KR101618243B1

KR101618243B1 - HRluc reporter vector inserted restriction site into open reading frame of HRluc

Info

Publication number: KR101618243B1
Application number: KR1020140027362A
Authority: KR
Inventors: 정면우; 임철주; 심선보; 지승완; 권오석; 조영민; 박송희; 김광제; 이운규; 김동화; 송기덕; 장혜정; 이수찬; 정재훈
Original assignee: 대한민국
Priority date: 2014-03-07
Filing date: 2014-03-07
Publication date: 2016-05-09
Also published as: KR20150105578A

Abstract

본 발명은 HRluc의 ORF (open reading frame)에 제한 효소 절단위치가 삽입된, HRluc 레포터 벡터 및 상기 벡터가 도입된 숙주세포에 관한 것이다. 또한, 본 발명은 상기 숙주세포를 이용한 실험 대상 유전자의 5＇ 플랭킹 부위 (5＇ flanking region) 활성의 확인 방법에 관한 것이다. 본 발명의 HRluc 레포터 벡터는 실험 대상 유전자의 5＇ 플랭킹 부위 (5＇ flanking region)와 접합되어 실험 대상 유전자의 5＇ 플랭킹 부위 (5＇ flanking region)에서 유전자 조절에서 중요한 핵산 영역들을 식별 및 분석하는데 매우 유용하게 사용될 수 있다.The present invention relates to an HRluc reporter vector in which a restriction enzyme cleavage site is inserted into an ORF (open reading frame) of HRluc and a host cell into which the vector is introduced. The present invention also relates to a method for confirming 5 'flanking region activity of a gene to be tested using the host cell. The HRluc reporter vector of the present invention is joined to the 5 'flanking region of the gene to be tested to identify regions of nucleic acid important in gene regulation in the 5' flanking region of the gene of the subject gene And can be very useful for analysis.

Description

The HRluc reporter vector {HRluc reporter vector inserted restriction site into open reading frame of HRluc}, in which the restriction enzyme cleavage site was inserted into the ORF of HRluc,

본 발명은 HRluc의 ORF (open reading frame)에 제한 효소 절단위치가 삽입된, HRluc레포터 벡터 및 상기 벡터가 도입된 숙주세포에 관한 것이다.The present invention relates to an HRluc reporter vector in which a restriction enzyme cleavage site is inserted into an ORF (open reading frame) of HRluc and a host cell into which the vector is introduced.

또한, 본 발명은 상기 숙주세포를 이용한 실험 대상 유전자의 5＇ 플랭킹 부위 (5＇ flanking region) 활성의 확인 방법에 관한 것이다.
The present invention also relates to a method for confirming 5 'flanking region activity of a gene to be tested using the host cell.

레포터 유전자 분석은 예컨대 DNA 서열, 전사 인자, RNA 서열, RNA-결합 단백질, 신호 전달 통로(signal transduction pathways) 및 특정 자극과 같은 중요 유전자 발현을 무엇이 조절하는 지에 대해 해석하여 유전자 발현 연구에 있어 중요한 도구를 나타낸다. 특히, 레포터 분석은 유전자 조절에서 중요한 핵산 영역들을 식별하는데 사용될 수 있다. 그러한 영역들 및/또는 상기 영역들을 결합하거나 변경하는 인자들은 인간 질병의 치료나 예방에 있어 치료적 개입(therapeutic intervention)을 위한 잠재적 목적으로서 역할을 할 수 있다. 레포터 분석은 약품을 선별하여 상기 약품의 효력이 유전자 발현을 변형하도록 하는 데 사용될 수 있다.Reporter gene analysis interprets what controls important gene expression, such as DNA sequences, transcription factors, RNA sequences, RNA-binding proteins, signal transduction pathways, and specific stimuli, Tool. In particular, reporter assays can be used to identify regions of nucleic acid important in gene regulation. Such regions and / or factors that combine or modify these regions may serve as potential targets for therapeutic intervention in the treatment or prevention of human disease. Reporter assays may be used to screen drugs to allow the effect of the drug to modify gene expression.

이상적인 레포터 유전자는 다음의 특징을 가져야 한다.The ideal reporter gene should have the following characteristics.

(1) 레포터 유전자는 면역반응을 피하기 위해 포유동물의 세포에 존재하되 발현은 하지 않아야 한다.(1) The reporter gene should be present in the cells of the mammal in order to avoid the immune response, but not expressed.

(2) 특정 레포터 탐색자는 특정 레포터 유전자가 발현된 세포에만 선택적으로 축적되어야 한다.(2) A specific reporter probe should selectively accumulate only in cells expressing a specific reporter gene.

(3) 레포터 유전자가 발현되지 않을 때는 레포터 탐색자가 축적되지 않아야 한다.(3) When the reporter gene is not expressed, the reporter probe should not accumulate.

(4) 레포터 유전자 생산물에 의해 면역 부작용이 일어나지 않아야 한다.(4) No immunosuppressive effects should be caused by reporter gene products.

(5) 레포터 탐색자는 생명체 내에서 안정해야 한다. (레포터 탐색자가 생명체 내에서 말단 대사물로 대사될 경우 이 탐색자 축적의 정량분석이 어렵기 때문이다.)(5) Reporter searchers must be stable in life. (This is because it is difficult to quantitate the accumulation of this probe when the reporter probe is metabolized to end-metabolism in living organisms.)

(6) 레포터 탐색자는 혈액 내에서나 비 특이적으로 섭취된 세포에서 빨리 배출되어야 한다.(6) Reporter probes should be rapidly released from blood or non-specific cells.

(7) 레포터 탐색자는 영상 목적의 용량에서 세포 독성을 가지지 않아야 한다.(7) Reporter probes should not be cytotoxic in imaging-purpose doses.

(8) 레포터 유전자의 크기는 매개체에 삽입할 수 있을 정도의 크기를 유지해야 한다.(8) The size of reporter gene should be large enough to be inserted into the medium.

(9) 레포터 탐색자는 표적 세포의 세포막을 용의하게 통과하여야 한다.(9) The reporter probe must pass through the cell membrane of target cells.

(10) 레포터 탐색자의 영상 신호는 레포터 유전자의 mRNA/ 단백질의 발현 정도와 높은 상관관계를 가져야 한다.(10) The signal of the reporter probe should have a high correlation with the expression level of the mRNA / protein of the reporter gene.

그 동안 사용되어 왔던 레포터 유전자로는 CAT (chloramphenicol acetyl transferase), ALP (alkaline phosphatase), lac Z등을 들 수 있다. 하지만 이들 레포터 유전자의 발현여부를 알기 위해서는 발현된 조직을 적출하거나 혈액을 채취해야 하는 번거로움이 있고, 반복 검사가 힘들거나, 발현된 세포나 조직의 위치, 발현 기간, 발현 강도 등을 생명체 내에서 종합적으로 모니터 하는데 한계가 있다.Reporter genes that have been used in the past include CAT (chloramphenicol acetyl transferase), ALP (alkaline phosphatase), and lac Z. However, in order to determine whether these reporter genes are expressed, it is troublesome to extract the expressed tissues or collect the blood, and it is difficult to repeatedly examine the reporter gene, and the position of the expressed cell or tissue, the period of expression, There is a limitation in monitoring them collectively.

생명체 내 실험에 사용되던 레포터 유전자로는 GFP (green fluorescent protein)과 루시퍼라아제 (luciferase)로, 이 유전자의 발현에 의한 형광(fluorescence) 또는 생물 발광(bioluminescence)으로 유전자 발현의 정보를 제공한다. 빛의 투과가 용이한 동물 모델에서는 적절한 장비에 의한 영상화가 가능하게 되었다. The reporter genes used in living organisms are GFP (green fluorescent protein) and luciferase, which provide information on gene expression by fluorescence or bioluminescence by expression of this gene . In an animal model that is easy to transmit light, imaging with appropriate equipment has become possible.

루시퍼라아제는 생체 외 분석 시스템에서 가장 일반적으로 사용되는 레포터 단백질이다. 루시퍼라아제는 생물 발광을 할 수 있는 효소이며, 유기체의 범위에서 발견된다. 시판중인 분석 시스템에서, 루시퍼라아제는 기질로서 D-루시페린을 사용하는 것과 기질로서 코엘렌테라진(coelenterazine)을 사용하는 것으로 나누어질수 있다. 전자의 가장 널리 채용되는 예는 반딧불이 루시퍼라아제 즉 세포내 효소이다. D-루시페린을 사용하는 루시퍼라아제의 다른 예는 방아벌레나 철도 벌레(railroad worms)와 같은 딱정벌레의 다른 종류를 포함한다. 루시퍼라아제는 상기 루시퍼라아제가 유도된(derived) 유기체가 육생 또는 수생(통상적으로 해양성)인지의 여부를 기초로 하여 구별될 수 있다. 통상적으로, 기질로서 코엘렌테라진을 사용하는 루시퍼라아제는 연산호류 레닐라 (soft coral Renilla) 또는 요각류 가우시아(copepod Gaussia)와 같은 해양성 동물로부터 유도되는 반면, D-루시페린을 사용하는 루시퍼라아제는 육색 동물로부터 유도되는 것이 일반적이다. 루시퍼라아제들을 구별하는 다른 수단은, 상기 루시퍼라아제들이 자연 상태 즉 상기 루시퍼라아제들이 유도된 유기체에서 분비성인지 비분비성인지의 여부를 기반으로 한다. 육생 유기체로부터 유도된 루시퍼라아제는 통상적으로 비 분비성(세포내)이며, 반면 해양성 유기체로부터 유도된 루시퍼라아제는 분비성 또는 비 분비성(세포내)일 수 있다. 예컨대, 레닐라 루시퍼라아제는 세포내에 있으며, 반면 가우시아 루시퍼라아제는 자연 상태에서 분비성 효소이다. 해양성 유기체에 의해 루시퍼라아제가 분비되는 것은 접근 중인 포식자를 교란시키도록 설계된 보호 감응으로 생각될 수 있다. 다른 분비성 루시퍼라아제들은 메트리디아 롱가(Metridia longa), 바르굴라 힐겐도르피아이(Vargula hilgendorfii), 오플로포루스 그라실리로스트리스(Oplophorus gracilirostris), 플류롬마 시피아스 (Pleuromamma xiphias), 키프리디나 녹틸루카(Cypridina noctiluca), 및 메트리딘 과(Metridinidae)의 다른 종류로부터의 루시퍼라아제들을 포함한다. 바르굴라 루시퍼라아제는 코엘렌테라진 또는 D-루시페린과 다른 기질을 사용한다. 루시퍼라아제의 다른 클래스(class)는 와편모조류(dinoflagellates)로부터 유도된 루시퍼라아제이다.Luciferase is the most commonly used reporter protein in an in vitro assay system. Luciferase is an enzyme capable of bioluminescence and is found in a range of organisms. In a commercially available assay system, luciferase can be divided into using D-luciferin as a substrate and using coelenterazine as a substrate. The most widely adopted example of the former is firefly luciferase, an intracellular enzyme. Other examples of luciferase using D-luciferin include other species of beetles, such as insect bugs and railroad worms. Luciferase may be distinguished on the basis of whether the organism from which the luciferase is derived is anthrax or aquatic (usually marine). Typically, luciferase using coelenterazine as a substrate is derived from marine animals such as soft coral Renilla or copepod Gaussia, while Luciferar using D-luciferin Agases are generally derived from colorectal animals. Another means of differentiating luciferases is based on whether the luciferases are naturally occurring, i.e., whether the luciferases are secreted in the induced organism or not. Luciferase derived from perennial organisms is typically non-secretable (intracellular) whereas luciferase derived from marine organisms may be secretory or non-secretable (intracellular). For example, Renilla luciferase is within a cell, whereas Gaussia luciferase is a secreted enzyme in its natural state. Secretion of luciferase by marine organisms can be thought of as a protective response designed to perturb an approaching predator. Other secretory luciferases include, but are not limited to, Metridia longa, Vargula hilgendorfii, Oplophorus gracilirostris, Pleuromamma xiphias, Cypridina noctiluca, and luciferases from other classes of methridine and Metridinidae. Vaglala luciferase uses a substrate other than coelenterazine or D-luciferin. Another class of luciferase is luciferase derived from dinoflagellates.

본 발명자는 루시퍼라아제를 포함하는 레포터 벡터에 제한 효소 절단 위치를 삽입하는 경우, 유전자의 5＇ 플랭킹 부위의 활성을 효과적으로 확인할 수 있음을 확인하고, 본 발명을 완성하기에 이르렀다.
The inventors of the present invention have confirmed that the activity of the 5 'flanking region of a gene can be effectively confirmed when a restriction enzyme cleavage site is inserted into a reporter vector comprising luciferase, and the present invention has been accomplished.

본 발명의 일 양상은 HRluc의 ORF (open reading frame)에 제한 효소 절단위치 (restriction site)가 삽입된, HRluc 레포터 벡터 및 상기 벡터가 도입된 숙주세포를 제공하는 것이다.One aspect of the present invention is to provide a HRluc reporter vector in which a restriction enzyme site is inserted into an ORF (open reading frame) of HRluc and a host cell into which the vector is introduced.

또한 본 발명의 다른 양상은 상기 숙주세포를 이용한 실험 대상 유전자의 5＇ 플랭킹 부위 (5＇ flanking region) 활성의 확인 방법을 제공하는 것이다.
Another aspect of the present invention is to provide a method for confirming 5 'flanking region activity of a gene to be tested using the host cell.

본 발명은 서열번호 1의 염기서열로 이루어진 HRluc 벡터에서 HRluc의 ORF (open reading frame)에 제한 효소 절단위치 (restriction site)가 삽입된, HRluc 레포터 벡터를 제공한다.The present invention provides an HRluc reporter vector in which a restriction site is inserted into an ORF (open reading frame) of HRluc in the HRluc vector consisting of the nucleotide sequence of SEQ ID NO: 1.

이하 본 발명에 대하여 상세히 설명한다.Hereinafter, the present invention will be described in detail.

본 발명에 있어서 레포터 유전자는 유전자의 발현물질 또는 기능 등을 검출할 수 있도록 하는 유전자를 의미하며, 예를 들면, 유전자 발현에 관여하는 촉진자/증강인자(promoter/enhancer), 유발촉진자(inducible promoter)에 의한 유전자 발현 여부, 내재 촉진자(endogenous promoter)의 하위에 레포터 유전자를 두고 내재 유전자(endogenous gene)의 발현을 연구하는 등에 활용될 수 있다.In the present invention, the reporter gene refers to a gene that can detect the expression substance or function of a gene and includes, for example, a promoter / enhancer involved in gene expression, an inducible promoter ), And to investigate the expression of endogenous genes by putting the reporter gene under the endogenous promoter.

본 발명에 있어서 벡터는 숙주 세포에서 목적 유전자를 발현시키기 위한 수단을 의미한다. In the present invention, the vector means a means for expressing a target gene in a host cell.

본 발명에 있어서 HRluc는 반디불이로부터 유래한, 프로모터가 제외된 Renilla luciferase를 코딩하는 유전자로서, 전사 인자 결합 부위 (transcription factor binding site)의 대부분이 제거되고, 코돈 최적화를 통하여 포유동물에서의 발현이 개선된 유전자이다. In the present invention, HRluc is derived from Rhodiola, a promoter-free Renilla A gene coding for luciferase is a gene in which most of the transcription factor binding sites are removed and the expression in the mammal is improved through codon optimization.

본 발명에 있어서, HRluc레포터 벡터는 HRluc유전자를 포함하는 벡터로 서열번호 1의 염기서열로 이루어진 것을 특징으로 한다. In the present invention, the HRluc reporter vector is a vector comprising the HRluc gene and is characterized by being composed of the nucleotide sequence of SEQ ID NO: 1.

본 발명에 있어서 ORF (open reading frame)는 코딩서열(coding sequence, CDS)을 나타내는 것으로 단백질 합성에서 주형(template)이 되는 부분을 말한다.In the present invention, an ORF (open reading frame) refers to a coding sequence (CDS), which is a template for protein synthesis.

본 발명에 있어서 제한 효소 절단위치 (restriction site)란 제한효소에 의해 자를 수 있는 DNA 부위를 의미하며, 상기 제한효소는 DNA의 특정한 염기배열을 식별하고 이중 사슬을 절단하는 효소로서, 그 종류를 제한하지 않는다. 예를 들면, EcoRI, BamHI, HindⅢ, kpnI, NotI, PstI, SmaI, XhoI, PmiI 등이 있으며, 바람직하게는 PmiI이다. 제한 효소 절단 위치는 HRluc유전자의 ORF 내에 임의적으로 위치할 수 있으며, 바람직하게는 서열번호 1의 109 내지 1114번째 염기서열 위치에 치환될 수 있다. 더욱 바람직하게는 상기 HRluc레포터 벡터는 서열번호 2의 염기서열로 이루어진 것 일 수 있다. 상기 HRluc레포터 벡터의 바람직한 개열지도는 도 1과 같다.
In the present invention, a restriction site refers to a DNA site that can be cleaved by a restriction enzyme. The restriction enzyme is an enzyme that recognizes a specific base sequence of DNA and cleaves double chains. I never do that. For example, EcoRI, BamHI, HindIII, kpnI, NotI, PstI, SmaI, XhoI, PmiI and the like, preferably PmiI. The restriction enzyme cleavage site may be arbitrarily located in the ORF of the HRluc gene, and preferably, it may be substituted at the 109th to 1114th nucleotide sequence positions of SEQ ID NO: 1. More preferably, the HRluc reporter vector may comprise the nucleotide sequence of SEQ ID NO: 2. A preferable cleavage map of the HRluc reporter vector is shown in Fig.

본 발명의 HRluc 레포터 벡터는 실험 대상 유전자의 5＇ 플랭킹 부위 (5＇ flanking region)이 접합될 수 있다. 상기 실험 대상 유전자는 그 종류를 제한하지 않으며, 예를 들면 KLF 10 유전자일 수 있다. The HRluc reporter vector of the present invention can be conjugated to the 5 'flanking region of the gene to be tested. The gene to be tested is not limited in its kind, and may be, for example, a KLF 10 gene.

상기의 5＇ 플랭킹 부위 (5＇ flanking region)는 유전자의 5＇ 말단에 인접해 있는 DNA 부위를 의미하며, 이 5＇ 플랭킹 부위 (5＇ flanking region)는 유전자의 발현 여부 및 정도를 조절할 수 있는 프로모터, 인핸서 또는 다른 단백질 결합 부위를 포함할 수 있다. 상기 5＇ 플랭킹 부위 (5＇ flanking region)는 예를 들면 서열번호 8의 염기서열 또는 이에 제한 효소가 처리한 형태, 예를 들면 서열번호 9의 염기서열로 접합될 수 있다. The 5 'flanking region refers to the DNA region adjacent to the 5' end of the gene, and the 5 'flanking region controls the expression and degree of the gene. Promoter, enhancer, or other protein-binding site that can be used. The 5 'flanking region may be conjugated to, for example, the nucleotide sequence of SEQ ID NO: 8 or a form treated with the restriction enzyme, for example, the nucleotide sequence of SEQ ID NO: 9.

본 발명의 벡터는 가장 바람직하게는 도 2의 개열지도와 같은 구성일 수 있으며, 서열번호 11의 염기서열로 이루어진 것일 수 있다.The vector of the present invention may most preferably have the same structure as the cleavage map of Fig. 2 and may be composed of the nucleotide sequence of SEQ ID NO: 11.

본 발명의 상기 HRluc 레포터 벡터는 실험 대상 유전자의 5＇ 플랭킹 부위 (5＇ flanking region)와 접합되어 실험 대상 유전자의 5＇ 플랭킹 부위 (5＇ flanking region)에서 유전자 조절에서 중요한 핵산 영역들을 식별 및 분석하는데 매우 유용하게 사용될 수 있다.
The HRluc reporter vector of the present invention is conjugated with the 5 'flanking region of the gene to be tested to identify regions of the nucleic acid important for gene regulation in the 5' flanking region of the subject gene It can be very useful for identification and analysis.

본 발명의 다른 양상은 상기 HRluc 레포터 벡터가 도입된 숙주 세포를 제공한다. Another aspect of the present invention provides a host cell into which the HRluc reporter vector is introduced.

상기 숙주세포는 당업계에 공지된 어떠한 숙주 세포도 이용할 수 있으며, 원핵 세포로는, 예를 들어, E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, 바실러스 서브틸리스, 바실러스 츄린겐시스와 같은 바실러스 속 균주, 그리고 살모넬라 티피무리움, 세라티아 마르세슨스 및 다양한 슈도모나스 종과 같은 장내균과 균주 등이 있으며, 진핵 세포에 형질 전환시키는 경우에는 숙주 세포로서, 효모(Saccharomyce cerevisiae), 곤충 세포, 식물 세포 및 동물 세포, 예를 들어, SP2/0, CHO(Chinese hamster ovary) K1, CHO DG44, PER.C6, W138, BHK, COS-7, 293, HepG2, Huh7, 3T3, RIN 및 MDCK 세포주 등이 이용될 수 있다.The host cell may be any host cell known in the art and includes, for example, E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B , E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus strain, and Enterobacteriaceae such as Salmonella typhimurium, Serratia marcesensis and various Pseudomonas spp. When transformed into eukaryotic cells, yeast (Saccharomyce cerevisiae), insect cells, plant cells and animal cells such as SP2 / 0, CHO (Chinese hamster ovary) K1, CHO DG44, PER.C6, W138, BHK, COS-7, 293, HepG2, Huh7, 3T3, RIN and MDCK cell lines.

상기 벡터의 숙주 세포 내로의 도입은, 당업계에 널리 알려진 삽입 방법을 사용할 수 있다. 상기 운반 방법은 예를 들어, 숙주 세포가 원핵 세포인 경우, CaCl₂ 방법 또는 전기 천공 방법 등을 사용할 수 있고, 숙주 세포가 진핵 세포인 경우에는, 미세 주입법, 칼슘 포스페이트 침전법, 전기 천공법, 리포좀-매개 형질감염법 및 유전자 밤바드먼트 등을 사용할 수 있으나, 이에 한정하지는 않는다.
The introduction of the vector into a host cell can be carried out using an insertion method well known in the art. For example, when the host cell is a prokaryotic cell, the CaCl ₂ method or the electroporation method can be used. When the host cell is a eukaryotic cell, the microinjection method, the calcium phosphate precipitation method, the electroporation method, Liposome-mediated transfection methods, and gene bombardment, but the present invention is not limited thereto.

본 발명의 또 다른 양상은 상기 HRluc 레포터 벡터가 도입된 숙주세포에서 HRluc의 발현 정도를 확인하는 단계를 포함하는, 실험 대상 유전자의 5＇ 플랭킹 부위 (5＇ flanking region) 활성의 확인 방법을 제공한다. 상기에 있어서, HRluc의 발현 정도를 확인하는 것은 당업계에 알려진 방법에 의할 수 있다. In another aspect of the present invention, there is provided a method for confirming the 5 'flanking region activity of a gene to be tested, which comprises confirming the expression level of HRluc in a host cell into which the HRluc reporter vector has been introduced to provide. In the above, the expression level of HRluc can be confirmed by a method known in the art.

또한, 본 발명은 상기 변이된 HRluc 레포터 벡터에 의하여 제조된 재조합 펩타이드를 제공할 수 있다.
In addition, the present invention can provide a recombinant peptide produced by the mutated HRluc reporter vector.

본 발명의 HRluc 레포터 벡터는 실험 대상 유전자의 5＇ 플랭킹 부위 (5＇ flanking region)와 접합되어 실험 대상 유전자의 5＇ 플랭킹 부위 (5＇ flanking region)에서 유전자 조절에서 중요한 핵산 영역들을 식별 및 분석하는데 매우 유용하게 사용될 수 있다.
The HRluc reporter vector of the present invention is joined to the 5 'flanking region of the gene to be tested to identify regions of nucleic acid important in gene regulation in the 5' flanking region of the gene of the subject gene And can be very useful for analysis.

도 1은 HRlucORF에 제한효소 절단 위치가 삽입된 HRluc레포터 벡터의 예시적인 개열지도를 나타낸 도이다.
도 2는 본 발명의 변이된 HRluc레포터 벡터에 실험 대상 유전자의 5＇ 플랭킹 부위가 접합된 형태의 예시적인 개열지도를 나타낸 도이다.
도 3은 KLF10/5＇아암/ hRluc_Neo_PmlI 벡터를 이용하여 KLF10 유전자의 활성을 확인한 도이다.BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram showing an exemplary cleavage map of HRluc reporter vector in which restriction enzyme cleavage sites are inserted in HRlucORF. FIG.
FIG. 2 is a diagram showing an exemplary cleavage map in which a 5 'flanking region of a subject gene is joined to a mutated HRluc reporter vector of the present invention.
FIG. 3 shows the activity of the KLF10 gene using the KLF10 / 5 'arm / hRluc_Neo_PmlI vector.

이하 본 발명을 실시예를 통하여 보다 상세하게 설명한다. 그러나, 이들 실시예는 본 발명을 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.
Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

실시예Example 1: One: hRluchRluc __ NeoNeo __ PmlI레포터PmlI Reporter 벡터의 제작 Production of vector

pGL4.79[hRluc_Neo] 벡터(Promega, Cat. number: E6971)로부터 돌연변이 (mutagenesis)를 통해 PmlI절단부위를 pGL4.79[hRluc_Neo] 벡터에 삽입하여 hRluc_Neo_PmlI레포터벡터를 제작하였다. hRluc_Neo_PmlI reporter vector was constructed by inserting the PmlI cleavage site into the pGL4.79 [hRluc_Neo] vector via mutagenesis from the pGL4.79 [hRluc_Neo] vector (Promega, Cat. number: E6971).

구체적으로, pGL4.79[hRluc_Neo] 벡터에 PmlI 절단부위를 첨가하기 위해 프라이머(서열번호 3 및 4)를 제작하였다. 또한, 돌연변이(mutagenesis)는 QuikChange^®Site-Directed Mutagenesis Kit(Stratagene, Cat. Number: 200518)를 사용하였으며 사용 설명서에 따라 실시하였다. 조건은 하기의 표 1과 같다.. PCR 산물을 DH5α 형질전환성 세포 (competent cell)에 형질전환하여 PmlI 절단부위가 성공적으로 만들어 진 클론을 확보하였고, 염기서열분석을 통해 최종적으로 확인하고, “hRluc_Neo_PmlI레포터벡터”라고 명명하였다. 변이되기 전 벡터 (pGL4.79[hRluc_Neo])의 염기서열은 서열번호 1로 나타내었으며, 변이된 후의 벡터 hRluc_Neo_PmII레포터벡터의 염기서열은 서열번호 2에 나타내었다.Specifically, primers (SEQ ID NOS: 3 and 4) were prepared in order to add a PmlI cleavage site to the pGL 4.79 [hRluc_Neo] vector. In addition, mutations (mutagenesis) is QuikChange ^® Site-Directed Mutagenesis Kit: was used (Stratagene, Cat Number. 200518) was carried out according to the manual. The conditions were as shown in Table 1. PCR product was transformed into competent cells to obtain a clone in which the PmlI cleavage site was successfully made. The clone was finally confirmed by sequencing and "hRluc_Neo_PmlI Reporter vector ". The nucleotide sequence of the vector before mutation (pGL4.79 [hRluc_Neo]) is shown in SEQ ID NO: 1, and the nucleotide sequence of the vector hRluc_Neo_PmII vector after mutation is shown in SEQ ID NO: 2.

제조된 hRluc_Neo_PmII레포터벡터의 구체적인 개열지도는 도 1과 같다. A specific cleavage map of the produced hRluc_Neo_PmII reporter vector is shown in Fig.

도 1과 같이, pGL4.79[hRluc_Neo]의 ORF에 PmlI 절단부위가 첨가된 새로운 레포터넉인벡터를 제조함으로써, hRluc_Neo를 이용하여 KLF10의 5＇ 플랭킹 부위 (5＇-flanking region)에 대한 활성을 보다 효과적으로 측정할 수 있는 레포터 벡터의 제작이 가능하도록 하였다. 이하 실시예 2를 통해 그 방법을 설명한다.
As shown in Fig. 1, a novel reporter knock-in vector to which a PmlI cleavage site was added to the ORF of pGL 4.79 [hRluc_Neo] was used to prepare a 5'-flanking region of KLF10 using hRluc_Neo It is possible to produce a reporter vector capable of measuring the activity more effectively. Hereinafter, the method will be described with reference to Example 2.

pGL4.79[hRluc_Neo]의 ORF 내에 PmlI 절단부위 첨가Addition of a PmlI cleavage site in the ORF of pGL4.79 [hRluc_Neo] 프라이머primer 서열번호 3(hRluc_Neo_PmlI-5＇):
5＇- CACCATGGCTTCCcacGTGTACGACCCCGAG-3＇SEQ ID NO: 3 (hRluc_Neo_PmlI-5 '):
5'-CACCATGGCTTCC cacGTG TACGACCCCGAG-3 ' 서열번호 4(hRluc_Neo_PmlI-3＇):
5＇- CTCGGGGTCGTAcacGTGGGAAGCCATGGTG-3＇SEQ ID NO: 4 (hRluc_Neo_PmlI-3 '):
5'-CTCGGGGTCGTA cacGTG GGAAGCCATGGTG-3 ' 밑줄 부분이 PmlI 제한효소가 인식하는 염기서열이며 이중 소문자가 변형시킨 염기서열임The underscore is the nucleotide sequence recognized by the PmlI restriction enzyme and the lower case is the modified base sequence PCR 을 위한 돌연변이 가닥 합성 반응액The mutant strand synthesis reaction solution for PCR 5㎕ 10×반응 완충액 5 [mu] l 10x reaction buffer PCR 조건PCR conditions 사이클 1: 95℃, 30초
사이클 16: 95℃, 30초/ 53℃, 1분/ 68℃, 5분
사이클 1: 4℃, 2분
(이때, 1㎕ Dpn I 제한효소를 첨가함)
사이클 1: 37℃, 1시간Cycle 1: 95 ° C, 30 seconds
Cycle 16: 95 ° C, 30 sec / 53 ° C, 1 min / 68 ° C, 5 min
Cycle 1: 4 캜, 2 min
(At this time, 1 [mu] l Dpn I restriction enzyme was added)
Cycle 1: 37 < 0 > C, 1 hour 2㎕ pGL4.79[hRluc_Neo]벡터(50ng) 2 [mu] l pGL4.79 [hRluc_Neo] vector (50 ng) 1㎕ hRluc_Neo_PmlI-5＇프라이머 (10pmole)1 [mu] l hRluc_Neo_PmlI-5 'primer (10 pmole) 1㎕ hRluc_Neo_PmlI-3＇프라이머 (10pmole)1 [mu] l hRluc_Neo_PmlI-3 'primer (10 pmole) 1㎕ dNTP mix1 μl dNTP mix 40㎕ dH₂O40 μl dH ₂ O 1㎕ PfuTurbo DNA 폴리머라아제 (2.5 U/㎕)1 μl PfuTurbo DNA polymerase (2.5 U / μl)

실시예Example 2: 상기 2: 레포터Reporter 벡터를 이용한 Using a vector KLF10KLF10 유전자 5＇ Gene 5 ' 플랭킹Flanking 부위 활성도 측정 Measurement of site activity

마우스의 KLF10 (Kruppel-like factor 10) 유전자의 5＇-flanking region에 대한 활성도를 측정하기 위하여 이에 대한 레포터로 hRluc_Neo 를 활용하는 벡터를 고안하였다. To measure the activity of 5'-flanking region of mouse KLP10 (Kruppel-like factor 10) gene, a vector using hRluc_Neo as a reporter was designed.

구체적으로, KLF10 유전자의 개시코돈으로부터 5＇-업스트림으로 약 1.2kb 되는 부분(1239bp 단편, 서열번호 5)을 상기 고안된 5＇-프라이머(서열번호 6: KLF10/5＇-1: 5＇-CACAAGCTGGCTTCCCTAAG-3＇)와 hRluc_Neo_PmlI 레포터벡터의 5＇말단 쪽의 결실된 폴리뉴클레오티드를 포함하도록 고안된 3＇-프라이머 (서열번호 7: KLF10/3＇-1: 5＇- CTTGGAAGCGATGGTTGGCTGCCTGGCCGCAGGTCGCGAGC-3＇)를 이용하여 주형 2㎕(플라스미드 DNA 10ng), 프라이머 쌍(10pmole) 1㎕/1㎕, Pfu-PreMix 5㎕ 및 dH₂O 41㎕로 총 50㎕의 반응액을 제조하여, 94℃, 5분; 94℃, 60초/72℃, 20초/ 72℃, 30초로 25 사이클; 72℃, 10분의 조건으로 PCR 증폭시켰다.5'-1: 5'-CACAAGCTGGCTTCCCTAAG (SEQ ID NO: 6), which is about 1.2 kb from the initiation codon of the KLF10 gene (about 12 kb fragment) Primer (SEQ ID NO: 7: KLF10 / 3'-1: 5'-3 ') designed to contain the deleted polynucleotide at the 5'end of the hRluc_Neo_PmlI reporter vector A total of 50 μl of the reaction mixture was prepared by using ₂ μl of template (10 ng of plasmid DNA), 1 μl / 1 μl of primer pair (10 pmole), 5 μl of Pfu-PreMix and 41 μl of dH ₂ O using CTTGGAAGCGATGGTTGGCTGCCTGGTGGCTGCCTGGCCGCGGTCGCGAGC-3 ' 94 캜, 5 min; 25 cycles at 94 占 폚, 60 seconds / 72 占 폚, 20 seconds / 72 占 폚, 30 seconds; Followed by PCR amplification at 72 ° C for 10 minutes.

PCR 산물(서열번호 8)을 KpnI 제한효소로 처리 (서열번호 9)하고, 실시예 1에서 제조한 hRluc_Neo_PmlI 레포터벡터를 KpnI/PmlI로 절단 (서열번호 10)한 후 클로닝 하였다. 이렇게 클로닝된 KLF10/5＇아암/ hRluc_Neo_PmlI 벡터의 개열지도는 도 2와 같다. 서열번호 11은 KLF10/5＇아암/ hRluc_Neo_PmlI 벡터의 염기서열이다. 이 벡터를 NIH3T3 세포주에 일시적으로 도입하여 KLF10 유전자의 활성을 확인하였으며, 그 결과를 도 3에 나타내었다. The PCR product (SEQ ID NO: 8) was treated with KpnI restriction enzyme (SEQ ID NO: 9), and the hRluc_Neo_PmlI reporter vector prepared in Example 1 was digested with KpnI / PmlI (SEQ ID NO: 10) and then cloned. The cleavage map of the cloned KLF10 / 5 'arm / hRluc_Neo_PmlI vector is shown in FIG. SEQ ID NO: 11 is the nucleotide sequence of the KLF10 / 5 'arm / hRluc_Neo_PmlI vector. This vector was transiently introduced into the NIH3T3 cell line to confirm the activity of the KLF10 gene. The results are shown in FIG.

도 3에 나타난 바와 같이, 본 발명의 벡터가 도입된 숙주세포에서는 KLF10 (Kruppel-like factor 10) 유전자의 5＇ 플랭킹 부위의 활성을 확인할 수 있었다.
As shown in FIG. 3, in the host cell into which the vector of the present invention was introduced, the activity of the 5 'flanking region of KLP10 (Kruppel-like factor 10) gene was confirmed.

<110> KOREA FOOD & DRUG ADMINISTRATION <120> HRluc reporter vector inserted restriction site into open reading frame of HRluc <130> 1-6 <160> 11 <170> KopatentIn 2.0 <210> 1 <211> 4879 <212> DNA <213> Artificial Sequence <220> <223> HRluc vector <400> 1 ggcctaactg gccggtacct gagctcgcta gcctcgagga tatcaagatc tggcctcggc 60 ggccaagctt ggcaatccgg tactgttggt aaagccacca tggcttccaa ggtgtacgac 120 cccgagcaac gcaaacgcat gatcactggg cctcagtggt gggctcgctg caagcaaatg 180 aacgtgctgg actccttcat caactactat gattccgaga agcacgccga gaacgccgtg 240 atttttctgc atggtaacgc tgcctccagc tacctgtgga ggcacgtcgt gcctcacatc 300 gagcccgtgg ctagatgcat catccctgat ctgatcggaa tgggtaagtc cggcaagagc 360 gggaatggct catatcgcct cctggatcac tacaagtacc tcaccgcttg gttcgagctg 420 ctgaaccttc caaagaaaat catctttgtg ggccacgact ggggggcttg tctggccttt 480 cactactcct acgagcacca agacaagatc aaggccatcg tccatgctga gagtgtcgtg 540 gacgtgatcg agtcctggga cgagtggcct gacatcgagg aggatatcgc cctgatcaag 600 agcgaagagg gcgagaaaat ggtgcttgag aataacttct tcgtcgagac catgctccca 660 agcaagatca tgcggaaact ggagcctgag gagttcgctg cctacctgga gccattcaag 720 gagaagggcg aggttagacg gcctaccctc tcctggcctc gcgagatccc tctcgttaag 780 ggaggcaagc ccgacgtcgt ccagattgtc cgcaactaca acgcctacct tcgggccagc 840 gacgatctgc ctaagatgtt catcgagtcc gaccctgggt tcttttccaa cgctattgtc 900 gagggagcta agaagttccc taacaccgag ttcgtgaagg tgaagggcct ccacttcagc 960 caggaggacg ctccagatga aatgggtaag tacatcaaga gcttcgtgga gcgcgtgctg 1020 aagaacgagc agtaattcta gagtcggggc ggccggccgc ttcgagcaga catgataaga 1080 tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt 1140 gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa acaagttaac 1200 aacaacaatt gcattcattt tatgtttcag gttcaggggg aggtgtggga ggttttttaa 1260 agcaagtaaa acctctacaa atgtggtaaa atcgataagg atccgtttgc gtattgggcg 1320 ctcttccgct gatctgcgca gcaccatggc ctgaaataac ctctgaaaga ggaacttggt 1380 tagctacctt ctgaggcgga aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa 1440 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa 1500 ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca 1560 attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca 1620 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg 1680 ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct 1740 tttgcaaaaa gctcgattct tctgacacta gcgccaccat gatcgaacaa gacggcctcc 1800 atgctggcag tcccgcagct tgggtcgaac gcttgttcgg gtacgactgg gcccagcaga 1860 ccatcggatg tagcgatgcg gccgtgttcc gtctaagcgc tcaaggccgg cccgtgctgt 1920 tcgtgaagac cgacctgagc ggcgccctga acgagcttca agacgaggct gcccgcctga 1980 gctggctggc caccaccggc gtaccctgcg ccgctgtgtt ggatgttgtg accgaagccg 2040 gccgggactg gctgctgctg ggcgaggtcc ctggccagga tctgctgagc agccaccttg 2100 cccccgctga gaaggtttct atcatggccg atgcaatgcg gcgcctgcac accctggacc 2160 ccgctacctg ccccttcgac caccaggcta agcatcggat cgagcgtgct cggacccgca 2220 tggaggccgg cctggtggac caggacgacc tggacgagga gcatcagggc ctggcccccg 2280 ctgaactgtt cgcccgactg aaagcccgca tgccggacgg tgaggacctg gttgtcacac 2340 acggagatgc ctgcctccct aacatcatgg tcgagaatgg ccgcttctcc ggcttcatcg 2400 actgcggtcg cctaggagtt gccgaccgct accaggacat cgccctggcc acccgcgaca 2460 tcgctgagga gcttggcggc gagtgggccg accgcttctt agtcttgtac ggcatcgcag 2520 ctcccgacag ccagcgcatc gccttctacc gcttgctcga cgagttcttt taatgatcta 2580 gaaccggtca tggccgcaat aaaatatctt tattttcatt acatctgtgt gttggttttt 2640 tgtgtgttcg aactagatgc tgtcgaccga tgcccttgag agccttcaac ccagtcagct 2700 ccttccggtg ggcgcggggc atgactatcg tcgccgcact tatgactgtc ttctttatca 2760 tgcaactcgt aggacaggtg ccggcagcgc tcttccgctt cctcgctcac tgactcgctg 2820 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta 2880 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc 2940 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag 3000 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac 3060 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc 3120 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt 3180 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc 3240 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga 3300 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta 3360 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta 3420 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga 3480 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg 3540 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag 3600 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc 3660 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact 3720 tggtctgaca gcggccgcaa atgctaaacc actgcagtgg ttaccagtgc ttgatcagtg 3780 aggcaccgat ctcagcgatc tgcctatttc gttcgtccat agtggcctga ctccccgtcg 3840 tgtagatcac tacgattcgt gagggcttac catcaggccc cagcgcagca atgatgccgc 3900 gagagccgcg ttcaccggcc cccgatttgt cagcaatgaa ccagccagca gggagggccg 3960 agcgaagaag tggtcctgct actttgtccg cctccatcca gtctatgagc tgctgtcgtg 4020 atgctagagt aagaagttcg ccagtgagta gtttccgaag agttgtggcc attgctactg 4080 gcatcgtggt atcacgctcg tcgttcggta tggcttcgtt caactctggt tcccagcggt 4140 caagccgggt cacatgatca cccatattat gaagaaatgc agtcagctcc ttagggcctc 4200 cgatcgttgt cagaagtaag ttggccgcgg tgttgtcgct catggtaatg gcagcactac 4260 acaattctct taccgtcatg ccatccgtaa gatgcttttc cgtgaccggc gagtactcaa 4320 ccaagtcgtt ttgtgagtag tgtatacggc gaccaagctg ctcttgcccg gcgtctatac 4380 gggacaacac cgcgccacat agcagtactt tgaaagtgct catcatcggg aatcgttctt 4440 cggggcggaa agactcaagg atcttgccgc tattgagatc cagttcgata tagcccactc 4500 ttgcacccag ttgatcttca gcatctttta ctttcaccag cgtttcgggg tgtgcaaaaa 4560 caggcaagca aaatgccgca aagaagggaa tgagtgcgac acgaaaatgt tggatgctca 4620 tactcgtcct ttttcaatat tattgaagca tttatcaggg ttactagtac gtctctcaag 4680 gataagtaag taatattaag gtacgggagg tattggacag gccgcaataa aatatcttta 4740 ttttcattac atctgtgtgt tggttttttg tgtgaatcga tagtactaac atacgctctc 4800 catcaaaaca aaacgaaaca aaacaaacta gcaaaatagg ctgtccccag tgcaagtgca 4860 ggtgccagaa catttctct 4879 <210> 2 <211> 4879 <212> DNA <213> Artificial Sequence <220> <223> HRluc report vector <400> 2 ggcctaactg gccggtacct gagctcgcta gcctcgagga tatcaagatc tggcctcggc 60 ggccaagctt ggcaatccgg tactgttggt aaagccacca tggcttccca cgtgtacgac 120 cccgagcaac gcaaacgcat gatcactggg cctcagtggt gggctcgctg caagcaaatg 180 aacgtgctgg actccttcat caactactat gattccgaga agcacgccga gaacgccgtg 240 atttttctgc atggtaacgc tgcctccagc tacctgtgga ggcacgtcgt gcctcacatc 300 gagcccgtgg ctagatgcat catccctgat ctgatcggaa tgggtaagtc cggcaagagc 360 gggaatggct catatcgcct cctggatcac tacaagtacc tcaccgcttg gttcgagctg 420 ctgaaccttc caaagaaaat catctttgtg ggccacgact ggggggcttg tctggccttt 480 cactactcct acgagcacca agacaagatc aaggccatcg tccatgctga gagtgtcgtg 540 gacgtgatcg agtcctggga cgagtggcct gacatcgagg aggatatcgc cctgatcaag 600 agcgaagagg gcgagaaaat ggtgcttgag aataacttct tcgtcgagac catgctccca 660 agcaagatca tgcggaaact ggagcctgag gagttcgctg cctacctgga gccattcaag 720 gagaagggcg aggttagacg gcctaccctc tcctggcctc gcgagatccc tctcgttaag 780 ggaggcaagc ccgacgtcgt ccagattgtc cgcaactaca acgcctacct tcgggccagc 840 gacgatctgc ctaagatgtt catcgagtcc gaccctgggt tcttttccaa cgctattgtc 900 gagggagcta agaagttccc taacaccgag ttcgtgaagg tgaagggcct ccacttcagc 960 caggaggacg ctccagatga aatgggtaag tacatcaaga gcttcgtgga gcgcgtgctg 1020 aagaacgagc agtaattcta gagtcggggc ggccggccgc ttcgagcaga catgataaga 1080 tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt 1140 gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa acaagttaac 1200 aacaacaatt gcattcattt tatgtttcag gttcaggggg aggtgtggga ggttttttaa 1260 agcaagtaaa acctctacaa atgtggtaaa atcgataagg atccgtttgc gtattgggcg 1320 ctcttccgct gatctgcgca gcaccatggc ctgaaataac ctctgaaaga ggaacttggt 1380 tagctacctt ctgaggcgga aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa 1440 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa 1500 ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca 1560 attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca 1620 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg 1680 ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct 1740 tttgcaaaaa gctcgattct tctgacacta gcgccaccat gatcgaacaa gacggcctcc 1800 atgctggcag tcccgcagct tgggtcgaac gcttgttcgg gtacgactgg gcccagcaga 1860 ccatcggatg tagcgatgcg gccgtgttcc gtctaagcgc tcaaggccgg cccgtgctgt 1920 tcgtgaagac cgacctgagc ggcgccctga acgagcttca agacgaggct gcccgcctga 1980 gctggctggc caccaccggc gtaccctgcg ccgctgtgtt ggatgttgtg accgaagccg 2040 gccgggactg gctgctgctg ggcgaggtcc ctggccagga tctgctgagc agccaccttg 2100 cccccgctga gaaggtttct atcatggccg atgcaatgcg gcgcctgcac accctggacc 2160 ccgctacctg ccccttcgac caccaggcta agcatcggat cgagcgtgct cggacccgca 2220 tggaggccgg cctggtggac caggacgacc tggacgagga gcatcagggc ctggcccccg 2280 ctgaactgtt cgcccgactg aaagcccgca tgccggacgg tgaggacctg gttgtcacac 2340 acggagatgc ctgcctccct aacatcatgg tcgagaatgg ccgcttctcc ggcttcatcg 2400 actgcggtcg cctaggagtt gccgaccgct accaggacat cgccctggcc acccgcgaca 2460 tcgctgagga gcttggcggc gagtgggccg accgcttctt agtcttgtac ggcatcgcag 2520 ctcccgacag ccagcgcatc gccttctacc gcttgctcga cgagttcttt taatgatcta 2580 gaaccggtca tggccgcaat aaaatatctt tattttcatt acatctgtgt gttggttttt 2640 tgtgtgttcg aactagatgc tgtcgaccga tgcccttgag agccttcaac ccagtcagct 2700 ccttccggtg ggcgcggggc atgactatcg tcgccgcact tatgactgtc ttctttatca 2760 tgcaactcgt aggacaggtg ccggcagcgc tcttccgctt cctcgctcac tgactcgctg 2820 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta 2880 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc 2940 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag 3000 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac 3060 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc 3120 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt 3180 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc 3240 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga 3300 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta 3360 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta 3420 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga 3480 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg 3540 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag 3600 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc 3660 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact 3720 tggtctgaca gcggccgcaa atgctaaacc actgcagtgg ttaccagtgc ttgatcagtg 3780 aggcaccgat ctcagcgatc tgcctatttc gttcgtccat agtggcctga ctccccgtcg 3840 tgtagatcac tacgattcgt gagggcttac catcaggccc cagcgcagca atgatgccgc 3900 gagagccgcg ttcaccggcc cccgatttgt cagcaatgaa ccagccagca gggagggccg 3960 agcgaagaag tggtcctgct actttgtccg cctccatcca gtctatgagc tgctgtcgtg 4020 atgctagagt aagaagttcg ccagtgagta gtttccgaag agttgtggcc attgctactg 4080 gcatcgtggt atcacgctcg tcgttcggta tggcttcgtt caactctggt tcccagcggt 4140 caagccgggt cacatgatca cccatattat gaagaaatgc agtcagctcc ttagggcctc 4200 cgatcgttgt cagaagtaag ttggccgcgg tgttgtcgct catggtaatg gcagcactac 4260 acaattctct taccgtcatg ccatccgtaa gatgcttttc cgtgaccggc gagtactcaa 4320 ccaagtcgtt ttgtgagtag tgtatacggc gaccaagctg ctcttgcccg gcgtctatac 4380 gggacaacac cgcgccacat agcagtactt tgaaagtgct catcatcggg aatcgttctt 4440 cggggcggaa agactcaagg atcttgccgc tattgagatc cagttcgata tagcccactc 4500 ttgcacccag ttgatcttca gcatctttta ctttcaccag cgtttcgggg tgtgcaaaaa 4560 caggcaagca aaatgccgca aagaagggaa tgagtgcgac acgaaaatgt tggatgctca 4620 tactcgtcct ttttcaatat tattgaagca tttatcaggg ttactagtac gtctctcaag 4680 gataagtaag taatattaag gtacgggagg tattggacag gccgcaataa aatatcttta 4740 ttttcattac atctgtgtgt tggttttttg tgtgaatcga tagtactaac atacgctctc 4800 catcaaaaca aaacgaaaca aaacaaacta gcaaaatagg ctgtccccag tgcaagtgca 4860 ggtgccagaa catttctct 4879 <210> 3 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> hRluc_Neo_PmlI-5' <400> 3 caccatggct tcccacgtgt acgaccccga g 31 <210> 4 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> hRluc_Neo_PmlI-3' <400> 4 ctcggggtcg tacacgtggg aagccatggt g 31 <210> 5 <211> 1239 <212> DNA <213> Artificial Sequence <220> <223> KLF10 genome fragment <400> 5 cacaagctgg cttccctaag aagctcagct tgccctcaat ccctggagcg gtggaggggg 60 cagggacagg aactcgatgt ttgaccgggt caggaatccc acctgcaagc atgctttcct 120 caccccatcc cctcaagcgg tttcttggta gggcgtcggc gaagactgtg gtcccgcggg 180 tgggggaagt ggagtccacc ttggaaatag ctcgaacatt gtattttaaa cttcaactac 240 atcaaactca aactcgattc tgccaccgag cgtcgggtct ggggctccct gccacgccac 300 tagctctttg gtaccggctt cccttagtcc ccactccaac cgcccagaag gacgggaggg 360 acgcgcagag gcgggccctg ctgctagtgc ctgtgctgca gatgggagga cgagccagcg 420 cctgccgcct tgggcgtggg cggtgagtgc gagcaggtgt ctgttccgtg acagggctcc 480 cgccgccacc atctcgccct ggttctcccc agtcctccgg cttagagtga caaaacccgg 540 caaatgctca gaaatgccgc tcagtggttc atccatccct tgcttccacg caagacccac 600 gagaaccacg ctgccaaaga acgtctggtg gctcggagcc gcgctcgcag cctccgcctc 660 cgcctcctgc tcggggtccc gcccactccc tggcgggccc agacgctgtg ggcgcgaagg 720 ggactgggcg gagcgggctt cccttcccac cccccacgtg gggccggctc tccgcaaagc 780 ctagtcgccg ccaccgcgcc tcccagctat gccccgccgc cggcgccgcc cccgggcatg 840 ggcggaggac tgaaggctag ggttgggcga ggggcgtcac ggagtcaggt ggtccccgga 900 gttccccggg gctggaccga gggaacacgg tgcctcgggt tgtgtacacg ctccactgac 960 agagcctctt gcagccgggc agccgctgat cacgcgtggc tctgccagct cattggctga 1020 ggcctcacac acctttgccg ctgattggtc gacactaggc cccgccctct accccgcccg 1080 cggcgcgggg caggcagagc ggctacctgc acggggaggg gggcagacag cgcggagcgc 1140 ggtggcgtcg acgtctagtg tctcagtgct cccgtctgtg gctaactaag cagccagcag 1200 ccaggcagct cgcgacctgc ggccaggcag ccaaccatg 1239 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> KLF10/5'-1 primer <400> 6 cacaagctgg cttccctaag 20 <210> 7 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> KLF10/3'-1 primer <400> 7 cttggaagcg atggttggct gcctggccgc aggtcgcgag c 41 <210> 8 <211> 1248 <212> DNA <213> Artificial Sequence <220> <223> PCR product <400> 8 cacaagctgg cttccctaag aagctcagct tgccctcaat ccctggagcg gtggaggggg 60 cagggacagg aactcgatgt ttgaccgggt caggaatccc acctgcaagc atgctttcct 120 caccccatcc cctcaagcgg tttcttggta gggcgtcggc gaagactgtg gtcccgcggg 180 tgggggaagt ggagtccacc ttggaaatag ctcgaacatt gtattttaaa cttcaactac 240 atcaaactca aactcgattc tgccaccgag cgtcgggtct ggggctccct gccacgccac 300 tagctctttg gtaccggctt cccttagtcc ccactccaac cgcccagaag gacgggaggg 360 acgcgcagag gcgggccctg ctgctagtgc ctgtgctgca gatgggagga cgagccagcg 420 cctgccgcct tgggcgtggg cggtgagtgc gagcaggtgt ctgttccgtg acagggctcc 480 cgccgccacc atctcgccct ggttctcccc agtcctccgg cttagagtga caaaacccgg 540 caaatgctca gaaatgccgc tcagtggttc atccatccct tgcttccacg caagacccac 600 gagaaccacg ctgccaaaga acgtctggtg gctcggagcc gcgctcgcag cctccgcctc 660 cgcctcctgc tcggggtccc gcccactccc tggcgggccc agacgctgtg ggcgcgaagg 720 ggactgggcg gagcgggctt cccttcccac cccccacgtg gggccggctc tccgcaaagc 780 ctagtcgccg ccaccgcgcc tcccagctat gccccgccgc cggcgccgcc cccgggcatg 840 ggcggaggac tgaaggctag ggttgggcga ggggcgtcac ggagtcaggt ggtccccgga 900 gttccccggg gctggaccga gggaacacgg tgcctcgggt tgtgtacacg ctccactgac 960 agagcctctt gcagccgggc agccgctgat cacgcgtggc tctgccagct cattggctga 1020 ggcctcacac acctttgccg ctgattggtc gacactaggc cccgccctct accccgcccg 1080 cggcgcgggg caggcagagc ggctacctgc acggggaggg gggcagacag cgcggagcgc 1140 ggtggcgtcg acgtctagtg tctcagtgct cccgtctgtg gctaactaag cagccagcag 1200 ccaggcagct cgcgacctgc ggccaggcag ccaaccatgg cttccaag 1248 <210> 9 <211> 934 <212> DNA <213> Artificial Sequence <220> <223> PCR product treated with KpnI <400> 9 cggcttccct tagtccccac tccaaccgcc cagaaggacg ggagggacgc gcagaggcgg 60 gccctgctgc tagtgcctgt gctgcagatg ggaggacgag ccagcgcctg ccgccttggg 120 cgtgggcggt gagtgcgagc aggtgtctgt tccgtgacag ggctcccgcc gccaccatct 180 cgccctggtt ctccccagtc ctccggctta gagtgacaaa acccggcaaa tgctcagaaa 240 tgccgctcag tggttcatcc atcccttgct tccacgcaag acccacgaga accacgctgc 300 caaagaacgt ctggtggctc ggagccgcgc tcgcagcctc cgcctccgcc tcctgctcgg 360 ggtcccgccc actccctggc gggcccagac gctgtgggcg cgaaggggac tgggcggagc 420 gggcttccct tcccaccccc cacgtggggc cggctctccg caaagcctag tcgccgccac 480 cgcgcctccc agctatgccc cgccgccggc gccgcccccg ggcatgggcg gaggactgaa 540 ggctagggtt gggcgagggg cgtcacggag tcaggtggtc cccggagttc cccggggctg 600 gaccgaggga acacggtgcc tcgggttgtg tacacgctcc actgacagag cctcttgcag 660 ccgggcagcc gctgatcacg cgtggctctg ccagctcatt ggctgaggcc tcacacacct 720 ttgccgctga ttggtcgaca ctaggccccg ccctctaccc cgcccgcggc gcggggcagg 780 cagagcggct acctgcacgg ggaggggggc agacagcgcg gagcgcggtg gcgtcgacgt 840 ctagtgtctc agtgctcccg tctgtggcta actaagcagc cagcagccag gcagctcgcg 900 acctgcggcc aggcagccaa ccatggcttc caag 934 <210> 10 <211> 4786 <212> DNA <213> Artificial Sequence <220> <223> hRluc_Neo_PmlI report vector treated with KpnI/PmlI <400> 10 ggcctaactg gccggtacgt gtacgacccc gagcaacgca aacgcatgat cactgggcct 60 cagtggtggg ctcgctgcaa gcaaatgaac gtgctggact ccttcatcaa ctactatgat 120 tccgagaagc acgccgagaa cgccgtgatt tttctgcatg gtaacgctgc ctccagctac 180 ctgtggaggc acgtcgtgcc tcacatcgag cccgtggcta gatgcatcat ccctgatctg 240 atcggaatgg gtaagtccgg caagagcggg aatggctcat atcgcctcct ggatcactac 300 aagtacctca ccgcttggtt cgagctgctg aaccttccaa agaaaatcat ctttgtgggc 360 cacgactggg gggcttgtct ggcctttcac tactcctacg agcaccaaga caagatcaag 420 gccatcgtcc atgctgagag tgtcgtggac gtgatcgagt cctgggacga gtggcctgac 480 atcgaggagg atatcgccct gatcaagagc gaagagggcg agaaaatggt gcttgagaat 540 aacttcttcg tcgagaccat gctcccaagc aagatcatgc ggaaactgga gcctgaggag 600 ttcgctgcct acctggagcc attcaaggag aagggcgagg ttagacggcc taccctctcc 660 tggcctcgcg agatccctct cgttaaggga ggcaagcccg acgtcgtcca gattgtccgc 720 aactacaacg cctaccttcg ggccagcgac gatctgccta agatgttcat cgagtccgac 780 cctgggttct tttccaacgc tattgtcgag ggagctaaga agttccctaa caccgagttc 840 gtgaaggtga agggcctcca cttcagccag gaggacgctc cagatgaaat gggtaagtac 900 atcaagagct tcgtggagcg cgtgctgaag aacgagcagt aattctagag tcggggcggc 960 cggccgcttc gagcagacat gataagatac attgatgagt ttggacaaac cacaactaga 1020 atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc 1080 attataagct gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt 1140 cagggggagg tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtaaaatc 1200 gataaggatc cgtttgcgta ttgggcgctc ttccgctgat ctgcgcagca ccatggcctg 1260 aaataacctc tgaaagagga acttggttag ctaccttctg aggcggaaag aaccagctgt 1320 ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc 1380 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag 1440 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc 1500 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa 1560 ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt 1620 gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cgattcttct gacactagcg 1680 ccaccatgat cgaacaagac ggcctccatg ctggcagtcc cgcagcttgg gtcgaacgct 1740 tgttcgggta cgactgggcc cagcagacca tcggatgtag cgatgcggcc gtgttccgtc 1800 taagcgctca aggccggccc gtgctgttcg tgaagaccga cctgagcggc gccctgaacg 1860 agcttcaaga cgaggctgcc cgcctgagct ggctggccac caccggcgta ccctgcgccg 1920 ctgtgttgga tgttgtgacc gaagccggcc gggactggct gctgctgggc gaggtccctg 1980 gccaggatct gctgagcagc caccttgccc ccgctgagaa ggtttctatc atggccgatg 2040 caatgcggcg cctgcacacc ctggaccccg ctacctgccc cttcgaccac caggctaagc 2100 atcggatcga gcgtgctcgg acccgcatgg aggccggcct ggtggaccag gacgacctgg 2160 acgaggagca tcagggcctg gcccccgctg aactgttcgc ccgactgaaa gcccgcatgc 2220 cggacggtga ggacctggtt gtcacacacg gagatgcctg cctccctaac atcatggtcg 2280 agaatggccg cttctccggc ttcatcgact gcggtcgcct aggagttgcc gaccgctacc 2340 aggacatcgc cctggccacc cgcgacatcg ctgaggagct tggcggcgag tgggccgacc 2400 gcttcttagt cttgtacggc atcgcagctc ccgacagcca gcgcatcgcc ttctaccgct 2460 tgctcgacga gttcttttaa tgatctagaa ccggtcatgg ccgcaataaa atatctttat 2520 tttcattaca tctgtgtgtt ggttttttgt gtgttcgaac tagatgctgt cgaccgatgc 2580 ccttgagagc cttcaaccca gtcagctcct tccggtgggc gcggggcatg actatcgtcg 2640 ccgcacttat gactgtcttc tttatcatgc aactcgtagg acaggtgccg gcagcgctct 2700 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca 2760 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac 2820 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt 2880 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg 2940 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc 3000 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc 3060 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc 3120 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac 3180 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt 3240 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct 3300 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc 3360 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt 3420 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg 3480 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc 3540 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa 3600 tcaatctaaa gtatatatga gtaaacttgg tctgacagcg gccgcaaatg ctaaaccact 3660 gcagtggtta ccagtgcttg atcagtgagg caccgatctc agcgatctgc ctatttcgtt 3720 cgtccatagt ggcctgactc cccgtcgtgt agatcactac gattcgtgag ggcttaccat 3780 caggccccag cgcagcaatg atgccgcgag agccgcgttc accggccccc gatttgtcag 3840 caatgaacca gccagcaggg agggccgagc gaagaagtgg tcctgctact ttgtccgcct 3900 ccatccagtc tatgagctgc tgtcgtgatg ctagagtaag aagttcgcca gtgagtagtt 3960 tccgaagagt tgtggccatt gctactggca tcgtggtatc acgctcgtcg ttcggtatgg 4020 cttcgttcaa ctctggttcc cagcggtcaa gccgggtcac atgatcaccc atattatgaa 4080 gaaatgcagt cagctcctta gggcctccga tcgttgtcag aagtaagttg gccgcggtgt 4140 tgtcgctcat ggtaatggca gcactacaca attctcttac cgtcatgcca tccgtaagat 4200 gcttttccgt gaccggcgag tactcaacca agtcgttttg tgagtagtgt atacggcgac 4260 caagctgctc ttgcccggcg tctatacggg acaacaccgc gccacatagc agtactttga 4320 aagtgctcat catcgggaat cgttcttcgg ggcggaaaga ctcaaggatc ttgccgctat 4380 tgagatccag ttcgatatag cccactcttg cacccagttg atcttcagca tcttttactt 4440 tcaccagcgt ttcggggtgt gcaaaaacag gcaagcaaaa tgccgcaaag aagggaatga 4500 gtgcgacacg aaaatgttgg atgctcatac tcgtcctttt tcaatattat tgaagcattt 4560 atcagggtta ctagtacgtc tctcaaggat aagtaagtaa tattaaggta cgggaggtat 4620 tggacaggcc gcaataaaat atctttattt tcattacatc tgtgtgttgg ttttttgtgt 4680 gaatcgatag tactaacata cgctctccat caaaacaaaa cgaaacaaaa caaactagca 4740 aaataggctg tccccagtgc aagtgcaggt gccagaacat ttctct 4786 <210> 11 <211> 5720 <212> DNA <213> Artificial Sequence <220> <223> KLF10/5'arm/hRluc_Neo_PmlI vector <400> 11 ggcctaactg gccggtaccg gcttccctta gtccccactc caaccgccca gaaggacggg 60 agggacgcgc agaggcgggc cctgctgcta gtgcctgtgc tgcagatggg aggacgagcc 120 agcgcctgcc gccttgggcg tgggcggtga gtgcgagcag gtgtctgttc cgtgacaggg 180 ctcccgccgc caccatctcg ccctggttct ccccagtcct ccggcttaga gtgacaaaac 240 ccggcaaatg ctcagaaatg ccgctcagtg gttcatccat cccttgcttc cacgcaagac 300 ccacgagaac cacgctgcca aagaacgtct ggtggctcgg agccgcgctc gcagcctccg 360 cctccgcctc ctgctcgggg tcccgcccac tccctggcgg gcccagacgc tgtgggcgcg 420 aaggggactg ggcggagcgg gcttcccttc ccacccccca cgtggggccg gctctccgca 480 aagcctagtc gccgccaccg cgcctcccag ctatgccccg ccgccggcgc cgcccccggg 540 catgggcgga ggactgaagg ctagggttgg gcgaggggcg tcacggagtc aggtggtccc 600 cggagttccc cggggctgga ccgagggaac acggtgcctc gggttgtgta cacgctccac 660 tgacagagcc tcttgcagcc gggcagccgc tgatcacgcg tggctctgcc agctcattgg 720 ctgaggcctc acacaccttt gccgctgatt ggtcgacact aggccccgcc ctctaccccg 780 cccgcggcgc ggggcaggca gagcggctac ctgcacgggg aggggggcag acagcgcgga 840 gcgcggtggc gtcgacgtct agtgtctcag tgctcccgtc tgtggctaac taagcagcca 900 gcagccaggc agctcgcgac ctgcggccag gcagccaacc atggcttcca aggtgtacga 960 ccccgagcaa cgcaaacgca tgatcactgg gcctcagtgg tgggctcgct gcaagcaaat 1020 gaacgtgctg gactccttca tcaactacta tgattccgag aagcacgccg agaacgccgt 1080 gatttttctg catggtaacg ctgcctccag ctacctgtgg aggcacgtcg tgcctcacat 1140 cgagcccgtg gctagatgca tcatccctga tctgatcgga atgggtaagt ccggcaagag 1200 cgggaatggc tcatatcgcc tcctggatca ctacaagtac ctcaccgctt ggttcgagct 1260 gctgaacctt ccaaagaaaa tcatctttgt gggccacgac tggggggctt gtctggcctt 1320 tcactactcc tacgagcacc aagacaagat caaggccatc gtccatgctg agagtgtcgt 1380 ggacgtgatc gagtcctggg acgagtggcc tgacatcgag gaggatatcg ccctgatcaa 1440 gagcgaagag ggcgagaaaa tggtgcttga gaataacttc ttcgtcgaga ccatgctccc 1500 aagcaagatc atgcggaaac tggagcctga ggagttcgct gcctacctgg agccattcaa 1560 ggagaagggc gaggttagac ggcctaccct ctcctggcct cgcgagatcc ctctcgttaa 1620 gggaggcaag cccgacgtcg tccagattgt ccgcaactac aacgcctacc ttcgggccag 1680 cgacgatctg cctaagatgt tcatcgagtc cgaccctggg ttcttttcca acgctattgt 1740 cgagggagct aagaagttcc ctaacaccga gttcgtgaag gtgaagggcc tccacttcag 1800 ccaggaggac gctccagatg aaatgggtaa gtacatcaag agcttcgtgg agcgcgtgct 1860 gaagaacgag cagtaattct agagtcgggg cggccggccg cttcgagcag acatgataag 1920 atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg 1980 tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa 2040 caacaacaat tgcattcatt ttatgtttca ggttcagggg gaggtgtggg aggtttttta 2100 aagcaagtaa aacctctaca aatgtggtaa aatcgataag gatccgtttg cgtattgggc 2160 gctcttccgc tgatctgcgc agcaccatgg cctgaaataa cctctgaaag aggaacttgg 2220 ttagctacct tctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga 2280 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca 2340 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc 2400 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc 2460 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag 2520 gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc 2580 ttttgcaaaa agctcgattc ttctgacact agcgccacca tgatcgaaca agacggcctc 2640 catgctggca gtcccgcagc ttgggtcgaa cgcttgttcg ggtacgactg ggcccagcag 2700 accatcggat gtagcgatgc ggccgtgttc cgtctaagcg ctcaaggccg gcccgtgctg 2760 ttcgtgaaga ccgacctgag cggcgccctg aacgagcttc aagacgaggc tgcccgcctg 2820 agctggctgg ccaccaccgg cgtaccctgc gccgctgtgt tggatgttgt gaccgaagcc 2880 ggccgggact ggctgctgct gggcgaggtc cctggccagg atctgctgag cagccacctt 2940 gcccccgctg agaaggtttc tatcatggcc gatgcaatgc ggcgcctgca caccctggac 3000 cccgctacct gccccttcga ccaccaggct aagcatcgga tcgagcgtgc tcggacccgc 3060 atggaggccg gcctggtgga ccaggacgac ctggacgagg agcatcaggg cctggccccc 3120 gctgaactgt tcgcccgact gaaagcccgc atgccggacg gtgaggacct ggttgtcaca 3180 cacggagatg cctgcctccc taacatcatg gtcgagaatg gccgcttctc cggcttcatc 3240 gactgcggtc gcctaggagt tgccgaccgc taccaggaca tcgccctggc cacccgcgac 3300 atcgctgagg agcttggcgg cgagtgggcc gaccgcttct tagtcttgta cggcatcgca 3360 gctcccgaca gccagcgcat cgccttctac cgcttgctcg acgagttctt ttaatgatct 3420 agaaccggtc atggccgcaa taaaatatct ttattttcat tacatctgtg tgttggtttt 3480 ttgtgtgttc gaactagatg ctgtcgaccg atgcccttga gagccttcaa cccagtcagc 3540 tccttccggt gggcgcgggg catgactatc gtcgccgcac ttatgactgt cttctttatc 3600 atgcaactcg taggacaggt gccggcagcg ctcttccgct tcctcgctca ctgactcgct 3660 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt 3720 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc 3780 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga 3840 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata 3900 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac 3960 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg 4020 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc 4080 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag 4140 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt 4200 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt 4260 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg 4320 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac 4380 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca 4440 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac 4500 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac 4560 ttggtctgac agcggccgca aatgctaaac cactgcagtg gttaccagtg cttgatcagt 4620 gaggcaccga tctcagcgat ctgcctattt cgttcgtcca tagtggcctg actccccgtc 4680 gtgtagatca ctacgattcg tgagggctta ccatcaggcc ccagcgcagc aatgatgccg 4740 cgagagccgc gttcaccggc ccccgatttg tcagcaatga accagccagc agggagggcc 4800 gagcgaagaa gtggtcctgc tactttgtcc gcctccatcc agtctatgag ctgctgtcgt 4860 gatgctagag taagaagttc gccagtgagt agtttccgaa gagttgtggc cattgctact 4920 ggcatcgtgg tatcacgctc gtcgttcggt atggcttcgt tcaactctgg ttcccagcgg 4980 tcaagccggg tcacatgatc acccatatta tgaagaaatg cagtcagctc cttagggcct 5040 ccgatcgttg tcagaagtaa gttggccgcg gtgttgtcgc tcatggtaat ggcagcacta 5100 cacaattctc ttaccgtcat gccatccgta agatgctttt ccgtgaccgg cgagtactca 5160 accaagtcgt tttgtgagta gtgtatacgg cgaccaagct gctcttgccc ggcgtctata 5220 cgggacaaca ccgcgccaca tagcagtact ttgaaagtgc tcatcatcgg gaatcgttct 5280 tcggggcgga aagactcaag gatcttgccg ctattgagat ccagttcgat atagcccact 5340 cttgcaccca gttgatcttc agcatctttt actttcacca gcgtttcggg gtgtgcaaaa 5400 acaggcaagc aaaatgccgc aaagaaggga atgagtgcga cacgaaaatg ttggatgctc 5460 atactcgtcc tttttcaata ttattgaagc atttatcagg gttactagta cgtctctcaa 5520 ggataagtaa gtaatattaa ggtacgggag gtattggaca ggccgcaata aaatatcttt 5580 attttcatta catctgtgtg ttggtttttt gtgtgaatcg atagtactaa catacgctct 5640 ccatcaaaac aaaacgaaac aaaacaaact agcaaaatag gctgtcccca gtgcaagtgc 5700 aggtgccaga acatttctct 5720 <110> KOREA FOOD & DRUG ADMINISTRATION <120> HRluc reporter vector inserted restriction site into open reading frame of HRluc <130> 1-6 <160> 11 <170> Kopatentin 2.0 <210> 1 <211> 4879 <212> DNA <213> Artificial Sequence <220> <223> HRluc vector <400> 1 ggcctaactg gccggtacct gagctcgcta gcctcgagga tatcaagatc tggcctcggc 60 ggccaagctt ggcaatccgg tactgttggt aaagccacca tggcttccaa ggtgtacgac 120 cccgagcaac gcaaacgcat gatcactggg cctcagtggt gggctcgctg caagcaaatg 180 aacgtgctgg actccttcat caactactat gattccgaga agcacgccga gaacgccgtg 240 atttttctgc atggtaacgc tgcctccagc tacctgtgga ggcacgtcgt gcctcacatc 300 gagcccgtgg ctagatgcat catccctgat ctgatcggaa tgggtaagtc cggcaagagc 360 gggaatggct catatcgcct cctggatcac tacaagtacc tcaccgcttg gttcgagctg 420 ctgaaccttc caaagaaaat catctttgtg ggccacgact ggggggcttg tctggccttt 480 cactactcct acgagcacca agacaagatc aaggccatcg tccatgctga gagtgtcgtg 540 gacgtgatcg agtcctggga cgagtggcct gacatcgagg aggatatcgc cctgatcaag 600 agcgaagagg gcgagaaaat ggtgcttgag aataacttct tcgtcgagac catgctccca 660 agcaagatca tgcggaaact ggagcctgag gagttcgctg cctacctgga gccattcaag 720 gagaagggcg aggttagacg gcctaccctc tcctggcctc gcgagatccc tctcgttaag 780 ggaggcaagc ccgacgtcgt ccagattgtc cgcaactaca acgcctacct tcgggccagc 840 gacgatctgc ctaagatgtt catcgagtcc gaccctgggt tcttttccaa cgctattgtc 900 gagggagcta agaagttccc taacaccgag ttcgtgaagg tgaagggcct ccacttcagc 960 caggaggacg ctccagatga aatgggtaag tacatcaaga gcttcgtgga gcgcgtgctg 1020 aagaacgagc agtaattcta gagtcggggc ggccggccgc ttcgagcaga catgataaga 1080 tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt 1140 gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa acaagttaac 1200 aacaacaatt gcattcattt tatgtttcag gttcaggggg aggtgtggga ggttttttaa 1260 agcaagtaaa acctctacaa atgtggtaaa atcgataagg atccgtttgc gtattgggcg 1320 ctcttccgct gatctgcgca gcaccatggc ctgaaataac ctctgaaaga ggaacttggt 1380 tagctacctt ctgaggcgga aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa 1440 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa 1500 ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca 1560 attacctcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca 1620 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg 1680 ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct 1740 tttgcaaaaa gctcgattct tctgacacta gcgccaccat gatcgaacaa gacggcctcc 1800 atgctggcag tcccgcagct tgggtcgaac gcttgttcgg gtacgactgg gcccagcaga 1860 ccatcggatg tagcgatgcg gccgtgttcc gtctaagcgc tcaaggccgg cccgtgctgt 1920 tcgtgaagac cgacctgagc ggcgccctga acgagcttca agacgaggct gcccgcctga 1980 gctggctggc caccaccggc gtaccctgcg ccgctgtgtt ggatgttgtg accgaagccg 2040 gccgggactg gctgctgctg ggcgaggtcc ctggccagga tctgctgagc agccaccttg 2100 cccccgctga gaaggtttct atcatggccg atgcaatgcg gcgcctgcac accctggacc 2160 ccgctacctg ccccttcgac caccaggcta agcatcggat cgagcgtgct cggacccgca 2220 tggaggccgg cctggtggac caggacgacc tggacgagga gcatcagggc ctggcccccg 2280 ctgaactgtt cgcccgactg aaagcccgca tgccggacgg tgaggacctg gttgtcacac 2340 acggagatgc ctgcctccct aacatcatgg tcgagaatgg ccgcttctcc ggcttcatcg 2400 actgcggtcg cctaggagtt gccgaccgct accaggacat cgccctggcc acccgcgaca 2460 tcgctgagga gcttggcggc gagtgggccg accgcttctt agtcttgtac ggcatcgcag 2520 ctcccgacag ccagcgcatc gccttctacc gcttgctcga cgagttcttt taatgatcta 2580 gaaccggtca tggccgcaat aaaatatctt tattttcatt acatctgtgt gttggttttt 2640 tgtgtgttcg aactagatgc tgtcgaccga tgcccttgag agccttcaac ccagtcagct 2700 ccttccggtg ggcgcggggc atgactatcg tcgccgcact tatgactgtc ttctttatca 2760 tgcaactcgt aggacaggtg ccggcagcgc tcttccgctt cctcgctcac tgactcgctg 2820 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta 2880 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc 2940 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag 3000 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac 3060 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc 3120 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt 3180 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc 3240 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga 3300 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta 3360 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta 3420 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga 3480 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg 3540 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag 3600 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc 3660 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact 3720 tggtctgaca gcggccgcaa atgctaaacc actgcagtgg ttaccagtgc ttgatcagtg 3780 aggcaccgat ctcagcgatc tgcctatttc gttcgtccat agtggcctga ctccccgtcg 3840 tgtagatcac tacgattcgt gagggcttac catcaggccc cagcgcagca atgatgccgc 3900 gagagccgcg ttcaccggcc cccgatttgt cagcaatgaa ccagccagca gggagggccg 3960 agcgaagaag tggtcctgct actttgtccg cctccatcca gtctatgagc tgctgtcgtg 4020 atgctagagt aagaagttcg ccagtgagta gtttccgaag agttgtggcc attgctactg 4080 gcatcgtggt atcacgctcg tcgttcggta tggcttcgtt caactctggt tcccagcggt 4140 caagccgggt cacatgatca cccatattat gaagaaatgc agtcagctcc ttagggcctc 4200 cgatcgttgt cagaagtaag ttggccgcgg tgttgtcgct catggtaatg gcagcactac 4260 acaattctct taccgtcatg ccatccgtaa gatgcttttc cgtgaccggc gagtactcaa 4320 ccaagtcgtt ttgtgagtag tgtatacggc gaccaagctg ctcttgcccg gcgtctatac 4380 gggacaacac cgcgccacat agcagtactt tgaaagtgct catcatcggg aatcgttctt 4440 cggggcggaa agactcaagg atcttgccgc tattgagatc cagttcgata tagcccactc 4500 ttgcacccag ttgatcttca gcatctttta ctttcaccag cgtttcgggg tgtgcaaaaa 4560 caggcaagca aaatgccgca aagaagggaa tgagtgcgac acgaaaatgt tggatgctca 4620 tactcgtcct ttttcaatat tattgaagca tttatcaggg ttactagtac gtctctcaag 4680 gataagtaag taatattaag gtacgggagg tattggacag gccgcaataa aatatcttta 4740 ttttcattac atctgtgtgt tggttttttg tgtgaatcga tagtactaac atacgctctc 4800 catcaaaaca aaacgaaaca aaacaaacta gcaaaatagg ctgtccccag tgcaagtgca 4860 ggtgccagaa catttctct 4879 <210> 2 <211> 4879 <212> DNA <213> Artificial Sequence <220> <223> HRluc report vector <400> 2 ggcctaactg gccggtacct gagctcgcta gcctcgagga tatcaagatc tggcctcggc 60 ggccaagctt ggcaatccgg tactgttggt aaagccacca tggcttccca cgtgtacgac 120 cccgagcaac gcaaacgcat gatcactggg cctcagtggt gggctcgctg caagcaaatg 180 aacgtgctgg actccttcat caactactat gattccgaga agcacgccga gaacgccgtg 240 atttttctgc atggtaacgc tgcctccagc tacctgtgga ggcacgtcgt gcctcacatc 300 gagcccgtgg ctagatgcat catccctgat ctgatcggaa tgggtaagtc cggcaagagc 360 gggaatggct catatcgcct cctggatcac tacaagtacc tcaccgcttg gttcgagctg 420 ctgaaccttc caaagaaaat catctttgtg ggccacgact ggggggcttg tctggccttt 480 cactactcct acgagcacca agacaagatc aaggccatcg tccatgctga gagtgtcgtg 540 gacgtgatcg agtcctggga cgagtggcct gacatcgagg aggatatcgc cctgatcaag 600 agcgaagagg gcgagaaaat ggtgcttgag aataacttct tcgtcgagac catgctccca 660 agcaagatca tgcggaaact ggagcctgag gagttcgctg cctacctgga gccattcaag 720 gagaagggcg aggttagacg gcctaccctc tcctggcctc gcgagatccc tctcgttaag 780 ggaggcaagc ccgacgtcgt ccagattgtc cgcaactaca acgcctacct tcgggccagc 840 gacgatctgc ctaagatgtt catcgagtcc gaccctgggt tcttttccaa cgctattgtc 900 gagggagcta agaagttccc taacaccgag ttcgtgaagg tgaagggcct ccacttcagc 960 caggaggacg ctccagatga aatgggtaag tacatcaaga gcttcgtgga gcgcgtgctg 1020 aagaacgagc agtaattcta gagtcggggc ggccggccgc ttcgagcaga catgataaga 1080 tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt 1140 gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa acaagttaac 1200 aacaacaatt gcattcattt tatgtttcag gttcaggggg aggtgtggga ggttttttaa 1260 agcaagtaaa acctctacaa atgtggtaaa atcgataagg atccgtttgc gtattgggcg 1320 ctcttccgct gatctgcgca gcaccatggc ctgaaataac ctctgaaaga ggaacttggt 1380 tagctacctt ctgaggcgga aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa 1440 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa 1500 ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca 1560 attacctcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca 1620 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg 1680 ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct 1740 tttgcaaaaa gctcgattct tctgacacta gcgccaccat gatcgaacaa gacggcctcc 1800 atgctggcag tcccgcagct tgggtcgaac gcttgttcgg gtacgactgg gcccagcaga 1860 ccatcggatg tagcgatgcg gccgtgttcc gtctaagcgc tcaaggccgg cccgtgctgt 1920 tcgtgaagac cgacctgagc ggcgccctga acgagcttca agacgaggct gcccgcctga 1980 gctggctggc caccaccggc gtaccctgcg ccgctgtgtt ggatgttgtg accgaagccg 2040 gccgggactg gctgctgctg ggcgaggtcc ctggccagga tctgctgagc agccaccttg 2100 cccccgctga gaaggtttct atcatggccg atgcaatgcg gcgcctgcac accctggacc 2160 ccgctacctg ccccttcgac caccaggcta agcatcggat cgagcgtgct cggacccgca 2220 tggaggccgg cctggtggac caggacgacc tggacgagga gcatcagggc ctggcccccg 2280 ctgaactgtt cgcccgactg aaagcccgca tgccggacgg tgaggacctg gttgtcacac 2340 acggagatgc ctgcctccct aacatcatgg tcgagaatgg ccgcttctcc ggcttcatcg 2400 actgcggtcg cctaggagtt gccgaccgct accaggacat cgccctggcc acccgcgaca 2460 tcgctgagga gcttggcggc gagtgggccg accgcttctt agtcttgtac ggcatcgcag 2520 ctcccgacag ccagcgcatc gccttctacc gcttgctcga cgagttcttt taatgatcta 2580 gaaccggtca tggccgcaat aaaatatctt tattttcatt acatctgtgt gttggttttt 2640 tgtgtgttcg aactagatgc tgtcgaccga tgcccttgag agccttcaac ccagtcagct 2700 ccttccggtg ggcgcggggc atgactatcg tcgccgcact tatgactgtc ttctttatca 2760 tgcaactcgt aggacaggtg ccggcagcgc tcttccgctt cctcgctcac tgactcgctg 2820 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta 2880 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc 2940 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag 3000 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac 3060 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc 3120 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt 3180 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc 3240 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga 3300 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta 3360 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta 3420 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga 3480 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg 3540 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag 3600 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc 3660 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact 3720 tggtctgaca gcggccgcaa atgctaaacc actgcagtgg ttaccagtgc ttgatcagtg 3780 aggcaccgat ctcagcgatc tgcctatttc gttcgtccat agtggcctga ctccccgtcg 3840 tgtagatcac tacgattcgt gagggcttac catcaggccc cagcgcagca atgatgccgc 3900 gagagccgcg ttcaccggcc cccgatttgt cagcaatgaa ccagccagca gggagggccg 3960 agcgaagaag tggtcctgct actttgtccg cctccatcca gtctatgagc tgctgtcgtg 4020 atgctagagt aagaagttcg ccagtgagta gtttccgaag agttgtggcc attgctactg 4080 gcatcgtggt atcacgctcg tcgttcggta tggcttcgtt caactctggt tcccagcggt 4140 caagccgggt cacatgatca cccatattat gaagaaatgc agtcagctcc ttagggcctc 4200 cgatcgttgt cagaagtaag ttggccgcgg tgttgtcgct catggtaatg gcagcactac 4260 acaattctct taccgtcatg ccatccgtaa gatgcttttc cgtgaccggc gagtactcaa 4320 ccaagtcgtt ttgtgagtag tgtatacggc gaccaagctg ctcttgcccg gcgtctatac 4380 gggacaacac cgcgccacat agcagtactt tgaaagtgct catcatcggg aatcgttctt 4440 cggggcggaa agactcaagg atcttgccgc tattgagatc cagttcgata tagcccactc 4500 ttgcacccag ttgatcttca gcatctttta ctttcaccag cgtttcgggg tgtgcaaaaa 4560 caggcaagca aaatgccgca aagaagggaa tgagtgcgac acgaaaatgt tggatgctca 4620 tactcgtcct ttttcaatat tattgaagca tttatcaggg ttactagtac gtctctcaag 4680 gataagtaag taatattaag gtacgggagg tattggacag gccgcaataa aatatcttta 4740 ttttcattac atctgtgtgt tggttttttg tgtgaatcga tagtactaac atacgctctc 4800 catcaaaaca aaacgaaaca aaacaaacta gcaaaatagg ctgtccccag tgcaagtgca 4860 ggtgccagaa catttctct 4879 <210> 3 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> hRluc_Neo_PmlI-5 ' <400> 3 caccatggct tcccacgtgt acgaccccga g 31 <210> 4 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> hRluc_Neo_PmlI-3 ' <400> 4 ctcggggtcg tacacgtggg aagccatggt g 31 <210> 5 <211> 1239 <212> DNA <213> Artificial Sequence <220> <223> KLF10 genome fragment <400> 5 cacaagctgg cttccctaag aagctcagct tgccctcaat ccctggagcg gtggaggggg 60 cagggacagg aactcgatgt ttgaccgggt caggaatccc acctgcaagc atgctttcct 120 caccccatcc cctcaagcgg tttcttggta gggcgtcggc gaagactgtg gtcccgcggg 180 tgggggaagt ggagtccacc ttggaaatag ctcgaacatt gtattttaaa cttcaactac 240 atcaaactca aactcgattc tgccaccgag cgtcgggtct ggggctccct gccacgccac 300 tagctctttg gtaccggctt cccttagtcc ccactccaac cgcccagaag gacgggaggg 360 acgcgcagag gcgggccctg ctgctagtgc ctgtgctgca gatgggagga cgagccagcg 420 cctgccgcct tgggcgtggg cggtgagtgc gagcaggtgt ctgttccgtg acagggctcc 480 cgccgccacc atctcgccct ggttctcccc agtcctccgg cttagagtga caaaaccccgg 540 caaatgctca gaaatgccgc tcagtggttc atccatccct tgcttccacg caagacccac 600 gagaaccacg ctgccaaaga acgtctggtg gctcggagcc gcgctcgcag cctccgcctc 660 cgcctcctgc tcggggtccc gcccactccc tggcgggccc agacgctgtg ggcgcgaagg 720 ggactgggcg gagcgggctt cccttcccac cccccacgtg gggccggctc tccgcaaagc 780 ctagtcgccg ccaccgcgcc tcccagctat gccccgccgc cggcgccgcc cccgggcatg 840 ggcggaggac tgaaggctag ggttgggcga ggggcgtcac ggagtcaggt ggtccccgga 900 gttccccggg gctggaccga gggaacacgg tgcctcgggt tgtgtacacg ctccactgac 960 agagcctctt gcagccgggc agccgctgat cacgcgtggc tctgccagct cattggctga 1020 ggcctcacac acctttgccg ctgattggtc gacactaggc cccgccctct accccgcccg 1080 cggcgcgggg caggcagagc ggctacctgc acggggaggg gggcagacag cgcggagcgc 1140 ggtggcgtcg acgtctagtg tctcagtgct cccgtctgtg gctaactaag cagccagcag 1200 ccaggcagct cgcgacctgc ggccaggcag ccaaccatg 1239 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> KLF10 / 5'-1 primer <400> 6 cacaagctgg cttccctaag 20 <210> 7 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> KLF10 / 3'-1 primer <400> 7 cttggaagcg atggttggct gcctggccgc aggtcgcgag c 41 <210> 8 <211> 1248 <212> DNA <213> Artificial Sequence <220> <223> PCR product <400> 8 cacaagctgg cttccctaag aagctcagct tgccctcaat ccctggagcg gtggaggggg 60 cagggacagg aactcgatgt ttgaccgggt caggaatccc acctgcaagc atgctttcct 120 caccccatcc cctcaagcgg tttcttggta gggcgtcggc gaagactgtg gtcccgcggg 180 tgggggaagt ggagtccacc ttggaaatag ctcgaacatt gtattttaaa cttcaactac 240 atcaaactca aactcgattc tgccaccgag cgtcgggtct ggggctccct gccacgccac 300 tagctctttg gtaccggctt cccttagtcc ccactccaac cgcccagaag gacgggaggg 360 acgcgcagag gcgggccctg ctgctagtgc ctgtgctgca gatgggagga cgagccagcg 420 cctgccgcct tgggcgtggg cggtgagtgc gagcaggtgt ctgttccgtg acagggctcc 480 cgccgccacc atctcgccct ggttctcccc agtcctccgg cttagagtga caaaaccccgg 540 caaatgctca gaaatgccgc tcagtggttc atccatccct tgcttccacg caagacccac 600 gagaaccacg ctgccaaaga acgtctggtg gctcggagcc gcgctcgcag cctccgcctc 660 cgcctcctgc tcggggtccc gcccactccc tggcgggccc agacgctgtg ggcgcgaagg 720 ggactgggcg gagcgggctt cccttcccac cccccacgtg gggccggctc tccgcaaagc 780 ctagtcgccg ccaccgcgcc tcccagctat gccccgccgc cggcgccgcc cccgggcatg 840 ggcggaggac tgaaggctag ggttgggcga ggggcgtcac ggagtcaggt ggtccccgga 900 gttccccggg gctggaccga gggaacacgg tgcctcgggt tgtgtacacg ctccactgac 960 agagcctctt gcagccgggc agccgctgat cacgcgtggc tctgccagct cattggctga 1020 ggcctcacac acctttgccg ctgattggtc gacactaggc cccgccctct accccgcccg 1080 cggcgcgggg caggcagagc ggctacctgc acggggaggg gggcagacag cgcggagcgc 1140 ggtggcgtcg acgtctagtg tctcagtgct cccgtctgtg gctaactaag cagccagcag 1200 ccaggcagct cgcgacctgc ggccaggcag ccaaccatgg cttccaag 1248 <210> 9 <211> 934 <212> DNA <213> Artificial Sequence <220> <223> PCR product treated with KpnI <400> 9 cggcttccct tagtccccac tccaaccgcc cagaaggacg ggagggacgc gcagaggcgg 60 gccctgctgc tagtgcctgt gctgcagatg ggaggacgag ccagcgcctg ccgccttggg 120 cgtgggcggt gagtgcgagc aggtgtctgt tccgtgacag ggctcccgcc gccaccatct 180 cgccctggtt ctccccagtc ctccggctta gagtgacaaa acccggcaaa tgctcagaaa 240 tgccgctcag tggttcatcc atcccttgct tccacgcaag acccacgaga accacgctgc 300 caaagaacgt ctggtggctc ggagccgcgc tcgcagcctc cgcctccgcc tcctgctcgg 360 ggtcccgccc actccctggc gggcccagac gctgtgggcg cgaaggggac tgggcggagc 420 gggcttccct tcccaccccc cacgtggggc cggctctccg caaagcctag tcgccgccac 480 cgcgcctccc agctatgccc cgccgccggc gccgcccccg ggcatgggcg gaggactgaa 540 ggctagggtt gggcgagggg cgtcacggag tcaggtggtc cccggagttc cccggggctg 600 gaccgaggga acacggtgcc tcgggttgtg tacacgctcc actgacagag cctcttgcag 660 ccgggcagcc gctgatcacg cgtggctctg ccagctcatt ggctgaggcc tcacacacct 720 ttgccgctga ttggtcgaca ctaggccccg ccctctaccc cgcccgcggc gcggggcagg 780 cagagcggct acctgcacgg ggaggggggc agacagcgcg gagcgcggtg gcgtcgacgt 840 ctagtgtctc agtgctcccg tctgtggcta actaagcagc cagcagccag gcagctcgcg 900 acctgcggcc aggcagccaa ccatggcttc caag 934 <210> 10 <211> 4786 <212> DNA <213> Artificial Sequence <220> <223> hRluc_Neo_PmlI report vector treated with KpnI / PmlI <400> 10 ggcctaactg gccggtacgt gtacgacccc gagcaacgca aacgcatgat cactgggcct 60 cagtggtggg ctcgctgcaa gcaaatgaac gtgctggact ccttcatcaa ctactatgat 120 tccgagaagc acgccgagaa cgccgtgatt tttctgcatg gtaacgctgc ctccagctac 180 ctgtggaggc acgtcgtgcc tcacatcgag cccgtggcta gatgcatcat ccctgatctg 240 atcggaatgg gtaagtccgg caagagcggg aatggctcat atcgcctcct ggatcactac 300 aagtacctca ccgcttggtt cgagctgctg aaccttccaa agaaaatcat ctttgtgggc 360 cacgactggg gggcttgtct ggcctttcac tactcctacg agcaccaaga caagatcaag 420 gccatcgtcc atgctgagag tgtcgtggac gtgatcgagt cctgggacga gtggcctgac 480 atcgaggagg atatcgccct gatcaagagc gaagagggcg agaaaatggt gcttgagaat 540 aacttcttcg tcgagaccat gctcccaagc aagatcatgc ggaaactgga gcctgaggag 600 ttcgctgcct acctggagcc attcaaggag aagggcgagg ttagacggcc taccctctcc 660 tggcctcgcg agatccctct cgttaaggga ggcaagcccg acgtcgtcca gattgtccgc 720 aactacaacg cctaccttcg ggccagcgac gatctgccta agatgttcat cgagtccgac 780 cctgggttct tttccaacgc tattgtcgag ggagctaaga agttccctaa caccgagttc 840 gtgaaggtga agggcctcca cttcagccag gaggacgctc cagatgaaat gggtaagtac 900 atcaagagct tcgtggagcg cgtgctgaag aacgagcagt aattctagag tcggggcggc 960 cggccgcttc gagcagacat gataagatac attgatgagt ttggacaaac cacaactaga 1020 atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc 1080 attataagct gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt 1140 cagggggagg tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtaaaatc 1200 gataaggatc cgtttgcgta ttgggcgctc ttccgctgat ctgcgcagca ccatggcctg 1260 aaataacctc tgaaagagga acttggttag ctaccttctg aggcggaaag aaccagctgt 1320 ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc 1380 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag 1440 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc 1500 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa 1560 ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc cagaagtagt 1620 gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cgattcttct gacactagcg 1680 ccaccatgat cgaacaagac ggcctccatg ctggcagtcc cgcagcttgg gtcgaacgct 1740 tgttcgggta cgactgggcc cagcagacca tcggatgtag cgatgcggcc gtgttccgtc 1800 taagcgctca aggccggccc gtgctgttcg tgaagaccga cctgagcggc gccctgaacg 1860 agcttcaaga cgaggctgcc cgcctgagct ggctggccac caccggcgta ccctgcgccg 1920 ctgtgttgga tgttgtgacc gaagccggcc gggactggct gctgctgggc gaggtccctg 1980 gccaggatct gctgagcagc caccttgccc ccgctgagaa ggtttctatc atggccgatg 2040 caatgcggcg cctgcacacc ctggaccccg ctacctgccc cttcgaccac caggctaagc 2100 atcggatcga gcgtgctcgg acccgcatgg aggccggcct ggtggaccag gacgacctgg 2160 acgaggagca tcagggcctg gcccccgctg aactgttcgc ccgactgaaa gcccgcatgc 2220 cggacggtga ggacctggtt gtcacacacg gagatgcctg cctccctaac atcatggtcg 2280 agaatggccg cttctccggc ttcatcgact gcggtcgcct aggagttgcc gaccgctacc 2340 aggacatcgc cctggccacc cgcgacatcg ctgaggagct tggcggcgag tgggccgacc 2400 gcttcttagt cttgtacggc atcgcagctc ccgacagcca gcgcatcgcc ttctaccgct 2460 tgctcgacga gttcttttaa tgatctagaa ccggtcatgg ccgcaataaa atatctttat 2520 tttcattaca tctgtgtgtt ggttttttgt gtgttcgaac tagatgctgt cgaccgatgc 2580 ccttgagagc cttcaaccca gtcagctcct tccggtgggc gcggggcatg actatcgtcg 2640 ccgcacttat gactgtcttc tttatcatgc aactcgtagg acaggtgccg gcagcgctct 2700 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca 2760 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac 2820 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt 2880 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg 2940 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc 3000 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc 3060 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc 3120 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac 3180 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt 3240 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct 3300 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc 3360 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt 3420 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg 3480 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc 3540 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa 3600 tcaatctaaa gtatatatga gtaaacttgg tctgacagcg gccgcaaatg ctaaaccact 3660 gcagtggtta ccagtgcttg atcagtgagg caccgatctc agcgatctgc ctatttcgtt 3720 cgtccatagt ggcctgactc cccgtcgtgt agatcactac gattcgtgag ggcttaccat 3780 caggccccag cgcagcaatg atgccgcgag agccgcgttc accggccccc gatttgtcag 3840 caatgaacca gccagcaggg agggccgagc gaagaagtgg tcctgctact ttgtccgcct 3900 ccatccagtc tatgagctgc tgtcgtgatg ctagagtaag aagttcgcca gtgagtagtt 3960 tccgaagagt tgtggccatt gctactggca tcgtggtatc acgctcgtcg ttcggtatgg 4020 cttcgttcaa ctctggttcc cagcggtcaa gccgggtcac atgatcaccc atattatgaa 4080 gaaatgcagt cagctcctta gggcctccga tcgttgtcag aagtaagttg gccgcggtgt 4140 tgtcgctcat ggtaatggca gcactacaca attctcttac cgtcatgcca tccgtaagat 4200 gcttttccgt gaccggcgag tactcaacca agtcgttttg tgagtagtgt atacggcgac 4260 caagctgctc ttgcccggcg tctatacggg acaacaccgc gccacatagc agtactttga 4320 aagtgctcat catcgggaat cgttcttcgg ggcggaaaga ctcaaggatc ttgccgctat 4380 tgagatccag ttcgatatag cccactcttg cacccagttg atcttcagca tcttttactt 4440 tcaccagcgt ttcggggtgt gcaaaaacag gcaagcaaaa tgccgcaaag aagggaatga 4500 gtgcgacacg aaaatgttgg atgctcatac tcgtcctttt tcaatattat tgaagcattt 4560 atcagggtta ctagtacgtc tctcaaggat aagtaagtaa tattaaggta cgggaggtat 4620 tggacaggcc gcaataaaat atctttattt tcattacatc tgtgtgttgg ttttttgtgt 4680 gaatcgatag tactaacata cgctctccat caaaacaaaa cgaaacaaaa caaactagca 4740 aaataggctg tccccagtgc aagtgcaggt gccagaacat ttctct 4786 <210> 11 <211> 5720 <212> DNA <213> Artificial Sequence <220> <223> KLF10 / 5'arm / hRluc_Neo_PmlI vector <400> 11 ggcctaactg gccggtaccg gcttccctta gtccccactc caaccgccca gaaggacggg 60 agggacgcgc agaggcgggc cctgctgcta gtgcctgtgc tgcagatggg aggacgagcc 120 agcgcctgcc gccttgggcg tgggcggtga gtgcgagcag gtgtctgttc cgtgacaggg 180 ctcccgccgc caccatctcg ccctggttct ccccagtcct ccggcttaga gtgacaaaac 240 ccggcaaatg ctcagaaatg ccgctcagtg gttcatccat cccttgcttc cacgcaagac 300 ccacgagaac cacgctgcca aagaacgtct ggtggctcgg agccgcgctc gcagcctccg 360 cctccgcctc ctgctcgggg tcccgcccac tccctggcgg gcccagacgc tgtgggcgcg 420 aaggggactg ggcggagcgg gcttcccttc ccacccccca cgtggggccg gctctccgca 480 aagcctagtc gccgccaccg cgcctcccag ctatgccccg ccgccggcgc cgcccccggg 540 catgggcgga ggactgaagg ctagggttgg gcgaggggcg tcacggagtc aggtggtccc 600 cggagttccc cggggctgga ccgagggaac acggtgcctc gggttgtgta cacgctccac 660 tgacagagcc tcttgcagcc gggcagccgc tgatcacgcg tggctctgcc agctcattgg 720 ctgaggcctc acacaccttt gccgctgatt ggtcgacact aggccccgcc ctctaccccg 780 cccgcggcgc ggggcaggca gagcggctac ctgcacgggg aggggggcag acagcgcgga 840 gt; gcagccaggc agctcgcgac ctgcggccag gcagccaacc atggcttcca aggtgtacga 960 ccccgagcaa cgcaaacgca tgatcactgg gcctcagtgg tgggctcgct gcaagcaaat 1020 gaacgtgctg gactccttca tcaactacta tgattccgag aagcacgccg agaacgccgt 1080 gatttttctg catggtaacg ctgcctccag ctacctgtgg aggcacgtcg tgcctcacat 1140 cgagcccgtg gctagatgca tcatccctga tctgatcgga atgggtaagt ccggcaagag 1200 cgggaatggc tcatatcgcc tcctggatca ctacaagtac ctcaccgctt ggttcgagct 1260 gctgaacctt ccaaagaaaa tcatctttgt gggccacgac tggggggctt gtctggcctt 1320 tcactactcc tacgagcacc aagacaagat caaggccatc gtccatgctg agagtgtcgt 1380 ggacgtgatc gagtcctggg acgagtggcc tgacatcgag gaggatatcg ccctgatcaa 1440 gagcgaagag ggcgagaaaa tggtgcttga gaataacttc ttcgtcgaga ccatgctccc 1500 aagcaagatc atgcggaaac tggagcctga ggagttcgct gcctacctgg agccattcaa 1560 gggaagggc gaggttagac ggcctaccct ctcctggcct cgcgagatcc ctctcgttaa 1620 gggaggcaag cccgacgtcg tccagattgt ccgcaactac aacgcctacc ttcgggccag 1680 cgacgatctg cctaagatgt tcatcgagtc cgaccctggg ttcttttcca acgctattgt 1740 cgagggagct aagaagttcc ctaacaccga gttcgtgaag gtgaagggcc tccacttcag 1800 ccaggaggac gctccagatg aaatgggtaa gtacatcaag agcttcgtgg agcgcgtgct 1860 gaagaacgag cagtaattct agagtcgggg cggccggccg cttcgagcag acatgataag 1920 atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg 1980 tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa 2040 caacaacaat tgcattcatt ttatgtttca ggttcagggg gaggtgtggg aggtttttta 2100 aagcaagtaa aacctctaca aatgtggtaa aatcgataag gatccgtttg cgtattgggc 2160 gctcttccgc tgatctgcgc agcaccatgg cctgaaataa cctctgaaag aggaacttgg 2220 ttagctacct tctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga 2280 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca 2340 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc 2400 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc 2460 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag 2520 gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc 2580 ttttgcaaaa agctcgattc ttctgacact agcgccacca tgatcgaaca agacggcctc 2640 catgctggca gtcccgcagc ttgggtcgaa cgcttgttcg ggtacgactg ggcccagcag 2700 accatcggat gtagcgatgc ggccgtgttc cgtctaagcg ctcaaggccg gcccgtgctg 2760 ttcgtgaaga ccgacctgag cggcgccctg aacgagcttc aagacgaggc tgcccgcctg 2820 agctggctgg ccaccaccgg cgtaccctgc gccgctgtgt tggatgttgt gaccgaagcc 2880 ggccgggact ggctgctgct gggcgaggtc cctggccagg atctgctgag cagccacctt 2940 gcccccgctg agaaggtttc tatcatggcc gatgcaatgc ggcgcctgca caccctggac 3000 cccgctacct gccccttcga ccaccaggct aagcatcgga tcgagcgtgc tcggacccgc 3060 atggaggccg gcctggtgga ccaggacgac ctggacgagg agcatcaggg cctggccccc 3120 gctgaactgt tcgcccgact gaaagcccgc atgccggacg gtgaggacct ggttgtcaca 3180 cacggagatg cctgcctccc taacatcatg gtcgagaatg gccgcttctc cggcttcatc 3240 gactgcggtc gcctaggagt tgccgaccgc taccaggaca tcgccctggc cacccgcgac 3300 atcgctgagg agcttggcgg cgagtgggcc gaccgcttct tagtcttgta cggcatcgca 3360 gctcccgaca gccagcgcat cgccttctac cgcttgctcg acgagttctt ttaatgatct 3420 agaaccggtc atggccgcaa taaaatatct ttattttcat tacatctgtg tgttggtttt 3480 ttgtgtgttc gaactagatg ctgtcgaccg atgcccttga gagccttcaa cccagtcagc 3540 tccttccggt gggcgcgggg catgactatc gtcgccgcac ttatgactgt cttctttatc 3600 atgcaactcg taggacaggt gccggcagcg ctcttccgct tcctcgctca ctgactcgct 3660 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt 3720 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc 3780 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga 3840 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata 3900 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac 3960 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg 4020 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc 4080 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag 4140 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt 4200 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt 4260 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg 4320 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac 4380 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca 4440 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac 4500 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac 4560 ttggtctgac agcggccgca aatgctaaac cactgcagtg gttaccagtg cttgatcagt 4620 gaggcaccga tctcagcgat ctgcctattt cgttcgtcca tagtggcctg actccccgtc 4680 gtgtagatca ctacgattcg tgagggctta ccatcaggcc ccagcgcagc aatgatgccg 4740 cgagagccgc gttcaccggc ccccgatttg tcagcaatga accagccagc agggagggcc 4800 gagcgaagaa gtggtcctgc tactttgtcc gcctccatcc agtctatgag ctgctgtcgt 4860 gatgctagag taagaagttc gccagtgagt agtttccgaa gagttgtggc cattgctact 4920 ggcatcgtgg tatcacgctc gtcgttcggt atggcttcgt tcaactctgg ttcccagcgg 4980 tcaagccggg tcacatgatc acccatatta tgaagaaatg cagtcagctc cttagggcct 5040 ccgatcgttg tcagaagtaa gttggccgcg gtgttgtcgc tcatggtaat ggcagcacta 5100 cacaattctc ttaccgtcat gccatccgta agatgctttt ccgtgaccgg cgagtactca 5160 accaagtcgt tttgtgagta gtgtatacgg cgaccaagct gctcttgccc ggcgtctata 5220 cgggacaaca ccgcgccaca tagcagtact ttgaaagtgc tcatcatcgg gaatcgttct 5280 tcggggcgga aagactcaag gatcttgccg ctattgagat ccagttcgat atagcccact 5340 cttgcaccca gttgatcttc agcatctttt actttcacca gcgtttcggg gtgtgcaaaa 5400 acaggcaagc aaaatgccgc aaagaaggga atgagtgcga cacgaaaatg ttggatgctc 5460 atactcgtcc tttttcaata ttattgaagc atttatcagg gttactagta cgtctctcaa 5520 ggataagtaa gtaatattaa ggtacgggag gtattggaca ggccgcaata aaatatcttt 5580 attttcatta catctgtgtg ttggtttttt gtgtgaatcg atagtactaa catacgctct 5640 ccatcaaaac aaaacgaaac aaaacaaact agcaaaatag gctgtcccca gtgcaagtgc 5700 aggtgccaga acatttctct 5720

Claims

A HRluc reporter vector comprising a nucleotide sequence in which the 109th to 114th nucleotide sequences of SEQ ID NO: 1 are substituted with a restriction enzyme restriction site.

2. The HRluc reporter vector of claim 1, wherein the restriction enzyme is PmlI.

delete

The HRluc reporter vector according to claim 1, wherein the HRluc reporter vector comprises the nucleotide sequence of SEQ ID NO: 2.

[Claim 4] The HRluc reporter vector according to claim 1, wherein the HRluc reporter vector is a 5 'flanking region of the gene to be tested.

[Claim 7] The HRluc reporter vector according to claim 5, wherein the gene to be tested is a KLF10 gene.

[Claim 6] The HRluc reporter vector according to claim 5, wherein the 5 'flanking region of the gene to be tested consists of the nucleotide sequence of SEQ ID NO: 8.

The HRluc reporter vector according to claim 7, wherein the HRluc reporter vector comprises the nucleotide sequence of SEQ ID NO: 11.

A host cell into which the HRluc reporter vector of any one of claims 1, 2 and 4 to 8 has been introduced.

Confirming the expression level of HRluc in the host cell into which the HRluc reporter vector of any one of claims 5 to 8 has been introduced, to determine the activity of the 5 'flanking region of the subject gene checking way.