CN113249399B - pAM gene, plant expression vector and application thereof - Google Patents
pAM gene, plant expression vector and application thereof Download PDFInfo
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Abstract
The invention discloses a pAM gene, a plant expression vector and application thereof, wherein the pAM gene has a nucleotide sequence shown in SEQ ID NO.1, and the plant expression vector is constructed by recombining the pAM gene and a plant vector and can be applied to preparation of transgenic plants. The pAM gene and the plant expression vector thereof can increase the expression quantity of the vector in expressing a target gene, and can also replace viruses which cause plant death in the vector.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a pAM gene, a plant expression vector thereof and application thereof.
Background
The transient expression system using the viral vector has an advantage of rapidly expressing the foreign protein at a high level within several days. Among the several plant virus-derived expression vector systems known, the most effective expression vectors described to date are based on DNA viruses, in particular double-type viruses or single-stranded positive-sense RNA viruses, such as tobacco viruses, potexviruses and comovirus. However, these techniques currently have the following drawbacks: (1) when most plant vectors express target genes (proteins or polypeptides), the expression level is extremely low, and the production requirement cannot be met; (2) part of viruses used by plant vectors constructed by using plant viruses have large damage to plants and are easy to cause death to the plants.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the defects of the prior art and provides a pAM gene which can increase the expression quantity of a vector expression target gene and is plant-friendly, a plant expression vector thereof and application thereof.
In order to solve the technical problems, the invention adopts the following technical scheme.
A pAM gene, which has a nucleotide sequence shown in SEQ ID NO. 1.
Preferably, the pAM gene consists of an open reading frame and LIR, C1: C2 and SIR genes in a chickpea chlorotic dwarf virus genome, and is arranged in a manner of LIR → C1: C2 → SIR → open reading frame → LIR, wherein the open reading frame comprises a promoter NOS terminator, a cloning site and a terminator CaMV 35S promoter, and the chickpea chlorotic dwarf virus has a nucleotide sequence shown in SEQ ID NO. 2.
As a general technical concept, the invention also provides a plant expression vector, which is constructed by recombining the pAM gene and the plant vector. Namely, any plant expression vector can be transformed by using pAM gene to obtain a new vector.
Preferably, the plant expression vector is pEGAD, and has a nucleotide sequence shown in SEQ ID NO.3, and the obtained plant expression vector is pEGM, and has a nucleotide sequence shown in SEQ ID NO. 4.
The above plant expression vector is preferably constructed by the following steps:
(1) carrying out double enzyme digestion on a plant vector pEGAD by using PacI and KpnI, and recovering a large fragment to obtain pEGAD-PacI/KpnI;
(2) adding an enzyme cutting site ttaattaa of PacI at the upstream of pAM, adding an enzyme cutting site ggtacc of KpnI at the downstream of pAM, synthesizing a plasmid pUC57-pAM, carrying out enzyme cutting reaction by using three enzymes of PacI, KpnI and BspQI, and recovering a fragment to obtain pAM-PacI/KpnI;
(3) the fragment pEGAD-PacI/KpnI and the fragment pAM-PacI/KpnI were ligated with DNA ligase to construct a plant expression vector pEGM.
In the above plant expression vector, preferably, the pAM gene is placed between RB and LB of the plant vector in the arrangement direction of RB → LIR → C1: C2 → SIR → terminator → cloning site → promoter → LIR → LB.
As a general technical concept, the invention also provides an application of the plant expression vector in preparing transgenic plants.
In the above application, preferably, the plant is tobacco leaves.
In the present invention, the Chickpea chlorotic dwarf virus (Chickpea dwarf virus; AM933135) used is a double-stranded virus having a single-stranded circular DNA genome that can replicate to very high copy numbers in a plasmid-like dsDNA format by a rolling circle replication mechanism in the plant nucleus.
Compared with the prior art, the invention has the advantages that:
the pAM gene and the plant expression vector thereof can increase the expression quantity of the vector in expressing a target gene (protein or polypeptide). After entering plant cells, pAM gene can be connected with LIRs at two ends to form circular dsDNA through a rolling circle replication mechanism in plant cell nuclei, and the copy number of the replication form is high, so that the target gene can be expressed in a large number of transient states, and the optimal accumulation amount can be achieved by expression in tobacco for 7 days.
Drawings
FIG. 1 is a genome map of a chick pea chlorotic dwarf virus according to example 1 of the present invention.
FIG. 2 is a map of pAM gene in example 1 of the present invention.
FIG. 3 is a map of the plant vector pEGAD in example 1 of the present invention.
FIG. 4 shows the diagram of the pEGAD double-restriction electrophoresis in example 1 of the present invention.
FIG. 5 is a map of plasmid puc57-pAM in example 1 of the present invention.
FIG. 6 shows the electrophoresis of puc57-pAM in example 1 of the present invention.
FIG. 7 is a map of plasmid pEGM in example 1 of the present invention.
FIG. 8 is a flow chart of the construction of the plant expression vector in example 1 of the present invention.
FIG. 9 shows the electrophoresis of pEGM cleavage in example 2 of the present invention.
FIG. 10 is a graph showing the results of PCR electrophoresis in examples 2 and 3 of the present invention.
FIG. 11 is a photograph showing the expression of protoplasmid and pEGM-DsRed in example 2 of the present invention, wherein A is the expression of protoplasmid and B is the expression of pEGM-DsRed.
FIG. 12 shows a Western blot development of hNGF in example 3 of the present invention.
Detailed Description
The invention is further described below with reference to the drawings and specific preferred embodiments of the description, without thereby limiting the scope of protection of the invention. The materials and instruments used in the following examples are commercially available.
Example 1
The pAM gene has a nucleotide sequence shown as SEQ ID NO. 1. The chickpea chlorotic dwarf virus has a nucleotide sequence shown as SEQ ID NO.2, and the genome of the chickpea chlorotic dwarf virus is shown as a figure 1. The pAM gene of the invention consists of an open reading frame and LIR, C1, C2 and SIR genes in a Chickpea chlorotic dwarf virus (AM 933135) genome, and the arrangement mode is LIR → C1, C2 → SIR → open reading frame → LIR, as shown in figure 2. The open reading frame comprises the promoter NOS terminator, a cloning site and the terminator CaMV 35S promoter.
The pAM gene sequence is as follows:
TTAATTAAatacgctctacaatgtagtgcgccttttataaagcaaaagcaaaagcaaaccctagtgggcgggaactttttccaattcaatctgaaccgctcgtgctaaagcactcgcgataagggggggccacgccggtaatattaatgcggcgtgggtccccccttatcttttacttaatcagtaaggaaatgccacatcatattccactgaccaacttcaattactgaaatacccctgcctccatgcctccacgcctggttataagatagagtttgaggcaacccaaggagtcacaacaaaaatgccttctgctagcatgaacttcagactccaatctaaatatgttttccttacctatcctaagtgctcatctcaaagagatgatctattcgagtttctctggcagaaactcacaccttttcttattttcttccttggtgttgctactgagcttcatcaagatggcactacccactatcatgctcttgtccagcttgataaaaagccttgtattagggacgcttctttttttgattttgagggaaatcaccctaatatccaaccagctagaaactctaaacaggtccttgactacatatcaaaggacggagatattaaaaccagaggagatttcagagatcataaggtctctcctcgcaaatctgacgcacgatggagaactattatccagactgcgacgtctaaggaggagtaccttaacatgattaaggatgaatttcctcatgaatgggcaacaaagctccaatggctggaatattcagccaataaactatttcctccacagccagaaccatatgtgtcgcccttcactgagtcagaactacgctgccatgaagatctggcctcatggagagacacacacctctaccatgtaagcatcgacgcctatactcttgtccatccagttacttatgaacaagctaagactgaccttgaatggatggctgatataaccaggacgagggaagggctggaaccagacaccccagcctctacatctgcggaccaactcgtaccggtaagaccacctgggctagaagtctcggacgacacaactactggaatgggaccatcgacttcaccacatatgatgaacatgccacatacaatgtcatcgacgacatccccttcaagttcgtcccattgtggaagcaattaataggttgtcagttagattttactgtcaaccctaagtatggaaaaaagaagaaaataaaaggtgggatcccttcaattatcctttgtaatccagatgaagactggatggcctcaatgtcaagtcaacagaaggattactttgaagaaaactgcatcacccactatatgtatgacggggagactttttttgctcggcaatcgtcgagtcactgaacgtgcctctcctcagacgagggattatagtgttatgttttgggcccgtttttttgatgtttttttagggggggttttttggaaatttcatgtgtattgcagattatgtaatttctacctacttgcacaatgtaaaatattcataataaaataattgatctagtaacatagatgacaccgcgcgcgataatttatcctagtttgcgcgctatattttgttttctatcgcgtattaaatgtataattgcgggactctaatcataaaaacccatctcataaataacgtcatgcattacatgttaattattacatgcttaacgtaattcaacagaaattatatgataatcatcgcaagaccggcaacaggattcaatcttaagaaactttattgccaaatgtttgaacgatcaATGAGAGACCCCATGGCCACCGGTcccgggCATCATCACCATCACCATTGACTGACTGAGTCGACGGTCTCTAAAGtgttctctccaaatgaaatgaacttccttatatagaggaagggtcttgcgaaggatagtgggattgtgcgtcatcccttacgtcagtggagatatcacatcaatccacttgctttgaagacgtggttggaacgtcttctttttccacgatgctcctcgtgggtgggggtccatctttgggaccactgtcggcagaggcatcttcaacgatggcctttcctttatcgcaatgatggcatttgtaggagccaccttccttttccactatcttcacaataaagtgacagatagctgggcaatggaatccgaggaggtttccggatattaccctttgttgaaaagtctcaatacgctctacaatgtagtgcgccttttataaagcaaaagcaaaagcaaaccctagtgggcgggaactttttccaattcaatctgaaccgctcgtgctaaagcactcgcgataagggggggccacgccggtaatattaatgcggcgtgggtccccccttatcttttacttaatcagtaaggaaatgccacatcatattccactgaccaacttcaattactgaaatacccctgcctccatgcctccacgcctggttataagatagagtttgaggcaacccaaggagtcacaacaaaaGGTACC
the sequence of the chick pea chlorotic dwarf virus genome (the genome of AM933135) is as follows:
accggcgtggcccccccttatcgcgagtgctttagcacgagcggttcagattgaattggaaaaagttcccgcccactagggtttgcttttgcttttgctttataaaaggcgcactacattgtagagcgtatcatggaacgtattttgtttcaggtatttccgtcgtatacgaactattcatacgaccctgttccagagactgttgctccacaggcatcatctcaaacggatttcggtaaggtggtcatcgccttggtggttattctggttagtgttggtgtgttctatctggcctataccttgttcttgaaggattgtatccttttgttcaaggcgaagaaacagagaaccaccactgaaatcgggtttggacaaacccctgctagaaaccaggatcatcctcaaccataggcggggtccatcatgtctactgtgacgtggggtaggaagcggaagcgtagcggaggagaatcaaaggcgaagagtagggcatctggttcctatgtgcccagatctgtgtcatccaggagggaatcattgcaggtggcaaccttttcctggacgagttctggatcgggtatcaagttctcagctggtggtgctgcctacctgataagcaactttccacagggtgccaatgataactgtcgtcacaccaacaagactgtgttatacaaattcatgtctaagaacacagtgtatctggatcctagccactattcgaaggtgtttaggtgtccatttactttttggctggtgtacgacaagtctcctggacccaatgttcctagtactggggacatctttgaaggcccatctctgttccctaacaatccatggacttggactgtgtctagggcttcctgtcatcgttttgttgtaaaaaaaacgtggacatgtatggtggagagcaatggtatagacccaggtaaggcccaaggatctacatactatgggcctggaccttgtaaccagattaagtcctgcaataagttctttaagagactgggtgtgtccacagagtggaagaattctgcaacgggtgatgttggtgacataaaggaaggagccctttatattgtaggtgctccttcccagaaatctgatgtatatgtaaatgggtatttcagagtgtacttcaagtctgttggaaatcaataaaattattttattatgaatattttacattgtgcaagtaggtagaaattacataatctgcaatacacatgaaatttccaaaaaaccccccctaaaaaaacatcaaaaaaacgggcccaaaacataacactataatccctcgtctgaggagaggcacgttcagtgactcgacgattgccgagcaaaaaaagtctccccgtcatacatatagtgggtgatgcagttttcttcaaagtaatccttctgttgacttgacattgaggccatccagtcttcatctggattacaaaggataattgaagggatcccaccttttattttcttcttttttccatacttagggttgacagtaaaatctaactgacaacctattaattgcttccacaatgggacgaacttgaaggggatgtcgtcgatgacattgtatgtggcatgttcatcatatgtggtgaagtcgatggtcccattccagtagttgtgtcgtccgagacttctagcccaggtggtcttaccggtacgagttggtccgcagatgtagaggctggggtgtctggttccagcccttccctcgtcctggttatatcagccatccattcaaggtcagtcttagcttgttcataagtaactggatggacaagagtataggcgtcgatgcttacatggtagaggtgtgtgtctctccatgaggccagatcttcatggcagcgtagttctgactcagtgaagggcgacacatatggttctggctgtggaggaaatagtttattggctgaatattccagccattggagctttgttgcccattcatgaggaaattcatccttaatcatgttaaggtactcctccttagacgtcgcagtctggataatagttctccatcgtgcgtcagatttgcgaggagagaccttatgatctctgaaatctcctctggttttaatatctccgtcctttgatatgtagtcaaggacctgtttagagtttctagctggttggatattagggtgatttccctcaaaatcaaaaaaagaagcgtccctaatacaaggctttttatcaagctggacaagagcatgatagtgggtagtgccatcttgatgaagctcagtagcaacaccaaggaagaaaataagaaaaggtgtgagtttctgccagagaaactcgaatagatcatctctttgagatgagcacttaggataggtaaggaaaacatatttagattggagtctgaagttcatgctagcagaaggcatttttgttgtgactccttgggttgcctcaaactctatcttataaccaggcgtggaggcatggaggcaggggtatttcagtaattgaagttggtcagtggaatatgatgtggcatttccttactgattaagtaaaagataaggggggacccacgccgcattaatatt
a plant expression vector of the present invention is constructed by inserting the pAM gene of the present embodiment into a plant vector to construct a novel vector, and the expression amount can be increased by expressing a target gene (protein or polypeptide) in a plant using the novel vector.
In this example, the plant vector is pEGAD having a nucleotide sequence shown in SEQ ID NO.3, and the obtained plant expression vector is pEGM having a nucleotide sequence shown in SEQ ID NO. 4.
As shown in FIG. 8, the construction process of the plant expression vector of this example is as follows:
(1) the plant vector used in this time is a common plant vector pEGAD, the plasmid map of which is shown in FIG. 3, PacI and KpnI are used for double digestion, a large fragment (9664bp) is recovered, the fragment is named as pEGAD-PacI/KpnI, and the electrophoresis chart is shown in FIG. 4.
(2) A client company synthesizes the gene pAM designed in the invention, and adds the restriction site ttaattaaa of PacI to the upstream of pAM and the restriction site ggtacc of KpnI to the downstream of pAM. The plasmid synthesized by the entrustment company was pUC57-pAM, as shown in FIG. 5. Since the sizes of the two fragments of the pUC57-pAM plasmid cut by PacI/KpnI double enzymes are close to each other, the two fragments were recovered to have great influence on subsequent experiments, the three enzymes PacI/KpnI/BspQI were used for digestion, and a 2523bp fragment was recovered and named pAM-PacI/KpnI, and the electrophoresis chart thereof is shown in FIG. 6.
(3) A novel plasmid pEGM was constructed by ligating the fragment pEGAD-PacI/KpnI with the fragment pAM-PacI/KpnI using DNA ligase, as shown in FIG. 7.
In the same way, pAM can also be placed between RB and LB of other plant vectors, i.e.the orientation of these several nucleic acid elements is RB, LIR, C1: C2, SIR, terminator, multiple cloning site, promoter, LIR, LB.
TABLE 1pEGAD plasmid double digestion reaction System
The reaction conditions are as follows: the reaction was carried out at 37 ℃ for 1 h.
Table 2 Synthesis of pUC57-pAM Gene restriction enzyme reaction System
The reaction conditions are as follows: after 1 hour at 37 ℃ and 0.5 hour at 50 ℃.
TABLE 3pEGM ligation reaction System
| pEGAD-PacI/KpnI | 1μL |
| pAM-PacI/KpnI | 1μL |
| T4 DNA Ligase Reaction Buffer(10×) | 1μL |
| T4 DNA Ligase | 1μL |
| ddH 2 O | 6μL |
The reaction conditions are as follows: 4 ℃ overnight.
Example 2 expression of Red fluorescent protein DsRed
The company was entrusted to synthesize a DsRed sequence, and the resulting plasmid was pDsRed and had the nucleotide sequence shown in SEQ ID NO. 5.
(1) BsaI cuts the plasmid pEGM, recovers two fragments of 10965bp and 1149bp, and the electrophoresis picture is shown in FIG. 9. These two fragments were recovered and named fragment # 1 and fragment # 2.
(2) Primers are designed for PCR amplification of the DsRed gene fragment, and then the three fragments are subjected to a ligation reaction. The designed primers are as follows:
the forward primer cgcgggtctcaaATGGCCTCCTCCGAGAACGT is the primer that is used for the forward primer,
reverse primer cgcgggtctcactttTCACAGGAACAGGTGGTGGC
TABLE 4PCR system
| 5 |
10μL |
| 2.5mM dNTPs | 4μL |
| Forward primer (10. mu.M) | 2.5μL |
| Reverse primer (10. mu.M) | 2.5μL |
| Plasmid pDsRed | 1μL |
| Q5 ultra-fidelity DNA polymerase | 0.5μL |
| ddH 2 O | 29.5μL |
The PCR procedure was: 98 ℃ for 10 sec; 30sec at 70 ℃; 72 ℃ for 1 min; 72 ℃, 10min, 30 cycles. The PCR electrophoresis results are shown in FIG. 10. The DsRed PCR amplified fragment was recovered and named 3# fragment.
TABLE 5 ligation reaction systems are as follows
The reaction conditions are as follows: controlling the temperature of the PCR instrument to be 37 ℃ for 5 min; at 16 ℃ for 2 min; 25 cycles. Finally reacting for 1h at 16 ℃. Finally, a new recombinant plasmid pEGM-DsRed is obtained.
(3) The pEGM-DsRed recombinant plasmid was transformed into DH 5. alpha. E.coli, and the recombinant plasmid was obtained by following the transformation procedure described in the specification.
(4) The recombinant plasmid is transformed into agrobacterium tumefaciens C58 by a chemical transformation method, and a positive single colony is screened by LB solid culture medium containing rifampicin and kanamycin.
(5) Positive single colonies were picked from the plate, inoculated into 3mL of LB liquid medium supplemented with the corresponding antibiotic (kan & Rif), and cultured at 28 ℃ at 180r/min on a constant temperature shaker until the OD600 became 0.6-0.8 (about 17 hours).
(6) Centrifuging at 3000rpm for 20min, and collecting thallus precipitate.
(7) Using AW buffer (10mM MES, pH 5.6; 10mM Mg) 2 Cl, 1mM acetosyringone) dissolved the pellet and adjusted OD660 to 0.1.
(8) And (4) extracting the bacterial liquid by using an injector with a needle removed, and injecting the bacterial liquid to the back of the tobacco leaves growing for 6 weeks. Agrobacterium was transfected into tobacco leaves and cultured for 7 days, and the expression effect is shown in FIG. 11.
As can be seen from fig. 11: the reconstructed vector directly expresses DsRed in a tobacco cytoplasm matrix, the tobacco leaves show macroscopic red spots, and the spots emit fluorescence under ultraviolet irradiation, so that the red fluorescent protein is proved to be red fluorescent protein. However, DsRed expressed on the original vector pEGAD was not visible to the naked eye, indicating that the expression level was not high.
Example 3
The company was entrusted with the synthesis of a hNGF sequence, and the resulting plasmid, designated phNGF, had the nucleotide sequence shown in SEQ ID NO.6, and was essentially identical to the procedure of example 2, except that: designing a primer according to the hNGF sequence (hNGF genebank access NO. NM-002506.3) and carrying out PCR amplification to obtain a hNGF sequence fragment.
The designed primers are as follows:
forward primer cgcgggtctcaaTCTTCTTCTCATCCTATTTTTCAT
Reverse primer cgcgggtctcactttTTAAGCTCTTCTAACAGCCTTTCT
TABLE 6PCR System
| 5 |
10μL |
| 2.5mM dNTPs | 4μL |
| Forward primer (10. mu.M) | 2.5μL |
| Reverse primer (10. mu.M) | 2.5μL |
| Plasmid phNGF | 1μL |
| Q5 ultra-fidelity DNA polymerase | 0.5μL |
| ddH 2 O | 29.5μL |
The PCR procedure was: 98 ℃ for 10 sec; 55 ℃ for 30 sec; at 65 ℃ for 2 min; 72 ℃, 10min, 30 cycles.
As shown in FIG. 10, the result of PCR electrophoresis was that about 390bp had NGF.
Tobacco transformed with Agrobacterium pEGM-hNGF was obtained by the preparation method described in example 2. For Western Blot (WB) assay, the primary antibody was Rb mAb to NGF EP1320Y and the secondary antibody was Goat anti-rabbitIgG-HRP (Santa Cruz, Cat. SC-2004). FIG. 12 is a development diagram of Western blot of hNGF, which demonstrates that the constructed plasmid can produce hNGF protein in tobacco, and the expression of hNGF protein in tobacco leaves, and the expression amount of the modified vector is larger than that of the original vector.
The foregoing is merely a preferred embodiment of the invention and is not intended to limit the invention in any manner. Although the present invention has been described with reference to the preferred embodiments, it is not intended to be limited thereto. Those skilled in the art can make many possible variations and modifications to the disclosed embodiments, or equivalent modifications, without departing from the spirit and scope of the invention, using the methods and techniques disclosed above. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention are still within the scope of the protection of the technical solution of the present invention.
Sequence listing
<110> Fissistigma Fischeriana Scott Biotech company Limited liability
<120> pAM gene, plant expression vector and application thereof
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2529
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(2529)
<223> nucleotide sequence of pAM Gene
<400> 1
ttaattaaat acgctctaca atgtagtgcg ccttttataa agcaaaagca aaagcaaacc 60
ctagtgggcg ggaacttttt ccaattcaat ctgaaccgct cgtgctaaag cactcgcgat 120
aagggggggc cacgccggta atattaatgc ggcgtgggtc cccccttatc ttttacttaa 180
tcagtaagga aatgccacat catattccac tgaccaactt caattactga aatacccctg 240
cctccatgcc tccacgcctg gttataagat agagtttgag gcaacccaag gagtcacaac 300
aaaaatgcct tctgctagca tgaacttcag actccaatct aaatatgttt tccttaccta 360
tcctaagtgc tcatctcaaa gagatgatct attcgagttt ctctggcaga aactcacacc 420
ttttcttatt ttcttccttg gtgttgctac tgagcttcat caagatggca ctacccacta 480
tcatgctctt gtccagcttg ataaaaagcc ttgtattagg gacgcttctt tttttgattt 540
tgagggaaat caccctaata tccaaccagc tagaaactct aaacaggtcc ttgactacat 600
atcaaaggac ggagatatta aaaccagagg agatttcaga gatcataagg tctctcctcg 660
caaatctgac gcacgatgga gaactattat ccagactgcg acgtctaagg aggagtacct 720
taacatgatt aaggatgaat ttcctcatga atgggcaaca aagctccaat ggctggaata 780
ttcagccaat aaactatttc ctccacagcc agaaccatat gtgtcgccct tcactgagtc 840
agaactacgc tgccatgaag atctggcctc atggagagac acacacctct accatgtaag 900
catcgacgcc tatactcttg tccatccagt tacttatgaa caagctaaga ctgaccttga 960
atggatggct gatataacca ggacgaggga agggctggaa ccagacaccc cagcctctac 1020
atctgcggac caactcgtac cggtaagacc acctgggcta gaagtctcgg acgacacaac 1080
tactggaatg ggaccatcga cttcaccaca tatgatgaac atgccacata caatgtcatc 1140
gacgacatcc ccttcaagtt cgtcccattg tggaagcaat taataggttg tcagttagat 1200
tttactgtca accctaagta tggaaaaaag aagaaaataa aaggtgggat cccttcaatt 1260
atcctttgta atccagatga agactggatg gcctcaatgt caagtcaaca gaaggattac 1320
tttgaagaaa actgcatcac ccactatatg tatgacgggg agactttttt tgctcggcaa 1380
tcgtcgagtc actgaacgtg cctctcctca gacgagggat tatagtgtta tgttttgggc 1440
ccgttttttt gatgtttttt tagggggggt tttttggaaa tttcatgtgt attgcagatt 1500
atgtaatttc tacctacttg cacaatgtaa aatattcata ataaaataat tgatctagta 1560
acatagatga caccgcgcgc gataatttat cctagtttgc gcgctatatt ttgttttcta 1620
tcgcgtatta aatgtataat tgcgggactc taatcataaa aacccatctc ataaataacg 1680
tcatgcatta catgttaatt attacatgct taacgtaatt caacagaaat tatatgataa 1740
tcatcgcaag accggcaaca ggattcaatc ttaagaaact ttattgccaa atgtttgaac 1800
gatcaatgag agaccccatg gccaccggtc ccgggcatca tcaccatcac cattgactga 1860
ctgagtcgac ggtctctaaa gtgttctctc caaatgaaat gaacttcctt atatagagga 1920
agggtcttgc gaaggatagt gggattgtgc gtcatccctt acgtcagtgg agatatcaca 1980
tcaatccact tgctttgaag acgtggttgg aacgtcttct ttttccacga tgctcctcgt 2040
gggtgggggt ccatctttgg gaccactgtc ggcagaggca tcttcaacga tggcctttcc 2100
tttatcgcaa tgatggcatt tgtaggagcc accttccttt tccactatct tcacaataaa 2160
gtgacagata gctgggcaat ggaatccgag gaggtttccg gatattaccc tttgttgaaa 2220
agtctcaata cgctctacaa tgtagtgcgc cttttataaa gcaaaagcaa aagcaaaccc 2280
tagtgggcgg gaactttttc caattcaatc tgaaccgctc gtgctaaagc actcgcgata 2340
agggggggcc acgccggtaa tattaatgcg gcgtgggtcc ccccttatct tttacttaat 2400
cagtaaggaa atgccacatc atattccact gaccaacttc aattactgaa atacccctgc 2460
ctccatgcct ccacgcctgg ttataagata gagtttgagg caacccaagg agtcacaaca 2520
<210> 2
<211> 2573
<212> DNA
<213> chick pea chlorotic dwarf virus (Chickpea dwarf virus)
<400> 2
accggcgtgg ccccccctta tcgcgagtgc tttagcacga gcggttcaga ttgaattgga 60
aaaagttccc gcccactagg gtttgctttt gcttttgctt tataaaaggc gcactacatt 120
gtagagcgta tcatggaacg tattttgttt caggtatttc cgtcgtatac gaactattca 180
tacgaccctg ttccagagac tgttgctcca caggcatcat ctcaaacgga tttcggtaag 240
gtggtcatcg ccttggtggt tattctggtt agtgttggtg tgttctatct ggcctatacc 300
ttgttcttga aggattgtat ccttttgttc aaggcgaaga aacagagaac caccactgaa 360
atcgggtttg gacaaacccc tgctagaaac caggatcatc ctcaaccata ggcggggtcc 420
atcatgtcta ctgtgacgtg gggtaggaag cggaagcgta gcggaggaga atcaaaggcg 480
aagagtaggg catctggttc ctatgtgccc agatctgtgt catccaggag ggaatcattg 540
caggtggcaa ccttttcctg gacgagttct ggatcgggta tcaagttctc agctggtggt 600
gctgcctacc tgataagcaa ctttccacag ggtgccaatg ataactgtcg tcacaccaac 660
aagactgtgt tatacaaatt catgtctaag aacacagtgt atctggatcc tagccactat 720
tcgaaggtgt ttaggtgtcc atttactttt tggctggtgt acgacaagtc tcctggaccc 780
aatgttccta gtactgggga catctttgaa ggcccatctc tgttccctaa caatccatgg 840
acttggactg tgtctagggc ttcctgtcat cgttttgttg taaaaaaaac gtggacatgt 900
atggtggaga gcaatggtat agacccaggt aaggcccaag gatctacata ctatgggcct 960
ggaccttgta accagattaa gtcctgcaat aagttcttta agagactggg tgtgtccaca 1020
gagtggaaga attctgcaac gggtgatgtt ggtgacataa aggaaggagc cctttatatt 1080
gtaggtgctc cttcccagaa atctgatgta tatgtaaatg ggtatttcag agtgtacttc 1140
aagtctgttg gaaatcaata aaattatttt attatgaata ttttacattg tgcaagtagg 1200
tagaaattac ataatctgca atacacatga aatttccaaa aaaccccccc taaaaaaaca 1260
tcaaaaaaac gggcccaaaa cataacacta taatccctcg tctgaggaga ggcacgttca 1320
gtgactcgac gattgccgag caaaaaaagt ctccccgtca tacatatagt gggtgatgca 1380
gttttcttca aagtaatcct tctgttgact tgacattgag gccatccagt cttcatctgg 1440
attacaaagg ataattgaag ggatcccacc ttttattttc ttcttttttc catacttagg 1500
gttgacagta aaatctaact gacaacctat taattgcttc cacaatggga cgaacttgaa 1560
ggggatgtcg tcgatgacat tgtatgtggc atgttcatca tatgtggtga agtcgatggt 1620
cccattccag tagttgtgtc gtccgagact tctagcccag gtggtcttac cggtacgagt 1680
tggtccgcag atgtagaggc tggggtgtct ggttccagcc cttccctcgt cctggttata 1740
tcagccatcc attcaaggtc agtcttagct tgttcataag taactggatg gacaagagta 1800
taggcgtcga tgcttacatg gtagaggtgt gtgtctctcc atgaggccag atcttcatgg 1860
cagcgtagtt ctgactcagt gaagggcgac acatatggtt ctggctgtgg aggaaatagt 1920
ttattggctg aatattccag ccattggagc tttgttgccc attcatgagg aaattcatcc 1980
ttaatcatgt taaggtactc ctccttagac gtcgcagtct ggataatagt tctccatcgt 2040
gcgtcagatt tgcgaggaga gaccttatga tctctgaaat ctcctctggt tttaatatct 2100
ccgtcctttg atatgtagtc aaggacctgt ttagagtttc tagctggttg gatattaggg 2160
tgatttccct caaaatcaaa aaaagaagcg tccctaatac aaggcttttt atcaagctgg 2220
acaagagcat gatagtgggt agtgccatct tgatgaagct cagtagcaac accaaggaag 2280
aaaataagaa aaggtgtgag tttctgccag agaaactcga atagatcatc tctttgagat 2340
gagcacttag gataggtaag gaaaacatat ttagattgga gtctgaagtt catgctagca 2400
gaaggcattt ttgttgtgac tccttgggtt gcctcaaact ctatcttata accaggcgtg 2460
gaggcatgga ggcaggggta tttcagtaat tgaagttggt cagtggaata tgatgtggca 2520
tttccttact gattaagtaa aagataaggg gggacccacg ccgcattaat att 2573
<210> 3
<211> 12594
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(12594)
<223> nucleotide sequence of pEGAD
<400> 3
ggtttacccg ccaatatatc ctgtcaaaca ctgatagttt aaactgaagg cgggaaacga 60
caatctgatc atgagcggag aattaaggga gtcaggcctt aattaagagc tcgcatgccc 120
tttcagaaag aatgctaacc cacagatggt tagagaggct tacgcagcag gtctcatcaa 180
gacgatctac ccgagcaata atctccagga aatcaaatac cttcccaaga aggttaaaga 240
tgcagtcaaa agattcagga ctaactgcat caagaacaca gagaaagata tatttctcaa 300
gatcagaagt actattccag tatggacgat tcaaggcttg cttcacaaac caaggcaagt 360
aatagagatt ggagtctcta aaaaggtagt tcccactgaa tcaaaggcca tggagtcaaa 420
gattcaaata gaggacctaa cagaactccc cgtaaagact ggcgaacagt tcatacagag 480
tctcttacga ctcaatgaca agaagaaaat cttcgtcaac atggtggagc acgacacgct 540
tgtctactcc aaaaatatca aagatacagt ctcagaagac caaagggcaa ttgagacttt 600
tcaacaaagg gtaatatccg gaaacctcct cggattccat tgcccagcta tctgtcactt 660
tattgtgaag atagtggaaa aggaaggtgg ctcctacaaa tgccatcatt gcgataaagg 720
aaaggccatc gttgaagatg cctctgccga cagtggtccc aaagatggac ccccacccac 780
gaggagcatc gtggaaaaag aagacgttcc aaccacgtct tcaaagcaag tggattgatg 840
tgatatctcc actgacgtaa gggatgacgc acaatcccac tatccttcgc aagacccttc 900
ctctatataa ggaagttcat ttcatttgga gagaacacgg ggactctagc gctaccggtc 960
gccaccatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag 1020
ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc 1080
acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg 1140
cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac 1200
atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc 1260
atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac 1320
accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg 1380
gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag 1440
aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag 1500
ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac 1560
aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac 1620
atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac 1680
aagtccggag ctgcggccgc tgccgctgcg gcagcggccg aattccccgg gctcgagaag 1740
cttggatcca ccggatctag ataactgatc tcgaggagct agctcgaatt tccccgatcg 1800
ttcaaacatt tggcaataaa gtttcttaag attgaatcct gttgccggtc ttgcgatgat 1860
tatcatataa tttctgttga attacgttaa gcatgtaata attaacatgt aatgcatgac 1920
gttatttatg agatgggttt ttatgattag agtcccgcaa ttatacattt aatacgcgat 1980
agaaaacaaa atatagcgcg caaactagga taaattatcg cgcgcggtgt catctatgtt 2040
actagatcgg gaattaattc gataagctag agatccgtca acatggtgga gcacgacacg 2100
cttgtctact ccaaaaatat caaagataca gtctcagaag accaaagggc aattgagact 2160
tttcaacaaa gggtaatatc cggaaacctc ctcggattcc attgcccagc tatctgtcac 2220
tttattgtga agatagtgga aaaggaaggt ggctcctaca aatgccatca ttgcgataaa 2280
ggaaaggcca tcgttgaaga tgcctctgcc gacagtggtc ccaaagatgg acccccaccc 2340
acgaggagca tcgtggaaaa agaagacgtt ccaaccacgt cttcaaagca agtggattga 2400
tgtgatatct ccactgacgt aagggatgac gcacaatccc actatccttc gcaagaccct 2460
tcctctatat aaggaagttc atttcatttg gagaggacag acctgcaggt cgatccatga 2520
gcccagaacg acgcccggcc gacatccgcc gtgccaccga ggcggacatg ccggcggtct 2580
gcaccatcgt caaccactac atcgagacaa gcacggtcaa cttccgtacc gagccgcagg 2640
aaccgcagga gtggacggac gacctcgtcc gtctgcggga gcgctatccc tggctcgtcg 2700
ccgaggtgga cggcgaggtc gccggcatcg cctacgcggg cccctggaag gcacgcaacg 2760
cctacgactg gacggccgag tcgaccgtgt acgtctcccc ccgccaccag cggacgggac 2820
tgggctccac gctctacacc cacctgctga agtccctgga ggcacagggc ttcaagagcg 2880
tggtcgctgt catcgggctg cccaacgacc cgagcgtgcg catgcacgag gcgctcggat 2940
atgccccccg cggcatgctg cgggcggccg gcttcaagca cgggaactgg catgacgtgg 3000
gtttctggca gctggacttc agcctgccgg taccgccccg tccggtcctg cccgtcaccg 3060
agatctgatc tcacgcgtct aggatcgacc tgcagatcgt tcaaacattt ggcaataaag 3120
tttcttaaga ttgaatcctg ttgccggtct tgcgatgatt atcatctaat ttctgttgaa 3180
ttacgttaag catgtaataa ttaacatgta atgcatgacg ttatttatga gatgggtttt 3240
tatgattaga gtcccgcaat tatacattta atacgcgata gaaaacaaaa tatagcgcgc 3300
aaactaggat aaattatcgc gcgcggtgtc atctatgtta ctagatctct agcttgatat 3360
cgaattaatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc 3420
caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc 3480
cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgcccgctc ctttcgcttt 3540
cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct 3600
ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgatttggg 3660
tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga 3720
gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc 3780
gggctattct tttgatttat aagggatttt gccgatttcg gaaccaccat caaacaggat 3840
tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc aactctctca gggccaggcg 3900
gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac cccagtacat 3960
taaaaacgtc cgcaatgtgt tattaagttg tctaagcgtc aatttgttta caccacaata 4020
tatcctgcca ccagccagcc aacagctccc cgaccggcag ctcggcacaa aatcaccact 4080
cgatacaggc agcccatcag tccgggacgg cgtcagcggg agagccgttg taaggcggca 4140
gactttgctc atgttaccga tgctattcgg aagaacggca actaagctgc cgggtttgaa 4200
acacggatga tctcgcggag ggtagcatgt tgattgtaac gatgacagag cgttgctgcc 4260
tgtgatcaaa tatcatctcc ctcgcagaga tccgaattat cagccttctt attcatttct 4320
cgcttaaccg tgacaggctg tcgatcttga gaactatgcc gacataatag gaaatcgctg 4380
gataaagccg ctgaggaagc tgagtggcgc tatttcttta gaagtgaacg ttgacgatat 4440
caactcccct atccattgct caccgaatgg tacaggtcgg ggacccgaag ttccgactgt 4500
cggcctgatg catccccggc tgatcgaccc cagatctggg gctgagaaag cccagtaagg 4560
aaacaactgt aggttcgagt cgcgagatcc cccggaacca aaggaagtag gttaaacccg 4620
ctccgatcag gccgagccac gccaggccga gaacattggt tcctgtaggc atcgggattg 4680
gcggatcaaa cactaaagct actggaacga gcagaagtcc tccggccgcc agttgccagg 4740
cggtaaaggt gagcagaggc acgggaggtt gccacttgcg ggtcagcacg gttccgaacg 4800
ccatggaaac cgcccccgcc aggcccgctg cgacgccgac aggatctagc gctgcgtttg 4860
gtgtcaacac caacagcgcc acgcccgcag ttccgcaaat agcccccagg accgccatca 4920
atcgtatcgg gctacctagc agagcggcag agatgaacac gaccatcagc ggctgcacag 4980
cgcctaccgt cgccgcgacc ccgcccggca ggcggtagac cgaaataaac aacaagctcc 5040
agaatagcga aatattaagt gcgccgagga tgaagatgcg catccaccag attcccgttg 5100
gaatctgtcg gacgatcatc acgagcaata aacccgccgg caacgcccgc agcagcatac 5160
cggcgacccc tcggcctcgc tgttcgggct ccacgaaaac gccggacaga tgcgccttgt 5220
gagcgtcctt ggggccgtcc tcctgtttga agaccgacag cccaatgatc tcgccgtcga 5280
tgtaggcgcc gaatgccacg gcatctcgca accgttcagc gaacgcctcc atgggctttt 5340
tctcctcgtg ctcgtaaacg gacccgaaca tctctggagc tttcttcagg gccgacaatc 5400
ggatctcgcg gaaatcctgc acgtcggccg ctccaagccg tcgaatctga gccttaatca 5460
caattgtcaa ttttaatcct ctgtttatcg gcagttcgta gagcgcgccg tgcgtcccga 5520
gcgatactga gcgaagcaag tgcgtcgagc agtgcccgct tgttcctgaa atgccagtaa 5580
agcgctggct gctgaacccc cagccggaac tgaccccaca aggccctagc gtttgcaatg 5640
caccaggtca tcattgaccc aggcgtgttc caccaggccg ctgcctcgca actcttcgca 5700
ggcttcgccg acctgctcgc gccacttctt cacgcgggtg gaatccgatc cgcacatgag 5760
gcggaaggtt tccagcttga gcgggtacgg ctcccggtgc gagctgaaat agtcgaacat 5820
ccgtcgggcc gtcggcgaca gcttgcggta cttctcccat atgaatttcg tgtagtggtc 5880
gccagcaaac agcacgacga tttcctcgtc gatcaggacc tggcaacggg acgttttctt 5940
gccacggtcc aggacgcgga agcggtgcag cagcgacacc gattccaggt gcccaacgcg 6000
gtcggacgtg aagcccatcg ccgtcgcctg taggcgcgac aggcattcct cggccttcgt 6060
gtaataccgg ccattgatcg accagcccag gtcctggcaa agctcgtaga acgtgaaggt 6120
gatcggctcg ccgatagggg tgcgcttcgc gtactccaac acctgctgcc acaccagttc 6180
gtcatcgtcg gcccgcagct cgacgccggt gtaggtgatc ttcacgtcct tgttgacgtg 6240
gaaaatgacc ttgttttgca gcgcctcgcg cgggattttc ttgttgcgcg tggtgaacag 6300
ggcagagcgg gccgtgtcgt ttggcatcgc tcgcatcgtg tccggccacg gcgcaatatc 6360
gaacaaggaa agctgcattt ccttgatctg ctgcttcgtg tgtttcagca acgcggcctg 6420
cttggcctcg ctgacctgtt ttgccaggtc ctcgccggcg gtttttcgct tcttggtcgt 6480
catagttcct cgcgtgtcga tggtcatcga cttcgccaaa cctgccgcct cctgttcgag 6540
acgacgcgaa cgctccacgg cggccgatgg cgcgggcagg gcagggggag ccagttgcac 6600
gctgtcgcgc tcgatcttgg ccgtagcttg ctggaccatc gagccgacgg actggaaggt 6660
ttcgcggggc gcacgcatga cggtgcggct tgcgatggtt tcggcatcct cggcggaaaa 6720
ccccgcgtcg atcagttctt gcctgtatgc cttccggtca aacgtccgat tcattcaccc 6780
tccttgcggg attgccccga ctcacgccgg ggcaatgtgc ccttattcct gatttgaccc 6840
gcctggtgcc ttggtgtcca gataatccac cttatcggca atgaagtcgg tcccgtagac 6900
cgtctggccg tccttctcgt acttggtatt ccgaatcttg ccctgcacga ataccagcga 6960
ccccttgccc aaatacttgc cgtgggcctc ggcctgagag ccaaaacact tgatgcggaa 7020
gaagtcggtg cgctcctgct tgtcgccggc atcgttgcgc cacatctagg tactaaaaca 7080
attcatccag taaaatataa tattttattt tctcccaatc aggcttgatc cccagtaagt 7140
caaaaaatag ctcgacatac tgttcttccc cgatatcctc cctgatcgac cggacgcaga 7200
aggcaatgtc ataccacttg tccgccctgc cgcttctccc aagatcaata aagccactta 7260
ctttgccatc tttcacaaag atgttgctgt ctcccaggtc gccgtgggaa aagacaagtt 7320
cctcttcggg cttttccgtc tttaaaaaat catacagctc gcgcggatct ttaaatggag 7380
tgtcttcttc ccagttttcg caatccacat cggccagatc gttattcagt aagtaatcca 7440
attcggctaa gcggctgtct aagctattcg tatagggaca atccgatatg tcgatggagt 7500
gaaagagcct gatgcactcc gcatacagct cgataatctt ttcagggctt tgttcatctt 7560
catactcttc cgagcaaagg acgccatcgg cctcactcat gagcagattg ctccagccat 7620
catgccgttc aaagtgcagg acctttggaa caggcagctt tccttccagc catagcatca 7680
tgtccttttc ccgttccaca tcataggtgg tccctttata ccggctgtcc gtcattttta 7740
aatataggtt ttcattttct cccaccagct tatatacctt agcaggagac attccttccg 7800
tatcttttac gcagcggtat ttttcgatca gttttttcaa ttccggtgat attctcattt 7860
tagccattta ttatttcctt cctcttttct acagtattta aagatacccc aagaagctaa 7920
ttataacaag acgaactcca attcactgtt ccttgcattc taaaacctta aataccagaa 7980
aacagctttt tcaaagttgt tttcaaagtt ggcgtataac atagtatcga cggagccgat 8040
tttgaaacca caattatggg tgatgctgcc aacttactga tttagtgtat gatggtgttt 8100
ttgaggtgct ccagtggctt ctgtgtctat cagctgtccc tcctgttcag ctactgacgg 8160
ggtggtgcgt aacggcaaaa gcaccgccgg acatcagcgc tatctctgct ctcactgccg 8220
taaaacatgg caactgcagt tcacttacac cgcttctcaa cccggtacgc accagaaaat 8280
cattgatatg gccatgaatg gcgttggatg ccgggcaaca gcccgcatta tgggcgttgg 8340
cctcaacacg attttacgtc acttaaaaaa ctcaggccgc agtcggtaac ctcgcgcata 8400
cagccgggca gtgacgtcat cgtctgcgcg gaaatggacg aacagtgggg ctatgtcggg 8460
gctaaatcgc gccagcgctg gctgttttac gcgtatgaca gtctccggaa gacggttgtt 8520
gcgcacgtat tcggtgaacg cactatggcg acgctggggc gtcttatgag cctgctgtca 8580
ccctttgacg tggtgatatg gatgacggat ggctggccgc tgtatgaatc ccgcctgaag 8640
ggaaagctgc acgtaatcag caagcgatat acgcagcgaa ttgagcggca taacctgaat 8700
ctgaggcagc acctggcacg gctgggacgg aagtcgctgt cgttctcaaa atcggtggag 8760
ctgcatgaca aagtcatcgg gcattatctg aacataaaac actatcaata agttggagtc 8820
attacccaat tatgatagaa tttacaagct ataaggttat tgtcctgggt ttcaagcatt 8880
agtccatgca agtttttatg ctttgcccat tctatagata tattgataag cgcgctgcct 8940
atgccttgcc ccctgaaatc cttacatacg gcgatatctt ctatataaaa gatatattat 9000
cttatcagta ttgtcaatat attcaaggca atctgcctcc tcatcctctt catcctcttc 9060
gtcttggtag ctttttaaat atggcgcttc atagagtaat tctgtaaagg tccaattctc 9120
gttttcatac ctcggtataa tcttacctat cacctcaaat ggttcgctgg gtttatcgca 9180
cccccgaaca cgagcacggc acccgcgacc actatgccaa gaatgcccaa ggtaaaaatt 9240
gccggccccg ccatgaagtc cgtgaatgcc ccgacggccg aagtgaaggg caggccgcca 9300
cccaggccgc cgccctcact gcccggcacc tggtcgctga atgtcgatgc cagcacctgc 9360
ggcacgtcaa tgcttccggg cgtcgcgctc gggctgatcg cccatcccgt tactgccccg 9420
atcccggcaa tggcaaggac tgccagcgct gccatttttg gggtgaggcc gttcgcggcc 9480
gaggggcgca gcccctgggg ggatgggagg cccgcgttag cgggccggga gggttcgaga 9540
agggggggca ccccccttcg gcgtgcgcgg tcacgcgcac agggcgcagc cctggttaaa 9600
aacaaggttt ataaatattg gtttaaaagc aggttaaaag acaggttagc ggtggccgaa 9660
aaacgggcgg aaacccttgc aaatgctgga ttttctgcct gtggacagcc cctcaaatgt 9720
caataggtgc gcccctcatc tgtcagcact ctgcccctca agtgtcaagg atcgcgcccc 9780
tcatctgtca gtagtcgcgc ccctcaagtg tcaataccgc agggcactta tccccaggct 9840
tgtccacatc atctgtggga aactcgcgta aaatcaggcg ttttcgccga tttgcgaggc 9900
tggccagctc cacgtcgccg gccgaaatcg agcctgcccc tcatctgtca acgccgcgcc 9960
gggtgagtcg gcccctcaag tgtcaacgtc cgcccctcat ctgtcagtga gggccaagtt 10020
ttccgcgagg tatccacaac gccggcggcc gcggtgtctc gcacacggct tcgacggcgt 10080
ttctggcgcg tttgcagggc catagacggc cgccagccca gcggcgaggg caaccagccc 10140
ggtgagcgtc gcaaaggcgc tcggtcttgc cttgctcgtc ggtgatgtac ttcaccagct 10200
ccgcgaagtc gctcttcttg atggagcgca tggggacgtg cttggcaatc acgcgcaccc 10260
cccggccgtt ttagcggcta aaaaagtcat ggctctgccc tcgggcggac cacgcccatc 10320
atgaccttgc caagctcgtc ctgcttctct tcgatcttcg ccagcagggc gaggatcgtg 10380
gcatcaccga accgcgccgt gcgcgggtcg tcggtgagcc agagtttcag caggccgccc 10440
aggcggccca ggtcgccatt gatgcgggcc agctcgcgga cgtgctcata gtccacgacg 10500
cccgtgattt tgtagccctg gccgacggcc agcaggtagg ccgacaggct catgccggcc 10560
gccgccgcct tttcctcaat cgctcttcgt tcgtctggaa ggcagtacac cttgataggt 10620
gggctgccct tcctggttgg cttggtttca tcagccatcc gcttgccctc atctgttacg 10680
ccggcggtag ccggccagcc tcgcagagca ggattcccgt tgagcaccgc caggtgcgaa 10740
taagggacag tgaagaagga acacccgctc gcgggtgggc ctacttcacc tatcctgccc 10800
ggctgacgcc gttggataca ccaaggaaag tctacacgaa ccctttggca aaatcctgta 10860
tatcgtgcga aaaaggatgg atataccgaa aaaatcgcta taatgacccc gaagcagggt 10920
tatgcagcgg aaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag 10980
cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct 11040
ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc 11100
aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt 11160
ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg 11220
tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga 11280
gtcagtgagc gaggaagcgg aagagcgcca gaaggccgcc agagaggccg agcgcggccg 11340
tgaggcttgg acgctagggc agggcatgaa aaagcccgta gcgggctgct acgggcgtct 11400
gacgcggtgg aaagggggag gggatgttgt ctacatggct ctgctgtagt gagtgggttg 11460
cgctccggca gcggtcctga tcaatcgtca ccctttctcg gtccttcaac gttcctgaca 11520
acgagcctcc ttttcgccaa tccatcgaca atcaccgcga gtccctgctc gaacgctgcg 11580
tccggaccgg cttcgtcgaa ggcgtctatc gcggcccgca acagcggcga gagcggagcc 11640
tgttcaacgg tgccgccgcg ctcgccggca tcgctgtcgc cggcctgctc ctcaagcacg 11700
gccccaacag tgaagtagct gattgtcatc agcgcattga cggcgtcccc ggccgaaaaa 11760
cccgcctcgc agaggaagcg aagctgcgcg tcggccgttt ccatctgcgg tgcgcccggt 11820
cgcgtgccgg catggatgcg cgcgccatcg cggtaggcga gcagcgcctg cctgaagctg 11880
cgggcattcc cgatcagaaa tgagcgccag tcgtcgtcgg ctctcggcac cgaatgcgta 11940
tgattctccg ccagcatggc ttcggccagt gcgtcgagca gcgcccgctt gttcctgaag 12000
tgccagtaaa gcgccggctg ctgaaccccc aaccgttccg ccagtttgcg tgtcgtcaga 12060
ccgtctacgc cgacctcgtt caacaggtcc agggcggcac ggatcactgt attcggctgc 12120
aactttgtca tgcttgacac tttatcactg ataaacataa tatgtccacc aacttatcag 12180
tgataaagaa tccgcgcgtt caatcggacc agcggaggct ggtccggagg ccagacgtga 12240
aacccaacat acccctgatc gtaattctga gcactgtcgc gctcgacgct gtcggcatcg 12300
gcctgattat gccggtgctg ccgggcctcc tgcgcgatct ggttcactcg aacgacgtca 12360
ccgcccacta tggcattctg ctggcgctgt atgcgttggt gcaatttgcc tgcgcacctg 12420
tgctgggcgc gctgtcggat cgtttcgggc ggcggccaat cttgctcgtc tcgctggccg 12480
gcgccagatc tggggaaccc tgtggttggc atgcacatac aaatggacga acggataaac 12540
cttttcacgc ccttttaaat atccgattat tctaataaac gctcttttct ctta 12594
<210> 4
<211> 12187
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(12187)
<223> nucleotide sequence of pEGM
<400> 4
ggtttacccg ccaatatatc ctgtcaaaca ctgatagttt aaactgaagg cgggaaacga 60
caatctgatc atgagcggag aattaaggga gtcaggcctt aattaaatac gctctacaat 120
gtagtgcgcc ttttataaag caaaagcaaa agcaaaccct agtgggcggg aactttttcc 180
aattcaatct gaaccgctcg tgctaaagca ctcgcgataa gggggggcca cgccggtaat 240
attaatgcgg cgtgggtccc cccttatctt ttacttaatc agtaaggaaa tgccacatca 300
tattccactg accaacttca attactgaaa tacccctgcc tccatgcctc cacgcctggt 360
tataagatag agtttgaggc aacccaagga gtcacaacaa aaatgccttc tgctagcatg 420
aacttcagac tccaatctaa atatgttttc cttacctatc ctaagtgctc atctcaaaga 480
gatgatctat tcgagtttct ctggcagaaa ctcacacctt ttcttatttt cttccttggt 540
gttgctactg agcttcatca agatggcact acccactatc atgctcttgt ccagcttgat 600
aaaaagcctt gtattaggga cgcttctttt tttgattttg agggaaatca ccctaatatc 660
caaccagcta gaaactctaa acaggtcctt gactacatat caaaggacgg agatattaaa 720
accagaggag atttcagaga tcataaggtc tctcctcgca aatctgacgc acgatggaga 780
actattatcc agactgcgac gtctaaggag gagtacctta acatgattaa ggatgaattt 840
cctcatgaat gggcaacaaa gctccaatgg ctggaatatt cagccaataa actatttcct 900
ccacagccag aaccatatgt gtcgcccttc actgagtcag aactacgctg ccatgaagat 960
ctggcctcat ggagagacac acacctctac catgtaagca tcgacgccta tactcttgtc 1020
catccagtta cttatgaaca agctaagact gaccttgaat ggatggctga tataaccagg 1080
acgagggaag ggctggaacc agacacccca gcctctacat ctgcggacca actcgtaccg 1140
gtaagaccac ctgggctaga agtctcggac gacacaacta ctggaatggg accatcgact 1200
tcaccacata tgatgaacat gccacataca atgtcatcga cgacatcccc ttcaagttcg 1260
tcccattgtg gaagcaatta ataggttgtc agttagattt tactgtcaac cctaagtatg 1320
gaaaaaagaa gaaaataaaa ggtgggatcc cttcaattat cctttgtaat ccagatgaag 1380
actggatggc ctcaatgtca agtcaacaga aggattactt tgaagaaaac tgcatcaccc 1440
actatatgta tgacggggag actttttttg ctcggcaatc gtcgagtcac tgaacgtgcc 1500
tctcctcaga cgagggatta tagtgttatg ttttgggccc gtttttttga tgttttttta 1560
gggggggttt tttggaaatt tcatgtgtat tgcagattat gtaatttcta cctacttgca 1620
caatgtaaaa tattcataat aaaataattg atctagtaac atagatgaca ccgcgcgcga 1680
taatttatcc tagtttgcgc gctatatttt gttttctatc gcgtattaaa tgtataattg 1740
cgggactcta atcataaaaa cccatctcat aaataacgtc atgcattaca tgttaattat 1800
tacatgctta acgtaattca acagaaatta tatgataatc atcgcaagac cggcaacagg 1860
attcaatctt aagaaacttt attgccaaat gtttgaacga tcaatgagag accccatggc 1920
caccggtccc gggcatcatc accatcacca ttgactgact gagtcgacgg tctctaaagt 1980
gttctctcca aatgaaatga acttccttat atagaggaag ggtcttgcga aggatagtgg 2040
gattgtgcgt catcccttac gtcagtggag atatcacatc aatccacttg ctttgaagac 2100
gtggttggaa cgtcttcttt ttccacgatg ctcctcgtgg gtgggggtcc atctttggga 2160
ccactgtcgg cagaggcatc ttcaacgatg gcctttcctt tatcgcaatg atggcatttg 2220
taggagccac cttccttttc cactatcttc acaataaagt gacagatagc tgggcaatgg 2280
aatccgagga ggtttccgga tattaccctt tgttgaaaag tctcaatacg ctctacaatg 2340
tagtgcgcct tttataaagc aaaagcaaaa gcaaacccta gtgggcggga actttttcca 2400
attcaatctg aaccgctcgt gctaaagcac tcgcgataag ggggggccac gccggtaata 2460
ttaatgcggc gtgggtcccc ccttatcttt tacttaatca gtaaggaaat gccacatcat 2520
attccactga ccaacttcaa ttactgaaat acccctgcct ccatgcctcc acgcctggtt 2580
ataagataga gtttgaggca acccaaggag tcacaacaaa aggtaccgcc ccgtccggtc 2640
ctgcccgtca ccgagatctg atctcacgcg tctaggatcg acctgcagat cgttcaaaca 2700
tttggcaata aagtttctta agattgaatc ctgttgccgg tcttgcgatg attatcatct 2760
aatttctgtt gaattacgtt aagcatgtaa taattaacat gtaatgcatg acgttattta 2820
tgagatgggt ttttatgatt agagtcccgc aattatacat ttaatacgcg atagaaaaca 2880
aaatatagcg cgcaaactag gataaattat cgcgcgcggt gtcatctatg ttactagatc 2940
tctagcttga tatcgaatta attcactggc cgtcgtttta caacgtcgtg actgggaaaa 3000
ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa 3060
tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgcccg 3120
ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc 3180
taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa 3240
aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc 3300
ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac 3360
tcaaccctat ctcgggctat tcttttgatt tataagggat tttgccgatt tcggaaccac 3420
catcaaacag gattttcgcc tgctggggca aaccagcgtg gaccgcttgc tgcaactctc 3480
tcagggccag gcggtgaagg gcaatcagct gttgcccgtc tcactggtga aaagaaaaac 3540
caccccagta cattaaaaac gtccgcaatg tgttattaag ttgtctaagc gtcaatttgt 3600
ttacaccaca atatatcctg ccaccagcca gccaacagct ccccgaccgg cagctcggca 3660
caaaatcacc actcgataca ggcagcccat cagtccggga cggcgtcagc gggagagccg 3720
ttgtaaggcg gcagactttg ctcatgttac cgatgctatt cggaagaacg gcaactaagc 3780
tgccgggttt gaaacacgga tgatctcgcg gagggtagca tgttgattgt aacgatgaca 3840
gagcgttgct gcctgtgatc aaatatcatc tccctcgcag agatccgaat tatcagcctt 3900
cttattcatt tctcgcttaa ccgtgacagg ctgtcgatct tgagaactat gccgacataa 3960
taggaaatcg ctggataaag ccgctgagga agctgagtgg cgctatttct ttagaagtga 4020
acgttgacga tatcaactcc cctatccatt gctcaccgaa tggtacaggt cggggacccg 4080
aagttccgac tgtcggcctg atgcatcccc ggctgatcga ccccagatct ggggctgaga 4140
aagcccagta aggaaacaac tgtaggttcg agtcgcgaga tcccccggaa ccaaaggaag 4200
taggttaaac ccgctccgat caggccgagc cacgccaggc cgagaacatt ggttcctgta 4260
ggcatcggga ttggcggatc aaacactaaa gctactggaa cgagcagaag tcctccggcc 4320
gccagttgcc aggcggtaaa ggtgagcaga ggcacgggag gttgccactt gcgggtcagc 4380
acggttccga acgccatgga aaccgccccc gccaggcccg ctgcgacgcc gacaggatct 4440
agcgctgcgt ttggtgtcaa caccaacagc gccacgcccg cagttccgca aatagccccc 4500
aggaccgcca tcaatcgtat cgggctacct agcagagcgg cagagatgaa cacgaccatc 4560
agcggctgca cagcgcctac cgtcgccgcg accccgcccg gcaggcggta gaccgaaata 4620
aacaacaagc tccagaatag cgaaatatta agtgcgccga ggatgaagat gcgcatccac 4680
cagattcccg ttggaatctg tcggacgatc atcacgagca ataaacccgc cggcaacgcc 4740
cgcagcagca taccggcgac ccctcggcct cgctgttcgg gctccacgaa aacgccggac 4800
agatgcgcct tgtgagcgtc cttggggccg tcctcctgtt tgaagaccga cagcccaatg 4860
atctcgccgt cgatgtaggc gccgaatgcc acggcatctc gcaaccgttc agcgaacgcc 4920
tccatgggct ttttctcctc gtgctcgtaa acggacccga acatctctgg agctttcttc 4980
agggccgaca atcggatctc gcggaaatcc tgcacgtcgg ccgctccaag ccgtcgaatc 5040
tgagccttaa tcacaattgt caattttaat cctctgttta tcggcagttc gtagagcgcg 5100
ccgtgcgtcc cgagcgatac tgagcgaagc aagtgcgtcg agcagtgccc gcttgttcct 5160
gaaatgccag taaagcgctg gctgctgaac ccccagccgg aactgacccc acaaggccct 5220
agcgtttgca atgcaccagg tcatcattga cccaggcgtg ttccaccagg ccgctgcctc 5280
gcaactcttc gcaggcttcg ccgacctgct cgcgccactt cttcacgcgg gtggaatccg 5340
atccgcacat gaggcggaag gtttccagct tgagcgggta cggctcccgg tgcgagctga 5400
aatagtcgaa catccgtcgg gccgtcggcg acagcttgcg gtacttctcc catatgaatt 5460
tcgtgtagtg gtcgccagca aacagcacga cgatttcctc gtcgatcagg acctggcaac 5520
gggacgtttt cttgccacgg tccaggacgc ggaagcggtg cagcagcgac accgattcca 5580
ggtgcccaac gcggtcggac gtgaagccca tcgccgtcgc ctgtaggcgc gacaggcatt 5640
cctcggcctt cgtgtaatac cggccattga tcgaccagcc caggtcctgg caaagctcgt 5700
agaacgtgaa ggtgatcggc tcgccgatag gggtgcgctt cgcgtactcc aacacctgct 5760
gccacaccag ttcgtcatcg tcggcccgca gctcgacgcc ggtgtaggtg atcttcacgt 5820
ccttgttgac gtggaaaatg accttgtttt gcagcgcctc gcgcgggatt ttcttgttgc 5880
gcgtggtgaa cagggcagag cgggccgtgt cgtttggcat cgctcgcatc gtgtccggcc 5940
acggcgcaat atcgaacaag gaaagctgca tttccttgat ctgctgcttc gtgtgtttca 6000
gcaacgcggc ctgcttggcc tcgctgacct gttttgccag gtcctcgccg gcggtttttc 6060
gcttcttggt cgtcatagtt cctcgcgtgt cgatggtcat cgacttcgcc aaacctgccg 6120
cctcctgttc gagacgacgc gaacgctcca cggcggccga tggcgcgggc agggcagggg 6180
gagccagttg cacgctgtcg cgctcgatct tggccgtagc ttgctggacc atcgagccga 6240
cggactggaa ggtttcgcgg ggcgcacgca tgacggtgcg gcttgcgatg gtttcggcat 6300
cctcggcgga aaaccccgcg tcgatcagtt cttgcctgta tgccttccgg tcaaacgtcc 6360
gattcattca ccctccttgc gggattgccc cgactcacgc cggggcaatg tgcccttatt 6420
cctgatttga cccgcctggt gccttggtgt ccagataatc caccttatcg gcaatgaagt 6480
cggtcccgta gaccgtctgg ccgtccttct cgtacttggt attccgaatc ttgccctgca 6540
cgaataccag cgaccccttg cccaaatact tgccgtgggc ctcggcctga gagccaaaac 6600
acttgatgcg gaagaagtcg gtgcgctcct gcttgtcgcc ggcatcgttg cgccacatct 6660
aggtactaaa acaattcatc cagtaaaata taatatttta ttttctccca atcaggcttg 6720
atccccagta agtcaaaaaa tagctcgaca tactgttctt ccccgatatc ctccctgatc 6780
gaccggacgc agaaggcaat gtcataccac ttgtccgccc tgccgcttct cccaagatca 6840
ataaagccac ttactttgcc atctttcaca aagatgttgc tgtctcccag gtcgccgtgg 6900
gaaaagacaa gttcctcttc gggcttttcc gtctttaaaa aatcatacag ctcgcgcgga 6960
tctttaaatg gagtgtcttc ttcccagttt tcgcaatcca catcggccag atcgttattc 7020
agtaagtaat ccaattcggc taagcggctg tctaagctat tcgtataggg acaatccgat 7080
atgtcgatgg agtgaaagag cctgatgcac tccgcataca gctcgataat cttttcaggg 7140
ctttgttcat cttcatactc ttccgagcaa aggacgccat cggcctcact catgagcaga 7200
ttgctccagc catcatgccg ttcaaagtgc aggacctttg gaacaggcag ctttccttcc 7260
agccatagca tcatgtcctt ttcccgttcc acatcatagg tggtcccttt ataccggctg 7320
tccgtcattt ttaaatatag gttttcattt tctcccacca gcttatatac cttagcagga 7380
gacattcctt ccgtatcttt tacgcagcgg tatttttcga tcagtttttt caattccggt 7440
gatattctca ttttagccat ttattatttc cttcctcttt tctacagtat ttaaagatac 7500
cccaagaagc taattataac aagacgaact ccaattcact gttccttgca ttctaaaacc 7560
ttaaatacca gaaaacagct ttttcaaagt tgttttcaaa gttggcgtat aacatagtat 7620
cgacggagcc gattttgaaa ccacaattat gggtgatgct gccaacttac tgatttagtg 7680
tatgatggtg tttttgaggt gctccagtgg cttctgtgtc tatcagctgt ccctcctgtt 7740
cagctactga cggggtggtg cgtaacggca aaagcaccgc cggacatcag cgctatctct 7800
gctctcactg ccgtaaaaca tggcaactgc agttcactta caccgcttct caacccggta 7860
cgcaccagaa aatcattgat atggccatga atggcgttgg atgccgggca acagcccgca 7920
ttatgggcgt tggcctcaac acgattttac gtcacttaaa aaactcaggc cgcagtcggt 7980
aacctcgcgc atacagccgg gcagtgacgt catcgtctgc gcggaaatgg acgaacagtg 8040
gggctatgtc ggggctaaat cgcgccagcg ctggctgttt tacgcgtatg acagtctccg 8100
gaagacggtt gttgcgcacg tattcggtga acgcactatg gcgacgctgg ggcgtcttat 8160
gagcctgctg tcaccctttg acgtggtgat atggatgacg gatggctggc cgctgtatga 8220
atcccgcctg aagggaaagc tgcacgtaat cagcaagcga tatacgcagc gaattgagcg 8280
gcataacctg aatctgaggc agcacctggc acggctggga cggaagtcgc tgtcgttctc 8340
aaaatcggtg gagctgcatg acaaagtcat cgggcattat ctgaacataa aacactatca 8400
ataagttgga gtcattaccc aattatgata gaatttacaa gctataaggt tattgtcctg 8460
ggtttcaagc attagtccat gcaagttttt atgctttgcc cattctatag atatattgat 8520
aagcgcgctg cctatgcctt gccccctgaa atccttacat acggcgatat cttctatata 8580
aaagatatat tatcttatca gtattgtcaa tatattcaag gcaatctgcc tcctcatcct 8640
cttcatcctc ttcgtcttgg tagcttttta aatatggcgc ttcatagagt aattctgtaa 8700
aggtccaatt ctcgttttca tacctcggta taatcttacc tatcacctca aatggttcgc 8760
tgggtttatc gcacccccga acacgagcac ggcacccgcg accactatgc caagaatgcc 8820
caaggtaaaa attgccggcc ccgccatgaa gtccgtgaat gccccgacgg ccgaagtgaa 8880
gggcaggccg ccacccaggc cgccgccctc actgcccggc acctggtcgc tgaatgtcga 8940
tgccagcacc tgcggcacgt caatgcttcc gggcgtcgcg ctcgggctga tcgcccatcc 9000
cgttactgcc ccgatcccgg caatggcaag gactgccagc gctgccattt ttggggtgag 9060
gccgttcgcg gccgaggggc gcagcccctg gggggatggg aggcccgcgt tagcgggccg 9120
ggagggttcg agaagggggg gcacccccct tcggcgtgcg cggtcacgcg cacagggcgc 9180
agccctggtt aaaaacaagg tttataaata ttggtttaaa agcaggttaa aagacaggtt 9240
agcggtggcc gaaaaacggg cggaaaccct tgcaaatgct ggattttctg cctgtggaca 9300
gcccctcaaa tgtcaatagg tgcgcccctc atctgtcagc actctgcccc tcaagtgtca 9360
aggatcgcgc ccctcatctg tcagtagtcg cgcccctcaa gtgtcaatac cgcagggcac 9420
ttatccccag gcttgtccac atcatctgtg ggaaactcgc gtaaaatcag gcgttttcgc 9480
cgatttgcga ggctggccag ctccacgtcg ccggccgaaa tcgagcctgc ccctcatctg 9540
tcaacgccgc gccgggtgag tcggcccctc aagtgtcaac gtccgcccct catctgtcag 9600
tgagggccaa gttttccgcg aggtatccac aacgccggcg gccgcggtgt ctcgcacacg 9660
gcttcgacgg cgtttctggc gcgtttgcag ggccatagac ggccgccagc ccagcggcga 9720
gggcaaccag cccggtgagc gtcgcaaagg cgctcggtct tgccttgctc gtcggtgatg 9780
tacttcacca gctccgcgaa gtcgctcttc ttgatggagc gcatggggac gtgcttggca 9840
atcacgcgca ccccccggcc gttttagcgg ctaaaaaagt catggctctg ccctcgggcg 9900
gaccacgccc atcatgacct tgccaagctc gtcctgcttc tcttcgatct tcgccagcag 9960
ggcgaggatc gtggcatcac cgaaccgcgc cgtgcgcggg tcgtcggtga gccagagttt 10020
cagcaggccg cccaggcggc ccaggtcgcc attgatgcgg gccagctcgc ggacgtgctc 10080
atagtccacg acgcccgtga ttttgtagcc ctggccgacg gccagcaggt aggccgacag 10140
gctcatgccg gccgccgccg ccttttcctc aatcgctctt cgttcgtctg gaaggcagta 10200
caccttgata ggtgggctgc ccttcctggt tggcttggtt tcatcagcca tccgcttgcc 10260
ctcatctgtt acgccggcgg tagccggcca gcctcgcaga gcaggattcc cgttgagcac 10320
cgccaggtgc gaataaggga cagtgaagaa ggaacacccg ctcgcgggtg ggcctacttc 10380
acctatcctg cccggctgac gccgttggat acaccaagga aagtctacac gaaccctttg 10440
gcaaaatcct gtatatcgtg cgaaaaagga tggatatacc gaaaaaatcg ctataatgac 10500
cccgaagcag ggttatgcag cggaaaagcg ccacgcttcc cgaagggaga aaggcggaca 10560
ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa 10620
acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt 10680
tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac 10740
ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt 10800
ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga 10860
ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg ccagaaggcc gccagagagg 10920
ccgagcgcgg ccgtgaggct tggacgctag ggcagggcat gaaaaagccc gtagcgggct 10980
gctacgggcg tctgacgcgg tggaaagggg gaggggatgt tgtctacatg gctctgctgt 11040
agtgagtggg ttgcgctccg gcagcggtcc tgatcaatcg tcaccctttc tcggtccttc 11100
aacgttcctg acaacgagcc tccttttcgc caatccatcg acaatcaccg cgagtccctg 11160
ctcgaacgct gcgtccggac cggcttcgtc gaaggcgtct atcgcggccc gcaacagcgg 11220
cgagagcgga gcctgttcaa cggtgccgcc gcgctcgccg gcatcgctgt cgccggcctg 11280
ctcctcaagc acggccccaa cagtgaagta gctgattgtc atcagcgcat tgacggcgtc 11340
cccggccgaa aaacccgcct cgcagaggaa gcgaagctgc gcgtcggccg tttccatctg 11400
cggtgcgccc ggtcgcgtgc cggcatggat gcgcgcgcca tcgcggtagg cgagcagcgc 11460
ctgcctgaag ctgcgggcat tcccgatcag aaatgagcgc cagtcgtcgt cggctctcgg 11520
caccgaatgc gtatgattct ccgccagcat ggcttcggcc agtgcgtcga gcagcgcccg 11580
cttgttcctg aagtgccagt aaagcgccgg ctgctgaacc cccaaccgtt ccgccagttt 11640
gcgtgtcgtc agaccgtcta cgccgacctc gttcaacagg tccagggcgg cacggatcac 11700
tgtattcggc tgcaactttg tcatgcttga cactttatca ctgataaaca taatatgtcc 11760
accaacttat cagtgataaa gaatccgcgc gttcaatcgg accagcggag gctggtccgg 11820
aggccagacg tgaaacccaa catacccctg atcgtaattc tgagcactgt cgcgctcgac 11880
gctgtcggca tcggcctgat tatgccggtg ctgccgggcc tcctgcgcga tctggttcac 11940
tcgaacgacg tcaccgccca ctatggcatt ctgctggcgc tgtatgcgtt ggtgcaattt 12000
gcctgcgcac ctgtgctggg cgcgctgtcg gatcgtttcg ggcggcggcc aatcttgctc 12060
gtctcgctgg ccggcgccag atctggggaa ccctgtggtt ggcatgcaca tacaaatgga 12120
cgaacggata aaccttttca cgccctttta aatatccgat tattctaata aacgctcttt 12180
tctctta 12187
<210> 5
<211> 3388
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(3388)
<223> nucleotide sequence of pDsRed
<400> 5
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60
cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180
accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240
attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300
tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360
tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acctcgcgaa 420
tgcatctaga tatggcctcc tccgagaacg tcatcaccga gttcatgcgc ttcaaggtgc 480
gcatggaggg caccgtgaac ggccacgagt tcgagatcga gggcgagggc gagggccgcc 540
cctacgaggg ccacaacacc gtgaagctga aggtgaccaa gggcggcccc ctgcccttcg 600
cctgggacat cctgtccccc cagttccagt acggctccaa ggtgtacgtg aagcaccccg 660
ccgacatccc cgactacaag aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga 720
tgaacttcga ggacggcggc gtggcgaccg tgacccagga ctcctccctg caggacggct 780
gcttcatcta caaggtgaag ttcatcggcg tgaacttccc ctccgacggc cccgtgatgc 840
agaagaagac gatgggctgg gaggcctcca ccgagcgcct gtacccccgc gacggcgtgc 900
tgaagggcga gacccacaag gccctgaagc tgaaggacgg cggccactac ctggtggagt 960
tcaagtccat ctacatggcc aagaagcccg tgcagctgcc cggctactac tacgtggacg 1020
ccaagctgga catcacctcc cacaacgagg actacaccat cgtggagcag tacgagcgca 1080
ccgagggccg ccaccacctg ttcctgtgaa tcggatcccg ggcccgtcga ctgcagaggc 1140
ctgcatgcaa gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg 1200
ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa 1260
tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac 1320
ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt 1380
gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga 1440
gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca 1500
ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg 1560
ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt 1620
cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc 1680
ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct 1740
tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc 1800
gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta 1860
tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca 1920
gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag 1980
tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag 2040
ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt 2100
agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa 2160
gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg 2220
attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga 2280
agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta 2340
atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc 2400
cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg 2460
ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga 2520
agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt 2580
tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt 2640
gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc 2700
caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc 2760
ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca 2820
gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag 2880
tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg 2940
tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa 3000
cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa 3060
cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga 3120
gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga 3180
atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg 3240
agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt 3300
ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa 3360
aataggcgta tcacgaggcc ctttcgtc 3388
<210> 6
<211> 3447
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(3447)
<223> nucleotide sequence of phNGF
<400> 6
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60
cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180
accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240
attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300
tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360
tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acctcgcgaa 420
tgcatctaga tcatgaagac taatcttttt ctctttctca tcttttcact tctcctatca 480
ttatcctcgg ccgaacctca ttctgaatct aatgttcctg ctggacatac tattcctcaa 540
gctcattgga ctaagttgca acattctttg gatactgctt tgagaagagc tagatctgct 600
cctgctgctg ctattgctgc tagagttgct ggacaaacta gaaatattac tgttgatcct 660
agattgttta agaagagaag attgagatct cctagagttt tgttttctac tcaacctcct 720
agagaagctg ctgatactca agatttggat tttgaagttg gaggagctgc tccttttaat 780
agaactcata gatctaagag atcttcttct catcctattt ttcatagagg agaattttct 840
gtttgtgatt ctgtttctgt ttgggttgga gataagacta ctgctactga tattaaggga 900
aaggaagtta tggttttggg agaagttaat attaataatt ctgtttttaa gcaatatttt 960
tttgaaacta agtgtagaga tcctaatcct gttgattctg gatgtagagg aattgattct 1020
aagcattgga attcttattg tactactact catacttttg ttaaggcttt gactatggat 1080
ggaaagcaag ctgcttggag atttattaga attgatactg cttgtgtttg tgttttgtct 1140
agaaaggctg ttagaagagc ttaaagctat cggatcccgg gcccgtcgac tgcagaggcc 1200
tgcatgcaag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc 1260
tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat 1320
gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc 1380
tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg 1440
ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag 1500
cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag 1560
gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 1620
tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc 1680
agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc 1740
tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt 1800
cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg 1860
ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat 1920
ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag 1980
ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt 2040
ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc 2100
cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta 2160
gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag 2220
atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga 2280
ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa 2340
gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa 2400
tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc 2460
ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga 2520
taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa 2580
gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt 2640
gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg 2700
ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc 2760
aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg 2820
gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag 2880
cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt 2940
actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt 3000
caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac 3060
gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac 3120
ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag 3180
caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa 3240
tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga 3300
gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc 3360
cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa 3420
ataggcgtat cacgaggccc tttcgtc 3447
Claims (8)
1. A pAM gene, wherein the nucleotide sequence of the pAM gene is shown as SEQ ID No. 1.
2. The pAM gene according to claim 1, wherein the pAM gene consists of an open reading frame and the LIR, C1: C2 and SIR genes in the genome of the chick pea chlorotic dwarf virus in an arrangement of LIR → C1: C2 → SIR → open reading frame → LIR, wherein the open reading frame comprises a promoter NOS terminator, a cloning site and a terminator CaMV 35S promoter, and wherein the nucleotide sequence of the chick pea chlorotic dwarf virus is shown in SEQ ID No. 2.
3. A plant expression vector constructed by recombining the pAM gene of claim 1 or 2 with a plant vector.
4. The plant expression vector of claim 3, wherein the plant vector is pEGAD, the nucleotide sequence of the plant vector is shown in SEQ ID No.3, the obtained plant expression vector is pEGM, and the nucleotide sequence of the plant expression vector is shown in SEQ ID No. 4.
5. The plant expression vector of claim 4, wherein the plant expression vector is constructed by:
(1) carrying out double enzyme digestion on a plant vector pEGAD by using PacI and KpnI, and recovering a large fragment to obtain pEGAD-PacI/KpnI;
(2) adding a restriction enzyme site ttaattaaa of PacI to the upstream of pAM, adding a restriction enzyme site ggtacc of KpnI to the downstream of pAM, synthesizing a plasmid pUC57-pAM, carrying out restriction enzyme digestion reaction by using three enzymes of PacI, KpnI and BspQI, and recovering a fragment to obtain pAM-PacI/KpnI;
(3) the fragment pEGAD-PacI/KpnI and the fragment pAM-PacI/KpnI were ligated with DNA ligase to construct a plant expression vector pEGM.
6. The plant expression vector of claim 3, wherein the pAM gene is located between RB and LB of the plant vector in the orientation RB → LIR → C1: C2 → SIR → terminator → cloning site → promoter → LIR → LB.
7. Use of a plant expression vector according to any one of claims 3 to 6 in the preparation of a transgenic plant.
8. Use according to claim 7, wherein the plant is tobacco leaf.
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