Drug-loaded DNA origami raft and preparation method and application thereof
阅读说明：本技术 负载药物的dna折纸筏及其制备方法、应用 (Drug-loaded DNA origami raft and preparation method and application thereof ) 是由 郑晓峰 李威 于 2021-06-17 设计创作，主要内容包括：本发明涉及生物医药领域,特别涉及负载药物的DNA折纸筏及其制备方法、应用。本发明利用DNA纳米技术,将IFN-α精确定量的负载于DNA折纸筏上,使载药量高度可控,并且使IFN-α缓慢释放。(The invention relates to the field of biomedicine, in particular to a DNA origami raft loaded with a medicine, and a preparation method and application thereof. The invention uses DNA nanometer technology to load IFN-alpha on DNA origami raft with accurate quantity, to control the drug-loading quantity and release IFN-alpha slowly.)
1. The DNA paper folding raft loaded with the medicine is characterized by comprising the medicine to be loaded modified by DNA and the DNA paper folding raft;
the DNA modified drug to be loaded comprises IFN-alpha, sulfhydryl modified DNA (SH-DNA) and the drug to be loaded; the IFN-alpha and the sulfhydryl modified DNA nucleotide sequence complementary pairing;
the DNA origami raft comprises M13mp 18DNA single-chain, staple chain and capture chain;
the staple chain is complementarily paired with the nucleotide sequence of the M13mp 18DNA single chain;
the capture strand is complementary paired with the nucleotide sequence of the single-stranded IFN-alpha modifying DNA.
2. The drug-loaded DNA origami raft of claim 1, wherein the nucleotide sequence of the staple chain is set forth in SEQ ID nos. 1 to 216.
3. The drug-loaded DNA origami raft of claim 1 or 2, wherein the nucleotide sequence of the capture strand is as set forth in SEQ ID nos. 217 to 258.
4. The drug-loaded DNA origami raft of any one of claims 1 to 3, wherein the DNA-modified drug to be loaded is present in a molar ratio to the DNA origami raft of not less than 5: 1.
5. The drug-loaded DNA origami raft of any one of claims 1 to 4, wherein the molar ratio of thiol-modified DNA to IFN- α is greater than 50: 1;
the molar ratio of the staple chain to the M13mp 18DNA single chain is more than 50: 1;
the molar ratio of the capture strand to the M13mp 18DNA single strand is greater than 100: 1.
6. The drug-loaded DNA origami raft of any one of claims 1 to 5, wherein the drug to be loaded comprises IFN- α.
7. Use of a drug-loaded DNA origami raft as claimed in any one of claims 1 to 6 in the preparation of a slow release drug.
8. The method of preparing a drug-loaded DNA origami raft as in any one of claims 1 to 6, comprising the steps of:
step 1, preparing a DNA modified drug to be loaded:
dissolving the IFN-alpha in a solvent;
taking IFN-alpha and 50 times of excessive SPDP to react in a buffer solution, and desalting to remove the excessive SPDP;
coupling the IFN-alpha modified by the SPDP with a DNA chain modified by sulfydryl;
step 2, preparing a DNA origami raft: mixing the M13mp 18DNA single strand with 50-fold molar excess of staple strand and 100-fold molar excess of capture strand;
step 3, preparing the drug-loaded DNA origami raft: mixing the DNA origami raft with 5-fold molar excess of the DNA modified drug to be loaded.
9. A sustained-release drug comprising the drug-loaded DNA origami raft of any one of claims 1 to 6, and a pharmaceutically acceptable excipient.
Technical Field
The invention relates to the field of biomedicine, in particular to a DNA origami raft loaded with a medicine, and a preparation method and application thereof.
Background
Cytokines are small proteins produced by immune and non-immune cells and play important roles in immune system regulation, tissue homeostasis maintenance, tissue injury repair, and anti-inflammation. Cytokines are highly potent and can function in micromolar or even picomolar. Cytokines act as molecular messengers, allowing immune system cells to communicate with each other to produce coordination of target antigens, with regulatory and effector functions in many diseases, and thus cytokines are useful in immunotherapy. For example, in the course of immunotherapy, cytokines directly stimulate immune effector cells and stromal cells at the tumor site, enhancing cytotoxicity. There are several cytokine drugs that have been approved by the FDA for marketing, such as high doses of IL-2 for the treatment of melanoma and renal cell carcinoma, and IFN- α for adjuvant treatment of stage III melanoma. Still more cytokines have entered clinical trials, such as GM-CSF, IL-7, IL-12, IL-15, IL-18, and IL-2.
However, cytokines have short half-lives and are cleared rapidly in vivo, thus requiring multiple continuous overdosing and increasing their toxic side effects.
Therefore, the method for synthesizing the cell factor drug based on the DNA origami raft is low in cost and simple in process, improves the stability of the cell factor and reduces the toxic and side effects of a cell factor therapy, and has important practical significance.
Disclosure of Invention
In view of the above, in order to improve the stability of the cytokine and reduce the toxic and side effects of the cytokine therapy, the invention provides a low-cost and simple-process method for synthesizing the cytokine drug based on the DNA origami raft.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a DNA paper folding raft loaded with a medicine, which comprises a DNA-modified medicine to be loaded and a DNA paper folding raft;
the DNA modified drug to be loaded comprises IFN-alpha, sulfhydryl modified DNA (SH-DNA) and the drug to be loaded; the IFN-alpha and the sulfhydryl modified DNA nucleotide sequence complementary pairing;
the DNA origami raft comprises M13mp 18DNA single chains, staple chains and capture chains;
the staple chain is complementarily paired with the nucleotide sequence of the M13mp 18DNA single strand;
the capture strand is complementary paired with the nucleotide sequence of the single-stranded IFN-alpha modifying DNA.
In some embodiments of the invention, the nucleotide sequence of the staple chain is as shown in SEQ ID Nos. 1-216.
In some embodiments of the invention, the capture strand has a nucleotide sequence as set forth in SEQ ID Nos. 217-258.
In some embodiments of the invention, the molar ratio of the DNA-modified drug to be loaded to the DNA origami raft is not less than 5: 1.
In some embodiments of the invention, the molar ratio of the sulfhydryl modified DNA to the IFN- α is greater than 50: 1;
the molar ratio of the staple chain to the M13mp 18DNA single chain is more than 50: 1;
the molar ratio of the capture strand to the M13mp 18DNA single strand is greater than 100: 1.
In some embodiments of the invention, the drug to be loaded comprises IFN- α.
The invention also provides application of the DNA origami raft loaded with the medicine in preparation of slow-release medicines.
The invention also provides a preparation method of the drug-loaded DNA origami raft, which comprises the following steps:
step 1, preparing a DNA modified drug to be loaded:
dissolving the IFN-alpha in a solvent;
taking IFN-alpha solution and 50 times of excessive SPDP to react in a buffer solution, and desalting to remove the excessive SPDP;
coupling the IFN-alpha modified by the SPDP with a DNA chain modified by sulfydryl;
step 2, preparing a DNA origami raft: mixing the M13mp 18DNA single strand with 50-fold molar excess of staple strand and 100-fold molar excess of capture strand;
step 3, preparing the drug-loaded DNA origami raft: mixing the DNA origami raft with 5-fold molar excess of the DNA modified drug to be loaded.
In addition, the invention also provides a slow-release medicament, which comprises the DNA origami raft loaded with the medicament and pharmaceutically acceptable auxiliary materials.
The invention uses DNA nanometer technology to load IFN-alpha on DNA origami raft with accurate quantity, to control the drug-loading quantity and release IFN-alpha slowly.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 shows a schematic diagram of DNA-modified INF- α and UV-visible spectrum of pyridine-2-thione (extinction coefficient: 8080M-1cm-1) in example 1 of the present invention; wherein FIG. 1a shows a schematic representation of the preparation of DNA-INF-alpha; FIG. 1b shows UV-VIS spectra of pyridine-2-thione before and after the reaction;
FIG. 2 shows the highly controllable loading of cytokines on DNA origami rafts to the precise point of each binding; wherein, fig. 2a shows a schematic diagram of a DAN origami raft and a corresponding topography representation; FIG. 2b shows a corresponding height map for DNA origami rafts;
FIG. 3 shows the DNA origami based IFN- α sustained release in example 3 of the present invention; wherein FIG. 3a shows a schematic representation of the release of IFN from DNA origami rafts; FIG. 3b shows a graph of the co-localization efficiency analysis of IFN- α in urine over time; FIG. 3c shows that the intensity of red and green fluorescence decreases with time and the co-localization efficiency of red and green fluorescence decreases in the graph of FIG. 3 b.
Detailed Description
The invention discloses a drug-loaded DNA origami raft and a preparation method and application thereof, and a person skilled in the art can realize the loading of the drug-loaded DNA origami raft by appropriately improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The invention discloses a method for synthesizing a cell factor drug based on a DNA origami raft by taking IFN-alpha as an example, which comprises the following steps:
(1) preparing materials: preparing DNA single strand (M13 mp 18DNA single strand, staple strand, capture strand), thiol-modified DNA (SH-DNA), recombinant IFN-. alpha.3- (2-pyridyldithio) propionic acid N-hydroxysuccinimide ester (SPDP), 1xTAE-Mg2+And (4) a buffer solution.
(2) Preparing DNA-IFN-alpha, separating and purifying through an ultrafiltration tube, and characterizing the preparation efficiency through ultraviolet-visible mass spectrum;
(3) synthesis of DNA origami raft A capture strand with complementarity to the DNA in step (1) was synthesized by PCR. Separating and purifying through an ultrafiltration tube.
(4) Assembling and synthesizing the DNA origami raft, namely mixing the DNA-IFN-alpha in the step (1) and the DNA origami raft in the step (2), assembling and synthesizing the DNA origami raft, separating and purifying through an ultrafiltration tube, and representing the combination efficiency through fluorescence co-localization;
the ultrafiltration tube in the step (1) is a 30kD cut-off filter membrane (Amicon);
the ultrafiltration tube in the step (3) and the step (4) is a 100kD cut-off filter membrane (Amicon).
TABLE 1 sequence of staple chains (5 '-3') required for the synthesis of DNA origami rafts in example 2 of the invention
TABLE 2 sequence of Capture Strand required for the Synthesis of DNA origami rafts in example 2 of the invention (5 '-3')
The beneficial effects of the invention include but are not limited to:
the invention uses DNA nanometer technology to load IFN-alpha on DNA origami raft with accurate quantity, to control the drug-loading quantity and release IFN-alpha slowly.
In the medicine-loaded DNA origami raft and the preparation method and application thereof, the raw materials and the reagents can be purchased from the market.
The invention is further illustrated by the following examples:
EXAMPLE 1 preparation of DNA-IFN-. alpha.
100 μ g of IFN-. alpha.powder was dissolved in 50 μ L of NaCl solution. mu.L of a 6.6. mu.M IFN-. alpha.solution was reacted with 50-fold excess of SPDP in 1XPBS buffer (pH 8.5) for 2 hours. Excess SPDP was removed by means of a Zeba spin desalting column (7K MWCO, 0.5 mL). IFN-. alpha.was then coupled to the thiol-modified DNA strand (3-fold excess) in 1XPBS buffer (pH 7.4) at room temperature for 8 hours. The efficiency of HS-DNA modification of IFN- α was assessed by monitoring the net increase in UV-vis spectrum at 343nm due to pyridine-2-thione production (extinction coefficient: 8080M-1 cm-1). Excess SH-DNA strands were removed by ultrafiltration (3000g, 10 min) 3 times in 1 × PBS (pH 7.4) with a 30kD cut-off filter (Amicon).
Example 2 Synthesis of DNA origami rafts
At 1x TAE-Mg2+In buffer, 10nM of single strand M13mp 18DNA was mixed with 50-fold excess of staple strands and 100-fold excess of capture strands. The mixture was annealed from 95 ℃ to 4 ℃.
TABLE 3 annealing sequence from 95 ℃ to 4 ℃
Temperature of
Gradient of gradient
95℃
2min
95-60℃
-0.1℃/12s
60℃
12min
60-25℃
-0.1℃/12s
4℃
Holding
At 1XTAE-Mg2+Ultrafiltration (3000g, 10 min) was performed twice in buffer using a 100kD ultrafiltration tube (Amicon) and once in 1xPBS (pH 7.4) (3000g, 10 min) to remove excess staple and capture strands.
Example 3 Assembly Synthesis of DNA origami rafts
A 5-fold molar excess of DNA-modified IFN- α was mixed with DNA origami in PBS (pH 7.4) for at least 2h at room temperature. Then, the mixture was ultrafiltered (100 kD cut-off membrane Amicon in an ultrafiltration tube) 3 times with 1 × PBS (pH 7.4) to remove excess IFN- α, and then DNA origami-cytokine drug was obtained. The assembly of DNA origami rafts was characterized by agarose gel electrophoresis. Alexa488 was labeled with IFN-alpha and Cy5 was labeled with DNA origami raft and its combinatorial efficiency was characterized by total internal reflection fluorescence microscopy.
Effect example 1
SPDP links DNA to IFN- α via amino groups on the IFN- α surface and thiol groups in the thiol DNA. If the DNA is successfully modified to the IFN-alpha surface, pyridine-2-thione is produced. Pyridine-2-thione has a strong absorption peak at 343nm, and the generation of pyridine-2-thione is detected to confirm that DNA is successfully modified to the IFN-alpha surface.
The method comprises the following steps: 100 μ g of IFN-. alpha.powder was dissolved in 50 μ L of NaCl solution. mu.L of a 6.6. mu.M IFN-. alpha.solution was reacted with 50-fold excess of SPDP in 1XPBS buffer (pH 8.5) for 2 hours. Excess SPDP was removed by means of a Zeba spin desalting column (7K MWCO, 0.5 mL). Then, IFN-. alpha.was mixed well with the thiol-modified DNA strand (3-fold excess) in 1XPBS buffer (pH 7.4) and coupled at room temperature for 8 hours. And detecting the spectrums of the mixed solution before and after reaction by using a UV-Vis spectrophotometer, and finding that the mixed solution has an absorption peak at 343nm after 8 hours of reaction, thereby indicating that pyridine-2-thioketone is generated and indirectly proving that DNA is successfully modified on the surface of IFN-alpha.
Effect example 2
The method comprises the following steps: 10nM of M13mp 18DNA was mixed with 50-fold molar excess of staple chains and 100-fold molar excess of different numbers of capture chains (0, 21 or 42) at 1 × TAE-Mg2+The buffer solution is mixed evenly, and DNA origami raft is synthesized by annealing in PCR from 95 ℃ to 4 ℃. The above mixed solution was then ultrafiltered twice in 1xTAE-Mg2+ buffer using a 100kD ultrafiltration tube (Amicon) and once in 1x PBS (pH 7.4) (3000g, 10 min). Then 5-fold excess of DNA-IFN-alpha was folded onto 1XTAE-Mg with synthetic DNA with different numbers of capture strands2+The mixture was mixed well in the buffer and then allowed to stand at room temperature for 2 hours. At 1XTAE-Mg2+Buffer solutionTwo ultrafiltration (3000g, 10 min) were performed using 100kD ultrafiltration tubes (Amicon) and one ultrafiltration (3000g, 10 min) in 1x PBS (pH 7.4) to remove excess staple and capture chains. Mu.l of DNA origami at a concentration of 2nM were dropped on the mica surface and adsorbed for 5 minutes. And then scanning by using an Atomic Force Microscope (AFM) to obtain a morphology graph of the DNA origami raft, and analyzing the height of the nano raft by using the self-contained software of the AFM.
As shown in FIG. 2a, by controlling the amount of the capturing chain, the amount of IFN-. alpha.loaded on the surface of the DNA origami can be precisely controlled.
Effect example 3
The method comprises the following steps: the DNA origami rafts were labeled with Cy5 and the IFN-. alpha.supported on the surface of the DNA origami rafts was labeled with Alexa488, and then incubated with mouse urine, and imaged with a total internal reflection fluorescence microscope after 0, 12, 24, 36, and 48 hours of incubation, respectively. As shown in fig. 3a, as the incubation time increased, the fluorescence spots of Cy5 and Alexa488 gradually overlapped and separated, and the two were gradually released from the DNA origami raft. As shown in fig. 3c, the red fluorescence and the green fluorescence gradually decreased, and the co-localization efficiency of the two gradually decreased, indicating that Cy5 and Alexa-488 were gradually released from the DNA origami raft, indirectly demonstrating that IFN- α can be slowly released from the DNA origami raft.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
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<120> drug-loaded DNA origami raft and preparation method and application thereof
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<210> 64
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 64
aatgcagacc gtttttattt tcatcttgcg gg 32
<210> 65
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 65
catatttaga aataccgacc gtgttacctt tt 32
<210> 66
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 66
aatggtttac aacgccaaca tgtagttcag ct 32
<210> 67
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 67
taacctccat atgtgagtga ataaacaaaa tc 32
<210> 68
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 68
aaatcaatgg cttaggttgg gttactaaat tt 32
<210> 69
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 69
gcgcagagat atcaaaatta tttgacatta tc 32
<210> 70
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 70
aacctaccgc gaattattca tttccagtac at 32
<210> 71
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 71
attttgcgtc tttaggagca ctaagcaaca gt 32
<210> 72
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 72
ctaaaataga acaaagaaac caccagggtt ag 32
<210> 73
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 73
gccacgctat acgtggcaca gacaacgctc at 32
<210> 74
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 74
gcgtaagaga gagccagcag caaaaaggtt at 32
<210> 75
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 75
ggaaatacct acattttgac gctcacctga aa 32
<210> 76
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 76
tatcaccgta ctcaggaggt ttagcggggt tt 32
<210> 77
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 77
tgctcagtca gtctctgaat ttaccaggag gt 32
<210> 78
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 78
ggaaagcgac caggcggata agtgaatagg tg 32
<210> 79
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 79
tgaggcaggc gtcagactgt agcgtagcaa gg 32
<210> 80
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 80
tgcctttagt cagacgattg gcctgccaga at 32
<210> 81
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 81
ccggaaacac accacggaat aagtaagact cc 32
<210> 82
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 82
acgcaaaggt caccaatgaa accaatcaag tt 32
<210> 83
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 83
ttattacggt cagagggtaa ttgaatagca gc 32
<210> 84
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 84
tgaacaaaca gtatgttagc aaactaaaag aa 32
<210> 85
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 85
ctttacagtt agcgaacctc ccgacgtagg aa 32
<210> 86
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 86
gaggcgttag agaataacat aaaagaacac cc 32
<210> 87
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 87
tcattacccg acaataaaca acatatttag gc 32
<210> 88
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 88
ccagacgagc gcccaatagc aagcaagaac gc 32
<210> 89
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 89
agaggcataa tttcatcttc tgactataac ta 32
<210> 90
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 90
ttttagtttt tcgagccagt aataaattct gt 32
<210> 91
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 91
tatgtaaacc ttttttaatg gaaaaattac ct 32
<210> 92
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 92
ttgaattatg ctgatgcaaa tccacaaata ta 32
<210> 93
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 93
gagcaaaaac ttctgaataa tggaagaagg ag 32
<210> 94
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 94
tggattatga agatgatgaa acaaaatttc at 32
<210> 95
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 95
cggaattatt gaaaggaatt gaggtgaaaa at 32
<210> 96
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 96
atcaacagtc atcatattcc tgattgattg tt 32
<210> 97
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 97
ctaaagcaag atagaaccct tctgaatcgt ct 32
<210> 98
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 98
gccaacagtc accttgctga acctgttggc aa 32
<210> 99
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 99
gaaatggatt atttacattg gcagacattc tg 32
<210> 100
<211> 28
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 100
tttttataag tatagcccgg ccgtcgag 28
<210> 101
<211> 36
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 101
agggttgatt ttataaatcc tcattaaatg atattc 36
<210> 102
<211> 36
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 102
acaaacaatt ttaatcagta gcgacagatc gatagc 36
<210> 103
<211> 36
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 103
agcaccgttt tttaaaggtg gcaacatagt agaaaa 36
<210> 104
<211> 36
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 104
tacatacatt ttgacgggag aattaactac agggaa 36
<210> 105
<211> 36
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 105
gcgcattatt ttgcttatcc ggtattctaa atcaga 36
<210> 106
<211> 36
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 106
tatagaagtt ttcgacaaaa ggtaaagtag agaata 36
<210> 107
<211> 36
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 107
taaagtactt ttcgcgagaa aactttttat cgcaag 36
<210> 108
<211> 36
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 108
acaaagaatt ttattaatta catttaacac atcaag 36
<210> 109
<211> 36
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 109
aaaacaaatt ttttcatcaa tataatccta tcagat 36
<210> 110
<211> 36
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 110
gatggcaatt ttaatcaata tctggtcaca aatatc 36
<210> 111
<211> 48
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 111
aaaccctctt ttaccagtaa taaaagggat tcaccagtca cacgtttt 48
<210> 112
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 112
ccgaaatccg aaaatcctgt ttgaagccgg aa 32
<210> 113
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 113
ccagcagggg caaaatccct tataaagccg gc 32
<210> 114
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 114
gcataaagtt ccacacaaca tacgaagcgc ca 32
<210> 115
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 115
gctcacaatg taaagcctgg ggtgggtttg cc 32
<210> 116
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 116
ttcgccattg ccggaaacca ggcattaaat ca 32
<210> 117
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 117
gcttctggtc aggctgcgca actgtgttat cc 32
<210> 118
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 118
gttaaaattt taaccaatag gaacccggca cc 32
<210> 119
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 119
agacagtcat tcaaaagggt gagaagctat at 32
<210> 120
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 120
aggtaaagaa atcaccatca atataatatt tt 32
<210> 121
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 121
tttcatttgg tcaataacct gtttatatcg cg 32
<210> 122
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 122
tcgcaaatgg ggcgcgagct gaaataatgt gt 32
<210> 123
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 123
ttttaattgc ccgaaagact tcaaaacact at 32
<210> 124
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 124
aagaggaacg agcttcaaag cgaagataca tt 32
<210> 125
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 125
ggaattactc gtttaccaga cgacaaaaga tt 32
<210> 126
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 126
gaataaggac gtaacaaagc tgctctaaaa ca 32
<210> 127
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 127
ccaaatcact tgccctgacg agaacgccaa aa 32
<210> 128
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 128
ctcatcttga ggcaaaagaa tacagtgaat tt 32
<210> 129
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 129
aaacgaaatg acccccagcg attattcatt ac 32
<210> 130
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 130
cttaaacatc agcttgcttt cgagcgtaac ac 32
<210> 131
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 131
tcggtttagc ttgataccga tagtccaacc ta 32
<210> 132
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 132
tgagtttcgt caccagtaca aacttaattg ta 32
<210> 133
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 133
ccccgattta gagcttgacg gggaaatcaa aa 32
<210> 134
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 134
gaatagccgc aagcggtcca cgctcctaat ga 32
<210> 135
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 135
gagttgcacg agatagggtt gagtaaggga gc 32
<210> 136
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 136
gtgagctagt ttcctgtgtg aaatttggga ag 32
<210> 137
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 137
tcatagctac tcacattaat tgcgccctga ga 32
<210> 138
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 138
ggcgatcgca ctccagccag ctttgccatc aa 32
<210> 139
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 139
gaagatcggt gcgggcctct tcgcaatcat gg 32
<210> 140
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 140
aaataatttt aaattgtaaa cgttgatatt ca 32
<210> 141
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 141
gcaaatatcg cgtctggcct tcctggcctc ag 32
<210> 142
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 142
accgttctaa atgcaatgcc tgagaggtgg ca 32
<210> 143
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 143
tatattttag ctgataaatt aatgttgtat aa 32
<210> 144
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 144
tcaattcttt tagtttgacc attaccagac cg 32
<210> 145
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 145
cgagtagaac taatagtagt agcaaaccct ca 32
<210> 146
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 146
gaagcaaaaa agcggattgc atcagataaa aa 32
<210> 147
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 147
tcagaagcct ccaacaggtc aggatctgcg aa 32
<210> 148
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 148
ccaaaatata atgcagatac ataaacacca ga 32
<210> 149
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 149
cattcaacgc gagaggcttt tgcatattat ag 32
<210> 150
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 150
acgagtagtg acaagaaccg gatataccaa gc 32
<210> 151
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 151
agtaatctta aattgggctt gagagaatac ca 32
<210> 152
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 152
gcgaaacatg ccactacgaa ggcatgcgcc ga 32
<210> 153
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 153
atacgtaaaa gtacaacgga gatttcatca ag 32
<210> 154
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 154
caatgacact ccaaaaggag ccttacaacg cc 32
<210> 155
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 155
aaaaaaggac aaccatcgcc cacgcgggta aa 32
<210> 156
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 156
tgtagcattc cacagacagc cctcatctcc aa 32
<210> 157
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 157
gtaaagcact aaatcggaac cctagttgtt cc 32
<210> 158
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 158
agtttggagc ccttcaccgc ctggttgcgc tc 32
<210> 159
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 159
agctgattac aagagtccac tattgaggtg cc 32
<210> 160
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 160
actgcccgcc gagctcgaat tcgttattac gc 32
<210> 161
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 161
cccgggtact ttccagtcgg gaaacgggca ac 32
<210> 162
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 162
cagctggcgg acgacgacag tatcgtagcc ag 32
<210> 163
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 163
gtttgaggga aagggggatg tgctagagga tc 32
<210> 164
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 164
ctttcatccc caaaaacagg aagaccggag ag 32
<210> 165
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 165
agaaaagcaa cattaaatgt gagcatctgc ca 32
<210> 166
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 166
ggtagctagg ataaaaattt ttagttaaca tc 32
<210> 167
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 167
caacgcaatt tttgagagat ctactgataa tc 32
<210> 168
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 168
caataaatac agttgattcc caatttagag ag 32
<210> 169
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 169
tccatataca tacaggcaag gcaactttat tt 32
<210> 170
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 170
tacctttaag gtctttaccc tgacaaagaa gt 32
<210> 171
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 171
caaaaatcat tgctcctttt gataagtttc at 32
<210> 172
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 172
tttgccagat cagttgagat ttagtggttt aa 32
<210> 173
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 173
aaagattcag ggggtaatag taaaccataa at 32
<210> 174
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 174
tttcaactat aggctggctg accttgtatc at 32
<210> 175
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 175
ccaggcgctt aatcattgtg aattacaggt ag 32
<210> 176
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 176
cgcctgatgg aagtttccat taaacataac cg 32
<210> 177
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 177
tttcatgaaa attgtgtcga aatctgtaca ga 32
<210> 178
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 178
atatattctt ttttcacgtt gaaaatagtt ag 32
<210> 179
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 179
aataataagg tcgctgaggc ttgcaaagac tt 32
<210> 180
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 180
cgtaacgatc taaagttttg tcgtgaattg cg 32
<210> 181
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 181
acccaaatca agttttttgg ggtcaaagaa cg 32
<210> 182
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 182
tggactccct tttcaccagt gagacctgtc gt 32
<210> 183
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 183
tggtttttaa cgtcaaaggg cgaagaacca tc 32
<210> 184
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 184
gccagctgcc tgcaggtcga ctctgcaagg cg 32
<210> 185
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 185
cttgcatgca ttaatgaatc ggcccgccag gg 32
<210> 186
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 186
attaagttcg catcgtaacc gtgcgagtaa ca 32
<210> 187
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 187
tagatggggg gtaacgccag ggttgtgcca ag 32
<210> 188
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 188
acccgtcgtc atatgtaccc cggtaaaggc ta 32
<210> 189
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 189
catgtcaaga ttctccgtgg gaaccgttgg tg 32
<210> 190
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 190
tcaggtcact tttgcgggag aagcagaatt ag 32
<210> 191
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 191
ctgtaatatt gcctgagagt ctggaaaact ag 32
<210> 192
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 192
caaaattaaa gtacggtgtc tggaagaggt ca 32
<210> 193
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 193
tgcaactaag caataaagcc tcagttatga cc 32
<210> 194
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 194
tttttgcgca gaaaacgaga atgaatgttt ag 32
<210> 195
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 195
aaacagttga tggcttagag cttatttaaa ta 32
<210> 196
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 196
actggataac ggaacaacat tattacctta tg 32
<210> 197
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 197
acgaactagc gtccaatact gcggaatgct tt 32
<210> 198
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 198
cgattttaga ggacagatga acggcgcgac ct 32
<210> 199
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 199
ctttgaaaag aactggctca ttatttaata aa 32
<210> 200
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 200
gctccatgag aggctttgag gactagggag tt 32
<210> 201
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 201
acggctactt acttagccgg aacgctgacc aa 32
<210> 202
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 202
aaaggccgaa aggaacaact aaagctttcc ag 32
<210> 203
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 203
gagaatagct tttgcgggat cgtcgggtag ca 32
<210> 204
<211> 32
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 204
acgttagtaa atgaattttc tgtaagcgga gt 32
<210> 205
<211> 28
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 205
ttttcgatgg cccactacgt aaaccgtc 28
<210> 206
<211> 36
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 206
tatcagggtt ttcggtttgc gtattgggaa cgcgcg 36
<210> 207
<211> 36
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 207
gggagaggtt tttgtaaaac gacggccatt cccagt 36
<210> 208
<211> 36
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 208
cacgacgttt ttgtaatggg ataggtcaaa acggcg 36
<210> 209
<211> 36
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 209
gattgacctt ttgatgaacg gtaatcgtag caaaca 36
<210> 210
<211> 36
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 210
agagaatctt ttggttgtac caaaaacaag cataaa 36
<210> 211
<211> 36
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 211
gctaaatctt ttctgtagct caacatgtat tgctga 36
<210> 212
<211> 36
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 212
atataatgtt ttcattgaat ccccctcaaa tcgtca 36
<210> 213
<211> 36
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 213
taaatatttt ttggaagaaa aatctacgac cagtca 36
<210> 214
<211> 36
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 214
ggacgttgtt tttcataagg gaaccgaaag gcgcag 36
<210> 215
<211> 36
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 215
acggtcaatt ttgacagcat cggaacgaac cctcag 36
<210> 216
<211> 48
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 216
cagcgaaaat tttactttca acagtttctg ggattttgct aaactttt 48
<210> 217
<211> 65
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 217
tgagatgata agtggattgg agtagaagtt tttgcataaa gttccacaca acatacgaag 60
cgcca 65
<210> 218
<211> 65
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 218
tgagatgata agtggattgg agtagaagtt tttttttaat tgcccgaaag acttcaaaac 60
actat 65
<210> 219
<211> 65
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 219
tgagatgata agtggattgg agtagaagtt tttctcatct tgaggcaaaa gaatacagtg 60
aattt 65
<210> 220
<211> 65
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 220
tgagatgata agtggattgg agtagaagtt tttgaatagc cgcaagcggt ccacgctcct 60
aatga 65
<210> 221
<211> 65
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 221
tgagatgata agtggattgg agtagaagtt tttggcgatc gcactccagc cagctttgcc 60
atcaa 65
<210> 222
<211> 65
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 222
tgagatgata agtggattgg agtagaagtt tttaccgttc taaatgcaat gcctgagagg 60
tggca 65
<210> 223
<211> 65
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 223
tgagatgata agtggattgg agtagaagtt tttgaagcaa aaaagcggat tgcatcagat 60
aaaaa 65
<210> 224
<211> 65
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 224
tgagatgata agtggattgg agtagaagtt tttacgagta gtgacaagaa ccggatatac 60
caagc 65
<210> 225
<211> 65
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 225
tgagatgata agtggattgg agtagaagtt tttcaatgac actccaaaag gagccttaca 60
acgcc 65
<210> 226
<211> 65
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 226
tgagatgata agtggattgg agtagaagtt tttgtaaagc actaaatcgg aaccctagtt 60
gttcc 65
<210> 227
<211> 65
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 227
tgagatgata agtggattgg agtagaagtt tttactgccc gccgagctcg aattcgttat 60
tacgc 65
<210> 228
<211> 65
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 228
tgagatgata agtggattgg agtagaagtt tttctttcat ccccaaaaac aggaagaccg 60
gagag 65
<210> 229
<211> 65
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 229
tgagatgata agtggattgg agtagaagtt tttcaataaa tacagttgat tcccaattta 60
gagag 65
<210> 230
<211> 65
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 230
tgagatgata agtggattgg agtagaagtt ttttttgcca gatcagttga gatttagtgg 60
tttaa 65
<210> 231
<211> 65
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 231
tgagatgata agtggattgg agtagaagtt tttcgcctga tggaagtttc cattaaacat 60
aaccg 65
<210> 232
<211> 65
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 232
tgagatgata agtggattgg agtagaagtt ttttggactc ccttttcacc agtgagacct 60
gtcgt 65
<210> 233
<211> 65
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 233
tgagatgata agtggattgg agtagaagtt tttattaagt tcgcatcgta accgtgcgag 60
taaca 65
<210> 234
<211> 65
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 234
tgagatgata agtggattgg agtagaagtt ttttcaggtc acttttgcgg gagaagcaga 60
attag 65
<210> 235
<211> 65
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 235
tgagatgata agtggattgg agtagaagtt ttttttttgc gcagaaaacg agaatgaatg 60
tttag 65
<210> 236
<211> 65
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 236
tgagatgata agtggattgg agtagaagtt tttcgatttt agaggacaga tgaacggcgc 60
gacct 65
<210> 237
<211> 65
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 237
tgagatgata agtggattgg agtagaagtt tttaaaggcc gaaaggaaca actaaagctt 60
tccag 65
<210> 238
<211> 65
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 238
tgagatgata agtggattgg agtagaagtt tttgagccgc cccaccaccg gaaccgcgac 60
ggaaa 65
<210> 239
<211> 65
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 239
tgagatgata agtggattgg agtagaagtt tttatcggct gcgagcatgt agaaacctat 60
catat 65
<210> 240
<211> 65
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 240
tgagatgata agtggattgg agtagaagtt tttcgacaac taagtattag actttacaat 60
accga 65
<210> 241
<211> 65
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 241
tgagatgata agtggattgg agtagaagtt tttctgaaac aggtaataag ttttaacccc 60
tcaga 65
<210> 242
<211> 65
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 242
tgagatgata agtggattgg agtagaagtt tttgacttga gagacaaaag ggcgacaagt 60
tacca 65
<210> 243
<211> 65
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 243
tgagatgata agtggattgg agtagaagtt tttattattt aacccagcta caattttcaa 60
gaacg 65
<210> 244
<211> 65
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 244
tgagatgata agtggattgg agtagaagtt tttacgctca aaataagaat aaacaccgtg 60
aattt 65
<210> 245
<211> 65
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 245
tgagatgata agtggattgg agtagaagtt tttcctgatt gaaagaaatt gcgtagaccc 60
gaacg 65
<210> 246
<211> 65
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 246
tgagatgata agtggattgg agtagaagtt tttaggcggt cattagtctt taatgcgcaa 60
tatta 65
<210> 247
<211> 65
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 247
tgagatgata agtggattgg agtagaagtt tttccctcag aaccgccacc ctcagaactg 60
agact 65
<210> 248
<211> 65
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 248
tgagatgata agtggattgg agtagaagtt tttcaccaga gttcggtcat agcccccgcc 60
agcaa 65
<210> 249
<211> 65
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 249
tgagatgata agtggattgg agtagaagtt tttataccca agataaccca caagaataaa 60
cgatt 65
<210> 250
<211> 65
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 250
tgagatgata agtggattgg agtagaagtt tttcaagcaa gacgcgcctg tttatcaaga 60
atcgc 65
<210> 251
<211> 65
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 251
tgagatgata agtggattgg agtagaagtt ttttaacctc catatgtgag tgaataaaca 60
aaatc 65
<210> 252
<211> 65
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 252
tgagatgata agtggattgg agtagaagtt tttattttgc gtctttagga gcactaagca 60
acagt 65
<210> 253
<211> 65
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 253
tgagatgata agtggattgg agtagaagtt ttttgctcag tcagtctctg aatttaccag 60
gaggt 65
<210> 254
<211> 65
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 254
tgagatgata agtggattgg agtagaagtt tttccggaaa cacaccacgg aataagtaag 60
actcc 65
<210> 255
<211> 65
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 255
tgagatgata agtggattgg agtagaagtt tttctttaca gttagcgaac ctcccgacgt 60
aggaa 65
<210> 256
<211> 65
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 256
tgagatgata agtggattgg agtagaagtt tttagaggca taatttcatc ttctgactat 60
aacta 65
<210> 257
<211> 65
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 257
tgagatgata agtggattgg agtagaagtt tttgagcaaa aacttctgaa taatggaaga 60
aggag 65
<210> 258
<211> 65
<212> DNA
<213> Artificial sequence (Artificial sequence;)
<400> 258
tgagatgata agtggattgg agtagaagtt tttctaaagc aagatagaac ccttctgaat 60
cgtct 65