CN118027162A - Polypeptide, polynucleotide encoding polypeptide, construct, expression system and battery thereof - Google Patents

Polypeptide, polynucleotide encoding polypeptide, construct, expression system and battery thereof Download PDF

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CN118027162A
CN118027162A CN202410356862.1A CN202410356862A CN118027162A CN 118027162 A CN118027162 A CN 118027162A CN 202410356862 A CN202410356862 A CN 202410356862A CN 118027162 A CN118027162 A CN 118027162A
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polypeptide
sequence
battery
amino acid
terminus
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王博祥
叶启当
刘家骏
刘锡蓓
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Shenzhen Lingspider Technology Co ltd
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01M—PROCESSES OR MEANS, e.g. BATTERIES, FOR THE DIRECT CONVERSION OF CHEMICAL ENERGY INTO ELECTRICAL ENERGY
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    • H01M8/16—Biochemical fuel cells, i.e. cells in which microorganisms function as catalysts
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Abstract

The application discloses a polypeptide, polynucleotide for encoding the polypeptide, a construct, an expression system and a battery thereof. The polypeptide, the polynucleotide and the battery thereof are characterized in that from the N end to the C end, the 1 st, 24 th and 27 th positions of the polypeptide are aromatic amino acids. The application discovers the sequence of the power generation material from microorganisms which do not produce the power generation material in nature, and utilizes escherichia coli to produce recombinant proteins of various microorganisms, and the recombinant proteins have excellent power generation function.

Description

Polypeptide, polynucleotide encoding polypeptide, construct, expression system and battery thereof
Technical Field
The application relates to the field of batteries, in particular to a polypeptide, a polynucleotide for encoding the polypeptide, a construct, an expression system of the polypeptide and a battery.
Background
The energy problem is a serious problem faced by human beings, and the existing new energy modes comprise nuclear energy, wind energy, solar energy and the like all have specific environmental requirements. The moist energy in the air is an energy source which is ignored by people, and is characterized in that weak energy can be extracted from the environment as long as humidity exists. In 2020, researchers have found that charged pili (e-PN) produced by extracting Geobacillus thioreductase (Geobacter sulfurreducens, G.s) can be made into a micro-film. The film can convert humidity gradient in air into electric energy and can generate voltage when an electric appliance is externally connected. The power generation material can be repeatedly charged and discharged, namely, the material can be discharged when an external electric appliance is connected with the material, and the material can automatically absorb the moist energy in the air to charge when the material is disconnected. The power generation material can stably supply power after being stored for 1 year in the environment.
The academy has only verified that the e-PN produced by the geobacillus thioreducens has the power generation capacity at present, but the geobacillus thioreducens is very high in large-scale culture cost due to the strict anaerobic characteristic of the geobacillus thioreducens. In 2020, researchers have produced e-PN of Geobacillus thioreductase by using Escherichia coli, and it is proved that the material has similar conductivity. However, the existing sulfur reduction geobacillus pilus material (G.s.E-PN) has poor electricity generating performance, and if the electricity utilization device is required to be directly driven, a great amount of pilus materials need to be provided, so that the cost is very high.
Disclosure of Invention
The object of the present application is to provide a novel polypeptide, a polynucleotide encoding the polypeptide, a construct, an expression system and a battery thereof.
In order to achieve the above purpose, the present application adopts the following technical scheme:
According to a first aspect, in one embodiment, there is provided a polypeptide comprising aromatic amino acids at positions 1, 24, 27 from the N-terminus to the C-terminus.
According to a second aspect, in an embodiment, there is provided an isolated polynucleotide encoding a polypeptide of the first aspect.
According to a third aspect, in an embodiment, there is provided a construct comprising the polynucleotide of the second aspect.
According to a fourth aspect, in an embodiment, there is provided an expression system comprising a construct or a polynucleotide of the second aspect having an exogenous source integrated into the genome of the construct or the polynucleotide of the second aspect.
According to a fifth aspect, in an embodiment, there is provided a battery comprising the polypeptide of the first aspect.
According to the polypeptide, the polynucleotide and the battery, the application discovers the sequence of the power generation material from microorganisms which do not produce the power generation material in nature, and utilizes escherichia coli to produce recombinant proteins of various microorganisms, and the recombinant proteins have excellent power generation function.
Drawings
FIG. 1 shows the amino acid sequence of each pilus;
FIG. 2 is a schematic diagram of an E-PN cell structure according to an embodiment;
FIG. 3 is a partial physical photograph of an E-PN battery;
FIG. 4 is a graph of current and voltage measurements for e-PN;
fig. 5 is a circuit construction process diagram of an embodiment.
The reference numerals in fig. 2 are explained as follows: 1. a Top Electrode (Top Electrode); 2. protein nanowire films (E-pili film); 3. a Bottom Electrode (Bottom Electrode); 4. a glass substrate (GLASS SLIDE).
Detailed Description
The application will be described in further detail below with reference to the drawings by means of specific embodiments. Wherein like elements in different embodiments are numbered alike in association. In the following embodiments, numerous specific details are set forth in order to provide a better understanding of the present application. However, one skilled in the art will readily recognize that some of the features may be omitted, or replaced by other elements, materials, or methods in different situations. In some instances, related operations of the present application have not been shown or described in the specification in order to avoid obscuring the core portions of the present application, and may be unnecessary to persons skilled in the art from a detailed description of the related operations, which may be presented in the description and general knowledge of one skilled in the art.
Furthermore, the described features, operations, or characteristics of the description may be combined in any suitable manner in various embodiments. Also, various steps or acts in the method descriptions may be interchanged or modified in a manner apparent to those of ordinary skill in the art. Thus, the various orders in the description and drawings are for clarity of description of only certain embodiments, and are not meant to be required orders unless otherwise indicated.
The numbering of the components itself, e.g. "first", "second", etc., is used herein merely to distinguish between the described objects and does not have any sequential or technical meaning. The term "coupled" as used herein includes both direct and indirect coupling (coupling), unless otherwise indicated.
As used herein, "peptide" refers to a compound in which the amino group of one amino acid is condensed with the carboxyl group of another amino acid, and a peptide may be "dehydrocondensed" from two, three or more amino acids.
As used herein, "polypeptide" refers to a compound that is "dehydrocondensed" from two or more amino acids.
As used herein, a "protein" is also referred to as a protein, and is a substance having a certain spatial structure formed by folding a polypeptide chain composed of amino acids in a "dehydration condensation" manner.
As used herein, the "N-terminus" of a polypeptide refers to the end of the polypeptide chain having a protruding amino group (-NH 2), and the "C-terminus" of a polypeptide refers to the end of the polypeptide chain having a protruding carboxyl group (-COOH).
As used herein, "aromatic amino acid" refers to an amino acid having a benzene ring structure in the molecular structure. Mainly comprises Tyrosine (Y), phenylalanine (PHENYLALANINE, F) and tryptophan (tryptophan, W).
According to a first aspect, in one embodiment, there is provided a polypeptide comprising aromatic amino acids at positions 1, 24, 27 from the N-terminus to the C-terminus.
In one embodiment, from the N-terminus to the C-terminus, the 32 nd position of the polypeptide is also an aromatic amino acid.
In one embodiment, the aromatic amino acid is selected from any one of Tyrosine (Y), phenylalanine (PHENYLALANINE, F), and tryptophan (tryptophan, W).
In one embodiment, from the N-terminus to the C-terminus, the 1 st position of the polypeptide is phenylalanine.
In one embodiment, from the N-terminus to the C-terminus, the 24 th position of the polypeptide is any one of Tyrosine (Y) and phenylalanine (PHENYLALANINE, F).
In one embodiment, the polypeptide has Tyrosine (Y) at position 27 from the N-terminus to the C-terminus.
In one embodiment, from the N-terminus to the C-terminus, the 32 nd position of the polypeptide is an aromatic amino acid.
In one embodiment, from N-terminus to C-terminus, the 32 nd position of the polypeptide is any one of Tyrosine (Y) and phenylalanine (PHENYLALANINE, F).
In one embodiment, from the N-terminus to the C-terminus, at least one of positions 50, 51, 55, 57, 59 of the polypeptide is also an aromatic amino acid.
In one embodiment, the aromatic amino acid is selected from any one of Tyrosine (Y), phenylalanine (PHENYLALANINE, F), and tryptophan (tryptophan, W).
In one embodiment, from the N-terminus to the C-terminus, the 51 st position of the polypeptide is also an aromatic amino acid.
In one embodiment, from the N-terminal to the C-terminal, the 51 st position of the polypeptide is any one of Tyrosine (Y) and phenylalanine (PHENYLALANINE, F).
In one embodiment, from the N-terminus to the C-terminus, the 57 th position of the polypeptide is an aromatic amino acid.
In one embodiment, from the N-terminal to the C-terminal, the 57 th position of the polypeptide is any one of Tyrosine (Y) and phenylalanine (PHENYLALANINE, F).
In one embodiment, from the N-terminus to the C-terminus, the polypeptide comprises the following characteristic sequences or mutants thereof at positions 2 to 23:
TLIELLIVVAIIGILAAIAIPQ。
in one embodiment, from the N-terminus to the C-terminus, the 2 nd to 23 rd polypeptides comprise at least one of the following sequences:
11)TLIELLIVVAIIGILAAIAIPQ；
12)TLIELMIVVAIIGILAAIAIPQ；
13)TLIELLVVVAIIGILAAIAIPQ；
14)TLVELMIVVAIIGILAAVAIPQ；
15)TLIELLVVVAIIAILAAIAIPQ。
In one embodiment, the polypeptide contains less than or equal to 61 amino acids. An excessive number of amino acids may make it more difficult to assemble into fibers.
In one embodiment, the polypeptide comprises at least one of the following amino acid sequences:
21)FTLIELLIVVAIIGILAAIAIPQFSAYRVKAYNSAASSDLRNLKTALESAFADDQTYPPES；
22)FTLIELMIVVAIIGILAAIAIPQYQNYVARSYGASALATINPLKTTVEESFSRGIAYSKIK；
23)FTLIELLVVVAIIGILAAIAIPQFAKYRQNAFNSAAQSDVRNSRSDVESFYAENFHYPY；
24)FTLVELMIVVAIIGILAAVAIPQFAQYRIRGFNSSALSDVRNLTTAQEAFFADWLRYAVTH；
25)FTLIELLVVVAIIAILAAIAIPQFAKYRENAAKASAVADAKNIATAIESYYADTQSFPSSI；
26)FTLIELLVVVAIIAILAAIAIPQFAKYRENAYKASAVADAKNIATAIESYYADTQSFPSSI；
27)FTLIELLVVVAIIAILAAIAIPQYAKYRENAYKASAVADAKNIATAIESYYADTQSFPSSI；
28)FTLIELLIVVAIIGILAAIAIPQFAAYRQKAFNSAAISDIRSTKTNLEAYYTDNNNYPY。
in one embodiment, the polypeptide has a signal peptide attached to the N-terminus.
In one embodiment, the polypeptide has a histidine tag attached to the C-terminus.
In one embodiment, the signal peptide comprises the following amino acid sequence from the N-terminus to the C-terminus: MDKQRG.
In one embodiment, the histidine tag contains 6 to 8 histidines.
According to a second aspect, in an embodiment, there is provided an isolated polynucleotide encoding the polypeptide of the first aspect. The same amino acid has two or more codons (i.e. the degeneracy of the codons), all polynucleotides encoding the polypeptides of the first aspect are within the scope of the application.
According to a third aspect, in an embodiment, there is provided a construct comprising a polynucleotide of the second aspect. The construct may generally be obtained by inserting the isolated polynucleotide into a suitable vector, which may be a phage, plasmid, viral vector, such as a bacterium, which may be selected by one of skill in the art. In other words, the vectors of embodiments of the application comprise a polynucleotide of interest capable of being expressed in a host cell or an isolated fraction thereof. Vectors are also generally suitable as cloning vectors, i.e.replicable in microbial systems; cloning vectors may be designed for replication in one host, while constructs are designed for expression in a different host. Vectors comprising the polypeptides and proteins of embodiments of the application may also comprise a selectable marker for propagation or selection in a host cell. The vector may be introduced into a prokaryotic or eukaryotic cell by conventional transformation or transfection techniques.
In one embodiment, the construct comprises the nucleotide sequence set forth in SEQ ID No. 9.
In one embodiment, the construct comprises the nucleotide sequence set forth in SEQ ID No. 10.
According to a fourth aspect, in an embodiment there is provided an expression system comprising a construct according to the third aspect or a polynucleotide according to the second aspect integrated with an exogenous source in the genome. The expression system may be a host cell which may express a polypeptide according to the first aspect. In another embodiment of the application, the host cell may be a eukaryotic cell and/or a prokaryotic cell.
According to a fifth aspect, in an embodiment, there is provided a battery comprising the polypeptide of the first aspect.
In one embodiment, the cell includes an upper electrode, a lower electrode, and a polypeptide attached between the upper electrode and the lower electrode.
In one embodiment, the lower electrode comprises graphene.
In one embodiment, the upper electrode comprises a printed circuit board (PCB board, printed Circuit Board).
In one embodiment, the PCB comprises a metal plated PCB.
In one embodiment, the metal comprises gold.
In one embodiment, the present application analyzes the basic amino acid structure of G.s.e-PN and finds the general sequence of e-PN (ELECTRICALLY CONDUCTIVE PROTEIN NANOWIRES, conductive protein nanofibers). And a series of e-PNs were found from NCBI database by amino acid sequence alignment. The e-PN amino acid similar sequences of D.t., F.s., P.a., G.u., D.a., C.n. are selected as raw materials, and a series of recombinant engineering such as peptide guide and histidine tag fusion, specific point mutation and the like are performed on the basis. Through molecular structure prediction, we found that these recombinant proteins have a high degree of similarity to the naturally occurring conducting proteins. By using recombinant expression of specific E.coli, we successfully extracted 8 e-PN materials and tested their charging properties, and found that these materials can both supply current and voltage. By selecting and designing the upper electrode and the lower electrode, the materials can generate an average voltage of 0.6V and a current of 100-300 mu A, and the current is 1000 times that reported in published literature. By comparison of material properties under the same conditions, we successfully produced a material d.a.pila with 15% higher voltage than the g.s.pila material reported in the literature, and a material d.t.pila with current increased to approximately 350%. Finally, we successfully lighted Led bulbs using 8 series connected e-PN cells. The materials can generate electricity by using wet energy in the future, provide electricity for equipment, reduce the dependence on traditional petrochemical energy sources, and provide assistance for human beings to realize carbon emission reduction and carbon neutralization in the early days.
Examples
1. Design and screening of e-PN
Some characteristic sequences are present in G.s.pilA and other found e-PN. As shown in FIG. 1, each pilus amino acid sequence, yellow is ppdD secretion signal peptide sequence, green is characteristic sequence (consensus sequence), black is unique sequence of each pilus, red is aromatic amino acid, and blue is 6xHistags sequence. It can be seen that there is a highly similar sequence (green letter designation) in each pilus amino acid sequence. We screened protein sequences from other microorganisms with this signature by BLASTP function of NCBI database and recorded. Then, we selected from these sequences that carry more aromatic amino acids (rose letter mark) and are uniformly distributed. For some natural proteins of 61 amino acids (excluding signal peptides), we retain the complete amino acid sequence. For some natural proteins greater than 61 amino acids, we only intercept up to 61 amino acids. After sequence interception, we add a secretion signal peptide (yellow letter mark) of E.coli (E.coli) ppdD to its N-terminal, helping the e-PN protein to secrete out of the cell membrane and form through typeIV pilus system. For better detection and extraction we add a 6x histidine tag (Histag, blue letter mark) downstream.
Finally, we selected 6 organisms of the 100 suspected e-PN proteins, and 2 amino acid mutations were made to one of the organisms of the e-PN proteins to alter the number and variety of aromatic amino acids (C.n.1pilA and C.n.2pilA).
The amino acid sequence in fig. 1 is as follows:
G.s.pilA
MDKQRGFTLIELLIVVAIIGILAAIAIPQFSAYRVKAYNSAASSDLRNLKTALESAFADDQTYPPESHHHHHH*(SEQ ID No.1)。
P.a.pilA(Modified)
MDKQRGFTLIELMIVVAIIGILAAIAIPQYQNYVARSYGASALATINPLKTTVEESFSRGIAYSKIKHHHHHH*(SEQ ID No.2)。
F.sinusarabici(F.s.pilA)
MDKQRGFTLIELLVVVAIIGILAAIAIPQFAKYRQNAFNSAAQSDVRNSRSDVESFYAENFHYPYHHHHHH*(SEQIDNo.3)。
D.alkaliphilus61(D.a.pilA)
MDKQRGFTLVELMIVVAIIGILAAVAIPQFAQYRIRGFNSSALSDVRNLTTAQEAFFADWLRYAVTHHHHHHH*(SEQIDNo.4)。
Calditerrivibrionitroreducens61(C.n.pilA)
MDKQRGFTLIELLVVVAIIAILAAIAIPQFAKYRENAAKASAVADAKNIATAIESYYADTQSFPSSIHHHHHH*(SEQIDNo.5)。
Calditerrivibrionitroreducens61mut1(C.n.1pilA)
MDKQRGFTLIELLVVVAIIAILAAIAIPQFAKYRENAYKASAVADAKNIATAIESYYADTQSFPSSIHHHHHH*(SEQIDNo.6)。
Calditerrivibrionitroreducens61mut2(C.n.2pilA)
MDKQRGFTLIELLVVVAIIAILAAIAIPQYAKYRENAYKASAVADAKNIATAIESYYADTQSFPSSIHHHHHH*(SEQIDNo.7)。
Desulfuromonasthiophila(D.tpilA)
MDKQRGFTLIELLIVVAIIGILAAIAIPQFAAYRQKAFNSAAISDIRSTKTNLEAYYTDNNNYPYHHHHHH*(SEQIDNo.8)。
Among the above 8 amino acid sequences, the single underlined amino acid is a signal peptide, the double underlined amino acid is an aromatic amino acid, the thick underlined amino acid is a characteristic sequence (functional sequence shared by the power generation functional proteins), the dot underlined amino acid is a unique sequence of each pilus, the wavy line marked amino acid is a 6x histidine tag, and "×" indicates an amino acid gap corresponding to a stop codon.
2. E-PN expression in E.coli
The final expression plasmid of e-PN is shown as SEQ ID No.9, and the synthesized e-PN sequence is inserted into the expression plasmid through BsaIGoldenGate assembly system.
The expression plasmid was transferred into E.coli BL21, and the monoclonal cells were placed in a 14mL test tube, and 5mLLB liquid and an appropriate amount of kanamycin antibiotic (50. Mu.g/mL in this example, that is, 50. Mu.g/mL final concentration of kanamycin antibiotic in a mixed solution) were added, and cultured overnight at 37℃in a shake flask at 220 rpm. mu.L of the bacterial liquid was spread on LB plates of 10cm standard diameter and kanamycin, 1.5% agar was solidified, and the plates were sealed with a sealing film (parfilm), and incubated overnight at 37 ℃. Cells were scraped from the plates with 300 μlm9 liquid medium and resuspended in 6mL of M9 medium (available from aledine). To the M9 solid medium were added 0.5% by volume of glycerol, 0.5mM IPTG, 2mMMgSO 4、0.1mMCaCl2, 0.4% by mass% by volume of glucose and kanamycin (50. Mu.g/mL), each concentration being the final concentration of the corresponding component in the M9 medium. Sealed with parfilm and incubated at 30℃for 48 hours.
Cells were scraped from M9 medium, 1.2 mM M9 medium (600. Mu.L) was added to each dish, about 10mL of the cell fluid was extracted, centrifuged at 7500rpm at 4℃for 15 minutes, and the cells were collected and the supernatant was discarded. Concentrated cells were resuspended in 20mL150mM ethanolamine buffer (ph=10.5), the suspended cells were poured into a 150mL ice-bath beaker, the tubes were rinsed 2 times with 10mL ethanolamine buffer, and added back into the beaker. The cells were whipped with a stirrer at high speed (about 20000 rpm/min) for 2 minutes. The resulting solution was transferred to a test tube for centrifugation. The beaker was rinsed twice with an additional 10mL of ethanolamine buffer. The collected bacterial liquid was centrifuged at 10,000Xg for 20 minutes at 4 ℃. After centrifugation, the supernatant, containing pilin, is collected, and Tritonx reagent is added to a final concentration of 6mM, stirred at 30℃at a speed of 100-150 rpm until Tritonx is dissolved.
The fungus Mao Rongye was diluted with twice the volume of distilled water, tritonx was diluted to a final concentration of 2mM, and then added to a centrifugal filtration device (100 kDaMilipore), and the pili were centrifuged at 10,000Xg for 20 minutes at 4 ℃. The e-PN solution was collected in the inner section of the tube and stored at 4℃for subsequent cell fabrication.
3. Electrode construction and detection effects of e-PN
Each protein solution was diluted with 150mM ethanolamine buffer to a final concentration of 1.5mg/mL, pH=2, stored at 4 ℃. The e-PN battery consists of three parts: the graphene lower electrode, the protein nanowire film and the mesh-shaped PCB upper electrode are all based on a piece of glass sheet. A thin graphene layer was coated as a lower electrode 3 (carbon nanotube layer) on a glass substrate 4 in a square of 15 x 15 mm. 20 mu L of 1.5mg/mL protein solution is directly added on the lower electrode for three times to form a three-layer round protein nanowire film 2 with the diameter of about 8-9 mm. The carbon nanotube electrode and the e-PN film were dried on a metal bath at 85℃to remove excess moisture. Ensure that the final e-PN film is coated with a layer after being completely dried. A perforated, gold-plated PCB board was chosen as the upper electrode 1. And covering the PCB above the film, and fixing the film on a glass slide by using a breathable medical adhesive tape to complete an e-PN battery unit. As shown in fig. 2, which is a schematic diagram of the e-PN battery structure, the golden section: a mesh-shaped upper electrode; intermediate portion E-pili film: e-pili protein film; gray portion: a graphene lower electrode; the lowest blue portion: a glass plate. As shown in fig. 3, a cell sample and a PCB mesh-shaped upper electrode, in fig. 3, a) a mesh-shaped upper electrode; b) A portion of the battery sample.
Current and voltage (15 x 15mm voltage current) were measured using a multimeter (FLUKE 8808A5-1/2digit multimeter). The results are shown in FIG. 4 as current and voltage measurements for e-PN. The red bar represents the voltage value (corresponding to the left y-axis, in V) and the green bar represents the current value (corresponding to the right y-axis, in mua). The higher the voltage and current of the power generation material under stable conditions, the more excellent the performance of the power generation material.
The current and voltage measurements for e-PN in FIG. 4 are shown in Table 1.
TABLE 1
Voltage/V Standard deviation of voltage Current/. Mu.A Standard deviation of current
Gs pilA 0.57775 0.036819662 116.8 14.94790955
Pa pilA 0.594 0.014508618 149.35 16.51582574
F.s PilA 0.60975 0.059751046 168.4 21.32103812
D.a pilA 0.6694 0.024984795 116.8333333 9.613300971
C.n pilA 0.571666667 0.037858288 148.475 4.938306896
C.n1 pilA 0.641 0.029810513 141.5333333 22.18548074
C.n2 pilA 0.610333333 0.031436444 191.6666667 4.988876516
D.t pilA 0.60925 0.021521791 308.3333333 31.96178273
4. Electric appliance driven by e-PN using damp energy
As shown in fig. 5, the battery materials are connected in series and an electric circuit is constructed. The Led bulb is successfully lit and maintained for a period of time. The multimeter measured the operating voltage to be maintained at 3.2V.
Fig. 5 shows that the e-PN material can drive a consumer. a shows the series connection mode of e-PN battery units, b shows the circuit principle of driving led, c is an actual circuit constructed by bread board, d shows a successful electric quantity bulb, and e is a stable voltage value measured when driving the bulb.
The DNA sequence of the expression vector is as follows:
ATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGCGCGCCGCTTCGTCAGGCCACATAGCTTTCTTGTTCTGATCGGAACGATCGTTGGCTGTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGCTCACAATTAGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAGAAAGAGGAGAAATACCATATGTGAGACCACGGATCAATGGTCTCACACCACCACCACCACCACTAATCTGAGCTCAGGAAGGAGCGGCAATGAATATTCCACAGCTCACTGCCCTGTGTCTGCGTTATCAGGGAGTCTTGCTGGATGCCAGCGAAGAGGTGGTTCATGTTGCGGTAGTCGATGCACCTTCGCATGAGCTACTGGACGCATTGCATTTCGCTACCACCAAACGTATTGAGATCACCTGCTGGACGCGCCAACAAATGGAAGGTCACGCCAGTCGCACACAACAGACATTGCCCGTAGCTGTTCAGGAGAAGCATCAGCCCAAAGCAGAGTTGCTGGCTCGAACGTTACAATCTGCGCTGGAACAACGCGCGTCTGATATTCATATCGAACCAGCGGACAATGCCTACCGCATCCGCTTGCGTATCGACGGCGTATTGCATCCTTTACCGGATGTTTCACCGGATGCCGGAGTCGCATTAACCGCCAGATTAAAAGTGCTGGGAAACCTGGATATTGCGGAACATCGCCTGCCGCAGGACGGGCAATTCACTGTCGAACTGGCAGGAAACGCCGTCTCATTTCGTATTGCGACCTTACCATGTCGGGGTGGTGAAAAGGTGGTATTAAGGTTGTTACAGCAGGTGAGTCAGGCACTGGATGTCAACACGCTTGGAATGCAGCCGTTACAACTGGCGGACTTTGCTCATGCCTTGCAACAACCACAGGGACTGGTGCTGGTAACTGGCCCTACCGGCAGCGGCAAAACGGTCACGCTTTATAGTGCCCTGCAAACGCTGAATACCGCTGACATTAATATTTGTAGCGTCGAAGATCCGGTTGAGATCCCCATAGCCGGACTAAACCAGACGCAAATCCATCCGCGTGCCGGGCTCACCTTTCAGGGCGTTTTGCGTGCGTTATTGCGCCAGGATCCTGACGTCATCATGATCGGAGAGATCCGCGATGGCGAAACAGCAGAGATCGCTATTAAAGCGGCGCAAACTGGTCACCTGGTGTTGTCTACCCTACACACTAATTCCACCTGCGAAACGCTGGTACGTTTACAGCAAATGGGAGTCGCCCGCTGGATGCTCTCATCAGCGCTTACGCTGGTAATAGCCCAGCGTCTGGTACGTAAACTTTGCCCACATTGTCGCCAGCAGCAAGGGGAGCCCATCCATATTCCAGACAATGTATGGCCGTCGCCGCTGCCCCACTGGCAGGCACCCGGTTGTGTACATTGCTACCACGGTTTTTATGGTCGTACGGCCTTATTTGAAGTTCTGCCCATAACGCCGGTCATTCGTCAGCTTATTTCCGCTAATACCGACGTTGAATCGCTGGAAACGCACGCACGACAGGCGGGTATGCGTACGCTTTTTGAAAACGGCTGCCTGGCCGTGGAGCAAGGCTTAACCACCTTTGAAGAGTTAATCCGCGTACTGGGGATGCCGCATGGCGAGTAAGCAACTCTGGCGCTGGCATGGCATAACCGGCGACGGCAATGCGCAAGATGGGATGCTATGGGCAGAGAGCCGTGCTTTGCTGCTCATGGCACTACAGCAACAGATGGTTACCCCACTTAGCCTGAAGCGAATCGCCATCAATTCTGCGCAGTGGCGAGGAGATAAAAGCGCGGAAGTCATTCATCAACTGGCGACGCTACTCAAAGCCGGGTTAACGCTTTCTGAAGGGCTGGCACTGCTGGCGGAACAGCATCCCAGTAAGCAATGGCAAGCGTTGCTGCAATCGCTGGCGCACGATCTCGAACAGGGCATTGCTTTTTCCAATGCCTTATTACCCTGGTCAGAGGTATTTCCGCCACTCTATCAGGCGATGATCCGCACGGGTGAACTGACCGGTAAGCTGGATGAATGCTGCTTTGAACTGGCGCGTCAGCAAAAAGCCCAGCGTCAGTTGACCGACAAAGTGAAATCAGCGTTACGTTATCCCATCATCATTTTAGCGATGGCAATCATGGTGGTTGTGGCAATGCTGCATTTTGTTCTGCCGGAGTTTGCCGCTATCTATAAGACCTTCAACACCCCACTACCGGCACTAACGCAGGGGATCATGACGCTGGCAGACTTTAGTGGCGAATGGAGCTGGCTGCTGGTGTTGTTCGGCTTTCTGCTGGCGATAGCCAATAAGTTGCTGATGCGCCGACCGACCTGGCTTATAGCGCGGCAGAAATTGCTGTTACGCATCCCGATTATGGGTTCACTGATGCGGGGACAAAAACTCACGCAGATTTTTACGATTCTGGCGCTGACACAAAGTGCAGGCATTACTTTTTTGCAGGGCGTAGAGAGCGTCAGAGAAACAATGCGCTGCCCGTACTGGGTGCAACTTCTGACACAAATCCAGCACGATATCAGTAACGGTCATCCCATCTGGCTGGCGCTAAAAAATGCCGGGGAGTTTAGCCCGCTCTGTTTGCAATTAGTGAGAACAGGAGAGGCATCCGGCTCGCTGGACCTCATGTTAGACAACCTCGCCCATCATCATCGGGATAACACAATGGCGCTGGCGGATAACCTCGCAGCCTTACTGGAACCGGCGTTGCTGATCATAACGGGAGGAATTATCGGTACGCTGGTGGTGGCGATGTATCTGCCAATTTTCCATTTAGGCGATGCGATGAGTGGGATGGGATAATCTAGAAGGCCGTCAGAGTGACGGGTGATAAGGAGATCATCACAATGGCATTTAAGATCTGGCAAATTGGTTTGCATTTACAACAGCAAGAAGCGGTAGCGGTTGCGATCGTACGGGGCGCAAAAGAATGCTTTTTGCAACGCTGGTGGCGGTTGCCGCTGGAGAACGACATTATCAAAGATGGGCGGATTGTTGATGCGCAGCAGCTGGCTAAAACGTTGTTACCTTGGAGTCGCGAACTGCCGCAGCGTCATCACATTATGTTGGCGTTTCCCGCCAGTCGCACATTACAGCGGTCATTTCCGCGCCCGTCGATGTCCCTTGGTGAGCGGGAGCAAACGGCCTGGCTGTCAGGGACGATGGCCCGCGAGCTGGATATGGATCCGGACTCCCTGCGCTTCGATTATAGCGAAGACTCACTCAGCCCCGCTTATAACGTGACTGCCGCGCAAAGCAAAGAGCTGGCAACGCTGCTTACGCTGGCAGAAAGGTTGCGTGTTCATGTTAGTGCGATCACCCCGGATGCCAGTGCATTACAGCGATTCCTGCCTTTTTTACCTTCTCATCAGCAATGTCTGGCCTGGCGTGATAACGAACAGTGGCTGTGGGCGACACGCTATCGCTGGGGGCGCAAACTGGCGGTAGGGATGACTAGCGCGAAGGAGCTGGCGGCAGCGTTATCCGTTGATCCCGAGAGCGTCGCGATATGTGGCGAAGGCGGATTTGATCCCTGGGAGGCCGTTTCTGTTCGTCAGCCGCCGCTACCGCCGAGCGGTGGAGACTTTGCCATCGCGCTGGGGCTGGCGCTTGGGAAGGCGTACTGATGAACCCGCCAATTAATTTTTTGCCCTGGCGACAGCAACGCCGGACCGCTTTTCTGCGTTTCTGGTTGCTGATGTTCGTTGCGCCTCTGCTGCTGGCCGTCGGGATAACGCTAATACTGCGTCTGACAGGCAGCGCCGAAGCTCGCATAGACGCCGTTTTGCTTCAGGCGGAACAACAACTCGCCCGCAGCTTACAGATAACGAAACCACGTTTGCTGGAGCAGCAACAATTGCGTGAGCAGCGTTCTCAAAGGCAGCGCCAGCGACAATTTACCCGCGACTGGCAATCTGCGCTGGAAGCACTGGCGGCTCTTTTACCTGAACACGCCTGGCTGACAACGATAAGCTGGCAGCAGGGAACGCTGGAGATCAAGGGGCTTACAACAAGCATTACCGCGTTAAACGCACTAGAAACGTCACTTCGCCAGGATGCTTCTTTTCATCTCAATCAGCGGGGGGCCACGCAGCAGGATGCGCAGGGACGCTGGCAATTTGAGTATCAGTTAACAAGGAAGGTTAGCGATGAACATGTTCTTTGACTGGTGGTTCGCCACATCACCCCGCCTCCGCCAGTTTTGCTGGGCAGTCTGGTTGCTGATGTTAGTTACGCTCATTTTTCTGTCATCGACACACCATGAAGAGCGCGACGCATTAATTCGACTACGGGCAAGTCATCACCAGCAGTGGGCCGCACTGTATCGCCTGGTAGACACCACTCCCTTCAGCGAGGAAAAAACGCTGCCCTTTTCGCCACTGGATTTTCAGTTATCCGGCGCGCAACTGGTTTCCTGGCATCCATCCGCGCAGGGAGGCGAGTTGGCGTTGAAAACGCTGTGGGAAGCAGTGCCGTCGGCATTTACACGGCTGGCAGAGCGCAACGTCAGCGTGAGCCGTTTTTCGTTAAGCGTGGAAGGTGATGATCTTTTGTTCACGCTACAACTGGAGACGCCGCATGAGGGTTAAACGCTGGTTGTTGGCAGGTATTGCATTGTGCCTTTTAACCGGTATGCGTGACCCTTTTAAACCGCCGGAAGATCTATGCCGGATTAGCGAACTTAGCCAGTGGCGCTATCAGGGGATGGTAGGGCGAGGCGAGCGCATCATCGGTGTAATAAAAGACGGGCAAAAGAAATGGCGACGGGTGCAGCAAAACGATGTGCTGGAAAACGGCTGGACAATTTTACAGCTGACGCCAGACGTACTAACGCTGGGTACCGGGACAAACTGCGAACCGCCACAATGGTTGTGGCAACGGCAAGGAGATACAAATGAAGCAATGGATAGCCGCACTACTGTTGATGCTGATACCCGGCGTACAGGCGGCAAAGCCGCAAAAAGTGACGCTGATGGTGGATGACGTTCCGGTAGCTCAGGTGTTGCAGGCGCTGGCTGAACAGGAGAAGTTGAACCTGGTCGTGTCGCCAGACGTCAGCGGTACGGTGTCGTTACATCTAACAGATGTTCCCTGGAAGCAGGCACTACAAACTGTAGTGAAAAGCGCCGGACTGATAACGCGGCAGGAAGGCAACATTCTCTCAGTGCATTCCATTGCCTGGCAGAATAACAATATCGCCCGCCAGGAGGCGGAGCAGGCGCGGGCGCAGGCAAATCTGCCGCTGGAAAATCGCAGTATAACCCTGCAATACGCCGACGCGGGAGAACTGGCGAAAGCGGGGGAGAAGCTACTGAGTGCCAAAGGGAGTATGACCGTCGATAAACGCACCAATCGCCTTTTGCTACGAGATAACAAAACGGCGTTAAGCGCGCTTGAACAGTGGGTAGCGCAAATGGATCTGCCGGTCGGGCAGGTTGAGCTGTCGGCGCATATTGTCACCATTAATGAAAAAAGTTTGCGTGAGTTAGGCGTGAAATGGACGCTGGCCGATGCGCAACACGCTGGTGGCGTTGGGCAAGTCACCACGCTTGGTAGCGACCTCTCCGTAGCGACGGCGACAACGCATGTCGGTTTTAACATTGGGCGCATCAACGGACGCTTGCTGGATCTTGAGCTTTCCGCGCTCGAACAAAAACAGCAGCTGGATATTATCGCCAGTCCGCGTCTGCTGGCCTCACATCTTCAGCCTGCCAGCATTAAACAGGGGAGCGAAATTCCATATCAGGTTTCCAGCGGGGAAAGTGGCGCGACGTCGGTGGAATTTAAAGAGGCCGTCCTGGGGATGGAGGTCACGCCCACGGTGTTACAAAAAGGTCGCATCCGGCTGAAATTACACATCAGCCAGAACGTTCCGGGGCAGGTGCTACAGCAGGCCGATGGCGAAGTGCTGGCGATTGATAAGCAGGAGATCGAAACGCAGGTCGAGGTCAAAAGCGGAGAAACGTTGGCGCTGGGCGGCATTTTTACCCGTAAAAATAAATCGGGTCAGGATAGCGTACCGTTGCTTGGCGACATTCCCTGGTTCGGGCAATTATTTCGTCATGACGGAAAAGAAGATGAACGACGCGAGTTAGTGGTGTTTATCACGCCACGACTGGTTTCCAGTGAGTAAGCTTAGGAGACTGCCGGCATGAAAACACAACGTGGTTATACGCTGATTGAAACGCTGGTCGCGATGCTGATTTTGGTCATGCTAAGCGCAAGTGGACTCTATGGCTGGCAATACTGGCAGCAGTCGCAACGGCTATGGCAAACCGCCAGCCAGGCGCGGGACTATTTGCTCTATTTACGTGAAGATGCCAACTGGCATAACCGCGACCACAGTATCAGCGTTATCAGGGAGGGGACGTTATGGTGCCTTGTGAGTTCCGCTGCTGGGGCCAATACCTGTCATGGCAGTTCACCATTGGTCTTTGTGCCACGCTGGCCCGAAGTCGAAATGAGCGACCTGACACCTTCGCTTGCTTTCTTTGGCCTGCGCAATACCGCATGGGCCGGGCATATTCGCTTCAAAAACTCAACGGGCGAGTGGTGGCTGGTGGTTTCGCCGTGGGGAAGACTCCGGCTTTGTCAGCAAGGAGAAACAGAAGGATGCCTGTAAAAGAGCAAGGTTTTTCTCTGCTGGAAGTGTTGATTGCTATGGCGATCAGTAGCGTATTGTTGCTGGGGGCTGCACGCTTTCTGCCTGCGTTACAGCGTGAAAGTTTAACGAGCACCCGTAAGCTGGCGCTGGAAGATGAAATCTGGCTGCGGGTATTTACCGTCGCGAAGCATCTCCAGAGGGCGGGTTATTGTCATGGCAGCTGTACCGGCGAAGGGCTGGAAATTGTCGGACAGGGTGACTGTATCATTGTGCAGTGGGATGCGAACAGTAACGGTATCTGGGATCGCGAACCGGTAAAAGAGTCTGACCAGATTGGATTTCGTCTGAAGGAGCATGTGCTGGAAACGCTACGCGGTGCGACATCCTGTGAAGGTAAGGGCTGGGATAAAGTCACTAATCCGGATGCCATCATTATCGACACTTTTCAGGTCGTACGTCAGGATGTCAGCGGCTTCTCGCCGGTGTTGACGGTTAATATGCGTGCTGCCAGTAAGTCTGAACCGCAAACCGTGGTGGATGCCAGCTATAGCGTGACAGGATTCAACCTGTGAACCGCGAAAAAGGTGTTTCGTCACTGGCTCTGGTTCTGATGCTGCTGGTTTTGGGTAGCTTGCTATTGCAAGGAATGAGTCAGCAGGATCGCAGTTTTGCCTCTCGCGTGAGCATGGAAAGTCAGTCATTGCGCCGCCAGGCCATCGTTCAGTCGGCGCTGGCGTGGGGGAAAATGCACTCCTGGCAGACGCAGACCGCAGTTCAGTGCTCGCAGTACGCTGGAACCGATGCCCAGGTTTGTTTGCGTTTACTGGCAGATAATGAAGCCTTATTGATTGCCGGTTATGAAGGCGTTTCGTTGTGGCGAACAGGCGAAGTCATTGATGGAAACATTGTTTTTTCGCCACGCGGCTGGAGCGATTTTTGTCCGCTGAAAGAGAGGGCGTTATGTCAGCTTCCCTGAAGAATCAACAAGGCTTTAGCCTGCCGGAGGTAATGGTTGCGATGGTGCTGATGGTGATGATTGTCACTGCGTTATCGGGTATCCAGCGAACATTAATGAACAGTCTCGCCAGCAGAAACCAGTACCAACAGCTCTGGCGGCATGGTTGGCAGCAAACGCAACTGCGCGCGATTTCGCCACCTGCCAACTGGCAGGTCAACCGAATGCAGACATCGCAGGCGGGATGTGTCAGCATCAGCGTTACGCTAGTTTCACCCGGGGGCAGAGAAGGCGAGATGACCCGCCTGCATTGTCCGAATCGTCAGTAGCAGGGAGCAACAATAATGACAATGCTACTGCCGCTCTTCATTCTGGTTGGTTTTATTGCAGATTATTTTGTTAATGCCATCGCCTATCACCTCTCGCCGTTGGAGGATAAAACGGCGTTAACGTTTCGCCAGGTATTGGTTCATTTCAGGCAAAAAAAATATGCCTGGCATGATACAGTGCCCCTGATACTTTGTGTTGCTGCTGCTATCGCCTGCGCCCTGGCACCTTTCACGCCCATCGTGACTGGCGCACTCTTTCTCTATTTCTGTTTCGTACTCACGCTCAGTGTTATTGATTTTCGCACTCAGCTCCTGCCCGACAAACTCACCTTACCGCTGCTCTGGCTTGGCTTGGTATTTAATGCGCAGTATGGATTAATTGATTTACATGATGCGGTTTACGGCGCGGTAGCGGGCTATGGGGTGCTGTGGTGTGTTTACTGGGGCGTCTGGTTAGTTTGTCACAAAGAGGGATTGGGCTACGGTGATTTCAAGCTACTGGCCGCTGCAGGCGCATGGTGTGGCTGGCAAACGTTGCCAATGATACTGCTGATTGCCTCGCTGGGTGGCATTGGTTACGCCATCGTTTCACAACTTCTGCAACGCCGGACTATAACCACTATCGCATTCGGCCCGTGGCTTGCGCTCGGCAGCATGATAAACCTGGGGTATCTGGCCTGGATCTCTTATTAAATAAGAGAGCAGAGGTTGATAAGTTTTCTCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATATACTAGAGCTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATTGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCAATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACTGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTATCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCCTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCAT(SEQ ID No.9).
The vector DNA sequence with d.t.pila is as follows:
ATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGCGCGCCGCTTCGTCAGGCCACATAGCTTTCTTGTTCTGATCGGAACGATCGTTGGCTGTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGCTCACAATTAGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGCCTCTACAAATAATTTTGTTTAAGAAAGAGGAGAAATACCATATGGATAAACAGCGTGGTTTTACCCTGATTGAACTGCTGATTGTGGTGGCGATTATTGGCATTCTGGCGGCGATTGCGATTCCGCAGTTTGCGGCGTATCGCCAGAAAGCGTTTAACAGCGCGGCGATTAGCGATATTCGCAGCACCAAAACCAACCTGGAAGCGTATTATACCGATAACAACAACTATCCGTATCACCACCACCACCACCACTAATCTGAGCTCAGGAAGGAGCGGCAATGAATATTCCACAGCTCACTGCCCTGTGTCTGCGTTATCAGGGAGTCTTGCTGGATGCCAGCGAAGAGGTGGTTCATGTTGCGGTAGTCGATGCACCTTCGCATGAGCTACTGGACGCATTGCATTTCGCTACCACCAAACGTATTGAGATCACCTGCTGGACGCGCCAACAAATGGAAGGTCACGCCAGTCGCACACAACAGACATTGCCCGTAGCTGTTCAGGAGAAGCATCAGCCCAAAGCAGAGTTGCTGGCTCGAACGTTACAATCTGCGCTGGAACAACGCGCGTCTGATATTCATATCGAACCAGCGGACAATGCCTACCGCATCCGCTTGCGTATCGACGGCGTATTGCATCCTTTACCGGATGTTTCACCGGATGCCGGAGTCGCATTAACCGCCAGATTAAAAGTGCTGGGAAACCTGGATATTGCGGAACATCGCCTGCCGCAGGACGGGCAATTCACTGTCGAACTGGCAGGAAACGCCGTCTCATTTCGTATTGCGACCTTACCATGTCGGGGTGGTGAAAAGGTGGTATTAAGGTTGTTACAGCAGGTGAGTCAGGCACTGGATGTCAACACGCTTGGAATGCAGCCGTTACAACTGGCGGACTTTGCTCATGCCTTGCAACAACCACAGGGACTGGTGCTGGTAACTGGCCCTACCGGCAGCGGCAAAACGGTCACGCTTTATAGTGCCCTGCAAACGCTGAATACCGCTGACATTAATATTTGTAGCGTCGAAGATCCGGTTGAGATCCCCATAGCCGGACTAAACCAGACGCAAATCCATCCGCGTGCCGGGCTCACCTTTCAGGGCGTTTTGCGTGCGTTATTGCGCCAGGATCCTGACGTCATCATGATCGGAGAGATCCGCGATGGCGAAACAGCAGAGATCGCTATTAAAGCGGCGCAAACTGGTCACCTGGTGTTGTCTACCCTACACACTAATTCCACCTGCGAAACGCTGGTACGTTTACAGCAAATGGGAGTCGCCCGCTGGATGCTCTCATCAGCGCTTACGCTGGTAATAGCCCAGCGTCTGGTACGTAAACTTTGCCCACATTGTCGCCAGCAGCAAGGGGAGCCCATCCATATTCCAGACAATGTATGGCCGTCGCCGCTGCCCCACTGGCAGGCACCCGGTTGTGTACATTGCTACCACGGTTTTTATGGTCGTACGGCCTTATTTGAAGTTCTGCCCATAACGCCGGTCATTCGTCAGCTTATTTCCGCTAATACCGACGTTGAATCGCTGGAAACGCACGCACGACAGGCGGGTATGCGTACGCTTTTTGAAAACGGCTGCCTGGCCGTGGAGCAAGGCTTAACCACCTTTGAAGAGTTAATCCGCGTACTGGGGATGCCGCATGGCGAGTAAGCAACTCTGGCGCTGGCATGGCATAACCGGCGACGGCAATGCGCAAGATGGGATGCTATGGGCAGAGAGCCGTGCTTTGCTGCTCATGGCACTACAGCAACAGATGGTTACCCCACTTAGCCTGAAGCGAATCGCCATCAATTCTGCGCAGTGGCGAGGAGATAAAAGCGCGGAAGTCATTCATCAACTGGCGACGCTACTCAAAGCCGGGTTAACGCTTTCTGAAGGGCTGGCACTGCTGGCGGAACAGCATCCCAGTAAGCAATGGCAAGCGTTGCTGCAATCGCTGGCGCACGATCTCGAACAGGGCATTGCTTTTTCCAATGCCTTATTACCCTGGTCAGAGGTATTTCCGCCACTCTATCAGGCGATGATCCGCACGGGTGAACTGACCGGTAAGCTGGATGAATGCTGCTTTGAACTGGCGCGTCAGCAAAAAGCCCAGCGTCAGTTGACCGACAAAGTGAAATCAGCGTTACGTTATCCCATCATCATTTTAGCGATGGCAATCATGGTGGTTGTGGCAATGCTGCATTTTGTTCTGCCGGAGTTTGCCGCTATCTATAAGACCTTCAACACCCCACTACCGGCACTAACGCAGGGGATCATGACGCTGGCAGACTTTAGTGGCGAATGGAGCTGGCTGCTGGTGTTGTTCGGCTTTCTGCTGGCGATAGCCAATAAGTTGCTGATGCGCCGACCGACCTGGCTTATAGCGCGGCAGAAATTGCTGTTACGCATCCCGATTATGGGTTCACTGATGCGGGGACAAAAACTCACGCAGATTTTTACGATTCTGGCGCTGACACAAAGTGCAGGCATTACTTTTTTGCAGGGCGTAGAGAGCGTCAGAGAAACAATGCGCTGCCCGTACTGGGTGCAACTTCTGACACAAATCCAGCACGATATCAGTAACGGTCATCCCATCTGGCTGGCGCTAAAAAATGCCGGGGAGTTTAGCCCGCTCTGTTTGCAATTAGTGAGAACAGGAGAGGCATCCGGCTCGCTGGACCTCATGTTAGACAACCTCGCCCATCATCATCGGGATAACACAATGGCGCTGGCGGATAACCTCGCAGCCTTACTGGAACCGGCGTTGCTGATCATAACGGGAGGAATTATCGGTACGCTGGTGGTGGCGATGTATCTGCCAATTTTCCATTTAGGCGATGCGATGAGTGGGATGGGATAATCTAGAAGGCCGTCAGAGTGACGGGTGATAAGGAGATCATCACAATGGCATTTAAGATCTGGCAAATTGGTTTGCATTTACAACAGCAAGAAGCGGTAGCGGTTGCGATCGTACGGGGCGCAAAAGAATGCTTTTTGCAACGCTGGTGGCGGTTGCCGCTGGAGAACGACATTATCAAAGATGGGCGGATTGTTGATGCGCAGCAGCTGGCTAAAACGTTGTTACCTTGGAGTCGCGAACTGCCGCAGCGTCATCACATTATGTTGGCGTTTCCCGCCAGTCGCACATTACAGCGGTCATTTCCGCGCCCGTCGATGTCCCTTGGTGAGCGGGAGCAAACGGCCTGGCTGTCAGGGACGATGGCCCGCGAGCTGGATATGGATCCGGACTCCCTGCGCTTCGATTATAGCGAAGACTCACTCAGCCCCGCTTATAACGTGACTGCCGCGCAAAGCAAAGAGCTGGCAACGCTGCTTACGCTGGCAGAAAGGTTGCGTGTTCATGTTAGTGCGATCACCCCGGATGCCAGTGCATTACAGCGATTCCTGCCTTTTTTACCTTCTCATCAGCAATGTCTGGCCTGGCGTGATAACGAACAGTGGCTGTGGGCGACACGCTATCGCTGGGGGCGCAAACTGGCGGTAGGGATGACTAGCGCGAAGGAGCTGGCGGCAGCGTTATCCGTTGATCCCGAGAGCGTCGCGATATGTGGCGAAGGCGGATTTGATCCCTGGGAGGCCGTTTCTGTTCGTCAGCCGCCGCTACCGCCGAGCGGTGGAGACTTTGCCATCGCGCTGGGGCTGGCGCTTGGGAAGGCGTACTGATGAACCCGCCAATTAATTTTTTGCCCTGGCGACAGCAACGCCGGACCGCTTTTCTGCGTTTCTGGTTGCTGATGTTCGTTGCGCCTCTGCTGCTGGCCGTCGGGATAACGCTAATACTGCGTCTGACAGGCAGCGCCGAAGCTCGCATAGACGCCGTTTTGCTTCAGGCGGAACAACAACTCGCCCGCAGCTTACAGATAACGAAACCACGTTTGCTGGAGCAGCAACAATTGCGTGAGCAGCGTTCTCAAAGGCAGCGCCAGCGACAATTTACCCGCGACTGGCAATCTGCGCTGGAAGCACTGGCGGCTCTTTTACCTGAACACGCCTGGCTGACAACGATAAGCTGGCAGCAGGGAACGCTGGAGATCAAGGGGCTTACAACAAGCATTACCGCGTTAAACGCACTAGAAACGTCACTTCGCCAGGATGCTTCTTTTCATCTCAATCAGCGGGGGGCCACGCAGCAGGATGCGCAGGGACGCTGGCAATTTGAGTATCAGTTAACAAGGAAGGTTAGCGATGAACATGTTCTTTGACTGGTGGTTCGCCACATCACCCCGCCTCCGCCAGTTTTGCTGGGCAGTCTGGTTGCTGATGTTAGTTACGCTCATTTTTCTGTCATCGACACACCATGAAGAGCGCGACGCATTAATTCGACTACGGGCAAGTCATCACCAGCAGTGGGCCGCACTGTATCGCCTGGTAGACACCACTCCCTTCAGCGAGGAAAAAACGCTGCCCTTTTCGCCACTGGATTTTCAGTTATCCGGCGCGCAACTGGTTTCCTGGCATCCATCCGCGCAGGGAGGCGAGTTGGCGTTGAAAACGCTGTGGGAAGCAGTGCCGTCGGCATTTACACGGCTGGCAGAGCGCAACGTCAGCGTGAGCCGTTTTTCGTTAAGCGTGGAAGGTGATGATCTTTTGTTCACGCTACAACTGGAGACGCCGCATGAGGGTTAAACGCTGGTTGTTGGCAGGTATTGCATTGTGCCTTTTAACCGGTATGCGTGACCCTTTTAAACCGCCGGAAGATCTATGCCGGATTAGCGAACTTAGCCAGTGGCGCTATCAGGGGATGGTAGGGCGAGGCGAGCGCATCATCGGTGTAATAAAAGACGGGCAAAAGAAATGGCGACGGGTGCAGCAAAACGATGTGCTGGAAAACGGCTGGACAATTTTACAGCTGACGCCAGACGTACTAACGCTGGGTACCGGGACAAACTGCGAACCGCCACAATGGTTGTGGCAACGGCAAGGAGATACAAATGAAGCAATGGATAGCCGCACTACTGTTGATGCTGATACCCGGCGTACAGGCGGCAAAGCCGCAAAAAGTGACGCTGATGGTGGATGACGTTCCGGTAGCTCAGGTGTTGCAGGCGCTGGCTGAACAGGAGAAGTTGAACCTGGTCGTGTCGCCAGACGTCAGCGGTACGGTGTCGTTACATCTAACAGATGTTCCCTGGAAGCAGGCACTACAAACTGTAGTGAAAAGCGCCGGACTGATAACGCGGCAGGAAGGCAACATTCTCTCAGTGCATTCCATTGCCTGGCAGAATAACAATATCGCCCGCCAGGAGGCGGAGCAGGCGCGGGCGCAGGCAAATCTGCCGCTGGAAAATCGCAGTATAACCCTGCAATACGCCGACGCGGGAGAACTGGCGAAAGCGGGGGAGAAGCTACTGAGTGCCAAAGGGAGTATGACCGTCGATAAACGCACCAATCGCCTTTTGCTACGAGATAACAAAACGGCGTTAAGCGCGCTTGAACAGTGGGTAGCGCAAATGGATCTGCCGGTCGGGCAGGTTGAGCTGTCGGCGCATATTGTCACCATTAATGAAAAAAGTTTGCGTGAGTTAGGCGTGAAATGGACGCTGGCCGATGCGCAACACGCTGGTGGCGTTGGGCAAGTCACCACGCTTGGTAGCGACCTCTCCGTAGCGACGGCGACAACGCATGTCGGTTTTAACATTGGGCGCATCAACGGACGCTTGCTGGATCTTGAGCTTTCCGCGCTCGAACAAAAACAGCAGCTGGATATTATCGCCAGTCCGCGTCTGCTGGCCTCACATCTTCAGCCTGCCAGCATTAAACAGGGGAGCGAAATTCCATATCAGGTTTCCAGCGGGGAAAGTGGCGCGACGTCGGTGGAATTTAAAGAGGCCGTCCTGGGGATGGAGGTCACGCCCACGGTGTTACAAAAAGGTCGCATCCGGCTGAAATTACACATCAGCCAGAACGTTCCGGGGCAGGTGCTACAGCAGGCCGATGGCGAAGTGCTGGCGATTGATAAGCAGGAGATCGAAACGCAGGTCGAGGTCAAAAGCGGAGAAACGTTGGCGCTGGGCGGCATTTTTACCCGTAAAAATAAATCGGGTCAGGATAGCGTACCGTTGCTTGGCGACATTCCCTGGTTCGGGCAATTATTTCGTCATGACGGAAAAGAAGATGAACGACGCGAGTTAGTGGTGTTTATCACGCCACGACTGGTTTCCAGTGAGTAAGCTTAGGAGACTGCCGGCATGAAAACACAACGTGGTTATACGCTGATTGAAACGCTGGTCGCGATGCTGATTTTGGTCATGCTAAGCGCAAGTGGACTCTATGGCTGGCAATACTGGCAGCAGTCGCAACGGCTATGGCAAACCGCCAGCCAGGCGCGGGACTATTTGCTCTATTTACGTGAAGATGCCAACTGGCATAACCGCGACCACAGTATCAGCGTTATCAGGGAGGGGACGTTATGGTGCCTTGTGAGTTCCGCTGCTGGGGCCAATACCTGTCATGGCAGTTCACCATTGGTCTTTGTGCCACGCTGGCCCGAAGTCGAAATGAGCGACCTGACACCTTCGCTTGCTTTCTTTGGCCTGCGCAATACCGCATGGGCCGGGCATATTCGCTTCAAAAACTCAACGGGCGAGTGGTGGCTGGTGGTTTCGCCGTGGGGAAGACTCCGGCTTTGTCAGCAAGGAGAAACAGAAGGATGCCTGTAAAAGAGCAAGGTTTTTCTCTGCTGGAAGTGTTGATTGCTATGGCGATCAGTAGCGTATTGTTGCTGGGGGCTGCACGCTTTCTGCCTGCGTTACAGCGTGAAAGTTTAACGAGCACCCGTAAGCTGGCGCTGGAAGATGAAATCTGGCTGCGGGTATTTACCGTCGCGAAGCATCTCCAGAGGGCGGGTTATTGTCATGGCAGCTGTACCGGCGAAGGGCTGGAAATTGTCGGACAGGGTGACTGTATCATTGTGCAGTGGGATGCGAACAGTAACGGTATCTGGGATCGCGAACCGGTAAAAGAGTCTGACCAGATTGGATTTCGTCTGAAGGAGCATGTGCTGGAAACGCTACGCGGTGCGACATCCTGTGAAGGTAAGGGCTGGGATAAAGTCACTAATCCGGATGCCATCATTATCGACACTTTTCAGGTCGTACGTCAGGATGTCAGCGGCTTCTCGCCGGTGTTGACGGTTAATATGCGTGCTGCCAGTAAGTCTGAACCGCAAACCGTGGTGGATGCCAGCTATAGCGTGACAGGATTCAACCTGTGAACCGCGAAAAAGGTGTTTCGTCACTGGCTCTGGTTCTGATGCTGCTGGTTTTGGGTAGCTTGCTATTGCAAGGAATGAGTCAGCAGGATCGCAGTTTTGCCTCTCGCGTGAGCATGGAAAGTCAGTCATTGCGCCGCCAGGCCATCGTTCAGTCGGCGCTGGCGTGGGGGAAAATGCACTCCTGGCAGACGCAGACCGCAGTTCAGTGCTCGCAGTACGCTGGAACCGATGCCCAGGTTTGTTTGCGTTTACTGGCAGATAATGAAGCCTTATTGATTGCCGGTTATGAAGGCGTTTCGTTGTGGCGAACAGGCGAAGTCATTGATGGAAACATTGTTTTTTCGCCACGCGGCTGGAGCGATTTTTGTCCGCTGAAAGAGAGGGCGTTATGTCAGCTTCCCTGAAGAATCAACAAGGCTTTAGCCTGCCGGAGGTAATGGTTGCGATGGTGCTGATGGTGATGATTGTCACTGCGTTATCGGGTATCCAGCGAACATTAATGAACAGTCTCGCCAGCAGAAACCAGTACCAACAGCTCTGGCGGCATGGTTGGCAGCAAACGCAACTGCGCGCGATTTCGCCACCTGCCAACTGGCAGGTCAACCGAATGCAGACATCGCAGGCGGGATGTGTCAGCATCAGCGTTACGCTAGTTTCACCCGGGGGCAGAGAAGGCGAGATGACCCGCCTGCATTGTCCGAATCGTCAGTAGCAGGGAGCAACAATAATGACAATGCTACTGCCGCTCTTCATTCTGGTTGGTTTTATTGCAGATTATTTTGTTAATGCCATCGCCTATCACCTCTCGCCGTTGGAGGATAAAACGGCGTTAACGTTTCGCCAGGTATTGGTTCATTTCAGGCAAAAAAAATATGCCTGGCATGATACAGTGCCCCTGATACTTTGTGTTGCTGCTGCTATCGCCTGCGCCCTGGCACCTTTCACGCCCATCGTGACTGGCGCACTCTTTCTCTATTTCTGTTTCGTACTCACGCTCAGTGTTATTGATTTTCGCACTCAGCTCCTGCCCGACAAACTCACCTTACCGCTGCTCTGGCTTGGCTTGGTATTTAATGCGCAGTATGGATTAATTGATTTACATGATGCGGTTTACGGCGCGGTAGCGGGCTATGGGGTGCTGTGGTGTGTTTACTGGGGCGTCTGGTTAGTTTGTCACAAAGAGGGATTGGGCTACGGTGATTTCAAGCTACTGGCCGCTGCAGGCGCATGGTGTGGCTGGCAAACGTTGCCAATGATACTGCTGATTGCCTCGCTGGGTGGCATTGGTTACGCCATCGTTTCACAACTTCTGCAACGCCGGACTATAACCACTATCGCATTCGGCCCGTGGCTTGCGCTCGGCAGCATGATAAACCTGGGGTATCTGGCCTGGATCTCTTATTAAATAAGAGAGCAGAGGTTGATAAGTTTTCTCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATATACTAGAGCTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATTGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCAATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACTGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTATCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCCTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCAT(SEQ ID No.10).
In one embodiment, the present application discovers the sequence of a power generating material from microorganisms that do not produce the power generating material in nature, and uses E.coli to produce recombinant proteins of various microorganisms, which verify the power generating function. Wherein D.t.e-PN reaches 0.6v voltage, 353 μA current, 3.5 times that of G.s.E-PN measured simultaneously; whereas the d.a. e-PN achieves a higher and more stable voltage supply.
In an embodiment, a new upper and lower electrode scheme is designed, namely, a graphene coating is used as a lower electrode, and a gold-plated reticular structure is used as an upper electrode. Using this electrode scheme we successfully detected a current magnitude reported to be 1000 times higher.
In one embodiment, the application provides the amino acid sequence of each recombinant protein, as shown in FIG. 1.
In one embodiment, the present application provides an electrode design as shown in fig. 3 and 4.
In one embodiment, the application provides a DNA sequence of an expression vector for producing e-PN as shown in SEQ ID No. 9.
In one embodiment, other strains may be used to produce the amino acid sequences of the present application, such as Top10, DH5a E.coli, etc., or Bacillus subtilis, vibrio natrii, etc. All strains produce materials which keep the amino acid sequences consistent and are protected by the application.
In one embodiment, other tag proteins can be added based on the amino acids of the present application, and the tags are chemically or enzymatically treated to achieve the same purpose of producing proteinaceous material. All power generating materials that maintain the identity of the core amino acid sequence (i.e., pilA sequence) are materials protected by this patent.
The foregoing description of the application has been presented for purposes of illustration and description, and is not intended to be limiting. Several simple deductions, modifications or substitutions may also be made by a person skilled in the art to which the application pertains, based on the idea of the application.

Claims (12)

1. A polypeptide, characterized in that the amino acid sequence of the polypeptide is a sequence I, a sequence II, a sequence III or a sequence IV,
Sequence one:
FTLIELLVVVAIIAILAAIAIPQFAKYRENAYKASAVADAKNIATAIESYYADTQSFPSSI；
Or alternatively
FTLIELLVVVAIIAILAAIAIPQYAKYRENAYKASAVADAKNIATAIESYYADTQSFPSSI；
Sequence two: the N end of the sequence I is connected with a signal peptide;
Sequence three: the C end of the sequence I is linked with a histidine tag;
sequence four: the N-terminal of sequence one is linked to a signal peptide, while the C-terminal is linked to a histidine tag.
2. The polypeptide of claim 1, wherein the amino acid sequence of the signal peptide is: MDKQRG.
3. The polypeptide of claim 1, wherein the histidine tag comprises from 6 to 8 histidines.
4. An isolated polynucleotide encoding the polypeptide of any one of claims 1-3.
5. A construct comprising the polynucleotide of claim 4.
6. An expression system comprising the construct of claim 5 or the polynucleotide of claim 4 integrated into the genome.
7. A battery comprising the polypeptide of any one of claims 1-3.
8. The battery of claim 7, comprising an upper electrode, a lower electrode, and the polypeptide of any one of claims 1-3 attached between the upper and lower electrodes.
9. The battery of claim 8, wherein the lower electrode comprises graphene.
10. The battery of claim 8 wherein the upper electrode comprises a printed circuit board.
11. The battery of claim 10, wherein the printed circuit board comprises a metal plated printed circuit board.
12. The battery of claim 11, wherein the metal is gold.
CN202410356862.1A 2022-01-07 2022-01-07 Polypeptide, polynucleotide encoding polypeptide, construct, expression system and battery thereof Pending CN118027162A (en)

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