Codon-optimized FAD-glucose dehydrogenase gene and application thereof
Technical Field
The invention relates to the technical field of biological enzyme genetic engineering and biosensing, in particular to a codon-optimized FAD-glucose dehydrogenase gene and application thereof in preparation of a biosensing element.
Background
Accurate and rapid monitoring of glucose is of great significance in the industries of medicine, food and the like, and therefore, the biosensor is an ideal method for monitoring glucose due to the characteristics of high specificity, short analysis time, capability of in-situ monitoring, low manufacturing cost and the like. Despite these advantages, there is still a need for biosensors that overcome the problems caused by enzyme molecule sensing elements for detection.
In biosensors using enzymes as sensing elements, oxidoreductases are the main biorecognition elements, among which Glucose Oxidase (GOX) is the most commonly used enzyme that can selectively oxidize glucose to gluconolactone in the presence of oxygen while producing H2O2. However, the response of such enzyme biosensors is susceptible to the concentration of oxygen in the measurement medium, whereas the use of artificial electron media is due to O2The presence of (a) results in a decrease in the glucose detection signal and thus underestimates the glucose content. Furthermore, if it is based on the determination of H2O2The levels reflect glucose content and often require very high operating potentials (typically in excess of +600mv compared to standard electrodes) versus H2O2Oxidation proceeds and thus many other electroactive compounds present in the biological fluid may also be oxidized causing a false current response. While biosensors based on nicotinamide adenine dinucleotide-dependent glucose dehydrogenase can avoid H2O2Is expected to solve the dependence on H2O2The problem of detecting glucose, but such enzymes require the additional addition of expensive coenzyme NAD to the reaction system+Limiting the use of such enzymes. Therefore, Glucose Dehydrogenase (GDH) having pyrroloquinoline quinone (PQQ) or Flavin Adenine Dinucleotide (FAD) as a coenzyme has been constructed without using O2As electron acceptors or NAD+As suitable candidate enzymes for biosensors of coenzymes, but a part of GDH-PQQ isolated from the outer cytoplasmic membrane requires suitable detergent solubilization, and another part of water-soluble GDH-PQQ has low specificity, which greatly limits the application of such enzymes in biosensors. GDH-FAD (FAD-glucose dehydrogenase gene) has high catalytic efficiency, strong substrate specificity, low redox potential and no O2The characteristic of the electron acceptor makes the construction independent of O2The glucose sensor has the most potential enzyme molecular element.
GDH-FAD of fungal origin (EC 1.1.99.10) was first discovered in Aspergillus oryzae in 1937, and the presence of GDH-FAD was later identified in Aspergillus terreus, Aspergillus niger, and Aspergillus flavus. However, most GDH-FAD is mainly separated and purified from wild strains at present, and is not easy to be recombined and expressed, which results in that GDH-FAD is used as an enzyme electrode biosensor element, and especially in China, only a small amount of research on GDH-FAD recombination and expression is carried out, so that GDH-FAD high-efficiency recombination and expression and application research in the field of sensors have important significance.
Disclosure of Invention
The invention provides a codon-optimized FAD-glucose dehydrogenase gene.
The codon-optimized FAD-glucose dehydrogenase gene provided by the invention is derived from Aspergillus niger An76, the original gene sequence is shown as SEQ ID No.1, and the sequence-optimized gene sequence is shown as SEQ ID No. 2. The original FAD-glucose dehydrogenase gene cannot be successfully expressed in pichia pastoris, can be smoothly expressed in the pichia pastoris through sequence optimization, and can be separated and purified to obtain a purified target protein.
In another aspect, the present invention also provides a recombinant vector comprising the FAD-glucose dehydrogenase gene. The vector of the invention comprises a cloning vector and an expression vector.
In one embodiment, the cloning vector comprises a vector of the pUC series, e.g., pUC18, pUC 19; the expression vector comprises a vector expressed in escherichia coli and a vector expressed in pichia pastoris, and preferably comprises a vector of pET series, such as pET-21 a; more preferably, the vector for expression in pichia is the pPIC9K vector.
On the other hand, the invention also provides a recombinant strain containing the recombinant vector, wherein the strain comprises escherichia coli or pichia pastoris, and preferably, the escherichia coli is escherichia coli DH5 α, and the pichia pastoris is pichia pastoris GS 115.
On the other hand, the invention also provides a method for preparing the recombinant FAD-glucose dehydrogenase, which comprises the steps of transforming the sequence-optimized FAD-glucose dehydrogenase gene into pichia pastoris, constructing the pichia pastoris for recombinant expression of the FAD-glucose dehydrogenase, culturing the recombinant pichia pastoris, and purifying the recombinant FAD-glucose dehydrogenase; preferably, the pichia is pichia GS 115.
In another aspect, the present invention also provides an immobilized FAD-glucose dehydrogenase derived from the FAD-glucose dehydrogenase recombinantly expressed by the above-described method.
On the other hand, the invention also provides application of the codon-optimized FAD-glucose dehydrogenase gene in preparation of FAD-glucose dehydrogenase electrodes.
Further, the FAD-glucose dehydrogenase electrode is a FAD-glucose dehydrogenase electrode obtained by immobilizing FAD-glucose dehydrogenase on a modified glassy carbon electrode.
Furthermore, the modified glassy carbon electrode is a glassy carbon electrode modified by adopting ferrocene and a multi-wall carbon nano tube; preferably, the ferrocene is hydroxymethyl ferrocene, and the multi-walled carbon nanotube is a carboxylated multi-walled carbon nanotube
On the other hand, the invention also provides application of the codon-optimized FAD-glucose dehydrogenase gene in preparation of a glucose biosensor.
According to the method, rare codons in an original FAD-glucose dehydrogenase gene are removed according to the difference of the use frequencies of different codons for coding the same amino acid in pichia pastoris, the occurrence of reverse repetitive sequences is avoided, the secondary structure of RNA is guaranteed to be stabilized, splice sites with intron attributes are removed, the FAD-glucose dehydrogenase gene is optimized, the optimized sequence can be successfully expressed in the pichia pastoris, and the purified target protein can be used for preparing FAD-glucose dehydrogenase electrodes.
Drawings
FIG. 1 SDS-PAGE detects codon optimized FAD-glucose dehydrogenase expressed by Pichia pastoris GS115 induced for six consecutive days. M is a protein standard, 1d, 2d, 3d, 4d, 5d and 6d respectively refer to fermentation crude enzyme liquid obtained by induction culture of Pichia pastoris GS115 for 1 day, 2 days, 3 days, 4 days, 5 days and 6 days.
FIG. 2 shows SDS-PAGE detection of codon-optimized FAD-glucose dehydrogenase expressed recombinantly in Pichia pastoris GS115 using imidazole elution at different concentrations, the left graph is a graph showing elution effects using 10mM imidazole, the right graph is a graph showing elution effects using 20mM imidazole, and lanes 1-10 show elution of codon-optimized FAD-glucose dehydrogenase obtained sequentially from 1mL collection tube at a time when recombinant protein is eluted using imidazole at a specific concentration.
FIG. 3 shows electrochemical characterization of recombinant FAD-glucose dehydrogenase electrode.
FIG. 4 shows the response curve of cyclic voltammetry for detecting glucose at different concentrations by using a recombinant FAD-glucose dehydrogenase electrode.
Detailed description of the preferred embodiments
The present invention will be further described with reference to the following examples, which are intended to be illustrative only and not to be limiting of the invention in any way, and any person skilled in the art can modify the present invention by applying the teachings disclosed above and applying them to equivalent embodiments with equivalent modifications. Any simple modification or equivalent changes made to the following embodiments according to the technical essence of the present invention, without departing from the technical spirit of the present invention, fall within the scope of the present invention.
Example 1 codon optimization and cloning of FAD-glucose dehydrogenase Gene
In this example, FAD-glucose dehydrogenase g5086.t1 gene in Aspergillus niger An76 genome was used as An original gene, and the original gene sequence is shown in SEQ ID No. 1. According to the difference of the using frequency of different codons for coding the same amino acid in pichia pastoris, rare codons in an FAD-glucose dehydrogenase g5086.t1 original gene are removed, the occurrence of a reverse repeat sequence is avoided, the secondary structure of RNA is ensured to be stabilized, splice sites of intron attributes are removed, the FAD-glucose dehydrogenase gene is optimized, and two sequences are optimized according to different strategies and are respectively shown as SEQ ID No.2 and SEQ ID No. 3.
Synthesizing optimized FAD-glucose dehydrogenase gene, and utilizing Nanjing Novozam according to principle of homologous recombination
The product of Ultra One Step Cloning Kit homologous recombines the codon optimized FAD-glucose dehydrogenase gene onto pPIC9K plasmid vector in the form of 10. mu.L (1. mu.L pPIC9K vector linearized with EcoRI and NotI, 2. mu.L PCR fragment of FAD-glucose dehydrogenase gene, 5. mu.
L 2 XClonexpress Mix, 2. mu.L ddH)
2O), sucking, pumping, uniformly mixing, reacting for 15min at 50 ℃, immediately placing on ice, and cooling to obtain a product of homologous recombination of the FAD-glucose dehydrogenase gene and the pPIC9K plasmid vector.
Example 2 transformation of Escherichia coli-enriched plasmid with FAD-glucose dehydrogenase Gene
Respectively mixing an original FAD-glucose dehydrogenase gene (SEQ ID No.1) and a codon-optimized FAD-glucose dehydrogenase gene (SEQ ID No.2 and SEQ ID No.3) and a pPIC9K homologous recombination product with E.coli DH5 α, thermally shocking for 90s, coating the mixture on a 100ug/mL LB agar culture plate with ampicillin resistance, culturing overnight at 37 ℃, selecting a single colony, extracting plasmid electrophoresis detection, storing the plasmid at-20 ℃, carrying out enzyme digestion detection on a target fragment by using EcoRI and NotI, then sending a bacterial suspension to a company for sequencing, and transforming the plasmid with correct sequencing into escherichia coli by the same method to realize plasmid enrichment.
Example 3 transformation of Pichia host bacteria with FAD-glucose dehydrogenase Gene
Inoculating Pichia pastoris GS115 single colony into a test tube containing 5mLYPD liquid culture medium, and culturing at 30 ℃ overnight. Inoculating into a triangular flask containing 50mL of YPD liquid culture medium according to the inoculation amount of 1%, and culturing overnight at 30 ℃ until OD600 is 1.3-1.5; 1500g, centrifuging the culture solution for 5min at 4 ℃, discarding the supernatant, and resuspending the cells by using 50mL ice bath double distilled water; centrifuging the culture solution at 4 ℃ for 5min at 1500g, removing the supernatant, and resuspending the cells by using 25mL ice-bath double distilled water; 1500g, centrifuging the culture solution at 4 ℃ for 5min, discarding the supernatant, and resuspending the cells with 2mL of ice-bath 1M sorbitol solution; 1500g, centrifuging the culture solution at 4 ℃ for 5min, discarding the supernatant, and resuspending the cells with 1mL of ice-bath 1M sorbitol solution to make the volume of the bacterial suspension about 1.5 mL; add 80. mu.L of treated competent cells and 5-20. mu.g of SacI-linearized plasmid containing recombinant FAD-glucose dehydrogenase obtained in example 1-2 into a 1.5mL precooling centrifuge tube, and mix well. Then the mixture was transferred into a transformation cup (type 0.2 cm) pre-iced; ice-bath the transformation cup containing transformation mixture for 5 min; an electric converter (Voltage (V): 2000; Capacitance (muF): 25; Resistance (omega): 200; Cuvette (mm):2) was set according to the electric conversion parameters of the Pichia Biorad, and an electric pulse was started, 1mL of ice-bath 1M sorbitol solution was added to the conversion cup immediately after the pulse, and then the conversion solution was transferred to a new 1.5mL centrifuge tube; standing and culturing for 2h at 30 ℃. mu.L of GS115 transformation solution was aspirated to coat MD plates and cultured at 30 ℃ until transformants appeared. Colony PCR verification is carried out on the monoclone on the transformed MD plate, and the integration of the exogenous gene is ensured.
Example 4 Induction of expression of FAD-glucose dehydrogenase Gene
Inoculating the screened pichia pastoris recombinant into 5mL of BMGY liquid medium, performing shaking culture at 30 ℃ and 250rpm overnight; 500 μ L of overnight culture was transferred to 50mL BMGY liquid medium at 30 ℃, 250rpm, and shake-cultured until OD600 was 2-6 (logarithmic growth phase, approximately 16-18 h); 3000g, centrifuge for 5min, discard the supernatant, resuspend the cells with BMMY broth to OD600 ═ 1.0, final methanol concentration of 1%; 4) placing in a 500mL triangular flask, sealing with 8 layers of sterilized gauze, culturing at 30 deg.C and 250rpm under continuous shaking for 6 days, and sampling 1mL per day; SDS-PAGE detects whether the foreign gene is expressed or not; the results show that the original FAD-glucose dehydrogenase gene (SEQ ID No.1) can not be successfully expressed in Pichia pastoris, the codon-optimized FAD-glucose dehydrogenase gene (SEQ ID No.2) can be successfully expressed in Pichia pastoris (FIG. 1), but the codon-optimized FAD-glucose dehydrogenase gene shown in SEQ ID No.3 can not be successfully expressed in Pichia pastoris; this is probably because the FAD-glucose dehydrogenase gene obtained by this codon optimization method cannot be expressed in pichia pastoris expression due to the influence of factors other than codons, and although the original gene sequence can be optimized according to codon preference, this is only a theoretical possibility, and it is closely related to various factors whether the gene can be successfully expressed in pichia pastoris or not in actual operation.
Example 5 isolation and purification of codon-optimized FAD-glucose dehydrogenase
Mixing the crude enzyme solution with target protein expression detected by SDS-PAGE with filler containing nickel, and rotating and combining for 6h in a refrigerator at 4 ℃. Then, the hetero-protein was eluted with an imidazole solution at a concentration of 5mM, the FAD-glucose dehydrogenase of interest was eluted with 10mM and 20mM imidazole solutions, and the protein of interest was detected by SDS-PAGE (FIG. 2). Ultrafiltering the protein solution eluted with 10mM and 20mM imidazole in disodium hydrogen phosphate-citric acid buffer solution of pH 5.0 at 4900rpm at 4 deg.C until the pH of the buffer solution flowing down is 5.0, stopping the ultrafiltration, and collecting the enzyme solution obtained by ultrafiltration with a 3K ultrafiltration tube. FIG. 2 shows the electrophoretogram of recombinant proteins eluted with imidazole of different concentrations after the successful expression of the codon-optimized FAD-glucose dehydrogenase gene (SEQ ID No.2) in Pichia pastoris, and it can be seen from FIG. 2 that the elution with 10mM and 20mM imidazole can yield purified recombinant FAD-glucose dehydrogenase (0.48 mg/mL).
Example 6 codon optimized immobilization of FAD-glucose dehydrogenase
Pretreating a glassy carbon electrode: polishing glassy carbon electrode firstly, the glassy carbon electrode is cleaned by deionized water and then is sequentially coated with Al with phi of 0.05 mu m and 50 mu m2O3Polishing the polishing powder until the surface of the electrode is mirror-finished, rinsing the electrode with distilled water, ultrasonically cleaning the electrode for 1min, taking out the electrode, naturally drying the electrode, ultrasonically cleaning the electrode for 1min by using nitric acid (V: V is 1:1) and ethanol (V: V is 1:1) sequentially, taking out the electrode, rinsing the electrode with distilled water, and naturally drying the electrode for later use.
Cyclic voltammetric scanning in potassium ferricyanide solution: 0.0329g of potassium ferricyanide and 2.022g of KNO were weighed out3Placing in a beaker, adding 80mL of distilled water, stirring to dissolve, transferring to a 100mL volumetric flask, shaking to constant volume, and making into 1.0 × 10-3mol/L potassium ferricyanide solution (containing 0.2mol/L KNO)3) And scanning the glassy carbon electrode in the prepared potassium ferricyanide solution by using cyclic voltammetry, wherein the potential difference is within 80mV and is close to 64 mV.
Modifying a glassy carbon electrode by using a carboxylated multi-wall carbon nanotube: 0.3g of carboxylated multi-walled carbon nanotubes was weighed and placed in a 50mL beaker, 0.5g of monoethyl 3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 0.5g N-hydroxysuccinimide (NHS) were added and dissolved in 10mL of distilled water, and the carbon nanotubes were activated by standing at room temperature for 6 h. Centrifuging at 13000 Xg for 10min, removing supernatant, taking out the precipitated carbon tube, redissolving with appropriate amount of distilled water, repeating the centrifugation step, adding distilled water to wash the carbon tube until the carbon tube is neutral, and drying for later use. 0.3g of the prepared carbon tube is weighed and dissolved in 100mL of distilled water by ultrasonic wave to prepare a carboxylated multi-walled carbon nanotube solution with the concentration of 3g/L, 7uL of the carboxylated multi-walled carbon nanotube is dripped on the surface of the glassy carbon electrode, and the glassy carbon electrode is naturally aired for later use.
The hydroxymethyl ferrocene-multiwalled carbon nanotube modified glassy carbon electrode comprises the following steps: and (3) in an environment of 4 ℃, soaking the glassy carbon electrode modified by the carboxylated multi-walled carbon nano tube in 1g/L hydroxymethyl ferrocene solution for 24h, and storing in a refrigerator for later use.
Cross-linking FAD-glucose dehydrogenase with cross-linker: 0.5g of chitosan is weighed and dissolved in 100mL of distilled water, and acetic acid is dropwise added while stirring until the chitosan is completely dissolved, so as to prepare a 0.5% chitosan solution. The FAD-glucose dehydrogenase (1mg/mL) purified in example 5, a 0.5% chitosan solution and a 25% glutaraldehyde solution were mixed at equal volume ratios to obtain a cross-linked enzyme solution containing FAD-glucose dehydrogenase, chitosan and glutaraldehyde, and the obtained cross-linked enzyme solution was stored at 4 ℃ for use.
Immobilization of FAD-glucose dehydrogenase: and taking out the glassy carbon electrode modified by the hydroxymethyl ferrocene-multi-walled carbon nano tube, washing with distilled water, and airing. And (3) dropwise adding 7 mu L of prepared crosslinking enzyme solution, airing at 4 ℃, then leaching by using phosphate buffer solution with pH of 7.0 to prepare the FAD-glucose dehydrogenase electrode, and storing at 4 ℃ for later use.
Example 7 electrochemical characterization of FAD-glucose dehydrogenase electrode
A three-electrode system is adopted to detect and explore the electrochemical properties of an enzyme electrode, when the potential range is-0.8-0.8V, the hydroxymethyl ferrocene-multi-walled carbon nanotube modified glassy carbon electrode is scanned and detected in double distilled water and 1mM Glucose in sequence and in FAD-Glucose dehydrogenase-chitosan-hydroxymethyl ferrocene-multi-walled carbon nanotube modified glassy carbon electrode is scanned and detected in 1mMGlucose by using a cyclic voltammetry method, the result shows that when the glassy carbon electrode only modifies the hydroxymethyl ferrocene-multi-walled carbon nanotube without modifying FAD-Glucose dehydrogenase, no redox peak exists in water or Glucose solution, and when the FAD-Glucose dehydrogenase is modified and detected in the Glucose solution, obvious redox peaks appear at the potentials of +0.4 and +0.2, which indicates that the FAD-Glucose dehydrogenase electrode can realize the detection of Glucose, and finally, performing cyclic voltammetry scanning on glucose solutions with different concentrations (3.8mmol.L-1, 5.8mmol.L-1, 6.3mmol.L-1, 6.9mmol.L-1, 7.2mmol.L-1, 8.0mmol.L-1) by using the glassy carbon electrode modified by the FAD-glucose dehydrogenase-chitosan-hydroxymethyl ferrocene-multi-walled carbon nanotube, wherein the result is shown in FIG. 4, and the current value corresponding to the oxidation peak is gradually increased along with the increase of the sugar concentration, which indicates that the prepared FAD-glucose dehydrogenase electrode can be applied to a glucose biosensor as a sensing element to realize the quantitative detection of glucose in a sample.
Sequence listing
<120> codon-optimized FAD-glucose dehydrogenase gene and application thereof
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<170>SIPOSequenceListing 1.0
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<213> Aspergillus niger (Aspergillus niger)
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gcattgaaca acttcaatgt gtctaatgtc atgggctaca gcacggcatt cgggacggag 180
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cgtgccggaa aggcacttgg aggtacaagc acaatcaatg gaatgtccta cactcgggcc 300
gaagacgtgc aaattgacaa ttgggaagtg ctcgggaacg aaggttggaa ctggaagaac 360
ctgtttcaat attataagaa gtcggaaggc ttccaggtgc cgaccaagga ccagatcgct 420
catggtgcca actacaacgc tagctatcat ggtttgaacg gccctctgaa ggttggctgg 480
cctacctcca tgaccaacag cagcgtcttc cctgtccttg aacagacctt cgagaaactg 540
ggcgttcagt acaaccccga ttccgaaggc ggcaagatgg tcggattcac tgttcacccc 600
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tatgaagccc gctcgaacct gaagatcatt tctaatactc gtgcaaacaa ggtcatctgg 720
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acgcaaacca tctacgctga caaggaggtc atcctgtcgg ccggcgcgct tagatcccct 840
gccctgcttg agctgtctgg tatcggaaac cccgacgtcc tcaataagca caatatcccc 900
gtcaaggtca acatcaccac cgtcggggag aacctacagg atcagaccaa caacgctctt 960
tcctgggaag gcgtcgacac cctcactggt ttggcaacct tctccgtcct gccatctgtg 1020
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gttttcgctc cgagcggaca atcgttcgcg gtcgaatact ggcctcttct tcccttctcc 1260
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gatctgatta aggaaagtta ccaccaccac caccaccact aa 1722
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<213> Artificial Sequence (Artificial Sequence)
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gctttgttgg aattgtctgg tattggtaat ccagatgttt tgaacaagca taacattcca 900
gttaaggtca acattactac tgttggtgaa aacttgcagg atcaaactaa caacgctttg 960
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aaccaattgt acggagataa tgttactgct ttggcttctt atgttaagtc ccaattggct 1080
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gaggcttttg aaagacaata tgatttgatt ttcaactccc aagttccata cgctgaggtt 1200
gtttttgccc catctggtca atcttttgca gttgaatatt ggccattgtt gccattttct 1260
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tactttatgt ttagacagga cgctgatgct caaattactg ttgctcagta cattagaaag 1380
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<210>3
<211>1722
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
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ggaagtcctg cccactacga ttttgtgatt gttggtggtg gcacttccgg acttgtcgtt 60
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gctcttaaca actttaatgt gtcaaatgta atgggctact ctactgcttt cggaactgaa 180
gtggattggg cataccaaac agaaaaccaa acttacgccg gtggtctaca acagactatt 240
cgtgctggaa aggccttggg aggaacctca actatcaatg gtatgtctta tactagagct 300
gaggacgtgc aaattgataa ttgggaagtc ttgggaaatg aaggatggaa ctggaaaaac 360
ttattccagt actacaaaaa atctgaagga tttcaggtac ctaccaagga tcagattgct 420
cacggtgcca actataacgc ttcatatcat ggactaaatg gtccattgaa ggtcggttgg 480
cctaccagta tgactaactc aagtgtgttc ccagttctag aacaaacatt cgaaaaactt 540
ggagtgcagt acaacccaga cagtgaagga ggtaaaatgg ttggattcac tgtccatcct 600
gacactttgg atcgtgagat gaatgtcaga gaggatgccg caagagccta ttactggcct 660
tacgaagcta gatcaaattt aaaaattatt tccaacacga gagctaacaa ggtcatctgg 720
gcagatacga cccaaggaga ggctgtggct gtcggtattg aagtcacaaa tgcttatgga 780
acacaaacta tatatgctga caaagaagtt atcctgtccg caggtgcttt gcgatctcca 840
gccttgttgg aattatccgg cattggtaat cctgacgttt taaacaagca caatatccct 900
gttaaggtga acataactac cgttggcgag aatttgcaag atcagactaa caacgcactt 960
tcatgggagg gtgttgatac attgactgga ttggcaacat tcagtgtact accctctgtt 1020
aatcaattgt acggtgataa tgttactgcc ctggcctcat atgtgaaatc tcaattggca 1080
tcatatgcta aaacggtagc cgacgcaagt aacggtgccg taaaagaagc taaccttatc 1140
gaagctttcg agagacaata cgatttaatc tttaacagtc aggttccata tgcagaggtc 1200
gtcttcgcac catcaggtca gtcattcgct gttgaatatt ggcccttatt gccattttca 1260
agaggaagtg tccacattca atctgcaaac gcctccgatt accctgctat aaaccctaat 1320
tactttatgt tccgtcagga tgcagacgct caaattactg ttgcccaata tataagaaaa 1380
gctctaggta cgacgccttt aaactcttta gttggtgagg aagtctcacc atctctggaa 1440
gttctgcctg caaacgctag ttcatctacc tggacaaagt gggtccagga gaactacaga 1500
accaactacc atccagtggg aaccaattct atgctgccaa gagaaaaagg aggagtagtc 1560
tcccctgagc ttcgtgtcta tggtaccaaa aacttgagag ttgttgatgc atctgtcttg 1620
ccattccagc tgtgtggcca ccttacctct actttgtacg ctgttgccga gagagcttca 1680
gacttgatca aggaatccta tcatcaccac caccatcatt aa 1722