Primer group for detecting pathogenic microorganisms
阅读说明：本技术 用于检测病原微生物的引物组 (Primer group for detecting pathogenic microorganisms ) 是由 余成鹏 邹树勇 朱鹏远 吴春求 陈丹 张东东 陈嘉昌 柳俊 胡朝晖 于 2020-12-31 设计创作，主要内容包括：本发明涉及核酸检测技术领域,具体而言,涉及一种用于检测病原微生物的引物组。本发明所提供的多重体系可最多同时检测98种病原微生物,检测准确率高,特异性好,范围外样本之间均不产生交叉污染现象,且引物之间几乎不产生引物二聚体,适用于对含有未知微生物样本的批量检测。(The invention relates to the technical field of nucleic acid detection, in particular to a primer group for detecting pathogenic microorganisms. The multi-system provided by the invention can detect 98 pathogenic microorganisms at most simultaneously, has high detection accuracy and good specificity, does not generate cross contamination among samples outside the range, hardly generates primer dimer among primers, and is suitable for batch detection of samples containing unknown microorganisms.)
1. A primer set, wherein the primer set comprises at least 50 of 175 primer pairs;
175 pairs of primer pairs have nucleotide sequences shown as SEQ ID NO: 1-175, and the nucleotide sequence corresponding to the upstream primer in sequence is shown as SEQ ID NO: 175-350 as shown in the specification.
2. The primer set of claim 1, wherein said primer set comprises at least 100 of 175 primer pairs.
3. The primer set of claim 2, wherein said primer set comprises at least 140 of 175 primer pairs.
4. A kit comprising the primer set according to any one of claims 1 to 3.
5. The kit according to claim 4, characterized in that it comprises at least one of the following reagents:
DNA polymerase, dNTPs, lysis and/or washing buffers, a solid support for enriching nucleic acids, nucleic acid elution reagents, dilution buffers, water, molecular weight marker, proteinase K and neutralization reagents.
6. The kit of claim 4 or 5, wherein the kit comprises a positive control for the nucleic acid to be detected of known sequence and concentration.
7. The kit of claim 4 or 5, wherein the kit comprises at least one of a terminal repair enzyme, a ligation buffer, a DNA ligase, and an adaptor fragment.
8. A method for sequencing a library of a plurality of target nucleic acids, said method comprising the steps of:
performing multiplex PCR amplification using the genomic DNA of an object to be detected as a template, using the primer set according to any one of claims 1 to 3, or the kit according to any one of claims 4 to 7.
9. A non-diagnostic method of interest for detecting a plurality of pathogenic microorganisms, comprising:
using the method of claim 8 for pooling, sequencing to determine the presence of pathogenic microorganisms.
10. The method of claim 9, wherein the sequencing method is high throughput sequencing.
Technical Field
The invention relates to the technical field of nucleic acid detection, in particular to a primer group for detecting pathogenic microorganisms.
Background
Infectious diseases caused by microorganisms are still the major diseases that currently threaten global human health. Infectious diseases are mainly controlled from the aspects of discovery, diagnosis, treatment and prevention. Among them, diagnosis is very important for early detection and early control of infectious diseases. At present, the diagnosis of the microorganism in China mainly depends on serological diagnosis and microorganism culture. These diagnostic methods often take several tens of hours to several days from sampling, culturing to identification, and in some remote areas, it takes a longer time, and it is more difficult to complete the relevant differential diagnosis in a short time. On the other hand, with the addition of WTO in China, foreign trade in China develops rapidly, and the import and export of various foods and cosmetics increases rapidly. Import and export quarantine faces tremendous pressure. Therefore, there is an urgent need for new technologies and devices to meet the high throughput and increasing low cost detection of various genetic information.
Polymerase Chain Reaction (PCR) has been widely used in medicine, genetics, microbiology, and even throughout life sciences. Multiplex PCR is a novel amplification technique developed on the basis of conventional PCR, i.e., two or more pairs of primers can be added into a reaction system to simultaneously amplify a plurality of nucleic acid fragments. The multiplex PCR has important application in the disciplines of microorganism, genetic disease, tumor, pharmacogenomics and the like.
A multiple PCR detection system and a method based on pathogenic microorganisms are urgently needed by departments such as clinical laboratories, disease control centers, import and export quarantine inspection and the like.
Disclosure of Invention
The first aspect of the present invention relates to a primer set comprising at least 50 of 175 primer pairs;
175 pairs of primer pairs have nucleotide sequences shown as SEQ ID NO: 1-175, and the nucleotide sequence corresponding to the upstream primer in sequence is shown as SEQ ID NO: 175-350 as shown in the specification.
Optionally, a primer set as described above, said primer set comprising at least 100 of the 175 primer pairs.
Optionally, a primer set as described above, said primer set comprising at least 140 of the 175 primer pairs.
A second aspect of the present invention relates to a kit comprising a primer set as described above.
Optionally, a kit as described above, comprising at least one of the following reagents:
DNA polymerase, dNTPs, lysis and/or washing buffers, a solid support for enriching nucleic acids, nucleic acid elution reagents, dilution buffers, water, molecular weight marker, proteinase K and neutralization reagents.
Alternatively, a kit as described above, comprising a positive control for the nucleic acid to be detected of known sequence and concentration.
Optionally, the kit as described above, comprising at least one of a terminal repair enzyme, a ligation buffer, a DNA ligase, and an adaptor fragment.
A third aspect of the invention relates to a method for sequencing and pooling a plurality of target nucleic acids, the method comprising the steps of:
the genomic DNA of the sample to be detected is used as a template, and the primer set or the kit is used for performing multiplex PCR amplification.
A fourth aspect of the invention relates to a method for non-diagnostic purposes of detecting a plurality of pathogenic microorganisms, characterized in that it comprises:
the method described above was used for pooling, sequencing to determine the presence of pathogenic microorganisms.
Alternatively, the method of sequencing is high throughput sequencing, as described above.
The invention has the beneficial effects that:
the multi-system provided by the invention can detect 98 pathogenic microorganisms at most simultaneously, has high detection accuracy and good specificity, does not generate cross contamination among samples outside the range, hardly generates primer dimer among primers, and is suitable for batch detection of samples containing unknown microorganisms.
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, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 shows the amplification effect of 6 samples of MT1-MT6 based on a primer system designed based on a penalty mechanism according to an embodiment of the present invention;
FIG. 2 shows the amplification effect of 6 samples of MT1-MT6 without a primer system designed based on a penalty mechanism in one embodiment of the present invention.
Detailed Description
Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
The first aspect of the present invention relates to a primer set comprising at least 50 of 175 primer pairs;
175 pairs of primer pairs have nucleotide sequences shown as SEQ ID NO: 1-175, and the nucleotide sequence corresponding to the upstream primer in sequence is shown as SEQ ID NO: 175-350 as shown in the specification.
In some embodiments, the primer set comprises at least 50, or at least 60, or at least 70, or at least 80, or at least 90, or at least 100, or at least 110, or at least 120, or at least 130, or at least 140, or at least 150, or at least 160, or at least 170 of the 175 primer pairs.
In some embodiments, the primer set comprises a primer for detecting any one of the microbial species defined in table 1.
In some embodiments, the primer set is capable of detecting at least 30, or at least 40, or at least 50, or at least 60, or at least 70, or at least 80, or at least 90 of the 98 pathogenic microorganism species defined in table 1.
A second aspect of the present invention relates to a kit comprising a primer set as described above.
The term "kit" refers to any article of manufacture (e.g., a package or container) comprising at least one device, the kit may further comprise instructions for use, supplemental reagents, and/or components or assemblies for use in the methods described herein or steps thereof.
In some embodiments, the kit comprises at least one of the following reagents:
DNA polymerase, dNTPs, lysis and/or washing buffers, a solid support for enriching nucleic acids, nucleic acid elution reagents, dilution buffers, water, molecular weight marker, proteinase K and neutralization reagents.
The term "buffer" as used herein refers to an aqueous solution or composition that resists changes in pH when an acid or base is added to the solution or composition. This resistance to pH changes is due to the buffer properties of such solutions. Thus, a solution or composition that exhibits buffering activity is referred to as a buffer or buffer solution. Buffers generally do not have the unlimited ability to maintain the pH of a solution or composition. Rather, they are generally capable of being maintained at a pH within a specified range, for example, pH 7-pH 9. Generally, Buffers are capable of maintaining a pH at their pKa and within the next logarithm (see, e.g., Mohan, Buffers, A guide for the preparation and use of Buffers in biological systems, CALBIOCHEM, 1999). Buffers and buffer solutions are generally prepared from buffered salts or preferably non-ionic buffer components such as TRIS and HEPES. The buffer which can be used in the method of the invention is preferably selected from the group consisting of phosphate buffer, phosphate buffered saline buffer (PBS), 2-amino-2 hydroxymethyl-1, 3-propanediol (TRIS) buffer, TRIS buffered saline solution (TBS) and TRIS/edta (te).
In some embodiments, the water is nuclease-free water, such as double distilled or deionized water.
In some embodiments, the DNA polymerase is selected from any of Taq, Bst, Vent, Phi29, Pfu, Tru, Tth, Tl1, Tac, Tne, Tma, Tih, Tf1, Pwo, Kod, Sac, Sso, Poc, Pab, Mth, Pho, ES4 DNA polymerase, Klenow fragment.
As used herein, a "solid support," preferably an "enrichment particle," can be made from any number of known materials. Examples of such materials include: minerals, natural polymers and synthetic polymers. Specific examples of these materials include: cellulose, cellulose derivatives, acrylics, glass, silica gel, polystyrene, gelatin, polyvinylpyrrolidone, copolymers of vinyl and acrylamide, polystyrene, polyacrylamide, latex gel, dextran, rubber, silica gel, plastic, nitrocellulose, natural sponge, silica gel, control pore glass (control pore glass), metal, cross-linked dextran (e.g., Sephadex TM), Sepharose (TM), and other solid supports known to those skilled in the art.
As used herein, "particle" refers to a discrete small object, such as a sphere (e.g., bead), capsule, polyhedron, etc., that can be of various shapes. The particles may be macroscopic or microscopic, such as microparticles or nanoparticles. The particles may be non-magnetic or magnetic. The magnetic particles may contain a ferromagnetic substance, and the ferromagnetic substance may be Fe, Ni, Co, iron oxide, or the like.
The kit may further contain a nucleic acid extraction reagent which can be used for extracting nucleic acid by performing phenol chloroform method, NaOH method, resin extraction method, salting-out method, hexadecyl trimethyl ammonium bromide method, silica gel membrane adsorption method, FTA card method, silica bead method or magnetic bead extraction method.
In some embodiments, the kit includes a positive control for the nucleic acid to be detected of known sequence and concentration.
In some embodiments, the kit comprises at least one of a terminal repair enzyme, a ligation buffer, a DNA ligase, and an adaptor fragment.
A third aspect of the invention relates to a method for sequencing and pooling a plurality of target nucleic acids, the method comprising the steps of:
the genomic DNA of the sample to be detected is used as a template, and the primer set or the kit is used for performing multiplex PCR amplification.
A fourth aspect of the present invention relates to a method for detecting a plurality of pathogenic microorganisms, comprising:
the method described above was used for pooling, sequencing to determine the presence of pathogenic microorganisms.
The term "detecting" and similar terms are used in this application to generally refer to a process or the discovery or determination of the presence or absence, as well as the degree, quantity or level, or probability of occurrence of something. For example, the term "detecting" when used in reference to a target nucleic acid sequence can refer to finding or determining the presence, absence, level, or amount, and the probability or likelihood of the presence or absence, of the sequence. It is to be understood that the expressions "detecting the presence or absence", "detecting the presence or absence" and related expressions include both qualitative and quantitative detections. For example, quantitative detection includes determining the level, amount, or quantity of a nucleic acid sequence associated with parainfluenza virus type 3 in a sample.
The term "test agent" refers to any composition that contains or is suspected of containing nucleic acid from a pathogenic microorganism. The term includes whole blood, plasma, serum, cells, throat swab, saliva, urine, feces, cerebrospinal fluid, pleural effusion, amniotic fluid, vaginal secretions.
In some embodiments of the invention, the sequencing method is high throughput sequencing, also known as next generation sequencing ("NGS"). Second generation sequencing produces thousands to millions of sequences simultaneously in a parallel sequencing process. NGS is distinguished from "Sanger sequencing" (one generation sequencing), which is based on electrophoretic separation of chain termination products in a single sequencing reaction. Sequencing platforms for NGS useful in the present invention may be commercially available, including but not limited to Illumina MiniSeq, NextSeq 550, and the like.
In some embodiments, the method is a method of non-diagnostic interest.
Embodiments of the present invention will be described in detail with reference to examples.
Example 1
The invention provides a method for detecting pathogens by high-throughput sequencing, which is used for a pathogenic microorganism ultra-multiplex PCR primer design device system output primer combination mode. The invention designs a specific primer combination scheme according to a specific sequence of a sample to be detected, the specific primer combination scheme is used for ultra-multiplex PCR amplification after verification, an amplification product is prepared and sequenced through a library, and the types of microorganisms and pathogenic/drug-resistant genes in the sample are detected. Take 98 kinds of pathogenic microorganism detection as an example.
98 genome sequences of 98 common pathogenic microorganisms are selected through literature search, and the sequences of the pathogenic microorganisms are downloaded on Genebank. Aiming at the functional regions, conserved regions and sequences of tandem repeat fragments supported by the existing literature of the pathogenic microorganisms, mega software is used for carrying out multi-sequence comparison analysis, and the obtained intergeneric sequence, interspecies specific sequence and subspecies conserved sequence of the subspecies under the classification are compared, and the sequences of pathogenic genes and drug-resistant genes related to the pathogenic microorganisms account for 175 target amplification regions.
Aiming at the sequences, designing primers, confirming no non-specific amplification risk through primer specificity comparison, and storing the primers in a primer pool, wherein the primer design rule is up to the following standard:
(1) the species specificity interval and the family conservation interval, the drug resistance gene and the virulence gene are selected, and 5 pairs of primers are designed for each pathogen to ensure the sensitivity and the specificity.
(2) The length of the primer fragment is between 18 and 24bp, and the length of the amplification product is between 110 and 120 bp;
(3) the annealing temperature of the primer is between 55 and 65 ℃;
(4) the GC content of the primer is between 40 and 60 percent;
(5) the 3' end of the primer is C or G; 6. each primer does not have 4 or more continuous A or T or C or G;
aiming at the obtained primer pool, screening and pairing are carried out according to the following conditions, and an optimal primer combination scheme is calculated:
(1) the head and the tail of each primer have the condition of complementary pairing of less than 2 continuous bases to obtain 2 points, the head and the tail of each primer have the condition of complementary pairing of 2-3 continuous bases to obtain 1 point, and the head and the tail of each primer have the condition of complementary pairing of more than 3 continuous bases to obtain 0 point;
(2) the deviation degree of the primer Tm value and the Tm average value is within 2 ℃, each primer is divided into +1, the deviation degree of the primer Tm value and the Tm average value is above 2 ℃, and each primer is divided into + 0;
(3) the GC content of the primers is within 5 percent of the average GC content, each primer is divided into +1 part, the GC content of the primers is more than 5 percent of the average GC content, and each primer is divided into +0 part;
(4) taking a primer pair with the minimum deviation Tm average value as a standard, constructing a binary matrix by using the standard primer pair, and respectively carrying out complementary analysis on the binary matrix and other primers, wherein the initial score of each 2 primers is 5 minutes, and the complementary pairing condition of more than 2 continuous bases exists between each primer and each primer, and the score is reduced by 2 when each base exceeds 1 base; and returning the primers with the scores of less than 3 points or the occurrence frequency of the primers with the scores of less than 3 points and other primers with the complementary analysis score of more than 50 percent to the primer pool, replacing the primers with the other primers and recalculating.
After the screening of the steps, a primer combination scheme with the highest score is output, and the obtained primers are shown in table 1.
SEQ ID NO: the upstream primers shown in 1 to 175 correspond to 1 to 175 in the sequence numbers in Table 1.
TABLE 1 primer information
The pathogenic microorganism ultra-multiplex PCR specific primers completely cover 175 regions of a target, the primer combination is used for ultra-multiplex PCR, and the amplification product is used for library construction and high-throughput sequencing.
The total of 846 known positive samples of 98 microorganisms within the primer design coverage and 48 known samples of 3 microorganisms outside the primer design coverage are selected, and 894 samples are subjected to DNA extraction, PCR amplification, library preparation and machine sequencing.
All samples covered by the primer design can be correctly detected, the corresponding pathogenic microorganisms have a positive coincidence rate of 100 percent, and the positive coincidence rate is shown in table 2. Pathogenic microorganisms are not detected in all samples outside the design coverage range of the primers, and the negative coincidence rate is 100 percent as shown in table 3. The cross contamination phenomenon does not occur between samples within the designed coverage range or outside the designed coverage range of the primers, and the specificity is better.
TABLE 2 results of sample detection within primer design coverage
Note: part of positive samples are difficult to collect, and the test adopts synthetic false viruses or commercial quality control products are purchased.
TABLE 3 detection results of samples outside the primer design coverage
Serial number
Pathogenic microorganism
Total number of
Negative count
Negative match rate (%)
1
Aspergillus terreus
5
5
100
2
Burkholderia cepacia
28
28
100
3
Pneumocystis yeri
15
15
100
Meanwhile, 8 positive samples of respiratory syncytial virus type B were selected, and the Sanger sequencing method and the tNGS method based on the combination of the ultra-multiple PCR primers described in this example were used respectively, and the Sanger sequencing was used as a standard to verify the consistency of the different methodologies. The results show that Sanger sequencing and tNGS can correctly detect respiratory syncytial virus B types in 8 samples, and the consistency is good.
The results are combined, and the primer combination designed by the ultra-multiplex PCR primer design device can accurately detect positive samples or negative samples in a detection range.
Example 2
The primer combination for the ultra-multiplex PCR described in example 1 was selected, and the Coxsackie virus A6, which is a closely related species of enterovirus not designed in the above primer design, was selected and detected, and it was revealed that 2 cases of Coxsackie virus A6 were not within the range of primer design, but the sequences of the universal primers suggested that the enterovirus was of another type. The results of the 2 cases are consistent with known positive results, which prove that the primer combination has the capability of detecting or predicting unknown samples, and the results are shown in Table 4.
TABLE 4 kindred species detection
Serial number
Pathogenic microorganism
Total number of
Positive counts
Positive match rate (%)
1
Coxsackie virus type A6
2
2
100
Example 3
The target sequence described in example 1 was selected for primer design without penalty mechanism to obtain primer combinations, and 6 samples were amplified with the primer combinations described in example 1, and the amplification effect was compared with the concentration of amplified product and distribution of amplified fragments.
The concentration of 6 samples after PCR amplification is analyzed by amplifying reaction systems of different primer systems with the same template amount and analyzing the fragment length of an amplification product by using Qsep100, and the result shows that the concentration of the primer combinations designed based on the penalty mechanism after amplification is higher than that of the primer combinations not designed based on the penalty mechanism, as shown in Table 5; the primer combination based on the penalty mechanism has no peak in the range of 40-60bp, and the primer combination not based on the penalty mechanism has a peak in the range of 40-60bp, i.e., the primer combination giving the penalty mechanism has significantly less primer dimer after amplification than the primer combination not designed based on the penalty mechanism, as shown in FIG. 1 and FIG. 2.
TABLE 5 comparison of concentrations of different primer combinations after amplification
In conclusion, the penalty mechanism described in this patent, especially the self-constructed binary matrix, can effectively eliminate primer dimers possibly generated between primer pairs, thereby effectively improving the primer amplification specificity and amplification efficiency of the super-multiplex PCR.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Sequence listing
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<213> artificial sequence
<400> 55
cttatgctta acttactaga taaa 24
<210> 56
<211> 21
<212> DNA
<213> artificial sequence
<400> 56
cacatccatg ggacgaagat g 21
<210> 57
<211> 21
<212> DNA
<213> artificial sequence
<400> 57
gctcagaatc tacgaagaac a 21
<210> 58
<211> 24
<212> DNA
<213> artificial sequence
<400> 58
actgtattca gtcaacacag ggct 24
<210> 59
<211> 20
<212> DNA
<213> artificial sequence
<400> 59
tggtgcaaag agtctrttga 20
<210> 60
<211> 24
<212> DNA
<213> artificial sequence
<400> 60
atatggtcac cctcacagcc acac 24
<210> 61
<211> 23
<212> DNA
<213> artificial sequence
<400> 61
cgatatggtc actctcacag caa 23
<210> 62
<211> 23
<212> DNA
<213> artificial sequence
<400> 62
atggtcactc tcacagcaac tct 23
<210> 63
<211> 23
<212> DNA
<213> artificial sequence
<400> 63
gcatagaaag caaggctatt cag 23
<210> 64
<211> 22
<212> DNA
<213> artificial sequence
<400> 64
ttaagcccag tgtggagaag ag 22
<210> 65
<211> 21
<212> DNA
<213> artificial sequence
<400> 65
gttcttagtg tgttcaagcc a 21
<210> 66
<211> 21
<212> DNA
<213> artificial sequence
<400> 66
aggcgcgtcg tcgagttkca a 21
<210> 67
<211> 21
<212> DNA
<213> artificial sequence
<400> 67
acaacaccta ctcgtacaaa g 21
<210> 68
<211> 20
<212> DNA
<213> artificial sequence
<400> 68
gatagaggtc ctagcttcaa 20
<210> 69
<211> 21
<212> DNA
<213> artificial sequence
<400> 69
cctactttta agccctactc c 21
<210> 70
<211> 22
<212> DNA
<213> artificial sequence
<400> 70
caccaacacc tacgagtaca tg 22
<210> 71
<211> 22
<212> DNA
<213> artificial sequence
<400> 71
accaggaaaa ttctgccaca ga 22
<210> 72
<211> 24
<212> DNA
<213> artificial sequence
<400> 72
aacttgtgga agagtggcct tttc 24
<210> 73
<211> 21
<212> DNA
<213> artificial sequence
<400> 73
atgtaaactc catgcattta a 21
<210> 74
<211> 23
<212> DNA
<213> artificial sequence
<400> 74
ttacaccaaa tgaaccaggg aaa 23
<210> 75
<211> 23
<212> DNA
<213> artificial sequence
<400> 75
cagacaaaaa ggactcatca tca 23
<210> 76
<211> 22
<212> DNA
<213> artificial sequence
<400> 76
tgcaaaataa gggaataagg tg 22
<210> 77
<211> 21
<212> DNA
<213> artificial sequence
<400> 77
taccatccag cttgtgactg c 21
<210> 78
<211> 21
<212> DNA
<213> artificial sequence
<400> 78
tggccgacaa ggagacatca t 21
<210> 79
<211> 23
<212> DNA
<213> artificial sequence
<400> 79
aggtccatca cgaccagggg gta 23
<210> 80
<211> 22
<212> DNA
<213> artificial sequence
<400> 80
agaactgaag ataaggccca ag 22
<210> 81
<211> 21
<212> DNA
<213> artificial sequence
<400> 81
cggccggcgg cacccccggg t 21
<210> 82
<211> 22
<212> DNA
<213> artificial sequence
<400> 82
cggtcagcgg gatgtcgtac ag 22
<210> 83
<211> 21
<212> DNA
<213> artificial sequence
<400> 83
ggtttccacc cgggtaattg c 21
<210> 84
<211> 24
<212> DNA
<213> artificial sequence
<400> 84
ggccgcaaaa tttctgacgc caca 24
<210> 85
<211> 21
<212> DNA
<213> artificial sequence
<400> 85
aatgtgtcat tctttggcat t 21
<210> 86
<211> 24
<212> DNA
<213> artificial sequence
<400> 86
tgctcaaatt tgatagcagt ctga 24
<210> 87
<211> 24
<212> DNA
<213> artificial sequence
<400> 87
agatcaacaa atcaatgcaa agct 24
<210> 88
<211> 24
<212> DNA
<213> artificial sequence
<400> 88
ctaatgttgg agaaaatcac aaac 24
<210> 89
<211> 23
<212> DNA
<213> artificial sequence
<400> 89
atccggaaag ttggctgatg acg 23
<210> 90
<211> 22
<212> DNA
<213> artificial sequence
<400> 90
tttccagaag tcctgttgcg tc 22
<210> 91
<211> 21
<212> DNA
<213> artificial sequence
<400> 91
ccgataatat gtccacctca c 21
<210> 92
<211> 22
<212> DNA
<213> artificial sequence
<400> 92
ctcgtgaaac actgaggcaa ta 22
<210> 93
<211> 24
<212> DNA
<213> artificial sequence
<400> 93
tgggatactg gctacgctaa ggtt 24
<210> 94
<211> 24
<212> DNA
<213> artificial sequence
<400> 94
ttacgacatg ccttgcgatg tttt 24
<210> 95
<211> 23
<212> DNA
<213> artificial sequence
<400> 95
gatgaacgcg aacaaacact taa 23
<210> 96
<211> 24
<212> DNA
<213> artificial sequence
<400> 96
taaaattccg acggcagctg tatt 24
<210> 97
<211> 22
<212> DNA
<213> artificial sequence
<400> 97
ggtatgattt cttgacccac aa 22
<210> 98
<211> 23
<212> DNA
<213> artificial sequence
<400> 98
cgcttctcgc caatcttttc ggg 23
<210> 99
<211> 22
<212> DNA
<213> artificial sequence
<400> 99
gaagctgctc gatcagttta ac 22
<210> 100
<211> 22
<212> DNA
<213> artificial sequence
<400> 100
tcagcggaat gctgtttgtg gt 22
<210> 101
<211> 22
<212> DNA
<213> artificial sequence
<400> 101
aaatacaaaa tgttaacgac gc 22
<210> 102
<211> 24
<212> DNA
<213> artificial sequence
<400> 102
ctccggttat cataaaacgg gaca 24
<210> 103
<211> 24
<212> DNA
<213> artificial sequence
<400> 103
agtttcttcg gatactatga tatt 24
<210> 104
<211> 22
<212> DNA
<213> artificial sequence
<400> 104
tgttgtggcg tttatcaatt ag 22
<210> 105
<211> 22
<212> DNA
<213> artificial sequence
<400> 105
tttgagacgc agcgtttatt gt 22
<210> 106
<211> 21
<212> DNA
<213> artificial sequence
<400> 106
tactttaaaa tcggctcgac t 21
<210> 107
<211> 23
<212> DNA
<213> artificial sequence
<400> 107
tatctcttat ggtttgcgtg gtg 23
<210> 108
<211> 22
<212> DNA
<213> artificial sequence
<400> 108
taagtgaata ccagcgaata tt 22
<210> 109
<211> 0
<212> DNA
<213> artificial sequence
<400> 109
<210> 110
<211> 23
<212> DNA
<213> artificial sequence
<400> 110
cccccgggtt cgacctcaac gtc 23
<210> 111
<211> 21
<212> DNA
<213> artificial sequence
<400> 111
gcagatgact cacctgttgg a 21
<210> 112
<211> 21
<212> DNA
<213> artificial sequence
<400> 112
cgactgcacc tggagcagct a 21
<210> 113
<211> 22
<212> DNA
<213> artificial sequence
<400> 113
ttttaaaaag gccaagcaaa ag 22
<210> 114
<211> 24
<212> DNA
<213> artificial sequence
<400> 114
tctttatcga tgaaattgat gctg 24
<210> 115
<211> 23
<212> DNA
<213> artificial sequence
<400> 115
tccttcgctt ttatggcaca agc 23
<210> 116
<211> 21
<212> DNA
<213> artificial sequence
<400> 116
gaacaggatg tcaaagatct a 21
<210> 117
<211> 21
<212> DNA
<213> artificial sequence
<400> 117
gtgtcaaggt gtctttcgga t 21
<210> 118
<211> 24
<212> DNA
<213> artificial sequence
<400> 118
gtgtagatag agagactgag gatg 24
<210> 119
<211> 22
<212> DNA
<213> artificial sequence
<400> 119
tgttagtgga gatccttcta ta 22
<210> 120
<211> 23
<212> DNA
<213> artificial sequence
<400> 120
gaggaacacc cggtgaatac cta 23
<210> 121
<211> 23
<212> DNA
<213> artificial sequence
<400> 121
tgggccgtga acaagactgg gct 23
<210> 122
<211> 21
<212> DNA
<213> artificial sequence
<400> 122
tttatatggt tgggtagtgg a 21
<210> 123
<211> 23
<212> DNA
<213> artificial sequence
<400> 123
aatcgtgcaa gtaaaccaaa taa 23
<210> 124
<211> 24
<212> DNA
<213> artificial sequence
<400> 124
caatgaatca agcgatgcag ctag 24
<210> 125
<211> 24
<212> DNA
<213> artificial sequence
<400> 125
attctcacag gcttgcttgc tgga 24
<210> 126
<211> 24
<212> DNA
<213> artificial sequence
<400> 126
agcgtaggcg tcggtgacaa aggc 24
<210> 127
<211> 21
<212> DNA
<213> artificial sequence
<400> 127
ccctcacggt tcagggttag c 21
<210> 128
<211> 24
<212> DNA
<213> artificial sequence
<400> 128
gatgtcaaga acttcgacgg ccgt 24
<210> 129
<211> 24
<212> DNA
<213> artificial sequence
<400> 129
gttcctttct tgggcagagc acgc 24
<210> 130
<211> 24
<212> DNA
<213> artificial sequence
<400> 130
ttgcttattt ggtttccacc cggg 24
<210> 131
<211> 21
<212> DNA
<213> artificial sequence
<400> 131
atcttcatct ggtccaggcc c 21
<210> 132
<211> 22
<212> DNA
<213> artificial sequence
<400> 132
attggtgaag agtttggacc ct 22
<210> 133
<211> 23
<212> DNA
<213> artificial sequence
<400> 133
tcgtgggctg cctgttgttt tac 23
<210> 134
<211> 22
<212> DNA
<213> artificial sequence
<400> 134
ttgtcgttgc agtgattacg at 22
<210> 135
<211> 21
<212> DNA
<213> artificial sequence
<400> 135
tggaaatgag ctggttcgtt t 21
<210> 136
<211> 24
<212> DNA
<213> artificial sequence
<400> 136
gcatgaagac ggattcaaac gtgc 24
<210> 137
<211> 22
<212> DNA
<213> artificial sequence
<400> 137
gaagtcttgg aggtatgcaa gc 22
<210> 138
<211> 24
<212> DNA
<213> artificial sequence
<400> 138
gggaccagcg gatatgaacg aaca 24
<210> 139
<211> 21
<212> DNA
<213> artificial sequence
<400> 139
ttcagctact tgcaagattg g 21
<210> 140
<211> 22
<212> DNA
<213> artificial sequence
<400> 140
ttcgggcaga caagctgctt tt 22
<210> 141
<211> 22
<212> DNA
<213> artificial sequence
<400> 141
aggcaaaatc gtgtttacgg ga 22
<210> 142
<211> 24
<212> DNA
<213> artificial sequence
<400> 142
gcctggtcaa aatgggcgcg aaaa 24
<210> 143
<211> 23
<212> DNA
<213> artificial sequence
<400> 143
aggctgcctg gaaggtgtcg ggt 23
<210> 144
<211> 22
<212> DNA
<213> artificial sequence
<400> 144
cctcggtgcg accccaactc ga 22
<210> 145
<211> 23
<212> DNA
<213> artificial sequence
<400> 145
gatatgccgt cgaaagtcct gag 23
<210> 146
<211> 24
<212> DNA
<213> artificial sequence
<400> 146
cgaaccggcg gattataaaa gtgt 24
<210> 147
<211> 22
<212> DNA
<213> artificial sequence
<400> 147
tgttgttcag gttttcataa gt 22
<210> 148
<211> 21
<212> DNA
<213> artificial sequence
<400> 148
tcgttttcgg cgcggcattc c 21
<210> 149
<211> 24
<212> DNA
<213> artificial sequence
<400> 149
tacaagaaag tcgtcctatt ttaa 24
<210> 150
<211> 23
<212> DNA
<213> artificial sequence
<400> 150
gaggcggtaa cgatgaacga gaa 23
<210> 151
<211> 23
<212> DNA
<213> artificial sequence
<400> 151
gatcattcgt tggcactgac atc 23
<210> 152
<211> 22
<212> DNA
<213> artificial sequence
<400> 152
cactgtatac cgatgaaact tt 22
<210> 153
<211> 23
<212> DNA
<213> artificial sequence
<400> 153
tatcaagggt cgatttttcc agc 23
<210> 154
<211> 22
<212> DNA
<213> artificial sequence
<400> 154
tgaatgtcac tagtaatgat gc 22
<210> 155
<211> 21
<212> DNA
<213> artificial sequence
<400> 155
cgctgctgat gctgctggac t 21
<210> 156
<211> 21
<212> DNA
<213> artificial sequence
<400> 156
aaaggtatcc atgccgcggc g 21
<210> 157
<211> 23
<212> DNA
<213> artificial sequence
<400> 157
ccatcatcca cagcgagaac tgg 23
<210> 158
<211> 22
<212> DNA
<213> artificial sequence
<400> 158
tgcggctgct gatagaacgc cg 22
<210> 159
<211> 24
<212> DNA
<213> artificial sequence
<400> 159
tcgtttggaa ggcttgactc ttgt 24
<210> 160
<211> 23
<212> DNA
<213> artificial sequence
<400> 160
tttaacattt cttgtgcaaa ctc 23
<210> 161
<211> 23
<212> DNA
<213> artificial sequence
<400> 161
gttatccctt tctcttctca agc 23
<210> 162
<211> 23
<212> DNA
<213> artificial sequence
<400> 162
agcgttttgt atttgcaatg cca 23
<210> 163
<211> 22
<212> DNA
<213> artificial sequence
<400> 163
gactcaggtc cctataggct tt 22
<210> 164
<211> 23
<212> DNA
<213> artificial sequence
<400> 164
aaccataatc cgaacgaagt cca 23
<210> 165
<211> 24
<212> DNA
<213> artificial sequence
<400> 165
tgataatgca ctgggggcat atct 24
<210> 166
<211> 23
<212> DNA
<213> artificial sequence
<400> 166
atctgctgct caatcgctcg ggt 23
<210> 167
<211> 24
<212> DNA
<213> artificial sequence
<400> 167
gacgcttaac atcttatcgt catg 24
<210> 168
<211> 21
<212> DNA
<213> artificial sequence
<400> 168
ttacaatgtc gagcctctag g 21
<210> 169
<211> 24
<212> DNA
<213> artificial sequence
<400> 169
tggttacggc taaaaatgtc agca 24
<210> 170
<211> 23
<212> DNA
<213> artificial sequence
<400> 170
attatcgtcc tactgatttg gcg 23
<210> 171
<211> 24
<212> DNA
<213> artificial sequence
<400> 171
ttgggccttt ttgagaatct gcac 24
<210> 172
<211> 22
<212> DNA
<213> artificial sequence
<400> 172
gcaactaaag attccagctc gg 22
<210> 173
<211> 22
<212> DNA
<213> artificial sequence
<400> 173
gaaagccgtc caatcttaac ag 22
<210> 174
<211> 24
<212> DNA
<213> artificial sequence
<400> 174
aggtgggaca tgtaaagttt tccc 24
<210> 175
<211> 22
<212> DNA
<213> artificial sequence
<400> 175
gttttagtaa tgttttagta gg 22
<210> 176
<211> 24
<212> DNA
<213> artificial sequence
<400> 176
aggatggtca aaagttatat cttc 24
<210> 177
<211> 24
<212> DNA
<213> artificial sequence
<400> 177
gcgtcgtgtt cgggagttgg tgga 24
<210> 178
<211> 23
<212> DNA
<213> artificial sequence
<400> 178
aagtcccttt aagagctcaa tga 23
<210> 179
<211> 21
<212> DNA
<213> artificial sequence
<400> 179
agcatgagag cttttaattt c 21
<210> 180
<211> 24
<212> DNA
<213> artificial sequence
<400> 180
atatcttcat atctgatttt atca 24
<210> 181
<211> 23
<212> DNA
<213> artificial sequence
<400> 181
aattccatat ctgattgtat tga 23
<210> 182
<211> 22
<212> DNA
<213> artificial sequence
<400> 182
ttttgggaca tcttccacat gt 22
<210> 183
<211> 22
<212> DNA
<213> artificial sequence
<400> 183
tcaagatcat ctgttatcaa tg 22
<210> 184
<211> 23
<212> DNA
<213> artificial sequence
<400> 184
cgattgcaga tccaacacct aac 23
<210> 185
<211> 23
<212> DNA
<213> artificial sequence
<400> 185
atctataaga tgagggtgtt ttt 23
<210> 186
<211> 23
<212> DNA
<213> artificial sequence
<400> 186
ttcaaaatgg taatcacctt ttg 23
<210> 187
<211> 23
<212> DNA
<213> artificial sequence
<400> 187
ccatttaggg cgttttggac aaa 23
<210> 188
<211> 23
<212> DNA
<213> artificial sequence
<400> 188
gggtgtctga caagttgtat tgc 23
<210> 189
<211> 21
<212> DNA
<213> artificial sequence
<400> 189
ggtaacagtt gctgtaggct t 21
<210> 190
<211> 23
<212> DNA
<213> artificial sequence
<400> 190
accgatttgc ttctctacct cat 23
<210> 191
<211> 21
<212> DNA
<213> artificial sequence
<400> 191
tattaccttg ctcctgccac t 21
<210> 192
<211> 24
<212> DNA
<213> artificial sequence
<400> 192
atcctagtgt cttgagaaaa tacc 24
<210> 193
<211> 24
<212> DNA
<213> artificial sequence
<400> 193
tccctaatcc caaagaggct aatg 24
<210> 194
<211> 22
<212> DNA
<213> artificial sequence
<400> 194
aagtatacac attcaccgtt at 22
<210> 195
<211> 23
<212> DNA
<213> artificial sequence
<400> 195
ttggagcttg ttgaatgggt tga 23
<210> 196
<211> 24
<212> DNA
<213> artificial sequence
<400> 196
aaacagtatt accataagta gtaa 24
<210> 197
<211> 23
<212> DNA
<213> artificial sequence
<400> 197
aaataataaa gaaattacaa aaa 23
<210> 198
<211> 22
<212> DNA
<213> artificial sequence
<400> 198
tgcattagtt tcaggattaa aa 22
<210> 199
<211> 23
<212> DNA
<213> artificial sequence
<400> 199
aaaacgattt tcataaaatg att 23
<210> 200
<211> 22
<212> DNA
<213> artificial sequence
<400> 200
taactatttc aagtctagcc gg 22
<210> 201
<211> 23
<212> DNA
<213> artificial sequence
<400> 201
aacagctaag aaaaccagtt tgt 23
<210> 202
<211> 23
<212> DNA
<213> artificial sequence
<400> 202
cgacatcagt actagtgcct gtg 23
<210> 203
<211> 21
<212> DNA
<213> artificial sequence
<400> 203
tttcttagtg acagtttggc c 21
<210> 204
<211> 23
<212> DNA
<213> artificial sequence
<400> 204
tcctaggtaa gctctaactt ctt 23
<210> 205
<211> 22
<212> DNA
<213> artificial sequence
<400> 205
aaatctatgt atgttagcac ag 22
<210> 206
<211> 21
<212> DNA
<213> artificial sequence
<400> 206
tgtgacactg ttatatggta t 21
<210> 207
<211> 23
<212> DNA
<213> artificial sequence
<400> 207
ataggtatgt cgagtaccgt cag 23
<210> 208
<211> 22
<212> DNA
<213> artificial sequence
<400> 208
ccatcccgca attactcrtt ac 22
<210> 209
<211> 21
<212> DNA
<213> artificial sequence
<400> 209
gcacatttca gtaaactttt t 21
<210> 210
<211> 23
<212> DNA
<213> artificial sequence
<400> 210
gcattgcatg catcattgaa ttt 23
<210> 211
<211> 23
<212> DNA
<213> artificial sequence
<400> 211
tcagtgaact tcttgagcca act 23
<210> 212
<211> 24
<212> DNA
<213> artificial sequence
<400> 212
ctgtgaactt cttaagccat gatt 24
<210> 213
<211> 21
<212> DNA
<213> artificial sequence
<400> 213
ttcaagatgt tgcaggcgag c 21
<210> 214
<211> 24
<212> DNA
<213> artificial sequence
<400> 214
tatggactcc tgattattaa taaa 24
<210> 215
<211> 21
<212> DNA
<213> artificial sequence
<400> 215
ttgggcaggc ttgaagcatc a 21
<210> 216
<211> 21
<212> DNA
<213> artificial sequence
<400> 216
tatgtttgga catgagttgc t 21
<210> 217
<211> 24
<212> DNA
<213> artificial sequence
<400> 217
ggcattgctc acacactcgg ggac 24
<210> 218
<211> 21
<212> DNA
<213> artificial sequence
<400> 218
gtctcgtacg tcgtgaccta c 21
<210> 219
<211> 22
<212> DNA
<213> artificial sequence
<400> 219
gtctcgtaca tcgtgaccta cc 22
<210> 220
<211> 24
<212> DNA
<213> artificial sequence
<400> 220
tggcggccgt gcacgtcgcc ttaa 24
<210> 221
<211> 24
<212> DNA
<213> artificial sequence
<400> 221
cgtctgtaca gctcgacgat atcg 24
<210> 222
<211> 24
<212> DNA
<213> artificial sequence
<400> 222
gttttccatc agaaaactat atac 24
<210> 223
<211> 22
<212> DNA
<213> artificial sequence
<400> 223
gggggtcggg ctgggccgcc ag 22
<210> 224
<211> 24
<212> DNA
<213> artificial sequence
<400> 224
ggggagaggg aaggcgactc gccc 24
<210> 225
<211> 21
<212> DNA
<213> artificial sequence
<400> 225
cgtgtgcgcg aaagcctgtt c 21
<210> 226
<211> 22
<212> DNA
<213> artificial sequence
<400> 226
ggtataggta atgctactgt ga 22
<210> 227
<211> 24
<212> DNA
<213> artificial sequence
<400> 227
aaaagcaaac agtattttgt ttgc 24
<210> 228
<211> 23
<212> DNA
<213> artificial sequence
<400> 228
atcactgttt tccagcatga aaa 23
<210> 229
<211> 22
<212> DNA
<213> artificial sequence
<400> 229
aaagggtatt tgtagtgcta ta 22
<210> 230
<211> 21
<212> DNA
<213> artificial sequence
<400> 230
acttgttcta ttaagcgtgc c 21
<210> 231
<211> 23
<212> DNA
<213> artificial sequence
<400> 231
tttcaagcat gaagaatggt atc 23
<210> 232
<211> 24
<212> DNA
<213> artificial sequence
<400> 232
ccaagttttt ctccccatgc cgtc 24
<210> 233
<211> 21
<212> DNA
<213> artificial sequence
<400> 233
tctatttgta gttcttgaaa t 21
<210> 234
<211> 21
<212> DNA
<213> artificial sequence
<400> 234
ataaaaggaa acacggmcac c 21
<210> 235
<211> 21
<212> DNA
<213> artificial sequence
<400> 235
aacttcttta gccatgaggc a 21
<210> 236
<211> 21
<212> DNA
<213> artificial sequence
<400> 236
gatgcgctct gtttctgagc a 21
<210> 237
<211> 24
<212> DNA
<213> artificial sequence
<400> 237
ttgaattttt ttagccatga tgcg 24
<210> 238
<211> 23
<212> DNA
<213> artificial sequence
<400> 238
acaatctgaa tacctttggt aga 23
<210> 239
<211> 21
<212> DNA
<213> artificial sequence
<400> 239
tttcattttt gtctggtcct t 21
<210> 240
<211> 22
<212> DNA
<213> artificial sequence
<400> 240
taatatcaga agcatagtct ag 22
<210> 241
<211> 24
<212> DNA
<213> artificial sequence
<400> 241
gaagtaggtg tctgtggcgc gsgc 24
<210> 242
<211> 21
<212> DNA
<213> artificial sequence
<400> 242
cgccgcggat gtcaaagtag g 21
<210> 243
<211> 22
<212> DNA
<213> artificial sequence
<400> 243
caatgccaaa tgtgtttgtg gt 22
<210> 244
<211> 20
<212> DNA
<213> artificial sequence
<400> 244
gcgttttgct cttcttcttc 20
<210> 245
<211> 21
<212> DNA
<213> artificial sequence
<400> 245
ggtggttgaa gggatttacg t 21
<210> 246
<211> 24
<212> DNA
<213> artificial sequence
<400> 246
tctcaaagct tctgccattt ctgc 24
<210> 247
<211> 22
<212> DNA
<213> artificial sequence
<400> 247
cctcgaatac cttcaacata tc 22
<210> 248
<211> 24
<212> DNA
<213> artificial sequence
<400> 248
acaataacca ccaggcatgt catt 24
<210> 249
<211> 21
<212> DNA
<213> artificial sequence
<400> 249
ttcttcaaag gttaagattg a 21
<210> 250
<211> 24
<212> DNA
<213> artificial sequence
<400> 250
ttgcagagtt tgtatacctt tgaa 24
<210> 251
<211> 23
<212> DNA
<213> artificial sequence
<400> 251
aagatcatcg tggttttcat ttt 23
<210> 252
<211> 23
<212> DNA
<213> artificial sequence
<400> 252
aagatctaga gatgtacaaa caa 23
<210> 253
<211> 23
<212> DNA
<213> artificial sequence
<400> 253
atggccgccc ttgaccaggt tgc 23
<210> 254
<211> 24
<212> DNA
<213> artificial sequence
<400> 254
tggcccccct gctggtggcc gcca 24
<210> 255
<211> 21
<212> DNA
<213> artificial sequence
<400> 255
ctttcaggtg agacgctttt g 21
<210> 256
<211> 24
<212> DNA
<213> artificial sequence
<400> 256
gatgtcctcg acctcgtcct tcag 24
<210> 257
<211> 21
<212> DNA
<213> artificial sequence
<400> 257
agtcgctgtt cgcggccgcg c 21
<210> 258
<211> 24
<212> DNA
<213> artificial sequence
<400> 258
agacgcccgt cggttgcccg taca 24
<210> 259
<211> 21
<212> DNA
<213> artificial sequence
<400> 259
agagtagttc cgtgttgccg a 21
<210> 260
<211> 24
<212> DNA
<213> artificial sequence
<400> 260
aatgccatct tgttctaatt cccg 24
<210> 261
<211> 24
<212> DNA
<213> artificial sequence
<400> 261
ttctttttta ttggataatc tcct 24
<210> 262
<211> 22
<212> DNA
<213> artificial sequence
<400> 262
aatttcattt tgcaacttct tt 22
<210> 263
<211> 23
<212> DNA
<213> artificial sequence
<400> 263
gtttgggtat tgggttgaca tat 23
<210> 264
<211> 23
<212> DNA
<213> artificial sequence
<400> 264
caacacactc tcctgcaaca cgc 23
<210> 265
<211> 22
<212> DNA
<213> artificial sequence
<400> 265
tgaaatgccg gctgtgtgat ca 22
<210> 266
<211> 23
<212> DNA
<213> artificial sequence
<400> 266
gacccatacc tcaattgaga cgc 23
<210> 267
<211> 24
<212> DNA
<213> artificial sequence
<400> 267
tcaactcgct tatcaactgt aaaa 24
<210> 268
<211> 21
<212> DNA
<213> artificial sequence
<400> 268
acccgctggg aacaaccaga t 21
<210> 269
<211> 21
<212> DNA
<213> artificial sequence
<400> 269
aattcttcgt caacagagat a 21
<210> 270
<211> 22
<212> DNA
<213> artificial sequence
<400> 270
gacctggacg aagaagggct gg 22
<210> 271
<211> 21
<212> DNA
<213> artificial sequence
<400> 271
ctttcctcaa gaatctgtct a 21
<210> 272
<211> 22
<212> DNA
<213> artificial sequence
<400> 272
atgaaatatt aactaataaa ca 22
<210> 273
<211> 22
<212> DNA
<213> artificial sequence
<400> 273
accggccgaa cgcggcggct tc 22
<210> 274
<211> 24
<212> DNA
<213> artificial sequence
<400> 274
ctcctgctcg cgcagaaacc ggga 24
<210> 275
<211> 24
<212> DNA
<213> artificial sequence
<400> 275
agaccgttat caccacaaac ggca 24
<210> 276
<211> 23
<212> DNA
<213> artificial sequence
<400> 276
cgatgtatca acctgcttag tat 23
<210> 277
<211> 23
<212> DNA
<213> artificial sequence
<400> 277
gatgaatgta tgtttacaat gtt 23
<210> 278
<211> 22
<212> DNA
<213> artificial sequence
<400> 278
gggttaaagc cataaagaaa at 22
<210> 279
<211> 23
<212> DNA
<213> artificial sequence
<400> 279
aattttaccc ctcaatttta aaa 23
<210> 280
<211> 23
<212> DNA
<213> artificial sequence
<400> 280
ccgaccatgg cgatttacag tta 23
<210> 281
<211> 23
<212> DNA
<213> artificial sequence
<400> 281
ttccctgtat agaacgggtg cga 23
<210> 282
<211> 21
<212> DNA
<213> artificial sequence
<400> 282
taatccgcgg cgaaaaaaga c 21
<210> 283
<211> 22
<212> DNA
<213> artificial sequence
<400> 283
tcgtcaaatt aatggcgaca tc 22
<210> 284
<211> 22
<212> DNA
<213> artificial sequence
<400> 284
gaaacgactg cgactacaag gg 22
<210> 285
<211> 23
<212> DNA
<213> artificial sequence
<400> 285
gacgtcctcg atctcgtcct tca 23
<210> 286
<211> 21
<212> DNA
<213> artificial sequence
<400> 286
tctcctcgcg cggcccgagg g 21
<210> 287
<211> 21
<212> DNA
<213> artificial sequence
<400> 287
aacgtaattt tatcggccac c 21
<210> 288
<211> 21
<212> DNA
<213> artificial sequence
<400> 288
atcttgatca tcatcatcag c 21
<210> 289
<211> 24
<212> DNA
<213> artificial sequence
<400> 289
gtcgcagcga tgacgataat ccct 24
<210> 290
<211> 22
<212> DNA
<213> artificial sequence
<400> 290
cgggatccca gcggtaggta gc 22
<210> 291
<211> 21
<212> DNA
<213> artificial sequence
<400> 291
ttcaatatgc catttagccc t 21
<210> 292
<211> 23
<212> DNA
<213> artificial sequence
<400> 292
ccttcaaaat ggctctcccc act 23
<210> 293
<211> 21
<212> DNA
<213> artificial sequence
<400> 293
acttctcctc gcaatccagg a 21
<210> 294
<211> 24
<212> DNA
<213> artificial sequence
<400> 294
ctgaatcgca atttttttcc ctct 24
<210> 295
<211> 22
<212> DNA
<213> artificial sequence
<400> 295
ccttactgtc cacttggaac gg 22
<210> 296
<211> 23
<212> DNA
<213> artificial sequence
<400> 296
tgcaacactt cataaaagta gtt 23
<210> 297
<211> 21
<212> DNA
<213> artificial sequence
<400> 297
gccaaccatt ctgatagagc t 21
<210> 298
<211> 23
<212> DNA
<213> artificial sequence
<400> 298
ttagttaaat agttccattc tac 23
<210> 299
<211> 24
<212> DNA
<213> artificial sequence
<400> 299
aaggcgcatc caccagatga taac 24
<210> 300
<211> 23
<212> DNA
<213> artificial sequence
<400> 300
tcctgaggag gtgatcccgc caa 23
<210> 301
<211> 21
<212> DNA
<213> artificial sequence
<400> 301
ggccacagcc cgtcccgccg a 21
<210> 302
<211> 21
<212> DNA
<213> artificial sequence
<400> 302
ccagccgccg cgagctgcgc g 21
<210> 303
<211> 21
<212> DNA
<213> artificial sequence
<400> 303
ccaacgtctc tatacaattg g 21
<210> 304
<211> 22
<212> DNA
<213> artificial sequence
<400> 304
tccggtgcac cagcgtctcg ac 22
<210> 305
<211> 21
<212> DNA
<213> artificial sequence
<400> 305
ggtcgtagac gcccgtcggt t 21
<210> 306
<211> 22
<212> DNA
<213> artificial sequence
<400> 306
acggtggagt actgcaacca gc 22
<210> 307
<211> 24
<212> DNA
<213> artificial sequence
<400> 307
gtattcaaag ggttttgagg tgaa 24
<210> 308
<211> 22
<212> DNA
<213> artificial sequence
<400> 308
gcctcgtgct tttaatgtct ca 22
<210> 309
<211> 23
<212> DNA
<213> artificial sequence
<400> 309
aggttggttt acgtaatttt att 23
<210> 310
<211> 21
<212> DNA
<213> artificial sequence
<400> 310
aacaagcttt ttattatcta a 21
<210> 311
<211> 21
<212> DNA
<213> artificial sequence
<400> 311
ttgatagata taattgctgt c 21
<210> 312
<211> 24
<212> DNA
<213> artificial sequence
<400> 312
ccttgcaatt tcattgaaag ctct 24
<210> 313
<211> 24
<212> DNA
<213> artificial sequence
<400> 313
cagaggcaga acgttgtccg aagc 24
<210> 314
<211> 24
<212> DNA
<213> artificial sequence
<400> 314
gcaattattg tcgggatctt agtt 24
<210> 315
<211> 23
<212> DNA
<213> artificial sequence
<400> 315
ggggtctatc aaatgatcat cta 23
<210> 316
<211> 24
<212> DNA
<213> artificial sequence
<400> 316
cgaagtgttt tcaaacagga tgtt 24
<210> 317
<211> 23
<212> DNA
<213> artificial sequence
<400> 317
ccaccgcaca caggaacgtc ccc 23
<210> 318
<211> 24
<212> DNA
<213> artificial sequence
<400> 318
gtgaggacgg cgctgcgtgc ggcg 24
<210> 319
<211> 23
<212> DNA
<213> artificial sequence
<400> 319
gacgaattgc gcgccgcgct ggc 23
<210> 320
<211> 23
<212> DNA
<213> artificial sequence
<400> 320
ggccgatgtg tgacttttcg gcc 23
<210> 321
<211> 21
<212> DNA
<213> artificial sequence
<400> 321
acccgggaag acggagaaac g 21
<210> 322
<211> 24
<212> DNA
<213> artificial sequence
<400> 322
cgtgtttacg ggaggccgga ccaa 24
<210> 323
<211> 21
<212> DNA
<213> artificial sequence
<400> 323
gtacgtccac aatgattgtg g 21
<210> 324
<211> 24
<212> DNA
<213> artificial sequence
<400> 324
tgaagtattg tccaaagtga ttaa 24
<210> 325
<211> 22
<212> DNA
<213> artificial sequence
<400> 325
aaagctggat ctaaaacatc ac 22
<210> 326
<211> 0
<212> DNA
<213> artificial sequence
<400> 326
<210> 327
<211> 22
<212> DNA
<213> artificial sequence
<400> 327
gatgttggat cattcgttct gc 22
<210> 328
<211> 21
<212> DNA
<213> artificial sequence
<400> 328
cctaaagtaa tgacaatcgt t 21
<210> 329
<211> 23
<212> DNA
<213> artificial sequence
<400> 329
cggcgctctc tagcggaacc aaa 23
<210> 330
<211> 21
<212> DNA
<213> artificial sequence
<400> 330
cagcaggccg atgtgcacca g 21
<210> 331
<211> 23
<212> DNA
<213> artificial sequence
<400> 331
cggcagcttg tcgtccggta cgt 23
<210> 332
<211> 22
<212> DNA
<213> artificial sequence
<400> 332
agccgcagcg ggcacggcaa tc 22
<210> 333
<211> 23
<212> DNA
<213> artificial sequence
<400> 333
ggcagccggg ctggaagtgg gag 23
<210> 334
<211> 23
<212> DNA
<213> artificial sequence
<400> 334
tctgccaatt tcacgatatc ttc 23
<210> 335
<211> 21
<212> DNA
<213> artificial sequence
<400> 335
tttcgtgaca agatgatcca g 21
<210> 336
<211> 23
<212> DNA
<213> artificial sequence
<400> 336
cctatccctt taaaagagcc tgt 23
<210> 337
<211> 23
<212> DNA
<213> artificial sequence
<400> 337
taaccctgcc ttcctggcat cat 23
<210> 338
<211> 22
<212> DNA
<213> artificial sequence
<400> 338
aaaataaata ttgggattta ac 22
<210> 339
<211> 24
<212> DNA
<213> artificial sequence
<400> 339
acacagacaa aaacacgcca gaaa 24
<210> 340
<211> 21
<212> DNA
<213> artificial sequence
<400> 340
gatccacacc cggcaaattc c 21
<210> 341
<211> 23
<212> DNA
<213> artificial sequence
<400> 341
tgaggagccg ccattgaaga tct 23
<210> 342
<211> 24
<212> DNA
<213> artificial sequence
<400> 342
acgtgcctca atataatgcc aata 24
<210> 343
<211> 24
<212> DNA
<213> artificial sequence
<400> 343
tattaattca attttataag cata 24
<210> 344
<211> 21
<212> DNA
<213> artificial sequence
<400> 344
gctcgtttga cgacttcacg a 21
<210> 345
<211> 21
<212> DNA
<213> artificial sequence
<400> 345
ttttagttca gctaccgtgg c 21
<210> 346
<211> 24
<212> DNA
<213> artificial sequence
<400> 346
aggaccggat gtattccctt gcgg 24
<210> 347
<211> 23
<212> DNA
<213> artificial sequence
<400> 347
gcggggctgt tggccacgac gga 23
<210> 348
<211> 24
<212> DNA
<213> artificial sequence
<400> 348
agggatttac ttggaacaac caaa 24
<210> 349
<211> 23
<212> DNA
<213> artificial sequence
<400> 349
taaccggtaa cgcaactgtg gca 23
<210> 350
<211> 22
<212> DNA
<213> artificial sequence
<400> 350
aactatagta gttagaacaa gt 22
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