A primer set, kit and method for detecting vector GATV3 transformation events

Abstract

The present invention relates to a safety assessment and detection method for transgenic plants in the field of bioengineering technology, and specifically to a primer set, a kit and a method for detecting a transformation event of a vector GATV3. The primer set includes a left border-specific primer and / or a right border-specific primer; wherein the left border-specific primer includes GATV3-LB-R1 / R2 / R3, and its sequence is shown in SEQ ID NOs: 1 to 3 respectively; the right border-specific primer includes GATV3-RB-F1 / F2 / F3, and its sequence is shown in SEQ ID NOs: 4 to 6 respectively. It can accurately and quickly find the left and right flanking sequences of GATV3, and no specific bands can be expanded in non-transgenic or other transgenic plants, and is suitable for detecting, monitoring and safety management of the rice genetic intelligent breeding technology (GAT) maintenance line GATV3 and its derivative lines.

Description

Technical Field

[0001] The present invention relates to a safety assessment and detection method for transgenic plants in the field of bioengineering technology, and in particular to a primer set, a kit and a method for detecting a transformation event of a vector GATV3. Background Art

[0002] In recent years, genetically modified crops such as soybeans, corn, cotton and rapeseed have been approved for planting and production in many countries, and some have been processed into food, feed or food additives. However, the ecological safety and food safety of genetically modified products are still controversial and need to be strictly regulated. Identifying the specific insertion site of the transformation event in the genome, judging the insertion inactivation and deletion of the host genome based on the site of insertion of the exogenous vector, and inferring the impact of the transformation event on the host and the resulting safety issues are one of the important links in supervision.

[0003] In transgenic crops obtained through Agrobacterium-mediated genetic transformation, the integration position of T-DNA in the recipient plant genome is random. The flanking sequence of the T-DNA insertion site, which is formed by splicing the left and right end sequences of the T-DNA of each transgenic event with the recipient genome sequence, is unique. It is a unique identifier to distinguish different transformation events and an important technical data for establishing a specific detection method for transgenic plant strains. At present, the isolation of exogenous T-DNA flanking sequences is mainly based on PCR technology. The established specific detection methods include reverse PCR, exogenous linker-mediated PCR, semi-random primer PCR, whole genome resequencing technology, etc. Among them, thermal asymmetric staggered PCR (Tail-PCR), high-efficiency thermal asymmetric PCR (hiTail-PCR) or chromosome walking in semi-random primer PCR are currently commonly used methods.

[0004] In order to clarify the molecular characteristics of GATV3 vector inserted into the genome and facilitate its detection, and promote its biosafety evaluation, the present invention designs specific detection primers according to the left and right end sequences of the T-DNA of the GATV3 vector, uses hiTail-PCR to separate its T-DNA flanking sequence, and establishes a method for detecting the insertion site of the transformation event of GATV3, providing a technical basis for the detection and identification of the transformation event created by GATV3 and its derivative products. Summary of the invention

[0005] The invention aims to provide a special PCR detection primer, a kit and a method for identifying the transformation event insertion site of a genetic intelligent breeding technology (GAT) vector pC0309-KhvMaauMCMK5400 (GATV3).

[0006] In the first aspect, the present invention first designs left border-specific primers GATV3-LB-R1 / 2 / 3 (SEQ ID NO: 1-3) and right border-specific primers GATV3-RB-F1 / 2 / 3 (SEQ ID NO: 4-6) based on the left border (LB) and right border (RB) sequences of the vector GATV3 (such as Figure 1 shown).

[0007] Accordingly, the present invention first provides a primer set for detecting a transformation event of a vector GATV3, which comprises a left border-specific primer and / or a right border-specific primer;

[0008] The left border specific primers include GATV3-LB-R1, GATV3-LB-R2 and GATV3-LB-R3, and their sequences are shown in SEQ ID NOs: 1 to 3 respectively;

[0009] The right border specific primers include GATV3-RB-F1, GATV3-RB-F2 and GATV3-RB-F3, and their sequences are shown in SEQ ID NOs: 4 to 6 respectively.

[0010] Preferably, the primer set further includes degenerate primers. In the present invention, preferably, the degenerate primers include LAD1, LAD3 and AC1, which are primers provided by Liu Yaoguang hi-PCR, and the specific sequences are shown in SEQ ID NOs: 7 to 9, respectively.

[0011] In a second aspect, the present invention further provides a kit comprising the primer set.

[0012] Preferably, the kit further comprises dNTPs, PCR buffer and thermostable DNA polymerase.

[0013] The kit described in the present invention can be divided into a kit for amplifying the left wing sequence and a kit for amplifying the right wing sequence, or a reagent for amplifying the left wing sequence and a reagent for amplifying the right wing sequence can be provided in one kit.

[0014] For example, as an embodiment of the present invention, a kit for amplifying a left wing sequence includes: left border-specific primers GATV3-LB-R1 / R2 / R3, dNTPs, PCR buffer and a thermostable DNA polymerase; a kit for amplifying a right wing sequence includes: right border-specific primers GATV3-RB-F1 / F2 / F3, dNTPs, PCR buffer and a thermostable DNA polymerase.

[0015] In a third aspect, the present invention also provides the use of the primer set or the kit in detecting transformation events of vector GATV3.

[0016] Preferably, the application specifically includes application in any of the following aspects:

[0017] (1) detecting whether there is a vector GATV3 transformation event in the sample to be tested;

[0018] (2) identification of flanking sequences specific to the transformation event of vector GATV3;

[0019] (3) Identify the insertion site of the vector GATV3 transformation event.

[0020] Furthermore, the present invention also provides a method for detecting a transformation event of a vector GATV3, which comprises: using the primer set or the kit and taking the DNA of the sample to be tested as a template to perform PCR amplification.

[0021] Preferably, the method comprises: performing hiTail-PCR amplification on the left border and / or the right border of GATV3;

[0022] When performing hiTail-PCR amplification on the left border of GATV3, the following steps are specifically included:

[0023] a) using the DNA of the sample to be tested as a template and LAD1, LAD3 and the GATV3-LB-R1 as primers to perform first-stage PCR amplification;

[0024] b) using the first-stage PCR amplification product as a template and AC1 and the GATV3-LB-R2 as primers to perform a second-stage PCR amplification;

[0025] c) using the second-stage PCR amplification product as a template and AC1 and the GATV3-LB-R3 as primers to perform a third-stage PCR amplification;

[0026] When performing hiTail-PCR amplification of the right border of GATV3, specifically including:

[0027] 1) using the DNA of the sample to be tested as a template, and using LAD1, LAD3 (preferably LAD1 and LAD3 are mixed in equal proportions) and the GATV3-RB-F1 as primers, performing the first stage PCR amplification;

[0028] b) using the first-stage PCR amplification product as a template and AC1 and the GATV3-RB-F2 as primers to perform a second-stage PCR amplification;

[0029] c) using the second-stage PCR amplification product as a template and AC1 and the GATV3-RB-F3 as primers, a third-stage PCR amplification was performed.

[0030] Preferably, the reaction procedure of the first-stage PCR amplification is: pre-denaturation at 93°C for 2 minutes, 95°C for 1 minute; (1) specific reaction at 94°C for 30 seconds, 60°C for 1 minute, 72°C for 3 minutes, 10 cycles; (2) degenerate primer hybridization reaction at 94°C for 30 seconds, 25°C for 2 minutes, 0.5°C / s to 72°C, 72°C for 3 minutes; (3) staggered thermal asymmetric PCR reaction at 94°C for 20 seconds, 58°C for 1 minute, 72°C for 3 minutes, 25 cycles; (4) 72°C for 5 minutes, 25°C for 2 minutes;

[0031] The reaction program of the second-stage PCR amplification is: 94°C 20s, 65°C 1min, 72 3min, 1 cycle; 94°C 20s, 68°C 1min, 72°C 3min, 94°C 20s, 68°C 1min, 72°C 3min, 94°C 20s, 50°C 1min, 72°C 3min, 13 cycles; 72°C 5min, 25°C 2min;

[0032] The reaction procedure of the third-stage PCR amplification is: 94°C 20s, 68°C 1min, 72°C 3min, 94°C 20s, 68°C 1min, 72°C 3min, 94°C 20s, 50°C 1min, 72°C 3min, 7 cycles; 72°C 5min, 25°C 2min.

[0033] Preferably, if a specific band can be amplified, it is determined that a vector GATV3 transformation event exists in the sample to be tested; otherwise, it is determined that a vector GATV3 transformation event does not exist in the sample to be tested.

[0034] Preferably, the method further comprises:

[0035] The final PCR amplification product was sequenced to determine the flanking sequence specific to the transformation event of the vector GATV3.

[0036] Preferably, the method further comprises:

[0037] The final PCR amplification product was compared with the sequence of the corresponding transformed plant genome to determine the insertion site of the vector GATV3 transformation event.

[0038] The objects of detection mentioned in the present invention include, but are not limited to, transgenic crops such as soybean, corn, cotton and rapeseed and their derivative products. As long as there is a vector GATV3 transformation event, it can be detected by the primer set, kit and method of the present invention.

[0039] Based on the above technical solution, the beneficial effects of the present invention are as follows:

[0040] The present invention discloses for the first time a primer set, a kit and a method for detecting transformation events of the vector GATV3, which can accurately and quickly find the left and right flanking sequences of GATV3, while no specific bands can be amplified in non-transgenic or other transgenic plants. The present invention is suitable for the detection, monitoring and safety management of the rice genetic intelligent breeding technology (GAT) maintenance line GATV3 and its derivative lines. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Specific primers for the left and right borders of GATV3 are distributed on the vector; GATV3-LB-R1 / 2 / 3 are left border specific primers; GATV3-RB-F1 / 2 / 3 are right border specific primers.

[0042] Figure 2 The hiTail-PCRIII electrophoresis diagram of the left border flanking sequence of the transformation events of GATV3-81-4, GATV3-438-2, and GATV3-1540-4; wherein, M, D2000 DNA Marker; 1-4, ddH2O, ZH11, 9311, GATV3 vector plasmid and other negative controls; 5-7, GAT maintenance lines GATV3-81-4, GATV3-438-2, and GATV3-1540-4, respectively.

[0043] Figure 3 The hiTail-PCRIII electrophoresis diagram of the right border flanking sequence of the transformation events of GATV3-81-4, GATV3-438-2, and GATV3-1540-4; wherein, M, D2000 DNA Marker; 1-4, ddH2O, ZH11, 9311, GATV3 vector plasmid and other negative controls; 5-7, GAT maintenance lines GATV3-81-4, GATV3-438-2, and GATV3-1540-4, respectively.

[0044] Figure 4 , 5 , 6 are the integration sites of the maintenance lines GATV3-81-4, GATV3-438-2, and GATV3-1540-4 in the rice genome, respectively; among them, the T-DNA of the rice GAT maintenance line GATV3-81-4 was inserted into the non-coding region of chromosome 4 of the rice genome at 11595116-11595154 bases, and the genome was deleted by 37bp; the T-DNA of GATV3-438-2 was inserted into the non-coding region of chromosome 12 of the rice genome at 23057219-23057260 bases, and the genome was deleted by 40bp; the T-DNA of GATV3-1540-4 was inserted into the non-coding region of chromosome 3 of the rice genome at 28491291-28491308 bases, and the genome was deleted by 16bp.

[0045] Figure 7 The diagram shows the comparison results of the left and right flanking sequences of rice GAT maintainer lines GATV3-81-4, GATV3-438-2, and GATV3-1540-4 with the transformation vector and the rice genome; LB is the left border of the GATV3 vector; RB is the right border of the GATV3 vector; the framed part is the rice genome sequence, and the italic part is the GATV3 vector sequence. DETAILED DESCRIPTION

[0046] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0047] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0048] Example 1 Design of specific primers for the left and right borders (LB / RB) of vector GATV3

[0049] In this embodiment, based on the genetic intelligent breeding technology GAT, pollen fertility restoration genes, pollen abortion genes, herbicide sensitive genes, screening marker genes, etc. are tightly linked in a specific order and direction to construct the vector pC0309-KhvMaauMCMK5400 (GATV3, sequence as shown in SEQ ID NO.16). For specific vector construction methods, refer to CN202010379287.9 ​​"A genetic intelligent breeding system for crop hybrid breeding and seed production and its application". Referring to the high-efficiency thermal asymmetric interlaced PCR (hiTail-PCR) method of Liu et al. (2007), three specific primers were designed at the left and right borders of GATV3, respectively. Three specific primers (GATV3-LB-R1 / R2 / R3) were designed for the left border (LB) sequence, and the sequences were shown in SEQ ID NOs: 1 to 3; three specific primers (GATV3-RB-F1 / F2 / F3) were designed for the right border (RB) sequence, and the sequences were shown in SEQ ID NOs: 4 to 6 (the specific primer sequences are shown in Table 1), and the specific primers were shown in Table 2. Figure 1 shown.

[0050] Example 2 Amplification of flanking fragments of the left and right borders of GATV3

[0051] According to Example 1, the GATV3 vector was transferred into Agrobacterium, and three different rice varieties, Zhonghua 11 (ZH11), 9311, and Regen 237, all carrying the homozygous recessive male sterility gene Oscyp704b2-3) were used as receptors, and the transformation event of the genetic intelligent breeding technology (GAT), i.e., the GATV3 maintainer line plants, was obtained through Agrobacterium-mediated genetic transformation. Taking the GAT rice maintainers GATV3-81-4, GATV3-438-2, and GATV3-1540-4, whose receptors were ZH11, 9311, and Regen 237, as examples, the left and right border flanking sequences of the transformation events were amplified and identified using the specific primers of the left and right borders of GATV3 in Example 1. The details are as follows:

[0052] (1) Extraction of rice genomic DNA using TPS method

[0053] ① Grinding sample: Take 3-4cm long young rice leaves (1-2cm for old leaves), put them into a 2ml centrifuge tube, add 800μl TPS extract, add steel beads, and grind them for 120 seconds using a cell disruptor (grinding machine);

[0054] ②After grinding, place in a 75℃ water bath and incubate for 30 minutes;

[0055] ③ Centrifuge at 13000 rpm for 10 min, take the supernatant (about 500 μl) into another 1.5 ml centrifuge tube;

[0056] ④ Add two times the volume of pre-cooled anhydrous ethanol or an equal volume of isopropanol, mix gently, and place in a -30℃ refrigerator for 2-3 hours (or place in a 4℃ refrigerator overnight; or place in a -80℃ refrigerator for 1-2 hours) until the DNA precipitates;

[0057] ⑤ Centrifuge at 13000rpm for 5min, pour off the supernatant, and place the centrifuge tube upside down on the table to air dry;

[0058] ⑥ After air drying, add 200 μl 1×TE solution or sterile ddH2O to dissolve; use 1.0% agarose gel electrophoresis to detect its integrity, and use a micro-UV spectrophotometer to determine the DNA concentration, and place in a 4°C refrigerator for later use.

[0059] (2) Separation of flanking sequences using hiTail-PCR

[0060] According to the specific primers of the left and right borders (LB / RB) of the vector GATV3 in Example 1, they were combined with the degenerate primers LAD1-1, LAD1-3 and AC1, and the sequences were respectively shown in SEQ ID NOs: 7 to 9, to separate the left and right flanking sequences of the T-DNA of the GATV3 transformation event. The specific primer sequences are shown in Table 1.

[0061] Table 1 hiTail-PCR primer list

[0062]

[0063]

[0064] Among them, B is G or C or A.

[0065] HiTail-PCR technology was used to amplify the left and right flanking sequences of the exogenous vector insertion site of the GAT rice maintainer lines GATV3-81-4, GATV3-438-2, and GATV3-1540-4 transformation events in three stages. The details are as follows:

[0066] ① Left flank sequence: hiTail-PCR stage 1 reaction used two long random primers (LAD1-1 and LAD1-3 mixed in equal proportions) combined with specific primer GATV3-LB-R1, and PCR amplification was performed using rice GAT maintainer lines GATV3-81-4, GATV3-438-2, and GATV3-1540-4 genomic DNA as templates, with ddH2O, non-transgenic rice Zhonghua 11 and 9311, and GATV3 plasmid as controls. The stage 1 PCR amplification reaction product was diluted 40 times and used as a template for the stage 2 hiTail-PCR reaction, and the primer combination for the stage 2 reaction was AC1 / GATV3-LB-R2. The stage 2 reaction product was diluted 10 times and used as a template for the stage 3 reaction, and the primer combination was AC1 / GATV3-LB-R3. The amplified products of the second and third stage reactions were separated by 1.0% agarose gel electrophoresis, and specific bands of approximately 1000 bp, 550 bp, and 480 bp were selected for sequencing (e.g. Figure 2 ). The PCR reaction system and procedure are shown in Tables 2 and 3.

[0067] ②Right flank sequence: hiTail-PCR stage 1 reaction used two long random primers (LAD1-1 and LAD1-3 mixed in equal proportions) combined with specific primer GATV3-RB-F1, and PCR amplification was performed using rice GAT maintainer lines GATV3-81-4, GATV3-438-2, and GATV3-1540-4 genomic DNA as templates, with ddH2O, non-transgenic rice Zhonghua 11 and 9311, and GATV3 plasmid as controls. The stage 1 PCR amplification reaction product was diluted 40 times and used as a template for the stage 2 hiTail-PCR reaction, and the primer combination for the stage 2 reaction was AC1 / GATV3-RB-F2. The stage 2 reaction product was diluted 10 times and used as a template for the stage 3 reaction, and the primer combination was AC1 / GATV3-RB-F3. The amplified products of the second and third stage reactions were separated by 1.0% agarose gel electrophoresis, and specific bands of approximately 520 bp, 380 bp, and 500 bp were selected for sequencing (e.g. Figure 3 ). The PCR reaction system and procedure are shown in Table 2 and Table 3:

[0068] Table 2 hiTail-PCR reaction system

[0069]

[0070]

[0071] Table 3 hiTail-PCR reaction procedure

[0072]

[0073]

[0074] Example 3 Identification of the integration site of GATV3 T-DNA in the rice genome

[0075] (1) Left wing sequence analysis

[0076] The specific band PCR products amplified by hiTail-PCR were sequenced to obtain left border fusion sequences of 1019 bp, 537 bp, and 465 bp, respectively, and the sequences are shown in SEQ ID NOs: 10 to 12. The NCBI website (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) was used to align the sequences with the rice genome and GATV3 vector. Figure 7 , the characteristics of the above three sequences are:

[0077] The left border fusion sequence of GATV3-81-4 (SEQ ID NO: 10) completely matches the left border sequence of the vector from position 1 to 107, and completely matches the published sequence on chromosome chr4 of rice (AP014960.1 (11594205-11595116)). That is, the T-DNA of the rice GAT maintenance line GATV3-81-4 is inserted into the non-coding region of chromosome 4 of the rice genome at base 11595116 (e.g. Figure 4 ).

[0078] The left border fusion sequence of GATV3-438-2 (SEQ ID NO: 11) completely matches the left border sequence of the vector from position 1 to 97, and completely matches the published sequence on chromosome chr12 of rice (AP014968.1 (23057260-23057699)). That is, the T-DNA of the rice GAT maintenance line GATV3-438-2 is inserted into the non-coding region of chromosome 12 of the rice genome at base 23057260 (e.g. Figure 5 ).

[0079] The left border fusion sequence of GATV3-1540-4 (SEQ ID NO: 12) completely matches the left border sequence of the vector from position 1 to 121, and completely matches the published sequence on chromosome chr3 of rice (AP014959.1 (28491308-28491651)). That is, the T-DNA of the rice GAT maintenance line GATV3-1540-4 is inserted into the non-coding region of chromosome 3 of the rice genome at base 28491308 (e.g. Figure 6 ).

[0080] (2) Right wing sequence analysis

[0081] The specific band PCR products amplified by hiTail-PCR were sequenced to obtain right border fusion sequences of 524 bp, 386 bp, and 485 bp, respectively, and the sequences are shown in SEQ ID NOs: 13 to 15. After comparison with the rice genome and the GATV3 vector sequence on the NCBI website (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), the characteristics of the above three sequences were analyzed as follows:

[0082] The right border fusion sequence of GATV3-81-4 (SEQ ID NO: 13) completely matches the right border sequence of the vector from position 1 to position 36, and completely matches the published sequence on chromosome chr4 of rice (AP014960.1 (11595154-11595641)) from position 37 to position 524. That is, the T-DNA of the rice GAT maintenance line GATV3-81-4 is inserted into the non-coding region of chromosome 4 of the rice genome at base 11595154 (e.g. Figure 4 ).

[0083] The right border fusion sequence of GATV3-438-2 (SEQ ID NO: 14) completely matches the right border sequence of the vector from position 1 to position 45, and completely matches the published sequence on chromosome chr12 of rice (AP014968.1 (23056879-23057219)). That is, the T-DNA of the rice GAT maintenance line GATV3-438-2 is inserted into the non-coding region of chromosome 12 of the rice genome at base 23057219 (e.g. Figure 5 ).

[0084] The right border fusion sequence of GATV3-1540-4 (SEQ ID NO: 15) completely matches the right border sequence of the vector from position 1 to position 39, and completely matches the published sequence on chromosome chr3 of rice (AP014959.1 (28490846-28491291)) from position 40 to position 485. That is, the T-DNA of the rice GAT maintenance line GATV3-1540-4 is inserted into the non-coding region of chromosome 3 of the rice genome at base 28491291 (e.g. Figure 6 ).

[0085] The results of hiTail-PCR verification of the left and right boundaries of the GAT maintenance line showed that:

[0086] The T-DNA of the rice GAT maintainer line GATV3-81-4 was inserted into the non-coding region of chromosome 4 of the rice genome at bases 11595116-11595154, causing a 37 bp deletion in the genome (AP014968.1 (11595117 to 11595153)).

[0087] The T-DNA of the rice GAT maintainer line GATV3-438-2 was inserted into the non-coding region of chromosome 12 of the rice genome at bases 23057219-23057260, causing a 40 bp deletion in the genome (AP014968.1 (23057220 to 23057259)).

[0088] The T-DNA of the rice GAT maintainer line GATV3-1540-4 was inserted into the non-coding region of chromosome 3 of the rice genome at bases 28491291-28491308, causing a 16 bp deletion in the genome (AP014959.1 (28491292-28491307)).

[0089] In summary, the present invention provides a kit for amplifying the left and right flanking sequences of the transformation event of the vector GATV3 in the genome, wherein the kit for amplifying the left flank sequence includes: left border specific primers GATV3-LB-R1 / R2 / R3, dNTPs, PCR buffer and high temperature resistant DNA polymerase and corresponding amplification procedures and methods; the kit for amplifying the right flank sequence includes: right border specific primers GATV3-RB-F1 / F2 / F3, dNTPs, PCR buffer and high temperature resistant DNA polymerase and corresponding amplification procedures and methods. The left and right flanking sequence kit can accurately and quickly find the left and right flanking sequences of GATV3, but no specific bands can be amplified in non-transgenic or other transgenic rice varieties. Therefore, the left and right flanking sequence kit is suitable for identifying the insertion sites of transformation events of other varieties produced by the GATV3 vector. The objects of detection mentioned in the present invention include but are not limited to transgenic crops such as soybeans, corn, cotton and rapeseed and their derivatives.

[0090] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention. Sequence Listing <110> Hainan Bolian Rice Gene Technology Co., Ltd. <120> A primer set, kit and method for detecting vector GATV3 transformation events <130> KHP211119423.4 <160> 16 <170> SIPOSequenceListing 1.0 <210> 1 <211> twenty four <212> DNA <213> Artificial Sequence <400> 1 attgttatcc gctcacaatt ccac 24 <210> 2 <211> 41 <212> DNA <213> Artificial Sequence <400> 2 cagtcgggaa acctgtcgtg ccccggactg gagtccatcg t 41 <210> 3 <211> twenty four <212> DNA <213> Artificial Sequence <400> 3 gagaggcggt ttgcgtattgcta 24 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <400> 4 actagatccc taggcacgtg 20 <210> 5 <211> 43 <212> DNA <213> Artificial Sequence <400> 5 acgatggact ccagtccggt taccaggtga actccaagtc ctg 43 <210> 6 <211> 25 <212> DNA <213> Artificial Sequence <400> 6 agattgtcgt ttcccgcctt cagtt 25 <210> 7 <211> 33 <212> DNA <213> Artificial Sequence <400> 7 acgatggact ccagagcggc cgcbnbnnng gaa 33 <210> 8 <211> 34 <212> DNA <213> Artificial Sequence <400> 8 acgatggact ccagagcggc cgcbbnbnnn ccaa 34 <210> 9 <211> 16 <212> DNA <213> Artificial Sequence <400> 9 acgatggact ccagag 16 <210> 10 <211> 1019 <212> DNA <213> Artificial Sequence <400> 10 gagaggcggt ttgcgtattg gctagagcag cttgccaaca tggtggagca tacattaaaa 60 acgtccgcaa tgtgttatta agttgtctaa gcgtcaattt gtttacatct gtgcatgacc 120 aaagagtact gacagaagca gtacggagct atccctttgc tttcccaagg ctaccatggg 180 gtaagtttta aatttaatct tacacaaatg caaagctgac tatctatttt cacaaaaaaa 240 aatgagtttg cttatttttt ggtgcaggga aatattttct tgtggactca ggatatgcaa 300 gccggtatgg attcctacca tcatacccac attttttgta ccacttggat gaatttgggg 360 gtaatacaga agcagccttg ccgcatggaa gggaagagac tttcaactac atgcacaatt 420 cactgtgtaa cacagtggaa agggcatttg gagtcatcaa aagccaatag agaatcctga 480 gggaattacc ttacttccct aggagtgacg atcataccaa gattatacat tctgccttcg 540 ctctccataa cttcaggatt gacagctccg acactcattt ccttacacaa aacccattgt 600 acaatgggta cccaatcatt tcagacgctc cacctctacg cggctggtac gatgcaccca 660 acagtgcgga agtaatgaat acagtccgcg acgctattgc cgatgaagtt tacaactagt 720 tggtttttag ggcgcaccgt ttaaaataaa taacatagcc atggctgttg tggatatgtt 780 atgtagcagt ctgtgtgttt ctatgatctg tctgttcctg catgtgttaa gtcagtgtgt 840 ttgtagtact gttttatttg tctaattttc ctgtacgaga ctattatgat taatgcgacc 900 acctttgtgt taatctctcc atttgatttc gtatcttgca taaattagct tttagcatcc 960 ttcattactg ccgtgattcc caccagcagt gcgtcggcct cagaacagct gctaagcag 1019 <210> 11 <211> 537 <212> DNA <213> Artificial Sequence <400> 11 ggtttgcgta ttggctagag cagcttgcca acatggtgga gcatacatta aaaacgtccg 60 caatgtgtta ttaagttgtc taagcgtcaa tttgtttcag tctctaaaat atatctttga 120 ctatgatttt ctattataat atataaaaaa attaagttta ttggagtatt tttcaagaat 180 agtgtttaca tcgtgtttta acgccttcaa actaaatatt ttaaattaat agctcaaaat 240 tttaattgtt tgacctcaac cttgttcaaa acattaaaaa ttatagaact ggaaagagta 300 ttttttgcat gagacttgtc aaccaatcaa attggtcaaa aactccaaat tgtaactgaa 360 aaattagtta tgcaatgatc gaagtgacat cttgcacggg ggtggtcaaa agagggaaat 420 aatcaatggc ctagaatcta gtttagtcat taggttgagt gagtgtgaca atcctgccag 480 aaaataatga cttttactta tttctaaggt ttacggctcc ttggatgcag taatttt 537 <210> 12 <211> 465 <212> DNA <213> Artificial Sequence <400> 12 ggcggtttgc gtattggcta gagcagcttg ccaacatggt ggagcataca ttaaaaacgt 60 ccgcaatgtg ttattaagtt gtctaagcgt caatttgttt acacagttag ttggtgtcca 120 cgctacccgc gggccgcggc gccagcatgc aagcgggaca cgtcccaccc gcggaccatg 180 catgtaaccg tctctccccg tagcgcgcgc cgcggggtgc gttgcattgg cagccgtccg 240 tcccgcgcgc cggatcgtgg caaacccatg tgacgttgtc gctggcctcg cgccccgcag 300 tacgtacgcc gttgcgccac ggttcgccgt gccgtcaccc caccttccga tctcacgcag 360 gcgcaggcgc gcagcgcatc atcgcgagcc cgtgaccgcg acacggggcg cggtggtgtg 420 ggtggcgtgg tgggggcgag gcgagcgcga cgggaacgtg cgcga 465 <210> 13 <211> 524 <212> DNA <213> Artificial Sequence <400> 13 cgtttcccgc cttcagttta aactatcagt gtttgatccc atccaacaac aaaaagaaca 60 ttcctagaag taatgtggtg acagttccta tgttgaatgg aatcatccac taccacaatg 120 caaggaatgt gtgttccatc cactgcacca gccgcaccag caaatggcag aaattcttcc 180 tcattaacaa cacgataatg aactctctca aagtttggat ctaccggctt atttttgtca 240 gcagccattg cataaatagc attaagtact ctatgaaagt acttagagat agtagaccca 300 gatcgcacca tcctattatt agtcgcccta tttgaattgc cccccgccat gatgtacaga 360 aatattccaa gtgcttccat taggcatacc tcaattgatc catgcagatt gtaccgatta 420 gtaagtaccc caaacagagc atggatttga tcaggcatca gacggaaaat tgcttggcat 480 tggatgggtt tgccattgtc tccatcatcc attccattcc agat 524 <210> 14 <211> 386 <212> DNA <213> Artificial Sequence <400> 14 agattgtcgt ttcccgcctt cagtttaaac tatcagtgtt tctcggctga gggagtatcc 60 caatccccag cactaattat gagcaaaatg gaccatgaaa cattggccac agcctgcaaa 120 tctgaatcca gcagagaaca cattgcactg ttcttcacca ctccttttct ccagccatca 180 cagcaaccac ttgagctgct gccacacaca agaaaaaagg ctaatacagc cacaggctgt 240 tcatggatgg aagaaagata tgatcttttc ctttgcatct tgaagagaaa gctggcatct 300 tggaaagcag cagcaaagcc atgtcatcac gagccaaaga aagctaatgg tggtggctgg 360 agctggaggt gtgtggatcc attcca 386 <210> 15 <211> 485 <212> DNA <213> Artificial Sequence <400> 15 agattgtcgt ttcccgcctt cagtttaaac tatcagtgtt tgcctgcgag atctcgatac 60 cgttcccatt catcgcataa ctccctgtat gctattgcta ctctactcct gcaggctgca 120 gcttctattt cttccagaat aattatagtc gtactagtac tcgcaagcta tattaggtta 180 gggtgcatta ttagcggcat caaaatctcc caatcgtaga atgctagtag atctgttgtc 240 agcgtgcaat ttttattcta ggattctcta aacaacaacc taagtgaaca tatttttgag 300 cggctgtggc tacggcagca caattcagat tcgctatatg tacatcggta gaccgtggcg 360 agctctacca atcttagctt agagtggcca ctgaccacac taccatgcgt tagtgcacga 420 caggctagga gctggagggg gggggggggg gggtacatgc tcgactcaaa cgttctatcc 480 ctggc 485 <210> 16 <211> 28723 <212> DNA <213> Artificial Sequence <400> 16 aattgacgct tagacaactt aataacacat tgcggacgtt tttaatgtat gctccaccat 60 gttggcaagc tgctctagcc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat 120 taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt 180 aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt 240 atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat 300 tacctgcagg ataatgacag cctaggcgga ggtgcggtaa agcttgccga aaacatgcag 360 aagagcaacg acggcaatga acccaatgct catgatgagg actgagttcg gggacatctt 420 gcgcccagca gcctcatcgg tgtagaactg gagcattgtg ctggcaccgc ctccaccagt 480 gccactgctg gtggttctac gcctgcgcaa gcttgcagca gctgctgcac tccctctagc 540 cggggcatct ccattggcca ccatcttgct ttatccctct gcatgataat atgagtttca 600 aatgtaaggt ttgcagcact aatattacag aaaaccaaca gaacaacaga gtttcatcca 660 aagtcgtatt gcatatacat aggaagtgtt aaaatatgtc tatcattttg gaagatacgg 720 tttatgctgt cacacagcat tttggaagtg actattttat aagcacagaa gtttcttcaa 780 tgtggaatat gtcaaaaggc aaaataagaa gcacagaagt ttcttcaatg tggaatatgt 840 cagaaggcag aataaggtac acatcttgga agtgtatgat agtactacac caataccagt 900 gaagttttag ttgtcacatt tgagtgctaa taaaaatata aaaaagaaat ggttgctgtt 960 gctcatgcct atatacattc ataatctatc aaactaactg ctcctggatg ctgcataact 1020 ataactaaac aagcttaagt taaatttacc acagaaaaag aaaaaatgac aactagtccc 1080 agaattctgc tgaaaaattt tggggctgtc ctgggcttgg ccaaaacaccc attgacatga 1140 tgctgcccaa gtgtaagaac tgtaaaacaa gtatagtgtc tgtgtatgta cagggatggc 1200 agcatattca ttgctgcaac acaagctacg ctacatgaaa ccaatttctt acgctgggaat 1260 atgaacaaac aacatggagg agagatttcg taatagaatt ttgagcaaat atgttggtac 1320 ggacaaaatg atcccccaca aaaatccgca gagaagatca tgagtgacac gcgatatatg 1380 aggtaacaca cgaacatctt atcaagaatt cagatccatt cccagatcct gacaaagcac 1440 tagaactaca acagaaatac ttcgataaaa caattcgatt tcccttcatg acacatccta 1500 acatcacatc aaaccccccg cagccaatct gaattctgaa cagcaagatc tggaacagaa 1560 gcggtaccca tcccagaatt ctaaatcggc caaaccaaac aagcccgatc taagacatcg 1620 attcaacatg aacgcgtacg gaatcaaagc aggctaatcg gagagatggc gaaaagagga 1680 tgattttcgc gcgcacctga tgaatctgcc ctgcgccaat cgctcgtgct cccgtcccaa 1740 cttggtcact cgtcttctcg cccgaaaatc tgagtgcgga attcagaatt ctctccgcgt 1800 ctgaacccgc gcgctgatat ctacccaact ggctggatta acgggttccg ttcaagatcc 1860 gatatcaagt gacgtggtcg gcgcgatctg attggccgga gcgcgtctcc gcgcgtcgat 1920 ctgagccgtc cgattcgttg ccgggtcccg atcgcgcggc ctggtgtgaa acgggtggcg 1980 tcaccgcgtg cggcgtggca ctgtgacgtg gcaacggtta tgcggttatg cacagtcatg 2040 ggctggacct tttggcccaa catctgtgga ctcgtggacc gggtttcggc ccttttatcc 2100 gctctacgga cgcagtccac gtcagccgac gtgggtccca ccacgaaggg cgtgcctccc 2160 tctaaaatt gccaatgacg ataagagcaa agacggacgg gaggggaggg gtccaaatta aaactccaaa atccattcga acagcgaagg aaatttgttg gaaaatttg agatttggat ttttgttcta ggagagggga aggttagaag aagttgagat cggtggagaa ctggagatcg aggggagatg ctgtgtctca cctcctcttc ctcctccgcg cccgctccgc tccttccctc tctcgctgat cgaccgagcc caggaatcgc gggcgggggt ggcaatgttc gcctgagcgt 2460 ggtttcttcg ccgcgccggt cgtggcctgg aaaggtcaag accaatttct cagttcctgc 2520 gactgcgcga aaaaacaaaa ccatggtgac tgttgtggag gagatcgcgt ccggccacca agtcctcttt caggggttca actgggagtc gtggaagcag agcggcgggt ggtacaacat gatgatgggc aaggtcgacg acatcgccgc tgccggagtc acccacgtct ggctgccacc 2700 gccgtcgcac tccgtctcca acgaaggtta catgcctggt cggctgtacg acatcgacgc gtccaagtac ggcaacgcgg cggagctcaa gtcgctcatc ggcgcgctcc acggcaaggg cgtgcaggcc atcgccgaca tcgtcatcaa ccaccgctgc gccgactaca aggatagccg 2880 cggcatctac tgcatcttcg agggcggcac ctccgacggc cgcctcgact ggggccccca 2940 catgatctgt cgcgacgaca ccaaatactc cgatggcacc gcaaacctcg acaccggagc 3000 cgacttcgcc gccgcgcccg acatcgacca cctcaacgac cgggtccagc gcgagctcaa 3060 ggagtggctc ctctggctca agagcgacct cggcttcgac gcgtggcgcc ttgacttcgc 3120 taggggctac tcgccggaga tggccaaggt gtacatcgac ggcacatccc cgagcctcgc 3180 cgtggccgag gtgtgggaca atatggccac cggcggcgac ggcaagccca actacgacca 3240 ggacgcgcac cggcagaatc tggtgaactg ggtggacaag gtgggcggcg cggcctcggc 3300 aggcatggtg ttcgacttca cgaccaaagg gatactgaac gctgccgtgg agggcgagct 3360 gtggaggctg atcgacccgc aggggaaggc ccccggcgtg atgggatggt ggccggccaa 3420 ggccgccacc ttcgtcgaca accacgatac aggctccacg caggccatgt ggccattccc 3480 ctccgacaag gtcatgcagg gctacgcgta catcctcacc caccccggca tcccatgcat 3540 cttctacgac catttcttca actgggggtt taggaccag atcgcggcgc tggtggcgat 3600 caggaagcgc aacggcatca cggcgacgag cgccctgaag atcctcatgc agaaggaga 3660 tgcctacgtc gccgagatag acggcaaggt ggtggtgaag atcgggtcca ggtacgacgt 3720 cggggcggtg atcccggccg ggttcgtgac ctcggcacac ggcaacgact acgccgtctg 3780 ggaagaac ggtgccgcgg caacactaca acggagctga agtctgcact gatccgtcat 3840 tcgatcggac aaagcagcat tagtccgttg atcggtggaa gaccactcgt cagtgttgag 3900 ttgaatgttt gatcaataa atacggcaat gctgtaaggg ttgtttttta tgccattgat 3960 aatacactgt actgttcagt tgttgaactc tatttcttag ccatgccaag tgcttttctt 4020 attttgaata acattacagc aaaaagttga aagacaaaaa aaaaaacccc cgaacagagt 4080 gctttggcga tcgcgcccgg gcttaattaa gacaaatttg tttgtcagat caaattttta 4140 agcaaaacat aagaaacagc acaaaagggt atagcagaag caaaatttgg acacaatgat 4200 tagggatcac ttatcatta tagttcttcc atgctgctct accacagcaa aacgtttcag 4260 4320. accagactta accagactta ttacaggtaa atagtacaca atgaaacggg acacgacca ggaccagacc agacaccat agctccattg gggagaca ataggcagac ttgtgaaccc tagaacagca gcagctctat ggctgaggac ggtacctcta gaggcgcgcc 4440 cactttgtga ggcctttaat acacagtcct gccatcacca tccaggatca tgtccttgaa tgcgccccca attgggatca taggcagcac atgctcctgg tgcgggacga tgatatccaa caagtatggc cctggagtct cgagcatctt cttgatggcg gcacggactt cactcttctt 4620 tgttacacgg actgccgga tattgaaccc cttagcaata gtcacaaaat ctggatatat ctcgctctca cattccgggt tgcccaagta tgtatgcgcc ctattcgcct tgtaaaacct 4740. atcctccaat tgcaccacca tacccaaatg ttggttgttc aacaccatca ccttcacagg gaggttctca atgcggatca atgccagctc ctgaatgttc atgaggaagc taccatcccc atcaatatca acaactgtga cacctgggtt agccacaga gcaccagctg cggcaggcag cccaaatccc attgcgccca cccagccga agacagccac tgccgtggcc gcttgtaggt 4980. gtaatattgt gccgcccaca tctggtgctg cccaacacca gtagcgatga ttgcctcacc 5040 tttcgtcagc tcatccagca cctgaatggc atattgcggt gggatctctt caccaaaagt 5100 tttgtacccc agaggaaact ccctcttctg ctggtccaac tcattgtgcc atgcactaaa 5160 atcagaactt gtctttgttg tgctctgttg tagcagagca ttcaagccct gtaaagcgag 5220 cttaacatct gcgcaaattg acacatgtgg ttgcttgttc tttccaatct ctgctggatc 5280 aatgtcaatg tgcacaatct tggccctgct tgcaaaagcc tcaattttcc ctgtcacacg 5340 atcatcaaac cgcacaccaa acgcaagcaa caggtcagcc ttatccacgg cataatttgc 5400 gtacaccgtg ccatgcatcc caagcatccg cagggacaac gggtcgtcac tggggaaatt 5460 gccgaggccc atcagagtgg ttgtaactgg gataccagtc agctcaacaa accagcgcaa 5520 ttcgtcacca gatgcagagc agccaccacc gacatagaga atcgggcgcc gtgactcgcc 5580 aaccagacgc aagacctgct caagcaattc tgtcgcgggt ggcttgggca ggcgtgcgat 5640 gtaccctggt agattcatcg aggtgtccca gaccggcacg gccatctgct gctggatgtc 5700 cttggggatg tccaccagca ccggggccagg acggcccgag gacgcgagga agaaggcttc 5760 ctgtatgacg cgggggatgt cctccacatc aaggacaagg taattgtgct tggtgatgga 5820 gcgggtgacc tcgactatgg gcgtctcctg gaaggcgtcg gtgccgatca tgcggcgggg 5880 gacctggccc gtgatggcga ccatcgggac ggagtcgagc agcgcgtcgg cgagcgcgga 5940 cacgaggttg gttgcgccgg ggccggaggt ggcgacgcag accccgacgc ggccggacgc 6000 gcgcgcgtac ccggacgccg cgaacgcctc gccctgctcg tggcggaaga ggtggttggt 6060 gatgaccggg gagcgcgtca gcgcctggtg gatctccatc gacgcgccgc ctgggtaggc 6120 gaacacgtcg ctgacgccgc accgctccag cgcctccacg aggatgtccg cgcccttgcg gggctcggcc ggcccccacg gccggagcgg cgtggccggc ggcgccgggg acggcggggt 6240 gaccggggac accgccgagc acctgaccgc cgccggcccc acccggcctc gagcgggag gacgtggtgt cgctggtggt tcttacggcc ggtcttggcc gtcgcggcgg cggacagggc 6360 ggcggccgcg gccgcggcgg tcgtagccat ggtgggtggt ggcggcggcg gcggcggcga 6420. gggtttctgg gtttgggcga gagggagaga ggggtggaga gtgtgggggt gtgggtgttt 6480 tgtccgcgcg cggtgacgca gcgtgaggcg atgtgtggct cggatgggat cgtgtggggc 6540 cacgtcacag tgggccccat atgtcggtgg gataggatga cacacagcca cgtcggggtc 6600 gatctgggcc cgcgtgtcag tcgattggat gaggatgaca ggtgggtcag tcccaataac 6660 gggtggaggg tacagaaccc gataaaaaat cgatctcaaa gtcggatgcg gatgcaaccg 6720 cgagacctag cagtctagca cccgcacccg cacccgcaca aacgcagcgc aagatgagca 6780 gtggcggcga gaagtccggc tcgggcggcg gcggcggtgg cggcggcggc ggggcggtga 6840 agacgccctc cgacttcctc aagtccatca gggggcgccc cgtcgtcgtc aagctcaact 6900 ccggcgtcga ctaccgcggt aaccccctgc tcaattccct cccccttttt gcctcaaccc 6960 tagacccgcc tcccgaaaac cctagcacga gacgaatgcg atgtttgttg taccccgtat 7020 ctctttctcc ttaattggaa ctctgcgttt cttttcgtta attagtgggg ggaaggaaag 7080 gcgtggactc tgtaggattt tcttttcgat ttgatgacca agggagatac ttattttagt 7140 gctgtttaag atcctgctag tgcccggtgt agctaggcca tttgagaagc agataaccgt 7200 gttttctgt aggaccttct ctaatcttga gctattttga tattctaatt acttatggtt 7260 aaaccctaga acctcagtat gttttaactt agtaagagat tgctacagta tttttatttg 7320 taggaacaac attgtaggtg gttatcatgc tactgactgt cttgtgtaga gccctcctat 7380 cattaagggg cttttatgtt tactaatagt tagttttagt acccattttt ctgtgagcta 7440 ctaaaaagaa tatttccaat tcaacctatg gtaatttaaa gtactttatg ctgtatgtcc 7500 attactgag gattatgatt aatatttaag agggaggaca tctgtgctta aggtttgcat 7560 tgcaatattt gatagattta tgctaaacaa aggtaattgt gcatgcttga gatagaaagg 7620 gatttccctt ctttgggttt gataataagg ggttggcatt ccatgatcag gagcatagtg 7680 catttatatt ttataatgtg gctatggta tgcagttctc aatttcacat tggcggtaca 7740 cccatgatat attaattgt tacccagaac catataatag cctaataaga gcatatggta 7800 ttccattgat tggggccttc ggaagtgtac ctgtgtatgt aactatgcat ataaggttta 7860 ctttctgta gttctttctt ctcctcatca cttagacat ctggatcgct gttatcaagt 7920 cgtagactag tcataacgtc agtcaagtca gacgactaga gggctacgac tcgaccaaat 7980 agcaagtcgc acaattagtc gtagattagt catagcaat catcatttca ccatgctat 8040 tggcttattg tgtttagtac ttaattta ttcttgtta acttattgtc ttgctggcta 8100 ctccataaac cgtagtttat atactatat atagtgctta gtacaataga atagtactag 8160 agactatata gtgcatccat tgtttcaca cgaccaaatc atggactagt cgtggactag 8220 attttgaatg gaacagtttt accagaaata cacttgttct ttttttct gatagtgtt 8280 gatcagtag ctgcaatgca tagcttgtgc aaaaatca ttatatacta tcaccaattt 8340 catggaatct tacttattgg aaatttgtaa caatctatt ttatcccctg agttaccgtg 8400 ttggtctaaa atcactcgc atgtgagaca agatgaagg gaccatgaag attgccctctt 8460 tcatttattc atttgctgac tgcttgcaat gccactaa acctaatcgg atgactggtt 8520 gtcatgtctc cgctaactag aggaaaggg ggaggagtga agacttacg tgtacacagt 8580 ctttcaccag gacggttgta tcggttcagc agtcgcgatc cgggcagcgc tgaagaactt 8640 ccccgatcta gtaacataga tgacaccgcg cgcgataatt tatcctagtt tgcgcgctat 8700 attttgtttt ctatcgcgta ttaaatgtat aattgcggga ctctaatcat aaaaacccat 8760 ctcataaata acgtcatgca ttacatgtta attattacat gcttaacgta attcaacaga 8820 aattatatga taatcatcgc aagaccggca acaggattca atcttaagaa accacctgca 8880 agtgacttcc tcagcaggcc tactagtttt attgccaaat gtttgaacga tcggggaaat 8940 tccagtgcac cagagtcaca gaaacacatc acacattcgt gagctcagct tagccatgga 9000 taacgcctac attattgcca ttctctctgt agctatcctc ttcttgctcc actactacct 9060 cctcggccgc ggcaatggcg gggcggcgcg gctgccgccg ggtccaccgg ccgtcccgat 9120 cctgggacac ctccacctcg tcaagaagcc gatgcacgcc accatgtccc gcctcgccga 9180 gcggtacggg ccggtgttct cgctgcgcct cgggtcgcgg cgcgccgtgg tggtgtcgtc 9240 gccggggtgc gccagggagt gcttcaccga gcacgacgtg accttcgcga accggcccag 9300 gttcgagtcg cagctgctgg tctcgttcaa cggcgccgcg ctcgccacgg cgagctacgg 9360 cgcgcactgg cgcaacctcc gccggatcgt cgccgtgcag ctgctctccg cgcaccgcgt 9420 cggcctcatg tctgaaaatc tcgaaacagc cgtgtcatag tcaatcatta ggtgttatag 9480 gaacaatcaa aggttttttc aagtgttaat cttcatacta atatatacag tgggtactct 9540 ttatctactg ccgtggaact gtcatatttg attatgaaat tttagctcta gaaaatattt 9600 gatcatcaat gtcaagactt tatgaccttg caaaatacat ttcctaattg agaacagggt 9660 aaaattatga actatgcctc tgaaccttca tacacaggca gcacattttt tgttgtaaaa 9720 ttcatcttaa tatcagcgga aagactggac cagagaaaga aaaagttaag acaggcatat 9780 actcttgatc ctctaaaaga gatgaggcgg tacaatgatc aaccatgaac attaaagtga 9840 tacgtggaac atgagaacac aaataattgt cactggaaca taaatacaaa aaggtttgta 9900 gtaacttacc gacatgaggc cgacgcggtg cgcggagagc agctgcacgg cgacgatccg 9960 gcggaggttg cgccagtgcg cgccgtagct cgccgtggcg agcgcggcgc cgttgaacga 10020 gaccagcagc tgcgactcga acctggggccg gttcgcgaag gtcacgtcgt gctcggtgaa 10080 gcactccctg gcgcaccccg gcgacgacc caccacggcg cgccgcgacc cgaggcgcag 10140 cgagaacacc gggcccgtacc gctcggcgag gcgggacatg gtggcgtgca tcggctctt 10200 gacgaggtgg aggtgtccca ggatcgggac ggccggtgga cccggcggca gccgcgccgc 10260 cccgccattg ccgcggccga ggaggtagta gtggagcaag aagaggatag ctacagagag 10320 aatggcaata atgtaggcgt tatccatggc taagctgagc tcacgaatgt gtgatgtgtt 10380 tctgtgactc tgtgcactg ggatcccctg agggtcacc gacttcaggt caagtacac 10440 caacacag ggtgagcatc ghaaaagaa acagtaccaa gcaataat agcgtgaa 10500 ggcagggcta aaaaaatcca catatagctg ctgcatatgc catcatccaa gtatatcaag 10560 atcaaaataa tttaaaaca tacttgttta ttataga taggtactca aggttagagc 10620 attgaatag atgctgcata tgccatcatg tatatgcatc agtaaaaccc acatcacat 10680 gtatacctat cctagatcga tattccatc catcttaaac tcgtaactat gagatgtat 10740 gadocacaca tacagttcca aaattaataa atacaccagg tagtttgaa cagtattcta 10800 ctccgatcta gaacgaatga acgaccgcccc aaccacacca catcatcaca accaagcgaa 10860 caaaagcat ctctgtatat gcatcagtaa aacccgcatc aacatgtata cctatcctag 10920 atcgatattt ccatccatca tctcaattc gtaactatga atatgtatgg cacacacata 10980 cagatccaaa attaataat ccaccaggta gtttgaaca gattctact ccgatctaga 11040 acgaccgccc aaccagacca catcatcaca accaaaaaagcat gaaaagatga 11100 cccgacaaac aagtgcacgg catatattga aaaaggaa aagggcaaac caaaccctat 11160 gcaacgaaac aaaaaaaatc atgaatcga tcccgtctgc ggaacggcta gagccatccc 11220 aggattcccc aaagagaaac actggcaagt tagcaatcag aacgtgctg acgtacaggt 11280 cgcatccgtg tacgaacgct agcagcacgg atctacaca aacacggatc taacacaac 11340 atgaacagaa gtagaactac cggggccctaa ccatggaccg gaacgccgat ctagagaagg 11400 taggagggg ggggggggg ggacgagcgg cgtaccttga agcggaggtg ccgacgggtg 11460 gattggggg agatctggtt gtgtgtgtgt gcgctccgaa caacacgagg ttggggaaag agggtgtgga gggggtgtct atttattacg gcgggcgagg aagggaaagc gaaggagcgg 11580 tgggaagga atcccccgta gctgccggtg ccgtgagagg aggaggaggc cgcctgccgt 11640 gccggctcac gtctgccgct ccgccacgca atttctggat gccgacagcg gagcaagtcc 11700 aacggtggag cggaactctc gagaggggtc cagaggcagc gacagagatg ccgtgccgtc tgcttcgctt ggcccgacgc gacgctgctg gttcgctggt tggtgtccgt tagactcgtc 11820 gacggcgttt aacaggctgg cattatctac tcgaaacaag aaaaatgttt ccttagtttt tttaattct taaagggtat ttgtttaatt tttagtcact ttattttatt ctatttata tctaattat taaataaaaa aactaaata gagttttagt tttcttaatt taggctaa aatagaataa aatagaata ctaaaaaaat tagtctaa aaaccataa ccctaaaccc 12120. ttgcaaaaaa 12120. ttgcaaaaaa aaaggagaac acatgcacac tAAaagata aactgtaga gtcctgttgt caaatactc 12180 aattgtcctt tagaccatgt ctaactgttc atttatatga ttctctaaaa cactgatatt 12240 attgtagtac tatagatta attattcgta gagtaagtt taaatatatg tataagata 12300 gataaactgc acttcaaca agtgtgacaa aaaaaatatg tgtatatttt ttataactta 12360 gatagcaat gctcattatc tctagagggg ggcacgaccg ggtcacgctg cacgttaacg 12420 acacctgtct ccggatcgca tgcgcattct agagatccc tcagtctc agtccactag 12480 ccttgtccac gtcgccaatg tttaaatcca gtcccat ttgtagcgta gatggaaca 12540 gctttcgata aaatttctca accatttaga tgaaattcgc accgttttcaa gcagggtcta 12600 ggaccaacaa atcctagaat caacatggt acatcaat ttcaattca aaccagatta 12660 cataggctta ttactactc cggtaagcga tacggcggc ggcggcggcgg ctgccggcga 12720 gcggcggcgg gggtcagacg gaggtggaga cgcggacctt gaggccgtga gccatggaga 12780 ggatggtcat catccggtac ttgacggggt ggtcctcgac gaggtcgaag gtgtagaagc 12840 ggaagaggat ggcgagcgcc atcttcatct ggaggtaggc ggagtccttg ccgaggcaga 12900 tccgcggccc ggcctggaac gcggtgaact tgaacggcga ggcgttgcgg aacgcgccgc 12960 cgtcgccgct gagccaccgc tccggccgga agctcgccgc gtcggggccc cagttgtact 13020 ccatcctccc catcgagtag ggcacgtacg tcaccatccc gccggcgcgc accttggtgc 13080 cgtcggggag cacgtcgtcc tccacgatcc ccttggggtc ctgcggcacc gccgggtaga 13140 ggcggagcgt ctccgtcacg caggcgtgca ggtacaccag cttccccacc gcgtcgtagc 13200 tcagcagcga cgcgaactgc gccacgcgcg ccgcgaatga cgcctcgccg gcggcgtcgg 13260 cgagcgcgac gccctcctcg cgcgcgcgct cagcctcgaa cgcggccagc tcgcgccgga 13320 gcttgtcggc gacggctggg tgcgtcatcg ccatgtacgt gaaccacgac agcgtcgtcg 13380 ccgtcgtgtc acgcccggcg atcacgaagt tgagcaccac gtcgcggagg ctcttgtcgt 13440 ccccgaagct gccgcccccc tcgtcgccgc cggcctcccc cagctcgatg aaccgcgaca 13500 gtatgtcgtg cttgatctgc tcgccattac aaatcaacca tatcaagaaa caaaaccttt 13560 tccgatctga tcatcgccat taccatgtca gttcagttct actgattctt tgagcaagag 13620 aggaaggatc accttctctt gcttgccgct ggctcgagcc tgcaagatct cagccttgcg 13680 gcggcggatc acgctgtagg tgaagtcatc aaccagcttc atgctctgct cgaggagagc 13740 ctctgatccg acgtgcaaga acttcttgag acgccacaga ggatcgatga accgcagcgt 13800 gacgatgatg ttggcagcgt cgaaggcctg ggcaaagctg ttctccggga gatcaggtga 13860 cagcgtcccg atctcaaccc caaacccgac cttgcagatc gagtccagtg tcatcctcat 13920 gaacaattcc tgaattttgg ttagttcttg catcacaatt ctgaacaatt tggtttctca 13980 agaaatgttt agatattagg caaggaaatc agttggttac ctgcatgtct acaactctgc 14040 cggccttgca cgcttggctc agaatgcttg atagcttcag ggagtactcc ctgaacacca 14100 cagtgctgaa gtctctcaag ttcttggagg caaactcgaa gctcgccgtc ttcctttgct 14160 tcctccacat ctcgccgtcg gcattgaata tgccatcacc gagcagcaca tccatgtaag 14220 acctgtagac ttcaccctgc atattttcag acatttttg tgtcagtgtt agtactgtgc 14280 aaggcacatt ttacagtaca ctgaagatcc tatggttctt ttaccttggg gtaattggtg 14340 aagttggtct tcaggacatg ctcgacgttc accgggtcgg caatgtaggt gtaggaggtg 14400 aaaggcatgt cgacggtcac cgtcctgtcc ttcgacaagt actcgacaag ccagtcatgc 14460 atcctgtggt agttcttcag ttgctccact gtcgcgccga tgattggcca tgatcttggc 14520 cctttctggt tcctcaggct ccacttgtgg accaagatcc atgagaggac aacaaggaag 14580 atagctatga gcttgtggat tcctgctact gggaagaatg atgtcactgg cattgcatga 14640 gcttcctcca tggggctctt catgaagggt tcagtctaca agagatgcta tgtagcattt 14700 tggatgccat agttaggtga tatattggga actgatgcta ccaaccttga gacaatcttt 14760 gtgtgaccaa gaattaagat tgtctcattc atatggtgtc cctaacctaa cacccttagg 14820 agttggatct tagctttagt tttctttttg tgatctgtcc acctagaaaa gaatcgttac 14880 tcatgtaaac acaaaatgaa tgctacagta tttttttccc ctgctcagcc aagttcgttg 14940 ttttgttcag ataactgaag cgtttgcagc gactaaaggt atggcacctt ctaatatcag 15000 aaccatatcc atctgaattc tgttaaattg tcaccaactg aaaccaggct taccattttc 15060 tgtcgatatt gtctagtctt tgatgacaac taaaatgaaa tggggtatcc tcaaaagaaa 15120 tataaattgt gaacccaaag tagttggttt ctacttcacc tgaattctac ttcatctgaa 15180 taggctattt taatgaagag aaaatccaag aaatgccatt tacaaacacc caaatccaag 15240 aaatgctatc gacgagtacg agttccataa aatgctattg tagaagcgat tttgtcccag 15300 aaatcagttg ccatctagtt aaaacattct cacataaaca agcaatataa aatactattg 15360 ccaagatata tccaatactt tgcatctcta aaacttctag ttattgacaa ctaatttctt 15420 ttccagtcaa tacaaataag ccatctatca aagatttgag aaattgcagg gcacaattct 15480 acagaaaatt aaagaacatc acttgaaaag gaagattttt tctatagaac ttatcctttg 15540 15600 tctgaaacca agatcagaag agcatgaaaa tagtgctctt atggattgac cttgattctt 15660 gcccacactc ttattacaac cttcgagcaa accggagcat ttgctgaata aatctctcat 15720 ctgttactga agatactagg taactaaata aacaatcaat caagagtac aaaacatttt 15780 gcaggatcta gtaacataga tgacaccgcg cgcgataatt tatcctagtt tgcgcgctat 15840 attttgtttt ctatcgcgta ttaaatgtat aattgcggga ctctaatcat aaaaaaccat 15900 ctcataaata acgtcatgca ttacatgtta attattacat gcttaacgta attcaacaga 15960 aattatatga taatcatcgc aagaccggca acaggattca atcttaagaa actttattgc 16020 caaatgtttg aacgatctca gctgtgcccc agtttgctag gcaggtcgca gtacctggcc 16080 acagccatct cgtgctgctc cacgtaggtc tctttgtcgg cctccttgat tctttccagt 16140 ctcctgtcca cgaaggaa gccgggcatc ttgaggttct tagcgggttt cttggatctg 16200 tatgtggtct tgagggagca gtgcaggtag cccccgccca cgagcttcag ggccatctgg 16260 ctatggcctc tcaggccgct gtcagcgggg tacagcatct cggtgctggc ctcccagccg 16320 agtgttttct tctgcatcac agggccgttg gatggggagt tgaccccgtt gatcttgacg ttgtagatga ggcagccgtt ctggaggctg gtgtcctggg tagcggtcag cacgcccccg 16440 tcttcgtatg tggtgatcct ctcccatgtg aagccctcag ggaaggactg cttaaagaag tcggggatgc cctgggtgtg gttgataaag gttttgctgc cgtacatgaa gctggtagcc 16560 aggatgtcga aggcgaaggg gagagggccg ccctcgacca ccttgatctt catggtctgg gtgccctcgt agggcttgcc ttcgccctcg gatgtgcact tgaagtggtg gtcgttcacg 16680 gtgccctcca tgtaggttt catgtgcatg ttctcggtga tcagcacgct atcctcaccc 16740 actgcggaag cgttgcatgc aacaatagca aggagagca atacgaaaat gatcttcatt tttgtaggat tctactacta tgcttcaact aattgtttg aattgtgtga aggatgagga 16860 gggttttcat tatgcttgtc tatttataga tgtttcccta tcggttgctt taggtgtta gttttgcttg atgctatatt aggatctaag tgagtcatat tatttggata ttctgttagg 16980 tgcacctaaa aatgttcttt gatatcatca tcaaagtaac tttatgccc atcatatata 17040 catgatggtg agcaaaaaaa taatgacatg actcatcgct cactttatgt gtcaatgtga 17100 taaggattgc aacttttgaa actaagtttg ttatgtttg taagctatta ttttgataat 17160 gcactacacg tggaacttta cgactgtgtg ggtttatgct ctttgaatta tataatttca 17220 gtaagaaaaa aaattatagt atgttcaatg gttttcattg ttccattaat actcttgtga 17280 aaacttgttt gtgatacgaa ataagttaat gcaataactt ttaaattaac tttgcagagc 17340 tttacaataa ctcaccaact tactctaatt gtggttgggg attgatttaa ttcatacttt 17400 tatagaccaa attgatattg tgttgctaaa taaaaattat agtggtgttg ctacacctac 17460 agacacag aaggaatcct gatatcatca gctatctact gcttcctcca catggactca 17520 agaagcagaa gcaaaaaaga agaaggcatt tgctccaaca aagttttccg gtgaaaggca 17580 acacattgtt ctgaacttcc tggcatgtat tatgtgaatc tagcaaatta accaaacatt 17640 gacacaatgc gacaccttgt aatgtagccg tgccgagaat tgtatatctc acttaccatt 17700 gtatatcaca cgtgccgaga ataggagatt gatttttgcc aaccaaaacg ctctaggcag 17760 accaattggg cgcccggccg gattaataaa ttcgaaacg cgtcggaccg gcaccggaca 17820 ctgtctggtg gcataccaga cagtccggtg tgccagatca gggcaccctt cggttccttt 17880 gctccttgc ttttgaaccc taactttgat cgtttattgg tttgtgttga acctttatgc 17940 acctgtggaa tatataatct agaacaaact agttagtcca atcatttgtg ttgggcattc 18000 aaccaccaaa attatttata ggaaaaggtt aaaccttatt tccctttcaa tctccccctt 18060 tttggtgatt gatgccaaca caaaccaaag aaaatatata agtgcagaat tgaactagtt 18120 tgcataaggt aagtgcatag gttacttaga attaaatcaa tttatacttt tacttgatat 18180 gcatggttgc tttcttttat tttaacattt tggaccacat ttgcaccact tgttttgttt 18240 tttgcaaatc tttttggaaa ttctttttca aagtcttttg caaatagtca aaggtatatg 18300 aataagattg taagaagcat tttcaagatt tgaaatttct ccccctgttt caaatgcttt 18360 tcctttgact aaacaaaact ccccctgaat aaaattctcc tcttagcttt caagagggtt 18420 ttaatagat atcaattgga aatatatta gatgctaatt ttgaaaatat accaattgaa 18480 aatcaacata ccaatttgaa attaaacata ccaatttaaa aaatttcaaa aagtggtggt 18540 gcggtccttt tgctttgggc ttaatatttc tccccctttg gcattaatcg ccaaaaaacgg 18600 agactttgtg agccatttat actttctccc cattggtaaa tgaaatatga gtgaaagatt 18660 18720 gagtggaagc cttgtcttcg ccgaagactc catttccctt tcaatctacg acttagcata 18780 gaatacact tgaaaacaca ttagtcgtag ccacgaaaga gatatgatca aaggtataca 18840 aatgagctat gtgtgtaatg tttcaatcaa agtttcgaga atcaagaata tttagctcat 18900 tcctaagttt gctaaaggtt ttatcatcta atggtttggt aaagatatcg actaattgtt 18960 ctttggtgct aacataagca atctcgatat cacccctttg ttggtgatcc ctcaaaaagt 19020 gataccgaat gtctatgtgc ttagtgcggc tgtgttcaac gggattatcc gccatgcaga tagcactctc attgtcacat aggagagga ctttgctcaa tttgtagcca tagtccctaa ggttttgcct catccaaagt aattgcacac aacaatgtcc tgcggcaata tacttggctt cggcggtaga aagagctatt gagttttgtt tctttgaagt ccaagacacc agggatctcc 19260. ctagaaactg acaagtccct gatgtgctct tcctatcaat tttacaccct gcccaatcgg catctgaata tcctattaaa tcaaaggtgg atcccttggg gtaccaaaga ccaaatttag gagtgtaaac taaatatctc atgattcttt tcacggccct aaggtgaact tccttaggat cggcttgga tcttgcacac atgcatatag aaagcatact atctggtcga gatgcacata aatagagtaa agatcctatc atcgaccggt ataccttttg gtctacggat ttacctcccg tgtcgaggtc gagatgccca ttagttccca tgggtgtcct gatgggcttg gcatccttca ttccaaactt gttgagtatg tcttgaatgt actttgtttg gctgatgaag gtgccatctt ggagtgctt gacttgaaat cctagaaaat atttcaactt ccccatcata gacatctcga 19740 atttcggaat catgatccta ctaaactctt cacaagtaga tttgttagta gacccaaata 19800 tatatcatc aacataaatt tggcatacaa acaaaacttt tgaaatggtt ttagtaaaga 19860 gagtaggatc ggctttactg actctgaagc cattagtgat aagaaaatct cttaggcatt 19920 cataccatgc tgttggggct tgcttgagcc cataaagcgc ctttgagagt ttataaacat 19980 ggttagggta ctcactatct tcaaagccga gaggttgctc aacatagacc tattcacccc 20040 atttgatcac ttttttggtc cttcaggatc tatagttat gtataattta gagtctcttg 20100 tttaatggcc agatatttct aattaatcta agaatttatg atatttttta attttttatc 20160 atgtctgatg agaattaaca taaaggctca attgggtcct gaattaataa tagagtgaaa 20220 attaatccag aggctctatt agaaccttca attagtaata ccaagatata tataagatag 20280 tagagtatag tttaaatgtt ggcattgttc attctttctt ttgttattta attatgctt 20340 tccacggtgg ttagtggtta cttctgaagg gtccaaataa tgcatgaaga gtttgaggac 20400 aagaagtctg ccctaaaaat agcgatgcaa aggcatggtg tccaagccat acatatagcg 20460 cactaatttt atcagcagaa caatggtatt tataggtcct agtgcccagg caacaagaga 20520 cacgaataaa gcatcgatca cgacaagatg ctgtgtctca cctcctcttc ctcctccgcg 20580 cccgctccgc tccttccctc tctcgctgat cgaccgagcc caggaatcgc gggcggggt 20640 ggcaatgttc gcctgagcgt ggtttcttcg ccgcggcggt cgtggcctgg aaaggtcaag 20700 accaatttct cagttcctgc gactgcgcga aaaaacaaaa ccatggtgac tgttgtggag 20760 gagatcgctt gttctggtac tggatctgga tttgaaatat tgtgtcaagg atttaactgg 20820 gaatctcata agtcaggaaa atggtatgtg gaacttggct caaaggccaa ggagttgtca 20880 tccatgggtt tcaccattgt ctggtcacca ccacctactg attctgtgtc gcctgaagga 20940 tacatgccaa gggatttgta taatctaaat tccagatatg ggaccatgga agagttgaag 21000 gaggctgtga aacgttttca tgaagccggt atgaaggttc ttggtgatgc cgtcctgaat 21060 cacaggtgtg ctcaatttca gaaccaaaat ggcgtctgga atatttttgg tggacgcctt 21120 aactgggatg atcgagcagt tgttgcagat gatccacatt tccagggaag aggaaacaag 21180 agcagtggag ataacttcca tgcagcccca aacattgatc actcgcaaga gtttgtgagg 21240 agtgatctta aagaatggct ttgttggatg agaaaggaag ttggatacga tggatggcga 21300 cttgattttg ttcgcggatt ttggggtgga tatgtccacg attacttgga agcaagcgaa 21360 ccatattttg footggaga gtactgggat tctctcagtt acacctatgg tgaaatggat 21420 tataatcaag atgcccacag gcagagaata gttgattgga taaatgctac aaatggaact 21480 gctggtgcat ttgatgttac cacgaaagga atacttcact ctgcactgga aagatctgag 21540 tactggcgtc tgtctgatga aaaaggaaaa ccccctggag tgttaggttg gtggccttcg 21600 cgtgctgtca catttataga aaatcatgac actggttcta ctcagggtca ttggagattc 21660 ccctttggta tggagttgca aggctatgtc tacatcttaa ctcacccagg cactcctgca 21720 atcttctatg atcatatatt ttcgcattta cagccagaga ttgctaaatt aatttctatt 21780 agaaatcgcc aaaagatcca ttgccgtagc aagatcaaga tactgaaagc agagggaaat 21840 ttatatgcgg cagagattga tgagagggta acaatgaaga ttggcgcagg acattttgag 21900 ccaagcggcc ccacaaactg ggtagttgct gccgagggac aggattacaa ggtctgggaa 21960 gtgtcatcgt agacttgccg cgctggtact tgcagaactt ctattgtaga cagtatacaa 22020 ccattcggta tagctcacaa aagggatcgt tcaaacattt ggcaataaag tttcttaaga 22080 ttgaatcctg ttgccggtct tgcgatgatt atkatataat ttctgttgaa ttacgttaag 22140 catgtaataa ttaacatgta atgcatgacg ttattatga gatgggtttt tatgattaga 22200 gtcccgcaat tatacattta atacgcgata gaaaacaaaa tatagcgcgc aaactaggat 22260 aaattatcgc gcgcggtgtc atctatgtta ctagatccct aggcacgtgt acgtattttt 22320 taccaggtga actccaagtc ctggaccctt tttttggcac tggccgtcgt tttaagctta 22380 gattgtcgtt tcccgccttc agtttaaact atcagtgttt gacaggatat attggcgggt 22440 aaacctaaga gaaagagcg tttattagaa taacggatat ttaaagggc gtgaaaaggt ttatccgttc gtccatttgt atgtgcatgc caaccacagg gttcccctcg ggatcaaagt 22560. actttgatcc aacccctccg ctgctatagt gcagtcggct tctgacgttc agtgcagccg 22620 tcttctgaaa acgacatgtc gcacaagtcc tagttacgc gacaggctgc cgccctgccc ttttcctggc gttttcttgt cgcgtgtttt agtcgcataa agtagac ttgcgactag 22740 aaccggagac attacgccat gaacaagagc gccgccgctg gcctgctggg ctatgcccgc 22800 gtcagcaccg acgaccagga cttgaccac caacgggccg aactgcacgc ggccggctgc accaagctgt tttccgagaa gatcaccggc accaggcgcg accgcccgga gctggccagg atgcttgacc acctacgccc tggcgacgtt gtgacagtga ccaggctaga ccgcctggcc 22980 cgcagcaccc gcgacctact ggacattgcc gagcgcatcc aggaggccgg cgcggggcctg 23040 cgtagcctgg cagagccgtg ggccgacacc accacgccgg ccggccgcat ggtgttgacc 23100 gtgttcgccg gcattgccga gttcgagcgt tccctaatca tcgaccgcac ccggagcggg 23160. cgcgaggccg ccaaggcccg aggcgtgaag tttggccccc gccctaccct caccccggca 23220 cagatcgcgc acgcccgcga gctgatcgac caggaaggcc gcaccgtgaa agaggcggct 23280 gcactgcttg gcgtgcatcg ctcgaccctg taccgcgcac ttgagcgcag cgaggaagtg 23340 acgcccaccg aggccaggcg gcgcggtgcc ttccgtgagg acgcattgac cgaggccgac 23400 gccctggcgg ccgccgagaa tgaacgccaa gaggaacaag catgaaaccg caccaggacg 23460 gccaggacga accgtttttc attaccgaag agatcgaggc ggagatgatc gcggccgggt 23520 acgtgttcga gccgcccgcg cacgtctcaa ccgtgcggct gcatgaaatc ctggccggtt 23580 tgtctgatgc caagctggcg gcctggccgg ccagcttggc cgctgaagaa accgagcgcc 23640 gccgtctaaa aaggtgatgt gtatttgagt aaaacagctt gcgtcatgcg gtcgctgcgt 23700 atatgatgcg atgagtaaat aaacaaatac gcaaggggaa cgcatgaagg ttatcgctgt 23760 acttaaccag aaaggcgggt caggcaagac gaccatcgca acccatctag cccgcgccct 23820 gcaactcgcc ggggccgatg ttctgttagt cgattccgat ccccagggca gtgcccgcga 23880 ttgggcggcc gtgcgggaag atcaaccgct aaccgttgtc ggcatcgacc gcccgacgat 23940 tgaccgcgac gtgaaggcca tcggccggcg cgacttcgta gtgatcgacg gagcgcccca 24000 ggcggcggac ttggctgtgt ccgcgatcaa ggcagccgac ttcgtgctga ttccggtgca 24060 gccaagccct tacgacatat gggccaccgc cgacctggtg gagctggtta agcagcgcat 24120 tgaggtcacg gatggaaggc tacaagcggc ctttgtcgtg tcgcgggcga tcaaaggcac 24180 gcgcatcggc ggtgaggttg ccgaggcgct ggccgggtac gagctgccca ttcttgagtc 24240 ccgtatcacg cagcgcgtga gctacccagg cactgccgcc gccggcacaa ccgttcttga 24300 atcagaaccc gagggcgacg ctgcccgcga ggtccaggcg ctggccgctg aaattaaatc 24360 aaaactcatt tgagttaatg aggtaaagag aaaatgagca aaagcacaaa cacgctaagt 24420 gccggccgtc cgagcgcacg cagcagcaag gctgcaacgt tggccagcct ggcagacacg 24480 ccagccatga agcgggtcaa ctttcagttg ccggcggagg atcacaccaa gctgaagatg 24540 tacgcggtac gccaaggcaa gaccattacc gagctgctat ctgaatacat cgcgcagcta 24600 ccagagtaaa tgagcaaatg aataatgag tagatgaatt ttagcggcta aaagggcgg 24660 catggaaaat caagaacaac caggcaccga cgccgtggaa tgccccatgt gtggaggaac 24720 gggcggttgg ccaggcgtaa gcggctgggt tgtctgccgg ccctgcaatg gcactggaac 24780 ccccaagccc gaggaatcgg cgtgacggtc gcaaaccatc cggcccggta caaatcggcg 24840 cggcgctggg tgatgacctg gtggagaagt tgaaggccgc gcaggccgcc cagcggcaac 24900 gcatcgaggc agaagcacgc cccggtgaat cgtggcaagc ggccgctgat cgaatccgca 24960 aagaatcccg gcaaccgccg gcagccggtg cgccgtcgat taggaagccg cccaagggcg 25020 acgagcaacc agattttttc gttccgatgc tctatgacgt gggcacccgc gatagtcgca 25080 gcatcatgga cgtggccgtt ttccgtctgt cgaagcgtga ccgacgagct ggcgaggtga 25140 tccgctacga gcttccagac gggcacgtag aggtttccgc agggccggcc ggcatggcca 25200 gtgtgtggga ttacgacctg gtactgatgg cggtttccca tctaaccgaa tccatgaacc 25260 gataccggga agggaggga gacaagcccg gccgcgtgtt ccgtccacac gttgcggacg 25320 tactcaagtt ctgccggcga gccgatggcg gaagcagaa agacgacctg gtagaaacct 25380 gcattcggtt aaacaccacg cacgttgcca tgcagcgtac gaagaggcc aagaacggcc 25440 gcctggtgac ggtatccgag ggtgaagcct tgattagccg ctacaagatc gtaaagagcg 25500 aaaccgggcg gccggagtac atcgagatcg agctagctga ttggatgtac cgcgagatca 25560 cagaaggcaa gaacccggac gtgctgacgg ttcaccccga ttactttttg atcgatcccg 25620 gcatcggccg ttttctctac cgcctggcac gccgcgccgc aggcaaggca gaagccagat 25680 ggttgttcaa gacgatctac gaacgcagtg gcagcgccgg agagttcaag aagttctgtt 25740 tcaccgtgcg caagctgatc gggtcaaatg acctgccgga gtacgatttg aaggaggga 25800 cggggcaggc tggcccgatc ctagtcatgc gctaccgcaa cctgatcgag ggcgaagcat 25860 ccgccggttc ctaatgtacg gagcagatgc tagggcaaat tgccctagca ggggaaaaag 25920 gtcgaaaagg tctctttcct gtggatagca cgtacattgg gaacccaaag ccgtacattg 25980 ggaaccggaa cccgtacatt gggaacccaa agccgtacat tgggaaccgg tcacacatgt 26040 aagtgactga tataaaagag aaaaaaggcg atttttccgc ctaaaactct ttaaaactta 26100 ttaaaactct taaaacccgc ctggcctgtg cataactgtc tggccagcgc acagccgaag 26160 agctgcaaaa agcgcctacc cttcggtcgc tgcgctccct acgccccgcc gcttcgcgtc 26220 ggcctatcgc ggccgctggc cgctcaaaaa tggctggcct acggccaggc aatctaccag 26280 ggcgcggaca agccgcgccg tcgccactcg accgccggcg cccacatcaa ggcaccctgc 26340 ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc 26400 acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt 26460 gttggcgggt gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtatact 26520 ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatg cggtgtgaaa 26580 taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct tcctcgctca 26640 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg 26700 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc 26760 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc 26820 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac 26880 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc 26940 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata 27000 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc 27060 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca 27120 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag 27180 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta 27240 gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg 27300 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc 27360 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt 27420 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgcat tctaggtact 27480 aaaacaattc atccagtaaa atataatatt ttattttctc ccaatcaggc ttgatcccca 27540 gtaagtcaaa aaatagctcg acatactgtt cttccccgat atcctccctg atcgaccgga 27600 cgcagaaggc aatgtcatac cacttgtccg ccctgccgct tctcccaaga tcaataaagc 27660 cacttacttt gccatctttc acaaagatgt tgctgtctcc caggtcgccg tgggaaaaga 27720 caagttcctc ttcgggcttt tccgtcttta aaaaatcata cagctcgcgc ggatctttaa 27780 atggagtgtc ttcttcccag ttttcgcaat ccacatcggc cagatcgtta ttcagtaagt 27840 aatccaattc ggctaagcgg ctgtctaagc tattcgtata gggacaatcc gatatgtcga 27900 tggagtgaaa gagcctgatg cactccgcat acagctcgat aatcttttca gggctttgtt 27960 catcttcata ctcttccgag caaaggacgc catcggcctc actcatgagc agattgctcc 28020 agccatcatg ccgttcaaag tgcaggacct ttggaacagg cagctttcct tccagccata 28080 gcatcatgtc cttttcccgt tccacatcat aggtggtccc tttataccgg ctgtccgtca 28140 tttttaaata taggttttca ttttctccca ccagcttata taccttagca ggagacattc 28200 cttccgtatc ttttacgcag cggtattttt cgatcagttt tttcaattcc ggtgatattc 28260 tcattttagc catttattat ttccttcctc ttttctacag tatttaaaga taccccaaga 28320 agctaattat aacaagacga actccaattc actgttcctt gcattctaaa accttaaata 28380 ccagaaaaca gctttttcaa agttgttttc aaagttggcg tataacatag tatcgacgga 28440 gccgattttg aaaccgcggt gatcacaggc agcaacgctc tgtcatcgtt acaatcaaca 28500 tgctaccctc cgcgagatca tccgtgtttc aaacccggca gcttagttgc cgttcttccg 28560 aatagcatcg gtaacatgag caaagtctgc cgccttacaa cggctctccc gctgacgccg 28620 tcccggactg atgggctgcc tgtatcgagt ggtgattttg tgccgagctg ccggtcgggg 28680 agctgttggc tggctggtgg caggatatat tgtggtgtaa aca 28723

Claims

1. A primer set for detecting transformation events of vector GATV3 in transgenic rice, characterized in that: It includes a right border-specific primer and a degenerate primer; The right border specific primers include GATV3-RB-F1, GATV3-RB-F2 and GATV3-RB-F3, and their sequences are shown in SEQ ID NO.4-6; The degenerate primers include LAD1, LAD3 and AC1, and the nucleotide sequences of LAD1, LAD3 and AC1 are shown in SEQ ID NOs. 7-9 respectively; The nucleotide sequence of the vector GATV3 is shown in SEQ ID NO.

16.

2. A kit for detecting transformation events of vector GATV3 in transgenic rice, characterized in that: It comprises the primer set described in claim 1; the nucleotide sequence of the vector GATV3 is shown as SEQ ID NO.

16.

3. The kit according to claim 2, characterized in that It also includes dNTPs, PCR buffer and a thermostable DNA polymerase.

4. Use of the primer set according to claim 1 or the kit according to claim 2 or 3 in detecting transformation events of vector GATV3 in transgenic rice, characterized in that: The application is to identify the right wing sequence specific to the transformation event of the vector GATV3; the nucleotide sequence of the vector GATV3 is shown in SEQ ID NO.

16.

5. A method for detecting the right flank sequence of the vector GATV3 transformation event in transgenic rice, characterized in that: It includes: Using the primer set of claim 1 or the kit of claim 2 or 3, and taking the DNA of the sample to be tested as a template, hiTail-PCR amplification is performed; The nucleotide sequence of the vector GATV3 is shown in SEQ ID NO.

16.

6. The method according to claim 5, characterized in that It includes: The right border of GATV3 was amplified by hiTail-PCR, including: a) using the DNA of the sample to be tested as a template and the LAD1, LAD3 and GATV3-RB-F1 as primers to perform first-stage PCR amplification; b) using the first-stage PCR amplification product diluted 40 times as a template and using the AC1 and the GATV3-RB-F2 as primers to perform the second-stage PCR amplification; c) Using the second-stage PCR amplification product diluted 20 times as a template and using the AC1 and the GATV3-RB-F3 as primers, a third-stage PCR amplification was performed.

7. The method according to claim 6, characterized in that The reaction procedure of the first-stage PCR amplification is as follows: pre-denaturation at 93°C for 2 min, 95°C for 1 min; (1) specific reaction at 94°C for 30 s, 60°C for 1 min, 72°C for 3 min, 10 cycles; (2) degenerate primer hybridization reaction at 94°C for 30 s, 25°C for 2 min, 0.5°C / s to 72°C, 72°C for 3 min; (3) staggered thermal asymmetric PCR reaction at 94°C for 20 s, 58°C for 1 min, 72°C for 3 min, 25 cycles; (4) 72°C for 5 min, 25°C for 2 min; The reaction program of the second-stage PCR amplification is: 94°C 20s, 65°C 1min, 72°C 3min, 1 cycle; 94°C 20s, 68°C 1min, 72°C 3min, 94°C 20s, 68°C 1min, 72°C 3min, 94°C 20s, 50°C 1min, 72°C 3min, 13 cycles; 72°C 5min, 25°C 2min; The reaction program of the third-stage PCR amplification is: 94°C 20s, 68°C 1min, 72°C 3min, 94°C 20s, 68°C 1min, 72°C 3min, 94°C 20s, 50°C 1min, 72°C 3min, 7 cycles; 72°C 5min, 25°C 2min.

8. The method according to any one of claims 5 to 7, characterized in that: The method further comprises: The final PCR amplification product was sequenced to determine the right flank sequence specific to the vector GATV3 transformation event.

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