CN114409746B

CN114409746B - Polypeptide and polynucleotide and battery thereof

Info

Publication number: CN114409746B
Application number: CN202210015980.7A
Authority: CN
Inventors: 王博祥; 叶启当; 刘家骏; 刘锡蓓
Original assignee: Shenzhen Lingspider Technology Co ltd
Current assignee: Shenzhen Lingspider Technology Co ltd
Priority date: 2022-01-07
Filing date: 2022-01-07
Publication date: 2024-02-02
Anticipated expiration: 2042-01-07
Also published as: CN114409746A

Abstract

A polypeptide, its polynucleotide and cell, from N-terminal to C-terminal, the 1 st, 24 th and 27 th positions of said polypeptide are aromatic amino acids. The invention 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 and polynucleotide and battery thereof

Technical Field

The invention relates to the field of batteries, in particular to a polypeptide, a polynucleotide thereof 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

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 the polypeptide of the first aspect.

According to a third aspect, in an embodiment, there is provided a construct comprising a polynucleotide of the second aspect.

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.

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 of the embodiment, the invention 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 functions.

Drawings

FIG. 1 shows the amino acid sequence of each pilus.

Fig. 2 is a schematic diagram of an E-PN battery 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 film (E-pili film); 3. a lower electrode (Bott om Electrode); 4. glass substrate (Glass Slide).

Detailed Description

The invention 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, some operations associated with the present application have not been shown or described in the specification to avoid obscuring the core portions of the present application, and may not be necessary for a person skilled in the art to describe in detail the relevant operations based on the description herein and the 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 terms "coupled" and "connected," as used herein, are intended to encompass 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 a plurality of (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 amino group (-NH) that is present in the polypeptide chain as a protrusion ₂ ) 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 (F) and tryptophan (W).

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 (F), and 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 N-terminal to C-terminal, the 24 th position of the polypeptide is any one of Tyrosine (Y) and 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-terminal to C-terminal, the 32 nd position of the polypeptide is any one of Tyrosine (Y) and 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, 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 N-terminal to C-terminal, the 51 st position of the polypeptide is any one of Tyrosine (Y) and 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 N-terminal to C-terminal, the 57 th position of the polypeptide is any one of Tyrosine (Y) and 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 invention.

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 invention 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 invention 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 invention, the host cell may be a eukaryotic cell and/or a prokaryotic cell.

In one embodiment, the cell includes an upper electrode, a lower electrode, and a polypeptide attached between the upper and lower electrodes.

In an embodiment, the lower electrode comprises graphene.

In an 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 invention 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 sequence of D.t.F.s.p.a.g.u.d.a.c.n. is selected as raw material, and a series of recombination modifications such as peptide guide and histidine tag fusion, specific point mutation, etc. 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.

Example 1

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, the amino acid sequences of the pili are shown, the ppdD secretion signal peptide sequence is yellow, the characteristic sequence is green (consensus sequence), the unique sequence of each pili is black, the aromatic amino acids are red, and the 6xHistags sequence is blue. 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 to its N-terminus a secretion signal peptide (yellow letter label) of E.coli (E.coli) ppdD, helping the e-PN protein to secrete out of the cell membrane and form through the 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

P.a.pilA(Modified)

F.sinusarabici(F.s.pilA)

D.alkaliphilus61(D.a.pilA)

Calditerrivibrionitroreducens61(C.n.pilA)

Calditerrivibrionitroreducens61mut1(C.n.1pilA)

Calditerrivibrionitroreducens61mut2(C.n.2pilA)

Desulfuromonasthiophila(D.tpilA)

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 BsaI Golden Gate assembly system.

The expression plasmid was transferred into E.coli BL21, and the monoclonal cells were placed in a 14mL test tube, 5mL of LB solution 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 the 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 closed with a sealing film (parfilm), and incubated overnight at 37 ℃. Cells were scraped from the plates with 300 μ L M liquid medium and resuspended in 6mL of M9 medium (available from aatin). 0.5% by volume of glycerol, 0.5mM IPTG, 2mM MgSO were added to the M9 solid medium ₄ 、0.1mM CaCl ₂ 0.4% (mass volume percent) glucose and kanamycin (50 μg/mL), each concentration being the final concentration of the corresponding component in M9 medium. Sealed with parfilm and incubated at 30℃for 48 hours.

Cells were scraped from M9 medium, 1.2mL of M9 medium (600. Mu.L once) was added to each dish, about 10mL of cell fluid was extracted, centrifuged at 7500rpm at 4℃for 15 minutes, the cells were collected and the supernatant was discarded. Concentrated cells were resuspended in 20mL 150mm 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 (approximately 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, was collected, and Triton X100 reagent was added to a final concentration of 6mM, and stirred at 30℃at a speed of 100-150 rpm until Triton X100 was dissolved.

The bacteria Mao Rongye were diluted with twice the volume of distilled water, triton x100 was diluted to a final concentration of 2mM, and then added to a centrifugal filtration device (100 kDa Milopore) 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 (carbon nanotube layer) 3 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 (excess moisture was removed) on a metal bath at 85 ℃. 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; middle part 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.

The current and voltage (15 x 15mm voltage current) were measured using a multimeter (FLUKE 8808A 5-1/2digit 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 invention 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 invention provides the amino acid sequence of each recombinant protein, as shown in FIG. 1.

In one embodiment, the present invention provides an electrode design as shown in fig. 3 and 4.

In one embodiment, the invention 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 invention, 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 invention.

In one embodiment, other tag proteins can be added based on the amino acids of the present invention, 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 invention 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 invention pertains, based on the idea of the invention.

SEQUENCE LISTING

<110> Shenzhen City Ling-spider science and technology Co., ltd

<120> a polypeptide, polynucleotide and battery

<130> 21I32839

<160> 9

<170> PatentIn version 3.3

<210> 1

<211> 73

<212> PRT

<213> artificial sequence

<400> 1

Met Asp Lys Gln Arg Gly Phe Thr Leu Ile Glu Leu Leu Ile Val Val

1 5 10 15

Ala Ile Ile Gly Ile Leu Ala Ala Ile Ala Ile Pro Gln Phe Ser Ala

20 25 30

Tyr Arg Val Lys Ala Tyr Asn Ser Ala Ala Ser Ser Asp Leu Arg Asn

35 40 45

Leu Lys Thr Ala Leu Glu Ser Ala Phe Ala Asp Asp Gln Thr Tyr Pro

50 55 60

Pro Glu Ser His His His His His His

65 70

<210> 2

<211> 73

<212> PRT

<213> artificial sequence

<400> 2

Met Asp Lys Gln Arg Gly Phe Thr Leu Ile Glu Leu Met Ile Val Val

1 5 10 15

Ala Ile Ile Gly Ile Leu Ala Ala Ile Ala Ile Pro Gln Tyr Gln Asn

20 25 30

Tyr Val Ala Arg Ser Tyr Gly Ala Ser Ala Leu Ala Thr Ile Asn Pro

35 40 45

Leu Lys Thr Thr Val Glu Glu Ser Phe Ser Arg Gly Ile Ala Tyr Ser

50 55 60

Lys Ile Lys His His His His His His

65 70

<210> 3

<211> 71

<212> PRT

<213> artificial sequence

<400> 3

Met Asp Lys Gln Arg Gly Phe Thr Leu Ile Glu Leu Leu Val Val Val

1 5 10 15

Ala Ile Ile Gly Ile Leu Ala Ala Ile Ala Ile Pro Gln Phe Ala Lys

20 25 30

Tyr Arg Gln Asn Ala Phe Asn Ser Ala Ala Gln Ser Asp Val Arg Asn

35 40 45

Ser Arg Ser Asp Val Glu Ser Phe Tyr Ala Glu Asn Phe His Tyr Pro

50 55 60

Tyr His His His His His His

65 70

<210> 4

<211> 73

<212> PRT

<213> artificial sequence

<400> 4

Met Asp Lys Gln Arg Gly Phe Thr Leu Val Glu Leu Met Ile Val Val

1 5 10 15

Ala Ile Ile Gly Ile Leu Ala Ala Val Ala Ile Pro Gln Phe Ala Gln

20 25 30

Tyr Arg Ile Arg Gly Phe Asn Ser Ser Ala Leu Ser Asp Val Arg Asn

35 40 45

Leu Thr Thr Ala Gln Glu Ala Phe Phe Ala Asp Trp Leu Arg Tyr Ala

50 55 60

Val Thr His His His His His His His

65 70

<210> 5

<211> 73

<212> PRT

<213> artificial sequence

<400> 5

Met Asp Lys Gln Arg Gly Phe Thr Leu Ile Glu Leu Leu Val Val Val

1 5 10 15

Ala Ile Ile Ala Ile Leu Ala Ala Ile Ala Ile Pro Gln Phe Ala Lys

20 25 30

Tyr Arg Glu Asn Ala Ala Lys Ala Ser Ala Val Ala Asp Ala Lys Asn

35 40 45

Ile Ala Thr Ala Ile Glu Ser Tyr Tyr Ala Asp Thr Gln Ser Phe Pro

50 55 60

Ser Ser Ile His His His His His His

65 70

<210> 6

<211> 73

<212> PRT

<213> artificial sequence

<400> 6

Met Asp Lys Gln Arg Gly Phe Thr Leu Ile Glu Leu Leu Val Val Val

1 5 10 15

Ala Ile Ile Ala Ile Leu Ala Ala Ile Ala Ile Pro Gln Phe Ala Lys

20 25 30

Tyr Arg Glu Asn Ala Tyr Lys Ala Ser Ala Val Ala Asp Ala Lys Asn

35 40 45

Ile Ala Thr Ala Ile Glu Ser Tyr Tyr Ala Asp Thr Gln Ser Phe Pro

50 55 60

Ser Ser Ile His His His His His His

65 70

<210> 7

<211> 73

<212> PRT

<213> artificial sequence

<400> 7

Met Asp Lys Gln Arg Gly Phe Thr Leu Ile Glu Leu Leu Val Val Val

1 5 10 15

Ala Ile Ile Ala Ile Leu Ala Ala Ile Ala Ile Pro Gln Tyr Ala Lys

20 25 30

Tyr Arg Glu Asn Ala Tyr Lys Ala Ser Ala Val Ala Asp Ala Lys Asn

35 40 45

Ile Ala Thr Ala Ile Glu Ser Tyr Tyr Ala Asp Thr Gln Ser Phe Pro

50 55 60

Ser Ser Ile His His His His His His

65 70

<210> 8

<211> 71

<212> PRT

<213> artificial sequence

<400> 8

Met Asp Lys Gln Arg Gly Phe Thr Leu Ile Glu Leu Leu Ile Val Val

1 5 10 15

Ala Ile Ile Gly Ile Leu Ala Ala Ile Ala Ile Pro Gln Phe Ala Ala

20 25 30

Tyr Arg Gln Lys Ala Phe Asn Ser Ala Ala Ile Ser Asp Ile Arg Ser

35 40 45

Thr Lys Thr Asn Leu Glu Ala Tyr Tyr Thr Asp Asn Asn Asn Tyr Pro

50 55 60

Tyr His His His His His His

65 70

<210> 9

<211> 13543

<212> DNA

<213> artificial sequence

<400> 9

attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt 60

gcgccattcg atggcgcgcc gcttcgtcag gccacatagc tttcttgttc tgatcggaac 120

gatcgttggc tgtgttgaca attaatcatc ggctcgtata atgtgtggaa ttgtgagcgc 180

tcacaattag ctgtcaccgg atgtgctttc cggtctgatg agtccgtgag gacgaaacag 240

cctctacaaa taattttgtt taagaaagag gagaaatacc atatgtgaga ccacggatca 300

atggtctcac accaccacca ccaccactaa tctgagctca ggaaggagcg gcaatgaata 360

ttccacagct cactgccctg tgtctgcgtt atcagggagt cttgctggat gccagcgaag 420

aggtggttca tgttgcggta gtcgatgcac cttcgcatga gctactggac gcattgcatt 480

tcgctaccac caaacgtatt gagatcacct gctggacgcg ccaacaaatg gaaggtcacg 540

ccagtcgcac acaacagaca ttgcccgtag ctgttcagga gaagcatcag cccaaagcag 600

agttgctggc tcgaacgtta caatctgcgc tggaacaacg cgcgtctgat attcatatcg 660

aaccagcgga caatgcctac cgcatccgct tgcgtatcga cggcgtattg catcctttac 720

cggatgtttc accggatgcc ggagtcgcat taaccgccag attaaaagtg ctgggaaacc 780

tggatattgc ggaacatcgc ctgccgcagg acgggcaatt cactgtcgaa ctggcaggaa 840

acgccgtctc atttcgtatt gcgaccttac catgtcgggg tggtgaaaag gtggtattaa 900

ggttgttaca gcaggtgagt caggcactgg atgtcaacac gcttggaatg cagccgttac 960

aactggcgga ctttgctcat gccttgcaac aaccacaggg actggtgctg gtaactggcc 1020

ctaccggcag cggcaaaacg gtcacgcttt atagtgccct gcaaacgctg aataccgctg 1080

acattaatat ttgtagcgtc gaagatccgg ttgagatccc catagccgga ctaaaccaga 1140

cgcaaatcca tccgcgtgcc gggctcacct ttcagggcgt tttgcgtgcg ttattgcgcc 1200

aggatcctga cgtcatcatg atcggagaga tccgcgatgg cgaaacagca gagatcgcta 1260

ttaaagcggc gcaaactggt cacctggtgt tgtctaccct acacactaat tccacctgcg 1320

aaacgctggt acgtttacag caaatgggag tcgcccgctg gatgctctca tcagcgctta 1380

cgctggtaat agcccagcgt ctggtacgta aactttgccc acattgtcgc cagcagcaag 1440

gggagcccat ccatattcca gacaatgtat ggccgtcgcc gctgccccac tggcaggcac 1500

ccggttgtgt acattgctac cacggttttt atggtcgtac ggccttattt gaagttctgc 1560

ccataacgcc ggtcattcgt cagcttattt ccgctaatac cgacgttgaa tcgctggaaa 1620

cgcacgcacg acaggcgggt atgcgtacgc tttttgaaaa cggctgcctg gccgtggagc 1680

aaggcttaac cacctttgaa gagttaatcc gcgtactggg gatgccgcat ggcgagtaag 1740

caactctggc gctggcatgg cataaccggc gacggcaatg cgcaagatgg gatgctatgg 1800

gcagagagcc gtgctttgct gctcatggca ctacagcaac agatggttac cccacttagc 1860

ctgaagcgaa tcgccatcaa ttctgcgcag tggcgaggag ataaaagcgc ggaagtcatt 1920

catcaactgg cgacgctact caaagccggg ttaacgcttt ctgaagggct ggcactgctg 1980

gcggaacagc atcccagtaa gcaatggcaa gcgttgctgc aatcgctggc gcacgatctc 2040

gaacagggca ttgctttttc caatgcctta ttaccctggt cagaggtatt tccgccactc 2100

tatcaggcga tgatccgcac gggtgaactg accggtaagc tggatgaatg ctgctttgaa 2160

ctggcgcgtc agcaaaaagc ccagcgtcag ttgaccgaca aagtgaaatc agcgttacgt 2220

tatcccatca tcattttagc gatggcaatc atggtggttg tggcaatgct gcattttgtt 2280

ctgccggagt ttgccgctat ctataagacc ttcaacaccc cactaccggc actaacgcag 2340

gggatcatga cgctggcaga ctttagtggc gaatggagct ggctgctggt gttgttcggc 2400

tttctgctgg cgatagccaa taagttgctg atgcgccgac cgacctggct tatagcgcgg 2460

cagaaattgc tgttacgcat cccgattatg ggttcactga tgcggggaca aaaactcacg 2520

cagattttta cgattctggc gctgacacaa agtgcaggca ttactttttt gcagggcgta 2580

gagagcgtca gagaaacaat gcgctgcccg tactgggtgc aacttctgac acaaatccag 2640

cacgatatca gtaacggtca tcccatctgg ctggcgctaa aaaatgccgg ggagtttagc 2700

ccgctctgtt tgcaattagt gagaacagga gaggcatccg gctcgctgga cctcatgtta 2760

gacaacctcg cccatcatca tcgggataac acaatggcgc tggcggataa cctcgcagcc 2820

ttactggaac cggcgttgct gatcataacg ggaggaatta tcggtacgct ggtggtggcg 2880

atgtatctgc caattttcca tttaggcgat gcgatgagtg ggatgggata atctagaagg 2940

ccgtcagagt gacgggtgat aaggagatca tcacaatggc atttaagatc tggcaaattg 3000

gtttgcattt acaacagcaa gaagcggtag cggttgcgat cgtacggggc gcaaaagaat 3060

gctttttgca acgctggtgg cggttgccgc tggagaacga cattatcaaa gatgggcgga 3120

ttgttgatgc gcagcagctg gctaaaacgt tgttaccttg gagtcgcgaa ctgccgcagc 3180

gtcatcacat tatgttggcg tttcccgcca gtcgcacatt acagcggtca tttccgcgcc 3240

cgtcgatgtc ccttggtgag cgggagcaaa cggcctggct gtcagggacg atggcccgcg 3300

agctggatat ggatccggac tccctgcgct tcgattatag cgaagactca ctcagccccg 3360

cttataacgt gactgccgcg caaagcaaag agctggcaac gctgcttacg ctggcagaaa 3420

ggttgcgtgt tcatgttagt gcgatcaccc cggatgccag tgcattacag cgattcctgc 3480

cttttttacc ttctcatcag caatgtctgg cctggcgtga taacgaacag tggctgtggg 3540

cgacacgcta tcgctggggg cgcaaactgg cggtagggat gactagcgcg aaggagctgg 3600

cggcagcgtt atccgttgat cccgagagcg tcgcgatatg tggcgaaggc ggatttgatc 3660

cctgggaggc cgtttctgtt cgtcagccgc cgctaccgcc gagcggtgga gactttgcca 3720

tcgcgctggg gctggcgctt gggaaggcgt actgatgaac ccgccaatta attttttgcc 3780

ctggcgacag caacgccgga ccgcttttct gcgtttctgg ttgctgatgt tcgttgcgcc 3840

tctgctgctg gccgtcggga taacgctaat actgcgtctg acaggcagcg ccgaagctcg 3900

catagacgcc gttttgcttc aggcggaaca acaactcgcc cgcagcttac agataacgaa 3960

accacgtttg ctggagcagc aacaattgcg tgagcagcgt tctcaaaggc agcgccagcg 4020

acaatttacc cgcgactggc aatctgcgct ggaagcactg gcggctcttt tacctgaaca 4080

cgcctggctg acaacgataa gctggcagca gggaacgctg gagatcaagg ggcttacaac 4140

aagcattacc gcgttaaacg cactagaaac gtcacttcgc caggatgctt cttttcatct 4200

caatcagcgg ggggccacgc agcaggatgc gcagggacgc tggcaatttg agtatcagtt 4260

aacaaggaag gttagcgatg aacatgttct ttgactggtg gttcgccaca tcaccccgcc 4320

tccgccagtt ttgctgggca gtctggttgc tgatgttagt tacgctcatt tttctgtcat 4380

cgacacacca tgaagagcgc gacgcattaa ttcgactacg ggcaagtcat caccagcagt 4440

gggccgcact gtatcgcctg gtagacacca ctcccttcag cgaggaaaaa acgctgccct 4500

tttcgccact ggattttcag ttatccggcg cgcaactggt ttcctggcat ccatccgcgc 4560

agggaggcga gttggcgttg aaaacgctgt gggaagcagt gccgtcggca tttacacggc 4620

tggcagagcg caacgtcagc gtgagccgtt tttcgttaag cgtggaaggt gatgatcttt 4680

tgttcacgct acaactggag acgccgcatg agggttaaac gctggttgtt ggcaggtatt 4740

gcattgtgcc ttttaaccgg tatgcgtgac ccttttaaac cgccggaaga tctatgccgg 4800

attagcgaac ttagccagtg gcgctatcag gggatggtag ggcgaggcga gcgcatcatc 4860

ggtgtaataa aagacgggca aaagaaatgg cgacgggtgc agcaaaacga tgtgctggaa 4920

aacggctgga caattttaca gctgacgcca gacgtactaa cgctgggtac cgggacaaac 4980

tgcgaaccgc cacaatggtt gtggcaacgg caaggagata caaatgaagc aatggatagc 5040

cgcactactg ttgatgctga tacccggcgt acaggcggca aagccgcaaa aagtgacgct 5100

gatggtggat gacgttccgg tagctcaggt gttgcaggcg ctggctgaac aggagaagtt 5160

gaacctggtc gtgtcgccag acgtcagcgg tacggtgtcg ttacatctaa cagatgttcc 5220

ctggaagcag gcactacaaa ctgtagtgaa aagcgccgga ctgataacgc ggcaggaagg 5280

caacattctc tcagtgcatt ccattgcctg gcagaataac aatatcgccc gccaggaggc 5340

ggagcaggcg cgggcgcagg caaatctgcc gctggaaaat cgcagtataa ccctgcaata 5400

cgccgacgcg ggagaactgg cgaaagcggg ggagaagcta ctgagtgcca aagggagtat 5460

gaccgtcgat aaacgcacca atcgcctttt gctacgagat aacaaaacgg cgttaagcgc 5520

gcttgaacag tgggtagcgc aaatggatct gccggtcggg caggttgagc tgtcggcgca 5580

tattgtcacc attaatgaaa aaagtttgcg tgagttaggc gtgaaatgga cgctggccga 5640

tgcgcaacac gctggtggcg ttgggcaagt caccacgctt ggtagcgacc tctccgtagc 5700

gacggcgaca acgcatgtcg gttttaacat tgggcgcatc aacggacgct tgctggatct 5760

tgagctttcc gcgctcgaac aaaaacagca gctggatatt atcgccagtc cgcgtctgct 5820

ggcctcacat cttcagcctg ccagcattaa acaggggagc gaaattccat atcaggtttc 5880

cagcggggaa agtggcgcga cgtcggtgga atttaaagag gccgtcctgg ggatggaggt 5940

cacgcccacg gtgttacaaa aaggtcgcat ccggctgaaa ttacacatca gccagaacgt 6000

tccggggcag gtgctacagc aggccgatgg cgaagtgctg gcgattgata agcaggagat 6060

cgaaacgcag gtcgaggtca aaagcggaga aacgttggcg ctgggcggca tttttacccg 6120

taaaaataaa tcgggtcagg atagcgtacc gttgcttggc gacattccct ggttcgggca 6180

attatttcgt catgacggaa aagaagatga acgacgcgag ttagtggtgt ttatcacgcc 6240

acgactggtt tccagtgagt aagcttagga gactgccggc atgaaaacac aacgtggtta 6300

tacgctgatt gaaacgctgg tcgcgatgct gattttggtc atgctaagcg caagtggact 6360

ctatggctgg caatactggc agcagtcgca acggctatgg caaaccgcca gccaggcgcg 6420

ggactatttg ctctatttac gtgaagatgc caactggcat aaccgcgacc acagtatcag 6480

cgttatcagg gaggggacgt tatggtgcct tgtgagttcc gctgctgggg ccaatacctg 6540

tcatggcagt tcaccattgg tctttgtgcc acgctggccc gaagtcgaaa tgagcgacct 6600

gacaccttcg cttgctttct ttggcctgcg caataccgca tgggccgggc atattcgctt 6660

caaaaactca acgggcgagt ggtggctggt ggtttcgccg tggggaagac tccggctttg 6720

tcagcaagga gaaacagaag gatgcctgta aaagagcaag gtttttctct gctggaagtg 6780

ttgattgcta tggcgatcag tagcgtattg ttgctggggg ctgcacgctt tctgcctgcg 6840

ttacagcgtg aaagtttaac gagcacccgt aagctggcgc tggaagatga aatctggctg 6900

cgggtattta ccgtcgcgaa gcatctccag agggcgggtt attgtcatgg cagctgtacc 6960

ggcgaagggc tggaaattgt cggacagggt gactgtatca ttgtgcagtg ggatgcgaac 7020

agtaacggta tctgggatcg cgaaccggta aaagagtctg accagattgg atttcgtctg 7080

aaggagcatg tgctggaaac gctacgcggt gcgacatcct gtgaaggtaa gggctgggat 7140

aaagtcacta atccggatgc catcattatc gacacttttc aggtcgtacg tcaggatgtc 7200

agcggcttct cgccggtgtt gacggttaat atgcgtgctg ccagtaagtc tgaaccgcaa 7260

accgtggtgg atgccagcta tagcgtgaca ggattcaacc tgtgaaccgc gaaaaaggtg 7320

tttcgtcact ggctctggtt ctgatgctgc tggttttggg tagcttgcta ttgcaaggaa 7380

tgagtcagca ggatcgcagt tttgcctctc gcgtgagcat ggaaagtcag tcattgcgcc 7440

gccaggccat cgttcagtcg gcgctggcgt gggggaaaat gcactcctgg cagacgcaga 7500

ccgcagttca gtgctcgcag tacgctggaa ccgatgccca ggtttgtttg cgtttactgg 7560

cagataatga agccttattg attgccggtt atgaaggcgt ttcgttgtgg cgaacaggcg 7620

aagtcattga tggaaacatt gttttttcgc cacgcggctg gagcgatttt tgtccgctga 7680

aagagagggc gttatgtcag cttccctgaa gaatcaacaa ggctttagcc tgccggaggt 7740

aatggttgcg atggtgctga tggtgatgat tgtcactgcg ttatcgggta tccagcgaac 7800

attaatgaac agtctcgcca gcagaaacca gtaccaacag ctctggcggc atggttggca 7860

gcaaacgcaa ctgcgcgcga tttcgccacc tgccaactgg caggtcaacc gaatgcagac 7920

atcgcaggcg ggatgtgtca gcatcagcgt tacgctagtt tcacccgggg gcagagaagg 7980

cgagatgacc cgcctgcatt gtccgaatcg tcagtagcag ggagcaacaa taatgacaat 8040

gctactgccg ctcttcattc tggttggttt tattgcagat tattttgtta atgccatcgc 8100

ctatcacctc tcgccgttgg aggataaaac ggcgttaacg tttcgccagg tattggttca 8160

tttcaggcaa aaaaaatatg cctggcatga tacagtgccc ctgatacttt gtgttgctgc 8220

tgctatcgcc tgcgccctgg cacctttcac gcccatcgtg actggcgcac tctttctcta 8280

tttctgtttc gtactcacgc tcagtgttat tgattttcgc actcagctcc tgcccgacaa 8340

actcacctta ccgctgctct ggcttggctt ggtatttaat gcgcagtatg gattaattga 8400

tttacatgat gcggtttacg gcgcggtagc gggctatggg gtgctgtggt gtgtttactg 8460

gggcgtctgg ttagtttgtc acaaagaggg attgggctac ggtgatttca agctactggc 8520

cgctgcaggc gcatggtgtg gctggcaaac gttgccaatg atactgctga ttgcctcgct 8580

gggtggcatt ggttacgcca tcgtttcaca acttctgcaa cgccggacta taaccactat 8640

cgcattcggc ccgtggcttg cgctcggcag catgataaac ctggggtatc tggcctggat 8700

ctcttattaa ataagagagc agaggttgat aagttttctc caggcatcaa ataaaacgaa 8760

aggctcagtc gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctct 8820

actagagtca cactggctca ccttcgggtg ggcctttctg cgtttatata ctagagctag 8880

cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta 8940

tatccggatt ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt 9000

ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt 9060

cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct 9120

ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg 9180

tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga 9240

gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc 9300

ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga 9360

gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta caatttcagg 9420

tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc 9480

aaatatgtat ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact 9540

gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg 9600

aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga 9660

ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat 9720

caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca 9780

tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat 9840

caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt 9900

taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat 9960

caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg 10020

ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg 10080

gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg 10140

caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc 10200

gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat 10260

cagcatccat gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc 10320

tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgaccaaa 10380

atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga 10440

tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg 10500

ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact 10560

ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac 10620

cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg 10680

gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg 10740

gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga 10800

acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc 10860

gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg 10920

agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc 10980

tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc 11040

agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt 11100

cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc 11160

gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc 11220

ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc 11280

tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg 11340

tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc 11400

ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg 11460

tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc 11520

gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt 11580

ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc 11640

ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc 11700

gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact 11760

ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac 11820

tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca 11880

gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag 11940

actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt 12000

gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag 12060

gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg 12120

ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt 12180

gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat 12240

cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg 12300

tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg 12360

cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg 12420

tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc 12480

gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt 12540

gcgtattggg cgccagggtg gtttttcttt tcaccagtga gactggcaac agctgattgc 12600

ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca 12660

ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagcta tcttcggtat 12720

cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc 12780

gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct 12840

cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt 12900

ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac 12960

gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga 13020

ccagatgctc cacgcccagt cgcgtaccgt cctcatggga gaaaataata ctgttgatgg 13080

gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag 13140

caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga 13200

gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca 13260

ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg 13320

cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt 13380

gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc 13440

gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga 13500

caccggcata ctctgcgaca tcgtataacg ttactggttt cat 13543

Claims

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:

FTLIELLIVVAIIGILAAIAIPQFAAYRQKAFNSAAISDIRSTKTNLEAYYTDNNNYPY；

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 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. The construct of claim 5, wherein the construct is the nucleotide sequence set forth in SEQ ID No. 10.

7. An expression system comprising a construct according to claim 5 or 6, or a polynucleotide according to claim 4 integrated into the genome as an exogenous source.

8. A battery comprising the polypeptide of any one of claims 1-3.

9. The battery of claim 8, comprising an upper electrode, a lower electrode, and the polypeptide of any one of claims 1-3 attached between the upper electrode and the lower electrode.

10. The battery of claim 9, wherein the lower electrode comprises graphene.

11. The battery of claim 9 wherein the upper electrode comprises a printed circuit board.

12. The battery of claim 11, wherein the printed circuit board comprises a metal plated printed circuit board.

13. The battery of claim 12, wherein the metal is gold.