KR20120083868A - Method of determining single nucleotide polymorphism of target gene using real-time pcr and kit for determining single nucleotide polymorphism of target gene using the same - Google Patents

Abstract

PURPOSE: A method for determining SNP of a target gene using real-time PCR is provided to quickly detect SNP. CONSTITUTION: A method for determining SNP of a target gene using real-time PCR comprises: a step of performing real time PCR using a common probe to the target gene to obtain a PCR product of the target gene; a step of calculating Ct value from allele-specific PCR products in each tube; a step of calculating delta Ct value from Ct value; and a step of determining SNP of the target gene from the delta Ct value. The probe is a Taqman probe.

Description

실시간 중합효소 연쇄반응을 이용한 표적 유전자의 단일 염기 다형성 판별 방법 및 이를 이용한 표적 유전자의 단일 염기 다형성 판별 키트{Method of Determining Single Nucleotide Polymorphism of Target Gene Using Real-Time PCR and Kit for Determining Single Nucleotide Polymorphism of Target Gene Using the Same}Method of Determining Single Nucleotide Polymorphism of Target Gene Using Real-Time PCR and Kit for Determining Single Nucleotide Polymorphism of Target Gene Using the Same}

본 발명은 표적 유전자의 단일 염기 다형성(SNP, Single Nucleotide Polymorphism)을 검출하는 방법 및 이를 이용한 표적 유전자의 단일 염기 다형성 판별 키트에 관한 것이다.The present invention relates to a method for detecting Single Nucleotide Polymorphism (SNP) of a target gene and a kit for determining a single nucleotide polymorphism of a target gene using the same.

단일 염기 다형성은 염색체 상에 유전자 특정 위치의 단일 염기(A, G, C, T)에 차이를 보인다. 단일 염기 다형성은 유전자의 3번째 코돈 위치에 단일 염기 차이를 보여 발현되는 단백질의 특성이 바뀌는 경우가 특징적이며, 단백질 발현의 특징 차이로 인해 약물에 대한 대사, 개인간 면역 체계의 차이를 보인다.Single base polymorphisms show differences in single bases (A, G, C, T) at gene specific locations on the chromosome. Single nucleotide polymorphism is characterized by a change in the characteristics of the protein expressed by a single base difference in the third codon position of the gene, and the metabolic to the drug, the difference in the immune system between individuals due to the characteristic differences in protein expression.

분자 유전학에서 많이 사용되고 있는 단일 염기 다형성을 검출하는 방법에는 ddNTP를 이용한 염기서열 분석법, 단일 염기 다형성 부분을 포함하여 중합효소 연쇄반응(PCR, Polymerase Chain Reaction) 후 특이적인 제한 효소를 이용하여 증폭 산물의 절편 크기 차이로 분석하는 제한효소 절편 길이 다형성(Polymerase Chain Reaction - Restriction Enzyme Length Polymorphism) 분석 방법이 있다. 그러나, 상기 염기서열 분석법 및 제한효소 길이 다형성 분석법은 실험에 필요한 시간이 오래 걸리며, 실험자가 직접 단일 염기 다형성을 검출된 정보를 판별해야 하는 단점이 있다.The method of detecting single nucleotide polymorphism, which is widely used in molecular genetics, includes sequencing using ddNTP, and amplification products using specific restriction enzymes after polymerase chain reaction (PCR) including single nucleotide polymorphism. There is a method of restriction chain fragment polymorphism (Polymerase Chain Reaction-Restriction Enzyme Length Polymorphism) analysis. However, the sequencing method and restriction enzyme length polymorphism analysis method takes a long time required for the experiment, and there is a disadvantage in that the experimenter must directly determine the information for detecting the single nucleotide polymorphism.

단일 염기 다형성을 분석하기 위한 중합효소 연쇄반응에는 SSP(Sequence-Specific Primed PCR)이 있다. 이는 대립형질에 특이적인 프라이머를 사용하여 아가로스 젤(agarose gel)에서 증폭 사이즈의 검출 여부로 확인 할 수 있는 방법이다. 이 방법은 유전자 증폭 후, 증폭 산물을 아가로스 젤에서 확인하는 2가지 실험 과정을 거치게 되며, 이 실험 과정 중 교차 오염(cross-contamination)의 위험성이 있어 정확한 검출이 어려울뿐더러, 실험자가 직접 증폭 사이즈에 대한 결과 확인까지의 실험시간이 오래 걸리는 단점을 가지고 있다.The polymerase chain reaction for analyzing single nucleotide polymorphisms includes Sequence-Specific Primed PCR (SSP). This is a method that can be confirmed by detecting the amplification size in the agarose gel (agarose gel) using a primer specific for the allele. This method involves two experiments to identify amplification products in agarose gel after gene amplification, and there is a risk of cross-contamination during the experiment, which makes it difficult to accurately detect the size of the experiment. The test takes a long time to confirm the results.

다른 한편으로, 실시간 유전자 분석법(Real-Time PCR)은 PCR의 일종으로 프라이머(primer) 및 프로브(probe)를 이용하여 형광 신호의 세기에 따라 증폭 여부를 확인하는 방법이다. 실시간 유전자 분석법으로 단일 염기 다형성을 분석하는 방법에는 검출하고자 하는 대립 형질(allele)의 형광 검출 신호의 강도 차이로 구분하는 방법, 증폭 절편의 녹는점(melting temperature)의 차이로 구분하는 방법이 상용화되어 있으며, 염기서열 분석법 및 제한효소 절편 길이 다형성 분석법에 비해 짧은 검출시간, 높은 민감도 및 특이도를 나타낸다. 그러나, 상용화된 실시간 유전자 분석법은 상기 염기서열 분석법 및 제한효소 절편 길이 다형성 분석법에 비해 단일 염기 다형성을 판별하는 시간이 짧고, 높은 민감도 및 특이도를 가지고 있지만, 실험자가 직접 단일 염기 다형성을 분석해야 하는 공통적인 단점이 있다.On the other hand, real-time PCR (Real-Time PCR) is a method of checking the amplification according to the intensity of the fluorescent signal using a primer (primer) and probe (probe) as a kind of PCR. The method of analyzing single nucleotide polymorphism by real-time genetic analysis method is commercially divided into the difference in the intensity of the fluorescence detection signal of the allele to be detected, and the difference in the melting temperature of the amplification fragments. Compared to sequencing and restriction fragment length polymorphism analysis, it shows shorter detection time, higher sensitivity and specificity. However, compared to the sequencing and restriction fragment length polymorphism assays, the commercialized real-time genetic analysis method has a shorter time for determining single nucleotide polymorphisms and has high sensitivity and specificity, but the experimenter has to directly analyze single nucleotide polymorphisms. There are common disadvantages.

본 발명은 염기서열 분석법 및 제한효소 절편 길이 다형성 분석법의 단점 및 유전자 연쇄 증폭 반응법의 실험 방법의 교차 오염 가능성과 실시간 유전자 분석법의 단점인 분석시간 및 직접 분석의 문제를 보완하는 표적 유전자의 단일 염기 다형성 판별 방법 및 자동 판별 프로그램, 이를 이용한 키트를 제공하는 것을 목적으로 한다.The present invention provides a single base of a target gene that compensates for the disadvantages of sequencing and restriction fragment length polymorphism analysis and the possibility of cross-contamination of the experimental method of the gene chain amplification reaction and the problem of analysis time and direct analysis, which are disadvantages of real-time gene analysis. An object of the present invention is to provide a polymorphism discrimination method, an automatic discrimination program, and a kit using the same.

본 발명은 형광프로브와 대립형질 특이적 프라이머(allele-specific primer, ASP)를 이용하여 자동으로 단일염기다형성을 판별하는 방법을 제공함에 있어 하기 단계의 방법을 실시한다.The present invention performs the following steps in providing a method for automatically determining monobasic polymorphism using a fluorescent probe and an allele-specific primer (ASP).

(1) 표적 유전자의 대립형질 특이적 프라이머를 서로 다른 튜브에 포함하고 표적 유전자에 공통적인 프로브를 각각 사용하여 실시간 중합효소 연쇄반응을 수행하여 상기 표적 유전자의 증폭산물을 생성하는 단계;(1) generating an amplification product of the target gene by including allele-specific primers of the target gene in different tubes and performing real-time polymerase chain reaction using probes common to the target gene, respectively;

(2) 상기 각각의 튜브의 대립형질 특이적 증폭산물로부터 Ct 값을 구하는 단계; (2) obtaining Ct values from allele specific amplification products of each tube;

(3) 상기 단계 (2)에서 얻어진 Ct 값으로부터 대립형질간의 ΔCt 값을 구하는 단계; 및(3) obtaining a ΔCt value between alleles from the Ct value obtained in step (2); And

(4) 상기 얻어진 ΔCt 값으로부터 상기 표적 유전자의 단일 염기 다형성을 판별하는 단계를 포함하는 실시간 중합 효소 연쇄반응을 이용하여 표적 유전자의 단일 염기 다형성 판별 방법을 제공한다.(4) it provides a method for determining the single nucleotide polymorphism of the target gene using a real-time polymerase chain reaction comprising the step of determining the single nucleotide polymorphism of the target gene from the obtained ΔCt value.

본 발명은 표적유전자의 대립형질을 판별하는 단계에 있어서 단일 유전자의 판별에만 한정되는 것은 아니며, 각 튜브에 2개 이상, 바람직하게는 2개 내지 5개 이하의 표적유전자에 해당하는 각각의 대립형질 특이적 프라이머를 포함하는 다중 검출 실시간 중합효소 연쇄반응인 것을 포함한다.The present invention is not limited to the identification of a single gene in the step of discriminating alleles of a target gene, and each allele corresponding to two or more, preferably two to five or less target genes in each tube. Multiple detection real time polymerase chain reaction comprising specific primers.

또한, 본 발명에 사용되는 형광프로브는 택맨 프로브인 것을 특징으로 한다.In addition, the fluorescent probe used in the present invention is characterized in that the Taekman probe.

본 발명에 있어서, "Ct"란 용어는 리포터 신호가 배경의 형광 수준을 초과하여 측정되는 역치 사이클(threshold cycle)을 나타내는 것으로서, ΔCt는 서로 다른 단일 염기 다형성을 특이적으로 증폭하는 프라이머를 이용하여 표적 유전자의 대립형질을 증폭한 경우의 Ct 값의 차를 나타낸다. 이 때 대립형질을 판단할 수 있는 ΔCt 값은 표적유전자 및 단일 염기 다형성 위치 등에 따라 달라질 수 있다. 예를 들어, 본 발명의 한 구현예에서 실시된 표적유전자 VKORC1와 CYP2C9의 ΔCt 값은 단일 염기 다형성 위치에 따라 VKORC1 3673G>A ΔCt 값이 ≤±4.1, 6484C>T ΔCt 값이 ≤±4.7, 6853G>A ΔCt 값이 ≤±7.8, 9041G>A ΔCt 값이 ≤±3.1, CYP2C9-430C>T ΔCt 값이 ≤±6.7, 1075A>C ΔCt 값이 ≤±3.0으로 달라지는 것을 확인할 수 있다.In the present invention, the term "Ct" refers to a threshold cycle in which the reporter signal is measured above the fluorescence level of the background, and ΔCt uses primers that specifically amplify different single base polymorphisms. The difference in Ct values when the allele of the target gene is amplified is shown. In this case, the ΔCt value from which the allele can be determined may vary depending on the target gene and the position of the single nucleotide polymorphism. For example, the ΔCt values of the target genes VKORC1 and CYP2C9 in one embodiment of the present invention are VKORC1 3673G> A ΔCt values ≤ ± 4.1, 6484C> T ΔCt values ≤ ± 4.7, 6853G It can be seen that> A ΔCt is ≤ ± 7.8, 9041G> A ΔCt is ≤ ± 3.1, CYP2C9-430C> T ΔCt is ≤ ± 6.7, and 1075A> C ΔCt is ≤ ± 3.0.

본 발명에 있어서, "단일 염기 다형성" 또는 "SNP"란 용어는 하나 이상의 변형 염기를 보유한 염기서열을 동반하는 임의의 위치를 의미한다. 단일 염기 다형성(SNP)은 인간 게놈에서 발견되는 DNA 서열 변형의 가장 공통적인 형태이며, 인간 게놈 프로젝트의 일부로 제작된 기준 참조 DNA 서열과의 차이로서 일반적으로 정의되거나, 또는 일반 집단에서 추출한 개체의 부분 집단 사이에서 발견되는 차이로서 정의된다. SNP는 무작위적으로 임의 선택한 2 종의 인간 염색체를 비교할 경우, 대략 1 SNP/1,000 염기쌍의 평균 비율로 발생한다. 특정 개체나 가계에 제한되는 매우 드문 SNP를 확인할 수 있으며, 반대로 일반 대중(다수의 연관성이 없는 개체들을 의미함)에서 매우 공통적인 것을 발견할 수도 있다. SNP는 DNA 복제 시 오류(즉, 자발적인 오류)에 의하거나, 또는 돌연변이 유발 제제(즉, 특정 DNA 유해 물질 등)에 의해 발생할 수 있으며, 유기체가 번식하는 동안 개별 후세에게 전달될 수도 있다.In the present invention, the term "single base polymorphism" or "SNP" means any position accompanied by a base sequence having one or more modified bases. Single nucleotide polymorphism (SNP) is the most common form of DNA sequence modification found in the human genome, and is generally defined as a difference from a reference reference DNA sequence produced as part of the Human Genome Project, or part of an individual extracted from a general population. It is defined as the difference found between groups. SNPs occur at an average ratio of approximately 1 SNP / 1,000 base pairs when comparing two randomly selected human chromosomes. Very rare SNPs can be identified that are limited to specific individuals or households, and conversely, they can be found to be very common among the general public (meaning many unrelated individuals). SNPs can be caused by errors in DNA replication (ie, spontaneous errors), or by mutagenic agents (ie, certain DNA harmful substances, etc.) and can also be passed on to individual posteriors during organism reproduction.

본 발명에 있어서, "핵산 분자"라는 용어는 DNA 또는 RNA를 포함하는 임의의 핵산 함유 분자를 의미하지만, 이에 한정되는 것은 아니다.In the present invention, the term "nucleic acid molecule" means, but is not limited to, any nucleic acid-containing molecule, including DNA or RNA.

본 발명에 있어서, "유전자"란 용어는 폴리펩티드, 전구체, 또는 RNA(예를 들어, rRNA, tRNA)의 생산을 위해 필수적인 코팅 서열을 포함하는 핵산(예를 들어, DNA) 서열을 의미한다. 상기 폴리펩티드는 전체 길이 코딩 서열로부터 인코딩되거나, 또는 전체 길이나 절편의 바람직한 활성이나 기능적 특성(예를 들어, 효소 활성, 리간드 결합, 신호 전달, 면역원성 등)을 유지하는 한 코딩 서열의 임의 부분으로부터 인코딩될 수 있다.In the present invention, the term "gene" refers to a nucleic acid (eg, DNA) sequence comprising a coating sequence necessary for the production of a polypeptide, precursor, or RNA (eg, rRNA, tRNA). The polypeptide is encoded from a full length coding sequence or from any portion of the coding sequence as long as it retains the desired activity or functional properties (eg, enzyme activity, ligand binding, signal transduction, immunogenicity, etc.) of the full length or fragment. Can be encoded.

본 발명에 있어서, "유전자 발현"이란 용어는 유전자의 "전사"를 통해(즉, RNA 중합효소의 효소 작용을 통해서) 유전자 내에 인코딩된 유전 정보를 RNA(예를 들어, mRNA, rRNA, tRNA, 또는 snRNA)로 전환시키고, mRNA의 "번역"을 통해서 단백질 인코딩 유전자로부터 단백질을 얻는 과정을 의미한다.In the present invention, the term "gene expression" refers to the genetic information encoded in the gene through "transcription" of the gene (ie, through the enzymatic action of RNA polymerase) RNA (eg, mRNA, rRNA, tRNA, Or snRNA), and a process of obtaining a protein from a protein encoding gene through "translation" of the mRNA.

본 발명에 있어서, "상보성 또는 상보적"이란 용어는 염기쌍 결합 규칙과 관련성이 있는 폴리뉴클레오티드(즉, 뉴클레오티드의 서열)에 대해 사용한다. 예를 들어, 서열 5'-A-G-T-3'은 서열 3'-T-C-A-5'과 상보적이다. 상보성은 "부분적"일 수 있는데, 즉 핵산 염기의 단지 일부만이 염기 쌍 결합 규칙에 따라 부합될 수 있다. 다른 한편으로, 핵산 사이에 "완전한" 또는 "전체" 상보성을 가질 수도 있다. 핵산 가닥 사이의 상보성 정도는 핵산 가닥 사이의 혼성화 강도 및 효율성에 상당한 영향이 있다. 이는 핵산 사이의 결합에 좌우되는 검출법뿐만 아니라 증폭 반응에서 특히 중요하다.In the present invention, the term “complementary or complementary” is used for polynucleotides (ie, sequences of nucleotides) that are relevant to base pair binding rules. For example, the sequence 5'-A-G-T-3 'is complementary to the sequence 3'-T-C-A-5'. Complementarity can be “partial,” that is, only some of the nucleic acid bases can be matched according to base pairing rules. On the other hand, they may have "complete" or "total" complementarity between nucleic acids. The degree of complementarity between nucleic acid strands has a significant impact on the strength and efficiency of hybridization between nucleic acid strands. This is particularly important in amplification reactions as well as detection methods that depend on binding between nucleic acids.

또한, 본 발명의 방법에서 사용하는 실시간 PCR에서는 형광물질이 화학적으로 결합하여 있는 프로브를 사용함으로써 반응 결과를 실시간으로 모니터하는 것이 바람직하다. 상기 프로브는 PCR 과정에서 두 개의 프라이머와 같이 검체의 핵산에 있는 상보적 서열에 결합하게 되는데, 결합 위치는 프라이머에서 약간 떨어진 부분이다. 본 발명의 프로브는 양 말단에 리포터(reporter)와 소광제(quencher)라는 형광물질이 붙어 있는 구조인 것이 바람직하다. 상기 프로브의 5' 말단에는 리포터가 결합되어 있고, 3' 말단에는 소광제가 결합되어 있는 것이 바람직하지만 이에 한정되는 것은 아니다. 본 발명의 한 구현예에 따르면, 상기 리포터와 소광제가 근접하여 존재하면 형광을 서로 상쇄하여 리포터의 형광이 감지되지 않으나, 증폭이 진행됨에 따라 리포터가 소광제로부터 떨어지면 리포터의 형광이 감지된다. 따라서, 형광의 강도는 증폭 사이클이 증가함에 따라 점점 증가하게 된다. 본 발명의 바람직한 구현예에 따르면, 상기 리포터는 FAM (6-carboxyfluorescein), Texas red, JOE, TAMRA, CY5 또는 CY3를 사용하고, 상기 소광제는 TAMRA (6-carboxytetramethyl-rhodamine), BHQ1, BHQ2 또는 Dabsyl을 사용하는 것이 바람직하지만 이에 한정되는 것은 아니다.In addition, in the real-time PCR used in the method of the present invention, it is preferable to monitor the reaction result in real time by using a probe to which the fluorescent substance is chemically bound. The probe binds to the complementary sequence in the nucleic acid of the sample like the two primers in the PCR process, where the binding position is slightly away from the primer. The probe of the present invention preferably has a structure in which both ends of a reporter and a quencher are attached. A reporter is bound to the 5 'end of the probe, and a quencher is bound to the 3' end, but is not limited thereto. According to one embodiment of the present invention, when the reporter and the quencher are present in close proximity to each other, the fluorescence of the reporter is not detected by the fluorescence of the reporter, but as the amplification proceeds, the reporter's fluorescence is detected when the reporter is dropped from the quencher. Thus, the intensity of fluorescence increases gradually as the amplification cycle increases. According to a preferred embodiment of the present invention, the reporter uses FAM (6-carboxyfluorescein), Texas red, JOE, TAMRA, CY5 or CY3, the quencher is TAMRA (6-carboxytetramethyl-rhodamine), BHQ1, BHQ2 or It is preferable to use Dabsyl, but is not limited thereto.

본 발명의 표적 유전자의 단일 염기 다형성 판별 방법에서는 PCR에 쓰이는 ASP를 실시간 PCR에 사용하였으며, 이를 위하여 상기 프라이머 이외에도 양 말단에 형광물질 및 소광제가 결합된 프로브를 사용한다. 종래 PCR에서는 ASP를 사용하여 단일 염기 다형성을 포함하는 부위를 증폭한 후 전기 영동 장치를 이용하여 증폭 산물의 증폭 정도를 비교함으로써 단일 염기 다형성을 판별하고 있다. 그러나, 이는 PCR 후 전기 영동법으로 확인해야만 하는 번거로움이 있을 뿐만 아니라, 민감도와 특이도가 낮다는 단점을 가지고 있다. 이에 본 발명에서는 상기 ASP와 형광프로브를 이용하여 실시간 유전자 연쇄 증폭 반응에 도입한 후, 타겟이 되는 단일 염기 다형성이 3'말단에 존재하는 특이적인 프라이머와 공통되게 사용하는 프라이머 및 형광 프로브를 한 쌍으로 하며, 2개 이상의 튜브에서 반응하도록 하였다. 또한, 본 발명에서는 판별 결과를 이용하여 단일 염기 다형성을 자동으로 분석해주는 프로그램을 제공한다(도 6 및 도 7 참조).In the method for determining the single nucleotide polymorphism of the target gene of the present invention, ASP used in PCR was used for real-time PCR, and for this purpose, a probe having a fluorescent material and a quencher coupled to both ends is used. In conventional PCR, a single base polymorphism is determined by amplifying a site including a single base polymorphism using ASP, and then comparing the amplification degree of the amplification product using an electrophoretic device. However, this has the disadvantage of having to check by electrophoresis after PCR, as well as low sensitivity and specificity. Therefore, in the present invention, after introducing into the real-time gene chain amplification reaction using the ASP and the fluorescence probe, a pair of primers and a fluorescence probe that use a single nucleotide polymorphism as a target in common with a specific primer present at the 3 'end It was made to react in two or more tubes. In addition, the present invention provides a program for automatically analyzing a single nucleotide polymorphism using the determination result (see FIGS. 6 and 7).

한편, VKORC1 (vitamin K epoxide reductase complex subunit 1) 및 CYP2C9 (cytochrome P450 2C9) 유전자의 다형성은 경구 항응고제인 와파린의 용량 조절에 있어서 가장 중요한 유전인자로 알려져 있다. 미 FDA와 국립 임상 생화학회(National Academy of Clinical Biochemistry)에서는 와파린 투여시 반드시 두 유전자의 유전형 검사를 하도록 권고하고 있다. 한국인에 있어서, 약물 유전형에 따라 와파린의 개인별 적정 사용량이 최대 46%까지 차이가 나며, 한국인 약물 유전형의 빈도는 백인 및 흑인과는 완전히 다른 양상을 보이고 있어 와파린의 사용량이 인종 간에 차이가 있으며, 동일한 인종이라 하더라도 개인별로 다를 수 있다는 사실이 밝혀져 있다. 와파린은 용량이 부족하면 혈관이 막히게 되고, 용량이 과도할 경우 내출혈 등으로 심각한 결과를 초래하기 때문에 개인별 사용량 조절이 매우 중요하다. 또한, 출혈 및 혈전형성의 치명적인 부작용을 줄이기 위해 신속한 와파린 용량 결정이 필수적이어서 검사소요시간이 짧고 경제적인 검사법이 필요하다. 따라서, 실시간 PCR을 통해 상기 VKORC1(3673G>A, 6484C>T, 6853G>C, 9041G>A) 및 CYP2C9(1075A>C, 430C>T) 유전자의 유전형을 감도 높고 재현성 있게 판별할 경우 와파린 용량 결정을 신속하게 결정할 수 있게 된다.Polymorphisms of the VKORC1 (vitamin K epoxide reductase complex subunit 1) and CYP2C9 (cytochrome P450 2C9) genes are known to be the most important genetic factors in dose regulation of warfarin, an oral anticoagulant. The FDA and the National Academy of Clinical Biochemistry recommend that genotypes of both genes be tested when warfarin is administered. For Koreans, the optimal use of warfarin varies by up to 46%, depending on the drug genotype, and the frequency of Korean drug genotypes is completely different from that of whites and blacks. It turns out that even races can vary from person to person. Warfarin is a blood vessel clogging when the dose is insufficient, and excessive doses cause serious consequences such as internal bleeding, it is very important to control the amount of individual use. In addition, rapid warfarin dose determination is essential to reduce the fatal side effects of bleeding and thrombus formation. Therefore, if the genotypes of the VKORC1 (3673G> A, 6484C> T, 6853G> C, 9041G> A) and CYP2C9 (1075A> C, 430C> T) genes are determined with high sensitivity and reproducibly, the warfarin dose is determined. Can be determined quickly.

본 발명의 한 구현 예에 따르면, 본 발명의 표적은 서열번호 1로 기재되는 VKORC1 유전자의 3673G>A(rs No. 9923231), 6484C>T(rs No. 9934438), 6853G>C(rs No. 8050894), 9041G>A(rs No. 7294) 및 서열번호 2로 기재되는 CYP2C9 유전자의 1075A>C(rs No. 1799853), 430C>T(rs No. 1057910)의 6개 단일 염기 다형성 부위 중 하나 이상으로 할 수 있으나, 반드시 상기 다형성 부위에 한정되어야 하는 것은 아니다. 바람직하게는, 상기 표적이 되는 단일 염기 다형성을 판별하기 위한 프라이머 및 프로브는 하기 표 1 및 표 2에 개시되어 있는 염기서열을 갖는다. 또한, 상기 프로브는 양 말단 중 어느 하나에 형광물질이 결합되어 있으며, 상기 형광물질로는 FAM, Texas-Red, TAMRA 등의 물질을 사용하는 것이 바람직하지만, 반드시 이에 한정되어야 하는 것은 아니다.According to one embodiment of the invention, the targets of the invention are 3673G> A (rs No. 9923231), 6484C> T (rs No. 9934438), 6853G> C (rs No. 8050894), 9041G> A (rs No. 7294) and one of six single base polymorphic sites of 1075A> C (rs No. 1799853), 430C> T (rs No. 1057910) of the CYP2C9 gene, set forth in SEQ ID NO: 2 Although it can be made as above, it is not necessarily limited to the said polymorphic site | part. Preferably, the primers and probes for determining the target single base polymorphism have the base sequences set forth in Tables 1 and 2 below. In addition, the probe is a fluorescent material is bonded to any one of both ends, it is preferable to use a material such as FAM, Texas-Red, TAMRA as the fluorescent material, but is not necessarily limited thereto.

VKORC1 및 CYP2C9 유전자에 대한 프라이머의 염기서열Sequences of Primers for the VKORC1 and CYP2C9 Genes 프라이머primer 염기서열Base sequence 길이Length 서열번호SEQ ID NO: VKORC1_3673_AVKORC1_3673_A ACCTGAAAAACAACCATTGGTCA ACCTGAAAAACAACCATTGGTC A 23-mer23-mer 33 VKORC1_3673_GVKORC1_3673_G ACCTGAAAAACAACCATTGGTCG ACCTGAAAAACAACCATTGGTC G 23-mer23-mer 44 VKORC1_3673_RevVKORC1_3673_Rev TCCTGACCTCAAGTGATCCACCTCCTGACCTCAAGTGATCCACC 22-mer22-mer 55 VKORC1_6484_CVKORC1_6484_C CGGTGCCAGGAGATCATCGTCCCGGTGCCAGGAGATCATCGTCC 22-mer22-mer 66 VKORC1_6484_TVKORC1_6484_T CGGTGCCAGGAGATCATCGTCTCGGTGCCAGGAGATCATCGTCT 22-mer22-mer 77 VKORC1_6484_RevVKORC1_6484_Rev CTGCCCGAGAAAGGTGATTTCCAAGCTGCCCGAGAAAGGTGATTTCCAAG 25-mer25-mer 88 VKORC1_6853_CVKORC1_6853_C CACCCGCAGGACGCACG CACCCGCAGGACGCAC G 17-mer17-mer 99 VKORC1_6853_GVKORC1_6853_G CACCCGCAGGACGCACC CACCCGCAGGACGCAC C 17-mer17-mer 1010 VKORC1_6853_revVKORC1_6853_rev CCCACTCCATGCAATCTTGGCCCACTCCATGCAATCTTGG 20-mer20-mer 1111 VKORC1_9041_AVKORC1_9041_A CCCTCCTCCTGCCATACTCA CCCTCCTCCTGCCATACTC A 20-mer20-mer 1212 VKORC1_9041_GVKORC1_9041_G CCCTCCTCCTGCCATACTCG CCCTCCTCCTGCCATACTC G 20-mer20-mer 1313 VKORC1_9041_RevVKORC1_9041_Rev GGCAATGGAAAGAGCTTTGGGGCAATGGAAAGAGCTTTGG 20-mer20-mer 1414 CYP2C9_430_CCYP2C9_430_C CGGGCTTCCTCTTGAACTCG CGGGCTTCCTCTTGAACTC G 20-mer20-mer 1515 CYP2C9_430_TCYP2C9_430_T CGGGCTTCCTCTTGAACTCA CGGGCTTCCTCTTGAACTC A 20-mer20-mer 1616 CYP2C9_430_REVCYP2C9_430_REV GGGAGGATGGAAAACAGAGACTTAGGGAGGATGGAAAACAGAGACTTA 24-mer24-mer 1717 CYP2C9_1075_ACYP2C9_1075_A GGTGCACGAGGTCCAGAGATTCA GGTGCACGAGGTCCAGAGATTC A 23-mer23-mer 1818 CYP2C9_1075_CCYP2C9_1075_C GTGCACGAGGTCCAGAGATTCC GTGCACGAGGTCCAGAGATTC C 22-mer22-mer 1919 CYP2C9_1075_REVCYP2C9_1075_REV GATACTATGAATTTGGGGACTTCGAAGATACTATGAATTTGGGGACTTCGAA 26-mer26-mer 2020

VKORC1 및 CYP2C9 유전자에 대한 프로브의 염기서열Sequences of Probes for the VKORC1 and CYP2C9 Genes 프로브Probe 염기서열Base sequence 형광Neon 소광제Matting agent 길이Length 서열번호SEQ ID NO: VKORC1_3673_probeVKORC1_3673_probe TGCGGTGGCTCACGCCTATAATCCTTGCGGTGGCTCACGCCTATAATCCT FAMFAM BHQ1BHQ1 25-mer25-mer 2121 VKORC1_6484_probeVKORC1_6484_probe TGGGAGGTCGGGGAACAGAGGATAGTGGGAGGTCGGGGAACAGAGGATAG FAMFAM BHQ1BHQ1 25-mer25-mer 2222 VKORC1_6853_probeVKORC1_6853_probe TGAGCAGCTAGCTGGCTGTCAGCTGTTGAGCAGCTAGCTGGCTGTCAGCTGT Texas-redTexas-red BHQ2BHQ2 26-mer26-mer 2323 VKORC1_9041_probeVKORC1_9041_probe ACCAAATGTGCCACACGCTCGCTACCAAATGTGCCACACGCTCGCT Texas-redTexas-red BHQ2BHQ2 23-mer23-mer 2424 CYP2C9_430_probeCYP2C9_430_probe TGGGCAGCCATGTGGATGGGCCTGGGCAGCCATGTGGATGGGCC FAMFAM BHQ1BHQ1 22-mer22-mer 2525 CYP2C9_1075_probeCYP2C9_1075_probe TCCCCACCAGCCTGCCCCATTCCCCACCAGCCTGCCCCAT FAMFAM BHQ1BHQ1 20-mer20-mer 2626

본 발명의 다른 구현예에 따르면, 상기 VKORC1과 CYP2C9 유전자의 단일 염기 다형성을 특이적으로 검출할 수 있는 프라이머 및 프로브를 디자인 및 합성한 후, 상기 프라이머와 프로브의 검증을 위해 단일 염기 다형성을 포함하는 400~500 염기서열의 크기로 합성하여, pGEM-T easy 벡터(Promega)에 클로닝하여 HIT™-DH5a 균주(RBC bioscience)에 형질전환하였다.According to another embodiment of the present invention, after designing and synthesizing a primer and a probe capable of specifically detecting a single base polymorphism of the VKORC1 and CYP2C9 genes, the method comprises a single base polymorphism for verification of the primer and the probe. It was synthesized in the size of 400 ~ 500 nucleotide sequence, cloned into pGEM-T easy vector (Promega) and transformed into HIT ™ -DH5a strain (RBC bioscience).

또한, 본 발명은 VKORC1 (vitamin K epoxide reductase complex subunit 1) 및 CYP2C9 (cytochrome P450 2C9)의 단일염기 다형성을 판별할 수 있는 키트를 제공한다. 보다 상세하게는, VKORC1 유전자 단일염기 다형성 판별을 위한 상기 서열번호 3 내지 서열번호 14의 프라이머와 서열번호 21 내지 서열번호 24의 프로브 및 CYP2C9 유전자 단일염기 다형성 판별을 위한 서열번호 15 내지 서열번호 20의 프라이머와 서열번호 25 내지 서열변호 26의 프로브 세트를 포함하는 실시간 중합효소 연쇄반응을 이용한 표적 유전자의 단일 염기 다형성 판별용 키트를 제공한다.The present invention also provides a kit that can determine the monobasic polymorphisms of vitamin K epoxide reductase complex subunit 1 (VKORC1) and CYP2C9 (cytochrome P450 2C9). More specifically, the primers of SEQ ID NO: 3 to SEQ ID NO: 14, the probes of SEQ ID NO: 21 to SEQ ID NO: 24, and the SEQ ID NO: 15 to SEQ ID NO: 20 of the CYP2C9 gene monobasic polymorphism for the determination of VKORC1 gene single nucleotide polymorphism Provided is a kit for determining a single nucleotide polymorphism of a target gene using a real time polymerase chain reaction comprising a primer and a probe set of SEQ ID NO: 25 to SEQ ID NO: 26.

본 발명의 바람직한 구현예에 따르면, 상기 키트는 (a) dNTP 혼합물(dATP, dCTP, dGTP, dTTP); (b) DNA 중합효소; 및 (c) 완충 용액을 추가로 포함할 수 있다. 본 발명의 다른 바람직한 구현예에 따르면, 상기 키트는 선택적으로 핵산 증폭에 필요한 시약, 예컨대, 완충액, DNA 중합효소 (예컨대, Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus 또는 Thermococcus literalis로부터 수득한 열 안정성 DNA 중합효소), DNA 중합 효소 조인자 및 dNTP를 포함할 수 있다.According to a preferred embodiment of the present invention, the kit comprises (a) a dNTP mixture (dATP, dCTP, dGTP, dTTP); (b) DNA polymerase; And (c) a buffer solution. According to another preferred embodiment of the invention, the kit optionally comprises reagents for nucleic acid amplification, such as buffers, DNA polymerases (eg, Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus or Thermococcus). thermally stable DNA polymerases obtained from literalis), DNA polymerase cofactors and dNTPs.

본 발명의 키트는 상기한 시약 성분을 포함하는 다수의 별도 패키징 또는 컴파트먼트로 제작될 수 있다. 또한, 본 발명의 키트는 최적의 반응 수행 조건을 기재한 사용자 안내서를 추가로 포함할 수 있다. 상기 안내서는 키트 사용법, 예를 들면, PCR 완충액 제조 방법, 제시되는 반응 조건 등을 설명하는 인쇄물로서, 팜플렛 또는 전단지 형태의 안내 책자, 키트에 부착된 라벨, 및 키트를 포함하는 패키지의 표면상에 설명을 포함할 수 있다. 또한, 상기 안내서는 인터넷과 같이 전기 매체를 통해 공개되거나 제공되는 정보를 포함할 수 있다.Kits of the invention can be prepared in a number of separate packaging or compartments containing the reagent components described above. In addition, the kits of the present invention may further comprise a user guide describing the optimal reaction performance conditions. The guide is a printed document that explains how to use the kit, e.g., how to prepare a PCR buffer, the reaction conditions presented, and the like, in the form of a brochure or leaflet, a label attached to the kit, and a surface of the package containing the kit. It may include a description. In addition, the guide may include information disclosed or provided through an electronic medium such as the Internet.

또한, 본 발명에서는 제공된 키트를 이용하여 실시간 중합효소 연쇄반응을 통해 얻어진 각각의 대립 형질 증폭 산물의 Ct 값을 이용하여 단일 염기 다형성을 자동으로 판별해주는 프로그램을 제공한다.In addition, the present invention provides a program for automatically determining a single nucleotide polymorphism using the Ct value of each allele amplification product obtained through real-time polymerase chain reaction using the kit provided.

본 발명의 표적 유전자의 단일 염기 다형성 판별 방법은 빠른 시간 내에 원하는 SNP를 검출할 수 있다는 장점이 있다.The method for determining single nucleotide polymorphism of the target gene of the present invention has an advantage of detecting a desired SNP in a short time.

도 1a 및 도 1b는 ASP 실험법의 기본적인 검출 방식에 대한 그림 및 실험 과정에서 판별까지의 과정을 보여주는 모식도이다.
도 2는 실시간 중합효소 연쇄반응에서의 동형 접합자와 이형 접합자의 검출 결과와 직접 염기 서열 분석법과의 비교결과에 대한 간단한 예이다.
도 3a 내지 도 3f는 실시예 3의 염기서열 분석 결과를 보여주는 그래프이다.
도 4a 내지 도 4f는 실시예 4의 SNP 판별결과에 대한 실시간 중합연쇄반응 실험 결과를 보여주는 그래프이다.
도 5는 실시예 4의 실험 결과에 대하여 민감도와 특이도를 나타내는 Receiver Operating Characteristic (ROC) curve를 그려서 동형 접합자와 이형 접합자에 대한 판별 검출 한계 값을 구한 결과를 보여주는 그래프이다.
도 6은 단일염기다형성을 자동으로 판별해 주는 프로그램의 흐름도를 나타낸 모식도이다.
도 7은 도 6의 과정을 통해 나타난 유전형 자동 판별 프로그램에 대한 형광 검출 신호 및 단일 염기 다형성 판별 결과이다.Figures 1a and 1b is a schematic diagram showing the process of the determination and the process from the experiment and the basic detection method of the ASP test method.
2 is a simple example of the results of detection of homozygotes and heterozygotes and direct sequencing in real time polymerase chain reaction.
3A to 3F are graphs showing the results of nucleotide sequence analysis of Example 3.
4A to 4F are graphs showing the results of real-time polymerization chain reaction experiments for the SNP determination result of Example 4. FIG.
FIG. 5 is a graph showing a result of obtaining a detection limit value for a homozygote and a heterozygote by drawing a Receiver Operating Characteristic (ROC) curve representing sensitivity and specificity with respect to the experimental result of Example 4. FIG.
6 is a schematic diagram showing a flowchart of a program for automatically determining a single nucleotide polymorphism.
7 is a fluorescence detection signal and a single nucleotide polymorphism determination result for the genotyping automatic identification program shown through the process of FIG. 6.

이하, 본 발명을 실시예에 의해 상세히 설명한다.Hereinafter, the present invention will be described in detail by way of examples.

단, 하기 실시예는 본 발명을 예시하기 위한 것일 뿐, 본 발명의 내용이 하기 실시예에 의해 한정되는 것은 아니다.
However, the following examples are only for illustrating the present invention, and the content of the present invention is not limited by the following examples.

실시예 1: 인간 혈액에서 DNA 추출Example 1 DNA Extraction from Human Blood

인간 혈액 검체 82개 검체에서 DNA를 추출하였다. 구체적으로, 인간 검체에서 DNA를 추출하기 위해 EDTA에 보관한 혈액 5 ㎖ 혈액중 200 ㎕를 취하여 1.5 ㎖ 튜브에 옮겼다. Accuprep　genomic DNA extraction kit(Bioneer, korea)를 사용하였으며, 추출 과정은 핵산 해리 혼합액을 200 ㎕ 넣고 단백질분해효소 K (25 ㎎/㎖)를 20 ㎕ 첨가하였다. 잘 혼합한 후 검체가 완전히 용해될 때까지 약 10분 동안 60℃ 항온수조에 방치하였다. 항온수조에서 반응 후 반응액을 genomic DNA를 결합할 수 있는 칼럼에 넣고 8,000 rpm에서 1분간 원심분리하였다. genomic DNA 결합 칼럼을 새로운 튜브에 장착시킨 후, 95% 에탄올이 들어있는 세척 용액 1을 500 ㎕ 넣고, 8,000 rpm에서 1분 동안 원심분리하였다. 다시 새로운 튜브를 장착하여 95% 에탄올이 들어 있는 세척 용액 2를 넣고 8,000 rpm에서 1분 동안 원심분리하였다. 추출된 용액을 버리고 12,000 rpm으로 원심 분리하여 남아 있는 용액을 제거하였다. 1분간 상온에서 건조한 후 증류수를 50 ㎕를 넣고, 12,000 rpm으로 원심분리 후 genomic DNA 추출액을 얻었다.
DNA was extracted from 82 human blood samples. Specifically, 200 μl of blood 5 ml of blood stored in EDTA to extract DNA from human specimens was transferred to a 1.5 ml tube. Accuprep genomic DNA extraction kit (Bioneer, Korea) was used, and the extraction process was carried out by adding 200 μl of the nucleic acid dissociation mixture and 20 μl of protease K (25 mg / ml). After mixing well, the sample was left in a 60 ° C. constant temperature water bath for about 10 minutes until the sample was completely dissolved. After the reaction in a water bath, the reaction solution was placed in a column capable of binding genomic DNA, and centrifuged at 8,000 rpm for 1 minute. After mounting the genomic DNA binding column in a new tube, 500 µl of Wash Solution 1 containing 95% ethanol was added and centrifuged at 8,000 rpm for 1 minute. Again, a new tube was added to wash solution 2 containing 95% ethanol and centrifuged for 1 minute at 8,000 rpm. The extracted solution was discarded and centrifuged at 12,000 rpm to remove the remaining solution. After drying at room temperature for 1 minute, 50 μl of distilled water was added and centrifuged at 12,000 rpm to obtain genomic DNA extract.

실시예 2: VKORC1, CYP2C9 SNP 프라이머 및 프로브 디자인Example 2: VKORC1, CYP2C9 SNP Primer and Probe Design

본 발명의 표적은 NCBI 인간(human) 데이터베이스에 등록된 VKORC1 유전자의 3673G>A(rs No. 9923231), 6484C>T(rs No. 9934438), 6853G>C(rs No. 8050894), 9041G>A(rs No. 7294) CYP2C9 1075A>C(rs No. 1799853), 430C>T(rs No. 1057910) 6개 단일 염기 다형성으로 하였으며, 표적이 되는 단일 염기 다형성을 판별하기 위한 프라이머 및 프로브를 디자인 하였고, 프라이머는 1개의 단일 염기 다형성에 대해 정방향 프라이머와 역방향 프라이머를 1 세트로 디자인 하였으며, 정방향이나 역방향 프라이머는 단일 염기 다형성이 3' 말단에 위치하도록 하여 20?30 base가 되도록 하였으며, 녹는점(Tm melting temperature)이 55℃에서 60℃사이에 있도록 하여 디자인 되었다. 타겟 프로브는 정방향, 역방향 프라이머가 증폭 되는 증폭 산물 안의 염기 서열내에서 디자인 되었으며, 정방향, 역방향 프라이머의 녹는점 보다 5~10℃ 높게 디자인 되었다. 이에 대해 VKORC1, CYP2C9 SNP의 디자인된 정방향, 역방향 프라이머, 프로브는 표 1 및 표 2에 나타내었다.The target of the present invention is 3673G> A (rs No. 9923231), 6484C> T (rs No. 9934438), 6853G> C (rs No. 8050894), 9041G> A of the VKORC1 gene registered in the NCBI human database. (rs No. 7294) CYP2C9 1075A> C (rs No. 1799853), 430C> T (rs No. 1057910) 6 single nucleotide polymorphisms, primers and probes were designed to determine the target single nucleotide polymorphism. The primers were designed with one set of forward and reverse primers for one single polymorphism, and the forward and reverse primers had a single base polymorphism located at the 3 'end to 20-30 bases, and the melting point (Tm). melting temperature) is designed to be between 55 ℃ and 60 ℃. The target probe was designed in the nucleotide sequence in the amplification product in which the forward and reverse primers were amplified, and was designed to be 5-10 ° C. higher than the melting point of the forward and reverse primers. The designed forward, reverse primers, probes of VKORC1, CYP2C9 SNPs are shown in Table 1 and Table 2 for this.

디자인된 프라이머 및 프로브는 VKORC1과 CYP2C9 유전자 단일 염기 다형성과 본 발명에서 하고자 하는 실험 방법에 맞게 프라이머 및 프로브를 디자인 및 합성(Bioneer, Korea) 하였으며, 프라이머와 프로브의 검증을 위해 단일 염기 다형성을 포함하는 400~500 base pair 크기로 합성하여, pGEM-T easy 벡터(Promega)에 클로닝한 후, HIT™-DH5a 균주(RBC bioscience)에 형질 전환하였다.
Designed primers and probes were designed and synthesized (Bioneer, Korea) primers and probes according to the VKORC1 and CYP2C9 gene single base polymorphism and the experimental method of the present invention, including a single base polymorphism for the verification of primers and probes 400-500 base pairs were synthesized, cloned into pGEM-T easy vector (Promega), and transformed into HIT ™ -DH5a strain (RBC bioscience).

실시예 3: 추출된 genomic DNA 염기서열 분석Example 3: Analysis of extracted genomic DNA sequencing

추출된 genomic DNA는 본 발명에서 하고자 하는 실험 방법과의 비교를 위해 염기서열 분석법을 이용하여 1차 분석을 진행하였다. 염기서열 분석을 위해 BigDye　Terminator v3.1 Cycle Sequencing Kit와 염기서열 분석 장비인 ABI 3730 장비를 이용하여 분석하였다.The extracted genomic DNA was subjected to the primary analysis using the sequencing method for comparison with the experimental method of the present invention. For sequencing, BigDye Terminator v3.1 Cycle Sequencing Kit and ABI 3730 equipment were used for sequencing.

분석 방법은 실시예 1에서 추출된 genomic DNA 150 ng/㎕~300 ng/㎕ 농도의 genomic DNA를 첨가하고, Terminator Ready Reaction Mix buffer 8 ㎕, 특이적인 프라이머 3.2 pmol 넣고 전체 volume 20 ㎕이 되도록 3차 증류수를 넣었다. 20 ㎕의 혼합물을 ABI3730 염기서열 분석 장비를 이용하여 1) 95℃ 5분 반응, 2)95℃ 30초, 50-55℃ 10초 동안 반응을 50번 수행하여 도 3a 내지 도 3f과 같은 염기서열 분석 결과를 얻었다.
Assay method was added to genomic DNA concentration of 150 ng / μl ~ 300 ng / μl genomic DNA extracted in Example 1, 8 μl of Terminator Ready Reaction Mix buffer, 3.2 pmol specific primer and 20 μl of total volume Distilled water was added. 20 μl of the mixture using ABI3730 sequencing equipment 1) 95 ℃ 5 minutes reaction, 2) 95 ℃ 30 seconds, 50-55 ℃ 10 times the reaction was carried out for 10 seconds 50 times the base sequence as shown in Figures 3a to 3f The analysis result was obtained.

실시예Example 4: 실시간  4: real time 중합연쇄Polymerization Chain 반응 실험 Reaction experiment

실시예 2에서 디자인한 프라이머/프로브를 이용하여 합성된 주형을 이용하여 1차 실시간 중합 연쇄 반응을 수행하였으며, 실험 과정은 표 3과 같은 위치에 프라이머/프로브 혼합물을 5 ㎕ 넣고, 그 혼합물의 조성 및 농도는 표 4와 같이 하였다.The primary real-time polymerization chain reaction was carried out using the template synthesized using the primer / probe designed in Example 2, and the experimental procedure was carried out with 5 µl of the primer / probe mixture at the positions shown in Table 3, and the composition of the mixture. And concentrations are as shown in Table 4.

프라이머/프로브 위치Primer / Probe Position well No.well No. FAMFAM Texas redTexas red TAMRATAMRA 1One 3673A3673A 9041A9041A GAPDHGAPDH 22 3673G3673G 9041G9041G GAPDHGAPDH 33 6484C6484C 6853C6853C GAPDHGAPDH 44 6484T6484T 6853G6853G GAPDHGAPDH 55 430C430C -- GAPDHGAPDH 66 430T430T -- GAPDHGAPDH 77 1075A1075A -- GAPDHGAPDH 88 1075C1075C -- GAPDHGAPDH

프라이머/프로브 사용 농도(단위 pmole/㎕)Primer / Probe Concentration (Units pmole / μl) 1One 22 33 44 55 66 77 88 GAPDH_F_primerGAPDH_F_primer 3030 3030 3030 3030 2020 2020 2020 2020 GAPDH_R_ primerGAPDH_R_ primer 3030 3030 3030 3030 2020 2020 2020 2020 GAPDH_probeGAPDH_probe 3030 3030 3030 3030 2020 2020 2020 2020 FAM_F_ primerFAM_F_ primer 4040 4040 3030 4040 2020 2020 1515 1515 FAM_R_ primerFAM_R_ primer 4040 4040 3030 3030 2020 2020 1515 1515 FAM_probeFAM_probe 3030 3030 1515 1515 2020 2020 1515 1515 Texas red_F_ primerTexas red_F_ primer 3030 3030 3030 3030 -- -- -- -- Texas red_R_ primerTexas red_R_ primer 3030 3030 3030 3030 -- -- -- -- Texas red_probeTexas red_probe 2020 2020 2020 2020 -- -- -- --

혼합된 프라이머 및 프로브 혼합물을 이용하여, 실시간 중합연쇄 반응을 위한 Mastermix 1x와 혼합 하여 3차 증류수를 이용하여 전체 volume 50 ㎕가 되도록 한다.
Using the mixed primer and probe mixture, it is mixed with Mastermix 1x for real-time polymerization chain reaction so that the total volume is 50 µl using tertiary distilled water.

실시간 PCR 장비를 이용하여, 1) 95℃ 10분, 2)95℃ 20초, 55℃ 30초를 40번 수행하였고, 그 결과 도 4a 내지 도 4f의 그래프를 얻었다. 요약된 내용은 표 5에 나타내었다. Using a real-time PCR equipment, 1) 95 ° C 10 minutes, 2) 95 ° C 20 seconds, 55 ° C 30 seconds were performed 40 times, and as a result, the graphs of FIGS. 4A to 4F were obtained. A summary is shown in Table 5.

단일 염기 다형성 표적Single Base Polymorphic Targets 　 단일 염기 다형성 비교 일치도(%)Single Base Polymorphism Comparison Concordance (%) 염기서열 분석법 vs. 실시간 유전자 증폭Sequencing vs. sequencing Real time gene amplification CYP2C9c.430 C>T(*2)CYP2C9c.430 C> T (* 2) CC(n=80)CC (n = 80) 80 (100)80 (100) CT (n=2)CT (n = 2) 2 (100)2 (100) TT(n=0)TT (n = 0) NANA C.1075 A>C(*3)C.1075 A> C (* 3) AA (n=71)AA (n = 71) 71 (100)71 (100) AC (n=10)AC (n = 10) 10 (100)10 (100) CC (n=1)CC (n = 1) 1 (100)1 (100) VKORC1g.3673 G>AVKORC1g.3673 G> A GG(n=7)GG (n = 7) 7 (100)7 (100) GA(n=20)GA (n = 20) 20 (100)20 (100) AA(n=55)AA (n = 55) 55 (100)55 (100) g.6484 C>Tg.6484 C> T CC (n=7)CC (n = 7) 7 (100)7 (100) CT(n=20)CT (n = 20) 20 (100)20 (100) TT(n=55)TT (n = 55) 55 (100)55 (100) g.6853 G>Cg.6853 G> C GG(n=7)GG (n = 7) 7 (100)7 (100) GC(n=20)GC (n = 20) 20 (100)20 (100) CC(n=55)CC (n = 55) 55 (100)55 (100) g.9041 G>Ag.9041 G> A GG (n=59)GG (n = 59) 59 (100)59 (100) GA(n=23)GA (n = 23) 23 (100)23 (100) AA(n=0)AA (n = 0) NANA

* NA: 미확인
* NA: Unidentified

실시예Example 5: 실시간 중합효소 판별  5: Real-time polymerase determination 기준값Reference value

본 발명에서 수행한 실험 결과에서 나온 Ct 값을 기준으로 동형 접합체와 이형 접합체를 구분하는 판별 기준값을 산출하여 도 5과 같은 결과를 얻었다.A determination criterion value for distinguishing a homozygote and a heterozygote was calculated based on the Ct value obtained from the experimental results performed in the present invention to obtain a result as shown in FIG. 5.

좀 더 정확한 동형 접합자와 이형 접합자를 판별하기 위한 검출 한계값을 구하기 위하여, 민감도와 특이도를 그래프로 나타내는 Receiver Operating Characteristic (ROC) curve를 그려서 동형 접합자와 이형 접합자에 대한 판별 검출 한계 값을 구하였다. In order to find more accurate detection limits for homozygotes and heterozygotes, we calculated the detection detection limits for homozygotes and heterozygotes by drawing a Receiver Operating Characteristic (ROC) curve that shows sensitivity and specificity. .

도 5는 실험에 사용한 검체는 타겟이 되는 VKORC1- 3673A>G, 6484C>T, 6853G>C, 9041G>A, CYP2C9 - 430C>T, 1075A>C 단일 염기 다형성에 대해 동형 접합자(homo type)와 이형 접합자(hetero type)의 형광 검출값 차이인 ΔCt를 나타내는 기준값이다. 예를 들어 3673A>G의 ΔCt가 도 4a 내지 도 4f에 나타낸 ±4.1 보다 큰 값을 나타내면, 먼저 검출된 형광값에 대해 동형 접합자(homo type)를 의미하며, ΔCt가 도 4a 내지 도 4f에 나타낸 ±4.1보다 작은 값을 나타내면 이형 접합자(hetero type)를 의미한다.
FIG. 5 shows homologous conjugates (homo type) for the VKORC1-3673A> G, 6484C> T, 6853G> C, 9041G> A, CYP2C9-430C> T, 1075A> C single base polymorphisms. It is the reference value which shows (DELTA) Ct which is the difference of the fluorescence detection value of a heterotype | zygote (hetero type). For example, if ΔCt of 3673A> G represents a value greater than ± 4.1 as shown in FIGS. 4A-4F, it means a homotype for the fluorescence value detected first, and ΔCt is shown in FIGS. 4A-4F. A value less than ± 4.1 means a heterotype.

실시예Example 6: 실시간 중합효소 자동 판별 프로그램 6: Real time polymerase automatic identification program

실시예 5에서 수행한 결과를 자동으로 판별해주는 프로그램을 개발하였다. 이 프로그램은 실시간 중합효소 연쇄 반응 장치에 적용하여, 타겟 유전자의 대립형질 증폭 및 단일 염기 다형성을 판별해주는 프로그램이다.A program for automatically determining the results performed in Example 5 was developed. This program is applied to the real-time polymerase chain reaction device to determine the allele amplification and single nucleotide polymorphism of the target gene.

본 발명에 있어서 실시간 중합효소 연쇄반응 장치는 바람직하게는 바이오니아의 실시간 중합효소 연쇄 반응 장치를 사용 하였다. 자동 판별 프로그램은 실시예 4에서 정해진 판별값을 적용하였다. 판별 방법은 도 1b에 나타낸 과정을 통해 상기 각각의 대립형질의 표적 유전자에 특이적인 튜브에서 검출된 형광 신호의 Ct 값을 구하여, 그 차이를 구하여 △Ct 값을 구하였고, 상기 실시예 4에서 구해진 판별 기준값 보다 △Ct 값 이상에 존재하면, Ct 값이 작은 형광 검출 신호의 대립 형질에 대해 동형 접합자(homozygote)로 판별되며, 기준값보다 △Ct 값이 이내에 존재하면, 대립 형질에 대해 이형 접합자(heterozygote)로 판별 하도록 설계 되었다. 상기 판별 과정을 검체 자동으로 판별하도록 단일 염기 다형성 판별 프로그램을 개발하였다(도 6 및 도 7).In the present invention, the real-time polymerase chain reaction device is preferably a Bioneer real-time polymerase chain reaction device. The automatic discrimination program applied the discrimination value defined in Example 4. In the determination method, the Ct value of the fluorescent signal detected in the tube specific to the target gene of each allele was obtained through the process shown in FIG. 1B, and the difference was determined to obtain the ΔCt value. If the Ct value is greater than the discrimination reference value, it is determined as a homozygote for the allele of the fluorescence detection signal having a smaller Ct value, and if the ΔCt value is less than the reference value, the heterozygote for the allele is present. Was designed to discriminate. A single nucleotide polymorphism discrimination program was developed to automatically discriminate the discrimination process (FIGS. 6 and 7).

<110> Bioneer Corporation <120> Method of Determining Single Nucleotide Polymorphism of Target Gene Using Polymerase Chain Reaction and Kit for Determining Single Nucleotide Polymorphism of Target Gene Using the Same <130> 2011-dpa-0120 <150> KR 10-2011-0004724 <151> 2011-01-18 <160> 26 <170> KopatentIn 1.71 <210> 1 <211> 12043 <212> DNA <213> Homo sapiens <400> 1 gtgcagtggt gtgatctcgg ctcactgcaa cctctgcctc ctgggttcaa gcaattctcc 60 tgcctcagcc tcctgagtag ctgggattac aggcatgcac caccatggcc agctaatttt 120 tgtattttta gttgagaagg ggtttcacca tgttgaccag gctggtctca aactcctaac 180 cttgtgatcc acctgccttg gcctcccaaa gtgctgggat tatgggcatg agccaccaca 240 ccccacagcc ttactctttc tccctctggg catgagctag ctgagctgtg tcttggcttc 300 cccgagatgg acagctctgg ctttctctct ttctctgggt acaagcacct gcacaagagc 360 catgtcccat gtcaagccat gttgggccga gccctgtgca cagtgtcagc agggcaatta 420 taccttttac agacaacagt ggctcagagc caagtgatga gccttcccat gttatggcta 480 cacggctgtg ataacaagtg gagctatatg cctgcactct aaactcactg agtcactctg 540 gatgtttact ttggcctatg cccacttgac tgcagcacac ccacgtttct tacagggcct 600 cagttcctca ctaaggagac ctctctccct agggttactt gagtgctttc atatcatggt 660 agctggcttt gtctagaatg agtgatctac aaaggagcaa gatggaagcc acaatatctt 720 catgatttag cctctaaagt cacacactgt catttctgca ttatcccaat gggccagttc 780 agcccctttc aatgtgagaa gagacttcac aatggtatca ataccaggaa gcaggaatca 840 ctgaggacca ttttggaagc tgactattaa agccttaagg ctgccatgct gtgaggaagc 900 ccaagctagc ccatgcagag gacaacatag ataggttcgg agactactca gagaaaggga 960 gagagaatgc taggttatct ttcagctgtt ttagctgctt attgttccag ctttagccac 1020 catctgactg caattgtatg agccagaaat agccaggtaa aattgtcttc ccaaattcct 1080 agtgcacaaa aactgtcaga ggctgggggc agtggctcac tcctgtaatc ccagcagcac 1140 tttgggaagc cagggtggga gaactgcttg agcccaggag ttagaggcaa cataacaaca 1200 actcatctct aatgaaaaaa aaaaaaaaag aaagaaaaag gccaggcaca gtggctcacg 1260 cctgtaatcc cagcacttta ggaagccaag gagggcggat catgaggtca ggagtttgag 1320 accagcctgg ccaatatggt gaaaccccgt ctctactaaa aacacaaaaa tcagccacgc 1380 atagtggctg gtgcctgtaa tcacagctac tcaggaggct aaggcaggag aatcacttga 1440 atccgggagg cagaggttgc agtgagccga gattgcacca ctgcatttca gcctgggcag 1500 cagagtggga ctgtctcaca ttaattaatt aattgattaa atgctagaag aaaatatggt 1560 caaaatcttt tataactctt ggagtgagga aggcaatctc ggctcactac aacttccacc 1620 tcccgggttc aaatgattct cctgcctcag cctcctgagt agctgggatt acaggcaccc 1680 accaccatgc ccggctaatt tttgtatttt tagtagagac gaggtttcgc catgttggcc 1740 aagcttgtct taaactcctg acctcaggtg atccacccac ctcagcctcc caaattgctg 1800 ggattacagg catgagccac catgccccgc ctaattttaa aaatgtttat aaagacagag 1860 tcttaccacg ttgtccagac ctgtctcaaa ctcctgggct caagtcattg tcttgtctca 1920 gcctcccaaa gttctgggac tacaggtgtg caccaccaca cctggctaat tttgtttatt 1980 ttttatagag tgagggtctc cctgtgttgt ccagggtgtc tcaaactcct gggctcaagt 2040 ggtcctcctg cctcagcctc ccaaagtgct gggattatag gcataagcca ctgcacctgg 2100 ccccagtgtg tgtcttctga ggctaagtca taaagagatt gcggcttcta ttttgcttcc 2160 tcttagatca ctcattctgg gggaaaccaa caataaataa caggaacaca ccaggagatc 2220 agagaaagct gagatacagg cctaatgccc agtcctctgt attcatttct aattgctgct 2280 gtaacaagtt actgcacatt taatgatgta aaacaacaca aatttatctt acagttctgt 2340 agcttagaag tctgacagag tctcactggg ctgaaatcaa gatgttgata gttccttccc 2400 ggtgactcta gagtgagaat ccagttcttt accctttctt gcctttagag gccacccgta 2460 tttattagct cacaatcccc ttcctccatc ttcaaaccag aaacattgca gctctctgtg 2520 tcttttttct ttactcacat ctccctctga ctttcttctg ccatcctcct tcatttttta 2580 aggacccctg tgggccaggc atggtggctt atgcctgtaa tcccagcact ttgagaggcg 2640 gaggcaggcg ggtcacctga ggtcaggagt tccagaccag cctggccaac atggcaaaac 2700 cccatctcca ctgaaaatac aaaaattggg ccaggcacgg cggctcacac ccgtaatccc 2760 catactttga gaggctgagg caggtggatc acttgaggtc aggagttcaa gaccagcctg 2820 gccaacatgg tgaaatcccg tctctactaa aaaaaaaaaa aattacaata attagtcagg 2880 cgtggtggcc agcacctata atcccagcta cttgggaggt tgaggcacaa gaattgcttg 2940 aacccgagag ggggaggttg cagtgagctg agattgcacc accgtactcc agcctggatg 3000 acagagcaag actctgtctc aaaataaaaa ttaagataaa taataaaaaa taaataaaaa 3060 tcagcaaatt aggccaggtg tggtggtggc tcatgcctgt actcccagca ctttgaggtg 3120 ggaaggttgc ttgaaaccag aagtttagac cagcctggga aacaaagtga gaccccatct 3180 ctacaaaaat aaaaataaat tagccaggtg tggtgggacg tacttgtact cctagctact 3240 agggaggctg aggtgcaaga atcgcttgaa cctgggagac ggaggttgca gtgagttatg 3300 acagtgccac tacactccag cctgggtgtc aagggtcttt gaaaaaataa acaaaatagg 3360 ctgggcgtgg tggcttacac ttgtaatccc agcactttgg gaggctgagg caggcagatc 3420 acaaagtcag gagttcgaga ccagcctggc caacacagtg aaaccccgtc tctactaaaa 3480 atacaaaaat tagccaggca tggttgcacg cgcctgtagt cccaactgct cgggaggctg 3540 aggcaggaga atggcttgaa cctggggggg ggcggaagtt ataatgagcc aagattgtgc 3600 cactgcactc cagcctaggc aacagagcga gactccgtct caaaaataaa aataaaaata 3660 aatcaataga gcctggtatg atggctcacg cctataatcc cagcactttg ggaggcccag 3720 gtgggtggat catctgaggt caggagtttg agaccagcct gaccaacatg gagaaacccc 3780 atctctacta aaaatacaaa aaattagcca ggcgtggtgg cacatgccta taatcccagc 3840 tactcaggag cctgaggcag gagaatcgct ttaaccgggg aggtggaggt tgcggtgagt 3900 cgagatagca ccattgcatt ctagcctggg caacaagagc aaaactccat ctcaaaaata 3960 aataaataaa tagatagata ggctgggcac ggtggctcac gcctgtaatc ccagcacttt 4020 gggaggccga ggtgggcgga tcacctgagg tcaggagttc aagaccagcc tggccaatat 4080 agcgaaaccc tgtctctact aaaaatttaa aaaattagcc aggcatgatg gcgggtgcct 4140 gtaatcccag ctactcggga ggctgaggca ggagaatctc tcgaacctgg caggcggagg 4200 ttgcagtgag ccgagatcac accattgcac tccagttggg caacaagagc gaaactccac 4260 ctcaaaaaca aaataaaata aaataaaatc aggagattgg tcagcttaat tccatctgca 4320 accttaattc ccttttggcc atttaaccta tcatattcac aagttccagg gattcatgca 4380 gggacatctt tggtgaccat tattctgtct accacactct ctagaagaga gtagatgtga 4440 gaaacagcat ctggagaggg aggagccagc aggagaggga aatatcacag acgccagagg 4500 aagagagttc ccagaagggt aggtgcaaca gtaagggatc cctctgggaa gtcaagcaag 4560 agaagacctg aaaaacaacc attggccggg tgcggtggct cacgcctata atcctagcat 4620 tttgggaggc cgaggtgggt ggatcacttg aggtcaggag tttaagacaa gcctggccaa 4680 catggtgaaa ccctgtctct actaaaaata caaaaattag ccagacatgg tggcaggcac 4740 ctgtagtcca aactacttgg gaggctgagg cacgagaacc acttgaaccc tggaggcgga 4800 agttgcagtg agccgagatg gcaccgctgc actccagcct gggcgacaga gtgagactcc 4860 gtctcaaaat aagtaaataa ataaataaat aaccattggg tctagcgtct tggtgacacc 4920 agccactagg tgtcagggca atgctggagg cagcagctag tttatggcgt gggcagcaag 4980 gaagaagtga ggaagtagag actgggagtg tagaatcttt ccaaaaactt gaatgtggag 5040 gggtagatgg gataatgccg ggagaggagg ctggtgaagg acctcttagt gaagggagat 5100 ttgttgctgt ttctgagatg ggaaagagaa acaagttgca gtgtagatgg ggaggatgga 5160 gaggttgaag gtgtaggaga gatgggtcac catcagatgg gacgtctgtg aaggagagac 5220 ctcatctggc ccacagcttg gaaaggagag actgactgtt gagttgatgc aagctcaggt 5280 gttgccaggc gggcgccatg atagtagaga ggttaggata ctgtcaaggg tgtgtgtggc 5340 caaaggagtg gttctgtgaa tgtatgggag aaagggagac cgaccaccag gaagcactgg 5400 tgaggcagga cccgggagga tgggaggctg cagcccgaat ggtgcctgaa atagtttcag 5460 gggaaatgct tggttcccga atcggatcgc cgtattcgct ggatcccctg atccgctggt 5520 ctctaggtcc cggatgctgc aattcttaca acaggacttg gcatagggta agcgcaaatg 5580 ctgttaacca cactaacaca cttttttttt tctttttttt ttttgagaca gagtctcact 5640 ctgtcggcct ggctggagtg cagtggcacg atctcggctc actgcaacct ccggctcccc 5700 ggctcaagca attctcctgc ctcagcctcc cgagtagctg ggattacagg catgtgccac 5760 cacgcccggc taatttttgt atttttagtt gagatggggt ttcaccatgt tggcgaggct 5820 ggtcttgaac tcctgacctc aggtaatccg ccagcctcgg cctcccaaag tgctgggatt 5880 acaagcgtga gccaccgtgc ccggccaaca gtttttaaat ctgtggagac ttcatttccc 5940 ttgatgcctt gcagccgcgc cgactacaac tcccatcatg cctggcagcc gctggggccg 6000 cgattccgca cgtcccttac ccgcttcact agtcccggca ttcttcgctg ttttcctaac 6060 tcgcccgctt gactagcgcc ctggaacagc catttgggtc gtggagtgcg agcacggccg 6120 gccaatcgcc gagtcagagg gccaggaggg gcgcggccat tcgccgcccg gcccctgctc 6180 cgtggctggt tttctccgcg ggcgcctcgg gcggaacctg gagataatgg gcagcacctg 6240 ggggagccct ggctgggtgc ggctcgctct ttgcctgacg ggcttagtgc tctcgctcta 6300 cgcgctgcac gtgaaggcgg cgcgcgcccg ggaccgggat taccgcgcgc tctgcgacgt 6360 gggcaccgcc atcagctgtt cgcgcgtctt ctcctccagg tgtgcacggg agtgggaggc 6420 gtggggcctc ggagcagggc ggccaggatg ccagatgatt attctggagt ctgggatcgg 6480 tgtgcccggg gaacggacac ggggctggac tgctcgcggg gtcgttgcac aggggctgag 6540 ctacccagcg atactggtgt tcgaaataag agtgcgaggc aagggaccag acagtgctgg 6600 ggactgggat tattccgggg actcgcacgt gaattggatg ccaaggaata acggtgacca 6660 ggaaaggcgg ggaggcagga tggcggtaga gattgacgat ggtctcaagg acggcgcgca 6720 ggtgaagggg ggtgttggcg atggctgcgc ccaggaacaa ggtggcccgg tctggctgtg 6780 cgtgatggcc aggcgttagc ataatgacgg aatacagagg aggcgagtga gtggccaggg 6840 agctggagat tctggggtcc agggcaaaga taatctgccc ccgactccca gtctctgatg 6900 caaaaccgag tgaaccgtta taccagcctt gccattttaa gaattactta agggccgggc 6960 gcggtggccc actcctgtaa tcccagcact ttgggaggcc gaggcggatg gatcacttga 7020 agtcaggagt tgaccagcct ggccaacatg gtgaaagcct gtctctacca aaaatagaaa 7080 aattaatcgg gcgctatggc gggtgcctta atcccagcta ctcggggggg ctaaggcagg 7140 agaatcgctt gaacccggga ggcggaggtt tcagtgagcc gagatcgcgc cactgcactc 7200 cagcctgggc cagagtgaga ctccgtctca aaaaaaaaaa aaaaaaaaaa aaaaaagaga 7260 cttacttaag gtctaagatg aaaagcaggg cctacggagt agccacgtcc gggcctggtc 7320 tggggagagg ggaggatagg gtcagtgaca tggaatcctg acgtggccaa aggtgcccgg 7380 tgccaggaga tcatcgaccc ttggactagg atgggaggtc ggggaacaga ggatagccca 7440 ggtggcttct tggaaatcac ctttctcggg cagggtccaa ggcactgggt tgacagtcct 7500 aacctggttc caccccaccc cacccctctg ccaggtgggg caggggtttc gggctggtgg 7560 agcatgtgct gggacaggac agcatcctca atcaatccaa cagcatattc ggttgcatct 7620 tctacacact acagctattg ttaggtgagt ggctccgccc cctccctgcc cgccccgccc 7680 cgcccctcat cccccttggt cagctcagcc ccactccatg caatcttggt gatccacaca 7740 gctgacagcc agctagctgc tcatcacgga gcgtcctgcg ggtggggatg tggggaggta 7800 actaacagga gtcttttaat tggtttaagt actgttagag gctgaagggc ccttaaagac 7860 atcctaggtc cccaggtttt ttgtttgttg ttgttttgag acagggtctg gctctgttgc 7920 ccaaagtgag gtctaggatg cccttagtgt gcactggcgt gatctcagtt catggcaacc 7980 tctgcctccc tgcccaaggg atcctcccac cttagcctcc caagcagctg gaatcacagg 8040 cgtgcaccac tatgcccagc taatttttgt ttttgttttt ttttggtaga gatggtgtct 8100 cgccatgttg cccaggctgg tctcaagcaa tctgtctgcc tcagcctccc aaagtgctgg 8160 ggggattaca ggcgtgagct accatgcccc accaacaccc cagttttgtg gaaaagatgc 8220 cgaaattcct ttttaaggag aagctgagca tgagctatct tttgtctcat ttagtgctca 8280 gcaggaaaat ttgtatctag tcccataaga acagagagag gaaccaaggg agtggaagac 8340 gatggcgccc caggccttgc tgatgccata tgccggagat gagactatcc attaccaccc 8400 ttcccagcag gctcccacgc tccctttgag tcacccttcc cagctccaga gaaggcatca 8460 ctgagggagg cccagcacca tggtcctggc tgacacatgg ttcagacttg gccgatttat 8520 ttaagaaatt ttattgctca gaactttccc tccctgggca atggcaagag cttcagagac 8580 cagtcccttg gaggggacct gttgaagcct tctttttttt tttttttaag aaataatctt 8640 gctctgttgc ccaggctgga gtgcagtggc acaatcatag ctcactgtaa cctggctcaa 8700 gcgatcctcc tgagtagcta ggactatagg catgtcactg cacccagcta attttttttt 8760 tttttttttt tttttttttg cgacatagtc tcgctctgtc accaggctgg agtgcagtgg 8820 cacgatcttg gctcactgca acctctgcct cccgggttca agcaattttc ctgcctcagc 8880 ctcctgagta gctgggacta caggcgcgtg tcaccacgcc cagctaattt ttgtattttt 8940 agtggagaca gggtttcacc atgttggcta ggatggtctc aatctcttga cctggtgatc 9000 catccgcctt ggcctcccaa agtgctagga ttacaggcgt gagtcaacct caccgggcat 9060 tttttttttg agacgaagtc ttgctcttgc tgcccaagct ggaatgtggt ggcatgatct 9120 cggctcactg caacctccac ctcctaggtt caagcgattc tccaccttag cctccccagc 9180 agctgggatt acaggtgccc atcaacacac ccggctaatt tttgtatttt tattagagat 9240 ggggttttgc catgttggcc aggctgctct cgaactccta acctcaggtg atccaccccc 9300 attggcctcc caaaatactg ggattacagg catgagccac cgtgcccagc tgaatttcta 9360 aatttttgat agagatcggg tctttctatg ttgcccaagc tggtcttgaa ctcctagcct 9420 aaagcagtct tcccacctcg gcctcccaga gtgtttggaa tacgtgcgta agccaccaca 9480 tctgccctgg agcctcttgt tttagagacc cttcccagca gctcctggca tctaggtagt 9540 gcagtgacat catggagtgt tcgggaggtg gccagtgcct gaagcccaca ccggaccctc 9600 ttctgccttg caggttgcct gcggacacgc tgggcctctg tcctgatgct gctgagctcc 9660 ctggtgtctc tcgctggttc tgtctacctg gcctggatcc tgttcttcgt gctctatgat 9720 ttctgcattg tttgtatcac cacctatgct atcaacgtga gcctgatgtg gctcagtttc 9780 cggaaggtcc aagaacccca gggcaaggct aagaggcact gagccctcaa cccaagccag 9840 gctgacctca tctgctttgc tttggcatgt gagccttgcc taagggggca tatctgggtc 9900 cctagaaggc cctagatgtg gggcttctag attaccccct cctcctgcca tacccgcaca 9960 tgacaatgga ccaaatgtgc cacacgctcg ctctttttta cacccagtgc ctctgactct 10020 gtccccatgg gctggtctcc aaagctcttt ccattgccca gggagggaag gttctgagca 10080 ataaagtttc ttagatcaat cagccaagtc tgaaccatgt gtctgccatg gactgtggtg 10140 ctgggcctcc ctcggtgttg ccttctctgg agctgggaag ggtgagtcag agggagagtg 10200 gagggcctgc tgggaagggt ggttatgggt agtctcatct ccagtgtgtg gagtcagcaa 10260 ggcctggggc accattggcc cccaccccca ggaaacaggc tggcagctcg ctcctgctgc 10320 ccacaggagc caggcctcct ctcctgggaa ggctgagcac acacctggaa gggcaggctg 10380 cccttctggt tctgtaaatg cttgctggga agttcttcct tgagtttaac tttaacccct 10440 ccagttgcct tatcgaccat tccaagccag tattggtagc cttggagggt cagggccagg 10500 ttgtgaaggt ttttgttttg cctattatgc cctgaccact tacctacatg ccaagcactg 10560 tttaagaact tgtgttggca gggtgcagtg gctcacacct gtaatccctg tactttggga 10620 ggccaaggca ggaggatcac ttgaggccag gagttccaga ccagcctggg caaaatagtg 10680 agacccctgt ctctacaaaa aaaaaaaaaa aaaaaaatta gccaggcatg gtggtgtatg 10740 tacctatagt cccaactaat cgggaagctg gcgggaagac tgcttgagcc cagaaggttg 10800 aggctgcagt gagccatgat cactgcactc cagcctgagc aacagagcaa gaccgtctcc 10860 aaaaaaaaac aaaaaacaaa aaaaaacttg tgttaacgtg ttaaactcgt ttaatcttta 10920 cagtgattta tgaggtgggt actattatta tccctatctt gatgataggg acagagtggc 10980 taattagtat gcctgagatc acacagctac tgcaggaggc tctcaggatt tgaatccacc 11040 tggtccatct ggctccagca tctatatgct tttttttttg ttggtttgtt tttgagacgg 11100 actttcgctc ttgttgtcca ggctggagtg caatggcaca atttcggctc accacaacct 11160 ccacctccca ggttcaagtg attctcctgc ctcagcctcc cgagtagctg ggattacagg 11220 catgcgccac caggcctggc taattttgta tttttagtag agacagtgtt tctctatgtt 11280 ggtcaggctg gtctcgcact cccgacctca ggtgatctgc ctgctttggc ctcccaaagt 11340 gctgggatta caggcgtgag ccaccatgcc cggccagcat ctataatata tgctaaacct 11400 tacccacagc tgcctctgtc ttggggtcag ggttcttgaa tggtctgggc cagcctgagt 11460 taggagtggg aagaggaaga ggaggcacgc gccaaagtgt tcttcccgcc tgaccgagtc 11520 ctctgcgcct atgaggaagg tgggcaccgc tgtcaccctg tttcccagga agccagcagg 11580 cccagggaga tactgggatt tgccaaaggc tcacagcgag caagcagcag tgggtgctgg 11640 ctcctcacca ctcagcctgg cacacactgt ctctgagcag acctgggaga aggcatgtga 11700 aacccaccta gccctggctg gaagagaagt gggggagtgg aaatgaaaag tggggactgg 11760 gaggagctgc ccacgtgccc tccatggttc caggatggca ctttcatgct gggagtgagc 11820 ttgccctgtg ccaggctgtc attgagacgt tttccattca acaactgttt ctcagacaag 11880 gcacaggcag gttagctctc ccaggccact caagtagtaa ggaaacagat ttaaacccag 11940 acaatttggc tccacggcta aggcccagag aggggtggtg acaagcctag ggtcacacag 12000 ccacagcccc agagctagga actgctggct agggcaagca gac 12043 <210> 2 <211> 50734 <212> DNA <213> Homo sapiens <400> 2 gtcttaacaa gaagagaagg cttcaatgga ttctcttgtg gtccttgtgc tctgtctctc 60 atgtttgctt ctcctttcac tctggagaca gagctctggg agaggaaaac tccctcctgg 120 ccccactcct ctcccagtga ttggaaatat cctacagata ggtattaagg acatcagcaa 180 atccttaacc aatgtaagta tgctccttca gtggcttgca aaaggtaagt aaattcacct 240 gtatttttta aataaagtgt atccctagag gtacatgtta caagaggtaa tggtaaagta 300 aaatactttg aaaggctttt gttgcctttt ccagtctgtc agtgtcagaa atagtggaat 360 gaaaccatgt attttgtgag tagagaaaga tttgggtctt tgcatgttag attcaaaata 420 acaagtgtca atagtttgaa aagctgtgtt ccttcttcat ttcataacca tttgctataa 480 tttttggctg aaggtaaatg gtaaggtatt gtgggatctg gtcagcagcc cacaaagcaa 540 ctgggctctc tcttttttcc caggtggatc ggcaggttga gaaataatag acacacaaga 600 tagtgaaagc tgggtccagg ggggtcaccg ccttctggtc ccatggtgcc aagaatgcac 660 tggatatacc agcatttatt attaagttta gtgagggcag gggtaggtta gtgagggatt 720 tagggtcatt tgattatgag gtgagatggt cacatgggga tgaagtaatt ctttaacata 780 acatttgtat gtagaagtac agtacatttg tatgtagaag tacagtatac ggagataaga 840 atttacaata tagtgtgtgc gtcagtaatt tctaacagag ccttaaaaca gaaacacaat 900 ctttccataa cctatgatta gcaagatatt aatcagcagt aacaattgca acaaaagctg 960 gttacaaaca atccatggaa ataggatgtg aagctagaca accagttaga ccagaaattc 1020 tcagaaggga gtatgcctta accctaaaga ggcctagaag agccatggca agatgagggc 1080 atttatagcc ctatcttatc catatggaca ggtgcccccc atgcgtccat ttataggttc 1140 tccacaaagg tcgcattcca ttcccagagc tatgaacatc tgcttttctg ggataggaat 1200 cttggtgatg tgaaacctct ctgactgcac gtccattcat aggctctctg cagggggaag 1260 cacatcacgc gctgttggct cattctggca gtccagcctg gcactgtcct tacacaatcc 1320 tgcatgcaat tttgtattta caataatcgg gagcatttca tcttttattc cgtagcaata 1380 gtttcagggg gggtctccct acagtaagga acatgtttgt tattagagat tttattaaat 1440 aagtcctcta ctatattagc catgtgtttt attcagaata gccatgaaaa ttaaacttct 1500 ctgaagaatt aatttatgcc tgttgtaaag gaaataactg tgggttcagg accagcaaaa 1560 gcagataagt ggtagaaaag aagttataga cttccatttt tagatccaca aagtactgtt 1620 ttaattatac tatagctaca atatataaac cccaaaatga ccttagcatt tagtaatggt 1680 aggaagaagg tcagaagatg tataagtatt agggtttttt ctcaagtgaa agatggttta 1740 gcagtgttaa tatgatatgt gaacacccct cgttgtgtaa taattaccat atacagagag 1800 taaagggctg caattagtat attaagtctt ataagcatga tggtatgatt agatcaagag 1860 aatgacgctg tagttatgaa gactactcct actagattga tggtaggggg cagggcaagg 1920 ttggcaagac tagctaggag tcatcacatg gctatcaggg agagaagtgt ttgaaggcct 1980 tgtgttagta gtatggtttg gctgtgaact cgtttgtagt ttgagtttgc aaggcagaat 2040 aatagggatg aggttagtct gtgggcagtt attagggaag ttgcactggt aaaacttcag 2100 ggggtttgaa tgagaaaagc tacgataaca agtgccatgt ggcttatgga aaagtaatga 2160 gtgattttag atcagtttgg cacagacaaa aggagcttgt tatgattatt tctcaaaagg 2220 atagtaagaa ggaggaataa gctataaact ctgttagggg gttaagaatt aaggtgattt 2280 gtataatcct atagccatct aattttagga gtactgctgt gaggactatt gacctggcaa 2340 ttgcggcctc tatgtgggct tttgggagtc ctaggtggag tccatagaga gatattttta 2400 gtataaatgc tatgatgcat gctaatcata aaaggctatt aaaccaggaa attgttagtt 2460 cttgaagctg ggtattggtc ttaaggtaga ctcgaaagtg ctgataaatt tctcaagcat 2520 cagtgtttga ataagcggag tttcaaattt tggtctgctg tacattagct gtgagacact 2580 gaaaatgaat ttgtcattct ctgagctcag tttttttttt tttttttttt ttttttgaga 2640 cagagtctta ctctgtagct caggctggag tgcagtggta caatcttggc tcactgcaac 2700 ctccatctcc caggtcccca ttcaagaaat tctcctgcct cagtccccca agtagctagc 2760 attacaggca tgcaccacca tgctcagcta atttttgtat ttttagtaga gacgtggtat 2820 caccttgttg gccaggctgg tcttgaactc ctgaccttgt gatccacctg ccttggcctc 2880 ccaaagtgtt gggattacag gcaggagcca ccacacctgg ccgtttgttt aaaatagagt 2940 aaatagacct gctgaatatg ttgatgtgag tattaattgt aatctgcata gcaattgtct 3000 gaccattgcc ttgaacatca caggccatct gagtggcaag tataatcatc atcatgtttc 3060 tatttaaaat tcagaaatat ttgaagcctg tgtggctgaa taaaagcata caaatacaat 3120 gaaaatatca tgctaaatca ggcttagcaa atggacaaaa tagtaacttc gtttgctgtt 3180 atctctgtct actttcctag ctctcaaagg tctatggccc tgtgttcact ctgtattttg 3240 gcctgaaacc catagtggtg ctgcatggat atgaagcagt gaaggaagcc ctgattgatc 3300 ttggagagga gttttctgga agaggcattt tcccactggc tgaaagagct aacagaggat 3360 ttggtaggtg tgcatgtgcc tgtttcagca tctgtcttgg ggatggggag gatggaaaac 3420 agagacttac agagctcctc gggcagagct tggcccatcc acatggctgc ccagtgtcag 3480 cttcctcttt cttgcctggg atctccctcc tagtttcgtt tctcttcctg ttaggaattg 3540 ttttcagcaa tggaaagaaa tggaaggaga tccggcgttt ctccctcatg acgctgcgga 3600 attttgggat ggggaagagg agcattgagg accgtgttca agaggaagcc cgctgccttg 3660 tggaggagtt gagaaaaacc aagggtgggt gaccctactc catatcactg accttactgg 3720 actactatct tctctactga cattcttgga aacatttcag gggtggccat atctttcatt 3780 atgagtcctg gttgttagct catgtgaagc gggggtttga agctgagagc caagggaatt 3840 tgcacatatt tgtgctgtgt gtgtacaggc atgattgtgc gtacagtgtg ggtataaaag 3900 gttcatttaa tcccatgttc tcctgaactt tgcttttttg ctttcaaata agaaatgatg 3960 aatatagatt ttgagttcat tttttgaaag agttaaagag cagtgttttt cccattacct 4020 attccagaac atgtcaccag agaatacttg acaagtcaac atggtgggaa tggccctatc 4080 atacccatat ggagcatgaa ccaaatggca tgtgctttta tttaattgga ctgtgtttgt 4140 atggtcagcc tcactgactt ctctggggtt tcttttaggc ccgtgcttgc cattctggcc 4200 agtaatgaca ttctacagtt tttattgctt aggcatatct tagtgcagtt ctcatcaatt 4260 attatttctc tgtaaacaca gcattatttt aaaaatagta ttaattattt cttgttactg 4320 tattgattta tatattttca gtaaatacat cctgtagcat aattctgtga aatacccaaa 4380 tgtcaattta taaaatgatt tatttaacaa gattttactt attagtaata actctgtaat 4440 ctgcattccc tatgtatgat ttggctctgt ttcagttttg cttatctctt tccaaccata 4500 tttatgaaat tttggcttag aaatttatgt taattatttt ttttccatgg ccaactctac 4560 tcatctatga agttttacaa tgaatctgtt tatcagcttg gataccaaat taccttgttt 4620 ttaaattctg ttttcccaat gaagttaaag actgaaaatc agatttatct gtgaatgaca 4680 cacacaaact aacagatttc caactgttca atgcctggcc attcattcag agtacttttg 4740 attaaagtca ctatttaggg cctatagata tcaggggaaa ttcagcccct gatatttcaa 4800 tgtgagtcct tttctattct ccctaagtgt tggctggtct gagaaataaa gggaaagagt 4860 acaaaagaga gaaattttaa agctgggtgt ccaggggaga catcacatgt cggcaggttc 4920 cgtgatgccc cctaaaccac aaaaccagca agttttcatt agtgattttc aaaagcggaa 4980 ggaatgtgtg aatagggtgt gagtcacaga gatcacatgc ttcacaaggt aataaaatat 5040 cacaaggcaa atggaggcag ggtgagatca caggaccgga gtgaaattaa aattgctaat 5100 gaagtttcag gcactcattg tcattgataa catctcatca ggagacaggg tttgagagaa 5160 gacaaccagt ctgaccaaaa tttattaggc aggaatttcc tcttcctaat aagcctggga 5220 gcactatggg agaccaggcc ttatttcatc ccttatctac aactgtaaaa gacagacgtt 5280 ctcaaaacag ccattttaga gaccacccct tgggaatgca ttcttctcag ggatgttcct 5340 tgctgagaaa aagaattcag tgatatttct cctacttgct tttgaaagaa gtgaaatatg 5400 gctctgttct gctcagccca caggcagcca gacttcaagg ttatctcact tgttccctga 5460 acatcgctgt tatcctgttt tttttttcaa ggtgcagatt tcatattgtt taaacaattt 5520 gtgcagttaa cacaatcatc acagggtcct gaggtgacat tcgtcctcag cttaggaaga 5580 tgatggtatt aagagattaa agtaaagaca ggcataggaa atcacaagag tattgattgg 5640 ggaagtggta agtgtccatg aaattttcac aatttatgtt cagagattgc agtaaagaca 5700 ggtgtaagaa attataaaag aatgaatttg gggaactaat aaatgtctgt gaaatcttca 5760 caatttatct tcttctgcca tggcttcagc tggtccctcc gttcggggtc cctgacttcc 5820 tgaaacatat agatgtgaat ttgggagctc tttaactata aagtttaata tctcaaaata 5880 ataagagcta tttatgacaa acccatagcc aatatcatat tgaatgggca aaagctggaa 5940 gcattccctt tgaaaaccag cacaagacaa ggatgccctc tctcaccact cttattcaac 6000 atagtattgg aagtcctggc cagagcaatc agtcaagggg gtattcaaat gggaagagaa 6060 gaagtcaaat tgtctctgtt tgcaggtgac atgactgtat acttagaaac cccatcatct 6120 cagccccaaa actgtttaag ctgataagca atttcagcaa gacctcagga tacaaaatca 6180 atgtgcaaaa ataacaagca ttcctataaa ccaataatag acaagcagag agccaaatca 6240 tgagtggact cccattcaca attgctacaa agggaataaa atgcctactt acacaactta 6300 caagcgatgt gaaggacctc ttcaaggaga actacaaacc acttctcaag gaaataagag 6360 gtgacacaaa tggaaaaaaa ttccgtgctc atgaatagaa agaatcaata ctgtgaaaat 6420 agccatactg cccaaagtaa ttcatagatt caatgctata cccgtcaaac tatcattgac 6480 tttcttcaca gaactagaaa agaataattt aaatttcata tgaagcaaaa aaagagcctg 6540 tatagccaag acaatcctaa gcaaaaacaa agctggaggc atcattctac ctgacttcaa 6600 acaatactac aaggctacag taaccaaaca gcatggtact gggaaaactg gctagccata 6660 tgcagaaaac agaaactgga ccctttcctt acaccttata caaaaattaa ctcaagatgg 6720 attaaacact taaacataaa accaaaaacc ataaaaaccc cagaagaaaa cctaggcaat 6780 accattcagg acataggcat gggccaagag tttgtgacta aaacacaaaa agcaatggca 6840 acaaaagcca aaattgacaa atgggatcta attaaactac agtgcttctg cacagccaaa 6900 gaaactgcat cagagtcaac aggcaaccta cagaatggga gaaatttttt gcaatctatc 6960 catctgacaa agggctaata tccagaatcc acaaagaact taaatttaca agaaaaaaag 7020 aaatatcccc atcaaaaatg gacaaaggat atgaacagat gcttctcaaa agaagacatt 7080 tatgcagcca acaaacatat ggaaaaaagc ttatcaacac tggtcattag agaatgcaaa 7140 tcaaaactac aatgagatac catctcatgt cagttagaat ggtgatcctt actaagtcag 7200 gaaacaacag gtgttggtga gaatgttgag aaatatgaac cctcttacac tgttggtagg 7260 agtgtaaatt agttcaacca ttgtggaaga cagtgtggtg attccttaag gatctagaac 7320 tagaaattcc acttgactca gcaatcccat tactgggtat atgcccaaag gattataaac 7380 cattctacta taatgacaca cgcccatgta tgtttatgtg gcaccgttca caatagcaaa 7440 gacttggaac caacccaaat gcccatcaat gatgtactgg ataaagaaaa tgtggcacat 7500 atacaccatg gaatactaca cagccataaa aaaggatgag ttcatgtcct ttgcaggaac 7560 atggatgaag ctggaaatca tcatcctcag caactaacac aggaacagaa aaccaaactc 7620 cacatgtttg cactcataaa tgggagttga acaatgagaa cacatggaca cagggagggg 7680 aacatcacac atgggacctg ttggggcacg gggggccagc agggggacag cattaggaca 7740 aatacctaat gcgtgtgggg cttaaaacct agatgacagg ttcataggtg cagcaaacca 7800 ccatggcaca tgtatacctc tgtaacaaac ctgcacgctc tgcacatgta tcctggaact 7860 taaagtaaaa taaaagaaaa tttaaaaaat ataaagttta tcgatcactt gattataaca 7920 acttcatttc agagatcctc caccaaacaa gaaaactttt cctataattt aattcattta 7980 gctattaaat gatacgctgg gcctaatcaa tatcaaatta aataggttaa atttgcagga 8040 tgattagaga tgagggcatt tagttcctaa agaagaaaat tcattttaaa aaaggtacag 8100 aagaaaaaaa ctccacttat caatttacac tttcttgttt gccttacctg ggatttatat 8160 ttaaaataaa agtttttggc caggcgcggt ggctcatgcc tgtaatccca caactttggg 8220 aggccaaggc tggtggatca tctgaggttg ggagttcgag accagcctga ccaacatgga 8280 ggaacctgtc tctactaaat atacaaaatt agctggatgt ggtggcacat gcctgtaatc 8340 ccagctacct gggaagctca ggtggagaat cacttgaacc taggaagcag aggttgtggt 8400 gagctgagat cgtgccatta cactccagcc caggcaacaa gagtgaaact ccatctcaaa 8460 acaaacaaat aaacaaaaaa caacaacaac aaaaaacaac aacaaagttt tcatttaatt 8520 ttttaacaaa ctaatctatg ttaatttgat attagtttgt taacataaca aactaataat 8580 atgttaggca tattattcat ttatatttgt tttccttctc ataaaacaca ttctgctctt 8640 acatttattt tcttaaatac atgaattcac caacagtctc tttggttcct ctctactggt 8700 tcaacacatg actctttcag ctattcatta tatataaaaa gctttgaaat ctccaactat 8760 tcttgccctt tccatctcag tgccttgctg tctactgact ttgcagactg atgtgattcc 8820 ctctgaaaca tgaattatta ggtttttaga aaatgccttt ttgttctttc caaagtaaaa 8880 gacaaatagg ctgggaatgt aaatttagca tttgaacaac cattatttaa ccagctaggt 8940 tgtaatggtc aactcaggat taatgtaaaa gtgaagtgtt gattttatgc atgccgaact 9000 cttttttgct gttaagggaa tttgtaggta agataatttc taaactacta ttatctgtta 9060 acaaatacag tgttttatat ctaaagttta atagtatttt aaattgtttc taattattta 9120 gcctcaccct gtgatcccac tttcatcctg ggctgtgctc cctgcaatgt gatctgctcc 9180 attattttcc ataaacgttt tgattataaa gatcagcaat ttcttaactt aatggaaaag 9240 ttgaatgaaa acatcaagat tttgagcagc ccctggatcc aggtaaggcc aagatttttg 9300 cttcctgaga aaccacttat tctctttttt ttctgacaaa tccaaaattc tacatggatc 9360 aagctctgaa gtgcattttt gaatactaca gtcttgccca gacagccttg gggtgaatat 9420 ctgggaaaga cggccaagcc ctttatttta tgcatgggaa ataaatgcct caatataggc 9480 ctgatttcta agcccattag ctccctcatc aatgtttttt ctgctaaact ccaaagccct 9540 gtttctgtaa agtactttgt tgacagccct aaagcgtgct catagcactc catggatatc 9600 caggcacttt ggaggctccc attactcaca agacttgtcc ttcaattaac actttgtcgt 9660 attatgtggc agaaatatcc taatttaaaa gacattatct ccttctagta gggagaatat 9720 ttgggaccat agaagctgcc aagaaacact gaatagggca ggggtgtttg atatctcagt 9780 tgggatccta gctgatgaga tagctgggtt aggaatgaca aaattattgg ttttatggtg 9840 tatgaaccat aaacagacat cacacttata ccctgtgctg agctggcatg ttttattctc 9900 tgcctaaata ataattgtgt gattttatag aagtcattta actgctctgg tgcacagttg 9960 gaatttgaag tatctttgag cccctcccac ttctaaaata ctaattccat ttcagaggct 10020 gcttgataga aatcaatata gtagggacta gctttgtact atcaatcagg ttgtccaaat 10080 tcttttaacc tatgctgtca tctacaaaac gtgaatgtag taattcatgc catcttatat 10140 ttcaagatta tagagaagaa ttgtaaaaag taacagaatt aacataaaga tgcttttata 10200 ctataaaaag gaggtctgag tctaggaaat gattatcatc tggttagaat tgatcctctg 10260 gtcagaattt tctttctcaa atcttttata atcagagaat tactacatat gtacaataaa 10320 aatttcccca tcaagatata caatatattt tatttatatt tatagctgta atttacaacc 10380 agagcttggt atatggtatg tatgctttta ttaaaatctt ttaatttaat aaattattgt 10440 tttctcttag atctgcaata atttttctcc tatcattgat tacttcccgg gaactcacaa 10500 caaattactt aaaaacgttg cttttatgaa aagttatatt ttggaaaaag taaaagaaca 10560 ccaagaatca atggacatga acaaccctca ggactttatt gattgcttcc tgatgaaaat 10620 ggagaaggta aaatgtaaac aaaagcttag ttatgtgact gcttgtgaat ttgtgatttg 10680 ttgactagtt ctgtgtttac taaggatgtt taactggtca atcagtaatg cttgagaagc 10740 actttaagtt tttattgtat gaatgaataa caaaatagac cactagctag actaataaag 10800 aagaaaaaag agaagaatca aatagacaca ataaaaatga taaaggggag atcaccactg 10860 agcccacaga aatataaact accatcagag aatagtataa acacctctat gctaataaac 10920 tagaaaatct agaagaaatg gataaattca tggacacata cactctccca agactaaaca 10980 aggaagaagt caaatccctg aatagaccaa taacaagttc tgaagttgag gcggtagtta 11040 atagcctaac aaccaaaaaa agtccaggac cagatggatt cacagccaaa ttctaccagg 11100 ggcacaaaga ggagctgaca ctattccttc tgaaactatt ccaaataata gaaaaagagg 11160 gactcctcct taactcattt tatgaggcta gcatcatcct gatacccaaa ccgggcagag 11220 acataacaga aaaagaacat ttcaggccaa tatccctgat gaacaccaat gcaaaaatcc 11280 acaacaaaat actgccaaac caaatccagc agcacatcaa aaagcttatc caccatgatc 11340 aagtagcctt catccctgag atgcaaggct gtttcaatat ttgcaaatca ataaatgtaa 11400 tccatcacat aaaaagaacc aatgacaaaa accacatgat tgtctcaata gatgcagaaa 11460 aggcctttga taaagttcaa tacccttcat gctaagaatt ctcaataaac taagtaatga 11520 tggaacatat ctcaaaatag taagagctgt ttatgacaaa cccacagcca atatcatatt 11580 gaatgggcag aagctggaag gattcccttt gaaaaccagc acaagacaag gatgccctct 11640 ctcaccactc ctattcaaca tagtattgga agctctggcc agggcagtca ggcaagagaa 11700 agaaatgaag tattgaaata ggaagagaga aagtcaaact gtctctattc gcagatgaca 11760 tgattgtata tttagaaaac ccctttgtct cagcccaaaa tctcaagttg ataagcaact 11820 tcagtatagt ctcaggttat aaaatcaatg tgcaaaaatc acaaccattt ctatacacca 11880 ataatagaca gacagccaaa taatgagtga actcccactc acaattgcta caaagagaat 11940 ataataccta ggacacaaac aaatggaaaa acattccatg ctcatggata ggaagaatca 12000 atatcatgaa aatggccact gcccaaagta atttatagat tcaattctat ccccatcaag 12060 ctatcattgc ctttcttcac agaactagaa aaatctactt tacattttat atggaacgaa 12120 agaagagccc acatagccaa gaaaatctaa gcaaaaagaa caaaaagttc ataatcactg 12180 ggacattgga gaaatgcaaa tcaaagtccc aatgaggtac catctcatgc cagttagaat 12240 ggtgatcatt aaaaagtcag gaaacaacag atgctggaga agatgtggag aaataggaat 12300 gcttttacac cgttagtggg agtgtaaatt agttcaacca ttatggaaga cagtgtgctg 12360 attcctcaag gatctggaac aagaaatacc atttgaccca gtaatcctgt tactgggtat 12420 atacccaaag gattataaat cattctacta taaagacaca tgtgcacgta tgtttattgc 12480 agtactattc acaatagcaa agacttggga ccaacccaaa tgctgatgaa tgacagtctg 12540 gataaagaaa atgtggcaca tatacaccat ggaatacagt gctgccataa aaaaggatga 12600 gttcatgtcc tctgcaggga tatgggtgaa gctggaaacc atcatcctca gcaaactaac 12660 acaggaacag aaaaccaaac attgtatgtt ctaactcatt aatgggagtt gaacaatgag 12720 aacacatgga cacatggagg ggaacatcaa caccggggcc tgtcaggggg tgggggactt 12780 ggggagggat agcattagaa gaaataccta atgtggatga caggttgatg ggtgcagcaa 12840 accaccatgg cgcgtgtata cctacgtaaa aaacctgcat gttctgcaca tgtatcccag 12900 aacttaaagt ataaatatat aaatctaccc aaggagaaac agaatgcaca ttccaggtga 12960 aatggaatca attactgaca aatatgaaaa cataaaatat gccctgtgat gaagatgggt 13020 gtcattattg cacacagttt atttgagaat gggaaagaat gacttaacct aggtgggttt 13080 cctgcattcc tgcaaattga aataggccat aaaatatttg cagtgagagt cagaggaggg 13140 gtttcagata ggttggaatg ctctacatgg aggtgcagag gtgggtgatg ggaaagagta 13200 ggtggatgga agagggaaag gaagccttta ttgagtgaga acaaaaatga tttgtacaga 13260 taacagttgg aatttagtgt ctatcagata aggcgtaaac atcaatgccg ttgtcatcgc 13320 accattgttg aaaacgctat tttttcccct attgaattgc cttggcaacc ttgtcaaaat 13380 ccaattgact ataaatgtgt gaaaggtttt tttctgtgtg ctctcaattg tactccattg 13440 atctatatgt ctgtcgtaat ccctatagtg caccatctta attactgtag tattgtatta 13500 atatcaagtt attatatcaa atcaaggagt gtgagtcttt gttgttcttt ttcaatacgt 13560 tttcactatt ctgggtccct tgaatcttca tatgaaattt agaatacact tgtcaatttt 13620 ctcaaaggag ccaatgggga ttttgacacg attgcattaa atcagtagat cagggaatta 13680 tgccattttt aaaattgaag tgaaattcac acaatataca attaaccatt ttaaagtgta 13740 gacttgagtg gtatttagtg tattcacaat gttgtacaac caccagcttt ctctggtttc 13800 aaaatgtttt tattacttca caaaaacata tcatattcat taaataatca ctccattcct 13860 ccaacccatc atgtaattac ctctaatctt ctttctgttt ctatggattt gcctgttatg 13920 gatatataaa ggactcacag catatgtaac ctgttatgtc tgagtccttt cacttataat 13980 gttttagagg ctcatctaag ttgcaacatg tatcagtact tcattctttt ttgtcgctga 14040 aatatatttt atttttccat gcacatatat cacaatttgt tattcatctg tttatggaca 14100 tttgcattgt ttccaacctt tggctcttat agataatctt gctttgatca tttgtgtatg 14160 tttctttatg ggcatatttt catcattctt gggtatattc tcaggagttg aattgctggg 14220 tcaaatggta attttatgct tatcttcttg tatataagag ccaatctttt ctacagtgaa 14280 tgtggcattt tatattcctt ccagcaatgt agaaggattt ctattttcca catagttgct 14340 aatacttgta actttctgta tttattgaag ccatcctagt gaatatagag tgttatctca 14400 ttgtggtttt atttatttat tcatttattt agagacagag tcttactctg tcacccaggc 14460 tggagtgcag tggcatgatc tcagctccct gcaacttcca cctcctgggt ttacacaatc 14520 ctcctgcctc agcccccaga gtagctggga ttacaggcat gtgtcaccat acctggttaa 14580 ttgttatatt tttggtagag acgggttttc tccactttgg ccaggctggt ctcgaactct 14640 tgacctcagg tgatctgccc atcttggcct ccctaagtgc tgggattaca ggtgtgagcc 14700 actgcgccca gctctcattg tggttttaac ttgtgtttaa tggccaatga ttttgaactt 14760 cttttaatat attattaccc atttgtatat cttctttgga taaatgccat tcaagtacat 14820 tttctgttta aattaagttg actttatttt tctttttaag ctgttagaat gatttatgta 14880 ttcaaaacat taaactccta catatacata atatgaagat atttcctccc attctgttgg 14940 ttgtcatttc acattctcaa ttatattctt gctgcacaaa gtttaatttt gatgaagttt 15000 ggtttatcta tctttccttt tgccactctg gtatcaaatt tataaatcta ttgccaaata 15060 tgaagtcatg aagatttacc cctacatttt attctaagag ttttatcgtt ttagctctta 15120 tatttaagtt tttccatcca ttttcagtta tatttttcat ttggagtgaa gtaagggaga 15180 tctcagcttc attcttctgc atttcgctgt cctgttgtct catcaccatt tgttgaagag 15240 actctttcct cagattgaat ggtctaggca cccttgatga aagtaaattt gccatagatc 15300 tttaggttta tttcgtgatt tcagttttat tccatttgtc ttgatgtcta tctttatgct 15360 agtaccacac tgttttgatt attacagttt gtagtaagtt tgaagttgga aactgagtac 15420 tcactgatac agaaattaag tagaaattac ttaggcaaat agtaagcatt tgggagtcct 15480 cagtaaggtt tttctttata ctggaaagca gccccaaatc attttctaac aaagagcagc 15540 ttgtaaaatc gagctgcaga catacacaag gaagctggaa acttgcacga gtgaaagctg 15600 gtagtaaaga actacctgtg accaggcaag ttcaaaatgg cggctccttc cccacccact 15660 atgtaaatgt cacacctgat taaaccaatc tctgggccat acgtaaatca gacactgctt 15720 cctccagcct ccctatgcaa tctgctgtgg tccacctcct tccccctttt ccgatgtccc 15780 tctctttcac aagaagctgc ttttttctct cctttcttct attaaacttt ctgctacata 15840 acccactcag atgtgtccgt gtcctaaatt tttctggggc atgatgacaa acctgagggt 15900 gtatatccca gacaacgtag ctgcttcatc accatgtagt tgcatattaa ttgaacaatc 15960 aactttccca tttctggaaa aaagaccatt ggtattgtgg tagggattgc ctgagttttt 16020 agatcacttt gaggagtact gccatcttaa cgatattaag tcttccaatc cctgaacatg 16080 agatagtttt caatttatgt aggtattaga tattcttttt tcttctgttg caagggtctg 16140 gtttagttta aaaggcaagt ttatcttaaa tttttttatt tagttatgac cattgaggat 16200 tttaaaatga taccatatga aatgccttct tttaaaaaaa atttcaactt ttatttattt 16260 atttatttat ttatttttat tatactttaa gttttagggt acatgtgcac tatgtgcagg 16320 tctgttacat atgtatacat gtgtcatgtt ggtgtgctgc atccatgaac tcgtcattta 16380 cattaggtat atctcctaat gctatccctc cccactctgc caccccacaa caggccccag 16440 tgtgatgttc cccttcctgt gaccatgtgt tctcattgtt caattcccac ctatgagtga 16500 gaacatgcgg tgtttggttt tttgtccttg agatagtttg ctgagagtga tggtttccag 16560 cttcatccat gtccctacaa aggacatgaa ctcatcattt tttatggctg catagtattc 16620 catggtgtat atgtgccaca ttttcttaat ccagtctatc attgttggac atttggcttg 16680 gttccaagtc gttgctattg tgaatagtgc cacaataaac atatgtgtgc atgtgtcttt 16740 acagcagcat ggtttataat cctttgggta tatacccagt aatgggatgg ctgggtcaaa 16800 tggtatttct agttctagat ccctgaggaa tcaccacacc gacttccaca atggttgaac 16860 tagtttacag tcccaccaac agtgtaaaag tgttcctatt tctccacatc ctctccagca 16920 ccttttgttt cctgactttt taatgatcgc cattctaact ggtgtgagat ggtatctcat 16980 tgtggttttg atttgcattt ctctgatggc cagtgatgat gagcattttt tcatgtgttt 17040 tttggctgca taaatgtctt cttttgagca gtgtctgttc atatcttttg cccacttttt 17100 gatggggttg tttgtttttt atcttgtaaa tttgtttgag ttctctgtag atcctggata 17160 ttagcccttt gtcagacaag agggttgcaa aaattttctc ccattctgta ggttgcttgt 17220 tcactctgat ggtagtttct tttgctgtgc agaagctctt tagtttaact agatcccatt 17280 tgtcattttg gcttttgttg ccattgcttt tggtgtatga gacaagaagt ccttgcccat 17340 gcctatgtcc tgaatagtat tgcctaggtt ttcttctagg gtttttatgg ttttaagtct 17400 aacatgtaag tcttgaatcc atcttgaatt aatttttgta taaggtgtaa ggaagggatc 17460 cagtttcagc tttctacata tggctagcca gttttcccag caccacttat taaatagtga 17520 atcctttccc catttcttgt ttttgtcagg tttgtcaaag atcagatagt tgtagatatg 17580 tggccttatt tctgagggct ctgttctgtt ccattggtct acatctctgt tttggtacca 17640 gtaccatgct gttttggtta ctgtagcctt ctagtatagt ttgaagtcag gtagctttgt 17700 tcttttggct taggattgac ttgacaatgc gggctctttt ttggttccat atgaacttta 17760 aagtagtttt ttccaaatct gtgaagaaag tcattggtag cttgatgggg atggtattga 17820 atctataaat taccttgggc agtatggcca ttttcacgat attgattctt cctacccatg 17880 agcatggaat gttcttccat ttgtttgtat cctcttttat ttcattgaac agtggtctct 17940 agttgtcctt gaagaggtca ttcacatccc ttgtaagttg gattcctagg tattttattc 18000 tctttgaagc aattatgaat gggagttcac tcatgatttg gctctctgtt tgtctgttat 18060 tggtgtataa gaatgcttgt gatttttgca cattgatttt gtatcctgag actttgctga 18120 agttgcttat cagcttaagg agattttggg ctgagatgat ggggttttct agatatacaa 18180 tcatgtcatc tgcaaacagg gacaatttga cttcctcttt tccttattga atacccttta 18240 tttctttctc ttgcctgact gccctggcca gaacttccaa cactgtgttg aataggagtg 18300 gtgagagagg gcatccctga cttgtgccag ttttcaaagg gattacttcc agtttttgtc 18360 cattcagtat gatattggct gtgggtttgt cataaataga tcttattatt ttgagatacg 18420 tcccatcaat acctaattta ttgagagttt ttagcatgaa gagctgttga attttctcaa 18480 aggccttttc tgtatctatt gagataatca tgtggttctt gtcattgatt ctgtttatat 18540 gctggattac atttattgat ttgcctatgt tgtaccagcc ttgcatccca gggatgaagc 18600 ccacttgatc atggtggata agctttttga tgtgctgctg gattcggttt gccagtattt 18660 tattgaggat ttttgcattg atgttcatca gggatattgg tctaaaattc tctttttttg 18720 ttgtgtctct gcccggcttt ggtatcagga tgatgctggc ctcataaaat gagttaggga 18780 gtattccctt tttctattga ttggaatagt ttcagaagga atggtaccag ctcctccttg 18840 tacctctggt agaattcggc tgtgaatccg tgtggttctg gacttttttt ggttggtaag 18900 ctattaatta ttgcctcaat ttcagagcct gttattggtc tattcagaga ttcaacttct 18960 tccttgttta atcttgggag agtgtatttg tcgaggaatt tatccatttc ttccagattt 19020 tctagtttat ttttgtacag gtgtttatag tattctctga tggtagtttg tatttctgtg 19080 ggatcggtga tgatatcccc ttcatcattt tttattgcgt gtgttggatt cttctctctt 19140 ttattcttta ttagtcttgc taacagtcta ccaattttgt tgatctttta aaaaacctgc 19200 tcctaaattc attgattttt tgaagggttt tttgtttcca tgtaattgag tttccattgt 19260 ttccatggaa tgcaaaaatt ccatgttttg agtgagtttc ttaatcctga gttctagttt 19320 gattgtgcta tggtctgaga gacagcttgt tataatttct gttcttttac atttgctgag 19380 gtgtgcttta cttccaacta tgtggtcaat tttggaatag gtgtggtgtg gcgaaaagaa 19440 tgtatattct gttgatttgg ggtggagact tctgcagatg tctattaggt ctgcttggtg 19500 cagaactgag ttcaattcct ggatatcctt gttaactttc tctctcgttg atctgtctaa 19560 tgttgacagt ggggtgttaa tgtctcccat tattattgtg tgggagtcta agtctctttg 19620 taggtctcta agcacttgct ttattaatct gggtgctccg ttattgggtg catatatatt 19680 taggatagtt agctgttctt gttgaattga tccctttacc tttatgtaat ggccttcttt 19740 gtcccttttg ttctttgttg gtttaaagtc tgttttatca tagagtagga ttgcaacctc 19800 tgcctttttt tgttttccat ttgcttggta gatctttctc catcccttta ttgtgagcct 19860 atgtgagtct ctgcatgtga gatgggtttt ctgtgtacag cacaccgatg ggtcttgact 19920 ctttatccaa tttgccactc tgtgtctttt aattggagca tttaacccat ttacatttaa 19980 cattaatatt gttatgtgtg aatttggtcc tgtcattatt atgttagctg gttattttgc 20040 tcattagttg atgcagtttc ttcctagcct tgatgatctt acaatttggc atgcttttgc 20100 catgggtggt accagttgtt cctttccatg tttaatgctt ccttcaggag ctcttttagg 20160 gcaggtctgg tggtgacaaa atctctcagc atttgcttgt gggtaaagga ttttgtttct 20220 ccttcactta tgaagcttag tttggctgga tatgaaattc tgggttgaaa attcttttct 20280 ttaagaatgt tgaatattgg cccccactct cttctggctt gtagagtttc tgccgagaga 20340 tcagctgtta atctgatcgg cttccctttg tgggtaaccc ggcctttctc tctggctgcc 20400 cttaacattt tttccttcat ttcaactttg gtgaatctga caattatgtg tcttggagct 20460 gctcttctcg aggagtatct ttgtggtgtt ctctgtattt cctgaatttg aatgttggcc 20520 tgccttgcta gattggggaa gttctcctgg atagtatcct gcagagtgtt ttccaacttg 20580 gttccattct cctcgtcact ttcaggtaca ccaatcagat gtagatttgg tcttttcaca 20640 taggcccata tttcttggag gctgtgttcg tttcttttta ttcttttttc tctaaacttc 20700 tcttctcact tcatttcatt catttcatct tccatcactt ataccctttc ttccagttga 20760 tcaaatcggc tatagaggct tatgcattca tcacgtagtt cttgtgccat gattttcagc 20820 tccatcaggt cctttaagga cttctctgca ttggttattc tagttagcca ttcatataat 20880 cttttttcaa ggttttttac ttctttgcca taggttcaaa cttcctcctt tagctcagac 20940 tagtttgatt gtctgaagac ttctctcaac tcatcaaagt cattctccat ccaactctgt 21000 tccattgctg gtgaggagct gtgttccttt ggaggaggag aggcactctg atttttagaa 21060 tattcagttt ttctgctgtt ttttccctgt ctttgtggtt tcatctgcct ttggtctttg 21120 atgatggtga catacagatg ggattttggt gtggatgtcc tttctgtttg ttagctttcc 21180 ttctaacagt caggaccctc agctgcagtc tgttggagtt tgccagaggt ccactgcaga 21240 ccttgtttgc ctgggtatca gcagcggagg ctgcagaaca gcggatattg gtgaacagca 21300 aatgttgctg cctgatcgtt cctctggaag ttttgtctca gaagagtacc cagccttgtg 21360 aggtgtcagt ctgcccctaa tggggggtgc ctcccagtta ggctacaagg ggatcaggga 21420 cccacttgag gaggcagtct gtccattctc aggtctcaag ctttgtgctg ggagaaccac 21480 tattctctcc aaagctgtca gactgggaca attaagtctg cagaattttc tgctgccttt 21540 tgtttggcta taccctgacc ccagaggtgg agtctacaga ggcaggcagg cctccttgag 21600 ctgcggtggg ctccacctgg ttcgagcttc ccagccgctt tgtttaccta ctcaagcttt 21660 ggcaatgggg ggcacccctc acccagcctc gctgctgcct tgctgtttga tctcagactg 21720 ctgtgttagg aatgagtgag cctcagtggg cataagaccc tctgagccag gcacgggata 21780 taatctcctg gtttgccatt tgctaagacc atcagaaaag cacagtatta gggtggaagt 21840 gaccttattt tccagatgcc ttctgtcctt ggctaagaaa gggaattctc tgaccccttg 21900 cacttcccag gtgaggtgat tcctcaccct gcttcagctc aggctccatg cactgcaccc 21960 actgtcctgt acccactgtc tcacaatccc cagtgagatg aacccagtac ctcagttgga 22020 aatgcaaaaa tcattcatct tctgcatcgc tcatgctggg agctgtagac tggagctgtt 22080 cctattcagc catcttggct ccaaatgttc tcaactttta ttttgaagtg cacgttcatg 22140 tgtcttacat aggtatattg tgtgatgctg aagtttaggg tacaaataat gccatcacac 22200 aggtagtgag actagtaccc aataggtagt ttttcagccc ttgcctttat atctctctac 22260 cctctatagt aattccttgt gtattttttc catctttgtg tcccactttt atatgagaac 22320 atgtggtatt tggatttctg ttgctgcatt aattccctca agataatggc ttccagctac 22380 atctatgttg ctgcaaagac atgatttttt ttaatggctg catagcattt catggtgtat 22440 atacaccaca ttttgtttat gtgacacatg attggttggt tctatgtctt tactattgtg 22500 aatagtgcag gaatggacat actagcacgt gtcgttttgg tagaacaatt tgttttcctc 22560 tgggtacata ctcagtagtg gaattgctgc atcgaatggt agttctgctt ttagttcttt 22620 gagaaatctc aaaactggtt tccatagtgg ctgaactaac ttacattccc accagtagta 22680 tataagtgtt ctgttttctc tgcagtctca ccaacatctg ttattttttg gctttttaat 22740 catatccatt ttgattggca tgagatgata tctcattgtg gttttgattt gcatttctct 22800 gatgattagt gatgttgagt gttttctcat atgcttgttg gccatgtgtg tgtcttcttt 22860 ggaaggacat atgtcctttg cccacttttt aatggggtta cttgtttttt aacttgttga 22920 attgtctaac ttccttatag attctcgcta ttgaaacatt gttggattca cagtttgcaa 22980 atattttgtc ccattctgta ggttgtctgt ttattctgtt gatagtttct cttgctgtgc 23040 agaagctctt taattaggtc ccacttgtca atattcattt tggttacaat tgcttttgag 23100 gagttattca taagttcttt gctaaagcct gtatccagaa tgttatttta tagatatttt 23160 tccatcagat tcttatagtt tgaggtctta aatttagatc attaatccat ttcgagttaa 23220 ttattgtata tggttcaagg aagtgtttca gtttcattct gcatatggct agtcagttat 23280 cctcattcaa tttactaaat agagaattct ttcctcattg attatttttt gtcaactttg 23340 ttgaagatca gatggctata ggtgtgtggc tttatttctg gcttctctac ttctgttcaa 23400 tttgtgtttt gtaccagtta catgctctct tggttactgt agcttacagt atagtttgaa 23460 gtcaatgtga agcttcccac tgttcttttt gtttaggatt gctttggatt tgggctcttc 23520 tttggttcca tatgaatttt agaataaaat aatattggta gtttgatagg aatagcattg 23580 aatctgtaga tagctttggg cagtctagcc attttaatga tattgattgt ttcaatccat 23640 aagcctgaac tgtttttcca tttgtttgtg tcatctatga ttttgttcat cagtattttg 23700 tagctctcct tgtagagacc tttttgcccc ctttgttagt tgtattccta ggttttttct 23760 tgtggctatt gtaaatggga ttgagttctt gattggcttt caggatgaac atttttggtg 23820 tttaaaaatg ctacagattt ttgcatattt attttgtgta ctgaaatctc actgaagttg 23880 tatgtcagtt ccaggagttt ttggaaagtt ctttagggtt ttctaggtct gaattgggtc 23940 ttctaggtct gagatgggtc ttctacttct actagtagga gatgggcctt gcaatcttga 24000 agacagtaga aggatgggtc ttatttttaa tccaacttcc tactctttgc cttttatgtg 24060 gagcatttag actttatctt caatgttaac actgatatat gaggttttga ttctattgtg 24120 aagttgttag ctggttgttt tgtagtttct attgtgtgat tgctttatag agtctgtggg 24180 ctatatactt aagtgtgttt tgtggtagca ggtattgttc tttcacttcc atatttagaa 24240 ctcccttaat gatctcttat aaggctggtc taatggtaac agattttcct ggtagttact 24300 tgtttggaca agattgtatt tcttccttga ttttgaagca tagtttggca aaatataaaa 24360 ttcttttttg taatttttat taaaatgaat gttgaaaata ggctgagcat ccttttagct 24420 tgacaaggtt ctctttgtat gtgatctgat ttttttctct agctgccttt aaaattattt 24480 tttagccttg actttggaca gcctggtgag taaatgcctt ggtgatgtta attttgtata 24540 gtgtctcaca tatattccct gggtttattg tatttggatg tctacctttc tagcaagttt 24600 gggaaaattt tctcaactta ttagcccaaa tgtattttct agattgttta ctttttcttc 24660 tctctcagga atgccaataa ttcctaggtt tactcctttt aaataatcag atatttcttg 24720 aagactgttc atttattaaa attatttttt ctttattttt gtctgacttg gttagttcaa 24780 aagactggtg ttcaagctct gaaattggtc cagtctattg ataaacgctt caattgaatt 24840 ctgaaattcc ttaagtgact ctttcaattc cagaagcttt gattgatttc tttttaagat 24900 gtttacttct cgctccattt cctggactgc tttagtagtt tgtttgcatt aattttcaac 24960 cctgccttgg atcactgagc ttacttgcag tccatatttt gaatagttta tctgtcattt 25020 ctgagttgcc agtttggtta gggttcattg ctggggagtt catgtgatcc tttggaggtt 25080 ttacgacatt cagacatttc atggtgccag aagtcttatg ctggtttctt cttatttgga 25140 gaggctgccg cttctgattt ttgaaattat ttttgtgcag atagaattgt ttttctctcc 25200 ctatttttgt tctttccttt ttctcttccc cttcctatgg gatgtgacta taaagtatgt 25260 tgggtagggc ctttgacttg gattctatag ctgtgttcac ttctgtgggt agttttatat 25320 tggcctgtgc agtttgacat acatgtcagt agatggcact tatgagcaaa agccagctgt 25380 gactaggaca acttgatata gcacagcttg atccttgttt tctgggggaa cttctctgtt 25440 tcctcagata atctgttcat ctgtggaatt cagagtagtc tgggctccct gctgagcccc 25500 agggggataa agatcggtgt cactggactg ggcagtcctg tctacaggtc ccctaatggc 25560 aggcactagc accagcactg agagacaact gatttttgca ttgatcttgt accctgaaat 25620 tatgctgaaa tcatttatta gctctactta ttttctatct gtattgcatg agatttttct 25680 atatagagag aatcatgttt cagtcaaata cacatgattt gaatttttcc tttcctattt 25740 gaatgtgttt tattttattt cttgcctaat tgctctggct agaacttcca atactatgtt 25800 taatagtagt aaaaatggac atccttgttt tttttctttt ctttctgatt ttaagttaca 25860 agttttagtc tttcacaatt tagtatgctt tagctttgca gttttcataa atatgcttta 25920 ttatgataaa ggagttttat ttcatttata gttttctgag tgtttttgtt atgatgggta 25980 ttaaaagttg tcaatttttt tttctgcatc taatgagatg aacatgattt tctcttttat 26040 tctgatatgg tgtagtgcac tgattgactt tcttgttgaa ccacttttga atttctggga 26100 taaatctcac ttgcttattg tgagatttat ttttataatg tttattatta taatttttta 26160 attttgtaaa aaattgttgc aaaatacaca taacataaaa gtttctgtat taatcatttt 26220 aagtgcatag tattttggta ttaattacat tcacattgtt gtgcaaatgt cactgccata 26280 catctgtaga attcttttca tcttgcaaaa cagaaacttt atacccattt aataataact 26340 cttggccggg cattgtagat catgtctgta atcccagcac tttgggaggt tgaggtgggc 26400 agattggatg agctcaggag ttcgagacta gcctgggtaa catggcaaaa ccctgtctct 26460 accaacaaag caatcaacaa acaaaatgaa agtaacataa tttcctcaac tgtgcaaact 26520 aattcatatt aataattcca tgaaatgtgt ccatagtaac acaagagcag aagatgggcc 26580 ttgtaaaggt ggcccaccac attatttgag gtctttgtca gtcttatctg ccagatactg 26640 tgtggactaa ctgcagttaa aattcccatt cccttatcac ttgccctgaa atttatctga 26700 aatcaaaggg aatgggttgc ccaaagagaa gtgtccagag acgtggatga tgcgacaaga 26760 aatgttgtag aaccacaccc ccacctcaaa tgataactct acatttctcc cttccaccac 26820 cccatggcaa ccaacattct actttgtgtc tctataaaat cgagtatgct gggtacctca 26880 taaaagtgga atcattacat atttgccttt tgtgactggt ttatttcact tagcctaatg 26940 ttatgttctc agggttcatt catgtttttg catgtgtcag atttttcttc ctttctaagg 27000 gtgaataata ttccgttgta tgtataaacc aagtttgctt acccatttat ctgttgatgg 27060 gcacttggat tactttcacc ttttgacttt ttgagtaatg ctattatgaa tatgggtaaa 27120 aaaatatctc tttgatgtgc tgctttcaat tcttttgggt ataaataacc caaaagaata 27180 acccaccacg cctggttaat tttttgtatc tttagtagaa actgagtttc accatgttgg 27240 ccaggctggt ctcaaactcc tgaccctgtg atccacccac ctcagcctcc caaagtgctg 27300 gaattatagg tgtgagccac gtcgcccggc ctacatttat tgatttgcat atgttgaacc 27360 aggcttgcat cccagggatg aagccaactt gatcgtggtg gataagcttt ttgatgtgct 27420 gctggattca gtttaccagt attttgttga ggattttcgc atggatgttc atcagggata 27480 ttggcctgaa attttctttt ttttgtttta tgtctgccaa gttttggtat caggatgatt 27540 cttcccttat aaaatgagtt aggaaagagt gcgtcttttt ctactgtttg gagtagtttc 27600 agaaggaatg gtaccagctc ctctttgtac ctctggtaga attcggctgt gaatccttca 27660 ggtcctgggc tttttttttg cttggtgagc tattccttgc tgcctcaatt tcagaacttg 27720 ttattggtct cttcacggat tcgacttctt cctggtttgg tcttgggagg gtgtgtctag 27780 gaatttatcc atttcttcta gattttctag tttatttgca tagaggtgtt tatagtattc 27840 tctgatggta gtttgtattc ctgtaggatc agtggtgata tcccctttat catttttttg 27900 ttctctctat ttgattcttc tttcttttct tctttattag tctggctagc agcctatttt 27960 gttaatcttt tcagaaaatt agctcctgta ttctttgata ttttgaaggg attttcctgt 28020 ctctatctcc ttcaattctg ctctgatctt agttattttt tgtcttctgc taggttttga 28080 atttgtttgc tcttggtttt ctagttcttt taaatgtgtt gttagggtgt tgattttaga 28140 tctttcctgc tttctcctct ggacatttag tgctataatt ttccctctaa acactgcttt 28200 agctgtgtct tagagattct ggtacactgt gtccttgttc tcattggttt caaagaacat 28260 ctttattttg ccttcatttt gtcatttacc cagtagtcat tcagagcagg ttgttcagtt 28320 tccatgtagt tgtgcagttt tgagtgagtt tcttaatcct gagttttaat ttgatggcac 28380 tgtggtctaa taggttgttt gctttaattt ctgttctttt gtatttgctg atgggtgttt 28440 tacttccaat tatgtggtca attttagaat aagtgtgaag tggtgctgag aagaatgcat 28500 attctgttga tttagggtgg agagttcttt agatgtctat tatgcccgct tggtccagag 28560 cttagttcaa gtcctggaga tcctagttaa ccttctgtct cattgatctg tctaatattg 28620 acagtggggt gttaaagcct ccatctatta ttgtgtggga gcctaagcct ctttgtaggt 28680 ctttaagaac ttgctttatg aatctgggtg ctcctgtatt gggtgtgtat ctatttagga 28740 tagttagctg ttctcgttgc attgatccct ttaacattat atgataccac cccttttttt 28800 aatctttgtt ggtttaaagt gcgttttatc agagactagg attgcaaccc ctgctttgtt 28860 ttgctttcca tttgcttggt aaatattcct ccatcccttt attttgagcc tatgtgtgtc 28920 ttttcacgtg agatgggtct cctgaataca gcacattgat gggtcttgac tctatccaat 28980 ttgccagtct gtgtctttta attggggcat ttagcctgtt tacatttaag gttaatattg 29040 tgtgtggatt taatcctgcc attatgatgc tagctgatta ttttgccttt tagttgatgc 29100 agtttttttt atagtgttga cggtctttac aaaatttggt atgtttttgc agtggctgtt 29160 cccagttgat cctttccata tttagtgttt ccttcaggag ctcttgtaag gcaggcttgg 29220 tgatgacaaa atccccttgt ggtgatgggc ttccctttgt ggtaacccga cctttctttc 29280 tggcttccct taacattttt cctttctttt aaccttggtg aatctgacaa ttatgtgtct 29340 tggggttgcc cttcttgagg agtatcattg tgggtgtttt ctgtatttcc tgaatttgaa 29400 tgttggtctg tcttgctaga ttgtggaagt tctcctggat aatatcctga agaatgtttt 29460 acaacttggt tccattctcc ccatcacttt caggtacacc aatcaaacgt agatttggtc 29520 ttttcacata gcgccacatt tcttggaggc tatgttcatt tctttttatt cttatttttt 29580 aacttttttc ttcacatttt atttcattaa gttgaccttg aatctctgat atcctttctt 29640 ctgctttatc aatttggcta ttgatattta tttatgcttc acaaagttct cattctgtgt 29700 tcttcagctc catcaggtca ctcatgttct tctctagtta gcaattcgtc taacattttt 29760 tcaaggttct tagcttcttt gcaatgggtt agaacatgct cctttagctc agtggagttt 29820 gttattacac accttctgaa gcctacttct gtcaattcat caaattcttt ctctgtccag 29880 ttttgtcccc ttgctggcaa ggagttgtga ttctttggag gagaagagat gttctggttt 29940 tttaaatttt cagccttttt gtgctgtttt tttttttctc atatttgtgg atttatctac 30000 ctcttgtctt tgatgttggt gacctttgaa tgggtttttt gcgtggacat cctttttgtt 30060 gatgttgatg ctattccttt ctgtttgcta gtttttttcc taacagtgag gcccttctgc 30120 tgcaggtctg ctggagtttg cttgaggtcc actccaaacc ttgtttgact gggtatcacc 30180 agaggaggcc acagaacagc aaagattgct gtgtgtttat tcctctggaa gcttcatcct 30240 agggggacac ttgccagatg ccagccaaag ctcttttgta tgaggtgtct gttgacccct 30300 gctgggaggt ctgctgttct ctttagagcc agcaggcagg aacatttaag tctgccgaag 30360 ctgcacccac tgccacccct tcttccaggt tctcagtaca gtgagatggg agttttatct 30420 ataagcccct gactgggact gttgcctgtc tttcacagat gcaactgctc agagagtagg 30480 aaactagagg ggcagtatgg ctacagtggc tttgcggagc tgaagtggac tccacctagt 30540 cctagcttcc tggaggcttt gtttacactg tgaggggaaa accacctact caagcctcaa 30600 caatggcaga ctcccctcac ccccccaccc cccccaagct cgaacatccc cagtcaactt 30660 cagactgctg tgctggcaaa cagaagttca agccagtgga tcttagcttg ctgggctcca 30720 tgggggtgag atcccctgag ctagatctct tggctccctg gcttcagtcc tctttccagg 30780 ggactgaatg gttctgactt gctggtgtta cagcaccact ggggcataaa aaaacccctc 30840 cagctatctt ggtgtctgcc cagatgacca cccagttttg tgcttgaaac ccagggccct 30900 ggtggtgtag gctctgaagg gaatcttctg gtctgtgggt tgtgaagacc atggaaaaac 30960 tgcagtattt gggccagagt gcaccgttct tcacagcaca ttccctcaag gctttccttg 31020 gctatgggag ggagttcacc aatcccttgg gcttccaagg tgagacaaca ttcctccctg 31080 cttcagttta gcctccatgc actgcaccta ctgtctaaac attcccagtg agatgagcca 31140 ggtacctcag ttggaaatgt agaaatcacc taccttcttc attgatctcg ctgggagctg 31200 ctgactggag cttttcttat tcagtcatct tgccagccac cctcctgatt gagtttcttt 31260 aaggtaatta ttttgaattc cttttttgaa tatttgtgga ttttttttca ttgaggtctg 31320 ttatttgaga gttattatgt ccctttggtg gtgccatatt tctttatttt tcatgttgtg 31380 tctctgaatt tatgtctatg catgtgatgg aacaattgct attccaaaat ctctagagtg 31440 aatttcatag agaaagactt ttatctgaag ttgagtgttt gtgtgcaggt gacgaagagt 31500 gggtaactca gtttttgata gatgcattgt gatagtatct gtgtagtttc tctagctatg 31560 gccaatatca gcaataattt tgggtgcctc agtggcctag gctgtagaag tttgcagcag 31620 tatcaggagc agtgtaggtt gttaatgtcc ttagtgtcaa agtatttggg cgtcctccta 31680 tttttatttt cctaaaattg gggtgactta gcctaaggga ttccttttgg tgtcatctct 31740 gacatggcat taactcagcg gcattggtgc tgggttacaa gtacagatcc ttgaagttgt 31800 catagaagca tcattctaga ctcagggtct tttaatcatg tattgtgaca cctgggtctt 31860 gcggtgcagg ttcactctct gtggcagtgt tgaatgcaga ttacccacag aaccaaggtc 31920 tatgactcta gcacacctta gcagctgaag cccacagact gagttgtggc tgtgaatctg 31980 tttctgaggg tgaggtgcag gaaaatgccg tggcctgcct ctgatgtaga aggggtgctc 32040 tttagatttg agcccagcaa gtagaatatg gctactatca ttaataagat aggatatact 32100 accttattaa tgctaagtaa ccaagctcaa gtcaagagat ggaaatgtta atactggttt 32160 gtgactgcac tgttcttctt acttaagagt aattttcaac atggcaagct tagaaaactg 32220 tgtgaagctg atgttaccaa gggagaggac tgtctttgtc tacacatgtt gagagaatct 32280 agtctatttc agtggtgttg attacacaat attcaacatg acacacatca cttggagact 32340 ctaaaagcat cattaattta ttgctgctgg ccttggggct ggttaaaaag taatacgttt 32400 tttatattga tgatctcttt caactaagta ttccttatcc attgctttct ttctcccaac 32460 ttccaaatta tgagaactcc agtctctaag ccttggcata tgcagtatcc ttactctctc 32520 ctttatcccc tgtggcaatt aatcccttta ttctccatct tcatcattgt tctttctctt 32580 cactttgtat atttttgttt attcctgcac tactgcaccc tagaacaagt tgtctgttgc 32640 ctcccatcta gattataata gtgtgggccc atcttgtcat tgtaaaccca ttctcttcct 32700 ctataattta tcttaggttt tgctgtcata ttgctttttt aatatttgtt tttacaaaat 32760 ttcaacttta taatgaatct gggggcaata ttgagtccat gtacctttct gacagaaaaa 32820 tataattgca gactcttgat tcaggctcac agtgtctaat gatatggccc caatttacgt 32880 tttctattaa tttagattgt ggttgtctac aggtaataat ggctttaaca cataatgtct 32940 ttatttatct caaatacagg gattctcttg gtggggcaat ctaggataat acagcagccc 33000 caaagcagca gagagtcaag tgccttcctt tttcccacta aatttagtat acagaccttc 33060 attttgatga ttaaatccca ggatggggtc tactctacct cctacttcac atttgtgttt 33120 tgagtatgaa agaggaaata gaggaaatgg acctagagac cttctgctta tatattttag 33180 caaaaaaaaa tatgctgtgt gactcagcta gctgcaaaga gcctgatgaa tggaattttt 33240 aggcaagcat ggaataaggg agtaggaaat aaagtttggg caagttggtc tacagcctct 33300 gctatacaag cagtattttt tttctagtac tgtactttcc agtttctatg ttggtaacta 33360 tataactatg tgaataattt tgaattcact gtaatcaaat atgctggtaa ataatttgtc 33420 agataattgc atcaaatcat tcctaggaaa agcacaacca accatctgaa tttactattg 33480 aaagcttgga aaacactgca gttgacttgt ttggagctgg gacagagacg acaagcacaa 33540 ccctgagata tgctctcctt ctcctgctga agcacccaga ggtcacaggt atgatcagag 33600 atgataagtt aattaatttt cagaaaagat tttgggaagg tgttgctagt gtcctccttc 33660 ctgtttctct tagagaagct tcattattta aacttttgtg cttccagctg taatctgttt 33720 caaactaatg gtgattacaa tgggatatct tggcctggca tggtggctca cacctgtaat 33780 cccagcactt tgggaggctg aggtgggtgg atcacttgag gctaggagtt caagaccagc 33840 ctggccaaca tggccaaatc ccatctgtag taaaaataca aaacttagcc aggcatggtg 33900 gcacatggct gtaatcccag ctactcgagg ggctgaggca ctggaatcaa ttgaacccag 33960 gtggtggagg ttgtagtgag ccaagatttt gccactatac tccagtctgg gcaacagagt 34020 gagactctgt ctcagaaaga aaaaaaaaga aaaaaagaaa aagaaaagat aaaaggggca 34080 tctttgcaca gtagaggaag ataactgaga gaaatgaaga cagcatggca gtagcaaaag 34140 cagtagaact ctggtccaat gtgtctggat ttatggcagg aagagacaag ataaattggc 34200 ctgggattgc atgttggttt tattatgaga aggccatttt aaatagcaag tatattcttc 34260 aagataactt ttctcattct caaaatttca ggttcgaatg ctggagtagg gaaacttgaa 34320 actctcctta ttgaaggata aatggtaatc ctaaaaatgt agtgatctgc ctgagaaaat 34380 tcctagtcca ataagtactc taaaaagtta gattcataag aaaggtgatt ctgtttacta 34440 gaagaggtat ataaagaatg ttccatttag gcagaatatg tacctgagac agtttccatg 34500 aaactgttgt tgggcaaaat aagatttttt tgaggaagtc aataatttta tcttttatga 34560 acaactttac tttagaaatt tacttttaag gacttttggt tatgctgcag ataagaaata 34620 ttcttttttt ctcctatgtc agtatccccc attgaaatga caataaccta attataaata 34680 agaattaggc ttttttttga acagttacta gcctatagag ttctagaaga ttttttgtca 34740 aattttatta tagattaaag ggtacaaata caggtttgtt acataggtaa gttgtgtaac 34800 actgaggctt ggggtcccag ctattccatc acactgacag taaacatatt acccaacaga 34860 tggttcttca gcccacatct cttctctccc tcccccatct agtgatcctc agtgtctatt 34920 gttcccatct ttatgttctt gtgtattcaa tgcttacctc ccacttataa gcaagaacat 34980 gtggtatttg gttttctgtt cttgtattag attgcttagg ataatggcct cctgcttcat 35040 ctataatgct gcaaaagaca taacttttgt tctttcttat ggctgcataa cattccatta 35100 tgtatgtact agtctgttct catgctgcta ataaagacat acctgagaca gggtaactca 35160 taaaggaaag aggtttaatg gactgtcaat tccacacggc tggaaaggcc tcacaataag 35220 tctctgatag agacttattc actatcacaa gaatggcatg ggaaagtcag gcctccatga 35280 ttaaattacc tcacattgag tacctcccgt gacgtgtggg aattatggga gctacaattc 35340 aagatgagat tttggtgggg acacagccta accatatcag tgtatatttg gtacattttc 35400 cttatacaat ccactgtcga tgggcacata agttgattcc atttctttgc tatcgtgaat 35460 agcactacat attggggtga acatattggg gtgtgtgtct tattgataga atgaatattt 35520 tgggggcata tactccatag tgggattgct gggtcaactg gtagttctat tttaagttct 35580 ttcagaaata tccaaactgg tttccacagt ggctgaacta gtttgcatac ccaccaacag 35640 tgtaaaagca tttccatttc tctgcaacct caccagcatc tgttgttttt ggacttttta 35700 atattaataa ttgctattct gactggtgtg agatggtacc tcattgtgat tttgatttgc 35760 atttctctga tgattagtga tgttgagcat tttttcataa gtttgttggc tgcttgtatg 35820 tcatctattg agaagtggct gttcatgtct tttgcccaat tttaattgat tttttttgtt 35880 tttttttttt gcttgttgat ttgtttaagt tttttataga ttttgtctgt tgatacatat 35940 ttagattttg gatattagac atttaggtcc tttaaggtag gaagagtaca ggaaatagtc 36000 aagaagactt taacaaattt ctaaaaacag agagcatata gaggaatgat atatctgtac 36060 agagagggta aacttataac caaaaggtca gaagagaaaa atttatcaag gatgaaaagt 36120 taatcagtgt cccttaaatc ctcccaatat tggtatttgg aagtaccgag ggtcaaagat 36180 ggtggagaat gaggctgaaa aaggaggctc tccctgccta cgtttcctcc acaactcttt 36240 tcatcaggta actgatttcc ttccacaaga agaaggaggc tcactctcta gagaaactga 36300 gcttaagaag ccctgtactc agagacacac agcagtgcca gaaaaggcat gaggtgtagg 36360 ctagaaagac agattaggta cacatccact ccttgaattc agagactctt agctctttta 36420 ttcatcttcc ttcctaaaat tcagcagcta gctctatcct acagaaaggg aactgatcta 36480 gagaaatggc atttttcttt tgccatctta acagacaatt ctcttcaatg tccaacagag 36540 tgaatgaaat gaaaacatat actgctagat acagggttat ggaatttcag gacatgagaa 36600 ttaaagggaa aatcacaaaa actttcagag ctgtaccagg ttatattgaa agaattagaa 36660 ttcagaattc tcaagaagaa ctattaatat tatcttgtat attaataaaa atagtgtttt 36720 cagatgtgca tgtttgccaa aaatttttac cattttccct ttctcagaaa aatactgaag 36780 aatgttcttc attcaaatgc ctgaaacctt gttagagaaa gtcctgagct ccagcaatca 36840 ggaaacctcg tgtaggagaa tgagagtgtc agggaggctg cctagcacca gacctagaaa 36900 acaactactt cagaacagac ctgggggata attcagaagt gagatgagac agcaagaaca 36960 aaagagatgt gacagatttt atgagagttt ccaataacta aaaaggagtt tcaaacatct 37020 gctggaacat ttgtaaataa ttctaacttg ggaaatggaa aattacaaat tgaaataaag 37080 agacattgtt aagcccagga aagtacaaaa ataaaattta atcatagttg tcttaccaga 37140 gaaccatatt tgtgtagtca gaatgatgta agcaatgaat actgatatca taaaaattgt 37200 catctaaata tattctggaa aaagcagtgt ggaaaagtga gtgtgtctat ctgagggtgt 37260 gttcaaatat gaggggaaga tggagattaa aattctcatc tctcttaata aaaaattatt 37320 aaatataatc tacaaaaaga taagtaagaa ttagaaacag agtatttaaa aatgtgaatg 37380 tatgttcaga agaagctgct agaatgttca aaatggtaga tttggaaagt gtgagaatga 37440 agagaatacc attgtgattt gtgacccctc ccacacacag atgcaccaat acagtcatat 37500 atatgcataa atctgatagt ataatttaac gtttaagcaa catatgttat tttgatatga 37560 aattaatctg tttttcaaag acagtaattt catgactaaa ttatatgctt actttatgtt 37620 ttcaaacatt ttattatttt tatttgtatt aattatcata tatactaatg tatatgtata 37680 tacatatctt ttaatttttt ttaattttta tgggtacaaa gtaggtatat atcaatatgt 37740 ttgggggtac atgagatgtt ttgatacagg catgcaatgt gaaataagca tgtcatggag 37800 aatggggtat ctattccctc aagcatttat tctttgagtt acaaacaatt caattacact 37860 ctttaagtta ttttaaaacg tacaattaag ttattattga ctatagtcac ccttttgtgc 37920 tatcatatag tagatcttat tcatctccac ctcccctccc cccacactac cctttcgagt 37980 ctctggtaac catctttcta cactttatgt ccattagttc aattctttta tttttagatc 38040 ccacaaataa gtgagaacat gctatgttcg tctttctgtg cctggcttat ttcacttaac 38100 gtaatgatct ccagttccct tcatgttgtt gcaaatgact gaatctcatt ctttttatgg 38160 ctgaatagaa ctacattgcg tatatgtact acattttctt tatacatttg tctgttgatg 38220 catatttagg ttgtttcaaa gtcttagcta ttgtaaacag tgctgcaaca aacaagagtg 38280 cagatacctc ttcgatatac ctatttcctt tcttttgggt atataccaag cagtagggtt 38340 gttggatcat atggtcgctt tatttttagg attttgagga acatttaaac agctctccat 38400 agaggtcaca ctaatttgca ttcccaccaa cactgtacga gggtccactt atctccacat 38460 cctcactagc atttgttatt gcctgtcttt tggatataag ccattttaac tggagtgaga 38520 tgacatctca ttgtagtttt gatttatatt tctctgagga tcagtggtgt tgaccacctt 38580 tttgtatgcc tgtaacccat ttatatatct tcttttgaga aacatctatt caaatctttt 38640 gcccactttt ggattggatt attagatttt ttttctatag agttgcttga atttcttata 38700 tattctgatt actaatctct tgtcagatga atagtttgca aatatttctc tccattctgt 38760 ggattgtctt ttccctttgt tgattgtatc tttggctgtg cagaagcttt ttaacttgat 38820 gtgatcccat ttgttcatgt ttgctttgaa tgcctgtgct tgtagggtat tgctcgaaaa 38880 atttttgcct agaccaatgt cctggagatt ttccctagtg ttttcctata gtagtttcac 38940 agtttgaggt cttagactta agtctttaat ccattttgag ttgatttttg tatatggcaa 39000 gggatatgag tccagtttca ttcttctctg taggatatta aattttccca gcacatttat 39060 tgaagagact gtctttcccc cagtgtacgt tcttggcacc tgtgttagtc tgttctcatg 39120 cctctaataa agccatacac aagactgggt aatttataaa ggaaagaagt ttaatggact 39180 cacagttcca catggctgga gaggcctcac aatcatggca gaagatgaat gaggagaaaa 39240 gtcatgtctt acgtggtagc aggcagatag ctttgcaggg gaactcacat ttataaaacc 39300 atcagatgtt gtgagattta gtcactatca tgagaaaaat atggaggaaa ccacccccat 39360 gattcaatga tccctacctg gcctccccct tgacttgtgg ggattatttc aattcaagat 39420 gagatttggg gtggggacac agccaaacca tatcagcccc tttgtcaaaa atgagttaac 39480 tctagatacg tgaacttgtt tctgaattct ctattctgtt tcattggtcc atgtgtttgt 39540 ttttatgtca gtaccatgct gttttagtta gtatagctct gtagtttaat ttgaagtgag 39600 ataatgtgat tcctccagtt ttgttccttt tgcttaggat agttttgact attctgggtc 39660 ttttgtgggt ccatatacat tttaggattt attttttcta tttctgtgaa gaatgccatt 39720 gatattttga taggaatttt attgaacctg tagattgttt tgatagtatg ggcactttaa 39780 caatattgat ttttccaatc catgaacatg aaatattttt ctgtttgttg ggtcctctta 39840 aatttctttc ctcagtgttt tatagttttc attatagaga tctttcaatt ctttggttaa 39900 attaatccta ggtatttaaa attatgtatg gctattttca atgggattat tttttaaatt 39960 tctttttctt ttttttcact gttggcatat aacaacacta ctgattttta tgttgattgt 40020 gtatccttca actttactga atttgttcat cagttctaat aattatcttg tagaatcttt 40080 agtttttcct actataaggt tatatcatct gcacacaagg atattttgac ttcttccttt 40140 ccaattttga tagcctttat atctttctct tgtctgattg ctcttgctag gtcatccagt 40200 actatgatga ataacaatgg tgacaatggg catccttgtt gtgttcctga tcttacagga 40260 aagacttcct attttctcca ttcaggatag tactagttgt gggtctgtca tatagctttt 40320 ttatgttgag gtatgttcct tctatctcaa gtttttttta tgaattttca tcaagaaggg 40380 atgttaaatt ttatcaaata ctttttcagc atcaactgaa atgatcatat ggcttttatc 40440 cttcattctg ttgatatgat gtatcacact gcttgatttg cacatgttga accattcttg 40500 catccaaggg ataaatccca cttggtcatt gtgaatgacc tttttattgt attgttgaat 40560 ttggtttgct agtgttttgt tgaagatttt tgcattagtg ttcatcaggt gtattggcct 40620 gtagtttttt tttttttttt tttttttttt gatgtgtctt agtctgattt tggtatcagg 40680 gtaatactgg cctcatagaa tgagtttgga agtattctct tctcctttat tttttggaat 40740 agtttgagta gggtcggtat tagttcttct ttaaatgttt ggtaaaattc agcagtgaag 40800 ctagccagtc ttgggatttt ctctactggg atgattttat ttctcgtttg atctttgtgc 40860 tggttattgc atgttcagat tttggatttc ttcctggttc aatcttgatg ggtcatattt 40920 gtgtagggat ttgtccattt cttctagatt ttccaattta ttggcatata gttgctcata 40980 gtaaccaata attattcttt gaatttctgc agtatcagtt ctaatgcctc ctttttaatt 41040 ttttatttat ctgaatctac tctttttttt tttcttagtc tggctaacgg tttgtcaatt 41100 ttgtttaact tctcaaaaaa gcaacttttt gtttcattga tcttttgttt tgttttcttc 41160 acttaaattt tatttatttc tgctctgatc tttattattc cttttcttct actaattttg 41220 gttttaattt gcttttgctt ttctaattct ttaggatgca tcattaaatt gtttgtttgt 41280 caattttctc catttttgat ataagctctt gtagctataa acttccctct tagtactggt 41340 cttgctgtaa cccctaggtt ttggtatgtt gtgtttccat tatcatttgt ttcaagaaat 41400 ttttcaattt tctttttaat ctcttcatgg gtccactgtt catttatgag catattttta 41460 aatttccatt tatttgtgta gtttccaaaa ttcctcttgt tattggttgc tggttttatt 41520 acattgtggt cagagaagat gcttgatatt atttcagttt ctttgaatat tttaagactt 41580 gttttgtgac ctaacatacg gtcaattctt gataacaatc catgtgctgt ggaaaagaat 41640 gtgtattctg tagcagttgg ataaaatatc ctgcaaatat ctatgagatc catttgatct 41700 atagtgcaga tgaatttcaa tgtttccttg ttgattttct atctggatga cctgtccaat 41760 gctgaaagtg gggtgttgaa gtctccaggt attattatat tggggcctat ctctctctag 41820 ttctaattat atgtctttta tatatctggg tgctgcatta ttggttgcat atatatttaa 41880 acttgttcca tcttcttgcc aagctgacca ctttatcacc aatagtgatc ttctttgtgt 41940 ctccttatgg tttttgtttt gaaatctact ttgtctgttt taaatatagt aactcatgct 42000 ctttttttca tttccattgg caggtactgt ctcattcaat tcctttattt tcagcctatg 42060 tgtgtcttta taagtgaagt gtgtttcttt taggcaacag attaataggt cttgtttttc 42120 catccaggtc agtaacaggt cagtatgtct tttgattgga gattttattc catttacatt 42180 cagtgttatt attgataagt aaggacttac ccatgcccct ttgttatttg ttttctggtt 42240 gttttgtgga cttctcttcc ttctttcatt tcttcctgtc ttcctttatt gaagagaatt 42300 ttctccactt atatgtgtac agatttttct taatatctgg tttatggcag ttacacattt 42360 gtgcatctgt aaccatcctc tctttaagtt tgcatatact tccagcacta taatttaaat 42420 ttataatgat gtttggatac cttcatgatt catatacccc tgaattgcta caacaaatgt 42480 gccatttttc tccttttcca tcagttttta cttgtgtctt atcagctaaa gtccaggaag 42540 agattgaacg tgtgattggc agaaaccgga gcccctgcat gcaagacagg agccacatgc 42600 cctacacaga tgctgtggtg cacgaggtcc agagatacat tgaccttctc cccaccagcc 42660 tgccccatgc agtgacctgt gacattaaat tcagaaacta tctcattccc aaggtaagtt 42720 tgtttctcct acactgcaac tccatgtttt cgaagtcccc aaattcatag tatcattttt 42780 aaacctctac catcaccggg tgagagaagt gcataactca tatgtatggc agtttaactg 42840 gactttctct tgtttccagt ttggggctat aaaggtttgt aacaggtcct agtgtctggc 42900 agtgtgtgtt ctccagattt attatctttc ttcaagattg gtttggctac tcttaggtgc 42960 ttatatttcc aaataatttt taaaggtatt agtttgtcaa tttcccaaaa ccttgggctg 43020 gaatttctgg cagggtgaca ctaaatttat aggctagttt ggaaagaact gaatcttgac 43080 acgttgaggc tttccattcc tgaatataat tatgcttcca atttgtttgg ggtttctttt 43140 atttaaccag gaatgttgtg aatttgttgt catggctttc gagtctttgg ttttccctag 43200 ataattaata tttttgttgt agaacataaa tagtttttat cattctgatg atgttaatct 43260 gtcaactttg ctaaatttac tagtcactat tcgtaattta tttctggatt cattgtaatt 43320 tctgtgtata ttatactgta tctgagttaa tattgtttta tttcttattt tccatttctc 43380 atgggcttaa tgtctcttta tcacattcat tattgcatta gctagaattt ctaggagagc 43440 attgaataga attggtgaca gtggggatcc ttgtttctca tttctaatct gcaggaagca 43500 gtggaagttt tccatttcaa tattgagaat gatgcttgaa gtagattttg gtagatattt 43560 tttatcagat tagagaagtt tgctgtcata tatatatatg tcataattgt gtgtaaaatc 43620 ttgtcaaatc aacactctgc atctatttat ataattgtgt gttagtccat ttccattgct 43680 ataaaggaat atctttgact gggtaattta taaataaaag agtttcattt tggctcatgg 43740 ctctgcaggc tgtgcaagag gcataatgtg ggcaggcatc tactcctagt aaggacttca 43800 ggtagctttc aatcttcgga gaaggtgaag gggaagcaag catgtcacat ggcaagagag 43860 ggagtaagag aaaaaggagt agaagtgact ttaaacaacc agctagtgtg tgaactaaca 43920 gaatatgaac tcacttatta tcatgggaag aggtttaagc cactcatgaa gaattcaccc 43980 ccatgtccta aatacttctc acaccagaac ccacctccaa cattgagaat cacatttcaa 44040 catgagattt ggaggggaca aatatccaca caacatcaga ttatatatat tgggcttaca 44100 tttctcacta aatagaaata agtatgcaca tagagatgaa ctggccttct tcatattgtt 44160 taggacttca gtgtttacat tcacaagaga tttggtttat agttttattt ctcagaattt 44220 tcaagatttt tgatattaag attatgccag ccttataaaa ctatgcaaga aatgtttcct 44280 aatgtgagac tgtttttatt gccgttccct ctccccactt ttcttcttgt gatcacttag 44340 ttgtatcttt gaaatgcctg ataacttttg gttaaatacc agatggtata tgtaagaaat 44400 tgcagaattg cagaggaggt ttatattctt caaagaaaga ttcattcctc tcccatcggc 44460 agtcataatg acatgatacc tgttgtcatg attcagaatt ggacataatg tgaatccagt 44520 tgaaaattga gtgtcagtgt tagcttagct ctctgaggtt ctcctacctt tggaagcttg 44580 agaccaattt ttgtcttgac agctttgcag tgccttctat cacatggttt ctgtatttat 44640 ccagacactc catttgccat ctcaaaccta ctcttttcca atcaaaacca atatatatta 44700 ttagtaaaat aaaaaataat ataactgaaa gttgtatttt caaggggaaa aagcaatgag 44760 acttggtaag gttgcaatta ctaatgtcat cttttgaaga agacagaatt gataatttac 44820 ttagagatta tggggcttac cctagtggtc actgagttag tacaagagaa aagacttcaa 44880 tgcactctgt tttcacaaac acaggagata accaggagac tttcgcttct caaaattagg 44940 gtctgagggt aatggtaatg aaacttactt cttctcctat aactaattaa atgagaaata 45000 tactgaagaa tcaggcacga gttgccagtc tccccagttc actgcatcag ctttggtgac 45060 ctcctggagg ggaacgctgc atctgatgag ctcatgctgt gcctgatgtc ttttgagccc 45120 tgagaagcca gagcccaagg ggattaccca ttgaatccct tcttcgtgga atagcccagt 45180 gcagaaaaaa gcagcactgt gtggccaatt ctaccctctt tacaaatcag atacatgtgt 45240 tcttctgcta caggctggat ccacttatct agctggagat gatagtctga gacaggagtt 45300 ggcaaactgt ctctgtaatg ggccagatag taaatatttt agacttttca ggcaatgtgg 45360 tttctgttgc aattcttgac atctgctatt gtagcatgat agcagtcaca aacaatctgc 45420 aaatgaatga ggattgatat gttccaattt ttatagaaac aaatgtaaat ttcctataaa 45480 atgtttgttt caaaaatatt gtttcttgtt tgcatttgtt caatgtaagt acttttagct 45540 cacatactat acaaaaacag gtggcagggt ggacttgtgc agggagtatt ttttattgat 45600 gcctaatata agacatccct gtccagtaga attttctgca aagatgtgcg attgatttgt 45660 gcttttatgt catatttaaa gaatatttgt cagcacaaga ttccaaagac tggctttcaa 45720 ctttcccaga atttttttgt tttagctctt atatctagat caatgacata tttcaaatta 45780 atttttattt cataatatga agtaatagtg gtcatgcttc aatgtctata aatttctact 45840 tattcaagta acatttgttg aaataaatct gctttggtat caataacatt ttaatatagt 45900 acttgtttat aaatattcaa gggaatattt ttaaaaaaca aaattataga ctatttatat 45960 gaaaatttag ggggtagaat ttttcctaac atttttatta taaaattttt taaaataaat 46020 aacattacac acacatatgt atatgtacat atggcagtgt gtatacatat ctactctgat 46080 atctgtccat ccatctattt acacctcaat acttcagtat gtatagcaca ataacaatgg 46140 ttttctccta cataataata gcattcatac tgtggagtta ttaatacaat aatattattt 46200 actatacagc ttttatttaa aatttcccaa ctttaaaata atattttaat cacttttttc 46260 ttgcaattca ctctccagtc aagaaataag cacatagttt cagtatcctg ttactttcca 46320 tctcctgtaa tcaagaagag tccatttcct ctgtttcctt tcagtatgtt gatctctgtg 46380 aagagttgag caattagtca tttaaatttt cctggggctc gttttgggta attgttttct 46440 aaatattata ttccagttat gactttggca agaaaactac atagtatttt gaatattctg 46500 ctgaatcaca gtggtgaggc aggggcacat aatgtcacat atgccattat tggttatgct 46560 aagatcctca cctaataagt tggaatctgc cagatctttc cattgaaggg gttctgcttt 46620 ttatgcgtga atgatgaaaa tctatggcat ggtacttcaa ggcagaacac cctgtttccc 46680 aagtcacaca tccaatagtt ttatctttca ttaatgatcc tcatctgacc tgatgatctt 46740 gacctgaatg gttacaaata ggtgattttt ctaatttcat cattccttcc gtatttttaa 46800 gctaggatta atctgtaatg aatatctctc ccatatcttt aaatgaagac aaacaaactg 46860 catggcaatg acaagaactc aatagtttta tccttgccac tacacttgtt accatcccag 46920 aagatgtcat ttgaaattca atcatatgtt tattcacaaa tgccctgtga acagatactg 46980 tggccatttc ccatgtcaac agagacttca acttttagtt atactctaca cataaggttg 47040 gtgaaaactg aagtgatgga atagagaagc aagtgtggac agccactagt ttccaagctt 47100 gatgaaaagg aggacagatt cagaaggttg catccaagta tccaagtact caggacttca 47160 aatgtgattg cagggcactt tagcaagatt attgtcatgg gccttaagtt catgactctt 47220 attacttggt ctatccatct ggaaatggta ctgcccttct ttggaacggg atttcctcat 47280 ctgcacatca aaagatttaa ctacatgatt accactgttt cttcaacctt catggcttct 47340 ttacagctca gttcatctat gtctcttgtt tctagggcac aaccatatta atttccctga 47400 cttctgtgct acatgacaac aaagaatttc ccaacccaga gatgtttgac cctcatcact 47460 ttctggatga aggtggcaat tttaagaaaa gtaaatactt catgcctttc tcagcaggta 47520 atataaattt atttccattt gtgtttcagg gtacaagata acttttttga tccattggaa 47580 cttacatgtg cctcctctgc agtggtacaa ttactctttg tacatgatca agagcactgt 47640 tctgaatgcc tgtgtacacc ctgctcatga tacatcctaa ttattgggcc agattagtgg 47700 actttgggga gttaatccaa ttcttccaaa ttgagaaagc tgaaatatag gttggttgaa 47760 ttctgcctct aggtacacca gtgaggtacc caagaactcc tcctggaaga taaaactaat 47820 tacattttcc tcactagcca tgaggaagtt atctcactcc agaacttcac tgagtgtctt 47880 ccacatggtg tccctcaccc cctaggctgg gcttgtagga taaaattatc cacaaacaca 47940 gaatagggtc ttaaaaggct cacttctgag tttggaaagc agagtaaaca gatcattgta 48000 gttcaatagg actgaggctg tgatatggaa agaacaggct gttgggggtt gggaggtaga 48060 cggaaaagct gtctgcttct tgtactctta taccccaaag tgaggcataa gtaatatttt 48120 aatagcagta aagacatttg agctacctca aaggaggcag agaggatgaa aagaagagaa 48180 gacagggcta ttaaaggaga taatgagcca caggagcagg acattggctc taaataaagg 48240 atattagaac ctttgccaaa tgaccatgga tgggatgagg gggacattgg gaatgtagca 48300 ggatactctg cagtgatgca gagcaccatg ctgttcccct agtcatggcc atgtatattg 48360 gatgttggat ctccatactt gaaatgtgtg tgctgagcat ctggtgatag aatctccttc 48420 ctttattcta tgacctttaa tgtctgcttt atatctgcca ctgtagatac caagacaaca 48480 agaaaagaaa ccttccttca agcattcaca tttagcacat gtattagtcc actcttacat 48540 tgccttaaag aactacctga gattgggtaa tttatgaaga aaagaggttt aattcactca 48600 cagatccaca ggctgtacag gaagcatggc tgggagacct caagaaactt gcaatcatgg 48660 tgggaggcag agcggaagca agcacatctt tccatggcag agcagaagag agagaacaat 48720 gaggaaaggc tacatacctt taaacaacca gatctcatga gaactcactc actatcatga 48780 gaacagaaag ggggaaatta tcccccatga ttcaatcacc tctcaccagg tatctccccc 48840 aacattggga attacagttt gacatgagat ttgagtaagg acacagagcc caaccatatc 48900 gcacactaat aaataaataa ataaatcagt aattattatt ctaggtgatc catgctgtct 48960 gtttcttctg ccataacaaa atatccaaga ctaggcaatt gataaataat ggaaatttat 49020 ttctcacagt tctgaaggct gggagttcca agatcgatgt gtcagcaagt tcagtgtctg 49080 atgtgggcct gttccttaca gataatgccc cctctgtatc cttatatggc agaagggcaa 49140 aaaggcaaaa ggggagaaat ggctcccttg catctcttga tgttattatt ttagcaagga 49200 cagagccctc ctgacttatt cacttcctaa aaggagccat ctctttagta atgttgcatt 49260 gggattatgt gtcaacatat gaaattgggg gaggcatatt cagaccatag cacatttttc 49320 aatggaaata taatgttgag gaaacccaga gaaggcaaca ttttcttgct caaggagatg 49380 agaaagaggg taaaaaagga gataaaattt gacctatgtc ctgactgtgg taatagaaaa 49440 gttcatcttg gctaaaagga gcagcatgat ataaaatttg aaacctcatg gtgtgttgga 49500 gactgatgat gagtggctat gcctagagtt gacagtatcg gatttgaaga gtgtaaggag 49560 tgatgtggat catcagactg gaaacagaat gtgagggtcc agatcaatcc attgggacct 49620 tatcctatag gacatacagg gaagccattt aaagttttaa agtgagaagg tgacatgttt 49680 agacatgtgc tcctgaaagt acctagagga aaaaaatctt tggctgcata ttgagccaga 49740 aatacaaagg gaaatacagt atgttagcct cctcctctaa gcccttctca gttcaaccca 49800 ctggacaaga aatgtatgtt tctaaagaaa gattgatgaa gacatttaaa gtctcttgaa 49860 agattttaat aaagtgcttg gcatgtagct ggtactcaac aaatatttgt tgaatacagg 49920 gtgcctgtta agatctgata ttaggtgaag agtaagtatg tccattcatt tttcagttgc 49980 ctatacatcc atccattcat ccatttatcc atccactcat ccatccattc attcatgcat 50040 gcacccatcc acccatctat ctcttcatct cttctacgat acactgaaca gttattgcat 50100 attctgtttg tgccagttac agagacagtg tttgtcactg tcacagttac gcatgaggag 50160 taactgctct ctgtgtttgc tattttcagg aaaacggatt tgtgtgggag aagccctggc 50220 cggcatggag ctgtttttat tcctgacctc cattttacag aactttaacc tgaaatctct 50280 ggttgaccca aagaaccttg acaccactcc agttgtcaat ggatttgcct ctgtgccgcc 50340 cttctaccag ctgtgcttca ttcctgtctg aagaagagca gatggcctgg ctgctgctgt 50400 gcagtccctg cagctctctt tcctctgggg cattatccat ctttcactat ctgtaatgcc 50460 ttttctcacc tgtcatctca cattttccct tccctgaaga tctagtgaac attcgacctc 50520 cattacggag agtttcctat gtttcactgt gcaaatatat ctgctattct ccatactctg 50580 taacagttgc attgactgtc acataatgct catacttatc taatgttgag ttattaatat 50640 gttattatta aatagagaaa tatgatttgt gtattataat tcaaaggcat ttcttttctg 50700 catgttctaa ataaaaagca ttattatttg ctga 50734 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> VKORC1_3673_A primer <400> 3 acctgaaaaa caaccattgg tca 23 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> VKORC1_3673_G primer <400> 4 acctgaaaaa caaccattgg tcg 23 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> VKORC1_3673_Rev primer <400> 5 tcctgacctc aagtgatcca cc 22 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> VKORC1_6484_C primer <400> 6 cggtgccagg agatcatcgt cc 22 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> VKORC1_6484_T primer <400> 7 cggtgccagg agatcatcgt ct 22 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> VKORC1_6484_Rev primer <400> 8 ctgcccgaga aaggtgattt ccaag 25 <210> 9 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> VKORC1_6853_C primer <400> 9 cacccgcagg acgcacg 17 <210> 10 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> VKORC1_6853_G primer <400> 10 cacccgcagg acgcacc 17 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> VKORC1_6853_rev primer <400> 11 cccactccat gcaatcttgg 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> VKORC1_9041_A primer <400> 12 ccctcctcct gccatactca 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> VKORC1_9041_G primer <400> 13 ccctcctcct gccatactcg 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> VKORC1_9041_Rev primer <400> 14 ggcaatggaa agagctttgg 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CYP2C9_430_C primer <400> 15 cgggcttcct cttgaactcg 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CYP2C9_430_T primer <400> 16 cgggcttcct cttgaactca 20 <210> 17 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> CYP2C9_430_REV primer <400> 17 gggaggatgg aaaacagaga ctta 24 <210> 18 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> CYP2C9_1075_A primer <400> 18 ggtgcacgag gtccagagat tca 23 <210> 19 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CYP2C9_1075_C primer <400> 19 gtgcacgagg tccagagatt cc 22 <210> 20 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> CYP2C9_1075_REV primer <400> 20 gatactatga atttggggac ttcgaa 26 <210> 21 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> VKORC1_3673_probe <400> 21 tgcggtggct cacgcctata atcct 25 <210> 22 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> VKORC1_6484_probe <400> 22 tgggaggtcg gggaacagag gatag 25 <210> 23 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> VKORC1_6853_probe <400> 23 tgagcagcta gctggctgtc agctgt 26 <210> 24 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> VKORC1_9041_probe <400> 24 accaaatgtg ccacacgctc gct 23 <210> 25 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CYP2C9_430_probe <400> 25 tgggcagcca tgtggatggg cc 22 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CYP2C9_1075_probe <400> 26 tccccaccag cctgccccat 20 <110> Bioneer Corporation <120> Method of Determining Single Nucleotide Polymorphism of Target          Gene Using Polymerase Chain Reaction and Kit for Determining          Single Nucleotide Polymorphism of Target Gene Using the Same <130> 2011-dpa-0120 <150> KR 10-2011-0004724 <151> 2011-01-18 <160> 26 <170> Kopatentin 1.71 <210> 1 <211> 12043 <212> DNA <213> Homo sapiens <400> 1 gtgcagtggt gtgatctcgg ctcactgcaa cctctgcctc ctgggttcaa gcaattctcc 60 tgcctcagcc tcctgagtag ctgggattac aggcatgcac caccatggcc agctaatttt 120 tgtattttta gttgagaagg ggtttcacca tgttgaccag gctggtctca aactcctaac 180 cttgtgatcc acctgccttg gcctcccaaa gtgctgggat tatgggcatg agccaccaca 240 ccccacagcc ttactctttc tccctctggg catgagctag ctgagctgtg tcttggcttc 300 cccgagatgg acagctctgg ctttctctct ttctctgggt acaagcacct gcacaagagc 360 catgtcccat gtcaagccat gttgggccga gccctgtgca cagtgtcagc agggcaatta 420 taccttttac agacaacagt ggctcagagc caagtgatga gccttcccat gttatggcta 480 cacggctgtg ataacaagtg gagctatatg cctgcactct aaactcactg agtcactctg 540 gatgtttact ttggcctatg cccacttgac tgcagcacac ccacgtttct tacagggcct 600 cagttcctca ctaaggagac ctctctccct agggttactt gagtgctttc atatcatggt 660 agctggcttt gtctagaatg agtgatctac aaaggagcaa gatggaagcc acaatatctt 720 catgatttag cctctaaagt cacacactgt catttctgca ttatcccaat gggccagttc 780 agcccctttc aatgtgagaa gagacttcac aatggtatca ataccaggaa gcaggaatca 840 ctgaggacca ttttggaagc tgactattaa agccttaagg ctgccatgct gtgaggaagc 900 ccaagctagc ccatgcagag gacaacatag ataggttcgg agactactca gagaaaggga 960 gagagaatgc taggttatct ttcagctgtt ttagctgctt attgttccag ctttagccac 1020 catctgactg caattgtatg agccagaaat agccaggtaa aattgtcttc ccaaattcct 1080 agtgcacaaa aactgtcaga ggctgggggc agtggctcac tcctgtaatc ccagcagcac 1140 tttgggaagc cagggtggga gaactgcttg agcccaggag ttagaggcaa cataacaaca 1200 actcatctct aatgaaaaaa aaaaaaaaag aaagaaaaag gccaggcaca gtggctcacg 1260 cctgtaatcc cagcacttta ggaagccaag gagggcggat catgaggtca ggagtttgag 1320 accagcctgg ccaatatggt gaaaccccgt ctctactaaa aacacaaaaa tcagccacgc 1380 atagtggctg gtgcctgtaa tcacagctac tcaggaggct aaggcaggag aatcacttga 1440 atccgggagg cagaggttgc agtgagccga gattgcacca ctgcatttca gcctgggcag 1500 cagagtggga ctgtctcaca ttaattaatt aattgattaa atgctagaag aaaatatggt 1560 caaaatcttt tataactctt ggagtgagga aggcaatctc ggctcactac aacttccacc 1620 tcccgggttc aaatgattct cctgcctcag cctcctgagt agctgggatt acaggcaccc 1680 accaccatgc ccggctaatt tttgtatttt tagtagagac gaggtttcgc catgttggcc 1740 aagcttgtct taaactcctg acctcaggtg atccacccac ctcagcctcc caaattgctg 1800 ggattacagg catgagccac catgccccgc ctaattttaa aaatgtttat aaagacagag 1860 tcttaccacg ttgtccagac ctgtctcaaa ctcctgggct caagtcattg tcttgtctca 1920 gcctcccaaa gttctgggac tacaggtgtg caccaccaca cctggctaat tttgtttatt 1980 ttttatagag tgagggtctc cctgtgttgt ccagggtgtc tcaaactcct gggctcaagt 2040 ggtcctcctg cctcagcctc ccaaagtgct gggattatag gcataagcca ctgcacctgg 2100 ccccagtgtg tgtcttctga ggctaagtca taaagagatt gcggcttcta ttttgcttcc 2160 tcttagatca ctcattctgg gggaaaccaa caataaataa caggaacaca ccaggagatc 2220 agagaaagct gagatacagg cctaatgccc agtcctctgt attcatttct aattgctgct 2280 gtaacaagtt actgcacatt taatgatgta aaacaacaca aatttatctt acagttctgt 2340 agcttagaag tctgacagag tctcactggg ctgaaatcaa gatgttgata gttccttccc 2400 ggtgactcta gagtgagaat ccagttcttt accctttctt gcctttagag gccacccgta 2460 tttattagct cacaatcccc ttcctccatc ttcaaaccag aaacattgca gctctctgtg 2520 tcttttttct ttactcacat ctccctctga ctttcttctg ccatcctcct tcatttttta 2580 aggacccctg tgggccaggc atggtggctt atgcctgtaa tcccagcact ttgagaggcg 2640 gaggcaggcg ggtcacctga ggtcaggagt tccagaccag cctggccaac atggcaaaac 2700 cccatctcca ctgaaaatac aaaaattggg ccaggcacgg cggctcacac ccgtaatccc 2760 catactttga gaggctgagg caggtggatc acttgaggtc aggagttcaa gaccagcctg 2820 gccaacatgg tgaaatcccg tctctactaa aaaaaaaaaa aattacaata attagtcagg 2880 cgtggtggcc agcacctata atcccagcta cttgggaggt tgaggcacaa gaattgcttg 2940 aacccgagag ggggaggttg cagtgagctg agattgcacc accgtactcc agcctggatg 3000 acagagcaag actctgtctc aaaataaaaa ttaagataaa taataaaaaa taaataaaaa 3060 tcagcaaatt aggccaggtg tggtggtggc tcatgcctgt actcccagca ctttgaggtg 3120 ggaaggttgc ttgaaaccag aagtttagac cagcctggga aacaaagtga gaccccatct 3180 ctacaaaaat aaaaataaat tagccaggtg tggtgggacg tacttgtact cctagctact 3240 agggaggctg aggtgcaaga atcgcttgaa cctgggagac ggaggttgca gtgagttatg 3300 acagtgccac tacactccag cctgggtgtc aagggtcttt gaaaaaataa acaaaatagg 3360 ctgggcgtgg tggcttacac ttgtaatccc agcactttgg gaggctgagg caggcagatc 3420 acaaagtcag gagttcgaga ccagcctggc caacacagtg aaaccccgtc tctactaaaa 3480 atacaaaaat tagccaggca tggttgcacg cgcctgtagt cccaactgct cgggaggctg 3540 aggcaggaga atggcttgaa cctggggggg ggcggaagtt ataatgagcc aagattgtgc 3600 cactgcactc cagcctaggc aacagagcga gactccgtct caaaaataaa aataaaaata 3660 aatcaataga gcctggtatg atggctcacg cctataatcc cagcactttg ggaggcccag 3720 gtgggtggat catctgaggt caggagtttg agaccagcct gaccaacatg gagaaacccc 3780 atctctacta aaaatacaaa aaattagcca ggcgtggtgg cacatgccta taatcccagc 3840 tactcaggag cctgaggcag gagaatcgct ttaaccgggg aggtggaggt tgcggtgagt 3900 cgagatagca ccattgcatt ctagcctggg caacaagagc aaaactccat ctcaaaaata 3960 aataaataaa tagatagata ggctgggcac ggtggctcac gcctgtaatc ccagcacttt 4020 gggaggccga ggtgggcgga tcacctgagg tcaggagttc aagaccagcc tggccaatat 4080 agcgaaaccc tgtctctact aaaaatttaa aaaattagcc aggcatgatg gcgggtgcct 4140 gtaatcccag ctactcggga ggctgaggca ggagaatctc tcgaacctgg caggcggagg 4200 ttgcagtgag ccgagatcac accattgcac tccagttggg caacaagagc gaaactccac 4260 ctcaaaaaca aaataaaata aaataaaatc aggagattgg tcagcttaat tccatctgca 4320 accttaattc ccttttggcc atttaaccta tcatattcac aagttccagg gattcatgca 4380 gggacatctt tggtgaccat tattctgtct accacactct ctagaagaga gtagatgtga 4440 gaaacagcat ctggagaggg aggagccagc aggagaggga aatatcacag acgccagagg 4500 aagagagttc ccagaagggt aggtgcaaca gtaagggatc cctctgggaa gtcaagcaag 4560 agaagacctg aaaaacaacc attggccggg tgcggtggct cacgcctata atcctagcat 4620 tttgggaggc cgaggtgggt ggatcacttg aggtcaggag tttaagacaa gcctggccaa 4680 catggtgaaa ccctgtctct actaaaaata caaaaattag ccagacatgg tggcaggcac 4740 ctgtagtcca aactacttgg gaggctgagg cacgagaacc acttgaaccc tggaggcgga 4800 agttgcagtg agccgagatg gcaccgctgc actccagcct gggcgacaga gtgagactcc 4860 gtctcaaaat aagtaaataa ataaataaat aaccattggg tctagcgtct tggtgacacc 4920 agccactagg tgtcagggca atgctggagg cagcagctag tttatggcgt gggcagcaag 4980 gaagaagtga ggaagtagag actgggagtg tagaatcttt ccaaaaactt gaatgtggag 5040 gggtagatgg gataatgccg ggagaggagg ctggtgaagg acctcttagt gaagggagat 5100 ttgttgctgt ttctgagatg ggaaagagaa acaagttgca gtgtagatgg ggaggatgga 5160 gaggttgaag gtgtaggaga gatgggtcac catcagatgg gacgtctgtg aaggagagac 5220 ctcatctggc ccacagcttg gaaaggagag actgactgtt gagttgatgc aagctcaggt 5280 gttgccaggc gggcgccatg atagtagaga ggttaggata ctgtcaaggg tgtgtgtggc 5340 caaaggagtg gttctgtgaa tgtatgggag aaagggagac cgaccaccag gaagcactgg 5400 tgaggcagga cccgggagga tgggaggctg cagcccgaat ggtgcctgaa atagtttcag 5460 gggaaatgct tggttcccga atcggatcgc cgtattcgct ggatcccctg atccgctggt 5520 ctctaggtcc cggatgctgc aattcttaca acaggacttg gcatagggta agcgcaaatg 5580 ctgttaacca cactaacaca cttttttttt tctttttttt ttttgagaca gagtctcact 5640 ctgtcggcct ggctggagtg cagtggcacg atctcggctc actgcaacct ccggctcccc 5700 ggctcaagca attctcctgc ctcagcctcc cgagtagctg ggattacagg catgtgccac 5760 cacgcccggc taatttttgt atttttagtt gagatggggt ttcaccatgt tggcgaggct 5820 ggtcttgaac tcctgacctc aggtaatccg ccagcctcgg cctcccaaag tgctgggatt 5880 acaagcgtga gccaccgtgc ccggccaaca gtttttaaat ctgtggagac ttcatttccc 5940 ttgatgcctt gcagccgcgc cgactacaac tcccatcatg cctggcagcc gctggggccg 6000 cgattccgca cgtcccttac ccgcttcact agtcccggca ttcttcgctg ttttcctaac 6060 tcgcccgctt gactagcgcc ctggaacagc catttgggtc gtggagtgcg agcacggccg 6120 gccaatcgcc gagtcagagg gccaggaggg gcgcggccat tcgccgcccg gcccctgctc 6180 cgtggctggt tttctccgcg ggcgcctcgg gcggaacctg gagataatgg gcagcacctg 6240 ggggagccct ggctgggtgc ggctcgctct ttgcctgacg ggcttagtgc tctcgctcta 6300 cgcgctgcac gtgaaggcgg cgcgcgcccg ggaccgggat taccgcgcgc tctgcgacgt 6360 gggcaccgcc atcagctgtt cgcgcgtctt ctcctccagg tgtgcacggg agtgggaggc 6420 gtggggcctc ggagcagggc ggccaggatg ccagatgatt attctggagt ctgggatcgg 6480 tgtgcccggg gaacggacac ggggctggac tgctcgcggg gtcgttgcac aggggctgag 6540 ctacccagcg atactggtgt tcgaaataag agtgcgaggc aagggaccag acagtgctgg 6600 ggactgggat tattccgggg actcgcacgt gaattggatg ccaaggaata acggtgacca 6660 ggaaaggcgg ggaggcagga tggcggtaga gattgacgat ggtctcaagg acggcgcgca 6720 ggtgaagggg ggtgttggcg atggctgcgc ccaggaacaa ggtggcccgg tctggctgtg 6780 cgtgatggcc aggcgttagc ataatgacgg aatacagagg aggcgagtga gtggccaggg 6840 agctggagat tctggggtcc agggcaaaga taatctgccc ccgactccca gtctctgatg 6900 caaaaccgag tgaaccgtta taccagcctt gccattttaa gaattactta agggccgggc 6960 gcggtggccc actcctgtaa tcccagcact ttgggaggcc gaggcggatg gatcacttga 7020 agtcaggagt tgaccagcct ggccaacatg gtgaaagcct gtctctacca aaaatagaaa 7080 aattaatcgg gcgctatggc gggtgcctta atcccagcta ctcggggggg ctaaggcagg 7140 agaatcgctt gaacccggga ggcggaggtt tcagtgagcc gagatcgcgc cactgcactc 7200 cagcctgggc cagagtgaga ctccgtctca aaaaaaaaaa aaaaaaaaaa aaaaaagaga 7260 cttacttaag gtctaagatg aaaagcaggg cctacggagt agccacgtcc gggcctggtc 7320 tggggagagg ggaggatagg gtcagtgaca tggaatcctg acgtggccaa aggtgcccgg 7380 tgccaggaga tcatcgaccc ttggactagg atgggaggtc ggggaacaga ggatagccca 7440 ggtggcttct tggaaatcac ctttctcggg cagggtccaa ggcactgggt tgacagtcct 7500 aacctggttc caccccaccc cacccctctg ccaggtgggg caggggtttc gggctggtgg 7560 agcatgtgct gggacaggac agcatcctca atcaatccaa cagcatattc ggttgcatct 7620 tctacacact acagctattg ttaggtgagt ggctccgccc cctccctgcc cgccccgccc 7680 cgcccctcat cccccttggt cagctcagcc ccactccatg caatcttggt gatccacaca 7740 gctgacagcc agctagctgc tcatcacgga gcgtcctgcg ggtggggatg tggggaggta 7800 actaacagga gtcttttaat tggtttaagt actgttagag gctgaagggc ccttaaagac 7860 atcctaggtc cccaggtttt ttgtttgttg ttgttttgag acagggtctg gctctgttgc 7920 ccaaagtgag gtctaggatg cccttagtgt gcactggcgt gatctcagtt catggcaacc 7980 tctgcctccc tgcccaaggg atcctcccac cttagcctcc caagcagctg gaatcacagg 8040 cgtgcaccac tatgcccagc taatttttgt ttttgttttt ttttggtaga gatggtgtct 8100 cgccatgttg cccaggctgg tctcaagcaa tctgtctgcc tcagcctccc aaagtgctgg 8160 ggggattaca ggcgtgagct accatgcccc accaacaccc cagttttgtg gaaaagatgc 8220 cgaaattcct ttttaaggag aagctgagca tgagctatct tttgtctcat ttagtgctca 8280 gcaggaaaat ttgtatctag tcccataaga acagagagag gaaccaaggg agtggaagac 8340 gatggcgccc caggccttgc tgatgccata tgccggagat gagactatcc attaccaccc 8400 ttcccagcag gctcccacgc tccctttgag tcacccttcc cagctccaga gaaggcatca 8460 ctgagggagg cccagcacca tggtcctggc tgacacatgg ttcagacttg gccgatttat 8520 ttaagaaatt ttattgctca gaactttccc tccctgggca atggcaagag cttcagagac 8580 cagtcccttg gaggggacct gttgaagcct tctttttttt tttttttaag aaataatctt 8640 gctctgttgc ccaggctgga gtgcagtggc acaatcatag ctcactgtaa cctggctcaa 8700 gcgatcctcc tgagtagcta ggactatagg catgtcactg cacccagcta attttttttt 8760 tttttttttt tttttttttg cgacatagtc tcgctctgtc accaggctgg agtgcagtgg 8820 cacgatcttg gctcactgca acctctgcct cccgggttca agcaattttc ctgcctcagc 8880 ctcctgagta gctgggacta caggcgcgtg tcaccacgcc cagctaattt ttgtattttt 8940 agtggagaca gggtttcacc atgttggcta ggatggtctc aatctcttga cctggtgatc 9000 catccgcctt ggcctcccaa agtgctagga ttacaggcgt gagtcaacct caccgggcat 9060 tttttttttg agacgaagtc ttgctcttgc tgcccaagct ggaatgtggt ggcatgatct 9120 cggctcactg caacctccac ctcctaggtt caagcgattc tccaccttag cctccccagc 9180 agctgggatt acaggtgccc atcaacacac ccggctaatt tttgtatttt tattagagat 9240 ggggttttgc catgttggcc aggctgctct cgaactccta acctcaggtg atccaccccc 9300 attggcctcc caaaatactg ggattacagg catgagccac cgtgcccagc tgaatttcta 9360 aatttttgat agagatcggg tctttctatg ttgcccaagc tggtcttgaa ctcctagcct 9420 aaagcagtct tcccacctcg gcctcccaga gtgtttggaa tacgtgcgta agccaccaca 9480 tctgccctgg agcctcttgt tttagagacc cttcccagca gctcctggca tctaggtagt 9540 gcagtgacat catggagtgt tcgggaggtg gccagtgcct gaagcccaca ccggaccctc 9600 ttctgccttg caggttgcct gcggacacgc tgggcctctg tcctgatgct gctgagctcc 9660 ctggtgtctc tcgctggttc tgtctacctg gcctggatcc tgttcttcgt gctctatgat 9720 ttctgcattg tttgtatcac cacctatgct atcaacgtga gcctgatgtg gctcagtttc 9780 cggaaggtcc aagaacccca gggcaaggct aagaggcact gagccctcaa cccaagccag 9840 gctgacctca tctgctttgc tttggcatgt gagccttgcc taagggggca tatctgggtc 9900 cctagaaggc cctagatgtg gggcttctag attaccccct cctcctgcca tacccgcaca 9960 tgacaatgga ccaaatgtgc cacacgctcg ctctttttta cacccagtgc ctctgactct 10020 gtccccatgg gctggtctcc aaagctcttt ccattgccca gggagggaag gttctgagca 10080 ataaagtttc ttagatcaat cagccaagtc tgaaccatgt gtctgccatg gactgtggtg 10140 ctgggcctcc ctcggtgttg ccttctctgg agctgggaag ggtgagtcag agggagagtg 10200 gagggcctgc tgggaagggt ggttatgggt agtctcatct ccagtgtgtg gagtcagcaa 10260 ggcctggggc accattggcc cccaccccca ggaaacaggc tggcagctcg ctcctgctgc 10320 ccacaggagc caggcctcct ctcctgggaa ggctgagcac acacctggaa gggcaggctg 10380 cccttctggt tctgtaaatg cttgctggga agttcttcct tgagtttaac tttaacccct 10440 ccagttgcct tatcgaccat tccaagccag tattggtagc cttggagggt cagggccagg 10500 ttgtgaaggt ttttgttttg cctattatgc cctgaccact tacctacatg ccaagcactg 10560 tttaagaact tgtgttggca gggtgcagtg gctcacacct gtaatccctg tactttggga 10620 ggccaaggca ggaggatcac ttgaggccag gagttccaga ccagcctggg caaaatagtg 10680 agacccctgt ctctacaaaa aaaaaaaaaa aaaaaaatta gccaggcatg gtggtgtatg 10740 tacctatagt cccaactaat cgggaagctg gcgggaagac tgcttgagcc cagaaggttg 10800 aggctgcagt gagccatgat cactgcactc cagcctgagc aacagagcaa gaccgtctcc 10860 aaaaaaaaac aaaaaacaaa aaaaaacttg tgttaacgtg ttaaactcgt ttaatcttta 10920 cagtgattta tgaggtgggt actattatta tccctatctt gatgataggg acagagtggc 10980 taattagtat gcctgagatc acacagctac tgcaggaggc tctcaggatt tgaatccacc 11040 tggtccatct ggctccagca tctatatgct tttttttttg ttggtttgtt tttgagacgg 11100 actttcgctc ttgttgtcca ggctggagtg caatggcaca atttcggctc accacaacct 11160 ccacctccca ggttcaagtg attctcctgc ctcagcctcc cgagtagctg ggattacagg 11220 catgcgccac caggcctggc taattttgta tttttagtag agacagtgtt tctctatgtt 11280 ggtcaggctg gtctcgcact cccgacctca ggtgatctgc ctgctttggc ctcccaaagt 11340 gctgggatta caggcgtgag ccaccatgcc cggccagcat ctataatata tgctaaacct 11400 tacccacagc tgcctctgtc ttggggtcag ggttcttgaa tggtctgggc cagcctgagt 11460 taggagtggg aagaggaaga ggaggcacgc gccaaagtgt tcttcccgcc tgaccgagtc 11520 ctctgcgcct atgaggaagg tgggcaccgc tgtcaccctg tttcccagga agccagcagg 11580 cccagggaga tactgggatt tgccaaaggc tcacagcgag caagcagcag tgggtgctgg 11640 ctcctcacca ctcagcctgg cacacactgt ctctgagcag acctgggaga aggcatgtga 11700 aacccaccta gccctggctg gaagagaagt gggggagtgg aaatgaaaag tggggactgg 11760 gaggagctgc ccacgtgccc tccatggttc caggatggca ctttcatgct gggagtgagc 11820 ttgccctgtg ccaggctgtc attgagacgt tttccattca acaactgttt ctcagacaag 11880 gcacaggcag gttagctctc ccaggccact caagtagtaa ggaaacagat ttaaacccag 11940 acaatttggc tccacggcta aggcccagag aggggtggtg acaagcctag ggtcacacag 12000 ccacagcccc agagctagga actgctggct agggcaagca gac 12043 <210> 2 <211> 50734 <212> DNA <213> Homo sapiens <400> 2 gtcttaacaa gaagagaagg cttcaatgga ttctcttgtg gtccttgtgc tctgtctctc 60 atgtttgctt ctcctttcac tctggagaca gagctctggg agaggaaaac tccctcctgg 120 ccccactcct ctcccagtga ttggaaatat cctacagata ggtattaagg acatcagcaa 180 atccttaacc aatgtaagta tgctccttca gtggcttgca aaaggtaagt aaattcacct 240 gtatttttta aataaagtgt atccctagag gtacatgtta caagaggtaa tggtaaagta 300 aaatactttg aaaggctttt gttgcctttt ccagtctgtc agtgtcagaa atagtggaat 360 gaaaccatgt attttgtgag tagagaaaga tttgggtctt tgcatgttag attcaaaata 420 acaagtgtca atagtttgaa aagctgtgtt ccttcttcat ttcataacca tttgctataa 480 tttttggctg aaggtaaatg gtaaggtatt gtgggatctg gtcagcagcc cacaaagcaa 540 ctgggctctc tcttttttcc caggtggatc ggcaggttga gaaataatag acacacaaga 600 tagtgaaagc tgggtccagg ggggtcaccg ccttctggtc ccatggtgcc aagaatgcac 660 tggatatacc agcatttatt attaagttta gtgagggcag gggtaggtta gtgagggatt 720 tagggtcatt tgattatgag gtgagatggt cacatgggga tgaagtaatt ctttaacata 780 acatttgtat gtagaagtac agtacatttg tatgtagaag tacagtatac ggagataaga 840 atttacaata tagtgtgtgc gtcagtaatt tctaacagag ccttaaaaca gaaacacaat 900 ctttccataa cctatgatta gcaagatatt aatcagcagt aacaattgca acaaaagctg 960 gttacaaaca atccatggaa ataggatgtg aagctagaca accagttaga ccagaaattc 1020 tcagaaggga gtatgcctta accctaaaga ggcctagaag agccatggca agatgagggc 1080 atttatagcc ctatcttatc catatggaca ggtgcccccc atgcgtccat ttataggttc 1140 tccacaaagg tcgcattcca ttcccagagc tatgaacatc tgcttttctg ggataggaat 1200 cttggtgatg tgaaacctct ctgactgcac gtccattcat aggctctctg cagggggaag 1260 cacatcacgc gctgttggct cattctggca gtccagcctg gcactgtcct tacacaatcc 1320 tgcatgcaat tttgtattta caataatcgg gagcatttca tcttttattc cgtagcaata 1380 gtttcagggg gggtctccct acagtaagga acatgtttgt tattagagat tttattaaat 1440 aagtcctcta ctatattagc catgtgtttt attcagaata gccatgaaaa ttaaacttct 1500 ctgaagaatt aatttatgcc tgttgtaaag gaaataactg tgggttcagg accagcaaaa 1560 gcagataagt ggtagaaaag aagttataga cttccatttt tagatccaca aagtactgtt 1620 ttaattatac tatagctaca atatataaac cccaaaatga ccttagcatt tagtaatggt 1680 aggaagaagg tcagaagatg tataagtatt agggtttttt ctcaagtgaa agatggttta 1740 gcagtgttaa tatgatatgt gaacacccct cgttgtgtaa taattaccat atacagagag 1800 taaagggctg caattagtat attaagtctt ataagcatga tggtatgatt agatcaagag 1860 aatgacgctg tagttatgaa gactactcct actagattga tggtaggggg cagggcaagg 1920 ttggcaagac tagctaggag tcatcacatg gctatcaggg agagaagtgt ttgaaggcct 1980 tgtgttagta gtatggtttg gctgtgaact cgtttgtagt ttgagtttgc aaggcagaat 2040 aatagggatg aggttagtct gtgggcagtt attagggaag ttgcactggt aaaacttcag 2100 ggggtttgaa tgagaaaagc tacgataaca agtgccatgt ggcttatgga aaagtaatga 2160 gtgattttag atcagtttgg cacagacaaa aggagcttgt tatgattatt tctcaaaagg 2220 atagtaagaa ggaggaataa gctataaact ctgttagggg gttaagaatt aaggtgattt 2280 gtataatcct atagccatct aattttagga gtactgctgt gaggactatt gacctggcaa 2340 ttgcggcctc tatgtgggct tttgggagtc ctaggtggag tccatagaga gatattttta 2400 gtataaatgc tatgatgcat gctaatcata aaaggctatt aaaccaggaa attgttagtt 2460 cttgaagctg ggtattggtc ttaaggtaga ctcgaaagtg ctgataaatt tctcaagcat 2520 cagtgtttga ataagcggag tttcaaattt tggtctgctg tacattagct gtgagacact 2580 gaaaatgaat ttgtcattct ctgagctcag tttttttttt tttttttttt ttttttgaga 2640 cagagtctta ctctgtagct caggctggag tgcagtggta caatcttggc tcactgcaac 2700 ctccatctcc caggtcccca ttcaagaaat tctcctgcct cagtccccca agtagctagc 2760 attacaggca tgcaccacca tgctcagcta atttttgtat ttttagtaga gacgtggtat 2820 caccttgttg gccaggctgg tcttgaactc ctgaccttgt gatccacctg ccttggcctc 2880 ccaaagtgtt gggattacag gcaggagcca ccacacctgg ccgtttgttt aaaatagagt 2940 aaatagacct gctgaatatg ttgatgtgag tattaattgt aatctgcata gcaattgtct 3000 gaccattgcc ttgaacatca caggccatct gagtggcaag tataatcatc atcatgtttc 3060 tatttaaaat tcagaaatat ttgaagcctg tgtggctgaa taaaagcata caaatacaat 3120 gaaaatatca tgctaaatca ggcttagcaa atggacaaaa tagtaacttc gtttgctgtt 3180 atctctgtct actttcctag ctctcaaagg tctatggccc tgtgttcact ctgtattttg 3240 gcctgaaacc catagtggtg ctgcatggat atgaagcagt gaaggaagcc ctgattgatc 3300 ttggagagga gttttctgga agaggcattt tcccactggc tgaaagagct aacagaggat 3360 ttggtaggtg tgcatgtgcc tgtttcagca tctgtcttgg ggatggggag gatggaaaac 3420 agagacttac agagctcctc gggcagagct tggcccatcc acatggctgc ccagtgtcag 3480 cttcctcttt cttgcctggg atctccctcc tagtttcgtt tctcttcctg ttaggaattg 3540 ttttcagcaa tggaaagaaa tggaaggaga tccggcgttt ctccctcatg acgctgcgga 3600 attttgggat ggggaagagg agcattgagg accgtgttca agaggaagcc cgctgccttg 3660 tggaggagtt gagaaaaacc aagggtgggt gaccctactc catatcactg accttactgg 3720 actactatct tctctactga cattcttgga aacatttcag gggtggccat atctttcatt 3780 atgagtcctg gttgttagct catgtgaagc gggggtttga agctgagagc caagggaatt 3840 tgcacatatt tgtgctgtgt gtgtacaggc atgattgtgc gtacagtgtg ggtataaaag 3900 gttcatttaa tcccatgttc tcctgaactt tgcttttttg ctttcaaata agaaatgatg 3960 aatatagatt ttgagttcat tttttgaaag agttaaagag cagtgttttt cccattacct 4020 attccagaac atgtcaccag agaatacttg acaagtcaac atggtgggaa tggccctatc 4080 atacccatat ggagcatgaa ccaaatggca tgtgctttta tttaattgga ctgtgtttgt 4140 atggtcagcc tcactgactt ctctggggtt tcttttaggc ccgtgcttgc cattctggcc 4200 agtaatgaca ttctacagtt tttattgctt aggcatatct tagtgcagtt ctcatcaatt 4260 attatttctc tgtaaacaca gcattatttt aaaaatagta ttaattattt cttgttactg 4320 tattgattta tatattttca gtaaatacat cctgtagcat aattctgtga aatacccaaa 4380 tgtcaattta taaaatgatt tatttaacaa gattttactt attagtaata actctgtaat 4440 ctgcattccc tatgtatgat ttggctctgt ttcagttttg cttatctctt tccaaccata 4500 tttatgaaat tttggcttag aaatttatgt taattatttt ttttccatgg ccaactctac 4560 tcatctatga agttttacaa tgaatctgtt tatcagcttg gataccaaat taccttgttt 4620 ttaaattctg ttttcccaat gaagttaaag actgaaaatc agatttatct gtgaatgaca 4680 cacacaaact aacagatttc caactgttca atgcctggcc attcattcag agtacttttg 4740 attaaagtca ctatttaggg cctatagata tcaggggaaa ttcagcccct gatatttcaa 4800 tgtgagtcct tttctattct ccctaagtgt tggctggtct gagaaataaa gggaaagagt 4860 acaaaagaga gaaattttaa agctgggtgt ccaggggaga catcacatgt cggcaggttc 4920 cgtgatgccc cctaaaccac aaaaccagca agttttcatt agtgattttc aaaagcggaa 4980 ggaatgtgtg aatagggtgt gagtcacaga gatcacatgc ttcacaaggt aataaaatat 5040 cacaaggcaa atggaggcag ggtgagatca caggaccgga gtgaaattaa aattgctaat 5100 gaagtttcag gcactcattg tcattgataa catctcatca ggagacaggg tttgagagaa 5160 gacaaccagt ctgaccaaaa tttattaggc aggaatttcc tcttcctaat aagcctggga 5220 gcactatggg agaccaggcc ttatttcatc ccttatctac aactgtaaaa gacagacgtt 5280 ctcaaaacag ccattttaga gaccacccct tgggaatgca ttcttctcag ggatgttcct 5340 tgctgagaaa aagaattcag tgatatttct cctacttgct tttgaaagaa gtgaaatatg 5400 gctctgttct gctcagccca caggcagcca gacttcaagg ttatctcact tgttccctga 5460 acatcgctgt tatcctgttt tttttttcaa ggtgcagatt tcatattgtt taaacaattt 5520 gtgcagttaa cacaatcatc acagggtcct gaggtgacat tcgtcctcag cttaggaaga 5580 tgatggtatt aagagattaa agtaaagaca ggcataggaa atcacaagag tattgattgg 5640 ggaagtggta agtgtccatg aaattttcac aatttatgtt cagagattgc agtaaagaca 5700 ggtgtaagaa attataaaag aatgaatttg gggaactaat aaatgtctgt gaaatcttca 5760 caatttatct tcttctgcca tggcttcagc tggtccctcc gttcggggtc cctgacttcc 5820 tgaaacatat agatgtgaat ttgggagctc tttaactata aagtttaata tctcaaaata 5880 ataagagcta tttatgacaa acccatagcc aatatcatat tgaatgggca aaagctggaa 5940 gcattccctt tgaaaaccag cacaagacaa ggatgccctc tctcaccact cttattcaac 6000 atagtattgg aagtcctggc cagagcaatc agtcaagggg gtattcaaat gggaagagaa 6060 gaagtcaaat tgtctctgtt tgcaggtgac atgactgtat acttagaaac cccatcatct 6120 cagccccaaa actgtttaag ctgataagca atttcagcaa gacctcagga tacaaaatca 6180 atgtgcaaaa ataacaagca ttcctataaa ccaataatag acaagcagag agccaaatca 6240 tgagtggact cccattcaca attgctacaa agggaataaa atgcctactt acacaactta 6300 caagcgatgt gaaggacctc ttcaaggaga actacaaacc acttctcaag gaaataagag 6360 gtgacacaaa tggaaaaaaa ttccgtgctc atgaatagaa agaatcaata ctgtgaaaat 6420 agccatactg cccaaagtaa ttcatagatt caatgctata cccgtcaaac tatcattgac 6480 tttcttcaca gaactagaaa agaataattt aaatttcata tgaagcaaaa aaagagcctg 6540 tatagccaag acaatcctaa gcaaaaacaa agctggaggc atcattctac ctgacttcaa 6600 acaatactac aaggctacag taaccaaaca gcatggtact gggaaaactg gctagccata 6660 tgcagaaaac agaaactgga ccctttcctt acaccttata caaaaattaa ctcaagatgg 6720 attaaacact taaacataaa accaaaaacc ataaaaaccc cagaagaaaa cctaggcaat 6780 accattcagg acataggcat gggccaagag tttgtgacta aaacacaaaa agcaatggca 6840 acaaaagcca aaattgacaa atgggatcta attaaactac agtgcttctg cacagccaaa 6900 gaaactgcat cagagtcaac aggcaaccta cagaatggga gaaatttttt gcaatctatc 6960 catctgacaa agggctaata tccagaatcc acaaagaact taaatttaca agaaaaaaag 7020 aaatatcccc atcaaaaatg gacaaaggat atgaacagat gcttctcaaa agaagacatt 7080 tatgcagcca acaaacatat ggaaaaaagc ttatcaacac tggtcattag agaatgcaaa 7140 tcaaaactac aatgagatac catctcatgt cagttagaat ggtgatcctt actaagtcag 7200 gaaacaacag gtgttggtga gaatgttgag aaatatgaac cctcttacac tgttggtagg 7260 agtgtaaatt agttcaacca ttgtggaaga cagtgtggtg attccttaag gatctagaac 7320 tagaaattcc acttgactca gcaatcccat tactgggtat atgcccaaag gattataaac 7380 cattctacta taatgacaca cgcccatgta tgtttatgtg gcaccgttca caatagcaaa 7440 gacttggaac caacccaaat gcccatcaat gatgtactgg ataaagaaaa tgtggcacat 7500 atacaccatg gaatactaca cagccataaa aaaggatgag ttcatgtcct ttgcaggaac 7560 atggatgaag ctggaaatca tcatcctcag caactaacac aggaacagaa aaccaaactc 7620 cacatgtttg cactcataaa tgggagttga acaatgagaa cacatggaca cagggagggg 7680 aacatcacac atgggacctg ttggggcacg gggggccagc agggggacag cattaggaca 7740 aatacctaat gcgtgtgggg cttaaaacct agatgacagg ttcataggtg cagcaaacca 7800 ccatggcaca tgtatacctc tgtaacaaac ctgcacgctc tgcacatgta tcctggaact 7860 taaagtaaaa taaaagaaaa tttaaaaaat ataaagttta tcgatcactt gattataaca 7920 acttcatttc agagatcctc caccaaacaa gaaaactttt cctataattt aattcattta 7980 gctattaaat gatacgctgg gcctaatcaa tatcaaatta aataggttaa atttgcagga 8040 tgattagaga tgagggcatt tagttcctaa agaagaaaat tcattttaaa aaaggtacag 8100 aagaaaaaaa ctccacttat caatttacac tttcttgttt gccttacctg ggatttatat 8160 ttaaaataaa agtttttggc caggcgcggt ggctcatgcc tgtaatccca caactttggg 8220 aggccaaggc tggtggatca tctgaggttg ggagttcgag accagcctga ccaacatgga 8280 ggaacctgtc tctactaaat atacaaaatt agctggatgt ggtggcacat gcctgtaatc 8340 ccagctacct gggaagctca ggtggagaat cacttgaacc taggaagcag aggttgtggt 8400 gagctgagat cgtgccatta cactccagcc caggcaacaa gagtgaaact ccatctcaaa 8460 acaaacaaat aaacaaaaaa caacaacaac aaaaaacaac aacaaagttt tcatttaatt 8520 ttttaacaaa ctaatctatg ttaatttgat attagtttgt taacataaca aactaataat 8580 atgttaggca tattattcat ttatatttgt tttccttctc ataaaacaca ttctgctctt 8640 acatttattt tcttaaatac atgaattcac caacagtctc tttggttcct ctctactggt 8700 tcaacacatg actctttcag ctattcatta tatataaaaa gctttgaaat ctccaactat 8760 tcttgccctt tccatctcag tgccttgctg tctactgact ttgcagactg atgtgattcc 8820 ctctgaaaca tgaattatta ggtttttaga aaatgccttt ttgttctttc caaagtaaaa 8880 gacaaatagg ctgggaatgt aaatttagca tttgaacaac cattatttaa ccagctaggt 8940 tgtaatggtc aactcaggat taatgtaaaa gtgaagtgtt gattttatgc atgccgaact 9000 cttttttgct gttaagggaa tttgtaggta agataatttc taaactacta ttatctgtta 9060 acaaatacag tgttttatat ctaaagttta atagtatttt aaattgtttc taattattta 9120 gcctcaccct gtgatcccac tttcatcctg ggctgtgctc cctgcaatgt gatctgctcc 9180 attattttcc ataaacgttt tgattataaa gatcagcaat ttcttaactt aatggaaaag 9240 ttgaatgaaa acatcaagat tttgagcagc ccctggatcc aggtaaggcc aagatttttg 9300 cttcctgaga aaccacttat tctctttttt ttctgacaaa tccaaaattc tacatggatc 9360 aagctctgaa gtgcattttt gaatactaca gtcttgccca gacagccttg gggtgaatat 9420 ctgggaaaga cggccaagcc ctttatttta tgcatgggaa ataaatgcct caatataggc 9480 ctgatttcta agcccattag ctccctcatc aatgtttttt ctgctaaact ccaaagccct 9540 gtttctgtaa agtactttgt tgacagccct aaagcgtgct catagcactc catggatatc 9600 caggcacttt ggaggctccc attactcaca agacttgtcc ttcaattaac actttgtcgt 9660 attatgtggc agaaatatcc taatttaaaa gacattatct ccttctagta gggagaatat 9720 ttgggaccat agaagctgcc aagaaacact gaatagggca ggggtgtttg atatctcagt 9780 tgggatccta gctgatgaga tagctgggtt aggaatgaca aaattattgg ttttatggtg 9840 tatgaaccat aaacagacat cacacttata ccctgtgctg agctggcatg ttttattctc 9900 tgcctaaata ataattgtgt gattttatag aagtcattta actgctctgg tgcacagttg 9960 gaatttgaag tatctttgag cccctcccac ttctaaaata ctaattccat ttcagaggct 10020 gcttgataga aatcaatata gtagggacta gctttgtact atcaatcagg ttgtccaaat 10080 tcttttaacc tatgctgtca tctacaaaac gtgaatgtag taattcatgc catcttatat 10140 ttcaagatta tagagaagaa ttgtaaaaag taacagaatt aacataaaga tgcttttata 10200 ctataaaaag gaggtctgag tctaggaaat gattatcatc tggttagaat tgatcctctg 10260 gtcagaattt tctttctcaa atcttttata atcagagaat tactacatat gtacaataaa 10320 aatttcccca tcaagatata caatatattt tatttatatt tatagctgta atttacaacc 10380 agagcttggt atatggtatg tatgctttta ttaaaatctt ttaatttaat aaattattgt 10440 tttctcttag atctgcaata atttttctcc tatcattgat tacttcccgg gaactcacaa 10500 caaattactt aaaaacgttg cttttatgaa aagttatatt ttggaaaaag taaaagaaca 10560 ccaagaatca atggacatga acaaccctca ggactttatt gattgcttcc tgatgaaaat 10620 ggagaaggta aaatgtaaac aaaagcttag ttatgtgact gcttgtgaat ttgtgatttg 10680 ttgactagtt ctgtgtttac taaggatgtt taactggtca atcagtaatg cttgagaagc 10740 actttaagtt tttattgtat gaatgaataa caaaatagac cactagctag actaataaag 10800 aagaaaaaag agaagaatca aatagacaca ataaaaatga taaaggggag atcaccactg 10860 agcccacaga aatataaact accatcagag aatagtataa acacctctat gctaataaac 10920 tagaaaatct agaagaaatg gataaattca tggacacata cactctccca agactaaaca 10980 aggaagaagt caaatccctg aatagaccaa taacaagttc tgaagttgag gcggtagtta 11040 atagcctaac aaccaaaaaa agtccaggac cagatggatt cacagccaaa ttctaccagg 11100 ggcacaaaga ggagctgaca ctattccttc tgaaactatt ccaaataata gaaaaagagg 11160 gactcctcct taactcattt tatgaggcta gcatcatcct gatacccaaa ccgggcagag 11220 acataacaga aaaagaacat ttcaggccaa tatccctgat gaacaccaat gcaaaaatcc 11280 acaacaaaat actgccaaac caaatccagc agcacatcaa aaagcttatc caccatgatc 11340 aagtagcctt catccctgag atgcaaggct gtttcaatat ttgcaaatca ataaatgtaa 11400 tccatcacat aaaaagaacc aatgacaaaa accacatgat tgtctcaata gatgcagaaa 11460 aggcctttga taaagttcaa tacccttcat gctaagaatt ctcaataaac taagtaatga 11520 tggaacatat ctcaaaatag taagagctgt ttatgacaaa cccacagcca atatcatatt 11580 gaatgggcag aagctggaag gattcccttt gaaaaccagc acaagacaag gatgccctct 11640 ctcaccactc ctattcaaca tagtattgga agctctggcc agggcagtca ggcaagagaa 11700 agaaatgaag tattgaaata ggaagagaga aagtcaaact gtctctattc gcagatgaca 11760 tgattgtata tttagaaaac ccctttgtct cagcccaaaa tctcaagttg ataagcaact 11820 tcagtatagt ctcaggttat aaaatcaatg tgcaaaaatc acaaccattt ctatacacca 11880 ataatagaca gacagccaaa taatgagtga actcccactc acaattgcta caaagagaat 11940 ataataccta ggacacaaac aaatggaaaa acattccatg ctcatggata ggaagaatca 12000 atatcatgaa aatggccact gcccaaagta atttatagat tcaattctat ccccatcaag 12060 ctatcattgc ctttcttcac agaactagaa aaatctactt tacattttat atggaacgaa 12120 agaagagccc acatagccaa gaaaatctaa gcaaaaagaa caaaaagttc ataatcactg 12180 ggacattgga gaaatgcaaa tcaaagtccc aatgaggtac catctcatgc cagttagaat 12240 ggtgatcatt aaaaagtcag gaaacaacag atgctggaga agatgtggag aaataggaat 12300 gcttttacac cgttagtggg agtgtaaatt agttcaacca ttatggaaga cagtgtgctg 12360 attcctcaag gatctggaac aagaaatacc atttgaccca gtaatcctgt tactgggtat 12420 atacccaaag gattataaat cattctacta taaagacaca tgtgcacgta tgtttattgc 12480 agtactattc acaatagcaa agacttggga ccaacccaaa tgctgatgaa tgacagtctg 12540 gataaagaaa atgtggcaca tatacaccat ggaatacagt gctgccataa aaaaggatga 12600 gttcatgtcc tctgcaggga tatgggtgaa gctggaaacc atcatcctca gcaaactaac 12660 acaggaacag aaaaccaaac attgtatgtt ctaactcatt aatgggagtt gaacaatgag 12720 aacacatgga cacatggagg ggaacatcaa caccggggcc tgtcaggggg tgggggactt 12780 ggggagggat agcattagaa gaaataccta atgtggatga caggttgatg ggtgcagcaa 12840 accaccatgg cgcgtgtata cctacgtaaa aaacctgcat gttctgcaca tgtatcccag 12900 aacttaaagt ataaatatat aaatctaccc aaggagaaac agaatgcaca ttccaggtga 12960 aatggaatca attactgaca aatatgaaaa cataaaatat gccctgtgat gaagatgggt 13020 gtcattattg cacacagttt atttgagaat gggaaagaat gacttaacct aggtgggttt 13080 cctgcattcc tgcaaattga aataggccat aaaatatttg cagtgagagt cagaggaggg 13140 gtttcagata ggttggaatg ctctacatgg aggtgcagag gtgggtgatg ggaaagagta 13200 ggtggatgga agagggaaag gaagccttta ttgagtgaga acaaaaatga tttgtacaga 13260 taacagttgg aatttagtgt ctatcagata aggcgtaaac atcaatgccg ttgtcatcgc 13320 accattgttg aaaacgctat tttttcccct attgaattgc cttggcaacc ttgtcaaaat 13380 ccaattgact ataaatgtgt gaaaggtttt tttctgtgtg ctctcaattg tactccattg 13440 atctatatgt ctgtcgtaat ccctatagtg caccatctta attactgtag tattgtatta 13500 atatcaagtt attatatcaa atcaaggagt gtgagtcttt gttgttcttt ttcaatacgt 13560 tttcactatt ctgggtccct tgaatcttca tatgaaattt agaatacact tgtcaatttt 13620 ctcaaaggag ccaatgggga ttttgacacg attgcattaa atcagtagat cagggaatta 13680 tgccattttt aaaattgaag tgaaattcac acaatataca attaaccatt ttaaagtgta 13740 gacttgagtg gtatttagtg tattcacaat gttgtacaac caccagcttt ctctggtttc 13800 aaaatgtttt tattacttca caaaaacata tcatattcat taaataatca ctccattcct 13860 ccaacccatc atgtaattac ctctaatctt ctttctgttt ctatggattt gcctgttatg 13920 gatatataaa ggactcacag catatgtaac ctgttatgtc tgagtccttt cacttataat 13980 gttttagagg ctcatctaag ttgcaacatg tatcagtact tcattctttt ttgtcgctga 14040 aatatatttt atttttccat gcacatatat cacaatttgt tattcatctg tttatggaca 14100 tttgcattgt ttccaacctt tggctcttat agataatctt gctttgatca tttgtgtatg 14160 tttctttatg ggcatatttt catcattctt gggtatattc tcaggagttg aattgctggg 14220 tcaaatggta attttatgct tatcttcttg tatataagag ccaatctttt ctacagtgaa 14280 tgtggcattt tatattcctt ccagcaatgt agaaggattt ctattttcca catagttgct 14340 aatacttgta actttctgta tttattgaag ccatcctagt gaatatagag tgttatctca 14400 ttgtggtttt atttatttat tcatttattt agagacagag tcttactctg tcacccaggc 14460 tggagtgcag tggcatgatc tcagctccct gcaacttcca cctcctgggt ttacacaatc 14520 ctcctgcctc agcccccaga gtagctggga ttacaggcat gtgtcaccat acctggttaa 14580 ttgttatatt tttggtagag acgggttttc tccactttgg ccaggctggt ctcgaactct 14640 tgacctcagg tgatctgccc atcttggcct ccctaagtgc tgggattaca ggtgtgagcc 14700 actgcgccca gctctcattg tggttttaac ttgtgtttaa tggccaatga ttttgaactt 14760 cttttaatat attattaccc atttgtatat cttctttgga taaatgccat tcaagtacat 14820 tttctgttta aattaagttg actttatttt tctttttaag ctgttagaat gatttatgta 14880 ttcaaaacat taaactccta catatacata atatgaagat atttcctccc attctgttgg 14940 ttgtcatttc acattctcaa ttatattctt gctgcacaaa gtttaatttt gatgaagttt 15000 ggtttatcta tctttccttt tgccactctg gtatcaaatt tataaatcta ttgccaaata 15060 tgaagtcatg aagatttacc cctacatttt attctaagag ttttatcgtt ttagctctta 15120 tatttaagtt tttccatcca ttttcagtta tatttttcat ttggagtgaa gtaagggaga 15180 tctcagcttc attcttctgc atttcgctgt cctgttgtct catcaccatt tgttgaagag 15240 actctttcct cagattgaat ggtctaggca cccttgatga aagtaaattt gccatagatc 15300 tttaggttta tttcgtgatt tcagttttat tccatttgtc ttgatgtcta tctttatgct 15360 agtaccacac tgttttgatt attacagttt gtagtaagtt tgaagttgga aactgagtac 15420 tcactgatac agaaattaag tagaaattac ttaggcaaat agtaagcatt tgggagtcct 15 480 cagtaaggtt tttctttata ctggaaagca gccccaaatc attttctaac aaagagcagc 15540 ttgtaaaatc gagctgcaga catacacaag gaagctggaa acttgcacga gtgaaagctg 15600 gtagtaaaga actacctgtg accaggcaag ttcaaaatgg cggctccttc cccacccact 15660 atgtaaatgt cacacctgat taaaccaatc tctgggccat acgtaaatca gacactgctt 15720 cctccagcct ccctatgcaa tctgctgtgg tccacctcct tccccctttt ccgatgtccc 15780 tctctttcac aagaagctgc ttttttctct cctttcttct attaaacttt ctgctacata 15840 acccactcag atgtgtccgt gtcctaaatt tttctggggc atgatgacaa acctgagggt 15900 gtatatccca gacaacgtag ctgcttcatc accatgtagt tgcatattaa ttgaacaatc 15960 aactttccca tttctggaaa aaagaccatt ggtattgtgg tagggattgc ctgagttttt 16020 agatcacttt gaggagtact gccatcttaa cgatattaag tcttccaatc cctgaacatg 16080 agatagtttt caatttatgt aggtattaga tattcttttt tcttctgttg caagggtctg 16140 gtttagttta aaaggcaagt ttatcttaaa tttttttatt tagttatgac cattgaggat 16200 tttaaaatga taccatatga aatgccttct tttaaaaaaa atttcaactt ttatttattt 16260 atttatttat ttatttttat tatactttaa gttttagggt acatgtgcac tatgtgcagg 16320 tctgttacat atgtatacat gtgtcatgtt ggtgtgctgc atccatgaac tcgtcattta 16380 cattaggtat atctcctaat gctatccctc cccactctgc caccccacaa caggccccag 16440 tgtgatgttc cccttcctgt gaccatgtgt tctcattgtt caattcccac ctatgagtga 16500 gaacatgcgg tgtttggttt tttgtccttg agatagtttg ctgagagtga tggtttccag 16560 cttcatccat gtccctacaa aggacatgaa ctcatcattt tttatggctg catagtattc 16620 catggtgtat atgtgccaca ttttcttaat ccagtctatc attgttggac atttggcttg 16680 gttccaagtc gttgctattg tgaatagtgc cacaataaac atatgtgtgc atgtgtcttt 16740 acagcagcat ggtttataat cctttgggta tatacccagt aatgggatgg ctgggtcaaa 16800 tggtatttct agttctagat ccctgaggaa tcaccacacc gacttccaca atggttgaac 16860 tagtttacag tcccaccaac agtgtaaaag tgttcctatt tctccacatc ctctccagca 16920 ccttttgttt cctgactttt taatgatcgc cattctaact ggtgtgagat ggtatctcat 16980 tgtggttttg atttgcattt ctctgatggc cagtgatgat gagcattttt tcatgtgttt 17040 tttggctgca taaatgtctt cttttgagca gtgtctgttc atatcttttg cccacttttt 17100 gatggggttg tttgtttttt atcttgtaaa tttgtttgag ttctctgtag atcctggata 17160 ttagcccttt gtcagacaag agggttgcaa aaattttctc ccattctgta ggttgcttgt 17220 tcactctgat ggtagtttct tttgctgtgc agaagctctt tagtttaact agatcccatt 17280 tgtcattttg gcttttgttg ccattgcttt tggtgtatga gacaagaagt ccttgcccat 17340 gcctatgtcc tgaatagtat tgcctaggtt ttcttctagg gtttttatgg ttttaagtct 17400 aacatgtaag tcttgaatcc atcttgaatt aatttttgta taaggtgtaa ggaagggatc 17460 cagtttcagc tttctacata tggctagcca gttttcccag caccacttat taaatagtga 17520 atcctttccc catttcttgt ttttgtcagg tttgtcaaag atcagatagt tgtagatatg 17580 tggccttatt tctgagggct ctgttctgtt ccattggtct acatctctgt tttggtacca 17640 gtaccatgct gttttggtta ctgtagcctt ctagtatagt ttgaagtcag gtagctttgt 17700 tcttttggct taggattgac ttgacaatgc gggctctttt ttggttccat atgaacttta 17760 aagtagtttt ttccaaatct gtgaagaaag tcattggtag cttgatgggg atggtattga 17820 atctataaat taccttgggc agtatggcca ttttcacgat attgattctt cctacccatg 17880 agcatggaat gttcttccat ttgtttgtat cctcttttat ttcattgaac agtggtctct 17940 agttgtcctt gaagaggtca ttcacatccc ttgtaagttg gattcctagg tattttattc 18000 tctttgaagc aattatgaat gggagttcac tcatgatttg gctctctgtt tgtctgttat 18060 tggtgtataa gaatgcttgt gatttttgca cattgatttt gtatcctgag actttgctga 18120 agttgcttat cagcttaagg agattttggg ctgagatgat ggggttttct agatatacaa 18180 tcatgtcatc tgcaaacagg gacaatttga cttcctcttt tccttattga atacccttta 18240 tttctttctc ttgcctgact gccctggcca gaacttccaa cactgtgttg aataggagtg 18300 gtgagagagg gcatccctga cttgtgccag ttttcaaagg gattacttcc agtttttgtc 18360 cattcagtat gatattggct gtgggtttgt cataaataga tcttattatt ttgagatacg 18420 tcccatcaat acctaattta ttgagagttt ttagcatgaa gagctgttga attttctcaa 18480 aggccttttc tgtatctatt gagataatca tgtggttctt gtcattgatt ctgtttatat 18540 gctggattac atttattgat ttgcctatgt tgtaccagcc ttgcatccca gggatgaagc 18600 ccacttgatc atggtggata agctttttga tgtgctgctg gattcggttt gccagtattt 18660 tattgaggat ttttgcattg atgttcatca gggatattgg tctaaaattc tctttttttg 18720 ttgtgtctct gcccggcttt ggtatcagga tgatgctggc ctcataaaat gagttaggga 18780 gtattccctt tttctattga ttggaatagt ttcagaagga atggtaccag ctcctccttg 18840 tacctctggt agaattcggc tgtgaatccg tgtggttctg gacttttttt ggttggtaag 18900 ctattaatta ttgcctcaat ttcagagcct gttattggtc tattcagaga ttcaacttct 18960 tccttgttta atcttgggag agtgtatttg tcgaggaatt tatccatttc ttccagattt 19020 tctagtttat ttttgtacag gtgtttatag tattctctga tggtagtttg tatttctgtg 19080 ggatcggtga tgatatcccc ttcatcattt tttattgcgt gtgttggatt cttctctctt 19140 ttattcttta ttagtcttgc taacagtcta ccaattttgt tgatctttta aaaaacctgc 19200 tcctaaattc attgattttt tgaagggttt tttgtttcca tgtaattgag tttccattgt 19260 ttccatggaa tgcaaaaatt ccatgttttg agtgagtttc ttaatcctga gttctagttt 19320 gattgtgcta tggtctgaga gacagcttgt tataatttct gttcttttac atttgctgag 19380 gtgtgcttta cttccaacta tgtggtcaat tttggaatag gtgtggtgtg gcgaaaagaa 19440 tgtatattct gttgatttgg ggtggagact tctgcagatg tctattaggt ctgcttggtg 19500 cagaactgag ttcaattcct ggatatcctt gttaactttc tctctcgttg atctgtctaa 19560 tgttgacagt ggggtgttaa tgtctcccat tattattgtg tgggagtcta agtctctttg 19620 taggtctcta agcacttgct ttattaatct gggtgctccg ttattgggtg catatatatt 19680 taggatagtt agctgttctt gttgaattga tccctttacc tttatgtaat ggccttcttt 19740 gtcccttttg ttctttgttg gtttaaagtc tgttttatca tagagtagga ttgcaacctc 19800 tgcctttttt tgttttccat ttgcttggta gatctttctc catcccttta ttgtgagcct 19860 atgtgagtct ctgcatgtga gatgggtttt ctgtgtacag cacaccgatg ggtcttgact 19920 ctttatccaa tttgccactc tgtgtctttt aattggagca tttaacccat ttacatttaa 19980 cattaatatt gttatgtgtg aatttggtcc tgtcattatt atgttagctg gttattttgc 20040 tcattagttg atgcagtttc ttcctagcct tgatgatctt acaatttggc atgcttttgc 20100 catgggtggt accagttgtt cctttccatg tttaatgctt ccttcaggag ctcttttagg 20160 gcaggtctgg tggtgacaaa atctctcagc atttgcttgt gggtaaagga ttttgtttct 20220 ccttcactta tgaagcttag tttggctgga tatgaaattc tgggttgaaa attcttttct 20280 ttaagaatgt tgaatattgg cccccactct cttctggctt gtagagtttc tgccgagaga 20340 tcagctgtta atctgatcgg cttccctttg tgggtaaccc ggcctttctc tctggctgcc 20400 cttaacattt tttccttcat ttcaactttg gtgaatctga caattatgtg tcttggagct 20460 gctcttctcg aggagtatct ttgtggtgtt ctctgtattt cctgaatttg aatgttggcc 20520 tgccttgcta gattggggaa gttctcctgg atagtatcct gcagagtgtt ttccaacttg 20580 gttccattct cctcgtcact ttcaggtaca ccaatcagat gtagatttgg tcttttcaca 20640 taggcccata tttcttggag gctgtgttcg tttcttttta ttcttttttc tctaaacttc 20700 tcttctcact tcatttcatt catttcatct tccatcactt ataccctttc ttccagttga 20760 tcaaatcggc tatagaggct tatgcattca tcacgtagtt cttgtgccat gattttcagc 20820 tccatcaggt cctttaagga cttctctgca ttggttattc tagttagcca ttcatataat 20880 cttttttcaa ggttttttac ttctttgcca taggttcaaa cttcctcctt tagctcagac 20940 tagtttgatt gtctgaagac ttctctcaac tcatcaaagt cattctccat ccaactctgt 21000 tccattgctg gtgaggagct gtgttccttt ggaggaggag aggcactctg atttttagaa 21060 tattcagttt ttctgctgtt ttttccctgt ctttgtggtt tcatctgcct ttggtctttg 21120 atgatggtga catacagatg ggattttggt gtggatgtcc tttctgtttg ttagctttcc 21180 ttctaacagt caggaccctc agctgcagtc tgttggagtt tgccagaggt ccactgcaga 21240 ccttgtttgc ctgggtatca gcagcggagg ctgcagaaca gcggatattg gtgaacagca 21300 aatgttgctg cctgatcgtt cctctggaag ttttgtctca gaagagtacc cagccttgtg 21360 aggtgtcagt ctgcccctaa tggggggtgc ctcccagtta ggctacaagg ggatcaggga 21420 cccacttgag gaggcagtct gtccattctc aggtctcaag ctttgtgctg ggagaaccac 21480 tattctctcc aaagctgtca gactgggaca attaagtctg cagaattttc tgctgccttt 21540 tgtttggcta taccctgacc ccagaggtgg agtctacaga ggcaggcagg cctccttgag 21600 ctgcggtggg ctccacctgg ttcgagcttc ccagccgctt tgtttaccta ctcaagcttt 21660 ggcaatgggg ggcacccctc acccagcctc gctgctgcct tgctgtttga tctcagactg 21720 ctgtgttagg aatgagtgag cctcagtggg cataagaccc tctgagccag gcacgggata 21780 taatctcctg gtttgccatt tgctaagacc atcagaaaag cacagtatta gggtggaagt 21840 gaccttattt tccagatgcc ttctgtcctt ggctaagaaa gggaattctc tgaccccttg 21900 cacttcccag gtgaggtgat tcctcaccct gcttcagctc aggctccatg cactgcaccc 21960 actgtcctgt acccactgtc tcacaatccc cagtgagatg aacccagtac ctcagttgga 22020 aatgcaaaaa tcattcatct tctgcatcgc tcatgctggg agctgtagac tggagctgtt 22080 cctattcagc catcttggct ccaaatgttc tcaactttta ttttgaagtg cacgttcatg 22140 tgtcttacat aggtatattg tgtgatgctg aagtttaggg tacaaataat gccatcacac 22200 aggtagtgag actagtaccc aataggtagt ttttcagccc ttgcctttat atctctctac 22260 cctctatagt aattccttgt gtattttttc catctttgtg tcccactttt atatgagaac 22320 atgtggtatt tggatttctg ttgctgcatt aattccctca agataatggc ttccagctac 22380 atctatgttg ctgcaaagac atgatttttt ttaatggctg catagcattt catggtgtat 22440 atacaccaca ttttgtttat gtgacacatg attggttggt tctatgtctt tactattgtg 22500 aatagtgcag gaatggacat actagcacgt gtcgttttgg tagaacaatt tgttttcctc 22560 tgggtacata ctcagtagtg gaattgctgc atcgaatggt agttctgctt ttagttcttt 22620 gagaaatctc aaaactggtt tccatagtgg ctgaactaac ttacattccc accagtagta 22680 tataagtgtt ctgttttctc tgcagtctca ccaacatctg ttattttttg gctttttaat 22740 catatccatt ttgattggca tgagatgata tctcattgtg gttttgattt gcatttctct 22800 gatgattagt gatgttgagt gttttctcat atgcttgttg gccatgtgtg tgtcttcttt 22860 ggaaggacat atgtcctttg cccacttttt aatggggtta cttgtttttt aacttgttga 22920 attgtctaac ttccttatag attctcgcta ttgaaacatt gttggattca cagtttgcaa 22980 atattttgtc ccattctgta ggttgtctgt ttattctgtt gatagtttct cttgctgtgc 23040 agaagctctt taattaggtc ccacttgtca atattcattt tggttacaat tgcttttgag 23100 gagttattca taagttcttt gctaaagcct gtatccagaa tgttatttta tagatatttt 23160 tccatcagat tcttatagtt tgaggtctta aatttagatc attaatccat ttcgagttaa 23220 ttattgtata tggttcaagg aagtgtttca gtttcattct gcatatggct agtcagttat 23280 cctcattcaa tttactaaat agagaattct ttcctcattg attatttttt gtcaactttg 23340 ttgaagatca gatggctata ggtgtgtggc tttatttctg gcttctctac ttctgttcaa 23400 tttgtgtttt gtaccagtta catgctctct tggttactgt agcttacagt atagtttgaa 23460 gtcaatgtga agcttcccac tgttcttttt gtttaggatt gctttggatt tgggctcttc 23520 tttggttcca tatgaatttt agaataaaat aatattggta gtttgatagg aatagcattg 23580 aatctgtaga tagctttggg cagtctagcc attttaatga tattgattgt ttcaatccat 23640 aagcctgaac tgtttttcca tttgtttgtg tcatctatga ttttgttcat cagtattttg 23700 tagctctcct tgtagagacc tttttgcccc ctttgttagt tgtattccta ggttttttct 23760 tgtggctatt gtaaatggga ttgagttctt gattggcttt caggatgaac atttttggtg 23820 tttaaaaatg ctacagattt ttgcatattt attttgtgta ctgaaatctc actgaagttg 23880 tatgtcagtt ccaggagttt ttggaaagtt ctttagggtt ttctaggtct gaattgggtc 23940 ttctaggtct gagatgggtc ttctacttct actagtagga gatgggcctt gcaatcttga 24000 agacagtaga aggatgggtc ttatttttaa tccaacttcc tactctttgc cttttatgtg 24060 gagcatttag actttatctt caatgttaac actgatatat gaggttttga ttctattgtg 24 120 aagttgttag ctggttgttt tgtagtttct attgtgtgat tgctttatag agtctgtggg 24180 ctatatactt aagtgtgttt tgtggtagca ggtattgttc tttcacttcc atatttagaa 24240 ctcccttaat gatctcttat aaggctggtc taatggtaac agattttcct ggtagttact 24300 tgtttggaca agattgtatt tcttccttga ttttgaagca tagtttggca aaatataaaa 24360 ttcttttttg taatttttat taaaatgaat gttgaaaata ggctgagcat ccttttagct 24420 tgacaaggtt ctctttgtat gtgatctgat ttttttctct agctgccttt aaaattattt 24480 tttagccttg actttggaca gcctggtgag taaatgcctt ggtgatgtta attttgtata 24540 gtgtctcaca tatattccct gggtttattg tatttggatg tctacctttc tagcaagttt 24600 gggaaaattt tctcaactta ttagcccaaa tgtattttct agattgttta ctttttcttc 24660 tctctcagga atgccaataa ttcctaggtt tactcctttt aaataatcag atatttcttg 24720 aagactgttc atttattaaa attatttttt ctttattttt gtctgacttg gttagttcaa 24780 aagactggtg ttcaagctct gaaattggtc cagtctattg ataaacgctt caattgaatt 24840 ctgaaattcc ttaagtgact ctttcaattc cagaagcttt gattgatttc tttttaagat 24900 gtttacttct cgctccattt cctggactgc tttagtagtt tgtttgcatt aattttcaac 24960 cctgccttgg atcactgagc ttacttgcag tccatatttt gaatagttta tctgtcattt 25020 ctgagttgcc agtttggtta gggttcattg ctggggagtt catgtgatcc tttggaggtt 25080 ttacgacatt cagacatttc atggtgccag aagtcttatg ctggtttctt cttatttgga 25140 gaggctgccg cttctgattt ttgaaattat ttttgtgcag atagaattgt ttttctctcc 25200 ctatttttgt tctttccttt ttctcttccc cttcctatgg gatgtgacta taaagtatgt 25260 tgggtagggc ctttgacttg gattctatag ctgtgttcac ttctgtgggt agttttatat 25320 tggcctgtgc agtttgacat acatgtcagt agatggcact tatgagcaaa agccagctgt 25380 gactaggaca acttgatata gcacagcttg atccttgttt tctgggggaa cttctctgtt 25440 tcctcagata atctgttcat ctgtggaatt cagagtagtc tgggctccct gctgagcccc 25500 agggggataa agatcggtgt cactggactg ggcagtcctg tctacaggtc ccctaatggc 25560 aggcactagc accagcactg agagacaact gatttttgca ttgatcttgt accctgaaat 25620 tatgctgaaa tcatttatta gctctactta ttttctatct gtattgcatg agatttttct 25680 atatagagag aatcatgttt cagtcaaata cacatgattt gaatttttcc tttcctattt 25740 gaatgtgttt tattttattt cttgcctaat tgctctggct agaacttcca atactatgtt 25800 taatagtagt aaaaatggac atccttgttt tttttctttt ctttctgatt ttaagttaca 25860 agttttagtc tttcacaatt tagtatgctt tagctttgca gttttcataa atatgcttta 25920 ttatgataaa ggagttttat ttcatttata gttttctgag tgtttttgtt atgatgggta 25980 ttaaaagttg tcaatttttt tttctgcatc taatgagatg aacatgattt tctcttttat 26040 tctgatatgg tgtagtgcac tgattgactt tcttgttgaa ccacttttga atttctggga 26100 taaatctcac ttgcttattg tgagatttat ttttataatg tttattatta taatttttta 26160 attttgtaaa aaattgttgc aaaatacaca taacataaaa gtttctgtat taatcatttt 26220 aagtgcatag tattttggta ttaattacat tcacattgtt gtgcaaatgt cactgccata 26280 catctgtaga attcttttca tcttgcaaaa cagaaacttt atacccattt aataataact 26340 cttggccggg cattgtagat catgtctgta atcccagcac tttgggaggt tgaggtgggc 26400 agattggatg agctcaggag ttcgagacta gcctgggtaa catggcaaaa ccctgtctct 26460 accaacaaag caatcaacaa acaaaatgaa agtaacataa tttcctcaac tgtgcaaact 26520 aattcatatt aataattcca tgaaatgtgt ccatagtaac acaagagcag aagatgggcc 26580 ttgtaaaggt ggcccaccac attatttgag gtctttgtca gtcttatctg ccagatactg 26640 tgtggactaa ctgcagttaa aattcccatt cccttatcac ttgccctgaa atttatctga 26700 aatcaaaggg aatgggttgc ccaaagagaa gtgtccagag acgtggatga tgcgacaaga 26760 aatgttgtag aaccacaccc ccacctcaaa tgataactct acatttctcc cttccaccac 26820 cccatggcaa ccaacattct actttgtgtc tctataaaat cgagtatgct gggtacctca 26880 taaaagtgga atcattacat atttgccttt tgtgactggt ttatttcact tagcctaatg 26940 ttatgttctc agggttcatt catgtttttg catgtgtcag atttttcttc ctttctaagg 27000 gtgaataata ttccgttgta tgtataaacc aagtttgctt acccatttat ctgttgatgg 27060 gcacttggat tactttcacc ttttgacttt ttgagtaatg ctattatgaa tatgggtaaa 27120 aaaatatctc tttgatgtgc tgctttcaat tcttttgggt ataaataacc caaaagaata 27180 acccaccacg cctggttaat tttttgtatc tttagtagaa actgagtttc accatgttgg 27240 ccaggctggt ctcaaactcc tgaccctgtg atccacccac ctcagcctcc caaagtgctg 27300 gaattatagg tgtgagccac gtcgcccggc ctacatttat tgatttgcat atgttgaacc 27360 aggcttgcat cccagggatg aagccaactt gatcgtggtg gataagcttt ttgatgtgct 27420 gctggattca gtttaccagt attttgttga ggattttcgc atggatgttc atcagggata 27480 ttggcctgaa attttctttt ttttgtttta tgtctgccaa gttttggtat caggatgatt 27540 cttcccttat aaaatgagtt aggaaagagt gcgtcttttt ctactgtttg gagtagtttc 27600 agaaggaatg gtaccagctc ctctttgtac ctctggtaga attcggctgt gaatccttca 27660 ggtcctgggc tttttttttg cttggtgagc tattccttgc tgcctcaatt tcagaacttg 27720 ttattggtct cttcacggat tcgacttctt cctggtttgg tcttgggagg gtgtgtctag 27780 gaatttatcc atttcttcta gattttctag tttatttgca tagaggtgtt tatagtattc 27840 tctgatggta gtttgtattc ctgtaggatc agtggtgata tcccctttat catttttttg 27900 ttctctctat ttgattcttc tttcttttct tctttattag tctggctagc agcctatttt 27960 gttaatcttt tcagaaaatt agctcctgta ttctttgata ttttgaaggg attttcctgt 28020 ctctatctcc ttcaattctg ctctgatctt agttattttt tgtcttctgc taggttttga 28080 atttgtttgc tcttggtttt ctagttcttt taaatgtgtt gttagggtgt tgattttaga 28140 tctttcctgc tttctcctct ggacatttag tgctataatt ttccctctaa acactgcttt 28200 agctgtgtct tagagattct ggtacactgt gtccttgttc tcattggttt caaagaacat 28260 ctttattttg ccttcatttt gtcatttacc cagtagtcat tcagagcagg ttgttcagtt 28320 tccatgtagt tgtgcagttt tgagtgagtt tcttaatcct gagttttaat ttgatggcac 28380 tgtggtctaa taggttgttt gctttaattt ctgttctttt gtatttgctg atgggtgttt 28440 tacttccaat tatgtggtca attttagaat aagtgtgaag tggtgctgag aagaatgcat 28500 attctgttga tttagggtgg agagttcttt agatgtctat tatgcccgct tggtccagag 28560 cttagttcaa gtcctggaga tcctagttaa ccttctgtct cattgatctg tctaatattg 28620 acagtggggt gttaaagcct ccatctatta ttgtgtggga gcctaagcct ctttgtaggt 28680 ctttaagaac ttgctttatg aatctgggtg ctcctgtatt gggtgtgtat ctatttagga 28740 tagttagctg ttctcgttgc attgatccct ttaacattat atgataccac cccttttttt 28800 aatctttgtt ggtttaaagt gcgttttatc agagactagg attgcaaccc ctgctttgtt 28860 ttgctttcca tttgcttggt aaatattcct ccatcccttt attttgagcc tatgtgtgtc 28920 ttttcacgtg agatgggtct cctgaataca gcacattgat gggtcttgac tctatccaat 28980 ttgccagtct gtgtctttta attggggcat ttagcctgtt tacatttaag gttaatattg 29040 tgtgtggatt taatcctgcc attatgatgc tagctgatta ttttgccttt tagttgatgc 29100 agtttttttt atagtgttga cggtctttac aaaatttggt atgtttttgc agtggctgtt 29160 cccagttgat cctttccata tttagtgttt ccttcaggag ctcttgtaag gcaggcttgg 29220 tgatgacaaa atccccttgt ggtgatgggc ttccctttgt ggtaacccga cctttctttc 29280 tggcttccct taacattttt cctttctttt aaccttggtg aatctgacaa ttatgtgtct 29340 tggggttgcc cttcttgagg agtatcattg tgggtgtttt ctgtatttcc tgaatttgaa 29400 tgttggtctg tcttgctaga ttgtggaagt tctcctggat aatatcctga agaatgtttt 29460 acaacttggt tccattctcc ccatcacttt caggtacacc aatcaaacgt agatttggtc 29520 ttttcacata gcgccacatt tcttggaggc tatgttcatt tctttttatt cttatttttt 29580 aacttttttc ttcacatttt atttcattaa gttgaccttg aatctctgat atcctttctt 29640 ctgctttatc aatttggcta ttgatattta tttatgcttc acaaagttct cattctgtgt 29700 tcttcagctc catcaggtca ctcatgttct tctctagtta gcaattcgtc taacattttt 29760 tcaaggttct tagcttcttt gcaatgggtt agaacatgct cctttagctc agtggagttt 29820 gttattacac accttctgaa gcctacttct gtcaattcat caaattcttt ctctgtccag 29880 ttttgtcccc ttgctggcaa ggagttgtga ttctttggag gagaagagat gttctggttt 29940 tttaaatttt cagccttttt gtgctgtttt tttttttctc atatttgtgg atttatctac 30000 ctcttgtctt tgatgttggt gacctttgaa tgggtttttt gcgtggacat cctttttgtt 30060 gatgttgatg ctattccttt ctgtttgcta gtttttttcc taacagtgag gcccttctgc 30120 tgcaggtctg ctggagtttg cttgaggtcc actccaaacc ttgtttgact gggtatcacc 30180 agaggaggcc acagaacagc aaagattgct gtgtgtttat tcctctggaa gcttcatcct 30240 agggggacac ttgccagatg ccagccaaag ctcttttgta tgaggtgtct gttgacccct 30300 gctgggaggt ctgctgttct ctttagagcc agcaggcagg aacatttaag tctgccgaag 30360 ctgcacccac tgccacccct tcttccaggt tctcagtaca gtgagatggg agttttatct 30420 ataagcccct gactgggact gttgcctgtc tttcacagat gcaactgctc agagagtagg 30480 aaactagagg ggcagtatgg ctacagtggc tttgcggagc tgaagtggac tccacctagt 30540 cctagcttcc tggaggcttt gtttacactg tgaggggaaa accacctact caagcctcaa 30600 caatggcaga ctcccctcac ccccccaccc cccccaagct cgaacatccc cagtcaactt 30660 cagactgctg tgctggcaaa cagaagttca agccagtgga tcttagcttg ctgggctcca 30720 tgggggtgag atcccctgag ctagatctct tggctccctg gcttcagtcc tctttccagg 30780 ggactgaatg gttctgactt gctggtgtta cagcaccact ggggcataaa aaaacccctc 30840 cagctatctt ggtgtctgcc cagatgacca cccagttttg tgcttgaaac ccagggccct 30900 ggtggtgtag gctctgaagg gaatcttctg gtctgtgggt tgtgaagacc atggaaaaac 30960 tgcagtattt gggccagagt gcaccgttct tcacagcaca ttccctcaag gctttccttg 31020 gctatgggag ggagttcacc aatcccttgg gcttccaagg tgagacaaca ttcctccctg 31080 cttcagttta gcctccatgc actgcaccta ctgtctaaac attcccagtg agatgagcca 31140 ggtacctcag ttggaaatgt agaaatcacc taccttcttc attgatctcg ctgggagctg 31200 ctgactggag cttttcttat tcagtcatct tgccagccac cctcctgatt gagtttcttt 31260 aaggtaatta ttttgaattc cttttttgaa tatttgtgga ttttttttca ttgaggtctg 31320 ttatttgaga gttattatgt ccctttggtg gtgccatatt tctttatttt tcatgttgtg 31380 tctctgaatt tatgtctatg catgtgatgg aacaattgct attccaaaat ctctagagtg 31440 aatttcatag agaaagactt ttatctgaag ttgagtgttt gtgtgcaggt gacgaagagt 31500 gggtaactca gtttttgata gatgcattgt gatagtatct gtgtagtttc tctagctatg 31560 gccaatatca gcaataattt tgggtgcctc agtggcctag gctgtagaag tttgcagcag 31620 tatcaggagc agtgtaggtt gttaatgtcc ttagtgtcaa agtatttggg cgtcctccta 31680 tttttatttt cctaaaattg gggtgactta gcctaaggga ttccttttgg tgtcatctct 31740 gacatggcat taactcagcg gcattggtgc tgggttacaa gtacagatcc ttgaagttgt 31800 catagaagca tcattctaga ctcagggtct tttaatcatg tattgtgaca cctgggtctt 31860 gcggtgcagg ttcactctct gtggcagtgt tgaatgcaga ttacccacag aaccaaggtc 31920 tatgactcta gcacacctta gcagctgaag cccacagact gagttgtggc tgtgaatctg 31980 tttctgaggg tgaggtgcag gaaaatgccg tggcctgcct ctgatgtaga aggggtgctc 32040 tttagatttg agcccagcaa gtagaatatg gctactatca ttaataagat aggatatact 32100 accttattaa tgctaagtaa ccaagctcaa gtcaagagat ggaaatgtta atactggttt 32160 gtgactgcac tgttcttctt acttaagagt aattttcaac atggcaagct tagaaaactg 32220 tgtgaagctg atgttaccaa gggagaggac tgtctttgtc tacacatgtt gagagaatct 32280 agtctatttc agtggtgttg attacacaat attcaacatg acacacatca cttggagact 32340 ctaaaagcat cattaattta ttgctgctgg ccttggggct ggttaaaaag taatacgttt 32400 tttatattga tgatctcttt caactaagta ttccttatcc attgctttct ttctcccaac 32460 ttccaaatta tgagaactcc agtctctaag ccttggcata tgcagtatcc ttactctctc 32520 ctttatcccc tgtggcaatt aatcccttta ttctccatct tcatcattgt tctttctctt 32580 cactttgtat atttttgttt attcctgcac tactgcaccc tagaacaagt tgtctgttgc 32640 ctcccatcta gattataata gtgtgggccc atcttgtcat tgtaaaccca ttctcttcct 32700 ctataattta tcttaggttt tgctgtcata ttgctttttt aatatttgtt tttacaaaat 32760 ttcaacttta taatgaatct gggggcaata ttgagtccat gtacctttct gacagaaaaa 32820 tataattgca gactcttgat tcaggctcac agtgtctaat gatatggccc caatttacgt 32880 tttctattaa tttagattgt ggttgtctac aggtaataat ggctttaaca cataatgtct 32940 ttatttatct caaatacagg gattctcttg gtggggcaat ctaggataat acagcagccc 33000 caaagcagca gagagtcaag tgccttcctt tttcccacta aatttagtat acagaccttc 33060 attttgatga ttaaatccca ggatggggtc tactctacct cctacttcac atttgtgttt 33120 tgagtatgaa agaggaaata gaggaaatgg acctagagac cttctgctta tatattttag 33180 caaaaaaaaa tatgctgtgt gactcagcta gctgcaaaga gcctgatgaa tggaattttt 33240 aggcaagcat ggaataaggg agtaggaaat aaagtttggg caagttggtc tacagcctct 33300 gctatacaag cagtattttt tttctagtac tgtactttcc agtttctatg ttggtaacta 33360 tataactatg tgaataattt tgaattcact gtaatcaaat atgctggtaa ataatttgtc 33420 agataattgc atcaaatcat tcctaggaaa agcacaacca accatctgaa tttactattg 33480 aaagcttgga aaacactgca gttgacttgt ttggagctgg gacagagacg acaagcacaa 33540 ccctgagata tgctctcctt ctcctgctga agcacccaga ggtcacaggt atgatcagag 33600 atgataagtt aattaatttt cagaaaagat tttgggaagg tgttgctagt gtcctccttc 33660 ctgtttctct tagagaagct tcattattta aacttttgtg cttccagctg taatctgttt 33720 caaactaatg gtgattacaa tgggatatct tggcctggca tggtggctca cacctgtaat 33780 cccagcactt tgggaggctg aggtgggtgg atcacttgag gctaggagtt caagaccagc 33840 ctggccaaca tggccaaatc ccatctgtag taaaaataca aaacttagcc aggcatggtg 33900 gcacatggct gtaatcccag ctactcgagg ggctgaggca ctggaatcaa ttgaacccag 33960 gtggtggagg ttgtagtgag ccaagatttt gccactatac tccagtctgg gcaacagagt 34020 gagactctgt ctcagaaaga aaaaaaaaga aaaaaagaaa aagaaaagat aaaaggggca 34080 tctttgcaca gtagaggaag ataactgaga gaaatgaaga cagcatggca gtagcaaaag 34140 cagtagaact ctggtccaat gtgtctggat ttatggcagg aagagacaag ataaattggc 34200 ctgggattgc atgttggttt tattatgaga aggccatttt aaatagcaag tatattcttc 34260 aagataactt ttctcattct caaaatttca ggttcgaatg ctggagtagg gaaacttgaa 34320 actctcctta ttgaaggata aatggtaatc ctaaaaatgt agtgatctgc ctgagaaaat 34380 tcctagtcca ataagtactc taaaaagtta gattcataag aaaggtgatt ctgtttacta 34440 gaagaggtat ataaagaatg ttccatttag gcagaatatg tacctgagac agtttccatg 34500 aaactgttgt tgggcaaaat aagatttttt tgaggaagtc aataatttta tcttttatga 34560 acaactttac tttagaaatt tacttttaag gacttttggt tatgctgcag ataagaaata 34620 ttcttttttt ctcctatgtc agtatccccc attgaaatga caataaccta attataaata 34680 agaattaggc ttttttttga acagttacta gcctatagag ttctagaaga ttttttgtca 34740 aattttatta tagattaaag ggtacaaata caggtttgtt acataggtaa gttgtgtaac 34800 actgaggctt ggggtcccag ctattccatc acactgacag taaacatatt acccaacaga 34860 tggttcttca gcccacatct cttctctccc tcccccatct agtgatcctc agtgtctatt 34920 gttcccatct ttatgttctt gtgtattcaa tgcttacctc ccacttataa gcaagaacat 34980 gtggtatttg gttttctgtt cttgtattag attgcttagg ataatggcct cctgcttcat 35040 ctataatgct gcaaaagaca taacttttgt tctttcttat ggctgcataa cattccatta 35100 tgtatgtact agtctgttct catgctgcta ataaagacat acctgagaca gggtaactca 35160 taaaggaaag aggtttaatg gactgtcaat tccacacggc tggaaaggcc tcacaataag 35220 tctctgatag agacttattc actatcacaa gaatggcatg ggaaagtcag gcctccatga 35280 ttaaattacc tcacattgag tacctcccgt gacgtgtggg aattatggga gctacaattc 35340 aagatgagat tttggtgggg acacagccta accatatcag tgtatatttg gtacattttc 35400 cttatacaat ccactgtcga tgggcacata agttgattcc atttctttgc tatcgtgaat 35460 agcactacat attggggtga acatattggg gtgtgtgtct tattgataga atgaatattt 35520 tgggggcata tactccatag tgggattgct gggtcaactg gtagttctat tttaagttct 35580 ttcagaaata tccaaactgg tttccacagt ggctgaacta gtttgcatac ccaccaacag 35640 tgtaaaagca tttccatttc tctgcaacct caccagcatc tgttgttttt ggacttttta 35700 atattaataa ttgctattct gactggtgtg agatggtacc tcattgtgat tttgatttgc 35760 atttctctga tgattagtga tgttgagcat tttttcataa gtttgttggc tgcttgtatg 35820 tcatctattg agaagtggct gttcatgtct tttgcccaat tttaattgat tttttttgtt 35880 tttttttttt gcttgttgat ttgtttaagt tttttataga ttttgtctgt tgatacatat 35940 ttagattttg gatattagac atttaggtcc tttaaggtag gaagagtaca ggaaatagtc 36000 aagaagactt taacaaattt ctaaaaacag agagcatata gaggaatgat atatctgtac 36060 agagagggta aacttataac caaaaggtca gaagagaaaa atttatcaag gatgaaaagt 36 120 taatcagtgt cccttaaatc ctcccaatat tggtatttgg aagtaccgag ggtcaaagat 36180 ggtggagaat gaggctgaaa aaggaggctc tccctgccta cgtttcctcc acaactcttt 36240 tcatcaggta actgatttcc ttccacaaga agaaggaggc tcactctcta gagaaactga 36300 gcttaagaag ccctgtactc agagacacac agcagtgcca gaaaaggcat gaggtgtagg 36360 ctagaaagac agattaggta cacatccact ccttgaattc agagactctt agctctttta 36420 ttcatcttcc ttcctaaaat tcagcagcta gctctatcct acagaaaggg aactgatcta 36 480 gagaaatggc atttttcttt tgccatctta acagacaatt ctcttcaatg tccaacagag 36540 tgaatgaaat gaaaacatat actgctagat acagggttat ggaatttcag gacatgagaa 36600 ttaaagggaa aatcacaaaa actttcagag ctgtaccagg ttatattgaa agaattagaa 36660 ttcagaattc tcaagaagaa ctattaatat tatcttgtat attaataaaa atagtgtttt 36720 cagatgtgca tgtttgccaa aaatttttac cattttccct ttctcagaaa aatactgaag 36780 aatgttcttc attcaaatgc ctgaaacctt gttagagaaa gtcctgagct ccagcaatca 36840 ggaaacctcg tgtaggagaa tgagagtgtc agggaggctg cctagcacca gacctagaaa 36900 acaactactt cagaacagac ctgggggata attcagaagt gagatgagac agcaagaaca 36960 aaagagatgt gacagatttt atgagagttt ccaataacta aaaaggagtt tcaaacatct 37020 gctggaacat ttgtaaataa ttctaacttg ggaaatggaa aattacaaat tgaaataaag 37080 agacattgtt aagcccagga aagtacaaaa ataaaattta atcatagttg tcttaccaga 37140 gaaccatatt tgtgtagtca gaatgatgta agcaatgaat actgatatca taaaaattgt 37200 catctaaata tattctggaa aaagcagtgt ggaaaagtga gtgtgtctat ctgagggtgt 37 260 gttcaaatat gaggggaaga tggagattaa aattctcatc tctcttaata aaaaattatt 37320 aaatataatc tacaaaaaga taagtaagaa ttagaaacag agtatttaaa aatgtgaatg 37380 tatgttcaga agaagctgct agaatgttca aaatggtaga tttggaaagt gtgagaatga 37440 agagaatacc attgtgattt gtgacccctc ccacacacag atgcaccaat acagtcatat 37500 atatgcataa atctgatagt ataatttaac gtttaagcaa catatgttat tttgatatga 37560 aattaatctg tttttcaaag acagtaattt catgactaaa ttatatgctt actttatgtt 37620 ttcaaacatt ttattatttt tatttgtatt aattatcata tatactaatg tatatgtata 37680 tacatatctt ttaatttttt ttaattttta tgggtacaaa gtaggtatat atcaatatgt 37740 ttgggggtac atgagatgtt ttgatacagg catgcaatgt gaaataagca tgtcatggag 37800 aatggggtat ctattccctc aagcatttat tctttgagtt acaaacaatt caattacact 37860 ctttaagtta ttttaaaacg tacaattaag ttattattga ctatagtcac ccttttgtgc 37920 tatcatatag tagatcttat tcatctccac ctcccctccc cccacactac cctttcgagt 37980 ctctggtaac catctttcta cactttatgt ccattagttc aattctttta tttttagatc 38040 ccacaaataa gtgagaacat gctatgttcg tctttctgtg cctggcttat ttcacttaac 38100 gtaatgatct ccagttccct tcatgttgtt gcaaatgact gaatctcatt ctttttatgg 38160 ctgaatagaa ctacattgcg tatatgtact acattttctt tatacatttg tctgttgatg 38220 catatttagg ttgtttcaaa gtcttagcta ttgtaaacag tgctgcaaca aacaagagtg 38280 cagatacctc ttcgatatac ctatttcctt tcttttgggt atataccaag cagtagggtt 38340 gttggatcat atggtcgctt tatttttagg attttgagga acatttaaac agctctccat 38400 agaggtcaca ctaatttgca ttcccaccaa cactgtacga gggtccactt atctccacat 38460 cctcactagc atttgttatt gcctgtcttt tggatataag ccattttaac tggagtgaga 38520 tgacatctca ttgtagtttt gatttatatt tctctgagga tcagtggtgt tgaccacctt 38580 tttgtatgcc tgtaacccat ttatatatct tcttttgaga aacatctatt caaatctttt 38640 gcccactttt ggattggatt attagatttt ttttctatag agttgcttga atttcttata 38700 tattctgatt actaatctct tgtcagatga atagtttgca aatatttctc tccattctgt 38760 ggattgtctt ttccctttgt tgattgtatc tttggctgtg cagaagcttt ttaacttgat 38820 gtgatcccat ttgttcatgt ttgctttgaa tgcctgtgct tgtagggtat tgctcgaaaa 38880 atttttgcct agaccaatgt cctggagatt ttccctagtg ttttcctata gtagtttcac 38940 agtttgaggt cttagactta agtctttaat ccattttgag ttgatttttg tatatggcaa 39000 gggatatgag tccagtttca ttcttctctg taggatatta aattttccca gcacatttat 39060 tgaagagact gtctttcccc cagtgtacgt tcttggcacc tgtgttagtc tgttctcatg 39120 cctctaataa agccatacac aagactgggt aatttataaa ggaaagaagt ttaatggact 39180 cacagttcca catggctgga gaggcctcac aatcatggca gaagatgaat gaggagaaaa 39240 gtcatgtctt acgtggtagc aggcagatag ctttgcaggg gaactcacat ttataaaacc 39300 atcagatgtt gtgagattta gtcactatca tgagaaaaat atggaggaaa ccacccccat 39360 gattcaatga tccctacctg gcctccccct tgacttgtgg ggattatttc aattcaagat 39420 gagatttggg gtggggacac agccaaacca tatcagcccc tttgtcaaaa atgagttaac 39 480 tctagatacg tgaacttgtt tctgaattct ctattctgtt tcattggtcc atgtgtttgt 39540 ttttatgtca gtaccatgct gttttagtta gtatagctct gtagtttaat ttgaagtgag 39600 ataatgtgat tcctccagtt ttgttccttt tgcttaggat agttttgact attctgggtc 39660 ttttgtgggt ccatatacat tttaggattt attttttcta tttctgtgaa gaatgccatt 39720 gatattttga taggaatttt attgaacctg tagattgttt tgatagtatg ggcactttaa 39780 caatattgat ttttccaatc catgaacatg aaatattttt ctgtttgttg ggtcctctta 39840 aatttctttc ctcagtgttt tatagttttc attatagaga tctttcaatt ctttggttaa 39900 attaatccta ggtatttaaa attatgtatg gctattttca atgggattat tttttaaatt 39960 tctttttctt ttttttcact gttggcatat aacaacacta ctgattttta tgttgattgt 40020 gtatccttca actttactga atttgttcat cagttctaat aattatcttg tagaatcttt 40080 agtttttcct actataaggt tatatcatct gcacacaagg atattttgac ttcttccttt 40140 ccaattttga tagcctttat atctttctct tgtctgattg ctcttgctag gtcatccagt 40200 actatgatga ataacaatgg tgacaatggg catccttgtt gtgttcctga tcttacagga 40260 aagacttcct attttctcca ttcaggatag tactagttgt gggtctgtca tatagctttt 40320 ttatgttgag gtatgttcct tctatctcaa gtttttttta tgaattttca tcaagaaggg 40380 atgttaaatt ttatcaaata ctttttcagc atcaactgaa atgatcatat ggcttttatc 40440 cttcattctg ttgatatgat gtatcacact gcttgatttg cacatgttga accattcttg 40500 catccaaggg ataaatccca cttggtcatt gtgaatgacc tttttattgt attgttgaat 40560 ttggtttgct agtgttttgt tgaagatttt tgcattagtg ttcatcaggt gtattggcct 40620 gtagtttttt tttttttttt tttttttttt gatgtgtctt agtctgattt tggtatcagg 40680 gtaatactgg cctcatagaa tgagtttgga agtattctct tctcctttat tttttggaat 40740 agtttgagta gggtcggtat tagttcttct ttaaatgttt ggtaaaattc agcagtgaag 40800 ctagccagtc ttgggatttt ctctactggg atgattttat ttctcgtttg atctttgtgc 40860 tggttattgc atgttcagat tttggatttc ttcctggttc aatcttgatg ggtcatattt 40920 gtgtagggat ttgtccattt cttctagatt ttccaattta ttggcatata gttgctcata 40980 gtaaccaata attattcttt gaatttctgc agtatcagtt ctaatgcctc ctttttaatt 41040 ttttatttat ctgaatctac tctttttttt tttcttagtc tggctaacgg tttgtcaatt 41100 ttgtttaact tctcaaaaaa gcaacttttt gtttcattga tcttttgttt tgttttcttc 41160 acttaaattt tatttatttc tgctctgatc tttattattc cttttcttct actaattttg 41220 gttttaattt gcttttgctt ttctaattct ttaggatgca tcattaaatt gtttgtttgt 41280 caattttctc catttttgat ataagctctt gtagctataa acttccctct tagtactggt 41340 cttgctgtaa cccctaggtt ttggtatgtt gtgtttccat tatcatttgt ttcaagaaat 41400 ttttcaattt tctttttaat ctcttcatgg gtccactgtt catttatgag catattttta 41460 aatttccatt tatttgtgta gtttccaaaa ttcctcttgt tattggttgc tggttttatt 41520 acattgtggt cagagaagat gcttgatatt atttcagttt ctttgaatat tttaagactt 41580 gttttgtgac ctaacatacg gtcaattctt gataacaatc catgtgctgt ggaaaagaat 41640 gtgtattctg tagcagttgg ataaaatatc ctgcaaatat ctatgagatc catttgatct 41700 atagtgcaga tgaatttcaa tgtttccttg ttgattttct atctggatga cctgtccaat 41760 gctgaaagtg gggtgttgaa gtctccaggt attattatat tggggcctat ctctctctag 41820 ttctaattat atgtctttta tatatctggg tgctgcatta ttggttgcat atatatttaa 41880 acttgttcca tcttcttgcc aagctgacca ctttatcacc aatagtgatc ttctttgtgt 41940 ctccttatgg tttttgtttt gaaatctact ttgtctgttt taaatatagt aactcatgct 42000 ctttttttca tttccattgg caggtactgt ctcattcaat tcctttattt tcagcctatg 42060 tgtgtcttta taagtgaagt gtgtttcttt taggcaacag attaataggt cttgtttttc 42120 catccaggtc agtaacaggt cagtatgtct tttgattgga gattttattc catttacatt 42180 cagtgttatt attgataagt aaggacttac ccatgcccct ttgttatttg ttttctggtt 42240 gttttgtgga cttctcttcc ttctttcatt tcttcctgtc ttcctttatt gaagagaatt 42300 ttctccactt atatgtgtac agatttttct taatatctgg tttatggcag ttacacattt 42360 gtgcatctgt aaccatcctc tctttaagtt tgcatatact tccagcacta taatttaaat 42420 ttataatgat gtttggatac cttcatgatt catatacccc tgaattgcta caacaaatgt 42 480 gccatttttc tccttttcca tcagttttta cttgtgtctt atcagctaaa gtccaggaag 42540 agattgaacg tgtgattggc agaaaccgga gcccctgcat gcaagacagg agccacatgc 42600 cctacacaga tgctgtggtg cacgaggtcc agagatacat tgaccttctc cccaccagcc 42660 tgccccatgc agtgacctgt gacattaaat tcagaaacta tctcattccc aaggtaagtt 42720 tgtttctcct acactgcaac tccatgtttt cgaagtcccc aaattcatag tatcattttt 42780 aaacctctac catcaccggg tgagagaagt gcataactca tatgtatggc agtttaactg 42840 gactttctct tgtttccagt ttggggctat aaaggtttgt aacaggtcct agtgtctggc 42900 agtgtgtgtt ctccagattt attatctttc ttcaagattg gtttggctac tcttaggtgc 42960 ttatatttcc aaataatttt taaaggtatt agtttgtcaa tttcccaaaa ccttgggctg 43020 gaatttctgg cagggtgaca ctaaatttat aggctagttt ggaaagaact gaatcttgac 43080 acgttgaggc tttccattcc tgaatataat tatgcttcca atttgtttgg ggtttctttt 43140 atttaaccag gaatgttgtg aatttgttgt catggctttc gagtctttgg ttttccctag 43200 ataattaata tttttgttgt agaacataaa tagtttttat cattctgatg atgttaatct 43260 gtcaactttg ctaaatttac tagtcactat tcgtaattta tttctggatt cattgtaatt 43320 tctgtgtata ttatactgta tctgagttaa tattgtttta tttcttattt tccatttctc 43380 atgggcttaa tgtctcttta tcacattcat tattgcatta gctagaattt ctaggagagc 43440 attgaataga attggtgaca gtggggatcc ttgtttctca tttctaatct gcaggaagca 43500 gtggaagttt tccatttcaa tattgagaat gatgcttgaa gtagattttg gtagatattt 43560 tttatcagat tagagaagtt tgctgtcata tatatatatg tcataattgt gtgtaaaatc 43620 ttgtcaaatc aacactctgc atctatttat ataattgtgt gttagtccat ttccattgct 43680 ataaaggaat atctttgact gggtaattta taaataaaag agtttcattt tggctcatgg 43740 ctctgcaggc tgtgcaagag gcataatgtg ggcaggcatc tactcctagt aaggacttca 43800 ggtagctttc aatcttcgga gaaggtgaag gggaagcaag catgtcacat ggcaagagag 43860 ggagtaagag aaaaaggagt agaagtgact ttaaacaacc agctagtgtg tgaactaaca 43920 gaatatgaac tcacttatta tcatgggaag aggtttaagc cactcatgaa gaattcaccc 43980 ccatgtccta aatacttctc acaccagaac ccacctccaa cattgagaat cacatttcaa 44040 catgagattt ggaggggaca aatatccaca caacatcaga ttatatatat tgggcttaca 44100 tttctcacta aatagaaata agtatgcaca tagagatgaa ctggccttct tcatattgtt 44160 taggacttca gtgtttacat tcacaagaga tttggtttat agttttattt ctcagaattt 44220 tcaagatttt tgatattaag attatgccag ccttataaaa ctatgcaaga aatgtttcct 44280 aatgtgagac tgtttttatt gccgttccct ctccccactt ttcttcttgt gatcacttag 44340 ttgtatcttt gaaatgcctg ataacttttg gttaaatacc agatggtata tgtaagaaat 44400 tgcagaattg cagaggaggt ttatattctt caaagaaaga ttcattcctc tcccatcggc 44460 agtcataatg acatgatacc tgttgtcatg attcagaatt ggacataatg tgaatccagt 44520 tgaaaattga gtgtcagtgt tagcttagct ctctgaggtt ctcctacctt tggaagcttg 44580 agaccaattt ttgtcttgac agctttgcag tgccttctat cacatggttt ctgtatttat 44640 ccagacactc catttgccat ctcaaaccta ctcttttcca atcaaaacca atatatatta 44700 ttagtaaaat aaaaaataat ataactgaaa gttgtatttt caaggggaaa aagcaatgag 44760 acttggtaag gttgcaatta ctaatgtcat cttttgaaga agacagaatt gataatttac 44820 ttagagatta tggggcttac cctagtggtc actgagttag tacaagagaa aagacttcaa 44880 tgcactctgt tttcacaaac acaggagata accaggagac tttcgcttct caaaattagg 44940 gtctgagggt aatggtaatg aaacttactt cttctcctat aactaattaa atgagaaata 45000 tactgaagaa tcaggcacga gttgccagtc tccccagttc actgcatcag ctttggtgac 45060 ctcctggagg ggaacgctgc atctgatgag ctcatgctgt gcctgatgtc ttttgagccc 45120 tgagaagcca gagcccaagg ggattaccca ttgaatccct tcttcgtgga atagcccagt 45180 gcagaaaaaa gcagcactgt gtggccaatt ctaccctctt tacaaatcag atacatgtgt 45240 tcttctgcta caggctggat ccacttatct agctggagat gatagtctga gacaggagtt 45300 ggcaaactgt ctctgtaatg ggccagatag taaatatttt agacttttca ggcaatgtgg 45360 tttctgttgc aattcttgac atctgctatt gtagcatgat agcagtcaca aacaatctgc 45420 aaatgaatga ggattgatat gttccaattt ttatagaaac aaatgtaaat ttcctataaa 45 480 atgtttgttt caaaaatatt gtttcttgtt tgcatttgtt caatgtaagt acttttagct 45540 cacatactat acaaaaacag gtggcagggt ggacttgtgc agggagtatt ttttattgat 45600 gcctaatata agacatccct gtccagtaga attttctgca aagatgtgcg attgatttgt 45660 gcttttatgt catatttaaa gaatatttgt cagcacaaga ttccaaagac tggctttcaa 45720 ctttcccaga atttttttgt tttagctctt atatctagat caatgacata tttcaaatta 45780 atttttattt cataatatga agtaatagtg gtcatgcttc aatgtctata aatttctact 45840 tattcaagta acatttgttg aaataaatct gctttggtat caataacatt ttaatatagt 45900 acttgtttat aaatattcaa gggaatattt ttaaaaaaca aaattataga ctatttatat 45960 gaaaatttag ggggtagaat ttttcctaac atttttatta taaaattttt taaaataaat 46020 aacattacac acacatatgt atatgtacat atggcagtgt gtatacatat ctactctgat 46080 atctgtccat ccatctattt acacctcaat acttcagtat gtatagcaca ataacaatgg 46140 ttttctccta cataataata gcattcatac tgtggagtta ttaatacaat aatattattt 46200 actatacagc ttttatttaa aatttcccaa ctttaaaata atattttaat cacttttttc 46260 ttgcaattca ctctccagtc aagaaataag cacatagttt cagtatcctg ttactttcca 46320 tctcctgtaa tcaagaagag tccatttcct ctgtttcctt tcagtatgtt gatctctgtg 46380 aagagttgag caattagtca tttaaatttt cctggggctc gttttgggta attgttttct 46440 aaatattata ttccagttat gactttggca agaaaactac atagtatttt gaatattctg 46500 ctgaatcaca gtggtgaggc aggggcacat aatgtcacat atgccattat tggttatgct 46560 aagatcctca cctaataagt tggaatctgc cagatctttc cattgaaggg gttctgcttt 46620 ttatgcgtga atgatgaaaa tctatggcat ggtacttcaa ggcagaacac cctgtttccc 46680 aagtcacaca tccaatagtt ttatctttca ttaatgatcc tcatctgacc tgatgatctt 46740 gacctgaatg gttacaaata ggtgattttt ctaatttcat cattccttcc gtatttttaa 46800 gctaggatta atctgtaatg aatatctctc ccatatcttt aaatgaagac aaacaaactg 46860 catggcaatg acaagaactc aatagtttta tccttgccac tacacttgtt accatcccag 46920 aagatgtcat ttgaaattca atcatatgtt tattcacaaa tgccctgtga acagatactg 46980 tggccatttc ccatgtcaac agagacttca acttttagtt atactctaca cataaggttg 47040 gtgaaaactg aagtgatgga atagagaagc aagtgtggac agccactagt ttccaagctt 47100 gatgaaaagg aggacagatt cagaaggttg catccaagta tccaagtact caggacttca 47160 aatgtgattg cagggcactt tagcaagatt attgtcatgg gccttaagtt catgactctt 47220 attacttggt ctatccatct ggaaatggta ctgcccttct ttggaacggg atttcctcat 47280 ctgcacatca aaagatttaa ctacatgatt accactgttt cttcaacctt catggcttct 47340 ttacagctca gttcatctat gtctcttgtt tctagggcac aaccatatta atttccctga 47400 cttctgtgct acatgacaac aaagaatttc ccaacccaga gatgtttgac cctcatcact 47460 ttctggatga aggtggcaat tttaagaaaa gtaaatactt catgcctttc tcagcaggta 47520 atataaattt atttccattt gtgtttcagg gtacaagata acttttttga tccattggaa 47580 cttacatgtg cctcctctgc agtggtacaa ttactctttg tacatgatca agagcactgt 47640 tctgaatgcc tgtgtacacc ctgctcatga tacatcctaa ttattgggcc agattagtgg 47700 actttgggga gttaatccaa ttcttccaaa ttgagaaagc tgaaatatag gttggttgaa 47760 ttctgcctct aggtacacca gtgaggtacc caagaactcc tcctggaaga taaaactaat 47820 tacattttcc tcactagcca tgaggaagtt atctcactcc agaacttcac tgagtgtctt 47880 ccacatggtg tccctcaccc cctaggctgg gcttgtagga taaaattatc cacaaacaca 47940 gaatagggtc ttaaaaggct cacttctgag tttggaaagc agagtaaaca gatcattgta 48000 gttcaatagg actgaggctg tgatatggaa agaacaggct gttgggggtt gggaggtaga 48060 cggaaaagct gtctgcttct tgtactctta taccccaaag tgaggcataa gtaatatttt 48120 aatagcagta aagacatttg agctacctca aaggaggcag agaggatgaa aagaagagaa 48180 gacagggcta ttaaaggaga taatgagcca caggagcagg acattggctc taaataaagg 48240 atattagaac ctttgccaaa tgaccatgga tgggatgagg gggacattgg gaatgtagca 48300 ggatactctg cagtgatgca gagcaccatg ctgttcccct agtcatggcc atgtatattg 48360 gatgttggat ctccatactt gaaatgtgtg tgctgagcat ctggtgatag aatctccttc 48420 ctttattcta tgacctttaa tgtctgcttt atatctgcca ctgtagatac caagacaaca 48480 agaaaagaaa ccttccttca agcattcaca tttagcacat gtattagtcc actcttacat 48540 tgccttaaag aactacctga gattgggtaa tttatgaaga aaagaggttt aattcactca 48600 cagatccaca ggctgtacag gaagcatggc tgggagacct caagaaactt gcaatcatgg 48660 tgggaggcag agcggaagca agcacatctt tccatggcag agcagaagag agagaacaat 48720 gaggaaaggc tacatacctt taaacaacca gatctcatga gaactcactc actatcatga 48780 gaacagaaag ggggaaatta tcccccatga ttcaatcacc tctcaccagg tatctccccc 48840 aacattggga attacagttt gacatgagat ttgagtaagg acacagagcc caaccatatc 48900 gcacactaat aaataaataa ataaatcagt aattattatt ctaggtgatc catgctgtct 48960 gtttcttctg ccataacaaa atatccaaga ctaggcaatt gataaataat ggaaatttat 49020 ttctcacagt tctgaaggct gggagttcca agatcgatgt gtcagcaagt tcagtgtctg 49080 atgtgggcct gttccttaca gataatgccc cctctgtatc cttatatggc agaagggcaa 49140 aaaggcaaaa ggggagaaat ggctcccttg catctcttga tgttattatt ttagcaagga 49200 cagagccctc ctgacttatt cacttcctaa aaggagccat ctctttagta atgttgcatt 49260 gggattatgt gtcaacatat gaaattgggg gaggcatatt cagaccatag cacatttttc 49320 aatggaaata taatgttgag gaaacccaga gaaggcaaca ttttcttgct caaggagatg 49380 agaaagaggg taaaaaagga gataaaattt gacctatgtc ctgactgtgg taatagaaaa 49440 gttcatcttg gctaaaagga gcagcatgat ataaaatttg aaacctcatg gtgtgttgga 49500 gactgatgat gagtggctat gcctagagtt gacagtatcg gatttgaaga gtgtaaggag 49560 tgatgtggat catcagactg gaaacagaat gtgagggtcc agatcaatcc attgggacct 49620 tatcctatag gacatacagg gaagccattt aaagttttaa agtgagaagg tgacatgttt 49680 agacatgtgc tcctgaaagt acctagagga aaaaaatctt tggctgcata ttgagccaga 49740 aatacaaagg gaaatacagt atgttagcct cctcctctaa gcccttctca gttcaaccca 49800 ctggacaaga aatgtatgtt tctaaagaaa gattgatgaa gacatttaaa gtctcttgaa 49860 agattttaat aaagtgcttg gcatgtagct ggtactcaac aaatatttgt tgaatacagg 49920 gtgcctgtta agatctgata ttaggtgaag agtaagtatg tccattcatt tttcagttgc 49980 ctatacatcc atccattcat ccatttatcc atccactcat ccatccattc attcatgcat 50040 gcacccatcc acccatctat ctcttcatct cttctacgat acactgaaca gttattgcat 50 100 attctgtttg tgccagttac agagacagtg tttgtcactg tcacagttac gcatgaggag 50160 taactgctct ctgtgtttgc tattttcagg aaaacggatt tgtgtgggag aagccctggc 50220 cggcatggag ctgtttttat tcctgacctc cattttacag aactttaacc tgaaatctct 50280 ggttgaccca aagaaccttg acaccactcc agttgtcaat ggatttgcct ctgtgccgcc 50340 cttctaccag ctgtgcttca ttcctgtctg aagaagagca gatggcctgg ctgctgctgt 50 400 gcagtccctg cagctctctt tcctctgggg cattatccat ctttcactat ctgtaatgcc 50460 ttttctcacc tgtcatctca cattttccct tccctgaaga tctagtgaac attcgacctc 50520 cattacggag agtttcctat gtttcactgt gcaaatatat ctgctattct ccatactctg 50580 taacagttgc attgactgtc acataatgct catacttatc taatgttgag ttattaatat 50640 gttattatta aatagagaaa tatgatttgt gtattataat tcaaaggcat ttcttttctg 50700 catgttctaa ataaaaagca ttattatttg ctga 50734 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> VKORC1_3673_A primer <400> 3 acctgaaaaa caaccattgg tca 23 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> VKORC1_3673_G primer <400> 4 acctgaaaaa caaccattgg tcg 23 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> VKORC1_3673_Rev primer <400> 5 tcctgacctc aagtgatcca cc 22 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> VKORC1_6484_C primer <400> 6 cggtgccagg agatcatcgt cc 22 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> VKORC1_6484_T primer <400> 7 cggtgccagg agatcatcgt ct 22 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> VKORC1_6484_Rev primer <400> 8 ctgcccgaga aaggtgattt ccaag 25 <210> 9 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> VKORC1_6853_C primer <400> 9 cacccgcagg acgcacg 17 <210> 10 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> VKORC1_6853_G primer <400> 10 cacccgcagg acgcacc 17 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> VKORC1_6853_rev primer <400> 11 cccactccat gcaatcttgg 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> VKORC1_9041_A primer <400> 12 ccctcctcct gccatactca 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> VKORC1_9041_G primer <400> 13 ccctcctcct gccatactcg 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> VKORC1_9041_Rev primer <400> 14 ggcaatggaa agagctttgg 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CYP2C9_430_C primer <400> 15 cgggcttcct cttgaactcg 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CYP2C9_430_T primer <400> 16 cgggcttcct cttgaactca 20 <210> 17 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> CYP2C9_430_REV primer <400> 17 gggaggatgg aaaacagaga ctta 24 <210> 18 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> CYP2C9_1075_A primer <400> 18 ggtgcacgag gtccagagat tca 23 <210> 19 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CYP2C9_1075_C primer <400> 19 gtgcacgagg tccagagatt cc 22 <210> 20 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> CYP2C9_1075_REV primer <400> 20 gatactatga atttggggac ttcgaa 26 <210> 21 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> VKORC1_3673_probe <400> 21 tgcggtggct cacgcctata atcct 25 <210> 22 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> VKORC1_6484_probe <400> 22 tgggaggtcg gggaacagag gatag 25 <210> 23 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> VKORC1_6853_probe <400> 23 tgagcagcta gctggctgtc agctgt 26 <210> 24 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> VKORC1_9041_probe <400> 24 accaaatgtg ccacacgctc gct 23 <210> 25 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CYP2C9_430_probe <400> 25 tgggcagcca tgtggatggg cc 22 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CYP2C9_1075_probe <400> 26 tccccaccag cctgccccat 20

Claims (15)

(1) 표적 유전자의 대립형질 특이적 프라이머를 서로 다른 튜브에 포함하고 표적 유전자에 공통적인 프로브를 각각 사용하여 실시간 중합효소 연쇄반응을 수행하여 상기 표적 유전자의 증폭산물을 생성하는 단계;
(2) 상기 각각의 튜브의 대립형질 특이적 증폭산물로부터 Ct 값을 구하는 단계;
(3) 상기 단계 (2)에서 얻어진 Ct 값으로부터 대립형질간의 ΔCt 값을 구하는 단계; 및
(4) 상기 얻어진 ΔCt 값으로부터 상기 표적 유전자의 단일 염기 다형성을 판별하는 단계를 포함하는 실시간 중합 효소 연쇄반응을 이용한 표적 유전자의 단일 염기 다형성 판별 방법.(1) generating an amplification product of the target gene by including allele-specific primers of the target gene in different tubes and performing real-time polymerase chain reaction using probes common to the target gene, respectively;
(2) obtaining Ct values from allele specific amplification products of each tube;
(3) obtaining a ΔCt value between alleles from the Ct value obtained in step (2); And
(4) A method for determining single nucleotide polymorphism of a target gene using a real-time polymerase chain reaction comprising determining a single nucleotide polymorphism of the target gene from the obtained ΔCt value. 청구항 1에 있어서,
상기의 튜브에 서로 다른 표적유전자 2개 내지 5개에 해당하는 각각의 대립형질 특이적 프라이머를 포함하는 다중 검출 실시간 중합효소 연쇄반응인 것을 특징으로 하는 단일 염기 다형성 판별 방법.The method according to claim 1,
Single-nucleotide polymorphism determination method characterized in that the multiple detection real-time polymerase chain reaction containing each allele specific primer corresponding to two to five different target genes in the tube. 청구항 1에 있어서,
상기의 프로브는 택맨 프로브인 것을 특징으로 하는 단일 염기 다형성 판별 방법.The method according to claim 1,
Single probe polymorphism determination method characterized in that the probe is a Taekman probe. 청구항 1에 있어서,
VKORC1 3673G>A ΔCt 값이 ≤±4.1, 6484C>T ΔCt 값이 ≤±4.7, 6853G>A ΔCt 값이 ≤±7.8, 9041G>A ΔCt 값이 ≤±3.1, CYP2C9-430C>T ΔCt 값이 ≤±6.7, 1075A>C ΔCt 값이 ≤±3.0을 특징으로 하는 단일 염기 다형성 판별 방법.The method according to claim 1,
VKORC1 3673G> A ΔCt value ≤ ± 4.1, 6484C> T ΔCt value ≤ ± 4.7, 6853G> A ΔCt value ≤ ± 7.8, 9041G> A ΔCt value ≤ ± 3.1, CYP2C9-430C> T ΔCt value ≤ Single base polymorphism determination method characterized in that ± 6.7, 1075A> C ΔCt value is ≤ ± 3.0. 청구항 1에 있어서,
상기 프로브의 5' 말단에는 리포터가 결합되어 있고, 3' 말단에는 소광제가 결합되어 있는 것을 특징으로 하는 단일 염기 다형성 판별 방법.The method according to claim 1,
The reporter is bound to the 5 'end of the probe, and the quencher is bound to the 3' end, characterized in that the single base polymorphism discrimination method. 청구항 5에 있어서,
상기 리포터는 FAM (6-carboxyfluorescein), Texas red, JOE, TAMRA, CY5 및 CY3로 이루어진 군으로부터 선택되는 것을 특징으로 하는 단일 염기 다형성 판별 방법.The method according to claim 5,
Wherein the reporter is selected from the group consisting of FAM (6-carboxyfluorescein), Texas red, JOE, TAMRA, CY5 and CY3. 청구항 5에 있어서,
상기 소광제는 TAMRA (6-carboxytetramethyl-rhodamine), BHQ1, BHQ2 및 Dabsyl로 이루어진 군으로부터 선택되는 것을 특징으로 하는 단일 염기 다형성 판별 방법.The method according to claim 5,
Said quencher is selected from the group consisting of TAMRA (6-carboxytetramethyl-rhodamine), BHQ1, BHQ2 and Dabsyl. 청구항 1에 있어서,
상기 표적 유전자는 서열번호 1로 기재되는 VKORC1 유전자 또는 서열번호 2로 기재되는 CYP2C9 유전자인 것을 특징으로 하는 단일 염기 다형성 판별 방법.The method according to claim 1,
The target gene is a single nucleotide polymorphism determination method, characterized in that the VKORC1 gene described in SEQ ID NO: 1 or CYP2C9 gene described in SEQ ID NO: 2. 청구항 1 또는 청구항 8에 있어서,
상기 표적 유전자의 대립형질은 서열번호 1로 기재되는 VKORC1 유전자의 3673G>A(rs No. 9923231), 6484C>T(rs No. 9934438), 6853G>C(rs No. 8050894), 9041G>A(rs No. 7294), 서열번호 2로 기재되는 CYP2C9 유전자의 1075A>C(rs No. 1799853) 및 430C>T(rs No. 1057910)로 이루어진 군으로부터 선택되는 6개 단일 염기 다형성 부위 중 하나 이상인 것을 특징으로 하는 단일 염기 다형성 판별 방법.The method according to claim 1 or 8,
Alleles of the target gene were 3673G> A (rs No. 9923231), 6484C> T (rs No. 9934438), 6853G> C (rs No. 8050894), 9041G> A (SEQ ID NO: 1) rs No. 7294), at least one of six single base polymorphic sites selected from the group consisting of 1075A> C (rs No. 1799853) and 430C> T (rs No. 1057910) of the CYP2C9 gene set forth in SEQ ID NO: 2 Single nucleotide polymorphism determination method characterized by. 청구항 1 또는 청구항 8에 있어서,
상기 프라이머는 서열번호 3 내지 서열번호 20으로 이루어진 군으로부터 선택되는 것을 특징으로 하는 단일 염기 다형성 판별 방법.The method according to claim 1 or 8,
The primer is a single nucleotide polymorphism determination method, characterized in that selected from the group consisting of SEQ ID NO: 3 to SEQ ID NO: 20. 청구항 1 또는 청구항 8에 있어서,
상기 프로브는 서열번호 21 내지 서열번호 26으로 이루어진 군으로부터 선택되는 것을 특징으로 하는 단일 염기 다형성 판별 방법.The method according to claim 1 or 8,
The probe is a single nucleotide polymorphism determination method, characterized in that selected from the group consisting of SEQ ID NO: 21 to SEQ ID NO: 26. 서열번호 3 내지 서열번호 20으로 이루어진 군으로부터 선택되는 프라이머를 포함하는, 서열번호 1로 기재되는 VKORC1 유전자 또는 서열번호 2로 기재되는 CYP2C9 유전자의 단일 염기 다형성을 PCR을 통해서 검출하기 위한 단일 염기 다형성 판별 키트.Single base polymorphism determination for detecting, by PCR, a single base polymorphism of the VKORC1 gene described in SEQ ID NO: 1 or the CYP2C9 gene described in SEQ ID NO: 2, comprising a primer selected from the group consisting of SEQ ID NO: 3 to SEQ ID NO: 20 Kit. 청구항 12에 있어서,
서열번호 21 내지 서열번호 26으로 이루어진 군으로부터 선택되는 프로브를 추가로 포함하는 것을 특징으로 하는 단일 염기 다형성 판별 키트.The method of claim 12,
Single base polymorphism determination kit, characterized in that it further comprises a probe selected from the group consisting of SEQ ID NO: 21 to SEQ ID NO: 26. 청구항 12에 있어서,
상기 키트는 (a) dNTP 혼합물(dATP, dCTP, dGTP, dTTP); (b) DNA 중합효소; 및 (c) 완충 용액을 추가로 포함하는 것을 특징으로 하는 단일 염기 다형성 판별 키트.The method of claim 12,
The kit comprises (a) a dNTP mixture (dATP, dCTP, dGTP, dTTP); (b) DNA polymerase; And (c) a buffer solution further comprising a single base polymorphism determination kit. 청구항 12에 있어서,
상기 DNA 중합효소는 Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus 또는 Thermococcus literalis로부터 수득한 열 안정성 DNA 중합효소로 이루어진 군으로부터 선택되는 것을 특징으로 하는 단일 염기 다형성 판별 키트.The method of claim 12,
Wherein said DNA polymerase is selected from the group consisting of thermally stable DNA polymerases obtained from Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus or Thermococcus literalis.

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