KR20230116388A - Novel transposase and trasnposon system using the same - Google Patents
Novel transposase and trasnposon system using the same Download PDFInfo
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- KR20230116388A KR20230116388A KR1020220013246A KR20220013246A KR20230116388A KR 20230116388 A KR20230116388 A KR 20230116388A KR 1020220013246 A KR1020220013246 A KR 1020220013246A KR 20220013246 A KR20220013246 A KR 20220013246A KR 20230116388 A KR20230116388 A KR 20230116388A
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- transposase
- promoter
- present
- tol2
- nucleotide sequence
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Abstract
Description
본 발명은 신규한 트랜스포사제 및 이를 이용한 트랜스포존 시스템에 관한 것으로, 더욱 상세하게는 히스톤 H2B를 포함하는Tol2 트랜스포사제 발현 벡터, 이로부터 발현된 트랜스포사제 및 트랜스포존 시스템에 관한 것이다.The present invention relates to a novel transposase and a transposon system using the same, and more particularly, to a Tol2 transposase expression vector containing histone H2B, a transposase and a transposon system expressed therefrom.
유전자 재조합 기술 기반 단백질의 생산기술은 단백질 의약, 식품가공물 등에 널리 이용되고 있다. 특히, 재조합 단백질 생산기술의 경우, 생산하고자 하는 목적 단백질을 인코딩하는 염기 서열을 포함하는 발현 벡터를 대장균, 효모, 곤충 세포, 식물 세포 및 동물 세포 등의 숙주에 도입하고, 이러한 세포주를 적절한 배양 조건으로 배양하여 목적 단백질을 발현시킴으로써 이루어진다. 하지만, 목적 단백질을 효율적으로 생산할 수 있는 숙주를 개발하기 위해서는, 목적으로 하는 단백질마다 생산성이 양호한 숙주 세포를 선정하는 것이 필요하며, 개개의 숙주에서의 목적 단백질 생산 기술에 있어서 한층 더 기술 혁신이 요망되고 있다. 구체적으로 동물 세포 숙주에서는 인간을 비롯한 고등 동물 유래의 단백질에 대하여 인산화, 당쇄 부가 및 폴딩(folding)등의 번역 후 변형(post-translation)을 생체에서 제조되는 것과 보다 동일하게 실시하는 것이 가능하다. 이러한 번역 후 변형은 단백질이 본래 갖는 생리 활성을 재조합 단백질로 재현하기 위해 필수적인 것이며, 그와 같은 생리 활성을 요구하는 의약품 등에는 포유 동물 세포를 숙주로 한 단백질 생산계가 이용되고 있다. 그러나, 동물 세포를 숙주로 한 단백질 발현계의 생산성은 일반적으로 낮고, 도입 유전자의 안정성에도 문제가 있는 경우가 많다. 포유 동물 배양 세포를 숙주로 한 단백질의 생산성 향상은, 치료용 의약품이나 진단약 등의 제조에 있어서 매우 중요할 뿐만 아니라, 이들의 개발 연구에도 많이 기여하고 있다. 그 때문에, 포유 동물 배양 세포, 특히 차이니즈ㆍ햄스터 난소 세포(CHO 세포)를 숙주로 하여 용이하게 고생산주의 획득을 가능하게 하는 유전자 발현계의 개발은 급선무가 되었다.Protein production technology based on gene recombination technology is widely used in protein medicine and food products. In particular, in the case of recombinant protein production technology, an expression vector containing a nucleotide sequence encoding a target protein to be produced is introduced into a host such as Escherichia coli, yeast, insect cells, plant cells, or animal cells, and these cell lines are cultured under appropriate culture conditions. It is achieved by culturing with and expressing the target protein. However, in order to develop a host that can efficiently produce a target protein, it is necessary to select host cells with good productivity for each target protein, and further technological innovation is required in the production technology of the target protein in each host. It is becoming. Specifically, in animal cell hosts, it is possible to perform post-translational modifications such as phosphorylation, sugar chain addition, and folding on proteins derived from higher animals, including humans, more the same as those produced in vivo. Such post-translational modification is essential for reproducing the original physiological activity of a protein as a recombinant protein, and a protein production system using mammalian cells as a host is used for pharmaceuticals requiring such physiological activity. However, the productivity of protein expression systems using animal cells as hosts is generally low, and there are often problems with the stability of transgenes. Improving the productivity of proteins using mammalian cultured cells as a host is not only very important in the production of therapeutic drugs and diagnostic drugs, but also contributes greatly to their development and research. Therefore, development of a gene expression system capable of easily acquiring high-producing strains using cultured mammalian cells, particularly Chinese hamster ovary cells (CHO cells) as a host, has become an urgent need.
트랜스포존(Transposon)은 염색체의 하나의 유전자좌로부터 별도의 유전자좌로 이동할 수 있는 전위성 유전자이다. 특히, DNA 트랜스포존은 수년간 포유류 세포에서 재조합 단백질 생산을 촉진하기 위해 사용되어 왔다. 이 트랜스포존 시스템은 유전자 치료분야 또는 형질 전환을 위한 비바이러스(non-viral) 전달 도구로 사용되어 왔다. 이 비바이러스성 벡터 시스템은 또한 원래의 DNA를 복제하여 여러 유전자좌에 통합할 수 있는 매우 우수한 능력을 가지고 있다. 트랜스포존 시스템은 "잘라서 붙이기" 메커니즘으로 작동한다. 이 시스템에는 두 가지 구성 요소가 필요하다. 트랜스포사제 부위 및 유전자 전이를 일으키는 트랜스포사제를 운반하는 것은 한 쌍의 역위 말단반복(ITR)이다. Transposons are transposable genes that can move from one locus to another locus on a chromosome. In particular, DNA transposons have been used to promote recombinant protein production in mammalian cells for many years. This transposon system has been used in the field of gene therapy or as a non-viral delivery tool for transformation. This non-viral vector system also has a very good ability to replicate the original DNA and integrate it into multiple loci. The transposon system works as a "cut and paste" mechanism. This system requires two components. Carrying the transposase site and the transposase responsible for gene transfer is a pair of inverted terminal repeats (ITRs).
주로 사용되는 트랜스포존 시스템은 Sleeping Beauty, PiggyBac, Tol2이다. 특히, Tol2 트랜스포존 시스템은 일본 쌀 물고기(Japanese rice fish)(Oryzias latipes) 또는 메다카로 알려진 물고기에서 유래한 hAT슈퍼 패밀리에 속하는 Tol2 트랜스포존은 자연에서 발견된 유일한 척추동물 트랜스포존이다. 이 도구는 주로 게놈을 수식하는데 사용되는 도구이며 닭, 마우스 및 인간 세포에서 잘 전치되는 것으로 나타났다. 본 발명자는 신규한 Tol2 트랜스포존 시스템을 발명했다.The mainly used transposon systems are Sleeping Beauty, PiggyBac, and Tol2. In particular, the Tol2 transposon system belongs to the hAT superfamily derived from a fish known as Japanese rice fish (Oryzias latipes) or medaka, and is the only vertebrate transposon found in nature. This tool is primarily used to modify genomes and has been shown to transpose well in chicken, mouse and human cells. We have invented a novel Tol2 transposon system.
본 발명이 이루고자 하는 기술적 과제는 신규한 트랜스포사제 및 이를 이용한 트랜스포존 시스템을 제공하는 것이고, 구체적으로 목적 단백질의 생산 효율을 높일 수 있는 트랜스포존 시스템을 제공하는 것이다.The technical problem to be achieved by the present invention is to provide a novel transposase and a transposon system using the same, and specifically, to provide a transposon system capable of increasing the production efficiency of a target protein.
본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 기술적 과제로 제한되지 않으며, 언급되지 않은 또 다른 기술적 과제들은 아래의 기재로부터 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자에게 명확하게 이해될 수 있을 것이다.The technical problem to be achieved by the present invention is not limited to the above-mentioned technical problem, and other technical problems not mentioned can be clearly understood by those skilled in the art from the description below. There will be.
상기 기술적 과제를 달성하기 위하여, 본 발명의 일실시예는 Tol2 트랜스포사제(Transposase)를 인코딩하는 염기서열 및 히스톤 H2B를 인코딩하는 염기서열을 포함하는 트랜스포사제 발현 벡터를 제공한다.In order to achieve the above technical problem, one embodiment of the present invention provides a transposase expression vector comprising a nucleotide sequence encoding Tol2 transposase and a nucleotide sequence encoding histone H2B.
본 발명의 실시예에 있어서, 상기 히스톤 H2B를 인코딩하는 염기서열은 상기 Tol2 트랜스포사제를 인코딩하는 염기서열의 5'방향에 위치할 수 있다.In an embodiment of the present invention, the nucleotide sequence encoding the histone H2B may be located in the 5' direction of the nucleotide sequence encoding the Tol2 transposase.
상기 기술적 과제를 달성하기 위하여, 본 발명의 다른 실시예는 Tol2 트랜스포사제(Transposase)를 인코딩하는 염기서열 및 히스톤 H2B를 인코딩하는 염기서열을 포함하는 트랜스포사제 발현 벡터로부터 발현된 트랜스포사제를 제공한다.In order to achieve the above technical problem, another embodiment of the present invention is a transposase expressed from a transposase expression vector comprising a nucleotide sequence encoding Tol2 transposase and a nucleotide sequence encoding histone H2B to provide.
상기 기술적 과제를 달성하기 위하여, 본 발명의 다른 실시예는 Tol2 트랜스포사제(Transposase)를 인코딩하는 염기서열 및 히스톤 H2B를 인코딩하는 염기서열을 포함하는 트랜스포사제 발현 벡터; 및 In order to achieve the above technical problem, another embodiment of the present invention is a transposase expression vector comprising a nucleotide sequence encoding Tol2 transposase and a nucleotide sequence encoding histone H2B; and
Tol2 우측 ITR(Inverted Terminal Repeat), Tol2 좌측 ITR, 프로모터(Promoter) 및 전이 유전자(Transgene)를 포함하는 트랜스포존(Transposon) 벡터;a transposon vector including a Tol2 right Inverted Terminal Repeat (ITR), a Tol2 left ITR, a promoter and a transgene;
를 이용하여 세포를 형질전환시키는 단계를 포함하는, 세포에 전이 유전자를 통합하는 방법을 제공한다.It provides a method for integrating a transgene into a cell, comprising transforming the cell using a.
본 발명의 실시예에 있어서, 상기 세포에 전이 유전자를 통합하는 방법은 임포틴(Importin)을 인코딩하는 염기서열을 포함하는 발현 벡터를 더 포함할 수 있다.In an embodiment of the present invention, the method of integrating the transgene into the cell may further include an expression vector containing a nucleotide sequence encoding importin.
본 발명의 실시예에 있어서, 상기 임포틴은 임포틴 알파 KPNA2일 수 있다.In an embodiment of the present invention, the importin may be importin alpha KPNA2.
본 발명의 실시예에 있어서, 상기 임포틴은 임포틴 베타 KPNB1일 수 있다.In an embodiment of the present invention, the importin may be importin beta KPNB1.
본 발명의 실시예에 있어서, 상기 트랜스포존 벡터 및 트랜스포사제 발현 벡터의 질량비율(트랜스포존 벡터 : 트랜스포사제 발현 벡터)은 1:0.05 내지 1:1일 수 있다.In an embodiment of the present invention, the mass ratio (transposon vector : transposase expression vector) of the transposon vector and the transposase expression vector may be 1:0.05 to 1:1.
본 발명의 실시예에 있어서, 상기 히스톤 H2B를 인코딩하는 염기서열은 상기 Tol2 트래스포사제를 인코딩하는 염기서열의 5'방향에 위치할 수 있다.In an embodiment of the present invention, the nucleotide sequence encoding the histone H2B may be located in the 5' direction of the nucleotide sequence encoding the Tol2 transportase.
본 발명의 실시예에 있어서, 상기 세포는 동물세포일 수 있다.In an embodiment of the present invention, the cells may be animal cells.
상기 기술적 과제를 달성하기 위하여, 본 발명의 또 다른 실시예는 상기 기술한 세포에 전이 유전자를 통합하는 방법에 따라서 전이 유전자가 통합된 세포를 배양하는 단계를 포함하는, 세포에서 전이 유전자가 인코딩하는 단백질을 생산하는 방법을 제공한다.In order to achieve the above technical problem, another embodiment of the present invention is a step of culturing cells into which the transgene is integrated according to the method for integrating the transgene into cells described above, wherein the transgene encodes in the cell. Provides methods for producing proteins.
본 발명의 실시예에 따르면, 본 발명의 트랜스포존 시스템을 이용하여 목적 단백질의 생산성을 높일 수 있다.According to an embodiment of the present invention, the productivity of a target protein can be increased using the transposon system of the present invention.
본 발명의 효과는 상기한 효과로 한정되는 것은 아니며, 본 발명의 상세한 설명 또는 특허청구범위에 기재된 발명의 구성으로부터 추론 가능한 모든 효과를 포함하는 것으로 이해되어야 한다.The effects of the present invention are not limited to the above effects, and should be understood to include all effects that can be inferred from the detailed description of the present invention or the configuration of the invention described in the claims.
도 1은 CMV프로모터, 전이 유전자로서 루시퍼라제(Luciferase)를 인코딩하는 염기서열, To2 우측 및 좌측ITR를 포함하는 트랜스포존 벡터(TP)를 나타낸 그림이다.
도 2는 전이 유전자로서 루시퍼라제(Luciferase)를 인코딩하는 염기서열, CMV프로모터를 포함하는 비트랜스포존 벡터(non-TP)를 나타낸 그림이다.
도 3은 Tol2 트랜스포사제를 발현하는 일반적인 Tol2 트랜스포사제 발현 벡터(Tpase)를 나타낸 그림이다.
도 4는 본 발명에 따른 Tol2 트랜스포사제 및 히스톤 H2B를 발현하는 트랜스포사제 발현 벡터(H2B Tpase)를 나타낸 그림이다.
도 5는 비트랜스포존(non-TP) 및 트랜스포존(TP)에 대해, 기존Tol2 트랜스포사제 벡터(Tpase) 와 본 발명에 따른 트랜스포사제 발현 벡터(H2B Tpase)의 양에 따른 형광발현값을 나타낸 그래프이다.
도 6은 단백질 생산성을 측정하기 위해, 본 발명에 따른 트랜스포사제와 일반적인 Tol2 트랜스포사제를 형광발현값으로 비교하여 나타낸 그래프이다.
도 7은 본 발명에 따른 트랜스포사제 발현 벡터를 포함하는 트랜스포존 시스템(TP+H2B Tpase)의 안정성을 세포 성장률(growth rate)기반으로 나타낸 그래프이다.
도 8은 대조군(Ctrl), 일반적인 Tol2 트랜스포사제(Tpase) 발현 벡터를 포함하는 트랜스포존 시스템, 본 발명에 따른 트랜스포사제 발현 벡터를 포함하는 트랜스포존 시스템이 세포핵에서 발현되는지 여부를 보여주는 그림이다.
도 9은 대조군(Ctrl), 일반적인 Tol2 트랜스포사제(Tpase) 발현 벡터를 포함하는 트랜스포존 시스템, 본 발명에 따른 트랜스포사제 발현 벡터를 포함하는 트랜스포존 시스템이 세포핵에서 발현되는지 여부를 수치로 보여주는 그래프이다.
도 10은 본 발명에 따른 트랜스포사제 발현 벡터를 포함하는 트랜스포존 시스템에 임포틴(Importin) 알파 KPNA2 및 임포틴 베타 KPNB1을 각각 넣어주었을 때에 대한 형광발현값을 나타낸 그래프이다.
도 11은 임포틴(Importin) 발현에 대한 대조군으로서의 벡터를 나타낸 그림이다.
도 12는 임포틴(Importin) 알파 KPNA2를 인코딩하는 염기서열을 포함하는 벡터를 나타낸 그림이다.
도 13은 임포틴(Importin) 베타 KPNB1를 인코딩하는 염기서열을 포함하는 벡터를 나타낸 그림이다.
도 14는 본 발명에 따른 트랜스포사제 발현 벡터를 포함하는 트랜스포존 시스템에 임포틴(Importin) 알파 KPNA2 및 임포틴 베타 KPNB1을 각각 넣어주었을 때에, Primer를 이용한 MYC-tag 발현 량 및 GAPDH 발현량을 나타낸 그림이다.1 is a diagram showing a transposon vector (TP) including a CMV promoter, a nucleotide sequence encoding luciferase as a transgene, and To2 right and left ITRs.
2 is a diagram showing a non-transposon vector (non-TP) containing a nucleotide sequence encoding luciferase as a transgene and a CMV promoter.
3 is a diagram showing a general Tol2 transposase expression vector (Tpase) expressing Tol2 transposase.
4 is a diagram showing a transposase expression vector (H2B Tpase) expressing Tol2 transposase and histone H2B according to the present invention.
Figure 5 shows the fluorescence expression values according to the amount of the existing Tol2 transposase vector (Tpase) and the transposase expression vector (H2B Tpase) according to the present invention for non-transposon (non-TP) and transposon (TP) it's a graph
6 is a graph showing a comparison between the fluorescence expression values of a transposase according to the present invention and a general Tol2 transposase in order to measure protein productivity.
7 is a graph showing the stability of the transposon system (TP+H2B Tpase) including the transposase expression vector according to the present invention based on cell growth rate.
8 is a diagram showing whether a control group (Ctrl), a transposon system including a general Tol2 transposase (Tpase) expression vector, and a transposon system including a transposase expression vector according to the present invention are expressed in cell nuclei.
9 is a graph showing numerically whether a control (Ctrl), a transposon system including a general Tol2 transposase (Tpase) expression vector, and a transposon system including a transposase expression vector according to the present invention are expressed in cell nuclei. .
10 is a graph showing fluorescence expression values when importin alpha KPNA2 and importin beta KPNB1 were added to a transposon system containing a transposase expression vector according to the present invention, respectively.
11 is a picture showing a vector as a control for importin expression.
12 is a diagram showing a vector including a nucleotide sequence encoding importin alpha KPNA2.
13 is a diagram showing a vector including a nucleotide sequence encoding importin beta KPNB1.
Figure 14 shows the MYC-tag expression level and GAPDH expression level using a primer when importin alpha KPNA2 and importin beta KPNB1 were added to the transposon system containing the transposase expression vector according to the present invention, respectively. It is a picture.
이하에서는 첨부한 도면을 참조하여 본 발명을 설명하기로 한다. 그러나 본 발명은 여러 가지 상이한 형태로 구현될 수 있으며, 따라서 여기에서 설명하는 실시예로 한정되는 것은 아니다. 그리고 도면에서 본 발명을 명확하게 설명하기 위해서 설명과 관계없는 부분은 생략하였으며, 명세서 전체를 통하여 유사한 부분에 대해서는 유사한 도면 부호를 붙였다.Hereinafter, the present invention will be described with reference to the accompanying drawings. However, the present invention may be embodied in many different forms and, therefore, is not limited to the embodiments described herein. And in order to clearly explain the present invention in the drawings, parts irrelevant to the description are omitted, and similar reference numerals are attached to similar parts throughout the specification.
명세서 전체에서, 어떤 부분이 다른 부분과 "연결(접속, 접촉, 결합)"되어 있다고 할 때, 이는 "직접적으로 연결"되어 있는 경우뿐 아니라, 그 중간에 다른 부재를 사이에 두고 "간접적으로 연결"되어 있는 경우도 포함한다. 또한 어떤 부분이 어떤 구성요소를 "포함"한다고 할 때, 이는 특별히 반대되는 기재가 없는 한 다른 구성요소를 제외하는 것이 아니라 다른 구성요소를 더 구비할 수 있다는 것을 의미한다.Throughout the specification, when a part is said to be "connected (connected, contacted, combined)" with another part, this is not only "directly connected", but also "indirectly connected" with another member in between. "Including cases where In addition, when a part "includes" a certain component, it means that it may further include other components without excluding other components unless otherwise stated.
본 명세서에서 사용한 용어는 단지 특정한 실시예를 설명하기 위해 사용된 것으로, 본 발명을 한정하려는 의도가 아니다. 단수의 표현은 문맥상 명백하게 다르게 뜻하지 않는 한, 복수의 표현을 포함한다. 본 명세서에서, "포함하다" 또는 "가지다" 등의 용어는 명세서상에 기재된 특징, 숫자, 단계, 동작, 구성요소, 부품 또는 이들을 조합한 것이 존재함을 지정하려는 것이지, 하나 또는 그 이상의 다른 특징들이나 숫자, 단계, 동작, 구성요소, 부품 또는 이들을 조합한 것들의 존재 또는 부가 가능성을 미리 배제하지 않는 것으로 이해되어야 한다.Terms used in this specification are only used to describe specific embodiments, and are not intended to limit the present invention. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this specification, terms such as "include" or "have" are intended to indicate that there is a feature, number, step, operation, component, part, or combination thereof described in the specification, but one or more other features It should be understood that the presence or addition of numbers, steps, operations, components, parts, or combinations thereof is not precluded.
본 발명의 일측면에 따르면, 본 발명의 트랜스포사제(Transposase) 발현 벡터는 Tol2 트랜스포사제를 인코딩하는 염기서열 및 히스톤 H2B를 인코딩하는 염기서열을 포함할 수 있다. According to one aspect of the present invention, the transposase expression vector of the present invention may include a nucleotide sequence encoding Tol2 transposase and a nucleotide sequence encoding histone H2B.
본 발명의 일실시예 따르면, 상기 트랜스포사제 발현 벡터는 트랜스포사제를 인코딩하는 염기서열을 포함하는 벡터일 수 있고, 상기 트랜스포사제 발현 벡터의 염기서열은 서열번호3을 포함할 수 있다. 또한, 상기 트랜스포사제 발현 벡터는 프로모터를 더 포함할 수 있다. According to one embodiment of the present invention, the transposase expression vector may be a vector including a nucleotide sequence encoding the transposase, and the nucleotide sequence of the transposase expression vector may include SEQ ID NO: 3. In addition, the transposase expression vector may further include a promoter.
여기서 벡터는 연결된 다른 핵산 분자를 수송할 수 있는 핵산 분자를 지칭할 수 있다. 구체적으로 박테리아, 플라스미드, 파지, 코스미드(cosmid), 에피솜, 바이러스, 및 삽입 가능한 DNA 단편, 즉 동종 재조합에 의해 숙주 세포 게놈에 삽입될 수 있는 단편을 포함할 수 있다. 본 발명의 일실시예에 따르면, 상기 벡터는 바람직하게 플라스미드일 수 있다. A vector herein may refer to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it is linked. Specifically, it may include bacteria, plasmids, phages, cosmids, episomes, viruses, and insertable DNA fragments, that is, fragments that can be inserted into the host cell genome by homologous recombination. According to one embodiment of the present invention, the vector may preferably be a plasmid.
본 발명의 일실시예 따르면, 상기 프로모터는 하나 이상의 핵산 서열의 발현을 위해 숙주 세포에 의해 인지되는 핵산 서열이다. 프로모터 서열은 폴리뉴클레오타이드의 발현을 조절하는 전사 제어 서열을 함유한다. 프로모터는 돌연변이체, 절단되고 하이브리드 프로모터를 포함하는 선택된 숙주 세포에서 전사 활성을 보여주는 임의의 핵산 서열일 수 있고 숙주 세포에 상동성 또는 이종성의 세포외 또는 세포내 폴리펩타이드를 암호화하는 유전자로부터 수득될 수 있다. 본 발명에 따른 프로모터는 유도성 및 비-유도성 프로모터를 포함할 수 있다. 핵산 서열은 프로모터의 제어하에 있고 프로모터는 상기 핵산에 대해 이의 기능을 발휘한다. 본 발명의 세포/벡터는 흔히 상기 프로모터를 포함할 수 있다. 본 발명의 일실시예에 따르면, 상기 프로모터는 포유동물 세포의 게놈으로부터 유래된 프로모터 또는 포유동물 바이러스로부터 유래된 프로모터일 수 있고, 보다 바람직하게 상기 프로모터는 U6 프로모터, H1 프로모터, CMV(cytomegalo virus) 프로모터, 아데노바이러스 후기 프로모터, 벡시니아 바이러스 7.5K 프로모터, SV40 프로모터, HSV의 tk 프로모터, RSV 프로모터, 인간 연장인자 1α(hEF1α) 프로모터, 메탈로티오닌 프로모터, 베타-액틴 프로모터, 인간 IL-2 유전자의 프로모 터, 인간 IFN 유전자의 프로모터, 인간 IL-4 유전자의 프로모터, 인간 림포톡신 유전자의 프로모터, 인간 GMCSF유전자의 프로모터, TERT 프로모터, PSA 프로모터, PSMA 프로모터, CEA 프로모터, E2F 프로모터 AFP 프로모터 또는 알부민 프로모터일 수 있고, 가장 바람직하게는 CMV(cytomegalo virus) 프로모터일 수 있다.According to one embodiment of the present invention, the promoter is a nucleic acid sequence recognized by a host cell for the expression of one or more nucleic acid sequences. Promoter sequences contain transcriptional control sequences that regulate expression of the polynucleotide. A promoter can be any nucleic acid sequence that exhibits transcriptional activity in a selected host cell, including mutant, truncated and hybrid promoters, and can be obtained from genes encoding extracellular or intracellular polypeptides homologous or heterologous to the host cell. there is. Promoters according to the present invention may include inducible and non-inducible promoters. A nucleic acid sequence is under the control of a promoter and the promoter exerts its function on the nucleic acid. Cells/vectors of the present invention may often contain such promoters. According to one embodiment of the present invention, the promoter may be a promoter derived from the genome of a mammalian cell or a promoter derived from a mammalian virus, more preferably the promoter is a U6 promoter, an H1 promoter, or a cytomegalo virus (CMV) promoter. promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, tk promoter of HSV, RSV promoter, human elongation factor 1α (hEF1α) promoter, metallothioneine promoter, beta-actin promoter, human IL-2 gene promoter of human IFN gene, promoter of human IL-4 gene, promoter of human lymphotoxin gene, promoter of human GMCSF gene, TERT promoter, PSA promoter, PSMA promoter, CEA promoter, E2F promoter, AFP promoter or albumin promoter It may be, and most preferably, it may be a CMV (cytomegalo virus) promoter.
본 발명의 일실시예에 따르면, 상기 Tol2 트랜스포사제는 Tol2 트랜스포존(Transposon)시스템에서 전이 유전자(Transgene) 또는 관심 유전자(Gene of Interest)의 양측 말단에 존재하는 역방향 반복 배열(Inverted Terminal Repeat)를 인식 후 결합하여 전이 유전자 또는 관심 유전자를 절단할 수 있다. 본 발명의 일실시예에 따르면, 상기 Tol2 트랜스포사제를 인코딩하는 염기서열은 서열번호1을 포함할 수 있다.According to one embodiment of the present invention, the Tol2 transposase is an inverted terminal repeat present at both ends of a transgene or a gene of interest in the Tol2 transposon system. After recognition, it can bind and cleave the transgene or gene of interest. According to one embodiment of the present invention, the nucleotide sequence encoding the Tol2 transposase may include SEQ ID NO: 1.
본 발명의 일실시예에 따르면, 상기 히스톤 H2B는 히스톤(Histone) 단백질 중 하나로서, 상기 히스톤은 염색질(chromatin)을 구성하는 기본단위인 뉴클레오솜(nucleosome)의 중심 단백질이다. 상기 히스톤은 DNA 가닥이 감기는 실패와 같은 역할을 하며 이는 진핵생물의 거대한 유전자가 세포 핵으로 들어가기 위해 필수적인 응축을 할 수 있도록 한다. 또한, 상기 히스톤은 NLSs (nuclear localization sequences)를 포함할 수 있다. 또한, 상기 히스톤 H2B를 인코딩하는 염기서열은 서열번호2를 포함할 수 있다.According to one embodiment of the present invention, the histone H2B is one of histone proteins, and the histone is a central protein of nucleosome, a basic unit constituting chromatin. These histones act as coiling failures for DNA strands, allowing eukaryotic giant genes to condense necessary for entry into the cell nucleus. In addition, the histones may include nuclear localization sequences (NLSs). In addition, the nucleotide sequence encoding the histone H2B may include SEQ ID NO: 2.
도3 및 4를 참고해서 보면, 본 발명의 일실시예에 따른H2B Tol2 트랜스포사제 발현 벡터는 NLS-H2B 서열을 기존의 Tol2 트랜포사제 발현 벡터(TPase 벡터)의 트랜스포사제 서열 앞에 붙임으로서 발명되었다. Referring to FIGS. 3 and 4, the H2B Tol2 transposase expression vector according to an embodiment of the present invention appends the NLS-H2B sequence in front of the transposase sequence of the existing Tol2 transposase expression vector (TPase vector). was invented
본 발명의 일실시예에 따르면, 상기 히스톤 H2B를 인코딩하는 염기서열은 상기 Tol2 트랜스포사제를 인코딩하는 염기서열의 5'방향에 위치할 수 있다. According to one embodiment of the present invention, the nucleotide sequence encoding the histone H2B may be located in the 5' direction of the nucleotide sequence encoding the Tol2 transposase.
본 발명의 일측면에 따르면, 본 발명의 트랜스포사제는 본 발명에 따른 발현 벡터로부터 발현될 수 있다. 본 발명의 일실시예에 따르면, 본 발명의 트랜스포사제는 Tol2 트랜스포사제에 히스톤 H2B가 융합된 상태일 수 있다. 본 발명의 일실시예에 따르면, 상기 트랜스포사제를 인코딩하는 염기서열은 서열번호4를 포함할 수 있다. According to one aspect of the present invention, the transposase of the present invention can be expressed from an expression vector according to the present invention. According to one embodiment of the present invention, the transposase of the present invention may be in a state in which histone H2B is fused to Tol2 transposase. According to one embodiment of the present invention, the nucleotide sequence encoding the transposase may include SEQ ID NO: 4.
도8을 참고해서 보면, 본 발명에 따른 트랜스포사제를 포함하고 있는 세포에서 가장 강한 형광발현이 일어났고 이는 본 발명에 따른 트랜스포사제가 Tol2 트랜스포사제보다 세포핵으로의 신호전달에 있어서 가장 효율적임을 보여주는 것이다. 또한, 도9를 참고해서 보면, 형광발현이 일어난 세포수를 전체 세포수에 대한 백분율로 살펴보았을 때, 본 발명에 따른 트랜스포사제가 다른 Tol2 트랜스포사제보다 훨씬 많다는 것을 알 수 있으며, 이는 본 발명에 따른 트랜스포사제가 세포내에서 활성을 가지고 작용하고 있다는 것을 보여준다.Referring to Figure 8, the strongest fluorescence expression occurred in cells containing the transposase according to the present invention, indicating that the transposase according to the present invention is the most efficient in signal transduction to the cell nucleus than the Tol2 transposase. it will show In addition, referring to FIG. 9, when the number of cells in which fluorescence expression has occurred as a percentage of the total number of cells, it can be seen that the transposase according to the present invention is much more than the other Tol2 transposase, which is the case of the present invention. It shows that the transposase according to is acting with activity in the cell.
본 발명의 다른 일측면에 따르면, 본 발명의 세포에 전이 유전자를 통합하는 방법은 Tol2 트랜스포사제(Transposase)를 인코딩하는 염기서열 및 히스톤 H2B를 인코딩하는 염기서열을 포함하는 트랜스포사제 발현 벡터; 및 Tol2 우측 ITR(Inverted Terminal Repeat), Tol2 좌측 ITR, 프로모터(Promoter) 및 전이 유전자(Transgene)를 포함하는 트랜스포존(Transposon) 벡터를 이용하여 세포를 형질전환시키는 단계를 포함할 수 있다. According to another aspect of the present invention, a method for integrating a transgene into a cell of the present invention comprises a transposase expression vector comprising a nucleotide sequence encoding Tol2 transposase and a nucleotide sequence encoding histone H2B; and transforming cells using a transposon vector including a Tol2 right inverted terminal repeat (ITR), a Tol2 left ITR, a promoter, and a transgene.
본 발명의 일실시예 따르면, 상기 Tol2 우측 ITR(Inverted Terminal Repeat) 및 Tol2 좌측 ITR은 본 발명의 트랜스포사제 발현 벡터에 발현된 트랜스포사제가 인식하는 부위로서, 상기 전이 유전자의 양말단에 위치할 수 있다. 또한, 상기 Tol2 우측 ITR 및 Tol2 좌측 ITR의 염기서열은 각각 서열번호6 및 서열번호7을 포함할 수 있다. According to one embodiment of the present invention, the Tol2 right Inverted Terminal Repeat (ITR) and the Tol2 left ITR are sites recognized by the transposase expressed in the transposase expression vector of the present invention, and may be located at both ends of the transgene. can In addition, the nucleotide sequences of the Tol2 right ITR and the Tol2 left ITR may include SEQ ID NO: 6 and SEQ ID NO: 7, respectively.
본 발명의 일실시예에 따르면, 상기 트랜스포존 벡터가 포함하는 프로모터는 포유동물 세포의 게놈으로부터 유래된 프로모터 또는 포유동물 바이러스로부터 유래된 프로모터일 수 있고, 보다 바람직하게 상기 프로모터는 U6 프로모터, H1 프로모터, CMV(cytomegalo virus) 프로모터, 아데노바이러스 후기 프로모터, 벡시니아 바이러스 7.5K 프로모터, SV40 프로모터, HSV의 tk 프로모터, RSV 프로모터, 인간 연장인자 1α(hEF1α) 프로모터, 메탈로티오닌 프로모터, 베타-액틴 프로모터, 인간 IL-2 유전자의 프로모 터, 인간 IFN 유전자의 프로모터, 인간 IL-4 유전자의 프로모터, 인간 림포톡신 유전자의 프로모터, 인간 GMCSF유전자의 프로모터, TERT 프로모터, PSA 프로모터, PSMA 프로모터, CEA 프로모터, E2F 프로모터 AFP 프로모터 또는 알부민 프로모터일 수 있고, 가장 바람직하게는 CMV(cytomegalo virus) 프로모터일 수 있다. 본 발명의 일실시예에 따르면, 상기 프로모터는 전이 유전자의 5'방향에 위치할 수 있다. According to one embodiment of the present invention, the promoter included in the transposon vector may be a promoter derived from the genome of a mammalian cell or a promoter derived from a mammalian virus, more preferably the promoter is a U6 promoter, an H1 promoter, cytomegalo virus (CMV) promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, HSV tk promoter, RSV promoter, human elongation factor 1α (hEF1α) promoter, metallothionein promoter, beta-actin promoter, Human IL-2 gene promoter, human IFN gene promoter, human IL-4 gene promoter, human lymphotoxin gene promoter, human GMCSF gene promoter, TERT promoter, PSA promoter, PSMA promoter, CEA promoter, E2F promoter It may be an AFP promoter or an albumin promoter, and most preferably a cytomegalo virus (CMV) promoter. According to one embodiment of the present invention, the promoter may be located in the 5' direction of the transgene.
상기 전이 유전자(Transgene)는 관심 유전자(Gene of Interest) 또는 목적 유전자로 지칭될 수 있고, 전이 유전자 단백질을 암호화하는DNA와 관련하여 RNA 전사 개시 신호, 프로모터 또는 인핸서와 같은 전사되지 않는 플랭킹 영역을 포함하지 않는다. 전이 유전자는 세포 내에서 목적 단백질을 생산하기 위하여 도입될 수 있으며 상기 관심 단백질을 인코딩하는 유전자를 의미하고, 기능성 RNA를 암호화하는 DNA 서열을 포함할 수 있다. 따라서, 전이 유전자는 형질감염을 통한 포유동물 숙주 세포와 같은 세포로 도입되고 목적하는 생성물(전이 유전자 발현 생성물, 예를 들어, 이종성 단백질)을 암호화하는 DNA 서열일 수 있다. 관심 유전자는 소포체 및/또는 세포질 막에 걸친 전좌 및/또는 분비를 매개하고/하거나 촉진시키는 신호 펩타이드를 암호화하고 분비 전 또는 분비 동안에 제거되는 신호 펩타이드 암호화 서열에 기능적으로 부착될 수 있다.The transgene may be referred to as a gene of interest or a target gene, and may include a non-transcribed flanking region such as an RNA transcription initiation signal, a promoter, or an enhancer in relation to DNA encoding the transgene protein. do not include. The transgene refers to a gene that can be introduced to produce a protein of interest in a cell and encodes the protein of interest, and may include a DNA sequence encoding a functional RNA. Thus, a transgene can be a DNA sequence that is introduced into a cell, such as a mammalian host cell, via transfection and encodes a desired product (transgene expression product, eg, a heterologous protein). The gene of interest may be functionally attached to a signal peptide coding sequence that encodes a signal peptide that mediates and/or promotes secretion and/or translocation across the endoplasmic reticulum and/or cytoplasmic membrane and is removed prior to or during secretion.
여기서 사용된 용어, 전이(Transposition)는 하나의 위치에서 트랜스포존의 이동 또는 복제와 다른 위치로의 삽입을 포함하는 복잡한 유전적 재배열(genetic rearrangement) 과정을 지칭하며, 이는 보통 게놈 내, 또는 게놈 간, DNA 구조물(예컨대 플라스미드, 박미드(bacmid), 및 코스미드)간, 또는 게놈과 DNA 구조물 간에서 발생한다. 본 발명의 일실시예에 따르면, 전이는 게놈과 DNA 구조물 사이에서 발생할 수 있다. As used herein, the term transposition refers to a complex genetic rearrangement process involving the movement or duplication of a transposon at one location and its insertion at another location, usually within or between genomes. , between DNA constructs (such as plasmids, bacmids, and cosmids), or between genomes and DNA constructs. According to one embodiment of the present invention, transition may occur between a genome and a DNA structure.
본 발명의 일실시예 따르면, 상기 트랜스포존 벡터의 염기서열은 서열번호5를 포함할 수 있다.According to one embodiment of the present invention, the nucleotide sequence of the transposon vector may include SEQ ID NO: 5.
본 발명의 일실시예 따르면, 상기 세포에 전이 유전자를 통합하는 방법은 임포틴(Importin)을 인코딩하는 염기서열을 포함하는 발현 벡터를 더 포함할 수 있다. 상기 임포틴은 단백질을 세포질에서 세포핵으로 이동시킬 수 있는 단백질로서, Nuclear Localization Sequences (NLS)라고 불리는 특정 인식부위에 결합할 수 있다. 상기 임포틴은 임포틴 알파 (importin α) KPNA2및 임포틴 베타(importin β KPNB1 라는 2종류의 서브유닛을 포함할 수 있다. 임포틴 알파 KPNA2는 이동시킬 단백질에 존재하는 NLS에 결합하는 어댑터(adaptor) 단백질이고, 임포틴 베타 KPNB1는 이동시킬 단백질에 그 자체가 결합하여 단백질을 이동시키거나, 또는 임포틴 알파 KPNA2와 결합하여 이종이합체(heterodimer)를 구성할 수 있다. 본 발명의 다른 일실시예에 따르면, 상기 임포틴은 바람직하게 임포틴 알파 KPNA2 또는 임포틴 베타 KPNB1일 수 있다. 도10을 참고해서 보면, 본 발명의 세포에 전이 유전자를 통합하는 방법에 있어서, 임포틴 베타 KPNB1를 추가로 처리해준 경우가 가장 뛰어난 전이 유전자 발현을 나타냈고 그 다음으로 임포틴 알파 KPNA2로 처리해준 경우였고, 마지막이 추가 임포틴 처리가 없는 경우였다. 또한, 도14를 참고해서 보면, 본 발명의 세포에 전이 유전자를 통합하는 방법에 있어서 임포틴 알파 KPNA2 또는 임포틴 베타 KPNB1를 처리해 준 경우, 각각 제대로 발현되었기에 트랜스포존 시스템에서 정상적으로 활성을 가지고 있음을 보여주었다. According to one embodiment of the present invention, the method of integrating the transgene into the cells may further include an expression vector containing a nucleotide sequence encoding importin. The importin is a protein capable of moving proteins from the cytoplasm to the cell nucleus, and can bind to specific recognition sites called Nuclear Localization Sequences (NLS). The importin may include two types of subunits, importin α KPNA2 and importin β KPNB1. Importin α KPNA2 is an adapter that binds to the NLS present in the protein to be moved. ) protein, and importin beta KPNB1 itself binds to the protein to be moved to move the protein, or it can combine with importin alpha KPNA2 to form a heterodimer. According to , the importin may preferably be importin alpha KPNA2 or importin beta KPNB1 Referring to Figure 10, in the method of integrating a transgene into cells of the present invention, importin beta KPNB1 is additionally added. The treated case showed the most excellent transgene expression, followed by the case of treatment with importin alpha KPNA2, and the last case was the case without additional importin treatment. In the method of integrating the transgene, when importin alpha KPNA2 or importin beta KPNB1 was treated, they were properly expressed, indicating that they were normally active in the transposon system.
본 발명의 일실시예 따르면, 상기 트랜스포존 벡터 및 트랜스포사제 발현 벡터의 질량비율(트랜스포존 벡터 : 트랜스포사제 발현 벡터)은 1:0.05 내지 1:1일 수 있다. 상기 질량비율이 1:1을 초과하는 경우, 즉, 트랜스포존 벡터에 비해 과도한 트랜스포사제 발현 벡터가 투입되면, 트랜스포사제-트랜스포존 복합체 생성을 방해할 수 있다. 반대로, 상기 질량비율이 1:0.05 미만일 경우, 즉, 트랜스포존 벡터에 비해 트랜스포사제 발현 벡터가 지나치게 덜 투입되면 트랜스포사제-트랜스포존 복합체 생성이 일어나지 않을 수 있다. 본 발명의 다른 일실시예에 따르면, 상기 질량비율은 1:0.05 내지 1:0.5일 수 있다. 본 발명의 또다른 일실시예에 따르면, 상기 질량비율은 1:0.08 내지 1:0.3일 수 있으며, 1:0.09 내지 1:0.2일수 있다. 바람직하게는 1:0.1일수 있다.According to one embodiment of the present invention, the mass ratio (transposon vector : transposase expression vector) of the transposon vector and the transposase expression vector may be 1:0.05 to 1:1. If the mass ratio exceeds 1:1, that is, if an excessive amount of the transposase expression vector is added compared to the transposon vector, the formation of the transposase-transposon complex may be hindered. Conversely, when the mass ratio is less than 1:0.05, that is, when too little transposase expression vector is added compared to the transposon vector, generation of the transposase-transposon complex may not occur. According to another embodiment of the present invention, the mass ratio may be 1:0.05 to 1:0.5. According to another embodiment of the present invention, the mass ratio may be 1:0.08 to 1:0.3, or 1:0.09 to 1:0.2. Preferably it may be 1:0.1.
도5를 참고해서 보면, 트랜스포존 벡터(TP) 및 Tol2 트랜스포사제 벡터 비율이 10:3인 경우가 비-트랜스포존 값과 비교해서, 루시퍼라제 발현을 증가시켰다. 하지만, 본 발명에 따른 발현 벡터로부터 발현된 Tol2 H2B 트랜스포사제 벡터 및 트랜스포존 벡터가 다른 경우들보다 월등히 루시퍼라제 발현을 증가시켰다. 이는 본 발명에 따른 발현 벡터로부터 발현된 Tol2 H2B 트랜스포사제가 단백질 생산성을 증가시키는 데 상당히 효과적이라는 것을 의미한다.Referring to FIG. 5, when the ratio between the transposon vector (TP) and the Tol2 transposase vector was 10:3, luciferase expression was increased compared to the non-transposon value. However, the Tol2 H2B transposase vector and the transposon vector expressed from the expression vector according to the present invention significantly increased luciferase expression compared to other cases. This means that the Tol2 H2B transposase expressed from the expression vector according to the present invention is quite effective in increasing protein productivity.
본 발명의 일실시예 따르면, 상기 세포는 동물세포일 수 있다. 바람직하게는 상기 세포는 포유동물 세포일 수 있으며, 상기 포유동물 세포는 COS 세포, CHO 세포, VERO 세포, MDCK 세포, WI38 세포, V79 세포, B14AF28-G3 세포, BHK 세포, HaK 세포, NS0 세포, SP2/0-Ag14 세포, HeLa 세포, HEK293 세포 및 PER.C6 세포로 이루어진 군으로부터 선택되는 하나일 수 있다. 바람직하게, 상기 세포는 CHO세포일 수 있다. According to one embodiment of the present invention, the cells may be animal cells. Preferably, the cells may be mammalian cells, and the mammalian cells include COS cells, CHO cells, VERO cells, MDCK cells, WI38 cells, V79 cells, B14AF28-G3 cells, BHK cells, HaK cells, NSO cells, It may be one selected from the group consisting of SP2/0-Ag14 cells, HeLa cells, HEK293 cells, and PER.C6 cells. Preferably, the cells may be CHO cells.
본 발명의 일실시예에 따르면, 상기 트랜스포존 벡터는 항생제 내성 유전자를 더 포함할 수 있다. 항생제 내성 유전자는 마커 유전자로 사용되고, 당업계에서 통상적으로 이용되는 항생제 내성 유전자를 포함하며, 예를 들어 암피실린, 겐타마이신, 카베니실린, 클로람페니콜, 스트렙토마이신, 카나마이신, 제네티신, 네오마이신 및 테트라사이클린에 대한 내성유전자가 있을 수 있다. According to one embodiment of the present invention, the transposon vector may further include an antibiotic resistance gene. Antibiotic resistance genes are used as marker genes and include antibiotic resistance genes commonly used in the art, such as ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetra There may be genes for resistance to cyclins.
본 발명의 일실시예에 따르면, 상기 트랜스포존 벡터는 전이 유전자 및 항생제 내성 유전자사이에 추가 프로모터를 더 포함할 수 있다. 상기 추가 프로모터는 포유동물 세포의 게놈으로부터 유래된 프로모터 또는 포유동물 바이러스로부터 유래된 프로모터일 수 있고, 보다 바람직하게 상기 프로모터는 U6 프로모터, H1 프로모터, CMV(cytomegalo virus) 프로모터, 아데노바이러스 후기 프로모터, 벡시니아 바이러스 7.5K 프로모터, SV40 프로모터, HSV의 tk 프로모터, RSV 프로모터, 인간 연장인자 1α(hEF1α) 프로모터, 메탈로티오닌 프로모터, 베타-액틴 프로모터, 인간 IL-2 유전자의 프로모 터, 인간 IFN 유전자의 프로모터, 인간 IL-4 유전자의 프로모터, 인간 림포톡신 유전자의 프로모터, 인간 GMCSF유전자의 프로모터, TERT 프로모터, PSA 프로모터, PSMA 프로모터, CEA 프로모터, E2F 프로모터 AFP 프로모터 또는 알부민 프로모터일 수 있고, 가장 바람직하게는 SV40 프로모터일 수 있다.According to one embodiment of the present invention, the transposon vector may further include an additional promoter between the transgene and the antibiotic resistance gene. The additional promoter may be a promoter derived from the genome of a mammalian cell or a promoter derived from a mammalian virus, more preferably the promoter is a U6 promoter, H1 promoter, CMV (cytomegalo virus) promoter, adenovirus late promoter, vexy nia virus 7.5K promoter, SV40 promoter, tk promoter of HSV, RSV promoter, human elongation factor 1α (hEF1α) promoter, metallothionein promoter, beta-actin promoter, promoter of human IL-2 gene, promoter of human IFN gene promoter, human IL-4 promoter, human lymphotoxin gene promoter, human GMCSF gene promoter, TERT promoter, PSA promoter, PSMA promoter, CEA promoter, E2F promoter, AFP promoter or albumin promoter, most preferably It may be the SV40 promoter.
본 발명의 또다른 일측면에 따르면, 본 발명의 세포에서 전이 유전자가 인코딩하는 단백질을 생산하는 방법은, 상기 세포에 전이 유전자를 통합하는 방법에 따라 전이 유전자가 통합된 세포를 배양하는 단계를 포함할 수 있다. According to another aspect of the present invention, a method for producing a protein encoded by a transgene in a cell of the present invention includes culturing a cell into which the transgene is integrated according to a method for integrating the transgene into the cell. can do.
본 발명의 일실시예에 따르면, 상기 단백질은 상기 전이 유전자에 의하여 인코딩되고, 상기 전이 유전자는 세포 내에서 전사 및 번역 단계를 거쳐 단백질을 생산할 수 있다. According to one embodiment of the present invention, the protein is encoded by the transgene, and the transgene can produce a protein through transcription and translation steps in a cell.
본 발명의 일실시예에 따르면, 상기 단백질을 생산하는 방법은 형질감염된 세포에 메틸화 억제제(methylationinhibitor) 또는 히스톤 디아세틸레이즈 억제제(histone deacetylase inhibitor)를 처리하는 단계를 더 포함할 수 있다. 상기 메틸화 억제제로의 처리는 풀림이 유지될 때 DNA 축합을 억제하고, 따라서 공동-형질감염된 트랜스포사제 벡터의 발현 수준이 증가하였고, 숙주 게놈 내부의 컷 및 페이스트로 인해 전이유전자가 더욱 안정적으로 통합되고 발현될 수 있다. 상기 메틸화 억제제는 5-아자시티딘(azacytidine), 5-아자-2'-데옥시시티딘, 제불라린, L-메티오닌, 아피시딘, 히드랄라진 및 프로카인아미드로 구성된 군으로부터 선택된 어느 하나 이상일 수 있다. 또한, 히스톤 디아세틸레이즈 억제제는 TSA(trichostatin A), NaBu(sodium butyrate), VAP(valproic acid) 및 SAHA으로 구성된 군으로부터 선택된 어느 하나 이상일 수 있다. According to one embodiment of the present invention, the method for producing the protein may further include treating the transfected cells with a methylation inhibitor or a histone deacetylase inhibitor. Treatment with the methylation inhibitor inhibits DNA condensation when unwinding is maintained, thus increasing the expression level of the co-transfected transposase vector, and more stable integration of the transgene due to cut and paste inside the host genome and can be expressed. The methylation inhibitor is any one selected from the group consisting of 5-azacytidine, 5-aza-2'-deoxycytidine, zebularin, L-methionine, apicidine, hydralazine and procainamide may be ideal In addition, the histone deacetylase inhibitor may be at least one selected from the group consisting of trichostatin A (TSA), sodium butyrate (NaBu), valproic acid (VAP), and SAHA.
도6을 참고해서 보면, 본 발명에 따른 트랜스포사제가 다른 Tol2 트랜스포사제보다 적은 양으로도 더욱 높은 단백질 생산성으로 보여준다는 것을 알 수 있다. 또한, 도7을 참고해서 보면, 본 발명에 따른 트랜스포사제는 세포 성장률 기반의 안정성 시험에서 다른 Tol2 트랜스포사제보다 훨씬 높은 성장률을 보여주어 상당히 높은 안정성을 가지고 있다. Referring to Figure 6, it can be seen that the transposase according to the present invention shows higher protein productivity with a smaller amount than other Tol2 transposases. In addition, referring to FIG. 7 , the transposase according to the present invention shows a much higher growth rate than other Tol2 transposases in a stability test based on cell growth rate, and thus has significantly high stability.
이하 첨부된 도면을 참고하여 본 발명의 실시예를 상세히 설명하기로 한다.Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings.
실시예1. 세포 배양Example 1. cell culture
CHO (Chinese hamster ovary) DG44 세포들 (A1100001; Thermo Fisher Scientific, Waltham, MA, USA)이 본 연구에서 사용되었다. 세포들은 100 U/ml 페니실린(penicillin) 및 100 μg/ml 스트렙토마이신(streptomycin) (SV30079.01; Hyclone)과 함께 10% 소태아혈청(fetal bovine serum), 10 mM 소듐 히포산틴(sodium hypoxanthine), 1.6 mM 티미딘(thymidine) (2068642; Gibco, Waltham, MA, USA)이 보충된 25 mM 글루코스(glucose)와 함께 DMEM 배지(Dulbecco's modified Eagle's medium Cells)에서 배양되었다. 세포들은 5% CO2가 보완된 외기(ambient air)(20% O2)에서 배양되었다. 세포들은 250 mL 삼각 플라스크(Erlenmeyer flask) (73250; SPL, Pocheon, Korea)에서 40 mL의 작업량(working volume)으로 배양되었고, 150 RPM 교반하에서 인큐베이팅 되었고, 세포 배지를 바꿔줄 때 계대되었다. 세포 밀도(density) 및 생존능력(viability)은 Cedex HiRes Analyzer (05650216001; Roche, Basel, Switzerland)로 측정되었다.Chinese hamster ovary (CHO) DG44 cells (A1100001; Thermo Fisher Scientific, Waltham, MA, USA) were used in this study. Cells were cultured in 10% fetal bovine serum, 10 mM sodium hypoxanthine, 100 U/ml penicillin and 100 μg/ml streptomycin (SV30079.01; Hyclone). They were cultured in DMEM medium (Dulbecco's modified Eagle's medium Cells) with 25 mM glucose supplemented with 1.6 mM thymidine (2068642; Gibco, Waltham, MA, USA). Cells were cultured in ambient air (20% O 2 ) supplemented with 5% CO 2 . Cells were cultured at a working volume of 40 mL in a 250 mL Erlenmeyer flask (73250; SPL, Pocheon, Korea), incubated under 150 RPM agitation, and passaged when the cell medium was changed. Cell density and viability were measured with Cedex HiRes Analyzer (05650216001; Roche, Basel, Switzerland).
실시예2. 플라스미드(Plasmid) 디자인 및 구축Example 2. Plasmid design and construction
재조합 플라스미드를 구축하기 위해서 Infusion cloning 기술을 사용했다. Tol2 트랜스포사제(transposase) 벡터는 Tol2 트랜스포사제를 인코딩하는 cDNA를 pcDNA6 벡터(V22020; Invitrogen, Carlsbad, CA, USA)에 삽입함으로써 만들어졌다. H2B Tol2 트랜스포사제는 이전에 만들어 놓은 Tol2 트랜스포사제 벡터를 Pmel (R0560s; NEB, Ipswich, MA, USA) 효소로 잘라내서 H2B 염기서열을 삽입함으로써 만들었다. TP 벡터는 Tol2 우측 및 좌측 ITRs가 CMV 프로모터 기반 루시퍼라제 유전자 옆에 위치하게끔 디자인되었다. 반면, non-TP벡터는 CMV 프로모터 기반 루시퍼라제 유전자만 포함되도록 디자인되었다. KPNA2 및 KPNB1은 각각 infusion cloning 기술을 통해 pcDNA3.1 벡터에 삽입되었다. EcoRI (R0101s; NEB)가 제한효소로서 사용되었다.Infusion cloning technology was used to construct recombinant plasmids. The Tol2 transposase vector was made by inserting the cDNA encoding the Tol2 transposase into the pcDNA6 vector (V22020; Invitrogen, Carlsbad, CA, USA). H2B Tol2 transposase was prepared by excising a previously prepared Tol2 transposase vector with Pmel (R0560s; NEB, Ipswich, MA, USA) enzyme and inserting the H2B nucleotide sequence. The TP vector was designed such that Tol2 right and left ITRs were flanked by the CMV promoter-based luciferase gene. On the other hand, the non-TP vector was designed to contain only the luciferase gene based on the CMV promoter. KPNA2 and KPNB1 were each inserted into pcDNA3.1 vector through infusion cloning technology. EcoRI (R0101s; NEB) was used as a restriction enzyme.
(서열번호8)GCTCTACAAAACCAT
(SEQ ID NO: 8)
(서열번호9)CCAGATCCTCTTCTG
(SEQ ID NO: 9)
Forwardimportin beta
Forward
(서열번호10)CAGCATTTGGGAAGG
(SEQ ID NO: 10)
(서열번호11)TGCCAGATCCTCTTC
(SEQ ID NO: 11)
실시예3. 형질감염(Transfection)Example 3. Transfection
CHO DG44 세포들은 제조사의 지시에 따라 SG 세포주 4D-NucleofectorTM X Kit L (V4XC-3024; Lonza, Basel, Switzerland)을 사용해서 형질감염되었다. 형질감염된 세포들은 10mg/mL 블라스티시딘(Blasticidin) (ant-bl-05; Invitrogen)을 사용해서 2주동안 선별되었다. 선별과정동안, 배지는 2일마다 바꿔주었다. 선별후, 단일세포들은 96-well 플레이트(353072; Falcon, Franklin Lakes, NJ, USA)에 150 μl 배지로 well당 seeding되었고 15일동안 인큐베이팅되었다. CHO DG44 cells were transfected using the SG cell line 4D-Nucleofector™ X Kit L (V4XC-3024; Lonza, Basel, Switzerland) according to the manufacturer's instructions. Transfected cells were selected for 2 weeks using 10 mg/mL Blasticidin (ant-bl-05; Invitrogen). During the selection process, the medium was changed every 2 days. After sorting, single cells were seeded per well in 96-well plates (353072; Falcon, Franklin Lakes, NJ, USA) in 150 μl medium and incubated for 15 days.
실시예4. 루시퍼라제(Luciferase) 활성 측정Example 4. Measurement of Luciferase activity
루시퍼라제 활성은 루시퍼라제 검사 시스템(luciferase assay system) (E1500; Promega, Madison, Wisconsin, USA)을 사용해서 분석되었다. 세포들(2 × 106)은 200 × g로 2분동안 원심분리되었고 PBS(10010023; Thermo Fisher Scientific)로 2번 세척되었다. 세포들은 100 μL의 1× 세포 배양 용해 시약(cell culture lysis reagent) 및 100 μL의 PBS (10010023; Thermo Fisher Scientific)에서 와류혼합(verotexed)시켜주었다. 그런 다음, 100 μL의 용해된 샘플은 96-well 플레이트(30396; SPL, Pocheon, Kyunggi, Korea)의 각 well에 형질주입되었고, 100 μL의 루시퍼라제 검사 시약II가 각 well에 추가되었다. 루시퍼라제 활성은 VICTOR multi-label plate reader (2030-0050; PerkinElmer, Waltham, MA, USA)를 사용해서 형광세기를 측정함으로써 구해졌다.Luciferase activity was assayed using the luciferase assay system (E1500; Promega, Madison, Wisconsin, USA). Cells (2×10 6 ) were centrifuged at 200×g for 2 minutes and washed twice with PBS (10010023; Thermo Fisher Scientific). Cells were verotexed in 100 μL of 1× cell culture lysis reagent and 100 μL of PBS (10010023; Thermo Fisher Scientific). Then, 100 μL of the lysed sample was transfected into each well of a 96-well plate (30396; SPL, Pocheon, Kyunggi, Korea), and 100 μL of luciferase test reagent II was added to each well. Luciferase activity was determined by measuring fluorescence intensity using a VICTOR multi-label plate reader (2030-0050; PerkinElmer, Waltham, MA, USA).
실시예5. 형광 측정(Immunoflourescence)Example 5. Immunoflourescence
형광측정을 위해서, 세포들은 세포 배양 슬라이드 4 well (30114; SPL)에 상온에서 15분동안 PBS에 4 % 파라포름알데하이드(paraformaldehyde)로 고정되었고 그런 다음 15분동안 PBS에 0.1 % 트리톤X-100(Triton X-100)으로 투과되도록 처리되었다(permeabilized). 상온에서 1시간동안 PBS에 2.5 % FBS로 차단을 수행하였다. 샘플들은 PBS에 2.5 % FBS 로 희석된 1차 항체와 함께 4 °C 에서 하룻밤동안(overnight) 인큐베이팅되었다. Myc-Tag (71D10) 토끼 단일클론 항체(Rabbit mAb) (2287s, 1:200 희석배율; Cell signaling Technology, Danvers, Massachusetts, USA)와 함께 인큐베이션한 후, 세포들은 얼음정도 차가운 PBS로 3번 세척되었고, 알렉사 플루오르(Alexa Fluor) 594-결합 항-마우스항체(A11005; 1:200 dilution; Invitrogen)로 1시간동안 상온에서 인큐베이팅되었다. 세포핵들은 Hoechst 33342 (H3570; Invitrogen)으로 염색되었고 샘플들은 Dako Fluorescence Mounting 배지 (S3023; Dako)를 사용해서 마운트(mount)되었다. 이미지들은 Carl Zeiss Axio Imager Z1 현미경을 사용해서 캡처되었다.For fluorescence measurement, cells were fixed on 4 wells (30114; SPL) of cell culture slides with 4% paraformaldehyde in PBS for 15 minutes at room temperature and then incubated with 0.1% TritonX-100 (0.1% TritonX-100) in PBS for 15 minutes. Triton X-100) was treated to be permeabilized (permeabilized). Blocking was performed with 2.5% FBS in PBS for 1 hour at room temperature. Samples were incubated overnight at 4 °C with primary antibodies diluted in 2.5% FBS in PBS. After incubation with Myc-Tag (71D10) rabbit monoclonal antibody (Rabbit mAb) (2287s, 1:200 dilution; Cell signaling Technology, Danvers, Massachusetts, USA), cells were washed 3 times with ice-cold PBS and , and incubated with Alexa Fluor 594-binding anti-mouse antibody (A11005; 1:200 dilution; Invitrogen) for 1 hour at room temperature. Cell nuclei were stained with Hoechst 33342 (H3570; Invitrogen) and samples were mounted using Dako Fluorescence Mounting Medium (S3023; Dako). Images were captured using a Carl Zeiss Axio Imager Z1 microscope.
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다. 예를 들어, 단일형으로 설명되어 있는 각 구성 요소는 분산되어 실시될 수도 있으며, 마찬가지로 분산된 것으로 설명되어 있는 구성 요소들도 결합된 형태로 실시될 수 있다.The above description of the present invention is for illustrative purposes, and those skilled in the art can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not limiting. For example, each component described as a single type may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined form.
본 발명의 범위는 후술하는 특허청구범위에 의하여 나타내어지며, 특허청구범위의 의미 및 범위 그리고 그 균등 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.The scope of the present invention is indicated by the following claims, and all changes or modifications derived from the meaning and scope of the claims and equivalent concepts should be interpreted as being included in the scope of the present invention.
<110> INCHEON NATIONAL UNIVERSITY RESEARCH AND BUSINESS FOUNDATION <120> NOVEL TRANSPOSASE AND TRASNPOSON SYSTEM USING THE SAME <130> P22-0004 <160> 11 <170> KoPatentIn 3.0 <210> 1 <211> 7098 <212> DNA <213> Artificial Sequence <220> <223> Tol2 transposase sequence <400> 1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900 gtttaaactt aagcttggta ccgagctcgg atccactagt aacggccgcc agtgtgctgg 960 aattcgccct tgatcagcta gcgccaccat ggaggaagta tgtgattcat cagcagctgc 1020 gagcagcaca gtccaaaatc agccacagga tcaagagcac ccgtggccgt atcttcgcga 1080 attcttttct ttaagtggtg taaataaaga ttcattcaag atgaaatgtg tcctctgtct 1140 cccgcttaat aaagaaatat cggccttcaa aagttcgcca tcaaacctaa ggaagcatat 1200 tgagagaatg cacccaaatt acctcaaaaa ctactctaaa ttgacagcac agaagagaaa 1260 gatcgggacc tccacccatg cttccagcag taagcaactg aaagttgact cagttttccc 1320 agtcaaacat gtgtctccag tcactgtgaa caaagctata ttaaggtaca tcattcaagg 1380 acttcatcct ttcagcactg ttgatctgcc atcatttaaa gagctgatta gtacactgca 1440 gcctggcatt tctgtcatta caaggcctac tttacgctcc aagatagctg aagctgctct 1500 gatcatgaaa cagaaagtga ctgctgccat gagtgaagtt gaatggattg caaccacaac 1560 ggattgttgg actgcacgta gaaagtcatt cattggtgta actgctcact ggatcaaccc 1620 tggaagtctt gaaagacatt ccgctgcact tgcctgcaaa agattaatgg gctctcatac 1680 ttttgaggta ctggccagtg ccatgaatga tatccactca gagtatgaaa tacgtgacaa 1740 ggttgtttgc acaaccacag acagtggttc caactttatg aaggctttca gagtttttgg 1800 tgtggaaaac aatgatatcg agactgaggc aagaaggtgt gaaagtgatg acactgattc 1860 tgaaggctgt ggtgagggaa gtgatggtgt ggaattccaa gatgcctcac gagtcctgga 1920 ccaagacgat ggcttcgaat tccagctacc aaaacatcaa aagtgtgcct gtcacttact 1980 taacctagtc tcaagcgttg atgcccaaaa agctctctca aatgaacact acaagaaact 2040 ctacagatct gtctttggca aatgccaagc tttatggaat aaaagcagcc gatcggctct 2100 agcagctgaa gctgttgaat cagaaagccg gcttcagctt ttaaggccaa accaaacgcg 2160 gtggaattca acttttatgg ctgttgacag aattcttcaa atttgcaaag aagcaggaga 2220 aggcgcactt cggaatatat gcacctctct tgaggttcca atgtttaatc cagcagaaat 2280 gctgttcttg acagagtggg ccaacacaat gcgtccagtt gcaaaagtac tcgacatctt 2340 gcaagcggaa acgaatacac agctggggtg gctgctgcct agtgtccatc agttaagctt 2400 gaaacttcag cgactccacc attctctcag gtactgtgac ccacttgtgg atgccctaca 2460 acaaggaatc caaacacgat tcaagcatat gtttgaagat cctgagatca tagcagctgc 2520 catccttctc cctaaatttc ggacctcttg gacaaatgat gaaaccatca taaaacgagg 2580 catggactac atcagagtgc atctggagcc tttggaccac aagaaggaat tggccaacag 2640 ttcatctgat gatgaagatt ttttcgcttc tttgaaaccg acaacacatg aagccagcaa 2700 agagttggat ggatatctgg cctgtgtttc agacaccagg gagtctctgc tcacgtttcc 2760 tgctatttgc agcctctcta tcaagactaa tacacctctt cccgcatcgg ctgcctgtga 2820 gaggcttttc agcactgcag gattgctttt cagccccaaa agagctaggc ttgacactaa 2880 caattttgag aatcagcttc tactgaagtt aaatctgagg ttttacaact ttgaggggct 2940 cgagtctaga gggcccttcg aaggtaagcc tatccctaac cctctcctcg gtctcgattc 3000 tacgcgtacc ggtcatcatc accatcacca ttgagtttaa acccgctgat cagcctcgac 3060 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct 3120 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct 3180 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg 3240 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag 3300 aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc 3360 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc 3420 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa 3480 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact 3540 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt 3600 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa 3660 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt 3720 aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag 3780 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc 3840 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa 3900 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc 3960 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat 4020 gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt 4080 ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat 4140 cagcacgtgt tgacaattaa tcatcggcat agtatatcgg catagtataa tacgacaagg 4200 tgaggaacta aaccatggcc aagcctttgt ctcaagaaga atccaccctc attgaaagag 4260 caacggctac aatcaacagc atccccatct ctgaagacta cagcgtcgcc agcgcagctc 4320 tctctagcga cggccgcatc ttcactggtg tcaatgtata tcattttact gggggacctt 4380 gtgcagaact cgtggtgctg ggcactgctg ctgctgcggc agctggcaac ctgacttgta 4440 tcgtcgcgat cggaaatgag aacaggggca tcttgagccc ctgcggacgg tgccgacagg 4500 tgcttctcga tctgcatcct gggatcaaag ccatagtgaa ggacagtgat ggacagccga 4560 cggcagttgg gattcgtgaa ttgctgccct ctggttatgt gtgggagggc taagcacttc 4620 gtggccgagg agcaggactg acacgtgcta cgagatttcg attccaccgc cgccttctat 4680 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg 4740 gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac 4800 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt 4860 tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc 4920 tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca 4980 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg 5040 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg 5100 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc 5160 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta 5220 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag 5280 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg 5340 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg 5400 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg 5460 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga 5520 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc 5580 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt 5640 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact 5700 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg 5760 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt 5820 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt 5880 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct 5940 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg 6000 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt 6060 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt 6120 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc 6180 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg 6240 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc 6300 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg 6360 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca 6420 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga 6480 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct 6540 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg 6600 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca 6660 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata 6720 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct 6780 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact 6840 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa 6900 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc 6960 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga 7020 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga 7080 aaagtgccac ctgacgtc 7098 <210> 2 <211> 381 <212> DNA <213> Artificial Sequence <220> <223> Histone H2B sequence <400> 2 atggatatgc ctgaaccagc gaagaccgcg cccaagaagg gctccaagaa agccgtcacc 60 aagaccgccg gtaaaggagg aaagaagcgt aagaggacca ggaaggagag ctacgctatc 120 tacgtgtaca aggttttgag gcaagttcat cccgacaccg gaatctcttc aaaggcgatg 180 ggcatcatga actcgttcgt caacgacatc ttcgagcgca tcgccggtga ggcgtctcgt 240 ctcgctcact acaacaagcg ctccaccatc acttccagag agatccagac cgccgtgcgt 300 ctgctgctac ccggagagtt ggccaaacac gccgtgtctg agggcacaaa ggccgtcacc 360 aagtacacca gctccaagac c 381 <210> 3 <211> 7359 <212> DNA <213> Artificial Sequence <220> <223> expression vector sequence for H2B Tol2 transposase <400> 3 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900 gtttggatcc gccaccatgc caaaaaagaa gagaaaggta atggatatgc ctgaaccagc 960 gaagaccgcg cccaagaagg gctccaagaa agccgtcacc aagaccgccg gtaaaggagg 1020 aaagaagcgt aagaggacca ggaaggagag ctacgctatc tacgtgtaca aggttttgag 1080 gcaagttcat cccgacaccg gaatctcttc aaaggcgatg ggcatcatga actcgttcgt 1140 caacgacatc ttcgagcgca tcgccggtga ggcgtctcgt ctcgctcact acaacaagcg 1200 ctccaccatc acttccagag agatccagac cgccgtgcgt ctgctgctac ccggagagtt 1260 ggccaaacac gccgtgtctg agggcacaaa ggccgtcacc aagtacacca gctccaagac 1320 cggatccatg gaggaagtat gtgattcatc agcagctgcg agcagcacag tccaaaatca 1380 gccacaggat caagagcacc cgtggccgta tcttcgcgaa ttcttttctt taagtggtgt 1440 aaataaagat tcattcaaga tgaaatgtgt cctctgtctc ccgcttaata aagaaatatc 1500 ggccttcaaa agttcgccat caaacctaag gaagcatatt gagagaatgc acccaaatta 1560 cctcaaaaac tactctaaat tgacagcaca gaagagaaag atcgggacct ccacccatgc 1620 ttccagcagt aagcaactga aagttgactc agttttccca gtcaaacatg tgtctccagt 1680 cactgtgaac aaagctatat taaggtacat cattcaagga cttcatcctt tcagcactgt 1740 tgatctgcca tcatttaaag agctgattag tacactgcag cctggcattt ctgtcattac 1800 aaggcctact ttacgctcca agatagctga agctgctctg atcatgaaac agaaagtgac 1860 tgctgccatg agtgaagttg aatggattgc aaccacaacg gattgttgga ctgcacgtag 1920 aaagtcattc attggtgtaa ctgctcactg gatcaaccct ggaagtcttg aaagacattc 1980 cgctgcactt gcctgcaaaa gattaatggg ctctcatact tttgaggtac tggccagtgc 2040 catgaatgat atccactcag agtatgaaat acgtgacaag gttgtttgca caaccacaga 2100 cagtggttcc aactttatga aggctttcag agtttttggt gtggaaaaca atgatatcga 2160 gactgaggca agaaggtgtg aaagtgatga cactgattct gaaggctgtg gtgagggaag 2220 tgatggtgtg gaattccaag atgcctcacg agtcctggac caagacgatg gcttcgaatt 2280 ccagctacca aaacatcaaa agtgtgcctg tcacttactt aacctagtct caagcgttga 2340 tgcccaaaaa gctctctcaa atgaacacta caagaaactc tacagatctg tctttggcaa 2400 atgccaagct ttatggaata aaagcagccg atcggctcta gcagctgaag ctgttgaatc 2460 agaaagccgg cttcagcttt taaggccaaa ccaaacgcgg tggaattcaa cttttatggc 2520 tgttgacaga attcttcaaa tttgcaaaga agcaggagaa ggcgcacttc ggaatatatg 2580 cacctctctt gaggttccaa tgtttaatcc agcagaaatg ctgttcttga cagagtgggc 2640 caacacaatg cgtccagttg caaaagtact cgacatcttg caagcggaaa cgaatacaca 2700 gctggggtgg ctgctgccta gtgtccatca gttaagcttg aaacttcagc gactccacca 2760 ttctctcagg tactgtgacc cacttgtgga tgccctacaa caaggaatcc aaacacgatt 2820 caagcatatg tttgaagatc ctgagatcat agcagctgcc atccttctcc ctaaatttcg 2880 gacctcttgg acaaatgatg aaaccatcat aaaacgaggc atggactaca tcagagtgca 2940 tctggagcct ttggaccaca agaaggaatt ggccaacagt tcatctgatg atgaagattt 3000 tttcgcttct ttgaaaccga caacacatga agccagcaaa gagttggatg gatatctggc 3060 ctgtgtttca gacaccaggg agtctctgct cacgtttcct gctatttgca gcctctctat 3120 caagactaat acacctcttc ccgcatcggc tgcctgtgag aggcttttca gcactgcagg 3180 attgcttttc agccccaaaa gagctaggct tgacactaac aattttgaga atcagcttct 3240 actgaagtta aatctgaggt tttacaactt tgagcatcat caccatcacc attgagttta 3300 aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc 3360 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga 3420 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca 3480 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc 3540 tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg 3600 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc 3660 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg 3720 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg 3780 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg 3840 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt 3900 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt 3960 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaatta 4020 attctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga 4080 agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc 4140 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc 4200 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc 4260 tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag 4320 aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt 4380 atatccattt tcggatctga tcagcacgtg ttgacaatta atcatcggca tagtatatcg 4440 gcatagtata atacgacaag gtgaggaact aaaccatggc caagcctttg tctcaagaag 4500 aatccaccct cattgaaaga gcaacggcta caatcaacag catccccatc tctgaagact 4560 acagcgtcgc cagcgcagct ctctctagcg acggccgcat cttcactggt gtcaatgtat 4620 atcattttac tgggggacct tgtgcagaac tcgtggtgct gggcactgct gctgctgcgg 4680 cagctggcaa cctgacttgt atcgtcgcga tcggaaatga gaacaggggc atcttgagcc 4740 cctgcggacg gtgccgacag gtgcttctcg atctgcatcc tgggatcaaa gccatagtga 4800 aggacagtga tggacagccg acggcagttg ggattcgtga attgctgccc tctggttatg 4860 tgtgggaggg ctaagcactt cgtggccgag gagcaggact gacacgtgct acgagatttc 4920 gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc 4980 tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt 5040 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca 5100 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc 5160 tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg 5220 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa 5280 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct 5340 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga 5400 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc 5460 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa 5520 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt 5580 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa 5640 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt 5700 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg 5760 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc 5820 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc 5880 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta 5940 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct 6000 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc 6060 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa 6120 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa 6180 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa 6240 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt 6300 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac 6360 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc 6420 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc 6480 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata 6540 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc 6600 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc 6660 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca 6720 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa 6780 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca 6840 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt 6900 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt 6960 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg 7020 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga 7080 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc 7140 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg 7200 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag 7260 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg 7320 gttccgcgca catttccccg aaaagtgcca cctgacgtc 7359 <210> 4 <211> 2334 <212> DNA <213> Artificial Sequence <220> <223> H2B Tol2 transposase sequence <400> 4 atggatatgc ctgaaccagc gaagaccgcg cccaagaagg gctccaagaa agccgtcacc 60 aagaccgccg gtaaaggagg aaagaagcgt aagaggacca ggaaggagag ctacgctatc 120 tacgtgtaca aggttttgag gcaagttcat cccgacaccg gaatctcttc aaaggcgatg 180 ggcatcatga actcgttcgt caacgacatc ttcgagcgca tcgccggtga ggcgtctcgt 240 ctcgctcact acaacaagcg ctccaccatc acttccagag agatccagac cgccgtgcgt 300 ctgctgctac ccggagagtt ggccaaacac gccgtgtctg agggcacaaa ggccgtcacc 360 aagtacacca gctccaagac cggatccatg gaggaagtat gtgattcatc agcagctgcg 420 agcagcacag tccaaaatca gccacaggat caagagcacc cgtggccgta tcttcgcgaa 480 ttcttttctt taagtggtgt aaataaagat tcattcaaga tgaaatgtgt cctctgtctc 540 ccgcttaata aagaaatatc ggccttcaaa agttcgccat caaacctaag gaagcatatt 600 gagagaatgc acccaaatta cctcaaaaac tactctaaat tgacagcaca gaagagaaag 660 atcgggacct ccacccatgc ttccagcagt aagcaactga aagttgactc agttttccca 720 gtcaaacatg tgtctccagt cactgtgaac aaagctatat taaggtacat cattcaagga 780 cttcatcctt tcagcactgt tgatctgcca tcatttaaag agctgattag tacactgcag 840 cctggcattt ctgtcattac aaggcctact ttacgctcca agatagctga agctgctctg 900 atcatgaaac agaaagtgac tgctgccatg agtgaagttg aatggattgc aaccacaacg 960 gattgttgga ctgcacgtag aaagtcattc attggtgtaa ctgctcactg gatcaaccct 1020 ggaagtcttg aaagacattc cgctgcactt gcctgcaaaa gattaatggg ctctcatact 1080 tttgaggtac tggccagtgc catgaatgat atccactcag agtatgaaat acgtgacaag 1140 gttgtttgca caaccacaga cagtggttcc aactttatga aggctttcag agtttttggt 1200 gtggaaaaca atgatatcga gactgaggca agaaggtgtg aaagtgatga cactgattct 1260 gaaggctgtg gtgagggaag tgatggtgtg gaattccaag atgcctcacg agtcctggac 1320 caagacgatg gcttcgaatt ccagctacca aaacatcaaa agtgtgcctg tcacttactt 1380 aacctagtct caagcgttga tgcccaaaaa gctctctcaa atgaacacta caagaaactc 1440 tacagatctg tctttggcaa atgccaagct ttatggaata aaagcagccg atcggctcta 1500 gcagctgaag ctgttgaatc agaaagccgg cttcagcttt taaggccaaa ccaaacgcgg 1560 tggaattcaa cttttatggc tgttgacaga attcttcaaa tttgcaaaga agcaggagaa 1620 ggcgcacttc ggaatatatg cacctctctt gaggttccaa tgtttaatcc agcagaaatg 1680 ctgttcttga cagagtgggc caacacaatg cgtccagttg caaaagtact cgacatcttg 1740 caagcggaaa cgaatacaca gctggggtgg ctgctgccta gtgtccatca gttaagcttg 1800 aaacttcagc gactccacca ttctctcagg tactgtgacc cacttgtgga tgccctacaa 1860 caaggaatcc aaacacgatt caagcatatg tttgaagatc ctgagatcat agcagctgcc 1920 atccttctcc ctaaatttcg gacctcttgg acaaatgatg aaaccatcat aaaacgaggc 1980 atggactaca tcagagtgca tctggagcct ttggaccaca agaaggaatt ggccaacagt 2040 tcatctgatg atgaagattt tttcgcttct ttgaaaccga caacacatga agccagcaaa 2100 gagttggatg gatatctggc ctgtgtttca gacaccaggg agtctctgct cacgtttcct 2160 gctatttgca gcctctctat caagactaat acacctcttc ccgcatcggc tgcctgtgag 2220 aggcttttca gcactgcagg attgcttttc agccccaaaa gagctaggct tgacactaac 2280 aattttgaga atcagcttct actgaagtta aatctgaggt tttacaactt tgag 2334 <210> 5 <211> 9272 <212> DNA <213> Artificial Sequence <220> <223> Tol2 TP (transposon vector) sequence <400> 5 aaaagtgcca cctgacgtcg acggatcggg agatctcccg atcccctatg gtgcactctc 60 agtacaatct gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg 120 gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa caaggcaagg cttgaccgac 180 aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgttt tgcgctgctt 240 cgcgatctgc gaagatacgg ccacgggtgc tcttgatcct gtggctgatt ttggactgtg 300 ctgctcgcag ctgctgatga atcacatact tcctccattt tcttccactg attgactgtt 360 ataatttccc taatttccag gtcaaggtgc tgtgcattgt ggtaatagat gtgacatgac 420 gtcacttcca aaggaccaat gaacatgtct gaccaatttc atataatgtg aaaacgattt 480 tcataggcag aataaataac atttaaatta aactgggcat cagcgcaatt caattggttt 540 ggtaatagca agggaaaata gaatgaagtg atctccaaaa aataagtact ttttgactgt 600 aaataaaatt gtaaggagta aaaagtactt ttttttctaa aaaaatgtaa ttaagtaaaa 660 gtaaaagtat tgatttttaa ttgtactcaa gtaaagtaaa aatccccaaa aataatactt 720 aagtacagta atcaagtaaa attactcaag tactttacac ctctggttct tgacccccta 780 ccttcattgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct 840 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat 900 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc 960 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg 1020 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg 1080 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga 1140 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta 1200 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc 1260 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc 1320 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg 1380 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc 1440 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca 1500 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct 1560 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat 1620 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg 1680 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat 1740 taagcattgg taactgtcag accaacccat atccactctg ttccacacag gtcagaggtt 1800 tgtccaggag ttcttgacag aggtgtaaaa agtactcaaa aattttactc aagtgaaagt 1860 acaagtactt agggaaaatt ttactcaatt aaaagtaaaa gtatctggct agaatcttac 1920 ttgagtaaaa gtaaaaaagt actccattaa aattgtactt gagtattaag gaagtaaaag 1980 taaaagcaag aaagaaaact agagattctt gtttaagctt ttaatctcaa aaaacattaa 2040 atgaaatgca tacaaggttt tatcctgctt tagaactgtt tgtatttaat tatcaaacta 2100 taagacagac aatctaatgc cagtacacgc tactcaaagt tgtaaaacct cagatttaac 2160 ttcagtagaa gctgattctc aaaattgtta gtgtcaagcc tagctctttt ggggctgaaa 2220 agcaatcctg cagtgctgaa aagcctctca caggcagccg atgcgggaag aggtgtatta 2280 gtcttgatag agaggctgca aatagcagga aacgtgagca gagactccct ggtgtctgaa 2340 acacaggcca gatgggcctc gcgatgtacg ggccagcgat gtacgggcca gatatacgcg 2400 ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag 2460 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc 2520 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg 2580 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca 2640 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc 2700 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt 2760 attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata 2820 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt 2880 ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca 2940 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctctct ggctaactag 3000 agaacccact gcttactggc ttatcgaaat taatacgact cactataggg agacccaagc 3060 tggctagcgc caccatggag tttgggctga gctgggtttt cctcgttgct ctttttagag 3120 gtgtccagtg tatggaagac gccaaaaaca taaagaaagg cccggcgcca ttctatccgc 3180 tggaagatgg aaccgctgga gagcaactgc ataaggctat gaagagatac gccctggttc 3240 ctggaacaat tgcttttaca gatgcacata tcgaggtgga catcacttac gctgagtact 3300 tcgaaatgtc cgttcggttg gcagaagcta tgaaacgata tgggctgaat acaaatcaca 3360 gaatcgtcgt atgcagtgaa aactctcttc aattctttat gccggtgttg ggcgcgttat 3420 ttatcggagt tgcagttgcg cccgcgaacg acatttataa tgaacgtgaa ttgctcaaca 3480 gtatgggcat ttcgcagcct accgtggtgt tcgtttccaa aaaggggttg caaaaaattt 3540 tgaacgtgca aaaaaagctc ccaatcatcc aaaaaattat tatcatggat tctaaaacgg 3600 attaccaggg atttcagtcg atgtacacgt tcgtcacatc tcatctacct cccggtttta 3660 atgaatacga ttttgtgcca gagtccttcg atagggacaa gacaattgca ctgatcatga 3720 actcctctgg atctactggt ctgcctaaag gtgtcgctct gcctcataga actgcctgcg 3780 tgagattctc gcatgccaga gatcctattt ttggcaatca aatcattccg gatactgcga 3840 ttttaagtgt tgttccattc catcacggtt ttggaatgtt tactacactc ggatatttga 3900 tatgtggatt tcgagtcgtc ttaatgtata gatttgaaga agagctgttt ctgaggagcc 3960 ttcaggatta caagattcaa agtgcgctgc tggtgccaac cctattctcc ttcttcgcca 4020 aaagcactct gattgacaaa tacgatttat ctaatttaca cgaaattgct tctggtggcg 4080 ctcccctctc taaggaagtc ggggaagcgg ttgccaagag gttccatctg ccaggtatca 4140 ggcaaggata tgggctcact gagactacat cagctattct gattacaccc gagggggatg 4200 ataaaccggg cgcggtcggt aaagttgttc cattttttga agcgaaggtt gtggatctgg 4260 ataccgggaa aacgctgggc gttaatcaaa gaggcgaact gtgtgtgaga ggtcctatga 4320 ttatgtccgg ttatgtaaac aatccggaag cgaccaacgc cttgattgac aaggatggat 4380 ggctacattc tggagacata gcttactggg acgaagacga acacttcttc atcgttgacc 4440 gcctgaagtc tctgattaag tacaaaggct atcaggtggc tcccgctgaa ttggaatcca 4500 tcttgctcca acaccccaac atcttcgacg caggtgtcgc aggtcttccc gacgatgacg 4560 ccggtgaact tcccgccgcc gttgttgttt tggagcacgg aaagacgatg acggaaaaag 4620 agatcgtgga ttacgtcgcc agtcaagtaa caaccgcgaa aaagttgcgc ggaggagttg 4680 tgtttgtgga cgaagtaccg aaaggtctta ccggaaaact cgacgcaaga aaaatcagag 4740 agatcctcat aaaggccaag aagggcggaa agatcgccgt gtaactcgag cggccgccac 4800 tgtgctggat atctgcagaa ttccaccaca ctggactagt ggatccgagc tcggtaccaa 4860 gcttaagttt aaaccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg 4920 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct 4980 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg 5040 gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg 5100 cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc 5160 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg 5220 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca 5280 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta 5340 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc 5400 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg 5460 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat 5520 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta 5580 acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc 5640 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc 5700 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat 5760 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc 5820 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga 5880 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc 5940 gggagcttgt atatccattt tcggatctga tcaagagaca ggatgaggat cgtttcgcat 6000 gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg 6060 ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc 6120 gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca 6180 ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct 6240 cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga 6300 tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg 6360 gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat 6420 cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga 6480 gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg 6540 cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg 6600 ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat 6660 agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct 6720 cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga 6780 cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg 6840 ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt 6900 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc 6960 caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat 7020 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat 7080 gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca 7140 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga 7200 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg 7260 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc 7320 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac 7380 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata 7440 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa 7500 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct 7560 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa 7620 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg 7680 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca 7740 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa 7800 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg 7860 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg 7920 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga 7980 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc 8040 tcttgatccg gcaaacaaac caccgctggt agcggttttt ttgtttgcaa gcagcagatt 8100 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct 8160 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc 8220 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa 8280 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta 8340 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc 8400 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat 8460 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta 8520 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt 8580 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt 8640 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg 8700 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc 8760 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc 8820 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg 8880 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga 8940 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta 9000 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct 9060 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag 9120 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga 9180 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat 9240 aaacaaatag gggttccgcg cacatttccc cg 9272 <210> 6 <211> 592 <212> DNA <213> Artificial Sequence <220> <223> Tol2 Right ITR sequence <400> 6 acccatatcc actctgttcc acacaggtca gaggtttgtc caggagttct tgacagaggt 60 gtaaaaagta ctcaaaaatt ttactcaagt gaaagtacaa gtacttaggg aaaattttac 120 tcaattaaaa gtaaaagtat ctggctagaa tcttacttga gtaaaagtaa aaaagtactc 180 cattaaaatt gtacttgagt attaaggaag taaaagtaaa agcaagaaag aaaactagag 240 attcttgttt aagcttttaa tctcaaaaaa cattaaatga aatgcataca aggttttatc 300 ctgctttaga actgtttgta tttaattatc aaactataag acagacaatc taatgccagt 360 acacgctact caaagttgta aaacctcaga tttaacttca gtagaagctg attctcaaaa 420 ttgttagtgt caagcctagc tcttttgggg ctgaaaagca atcctgcagt gctgaaaagc 480 ctctcacagg cagccgatgc gggaagaggt gtattagtct tgatagagag gctgcaaata 540 gcaggaaacg tgagcagaga ctccctggtg tctgaaacac aggccagatg gg 592 <210> 7 <211> 539 <212> DNA <213> Artificial Sequence <220> <223> Tol2 Left ITR sequence <400> 7 tgcgaagata cggccacggg tgctcttgat cctgtggctg attttggact gtgctgctcg 60 cagctgctga tgaatcacat acttcctcca ttttcttcca ctgattgact gttataattt 120 ccctaatttc caggtcaagg tgctgtgcat tgtggtaata gatgtgacat gacgtcactt 180 ccaaaggacc aatgaacatg tctgaccaat ttcatataat gtgaaaacga ttttcatagg 240 cagaataaat aacatttaaa ttaaactggg catcagcgca attcaattgg tttggtaata 300 gcaagggaaa atagaatgaa gtgatctcca aaaaataagt actttttgac tgtaaataaa 360 attgtaagga gtaaaaagta cttttttttc taaaaaaatg taattaagta aaagtaaaag 420 tattgatttt taattgtact caagtaaagt aaaaatcccc aaaaataata cttaagtaca 480 gtaatcaagt aaaattactc aagtacttta cacctctggt tcttgacccc ctaccttca 539 <210> 8 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> forward primer sequence for Importin alpha <400> 8 gctctacaaa accat 15 <210> 9 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> reverse primer sequence for Importin alpha <400> 9 ccagatcctc ttctg 15 <210> 10 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> forward primer sequence for Importin beta <400> 10 cagcatttgg gaagg 15 <210> 11 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> reverse primer sequence for Importin beta <400> 11 tgccagatcc tcttc 15 <110> INCHEON NATIONAL UNIVERSITY RESEARCH AND BUSINESS FOUNDATION <120> NOVEL TRANSPOSASE AND TRASNPOSON SYSTEM USING THE SAME <130> P22-0004 <160> 11 <170> KoPatentIn 3.0 <210> 1 <211> 7098 <212> DNA <213 > Artificial Sequence <220> <223> Tol2 transposase sequence <400> 1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggttt g 660 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840 ctgcttactg gctttcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900 gtttaaactt aagcttggta ccgagctcgg atccactagt aacggccgcc agtgtgctgg 960 aattcgccct tgatcagcta gcgccaccat ggaggaagta tgtgattcat cagcagctgc 1020 gagcagcaca gtccaaaatc agccacagga tcaagagcac ccgtggccgt atct tcgcga 1080 attcttttct ttaagtggtg taaataaaga ttcattcaag atgaaatgg tcctctgtct 1140 cccgcttaat aaagaaatat cggccttcaa aagttcgcca tcaaacctaa ggaagcatat 1200 tgagagaatg cacccaaatt acctcaaaaa ctactctaaa ttgacagcac agaagagaaa 1260 gatcgggacc tccacccatg cttccagcag taagcaactg aaagttgact cagttt tccc 1320 agtcaaacat gtgtctccag tcactgtgaa caaagctata ttaaggtaca tcattcaagg 1380 acttcatcct ttcagcactg ttgatctgcc atcatttaaa gagctgatta gtacactgca 1440 gcctggcatt tctgtcatta caaggcctac tttacgctcc aagatagctg aagctgctct 1500 gatcatgaaa cagaaagtga ctgctgccat gagtgaagtt gaatggattg caaccacaac 1560 ggattgttgg actgcacgta gaaagtcatt cattggtgta actgctcact ggatcaaccc 1620 tggaagtctt gaaagacatt ccgctgcact tgcctgcaaa agattaatgg gctctcatac 1680 ttttgaggta ctggccagtg ccatgaatga tatccactca gagtatgaaa tacgtgacaa 1740 ggttgt ttgc acaaccag acagtggttc caactttatg aaggctttca gagtttttgg 1800 tgtggaaaac aatgatatcg agactgaggc aagaaggtgt gaaagtgatg acactgattc 1860 tgaaggctgt ggtgagggaa gtgatggtgt ggaattccaa gatgcctcac ga gtcctgga 1920 ccaagacgat ggcttcgaat tccagctacc aaaacatcaa aagtgtgcct gtcacttact 1980 taacctagtc tcaagcgttg atgcccaaaa agctctctca aatgaacact acaagaaact 2040 ctacagatct gtctttggca aatgccaagc tttatggaat aaaagcagcc gatcggctct 2100 agcagctgaa gctgttgaat cagaaagccg gcttcagctt ttaaggccaa accaaacgcg 2160 gtggaattca acttttatgg ctgttgacag aattcttcaa atttgcaaag aagcaggaga 2220 aggcgcactt cggaatatat gcacctctct tgaggttcca atgtttaatc cagcagaaat 2280 gctgttcttg acagagtggg ccaacacaat gcgtccagtt gcaaaagtac tcgacatctt 2340 gcaagcggaa acgaatacac agctggggtg gctgctgcct agtgtccatc agttaagctt 2400 gaaacttcag cgactccacc attctctcag gtactgtgac ccacttgtgg atgccctaca 2460 acaaggaatc caaacacgat tcaagcatat gtttgaagat cctgagatca tagcagctgc 2520 catccttctc cctaaatttc ggacctcttg gacaaatgat gaaaccatca taaaacgagg 2580 catggactac atcagagtgc atctggagcc tttg gaccac aagaaggaat tggccaacag 2640 ttcatctgat gatgaagatt ttttcgcttc tttgaaaccg acaacacatg aagccagcaa 2700 agagttggat ggatatctgg cctgtgtttc agacaccagg gagtctctgc tcacgtttcc 2760 tgctatttgc agcc tctcta tcaagactaa tacacctctt cccgcatcgg ctgcctgtga 2820 gaggcttttc agcactgcag gattgctttt cagccccaaa agagctaggc ttgacactaa 2880 caattttgag aatcagcttc tactgaagtt aaatctgagg ttttacaact ttgaggggct 2940 cgagtctaga gggcccttcg aaggtaagcc tatccctaac cctctcctcg gtctcgattc 3000 tacgcgtacc ggtcatcatc accatcacca tt gagtttaa acccgctgat cagcctcgac 3060 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct 3120 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct 3180 gagtaggtgt cattctattc t ggggggtgg ggtggggcag gacagcaagg gggaggattg 3240 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag 3300 aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc 3360 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc 3420 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa 3480 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact 3540 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt 3600 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa 3660 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt 3720 aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag 3780 ttagggtgg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc 3840 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggca g aagtatgcaa 3900 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc 3960 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat 4020 gcagaggccg aggccgcctc tgcctctgag ctattccaga a gtagtgagg aggctttttt 4080 ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat 4140 cagcacgtgt tgacaattaa tcatcggcat agtatatcgg catagtataa tacgacaagg 4200 tgaggaacta aaccatggcc aagcctttgt ctcaagaaga atccaccctc attgaaagag 4260 caacggctac aatcaacagc atccccatct ctgaagacta cagcgtcgcc agcgcagctc 4320 tct ctagcga cggccgcatc ttcactggtg tcaatgtata tcattttact gggggacctt 4380 gtgcagaact cgtggtgctg ggcactgctg ctgctgcggc agctggcaac ctgacttgta 4440 tcgtcgcgat cggaaatgag aacaggggca tcttgagccc ctgcggacgg tgccgacagg 4500 tgcttctcga tctgcatcct gggatcaaag ccatagtgaa ggacagtgat ggacagccga 4560 cggcagttgg gattcgtgaa ttgctgccct ctggttatgt gtgggagggc taagcacttc 4620 gtggccgagg agcaggactg acacgtgcta cgagatttcg attccaccgc cgccttctat 4680 gAAaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg 474 0 gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac 4800 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt 4860 tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc 4920 tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca 4980 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg 5040 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg 5100 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc 5160 t cttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta 5220 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag 5280 aacatgtgag caaaaggcca gcaaaaggcc agggaaccgta a aaaggccgc gttgctggcg 5340 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg 5400 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg 5460 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga 5520 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc 5580 tccaagctgg g ctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt 5640 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact 5700 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg 57 60 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt 5820 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt 5880 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct 5940 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta aggattttg 6000 gtcatgagat tatcaaaa ag gatcttcacc tagatccttt taaattaaaa atgaagtttt 6060 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt 6120 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc 6180 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg 6240 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc 6300 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg 6360 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca 6420 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tca gctccgg ttcccaacga 6480 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct 6540 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg 6600 cataattctc ttactgtcat gc catccgta agatgctttt ctgtgactgg tgagtactca 6660 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata 6720 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct 6780 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact 6840 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagca t tccgcgcac atttccccga 7080 aaagtgccac ctgacgtc 7098 <210> 2 < 211> 381 <212> DNA <213> Artificial Sequence <220> <223> Histone H2B sequence <400> 2 atggatatgc ctgaaccagc gaagaccgcg cccaagaagg gctccaagaa agccgtcacc 60 aagaccgccg gtaaaggagg aaagaagcgt aagaggacca ggaaggagg ctacgct atc 120 tacgtgtaca aggttttgag gcaagttcat cccgacaccg gaatctcttc aaaggcgatg 180 ggcatcatga actcgttcgt caacgacatc ttcgagcgca tcgccggtga ggcgtctcgt 240 ctcgctcact acaacaagcg ctccaccatc acttccagag agatccagac cgccgtgcgt 300 ctgctgctac ccggagagtt ggccaaacac gccgtgtctg agggcacaaa ggccgtcacc 360 aagtacacca gctccaagac c 381 <2 10> 3 <211> 7359 <212> DNA <213> Artificial Sequence <220> <223> expression vector sequence for H2B Tol2 transposase <400> 3 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga ca attgcatg aagaatctgc 180 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300 tggagttccg cgttacataa cttacggtaa atggcccgcc tggct gaccg cccaacgacc 360 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720 aaaatcaacg ggacttt cca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900 gtttggatcc gccaccatgc caaaaaagaa gagaaaggta atggatatgc ctgaaccagc 960 gaagaccgcg cccaagaagg gctccaagaa ag ccgtcacc aagaccgccg gtaaaggagg 1020 aaagaagcgt aagaggacca ggaaggagag ctacgctatc tacgtgtaca aggttttgag 1080 gcaagttcat cccgacaccg gaatctcttc aaaggcgatg ggcatcatga actcgttcgt 1140 caacgacatc ttcgagcgca t cgccggtga ggcgtctcgt ctcgctcact acaacaagcg 1200 ctccaccatc acttccagag agatccagac cgccgtgcgt ctgctgctac ccggagagtt 1260 ggccaaacac gccgtgtctg agggcacaaa ggccgtcacc aagtacacca gctccaagac 1320 cggatccatg gaggaagtat gtgattcatc agcagctgcg agcagcacag tccaaaatca 1380 gccacaggat caagagcacc cgtggccgta tcttc gcgaa ttcttttctt taagtggtgt 1440 aaataaagat tcattcaaga tgaaatgtgt cctctgtctc ccgcttaata aagaaatatc 1500 ggccttcaaa agttcgccat caaacctaag gaagcatatt gagagaatgc acccaaatta 1560 cctcaaaaac tactctaaat tgacagcaca gaagagaaag atcgggacct ccacccatgc 1620 ttccagcagt aagcaactga aagttgactc agttttccca gtcaaacatg tgtctccagt 1680 cactgtgaac aaagctatat taaggtacat cattcaagga cttcatcctt tcagcactgt 1740 tgatctgcca tcatttaaag agctgattag tacactgcag cctggcattt ctgtcattac 1800 aaggcctact ttacgctcca agatagctga agctgctctg atcatgaaac agaaag tgac 1860 tgctgccatg agtgaagttg aatggattgc aaccacaacg gattgttgga ctgcacgtag 1920 aaagtcattc attggtgtaa ctgctcactg gatcaaccct ggaagtcttg aaagacattc 1980 cgctgcactt gcctgcaaaa gattaatggg ctctcat act tttgaggtac tggccagtgc 2040 catgaatgat atccactcag agtatgaaat acgtgacaag gttgtttgca caaccacaga 2100 cagtggttcc aactttatga aggctttcag agtttttggt gtgggaaaca atgatatcga 2160 gactgaggca agaaggtgg aaagtgatga cactgattct gaaggctgtg gtgagggaag 2220 tgatggtgtg gaattccaag atgcctcacg agtcctggac caagacgatg gct tcgaatt 2280 ccagctacca aaacatcaaa agtgtgcctg tcacttactt aacctagtct caagcgttga 2340 tgcccaaaaa gctctctcaa atgaacacta caagaaactc tacagatctg tctttggcaa 2400 atgccaagct ttatggaata aaagcagccg atcggctc ta gcagctgaag ctgttgaatc 2460 agaaagccgg cttcagcttt taaggccaaa ccaaacgcgg tggaattcaa cttttatggc 2520 tgttgacaga attcttcaaa tttgcaaaga agcaggagaa ggcgcacttc ggaatatatg 2580 cacctctctt gaggttccaa tgtttaatcc agcagaaatg ctgttcttga cagagtgggc 2640 caacacaatg cgtccagttg caaaagtact cgacatcttg caagcggaaa cgaata caca 2700 gctggggtgg ctgctgccta gtgtccatca gttaagcttg aaacttcagc gactccacca 2760 ttctctcagg tactgtgacc cacttgtgga tgccctacaa caaggaatcc aaacacgatt 2820 caagcatatg tttgaagatc ctgagatcat agcagctgcc atcctt ctcc ctaaatttcg 2880 gacctcttgg acaaatgatg aaaccatcat aaaacgaggc atggactaca tcagagtgca 2940 tctggagcct ttggaccaca agaaggaatt ggccaacagt tcatctgatg atgaagattt 3000 tttcgcttct ttgaaaccga caacacatga agccagcaaa gagttggatg gatatctggc 3060 ctgtgtttca gacaccaggg agtctctgct cacgtttcct gctatttgca gcctctctat 3120 caagacta at acacctcttc ccgcatcggc tgcctgtgag aggcttttca gcactgcagg 3180 attgcttttc agccccaaaa gagctaggct tgacactaac aattttgaga atcagcttct 3240 actgaagtta aatctgaggt tttacaactt tgagcatcat caccatcacc attgagttta 330 0 aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc 3360 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga 3420 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca 3480 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc 3540 tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg 3600 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc 3660 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgcc gg 3720 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg 3780 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg 3840 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt 3900 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt 3960 gccgatttcg gcctattggt taaa aaatga gctgatttaa caaaaattta acgcgaatta 4020 attctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga 4080 agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc 4140 ccagcaggca gaagtatg ca aagcatgcat ctcaattagt cagcaaccat agtcccgccc 4200 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gcccccatggc 4260 tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag 4320 aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt 4380 atatccattt tcggatctga tcagcacgt g ttgacaatta atcatcggca tagtatatcg 4440 gcatagtata atacgacaag gtgaggaact aaaccatggc caagcctttg tctcaagaag 4500 aatccaccct cattgaaaga gcaacggcta caatcaacag catccccatc tctgaagact 4560 acagcgtcgc cagcgcagct ctctctagc g acggccgcat cttcactggt gtcaatgtat 4620 atcattttac tgggggacct tgtgcagaac tcgtggtgct gggcactgct gctgctgcgg 4680 cagctggcaa cctgacttgt atcgtcgcga tcggaaatga gaacaggggc atcttgagcc 4740 cctgcggacg gtgccgacag gtgcttctcg atctgcatcc tgggatcaaa gccatagtga 4800 aggacagtga tggacagccg acggcagttg ggattcgtga attgct gccc tctggttatg 4860 tgtgggaggg ctaagcactt cgtggccgag gagcaggact gacacgtgct acgagatttc 4920 gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc 4980 tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc caacttgttt 5040 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca 5100 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc 5160 tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg 5220 tgaaattgtt atccgctcac aattccacac aacatacgag ccgga ctct tccgct tcctcgctca ctgactcgct gcgctcggtc 5460 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa 5520 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt 5580 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa 5640 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgt tt 5700 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg 5760 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc 5820 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc 5880 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta 5940 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct 6000 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc 60 tct acggggt ctgacgctca gtggaacgaa 6240 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt 6300 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac 6360 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc 6420 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc 6480 ccca gtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata 6540 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc 6600 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc 6 660 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca 6720 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa 6780 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca 6840 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt 6900 tctgtg actg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt 6960 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg 7020 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga 7080 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc 7140 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg 7200 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag 7260 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg 7320 gttccgcgca catttcc ccg aaaagtgcca cctgacgtc 7359 <210> 4 <211> 2334 <212> DNA <213> Artificial Sequence <220> <223> H2B Tol2 transposase sequence <400> 4 atggatatgc ctgaaccagc gaagaccgcg cccaagaagg gctccaagaa agccgtcacc 60 aagaccgccg gtaaaggagg aaagaagcgt aagaggacca ggaaggagag ctacgctatc 120 tacgtgtaca aggttttgag gcaagttcat cccgacaccg gaatctcttc aa aggcgatg a gggcacaaa ggccgtcacc 360 aagtacacca gctccaagac cggatccatg gaggaagtat ggcc ttcaaa agttcgccat caaacctaag gaagcatatt 600 gagagaatgc acccaaatta cctcaaaaac tactctaaat tgacagcaca gaagagaaag 660 atcgggacct ccacccatgc ttccagcagt aagcaactga aagttgactc agttttccca 720 gtcaaacatg tgtct ccagt cactgtgaac aaagctatat taaggtacat cattcaagga 780 cttcatcctt tcagcactgt tgatctgcca tcatttaaag agctgattag tacactgcag 840 cctggcattt ctgtcattac aaggcctact ttacgctcca agatagctga agctgctctg 900 atcatgaaac agaaagtgac tgctgccatg agtgaagttg aatggattgc aaccacaacg 960 gattgttgga ctgcacgtag aaagtcattc attggtgtaa ctgctcactg gatcaaccct 1020 ggaagtcttg aaagacattc cgctgcactt gcctgcaaaa gattaatggg ctctcatact 1080 tttgaggtac tggccagtgc catgaatgat atccactcag agtatgaaat acgtgacaag 1140 gttgtttgca caaccacaga cagtggtt cc aactttatga aggctttcag agtttttggt 1200 gtggaaaaca atgatatcga gactgaggca agaaggtggg aaagtgatga cactgattct 1260 gaaggctgtg gtgagggaag tgatggtgtg gaattccaag atgcctcacg agtcctggac 1320 caagacgatg gcttcgaatt ccagctacca aaacatcaaa agtgtgcctg tcacttactt 1380 aacctagtct caagcgttga tgcccaaaaa gctctctca a atgaacacta caagaaactc 1440 taacagatctg tctttggcaa atgccaagct ttatggaata aaagcagccg atcggctcta 1500 gcagctgaag ctgttgaatc agaaagccgg cttcagcttt taaggccaaa ccaaacgcgg 1560 tggaattcaa cttttatggc t gttgacaga attcttcaaa tttgcaaaga agcaggagaa 1620 ggcgcacttc ggaatatatg cacctctctt gaggttccaa tgtttaatcc agcagaaatg 1680 ctgttcttga cagagtgggc caacacaatg cgtccagttg caaaagtact cgacatcttg 1740 caagcggaaa cgaatacaca gctggggtgg ctgctgccta gtgtccatca gttaagcttg 1800 aaacttcagc gactccacca ttctctcagg tactgtgacc cacttgtgga tgcc ctacaa 1860 caaggaatcc aaacacgatt caagcatatg tttgaagatc ctgagatcat agcagctgcc 1920 atccttctcc ctaaatttcg gacctcttgg acaaatgatg aaaccatcat aaaacgaggc 1980 atggactaca tcagagtgca tctggagcct ttggaccaca agaag gaatt ggccaacagt 2040 tcatctgatg atgaagattt tttcgcttct ttgaaaccga caacacatga agccagcaaa 2100 gagttggatg gatatctggc ctgtgtttca gacaccaggg agtctctgct cacgtttcct 2160 gctatttgca gcctctctat caagactaat acacctcttc ccgcatcggc tgcctgtgag 2220 aggcttttca gcactgcagg attgcttttc agccccaaaa gagctaggct tgacacta ac 2280 aattttgaga atcagcttct actgaagtta aatctgaggt tttacaactt tgag 2334 <210> 5 <211> 9272 <212> DNA <213> Artificial Sequence <220> <223> Tol2 TP (transposon vector) sequence <400> 5 aaaagtgcca cctgacgtcg acggatcggg agatctcccg atcccctatg gtgcactctc 60 agtacaatct gctctgatgc cgcatagtta agccagtatc tgctccctgc ttgtgtgttg 120 gaggtcgctg agtagtgcg c gagcaaaatt taagctacaa caaggcaagg cttgaccgac 180 aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgttt tgcgctgctt 240 cgcgatctgc gaagatacgg ccacgggtgc tcttgatcct gtggctgatt ttggactgtg 300 ctgctcgcag ctgctgatga atcacatact tcctccattt cag cgcaatt caattggttt 540 ggtaatagca agggaaaata gaatgaagtg atctccaaaa aataagtact ttttgactgt 600 aaataaaatt gtaaggagta aaaagtactt ttttttctaa aaaaatgtaa ttaagtaaaa 660 gtaaaagtat tgatttttaa tt gtactcaa gtaaagtaaa aatccccaaa aataatactt 720 aagtacagta atcaagtaaa attactcaag tactttacac ctctggttct tgacccccta 780 ccttcattgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct 840 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat 900 tcaacatttc cgtgtcgccc ttattccctt ttttg cggca ttttgccttc ctgtttttgc 960 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg 1020 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg 1080 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga 1140 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta 1200 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc 1260 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc 1320 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg 1380 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc 1440 aatggcaaca ac gttgcgca aactattaac tggcgaacta cttactctag cttcccggca 1500 acaattaata gactggatgg aggcggataa agttgcagga cc acttctgc gctcggccct 1560 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat 1620 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg 1680 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat 1740 taagcattgg taactgtcag accaacccat atccactctg ttccacacag gtcagaggtt 1800 tgtcc aggag ttcttgacag aggtgtaaaa agtactcaaa aattttactc aagtgaaagt 1860 acaagtactt agggaaaatt ttactcaatt aaaagtaaaa gtatctggct agaatcttac 1920 ttgagtaaaa gtaaaaaagt actccattaa aattgtactt gagtattaag gaagtaaaag 1980 taaaagcaag aaagaaaact agagattctt gtttaagctt ttaatctcaa aaaacattaa 2040 atgaaatgca tacaaggttt tatcctgctt tagaactgtt tgtatttaat tatcaaacta 2100 taagacagac aatctaatgc cagtacacgc tactcaaagt tgtaaaacct cagatttaac 2160 ttcagtagaa gctgattctc aaaattgtta gtgtcaagcc tagctctttt ggggctgaaa 2220 agcaatcctg cagtgctgaa aagcctctca caggcagccg atgcgggaag aggtgtatta 2280 gtcttgatag agaggctgca aatagcagga aacgtgagca gagactccct ggtgtctgaa 2340 acacaggcca gatgggcctc gcgatgtacg ggccagcgat gtacgggcca gatatacg cg 2400 ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag 2460 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc 2520 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg 2580 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca 2640 tcaagtgtat catatgcc aa gtacgccccc tattgacgtc aatgacggta aatggcccgc 2700 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt 2760 attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata 2820 gcggttt gac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt 2880 ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca 2940 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctctct ggctaactag 3000 agaacccact gcttactggc ttatcgaaat taatacgact cactataggg agacccaagc 3060 tggctagcgc caccatggag ttt gggctga gctgggtttt cctcgttgct ctttttagag 3120 gtgtccagtg tatggaagac gccaaaaca taaagaaagg cccggcgcca ttctatccgc 3180 tggaagatgg aaccgctgga gagcaactgc ataaggctat gaagagatac gccctggttc 3240 ctg gaacaat tgcttttaca gatgcacata tcgaggtgga catcacttac gctgagtact 3300 tcgaaatgtc cgttcggttg gcagaagcta tgaaacgata tgggctgaat acaaatcaca 3360 gaatcgtcgt atgcagtgaa aactctcttc aattctttat gccggtgttg ggcgcgttat 3420 ttatcggagt tgcagttgcg cccgcgaacg acatttataa tgaacgtgaa ttgctcaaca 3480 gtatgggcat ttcgcag cct accgtggtgt tcgtttccaa aaaggggttg caaaaaattt 3540 tgaacgtgca aaaaaagctc ccaatcatcc aaaaaattat tatcatggat tctaaaacgg 3600 attaccaggg atttcagtcg atgtacacgt tcgtcacatc tcatctacct cccggtttta 3660 at gaatacga ttttgtgcca gagtccttcg atagggacaa gacaattgca ctgatcatga 3720 actcctctgg atctactggt ctgcctaaag gtgtcgctct gcctcataga actgcctgcg 3780 tgagattctc gcatgccaga gatcctattt ttggcaatca aatcattccg gatactgcga 3840 ttttaagtgt tgttccattc catcacggtt ttggaatgtt tactacactc ggatatttga 3900 tatgtggatt tcgagtcgtc ttaatgtata gatttgaaga agagctgttt ctgaggagcc 3960 ttcaggatta caagattcaa agtgcgctgc tggtgccaac cctattctcc ttcttcgcca 4020 aaagcactct gattgacaaa tacgatttat ctaatttaca cgaaattgct tctggtggcg 4080 ctcccctctc taaggaagtc ggg gaagcgg ttgccaagag gttccatctg ccaggtatca 4140 ggcaaggata tgggctcact gagactacat cagctattct gattacaccc gagggggatg 4200 ataaaccggg cgcggtcggt aaagttgttc cattttttga agcgaaggtt gtggatctgg 4260 ataccgggaa aacgctgggc gttaatcaaa gaggcgaact gtgtgtgaga ggtcctatga 4320 ttatgtccgg ttatgtaaac aatccggaag cgaccaacgc cttgattgac aaggatggat 4380 ggctacattc tggagacata gcttactggg acgaagacga acacttcttc atcgttgacc 4440 gcctgaagtc tctgattaag tacaaaggct atcaggtggc tcccgctgaa ttggaatcca 4500 tcttgctcca acaccccaac atcttcgacg caggtgtcgc aggtcttccc gacgatgacg 4560 ccggtgaact tcccgccgcc gttgttgttt tggagcacgg aaagacgatg acggaaaaag 4620 agatcgtgga ttacgtcgcc agtcaagtaa caaccgcgaa aaagttgcgc ggaggagttg 4680 tgtttgtgga cgaagtaccg aaaggtctta ccggaaaact cgacgcaaga aaaatcagag 4740 agatcctcat aaaggccaag aagggcggaa agatcgccgt g taactcgag cggccgccac 4800 tgtgctggat atctgcagaa ttccaccaca ctggactagt ggatccgagc tcggtaccaa 4860 gcttaagttt aaaccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg 4920 tttgcccctc cc ccgtg cct tccttgaccc tggaaggtgc cactcccact gtcctttcct 4980 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg 5040 gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg 5100 cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc 5160 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg 522 0 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca 5280 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta 5340 gtgctttacg gcacctcgac cccaaaaaac tt gattaggg tgatggttca cgtagtgggc 5400 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg 5460 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat 5520 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta 5580 acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc 5640 agcaggcaga agtat gcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc 5700 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat 5760 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc 5820 gcccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga 5880 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc 5940 gggagcttgt atatccattt tcggatctga tcaagagaca ggatgaggat cgtttcgcat 6000 gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg 6060 ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc 6120 gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca 6180 ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct 6240 cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga 6300 tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg 6360 gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat 6420 cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga 6480 gcatcagggg ctcgcgccag ccgaact gtt cgccaggctc aaggcgcgca tgcccgacgg 6540 cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg 6600 ccgcttttct ggattcatcggt actggccg gctgggtgtg gcggaccgct atcaggacat 6660 agcgtt ggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct 6720 cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga 6780 cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg 6840 ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt 6900 ttccgggacg ccggctggat g atcctccag cgcggggatc tcatgctgga gttcttcgcc 6960 caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat 7020 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat 7080 gtatc ttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca 7140 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacacat acgagccgga 7200 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg 7260 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc 7320 caacgcgcgg ggagaggcgg tttgcgtatt gggcgct ctt ccgcttcctc gctcactgac 7380 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata 7440 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa 7500 aaggccagga accgtaaaa a ggccgcgttg ctggcgtttt tccataggct ccgcccccct 7560 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa 7620 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg 7680 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca 7740 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca ag ctgggctg tgtgcacgaa 7800 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg 7860 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg 7920 tatgtaggcg gtgctacaga gttcttgaag t ggtggccta actacggcta cactagaaga 7980 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc 8040 tcttgatccg gcaaacaaac caccgctggt agcggttttt ttgtttgcaa gcagcagatt 8100 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct 8160 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc 8220 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa 8280 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta 8340 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc 8400 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat 8460 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta 8520 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt 8580 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt 8 640 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg 8700 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc 8760 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catg ccatcc 8820 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg 8880 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga 8940 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta 9000 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct 9060 tttaactttca ccagc gtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag 9120 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga 9180 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat 9240 a aacaaatag gggttccgcg cacatttccc cg 9272 <210> 6 <211 > 592 <212> DNA <213> Artificial Sequence <220> <223> Tol2 Right ITR sequence <400> 6 acccatatcc actctgttcc acacaggtca gaggtttgtc caggagttct tgacagaggt 60 gtaaaaagta ctcaaaaatt ttactcaagt gaaagtacaa gtacttaggg aaaattttac 120 tcaattaaaa gtaaaagtat ctggctagaa tcttacttga gtaaaagtaa aaaagtactc 180 cattaaaatt gtacttgagt attaaggaag taaaagtaaa agcaagaaag aaaactagag 240 attcttgttt aagcttttaa tctcaaaaaa cattaaatga aatgcataca aggttttatc 300 ctgctttaga actgtttgta tttaattatc aaactataag acagacaatc taatgccagt 360 acacgctact caaagttgta aaacctcaga tttaacttca gtagaagctg attctcaaaa 420 ttgttagtgt caagcctagc tcttttgggg ctgaaaagca atcctgcagt gctgaaaagc 480 ctctcacagg cagccgatgc gggaagaggt gtattagtct tgatagagag gctgcaaata 540 gcaggaaacg tgagcagaga ctccctggtg tctgaaacac aggccagatg gg 592 <210> 7 <211> 539 <212> DNA <213> Artificial Sequence <220> <223> Tol2 Left ITR sequence <400 > 7 tgcgaagata cggccacggg tgctcttgat cctgtggctg attttggact gtgctgctcg 60 cagctgctga tgaatcacat acttcctcca ttttcttcca ctgattgact gttataattt 120 ccctaatttc caggtcaagg tgctgtgcat tgtggtaata gatgtgacat gac gtcactt 180 ccaaaggacc aatgaacatg tctgaccaat ttcatataat gtgaaaacga ttttcatagg 240 cagaataaat aacatttaaa ttaaactggg catcagcgca attcaattgg tttggtaata 300 gcaagggaaa atagaatgaa gtgatctcca aaaaataagt acttttgac tgtaaataaa 360 attgtaagga gtaaaaagta cttttttttc taaaaaaatg taattaagta aaagtaaaag 539 <210> 8 <211> 15 <212> DNA <213> Artificial Sequence <220> <22 3> forward primer sequence for Importin alpha <400> 8 gctctacaaa accat 15 <210> 9 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> reverse primer sequence for Importin alpha <400> 9 ccagatcctc ttctg 15 <210> 10 <211> 15 <212> DNA < 213> Artificial Sequence <220> <223> forward primer sequence for Importin beta <400> 10 cagcatttgg gaagg 15 <210> 11 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> reverse primer sequence for Importin beta<400> 11 tgccagatcc tcttc 15
Claims (11)
A transposase expression vector comprising a nucleotide sequence encoding Tol2 transposase and a nucleotide sequence encoding histone H2B.
상기 히스톤 H2B를 인코딩하는 염기서열은 상기 Tol2 트랜스포사제를 인코딩하는 염기서열의 5'방향에 위치하는 것을 특징으로 하는 트랜스포사제 발현 벡터.
According to claim 1,
The transposase expression vector, characterized in that the nucleotide sequence encoding the histone H2B is located in the 5' direction of the nucleotide sequence encoding the Tol2 transposase.
A transposase expressed from the expression vector according to claim 1 .
Tol2 우측 ITR(Inverted Terminal Repeat), Tol2 좌측 ITR, 프로모터(Promoter) 및 전이 유전자(Transgene)를 포함하는 트랜스포존(Transposon) 벡터;
를 이용하여 세포를 형질전환시키는 단계를 포함하는, 세포에 전이 유전자를 통합하는 방법.
a transposase expression vector comprising a nucleotide sequence encoding Tol2 transposase and a nucleotide sequence encoding histone H2B; and
a transposon vector including a Tol2 right Inverted Terminal Repeat (ITR), a Tol2 left ITR, a promoter and a transgene;
A method of integrating a transgene into a cell, comprising transforming the cell using
상기 방법은 임포틴(Importin)을 인코딩하는 염기서열을 포함하는 발현 벡터를 더 포함하는 것을 특징으로 하는 방법.
According to claim 4,
The method further comprises an expression vector comprising a nucleotide sequence encoding importin.
상기 임포틴은 임포틴 알파 KPNA2인 것을 특징으로 하는 방법.
According to claim 5,
The method of claim 1, wherein the importin is importin alpha KPNA2.
상기 임포틴은 임포틴 베타 KPNB1인 것을 특징으로 하는 방법.
According to claim 5,
The method of claim 1, wherein the importin is importin beta KPNB1.
상기 트랜스포존 벡터 및 트랜스포사제 발현 벡터의 질량비율(트랜스포존 벡터 : 트랜스포사제 발현 벡터)은 1:0.05 내지 1:1인 것을 특징으로 하는 방법.
According to claim 4,
Wherein the mass ratio of the transposon vector and the transposase expression vector (transposon vector: transposase expression vector) is 1:0.05 to 1:1.
상기 히스톤 H2B를 인코딩하는 염기서열은 상기 Tol2 트래스포사제를 인코딩하는 염기서열의 5'방향에 위치하는 것을 특징으로 하는 방법.
According to claim 4,
Wherein the nucleotide sequence encoding the histone H2B is located in the 5' direction of the nucleotide sequence encoding the Tol2 transportase.
상기 세포는 동물세포인 것을 특징으로 하는 방법.
According to claim 4,
The method characterized in that the cells are animal cells.
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