CN107400714A - The multiple PCR primer group and kit of colorectal cancer medication related gene detection - Google Patents

The multiple PCR primer group and kit of colorectal cancer medication related gene detection Download PDF

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CN107400714A
CN107400714A CN201710721477.2A CN201710721477A CN107400714A CN 107400714 A CN107400714 A CN 107400714A CN 201710721477 A CN201710721477 A CN 201710721477A CN 107400714 A CN107400714 A CN 107400714A
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CN107400714B (en
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赖炳权
何广良
唐毅
张纪斌
李伟琴
林钊
许少飞
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Guangzhou Forevergendx Technology Co ltd
Guangzhou Forevergen Biotechnology Co ltd
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Abstract

The invention discloses a kind of multiple PCR primer group of colorectal cancer medication related gene detection, primer sets include sense primer group and anti-sense primer group, wherein, 5 ' terminal sequences of sense primer and the partial sequence complementarity of sequencing primer, 3 ' ends of sense primer are the specific sequence for being attached to target area；5 ' terminal sequences of anti-sense primer and the partial sequence complementarity of sequencing primer, 3 ' ends of anti-sense primer are the specific sequence for being attached to target area, and the centre position sequence of anti-sense primer is molecular label sequence.Also disclose the detection kit containing multiple PCR primer group and the detection method of colorectal cancer medication related gene, using the mutation of the kit of the present invention 8 gene extron subregions related to the targeted drug that detection method can detect treatment colorectal cancer simultaneously 23 pleomorphism sites related to chemotherapeutics, adjuvant clinical colorectal cancer medication is used directly for, and is monitored more afterwards for early diagnosis of cancer, auxiliary diagnosis and examination or cancer.

Description

The multiple PCR primer group and kit of colorectal cancer medication related gene detection
Technical field
The present invention relates to dihydrate gypsum preparing technical field, especially a kind of colorectal cancer personalized medicine related gene inspection Multiple PCR primer group, kit and the detection method of survey.
Background technology
Colorectal cancer (Colorectal Cancer, CRC) is the general name of colon cancer and the carcinoma of the rectum, refers to colorectal mucosa epithelium The canceration occurred under a variety of carcinogenic factor effects such as environment or heredity, is the third-largest common cancer in the whole world, it is dead Rate is higher.Treatment of cancer at present is in addition to the modes such as surgery excision, radiotherapy, also chemotherapy and for cancer cell The targeted drug treatment attacked.Targeted drug treatment is to utilize structure unique in cancer cell or molecule, with selectivity These special constructions of drug attack and then kill cancer cell.Most of all, target medicine less likely attacks normal cell, align Normal cell damage is smaller, can reduce many side effects phenomena such as (such as reduction of white blood cell, blood platelet, anaemia, vomitting, losing hair), Also cancer cell can be suppressed to increase, shift and drug-fast vicious transformation ability.Targeted drug treatment and treatment of cancer in recent years New trend, and the research emphasis that medical field is most popular, during many clinical experimental studies as target are just persistently carried out.
At present, U.S.'s food and medicine Surveillance Authority (FDA) ratifies to have for the targeted drug for the treatment of of colorectal cancer：It is western appropriate Former times monoclonal antibody, bevacizumab, bevacizumab, Rui Gefeini, thunder not Lu Dankang and VEGF Trap.The base related to said medicine drug effect Because of totally 8, including：KRAS、NRAS、BRAF、EGFR、PIK3CA、MET、HER2、PTEN.Meanwhile the change clinically used at present Treating medicine includes：Cis-platinum, carboplatin, oxaliplatin, taxol, Docetaxel, vinorelbine, vinblastine, vincristine, according to Support pool glycosides, gemcitabine, fluorouracil, capecitabine, endoxan, Methotrexate/ methotrexate (MTX)s, Irinotecan, training Beautiful Qu Sai, Doxorubicin, epirubicin, TAM, Letrozole, Temozolomide, the polymorphism related to said medicine drug effect Totally 23, site, including：rs1042522、rs11615、rs12132152、rs13181、rs1695、rs1799782、 rs1799983、rs1801131、rs1801133、rs2032582、rs2070744、rs2297595、rs25487、 rs3212986、rs34743033、rs3918290、rs4148323、rs8175347、BAT-25、BAT-26、D2S123、 D5S346、D17S250。
High throughput sequencing technologies (high throughput sequencing) are also known as sequencing (next of future generation Generation sequencing, NGS) or large-scale parallel sequencing (massively parallel sequencing, MPS), sequencing once can be carried out to millions of DNA moleculars to hundreds of thousands parallel, substantially increases the flux of sequencing.Together When, with the fast development of bioinformatics, the analyses of high-flux sequence data has been realized in increasingly automated.Based on this, In the market emerges the more and more gene tester based on high-flux sequence, including hereditary breast cancer BRCA1/2 inspections Survey.
Human haploid Genome Size is 3,000,000,000 base-pairs or so, and data volume is huge.Therefore, consider research purpose and Economic benefit, generally require and target sequence enrichment interested is first carried out before high-flux sequence.Currently used sequence is rich Diversity method include chip hybridization capture and multiplexed PCR amplification, core technology mainly grasp abroad big biotechnology company on hand, Such as Roche, Agilent, Life Technologies.Wherein, it is that externally can show subgroup even more the characteristics of hybrid capture Big target area is captured, but operating process is more complicated, it is necessary to rely on specialized equipment equipment；Multiplex PCR spirit simple to operate It is living, it is only necessary to which that PCR instrument can be carried out, and target sequence enrichment and library construction can be completed within several hours, suitable for relatively Small target sequence enrichment.
The content of the invention
Based on this, one kind is provided and can be used for instructing it is an object of the invention to overcome above-mentioned the deficiencies in the prior art part 8 gene extron region mutagenesis of colorectal cancer personalized medicine and the multiple PCR primer group of 23 pleomorphism site detections, 8 gene extron subregions and 23 pleomorphism site correlated serieses can be quickly and accurately amplified by the primer sets, so as to The mutation of 8 gene extron subregions of detection and 23 pleomorphism sites, to instruct the personalized medicine of colorectal cancer patients (targeted drug treatment), improve therapeutic effect.
To achieve the above object, the technical scheme taken of the present invention is：A kind of colorectal cancer medication related gene detection Multiple PCR primer group, the primer sets include sense primer group and anti-sense primer group, wherein, 5 ' end sequences of the sense primer The partial sequence complementarity of row and sequencing primer, 3 ' ends of the sense primer are the specific sequence for being attached to target area；Institute 5 ' terminal sequences of anti-sense primer and the partial sequence complementarity of sequencing primer are stated, 3 ' ends of the anti-sense primer are to be attached to target area The specific sequence in domain, the centre position sequence of the anti-sense primer is molecular label sequence；The multiple PCR primer group is used for The nucleotide sequence of multiple target areas in testing sample is expanded；The target area that the primer sets are used to expand includes Sequence around 8 targeted drug related gene exon regions and 23 chemotherapeutics pleomorphism sites, wherein, 8 targets It is to pharmaceutical relevant gene：KRAS, NRAS, BRAF, EGFR, PIK3CA, MET, HER2 and PTEN；23 chemotherapeutics are more State property site is：rs1042522、rs11615、rs12132152、rs13181、rs1695、rs1799782、rs1799983、 rs1801131、rs1801133、rs2032582、rs2070744、rs2297595、rs25487、rs3212986、 Rs34743033, rs3918290, rs4148323, rs8175347, BAT-25, BAT-26, D2S123, D5S346 and D17S250。
It should be noted that surrounding sequence refers to the sequence of each 100bp or so before and after pleomorphism site；Sequencing primer refers to The adapter-primer of Illuminate sequencer library constructions, the adapter-primer are SEQ ID NO:432 and SEQ ID NO:Nucleotide sequence shown in 433；And anti-sense primer 5 ' end also with SEQ ID NO:Nucleotide sequence shown in 431 is complementary.
Preferably, the sense primer group is SEQ ID NO:216~SEQ ID NO:Nucleotide sequence shown in 430. It should be noted that SEQ ID NO:As general anti-sense primer (URS), itself is nucleotide sequence shown in 431 A part for Illuminate sequenator adapter-primers, the product downstream of first round PCR amplifications can be specifically bound to.
Preferably, the anti-sense primer group is SEQ ID NO:1~SEQ ID NO:Nucleotide sequence shown in 215.Should When explanation, multiple PCR primer group of the invention includes but is not limited to SEQ ID NO:1~SEQ ID NO:Shown in 431 Nucleotide sequence, in addition to SEQ ID NO:1~SEQ ID NO:Nucleotide sequence identity shown in 431 reaches 90%, 91%th, 92%, 93%, 95%, 96%, 97%, 98%, 99% nucleotide sequence, as long as primer can be by the core of target area Nucleotide sequence, which amplifies, to be come, and is within the scope of the present invention；" n " in primer sets is any one in A, C, G, T Kind base.
Preferably, the primer sets also include SEQ ID NO:Nucleotide sequence shown in 431 and for Illuminate Adapter-primer (the i.e. SEQ ID NO of sequencer library construction:432~SEQ ID NO:Nucleotide sequence shown in 433).
Preferably, the molecular label sequence is one section of 8~10 random nucleotide sequence.Molecular label sequence is (i.e. Barcode it) can be used for the molecular weight of accurate statistics primary template.
As another aspect of the present invention, present invention also offers the detection reagent of colorectal cancer medication related gene Box, the kit include above-mentioned primer sets.
Preferably, the sense primer group includes F1- without molecular label primer pond and F2- without molecular label primer pond；Institute Stating anti-sense primer group includes R1- molecular label primer ponds and R2- molecular label primers pond；
Wherein, the R1- molecular labels primer pond includes SEQ ID NO:1~SEQ ID NO:Nucleotides shown in 118 Sequence；The R2- molecular labels primer pond includes SEQ ID NO:119~SEQ ID NO:Nucleotide sequence shown in 215；Institute State F1- and include SEQ ID NO without molecular label primer pond:216~SEQ ID NO:Nucleotide sequence shown in 333；The F2- SEQ ID NO are included without molecular label primer pond:334~SEQ ID NO:Nucleotide sequence shown in 430.
It should be noted that 8 targeted drug related gene exon regions and 23 chemotherapeutics pleomorphism site weeks Enclose sequence overall length and have about 36.7kb altogether, and Illuminate sequencers read a length of 150bp, therefore, we devise 430 Primer is to cover target complete sequence；Because the most long codes extron of portion gene is up to several K, single pair primer can not expand this The region of length, and sequencing reading length does not reach the length, so needing to be divided the excessive extron of length (by amplification region The primer that domain part repeats is divided into different primer ponds), primer sets are divided into each 2 primer ponds of upstream and downstream, it is anti-in multiple PCR System is answered to carry out the enrichment and amplification of target area nucleotide sequence；This kit uses special design of primers algorithm, can protect Amplification efficiency of the card per pair of primers, and significantly reduce the interaction between primer, it is only necessary to upper (lower) trip primer sets are divided into 2 Individual primer pond, the nucleotide sequence in target complete region is enriched with and expanded in 2 PCR reaction systems.
It is highly preferred that the kit also includes：General anti-sense primer (URS, i.e. SEQ ID NO:Core shown in 431 Nucleotide sequence), adapter-primer (the i.e. SEQ ID NO for Illuminate sequencer library constructions:432~SEQ ID NO:Nucleotide sequence shown in 433), PCR Buffer and KOD enzymes.
As the third aspect of the invention, present invention also offers a kind of detection side of colorectal cancer medication related gene Method, the detection method comprise the following steps：
(1) sample gene to be detected is entered respectively using above-mentioned R1- molecular labels primer pond, R2- molecular label primer ponds Performing PCR expands, and obtains first round PCR primer；
(2) first round PCR primer for obtaining step (1) is respectively with above-mentioned F1- without molecular label primer pond and SEQ ID NO:Nucleotide sequence shown in 431, F2- is without molecular label primer pond and SEQ ID NO:Nucleotide sequence shown in 431 is carried out Second wheel PCR amplifications；
(3) sequencing analysis are carried out to the second wheel PCR primer, to obtain the mutation of sample gene and loci polymorphism information. It should be noted that the type of detection mutation includes：Displacement, insertion and/or the missing of one or more bases.
Thus, first round PCR only uses anti-sense primer group, the original nucleotide sequences of target area is enriched with, most Whole first round pcr amplification product carries molecular label；Second wheel PCR uses sense primer group and SEQ ID NO:Shown in 431 Nucleotide sequence, using first round pcr amplification product as template, further enrichment and amplification target area nucleotide sequence.
Preferably, the sequencing analysis in the step (3) comprise the following steps：
(31) the second wheel PCR primer for obtaining step (2) uses magnetic beads for purifying after mixing, and reuses for Illuminate Adapter-primer (the i.e. SEQ ID NO of sequencer library construction:432 and SEQ ID NO:Nucleotide sequence shown in 433) Carry out third round PCR amplifications；
(32) by the sequencing library constructed by step (31) it is qualified after testing after, entered using Illumina second generation sequenators Row sequencing analysis.
It should be noted that the qualified concentration for referring to sequencing library of detection is more than 0.5ng/ μ L；Nucleic acid fragment in library In 80~90% be distributed within 200~400bp；The instrument detected can be Qubit fluorescent quantitation instruments and Agilent 2100bioanalyzer；
Different from existing multiplex PCR, first round PCR only takes a wheel PCR, final first round PCR expansions in the application Volume increase thing carries molecular label；Primer uses special algorithm for design (referring to embodiment 1) in the detection method, can guarantee that every The amplification efficiency and specificity of pair of primers, and significantly reduce the interaction between primer, it is only necessary to upper (lower) trip primer sets It is divided into 2 primer ponds, the nucleotide sequence in target complete region is enriched with and expanded in 2 PCR reaction systems, it is possible to realize To the high degree of specificity of target sequence enrichment；Meanwhile the sequencing library construction method in the detection method is more simplified, quickly, sample This initial amount is significantly reduced, it is only necessary to 20ng DNA.
In summary, beneficial effects of the present invention are：
(1) the target area sequence obtained using multiple PCR primer group and the detection method enrichment of the present invention has height Specificity；
(2) present invention has reached 100% coverage to the concentration effect of target area sequence, and target area is averagely surveyed Sequence depth is up to more than 10,000 times；
(3) detection method of the invention need to only be carried out in 2 PCR reaction systems, on the one hand reduce laboratory operating procedures, Sample initial amount is also reduced to 20ng simultaneously；
(4) targeted drug related 8 for the treatment of colorectal cancer can be detected simultaneously to detection method using the kit of the present invention The mutation of individual gene extron subregion 23 pleomorphism sites related to chemotherapeutics, is used directly for adjuvant clinical Colon and rectum Cancer medication, and monitored more afterwards for early diagnosis of cancer, auxiliary diagnosis and examination or cancer.
Brief description of the drawings
Fig. 1 is the result of normal human blood sample library 2100 in the embodiment of the present invention 2；
Fig. 2 is the result of MCF10A cell line samples library 2100 in the embodiment of the present invention 2；
Fig. 3 is the result of colorectal cancer FFPE samples library 2100 in the embodiment of the present invention 2；
Fig. 4 is the principle schematic of multiplex PCR sequence enrichment method in the present invention.
Embodiment
To better illustrate the object, technical solutions and advantages of the present invention, below in conjunction with the drawings and specific embodiments pair The present invention is described further.
The multiple PCR primer design and synthesis of the colorectal cancer personalized medicine detection in Gene Mutation of embodiment 1
The use of name of patent application is a kind of multiple PCR primer and its design method (application number of amplification BRCA1/2 genes Design of primers is carried out for primer-design software 201610737156.7), is inputted to need to carry out the genomic region of design of primers Domain (bed file formats, often row content is " chromosome-tab-origin coordinates-tab-termination coordinate "), export as design The multipair primer that can be expanded in a reaction well.It is as follows that it implements thinking：1) Primer3 softwares are used, for Design available primer as much as possible alternately in each target area；2) according to primer expand specificity, primer whether (it is defined as smaller gene frequency in dbSNP to exist comprising the SNP site that whether there is high frequency on simple repeated sequence, primer More than 5/1000ths site) etc. condition designed primer in step 1 is filtered；3) for meeting previous step filtering rod Primer3 score value highest pair of primers is selected in the primer of part, each target area, between the primer that different target region is selected Meet the principle of " primer interphase interaction minimum ", that is, minimize the end of primer 3 ' and primer integrally complementary rate.Specifically The mutual complement of 3 ' terminal bases is not higher than 10 between seeking generally primer-primer, and overall complementary rate is not higher than between primer-primer 75%, the principle can effectively avoid the formation of primer dimer.
The target area of this kit for 8 related genes of colorectal cancer targeted drug encoded exon and extron- The base of introne border 10, and related 23 pleomorphism sites of chemotherapeutics.Because the most long codes extron of portion gene Up to several K, single pair primer can not expand the region of the length, and sequencing reading length does not reach the length, so needing to spend length Big extron is divided, and is assigned to and is carried out in two PCR reaction systems.Final colorectal cancer personalized medicine gene mutation It is as shown in table 1 to detect multiple PCR primer Survey design.
The colorectal cancer personalized medicine detection in Gene Mutation multiple PCR primer Survey design of table 1
The different type sample library construction of embodiment 2 and abrupt climatic change
First, experiment material
The sample that this experiment is related to has respectively：Genomic DNA, MCF10A cell lines genomic DNA, the knot of normal human blood Carcinoma of the rectum FFPE genomic DNAs, the genomic DNA of all types of samples is extracted using DNA extraction kit.
Extracting genome DNA：Using Qiagen DNeasy Blood＆Tissue Kit, carried out with reference to kit specification The extraction of genomic DNA.The genomic DNA of acquisition carries out gel electrophoresis and carries out quality testing using NanoDrop100, it is desirable to Genomic DNA is not degraded significantly, and concentration is more than 20ng/ μ L, A260/A280>1.8, A260/A230>2.0, and use Qubit 3.0 carries out accurate quantification.
The detection kit of colorectal cancer medication related gene, including the primer sets in embodiment 1, general anti-sense primer (URS), adapter-primer (the i.e. SEQ ID NO for Illuminate sequencer library constructions:432~SEQ ID NO: Nucleotide sequence shown in 433), PCR Buffer and KOD enzymes；
Primer sets are divided into four primer ponds：R1- molecular label primers pond, R2- molecular label primers pond, F1- are without molecule mark Primer pond and F2- are signed without molecular label primer pond；Four primer ponds are formulated as four primer pond mixed liquors respectively；
Wherein, R1- molecular labels primer pond includes SEQ ID NO:1~SEQ ID NO:Nucleotide sequence shown in 118； R2- molecular label primers pond includes SEQ ID NO:119~SEQ ID NO:Nucleotide sequence shown in 215；F1- is without molecule mark Label primer pond includes SEQ ID NO:216~SEQ ID NO:Nucleotide sequence shown in 333；F2- is without molecular label primer pond Include SEQ ID NO:334~SEQ ID NO:Nucleotide sequence shown in 430.
All primer purity should reach electrophoresis level (PAGE) level, without miscellaneous band.The synthetic product that combination mechanism is provided is provided Quality inspection prove, such as PAGE electrophoresis results, it was demonstrated that should have obvious unimodal PAGE collection of illustrative plates after purification using PAGE.
All reagents are bought from regular producer：KOD(TOYOBO)、2×PCR Buffer(TOYOBO)、VAHTS DNA Adapters set1/set2for Illumina (promise is only praised), VAHTS DNA Clean Beads (promise is only praised), DsDNA HS Assay Kit (life technologies), Agilent DNA 1000Kit (Agilent) etc..
2nd, key instrument
The sequenators of Illumina Nextseq 500, Agilent 2100bioanalyzer, Qubit fluorescent quantitation instruments, PCR instrument, supercentrifuge, water-bath, whirlpool concussion instrument, refrigerator, pipettor.
3rd, multiplexed PCR amplification, library construction and NGS sequencings
(1) first round multiplexed PCR amplifications
1. primer adds sterilizing purified water dissolving, final concentration of 400nM.SEQ ID NO:1~SEQ ID NO:118 grade things The amount mixing of matter, is configured to amplimer mixed liquor R1-barcode, described SEQ ID NO:119~SEQ ID NO:215 Amount Deng material mixes, and is configured to amplimer mixed liquor R2-barcode.SEQ ID NO:216~SEQ ID NO:333 etc. The amount mixing of material, is configured to amplimer mixed liquor F1-non barcode, described SEQ ID NO:334~SEQ ID NO:The amount mixing of 430 grade materials, is configured to amplimer mixed liquor F2-non barcode.The final concentration of primer mixed liquor is equal For 50nM.
2. same sample to be tested is divided into the pipe of A, B two to be reacted.Amplification system is：
PCR amplification programs are：
Temperature Time
98℃ 2min
58℃ 15min
68℃ 15min
3. pcr amplification product is purified using magnetic bead and fragment is screened.
(2) second takes turns PCR amplifications
1. using purified first round pcr amplification product as DNA profiling, respectively with amplimer mixed liquor F1-non Barcode, F2-non barcode, carry out the second wheel pcr amplification reaction.Amplification system is：
PCR amplification programs are：
2. after the amount mixing of the materials such as the pcr amplification product of same sample, it is purified using magnetic bead and fragment is sieved Choosing.
(3) library constructions
1. using the second purified wheel pcr amplification product as DNA profiling, drawn with Illuminate sequencer joints Thing, third round pcr amplification reaction is carried out, obtains library.Amplification system is：
PCR amplification programs are：
2. the library after amplification is purified using magnetic bead and fragment is screened.
(4) quality inspection of libraries and sequencing
1. library concentration is quantified using Qubit fluorescent quantitation instruments.
2. quality inspection is carried out to Library Quality using Agilent 2100bioanalyzer.
3. sample library is sequenced using the sequenators of Nextseq 500.
4th, experimental result and data analysis
Above-mentioned library machine on Illumina Nextseq 500 are carried out is sequenced, and sequencing pattern is PE150.For number is sequenced According to by analysis of biological information, obtained statistics index is used to evaluate sequence enrichment effect.
Normal human blood, the high-throughput sequencing library 2100 of tri- kinds of samples of MCF10A cell lines and colorectal cancer FFPE are analyzed As a result such as Fig. 1,2,3, the sequencing library of high quality, and the distribution of lengths in library and respective expected design phase are finally generated Symbol.
The sequencing data of table 2 counts
As shown in table 2, target area size is about 63Kb, and three library sequencings of the present embodiment are generated more than 10,000,000 Bar read sequences, sequencing data amount are respectively 1567Mb, 2828Mb and 1533Mb.Short sequence energy in sequencing data more than 94% Enough to compare upper genome, target area has been arrived in more than 96% comparison in the data of genome in comparisons, it was demonstrated that multiple PCR primer To the high degree of specificity of target sequence enrichment.Covered in addition, the present embodiment has reached 100% to the concentration effect of target area sequence Cover degree, and target area average sequencing depth is up to 10,000 more than X.The present embodiment need to only be carried out in 2 PCR reaction systems, a side Face reduces laboratory operating procedures, while sample initial amount also is reduced into 20ng (table 1).
To sum up, the multiplex PCR sequence enrichment technology (technical principle is as shown in Figure 4) in the present embodiment be it is a kind of efficiently, can The high-flux sequence sequence enrichment method leaned on.Kit in the present embodiment can detect the targeted drug for the treatment of colorectal cancer simultaneously The mutation of 8 gene extron subregions of correlation, 23 pleomorphism sites related to chemotherapeutics, it is used directly for instructing clinic Colorectal cancer medication, and monitored more afterwards for early diagnosis of cancer, auxiliary diagnosis and examination and cancer.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than the present invention is protected The limitation of scope is protected, although being explained in detail with reference to preferred embodiment to the present invention, one of ordinary skill in the art should Understand, technical scheme can be modified or equivalent substitution, without departing from the essence of technical solution of the present invention And scope.
<110>Guangzhou Yong Nuo bio tech ltd, Guangzhou promise health Science and Technology Ltd. forever
<120>The multiple PCR primer group and kit of colorectal cancer medication related gene detection
<130> 2017
<160> 860
<170> PatentIn version 3.3
<210> 1
<211> 51
<212> DNA
<213>Artificial sequence
<400> 1
agacgtgtgc tcttccgatc tnnnnnnnnn ntggttttcc caccacatcc t 51
<210> 2
<211> 51
<212> DNA
<213>Artificial sequence
<400> 2
agacgtgtgc tcttccgatc tnnnnnnnnn ngtgggcagg taggtgagtt c 51
<210> 3
<211> 51
<212> DNA
<213>Artificial sequence
<400> 3
agacgtgtgc tcttccgatc tnnnnnnnnn ncattgtcta gcacggccag g 51
<210> 4
<211> 51
<212> DNA
<213>Artificial sequence
<400> 4
agacgtgtgc tcttccgatc tnnnnnnnnn natcagtgtg agagccagct g 51
<210> 5
<211> 50
<212> DNA
<213>Artificial sequence
<400> 5
agacgtgtgc tcttccgatc tnnnnnnnnn ntccccttcc ctgactccag 50
<210> 6
<211> 51
<212> DNA
<213>Artificial sequence
<400> 6
agacgtgtgc tcttccgatc tnnnnnnnnn ncgaatgtat accggccctc g 51
<210> 7
<211> 49
<212> DNA
<213>Artificial sequence
<400> 7
agacgtgtgc tcttccgatc tnnnnnnnnn nactgcagct ggcctcggg 49
<210> 8
<211> 52
<212> DNA
<213>Artificial sequence
<400> 8
agacgtgtgc tcttccgatc tnnnnnnnnn ntggatcctc aggactctgt ct 52
<210> 9
<211> 51
<212> DNA
<213>Artificial sequence
<400> 9
agacgtgtgc tcttccgatc tnnnnnnnnn ncatcaggcc ccctctttct c 51
<210> 10
<211> 49
<212> DNA
<213>Artificial sequence
<400> 10
agacgtgtgc tcttccgatc tnnnnnnnnn ntggcagatg gggagtgcg 49
<210> 11
<211> 49
<212> DNA
<213>Artificial sequence
<400> 11
agacgtgtgc tcttccgatc tnnnnnnnnn nccctccagc caccctctc 49
<210> 12
<211> 52
<212> DNA
<213>Artificial sequence
<400> 12
agacgtgtgc tcttccgatc tnnnnnnnnn nacttccaga tgggcatgta gg 52
<210> 13
<211> 52
<212> DNA
<213>Artificial sequence
<400> 13
agacgtgtgc tcttccgatc tnnnnnnnnn ngggcaatga agggtacatc ct 52
<210> 14
<211> 49
<212> DNA
<213>Artificial sequence
<400> 14
agacgtgtgc tcttccgatc tnnnnnnnnn ngctgggagg acttcaccc 49
<210> 15
<211> 49
<212> DNA
<213>Artificial sequence
<400> 15
agacgtgtgc tcttccgatc tnnnnnnnnn ncgaccacac ccctccagg 49
<210> 16
<211> 52
<212> DNA
<213>Artificial sequence
<400> 16
agacgtgtgc tcttccgatc tnnnnnnnnn ncatggtcta agaggcagcc at 52
<210> 17
<211> 51
<212> DNA
<213>Artificial sequence
<400> 17
agacgtgtgc tcttccgatc tnnnnnnnnn ntgtctcgtc aatgtccagc a 51
<210> 18
<211> 49
<212> DNA
<213>Artificial sequence
<400> 18
agacgtgtgc tcttccgatc tnnnnnnnnn ntcacccacc cctcccaac 49
<210> 19
<211> 49
<212> DNA
<213>Artificial sequence
<400> 19
agacgtgtgc tcttccgatc tnnnnnnnnn ngacctccca ccctcctcc 49
<210> 20
<211> 51
<212> DNA
<213>Artificial sequence
<400> 20
agacgtgtgc tcttccgatc tnnnnnnnnn nagggtctgg acagaagaag c 51
<210> 21
<211> 50
<212> DNA
<213>Artificial sequence
<400> 21
agacgtgtgc tcttccgatc tnnnnnnnnn nggtcatgtg tggggaggct 50
<210> 22
<211> 51
<212> DNA
<213>Artificial sequence
<400> 22
agacgtgtgc tcttccgatc tnnnnnnnnn ntgtctctgc cactccctct g 51
<210> 23
<211> 52
<212> DNA
<213>Artificial sequence
<400> 23
agacgtgtgc tcttccgatc tnnnnnnnnn ngggtcctgg tcccagtaat ag 52
<210> 24
<211> 49
<212> DNA
<213>Artificial sequence
<400> 24
agacgtgtgc tcttccgatc tnnnnnnnnn nctggaagtg gtcggaggg 49
<210> 25
<211> 57
<212> DNA
<213>Artificial sequence
<400> 25
agacgtgtgc tcttccgatc tnnnnnnnnn ntcatgcttt atggttatta atgtagc 57
<210> 26
<211> 57
<212> DNA
<213>Artificial sequence
<400> 26
agacgtgtgc tcttccgatc tnnnnnnnnn ntggttattt gcattttaga ataggat 57
<210> 27
<211> 53
<212> DNA
<213>Artificial sequence
<400> 27
agacgtgtgc tcttccgatc tnnnnnnnnn nctatgaggt gaattgaggt ccc 53
<210> 28
<211> 55
<212> DNA
<213>Artificial sequence
<400> 28
agacgtgtgc tcttccgatc tnnnnnnnnn nagaggatag caacatactt cgagt 55
<210> 29
<211> 58
<212> DNA
<213>Artificial sequence
<400> 29
agacgtgtgc tcttccgatc tnnnnnnnnn nacagatact catcctcaat gtgattac 58
<210> 30
<211> 54
<212> DNA
<213>Artificial sequence
<400> 30
agacgtgtgc tcttccgatc tnnnnnnnnn nagcatcagc atttgacttt acct 54
<210> 31
<211> 58
<212> DNA
<213>Artificial sequence
<400> 31
agacgtgtgc tcttccgatc tnnnnnnnnn nactgctaaa cactaatata acctttgg 58
<210> 32
<211> 58
<212> DNA
<213>Artificial sequence
<400> 32
agacgtgtgc tcttccgatc tnnnnnnnnn ngcaaatctt ctaatccatg aggtactg 58
<210> 33
<211> 52
<212> DNA
<213>Artificial sequence
<400> 33
agacgtgtgc tcttccgatc tnnnnnnnnn nacagaccaa ttggcatgct ct 52
<210> 34
<211> 53
<212> DNA
<213>Artificial sequence
<400> 34
agacgtgtgc tcttccgatc tnnnnnnnnn ntctttctcc tgctcagtga ttt 53
<210> 35
<211> 56
<212> DNA
<213>Artificial sequence
<400> 35
agacgtgtgc tcttccgatc tnnnnnnnnn nagtgacata ccaatttgta caacag 56
<210> 36
<211> 59
<212> DNA
<213>Artificial sequence
<400> 36
agacgtgtgc tcttccgatc tnnnnnnnnn ntttaaaagg aagttgtatg gatctagaa 59
<210> 37
<211> 60
<212> DNA
<213>Artificial sequence
<400> 37
agacgtgtgc tcttccgatc tnnnnnnnnn ntgagaatgt cagttaagtt aatgagcttt 60
<210> 38
<211> 55
<212> DNA
<213>Artificial sequence
<400> 38
agacgtgtgc tcttccgatc tnnnnnnnnn naaatatatc aatgaaaccc ccaag 55
<210> 39
<211> 55
<212> DNA
<213>Artificial sequence
<400> 39
agacgtgtgc tcttccgatc tnnnnnnnnn naactttcaa catacaggtt gcctt 55
<210> 40
<211> 59
<212> DNA
<213>Artificial sequence
<400> 40
agacgtgtgc tcttccgatc tnnnnnnnnn nacagactca tctaacaaaa acttagatt 59
<210> 41
<211> 51
<212> DNA
<213>Artificial sequence
<400> 41
agacgtgtgc tcttccgatc tnnnnnnnnn ntgtgcattc ttgggctcct t 51
<210> 42
<211> 55
<212> DNA
<213>Artificial sequence
<400> 42
agacgtgtgc tcttccgatc tnnnnnnnnn ntcttgctca gttttatcta aggct 55
<210> 43
<211> 51
<212> DNA
<213>Artificial sequence
<400> 43
agacgtgtgc tcttccgatc tnnnnnnnnn nggtctttgc ctgctgagag t 51
<210> 44
<211> 57
<212> DNA
<213>Artificial sequence
<400> 44
agacgtgtgc tcttccgatc tnnnnnnnnn ncagattttc attcttcttt ctgtttt 57
<210> 45
<211> 57
<212> DNA
<213>Artificial sequence
<400> 45
agacgtgtgc tcttccgatc tnnnnnnnnn ntctacaagg ttgaattatt tcccttt 57
<210> 46
<211> 58
<212> DNA
<213>Artificial sequence
<400> 46
agacgtgtgc tcttccgatc tnnnnnnnnn nagtaggttg tttttcagtc tttattca 58
<210> 47
<211> 51
<212> DNA
<213>Artificial sequence
<400> 47
agacgtgtgc tcttccgatc tnnnnnnnnn ngatttcgag gccagagtcc t 51
<210> 48
<211> 59
<212> DNA
<213>Artificial sequence
<400> 48
agacgtgtgc tcttccgatc tnnnnnnnnn ntgtcgacat ttaatgttta ctgtcacat 59
<210> 49
<211> 54
<212> DNA
<213>Artificial sequence
<400> 49
agacgtgtgc tcttccgatc tnnnnnnnnn ntctgtttgg cttgacttga cttt 54
<210> 50
<211> 52
<212> DNA
<213>Artificial sequence
<400> 50
agacgtgtgc tcttccgatc tnnnnnnnnn ncccaacctt ctacccctga ta 52
<210> 51
<211> 52
<212> DNA
<213>Artificial sequence
<400> 51
agacgtgtgc tcttccgatc tnnnnnnnnn nttccaattc cacagccctt cc 52
<210> 52
<211> 57
<212> DNA
<213>Artificial sequence
<400> 52
agacgtgtgc tcttccgatc tnnnnnnnnn ncacaagtta ggtttgtttt gttttgc 57
<210> 53
<211> 56
<212> DNA
<213>Artificial sequence
<400> 53
agacgtgtgc tcttccgatc tnnnnnnnnn ntgtgagttt ttgaaatctc tgtgat 56
<210> 54
<211> 54
<212> DNA
<213>Artificial sequence
<400> 54
agacgtgtgc tcttccgatc tnnnnnnnnn ntgtaatttg ctccctttac ctct 54
<210> 55
<211> 54
<212> DNA
<213>Artificial sequence
<400> 55
agacgtgtgc tcttccgatc tnnnnnnnnn nggcctatga agaatacacc agca 54
<210> 56
<211> 62
<212> DNA
<213>Artificial sequence
<400> 56
agacgtgtgc tcttccgatc tnnnnnnnnn nactaagtga tttttattta aattcttttg aa 62
<210> 57
<211> 49
<212> DNA
<213>Artificial sequence
<400> 57
agacgtgtgc tcttccgatc tnnnnnnnnn nagctctccg cctcccttc 49
<210> 58
<211> 49
<212> DNA
<213>Artificial sequence
<400> 58
agacgtgtgc tcttccgatc tnnnnnnnnn ngggagccgg cgagacacg 49
<210> 59
<211> 57
<212> DNA
<213>Artificial sequence
<400> 59
agacgtgtgc tcttccgatc tnnnnnnnnn nttctccact tagattttct ccaattt 57
<210> 60
<211> 49
<212> DNA
<213>Artificial sequence
<400> 60
agacgtgtgc tcttccgatc tnnnnnnnnn natgaacccc cagccttgg 49
<210> 61
<211> 51
<212> DNA
<213>Artificial sequence
<400> 61
agacgtgtgc tcttccgatc tnnnnnnnnn naggagctgg aggcagagat a 51
<210> 62
<211> 49
<212> DNA
<213>Artificial sequence
<400> 62
agacgtgtgc tcttccgatc tnnnnnnnnn nctgctccag ggaggctgc 49
<210> 63
<211> 50
<212> DNA
<213>Artificial sequence
<400> 63
agacgtgtgc tcttccgatc tnnnnnnnnn nggagacaga gcgggacaag 50
<210> 64
<211> 50
<212> DNA
<213>Artificial sequence
<400> 64
agacgtgtgc tcttccgatc tnnnnnnnnn ncggcaggat gtggagatcg 50
<210> 65
<211> 57
<212> DNA
<213>Artificial sequence
<400> 65
agacgtgtgc tcttccgatc tnnnnnnnnn ntctctctct aaaacactga tttccca 57
<210> 66
<211> 58
<212> DNA
<213>Artificial sequence
<400> 66
agacgtgtgc tcttccgatc tnnnnnnnnn nccagtttat tgtatttgca tagcacaa 58
<210> 67
<211> 51
<212> DNA
<213>Artificial sequence
<400> 67
agacgtgtgc tcttccgatc tnnnnnnnnn ngaaggttgc acttgtccac g 51
<210> 68
<211> 50
<212> DNA
<213>Artificial sequence
<400> 68
agacgtgtgc tcttccgatc tnnnnnnnnn ngtgaaggcc cttcccatga 50
<210> 69
<211> 57
<212> DNA
<213>Artificial sequence
<400> 69
agacgtgtgc tcttccgatc tnnnnnnnnn ngaaatgttc tgttctcctt cactttc 57
<210> 70
<211> 53
<212> DNA
<213>Artificial sequence
<400> 70
agacgtgtgc tcttccgatc tnnnnnnnnn ngagaaggga tccatcctaa gca 53
<210> 71
<211> 53
<212> DNA
<213>Artificial sequence
<400> 71
agacgtgtgc tcttccgatc tnnnnnnnnn ntggaaggaa ggaaatgggt agg 53
<210> 72
<211> 49
<212> DNA
<213>Artificial sequence
<400> 72
agacgtgtgc tcttccgatc tnnnnnnnnn ngaacgatgt ggcgccttc 49
<210> 73
<211> 51
<212> DNA
<213>Artificial sequence
<400> 73
agacgtgtgc tcttccgatc tnnnnnnnnn nggagcccag cactttgatc t 51
<210> 74
<211> 49
<212> DNA
<213>Artificial sequence
<400> 74
agacgtgtgc tcttccgatc tnnnnnnnnn nacccccaca cagcaaagc 49
<210> 75
<211> 51
<212> DNA
<213>Artificial sequence
<400> 75
agacgtgtgc tcttccgatc tnnnnnnnnn nctccccgta tctcccttcc c 51
<210> 76
<211> 52
<212> DNA
<213>Artificial sequence
<400> 76
agacgtgtgc tcttccgatc tnnnnnnnnn ngtgtcagga aaatgctggc tg 52
<210> 77
<211> 52
<212> DNA
<213>Artificial sequence
<400> 77
agacgtgtgc tcttccgatc tnnnnnnnnn nggtacatat gggtggctga gg 52
<210> 78
<211> 51
<212> DNA
<213>Artificial sequence
<400> 78
agacgtgtgc tcttccgatc tnnnnnnnnn ncccaatgga agcacagact g 51
<210> 79
<211> 56
<212> DNA
<213>Artificial sequence
<400> 79
agacgtgtgc tcttccgatc tnnnnnnnnn nttcaaatga gtagacacag cttgag 56
<210> 80
<211> 51
<212> DNA
<213>Artificial sequence
<400> 80
agacgtgtgc tcttccgatc tnnnnnnnnn ntgaggagca ggactgtttc c 51
<210> 81
<211> 53
<212> DNA
<213>Artificial sequence
<400> 81
agacgtgtgc tcttccgatc tnnnnnnnnn ngaaagaagt cctgctggta gtc 53
<210> 82
<211> 50
<212> DNA
<213>Artificial sequence
<400> 82
agacgtgtgc tcttccgatc tnnnnnnnnn nctaatgcgg gcatggctgt 50
<210> 83
<211> 53
<212> DNA
<213>Artificial sequence
<400> 83
agacgtgtgc tcttccgatc tnnnnnnnnn nttgagctag ctctctcatt ttt 53
<210> 84
<211> 56
<212> DNA
<213>Artificial sequence
<400> 84
agacgtgtgc tcttccgatc tnnnnnnnnn ngtacccagc ctaatcttgt ttttct 56
<210> 85
<211> 49
<212> DNA
<213>Artificial sequence
<400> 85
agacgtgtgc tcttccgatc tnnnnnnnnn nctgccccct taccctcca 49
<210> 86
<211> 51
<212> DNA
<213>Artificial sequence
<400> 86
agacgtgtgc tcttccgatc tnnnnnnnnn ngttggaggc agtagaaggg g 51
<210> 87
<211> 49
<212> DNA
<213>Artificial sequence
<400> 87
agacgtgtgc tcttccgatc tnnnnnnnnn ncgcttggct ctggaccgc 49
<210> 88
<211> 51
<212> DNA
<213>Artificial sequence
<400> 88
agacgtgtgc tcttccgatc tnnnnnnnnn ngcagccgca gaaatggata c 51
<210> 89
<211> 59
<212> DNA
<213>Artificial sequence
<400> 89
agacgtgtgc tcttccgatc tnnnnnnnnn ntgaaaacac aacatgaata taaacatca 59
<210> 90
<211> 56
<212> DNA
<213>Artificial sequence
<400> 90
agacgtgtgc tcttccgatc tnnnnnnnnn ntgacagtaa gatacagtct atcggg 56
<210> 91
<211> 55
<212> DNA
<213>Artificial sequence
<400> 91
agacgtgtgc tcttccgatc tnnnnnnnnn ntccaataaa ttctcagatc cagga 55
<210> 92
<211> 53
<212> DNA
<213>Artificial sequence
<400> 92
agacgtgtgc tcttccgatc tnnnnnnnnn ntcttgtgaa acaacagtgc cac 53
<210> 93
<211> 51
<212> DNA
<213>Artificial sequence
<400> 93
agacgtgtgc tcttccgatc tnnnnnnnnn nacacacagg taacggctga g 51
<210> 94
<211> 53
<212> DNA
<213>Artificial sequence
<400> 94
agacgtgtgc tcttccgatc tnnnnnnnnn ntccttgtca ttatctgcac gct 53
<210> 95
<211> 52
<212> DNA
<213>Artificial sequence
<400> 95
agacgtgtgc tcttccgatc tnnnnnnnnn ntggatcaga gtcagtggtg tc 52
<210> 96
<211> 56
<212> DNA
<213>Artificial sequence
<400> 96
agacgtgtgc tcttccgatc tnnnnnnnnn nttaccaatg caacagactt taaaga 56
<210> 97
<211> 58
<212> DNA
<213>Artificial sequence
<400> 97
agacgtgtgc tcttccgatc tnnnnnnnnn ntggacaggt tttgaaagat atttgtgt 58
<210> 98
<211> 55
<212> DNA
<213>Artificial sequence
<400> 98
agacgtgtgc tcttccgatc tnnnnnnnnn nggtgcactg taataatcca gactg 55
<210> 99
<211> 55
<212> DNA
<213>Artificial sequence
<400> 99
agacgtgtgc tcttccgatc tnnnnnnnnn ngctggccat atatatattt aaacc 55
<210> 100
<211> 51
<212> DNA
<213>Artificial sequence
<400> 100
agacgtgtgc tcttccgatc tnnnnnnnnn ntcactgaag acccaggtcc a 51
<210> 101
<211> 49
<212> DNA
<213>Artificial sequence
<400> 101
agacgtgtgc tcttccgatc tnnnnnnnnn nccacaggca tggcgcggc 49
<210> 102
<211> 49
<212> DNA
<213>Artificial sequence
<400> 102
agacgtgtgc tcttccgatc tnnnnnnnnn nggtcctagg cctgggagg 49
<210> 103
<211> 50
<212> DNA
<213>Artificial sequence
<400> 103
agacgtgtgc tcttccgatc tnnnnnnnnn ncaagcttgg ccagttccgt 50
<210> 104
<211> 50
<212> DNA
<213>Artificial sequence
<400> 104
agacgtgtgc tcttccgatc tnnnnnnnnn nctgggggtg tcaccttctg 50
<210> 105
<211> 52
<212> DNA
<213>Artificial sequence
<400> 105
agacgtgtgc tcttccgatc tnnnnnnnnn nccctgtggt tatcaagggt ca 52
<210> 106
<211> 49
<212> DNA
<213>Artificial sequence
<400> 106
agacgtgtgc tcttccgatc tnnnnnnnnn ncagcaggcc caggacaag 49
<210> 107
<211> 55
<212> DNA
<213>Artificial sequence
<400> 107
agacgtgtgc tcttccgatc tnnnnnnnnn nagactcttt cacatcctcc ctttg 55
<210> 108
<211> 53
<212> DNA
<213>Artificial sequence
<400> 108
agacgtgtgc tcttccgatc tnnnnnnnnn naaccattca acatttttaa ccc 53
<210> 109
<211> 51
<212> DNA
<213>Artificial sequence
<400> 109
agacgtgtgc tcttccgatc tnnnnnnnnn nggactttcc acctatggga c 51
<210> 110
<211> 52
<212> DNA
<213>Artificial sequence
<400> 110
agacgtgtgc tcttccgatc tnnnnnnnnn ntctgcattt taactatggc tc 52
<210> 111
<211> 56
<212> DNA
<213>Artificial sequence
<400> 111
agacgtgtgc tcttccgatc tnnnnnnnnn nagcagataa gacagtatta ctagtt 56
<210> 112
<211> 49
<212> DNA
<213>Artificial sequence
<400> 112
agacgtgtgc tcttccgatc tnnnnnnnnn ncttccagtg cctccaccc 49
<210> 113
<211> 51
<212> DNA
<213>Artificial sequence
<400> 113
agacgtgtgc tcttccgatc tnnnnnnnnn naatcccttt ggtgctcacg t 51
<210> 114
<211> 59
<212> DNA
<213>Artificial sequence
<400> 114
agacgtgtgc tcttccgatc tnnnnnnnnn nggagttgtt gaaatgaaaa tgttgtctg 59
<210> 115
<211> 53
<212> DNA
<213>Artificial sequence
<400> 115
agacgtgtgc tcttccgatc tnnnnnnnnn nttctacctg aagagcaagt ccc 53
<210> 116
<211> 50
<212> DNA
<213>Artificial sequence
<400> 116
agacgtgtgc tcttccgatc tnnnnnnnnn ncaggttacc ccaaaggcca 50
<210> 117
<211> 57
<212> DNA
<213>Artificial sequence
<400> 117
agacgtgtgc tcttccgatc tnnnnnnnnn ncacagccaa accatatcac tcctttc 57
<210> 118
<211> 52
<212> DNA
<213>Artificial sequence
<400> 118
agacgtgtgc tcttccgatc tnnnnnnnnn nctttctttg ttcagccccc ag 52
<210> 119
<211> 49
<212> DNA
<213>Artificial sequence
<400> 119
agacgtgtgc tcttccgatc tnnnnnnnnn ncatccacag ggcagggtc 49
<210> 120
<211> 52
<212> DNA
<213>Artificial sequence
<400> 120
agacgtgtgc tcttccgatc tnnnnnnnnn ncctgtaaac agagggctca gc 52
<210> 121
<211> 54
<212> DNA
<213>Artificial sequence
<400> 121
agacgtgtgc tcttccgatc tnnnnnnnnn nccaagagaa ggtttcaatg acgg 54
<210> 122
<211> 51
<212> DNA
<213>Artificial sequence
<400> 122
agacgtgtgc tcttccgatc tnnnnnnnnn ntgtaagttg ggagtttgcg g 51
<210> 123
<211> 51
<212> DNA
<213>Artificial sequence
<400> 123
agacgtgtgc tcttccgatc tnnnnnnnnn nggtagagca cattgggcac a 51
<210> 124
<211> 52
<212> DNA
<213>Artificial sequence
<400> 124
agacgtgtgc tcttccgatc tnnnnnnnnn nacagaaaca aacctcccca cc 52
<210> 125
<211> 51
<212> DNA
<213>Artificial sequence
<400> 125
agacgtgtgc tcttccgatc tnnnnnnnnn naaatgccag gctcccaaag a 51
<210> 126
<211> 52
<212> DNA
<213>Artificial sequence
<400> 126
agacgtgtgc tcttccgatc tnnnnnnnnn ntgcagaaca ggatgcagag ac 52
<210> 127
<211> 51
<212> DNA
<213>Artificial sequence
<400> 127
agacgtgtgc tcttccgatc tnnnnnnnnn ncgaaagagc tggtcccagg g 51
<210> 128
<211> 51
<212> DNA
<213>Artificial sequence
<400> 128
agacgtgtgc tcttccgatc tnnnnnnnnn ngaactggct gcagttgaca c 51
<210> 129
<211> 49
<212> DNA
<213>Artificial sequence
<400> 129
agacgtgtgc tcttccgatc tnnnnnnnnn nctggcaatc cccctcgct 49
<210> 130
<211> 53
<212> DNA
<213>Artificial sequence
<400> 130
agacgtgtgc tcttccgatc tnnnnnnnnn ncctcctttc tgcagaaaag acc 53
<210> 131
<211> 51
<212> DNA
<213>Artificial sequence
<400> 131
agacgtgtgc tcttccgatc tnnnnnnnnn ncgcatcgtg tacttccgga t 51
<210> 132
<211> 51
<212> DNA
<213>Artificial sequence
<400> 132
agacgtgtgc tcttccgatc tnnnnnnnnn ntggccctga ccttgtagac t 51
<210> 133
<211> 51
<212> DNA
<213>Artificial sequence
<400> 133
agacgtgtgc tcttccgatc tnnnnnnnnn ncctagcagg agagggtgga g 51
<210> 134
<211> 51
<212> DNA
<213>Artificial sequence
<400> 134
agacgtgtgc tcttccgatc tnnnnnnnnn nctccggaga gacctgcaaa g 51
<210> 135
<211> 51
<212> DNA
<213>Artificial sequence
<400> 135
agacgtgtgc tcttccgatc tnnnnnnnnn nccatgggct agacaccact c 51
<210> 136
<211> 49
<212> DNA
<213>Artificial sequence
<400> 136
agacgtgtgc tcttccgatc tnnnnnnnnn ncagccgctc ccccttttc 49
<210> 137
<211> 51
<212> DNA
<213>Artificial sequence
<400> 137
agacgtgtgc tcttccgatc tnnnnnnnnn ngagggtgct cttagccaca g 51
<210> 138
<211> 51
<212> DNA
<213>Artificial sequence
<400> 138
agacgtgtgc tcttccgatc tnnnnnnnnn ntgggggagg taagcagaca g 51
<210> 139
<211> 50
<212> DNA
<213>Artificial sequence
<400> 139
agacgtgtgc tcttccgatc tnnnnnnnnn nctggactcc ataccaggct 50
<210> 140
<211> 53
<212> DNA
<213>Artificial sequence
<400> 140
agacgtgtgc tcttccgatc tnnnnnnnnn ncaaaggcaa aaacgtcttt gac 53
<210> 141
<211> 49
<212> DNA
<213>Artificial sequence
<400> 141
agacgtgtgc tcttccgatc tnnnnnnnnn ntgcggactt ggccttctg 49
<210> 142
<211> 54
<212> DNA
<213>Artificial sequence
<400> 142
agacgtgtgc tcttccgatc tnnnnnnnnn nggtcacaaa gtcgtcttgt ttca 54
<210> 143
<211> 59
<212> DNA
<213>Artificial sequence
<400> 143
agacgtgtgc tcttccgatc tnnnnnnnnn nactgcacta tagtaatgat tagagtaga 59
<210> 144
<211> 58
<212> DNA
<213>Artificial sequence
<400> 144
agacgtgtgc tcttccgatc tnnnnnnnnn nacttgttac tcaccttata ctgactca 58
<210> 145
<211> 58
<212> DNA
<213>Artificial sequence
<400> 145
agacgtgtgc tcttccgatc tnnnnnnnnn ntgtttctcc attcatatat ggtgtagc 58
<210> 146
<211> 58
<212> DNA
<213>Artificial sequence
<400> 146
agacgtgtgc tcttccgatc tnnnnnnnnn nccatctgaa aacatactac aggtcaac 58
<210> 147
<211> 56
<212> DNA
<213>Artificial sequence
<400> 147
agacgtgtgc tcttccgatc tnnnnnnnnn naggtaaaca taattgttcc ttctga 56
<210> 148
<211> 56
<212> DNA
<213>Artificial sequence
<400> 148
agacgtgtgc tcttccgatc tnnnnnnnnn ngagagaagg tttgactgcc ataaaa 56
<210> 149
<211> 58
<212> DNA
<213>Artificial sequence
<400> 149
agacgtgtgc tcttccgatc tnnnnnnnnn nagagaaagt atctacctaa atccacag 58
<210> 150
<211> 52
<212> DNA
<213>Artificial sequence
<400> 150
agacgtgtgc tcttccgatc tnnnnnnnnn nctgagatca gccaaattca gt 52
<210> 151
<211> 51
<212> DNA
<213>Artificial sequence
<400> 151
agacgtgtgc tcttccgatc tnnnnnnnnn naagcaccga acagcaaaac c 51
<210> 152
<211> 58
<212> DNA
<213>Artificial sequence
<400> 152
agacgtgtgc tcttccgatc tnnnnnnnnn nagaaagcaa gtaatatgta ccttaaga 58
<210> 153
<211> 56
<212> DNA
<213>Artificial sequence
<400> 153
agacgtgtgc tcttccgatc tnnnnnnnnn ntgaaaagag tctcaaacac aaacta 56
<210> 154
<211> 58
<212> DNA
<213>Artificial sequence
<400> 154
agacgtgtgc tcttccgatc tnnnnnnnnn ntcatgcatc ataagctcat taatactc 58
<210> 155
<211> 50
<212> DNA
<213>Artificial sequence
<400> 155
agacgtgtgc tcttccgatc tnnnnnnnnn nggctgttga actgcagtgc 50
<210> 156
<211> 57
<212> DNA
<213>Artificial sequence
<400> 156
agacgtgtgc tcttccgatc tnnnnnnnnn nagaacacca aaacatttta aacagag 57
<210> 157
<211> 54
<212> DNA
<213>Artificial sequence
<400> 157
agacgtgtgc tcttccgatc tnnnnnnnnn nttgattgtt tctaatagag cagc 54
<210> 158
<211> 54
<212> DNA
<213>Artificial sequence
<400> 158
agacgtgtgc tcttccgatc tnnnnnnnnn ntgcatgctg tttaattgtg tgga 54
<210> 159
<211> 52
<212> DNA
<213>Artificial sequence
<400> 159
agacgtgtgc tcttccgatc tnnnnnnnnn nagtgcatca gaaccctcct tg 52
<210> 160
<211> 54
<212> DNA
<213>Artificial sequence
<400> 160
agacgtgtgc tcttccgatc tnnnnnnnnn naggatacct gtctccagat ctca 54
<210> 161
<211> 56
<212> DNA
<213>Artificial sequence
<400> 161
agacgtgtgc tcttccgatc tnnnnnnnnn ntccatacag ctttcagtca gatgta 56
<210> 162
<211> 56
<212> DNA
<213>Artificial sequence
<400> 162
agacgtgtgc tcttccgatc tnnnnnnnnn naggaaaatg agatctactg ttttcc 56
<210> 163
<211> 49
<212> DNA
<213>Artificial sequence
<400> 163
agacgtgtgc tcttccgatc tnnnnnnnnn ncccagtggt gtgagggct 49
<210> 164
<211> 53
<212> DNA
<213>Artificial sequence
<400> 164
agacgtgtgc tcttccgatc tnnnnnnnnn ntgacacttg gagtaacaat tgc 53
<210> 165
<211> 53
<212> DNA
<213>Artificial sequence
<400> 165
agacgtgtgc tcttccgatc tnnnnnnnnn nacctggctc actaactaac gtg 53
<210> 166
<211> 56
<212> DNA
<213>Artificial sequence
<400> 166
agacgtgtgc tcttccgatc tnnnnnnnnn ntcagtgtta tcgctactct ctgatt 56
<210> 167
<211> 59
<212> DNA
<213>Artificial sequence
<400> 167
agacgtgtgc tcttccgatc tnnnnnnnnn ncgttactct gaatcttatc ttccaatga 59
<210> 168
<211> 51
<212> DNA
<213>Artificial sequence
<400> 168
agacgtgtgc tcttccgatc tnnnnnnnnn ncgaaagctg cttttccagg g 51
<210> 169
<211> 52
<212> DNA
<213>Artificial sequence
<400> 169
agacgtgtgc tcttccgatc tnnnnnnnnn naagaaaggc ttgtgtctac at 52
<210> 170
<211> 55
<212> DNA
<213>Artificial sequence
<400> 170
agacgtgtgc tcttccgatc tnnnnnnnnn ntcttcttcc tcttctagcc tttca 55
<210> 171
<211> 59
<212> DNA
<213>Artificial sequence
<400> 171
agacgtgtgc tcttccgatc tnnnnnnnnn nttctctttt gttaatgtca ggacaaagt 59
<210> 172
<211> 50
<212> DNA
<213>Artificial sequence
<400> 172
agacgtgtgc tcttccgatc tnnnnnnnnn nggctctgtt caacggggac 50
<210> 173
<211> 52
<212> DNA
<213>Artificial sequence
<400> 173
agacgtgtgc tcttccgatc tnnnnnnnnn ntccaaattc ccaaggacca cc 52
<210> 174
<211> 56
<212> DNA
<213>Artificial sequence
<400> 174
agacgtgtgc tcttccgatc tnnnnnnnnn ntgctaaggc ataggaattt tcgtag 56
<210> 175
<211> 54
<212> DNA
<213>Artificial sequence
<400> 175
agacgtgtgc tcttccgatc tnnnnnnnnn nacatgttgc tgagaaagtc actg 54
<210> 176
<211> 51
<212> DNA
<213>Artificial sequence
<400> 176
agacgtgtgc tcttccgatc tnnnnnnnnn ncatgggtct gaggctgttc a 51
<210> 177
<211> 51
<212> DNA
<213>Artificial sequence
<400> 177
agacgtgtgc tcttccgatc tnnnnnnnnn nctgtatttg ccctcggggt t 51
<210> 178
<211> 49
<212> DNA
<213>Artificial sequence
<400> 178
agacgtgtgc tcttccgatc tnnnnnnnnn ncaagcctcg tccgcacac 49
<210> 179
<211> 58
<212> DNA
<213>Artificial sequence
<400> 179
agacgtgtgc tcttccgatc tnnnnnnnnn nctctaccag ttaaaaaggc agattttg 58
<210> 180
<211> 53
<212> DNA
<213>Artificial sequence
<400> 180
agacgtgtgc tcttccgatc tnnnnnnnnn nggcaaatgt aatctaccag gct 53
<210> 181
<211> 56
<212> DNA
<213>Artificial sequence
<400> 181
agacgtgtgc tcttccgatc tnnnnnnnnn nggacccatt agaaccaact ccataa 56
<210> 182
<211> 54
<212> DNA
<213>Artificial sequence
<400> 182
agacgtgtgc tcttccgatc tnnnnnnnnn ntgatcaacg caaggggatt aaag 54
<210> 183
<211> 51
<212> DNA
<213>Artificial sequence
<400> 183
agacgtgtgc tcttccgatc tnnnnnnnnn ntccagacca gggtgttgtt t 51
<210> 184
<211> 57
<212> DNA
<213>Artificial sequence
<400> 184
agacgtgtgc tcttccgatc tnnnnnnnnn ngcaaaggga gaaattgaaa ttgtaga 57
<210> 185
<211> 51
<212> DNA
<213>Artificial sequence
<400> 185
agacgtgtgc tcttccgatc tnnnnnnnnn nacagacacg tgaaggcatg a 51
<210> 186
<211> 51
<212> DNA
<213>Artificial sequence
<400> 186
agacgtgtgc tcttccgatc tnnnnnnnnn ngcaggaaag catcttcacc c 51
<210> 187
<211> 50
<212> DNA
<213>Artificial sequence
<400> 187
agacgtgtgc tcttccgatc tnnnnnnnnn ncttgttcct gtcggggtgc 50
<210> 188
<211> 49
<212> DNA
<213>Artificial sequence
<400> 188
agacgtgtgc tcttccgatc tnnnnnnnnn ngaccatgag aggccctgc 49
<210> 189
<211> 49
<212> DNA
<213>Artificial sequence
<400> 189
agacgtgtgc tcttccgatc tnnnnnnnnn ntccaggagg cagccgaag 49
<210> 190
<211> 51
<212> DNA
<213>Artificial sequence
<400> 190
agacgtgtgc tcttccgatc tnnnnnnnnn nggccagccc aaaatctgtg a 51
<210> 191
<211> 57
<212> DNA
<213>Artificial sequence
<400> 191
agacgtgtgc tcttccgatc tnnnnnnnnn ntcagtacaa actcattagc atcagga 57
<210> 192
<211> 51
<212> DNA
<213>Artificial sequence
<400> 192
agacgtgtgc tcttccgatc tnnnnnnnnn nctcagctag gcagtgtgga c 51
<210> 193
<211> 50
<212> DNA
<213>Artificial sequence
<400> 193
agacgtgtgc tcttccgatc tnnnnnnnnn ngaagccctg ctgtgggatg 50
<210> 194
<211> 57
<212> DNA
<213>Artificial sequence
<400> 194
agacgtgtgc tcttccgatc tnnnnnnnnn nccatgtaaa atagagccat agtggag 57
<210> 195
<211> 52
<212> DNA
<213>Artificial sequence
<400> 195
agacgtgtgc tcttccgatc tnnnnnnnnn nagtgttgag atactcgggg tt 52
<210> 196
<211> 52
<212> DNA
<213>Artificial sequence
<400> 196
agacgtgtgc tcttccgatc tnnnnnnnnn nggctcatac tatcctccgt gg 52
<210> 197
<211> 55
<212> DNA
<213>Artificial sequence
<400> 197
agacgtgtgc tcttccgatc tnnnnnnnnn ngaaacaagt gtgatttgcc aacaa 55
<210> 198
<211> 53
<212> DNA
<213>Artificial sequence
<400> 198
agacgtgtgc tcttccgatc tnnnnnnnnn ntggacaaga agagtacagt gcc 53
<210> 199
<211> 52
<212> DNA
<213>Artificial sequence
<400> 199
agacgtgtgc tcttccgatc tnnnnnnnnn ntggtttcca acaggttctt gc 52
<210> 200
<211> 51
<212> DNA
<213>Artificial sequence
<400> 200
agacgtgtgc tcttccgatc tnnnnnnnnn nataatcctc cgaacggctg c 51
<210> 201
<211> 51
<212> DNA
<213>Artificial sequence
<400> 201
agacgtgtgc tcttccgatc tnnnnnnnnn nttgatgatg gctgtcatgt c 51
<210> 202
<211> 51
<212> DNA
<213>Artificial sequence
<400> 202
agacgtgtgc tcttccgatc tnnnnnnnnn nagtcaccca aactacggac a 51
<210> 203
<211> 58
<212> DNA
<213>Artificial sequence
<400> 203
agacgtgtgc tcttccgatc tnnnnnnnnn ncactctaac aagcagataa ctttcact 58
<210> 204
<211> 51
<212> DNA
<213>Artificial sequence
<400> 204
agacgtgtgc tcttccgatc tnnnnnnnnn nacaccagtt cgtccctttc c 51
<210> 205
<211> 58
<212> DNA
<213>Artificial sequence
<400> 205
agacgtgtgc tcttccgatc tnnnnnnnnn nggttaagaa aactgttcca atacatgg 58
<210> 206
<211> 58
<212> DNA
<213>Artificial sequence
<400> 206
agacgtgtgc tcttccgatc tnnnnnnnnn nctgtcctta ttttggatat ttctccca 58
<210> 207
<211> 56
<212> DNA
<213>Artificial sequence
<400> 207
agacgtgtgc tcttccgatc tnnnnnnnnn nacacacatc acatacatac aagtca 56
<210> 208
<211> 58
<212> DNA
<213>Artificial sequence
<400> 208
agacgtgtgc tcttccgatc tnnnnnnnnn nattttcatg gtgttttatc cctcttga 58
<210> 209
<211> 54
<212> DNA
<213>Artificial sequence
<400> 209
agacgtgtgc tcttccgatc tnnnnnnnnn ntgaagccat cgtatatatt caca 54
<210> 210
<211> 55
<212> DNA
<213>Artificial sequence
<400> 210
agacgtgtgc tcttccgatc tnnnnnnnnn naggactctg aagatgtacc tatgg 55
<210> 211
<211> 51
<212> DNA
<213>Artificial sequence
<400> 211
agacgtgtgc tcttccgatc tnnnnnnnnn nacacagcag gtcaagagga g 51
<210> 212
<211> 58
<212> DNA
<213>Artificial sequence
<400> 212
agacgtgtgc tcttccgatc tnnnnnnnnn nacatgttct aatatagtca cattttca 58
<210> 213
<211> 52
<212> DNA
<213>Artificial sequence
<400> 213
agacgtgtgc tcttccgatc tnnnnnnnnn nccctctccc tttcctctgt tc 52
<210> 214
<211> 57
<212> DNA
<213>Artificial sequence
<400> 214
agacgtgtgc tcttccgatc tnnnnnnnnn nggacaaagc tcctttctga atattga 57
<210> 215
<211> 59
<212> DNA
<213>Artificial sequence
<400> 215
agacgtgtgc tcttccgatc tnnnnnnnnn natatgtttg aggatgtaag ctagtttca 59
<210> 216
<211> 44
<212> DNA
<213>Artificial sequence
<400> 216
tacacgacgc tcttccgatc ttctcctccc aaatttgtag accc 44
<210> 217
<211> 41
<212> DNA
<213>Artificial sequence
<400> 217
tacacgacgc tcttccgatc ttcctctgac ccatctgctc t 41
<210> 218
<211> 42
<212> DNA
<213>Artificial sequence
<400> 218
tacacgacgc tcttccgatc ttcctatttc agccccactc tg 42
<210> 219
<211> 41
<212> DNA
<213>Artificial sequence
<400> 219
tacacgacgc tcttccgatc tggtgacaga aggggaaagg g 41
<210> 220
<211> 42
<212> DNA
<213>Artificial sequence
<400> 220
tacacgacgc tcttccgatc tcgtcctctc gctgttagac at 42
<210> 221
<211> 42
<212> DNA
<213>Artificial sequence
<400> 221
tacacgacgc tcttccgatc taaacagcac agtgaaagcc ag 42
<210> 222
<211> 40
<212> DNA
<213>Artificial sequence
<400> 222
tacacgacgc tcttccgatc tcgaagggcc agggtatgtg 40
<210> 223
<211> 41
<212> DNA
<213>Artificial sequence
<400> 223
tacacgacgc tcttccgatc tcgagaggtg agggcagtta c 41
<210> 224
<211> 43
<212> DNA
<213>Artificial sequence
<400> 224
tacacgacgc tcttccgatc tgtcctgtcc ccactccttt aat 43
<210> 225
<211> 40
<212> DNA
<213>Artificial sequence
<400> 225
tacacgacgc tcttccgatc tggaactggg cagtggactg 40
<210> 226
<211> 40
<212> DNA
<213>Artificial sequence
<400> 226
tacacgacgc tcttccgatc tcggtccctt cctcctcact 40
<210> 227
<211> 39
<212> DNA
<213>Artificial sequence
<400> 227
tacacgacgc tcttccgatc ttgggggtgt cagtgccag 39
<210> 228
<211> 41
<212> DNA
<213>Artificial sequence
<400> 228
tacacgacgc tcttccgatc ttgaggctgg aaaggtggtt c 41
<210> 229
<211> 40
<212> DNA
<213>Artificial sequence
<400> 229
tacacgacgc tcttccgatc tcagccctct gacgtccatc 40
<210> 230
<211> 39
<212> DNA
<213>Artificial sequence
<400> 230
tacacgacgc tcttccgatc tgagcgatgc ccaaccagg 39
<210> 231
<211> 41
<212> DNA
<213>Artificial sequence
<400> 231
tacacgacgc tcttccgatc ttcccatacc ctctcagcgt a 41
<210> 232
<211> 41
<212> DNA
<213>Artificial sequence
<400> 232
tacacgacgc tcttccgatc taggtctaca tgggtgcttc c 41
<210> 233
<211> 41
<212> DNA
<213>Artificial sequence
<400> 233
tacacgacgc tcttccgatc tcctccccac aacacacagt t 41
<210> 234
<211> 42
<212> DNA
<213>Artificial sequence
<400> 234
tacacgacgc tcttccgatc tacttttggg gccaaacctt ac 42
<210> 235
<211> 41
<212> DNA
<213>Artificial sequence
<400> 235
tacacgacgc tcttccgatc tcaccatcct gcctctcctt c 41
<210> 236
<211> 41
<212> DNA
<213>Artificial sequence
<400> 236
tacacgacgc tcttccgatc ttgagtgacc cccatcatga c 41
<210> 237
<211> 40
<212> DNA
<213>Artificial sequence
<400> 237
tacacgacgc tcttccgatc tagtacccct gccctctgag 40
<210> 238
<211> 40
<212> DNA
<213>Artificial sequence
<400> 238
tacacgacgc tcttccgatc tgctggtgcc actctggaaa 40
<210> 239
<211> 41
<212> DNA
<213>Artificial sequence
<400> 239
tacacgacgc tcttccgatc tgacacctac ggcagagaac c 41
<210> 240
<211> 45
<212> DNA
<213>Artificial sequence
<400> 240
tacacgacgc tcttccgatc ttgggacaac catacatcta attcc 45
<210> 241
<211> 44
<212> DNA
<213>Artificial sequence
<400> 241
tacacgacgc tcttccgatc ttcgtaagtg ttactcaaga agca 44
<210> 242
<211> 45
<212> DNA
<213>Artificial sequence
<400> 242
tacacgacgc tcttccgatc taaacatgtt catgctgtgt atgta 45
<210> 243
<211> 46
<212> DNA
<213>Artificial sequence
<400> 243
tacacgacgc tcttccgatc ttggtgattg catctaatgt tttcct 46
<210> 244
<211> 45
<212> DNA
<213>Artificial sequence
<400> 244
tacacgacgc tcttccgatc ttcctctgaa caactaaaac tctgt 45
<210> 245
<211> 47
<212> DNA
<213>Artificial sequence
<400> 245
tacacgacgc tcttccgatc ttgttttaca atgccatctt attccag 47
<210> 246
<211> 46
<212> DNA
<213>Artificial sequence
<400> 246
tacacgacgc tcttccgatc tgtgtgcata tgtgtatgtt gagtgt 46
<210> 247
<211> 43
<212> DNA
<213>Artificial sequence
<400> 247
tacacgacgc tcttccgatc tccttttggg gaagaaaagt gtt 43
<210> 248
<211> 43
<212> DNA
<213>Artificial sequence
<400> 248
tacacgacgc tcttccgatc ttggcagtca aaccttctct ctt 43
<210> 249
<211> 48
<212> DNA
<213>Artificial sequence
<400> 249
tacacgacgc tcttccgatc ttgctttttc tgtaaatcat ctgtgaat 48
<210> 250
<211> 43
<212> DNA
<213>Artificial sequence
<400> 250
tacacgacgc tcttccgatc ttcaaccttt tgaacagcat gca 43
<210> 251
<211> 43
<212> DNA
<213>Artificial sequence
<400> 251
tacacgacgc tcttccgatc tggctatgga acttctggac tgt 43
<210> 252
<211> 53
<212> DNA
<213>Artificial sequence
<400> 252
tacacgacgc tcttccgatc tcaaagatta tttgtatact gatttaagac tat 53
<210> 253
<211> 45
<212> DNA
<213>Artificial sequence
<400> 253
tacacgacgc tcttccgatc tagtttctta ctgtgactat ccttt 45
<210> 254
<211> 46
<212> DNA
<213>Artificial sequence
<400> 254
tacacgacgc tcttccgatc tgcatctgtg gcattaaatg gtgata 46
<210> 255
<211> 42
<212> DNA
<213>Artificial sequence
<400> 255
tacacgacgc tcttccgatc ttgggactta ttgaggtggt gc 42
<210> 256
<211> 49
<212> DNA
<213>Artificial sequence
<400> 256
tacacgacgc tcttccgatc tatttattca agacattttg tatctgcat 49
<210> 257
<211> 44
<212> DNA
<213>Artificial sequence
<400> 257
tacacgacgc tcttccgatc tctccaaact gaccaaactg ttct 44
<210> 258
<211> 41
<212> DNA
<213>Artificial sequence
<400> 258
tacacgacgc tcttccgatc tgatgcacat catggtggct g 41
<210> 259
<211> 46
<212> DNA
<213>Artificial sequence
<400> 259
tacacgacgc tcttccgatc tagcatatag actaaaatcc tctgtt 46
<210> 260
<211> 42
<212> DNA
<213>Artificial sequence
<400> 260
tacacgacgc tcttccgatc tacttgggga aagagtggtc tc 42
<210> 261
<211> 42
<212> DNA
<213>Artificial sequence
<400> 261
tacacgacgc tcttccgatc tacctggtcc ctgttgttga tg 42
<210> 262
<211> 44
<212> DNA
<213>Artificial sequence
<400> 262
tacacgacgc tcttccgatc tgtcaggatg ttttcaaact tcgc 44
<210> 263
<211> 48
<212> DNA
<213>Artificial sequence
<400> 263
tacacgacgc tcttccgatc tggtctcaat gatatggaga tggtgata 48
<210> 264
<211> 43
<212> DNA
<213>Artificial sequence
<400> 264
tacacgacgc tcttccgatc ttgacttgtc acaatgtcac cac 43
<210> 265
<211> 41
<212> DNA
<213>Artificial sequence
<400> 265
tacacgacgc tcttccgatc tcgctgaggt cctggagatt t 41
<210> 266
<211> 49
<212> DNA
<213>Artificial sequence
<400> 266
tacacgacgc tcttccgatc tgcagaaaaa taaagataca tacttggtt 49
<210> 267
<211> 41
<212> DNA
<213>Artificial sequence
<400> 267
tacacgacgc tcttccgatc taagcggttc aagtagcatg t 41
<210> 268
<211> 42
<212> DNA
<213>Artificial sequence
<400> 268
tacacgacgc tcttccgatc ttctgtacta caacgctggt ga 42
<210> 269
<211> 48
<212> DNA
<213>Artificial sequence
<400> 269
tacacgacgc tcttccgatc tttaaacaaa atttcacgtc acatacaa 48
<210> 270
<211> 45
<212> DNA
<213>Artificial sequence
<400> 270
tacacgacgc tcttccgatc tatctgtggg attttgaaaa actga 45
<210> 271
<211> 51
<212> DNA
<213>Artificial sequence
<400> 271
tacacgacgc tcttccgatc ttcttttcaa aattactaga tatgatactc a 51
<210> 272
<211> 39
<212> DNA
<213>Artificial sequence
<400> 272
tacacgacgc tcttccgatc tgaatggcag ggtccgcag 39
<210> 273
<211> 41
<212> DNA
<213>Artificial sequence
<400> 273
tacacgacgc tcttccgatc tagtattgat cgggagagcc g 41
<210> 274
<211> 41
<212> DNA
<213>Artificial sequence
<400> 274
tacacgacgc tcttccgatc tacgcagttg ggcacttttg a 41
<210> 275
<211> 45
<212> DNA
<213>Artificial sequence
<400> 275
tacacgacgc tcttccgatc tctttggaaa acctgcagat catca 45
<210> 276
<211> 39
<212> DNA
<213>Artificial sequence
<400> 276
tacacgacgc tcttccgatc tgcggttcag caacaaccc 39
<210> 277
<211> 42
<212> DNA
<213>Artificial sequence
<400> 277
tacacgacgc tcttccgatc tctaccctca ctcttcagct ca 42
<210> 278
<211> 42
<212> DNA
<213>Artificial sequence
<400> 278
tacacgacgc tcttccgatc tcactcatgc tctacaaccc ca 42
<210> 279
<211> 44
<212> DNA
<213>Artificial sequence
<400> 279
tacacgacgc tcttccgatc taatgtgaac ggaatacacg tctc 44
<210> 280
<211> 48
<212> DNA
<213>Artificial sequence
<400> 280
tacacgacgc tcttccgatc tctggtagag attggtgatc aataatca 48
<210> 281
<211> 49
<212> DNA
<213>Artificial sequence
<400> 281
tacacgacgc tcttccgatc tatttttcac cacatgattt ttcttctct 49
<210> 282
<211> 41
<212> DNA
<213>Artificial sequence
<400> 282
tacacgacgc tcttccgatc tgactgctgt gacccactct g 41
<210> 283
<211> 41
<212> DNA
<213>Artificial sequence
<400> 283
tacacgacgc tcttccgatc tctgacgggt ttcctcttcc t 41
<210> 284
<211> 40
<212> DNA
<213>Artificial sequence
<400> 284
tacacgacgc tcttccgatc tctacattga cggcccccac 40
<210> 285
<211> 40
<212> DNA
<213>Artificial sequence
<400> 285
tacacgacgc tcttccgatc tctccccagg cctctcacat 40
<210> 286
<211> 40
<212> DNA
<213>Artificial sequence
<400> 286
tacacgacgc tcttccgatc tcctggcctt ctgcatctgt 40
<210> 287
<211> 41
<212> DNA
<213>Artificial sequence
<400> 287
tacacgacgc tcttccgatc tatctgtcag caacctcacc c 41
<210> 288
<211> 40
<212> DNA
<213>Artificial sequence
<400> 288
tacacgacgc tcttccgatc ttttccagca tggtgagggc 40
<210> 289
<211> 45
<212> DNA
<213>Artificial sequence
<400> 289
tacacgacgc tcttccgatc tccagttaac gtcttccttc tctct 45
<210> 290
<211> 40
<212> DNA
<213>Artificial sequence
<400> 290
tacacgacgc tcttccgatc tcatctgcct cacctccacc 40
<210> 291
<211> 40
<212> DNA
<213>Artificial sequence
<400> 291
tacacgacgc tcttccgatc tggcagccag gaacgtactg 40
<210> 292
<211> 42
<212> DNA
<213>Artificial sequence
<400> 292
tacacgacgc tcttccgatc ttcatgatcc cactgccttc tt 42
<210> 293
<211> 44
<212> DNA
<213>Artificial sequence
<400> 293
tacacgacgc tcttccgatc tttcttccag tgttctaatt gcac 44
<210> 294
<211> 43
<212> DNA
<213>Artificial sequence
<400> 294
tacacgacgc tcttccgatc tgatgaagaa gacatggacg acg 43
<210> 295
<211> 41
<212> DNA
<213>Artificial sequence
<400> 295
tacacgacgc tcttccgatc ttgaccggag taaccttccc t 41
<210> 296
<211> 41
<212> DNA
<213>Artificial sequence
<400> 296
tacacgacgc tcttccgatc tagacccaca ctaccaggac c 41
<210> 297
<211> 42
<212> DNA
<213>Artificial sequence
<400> 297
tacacgacgc tcttccgatc ttgcagaata cctaagggtc gc 42
<210> 298
<211> 41
<212> DNA
<213>Artificial sequence
<400> 298
tacacgacgc tcttccgatc tatgccaaga aaccatatgc t 41
<210> 299
<211> 44
<212> DNA
<213>Artificial sequence
<400> 299
tacacgacgc tcttccgatc ttcgtgggct tgttttgtat caac 44
<210> 300
<211> 41
<212> DNA
<213>Artificial sequence
<400> 300
tacacgacgc tcttccgatc ttacacagag gaagccttcg c 41
<210> 301
<211> 39
<212> DNA
<213>Artificial sequence
<400> 301
tacacgacgc tcttccgatc tcgtgggctc gggcgggag 39
<210> 302
<211> 39
<212> DNA
<213>Artificial sequence
<400> 302
tacacgacgc tcttccgatc taacctcccg tgcgccgcc 39
<210> 303
<211> 41
<212> DNA
<213>Artificial sequence
<400> 303
tacacgacgc tcttccgatc ttccatcctg cagaagaagc c 41
<210> 304
<211> 49
<212> DNA
<213>Artificial sequence
<400> 304
tacacgacgc tcttccgatc ttgctgcata tttcagatat ttctttcct 49
<210> 305
<211> 49
<212> DNA
<213>Artificial sequence
<400> 305
tacacgacgc tcttccgatc ttttcttcct aagtgcaaaa gataacttt 49
<210> 306
<211> 43
<212> DNA
<213>Artificial sequence
<400> 306
tacacgacgc tcttccgatc tagatgacaa tcatgttgca gca 43
<210> 307
<211> 41
<212> DNA
<213>Artificial sequence
<400> 307
tacacgacgc tcttccgatc ttggctacga cccagttacc a 41
<210> 308
<211> 45
<212> DNA
<213>Artificial sequence
<400> 308
tacacgacgc tcttccgatc ttgacagtta aaggcatttc ctgtg 45
<210> 309
<211> 51
<212> DNA
<213>Artificial sequence
<400> 309
tacacgacgc tcttccgatc ttgtcatttc atttcttttt cttttctttt t 51
<210> 310
<211> 47
<212> DNA
<213>Artificial sequence
<400> 310
tacacgacgc tcttccgatc tagatgagtc atatttgtgg gttttca 47
<210> 311
<211> 48
<212> DNA
<213>Artificial sequence
<400> 311
tacacgacgc tcttccgatc tgccaaaatt aatgtgctga acttaaac 48
<210> 312
<211> 42
<212> DNA
<213>Artificial sequence
<400> 312
tacacgacgc tcttccgatc ttcctgagcc tgttttgtgt ct 42
<210> 313
<211> 41
<212> DNA
<213>Artificial sequence
<400> 313
tacacgacgc tcttccgatc tccctcccca gtcctcatgt a 41
<210> 314
<211> 41
<212> DNA
<213>Artificial sequence
<400> 314
tacacgacgc tcttccgatc tggaagaatc aaatagacaa t 41
<210> 315
<211> 41
<212> DNA
<213>Artificial sequence
<400> 315
tacacgacgc tcttccgatc tgccctggta ggttttctgg g 41
<210> 316
<211> 39
<212> DNA
<213>Artificial sequence
<400> 316
tacacgacgc tcttccgatc tgagcaggaa gaggcggag 39
<210> 317
<211> 41
<212> DNA
<213>Artificial sequence
<400> 317
tacacgacgc tcttccgatc tcagtctgac tcccctccag a 41
<210> 318
<211> 41
<212> DNA
<213>Artificial sequence
<400> 318
tacacgacgc tcttccgatc tagacccacg agtctaggtc t 41
<210> 319
<211> 42
<212> DNA
<213>Artificial sequence
<400> 319
tacacgacgc tcttccgatc tccgccactg aattcagagt ct 42
<210> 320
<211> 40
<212> DNA
<213>Artificial sequence
<400> 320
tacacgacgc tcttccgatc tgaggaacag ggcacaggtg 40
<210> 321
<211> 42
<212> DNA
<213>Artificial sequence
<400> 321
tacacgacgc tcttccgatc tgtcacgtga cacagtcaaa ca 42
<210> 322
<211> 39
<212> DNA
<213>Artificial sequence
<400> 322
tacacgacgc tcttccgatc tgagcatgct tggggccat 39
<210> 323
<211> 42
<212> DNA
<213>Artificial sequence
<400> 323
tacacgacgc tcttccgatc ttgactactt ttgacttcag cc 42
<210> 324
<211> 41
<212> DNA
<213>Artificial sequence
<400> 324
tacacgacgc tcttccgatc taaacaggat gcctgccttt a 41
<210> 325
<211> 41
<212> DNA
<213>Artificial sequence
<400> 325
tacacgacgc tcttccgatc ttcgcctcca agaatgtaag t 41
<210> 326
<211> 44
<212> DNA
<213>Artificial sequence
<400> 326
tacacgacgc tcttccgatc tactcactct agtgataaat cggg 44
<210> 327
<211> 43
<212> DNA
<213>Artificial sequence
<400> 327
tacacgacgc tcttccgatc ttgtagtttc cctagtcccc cat 43
<210> 328
<211> 41
<212> DNA
<213>Artificial sequence
<400> 328
tacacgacgc tcttccgatc tctccccaca gctctgcatt c 41
<210> 329
<211> 48
<212> DNA
<213>Artificial sequence
<400> 329
tacacgacgc tcttccgatc tcagtaggga gtaacaaaat aacactga 48
<210> 330
<211> 41
<212> DNA
<213>Artificial sequence
<400> 330
tacacgacgc tcttccgatc tccactccag catcactcac t 41
<210> 331
<211> 40
<212> DNA
<213>Artificial sequence
<400> 331
tacacgacgc tcttccgatc tcatgtcggt gcatgccttc 40
<210> 332
<211> 52
<212> DNA
<213>Artificial sequence
<400> 332
tacacgacgc tcttccgatc ttcttagata agactttgga cttagacttt tg 52
<210> 333
<211> 41
<212> DNA
<213>Artificial sequence
<400> 333
tacacgacgc tcttccgatc tgggctctat gggaaggacc a 41
<210> 334
<211> 39
<212> DNA
<213>Artificial sequence
<400> 334
tacacgacgc tcttccgatc ttccagccgg agccatggg 39
<210> 335
<211> 39
<212> DNA
<213>Artificial sequence
<400> 335
tacacgacgc tcttccgatc tgacatgctc cgccacctc 39
<210> 336
<211> 39
<212> DNA
<213>Artificial sequence
<400> 336
tacacgacgc tcttccgatc tctgcggatt gtgcgaggc 39
<210> 337
<211> 41
<212> DNA
<213>Artificial sequence
<400> 337
tacacgacgc tcttccgatc tcagctctgc taccaggaca c 41
<210> 338
<211> 40
<212> DNA
<213>Artificial sequence
<400> 338
tacacgacgc tcttccgatc tcactcagcc ctcatcctgc 40
<210> 339
<211> 41
<212> DNA
<213>Artificial sequence
<400> 339
tacacgacgc tcttccgatc tgccctggtc acctacaaca c 41
<210> 340
<211> 41
<212> DNA
<213>Artificial sequence
<400> 340
tacacgacgc tcttccgatc tcccggtatg aaggctgaga c 41
<210> 341
<211> 41
<212> DNA
<213>Artificial sequence
<400> 341
tacacgacgc tcttccgatc tgatgtccac cctgttcctg g 41
<210> 342
<211> 39
<212> DNA
<213>Artificial sequence
<400> 342
tacacgacgc tcttccgatc ttccgtggta aggtgccca 39
<210> 343
<211> 41
<212> DNA
<213>Artificial sequence
<400> 343
tacacgacgc tcttccgatc tcctggttca cttggacctg g 41
<210> 344
<211> 40
<212> DNA
<213>Artificial sequence
<400> 344
tacacgacgc tcttccgatc ttcagacccc ctcaccactg 40
<210> 345
<211> 39
<212> DNA
<213>Artificial sequence
<400> 345
tacacgacgc tcttccgatc tgaccctccc ttctgcgtg 39
<210> 346
<211> 41
<212> DNA
<213>Artificial sequence
<400> 346
tacacgacgc tcttccgatc ttagccctca atccctgacc c 41
<210> 347
<211> 39
<212> DNA
<213>Artificial sequence
<400> 347
tacacgacgc tcttccgatc tctgacccac caccccctc 39
<210> 348
<211> 47
<212> DNA
<213>Artificial sequence
<400> 348
tacacgacgc tcttccgatc ttcttctcac tcatatcctc ctctttc 47
<210> 349
<211> 39
<212> DNA
<213>Artificial sequence
<400> 349
tacacgacgc tcttccgatc tggtgcagct ggtgacaca 39
<210> 350
<211> 41
<212> DNA
<213>Artificial sequence
<400> 350
tacacgacgc tcttccgatc tgtgctggtc aagagtccca a 41
<210> 351
<211> 43
<212> DNA
<213>Artificial sequence
<400> 351
tacacgacgc tcttccgatc tggctcagta cactaaagct ccc 43
<210> 352
<211> 41
<212> DNA
<213>Artificial sequence
<400> 352
tacacgacgc tcttccgatc tgggactagc atgctgacct c 41
<210> 353
<211> 40
<212> DNA
<213>Artificial sequence
<400> 353
tacacgacgc tcttccgatc tggacgatga catgggggac 40
<210> 354
<211> 43
<212> DNA
<213>Artificial sequence
<400> 354
tacacgacgc tcttccgatc ttccgatgta tttgatggtg acc 43
<210> 355
<211> 39
<212> DNA
<213>Artificial sequence
<400> 355
tacacgacgc tcttccgatc tagacaccgg ggttccttc 39
<210> 356
<211> 39
<212> DNA
<213>Artificial sequence
<400> 356
tacacgacgc tcttccgatc tcctcctcct gccttcagc 39
<210> 357
<211> 46
<212> DNA
<213>Artificial sequence
<400> 357
tacacgacgc tcttccgatc tggaatgata gtgactttag aatgcc 46
<210> 358
<211> 47
<212> DNA
<213>Artificial sequence
<400> 358
tacacgacgc tcttccgatc tccgaagaaa tattctgaac gtttgta 47
<210> 359
<211> 44
<212> DNA
<213>Artificial sequence
<400> 359
tacacgacgc tcttccgatc tcatgactgt gtaccagaac aagt 44
<210> 360
<211> 41
<212> DNA
<213>Artificial sequence
<400> 360
tacacgacgc tcttccgatc tatggctcgc ccccttaatc t 41
<210> 361
<211> 44
<212> DNA
<213>Artificial sequence
<400> 361
tacacgacgc tcttccgatc ttctttgtgc aacctacgtg aatg 44
<210> 362
<211> 49
<212> DNA
<213>Artificial sequence
<400> 362
tacacgacgc tcttccgatc taggagtcat ttatatactt tgatgaaga 49
<210> 363
<211> 47
<212> DNA
<213>Artificial sequence
<400> 363
tacacgacgc tcttccgatc tacactctag tatctggaaa aatggct 47
<210> 364
<211> 42
<212> DNA
<213>Artificial sequence
<400> 364
tacacgacgc tcttccgatc tagcagtgtg gtaaagttcc ca 42
<210> 365
<211> 42
<212> DNA
<213>Artificial sequence
<400> 365
tacacgacgc tcttccgatc ttgacaaaga acagctcaaa gc 42
<210> 366
<211> 43
<212> DNA
<213>Artificial sequence
<400> 366
tacacgacgc tcttccgatc taggcagtgt tttagatggc tca 43
<210> 367
<211> 41
<212> DNA
<213>Artificial sequence
<400> 367
tacacgacgc tcttccgatc tgccatgcag aaactgaccc t 41
<210> 368
<211> 46
<212> DNA
<213>Artificial sequence
<400> 368
tacacgacgc tcttccgatc ttgcacaata aaacagttag ccagag 46
<210> 369
<211> 49
<212> DNA
<213>Artificial sequence
<400> 369
tacacgacgc tcttccgatc tgcaaaggta cctagtaaag tttttaact 49
<210> 370
<211> 49
<212> DNA
<213>Artificial sequence
<400> 370
tacacgacgc tcttccgatc tccatcattt aattgtaaac gtgttactc 49
<210> 371
<211> 46
<212> DNA
<213>Artificial sequence
<400> 371
tacacgacgc tcttccgatc tttccatact actcatgagg tgttta 46
<210> 372
<211> 44
<212> DNA
<213>Artificial sequence
<400> 372
tacacgacgc tcttccgatc ttggacactt tttggatcac aaga 44
<210> 373
<211> 46
<212> DNA
<213>Artificial sequence
<400> 373
tacacgacgc tcttccgatc tggaatgcca gaactacaat cttttg 46
<210> 374
<211> 45
<212> DNA
<213>Artificial sequence
<400> 374
tacacgacgc tcttccgatc tgctactctc ctgaactctc tcact 45
<210> 375
<211> 47
<212> DNA
<213>Artificial sequence
<400> 375
tacacgacgc tcttccgatc tagtctgttc ttttggatag catgaag 47
<210> 376
<211> 44
<212> DNA
<213>Artificial sequence
<400> 376
tacacgacgc tcttccgatc tactggagcc ttgtatatag acgg 44
<210> 377
<211> 47
<212> DNA
<213>Artificial sequence
<400> 377
tacacgacgc tcttccgatc tatttaatca gtggaaaaat agcctca 47
<210> 378
<211> 48
<212> DNA
<213>Artificial sequence
<400> 378
tacacgacgc tcttccgatc ttaaaacatc ctcaatggtc ttcaaaaa 48
<210> 379
<211> 47
<212> DNA
<213>Artificial sequence
<400> 379
tacacgacgc tcttccgatc tggaaccagg agctaataaa aataact 47
<210> 380
<211> 46
<212> DNA
<213>Artificial sequence
<400> 380
tacacgacgc tcttccgatc tcatttggct gtgacttcta agaaga 46
<210> 381
<211> 45
<212> DNA
<213>Artificial sequence
<400> 381
tacacgacgc tcttccgatc tagcaattgc agtttccttg agttt 45
<210> 382
<211> 41
<212> DNA
<213>Artificial sequence
<400> 382
tacacgacgc tcttccgatc ttgggagctg atgaggatcg g 41
<210> 383
<211> 48
<212> DNA
<213>Artificial sequence
<400> 383
tacacgacgc tcttccgatc tagtgtaaaa tggtaggtag aaaagaga 48
<210> 384
<211> 49
<212> DNA
<213>Artificial sequence
<400> 384
tacacgacgc tcttccgatc tacattccct aaataaaaat tcattcatt 49
<210> 385
<211> 48
<212> DNA
<213>Artificial sequence
<400> 385
tacacgacgc tcttccgatc ttctgtctga aaaatacaaa gaaacagc 48
<210> 386
<211> 46
<212> DNA
<213>Artificial sequence
<400> 386
tacacgacgc tcttccgatc tgaaaaatca gttccgttcc ccagag 46
<210> 387
<211> 41
<212> DNA
<213>Artificial sequence
<400> 387
tacacgacgc tcttccgatc taaataaaca ccagccagcc g 41
<210> 388
<211> 47
<212> DNA
<213>Artificial sequence
<400> 388
tacacgacgc tcttccgatc ttctgcattt ctcagtattt catgtga 47
<210> 389
<211> 48
<212> DNA
<213>Artificial sequence
<400> 389
tacacgacgc tcttccgatc tctcaaaaga gaaatcacgc atttatgt 48
<210> 390
<211> 42
<212> DNA
<213>Artificial sequence
<400> 390
tacacgacgc tcttccgatc taaaggagct ggaaagagtg ct 42
<210> 391
<211> 41
<212> DNA
<213>Artificial sequence
<400> 391
tacacgacgc tcttccgatc tagggcgtca tcagtttctc a 41
<210> 392
<211> 43
<212> DNA
<213>Artificial sequence
<400> 392
tacacgacgc tcttccgatc tgtgtactta cctcacttgc cca 43
<210> 393
<211> 41
<212> DNA
<213>Artificial sequence
<400> 393
tacacgacgc tcttccgatc tccaccgtca tcaccttcct t 41
<210> 394
<211> 47
<212> DNA
<213>Artificial sequence
<400> 394
tacacgacgc tcttccgatc tactcactct ccataaatgc tacgaat 47
<210> 395
<211> 41
<212> DNA
<213>Artificial sequence
<400> 395
tacacgacgc tcttccgatc tacgtggtgt gtgtctgaag t 41
<210> 396
<211> 47
<212> DNA
<213>Artificial sequence
<400> 396
tacacgacgc tcttccgatc tgtgatggag atgtgataat ttcagga 47
<210> 397
<211> 40
<212> DNA
<213>Artificial sequence
<400> 397
tacacgacgc tcttccgatc tgtctgccat gccttgtgct 40
<210> 398
<211> 47
<212> DNA
<213>Artificial sequence
<400> 398
tacacgacgc tcttccgatc taagagaaaa gaaagagaca tgcatga 47
<210> 399
<211> 46
<212> DNA
<213>Artificial sequence
<400> 399
tacacgacgc tcttccgatc ttgccaaata tagaaagagg ggattt 46
<210> 400
<211> 41
<212> DNA
<213>Artificial sequence
<400> 400
tacacgacgc tcttccgatc ttaatgatgg cagcgtgtcc c 41
<210> 401
<211> 49
<212> DNA
<213>Artificial sequence
<400> 401
tacacgacgc tcttccgatc tgccaaagaa gtagaatgag aaaaatgta 49
<210> 402
<211> 39
<212> DNA
<213>Artificial sequence
<400> 402
tacacgacgc tcttccgatc tccctcctct tgctgctgg 39
<210> 403
<211> 41
<212> DNA
<213>Artificial sequence
<400> 403
tacacgacgc tcttccgatc tagcctctta cacccagtgg a 41
<210> 404
<211> 40
<212> DNA
<213>Artificial sequence
<400> 404
tacacgacgc tcttccgatc tgaagccaca ctgacgtgcc 40
<210> 405
<211> 43
<212> DNA
<213>Artificial sequence
<400> 405
tacacgacgc tcttccgatc ttcccatgat gatctgtccc tca 43
<210> 406
<211> 46
<212> DNA
<213>Artificial sequence
<400> 406
tacacgacgc tcttccgatc tggaaactaa tagttgtctc actgcc 46
<210> 407
<211> 42
<212> DNA
<213>Artificial sequence
<400> 407
tacacgacgc tcttccgatc ttggatccaa gccatatgac gg 42
<210> 408
<211> 42
<212> DNA
<213>Artificial sequence
<400> 408
tacacgacgc tcttccgatc tcgggccatt ctaatagcct ca 42
<210> 409
<211> 41
<212> DNA
<213>Artificial sequence
<400> 409
tacacgacgc tcttccgatc tacaagcatt ccatgggcaa c 41
<210> 410
<211> 44
<212> DNA
<213>Artificial sequence
<400> 410
tacacgacgc tcttccgatc ttgctgatta cttcacctct gatt 44
<210> 411
<211> 41
<212> DNA
<213>Artificial sequence
<400> 411
tacacgacgc tcttccgatc tgccaccaaa ttagcctgga c 41
<210> 412
<211> 48
<212> DNA
<213>Artificial sequence
<400> 412
tacacgacgc tcttccgatc tgagttaatc aactgatgca aactcttg 48
<210> 413
<211> 47
<212> DNA
<213>Artificial sequence
<400> 413
tacacgacgc tcttccgatc tgaacacaaa gatcatcctt tcagaga 47
<210> 414
<211> 39
<212> DNA
<213>Artificial sequence
<400> 414
tacacgacgc tcttccgatc tgtcagcggg ctaccactg 39
<210> 415
<211> 41
<212> DNA
<213>Artificial sequence
<400> 415
tacacgacgc tcttccgatc tctgagtcgc ctgtcaccat t 41
<210> 416
<211> 40
<212> DNA
<213>Artificial sequence
<400> 416
tacacgacgc tcttccgatc tcagtcgctg caaccatcca 40
<210> 417
<211> 45
<212> DNA
<213>Artificial sequence
<400> 417
tacacgacgc tcttccgatc tagatcgtta gcagaaacaa aagga 45
<210> 418
<211> 46
<212> DNA
<213>Artificial sequence
<400> 418
tacacgacgc tcttccgatc tatggtggct ttttgtttgt ttgttt 46
<210> 419
<211> 49
<212> DNA
<213>Artificial sequence
<400> 419
tacacgacgc tcttccgatc tcctacttgt taattaaaaa ttcaagagt 49
<210> 420
<211> 47
<212> DNA
<213>Artificial sequence
<400> 420
tacacgacgc tcttccgatc tagctacctg ttaaagaatc atctgga 47
<210> 421
<211> 41
<212> DNA
<213>Artificial sequence
<400> 421
tacacgacgc tcttccgatc tcacacgacg ggaagacaag t 41
<210> 422
<211> 47
<212> DNA
<213>Artificial sequence
<400> 422
tacacgacgc tcttccgatc tgtgatcaag aaatcgatag catttgc 47
<210> 423
<211> 41
<212> DNA
<213>Artificial sequence
<400> 423
tacacgacgc tcttccgatc tgccgtcaaa tccagaggct a 41
<210> 424
<211> 42
<212> DNA
<213>Artificial sequence
<400> 424
tacacgacgc tcttccgatc taccacttgt actagtatgc ct 42
<210> 425
<211> 49
<212> DNA
<213>Artificial sequence
<400> 425
tacacgacgc tcttccgatc ttgcagaaaa cagatctgta tttatttca 49
<210> 426
<211> 49
<212> DNA
<213>Artificial sequence
<400> 426
tacacgacgc tcttccgatc taactataat tactccttaa tgtcagctt 49
<210> 427
<211> 48
<212> DNA
<213>Artificial sequence
<400> 427
tacacgacgc tcttccgatc ttgcaccagt aatatgcata ttaaaaca 48
<210> 428
<211> 40
<212> DNA
<213>Artificial sequence
<400> 428
tacacgacgc tcttccgatc taggaaccgt ttatggcccc 40
<210> 429
<211> 49
<212> DNA
<213>Artificial sequence
<400> 429
tacacgacgc tcttccgatc taaataaaca ttcaccaact tatgccaat 49
<210> 430
<211> 40
<212> DNA
<213>Artificial sequence
<400> 430
tacacgacgc tcttccgatc tgcccagcac caaaaagagc 40
<210> 431
<211> 21
<212> DNA
<213>Artificial sequence
<400> 431
agacgtgtgc tcttccgatc t 21
<210> 432
<211> 58
<212> DNA
<213>Artificial sequence
<400> 432
aatgatacgg cgaccaccga gatctacact ctttccctac acgacgctct tccgatct 58
<210> 433
<211> 64
<212> DNA
<213>Artificial sequence
<400> 433
caagcagaag acggcatacg agatnnnnnn gtgactggag ttcagacgtg tgctcttccg atct 64

Claims (9)

1. a kind of multiple PCR primer group of colorectal cancer medication related gene detection, it is characterised in that the primer sets include upper Primer sets and anti-sense primer group are swum,
Wherein, 5 ' terminal sequences of the sense primer and the partial sequence complementarity of sequencing primer, 3 ' ends of the sense primer are It is attached to the specific sequence of target area；
5 ' terminal sequences of the anti-sense primer and the partial sequence complementarity of sequencing primer, 3 ' ends of the anti-sense primer are to be attached to The specific sequence of target area, the centre position sequence of the anti-sense primer is molecular label sequence；
The target area that the primer sets are used to expand includes 8 targeted drug related gene exon regions and 23 chemotherapeutics Sequence around thing pleomorphism site, 8 targeted drug related genes are：KRAS、NRAS、BRAF、EGFR、PIK3CA、 MET, HER2 and PTEN；23 chemotherapeutics pleomorphism sites are：rs1042522、rs11615、rs12132152、 rs13181、rs1695、rs1799782、rs1799983、rs1801131、rs1801133、rs2032582、rs2070744、 rs2297595、rs25487、rs3212986、rs34743033、rs3918290、rs4148323、rs8175347、BAT-25、 BAT-26, D2S123, D5S346 and D17S250.
2. primer sets according to claim 1, it is characterised in that the sense primer group is SEQ ID NO:216~SEQ ID NO:Nucleotide sequence shown in 430.
3. primer sets according to claim 1, it is characterised in that the anti-sense primer group is SEQ ID NO:1~SEQ ID NO:Nucleotide sequence shown in 215.
4. primer sets according to claim 1, it is characterised in that the primer sets also include SEQ ID NO:Shown in 431 Nucleotide sequence and adapter-primer for Illuminate sequencer library constructions.
5. primer sets according to claim 1, it is characterised in that the molecular label sequence is one section of random 8~10 Individual nucleotide sequence.
6. the detection kit of colorectal cancer medication related gene, it is characterised in that the kit include as claim 1~ Primer sets described in 5 any one.
7. detection kit according to claim 6, it is characterised in that the sense primer group includes F1- without molecule mark Primer pond and F2- are signed without molecular label primer pond；The anti-sense primer group includes R1- molecular label primer ponds and R2- molecule marks Sign primer pond；
Wherein, the R1- molecular labels primer pond includes SEQ ID NO:1~SEQ ID NO:Nucleotide sequence shown in 118； The R2- molecular labels primer pond includes SEQ ID NO:119~SEQ ID NO:Nucleotide sequence shown in 215；The F1- SEQ ID NO are included without molecular label primer pond:216~SEQ ID NO:Nucleotide sequence shown in 333；The F2- without point Subtab primer pond includes SEQ ID NO:334~SEQ ID NO:Nucleotide sequence shown in 430.
8. a kind of detection method of colorectal cancer medication related gene, it is characterised in that the detection method comprises the following steps：
(1) the R1- molecular label primers pond in claim 7, R2- molecular label primer ponds is used to divide sample gene to be detected Do not enter performing PCR amplification, obtain first round PCR primer；
(2) first round PCR primer for obtaining step (1) use the F1- in claim 7 without molecular label primer pond respectively and SEQ ID NO:Nucleotide sequence shown in 431, F2- is without molecular label primer pond and SEQ ID NO:Nucleotides shown in 431 Sequence carries out the second wheel PCR amplifications；
(3) sequencing analysis are carried out to the second wheel PCR primer, to obtain the mutation of sample gene and loci polymorphism information.
9. detection method according to claim 8, it is characterised in that the sequencing analysis in the step (3) include as follows Step：
(31) the second wheel PCR primer for obtaining step (2) uses magnetic beads for purifying after mixing, and reuses and is sequenced for Illuminate The adapter-primer of instrument sequencing library structure carries out third round PCR amplifications；
(32) by the sequencing library constructed by step (31) it is qualified after testing after, surveyed using Illumina second generation sequenators Sequence is analyzed.
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CN108570499A (en) * 2018-01-15 2018-09-25 新开源禄西（南京）生物科技有限公司 A kind of the rapid amplifying kit and amplification method of UGT1A1 genes
CN108998515A (en) * 2018-08-24 2018-12-14 北京青航基因科技有限公司 SNP combination and the application in the product of preparation detection drug response related gene
CN109295171A (en) * 2018-10-22 2019-02-01 北京华夏时代生物工程有限公司 The nucleic acid molecule combination and application of detection SNP are sequenced for fluorescence in situ hybridization
CN109486951A (en) * 2018-12-04 2019-03-19 东莞博奥木华基因科技有限公司 A kind of lung cancer targeting medication gene detecting kit, primer and method
CN109486923A (en) * 2017-09-11 2019-03-19 广州永诺生物科技有限公司 The construction method of multiplex amplification sequencing primer system, its application and sequencing library
CN110283937A (en) * 2019-05-31 2019-09-27 上海奥根诊断技术有限公司 Detection primer group, detection reagent and sequencing library for the infection of sense organ post-transplantation
CN110592210A (en) * 2019-08-13 2019-12-20 上海艾汭得基因科技有限公司 Sequence primer for detecting BRCA1/2 full exonic sequence and design method
CN112553339A (en) * 2020-12-29 2021-03-26 广东南芯医疗科技有限公司 Method for guiding gene for individualized administration of irinotecan and kit
CN113005197A (en) * 2021-01-06 2021-06-22 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） Kit for detecting 18 gene mutation sites related to sensitivity of radiotherapy and chemotherapy of rectal cancer and application thereof
CN113278611A (en) * 2021-03-07 2021-08-20 华中科技大学同济医学院附属协和医院 Capture sequencing probes and uses thereof
CN117844933A (en) * 2024-03-07 2024-04-09 上海复迪生生命科学有限公司 Multiplex PCR primer group for detecting lung tumor related gene variation and application thereof

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CN109486923A (en) * 2017-09-11 2019-03-19 广州永诺生物科技有限公司 The construction method of multiplex amplification sequencing primer system, its application and sequencing library
CN108570499A (en) * 2018-01-15 2018-09-25 新开源禄西（南京）生物科技有限公司 A kind of the rapid amplifying kit and amplification method of UGT1A1 genes
CN108570499B (en) * 2018-01-15 2020-08-28 新开源禄西（南京）生物科技有限公司 Rapid amplification kit and amplification method for UGT1A1 gene
CN108048561A (en) * 2018-01-29 2018-05-18 为朔医学数据科技（北京）有限公司 A kind of primer sets, kit and detection method for instructing personalized medicine for detecting pharmacogenomics genotype
CN108998515A (en) * 2018-08-24 2018-12-14 北京青航基因科技有限公司 SNP combination and the application in the product of preparation detection drug response related gene
CN109295171A (en) * 2018-10-22 2019-02-01 北京华夏时代生物工程有限公司 The nucleic acid molecule combination and application of detection SNP are sequenced for fluorescence in situ hybridization
CN109486951B (en) * 2018-12-04 2020-01-07 东莞博奥木华基因科技有限公司 Lung cancer targeted drug gene detection kit, primers and method
CN109486951A (en) * 2018-12-04 2019-03-19 东莞博奥木华基因科技有限公司 A kind of lung cancer targeting medication gene detecting kit, primer and method
CN110283937A (en) * 2019-05-31 2019-09-27 上海奥根诊断技术有限公司 Detection primer group, detection reagent and sequencing library for the infection of sense organ post-transplantation
CN110592210A (en) * 2019-08-13 2019-12-20 上海艾汭得基因科技有限公司 Sequence primer for detecting BRCA1/2 full exonic sequence and design method
CN110592210B (en) * 2019-08-13 2023-10-31 阔然生物医药科技(上海)有限公司 Sequence primer for detecting BRCA1/2 total exon and design method
CN112553339A (en) * 2020-12-29 2021-03-26 广东南芯医疗科技有限公司 Method for guiding gene for individualized administration of irinotecan and kit
CN112553339B (en) * 2020-12-29 2024-08-09 广东南芯医疗科技有限公司 Guiding method and kit for irinotecan personalized medicine genes
CN113005197A (en) * 2021-01-06 2021-06-22 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） Kit for detecting 18 gene mutation sites related to sensitivity of radiotherapy and chemotherapy of rectal cancer and application thereof
CN113278611A (en) * 2021-03-07 2021-08-20 华中科技大学同济医学院附属协和医院 Capture sequencing probes and uses thereof
CN113278611B (en) * 2021-03-07 2022-11-25 华中科技大学同济医学院附属协和医院 Capture sequencing probes and uses thereof
CN117844933A (en) * 2024-03-07 2024-04-09 上海复迪生生命科学有限公司 Multiplex PCR primer group for detecting lung tumor related gene variation and application thereof
CN117844933B (en) * 2024-03-07 2024-06-11 上海复迪生生命科学有限公司 Multiplex PCR primer group for detecting lung tumor related gene variation and application thereof

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