr Lys Glu Ile Leu Ser Arg Val Asn 
        835                 840                 845 

Phe Asp Glu Asp Leu Ile Leu Val Pro Gly Lys Gln Ala Ala Glu Met 
    850                 855                 860 

Ile Arg Arg Arg Ala Asn Ser Ser Gly Ile Ile Val Ala Thr Lys Asp 
865                 870                 875                 880 

Asn Val Lys Thr Val Asp Ser Phe Met Met Asn Phe Gly Lys Ser Thr 
                885                 890                 895 

Arg Cys Gln Phe Lys Arg Leu Phe Ile Asp Glu Gly Leu Met Leu His 
            900                 905                 910 

Thr Gly Cys Val Asn Phe Leu Val Ala Met Ser Leu Cys Glu Ile Ala 
        915                 920                 925 

Tyr Val Tyr Gly Asp Thr Gln Gln Ile Pro Tyr Ile Asn Arg Val Ser 
    930                 935                 940 

Gly Phe Pro Tyr Pro Ala His Phe Ala Lys Leu Glu Val Asp Glu Val 
945                 950                 955                 960 

Glu Thr Arg Arg Thr Thr Leu Arg Cys Pro Ala Asp Val Thr His Tyr 
                965                 970                 975 

Leu Asn Arg Arg Tyr Glu Gly Phe Val Met Ser Thr Ser Ser Val Lys 
            980                 985                 990 

Lys Ser Val Ser Gln Glu Met Val  Gly Gly Ala Ala Val  Ile Asn Pro 
        995                 1000                 1005 

Ile Ser  Lys Pro Leu His Gly  Lys Ile Leu Thr Phe  Thr Gln Ser 
    1010                 1015                 1020 

Asp Lys  Glu Ala Leu Leu Ser  Arg Gly Tyr Ser Asp  Val His Thr 
    1025                 1030                 1035 

Val His  Glu Val Gln Gly Glu  Thr Tyr Ser Asp Val  Ser Leu Val 
    1040                 1045                 1050 

Arg Leu  Thr Pro Thr Pro Val  Ser Ile Ile Ala Gly  Asp Ser Pro 
    1055                 1060                 1065 

His Val  Leu Val Ala Leu Ser  Arg His Thr Cys Ser  Leu Lys Tyr 
    1070                 1075                 1080 

Tyr Thr  Val Val Met Asp Pro  Leu Val Ser Ile Ile  Arg Asp Leu 
    1085                 1090                 1095 

Glu Lys  Leu Ser Ser Tyr Leu  Leu Asp Met Tyr Lys  Val Asp Ala 
    1100                 1105                 1110 

Gly Thr  Gln Xaa Gln Leu Gln  Ile Asp Ser Val Phe  Lys Gly Ser 
    1115                 1120                 1125 

Asn Leu  Phe Val Ala Ala Pro  Lys Thr Gly Asp Ile  Ser Asp Met 
    1130                 1135                 1140 

Gln Phe  Tyr Tyr Asp Lys Cys  Leu Pro Gly Asn Ser  Thr Met Met 
    1145                 1150                 1155 

Asn Asn  Phe Asp Ala Val Thr  Met Arg Leu Thr Asp  Ile Ser Leu 
    1160                 1165                 1170 

Asn Val  Lys Asp Cys Ile Leu  Asp Met Ser Lys Ser  Val Ala Ala 
    1175                 1180                 1185 

Pro Lys  Asp Gln Ile Lys Pro  Leu Ile Pro Met Val  Arg Thr Ala 
    1190                 1195                 1200 

Ala Glu  Met Pro Arg Gln Thr  Gly Leu Leu Glu Asn  Leu Val Ala 
    1205                 1210                 1215 

Met Ile  Lys Arg Asn Phe Asn  Ala Pro Glu Leu Ser  Gly Ile Ile 
    1220                 1225                 1230 

Asp Ile  Glu Asn Thr Ala Ser  Leu Val Val Asp Lys  Phe Phe Asp 
    1235                 1240                 1245 

Ser Tyr  Leu Leu Lys Glu Lys  Arg Lys Pro Asn Lys  Asn Val Ser 
    1250                 1255                 1260 

Leu Phe  Ser Arg Glu Ser Leu  Asn Arg Trp Leu Glu  Lys Gln Glu 
    1265                 1270                 1275 

Gln Val  Thr Ile Gly Gln Leu  Ala Asp Phe Asp Phe  Val Asp Leu 
    1280                 1285                 1290 

Pro Ala  Val Asp Gln Tyr Arg  His Met Ile Lys Ala  Gln Pro Lys 
    1295                 1300                 1305 

Gln Lys  Leu Asp Thr Ser Ile  Gln Thr Glu Tyr Pro  Ala Leu Gln 
    1310                 1315                 1320 

Thr Ile  Val Tyr His Ser Lys  Lys Ile Asn Ala Ile  Phe Gly Pro 
    1325                 1330                 1335 

Leu Phe  Ser Glu Leu Thr Arg  Gln Leu Leu Asp Ser  Val Asp Ser 
    1340                 1345                 1350 

Ser Arg  Phe Leu Phe Phe Thr  Arg Lys Thr Pro Ala  Gln Ile Glu 
    1355                 1360                 1365 

Asp Phe  Phe Gly Asp Leu Asp  Ser His Val Pro Met  Asp Val Leu 
    1370                 1375                 1380 

Glu Leu  Asp Ile Ser Lys Tyr  Asp Lys Ser Gln Asn  Glu Phe His 
    1385                 1390                 1395 

Cys Ala  Val Glu Tyr Glu Ile  Trp Arg Arg Leu Gly  Phe Glu Asp 
    1400                 1405                 1410 

Phe Leu  Gly Glu Val Trp Lys  Gln Gly His Arg Lys  Thr Thr Leu 
    1415                 1420                 1425 

Lys Asp  Tyr Thr Ala Gly Ile  Lys Thr Cys Ile Trp  Tyr Gln Arg 
    1430                 1435                 1440 

Lys Ser  Gly Asp Val Thr Thr  Phe Ile Gly Asn Thr  Val Ile Ile 
    1445                 1450                 1455 

Ala Ala  Cys Leu Ala Ser Met  Leu Pro Met Glu Lys  Ile Ile Lys 
    1460                 1465                 1470 

Gly Ala  Phe Cys Gly Asp Asp  Ser Leu Leu Tyr Phe  Pro Lys Gly 
    1475                 1480                 1485 

Cys Glu  Phe Pro Asp Val Gln  His Ser Ala Asn Leu  Met Trp Asn 
    1490                 1495                 1500 

Phe Glu  Ala Lys Leu Phe Lys  Lys Gln Tyr Gly Tyr  Phe Cys Gly 
    1505                 1510                 1515 

Arg Tyr  Val Ile His His Asp  Arg Gly Cys Ile Val  Tyr Tyr Asp 
    1520                 1525                 1530 

Pro Leu  Lys Leu Ile Ser Lys  Leu Gly Ala Lys His  Ile Lys Asp 
    1535                 1540                 1545 

Trp Glu  His Leu Glu Glu Phe  Arg Arg Ser Leu Cys  Asp Val Ala 
    1550                 1555                 1560 

Val Ser  Leu Asn Asn Cys Ala  Tyr Tyr Thr Gln Leu  Asp Asp Ala 
    1565                 1570                 1575 

Val Trp  Glu Val His Lys Thr  Ala Pro Pro Gly Ser  Phe Val Tyr 
    1580                 1585                 1590 

Lys Ser  Leu Val Lys Tyr Leu  Ser Asp Lys Val Leu  Phe Arg Ser 
    1595                 1600                 1605 

Leu Phe  Ile Asp Gly Ser Ser  Cys 
    1610                 1615 

 
           
             5  
             3351  
             DNA  
             Tobacco mosaic virus  
             
               CDS  
               (1)..(3348)  
             
             
               misc_feature  
               (1096)..(1096)  
               n is “t”, “c”, “a” or “g”,  except when 
      nucleotide 1097 is “t” and nucleotide 1098 is “t” or “c”, n cannot 
      be “t” 
             
           
            5 

atg gca tac aca cag aca gct acc aca tca gct ttg ctg gac act gtc       48 
Met Ala Tyr Thr Gln Thr Ala Thr Thr Ser Ala Leu Leu Asp Thr Val 
1               5                   10                  15 

cga gga aac aac tcc ttg gtc aat gat cta gca aag cgt cgt ctt tac       96 
Arg Gly Asn Asn Ser Leu Val Asn Asp Leu Ala Lys Arg Arg Leu Tyr 
            20                  25                  30 

gac aca gcg gtt gaa gag ttt aac gct cgt gac cgc agg ccc aaa gtg      144 
Asp Thr Ala Val Glu Glu Phe Asn Ala Arg Asp Arg Arg Pro Lys Val 
        35                  40                  45 

aac ttt tca aaa gta ata agc gag gag cag acg ctt att gct acc cgg      192 
Asn Phe Ser Lys Val Ile Ser Glu Glu Gln Thr Leu Ile Ala Thr Arg 
    50                  55                  60 

gcg tat cca gaa ttc caa att aca ttt tat aac acg caa aat gcc gtg      240 
Ala Tyr Pro Glu Phe Gln Ile Thr Phe Tyr Asn Thr Gln Asn Ala Val 
65                  70                  75                  80 

cat tcg ctt gca ggt gga ttg cga tct tta gaa ctg gaa tat ctg atg      288 
His Ser Leu Ala Gly Gly Leu Arg Ser Leu Glu Leu Glu Tyr Leu Met 
                85                  90                  95 

atg caa att ccc tac gga tca ttg act tat gac ata ggc ggg aat ttt      336 
Met Gln Ile Pro Tyr Gly Ser Leu Thr Tyr Asp Ile Gly Gly Asn Phe 
            100                 105                 110 

gca tcg cat ctg ttc aag gga cga gca tat gta cac tgc tgc atg ccc      384 
Ala Ser His Leu Phe Lys Gly Arg Ala Tyr Val His Cys Cys Met Pro 
        115                 120                 125 

aac ctg gac gtt cga gac atc atg cgg cat gaa ggc cag aaa gac agt      432 
Asn Leu Asp Val Arg Asp Ile Met Arg His Glu Gly Gln Lys Asp Ser 
    130                 135                 140 

att gaa cta tac ctt tct agg cta gag aga ggg gga aaa aca gtc ccc      480 
Ile Glu Leu Tyr Leu Ser Arg Leu Glu Arg Gly Gly Lys Thr Val Pro 
145                 150                 155                 160 

aac ttc caa aag gaa gca ttt gac aga tac gca gaa att cct gaa gac      528 
Asn Phe Gln Lys Glu Ala Phe Asp Arg Tyr Ala Glu Ile Pro Glu Asp 
                165                 170                 175 

gct gtc tgt cac aat act ttc cag aca tgc gaa cat cag ccg atg caa      576 
Ala Val Cys His Asn Thr Phe Gln Thr Cys Glu His Gln Pro Met Gln 
            180                 185                 190 

caa tca ggc aga gtg tat gcc att gcg cta cac agc ata tat gac ata      624 
Gln Ser Gly Arg Val Tyr Ala Ile Ala Leu His Ser Ile Tyr Asp Ile 
        195                 200                 205 

ccc gct gat gag ttc ggg gca gca ctc ttg agg aaa aat gtc cat acg      672 
Pro Ala Asp Glu Phe Gly Ala Ala Leu Leu Arg Lys Asn Val His Thr 
    210                 215                 220 

tgc tat gcc gct ttc cac ttc tct gag aac ctg ctt ctt gaa gat tca      720 
Cys Tyr Ala Ala Phe His Phe Ser Glu Asn Leu Leu Leu Glu Asp Ser 
225                 230                 235                 240 

tac gtc aat ctg gac gaa atc aac gcg tgt ttt tcg cgc gat gga gac      768 
Tyr Val Asn Leu Asp Glu Ile Asn Ala Cys Phe Ser Arg Asp Gly Asp 
                245                 250                 255 

aag ttg acc ttt tct ttt gca tca gag agt act ctt aat tac tgt cat      816 
Lys Leu Thr Phe Ser Phe Ala Ser Glu Ser Thr Leu Asn Tyr Cys His 
            260                 265                 270 

agt tat tct aat att ctt aag tat gtg tgc aaa act tac ttc ccg gcc      864 
Ser Tyr Ser Asn Ile Leu Lys Tyr Val Cys Lys Thr Tyr Phe Pro Ala 
        275                 280                 285 

tct aat aga gag gtt tac atg aag gag ttt tta gtc acc agg gtt aat      912 
Ser Asn Arg Glu Val Tyr Met Lys Glu Phe Leu Val Thr Arg Val Asn 
    290                 295                 300 

acc tgg ttt tgt aag ttt tct aga ata gat act ttt ctt ttg tac aaa      960 
Thr Trp Phe Cys Lys Phe Ser Arg Ile Asp Thr Phe Leu Leu Tyr Lys 
305                 310                 315                 320 

ggt gtg gcc cat aaa ggt gta gat agt gag cag ttt tat act gca atg     1008 
Gly Val Ala His Lys Gly Val Asp Ser Glu Gln Phe Tyr Thr Ala Met 
                325                 330                 335 

gaa gac gca tgg cat tac aaa aag act ctt gca atg tgc aac agc gag     1056 
Glu Asp Ala Trp His Tyr Lys Lys Thr Leu Ala Met Cys Asn Ser Glu 
            340                 345                 350 

aga atc ctc ctt gag gat tca tca aca gtc aat tac tgg nnn ccc gaa     1104 
Arg Ile Leu Leu Glu Asp Ser Ser Thr Val Asn Tyr Trp Xaa Pro Glu 
        355                 360                 365 

atg agg gat atg gtc atc gta cca tta ttc gac att tct ttg gag act     1152 
Met Arg Asp Met Val Ile Val Pro Leu Phe Asp Ile Ser Leu Glu Thr 
    370                 375                 380 

agt aag agg acg cgc aag gaa gtc tta gtg tcc aag gat ttc gtg ttt     1200 
Ser Lys Arg Thr Arg Lys Glu Val Leu Val Ser Lys Asp Phe Val Phe 
385                 390                 395                 400 

aca gtg ctt aac cac att cga aca tac cag gca aaa gct ctt aca tac     1248 
Thr Val Leu Asn His Ile Arg Thr Tyr Gln Ala Lys Ala Leu Thr Tyr 
                405                 410                 415 

gta aat gtt ttg tcc ttc gtc gaa tcg att cga tcg agg gta atc att     1296 
Val Asn Val Leu Ser Phe Val Glu Ser Ile Arg Ser Arg Val Ile Ile 
            420                 425                 430 

aac ggt gtg aca gcg agg tcc gaa tgg gat gtg gac aaa tct ttg tta     1344 
Asn Gly Val Thr Ala Arg Ser Glu Trp Asp Val Asp Lys Ser Leu Leu 
        435                 440                 445 

caa tcc ttg tcc atg acg ttt tac ctg cat act aag ctt gcc gtt cta     1392 
Gln Ser Leu Ser Met Thr Phe Tyr Leu His Thr Lys Leu Ala Val Leu 
    450                 455                 460 

aag gat gac tta ctg att agc aag ttt agt ctc ggt tcg aaa acg gtg     1440 
Lys Asp Asp Leu Leu Ile Ser Lys Phe Ser Leu Gly Ser Lys Thr Val 
465                 470                 475                 480 

tgc cag cat gtg tgg gat gag att tca ctg gcg ttt ggg aac gca ttt     1488 
Cys Gln His Val Trp Asp Glu Ile Ser Leu Ala Phe Gly Asn Ala Phe 
                485                 490                 495 

ccc tcc gtg aaa gag agg ctc ttg aac agg aaa ctt atc aga gtg gca     1536 
Pro Ser Val Lys Glu Arg Leu Leu Asn Arg Lys Leu Ile Arg Val Ala 
            500                 505                 510 

ggc gac gca cta gag atc agg gtg cct gat cta tat gtg acc ttc cac     1584 
Gly Asp Ala Leu Glu Ile Arg Val Pro Asp Leu Tyr Val Thr Phe His 
        515                 520                 525 

gac cga tta gtg act gag tac aag gcc tct gtg gac atg cct gcg ctt     1632 
Asp Arg Leu Val Thr Glu Tyr Lys Ala Ser Val Asp Met Pro Ala Leu 
    530                 535                 540 

gac att agg aag aag atg gaa gaa acg gaa gtg atg tac aat gca ctt     1680 
Asp Ile Arg Lys Lys Met Glu Glu Thr Glu Val Met Tyr Asn Ala Leu 
545                 550                 555                 560 

tca gag tta tcg gtg tta agg gag tct gac aaa ttc gat gtt gat gtt     1728 
Ser Glu Leu Ser Val Leu Arg Glu Ser Asp Lys Phe Asp Val Asp Val 
                565                 570                 575 

ttt tcc cag atg tgc caa tct ttg gaa gtt gac gca atg acg gca gcg     1776 
Phe Ser Gln Met Cys Gln Ser Leu Glu Val Asp Ala Met Thr Ala Ala 
            580                 585                 590 

aag gtt ata gtc gcg gtc atg agc aat aag agc ggt ctg act ctc aca     1824 
Lys Val Ile Val Ala Val Met Ser Asn Lys Ser Gly Leu Thr Leu Thr 
        595                 600                 605 

ttt gaa cga cct act gag gcg aat gtt gcg cta gct tta cag gat caa     1872 
Phe Glu Arg Pro Thr Glu Ala Asn Val Ala Leu Ala Leu Gln Asp Gln 
    610                 615                 620 

gaa aag gct tca gaa ggt gct ttg gta gtt acc tca aga gaa gtt gaa     1920 
Glu Lys Ala Ser Glu Gly Ala Leu Val Val Thr Ser Arg Glu Val Glu 
625                 630                 635                 640 

gaa ccg tcc atg aag ggt tcg atg gcc aga gga gag tta caa tta gct     1968 
Glu Pro Ser Met Lys Gly Ser Met Ala Arg Gly Glu Leu Gln Leu Ala 
                645                 650                 655 

ggt ctt gct gga gat cat ccg gag tcg tcc tat tct agg aac gag gag     2016 
Gly Leu Ala Gly Asp His Pro Glu Ser Ser Tyr Ser Arg Asn Glu Glu 
            660                 665                 670 

ata gag tct tta gag cag ttt cat atg gca acg gca gat tcg tta att     2064 
Ile Glu Ser Leu Glu Gln Phe His Met Ala Thr Ala Asp Ser Leu Ile 
        675                 680                 685 

cgt aag cag atg agc tcg att gtg tac acg ggt ccg att aaa gtt cag     2112 
Arg Lys Gln Met Ser Ser Ile Val Tyr Thr Gly Pro Ile Lys Val Gln 
    690                 695                 700 

caa atg aaa aac ttt atc gat agc ctg gta gca tca cta tct gct gcg     2160 
Gln Met Lys Asn Phe Ile Asp Ser Leu Val Ala Ser Leu Ser Ala Ala 
705                 710                 715                 720 

gtg tcg aat ctc gtc aag atc ctc aaa gat aca gct gct att gac ctt     2208 
Val Ser Asn Leu Val Lys Ile Leu Lys Asp Thr Ala Ala Ile Asp Leu 
                725                 730                 735 

gaa acc cgt caa aag ttt gga gtc ttg gat gtt aca tct agg aag tgg     2256 
Glu Thr Arg Gln Lys Phe Gly Val Leu Asp Val Thr Ser Arg Lys Trp 
            740                 745                 750 

tta att aaa cca acg gcc aag agt cat gca tgg ggt gtt gtt gaa acc     2304 
Leu Ile Lys Pro Thr Ala Lys Ser His Ala Trp Gly Val Val Glu Thr 
        755                 760                 765 

cac gcg agg aag tat cat gtg gcg ctt ctg gaa tat gat gag cag ggt     2352 
His Ala Arg Lys Tyr His Val Ala Leu Leu Glu Tyr Asp Glu Gln Gly 
    770                 775                 780 

gtg gtg aca tgc gat gat tgg aga aga gta gct gtc agc tct gag tct     2400 
Val Val Thr Cys Asp Asp Trp Arg Arg Val Ala Val Ser Ser Glu Ser 
785                 790                 795                 800 

gtt gtt tat tcc gac atg gcg aaa ctc aga act ctg cgc aga ctg ctt     2448 
Val Val Tyr Ser Asp Met Ala Lys Leu Arg Thr Leu Arg Arg Leu Leu 
                805                 810                 815 

cga aac gga gaa ccg cat gtc agt agc gca aag gtt gtt ctt gtg gac     2496 
Arg Asn Gly Glu Pro His Val Ser Ser Ala Lys Val Val Leu Val Asp 
            820                 825                 830 

gga gtt ccg ggc tgt gga aaa acc aaa gaa att ctt tcc agg gtt aat     2544 
Gly Val Pro Gly Cys Gly Lys Thr Lys Glu Ile Leu Ser Arg Val Asn 
        835                 840                 845 

ttt gat gaa gat cta att tta gta cct ggg aag caa gct gct gaa atg     2592 
Phe Asp Glu Asp Leu Ile Leu Val Pro Gly Lys Gln Ala Ala Glu Met 
    850                 855                 860 

atc aga aga cgt gcg aat tcc tca ggg att att gtg gcc acg aag gac     2640 
Ile Arg Arg Arg Ala Asn Ser Ser Gly Ile Ile Val Ala Thr Lys Asp 
865                 870                 875                 880 

aac gtt aaa acc gtt gat tct ttc atg atg aat ttt ggg aaa agc aca     2688 
Asn Val Lys Thr Val Asp Ser Phe Met Met Asn Phe Gly Lys Ser Thr 
                885                 890                 895 

cgc tgt cag ttc aag agg tta ttc att gat gaa ggg ttg atg ttg cat     2736 
Arg Cys Gln Phe Lys Arg Leu Phe Ile Asp Glu Gly Leu Met Leu His 
            900                 905                 910 

act ggt tgt gtt aat ttt ctt gtg gcg atg tca ttg tgc gaa att gca     2784 
Thr Gly Cys Val Asn Phe Leu Val Ala Met Ser Leu Cys Glu Ile Ala 
        915                 920                 925 

tat gtt tac gga gac aca cag cag att cca tac atc aat aga gtt tca     2832 
Tyr Val Tyr Gly Asp Thr Gln Gln Ile Pro Tyr Ile Asn Arg Val Ser 
    930                 935                 940 

gga ttc ccg tac ccc gcc cat ttt gcc aaa ttg gaa gtt gac gag gtg     2880 
Gly Phe Pro Tyr Pro Ala His Phe Ala Lys Leu Glu Val Asp Glu Val 
945                 950                 955                 960 

gag aca cgc aga act act ctc cgt tgt cca gcc gat gtc aca cat tat     2928 
Glu Thr Arg Arg Thr Thr Leu Arg Cys Pro Ala Asp Val Thr His Tyr 
                965                 970                 975 

ctg aac agg aga tat gag ggc ttt gtc atg agc act tct tcg gtt aaa     2976 
Leu Asn Arg Arg Tyr Glu Gly Phe Val Met Ser Thr Ser Ser Val Lys 
            980                 985                 990 

aag tct gtt tcg cag gag atg gtc  ggc gga gcc gcc gtg  atc aat ccg   3024 
Lys Ser Val Ser Gln Glu Met Val  Gly Gly Ala Ala Val  Ile Asn Pro 
        995                 1000                 1005 

atc tca  aaa ccc ttg cat ggc  aag atc ctg act ttt  acc caa tcg      3069 
Ile Ser  Lys Pro Leu His Gly  Lys Ile Leu Thr Phe  Thr Gln Ser 
    1010                 1015                 1020 

gat aaa  gaa gct ctg ctt tca  aga ggg tat tca gat  gtt cac act      3114 
Asp Lys  Glu Ala Leu Leu Ser  Arg Gly Tyr Ser Asp  Val His Thr 
    1025                 1030                 1035 

gtg cat  gaa gtg caa ggc gag  aca tac tct gat gtt  tca cta gtt      3159 
Val His  Glu Val Gln Gly Glu  Thr Tyr Ser Asp Val  Ser Leu Val 
    1040                 1045                 1050 

agg cta  acc cct aca cca gtc  tcc atc att gca gga  gac agc ccg      3204 
Arg Leu  Thr Pro Thr Pro Val  Ser Ile Ile Ala Gly  Asp Ser Pro 
    1055                 1060                 1065 

cat gtt  ttg gtc gca ttg tca  agg cac acc tgt tcg  ctc aag tac      3249 
His Val  Leu Val Ala Leu Ser  Arg His Thr Cys Ser  Leu Lys Tyr 
    1070                 1075                 1080 

tac act  gtt gtt atg gat cct  tta gtt agt atc att  aga gat cta      3294 
Tyr Thr  Val Val Met Asp Pro  Leu Val Ser Ile Ile  Arg Asp Leu 
    1085                 1090                 1095 

gag aaa  ctt agc tcg tac ttg  tta gat atg tat aag  gtc gat gca      3339 
Glu Lys  Leu Ser Ser Tyr Leu  Leu Asp Met Tyr Lys  Val Asp Ala 
    1100                 1105                 1110 

gga aca  caa tag                                                    3351 
Gly Thr  Gln 
    1115 

 
           
             6  
             1116  
             PRT  
             Tobacco mosaic virus  
             
               misc_feature  
               (366)..(366)  
               The ′Xaa′ at location 366 stands for any amino 
      acid except Phe.  
             
           
            6 

Met Ala Tyr Thr Gln Thr Ala Thr Thr Ser Ala Leu Leu Asp Thr Val 
1               5                   10                  15 

Arg Gly Asn Asn Ser Leu Val Asn Asp Leu Ala Lys Arg Arg Leu Tyr 
            20                  25                  30 

Asp Thr Ala Val Glu Glu Phe Asn Ala Arg Asp Arg Arg Pro Lys Val 
        35                  40                  45 

Asn Phe Ser Lys Val Ile Ser Glu Glu Gln Thr Leu Ile Ala Thr Arg 
    50                  55                  60 

Ala Tyr Pro Glu Phe Gln Ile Thr Phe Tyr Asn Thr Gln Asn Ala Val 
65                  70                  75                  80 

His Ser Leu Ala Gly Gly Leu Arg Ser Leu Glu Leu Glu Tyr Leu Met 
                85                  90                  95 

Met Gln Ile Pro Tyr Gly Ser Leu Thr Tyr Asp Ile Gly Gly Asn Phe 
            100                 105                 110 

Ala Ser His Leu Phe Lys Gly Arg Ala Tyr Val His Cys Cys Met Pro 
        115                 120                 125 

Asn Leu Asp Val Arg Asp Ile Met Arg His Glu Gly Gln Lys Asp Ser 
    130                 135                 140 

Ile Glu Leu Tyr Leu Ser Arg Leu Glu Arg Gly Gly Lys Thr Val Pro 
145                 150                 155                 160 

Asn Phe Gln Lys Glu Ala Phe Asp Arg Tyr Ala Glu Ile Pro Glu Asp 
                165                 170                 175 

Ala Val Cys His Asn Thr Phe Gln Thr Cys Glu His Gln Pro Met Gln 
            180                 185                 190 

Gln Ser Gly Arg Val Tyr Ala Ile Ala Leu His Ser Ile Tyr Asp Ile 
        195                 200                 205 

Pro Ala Asp Glu Phe Gly Ala Ala Leu Leu Arg Lys Asn Val His Thr 
    210                 215                 220 

Cys Tyr Ala Ala Phe His Phe Ser Glu Asn Leu Leu Leu Glu Asp Ser 
225                 230                 235                 240 

Tyr Val Asn Leu Asp Glu Ile Asn Ala Cys Phe Ser Arg Asp Gly Asp 
                245                 250                 255 

Lys Leu Thr Phe Ser Phe Ala Ser Glu Ser Thr Leu Asn Tyr Cys His 
            260                 265                 270 

Ser Tyr Ser Asn Ile Leu Lys Tyr Val Cys Lys Thr Tyr Phe Pro Ala 
        275                 280                 285 

Ser Asn Arg Glu Val Tyr Met Lys Glu Phe Leu Val Thr Arg Val Asn 
    290                 295                 300 

Thr Trp Phe Cys Lys Phe Ser Arg Ile Asp Thr Phe Leu Leu Tyr Lys 
305                 310                 315                 320 

Gly Val Ala His Lys Gly Val Asp Ser Glu Gln Phe Tyr Thr Ala Met 
                325                 330                 335 

Glu Asp Ala Trp His Tyr Lys Lys Thr Leu Ala Met Cys Asn Ser Glu 
            340                 345                 350 

Arg Ile Leu Leu Glu Asp Ser Ser Thr Val Asn Tyr Trp Xaa Pro Glu 
        355                 360                 365 

Met Arg Asp Met Val Ile Val Pro Leu Phe Asp Ile Ser Leu Glu Thr 
    370                 375                 380 

Ser Lys Arg Thr Arg Lys Glu Val Leu Val Ser Lys Asp Phe Val Phe 
385                 390                 395                 400 

Thr Val Leu Asn His Ile Arg Thr Tyr Gln Ala Lys Ala Leu Thr Tyr 
                405                 410                 415 

Val Asn Val Leu Ser Phe Val Glu Ser Ile Arg Ser Arg Val Ile Ile 
            420                 425                 430 

Asn Gly Val Thr Ala Arg Ser Glu Trp Asp Val Asp Lys Ser Leu Leu 
        435                 440                 445 

Gln Ser Leu Ser Met Thr Phe Tyr Leu His Thr Lys Leu Ala Val Leu 
    450                 455                 460 

Lys Asp Asp Leu Leu Ile Ser Lys Phe Ser Leu Gly Ser Lys Thr Val 
465                 470                 475                 480 

Cys Gln His Val Trp Asp Glu Ile Ser Leu Ala Phe Gly Asn Ala Phe 
                485                 490                 495 

Pro Ser Val Lys Glu Arg Leu Leu Asn Arg Lys Leu Ile Arg Val Ala 
            500                 505                 510 

Gly Asp Ala Leu Glu Ile Arg Val Pro Asp Leu Tyr Val Thr Phe His 
        515                 520                 525 

Asp Arg Leu Val Thr Glu Tyr Lys Ala Ser Val Asp Met Pro Ala Leu 
    530                 535                 540 

Asp Ile Arg Lys Lys Met Glu Glu Thr Glu Val Met Tyr Asn Ala Leu 
545                 550                 555                 560 

Ser Glu Leu Ser Val Leu Arg Glu Ser Asp Lys Phe Asp Val Asp Val 
                565                 570                 575 

Phe Ser Gln Met Cys Gln Ser Leu Glu Val Asp Ala Met Thr Ala Ala 
            580                 585                 590 

Lys Val Ile Val Ala Val Met Ser Asn Lys Ser Gly Leu Thr Leu Thr 
        595                 600                 605 

Phe Glu Arg Pro Thr Glu Ala Asn Val Ala Leu Ala Leu Gln Asp Gln 
    610                 615                 620 

Glu Lys Ala Ser Glu Gly Ala Leu Val Val Thr Ser Arg Glu Val Glu 
625                 630                 635                 640 

Glu Pro Ser Met Lys Gly Ser Met Ala Arg Gly Glu Leu Gln Leu Ala 
                645                 650                 655 

Gly Leu Ala Gly Asp His Pro Glu Ser Ser Tyr Ser Arg Asn Glu Glu 
            660                 665                 670 

Ile Glu Ser Leu Glu Gln Phe His Met Ala Thr Ala Asp Ser Leu Ile 
        675                 680                 685 

Arg Lys Gln Met Ser Ser Ile Val Tyr Thr Gly Pro Ile Lys Val Gln 
    690                 695                 700 

Gln Met Lys Asn Phe Ile Asp Ser Leu Val Ala Ser Leu Ser Ala Ala 
705                 710                 715                 720 

Val Ser Asn Leu Val Lys Ile Leu Lys Asp Thr Ala Ala Ile Asp Leu 
                725                 730                 735 

Glu Thr Arg Gln Lys Phe Gly Val Leu Asp Val Thr Ser Arg Lys Trp 
            740                 745                 750 

Leu Ile Lys Pro Thr Ala Lys Ser His Ala Trp Gly Val Val Glu Thr 
        755                 760                 765 

His Ala Arg Lys Tyr His Val Ala Leu Leu Glu Tyr Asp Glu Gln Gly 
    770                 775                 780 

Val Val Thr Cys Asp Asp Trp Arg Arg Val Ala Val Ser Ser Glu Ser 
785                 790                 795                 800 

Val Val Tyr Ser Asp Met Ala Lys Leu Arg Thr Leu Arg Arg Leu Leu 
                805                 810                 815 

Arg Asn Gly Glu Pro His Val Ser Ser Ala Lys Val Val Leu Val Asp 
            820                 825                 830 

Gly Val Pro Gly Cys Gly Lys Thr Lys Glu Ile Leu Ser Arg Val Asn 
        835                 840                 845 

Phe Asp Glu Asp Leu Ile Leu Val Pro Gly Lys Gln Ala Ala Glu Met 
    850                 855                 860 

Ile Arg Arg Arg Ala Asn Ser Ser Gly Ile Ile Val Ala Thr Lys Asp 
865                 870                 875                 880 

Asn Val Lys Thr Val Asp Ser Phe Met Met Asn Phe Gly Lys Ser Thr 
                885                 890                 895 

Arg Cys Gln Phe Lys Arg Leu Phe Ile Asp Glu Gly Leu Met Leu His 
            900                 905                 910 

Thr Gly Cys Val Asn Phe Leu Val Ala Met Ser Leu Cys Glu Ile Ala 
        915                 920                 925 

Tyr Val Tyr Gly Asp Thr Gln Gln Ile Pro Tyr Ile Asn Arg Val Ser 
    930                 935                 940 

Gly Phe Pro Tyr Pro Ala His Phe Ala Lys Leu Glu Val Asp Glu Val 
945                 950                 955                 960 

Glu Thr Arg Arg Thr Thr Leu Arg Cys Pro Ala Asp Val Thr His Tyr 
                965                 970                 975 

Leu Asn Arg Arg Tyr Glu Gly Phe Val Met Ser Thr Ser Ser Val Lys 
            980                 985                 990 

Lys Ser Val Ser Gln Glu Met Val  Gly Gly Ala Ala Val  Ile Asn Pro 
        995                 1000                 1005 

Ile Ser  Lys Pro Leu His Gly  Lys Ile Leu Thr Phe  Thr Gln Ser 
    1010                 1015                 1020 

Asp Lys  Glu Ala Leu Leu Ser  Arg Gly Tyr Ser Asp  Val His Thr 
    1025                 1030                 1035 

Val His  Glu Val Gln Gly Glu  Thr Tyr Ser Asp Val  Ser Leu Val 
    1040                 1045                 1050 

Arg Leu  Thr Pro Thr Pro Val  Ser Ile Ile Ala Gly  Asp Ser Pro 
    1055                 1060                 1065 

His Val  Leu Val Ala Leu Ser  Arg His Thr Cys Ser  Leu Lys Tyr 
    1070                 1075                 1080 

Tyr Thr  Val Val Met Asp Pro  Leu Val Ser Ile Ile  Arg Asp Leu 
    1085                 1090                 1095 

Glu Lys  Leu Ser Ser Tyr Leu  Leu Asp Met Tyr Lys  Val Asp Ala 
    1100                 1105                 1110 

Gly Thr  Gln 
    1115 

 
           
             7  
             4834  
             DNA  
             Tobacco mosaic virus  
             
               misc_feature  
               (1096)..(1096)  
               n is “t”, “c”, “a” or “g”, except when 
      nucleotide 1097 is “t” and nucleotide 1098 is “t” or “c”, n cannot 
      be “t” 
             
           
            7 

atggcataca cacagacagc taccacatca gctttgctgg acactgtccg aggaaacaac     60 

tccttggtca atgatctagc aaagcgtcgt ctttacgaca cagcggttga agagtttaac    120 

gctcgtgacc gcaggcccaa agtgaacttt tcaaaagtaa taagcgagga gcagacgctt    180 

attgctaccc gggcgtatcc agaattccaa attacatttt ataacacgca aaatgccgtg    240 

cattcgcttg caggtggatt gcgatcttta gaactggaat atctgatgat gcaaattccc    300 

tacggatcat tgacttatga cataggcggg aattttgcat cgcatctgtt caagggacga    360 

gcatatgtac actgctgcat gcccaacctg gacgttcgag acatcatgcg gcatgaaggc    420 

cagaaagaca gtattgaact atacctttct aggctagaga gagggggaaa aacagtcccc    480 

aacttccaaa aggaagcatt tgacagatac gcagaaattc ctgaagacgc tgtctgtcac    540 

aatactttcc agacatgcga acatcagccg atgcaacaat caggcagagt gtatgccatt    600 

gcgctacaca gcatatatga catacccgct gatgagttcg gggcagcact cttgaggaaa    660 

aatgtccata cgtgctatgc cgctttccac ttctctgaga acctgcttct tgaagattca    720 

tacgtcaatc tggacgaaat caacgcgtgt ttttcgcgcg atggagacaa gttgaccttt    780 

tcttttgcat cagagagtac tcttaattac tgtcatagtt attctaatat tcttaagtat    840 

gtgtgcaaaa cttacttccc ggcctctaat agagaggttt acatgaagga gtttttagtc    900 

accagggtta atacctggtt ttgtaagttt tctagaatag atacttttct tttgtacaaa    960 

ggtgtggccc ataaaggtgt agatagtgag cagttttata ctgcaatgga agacgcatgg   1020 

cattacaaaa agactcttgc aatgtgcaac agcgagagaa tcctccttga ggattcatca   1080 

acagtcaatt actggnnncc cgaaatgagg gatatggtca tcgtaccatt attcgacatt   1140 

tctttggaga ctagtaagag gacgcgcaag gaagtcttag tgtccaagga tttcgtgttt   1200 

acagtgctta accacattcg aacataccag gcaaaagctc ttacatacgt aaatgttttg   1260 

tccttcgtcg aatcgattcg atcgagggta atcattaacg gtgtgacagc gaggtccgaa   1320 

tgggatgtgg acaaatcttt gttacaatcc ttgtccatga cgttttacct gcatactaag   1380 

cttgccgttc taaaggatga cttactgatt agcaagttta gtctcggttc gaaaacggtg   1440 

tgccagcatg tgtgggatga gatttcactg gcgtttggga acgcatttcc ctccgtgaaa   1500 

gagaggctct tgaacaggaa acttatcaga gtggcaggcg acgcactaga gatcagggtg   1560 

cctgatctat atgtgacctt ccacgaccga ttagtgactg agtacaaggc ctctgtggac   1620 

atgcctgcgc ttgacattag gaagaagatg gaagaaacgg aagtgatgta caatgcactt   1680 

tcagagttat cggtgttaag ggagtctgac aaattcgatg ttgatgtttt ttcccagatg   1740 

tgccaatctt tggaagttga cgcaatgacg gcagcgaagg ttatagtcgc ggtcatgagc   1800 

aataagagcg gtctgactct cacatttgaa cgacctactg aggcgaatgt tgcgctagct   1860 

ttacaggatc aagaaaaggc ttcagaaggt gctttggtag ttacctcaag agaagttgaa   1920 

gaaccgtcca tgaagggttc gatggccaga ggagagttac aattagctgg tcttgctgga   1980 

gatcatccgg agtcgtccta ttctaggaac gaggagatag agtctttaga gcagtttcat   2040 

atggcaacgg cagattcgtt aattcgtaag cagatgagct cgattgtgta cacgggtccg   2100 

attaaagttc agcaaatgaa aaactttatc gatagcctgg tagcatcact atctgctgcg   2160 

gtgtcgaatc tcgtcaagat cctcaaagat acagctgcta ttgaccttga aacccgtcaa   2220 

aagtttggag tcttggatgt tacatctagg aagtggttaa ttaaaccaac ggccaagagt   2280 

catgcatggg gtgttgttga aacccacgcg aggaagtatc atgtggcgct tctggaatat   2340 

gatgagcagg gtgtggtgac atgcgatgat tggagaagag tagctgtcag ctctgagtct   2400 

gttgtttatt ccgacatggc gaaactcaga actctgcgca gactgcttcg aaacggagaa   2460 

ccgcatgtca gtagcgcaaa ggttgttctt gtggacggag ttccgggctg tggaaaaacc   2520 

aaagaaattc tttccagggt taattttgat gaagatctaa ttttagtacc tgggaagcaa   2580 

gctgctgaaa tgatcagaag acgtgcgaat tcctcaggga ttattgtggc cacgaaggac   2640 

aacgttaaaa ccgttgattc tttcatgatg aattttggga aaagcacacg ctgtcagttc   2700 

aagaggttat tcattgatga agggttgatg ttgcatactg gttgtgttaa ttttcttgtg   2760 

gcgatgtcat tgtgcgaaat tgcatatgtt tacggagaca cacagcagat tccatacatc   2820 

aatagagttt caggattccc gtaccccgcc cattttgcca aattggaagt tgacgaggtg   2880 

gagacacgca gaactactct ccgttgtcca gccgatgtca cacattatct gaacaggaga   2940 

tatgagggct ttgtcatgag cacttcttcg gttaaaaagt ctgtttcgca ggagatggtc   3000 

ggcggagccg ccgtgatcaa tccgatctca aaacccttgc atggcaagat cctgactttt   3060 

acccaatcgg ataaagaagc tctgctttca agagggtatt cagatgttca cactgtgcat   3120 

gaagtgcaag gcgagacata ctctgatgtt tcactagtta ggctaacccc tacaccagtc   3180 

tccatcattg caggagacag cccgcatgtt ttggtcgcat tgtcaaggca cacctgttcg   3240 

ctcaagtact acactgttgt tatggatcct ttagttagta tcattagaga tctagagaaa   3300 

cttagctcgt acttgttaga tatgtataag gtcgatgcag gaacacaata gcaattacag   3360 

attgactcgg tgttcaaagg ttccaatctt tttgtggcag cgccaaagac tggtgatatt   3420 

tctgatatgc agttttacta tgataagtgt ctcccaggca acagcaccat gatgaataat   3480 

tttgatgctg ttaccatgag gttgactgac atttcattga atgtcaaaga ttgcatattg   3540 

gatatgtcta agtctgttgc tgcgcctaag gatcaaatca aaccactaat acctatggta   3600 

cgaacggcgg cagaaatgcc acgccagact ggactattgg aaaatttagt ggcgatgatt   3660 

aaaaggaact ttaacgcacc cgagttgtct ggcatcattg atattgaaaa tactgcatct   3720 

ttagttgtag ataagttttt cgatagttat ttgcttaaag aaaaaagaaa accaaataaa   3780 

aatgtttctt tgttcagtag agagtctctc aatagatggt tagaaaagca ggaacaggta   3840 

acaataggcc agctcgcaga ttttgatttt gtagatttgc cagcagttga tcagtacaga   3900 

cacatgatca aagcacaacc caagcaaaaa ttggacactt caatccaaac ggagtacccg   3960 

gctttgcaga cgattgtgta ccattcgaaa aagatcaatg caatatttgg cccgttgttt   4020 

agtgagctta ctaggcaatt actggacagt gttgattcga gcagattttt gtttttcaca   4080 

agaaagacac cagcgcagat tgaggatttc ttcggagatc tcgacagtca tgtgccgatg   4140 

gatgtcttgg agctggatat atcaaaatac gacaaatctc agaatgaatt ccactgtgca   4200 

gtagaatacg agatttggcg aagattgggt tttgaagact tcttgggaga agtttggaaa   4260 

caagggcata gaaagaccac cctcaaggat tataccgcag gtatcaaaac ttgcatctgg   4320 

tatcaaagaa agagtgggga cgtcacgaca ttcattggaa acactgtgat cattgctgca   4380 

tgtttggcct cgatgcttcc gatggagaaa ataatcaaag gagccttttg tggtgacgat   4440 

agtctgctgt acttcccaaa gggttgtgag tttccggatg tgcaacactc cgcgaatctt   4500 

atgtggaatt ttgaagcaaa actgtttaaa aaacagtatg gatacttttg cggaagatat   4560 

gtaatacatc acgacagagg atgcattgtg tattacgatc ccctaaagtt gatctcgaaa   4620 

cttggcgcta aacacatcaa ggattgggaa cacttggagg agttcagaag gtctctttgt   4680 

gatgttgctg tttcgttgaa caattgtgcg tattatacac agttggacga cgctgtatgg   4740 

gaggttcata agaccgcccc tccaggttcg tttgtttata aaagtctggt gaagtatttg   4800 

tctgataaag ttctttttag aagtttgttt atag                               4834 

 
           
             8  
             1616  
             PRT  
             Tobacco mosaic virus  
             
               MISC_FEATURE  
               (366)..(366)  
               The ′Xaa′ at location 366 stands for any amino 
      acid except Phe.  
             
           
            8 

Met Ala Tyr Thr Gln Thr Ala Thr Thr Ser Ala Leu Leu Asp Thr Val 
1               5                   10                  15 

Arg Gly Asn Asn Ser Leu Val Asn Asp Leu Ala Lys Arg Arg Leu Tyr 
            20                  25                  30 

Asp Thr Ala Val Glu Glu Phe Asn Ala Arg Asp Arg Arg Pro Lys Val 
        35                  40                  45 

Asn Phe Ser Lys Val Ile Ser Glu Glu Gln Thr Leu Ile Ala Thr Arg 
    50                  55                  60 

Ala Tyr Pro Glu Phe Gln Ile Thr Phe Tyr Asn Thr Gln Asn Ala Val 
65                  70                  75                  80 

His Ser Leu Ala Gly Gly Leu Arg Ser Leu Glu Leu Glu Tyr Leu Met 
                85                  90                  95 

Met Gln Ile Pro Tyr Gly Ser Leu Thr Tyr Asp Ile Gly Gly Asn Phe 
            100                 105                 110 

Ala Ser His Leu Phe Lys Gly Arg Ala Tyr Val His Cys Cys Met Pro 
        115                 120                 125 

Asn Leu Asp Val Arg Asp Ile Met Arg His Glu Gly Gln Lys Asp Ser 
    130                 135                 140 

Ile Glu Leu Tyr Leu Ser Arg Leu Glu Arg Gly Gly Lys Thr Val Pro 
145                 150                 155                 160 

Asn Phe Gln Lys Glu Ala Phe Asp Arg Tyr Ala Glu Ile Pro Glu Asp 
                165                 170                 175 

Ala Val Cys His Asn Thr Phe Gln Thr Cys Glu His Gln Pro Met Gln 
            180                 185                 190 

Gln Ser Gly Arg Val Tyr Ala Ile Ala Leu His Ser Ile Tyr Asp Ile 
        195                 200                 205 

Pro Ala Asp Glu Phe Gly Ala Ala Leu Leu Arg Lys Asn Val His Thr 
    210                 215                 220 

Cys Tyr Ala Ala Phe His Phe Ser Glu Asn Leu Leu Leu Glu Asp Ser 
225                 230                 235                 240 

Tyr Val Asn Leu Asp Glu Ile Asn Ala Cys Phe Ser Arg Asp Gly Asp 
                245                 250                 255 

Lys Leu Thr Phe Ser Phe Ala Ser Glu Ser Thr Leu Asn Tyr Cys His 
            260                 265                 270 

Ser Tyr Ser Asn Ile Leu Lys Tyr Val Cys Lys Thr Tyr Phe Pro Ala 
        275                 280                 285 

Ser Asn Arg Glu Val Tyr Met Lys Glu Phe Leu Val Thr Arg Val Asn 
    290                 295                 300 

Thr Trp Phe Cys Lys Phe Ser Arg Ile Asp Thr Phe Leu Leu Tyr Lys 
305                 310                 315                 320 

Gly Val Ala His Lys Gly Val Asp Ser Glu Gln Phe Tyr Thr Ala Met 
                325                 330                 335 

Glu Asp Ala Trp His Tyr Lys Lys Thr Leu Ala Met Cys Asn Ser Glu 
            340                 345                 350 

Arg Ile Leu Leu Glu Asp Ser Ser Thr Val Asn Tyr Trp Xaa Pro Glu 
        355                 360                 365 

Met Arg Asp Met Val Ile Val Pro Leu Phe Asp Ile Ser Leu Glu Thr 
    370                 375                 380 

Ser Lys Arg Thr Arg Lys Glu Val Leu Val Ser Lys Asp Phe Val Phe 
385                 390                 395                 400 

Thr Val Leu Asn His Ile Arg Thr Tyr Gln Ala Lys Ala Leu Thr Tyr 
                405                 410                 415 

Val Asn Val Leu Ser Phe Val Glu Ser Ile Arg Ser Arg Val Ile Ile 
            420                 425                 430 

Asn Gly Val Thr Ala Arg Ser Glu Trp Asp Val Asp Lys Ser Leu Leu 
        435                 440                 445 

Gln Ser Leu Ser Met Thr Phe Tyr Leu His Thr Lys Leu Ala Val Leu 
    450                 455                 460 

Lys Asp Asp Leu Leu Ile Ser Lys Phe Ser Leu Gly Ser Lys Thr Val 
465                 470                 475                 480 

Cys Gln His Val Trp Asp Glu Ile Ser Leu Ala Phe Gly Asn Ala Phe 
                485                 490                 495 

Pro Ser Val Lys Glu Arg Leu Leu Asn Arg Lys Leu Ile Arg Val Ala 
            500                 505                 510 

Gly Asp Ala Leu Glu Ile Arg Val Pro Asp Leu Tyr Val Thr Phe His 
        515                 520                 525 

Asp Arg Leu Val Thr Glu Tyr Lys Ala Ser Val Asp Met Pro Ala Leu 
    530                 535                 540 

Asp Ile Arg Lys Lys Met Glu Glu Thr Glu Val Met Tyr Asn Ala Leu 
545                 550                 555                 560 

Ser Glu Leu Ser Val Leu Arg Glu Ser Asp Lys Phe Asp Val Asp Val 
                565                 570                 575 

Phe Ser Gln Met Cys Gln Ser Leu Glu Val Asp Ala Met Thr Ala Ala 
            580                 585                 590 

Lys Val Ile Val Ala Val Met Ser Asn Lys Ser Gly Leu Thr Leu Thr 
        595                 600                 605 

Phe Glu Arg Pro Thr Glu Ala Asn Val Ala Leu Ala Leu Gln Asp Gln 
    610                 615                 620 

Glu Lys Ala Ser Glu Gly Ala Leu Val Val Thr Ser Arg Glu Val Glu 
625                 630                 635                 640 

Glu Pro Ser Met Lys Gly Ser Met Ala Arg Gly Glu Leu Gln Leu Ala 
                645                 650                 655 

Gly Leu Ala Gly Asp His Pro Glu Ser Ser Tyr Ser Arg Asn Glu Glu 
            660                 665                 670 

Ile Glu Ser Leu Glu Gln Phe His Met Ala Thr Ala Asp Ser Leu Ile 
        675                 680                 685 

Arg Lys Gln Met Ser Ser Ile Val Tyr Thr Gly Pro Ile Lys Val Gln 
    690                 695                 700 

Gln Met Lys Asn Phe Ile Asp Ser Leu Val Ala Ser Leu Ser Ala Ala 
705                 710                 715                 720 

Val Ser Asn Leu Val Lys Ile Leu Lys Asp Thr Ala Ala Ile Asp Leu 
                725                 730                 735 

Glu Thr Arg Gln Lys Phe Gly Val Leu Asp Val Thr Ser Arg Lys Trp 
            740                 745                 750 

Leu Ile Lys Pro Thr Ala Lys Ser His Ala Trp Gly Val Val Glu Thr 
        755                 760                 765 

His Ala Arg Lys Tyr His Val Ala Leu Leu Glu Tyr Asp Glu Gln Gly 
    770                 775                 780 

Val Val Thr Cys Asp Asp Trp Arg Arg Val Ala Val Ser Ser Glu Ser 
785                 790                 795                 800 

Val Val Tyr Ser Asp Met Ala Lys Leu Arg Thr Leu Arg Arg Leu Leu 
                805                 810                 815 

Arg Asn Gly Glu Pro His Val Ser Ser Ala Lys Val Val Leu Val Asp 
            820                 825                 830 

Gly Val Pro Gly Cys Gly Lys Thr Lys Glu Ile Leu Ser Arg Val Asn 
        835                 840                 845 

Phe Asp Glu Asp Leu Ile Leu Val Pro Gly Lys Gln Ala Ala Glu Met 
    850                 855                 860 

Ile Arg Arg Arg Ala Asn Ser Ser Gly Ile Ile Val Ala Thr Lys Asp 
865                 870                 875                 880 

Asn Val Lys Thr Val Asp Ser Phe Met Met Asn Phe Gly Lys Ser Thr 
                885                 890                 895 

Arg Cys Gln Phe Lys Arg Leu Phe Ile Asp Glu Gly Leu Met Leu His 
            900                 905                 910 

Thr Gly Cys Val Asn Phe Leu Val Ala Met Ser Leu Cys Glu Ile Ala 
        915                 920                 925 

Tyr Val Tyr Gly Asp Thr Gln Gln Ile Pro Tyr Ile Asn Arg Val Ser 
    930                 935                 940 

Gly Phe Pro Tyr Pro Ala His Phe Ala Lys Leu Glu Val Asp Glu Val 
945                 950                 955                 960 

Glu Thr Arg Arg Thr Thr Leu Arg Cys Pro Ala Asp Val Thr His Tyr 
                965                 970                 975 

Leu Asn Arg Arg Tyr Glu Gly Phe Val Met Ser Thr Ser Ser Val Lys 
            980                 985                 990 

Lys Ser Val Ser Gln Glu Met Val  Gly Gly Ala Ala Val  Ile Asn Pro 
        995                 1000                 1005 

Ile Ser  Lys Pro Leu His Gly  Lys Ile Leu Thr Phe  Thr Gln Ser 
    1010                 1015                 1020 

Asp Lys  Glu Ala Leu Leu Ser  Arg Gly Tyr Ser Asp  Val His Thr 
    1025                 1030                 1035 

Val His  Glu Val Gln Gly Glu  Thr Tyr Ser Asp Val  Ser Leu Val 
    1040                 1045                 1050 

Arg Leu  Thr Pro Thr Pro Val  Ser Ile Ile Ala Gly  Asp Ser Pro 
    1055                 1060                 1065 

His Val  Leu Val Ala Leu Ser  Arg His Thr Cys Ser  Leu Lys Tyr 
    1070                 1075                 1080 

Tyr Thr  Val Val Met Asp Pro  Leu Val Ser Ile Ile  Arg Asp Leu 
    1085                 1090                 1095 

Glu Lys  Leu Ser Ser Tyr Leu  Leu Asp Met Tyr Lys  Val Asp Ala 
    1100                 1105                 1110 

Gly Thr  Gln Xaa Gln Leu Gln  Ile Asp Ser Val Phe  Lys Gly Ser 
    1115                 1120                 1125 

Asn Leu  Phe Val Ala Ala Pro  Lys Thr Gly Asp Ile  Ser Asp Met 
    1130                 1135                 1140 

Gln Phe  Tyr Tyr Asp Lys Cys  Leu Pro Gly Asn Ser  Thr Met Met 
    1145                 1150                 1155 

Asn Asn  Phe Asp Ala Val Thr  Met Arg Leu Thr Asp  Ile Ser Leu 
    1160                 1165                 1170 

Asn Val  Lys Asp Cys Ile Leu  Asp Met Ser Lys Ser  Val Ala Ala 
    1175                 1180                 1185 

Pro Lys  Asp Gln Ile Lys Pro  Leu Ile Pro Met Val  Arg Thr Ala 
    1190                 1195                 1200 

Ala Glu  Met Pro Arg Gln Thr  Gly Leu Leu Glu Asn  Leu Val Ala 
    1205                 1210                 1215 

Met Ile  Lys Arg Asn Phe Asn  Ala Pro Glu Leu Ser  Gly Ile Ile 
    1220                 1225                 1230 

Asp Ile  Glu Asn Thr Ala Ser  Leu Val Val Asp Lys  Phe Phe Asp 
    1235                 1240                 1245 

Ser Tyr  Leu Leu Lys Glu Lys  Arg Lys Pro Asn Lys  Asn Val Ser 
    1250                 1255                 1260 

Leu Phe  Ser Arg Glu Ser Leu  Asn Arg Trp Leu Glu  Lys Gln Glu 
    1265                 1270                 1275 

Gln Val  Thr Ile Gly Gln Leu  Ala Asp Phe Asp Phe  Val Asp Leu 
    1280                 1285                 1290 

Pro Ala  Val Asp Gln Tyr Arg  His Met Ile Lys Ala  Gln Pro Lys 
    1295                 1300                 1305 

Gln Lys  Leu Asp Thr Ser Ile  Gln Thr Glu Tyr Pro  Ala Leu Gln 
    1310                 1315                 1320 

Thr Ile  Val Tyr His Ser Lys  Lys Ile Asn Ala Ile  Phe Gly Pro 
    1325                 1330                 1335 

Leu Phe  Ser Glu Leu Thr Arg  Gln Leu Leu Asp Ser  Val Asp Ser 
    1340                 1345                 1350 

Ser Arg  Phe Leu Phe Phe Thr  Arg Lys Thr Pro Ala  Gln Ile Glu 
    1355                 1360                 1365 

Asp Phe  Phe Gly Asp Leu Asp  Ser His Val Pro Met  Asp Val Leu 
    1370                 1375                 1380 

Glu Leu  Asp Ile Ser Lys Tyr  Asp Lys Ser Gln Asn  Glu Phe His 
    1385                 1390                 1395 

Cys Ala  Val Glu Tyr Glu Ile  Trp Arg Arg Leu Gly  Phe Glu Asp 
    1400                 1405                 1410 

Phe Leu  Gly Glu Val Trp Lys  Gln Gly His Arg Lys  Thr Thr Leu 
    1415                 1420                 1425 

Lys Asp  Tyr Thr Ala Gly Ile  Lys Thr Cys Ile Trp  Tyr Gln Arg 
    1430                 1435                 1440 

Lys Ser  Gly Asp Val Thr Thr  Phe Ile Gly Asn Thr  Val Ile Ile 
    1445                 1450                 1455 

Ala Ala  Cys Leu Ala Ser Met  Leu Pro Met Glu Lys  Ile Ile Lys 
    1460                 1465                 1470 

Gly Ala  Phe Cys Gly Asp Asp  Ser Leu Leu Tyr Phe  Pro Lys Gly 
    1475                 1480                 1485 

Cys Glu  Phe Pro Asp Val Gln  His Ser Ala Asn Leu  Met Trp Asn 
    1490                 1495                 1500 

Phe Glu  Ala Lys Leu Phe Lys  Lys Gln Tyr Gly Tyr  Phe Cys Gly 
    1505                 1510                 1515 

Arg Tyr  Val Ile His His Asp  Arg Gly Cys Ile Val  Tyr Tyr Asp 
    1520                 1525                 1530 

Pro Leu  Lys Leu Ile Ser Lys  Leu Gly Ala Lys His  Ile Lys Asp 
    1535                 1540                 1545 

Trp Glu  His Leu Glu Glu Phe  Arg Arg Ser Leu Cys  Asp Val Ala 
    1550                 1555                 1560 

Val Ser  Leu Asn Asn Cys Ala  Tyr Tyr Thr Gln Leu  Asp Asp Ala 
    1565                 1570                 1575 

Val Trp  Glu Val His Lys Thr  Ala Pro Pro Gly Ser  Phe Val Tyr 
    1580                 1585                 1590 

Lys Ser  Leu Val Lys Tyr Leu  Ser Asp Lys Val Leu  Phe Arg Ser 
    1595                 1600                 1605 

Leu Phe  Ile Asp Gly Ser Ser  Cys 
    1610                 1615 

 
           
             9  
             9  
             PRT  
             Alfalfa mosaic virus  
             
               PEPTIDE  
               (1)..(9)  
             
           
            9 

Ser Cys Ala Trp Tyr Asn Arg Val Lys 
1               5 

 
           
             10  
             9  
             PRT  
             Brome mosaic virus  
             
               PEPTIDE  
               (1)..(9)  
             
           
            10 

His Cys Val Trp Phe Glu Asp Ile Ser 
1               5 

 
           
             11  
             9  
             PRT  
             Citrus leaf rugose virus  
             
               PEPTIDE  
               (1)..(9)  
             
           
            11 

Ser Cys Ala Trp Leu Ser Ser Leu Arg 
1               5 

 
           
             12  
             9  
             PRT  
             cucumber mosaic virus  
             
               PEPTIDE  
               (1)..(9)  
             
           
            12 

His Cys Ile Trp Phe Pro Ser Met Lys 
1               5 

 
           
             13  
             9  
             PRT  
             Sunn-hemp mosaic virus  
             
               PEPTIDE  
               (1)..(9)  
             
           
            13 

Phe Asn Val Tyr Phe Pro Asn Ala Lys 
1               5 

 
           
             14  
             10  
             PRT  
             Tobacco mosaic virus  
             
               PEPTIDE  
               (1)..(10)  
             
           
            14 

Ser Val Asn Tyr Trp Phe Pro Lys Met Arg 
1               5                   10 

 
           
             15  
             9  
             PRT  
             Tobacco rattle virus  
             
               PEPTIDE  
               (1)..(9)  
             
           
            15 

Val Glu Lys Gln Phe Met Asp Lys Cys 
1               5 

 
           
             16  
             9  
             PRT  
             Turnip vein-clearing virus  
             
               PEPTIDE  
               (1)..(9)  
             
           
            16 

Leu Asn Phe Trp Phe Pro Lys Val Arg 
1               5 

 
           
             17  
             16  
             PRT  
             Tobacco mosaic virus  
             
               PEPTIDE  
               (1)..(16)  
             
           
            17 

Ser Ser Val Asn Tyr Trp Phe Pro Lys Met Arg Ala Pro Glu Lys Ala 
1               5                   10                  15 

 
           
             18  
             16  
             PRT  
             Tobacco mosaic virus  
             
               PEPTIDE  
               (1)..(16)  
             
           
            18 

Gly Thr Val Asn Tyr Trp Phe Pro Glu Met Arg Val Ala Lys Arg Thr 
1               5                   10                  15 

 
           
             19  
             16  
             PRT  
             Tobacco mosaic virus  
             
               PEPTIDE  
               (1)..(16)  
             
           
            19 

Gly Ser Val Asn Tyr Trp Phe Pro Glu Met Arg Val Ala Lys Arg Thr 
1               5                   10                  15 

 
           
             20  
             16  
             PRT  
             Tobacco mosaic virus  
             
               PEPTIDE  
               (1)..(16)  
             
           
            20 

Gly Ser Val Asn Tyr Trp Ala Pro Glu Met Arg Val Ala Lys Arg Thr 
1               5                   10                  15 

 
           
             21  
             16  
             PRT  
             Tobacco mosaic virus  
             
               PEPTIDE  
               (1)..(16)  
             
           
            21 

Gly Ser Val Asn Tyr Trp Tyr Pro Glu Met Arg Val Ala Lys Arg Thr 
1               5                   10                  15 

 
           
             22  
             37  
             DNA  
             Tobacco mosaic virus  
             
               misc_feature  
               (1)..(37)  
               PCR primer  
             
           
            22 

ctcatttcgg gagcccagta attgactgat gatgaat                              37 

 
           
             23  
             33  
             DNA  
             Tobacco mosaic virus  
             
               misc_feature  
               (1)..(33)  
               PCR primer  
             
           
            23 

tttcgggata ccagtaattg actgatgatg aat                                  33 

 
           
             24  
             37  
             DNA  
             Tobacco mosaic virus  
             
               misc_feature  
               (1)..(37)  
               PCR primer  
             
           
            24 

ccatgccatg gcgctcgaga tggcatacac acagaca                              37 

 
           
             25  
             30  
             DNA  
             Tobacco mosaic virus  
             
               misc_feature  
               (1)..(30)  
               PCR primer  
             
           
            25 

cccttgctca ccatttgtgt tcctgcatcg                                      30 

 
           
             26  
             30  
             DNA  
             Plasmid pEGFP  
             
               misc_feature  
               (1)..(30)  
               PCR primer  
             
           
            26 

atgcaggaac acaaatggtg agcaagggcg                                      30 

 
           
             27  
             34  
             DNA  
             Plasmid pEGFP  
             
               misc_feature  
               (1)..(34)  
               PCR primer  
             
           
            27 

ccatgccatg gctcgagtta cttgtacagc tcgt                                 34