KR102283918B1 - The vectors for expressing hIGF1 transformed microalgae for expression those growth factors - Google Patents
The vectors for expressing hIGF1 transformed microalgae for expression those growth factors Download PDFInfo
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Abstract
본 발명은 인간인슐린유사성장인자 타입1 (hIGF1) 생산 발현용 벡터 및 상기벡터로 형질전환된 미세조류를 제공한다. The present invention provides a vector for expression of human insulin-like growth factor type 1 (hIGF1) and microalgae transformed with the vector.
Description
본 발명은 인간섬유아세포성장인자 타입1 (hFGF1), 인간섬유아세포성장인자 타입2 (hFGF2), 인간인슐린유사성장인자 타입1 (hIGF1) 생산 발현용 벡터 및 상기벡터로 형질전환된 미세조류에 관한 것이다.The present invention relates to a vector for expression of human fibroblast growth factor type 1 (hFGF1), human fibroblast growth factor type 2 (hFGF2), human insulin-like growth factor type 1 (hIGF1) and microalgae transformed with the vector will be.
인간섬유아세포성장인자 타입1 (hFGF1), 인간섬유아세포성장인자 타입2 (hFGF2), 인간인슐린유사성장인자 타입1 (hIGF1) 는 주름개선 화장품의 원료로 사용되는 기능성 단백질이다. 현재 대부분의 상기 성장인자류는 대장균에서 생산되어 사용되고 있다. 그러나 대장균은 내성독소(endotoxin)를 포함하고 있기 때문에 이에 따른 위험성을 내포하고 있다.Human fibroblast growth factor type 1 (hFGF1), human fibroblast growth factor type 2 (hFGF2), and human insulin-like growth factor type 1 (hIGF1) are functional proteins used as raw materials for wrinkle-improving cosmetics. Currently, most of the growth factors are produced and used in E. coli. However, since E. coli contains endotoxin, there is a risk associated with it.
따라서, 내성독소에 따른 위험성을 원칙적으로 제거한 기존의 대장균 생산 hFGF1, hFGF2, hIGF1 원료의 대체제가 필요한 실정이다.Therefore, there is a need for substitutes for the existing raw materials for E. coli production hFGF1, hFGF2, and hIGF1, which in principle have eliminated the risk of resistant toxins.
상기와 같은 문제점에 착안하여, 본 발명의 발명자들은 내성독성의 가능성을 원천 봉쇄하면서, 효과적으로 hFGF1, hFGF2, hIGF1 단백질을 생산할 수 있는 방법에 관한 연구를 하여 본 발명에 이르게 되었다.In view of the above problems, the inventors of the present invention conducted a study on a method capable of effectively producing hFGF1, hFGF2, and hIGF1 proteins while blocking the source of the possibility of resistant toxicity, leading to the present invention.
본 발명의 일 측면은, 서열번호 2의 인간섬유아세포성장인자 타입1 (hFGF1)을 코딩하는 염기서열을 포함하는 벡터를 제공하고자 한다.One aspect of the present invention is to provide a vector comprising a nucleotide sequence encoding human fibroblast growth factor type 1 (hFGF1) of SEQ ID NO: 2.
본 발명의 일 측면은, 서열변호 3의 인간섬유아세포성장인자 타입2 (hFGF2)를 코딩하는 염기서열을 포함하는 벡터를 제공하고자 한다.One aspect of the present invention is to provide a vector comprising a nucleotide sequence encoding human fibroblast growth factor type 2 (hFGF2) of SEQ ID NO: 3.
본 발명의 일 측면은, 서열번호 4의 인간인슐린유사성장인자 (hIGF1)를 코딩하는 염기서열을 포함하는 벡터를 제공하고자 한다. One aspect of the present invention is to provide a vector comprising a nucleotide sequence encoding human insulin-like growth factor (hIGF1) of SEQ ID NO: 4.
본 발명의 일 측면에서, 상기 벡터는 서열번호 8-10 으로 표시되는 염기서열을 포함하는 벡터일 수 있다.In one aspect of the present invention, the vector may be a vector comprising the nucleotide sequence represented by SEQ ID NOs: 8-10.
본 발명의 다른 측면은, 상기 벡터로 형질전환된 미세조류를 제공하고자 한다.Another aspect of the present invention is to provide microalgae transformed with the vector.
본 발명의 또 다른 측면은, 상기 형질전환된 미세조류를 이용하여 FGF1, hFGF2, hIGF1 단백질 생산하는 방법을 제공하고자 한다.Another aspect of the present invention is to provide a method for producing FGF1, hFGF2, and hIGF1 proteins using the transformed microalgae.
본 발명의 일 측면은, 서열번호 1의 프로모터 및 hFGF1, hFGF2, hIGF1을 코딩하는 유전자를 포함하는 상기 성장인자 발현구역을 포함하는 벡터를 제공한다.One aspect of the present invention provides a vector comprising the promoter of SEQ ID NO: 1 and the growth factor expression region comprising genes encoding hFGF1, hFGF2, and hIGF1.
본 발명의 일 측면에서, 상기 성장인자 발현구역은 His-tag, GST-tag, GFP, Tobacco etch virus (TEV) cleavage site서열을 더 포함하는, 벡터일 수 있다.In one aspect of the present invention, the growth factor expression region may be a vector, further comprising a His-tag, GST-tag, GFP, Tobacco etch virus (TEV) cleavage site sequence.
본 발명의 일 측면에서, 상기 성장인자 발현구역은 서열번호 2-4 로 표시되는 염기서열인, 벡터일 수 있다.In one aspect of the present invention, the growth factor expression region may be a vector, which is a nucleotide sequence represented by SEQ ID NOs: 2-4.
본 발명의 일 측면에서, 상기 벡터는 서열번호 8-10 으로 표시되는 염기서열을 포함하는 벡터일 수 있다. In one aspect of the present invention, the vector may be a vector comprising the nucleotide sequence represented by SEQ ID NOs: 8-10.
본 발명의 다른 측면은 상기 어느 하나에 따른 벡터로 형질전환된 미세조류를 제공한다.Another aspect of the present invention provides a microalgae transformed with the vector according to any one of the above.
본 발명의 다른 측면에서, 상기 미세조류는 페오닥틸룸 트리코뉴튬 (Phaeodactylum tricornutum) 인 미세조류일 수 있다.In another aspect of the present invention, the microalgae may be microalgae that is Phaeodactylum tricornutum.
본 발명의 또 다른 측면은 상기 생산 방법에 따라 생산된 hFGF1, hFGF2, hIGF1 단백질을 포함하는 화장품원료 조성물을 제공한다.Another aspect of the present invention provides a cosmetic raw material composition comprising hFGF1, hFGF2, and hIGF1 proteins produced according to the above production method.
본 발명의 또다른 측면에서, 상기 화장품원료 조정물은 주름개선용 화장품원료 조성물일 수 있다.In another aspect of the present invention, the cosmetic raw material adjustment may be a cosmetic raw material composition for wrinkle improvement.
본 발명의 또 다른 측면은 상기 형질전환된 미세조류를 포함하는 키트를 제공한다.Another aspect of the present invention provides a kit comprising the transformed microalgae.
본 발명의 일 측면에 따른 미세균주는 내성독성 없이 hFGF1, hFGF2, hIGF1 단백질을 생산할 수 있다.The microstrain according to one aspect of the present invention can produce hFGF1, hFGF2, and hIGF1 proteins without resistance toxicity.
본 발명의 일 측면에 따라 생산된 없이 hFGF1, hFGF2, hIGF1 단백질은 기존의 대장균에 의해 생산된 상기 성장인자류 단백질을 대체할 수 있다.The hFGF1, hFGF2, and hIGF1 proteins without production according to one aspect of the present invention can replace the growth factor protein produced by the existing E. coli.
본 발명의 일 측면에 따라 생산된 hFGF1, hFGF2, hIGF1 단백질을 포함하는 화장품원료 조성물은 주름개선 효과가 우수하다.The cosmetic raw material composition comprising hFGF1, hFGF2, and hIGF1 proteins produced according to an aspect of the present invention has excellent anti-wrinkle effect.
도 1은 본 발명 실시예에 따른 벡터 구성으로, HSPA1p: HSPA1 promoter, TEV: tobacco etch virus (TEV cleavage site), 3'-UTR: 3 terminal untranslated region, FcpB: fucoxanthin/chlorophyll binding protein B promoter, shble: zeocin resistance gene 이다.
도 2는 유전자 삽입여부를 gDNA-PCR로 확인한 결과이다.
도 3은 GFP 형광측정을 통해 배양 중 목적 단백질 발현 추이를 확인한 결과이다.
도 4는 ELISA를 통해 단백질 발현결과를 확인한 결과이다.
도 5는 목적 단백질 정제 결과이다.
도 6은 서열번호 1의 HASP1 promoter의 염기서열이다.
도 7은 서열번호 2의 His-GST-GFP-TEV-hFGF1 (HGGTF1)의 염기서열이다[노란색 박스: 제한효소위치, 하늘색 박스: hFGF1, 파란색 굵은글씨: kozak sequence, 빨간색 글씨: 시작 코돈 및 스탑 코돈, 보라색 글씨: His-tag, 갈색 글씨: GST-tag, 녹색 글씨: GFP, 파란색 글씨: TEV site]
도 8은 서열번호 3의 His-GST-GFP-TEV-hFGF2 (HGGTF2)의 염기서열이다[노란색 박스: 제한효소위치, 하늘색 박스: hFGF2, 파란색 굵은글씨: kozak sequence, 빨간색 글씨: 시작 코돈 및 스탑 코돈, 보라색 글씨: His-tag, 갈색 글씨: GST-tag, 녹색 글씨: GFP, 파란색 글씨: TEV site]
도 9는 서열번호 4의 His-GST-GFP-TEV-IGF1 (HGGTI1)의 염기서열이다[노란색 박스: 제한효소위치, 하늘색 박스: hIGF1, 파란색 굵은글씨: kozak sequence, 빨간색 글씨: 시작 코돈 및 스탑 코돈, 보라색 글씨: His-tag, 갈색 글씨: GST-tag, 녹색 글씨: GFP, 파란색 글씨: TEV site]
도 10은 서열번호 5의 3'-UTR의 염기서열이다.
도 11은 서열번호 6의 Shble cassete의 염기서열이다.
도 12는 서열번호 7의 pPha-HASP1p vector full sequence의 염기서열이다[녹색: HASP1 promoter, 갈색: 3'-UTR, 자주색: shble cassete, 노란색 박스: multi cloning site]
도 13은 서열번호 8의 pPha-HASP1p-HGGTF1의 염기서열이다.
도 14는 서열번호 9의 pPha-HASP1p-HGGTF2의 염기서열이다.
도 15는 서열번호 10의 pPha-HASP1p-HGGTI1의 염기서열이다.1 is a vector configuration according to an embodiment of the present invention, HSPA1p: HSPA1 promoter, TEV: tobacco etch virus (TEV cleavage site), 3'-UTR: 3 terminal untranslated region, FcpB: fucoxanthin / chlorophyll binding protein B promoter, shble : It is a zeocin resistance gene.
2 is a result of confirming whether a gene is inserted by gDNA-PCR.
3 is a result of confirming the expression trend of the target protein during culture through GFP fluorescence measurement.
4 is a result of confirming the protein expression results through ELISA.
5 is a result of purification of the target protein.
6 is a nucleotide sequence of the HASP1 promoter of SEQ ID NO: 1.
7 is a nucleotide sequence of His-GST-GFP-TEV-hFGF1 (HGGTF1) of SEQ ID NO: 2 [yellow box: restriction enzyme site, light blue box: hFGF1, blue bold text: kozak sequence, red text: start codon and stop codon, purple text: His-tag, brown text: GST-tag, green text: GFP, blue text: TEV site]
8 is a nucleotide sequence of His-GST-GFP-TEV-hFGF2 (HGGTF2) of SEQ ID NO: 3 [yellow box: restriction enzyme site, light blue box: hFGF2, blue bold text: kozak sequence, red text: start codon and stop codon, purple text: His-tag, brown text: GST-tag, green text: GFP, blue text: TEV site]
9 is a nucleotide sequence of His-GST-GFP-TEV-IGF1 (HGGTI1) of SEQ ID NO: 4 [yellow box: restriction enzyme site, light blue box: hIGF1, blue bold text: kozak sequence, red text: start codon and stop codon, purple text: His-tag, brown text: GST-tag, green text: GFP, blue text: TEV site]
10 is a base sequence of 3'-UTR of SEQ ID NO: 5.
11 is a nucleotide sequence of Shble cassette of SEQ ID NO: 6.
12 is a base sequence of the pPha-HASP1p vector full sequence of SEQ ID NO: 7 [green: HASP1 promoter, brown: 3'-UTR, purple: shble cassette, yellow box: multi cloning site]
13 is a nucleotide sequence of pPha-HASP1p-HGGTF1 of SEQ ID NO: 8.
14 is a nucleotide sequence of pPha-HASP1p-HGGTF2 of SEQ ID NO: 9.
15 is a nucleotide sequence of pPha-HASP1p-HGGTI1 of SEQ ID NO: 10.
본 발명의 이점 및 특징, 그리고 그것들을 달성하는 방법은 첨부되는 도면과 함께 상세하게 후술되어 있는 실시예들을 참조하면 명확해질 것이다. 그러나 본 발명은 이하에서 개시되는 실시예들에 한정되는 것이 아니라 서로 다른 다양한 형태로 구현될 수 있으며, 단지 본 실시예들은 본 발명의 개시가 완전하도록 하고, 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자에게 발명의 범주를 완전하게 알려주기 위해 제공되는 것이며, 본 발명은 청구항의 범주에 의해 정의될 뿐이다.Advantages and features of the present invention and methods of achieving them will become apparent with reference to the embodiments described below in detail in conjunction with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below, but may be implemented in various different forms, and only these embodiments allow the disclosure of the present invention to be complete, and common knowledge in the art to which the present invention pertains It is provided to fully inform those who have the scope of the invention, and the present invention is only defined by the scope of the claims.
본 발명의 실시예들을 설명함에 있어서 공지 기능 또는 구성에 대한 구체적인 설명이 본 발명의 요지를 불필요하게 흐릴 수 있다고 판단되는 경우에는 그 상세한 설명을 생략할 것이다. 그리고 후술되는 용어들은 본 발명의 실시예에서의 기능을 고려하여 정의된 용어들로서 이는 사용자, 운용자의 의도 또는 관례 등에 따라 달라질 수 있다. 그러므로 그 정의는 본 명세서 전반에 걸친 내용을 토대로 내려져야 할 것이다. In describing the embodiments of the present invention, if it is determined that a detailed description of a well-known function or configuration may unnecessarily obscure the gist of the present invention, the detailed description thereof will be omitted. In addition, the terms to be described later are terms defined in consideration of functions in an embodiment of the present invention, which may vary according to intentions or customs of users and operators. Therefore, the definition should be made based on the content throughout this specification.
본 발명에서 "인간섬유아세포성장인자 타입1 (human fibroblast growth factor type 1, hFGF1 또는 FGF1, aFGF)"은 155개의 아미노산으로 이루어진 17-18 kDa의 폴리펩타이드로 성장인자 및 신호단백질 역할을 한다.In the present invention, "human fibroblast growth factor type 1 (human fibroblast
본 발명에서 "인간섬유아세포성장인자 타입2 (human fibroblast growth factor type 2, hFGF2 또는 FGF2, bFGF)"은 155개의 아미노산으로 이루어진 18 kDa의 폴리펩타이드로 성자인자 및 신호단백질 역할을 한다.In the present invention, "human fibroblast growth factor type 2 (human fibroblast
본 발명에서 "인간인슐린유사성장인자 타입1 (human insulin like growth factor type 1, hIGF1 또는 IGF1)"은 70개의 아미노산으로 이루어진 약 8 kDa에 이르는 폴리펩타이드이다.In the present invention, "human insulin like growth factor type 1 (human insulin like
본 발명의 일측면에 따르면, 미세조류로부터 hFGF1, hFGF2, hIGF1을 생산할 경우 내성독소에 의한 위험성을 원천적으로 제거할 수 있으며, 기존의 대장균 생산 hFGF1, hFGF2, hIGF1 원료의 대체제로 사용할 수 있다. 이러한 미세조류 생산 hFGF1, hFGF2, hIGF1는 최근 화장품 트렌드인 친환경적 및 천연화 추세에 부합하는 화장품 원료로 사용할 수 있다.According to one aspect of the present invention, when producing hFGF1, hFGF2, hIGF1 from microalgae, the risk caused by resistant toxins can be fundamentally eliminated, and it can be used as a substitute for the existing E. coli production hFGF1, hFGF2, hIGF1 raw materials. These microalgae-producing hFGF1, hFGF2, and hIGF1 can be used as cosmetic raw materials in line with the recent cosmetic trend, eco-friendly and natural.
이하 본 발명에 대하여 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명의 일측면은, 서열번호 1의 프로모터; 및 인간섬유아세포성장인자 타입1 (hFGF1), 인간섬유아세포성장인자 타입2 (hFGF2), 인간인슐린유사성장인자 타입1 (hIGF1)을 코딩하는 유전자를 포함하는 상기 성장인자의 발현구역;을 포함하는, 벡터를 제공한다.One aspect of the present invention, the promoter of SEQ ID NO: 1; And human fibroblast growth factor type 1 (hFGF1), human fibroblast growth factor type 2 (hFGF2), human insulin-like growth factor type 1 (hIGF1) the expression region of the growth factor comprising a gene encoding a; , gives a vector.
본 발명의 일 측면에서, 상기 성장인자 발현구역은 His-tag, GST-tag, GFP 및 TEV site 서열을 더 포함하는, 벡터일 수 있다.In one aspect of the present invention, the growth factor expression region may be a vector, further comprising a His-tag, GST-tag, GFP and TEV site sequence.
본 발명의 일 측면에서, 상기 성장인자 발현구역은 서열번호 2-4로 표시되는 염기서열인, 벡터일 수 있다.In one aspect of the present invention, the growth factor expression region may be a vector, which is a nucleotide sequence represented by SEQ ID NOs: 2-4.
본 발명의 일 측면에서, 상기 벡터는 서열번호 8-10으로 표시되는 염기서열을 포함하는 벡터일 수 있다.In one aspect of the present invention, the vector may be a vector comprising the nucleotide sequence represented by SEQ ID NOs: 8-10.
본 발명의 일 측면에서, 상기 벡터는 서열번호 8-10으로 표시되는 염기서열을 포함하는 코딩된, 벡터일 수 있다. 구체적으로 상기 서열번호 8-10은 HASP1 promotor서열, His-tag서열, GFP, TEV 서열, FGF1 또는 FGF2, IGF1 서열, 3'-UTR 서열 및 Shble cassete를 포함한다.In one aspect of the present invention, the vector may be an encoded vector comprising the nucleotide sequence represented by SEQ ID NOs: 8-10. Specifically, SEQ ID NOs: 8-10 include HASP1 promoter sequence, His-tag sequence, GFP, TEV sequence, FGF1 or FGF2, IGF1 sequence, 3'-UTR sequence, and Shble cassette.
상기 Shble cassete는 FcpB, Shble 및 3'-UTR 포함를 포함할 수 있다.The Shble Cassette may include FcpB, Shble and 3'-UTR.
본 발명의 다른 측면은 상기 어느 하나에 따른 벡터로 형질전환된 미세조류를 제공한다.Another aspect of the present invention provides a microalgae transformed with the vector according to any one of the above.
본 발명의 다른 측면에서, 상기 미세조류는 페오닥틸룸 트리코뉴튬(Phaeodactylum tricornutum)인, 미세조류일 수 있다. 상기 페오닥틸룸 트리코뉴튬은 국외 미세조류 은행으로부터 용이하게 입수가 가능하다. 구체적으로, 분양기관인 UTEX에서 분양번호 UTEX 646으로 Phaeodactylum tricornutum (Bohlin)을 분양받아 사용할 수 있으며, 분양기관인 CCAP에서 분양번호 CCAP1052/6로 Phaeodactylum tricornutum (Bohlin)을 분양받아 사용할 수 있으며, 분양기관인 CCMP에서 분양번호 CCMP2559로 Phaeodactylum tricornutum을 분양받아 사용할 수 있다.In another aspect of the present invention, the microalgae may be microalgae, which is Phaeodactylum tricornutum. The pheodactylum triconutium can be easily obtained from foreign microalgae banks. Specifically, Phaeodactylum tricornutum (Bohlin) can be purchased and used from UTEX, a sales agency, with the sales number UTEX 646. You can purchase and use Phaeodactylum tricornutum under the sale number CCMP2559.
본 발명의 또 다른 측면은 상기 형질전환된 미세조류를 이용하여 hFGF1, hFGF2 및 hIGF1 단백질을 생산하는 방법을 제공한다.Another aspect of the present invention provides a method for producing hFGF1, hFGF2 and hIGF1 proteins using the transformed microalgae.
본 발명의 또 다른 측면은 상기 생산 방법에 따라 생산된 hFGF1, hFGF2 및 hIGF1 단백질을 포함하는 화장료 조성물을 제공한다.Another aspect of the present invention provides a cosmetic composition comprising hFGF1, hFGF2 and hIGF1 protein produced according to the above production method.
본 발명의 또 다른 측면에서, 상기 화장료 조성물은 주름개선용 화장료 조성물일 수 있다.In another aspect of the present invention, the cosmetic composition may be a cosmetic composition for improving wrinkles.
상기 hFGF1, hFGF2 및 hIGF1 단백질을 포함하는 화장료 제조시에 함유되는 hFGF1, hFGF2 및 hIGF1 단백질의 함량은 조성물 총량에 대하여 건조 중량으로 0.00001 내지 10.0 중량%, 바람직하게는 0.001 내지 1.0 중량%이다. hFGF1, hFGF2 및 hIGF1 단백질의 함량이 조성물 총량에 대하여 건조 중량으로 0.00001 중량% 미만이면 주름개선의 뚜렷한 효과를 기대할 수 없었고, 10.0 중량%를 초과하면 함유량의 증가에 따라 뚜렷한 효과의 증가를 기대하기 어렵다.The content of hFGF1, hFGF2 and hIGF1 proteins contained in the preparation of a cosmetic comprising the hFGF1, hFGF2 and hIGF1 protein is 0.00001 to 10.0 wt%, preferably 0.001 to 1.0 wt%, based on the total amount of the composition by dry weight. If the content of hFGF1, hFGF2, and hIGF1 protein is less than 0.00001 wt% by dry weight based on the total amount of the composition, a clear effect of wrinkle improvement could not be expected, and if it exceeds 10.0 wt%, it is difficult to expect a distinct effect to increase as the content increases. .
본 발명의 화장료 조성물에 포함되는 성분은 유효 성분으로서의 상기 성분 이외에 화장료 조성물에 통상적으로 이용되는 성분들을 포함하며, 예컨대 항산화제, 안정화제, 용해화제, 비타민, 안료 및 향료와 같은 통상적인 보조제, 그리고 담체를 포함한다.The ingredients included in the cosmetic composition of the present invention include ingredients commonly used in cosmetic compositions in addition to the above ingredients as active ingredients, for example, conventional adjuvants such as antioxidants, stabilizers, solubilizers, vitamins, pigments and fragrances, and carrier.
본 발명의 화장료 조성물은 당업계에서 통상적으로 제조되는 어떠한 제형으로도 제조될 수 있으며, 예를 들어, 용액, 현탁액, 유탁액, 페이스트, 겔, 크림, 로션, 파우더, 비누, 계면활성제-함유 클린싱, 오일, 분말 파운데이션, 유탁액 파운데이션, 왁스 파운데이션 및 스프레이 등으로 제형화될 수 있으나, 이에 한정되는 것은 아니다. 보다 상세하게는, 유연 화장수, 수렴 화장수, 영양 화장수, 영양 크림, 마사지 크림, 에센스, 아이 크림, 클렌징 크림, 클렌징 포옴, 클렌징워터, 팩, 스프레이 또는 파우더의 제형으로 제조될 수 있다.The cosmetic composition of the present invention may be prepared in any formulation conventionally prepared in the art, for example, solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing , oil, powder foundation, emulsion foundation, wax foundation, spray, etc., but is not limited thereto. More specifically, it may be prepared in the form of a flexible lotion, astringent lotion, nourishing lotion, nourishing cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray or powder.
본 발명의 제형이 페이스트, 크림 또는 겔인 경우에는 담체 성분으로서 동물성유, 식물성유, 왁스, 파라핀, 전분, 트라칸트, 셀룰로오스 유도체, 폴리에틸렌 글리콜, 실리콘, 벤토나이트, 실리카, 탈크 또는 산화아연 등이 이용될 수 있다.When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as a carrier component. can
본 발명의 제형이 파우더 또는 스프레이인 경우에는 담체 성분으로서 락토스, 탈크, 실리카, 알루미늄 히드록시드, 칼슘 실리케이트 또는 폴리아미드 파우더가 이용될 수 있고, 특히 스프레이인 경우에는 추가적으로 클로로플루오로히드로카본, 프로판/부탄 또는 디메틸 에테르와 같은 추진체를 포함할 수 있다.When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and in particular, in the case of a spray, additional chlorofluorohydrocarbon, propane /may contain propellants such as butane or dimethyl ether.
본 발명의 제형이 용액 또는 유탁액인 경우에는 담체 성분으로서 용매, 용해화제 또는 유탁화제가 이용되고, 예컨대 물, 에탄올, 이소프로판올, 에틸 카보네이트, 에틸 아세테이트, 벤질 알코올, 벤질 벤조에이트, 프로필렌 글리콜, 1,3-부틸글리콜 오일, 글리세롤 지방족 에스테르, 폴리에틸렌 글리콜 또는 소르비탄의 지방산 에스테르가 있다.When the formulation of the present invention is a solution or emulsion, a solvent, solubilizer or emulsifier is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylglycol oil, glycerol fatty esters, fatty acid esters of polyethylene glycol or sorbitan.
본 발명의 제형이 현탁액인 경우에는 담체 성분으로서 물, 에탄올 또는 프로필렌 글리콜과 같은 액상의 희석제, 에톡실화 이소스테아릴 알코올, 폴리옥시에틸렌 소르비톨 에스테르 및 폴리옥시에틸렌 소르비탄 에스테르와 같은 현탁제, 미소결정성 셀룰로오스, 알루미늄 메타히드록시드, 벤토나이트, 아가 또는 트라칸트 등이 이용될 수 있다.When the formulation of the present invention is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystals Adult cellulose, aluminum metahydroxide, bentonite, agar or tracanth may be used.
본 발명의 제형이 계면-활성제 함유 클린징인 경우에는 담체 성분으로서 지방족 알코올 설페이트, 지방족 알코올 에테르 설페이트, 설포숙신산 모노에스테르, 이세티오네이트, 이미다졸리늄 유도체, 메틸타우레이트, 사르코시네이트, 지방산 아미드 에테르 설페이트, 알킬아미도베타인, 지방족 알코올, 지방산 글리세리드, 지방산 디에탄올아미드, 식물성 유, 라놀린 유도체 또는 에톡실화 글리세롤 지방산 에스테르 등이 이용될 수 있다.When the formulation of the present invention is a surfactant-containing cleansing agent, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide as carrier components Ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative or ethoxylated glycerol fatty acid ester and the like can be used.
본 발명의 또 다른 측면은 상기 형질전환된 미세조류를 포함하는 키트를 제공한다.Another aspect of the present invention provides a kit comprising the transformed microalgae.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these examples.
[실시예 1] 벡터 구성[Example 1] Vector construction
벡터 구성은 하기와 같은 방법으로 준비하였다.Vector construction was prepared in the following way.
■ F/2배지 (50%인공해수)에서 배양한 P. tricornutum 배양액 50 mL를 취하여 원심분리 (3500 rpm, 15분, 20℃) cell pellet을 얻은 후, 이로부터 genomic DNA (gDNA)를 추출하였다. ■ 50 mL of P. tricornutum culture solution cultured in F/2 medium (50% artificial seawater) was centrifuged (3500 rpm, 15 minutes, 20℃) to obtain a cell pellet, and genomic DNA (gDNA) was extracted from it. .
■ 추출한 gDNA를 template로 사용하여 HASP1-F, HASP1-R 프라이머 및 pfu polymerase 로부터 PCR을 통해 HASP1 프로모터 부분의 DNA를 확보하였다. NdeI 및 EcoRI 제한효소를 사용하여 pPha-T1 벡터의 Fcp a 프로모터를 교체하여 pPha-HASP1p 벡터를 구성하였다.■ Using the extracted gDNA as a template, the HASP1 promoter DNA was obtained through PCR from HASP1-F, HASP1-R primers and pfu polymerase. The pPha-HASP1p vector was constructed by replacing the Fcp a promoter of the pPha-T1 vector using NdeI and EcoRI restriction enzymes.
■ 상기 프로모터 부분을 포함하여 hFGF1, hFGF2 및 hIGF1 발현 벡터를 도 1과 같이 구성하여, 목적 단백질인 상기 성장인자류를 생산할 수 있는 벡터를 구성하였다.■ hFGF1, hFGF2 and hIGF1 expression vectors including the promoter part were constructed as shown in FIG. 1 to construct a vector capable of producing the growth factors, the target protein.
■ 상기 벡터를 구성하는 서열을 하기 표 1와 같았다.■ The sequences constituting the vector are shown in Table 1 below.
(FcpB, Shble, 3'-UTR을 포함)Shble Cassette
(Including FcpB, Shble, 3'-UTR)
[실시예 2] 미세조류 형질전환[Example 2] Transformation of microalgae
미세조류 형질전환은 하기와 같은 방법으로 실시하였다.Microalgae transformation was carried out in the following way.
■ 상기 실시예 1에서 목적 단백질 생산용 벡터들은 E.coli (DH5a (NEBㄾ 5-alpha Competent E.coli) 에 형질전환 후 형질전환된 E.coli를 LB (100 μg/mL ampicillin) 배지에서 배양함.■ the culture in Example 1 in the desired protein production vectors are for E.coli (DH5a (ㄾ NEB 5-alpha Competent E.coli) E.coli transformant transformed the LB (100 μg / mL ampicillin and then switched to) medium box.
■ 배양한 E.coli로부터 Midiprep 법으로 목적 벡터들을 추출하고, phenol/chloroform 유전자 정제법 및 Ethanol 침전법으로 P. tricornutum 형질전환에 필요한 고순도 벡터 (농도 1.0 μg/μL) 를 확보하였다.■ The target vectors were extracted from the cultured E. coli by the Midiprep method, and a high-purity vector (concentration 1.0 μg/μL) required for P. tricornutum transformation was obtained by phenol/chloroform gene purification and ethanol precipitation.
■ 벡터를 금나노입자에 코팅한 후, 유전자총 방법 (Particle bombardment) 를 통해 P. tricornutum 으로 목적 벡터를 전달함으로써 형질전환 시켰다.■ After the vector was coated on gold nanoparticles, the target vector was transferred to P. tricornutum through the particle bombardment method to transform it.
■ 상기 형질전환체는 zeocin (100 μg/mL) 이 함유된 F/2 한천배지에서 선별과정을 진행하였다.■ The transformants were selected in F/2 agar medium containing zeocin (100 μg/mL).
상기와 같은 과정에 의해 얻어진 형질전환체는 P. tricornutum (PT-HASP1p-HGGTF1), P. tricornutum (PT-HASP1p-HGGTF2), P. tricornutum (PT-HASP1p-HGGTI1)으로 명명하고, 2019.12.18.자로 각각 한국생명공학 연구원 생물자원센터 (Korean Collection for Type Culture; KCTC)에 수탁 번호 KCTC 14080BP, 14081BP, 14082BP로 기탁되었다.The transformants obtained by the above process were named P. tricornutum (PT-HASP1p-HGGTF1), P. tricornutum (PT-HASP1p-HGGTF2), and P. tricornutum (PT-HASP1p-HGGTI1), 2019.12.18 It was deposited with the Korean Collection for Type Culture (KCTC) with accession numbers KCTC 14080BP, 14081BP, and 14082BP, respectively.
[실험예 1] hFGF1, hFGF2 및 hIGF1 발현 확인[Experimental Example 1] hFGF1, hFGF2 and hIGF1 expression confirmation
상기 실시예 3을 통해 선별된 P. tricornutum 형질전환체에 hFGF1, hFGF2 및 hIGF1 코딩유전자의 삽입 여부는 gDNA-PCR 통해 확인하였고, 목적 단백질의 발현여부는 ELISA를 통해 확인하였으며, 구체적으로 하기 실험예 1 내지 2를 통해 확인하였다. The insertion of hFGF1, hFGF2 and hIGF1 coding genes into the P. tricornutum transformants selected in Example 3 was confirmed through gDNA-PCR, and the expression of the target protein was confirmed through ELISA, specifically, the following Experimental Example It was confirmed through 1 and 2.
[실험예 2] gDNA-PCR을 통해 유전자 삽입 여부 확인[Experimental Example 2] Confirmation of gene insertion through gDNA-PCR
(1) 실험방법(1) Experimental method
■ 상기 실시예 2에서 hFGF1, hFGF2, hIGF1발현 형질전환체 콜로니를 확인하였다.■ In Example 2, hFGF1, hFGF2, hIGF1 expressing transformant colonies were confirmed.
■ P. tricornutum의 genomic DNA 로 삽입여부의 확인은 gDNA-PCR을 통해 확인하였다.■ Confirmation of insertion into genomic DNA of P. tricornutum was confirmed by gDNA-PCR.
■ 각 형질전환체 콜로니를 F/2 (50% 인공해수) 배지 200 μL 에 접균하여 5일간 배양한 후, 원심분리 (3500 rpm, 15분, 20 ℃) 후 상등액 제거하였다.■ Each transformant colony was inoculated in 200 μL of F/2 (50% artificial seawater) medium, cultured for 5 days, centrifuged (3500 rpm, 15 minutes, 20 ℃), and the supernatant was removed.
■ 10 μL 용해버퍼 (1% Nonidet P-40) 를 첨가한후 10분 후 용액을 PCR에 사용하였다.■ After adding 10 μL of lysis buffer (1% Nonidet P-40), the solution was used for
■ pPha-Up-NdeI-F 및 pPha-Down-MCS-R 프라이머 및 taq polymerase 사용하여 PCR 진행하여 유전자 삽입을 확인하였다.■ PCR was performed using pPha-Up-NdeI-F and pPha-Down-MCS-R primers and taq polymerase to confirm gene insertion.
(2) 실험결과(2) Experimental results
■ DNA 전기영동 결과는 도 2에 도시하였다. 도 2a가 hFGF1이며, 도 2b가 hFGF2, 도 2c가 hIGF1의 gDNA-PCR 결과이다. 확인 결과 비슷한 위치에서 목적 유전자 띠를 확인할 수 있었다.■ DNA electrophoresis results are shown in FIG. 2 . Figure 2a is hFGF1, Figure 2b is hFGF2, Figure 2c is the gDNA-PCR results of hIGF1. As a result of the confirmation, the target gene band could be identified at a similar location.
■ 대조군으로는 목적 유전자가 삽입되지 않은 빈 벡터를 삽입한 형질전환체 (Blank)사용하였는데, 여기서는 목적 유전자 띠를 확인하지 못하였다.■ As a control group, an empty vector into which the target gene was not inserted was used as a transformant (Blank), but the target gene band could not be identified.
[실험예 3] GFP 형광측정을 통한 hFGF1, hFGF2 및 hIGF1 발현 확인[Experimental Example 3] Confirmation of hFGF1, hFGF2 and hIGF1 expression through GFP fluorescence measurement
(1) 실험방법(1) Experimental method
■ 목적 단백질 hFGF1, hFGF2 및 hIGF1 N-terminal 에 결합된 GFP를 통해 미세조류 내 목적 단백질 발현량을 확인하였다.■ Target proteins hFGF1, hFGF2 and hIGF1 The expression level of the target protein in microalgae was confirmed through GFP bound to the N-terminal.
■ 미세조류 배양액을 96 well plate 에 200 μL 씩 취한 후, microplate reader (Biotek, Synergy HTX)를 통해 측정하였다 (excitation: 485/20, emission: 528/20).■ After taking 200 μL of microalgal culture solution to a 96 well plate, it was measured through a microplate reader (Biotek, Synergy HTX) (excitation: 485/20, emission: 528/20).
■ 동일한 방법으로 standard curve를 통해 미세조류 내 단백질 발현량 계산을 진행하였다.■ Protein expression level in microalgae was calculated using the standard curve in the same way.
(2) 실험결과(2) Experimental results
■ 형질전환 미세조류에서 목적단백질이 성공적으로 발현되는 것을 확인하였다.■ It was confirmed that the target protein was successfully expressed in the transgenic microalgae.
■ hFGF1 및 hFGF2의 발현은 배양 중 5일차에 발현이 나타나기 시작하며, 7일차 이후 발현 농도가 각각 23.0 mg/L 및 7.57 mg/L 로 측정되었다.■ Expressions of hFGF1 and hFGF2 started to appear on the 5th day of culture, and after the 7th day, the expression concentrations were measured to be 23.0 mg/L and 7.57 mg/L, respectively.
■ hIGF1의 경우 배양 5일차에 발현이 나타나며, 5일까지 발현농도 3.01 mg/L 로 측정되었다.■ In the case of hIGF1, expression appeared on the 5th day of culture, and the expression concentration was measured to be 3.01 mg/L until the 5th day.
[실험예 4] ELISA를 통한 hFGF1, hFGF2 및 hIGF1 발현 확인[Experimental Example 4] Confirmation of hFGF1, hFGF2 and hIGF1 expression through ELISA
(1) 실험방법(1) Experimental method
■ hFGF1, hFGF2, hIGF1의 함량은 각각의 효소에 대한 ELISA kit (abchem, ab219636, ab99979, ab211651)에 의해 진행하였다.■ The contents of hFGF1, hFGF2, and hIGF1 were determined by ELISA kit (abchem, ab219636, ab99979, ab211651) for each enzyme.
■ 각각의 형질전환체는 10일간 F/2 (50% 인공해수) 배양한 후 20 mL를 취하여 원심분리를 통해 cell pellet 얻은 후, 5 mL 용해버퍼 (50 mL Tris-HCl, 150 mM NaCl, 1 mM PMSF, 0.1% Tween 20, 1 mM EDTA, 1% Nonidet P40)를 첨가하였다.■ Each transformant is cultured in F/2 (50% artificial seawater) for 10 days, then 20 mL is taken, centrifuged to obtain a cell pellet, and 5 mL lysis buffer (50 mL Tris-HCl, 150 mM NaCl, 1 mM PMSF, 0.1
■ 초음파파쇄기 (sonicator, 10% power, 10 초 초음파 - 50초 휴식 및 냉각, 10회) 로 미세조류 세포의 파쇄를 진행하였다.■ Microalgae cells were disrupted with a sonicator (sonicator, 10% power, 10 seconds ultrasonic - 50 seconds rest and cooling, 10 times).
■ 원심분리 (10,000 rpm, 20분, 4 ℃)를 통해 찌꺼기 제거 후 상등액을 ELISA 샘플로 사용하였다.■ After removing debris through centrifugation (10,000 rpm, 20 minutes, 4 ℃), the supernatant was used as an ELISA sample.
(2) 실험결과(2) Experimental results
■ hFGF1는 15.4 mg/L의 발현량을 확인하였다.■ hFGF1 was confirmed to have an expression level of 15.4 mg/L.
■ hFGF2는 8.1 mg/L 이상 발현량을 확인하였다.■ hFGF2 was found to have an expression level of 8.1 mg/L or more.
■ hIGF1는 2.8 mg/L 이상 발현량을 확인하였다.■ hIGF1 was found to have an expression level of 2.8 mg/L or more.
[실험예 5] hFGF1, hFGF2 및 hIGF1 정제 및 SDS-PAGE 확인[Experimental Example 5] hFGF1, hFGF2 and hIGF1 purification and SDS-PAGE confirmation
(1) 실험방법(1) Experimental method
■ 각각의 형질전환체는 10일간 F/2 (50% 인공해수) 배양한 후 2 L를 취하여 원심분리를 통해 cell pellet 얻은 후, 20 mL 용해버퍼 (50 mL Tris-HCl, 150 mM NaCl, 1 mM PMSF, 0.1% Tween 20, 1 mM EDTA, 1% Nonidet P40)를 첨가하였다.■ Each transformant is cultured in F/2 (50% artificial seawater) for 10 days, then 2 L is taken and a cell pellet is obtained through centrifugation. Then, 20 mL lysis buffer (50 mL Tris-HCl, 150 mM NaCl, 1 mM PMSF, 0.1
■ 초음파파쇄기 (sonicator, 30% power, 10 초 초음파 - 50초 휴식 및 냉각, 10회)로 미세조류 세포의 파쇄를 진행하였다.■ Microalgal cells were disrupted with a sonicator (sonicator, 30% power, 10 sec ultrasound - 50 sec rest and cooling, 10 times).
■ 원심분리 (10,000 rpm, 20분, 4 ℃)를 통해 찌꺼기 제거 후 상등액을 정제에 사용하였다.■ After removing residues through centrifugation (10,000 rpm, 20 minutes, 4 ℃), the supernatant was used for purification.
■ Ni-resin 5 mL (His-trap 5 mL, GE healthcare)으로 1차 흡착 크로마토그래피를 진행하였다.■ Primary adsorption chromatography was performed with Ni-
■ 12% Tricine-SDS-PAGE 를 통해 결과를 확인하였다.■ The results were confirmed through 12% Tricine-SDS-PAGE.
■ PD10 column (Hi-desalt 5 mL, GE healthcare)을 통해 염 제거 후, TEV protease 5-10 μg/mL 농도로 처리하여 4℃ 에서 9시간 반응시켰다.■ After removing salt through PD10 column (Hi-
■ Ni-resin 5 mL (His-trap 5 mL, GE healthcare)로 2차 흡착 크로마토그래피 진행하여, TEV 제거 및 tag 서열 제거된 목적 단백질을 확보하였다.■ Secondary adsorption chromatography was performed with Ni-
■ 15% Tricine-SDS-PAGE를 통해 결과를 확인하였다.■ The results were confirmed through 15% Tricine-SDS-PAGE.
(2) 실험결과(2) Experimental results
■ HGGTF1 (His-GST-GFP-TEV-FGF1, 71.1 kDa) 단백질 밴드를 확인하였다.■ HGGTF1 (His-GST-GFP-TEV-FGF1, 71.1 kDa) protein band was confirmed.
■ HGGTF2 (His-GST-GFP-TEV-FGF1, 72.7 kDa) 단백질 밴드를 확인하였다.■ HGGTF2 (His-GST-GFP-TEV-FGF1, 72.7 kDa) protein band was confirmed.
■ HGGTI1 (His-GST-GFP-TEV-IGF1, 62.9 kDa) 단백질 밴드를 확인하였다.■ HGGTI1 (His-GST-GFP-TEV-IGF1, 62.9 kDa) protein band was identified.
■ 최종 hFGF1 (15.8 kDa) 단백질 밴드를 확인하였다.■ The final hFGF1 (15.8 kDa) protein band was identified.
■ 최종 hFGF2 (17.4 kDa) 단백질 밴드를 확인하였다.■ The final hFGF2 (17.4 kDa) protein band was identified.
■ 최종 hIGF1 (7.76 kDa) 단백질 밴드를 확인하였다.■ The final hIGF1 (7.76 kDa) protein band was identified.
[실험예 6] 주름개선 효과 시험[Experimental Example 6] Wrinkle improvement effect test
주름개선효과는 통상 콜라겐 생합성능과 콜라게네이즈 분해 억제능 및 사람에 대한 임상시험으로서 효과를 측정할 수 있다.The anti-wrinkle effect can be measured as a clinical test for collagen biosynthesis and collagenase decomposition inhibition and human clinical trials.
사람의 정상 섬유아세포(human normal fibroblast)를 DMEM배지가 들어있는 6-웰 마이크로플레이트에 접종시키고(2ㅧ105세포/웰) 5%농도의 CO2 배양기에 37℃로 24시간 배양하였다. 24시간 후, 각 웰에서 배지를 제거하고 농도별로 시료를 처리한 다음 24시간 동안 다시 배양하였다. 24시간 후, 세포배지를 수집하여 샘플을 제조하였다. Human normal fibroblasts (human normal fibroblasts) were inoculated into a 6-well microplate containing DMEM medium (2ㅧ10 5 cells/well) and incubated for 24 hours at 37°C in a 5% CO 2 incubator. After 24 hours, the medium was removed from each well, samples were treated by concentration, and then incubated again for 24 hours. After 24 hours, the cell medium was collected to prepare a sample.
콜라겐 합성량은 콜라겐 측정 키트(Procollagen type I C-peptide EIA kit(MK101), 다카라, 교토, 일본)를 이용하여 세포배지 내에서의 프로콜라겐(procollagen)타입 I C-펩타이드(type I C-peptide : PICP)의 양을 측정하고 분석하였다.The amount of collagen synthesis was measured using a collagen measurement kit (Procollagen type I C-peptide EIA kit (MK101), Takara, Kyoto, Japan) in the cell medium for procollagen type I C-peptide (type I C-peptide). : PICP) was measured and analyzed.
콜라겐을 분해하는 콜라게네이즈의 활성정도를 측정하는 방법으로 콜라게네이즈에 대한 항체를 이용하였다. 콜라게네이즈 활성은 타입 I 콜라게네이즈 어세이 키트(Amersham Biosciences, RPN2629)를 이용하였으며, ELISA 판독기로 흡광도를 측정하였다. 측정된 표준치는 평균 ㅁ표준편차의 형태로 표시하였고 SPSS/PC+프로그램을 이용하여 t-test로 유의성을 검정하였고, 그 결과를 하기 표 3에 나타내었다.An antibody against collagenase was used as a method of measuring the activity level of collagenase that decomposes collagen. Collagenase activity was measured using a type I collagenase assay kit (Amersham Biosciences, RPN2629), and absorbance was measured with an ELISA reader. The measured standard values were expressed in the form of mean ㅁ standard deviation, and significance was tested by t-test using the SPSS/PC+ program, and the results are shown in Table 3 below.
표 3에 나타난 바와 같이, 실험예 5에 의해 정제된 hFGF1, hFGF2 및 hIGF1의 첨가를 통해 콜라겐합성이 증가되었으며, 또한 콜라게네이즈 활성은 억제되었다. 따라서 본 발명에 따른 hFGF1, hFGF2 및 hIGF1은 주름개선 효능을 가지고 있음을 알 수 있다.As shown in Table 3, collagen synthesis was increased through the addition of hFGF1, hFGF2 and hIGF1 purified according to Experimental Example 5, and collagenase activity was also inhibited. Therefore, it can be seen that hFGF1, hFGF2 and hIGF1 according to the present invention have anti-wrinkle effects.
이상의 설명은 본 발명의 기술 사상을 예시적으로 설명한 것에 불과한 것으로서, 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자라면 본 발명의 본질적인 특성에서 벗어나지 않는 범위에서 다양한 수정 및 변형이 가능할 것이다. 따라서, 본 발명에 개시된 실시예들은 본 발명의 기술 사상을 한정하기 위한 것이 아니라 설명하기 위한 것이고, 이러한 실시예에 의하여 본 발명의 기술 사상의 범위가 한정되는 것은 아니다. 본 발명의 보호 범위는 아래의 청구범위에 의하여 해석되어야 하며, 그와 동등한 범위 내에 있는 모든 기술사상은 본 발명의 권리범위에 포함되는 것으로 해석되어야 할 것이다.The above description is merely illustrative of the technical spirit of the present invention, and various modifications and variations will be possible without departing from the essential characteristics of the present invention by those skilled in the art to which the present invention pertains. Therefore, the embodiments disclosed in the present invention are not intended to limit the technical spirit of the present invention, but to explain, and the scope of the technical spirit of the present invention is not limited by these embodiments. The protection scope of the present invention should be construed by the following claims, and all technical ideas within the scope equivalent thereto should be construed as being included in the scope of the present invention.
<110> ALGAEPRONA INC. <120> The vectors for expressing hIGF1 transformed microalgae for expression those growth factors <130> SP1102 <160> 10 <170> KoPatentIn 3.0 <210> 1 <211> 499 <212> DNA <213> Phaeodactylum tricornutum <220> <221> promoter <222> (1)..(499) <223> HASP1 promoter <400> 1 catacagtga atgtaacttt cgaattgaca gtattagtag tcgtattgac agtgaggcac 60 gcccctcaat gtgcgaggtg gaaaatatac cagcatgaca atgaatcttg gagattcttt 120 tgctgtcatc aagattcacc gccaaatctt caggaaccta tcacgtccac aggcgatgtt 180 aattcttgag tcgtcaaaac aaagtcctgt cctacctgta gaagttgaca gcgagcaatt 240 gtatgcaaac ttctgacttt gttataataa cattaaaggt aattaagtat cttcaattag 300 gcattttgtc actgtcagtc cgttccgaca atataggtag atttggaatg aatcttttct 360 atgctgctgc gaatcttgta cacctttgag gccgtagatt ctgtccgacg aagcgataat 420 tattgcaaaa tacatggact cattattttg attcgatttc tttttggtat ccgactcgaa 480 aagatccatc acggcgagc 499 <210> 2 <211> 1890 <212> DNA <213> Artificial Sequence <220> <223> His-GST-GFP-TEV-hFGF1 (HGGTF1) <400> 2 gatatcacca tgcaccacca ccaccaccac gccggaatgt cccccattct cggatactgg 60 aagattaagg gactcgtcca gcccacccgt ctcctcctcg aatacctcga agaaaagtac 120 gaagaacacc tctacgaacg tgacgaagga gacaagtggc gtaacaagaa gttcgaactc 180 ggactcgaat tccccaacct cccctactac attgacggag acgtcaagct cacccagtcc 240 atggccatta ttcgttacat tgccgacaag cacaacatgc tcggaggatg ccccaaggaa 300 cgtgccgaaa tttccatgct cgaaggagcc gtcctcgaca ttcgttacgg agtctcccgt 360 attgcctact ccaaggactt cgaaaccctc aaggtcgact tcctctccaa gctccccgaa 420 atgctcaaga tgttcgaaga ccgtctctgc cacaagacct acctcaacgg agaccacgtc 480 acccaccccg acttcatgct ctacgacgcc ctcgacgtcg tcctctacat ggaccccatg 540 tgcctcgacg ccttccccaa gctcgtctgc ttcaagaagc gtattgaagc cattccccag 600 attgacaagt acctcaagtc ctccaagtac attgcctggc ccctccaggg atggcaggcc 660 accttcggag gaggagacca cccccccaag tccgacggag gacgtgtctc caagggagaa 720 gaactcttca ccggagtcgt ccccattctc gtcgaactcg acggagacgt caacggacac 780 aagttctccg tctccggaga aggagaagga gacgccacct acggaaagct caccctcaag 840 ttcatttgca ccaccggaaa gctccccgtc ccctggccca ccctcgtcac caccctcacc 900 tacggagtcc agtgcttctc ccgttacccc gaccacatga agcagcacga cttcttcaag 960 tccgccatgc ccgaaggata cgtccaggaa cgtaccattt tcttcaagga cgacggaaac 1020 tacaagaccc gtgccgaagt caagttcgaa ggagacaccc tcgtcaaccg tattgaactc 1080 aagggaattg acttcaagga agacggaaac attctcggac acaagctcga atacaactac 1140 aactcccaca acgtctacat tatggccgac aagcagaaga acggaattaa ggtcaacttc 1200 aagattcgtc acaacattga agacggatcc gtccagctcg ccgaccacta ccagcagaac 1260 acccccattg gagacggacc cgtcctcctc cccgacaacc actacctctc cacccagtcc 1320 gccctctcca aggaccccaa cgaaaagcgt gaccacatgg tcctcctcga attcgtcacc 1380 gccgccggaa ttaccctcgg aatggacgaa ctctacaagt ccgacggagg acgtaccgga 1440 gaaaacctct acttccaggg attcaacctc ccccccggaa actacaagaa gcccaagctc 1500 ctctactgct ccaacggagg acacttcctc cgtattctcc ccgacggaac cgtcgacgga 1560 acccgtgacc gttccgacca gcacattcag ctccagctct ccgccgaatc cgtcggagaa 1620 gtctacatta agtccaccga aaccggacag tacctcgcca tggacaccga cggactcctc 1680 tacggatccc agacccccaa cgaagaatgc ctcttcctcg aacgtctcga agaaaaccac 1740 tacaacacct acatttccaa gaagcacgcc gaaaagaact ggttcgtcgg actcaagaag 1800 aacggatcct gcaagcgtgg accccgtacc cactacggac agaaggccat tctcttcctc 1860 cccctccccg tctcctccga ctaatctaga 1890 <210> 3 <211> 1941 <212> DNA <213> Artificial Sequence <220> <223> His-GST-GFP-TEV-hFGF2 (HGGTF2) <400> 3 gatatcacca tgcaccacca ccaccaccac gccggaatgt cccccattct cggatactgg 60 aagattaagg gactcgtcca gcccacccgt ctcctcctcg aatacctcga agaaaagtac 120 gaagaacacc tctacgaacg tgacgaagga gacaagtggc gtaacaagaa gttcgaactc 180 ggactcgaat tccccaacct cccctactac attgacggag acgtcaagct cacccagtcc 240 atggccatta ttcgttacat tgccgacaag cacaacatgc tcggaggatg ccccaaggaa 300 cgtgccgaaa tttccatgct cgaaggagcc gtcctcgaca ttcgttacgg agtctcccgt 360 attgcctact ccaaggactt cgaaaccctc aaggtcgact tcctctccaa gctccccgaa 420 atgctcaaga tgttcgaaga ccgtctctgc cacaagacct acctcaacgg agaccacgtc 480 acccaccccg acttcatgct ctacgacgcc ctcgacgtcg tcctctacat ggaccccatg 540 tgcctcgacg ccttccccaa gctcgtctgc ttcaagaagc gtattgaagc cattccccag 600 attgacaagt acctcaagtc ctccaagtac attgcctggc ccctccaggg atggcaggcc 660 accttcggag gaggagacca cccccccaag tccgacggag gacgtgtctc caagggagaa 720 gaactcttca ccggagtcgt ccccattctc gtcgaactcg acggagacgt caacggacac 780 aagttctccg tctccggaga aggagaagga gacgccacct acggaaagct caccctcaag 840 ttcatttgca ccaccggaaa gctccccgtc ccctggccca ccctcgtcac caccctcacc 900 tacggagtcc agtgcttctc ccgttacccc gaccacatga agcagcacga cttcttcaag 960 tccgccatgc ccgaaggata cgtccaggaa cgtaccattt tcttcaagga cgacggaaac 1020 tacaagaccc gtgccgaagt caagttcgaa ggagacaccc tcgtcaaccg tattgaactc 1080 aagggaattg acttcaagga agacggaaac attctcggac acaagctcga atacaactac 1140 aactcccaca acgtctacat tatggccgac aagcagaaga acggaattaa ggtcaacttc 1200 aagattcgtc acaacattga agacggatcc gtccagctcg ccgaccacta ccagcagaac 1260 acccccattg gagacggacc cgtcctcctc cccgacaacc actacctctc cacccagtcc 1320 gccctctcca aggaccccaa cgaaaagcgt gaccacatgg tcctcctcga attcgtcacc 1380 gccgccggaa ttaccctcgg aatggacgaa ctctacaagt ccgacggagg acgtaccgga 1440 gaaaacctct acttccaggg aggacacatg gccgccggat ccattaccac cctccccgcc 1500 ctccccgaag acggaggatc cggagccttc ccccccggac acttcaagga ccccaagcgt 1560 ctctactgca agaacggagg attcttcctc cgtattcacc ccgacggacg tgtcgacgga 1620 gtccgtgaaa agtccgaccc ccacattaag ctccagctcc aggccgaaga acgtggagtc 1680 gtctccatta agggagtctc cgccaaccgt tacctcgcca tgaaggaaga cggacgtctc 1740 ctcgcctcca agtccgtcac cgacgaatgc ttcttcttcg aacgtctcga atccaacaac 1800 tacaacacct accgttcccg taagtacacc tcctggtacg tcgccctcaa gcgtaccgga 1860 cagtacaagc tcggatccaa gaccggaccc ggacagaagg ccattctctt cctccccatg 1920 tccgccaagt cctaatctag a 1941 <210> 4 <211> 1680 <212> DNA <213> Artificial Sequence <220> <223> His-GST-GFP-TEV-IGF1 (HGGTI1) <400> 4 gatatcacca tgcaccacca ccaccaccac gccggaatgt cccccattct cggatactgg 60 aagattaagg gactcgtcca gcccacccgt ctcctcctcg aatacctcga agaaaagtac 120 gaagaacacc tctacgaacg tgacgaagga gacaagtggc gtaacaagaa gttcgaactc 180 ggactcgaat tccccaacct cccctactac attgacggag acgtcaagct cacccagtcc 240 atggccatta ttcgttacat tgccgacaag cacaacatgc tcggaggatg ccccaaggaa 300 cgtgccgaaa tttccatgct cgaaggagcc gtcctcgaca ttcgttacgg agtctcccgt 360 attgcctact ccaaggactt cgaaaccctc aaggtcgact tcctctccaa gctccccgaa 420 atgctcaaga tgttcgaaga ccgtctctgc cacaagacct acctcaacgg agaccacgtc 480 acccaccccg acttcatgct ctacgacgcc ctcgacgtcg tcctctacat ggaccccatg 540 tgcctcgacg ccttccccaa gctcgtctgc ttcaagaagc gtattgaagc cattccccag 600 attgacaagt acctcaagtc ctccaagtac attgcctggc ccctccaggg atggcaggcc 660 accttcggag gaggagacca cccccccaag tccgacggag gacgtgtctc caagggagaa 720 gaactcttca ccggagtcgt ccccattctc gtcgaactcg acggagacgt caacggacac 780 aagttctccg tctccggaga aggagaagga gacgccacct acggaaagct caccctcaag 840 ttcatttgca ccaccggaaa gctccccgtc ccctggccca ccctcgtcac caccctcacc 900 tacggagtcc agtgcttctc ccgttacccc gaccacatga agcagcacga cttcttcaag 960 tccgccatgc ccgaaggata cgtccaggaa cgtaccattt tcttcaagga cgacggaaac 1020 tacaagaccc gtgccgaagt caagttcgaa ggagacaccc tcgtcaaccg tattgaactc 1080 aagggaattg acttcaagga agacggaaac attctcggac acaagctcga atacaactac 1140 aactcccaca acgtctacat tatggccgac aagcagaaga acggaattaa ggtcaacttc 1200 aagattcgtc acaacattga agacggatcc gtccagctcg ccgaccacta ccagcagaac 1260 acccccattg gagacggacc cgtcctcctc cccgacaacc actacctctc cacccagtcc 1320 gccctctcca aggaccccaa cgaaaagcgt gaccacatgg tcctcctcga attcgtcacc 1380 gccgccggaa ttaccctcgg aatggacgaa ctctacaagt ccgacggagg acgtaccgga 1440 gaaaacctct acttccaggg aggacccgaa accctctgcg gagccgaact cgtcgacgcc 1500 ctccagttcg tctgcggaga ccgtggattc tacttcaaca agcccaccgg atacggatcc 1560 tcctcccgtc gtgcccccca gaccggaatt gtcgacgaat gctgcttccg ttcctgcgac 1620 ctccgtcgtc tcgaaatgta ctgcgccccc ctcaagcccg ccaagtccgc ctaatctaga 1680 1680 <210> 5 <211> 330 <212> DNA <213> Artificial Sequence <220> <223> pPha-HASP1p-HGGTI1 <400> 5 cagaagcgtg ctatcgaact caaccaggga cgtgcggcac aaatgggcat ccttgctctc 60 atggtgcacg aacagttggg agtctctatc cttccttaaa aatttaattt tcattagttg 120 cagtcactcc gctttggttt cacagtcagg aataacacta gctcgtcttc accatggatg 180 ccaatctcgc ctattcatgg tgtataaaag ttcaacatcc aaagctagaa cttttggaaa 240 gagaaagaat atccgaatag ggcacggcgt gccgtattgt tggagtggac tagcagaaag 300 tgaggaaggc acaggatgag ttttctcgag 330 <210> 6 <211> 862 <212> DNA <213> Artificial Sequence <220> <223> Shble cassete <400> 6 acataccttc agcgtcgtct tcactgtcac agtcaactga cagtaatcgt tgatccggag 60 agattcaaaa ttcaatctgt ttggacctgg ataagacaca agagcgacat cctgacatga 120 acgccgtaaa cagcaaatcc tggttgaaca cgtatccttt tgggggcctc cgctacgacg 180 ctcgctccag ctggggcttc cttactatac acagcgcgca tatttcacgg ttgccagatg 240 tcaagatggc caagttgacc agtgccgttc cggtgctcac cgcgcgcgac gtcgccggag 300 cggtcgagtt ctggaccgac cggctcgggt tctcccggga cttcgtggag gacgacttcg 360 ccggtgtggt ccgggacgac gtgaccctgt tcatcagcgc ggtccaggac caggtggtgc 420 cggacaacac cctggcctgg gtgtgggtgc gcggcctgga cgagctgtac gccgagtggt 480 cggaggtcgt gtccacgaac ttccgggacg cctccgggcc ggccatgacc gagatcggcg 540 agcagccgtg ggggcgggag ttcgccctgc gcgacccggc cggcaactgc gtgcacttcg 600 tggccgagga gcaggactga accttcctta aaaatttaat tttcattagt tgcagtcact 660 ccgctttggt ttcacagtca ggaataacac tagctcgtct tcaccatgga tgccaatctc 720 gcctattcat ggtgtataaa agttcaacat ccaaagctag aacttttgga aagagaaaga 780 atatccgaat agggcacggc gtgccgtatt gttggagtgg actagcagaa agtgaggaag 840 gcacaggatg agttttctcg ag 862 <210> 7 <211> 4059 <212> DNA <213> Artificial Sequence <220> <223> pPha-HASP1p vector full sequence <400> 7 catatgcata cagtgaatgt aactttcgaa ttgacagtat tagtagtcgt attgacagtg 60 aggcacgccc ctcaatgtgc gaggtggaaa atataccagc atgacaatga atcttggaga 120 ttcttttgct gtcatcaaga ttcaccgcca aatcttcagg aacctatcac gtccacaggc 180 gatgttaatt cttgagtcgt caaaacaaag tcctgtccta cctgtagaag ttgacagcga 240 gcaattgtat gcaaacttct gactttgtta taataacatt aaaggtaatt aagtatcttc 300 aattaggcat tttgtcactg tcagtccgtt ccgacaatat aggtagattt ggaatgaatc 360 ttttctatgc tgctgcgaat cttgtacacc tttgaggccg tagattctgt ccgacgaagc 420 gataattatt gcaaaataca tggactcatt attttgattc gatttctttt tggtatccga 480 ctcgaaaaga tccatcacgg cgagcgaatt cgatatcatc gactaattcg agctcggtac 540 ccggggatcc tctagagtcg acctgcaggc atgcaagctt cagaagcgtg ctatcgaact 600 caaccaggga cgtgcggcac aaatgggcat ccttgctctc atggtgcacg aacagttggg 660 agtctctatc cttccttaaa aatttaattt tcattagttg cagtcactcc gctttggttt 720 cacagtcagg aataacacta gctcgtcttc accatggatg ccaatctcgc ctattcatgg 780 tgtataaaag ttcaacatcc aaagctagaa cttttggaaa gagaaagaat atccgaatag 840 ggcacggcgt gccgtattgt tggagtggac tagcagaaag tgaggaaggc acaggatgag 900 ttttctcgag acataccttc agcgtcgtct tcactgtcac agtcaactga cagtaatcgt 960 tgatccggag agattcaaaa ttcaatctgt ttggacctgg ataagacaca agagcgacat 1020 cctgacatga acgccgtaaa cagcaaatcc tggttgaaca cgtatccttt tgggggcctc 1080 cgctacgacg ctcgctccag ctggggcttc cttactatac acagcgcgca tatttcacgg 1140 ttgccagatg tcaagatggc caagttgacc agtgccgttc cggtgctcac cgcgcgcgac 1200 gtcgccggag cggtcgagtt ctggaccgac cggctcgggt tctcccggga cttcgtggag 1260 gacgacttcg ccggtgtggt ccgggacgac gtgaccctgt tcatcagcgc ggtccaggac 1320 caggtggtgc cggacaacac cctggcctgg gtgtgggtgc gcggcctgga cgagctgtac 1380 gccgagtggt cggaggtcgt gtccacgaac ttccgggacg cctccgggcc ggccatgacc 1440 gagatcggcg agcagccgtg ggggcgggag ttcgccctgc gcgacccggc cggcaactgc 1500 gtgcacttcg tggccgagga gcaggactga accttcctta aaaatttaat tttcattagt 1560 tgcagtcact ccgctttggt ttcacagtca ggaataacac tagctcgtct tcaccatgga 1620 tgccaatctc gcctattcat ggtgtataaa agttcaacat ccaaagctag aacttttgga 1680 aagagaaaga atatccgaat agggcacggc gtgccgtatt gttggagtgg actagcagaa 1740 agtgaggaag gcacaggatg agttttctcg aggccggtct ccctatagtg agtcgtatta 1800 atttcgataa gccaggttaa cctgcattaa tgaatcggcc aacgcgcggg gagaggcggt 1860 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg 1920 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg 1980 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 2040 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga 2100 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 2160 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc 2220 tttctccctt cgggaagcgt ggcgctttct caatgctcac gctgtaggta tctcagttcg 2280 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc 2340 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca 2400 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag 2460 ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct 2520 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc 2580 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga 2640 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca 2700 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat 2760 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac 2820 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt 2880 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt 2940 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag 3000 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct 3060 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt 3120 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc 3180 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt 3240 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg 3300 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg 3360 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct 3420 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc 3480 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt 3540 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt 3600 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg 3660 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat 3720 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg 3780 cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta 3840 acctataaaa ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt 3900 gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc 3960 gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt 4020 aactatgcgg catcagagca gattgtactg agagtgcac 4059 <210> 8 <211> 5904 <212> DNA <213> Artificial Sequence <220> <223> pPha-HASP1p-HGGTF1 <400> 8 catatgcata cagtgaatgt aactttcgaa ttgacagtat tagtagtcgt attgacagtg 60 aggcacgccc ctcaatgtgc gaggtggaaa atataccagc atgacaatga atcttggaga 120 ttcttttgct gtcatcaaga ttcaccgcca aatcttcagg aacctatcac gtccacaggc 180 gatgttaatt cttgagtcgt caaaacaaag tcctgtccta cctgtagaag ttgacagcga 240 gcaattgtat gcaaacttct gactttgtta taataacatt aaaggtaatt aagtatcttc 300 aattaggcat tttgtcactg tcagtccgtt ccgacaatat aggtagattt ggaatgaatc 360 ttttctatgc tgctgcgaat cttgtacacc tttgaggccg tagattctgt ccgacgaagc 420 gataattatt gcaaaataca tggactcatt attttgattc gatttctttt tggtatccga 480 ctcgaaaaga tccatcacgg cgagcgaatt cgatatcacc atgcaccacc accaccacca 540 cgccggaatg tcccccattc tcggatactg gaagattaag ggactcgtcc agcccacccg 600 tctcctcctc gaatacctcg aagaaaagta cgaagaacac ctctacgaac gtgacgaagg 660 agacaagtgg cgtaacaaga agttcgaact cggactcgaa ttccccaacc tcccctacta 720 cattgacgga gacgtcaagc tcacccagtc catggccatt attcgttaca ttgccgacaa 780 gcacaacatg ctcggaggat gccccaagga acgtgccgaa atttccatgc tcgaaggagc 840 cgtcctcgac attcgttacg gagtctcccg tattgcctac tccaaggact tcgaaaccct 900 caaggtcgac ttcctctcca agctccccga aatgctcaag atgttcgaag accgtctctg 960 ccacaagacc tacctcaacg gagaccacgt cacccacccc gacttcatgc tctacgacgc 1020 cctcgacgtc gtcctctaca tggaccccat gtgcctcgac gccttcccca agctcgtctg 1080 cttcaagaag cgtattgaag ccattcccca gattgacaag tacctcaagt cctccaagta 1140 cattgcctgg cccctccagg gatggcaggc caccttcgga ggaggagacc acccccccaa 1200 gtccgacgga ggacgtgtct ccaagggaga agaactcttc accggagtcg tccccattct 1260 cgtcgaactc gacggagacg tcaacggaca caagttctcc gtctccggag aaggagaagg 1320 agacgccacc tacggaaagc tcaccctcaa gttcatttgc accaccggaa agctccccgt 1380 cccctggccc accctcgtca ccaccctcac ctacggagtc cagtgcttct cccgttaccc 1440 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggat acgtccagga 1500 acgtaccatt ttcttcaagg acgacggaaa ctacaagacc cgtgccgaag tcaagttcga 1560 aggagacacc ctcgtcaacc gtattgaact caagggaatt gacttcaagg aagacggaaa 1620 cattctcgga cacaagctcg aatacaacta caactcccac aacgtctaca ttatggccga 1680 caagcagaag aacggaatta aggtcaactt caagattcgt cacaacattg aagacggatc 1740 cgtccagctc gccgaccact accagcagaa cacccccatt ggagacggac ccgtcctcct 1800 ccccgacaac cactacctct ccacccagtc cgccctctcc aaggacccca acgaaaagcg 1860 tgaccacatg gtcctcctcg aattcgtcac cgccgccgga attaccctcg gaatggacga 1920 actctacaag tccgacggag gacgtaccgg agaaaacctc tacttccagg gattcaacct 1980 cccccccgga aactacaaga agcccaagct cctctactgc tccaacggag gacacttcct 2040 ccgtattctc cccgacggaa ccgtcgacgg aacccgtgac cgttccgacc agcacattca 2100 gctccagctc tccgccgaat ccgtcggaga agtctacatt aagtccaccg aaaccggaca 2160 gtacctcgcc atggacaccg acggactcct ctacggatcc cagaccccca acgaagaatg 2220 cctcttcctc gaacgtctcg aagaaaacca ctacaacacc tacatttcca agaagcacgc 2280 cgaaaagaac tggttcgtcg gactcaagaa gaacggatcc tgcaagcgtg gaccccgtac 2340 ccactacgga cagaaggcca ttctcttcct ccccctcccc gtctcctccg actaatctag 2400 agtcgacctg caggcatgca agcttcagaa gcgtgctatc gaactcaacc agggacgtgc 2460 ggcacaaatg ggcatccttg ctctcatggt gcacgaacag ttgggagtct ctatccttcc 2520 ttaaaaattt aattttcatt agttgcagtc actccgcttt ggtttcacag tcaggaataa 2580 cactagctcg tcttcaccat ggatgccaat ctcgcctatt catggtgtat aaaagttcaa 2640 catccaaagc tagaactttt ggaaagagaa agaatatccg aatagggcac ggcgtgccgt 2700 attgttggag tggactagca gaaagtgagg aaggcacagg atgagttttc tcgagacata 2760 ccttcagcgt cgtcttcact gtcacagtca actgacagta atcgttgatc cggagagatt 2820 caaaattcaa tctgtttgga cctggataag acacaagagc gacatcctga catgaacgcc 2880 gtaaacagca aatcctggtt gaacacgtat ccttttgggg gcctccgcta cgacgctcgc 2940 tccagctggg gcttccttac tatacacagc gcgcatattt cacggttgcc agatgtcaag 3000 atggccaagt tgaccagtgc cgttccggtg ctcaccgcgc gcgacgtcgc cggagcggtc 3060 gagttctgga ccgaccggct cgggttctcc cgggacttcg tggaggacga cttcgccggt 3120 gtggtccggg acgacgtgac cctgttcatc agcgcggtcc aggaccaggt ggtgccggac 3180 aacaccctgg cctgggtgtg ggtgcgcggc ctggacgagc tgtacgccga gtggtcggag 3240 gtcgtgtcca cgaacttccg ggacgcctcc gggccggcca tgaccgagat cggcgagcag 3300 ccgtgggggc gggagttcgc cctgcgcgac ccggccggca actgcgtgca cttcgtggcc 3360 gaggagcagg actgaacctt ccttaaaaat ttaattttca ttagttgcag tcactccgct 3420 ttggtttcac agtcaggaat aacactagct cgtcttcacc atggatgcca atctcgccta 3480 ttcatggtgt ataaaagttc aacatccaaa gctagaactt ttggaaagag aaagaatatc 3540 cgaatagggc acggcgtgcc gtattgttgg agtggactag cagaaagtga ggaaggcaca 3600 ggatgagttt tctcgaggcc ggtctcccta tagtgagtcg tattaatttc gataagccag 3660 gttaacctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc 3720 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta 3780 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag 3840 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg 3900 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg 3960 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg 4020 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga 4080 agcgtggcgc tttctcaatg ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc 4140 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt 4200 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact 4260 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg 4320 cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt 4380 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt 4440 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct 4500 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg 4560 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt 4620 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt 4680 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc 4740 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg 4800 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc 4860 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg 4920 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca 4980 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga 5040 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct 5100 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg 5160 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca 5220 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata 5280 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct 5340 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact 5400 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa 5460 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc 5520 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga 5580 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga 5640 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg 5700 cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac 5760 atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc 5820 cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta tgcggcatca 5880 gagcagattg tactgagagt gcac 5904 <210> 9 <211> 5955 <212> DNA <213> Artificial Sequence <220> <223> pPha-HASP1p-HGGTF2 <400> 9 catatgcata cagtgaatgt aactttcgaa ttgacagtat tagtagtcgt attgacagtg 60 aggcacgccc ctcaatgtgc gaggtggaaa atataccagc atgacaatga atcttggaga 120 ttcttttgct gtcatcaaga ttcaccgcca aatcttcagg aacctatcac gtccacaggc 180 gatgttaatt cttgagtcgt caaaacaaag tcctgtccta cctgtagaag ttgacagcga 240 gcaattgtat gcaaacttct gactttgtta taataacatt aaaggtaatt aagtatcttc 300 aattaggcat tttgtcactg tcagtccgtt ccgacaatat aggtagattt ggaatgaatc 360 ttttctatgc tgctgcgaat cttgtacacc tttgaggccg tagattctgt ccgacgaagc 420 gataattatt gcaaaataca tggactcatt attttgattc gatttctttt tggtatccga 480 ctcgaaaaga tccatcacgg cgagcgaatt cgatatcacc atgcaccacc accaccacca 540 cgccggaatg tcccccattc tcggatactg gaagattaag ggactcgtcc agcccacccg 600 tctcctcctc gaatacctcg aagaaaagta cgaagaacac ctctacgaac gtgacgaagg 660 agacaagtgg cgtaacaaga agttcgaact cggactcgaa ttccccaacc tcccctacta 720 cattgacgga gacgtcaagc tcacccagtc catggccatt attcgttaca ttgccgacaa 780 gcacaacatg ctcggaggat gccccaagga acgtgccgaa atttccatgc tcgaaggagc 840 cgtcctcgac attcgttacg gagtctcccg tattgcctac tccaaggact tcgaaaccct 900 caaggtcgac ttcctctcca agctccccga aatgctcaag atgttcgaag accgtctctg 960 ccacaagacc tacctcaacg gagaccacgt cacccacccc gacttcatgc tctacgacgc 1020 cctcgacgtc gtcctctaca tggaccccat gtgcctcgac gccttcccca agctcgtctg 1080 cttcaagaag cgtattgaag ccattcccca gattgacaag tacctcaagt cctccaagta 1140 cattgcctgg cccctccagg gatggcaggc caccttcgga ggaggagacc acccccccaa 1200 gtccgacgga ggacgtgtct ccaagggaga agaactcttc accggagtcg tccccattct 1260 cgtcgaactc gacggagacg tcaacggaca caagttctcc gtctccggag aaggagaagg 1320 agacgccacc tacggaaagc tcaccctcaa gttcatttgc accaccggaa agctccccgt 1380 cccctggccc accctcgtca ccaccctcac ctacggagtc cagtgcttct cccgttaccc 1440 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggat acgtccagga 1500 acgtaccatt ttcttcaagg acgacggaaa ctacaagacc cgtgccgaag tcaagttcga 1560 aggagacacc ctcgtcaacc gtattgaact caagggaatt gacttcaagg aagacggaaa 1620 cattctcgga cacaagctcg aatacaacta caactcccac aacgtctaca ttatggccga 1680 caagcagaag aacggaatta aggtcaactt caagattcgt cacaacattg aagacggatc 1740 cgtccagctc gccgaccact accagcagaa cacccccatt ggagacggac ccgtcctcct 1800 ccccgacaac cactacctct ccacccagtc cgccctctcc aaggacccca acgaaaagcg 1860 tgaccacatg gtcctcctcg aattcgtcac cgccgccgga attaccctcg gaatggacga 1920 actctacaag tccgacggag gacgtaccgg agaaaacctc tacttccagg gaggacacat 1980 ggccgccgga tccattacca ccctccccgc cctccccgaa gacggaggat ccggagcctt 2040 cccccccgga cacttcaagg accccaagcg tctctactgc aagaacggag gattcttcct 2100 ccgtattcac cccgacggac gtgtcgacgg agtccgtgaa aagtccgacc cccacattaa 2160 gctccagctc caggccgaag aacgtggagt cgtctccatt aagggagtct ccgccaaccg 2220 ttacctcgcc atgaaggaag acggacgtct cctcgcctcc aagtccgtca ccgacgaatg 2280 cttcttcttc gaacgtctcg aatccaacaa ctacaacacc taccgttccc gtaagtacac 2340 ctcctggtac gtcgccctca agcgtaccgg acagtacaag ctcggatcca agaccggacc 2400 cggacagaag gccattctct tcctccccat gtccgccaag tcctaatcta gagtcgacct 2460 gcaggcatgc aagcttcaga agcgtgctat cgaactcaac cagggacgtg cggcacaaat 2520 gggcatcctt gctctcatgg tgcacgaaca gttgggagtc tctatccttc cttaaaaatt 2580 taattttcat tagttgcagt cactccgctt tggtttcaca gtcaggaata acactagctc 2640 gtcttcacca tggatgccaa tctcgcctat tcatggtgta taaaagttca acatccaaag 2700 ctagaacttt tggaaagaga aagaatatcc gaatagggca cggcgtgccg tattgttgga 2760 gtggactagc agaaagtgag gaaggcacag gatgagtttt ctcgagacat accttcagcg 2820 tcgtcttcac tgtcacagtc aactgacagt aatcgttgat ccggagagat tcaaaattca 2880 atctgtttgg acctggataa gacacaagag cgacatcctg acatgaacgc cgtaaacagc 2940 aaatcctggt tgaacacgta tccttttggg ggcctccgct acgacgctcg ctccagctgg 3000 ggcttcctta ctatacacag cgcgcatatt tcacggttgc cagatgtcaa gatggccaag 3060 ttgaccagtg ccgttccggt gctcaccgcg cgcgacgtcg ccggagcggt cgagttctgg 3120 accgaccggc tcgggttctc ccgggacttc gtggaggacg acttcgccgg tgtggtccgg 3180 gacgacgtga ccctgttcat cagcgcggtc caggaccagg tggtgccgga caacaccctg 3240 gcctgggtgt gggtgcgcgg cctggacgag ctgtacgccg agtggtcgga ggtcgtgtcc 3300 acgaacttcc gggacgcctc cgggccggcc atgaccgaga tcggcgagca gccgtggggg 3360 cgggagttcg ccctgcgcga cccggccggc aactgcgtgc acttcgtggc cgaggagcag 3420 gactgaacct tccttaaaaa tttaattttc attagttgca gtcactccgc tttggtttca 3480 cagtcaggaa taacactagc tcgtcttcac catggatgcc aatctcgcct attcatggtg 3540 tataaaagtt caacatccaa agctagaact tttggaaaga gaaagaatat ccgaataggg 3600 cacggcgtgc cgtattgttg gagtggacta gcagaaagtg aggaaggcac aggatgagtt 3660 ttctcgaggc cggtctccct atagtgagtc gtattaattt cgataagcca ggttaacctg 3720 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct 3780 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac 3840 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga 3900 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat 3960 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac 4020 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct 4080 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg 4140 ctttctcaat gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg 4200 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt 4260 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg 4320 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac 4380 ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga 4440 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt 4500 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt 4560 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga 4620 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc 4680 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct 4740 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata 4800 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca 4860 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga 4920 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga 4980 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg 5040 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga 5100 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt 5160 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct 5220 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca 5280 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat 5340 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga 5400 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc 5460 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg 5520 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc 5580 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt 5640 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca 5700 cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg 5760 aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc 5820 ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc 5880 gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact atgcggcatc agagcagatt 5940 gtactgagag tgcac 5955 <210> 10 <211> 5694 <212> DNA <213> Artificial Sequence <220> <223> pPha-HASP1p-HGGTI1 <400> 10 catatgcata cagtgaatgt aactttcgaa ttgacagtat tagtagtcgt attgacagtg 60 aggcacgccc ctcaatgtgc gaggtggaaa atataccagc atgacaatga atcttggaga 120 ttcttttgct gtcatcaaga ttcaccgcca aatcttcagg aacctatcac gtccacaggc 180 gatgttaatt cttgagtcgt caaaacaaag tcctgtccta cctgtagaag ttgacagcga 240 gcaattgtat gcaaacttct gactttgtta taataacatt aaaggtaatt aagtatcttc 300 aattaggcat tttgtcactg tcagtccgtt ccgacaatat aggtagattt ggaatgaatc 360 ttttctatgc tgctgcgaat cttgtacacc tttgaggccg tagattctgt ccgacgaagc 420 gataattatt gcaaaataca tggactcatt attttgattc gatttctttt tggtatccga 480 ctcgaaaaga tccatcacgg cgagcgaatt cgatatcacc atgcaccacc accaccacca 540 cgccggaatg tcccccattc tcggatactg gaagattaag ggactcgtcc agcccacccg 600 tctcctcctc gaatacctcg aagaaaagta cgaagaacac ctctacgaac gtgacgaagg 660 agacaagtgg cgtaacaaga agttcgaact cggactcgaa ttccccaacc tcccctacta 720 cattgacgga gacgtcaagc tcacccagtc catggccatt attcgttaca ttgccgacaa 780 gcacaacatg ctcggaggat gccccaagga acgtgccgaa atttccatgc tcgaaggagc 840 cgtcctcgac attcgttacg gagtctcccg tattgcctac tccaaggact tcgaaaccct 900 caaggtcgac ttcctctcca agctccccga aatgctcaag atgttcgaag accgtctctg 960 ccacaagacc tacctcaacg gagaccacgt cacccacccc gacttcatgc tctacgacgc 1020 cctcgacgtc gtcctctaca tggaccccat gtgcctcgac gccttcccca agctcgtctg 1080 cttcaagaag cgtattgaag ccattcccca gattgacaag tacctcaagt cctccaagta 1140 cattgcctgg cccctccagg gatggcaggc caccttcgga ggaggagacc acccccccaa 1200 gtccgacgga ggacgtgtct ccaagggaga agaactcttc accggagtcg tccccattct 1260 cgtcgaactc gacggagacg tcaacggaca caagttctcc gtctccggag aaggagaagg 1320 agacgccacc tacggaaagc tcaccctcaa gttcatttgc accaccggaa agctccccgt 1380 cccctggccc accctcgtca ccaccctcac ctacggagtc cagtgcttct cccgttaccc 1440 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggat acgtccagga 1500 acgtaccatt ttcttcaagg acgacggaaa ctacaagacc cgtgccgaag tcaagttcga 1560 aggagacacc ctcgtcaacc gtattgaact caagggaatt gacttcaagg aagacggaaa 1620 cattctcgga cacaagctcg aatacaacta caactcccac aacgtctaca ttatggccga 1680 caagcagaag aacggaatta aggtcaactt caagattcgt cacaacattg aagacggatc 1740 cgtccagctc gccgaccact accagcagaa cacccccatt ggagacggac ccgtcctcct 1800 ccccgacaac cactacctct ccacccagtc cgccctctcc aaggacccca acgaaaagcg 1860 tgaccacatg gtcctcctcg aattcgtcac cgccgccgga attaccctcg gaatggacga 1920 actctacaag tccgacggag gacgtaccgg agaaaacctc tacttccagg gaggacccga 1980 aaccctctgc ggagccgaac tcgtcgacgc cctccagttc gtctgcggag accgtggatt 2040 ctacttcaac aagcccaccg gatacggatc ctcctcccgt cgtgcccccc agaccggaat 2100 tgtcgacgaa tgctgcttcc gttcctgcga cctccgtcgt ctcgaaatgt actgcgcccc 2160 cctcaagccc gccaagtccg cctaatctag agtcgacctg caggcatgca agcttcagaa 2220 gcgtgctatc gaactcaacc agggacgtgc ggcacaaatg ggcatccttg ctctcatggt 2280 gcacgaacag ttgggagtct ctatccttcc ttaaaaattt aattttcatt agttgcagtc 2340 actccgcttt ggtttcacag tcaggaataa cactagctcg tcttcaccat ggatgccaat 2400 ctcgcctatt catggtgtat aaaagttcaa catccaaagc tagaactttt ggaaagagaa 2460 agaatatccg aatagggcac ggcgtgccgt attgttggag tggactagca gaaagtgagg 2520 aaggcacagg atgagttttc tcgagacata ccttcagcgt cgtcttcact gtcacagtca 2580 actgacagta atcgttgatc cggagagatt caaaattcaa tctgtttgga cctggataag 2640 acacaagagc gacatcctga catgaacgcc gtaaacagca aatcctggtt gaacacgtat 2700 ccttttgggg gcctccgcta cgacgctcgc tccagctggg gcttccttac tatacacagc 2760 gcgcatattt cacggttgcc agatgtcaag atggccaagt tgaccagtgc cgttccggtg 2820 ctcaccgcgc gcgacgtcgc cggagcggtc gagttctgga ccgaccggct cgggttctcc 2880 cgggacttcg tggaggacga cttcgccggt gtggtccggg acgacgtgac cctgttcatc 2940 agcgcggtcc aggaccaggt ggtgccggac aacaccctgg cctgggtgtg ggtgcgcggc 3000 ctggacgagc tgtacgccga gtggtcggag gtcgtgtcca cgaacttccg ggacgcctcc 3060 gggccggcca tgaccgagat cggcgagcag ccgtgggggc gggagttcgc cctgcgcgac 3120 ccggccggca actgcgtgca cttcgtggcc gaggagcagg actgaacctt ccttaaaaat 3180 ttaattttca ttagttgcag tcactccgct ttggtttcac agtcaggaat aacactagct 3240 cgtcttcacc atggatgcca atctcgccta ttcatggtgt ataaaagttc aacatccaaa 3300 gctagaactt ttggaaagag aaagaatatc cgaatagggc acggcgtgcc gtattgttgg 3360 agtggactag cagaaagtga ggaaggcaca ggatgagttt tctcgaggcc ggtctcccta 3420 tagtgagtcg tattaatttc gataagccag gttaacctgc attaatgaat cggccaacgc 3480 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg 3540 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta 3600 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc 3660 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag 3720 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac 3780 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc 3840 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcaatg ctcacgctgt 3900 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc 3960 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga 4020 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta 4080 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta 4140 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga 4200 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg 4260 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag 4320 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc 4380 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact 4440 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt 4500 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta 4560 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta 4620 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc 4680 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat 4740 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt 4800 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg 4860 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca 4920 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta 4980 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg 5040 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact 5100 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg 5160 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt 5220 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga 5280 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc 5340 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa 5400 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt 5460 attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt 5520 ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt 5580 ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg 5640 tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt gcac 5694 <110> ALGAEPRONA INC. <120> The vectors for expressing hIGF1 transformed microalgae for expression those growth factors <130> SP1102 <160> 10 <170> KoPatentIn 3.0 <210> 1 <211> 499 <212> DNA <213> Phaeodactylum tricornutum <220> <221> promoter <222> (1)..(499) <223> HASP1 promoter <400> 1 catacagtga atgtaacttt cgaattgaca gtattagtag tcgtattgac agtgaggcac 60 gcccctcaat gtgcgaggtg gaaaatatac cagcatgaca atgaatcttg gagattcttt 120 tgctgtcatc aagattcacc gccaaatctt caggaaccta tcacgtccac aggcgatgtt 180 aattcttgag tcgtcaaaac aaagtcctgt cctacctgta gaagttgaca gcgagcaatt 240 gtatgcaaac ttctgacttt gttataataa cattaaaggt aattaagtat cttcaattag 300 gcattttgtc actgtcagtc cgttccgaca atataggtag atttggaatg aatcttttct 360 atgctgctgc gaatcttgta cacctttgag gccgtagatt ctgtccgacg aagcgataat 420 tattgcaaaa tacatggact cattattttg attcgatttc tttttggtat ccgactcgaa 480 aagatccatc acggcgagc 499 <210> 2 <211> 1890 <212> DNA <213> Artificial Sequence <220> <223> His-GST-GFP-TEV-hFGF1 (HGGTF1) <400> 2 gatatcacca tgcaccacca ccaccaccac gccggaatgt cccccattct cggatactgg 60 aagattaagg gactcgtcca gcccacccgt ctcctcctcg aatacctcga agaaaagtac 120 gaagaacacc tctacgaacg tgacgaagga gacaagtggc gtaacaagaa gttcgaactc 180 ggactcgaat tccccaacct cccctactac attgacggag acgtcaagct cacccagtcc 240 atggccatta ttcgttacat tgccgacaag cacaacatgc tcggaggatg ccccaaggaa 300 cgtgccgaaa tttccatgct cgaaggagcc gtcctcgaca ttcgttacgg agtctcccgt 360 attgcctact ccaaggactt cgaaaccctc aaggtcgact tcctctccaa gctccccgaa 420 atgctcaaga tgttcgaaga ccgtctctgc cacaagacct acctcaacgg agaccacgtc 480 acccaccccg acttcatgct ctacgacgcc ctcgacgtcg tcctctacat ggaccccatg 540 tgcctcgacg ccttccccaa gctcgtctgc ttcaagaagc gtattgaagc cattccccag 600 attgacaagt acctcaagtc ctccaagtac attgcctggc ccctccaggg atggcaggcc 660 accttcggag gaggagacca cccccccaag tccgacggag gacgtgtctc caagggagaa 720 gaactcttca ccggagtcgt ccccattctc gtcgaactcg acggagacgt caacggacac 780 aagttctccg tctccggaga aggagaagga gacgccacct acggaaagct caccctcaag 840 ttcatttgca ccaccggaaa gctccccgtc ccctggccca ccctcgtcac caccctcacc 900 tacggagtcc agtgcttctc ccgttacccc gaccacatga agcagcacga cttcttcaag 960 tccgccatgc ccgaaggata cgtccaggaa cgtaccattt tcttcaagga cgacggaaac 1020 tacaagaccc gtgccgaagt caagttcgaa ggagacaccc tcgtcaaccg tattgaactc 1080 aagggaattg acttcaagga agacggaaac attctcggac acaagctcga atacaactac 1140 aactcccaca acgtctacat tatggccgac aagcagaaga acggaattaa ggtcaacttc 1200 aagattcgtc acaacattga agacggatcc gtccagctcg ccgaccacta ccagcagaac 1260 acccccattg gagacggacc cgtcctcctc cccgacaacc actacctctc cacccagtcc 1320 gccctctcca aggaccccaa cgaaaagcgt gaccacatgg tcctcctcga attcgtcacc 1380 gccgccggaa ttaccctcgg aatggacgaa ctctacaagt ccgacggagg acgtaccgga 1440 gaaaacctct acttccaggg attcaacctc ccccccggaa actacaagaa gcccaagctc 1500 ctctactgct ccaacggagg acacttcctc cgtattctcc ccgacggaac cgtcgacgga 1560 acccgtgacc gttccgacca gcacattcag ctccagctct ccgccgaatc cgtcggagaa 1620 gtctacatta agtccaccga aaccggacag tacctcgcca tggacaccga cggactcctc 1680 tacggatccc agacccccaa cgaagaatgc ctcttcctcg aacgtctcga agaaaaccac 1740 tacaacacct acatttccaa gaagcacgcc gaaaagaact ggttcgtcgg actcaagaag 1800 aacggatcct gcaagcgtgg accccgtacc cactacggac agaaggccat tctcttcctc 1860 cccctccccg tctcctccga ctaatctaga 1890 <210> 3 <211> 1941 <212> DNA <213> Artificial Sequence <220> <223> His-GST-GFP-TEV-hFGF2 (HGGTF2) <400> 3 gatatcacca tgcaccacca ccaccaccac gccggaatgt cccccattct cggatactgg 60 aagattaagg gactcgtcca gcccacccgt ctcctcctcg aatacctcga agaaaagtac 120 gaagaacacc tctacgaacg tgacgaagga gacaagtggc gtaacaagaa gttcgaactc 180 ggactcgaat tccccaacct cccctactac attgacggag acgtcaagct cacccagtcc 240 atggccatta ttcgttacat tgccgacaag cacaacatgc tcggaggatg ccccaaggaa 300 cgtgccgaaa tttccatgct cgaaggagcc gtcctcgaca ttcgttacgg agtctcccgt 360 attgcctact ccaaggactt cgaaaccctc aaggtcgact tcctctccaa gctccccgaa 420 atgctcaaga tgttcgaaga ccgtctctgc cacaagacct acctcaacgg agaccacgtc 480 acccaccccg acttcatgct ctacgacgcc ctcgacgtcg tcctctacat ggaccccatg 540 tgcctcgacg ccttccccaa gctcgtctgc ttcaagaagc gtattgaagc cattccccag 600 attgacaagt acctcaagtc ctccaagtac attgcctggc ccctccaggg atggcaggcc 660 accttcggag gaggagacca cccccccaag tccgacggag gacgtgtctc caagggagaa 720 gaactcttca ccggagtcgt ccccattctc gtcgaactcg acggagacgt caacggacac 780 aagttctccg tctccggaga aggagaagga gacgccacct acggaaagct caccctcaag 840 ttcatttgca ccaccggaaa gctccccgtc ccctggccca ccctcgtcac caccctcacc 900 tacggagtcc agtgcttctc ccgttacccc gaccacatga agcagcacga cttcttcaag 960 tccgccatgc ccgaaggata cgtccaggaa cgtaccattt tcttcaagga cgacggaaac 1020 tacaagaccc gtgccgaagt caagttcgaa ggagacaccc tcgtcaaccg tattgaactc 1080 aagggaattg acttcaagga agacggaaac attctcggac acaagctcga atacaactac 1140 aactcccaca acgtctacat tatggccgac aagcagaaga acggaattaa ggtcaacttc 1200 aagattcgtc acaacattga agacggatcc gtccagctcg ccgaccacta ccagcagaac 1260 acccccattg gagacggacc cgtcctcctc cccgacaacc actacctctc cacccagtcc 1320 gccctctcca aggaccccaa cgaaaagcgt gaccacatgg tcctcctcga attcgtcacc 1380 gccgccggaa ttaccctcgg aatggacgaa ctctacaagt ccgacggagg acgtaccgga 1440 gaaaacctct acttccaggg aggacacatg gccgccggat ccattaccac cctccccgcc 1500 ctccccgaag acggaggatc cggagccttc ccccccggac acttcaagga ccccaagcgt 1560 ctctactgca agaacggagg attcttcctc cgtattcacc ccgacggacg tgtcgacgga 1620 gtccgtgaaa agtccgaccc ccacattaag ctccagctcc aggccgaaga acgtggagtc 1680 gtctccatta agggagtctc cgccaaccgt tacctcgcca tgaaggaaga cggacgtctc 1740 ctcgcctcca agtccgtcac cgacgaatgc ttcttcttcg aacgtctcga atccaacaac 1800 tacaacacct accgttcccg taagtacacc tcctggtacg tcgccctcaa gcgtaccgga 1860 cagtacaagc tcggatccaa gaccggaccc ggacagaagg ccattctctt cctcccccatg 1920 tccgccaagt cctaatctag a 1941 <210> 4 <211> 1680 <212> DNA <213> Artificial Sequence <220> <223> His-GST-GFP-TEV-IGF1 (HGGTI1) <400> 4 gatatcacca tgcaccacca ccaccaccac gccggaatgt cccccattct cggatactgg 60 aagattaagg gactcgtcca gcccacccgt ctcctcctcg aatacctcga agaaaagtac 120 gaagaacacc tctacgaacg tgacgaagga gacaagtggc gtaacaagaa gttcgaactc 180 ggactcgaat tccccaacct cccctactac attgacggag acgtcaagct cacccagtcc 240 atggccatta ttcgttacat tgccgacaag cacaacatgc tcggaggatg ccccaaggaa 300 cgtgccgaaa tttccatgct cgaaggagcc gtcctcgaca ttcgttacgg agtctcccgt 360 attgcctact ccaaggactt cgaaaccctc aaggtcgact tcctctccaa gctccccgaa 420 atgctcaaga tgttcgaaga ccgtctctgc cacaagacct acctcaacgg agaccacgtc 480 acccaccccg acttcatgct ctacgacgcc ctcgacgtcg tcctctacat ggaccccatg 540 tgcctcgacg ccttccccaa gctcgtctgc ttcaagaagc gtattgaagc cattccccag 600 attgacaagt acctcaagtc ctccaagtac attgcctggc ccctccaggg atggcaggcc 660 accttcggag gaggagacca cccccccaag tccgacggag gacgtgtctc caagggagaa 720 gaactcttca ccggagtcgt ccccattctc gtcgaactcg acggagacgt caacggacac 780 aagttctccg tctccggaga aggagaagga gacgccacct acggaaagct caccctcaag 840 ttcatttgca ccaccggaaa gctccccgtc ccctggccca ccctcgtcac caccctcacc 900 tacggagtcc agtgcttctc ccgttacccc gaccacatga agcagcacga cttcttcaag 960 tccgccatgc ccgaaggata cgtccaggaa cgtaccattt tcttcaagga cgacggaaac 1020 tacaagaccc gtgccgaagt caagttcgaa ggagacaccc tcgtcaaccg tattgaactc 1080 aagggaattg acttcaagga agacggaaac attctcggac acaagctcga atacaactac 1140 aactcccaca acgtctacat tatggccgac aagcagaaga acggaattaa ggtcaacttc 1200 aagattcgtc acaacattga agacggatcc gtccagctcg ccgaccacta ccagcagaac 1260 acccccattg gagacggacc cgtcctcctc cccgacaacc actacctctc cacccagtcc 1320 gccctctcca aggaccccaa cgaaaagcgt gaccacatgg tcctcctcga attcgtcacc 1380 gccgccggaa ttaccctcgg aatggacgaa ctctacaagt ccgacggagg acgtaccgga 1440 gaaaacctct acttccaggg aggacccgaa accctctgcg gagccgaact cgtcgacgcc 1500 ctccagttcg tctgcggaga ccgtggattc tacttcaaca agcccaccgg atacggatcc 1560 tcctcccgtc gtgcccccca gaccggaatt gtcgacgaat gctgcttccg ttcctgcgac 1620 ctccgtcgtc tcgaaatgta ctgcgccccc ctcaagcccg ccaagtccgc ctaatctaga 1680 1680 <210> 5 <211> 330 <212> DNA <213> Artificial Sequence <220> <223> pPha-HASP1p-HGGTI1 <400> 5 cagaagcgtg ctatcgaact caaccaggga cgtgcggcac aaatgggcat ccttgctctc 60 atggtgcacg aacagttggg agtctctatc cttccttaaa aatttaattt tcattagttg 120 cagtcactcc gctttggttt cacagtcagg aataacacta gctcgtcttc accatggatg 180 ccaatctcgc ctattcatgg tgtataaaag ttcaacatcc aaagctagaa cttttggaaa 240 gagaaagaat atccgaatag ggcacggcgt gccgtattgt tggagtggac tagcagaaag 300 tgaggaaggc acaggatgag ttttctcgag 330 <210> 6 <211> 862 <212> DNA <213> Artificial Sequence <220> <223> Shble casete <400> 6 acataccttc agcgtcgtct tcactgtcac agtcaactga cagtaatcgt tgatccggag 60 agattcaaaa ttcaatctgt ttggacctgg ataagacaca agagcgacat cctgacatga 120 acgccgtaaa cagcaaatcc tggttgaaca cgtatccttt tgggggcctc cgctacgacg 180 ctcgctccag ctggggcttc cttactatac acagcgcgca tatttcacgg ttgccagatg 240 tcaagatggc caagttgacc agtgccgttc cggtgctcac cgcgcgcgac gtcgccggag 300 cggtcgagtt ctggaccgac cggctcgggt tctcccggga cttcgtggag gacgacttcg 360 ccggtgtggt ccgggacgac gtgaccctgt tcatcagcgc ggtccaggac caggtggtgc 420 cggacaacac cctggcctgg gtgtgggtgc gcggcctgga cgagctgtac gccgagtggt 480 cggaggtcgt gtccacgaac ttccgggacg cctccgggcc ggccatgacc gagatcggcg 540 agcagccgtg ggggcgggag ttcgccctgc gcgacccggc cggcaactgc gtgcacttcg 600 tggccgagga gcaggactga accttcctta aaaatttaat tttcattagt tgcagtcact 660 ccgctttggt ttcacagtca ggaataacac tagctcgtct tcaccatgga tgccaatctc 720 gcctattcat ggtgtataaa agttcaacat ccaaagctag aacttttgga aagagaaaga 780 atatccgaat agggcacggc gtgccgtatt gttggagtgg actagcagaa agtgaggaag 840 gcacaggatg agttttctcg ag 862 <210> 7 <211> 4059 <212> DNA <213> Artificial Sequence <220> <223> pPha-HASP1p vector full sequence <400> 7 catatgcata cagtgaatgt aactttcgaa ttgacagtat tagtagtcgt attgacagtg 60 aggcacgccc ctcaatgtgc gaggtggaaa atataccagc atgacaatga atcttggaga 120 ttcttttgct gtcatcaaga ttcaccgcca aatcttcagg aacctatcac gtccacaggc 180 gatgttaatt cttgagtcgt caaaacaaag tcctgtccta cctgtagaag ttgacagcga 240 gcaattgtat gcaaacttct gactttgtta taataacatt aaaggtaatt aagtatcttc 300 aattaggcat tttgtcactg tcagtccgtt ccgacaatat aggtagattt ggaatgaatc 360 ttttctatgc tgctgcgaat cttgtacacc tttgaggccg tagattctgt ccgacgaagc 420 gataattatt gcaaaataca tggactcatt attttgattc gatttctttt tggtatccga 480 ctcgaaaaga tccatcacgg cgagcgaatt cgatatcatc gactaattcg agctcggtac 540 ccggggatcc tctagagtcg acctgcaggc atgcaagctt cagaagcgtg ctatcgaact 600 caaccaggga cgtgcggcac aaatgggcat ccttgctctc atggtgcacg aacagttggg 660 agtctctatc cttccttaaa aatttaattt tcattagttg cagtcactcc gctttggttt 720 cacagtcagg aataacacta gctcgtcttc accatggatg ccaatctcgc ctattcatgg 780 tgtataaaag ttcaacatcc aaagctagaa cttttggaaa gagaaagaat atccgaatag 840 ggcacggcgt gccgtattgt tggagtggac tagcagaaag tgaggaaggc acaggatgag 900 ttttctcgag acataccttc agcgtcgtct tcactgtcac agtcaactga cagtaatcgt 960 tgatccggag agattcaaaa ttcaatctgt ttggacctgg ataagacaca agagcgacat 1020 cctgacatga acgccgtaaa cagcaaatcc tggttgaaca cgtatccttt tgggggcctc 1080 cgctacgacg ctcgctccag ctggggcttc cttactatac acagcgcgca tatttcacgg 1140 ttgccagatg tcaagatggc caagttgacc agtgccgttc cggtgctcac cgcgcgcgac 1200 gtcgccggag cggtcgagtt ctggaccgac cggctcgggt tctcccggga cttcgtggag 1260 gacgacttcg ccggtgtggt ccgggacgac gtgaccctgt tcatcagcgc ggtccaggac 1320 caggtggtgc cggacaacac cctggcctgg gtgtgggtgc gcggcctgga cgagctgtac 1380 gccgagtggt cggaggtcgt gtccacgaac ttccgggacg cctccgggcc ggccatgacc 1440 gagatcggcg agcagccgtg ggggcgggag ttcgccctgc gcgacccggc cggcaactgc 1500 gtgcacttcg tggccgagga gcaggactga accttcctta aaaatttaat tttcattagt 1560 tgcagtcact ccgctttggt ttcacagtca ggaataacac tagctcgtct tcaccatgga 1620 tgccaatctc gcctattcat ggtgtataaa agttcaacat ccaaagctag aacttttgga 1680 aagagaaaga atatccgaat agggcacggc gtgccgtatt gttggagtgg actagcagaa 1740 agtgaggaag gcacaggatg agttttctcg aggccggtct ccctatagtg agtcgtatta 1800 atttcgataa gccaggttaa cctgcattaa tgaatcggcc aacgcgcggg gagaggcggt 1860 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg 1920 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg 1980 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 2040 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga 2100 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 2160 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc 2220 tttctccctt cgggaagcgt ggcgctttct caatgctcac gctgtaggta tctcagttcg 2280 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc 2340 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca 2400 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag 2460 ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct 2520 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc 2580 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga 2640 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca 2700 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat 2760 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac 2820 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt 2880 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt 2940 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag 3000 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct 3060 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt 3120 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc 3180 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt 3240 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg 3300 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg 3360 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct 3420 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc 3480 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt 3540 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt 3600 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg 3660 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat 3720 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg 3780 cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta 3840 acctataaaa ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt 3900 gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc 3960 gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt 4020 aactatgcgg catcagagca gattgtactg agagtgcac 4059 <210> 8 <211> 5904 <212> DNA <213> Artificial Sequence <220> <223> pPha-HASP1p-HGGTF1 <400> 8 catatgcata cagtgaatgt aactttcgaa ttgacagtat tagtagtcgt attgacagtg 60 aggcacgccc ctcaatgtgc gaggtggaaa atataccagc atgacaatga atcttggaga 120 ttcttttgct gtcatcaaga ttcaccgcca aatcttcagg aacctatcac gtccacaggc 180 gatgttaatt cttgagtcgt caaaacaaag tcctgtccta cctgtagaag ttgacagcga 240 gcaattgtat gcaaacttct gactttgtta taataacatt aaaggtaatt aagtatcttc 300 aattaggcat tttgtcactg tcagtccgtt ccgacaatat aggtagattt ggaatgaatc 360 ttttctatgc tgctgcgaat cttgtacacc tttgaggccg tagattctgt ccgacgaagc 420 gataattatt gcaaaataca tggactcatt attttgattc gatttctttt tggtatccga 480 ctcgaaaaga tccatcacgg cgagcgaatt cgatatcacc atgcaccacc accaccacca 540 cgccggaatg tcccccattc tcggatactg gaagattaag ggactcgtcc agcccacccg 600 tctcctcctc gaatacctcg aagaaaagta cgaagaacac ctctacgaac gtgacgaagg 660 agacaagtgg cgtaacaaga agttcgaact cggactcgaa ttccccaacc tcccctacta 720 cattgacgga gacgtcaagc tcacccagtc catggccatt attcgttaca ttgccgacaa 780 gcacaacatg ctcggaggat gccccaagga acgtgccgaa atttccatgc tcgaaggagc 840 cgtcctcgac attcgttacg gagtctcccg tattgcctac tccaaggact tcgaaaccct 900 caaggtcgac ttcctctcca agctccccga aatgctcaag atgttcgaag accgtctctg 960 ccacaagacc tacctcaacg gagaccacgt cacccacccc gacttcatgc tctacgacgc 1020 cctcgacgtc gtcctctaca tggaccccat gtgcctcgac gccttcccca agctcgtctg 1080 cttcaagaag cgtattgaag ccattcccca gattgacaag tacctcaagt cctccaagta 1140 cattgcctgg cccctccagg gatggcaggc caccttcgga ggaggagacc acccccccaa 1200 gtccgacgga ggacgtgtct ccaagggaga agaactcttc accggagtcg tccccattct 1260 cgtcgaactc gacggagacg tcaacggaca caagttctcc gtctccggag aaggagaagg 1320 agacgccacc tacggaaagc tcaccctcaa gttcatttgc accaccggaa agctccccgt 1380 cccctggccc accctcgtca ccaccctcac ctacggagtc cagtgcttct cccgttaccc 1440 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggat acgtccagga 1500 acgtaccatt ttcttcaagg acgacggaaa ctacaagacc cgtgccgaag tcaagttcga 1560 aggagacacc ctcgtcaacc gtattgaact caagggaatt gacttcaagg aagacggaaa 1620 cattctcgga cacaagctcg aatacaacta caactcccac aacgtctaca ttatggccga 1680 caagcagaag aacggaatta aggtcaactt caagattcgt cacaacattg aagacggatc 1740 cgtccagctc gccgaccact accagcagaa cacccccatt ggagacggac ccgtcctcct 1800 ccccgacaac cactacctct ccacccagtc cgccctctcc aaggacccca acgaaaagcg 1860 tgaccacatg gtcctcctcg aattcgtcac cgccgccgga attaccctcg gaatggacga 1920 actctacaag tccgacggag gacgtaccgg agaaaacctc tacttccagg gattcaacct 1980 cccccccgga aactacaaga agcccaagct cctctactgc tccaacggag gacacttcct 2040 ccgtattctc cccgacggaa ccgtcgacgg aacccgtgac cgttccgacc agcacattca 2100 gctccagctc tccgccgaat ccgtcggaga agtctacatt aagtccaccg aaaccggaca 2160 gtacctcgcc atggacaccg acggactcct ctacggatcc cagaccccca acgaagaatg 2220 cctcttcctc gaacgtctcg aagaaaacca ctacaacacc tacatttcca agaagcacgc 2280 cgaaaagaac tggttcgtcg gactcaagaa gaacggatcc tgcaagcgtg gaccccgtac 2340 ccactacgga cagaaggcca ttctcttcct ccccctcccc gtctcctccg actaatctag 2400 agtcgacctg caggcatgca agcttcagaa gcgtgctatc gaactcaacc agggacgtgc 2460 ggcacaaatg ggcatccttg ctctcatggt gcacgaacag ttgggagtct ctatccttcc 2520 ttaaaaattt aattttcatt agttgcagtc actccgcttt ggtttcacag tcaggaataa 2580 cactagctcg tcttcaccat ggatgccaat ctcgcctatt catggtgtat aaaagttcaa 2640 catccaaagc tagaactttt ggaaagagaa agaatatccg aatagggcac ggcgtgccgt 2700 attgttggag tggactagca gaaagtgagg aaggcacagg atgagttttc tcgagacata 2760 ccttcagcgt cgtcttcact gtcacagtca actgacagta atcgttgatc cggagagatt 2820 caaaattcaa tctgtttgga cctggataag acacaagagc gacatcctga catgaacgcc 2880 gtaaacagca aatcctggtt gaacacgtat ccttttgggg gcctccgcta cgacgctcgc 2940 tccagctggg gcttccttac tatacacagc gcgcatattt cacggttgcc agatgtcaag 3000 atggccaagt tgaccagtgc cgttccggtg ctcaccgcgc gcgacgtcgc cggagcggtc 3060 gagttctgga ccgaccggct cgggttctcc cgggacttcg tggaggacga cttcgccggt 3120 gtggtccggg acgacgtgac cctgttcatc agcgcggtcc aggaccaggt ggtgccggac 3180 aacaccctgg cctgggtgtg ggtgcgcggc ctggacgagc tgtacgccga gtggtcggag 3240 gtcgtgtcca cgaacttccg ggacgcctcc gggccggcca tgaccgagat cggcgagcag 3300 ccgtgggggc gggagttcgc cctgcgcgac ccggccggca actgcgtgca cttcgtggcc 3360 gaggagcagg actgaacctt ccttaaaaat ttaattttca ttagttgcag tcactccgct 3420 ttggtttcac agtcaggaat aacactagct cgtcttcacc atggatgcca atctcgccta 3480 ttcatggtgt ataaaagttc aacatccaaa gctagaactt ttggaaagag aaagaatatc 3540 cgaatagggc acggcgtgcc gtattgttgg agtggactag cagaaagtga ggaaggcaca 3600 ggatgagttt tctcgaggcc ggtctcccta tagtgagtcg tattaatttc gataagccag 3660 gttaacctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc 3720 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta 3780 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag 3840 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg 3900 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg 3960 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg 4020 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga 4080 agcgtggcgc tttctcaatg ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc 4140 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt 4200 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact 4260 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg 4320 cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt 4380 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt 4440 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct 4500 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg 4560 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt 4620 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt 4680 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc 4740 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg 4800 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc 4860 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctatta ttgttgccgg 4920 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca 4980 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga 5040 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct 5100 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg 5160 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca 5220 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata 5280 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct 5340 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact 5400 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa 5460 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc 5520 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga 5580 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga 5640 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg 5700 cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac 5760 atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc 5820 cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta tgcggcatca 5880 gagcagattg tactgagagt gcac 5904 <210> 9 <211> 5955 <212> DNA <213> Artificial Sequence <220> <223> pPha-HASP1p-HGGTF2 <400> 9 catatgcata cagtgaatgt aactttcgaa ttgacagtat tagtagtcgt attgacagtg 60 aggcacgccc ctcaatgtgc gaggtggaaa atataccagc atgacaatga atcttggaga 120 ttcttttgct gtcatcaaga ttcaccgcca aatcttcagg aacctatcac gtccacaggc 180 gatgttaatt cttgagtcgt caaaacaaag tcctgtccta cctgtagaag ttgacagcga 240 gcaattgtat gcaaacttct gactttgtta taataacatt aaaggtaatt aagtatcttc 300 aattaggcat tttgtcactg tcagtccgtt ccgacaatat aggtagattt ggaatgaatc 360 ttttctatgc tgctgcgaat cttgtacacc tttgaggccg tagattctgt ccgacgaagc 420 gataattatt gcaaaataca tggactcatt attttgattc gatttctttt tggtatccga 480 ctcgaaaaga tccatcacgg cgagcgaatt cgatatcacc atgcaccacc accaccacca 540 cgccggaatg tcccccattc tcggatactg gaagattaag ggactcgtcc agcccacccg 600 tctcctcctc gaatacctcg aagaaaagta cgaagaacac ctctacgaac gtgacgaagg 660 agacaagtgg cgtaacaaga agttcgaact cggactcgaa ttccccaacc tcccctacta 720 cattgacgga gacgtcaagc tcacccagtc catggccatt attcgttaca ttgccgacaa 780 gcacaacatg ctcggaggat gccccaagga acgtgccgaa atttccatgc tcgaaggagc 840 cgtcctcgac attcgttacg gagtctcccg tattgcctac tccaaggact tcgaaaccct 900 caaggtcgac ttcctctcca agctccccga aatgctcaag atgttcgaag accgtctctg 960 ccacaagacc tacctcaacg gagaccacgt cacccacccc gacttcatgc tctacgacgc 1020 cctcgacgtc gtcctctaca tggaccccat gtgcctcgac gccttcccca agctcgtctg 1080 cttcaagaag cgtattgaag ccattcccca gattgacaag tacctcaagt cctccaagta 1140 cattgcctgg cccctccagg gatggcaggc caccttcgga ggaggagacc acccccccaa 1200 gtccgacgga ggacgtgtct ccaagggaga agaactcttc accggagtcg tccccattct 1260 cgtcgaactc gacggagacg tcaacggaca caagttctcc gtctccggag aaggagaagg 1320 agacgccacc tacggaaagc tcaccctcaa gttcatttgc accaccggaa agctccccgt 1380 cccctggccc accctcgtca ccaccctcac ctacggagtc cagtgcttct cccgttaccc 1440 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggat acgtccagga 1500 acgtaccatt ttcttcaagg acgacggaaa ctacaagacc cgtgccgaag tcaagttcga 1560 aggagacacc ctcgtcaacc gtattgaact caagggaatt gacttcaagg aagacggaaa 1620 cattctcgga cacaagctcg aatacaacta caactcccac aacgtctaca ttatggccga 1680 caagcagaag aacggaatta aggtcaactt caagattcgt cacaacattg aagacggatc 1740 cgtccagctc gccgaccact accagcagaa cacccccatt ggagacggac ccgtcctcct 1800 ccccgacaac cactacctct ccacccagtc cgccctctcc aaggacccca acgaaaagcg 1860 tgaccacatg gtcctcctcg aattcgtcac cgccgccgga attaccctcg gaatggacga 1920 actctacaag tccgacggag gacgtaccgg agaaaacctc tacttccagg gaggacacat 1980 ggccgccgga tccattacca ccctccccgc cctccccgaa gacggaggat ccggagcctt 2040 cccccccgga cacttcaagg accccaagcg tctctactgc aagaacggag gattcttcct 2100 ccgtattcac cccgacggac gtgtcgacgg agtccgtgaa aagtccgacc cccacattaa 2160 gctccagctc caggccgaag aacgtggagt cgtctccatt aagggagtct ccgccaaccg 2220 ttacctcgcc atgaaggaag acggacgtct cctcgcctcc aagtccgtca ccgacgaatg 2280 cttcttcttc gaacgtctcg aatccaacaa ctacaacacc taccgttccc gtaagtacac 2340 ctcctggtac gtcgccctca agcgtaccgg acagtacaag ctcggatcca agaccggacc 2400 cggacagaag gccattctct tcctccccat gtccgccaag tcctaatcta gagtcgacct 2460 gcaggcatgc aagcttcaga agcgtgctat cgaactcaac cagggacgtg cggcacaaat 2520 gggcatcctt gctctcatgg tgcacgaaca gttgggagtc tctatccttc cttaaaaatt 2580 taattttcat tagttgcagt cactccgctt tggtttcaca gtcaggaata acactagctc 2640 gtcttcacca tggatgccaa tctcgcctat tcatggtgta taaaagttca acatccaaag 2700 ctagaacttt tggaaagaga aagaatatcc gaatagggca cggcgtgccg tattgttgga 2760 gtggactagc agaaagtgag gaaggcacag gatgagtttt ctcgagacat accttcagcg 2820 tcgtcttcac tgtcacagtc aactgacagt aatcgttgat ccggagagat tcaaaattca 2880 atctgtttgg acctggataa gacacaagag cgacatcctg acatgaacgc cgtaaacagc 2940 aaatcctggt tgaacacgta tccttttggg ggcctccgct acgacgctcg ctccagctgg 3000 ggcttcctta ctatacacag cgcgcatatt tcacggttgc cagatgtcaa gatggccaag 3060 ttgaccagtg ccgttccggt gctcaccgcg cgcgacgtcg ccggagcggt cgagttctgg 3120 accgaccggc tcgggttctc ccgggacttc gtggaggacg acttcgccgg tgtggtccgg 3180 gacgacgtga ccctgttcat cagcgcggtc caggaccagg tggtgccgga caacaccctg 3240 gcctgggtgt gggtgcgcgg cctggacgag ctgtacgccg agtggtcgga ggtcgtgtcc 3300 acgaacttcc gggacgcctc cgggccggcc atgaccgaga tcggcgagca gccgtggggg 3360 cgggagttcg ccctgcgcga cccggccggc aactgcgtgc acttcgtggc cgaggagcag 3420 gactgaacct tccttaaaaa tttaattttc attagttgca gtcactccgc tttggtttca 3480 cagtcaggaa taacactagc tcgtcttcac catggatgcc aatctcgcct attcatggtg 3540 tataaaagtt caacatccaa agctagaact tttggaaaga gaaagaatat ccgaataggg 3600 cacggcgtgc cgtattgttg gagtggacta gcagaaagtg aggaaggcac aggatgagtt 3660 ttctcgaggc cggtctccct atagtgagtc gtattaattt cgataagcca ggttaacctg 3720 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct 3780 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac 3840 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga 3900 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat 3960 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac 4020 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct 4080 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg 4140 ctttctcaat gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg 4200 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt 4260 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg 4320 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac 4380 ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga 4440 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt 4500 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt 4560 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga 4620 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc 4680 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct 4740 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata 4800 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca 4860 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga 4920 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga 4980 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg 5040 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga 5100 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt 5160 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct 5220 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca 5280 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat 5340 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga 5400 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc 5460 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg 5520 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc 5580 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt 5640 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca 5700 cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg 5760 aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc 5820 ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc 5880 gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact atgcggcatc agagcagatt 5940 gtactgagag tgcac 5955 <210> 10 <211> 5694 <212> DNA <213> Artificial Sequence <220> <223> pPha-HASP1p-HGGTI1 <400> 10 catatgcata cagtgaatgt aactttcgaa ttgacagtat tagtagtcgt attgacagtg 60 aggcacgccc ctcaatgtgc gaggtggaaa atataccagc atgacaatga atcttggaga 120 ttcttttgct gtcatcaaga ttcaccgcca aatcttcagg aacctatcac gtccacaggc 180 gatgttaatt cttgagtcgt caaaacaaag tcctgtccta cctgtagaag ttgacagcga 240 gcaattgtat gcaaacttct gactttgtta taataacatt aaaggtaatt aagtatcttc 300 aattaggcat tttgtcactg tcagtccgtt ccgacaatat aggtagattt ggaatgaatc 360 ttttctatgc tgctgcgaat cttgtacacc tttgaggccg tagattctgt ccgacgaagc 420 gataattatt gcaaaataca tggactcatt attttgattc gatttctttt tggtatccga 480 ctcgaaaaga tccatcacgg cgagcgaatt cgatatcacc atgcaccacc accaccacca 540 cgccggaatg tcccccattc tcggatactg gaagattaag ggactcgtcc agcccacccg 600 tctcctcctc gaatacctcg aagaaaagta cgaagaacac ctctacgaac gtgacgaagg 660 agacaagtgg cgtaacaaga agttcgaact cggactcgaa ttccccaacc tcccctacta 720 cattgacgga gacgtcaagc tcacccagtc catggccatt attcgttaca ttgccgacaa 780 gcacaacatg ctcggaggat gccccaagga acgtgccgaa atttccatgc tcgaaggagc 840 cgtcctcgac attcgttacg gagtctcccg tattgcctac tccaaggact tcgaaaccct 900 caaggtcgac ttcctctcca agctccccga aatgctcaag atgttcgaag accgtctctg 960 ccacaagacc tacctcaacg gagaccacgt cacccacccc gacttcatgc tctacgacgc 1020 cctcgacgtc gtcctctaca tggaccccat gtgcctcgac gccttcccca agctcgtctg 1080 cttcaagaag cgtattgaag ccattcccca gattgacaag tacctcaagt cctccaagta 1140 cattgcctgg cccctccagg gatggcaggc caccttcgga ggaggagacc acccccccaa 1200 gtccgacgga ggacgtgtct ccaagggaga agaactcttc accggagtcg tccccattct 1260 cgtcgaactc gacggagacg tcaacggaca caagttctcc gtctccggag aaggagaagg 1320 agacgccacc tacggaaagc tcaccctcaa gttcatttgc accaccggaa agctccccgt 1380 cccctggccc accctcgtca ccaccctcac ctacggagtc cagtgcttct cccgttaccc 1440 cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggat acgtccagga 1500 acgtaccatt ttcttcaagg acgacggaaa ctacaagacc cgtgccgaag tcaagttcga 1560 aggagacacc ctcgtcaacc gtattgaact caagggaatt gacttcaagg aagacggaaa 1620 cattctcgga cacaagctcg aatacaacta caactcccac aacgtctaca ttatggccga 1680 caagcagaag aacggaatta aggtcaactt caagattcgt cacaacattg aagacggatc 1740 cgtccagctc gccgaccact accagcagaa cacccccatt ggagacggac ccgtcctcct 1800 ccccgacaac cactacctct ccacccagtc cgccctctcc aaggacccca acgaaaagcg 1860 tgaccacatg gtcctcctcg aattcgtcac cgccgccgga attaccctcg gaatggacga 1920 actctacaag tccgacggag gacgtaccgg agaaaacctc tacttccagg gaggacccga 1980 aaccctctgc ggagccgaac tcgtcgacgc cctccagttc gtctgcggag accgtggatt 2040 ctacttcaac aagcccaccg gatacggatc ctcctcccgt cgtgcccccc agaccggaat 2100 tgtcgacgaa tgctgcttcc gttcctgcga cctccgtcgt ctcgaaatgt actgcgcccc 2160 cctcaagccc gccaagtccg cctaatctag agtcgacctg caggcatgca agcttcagaa 2220 gcgtgctatc gaactcaacc agggacgtgc ggcacaaatg ggcatccttg ctctcatggt 2280 gcacgaacag ttgggagtct ctatccttcc ttaaaaattt aattttcatt agttgcagtc 2340 actccgcttt ggtttcacag tcaggaataa cactagctcg tcttcaccat ggatgccaat 2400 ctcgcctatt catggtgtat aaaagttcaa catccaaagc tagaactttt ggaaagagaa 2460 agaatatccg aatagggcac ggcgtgccgt attgttggag tggactagca gaaagtgagg 2520 aaggcacagg atgagttttc tcgagacata ccttcagcgt cgtcttcact gtcacagtca 2580 actgacagta atcgttgatc cggagagatt caaaattcaa tctgtttgga cctggataag 2640 acacaagagc gacatcctga catgaacgcc gtaaacagca aatcctggtt gaacacgtat 2700 ccttttgggg gcctccgcta cgacgctcgc tccagctggg gcttccttac tatacacagc 2760 gcgcatattt cacggttgcc agatgtcaag atggccaagt tgaccagtgc cgttccggtg 2820 ctcaccgcgc gcgacgtcgc cggagcggtc gagttctgga ccgaccggct cgggttctcc 2880 cgggacttcg tggaggacga cttcgccggt gtggtccggg acgacgtgac cctgttcatc 2940 agcgcggtcc aggaccaggt ggtgccggac aacaccctgg cctgggtgtg ggtgcgcggc 3000 ctggacgagc tgtacgccga gtggtcggag gtcgtgtcca cgaacttccg ggacgcctcc 3060 gggccggcca tgaccgagat cggcgagcag ccgtgggggc gggagttcgc cctgcgcgac 3120 ccggccggca actgcgtgca cttcgtggcc gaggagcagg actgaacctt ccttaaaaat 3180 ttaattttca ttagttgcag tcactccgct ttggtttcac agtcaggaat aacactagct 3240 cgtcttcacc atggatgcca atctcgccta ttcatggtgt ataaaagttc aacatccaaa 3300 gctagaactt ttggaaagag aaagaatatc cgaatagggc acggcgtgcc gtattgttgg 3360 agtggactag cagaaagtga ggaaggcaca ggatgagttt tctcgaggcc ggtctcccta 3420 tagtgagtcg tattaatttc gataagccag gttaacctgc attaatgaat cggccaacgc 3480 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg 3540 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta 3600 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc 3660 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag 3720 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac 3780 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc 3840 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcaatg ctcacgctgt 3900 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc 3960 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga 4020 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta 4080 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta 4140 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga 4200 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg 4260 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag 4320 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc 4380 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact 4440 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt 4500 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta 4560 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta 4620 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc 4680 gcctccatcc agtctattata ttgttgccgg gaagctagag taagtagttc gccagttaat 4740 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt 4800 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg 4860 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca 4920 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta 4980 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg 5040 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact 5100 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg 5160 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt 5220 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga 5280 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc 5340 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa 5400 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt 5460 attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt 5520 ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt 5580 ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg 5640 tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt gcac 5694
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| BIOLOGY OF REPRODUCTION, 제86권, 제5호, 163, 제1-10면* |
| 국가 R&D 과제, 식물성 플랑크톤 Phaeodactylym tricornutum 유래 주름개선 화장품 원료 hEGF 및 PTD-hEGF 생산 시스템 구축 및 제품화(인터넷 URL<https://www.ntis.go.kr/project/pjtInfo.do?pjtId=1425113736& pageCode=TH_TOTAL_PJT_DTL> 2017년).* |
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